Design, synthesis and in vitro evaluation of amidoximes as histone deacetylase inhibitors for cancer therapy

Article abstract of DOI:10.1016/j.bmcl.2016.08.073

Amindoximes are geometric isomers of N-hydroxyamidines which are bioisosteres of hydroxamates. Since amindoxime group is capable of chelating transition metal ions including zinc ion, amindoximes should possess histone deacetylases (HDACs) inhibitory activity. In this work, we designed and synthesized a series of amindoximes, examined their inhibitory activities against HDACs, and investigated their cytotoxicity to human cancer cells. Preliminary results demonstrated that amindoximes possessed submicromolar HDACs inhibitory activity, with noteworthy enhancement compared with hydroxamates. Furthermore, the amindoximes arrested HCT116 and A549 cells in G2/M phase and showed good efficacy in inducing cells death. We provided a proof-of-concept that amindoximes could be used as HDACs inhibitors and hold great promise as epigenetic drugs.

Full text of DOI:10.1016/j.bmcl.2016.08.073

Bioorganic & Medicinal Chemistry Letters 26 (2016) 4679–4683
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and in vitro evaluation of amidoximes as histone
deacetylase inhibitors for cancer therapy
Peifu Jiao a, Peng Jin a, Chencan Li a, Lechao Cui a, Lihua Dong a, Bin Pan c, Wentong Song c, Liang Ma c,
Jinlong Dong a, Lei Song a, Xinjie Jin a, Faming Li a, Maosheng Wan b, , Zhitao Lv c, , Qiaohong Geng a,
⇑
⇑
⇑
a Department of Chemistry, Qilu Normal University, Jinan, Shandong 250200, China
b School of Chemistry and Biological Engineering, Hezhou University, Hezhou, Guangxi 542899, China
c Shandong Chengchuang Pharmaceutical R&D Co., Ltd, Jinan, Shandong 250101, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 21 June 2016
Revised 28 July 2016
Accepted 18 August 2016
Available online 24 August 2016
Amindoximes are geometric isomers of N-hydroxyamidines which are bioisosteres of hydroxamates.
Since amindoxime group is capable of chelating transition metal ions including zinc ion, amindoximes
should possess histone deacetylases (HDACs) inhibitory activity. In this work, we designed and synthe-
sized a series of amindoximes, examined their inhibitory activities against HDACs, and investigated their
cytotoxicity to human cancer cells. Preliminary results demonstrated that amindoximes possessed sub-
micromolar HDACs inhibitory activity, with noteworthy enhancement compared with hydroxamates.
Furthermore, the amindoximes arrested HCT116 and A549 cells in G2/M phase and showed good efficacy
in inducing cells death. We provided a proof-of-concept that amindoximes could be used as HDACs inhi-
bitors and hold great promise as epigenetic drugs.
Keywords:
Amindoximes
HDACs
Cytotoxicity
G2/M arrest
Ó 2016 Elsevier Ltd. All rights reserved.
The initiation and progression of cancer is controlled by both
genetic and epigenetic events,1,2 and epigenetic proteins have been
considered to be among the most promising targets for cancer
therapy.3 HDACs are a family of epigenetic proteins with critical
roles in the chromatin remolding and regulation of gene expression
by removing acetyl groups from lysine residues on the surface of
histone proteins.4,5 In cancer cells, a common finding is high level
expression of HDACs and a corresponding hypoacetylation of his-
tones.6 Inhibition of HDACs may cause differentiation, apoptosis,
anti-angiogenesis, migration suppression, susceptibility to radio-
therapy, cancer stem cell self-renewal pathway inhibition, and
enhancement of cancer cell recognition by T cells via suppressing
the expression of Program Death Receptor Ligand 1 (PD-L1).7–13
HDACs consist of four classes (class I, II, III, IV) and eighteen iso-
forms (HDAC1–11, SIRT1–7) based on their homology to yeast pro-
teins.14–17 Class I, II and IV of HDACs (HDAC1–11) are Zn2+
dependent enzymes whereas class III of HDACs (SIRT1–7) utilize
NAD+ as a cofactor as opposed to zinc ion.18 The term of ‘HDACs
inhibitors’ used in this work refers to compounds that target the
Zn2+ dependent HDACs. These HDACs inhibitors work by binding
to the active site pocket and chelating the catalytic zinc ion located
at its base.19–22
Conventional HDACs inhibitors are composed of three regions:
cap motif, linker and zinc-binding group (ZBG). The cap motif
occludes the entrance of the zinc ion containing pocket, and are
required for potent and selective binding of HDACs.18,20 The ZBG
chelates the zinc ion in the pocket, and plays a vital role in inhibit-
ing HDACs activities. The linker is the bridge of two functional
groups and is important for flexibility of the molecule. Current
ZBGs include hydroxamates, benzamides, thiols, sulfamides,
ketones and trithiocarbonates,18 among which, hydroxamates are
potent in inhibiting HDACs activity at micromolar concentration.
Although three hydroxamates have been approved by regulatory
agencies for cancer therapy (Fig. 1), these compounds also have
some intrinsic shortcomings. For example, the structure of hydrox-
amates is unstable in plasma and prone to hydrolysis producing
hydroxylamine which has potential mutagenic properties.23,24 As
a result, hydroxamates have a relatively short half-life in plasma
and potential mutagenic properties.25,26
Amindoximes are geometric isomers of N-hydroxyamidines
which are bioisosteres of hydroxamates (Fig. 2). Since these com-
pounds are capable of chelating transition metal ions and relatively
resistant to hydrolysis,27 they have been used as absorbents to
remove polluting metal ions including zinc ions from industrial
effluents, water supplies and mine waters.28,29 In the field of phar-
maceuticals, amindoximes have been developed as Lysine-Specific
Demethylase 1 (LSD1) inhibitors, which cause dramatic increases
⇑
Corresponding authors.
E-mail addresses: shmw11258@163.com (M. Wan), rorry-1@163.com (Z. Lv),
gengqh2006@163.com (Q. Geng).
http://dx.doi.org/10.1016/j.bmcl.2016.08.073
0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.

4680
P. Jiao et al. / Bioorg. Med. Chem. Lett. 26 (2016) 4679–4683
H
O
Cap
ZBG
O
Linker
O
S
N
OH
H
N
N
H
O
OH
N
H
O
Belinostat
Vorinostat
Approved, 2014, Spectrum
Approved, 2006, MSD
N
N
HN OH
O
N
O
N
N
H
N
OH
N
H
HN
JNJ-26481585
Panobinostat
Phase II, 2011, Janssen Research & Development
Approved, 2015, Novartis
O
O
OH
OH
O
N
H
O
N
H
N
H
O
N
H
N
ITF2357
Phase II, 2012, Italfarmaco
AR-42
Phase I, 2013, Alison Walker
Figure 1. Hydroxamates as HDAC inhibitors for cancer therapy.
O
NH
NH2
N
Bioisosteres
OH
OH
OH
N
H
N
H
R
R
R
hydroxamates
Figure 2. Design of amindoximes as HDAC inhibitors by bioisosterism.
N-hydroxyamidines
amindoximes
in global H3K4me2 levels and also promote the re-expression of
aberrantly silenced tumor suppressor genes in Calu-6 cell line.30
However, the application of these compounds as HDACs inhibitors
is still unexplored. In the current work, we designed and synthe-
sized a series of amindoximes, examined their inhibitory activities
against HDACs, and investigated their cytotoxicity to human can-
cer cells.
simply by phenyl chloroformate chemistry. In order to synthesize
benzonitriles 3d–3o, a variety of amides 2d–2o were prepared by
reactions of 4-aminobenzonitrile 1 with bromocarboxylic acid
chlorides. Amides 2d–2o bearing a bromide atom were subjected
to a nucleophilic substitution reaction employing a series of ami-
nes to form the desired benzonitriles 3d–3o. This chemistry gener-
ated a series of benzonitriles displaying substitutions on the distal
nitrogen atom as cap motifs. Finally, the amindoxime group was
installed using hydroxylamine under basic conditions to provide
Amindoximes 5a–5o could be readily synthesized using the
route described in Scheme 1. Benzonitriles 3a–3c were synthesized
v
O
n
vi
O
H
N
80-92%, two steps
41-79%
Br
CN
H2N
CN
R
N
H
CN
N
H
O
n
R-NH2
Br
1
Cl
2d-2o
3d-3o
n
i
iv
67%
vii
36-85%
O
n
Br
O
OH
CN
O
N
H
O
n
H
NH
HN OH
2
n = 0, 1, 2, 3, 4
N
R
N
H
ii
73-85%
CH3CH2CH2CH2-
R-NH2
O
4d-4o
R =
CH3CH2CH2CH2CH2CH2-
PhCH2CH2-
R
CN
N
H
N
H
3a-3c
iii
66-79%
O
R
O
H
N
NH2
OH
NH2
OH
O
NH
R
N
H
N
H
N
H
R
n
N
N
H
N
N
H
HN OH
5d-5o
5a-5c
4a-4c
Scheme 1. Synthesis of amindoximes 5a–5o. Reagents and conditions: (i) Phenyl chloroformate, Na2CO3, CH3CN, rt; (ii) Na2CO3, CH3CN, 50 °C; (iii) NH2OHÁHCl, Et3N, EtOH–
H2O, reflux; (iv) Oxalyl chloride, CH2Cl2, DMF, rt; (v) Et3N, CH3CN, rt; (vi) Et3N, CH2Cl2, rt; (vii) NH2OHÁHCl, Et3N, EtOH–H2O, reflux.

P. Jiao et al. / Bioorg. Med. Chem. Lett. 26 (2016) 4679–4683
4681
the amindoximes 5a–5o. All amindoximes were characterized by
1H NMR, 13C NMR, MS, melting point and elemental analysis (see
SI for details). 1HNMR spectrum showed the presence of –NH2
group at 5.7 ppm, which confirmed that the obtained compounds
were amindoximes 5a–5o instead of N-hydroxyamidines 4a–4o
(Fig. 3).
amindoximes 5c, 5f, 5i bearing phenyl ring in R group showed bet-
ter HDACs inhibitory activities compared with amindoximes con-
taining n-butyl and n-hexyl groups, indicated that the
introduction of distal aromatic rings increased inhibitory activity
of amindoximes to HDACs.
We next tested whether amindoximes 5a–5o showed anti-pro-
liferative activities. Since human colon carcinoma cell line HCT116
and non-small cell lung cancer cell line A549 worked very well in
the screening of HDACs inhinitors,20,31 we selected them for anti-
cancer activity determination. As shown in Table 1, most amin-
doximes (5a–5i) have good efficacy in inducing HCT116 cell death
and the general trend of cell growth inhibition correlates with
potency for HDACs. However, these compounds show poor efficacy
in inducing A549 cell death. Since the HDACs expression in HCT116
is much higher than that in A549,31 this poor efficacy is likely due
to the relative low HDACs expression in A549 cells.
To compare the HDACs inhibition activities of amindoximes
with those of hydroxamates, hydroxamate 10 was synthesized
according to Scheme 2. Carbamate 8 was also synthesized by phe-
nyl chloroformate chemistry, and converted to its methyl ester 9
by Fischer esterification. Finally, the hydroxamate group was also
installed using hydroxylamine under basic condition to give
hydroxamate 10. A variety of basic conditions were examined to
optimize the preparing process of hydroxamate 10. The employ-
ment of bases such as sodium hydroxide, potassium hydroxide,
sodium carbonate, and potassium carbonate could lead to signifi-
cant hydrolysis of methyl ester 9, which accounted for both low
yield (13–28% by HPLC, data not shown) of hydroxamate 10 and
the following annoying reaction work-up. In contrast, replacement
of these bases with sodium methoxide not only increased the reac-
tion yield up to 43%, but also facilitated the reaction work-up by
avoiding flash chromatography.
HDACs inhibitors can bring cancer cells to either G1/S phase or
G2/M phase, and trigger apoptotic cell death pathway.31,32 We next
turned our attention to the effects of amindoximes on cell-cycle
checkpoints. Our results demonstrated that A549 and HCT116 cells
were pushed into the G2/M phase but not G1/S phase when being
treated with amindoxime 5f. As shown in Figure 4, amindoxime 5f
With compounds synthesized, we firstly determined whether
amindoximes could inhibit the activity of HDACs. Our current
amindoximes were designed as unspecific HDACs inhibitors.
Therefore, HeLa nuclear extracts containing HDAC1 and HDAC2
were used as HDAC-containing test samples. HDACs activities were
measured using an AmpliteTM fluorimetric HDACs activity assay kit
(see SI for details). Table 1 showed that amindoximes did possess
submicromolar HDACs inhibitory activity. Compared to hydroxam-
ate 10, amindoximes 5c displayed an approximately 1-fold
enhancement in inhibition of HDACs activity, which suggested that
amindoximes might be more potent HDACs inhibitors than
hydroxamates. Since the length of the linker (n) and the type of
the cap (R) also play important roles in inhibitory potency,21 we
synthesized amindoximes 5a–5o containing different linker
lengths and R groups to examine their effects on the activity for
HDACs. Amindoximes 5d–5f containing one methylene group
showed better HDACs inhibitory activity in nanomolar range.
Decreasing and increasing the distance between the amindoxime
arrested 36% and 68% of A549 cells in G2/M phase at 26
lM and
157 M, respectively, suggesting amindoxime 5f caused G2/M
l
arrest of A549 cells in a dose dependent manner. Similar results
were found in HCT116 cells. Moreover, the proportion of HCT116
cells arrested in G2/M phase were higher than that of A549 cells,
indicating HCT116 cells were more sensitive to amindoximes than
A549 cells.
Regulation of gene expression by epigenetic approaches is a
promising strategy both in small molecules targeted cancer ther-
apy and in cancer immunotherapy. Concurrently, HDACs inhibitors
have been developed as important epigenetic drugs, and have
shown great potential in cancer therapies. As a result, the discovery
of novel HDACs inhibitors containing novel functional groups to
improve potency and selectivity is continuing. In this work, we
designed and synthesized a series of amindoximes with different
cap motifs and linkers, while amindoxime group is used as zinc
ion binding group. The amindoximes possessed submicromolar
HDACs inhibitory activity and displayed noteworthy enhancement
in inhibition of HDACs activity compared with hydroxamates.
Amindoximes containing the distal aromatic ring in the R group
group and
R group by regulating methylene groups led to
decreased HDACs inhibitory activity (5a–5c, 5g–5o). In addition,
Figure 3. 1H NMR spectrum of amindoxime 5c suggested the presence of –NH2 group which indicated that the exact structures of our compounds were amindoximes but not
N-hydroxyamidines. 1H NMR spectrum of the other compounds showed similar signals at the chemical shift of near 5.7 ppm.

4682
P. Jiao et al. / Bioorg. Med. Chem. Lett. 26 (2016) 4679–4683
O
O
i
ii
COOH
H2N
68%
COOH
COOH
N
H
N
H
O
N
H
71%
7
8
6
O
O
iii
57%
iv
O
COOCH3
N
N
H
N
H
N
H
43%
H
HN OH
9
10
Scheme 2. Synthesis of hydroxamate 10. Reagents and conditions: (i) Phenyl chloroformate, Et3N, DMF, rt; (ii) 2-phenylethanamine, Et3N, DMF, 70 °C; (iii) MeOH, concd
H2SO4, reflux; (iv) NH2OHÁHCl, CH3ONa/CH3OH, reflux.
Table 1
HDACs inhibition and anti-cancer activity of amindoximes with diverse substitutions
O
H
N
NH2
R
N
H
n
OH
N
Compds
R
n
HDACs IC50 (nM)
HCT116 EC50
(l
M)**
A549 EC50 (l
M)**
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
10
CH3CH2CH2CH2–
CH3CH2CH2CH2CH2CH2–
PhCH2CH2–
CH3CH2CH2CH2–
CH3CH2CH2CH2CH2CH2–
PhCH2CH2–
CH3CH2CH2CH2–
CH3CH2CH2CH2CH2CH2–
PhCH2CH2–
CH3CH2CH2CH2–
CH3CH2CH2CH2CH2CH2–
PhCH2CH2–
CH3CH2CH2CH2–
CH3CH2CH2CH2CH2CH2–
PhCH2CH2–
—
—
0
0
0
1
1
1
2
2
2
3
3
3
4
4
4
—
—
591
599
304
282
381
93
528
602
186
195
91
83
124
26
153
193
69
267
312
218
516
>1000
691
175
3
>1000
>1000
463
395
658
157
881
>1000
330
>1000
>1000
>1000
>1000
>1000
>1000
>1000
12
273
723*
>1000*
697*
>1000*
>1000*
>1000*
582
Vorinostat
11
*
Data is from a 4-point screen (0.1, 0.5, 1 and 5
EC50 is the concentration of compounds required to inhibit the cell growth by 50% compared to control cells (72 h after treatment).
l
M).
**
Figure 4. Induction of G2/M arrest by amindoxime 5f (26 lM and 157 lM) in A549 and HCT116 cells.

P. Jiao et al. / Bioorg. Med. Chem. Lett. 26 (2016) 4679–4683
4683
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Supplementary data
Supplementary data (experimental section, characterization,
NMR spectra, and in vitro methods) associated with this article
can be found, in the online version, at http://dx.doi.org/10.1016/j.
bmcl.2016.08.073.
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