Development of novel β-carboline-based hydroxamate derivatives as HDAC inhibitors with DNA damage and apoptosis inducing abilities

Article abstract of DOI:10.1039/c6md00681g

A series of novel β-carboline-based hydroxamate derivatives (8a-n) as HDAC inhibitors have been designed and synthesized. Most of these compounds displayed potent histone deacetylase inhibitory effects and good antiproliferative activity with IC₅₀

Full text of DOI:10.1039/c6md00681g

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Development of novel βꢀcarbolineꢀbased hydroxamate derivatives as HDAC
inhibitors with DNA damage and apoptosis inducing ability‡
Ji Liua,†, Tingting Wanga,†, Xinyang Wanga,b,†, Lin Luoa, Jing Guoa, Yanfu Penga, Qibing Xua,
Jiefei Miaoa,c, Yanan Zhanga,*, Yong Linga,b,*
aSchool of Pharmacy and Jiangsu Province Key Laboratory for Inflammation and Molecular Drug
Target, Nantong University, Nantong 226001, P.R. China;
bState Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009,
P.R. China;
cTumorꢀchemotherapy Department, Affiliated Hospital, Nantong University, Nantong 226001,
P.R. China
†These authors contributed equally to this work.
*Correspondence and requests for materials should be addressed to Yong Ling and Yanan Zhang,
Email: Lyyy111@sina.com, zhangyanan@gmail.com.
‡The authors declare no competing interests.
Abstract: A series of novel βꢀcarbolineꢀbased hydroxamate derivatives (8a−n) as HDAC
inhibitors have been designed and synthesized. Most of these compounds displayed potent histone
deacetylase inhibitory effects and good antiproliferative activity with IC50’s in the low micromole
range. One of the most potent compounds 8k showed the strongest inhibition of the proliferation
of human hepatocellular carcinoma (HCC) cells in vitro, with IC50 values lower than currently
approved HDAC inhibitor SAHA. Compound 8k also increased acetylation of histone H3 and
αꢀtubulin, consistent with its potent HDAC inhibition. Importantly, 8k induced hypochromism by
electrostatical interaction with CTꢀDNA, suggesting potential induction of DNA damage. Finally,
8k significantly induced HepG2 cell apoptosis by regulating apoptotic relative proteins expression.
Together, our findings suggest that these novel βꢀcarbolineꢀbased hydroxamate derivatives may
provide a new framework for the discovery of novel antitumor agents for the intervention of
human carcinoma cells.
Keywords: Synthesis; Antitumor activities; Apoptosis; DNA damage; βꢀCarboline derivatives;
Histone deacetylases inhibitors
Introduction
Many small molecules could interact with DNA through intercalation; surface binding; minor
groove, or major groove binding.1 Studies on DNA interacting compounds in cancer
chemotherapy have led to the development of many novel heterocyclic compounds as
potential anticancer agents. In fact, anticancer drugs that target DNA are one of the most
effective agents in clinical usage and have enhanced the survival rates of patients when used
alone or in combination with other drugs.2 These agents exert their biological profile by
nonꢀcovalent or covalent interactions in either the minor or major groove or between the base
pairs (intercalation) of the double helix. As a result, a number of novel DNA interacting
scaffolds such as anthracyclines, acridines, anthraquinones, distamycins, βꢀcarbolines have
been developed in antiꢀcancer drug discovery efforts.3

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βꢀCarbolines are alkaloids that contain a planar tricyclic pyridoꢀ[3,4ꢀb]indole ring structure.
They are widely distributed in plants, marine creatures, and mammals, as well as human
tissues and bodily fluids.4 Many naturally occurring βꢀcarboline alkaloids and synthetic
analogues containing βꢀcarboline subunit have been reported to inhibit cancer cell growth,
and lead to apoptosis through multiple mechanisms such as DNA intercalation, inhibition of
DNA topoisomerases I and II, CDK, PLK, and ABCG2.5ꢀ10 DNA intercalation is one of the
important mode of action in clinical oncology and many drugs with this mechanism are
currently used for the treatment of various cancers.11 Several βꢀcarbolines including harmane,
norharman, and βꢀcarboline–benzimidazole conjugates have been reported to intercalate with
DNA, alter DNA replication fidelity, and influence enzymatic activities in DNA repair
processes due to the presence of planar polycyclic aromatic pharmacophore, which is capable
of stacking between DNA base pairs.5ꢀ7,12,13
Inhibition of histone deacetylases (HDACs) has long been considered a promising
therapeutic approach to the treatment of cancer. It has been shown that HDAC inhibitors
(HDACi) significantly suppress cancer cell proliferation, angiogenesis, and metastasis, and
induce apoptosis through multiple mechanisms, including changes in gene expression and
alterations of both histone and nonꢀhistone proteins.14,15 Five HDACi have been approved for
cancer treatment to date. Despite promising results in the treatment of cutaneous Tꢀcell
lymphoma, the two US FDA approved HDACi’s, Vorinostat (SAHA)16 and romidepsin,17
have not been proven effective in clinical trials involving solid tumors (Fig. 1).18,19 Other
HDAC inhibitors that have recently been approved include belinostat (PXD101), panobinostat
(LBH589), and chidamide (Epidaza), for the treatment of PTCL or multiple myeloma,20ꢀ22
although their effects on solid tumors have not been examined.
O
H
H
H
N
N
N
N
H
OH
S
OH
O
O
O
O
PXD101
Cap group
Linker
SAHA, vorinostat
O
ZBG
HN
N
H
N
H
N
H
NH2
H
N
OH
N
O
LBH589
O
Chidamide
O
N
F
O
OH
OH
N
N
H
H
H
N
N
N
O
Nexturastat A
Tubastatin A
Fig. 1 Structures of clinically approved HDAC inhibitors in clinical trials, and some
representative HDAC inhibitors with the benzylic linker.
It has been widely reported that HDACi can synergistically enhance the inhibitory effect of
other antitumor agents, which target tubulin, EGFR, DNA, or topoisomerase,23ꢀ30 in
suppressing proliferation and inducing apoptosis in tumor cells. HDAC inhibitors have
therefore been pursued in the design of multifunctional inhibitors to simultaneously interact

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with multiple targets with high potency and low toxicity.26ꢀ28 Given the DNA damage effects
of βꢀcarbolines, the combination of HDACi and βꢀcarboline alkaloids may represent a
promising strategy to achieve increased anticancer efficacy. A key feature of many potent and
selective HDAC inhibitors is the presence of the benzylic linker built into the canonical
inhibitor structure of the common CAPꢀlinkerꢀzinc binding group scaffold, as seen in
nexturastat A, tubastatin A, chidamide (Fig. 1). Thus, we aimed to integrate the key structural
elements of βꢀcarboline alkaloids and HDACi into one single structure to form novel
βꢀcarboline derivatives that may simultaneously inhibit DNA and HDAC and provide
improved antitumor activity (Fig. 2). We herein report the synthesis and biological evaluation
of a series of novel hydroxamic acidꢀbased βꢀcarboline derivatives (8a−n), as well as the
investigation on their antiꢀtumor mechanisms in multiple cancer cell lines.
Fig. 2 The design of novel hydroxamateꢀbased βꢀcarboline derivatives 8aꢀn.
Results and Discussion
Chemistry. The synthetic route to compounds 8aꢀn is depicted in Scheme 1. The starting
methyl 4ꢀformylbenzoate 1 reacted with different alkyl amines in the presence of acetic acid
to afford the imines, which were then reduced by NaBH4 to give the secondary amines 2aꢀd.
Substituted βꢀcarbolines 6aꢀd were prepared in a twoꢀstep sequence. First, commercially
available Lꢀtryptophan 4 was converted to 1ꢀsubstitutedꢀ1,2,3,4ꢀtetrahydroꢀβꢀcarbolineꢀ
3ꢀcarboxylic acid 5aꢀd in a PictetꢀSpengler reaction with the treatment of differently
substituted aldehydes (formaldehyde, acetaldehyde or 4ꢀmethoxyphenaldehyde).
Intermediates 5aꢀd were then oxidized by KMnO4 in DMF to afford compounds 6aꢀd, which
then reacted with secondary amines 3aꢀd in the presence of 1ꢀethylꢀ3ꢀ
(3ꢀdimethylaminopropyl)carbodiimide hydrochloride (EDCI) and 4ꢀdimethylꢀ aminoꢀpyridine
(DMAP) to produce amides 7aꢀn. Finally, intermediates 7aꢀn were treated with NH2OK to
give the target compounds 8aꢀn. All target compounds were purified by column
chromatography, and their structures were confirmed by 1H NMR, MS, and HRMS.

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Scheme 1. Synthetic routes of target compounds 8aꢀn. Reagents and conditions: (a) (i) HOAc,
THF, rt, 1ꢀ2h, (ii) NaBH4, THF, rt, 2h, 56ꢀ73%; (b) H+ or OHꢀ, RCHO, reflux, 2ꢀ4 h, 81ꢀ86%; (c)
KMnO4, DMF, rt, 6h, 67ꢀ75%; (d) EDCI, DMAP, CH2Cl2, rt, 5ꢀ8h; (e) NH2OK, MeOH, rt, 10ꢀ15h,
52−67%.
In vitro biological evaluations. All target compounds were first tested against HeLa cell
nuclear extract, a rich source of HDAC activity, for their HDAC inhibitory potencies,31 using
SAHA, an FDA approved HDAC inhibitor, as the positive control. The IC50 values of compounds
8aꢀn against HDAC in HeLa cell nuclear extract are listed in Table 2. Harmine showed IC50 value
greater than 10 ꢁM. All target compounds showed HDAC inhibitory effects with IC50 values in the
low micromolar range.
All the synthesized compounds 8aꢀn were then screened for their cancer cell growth
inhibitory activitiy against human hepatocellular carcinoma cells (HepG2, SMMCꢀ7721) and
human colon cancer cells (HCT116, LOVO) using MTT assay. βꢀCarboline analog harmine
and SAHA were used as reference compounds. The IC50 values of 8aꢀn against the four
human cancer cell lines are listed in Table 1. SAHA significantly inhibited the proliferation of
all four types of cells, while harmine showed limited potency.
These compounds were designed to evaluate the structureꢀactivity relationships (SARs) at
the C1 position of the βꢀcarbolines, as well as the Nꢀalkylation of the C3 amide. The results
clearly demonstrate the impact of C1 substitution on both the HDAC inhibition and
antiproliferation. For HDAC inhibition (Table 1), compounds 8aꢀd with R = H as in
norhamane were less potent than 8eꢀh with R = Me as in harmane. The methoxyphenyl series
(8iꢀn) were more potent than the previous two series. In anticancer proliferation, a similar
trend is present. Compounds 8aꢀd (R = H) and 8eꢀh (R = Me) all showed limted
antiproliferative activities. Compounds derivatives (8iꢀn) with a methoxyphenyl group at the
C1 positions, as present in the previously reported βꢀcarbolineꢀbenzimidazole conjugates,10
markedly inhibited human cancer cell growth, showing greater antiproliferative activity
against the tested cells than harmine.

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Within each subseries (R = H, Me, or methoxyphenyl), IC50 values in HDAC inhibition
first increased then decreased with the elongation of the Nꢀalkyl substitution, with propyl
being the most potent compounds of each subseries. Compounds 8k and 8m were the most
active analogues, with an IC50 value of 0.26 ꢁM and 0.23 ꢁM against the nuclear extract,
respectively. In antiproliferation against 4 cancer cells, the propyl analogs (8k and 8m) were
also the most active compounds. Compound 8k exhibited antiꢀproliferative activities against
HepG2 cells with IC50 values of 2.15 ꢁM, which is threeꢀfold lower than SAHA (IC50 = 6.26
ꢁM). Although 8k was slightly less potent than 8m in HDAC inhibition, it was more potent in
antiꢀproliferation.
Table 1. Compounds 8aꢀn and their HDAC inhibition
nuclear extract
Compd.
R
R’
(IC50a, ꢁM)
0.48 ± 0.06
>10
SAHA
harmine
8a
ꢀ
ꢀ
ꢀ
ꢀ
H
CH3
5.63 ± 0.65
4.78 ± 0.61
2.82 ± 0.37
2.69 ± 0.30
3.06 ± 0.33
1.75 ± 0.28
0.85 ± 0.31
0.93 ± 0.22
0.96 ± 0.13
0.83 ± 0.10
0.26 ± 0.03
0.34 ± 0.05
0.23 ± 0.04
0.37 ± 0.08
8b
H
CH2CH3
8c
H
CH2CH2CH3
CH2CH2CH2CH3
CH3
8d
H
8e
Me
8f
Me
CH2CH3
8g
Me
CH2CH2CH3
CH2CH2CH2CH3
CH3
8h
Me
8i
pꢀMeOPh
pꢀMeOPh
pꢀMeOPh
pꢀMeOPh
mꢀMeOPh
mꢀMeOPh
8j
CH2CH3
8k
CH2CH2CH3
CH2CH2CH2CH3
CH2CH2CH3
CH2CH2CH2CH3
8l
8m
8n
a The data are expressed as the mean ± SD of three independent experiments data.
Table 2. IC50 values of compounds 5kꢀo and 11aꢀj against four CRC cell linesa
In vitro antiproliferative activity (IC50a, ꢁM)
Compd.
HCT116
5.53 ± 0.68
46.7 ± 3.92
>12.5
HepG2
6.26 ± 0.51
51.2 ± 4.76
>12.5
SUMMꢀ7721
5.61 ± 0.57
55.3 ± 5.01
>12.5
LOVO
6.75 ± 0.58
53.2 ± 4.35
bND
SAHA
harmine
8a
8b
>12.5
>12.5
>12.5
ND
8c
>12.5
>12.5
>12.5
ND
8d
>12.5
>12.5
>12.5
ND
8e
>12.5
>12.5
>12.5
>12.5
8f
>12.5
>12.5
>12.5
>12.5
8g
10.5 ± 0.93
9.83 ± 0.86
11.6 ± 1.28
>12.5

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8h
8i
>12.5
11.7 ± 1.35
10.5 ± 1.21
8.34 ± 0.76
2.15 ± 0.36
3.37 ± 0.52
3.64 ± 0.59
5.16 ± 0.62
>12.5
>12.5
9.76 ± 1.09
6.07 ± 0.58
2.24 ± 0.21
3.02 ± 0.26
2.96 ± 0.30
4.32 ± 0.41
>12.5
>12.5
8j
10.3 ± 0.87
3.42 ± 0.31
5.25 ± 0.60
4.12 ± 0.36
5.75 ± 0.58
9.24 ± 1.10
5.07 ± 0.63
4.76 ± 0.55
5.31 ± 0.52
7.63 ± 0.67
8k
8l
8m
8n
a
The inhibitory effects of individual compounds on the proliferation of cancer cell lines were
determined by the MTT assay. The data are expressed as the mean ± SD of three independent
experiments data. bND: not detected.
Acetylation of histone H3 and αꢀtubulin. Given that the inhibition of HDACs by
βꢀcarboline derivative 8k enhanced the tumor cell antiproliferative activity, we further
investigated whether 8k induces acetylation of histones in HCC cells. The cells were treated
with different concentrations of 8k, the levels of acetylation of histone H3 and αꢀtubulin were
analyzed by western blotting assays using βꢀactin as the negative control (Fig. 3). HepG2 cells
were incubated with the vehicle alone, SAHA (5.0 ꢁM), or 8k (1.0, 2.0, and 4.0 ꢁM) for 48h.
Compared to the control group, compound 8k demonstrated the ability to increase the
expression of Acꢀhistone H3 and Acꢀαꢀtubulin correlating with 8k dose of treatment. Level of
Acꢀhistone H3 and Acꢀαꢀtubulin in 8k treated groups (2.0 and 4.0 uM) were higher than or
equivalent to the values from the 5.0 µM SAHA treated group, which was consistent with the
results from the HDAC fluorimetric activity assay. The above results about Acꢀhistone H3
and Acꢀαꢀtubulin clearly showed that 8k was not selective against HDAC1/2/3/6. Therefore,
the selective profile of 8k against other representative HDACs isoforms, such as HDAC4 and
HDAC8, was tested. However, 8k showed rather weak HDAC4 inhibition with the IC50
values of >10 ꢁM and moderate HDAC8 inhibitory activity (IC50 = 0.76 ꢁM), which were
similar to SAHA.
Fig. 3 The expression of the acetylation for histone H3 and αꢀtubulin was examined by western
blot analysis in vitro. (A) HepG2 cells were treated with 8k and SAHA for 48 h at the indicated
concentrations, and the levels of protein expression were detected using antiꢀacetylꢀhistone H3,
antiꢀacetylꢀαꢀtubulin, and antiꢀβꢀactin antibodies, respectively. βꢀActin was used as the loading
control. Data shown are representative images of each protein for three separate experiments. (B)
Quantitative analysis. The relative levels of AcꢀH3 and Acꢀαꢀtubulin used to control βꢀactin were
determined by densimetric scanning. The data are expressed as means ± SD of three separate
experiments. *P< 0.01 vs control.

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0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
with increasing DNA
200
250
300
350
400
450
500
Wavelength (nm)
Fig. 4 UVꢀvisible absorption spectra of 8k in the presence of increasing amounts of CT DNA.
Arrows indicate the changes in absorbance with increasing the concentration of DNA. [8k] = 20
ꢁM.
DNA binding studies by UVꢀvisible spectroscopic analysis. It is generally accepted that
the planar structured βꢀcarbolines can bind to DNA and induce DNA damage.11,12 The SAR
results clearly showed the contribution of HDAC inhibition to the antitumor activities. To
investigate whether the antitumor activities of these active βꢀcarboline derivatives also arise
partly from their interactions with DNA, UV–visible spectroscopic titration studies were
performed. In general, hypochromism, or reduction in absorption, is observed when a small
molecule binds to DNA through intercalation, resulting from a strong π–π stacking interaction
between an aromatic chromophore from the molecule and the base pairs of DNA.24 In our
study, the 8kꢀCTꢀDNA system showed absorption bands at 260 nm, 300 nm and 365 nm (Fig.
4). Upon addition of equal increments of CTꢀDNA to 8k solution, the intensity of all 3
absorption bands gradually decreased. The maximum absorption was at 300 nm, and the
strongest hypochromicity was also observed at this wave length. A similar trend was observed
in harmine, with the maximum absorption at 320 nm. However, the absorption slightly
increased with SAHA upon addition of incremental CTꢀDNA (see Supporting Information).
These results clearly indicate electrostatic binding of compound 8k to CTꢀDNA with
intercalation.
Compound 8k Induces Apoptosis in HepG2 Cells. To determine whether the inhibitory
effects of 8k on liver cancer cellular proliferation are accompanied by enhanced cancer cell
apoptosis, FITCꢀAnnexin V/Propidium Iodide (PI) staining and flow cytometry assay were
performed, and the percentages of apoptotic cells were determined. HepG2 cells were
incubated with different concentrations of 8k or SAHA for 48h. As shown in Fig. 5, 8k
treated HepG2 cells exhibited a doseꢀdependent increase (P < 0.01) of apoptosis by 18.2%,
35.2%, and 61.8% at 1.0 µM, 2.0 µM, and 4.0 µM, respectively. The 61.8% induction of
HepG2 cell apoptosis with 4.0 ꢁM of 8k was significantly higher than SAHA of 5.0 ꢁM
(34.5% apoptotic cells).

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To further investigate the apoptosis induction of 8k, we examined the expression of
apoptotic proteins Bax, Bclꢀ2, and the cleavage states of caspaseꢀ3 and PARP, in response to
8k treatment (Fig. 6). Subꢀconfluent HepG2 liver cancer cells were treated with or without 8k
for 48h, and then lysed and analyzed by western blot. βꢀActin expression was used as an
internal control. It was revealed that treatment with 8k dramatically increased the relative
levels of proꢀapoptotic Bax expression, but reduced the levels of antiꢀapoptotic Bclꢀ2
expression (Fig. 6A) in a doseꢀdependent manner. Furthermore, compound 8k resulted in
more significant cleavage of both PARP and caspaseꢀ3 than the control group in Fig. 6B.
Importantly, 8k treatment also induced more cleavage of PARP and caspaseꢀ3 than the SAHA
treated group. Taken together, these results confirm that 8k treatment induced apoptosis
associated with cleavage of caspases 3 and PARP in HepG2 cells.
Fig. 5 Compound 8k induces HepG2 cell apoptosis in vitro. HepG2 cells were incubated with the
indicated concentrations of 8k, or SAHA (5.0 ꢁM) for 48 h, and the cells were stained with
FITCꢀAnnexin V/PI, followed by flow cytometry analysis. (A) Flow cytometry analysis. (B)
Quantitative analysis of apoptotic cells. Data are expressed as means ± SD of the percentages of
apoptotic cells from three independent experiments. *P< 0.01 vs control.
Fig. 6 The expression of Bax, Bclꢀ2, cleaved Caspase 3, PARP, and βꢀactin was examined by
western blot analysis. (A) HepG2 cells were incubated with, or without, 8k and SAHA at the
indicated concentrations for 48 h and the levels of protein expression were detected using specific
antibodies. Data shown are representative images of each protein for three separate experiments.
(B) Quantitative analysis of Bax, Bclꢀ2, cleaved Caspase 3, and cleaved PARP. The relative levels

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of each protein compared to control βꢀactin were determined by densimetric scanning. Data are
expressed as means ± SD from three separate experiments. *P < 0.01 vs respective control.
Preliminary in vitro pharmacokinetic Studies. In order to assess the drugꢀlikeness of this
class, we have conducted preliminary in vitro pharmacokinetic studies on compound 8k.
Solubility is an important parameter to predict drug absorption and it is commonly accepted
that insufficient drug solubility can lead to poor oral absorption. The most potent compound
8k of the series demonstrated moderate aqueous solubility (11.8 mg·mL−1). We also evaluated
the metabolic stability of 8k in incubation with rat liver microsomes (BD Gentest). The
compound concentration was 1 ꢁM and the final liver microsomal protein concentration was
0.5 mg/mL. The percentage remaining of 8k was followed using LCꢀMS and the halfꢀlife (t1/2)
was determined using the slope of the linear regression method. In this assay, 8k showed a t1/2
of 20.43 min, which is comparable to the reported t1/2 of SAHA (44 min) with a test
concentration of 5 ꢁM. These results are consistent with the fact that 8k and SAHA share
similar sites of metabolism such as the hydroxamic acid moiety and the amide group.
Conclusion
In summary, we designed and synthesized a series of βꢀcarbolineꢀbased hydroxamate
derivatives (8a−n) and investigated their in vitro biological activities. Most of these
compounds displayed significant histone deacetylase inhibitory effects and good
antiproliferative activity with IC50 values in the low micromole range. Their activities are
clearly impacted by the substitution at the C1 position and the amide Nꢀalkylation. Compound
8k with a C1ꢀmethoxyphenyl group and Nꢀpropyl was the most potent of the series and
possess greater antiproliferative activity against cancer cells than SAHA. In addition,
compound 8k doseꢀdependently increased the acetylation of histone H3 and αꢀtubulin, as
expected with HDAC inhibitors. The increased antiproliferative activity of 8k most likely
resulted partly from induced DNA damage by the βꢀcarboline structural moiety, as evidenced
by the hypochromism observed in the DNA binding studies by UVꢀvisible spectroscopic
analysis. Further, 8k could significantly induce cell apoptosis by reducing the antiꢀapoptotic
protein levels of Bclꢀ2 and upꢀregulating the proꢀapoptotic proteins of Bax and the cleavage
of caspases 3 and PARP. Finally, 8k showed reasonable solubility and metabolic stability. Our
findings suggest that βꢀcarbolineꢀbased hydroxamates such as 8k may hold promise as potential
therapeutic agents for the intervention of human cancers.
Acknowledgements
We gratefully acknowledge the financial support by the Natural Science Foundation of China
(Grant Nos. 81302628 and 81473089), the Project of “Jiangsu Six Peaks of Talent”
(2014ꢀSWYYꢀ044 and SWYYꢀCXTDꢀ008), China Pharmaceutical University for the Open
Project Program of State Key Laboratory of Natural Medicines (SKLNMKF201611), China and
Jiangsu Province Postdoctoral Science Foundation (2016M590488 and 1601136B), Jiangsu
Government Scholarship for Overseas Studies (JSꢀ2014ꢀ212), Applied Research Projects of
Nantong City (MS12015060), and also thank a project funded by the Priority Academic Programs
Development of Jiangsu Higher Education Institutions (PAPD).

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