Reactive oxygen species (ROS) inducible DNA cross-linking agents and their effect on cancer cells and normal lymphocytes

Article abstract of DOI:10.1021/jm401349g

Reducing host toxicity is one of the main challenges of cancer chemotherapy. Many tumor cells contain high levels of ROS that make them distinctively different from normal cells. We report a series of ROS-activated aromatic nitrogen mustards that selectively kill chronic lymphocytic leukemia (CLL) over normal lymphocytes. These agents showed powerful DNA cross-linking abilities when coupled with H₂O₂, one of the most common ROS in cancer cells, whereas little DNA cross-linking was detected without H₂O₂. Consistent with chemistry observation, in vitro cytotoxicity assay demonstrated that these agents induced 40-80% apoptosis in primary leukemic lymphocytes isolated from CLL patients but less than 25% cell death to normal lymphocytes from healthy donors. The IC₅₀ for the most potent compound (2) was ～5 μM in CLL cells, while the IC ₅₀ was not achieved in normal lymphocytes. Collectively, these data provide utility and selectivity of these agents that will inspire further and effective applications.

Full text of DOI:10.1021/jm401349g

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Article
Reactive oxygen species (ROS)-inducible DNA cross-linking
agents and their effect on cancer cells and normal lymphocytes
WENBING CHEN, Kumudha Balakrishnanb, Yunyan Kuang,
Yanyan Han, Min Fu, Varsha Gandhi, and Xiaohua Peng
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm401349g • Publication Date (Web): 06 May 2014
Downloaded from http://pubs.acs.org on May 13, 2014
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Reactive oxygen species (ROS)-inducible DNA
cross-linking agents and their effect on cancer
cells and normal lymphocytes
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Wenbing Chena, Kumudha Balakrishnanb, Yunyan Kuanga, Yanyan Hana, Min Fub, Varsha
Gandhib, Xiaohua Peng*a
aDepartment of Chemistry and Biochemistry, University of Wisconsin Milwaukee, 3210 N.
Cramer St., Milwaukee. WI 53211, USA.
bDepartment of Experimental Therapeutics, MD Anderson Cancer Center, Houston, TX 77030,
USA.
Keywords: ROSꢀactivated prodrugs; DNA crossꢀlinking; reactive oxygen species; arylboronates;
targeted anticancer drugs
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ABSTRACT
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Reducing host toxicity is one of the main challenges of cancer chemotherapy. Many tumor cells
contain high levels of ROS which makes them distinctively different from normal cells. We
report a series of ROSꢀactivated aromatic nitrogen mustards which selectively kill chronic
lymphocytic leukemia (CLL) over normal lymphocytes. These agents showed powerful DNA
crossꢀlinking abilities when coupled with H2O2, one of the most common ROS in cancer cells,
whereas little DNA crossꢀlinking was detected without H2O2. Consistent with chemistry
observation, in vitro cytotoxicity assay demonstrated that these agents induced 40ꢀ80%
apoptosis in primary leukemic lymphocytes isolated from CLL patients while less than 25% cell
death to normal lymphocytes from healthy donors. The IC50 for the most potent compound (2)
was ~5 ꢁM in CLL cells, while the IC50 was not achieved in normal lymphocytes. Collectively,
these data provide utility and selectivity of these agents which will inspire further and effective
applications.
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INTRODUCTION
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Making use of the unique property of cancer cells is one of the most important avenues to design
targeted anticancer drugs. Many types of cancer cells are under oxidative stress because of their
disturbed intracellular redox balance, which make them distinct from their ‘healthy’
counterparts.1ꢀ5 The increased amounts of reactive oxygen species (ROS) can be a therapeutic
advantage because it is an intrinsic feature of cancer cells.6ꢀ9 Recently, several anticancer agents
based on the ROSꢀmediated mechanisms have been developed to target these specific tumor cells
and have shown selective killing of cancer cells.10ꢀ14 For example, Huang and coworkers
reported that βꢀphenethyl isothiocyanate10 and 2ꢀmethoxyoestradiol11 selectively killed human
leukemia cells but not normal lymphocytes by causing further ROS stress in cancer cells.
Piperlongumine was also found to selectively kill cancer cells by increasing ROS levels but had
little effect on primary normal cells.13,14 Most of the existing ROSꢀtargeting drugs focus on
enhancing ROS production to inflict lethal damage. To the best of our knowledge, the drug
design for targeting tumor cells containing high levels of ROS via inducing DNA interstrand
crossꢀlinks (ICLs) is rarely reported.
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DNA ICLs are recognized as the primary mechanism for the cytotoxic activity of many
clinically useful antitumor drugs, such as chlorambucil, cyclophosphamide, bendamustine, and
cisplatin. However, the severe host toxicity exhibited by these anticancer drugs continues to be a
major problem in cancer chemotherapy. Prodrugs that are activated specifically in tumor cells
have the potential to reduce the toxicity of the crossꢀlinking agents for normal cells. Gates and
coworkers demonstrated that several anticancer drugs displayed selective toxicity by releasing
DNA damaging species selectively in tumor cells.15ꢀ17 Over the past few decades, several
research groups have developed novel DNA crossꢀlinking or alkylating agents that can induce
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ICL formation either by oxidation, reduction, or photolysis.18ꢀ25 Recently, our group has shown
that H2O2ꢀinduced DNA crossꢀlinking behaviors provided a novel strategy for tumorꢀspecific
damage.26,27 H2O2 is one of the most common ROS, which is believed to be produced in large
amounts in several human tumor cells.1ꢀ5 The transformed cells showed more than 10ꢀfold
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●ꢀ
increase in H2O2 levels.28a Different from O2 or hydroxyl radicals that are extremely unstable,
H2O2 has the chemical stability required to establish significant steadyꢀstate concentrations in
vivo and is uncharged. These properties allow H2O2 to freely diffuse across plasma membranes
and to travel to the cells. In addition, other ROS such as O2 can also be reduced to H2O2 in the
●ꢀ
oxygen metabolism via O2 generation involved in hypoxiaꢀinducible factor 1 (HIFꢀ1)
regulation.28bꢀc Thus, developing H2O2ꢀactivated prodrugs to selectively kill ROSꢀcontaining
cancer cells can be a potent strategy in cancer chemotherapies.
Scheme 1. Selective DNA crossꢀlinking agent with a ROSꢀresponsive ‘trigger’ and an ‘effector’.
DNA ICL was not formed in the absence of ROS as the ‘effector’ was deactivated in the
prodrug, while in a ROSꢀcontaining environment, the reaction of the trigger unit with ROS
activated the “triggerꢀeffector” system resulting in a potent DNA crossꢀlinking agent (‘trigger’
changes from a grey triangle to a red sector leading to a complete red circle).
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Such agents should consist of two separate functional domains: an efficient H2O2ꢀresponsive
moiety ‘trigger’ and a potent cellꢀdamaging functional group ‘effector’, joined by a linker system
in such a way that the reaction of the trigger with H2O2 causes a large increase in the cytotoxic
potency of the effector (Scheme 1). The selective reaction of boronic acid or ester derivatives
with H2O2 has been applied for fluorescent detection of H2O2, gene expression, pointꢀofꢀcare
assay, and prodrug development.26,27,29ꢀ37 Recently, we have developed two types of H2O2ꢀ
activated DNA crossꢀlinking agents using boronic acid or ester as ‘trigger’. One class can release
a nitrogen mustard effector upon treatment with H2O2 while the other can produce quinone
methides crossꢀlinking DNA. However, both did not show potent anticancer activity. We
speculate that these charged molecules may not be suitable for drug development because it is
wellꢀknown that charged molecules cannot diffuse across cell membrane. Here, we report a
novel strategy for creating neutral H2O2ꢀactivated prodrugs which showed dramatically increased
potency and selective cytotoxicity towards various cancer cells. For the first time we
demonstrated that the direct attachment of a boron group to an aromatic ring is sufficient to mask
the toxicity of the nitrogen mustard. The potential therapeutic utility has been demonstrated by
determining their toxicity and selectivity towards primary leukemic lymphocytes from CLL
patients and comparing that with normal lymphocytes from healthy donors.
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We designed and synthesized a series of H2O2ꢀactivated boronꢀcontaining aromatic nitrogen
mustard prodrugs (1-3) with two linker systems and various leaving groups. Compounds 1a-f
and 2 contain a nitrogen mustard group directly bonded to the aromatic ring (Scheme 2A). The
electronꢀwithdrawing boronate group decreases the electron density of the benzene ring and
makes the loneꢀpair of the nitrogen mustard delocalize to boron (D). Therefore, these prodrugs
do not form the electrophilic aziridinium ring E and are not deleterious to cells with low ROS
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levels (Scheme 2B). However, the oxidation of the carbonꢀboron bond by H2O2 followed by a
transformation to a hydroxyl group can trigger an increased electron release to the nitrogen of the
mustard moiety (B),29 this facilitates the formation of a highly electrophilic aziridinium ring C
capable of crossꢀlinking DNA. Compound 3 contains a withdrawing carbonyl group which can
reduce the toxicity of the nitrogen mustard. We assumed that release of the amine effector would
occur upon activation of 3 by H2O2.30ꢀ32
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(A)
(B)
Scheme 2. (A) Structures of the designed prodrugs. (B) Mechanism of targeting ROSꢀcontaining
cancer cells.
RESULTS
Synthesis of 1a-1f, 2 and 3
The synthesis of compounds 1-3 is shown in Scheme 3. Compounds 1aꢀ1f and 2 were
synthesized starting from paraꢀbromoaniline (4). 2ꢀChloroethanol was first coupled with 4 using
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calcium carbonate as a base yielding 6. Palladiumꢀcatalyzed borylation of 6 provided boronate
intermediate 8 which reacted with MsCl resulting in dimesylate mustard 1f at 80% yield.
Nucleophilic substitution of 1f with 1.0 equivalent of lithium chloride or lithium bromide
afforded 1a, 1d or 1b, 1e respectively. Compound 1d was converted to 1c by further treatment
with lithium bromide (Scheme 3A). For the synthesis of boronic acid 2, compound 6 was first
converted to dichloromustard 9 via mesylation and chlorination (Scheme 3B), and treatment of 9
with butyllithium and triisopropyl borate was followed by hydrolysis which yielded boronic acid
2. Compound 3 was obtained via an amidation reaction of bis(2ꢀchloroethyl)amine hydrochloride
and chloroformate 11 which was synthesized from 10 and triphosgene (Scheme 3C).
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NH2
Br
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O
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NaI, CaCO3,
H2O, reflux
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CH2Cl2
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80%
AcOK, PdCl2(dppf),
dioxane, 110 oC
N
+
OH
HO
OH
O
Cl
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Scheme 3. Synthesis of compounds 1a-1f (A), 2 (B), and 3 (C).
Selective DNA cross-linking ability
Initially we investigated their DNA crossꢀlinking abilities and selectivity by allowing crossꢀ
linkers to react with DNA duplex 12 which contains GNC sequences at the terminus. First, we
studied the effect of the carbamate linker (3) on the activity of nitrogen mustard. As we expected,
3 did not induce ICL formation in the absence of H2O2, which indicated that a carbamate linker
is sufficient to deactivate the nitrogen mustard. To our surprise, DNA ICLs were not formed in
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the presence of 3 and H2O2. Obviously, a carbamate linker is not suitable for constructing H2O2ꢀ
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inducible DNA crossꢀlinking agents.
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Next, we studied the reactivity of 1a-f and 2 towards 12 (Figure 1). In the absence of H2O2, no
obvious DNA ICLs were observed for 1a-e and 2, while 1f induced 22% DNA crossꢀlinking.
Compound 1f contains two mesylate groups while the others have one (1d and 1e) or no
mesylate group (1aꢀ1c and 2). The better leaving property of the mesylate group may cause the
higher reactivity of 1f compared to others in a H2O2ꢀfree system. These data indicated that boron
groups are sufficient to deactivate dihalogen or halogenmesylate mustards but cannot completely
mask the reactivity of dimesylate mustard 1f. The addition of H2O2 triggered the activity of 1aꢀe
and 2 leading to efficient ICL formation (37%ꢀ49%). Similarly, the crossꢀlinking yield of 1f was
increased three fold upon H2O2ꢀactivation. It is worth mentioning that the ICL was not observed
when the DNAs were treated with H2O2 only (Figure 1, lane 2). As we proposed, the conversion
of an electronꢀwithdrawing boron group to a donating hydroxyl group by H2O2 increases the
electron density of nitrogen mustard and therefore facilitates the ICL formation (Scheme 2B).
DNA ICL induced by 1a-f and 2 were observed at a concentration as low as 50 ꢀM (~ 3% ICL
yield) and the optimum ratio of drug to H2O2 was 1:2 (Tables S1ꢀS7 and Figures S2ꢀS3). The
best selectivity and activity were observed under physiological pH and temperature (pH 7.0 – 8.0
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o
and 37.0 – 38.0 C) (Figure S4ꢀS5). The ICL production induced by these compounds followed
firstꢀorder kinetics with a rate constant (kICL) ranging from 2.45 ± 0.25 × 10ꢀ5 sꢀ1 to 5.42 ± 0.15 ×
10ꢀ5 sꢀ1) (Figure S6). Among these agents with different leaving groups, compounds with the
methyl mesylate group showed a higher H2O2ꢀinducible DNA crossꢀlinking ability than those
having halogen groups with an order of 1f > 1d ≈ 1e > 1a ≈ 1b ≈1c. Upon treatment of H2O2, 1f
with two mesylate groups resulted in 66% DNA ICLs, while 1d and 1e with one mesylate group
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and one halogen group produced 47% and 48% ICL respectively, lastly only 35%ꢀ37% yields
were observed with 1a, 1b and 1c with two halogen groups. Besides that, no obvious different
reactivity was found in compounds with bromine or chlorine groups (1d vs 1e; 1a vs 1b vs 1c).
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In order to understand whether the location of GNC sequences affects the crossꢀlinking
efficiency, we synthesized duplex 12’ with GNCs in the middle of the sequence and investigated
ICL formation of duplex 12’ induced by 1a/H2O2 and 1b/H2O2 (1 mM). The crossꢀlinking yield
of 12’ (27% ± 3.5) was slightly lower than that of duplex 12 (35% ± 4.0), but both are within the
experimental error of each other (Figure S7). These data showed that the activity and selectivity
of these agents can be achieved with a variety of DNA sequences. It is necessary to point out that
smearing bands were observed when the crossꢀlinking yield was high and/or when there were
multiple crossꢀlinking sites or alkylating sites, while such phenomena was not observed when the
yields were low (Figures S6F and S8).
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5'-dGCCTAGTTCTTTTAA1T6 T17A1C9T20TGCAATGCAAGTAATTAAAGCTT4G4A45TCTG(12a)
3'-dCGGATCAAGAAAATTAATGAACGTTACGTTCATTAATTTCGAACTAGAC(12b)
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Figure 1. Comparison of the H2O2ꢀinducible activity and selectivity of 1a-f and 2.
Phosphorimage autoradiogram of denaturing PAGE analysis of the crossꢀlinking reaction of
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DNA duplex 12 in the presence of 1a-f or 2 (1.0 mM) (all reactions were carried out at room
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temperature).
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Subsequently, we investigated the activity of 1a, 1d, and 2 towards other ROS including tertꢀ
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t
ꢀ
butylhydroperoxide (TBHP), OClꢀ, HOꢂ, BuOꢂ, O2 , and NO (Figure S8). Among these, H2O2 is
the most efficient ROS that triggers the activity of these prodrugs, while TBHP, OClꢀ, and O2
ꢀ
also slightly activate 1a, 1d, and 2. In the presence of H2O2, these compounds induced 30ꢀ47%
ICL formation, while much lower ICL yields were observed with other ROS (0.9ꢀ6.6% for OClꢀ,
ꢀ
1.0ꢀ3.6% for TBHP, and 5ꢀ15% for O2 ) (Figure 2). This is consistent with previous reports about
the selective reaction of boronic acids and their esters towards H2O2.26,30
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1d
2
0
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2
-
t
H O
2 2
TBHP
O
HO
NO
OCl
BuO
ROS
Figure 2. ICL formation induced by 1a, 1d and 2 upon treatment with various ROS (1 mM of
drugs and ROS were used).
NMR detection of activation of 1a and 2 by H2O2
Activation of 1a and 2 by H2O2 and the formation of hydroxyl analogue 13 were confirmed by
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NMR analysis (Figures 3ꢀ5). The reaction of 1a (20 ꢃmol) or 2 (20 ꢃmol) with H2O2 (30 ꢃmol)
was carried out in a mixture of 400 mM deuterated potassium phosphate buffer (pH 8.0) (50 ꢀL)
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and d6ꢀDMSO (450 ꢀL). In the presence of H2O2, oxidative deboronation of 1a occurred yielding
alkylating agent 13 and boronic acid (1C), which was evidenced by the appearance of C1’ꢀH (δ
1.26 for 1a and δ 1.14 for 1C) (Figure 4). Compound 1C was further hydrolyzed to pinacol (1D,
δ 1.06). The intermediate 1B was too active to be detected. The conversion of 1a to 13 was so
fast that more than 80% of 1a to 13 was completed within 30 min, and >95% of 13 was formed
after 2 h, which showed that the arylboronates developed in this work are efficient H2O2ꢀ
responsive trigger units. To ensure the role of H2O2 in activation, a control experiment was
performed by incubating 1a in potassium phosphate buffer in the absence of H2O2 (Figure 4C).
The activated product 13 was not detected while hydrolysis of 1a occurred leading to 2 and
pinacol. Compound 2 can also be efficiently converted to 13 by H2O2, 93% of 2 was converted to
13 within 2 h (Figure 5). Over all, the prodrugs developed in this work are sensitive to H2O2
under physiological condition.
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Figure 3. Activation of 1a and 2 by H2O2.
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Figure 4. H NMR analysis of the activation of 1a (40 mM) by H2O2 (60 mM). A, H NMR
analysis of 1a in 400 mM phosphate buffer (pH 8) (50 ꢀL)/d6ꢀDMSO (450 ꢀL); B, 30 min after
the addition of H2O2; C, 24 h after 1a was incubated in 4.0 mM deuterated potassium phosphate
buffer in the absence of H2O2 (1.0 M deuterated potassium phosphate buffer/d6ꢀDMSO/D2O =
2:450:48).
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Figure 5. H NMR analysis of the activation of 2 (40 mM) by H2O2 (60 mM). A, 5 min after the
addition of H2O2; B, 2 h after the addition of H2O2 [The reaction was carried out in 1.0 M
deuterated phosphate buffer (pH 8) (100 ꢀL)/d6ꢀDMSO (450 ꢀL)]; C, An authentic sample of
compound 13 in D2O (100 ꢀL)/d6ꢀDMSO (450 ꢀL).
In order to confirm that 13 was the direct alkylating agent generated from 1a or 2 with H2O2, we
isolated 13 from the reaction of 2 with H2O2 in potassium phosphate buffer (Figure S1). The
reaction was so efficient that 85% (isolated yield) of 13 was obtained after 2 h. Compound 13
was characterized by 1H NMR, 13C NMR, and high resolution mass (Supporting information). Its
reactivity with DNA was also studied. The crossꢀlinking efficiency of 1a/H2O2 (50%) or 2/H2O2
(55%) was close to that of 13 (52%) (Figure S9), which also supported our conclusion.
Determination of specific cross-linking sites and the stability of cross-linked products
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The stability and reaction sites of the ICL products were examined to provide further insight
into the reactivity of compounds 1a-e and 2. The reaction sites of DNA alkylation can be
determined by investigating their heating stability under basic and/or neutral conditions. It was
reported that the ICL induced by nitrogen mustards usually occurred at Nꢀ7 of dGs.38 Piperidine
is known to induce cleavage with Nꢀ7 alkylated purines upon heating.39 Thus, we examined the
stability of DNA crossꢀlinking products formed by these compounds in phosphate buffer (pH 7)
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or in 1.0 M piperidine (90 C). The DNA ICLs were completely destroyed after heating for 30
min which led to obvious cleavage bands at dGs and dAs (Figure 6 and Figures S10ꢀS13). These
results are consistent with the observation that the reaction of nitrogen mustard mainly occurred
at N7 of purines. However, in addition to major cleavage bands at the purine sites, we also
observed some week ones at pyrimidine nucleotides (e.g. 7ꢀ9, 16, 17, 19, 20, 59, 60, 65, 66, 68,
and 72) as shown in Figure 6 and Figure S9ꢀS11. Compared with other nitrogen mustards
compounds,22 such as mechlorethamine (Figure 6, lanes 7ꢀ9) and 13 (Figure 6, lanes 4ꢀ6), the
crossꢀlinking products induced by 1aꢀe and 2 in the presence of H2O2 showed similar cleavage
patterns as those induced by 13 (lanes 4ꢀ6), but were a little different from mechlorethamine. In a
separate experiment, the ICL products and the drugꢀtreated single stranded DNA were isolated
from the reaction mixture and heated in neutral phosphate buffer or 1.0 M piperidine. Similar
cleavage patterns were observed for ICL products and single stranded DNA (Figures S14ꢀS15).
These data showed that apart from ICL formation, intrastrand crossꢀlinking and/or mono
alkylations were also possible.
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5'-dGCCTAGTTCTTTTAA1T6 T17A1C9T20TGCAATGCAAGTAATTAAAGCTTGATCTG(12a)
3'-dCGGATCAAGAAAATTAATGAACGTTACGTTCATTAATTTCGAACTAGAC(12b)
1
5
6 7 8 9
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39 40
44 45
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58 5756
60 59
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97 96
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72 71
68 66 65
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Figure 6. Comparison of specific crossꢀlinking sites caused by 1a/H2O2, 13 and
mechlorethamine. Phosphorimage autoradiogram of 20% denaturing PAGE analysis of the ICL
products upon heating in piperidineor phosphate buffer (The ICL products were produced by
incubation of duplex 12 with 1a/H2O2, 13 or mechlorethamine. 12a was radiolabeled at 5’ꢀ
terminus): Lanes 1ꢀ3: 1a/H2O2; lanes 4ꢀ6: compound 13; lanes 7ꢀ9: mechlorethamine; lanes 1, 4,
7: control (no treatment); lanes 2, 5, 8: treated by heating at 90 oC in buffer (pH 7.0); lanes 3, 6,
o
9: treated by heating at 90 C in piperidine; lane 10: G+A sequencing; lane 11: FeꢂEDTA
treatment of 12.
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Evaluation of the cytotoxicities in cell lines
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Since the activity of nitrogen mustards were effectively masked in 1a-e and 2 but can be
selectively triggered by H2O2 to induce efficient ICL formation, their cytotoxicity and selectivity
were further evaluated in biological systems.40 All of these agents showed significant growth
inhibition of the cell lines tested. The growth percentages of most cell lines were less than 50%
at a single dose of 10 ꢀM. For comparison, 3 was also tested. However, no obvious toxicity was
observed, which is consistent with the DNA crossꢀlinking study. Furthermore, we compared the
anticancer activity of the aromatic nitrogen mustards 1a and 2 with that of 14 and 15 which
released the simplest nitrogen mustard mechlorethamine.26 Compounds 1a and 2 showed a much
higher growthꢀinhibitory effect on tumor cells than 14 and 15 (Figure 7). Although the precise
mechanism underlying the higher toxicity of 1a and 2 is not clearly understood yet, one of the
possible reasons we considered is that 1aꢀe and 2 are neutral molecules which are expected to
diffuse across a cell membrane better than positively charged 14 and 15.
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(A)
(B)
Figure 7. Comparison of the anticancer activities of prodrugs with the ones releasing
mechlorethamine. Each cell line was grown in two plates and treated with drug (10 ꢃM) for 48 h
at 37°C, 5 % CO2, 95 % air, and 100 % relative humidity. The growth percents were determined
by NCIꢀ60 DTP Human Tumor Cell Line Screen40: (A) 1a vs.14; (B) 2 vs. 15.
Considering that different halogen groups (Br and Cl) in these compounds didn’t show much
difference on the reactivity towards DNA and cytotoxicity towards cancer cell lines, compounds
1a, 1c, 1d, and 2 were chosen as representative compounds to evaluate their GI50 (Table 1). All
four compounds exhibited a high level of toxicity to the cell lines tested, such as leukemia, nonꢀ
small cell lung cancer, colon cancer, CNS cancer, melanoma, ovarian cancer, renal cancer,
prostate cancer, and breast cancer. Although the GI50 towards these cell lines range from 0.23
ꢃM to 31.4 ꢃM, most of these compounds have a GI50 of less than 5 ꢃM. In particular, they are
more toxic towards leukemia, nonꢀsmall cell lung cancer, CNS cancer, renal cancer and breast
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cancer than colon cancer, melanoma and ovarian cancer. For example, a GI50 of about 1 ꢃM was
observed with cell lines SR (Leukemia), NCIꢀH460 (NonꢀSmall Cell Lung Cancer), and MDAꢀ
MBꢀ468 (breast cancer). It was reported that leukemia, lung cancer, and breast cancer contain
cells which proliferate under conditions of oxidative stress and have high intracellular
concentrations of ROS.41ꢀ44 It is very likely that these compounds can be more efficiently
activated in these cells and therefore can lead to higher toxicity. In our initial DNA ICL study,
compounds 1d and 2 showed a little higher inducible DNA crossꢀlinking ability than 1a, but no
obvious difference was observed with their cytotoxicity. Compound 2 with a boronic acid group
had a little higher activity towards most cell lines than the other three with boronic esters (1a, 1c
and 1d), possibly due to its better water solubility (logP1a = 2.8, logP2 = 2.5. LopP was
determined in 1ꢀOctanol and PBS).
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Table1. Cytotoxicities of 1a, 1c, 1d and 2.
GI50 (ꢃM)
Tumor Type
Cell Line
1a
3.34
4.66
17.2
3.48
10.90
0.63
2.69
18.6
10.9
9.24
12.9
4.57
14.7
0.33
6.59
11.60
14.9
11.60
13.20
13.8
14.9
11.30
4.61
1c
5.03
5.11
22.6
3.69
19.40
0.66
4.88
22.5
10.9
12.80
11.9
5.38
32.5
0.42
11.70
11.40
24.0
13.90
17.10
18.8
31.3
13.90
4.90
1d
4.01
3.88
19.0
3.74
14.7
0.63
4.98
24.5
8.85
11.5
10.4
4.70
28.2
0.49
5.99
11.00
14.6
11.10
13.50
14.6
25.8
11.90
5.39
2
CCRFꢀCEM
HLꢀ60(TB)
Kꢀ562
MOLTꢀ4
RPMIꢀ8226
SR
A549/ATCC
EKVX
HOPꢀ62
3.27
2.88
15.8
2.90
8.59
0.48
0.89
15.4
8.48
10.50
10.3
3.36
15.1
0.23
3.53
7.26
12.1
9.37
9.46
11.9
15.1
8.39
4.72
Leukemia
HOPꢀ92
NonꢀSmall Cell
Lung
NCIꢀH226
NCIꢀH23
NCIꢀH322M
NCIꢀH460
NCIꢀH522
COLO 205
HCCꢀ2998
HCTꢀ116
HCTꢀ15
HT29
KM12
SWꢀ620
SFꢀ268
Colon Cancer
CNS
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SFꢀ295
SFꢀ539
SNBꢀ19
SNBꢀ75
U251
2.11
5.70
8.06
7.98
3.75
5.17
17.5
5.10
14.2
22.1
21.0
14.5
11.6
6.12
15.6
14.6
17.4
21.6
7.83
5.33
8.03
5.35
2.81
3.64
2.78
10.3
4.11
16.7
5.78
14.30
4.25
4.12
2.64
6.37
10.60
10.70
5.07
6.60
18.4
7.86
16.5
20.8
20.6
13.7
13.4
9.38
19.1
15.0
14.4
25.9
11.90
6.69
9.25
8.95
5.87
3.40
3.52
10.4
4.45
22.6
6.51
17.70
6.69
5.03
2.99
4.83
10.20
5.85
0.70
4.72
14.0
5.68
15.3
21.5
15.6
12.8
13.0
5.83
13.6
13.4
13.9
22.9
8.20
6.52
8.96
6.55
8.59
3.03
3.10
5.71
4.40
18.0
6.18
15.10
4.52
4.44
1.36
3.35
8.00
3.21
3.49
3.13
19.7
4.77
13.0
19.0
13.5
10.9
11.6
4.71
18.1
12.0
12.1
16.0
4.20
2.69
3.81
4.04
3.43
1.75
1.44
2.55
2.08
15.5
6.05
15.10
4.89
1.89
LOX IMVI
MALMEꢀ3M
M14
10
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12
13
14
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60
MDAꢀMBꢀ435
SKꢀMELꢀ2
SKꢀMELꢀ28
SKꢀMELꢀ5
UACCꢀ257
UACCꢀ62
IGROV1
OVCARꢀ3
OVCARꢀ4
OVCARꢀ5
OVCARꢀ8
NCI/ADRꢀRES
SKꢀOVꢀ3
786ꢀ0
Melanoma
Ovarian
A498
ACHN
CAKIꢀ1
RXF 393
SN12C
TKꢀ10
UOꢀ31
PCꢀ3
DUꢀ145
Renal
Prostate
Breast
MCF7
MDAꢀMBꢀ
231/ATCC
HS 578T
BTꢀ549
Tꢀ47D
MDAꢀMBꢀ468
16.5
22.8
22.1
16.6
26.2
10.5
10.70
27.4
12.1
12.00
24.1
12.0
8.49
1.07
31.4
6.63
6.29
0.51
1.60
1.49
Apoptosis of CLL cells or normal lymphocytes
Given that these compounds showed significant cytotoxicity in several cell lines, we investigated
the selectivity of representative compounds (1a, 1c, and 2) in primary samples obtained from
patients with CLL. CLL cells contain high levels of ROS and therefore should be effectively
targeted by these agents. As expected, all 3 compounds (1a, 1c and 2) induced significant
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amount of apoptosis in all samples tested. We observed a time (Figure 8A; 24 h and 48 h; n=4)
and dose dependent apoptosis (Figure 9; 24 h; n=3) in these samples. Compared to compounds
1a and 1c, compound 2 demonstrated increased activities in CLL samples. The IC50 for the most
potent compound (compound 2) was between 5 & 6 ꢁM.
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A
100
80
60
40
20
0
100
80
60
40
20
0
100
80
60
40
20
0
24 hr
24 hr
24 hr
P value
0.1171
Compound 1a
P value
0.0088
Compound 2
P value
0.0040
Compound 1c
Con
Con
Con
100
80
60
40
20
0
100
80
60
40
20
0
100
80
60
40
20
0
48 hr
48 hr
48 hr
P value
0.0109
Compound 2
P value
0.0095
Compound 1c
P value
0.0132
Con
Con
Con
Compound 1a
B
100
80
60
40
20
0
100
80
60
40
20
0
100
80
60
40
20
0
24 hr
24 hr
24 hr
P value
Con
0.2254
P value
0.1107
P value
0.0742
Compound 1a
Con
Compound 1c
Con
Compound 2
100
80
60
40
20
0
100
80
60
40
20
0
100
80
60
40
20
0
48 hr
48 hr
48 hr
P value
0.1140
P value
0.1112
P value
0.1373
Con
Compound 1c
Con
Compound 2
Con
Compound 1a
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Figure 8. Evaluation of cytotoxicity in primary leukemia cells (A) and normal lymphocytes (B)
at 24 h (upper panel) and 48 h (lower panel). Leukemic lymphocytes were obtained from
peripheral blood of patients with CLL (n = 4); Normal lymphocytes were obtained from
peripheral blood of ageꢀmatched healthy donors (n = 3). Incubations were carried with or without
(con) 10 ꢁM of compounds 1a, 1c, or 2 for 24 h or 48 h and the apoptosis induction were
measured by annexin/PI binding assay. Each line represents one patient. The p value was
obtained from student tꢀtests (two tailed) performed using the GraphPad Prism5 software
(GraphPad Software, Inc. San Diego, CA).
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To further assess the selectivity of these compounds towards cancer cells, we evaluated their
toxicity towards normal lymphocytes isolated from peripheral blood of ageꢀmatched healthy
donors (Figure 8B; 24 h and 48 h; n=3). Interestingly, 1a, 1c, and 2 resulted in comparatively
less apoptosis suggesting the selective action towards cancer cells and the IC50 was not achieved
in normal lymphocytes. Doseꢀdependent apoptosis of normal lymphocytes with prodrug 2 was
achieved at 24 h. Compound 1a at 24 h demonstrated % median for conꢀ95, treatedꢀ93 (p =
0.226); at 48h the % median for conꢀ91, treatedꢀ83 (p = 0.138). Compound 1c at 24 h
demonstrated % median for conꢀ95, treatedꢀ90 (p = 0.111); at 48h % median for conꢀ91, treatedꢀ
81 (p = 0.114) and compound 2 at 24 h demonstrated % median for conꢀ95, treatedꢀ92
(p=0.074); at 48 h % median for conꢀ91, treatedꢀ83 (p = 0.111). CLL lymphocytes are known to
have high ROS compared to normal lymphocytes.45 This may be one of reasons why these
compounds specifically kill leukemia cells while they spare normal lymphocytes.
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Figure 9. Doseꢀdependent apoptosis of CLL cells or normal lymphocytes with prodrugs 1a, 1c,
2. Leukemic lymphocytes obtained from peripheral blood of patients with CLL (n = 3). Normal
lymphocytes obtained from peripheral blood of ageꢀmatched healthy donors (n = 3). Incubations
were carried with compounds 1a, 1c, or 2 for 24 h and the apoptosis induction was measured by
annexin/PI binding assay. (A) CLL cells with 1a; (B) CLL cells with 1c; (C) CLL cells with 2;
(D) normal lymphocytes with 2 for 24 h.
Given that the H2O2ꢀactivated prodrugs are converted to active analogs in presence of ROS, we
postulated that blocking the ROS production by Nꢀacetyl cysteine (NAC) should inhibit the
cytotoxicity induced by these agents. To test our hypothesis, we incubated CLL lymphocytes
with compound 1c in the presence or absence of NAC and measured the end points such as
cytotoxicity (by annexin/PI binding assay) and ROS production (by DCDFA assay). Our data
showed that in the presence of NAC (100 mM) the apoptosis induced by 1c (10 ꢁM) was
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completely abrogated in 8 patients tested (Figure 10A). Consistent with these results, incubation
of 1c in the presence of NAC further blocked the production of ROS (Figure 10B), suggesting
that these agents function through ROSꢀdependent mechanisms. Data from one representative
patient sample is shown in the right panel but was done in six patient samples.
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Figure 10. (A) Comparison of the apoptosis induced by ROSꢀactivated prodrug 1c in the
presence or absence of Nꢀacetyl cysteine (NAC). Primary CLL cells were incubated with
compound 1c in the absence or presence of NAC to block the production of ROS and the cells
were harvested at the end of 24 h. Apoptosis was measured by annexin/PI binding assay as
described in the methods section. The error bars depict the mean SD for n=8; (B) Comparison of
the ROS level in CLL cells in the presence or absence of compound 1c and/or NAC. Primary
CLL cells were incubated with compound 1c in the absence or presence of NAC (Nꢀacetyl
cysteine; 100 mM) to block the production of ROS. The cells were harvested at the end of 24 h
and the global ROS levels were measured by DCFDA staining as described in the materials and
methods section. The histograms are provided for one representative patient sample (n=8).
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CONCLUSION
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In summary, a series of ROSꢀactivated aromatic nitrogen mustards with different leaving
groups have been successfully synthesized. The boronate ester group sufficiently masks the
activity of the aromatic nitrogen mustards which can be restored upon H2O2 treatment. The
activation mechanism of these prodrugs by hydrogen peroxide was determined by NMR analysis.
Among these agents with different leaving groups, compounds with methyl mesylate group
showed more potent inducible DNA crossꢀlinking ability than that with halogen groups, while
there was no obvious difference in the reactivity of compounds with bromine or chlorine group.
The stability study revealed that DNA crossꢀlinking and/or alkylation induced by these agents
mainly occurred with purine nucleotides. Consistent with the chemistry observation, in vitro
cytotoxicity assay in respective cell lines demonstrated that these reagents exhibited effective
killing of cancer cells with the concentration as low as or less than 1.0 ꢃM. Higher toxicities
were observed in cell lines, such as SR (Leukemia), NCIꢀH460 (NonꢀSmall Cell Lung Cancer),
and MDAꢀMBꢀ468 (breast cancer). In addition, these compounds showed selective toxicity
towards primary leukemic lymphocytes from patients with chronic lymphocytic leukemia (40%
to 80% apoptosis) while less toxic to normal lymphocytes from healthy donors (less than 25%
cell death). The cellular study with or without an ROS quencher showed that these agents
function through ROSꢀdependent mechanisms. Collectively, these data provide utility and
selectivity of these agents which should inspire further and effective application in potential
cancer chemotherapies.
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EXPERIMENTAL SECTION
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General Information. Unless otherwise specified, chemicals were purchased from Aldrich or
Fisher Scientific and were used as received without further purification. T4 polynucleotide kinase
was purchased from New England Biolabs. Oligonucleotides were synthesized via standard
automated DNA synthesis techniques using an Applied Biosystems model 394 instrument in a
1.0 ꢃmole scale using commercial 1000Å CPGꢀsuccinylꢀnucleoside supports. Deprotection of
the nucleobases and phosphate moieties as well as cleavage of the linker were carried out under
mild deprotection conditions using a mixture of 40% aq. MeNH2 and 28% aq. NH3 (1:1) at room
temperature for 2 h. [γꢀ32P]ATP were purchased from PerkinꢀElmer Life Sciences. Quantification
of radiolabeled oligonucleotides was carried out using a Molecular Dynamics Phosphorimager
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equipped with ImageQuant Version 5.2 software. H NMR and C NMR spectra were taken on
either a Bruker DRX 300 or DRX 500 MHz spectrophotometer. Silicon reagents were used in
CDCl3 as internal standard. High resolution mass spectrometry was performed at the University
of Kansas Mass Spectrometry Lab or University of CaliforniaꢀRiverside Mass Spectrometry Lab.
The purity was determined by RPꢀHPLC on a 4.6 x 250 mm RPꢀC18 column with 277 nm
detection, which confirmed that all compounds had ≥95% purity.
Synthesis. 2,2'-(4-Bromophenylazanediyl)diethanol (6). A mixture of 4ꢀbromoaniline (17.1 g,
0.1 mol), 2ꢀchloroethanol (20 mL), CaCO3 (20.0 g), and NaI (1.4 g) in 250 mL water was heated
to reflux overnight, then extracted with dichloromethane and washed with water. After
evaporation of the solvent, the residue was purified by column chromatography (Hexane/Ethyl
Acetate = 1:2) to afford white solid 6 (18 g, 70%). mp 78ꢀ80 ºC; 1H NMR (CDCl3, 300 MHz): δ
3.62 (t, J = 4.8 Hz, 4H), 3.88 (t, J = 4.8 Hz, 4H), 6.76 (d, J = 8.4 Hz, 2H), 7.38 (d, J = 8.4 Hz,
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2H); 13C NMR (CDCl3, 75 MHz): δ 55.3, 60.5, 108.8, 114.2, 131.9, 146.8; HRMSꢀES (m/z)
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[M+H]+ calcd for C10H14BrNO2: 260.0286, found: 260.0302.
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2,2'-(4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)phenylazanediyl)diethanol
(8).
A
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mixture of 6 (3.8 g, 14.7 mmol), bis(pinacolato)diboron (7.4 g, 29.4 mmol), KOAc (4.3 g, 43.9
mol), and PdCl2(dppf) (1.1 g, 1.5 mol) in dioxane (100 ml) was flushed with argon for 10
minutes and heated to reflux overnight under argon. After cooling to room temperature, the
mixture was extracted with ethyl acetate and washed with brine. The organic layers were
collected and dried over Na2SO4. After evaporation of the solvent under vacuum, the residue
was purified by column chromatography (Hexane/Ethyl Acetate = 1:2) to afford white foam 8
1
(2.52 g, 50%). mp 130ꢀ132 ºC; H NMR (CDCl3, 300 MHz): δ 1.34 (s, 12H), 3.30 (br s, 2H),
3.65 (t, J = 4.5 Hz, 4H), 3.89 (t, J = 4.5 Hz, 4H), 4.08 (s, 2H), 6.71 (d, J = 8.4 Hz, 2H), 7.70 (d,
J = 8.4 Hz, 2H); 13C NMR (CDCl3, 75 MHz): δ 24.8, 55.1, 60.6, 83.3, 111.4, 136.3, 150.1;
HRMSꢀES (m/z) [M+H]+ calcd for C16H27NO4B: 308.2033, found: 308.2013.
2,2'-(4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)phenylazanediyl)bis(ethane-2,1-diyl)
dimethanesulfonate (1f). To a solution of 8 (2.0 g, 5.83 mmol) and Et3N (2.3 mL, 17.5 mmol) in
dry CH2Cl2 (50mL), MsCl (1.4 mL, 17.5 mmol) was added dropwise at 0 °C. After 30 min, the
mixture was extracted with CH2Cl2 and washed with brine water, dried over Na2SO4, and
concentrated under vacuum. The residue was purified by column chromatography (Hexane/Ethyl
Acetate = 1:1) followed by recrystallization from EtOAc to afford white crystal solid 1f (2.1 g,
1
80%). mp 85ꢀ86 ºC; H NMR (CDCl3, 300 MHz): δ 1.33 (s, 12H), 2.96 (s, 6H), 3.81 (t, J = 5.7
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Hz, 4H), 4.37 (t, J = 5.7 Hz, 4H), 6.71 (d, J = 9.0 Hz, 2H), 7.71 (d, J = 9.0 Hz, 2H); C NMR
(CDCl3, 75 MHz): δ 24.9, 37.4, 50.4, 66.4, 83.4, 111.4, 136.7, 137.0, 148.5; HRMSꢀES (m/z)
[M+Na]+ calcd for C18H30NO8S2BNa: 486.1404, found: 486.1387.
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N,N-Bis(2-chloroethyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (1a). A mixture
of 1f (926 mg, 2 mmol) and LiCl (84 mg, 2 mmol) in acetonitrile (5 mL) was stirred at 60 °C for
18 h. After removal of solvent, the residue was purified by column chromatography
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(Hexane/Ethyl Acetate = 6:1) to afford white solid 1a (233 mg, 34%). mp 79ꢀ80 ºC; H NMR
(CDCl3, 300 MHz): δ 1.35 (s, 12H), 3.66 (t, J = 6.9 Hz, 4H), 4.37 (t, J = 6.9 Hz, 4H), 6.68 (d, J =
8.7 Hz, 2H), 7.73 (d, J = 8.7 Hz, 2H); 13C NMR (CDCl3, 75 MHz): δ 24.8, 40.3, 53.3, 83.5,
111.0, 136.7, 148.3; HRMSꢀES (m/z) [M+H]+ calcd for C16H25NO2Cl2B: 344.1355, found:
344.1365.
1
Further elution with Hexane/Ethyl Acetate 3:1 gave colorless oil 1d (226mg, 28%). H NMR
(CDCl3, 300 MHz): δ 1.34 (s, 12H), 2.95 (s, 3H), 3.66 (t, J = 6.3 Hz, 2H), 3.75ꢀ3.83 (m, 4H),
4.34 (t, J = 5.7 Hz, 2H), 6.70 (d, J = 8.7 Hz, 2H), 7.72 (d, J = 8.7 Hz, 2H); 13C NMR (CDCl3, 75
MHz): δ 24.9, 37.5, 40.4, 50.3, 53.2, 66.4, 83.4, 111.2, 136.7, 148.5; HRMSꢀES (m/z) [M+H]+
calcd for C17H28NO5SBCl: 404.1470, found:404.1497.
2-(2-Bromoethyl(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)amino)ethyl
methanesulfonate (1b). A mixture of 1f (926 mg, 2 mmol) and LiBr (170 mg, 2 mmol) in
acetonitrile (5 mL) was stirred at 60 °C for 20 h. After removal of solvent, the residue was
purified by column chromatography (Hexane/Ethyl Acetate = 6:1) to afford white solid 1b (276
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mg, 32%). mp 82ꢀ83 ºC; H NMR (CDCl3, 300 MHz): δ 1.35 (s, 12H), 3.49 (t, J = 7.5 Hz, 4H),
3.83 (t, J = 7.5 Hz, 4H), 6.68 (d, J = 8.4 Hz, 2H), 7.74 (d, J = 8.4 Hz, 2H); 13C NMR (CDCl3, 75
MHz): δ 24.8, 28.1, 53.1, 83.4, 110.1, 136.8, 148.1; HRMSꢀEI (m/z) [M+H]+ calcd for
C16H25BNO2Br2: 432.0340, found: 432.0340.
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Further elution with Hexane/Ethyl Acetate 3:1 gave colorless oil 1e (277 mg, 31%). H NMR
(DMSOꢀd6, 300 MHz): δ 1.24 (s, 12H), 3.10 (s, 3H), 3.71ꢀ3.72 (m, 4H), 3.77 (t, J = 5.4 Hz, 2H),
4.30(t, J = 5.4 Hz, 2H), 6.75 (d, J = 8.4 Hz, 2H), 7.49 (d, J = 8.4 Hz, 2H); 13C NMR (CDCl3, 75
MHz): δ 24.9, 37.5, 40.4, 50.3, 53.2, 66.4, 83.4, 111.2, 136.7, 148.5; HRMSꢀES (m/z) [Mꢀ
H+Na]+ calcd for C17H26NO5SBrBNa: 469.0706, found: 469.0721.
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N-(2-Bromoethyl)-N-(2-chloroethyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline
(1c). A mixture of 1d (806 mg, 2.0 mmol) and LiBr (170 mg, 2.0 mmol) in DMF (2 mL) was
stirred at 60 °C for 4 h. The mixture was extracted with CH2Cl2, washed with brine water, dried
over Na2SO4 and concentrated under vacuum. The residue was purified by column
chromatography (Hexane/Ethyl Acetate = 10:1) to afford white foam 1c (696 mg, 90%). mp 80ꢀ
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82 ºC; H NMR (CDCl3, 500 MHz): δ 1.36 (s, 12H), 3.50 (t, J = 6.9 Hz, 2H), 3.66 (t, J = 6.9
Hz, 2H), 3.78 (t, J = 6.9 Hz, 2H), 3.84 (t, J = 6.9 Hz, 2H), 6.69 (d, J = 8.5 Hz, 2H), 7.75 (d, J =
8.5 Hz, 2H); 13C NMR (CDCl3, 75 MHz): δ 24.9, 28.2, 40.4, 53.0, 53.3, 83.4, 110.9, 136.6,
148.2; HRMSꢀEI (m/z) [M]+ calcd for C16H24BNO2ClBr: 368.0807, found: 368.0803.
4-Bromo-N,N-bis(2-chloroethyl)aniline (9). A solution of MsCl (0.9 mL, 11.5 mmol) in DCM
(5 mL) was added dropwise to a mixture of 6 (1.0 g, 3.85 mmol) and Et3N (1.5 mL, 11.5 mmol)
in dry CH2Cl2 (20mL) at 0 °C. After 30 min, the mixture was extracted with CH2Cl2 twice and
the combined organic phase was washed with brine water, dried over Na2SO4 and then
concentrated under vacuum. The residue was used in the next step without further purification.
The residue was dissolved in DMF (8 mL) and LiCl (966 mg, 23.0 mmol) was added. After
stirred at 70 °C for 5 h, the mixture was extracted with CH2Cl2 and washed with brine water,
dried over Na2SO4 and concentrated under vacuum. The residue was purified by column
chromatography (Hexane/Ethyl Acetate = 10:1) to afford white foam 9 (1.1 g, 91%). mp 65ꢀ67
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