Hybrid inhibitors of DNA and HDACs remarkably enhance cytotoxicity in leukaemia cells

Article abstract of DOI:10.1080/14756366.2020.1754812

Chlorambucil is a nitrogen mustard-based DNA alkylating drug, which is widely used as a front-line treatment of chronic lymphocytic leukaemia (CLL). Despite its widespread application and success for the initial treatment of leukaemia, a majority of patients eventually develop acquired resistance to chlorambucil. In this regard, we have designed and synthesised a novel hybrid molecule, chloram-HDi that simultaneously impairs DNA and HDAC enzymes. Chloram-HDi efficiently inhibits the proliferation of HL-60 and U937 leukaemia cells with GI₅₀ values of 1.24 μM and 1.75 μM, whereas chlorambucil exhibits GI₅₀ values of 21.1 μM and 37.7 μM against HL-60 and U937 leukaemia cells, respectively. The mechanism behind its remarkably enhanced cytotoxicity is that chloram-HDi not only causes a significant DNA damage of leukaemia cells but also downregulates DNA repair protein, Rad52, resulting in the escalation of its DNA-damaging effect. Furthermore, chloram-HDi inhibits HDAC enzymes to induce the acetylation of α-tubulin and histone H3.

Full text of DOI:10.1080/14756366.2020.1754812

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Hybrid inhibitors of DNA and HDACs remarkably
enhance cytotoxicity in leukaemia cells
Yoojin Song, Sun You Park, Zhexue Wu, Kwang-Hyeon Liu & Young Ho Seo
To cite this article: Yoojin Song, Sun You Park, Zhexue Wu, Kwang-Hyeon Liu & Young
Ho Seo (2020) Hybrid inhibitors of DNA and HDACs remarkably enhance cytotoxicity in
leukaemia cells, Journal of Enzyme Inhibition and Medicinal Chemistry, 35:1, 1069-1079, DOI:
10.1080/14756366.2020.1754812
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2020, VOL. 35, NO. 1, 1069–1079
https://doi.org/10.1080/14756366.2020.1754812
RESEARCH PAPER
Hybrid inhibitors of DNA and HDACs remarkably enhance cytotoxicity in
leukaemia cells
Yoojin Songa, Sun You Parka, Zhexue Wub, Kwang-Hyeon Liub
and Young Ho Seoa
b
aCollege of Pharmacy, Keimyung University, Daegu, Republic of Korea; BK21 Plus KNU Multi-Omics based Creative Drug Research Team,
College of Pharmacy and Research Institute of Pharmaceutical Sciences, Kyungpook National University, Daegu, South Korea
ABSTRACT
ARTICLE HISTORY
Received 16 January 2020
Revised 24 March 2020
Accepted 7 April 2020
Chlorambucil is a nitrogen mustard-based DNA alkylating drug, which is widely used as a front-line treat-
ment of chronic lymphocytic leukaemia (CLL). Despite its widespread application and success for the initial
treatment of leukaemia, a majority of patients eventually develop acquired resistance to chlorambucil. In
this regard, we have designed and synthesised a novel hybrid molecule, chloram-HDi that simultaneously
impairs DNA and HDAC enzymes. Chloram-HDi efficiently inhibits the proliferation of HL-60 and U937 leu-
kaemia cells with GI50 values of 1.24 mM and 1.75 mM, whereas chlorambucil exhibits GI50 values of
21.1 mM and 37.7 mM against HL-60 and U937 leukaemia cells, respectively. The mechanism behind its
remarkably enhanced cytotoxicity is that chloram-HDi not only causes a significant DNA damage of leu-
kaemia cells but also downregulates DNA repair protein, Rad52, resulting in the escalation of its DNA-
damaging effect. Furthermore, chloram-HDi inhibits HDAC enzymes to induce the acetylation of a-tubulin
and histone H3.
KEYWORDS
DNA damage; histone
deacetylases; can-
cer; leukaemia
1. Introduction
inhibitor, olaparib (tradename; Lynparza) as an anticancer drug
in 20149,10
.
Genotoxic drugs represent an important class of chemotherapy
and constitute a major treatment modality of human cancers.
Genotoxic drugs such as cisplatin, busulfan, cyclophosphamide,
chlorambucil, and temozolomide, cause various types of DNA
damage, exerting their anticancer activity (Figure 1). Among many
different DNA lesions resulting from genotoxic drugs, double-
strand breaks (DSBs) are the most deleterious and a single irrepar-
able DSB is sufficient to induce apoptosis1. Despite such potent
lethality of genotoxic drugs, cancer cells often mitigate DNA dam-
age by their intrinsic DNA repair capability, render drug resistance
and ultimately lead to treatment failure. DNA DSBs are repaired
by two distinct pathways, non-homologous end joining (NHEJ)
and homologous recombination (HR). NHEJ allows for direct liga-
tion of the DSB ends in the G0/G1 phase of the cell cycle, involv-
ing KU70/80, DNAPK and DNA ligase IV, while HR utilises
homologous DNA sequences as a template accurately to restore
the genomic sequence in the S/G2 phase of the cell cycle, medi-
ated by BRCA1, BRCA2 and Rad522,3. In eukaryotic cells, DNA dam-
age triggers a sophisticated network of DNA damage response
(DDR), which includes a sensing of DNA damage, the assembly of
DNA repair factors, cell cycle transit arrest, and the activation of
DNA repair for maintaining genomic integrity4,5. Conversely, dysre-
gulation of DDR causes genomic instability, leading to activation
of cell death pathway. The physiological importance of DDR is
clearly illustrated by the severe cancer susceptibility of inherited
Chlorambucil is a bifunctional DNA alkylating drug, belonging
to a member of the nitrogen mustard. First synthesised in 1953,
chlorambucil still remains one of the front-line treatment of
chronic lymphocytic leukaemia (CLL) and malignant lympho-
mas11–13. The nitrogen mustards share a common mechanism of
action stemming from the presence of N,N-bis(2-chloroehtyl)-
amine moiety. Owing to the high electrophilic reactivity of the
nitrogen mustard moiety, chlorambucil readily reacts with separ-
ate DNA bases, forms DNA interstrand crosslinks and causes
DSBs14. In this regard, exposure of chlorambucil promotes apop-
totic cell death of human cancers via the accumulation of persist-
ent DNA damage.
Although approximately 60–80% of patients respond to chlor-
mabucil for years, eventually all patients become resistant to this
drug11,15. The mechanism of chlorambucil resistance is poorly
understood. However, there are several reports suggesting that
DNA repair system including homologous recombination (HR) are
the major culprit to deactivate its therapeutic potency15–17
.
Accordingly, the occurrence of drug resistance is a serious impedi-
ment to the successful treatment of chronic lymphocytic leukae-
mia (CLL) and malignant lymphomas with chlorambucil.
Histone deacetylases (HDACs) are enzymes responsible for cata-
lysing the removal of acetyl groups from lysine residues on his-
tone, leading to gene transcription or silencing18. Apart from
histone modification, HDACs also regulate the deacetylation of
non-histone proteins, including transcription factors, chaperones,
signalling molecules, and DNA repair proteins19–21. In recent years,
inhibition of HDACs has emerged as a promising therapeutic tar-
defects in DDR factors, such as BRCA1 and BRCA26–8
.
Consequently, drugs that interfere DNA damage response (DDR)
network, such as PARP1, ATM, and DNAPK, have been intensely
investigated, resulting in the first clinical approval of PARP1 get for cancers and several other diseases22–26. As a result,
CONTACT Young Ho Seo
seoyho@kmu.ac.kr
College of Pharmacy, Keimyung University, Daegu, Republic of Korea
ß 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

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Y. SONG ET AL.
coupling reaction of chlorambucil (1) with NH2OTHP was carried
out in the presence of EDC, HOBt, and DIPEA in DMF to afford
compound 2. Subsequent cleavage of THP group with 1 N HCl
successfully provided compound 3 in 34% yield for two steps.
We next embarked on synthesis of compound 7 and 8a-b,
which did not have electrophilic chloro group unlike chloram-HDi
(Scheme 2). The synthesis of 7 and 8a-b commenced with the
esterification of carboxylic acid 4 in the presence of sulphuric acid
in methanol to give ester 5 in 86% yield. Methyl Ester 5 was then
alkylated with ethyl iodide or propyl iodide to afford 6a-b in
75–76% yield. Finally, methyl ester 5 and 6a-b were treated with
hydroxylamine in the presence of potassium hydroxide in metha-
nol to afford 7 and 8a-b in 40–46% yield.
2.3. Biological evaluations of compounds
Upon completion of synthesis, compound 1, 3, 7, and 8a-b were
evaluated for their anti-proliferative activity against various cancer
cell lines, including human acute myeloid leukaemia (AML) cell
lines (U-937 and HL-60), human breast cancer cell lines (MDA-MB-
231 and MCF-7), and a human ovarian cancer cell line (A2780).
Each cancer cell line was treated with the indicated compound for
Figure 1. Structure of representative genotoxic drugs.
significant efforts have been made to identify HDAC inhibitors,
leading to the approval of SAHA (vorinostat), FK228 (romidepsin),
LBH589 (panobinostat), and PXD101 (belinostat) as anticancer 3 days and its anti-proliferative effect on cancer cell lines was
drugs (Figure 2)27–36
.
measure using MTS colorimetric assay. As shown in Table 1,
chloram-HDi (3) displayed greater anti-proliferative activities than
its parent drug, chlorambucil (1). Interestingly, chloram-HDi
exerted the most potent anti-proliferative activity against AML cell
lines, U-937 and HL-60 with GI50 values of 1.75 mM and 1.24 mM
respectively, which were 17–21 fold lower than GI50 values of
chlorambucil. In contrast, chloram-HDi showed relatively poor
anti-proliferative activity against human breast cancer cell lines,
MDA-MB-231 and MCF-7, in that its GI50 values against MDA-MB-
231 and MCF-7 were 95.9 mM and 244.9 mM, respectively.
Compound 7 and 8a-b, which are lack of nitrogen mustard moi-
ety furnished either poor or mediocre anti-proliferative activities
against tested cancer cell lines, indicating that nitrogen mustard
moiety is an essential warhead in this anticancer drug design.
We next determined the effect of chlorambucil and chloram-
HDi on the growth inhibition of various cancer cell lines (HL60,
U937, acute myeloid leukaemia; A549, non-small cell lung carcin-
oma; H1975, gefitinib-resistant non-small cell lung carcinoma
(NSCLC); MCF-7/ADR, multidrug-resistant breast adenocarcinoma;
MDA-MD-231, mammary carcinoma; U266, multiple myeloma;
A2780, ovarian carcinoma). As shown in Figure 3, chloram-HDi dis-
played significantly enhanced anti-proliferative activities against
various cancer cell lines, compared to its parent drug, chlorambu-
cil. Especially, the GI50 values (concentration in 50% growth inhib-
ition) of chloram-HDi (1.24 mM and 1.75 mM) against HL-60 and
U937 cancer cell lines were obviously much lower than those of
chlorambucil (21.1 mM and 37.7 mM), indicating that chloram-HDi
was superior to the clinical drug, chlorambucil in cellular potency
(Table 2).
HDAC inhibitors are grouped structurally into four classes,
hydroxamic acids, cyclic tetrapeptides, benzamides, and short-
chain fatty acids20,21. Most classes of HDAC inhibitors contain the
common pharmacophore, which is composed of three distinct
domains, a zinc-binding domain, a linker domain, and a cap
domain37,38. The zinc-binding domain chelates the catalytic Zn2þ
ion in the active site, which is critical for catalytic function of
HDACs, the cap domain is a surface recognition group that inter-
acts with the entrance of the active site pocket, and the linker
domain connects the cap domain to the zinc-binding domain.
2. Results and discussion
2.1. Drug design and synthesis
Recently, it has been reported that the inhibition of HDAC
enzymes suppress DNA repair machinery and sensitises cancers to
genotoxic drugs39,40. Based on this, we determined to develop
small molecule inhibitors, which could simultaneously attack DNA
and HDACs. To this end, a novel hybrid, chloram-HDi (3) was
designed by coupling chlorambucil with hydroxamic acid as a
zinc-binding group, anticipating that chloram-HDi (3) could inhibit
HDACs function by chelating the catalytic Zn2þ ion in the active
site of HDACs without losing the inherent DNA damaging capabil-
ity of chlrambucil. We hypothesised that chloram-HDi (3) impaired
DNA HR repair system via inactivation of HDACs, while nitrogen
mustard moiety of 3 caused DSBs by alkylating DNA, and accumu-
lated DNA damage. Besides, the fact that DNA and HDACs mostly
exist in a same nuclear compartment, makes them more prone to
dual inhibition by chloram-HDi (3), circumventing difficult pharma-
cokinetics of dual targeting. During our investigation of novel
hybrid DNA-HDACs inhibitors, Yuan’s group reported a DNA-HDAC
inhibitor by attaching a benzamide zinc-binding group to chlor-
ambucil. However, their inhibitor did not show good HDACs
inhibitory activities as well as potent anti-proliferative activities
against cancer cell lines41.
The comet assay is a sensitive and rapid technique for deter-
mining the amount of DNA damage in a single cell42. This assay
directly allows the microscopic observation of the “comet tail”
that is associated with the damaged DNA content. Therefore, we
investigated the impact of chloram-HDi and chlorambucil on the
integrity of DNA in HL-60 cells using the comet assay (Figure 4).
We first treated HL-60 cells with chloram-HDi or chlorambucil for
24 h and then measured DNA damage using the comet assay. The
assay indicated that chloram-HDi (10 mM) caused more noticeable
We first pursued the synthesis of chloram-HDi (3) from chlor-
ambucil (1), which was equipped with hydroxamic acid moiety as
zinc-binding group (ZBG), as shown in Scheme 1. To do so, amide DNA damage than chlorambucil (10 mM), in that % DNA content

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1071
Figure 2. Structure of HDAC inhibitors.
Cl
Cl
Cl
a
b
N
N
N
Cl
O
Cl
O
Cl
O
OTHP
OH
N
H
N
H
OH
1
2
3
Scheme 1. Synthesis of compound 3. Reagents and conditions: (a) NH2OTHP, EDC, DIPEA, DMF, rt, 12 h; (b) 1 N HCl, CH2Cl2, rt, 1 h, 34% (2 steps).
R
H2N
H2N
N
a
b
O
O
R
O
OH
OMe
OMe
4
5
6a-b
6a
6b
R = Et
R = Pr
R
N
c
R
O
5, 6a-b
OH
N
H
7, 8a-b
7
6a
6b
R = H
R = Et
R = Pr
Scheme 2. Synthesis of compound 7 and 8a-b. Reagents and conditions: (a) H2SO4, MeOH, rt, 12h, 86%; (b) Ethyl iodide for 6a or propyl iodide for 6b, K2CO3, DMF,
rt, 24 h, 75-76%; (c) NH2OH, KOH, MeOH, 0 ꢀC, 3 h, 40-46%.
in the tail induced by chloram-HDi and chlorambucil were 25.5% downregulated the protein level of Rad52 in a dose-dependent
and 2.1%, respectively (Figure 2(B)).
manner. It has been reported that the inhibition of HDACs leads
to the reduction of Rad52 protein level. Nevertheless, the reduc-
tion of the DNA repair protein, Rad52 contributed to the increased
DNA-damaging effect of chloram-HDi, offering a distinct advan-
tage over chlorambucil.
We next investigated the precise mechanism of action behind
the increased DNA-damaging effect of chloram-HDi compared
with its parent drug, chlorambucil. Hence, we first treated HL-60
cells with the indicated concentrations of chlorambucil and
chloram-HDi for 24 h and measured the expression levels of
To assess the comparative effect of chlorambucil and chloram-
Rad52, H2AX, c-H2AX, and b-actin. c-H2AX is a well- established HDi on the acetylation status of a-tubulin and histone H3, we also
hallmark of DNA double-strand breaks and Rad52 is an important measured the expression levels of a-tubulin, Ac-a-tubulin, histone
protein in the homologous recombination repair (HRR) of DNA H3, and Ac-histone H3 (Figure 5(B)). a-Tubulin and histone H3 are
double-strand breaks43–45. As shown in Figure 5(A), the treatment well documented substrates of HDAC6 and HDAC146,47
.
of cells with chloram-HDi more significantly increased the expres- Accordingly, the inhibition of HDAC6 and HDAC1 enzymes epige-
sion of c-H2AX than chlorambucil in a dose-dependent manner, netically induced the acetylation of a-tubulin and histone H3,
indicating that chloram-HDi effectively induced more DNA dam- respectively. The treatment of HL-60 cells with chloram-HDi dose-
age than chlorambucil, which was consistent with the result that dependently increased the acetylation of a-tubulin and histone
we observed in the comet assay. Furthermore, chlrom-HDi H3, while total amount of histone H3 remained unchanged. It is

1072
Y. SONG ET AL.
Table 1. Growth inhibition of compound 1, 3, 7, and 8a-b in various cancer cell lines
R1
O
R2
GI50 values (lM)
Compound
R1
R2
OH
U-937
37.7
HL-60
21.1
MDA-MB-231
MCF-7
393
A2780
Chlorambucil
(1)
>100
>100
Cl
N
N
Cl
Cl
Chloram-HDi
(3)
NHOH
1.75
1.24
95.9
244.91
21.2
Cl
7
NHOH
NHOH
93.0
60.5
70.3
>100
393
282
>100
NH2
8a
>100
85.1
81
N
8b
NHOH
40.2
34.4
66.8
37.8
7.79
N
Figure 3. Effects of (A) chlorambucil and (B) chloram-HDi on the growth inhibition of various cancer cell lines. Cells were incubated with various concentrations of
chlorambucil or chloram-HDi for 3 days, and cell proliferation was measured using the MTS assay. Data are presented as mean SD (n ¼ 4). (C) GI50 values of chloram-
bucil and chloram-HDi against various cancer cell lines.
Table 2. GI50 values of chlorambucil and chloram-HDi against various cancer
cell lines
interesting to note that chloram-HDi decreased the expression
level of a-tubulin in a dose-dependent manner. In contrast, the
Chlorambucil
Chloram-HDi
(GI50; lM)
(GI50; lM)a
treatment of HL-60 cells with chlorambucil (5 and 10 mM) failed to
induce the acetylation of a-tubulin and histone H3, whereas
30 mM of chlorambucil resulted in little acetylation of a-tubulin
and histone H3. Taken together, the result undoubtedly demon-
strated that chloram-HDi efficiently inhibited the function of
HDAC6 and HDAC1 enzymes and promoted the cellular acetyl-
ation status of a-tubulin and histone H3.
Cancer cell lines
HL60
U937
A549
H1975
MCF-7/ADR
MDA-MB-231
U266
21.1
37.7
1.24
1.75
65.3
76.7
62.1
83.5
38.1
21.2
>100
>100
>100
>100
>100
>100
A2780
The activation of apoptotic signals can suppress the prolifer-
ation of cancer cells, as it is regulated by the avoidance of the
aGI50 values were obtained after the treatment of cells for 72 h.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1073
Figure 4. Effect of chloram-HDi on DNA damage in HL-60 cells. (A) Fluorescent images of DNA from chlorambucil or chloram-HDi treated HL-60 cells in comet assay.
HL-60 cells were treated with chlorambucil (10lM) and chloram-HDi (10lM) for 24 h and then subjected to comet assay. DMSO is used as a negative control. (B)
Comet assay results are graphed as median % DNA in the tail SD (n ¼ 4).
Figure 5. Treatment of HL-60 cells with chloram-HDi enhances DNA damage and induces apoptosis of HL-60 cancer cells. (A) Effect of chloram-HDi on the DNA dam-
age markers, Rad52, H2AX, and c-H2AX. HL-60 cells were treated with chlorambucil (10lM) and chloram-HDi (10lM) for 24h and then subjected to western blot ana-
lysis. (B) Chloram-HDi promotes the acetylation of a-tubulin and Histone H3 though inhibition of HDAC6 and HDAC1. (C) Chloram-HDi induces the cleavage of PARP,
caspase 3, and caspase 8.
apoptotic mechanism. To determine if the anti-proliferative cleavage of PARP, caspase 3, and caspase 8, clearly indicating
effect of chloram-HDi on HL-60 cells is associated with the that chloram-HDi more potently led to the apoptotic cancer
induction of apoptosis, we therefore measured the cleavage of cells death than chlorambucil.
apoptotic biomarkers, including PARP, caspase 3, and caspase 8.
To determine whether chloram-HDi affected the cell cycle, we
As shown in Figure 5(C), the exposure of HL-60 cells with evaluated the effect of chloram-HDi on the cell cycle distribution
chloram-HDi significantly promoted the cleavage of PARP, cas- of HL-60 cells using flow cytometry. After exposed to the indi-
pase 3, and caspase 8 in a dose-dependent manner. Conversely, cated concentrations of chlorambucil and chloram-HDi for 24 h,
when HL-60 cells were treated with chlorambucil, the adminis- the cell cycle distribution of HL-60 cells was measured. As shown
tration of 30 mM concentration was required to induce the in Figure 6, the exposure of HL-60 cells with chloram-HDi

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Y. SONG ET AL.
Table 3. IC50 values of chloram-HDi against HDAC enzymes
IC50 values (nM)
Chloram-HDi
Class
I
Enzymes
SAHA
HDAC1
HDAC3
HDAC6
HDAC7
4,497
6,201
694
219
107
49
IIb
IIa
74,568
61,020
Figure 6. Chloram-HDi induces G2/M cell cycle arrest. The effect of chlorambucil
and chloram-HDi on the cell cycle was analysed by flow cytometry. HL-60 cells
were treated with the indicated concentrations of chlorambucil and chloram-HDi
for 24 h. HL-60 cells were stained with propidium iodide and cell cycle distribu-
tion was analysed by flow cytometry. DMSO (D) was used as a negative control.
remarkably promoted the cell cycle arrest in the G2/M phases,
compared with the DMSO control and the reference drug chlor-
ambucil in that the prominence of G2/M cell cycle arrest is a
reported feature of the nitrogen mustards48,49. The result indi-
cated that chloram-HDi caused the DNA double-strand breaks of
HL-60 cells, leading to the activation of the homologous recom-
bination repair (HRR) pathway through G2/M cell cycle arrest.
Conversely, another DNA DSB repair pathway, Non-homologous
end joining (NHEJ) is reported to primarily occur in G0/G1 phase2.
The treatment of HL-60 cells with 0.1 mM of chloram-HDi increased
G2/M fraction from 21.8% to 30.8%, while the treatment of HL-60
cells with 1 mM of chlorambucil led to 32.2% G2/M arrest of HL-60
cells, which indicated that chloram-HDi was more efficacious than
chlorambucil in the cell cycle arrest. When the cells were treated
with the increased concentration of chloram-HDi, the G2/M arrest
was more remarkable (52% G2/M fraction at 0.5 mM and 62.4% G2/
M fraction at 1 mM). Conversely, the treatment of cells with
chloram-HDi resulted in a reduction of the cell population at the
G0/G1 phases in a dose-dependent manner. It is also interesting to
note that chloram-HDi increased the cell population at sub G0/G1
phases in a dose-dependent manner. Taken together, the results
suggested that chloram-HDi effectively induced the cell cycle
arrest at the G2/M phases in HL-60 cells in a dose-depend-
ent manner.
We next investigated the inhibitory activity of chloram-HDi
against HDAC1, 3, 6, and 7 isoforms, in that the clinically approved
HDAC inhibitor, SAHA was used as a reference drug (Table 3).
Although chloram-HDi displayed less efficacious inhibitory activ-
ities than the reference drug SAHA against all tested HDACs
enzymes, chloram-HDi still displayed modest inhibitory activities
against HDACs enzymes. Interestingly, chloram-HDi more select-
ively inhibited HDAC6 enzyme with an IC50 value of 694 nM than
other HDAC enzymes (IC50 of HDAC1¼4.497 mM, IC50 of
HDAC3¼6.201 mM, and IC50 of HDAC7¼74.568 mM).
Figure 7. Effect of chloram-HDi on the cell viability of PBMCs. Cells were incu-
bated with chloram-HDi (0.01, 0.05, 0.1, 0.5, 1, 5, and 10 lM) for 3 days and cell
viability was measured using the colorimetric MTS assay. Data are presented as
mean SD (n ¼ 4).
Cytochrome P450 (P450) enzymes have emerged as an important
determinant in the occurrence of drug interactions that can lead to
adverse drug reactions. Therefore, we investigated the effect of
chloram-HDi (3) on the catalytic activities of clinically significant human
P450s such as 1A2, 2C9, 2C19, 2D6, and 3A in human liver micro-
somes50,51. To do so, the inhibitory potency of chloram-HDi (3) was
determined with cytochrome P450 assays in the absence and the pres-
ence of chloram-HDi (3) up to 50mM final concentration using pooled
human liver microsomes (Table 4). The assay indicated that chloram-
HDi (3) showed weak inhibitory effect (>50 mM) against five P450 iso-
forms. These findings suggest that clinical interactions between
chloram-HDi (3) and substrate drugs of five P450 isoforms would not
be expected.
2.3. Molecular modelling of chloram-HDi (3)
To investigate the binding mode of chloram-HDi (3), in silico
docking studies were performed with HDAC6 enzymes (Figure 8).
Modelling chloram-HDi (3) in the substrate-binding pocket of
HDAC6 (PBD code: 5EF7) indicated that chloram-HDi (3) effect-
ively bound into a deep substrate-binding pocket of HDAC6
(Figure 8(A)). The middle phenylpropyl group of chloram-HDi (3)
well occupied the lipophilic channel and formed favourable p-p
stacking interactions with F643 and F583 residues (Figure 8(B)).
The hydroxamate C¼O and OH groups of chloram-HDi (3) che-
lated the active site Zn2þ ion in a bidentate manner and formed
additional hydrogen bond interactions with Y745, D612, H573,
and G743 residues. In contrast, the nitrogen mustard moiety of
chloram-HDi (3) was located in the rim of the substrate-binding
pocket, participating in Van der Waals interactions with the
hydrophobic patches of the rim, composed of F643 and
F583 residues.
To determine whether chloram-HDi was toxic to the normal cells,
we isolated primary human peripheral blood mononuclear cells
(PBMCs) and examined the cytotoxicity of chloram-HDi on normal
human PBMCs. Cells were first treated with various concentrations of
chloram-HDi for 3 days and cell viability was measured using the col-
orimetric MTS assay. As shown in Figure 7, the result indicated that
chloram-HDi was not toxic to normal human PBMCs up to 10 mM
concentration, suggesting that chloram-HDi selectively targets the
malignant leukaemia cell lines HL-60 (IC50 ¼ 1.24 mM) and U937 (IC50
¼ 1.75 mM) over the normal PBMCs (IC50 > 10 mM).

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
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(0.28 g, 1.48 mmol) and N,N-diisopropylethylamine (0.35 ml,
1.97 mmol) in DMF (5 ml) was stirred at room temperature for 12 h
under argon. The solution was diluted with ethyl acetate and
washed with brine. The organic layer was dried over Na2SO4, fil-
tered and concentrated under reduced pressure. The resulting
intermediate was dissolved in CH2Cl2 (8 ml) and treated with 1 N
HCl in Et2O (8 ml) at room temperature for 1 h. The precipitating
solid was filtered off and purified by MPLC (Biotage SNAP KP-C18
column) to afford compound 3 in 34% yield. Rf ¼ 0.26 (7:3 ethyl
acetate: hexanes). 1H NMR (500 MHz, MeOD) d 7.05 (d, J¼8.5 Hz,
2 H), 6.68 (d, J¼8.0 Hz, 2 H), 3.72 (t, J¼6.5 Hz, 4 H), 3.51 (t, J¼6.5 Hz,
4 H), 2.52 (t, J¼7.5 Hz, 2 H), 2.30 (t, J¼7.5 Hz, 2 H), 1.89–1.83 (m,
2 H). 13 C NMR (125 MHz, MeOD) d 172.8, 145.9, 131.5, 13.5, 113.4,
54.5, 48.4, 35.0, 33.1, 28.8 ESI MS (m/z) 320.25 [M þ 1]þ.
3. Experimental
3.1. Chemistry
3.1.1. General methods and materials
Unless otherwise noted, all reactions were performed under argon
atmosphere in oven-dried glassware. All purchased reagents and
solvents were used without further purification. Thin layer chroma-
tography (TLC) was carried out using Merck silica gel 60 F254 plates.
TLC plates were visualised using a combination of UV light and iod-
ine staining. Column chromatography was conducted under
medium pressure on silica (Merck Silica Gel 40–63 mm) or performed
by MPLC (Biotage Isolera One instrument) on a silica column
(Biotage SNAP HP-Sil) or C18 column (Biotage SNAP Ultra C18). NMR
analyses were carried out using a JNM-ECZ500R (500 MHz) manufac-
tured by Jeol resonance. 1H and 13 C NMR chemical shifts are
reported in parts per million (ppm). The deuterium lock signal of
the sample solvent was used as a reference, and coupling constants
(J) are given in hertz (Hz). The splitting pattern abbreviations are as
follows: s, singlet; d, doublet; t, triplet; q, quartette; dd, doublet of
doublets; m, multiplet. The purity of all tested compounds was con-
firmed to be higher than 95% by HPLC analysis performed with a
dual pump Shimadzu LC-6AD system equipped with VP-ODS C18
column (4.6 mm ꢁ 250 mm, 5 mm, Shimadzu).
3.1.3. Methyl 4–(4-aminophenyl)butanoate (5)
To a solution of 4–(4-aminophenyl)butyric acid (1.0 g, 5.58 mmol)
in methanol (40 ml), was added sulphuric acid (4 ml) and it was
stirred at room temperature for 12 h under argon. The mixture
was neutralised with 10% NaOH to pH 8, concentrated under
reduced pressure and then extracted with ethyl acetate. The
organic layer was dried over Na2SO4, filtered, concentrated under
reduced pressure to afford compound 5 as yellow oil in 86% yield.
1
Rf ¼ 0.27 (3:7 ethyl acetate: hexanes). H NMR (500 MHz, MeOD) d
6.96 (d, J¼8.0 Hz, 2 H), 6.70 (dd, J¼6.3 Hz, 2.0 Hz, 2 H), 3.67 (s, 3 H),
2.53 (t, J¼7.0 Hz, 2 H), 2.32 (t, J¼7.5 Hz, 2 H), 1.91–1.85 (m, 2 H).
13 C NMR (125 MHz, MeOD) d 175.8, 146.3, 132.5, 130.1, 116.9, 51.9,
35.2, 34.0, 28.0. ESI MS (m/z) 194.11 [M þ 1]þ.
3.1.2. 4–(4-(Bis(2-chloroethyl)amino)phenyl)-N-hydroxybutanamide (3)
A mixture of chlorambucil (0.30 g, 1.00 mmol), NH2OTHP (0.13 g,
1.10 mmol),
1-ethyl-3–(3-dimethylaminopropyl)carbodiimide
Table 4. Inhibitory potency of chloram-HDi on specific P450 activities in human
liver microsomes
3.1.4. Methyl 4–(4-(diethylamino)phenyl)butanoate (6a)
Enzyme activity
P450
IC50 (lM)
A mixture of compound 5 (0.40 g, 2.07 mmol), iodoethane (0.42 ml,
5.17 mmol) and potassium carbonate (0.85 g, 6.21 mmol) in DMF
was stirred at room temperature for 24 h under argon. The mix-
ture was concentrated under reduced pressure, and then
extracted with ethyl aceatate. The organic layer was washed with
Phenacetin O-deethylation
Tolbutamide 4-methylhydroxylation
Omepraozle hydroxylation
Dextromethorphan O-demethylation
Midazolam 10-hydroxylation
1A2
2C9
2C19
2D6
3A
>50
>50
>50
>50
>50
Figure 8. Molecular docking model of chloram-HDi (3) bound in the substrate-binding pocket of HDAC6 (PBD code: 5EF7). (A) Surface representation of HDAC6 and
chloram-HDi (3) complex. (B) Cartoon and sticks representation of HDAC6 and chloram-HDi (3) complex. The carbon, oxygen, nitrogen and chlorine atoms of chloram-
HDi (3) are shown in lime, red, blue, and green, respectively. The side chains of the substrate-binding site are coloured according to the atom types (carbon, light
blue; oxygen, red; nitrogen, blue) and labelled with their residue name. The hydrogen bonds are shown as dashed red lines. The docking poses are visualised
using PyMOL1.3.

1076
Y. SONG ET AL.
saturated NaHCO3 solution, dried over Na2SO4, concentrated (125 MHz, CDCl3) d 171.9, 146.3, 129.4, 128.2, 112.5, 44.6, 34.0,
under reduced pressure and purified by MPLC (Biotage SNAP 32.3, 27.2, 12.6. ESI MS (m/z) 251.17 [M þ 1]þ.
HP-Sil column) to afford compound 6a as white oil in 76% yield.
Rf ¼ 0.39 (1:9 ethyl acetate: hexanes). 1H NMR (500 MHz, CDCl3) d
3.1.8. 4–(4-(Dipropylamino)phenyl)-N-hydroxybutanamide (8 b)
7.07 (d, J¼8.5 Hz, 2 H), 6.67 (d, J¼8.5 Hz, 2 H), 3.70 (s, 3H), 3.38–3.34
Hydroxylamine hydrochloride (2.25 g, 32.44 mmol) in methanol
(m, 4H), 2.59 (t, J¼7.5 Hz, 2 H), 2.37 (t, J¼7.5 Hz, 2 H), 1.99–1.93 (m,
2 H), 1.19 (t, J¼7.5Hz, 6H). 13 C NMR (125 MHz, CDCl3) d 174.1, 146.2,
(5 ml) was added to a solution of potassium hydroxide (1.82 g,
32.44 mmol) in methanol (5 ml) at 0 ꢀC. The mixture was stirred for
129.3, 128.2, 112.2, 51.4, 44.4, 34.1, 33.5, 26.9, 12.6.
15 min at 0 ꢀC and the precipitated potassium chloride was
removed and the filtrate was used as such; To a solution of the
compound 6b (0.20 g, 0.72 mmol) in tetrahydrofuran (10 ml) was
added to freshly prepared hydroxylamine at 0 ꢀC and stirred at
the same temperature for 2 h. The mixture was neutralised with
acetic acid to pH 7 and extracted with ethyl acetate. The organic
layer was dried over Na2SO4, concentrated under reduced pressure
and purified by MPLC (Biotage SNAP KP-C18 column) to afford
compound 8b in 46% yield. Rf ¼ 0.27 (6:4 ethyl acetate: hexanes).
1H NMR (500 MHz, CDCl3) d 6.98 (d, J¼8.5 Hz, 2 H), 6.57 (d,
J¼8.5 Hz, 2 H), 3.20 (t, J¼7.5 Hz, 4 H), 2.50 (t, J¼7.5 Hz, 2 H),
2.12–2.06 (m, 2 H), 1.92 (m, 2 H), 1.63–1.55 (m, 4 H), 0.92 (t,
J¼7.5 Hz, 6 H). 13 C NMR (125 MHz, CDCl3) d 172.0, 146.7, 129.3,
127.7, 112.1, 53.1, 33.9, 32.3, 27.2, 20.5, 11.6. ESI MS (m/z)
279.20 [M þ 1]þ.
3.1.5. Methyl 4–(4-(dipropylamino)phenyl)butanoate (6b)
A mixture of compound 5 (0.36 g, 1.87 mmol), iodopropane
(0.46 ml, 4.67 mmol) and potassium carbonate (0.78 g, 5.61 mmol)
in DMF was stirred at room temperature for 24 h under argon.
The mixture was concentrated under reduced pressure, and then
extracted with ethyl aceatate. The organic layer was washed with
saturated NaHCO3 solution, dried over Na2SO4, concentrated
under reduced pressure and purified by MPLC (Biotage SNAP HP-
Sil column) to afford compound 6b as white oil in 76% yield. Rf ¼
0.38 (1:9 ethyl acetate: hexanes). 1H NMR (500 MHz, CDCl3) d 7.03
(dd, J¼11.5 Hz, 3.0 Hz, 2 H), 6.60 (dd, J¼11.5 Hz, 2.0 Hz, 2 H), 3.68 (s,
3H), 3.22 (t, J¼7.5 Hz, 4 H), 2.55 (t, J¼7.5 Hz, 2 H), 2.35 (t, J¼7.5 Hz,
2 H), 1.96–1.90 (m, 2H), 1.65–1.58 (m, 4H), 0.94 (t, J¼7.5 Hz, 6 H).
13 C NMR (125 MHz, CDCl3) d 174.3, 146.7, 129.3, 127.9, 112.0, 53.1,
51.5, 34.1, 33.6, 26.9, 20.5, 11.6. ESI MS (m/z) 279.20 [M þ 1]þ.
3.2. Biology
3.2.1. Materials
3.1.6. 4–(4-Aminophenyl)-N-hydroxybutanamide (7)
Dulbecco’s Modified Eagle’s medium (DMEM) with L-glutamine
was purchased from GenDEPOT (Barker, TX, USA) and RPMI 1640
medium, fatal bovine serum (FBS), and penicillin/streptomycin
were purchased from Gibco BRL (Gaithersburg, MD, USA).
Antibodies specific for a-tubulin, Ac-a-tubulin, Histone H3, Ac-his-
tone H3, PARP, caspase 3, cleaved caspase 8, b-actin HDAC1, and
HDAC6 were purchased from Cell Signalling Technology (Boston,
MA, USA). Rad52 antibody and goat anti-rabbit IgG horseradish
peroxidase conjugate were purchased from Santa Cruz
Biotechnology Inc. (Dallas, TX, USA). Cell Titre 96 Aqueous One
Solution cell proliferation assay kit was purchased from Promega
(Madison, WI, USA). Amersham ECL select Western blotting detec-
tion reagent was purchased from GE Healthcare (Waukesha, WI,
USA). HDAC fluorogenic assay kits (HDAC1, HDAC3, HDAC6, and
HDAC8) were purchased from BPS Bioscience (San Diego, CA,
USA). OxiSelectTM Comet Assay Kit was purchased from Cell
Biolabs, Inc (San Diego, CA, USA).
Hydroxylamine hydrochloride (0.95 g, 13.7 mmol) in methanol
(4 ml) was added to a solution of potassium hydroxide (0.78 g,
13.7 mmol) in methanol (4 ml) at 0 ꢀC. The mixture was stirred for
15 min at 0 ꢀC and the precipitated potassium chloride was
removed and the filtrate was used as such; To a solution of the
compound 5 (0.13 g, 0.34 mmol) in tetrahydrofuran (4 ml) was
added to freshly prepared hydroxylamine at 0 ꢀC and stirred at
the same temperature for 2 h. The mixture was neutralised with
3 N HCl to pH 7 and extracted with ethyl acetate. The organic
layer was dried over Na2SO4, concentrated under reduced pressure
and purified by HPLC to afford compound 7 in 40% yield. Rf ¼
1
0.27 (9:1 ethyl acetate: methanol). H NMR (500 MHz, DMSO-D6) d
10.33 (s, 1 H), 8.66 (s, 1 H), 6.81 (d, J¼8.5 Hz, 2 H), d 6.47 (dd,
J¼10.8 Hz, 2.0 Hz, 2 H), 4.80 (s, 2 H), 2.36 (t, J¼7.5 Hz, 2 H), 1.92 (t,
J¼7.5 Hz, 2 H), 1.71–1.65 (m, 2H). 13 C NMR (125 MHz, DMSO-D6) d
169.6, 147.0, 129.2, 129.1, 114.5, 34.4, 32.4, 28.0. ESI MS (m/z)
195.11 [M þ 1]þ.
3.2.2. Cell culture
3.1.7. 4–(4-(Diethylamino)phenyl)-N-hydroxybutanamide (8a)
Hydroxylamine hydrochloride (4.11 g, 59.19 mmol) in methanol
(5 ml) was added to a solution of potassium hydroxide (3.32 g,
59.19 mmol) in methanol (5 ml) at 0 ꢀC. The mixture was stirred for
15 min at 0 ꢀC and the precipitated potassium chloride was
removed and the filtrate was used as such; To a solution of the
compound 6a (0.33 g, 1.32 mmol) in tetrahydrofuran (10 ml) was
added to freshly prepared hydroxylamine at 0 ꢀC and stirred at
the same temperature for 2 h. The mixture was neutralised with
acetic acid to pH 7 and extracted with ethyl acetate. The organic
layer was dried over Na2SO4, concentrated under reduced pressure
and purified by MPLC (Biotage SNAP KP-C18 column) to afford
compound 8a in 40% yield. Rf ¼ 0.18 (3:2 ethyl acetate: hexanes).
MDA-MB-231 cells were grown in DMEM with L-glutamine supple-
mented with streptomycin (500 mg/mL), penicillin (100 units/mL),
and 10% foetal bovine serum (FBS). HL-60, H1975, U937, A549,
U266, MCF-7/ADR and A2780 cells were grown in RPMI 1640 with
L-glutamine supplemented with streptomycin (500 mg/mL), peni-
cillin (100 units/mL), and 10% FBS. Cells were grown to confluence
in a humidified atmosphere (37 ꢀC, 5% CO2).
3.2.3. Cell proliferation assay
Cells were seeded at a clear 96-well plate, the medium volume
was brought to 100 mL, and cells were allowed to attach over-
night. Various concentrations of compounds (1, 3, 7, 8a, 8b or
1H NMR (500 MHz, CDCl3) d 6.99 (d, J¼8.0 Hz, 2 H), 6.62 (d, DMSO) were added to the wells. Cells were then incubated at
J¼9.0 Hz, 2 H), 3.32–3.28 (m, 4 H), 2.50 (t, J¼7.5 Hz, 2 H), 2.09 (s, 37 ꢀC for 3 days. Cell viability was determined using the Promega
2H), 1.88 (t, J¼7.0 Hz, 2 H), 1.13 (t, J¼6.5 Hz, 6 H). 13 C NMR Cell Titre 96 Aqueous One solution cell proliferation assay.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1077
Absorbance at 490 nm and 690 nm as reference wavelength was was added to the well, followed by various concentrations of
read on Tecan Infinite F200 Pro plate reader, and values were
expressed as percent of absorbance from cells incubated in
DMSO alone.
compounds (5 mL) or SAHA (5 mL) as a positive control, and then
the resulting mixture was incubated at 37 ꢀC for 30 min. After the
incubation, 50 mL of undiluted 2ꢁ HDAC developer was added to
each well. After the mixture was incubated at rt for 15 min, fluor-
escence intensity was measured using a microplate reader at
360 nm excitation and 460 nm emission wavelengths.
3.2.4. Western blot
HL-60 cells were seeded in 50 mm culture dish (1 ꢁ 106 cells/dish),
and allowed to attach overnight. Cells were then treated with the
indicated concentrations of chlorambucil or chloram-HDi for 24 h. 3.3. Molecular docking
Cells were harvested in ice-cold lysis buffer (23 mM Tris-HCl pH
In silico docking of chloram-HDi with the 3 D coordinates of the
7.6, 130 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS)
and 30 mg of lysate per lane was separated by SDS-PAGE, followed
by transferring to a PVDF membrane (Bio-Rad, Hercules, USA). The
membrane was blocked with 5% skim milk in TBST, and then incu-
bated with the corresponding primary antibody (a-tubulin, Ac-
a-tubulin, HDAC1, HDAC6, Histone H3, Ac-histone H3, PARP, cas-
pase 3, cleaved caspase 8, Rad52, H2AX, cH2AX, or b-actin). After
being treated with the secondary antibody coupled to horseradish
peroxidase (Santa Cruz, CA, USA), proteins were visualised by ECL
chemiluminescence according to the manufacturer’s instruction
(GE healthcare, USA).
X-ray crystal structure of HDAC6 (PDB code: 5EF7) was accom-
plished using the AutoDock 4.2 programme downloaded from the
Molecular Graphics Laboratory of the Scripps Research Institute. In
the docking experiments carried out, water was removed from the
3 D X-ray coordinates while Gasteiger charges were placed on the
X-ray structures of HDAC6 along with chloram-HDi using tools
from the AutoDock suite. A grid box centred on the substrate
binding pocket of HDAC6 enzyme with definitions of 60 ꢁ 60 ꢁ 60
points and 0.375 Å spacing was chosen for ligand docking experi-
ments. The docking parameters consisted of setting the popula-
tion size to 150, the number of generations to 27,000, and the
number of evaluations to 2,500,000, while the number of docking
runs was set to 100 with a cut-off of 1 Å for the root-meansquare
tolerance for the grouping of each docking run. The docking
model of HDAC6 with chloram-HDi was depicted in Figure 8 and
rendering of the picture was generated using PyMol software
(DeLanoScientific).
3.2.5. Comet assay
Comet assay was performed using OxiSelectTM comet assay kit (Cell
Biolabs Inc., San Diego, CA, USA). The alkaline electrophoresis was
conducted to detect any single-stranded and double-stranded DNA
breaks (SSBs and DSBs) according to the manufacturer’s instruction.
HL-60 cells were seeded in 50mm culture dish (1ꢁ 106 cells/dish),
and allowed to attach overnight. Cells were then incubated with
DMSO, chlorambucil (10mM) or chloram-HDi (10 mM) for 24h. Cells
were harvested into 15 ml falcon tube and washed with Dulbecco’s
phosphate-buffered saline (DPBS). Cell suspension was mixed with
molten agarose, 75mL of the mixture was added to the slides, and
dried at 4 ꢀC for 15min in dark. The embedded cells were treated
with lysis buffer at 4 ꢀC for 30min in dark, which was then replaced
with pre-chilled alkaline buffer and placed at 4˚C for 30 min. After
replacing alkaline buffer with TBE buffer, the embedded cells were
incubated for 5 min, twice. Finally, the samples were electrophor-
esed in a horizontal chamber to separate intact DNA from the dam-
aged fractions and washed with deionised water. After being
incubated in 70% ethanol for 5 min, the slides were dried, stained
with DNA dye for 15 min at rt and visualised by invert microscope
Eclipse-TiU (Nickon, Tokyo, Japan). DNA tail and DNA head were
measured with Image J software.
3.4. Cytochrome P450 inhibition assay
The inhibitory potency of chloram-HDi was determined with cyto-
chrome P450 assays in the absence and presence of chloram-HDi
(final concentrations of 0–50 mM with acetonitrile concentration
less than 0.5%) using pooled human liver microsomes (Xenotech
H0630). All experiments were performed in duplicate. Phenacetin
O-deethylase, tolbutamide 4-hydroxylase, omeprazole hydroxylase,
dextromethorphan O-demethylase, and midazolam 10-hydroxylase
activities were determined as probe activities for CYP1A2, CYP2C9,
CYP2C19, CYP2D6, and CYP3A, respectively, using cocktail incuba-
tion and tandem mass spectrometry, as described previously50,51
.
4. Conclusion
Although chlorambucil remains one of the front-line treatment of
CLL and malignant lymphomas, the occurrence of drug resistance
is still a major hurdle to the successful cancer treatment. Among
the mechanisms of drug resistance, it has been suggested that
the activation of DNA repair machinery such as homologous
recombination (HR) plays a critical role in the drug resistance of
3.2.6. Cell cycle arrest
HL-60 cells were treated with the indicated concentrations of
chlorambucil or chloram-HDi for 24 h. Cells were resuspended in
300 mL of PBS, treated with 700 mL of ethanol, and gently vortexed.
Cells were then incubated at 4 ꢀC for 2 h, washed with PBS, resus-
pended in 500 mL of PBS containing 50 mg/mL of propidium iodide
and 1 mg/mL of Rnase A. After being incubated for an additional
30 min at rt in the dark, cells were analysed by FACS flow cytome-
ter and BD CellQuest Pro software.
chlorambucil17,52
.
In this regard, we designed a novel hybrid molecule, chloram-
HDi that simultaneously impaired DNA and HDAC enzymes (Figure
9). Chloram-HDi caused a remarkable DNA damage of HL-60 cells,
in that % DNA content in the tail was xx% in comet assay and the
DNA damage marker, c-H2AX was significantly upregulated in
western blot analysis. Chloram-HDi also caused the reduction of
the DNA repair protein, Rad52, magnifying its DNA-damaging
effect by disrupting Rad52-mediated homologous recombination
(HR). Furthermore, chloram-HDi inhibited HDAC enzymes to
induce the acetylation of a-tubulin and histone H3. It has been
3.2.7. Hdac assay
Enzymatic HDAC assaywas performed following manufacturer’s
protocol (BPS Bioscience). Briefly, HDAC assay buffer (35 mL) was
mixed with 5 ml of BSA (1 mg/mL) and 5 mL of HDAC substrate
(200 mM) in 96-well black plate. 5 mL of HDAC enzyme (7 ng/mL) reported that the inhibition of HDACs induces the acetylation of

1078
Y. SONG ET AL.
Figure 9. Mode of action of the hybrid molecule, chloram-HDi.
Hsp90, which impairs the chaperone activity of Hsp90 and conse-
quently causes the depletion of its client protein, Rad5253. As a
result, chloram-HDi efficiently promoted the cell cycle arrest at the
G2/M phases and resulted in apoptotic cell death, evidenced by
the cleavage of PARP, caspase 3, and caspase 8. Overall, these
findings firmly supported that chloram-HDi could serve as a
potential drug candidate for the treatment of leukaemia, warrant-
ing further studies on in vivo evaluation as well as ADMET profiles.
These studies will be reported in due course.
constitutive activation of DNA damage signalling in human
gliomas. Oncogene 2010;29:5095–102.
5. Gorgoulis VG, Vassiliou LV, Karakaidos P, et al. Activation of
the DNA damage checkpoint and genomic instability in
human precancerous lesions. Nature 2005;434:907–13.
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Disclosure statement
9. O’Connor MJ. Targeting the DNA damage response in can-
cer. Mol Cell 2015;60:547–60.
No potential conflict of interest was reported by the author(s).
10. Gao CZ, Dong W, Cui ZW, et al. Synthesis, preliminarily bio-
logical evaluation and molecular docking study of new ola-
Funding
parib
analogues
as
multifunctional
parp-1
and
This research was supported by Basic Science Research
Programme through the National Research Foundation of Korea
(NRF) funded by the Ministry of Education (NRF-
2016R1A6A1A03011325 and 2016R1D1A1B01009559).
cholinesterase inhibitors. J Enzyme Inhib Med Chem 2019;
34:150–62.
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(di-2-chloroethylamino)-phenylbutyric acid (cb 1348) in
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Part xii. Some carboxylic derivatives of nn-di-2-chloroethyla-
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ORCID
Kwang-Hyeon Liu
http://orcid.org/0000-0002-3285-5594
14. Noll DM, Mason TM, Miller PS. Formation and repair of inter-
strand cross-links in DNA. Chem Rev 2006;106:277–301.
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