Design of a hydrogen peroxide-activatable agent that specifically targets cancer cells

Article abstract of DOI:10.1016/j.bmc.2014.10.029

Some cancers, like acute myeloid leukemia (AML), use reactive oxygen species to endogenously activate cell proliferation and angiogenic signaling cascades. Thus many cancers display increases in reactive oxygen like hydrogen peroxide concentrations. To translate this finding into a therapeutic strategy we designed new hydrogen peroxide-activated agents with two key molecular pharmacophores. The first pharmacophore is a peroxide-acceptor and the second is a pendant amine. The acceptor is an N-(2,5-dihydroxyphenyl)acetamide susceptible to hydrogen peroxide oxidation. We hypothesized that selectivity between AML and normal cells could be achieved by tuning the pendant amine. Synthesis and testing of fourteen compounds that differed at the pendent amine led to the identification of an agent (14) with 2 μM activity against AML cancer cells and an eleven fold-lower activity in healthy CD34+ blood stem cells. Interestingly, analysis shows that upon oxidation the pendant amine cyclizes, ejecting water, with the acceptor to give a bicyclic compound capable of reacting with nucleophiles. Preliminary mechanistic investigations show that AML cells made from addition of two oncogenes (NrasG12D and MLL-AF9) increase the ROS-status, is initially an anti-oxidant as hydrogen peroxide is consumed to activate the pro-drug, and cells respond by upregulating electrophilic defense as visualized by Western blotting of KEAP1. Thus, using this chemical approach we have obtained a simple, potent, and selective ROS-activated anti-AML agent.

Full text of DOI:10.1016/j.bmc.2014.10.029

Bioorganic & Medicinal Chemistry 22 (2014) 6885–6892
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design of a hydrogen peroxide-activatable agent that specifically
targets cancer cells
Anish K. Vadukoot a, Safnas F. AbdulSalam a, Mark Wunderlich b, Eboni D. Pullen a,
Julio Landero-Figueroa a, James C. Mulloy b, Eddie J. Merino a,
⇑
a Department of Chemistry, University of Cincinnati, Cincinnati, OH 45221-0172, United States
b Division of Experimental Hematology and Cancer Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Some cancers, like acute myeloid leukemia (AML), use reactive oxygen species to endogenously activate
cell proliferation and angiogenic signaling cascades. Thus many cancers display increases in reactive
oxygen like hydrogen peroxide concentrations. To translate this finding into a therapeutic strategy we
designed new hydrogen peroxide-activated agents with two key molecular pharmacophores. The first
Received 25 August 2014
Revised 10 October 2014
Accepted 19 October 2014
Available online 30 October 2014
pharmacophore is
a peroxide-acceptor and the second is a pendant amine. The acceptor is an
N-(2,5-dihydroxyphenyl)acetamide susceptible to hydrogen peroxide oxidation. We hypothesized that
selectivity between AML and normal cells could be achieved by tuning the pendant amine. Synthesis
and testing of fourteen compounds that differed at the pendent amine led to the identification of an agent
Keywords:
Reactive oxygen species
Conjugate addition
Acute myeloid leukemia
Oxidative stress
(14) with 2 lM activity against AML cancer cells and an eleven fold-lower activity in healthy CD34+ blood
stem cells. Interestingly, analysis shows that upon oxidation the pendant amine cyclizes, ejecting water,
with the acceptor to give a bicyclic compound capable of reacting with nucleophiles. Preliminary
mechanistic investigations show that AML cells made from addition of two oncogenes (NrasG12D and
MLL-AF9) increase the ROS-status, is initially an anti-oxidant as hydrogen peroxide is consumed to
activate the pro-drug, and cells respond by upregulating electrophilic defense as visualized by Western
blotting of KEAP1. Thus, using this chemical approach we have obtained a simple, potent, and selective
ROS-activated anti-AML agent.
Anti-cancer agent
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
We hypothesized that simple anti-cancer agents that simply act
through a conjugate addition would be more difficult to design due
Cytotoxic molecules hold a privileged position as anti-cancer
agents. These agents include compounds that damage DNA to
modify cellular genetic information and natural products that
induce cytotoxicity in cancer cells.1,2 Several natural products are
reactive with cellular biomolecules at very low concentrations
and selectively affect cancer cells without harming normal cells.3
Two recently identified reactive natural products, parthenolide4,5
(PTL) and piperlongmine6,7 (PLG), extend life in mouse models of
aggressive cancers. Both these natural products modify protein
targets through a conjugate addition reaction as part of their
mechanism of action. It is thought that these natural products react
with proteins involved in anti-oxidant synthesis and thereby selec-
tively eliminate cells with high concentrations of reactive oxygen
species (ROS).4–7 For this project these two natural products are
a benchmark from which to compare new designs.
to in vivo metabolism and off-target effects thus we focused on a
pro-drug strategy.8–10 Certain cancer cells, for example acute mye-
loid leukemia (AML) cells, have elevated levels of ROS.11,12 At the
biochemical level, ROS molecules catalyze formation of disulfide
bonds or sulfinic acids to alter proteins and subsequent activity
responsible for growth and angiogenesis.13–17 For example, tumor
cells from approximately 60% of patients with AML show elevated
ROS; in some cases, a 100-fold elevation is observed.18 This
increased ROS is ripe for exploitation via a pro-drug strategy
(Fig. 1).19–21 In this manuscript we have designed novel pro-drugs
that are unreactive until oxidized by hydrogen peroxide. Once oxi-
dized, the molecules, like the two natural products, are capable of a
conjugate addition reaction.9,19
We altered the design of our previous superoxide-sensitive
agents such that they would react via conjugate addition upon
hydrogen peroxide oxidation. A recent manuscript shows that
induction of novel adducts changes anti-cancer activity and
targets cancers.22 The previous agents we designed possess
⇑
Corresponding author.
http://dx.doi.org/10.1016/j.bmc.2014.10.029
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

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A.K. Vadukoot et al. / Bioorg. Med. Chem. 22 (2014) 6885–6892
generated upon hydrogen peroxide activation to be C5. This
change thereby facilitates hydrogen peroxide-activated conjugate
addition since it utilizes quinone chemistry. In order to facilitate
synthesis and identify an agent with low micromolar activity we
chose an amide linker. As you will see this design led to a lead
compound with a surprising mechanism of activation but still is
a hydrogen peroxide-activatable agent that reacts via formation
of a conjugate addition product.
As a starting point for our analysis, we synthesized compounds
1–14 (Table 1). Compound 14 has an IC50 value of 2 lM against
Figure 1. Design of hydrogen peroxide-activatable anti-cancer agents based on
conjugate addition. AML is thought to be addicted to elevated levels of ROS, thus we
have designed new agents that are activated by hydrogen peroxide. In this
manuscript, blood stem cells from healthy donors are transformed into faithful
models of AML by addition of two genes: an MLL-AF9 fusion and active NrasG12D.
To implement the design of hydrogen peroxide-activatable agents a linker (red) on
an oxidation-prone hydroquinone was installed to enforce conjugate addition after
AML cancer cells. Upon hydrogen-peroxide induced oxidation, 14
formed a bicyclic ring that equilibrated between an electrophilic
oxidized and a reduced relatively stable state. Hydrogen peroxide
activated the compound by more than 150-fold. Incubation with
N-acetylcysteine leads to formation of a conjugate addition prod-
uct. Cellular testing showed that our AML model system possesses
more ROS than normal blood stem cells from healthy donors and
the addition of 14 consumes ROS as part of its mechanism since
it is essentially an anti-oxidant. Importantly, 14 is 11-fold selective
and activates AML cells electrophilic defense system consistent
with the hypothesized mechanism of action.
hydrogen peroxide oxidation. ‘‘X’’ represents a-substituent of the acetamide.
mid-nanomolar activity against some tumor cells and up to
10-fold selectivity for AML cells.8,9 Upon oxidation, the compound
reacts by 1,2-addition with an amine identified as essential. We
envisioned a hydrogen peroxide-activatable agent that reacts via
conjugate addition could be designed by migrating this amine
and its tether ortho to the hydroxyl group of the hydroquinone
(Fig. 1 red structure). This would cause the electrophilic center
2. Results
2.1. Design and reactivity of compound 1
To begin the molecular design process we synthesized com-
pound 1 (Fig. 1). After synthesizing 1 we examined its oxidation.
Two aspects of the design were of concern based on previously
published studies of similar compounds. First, oxidation could
Table 1
Activity of compounds against AML cell line MA9.3RAS
Structure
IC50
Structure
IC50
M)
(l
M)
(
l
form either the 1,4-quinone or an a-substituted-N-arylacetamide.
Second, because of the many tautomers, several positions could
serve as the electrophilic center. To identify the oxidation product
of 1, (diacetoxyiodo)benzene was used as
a model oxidizer
1
2
3
6
1
8
12
13
3
3
2
1
(Fig. 2B). The product after two-electron and concurrent two-
proton oxidation was isolated and found to be 2 based on 1H
NMR. 1H NMR showed a broad singlet at 9.75 ppm confirming
the presence of amide proton and the formation of 1,4 quinone
21
4
9
rather than
a-substituted-N-arylacetamide. The next step in the
reaction is the conjugate addition. As a test reaction the substrate
N-acetylcysteine was incubated in dimethylsulfoxide (DMSO)
along with 2 for 2 h. After isolation, NAC-2 was characterized by
1H NMR (Fig. 2B). The spectrum revealed that addition occurred
at C5. Singlets were observed at 7.83 ppm for H1 and 6.85 ppm
for H2. If the addition had happened at C6, doublets due to meta
coupling between H3 and H5 protons would have been present in
the spectrum. Figure 2A shows the proposed reaction pathway:
oxidation, conjugate addition, and regeneration of aromaticity.
To mimic cellular addition, 1 was incubated in PBS with limit-
ing N-acetylcysteine and the products were analyzed by HPLC
(Fig. 2C). The limiting N-acetylcysteine was necessary due to
the formation of multiple addition products since addition of a
thiol to the aromatic ring makes further oxidation and addition
easier. Compound 1 was converted to 2 in the absence of exoge-
nous oxidant at a rate of 0.011 0.002 minꢀ1 (Fig. 2D, grey line),
which leads to approximately 50% conversion in one hour. Please
note that background oxidation is dependent on molecular oxy-
gen as reactions under argon show no oxidation (Supporting
information, Fig. 5). We then assessed the ROS-induced activation
of 1. In the presence of 10 mM H2O2, a ten-fold excess, the rate of
> 100
10
4
5
6
> 100
>100
11
12
13
5
18
9
1
2
2
35
5
7
16
2
2
14
2
3
1
1
conversion was enhanced by more than 60-fold to 0.6 0.1 minꢀ1
.
Once 2 was formed N-acetylcysteine was added and NAC-2 was
rapidly produced (Fig. 2C, black line) as confirmed by FTMS
(Fig. 2E). Thus, 1 is activated by ROS, supporting our hypothesis
that this agent will react with biomolecules in cells under higher
oxidative stress.
PLG
1
PTL

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6887
studies were performed in triplicates using an MTT cell prolifera-
tion assay. All MTT assays were repeated twice.
We evaluated the ROS-activated portion of the molecule as
shown in Table 1. Compound 1 had an IC50 value (i.e., concentra-
tion at which only 50% of the AML cells survive) of 6
Oxidation within cells was necessary for activity since the IC50 of
2 was 21 M. In support of the requirement for cellular
1 lM.
4
l
oxidation, compound 3, which is not readily oxidized into the
corresponding quinone due to the presence of methoxy groups
had an IC50 greater than 100
a single phenol at positions 2 and 5, respectively, also had IC50
values greater than 100 M, showing the importance of both
lM. Compounds 4 and 5, each lacking
l
hydroxyl groups. Replacement of a hydroxyl group with hydrogen
makes oxidization much less favorable. Analysis of these com-
pounds indicated that oxidation was a prerequisite for high activ-
ity. Thus, these data support that to obtain a cytotoxic compound a
key requirement is the ability of the compound to be oxidized.
Molecular requirements of ‘X’ functionality (a-substituent of
the acetamide, Fig. 1) were next assessed in compounds 6–14
(Table 1). In compound 6, the N-methylaniline of 1 was replaced
with hydrogen; this substitution reduced the IC50 by approxi-
mately 6 fold to 35
of 16 M, whereas replacement of the N-methylaniline with a
hydroxyl group (compound 8) resulted in an IC50 of 12 M.
5 lM. The amine-substituted 7 had an IC50
2
l
3
l
We then synthesized a derivative lacking the methyl on the aniline,
9. Compared to compound 1, 9 also had a moderate reduction in
activity (IC50 of 13
a phenol and thiophenol, respectively had greater activity than 9
with IC50 values of 3 M and 5 M. We then replaced the
distal aromatic ring with alkyl groups. In 12, substitution with an
N-methylpropylamine reduced activity to 18 M. Compound
13, an N-methylcyclohexylamine derivative, showed slight
reduction in potency to 9 M compared to 1, but its activity
2 lM). Two isosteres, 10 and 11, containing
1
l
1 l
2
l
a
2
l
is improved over 12. The most active compound was 14, in which
the aromatic ring was replaced with a cyclohexane. Compound 14
had an IC50 of 2
1 lM against AML cells. We chose 14 for further
chemical and biochemical analysis since it met out criterion of hav-
ing similar IC50 as the two natural products that showed efficacy.
2.3. Compound 14 displays a propensity to cyclize upon
oxidation
We then examined chemical reactivity of 14. A potential reac-
tion scheme is shown in Figure 3A. We hypothesized that the
chemistry of 14 would differ significantly from 1. First, as a second-
ary aliphatic amine we envisioned a possible cyclization reaction
upon oxidation. The best possibilities for cyclization via amine
addition was to C1 or C3 of the phenol to generate a new six-
membered ring. Additionally nucleophilic attack could occur at
C2 or by a second molecule to yield a dimer. The two six membered
ring possibilities are shown in Figure 3B. Thus, we probed the reac-
tion. Representative HPLC chromatograms in the presence and
absence of hydrogen peroxide are shown in Figure 3C (left hand
side). Interestingly, by comparing the dark and light grey chro-
matograms it is observed that two products were formed (11.2
and 15.9 min elution times). These products were produced via
both spontaneous and hydrogen-peroxide induced oxidation but
not under an argon atmosphere (Fig. 3C right hand side). It was
found that only 4 3% of 14 was converted to its oxidation product
in two hours under argon atmosphere. Under normal atmosphere
the reaction proceeds under pseudo-zero order reaction kinetics
with a t1/2 of 155 13 min (Supporting info). Importantly, addition
of one molar equivalent hydrogen peroxide lead to a near instanta-
neous conversion to the oxidized products. Using the lower limit of
t1/2 less than 1 min leads to a large rate enhancement. Initially the
two products were isolated and a high-resolution MS was obtained
Figure 2. Reactivity of 1. (A) The proposed reaction pathway. Oxidation of 1 leads
to a quinone. In the presence of a nucleophile, like N-acetylcysteine, a conjugate
addition reaction followed by an elimination results in the aromatic NAC-2 species.
(B) 1H NMR of 2 (upper) and NAC-2 (lower). The two red proton shifts do not show
multiplicity indicating that N-acetylcysteine has added to C5. (C) HPLC analysis of
reaction products in the absence of NAC (grey) and upon addition of NAC (black).
The peak at 11.2 mins was identified as the NAC-2 adduct. (D) Hydrogen peroxide
increases the rate of conversion of 1 into 2 to 0.6 minꢀ1, an enhancement of at least
60-fold. (E) FTMS of NAC-2.
2.2. Structure–activity relationship
We then began to probe structural components within 1 and
optimize its potency. The goal was to obtain activity that matched
the two natural products, PTL and PLG that show efficacy in tumor
xenografts in mice.4,6 As a model system we evaluated cytotoxicity
of synthesized agents in the MA9.3RAS AML cell line. These cells
are transformed CD34+ human blood stem cells engineered to
express both an MLL-AF9 fusion protein and the common onco-
gene NrasG12D. Culturing was accomplished as previously
described.23 The molecular signatures associated with these cells
closely mimic those identified in primary AML patient samples
and the MLL-fusion is associated with poor survival.24 Cytotoxicity

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A.K. Vadukoot et al. / Bioorg. Med. Chem. 22 (2014) 6885–6892
Figure 3. Oxidation of 14 leads to cyclization. (A) Proposed reaction pathway.
Oxidation of 14 leads to cyclization or dimerization (not shown) to produce 14-ox.
This oxidation product can potentially reduce giving 14-red. (B) The 14-ox can
cyclize by attack at either C1 or C3 giving products of distinct molecular weight. (C)
HPLC analysis of the 14 (lower grey) and its oxidation (upper black) shows two
distinct product. 14 in the absence of oxygen and peroxide is not oxidized. Two-
hour exposure to the atmosphere leads to some conversion to 14-ox while
hydrogen peroxide converts all 14 in less than 0.1 h (D) Isolation and MS spectra of
14-ox and 14-red.
on both of them. The corresponding FTMS is shown in Figure 3D.
Figure 4. Oxidation of 14 leads to cyclization. (A) Proposed tautomeric structures of
14. (B) This oxidation product can potentially reduce giving 14-red. (Fig B first
NMR) The 14-ox can be predicted from the 1H NMR of the equilibrated species
(Fig. B second NMR). (C) HPLC traces of NAC- addition product with 14 and ITMS of
the NAC adduct (Fig 4C left hand side).
After isolation it was found that the 14-ox product that eluted at
11.2 min had an elemental composition of
C
14H17N2O+2, m/z
245.148 with an error less than 100 ppb, which inferred oxidation
and additionally lost H3O+. Please note there are several tautomers
of 14-ox possible (see Fig. 4A). Importantly, the second product
(14-red) that eluted at 15.9 min had an elemental composition of
C
14H19N2O+2, m/z 247.144 with an error less than 100 ppb, which
was a reduction of 14-ox.
Several critical questions remained including the tautomeric
state and if thiol could add to 14-ox. Figure 4A details six key
tautomers possible from 14-ox (numbered T1–T6). It was possible
to precipitate 14-red from aqueous solutions as neither 14-ox nor
14-red is stable to chromatography or heating. Thus the structure
of 14-red was identified. We then allowed 14-red to equilibrate
back to 14-ox to identify its structure.
Important proton resonances for 14-red are as follows. Two
singlets at d 10.24 (s, 1H) and d 8.87 (s, 1H) correspond to the
amido proton of the tether and hydroxyl group of the quinone. Aro-
matic protons showed up at d 6.59 (d, J = 9.0 Hz, 1H), d 6.35–6.30
(m, 2H). A singlet at d 3.42 (s, 2H) ppm corresponds to protons
on
a-carbon next to the carbonyl group. Cyclohexylamine pro-
Figure 5. Compound 14 displays highly selective anti-AML activity. (A) Ratio of
blood stem cell to AML activity of select synthesized agents and natural products
piperlongumine (PLG) and parthenolide (PTL). (B) Viability of AML (black) and
CD34+ normal blood stem cells (grey) in the presence of compound 14.
tons showed up at d 3.31–3.21 (m, 1H), d 1.77–1.59 (m, 5 H), d
1.40–1.33(m, 4 H), d 1.11–1.08 (m, 1H).The 1H NMR is consistent
with the tautomer shown in Figure 3A. Importantly, the structure

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6889
of 14-ox could be assigned by allowing pure 14-red to equilibrate
back into a ꢁ25% mixture of 14-ox. We found that it takes 1 hr for
this equilibration to occur. The spectrum was normalized to the
most downfield 14-red aromatic shift at d 6.59 ppm. By doing this
it becomes apparent that there is a set of shifts corresponding to
14-red at single proton counts and a smaller set corresponding to
14-ox. There should be a total integration, assuming 25% 14-ox
and 75% 14-red of 23.4 proton integration sum between the two
species and we observe 23.0, well within error. The proton shifts
for the 14-ox are given as follows. Amido protons and phenolic
hydroxyl are present as singlets at 10.3 and 8.48 ppm respectively
with an integration of 0.3. There are three aromatic protons
observed with one doublet that is shifted up-field to d 5.58 ppm
since it is adjacent to the cationic site. Since 14-red has a total
11 protons in cyclohexyl ring, then the proton integration sum
for both species should be 14.3. They are well accounted for in
the spectrum. The most important and defining proton shift is
Figure 6. Compound 14 displays ROS-dependent and selective anti-AML activity.
Blood stem cells from three healthy donors each show less ROS then the model AML
cell line (p < 0.01 in all cases) as measured by a DCF fluorescence assay. (B) Addition
of 20 lM 14 to AML cells leads to a time dependent loss in cellular ROS levels as
measured by a DCF fluorescence assay.
the a-CH2 group (at d 3.39 ppm). There are supposed to be a total
of 2.6 proton integration sum combined between 14-red and 14-ox
but integrated value is 1.9 (bold and underlined in Fig. 4B, bottom
NMR). This indicates that there is no a-CH2 group present in 14-ox.
mice.4,6 PLG is thought to be reactive via a conjugate addition
reaction leading to a depletion of cellular antioxidant capacity.
Thus, selective targeting of cancers with elevated ROS status
occurs. Similarly PTL is a sesquiterpene lactone that utilizes conju-
gate addition. Table 1 shows that 14 has similar anti-AML activity
Interestingly, we observe a new shift at d 4.98 ppm (bold and
underlined in Fig. 4B, bottom NMR) similar to an allylic proton shift
with an intensity of 0.3. Based on this information we can elimi-
nate 14-ox-T3, 14-ox-T4, 14-ox-T5, and 14-ox-T6 (Figure 4A) as
likely structures since they lack this characteristic proton shifts.
This leaves the only candidate tautomer as 14-ox-T2. Thus, 14-ox
is likely not in a quinone state but in-fact an aromatic bicyclic ring
(14-ox-T2). We then needed to validate that such a compound
would be reactive toward nucleophile addition. As before we
examined addition using N-acetylcysteine via HPLC (Fig. 4C). Lim-
iting N-acetylcysteine was necessary due to the formation of multi-
ple addition products as observed in HPLC and MS since the first
addition leads to easier oxidation and capture of the next (thiol)
nucleophile. When N-acetylcysteine is present a new product is
observed at 12 min (14-S in Fig. 4C). Upon isolation and MS of
14-S a single m/z value of 406.0 by ITMS is observed. Thus, 14 is
oxidatively activated and reacts with nucleophiles.
as both these natural products. PLG has an IC50 of 2
1 lM, while
PTL has an IC50 of 3 M. Strikingly, 14 was more selective than
1
l
either PLG or PTL in the MA9.3RAS AML cells as their respective
selectivity were 3.7 1 and 2.1 0.4. When AML and control cells
were treated with compound 14, AML cells were eradicated at a
concentration that did not affect viability of normal blood stem
cells (Fig. 5B).
2.5. Model AML cancer cells display high ROS compared to
normal cells
Though it is widely accepted that bulk AML cells have elevated
concentrations of reactive oxygen, we wanted to be sure that our
models had higher levels of ROS than healthy cells. CD34+ blood
stem cells were harvested from three donors. Cells were counted,
incubated with 2,7-dihydrodichlorofluorescein (DCF) diacetate,
and their respective fluorescence compared after 30 min (Fig. 6).
After deacetylation DCF is oxidized by a broad spectrum of ROS
species and oxidases. Thus is a marker of general ROS and oxida-
tion within cells. The three CD34+ selected UCB cell populations
each derived from different healthy donors referred as #1 through
#3 (Fig. 6) had fluorescence intensity of 0.69 0.03 (p < 0.002),
0.60 0.03 (p < 10ꢀ6), 0.54 0.02 (p < 10ꢀ6) compared to 1.00
0.02 in the AML cell model.
2.4. Compound 14 has the highest selectivity among the analogs
tested
The selectivity of select compounds was examined by treatment
of primary CD34+ normal human blood stem cells. These are
untransformed and are genetically similar to MA9.3RAS cells
except they lack the MLL-AF9 oncogene and active NrasG12D
oncogene. It is essential that reactive agents be selective since
off-target reactivity is of great concern. In anti-cancer compounds,
a selectivity factor, which in our case is the ratio between the IC50
of normal cells over the IC50 of the AML cells, of one log unit is
desired. The selectivity factors for selected compounds are given
in Figure 5A. Please note that MA9.3RAS and primary CD34+
normal human blood stem cells do not show different rates of cell
proliferation (Supporting information, Fig. 6). Compound 1 is mar-
ginally selective with a ratio of 2.0 0.2. In addition compound 2
showed similar selectivity. Active compounds, which are isosteres
to 1, 10 and 11 also had limited selectivity. Neither compound 6
nor compound 7 were selective indicating that both linker and
nitrogen affect selectivity. Replacement of the aniline of 1 signifi-
cantly enhanced selectivity. Conversion of the aniline to an alkyl
amine led to improved selectivity. Compound 13 had a selectivity
factor of 3.8 1.4. Importantly, 14 was highly selective with a ratio
of IC50 values of 11.5 0.5.
2.6. Compound 14 reacts with cellular ROS leading to
electrophilic stress
With a ROS-activatable molecule with a unique mode of reac-
tivity, 14, we ensured that similar events occurred within cancer
cells. If 14 is ROS-activated in a cellular context it will enter a cell
and react with hydrogen peroxide. Upon reaction the concentra-
tion of hydrogen peroxide will decrease. Thus examination by
DCF fluorescence would show a similar effect as an anti-oxidant:
lowering fluorescent signal over time (Fig. 7A). It should be noted
that because 14 is highly lethal, concentrations ten-fold higher
than the IC50 are required to lower a cell’s ROS level. When 14
was added to AML cells the fluorescence intensity was set to
1.06 0.13 and it smoothly increased to 4.31 0.08 over time.
Similar experiments lacking addition of 14 show statistically sig-
nificant increases in fluorescence intensities (p < 0.001 for all time
points while pretreated data set was not statistically significant
We next compared 14, the best compound, to the selectivity of
two natural products that show efficacy in tumor xenografts in

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A.K. Vadukoot et al. / Bioorg. Med. Chem. 22 (2014) 6885–6892
4.1.2. N-(3,6-Dioxocyclohexa-1,4-dienyl)-2-
(methyl(phenyl)amino)acetamide (2)
1H NMR (DMSO-d6,400 MHz) d 9.75 (s, 1H), 7.34 (d, J = 2.4 Hz,
1H), 7.17 (t, J = 8.0 Hz, 2H), 6.92 (d, J = 10.0 Hz, 1H), 6.79 (dd,
J = 2.8, 10.4 Hz, 1H), 6.68–6.64 (m, 3H), 4.33 (s, 2H), 3.01 (s, 3H).
13C NMR (DMSO-d6, 400 MHz) d 187.85 (C), 182.33 (C), 170.84
(C), 149.00 (C), 138.04 (CH), 137.79 (C), 133.25 (CH), 129.53 (CH),
119.72 (CH), 115.28 (CH), 113.90 (CH), 59.88 (CH2), 40.41 (CH3).
HRMS (ESI) for [MH]+ calculated: 271.10772, observed: 271.10771.
4.1.3. N-(2,5-Dimethoxyphenyl)-2-(methyl(phenyl)amino)
acetamide (3)
1H NMR (CDCl3, 400 MHz) d 8.96 (br s, 1H), 8.13 (dd, J = 3.2 Hz,
1H), 7.32 (d, J = 7.6 Hz, 2H), 6.89–6.84 (m, 3H), 6.76 (d, J = 9.2 Hz,
1H), 6.59 (dd, J = 3.2,8.8 Hz, 1H),4.02 (s, 2H), 3.82 (s, 3H), 3.63 (s,
3H), 3.12 (s, 3H). 13C NMR (CDCl3, 400 MHz) d 169.85 (C), 153.77
(C), 149.19 (C), 143.06 (C), 128.90 (CH), 127.30 (C), 118.16 (CH),
112.93 (CH), 111.31 (CH), 108.35 (CH), 106.55 (CH), 58.20 (CH2),
55.64 (CH3), 54.67 (CH3), 38.82 (CH3). HRMS (ESI) for [MH] + calcu-
lated: 301.15467, observed: 301.15468.
Figure 7. Compound 14 lowers ROS and leads to electrophilic stress. (A) Addition of
20 lM 14 to AML cells leads to a time dependent loss in cellular ROS levels as
measured by a DCF fluorescence assay. (B) Electrophilic molecules, like 14, are
detected by a loss in concentration of the sensor protein KEAP1 (p < 0.05 in all
cases).
4.1.4. N-(2-Hydroxyphenyl)-2-(methyl(phenyl)amino)
acetamide (4)
1H NMR (CDCl3, 400 MHz) d 9.08 (s, 1H), 8.70 (s, 1H), 7.36–7.32
(m, 2H), 7.17–7.13 (s, 1H), 7.05 (d, J = 8.4 Hz, 1H), 6.95 (t, J = 7.6 Hz,
1H), 6.87–6.81 (m, 4H), 4.04 (s, 2H), 3.12 (s, 3H).
from each other) with the endpoint having an intensity of
5.02 0.08. Thus, 14 enters AML cells and lowers total ROS as it
is activated. Finally, we wanted to assess if 14 was a reactive
molecule within a cell (Fig. 7B). To accomplish this western blot
against KEAP1, normalized to beta-actin was performed. KEAP1 is
a signaling molecule that inhibits NRF2,25 the transcription factor
that regulates anti-oxidant concentrations in cells. Loss of KEAP1
is dependent on electrophilic and reactive stressors since it
possesses several easy to modify thiols. Addition of 14 lead to a rel-
ative concentration of 0.65 0.09 (p < 0.04) after 24 h.
13C NMR (CDCl3, 400 MHz) d 170.65 (C), 149.15 (C), 148.82 (C),
129.62 (CH), 127.44 (CH), 124.71 (C), 122.24 (CH), 122.15 (CH),
120.39 (CH), 119.87 (CH), 113.89 (CH), 59.44 (CH2), 40.18 (CH3).
HRMS (ESI) for [MH]+ calculated: 257.12845, observed: 257.12845.
4.1.5. N-(3-Hydroxyphenyl)-2-(methyl(phenyl)amino)
acetamide (5)
1H NMR (CD3OD, 400 MHz) d 7.24–7.19 (m, 2H), 7.15–7.07 (m,
2H), 6.92 (d, J = 8.0 Hz, 1H), 6.78–6.71 (m, 3H), 6.54 (m, 1H), 4.05 (s,
2H), 3.08 (s, 3H). 13C NMR (CD3OD, 400 MHz) d 170.36 (C), 157.51
(C), 149.52 (C), 138.86 (C), 129.10 (CH), 128.71 (CH), 117.37 (CH),
112.52 (CH), 111.24 (CH), 111.08 (CH), 107.30 (CH), 57.24 (CH2),
38.85 (CH3). HRMS (ESI) for [MH]+ calculated: 257.12845,
observed: 257.12845.
3. Conclusion
In conclusion, we have synthesized a series of ROS-activatable
compounds. The best compounds are cytotoxic to AML cancer cells
but much less so to normal cells. The ROS-activatable portion is a
simple hydroquinone. When oxidized, this module reacts with
the pendant amine to form an unusual bicyclic ring. Interestingly
14 displays unique selectivity compared to its peers. We propose
that this complex chemistry is integral to selectivity. Current
efforts in our laboratory are focusing on optimization of this design
and making this design suitable for in vivo administration via tun-
ing of pharmacokinetic properties.
4.1.6. N-(2,5-Dihydroxyphenyl)acetamide (6)
1H NMR (CD3OD, 400 MHz) d 7.20 (t, J = 1.6 Hz, 1H), 6.71 (dd,
J = 1.6 Hz, 8.8 Hz, 1H), 6.49–6.46 (m, 1H), 2.16 (s, 3H). 13C NMR
(CD3OD, 400 MHz) d 170.70 (C), 149.86 (C), 140.85 (C), 126.21
(C), 116.36 (CH), 111.68 (CH), 109.10 (CH), 22.14 (CH3). HRMS
+
(ESI) for [MH] calculated: 168.06552, observed: 168.06551.
4. Material and methods for the synthesis
4.1. Synthesis
4.1.7. 2-Aminocyclohexa-2,5-diene-1,4-dione (7)
1H NMR (DMSO-d6, 400 MHz) d 9.22 (s, 1H), 8.96 (s, 1H), 6.67 (d,
J = 8.4 Hz, 1H), 6.40 (dd, J = 2.8, 8.4 Hz, 1H), 6.33 (d, J = 2.8 Hz, 1H).
13C NMR (CD3OD, 400 MHz) d 150.67 (C), 142.39 (C), 126.73 (C),
116.74 (CH), 112.04 (CH), 106.38 (CH). HRMS (ESI) for [MH]+ calcu-
lated: 124.03930, observed: 124.03929.
All compounds were made by divergent routes. Final com-
pounds were prepared with more than 98% purity and character-
ized by 1H NMR, 13C NMR and HRMS. Structural characterizations
for the final compounds are given below.
4.1.8. N-(2,5-Dihydroxyphenyl)-2-hydroxyacetamide (8)
1H NMR (CD3OD, 400 MHz) d 7.72 (d, J = 2.4, 1H), 6.71 (dd,
J = 1.2, 8.8 Hz, 1H), 6.46–6.43 (m, 1H), 4.13 (s, 2H).13C NMR (MeOD,
400 MHz) d 171.49 (C), 149.76 (C), 139.66 (C), 125.91 (C), 115.06
(CH), 110.57 (CH), 107.37 (CH), 61.66 (CH2). HRMS (ESI) for [MH]
calculated: 184.06043, observed: 184.06043.
4.1.1. N-(2,5-Dihydroxyphenyl)-2-(methyl(phenyl)amino)
acetamide (1)
1H NMR (CD3OD, 400 MHz) d 7.64 (d, J = 2.91 Hz, 1H), 7.25 (dd,
J = 7.12, 8.69 Hz, 2H), 6.81 (d, J = 8.30 Hz, 2H), 6.65 (d, J = 8.56, 1H),
6.41 (dd, J = 2.86, 8.61, 1H), 4.03 (s, 2H), 3.12 (s, 3H). 13C NMR (CD3-
OD, 400 MHz) d 169.13 (C), 149.82 (C), 148.59 (C), 139.37 (C),
129.03 (CH), 128.98 (CH), 128.91 (CH), 125.96 (C), 119.41 (CH),
115.13 (CH), 113.83 (CH), 110.74 (CH), 107.70 (CH), 58.59 (CH2),
39.54 (CH3).HRMS (ESI) for [MH]+ calculated: 273.12337, observed:
273.12338.
+
4.1.9. N-(2,5-Dihydroxyphenyl)-2-(phenylamino)acetamide (9)
1H NMR (CD3OD, 400 MHz) d 7.68 (s, 1H), 7.15 (t, J = 7.6 Hz, 2H),
6.73–6.60 (m, 4 H), 6.38 (d, J = 8.8 Hz, 1H), 3.85 (s, 2H). 13C NMR
(CD3OD, 400 MHz) d 170.79 (C), 149.81 (C), 147.92 (C), 139.66

A.K. Vadukoot et al. / Bioorg. Med. Chem. 22 (2014) 6885–6892
6891
(C), 128.82 (CH), 126.14 (C), 118.12 (CH), 115.09 (CH), 112.78 (CH),
110.49 (CH), 107.40 (CH), 48.86 (CH2). HRMS (ESI) for [MH]+ calcu-
lated: 259.10772, observed: 259.10773.
for 48 h with indicated concentrations of freshly dissolved com-
pounds. The plates were centrifuged at 1500 ꢂ g for 5 min at 4 °C
to pellet the cells and the medium containing compounds was dis-
carded; fresh medium containing 20
added to each well and incubated for an additional 4 h. The med-
ium was removed. After adding 200 L of DMSO to each well, the
optical densities at 575 nm were determined. Cytotoxicity data
are expressed as IC50 values obtained from the fit to a four param-
eter sigmoid using the graphing software KaleidaGraph. All R2 val-
ues were greater than 0.98 and standard errors of the three
replicates were less than 20%.
lL of MTT (5 mg/mL) was
4.1.10. N-(2,5-Dihydroxyphenyl)-2-phenoxyacetamide (10)
1H NMR (CD3OD, 400 MHz) d 7.69 (d, J = 2.98 Hz, 1H), 7.37–7.33
(m, 2H), 7.07–7.02 (m, 3H), 6.71 (d, J = 8.8 Hz, 1H), 6.44 (dd, J = 2.8,
8.4 Hz, 1H), 4.68 (s, 2H). 13C NMR (CD3OD, 400 MHz) d 167.24 (C),
157.49 (C), 149.87 (C), 139.76 (C), 129.37 (CH), 125.76 (C), 121.77
(CH), 115.06 (CH), 114.59 (CH), 110.79 (CH), 107.68 (CH), 67.24
(CH2). HRMS (ESI) for [MH]+ calculated: 260.09173, observed:
260.09175.
l
4.2.2. DCF diacetate fluorescence assay
4.1.11. N-(2,5-Dihydroxyphenyl)-2-(phenylthio)acetamide (11)
1H NMR (CD3OD, 400 MHz) d 7.49–7.46 (m, 3H), 7.33 (t,
J = 7.12 Hz, 2H), 7.27–7.23 (m, 1H), 6.67 (d, J = 8.8 Hz, 1H), 6.42
(dd, J = 3.2, 8.2 Hz, 1H), 3.84 (s, 2H). 13C NMR (CD3OD, 400 MHz)
d 167.84 (C), 149.78 (C), 140.12 (C), 134.62 (C), 129.66 (CH),
128.88 (CH), 126.82 (CH), 126.09 (C), 115.31 (CH), 110.96 (CH),
107.84 (CH), 38.52 (CH2). HRMS (ESI) for [MH]+ calculated:
276.06889, observed: 276.06892.
Cells in active growth phase were pelleted and dissolved in
1X HBSS to a final concentration of 4 ꢂ 105 cells/ml. 100
ll of
cells were added to each well in a 96 well plate. 100 ll 1X HBSS
was used as blank. DCF-DA (20,70-dichlorofluorescein diacetate,
DCFH-DA) in 1X HBSS was added to each well to a final concen-
tration of 10 lM and the fluorescence increase was measured
(kexc = 485 nm; kem = 535 nm) with a microplate reader immedi-
ately and after 30 min incubation in 37 °C. While measuring
the H2O2 level changes in the presence of 14, measurements
were taken after adding 14 dissolved in HBSS to a final concen-
4.1.12. N-(2,5-Dihydroxyphenyl)-2-(methyl(propyl)amino)
acetamide (12)
tration of 20 lM. Cells were incubated at 37 °C and measure-
1H NMR (CD3OD, 400 MHz) d 7.72 (dd, J = 1.2, 2.4 Hz, 1H), 6.68
(dd, J = 1.6, 8.8 Hz, 1H), 6.42–6.39 (m, 1H), 3.14 (s, 2H), 2.48 (t,
J = 7.2 Hz, 2H), 2.38 (s, 3H), 1.63–1.54 (m, 2H), 0.99 (t, J = 7.2 Hz,
3H).13C NMR (CD3OD, 400 MHz) d 170.30 (C), 149.76 (C), 139.57
(C), 126.21 (C), 114.81 (CH), 110.16 (CH), 107.13 (CH), 61.74
(CH2), 59.71 (CH2), 41.93 (CH3), 20.47 (CH2), 10.53 (CH3).HRMS
(ESI) for [MH]+ calculated: 239.13902, observed: 239.13900.
ments were taken in 15 min intervals for one hour. Care has
been taken to control extended light exposure since DCF can
undergo light induced oxidation. Data are reported as the
mean standard deviation of six replicates. Unpaired t-test was
performed to determine the significance. The flow rate was
1 mL/min. The injection volume for each sample was kept con-
stant at 20 ll.
4.1.13. 2-(Cyclohexyl(methyl)amino)-N-(2,5-dihydroxyphenyl)
acetamide (13)
4.2.3. Western blot analysis
MA9.3RAS cells were grown to obtain a density of 1 ꢂ 106 cells/
1H NMR (CD3OD, 400 MHz) d 7.71 (d, J = 2.8 Hz, 1H), 6.67 (d,
J = 8.4 Hz, 1H), 6.38 (dd, J = 2.8 Hz, 8.4, 1H), 3.18 (s, 2H), 2.49–
2.44 (m, 1H), 2.39 (s, 3H), 1.89–1.80 (m, 4 H), 1.64 (d, J = 12.4 Hz,
1H), 1.35–1.11 (m, 5 H). 13C NMR (CD3OD, 400 MHz) d 170.98
(C), 149.78 (C), 139.58 (C), 126.24 (C), 114.80 (CH), 110.08 (CH),
107.04 (CH), 62.96 (CH), 57.69 (CH2), 38.02 (CH3), 28.68 (CH2),
25.82 (CH2), 25.47 (CH2). HRMS (ESI) for [MH]+ calculated:
279.17032, observed: 279.17031.
ml, splitted into two and one was treated with 0.7 lM 14 over-
night. Total protein was extracted from both treated and untreated
cell populations using M-PER mammalian protein extraction
reagent (Thermo scientific) according to the manufacturer’s proto-
col. The proteins were separated by 10% SDS polyacrylamide gel
(precast gel purchased from Genscript) electrophoresis with a
quantity of 20 lg loaded per well and transferred onto nitrocellu-
lose membrane using Invitrogen iBlot instrument according the
manufacturer’s instructions. The nitrocellulose was treated accord-
ing to the Western blot analysis procedure developed by LI-COR
imagers. The membrane was first blocked with Odyssey blocking
buffer (purchased from LI-COR) for 1 h and incubated with primary
antibody mixture including rabbit anti KEAP1 mAb and mouse anti
b-actin mAb for one hour followed by washing with PBS+0.1%
Tween 20. The membrane was then incubated with secondary
antibody mixture containing fluorescently labelled Goat anti-
mouse and goat anti-rabbit antibodies and imaged by LI-COR odys-
sey infrared imager. Unpaired t-test was performed to determine
the significance.
4.1.14. 2-(Cyclohexylamino)-N-(2,5-dihydroxyphenyl)
acetamide (14)
1H NMR (CD3OD, 400 MHz) d 7.59 (s, 1H), 6.70–6.68 (m, 1H),
6.44–6.41 (m, 1H), 3.65 (s, 2H), 2.71 (t, J = 10.14 Hz, 1H),
2.03–1.99 (m, 2H), 1.83–1.80 (m, 2H), 1.68 (d, J = 12.05 Hz, 1H),
1.36–1.24 (m, 5 H). 13C NMR (CD3OD, 400 MHz) d 163.57 (C),
149.71 (C), 140.68 (C), 125.36 (C), 115.54 (CH), 111.65 (CH),
109.16 (CH), 57.21 (CH), 45.54 (CH2), 28.83 (CH2), 24.61 (CH2),
+
24.11 (CH2). HRMS (ESI) for [MH]
observed: 265.15469.
calculated: 265.15467,
4.2. Cellular assays
4.3. Analytical studies
CD34+ selected Human umbilical cord blood (UCB) and
MA9.3RAS AML cells were obtained from Mulloy lab at CCHMC.
MA9.3RAS AML cells were cultured in IMDM 20% bovine calf serum
and Human umbilical cord blood (UCB) cells were cultured in
IMDM 20% bovine calf serum and supplemented with SCF, IL-3,
IL-6, Flt-3L and TPO.
4.3.1. HPLC Conditions
A Beckman Coulter’s HPLC system consisting of a dual pump
Model 126 with 32 Karat Software, an System Gold 168 detector
and an System Gold 508 AutoSampler was used for kinetic studies.
A reverse phase C-18 column (Synergi™ 4 lm Hydro-RP 80 Å, LC
Column 150 ꢂ 4.6 mm, Ea) was used. The mobile phase consisted
of HPLC grade Acetonitrile (Fisher Scientific) and distilled water fil-
tered through a Millipore Milli-Q water purification system. A
detection wavelength of 250 nm was used.
4.2.1. Cell cytotoxicity assay (MTT)
Cells were seeded at a density of 4 ꢂ 104 cells/well in a 96-well
plate and incubated at 37 °C for overnight. Then cells were treated

6892
A.K. Vadukoot et al. / Bioorg. Med. Chem. 22 (2014) 6885–6892
4.3.2. NAC-2 addition conditions
Supplementary data
To
a
solution of compound
2
(0.1 g, 0.369 mmol) in
dimethylsulfoxide (4.0 mL) was added N-acetylcysteine (0.072 g,
0.443 mmol) portion-wise. After the addition was complete the
reaction was stirred at rt for 2 h. The completion of the reaction
was monitored by TLC. The reaction mixture was concentrated,
purified by column chromatography (10% MeOH:CH2Cl2) to isolate
the required compound as an white solid (0.08 g, 50.0%). 1H NMR
(CD3OD, 400 MHz) d 7.83 (s, 1H), 7.26–7.23 (m, 2H), 6.85 (s, 1H),
6.83–6.78 (m, 3H), 4.49–4.44 (m, 1H), 4.04 (s, 2H), 3.14–3.01 (m,
5 H), 1.95 (s, 3H). HRMS (ESI) for [MH]+ calculated: 434.13800,
observed: 434.13803.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2014.10.029.
References and notes
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Compound 1 (2 mM) was freshly dissolved in 1 mL of 1XPBS.
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4.3.4. Isolation of 14-red
10 mM concentration of compound 14 in 1XPBS was allowed to
sit for 2 h, and the precipitate formed was isolated by filtration,
dried under vacuum and characterized by 1H NMR, 13C NMR and
HRMS. 1H NMR (DMSO-d6, 400 MHz) d 10.24 (s, 1H), 8.87 (s, 1H),
6.59 (d, J = 9.0 Hz, 1H), 6.35–6.30 (m, 2H), 3.42 (s, 2H), 3.31–3.21
(m, 1H), 1.77–1.59 (m, 5H), 1.40–1.33 (m, 4H), 1.11–1.08 (m,
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130.07 (C), 128.08 (C), 115.14 (CH), 109.72 (CH), 108.72 (CH),
56.90 (CH), 55.49 (CH2), 47.35 (CH2), 28.67 (CH2), 26.02 (CH2),
25.975 (CH2). HRMS (ESI) for [MH]
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This work is supported by an NIH NCI award (R21CA185370) to
E.J.M. We would also like to thank the UC Technology Accelerator
for funding as well as an Institutional Clinical and Translational
Science Award, NIH/NCR R Grant Number 1UL1RR026314-01. This
work was supported by a Translational Trials Development and
Support Laboratory award (USPHS, MO1 RR 08084) and a Center
of Excellence in Molecular Hematology P30 award (DK090971) to
J.C.M. J.C.M. is a Leukemia and Lymphoma Society Scholar.