Discovery of a series of small molecules as potent histone deacetylase inhibitors

Article abstract of DOI:10.3109/14756366.2013.780237

A series of small molecules were designed and synthesized based on our previous virtual screening approach, which was performed to discover potent histone deacetylase inhibitors (HDACIs) with novel structures. The derived compounds were tested by Hela cell nucleus extract for enzyme inhibition assay. Tumor cell growth inhibition assays were performed using a series of tumor cell lines. Molecule 4h has the best performance among these compounds with enzyme inhibition IC50 of 0.14 μM and tumor cell growth inhibition IC50 of 1.85 (U937), 2.02 (HL60), 2.67 (K562). Docking studies showed that multiple H-bonds and hydrophobic interactions make 4h binding to the active site of HDAC. 4h has the advantage of low molecular weight, so a variety of structural modifications can be performed in our further studies.

Full text of DOI:10.3109/14756366.2013.780237

http://informahealthcare.com/enz
ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, 2014; 29(3): 333–337
2014 Informa UK Ltd. DOI: 10.3109/14756366.2013.780237
!
ORIGINAL ARTICLE
Discovery of a series of small molecules as potent histone
deacetylase inhibitors
Lei Zhang1, Xuejian Wang2, Xiaoguang Li1, and Wenfang Xu1
2
1Department of Medicinal Chemistry, School of Pharmacy, Shandong University, Jinan, Shandong, China and School of Pharmacy and Biology
Science, Weifang Medicinal University, Weifang, Shandong, China
Abstract
Keywords
A series of small molecules were designed and synthesized based on our previous virtual
screening approach, which was performed to discover potent histone deacetylase inhibitors
(HDACIs) with novel structures. The derived compounds were tested by Hela cell nucleus
extract for enzyme inhibition assay. Tumor cell growth inhibition assays were performed using
a series of tumor cell lines. Molecule 4h has the best performance among these compounds
with enzyme inhibition IC50 of 0.14 mM and tumor cell growth inhibition IC50 of 1.85 (U937), 2.02
(HL60), 2.67 (K562). Docking studies showed that multiple H-bonds and hydrophobic
interactions make 4h binding to the active site of HDAC. 4h has the advantage of low
molecular weight, so a variety of structural modifications can be performed in our further
studies.
Docking, histone deacetylase inhibitors,
phenylglycine, tumor
History
Received 31 January 2013
Revised 24 February 2013
Accepted 24 February 2013
Published online 25 March 2013
Introduction
were introduced to the linker to improve H-bond interactions.
A series of novel hydroximic acid-based compounds were
synthesized and tested by enzyme inhibition and tumor cell
growth inhibition activity assay. A phenylglycine containing
structure (4h) was discovered to have good performance in these
activity assays.
Histone deacetylases (HDACs) are a family of 18 enzymes
responsible for the regulation of transcription and other nuclear
events by regulating the deacetylation of N-terminal tails of
histones. They are subdivided into four classes, class I (HDAC1,
2, 3 and 8), class II (IIa: HDAC4, 5, 7 and 9; IIb: HDAC6 and 10)
and class IV (HDAC11) are zinc-dependent enzymes. Class
III (known as sirtuins, Sirt1-7) consists of NAD-dependent
enzymes1–3.Overexpression and aberrant recruitment of HDACs
Materials and methods
Chemistry
have a significant role in tumorigenesis4–6
.
1H NMR spectra were recorded on a Bruker DRX spectrometer at
600 MHz, d in parts per million and J in Hertz, using TMS as an
internal standard. High-resolution mass spectra were conducted
by Shandong Analysis and Test Center in Ji’nan, China. ESI-MS
were determined on an API 4000 spectrometer (AB SCIEX,
Framingham, MA). Melting points were determined uncorrected
on an electrothermal melting point apparatus. Optical rotation
values were measured at room temperature using a modular
circular polarimeter 200 operating at ꢀ ¼ 589 nm.
Histone deacetylase inhibitors (HDACIs) have been proved
to have the effects of apoptosis, cell cycle arrest and differenti-
ation7–9. Suberoyl anilide hydroxamic acid (SAHA)10 and
FK22811 have been approved by FDA for treatment of advanced
cutaneous T-cell lymphoma (CTCL). Many HDACIs are still
under investigation in various stages of clinical trials for treatment
of cancer by administration individually or in combination.
In order to discover new scaffold of HDACIs, a virtual
screening approach was performed in our previous work12.
A potent small molecule HDAC inhibitor (02) was derived as a
lead structure. Although 02 has similar enzyme inhibition activity
compared with SAHA, it has no activity in the tumor cell growth
inhibition assays. Thus, structural modification was performed to
discover more potent HDACIs (Figure 1). Hydroximic acid group
was used to bind to zinc ion, sulphonamide and amide groups
Methyl 4-aminobenzoate hydrochloric acid (2)
4-aminobenzoic acid (1) (13.7 g, 100 mmol) was dissolved in
100 mL of MeOH, acetyl chloride (23.6 g, 300 mmol) was added
under ice bath. The solution was stirred at 75 ꢀC for 5 h. Then, the
solvent was evaporated with the desired compound 2 (14.4 g, 77%
yield) being recrystallized as white powder by ester. ESI-MS:
m/z: 152.1 [M þ H]þ.
(S)-2-((tert-butoxycarbonyl)amino)-2-phenylacetic acid (h2)
Address for correspondence: Wenfang Xu, Department of Medicinal
Chemistry, School of Pharmacy, Shandong University, Jinan, Shandong
250012, China. Tel/Fax: +86-531-88382264. E-mail: xuwenf@sdu.
edu.cn
(S)-2-amino-2-phenylacetic acid (h1) (15.1 g, 100 mmol) was
dissolved in 200 mL of MeOH/H2O (3:1), Et3N (15.1 g,
150 mmol) and (Boc)2O (32,7 g, 150 mmol) were added in turn.

334 L. Zhang et al.
J Enzyme Inhib Med Chem, 2014; 29(3): 333–337
4-(4-acetamidophenylsulfonamido)-N-hydroxybenzamide (4b)
Title compound was obtained as an amorphous white solid
(0.35 g, 64% yield); m.p. ¼ 214–216 ꢀC; 1H NMR (600 MHz,
(CD3)2SO) d 11.02 (s, 1H), 10.68 (s, 1H), 10.30 (s, 1H), 8.91
(s, 1H), 7.82 (d, J ¼ 8.4 Hz, 2H), 7.74 (d, J ¼ 10.8 Hz, 2H), 7.71
(d, J ¼ 5.4 Hz, 2H), 7.19 (d, J ¼ 6 Hz, 2H), 2.06 (s, 3H). HRMS
(AP-ESI) m/z calcd for C15H16N3O5S [M þ H]þ ¼ 350.0810,
found ¼ 350.0805. Retention time: 2.9 min.
Figure 1. Design of new scaffold from the lead structure.
4-(4-chlorophenylsulfonamido)-N-hydroxybenzamide (4c)
Title compound was obtained as an amorphous white solid
(0.32 g, 61% yield); m.p. ¼ 202–204 ꢀC; 1H NMR (600 MHz,
(CD3)2SO) d 11.06 (s, 1H), 10.71 (s, 1H), 8.94 (s, 1H), 7.80
(d, J ¼ 8.4 Hz, 2H), 7.65 (d, J ¼ 7.2 Hz, 2H), 7.63 (d, J ¼ 8.4 Hz,
2H), 7.14 (d, J ¼ 9 Hz, 2H). HRMS (AP-ESI) m/z calcd for
After stirred at room temperature for 8 h, the solvent was
evaporated with the residue being dissoved in saturated citric
acid (100 mL). The solution was extracted with EtOAc
(3 ꢁ 60 mL). The EtOAc solution was washed with saturated
brine (3 ꢁ 50 mL), dried over MgSO4, and evaporated under
vacuum. The desired compound h2 (21.6 g, 86% yield) was
derived by crystallization in hexane as white powder. ESI-MS:
m/z: 252.1 [M þ H]þ.
C13H12ClN2O4S
[M þ H]þ ¼ 327.0200,
found ¼ 327.0202.
Retention time: 2.9 min.
4-(4-fluorophenylsulfonamido)-N-hydroxybenzamide (4d)
N-hydroxy-4-(4-nitrophenylsulfonamido)benzamide (3a)
Title compound was obtained as an amorphous white solid
(0.28 g, 57% yield); m.p. ¼ 181–183 ꢀC; 1H NMR (600 MHz,
(CD3)2SO) d 11.05 (s, 1H), 10.66 (s, 1H), 8.93 (s, 1H), 7.87
(d, J ¼ 8.4 Hz, 2H), 7.63 (d, J ¼ 8.4 Hz, 2H), 7.42 (d, J ¼ 8.4 Hz,
2H), 7.14 (d, J ¼ 8.4 Hz, 2H). HRMS (AP-ESI) m/z calcd
for C13H12FN2O4S [M þ H]þ ¼ 311.0502, found ¼ 311.0496.
Retention time: 2.9 min.
To a solution of compound 2 (0.56 g, 3.0 mmol) in THF (50 mL)/
H2O (1.0 mL), NaHCO3 (1.01 g, 12 mmol) was added. After
10 min, 4-nitrobenzene-1-sulfonyl chloride (0.73 g, 3.3 mmol) was
added. The 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 3a (0.68 g, 70% yield) was derived by
crystallization in EtOAc as white powder. ESI-MS: m/z: 337.1
[M þ H]þ.
N-hydroxy-4-(naphthalene-2-sulfonamido)benzamide (4e)
Title compound was obtained as an amorphous white solid
(0.36 g, 66% yield); m.p. ¼ 237–239 ꢀC; 1H NMR (600 MHz,
(CD3)2SO) d 11.06 (s, 1H), 10.97 (s, 1H), 8.88 (s, 1H), 8.72
(d, J ¼ 8.4 Hz, 1H), 8.28 (d, J ¼ 7.2 Hz, 1H), 8.23 (d, J ¼ 8.4 Hz,
1H), 8.08 (d, J ¼ 8.4 Hz, 1H), 7.75 (t, J ¼ 7.8 Hz, 1H), 7.68
(d, J ¼ 7.2 Hz, 1H), 7.65 (t, J ¼ 7.8 Hz, 1H), 7.53 (d, J ¼ 8.4 Hz,
2H), 7.08 (d, J ¼ 9 Hz, 1H). HRMS (AP-ESI) m/z calcd
for C17H15N2O4S [M þ H]þ ¼ 343.0752, found ¼ 343.0746.
Retention time: 2.9 min.
The other compounds (3b–3g) were prepared in the same
procedure as described above.
(S)-methyl 4-(2-((tert-butoxycarbonyl)amino)-2-
phenylacetamido)benzoate (3h)
To a solution of h2 (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, compound 2 (0.94 g, 5 mmol) and Et3N
(0.50 g, 5 mmol) were added. The 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 3h (1.44 g, 75%
yield) was derived by crystallization in EtOAc as white powder.
ESI-MS: m/z: 385.2 [M þ H]þ.
4-(2,6-difluorophenylsulfonamido)-N-hydroxybenzamide (4f)
Title compound was obtained as an amorphous white solid
(0.35 g, 63% yield); m.p. ¼ 210–212 ꢀC; 1H NMR (600 MHz,
(CD3)2SO) d 11.24 (s, 1H), 11.05 (s, 1H), 8.94 (s, 1H), 7.20
(d, J ¼ 8.4 Hz, 1H), 7.65 (d, J ¼ 9 Hz, 2H), 7.30 (d, J ¼ 9 Hz,
2H), 7.18 (d, J ¼ 8.4 Hz, 2H). HRMS (AP-ESI) m/z calcd
for C13H11F2N2O4S [M þ H]þ ¼ 329.0407, found ¼ 329.0404.
Retention time: 2.9 min.
N-hydroxy-4-(4-(trifluoromethyl)phenylsulfonamido)benzamide
(4g)
N-hydroxy-4-(4-nitrophenylsulfonamido)benzamide (4a)
Compound 3a (0.5 g, 1.6 mmol) was dissolved in 14 mL of 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 4a (0.33 g, 62% yield)
was derived by crystallization in EtOAc as white powder;
m.p. ¼ 190–191 ꢀC; 1H NMR (600 MHz, (CD3)2SO) d 11.08
(s, 1H), 10.63 (s, 1H), 8.95 (s, 1H), 8.38 (d, J ¼ 9 Hz, 2H), 8.05
(d, J ¼ 8.4 Hz, 2H), 7.65 (d, J ¼ 8.4 Hz, 2H), 7.16 (d, J ¼ 6 Hz, 2H).
HRMS (AP-ESI) m/z calcd for C13H12N3O6S [M þ H]þ ¼
337.0447, found ¼ 338.0442. Retention time: 2.9 min.
Title compound was obtained as an amorphous white solid
(0.34 g, 62% yield); m.p. ¼ 249–250 ꢀC; 1H NMR (600 MHz,
(CD3)2SO) d 11.06 (s, 1H), 10.87 (s, 1H), 8.94 (s, 1H), 8.01
(d, J ¼ 8.4 Hz, 2H), 7.98 (d, J ¼ 9 Hz, 2H), 7.64 (d, J ¼ 8.4 Hz,
2H), 7.16 (d, J ¼ 9 Hz, 2H). HRMS (AP-ESI) m/z calcd for
C14H12F3N2O4S
[M þ H]þ ¼ 361.0470,
found ¼ 361.0463.
Retention time: 2.9 min.
(S)-tert-butyl (2-((4-(hydroxycarbamoyl)phenyl)amino)-2-oxo-
1-phenylethyl)carbamate (4h)
Title compound was obtained as an amorphous white solid
20
The other compounds (4b–4h and h3) were prepared in the (0.37 g, 63% yield). ½ꢁꢂD ¼ ꢃ6.26 (c ¼ 1.0, MeOH); m.p. ¼ 200–
1
same procedure as described above.
202 ꢀC; H NMR (600 MHz, (CD3)2SO) d 11.10 (s, 1H), 10.44

DOI: 10.3109/14756366.2013.780237
Histone deacetylase inhibitors 335
(s, 1H), 8.93 (s, 1H), 7.70 (d, J ¼ 9 Hz, 2H), 7.63 (d, J ¼ 9 Hz, (Amresco, Solon, OH) solution was added to each well. Then
2H), 7.56 (d, J ¼ 8.4 Hz, 1H), 7.49 (d, J ¼ 7.2 Hz, 2H), 7.37–7.35 a further 4 h of incubation was performed. After that the culture
(m, 2H), 7.31 (d, J ¼ 7.2 Hz, 1H), 5.36 (d, J ¼ 7.2 Hz, 1H), 1.40 medium was removed, and 200 mL of DMSO was added to
(s, 9H). HRMS (AP-ESI) m/z calcd for C20H23N3NaO5 dissolve the formazan. After mixing for 5 min, optical density
[M þ Na]þ ¼ 408.1536, found ¼ 408.1529. Retention time: values were detected at 570 nm on a microplate reader (Thermo
2.9 min.
Fisher Scientific, Waltham, MA).
Molecular docking
(S)-tert-butyl (2-(hydroxyamino)-2-oxo-1-phenylethyl)carbamate
(h3)
The molecular docking process was performed using Glide (www.
schrodinger.com). Crystal structure of HDAC2 (PDB Entry:
3MAX) was selected as a receptor in the docking study. Structural
modifications were performed to make the protein suitable for
docking. The water molecules and the ligand crystallized in the
protein structure were removed. OPLS 2005 force field was
assigned to the refined structure. The ligands used in the docking
approach were sketched by maestro and prepared by LigPrep
(www.schrodinger.com). The active site was defined as a cubic
Title compound was obtained as an amorphous white solid
20
(0.34 g, 61% yield); m.p. ¼ 154–155 ꢀC; ½ꢁꢂD ¼ ꢃ6.78 (c ¼ 1.0,
1
MeOH). H NMR (600 MHz, (CD3)2SO) d 10.88 (s, 1H), 8.96
(s, 1H), 7.40 (d, J ¼ 7.8 Hz, 2H), 7.37 (d, J ¼ 8.4 Hz, 1H), 7.33
(d, J ¼ 7.2 Hz, 2H), 7.27 (t, J ¼ 7.2 Hz, 1H), 5.05 (d, J ¼ 8.4 Hz,
1H), 1.38 (s, 9H). HRMS (AP-ESI) m/z calcd for C13H18N2NaO4
[M þ Na]þ ¼ 289.1165, found ¼ 289.1158. Retention time:
2.9 min.
˚
box containing residues around Zn ion at a distance of 20 A. Extra
precision was applied in the docking process, other parameters
were set as default.
(S)-4-(2-amino-2-phenylacetamido)-N-hydroxybenzamide
hydrochloride (4hb)
To a solution of compound 4h (0.38 g, 1 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 filtered, with the filter
being washed with ether, to give desired compound 4hb (0.19 g,
68% yield). Title compound was obtained as an amorphous
Results and discussion
Chemistry
The target compounds were synthesized following the route
shown in Scheme 1. The starting material 4-aminobenzoic acid
was converted to be compound 2 by esterification. The key
intermediates (3a–3g) were generated by nucleophilic substitua-
20
white solid; m.p. ¼ 187–188 ꢀC; ½ꢁꢂD ¼ ꢃ6.53 (c ¼ 1.0, MeOH).
1H NMR (600 MHz, (CD3)2SO) d 11.45 (s, 1H), 10.31 (s, 1H),
8.92–8.91 (m, 3H), 7.73 (d, J ¼ 8.4 Hz, 2H), 7.70 (d, J ¼ 8.4 Hz,
2H), 7.69 (d, J ¼ 7.2 Hz, 2H), 7.46 (d, J ¼ 7.8 Hz, 2H), 7.43
(t, J ¼ 7.2 Hz, 1H), 5.33 (d, J ¼ 5.4 Hz, 1H). HRMS (AP-ESI) m/z
calcd for C15H16N3O3 [M þ H]þ ¼ 286.1191, found ¼ 286.1186.
Retention time: 2.9 min.
tion of compound 2 with corresponding sulfuryl chloride13–18
.
L-phenylglycine was protected by Boc, followed by coupling with
compound 2 to yield 3h. Compounds 4a–4h were derived by
treating the corresponding intermediates (3a–3h) with NH2OK
in methanol19. Compound 4hb was derived by deprotection of the
amino group of 4h. Compounds h3 and h4 were prepared via the
same route as 4hb.
Compound h4 was prepared in the same procedure as
described above.
Activity assay
(S)-2-amino-N-hydroxy-2-phenylacetamide hydrochloride (h4)
The derived compounds were assayed by Hela cell nucleus extract
using SAHA as a positive control (Table 1). Molecule 4h has the
best performance in the enzyme inhibition assay. Among com-
pounds 4a–4g, 4e has the best enzyme inhibition activity. The result
means bulky R groups are beneficial for these molecules binding to
HDACs. Protection of the amino group by Boc group in the
phenylglycine improved the activity, which was proved by
comparing the IC50 values between 4h (0.14 mM) and 4hb
(0.18 mM), h3 (1.60 mM) and h4 (3.79 mM). The results indicate
that substituent in the amino group improves the interaction
between the ligands and the receptor. Molecules h3 and h4 were
subsequently synthesized to find out if the phenylglycine structure
has enzyme inhibition activity without a linker. The results suggest
that removal of the linker results in the reduction of the activity.
The results of the tumor cell growth inhibition assay have
correlation with the results of the enzyme inhibition assay
(Table 1). K562 cells are more sensitive to these molecules
than other cells. In these assays, 4h is also the most potent
inhibitor. Deprotection of the amino group in the phenylglycine
Title compound was obtained as an amorphous white solid
20
(0.21 g, 70% yield); m.p. ¼ 209–210 ꢀC; ½ꢁꢂD ¼ ꢃ7.21 (c ¼ 1.0,
MeOH). 1H NMR (600 MHz, (CD3)2SO) d 11.10 (s, 1H), 9.30 (s,
1H), 8.87 (s, 3H), 7.56 (d, J ¼ 6.6 Hz, 2H), 7.45 (d, J ¼ 7.2 Hz,
2H), 7.43–7.41 (m, 5H), 4.85 (s, 1H). HRMS (AP-ESI) m/z calcd
for C8H11N2O2 [M þ H]þ ¼ 167.0820, found ¼ 167.0810.
Retention time: 2.9 min.
Enzyme inhibition assay
The HDAC enzyme was extracted from Hela cells. Boc-Lys
(acetyl)-AMC was used as the substrate of HDAC. SAHA which
is the HDAC inhibitor on market was used as a positive control.
The compounds were diluted to six concentrations (25, 5, 1, 0.2,
0.04 and 0.008 uM/L) to investigate their ability of inhibiting
HDAC activity.
Tumor cell inhibition assay
HCT116, K562, MDA-MB-231, ES-2, U937, HL60, MCF7 and and removal of the linker have significant influence on the
PC3 cells were used in the cell proliferation assays. A total of inhibitory activity.
5000 cells were seeded into each well of 96-well plates which
The IC50 values of 4h in inhibition of U937, HCT116, K562,
were incubated at 37 ꢀC, 5% CO2 for overnight. Then cells HL60, MCF7 and PC3 cell growth were derived compared with
(HCT116, K562, MDA-MB-231, ES-2) were treated with 5 mM SAHA (Table 2). The results showed that 4h could efficiently
of all these molecules. In order to calculate the IC50 values, cells inhibit the growth of these cell lines. In inhibition, the growth
(U937, HCT116, K562, HL60, MCF7 and PC3) were treated of K562, HL60 and PC3 cells, 4h even has similar performance
with compound 4h and SAHA at final concentrations ranging with SAHA. Our results also indicate that 4h can selectively
from 0.25 to 20 mM. After 48 h of incubation, 0.5% MTT inhibit the growth lymphoma cells and hematological tumor cells.

336 L. Zhang et al.
J Enzyme Inhib Med Chem, 2014; 29(3): 333–337
Scheme 1. Reagents and conditions: (a) CH3COCl, CH3OH; (b) (Boc)2O, Et3N, MeOH/H2O; (c) RSO2Cl, NaHCO3, THF/H2O; (d) TBTU, Et3N, THF;
(e) NH2OK, MeOH; (f) EtOAc, HCl.
Table 1. The inhibitory activities of derived molecules.*
IC50
z
HCT116
K562
MDA-MB-231
(inhib%)
ES-2
(inhib%)
Compounds (mM) (inhib%)y,z (inhib%)
4a
4b
4c
4d
4e
4f
4g
4h
4hb
h3
h4
SAHA
0.28
0.74
0.33
1.42
0.23
1.05
2.11
0.14
0.18
2.60
3.79
0.17
3.36
3.77
17.49
20.51
8.12
8.31
3.04
22.32
8.66
7.33
24.59
22.75
50.79
55.63
56.77
8.22
27.86
57.79
50.44
41.35
34.88
56.84
24.35
11.40
28.55
21.98
36.99
3.05
1.48
5.07
23.49
13.33
44.16
16.73
13.59
50.01
32.71
12.33
9.55
3.71
35.98
9.73
8.21
5.06
44.68
4.65
35.11
Figure 2. Binding patterns of 4h and 4hb in the active site of HDAC2:
(a) 4h and 4hb were shown using the wire model, the residues were
displayed as sticks; carbon atoms of 4h were colored magenta, carbon
atoms of 4hb were colored green, the gray ball is zinc ion, the H-bonds
were represented as dash lines (in black–white mode, 4h is the structure
containing Boc group); (b) 4h was shown using the ball–stick model.
47.19
*The method for in vitro activity assay has been described in our previous
work20
.
yEach value is the mean of at least three experiments (all SDs are within
10% of the mean).
zThe acitivity was described as inhibition%, the concentration of the
molecules is 5 mM.
that 4hb has similar binding pattern with 4h (Figure 2a). The
hydroximic acid groups of both molecules play a significant role
in binding the ligands to the receptor by chelating to the zinc ion
and forming H-bond interactions with residues around zinc ion
(Figure 2b). The ketonic oxygen of the hydroximic acid group has
H-bond interaction with the phenolic hydroxyl group of Tyr308.
Nevertheless, the amino group binds to the NE2 of His164 by
forming H-bond interactions. The hydroxyl group of Asp104 has
H-bond binding with the other two amino groups, which also
make important contributions to the binding. The benzene ring of
the linker has ꢂ–ꢂ stacking interactions with the phenyl groups
of Phe155 and Phe210, which improves the binding of these two
molecules to the active site of HDAC.
Table 2. The tumor cell growth inhibitory activities of 4h compared with
SAHA.*
Compounds
U937
HCT116
K562
HL60
MCF7
PC3
4h
SAHA
1.85
0.99
12.12
4.21
2.67
2.99
2.02
1.86
12.70
5.09
4.93
6.33
*Each value is the mean of at least three experiments (all SDs are within
10% of the mean).
Binding pattern analysis
Docking study was performed to simulate the binding pattern
between 4h and HDAC. In order to seek evidence for the
Conclusion
difference of the activity between 4h and 4hb, 4hb was also In order to develop potent HDAC inhibitors with efficient
docked to the active site of HDAC2. The docking result shows tumor cell inhibitory activities, a series of small molecules were

DOI: 10.3109/14756366.2013.780237
Histone deacetylase inhibitors 337
9. Insinga A, Monestiroli S, Ronzoni S, et al. Inhibitors of histone
deacetylases induce tumor-selective apoptosis through activation of
the death receptor pathway. Nat Med 2005;11:233.
10. Richon VM, Emiliani S, Verdin E, et al. A class of hybrid polar
inducers of transformed cell differentiation inhibits histone deace-
tylases. P Natl Acad Sci USA 1998;95:3003–7.
11. Ueda H, Nakajima H, Hori Y, 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 1994;47:
301–10.
12. Zhang L, Li MY, Feng JH, et al. Discovery of a novel histone
deacetylase 8 inhibitor by virtual screening. Med Chem Res 2012;
21:152–6.
synthesized based on our previous virtual screening results.
The enzyme inhibition assay and tumor cell growth inhibition
assay results show that 4h is the most potent HDAC inhibitor.
Moreover, 4h also has good performance in inhibiting the growth of
a series of tumor cell lines compared with SAHA. An important
finding is introduction of bulky group to the amino group of the
phenylglycine part that can obviously improve the inhibitory
activity. Our work emphasizes the prospect and necessity of
development HDAC inhibitors with various substituent groups
in the amino group of phenylglycine in our further work.
Declaration of interest
13. D’Ambrosio K, Smaine F-Z, Carta F, et al. Development of potent
carbonic anhydrase inhibitors incorporating both sulfonamide and
sulfamide groups. J Med Chem 2012;55:6776–83.
14. Akdemir A, Vullo D, Luca VD, et al. The extremo-alpha-carbonic
anhydrase (CA) from Sulfurihydrogenibium azorense, the fastest CA
known, is highly activated by amino acids and amines. Bioorg Med
Chem Lett 2013;23:1087–90.
The authors report no conflicts of interest. The authors alone are
responsible for the content and writing of this article.
This work was supported by National Nature Science Foundation
of China (Grant No. 90713041), National High Technology Research
and Development Program of China (Grant No. 2007AA02Z314 and
2009ZX09103-118) and Graduate Innovation Foundation of Shandong
University (GIFSDU) (No. yyx10020).
15. Allouche F, Chabchoub F, Carta F, Supuran CT. Synthesis of
aminocyanopyrazoles via a multi-component reaction and anti-
carbonic anhydrase inhibitory activity of their sulfamide derivatives
against cytosolic and transmembrane isoforms. J Enzym Inhib Med
Chem 2013;28:343–9.
16. Alp C, Maresca A, Alp NA, et al. Secondary/tertiary benzenesulfo-
namides with inhibitory action against the cytosolic human carbonic
anhydrase isoforms I and II. J Enzym Inhib Med Chem 2013;28:
294–8.
References
1. Bernstein BE, Tong JK, Schreiber SL. Genomewide studies of
histone deacetylase function in yeast. P Natl Acad Sci USA 2000;97:
13708–13.
2. De Ruijter AJM, Van Gennip AH, Caron HN, et al. Histone
deacetylases (HDACs): characterization of the classical HDAC
family. Biochem J 2003;370:737–49.
17. Maresca A, Scozzafava A, Vullo D, Supuran CT. Dihalogenated
sulfanilamides and benzolamides are effective inhibitors of the three
beta-class carbonic anhydrases from Mycobacterium tuberculosis.
J Enzym Inhib Med Chem 2013;28:384–7.
18. Supuran CT, Maresca A, Gregan F, Remko M. Three new aromatic
sulfonamide inhibitors of carbonic anhydrases I, II, IV and XII.
J Enzym Inhib Med Chem 2013;28:289–93.
3. Foglietti C, Filocamo G, Cundari E, et al. Dissecting the biological
functions of Drosophila histone deacetylases by RNA interference
and transcriptional profiling. J Biol Chem 2006;281:17968–76.
4. Bode AM, Dong ZG. Post-translational modification of p53 in
tumorigenesis. Nat Rev Cancer 2004;4:793–805.
5. Chen LF, Greene WC. Shaping the nuclear action of NF-kappa B.
Nat Rev Mol Cell Bio 2004;5:392–401.
19. Su L, Cao J, Jia Y, et al. Development of synthetic aminopeptidase
N/CD13 inhibitors to overcome cancer metastasis and angiogenesis.
ACS Med Chem Lett 2012;3:959–64.
6. Kovacs JJ, Murphy PJM, Gaillard S, et al. HDAC6 regulates Hsp90
acetylation and chaperone-dependent activation of glucocorticoid
receptor. Mol Cell 2005;18:601–7.
20. Zhang YJ, Feng JH, Liu CX, 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;18:1761–72.
7. Marks PA, Rifkind RA, Richon VM, et al. Histone deacetylases and
cancer: causes and therapies. Nat Rev Cancer 2001;1:194–202.
8. Johnstone RW. Histone-deacetylase inhibitors: novel drugs for the
treatment of cancer. Nat Rev Drug Discov 2002;1:287–99.