ROS-inducible DNA cross-linking agent as a new anticancer prodrug building block

Article abstract of DOI:10.1002/chem.201200075

Down at the crossed code: Novel arylboronic ester and biarylboronic ester derivatives were synthesized. Compound 1 can be activated by hydrogen peroxide to release 2,5-bis(trimethylammonium) benzyl-1,4-diol (2), which leads to interstrand cross-link formation and DNA alkylation (see scheme). Compound 1 provides a novel building block for the development of H₂O ₂-targeting anticancer prodrugs. Copyright

Full text of DOI:10.1002/chem.201200075

DOI: 10.1002/chem.201200075
ROS-Inducible DNA Cross-Linking Agent as a New Anticancer Prodrug
Building Block
Sheng Cao, Yibin Wang, and Xiaohua Peng*[a]
Some antitumor drugs react with DNA by inducing DNA
interstrand cross-links (ICLs), which can block DNA tran-
scription and replication.[1] ICL-inducing agents, such as ni-
trogen mustard, mitomycin C, cisplatin, and psoralens have
been used in cancer therapy.[2] However, the major disad-
vantage of these agents is their poor selectivity for cancer
cells. One novel approach to reduce the toxicity of cross-
linking agents for normal cells would be the creation of pro-
drugs that undergo tumor-specific activation. Inducible
DNA cross-linking or alkylating agents have been devel-
oped by several research groups.[3–6] However, few of them
can induce DNA cross-links selectively under tumor-specific
conditions. One of the exclusive features of cancer cells is
the high level of oxidative stress that is associated with the
increased amounts of reactive oxygen species (ROS).[7–10]
Therefore, it would be advantageous to develop novel cross-
linking agents that can be activated by the high level of
ROS in cancer cells.
with boronic ester as the H2O2-sensitive trigger.[15] Boro-
nate-based probes have been developed by Chang and co-
workers, Loꢀs group, and others, for selective detection and
imaging of hydrogen peroxide in cells.[16] Recently, our
group has shown that a prodrug of nitrogen mustard cou-
pled with an arylboronate can be triggered by H2O2 to re-
lease active drugs that can kill cancer cells.[17] However,
therapeutic utility would require a more efficient trigger
that can be coupled with multiple potent effectors to maxi-
mize the ROS-inducible cytotoxicity of prodrugs. We expect
that the arylboronic ester and biarylboronic ester derivatives
1–3 can be activated by H2O2 to release biquinone methide
and two amine effectors. This work describes the synthesis
and biological studies of 1–3.
Among different ROS, such as H2O2, hydroxyl radical
C
Cꢀ
( OH), and superoxide radical anions (O2 ), H2O2 has a piv-
otal role because it is a stable ROS and generated from
nearly all sources of oxygen radicals.[11] Increased levels of
H2O2 in cancer cells compared to normal cells have been re-
ported.[7,12] These factors make H2O2 an ideal candidate as
a target to develop new ROS-inducible prodrugs with high
selectivity to cancer cells. Arylboronic esters are particularly
suitable to this prodrug approach because H2O2 can readily
cleave the boronic ester to release the quinone methide
(QM).[13] QMs are important intermediates in a large
number of DNA cross-linking and alkylating processes.
With simple modifications suggested by Rokitaꢀs group,[4]
Freccero and colleagues,[5] Zhou et al.[6] and others,[14] QM
can induce DNA ICLs through different strategies for initia-
tion, including UV irradiation, fluoride ions, heating, or oxi-
dation, etc. Meanwhile, Cohenꢀs group reported the activa-
tion of matrix metalloproteinase inhibitor in situ by protect-
ing the hydroxyl group of the zinc-binding group (ZBG)
Compound 2 was synthesized starting from commercially
available 2,5-dibromo-p-xylene (2a; Scheme 1). Palladium-
catalyzed borylation of 2a provided boronated intermediate
2b, which reacted with NBS to yield brominated analogue
2c. Compound 2c was converted to 2 by using trimethyl-
[a] Dr. S. Cao, Y. Wang, Prof. X. Peng
Department of Chemistry and Biochemistry
University of Wisconsin-Milwaukee
3210 N. Cramer St., Milwaukee, WI 53211 (USA)
Fax : (+1)414-2295530
E-mail: pengx@uwm.edu
AHCTUNGTRENNUNG
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201200075.
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COMMUNICATION
Figure 1. Concentration dependence of compounds 1–3 for DNA cross-
link formation upon H2O2 activation. Lane 1: DNA only (cross-linking
yield 0%); lane 2: DNA with 1 (2 mm) only (cross-linking yield 0%);
lane 3: DNA with 2 (2 mm) only (cross-linking yield 0%); lane 4: DNA
with 3 (2 mm) only (cross-linking yield 0%); lane 5: DNA with H2O2
(10 mm) only (cross-linking yield 0%); lane 6: 10 mm 1+5 mm H2O2 (0%);
lane 7: 100 mm 1+50 mm H2O2 (0%); lane 8: 2 mm 1+1 mm H2O2 (0%);
lane 9: 10 mm 2+5 mm H2O2 (2%); lane 10: 100 mm 2+50 mm H2O2 (8%);
lane 11: 2 mm 2+1 mm H2O2 (24%); lane 12: 10 mm 3+5 mm H2O2 (0%);
lanes 13: 100 mm 3+50 mm H2O2 (0%); lane 14: 2 mm 3+1 mm H2O2
(0%).
The deboronation and activation of 2 was selective for
H2O2 over other reactive oxygen species (Figure 2). The
treatment of 2 with H2O2 at 200 mm induced about 8% ICL
formation, whereas the cross-linking yield with tert-butylhy-
droperoxide (TBHP), hydroxyl radical, tert-butoxy radical,
superoxide, and nitric oxide was less than 1% and less than
5% ICL was observed for hypochlorite.
Scheme 1. Synthesis of compound 2.
Initially, the activity of compounds 1–3 towards DNA was
investigated by using a 49-mer DNA duplex (4) by measur-
ing DNA ICL formation. The treatment of the DNA with 1–
3 were carried out in phosphate buffer (pH 8.0) at 378C.
ICL formation and cross-linking yields were analyzed with
denaturing polyacrylamide gel electrophoresis (PAGE) with
phosphorimager analysis (Image Quant 5.2). In the absence
of H2O2, ICLs were not observed with 1–3 (Figure 1,
lanes 2–4), which indicated that QM was not formed. In the
presence of H2O2, compound 2 efficiently induced ICL for-
mation (Figure 1, lanes 9–11). However, no ICL was ob-
served with 1 and 3 (Figure 1, lanes 6–8 and 12–14). The
cross-linking efficiency of 2 was affected by the concentra-
tion of the drug, the ratio of drug to H2O2, and the pH
value of the buffer solution. DNA cross-linking by 2 was ob-
served at a concentration as low as 10 mm (Figure 1, lane 9),
and 2 mm of 2 led to 24% ICLs (Figure 1, lane 11). The best
ratio of drug to H2O2 was 2:1 (Figure S1 in the Supporting
Information). Higher cross-linking yields were observed
under basic conditions (pH 8 and 9; Figure S2 in the Sup-
porting Information). This is consistent with the earlier ob-
servation that the reaction between arylboronic acid and hy-
drogen peroxide is pH dependent.[13,17,18] The ICL formation
induced by 2 displayed first-order kinetics with a rate con-
Figure 2. Selectivity of 2 (400 mm) with various reactive oxygen species
(ROS) at 200 mm. Data were acquired at 258C in phosphate buffer
(10 mm, pH 8) after 48 h. H2O2: hydrogen peroxide, TBHP: tert-butylhy-
ꢀ
C
C
droperoxide, OCl : hypochlorite anion, OH : hydroxyl radical, OtBu :
tert-butoxy radical, O2ꢀ: superoxide, NO: nitric oxide.
In order to acquire further insight into the reactivity of 2,
we determined DNA monoalkylations by examining the sta-
bility and reactivity of the purified ICL products and the
treated single stranded DNA. The stability and alkaline la-
bility of DNA alkylation products depend upon the reaction
site. The N7-alkylated products of purines are labile to pi-
peridine treatment, which can result in DNA cleavage.[3c,19]
stant (kobs) of 4.9ACHTUNGTRENNUNG
Supporting Information).
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X. Peng et al.
The ICLs formed from 2 are stable at high temperatures in
phosphate buffer (908C, pH 7.2, 30 min) and partially stable
in piperidine (1m; 908C, 30 min; Figure S4, lanes 3 and 4, in
the Supporting Information). DNA cleavage bands were ob-
served with all dGs and dAs when the purified ICLs were
treated with piperidine (1m; Figure S4, lanes 4, 11, 18, and
25, in the Supporting Information). Weak cleavage bands
were observed with dCs flanked by dAs and/or dGs. These
results indicated that the alkylations occurred at N7 of dG
and dA, and partially with dC. The stable ICLs observed
after piperidine treatment suggested that N7 is not the only
alkylation site (Figure S4, lanes 4, 11, 18, and 25, in the Sup-
porting Information). It is highly likely that the alkylation
took place at exocyclic amines. Monoalkylation of all gua-
nine and adenine units was also observed with single-strand-
ed DNA 4a or 4b (Figure S4, lanes 2, 9, 16, and 23, in the
Supporting Information). In a control experiment, oligonu-
cleotide 4 was treated with 10 mm, 100 mm, and 1.0 mm H2O2
alone; weak cleavage bands were observed at the purine
and pyrimidine sites (Figure S5 in the Supporting Informa-
tion). This indicated that H2O2 alone induced trace amounts
of hydroxyl radical upon heating, which led to the equal
cleavages with purine and pyrimidine nucleotides.[20]
In an effort to identify the interstrand cross-linking site of
compound 2, we used hydroxyl radical cleavage analysis of
the purified cross-linking product.[20] However, a completely
cleaved ladder similar to the hydroxyl radical control experi-
ment was observed with stronger cleavage bands at the posi-
tions of dGs and dAs (Figure S4, lanes 5, 12, 19, and 26, in
the Supporting Information). This further confirmed that
the interstrand cross-linking should occur at these positions.
However, the exact interstrand cross-linking site and pattern
are still unknown. This is caused by three factors: 1) high re-
activity of the quinone methide generated from 2 upon
H2O2 activation; 2) the complexity of the reaction products,
which include the interstrand cross-linking product, intra-
strand cross-linking and monoalkylated products, and ad-
ducts formed with dG, dA, and dC; 3) the cross-linking and
alkylated products are labile in piperidine.
To understand the difference for ICL formation between
compounds 2 and 1 or 3, their reaction mechanism was in-
vestigated by NMR spectroscopy analysis of the monomer
reactions. The treatment of 1–3 with H2O2 was performed in
deuterated potassium phosphate buffer (10 mm; pH 8.0) as
well as in DMSO/D2O. When phosphate buffer was used as
a solvent, the reaction was too fast to observe any inter-
mediates. Compounds 1–3 were completely consumed
within 5 min and converted to the phenol derivatives 6, 8,
and 10 (Figures S6B, S7B, and S9B in the Supporting Infor-
mation). The formation of 6, 8, and 10 was confirmed by
1H,13C NMR, and HRMS-ESI analysis of the isolated mate-
rials (Figures S21–S27 in the Supporting Information).
Scheme 2. Tandem QM generation and ICL formation induced by 2 upon
H2O2 activation.
phenol derivative 6. Analysis 3 h after addition of H2O2 re-
vealed that compound 2 was completely consumed (Fig-
ure 3C), as evidenced by the absence of diagnostic resonan-
ces at d=7.99 ppm (singlet) and d=4.86 ppm (singlet) cor-
responding to the vinyl and methylene protons of 2, respec-
tively. The resonances corresponding to 2 were replaced by
When the treatment of 2 with H2O2 was carried out in
a mixture of DMSO/D2O, we were able to observe all inter-
mediates formed (Scheme 2 and Figure 3A–D). The integral
ꢀ
ꢀ
ꢀ
ꢀ
change of protons C3 H, C6 H, C2’ H and C5’ H charac-
terized the kinetic transformation of compound 2 into the
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A Novel Anticancer Prodrug Building Block
COMMUNICATION
hinders the formation of QM.[4d,e,5b] This could explain the
obvious difference between compounds 1/3 and 2 regarding
the cross-linking properties.
In order to determine whether QM was generated from
the oxidation process or from the intermediate 6, QM trap-
ping experiments with a large excess of ethyl vinyl ether
(EVE) were performed. When 2 was incubated at 378C for
3 h in the presence of H2O2 and EVE, no detectable QM–
EVE adduct (11) was observed and 2 was completely con-
verted to 6 (Scheme 3). However, 11 was detected if 6 was
incubated at the same temperature for 48 h. Compound 11
was confirmed by 1H and 13C NMR spectroscopy analysis.
The generation of QM via 6 was further supported by the
cross-linking capability of 6 (Figure S11 in the Supporting
Information).
Figure 3. 1H NMR spectroscopic kinetic analysis of 2 in a mixture of deu-
terated DMSO (550 mL) and D2O (96 mL) with H2O2 (57.7 mm) and 2
(23.1 mm). A) Sample
2 in deuterated DMSO only, as reference;
B) 30 min after addition of H2O2; C) 3 h after addition of H2O2; D) 24 h
after addition of H2O2.
Scheme 3. Trapping reactions in the presence of EVE.
those that were indicative of molecule 5. Evidence for these
intermediates was obtained from the appearance of two dis-
tinct aromatic protons (dꢂ7.85, 7.11 ppm; Figure 3B and C)
and two different methylene protons (dꢂ4.72, 4.46 ppm).
After incubating the sample at room temperature for 24 h
(Figure 3D), the resonances corresponding to 5 were re-
placed by those of 6 (dꢂ6.95, 4.37 ppm). Similarly, the acti-
vation of 1 and 3 with H2O2 followed by the formation of
phenol derivatives were observed by NMR spectroscopy
analysis (Figures S8 and S10, and Schemes S4 and S5 in the
Supporting Information).
In conclusion, we have developed three H2O2-inducible
DNA bisalkylating and/or cross-linking agents (1–3), which
contain boronic ester. Among these compounds, the non-
toxic compound 2 can be selectively activated by H2O2 to
generate a powerful and reversible DNA alkylating agent 6,
which directly produces QMs under physiological conditions
and releases the leaving group trimethyl amine. The mecha-
nism of H2O2 activation, ICL formation, and DNA alkyla-
tion was determined by NMR spectroscopy analysis as well
as by a QM trapping experiment. Although the ICL yield by
2 is moderate, the alkylating and cross-linking potency can
be improved by introducing an alkylating, DNA binding, or
intercalating agent in the position of trimethyl amine. There-
fore, an effective strategy has been developed to design and
synthesize novel potent anticancer prodrugs that can be acti-
vated under tumor-specific conditions (high level of ROS)
to release multiple active species by using compound 2 as
a building block. Such a model will also be equally applica-
ble to the development of prodrugs for the treatment of
other diseases that are associated with H2O2. The synthesis
of boronate-based prodrugs with different functional leaving
groups is underway in our laboratory.
The NMR spectroscopic analysis of the monomer reaction
indicated that compound 2 can be efficiently activated by
H2O2 to give the intermediate 6. The formation of 6 was
ꢀ
triggered by the stepwise oxidation of the carbon boron
bond initiated by nucleophilic attack by H2O2 (2!2d and
5!2e) followed by deboronation (2d!5 and 2e!6;
Scheme 2). We believed that compound 6 directly generated
o-QMs (2 f and 2h) under physiological conditions that
alkylACHTUNGTRENNUNGate and cross-link DNA. By comparison with com-
pounds 8 and 10, an additional hydroxyl group in 6 leads to
an electron-rich aromatic ring, which is an important factor
for efficient ICL formation induced by 2. Rokita et al. have
shown that the electron-donating group favors QM genera-
tion and regeneration, while the electron-withdrawing group
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X. Peng et al.
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Experimental Section
1,4-Dimethyl-2,5-di(4,4,5,5-tetramethyl
(2b): 2,5-Dibromo-p-xylene (2a; 1.06 g, 4 mmol), bis(pinacolato)diboron
(3.05 g, 12 mmol), KOAc (2.36 g, 24 mmol), and PdCl2A(dppf) (196 mg,
ACHTUNGTRNE[NUNG 1,3,2]dioxaborolan-2-yl)phenyl
CTHUNGTRENNUNG
0.24 mmol) were dissolved in DMF (40 mL) under argon atmosphere.
The mixture was heated at 858C for 48 h, cooled and then water
(100 mL) was added, the mixture was extracted with CH2Cl2 (3ꢂ50 mL).
The combined organic layer was washed with water and brine, and then
dried over Na2SO4, and the solvent was evaporated. The crude product
was purified through column chromatography with 0–50% EtOAc in
hexane to provide compound 2b as white solid (1.28 g, 90%). 1H NMR
(300 MHz, CDCl3): d=1.39 (s, 24H), 2.51 (s, 6H), 7.57 ppm (s, 2H);
13C NMR (500 MHz, CDCl3): d=140.58, 136.94, 83.43, 24.91, 21.52 ppm.
1,4-Dibromomethyl-2,5-di(4,4,5,5-tetramethylACTHNUTRGENUG[N 1,3,2]dioxaborolan-2-yl)-
phenyl (2c): Compound 2b (1.08 g, 3 mmol) was dissolved in CH3CN
(45 mL), and NBS (1.34 g, 7.5 mmol) and AIBN (52.4 mg) were added.
The mixture was refluxed at 908C for 6 h. Then the mixture was concen-
trated and dissolved in DCM (100 mL). The organic phase was washed
with H2O (3ꢂ50 mL) and dried with anhydrous Na2SO4. The solution
was evaporated and the residue was subjected to column chromatography
on silica gel with 0–50% DCM in hexane to give the desired product 2c
as a white solid (0.71 g, 60%). 1H NMR (300 MHz, CDCl3): d=1.38 (s,
24H), 4.88 (s, 4H), 7.79 ppm (s, 2H); 13C NMR (500 MHz, CDCl3): d=
143.36, 137.85, 84.13, 33.43, 24.90 ppm.
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1,4-Di(trimethylammonium bromide-methyl)-2,5-di(4,4,5,5-tetramethyl-
ACHTUNGTRENNUNG[1,3,2]dioxaborolan-2’-yl)phenyl (2): Compound 2c (0.1 g, 0.19 mmol)
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was suspended in CH3CN (5 mL) and trimethylamine (4.2m; 0.14 mL,
0.57 mmol) in ethanol was added dropwise with stirring. The reaction
mixture was concentrated after 12 h at room temperature and gave 2 as
a white solid (0.12 g, 95%). 1H NMR (300 MHz, DMSO): d=1.35 (s,
24H), 3.05 (s, 18H), 4.86 (s, 4H), 8.02 ppm (s, 2H); 13C NMR (500 MHz,
DMSO): d=141.57, 134.66, 84.76, 65.84, 52.10, 24.49 ppm; HRMS (ESI)
m/z calcd (%) for C26H48B2Br2N2O4 [(Mꢀ2Br)/2]+: 237.1900; found:
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We are grateful for financial support of this research from the National
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Keywords: antitumor agents
alkylation · DNA cross-linking · prodrugs
·
arylboronates
·
DNA
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Received: January 8, 2012
Published online: February 29, 2012
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