Design, synthesis and biological evaluation of isoquinoline-based derivatives as novel histone deacetylase inhibitors

Article abstract of DOI:10.1016/j.bmc.2015.06.071

The design, synthesis and biological evaluation of a series of isoquinoline-based hydroxamic acid compounds as novel HDACs inhibitors were reported herein. A detailed SAR study showed most of the compounds displayed good to excellent inhibitory activities against HDAC1, 3, 6. The IC₅₀ values of compound 10c against HDAC1, 3, 6 were 4.17 ± 0.11 nM, 4.00 ± 0.10 nM, 3.77 ± 0.07 nM, respectively. Most of the compounds showed great anti-proliferative activities against RPMI 8226, HCT 116 and Hep G2 cells. The IC₅₀ values of compounds 10a-h against RPMI 8226 cancer cell proliferation were all below 1 μM. HCT 116 cell was sensitive to the compounds 10a, 10f-g and 18a with the IC₅₀ values <0.3 μM. The active compounds 10a-d did not show inhibitory activity against hERG channel. All these evidence indicated these compounds had great potential as HDACs inhibitors for the further development.

Full text of DOI:10.1016/j.bmc.2015.06.071

Accepted Manuscript
Design, synthesis and biological evaluation of isoquinoline-based derivatives as
novel histone deacetylase inhibitors
Wei Yang, Lixuan Li, Yulan Wang, Xiaowei Wu, Tingting Li, Nan Yang,
Mingbo Su, Li Sheng, Mingyue Zheng, Yi Zang, Jia Li, Hong Liu
PII:
S0968-0896(15)00565-9
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.06.071
BMC 12427
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
18 May 2015
25 June 2015
26 June 2015
Please cite this article as: Yang, W., Li, L., Wang, Y., Wu, X., Li, T., Yang, N., Su, M., Sheng, L., Zheng, M., Zang,
Y., Li, J., Liu, H., Design, synthesis and biological evaluation of isoquinoline-based derivatives as novel histone
deacetylase inhibitors, Bioorganic & Medicinal Chemistry(2015), doi: http://dx.doi.org/10.1016/j.bmc.2015.06.071
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Design, synthesis and biological evaluation of
isoquinoline-based derivatives as novel histone
deacetylase inhibitors
Leave this area blank for abstract info.
Wei Yanga, Lixuan Lib,c, Yulan Wangb, Xiaowei Wua,, Tingting Lia, Nan Yanga,Mingbo Sub, Li Shengb,
Mingyue Zhengb, Yi Zangb, Jia Lib,c,*, Hong Liua,*
a CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of
Sciences, 555 Zuchongzhi Road, Shanghai 201203, People’s Republic of China
b State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences,
Shanghai 201203, PR China
c East China Normal University, Institutes for Advanced Interdisciplinary Research, North Zhongshan Road
Campus: 3663 N. Zhongshan Rd., Shanghai 200062, PR China
1

Bioorganic & Medicinal Chemistry
journal homepage: www.els evier.com
Design, synthesis and biological evaluation of isoquinoline-based derivatives as novel
histone deacetylase inhibitors
Wei Yanga, Lixuan Lib,c,, Yulan Wangb, Xiaowei Wua, Tingting Lia, Nan Yanga, Mingbo Sub, Li Shengb,
Mingyue Zhengb,Yi Zangb, Jia Lib,c,*, Hong Liua,*
a CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, PR
China
b State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, PR China
c East China Normal University, Institutes for Advanced Interdisciplinary Research, North Zhongshan Road Campus: 3663 N. Zhongshan Rd., Shanghai 200062,
PR China
ARTICLE INFO
ABSTRACT
Article history:
Received
The design, synthesis and biological evaluation of a series of isoquinoline-based hydroxamic
acid compounds as novel HDACs inhibitors were reported herein. A detailed SAR study showed
most of the compounds displayed good to excellent inhibitory activities against HDAC1, 3, 6.
The IC50 values of compound 10c against HDAC1, 3, 6 were 4.17 0.11 nM, 4.00 0.10 nM,
3.77 0.07 nM respectively. Most of the compounds showed great anti-proliferative activities
against RPMI8226, HCT-116 and HepG2 cells. The IC50 values of compounds 10a-h against
RPMI8226 cancer cell proliferation were all below 1 µM. HCT-116 cell was sensitive to the
compounds 10a, 10f-g and 18a with the IC50 values < 0.3 µM. The active compounds 10a-d did
not show inhibitory activity against hERG channel. All these evidence indicated these
compounds had great potential as HDACs inhibitors for the further development.
Received in revised form
Accepted
Available online
Keywords:
HDACs
HDACs inhibitor
Hydroxamic acid
Isoquinoline
2009 Elsevier Ltd. All rights reserved.
1. Introduction
cancer therapy. Among of the reported HDACs inhibitors,
CUDC-101 is a first-in-class multi-targeted hybrid which was
Histone deacetylases (HDACs) are a family of epigenetic
enzymes that remove acetyl groups from the lysine residues at
the N-terminal tail of histone and non-histone proteins regulating
numerous cellular processes and gene expression.1 These
enzymes can be divided into the NAD+-dependent sirtuins (class
III) and the Zn+-dependent HDACs which can be further divided
into three classes named class I (HDAC1, 2, 3, 8), IIa (HDAC4, 5,
7, 9), IIb (HDAC6, 10) and IV (HDAC11).2 Currently, HDACs
have been found to associate with many oncogenes and tumor
suppressors, altered gene expression and validated as a promising
therapeutic strategy for the treatment of cancer,3 neurological
disorders4 and infectious diseases.5 Histone deacetylases
inhibitors (HDACi) have been shown to cause transformed cell
growth arrest, cell death and angiogenesis repression by
increasing the levels of histone and non-histone proteins
acetylation such as α-tubulin, HIF-1α, HSP90.6-9 A large number
of the known HDACi have been developed and exhibited
powerful efficacy in animal models of human diseases.10 To date,
Suberoylanilide hydroxamic acid (SAHA)11, Depsipeptide
(FK228)12 and Belinostat13 have been approved by FDA for the
treatment of cutaneous T-cell lymphoma. These three approved
drugs have validated the therapeutic potentials of HDACi in the
designed to simultaneously inhibit HDACs, epidermal growth
factor receptor (EGFR) and human epidermal growth factor
receptor 2 (HER2), inducing significantly synergistic tumor
regression or inhibition in various cancer xenograft models
including non-small cell lung cancer (NSCLC), liver, breast,
head and neck, colon and pancreatic cancers.14 In our previous
studies, we have reported the replacement of quinazoline core in
the CUDC-101 with thieno[2,3-d]pyrimidine fragment.15 As a
continuation in this project, we aimed at further modifying
CUDC-101 to obtain novel HDACs inhibitors with powerful
inhibitory activities against HDACs. Based on the
pharmacophore of HDACs inhibitors16, it was deduced that the
R
NH
H
N
H
N
Linker
OH
N
O
O
s
i
r
e
t
s
o
s
i
o
10a-h R = substituted aniline
14a-d R = aliphatic amines
24,27a-c Linker = Phenyl group
i
B
NH
N
O
O
O
OH
N
N
H
4
S
c
a
f
f
o
l
d
H
o
p
O
O
p
1. CUDC-101
EGFR and HDAC inhibitors
Phase I
i
n
g
H
N
R
OH
N
N
H
5
O
18a-d R = aliphatic group
Corresponding author: Hong Liu: Tel.: +86-21-50807042; fax:
+86-21-50807042; e-mail: hliu@mail.shcnc.ac.cn. Jia Li: +86-21-50801552:
+86-21-50800721; e-mail: jli@mail.shcnc.ac.cn.
Fig 1. Design strategy and structural modifications

4-aniline quinazoline motif extended to the outside of the
HDACs catalytic pocket without significantly affecting its
binding to HDACs. Therefore, we employed the bioisosterism
principle to modify the CUDC-101 by using the isoquinoline
scaffold and designed a new series of isoquinoline-based
compounds to conduct extensive structure-activity relationship
(SAR) studies (Fig 1). Herein, we reported the novel
isoquinoline-based CUDC-101 derivatives function as HDACs
inhibitors. Most of these derivatives exhibited the powerful
enzymatic activities and excellent anti-proliferative potential
against three cancer cell lines.
formed the amide products 13. Conversion of ester group to
hydroxamic acids using freshly prepared hydroxylamine yielded
the final products 14a-14d.
The synthesis of isoquinoline-1(2H)-one-based compounds
18a-18d was shown in Scheme 3. Treatment of intermediate 4
with various aliphatic amines, followed by reduction of nitro
gave
intermediate
16
which
was
coupled
with
8-methoxy-8-oxooctanoic acid to yield intermediate 17. The
intermediate 17 was converted to targeted compounds 18a-18d
using the freshly prepared hydroxylamine.
2. Results and discussion
2.1 Chemistry
The synthetic route for preparing compounds 10a-10h was
1 was treated with
depicted in Scheme 1. Compound
iodomethane to afford 2 which was treated with DMA-DMF,
followed by cyclization with 3,4-dimethoxylbenzylamine to
generate 4. Deprotection and chlorination gave 6 which was
coupled with various amines to yield 7. Reduction of nitro group
using iron/NH4Cl, then couple with 8-methoxy-8-oxooctanoic
acid afforded the amide 9 which were finally treated with the
freshly prepared hydroxylamine to form targeted products
10a-10h.
Scheme 3 Reagents and conditions: a) Various aliphatic amines, Toluene,
110 oC; b) Fe, NH4Cl(aq), EtOH; c) EDCI,HOBT, HOOC(CH2)6COOCH3, RT;
d) NH2OH, MeOH, THF, 0 oC to RT.
As shown in Scheme 4, compound 21 was prepared through
the coupling of N1-phenylisoquinoline-1,7-diamine with
2-(4-(methoxycarbonyl)phenoxy)acetic acid, followed by
treatment with freshly prepared hydroxylamine.
Scheme
1
Reagents and conditions: a) CH3I, K2CO3, DMF, RT; b)
Scheme 4 Reagents and conditions: a) EDCI, HOBT, RT; b) NH2OH, MeOH,
THF, 0 oC to RT.
DMA-DMF, 100 oC; c) 3,4-dimethoxylbenzylamine, toluene, 125 oC; d)
CF3COOH, 85oC; e) POCl3, 100 oC; f) Substituted aromatic amines, conc HCl,
n-BuOH; g) Fe, NH4Cl, EtOH, 50 oC; h) EDCI, HOBT, DMF,
HOOC(CH2)6COOCH3, RT; i) NH2OH, MeOH, THF, 0 oC to RT.
Scheme 2 Reagents and conditions: a) Various aliphatic amines, Microwave,
30 min; b) Pd/C, H2, MeOH, RT; c) HOOC(CH2)6COOCH3 EDCI, HOBT,
DMF, RT; d) NH2OH, MeOH, THF, 0 oC to RT.
o
Scheme 5 Reagents and conditions: a) NaI, DMF, 60 C; b) NH2OH, MeOH,
THF, 0 oC to RT.
The preparation of 1-aliphatic amines isoquinoline compounds
14a-14d was shown in Scheme 2. Assistance with microwave,
the intermediate 6 was coupled with aliphatic amines to form
intermediate 11. Reduction of nitro group gave the corresponding
amine 12. Coupling of 12 with 8-methoxy-8-oxooctanoic acid
The compounds 24 and 27a-c were synthesized according to
Scheme 5. Reaction of benzyl bromo 22 and 25 with intermediate
8 respectively formed the intermediate 23 and 26. The final
compounds were achieved through the conversion of esters 23
3

and 26 using freshly prepared hydroxylamine to hydroxamic
acid.
spatial orientation of the N-substituents on inhibitory activities
towards HDACs enzymes and cancer cell viability. The
inhibitory activities against HDAC1, 3, 6 isoforms were
evaluated and the results were summarized in Table 3. This series
of compounds exhibited weaker inhibitory activities against
HDACs indicating that the binding affinity between
N-substituent isoquinoline-1-one scaffold and HDACs surface
was decreased, although compounds 18a and 18c showed not too
bad anti-proliferative activities against RPMI8226 cell with the
IC50 values below 2 µM.
2.2. In vitro HDAC inhibition and anti-proliferative activities
of compounds 10a-h
We initially evaluated the inhibitory activities of 1-anilines
isoquinoline derivatives 10a-h against recombinant human
HDAC1, HDAC3 and HDAC6 isoforms using SAHA as a
positive control (Table 1). Based on our previous study,15 we
directly fixed the carbon chain length between the two amides
was 6 carbons. Overall, the first designed compounds 10a-h
exhibited good to excellent inhibitory activities against a wide
range of HDACs subtypes. For example, the IC50 values of
compounds 10c against HDAC1, HDAC3, and HDAC6 were
4.17 nM, 4.0 nM and 3.77 nM respectively. This excellent
inhibitory potency indicated that the design strategy was rational
and successful. Furthermore, we assessed the anti-proliferative
activities of these compounds against multiple myeloma cell line
RPMI8226. As shown in Table 1, most of the compounds
exhibited stronger anti-proliferative activities against RPMI8226
cells than SAHA. The IC50 values of compounds 10a, 10f, 10g
were 0.46 µM, 0.52 µM and 0.47 µM respectively.
Table 3. In vitro inhibitory activities of isoquinoline-1(2H)-one-based
compounds 18a-d
IC50 (nM)
HDAC3
IC50 (µM)
RPMI8226
1.88
Compd
HDAC1
HDAC6
18a
18b
18c
18d
65.77 6.85
68.98 12.69
66.02 8.71
98.08 4.73
93.34 2.78
92.03 6.99
74.42 2.56
69.19 4.65
107.7 10.1
32.06 1.15
38.70 3.35
54.25 0.12
100 14.64
2.16
1.80
2.64
SAHA
158.17 6.66 78.98 13.19 2.39 0.08
2.5 In vitro HDAC inhibition and anti-proliferative activities of
compounds with Aryl as linker
Based on the reports HDACs inhibitors using aryl group as
linker showed stronger anti-HDACs activities than SAHA due to
the π-π stack between the aryl linker and phenylalanine residue in
the rim of the catalytic site,17 we switched the linker from carbon
chain to phenyl group and synthesized compounds 21, 24 and
27a-c as well as evaluated their inhibitory activities against
HDACs and the proliferation of RPMI8226 cell (Table 4).
Unexpectedly, all compounds displayed poor inhibitory activities
against HDACs. The IC50 values of these compounds against
HDAC1 and 3 ranged from 200 nM to 570 nM, while, compound
27a could show good anti-proliferative activity against
RPMI8226 with IC50 of 2.04 µM.
Table 1. In vitro inhibitory activities of 1-anilines isoquinoline derivatives
10a-h
IC50 (nM)
HDAC3
IC50 (µM)
RPMI8226
0.46 0.01
0.72 0.13
0.77 0.24
0.74 0.02
2.04 0.11
0.52 0.02
0.47 0.01
0.94 0.08
2.39 0.08
Compd
HDAC1
HDAC6
10a
10b
15.46 1.16
17.47 0.43
4.17 0.11
5.75 0.10
35.41 6.02
19.49 0.79
15.43 0.39
17.98 0.99
93.34 2.78
16.65 0.33
21.62 3.10
4.00 0.10
5.42 0.07
39.67 13.28
21.20 1.34
17.63 2.06
18.88 0.09
158.17 6.66
6.83 0.09
7.96 0.33
3.77 0.07
5.15 0.04
40.88 6.89
29.91 0.92
6.26 0.17
10.95 0.35
78.98 13.19
10c
10d
10e
10f
10g
10h
SAHA
Table 4. In vitro inhibitory activities of compounds 21, 24 and 27a-c using
2.3. In vitro HDAC inhibition and anti-proliferative activities
of compounds 14a-d
aryl group as linker.
IC50 (nM)
HDAC3
275.74
263.98
176.48
474.74
632.54
IC50 (µM)
RPMI8226
7.30 0.49
> 20
Next, the substituents at the C-1 position were switched to
aliphatic amines aimed at testing its effects on the inhibitory
activities. We synthesized compounds 14a-d and tested their
inhibitory activity on HDACs enzyme and the viability of the
cancer cells. As shown in Table 2, compound 14a with a large
substituent at C-1 position significantly decreased the inhibitory
activities against HDACs as well as the proliferation of
RPMI8226 cells. The other compounds 14b-d containing a
smaller group at the C-1 position displayed similar enzymatic
activities although a slight decrease in anti-proliferative activity
against RPMI 8266 cells was observed. These observed results
proved that smaller aliphatic amines at the C-1 position were
compatible without significantly affecting the inhibitory activities
against HDACs enzyme in vitro and the proliferation of the
cancer cells.
Compd
HDAC1
272.08
323.23
205.34
570.99
439.06
HDAC6
545.42
351.85
58.90
21
24
27a
2.04 0.23
2.71 0.53
5.22 0.56
2.39 0.08
27b
27c
286.26
304.02
SAHA
93.34 2.78 158.17 6.66 78.98 13.19
2.6 Anti-proliferation of representative compounds against
HepG2 and HCT-116
We selected compounds 10a, 10f, 10g, 14b and 18a which
possessed good to excellent inhibitory activities against HDACs
and the proliferation of RPMI8266 to further assess their
anti-proliferative activities towards human colonic carcinoma cell
line (HCT-116) and human liver hepatocellular carcinoma cell
line (HepG2). As shown in Table 5, compounds 10a, 10f, 10g
and 18a exhibited more robust anti-proliferative activities against
Table 2. In vitro inhibitory activities of 1-aliphatic amines isoquinoline
derivatives 14
Table 5. In vitro anti-proliferative activity against HepG2 and HCT-116 cells
of the representative compounds
IC50 (nM)
HDAC3
IC50 (µM)
RPMI8226
7.712
Compd
HDAC1
HDAC6
IC50 (µM)
14a
14b
263.0 21.5
45.85 7.46
41.63 1.51
27.91 12.15
93.34 2.78
192.0 3.9
40.78 1.38
36.00 0.62
13.95 0.19
158.17 6.66
147.6 0.21
29.50 4.86
25.00 0.90
13.59 2.20
78.98 13.19
Compound
HCT-116
0.19 0.02
0.17 0.02
0.26 0.07
1.81 0.15
0.28 0.03
1.72 0.06
HepG2
1.33
1.35
10a
10f
5.87 1.11
10.00 3.93
8.45 1.65
23.62 2.29
14.01 2.46
18.54 1.82
14c
14d
SAHA
2.71
2.39 0.08
10g
14b
2.4 In vitro HDAC inhibition and anti-proliferative activities
of compounds 18a-d
18a
SAHA
HCT-116 cell with the IC50 values of 0.19 µM, 0.17 µM, 0.26 µM
and 0.28 µM respectively. While, HepG2 cell was not sensitive to
Through scaffold hopping strategy, we synthesized
isoquinoline-1-one-based compounds 18a-d to test the effects of
4

our synthesized compounds with the IC50 values over 5 µM.
2.7 α-Ac-tubulin and Ac-histone H3 levels in RPMI8226 cells
ensuring drug safety, the most potent HDAC inhibitors among
the isoquinoline-based series, compounds 10a-d were selected to
further evaluate the side effect on the human
ether-a-go-go-related (hERG) channel. The inhibitory activities
of compounds 10a-d towards hERG at concentration of up to 40
µM were summarized in Table 6, showing no obvious inhibitory
activities against hERG was observed. The above results
provided further evidence that these novel compounds could be
non-cardiatoxic lead compounds for the development of novel
anticancer drugs.
Two representative compounds 10a and 10b were selected to
validate the observed biochemical potencies by assessing their
effects on the levels of α-tubulin and histone H3 acetylation in
the multiple myeloma cancer cell RPMI8226 (Fig 2). Western
blot analysis revealed that compounds 10a and 10b caused a
substantial increase in the level of acetylated α-tubulin and
histone H3 in a dose-dependent manner which was consistent
with their strong inhibitory activities against HDACs and cancer
cells. Compound 10a noticeably up-regulated the level of H3
acetylation at the low concentration of 0.03 µM. Additionally, the
western blot analysis showed that our designed compounds, like
the reference compound SAHA, were pan-HDACs inhibitors.
Table 6. Inhibitory activities of compounds 10a-d against hERG channel
Compd
10a
10b
10c
10d
Concentration (µM)
Inhibition (%)
43.69
26.36
IC50 (µM)
> 40
> 40
> 40
> 40
40
40
40
40
3
40.85
17.51
98.67
Cisapride
0.098
3.Conclusion
Based on the HDACs inhibitor CUDC-101, novel amine
substituted isoquinoline hydroxamic acid derivatives were
designed, synthesized and evaluated using the bioisosterism
principle. Most of the compounds were found to effectively
inhibit the HDACs catalytic activities and the proliferation of
multiple cancer cells with low IC50 values. The representative
compounds 10a, 10f, 10g, 14b and 18a also showed
anti-proliferative activity against colon cancer cells and liver
carcinoma cells. These compounds could dramatically increase
the levels of the Histone H3 and α-tubulin acetylation in low
concentration. These results proved the design strategy was
rational and the amines substituted isoquinoline was an
outstanding cap group. The study also showed compounds 10a-d
were not toxic to cardiac function and could be as lead
compounds for further optimization. The assessments of PK
profile and efficacy in animal are now undertaken in our lab.
Fig 2. Western blot analysis of Ac-tubulin and Ac-histone H3 from RPMI
8226 cell cultured for 24h with DMSO control and compounds 10a, 10b,
SAHA and Chidamide
2.8 Molecular docking
In order to understand the interaction of these inhibitors with
HDACs, we docked the compound 10c and SAHA in the active
site of HDAC2 (PDB code: 4LXZ) using induced Fit Docking
module of Schrӧdinger software package (Schrӧdinger, LLC:
New York, NY, 2010). As shown in Fig 3, compound 10c
displayed the similar binding mode to SAHA in the active site of
HDAC2. The hydroxamic acid of the compound 10c could
chelate Zn2+ and form the hydrogen bonds with Y308, H145 and
H146 residues. Additionally, the phenyl moiety had a cation-π
interaction with R275 residue that could enhance the binding
affinity of compound 10c with HDAC2.
4. Experimental section
Chemistry. The reagents (chemicals) were commercially
available and used without further purification. Analytical
thin-layer chromatography (TLC) was performed on HSGF 254
(0.15-0.2 mm thickness). Column chromatography was
performed on silica gel 300-400 mesh to purify the compounds.
Nuclear magnetic resonance (NMR) spectra were performed on
a Brucker AMX-400 (TMS as IS). Chemical shifts were reported
in parts per million (ppm, δ) downfield from tetramethylsilane.
Proton coupling patterns were described as singlet (s), doublet
(d), triplet (t), quartet (q), multiplet (m), and broad (br). Low-
and high-resolution mass spectra (LRMS and HRMS) were given
with electric, electrospray and matrix-assisted laser desorption
ionization (EI, ESI and MALDI) produced by Finnigan
MAT-95, LCQ-DECA spectrometer and IonSpec4.7 T.
4.1. General procedures for synthesis of compounds 10a-h
4.1.1 Methyl-2-methyl-5-nitrobenzoate (2). To a mixture of
2-methyl-5-nitrobenzoic acid (15 g, 82.8 mmol) and K2CO3 (17.2
g, 124.2 mmol) in 75 mL DMF was added iodomethane (6.7 mL).
The mixture was stirred at room temperature overnight. Then,
500 mL water was added into the mixture, the aqueous phase was
extracted with EA for three times. The combined organic phase
was dried with Na2SO4, concentrated to afford the product 2
1
(15.3 g, 94%) as yellow solid. H NMR (400 MHz, CDCl3) δ
8.52 (s, 1H), 8.05 (s, 1H), 7.30 (s, 1H), 3.83 (s, 3H), 2.56 (s, 3H).
ESI-MS m/z 196 [M + H]+.
Fig 3. Molecular docking of compound 10c (green) and SAHA
(purple) in the active site of HDAC2.
2.9 In vitro Drug safety assessment
4.1.2. (E)-methyl 2-(2-(dimethylamino)vinyl)-5-nitrobenzoate
(3). A mixture of intermediate 2 (2 g, 10 mmol) and DMA (4.8 g,
40 mmol) in 15 mL DMF was heated at 100 oC for 5 h. The
To assess the cardiac toxicity of the designed compounds for

mixture was poured into ice-water and the precipitated solid was
filtered and washed with water to give (E)-methyl
2-(2-(dimethylamino)vinyl)-5-nitrobenzoate 3 (2.0 g, 80%) as
yellow solid . 1H NMR (400 MHz, CDCl3) δ 8.70 (d, J = 2.64 Hz,
1 H)，8.03 (dd, J = 9.23, 2.64 Hz, 1 H)，7.43 (d, J = 9.23 Hz, 1
H)，7.17 (d, J = 13.62 Hz, 1 H)，6.39 (d, J = 13.18 Hz, 1 H),
3.89 (s, 3 H)，2.99 (s, 6 H). ESI-MS m/z 251 [M + H]+.
The reaction mixture was warmed to room temperature and
o
stirred at 25 C for 30 mins. The mixture was neutralized with
acetic acid and concentrated. The residue was purified by column
chromatagraphy on silica gel (DCM/MeOH, v/v = 20/1-10/1) to
give the desired product 10a (40 mg, 87%) as oil. 1H NMR (400
MHz, DMSO) δ 10.34 (s, 1H), 10.20 (s, 1H), 8.94 (s, 1H), 8.67
(s, 1H), 8.66 (s, 1H), 7.82-7.73 (m, 3H), 7.59-7.55 (m, 2H), 7.38
(d, J = 7.2 Hz, 1H), 7.16 (d, J = 5.7 Hz, 1H), 2.37 (t, J = 7.3 Hz,
2H), 1.94 (t, J = 7.4 Hz, 2H), 1.66-1.58 (m, 2H), 1.53-1.46 (m,
2H), 1.36-1.23 (m, 4H); 13C NMR (100 MHz, DMSO) δ 172.5,
171.9, 169.5, 157.0, 155.0, 152.8, 139.5, 137.8, 133.8, 129.6,
127.7, 126.9, 124.6, 119.6, 119.2, 115.0, 113.7, 112.4, 58.4, 36.8,
32.7, 28.9, 25.5, 25.4, 21.5. HRMS calcd for C23H25BrFN4O3
[M + H]+ 503.1094, found 503.1098.
4.1.3. 2-(2,5-Dimethoxybenzyl)-7-nitroisoquinolin-1(2H)-one
(4).
A mixture of intermediate 3 (2 g, 8 mmol) and
2,4-dimethoxylbenzylamine (1.87 g, 11.2 mmol) was heated at
125 oC until the starting material was consumed completely.
After the mixture was cooled to rt, it was triturated with
hexanes/ethyl acetate (v/v = 2:1). After standing at room
temperature overnight, the precipitate was collected by filtration
1
to give 4 (2.5 g, 92%) as yellow solid. H NMR (400 MHz,
4.1.10 N1-(1-((3-chloro-2-fluorophenyl)amino)isoquinolin-7-yl
)-N8-hydroxyoctanediamide 10b. Compound 10b was prepared
from 9b according the same procedure described for compound
CDCl3) δ 9.29 (d, J=2.64 Hz, 1 H), 8.37 (dd, J = 8.79, 2.20 Hz, 1
H), 7.58 (d, J = 8.79 Hz, 1 H), 7.39-7.48 (m, 2 H), 6.47-6.51 (m,
3 H), 5.13 (s, 2 H), 3.84 (s, 3 H), 3.79 (s, 3 H). ESI-MS m/z 341
[M + H]+.
1
10a. Yield: 82%, 375 mg. H NMR (400 MHz, DMSO) δ 10.36
(s, 1H), 10.22 (s, 1H), 9.08 (s, 1H), 8.68 (s, 2H), 7.84 (d, J = 5.7
Hz, 1H), 7.79 (t, J = 9.0 Hz, 1H), 7.54 (t, J = 8.2 Hz, 1H), 7.30 (t,
J = 6.8 Hz, 1H), 7.19 (dd, J = 9.8, 6.7 Hz, 2H), 2.38 (t, J = 7.4 Hz,
2H), 1.95 (t, J = 7.4 Hz, 2H), 1.69-1.58 (m, 2H), 1.54-1.46 (m,
2H), 1.36-1.25 (m, 4H). HRMS calcd for C23H25ClFN4O3 [M +
H]+ 459.1599, found 459.1603.
4.1.4 7-nitroisoquinolin-1(2H)-one (5). A mixture of 4 (2.5 g,
7.33 mmol) in CF3COOH (42 mL) was stirred at 85 C for 5 h.
o
The mixture was cooled to room temperature and concentreated.
The residue was suspended in EtOAc and stirred for 30 min,
collected the solid and washed with EtOAc to afford off-white
1
solid 5 (1.1 g, 80%). H NMR (400 MHz, DMSO-d6) δ 11.77 (s,
4.1.11 N1-hydroxy- N8-(1-(p-tolylamino)isoquinolin-7-yl)octa-
nediamide 10c. Compound 10c was prepared from the 9c
according the same procedure described for compound 10a.
1 H), 8.88 (d, J = 2.20 Hz, 1 H), 8.43 (dd, J = 8.79, 2.64 Hz, 1
H), 7.90 (d, J = 8.79 Hz, 1 H), 7.42 - 7.48 (m, 1 H), 6.72 (d, J =
7.03 Hz, 1 H). ESI-MS m/z 191 [M + H]+.
1
Yield: 80%, 337 mg. H NMR (400 MHz, DMSO) δ 10.36 (s,
4.1.5 1-chloro-7-nitroisoquinoline (6). The intermediate 5 (1.9 g,
10 mmol) was suspended in phosphoryl trichloride (20 mL) and
1H), 10.17 (s, 1H), 9.05 (s, 1H), 8.69 (s, 1H), 8.59 (s, 1H), 7.85
(d, J = 5.6 Hz, 1H), 7.82-7.74 (m, 2H), 7.63 (d, J = 8.1 Hz, 2H),
7.16-7.07 (m, 3H), 2.38 (t, J = 7.3 Hz, 2H), 2.28 (s, 3H), 1.95 (t,
J = 7.2 Hz, 2H), 1.67-1.58 (m, 2H), 1.55-1.45 (m, 2H), 1.36-1.23
(m, 4H). 13C NMR (100 MHz, DMSO) δ 172.0, 170.0, 152.6,
138.3, 133.8, 129.7, 128.0, 127.7, 125.8, 124.9, 122.5, 119.6,
113.3, 112.7, 110.1, 49.1, 36.7, 32.7, 28.9, 25.5, 25.4, 21.0.
HRMS calcd for C24H29N4O3 [M + H]+ 421.2240, found
421.2236.
o
the mixture was heated at 100 C until the intermediate 5 was
consumed completely. The mixture was poured into ice-water
and extracted with EtOAc for three times. The combined organic
phase was washed with saturated NaHCO3 solution and brine,
dried over Na2SO4 andconcentrated to afford yellow solid which
was purified by column chromatography on silica gel
(hexane/ethyl acetate, v/v = 4/1) to give 6 (1.5 g, 72%) as yellow
solid.
4.1.12 N1-hydroxy- N8-(1-(naphthalen-2-ylamino)isoquinolin-
7-yl)octanediamide 10d. Compound 10d was prepared from the
9d according the same procedure described for compound 10a.
4.1.6 General procedure for synthesis of Intermediate 7. A
mixture of 1-chloro-7-nitroisoquinoline (100 mg, 0.48 mmol),
aniline derivatives (0.72 mmol) and 50 µL conc HCl in n-BuOH
1
Yield: 82%, 374 mg. H NMR (400 MHz, DMSO) δ 10.37 (s,
o
(3 mL) was stirred at 90 C overnight. The mixture was poured
1H), 10.22 (s, 1H), 9.36 (s, 1H), 8.69 (s, 2H), 8.34 (s, 1H), 7.97
(d, J = 5.6 Hz, 1H), 7.90-7.75 (m, 6H), 7.44 (t, J = 7.4 Hz, 1H),
7.35 (t, J = 7.4 Hz, 1H), 7.20 (d, J = 5.7 Hz, 1H), 2.39 (t, J = 7.4
Hz, 2H), 1.96 (t, J = 7.3 Hz, 2H), 1.68 -1.59 (m, 2H), 1.55-1.46
(m, 2H), 1.38-1.26 (m, 4H); 13C NMR (100 MHz, DMSO) δ
172.5, 171.9, 169.6, 152.7, 139.7, 137.8, 134.3, 133.9, 129.6,
128.2, 127.9, 127.8, 127.3, 126.5, 125.0, 124.2, 122.5, 120.0,
115.6, 113.5, 112.9, 49.1, 36.7, 32.7, 28.9, 28.8, 25.5, 25.4.
HRMS calcd for C27H29N4O3 [M + H]+ 457.2240, found
457.2245.
4.1.13 N1-(1-((2-fluoro-4-methylphenyl)amino)isoquinolin-7-
yl)-N8-hydroxyoctanediamide 10e. Compound 10e was
prepared from the 9e according the same procedure described for
compound 10a. Yield: 68%, 297 mg. 1H NMR (400 MHz,
DMSO) δ 10.35 (s, 1H), 10.22 (s, 1H), 8.68 (s, 2H), 7.76 (d, J =
10.5 Hz, 3H), 7.46 (s, 1H), 7.09 (d, J = 12.1 Hz, 2H), 7.02 (d, J =
7.7 Hz, 1H), 2.38 (t, J = 7.3 Hz, 2H), 2.34 (s, 3H), 1.95 (t, J = 7.2
Hz, 2H), 1.68-1.58 (m, 2H), 1.55-1.46 (m, 2H), 1.31 (m, 4H).
HRMS calcd for C24H28FN4O3 [M + H]+ 439.2145, found
439.2150.
into ice-water, extracted with EtOAc. The combined organic
phase was washed with brine, dried over Na2SO4, concentrated to
afford the desired product as a solid which was used without
further purification.
4.1.7 General procedure for synthesis of intermediate 8. To a
solution of 7 (0.48 mmol), iron powder (268 mg, 4.8 mmol) in
EtOH (3 mL) was added saturated NH4Cl solution (3 mL). The
o
mixture was stirred at 50 C for 2 h, then filtered through celite,
washed with MeOH, and concentrated. The residue was purified
by column chromatagraphy on silica gel to give 8 as yellow solid.
4.1.8 General procedure for synthesis of intermediate 9. To a
solution of 8 (0.35 mmol), DIPEA (90.3 mg, 0.7 mmol) in dried
THF (2 mL) was added methyl 8-chloro-8-oxooctanoate (0.52
mmol) slowly at 0 oC. The mixture was stirred at room
temperature for 2 h, then poured into water, extracted with
EtOAc. The combined organic phase was dried over Na2SO4,
concentrated and purified by column chromatagraphy
(hexane/ethyl acetate, v/v = 2/1) to offer 9 as oil.
General procedure for the synthesis of targeted compounds
10a-h
4.1.15 N1-(1-((2-Chloro-4-methylphenyl)amino)isoquinolin-7-
yl)-N8-hydroxyoctanediamide 10f. Compound 10f was prepared
from the 9f according the same procedure described for
compound 10a. Yield: 65%, 296 mg. 1H NMR (400 MHz,
DMSO) δ 10.35 (s, 1H), 10.25 (s, 1H), 8.67 (s, 2H), 7.81 (s, 3H),
4.1.9 N1-(1-((4-bromo-2-fluorophenyl)amino)isoquinolin-7-yl)
-N8-hydroxyoctanediamide (10a). To
appropriate esters (1 mmol) in THF (3 mL) was added to the
a solution of the
o
freshly prepared hydroxylamine solution (2 M, 3 mL) at 0 C.
6

1
7.69 (s, 1H), 7.37 (s, 1H), 7.18 (d, J = 7.4 Hz, 1H), 7.16-7.10 (m,
1H), 2.38 (t, J = 7.4 Hz, 2H), 2.33 (s, 3H), 1.95 (t, J = 7.4 Hz,
2H), 1.62 (d, J = 7.3 Hz, 2H), 1.55-1.46 (m, 2H), 1.31 (m, 4H);
13C NMR (100 MHz, DMSO) δ 172.0, 169.5, 152.8, 138.2,
135.4, 133.7, 130.2, 128.7, 127.9, 126.4, 124.9, 119.4, 122.8,
112.0, 40.6, 36.8, 32.7, 28.9, 25.5, 25.4, 20.6. HRMS calcd for
C24H28ClN4O3 [M + H]+ 455.1850, found 455.1858.
10a. Yield: 86%, 379 mg. H NMR (400 MHz, DMSO) δ 10.35
(s, 1H), 10.24 (s, 1H), 8.70 (s, 1H), 8.48 (s, 1H), 8.01 (d, J = 5.7
Hz, 1H), 7.88 -7.78 (m, 2H), 7.31 (d, J = 5.7 Hz, 1H), 3.37-3.29
(m, 4H), 2.93-2.79 (m, 5H), 2.37 (t, J = 7.5 Hz, 2H), 1.95 (t, J =
7.3 Hz, 2H), 1.67-1.58 (m, 2H), 1.55-1.46 (m, 2H), 1.37-1.25 (m,
4H), 1.11 (d, J = 6.5 Hz, 6H). HRMS calcd for C24H36N5O3 [M +
H]+ 442.2818, found 442.2822.
4.1.16 N1-(1-((2-Bromo-4-methylphenyl)amino)isoquinolin-7-
yl)-N8-hydroxyoctanediamide 10g. Compound 10g was
prepared from the 9g according the same procedure described for
compound 10a. Yield: 70%, 348 mg. 1H NMR (400 MHz,
DMSO) δ 10.35 (s, 1H), 10.21 (s, 1H), 8.67 (s, 1H), 8.64 (s, 1H),
8.55 (s, 1H), 7.83 -7.77 (m, 3H), 7.71 (d, J = 8.3 Hz, 1H), 7.50 (s,
1H), 7.20 (d, J = 8.4 Hz, 1H), 7.10 (d, J = 5.8 Hz, 1H), 2.42 –
2.35 (m, 2H), 1.95 (t, J = 7.3 Hz, 2H), 1.63 (m, 2H), 1.50 (m,
2H), 1.31 (m, 4H). HRMS calcd for C24H28BrN4O3 [M + H]+
499.1345, found 499.1338.
4.1.24 N1-Hydroxy-N8-(1-((3-methoxypropyl)amino)isoquino-
lin-7-yl)octanediamide 14d. Compound 14d was prepared from
the 13d according the same procedure described for compound
1
10a. Yield: 82%, 329 mg. H NMR (400 MHz, DMSO) δ 10.35
(s, 1H), 10.05 (s, 1H), 8.67 (s, 1H), 8.35 (s, 1H), 7.77 (d, J = 5.8
Hz, 1H), 7.63 (s, 2H), 7.10 (s, 1H), 6.82 (d, J = 5.7 Hz, 1H), 3.50
(q, J = 12.2, 6.5 Hz, 2H), 3.44 (t, J = 6.2 Hz, 2H), 3.27 (s, 3H),
2.35 (t, J = 7.5 Hz, 2H), 1.95 (t, J = 7.2 Hz, 2H), 1.90-1.84 (m,
2H), 1.61 (t, J = 7.0 Hz, 2H), 1.55-1.46 (m, 2H), 1.30 (m, 4H);
13C NMR (100 MHz, DMSO) δ 171.7, 169.6, 155.4, 140.5,
136.9, 133.4, 127.4, 124.3, 118.6, 112.5, 109.6, 71.0, 58.4, 40.5,
39.2, 36.6, 32.7, 29.3, 28.9, 25.5, 25.4. HRMS calcd for
C21H31N4O4 [M + H]+ 403.2345, found 403.2350.
4.1.17 N1-(1-((2-fluorophenyl)amino)isoquinolin-7-yl)-N8-hyd-
roxyoctanediamide 10h. Compound 10h was prepared from the
9h according the same procedure described for compound 10a.
1
Yield: 82%, 347 mg. H NMR (400 MHz, DMSO) δ 10.35 (s,
4.1.25 General procedure for syntheis of intermediate 15
Intermediate 15 was prepared according the same procedure
described for the intermediate 4.
1H), 10.19 (s, 1H), 8.82 (s, 1H), 8.66 (d, J = 11.8 Hz, 2H), 7.81
(d, J = 5.6 Hz, 1H), 7.78 (s, 2H), 7.64 (t, J = 7.5 Hz, 1H),
7.26-7.10 (m, 4H), 2.38 (t, J = 7.4 Hz, 2H), 1.95 (t, J = 7.3 Hz,
2H), 1.62 (d, J = 7.1 Hz, 2H), 1.56-1.46 (m, 2H), 1.32 (m, 4H).
HRMS calcd for C23H26FN4O3 [M + H]+ 425.1989, found
425.1980.
4.1.26 General procedure for syntheis of intermediate 16
Intermediate 16 was prepared according the same procedure
described for the intermediate 8.
4.1.27 General procedure for syntheis of intermediate 17
Intermediate 17 was prepared according the same procedure
described for the intermediate 9.
General procedure for synthesis of targeted compounds 18
4.1.28 N1-(2-butyl-1-oxo-1,2-dihydroisoquinolin-7-yl)-N8-hyd-
roxyoctanediamide 18a. Compound 18a was prepared from the
17a according the same procedure described for compound 10a.
4.1.18 General procedure for the synthesis of intermediate 11
Intermediate 6 (416 mg, 2 mmol) was dissolved in 2.5 mL
aliphatic amines. The solution was microwave irradiated at 110
oC for 30 min. The reaction was cooled to room temperature. To
the solution was added water, extracted with EtOAc for three
times. The combined organic phase was washed with brine, dried
with Na2SO4, concentrated to afford the oil residue, which was
used for the next step without purification.
1
Yield: 62%, 240 mg. H NMR (400 MHz, DMSO) δ 10.35 (s,
1H), 10.16 (s, 1H), 8.69 (s, 1H), 8.49 (d, J = 1.7 Hz, 1H), 7.91
(dd, J = 8.6, 2.1 Hz, 1H), 7.58 (d, J = 8.6 Hz, 1H), 7.35 (d, J =
7.3 Hz, 1H), 6.55 (d, J = 7.3 Hz, 1H), 3.94 (t, J = 7.2 Hz, 2H),
2.33 (t, J = 7.4 Hz, 2H), 1.94 (t, J = 7.4 Hz, 2H), 1.68-1.55 (m,
4H), 1.54-1.44 (m, 2H), 1.35-1.23 (m, 6H), 0.90 (t, J = 7.4 Hz,
3H); 13C NMR (100 MHz, DMSO) δ 171.8, 169.5, 161.1, 138.4,
132.9, 131.9, 127.1, 126.4, 124.5, 116.2, 105.1, 48.3, 40.4, 36.8,
32.7, 31.3, 28.9, 25.5, 25.4, 19.8, 14.1. HRMS calcd for
C21H30N3O4 [M + H]+ 388.2236, found 388.2246.
4.1.19 General procedure for the synthesis of intermediate 12
Intermediate 12 was prepared according the same procedure
described for the intermediate 8.
4.1.20 General procedure for the synthesis of intermediate 13
Intermediate 13 was prepared according the same procedure
described for the intermediate 9.
General procedure for the synthesis of targeted compounds
14
4.1.21 N1-(1-(Bis(2-methoxyethyl)amino)isoquinolin-7-yl)-N8-
hydroxyoctanediamide 14a. Compound 14a was prepared from
the 13a according the same procedure described for compound
4.1.29 N1-(2-(2-(diethylamino)ethyl)-1-oxo-1,2-dihydroisoqui-
nolin-7-yl)-N8-hydroxyoctanediamide 18b. Compound 18b was
prepared from the 17b according the same procedure described
1
1
10a. Yield: 76%, 362 mg. H NMR (400 MHz, DMSO) δ 10.34
for compound 10a. Yield: 66%, 284 mg. H NMR (400 MHz,
(s, 1H), 10.20 (s, 1H), 8.67 (s, 1H), 8.54 (s, 1H), 7.97 (d, J = 5.7
Hz, 1H), 7.76 (dd, J = 20.0, 8.8 Hz, 2H), 7.24 (d, J = 5.6 Hz, 1H),
3.61 (t, J = 5.8 Hz, 4H), 3.55 (t, J = 5.7 Hz, 4H), 3.19 (s, 6H),
2.37 (t, J = 7.3 Hz, 2H), 1.95 (t, J = 7.3 Hz, 2H), 1.67-1.57 (m,
2H), 1.55-1.45 (m, 2H), 1.36-1.25 (m, 4H). HRMS calcd for
C23H35N4O5 [M + H]+ 477.2607, found 477.2600.
DMSO) δ 10.34 (s, 1H), 10.14 (s, 1H), 8.67 (s, 1H), 8.49 (s, 1H),
7.89 (d, J = 8.6 Hz, 1H), 7.57 (d, J = 8.7 Hz, 1H), 7.32 (d, J = 7.1
Hz, 1H), 6.61-6.35 (m, 1H), 3.97 (t, J = 6.3 Hz, 2H), 2.66 (t, J =
6.3 Hz, 2H), 2.50-2.45 (m, 4H), 2.33 (t, J = 7.3 Hz, 2H), 1.94 (t,
J = 7.3 Hz, 2H), 1.59 (d, J = 7.0 Hz, 2H), 1.55-1.44 (m, 2H), 1.29
(m, 4H), 0.88 (t, J = 7.0 Hz, 6H). HRMS calcd for C23H34N4O4
[M + H]+ 431.2580, found 431.2584.
4.1.22 N1-Hydroxy-N8-(1-morpholinoisoquinolin-7-yl)octane-
diamide 14b. Compound 14b was prepared from the 13b
according the same procedure described for compound 10a.
4.1.30 N1-hydroxy-N8-(2-isopentyl-1-oxo-1,2-dihydroisoquino-
lin-7-yl)octanediamide 18c. Compound 18c was prepared from
the 17c according the same procedure described for compound
1
Yield: 70%, 280 mg. H NMR (400 MHz, DMSO) δ 10.35 (s,
1
1H), 10.22 (s, 1H), 8.68 (s, 1H), 8.53 (s, 1H), 8.02 (d, J = 5.6 Hz,
1H), 7.83 (s, 2H), 7.33 (d, J = 5.6 Hz, 1H), 3.86 (t, J = 4.2 Hz,
4H), 3.28 (t, J = 4.2 Hz , 4H), 2.37 (t, J = 7.5 Hz, 2H), 1.95 (t, J
= 7.4 Hz, 2H), 1.66-1.58 (m, 2H), 1.54-1.46 (m, 2H), 1.34-1.25
(m, 4H). HRMS calcd for C21H29N4O4 [M + H]+ 401.2189, found
401.2194.
4.1.23 N1-Hydroxy-N8-(1-(4-isopropylpiperazin-1-yl)isoquino-
lin-7-yl)octanediamide 14c. Compound 14c was prepared from
the 13c according the same procedure described for compound
10a. Yield: 72%, 288 mg. H NMR (400 MHz, DMSO) δ 10.34
(s, 1H), 10.16 (s, 1H), 8.67 (s, 1H), 8.48 (s, 1H), 7.91 (d, J = 8.6
Hz, 1H), 7.59 (d, J = 8.6 Hz, 1H), 7.32 (d, J = 7.4 Hz, 1H), 6.54
(d, J = 7.2 Hz, 1H), 3.77 (d, J = 7.5 Hz, 2H), 2.33 (t, J = 7.3 Hz,
2H), 2.15-2.04 (m, 1H), 1.93 (dd, J = 14.0, 6.4 Hz, 2H), 1.60 (s,
2H), 1.54-1.45 (m, 2H), 1.29 (m, 4H), 0.87 (d, J = 6.7 Hz, 6H).
HRMS calcd for C22H32N3O4 [M + H]+ 402.2393, found
402.2390.
4.1.31 N1-(2-(2,4-dimethoxybenzyl)-1-oxo-1,2-dihydroisoquin-
7

olin-7-yl)-N8-hydroxyoctanediamide 18d. Compound 18d was
prepared from the 17d according the same procedure described
for compound 10a. Yield: 86%, 413 mg. H NMR (400 MHz,
(OptiPlateTM-384F, PerkinElmer). In brief, the HDAC assay
mixture contained the substrate (5-50 µM, 5 µL), rhHDAC
isoforms (20-200 nM) and inhibitor (1 µL). Positive controls
contained all the above components except the inhibitor. The
negative controls contained neither enzyme nor inhibitor. The
HDAC6 assay components were incubated at room temperature
for 3 h, and HDAC1,3 were incubated for 24 h . The reaction was
quenched with the addition of 25 µL Trypsin with the final
concentration of 0.3125%. After 30 min incubation at room
temperature, the 384 micro-well plates were read at wavelengths
355nm(excitation) and 460nm(emission) using Envision
(PerkinElmer). Each experiment was done in triplicate.
1
DMSO) δ 10.34 (s, 1H), 10.16 (s, 1H), 8.68 (s, 1H), 8.49 (s, 1H),
7.91 (d, J = 8.8 Hz, 1H), 7.58 (d, J = 8.6 Hz, 1H), 7.29 (d, J = 7.4
Hz, 1H), 6.96 (d, J = 8.3 Hz, 1H), 6.59 (s, 1H), 6.55 (d, J = 7.4
Hz, 1H), 6.47 (d, J = 8.0 Hz, 1H), 5.02 (s, 2H), 3.82 (s, 3H), 3.74
(d, J = 4.2 Hz, 3H), 2.33 (t, J = 7.4 Hz, 2H), 1.94 (t, J = 7.2 Hz,
2H), 1.59 (d, J = 7.2 Hz, 2H), 1.54-1.45 (m, 2H), 1.29 (m, 4H).
HRMS calcd for C26H32N3O6 [M + H]+ 482.2291, found
482.2297.
General procedure for synthesis of compound 21, 24, 27a-c
The procedure for synthesis of 21, 24, 27a-c was similar to the
procedure described for compound 10a.
4.2.2. Cell proliferation assay
Cell proliferation assay was determined with the Cell Counting
Kit-8 (CCK-8) which utilized Dojindo’s highly water-soluble
tetrazolium salt. WST-8. WST-8 is reduced by dehydrogenases in
cells to give an orange colored product (formazan), which is
soluble in the tissue culture medium. The amount of the
formazan dye generated by dehydrogenases in cells is directly
proportional to the number of living cells. According to the
manufacturer’s instructions, cells were plated overnight on
96-well plates at 10000 cells per well in RPMI1640 growth
medium. Then, the cells were treated with the compounds at
indicated concentrations (0.00064 µM, 0.032 µM, 0.16 µM,
0.8 µM, 4 µM and 20 µM). After 72 hr treatment, 10 µL of
CCK-8 solution was added to each well. Plates were returned to
the incubator and left in the dark for 3 hour. The absorbance was
measured on a SpectraMax 340 microplate reader (Molecular
Devices, USA) at 450 nm with a reference at 690 nm. Each
experiment was done in triplicate.
4.1.32 N-hydroxy-4-(2-((1-(naphthalen-2-ylamino)isoquinolin
-7-yl)amino)-2-oxoethoxy)benzamide 21. Yield: 74%, 353 mg.
1H NMR (400 MHz, DMSO) δ 11.12 (s, 1H), 10.52 (s, 1H),
9.49-9.33 (m, 1H), 8.95 (s, 1H), 8.75 (s, 1H), 8.35 (s, 1H), 7.98
(s, 1H), 7.95-7.72 (m, 8H), 7.45 (t, J = 7.4 Hz, 1H), 7.36 (t, J =
7.2 Hz, 1H), 7.23 (d, J = 5.4 Hz, 1H), 7.11 (d, J = 8.8 Hz, 2H),
4.88 (s, 2H). HRMS calcd for C28H23N4O4 [M + H]+ 479.1719,
found 479.1724.
4.1.33 N-Hydroxy-4-(((1-(p-tolylamino)isoquinolin-7-yl)Ami-
1
no)methyl)benzamide 24. Yield: 82%, 326 mg. H NMR (400
MHz, DMSO) δ10.80 (s, 1H), 9.14 (s, 1H), 8.58 (s, 1H), 7.72 (d,
J = 8.2 Hz, 2H), 7.66 (d, J = 8.3 Hz, 3H), 7.52 (dd, J = 8.4, 3.7
Hz, 3H), 7.29 (s, 1H), 7.19 (d, J = 7.7 Hz, 1H), 7.10 (d, J = 8.3
Hz, 2H), 6.94 (d, J = 5.6 Hz, 1H), 6.66 (s, 1H), 4.53 (d, J = 5.9
Hz, 2H), 2.27 (s, 3H). HRMS calcd for C24H23N4O2 [M + H]+
399.1821, found 399.1827.
4.1.34 (E)-3-(4-(((1-((2-Bromo-4-methylphenyl)amino)isoqui-
nolin-7-yl)amino)methyl)phenyl)-N-hydroxyacrylamide 27c.
Yield: 68%, 341 mg. H NMR (400 MHz, DMSO) δ 10.78 (s,
1H), 9.02 (s, 1H), 8.06 (s, 1H), 7.79 (t, J = 12.9 Hz, 1H),
7.59-7.51 (m, 4H), 7.50-7.39 (m, 4H), 7.23 (d, J = 7.7 Hz, 1H),
7.17 (t, J = 7.9 Hz, 1H), 7.03 (d, J = 10.9 Hz, 1H), 6.95 (t, J = 6.9
Hz, 1H), 6.80 (d, J = 10.5 Hz, 1H), 6.42 (dd, J = 14.3, 8.0 Hz,
1H), 4.46 (d, J = 18.3 Hz, 2H), 2.30 (s, 3H). HRMS calcd for
C26H24BrN4O2 [M + H]+ 503.1083, found 503.1079.
4.2.3. Western blot analysis
20-100 µg of protein per lane was loaded onto a 10%
SDS-polyacrylamide gel and then transferred to a PVDF
membrane. The membrane was incubated with the following
primary antibodies: anti-tubulin, anti-ac tubulin, anti-actin,
anti-H3 and anti-ac H3 at 4°C overnight. Then, the membrane was
washed with TBST three times and incubated with Anti-Rabbit or
mouse IgG (H+L), DyLight 800 labeled secondary antibody for 60
min at room temperature. After three times washing with TBST,
The immunoblots were visualized by Odyssey® CLx Infrared
Imaging System (LI-COR Biosciences).
1
4.2. Biological evaluation
4.2.4 Molecular docking
Materials：
In molecular docking study, the crystal structure of HDAC2
(PDB code: 4LXZ) was prepared with the Protein Preparation
Wizard module in the Maestro program (Maestro, version 9.1;
Schrӧdinger, LLC: New York, NY, 2010) with standard Glide
protocols18. The compounds for docking were prepared with
LigPrep (LigPrep, version 2.4; Schrӧdinger, LLC: New York,
NY, 2010) and protonated with Epik (Epik, version 2.1;
Schrӧdinger, LLC: New York, NY, 2010). Finally, molecular
docking was performed with induced Fit Docking (Schrӧdinger,
LLC: New York, NY, 2010) adding metal constraints with Zn2+
and other parameters using default setting.
Cell counting kit-8 was purchased from Dojindo; anti-histone H3
primary antibody (#4499) was purchased from Cell Signaling
Technology Corporation. Anti-tubulinprimary antibody (#1878-1)
was purchased from epitomics. Anti-acetyl-histone H3primary
antibody (#06-599) was purchased from millipore.
Anti-acetyl-tubulin primary antibody (#T7451) was purchased
from Sigma-Aldrich. Both anti-Rabbit and anti-mouse IgG
(H+L), DyLight 800 labeled secondary antibodies, DyLight 800
labeled were both purchased from KPL (Kirkegaard & Perry
Laboratories, Inc). PVDF transfer membrane was purchased from
Amersham Biosciences Corporation.
4.2.5. hERG inhibition assay
4.2.1. In vitro HDAC activity assay
The hERG inhibition assays were performed in the cultured
Chinese hamster ovary (CHO) cells with the overexpression of
hERG channel protein. The cells were treated with compounds at
indicated concentrations (0.0128 µM, 0.064 µM, 0.32 µM,
1.6 µM, 8 µM and 40 µM). Cisapride served as the control. The
recordings were made at 21-23 oC using a Qpatch. The
intracellular solution contains the following components (in
mmol/L): KCl 140, CaCl2 0.1, MgCl2 1, EGTA 5, and HEPES 10
(pH=7.3 with KOH). The extra-cellular solution is made up of the
following components (in mmol/L): NaCl 145, KCl 5, CaCl2 2,
MgCl2 1, HEPES 10, and glucose 10 (pH=7.4 with NaOH).
All three full-length recombinant human HDACs (rhHDACs) 1,
3 and 6 were expressed in insect High5 cells using a baculoviral
expression system, and all His6-tagged and GST-fusion proteins
was purified using Ni-NTA (QIAGEN). The deacetylase activity
of rhHDACs 1and 3 were assayed with a HDAC substrate
(Ac-Lys-Tyr-Lys(ε-acetyl)-AMC), and HDAC6 was assayed
with another HDAC substrate(Boc-Lys(ε-acetyl)-AMC).The
total HDAC assay volume was 25 µL and all the assay
components were diluted in Hepes buffer (25 mM Hepes, 137
mM NaCl, 2.7 mM KCl and 4.9 mM MgCl2, pH ＝ 8.0). The
reaction was carried out in black 384-well plates
8

17. (a) Zhang, Y.; Yang, P.; Chou, C. J.; Liu, C.; Wang, X.; Xu, W. ACS
Med. Chem. Lett. 2013, 4, 235; (b) Pairs, M.; Porcelloni, Marina.;
Binaschi, M.; Fattori, D. J. Med. Chem. 2008, 51, 1505.
5.Achowledgements
We gratefully acknowledge financial support from the
National Natural Science Foundation of China (Grants 21021063,
91229204, 81125023, 81173033 and 81025017), National S&T
Major Projects (2012ZX09103101-072, 2012ZX09301001-005,
and 2013ZX09507-001), Chinese Academy of Science ‘‘strategic
leader in science and technology projects’’ (Grant
XDA01040303), Sponsored by Program of Shanghai Subject
Chief Scientist (Grant 12XD1407100, 13XD1404300), the
National Science & Technology Major Project "Key New Drug
Creation and Manufacturing Program" of China (Grant No.
2014ZX09507-002.
18. Friesner, R. A.; Banks, J. L.; Murphy, R. B.; Halgren, T. A.; Klicic, J.
J.; Mainz, D. T.; Repasky, M. P.; Knoll, E. H.; Shelley, M.; Perry, J. K.;
Shaw, D. E.; Francis, P.; Shenkin, P. S. J. Med. Chem. 2004, 47, 1739.
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