Discovery of Novel Class i Histone Deacetylase Inhibitors with Promising in Vitro and in Vivo Antitumor Activities

Article abstract of DOI:10.1021/acs.jmedchem.5b01044

A successful structure-based design of novel cyclic depsipeptides that selectively target class I HDAC isoforms is described. Compound 11 has an IC₅₀ of 2.78 nM for binding to the HDAC1 protein, and the prodrugs 12 and 13 also exhibit promising antiproliferative activities in the nanomolar range against various cancer cell lines. Compounds 12 and 13 show more than 20-fold selectivity toward human cancer cells over human normal cells in comparison with romidepsin (FK228), demonstrating low probability of toxic side effects. In addition, compound 13 exhibits excellent in vivo anticancer activities in a human prostate carcinoma (Du145) xenograft model with no observed toxicity. Thus, prodrug 13 has therapeutic potential as a new class of anticancer agent for further clinical translation.

Full text of DOI:10.1021/acs.jmedchem.5b01044

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Article
Discovery of Novel Class I Histone Deacetylase Inhibitors
with Promising in vitro and in vivo Antitumor Activities
yiwu yao, Zheng-Chao Tu, Chenzhong Liao, zhen wang, Shang Li, Hequan Yao, Zheng Li, and Sheng Jiang
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01044 • Publication Date (Web): 02 Sep 2015
Downloaded from http://pubs.acs.org on September 6, 2015
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Discovery of Novel Class I Histone Deacetylase Inhibitors with Promising in vitro
and in vivo Antitumor Activities
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Yiwu Yao,a Zhengchao Tu,a Chenzhong Liao,b Zhen Wang,a Shang Li,a Hequan Yao,d
Zheng Li,c,* Sheng Jianga,*
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aLaboratory of Medicinal Chemistry, Guangzhou Institute of Biomedicine and Health,
Chinese Academy of Sciences, Guangzhou 510530, China
bSchool of Medical Engineering, Hefei University of Technology, Hefei, Anhui
230009, China
cDepartment of Nanomedicine, Houston Methodist Hospital Research Institute,
Houston, Texas 77030, United States
dState Key Laboratory of Natural Medicines and Department of Medicinal Chemistry,
China Pharmaceutical University, Nanjing, 210009, China
*Corresponding
author:
jiang_sheng@gibh.ac.cn
(S.
Jiang);
or
zli@HoustonMethodist.org (Z. Li).
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Abstract
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A successful structure-based design of novel cyclic depsipeptides that selectively
target class I HDAC isoforms is described. Compound 11 has an IC50 of 2.78 nM for
binding to the HDAC1 protein and the pro-drugs 12 and 13 also exhibit promising
antiproliferative activities in the nanomolar range against various cancer cell lines.
Compounds 12 and 13 show more than 20-fold selectivity toward human cancer cells
over human normal cells in comparison with Romidepsin (FK228) demonstrating low
probability of toxic side effects. In addition, compound 13 exhibits excellent in vivo
anticancer activities in a human prostate carcinoma (Du145) xenograft model with no
observed toxicity. Thus, prodrug 13 has therapeutic potential as a new class of
anticancer agent for further clinical translation.
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Keywords
:
class I histone deacetylases, inhibitor, cyclic depsipeptides, macrocycles
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Graphical abstract
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O
N
O
O
Human cancer cell lines
Du145: GI50 = 2.55 nM
Molt-4: GI50 = 2.06 nM
U937: GI50 = 3.41 nM
N
HN
H
H
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S
SH
NH
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reduction
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in vivo
O
Human normal cell lines
HLF:
Wi38:
NH
GI50 = 4.89 nM
GI50 = 5.39 nM
O
O
HS
N
H
O
1
Romidepsin (FK228) 1
redFK228
Conformational intramolecular
constrain
cyclization
O
O
O
O
N
H
N
H
Human cancer cell lines
Du145: GI50 = 11.02 nM
Molt-4: GI50 = 7.22 nM
U937: GI50 = 10.52 nM
NH
O
NH
N
S
N
S
O
O
O
O
O
Human normal cell lines
GI50 = 321 nM
GI50 = 258 nM
R
HLF:
Wi38:
R
deletion
S
N
H
S
N
H
13
10: R = C7H15CO-
12: R = C7H15CO-
13: R = CH3CO-
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Introduction
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Traditional anticancer chemotherapeutics targeting DNA replication and cell
division are effective but have systemic side effects due to the lack of selectivity
toward cancer cells over normal cells. Development of a new generation of
compounds that selectively kill cancer cells without affecting normal tissues and have
increased potency and minimal toxic side effects is extremely urgent and necessary.1
Epigenetics is classically defined as reversible changes in gene expression that are not
a result of changes in DNA sequences such as genetic mutations or deletions. The
epigenetic regulators of gene expression are cellular support systems and include
transcription factors, RNA silencing, DNA methylation, histone modification,
chromatin remodeling and nuclear architecture. Cancer cells exploit epigenetic
processes to their advantage to support uncontrolled growth and abnormal epigenetic
silencing of tumor suppressor genes is a hallmark of many cancers.2 Currently,
targeting epigenetic misregulations, which are as important as genetic defects in the
origin of cancers have initiated a new era in cancer treatment. In particular, targeting
histone modifiers such as histone deacetylases (HDAC) to restore the expression of
tumor suppressor genes has shown clinical benefits. Histone deacetylases are a family
of enzymes that catalyze the deacetylation of lysine side chains in chromatin, and
these enzymes are involved in a wide range of biological processes, including cell
differentiation, proliferation, angiogenesis, and apoptosis.3-5
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So far, 18 members of the human HDAC family have been identified, and are
divided into four distinct classes on the basis of their size, number of catalytically
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active sites, subcellular locations, and sequence homology with respect to yeast
counterparts.6-9 Classes I HDACs (1, 2, 3, and 8), class IIa HDACs (4, 5, 7, and 9),
class IIb HDACs (6 and 10), and the class IV HDAC (11) are Zn2+-dependent
proteases, while class III HDACs (sirtuins 1-7) are NAD+ -dependent Sir2-like
deacetylases.6 Class I HDAC isoforms have been intensively studied due to their
importance in tumorigenesis and development. They are highly expressed in various
cancers, including gastric, pancreatic, colorectal, and prostate cancers and
hepatocellular carcinoma,10-13 but not in resting endothelial cells and normal organs.
Therefore, selectively targeting class I HDACs by directly inhibiting their function
has recently become a major area of research in cancer therapy.14-18
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Numerous structurally diverse HDACs inhibitors (HDACi) against Zn2+-dependent
HDACs have been exploited for a broad range of tumor indications.19,20 Currently,
over 12 HDACi are in clinical trials against different cancers,8 and four of which,
Romidepsin (FK228)21,22 (1, Figure 1), SAHA (2, Figure 1),23 Belinostat (3, Figure
1),24 and Panobinostat (4, Figure 1) 25 have been approved by the US Food and Drug
Administration (FDA) for the treatment of cutaneous T-cell lymphoma (CTCL),
peripheral T-cell lymphoma (PTCL) and multiple myeloma, respectively. Chidamide,
an HDACi developed in China, has been recently approved by the China Food and
Drug Administration for the treatment of PTCL.25
Due to the complexity and diversity of the chemical structures, macrocyclic HDAC
inhibitors such as romidepsin, spiruchostatins, thailandepsin A and largazole, were
capable of making numerous interactions with the outer rim of the enzyme and
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demonstrated excellent HDAC affinity and isoform selectivity. A few reviews
summarized natural HDAC inhibitors from the discovery to the synthesis of various
analogues.26-28 The most potent macrocyclic HDAC inhibitor is FK228
(romidepsin),29 which was isolated from the fermentation broth of Chromobacterium
Violaceum (No. 968) by Fujisawa Pharmaceutical Co., Ltd. in 1991. The total
syntheses and SAR studies of FK228 were conducted by several research groups,
providing additional insight into the biochemistry, and the toxicity of FK228.30-33
Recently, largazole, isolated from the marine cyanobacterium Symploca sp. in 2008,
showed promising HDAC1 inhibitory activity and selectivity.34 The unique structural
features and excellent biological properties of largazole have attracted significant
attention from the synthetic chemists and medicinal chemists to obtain new molecules
of higher potency or selectivity through the investigation of its structure activity
relationship (SAR).35 Mechanistic studies showed that FK228 and largazole are
prodrugs due to the fact that the 4-heptenoic acid side chain may be hydrolyzed by
cellular esterases or lipases in vitro and in vivo.33,35a The free thiol functional group
could chelate with the Zn2+ cation of the HDACs, resulting in potent inhibitory effect.
Romidepsin acts as a natural prodrug whose intramolecular disulfide bond is
reduced in vivo by glutathione reductase to produce a reduced form whose butenyl
thiol interacts with zinc ions in the binding pocket of class I histone deacetylases, thus
obstructing access of the substrate.36,37 Although HDACi, like romidepsin, showed
promising results in the treatment of cancers of the blood such as CTCL and multiple
myeloma, their application in solid tumors has been limited as a result of cardiac
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toxicity and ineffective low concentrations in solid tumors.14 For example, a phase II
study of FK228 in gastrointestinal neuroendocrine tumors was recently terminated
due to a sudden cardiac death.38
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Development of HDACi with increased selectivity toward cancer cells over normal
cells and consequent reduction of off-target toxicity is highly desirable. In this paper,
we report the design of novel cyclic depsipeptides selectively targeting class I HDAC
isoforms based on analysis of structure activity relationships (SAR) and a small
library of analogs has been synthesized. The lead compound from this library exhibits
potent class I HDAC selectivity, improved selectivity towards tumors over normal
tissue and promising therapeutic effects in a human prostate carcinoma (Du145)
xenograft model.
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O
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2: SAHA (approved in 2006)
1: Romidepsin (FK228)
(approved in 2009)
O
OH
O
N
H
O
O
H
N
S
OH
N
H
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H
HN
3: Belinostat (approved in 2014)
4: Panobinostat (approved in 2015)
R
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H
HN
HN
H
OH
O
OH
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S
S
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NH
O
NH
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MeS
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5a: Spiruchostatin A (R = Me)
5b: Spiruchostatin B (R = Et)
6: Thailandepsin A
S
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NH
N
S
O
5
O
O
O
S
N
H
7: Largazole
Figure 1. Selected HDAC inhibitors.
Results and discussion
The basic design strategy for the development of selective class I HDAC isoform
inhibitors based on FK228 is shown in Figure 2. FK228 is converted to its active form
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(redFK228) by reduction in vivo of the disulfide bond by glutathione reductase (Fig.
2). It has been shown that butenyl thiol group of redFK228 interacts reversibly with
zinc ions localized in the active site of HDACs, and its cysteine residue seems to have
a weak interaction with enzymes.36 Our hypothesis is that the toxicity of FK228 may
be caused by the relatively high nucleophilic reactivity of its cysteine residue, which
may underlie the lack of selectivity of FK228 for cancer cells over normal cells.
Accordingly, we designed a class of novel cyclic depsipeptides using a thiol
cyclization strategy which removes the cysteine residue of redFK228 and potentially
increases the selectivity toward cancer cells over normal cells thus reducing the
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toxicity associated with FK228.
O
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redFK228 8
Conformational intramolecular
constrain
cyclization
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12: R = C7H15CO-
13: R = CH3CO-
9: R = H
10: R = C7H15CO-
Figure 2. Design of a new class of conformationally constrained FK228 mimetics as
selective class I HDAC isoform inhibitors.
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Table 1. Inhibition Activities (IC50) of Designed Compounds and SAHA against
HDAC Isoforms 1-11.
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8
IC50 (μM)
9
Compd
class I
class IIa
class IIb
class IV
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HDAC1 HDAC2 HDAC3 HDAC8 HDAC4 HDAC5 HDAC7 HDAC9 HDAC6 HDAC10 HDAC11
1 (FK228)
1.05
6.07
0.062
>10
>10
>10
>10
>10
>10
>10
1.22
0.011
>10
8
0.00012 0.0014
0.00218 0.0366
0.0048
0.025
9.57
1.76
0.149
0.0039
2.36
(redFK228)
9
10
0.00977 2.55
3.15 >10
0.00767 3.55
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
2.58
>10
>10
>10
>10
>10
>10
6.11
>10
>10
>10
>10
>10
>10
3.57
0.459
>10
0.138
0.395
0.0374
1.93
>10
>10
>10
>10
>10
>10
>10
0.165
2.74
11
0.00278 0.045
0.331
6.95
12
0.158
2.71
0.328
1.01
0.76
1.68
>10
>10
>10
4.89
13
0.0216
0.0698
0.0885
0.073
0.859
0.323
4.63
0.751
2.21
Largazole
SAHA
1.85
0.0256
0.686
SAHA was used as the positive control. Values are means of three experiments, the standard error of the IC50 was generally less than 10%.
First, FK228, redFK228 and compounds 9-13 were tested against HDAC isoforms
1-11 using SAHA and largazole as the positive control. The results are outlined in
table 1 and exhibited the interesting HDACs isoform selectivity. In our fluorescence
fret-based binding assay, compound 9 strongly inhibited HDAC1, 2 or 3 with IC50
values of 2.18 nM, 36.6 nM, and 9.77 nM, respectively, more effectively than
HDAC4, 5, 7, 9 or 11 (IC50 > 10 µM), confirming that this cyclization strategy is
feasible. However, compound 9 is substantially less potent than redFK228, due to
slight conformational differences when bound to HDAC1. We masked the thiol group
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of 9 with octanoyl chloride, and the resulting prodrug 10 is less potent than 9 against
HDAC1, HDAC2 or HDAC3. In view of the disadvantage of a hydrophobic group,
we investigated the possibility of removing the isopropyl group from the 14 position
of 9, leading to the design of 11 (Figure 2). Indeed, it was determined that compound
11 has IC50 values of 2.78, 45.0 and 7.67 nM, respectively, against HDAC1, 2 and 3
(Table 1). The binding data thus confirm our assumption that the isopropyl group of 9
does not play key role in the binding with HDAC1 and consequently compound 11
represents a potent, novel lead compound for HDAC1 inhibition. In addition,
compound 11 was synthesized in much higher yield than that of compound 9.
Subsequently, acetylation of the thiol group37 in 11 led to the prodrug 13, which is 48
and 18 times more potent than FK228 against HDAC1 and 2, respectively. When the
thiol group of 11 is masked with octanoyl chloride, the resulting prodrug 12 is 6 and 2
times more potent than FK228 against HDAC1 and HDAC2. The acetyl and octanoyl
groups in prodrug 12 and 13 could make the compounds more membrane permeable
and more stable under physiological conditions than the free thiol, which would be
released by the action of a thioesterase within the cells.
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The chemical synthesis of compounds 9-13 is shown in Schemes 1 and 2. The
intermediates 16 and 17 were synthesized from t-Boc protected glycine 14 and
D-valine 15, respectively, using the modified Hantzsch procedure.38 Hydrolysis of the
ethyl ester group in 16 or 17 with 1M aqueous LiOH gave the corresponding acid.
The resulting acids were then coupled with L-threonine methyl ester under PyBOP
conditions in the presence of diisopropylethylamine to furnish dipeptides 18 and 19,
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which were mesylated, and subsequently by eliminated and saponified to provide the
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acids 20 and 21, respectively.
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Scheme 1. Synthesis of the Key Fragments 20 and 21
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O
S
1.LiOH,C2H5OH
Ref. 26
O
.
BocHN
BocHN
OH
N
2.L-Thr-OMe,PyBOP
DIPEA, DCM, 90%
O
R
R
14: R = i-Pr;
15: R = H
16: R = i-Pr;
17: R = H
O
O
S
H
N
1.MsCl,DMAP,Et3N
2.DABCO
H
N
S
BocHN
O
BocHN
OH
N
N
O
O
3. LiOH,THF,H2O,
90% (3 steps)
R
OH
R
18: R = i-Pr;
19: R = H
20: R = i-Pr;
21: R = H
The intermediate 23 was synthesized from commercially available (-)-malic acid 22
according to our previously reported method (Scheme 2).35k Swern oxidation led to
the aldehyde 23 which was subjected to Julia-Kocienski olefination coupling with the
sulfone 24 to obtain the olefin 25. For the synthesis of olefin 25, several bases (e.g.,
LiHMDS, NaHMDS, KHMDS, or LDA) and solvents (THF, DCM, DMF and toluene)
were tested at temperatures ranging from -78 °C to room temperature. We found that
utilization of NaHMDS in DMF gave the most favorable E/Z (20/1) ratio, which is
better than our previous reported method.35k After selective removal of the TBS
protecting group, condensation of 26 with Fmoc-L-valine in the presence of EDCI
and HOAt at room temperature gave intermediate 27. Removal of the Fmoc group
followed by EDC-mediated coupling to the acids 20 and 21 in different combinations
Subsequently, TFA-mediated removal of the t-Boc and 2-(trimethylsilyl)ethoxy
groups were achieved. After examining several macrocyclization conditions, 10 and
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12 were obtained in 60-80% yield, respectively, using HATU as the condensation
reagent in anhydrous DCM solution (0.7 mM). In addition, aminolysis of 10 or 12,
using aqueous NH3 or CH3CN, proceeded smoothly to afford the thiol analogue 9 and
11 in 72% and 85% yield, respectively. Acetylation of the thiol group of 11 with
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
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60
acetyl chloride under standard conditions led to the prodrug 13 in 98% yield.
Scheme 2. Synthesis of compounds 9-13
OH
O
O
TBSO
NaHMDS, 24, DMF, 80%
Ref. 27
.
HO
H
Si
O
OH
O
O
23
(-)-Malic acid 22
O
OH
O
O
OTBS
O
CSA, CH2Cl2/MeOH (1/1)
80%
Si
Si
S
O
S
5
O
5
26
25
NHFmoc
1) piperidine, MeCN,
20 min, r.t.;
Fmoc-L-valine, EDCI,
O
5
O
O
O
Si
DIPEA, CH2Cl2, r. t., 90%;
2) 20 or 21, HATU, HOAt,
DIPEA, CH2Cl2, r.t., 76-80%;
S
O
27
O
O
O
O
N
H
N
H
S
NH
NH
O
R
N
1) TFA, CH2Cl2, 24 h
N
S
O
O
O
O
5
NHBoc
2) HATU, HOAt, DIPEA,
CH2Cl2, 2 days, r.t.,
60%-80% (2 steps)
O
O
O
5
Si
S
O
S
N
H
R
28: R = i-Pr;
29: R = H
10: R = i-Pr;
12: R = H
O
O
N
H
O
O
NH
N
aq.NH3, CH3CN
25 oC, 12 h, 72%-85%
CH3COCl, Et3N
H
N
S
NH
O
O
O
O
DCM, 25 oC, 98%
N
S
O
O
O
HS
N
H
R
S
N
H
9: R = i-Pr;
11: R = H
13
O
5
O
Ph
N
S
S
N
O
N
N
24
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9
To further investigate the antiproliferative activities of these derivatives, correlate
them with their HDAC inhibitory activity and test their selectivity toward cancer cells
over normal cells, compounds 10, 12 and 13 were evaluated with CCK8 assays for
their activity in inhibition of cell growth against the human prostate carcinoma cells
Du145, the human acute lymphoblastic leukemia cells Molt-4, and the human
leukemic monocyte lymphoma cells U937, and human normal cell lines lung
fibroblast cells HLF and fibroblast-like fetal lung cells Wi38, with SAHA, largazole
and FK-228 as the positive controls. The results are summarized in Table 2.
Compounds 12 and 13 showed promising anti-proliferative activities and good cancer
cell selectivity.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
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59
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Table 2. Antiproliferative activity of SAHA, largazole, FK-228 and the designed
compounds 10, 12 and 13
GI50 (nM)
Cancer cell lines
Molt-4
Normal cell lines
Sample
Selectivity
Du145
U937
HLF
Wi38
(Du145/HLF)
FK-228
10
2.55
20.5
2.06
22
3.41
31.7
4.89
710
312
321
5.39
561
157
258
0.52
0.029
6.99
5.87
7.22
6.59
0.022
12
11.02
10.52
0.034
13
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Largazole
SAHA
11
16.2
542
16.8
39.7
82.5
0.28
0.96
4
5
6
7
1590
1460
1650
6080
8
9
b
aSAHA largazole and FK-228 were used as a positive control. Inhibition of cell
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
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39
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47
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50
51
52
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60
c
growth by the listed compounds was determined by using CCK8 assay. Values are
means of three experiments, and standard error of the GI50 was generally less than
20%.
In our cellular assay (Table 2), FK-228 exhibits very potent activities not only for
the cancer cell lines (Du145, GI50 = 2.55 nM; Molt-4, GI50 = 2.06 nM; U937, GI50 =
3.41 nM) but also for the normal cell lines (HLF, GI50 = 4.89 nM; Wi38, GI50 = 5.39
nM). Our assay showed that compound 10 is 8-11 times less potent against cancer cell
lines in comparison to FK-228 but has improved selectivity for cancer cells over
normal cells. Dramatically increased selectivity toward human cancer cells over
normal cells was observed with compounds 12 and 13. Intramolecular cyclization and
removal of the isopropyl group at the 14 position led to compound 12 (selectivity
0.022) with 22-fold and 20 fold more selective to cancer cells when compared to
FK228 (selectivity 0.52) and largazole (selectivity 0.28), respectively, with similar
inhibitory effect on cancer cell lines, on the order of nM, compared to FK228, further
confirming our design strategy. Compound 13, with an acetyl side chain, also retains
similar selectivity toward human cancer cells over normal cells with potency in the
nanomolar range against cancer cells.
To further investigate selectivity of these subtypes at the cellular level, compounds
12 and 13 were evaluated for the expression of the acetylation status of histone H3
and H4 in comparison to that of the HDAC6-specific substrate α-tubulin in U937
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cancer cell lines, which are important biomarkers associated with intracellular HDAC
inhibition. FK228 and SAHA were used as positive control. We found that both
compounds 12 and 13 have significant, dose-dependent effect on histone H3 and H4
acetylation, whereas much less effect to induce significant acetylation of α-tubulin
comparing to SAHA and FK228 confirmed that they are not very effective inhibitors
of HDAC6 (Figure 3). This result further suggests that both compounds 12 and 13 are
class I selective inhibitors.
10
11
12
13
14
15
16
17
18
19
20
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22
23
24
25
26
27
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Compd 13 Compd 12
FK228
SAHA
Control
5nM
5nM
5ꢀM
5ꢀM
5ꢀM
50nM 5ꢀM 5nM 50nM
50nM
5nM 50nM
Ac HisH3
Ac HisH4
AC-tubulin
β-actin
HisH3
Figure 3. Effect of histone H3 and H4 acetylation and α-tubulin acetylation in
cultured human lymphoma cells (U937) after 24 h treatment with compounds 12, 13,
FK228 and SAHA using Western blot analysis. β-actin and histone H3 (bottom)
serves as a control for protein loading.
Compound 13 was chosen for a further in vivo efficacy study of its high binding
affinities to class I HDACs, potent cell growth inhibitory activity and good aqueous
solubility. The human prostate cancer cell line (Du145) obtained from ATCC was
tested negative for rodent pathogens. The BALB/c nude mice (6 weeks old) were
inoculated with suspensions of 5×106 Du145 cells at the right flank. When the mean
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tumor size reached approximately 110 mm3 14 days after inoculation, compound 13
was administered via i.v. injection 5 days a week for 4 weeks at dosages of 20, 40,
and 80 mg/kg with SAHA (50 mg/kg) as a positive control. The result of tumor
growth inhibition in different groups at different time points after treatment is shown
in Figure 4. Compound 13 causes tumor growth delay in a dose-dependent manner,
such that doses of 20, 40, and 80 mg/kg/day cause reductions of 51.7%, 75.2%, and
81.2%, respectively, compared with untreated control group. The activity of 13 at the
low dosage (20 mg/kg, tumor growth inhibition (TGI) = 51.7%) was greater than that
of the control compound SAHA (50 mg/kg, TGI = 41.7%). The toxicity of 13 was
assessed by monitoring the body weight and survival of the mice. Before sacrifice, the
mouse body weights showed no significant change in different groups. All doses of 13
are well-tolerated, with no mortality or significant loss of body weight observed
during treatment (Figure 5).
10
11
12
13
14
15
16
17
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19
20
21
22
23
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25
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44
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50
51
52
53
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56
57
58
59
60
1800
1600
Vehicle
Compound 13, 20mg/kg
1400
Compound 13, 40mg/kg
Compound 13, 80mg/kg
1200
SAHA, 50mg/kg
1000
800
600
400
200
0
0
2
4
6
8
10 12 14 16 18 20 22 24 26 28 30
Days
Figure 4. Growth curve of the Du145 prostate cancer xenograft in nude mice treated
with i.v. injections of vehicle alone or compound 13 (20, 40, or 80 mg/kg) with
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SAHA (50 mg/kg) as positive control (p < 0.01). Data are expressed as the mean
4
5
tumor volume SE of the animals in each treatment group.
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
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50
51
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56
57
58
59
60
40
Vehicle
Compound 13, 20mg/kg
Compound 13, 40mg/kg
Compound 13, 80mg/kg
SAHA, 50mg/kg
30
20
10
0
0
2
4
6
8
10 12 14 16 18 20 22 24 26 28 30
Days
Figure 5. Body weight of mice during treatment with compound 13 and SAHA (p <
0.01).
Conclusion
A new class of histone deacetylase inhibitors (9-13) has been designed based upon
the thiol cyclization strategy. The lead drug candidates, compounds 12 and 13 have
good HDAC class I selectivity in the inhibition assay against HDAC isoforms and
sub-nanomolar inhibitory efficacy on cancer cell lines. These two compounds showed
improved selectivity toward human cancer cells over normal cells in comparison with
FK228, which makes them attractive lead compounds for cancer therapy. More
importantly, compound 13 exhibited potent in vivo antitumor activities with no
observed toxicity in prostate cancer Du145 xenograft model. In addition, the synthesis
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of compound 13 is well-suited to scale-up and much easier than that of FK-228,
which needs over 12 synthetic steps with low overall yield. Taken together, our
results indicate that the compound 13 is a promising new class of HDAC targeted
anticancer agents and has potential for clinical translation. Further evaluation of safety
and toxicology of compound 13 is ongoing. The strategy for the design of novel
HDAC drug candidate 13 presented in this report may accelerate further discovery of
more potent and selective anti-tumor agents.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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Experimental procedure
1. Chemistry. General. Reagents and solvents were used as purchased without
further purification. The progress of all reactions was monitored by TLC using ethyl
acetate/n-hexane as solvent system, and spots were visualized by irradiation with
ultraviolet light (254 nm). Flash chromatography was performed using silica gel
1
(300−400 mesh). H NMR and 13C NMR spectra were recorded on Bruker Avance
ARX-400 (or Bruker Avance ARX-500). The low or high resolution of ESIMS was
recorded on an Agilent 1200 HPLC-MSD mass spectrometer or Applied Biosystems
Q-STAR Elite ESI-LC-MS/MS mass spectrometer, respectively. Anhydrous solvents
were obtained as follows: THF by distillation from sodium and benzophenone;
dichloromethane, toluene and N, N- dimethylformamide from CaH2. All other
solvents were reagent grade. All moisture sensitive reactions were carried out in flame
dried flask under argon atmosphere. The purity of the final compounds was
determined by Agilent 1260 series HPLC system using the following conditions: C-18
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column (DicKma, 4.6 mm × 250 mm) with the solvent system (elution conditions:
mobile phase A consisting of MeOH; mobile phase B consisting of water containing
0.1% ammonia), with monitoring between 190 and 800 nm. A flow rate of 1.0
mL/min was used. The retention time was reported as tR (min). The purity of final
compounds is >95%.
10
11
12
13
14
15
16
17
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(R,Z)-2-(2-(1-(tert-butoxycarbonyl)-2-methylpropyl)thiazole-4-carboxamido)but
-2-enoic acid (20)
To a solution of 18 (785 mg, 1.9 mmol) in anhydrous CH2Cl2 (10 mL) was added
DMAP (46 mg, 0.4 mmol), Et3N (0.56 mL, 4 mmol), and MsCl (0.24 ml, 2.9 mmol)
o
at 0 C. After stirring for 1 h, sat aq NaHCO3 (10 mL) was added followed by
dichloromenthane (10 mL). The phase was separated and the aqueous phase extracted
with dichloromenthane (3 × 10 mL). The combined organic phases were dried over
Na2SO4, filtered, evaporated in vacuo. The result oil was dissolved in anhydrous
o
CH2Cl2 (10 ml) and cooled to 0 C, DABCO (448 mg, 4 mmol) was added. After
stirring for 30 min, the solution was warmed to rt, and stirred for 2 h. The solution
was washed with sat aq NaHCO3, saturated NaCl solution and dried over Na2SO4,
filtered, evaporated in vacuo and purified by chromatography to give a white solid. To
a solution of above solid in MeOH (10 mL) was added an aqueous solution of LiOH
o
(161 mg, 3.8 mmol) in H2O (5 ml) at 0 C. After stirring for 2 h, the solution was
neutralized by adding 1M aq HCl and evaporated in vacuo. The residue was diluted
with ethyl acetate and saturated NaCl solution, separated aqueous layer was extracted
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with ethyl acetate (3 × 10 mL). The combined organic layers dried over Na2SO4, and
evaporated to afford 20 as white solid (655 mg, 90%). [α]23D: 4.68 (c 0.83 , CHCl3).
1H NMR (400MHz, CDCl3): δ 8.68 (s, 1H), 8.01 (s, 1H), 7.00 (q, J = 6.8 Hz, 1H),
5.16 (d, J = 8.8 Hz, 1H), 4.89 (d, J = 7.2 Hz, 1H), 2.37 (brs, 1H), 1.90 (d, J = 7.2 Hz,
3H), 1.47 (s, 9H), 1.24 (m, 1H), 1.00 (d, J = 6.4 Hz, 3H), 0.94 (d, J = 6.4 Hz, 3H)
ppm. 13C NMR (125 MHz, CDCl3): δ 173.0, 167.9, 159.0, 155.5, 149.3, 135.9, 125.7,
124.0, 80.4, 58.0, 33.2, 28.3, 19.3, 17.4, 15.1 ppm. MS (EI, m/z): 383.1 [M+H]+.
HRMS (ESI) Calcd m/z for C17H25N3O5S [M+H]+ 384.1588, found 384.1586.
(Z)-2-(2-((tert-butoxycarbonyl)methyl) thiazole-4-carboxamido)but-2-enoic acid
(21)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
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1
The procedure was the same as described above. H NMR (400 MHz, MeOD-D4):
δ 8.19 (s, 1H), 6.96 (q, J = 7.2Hz, 1H), 5.56(s, 2H), 1.82 (d, J = 7.2Hz, 3H), 1.47 (s,
9H) ppm. 13C NMR (125 MHz, MeOD-D4): δ 173.0, 167.4, 161.9, 158.4, 150.0,
137.1, 128.1, 125.9, 81.2, 43.1, 28.7, 14.5 ppm. MS (EI, m/z): 341.0 [M]+. HRMS
(ESI) Calcd m/z for C14H19N3NaO5S [(M + Na)+] 364.1251, found 364.1251.
(S,
E)-2-(trimethylsilyl)ethyl
3-((S)-2-(2-((R)-1-(tert-butoxycarbonyl)-2-
methylpropyl)
thiazole-4-carboxamido)but-2-enamido)-3-methyl
butanoyloxy)-7-(octanoylthio)hept-4-enoate (28).
o
To the solution of 27 (860 mg, 1.19 mmol) in MeCN (10 mL) at 0 C was added
piperidine (0.5 mL). After stirring for 1 h at rt, the reaction mixture was carefully
quenched with sat aq NH4Cl (10 ml). The aqueous layer was extracted with ethyl
acetate (3 × 10 mL). The combined organic layer was dried over anhydrous Na2SO4,
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concentrated in vacuo, and purified by chromatography to give the amine as oil for
the next step.
7
8
9
To the solution of acid 20 (455 mg, 1.19 mmol) in CH2Cl2 (15 mL) was added
HATU (542 mg, 1.43 mmol), HOAt (194 mg, 1.43 mmol) followed by addition of the
above amine (475 mg, 0.95 mmol) in CH2Cl2 (5 mL), DIEA (0.47 mL, 2.85 mmol) at
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
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44
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46
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48
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50
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60
o
0 C. And the reaction mixture was allowed to warm to rt and stirred for 12 h. The
reaction mixture was concentrated in vacuo and subjected to flash column
1
chromatography to give 28 (783 mg, 76%) as an oil. [α]23D: -2.2 (c 0.50, CHCl3). H
NMR (400 MHz, CDCl3): δ 8.61 (s, 1H), 8.07 (s, 1H), 6.58 (m, 1H), 6.56 (m, 1H),
5.77 (m, 1H), 5.62 (q, J = 6.8 Hz, 1H), 5.50 (dd, J = 15.6, 7.2 Hz, 1H), 5.28 (brs, 1H),
4.86 (brs, 1H), 4.60 (q, J = 4.0 Hz, 1H), 4.15 (m, 2H), 2.85 (t, J = 7.2 Hz, 2H), 2.72
(dd, J = 15.6, 8.0 Hz, 1H), 2.66 (dd, J = 15.6, 5.2 Hz, 1H), 2.56 (t, J = 7.6 Hz, 2H),
2.48 (brs, 2H), 2.27 (q, J = 7.2 Hz, 2H), 2.18 (m, 1H), 1.89 (d, J = 7.2 Hz, 3H), 1.63
(m, 2H), 1.44 (s, 9H), 1.27 (m, 8H), 0.95 (t, J = 4.0 Hz, 2H), 0.90 (t, 6H), 0.87 (t, 3H),
13
0.03 (s, 9H) ppm. C NMR (125 MHz, CDCl3): δ 199.2, 173.0, 170.7, 169.5, 164.4,
159.2, 155.4, 149.2, 132.3, 129.5, 128.8, 128.7, 128.3, 123.9, 80.2, 71.8, 63.1, 60.3,
57.9, 57.1, 44.1, 39.6, 38.5, 33.1, 32.1, 31.6, 31.5, 28.8, 28.3 27.8, 25.6, 22.5, 19.3,
18.9, 17.5, 17.3, 14.1, 14.0, 13.9, 0.95 ppm. MS (EI, m/z): 867.3 [M+H]+. HRMS
(ESI) Calcd m/z for C42H70N4O9S2Si [M+H]+ 867.3519, found 867.3521.
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S-(E)-4-((7S, 10S, 14R, Z)-4-ethylidene-7, 14-diisopropyl-2, 5, 8, 12-tetraoxo-9-16
–thia-3, 6, 13, 18-tetraaza-bicyclo [13.2.1]octadec-1(17)-en-10-yl) but-3-enyl
octanethioate (10).
10
11
12
13
14
15
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17
18
19
20
21
22
23
24
25
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To a solution of 28 (693 mg, 0.8 mmol) in CH2Cl2 (10 mL) was added TFA (2 mL)
o
at 0 C, and the mixture was stirred at rt for 24 h. The reaction mixture was
concentrated in vacuo to give the crude amino acid. This amino acid was then
dissolved in DMF (800 mL), and HATU (1.52 g, 4 mmol), HOAt (0.55 g, 4 mmol)
and DIEA (1.3 mL, 8 mmol) were added to the solution successively. The reaction
mixture was stirred for 48 h at rt and then the solvent was evaporated under reduced
pressue and dissolved in ethyl acetate (20 ml). This solution was washed with sat aq
NaCl and the aqueous layer was extracted with ethyl acetate (3 × 20 mL). The
combined organic layer was dried over anhydrous Na2SO4 and evaporated in vacuo to
give an oil which was subjected to column chromatography to give 10 (311 mg, 60%)
as a white solid. [α]23D: 12.5 (c 0.30 , CHCl3). 1H NMR (400 MHz, CDCl3): δ 8.50 (s,
1H), 8.20 (s, 1H), 7.07 (q, J = 7.2 Hz, 1H), 6.36 (d, J = 10.4 Hz, 1H), 6.03 (d, J = 9.2
Hz, 1H), 5.76 (m, 1H), 5.69 (dd, J = 15.6, 7.2 Hz, 1H), 5.50 (dd, J = 15.6, 7.2 Hz, 1H),
5.35 (dd, J = 9.2, 3.6 Hz, 1H), 4.78 (dd, J = 10.4, 3.6 Hz, 1H), 2.89 (t, J = 7.2 Hz, 2H),
2.73 (d, J = 6.4 Hz, 2H), 2.52 (t, J = 7.2 Hz, 2H), 2.54 (m, 2H), 2.29 (m, 4H), 1.92 (d,
J = 7.2 Hz, 3H), 1.63 (m, 2H), 1.28 (m, 8H), 1.10 (d, J = 7.2 Hz, 3H), 0.92 (d, J = 7.2
Hz, 3H), 0.87 (t, J = 7.2 Hz, 3H), 0.78 (d, J = 7.2 Hz, 3H), 0.54 (d, J = 7.2 Hz, 3H)
ppm. 13C NMR (125 MHz, CDCl3): δ 199.4, 171.4, 169.8, 168.9, 163.3, 158.6, 147.8,
135.0, 132.6, 128.4, 127.1, 125.1, 71.8, 57.0, 56.4, 44.1, 40.8, 33.6, 32.2, 31.6, 28.9,
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27.8, 25.6, 22.6, 19.5, 18.9, 16.5, 16.2, 14.7, 14.0 ppm. MS (EI, m/z): 648.7 [M+H]+.
HRMS (ESI) Calcd m/z for C32H48N4NaO6S2 [M + Na] + 671.2907, found 671.2908.
HPLC analysis: 97.2% in purity.
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(S, E)-2-(trimethylsilyl)ethyl 3-((S)-2-(2-((Z)-2-(2-((tert-butoxycarbonyl)methyl)
thiazole-4-carboxamido)but-2-enamido)-3-methylbutanoyloxy)-7-(octanoylthio)
hept-4-enoate (29).
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To a stirred solution of 29 (3.5 g, 4.7 mmol) in MeCN (30 mL) at 0 C was added
piperidine (2 mL). After stirring for 2 h at rt, the reaction mixture was concentrated
under reduce pressure to afford the amine for the next step.
To the solution of acid 21 (1.92 g, 5.63 mmol) in CH2Cl2 (50 mL) was added
HATU (2.68 g, 7.04 mmol), HOAt (0.96 g, 7.04 mmol) at 0 oC, followed by addition
of the above amine in CH2Cl2 (10 mL), DIEA (2.33 mL, 14.07 mmol) and the reaction
mixture was allowed to warm to rt and stirred for 12 h. The reaction mixture was
concentrated in vacuo and subjected to flash column chromatography to give 29 (3.10
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g, 80%) as an oil. [α]23D: -7.57 (c 7.5, CHCl3). H NMR (400MHz, CDCl3, ppm): δ
8.60 (s, 1H), 8.10 (s, 1H), 6.60 (m, 2H), 5.77 (m, 1H), 5.62 (dd, J = 13.7, 7.2 Hz, 1H),
5.49 (dd, J = 13.7, 7.5 Hz, 1H), 5.42 (m, 1H), 4.58 (m, 3H), 4.14 (t, J = 8.6Hz, 2H),
2.85 (t, J = 7.2Hz, 2H), 2.68 (dd, J = 15.7, 7.7 Hz, 2H), 2.50 (t, J = 7.5 Hz, 2H), 2.26
(q, J = 7.1 Hz, 2H), 2.18 (m, 1H), 1.80 (d, J = 7.2Hz, 3H), 1.60 (m, 2H), 1.46 (m, 8H),
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1.26 (m, 9H), 0.96 (m, 3H), 0.86 (t, J = 6.4 Hz, 6H), 0.02 (s, 9H). C NMR (125
MHz, CDCl3): δ 199.2, 170.7, 169.6, 164.3, 159.3, 148.9, 133.3, 129.4, 129.2, 128.3,
124.8, 71.8, 63.1, 57.2, 44.1, 39.6, 38.6, 32.1, 31.6, 31.6, 28.8, 28.3, 27.8, 25.6, 22.5,
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18.9, 17.6, 17.3, 14.0, 13.9, 0.96, -1.57. MS (EI, m/z): 847 [M+Na]+. HRMS (ESI):
calcd m/z for C39H64N4NaO9S2Si [M+Na] + 847.3779, found 847.3777.
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S-(E)-4-((7S,10S,Z)-4-ethylidene-7-isopropyl-2,5,8,12-tetraoxo-9-oxa-16-thia-3,6,
13,18-tetraaza-bicyclo[13.2.1]octadec-1(17)-en-10-yl)but-3-enyl
(12).
octanethioate
To a solution of 29 (659 mg, 0.8 mmol) in CH2Cl2 (10 mL) was added TFA (2 mL)
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at 0 C, and the mixture was stirred at room temperature for 24 h. The reaction
mixture was concentrated in vacuo to give the crude amino acid. This amino acid was
then dissolved in DCM (800 mL), and HATU (1.52 g, 4 mmol), HOAt (0.55 g, 4
mmol) and DIEA (1.3 mL, 8 mmol) were added to the solution successively. The
reaction mixture was stirred for 48 h at rt and then the solvent was evaporated under
reduced pressue and dissolved in ethyl acetate (20 mL). This solution was washed
with sat aq NaCl and the aqueous layer was extracted with ethyl acetate (3 × 20 mL).
The combined organic layer was dried over anhydrous Na2SO4 and evaporated in
vacuo to give an oil which was subjected to column chromatography to give 12 (388
mg, 80%) as white solid. [α]23D: 19.2 (c 0.9, CHCl3). 1H NMR (400MHz, CDCl3):
δ 8.54 (s, 1H), 8.08 (s, 1H), 6.97 (q, J = 7.2 Hz, 1H), 6.82 (m, 1H), 6.51 (d, J = 10.2
Hz, 1H), 5.74-5.6 (m, 2H), 5.47 (dd, J = 15.5, 6.8 Hz, 1H), 5.14 (dd, J = 16, 8.2
Hz,1H), 4.70 (dd, J = 10.1, 3.2 Hz ,1H), 4.32 (dd, J = 17.4, 3.5 Hz, 1H), 2.82 (t, J =
7.2 Hz, 2H), 2.75-2.59 (m, 2H), 2.47 (t, J = 7.5 Hz, 2H), 2.24 (m, 2H), 1.82 (d, J =
7.2 Hz, 3H), 1.58 (m, 2H), 1.24 (m, 8H), 0.83 (m, 3H), 0.74 (d, J = 6.7 Hz, 3H), 0.55
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(d, J = 6.7 Hz, 3H) ppm. 13C NMR (125 MHz, CDCl3): δ 199.4, 169.7, 169.0, 167.5,
163.4, 158.8, 147.8, 134.6, 132.5, 128.2, 127.1, 124.4, 71.8, 57.0, 44.0, 40.7, 40.0,
32.1, 31.5, 28.7, 27.7, 25.5, 22.4, 18.9, 16.3, 14.5, 13.9 ppm. MS (EI, m/z): 607.1
[M+H]+. HRMS (ESI): calcd m/z for C29H42N4NaO6S2 [(M+Na)+] 629.2438, found
629.2440. HPLC analysis: 98.8% in purity.
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(7S,10S,14R,Z)-4-ethylidene-7,14-diisopropyl-10-((E)-4-mercaptobut-1-en-1-yl)-9
-oxa-16-thia-3,6,13,18-tetraazabicyclo[13.2.1]octadeca-1(17),15(18)-diene-2,5,8,12
-tetraone (9).
To a solution of 10 (40 mg, 0.06 mmol) in CH3CN (5 mL) was added aqueous NH3
(28.9%, 0.5 mL). The resulting mixture was stirred at room temperature for 18 h and
concentrated in vacuo. The residue was purified by column chromatography to afford
thiol 9 (23 mg, 72%) as a white solid. [α]23D: 19.8 (c 0.17 , CHCl3). 1H NMR (CDCl3,
400 MHz, ppm): δ 8.49 (s, 1H), 8.20 (s, 1H), 7.09 (q, J = 7.2 Hz, 1H), 6.37 (d, J =
10.0 Hz, 1H), 5.99 (d, J = 9.2 Hz, 1H), 5.83-5.69 (m, 2H), 5.56 (dd, J = 15.6, 6.8 Hz,
1H), 4.80 (dd, J = 10.4, 3.6 Hz, 1H), 4.79 (dd, J = 10.4, 3.6 Hz, 1H), 2.77 (s, 1H),
2.75 (d, J = 2.4 Hz, 1H), 2.57 (q, J = 7.2 Hz, 2H), 2.38-2.28 (m, 5H), 1.93 (d, J = 7.2
Hz, 3H), 1.38 (t, J = 8.0 Hz, 1H), 1.11 (d, J = 6.8 Hz, 3H), 0.92 (d, J = 6.8Hz, 3H),
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0.80 (d, J = 6.8Hz, 3H), 0.55 (d, J = 6.8 Hz, 3H); C NMR (125 MHz, CDCl3): δ
171.4, 169.9, 168.9, 163.3, 158.6, 147.8, 135.1, 132.4, 130.9, 128.8, 127.0, 125.1,
71.9, 57.0, 56.4, 40.9, 38.7, 36.2, 31.6, 23.0, 19.6, 19.0, 16.5, 16.2, 14.7 ppm. MS (EI,
m/z): 523.2 [M+H]+. HRMS (ESI) Calcd m/z for C24H34N4O5S2 M+ 522.1971, found
522.1973. HPLC analysis: 97.7% in purity.
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(7S,10S,Z)-4-ethylidene-7-isopropyl-10-((E)-4-mercaptobut-1-en-1-yl)-9-oxa-16-t
hia-3,6,13,18-tetraazabicyclo[13.2.1]octadeca-1(17),15(18)-diene-2,5,8,12-tetraon
e (11).
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To a solution of 12 (606 mg, 1 mmol) in CH3CN (5 mL) was added aqueous NH3
(28.9%, 0.5 mL). The resulting mixture was stirred at room temperature for 18 h and
concentrated in vacuo. The residue was purified by column chromatography to afford
thiol 11 (407 mg, 85%) as white solid. [α]23D: 15.9 (c 0.26 , CHCl3). 1H NMR (CDCl3,
400 MHz, ppm): δ 8.53 (s, 1H), 8.13 (s, 1H), 7.03 (q, J = 7.2 Hz, 1H), 6.56 (brs, 1H),
6.48 (d, J = 10.4 Hz, 1H), 5.78-5.67 (m, 2H), 5.55 (dd, J = 15.6, 7.2 Hz, 1H), 5.19 (dd,
J = 15.6, 7.2 Hz, 1H), 4.77 (dd, J = 10.4, 3.2 Hz, 1H), 4.35 (dd, J = 17.6, 4.0 Hz, 1H),
2.78-2.72 (m, 2H), 2.55 (q, J = 7.2 Hz, 2H), 2.33(q, J = 6.4 Hz, 3H), 1.88 (d, J = 7.2
Hz, 3H), 1.37 (t, J = 8.0 Hz, 1H), 0.82 (d, J = 6.8 Hz, 3H), 0.62 (d, J = 6.8 Hz, 3H);
13C NMR (125 MHz, CDCl3): δ 169.8, 168.9, 167.4, 163.4, 158.5, 148.0, 134.6, 132.4,
128.8, 127.0, 124.5, 71.7, 57.1, 40.8, 40.5, 36.1, 31.6, 23.7, 19.0, 16.5, 14.7 ppm; MS
(EI, m/z): 480.2 [M+H]+. HRMS (ESI) Calcd m/z for C21H28N4O5S2 [(M + H)+]
481.1571, found 481.1574. HPLC analysis: 98.2% in purity.
S-((E)-4-((7S,10S,Z)-4-ethylidene-7-isopropyl-2,5,8,12-tetraoxo-9-oxa-16-thia-3,6,
13,18-tetraazabicyclo[13.2.1]octadeca-1(17),15(18)-dien-10-yl)but-3-en-1-yl)
ethanethioate (13).
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To a solution of 11 (208 mg, 0.43 mmol) in CH2Cl2 (10 ml) cooled to 0 C was
added Et3N (0.1 ml, 1.3 mmol), AcCl (0.2 ml, 1.3 mmol). The reaction mixture was
stirred at room temperature for 4 h and concentrate in vacuo. The residue was purified
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by column chromatography to afford 13 (220 mg, 98%) as white solid. [α]23D: 19.6
(c 0.16 , CHCl3). 1H NMR (CDCl3, 400 MHz, ppm): δ 8.50 (s, 1H), 8.15 (s, 1H), 7.06
(q, J = 7.2 Hz, 1H), 6.41 (d, J = 10.4 Hz, 1H), 6.27 (s, 1H), 5.78-5.67 (m, 2H), 5.55
(dd, J = 15.6, 7.2 Hz, 1H), 5.26 (dd, J = 15.6, 7.2 Hz, 1H), 4.82 (dd, J = 10.4, 3.2 Hz,
1H), 4.38 (dd, J = 17.6, 4.0 Hz, 1H), 2.90 (t, J = 7.2 Hz, 2H), 2.72 (d, J = 6.4 Hz, 2H),
2.30 (m, 6H), 1.91 (d, J = 7.2 Hz, 3H), 0.82 (d, J = 6.8 Hz, 3H), 0.62 (d, J = 6.4 Hz,
3H); 13C NMR (125 MHz, CDCl3): δ 195.6, 169.7, 170.0, 167.4, 163.3, 158.5, 148.0,
134.6, 132.5, 128.3, 127.0, 124.5, 71.6, 57.0, 40.8, 40.3, 32.1, 31.6, 30.6, 28.1, 19.0,
16.4, 14.7 ppm; MS (EI, m/z): 523.2 [M+H]+. HRMS (ESI) Calcd m/z for
C23H30N4NaO6S2 [(M + Na)+] 545.1499, found 545.1499. HPLC analysis: 98.9% in
purity.
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2. Biological Assays.
In Vitro HDAC Inhibition Assay. The Purified recombinant Human HDACs 1-11
and their corresponding substrates were purchased from BPS Bioscience (BPS
Bioscience Inc., USA). The assays were carried out in 384-well format using the BPS
fluorescent-based HDAC activity assay according to the manufacturer’s protocol
(BPS Bioscience Inc., USA). The HDAC reaction mixture was composed of HDAC
assay buffer (BPS Bioscience Inc., USA), BSA, serial diluted test compounds,
appropriate concentration of HDACs, and 20 ꢀM fluorogenic substrate, the mixture
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was incubated at 37 C for 60 min, and then stopped by addition of developer
containing trypsin and TSA. After 20 min incubation, the fluorescence was detected at
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the excitation wavelength of 360 nm and the emission wave length of 460 nm using
EnVision Multilabel Reader (PerkinElmer Inc., USA). The analytical software,
GraphPad Prism 5.0 (GraphPad Software, Inc., USA) was used to generate IC50 value
via non-linear regression analysis.
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Cell-Based anti-proliferation Assay. Cell lines, DU145, Molt-4, U937, Wi38 and
HLF were purchased from Shanghai Cell Bank, Chinese Academy of Sciences. Cells
were grown and maintained in mediums, RPMI for Molt-4 and U937, HAM’S/F-12
for DU145, DMEM for HLF, and MEM for Wi38, with 10% FBS and with 1%
penicillin/streptomycin or without antibiotics (only for Wi38). All cell lines were
incubated in a Thermo/Forma Scientific CO2 Water Jacketed Incubator with 5% CO2
in air at 37 ◦C. Cell proliferation assay was determined by the CCK8 (DOjinDo, Japan)
assay. Cells were seeded at a density of 800-1000 cells/ well in 384 well plates and
treated with various concentration of compounds or solvent control. After 72 h
incubation, CCk8 reagent was added, and absorbance was measured at 450 nm using
Envision 2104 multi-label Reader (Perkin Elmer, USA). Dose response curves were
plotted to determine the IC50 values using Prism 5.0 (GraphPad Software Inc., USA).
Western blot Analysis. Human leukemic monocyte lymphoma cell lines (U937)
were cultivated in 1640 culture medium with antibiotics and 10% fetal bovine serum.
Cells were seeded at a destiny of 6×104 /well in six well plates and incubated
overnight. The U937 cells were treated with various concentrations of compounds 13,
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12, FK228 and SAHA for 24h at 37 C. After treatment, the cells were washed,
harvested and lysed. The 10 ꢀg total proteins for each sample were loaded on 15%
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