Design and Synthesis of C3-Substituted β-Carboline-Based Histone Deacetylase Inhibitors with Potent Antitumor Activities

Article abstract of DOI:10.1002/cmdc.201700133

A series of hydroxamic acid histone deacetylase (HDAC) inhibitors in which the β-carboline motif has been incorporated were designed and synthesized. The effect of substitution at the C3 amide on HDAC inhibition and antiproliferative activities was invest

Full text of DOI:10.1002/cmdc.201700133

DOI: 10.1002/cmdc.201700133
Communications
Design and Synthesis of C3-Substituted b-Carboline-Based
Histone Deacetylase Inhibitors with Potent Antitumor
Activities
Yong Ling+,[a, b] Jiao Feng+,[a] Lin Luo,[a] Jing Guo,[a] Yanfu Peng,[a] Tingting Wang,[a]
Xiang Ge,[a] Qibing Xu,[a] Xinyang Wang,[a, b] Hong Dai,*[a, c] and Yanan Zhang*[a]
A series of hydroxamic acid histone deacetylase (HDAC) inhibi-
tors in which the b-carboline motif has been incorporated
were designed and synthesized. The effect of substitution at
the C3 amide on HDAC inhibition and antiproliferative activi-
ties was investigated. Most of these compounds were found to
display significant HDAC inhibitory effects and good antiproli-
ferative activity, with IC50 values in the low-micromolar range.
In particular, the HDAC inhibition IC50 value of N-(2-(dimethyla-
mino)ethyl)-N-(4-(hydroxylcarbamoyl)benzyl)-1-(4-methoxy-
phenyl)-9H-pyrido[3,4-b]indole-3-carboxamide (9h) is five-fold
lower than that of suberoylanilide hydroxamic acid (SAHA, vor-
inostat). Furthermore, 9h was found to increase the acetylation
of histone H3 and a-tubulin, and to induce DNA damage as
evidenced by hypochromism and enhanced phosphorylation
of histone H2AX. Compound 9h inhibits Stat3, Akt, and ERK
signaling, important cell-growth-promoting pathways that are
aberrantly activated in most cancers. Finally, 9h showed rea-
sonable solubility and permeability in Caco-2 cells. Our findings
suggest that these novel b-carboline-based HDAC inhibitors
may hold great promise as therapeutic agents for the treat-
ment of human cancers.
drugs may also be effective against a wider range of cancers.
Moreover, multitarget drugs as single chemical entities may
achieve effective combinatorial therapies without the common
disadvantages of combining multiple agents together, such as
drug–drug interactions, difficulty in achieving sufficient poten-
cies against tumor cells, and potentiation of adverse effects.[6,7]
Histone acetyltransferases (HATs) and histone deacetylases
(HDACs) are enzymes that control the reversible acetylation
and deacetylation of histones. DNA methylation or post-trans-
lational modification of histone proteins often leads to chro-
matin remodeling, which partly controls the epigenetic regula-
tion of gene expression.[8,9] Several HDACs are overexpressed
in various tumor types, indicating their importance in tumor
growth.[10,11] HDACs have been shown to play an important
role in carcinogenesis, including the transcription and regula-
tion of genes involved in cell proliferation, cell-cycle regulation,
and apoptosis.[10,11] HDAC inhibitors (HDACis) can induce tumor
cell apoptosis, growth arrest, senescence, differentiation, and
immunogenicity, and can inhibit angiogenesis.[12] Moreover,
HDACis have also been reported to synergistically enhance the
inhibitory effects of a number of other antitumor agents, in-
cluding those targeting EGFR, DNA, or topoisomerase, in sup-
pressing proliferation and inducing apoptosis in tumor
cells.[13–16]
The design and development of single drug molecules that si-
multaneously interact with multiple drug targets have recently
gained considerable interest in drug discovery.[1–4] This ap-
proach aims to alleviate issues commonly associated with
single-target drugs, such as limited efficacies and development
of resistance.[4,5] Given that different types of tumors may origi-
nate from different cell types and are frequently driven by vari-
ous combinations of genetic alterations, multitarget antitumor
Several HDACis are currently on the market, the majority of
which primarily target hematological malignancies, and about
20 others are in clinical trials. Based on the structural character-
istics of the Zn2+ binding domain, HDACis can be classified
into four main categories: hydroxamic acids, cyclic peptides,
short-chain fatty acids, and benzamides. The most widely ex-
plored class of HDACis that have entered pre-clinical or clinical
studies as anticancer agents are the hydroxamic acid based
compounds, including suberoylanilide hydroxamic acid (SAHA,
vorinostat; Figure 1), belinostat (PXD101), and panobinostat
(LBH589), three FDA-approved drugs for the treatment of cuta-
neous T-cell lymphoma (CTCL) or multiple myeloma.[17–19]
Hydroxamic acid based HDACis such as SAHA commonly
have an aromatic amide (CAP) group and a hydroxamic acid
(ZBG) group connected through a linker. While structure–activi-
ty relationship (SAR) studies have shown that the hydroxamic
acid group is required for HDAC activity, the aromatic group,
such as the phenyl group in SAHA, can be replaced with vari-
ous aromatic groups. We recently demonstrated that merging
the key structural elements of b-carboline and SAHA resulted
in hybrid compounds with increased antitumor potency and
low acute toxicity.[20] Naturally occurring b-carboline alkaloids
[a] Prof. Dr. Y. Ling,+ J. Feng,+ L. Luo, J. Guo, Y. Peng, T. Wang, X. Ge, Q. Xu,
X. Wang, Prof. H. Dai, Prof. Y. Zhang
School of Pharmacy and Jiangsu Province Key Laboratory for Inflammation
and Molecular Drug Targets, Nantong University, Nantong, 226001 (P.R.
China)
E-mail: zhangyanan@gmail.com
dh123@ntu.edu.cn
[b] Prof. Dr. Y. Ling,+ X. Wang
State Key Laboratory of Natural Medicines, China Pharmaceutical Universi-
ty, Nanjing, 210009 (P.R. China)
[c] Prof. H. Dai
College of Chemistry and Chemical Engineering, Nantong University, Nan-
tong, 226001 (P.R. China)
[+] These authors contributed equally to this work.
Supporting information for this article can be found under:
https://doi.org/10.1002/cmdc.201700133.
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Figure 1. Structures of representative hydroxamic acid based HDAC inhibitors on the market or in clinical trials.
and synthetic analogues containing b-carboline subunits are
known to have antitumor activities,[21,22] likely by DNA interca-
lation and inhibition of topoisomerase, cyclin-dependent
kinase (CDK), or the IkB kinase complex (IKK).[20,23–26] This ob-
served increase in antitumor potency of the SAHA–b-carboline
hybrids is most likely the result of simultaneous inhibition of
HDAC and induction of DNA damage. In these hybrid com-
pounds, the b-carboline and hydroxamic acid motifs were con-
nected via alkyl linkers of various length, as seen in SAHA. Con-
sidering that other HDACis such as PCI-24781 and ITF2357 con-
tain aromatic rings in the linker region (Figure 1), we reasoned
that connecting substituted b-carbolines and hydroxamic acids
with aromatic-ring-containing linkers may lead to similar in-
creased anticancer efficacy. Herein we report our efforts in the
design, synthesis, and biological evaluation of a series of novel
C3-substituted b-carboline-based hydroxamic acids (9a–l), and
investigations into the antitumor mechanism of these com-
pounds in multiple cancer cell lines. We also investigated the
effects of substitution at the amide nitrogen atom on HDAC in-
hibition and antitumor activities (Figure 2).
The general route for the synthesis of the target compounds
9a–l is depicted in Scheme 1. Reductive amination between
methyl 4-formylbenzoate (1) and various primary amines 2a–
l in the presence of acetic acid yielded imines 3a–l, which
were subsequently reduced by sodium borohydride to afford
amines 4a–l. 1-(4-Methoxylphenyl)-3-carboxyl-b-carboline
7
was prepared in a two-step sequence. First, treatment of com-
mercially available l-tryptophan 5 with a solution of 4-methox-
yphenaldehyde in acetic acid produced the intermediate 1-(4-
methoxyphenyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-
carboxylic acid (6) via Pictet–Spengler reaction, which was
then oxidized by potassium permanganate in N,N-dimethylfor-
mamide to furnish compound 7. Reaction of 7 with amines
4a–l in the presence of 1-ethyl-3-(3-dimethylaminopropyl)car-
bodiimide hydrochloride (EDCI) and 4-dimethylaminopyridine
(DMAP) afforded amide derivatives 8a–l. Finally, treatment of
Figure 2. Design of b-carboline-based hydroxamic acids 9a–l.
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Scheme 1. Synthesis of target compounds 9a–l. Reagents and conditions: a) primary amines 2a–l, HOAc, THF, RT, 1–2 h; b) NaBH4, THF, RT, 2 h, 56–73%; c) 4-
methoxybenzaldehyde, HOAc, reflux, 2–4 h, 86%; d) KMnO4, DMF, RT, 6 h, 70%; e) EDCI, DMAP, CH2Cl2, RT, 5–8 h; f) NH2OK, MeOH, RT, 10–15 h, 52–67%.
intermediates 8a–l with NH2OK gave the target compounds
9a–l. All target compounds 9a–l were purified by column
chromatography, and their structures were confirmed by IR,
1H NMR, MS, and HRMS. All compounds were of >95% purity,
as determined by high-performance liquid chromatography
(HPLC).
acid via aromatic-ring-containing linkers permits the mainte-
nance of HDAC activities. Compounds 9a, 9c, and 9h–j all dis-
played IC50 values (0.092–0.34 mm) lower than that of SAHA
(IC50 =0.48 mm) in HeLa nuclear extracts. In particular, the IC50
value of 9h (0.092 mm), the most potent compound of the
series, was five-fold lower than that of SAHA (IC50 =0.48 mm).
The inhibitory activities of these compounds against human
hepatocellular carcinoma cells (HepG2 and SMMC-7721),
human colon cancer cells (HCT116), and human lung cancer
cells (H1299) were evaluated in vitro in MTT assays. SAHA and
harmine were used as positive controls. The IC50 values of 9a–
l against the four human cancer cell lines are listed in Table 2.
Most of the target compounds displayed similar or better anti-
All target compounds were first tested against HeLa cell nu-
clear extracts, a rich source of HDAC activity, for their HDAC in-
hibitory potencies (Table 1).[27] SAHA was used as a positive
control. As expected, harmine, a b-carboline analogue with an-
titumor activity, showed no HDAC activity at concentrations up
to 10 mm. All target compounds show moderate to good po-
tency in the inhibition of HDAC, similar to that of SAHA. These
results suggest that connecting b-carboline and hydroxamic
Table 2. In vitro antiproliferative activity of compounds 9a–l against four
human cancer cell lines.
Table 1. HDAC inhibition by compounds 9a–l.
Compound
IC50 [mm][a]
Compound
R
IC50 [mm][a]
0.48ꢀ0.06
HCT116
HepG2
SMMC-7721
H1299
SAHA
harmine
9a
–
–
SAHA
harmine
9a
9b
9c
9d
9e
9 f
9g
9h
9i
9j
9k
5.53ꢀ0.68
46.7ꢀ3.92
2.34ꢀ0.32
5.60ꢀ0.64
2.24ꢀ0.21
6.26ꢀ0.51
51.2ꢀ4.76
4.10ꢀ0.37
6.73ꢀ0.71
2.15ꢀ0.36
5.61ꢀ0.57
55.3ꢀ5.01
5.78ꢀ0.65
8.94ꢀ1.02
3.42ꢀ0.31
>12.5
7.78ꢀ0.63
ND[b]
>10
(CH2)2OH
0.41ꢀ0.05
0.65ꢀ0.09
0.26ꢀ0.03
1.23ꢀ0.15
0.64ꢀ0.08
0.97ꢀ0.12
1.35ꢀ0.18
0.092ꢀ0.01
0.12ꢀ0.02
0.34ꢀ0.05
0.93ꢀ0.11
1.56ꢀ0.19
7.36ꢀ0.82
11.1ꢀ1.05
5.56ꢀ0.68
9b
9c
(CH2)2OCH3
(CH2)2CH3
9d
9e
cyclopropyl
(CH2)3OH
>12.5
>12.5
>12.5
4.89ꢀ0.13
>12.5
>12.5
6.06ꢀ0.48
>12.5
>12.5
7.89ꢀ0.85 >12.5
9 f
CH(CH3)2
>12.5
>12.5
>12.5
>12.5
9g
9h
9i
9j
CH2CH(CH3)2
(CH2)2N(CH3)2
(CH2)2N(CH2CH3)2
(CH2)3CH3
1.79ꢀ0.15
1.60ꢀ0.18
2.46ꢀ0.31
4.25ꢀ0.58
1.81ꢀ0.26
3.02ꢀ0.26
3.27ꢀ0.29
3.37ꢀ0.52
4.05ꢀ0.38
5.25ꢀ0.60
6.63ꢀ0.70
5.37ꢀ0.61
9k
(CH2)4OH
>12.5
>12.5
>12.5
>12.5
>12.5
>12.5
>12.5
>12.5
9l
(CH2)2O(CH2)2OH
9l
[a] Determined in HeLa cell nuclear extracts; data are the meanꢀSD of
[a] Determined by MTT assay; data are the meanꢀSD of three independ-
three independent experiments.
ent experiments. [b] Not detected.
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proliferative activity against the tested cells than that of SAHA,
and significantly more potent than harmine. The IC50 values of
9a, 9c, and 9h–j, compounds with low-micromolar potencies
in HDAC inhibition, were also in the low-micromolar range
(1.60–7.36 mm) in antiproliferation. These values were signifi-
cantly lower than both SAHA and harmine. Consistent with the
HDAC inhibitory activities, 9h [R=(CH2)2NMe2] was also the
most potent compound among the group in antiproliferative
activities, with IC50 values of 1.60–4.25 mm, are were lower than
those of SAHA (IC50: 5.53–7.78 mm) in all four cell lines.
Considering the significant growth inhibitory activity of 9h
in vitro, the general toxicity of 9h was further evaluated in
normal human LO2 cells. The treatment of non-tumor LO2
cells with 9h showed no apparent growth inhibitory activity at
doses up to 25 mm, the same dose that induced the majority
of HepG2 cell death, suggesting 9h possess no significant gen-
eral toxicity to normal human cells.
The fact that all target compounds are active in HDAC inhib-
ition and antitumor assays suggests that linkers with aromatic
rings, like the alkyl linkers previously studied, are well tolerated
for activity. In general, the potencies of these compounds are
similar to that of SAHA, but significantly more potent than har-
mine, confirming the importance of the hydroxamic acid for
activity. The SARs of this compound series show remarkable re-
semblance in their inhibition of HDAC and cancer cell prolifera-
tion. The size and composition of the substituents on the
amide nitrogen atom have a clear impact on the activities of
these compounds. Compounds with straight-chain alkyl
groups (9c, 9j) are favored. However, if a terminal hydroxy
group is introduced, a decrease in potency is observed (9c vs.
9e, 9j vs. 9k). Introduction of an oxygen atom within the
chain slightly decreases potency (9b vs. 9c vs. 9j). Bulkier sub-
stituents such as those of 9d, 9 f, and 9g also resulted in de-
creased potency in both HDAC and antiproliferation assays. In-
terestingly, if a nitrogen atom was introduced, the potency sig-
nificantly increased (9h, 9i), particularly in the inhibition of
HDAC activity. This is likely the result of hydrogen bonding in-
teractions between the hydrogen and the hydrogen bonding
donating residue(s) of the target.
Figure 3. Immunoblot analysis of the acetylation of histone H3 and a-tubu-
lin in vitro. a) HepG2 cells were treated with 9h and SAHA for 48 h at the in-
dicated concentrations. Cell lysates were prepared and subjected to SDS-
PAGE and immunoblot analysis using anti-acetyl-histone H3, anti-acetyl-a-tu-
bulin, and anti-b-actin antibodies. b-Actin was used as a loading control.
b) Quantitative analysis. The levels of Ac-H3 and Ac-a-tubulin relative to b-
actin control were determined by densitometric scanning. Data are the
meansꢀSD of three separate experiments; *p<0.01 vs. control.
decrease in absorption, is observed when a small molecule
binds to DNA by intercalation, resulting from a strong p–p
stacking interaction between an aromatic chromophore from
the molecule and the base pairs of DNA.[24] In our study, the
9h–CT-DNA system showed absorption bands at l 250, 300,
and 360 nm, with the maximum absorption at 300 nm
(Figure 4). Upon addition of equal increments of CT-DNA to
a solution of 9h, the intensity of all three absorption bands
gradually decreased, with the strongest hypochromicity ob-
served at 300 nm. These results clearly indicate electrostatic
binding of compound 9h to CT-DNA via intercalation.
Given that the inhibition of HDACs by these compounds en-
hances the antiproliferative activity against tumor cells, the
HDAC inhibitory effects of the most active compound 9h on
the levels of acetylation of histone H3 and a-tubulin were de-
termined by immunoblotting assays using b-actin as a negative
control (Figure 3). HepG2 cells were incubated with the vehicle
alone, SAHA (5.0 mm), or 9h (0.8, 1.6, and 3.2 mm). Compared
with the control group, compound 9h increased the expres-
sion of acetyl-histone H3 and acetyl-a-tubulin in a dose-depen-
dent manner. Levels of acetyl-histone H3 in the groups treated
with 9h at 1.6 and 3.2 mm were higher than the values from
the group treated with 5.0 mm SAHA, consistent with the re-
sults from the HDAC fluorimetric activity assay.
It is known that the planar structure of b-carbolines can
bind to DNA and induce DNA damage.[23,24] To investigate
whether the anticancer activities of these b-carboline deriva-
tives resulted partly from DNA binding, UV/Vis spectroscopic ti-
tration studies were performed. In general, hypochromism, or
Figure 4. UV/Vis absorption spectra of 9h (20 mm) in the presence of increas-
ing amounts of calf thymus (CT)-DNA. Arrows indicate the changes in ab-
sorbance with increasing DNA concentrations.
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To further investigate whether DNA damage contributes to
the anticancer activities of these b-carboline derivatives, we ex-
amined the extent of DNA damage caused by 9h in HepG2
cells. We used histone H2AX phosphorylation as the DNA
damage marker. Harmine and SAHA were used as positive con-
trols. HepG2 cells were treated with vehicle or 9h for 48 h.
H2AX (S139ph) levels were then detected by western blot anal-
ysis. As shown in Figure 5, treatment with 9h dose-dependent-
ly increased the levels of H2AX phosphorylation in HepG2
cells, significantly higher than that of the SAHA group. Because
DNA damage induced by the b-carboline complex is associated
with H2AX (S139ph) activation, these results clearly indicate
that these new b-carboline-based hydroxamic acid derivatives
are involved in DNA damage.
It is known that the Stat3, Akt, and ERK signaling pathways
are important cell-growth-promoting pathways which are aber-
rantly activated in most cancers.[28,29] To gain greater insight
into the molecular mechanisms that underlie the activity of
9h, we examined the regulatory effects of 9h on Stat3, Akt,
and ERK signaling in HepG2 cells using SAHA as a control. The
cells were treated with various concentrations of 9h or SAHA.
The expression and activation of Stat3, AKT, and ERK1/2 were
determined by western blotting. As shown in Figure 6, treat-
ment with 9h significantly inhibited Stat3, Akt, and ERK1/2
phosphorylation in a dose-dependent manner, although it did
not alter the levels of Stat3, Akt, and ERK1/2 expression in
cancer cells. Similarly, treatment with SAHA also significantly
decreased the levels of Stat3, Akt, and ERK1/2 phosphorylation,
although to a lesser extent than 9h. Previous studies have
shown that aberrant activation of Stat3 and Akt are associated
with the development of human cancer, and SAHA can inhibit
the proliferation of cancer cells by downregulating Stat3 and
Akt activation in cancer cells.[30–32] Compound 9h may share
the same mechanisms with SAHA, but further investigations
are needed.
Figure 5. Immunoblot analysis of DNA damage in vitro. a) HepG2 cells were
treated with vehicle (control), various doses of 9h, or SAHA. Cells were ho-
mogenized, and their lysates were subjected to immunoblot analysis using
anti-H2AX (S139ph) and anti-b-actin antibodies. b-Actin was used a loading
control. b) Quantitative analysis. The levels of each signaling event relative
to b-actin control were determined by densitometric scanning. Data are the
meansꢀSD of three separate experiments; *p<0.01 vs. control.
tion, and it is commonly accepted that insufficient drug solu-
bility can lead to poor oral absorption.[33] The most potent
compound of the series, 9h, demonstrated good aqueous sol-
ubility (32.9 mgmLꢁ1). SAHA was previously reported to have
an approximate solubility of 0.5 mgmLꢁ1 in a 1:1 solution of
DMSO/PBS (pH 7.2).[34] The transport of compound 9 was mea-
sured to evaluate the intestinal permeation of these com-
pounds. The human colon adenocarcinoma Caco-2 cell line is
the most widely used in vitro model that mimics the absorp-
tive properties of the intestinal epithelium.[35] Experiments
were conducted at 378C, and measurements were performed
over a 90 min incubation period. Compound 9h showed mod-
erate permeability, with the rate of transport (Papp A!B) of
To assess the drug-likeness of this class, we conducted pre-
liminary in vitro pharmacokinetics studies on compound 9h.
Solubility is an important parameter to predict drug absorp-
Figure 6. Effects of 9h and SAHA on Stat3, Akt, and ERK signaling in HepG2 cells. Cells were treated with vehicle or the indicated test compound for 48 h,
and the relative levels of Stat3, Akt, and ERK1/2 expression and phosphorylation were determined by western blot assays using b-actin as a control. a) West-
ern blot analysis of the relative levels of Stat3, Akt, and ERK expression and phosphorylation. b) Quantitative analysis. Data are the meansꢀSD of each group
of cells from three separate experiments; *p<0.01 vs. vehicle-treated control.
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0.58ꢁ10ꢁ6 cmsꢁ1 at 10 mm, which is three-fold lower than the
reported value for SAHA (1.70ꢁ10ꢁ6 cmsꢁ1).[36]
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line structural moiety, as evidenced by hypochromism and en-
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also found to inhibit Stat3, Akt, and ERK signaling, pathways
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Acknowledgements
We gratefully acknowledge financial support by the Natural Sci-
ence Foundation of China (Grant Nos. 81302628 and 81473089),
the Project of “Jiangsu Six Peaks of Talent” (2014-SWYY-044 and
SWYY-CXTD-008), the China Pharmaceutical University for the
Open Project Program of State Key Laboratory of Natural Medi-
cines (SKLNMKF201611), the China and Jiangsu Province Postdoc-
toral Science Foundation (2016M590488 and 1601136B), the
Jiangsu Government Scholarship for Overseas Studies (JS-2014-
212), Jiangsu Key Training Programs of Innovation and Entrepre-
neurship for Undergraduates (201610304055Z), Applied Research
Projects of Nantong City (MS12015060), and we are also thankful
for a project funded by the Priority Academic Programs Develop-
ment of Jiangsu Higher Education Institutions (PAPD).
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Conflict of interest
The authors declare no conflict of interest.
Manuscript received: February 26, 2017
Revised: April 4, 2017
Keywords: antitumor agents · b-carboline · DNA damage ·
histone deacetylase · inhibitors
Accepted Article published: April 4, 2017
Final Article published: && &&, 0000
&
ChemMedChem 2017, 12, 1 – 7
www.chemmedchem.org
6
ꢀ 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!

COMMUNICATIONS
It takes two: A series of hydroxamic
acid histone deacetylase (HDAC) inhibi-
tors containing b-carboline motifs were
designed and synthesized. The most
potent compound was found to in-
crease the acetylation of histone H3 and
a-tubulin, to induce DNA damage, and
to inhibit important cell growth signal-
ing pathways such as Stat3, Akt, and
ERK. Our findings suggest that these
new b-carboline-based HDAC inhibitors
have great potential as therapeutic
agents for the treatment of human can-
cers.
Y. Ling, J. Feng, L. Luo, J. Guo, Y. Peng,
T. Wang, X. Ge, Q. Xu, X. Wang, H. Dai,*
Y. Zhang*
&& – &&
Design and Synthesis of C3-
Substituted b-Carboline-Based Histone
Deacetylase Inhibitors with Potent
Antitumor Activities
ChemMedChem 2017, 12, 1 – 7
www.chemmedchem.org
7
ꢀ 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
These are not the final page numbers! ÞÞ