Structural modification of histone deacetylase inhibitors with a phenylglycine scaffold

Article abstract of DOI:10.1097/CAD.0000000000000579

During the discovery of histone deacetylase inhibitors (HDACIs) as antitumor drugs, a series of potent phenylglycine-based HDACIs were developed. However, further development is restricted by the poor solubility. Therefore, structural modifications were performed in the present study in the development of potent HDACIs with improved pharmacokinetic properties. The synthesized molecules were designed by the substitution of fatty linkers for aromatic linkers, and showed good solubility profiles. Among the compounds derived, molecule HD9 showed a potent enzyme-inhibitory effect (IC₅₀ values of 76 nmol/l) and in-vitro antiproliferative activities (IC₅₀ values of 0.51, 0.83, and 0.76 μmol/l against U937, K562, and HL60 cells, respectively). Molecule HD9 showed selectivity of HDAC3 over HDAC6 in the isoform selectivity assays. Molecular docking studies showed good binding patterns of molecule HD9 to the active site of HDAC3. Results from the present work indicated that molecule HD9 is a promising lead compound for the tumor therapy.

Full text of DOI:10.1097/CAD.0000000000000579

Preclinical report
1
Structural modification of histone deacetylase inhibitors with
a phenylglycine scaffold
Lei Zhanga, Li Zhangb, Jiang Bianb, Qixiao Jiangb, Jianjun Gaob
and Weiguo Songa
During the discovery of histone deacetylase inhibitors
(HDACIs) as antitumor drugs, a series of potent
compound for the tumor therapy. Anti-Cancer Drugs
00:000–000 Copyright © 2017 Wolters Kluwer Health, Inc.
All rights reserved.
phenylglycine-based HDACIs were developed. However,
further development is restricted by the poor solubility.
Therefore, structural modifications were performed in the
present study in the development of potent HDACIs with
improved pharmacokinetic properties. The synthesized
molecules were designed by the substitution of fatty linkers
for aromatic linkers, and showed good solubility profiles.
Among the compounds derived, molecule HD9 showed a
potent enzyme-inhibitory effect (IC50 values of 76 nmol/l)
and in-vitro antiproliferative activities (IC50 values of 0.51,
0.83, and 0.76 µmol/l against U937, K562, and HL60 cells,
respectively). Molecule HD9 showed selectivity of HDAC3
over HDAC6 in the isoform selectivity assays. Molecular
docking studies showed good binding patterns of molecule
HD9 to the active site of HDAC3. Results from the present
work indicated that molecule HD9 is a promising lead
Anti-Cancer Drugs 2017, 00:000–000
Keywords: antitumor, fatty linker, histone deacetylase, inhibitors,
phenylglycine, structural modification
aDepartment of Medicinal Chemistry, School of Pharmacy, Weifang Medical
University, Weifang and bDepartment of Medicinal Chemistry, School of Pharmacy,
Qingdao University, Qingdao 261053, China
Correspondence to Dr Lei Zhang, Department of Medicinal Chemistry,
School of Pharmacy, Weifang Medical University, Weifang 261053, China
Tel: + 86 150 9208 5925; fax: + 86 053 6846 2014;
e-mail: leiqdu@foxmail.com
Correspondence to Dr Weiguo Song, Department of Medicinal Chemistry, School
of Pharmacy, Weifang Medical University, Weifang 261053, China
Fax: +86 053 6846 2014
Received 13 February 2017 Revised form accepted 8 November 2017
Introduction
approved by FDA for the treatment of advanced cuta-
neous T-cell lymphoma. PXD101 [16] and LBH589 [17]
were approved for the treatment of peripheral T-cell
lymphoma and multiple myeloma, respectively.
Histone deacetylases (HDACs) are a class of enzymes
responsible for removing acetyl groups from the ε-
N-acetyl lysine on a histone. A total of 18 isoforms of
HDACs (divided into four classes) have been discovered
[1–3]. Class I (HDAC 1, 2, 3, and 8), II (IIa: HDAC 4, 5,
7, and 9; IIb: HDAC 6 and 10), and VI (HDAC11)
HDACs are zinc-dependent enzymes with a zinc ion in
the catalytic site, whereas class III HDACs (known as
sirtuins, Sirt1–7) are a set of NAD-dependent enzymes.
To develop novel HDACIs as antitumor agents, a series
of small molecules were designed and synthesized on the
basis of virtual screening results [18,19]. Molecules with
improved activities were developed by structural mod-
ification of the lead compound 4h [20]. However, the
drug ability of the derived molecules was restricted by
the poor solubility because of the existence of three
aromatic groups. To improve the soluble properties and
the antitumor activities, a fatty linker was introduced into
the current phenylglycine-based HDACIs (Fig. 1). The
Acetylation and deacetylation of histones and nonhistone
proteins by histone acetylases and HDACs play a sig-
nificant role in the regulation of various gene expressions
[4–6]. Affected genes include those involved in signal
transduction, protein phosphorylation, cell cycle, pro-
liferation, apoptosis, and cardiac development [7–9].
Overexpression and aberrant recruitment of HDACs play
a critical role in tumorigenesis and tumor develop-
ment [10].
Fig. 1
Histone deacetylase inhibitors (HDACIs) have been
proven to regulate gene expression by blocking the
function of HDACs and inducing hyperacetylation of
histones [11,12]. A number of HDACIs have shown
enormous potential as chemotherapeutic agents and are
currently under clinical trials [13]. Suberoyl anilide
hydroxamic acid (SAHA) [14] and FK228 [15] have been
Design of phenylglycine-based HDACIs by structural modification.
HDACI, histone deacetylase inhibitors.
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DOI: 10.1097/CAD.0000000000000579
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2
Anti-Cancer Drugs 2017, Vol 00 No 00
designed target molecules were synthesized and assessed
with the enzyme-inhibitory and antiproliferative assays.
reaction solution was stirred at room temperature for 8 h.
Then, the solvent was evaporated, with the residue being
taken up in EtOAc (50 ml). The EtOAc solution was
washed with saturated citric acid (3 × 20 ml), NaHCO3
(3 × 20 ml), and brine (3 × 20 ml), dried over MgSO4, and
evaporated under vacuum. The desired compound c1
(1.33 g, 76% yield) was derived by crystallization in
EtOAc as a white powder. ESI-MS: m/z: 351.4 [M + H]+.
Chemistry
On the basis of the previous lead structure, a series of
phenylglycine-based HDACIs with fatty linkers were syn-
thesized according to the procedures described in Scheme 1.
The starting materials (S)-2-amino-2-phenylacetic acid and
4-aminobutyic acid were protected by the Boc group and
methyl ester, respectively. Intermediate (S)-methyl 4-(2-
((tert-butoxycarbonyl)amino)-2-phenylacetamido) butanoate
(a) was derived by coupling these two products. Subsequent
N-deprotection and condensation with various acyl chloride
and sulfonyl chloride yielded c1–c22. Target compounds
HD1–HD22 were derived by treating the corresponding
intermediates (c1–c22) with NH2OK in methanol.
(S)-2-((4-Methoxy-4-oxobutyl)amino)-2-oxo-
1-phenylethanaminium chloride (b)
To a solution of compound a (1.0 g, 2.8 mmol) in EtOAc
(10 ml), a solution of EtOAc (15 ml) saturated by dry HCl
gas was added. The reaction solution was stirred at room
temperature for 5 h. Precipitates appeared and were fil-
tered, with the filter being washed with ether, to yield
the desired compound b (0.48 g, 67% yield). The title
compound b was obtained as an amorphous white solid.
ESI-MS: m/z: 251.4 [M + H]+.
Materials and methods
Chemistry
1H NMR spectra were recorded on a Bruker DRX
spectrometer at 500 MHz, δ in parts per million and J in
hertz, using TMS as an internal standard. ESI-MS were
determined on an API 4000 spectrometer. Optical rota-
tion values were measured at room temperature using a
modular circular polarimeter 200 operating at λ = 589 nm.
(S)-Methyl 4-(2-(4-bromobenzamido)-
2-phenylacetamido)butanoate (c1)
To a solution of b (0.86 g, 3.0 mmol) in THF (50 ml)/H2O
(1.0 ml), NaHCO3 (1.01 g, 12 mmol) was added. After
10 min, 4-bromobenzoyl chloride (0.72 g, 3.3 mmol) was
added. The reaction solution was stirred at room tem-
perature for 8 h. Then, the solvent was evaporated with
the residue was taken up in EtOAc (50 ml). The EtOAc
solution was washed with saturated citric acid (3 × 20 ml),
NaHCO3 (3 × 20 ml), and brine (3 × 20 ml), dried over
MgSO4, and evaporated under vacuum. The desired
compound c1 (0.77 g, 59% yield) was derived by
(S)-2-((Tert-butoxycarbonyl)amino)-2-phenylacetic
acid
and 4-methoxy-4-oxobutan-1-aminium chloride were syn-
thesized as described in the previously reported work [19].
(S)-Methyl 4-(2-((tert-butoxycarbonyl)amino)-
2-phenylacetamido)butanoate (a)
To a solution of (S)-2-((tert-butoxycarbonyl)amino)-
2-phenylacetic acid (1.26 g, 5 mmol) in DCM (50 ml),
Et3N (0.55 g, 5.5 mmol) and TBTU (1.8 g, 5.5 mmol)
were added in turn. After 20 min, 4-methoxy-4-oxobutan-
1-aminium chloride (0.77 g, 5 mmol) was added. The
crystallization in EtOAc as
a
white powder. 1H
NMR (500 MHz, CDCl3) δ 7.59 (d, J = 7.9 Hz, 1H), 7.53
(t, J = 6.7 Hz, 1H), 7.52–7.45 (m, 2H), 7.42–7.30 (m, 4H),
7.30–7.23 (m, 1H), 5.67 (d, J = 6.7 Hz, 1H), 3.39–3.13
Scheme 1
Synthesis of compounds HD1–HD20. Reagents and conditions: (1) (Boc)2O, Et3N, MeOH/H2O; (2) CH3COCl, CH3OH; (3) TBTU, Et3N, THF;
(4) EtOAc, HCl; (5) RSO2Cl, RCOCl, NaHCO3, THF/H2O; (6) NH2OK, MeOH.
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Structural modification of HDACIs Zhang et al.
3
(m, 2H), 2.36–2.16 (m, 2H), 1.85–1.66 (m, 2H), and 1.35
(s, 3H).
3.13–2.97 (m, 2H), 2.29 (s, 3H), 1.92 (t, J=7.5 Hz, 2H), 1.60
(p, J=7.3 Hz, 2H).
The other compounds (c2–c21) were prepared using the
same procedure as described above.
(S)-N-(2-((4-(Hydroxyamino)-4-oxobutyl)amino)-2-oxo-
1-phenylethyl)-3,5-dimethylbenzamide (HD5)
The title compound HD5 was obtained as an amorphous
white solid (0.28 g, 56% yield). [α]20D −6.63 (c 1.0, MeOH).
1H NMR (500 MHz, dmso) δ 10.25 (s, 1H), 8.64 (s, 1H), 8.61
(d, J=9.3 Hz, 1H), 8.30 (s, 1H), 8.17 (t, J=5.4 Hz, 1H), 8.03
(d, J=8.0 Hz, 1H), 7.97 (dd, J=8.3, 4.0 Hz, 2H), 7.74
(d, J=8.7 Hz, 1H), 7.64 (dt, J=14.5, 6.9 Hz, 2H), 7.27
(d, J=7.5 Hz, 2H), 7.16 (t, J=7.3 Hz, 2H), 4.98
(d, J=9.2 Hz, 1H), 2.69 (dd, J=13.2, 6.4 Hz, 2H), 1.72
(t, J=7.5 Hz, 2H), 1.31 (p, J=7.3 Hz, 2H).
(S)-4-Bromo-N-(2-((4-(hydroxyamino)-4-oxobutyl)
amino)-2-oxo-1-phenylethyl)benzamide (HD1)
Compound c1 (0.5 g, 1.15 mmol) was dissolved in 14 ml
of an NH2OK (0.56 g, 24 mmol) methanol solution. After
2 h, the solvent was evaporated under vacuum. The
residue was acidified with saturated citric acid and then
extracted with EtOAc (3 × 20 ml). The organic layers
were combined, washed with brine (3 × 20 ml), and dried
over MgSO4. The desired compound HD1 (0.21 g, 42%
yield) was derived by crystallization in EtOAc as a
(S)-N-(2-((4-(Hydroxyamino)-4-oxobutyl)amino)-2-oxo-
1-phenylethyl)-3,5-dimethoxybenzamide (HD6)
white powder. [α]20 −6.88 (c 1.0, MeOH). 1H NMR
D
(500 MHz, dmso) δ 10.33 (s, 1H), 8.99 (d, J=8.1 Hz, 1H),
8.68 (s, 1H), 8.29 (t, J=5.4 Hz, 1H), 7.62 (d, J=7.9 Hz, 1H),
7.48 (d, J=7.5 Hz, 2H), 7.41 (d, J=4.4 Hz, 2H), 7.34
(t, J=7.4 Hz, 3H), 7.28 (t, J=7.2 Hz, 1H), 5.61
(d, J=8.1 Hz, 1H), 3.13–2.95 (m, 2H), 1.93 (t, J=7.5 Hz,
2H), 1.66–1.52 (m, 2H).
The title compound HD6 was obtained as an amorphous
white solid (0.27 g, 54% yield). [α]20 − 6.11 (c 1.0,
D
MeOH). 1H NMR (500 MHz, dmso) δ 10.42 (s, 1H),
8.71 (d, J = 8.1 Hz, 2H), 8.44 (t, J = 5.4 Hz, 1H), 7.57
(d, J = 8.4 Hz, 1H), 7.52–7.43 (m, 3H), 7.33 (t, J = 7.5 Hz,
2H), 7.27 (s, 1H), 6.99 (d, J = 8.5 Hz, 1H), 5.66
(d, J = 8.0 Hz, 1H), 3.79 (d, J = 2.2 Hz, 6H), 3.12–2.95
(m, 2H), 1.93 (d, J = 7.5 Hz, 2H), 1.61 (d, J = 7.3 Hz, 2H).
The other compounds (HD2–HD21) were prepared
using the same procedure as described above.
(S)-2-Bromo-N-(2-((4-(hydroxyamino)-4-oxobutyl)
amino)-2-oxo-1-phenylethyl)benzamide (HD2)
The title compound HD2 was obtained as an amorphous
(S)-4-Chloro-N-(2-((4-(hydroxyamino)-4-oxobutyl)
amino)-2-oxo-1-phenylethyl)-3-nitrobenzamide (HD7)
The title compound HD7 was obtained as an amorphous
white solid (0.24 g, 48% yield). [α]20 − 6.52 (c 1.0,
white solid (0.22 g, 44% yield). [α]20 − 6.38 (c 1.0,
D
D
MeOH). 1H NMR (500 MHz, dmso) δ 10.32 (s, 1H), 8.90
(d, J = 7.9 Hz, 1H), 8.67 (s, 1H), 8.30 (t, J = 5.6 Hz, 1H),
7.89–7.82 (m, 2H), 7.68–7.63 (m, 2H), 7.47 (d, J = 7.3 Hz,
2H), 7.34 (t, J = 7.4 Hz, 2H), 7.29 (d, J = 7.3 Hz, 1H), 5.62
(d, J = 7.8 Hz, 1H), 3.06 (td, J = 12.9, 7.2 Hz, 2H), 1.92
(t, J = 7.5 Hz, 2H), 1.66–1.52 (m, 2H).
MeOH). 1H NMR (500 MHz, dmso) δ 10.32 (s, 1H), 9.46
(d, J = 8.3 Hz, 1H), 8.68 (s, 1H), 8.37 (t, J = 5.5 Hz, 1H),
7.46 (d, J = 7.4 Hz, 2H), 7.46–7.41 (m, 1H), 7.35
(t, J = 7.5 Hz, 2H), 7.28 (ddd, J = 20.3, 16.5, 8.5 Hz, 3H),
5.68 (d, J = 8.3 Hz, 1H), 3.06 (dd, J = 13.1, 5.7 Hz, 2H),
1.93 (t, J = 7.5 Hz, 2H), 1.66–1.54 (m, 2H).
(S)-N-(2-((4-(Hydroxyamino)-4-oxobutyl)amino)-2-oxo-
1-phenylethyl)-1-naphthamide (HD3)
(S)-2-Chloro-6-fluoro-N-(2-((4-(hydroxyamino)-
4-oxobutyl)amino)-2-oxo-1-phenylethyl)benzamide
(HD8)
The title compound HD3 was obtained as an amorphous
white solid (0.23 g, 46% yield). [α]20 − 6.71 (c 1.0,
The title compound HD8 was obtained as an amorphous
D
MeOH). 1H NMR (500 MHz, dmso) δ 10.44 (s, 1H),
9.01 (d, J = 7.9 Hz, 1H), 8.44 (t, J = 5.2 Hz, 1H), 8.15
(dd, J = 6.1, 3.5 Hz, 1H), 8.00 (d, J = 8.2 Hz, 1H), 7.96
(dd, J = 6.1, 3.2 Hz, 1H), 7.66 (d, J = 6.9 Hz, 1H), 7.52
(dd, J = 9.4, 7.4 Hz, 5H), 7.36 (t, J = 7.5 Hz, 2H), 7.29
(t, J = 7.2 Hz, 1H), 5.73 (d, J = 7.8 Hz, 1H), 3.16–3.02
(m, 2H), 1.99–1.89 (m, 2H), 1.63 (p, J = 7.2 Hz, 2H).
white solid (0.24 g, 48% yield). [α]20 − 8.21 (c 1.0,
D
MeOH). 1H NMR (500 MHz, dmso) δ 10.32 (s, 1H), 9.46
(d, J = 8.3 Hz, 1H), 8.68 (s, 1H), 8.37 (t, J = 5.5 Hz, 1H),
7.46 (d, J = 7.4 Hz, 2H), 7.46–7.41 (m, 1H), 7.35
(t, J = 7.5 Hz, 2H), 7.28 (ddd, J = 20.3, 16.5, 8.5 Hz, 3H),
5.68 (d, J = 8.3 Hz, 1H), 3.06 (dd, J = 13.1, 5.7 Hz, 2H),
1.93 (t, J = 7.5 Hz, 2H), 1.66–1.54 (m, 2H).
(S)-N-(2-((4-(Hydroxyamino)-4-oxobutyl)amino)-2-oxo-
1-phenylethyl)-2-methylbenzamide (HD4)
(S)-N-Hydroxy-4-(2-(2-(naphthalen-1-yl)acetamido)-
2-phenylacetamido)butanamide (HD9)
The title compound HD4 was obtained as an amorphous
white solid (0.32 g, 54% yield). [α]20D −7.88 (c 1.0, MeOH).
1H NMR (500 MHz, dmso) δ 10.32 (s, 1H), 8.68
(d, J=8.2 Hz, 2H), 8.25 (t, J=5.6 Hz, 1H), 7.49–7.45
(m, 2H), 7.38–7.34 (m, 2H), 7.29 (ddd, J=11.2, 9.4, 7.0 Hz,
3H), 7.20 (t, J=7.4 Hz, 2H), 5.61 (d, J=8.1 Hz, 1H),
The title compound HD9 was obtained as an amorphous
white solid (0.24 g, 48% yield). [α]20 − 7.35 (c 1.0,
D
MeOH). 1H NMR (500 MHz, dmso) δ 10.37 (s, 1H), 8.87
(d, J = 8.2 Hz, 1H), 8.68 (s, 1H), 8.39 (t, J = 5.4 Hz, 1H),
8.13–8.03 (m, 1H), 7.89 (dd, J=6.2, 3.0 Hz, 1H), 7.79
(dd, J=5.8, 3.5 Hz, 1H), 7.53–7.45 (m, 2H), 7.45–7.38
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4
Anti-Cancer Drugs 2017, Vol 00 No 00
Table 1 Structure and enzyme-inhibitory activities of target compounds
Compounds
R
IC50 (nmol/l)a
537 25
HD1
HD2
HD3
851 32
394 17
HD4
HD5
2930 155
681 33
HD6
238 18
HD7
HD8
2977 98
967 40
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Structural modification of HDACIs Zhang et al.
5
Table 1 (continued)
Compounds
R
IC50 (nmol/l)a
HD9
76
5
HD10
HD11
108 9
213 14
HD12
318 19
HD13
HD14
HD15
286 22
351 29
195 23
HD16
HD17
471 27
1842 123
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6
Anti-Cancer Drugs 2017, Vol 00 No 00
Table 1 (continued)
Compounds
R
IC50 (nmol/l)a
HD18
HD19
HD20
1346 144
526 35
173 12
HD21
274 21
SAHA
158 12
SAHA, suberoyl anilide hydroxamic acid.
aEach value is the mean of at least three experiments.
(m, 4H), 7.31 (t, J=7.5 Hz, 2H), 7.25 (t, J=7.3 Hz, 1H), 5.43
(d, J=8.1 Hz, 1H), 4.06 (dd, J=44.0, 15.0 Hz, 2H), 3.09–2.95
(m, 2H), 1.91 (t, J=7.5 Hz, 2H), 1.58 (p, J=7.3 Hz, 2H).
MeOH). 1H NMR (500 MHz, dmso) δ 11.53 (s, 1H),
10.33 (s, 1H), 9.61 (d, J = 7.4 Hz, 1H), 8.67 (s, 1H), 8.56
(s, 1H), 8.48 (t, J = 5.5 Hz, 1H), 7.89 (d, J = 8.2 Hz, 1H),
7.74 (d, J = 8.3 Hz, 1H), 7.52–7.45 (m, 3H), 7.40–7.31 (m,
3H), 7.31–7.26 (m, 2H), 5.69 (d, J = 7.4 Hz, 1H),
3.16–2.98 (m, 2H), 1.93 (t, J = 7.5 Hz, 2H), 1.66–1.55
(m, 2H).
(S)-N-Hydroxy-4-(2-(2-(naphthalen-2-yl)acetamido)-
2-phenylacetamido)butanamide (HD10)
The title compound HD10 was obtained as an amor-
phous white solid (0.25 g, 50% yield). [α]20 −6.73 (c 1.0,
D
MeOH). 1H NMR (500 MHz, dmso) δ 10.38 (s, 1H),
8.83 (d, J=8.1 Hz, 1H), 8.40 (t, J=5.3 Hz, 1H), 7.85
(d, J=7.8 Hz, 1H), 7.81 (d, J=8.2 Hz, 2H), 7.74 (s, 1H), 7.44
(ddd, J=24.3, 13.0, 7.2 Hz, 5H), 7.31 (t, J=7.4 Hz, 2H), 7.25
(t, J=7.2 Hz, 1H), 5.43 (d, J=8.1 Hz, 1H), 3.77–3.70
(m, 2H), 3.05–2.95 (m, 2H), 1.91 (t, J=7.5 Hz, 2H), 1.57
(p, J=7.2 Hz, 2H).
(S)-3-Hydroxy-N-(2-((4-(hydroxyamino)-4-oxobutyl)
amino)-2-oxo-1-phenylethyl)-2-naphthamide (HD12)
The title compound HD12 was obtained as an amor-
phous white solid (0.26 g, 52% yield). [α]20D − 6.55 (c 1.0,
MeOH). 1H NMR (500 MHz, dmso) δ 11.53 (s, 1H),
10.33 (s, 1H), 9.61 (d, J = 7.4 Hz, 1H), 8.67 (s, 1H), 8.56
(s, 1H), 8.48 (t, J = 5.5 Hz, 1H), 7.89 (d, J = 8.2 Hz, 1H),
7.74 (d, J = 8.3 Hz, 1H), 7.52–7.45 (m, 3H), 7.40–7.31 (m,
3H), 7.31–7.26 (m, 2H), 5.69 (d, J = 7.4 Hz, 1H),
3.16–2.98 (m, 2H), 1.93 (t, J = 7.5 Hz, 2H), 1.66–1.55
(m, 2H).
(S)-6-Hydroxy-N-(2-((4-(hydroxyamino)-4-oxobutyl)
amino)-2-oxo-1-phenylethyl)-2-naphthamide (HD11)
The title compound HD11 was obtained as an amor-
phous white solid (0.25 g, 50% yield). [α]20D − 6.84 (c 1.0,
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Structural modification of HDACIs Zhang et al.
7
Table 2 In-vitro activities of representative compounds (µmol/l)a
Compounds
HDAC3
HDAC6
U937
K562
HL60
MDA-MB-231
PC3
SK-OV-3
L02
HD9
HD10
SAHA
0.12 0.01
0.23 0.01
0.20 0.01
0.39 0.02
0.42 0.02
0.44 0.03
0.51 0.05
0.94 0.06
1.34 0.08
0.83 0.04
0.97 0.06
1.62 0.05
0.76 0.05
0.91 0.08
1.53 0.03
2.94 0.12
3.35 0.15
3.73 0.17
3.66 0.22
5.03 0.23
4.58 0.38
3.17 0.18
7.52 0.33
3.98 0.33
5.58 0.35
7.96 0.46
1.62 0.08
SAHA, suberoyl anilide hydroxamic acid.
aEach value is the mean of at least three experiments.
Fig. 2
(s, 3H), 4.93 (d, J = 9.2 Hz, 1H), 2.84–2.64 (m, 2H), 1.77
(t, J = 7.2 Hz, 2H), 1.44 –1.26 (m, 2H).
(S)-N-Hydroxy-4-(2-(4-methylphenylsulfonamido)-
2-phenylacetamido)butanamide (HD16)
The title compound HD16 was obtained as an amorphous
white solid (0.23 g, 46% yield). Melting point: 196–198°C.
1
[α]20 − 6.86 (c 1.0, MeOH). H NMR (500 MHz, dmso)
D
δ 10.30 (s, 1H), 8.15 (t, J= 5.5 Hz, 1H), 7.58 (d, J = 8.1 Hz,
2H), 7.30–7.24 (m, 3H), 7.24–7.16 (m, 4H), 4.88 (s, 1H),
2.80 (dtd, J= 19.2, 13.1, 6.2 Hz, 2H), 2.32 (s, 3H), 1.81
(t, J = 7.5 Hz, 2H), 1.42 (dd, J = 13.8, 6.9 Hz, 2H).
(a) Western blotting analysis of acetylated histone H3 (Ac-HH3) and
acetylated histone H4 (Ac-HH4) in U937 cells after 6 h of treatment
with compounds at 1 µmol/l. β-actin was used as a loading control.
(b) Quantification of tumor weight in the negative control group, SAHA
(positive control group), and HD9 group.*Statistically different from the
data of the negative control group (N = 6). Ctrl, control; SAHA, suberoyl
anilide hydroxamic acid.
(S)-N-Hydroxy-4-(2-(4-nitrophenylsulfonamido)-
2-phenylacetamido)butanamide (HD17)
The title compound HD17 was obtained as an amorphous
white solid (0.28 g, 56% yield). [α]20D −7.08 (c 1.0, MeOH).
1H NMR (500 MHz, dmso) δ 10.44 (s, 8H), 9.01
(d, J=7.9 Hz, 5H), 8.52–8.10 (m, 9H), 8.04–7.87 (m, 8H),
7.66 (d, J=6.9 Hz, 4H), 7.52 (dd, J= 9.4, 7.4 Hz, 20H), 7.33
(dt, J=31.6, 7.4 Hz, 13H), 5.73 (d, J=7.8 Hz, 4H), 4.01 (d,
J=7.1 Hz, 4H), 3.33 (s, 1H), 2.50 (d, J=13.6 Hz, 11H), 2.29
(s, 2H), 3.14–1.07 (m, 64H), 2.00–1.84 (m, 9H), 2.54–1.07
(m, 34H), 1.73–1.45 (m, 9H), 1.16 (t, J=7.1 Hz, 1H).
(S)-1-Hydroxy-N-(2-((4-(hydroxyamino)-4-oxobutyl)
amino)-2-oxo-1-phenylethyl)-2-naphthamide (HD13)
The title compound HD13 was obtained as an amorphous
white solid (0.23 g, 46% yield). [α]20 − 7.88 (c 1.0,
D
MeOH). 1H NMR (500 MHz, dmso) δ 11.53 (s, 1H), 10.33
(s, 1H), 9.61 (d, J = 7.4 Hz, 1H), 8.67 (s, 1H), 8.56 (s, 1H),
8.48 (t, J = 5.5 Hz, 1H), 7.89 (d, J = 8.2 Hz, 1H), 7.74 (d,
J = 8.3 Hz, 1H), 7.52–7.45 (m, 3H), 7.40–7.31 (m, 3H),
7.31–7.26 (m, 2H), 5.69 (d, J = 7.4 Hz, 1H), 3.16–2.98 (m,
2H), 1.93 (t, J = 7.5 Hz, 2H), 1.66–1.55 (m, 2H).
(S)-4-(2-(4-Fluorophenylsulfonamido)-
2-phenylacetamido)-N-hydroxybutanamide (HD18)
The title compound HD18 was obtained as an amorphous
white solid (0.22 g, 44% yield). [α]20D −5.97 (c 1.0, MeOH).
1H NMR (500 MHz, dmso) δ 10.44 (s, 8H), 9.01 (d,
J=7.9 Hz, 5H), 8.52–8.10 (m, 9H), 8.04–7.87 (m, 8H), 7.66
(d, J=6.9 Hz, 4H), 7.52 (dd, J=9.4, 7.4 Hz, 20H), 7.33
(dt, J=31.6, 7.4 Hz, 13H), 5.73 (d, J=7.8 Hz, 4H), 4.01 (d,
J=7.1 Hz, 4H), 3.33 (s, 1H), 2.50 (d, J=13.6 Hz, 11H), 2.29
(s, 2H), 3.14–1.07 (m, 64H), 2.00–1.84 (m, 9H), 2.54–1.07
(m, 34H), 1.73–1.45 (m, 9H), 1.16 (t, J=7.1 Hz, 1H).
(S)-4-(2-(4-Bromophenylsulfonamido)-
2-phenylacetamido)-N-hydroxybutanamide (HD14)
The title compound HD14 was obtained as an amorphous
white solid (0.20 g, 40% yield). [α]20D −8.12 (c 1.0, MeOH).
1H NMR (500 MHz, dmso) δ 10.44 (s, 8H), 9.01 (d,
J=7.9 Hz, 5H), 8.52–8.10 (m, 9H), 8.04–7.87 (m, 8H), 7.66
(d, J=6.9 Hz, 4H), 7.52 (dd, J=9.4, 7.4 Hz, 20H), 7.33
(dt, J= 31.6, 7.4 Hz, 13H), 5.73 (d, J=7.8 Hz, 4H), 4.01 (d,
J=7.1 Hz, 4H), 3.33 (s, 1H), 2.50 (d, J=13.6 Hz, 11H), 2.29
(s, 2H), 3.14–1.07 (m, 64H), 2.00–1.84 (m, 9H), 2.54–1.07
(m, 34H), 1.73–1.45 (m, 9H), 1.16 (t, J=7.1 Hz, 1H).
(S)-4-(2-(4-Chlorophenylsulfonamido)-
2-phenylacetamido)-N-hydroxybutanamide (HD19)
The title compound HD19 was obtained as an amorphous
white solid (0.22 g, 44% yield). [α]20D −6.12 (c 1.0, MeOH).
1H NMR (500 MHz, dmso) δ 10.44 (s, 8H), 9.01 (d,
J=7.9 Hz, 5H), 8.52–8.10 (m, 9H), 8.04–7.87 (m, 8H), 7.66
(d, J=6.9 Hz, 4H), 7.52 (dd, J=9.4, 7.4 Hz, 20H), 7.33
(dt, J=31.6, 7.4 Hz, 13H), 5.73 (d, J=7.8 Hz, 4H), 4.01
(d, J=7.1 Hz, 4H), 3.33 (s, 1H), 2.50 (d, J=13.6 Hz, 11H),
2.29 (s, 2H), 3.14–1.07 (m, 64H), 2.00–1.84 (m, 9H),
2.54–1.07 (m, 34H), 1.73–1.45 (m, 9H), 1.16 (t,
J=7.1 Hz, 1H).
(S)-N-Hydroxy-4-(2-(naphthalene-1-sulfonamido)-
2-phenylacetamido)butanamide (HD15)
The title compound HD15 was obtained as an amor-
phous white solid (0.23 g, 46% yield). [α]20D − 7.67 (c 1.0,
MeOH). 1H NMR (500 MHz, dmso) δ 10.27 (s, 1H), 8.81
(d, J = 9.4 Hz, 1H), 8.75–8.61 (m, 2H), 8.09 (dd, J = 24.1,
7.3 Hz, 3H), 7.99 (d, J = 7.9 Hz, 1H), 7.62 (dt, J = 14.5,
7.0 Hz, 2H), 7.54 (t, J = 7.7 Hz, 1H), 7.19 (s, 2H), 7.10
Copyright r 2017 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.

8
Anti-Cancer Drugs 2017, Vol 00 No 00
Fig. 3
Binding patterns of molecule HD9 (b), HD10 (c), and HD3 (d) in the active site of HDAC3; a: surface representation of HD3, HD9, and HD10, in the
active site. HDAC, histone deacetylase.
1H NMR (500 MHz, dmso) δ 10.44 (s, 8H), 9.01 (d,
J=7.9 Hz, 5H), 8.52–8.10 (m, 9H), 8.04–7.87 (m, 8H), 7.66
Table 3 Time-dependent and dose-dependent U937 cell-inhibitory
ratio of representative compounds (%)a
(d, J=6.9 Hz, 4H), 7.52 (dd, J=9.4, 7.4 Hz, 20H), 7.33
0.125
µmol/l
0.25 µmol/l 0.5 µmol/l
1 µmol/l
2 µmol/l
(dt, J=31.6, 7.4 Hz, 13H), 5.73 (d, J=7.8 Hz, 4H), 4.01 (d,
J=7.1 Hz, 4H), 3.33 (s, 1H), 2.50 (d, J=13.6 Hz, 11H), 2.29
(s, 2H), 3.14–1.07 (m, 64H), 2.00–1.84 (m, 9H), 2.54–1.07
(m, 34H), 1.73–1.45 (m, 9H), 1.16 (t, J=7.1 Hz, 1H).
HD9–24 h
HD9–48 h
HD9–72 h
HD10–24 h
HD10–48 h
HD10–72 h
SAHA–24 h
SAHA–48 h
SAHA–72 h
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
10.6 0.98
12.2 1.1 48.6 2.1 56.6 3.1
22.3 1.1
NA
NA
45.6 3.6 64.2 3.2
NA
NA
87.1 2.8
NA
47.8 2.6
NA
21.9 1.8
15.7 1.3
NA
NA
36 2.9 55.7 4.3 76.4 5.2
NA
NA
(S)-N-Hydroxy-4-(2-(naphthalene-2-sulfonamido)-
2-phenylacetamido)butanamide (HD21)
NA
NA
18.3 1.2 43.6 3.3
26.7 2.5
41.4 2.7 52.5 3.4 79.8 5.8
The title compound HD21 was obtained as an amorphous
white solid (0.24 g, 48% yield). [α]20 − 6.12 (c 1.0,
D
NA, not available; SAHA, suberoyl anilide hydroxamic acid.
aEach value is the mean of at least three experiments.
1
MeOH). H NMR (500 MHz, dmso) δ 10.44 (s, 8H), 9.01
(d, J = 7.9 Hz, 5H), 8.52–8.10 (m, 9H), 8.04–7.87 (m, 8H),
7.66 (d, J = 6.9 Hz, 4H), 7.52 (dd, J = 9.4, 7.4 Hz, 20H), 7.33
(dt, J = 31.6, 7.4 Hz, 13H), 5.73 (d, J = 7.8 Hz, 4H), 4.01
(d, J = 7.1 Hz, 4H), 3.33 (s, 1H), 2.50 (d, J = 13.6 Hz, 11H),
2.29 (s, 2H), 3.14–1.07 (m, 64H), 2.00–1.84 (m, 9H),
(S)-4-(2-(4-(Tert-butyl)phenylsulfonamido)-
2-phenylacetamido)-N-hydroxybutanamide (HD20)
The title compound HD20 was obtained as an amorphous
white solid (0.20 g, 40% yield). [α]20D −6.12 (c 1.0, MeOH).
Copyright r 2017 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.

Structural modification of HDACIs Zhang et al.
9
Fig. 4
Quantification of tumor volume from the negative control, SAHA, and HD9 groups at different time points (week 1–6). N = 6/group. (a) Quantification
of tumor volume at week 1. (b) Quantification of tumor volume at week 2. (c) Quantification of tumor volume at week 3. (d) Quantification of tumor
volume at week 4. (e) Quantification of tumor volume at week 5. (f) Quantification of tumor volume at week 6.*Statistically different from the negative
control data of the same time point. SAHA, suberoyl anilide hydroxamic acid.
Fig. 5
0.2, 0.04, and 0.008 µmol/l/l), at which their HDAC
inhibitory effects were investigated.
Western blot analysis
Briefly, U937 cells with different treatments were col-
lected and lysed with lysis buffer for 30 min, centrifuged
for 15 min at 4000 rpm at 4°C, and the supernatant was
collected as total protein extracts. Total protein extracts
were then separated by 12% SDS polyacrylamide gel
electrophoresis and transferred onto PVDF membranes
Picture of dissected tumor tissues. SAHA, suberoyl anilide
hydroxamic acid.
(cat. IPVH00010; Millipore, Billerica, Massachusetts,
USA). The membrane was blocked with 5% milk in
TBS-T for 1 h at room temperature, and then incubated
with a 1 : 1000 or a 1 : 2000 dilution of primary antibodies
2.54–1.07 (m, 34H), 1.73–1.45 (m, 9H), 1.16 (t,
J = 7.1 Hz, 1H).
[antibodies against acetylated histone H3 (Sigma, St.
Louis, Missouri, USA) and H4 (Sigma)] overnight at 4°C.
Then, the membrane was washed three times and incu-
bated with a 1 : 2000 dilution of anti-mouse or anti-rabbit
goat-HRP-conjugated secondary antibodies for 2 h at
room temperature. Finally, the membrane was washed
another three times and developed with enhanced che-
miluminescence (ECL, cat. WBKL S0050; Millipore).
Enzyme inhibition assay
The method of enzymatic inhibition assay has been
described in our previous work [21]. The nucleus extract
was prepared from Hela cells. Recombinant HDAC3
and HDAC6 were purchased from BPS Bioscience
(bpsbioscience.com). Boc-Lys (acetyl)-AMC was used as
the substrate of HDAC. SAHA, the well-established
HDAC inhibitor, was used as a positive control. The
compounds were diluted to six concentrations (25, 5, 1,
Molecular docking
The molecular docking process was performed using
Glide (Schrodinger Inc., supported by Shanghai Institute
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10 Anti-Cancer Drugs 2017, Vol 00 No 00
Fig. 6
The body weight of animals in the negative control, SAHA, and HD9 groups at different time points (week 1–6). N = 6/group. (a) Animal body weight
at week 1. (b) Animal body weight at week 2. (c) Animal body weight at week 3. (d) Animal body weight at week 4. (e) Animal body weight at week 5.
(f) Animal body weight at week 6.*Statistically different from the negative control data of the same time point. SAHA, suberoyl anilide hydroxamic acid.
of Materia Medica Chinese Academy of Sciences). The
crystal structure of HDAC3 (PDB Entry: 4A69) was
selected as a receptor in the docking study. Structural
modifications were performed to make the protein sui-
table for docking. The water molecules and the ligand
crystallized in the protein structure were removed. The
OPLS 2005 force field was assigned to the refined
structure. The ligands used in the docking approach were
sketched by Maestro and refined by LigPrep. The active
site was defined as a cubic box containing residues
around the Zn ion at a distance of 20 Å. Extra precision
was applied in the docking process and other parameters
were set as default.
density values were detected at 570 nm on a microplate
reader (Thermo, Waltham, Massachusetts, USA).
In-vivo antitumor assay against the U937 xenograft
For the in-vivo antitumor efficacy study, 1.8 × 107 U937
cells were inoculated subcutaneously into the right
shoulder of male athymic nude mice (5–6 weeks old, Slac
Laboratory Animal, Shanghai). Ten days after the injec-
tion, tumors were palpable and mice were assigned ran-
domly into treatment and control groups (six mice/
group). The animals in the treatment group were admi-
nistered 100 mg/kg/day HD9 and SAHA through gavage,
whereas the animals in the control group were adminis-
tered an equal volume of PBS. During treatment, the
body weight of mice was monitored regularly. After
16 days of treatment, the mice were killed and the tumor
weight was measured using an electronic balance.
Tumor cell inhibition assay
U937, K562, HL60, MDA-MB-231, PC3, and SK-OV-3
cells were used in the cell proliferation assays. 5000 cells
were seeded into each well of 96-well plates in a standard
medium and incubated at 37°C, 5% CO2 overnight.
Then, cells were treated with 5 μmol/l of all the mole-
cules. After 72 h of incubation, a 0.5% MTT (Amresco,
Solon, Ohio, USA) solution was added to each well, and
the cells were incubated for another 4 h. Then, the cul-
ture medium was removed, and 200 μl DMSO was added
to each well. After thoroughly mixing for 5 min, optical
Results and discussion
The enzymatic inhibition assay was performed against
the Hela cell nucleus extract, which provided a mixture
of HDAC isoforms. Compared with the previously
reported D series molecules, HDACIs with the fatty
linker showed reduced inhibitory activities (Table 1).
The reason for the reduction in activity was deduced to
be the lack of π–π stacking interactions formed between
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Structural modification of HDACIs Zhang et al. 11
the aromatic linkers and the surrounding residues, which
was discovered in our previous work [22]. In the enzy-
matic assay, molecule HD9 showed the most potent
inhibitory activity, with an IC50 value of 76 nmol/l, a
value superior to SAHA (158 nmol/l). Structure–activity
relationship analysis shows that bulky R groups can
enhance the enzyme-inhibitory activities, as observed in
the case of molecule HD9–HD13. Moreover, increased
flexibility in the R groups significantly improved the
inhibitory activities, such as HD9 and HD10 (IC50
values of 76 and 108 nmol/l, compared with HD1 of
394 nmol/l). Molecules with the strong electron-
withdrawing nitro group substituted into the R group
showed limited activities (HD7 and HD17), suggesting
that such groups are not favored in the ligand–receptor
interactions. The hydroxyl group substituted into the
naphthalene ring of the R group can increase the inhi-
bitory activities, but the effect is not obvious. No sig-
nificant rules of haloid or alkyl substitution were
observed in the R groups. The isoform selectivity assay
was performed on the basis of HDAC3 and HDAC6
(Table 2), and the results showed that both HD9 and
HD10 are more potent in the inhibition of HDAC3 (0.12
and 0.23 µmol/l) than that of HDAC6 (0.39 and 0.42
µmol/l) compared with SAHA.
HD10 and HD11 bind to the hydrophobic regions in the
opening of the site (Figs 3c and 3d). Moreover, the hydro-
phobic interactions with the surrounding residues (such as
Tyr197, Phe198, and Phe199) play significant roles in the
binding of HD10 and HD11 to HDAC3. In the scoring
analysis, molecule HD9 showed the smallest glide evdw
(−36.4), glide ecoul (−29.54), and glide energy (−66.0)
values compared with HD10 (−32.5, −27.61, and −60.1)
and HD3 (−30.8, −29.53, and −60.4). All the docking
analyses indicated that molecule HD9 is a more potent
HDAC inhibitor, which is in line with the enzyme-
inhibitory assay results.
To evaluate the antiproliferation activity of the synthe-
sized molecules, MTT assays were performed using
U937, K562, HL60, MDA-MB-231, PC3, and SK-OV-3
cell lines (Table 2). The initial screening in 1 µmol/l of
each derived target molecule against U937 cells sug-
gested that only molecules HD9 and HD10 showed
similar activities compared with SAHA. Therefore, only
HD9 and HD10 were tested for the IC50 value.
Generally, lymphomatous and leukemic cells lines
(U937, K562, and HL60) are more sensitive to the
representative molecules than the solid tumor cell lines
(MDA-MB-231, PC3, and SK-OV-3), which is in line
with our previously reported results and the published
results that the therapeutic efficacy of HDACIs is
restricted predominantly to hematological malignancies
[23,24]. Molecule HD9 showed good performance inhi-
biting the growth of tested tumor cell lines, with mean
IC50 values of 0.7 µmol/l against hematologic tumor cell
lines and 3.26 µmol/l against solid tumor cell lines,
respectively. The antiproliferative activity of HD10 is
not as good as HD9, especially when used against the
growth of solid tumor cells (PC3 and SK-OV-3).
Molecules HD9 and HD10 showed improved safety
properties over SAHA in the growth inhibition of the L02
cell line, with IC50 values of 5.58 and 7.96 µmol/l (SAHA:
1.62 µmol/l), respectively. Viability tests on U937 cells
showed time-dependent and dose-dependent effects of
the tested molecules (HD9 and HD10, Table 3). The
results suggested that molecule HD9 is valuable for
further development as a lead compound.
The intracellular acetylation abilities of representative
molecules were assessed by western blotting (Fig. 2a).
Significantly elevated acetylation levels of histone H3 and
H4 (targets of HDAC1 and HDAC2) were observed in the
U937 cells treated with all the three tested molecules rela-
tive to the control, indicating effective inhibition of HDAC1
and HDAC2 by these molecules. Among the three mole-
cules, HD9 showed best acetylation-enhancement effect at
both histone H3 and H4, indicating its stability and effec-
tiveness as a class I HDAC inhibitor. Docking studies were
carried out to investigate the binding patterns of repre-
sentative molecules (HD3, HD9, and HD10) to the active
site of human HDAC3. The docking results are of guiding
significance for the explanation of structure–activity rela-
tionship and further drug design. The docking results
indicated that hydroxamic acid groups of molecule HD3,
HD9, and HD10 bind to the zinc ion and form H-bond
interactions with His135 (Fig. 3). The fatty linkers locate in
the narrow channel of the site in which aromatic linkers can
form π–π stacking interactions with the surrounding resi-
dues. The aromatic groups in the cap region of the three
molecules bind to different regions in the opening of the site
(Fig. 3a). Good spatial match of HD9 can be observed by
stretching of the naphthalene ring to a pocket below the
opening of the tunnel (Fig. 3b). The phenyl ring of HD9
can form π–π stacking interactions with Phe199 of HDAC3.
H-bond interactions formed between HD9 and Asp193 also
contribute to the binding of HD9 to the active site. Because
of the strong interactions involved, molecule HD9 was
found to show the best performance in the enzyme-based
assay. Meanwhile, the naphthalene and phenyl rings of both
To investigate the in-vivo antiproliferation ability of
molecule HD9, the U937 cell line was selected for the
in-vivo antitumor assay. After 16 days of intragastric
administration, the mice were killed and the tumor
weight was measured. The mean weight of tumors in the
negative control group was 3.60 g (SD 32%), and the
values of the SAHA-treated group and the HD9-treated
group were 1.79 g (SD 23%) and 2.02 g (SD 27%),
respectively (Fig. 2b). The inhibitory ratio of molecule
HD9 was 43.9%, whereas SAHA achieved a value of
50.4%. The result showed that HD9 is less effective than
SAHA in the in-vivo test. The tumor volume was mea-
sured every other day, and the result was correlated with
the tumor weight (Figs 4 and 5). The absence of body
Copyright r 2017 Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited.

12 Anti-Cancer Drugs 2017, Vol 00 No 00
weight loss (as indicated by the body weight data
acquired every other day) and signs of hepatotoxicity or
splenotoxicity suggested the relative safety of molecule
HD9 (Fig. 6). The in-vitro and in-vivo results suggest
that molecule HD9 could be used as a lead compound
for further design of HDAC inhibitors.
3
Foglietti C, Filocamo G, Cundari E, De Rinaldis E, Lahm A, Cortese R, et al.
Dissecting the biological functions of Drosophila histone deacetylases by
RNA interference and transcriptional profiling. J Biol Chem 2006;
281:17968–17976.
Gronroos E, Hellman U, Heldin CH, Ericsson J. Control of Smad7 stability
by competition between acetylation and ubiquitination. Mol Cell 2002;
10:483–493.
Giandomenico V, Simonsson M, Gronroos E, Ericsson J. Coactivator-
dependent acetylation stabilizes members of the SREBP family of
transcription factors. Mol Cell Biol 2003; 23:2587–2599.
Jin YH, Jeon EJ, Li QL, Lee YH, Choi JK, Kim WJ, et al. Transforming growth
factor-beta stimulates p300-dependent RUNX3 acetylation, which inhibits
ubiquitination-mediated degradation. J Biol Chem 2004;
279:29409–29417.
4
5
6
Conclusion
To improve the solubility of previously derived
phenylglycine-based HDAC inhibitors, structural mod-
ification was performed by the substitution of fatty lin-
kers for aromatic linkers. In the enzymatic inhibition
assay, most of the target molecules showed reduced
in-vitro inhibitory activities. Nevertheless, molecules
HD9 and HD10 showed potent HDAC-inhibitory
activities (with IC50 values of 76 and 108 nmol/l,
respectively) compared with SAHA (IC50 values of
158 nmol/l). In the docking analysis, molecules HD3,
HD9, and HD10 were docked to the active site of
HDAC3 to predict the structural factors that influence
the activities. Molecule HD9 was discovered to have a
good structural match with the surface of the binding site,
and showed the lowest binding energies compared with
the other two molecules. Multiple H-bonds and hydro-
phobic interactions make HD9 bind tightly to the active
site of HDAC3. Molecule HD9 also showed good per-
formance in the in-vitro antiproliferative test, and showed
a hematological malignancy cell line-selective inhibitory
pattern similar to previously reported HDACIs. Although
molecule HD9 showed slightly less efficacy compared
with SAHA in the in-vivo tumor suppression assay,
improved solubility as well as pharmacokinetic profiles
guaranteed its potential to be a promising lead compound
for further design of HDACIs.
7
8
9
Bode AM, Dong ZG. Post-translational modification of p53 in tumorigenesis.
Nat Rev Cancer 2004; 4:793–805.
Chen LF, Greene WC. Shaping the nuclear action of NF-kappa B. Nat Rev
Mol Cell Biol 2004; 5:392–401.
Kovacs JJ, Murphy PJ, Gaillard S, Zhao XA, Wu JT, Nicchitta CV, et al.
HDAC6 regulates Hsp90 acetylation and chaperone-dependent activation of
glucocorticoid receptor. Mol Cell 2005; 18:601–607.
10 Marks PA, Rifkind RA, Richon VM, Breslow R, Miller T, Kelly WK. Histone
deacetylases and cancer: causes and therapies. Nat Rev Cancer 2001;
1:194–202.
11 Thiagalingam S, Cheng KH, Lee HJ, Mineva N, Thiagalingam A, Ponte JF.
Histone deacetylases: unique players in shaping the epigenetic histone code.
Ann N Y Acad Sci 2003; 983:84–100.
12 Marks PA, Richon VM, Rifkind RA. Histone deacetylase inhibitors: inducers of
differentiation or apoptosis of transformed cells. J Natl Cancer Inst 2000;
92:1210–1216.
13 Zhang L, Han Y, Jiang Q, Wang C, Chen X, Li X, et al. Trend of histone
deacetylase inhibitors in cancer therapy: isoform selectivity or multitargeted
strategy. Med Res Rev 2015; 35:63–84.
14 Richon VM, Emiliani S, Verdin E, Webb Y, Breslow R, Rifkind RA, et al.
A class of hybrid polar inducers of transformed cell differentiation inhibits
histone deacetylases. Proc Natl Acad Sci USA 1998; 95:3003–3007.
15 Ueda H, Nakajima H, Hori Y, Fujita T, Nishimura M, Goto T, et al. Fr901228,
a novel antitumor bicyclic depsipeptide produced by chromobacterium-
violaceum No-968 .1. Taxonomy, fermentation, isolation, physicochemical
and biological properties, and antitumor-activity. J Antibiot (Tokyo) 1994;
47:301–310.
16 Yang L, Xue XW, Zhang YH. Simple and efficient synthesis of belinostat.
Synthetic Commun 2010; 40:2520–2524.
17 Neri P, Bahlis NJ, Lonial S. Panobinostat for the treatment of multiple
myeloma. Expert Opin Investig Drugs 2012; 21:733–747.
18 Zhang L, Li MY, Feng JH, Fang H, Xu WF. Discovery of a novel histone
deacetylase 8 inhibitor by virtual screening. Med Chem Res 2012;
21:152–156.
19 Zhang L, Wang X, Li X, Xu W. Discovery of a series of small molecules as
potent histone deacetylase inhibitors. J Enzyme Inhib Med Chem 2013;
1:1–7.
20 Zhang L, Zhang Y, Chou CJ, Inks ES, Wang X, Li X, et al. Histone
deacetylase inhibitors with enhanced enzymatic inhibition effects and potent
in vitro and in vivo antitumor activities. Chem Med Chem 2014; 9:638–648.
21 Zhang YJ, Feng JH, Liu CX, Zhang L, Jiao J, Fang H, et al. Design, synthesis
and preliminary activity assay of 1,2,3,4-tetrahydroisoquinoline-3-carboxylic
acid derivatives as novel Histone deacetylases (HDACs) inhibitors. Bioorg
Med Chem 2010; 18:1761–1772.
Acknowledgements
This work was supported by the Qingdao Applied Basic
Research Program (Youth Special, no. 16-5-1-60-jch).
Structural modification of histone deacetylase inhibitors
with a phenylglycine scaffold.
Conflicts of interest
There are no conflicts of interest.
22 Zhang L, Zhang L. Molecular simulation of HDAC1/3 Inhibitor: insights into
the structural basis of selectivity. Lett Drug Des Discov 2016; 13:43–46.
23 Verbrugge I, Johnstone RW, Bots M. Promises and challenges of anticancer
drugs that target the epigenome. Epigenomics 2011; 3:547–565.
24 Gryder BE, Sodji QH, Oyelere AK. Targeted cancer therapy: giving histone
deacetylase inhibitors all they need to succeed. Future Med Chem 2012;
4:505–524.
References
1
2
Bernstein BE, Tong JK, Schreiber SL. Genomewide studies of histone
deacetylase function in yeast. Proc Natl Acad Sci USA 2000;
97:13708–13713.
De Ruijter AJ, van Gennip AH, Caron HN, Kemp S, van Kuilenburg AB.
Histone deacetylases (HDACs): characterization of the classical
HDAC family. Biochem J 2003; 370:737–749.
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