Enhanced Tumor Selectivity of 5-Fluorouracil Using a Reactive Oxygen Species-Activated Prodrug Approach

Article abstract of DOI:10.1021/acsmedchemlett.8b00539

We report the design, synthesis, and evaluation of novel 5-fluorouracil (5FU) prodrugs 1a,1b that are efficiently activated by the high level of reactive oxygen species (ROS) in cancer cells. Prodrugs 1a,1b selectively kill cancer cells over normal cells and are well-tolerated in mice. The strategy described herein can extend application of chemotherapeutic drugs.

Full text of DOI:10.1021/acsmedchemlett.8b00539

Letter
pubs.acs.org/acsmedchemlett
Cite This: ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX
Enhanced Tumor Selectivity of 5‑Fluorouracil Using a Reactive
Oxygen Species-Activated Prodrug Approach
Yong Ai,†,∥ Obinna N. Obianom,†,∥ Meredith Kuser,† Yue Li,‡ Yan Shu,*,†,§ and Fengtian Xue*,†
†Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, Maryland 21201, United States
‡Department of Chemistry and Biochemistry, University of Maryland College Park, College Park, Maryland 20740, United States
§School and Hospital of Stomatology, Guangzhou Medical University, Guangzhou 510140, China
S
* Supporting Information
ABSTRACT: We report the design, synthesis, and evaluation of novel 5-fluorouracil (5FU) prodrugs 1a,1b that are efficiently
activated by the high level of reactive oxygen species (ROS) in cancer cells. Prodrugs 1a,1b selectively kill cancer cells over
normal cells and are well-tolerated in mice. The strategy described herein can extend application of chemotherapeutic drugs.
KEYWORDS: 5-Fluorouracil, prodrugs, reactive oxygen species, anticancer
he FDA-approved chemotherapy drug 5-fluorouracil
(5FU, Figure 1) was first introduced by Heidelberger et
cancer as a cream. While remarkably benefiting cancer patients,
5FU also possesses major side effects such as myelosuppres-
sion, central neurotoxicity, and gastrointestinal toxicity.3
Moreover, 5FU is metabolically unstable with the majority of
the dose converted to 3-fluoroalanine by the pyrimidine
degrading enzyme dihydropyrimidine dehydrogenase (DPD).4
To overcome the limitation of 5FU, prodrug strategies have
been actively pursued. Among numerous small molecule 5FU-
prodrugs described in the literature,5 four have been
successfully used in the clinic, including tegafur, doxifluridine,
carmofur, and capecitabine (Figure 1). These compounds are
orally available and activated in the human body via different
mechanisms. Activation of tegafur involves either a 5′-
hydroxylation catalyzed by the cytochrome P450 enzyme
CYP2A66 or an enzymatic cleavage of the N1−C2′ bond of the
molecule.7 Doxifluridine is a second-generation of nucleotide-
based prodrug of 5FU that has been approved in several Asian
countries.8 Its activation in tumors is achieved by either
thymidine phosphorylase9 or pyrimidine-nucleoside phosphor-
ylase.10 The lipophilic 5FU analogue carmofur overcomes the
problem of 5FU degradation by DPD.11 The carbamoyl group
of carmofur is enzymatically removed in the human body to
release 5FU. Finally, capecitabine is another carbamate-based
5FU-prodrug that is activated in the liver and cancer cells
T
Figure 1. Chemical structures of 5FU and clinically used 5FU-
prodrugs.
al. in 1957 as an antineoplastic antimetabolite of the uracil
anabolic pathway.1 In the human body, 5FU interferes with
DNA synthesis by blocking the activity of the nucleotide
synthesis enzyme thymidylate synthase (TS), which catalyzes
the conversion of deoxyuridylic acid to thymidylic acid.2 As a
single agent or in combination with other chemotherapeutics,
5FU has been widely prescribed for a variety of solid tumors
including breast cancer, colorectal cancer, stomach cancer,
pancreatic cancer, and cervical cancer by injection and skin
Received: November 7, 2018
Accepted: December 3, 2018
Published: December 3, 2018
© XXXX American Chemical Society
A
DOI: 10.1021/acsmedchemlett.8b00539
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
Figure 2. ROS-activated 5FU prodrugs 1a,1b.
a
through sequential reactions catalyzed by carboxylesterase and
cytidine deaminase.12
Scheme 1. Synthesis of Compounds 1a−1c
Reactive oxygen species (ROS), the metabolic byproducts of
oxygen metabolism, play an important role in maintaining
cellular redox homeostasis. Unlike the normal cell environment
where the ROS level is controlled by balancing its production
and elimination, cancer cells exhibit enhanced levels of ROS
such as superoxide (O2•−),13 hydrogen peroxide (H2O2),14
and hydroxyl radical (HO•)15 due to increased metabolic
activities and mitochondrial malfunction. It has been shown
that high levels of ROS in cancer cells are also associated with
DNA mutations that lead to tumor angiogenesis16 and
metastasis17 and drug resistance.18 At the same time, the
growth and transformation of cancer cells are promoted with
the condition of a modest rise of intracellular ROS.19
a
Reagents and conditions: (a) 5FU, DBU, DMF, rt, 12 h, 37% for 1a
and 49% for 1c; (b) NaIO4, NH4OAc, rt, 16 h, 69%.
While ROS themselves have been exploited for the
development of tumor-selective therapeutics by amplifying
oxidative stress,20−22 ROS-activated prodrugs have also been
developed to obtain tumor selective agents.23,24 As commonly
used bioprecursors, arylboronic acids and esters have been
widely employed as specific ROS-activated groups for various
drugs including SAHA,25 aminopterin and methotrexate,26 SN-
38,27 nitrogen mustards,28,29 nitric oxide donors,30,31 amino-
ferrocene,32−34 and matrix metalloproteinases (MMP) inhib-
itors.35 To the best of our knowledge, a similar strategy has not
been described for 5FU.
Herein we report the design and evaluation of novel
arylboronate-based prodrugs 1a,1b of 5FU (Figure 2). The p-
boronate-benzyl group was introduced to the N1 position to
limit the metabolic degradation by DPD. The arylboronate
groups are designed as ROS-sensitive triggers, which react with
H2O2 to generate the corresponding phenol intermediate.
Spontaneous breakdown of this intermediate will release 5FU,
along with a highly reactive byproduct 4-methylenecyclohexa-
2,5-dien-1-one, which is rapidly hydrolyzed in aqueous
environment to give the nontoxic 4-(hydroxymethyl)phenol.36
The synthesis of prodrugs 1a,1b and a control compound 1c
is detailed in Scheme 1. The N1-selective benzylation of 5FU
using either compound 2 or compound 3 in the presence of
1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) as the base gen-
erated phenylboronate ester 1a and control 1c in modest
yields. Then compound 1a was treated with NaIO4 in the
presence of NH4OAc to yield compound 1b in modest yields.
We first tested the efficiency of new prodrugs 1a,1b in
releasing the parent drug 5FU in the presence of the ROS
H2O2 (Figure 3). Compounds 1a,1b were incubated in a
mixture of DMSO in PBS buffer (30%) with the addition of
H2O2 (5 equiv) at 37 °C. The conversion of prodrugs 1a,1b
and the production of 5FU were followed using RP-UPLC-MS.
Both prodrugs were activated efficiently by H2O2 to release
5FU. Interestingly, under the assay conditions, the boronic
Figure 3. Release of 5FU from compounds 1a,1b. The incubation of
each prodrugs (100 μM) was carried out in the presence of 5 equiv of
H2O2: (A) prodrug 1a and (B) 1b. The conversion of prodrugs 1a,1b
and release of 5FU was determined. The data represent mean SD of
triplicates.
ester 1a hydrolyzed rapidly to generate boronic acid 1b (Figure
3A).
Next, we studied the anticancer effects of compounds 1a,1b
against 60 human cancer cell lines (the Developmental
Therapeutics Program of NCI). The cell lines were derived
from different cancer types including leukemia, nonsmall-cell
lung cancer, colon cancer, CNS cancer, melanoma, ovarian
cancer, renal cancer, prostate cancer, and breast cancer
(Supporting Information (SI), Table S1). The assay protocol
can be found at: https://dtp.cancer.gov/default.htm. Com-
pound 1a caused over 50% growth inhibition for most of the
tested cell lines. In particular, it exhibited the most significant
B
DOI: 10.1021/acsmedchemlett.8b00539
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
inhibition (81−96%) against the lung cancer NCI-H522 cells,
melanoma MDA-MB-435 cells, ovarian cancer OVCAR-3 cells,
and breast cancer MCF7 and MDA-MB-468 cells (Table 1). In
prodrugs 1a,1b for cancer cells over normal cells. Moreover,
the control compound 1c, without the ROS-reactive group in
its structure, exhibited no obvious antiproliferative effects
against the MCF7 cells, indicating that prodrugs 1a,1b
achieved their anticancer effects through the reactivity of the
boronic acid or ester groups toward the ROS. To further verify
the role of ROS in the drug release process, we performed the
MTT assays using MCF7 cells pretreated with the ROS
scavenger N-acetyl-cysteine (NAC). Upon pretreatment of
NAC, the anticancer effects of prodrugs 1a,1b decreased
significantly (SI, Figure S1), confirming that the new prodrugs
exerted their cytotoxicity depending on ROS activation.
Table 1. Anticancer Effects of Compounds 1a,1b
a
inhibition (%)
tumor type
cell line
1a
1b
lung cancer
melanoma
ovarian cancer
breast cancer
NCI-H522
MDA-MB-435
OVCAR-3
MCF7
90
96
95
81
87
44
65
30
33
22
MDA-MB-468
To understand the mechanism of action for prodrugs 1a,1b,
we examined the impact of the treatment of compounds 1a,1b
on γH2AX formation in MCF7 cells using an immunofluor-
escence assay. The γH2AX formation is a sensitive marker to
visualize and quantify DNA double-strand breaks and the
consequential cell apoptosis.38,39 As shown in Figure 5A, upon
treatment of compounds 1a,1b, the cells showed dramatical
increases in γH2AX formation, which was consistent with the
effect of their parent 5FU. In addition, we also evaluated the
effects of prodrugs 1a,1b on the PARP1 and caspase 3 cleavage
in MCF7 cells using Western blots assays (Figure 5B).
Activation of PARP1 and caspase 3, through cleavage, leads to
a
Each cell line was treated with either compound 1a or compound 1b
at the concentration of 50 μM for a period of 48 h. Then the growth
inhibition (%) values were determined.
general, boronic ester 1a demonstrated larger effects than the
boronic acid analogue 1b (SI, Table S1). We reasoned that the
differences in anticancer effects between compounds 1a,1b
may be due to the more favorable membrane permeability of
the boronic ester 1a.
We further evaluated the anticancer effects of compounds
1a,1b in detail using the breast cancer MCF7 cells and normal
MEC1 cells (Figure 4). Compound 1a exerted potent but
Figure 4. Anticancer effects and selectivity of 5FU and compounds
1a−1c. Cell viability was analyzed after the breast cancer MCF7 cells
(A), and normal MEC1 cells (B) were treated with 5FU or
compounds 1a−1c at different concentrations. Data are presented
as mean SD, n = 4.
lower antiproliferative activity as compared to 5FU, while
compound 1b indicated much weaker effects. We reasoned
that the decreased anticancer effects of the prodrugs may be
due to the diminished cell membrane permeability. Cellular
uptake of 5FU is facilitated by organic anion transporter 2
(OAT2) and reduced folate carrier 1 (RFC1),37 which would
probably not contribute to the penetration of compounds
1a,1b. On the other hand, compared to 5FU, both prodrugs
1a,1b demonstrated no toxicity toward the normal MEC1 cells
(cell viability >95%) at concentrations up to 100 μM. These
differential killing results highlight the specificity of the new
Figure 5. Effects of compounds 1a,1b in causing cell apoptosis. (A)
Immunofluorescence of DNA cleavage protein γH2Ax was enhanced
by using 5FU and prodrugs 1a,1b. (B) Immunoblot analysis of MCF-
7 cells treated with 5FU, prodrugs 1a,1b for apoptosis markers
PARP1, and caspase 3. The cells were treated for 48 h with the
indicated concentrations of the compounds.
C
DOI: 10.1021/acsmedchemlett.8b00539
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
cell apoptosis.40,41 Compared to vehicle-treated cells, treat-
ment of prodrugs 1a,1b, similar to 5FU, reduced the levels of
both PARP1 and caspase 3 in MCF7 cells. Interestingly, the
same treatments of compounds 1a,1b, similar to that of 5FU,
significantly increased the levels of cleaved PARP1 (CL-
PARP1) and cleaved caspase 3 (CL-caspase 3) in MCF7 cells,
as compared with those in the vehicle-treated controls.
Therefore, prodrugs 1a,1b, similar to 5FU, can induce
MCF7 cell apoptosis, which might contribute to their
pharmacological actions.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.8b00539.
■
S
Experimental procedures and compound characteriza-
tion for all new compounds (PDF)
AUTHOR INFORMATION
Corresponding Authors
*For F.X.: phone, 410-706-8521; E-mail, fxue@rx.umaryland.
■
To evaluate the safety profile of the new prodrugs, we tested
the acute toxicity of compound 1a in wild-type C57BL/6 male
mice (Figure 6). While treatment with a high dose (100 mg/
edu.
*For Y.S.: phone, 410-706-7358; E-mail, yshu@rx.umaryland.
edu.
ORCID
Yan Shu: 0000-0001-8492-1437
Fengtian Xue: 0000-0002-4132-9887
Author Contributions
∥Y.A. and O.N.B. contributed equally to the work.
Notes
The authors declare the following competing financial
interest(s): Y.S. is a co-founder for and owns equity in Optivia
Biotechnology.
ACKNOWLEDGMENTS
■
We thank the American Association for Cancer Research grant
00167155 to F.X., the Samuel Waxman Cancer Research
Foundation, and the Leukemia Lymphoma Society for grant to
F.X. The National Institute of General Medical Sciences of the
US National Institutes of Health (NIH) supported the
research by Y.S. [R01GM099742]. Y.S. is a cofounder for
and owns equity in Optivia Biotechnology.
Figure 6. Survival rate (A) and body weight (B) curves of the mice
received vehicle, 5FU, or 1a. The 21 mice were randomly distributed
into three groups (seven mice in each group): vehicle (5% DMSO
and 15% polyethylene glycol in saline), 100 mg/kg of 5FU, and 100
mg/kg of prodrug 1a. Mice were treated every other day by
intraperitoneal (ip) injection for 15 days, and the survival and body
weights of the mice were recorded during the treatment.
REFERENCES
■
(1) Heidelberger, C.; Chaudhuri, N. K.; Danneberg, P.; Mooren, D.;
Griesbach, L.; Duschinsky, R.; Schnitzer, R. J.; Pleven, E.; Scheiner, J.
Fluorinated pyrimidines, a new class of tumour-inhibitory com-
pounds. Nature 1957, 179, 663−666.
(2) Longley, D. B.; Harkin, D. P.; Johnston, P. G. 5-fluorouracil:
mechanisms of action and clinical strategies. Nat. Rev. Cancer 2003, 3,
330−338.
(3) Macdonald, J. S. Toxicity of 5-fluorouracil. Oncology (Williston
Park) 1999, 13, 33−34.
(4) Heggie, G. D.; Sommadossi, J. P.; Cross, D. S.; Huster, W. J.;
Diasio, R. B. Clinical pharmacokinetics of 5-fluorouracil and its
metabolites in plasma, urine, and bile. Cancer Res. 1987, 47, 2203−
2206.
(5) Shelton, J.; Lu, X.; Hollenbaugh, J. A.; Cho, J. H.; Amblard, F.;
Schinazi, R. F. Metabolism, biochemical actions, and chemical
synthesis of anticancer nucleosides, nucleotides, and base analogs.
Chem. Rev. 2016, 116, 14379−14455.
(6) Ikeda, K.; Yoshisue, K.; Matsushima, E.; Nagayama, S.;
Kobayashi, K.; Tyson, C. A.; Chiba, K.; Kawaguchi, Y. Bioactivation
of tegafur to 5-fluorouracil is catalyzed by cytochrome P-450 2A6 in
human liver microsomes in vitro. Clin. Cancer Res. 2000, 6, 4409−
4415.
kg) of 5FU killed all mice on the twelfth day (Figure 6A), the
same dose of compound 1a did not cause animal death or
abnormality in eating, drinking, or activity throughout the
period. While body weights of the mice in the 5FU treated
group significantly decreased as compared to those of the
vehicle group, no obvious changes were observed in the
prodrug 1a treated group (Figure 6B). These results indicate
that, compared with the parent 5FU, prodrug 1a had more
favorable drug safety, exhibiting reduced toxicity in normal
tissues. This prodrug would benefit further research on its
potential as an effective targeted therapy.
In conclusion, we have designed, synthesized, and evaluated
novel arylboronate-based 5FU prodrugs 1a,1b. These prodrugs
are activated by high levels of ROS in cancer cells and
efficiently release 5FU. Prodrugs 1a,1b exhibited high
specificity for cancer cells over normal cells. Furthermore,
prodrug 1a demonstrated a more favorable safety profile as
compared to parent 5FU. These findings suggest that ROS-
activated prodrugs offer an effective way to improve the
therapeutic effectiveness and selectivity of current anticancer
chemotherapeutic agents.
́
(7) El Sayed, Y. M.; Sadee, W. Metabolic activation of R,S-1-
(tetrahydro-2-furanyl)-5-fluorouracil (ftorafur) to 5-fluorouracil by
soluble enzymes. Cancer Res. 1983, 43, 4039−4044.
(8) Schoffski, P. The modulated oral fluoropyrimidine prodrug S-1,
and its use in gastrointestinal cancer and other solid tumors. Anti-
Cancer Drugs 2004, 15, 85−106.
(9) Kono, A.; Hara, Y.; Sugata, S.; Karube, Y.; Matsushima, Y.;
Ishitsuka, H. Activation of 5′-deoxy-5-fluorouridine by thymidine
D
DOI: 10.1021/acsmedchemlett.8b00539
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
phosphorylase in human tumors. Chem. Pharm. Bull. 1983, 31, 175−
178.
cross-linking agents and their effect on cancer cells and normal
lymphocytes. J. Med. Chem. 2014, 57, 4498−4510.
(10) Nishioka, M.; Miyamoto, H.; Kurita, N.; Higashijima, J.;
Yoshikawa, K.; Miyatani, T.; Shimada, M. Pyrimidine nucleoside
phosphorylase and dihydropyrimidine dihydrogenase activities as
predictive factors for the efficacy of doxifluridine together with
mitomycin C as adjuvant chemotherapy in primary colorectal cancer.
Hepatogastroenterology 2007, 54, 1089−1093.
(11) Sakamoto, J.; Oba, K.; Matsui, T.; Kobayashi, M. Efficacy of
oral anticancer agents for colorectal cancer. Dis. Colon Rectum 2006,
49, S82−91.
(12) Shimma, N.; Umeda, I.; Arasaki, M.; Murasaki, C.; Masubuchi,
K.; Kohchi, Y.; Miwa, M.; Ura, M.; Sawada, N.; Tahara, H.; Kuruma,
I.; Horii, I.; Ishitsuka, H. The design and synthesis of a new tumor-
selective fluoropyrimidine carbamate, capecitabine. Bioorg. Med.
Chem. 2000, 8, 1697−1706.
(13) Hileman, E. O.; Liu, J.; Albitar, M.; Keating, M. J.; Huang, P.
Intrinsic oxidative stress in cancer cells: a biochemical basis for
therapeutic selectivity. Cancer Chemother. Pharmacol. 2004, 53, 209−
219.
(14) Szatrowski, T. P.; Nathan, C. F. Production of large amounts of
hydrogen peroxide by human tumor cells. Cancer Res. 1991, 51, 794−
798.
(15) Toyokuni, S.; Okamoto, K.; Yodoi, J.; Hiai, H. Persistent
oxidative stress in cancer. FEBS Lett. 1995, 358, 1−3.
(16) Ushio-Fukai, M.; Alexander, R. W. Reactive oxygen species as
mediators of angiogenesis signaling: role of NAD(P)H oxidase. Mol.
Cell. Biochem. 2004, 264, 85−97.
(17) Ishikawa, K.; Takenaga, K.; Akimoto, M.; Koshikawa, N.;
Yamaguchi, A.; Imanishi, H.; Nakada, K.; Honma, Y.; Hayashi, J.
ROS-generating mitochondrial DNA mutations can regulate tumor
cell metastasis. Science 2008, 320, 661−664.
(18) Dharmaraja, A. T. Role of reactive oxygen species (ROS) in
therapeutics and drug resistance in cancer and bacteria. J. Med. Chem.
2017, 60, 3221−3240.
(19) Arnold, R. S.; Shi, J.; Murad, E.; Whalen, A. M.; Sun, C. Q.;
Polavarapu, R.; Parthasarathy, S.; Petros, J. A.; Lambeth, J. D.
Hydrogen peroxide mediates the cell growth and transformation
caused by the mitogenic oxidase Nox1. Proc. Natl. Acad. Sci. U. S. A.
2001, 98, 5550−5555.
(20) Pelicano, H.; Feng, L.; Zhou, Y.; Carew, J. S.; Hileman, E. O.;
Plunkett, W.; Keating, M. J.; Huang, P. Inhibition of mitochondrial
respiration: a novel strategy to enhance drug-induced apoptosis in
human leukemia cells by a reactive oxygen species-mediated
mechanism. J. Biol. Chem. 2003, 278, 37832−37839.
(21) Trachootham, D.; Alexandre, J.; Huang, P. Targeting cancer
cells by ROS-mediated mechanisms: a radical therapeutic approach?
Nat. Rev. Drug Discovery 2009, 8, 579−591.
(22) Fang, J.; Seki, T.; Maeda, H. Therapeutic strategies by
modulating oxygen stress in cancer and inflammation. Adv. Drug
Delivery Rev. 2009, 61, 290−302.
(29) Kuang, Y.; Balakrishnan, K.; Gandhi, V.; Peng, X. Hydrogen
peroxide inducible DNA cross-linking agents: targeted anticancer
prodrugs. J. Am. Chem. Soc. 2011, 133, 19278−19281.
(30) Dharmaraja, A. T.; Ravikumar, G.; Chakrapani, H.
Arylboronate ester based diazeniumdiolates (BORO/NO), a class
of hydrogen peroxide inducible nitric oxide (NO) donors. Org. Lett.
2014, 16, 2610−2613.
(31) Ravikumar, G.; Bagheri, M.; Saini, D. K.; Chakrapani, H. A
small molecule for theraNOstic targeting of cancer cells. Chem.
Commun. 2017, 53, 13352−13355.
(32) Marzenell, P.; Hagen, H.; Sellner, L.; Zenz, T.; Grinyte, R.;
Pavlov, V.; Daum, S.; Mokhir, A. Aminoferrocene-based prodrugs and
their effects on human normal and cancer cells as well as bacterial
cells. J. Med. Chem. 2013, 56, 6935−6944.
(33) Hagen, H.; Marzenell, P.; Jentzsch, E.; Wenz, F.; Veldwijk, M.
R.; Mokhir, A. Aminoferrocene-based prodrugs activated by reactive
oxygen species. J. Med. Chem. 2012, 55, 924−934.
(34) Daum, S.; Chekhun, V. F.; Todor, I. N.; Lukianova, N. Y.;
Shvets, Y. V.; Sellner, L.; Putzker, K.; Lewis, J.; Zenz, T.; de Graaf, I.
A.; Groothuis, G. M.; Casini, A.; Zozulia, O.; Hampel, F.; Mokhir, A.
Improved synthesis of N-benzylaminoferrocene-based prodrugs and
evaluation of their toxicity and antileukemic activity. J. Med. Chem.
2015, 58, 2015−2024.
(35) Major Jourden, J. L.; Cohen, S. M. Hydrogen peroxide
activated matrix metalloproteinase inhibitors: a prodrug approach.
Angew. Chem., Int. Ed. 2010, 49, 6795−6797.
(36) Degner, A.; Carlsson, H.; Karlsson, I.; Eriksson, J.; Pujari, S. S.;
̈
Tretyakova, N. Y.; Tornqvist, M. Discovery of novel N-(4-
Hydroxybenzyl)valine hemoglobin adducts in human blood. Chem.
Res. Toxicol. 2018, DOI: 10.1021/acs.chemrestox.8b00173.
(37) Nishino, S.; Itoh, A.; Matsuoka, H.; Maeda, K.; Kamoshida, S.
Immunohistochemical analysis of organic anion transporter 2 and
reduced folate carrier 1 in colorectal cancer: Significance as a
predictor of response to oral uracil/ftorafur plus leucovorin
chemotherapy. Mol. Clin. Oncol. 2013, 1, 661−667.
(38) Podhorecka, M.; Skladanowski, A.; Bozko, P. H2AX
Phosphorylation: its role in DNA damage response and cancer
therapy. J. Nucleic Acids 2010, 2010, 920161.
(39) Chen, W.; Fan, H.; Balakrishnan, K.; Wang, Y.; Sun, H.; Fan,
Y.; Gandhi, V.; Arnold, L. A.; Peng, X. Discovery and optimization of
novel hydrogen peroxide activated aromatic nitrogen mustard
derivatives as highly potent anticancer agents. J. Med. Chem. 2018,
61, 9132−9145.
(40) Strasser, A.; Cory, S.; Adams, J. M. Deciphering the rules of
programmed cell death to improve therapy of cancer and other
diseases. EMBO J. 2011, 30, 3667−3683.
(41) Malanga, M.; Althaus, F. R. The role of poly(ADP-ribose) in
the DNA damage signaling network. Biochem. Cell Biol. 2005, 83,
354−364.
(23) Brown, J. M.; Wilson, W. R. Exploiting tumour hypoxia in
cancer treatment. Nat. Rev. Cancer 2004, 4, 437−447.
(24) Peng, X.; Gandhi, V. ROS-activated anticancer prodrugs: a new
strategy for tumor-specific damage. Ther. Delivery 2012, 3, 823−833.
(25) Liao, Y.; Xu, L.; Ou, S.; Edwards, H.; Luedtke, D.; Ge, Y.; Qin,
Z. H2O2/Peroxynitrite-Activated Hydroxamic Acid HDAC Inhibitor
Prodrugs Show Antileukemic Activities against AML Cells. ACS Med.
Chem. Lett. 2018, 9, 635−640.
(26) Peiro Cadahia, J.; Bondebjerg, J.; Hansen, C. A.; Previtali, V.;
Hansen, A. E.; Andresen, T. L.; Clausen, M. H. Synthesis and
evaluation of hydrogen peroxide sensitive prodrugs of methotrexate
and aminopterin for the treatment of rheumatoid arthritis. J. Med.
Chem. 2018, 61, 3503−3515.
(27) Kim, E. J.; Bhuniya, S.; Lee, H.; Kim, H. M.; Cheong, C.; Maiti,
S.; Hong, K. S.; Kim, J. S. An activatable prodrug for the treatment of
metastatic tumors. J. Am. Chem. Soc. 2014, 136, 13888−13894.
(28) Chen, W.; Balakrishnan, K.; Kuang, Y.; Han, Y.; Fu, M.;
Gandhi, V.; Peng, X. Reactive oxygen species (ROS) inducible DNA
E
DOI: 10.1021/acsmedchemlett.8b00539
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX