Design, Synthesis, and Blood-Brain Barrier Transport Study of Pyrilamine Derivatives as Histone Deacetylase Inhibitors

Article abstract of DOI:10.1021/acsmedchemlett.8b00099

We designed and synthesized a pyrilamine derivative 1 as a selective class I HDAC inhibitor that targets pyrilamine-sensitive proton-coupled organic cation antiporter (PYSOCA) at the blood-brain barrier (BBB). Introduction of pyrilamine moiety to benzamid

Full text of DOI:10.1021/acsmedchemlett.8b00099

Subscriber access provided by Kaohsiung Medical University
Letter
Design, Synthesis and Blood-Brain Barrier Transport Study
of Pyrilamine Derivatives as Histone Deacetylase Inhibitors
Seiya Hiranaka, Yuma Tega, Kei Higuchi, Toshiki Kurosawa, Yoshiharu Deguchi,
Mayumi Arata, Akihiro Ito, Minoru Yoshida, Yasuo Nagaoka, and Takaaki Sumiyoshi
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.8b00099 • Publication Date (Web): 23 Aug 2018
Downloaded from http://pubs.acs.org on August 24, 2018
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 6
ACS Medicinal Chemistry Letters
1
2
3
4
5
6
7
8
9
Design, Synthesis and Blood-Brain Barrier Transport Study of
Pyrilamine Derivatives as Histone Deacetylase Inhibitors
Seiya Hiranaka,† Yuma Tega,‡ Kei Higuchi,‡ Toshiki Kurosawa,‡ Yoshiharu Deguchi,‡ Mayumi Arata,ǁ
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Akihiro Ito, § Minoru Yoshida,⸸,ǁ Yasuo Nagaoka,† and Takaaki Sumiyoshi*,†
,
† Department of Life Science and Biotechnology, Faculty of Chemistry, Materials and Bioengineering, Kansai University,
Yamateꢀcho 3ꢀ3ꢀ35, Suita, Osaka, 564ꢀ8680, Japan
‡ Faculty of PharmaꢀSciences, Teikyo University, 2ꢀ11ꢀ1 Kaga, Itabashiꢀku, Tokyo 173ꢀ8605, Japan
⸸ Chemical Genomics Research Group, RIKEN Center for Sustainable Resource Science, 2ꢀ1 Hirosawa, Wako, Saitama
351ꢀ0198, Japan
ǁ Seed Compounds Exploratory Unit for Drug Discovery Platform, RIKEN Center for Sustainable Resource Science, 2ꢀ1
Hirosawa, Wako, Saitama 351ꢀ0198, Japan
§ School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, 1432ꢀ1 Horinouchi, Hachioji, Tokyo 192ꢀ0392,
Japan
KEYWORDS: Histone deacetylase, HDAC inhibitor, Blood brain barrier, Pyrilamine sensitive proton coupled organic cati-
on antiporter, Selective inhibitor
ABSTRACT: We designed and synthesized a pyrilamine derivative 1 as a selective class I HDAC inhibitor that targets pyrilamine
sensitive organic cation antiporter (PYSOCA) at the blood brain barrier (BBB). Introduction of pyrilamine moiety to benzamide
type HDAC inhibitors kept selective class I HDAC inhibitory activity and increased BBB permeability. Our BBB transport study
showed that compound 1 is a substrate of PYSOCA. Thus, our findings suggest that the hybrid method of HDAC inhibitor and subꢀ
strate of PYSOCA such as pyrilamine is useful for development of HDAC inhibitors with increased BBB permeability.
Histone deacetylases (HDACs) are a family of enzymes that
repress gene expression by catalyzing the post translational
hydrolytic cleavage of acetyl groups present on lysine residues
of nuclear histones.1,2 Excluding the nonꢀzincꢀdependent
HDACs, HDACs consist of eleven isoforms categorized into
four classes as follows: class I (HDAC1, 2, 3, 8), class IIa
(HDAC4, 5, 7, 9), class IIb (HDAC6, 10) and class IV
(HDAC11).3 Since the discovery that transcription is activated
by the HDAC inhibitor trichostatin A (TSA)4, medicinal
chemists have discovered a variety of HDAC inhibitors (Figꢀ
ure S1, see Supporting Information). Among them, romidepsin
(FK228), vorinostat (SAHA), and panovinostat have been
approved as antiꢀcancer drugs by the United States Food and
Drug Administration (Figure S1). Also, HDACs are the attracꢀ
tive drug targets in many central nervous system (CNS) disꢀ
eases.5 In fact, HDAC inhibitors showed diseaseꢀmodifying
effects in CNS disease models.6 For example, class I HDAC
inhibitors and HDAC6 inhibitors improved diseaseꢀlike behavꢀ
iors in Alzheimer’s disease.7ꢀ10 In addition, HDAC inhibitor
improved disease phenotype in Huntington’s disease model.11ꢀ
14 Moreover, CIꢀ994 was exposed to brain and improved postꢀ
traumatic stress disorder (PTSD)ꢀlike behavior in rats.15 Altꢀ
hough a variety of CNSꢀpenetrant HDAC inhibitors have been
identified (Figure S2, see Supporting Information), there is no
HDAC inhibitors approved as CNS drugs. One of the hurdles
of applying HDAC inhibitors to CNS drugs is that they generꢀ
ally show poor bloodꢀbrain barrier (BBB) permeability due to
the presence of polar hydroxamic acid or anilinic amino group
as zincꢀbinding region (ZBN). In fact, positron emission toꢀ
mography (PET) studies using radiolabeled HDAC inhibitors
indicate that brain penetration of MSꢀ275, butyric acid, 4ꢀ
phenyl butyric acid, valproic acid and SAHA were poor.16ꢀ20
Efforts to discover HDAC inhibitors with improved BBB
permeability have mainly focused on increasing the lipophilicꢀ
ity. For example, Hooker and coꢀworkers reported that some
HDAC inhibitors showed good CNS distribution, based on
PET studies.21ꢀ25 However, increasing the lipophilicity generalꢀ
ly leads to metabolic instability and increases affinities to othꢀ
er offꢀtarget proteins and protein binding.26 Therefore, a new
strategy is needed for delivery of HDAC inhibitors to the CNS.
One approach would be targeting compounds to transporters
expressed in brain capillary endothelial cells, which form the
BBB. Polt and coꢀworkers designed prodrugs targeting gluꢀ
cose transporter 1.27 In addition, large amino acid transporter
128 and vitamin C transporter 229 have been used to deliver
compounds to the brain. On the other hand, Wang and coꢀ
workers focused on pyrilamineꢀsensitive protonꢀcoupled orꢀ
ganic cation (H+/OC) antiporter (PYSOCA); they designed
conjugates of naproxen and cyclic tertiary amines, and conꢀ
firmed that they were distributed to CNS.30 Nevertheless, no
BBB permeable shuttle moiety is yet available for targeting
HDAC inhibitors to transporters at the BBB. Here, we deꢀ
scribe the design, synthesis and pharmacokinetic evaluations
of compound 1 as a BBB permeable HDAC inhibitor targeting
PYSOCA. A transport study using human immortalized brain
capillary endothelial cells (hCMEC/D3) confirmed that comꢀ
pound 1 is recognized by PYSOCA. In addition, we confirmed
that compound 1 showed both HDACꢀinhibitory activity in
vitro and increased BBB permeability in rats.
We focused on pyrilamine as a shuttle moiety to increase
BBB permeability because PYSOCA recognizes a variety of
centrally acting drugs.31,32 Indeed, Yamazaki and coꢀworkers
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
have reported that PYSOCA mediates BBB penetration and
brain accumulation of some pyrilamineꢀcontaining histamine
H1 receptor antagonists,33,34 including compounds containing a
polar moiety. Thus, we adopted a hybrid strategy based on
linking benzamideꢀtype HDAC inhibitors with substrates of
PYSOCA. Class I selective HDAC inhibitors generally inꢀ
Page 2 of 6
inhibitory activity (Figure S3, see Supporting Information) by
monitoring acetylated histone protein. Evaluation of timeꢀ
dependent inhibition is necessary to clarify the potency of
compound 1 because benzamideꢀtype HDAC inhibitors show
slow and tight binding.37 As a result, both compound 1 and CIꢀ
994 increased acetylated histone H3K9 in HeLaS3 cells at 10
ꢁM (Figure S3A). In addition, in vitro kinetic analysis suggest
that compound 1 inhibited HDAC timeꢀdependently (Figure
S3B). Treatment of both compound 1 and CIꢀ994 increased
acetylated histone H3K9 from six hours after the addition of
the compounds. On the other hand, TSA increased acetylated
histone immediately (Figure S3B). Especially, compound 1
showed more continuous HDAC inhibitory activity than CIꢀ
994 and TSA (Figures S3C, S3D and S3E). These results indiꢀ
cate that compound 1 and CIꢀ994 inhibited HDAC at the same
content in HeLaK3 cells.
1
2
3
4
5
6
7
8
clude
Nꢀ(2ꢀaminophenyl)pyridylamide
or
Nꢀ(2ꢀ
aminophenyl)benzamide as the ZBN (Figure 1). Since the 2ꢀ
aminopyridine structure is a known linker moiety,35 we deꢀ
signed the pyrilamineꢀbased hybrid compound 1 (Figure 1).
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
O
N
H
O
NH2
X = CH or N
Pharmacophore of class I HDAC inhibitors
X
CAP region
N
H
N
N
NH2
N
N
MeO
MeO
N
N
Pyrilamine
1
Figure 1. Hybrid design strategy for compound 1.
Table 1. Structure-activity relationship study of compound
1 and its derivatives.
R
5
4
O
3
1
N
2
H
NH2
N
N
MeO
NMe2
HDAC inhibition
(%, 100 ꢁM)
entry
compound
R
HDAC1
HDAC6
1
2
3
4
5
6
7
8
9
1
2
3
4
5
6
7
H
Cl
Br
61
62
52
45
64
62
64
67
100
11
7
21
16
15
14
15
18
I
thiophenꢀ2ꢀyl
phenyl
furanꢀ2ꢀyl
CIꢀ994
TSA
98
The SAR study of compound 1 and its derivatives are sumꢀ
marized in Table 1. Compound 1 inhibited HDAC1 (61 % at
100 ꢁM), but did not inhibit HDAC6 (11% at 100 ꢁM). The
inhibitory activities against HDACs are similar to those of CIꢀ
994 (entries 1 and 9). Replacement of hydrogen atom at 5ꢀ
position of benzene ring by chloride kept HDAC1 inhibitory
activity (entry 2). On the other hand, introduction of bromide
or iodide at 5ꢀposition decreased HDAC1 inhibitory activity
(entries 3 and 4). The compounds with thiophenꢀ2ꢀyl, furanꢀ2ꢀ
yl or phenyl group at 5ꢀposition of benzene ring inhibited
HDAC1 at the same content (entries 5ꢀ7). These results indiꢀ
cate that introduction of internal cavity motif does not signifiꢀ
cantly increase HDAC1 inhibitory activity in spite of the inꢀ
crease of molecular weight. Based on our criteria that molecuꢀ
lar weight is less than 430 which is generally advantageous for
CNS drugs,36 we picked up compound 1 for further evaluation.
After that, we evaluated IC50 values and isozyme selectivity to
clarify pharmacological potential of compound 1. As a result,
compound 1 showed selectivity for class I HDACs over other
HDAC isozymes (HDAC1 IC50 = 1.5 µM, HDAC2 IC50 = 5.5
µM, HDAC3 IC50 = 12.0 µM, HDAC10 IC50 = 15.5 µM, other
HDACs: < 10% inhibition at 30 µM, Tables S1 and S2). These
results indicate that pyrilamine moiety is acceptable as a CAP
region.
Figure 2. Timeꢀcourses of uptake of (A) compound 1 and (B) CIꢀ
994 by hCMEC/D3 cells. Uptake were measured at 37 °C (open
circles) or 4 °C (closed squares) for the indicated times. Each
point represents the mean ± S.E. (n = 4). Where the S.E. is not
shown, it is smaller than the symbol. Uptake of 1 and CIꢀ994 by
hCMEC/D3 cells increased linearly (black lines) for at least 2 min
and 1 min, respectively. *p < 0.01, significantly different from the
uptake at 4 °C at the corresponding time. (C) Concentrationꢀ
dependency of the uptake of compound 1 by hCMEC/D3 cells.
The concentrationꢀdependency of the uptake of compound 1 was
examined at 37 °C for 2 min. Kinetic parameters obtained from
Eq. 1 were Km 326 ± 72 µM and Vmax 5.17 ± 0.86 nmol/mg proꢀ
tein/min. The solid line was calculated according to Eq. 1 with
these parameters. Each point represents the mean ± S.E. (n = 4).
Where the S.E. is not shown, it is smaller than the symbol. (D),
(E) Dependency of the uptake of compound 1 by hCMEC/D3
cells on metabolic energy (D) and intracellular pH (E). (D)
hCMEC/D3 cells were pretreated with NaN3 (0.1%) for 20 min to
deplete cellular ATP, and uptake of compound 1 was measured at
37 °C for 2 min. (E) hCMEC/D3 cells were pretreated with
transport buffer in the absence (Control and Acute) or presence
(Pre) of 30 mM NH4Cl, and then uptake of compound 1 was
measured in the absence (Control and Pre) or presence (Acute) of
Next, we evaluated functional cellꢀbased assay for HDAC
ACS Paragon Plus Environment

Page 3 of 6
ACS Medicinal Chemistry Letters
known substrate of PYSOCA.34 The uptake of compound 1
was competitively inhibited by diphenhydramine with a Ki
value of 104 ± 28 µM, again suggesting that PYSOCA is inꢀ
volved in the uptake of compound 1 by hCMEC/D3 cells
(Figure 3).
30 mM NH4Cl at 37 °C for 1 min. Each column represents the
mean ± S.E. (n = 4). *p < 0.01, significantly different from conꢀ
trol.
1
2
3
4
5
6
7
8
To examine whether compound 1 and CIꢀ994 are taken up
via PYSOCA, we first evaluated their uptake by hCMEC/D3
cells at 37°C or 4 °C (Figures 2A and 2B). The uptake clearꢀ
ance of compound 1, which was calculated from the initial
slope, was estimated as 107 µL/min/mg protein. This clearꢀ
ance was 85ꢀfold greater than that of CIꢀ994 (1.26 µL/min/mg
protein). The uptake of compound 1 was significantly lower at
4 °C for up to 10 min, while CIꢀ994 uptake at 4 °C slowly
increased and reached a similar cellꢀtoꢀmedium ratio to that at
37 °C by 30 min. Uptake of compound 1 was concentrationꢀ
dependent with a Km of 326 ± 72 µM and a Vmax of 5.17 ± 0.86
nmol/mg protein/min (Figure 2C). These results suggest that
compound 1 is taken up by hCMEC/D3 cells via a carrierꢀ
mediated transport process.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Next, we investigated whether the above transport properꢀ
ties are consistent with those previously reported for PYSOCA.
To examine the effect of ATP depletion, hCMEC/D3 cells
were pretreated with NaN3 as a metabolic inhibitor. The upꢀ
take of compound 1 was significantly inhibited by NaN3 (Figꢀ
ure 2D). On the other hand, uptake was increased by intracelꢀ
lular acidification (Figure 2E, preꢀtreatment with NH4Cl), and
decreased by intracellular alkalization (Figure 2E, acute treatꢀ
ment with NH4Cl). These results suggest that the uptake is
dependent on metabolic energy and an outward H+ꢀgradient.
These properties are consistent with those of PYSOCA reportꢀ
ed previously.34,38
Figure 4. BBB permeability of compound 1 and CIꢀ994 across
the rat BBB. The right cerebral hemisphere of the rat was perꢀ
fused with buffer containing compound 1 or CIꢀ994 (20 µM) at a
rate 4.9 mL/min for 1 min. Each column represents the mean ±
S.E. (n = 3). *p < 0.01, significantly different from the PSBBB of
CIꢀ994.
Finally, in situ brain perfusion was performed to measure
bloodꢀtoꢀbrain transport of compound 1 and CIꢀ994 across the
BBB. The results are shown in Figure 4. The BBB permeabilꢀ
ity (PSBBB) of compound 1 was determined to be 42.4 ± 4.9
µL/min/g brain, which is 3.3ꢀfold greater than that of CIꢀ994
(12.7 ± 0.1 µL/min/g brain). Discrepancy in the BBB permeaꢀ
bility between in vitro and in vivo may be due to species difꢀ
ference of efflux transporter activity such as Pꢀgp. In fact, it
has been reported that the protein expression level of Pꢀgp
(mdra1) in rodent animal microvessels is significantly higher
than that in humans.41
Preparation of compound 1 is shown in Scheme 1. Reaction
of compound 942 with commercially available compound 8
under microwave irradiation afforded compound 10 in 61%
yield, and then deprotection of the Boc group afforded 1 in
90% yield. Synthesis of compounds 2‒7 are provided in Supꢀ
porting Information (Schemes S2, S3 and S4, see Supporting
Information).
Third, we determined the effect of several known substrates
of PYSOCA on uptake of compound 1 and CIꢀ994 by
hCMEC/D3 cells. Indeed, uptake of compound 1 was signifiꢀ
cantly inhibited by pyrilamine33, diphenhydramine34, memanꢀ
tine39, tramadol32, clonidine40 and varenicline31. In contrast,
TEA, MPP+, decyniumꢀ22,
Lꢀcarnitine, and choline, which are
typical substrates of OCTs, OCTNs, and choline transporter,
had little effect on the uptake of compound 1 (Table S4, see
Supporting Information). On the other hand, CIꢀ994 uptake
was not significantly decreased by these inhibitors (Table S4).
Scheme 1. Synthesis of the hybrid compound 1a
O
N
H
HN
O
NH
N
N
Boc
a
+
N
MeO
MeO
H
HN
NMe2
NMe2
Cl
N
O
Boc
8
9
10
Figure 3. Inhibition by diphenhydramine of the uptake of comꢀ
pound 1 into hCMEC/D3 cells. Uptake of compound 1 at various
concentrations was measured at 37 °C for 2 min in the absence
(open circles) or presence (closed circles) of diphenhydramine
(100 µM). Where the S.E. is not shown, it is smaller than the
symbol. Data were subjected to MichaelisꢀMenten plot (A) and
LineweaverꢀBurk plot (B) analysis. Kinetic parameters were obꢀ
tained by nonlinear leastꢀsquares regression according to Eqs. 1
and 2 (see Supporting Information). The straight lines were drawn
using the kinetic parameters obtained. Each point shows the mean
± S.E. (n = 4).
N
b
H
NH2
N
N
MeO
NMe2
1
aReagents and conditions: (a) pyridine, DMSO, 110 °C, 4.5 h,
microwave, 61%; (b) TFA, 0.5 h, 90%.
In summary, to overcome the low BBB permeability of exꢀ
isting HDAC inhibitors, we designed compound 1 using a
pyrilamine moiety as a shuttle targeting PYSOCA for drug
delivery to the CNS. The synthesized hybrid compound 1
showed selective class I HDAC inhibitory activity. Transport
studies using hCMEC/D3 cells confirmed that compound 1 is
The above results suggest that organic cation transport via
PYSOCA is primarily involved in the uptake of compound 1
by hCMEC/D3 cells, but not in CIꢀ994. Diphenhydramine is a
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 4 of 6
(3) Gregoretti, I. V.; Lee, Y. ꢀM.; Goodson, H. V. Molecular evoꢀ
a substrate of PYSOCA, and in situ brain perfusion confirmed
increased BBB permeability of compound 1 in the brain comꢀ
pared with CIꢀ994. This design strategy appears to promising
for development of potent BBBꢀpermeable HDAC inhibitors
to treat CNS diseases.
lution of the histone deacetylase family: functional implications of
phylogenetic analysis. J. Mol. Biol. 2004, 338, 17–31.
(4) Yoshida. M.; Kijima, M.; Akita, M.; Beppu, T.; Potent and
Specific Inhibition of mammalian histone deacetylase both in vivo
and in vitro by trichostatin A. J. Biol. Chem. 1990, 265, 17174–
17179.
(5) Kazantsev, A. G.; Thompson, L. M. Therapeutic application of
histone deacetylase inhibitors for central nervous system disorꢀ
ders. Nat. Rev. Drug Discov., 2008, 7, 854–868.
1
2
3
4
5
6
7
8
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI:
(6) Didonna, A.; Opal, P. The promise and perils of HDAC inhibiꢀ
tors in neurodegeneration. Ann. Clin. Transl. Neurol., 2015, 2, 79–
101.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Experimental procedures for the biological experiments
and synthetic procedures and characterization data for the
compounds.
(7) Kilgore, M.; Miller, C. A.; Fass, D. M.; Hennig, K. M.; Hagꢀ
garty, S. J.; Sweatt, J. D.; Rumbaugh, G. Inhibitors of class 1 hisꢀ
tone deacetylases reverse contextual memory deficits in a mouse
model of Alzheimer’s disease. Neuropsychopharmacology, 2010,
35, 870–880.
(8) Rumbaugh, G.; Sillivan, S. E.; Ozkan, E. D.; Rojas, C. S.; Hubbs,
C. R.; Aceti, M.; Kilgore, M.; Kudugunti, S.; Puthanveettil, S. V.;
Sweatt, J. D.; Rusche, J.; Miller, C. A. Pharmacological selectivity
within class I histone deacetylases predicts effects on synaptic funcꢀ
tion and memory rescue. Neuropsychopharmacology, 2015, 40,
2307–2316.
(9) Majid, T.; Griffin, D.; Criss II, Z.; Jarpe, M.; Pautler, R. G. Pharꢀ
macologic treatment with histone deacetylase 6 inhibitor (ACYꢀ738)
recovers Alzheimer's disease phenotype in amyloid precursor proꢀ
tein/presenilin 1 (APP/PS1) mice. Alzheimer’s Dement., 2015, 1, 170–
181.
(10) Zhang, L.; Liu, C.; Wu, J.; Tao, J. –J.; Sui, X. ꢀL.; Yao, Z. –G.;
Xu, Y. –F. Huang, L.; Zhu, H.; Sheng, S. –L.; Qin, C. Tubastatin
A/ACYꢀ1215 improves cognition in Alzheimer's disease transgenic
mice. J. Alzheimer’s Dis., 2014, 41, 1193–1205.
(11) Jia, H.; Kast, R. J.; Steffan. J. S.; Thomas, E. A. Selective
histone deacetylase (HDAC) inhibition imparts beneficial effects
in Huntington’s disease mice: implications for the ubiquitin–
proteasomal and autophagy systems. Hum. Mol. Genet. 2012, 21,
5280–5293.
(12) Thomas, E. A.; Coppola, G.; Desplats, P. A.; Tang, B.; Soragni,
E.; Burnett, R.; Gao, F.; Fitzgerald, K. M.; Borok, J. F.; Herman, D.;
Geschwind, D. H.; Gottesfeld, J. M. The HDAC inhibitor 4b amelioꢀ
rates the disease phenotype and transcriptional abnormalities in Hunꢀ
tington’s disease transgenic mice. Proc. Natl. Acad. Sci. U.S.A., 2008,
105, 15564–15569.
(13) Jia, H.; Morris, C. D.; Williams, R. M.; Loring, J. F.; Thomas, E.
A. HDAC inhibition imparts beneficial transgenerational effects in
Huntington’s disease mice via altered DNA and histone methylation.
Proc. Natl. Acad. Sci. U.S.A., 2014, E56–E64.
(14) Jia, H.; Wang, Y.; Morris, C. D.; Jacques, V.; Gottesfeld, J. M.;
Rusche, J. R.; Thomas, E. A. The effects of pharmacological inhibiꢀ
tion of histone deacetylase 3 (HDAC3) in Huntington’s disease mice.
PLOS One, 2016, 11, e0152498.
(15) Gräff, J.; Joseph, N. F.; Horn, M. E.; Samiei, A.; Meng, J.;
Seo, J.; Rei, D.; Bero, A. W.; Phan, T. X.; Wagner, F.; Holson, E.;
Xu, J.; Sun J.; Neve, R. L.; March, R. H.; Haggarty, S. J.; Tsai,
L.ꢀH. Epigenetic priming of memory updating during reconsolidaꢀ
tion to attenuate remote fear memories. Cell, 2014, 156, 261–276.
(16) Hooker, J. M.; Kim, S. W.; Alexoff, D.; Xu, Y.; Shea, C.;
Reid, A.; Volkow, N.; Fowler, J. S. Histone deacetylase inhibitor
MSꢀ275 exhibits poor brain penetration: pharmacokinetic studies
of [11C]MSꢀ275 using positron emission tomography. ACS Chem.
Neurosci., 2010, 1, 65–73.
(17) Hendricks, J. A.; Keliher, E. J.; Mrinelli, B.; Reiner, T.; Weisꢀ
sleder, R.; Mazitschek, R. In vivo PET imaging of histone
deacetylases by 18Fꢀsuberoylanilide hydroxamic acid (18FꢀSAHA).
J. Med. Chem., 2011, 54, 5576–5582.
(18) Kim, S. W.; Hooker, J. M.; Otto, N.; Win, K.; Muench, I.;
Shea. C.; Carter, P.; King, P.; Reid, A. E.; Volkow, N. D.; Fowler,
J. S. Wholeꢀbody pharmacokinetics of HDAC inhibitor drugs,
AUTHOR INFORMATION
Corresponding Author
* Tel: +81ꢀ6ꢀ6368ꢀ1773. Fax: +81ꢀ6ꢀ6388ꢀ8609.
Eꢀmail: tꢀsumiyo@kansaiꢀu.ac.jp
Author Contributions
T.S. and S.H. conceived and directed medicinal chemistry.
M.Y. and A.I. directed pharmacology. M.A. contributed hisꢀ
tone acetylation assay. Y.D. directed pharmacokinetic study.
Y.T., K.H. and T.K. contributed PK studies. The manuscript
was written through contributions of all authors.
Funding Sources
Part of this work was supported by the Kansai University Subsidy
for Supporting Young Scholars, 2015 “Development of macrocyꢀ
clization reaction via a photoaffinity reaction”, JSPS KAKENHI
Grant Number 15K18903, GrantꢀinꢀAid for Young Scientists (B)
– Japan and MEXT – Supported Program for the Strategic Reꢀ
search Foundation at Private Universities (2013–2017) – Japan.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
We thank Dr. PierreꢀOlivier Couraud (Institut Cochin, Paris,
France) for supplying hCMEC/D3 cells under license from
INSERM. The authors also gratitude to Dr. Teruki Honma for
calculations of physicochemical properties and Dr. Shinichi
Uesato for useful suggestions.
ABBREVIATIONS
HDAC, histone deacetylase; BBB, blood brain barrier; CNS, cenꢀ
tral nervous system; SAHA, suberoylanilide hydroxamic acid;
TSA, trichostatin A; PET, positron emission tomography;
PYSOCA, pyrilamine sensitive proton coupled organic cation
antiporter; hCMEC, human cardiac microvascular endothelial
cells; ATP, adenosine triphosphate; DMSO, dimethyl sulfoxide;
TFA, trifluoroacetic acid; TEA, tetraethylammonium; MPP+, 1ꢀ
methylꢀ4ꢀphenylpyridinium; OCTs, organic cation transporters;
OSTNs, organic cation/carnitine transporters
REFERENCES
(1) Walsh, C. T.; GarneauꢀTsodikova, S.; Gatto, G. J. Protein
posttranslational modifications: the chemistry of proteome diverꢀ
sifications. Angew. Chem., Int. Ed. 2005, 44, 7342−7372.
(2) Bradner, J. E.; West, N.; Grachan, M. L.; Greenberg, E. F.;
Haggarty, S. J.; Warnow, T.; Mazitschek, R.; Chemical phylogeꢀ
netics of histone deacetylases. Nat. Chem. Biol. 2010, 6, 238–243.
ACS Paragon Plus Environment

Page 5 of 6
ACS Medicinal Chemistry Letters
butyric acid, valproic acid and 4ꢀphenylbutyric acid and measured
in” function. Eur. J. Med. Chem., 2014, 82, 314–323.
with carbonꢀ11 labeled analogs by PET. Nucl. Med. Biol., 2013,
40, 912–918.
(30) Wang, X.; Qi, B.; Su, H.; Li, J.; Sun, X.; He, Q.; Fu, Y.;
Zhang, Z. Pyrilamineꢀsensitive protonꢀcoupled organic cation
(H+/OC) antiporter for brainꢀspecific drug delivery. J. Control.
Release, 2017, 254, 34–43.
(31) Kurosawa, T.; Higuchi, K.; Okura, T.; Kobayashi, K.; Kusuꢀ
hara, H.; Deguchi, Y. Involvement of protonꢀcoupled organic
cation antiporter in varenicline transport at bloodꢀbrain barrier of
rats and in human brain capillary Endothelial Cells. J. Pharm.
Sci., 2017, 106, 2576–2582.
(32) Kitamura, A.; Higuchi, K.; Okura, T.; Deguchi, Y. Cocktailꢀ
dosing microdialysis study to simultaneously assess delivery of
multiple organicꢀcationic drugs to the brain. J. Pharm. Sci., 2016,
105, 935–940.
(33) Yamazaki, M.; Terasaki, T.; Yoshioka, K.; Nagata, O.; Kato,
H.; Ito, Y.; Tsuji, A. Carrierꢀmediated transport of H1ꢀantagonist at
the bloodꢀbrain barrier: Mepyramine uptake into bovine brain
capillary endothelial cells in primary monolayer cultures. Pharm.
Res., 1994, 11, 975–978.
(34) Shimomura, K.; Okura, T.; Kato, S.; Couraud, P. O.; Scherꢀ
mann, J. M.; Terasaki, T.; Deguchi, Y. Functional expression of a
protonꢀcoupled organic cation (H+/OC) antiporter in human brain.
Fluids and Barriers of CNS., 2013, 10, 1–10.
(35) Newbold, A.; Matthews, G. M.; Bots, M.; Cluse, L. A.;
Clarke, C. J. P.; Banks, K. M.; Cullinane, C.; Bolden, J. E.; Chrisꢀ
tiansen, A. J.; Dickins, R. A.; Miccolo, C.; Chiocca, S.; Kral, A.
M.; Ozerova, N. D.; Miller, T. A.; Methot, J. L.; Richon, V. M.;
Secrist, J. P.; Minucci, S.; Johnstone, R. W. Molecular and biologꢀ
ic analysis of histone deacetylase inhibitors with diverse specificiꢀ
ties. Mol. Cancer Ther. 2013, 12(12), 2709–2721.
(36) Rankovic, Z. CNS physicochemical property space shaped
by a diverse set of molecules with experimentally determined
exposure in the mouse brain. J. Med. Chem., 2017, 60, 5943–5954.
(37) Chou, C. J.; Herman, D.; Gottesfeld, J. M.; Pimelic diphenylaꢀ
mide 106 is a slow, tightꢀbinding inhibitor of class I histone deacetyꢀ
lases. J. Biol. Chem., 2008, 283, 35402–35409.
(38) Okura, T.; Hattori, A.; Takano, Y.; Sato, T.; Hammarlundꢀ
Udenaes, M.; Terasaki, T.; Deguchi Y. Involvement of the
pyrilamine transporter, a putative organic cation transporter, in
bloodꢀbrain barrier transport of oxycodone. Drug Metab. Dispos.
2008, 36, 2005–2013.
(39) Higuchi, K.; Kitamura, A.; Okura, T.; Deguchi, Y. Memanꢀ
tine transport by a protonꢀcoupled organic cation antiporter in
hCMEC/D3 cells, an in vitro human bloodꢀbrain barrier model.
Drug Metab. Pharmacokinet., 2015, 30, 182–187.
(40) Chapy, H.; Andre, P.; Decleves, X.; Scherrmann, J. M.;
Cisternino, S. A. polyspecific drug/proton antiporter mediates
diphenhydramine and clonidine transport at the mouse bloodꢀ
brain barrier. Br. J. Pharmacol., 2015, 172, 4714–4725.
(41) Uchida, Y.; Ohtsuki, S.; Katsukura, Y.; Ideda, C.; Suzuki, T.;
Kamiie, J.; Terasaki, T. Quantitative targeted absolute proteomics
of human bloodꢀbrain barrier transporters and receptors. J. Neuro-
chem, 2011, 117, 335–345.
(42) Hamblett, C. L.; Methot, J. L.; Mampreian, D. M.; Sloman, D.
L.; Stanton, M. G.; Kral, A. M.; Fleming, J. C.; Cruz, J. C.;
Chenard, M.; Ozerova, N.; Hitz, A. M.; Wang, H.; Deshmukh, S.
V.; Nazef, N.; Harsch, A.; Hughes, B.; Dahlberg, W. K.; Szewczak,
A. A; Middleton, R. E.; Mosley, R. T.; Secrist, J. P.; Millera, T. A.
The discovery of 6ꢀamino nicotinamides as potent and selective
histone deacetylase inhibitors. Bioorg. Med. Chem. Lett., 2007, 17,
5300–5309.
1
2
3
4
5
6
7
8
(19) Seo, Y. J.; Muench, L.; Reid, A.; Chen, J.; Kang, Y.; Hooker,
J. M.; Volkow, N. D.; Fowler, J. S.; Kim, S. W. Radionuclide laꢀ
beling and evaluation of candidate radioligands for PET imaging
of histone deacetylase in the brain. Bioorg. Med. Chem. Lett.,
2013, 23, 6700–6705.
(20) Strebl, M. G.; Campbell, A. J.; Zhao. N. W.; Schroeder, F. A.;
Riley, M. M.; Chindavong, P. S.; Morin, T. M.; Haggarty, S. J.;
Wagner, F. F.; Ritter, T.; Hooker, J. M. HDAC6 brain mapping
with [18F]bavarostat enabled by a Ruꢀmediated deoxyfluorination.
ACS Cent. Sci., 2017, 3, 1006–1014.
(21) Wang, C.; Schroeder, A; Wey, H. Y.; Borra, R.; Wagner, F. F.;
Reis, S.; Kim, W. S.; Holson, B. E.; Haggarty, J. S.; Hooker, M. J.
In vivo imaging of histone deacetylases (HDACs) in the central
nervous system and major peripheral organs. J. Med. Chem., 2014,
57, 7999–8009.
(22) Reid, A. E.; Hooker, J.; Shumay, E.; Logan, J.; Shea, C.; Kim,
S. W.; Collins, S.; Xu, Y.; Volkow, N.; Fowler, J. S. Evaluation of
6ꢀ([18F]fluoroacetamido)ꢀ1ꢀhexanoicanilide for PET imaging of
histone deacetylase in the baboon brain. Nucl. Med. Biol., 2009,
36, 247–258.
(23) Wang. C.; Eessalu, T. E.; Barth, V. N.; Mitch, C. H.; Wagner,
F. F.; Hong, Y.; Neelamegam, R.; Schroeder, F. A.; Holson, E. B.;
Haggarty, S. J.; Hooker, J. M. Design, synthesis, and evaluation of
hydroxamic acidꢀbased molecular probes for in vivo imaging of
histone deacetylase (HDAC) in brain. Am. J. Nucl. Med. Mol.
Imaging, 2014, 4, 29–38.
(24) Schroeder, F. A.; Wang, S.; Van de Bittner, G. C.;
Neelamegam, R.; Takakura, W. R.; Karunakaran, A.; Wey, H. Y.;
Reis, S. A.; Gale, J.; Zhang, Y. L.; Holson E. B.; Haggarty, S. J.;
Hooker, J. M. PET imaging demonstrates histone deacetylase
target engagement and clarifies brain penetrance of known and
novel small molecule inhibitors in rat. ACS Chem. Neurosci.,
2014, 5, 1055–1062.
(25) Seo, Y. J.; Kang, Y.; Muench, L.; Reid, A.; Caesar, S.; Jean,
L.; Wagner, F.; Holson, E.; Haggarty, S. J.; Weiss, P.; King. P.;
Carter, P.; Volkow, N. D.; Fowler, J. S.; Hooker, J. M.; Kim. W. S.
Imageꢀguided synthesis reveals potent bloodꢀbrain barrier permeꢀ
able histone deacetylase inhibitors. ACS Chem. Neurosci., 2014, 5,
588–596.
(26) Di, L.; Rong, H.; Feng, B. Demystifying brain penetration in
central nervous system drug discovery. J, Med. Chem., 2013, 56,
2–12.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(27) (a) Bilsky, E. J.; Egleton, R. D.; Mitchell, S. A.; Palian, M.
M.; Davis, P.; Huber, J. D.; Jones, H.; Yamamura, H. I.; Janders,
J.; Davis, T. P.; Porreca, F.; Hruby, V. J.; Polt, R. Enkephalin glyꢀ
copeptide analogues produce analgesia with reduced dependence
liability. J. Med. Chem., 2000, 43, 2586–2590. (b) Gynther, M.;
Ropponen, J.; Laine, K.; Leppänen, J.; Haapakoski, P.; Peura, L.;
Järvinen, T.; Rautio, J. Glucose promoiety enables glucose transꢀ
porter mediated brain uptake of ketoprofen and indomethacin
prodrugs in rats. J. Med. Chem., 2009, 52, 3348–3353.
(28) Peura, L.; Malmioja, K.; Huttunen, K.; Leppänen, J.; Hämäꢀ
läinen, M.; Forsberg, M. M.; Rautio, J.; Laine, K. Design, syntheꢀ
sis and brain uptake of LAT1ꢀtargeted amino acid prodrugs of
dopamine. Pharm. Res., 2013, 30, 2523–2537.
(29) Zhao, Y.; Qu, B.; Wu, X.; Li, X.; Liu, O.; Jin, X.; Guo, L.;
Hai. L.; Wu, Y. Design, synthesis and biological evaluation of
brain targeting Lꢀascorbic acid prodrugs of ibuprofen with “lockꢀ
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 6 of 6
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Insert Table of Contents artwork here
ACS Paragon Plus Environment
6