Design and optimization of novel hydroxamate-based histone deacetylase inhibitors of bis-substituted aromatic amides bearing potent activities against tumor growth and metastasis

Article abstract of DOI:10.1021/jm5012148

Histone deacetylases (HDACs) are one of the most promising drug targets for cancer therapy, and since more than 90% of all cancer-related deaths are associated with tumor metastasis, developing strategies to inhibit tumor metastasis while retaining anti-t

Full text of DOI:10.1021/jm5012148

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Article
Design and Optimization of Novel Hydroxamate-Based
HDAC Inhibitors of Bis-substituted Aromatic Amides Bearing
Potent Activities against Tumor Growth and Metastasis
Feifei Yang, Tao Zhang, Haigang Wu, Yang Yang, Ning Liu, Ang Chen, Qiang Li, Jingjie Li,
Liwen Qin, Beier Jiang, XIN WANG, Xiufeng Pang, Zhengfang Yi, Mingyao Liu, and Yihua Chen
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm5012148 • Publication Date (Web): 31 Oct 2014
Downloaded from http://pubs.acs.org on November 5, 2014
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Design and Optimization of Novel Hydroxamateꢀ
Based HDAC Inhibitors of Bisꢀsubstituted Aromatic
Amides Bearing Potent Activities against Tumor
Growth and Metastasis
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Feifei Yang,†, # Tao Zhang,†, # Haigang Wu,† Yang Yang,† Ning Liu,† Ang Chen,† Qiang Li,†
Jingjie Li,† Liwen Qin,† Beier Jiang,† Xin Wang,† Xiufeng Pang,† Zhengfang Yi,†,* Mingyao
Liu†,* and Yihua Chen†,*
†Shanghai Key Laboratory of Regulatory Biology, The Institute of Biomedical Sciences and
School of Life Sciences, East China Normal University, Shanghai, 200241, China.
ABSTRACT HDACs are one of the most promising drug targets for cancer therapy, and since
more than 90% of all cancerꢀrelated deaths are associated with tumor metastasis, therefore
developing strategies to inhibit tumor metastasis while retaining antitumor growth activity are of
great interest. Herein we demonstrated the design and identification of a series of novel
hydroxamateꢀbased HDAC inhibitors bearing potent activities against tumor growth and
metastasis. Optimization of the initial hit resulted in the discovery of new HDAC inhibitors
through studying the SAR. Among them, compound 11b, one of the most potent leads, exhibited
nanomolar IC50 values towards inhibition of Class I and IIb HDACs as well as submicromolar
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activity against proliferation and migration of breast cancer cells in vitro. More importantly, it
also significantly suppressed tumor growth in a breast tumor xenograft mouse model and doseꢀ
dependently blocked in vivo tumor metastasis in a mouse pulmonary metastasis model.
■ INTRODUCTION
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Posttranscriptional modifications of histone play a pivotal role in the epigenetic regulation of
gene expression.1, 2 Among them, the histone acetylation process, as one of the most important
and thoroughly investigated modes of regulation, is mediated by two counteracting enzyme
families: histone acetyl transferases (HATs) and histone deacetylases (HDACs), which maintain
the balance between the acetylated and deacetylated states of histones.3, 4 HATs add acetyl groups
to lysine residues and acetylated histones adapt an open and transcriptionally active chromatin
structure, whereas HDACs remove acetyl groups and cause chromatin condensation and
transcriptional repression.5ꢀ8 18 known human HDAC isoforms are classified into four classes on
the basis of structural characteristics and catalytic mechanisms, among which HDACs 1ꢀ11 are
zinc dependent enzymes,9 subdivided among class I (HDAC1, 2, 3 and 8), class IIa (HDAC4, 5,
7 and 9), class IIb (HDAC6 and 10), and class IV (HDAC11).10, 11 SIRT1ꢀ7, the class III HDACs,
are zincꢀindependent and NAD+ꢀdependent.12, 13 HDACs have key effects on numerous cellular
functions such as cell proliferation, differentiation, apoptosis and angiogenesis.8 Dysregulation of
HDACs is responsible for the pathogenesis of many diseases’ pathogenesis, cancer being one of
the most clinically challenging ones.14, 15 Many HDACs, especially Class I HDACs, are found to
be overexpressed in various human cancers and associated with tumorigenesis and tumor
development, therefore HDACs are regarded as one of the most promising drug targets for novel
cancer therapies.9, 13, 16, 17 To date, several kinds of HDAC inhibitors have been reported,
including shortꢀchain fatty acids, benzamides, epoxyketones, hydroxamic acid and macrocyclic
peptides.17ꢀ20 Suberoylanilide hydroxamic acid 1 (SAHA, Vorinostat, Merck Research
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Laboratories, Figure 1) was the first HDAC inhibitor approved by the FDA for the treatment of
cutaneous Tꢀcell lymphoma (CTCL) in 2006.21, 22 The microbial natural product 2 (FK228,
Romidepsin, Celgene, Figure 1) was subsequently approved for CTCL and peripheral Tꢀcell
lymphoma (PTCL) in 2009 and 2011 respectively.11, 13 And on July 3, 2014 the FDA approved
the third HDAC inhibitor, another hydroxamic acid 3 (Belinostat, Beleodaq, Spectrum Pharma,
Inc, Figure 1), for the treatment of relapsed or refractory PTCL.23 In addition, over 20 HDAC
inhibitors are now being developed as drug candidates against various cancers under different
stages of clinical trials either as a single agent or in combination therapies.24 All of the clinical
progress validates the principle that potent HDAC inhibitors, especially hydroxamateꢀbased
analogues, could be accepted as novel anticancer drug candidates.13, 24 However, most of the
current HDAC inhibitors show lack of visible efficacy against solid tumors despite the promising
results in the treatment of CTCL.25 In view of the fact that tumor metastasis is responsible for the
death of above 90% of solid cancer patients mortality,26ꢀ28 our top strategy for discovering novel
multifunctional HDAC inhibitors is to design and synthesize novel HDAC inhibitors combining
elements of typical HDAC inhibitors and other pharmacophores to achieve both deep inhibition
of metastasis and high potency against solid tumors.
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At present, two main strategies (developing highly selective HDAC inhibitors or panꢀHDAC
ones) exist for the discovery of novel HDAC inhibitors in the field of cancer treatment.13
Although the selective HDAC inhibitors were supposed to produce fewer undesirable side effects,
their therapeutic advantages have not yet been confirmed in clinical research.11, 29 Most of the
HDAC inhibitors approved or in clinical trials are panꢀHDAC inhibitors and HDACs regulate not
only the acetylation of histone proteins but also nonꢀhistone proteins, suggesting potential
problems with specificity.25 Whether or not the selective HDAC inhibitors showed improved
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therapeutic effects and decreased toxicities compared to panꢀHDAC inhibitors, especially for
those candidates with excellent potency, is still under investigation.11, 13, 29
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The common structural features of classic HDAC inhibitors consist of three domains: a zincꢀ
binding group (ZBG), a CAP group and a linker.30, 31 Given the topological differences and
diverse amino acids sequences present in the CAP regions of HDACs,25 as well as the large size
of the hydrophobic CAP that probably contributes to the high inhibitory potency against
HDACs,8 focused structural modifications through targeting the CAP region are one of the
successful and effective strategies in the design of novel HDAC inhibitors.32 The actinꢀrelated
protein (Arp) 2/3 complex, as a sevenꢀsubunit protein, directly regulates polymerization of actin
filaments which support various types of cellular movements.33 A small molecule 5 (CKꢀ548)
was reported to insert into the hydrophobic core of Arp3, which would alter its conformation and
suppress cellular movement (Figure 2A).33, 34 In order to retain the antiꢀproliferative and antiꢀ
metastatic activities in one synthetic small molecule, the scaffold of compound 5 was
incorporated into the design of the CAP region for the nextꢀgeneration of HDAC inhibitors. The
thiazolidinone ring of compound 5 was “scissored” at the CꢀS site, and then the thiol group was
replaced by the more common hydroxamate at the end of the linker as the ZBG. Thus a novel
scaffold of HDAC inhibitor of bisꢀsubstituted aromatic amides was developed and endowed with
excellent antiꢀproliferation and antiꢀmigration activities (Figure 2A). Moreover, several novel
effective HDAC inhibitors were validated and investigated with multiꢀsubstitution on the
nitrogen atom such as tertiary amines (6) or bisꢀamides (7) as the cap groups in recent years
(Figure 2B), which demonstrated that the two aromatic regions can form the necessary
hydrophobic interactions with the external areas of the enzymes and result in increasing
inhibitory activities.8, 35ꢀ38 All these research findings also support the rationality of our design
strategy to develop novel hydroxamateꢀbased HDAC inhibitors. In the present study, a series of
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novel hydroxamateꢀbased HDAC inhibitors of bisꢀsubstituted aromatic amide derivatives was
synthesized and tested for high potency of HDAC inhibition, blocking cancer cell proliferation
and migration, and inhibiting tumor growth and metastasis through inꢀdepth study of the
structureꢀactivity relationship (SAR). Many of these compounds, especially 11b, had much better
inhibitory activities in vitro and in vivo compared to compound 1.
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■ CHEMISTRY
Compounds 8a–e, 9a–f, 10a–i and 11a were prepared by following the procedures described
in Scheme 1. The intermediates of secondary amines were constructed from substituted anilines
with a series of aromatic aldehydes through reductive amination, which were further condensed
with different anhydrides in 1,4ꢀdioxane to yield acids according to procedure c. Esterification of
these acids in methanol at the presence of catalytic amounts of thionyl chloride generated the
corresponding esters, which were at last stirred with the freshly prepared hydroxylamine in
methanol at room temperature for 1 h to generally give the desired hydroxamates 8a–e, 9a–f,
10a–i and 11a.39
Scheme 1. Synthesis of compounds 8a–e, 9a–f, 10a–i and 11aa.
Ar
Ar
Ar
R
NH
2
NH
N
OH
N
OCH
3
a, b
c
d
n
O
n
O
O
O
R
R
R
Br
Br
Ar
N
NHOH
e
5
O
or
or
N
NHOH
N
NHOH
O
n
5
OMe
O
O
O
O
OCH
3
R
8aꢀe
9aꢀf, 11a
10aꢀi
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aReagents and conditions: (a) ArCHO, EtOH, 0 C then to rt; (b) NaBH4, 0 C then to rt; (c)
anhydride, 1,4ꢀdioxane, reflux; (d) MeOH, Cat.SOCl2, reflux; (e) NH2OH·HCl, KOH, MeOH.
Compounds 9g–i and 11b were obtained according to the procedures described in Scheme 2. The
key intermediate esters were synthesized using similar protocols as outlined in Scheme 1.
Subsequent transformations were obtained by Suzuki coupling reactions of the intermediates
with different arylboronic acids in the presence of Pd(dba)2 under the corresponding conditions
(procedure e).40, 41 At last, the desired compounds 9g–i and 11b were achieved by transforming
ester to hydroxamate group as usual.
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Scheme 2. Synthesis of Compounds 9g–i and 11ba
Br
Br
S
S
a, b
c
NH
OMe
N
OH
CHO
5
O
S
Br
O
OMe
d
R2
R2
Br
S
S
S
e
N
NHOH
f
N
OCH3
N
OCH3
5
O
5
O
5
O
O
O
O
OMe
OMe
OMe
9g-i, 11b
aReagents and conditions: (a) substituted aniline, EtOH, 0 oC then to rt; (b) NaBH4, 0 oC then to
rt; (c) pimelic acid anhydride, 1,4ꢀdioxane, reflux; (d) MeOH, Cat. SOCl2, reflux; (e) Pd(dba)2,
PPh3, K3PO4, 1,4ꢀdioxane/H2O; (f) NH2OH·HCl, KOH, MeOH.
■ RESULTS AND DISCUSSION
HDAC Inhibition Assay. All the target compounds were initially measured for their HDAC
inhibitory activities against HeLa cell nuclear extract (which mainly contains HDACs 1 and 2),7
and compound 1 (SAHA) was selected as a positive control. For insight into validating the
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structureꢀactivity relationship between the linker length of this scaffold and HDAC inhibitory
activities, compounds 8a–e were first synthesized and then evaluated through testing their IC50
values against the nuclear extract, as shown in Table 1. Being consistent with classical HDAC
inhibitors, this new series of compounds indicated a linkerꢀlengthꢀdependent inhibition of nuclear
extract HDACs. Nevertheless, compound 8d, containing a fiveꢀmethylene linker instead of the
sixꢀmethylene group existed in typical HDAC inhibitors in the linker part, was the most active
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analogue with an IC50 value of 0.80
ꢀM, which revealed that the variation in the cap zone
probably exerted some influence on the effect of linker length on HDAC inhibition. From the
docking assays, it was indicated the structural difference of binding mode with active domain of
HDAC2 or 8 existed between this series of compounds and 1, which partially explained the
possible reasons that the linker of this novel hydroxamateꢀbased HDAC inhibitors of bisꢀ
substituted aromatic amides were fiveꢀmethene whereas 1 was 6ꢀmethylene toward retaining
their high potency, as shown in Figure S1 and S2 in supporting information. Similar results with
fiveꢀmethylene linker were also observed in other reported HDAC inhibitors with a macrocyclic
scaffold.8, 42
Table 1. IC50 values of HDAC Inhibition of compounds 8a–e
Br
N
NHOH
n
O
O
OCH3
Nuclear extract
IC50 ( M)
Compd.
n
ꢀ
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>1
>1
8a
8b
>1
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8d
0.80
>1
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8e
6
0.49
1 (SAHA)
Next we focused our interest on modifications of the cap group to enhance HDAC inhibition.
First, we replaced the 3ꢀbromophenyl (A ring, Figure 2A) with different heteroaromatic rings.
The HDAC inhibitory activities of 3ꢀpyridinyl analogue (9a) and 6ꢀbromoꢀ2ꢀpyridinyl analogue
(9b) were decreased compared to 8d, while compounds with bromofuranyl (9c) or
bromothiopheneyl (9d–e) displayed comparable inhibitions in comparison to 8d. Thus the
thiopheneyl group was retained in subsequent structural modifications and the bromo atom was
further converted to pyridinyl (9g–h) or pyrimidinyl (9i) via palladium catalyzed coupling
reactions. Introduction of a chlorine atom to the 5ꢀposition of thiophene (9f) seemed to slightly
decrease the inhibitory activity compared to 9d. Interestingly, compound 9i with replacement of
bromo by pyrimidinyl at 5ꢀposition of thiopheneyl increased potency to almost threeꢀfold that of
9d and twice that of 1. These results indicated that the heteroaromatic groups such as
pyrimidinylthiopheneyl were more suitable for enhancing inhibitory activities among the
investigated compounds.
Table 2. HDAC Inhibition Activity of compounds 9a–i
R
1
N
NHOH
O
O
OMe
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Compd.
R1
Nuclear extract Compd.
R1
Nuclear extract
IC50 ( M)
IC50 (ꢀM)
ꢀ
6
7
8
9
3ꢀpyridinyl
>1
>1
5ꢀchlorothiopheneyl
5ꢀ(3ꢀpyridinyl)thiopheneyl
5ꢀ(4ꢀpyridinyl)thiopheneyl
5ꢀ(5ꢀpyrimidinyl)thiopheneyl
ꢀꢀꢀ
0.81
0.67
1.00
0.20
0.49
9a
9b
9c
9d
9e
9f
9g
9h
9i
1
6ꢀbromopyridinyl
5ꢀbromofuranyl
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0.69
0.63
0.65
5ꢀbromothiopheneyl
4ꢀbromothiopheneyl
Substitutions of the B ring of the cap group (Figure 2A) were also meantime investigated. A
methoxy (8d, 10b–c) and a chloro group (10d–f) were examined at different positions of the
phenyl ring as well as no substitution (10a) in order to explore the best substitution position, as
displayed in Table 3. The results demonstrated that the substitution with MeO– or Cl– in the B
ring (10b–f) was superior to no substitution (10a). And more importantly, compound efficacy
was affected significantly by the substituted positions. Compounds with p–substitution (10c and
10f) showed the most potent activities among the three investigated ones (o–, m– and p–
positions). Furthermore, compound (10c) with p–OMe substitution was approximately 5ꢀfold
more active than compound 1, and then diverse substitutions on the pꢀposition were naturally
carried out for subsequent structural optimization against HDACs. When the methoxy of 10c was
replaced with electron withdrawing groups, such as –F and –Br, it resulted in a decrease in
inhibitory activities. However, compound 10i with iodide atom at the p–position was the
exception with the most potent nuclear extract inhibition activity compared to other investigated
halogenꢀsubstituted compounds. In light of the results derived from the A ring based on previous
research, two compounds (11a–b) were chosen to be synthesized and evaluated, and a dramatic
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increase of potency was observed in compound 11b, with an IC50 of 0.12
potent than 1.
ꢀM, almost 4ꢀfold more
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8
9
Table 3. HDAC Inhibition Activity of compounds 10a–i
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R3
N
NHOH
O
O
2'
R2
4'
3'
Compd.
R2
R3
Nuclear extract Compd.
IC50 ( M)
R2
R3
Nuclear extract
ꢀ
IC50 (ꢀM)
ꢀH
3ꢀBrꢀPh
>1
4’– F
3ꢀBrꢀPh
3ꢀBrꢀPh
3ꢀBrꢀPh
>1
10g
10h
10i
10a
8d
2’–OMe 3ꢀBrꢀPh
3’–OMe 3ꢀBrꢀPh
0.80
0.66
4’–Br
4’–I
0.50
0.035
0.20
10b
10c
5ꢀbromoꢀ
thiopheneyl
4’–OMe 3ꢀBrꢀPh
0.11
0.90
4’–OMe
4’–OMe
11a
5ꢀ(5ꢀpyrimidinyl)ꢀ
thiopheneyl
2’–Cl
3ꢀBrꢀPh
0.12
10d
11b
1
3’–Cl
4’–Cl
3ꢀBrꢀPh
3ꢀBrꢀPh
0.79
0.40
ꢀꢀꢀ
ꢀꢀꢀ
ꢀꢀꢀ
ꢀꢀꢀ
0.49
ꢀꢀꢀ
10e
10f
ꢀꢀꢀ
Determination of potent compound in vitro inhibition activity (IC50) toward HDACs
isoforms.
Based on our previous results with highly potent inhibition of nuclear extract HDACs,
compounds 10c, 10i, 11a and 11b were chosen for testing their capabilities against HDACs 1ꢀ11
in order to elucidate the selectivity of this series of compounds against HDAC isoforms.7 The
results indicated that these compounds were panꢀHDAC inhibitors, which were similar to
compound 1. And all four of the compounds showed more potent inhibition against Class I and
IIb HDAC isoforms than class IV and IIa ones, especially for 11b. It showed about 5ꢀ10 fold
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enhanced potency against Class I HDACs (which are mainly associated with cancer and
inflammation) compared to 1.6, 43 The similar results of hydroxamateꢀbased HDAC inhibitors
with poor activities against Class IIa HDACs were also observed by other research groups.12 In
addition, this series of compounds have shown progressive isoformꢀselectivity for HDAC1, 2 and
3 over HDAC8.
Table 4. In vitro inhibition activity (IC50) of tested compounds on different HDACs isoforms
IC50 (nM) for HDACs
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Classes
isoforms
10c
Class I
Class IIa
Class IIb
Class IV
11
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2
3
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7
9
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10
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69
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4.6
2.2
5.0
1.8
11.2
5.5
4.5
4.4
2.8
15
5.3
81.0 > 10000 > 10000 > 10000 >10000 11.7
2308
1104
2558
3339
35540
1.2 125.7 > 10000 > 10000 > 10000 >10000 6.2
4.4 316.6 > 10000 > 10000 > 10000 >10000 10.5
10i
11a
2.2
20.1 > 10000 > 10000 > 10000 >10000 8.2
ND ND ND ND
11b
20.2 172.2
13.5 175
1
Hydroxamateꢀbased HDAC inhibitors of bisꢀsubstituted aromatic amides suppress
proliferation of breast cancer cells in vitro.
Breast cancer is one of the most common cancers and is responsible for about 29% of new
cases and 15% of deaths among women in the United States in 2014.44 Therefore the four
compounds with high potency in HDACs inhibitions were further investigated against a panel of
breast cancer cell lines (MDAꢀMBꢀ231, 4T1, MCFꢀ7, T47D, HCC1937, MDAꢀMBꢀ468, BTꢀ549
and Bcap37) for measuring their antiꢀproliferative effects via MTT assay. Their respective IC50
values against these cancer cell lines were summarized in Table 5, and compound 1 was also
used as positive control. The results showed that most compounds exhibited potent antiꢀ
proliferation efficacy with IC50 values ranging from 0.18–4.01 ꢀM, which were consistent with
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their antiꢀHDACs activities. These compounds (10c, 10i, and 11a–b) have better IC50 values than
1, especially for compound 11b, which was the most active analogue with almost tenꢀfold more
potency than 1.
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Table 5. Antiꢀproliferative activities of representative compounds against different breast
cancer cell lines
IC50 (ꢀM)
Compd.
MDAꢀMBꢀ231 4T1 MCFꢀ7 T47D HCC1937 MDAꢀMBꢀ468 BTꢀ549 Bcap37
1.30
1.76
1.99
0.48
4.10
0.53
0.82
0.59
0.18
2.85
2.12
2.34
3.13
0.53
4.47
1.55
1.52
2.33
0.48
3.25
4.01
2.53
3.21
1.25
> 10
2.73
2.34
3.25
0.42
6.04
2.35
1.82
2.99
0.92
> 10
2.91
2.64
1.46
1.31
6.14
10c
10i
11a
11b
1
Efficacy of compounds on tumor cell acetylated histone levels.
Global hypoꢀacetylation of histone H4 has been reported as a common hallmark of human
tumors occurring early in the tumorigenic process.35 The anticancer effect of HDAC inhibitors
could be also assessed by their potency in inducing hyperꢀacetylation of histone H3 or H4.9, 45 To
further confirm the HDAC inhibitory activity of these compounds in cell based assays, three
potent compounds were chosen to examine their efficacy on histone acetylation in MDAꢀMBꢀ
231 cells. As shown in Figure 3, the tumor cells were cultured with selected compounds at two
different concentrations (0.2 and 0.5 ꢀM), and the results demonstrated that compounds 10c, 11a,
and 11b significantly upregulated the level of acetylated histone H3 and H4 in a doseꢀdependent
manner compared to 1. As expected, compound 11b displayed the most potent activity to induce
histone acetylation among the investigated compounds.
Hydroxamateꢀbased HDAC inhibitors with bisꢀsubstituted aromatic amides suppress
metastatic activities of breast cancer cells in vitro.
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In order to develop a broad spectrum of novel HDAC inhibitors usable as cancer therapeutics,
especially for metastatic or advanced cancer, it is valuable to investigate the effect of these
compounds on cell migration. This is also in accordance with our initial objective of developing
next generation HDAC inhibitors with high potency against cell proliferation as well as
migration. We evaluated four compounds with the best antiꢀproliferative activities and HDAC
inhibitions potencies to determine their antiꢀmetastatic effects in MDAꢀMBꢀ231 and 4T1 cell
lines, as shown in Table 6 and Figure 4. We chose 1 as a positive control once again. In wound
healing migration assay and invasion assay, the results showed that these four compounds
decreased MDAꢀMBꢀ231 cell migration and invasion in a doseꢀdependent manner (Figure 4A
and 4B). We observed similar results were observed in the 4T1 cell line. The IC50 values of 10c,
10i, 11a and 11b were listed in Table 6, and we found that 11b was about 6ꢀ to 11ꢀfold more
potent than 1 either in migration or invasion models.
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Table 6. Antiꢀmetastatic Activities of Representative Compounds
IC50 (ꢀM) of Migration assay
IC50 (ꢀM) of Invasion assay
Compd.
MDAꢀMBꢀ231
1.81
4T1
3.16
2.74
1.74
1.00
6.02
MDAꢀMBꢀ231
1.34
4T1
0.74
0.95
0.97
0.81
9.20
10c
10i
11a
11b
1
1.73
1.56
0.83
1.37
1.14
0.90
10.60
9.80
To understand the possible mechanism of these compounds on cell invasion, we chose to
determine the potential downstream effectors of compound 11b against tumor metastasis.
Paxillin plays a critical role in tumor metastasis related behaviors, such as tumor cell morphology,
cell migration and invasion.46, 47 Our immunofluorescence analysis showed that 11b significantly
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downregulated paxillin expression (Figure 5A). In addition, EpithelialꢀMesenchymalꢀtransition
(EMT), a process involves the adherent epithelial cells to a migratory mesenchymal state, has
been widely considered to be essential in tumor metastasis in preclinical models.48 Western blot
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analysis revealed that an upregulation of epithelial markers (EꢀCadherin and βꢀcatenin) and a
downregulation of mesenchymal marker (vimentin) were observed after treatment with 11b
(Figure 5B). Previous studies reported that HDAC inhibitors inhibit cell motility via modulating
tubulin acetylation.49 Compound 11b was found to significantly induce the acetylation level of
tubulin (Figure 5C). Matrix metalloproteinases (MMPs) also play key roles in tumor invasion
and metastasis and their natural inhibitors (tissue inhibitor of metalloproteinase 1: TIMPs) can
decrease tumor cell motility.50 Our data showed that 11b reduced MMP2 and MMP9 expression,
whereas it increased TIMP1 and TIMP2 expression (Figure 5D). These results implied that our
target compound may inhibit tumor metastasis via regulating the expression of key tumor
metastasis related proteins (paxillin, MMP2, MMP9, TIMP1 and TIMP2 etc.) and modulating
EMT.
Preliminary pharmacokinetic studies of compound 11b in vivo.
To characterize the preclinical pharmacokinetic profiles of 11b in rats after oral and
intravenous administration, an analysis of the concentration of 11b was performed in rat plasma
and pharmacokinetic parameters were calculated, as shown in Table 7. The t1/2 values of oral
administration of 30 mg/kg 11b and intravenous administration of 5.0 mg/kg 11b were 128.23 ±
33.58 min and 33.21 ± 9.38 min, respectively. The difference between them may be due to slow
dissolution and absorption rate of 11b, irregular gastric emptying, as well as enterohepatic
circulation which might also be responsible for the doubleꢀpeak phenomenon in the mean plasma
concentrationꢀtime curve of oral administration.51 Indeed, the mean values of apparent volume of
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distribution (Vd) were extremely high for both oral and intravenous administration, indicating
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that 11b was widely distributed in tissues.
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Table 7. Pharmacokinetics of parameters of 11b after oral administration or intravenous
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injection in rats (mean ± SD)
p.o. (n = 3)
30
i.v. (n = 5)
5.0
Dose (mg/kg)
Cmax (nM)
189.60 ± 43.67
28.34 ± 17.55
Tmax (min)
10.78 ± 2.38
33.21 ± 9.38
146.46 ± 11.86
146.60 ± 11.82
3.62 ± 1.11
C0 (ꢀM)
T
1/2 (min)
128.23 ± 33.58
22.54 ± 11.22
22.90 ± 11.28
644.23 ± 387.93
3.31 ± 1.32
AUC0~t
AUC0~∞
(
ꢀ
M·min)
(
ꢀM·min)
Vd (L/kg)
CL (L/min/kg)
MRT (min)
0.08 ± 0.01
133.92 ± 17.36
17.97 ± 5.72
Hydroxamateꢀbased HDAC inhibitor 11b blocks breast cancer growth and metastasis in
vivo.
Because compound 11b showed the best inhibition of HDAC activity as well as antiꢀ
proliferative and antiꢀmigratory effects on different breast cancer cells in vitro, we investigated
the preliminary anticancer efficacy of compound 11b in vivo.
A mouse MDAꢀMBꢀ231 xenograft model was established to investigate its antiꢀtumor
growth activity. Following injection of 1x 106 MDAꢀMBꢀ231ꢀluc cells into the #4 mammary
gland, mice were dosed intraperitoneally with either 11b (10 mg/kg/day or 30 mg/kg/day) or 1
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(30 mg/kg/day) for 30 days. As presented in Figure 6, compound 11b significantly suppressed the
tumor size and weight in a doseꢀdependent manner. In addition, compound 11b also showed
better antitumor growth efficacy in vivo compared to 1.
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In order to further verify the antiꢀmetastatic activity of our compounds in vivo, a mouse
pulmonary metastasis model using MDAꢀMBꢀ231ꢀluciferase was used. Mice were randomly
assigned into four groups according to the dosing regimen: control group, 1 (30 mg/kg/day), and
11b (10 mg/kg/day or 30 mg/kg/day). Tested compounds were administered intraperitoneally
during treatment for 30 consecutive days. Compound 11b showed statistically significant antiꢀ
metastatic activity and more potent antiꢀmetastatic activity compared to 1 even at the lower
dosage (Figure 7A and 7B). In addition, no significant difference in body weight or obvious signs
of evident toxicity were detected among these groups of mice during the treatment (Figure 7C).
Taken together, all these results validated our rationale for designing and synthesis of novel
HDAC inhibitors bearing potent activities against tumor growth and metastasis.
■ CONCLUSION
With the aim of developing a new generation of HDAC inhibitors with potent activities
against tumor growth and metastasis, a series of novel hydroxamateꢀbased HDAC inhibitors of
bisꢀsubstituted aromatic amides was designed, synthesized and modified. Some of the
compounds exhibited nanomolar IC50 values in inhibiting class I and IIb HDACs in vitro as well
as submicro molar grade activity in blocking cancer cell proliferation and migration. In vitro
potency was related to linker length and compounds containing a fiveꢀmethylene in the linker
part were the most active among the investigated analogues. Testing further modifications of the
cap region indicated that aromatic or heteroaromatic residues could contribute to stronger
hydrophobic interactions with HDAC enzymes, which was in consistent with the conclusion that
aromatic rings in the cap regions were appeared to be optimal for compound activities.8 Detailed
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SAR analysis also revealed that the inhibitory potency could be further increased by pꢀ
substitution on the phenyl and the two linked heteroaromatic rings. For this series of
hydroxamateꢀbased HDAC inhibitors, the branched cap region of our pharmacophore contained a
pꢀsubstituted phenyl and two linked heteroaromatic groups which formed the necessary
hydrophobic interactions between inhibitors and enzymes, and therefore was responsible for the
efficacy of the target compounds. Furthermore, one compound, 11b, showed much better efficacy
compared to that of the known HDAC inhibitor 1 in inhibiting xenograft tumor growth in vivo.
And more importantly, our compound 11b also possessed significant antiꢀmetastatic activity at
the dose of 10 mg/kg/day (i.p). All of our above results revealed that these new generation
hydroxamateꢀbased, bisꢀsubstituted aromatic amide HDAC inhibitors could be developed as
novel potential antitumor growth and metastasis agents against breast cancer growth and
metastasis in the future.
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■ EXPERIMENTAL SECTION
General Methods for Chemistry. Reagents were purchased from Adamasꢀbeta Ltd, Sigmaꢀ
Aldrich Inc., J&K Inc., or Aladdinꢀreagents Inc., and used without further purification unless
otherwise specified. All reactions were carried out with the use of standard techniques under an
1
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inert atmosphere (Ar or N2). H and C NMR spectra were generated on a Varian 300 MHz or
Bruker 400 or 500 MHz instruments and obtained as CDCl3 or DMSOꢀd6 solutions (reported in
ppm), using CDCl3 as the reference standard (7.26 and 77.00 ppm) or DMSOꢀd6 (2.50 and 39.51
ppm). Coupling constants (J) were reported in Hertz (Hz). Standard abbreviations indicating spin
multiplicities were given as follow: s (singlet), d (doublet), t (triplet), q (quartet), br (broad) or m
(multiplet). High resolution mass spectra were gathered on a Bruker MicroTOFꢀQ II LCMS
instrument operating in electrospray ionization (ESI). HPLC (Agilent Technologies 1200 Series)
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was employed for purity determination, using the following method: Eclipse XDB C18 column,
5
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7
5 ꢀ
m, 4.6 mm×150 mm, column temperature 40 oC; solvent A: water; solvent B: methanol;
8
9
gradient of 40−70% B (0−10 min), 70−90% B (10−15 min), 90−40% B (15−20 min); flow rate
of 1.5 mL/min. Compounds purities were determined by high pressure liquid chromatography
(HPLC) with a confirmed purity of ≥95% for all of the final biologically tested compounds.
N1ꢀ(3ꢀbromobenzyl)ꢀN4ꢀhydroxyꢀN1ꢀ(2ꢀmethoxyphenyl)succinamide (8a). To a solution of
hydroxyl amine hydrochloride (1.39 g, 20 mmol) in 10 mL MeOH was added KOH (1.12 g, 20
mmol) and the mixture was stirred for 10 min at 40 oC, then the reaction mixture was cooled to 0
°C and filtered. Compound Nꢀ(3ꢀbromobenzyl)ꢀNꢀ(2ꢀmethoxyphenyl) succinamic acid methyl
ester (410 mg, 1.0 mmol) was added to the filtrate followed by KOH (112 mg, 2.0 mmol) at
room temperature for 30 min. The reaction mixture was concentrated under reduced pressure,
diluted with saturated NH4Cl aqueous solution and extracted with EtOAc. The organic layer was
then washed with brine, dried over Na2SO4, and concentrated, then the residue was purified by
column chromatography [eluting with EA followed by 20:1 CHCl3/MeOH followed by 10:1
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1
CHCl3/MeOH] to give compound 8a (225 mg, 55% yield). H NMR (CD3OD, 400 MHz):
δ
7.36–7.32 (m, 3H), 7.15–7.13 (m, 2H), 7.06 (d, J = 8.0 Hz, 1H), 7.01 (dd, J = 1.6, 8.0 Hz, 1H),
6.92 (dd, J = 8.0, 8.0 Hz, 1H), 4.95 (d, J = 14.4 Hz, 1H), 4.58 (d, J = 14.4 Hz, 1H), 3.74 (s, 3H),
2.50–2.42 (m, 2H), 2.40–2.33 (m, 2H). HR MS (ESI) cacld for [C18H19BrN2O4 + Na]+ 429.0420,
found 429.0405. HPLC purity: 99.5%, Rt = 6.944 min.
N1ꢀ(3ꢀbromobenzyl)ꢀN5ꢀhydroxyꢀN1ꢀ(2ꢀmethoxyphenyl)glutaramide (8b). Compound 8b
(63% yield) was prepared according to the method described for the preparation of compound 8a
1
except using glutaric anhydride instead of succinic anhydride. H NMR (CD3OD, 400 MHz):
δ
7.35–7.30 (m, 3H), 7.14–7.11 (m, 2H), 7.03 (d, J = 7.2 Hz, 1H), 6.96 (d, J = 7.2 Hz, 1H), 6.89
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(dd, J = 7.2, 7.2 Hz, 1H), 4.95 (d, J = 14.4 Hz, 1H), 4.56 (d, J = 14.4 Hz, 1H), 3.73 (s, 3H), 2.16–
2.04 (m, 4H), 1.87–1.84 (m, 2H). HR MS (ESI) cacld for [C19H21BrN2O4 + Na]+ 443.0577,
found 443.0551. HPLC purity: 95.8%, Rt = 8.613 min.
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N1ꢀ(3ꢀbromobenzyl)ꢀN6ꢀhydroxyꢀN1ꢀ(2ꢀmethoxyphenyl)adipamide (8c). Compound 8c (35%
yield) was prepared according to the method described for the preparation of compound 8a
1
except using adipic anhydride instead of succinic anhydride. H NMR (CDCl3, 300 MHz):
δ
7.34–7.27 (m, 3H), 7.11–7.09 (m, 2H), 6.91 (dd, J = 7.8, 8.4 Hz, 2H), 6.86–6.84 (m, 1H), 5.06
(d, J = 14.4 Hz, 1H), 4.44 (d, J = 14.4 Hz, 1H), 3.73 (s, 3H), 2.21 (br s, 2H), 2.11–2.04 (m, 2H),
1.58 (br s, 4H). HR MS (ESI) cacld for [C20H23BrN2O4 + Na]+ 457.0733, found 457.0743. HPLC
purity: 98.9%, Rt = 8.874 min.
N1ꢀ(3ꢀbromobenzyl)ꢀN7ꢀhydroxyꢀN1ꢀ(2ꢀmethoxyphenyl)heptanediamide (8d). Compound 8d
(53% yield) was prepared according to the method described for the preparation of compound 8a
1
except using pimelic acid anhydride instead of succinic anhydride. H NMR (CD3OD, 400
MHz):
δ 7.35–7.32 ( m, 3H), 7.10–7.03 (m, 3H), 6.94–6.89 (m, 2H), 4.90 (d, J = 14.4 Hz, 1H),
4.52 (d, J = 14.4 Hz, 1H), 3.73 (s, 3H), 2.03 (br s, 4H), 1.53–1.50 (m, 4H), 1.26–1.19 (m, 2H).
HR MS (ESI) cacld for [C21H25BrN2O4 + Na]+ 471.0890, found 471.0911. HPLC purity: 96.8%,
Rt = 7.880 min.
N1ꢀ(3ꢀbromobenzyl)ꢀN8ꢀhydroxyꢀN1ꢀ(2ꢀmethoxyphenyl)octanediamide (8e). Compound 8e
(42% yield) was prepared according to the method described for the preparation of compound 8a
1
except using suberic anhydride instead of succinic anhydride. H NMR (CD3OD, 400 MHz):
δ
7.37–7.36 (m, 3H), 7.11–7.06 (m, 3H), 6.94–6.90 (m, 2H), 4.98 (d, J = 12.8 Hz, 1H), 3.78 (d, J =
12.8 Hz, 1H), 3.74 (s, 3H), 2.04–2.00 (br s, 4H), 1.53–1.45 (m, 4H), 1.27–1.20 (m, 4H). HR MS
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(ESI) cacld for [C22H27BrN2O4 + Na]+ 485.1046, found 486.1052. HPLC purity: 97.9%, Rt =
8.160 min.
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N1ꢀ(2ꢀmethoxyphenyl)ꢀN7ꢀhydroxyꢀN1ꢀ((3ꢀpyridinyl)methyl)heptanediamide
(9a).
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Compound 9a (50% yield) was prepared according to the method described for the preparation of
compound 8d except using 3ꢀpyridinecarboxaldehyde instead of 3ꢀbromobenzaldehyde. 1H NMR
(DMSOꢀd6, 300 MHz):
δ 10.28 (br s, 1H), 8.65 (br s, 1H), 8.41 (dd, J = 1.2, 6.0 Hz, 1H), 8.31 (s,
1H), 7.57 (d, J = 7.5 Hz, 1H), 7.35–7.27 (m, 2H), 7.09 (d, J = 7.8 Hz, 1H), 6.99 (d, J = 7.5 Hz,
1H), 6.90 (dd, J = 7.5, 7.5 Hz, 1H), 4.99 (d, J = 15.0 Hz, 1H), 4.45 (d, J = 15.0 Hz, 1H), 3.70 (s,
3H), 2.02–1.97 (m, 2H), 1.87 (t, J = 7.5 Hz, 2H), 1.45–1.33 (m, 4H), 1.11–1.06 (m, 2H). HR MS
(ESI) cacld for [C20H25N3O4 + H]+ 372.1918, found 372.1930. HPLC purity: 98.7%, Rt = 3.742
min.
N1ꢀ(2ꢀmethoxyphenyl)ꢀN7ꢀhydroxyꢀN1ꢀ((6ꢀbromoꢀ2ꢀpyridinyl)methyl)heptanediamide (9b).
Compound 9b (76% yield) was prepared according to the method described for the preparation of
compound 8d except using 6ꢀbromoꢀ2ꢀpyridinecarboxaldehyde instead of 3ꢀbromobenzaldehyde.
1H NMR (DMSOꢀd6, 300 MHz):
δ 10.29 (br s, 1H), 8.66 (br s, 1H), 7.71 (dd, J = 7.8, 7.8 Hz,
1H), 7.47 (d, J = 7.5 Hz, 1H), 7.43 (d, J = 7.8 Hz, 1H), 7.33 (dd, J = 7.5, 7.5 Hz, 1H), 7.20 (d, J
= 7.8 Hz, 1H), 7.12 (d, J = 7.8 Hz, 1H), 6.93 (dd, J = 7.5, 7.5 Hz, 1H), 5.11 (d, J = 15.9 Hz, 1H),
4.38 (d, J = 15.9 Hz, 1H), 3.78 (s, 3H), 2.09–2.02 (m, 2H), 1.87 (t, J = 7.2 Hz, 2H), 1.43–1.34
(m, 4H),1.15–1.09 (m, 2H). HR MS (ESI) cacld for [C20H24BrN3O4 + Na]+ 472.0842, found
472.0856. HPLC purity: 95.5%, Rt = 7.133 min.
N1ꢀ(2ꢀmethoxyphenyl)ꢀN7ꢀhydroxyꢀN1ꢀ((5ꢀbromoꢀ2ꢀfuranyl)methyl)heptanediamide
(9c).
Compound 9c (48% yield) was prepared according to the method described for the preparation of
1
compound 8d except using 5ꢀbromoꢀ2ꢀfuraldehyde instead of 3ꢀbromobenzaldehyde. H NMR
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(DMSOꢀd6, 300 MHz):
δ
10.29 (br s, 1H), 8.64 (br s, 1H), 7.35 (dd, J = 7.8, 7.8 Hz, 1H), 7.11 (d,
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J = 7.8 Hz, 1H), 7.03 (d, J = 7.8 Hz, 1H), 6.95 (dd, J = 7.5, 7.5 Hz, 1H), 6.39 (d, J = 3.3 Hz, 1H),
6.16 (d, J = 3.3 Hz, 1H), 5.11 (d, J = 15.6 Hz, 1H), 4.38 (d, J = 15.6 Hz, 1H), 3.76 (s, 3H), 1.99–
1.96 (m, 2H), 1.93–1.87 (m, 2H), 1.43–1.33 (m, 4H), 1.15–1.07 (m, 2H). HR MS (ESI) cacld for
[C19H23BrN2O5 + Na]+ 461.0683, found 461.0699. HPLC purity: 99.7%, Rt = 6.794 min.
N1ꢀ(2ꢀmethoxyphenyl)ꢀN7ꢀhydroxyꢀN1ꢀ((5ꢀbromoꢀ2ꢀthiopheneyl)methyl)heptanediamide
(9d). Compound 9d (74% yield) was prepared according to the method described for the
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preparation of compound 8d except using 5ꢀbromoꢀ2ꢀthiophenecarboxaldehyde instead of 3ꢀ
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bromobenzaldehyde. H NMR (DMSOꢀd6, 300 MHz):
δ 10.30 (br s, 1H), 8.65 (br s, 1H), 7.35
(dd, J = 7.5, 7.5 Hz, 1H), 7.13 (d, J = 7.8 Hz, 1H), 6.98–6.93 (m, 3H), 6.61 (d, J = 3.6 Hz, 1H),
5.08 (d, J = 15.3 Hz, 1H), 4.38 (d, J = 15.3 Hz, 1H), 3.75 (s, 3H), 1.98–1.92 (m, 2H), 1.86 (t, J =
7.2 Hz, 2H), 1.43–1.32 (m, 4H), 1.10–1.06 (m, 2H). HR MS (ESI) cacld for [C19H23BrN2O4S +
Na]+ 477.0454, found 477.0468. HPLC purity: 99.6%, Rt = 7.833 min.
N1ꢀ(2ꢀmethoxyphenyl)ꢀN7ꢀhydroxyꢀN1ꢀ((4ꢀbromoꢀ2ꢀthiopheneyl)methyl)heptanediamide
(9e). Compound 9e (70% yield) was prepared according to the method described for the
preparation of compound 8d except using 4ꢀbromoꢀ2ꢀthiophenecarboxaldehyde instead of 3ꢀ
bromobenzaldehyde. 1H NMR (DMSOꢀd6, 300 MHz):
δ 10.32 (br s, 1H), 8.67 (br s, 1H), 7.52 (s,
1H), 7.36 (dd, J = 7.8, 7.5 Hz, 1H), 7.13 (d, J = 7.8 Hz, 1H), 7.00–6.95 (m, 2H), 6.79 (s, 1H),
5.15 (d, J = 15.0 Hz, 1H), 4.41 (d, J = 15.0 Hz, 1H), 3.75 (s, 3H), 2.01–1.93 (m, 2H), 1.86 (t, J =
7.5 Hz, 2H), 1.45–1.32 (m, 4H), 1.12–1.06 (m, 2H). HR MS (ESI) cacld for [C19H23BrN2O4S +
Na]+ 477.0454, found 477.0469. HPLC purity: 95.2%, Rt = 9.274 min.
N1ꢀ(2ꢀmethoxyphenyl)ꢀN7ꢀhydroxyꢀN1ꢀ((5ꢀchloroꢀ2ꢀthiopheneyl)methyl)heptanediamide
(9f). Compound 9f (71% yield) was prepared according to the method described for the
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preparation of compound 8d except using 5ꢀchloroꢀ2ꢀthiophenecarboxaldehyde instead of 3ꢀ
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bromoꢀbenzaldehyde. H NMR (DMSOꢀd6, 300 MHz):
δ 10.33 (br s, 1H), 8.73 (br s, 1H), 7.35
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(dd, J = 7.8, 7.5 Hz, 1H), 7.13 (d, J = 7.8 Hz, 1H), 6.98–6.92 (m, 2H), 6.83 (d, J = 3.6 Hz, 1H),
6.61 (d, J = 3.6 Hz, 1H), 5.07 (d, J = 15.3 Hz, 1H), 4.35 (d, J = 15.3 Hz, 1H), 3.75 (s, 3H), 1.95–
1.84 (m, 4H), 1.43–1.35 (m, 4H), 1.11–1.08 (m, 2H). HR MS (ESI) cacld for [C19H23ClN2O4S +
H]+ 411.1140, found 411.1155. HPLC purity: 96.7%, Rt = 9.275 min.
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Nꢀ((5ꢀ(3ꢀpyridinyl)ꢀ2ꢀthiopheneyl)methyl)ꢀNꢀ(2ꢀmethoxyphenyl) pimelic acid methyl ester.
To a mixture of Nꢀ((5ꢀbromoꢀ2ꢀthiopheneyl)methyl)ꢀNꢀ(2ꢀmethoxyphenyl) pimelic acid methyl
ester (300 mg, 0.66 mmol) and triphenylphosphine (4 mg, 0.016 mmol) in dioxane was added
bis(dibenzylideneacetone)palladium (4 mg, 0.007 mmol) at room temperature, then the vial was
purged with nitrogen and stirred for 5 min. 3ꢀpyridinylboronic acid (81 mg, 0.66 mmol) and a
aqueous solution of K3PO4 (238 mg in 0.9 mL of H2O) were added to the mixture, the vial was
purged again and the resulting mixture was heated to reflux for 12 h. Then the reaction mixture
was concentrated under reduced pressure and extracted with EtOAc and H2O. The extract was
washed with brine, dried over Na2SO4, and concentrated, the residue was purified by column
chromatography (PE/EtOAc = 1:1) to give Nꢀ((5ꢀ(3ꢀpyridinyl)ꢀ2ꢀthiopheneyl)methyl)ꢀNꢀ(2ꢀ
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methoxyphenyl) pimelic acid methyl ester (63 mg, 21%) as a colorless oil. H NMR (DMSOꢀd6,
400 MHz):
δ 8.85–8.81 (m, 1H), 8.47 (d, J = 4.5 Hz, 1H), 7.97 (ddd, J = 8.0, 2.4, 1.5 Hz, 1H),
7.43– 7.33 (m, 3H), 7.16 (d, J = 7.5 Hz, 1H), 7.01 (dd, J = 7.5, 1.5 Hz, 1H), 6.93 (ddd, J = 7.5,
7.5, 1.2 Hz, 1H), 6.81 (d, J = 3.6 Hz, 1H), 5.26 (d, J = 15.0 Hz, 1H), 4.43 (d, J = 15.0 Hz, 1H),
3.76 (s, 3H), 3.54 (s, 3H), 2.21 (t, J = 7.5 Hz, 2H), 1.99–1.85 (m, 2H), 1.43–1.35 (m, 4H), 1.15–
1.11 (m, 2H). The intermediate was used in the following procedure directly for preparing
compound 9g.
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N1ꢀ(2ꢀmethoxyphenyl)ꢀN7ꢀhydroxyꢀN1ꢀ((5ꢀ(3ꢀpyridinyl)ꢀ2ꢀthiopheneyl)methyl)heptanediꢀ
amide (9g). Compound 9g (53% yield) was prepared according to the method described for the
preparation of compound 8a except using Nꢀ((5ꢀ(3ꢀpyridinyl)ꢀ2ꢀthiopheneyl)methyl)ꢀNꢀ(2ꢀ
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methoxyphenyl) pimelic acid methyl ester instead of Nꢀ(3ꢀbromobenzyl)ꢀNꢀ(2ꢀmethoxyphenyl)ꢀ
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succinamic acid methyl ester. H NMR (DMSOꢀd6, 300 MHz):
δ 10.32 (br s, 1H), 8.84 (s, 1H),
8.66 (br s, 1H), 8.47 (d, J = 4.5 Hz, 1H), 7.98 (d, J = 8.1 Hz, 1H), 7.46–7.33 (m, 3H), 7.16 (d, J =
7.5 Hz, 1H), 7.01 (d, J = 7.8 Hz, 1H), 6.95 (dd, J = 7.5, 7.5 Hz, 1H), 6.81 (d, J = 3.6 Hz, 1H),
5.25 (d, J = 15.0 Hz, 1H), 4.41 (d, J = 15.0 Hz, 1H), 3.77 (s, 3H), 1.98–1.85 (m, 4H), 1.42–1.32
(m, 4H), 1.13–1.07 (m, 2H). HR MS (ESI) cacld for [C24H27N3O4S + H]+ 454.1795, found
454.1805. HPLC purity: 96.2%, Rt = 7.208 min.
N1ꢀ(2ꢀmethoxyphenyl)ꢀN7ꢀhydroxyꢀN1ꢀ((5ꢀ(4ꢀpyridinyl)ꢀ2ꢀthiopheneyl)methyl)heptanediꢀ
amide (9h). Compound 9h (37% yield) was prepared according to the method described for the
preparation of compound 9g except using 4ꢀpyridinylboronic acid instead of 3ꢀpyridinylboronic
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acid. H NMR (DMSOꢀd6, 300 MHz):
δ 10.30 (br s, 1H), 8.68 (br s, 1H), 8.55–8.52 (m, 2H),
7.59–7.56 (m, 3H), 7.36 (dd, J = 7.8, 7.8 Hz, 1H), 7.14 (d, J = 8.1 Hz, 1H), 7.02 (d, J = 7.5 Hz,
1H), 6.92 (dd, J = 7.5, 7.5 Hz, 1H), 6.85 ( d, J = 3.6 Hz, 1H), 5.25 (d, J = 15.3 Hz, 1H), 4.45 (d, J
= 15.3 Hz, 1H), 3.76 (s, 3H), 1.92–1.84 (m, 4H), 1.45–1.34 (m, 4H), 1.16–1.09 (m, 2H). HR MS
(ESI) cacld for [C24H27N3O4S + H]+ 454.1795, found 454.1816. HPLC purity: 95.4%, Rt = 8.642
min.
N1ꢀ(2ꢀmethoxyphenyl)ꢀN7ꢀhydroxyꢀN1ꢀ((5ꢀ(5ꢀpyrimidinyl)ꢀ2ꢀthiopheneyl)methyl)heptanediꢀ
amide (9i). Compound 9i (37% yield) was prepared according to the method described for the
preparation of compound 9g except using 5ꢀpyrimidinylboronic acid instead of 3ꢀpyridinylꢀ
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boronic acid. H NMR (DMSOꢀd6, 300 MHz):
δ 10.30 (br s, 1H), 9.07 (s, 1H), 9.05 (br s, 2H),
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8.65 (br s, 1H), 7.51 (d, J = 3.3 Hz, 1H), 7.36 (dd, J = 8.1, 7.2 Hz, 1H), 7.14 (d, J = 8.1 Hz, 1H),
7.02 (d, J = 7.2 Hz, 1H), 6.93 (dd, J = 7.5, 7.5 Hz, 1H), 6.86 ( d, J = 3.0 Hz, 1H), 5.25 (d, J =
15.3 Hz, 1H), 4.45 (d, J = 15.3 Hz, 1H), 3.76 (s, 3H), 1.95–1.84 (m, 4H), 1.45–1.36 (m, 4H),
1.12–1.09 (m, 2H). HR MS (ESI) cacld for [C23H26N4O4S + H]+ 455.1748, found 455.1760.
HPLC purity: 99.6%, Rt = 5.604 min.
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N1ꢀ(3ꢀbromobenzyl)ꢀN7ꢀhydroxyꢀN1ꢀphenyl heptanediamide (10a). Compound 10a (51%
yield) was prepared according to the method described for the preparation of compound 8d
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except using aniline instead of 2ꢀmethoxyaniline. H NMR (DMSOꢀd6, 300 MHz):
δ 10.33 (br s,
1H), 8.67 (br s, 1H), 7.45–7.39 (m, 4H), 7.33 (d, J = 7. 2 Hz, 1H), 7.26 (dd, J = 7.8, 7.5 Hz, 1H),
7.19–7.16 (m, 3H), 4.86 (s, 2H), 2.07–2.04 (m, 2H), 1.88 (t, J = 7. 2 Hz, 2H), 1.49–1.46 (m, 2H),
1.38–1.35 (m, 2H), 1.14–1.11 (m, 2H). HR MS (ESI) cacld for [C20H23BrN2O3 + Na]+ 441.0784,
found 441.0791. HPLC purity: 95.4%, Rt = 7.653 min.
N1ꢀ(3ꢀbromobenzyl)ꢀN7ꢀhydroxyꢀN1ꢀ(3ꢀmethoxyphenyl)heptanediamide (10b). Compound
10b (51% yield) was prepared according to the method described for the preparation of
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compound 8d except using 3ꢀmethoxyaniline instead of 2ꢀmethoxyaniline. H NMR (DMSOꢀd6,
300 MHz):
δ 10.31 (br s, 1H), 8.65 (br s, 1H), 7.42–7.39 (m, 2H), 7.31–7.29 (m, 2H), 7.18 (dd, J
= 7.8, 7.5 Hz, 1H), 6.91 (d, J = 7.5 Hz, 1H), 6.73–6.70 (m, 2H), 4.84 (s, 2H), 3.72 (s, 3H), 2.13–
2.11 (m, 2H), 1.87 (t, J = 7.5 Hz, 2H), 1.49–1.43 (m, 4H), 1.15–1.12 (m, 2H). HR MS (ESI)
cacld for [C21H25BrN2O4 + Na]+ 471.0890, found 471.0884. HPLC purity: 96.1%, Rt = 7.846
min.
N1ꢀ(3ꢀbromobenzyl)ꢀN7ꢀhydroxyꢀN1ꢀ(4ꢀmethoxyphenyl)heptanediamide (10c). Compound
10c (55% yield) was prepared according to the method described for the preparation of
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compound 8d except using 4ꢀmethoxyaniline instead of 2ꢀmethoxyaniline. H NMR (DMSOꢀd6,
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300 MHz):
δ
10.33 (br s, 1H), 8.67 (br s, 1H), 7.43 (d, J = 7.8 Hz, 1H), 7.37 (s, 1H), 7.26 (dd, J =
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7.5, 7.5 Hz, 1H), 7.17 (d, J = 7.5 Hz, 1H), 7.06 (d, J = 9.0 Hz, 2H), 6.92 (d, J = 8.7 Hz, 2H), 4.87
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(s, 2H), 3.74 (s, 3H), 2.03 (t, J = 7.5 Hz, 2H), 1.89 (t, J = 7.5 Hz, 2H), 1.49–1.45 (m, 2H), 1.39–
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1.37 (m, 2H), 1.15–1.13 (m, 2H). C NMR (DMSOꢀd6, 125 MHz):
δ 172.23, 169.01, 158.34,
140.72, 134.69, 130.62, 130.47, 129.90, 129.21, 126.98, 121.54, 114.63, 55.23, 51.51, 33.22,
32.12, 28.19, 24.92, 24.62. HR MS (ESI) cacld for [C21H25BrN2O4 + Na]+ 471.0890, found
471.0888. HPLC purity: 99.3%, Rt = 7.817 min.
N1ꢀ(3ꢀbromobenzyl)ꢀN7ꢀhydroxyꢀN1ꢀ(2ꢀchlorophenyl)heptanediamide (10d). Compound 10d
(46% yield) was prepared according to the method described for the preparation of compound 8d
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except using 2ꢀchloroaniline instead of 2ꢀmethoxyaniline. H NMR (DMSOꢀd6, 300 MHz):
δ
10.32 (br s, 1H), 8.67 (br s, 1H), 7.46–7.42 (m, 3H), 7.38 (s, 1H), 7.27 (d, J = 7.8 Hz, 1H), 7.24–
7.21 (m, 2H), 7.18 (d, J = 8.1 Hz, 1H), 4.85 (s, 2H), 2.06–2.04 (m, 2H), 1.89 (t, J = 7.5 Hz, 2H),
1.48–1.45 (m, 2H), 1.41–1.38 (m, 2H), 1.24–1.21 (m, 2H). HR MS (ESI) cacld for
[C20H22BrClN2O3 + Na]+ 475.0395, found 475.0374. HPLC purity: 97.3%, Rt = 4.206 min.
N1ꢀ(3ꢀbromobenzyl)ꢀN7ꢀhydroxyꢀN1ꢀ(3ꢀchlorophenyl)heptanediamide (10e). Compound 10e
(35% yield) was prepared according to the method described for the preparation of compound 8d
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except using 3ꢀchloroaniline instead of 2ꢀmethoxyaniline. H NMR (DMSOꢀd6, 300 MHz):
δ
10.33 (br s, 1H), 8.67 (br s, 1H), 7.44–7.38 (m, 5H), 7.26 (dd, J = 7.5, 7.5 Hz, 1H), 7.17ꢀ7.14 (m,
2H), 4.87 (s, 2H), 2.11–2.09 (m, 2H), 1.90 (t, J = 7.5 Hz, 2H), 1.52–1.50 (m, 2H), 1.45–1.41 (m,
2H), 1.17–1.14 (m, 2H). HR MS (ESI) cacld for [C20H22BrClN2O3 + Na]+ 475.0395, found
475.0407. HPLC purity: 98.8%, Rt = 8.408 min.
N1ꢀ(3ꢀbromobenzyl)ꢀN7ꢀhydroxyꢀN1ꢀ(4ꢀchlorophenyl)heptanediamide (10f). Compound 10f
(41% yield) was prepared according to the method described for the preparation of compound 8d
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except using 4ꢀchloroaniline instead of 2ꢀmethoxyaniline. H NMR (DMSOꢀd6, 300 MHz):
δ
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10.31 (br s, 1H), 8.66 (br s, 1H), 7.46–7.41 (m, 3H), 7.37 (s, 1H), 7.26 (d, J = 7.8 Hz, 1H), 7.21
(d, J = 8.7 Hz, 2H), 7.17 (d, J = 8.1 Hz, 1H), 4.83 (s, 2H), 2.08–2.03 (m, 2H), 1.88 (t, J = 7.2 Hz,
2H), 1.49–1.44 (m, 2H), 1.40–1.36 (m, 2H), 1.14–1.09 (m, 2H). HR MS (ESI) cacld for
[C20H22BrClN2O3 + Na]+ 475.0395, found 475.0411. HPLC purity: 95.3%, Rt = 8.789 min.
N1ꢀ(3ꢀbromobenzyl)ꢀN7ꢀhydroxyꢀN1ꢀ(4ꢀfluorophenyl)heptanediamide (10g). Compound 10g
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(44% yield) was prepared according to the method described for the preparation of compound 8d
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except using 4ꢀfluoroaniline instead of 2ꢀmethoxyaniline. H NMR (DMSOꢀd6, 300 MHz):
δ
10.29 (br s, 1H), 8.65 (br s, 1H), 7.40 (d, J = 7. 8 Hz, 1H), 7.35 (s, 1H), 7.24 (d, J = 7. 8 Hz, 1H),
7.20–7.18 (m, 2H), 7.17–7.14 (m, 3H), 4.80 (s, 2H), 2.01 (t, J = 7. 2 Hz, 2H), 1.86 (t, J = 7. 2 Hz,
2H), 1.47–1.42 (m, 2H), 1.39–1.34 (m, 2H), 1.14–1.10 (m, 2H). HR MS (ESI) cacld for
[C20H22BrFN2O3 + Na]+ 459.0690, found 459.0710. HPLC purity: 99.7%, Rt = 9.206 min.
N1ꢀ(3ꢀbromobenzyl)ꢀN7ꢀhydroxyꢀN1ꢀ(4ꢀbromophenyl)heptanediamide (10h). Compound 10h
(83% yield) was prepared according to the method described for the preparation of compound 8d
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except using 4ꢀbromoaniline instead of 2ꢀmethoxyaniline. H NMR (DMSOꢀd6, 300 MHz):
δ
10.28 (br s, 1H), 8.63 (br s, 1H), 7.56 (d, J = 8.7 Hz, 2H), 7.40 (d, J = 7. 5 Hz, 1H), 7.36 (s, 1H),
7.23 (dd, J = 7. 8, 7. 8 Hz, 1H), 7.17–7.15 (m, 1H), 7.13 (d, J = 8.7 Hz, 2H), 4.82 (s, 2H), 2.07–
2.04 (m, 2H), 1.86 (t, J = 7. 2 Hz, 2H), 1.48–1.45 (m, 2H), 1.39–1.34 (m, 2H), 1.15–1.09 (m,
2H). HR MS (ESI) cacld for [C20H22Br2N2O3 + Na]+ 518.9889, found 518.9899. HPLC purity:
97.3%, Rt = 10.490 min.
N1ꢀ(3ꢀbromobenzyl)ꢀN7ꢀhydroxyꢀN1ꢀ(4ꢀiodophenyl)heptanediamide (10i). Compound 10i
(35% yield) was prepared according to the method described for the preparation of compound 8d
except using 4ꢀiodoaniline instead of 2ꢀmethoxyaniline. 1H NMR (DMSOꢀd6, 300 MHz):
δ
10.30
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(br s, 1H), 8.67 (br s, 1H), 7.71 (d, J = 8.4 Hz, 2H), 7.40 (d, J = 7.8 Hz, 1H), 7.35 (s, 1H), 7.23
(dd, J = 7. 8, 7. 8 Hz, 1H), 7.15 (d, J = 7.8 Hz, 1H), 6.95 (d, J = 8.4 Hz, 2H), 4.81 (s, 2H), 2.06–
2.04 (m, 2H), 1.86 (t, J = 7. 2 Hz, 2H), 1.47–1.42 (m, 2H), 1.39–1.34 (m, 2H), 1.15–1.10 (m,
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2H). 13C NMR (DMSOꢀd6, 125 MHz):
δ 171.76, 168.98, 141.81, 140.38, 138.36, 130.53, 130.36,
130.00, 126.87, 121.61, 93.75, 51.20, 33.34, 32.12, 28.14, 24.92, 24.56. HR MS (ESI) cacld for
[C20H22BrIN2O3 + Na]+ 566.9751, found 566.9765. HPLC purity: 95.7%, Rt = 10.576 min.
N1ꢀ((5ꢀbromoꢀ2ꢀthiopheneyl)methyl)ꢀN7ꢀhydroxyꢀN1ꢀ(4ꢀmethoxyphenyl)heptanediamide
(11a). Compound 11a (46% yield) was prepared according to the method described for the
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preparation of compound 9d except using 4ꢀmethoxyaniline instead of 2ꢀmethoxyaniline. H
NMR (DMSOꢀd6, 300 MHz):
δ 10.29 (br s, 1H), 8.64 (br s, 1H), 7.03 (d, J = 9.0 Hz, 2H), 6.96
(d, J = 3.6 Hz, 1H), 6.93 (d, J = 9.0 Hz, 2H), 6.62 (d, J = 3.6 Hz, 1H), 4.79 (s, 2H), 3.73 (s, 3H),
1.94–1.91 (m, 2H), 1.84 (t, J = 7.5 Hz, 2H), 1.41–1.38 (m, 2H), 1.34–1.32 (m, 2H), 1.07–1.04
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(m, 2H). C NMR (DMSOꢀd6, 125 MHz):
δ 172.02, 168.92, 158.55, 142.37, 134.32, 129.41,
129.21, 127.78, 114.71, 111.02, 55.28, 47.65, 33.15, 32.11, 28.15, 24.89, 24.54. HR MS (ESI)
cacld for [C19H23BrN2O4S + Na]+ 477.0454, found 477.0450. HPLC purity: 98.4%, Rt = 7.712
min.
N1ꢀ((5ꢀ(5ꢀpyrimidinyl)ꢀ2ꢀthiopheneyl)methyl)ꢀN7ꢀhydroxyꢀN1ꢀ(4ꢀmethoxyphenyl)heptaneꢀ
diamide (11b). Compound 11b (46% yield) was prepared according to the method described for
1
the preparation of compound 9i except using 4ꢀmethoxyaniline instead of 2ꢀmethoxyaniline. H
NMR (DMSOꢀd6, 300 MHz):
δ 10.31 (br s, 1H), 9.09 (s, 1H), 9.07 (s, 2H), 8.66 (br s, 1H), 7.55
(d, J = 2.6 Hz, 1H), 7.09 (d, J = 9.0 Hz, 2H), 6.94 (d, J = 9.0 Hz, 2H), 6.89 (d, J = 2.6 Hz, 1H),
4.95 (s, 2H), 3.76 (s, 3H), 1.99 (t, J = 7.5 Hz, 2H), 1.87 (t, J = 7.5 Hz, 2H), 1.48–1.43 (m, 2H),
1.40–1.35 (m, 2H), 1.13–1.09 (m, 2H). 13C NMR (DMSOꢀd6, 125 MHz):
δ 171.94, 169.04,
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158.54, 156.87, 152.86, 142.56, 135.47, 134.39, 129.30, 128.50, 128.05, 125.59, 114.71, 55.28,
47.44, 33.24, 32.14, 28.19, 24.92, 24.58. HR MS (ESI) cacld for [C23H26N4O4S + Na]+ 477.1567,
found 477.1582. HPLC purity: 98.4%, Rt = 7.037 min.
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■ ASSOCIATED CONTENT
Supporting Information
The detailed docking studies of selected compounds with HDAC2 and 8. Additional details of
the procedures used for the in vitro and in vivo related biological studies as well as PK studies.
This material is available free of charge via the Internet at http://pubs.acs.org.
■ AUTHOR INFORMATION
Corresponding Authors:
* For Y. C. Phone: +86ꢀ21ꢀ2420ꢀ6647. Fax: +86ꢀ21ꢀ54344922. Eꢀmail: yhchen@bio.ecnu.edu.cn;
* For Z. Y. Phone: +86ꢀ21ꢀ5434ꢀ5016. Fax: +86ꢀ21ꢀ54344922. Eꢀmail: zfyi@bio.ecnu.edu.cn;
* For M. Y. Phone: +86ꢀ21ꢀ5434ꢀ5124. Fax: +86ꢀ21ꢀ54344922. Eꢀmail: myliu@bio.ecnu.edu.cn.
Author Contributions:
#These authors contributed equally to this work.
■ ACKNOWLEDGMENTS
We thank Dr. Stefan Siwko for comments and advice. This work was partially supported by the
grants from National Basic Research Program of China (2012CB910404), National Major
Scientific and Technological Special Project for "Significant New Drugs Development"
(2013ZX09507001), National Natural Science Foundation of China (81202407, 81330049 and
81272463), Research Fund for the Doctoral Program of Higher Education of China
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(20120076120029), The Science and Technology Commission of Shanghai Municipality
(14DZ2270100), Large Instruments Open Foundation of East China Normal University (2013ꢀ76
and 2014ꢀ13) and Innovation Program of Shanghai Municipal Education Commission (13zz034).
■ ABBREVIATIONS USED
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HDAC, histone deacetylase; HAT, histone acetyl transferase; ZBG, zincꢀbinding group; SAR,
structureꢀactivity relationship; CTCL, cutaneous Tꢀcell lymphoma; PTCL, peripheral Tꢀcell
lymphoma; Arp, actinꢀrelated protein; EMT, epithelialꢀMesenchymalꢀtransition; EDC, Nꢀ(3ꢀ
dimethylꢀaminopropyl)ꢀN'ꢀethylcarbodiimide hydrochloride; HOBt, 1ꢀHydroxyꢀbenzotriazole;
PE, petroleum ether; EA, ethyl acetate; MDAꢀMBꢀ231, MCFꢀ7, T47D, HCC1937, MDAꢀMBꢀ
468, BTꢀ549 and Bcap37, human breast cancer cell lines; 4T1, mouse breast cancer cell line.
■ REFERENCES
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