Discovery of a series of hydroximic acid derivatives as potent histone deacetylase inhibitors

Article abstract of DOI:10.3109/14756366.2013.827678

To develop potent histone deacetylase inhibitors as antitumor agents, structural modification was performed. The synthesized molecules were tested by enzymatic inhibition assay and anti-proliferation assay. Several molecules show improved activities in the enzymatic inhibition assay. However, in the MTT assays, all these derived molecules have limited performance compared with SAHA. The IC50 values of molecule ((S)-N-(6-(hydroxyamino)-6-oxohexyl)-4-(3-(2-oxo-1-phenyl-2-((3-(trifluoromethyl)phenyl)amino)ethyl)ureido)benzamide, L8) which has the best enzymatic inhibition activity (with an IC50 value of 11.7nm and 967nm against Hela nucleus extract and HDAC8, respectively) were calculated compared with SAHA. Molecular docking was performed to predict the binding mode of molecule L8 in the active site of HDAC2 and HDAC8. Hydrophobic interaction, chelate binding, electrostatic attraction and H-bond interaction in combination make contribution to the ligand-receptor interactions.

Full text of DOI:10.3109/14756366.2013.827678

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ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, Early Online: 1–8
2013 Informa UK Ltd. DOI: 10.3109/14756366.2013.827678
!
ORIGINAL ARTICLE
Discovery of a series of hydroximic acid derivatives as potent histone
deacetylase inhibitors
Lei Zhang1, Xuejian Wang2, Xiaoguang Li3, Lihui Zhang4, and Wenfang Xu3
1Department of Pharmacy, School of Medicine, Qingdao University, Qingdao, Shandong, China, 2School of Pharmacy and Biology Science, Weifang
Medicinal University, Weifang, Shandong, China, 3Department of Medicinal Chemistry, School of Pharmacy, Shandong University, Jinan, Shandong,
4
China, and Department of Stomatology, Jiaotong Hospital, Qingdao, Shandong, China
Abstract
Keywords
To develop potent histone deacetylase inhibitors as antitumor agents, structural modification
was performed. The synthesized molecules were tested by enzymatic inhibition assay and anti-
proliferation assay. Several molecules show improved activities in the enzymatic inhibition
assay. However, in the MTT assays, all these derived molecules have limited performance
compared with SAHA. The IC50 values of molecule ((S)-N-(6-(hydroxyamino)-6-oxohexyl)-4-(3-
(2-oxo-1-phenyl-2-((3-(trifluoromethyl)phenyl)amino)ethyl)ureido)benzamide, L8) which has the
best enzymatic inhibition activity (with an IC50 value of 11.7 nm and 967 nm against Hela
nucleus extract and HDAC8, respectively) were calculated compared with SAHA. Molecular
docking was performed to predict the binding mode of molecule L8 in the active site of HDAC2
and HDAC8. Hydrophobic interaction, chelate binding, electrostatic attraction and H-bond
interaction in combination make contribution to the ligand–receptor interactions.
Anti-tumor, inhibitor, histone deacetylase,
structural modification
History
Received 3 April 2013
Revised 4 July 2013
Accepted 11 July 2013
Published online 23 September 2013
Introduction
antitumor efficacy in various stages of clinical trials. Suberoyl
anilide hydroxamic acid (SAHA)10 and FK22811 have been
approved by FDA for the treatment of advanced cutaneous T-cell
lymphoma (CTCL).
Histone deacetylases (HDACs) are a class of enzymes responsible
for removing the acetyl groups from the N-terminal lysine of
histones. There are 18 isforms of HDACs which are subdivided
into 4 classes that have been discovered until now1–3. Class I
HDACs (HDAC 1, 2, 3 and 8) are primarily found in nucleus,
HDAC 3 is found in both nucleus and cytoplasm. Class II HDACs
(IIa: HDAC4, 5, 7 and 9; IIb: HDAC6 and 10) are able to shuttle
in and out of the nucleus (with the possible exception of HDAC6
which is cytosolic). The zinc ion-dependent class I and II HDACs
are also called ‘‘classical’’ HDACs. Class III HDACs (known as
sirtuins, Sirt1-7) are a set of NAD-dependent enzymes. Class IV
HDAC (HDAC11) which also exists in both nucleus and
cytoplasm is considered as an atypical category of its own
based on sequence similarity to the others.
To develop potent HDACIs as antitumor agents, a series of
small molecules were designed and synthesized based on virtual
screening results12,13. A phenylglycine containing lead compound
(4 h) was discovered to have good performance in the enzyme
inhibition assays and tumor cell growth inhibition assays.
Structural modifications were performed in the present work in
discovery of more potent molecules. A long fatty linker was
introduced to the linker referring to the structure of SAHA, and
carbamido group was used to connect the linker to the external
motif (Figure 1). Hydrophobic interactions play significant role in
the HDACIs-HDACs bind, therefore substituted aromatic groups
were introduced to the external motifs of the designed molecules.
Acetylation and deacetylation of histones and non-histone
proteins by histone acetylases (HATs) and HDACs play signifi-
cant role in the regulation of gene expression and transcription4–6
The effects include signal transduction, protein phosphorylation, Chemistry
Materials and methods
.
cell cycle, proliferation, apoptosis, cardiac development and so
1H NMR spectra were recorded on a Bruker DRX spectrometer at
on. Overexpression and aberrant recruitment of HDACs (espe-
cially Class I and II HDACs) have a significant role in
tumorigenesis and development. Histone deacetylase inhibitors
(HDACIs) have been proved to have the function of blocking the
600 MHz, ꢀ 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. 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 l ¼ 589 nm.
action of HDACs and inducing hyperacetylation of histones7–9
.
A number of structurally diverse HDACIs have shown potent
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-((tert-butoxycarbonyl)amino)-2-phenylacetic acid (b) has
been synthesized and described in our previous work13.

2
L. Zhang et al.
J Enzyme Inhib Med Chem, Early Online: 1–8
(S)-methyl 6-(4-(3-(2-(benzylamino)-2-oxo-1-
phenylethyl)ureido)benzamido)hexanoate (k1)
To a solution of triphosgene (1.5 g, 5.0 mmol) in dioxane (40 mL),
intermediate j (3.00 g, 10 mmol) was added. The reaction solution
was stirred at 110 ꢁC for 8 h. Then, the solvent was evaporated
sufficiently without further purification.
The residue was dissolved in DCM, compound d1 (1.4 g,
5.0 mmol) and Et3N (0.61 g, 6.0 mmol) were added. The reaction
solution was stirred at room temperature for 5 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 k1 (1.11 g, 42% yield) was derived by crystallization in
EtOAc as white powder. ESI-MS: m/z: 531.3 [M þ H]þ.
The other compounds (k2–k19) were prepared using the same
procedure as described above.
(S)-4-(3-(2-(benzylamino)-2-oxo-1-phenylethyl)ureido)-N-
(6-(hydroxyamino)-6-oxohexyl)benzamide (L1)
Figure 1. Design of novel HDAC inhibitors by structural modification of
lead compound 4 h.
Compound k1 (0.53 g, 1.0 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
L1 (0.23 g, 43% yield) was derived by crystallization in EtOAc as
(S)-tert-butyl (2-(benzylamino)-2-oxo-1-phenylethyl)carbamate
(c1)
To a solution of compound b (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, benzylamine (0.54 g, 5 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 c1 (1.25 g, 71% yield) was
derived by crystallization in EtOAc as white powder. ESI-MS:
m/z: 341.4 [M þ H]þ.
20
white powder. M.p.: 197–198 ꢁC. ½ꢁꢂD ꢃ6.93 (c 1.0, MeOH). 1H
NMR (600 MHz, (CD3)2SO) ꢀ 10.35 (s, 1H), 9.21 (s, 1H), 8.95
(d, J ¼ 5.4 Hz, 1H), 8.65 (d, J ¼ 1.2 Hz, 1H), 8.25 (t, J ¼ 5.4 Hz,
1H), 7.73 (d, J ¼ 9 Hz, 2H), 7.46 (d, J ¼ 7.2 Hz, 2H), 7.41
(d, J ¼ 9 Hz, 2H), 7.38–7.36 (m, 2H), 7.31 (d, J ¼ 7.2 Hz, 1H),
7.28 (s, 1H), 7.26 (d, J ¼ 7.2 Hz, 1H), 7.24–7.21 (m, 2H), 7.15
(d, J ¼ 7.8 Hz, 2H), 5.41 (d, J ¼ 7.8 Hz, 1H), 4.29 (d, J ¼ 6 Hz,
2H), 3.20 (d, J ¼ 6 Hz, 2H), 1.65 (t, J ¼ 7.2 Hz, 2H), 1.52
(t, J ¼ 7.8 Hz, 2H), 1.49–1.47 (m, 2H), 1.31–1.24 (m, 2H). HRMS
(AP-ESI) m/z calculated for C29H34N5O5 [M þ H]þ 532.2560
was found to be 532.2556. Retention time: 3.9 min.
Other compounds (c2–c19) were prepared using the same
procedure as described above.
The other compounds (L2–L19) were prepared using the same
procedure as described above.
(S)-2-amino-N-benzyl-2-phenylacetamide hydrochloride (d1)
To a solution of compound c1 (0.68 g, 2 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 d1 (0.34 g, 62%
yield). Title compound was obtained as an amorphous white solid.
ESI-MS: m/z: 241.4 [M þ H]þ.
(S)-4-(3-(2-((2-fluorophenyl)amino)-2-oxo-1-phenylethyl)ureido)-
N-(6-(hydroxyamino)-6-oxohexyl)benzamide (L2)
Title compound was obtained as an amorphous white solid
20
(0.23 g, 44% yield). M.p.: 200–202 ꢁC. ½ꢁꢂD ꢃ6.52 (c 1.0,
1
MeOH). H NMR (600 MHz, (CD3)2SO) ꢀ 10.35 (s, 1H), 10.28
(s, 1H), 9.26 (s, 1H), 8.66 (s, 1H), 8.27 (m, 1H), 7.80–7.79
(m, 1H), 7.74 (d, J ¼ 9.0 Hz, 2H), 7.54 (d, J ¼ 7.2 Hz, 2H), 7.43
(d, J ¼ 8.4 Hz, 1H), 7.41–7.39 (m, 1H), 7.33 (d, J ¼ 7.8 Hz, 1H),
7.31 (d, J ¼ 8.4 Hz, 1H), 7.27–7.24 (m, 2H), 7.24–7.18 (m, 1H),
7.15 (d, J ¼ 7.8 Hz, 2H), 5.74 (d, J ¼ 7.8 Hz, 1H), 3.20
(d, J ¼ 6 Hz, 2H), 1.96–1.94 (m, 2H), 1.53–1.51 (m, 2H), 1.50–
1.47 (m, 2H), 1.29–1.24 (m, 2H). HRMS (AP-ESI) m/z calculated
for C28H31FN5O5 [M þ H]þ 536.2309 was found to be 536.2300.
Retention time: 3.9 min.
The other compounds (d2–d19) were prepared using the same
procedure as described above.
Compound f was prepared using the same procedure as
molecule b.
Methyl 6-aminohexanoate hydrochloride (h)
6-Aminohexanoic acid (g) (13.1 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 h (15.7 g, 87%
yield) being recrystallized as white powder by ester. ESI-MS: m/z:
146.1 [M þ H]þ.
(S)-4-(3-(2-((4-fluorophenyl)amino)-2-oxo-1-phenylethyl)ureido)-
N-(6-(hydroxyamino)-6-oxohexyl)benzamide (L3)
Title compound was obtained as an amorphous white solid
20
(0.26 g, 49% yield). M.p.: 202–204 ꢁC. ½ꢁꢂD ꢃ6.71 (c 1.0,
Compound i was prepared using the same procedure as
molecule c1.
1
MeOH). H NMR (600 MHz, (CD3)2SO) ꢀ 10.63 (s, 1H), 10.36
Compound j was prepared using the same procedure as
molecule d1.
(s, 1H), 9.72 (s, 1H), 9.30 (s, 1H), 9.03 (s, 1H), 8.66
(d, J ¼ 1.8 Hz, 2H), 8.27–8.25 (m, 1H), 7.74 (d, J ¼ 8.4 Hz, 2H),

DOI: 10.3109/14756366.2013.827678
Discovery of a series of hydroximic acid derivatives as potent histone deacetylase inhibitors
3
7.65–7.63 (m, 2H), 7.53 (d, J ¼ 7.8 Hz, 1H), 7.43 (d, J ¼ 9.0 Hz, (m, 2H). HRMS (AP-ESI) m/z calcd for C32H34N5O5 [M þ H]þ
1H), 7.39 (t, J ¼ 7.8 Hz, 1H), 7.35 (d, J ¼ 7.8 Hz, 1H), 7.32 568.2550 was found to be 568.2553. Retention time: 3.9 min.
(d, J ¼ 7.2 Hz, 1H), 7.15 (d, J ¼ 6.6 Hz, 2H), 5.56 (d, J ¼ 7.8 Hz,
1H), 3.20 (t, J ¼ 6.6 Hz, 2H), 1.96–1.92 (m, 2H), 1.52
(S)-N-(6-(hydroxyamino)-6-oxohexyl)-4-(3-(2-oxo-1-phenyl-2-
(t, J ¼ 7.8 Hz, 2H), 1.49–1.47 (m, 2H), 1.29–1.24 (m, 2H).
((3-(trifluoromethyl)phenyl)amino)ethyl)ureido)benzamide (L8)
HRMS (AP-ESI) m/z calculated for C28H31FN5O5 [M þ H]þ
536.2309 was found to be 536.2303. Retention time: 3.9 min.
Title compound was obtained as an amorphous white solid
20
(0.24 g, 40% yield). M.p.: 208–209 ꢁC. ½ꢁꢂD ꢃ8.21 (c 1.0,
1
MeOH). H NMR (600 MHz, (CD3)2SO) ꢀ 10.78 (s, 1H), 10.32
(S)-4-(3-(2-((4-(tert-butyl)phenyl)amino)-2-oxo-1-phenylethyl)
ureido)-N-(6-(hydroxyamino)-6-oxohexyl)benzamide (L4)
(s, 1H), 9.72 (s, 1H), 9.04 (s, 1H), 8.64 (s, 1H), 8.24 (t, J ¼ 5.4 Hz,
1H), 8.09 (s, 1H), 7.81 (d, J ¼ 8.4 Hz, 2H), 7.74 (d, J ¼ 8.4 Hz,
1H), 7.57 (t, J ¼ 7.8 Hz, 1H), 7.52 (d, J ¼ 7.8 Hz, 2), 7.44–7.41
(m, 2H), 7.34 (d, J ¼ 7.2 Hz, 1H), 7.24 (d, J ¼ 7.2 Hz, 1H), 5.54
(d, J ¼ 7.2 Hz, 1H), 3.20 (d, J ¼ 6 Hz, 2H), 1.99–1.92 (m, 2H),
1.51 (t, J ¼ 7.8 Hz, 2H), 1.49–1.47 (m, 2H), 1.30–1.25 (m, 2H).
HRMS (AP-ESI) m/z calculated for C29H31F3N5O5 [M þ H]þ
586.2277 was found to be 586.2272. Retention time: 3.9 min.
Title compound was obtained as an amorphous white solid
20
(0.30 g, 53% yield). M.p.: 198–200 ꢁC. ½ꢁꢂD ꢃ7.88 (c 1.0,
1
MeOH). H NMR (600 MHz, (CD3)2SO) ꢀ 10.41 (s, 1H), 10.33
(s, 1H), 9.12 (s, 1H), 8.65 (d, J ¼ 1.2 Hz, 1H), 8.25–8.23 (m, 1H),
7.73 (d, J ¼ 9.0 Hz, 2H), 7.51 (d, J ¼ 8.4 Hz, 4H), 7.42
(d, J ¼ 9.0 Hz, 2H), 7.41–7.38 (m, 2H), 7.33 (s, 1H), 7.31
(d, J ¼ 7.8 Hz, 2H), 7.26 (d, J ¼ 7.8 Hz, 1H), 5.56 (d, J ¼ 7.8 Hz,
1H), 3.20 (d, J ¼ 6.0 Hz, 2H), 1.96–1.93 (m, 2H), 1.51
(t, J ¼ 7.8 Hz, 2H), 1.50–1.48 (m, 2H), 1.28–1.25 (m, 11H).
HRMS (AP-ESI) m/z calculated for C32H40N5O5 [M þ H]þ
574.3029 was found to be 574.3024. Retention time: 3.9 min.
(S)-N-(6-(hydroxyamino)-6-oxohexyl)-4-(3-(2-oxo-1-phenyl-2-
(p-tolylamino)ethyl)ureido)benzamide (L9)
Title compound was obtained as an amorphous white solid
20
(0.24 g, 45% yield). M.p.: 203–205 ꢁC. ½ꢁꢂD ꢃ7.35 (c 1.0,
1
MeOH). H NMR (600 MHz, (CD3)2SO) ꢀ 10.45 (s, 1H), 10.32
(S)-4-(3-(2-(furan-2-ylamino)-2-oxo-1-phenylethyl)ureido)-N-
(6-(hydroxyamino)-6-oxohexyl)benzamide (L5)
(s, 1H), 9.12 (s, 1H), 8.25–8.23 (m, 1H), 7.94 (d, J ¼ 7.8 Hz, 1H),
7.85 (d, J ¼ 8.4 Hz, 2H), 7.79 (d, J ¼ 8.4 Hz, 2H), 7.63
(d, J ¼ 7.2 Hz, 2H), 7.53 (d, J ¼ 7.2 Hz, 2H), 7.51–7.47 (m, 2H),
7.45 (d, J ¼ 8.4 Hz, 2H), 7.38–7.36 (m, 1H), 7.30 (d, J ¼ 7.8 Hz,
1H), 5.82 (d, J ¼ 7.8 Hz, 1H), 3.21 (d, J ¼ 6 Hz, 2H), 2.34
(m, 3H), 1.53 (t, J ¼ 7.8 Hz, 2H), 1.50–1.48 (m, 2H), 1.32–1.26
(m, 2H). HRMS (AP-ESI) m/z calculated for C29H34N5O5
[M þ H]þ 532.2560 was found to be 532.2554. Retention time:
3.9 min.
Title compound was obtained as an amorphous white solid
20
(0.24 g, 46% yield). M.p.: 173–175 ꢁC. ½ꢁꢂD ꢃ6.63 (c 1.0,
1
MeOH). H NMR (600 MHz, (CD3)2SO) ꢀ 10.32 (s, 1H), 9.05
(s, 1H), 8.90 (t, J ¼ 5.4 Hz, 1H), 8.65 (s, 1H), 8.24–8.22 (m, 1H),
7.73 (d, J ¼ 8.4 Hz, 2H), 7.56 (s, 1H), 7.42 (d, J ¼ 7.8 Hz, 2H),
7.40 (d, J ¼ 8.4 Hz, 2H), 7.37–7.35 (m, 2H), 7.31–7.28
(t, J ¼ 7.2 Hz, 1H), 7.16 (d, J ¼ 7.8 Hz, 1H), 6.37–6.36 (m, 1H),
6.13 (d, J ¼ 3 Hz, 1H), 5.39 (d, J ¼ 7.8 Hz, 1H), 3.20 (d, J ¼ 6 Hz,
2H), 1.96–1.93 (m, 2H), 1.51 (t, J ¼ 9 Hz, 2H), 1.29–1.25 (m, 2H),
1.19–1.17 (m, 2H). HRMS (AP-ESI) m/z calculated for
C27H32N5O6 [M þ H]þ 522.2362 was found to be 522.2344.
Retention time: 3.9 min.
(S)-N-(6-(hydroxyamino)-6-oxohexyl)-4-(3-(2-((2-methoxyphenyl)
amino)-2-oxo-1-phenylethyl)ureido)benzamide (L10)
Title compound was obtained as an amorphous white solid
20
(0.24 g, 43% yield). M.p.: 186–188 ꢁC. ½ꢁꢂD ꢃ6.73 (c 1.0,
1
MeOH). H NMR (600 MHz, (CD3)2SO) ꢀ 10.38 (s, 1H), 9.64
(S)-N-(6 -(hydroxyamino)-6-oxohexyl)-4-(3-(2-oxo-1-phenyl-2-
(o-tolylamino)ethyl)ureido)benzamide (L6)
(s, 1H), 9.46 (s, 1H), 8.66 (d, J ¼ 1.2 Hz, 1H), 8.27 (t, J ¼ 5.4 Hz,
1H), 7.85 (d, J ¼ 7.2 Hz, 1H), 7.75 (d, J ¼ 9.0 Hz, 1H), 7.55
(d, J ¼ 7.2 Hz, 2H), 7.45 (d, J ¼ 8.4 Hz, 2H), 7.42 (d, J ¼ 9.0 Hz,
2H), 7.39 (t, J ¼ 7.8 Hz, 2H), 7.31 (t, J ¼ 7.2 Hz, 1H), 7.09
(t, J ¼ 7.2 Hz, 1H), 7.03 (d, J ¼ 7.2 Hz, 1H), 6.90 (d, J ¼ 8.4 Hz,
1H), 5.79 (d, J ¼ 7.8 Hz, 1H), 3.80 (s, 1H), 3.21 (d, J ¼ 6 Hz, 2H),
1.97–1.94 (m, 2H), 1.51 (t, J ¼ 7.2 Hz, 2H), 1.49–1.47 (m, 2H),
1.29-1.24 (m, 2H). HRMS (AP-ESI) m/z calculated for
C29H34N5O6 [M þ H]þ 548.2509 was found to be 548.2502.
Retention time: 3.9 min.
Title compound was obtained as an amorphous white solid
20
(0.25 g, 47% yield). M.p.: 198–200 ꢁC. ½ꢁꢂD ꢃ6.11 (c 1.0,
1
MeOH). H NMR (600 MHz, (CD3)2SO) ꢀ 10.46 (s, 1H), 9.97
(s, 1H), 9.70 (s, 1H), 8.69 (s, 1H), 8.33 (d, J ¼ 4.8 Hz, 1H), 7.76
(d, J ¼ 8.4 Hz, 2H), 7.59 (d, J ¼ 7.2 Hz, 2H), 7.51 (d, J ¼ 7.8 Hz,
1H), 7.46 (d, J ¼ 8.4 Hz, 2H), 7.39 (t, J ¼ 7.8 Hz, 2H), 7.33–7.29
(m, 2H), 7.18–7.13 (m, 2H), 7.08 (t, J ¼ 7.2 Hz, 1H), 5.68
(d, J ¼ 7.8 Hz, 1H), 3.20 (d, J ¼ 6 Hz, 2H), 2.04 (s, 3H), 1.96
(t, J ¼ 7.2 Hz, 2H), 1.53–1.48 (m, 4H), 1.31–1.24 (m, 2H). HRMS
(AP-ESI) m/z calculated for C29H34N5O5 [M þ H]þ 532.2560 was
found to be 532.2553. Retention time: 3.9 min.
(S)-N-(6-(hydroxyamino)-6-oxohexyl)-4-(3-(2-((2-hydroxyphenyl)
amino)-2-oxo-1-phenylethyl)ureido)benzamide (L11)
(S)-N-(6-(hydroxyamino)-6-oxohexyl)-4-(3-(2-(naphthalen-1-
ylamino)-2-oxo-1-phenylethyl)ureido)benzamide (L7)
Title compound was obtained as an amorphous white solid
20
(0.25 g, 48% yield). M.p.: 175–177 ꢁC. ½ꢁꢂD ꢃ6.84 (c 1.0,
1
Title compound was obtained as an amorphous white solid MeOH). H NMR (600 MHz, (CD3)2SO) ꢀ 10.32 (s, 1H), 9.80
20
(0.24 g, 42% yield). M.p.: 204–206 ꢁC. ½ꢁꢂD ꢃ6.38 (c 1.0, (s, 1H), 9.65 (s, 1H), 9.06 (s, 1H), 8.64 (s, 1H), 8.23 (t, J ¼ 5.4 Hz,
1
MeOH). H NMR (400 MHz, (CD3)2SO) ꢀ 10.32 (s, 1H), 9.10 1H), 7.77 (d, J ¼ 2.4 Hz, 1H), 7.74 (d, J ¼ 8.4 Hz, 2H), 7.53
(s, 1H), 8.65 (s, 1H), 8.26–8.23 (m, 1H), 7.93 (d, J ¼ 7.6 Hz, 1H), (d, J ¼ 8.4 Hz, 2H), 7.3 (d, J ¼ 9.0 Hz, 2H), 7.39 (t, J ¼ 7.8 Hz,
7.84 (d, J ¼ 7.6 Hz, 1H), 7.79 (d, J ¼ 8 Hz, 1H), 7.76 (s, 1H), 7.73 2H), 7.32 (t, J ¼ 7.2 Hz, 1H), 7.21 (d, J ¼ 7.8 Hz, 1H), 6.95–6.93
(s, 1H), 7.62 (d, J ¼ 7.2 Hz, 1H), 7.60 (d, J ¼ 7.2 Hz, 2H), 7.53 (m, 1H), 6.86 (d, J ¼ 7.2 Hz, 1H), 6.75 (d, J ¼ 7.2 Hz, 1H), 5.77
(d, J ¼ 6.8 Hz, 2H), 7.51–7.50 (m, 2H), 7.48 (d, J ¼ 8.0 Hz, 2H), (d, J ¼ 7.8 Hz, 1H), 3.21 (d, J ¼ 6 Hz, 2H), 1.96–1.94 (m, 2H),
7.44 (d, J ¼ 8 Hz, 1H), 7.38–7.34 (m, 1H), 7.29 (d, J ¼ 8 Hz, 1H), 1.53 (t, J ¼ 7.8 Hz, 2H), 1.50–1.47 (m, 2H), 1.30–1.24 (m, 2H).
5.81 (d, J ¼ 7.6 Hz, 1H), 3.21 (d, J ¼ 7.2 Hz, 2H), 1.96–1.92 HRMS (AP-ESI) m/z calculated for C28H32N5O6 [M þ H]þ
(m, 2H), 1.53 (t, J ¼ 7.2 Hz, 2H), 1.49–1.46 (m, 2H), 1.30–1.24 534.2352 was found to be 534.2345. Retention time: 3.9 min.

4
L. Zhang et al.
J Enzyme Inhib Med Chem, Early Online: 1–8
1
(S)-4-(3-(2-((3-chlorophenyl)amino)-2-oxo-1-phenylethyl)
ureido)-N-(6-(hydroxyamino)-6-oxohexyl)benzamide (L12)
MeOH). H NMR (600 MHz, (CD3)2SO) ꢀ 10.67 (s, 1H), 10.33
(s, 1H), 9.13 (s, 1H), 8.64 (s, 1H), 8.25–8.24 (m, 1H), 7.95
(d, J ¼ 1.8 Hz, 1H), 7.74 (d, J ¼ 9.0 Hz, 2H), 7.54 (t, J ¼ 1.8 Hz,
2H), 7.52 (d, J ¼ 7.2 Hz, 1H), 7.43–7.40 (m, 4H), 7.34
(d, J ¼ 7.8 Hz, 1H), 7.29 (d, J ¼ 7.8 Hz, 1H), 7.27 (s, 1H), 7.26
(d, J ¼ 8.4 Hz, 1H), 5.54 (d, J ¼ 7.8 Hz, 1H), 3.20 (t, J ¼ 6.6 Hz,
2H), 1.95 (t, J ¼ 7.2 Hz, 2H), 1.52–1.48 (m, 4H), 1.28–1.25
(m, 2H). HRMS (AP-ESI) m/z calculated for C28H31BrN5O5
[M þ H]þ 596.1508 was found to be 596.1501. Retention time:
3.9 min.
Title compound was obtained as an amorphous white solid
20
(0.23 g, 41% yield). M.p.: 192–193 ꢁC. ½ꢁꢂD ꢃ6.55 (c 1.0,
1
MeOH). H NMR (600 MHz, (CD3)2SO) ꢀ 10.71 (s, 1H), 10.35
(s, 1H), 9.17 (s, 1H), 8.66 (s, 1H), 8.26–8.25 (m, 1H), 7.82
(t, J ¼ 1.8 Hz, 1H), 7.78 (s, 1H), 7.74 (s, 1H), 7.66 (d, J ¼ 7.8 Hz,
1H), 7.53 (d, J ¼ 7.2 Hz, 2H), 7.49 (d, J ¼ 7.2 Hz, 1H), 7.44
(d, J ¼ 9.0 Hz, 2H), 7.41 (t, J ¼ 7.8 Hz, 2H), 7.35 (t, J ¼ 7.8 Hz,
1H), 7.31 (d, J ¼ 7.2 Hz, 2H), 7.12 (d, J ¼ 7.8 Hz, 1H), 5.56
(d, J ¼ 7.8 Hz, 1H), 3.21 (d, J ¼ 6 Hz, 2H), 1.97–1.94 (m, 2H),
1.52 (t, J ¼ 7.8 Hz, 2H), 1.49–1.48 (m, 2H), 1.30–1.35 (m, 2H).
HRMS (AP-ESI) m/z calculated for C28H31ClN5O5 [M þ H]þ
552.2013 was found to be 552.2010. Retention time: 3.9 min.
(S)-4-(3-(2-((2,6-diisopropylphenyl)amino)-2-oxo-1-phenylethyl)
ureido)-N-(6-(hydroxyamino)-6-oxohexyl)benzamide (L17)
Title compound was obtained as an amorphous white solid
20
(0.26 g, 43% yield). M.p.: 252–254 ꢁC. ½ꢁꢂD ꢃ7.08 (c 1.0,
1
MeOH). H NMR (600 MHz, (CD3)2SO) ꢀ 10.44 (s, 1H), 9.82
(S)-4-(3-(2-((3,5-dimethylphenyl)amino)-2-oxo-1-phenylethyl)
ureido)-N-(6-(hydroxyamino)-6-oxohexyl)benzamide (L13)
(s, 1H), 9.56 (s, 1H), 8.67 (s, 1H), 8.31 (t, J ¼ 5.4 Hz, 1H), 7.75
(d, J ¼ 8.4 Hz, 2H), 7.61 (d, J ¼ 7.2 Hz, 2H), 7.47 (d, J ¼ 7.8 Hz,
1H), 7.45 (d, J ¼ 9.0 Hz, 2H), 7.41–7.38 (m, 2H), 7.33
(t, J ¼ 7.2 Hz, 1H), 7.21 (t, J ¼ 7.8 Hz, 1H), 7.20 (d, J ¼ 6.0 Hz,
2H), 5.63 (d, J ¼ 7.8 Hz, 1H), 3.31 (d, J ¼ 6.0 Hz, 2H), 1.97–1.95
(m, 2H), 1.53–1.51 (m, 2H), 1.50–1.48 (m, 2H), 1.29–1.26
(m, 2H), 0.25–1.24 (m, 2H), 1.17 (d, J ¼ 13.2 Hz, 6H). HRMS
(AP-ESI) m/z calculated for C34H44N5O5 [M þ H]þ 602.3342 was
found to be 602.3339. Retention time: 3.9 min.
Title compound was obtained as an amorphous white solid
20
(0.25 g, 46% yield). M.p.: 207–209 ꢁC. ½ꢁꢂD ꢃ7.88 (c 1.0,
1
MeOH). H NMR (600 MHz, (CD3)2SO) ꢀ 10.36 (s, 1H), 10.33
(s, 1H), 9.29 (s, 1H), 8.66 (s, 1H), 8.27–8.25 (m, 1H), 7.74
(d, J ¼ 8.4 Hz, 2H), 7.53 (d, J ¼ 7.2 Hz, 2H), 7.44 (d, J ¼ 8.4 Hz,
2H), 7.39 (t, J ¼ 7.8 Hz, 2H), 7.32 (d, J ¼ 7.2 Hz, 1H), 7.30
(d, J ¼ 7.2 Hz, 1H), 7.23 (s, 2H), 6.70 (s, 1H), 5.55 (d, J ¼ 7.8 Hz,
1H), 3.21 (d, J ¼ 6.6 Hz, 2H), 2.22 (s, 6H), 1.96–1.94 (m, 2H),
1.52 (t, J ¼ 7.8 Hz, 2H), 1.50–1.47 (m, 2H), 1.29–1.24 (m, 2H).
HRMS (AP-ESI) m/z calculated for C30H36N5O5 [M þ H]þ
546.2716 was found to be 546.2710. Retention time: 3.9 min.
(S)-N-(6-(hydroxyamino)-6-oxohexyl)-4-(3-(2-oxo-1-phenyl-2-(m-
tolylamino)ethyl)ureido)benzamide (L18)
Title compound was obtained as an amorphous white solid
20
(S)-4-(3-(2-((3-fluoro-4-methoxyphenyl)amino)-2-oxo-1-pheny-
lethyl)ureido)-N-(6-(hydroxyamino)-6-oxohexyl)benzamide (L14)
(0.22 g, 41% yield). M.p.: 195–197 ꢁC. ½ꢁꢂD ꢃ5.97 (c 1.0,
1
MeOH). H NMR (600 MHz, (CD3)2SO) ꢀ 10.42 (s, 1H), 10.35
(s, 1H), 9.26 (s, 1H), 8.65 (s, 1H), 8.26–8.24 (m, 1H), 7.74
(d, J ¼ 9 Hz, 2H), 7.53 (d, J ¼ 7.8 Hz, 2H), 7.44 (d, J ¼ 7.2 Hz,
1H), 7.42 (d, J ¼ 9.0 Hz, 1H), 7.41–7.38 (m, 2H), 7.32
(d, J ¼ 7.2 Hz, 1H), 7.31 (d, J ¼ 6 Hz, 1H), 7.30 (s, 1H), 7.18
(t, J ¼ 7.8 Hz, 1H), 6.88 (d, J ¼ 7.2 Hz, 1H), 5.57 (d, J ¼ 7.8 Hz,
1H), 3.20 (t, J ¼ 6 Hz, 2H), 2.26 (s, 3H), 1.95 (t, J ¼ 6.6 Hz, 2H),
1.54–1.51 (m, 2H), 1.50–1.47 (m, 2H), 1.29–1.25 (m, 2H). HRMS
(AP-ESI) m/z calculated for C29H34N5O5 [M þ H]þ 532.2560 was
found to be 532.2554. Retention time: 3.9 min.
Title compound was obtained as an amorphous white solid
20
(0.24 g, 43% yield). M.p.: 198–200 ꢁC. ½ꢁꢂD ꢃ8.12 (c 1.0,
1
MeOH). H NMR (600 MHz, (CD3)2SO) ꢀ 10.55 (s, 1H), 10.33
(s, 1H), 9.18 (s, 1H), 8.65 (s, 1H), 8.25 (t, J ¼ 5.4 Hz, 1H), 7.74
(d, J ¼ 8.4 Hz, 1H), 7.60 (d, J ¼ 2.4 Hz, 2H), 7.58 (d, J ¼ 2.4 Hz,
1H), 7.51 (d, J ¼ 7.2 Hz, 2H), 7.43 (d, J ¼ 9.0 Hz, 2H), 7.40
(t, J ¼ 7.2 Hz, 2H), 7.33 (d, J ¼ 7.2 Hz, 1H), 7.31 (s, 1H), 7.29
(t, J ¼ 3.6 Hz, 1H), 7.11 (d, J ¼ 9.0 Hz, 1H), 5.52 (d, J ¼ 7.8 Hz,
1H), 3.79 (s, 1H), 3.20 (t, J ¼ 6.6 Hz, 2H), 1.95 (t, J ¼ 7.2 Hz, 2H),
1.54–1.51 (m, 2H), 1.50–1.47 (m, 2H), 1.29–1.24 (m, 2H). HRMS
(AP-ESI) m/z calculated for C29H33FN5O6 [M þ H]þ 566.2415
was found to be 566.2411. Retention time: 3.9 min.
(S)-4-(3-(2-((2,4-dichlorophenyl)amino)-2-oxo-1-phenylethyl)
ureido)-N-(6-(hydroxyamino)-6-oxohexyl)benzamide (L19)
Title compound was obtained as an amorphous white solid
20
(0.29 g, 49% yield). M.p.: 205–206 ꢁC. ½ꢁꢂD ꢃ6.12 (c 1.0,
(S)-4-(3-(2-((4-fluorobenzyl)amino)-2-oxo-1-phenylethyl)ureido)-
N-(6-(hydroxyamino)-6-oxohexyl)benzamide (L15)
1
MeOH). H NMR (600 MHz, (CD3)2SO) ꢀ 10.33 (s, 1H), 10.15
(s, 1H), 9.05 (s, 1H), 8.65 (s, 1H), 8.25–8.23 (m, 1H), 7.78
(d, J ¼ 8.4 Hz, 1H), 7.73 (d, J ¼ 8.4 Hz, 1H), 7.65 (d, J ¼ 8.4 Hz,
2H), 7.54 (d, J ¼ 7.2 Hz, 2H), 7.42 (d, J ¼ 6.6 Hz, 1H), 7.37
(t, J ¼ 7.8 Hz, 2H), 7.34 (t, J ¼ 7.2 Hz, 1H), 7.30 (d, J ¼ 7.8 Hz,
1H), 7.21 (d, J ¼ 8.4 Hz, 1H), 5.74 (d, J ¼ 7.8 Hz, 1H), 3.21
(d, J ¼ 6.0 Hz, 2H), 1.96–1.94 (m, 2H), 1.51 (t, J ¼ 7.8 Hz, 2H),
1.49–1.47 (m, 2H), 1.29–1.24 (m, 2H). HRMS (AP-ESI) m/z
calculated for C28H30Cl2N5O5 [M þ H]þ 586.1624 was found to
be 586.1618. Retention time: 3.9 min.
Title compound was obtained as an amorphous white solid
20
(0.27 g, 49% yield). M.p.: 185–187 ꢁC. ½ꢁꢂD ꢃ7.67 (c 1.0,
1
MeOH). H NMR (600 MHz, (CD3)2SO) ꢀ 10.41 (s, 1H), 9.40
(s, 1H), 8.99 (t, J ¼ 6 Hz, 1H), 8.68 (s, 1H), 8.29–8.27 (m, 1H),
7.74 (d, J ¼ 8.4 Hz, 2H), 7.46 (d, J ¼ 7.8 Hz, 2H), 7.42
(d, J ¼ 8.4 Hz, 2H), 7.35 (d, J ¼ 7.8 Hz, 2H), 7.29 (t, J ¼ 7.2 Hz,
2H), 7.20 (d, J ¼ 9.0 Hz, 1H), 7.19 (d, J ¼ 6 Hz, 1H), 7.07
(d, J ¼ 8.4 Hz, 2H), 5.39 (d, J ¼ 7.8 Hz, 1H), 4.26 (t, J ¼ 6.0 Hz,
2H), 3.20 (t, J ¼ 6.6 Hz, 2H), 1.96–1.94 (m, 2H), 1.53–1.51
(m, 2H), 1.49–1.48 (m, 2H), 1.29–1.25 (m, 2H). HRMS (AP-ESI)
m/z calculated for C29H33FN5O5 [M þ H]þ 550.2465 was found to
be 550.2460. Retention time: 3.9 min.
Enzyme inhibition assay
The method of enzymatic inhibition assay has been described in
our previous work14. The nucleus extract was extracted from Hela
cells and the HDAC8 enzyme was expressed in Escherichia coli.
Boc-Lys (acetyl)-AMC was used as the substrate of HDAC.
(S)-4-(3-(2-((3-bromophenyl)amino)-2-oxo-1-phenylethyl)
ureido)-N-(6-(hydroxyamino)-6-oxohexyl)benzamide (L16)
Title compound was obtained as an amorphous white solid SAHA which is the HDAC inhibitor on market was used
20
(0.26 g, 44% yield). M.p.: 196–198 ꢁC. ½ꢁꢂD ꢃ6.86 (c 1.0, as a positive control. The compounds were diluted to six

DOI: 10.3109/14756366.2013.827678
Discovery of a series of hydroximic acid derivatives as potent histone deacetylase inhibitors
5
concentrations (25, 5, 1, 0.2, 0.04 and 0.008 mM/L) to investigate Chemistry
their ability of inhibiting HDAC activity.
Based on the lead structure 4 h, the target compounds designed by
structural modification were synthesized according to the pro-
cedures described in Scheme 1. The starting material (S)-2-
amino-2-phenylacetic acid was firstly protected by Boc group;
subsequent condensation with various substituted aromatic
amides and N-deprotection afforded d1–d19. Intermediate i was
derived by coupling of the Boc protected 4-aminobenzoic acid
and methyl ester protected 6-aminohexanoic acid. N-deprotection
of intermediate i and condensation with d1–d19 afforded k1–k19.
Target compounds L1–L19 were derived by treating the corres-
ponding intermediates (k1–k19) with NH2OK in methanol.
Tumor cell inhibition assay
HCT116, K562, MDA-MB-231 and ES-2 cells were used in the
cell proliferation assays. Five thousand cells were seeded into
each well of 96-well plates which were incubated at 37 ꢁC, 5%
CO2 for overnight. Then cells were treated with 5 mM of all these
molecules. After 48 hours of incubation, 0.5% MTT (Amresco,
Solon, OH) solution was added to each well. Then a further four
hours of incubation was performed. After that the culture medium
was removed, and 200 mL DMSO was added to dissolve the
formazan. After mixing for five minutes, optical density values
were detected at 570 nm on a microplate reader (Thermo Fisher
Scientific, Waltham, MA).
Results and discussion
The synthesized molecules were tested by enzymatic inhibition
assay against Hela cell nucleus extract using SAHA as a positive
control (Table 1). There are seven derived molecules that show
lower IC50 values than SAHA in the enzymatic inhibition assay.
Molecule L8 shows best enzymatic inhibition activity with IC50
values of 11.7 nM. Molecules with polar groups substituted in the
phenyl ring of the R group have small IC50 values such as L8
(11.7 nM), L11 (45.8 nM), L14 (43.1 nM), L15 (19.2 nM), L16
(69.3 nM). Fatty groups substituted in the phenyl ring of the R
group reduce the activity significantly such as L4 (IC50 values of
1910 nM) and L17 (IC50 values of 1292 nM). Chlorine and
fluorine substitutions (L2, L3, L12 and L19) make negative
contribution to the inhibitory activity, and bromine substitution
(L16) makes positive contribution to the activity. However, the
4-fluorobenzyl substitution (L15) improves the activity obviously.
The ortho position (L6), para position (L9) methyl substitution
Molecular docking
The molecular docking process was performed using Glide
(Schrodinger Inc, supported by Shanghai Institute of Materia
Medica Chinese Academy of Sciences). Crystal structures of
HDAC2 (PDB Entry: 3MAX) and HDAC8 (PDB Entry: 1T69)
were selected as receptors 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. The
active site was defined as a cubic 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.
Scheme 1. Synthesis of target compounds L1–L19. Reagents and conditions: (1) (Boc)2O, Et3N, MeOH/H2O; (2) TBTU, Et3N, THF; (3) EtOAc, HCl;
(4) (Boc)2O, Et3N, MeOH/H2O; EtOAc, HCl; (5) CH3COCl, CH3OH; (6) TBTU, Et3N, THF; (7) EtOAc, HCl; (8) triphosgene, dioxane, d1–d19, Et3N,
DCM; (9) NH2OK, MeOH.

6
L. Zhang et al.
J Enzyme Inhib Med Chem, Early Online: 1–8
Table 1. The enzyme inhibitory activities of the derived molecules
(IC50, nM)*.
Table 1. Continued
O
H
H
N
O
R
N
H
H
N
N
O
R
N
H
H
N
O
O
N
H
H
NHOH
O
N
NHOH
O
O
Compound
R
Hela nucleus extract
332
HDAC8
1655
Compound
R
Hela nucleus extract
853
HDAC8
L13
L1
1506
L2
L3
L4
1305
386
1325
L14
43.1
1544
F
O
1766
2344
F
L15
L16
19.2
69.3
1067
1211
F
1910
F
Br
L5
L6
126
335
1455
1340
O
L17
1292
1789
L7
L8
274
1788
967
L18
L19
134
254
1324
1567
11.7
CF
3
Cl
Cl
L9
496
225
1866
1767
4 h
SAHA
140
174
1522
1406
*Each value is the mean of at least three experiments (all SD are within
10% of the mean).
L10
O
and 3, 5-dimethy (L13) substitution are disfavored for the activity,
but the meta position (L18) methyl substitution is favored for the
inhibitory activity. The 2-methoxy group substitution (L10) has
negative effect to the activity, but the 3-fluoro-4-methoxy
substitution (L14) improves the activity obviously. In conclusion,
seven molecules show better IC50 values than SAHA (174 nM)
and compound 4 h (140 nM). Polar groups substituted in the
phenyl ring of the R group make positive contribution to the
enzymatic inhibition activity and fatty groups make negative
effect to the activity.
L11
L12
45.8
314
1305
1277
OH
Cl
The inhibitory activities of derived molecules against HDAC8
were also investigated. The synthesized molecules show reduced
enzymatic inhibition activities against HDAC8 comparing with
(continued )

DOI: 10.3109/14756366.2013.827678
Discovery of a series of hydroximic acid derivatives as potent histone deacetylase inhibitors
7
Figure 2. Binding pattern of molecule L8 in the active site of HDAC2; (a) surface plot of molecule L8 in the catalytic center of HDAC2;
(b) representation of H-bond interactions between molecule L8 and HDAC2.
Figure 3. Binding pattern of molecule L8 in the active site of HDAC8; (a) surface plot of molecule L8 in the catalytic center of HDAC8;
(b) representation of hydrophobic and H-bond interactions between molecule L8 and HDAC8.
activities against Hela cell nucleus extract. The difference of ketonic oxygen have H-bond interactions with His146 and
activities between these compounds is not significant, and Gly306, respectively. The ketonic oxygen of the peptide bond
molecule L8 is also the most potent inhibitor with an IC50 connecting the linker and external motif binds to Tyr308, and the
value of 967 nM.
The binding pattern of the most active molecule (L8) in the
NH can form H-bond binding with Gly154.
The binding mode of molecule L8 in the active site of HDAC8
active site of HDAC2 (PDB entry: 3MAX) was investigated by (PDB entry: 1T69) was also investigated by docking analysis
docking analysis. The result shows that the fatty linker stretches to (Figure 3). Significant hydrophobic interactions are also dis-
the tunnel of the site, and the hydroximic acid group chelates to covered to make contributions to the ligand–receptor bindings.
the zinc ion (Figure 2). Hydrophobic interactions were discovered These two aromatic ring in the external motif of molecule L8 also
to enhance the ligand–receptor bindings from the surface plot. binds to the hydrophobic region of the active site of HDAC8
These two phenyl rings of the external motif bind to the (Figure 3a). The phenyl ring in the linker also has hydrophobic
hydrophobic region in the opening of tunnel (Figure 2a). The interactions with surrounding residues, such as Phe152 and
trifluoromehtyl group which binds to the polar area of the pocket Phe208 (Figure 3b). Similar to the binding pattern of L8-HDAC2,
under the tunnel makes significant contribution to the binding of the hydroxamic acid group of molecule L8 not only chelates to the
molecule L8 to the active site of HDAC2 by electrostatic zinc ion, but also has H-bond interaction with surrounding
attraction. Fatty group substitutions which locate in this region are residues. The OH group has H-bond interactions with His143 and
supposed to make negative effect to the binding such as molecule His180; the NH binds to His142 and His143 with H-bond; and the
L4, L13 and L17. The H-bond interactions also play important ketonic oxygen can form H-bond interaction with the OH of
role in the ligand–receptor bindings (Figure 2b). The OH and Tyr306.

8
L. Zhang et al.
J Enzyme Inhib Med Chem, Early Online: 1–8
Table 2. The tumor cell growth inhibitory activities of synthesized
molecules (IC50)*y.
evaluated by enzymatic inhibition assay and tumor cell growth
inhibition assay. Significant improvement in the enzymatic
inhibition activities was discovered by comparing with SAHA
and 4 h. Molecule L8 has the IC50 value of 11.7 nM. However,
obvious loss of antiproliferation activities was detected in the
MTT assays. Molecular docking analysis shows that hydrophobic
interactions, chelate binding, electrostatic attraction and H-bond
interaction make important contribution to the L8-HDAC2 and
L8-HDAC8 interactions.
HCT116
(inhib%)
K562
(inhib%)
MDA-MB-231
(inhib%)
ES-2
(inhib%)
Compound
L1
L2
L3
L4
L5
L6
L7
L8
7.12
10.4
12.6
8.74
6.55
7.56
5.34
18.5
11.3
12.4
11.7
14.6
16.1
17.1
10.4
9.41
3.67
4.82
6.34
35.1
22.3
9.92
43.8
11.64
22.4
13.45
11.2
48.9
30.3
27.2
31.1
34.3
14.7
12.1
22.7
10.9
3.61
9.70
24.1
56.8
6.27
15.19
12.5
14.37
2.61
8.16
13.51
29.6
12.7
20.7
1.56
23.3
17.4
19.0
20.7
24.9
8.21
10.2
9.29
44.7
2.75
8.40
7.63
3.27
2.50
2.20
2.41
17.4
2.08
9.02
10.9
6.68
6.93
3.79
8.24
5.10
1.30
4.32
4.16
47.2
Declaration of interest
L9
The authors report no conflicts of interest. The authors alone are
responsible for the content and writing of this article.
L10
L11
L12
L13
L14
L15
L16
L17
L18
L19
SAHA
This work was supported by National Nature Science Foundation of
China (Grant No. 21172134), National High Technology Research and
Development Program of China (Grant No. 2011ZX09401-015) and
Doctoral Foundation of Ministry of Education of China (Grant No.
20110131110037).
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Although several molecules have better activities than SAHA
and 4 h in the enzymatic inhibition assay, the anti-proliferation
activities of the derived molecules are limited. The inhibitory
percentages of the synthesized molecules were primarily screened
at the concentration of 5 mM (Table 2). The results show that
molecule L8 which has the smallest IC50 values also has the best
performance in the tumor cell growth inhibition assays.
The inhibitory percentages of molecule L8 are 18.5, 48.9, 29.6
and 17.4 against HCT116, K562, MDA-MB-231 and ES-2
compared with SAHA, respectively. Therefore, the IC50 values
of molecule L8 against a series of tumor cell lines were derived
by comparing with SAHA (Table 3). Although molecule L8
shows relatively reduced activities compared with SAHA, the
antiproliferation activities of L8 against non-solid tumor cell lines
(U937: 2.67 mM; K562: 2.89 mM; HL60: 2.79 mM) is better than
activities against solid tumor cells (HCT116: 20.56 mM; MCF7:
25.42 mM; PC3: 17.64 mM).
Conclusion
14. 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.
In this work, structural modification was performed based on the
lead compound 4 h which was derived by structural optimization
of the virtual screening results. The synthesized molecules were