Radionuclide labeling and evaluation of candidate radioligands for PET imaging of histone deacetylase in the brain

Article abstract of DOI:10.1016/j.bmcl.2013.10.038

Histone deacetylases (HDACs) regulate gene expression by inducing conformational changes in chromatin. Ever since the discovery of a naturally occurring HDAC inhibitor, trichostatin A (TSA) stimulated the recent development of suberoylanilide (SAHA, Zolinza), HDAC has become an important molecular target for drug development. This has created the need to develop specific in vivo radioligands to study epigenetic regulation and HDAC engagement for drug development for diseases including cancer and psychiatric disorders. 6-([ ¹⁸F]Fluoroacetamido)-1-hexanoicanilide ([¹⁸F]FAHA) was recently developed as a HDAC substrate and shows moderate blood-brain barrier (BBB) permeability and specific signal (by metabolic trapping/or deacetylation) but rapid metabolism. Here, we report the radiosynthesis of two carbon-11 labeled candidate radiotracers (substrate- and inhibitor-based radioligand) for HDAC and their evaluation in non-human primate brain. PET studies showed very low brain uptake and rapid metabolism of both labeled compounds but revealed a surprising enhancement of brain penetration by F for H substitution when comparing one of these to [¹⁸F]FAHA. Further structural refinement is needed for the development of brain-penetrant, metabolically stable HDAC radiotracers and to understand the role of fluorine substitution on brain penetration.

Full text of DOI:10.1016/j.bmcl.2013.10.038

Bioorganic & Medicinal Chemistry Letters 23 (2013) 6700–6705
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Radionuclide labeling and evaluation of candidate radioligands
for PET imaging of histone deacetylase in the brain
Young Jun Seo a,b, Lisa Muench c, Alicia Reid d, Jinzhu Chen a, Yeona Kang a, Jacob M. Hooker e,
,
⇑
Nora D. Volkow c,f, Joanna S. Fowler a, Sung Won Kim c,
⇑
a Biosciences Department, Brookhaven National Laboratory, Upton, NY 11973, USA
b Department of Chemistry, Chonbuk National University, Jeonju 561-756, South Korea
c Laboratory of Neuroimaging, National Institute on Alcohol Abuse and Alcoholism, Bethesda, MD 20892, USA
d Department of Physical, Environmental and Computer Sciences, Medgar Evers College, The City University of New York, Brooklyn, NY 11225, USA
e Athinoula A. Martinos Center for Biomedical Imaging, Department of Radiology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA 02129, USA
f National Institute on Drug Abuse, 6001 Executive Blvd, Rockville, MD 20852, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Histone deacetylases (HDACs) regulate gene expression by inducing conformational changes in chroma-
tin. Ever since the discovery of a naturally occurring HDAC inhibitor, trichostatin A (TSA) stimulated the
recent development of suberoylanilide (SAHA, ZolinzaÒ), HDAC has become an important molecular tar-
get for drug development. This has created the need to develop specific in vivo radioligands to study epi-
genetic regulation and HDAC engagement for drug development for diseases including cancer and
psychiatric disorders. 6-([18F]Fluoroacetamido)-1-hexanoicanilide ([18F]FAHA) was recently developed
as a HDAC substrate and shows moderate blood–brain barrier (BBB) permeability and specific signal
(by metabolic trapping/or deacetylation) but rapid metabolism. Here, we report the radiosynthesis of
two carbon-11 labeled candidate radiotracers (substrate- and inhibitor-based radioligand) for HDAC
and their evaluation in non-human primate brain. PET studies showed very low brain uptake and rapid
metabolism of both labeled compounds but revealed a surprising enhancement of brain penetration by F
for H substitution when comparing one of these to [18F]FAHA. Further structural refinement is needed for
the development of brain-penetrant, metabolically stable HDAC radiotracers and to understand the role
of fluorine substitution on brain penetration.
Received 1 August 2013
Revised 16 October 2013
Accepted 21 October 2013
Available online 30 October 2013
Keywords:
Carbon-11
Positron emission tomography
SAHA
Brain
Epigenetics
Brain permeability
Published by Elsevier Ltd.
Gene expression is regulated, in part, through enzyme catalyzed
epigenetic modifications targeting both the DNA and its associated
histone proteins.1 For example, histone acetyl transferases (HATs)
catalyze the acetylation of the lysine residues of histone proteins
removing the positive charge and rendering the DNA more accessi-
ble to transcription initiation complexes and RNA polymerase. In
contrast, histone deacetylases (HDACs) typically catalyze the
hydrolysis of the acetyl groups from lysine residues of histone pro-
teins condensing the chromatin and repressing gene expression.2
Because the folding process induced by the deacetylation of
histones represses the expression of genes which are involved in
critical metabolic processes such as apoptosis, cell-cycle arrest
and differentiation, HDAC has become an important molecular
target in drug development.3–6
The discovery of a naturally occurring HDAC inhibitor, trichos-
tatin A (TSA, Fig. 1),7,8 an antifungal antibiotic with anticancer
activity, has stimulated the development of inhibitors of HDAC
including Vorinostat (SAHA, Fig. 1) which was recently approved
by FDA for the treatment of cutaneous T cell lymphoma (CTCL)
and other cancers.9 HDAC inhibitors are also of interest in the
study and treatment of a number of CNS disorders including
depression, addiction and neurodegenerative diseases.4,10,11 In fact,
valproic acid, a class I HDAC inhibitor,6 is one of the most widely
used and effective antiseizure medications, which we recently
showed its poor blood–brain barrier (BBB) penetration explaining
the high doses needed for therapeutic efficacy.12 For this reason,
the development of brain penetrant radiotracers for positron emis-
sion tomography (PET) imaging of HDAC in the brain would ad-
vance the study of this important epigenetic marker as well as
the measurement of target engagement during HDAC inhibitor
therapy.
⇑
Corresponding authors. Address: National Institute on Alcohol Abuse and
Alcoholism, 10 Center Dr, rm B2L304, Bethesda, MD 20892-1013, USA. Tel.: +1 301
496 5548 (S. Kim); Tel.: +1 617 726 6596 (J. M. Hooker).
E-mail addresses: hooker@nmr.mgh.harvard.edu (J.M. Hooker), sunny.kim@nih.
gov (S.W. Kim).
Only a handful of HDAC active compounds including HDAC
inhibitor drugs have been radiolabeled with either F-18 (t1/2
:
110 min) or C-11 (t1/2: 20.4 min) and evaluated for specificity for
0960-894X/$ - see front matter Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.bmcl.2013.10.038

Y. J. Seo et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6700–6705
6701
[
18F]fluoroacetic acid and [11C]acetic acid respectively as labeled
HO
NH
O
metabolites. In addition, we attempted to label M344, which has
4 times higher potency22 than SAHA, and, although we were
not successful, we describe our experience with this difficult-
to-alkylate aromatic amine.23
O
H
N
OH
O
N
H
3
O
N
The syntheses of the two hydroxamate reference compounds, 1
and 2, were achieved according to known procedures21,22 and their
precursors for C-11 labeling were also synthesized as shown
Scheme 1. Labeling 1 with [11C]methyl iodide required a 2-step
one-pot reaction, first forming the methyl ether followed by con-
version to the hydroxamate. This was necessary to avoid preferen-
tial [11C]methylation of the hydroxamate moiety under basic
conditions. Our initial attempt using a hydroxylamine hydrochlo-
ride and strong bases such as sodium hydroxide24 was not success-
ful due to slow reaction rate and side product formation. However,
treatment of the ester with hydroxylamine hydrochloride, using
potassium cyanide as a catalyst,25,26 converted the ester to the
hydroxamate within 5 min. Crude [11C]1 was subsequently puri-
fied by high performance liquid chromatography (HPLC). After
optimization, the average decay-corrected radiochemical yield
(RCY), calculated from 11CH3I, was 23 3% (decay-corrected,
n = 3) in a total synthesis time of 50 min. Analytical HPLC and
TLC demonstrated that the radiochemical purity was >99% and
SAHA
Trichostatin A
Figure 1. Structures of SAHA and trichostatin A (TSA).
PET imaging (Fig. 2). The first was [18F]FAHA13 which has a similar
structure to SAHA except that the zinc binding hydroxamate moi-
ety is replaced by a fluoroacetylamido group. [18F]FAHA shows
good brain penetration, its deacetylation process in brain is
blocked by SAHA in a dose-dependent manner14,15 and its distribu-
tion as measured by PET parallels the HDAC IIa distribution in the
primate brain as measured by immunohistochemical analysis of
post-mortem tissue.15 18F]FAHA is a HDAC substrate rather than
[
an inhibitor and manifests very rapid metabolism to [18F]fluoroac-
etate which can be trapped in the brain.14 More recently, hyroxa-
mate inhibitors, F-18 labeled SAHA derivatives ([18F]SAHA,16
[
18F]FESAHA17) were synthesized and evaluated in a mouse tumor
model. While [18F]SAHA16 showed HDAC specific binding in tumor,
both of them showed very poor BBB permeability. In another study,
we radiolabeled the benzamide HDAC inhibitor, MS-27518 with
carbon-11 for PET studies and reported that it showed very poor
BBB penetration as a radiotracer.19 Most recently, Pike and co-
workers labeled the b-carboline hydroxamate (KB631, Fig. 2),
which has high affinity for HDAC6, with carbon-11 and also
observed low brain penetration.20
the specific activity was 280 50 mCi/lmol (n = 3) at the time of
administration to the baboon.
For [11C]2 ([11C]M344), we anticipated that N-methylation of
precursor, 9, using [11C]methyl iodide/or triflate would be difficult
due to inherent low nucleophilicity of the aromatic amine and the
presence of a p-carbonyl group. In addition, the hydroxamate, if
unprotected, would alkylate preferentially to the aromatic nitrogen
in basic condition, we explored reductive amination with
11C]formaldehyde (Scheme 1). We used N-methylaniline as a
Based on the [18F]FESAHA17 and [18F]FAHA PET studies, we set
out to develop a high affinity radiotracer which also crosses the
BBB freely and is metabolically stable for PET studies of brain
HDAC in humans. Our preference has been to radiolabel with car-
bon-11 since its short half-life (20.4 min) enables the performance
of serial studies in the same day wherein a subject or patients can
serve as his/her own control. Herein we report the synthesis of
precursors and carbon-11 radiolabeling of two different types of
radiotracer candidates, an inhibitor and a substrate (Fig. 3), and
their PET studies in the baboon brain and an analysis of plasma
clearance. We predicted that the potent TSA-like HDAC inhibitor
1 (IC50, 15 nM for HDAC1)21 would be more stable in vivo than
HDAC substrate [18F]FAHA. We also predicted that HDAC substrate
compound 3 would be rapidly deacetylated similar to FAHA and
would have similar brain uptake and kinetics since the
deacetylation of [18F]FAHA and [11C]3 by HDAC would produce
[
model compound and [11C]formaldehyde formed in situ from the
reaction of trimethylamine oxide and [11C]methyl iodide.27 Unfor-
tunately the yield of [11C]dimethylaniline was unacceptably low.
Nonetheless we proceeded to try these reductive alkylation condi-
tions of 9 since reductive alkylation with in situ generated
[
11C]formaldehyde would have the advantage of avoiding the pro-
tection/deprotection of the hydroxamate group which would be re-
quired for alkylation with [11C]methyl iodide. More specifically, the
hydroxamate is acidic (pKa = 8–9) and thus anionic under basic
conditions and would be predicted to be alkylated with
[
11C]methyl iodide. For the reductive methylation, we used ‘mild’
reductants such as sodium cyanoborohydride. We obtained an
even poorer labeling efficiency for [11C]2 than for N-methyl aniline,
which we attributed to reduced nucleophilicity of the substrate
due to the presence of the p-carbonyl group.
O
O
N
H
*
NH
N
F
H
N
H
N
NH2
O
2
*
O
O
MS-275
[18F]FAHA
O
O
O
H
N
H
N
OH
OH
N
NH-OH
N
H
N
H
3
3
O
O
*
*
F
F
N
*
[18F]SAHA
[18F]FESAHA
[11C]KB631
Figure 2. Structures of radiolabeled HDAC inhibitors. ⁄C-11 or F-18.

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Y. J. Seo et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6700–6705
O
O
O
3
H
N
O
OH
H
N
OH
N
H
N
H
3
N
H
O
*
*
2
O
N
O
*
[11C]1
[11C]2, [11C]M344
[11C]3
Figure 3. Structures of C-11 labeled trichostatin A, SAHA, and FAHA derivatives. ⁄C-11 or F-18.
O
O
A
B
O
NHOH
i
ii
OMe
OMe
3
Cl
3
O
3
iii
O
O
O
HO
Bn
11CH3
[11C]1
4
6
5
O
O
O
O
iv
v
OMe
OMe
N
H
OMe
OMe
3
Bn
HN
N
N
7
8
O
O
O
O
vii
vi
N
N
H
OMe
O
3
H
H311C
3
N
HN
9
10
O
viii
N
H
NHOH
3
H311C
N
[11C]2, [11C]M344
C
O
H
N
H
ix
N
11
NH2
N
H
CCH3
2
2
O
O
[11C]3
11
Scheme 1. Synthesis of C-11 labeled HDAC radioligands. Reagents and conditions: (i) phenol, aluminum chloride/ DCM; (ii) NaH/DMF, [11C]methyl iodide, 5 min, 70 °C; (iii)
NH2OHÁHCl, NaOH, KCN, MeOH/THF/water (1:1:0.4); (iv) benzyl bromide, potassium carbonate/DMF; (v) methyl 7-aminoheptanoate; NaH/DMF; (vi) H2, Pd/C/EtOAc; (vii)
[
11C]formaldehyde, then NaCNBH3; (viii) NH2OHÁHCl, NaOH, KCN, MeOH/THF/water (1:1:0.4); (ix) [11C]acetyl chloride, triethylamine, THF.
Radiolabeling of the FAHA analog 3 with C-11 required 2 steps,
samples were analyzed using HPLC to measure the fraction of par-
first formation of [11C]acetyl chloride using from [11C]carboxyla-
tion of methylmagnesium bromide followed by treatment with
phthaloyl chloride as described previously.28 Distillation of
ent labeled compound at different time points during the scan. This
information was used to measure the AUC for the parent labeled
compounds.
[
11C]acetyl chloride into amine, 11,13 in the presence of triethyl-
As shown Figures 4 and 5, [11C]1 showed lack of brain uptake,
compared with [18F]FAHA, even though measured plasma protein
binding (91.5%, n = 5) and lipophilicity (logD7.4 = 0.5, n = 5) were
in the acceptable range for brain penetration.29 The pattern biodis-
tribution of [11C]1 was homogenous in various brain regions indi-
cating a lack of specificity (Fig. 6). Time–activity curves for [11C]1
in peripheral organs at baseline and after pretreatment with SAHA
(1 mg/kg, 5 min before injection) showed no significant differences
indicating that, similar to brain, there is no binding specificity in
the major peripheral organs (Fig. 7).
amine in THF resulted in the formation of [11C]3 which was puri-
fied by HPLC. Isolated radiochemical yield and radiochemical
purity were 48 7% (decay-corrected, n = 5), >99% measured by
radioTLC, respectively. Specific activity was 163 38 mCi/lmol
(n = 2) at the time of the injection.
PET studies in baboons were performed with [11C]1 and [11C]3
to determine organ uptake and clearance and regional distribution
over 90 min scanning period after intravenous administration.
Arterial blood plasma were also collected and counted and selected

Y. J. Seo et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6700–6705
6703
0.010
WB
PU
CB
CD
0.008
0.006
0.004
0.002
0.000
0
20
40
60
80
Time (min)
Figure 4. Time–activity curves in baboon brain for [11C]1, [11C]3, [18F]FAHA (data
from Reid et al., 2009). Similar data for [11C]acetate, a potential metabolite of [11C]3
is shown for comparison.
Figure 6. Time course for C-11 in different regions of the baboon brain after the
administration of [11C]1; WB (whole brain); CB (cerebellum); PU (putamen); CD
(caudate). %ID/cc, 100Â the radioactivity concentration in tissue/injected
radioactivity.
0.200
[11C]1
A
liver
heart
0.160
kidney
liver (SAHA)
BEJ289dy2
0.120
0.080
0.040
0.000
heart (SAHA)
kidney (SAHA)
[11C]3
B
0
20
40
60
80
Time (min)
BEJ436dy3
Figure 7. Comparison of time-activity curves in peripheral organs between
baseline and after pretreatment with SAHA (1 mg/kg) of [11C]1.
[18F]FAHA
a much larger impact than would be predicted based on their
structural similarities. We have made a similar observation for a
series of lipophilic [11C]benzamides where the substitution of a
fluorine for a hydrogen atom significantly increases brain penetra-
tion (unpublished). In addition, when compared with [11C]acetic
acid, the brain uptake of [18F]fluoroacetic acid was also 2–3 times
higher than that of [11C]acetic acid. Understanding the mecha-
nisms(s) underlying the effect of fluorine substitution on brain
penetration is important for CNS drug development and merits fur-
ther investigation.
C
BEH193dy1
Figure 5. Averaged PET images for baboon brain over a 90 min scanning period for
[
11C]1 (panel A), [11C]3 (Panel B) and [18F]FAHA (panel C) over a 90 min scan. Each
panel shows three views: transaxial (left); sagittal (middle) and; coronal (right).
Based on our results and prior studies, the most important
physiological criterion to be optimized for the future development
of HDAC radiotracers for brain studies is BBB permeability. In order
to set the guideline of each molecular property known to affect BBB
permeability, we compared lipophilicity (clogP) and polar surface
area (PSA) for known HDAC inhibitors and radioligands as shown
in Table 1. Both molecular weight and clogP for all selected com-
pounds were within acceptable ranges, where many conventional
PET radiotracers also fall.30 However, the PSA range is broad
(73.6–107.1) and relatively high compared with conventional
radiotracers. Lower PSA values are known to be associated with
higher BBB permeability. For example, in a class-by-class compar-
ison, the PSA of BBB permeable hydroxamate should be lower than
Prior studies have shown that [18F]FAHA has moderate brain
penetration which is reduced by treatment with SAHA indicating
that radioactivity accumulation in the brain is HDAC-specific.14,15
However, contrary to our prediction, the brain uptake of [11C]3,
which only differs from [18F]FAHA by the substitution of a hydro-
gen atom for the fluorine atom in the acetyl group, was very poor
(Fig. 4). The difference is not due to different rates of metabolism;
the fraction of [18F]FAHA in plasma at 10 min post is actually lower
than that for [11C]3 (1% vs 10%) (Fig. 8). This can be clearly seen in
the plasma AUC’s which clearly show a lower AUC for [18F]FAHA
(Fig. 8) than for [11C]3. Thus the presence of a fluorine atom has

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Y. J. Seo et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6700–6705
100
90
80
70
60
50
40
30
20
10
0
4000
[¹¹C]1 (n=2)
3500
[¹¹C]3 (n=2)
[¹8F. ]FAHA
[¹¹C]1 (n=2)
3000
[¹¹C]3 (n=2)
2500
2000
1500
1000
500
0
0
20
40
60
80
0
5
10
15
20
25
30
Time (min)
Time (min)
Figure 8. (left panel)% of unchanged (parent) labeled compound in plasma at different times after injection of [11C]1, [1111C]3 and [18F]FAHA; (right panel) AUC for [11C]1,
11C]3 and [18F]FAHA.
[
Table 1
Calculated molecular descriptors for HDAC inhibitors
Type
Class
Compound
MWa
clogBB
PSAb
clogPb
Sb
À1.95
Mc
Inhibitor
Benzamide
Hydroxamate
[
11C]MS-275
376.4
302.4
264.3
282.3
310.4
337.4
279.3
307.4
266.3
248.3
À0.75
À0.48
À0.87
À0.85
À0.91
À0.17
À0.77
À0.73
À0.64
À0.54
118.8
93.1
102.5
102.5
102.5
73.6
101.5
107.1
77.0
2.5 (1.79)d
2.2
1.8
2.0
2.2
TSA
SAHA
À1.60
À1.85
À1.75
À1.90
ÀÀ0.19
À1.84
À2.08
À0.94
À1.05
[
[
[
[
[
[
[
18F]SAHA
18F]FESAHA
11C]KB631
11C]1
2.5
2.4 (0.5)d
1.8
11C]2, M344
18F]FAHA
11C]3
Substrate
Acetamide
1.7 (1.39)d
1.7
77.6
a
b
c
Molecular weight.
Calculated (QuikProp, version 2.0, Schrödinger).
Calculated (MOE, version 2012. 10, chemical computing group).
d
Measured logD7.4
.
that of the lowest PSA hydroxamate, [11C]KB631 which showed
little brain uptake.20 In case of HDAC substrates, the PSA of com-
pound [11C]3 is slightly higher than that of [18F]FAHA which has
shown suitable BBB permeability14 as a imaging probe. However,
this small difference affected the clogBB value (BB is ratio of brain
to plasma concentration of drug in steady state) and may affect
brain entry rate. Therefore, we suggest that potential acetamide
types of HDAC radiotracers should have a PSA 677.
In summary, we successfully synthesized C-11 labeled trichos-
tatin A derivative, [11C]1, and C-11 version of [18F]FAHA-like HDAC
substrate, [11C]3, to evaluate and compare their potential as a
HDAC imaging probes. However, their lack of BBB permeability
and HDAC-specific signal limited their utility as a CNS radiotracers
for HDAC.
Don Warner) for carrying out primate imaging experiments. We
used two computational chemistry programs available in the Cen-
ter 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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