Introduction of the α-ketoamide structure: En route to develop hydrogen peroxide responsive prodrugs

Article abstract of DOI:10.1039/c9sc00910h

Leveraging the elevated levels of hydrogen peroxide (H₂O₂) in cancer, inflammatory diseases and cardiovascular disorders, H₂O₂-activated promoieties have been widely used in drugs and biomaterials design. However, the overwhelming majority of the promoieties only share the common structure of a H₂O₂-responsive arylboronic acid/ester moiety with low diversity. We report here an unprecedented strategy to construct novel H₂O₂-responsive prodrugs based on an α-ketoamide structure. As a proof of concept, we designed and synthesized a panel of α-ketoamide based nitrogen mustard prodrugs, among which KAM-2 showed potent growth inhibitory activity and high selectivity toward cancer cells. The H₂O₂-trigged decomposition of KAM-2 was validated, and the DNA damaging and apoptosis promoting activity attributed to the released nitrogen mustard were demonstrated. Our work unveils α-ketoamide as a new scaffold for prodrug design and may quickly inspire future developments.

Full text of DOI:10.1039/c9sc00910h

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DOI: 10.1039/C9SC00910H
ARTICLE
Introduction of the α-ketoamide structure: En route to develop
hydrogen peroxide responsive prodrugs†
Tingting Meng,‡a,b Jing Han,‡c Pengfei Zhang,a Jing Hu,a Junjie Fu*a,b and Jian Yin*a
Received 00th January 20xx,
Accepted 00th January 20xx
Leveraging the elevated levels of hydrogen peroxide (H2O2) in cancer, inflammatory diseases and cardiovascular disorders,
H2O2-activated promoieties have been widely used in drugs and biomaterials design. However, the overwhelming majority
of the promoieties only share the common structure of a H2O2-responsive arylboronic acid/ester moiety with low diversity.
We report here an unprecedented strategy to construct novel H2O2-responsive prodrugs based on an α-ketoamide structure.
As a proof of concept, we designed and synthesized a panel of α-ketoamide based nitrogen mustard prodrugs, among which
KAM-2 showed potent growth inhibitory activity and high selectivity toward cancer cells. The H2O2-trigged decomposition
of KAM-2 was validated, and the DNA damaging and apoptosis promoting activity attributed to the released nitrogen
mustard were demonstrated. Our work unveils α-ketoamide as a new scaffold for prodrug design and may quickly inspire
future developments.
DOI: 10.1039/x0xx00000x
precursors as selective anticancer agents in 2011, followed by
the blooming of various prodrugs (over 50 published papers)
bearing an arylboronic acid/ester as the H2O2-cleavable trigger
Introduction
Prodrugs and probes, two important and vibrant research
subjects in medicinal chemistry and analytical chemistry, are
indeed closely related. Probes are usually trigged by specific
analytes to release fluorescent signals, and prodrugs are
between 2012–2019 (Table S1). Other H2O2-responsive
moieties, such as arylsulfonyl ester,19 thiazolidinone,20 and
benzothiazole21 have also been reported for constructing small
molecule prodrugs, but with very limited examples of each. In
addition, sulfide, thioether, thioketal, selenium, and peroxalate
ester are also known as H2O2-senstive scaffolds, but in most
cases, their applications are confined to polymers or
nanoparticles.22, 23 As yet, currently over 90% H2O2-activated
small molecule prodrugs still rely on the long-known boron-
based triggers. Moreover, several disadvantages have been
reported for the boron moiety, such as the high susceptibility of
boronic esters to hydrolysis,24 the off-target reactions between
boronic acids and biological diols,25 the moderate selectivity
toward H2O2 and interference from ONOO−, as well as the
potential unknown bioeffects from the by-produced boric
acid.21 Considering the extremely low structural diversity of the
current H2O2-responsive prodrugs, we have been highly
interested in searching for novel chemical structures with H2O2-
cleavable property.
We were recently enlightened by the elegant work from
Tang et al., who used α-ketoamide-based fluorescent probes for
intracellular H2O2 visualization.26-28 The α-ketoamide moiety
was demonstrated as a highly specific reaction switch for H2O2.
Herein, as a proof-of-concept, we introduce the α-ketoamide
group into prodrug design by hybridizing it with nitrogen
mustards, affording a panel of α-ketoamide-based nitrogen
mustard (KAM) prodrugs. Multiple assays confirmed that one of
the prodrugs, KAM-2, was efficiently and preferentially
activated by H2O2, and showed potent and selective cytotoxicity
to several cancer cell lines, particularly for HL-60 human
leukemia cells. The ability of KAM-2 to induce DNA damage and
activated by pathological stimuli to liberate drugs.1,
2
Importantly, chemists are always encouraged by the analyte-
responsive moieties of probes and expand their applications to
prodrug design to achieve spatiotemporal control of drug
release. Recent examples include the intriguing discoveries of
prodrugs activated by β-secretase,3 histone deacetylase
(HDAC)/cathepsin L (CTSL),4 matrix metalloproteinase-7 (MMP-
7),5 Caspase-3,6 γ-glutamyl transferase,7 thioredoxin
reductase,8 lysyloxidase,9 photocleavage,10 hypochlorous
acid,11 etc.
H2O2 has been taken as an important molecule due to its
elevated levels in many pathological conditions.12,
13
The
pioneer work from Chang et al.,14, 15 who initiated fluorescent
probes bearing a H2O2-reponsive boronate trigger in 2004–
2005, led to the rapid expansion of this strategy to the design of
H2O2-activated prochelators and MMP proinhibitors.16, 17 Given
the significantly high H2O2 concentrations (5–1000 μM) in
cancer cells relative to normal cells (0.001–0.7 μM), Peng et
al.,18 for the first time, reported boron-based nitrogen mustard
a. Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of
Education, School of Biotechnology, Jiangnan University, Wuxi 214122, P. R. China
b. School of Pharmacy, Nanjing Medical University, Nanjing 211166, P. R. China
c. School of Chemistry & Materials Science, Jiangsu Key Laboratory of Green
Synthetic Chemistry for Functional Materials, Jiangsu Normal University, Xuzhou
221116, P. R. China
* E-mail: jfu@jiangnan.edu.cn (J. F.), jianyin@jiangnan.edu.cn (J. Y.)
†Electronic
Supplementary
Information
(ESI)
available.
See
DOI: 10.1039/x0xx00000x
‡These authors contributed equally.
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OH
apoptosis was thoroughly studied. To the best of our
knowledge, this is the first example to utilize the α-ketoamide
moiety as an alternative non-boron H2O2-responsive element
for prodrug development.
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d
KAM-1
DOI: 10.1039/C9SC00910H
KAM-3
O2N
N
c
e
O
KAM-2
KAM-4
b
f
N
H
OH
O
O
O
2
OH
O
R1
H
N
O2N
NH2
1
g
KAM-1: R1=R2=Cl
H2O2 cleavage
O2
N
N
O
R
O
KAM-2: R1=R2=Br
O2N
a
i
N
R2
KAM-3: R1=R2=OMs
KAM-4: R1=OMs, R2=Cl
O
N
H
KAM-5
R1
3
4
: R = OH
: R = Br
R2
O
h
Cl
O2N
Br
Cl
O2N
OH
k
O
N
N
Br
j
1) H2O2 cleavage
2) 1,6-elimination
KAM-6
N
O2
N
DNA alkylation
O
N
Cl
H
O
OH
5
N
Cl
N
H
O
Scheme 2. Synthesis of KAM prodrugs. Reactants and conditions: a) SeO2, pyridine, 90
°C, N2, 35%; b) N-4-aminophenyl diethanolamine, EDCI, DIEA, DMF, 42%; c) MsCl, DMAP,
TEA, DCM, reflux, 70%; d) LiCl (5 eq.), DMF, 60 °C, 65%; e) NaBr (5 eq.), DMF, 60 °C, 67%;
f) LiCl (1 eq.), MeCN, 60 °C, 23%; g) (4-aminophenyl)methanol, EDCI, DIEA, DMF, 33%; h)
KAM-5
O
Cl
Cl
H
1) H2O2 cleavage
2) 1,6-elimination
N
O
N
N
O2
N
Cl
Cl
CBr4, PPh3, DCM,
0
°C–r.t., 50%; i) N-(4-N’,N’-dimethylaminophenyl)-bis(2-
Cl
chloroethyl)amine, MeCN, 43%; j) N-methyl-diethanolamine, CHCl3, reflux, 76%; k) SOCl2,
r.t., 62%.
KAM-6
N
H2O2 cleavage
O2
1,6-elimination
O2
N
O
O
O
H2O2
OH
N
H
N
H
Anticancer activity screening
O
NR3
NR3
O
O
O
OH
H2
N
O2
N
H2
O
The antiproliferative activity of KAM prodrugs was screened
against four human cancer cell lines (A549, HepG2, HCT-116,
and HL-60), which have all been well-employed in H2O2
responsive prodrug studies.33-35 The classical nitrogen mustard
mechlorethamine (MEC) was selected as the positive control
Evidently, KAM-2 exhibited the most potent activity among all
the candidates (Fig. 1). At a dose of 10 μM, the inhibition rates
of KAM-2 in all cancer cells were >50%, which further exceeded
80% at 20 μM. Structurally similar prodrugs KAM-3 and KAM-4
showed decreased cytotoxicity compared with KAM-2. The
activity of chlorine counterpart, KAM-1, was further
compromised. These results suggest that the leaving groups of
mustard significantly affect the anticancer activity of KAM
prodrugs. Ammonia salts KAM-5 and KAM-6 showed limited
inhibition effects, particularly for KAM-5, possibly due to their
lower membrane permeability and absorption.30 This
assumption was preliminarily tested by calculating the LogP and
membrane permeability values of KAM prodrugs, and the
results are summarized in Table S2. Considering the essential
role of the nitro group of KAM in H2O2-initiated nucleophilic
attack,26, 27 a non-nitro analogue KAM-7 was synthesized (ESI)
as a negative control, which was supposed to be resistant to
H2O2. As expected, KAM-7 displayed diminished cytotoxicity
against cancer cells (Fig. 1), demonstrating the role of H2O2 in
prodrug activation.
H
N
H2N
H2
N
O
4-NO2-PhCOOH, CO2
Scheme 1. Structures of KAM prodrugs and the mechanisms of H2O2-responsive
activation to generate DNA alkylating agents.
Results and Discussion
Design and synthesis of KAM prodrugs
The structures of KAM prodrugs are illustrated in Scheme 1. It is
speculated that the α-ketoamide moiety in KAM-1–KAM-4 acts
as an electron-withdrawing group and shields the activity of
nitrogen mustards.18, 29-31 Upon the nucleophilic attack by H2O2
anion and the following Baeyer-Villiger rearrangement,27 the
promoiety finally hydrolyses into aniline nitrogen mustards with
increased nitrogen electron density and restored DNA alkylating
activity. To enrich the structural diversity, quaternary ammonia
salts KAM-5 and KAM-6 were also designed as masked mustard
prodrugs. The activation process of KAM-5 and KAM-6 are
similar to the mechanism described above but require an
additional spontaneous 1,6-elimination.32
The synthetic route (Scheme 2) of the target compounds
commenced with a SeO2-mediated oxidation of acetophenone
in pyridine to afford phenylglyoxylic acid 1, which was coupled
with
N-4-aminophenyl-diethanolamine
or
(4-
aminophenyl)methanol in the presence of EDCI and DIEA to give
α-ketoamides 2 or 3, respectively. The two hydroxy groups of 2
were methylated to generate KAM-3. Further dichlorination,
dibromination, and monochlorination of KAM-3 furnished
KAM-1, KAM-2, and KAM-4, respectively. Bromination of the
hydroxy group of 3 using CBr4 and PPh3 yielded 4, which was
then directly reacted with aniline nitrogen mustard to give
KAM-5. Alternatively, nucleophilic substitution of 4 with N-
methyl-diethanolamine, followed by chlorination with SOCl2
generated KAM-6. Detailed synthetic procedures and
spectroscopic data are provided in the Supporting Information.
2 | J. Name., 2012, 00, 1-3
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were in-depth studied. KAM-2 exhibited a decent stability in the
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absence of H2O2 at pH 4, 7, 10, respectivDeOlyI:(1F0i.g10. 3S92/BC9aSnCd00S921C0H).
H2O2 rapidly trigged the decomposition of KAM-2 in a H2O2
equivalent-dependent way (Fig. S2D). As monitored by HPLC,
the released nitrogen mustard N,N-bis(2-bromoethyl)benzene-
1,4-diamine (BrM), as well as the by-product p-nitrobenzoic acid
(PNB), were clearly observed 60 min after H2O2 treatment (Fig.
2A). In sharp contrast, KAM-7 remained intact regardless of
H2O2 presence (Fig. 2B and S2E). The decomposition of KAM-2
in H2O2 was further studied by 1H NMR and mass spectroscopy,
both of which unambiguously confirmed the prodrug activation
behaviour (Fig. S3 and S4). It is worth noting that in both HPLC
and NMR studies, the signal of BrM shrank with time and
became barely visible after long time treatment with H2O2. This
is highly possible due to the instability of the phenylenediamine
mustard toward H2O2 oxidation39, 40 under the in vitro study
conditions, as shown in Fig. S5 and S6. To provide a visual
demonstration of the mustard release from KAM-2, BrM was
Fig.1 Cytotoxicity of KAM prodrugs against four human cancer cell lines. Cells were prepared as a yellow solution, which turned dark upon H2O2
incubated with indicated compounds at 10 (A) and 20 μM (B) for 72 h, and the
percentages of cell death were assessed by MTT and CCK-8 assays. Mean ± SD, n = 3.
addition. The same colour change was observed with KAM-
2/H2O2, but not with KAM-7/H2O2 (Fig. S7). Moreover, in HL-60
cells, H2O2 scavenger N-acetyl-cysteine (NAC) pretreatment
remarkably decreased the cytotoxicity of KAM-2, but not KAM-
The anticancer activity of KAM-2 was then more closely
7 (Fig. 2C). All the above results clearly demonstrated the H2O2-
studied by measuring its IC50 values, employing three additional
H2O2- overproducing cancer cell lines (SW620, MCF-7, PANC-
responsive activation of KAM-2. In regards of selectivity, KAM-
t
2 was resistant to HO•, BuO•, TBHB, NO, GSH, and Fe2+, very
1)36-38 plus one normal line, peripheral blood mononuclear cell
(PBMC) (Fig. S1). As summarized in Table 1, KAM-2 showed
lower IC50 values than MEC in cancer cells, except for PANC-1,
which was particularly sensitive to MEC. The lowest IC50 (5.942
μM) of KAM-2 was seen in HL-60 cell. Encouragingly, KAM-2
proved its excellent selectivity toward cancer cells since the IC50
value in PBMC was larger than 50 μM. In contrast, MEC
remained cytotoxic in PBMC with an IC50 value of 11.50 μM.
stable in ClO−, Cys and Hcy. Slight decomposition was observed
in ONOO−, but to a much lesser extent than in H2O2 (Fig. 2D).
Table 1. IC50 values (μM) of KAM-2 and MEC against selected cell lines.[a]
IC50 (μM)
Cell lines
KAM-2
8.82 ± 0.83
8.01 ± 0.85
8.84 ± 1.24
5.94 ± 0.94
12.19 ± 1.55
16.25 ± 1.15
9.10 ± 0.27
> 50
MEC
A549
HCT-116
HepG2
HL-60
10.07 ± 0.71
13.25 ± 1.57
10.68 ± 1.52
12.60 ± 3.47
24.26 ± 1.23
2.19 ± 0.08
18.50 ± 0.07
11.50 ± 0.94
Fig. 2 H2O2-responsive behaviours of KAM-2. (A) Decomposition of 100 μM KAM-2 in
presence of H2O2 (2 eq.) as monitored by RP-HPLC (254 nm). (B) Decomposition of KAM-2
and KAM-7 (100 μM) with or without H2O2 (10 eq.). (C) The effects of NAC on the
cytotoxicity of KAM-2 and KAM-7 in HL-60 cells after 72 h incubation. (D) Selectivity of
KAM-2 toward H2O2 as determined by measuring its decomposition ratios under various
stimuli.
MCF-7
PANC-1
SW620
PBMC
[a]Cells were incubated with indicated compounds at different concentrations for
72 h, and IC50 values were measured by MTT and CCK-8 assays. Mean ± SD, n = 3.
Proapoptotic mechanism of KAM-2
The anticancer activity of KAM-2 was further evaluated in HL-
60 cells using MEC as positive control. It was found that KAM-2
treatment for 48 h induced apoptosis (Fig. 3A and S8A) of HL-60
cells in a dose-dependent manner. Notably, NAC pretreatment
significantly decreased the activity of KAM-2. Neither KAM-7
H2O2-responsive behaviours
All the KAM prodrugs decomposed quickly upon H2O2 treatment
(Fig. S2A). Considering the good cytotoxicity and selectivity of
KAM-2 toward cancer cells, its H2O2-responsive behaviours
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cytometry, Mean ± SD, n = 3. (C) Dose-dependent effects of KAM-2 on the expression of
nor NAC showed apoptosis-inducing activity under the same
condition (Fig S8B). Moreover, KAM-2 arrested cell cycle in
G0/G1 phase and NAC reversed the cell cycle arrest (Fig. 3B and
S9). Modest effects of KAM-2 on the loss of mitochondrial
membrane potential were also observed (Fig. S10). Western
blot studies revealed that KAM-2 induced the cleavage of
Caspase 3 and Caspase 9, elevated the expression of Bax, and
suppressed the expression of Bcl-2 in a dose (Fig. 3C) and time
(Fig. 3D) dependent way, similar to the effects of MEC. The
above results suggest that KAM-2 acts at least partially through
the mitochondria-mediated apoptosis pathway.
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apoptosis-related proteins by Western blot. HL-60 cells were treated with compounds at
DOI: 10.1039/C9SC00910H
indicated concentrations for 48 h or pre-incubated with NAC (20 mM) for 1 h, followed
by treatment with compounds for 48 h. Mean ± SD, n = 3. **p < 0.01 vs. blank; #p < 0.05
vs. 20 μM KAM-2 treatment; ##p < 0.01 vs. 20 μM KAM-2 treatment; ap < 0.05 vs. 10 μM
MEC treatment; aap < 0.01 vs. 10 μM MEC treatment. (D) Time-dependent effects of
KAM-2 on the expression of apoptosis-related proteins by Western blot. HL-60 cells were
treated with 10 μM KAM-2 for 0, 6, 12, 24, and 48 h. Mean ± SD, n = 3. *p < 0.05 vs. 0 h;
**p < 0.01 vs. 0 h.
DNA cross-linking and damaging effects of KAM-2
In addition to the above disclosed mechanism, the nitrogen
mustard released from KAM-2 upon H2O2 activation is supposed
to exert its cytotoxicity by DNA interstrand cross-linking (ICL).41
This was first examined by
a fluorescence quenching
experiment. It is known that ethidium bromide (EB) interacts Fig. 4 (A) Fluorescence quenching of EB/DNA by KAM-2. Various concentrations of KAM-
2 was added into EB-DNA solution (2.5 μM EB and 50 μM linearized pBR322 plasmid
DNA) with or without H2O2 (10 mM) in Tris-HCl/NaCl buffer. Ex = 300 nm (B) Stern-Volmer
plots of fluorescence quenching of EB-DNA by KAM-2. I0 and I are the emission intensity
in the absence and presence of the KAM-2, respectively. Slope (Ksv) is the quenching
with DNA to yield maximum fluorescence emission at 600 nm
(Ex = 300 nm), which is supposed to be quenched due to
competitive binding of nitrogen mustard to DNA. As expected,
KAM-2 decreased the fluorescence intensity at 600 nm in the
rate constant, and [Q] is the concentration of the quencher KAM-2.
presence of H2O2 (Fig. 4).
Fig. 5 DNA interstrand cross-linking (ICL) ability of KAM-2. (A) Denaturing alkaline
agarose gel electrophoresis performed with 1 μg linearized pBR322 plasmid DNA and
indicated concentrations of KAM-2 or MEC with or without H2O2 (10 mM) for 2 h at 37
°C. (B) Denaturing alkaline agarose gel electrophoresis performed with 1 μg linearized
pBR322 plasmid DNA, 50 μM KAM-2, and 10 mM H2O2 at 37 °C for indicated time. (C)
Denaturing alkaline agarose gel electrophoresis performed with 1 μg pBR322, 50 μM
KAM-2 or KAM-7 with or without H2O2 (10 mM) for 2 h at 37 °C. CLY: cross-linking yield.
Fig. 3 (A) Apoptotic effects of KAM-2 measured by flow cytometry using annexin V-
FITC/PI staining. HL-60 cells were treated with compounds at indicated concentrations
for 48 h, or pre-incubated with ROS scavenger NAC (20 mM) for 1 h, followed by
treatment with 10 μM KAM-2 for 48 h. Mean ± SD, n = 3. (B) Effects of KAM-2 on cell
cycle arrest. HL-60 cells were treated with compounds at indicated concentrations for 48
h, or pre-incubated with NAC (20 mM) for 1 h, followed by treatment with 10 μM KAM-
2 for 48 h. The percentage of cells in each cell cycle phase was analyzed by flow
The DCL ability of activated KAM-2 was further confirmed
by denaturing alkaline agarose gel electrophoresis.42 As shown
in Fig. 5A and B, KAM-2 or H2O2 alone had no effects on
linearized pBR322 plasmid DNA. However, in the presence of 10
mM H2O2, KAM-2 treatment induced DNA ICL (from 3.3% to
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94.8%) as indicated by the intensified bands of double strand
DNA (dsDNA) in both a dose and time dependent way. In
contrast, KAM-7 combined with H2O2 was unable to yield dsDNA
(Fig. 5C). These results again, strongly support the H2O2-
triggered activation of KAM-2 and consequent generation of
nitrogen mustard.
View Article Online
DOI: 10.1039/C9SC00910H
Next, the DNA damaging activity of KAM-2 was assessed in
HL-60 cells using comet assay. KAM-2 treatment resulted in
obvious cellular DNA damage as visually shown in Fig. 6. NAC
preincubation protected HL-60 cells from tail DNA formation
due to diminished activation of KAM-2. KAM-7 or NAC alone
had no effects on DNA damage (Fig. S11).
Fig. 6 Effects of KAM-2 on cell damage assessed by Comet assay. HL-60 cells were treated
with 20 μM KAM-2 or 10 μM MEC for 48 h, or pre-incubated with NAC (20 mM) for 1 h,
followed by treatment with 10 μM KAM-2 for 48 h. Alkaline comet electrophoresis was
performed. Quantitative analysis was conducted with the comet analysis software CASP.
The tail DNA% and olive tail moment were employed to evaluate DNA damage. Mean ±
SD, n = 3. **p < 0.01 vs. blank; ##p < 0.01 vs. 20 μM KAM-2 treatment.
Fig. 7 Immunofluorescence of DNA cleavage protein γH2AX. HL-60 cells were treated with
KAM-2 or MEC (10 μM) for 12 h, or pre-incubated with ROS scavenger NAC (20 mM) for
1 h, followed by treatment with KAM-2 for 12 h. For some groups, cells were incubated
for an additional 6 or 12 h after removal of KAM-2.
Following DNA double-strand break (DSB), histone H2AX is
rapidly phosphorylated at Ser139 to yield γ-H2AX as a golden
biomarker of DNA damage response (DDR).43 The expression of
γ-H2AX upon KAM-2 treatment was therefore examined using
immunofluorescence microscopy. Incubation of HL-60 cells with
KAM-2 for 12 h remarkably increased the level of γ-H2AX (Fig.
7). Notably, when KAM-2 was removed and cells were allowed
to grow without KAM-2 for an additional 6 or 12 h, elevated
expression of γ-H2AX was still observed. However, NAC
pretreatment mitigated the effects of KAM-2 on γ-H2AX
formation. Compared with KAM-2, KAM-7 and NAC had
negliable effects on γ-H2AX formation as shown in Fig. S12.
In addition to γ-H2AX, poly(ADP-ribose) polymerase-1
(PARP1), RAD51 and BRCA1 are activated by DNA DSB and
involved in DNA repair.44 The changes of PARP1, RAD51, BRCA1,
and γ-H2AX were quantified by Western blot. Clearly, KAM-2
induced the cleavage of PARP1 and increased the expression of
γ-H2AX, RAD51 and BRCA1 in a dose (Fig. 8A) and time (Fig. 8B)
dependent way. In contrast, KAM-7 or NAC treatment led to no
alterations of the expressions of cleaved PARP1 or γ-H2AX (Fig.
S13).
Fig. 8 (A) Concentration-dependent effects of KAM-2 on the expression of DDR-related
proteins by Western blot. HL-60 cells were treated with compounds at indicated
concentrations for 48 h or pre-incubated with NAC (20 mM) for 1 h, followed by
treatment with compounds for 48 h. Mean ± SD, n = 3. **p < 0.01 vs. blank; #p < 0.05 vs.
20 μM KAM-2 treatment; ##p < 0.01 vs. 20 μM KAM-2 treatment. (B) Time-dependent
effects of KAM-2 on the expression of DDR-related proteins by Western blot. HL-60 cells
were treated with 10 μM KAM-2 for 0, 6, 12, 24, and 48 h. Mean ± SD, n = 3. *p < 0.05 vs.
0 h; **p < 0.01 vs. 0 h.
Finally, the effects of KAM-2 on the expression of Parp1,
Rad51 and Brca1 mRNAs were investigated in HL-60 cells. Real-
time PCR assay confirmed that KAM-2 concentration (Fig. 9A)
and time (Fig. 9B) dependently elevated the mRNA expression
levels of Parp-1, Rad51 and Brca1. Importantly, NAC
pretreatment mitigated the effects of KAM-2 on gene
expression.
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6.
S. W. Chung, J. U. Choi, Y. S. Cho, H. R. Kim, T. H. Won, P.
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Dimitrion, O. C. Jeon, S. W. Kim, I. S. Kim, S. Y. Kim and Y. Byun,
DOI: 10.1039/C9SC00910H
Adv. Sci., 2018, 5, 1800368.
7.
8.
9.
S. Bakthavatsalam, M. L. Sleeper, A. Dharani, D. J. George, T.
Zhang and K. J. Franz, Angew. Chem. Int. Ed., 2018, 57, 12780.
X. Li, Y. Hou, X. Meng, C. Ge, H. Ma, J. Li and J. Fang, Angew.
Chem. Int. Ed., 2018, 57, 6141.
F. Kang, J. Zhu, J. Wu, T. Lv, H. Xiang, J. Tian, Y. Zhang and Z.
Huang, Chem. Sci., 2018, 9, 6893.
Fig. 9 The effects of 20 μM KAM-2 on the expression DDR-related mRNA levels tested by
real-time PCR. Mean ± SD, n = 3. **p < 0.01 vs. control or 0 h treatment; ##p < 0.01 vs. 20
μM KAM-2 treatment.
10. S. B. Salunke, J. A. Malla and P. Talukdar, Angew. Chem. Int. Ed.
Engl., 2019, 58, 5354.
11. P. Wei, L. Liu, Y. Wen, G. Zhao, F. Xue, W. Yuan, R. Li, Y. Zhong,
M. Zhang and T. Yi, Angew. Chem. Int. Ed., 2019, 58, 4547.
12. R. Radi, Proc. Natl. Acad. Sci. U. S. A., 2018, 115, 5839.
13. B. D'Autreaux and M. B. Toledano, Nat. Rev. Mol. Cell Biol.,
2007, 8, 813.
14. M. C. Chang, A. Pralle, E. Y. Isacoff and C. J. Chang, J. Am. Chem.
Soc., 2004, 126, 15392.
15. E. W. Miller, A. E. Albers, A. Pralle, E. Y. Isacoff and C. J. Chang,
J. Am. Chem. Soc., 2005, 127, 16652.
16. L. K. Charkoudian, D. M. Pham and K. J. Franz, J. Am. Chem. Soc.,
2006, 128, 12424.
17. J. L. M. Jourden and S. M. Cohen, Angew. Chem., Int. Ed., 2010,
49, 6795.
18. Y. Kuang, K. Baakrishnan, V. Gandhi and X. Peng, J. Am. Chem.
Soc., 2011, 133, 19278.
19. K. B. Daniel, J. L. M. Jourden, K. E. Negoescu and S. M. Cohen,
J. Biol. Inorg. Chem., 2011, 16, 313.
20. C. Perez, J.-P. Monserrat, Y. Chen and S. M. Cohen, Chem.
Commun., 2015, 51, 7116.
Conclusions
In summary, we for the first time present an α-ketoamide
scaffold to build up H2O2-activated prodrugs, using nitrogen
mustard as the model drug. Our studies identified KAM-2 as a
novel anticancer prodrug deserving further investigations.
KAM-2 obviously inhibited the growth of different cancer cell
lines with diminished cytotoxicity in normal PBMC cells. The
H2O2-induced decomposition of KAM-2 was confirmed using
various analytical methods. The nitrogen mustard released
from KAM-2 exhibited DNA ICL ability, led to DNA damage in HL-
60 cells, and increased the expression of DDR related proteins
and mRNAs. Additional mechanism studies suggested that
KAM-2 induced HL-60 apoptosis in a mitochondrial-dependent
manner. In addition to the emerged boron-based H2O2-
responsive prodrugs, our work showcases the establishment of
a new platform for prodrug design inspired from analytical
probes. Considering the close involvement of H2O2 in many
pathological diseases, it is anticipated that our results will
actively promote the development of both small molecule and
nanoparticle-based agents in related fields.
21. C. K. Wang, J. Cheng, X. G. Liang, C. Tan, Q. Jiang, Y. Z. Hu, Y. M.
Lu, K. Fukunaga, F. Han and X. Li, Theranostics, 2017, 7, 3803.
22. C. Tapeinos and A. Pandit, Adv. Mater., 2016, 28, 5553.
23. G. Saravanakumar, J. Kim and W. J. Kim, Adv. Sci., 2017, 4,
1600124.
24. C. Achilli, A. Ciana, M. Fagnoni, C. Balduini and G. Minetti, Cent.
Eur. J. Chem., 2013, 11, 137.
25. Y. Furikado, T. Nagahata, T. Okamoto, T. Sugaya, S. Iwatsuki, M.
Inamo, H. D. Takagi, A. Odani and K. Ishihara, Chem. Eur. J.,
2014, 20, 13194.
Conflicts of interest
There are no conflicts to declare.
26. C. Gao, Y. Tian, R. Zhang, J. Jing and X. Zhang, Anal. Chem., 2017,
89, 12945.
27. X. Xie, X. Yang, T. Wu, Y. Li, M. Li, Q. Tan, X. Wang and B. Tang,
Anal. Chem., 2016, 88, 8019.
28. P. Gao, W. Pan, N. Li and B. Tang, Chem Sci, 2019, DOI:
10.1039/C9SC01652J.
29. S. Cao, Y. Wang and X. Peng, Chem. Eur. J., 2012, 18, 3850.
30. W. Chen, K. Balakrishnan, Y. Kuang, Y. Han, M. Fu, V. Gandhi
and X. Peng, J. Med. Chem., 2014, 57, 4498.
Acknowledgements
This work is supported by National Natural Science Foundation
of China (21877052, 81602960), Natural Science Foundation of
Jiangsu Province (BK20180030, BK20161028), and the
Fundamental Research Funds for the Central Universities
(JUSRP51712B).
31. W. Chen, H. Fan, K. Balakrishnan, Y. Wang, H. Sun, Y. Fan, V.
Gandhi, L. A. Arnold and X. Peng, J. Med. Chem., 2018, 61, 9132.
32. A. Alouane, R. Labruere, T. Le Saux, F. Schmidt and L. Jullien,
Angew. Chem. Int. Ed., 2015, 54, 7492.
33. R. Kumar, J. Han, H. J. Lim, W. X. Ren, J. Y. Lim, J. H. Kim and J.
S. Kim, J. Am. Chem. Soc., 2014, 136, 17836.
34. H. W. Liu, X. X. Hu, K. Li, Y. Liu, Q. Rong, L. Zhu, L. Yuan, F. L. Qu,
X. B. Zhang and W. Tan, Chem Sci, 2017, 8, 7689.
35. H. Hagen, P. Marzenell, E. Jentzsch, F. Wenz, M. R. Veldwijk and
A. Mokhir, J. Med. Chem., 2012, 55, 924.
36. J. Noh, B. Kwon, E. Han, M. Park, W. Yang, W. Cho, W. Yoo, G.
Khang and D. Lee, Nat. Commun., 2015, 6, 6907.
Notes and references
1.
J. Rautio, N. A. Meanwell, L. Di and M. J. Hageman, Nat. Rev.
Drug Discov., 2018, 17, 559.
2.
S. H. Alamudi and Y. T. Chang, Chem. Commun., 2018, 54,
13641.
3.
4.
D. S. Folk and K. J. Franz, J. Am. Chem. Soc., 2010, 132, 4994.
N. Ueki, S. Lee, N. S. Sampson and M. J. Hayman, Nat. Commun.,
2013, 4, 2735.
5.
A. Tanaka, Y. Fukuoka, Y. Morimoto, T. Honjo, D. Koda, M. Goto
and T. Maruyama, J. Am. Chem. Soc., 2015, 137, 770.
6 | J. Name., 2012, 00, 1-3
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Page 7 of 8
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Please do not adjust margins
Journal Name
ARTICLE
37. C. Q. Luo, Y. X. Zhou, T. J. Zhou, L. Xing, P. F. Cui, M. Sun, L. Jin,
N. Lu and H. L. Jiang, J. Control. Release, 2018, 274, 56.
38. T. P. Szatrowski and C. F. Nathan, Cancer Res., 1991, 51, 794.
39. Z. Can, A. Uzer, K. Turkekul, E. Ercag and R. Apak, Anal. Chem.,
2015, 87, 9589.
View Article Online
DOI: 10.1039/C9SC00910H
40. T. B. Zanoni, F. Hudari, A. Munnia, M. Peluso, R. W. Godschalk,
M. V. Zanoni, G. J. den Hartog, A. Bast, S. B. Barros, S. S. Maria-
Engler, G. J. Hageman and D. P. de Oliveira, Toxicol. Lett., 2015,
239, 194.
41. R. K. Singh, S. Kumar, D. N. Prasad and T. R. Bhardwaj, Eur. J.
Med. Chem., 2018, 151, 401.
42. J. J. Tepe and R. M. Williams, J. Am. Chem. Soc., 1999, 121, 2951.
43. E. P. Rogakou, D. R. Pilch, A. H. Orr, V. S. Ivanova and W. M.
Bonner, J. Biol. Chem., 1998, 273, 5858.
44. S. Ganesan and A. F. Keating, Toxicol. Appl. Pharmacol., 2015,
282, 252.
This journal is © The Royal Society of Chemistry 20xx
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DOI: 10.1039/C9SC00910H
Table of contents entry
New light on H2O2-activated prodrugs: the first α-ketoamide based prodrug opens up new
alternatives for designing non-boron based H2O2-responsive promoieties.