Rational design, synthesis and preliminary antitumor activity evaluation of a chlorambucil derivative with potent DNA/HDAC dual-targeting inhibitory activity

Article abstract of DOI:10.1016/j.bmcl.2017.08.011

Histone deacetylases (HDACs) play a pivotal role not only in gene expression but also in DNA repair. Herein, we report the successful design, synthesis and evaluation of a chlorambucil derivative named vorambucil with a hydroxamic acid tail as a DNA/HDAC dual-targeting inhibitor. Vorambucil obtained both potent DNA and HDACs inhibitory activities. Molecular docking results supported the initial pharmacophoric hypothesis and rationalized the potent inhibitory activity of vorambucil against HDAC1, HDAC2 and HDAC6. Vorambucil showed potent antiproliferative activity against all the test four cancer cell lines with IC₅₀ values of as low as 3.2–6.2 μM and exhibited 5.0–18.3-fold enhanced antiproliferative activity than chlorambucil. Vorambucil also significantly inhibits colony formation of A375 cancer cells. Further investigation showed that vorambucil remarkably induced apoptosis and arrested the cell cycle of A375 cells at G2/M phase. Vorambucil could be a promising candidate and a useful tool to elucidate the role of those DNA/HDAC dual-targeting inhibitors for cancer therapy.

Full text of DOI:10.1016/j.bmcl.2017.08.011

Accepted Manuscript
Rational design, synthesis and preliminary antitumor activity evaluation of a
chlorambucil derivative with potent DNA/HDAC dual-targeting inhibitory ac-
tivity
Rui Xie, Yan Li, Pingwah Tang, Qipeng Yuan
PII:
S0960-894X(17)30801-6
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.08.011
BMCL 25205
Reference:
Bioorganic & Medicinal Chemistry Letters
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Received Date:
Revised Date:
Accepted Date:
2 July 2017
6 August 2017
7 August 2017
Please cite this article as: Xie, R., Li, Y., Tang, P., Yuan, Q., Rational design, synthesis and preliminary antitumor
activity evaluation of a chlorambucil derivative with potent DNA/HDAC dual-targeting inhibitory activity,
Bioorganic & Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.08.011
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Rational design, synthesis and preliminary antitumor activity evaluation of a
chlorambucil derivative with potent DNA/HDAC dual-targeting inhibitory activity
Rui Xie, Yan Li, Pingwah Tang and Qipeng Yuan*
Beijing Laboratory of Biomedical Materials, College of Life Science and Technology, Beijing University of Chemical
Technology, 15 Beisanhuan East Road, Beijing 100029, China
*Corresponding author, E-mail addresses: yuanqp@mail.buct.edu.cn
ABSTRACT:
Histone deacetylases (HDACs) play a pivotal role not only in gene expression but
also in DNA repair. Herein, we report the successful design, synthesis and evaluation
of a chlorambucil derivative named vorambucil with a hydroxamic acid tail as a
DNA/HDAC dual-targeting inhibitor. Vorambucil obtained both potent DNA and
HDACs inhibitory activities. Molecular docking results supported the initial
pharmacophoric hypothesis and rationalized the potent inhibitory activity of
vorambucil against HDAC1, HDAC2 and HDAC6. Vorambucil showed potent
antiproliferative activity against all the test four cancer cell lines with IC50 values of as
low as 3.2~6.2 µM and exhibited 5.0~18.3 fold enhanced antiproliferative activity
than chlorambucil. Vorambucil also significantly inhibits colony formation of A375
cancer cells. Further investigation showed that vorambucil remarkably induced
apoptosis and arrested the cell cycle of A375 cells at G2/M phase. Vorambucil could
be a promising candidate and a useful tool to elucidate the role of those DNA/HDAC
dual-targeting inhibitors for cancer therapy.
Keywords: Chlorambucil; Nitrogen mustard; Hydroxamic acid; Anticancer
Genotoxic anticancer drugs play a key role in cancer treatment for many years.
Among them, the nitrogen mustards represent a major class of genotoxic anticancer
drugs for the treatment of various cancers. Chlorambucil as one of the best tolerated
nitrogen mustards is in worldwide clinical use (Fig. 1A). The mechanism of action of
chlorambucil is based on attacking cellular DNA and causing DNA damage.1-3
However, the DNA damage caused by genotoxic drugs can be alleviated by cellular
DNA repair machinery, so that some cancer cells can survive and eventually lead to
treatment failure.4-6 As a result, nitrogen mustards exhibited poor potency and
limited treatment success. Inhibition of DNA repair machinery may be an effective
strategy to enhance the efficacy of nitrogen mustards.
Histone deacetylases (HDACs) are a family of epigenetic enzymes with critical roles
in chromatin condensation and gene expression by catalyzing the removal of acetyl
groups from histones.7-11 It has been shown that acetylation of the core histones
weakens the histone-DNA interactions, thereby increasing DNA accessibility.12 Recent
evidence demonstrates that the structural alterations in chromatin induced by HDAC
inhibitors could expose DNA to DNA-damaging agents such as ultraviolet rays, x-ray
and genotoxic drugs, eventually leading to double strand breaks in DNA.13-15
Inhibition of HDAC could down-regulate the DNA repair machinery.16 The critical role
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of HDACs in DNA repair has provide new opportunities for improving traditional
genotoxic drugs.17 Therefore, we devised a strategy to design a novel chlorambucil
derivative with the ability to not only bind DNA in much the same way as classical
nitrogen mustards drugs but also inhibit HDACs.
In general, the pharmacophore model for HDAC inhibitors is composed of three
regions: a zinc binding group (ZBG), a linker and a cap group. The zinc binding group
chelates the zinc ion in the active pocket of HDAC. The cap group interacts with the
entrance surface of HDAC active pocket. The linker accommodates the tubular access
of the active pocket.18-20 The common ZBG of HDAC inhibitors is hydroxamic acid.
There are many HDAC inhibitors with hydroxamic acid as ZBG, such as SAHA and
LBH589 (Fig. 1B), which represents the most studied and potent HDAC inhibitor with
a hydroxamic acid ZBG. Liu et al. synthesized a bendamustine derivative CY190602 as
DNA/HDAC dual-targeting drug.21 However, CY190602 contains a nitrogen
heterocycle (1-methyl-1H-benzimidazole) which is difficult to synthesize. Moreover
the nitrogen heterocyclic structure may not be a necessary group to obtain potent
antitumor drug. Herein, we report our effort in developing and characterizing a
chlorambucil derivative vorambucil (Fig. 1C) with a hydroxamate moiety displaying
potent HDAC inhibitory activity and antitumor activity. Vorambucil possesses simple
structure, potent antitumor activity and special HDAC isoform inhibitory activity,
which may be a promising candidate for cancer therapy.
Fig. 1. （A）Structure of chlorambucil；（B）Structure of SAHA and LBH589 ; (C) Design of
vorambucil as DNA/HDAC dual-targeting inhibitor.
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The preparation of vorambucil was depicted in Scheme 1. Firstly, commercially
available 1-fluoro-4-nitrobenzene was treated with 2,2'-iminodiethanol to provide
2,2'-[(4-nitrophenyl)imino]diethanol (intermediate 1). Intermediate 1 was treated
with SOCl2 to obtain N,N-bis(2-chloroethyl)-4-nitroaniline (Intermediate 2).
Intermediate 2 was reduced with zinc and hydrochloric acid to obtain
N,N-bis(2-chloroethyl)benzene-1,4-diamine (Intermediate 3). Then, intermediate 3
coupled with 8-methoxy-8-oxooctanoic acid to obtain intermediate 4. Finally,
conversion of methyl ester (intermediate 4) to hydroxamic acid using freshly
prepared hydroxylamine furnished vorambucil.
Scheme 1. Synthesis of vorambucil. Reagents and conditions: (a) 2,2'-iminodiethanol, 118 ℃，
reflux, 87%; (b) SOCl2, THF, 0 ℃ to r.t., 91%; (c) Zn, HCl, CH3OH, 78%; (d)
8-methoxy-8-oxooctanoic acid, ethanedioyl dichloride, DMF, triethylamine, THF, 0℃to r.t., 64%;
(e) KOH, NH2OH.HCl/MeOH, r.t.,57%.
The HDACs family are classified in four classes according to their homology to
yeast proteins and catalytic mechanism: classＩ(HDAC 1, 2, 3, and 8), classⅡ (HDAC
4, 5, 6, 7, 9 and 10), classⅣ (HDAC 11) and class Ⅲ (sirtuin 1-7).22, 23 It has been
shown that the dysregulation of classＩand classⅡHDACs, especially classＩ
isozymes, has been associated with the process of tumor cells proliferation and DNA
repair.24-27 We firstly tested the total HDACs inhibitory activity of vorambucil using a
HDACs Assay kit (BML-AK530, Enzo® Life Sciences) to investigate whether vorambucil
obtained HDACs inhibitory activity or not. As expected pharmacophoric hypothesis,
vorambucil achieve potent HDACs inhibitory activity (Fig. 2). Since classＩand classⅡ
HDACs were most closely related to tumor cells proliferation and DNA repair,
vorambucil was then assessed for inhibitory activity against HDAC isoforms 1 and 2
(classⅠ), and 6 (classⅡ). As shown in Table 1, vorambucil displayed potent HDAC1
and HDAC2 inhibitory activity, which was comparable to SAHA. Interestingly,
vorambucil showed approximately 20-fold weaker inhibitory activity against HDAC6
than SAHA. The results of HDAC isoforms inhibitory activity demonstrated that
vorambucil showed moderately more potent inhibitory activity against HDAC1 and
HDAC2 over HDAC6.
In order to understand the potent HDACs inhibitory activity of vorambucil, we
carried out docking study of vorambucil to the active pockets of HDAC1 (PDB code:
4BKX), HDAC2 (PDB code: 4LXZ) and HDAC6 (PDB code: 5EDU) using Surflex-dock. As
3

shown in Fig. 3, Vorambucil could form five hydrogen bonds with HIS145, HIS146,
ASP104 and TYR308 in the active site of HDAC2. The binding affinity of vorambucil for
HDAC2 was -8.2 kcal/mol. Vorambucil could also form five hydrogen bonds with
ARG34, TRP135, ALA136 and GLY137 in the active site of HDAC1. The binding
affinity of vorambucil for HDAC1 was -5.5 kcal/mol. Vorambucil could only form three
hydrogen bonds with HIS610, GLY619 and SER568 in the active site of HDAC6 and the
binding affinity was only -4.5 kcal/mol. The docking results could well explain the
potent HDACs inhibitory activity and moderate selectivity of vorambucil against
HDAC1 and HDAC2 over HDAC6.
Fig. 2. Total HDACs inhibitory activity of vorambucil and chlorambucil
Table 1 Inhibition of individual HDAC isoforms, IC50 (µM).
IC50 (µM) a
Isoform selectivity
classⅠ
classⅡ
Compd
HDAC6/HDA HDAC6/HDA
HDAC1
HDAC2
HDAC6
C1
2.3
C2
vorambucil
SAHA
2.6
0.27±0.04
0.19±0.02
0.24±0.05
0.18±0.03
0.62±0.11
0.16
0.17
0.03±0.004
a Values are the mean of at least two separate determinations.
4

Fig. 3. Predicted binding modes of vorambucil on HDAC1 (PDB code: 4BKX) , HDAC2 (PDB code:
4LXZ) and HDAC6 (PDB code: 5EDU). Interactions between the protein and the ligand are shown
as yellow dotted lines. (A) Molecular surface of the HDAC1 binding pocket (B) vorambucil
interacted with the actives of HDAC1. (C) Molecular surface of the HDAC2 binding pocket (D)
vorambucil interacted with the actives of HDAC2. (E) Molecular surface of the HDAC6 binding
pocket (F) vorambucil interacted with the actives of HDAC6.
To examine whether vorambucil triggers DNA damage in cancer cells, we
performed DNA damage assay of vorambucil against A375 cancer cells by a DNA
damage assay kit (Epigentek, NY, USA). The kit is able to determine the
phosphorylation of histone H2AX (γH2AX), which has been regarded as a marker for
DNA-double strand breaks. As shown in Fig. 4, after 48 h exposure, vorambucil
5

significantly induced more DNA damage (224.1%±2.4% at 16 µM) compared with
chlorambucil (128.6%±24.3% at 16 µM). Taken together, those results indicated that
vorambucil obtained both enhanced DNA-damaging activity and potent HDAC
inhibitory activity.
Does the enhanced DNA-damaging activity and potent HDAC inhibitory activity of
vorambucil bring enhanced anticancer activities or not? The antiproliferative
activities of vorambucil against four cancer cell lines were evaluated by CCK-8-based
assay. As shown in Table 2, vorambucil exhibited significantly enhanced anticancer
potency with IC50 values (concentration required to reduce viability to 50%) of as low
as 3.2~6.2 µM against four human cancer cell lines, more than 5.0~18.3 fold lower
than chlorambucil. The dose-dependent antiproliferative activities of vorambucil on
cancer cell lines were displayed in Fig. 5, which revealed that the antiproliferative
activities of vorambucil showed obvious dose-dependent manner.
To further explore the antitumor ability of vorambucil, we performed colony
formation assay of vorambucil against A375 cells with chlorambucil as positive
control. Results were summarized in Fig. 6. Vorambucil inhibited the colony
formation of A375 cancer cells in a dose dependent manner, and remarkably more
potent than chlorambucil. To further investigated whether the potent
antiproliferative activities of vorambucil was related to enhanced apoptosis of cancer
cells, we carried out flow cytometry assay on A375 cancer cell line and the
percentages of apoptotic cells were determined. As shown in Fig. 7, vorambucil
significantly induced apoptosis of A375 cancer cells in a concentration-dependent
manner. vorambucil induced 15.64%, 49.24% and 69.58% apoptosis of A375 cancer
cells at 2 µM, 8 µM and 16 µM respectively. However, chlorambucil only induced
9.93%, 12.85% and 28.86% apoptotic cells at the same concentration. Since
genotoxic anticancer drugs block the DNA synthesis, the effect of vorambucil on
A375 cell cycle progression was determined. As showed in Fig. 8, vorambucil
remarkably induced the accumulation of A375 cells at the G2/M phase (66.5% at 8
µM, Fig. 8), which were obviously more potent than chlorambucil (47.6% at 8 µM, Fig.
8).
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Fig. 4. Effects of chlorambucil and vorambucil on DNA damage in A375 cells. A375 cells were
treated with vorambucil or chlorambucil at 8 µM and 16 µM for 48 hours. The DNA damage was
detected using the EpiQuik In Situ DNA Damage Assay kit (Epigentek, NY, USA) according to the
manufacturer’s instructions. Data were expressed as the percent of the control (no drug
treatment), as mean±SD (n=3). P-values were determined using a t-test (* indicates p<0.05, **
indicates P <0.01, *** indicates P<0.001).
Table 2 Antiproliferative activities data (IC50, µM) of vorambucil and chlorambucil against four
cancer cell lines
Compd
HepG2
111.8±17.0
6.1±1.5
A549
HCT116
57.6±11.0
3.2±0.4
A375
Chlorambucil
Vorambucil
22.2±3.7
4.4±0.6
69.2±9.9
6.2±0.6
7

Fig. 5. Dose-dependent antiproliferative activities of vorambucil against four cancer cell lines. The
values represent the means of three experiments. (A) treatment in HepG2 cells. (B) treatment in
A549 cells. (C) treatment in HCT116 cells. (D) treatment in A375 cells.
Fig. 6. Vorambucil and chlorambucil inhibit A375 cancer cell colony formation.
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Fig. 7. Induction of apoptosis by vorambucil and chlorambucil in different concentrations in A375
cancer cells. The values represent the means of three separate experiments with SD less than
10%.
Fig. 8. Effect of vorambucil and chlorambucil on A375 cell cycle progression. The values represent
the means of three separate experiments with SD less than 10%.
9

In conclusion, using structure-based design approach, we have successfully
generated a novel chlorambucil derivative named vorambucil with a hydroxamate
acid tail as DNA/HDAC dual inhibitor. As expected pharmacophoric hypothesis,
vorambucil displayed potent inhibitory activity against both DNA and HDACs. Notably,
vorambucil showed slightly selective inhibition against HDAC1 and HDAC2 over
HDAC6. Molecular docking results could well support the potent HDACs inhibitory
activity and moderate selectivity of vorambucil. Vorambucil caused increased DNA
damage than chlorambucil. In vitro cell growth inhibition assays indicated that
vorambucil exhibited significantly enhanced anticancer potency than chlorambucil
against all the tested cancer cell line with IC50 of 3.2-6.2 µM. Moreover, vorambucil
was able to inhibit colony formation of A375 cells. Furthermore, vorambucil was
found to potently induce A375 cells apoptosis and arrest cell cycle at G2/M phase.
Together, vorambucil possessed potent antitumor activity and would be a useful tool
to elucidate the role of those DNA/HDAC dual-targeting inhibitors in cancer therapy.
More detailed antitumor mechanisms and in vivo activity evaluation of vorambucil
are underway in our lab.
Acknowledgements
This work was supported by the National Science Foundation of China (Grant No.
21176018).
Supplementary data
Supplementary data associated with this article can be found in the online version
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1. A chlorambucil derivative named vorambucil with a hydroxamic acid tail was
synthesized.
2. Vorambucil obtained both potent DNA damage and HDACs inhibitory activities.
3. Vorambucil displayed potent antiproliferative activity with IC50 values of as low as
3.2~6.2 µM against 4 kinds of cancer cells.
4. Vorambucil was able to induce apoptosis and arrest cell cycle at G2/M phase of
A375 cancer cells.
5. Vorambucil would be an important lead compound to guide the design of novel
DNA/HDAC dual-targeting inhibitors.
13