Development of N-hydroxycinnamamide-based histone deacetylase inhibitors with an indole-containing Cap group

Article abstract of DOI:10.1021/ml300366t

A novel series of histone deacetylase inhibitors combining N-hydroxycinnamamide bioactive fragment and indole bioactive fragment was designed and synthesized. Several compounds (17c, 17g, 17h, 17j, and 17k) exhibited comparable, even superior, total HDACs inhibitory activity and in vitro antiproliferative activities relative to the approved drug SAHA. A representative compound 17a with moderate HDACs inhibition was progressed to isoform selectivity profile, Western blot analysis, and in vivo antitumor assay. Although HDACs isoform selectivity of 17a was similar to that of SAHA, our Western blot results indicated that intracellular effects of 17a at 1 μM were class I selective. It was noteworthy that the effect on histone H4 acetylation of SAHA decreased with time, while the effect on histone H4 acetylation of 17a was maintained and even increased. Most importantly, compound 17a exhibited promising in vivo antitumor activity in a U937 xenograft model.

Full text of DOI:10.1021/ml300366t

Letter
pubs.acs.org/acsmedchemlett
Development of N‑Hydroxycinnamamide-Based Histone Deacetylase
Inhibitors with an Indole-Containing Cap Group
Yingjie Zhang,†,⊥ Penghui Yang,†,⊥ C. James Chou,‡ Chunxi Liu,§ Xuejian Wang,∥ and Wenfang Xu*,†
†Department of Medicinal Chemistry, School of Pharmacy, Shandong University, Ji’nan, Shandong, 250012, People’s Republic of
China
‡Department of Drug Discovery and Biomedical Sciences, South Carolina College of Pharmacy, Medical University of South Carolina,
Charleston, South Carolina, 29425, United States United States
§Department of Pharmaceutics, School of Pharmacy, Shandong University, Ji’nan, Shandong, 250012, People’s Republic of China
∥Postdoctoral Workstation, Biomedical Industry Park Management Office, Weifang, Shandong, 261205, People’s Republic of China
S
* Supporting Information
ABSTRACT: A novel series of histone deacetylase inhibitors combining N-
hydroxycinnamamide bioactive fragment and indole bioactive fragment was
designed and synthesized. Several compounds (17c, 17g, 17h, 17j, and 17k)
exhibited comparable, even superior, total HDACs inhibitory activity and in
vitro antiproliferative activities relative to the approved drug SAHA. A
representative compound 17a with moderate HDACs inhibition was
progressed to isoform selectivity profile, Western blot analysis, and in vivo
antitumor assay. Although HDACs isoform selectivity of 17a was similar to
that of SAHA, our Western blot results indicated that intracellular effects of 17a at 1 μM were class I selective. It was noteworthy
that the effect on histone H4 acetylation of SAHA decreased with time, while the effect on histone H4 acetylation of 17a was
maintained and even increased. Most importantly, compound 17a exhibited promising in vivo antitumor activity in a U937
xenograft model.
KEYWORDS: histone deacetylases, inhibitor, N-hydroxycinnamamide, indole
he epigenetic regulation of histone and DNA plays an
Timportant role in chromatin structure and control of gene
expression. Altered patterns of epigenetic modification are very
common in many diseases, including cancer.1 As with genetic
information, epigenetic modifications are heritable; in contrast,
however, they are reversible and catalyzed by pairs of enzymes
with converse activity. Among these epigenetic enzymes,
histone deacetylases (HDACs) have now emerged as a
promising new class of therapeutic targets.2
Four classes were identified in the HDACs family,
characterized by different cellular localization and substrate
specificity. The common feature of classes I (HDAC1−3 and
-8), II (HDAC4−7, -9, and -10) and IV (HDAC11) active sites
are characterized by the presence of a catalytic Zn2+ ion, and
the class III HDACs (sirtuins 1−7) are NAD+-dependent.
These HDACs isoforms perform their multiple functions by
either epigenetic mechanism (deacetylation of histones) or
nonepigenetic mechanism (deacetylation of nonhistone sub-
strates). It has been revealed that Zn2+-dependent isozymes,
especially class I and class II, are closely related to
tumorigenesis and development.3 In the past 10 years, over
490 clinical trials of more than 20 histone deacetylase inhibitor
(HDACI) candidates have been initiated, culminating in the
approval of two antitumor drugs, vorinostat (SAHA) and
romidepsin (FK228) (Figure 1).4,5
Figure 1. Approved and clinical HDACIs with the N-hydroxycinna-
mamide fragment highlighted in red.
N-Hydroxycinnamamide (Figure 1) is a very common active
fragment in HDACI design.6,7 This fragment could not only
form bichelation with the active site Zn2+ by its hydroxamic
acid group but also form a sandwichlike π−π interaction by
inserting its vinyl benzene group into two parallel phenylalanine
residues of HDAC. Currently, there are three HDACIs
Received: October 27, 2012
Accepted: January 8, 2013
Published: January 8, 2013
© 2013 American Chemical Society
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ACS Medicinal Chemistry Letters
Letter
containing N-hydroxycinnamamide fragment in clinical trials
(Figure 1). Indole is recognized as a privileged structure in drug
design,8,9 and its derivatives have been found to exhibit
anticancer activity by interacting with different targets.10 Kinds
of natural and synthetic HDACIs containing an indole scaffold,
especially in the cap group, possessed promising in vitro and in
vivo potency.11−14 Recently, comprehensive HDACIs struc-
ture−activity relationship (SAR) studies revealed that N-
hydroxycinnamamide-based compounds were more stable
than their straight chain analogues, and compounds having
indole groups exhibited the best in vivo efficacy and
tolerability.15 On the basis of the aforementioned information,
we designed a novel series of N-hydroxycinnamamide-based
HDACIs with an indole-containing cap group.
Table 1. HeLa Cell Nuclear Extract Inhibitory Activity
a
a
compd
IC50 (μM)
compd
IC50 (μM)
14a
14b
15
0.65 0.12
2.09 0.31
2.91 0.34
1.08 0.20
0.49 0.09
0.17 0.04
0.62 0.13
0.57 0.09
0.38 0.05
0.16 0.04
0.18 0.04
0.43 0.05
0.19 0.03
17j
0.18 0.03
0.14 0.03
0.32 0.05
0.43 0.05
1.17 0.21
0.80 0.17
1.12 0.23
0.73 0.16
b
17k
17l
17a
17b
17c
17d
17e
17f
17m
17n
17o
17p
17q
17r
17s
17t
17g
17h
17i
1.06 0.20
1.47 0.36
0.71 0.19
17u
Compounds 14a, 14b, and 15 were synthesized following the
procedures described in Scheme 1. Methyl ester protection and
SAHA
a
Assays were performed in replicate (n ≥ 2). Data are shown as means
b
a
SDs. Compound 17r was undissolved under our test condition.
Scheme 1. Synthesis of Compounds 14a, 14b, and 15
a
Scheme 2. Synthesis of Compounds 17a−u
a
Reagents and conditions: (a) SOCI2, CH3OH, 98%. (b) (Boc)2O,
Et3N, DCM, 80%. (c) LiAIH4, anhydrous THF, 0 °C, 86%. (d) PTSA,
CH3OH, 80 °C, 96%. (e) H2, 10% Pd−C, CH3OH. (f) PPh3, DEAD,
anhydrous THF, 0 °C, 75% for 8a, 79% for 8b. (g) NH2OK, CH3OH,
51% for 9a, 49% for 9b. (h) HCI, anhydrous EtOAc, 90%.
tert-butyloxycarbonyl (Boc) protection of L-tryptophan (1)
followed by LiAlH4 reduction provided the intermediate 9.
Methyl ester protection of ferulic acid (10) afforded compound
11, which was hydrogenized to give compound 12. Both 11 and
12 could be connected with 9 under Mitsunobu reaction
conditions to get 13a and 13b, which were converted to
corresponding hydroxamic acid compounds 14a and 14b,
respectively. Subsequent N-deprotection of 14a afforded
compound 15.
The preliminary HDACs inhibitory assay was tested against
HeLa cell nuclear extract, mainly containing HDAC1 and
HDAC2. Results listed in Table 1 revealed that compound 14a
(IC50 = 0.65 μM) was more potent than its analogues 14b
(IC50 = 2.09 μM) and 15 (IC50 = 2.91 μM), which indicated
that both the vinyl benzene group and the substituent located
on the amine atom (the Boc group in 14a) were advantageous
to HDACs inhibition. Therefore, using 14a as lead, we kept its
N-hydroxycinnamamide group unchanged and derivatized its
Boc group to other functional groups. Such derivatizations were
performed according to the methods in Scheme 2. N-
Deprotection of 13a with trifluoroacetic acid (TFA) and
subsequent amide condensation or sulfonylation gave inter-
mediates 16a−u, which were treated with NH2OK to get target
compounds 17a−u.
a
Reagents and conditions: (a) (i) TFA, Et3N, DCM; (ii) R′COOH,
TBTU, Et3N, THF, 52−75% for two steps. (b) (i) TFA, Et3N, DCM;
(ii) R′SO2CI, Et3N, DCM, 60−76% for two steps. (c) NH2OK,
CH3OH, 31−54%.
activities of compounds 17c, 17g, 17h, 17j, and 17k were
comparable, even more potent than that of SAHA. However,
replacing the Boc group of 14a with valproyl group (17a),
unnatural amino acid residues (17o−q) and sulfuryl group
(17r−u) were detrimental to activity.
To characterize HDACs isoform selectivity of these
analogues, representative compound 17a with moderate total
HDACs inhibitory activity was tested against HDAC1,
HDAC2, HDAC3, and HDAC6 using acetylated substrate.
Besides, the class IIa inhibitory activity was evaluated against
MDA-MB-231 cell lysate using class IIa-specific triflouroacety-
lated substrate.16 Results in Table 2 showed that 17a displayed
modest preference for HDAC1 and HDAC3 over HDAC2 and
HDAC6 but exhibited no obvious inhibition against class IIa
HDACs up to 10 μM. The overall selectivity profile of 17a was
As shown in Table 1, we found that compounds 17b−n with
N-Boc-protected natural α-amino acid residues as R group
except 17n exhibited more potent activity than their parent
compound 14a. Most importantly, the total HDACs inhibitory
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ACS Medicinal Chemistry Letters
Letter
a
Table 2. HDACs Isoform Selectivity of 17a and SAHA
class I
class IIb
HDAC6
class IIa
compd
HDAC1
HDAC2
HDAC3
cell lysate
b
17a
0.39 0.12
1.42 0.06
0.28 0.13
0.94 0.14
NA
b
SAHA
0.076 0.011
0.256 0.003
0.028 0.011
0.118 0.012
NA
a
b
Assays were performed in replicate (n ≥ 2). IC50 (μM) values are shown as means SDs. NA, not active at 10 μM.
similar to that of SAHA, which was in line with literature
information.17
Table 3. In Vitro Antiproliferative Activity of Representative
Compounds
a
IC50 (μM)
compd
U937
PC-3
A549
ES-2
MDA-MB-231 HCT116
17a
1.8
3.1
2.2
2.2
2.7
3.9
2.3
3.7
10.5
10.4
5.8
4.4
11.8
4.2
5.4
29.2
25.1
4.4
3.1
7.2
5.5
6.0
3.8
5.9
2.4
9.4
6.0
17c
17g
17h
17j
4.5
1.6
6.8
5.4
7.0
8.9
7.2
17k
SAHA
8.2
13.5
3.8
9.5
11.7
5.6
9.9
12.7
a
Values are the means of at least two experiments. The SD values are
<20% of the mean.
was comparable and even superior, which was in line with our
Western blot analysis results.
Figure 2. Western blot analysis of acetylated tubulin, acetylated
histone H3, acetylated histone H4, and p21 in MDA-MB-231 cell lines
after 3 (a) and 24 h (b) of treatment with compounds at 1 μM. β-
Actin was used as a loading control.
Among these tested tumor cell lines, human leukemic
monocyte lymphoma (U937) was the most sensitive to our
HDACI. To preliminarily investigate if our newly designed
compounds were active in vivo, representative compound 17a
was progressed to a U937 xenograft model using Tamibarotene
as a positive control. Mice were treated once daily by oral
gavage for 3 weeks. The tumor growth curve depicted in Figure
3 and the final tumor tissue size visualized in Figure 4 explicitly
Compound 17a was confirmed to be cell permeable and able
to inhibit intracellular and even nuclear HDACs by monitoring
the acetylation levels of tubulin (target of HDAC6) and
histones H3 and H4 (targets of HDAC1 and HDAC2) in the
MDA-MB-231 cell line. Moreover, the effect on the expression
level of the cyclin-dependent kinase (CDK) inhibitor p21 was
also investigated (Figure 2). Although the inhibition against
class I HDACs of 17a was inferior to that of SAHA (Table 2),
its effects on the levels of histone acetylation and p21
expression at the concentration of 1 μM were comparable
and even superior to SAHA, especially after 24 h of treatment.
However, 17a had almost no effect on acetylated tubulin level
as compared with SAHA at both time points. There was a
possible explanation that the intracellular amide hydrolysis of
17a could release compound 15 and valproic acid (VPA),
which is a clinical HDACI with class I selectivity.18 Therefore,
17a and its intracellular metabolites (15 and VPA) could
exhibit synergistic inhibition against class I HDACs but not
HDAC6. It was noteworthy that the effect on histone H4
acetylation of SAHA decreased with time, while the effect on
histone H4 acetylation of 17a was maintained and even
increased. This indicated that 17a might be promising in tumor
prevention and treatment because it has been shown that global
hypoacetylation of histone H4 is a common hallmark of cancer
and changes in H4 acetylation happen early in tumorigenesis.19
The exact intracellular mechanism of 17a needs further
research.
Figure 3. Growth curve of implanted U937 xenograft in nude mice
(seven mice per group). Data are expressed as the mean SD.
showed that compound 17a exhibited potent oral antitumor
activity. Tumor growth inhibition (TGI) and relative increment
Compound 17a and five other analogues with comparable
total HDACs inhibitory activity to SAHA were selected to test
their effects on tumor cell viability. The results in Table 3
showed that the antiproliferative activities of these derivatives
were similar to those of SAHA. It was intriguing that although
total HDACs inhibition of 17a was inferior, its cellular potency
Figure 4. Picture of dissected U937 tumor tissues.
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In conclusion, we designed and synthesized a novel series of
N-hydroxycinnamamide-based HDACIs with an indole-con-
taining cap group, among which compounds 17c, 17g, 17h, 17j,
and 17k exhibited similar HDACs inhibition and in vitro
antitumor potency to SAHA. Our further research focused on
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ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental procedures for compound synthesis, HDACs
inhibition fluorescence assay, in vitro antiproliferative assay,
Western blot analysis, in vivo antitumor assay, and character-
ization data for new compounds. This material is available free
of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Tel/Fax: 86-531-88382264. E-mail: wfxu@yahoo.cn.
Author Contributions
⊥These two authors contributed equally.
Funding
This work was supported by the National Scientific and
Technological Major Project of Ministry of Science and
Technology of China (Grant No. 2011ZX09401-015), National
Natural Science Foundation of China (Grant No. 21172134),
Doctoral Foundation of Ministry of Education of China (Grant
No. 20110131110037), the National Center for Research
Resources (Grant No. 5 P20 RR024485-02), and the National
Institute of General Medical Sciences (Grant No. 8 P20
GM103542-02) from the National Institutes of Health.
Notes
The authors declare no competing financial interest.
ABBREVIATIONS
■
HDACs, histone deacetylases; HDACIs, histone deacetylase
inhibitors; SAR, structure−activity relationship; Boc, tert-
butyloxycarbonyl; DCM, dichloromethane; THF, tetrahydro-
furan; PTSA, p-toluenesulfonic acid; DEAD, diethyl azodicar-
boxylate; TFA, trifluoroacetic acid; TBTU, O-(benzotriazol-1-
yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate; CDK,
cyclin-dependent kinase; VPA, valproic acid
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