Floxuridine Oligomers Activated under Hypoxic Environment

Floxuridine Oligomers Activated under Hypoxic Environment

Article

Kunihiko Morihiro, Takuro Ishinabe, Masako Takatsu, Hiraki Osumi, Tsuyoshi Osawa,

and Akimitsu Okamoto *

Cite This: J. Am. Chem. Soc. 2021, 143, 3340 −3347

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sı Supporting Information

ABSTRACT: Floxuridine oligomers are anticancer oligonucleo-

tide drugs composed of a number of ﬂoxuridine residues. They

show enhanced cytotoxicity per ﬂoxuridine monomer because the

nuclease degradation of ﬂoxuridine oligomers directly releases

highly active ﬂoxuridine monophosphate in cells. However, their

clinical use is limited by the low selectivity against cancer cells. To

address this limitation, we herein report ﬂoxuridine oligomer

prodrugs that are active under hypoxia conditions, which is one of

the distinguishing features of the microenvironment of all solid

tumors. We designed and synthesized two types of ﬂoxuridine

oligomer prodrugs that possess hypoxia-responsive moieties on

nucleobases. The ﬂoxuridine oligomer prodrugs showed lower

cytotoxicity under normoxia conditions (O = 20%), while the parent ﬂoxuridine oligomer showed similar anticancer eﬀects under

hypoxia conditions (O = 1%). The ﬂoxuridine oligomer prodrug enabled tumor growth suppression in live mice. This would be the

ﬁrst example demonstrating the conditional control of the medicinal eﬃcacy of oligomerized nucleoside anticancer drugs.

INTRODUCTION

concentration is around 4%. Hence, hypoxia-mediated

activation of anticancer prodrugs would contribute to the

selective treatment of tumors and minimization of the adverse

events. Tanabe and co-workers demonstrated the hypoxia-

mediated activation of ﬂoxuridine prodrug bearing an

■

Chemotherapy is a fundamental treatment of cancer as well as

surgery and radiation therapy. Fluoropyrimidines are tradi-

tional antimetabolites that are widely applied for the

chemotherapy treatment of various solid tumors. The

indolequinone moiety, which is a substrate of reductases.

Nitro and azo groups are also known to be reduced by some

reductases in cells and work as hypoxia-sensitive moieties.

The one-electron reduction of nitro and azo groups generates

the radical anion, which can be reoxidized by oxygen under

normoxia conditions (Scheme 1A,B). Meanwhile, under

hypoxia conditions, the reoxidation is very slow and both

moieties are reduced to amino groups. These hypoxia-sensitive

moieties have been used to temporarily deactivate not only

compound 2′-deoxy-5-ﬂuorouridine (ﬂoxuridine) is a clinically

used ﬂuoropyrimidine drug that was approved by the US FDA

in 1970 under the brand name FUDR. Floxuridine undergoes

catabolism to form 5-ﬂuorouracil, mediated by DNA

glycosylases in the blood, and interferes with DNA and RNA

synthesis. Floxuridine is also intracellularly monophosphory-

lated at its 5′-hydroxyl group by thymidine kinase and causes

the inhibition of thymidylate synthetase, leading to thymineless

cell death. Although ﬂoxuridine and its derivatives have

eﬀective anticancer activity, the systemic adverse eﬀects,

including bone marrow suppression, diarrhea, and stomach

anticancer agents but also tumor imaging probes. In this

study, we install azo and/or nitro functionality on the

nucleobase of ﬂoxuridine and realize the hypoxia-selective

inhibition of cancer cell proliferation. Further application

includes the hypoxia-induced activation of ﬂoxuridine

oligomers. This approach represents the ﬁrst time oligomerized

nucleoside anticancer drugs have been conditionally controlled

in vitro and in vivo.

pain, need to be reduced to prevent therapy discontinuation.

These issues stem from their low tumor selectivity; hence,

designing an anticancer agent that is selective for killing tumor

cells would enable signiﬁcant progress to be made in cancer

chemotherapy.

To improve the tumor selectivity of anticancer agents,

prodrug strategies have been successfully applied. Prodrugs

are masked derivatives of drug molecules that undergo

enzymatic or chemical transformations to release the active

parent drugs, which can exert the therapeutic activity. The

cancer-speciﬁc microenvironment can be used as a trigger for

selective prodrug activation, and hypoxia is a distinguishing

feature of solid tumors wherein the median oxygen

Received: October 9, 2020

Published: March 2, 2021

2021 American Chemical Society

J. Am. Chem. Soc. 2021, 143, 3340−3347

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Article

Scheme 1. Reduction Mechanisms of (A) Nitro and (B) Azo

Functionality and (C) Hypoxia-Activated Floxuridine

Prodrugs

Scheme 2. Synthesis of Floxuridine Prodrugs

Reagents and conditions: (a) 1,2,4-triazole, POCl , Et N, 0 °C to rt,

6 h, 88%; (b) 4-nitrobenzyl alcohol, DBU, MeCN, rt, 1.5 h, 92%

3); 4-[(4-dimethylaminophenyl)azo]benzyl alcohol, DBU, MeCN, 2

h, 78% (4); 4-[(4-nitrophenyl)azo]benzyl alcohol, DBU, MeCN, rt,

.5 h, 24% (5); 4-[2-(4-dimethylaminophenyl)ethynyl]benzyl alcohol,

(

DBU, MeCN, rt, 3 h, 61% (6); (c) TBAF, THF, rt, 8 h, 86% (7);

TBAF, THF, rt, 2 h, quant. (8); TBAF, THF, rt, 4 h, 65% (9); TBAF,

THF, rt, 4 h, 89% (10).

11−13

reducing agent (Figure S1).

Due to the excess amount of

Na S O , all caged monomers were reduced to ﬂoxuridine

within 1 h, indicating that the designed compounds have

excellent sensitivity toward reducing environments. We

conﬁrmed that FdU , which has a CC bond instead of a

Azo

NN bond in FdU , did not react with Na S O . In

contrast, ﬂoxuridine prodrugs did not react with biologically

relevant reducing agent glutathione (GSH) and under weakly

acidic conditions (pH 5.5), which are other unique character-

istics of the microenvironment of tumors (Figure S1 ),

indicating that the decaging reaction of nitro and azo

functionality is hypoxia-selective. To further study the

RESULTS AND DISCUSSION

NO2

Azo

■

bioreduction of ﬂoxuridine prodrugs, FdU , FdU , and

FdU were incubated with rat liver microsomes and NADPH

We designed three types of caged ﬂoxuridine monomers,

NO2

Azo

FdU , FdU , and FdU , bearing a nitrobenzyl group, 4-

at 37 °C in argon-bubbled media (in vitro conditions that

14−20

(

dimethylamino)azobenzyl group, and 4-nitroazobenzyl group,

mimic those of hypoxia).

As shown in Figure 1A, the

Azo

respectively (Scheme 1C). These hypoxia-sensitive moieties

were installed on the 4-oxygen atom of ﬂoxuridine because the

hydrogen-bonding interactions between thymidine kinase with

the carbonyl oxygen in the 4-position are crucial for 5′-

amount of FdU

and FdU

decreased under hypoxia-

simulating conditions, whereas no consumption was observed

NO2

in argon-unbubbled media. In contrast, FdU

was not

reduced under either normoxic- or hypoxia-mimicking

NO2

Azo

NO2

phosphorylation. FdU

and FdU are directly reduced to

conditions, indicating that FdU

bioreduction than FdU

is more tolerant toward

Azo

the hydroxyl amine or aniline intermediate, and a subsequent

a two-phased reduction; the nitro group should be reduced

ﬁrst because the 4-nitroazobenzyl moiety is not a substrate of

azoreductases. After the reduction of the nitro group, the azo

moiety of FdU is reduced to give the aniline intermediate.

We can conclude that FdU , FdU , and FdU require at

and FdU . Although rat liver

,6-elimination gives ﬂoxuridine. In contrast, FdU requires

microsomes do not completely reproduce the reductive

environment in human cancer cells, the slow activation of 4-

nitrobenzyl-modiﬁed ﬂuorescent proven by them was

reported. It is suggested that 4-nitrobenzyl moiety in small

molecules is not a good substrate of rat liver microsomes.

NO2

Azo

NO2

However, we cannot conclude that FdU

does not work as a

most 6, 4, and 10 electrons, respectively, for the complete

bioreduction to release ﬂoxuridine. FdU was considered a

hypoxia-prodrug because there are many types of reductases in

human cells such as methionine synthase reductase,

hypoxia-insensitive compound because of its CC bond

NADPH-dependent diﬂavin oxidoreductase 1, and nitric

instead of azo functionality.

oxide synthase, and their intracellular concentrations and

localizations are complicated.

The synthetic details for each ﬂoxuridine prodrug are

summarized in Scheme 2. In short, hypoxia-responsive and

Hypoxic conditions make cancer cells more tolerant toward

anticancer drugs by slowing the cell proliferation rate and

inhibiting the cell death induced by p53. Moreover, the α-

subunit of hypoxia-inducible factor-1 (HIF-1α) protects cancer

unresponsive moieties were introduced by S Ar reactions

between O4-triazolyl-ﬂoxuridine analogue 2 and the

corresponding benzyl alcohol. With the caged ﬂoxuridine

monomers 7−10 in hand, we used high-performance liquid

chromatography (HPLC) to measure the reactivity of each

ﬂoxuridine prodrug against sodium dithionite (Na S O ) as a

cells from drug-induced senescence. Hence, we ﬁrst checked

the sensitivity of various human cancer cell lines against

ﬂoxuridine under hypoxic conditions using a cell viability assay

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normalized relative to ﬂoxuridine. FdU^N^O², FdU^A^z^o, and

FdU showed much less cytotoxicity than ﬂoxuridine under

normoxia conditions (3.8, 3.2, and 3.2 times, respectively),

while they showed a similar cell viability as that of ﬂoxuridine

under hypoxia conditions (1.6, 1.3, and 1.1 times, respec-

tively). Together, the anticancer activity of our designed

ﬂoxuridine prodrugs with nitro and/or azo functionality was

turned on through hypoxia-mediated deprotection in human

cancer cells. We conﬁrmed that a negligible hypoxia activation

of prodrugs was observed in HCT116 cells as expected (Figure

S4).

Recently, ﬂoxuridine has been employed to construct

ﬂoxuridine oligomers, which release highly cytotoxic ﬂoxur-

idine monophosphates and inhibit cell proliferation. Floxur-

idine oligomers show a greater cytotoxicity to human cancer

cells per residue than monomeric ﬂoxuridine. The enhance-

ment in the cytotoxicity of ﬂoxuridine oligomers is attributed

to a more eﬃcient metabolism to the monophosphate than the

ﬂoxuridine monomer. Their eﬀective delivery to tumors has

28,29

been achieved using programmable DNA self-assembly,

and a recent application has demonstrated the use of

combinatorial chemo and gene therapy to treat drug-resistant

0,31

cancers.

The results of cell viability assay of caged

NO2

ﬂoxuridine monomers prompted us to employ FdU

Azo

FdU , and FdU to construct hypoxia-activated ﬂoxuridine

oligomers. An oligomeric ﬂoxuridine of 6-mer length (F6) was

chosen as the active form of the prodrugs because of its

synthetic simplicity and higher cytotoxicity per residue than

monomeric ﬂoxuridine. Moreover, better pharmacokinetics

in the body can be expected with shorter oligomers.

We synthesized hypoxia-activated F6 using an automated

DNA synthesizer by a standard phosphoramidite method

Figure 1. Hypoxia-induced activation of ﬂoxuridine monomer

prodrugs. (A) Reduction of ﬂoxuridine monomer prodrugs by rat

microsomes/NADPH for 1 h in argon-bubbled media. (B) Eﬀects of

ﬂoxuridine monomer prodrugs (1 μM) on the viability of A549 cells

under normoxia (O = 20%) and hypoxia conditions (O = 1%). Six

independent experiments were averaged, and the error bars represent

standard deviations. * p ≤ 0.05, ** p ≤ 0.01, and *** p ≤ 0.001 by an

unpaired Student’s t test.

(

Figure S5 ). The synthetic details of each modi ﬁ ed

phosphoramidite are described in the Supporting Information.

Caged ﬂoxuridine nucleotides were introduced into both the

5′- and 3′-ends to protect F6 from exonucleases, and two

nucleotides at the center position were also replaced for

Azo

protection from endonucleases. Unfortunately, FdU decom-

posed under the DNA synthesis conditions; hence, we

NO2

Figure S2 ). Floxuridine was added to human lung cancer

obtained F6

and F6 , which have four FdU

and

NO2

A549 cells, cervical cancer HeLa cells, ﬁbrosarcoma HT1080,

and colon cancer HCT116 and SW480 cells and incubated

under normoxia (O = 20%) or hypoxia (O = 1%) conditions

FdU nucleotides in the sequence, respectively (Scheme 3).

and F6 have nuclease resistance because the presence

of unnatural nucleobases hampers nuclease access to the

at 37 °C for 72 h using a hypoxic culture kit. Among the cancer

cell lines we tested, A549 cells were the most sensitive to

ﬂoxuridine under hypoxia, and therefore, we decided to use

them in subsequent in vitro and in vivo experiments.

phosphodiester backbone, and these compounds show no

cytotoxicity under normoxia conditions. In contrast, after

reduction to the parent F6 under hypoxia conditions, multiple

highly cytotoxic ﬂoxuridine monophosphates are released by

nuclease degradation, inducing eﬀective anticancer activity.

To conﬁrm the fate of the modiﬁed ﬂoxuridine oligomers,

Azo

NO2

FdU , FdU , and FdU

were added to A549 cells at 1

μM, and cell viability assays were conducted (Figure 1B).

Importantly, ﬂoxuridine was less cytotoxic under hypoxia (O₂

NO2

F6, F6 , and F6 were incubated at 37 °C in 10% fetal

bovine serum (FBS), a blood product known to contain a

variety of nucleases, and the mixtures were analyzed at several

incubation time points by HPLC. Unmodiﬁed F6 was mostly

1%) than under normoxia (O = 20%) conditions, while all

the prodrugs showed a higher cytotoxicity under hypoxia than

under normoxia conditions. The smaller hypoxia-induced

NO2

Azo

NO2

activation of FdU

compared with FdU

and FdU

degraded within 1 h, while almost all F6

and F6

may be explained by the in vitro experimental results that

showed that FdU^N^O²was more resistant to bioreductive

conditions (Figure 1A). We conﬁrmed that negative control

remained (Figure 2A). F6 showed a longer half-life than

F6 , probably because the larger caging groups more

NO2

eﬀectively prevented nuclease access to the phosphodiester

FdU did not show cytotoxicity under either normoxic or

backbone. We detected intact F6

even after 48 h of

NO2

hypoxic conditions because the CC bond in FdU is

incubation. The half-lives of F6, F6 , and F6 in 10% FBS

were calculated to be 0.6, 3.4, and 15.3 h, respectively. This

indicates that caging groups can enhance F6 retention in

nonbiodegradable. The concentration-dependent anticancer

activity of each prodrug was also examined (Figure S3 ). To

evaluate the hypoxia selectivity of each ﬂoxuridine prodrug

more clearly, the cell viability at 1 μM drug concentration was

NO2

blood. Both F6

and F6 showed a rapid response to

Na S O and generated parent F6 in accordance with the

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Scheme 3. Chemical Structure and Hypoxia-Mediated

Activation of Caged Floxuridine Oligomers F6^N^O²and F6^N^A

reductive property of the corresponding nucleoside (Figure

NO2

S6). Overall, caged ﬂoxuridine oligomers F6

and F6 have

a high nuclease resistance and would not exert their

cytotoxicity until hypoxia-mediated bioreduction occurs.

To evaluatethe hypoxia-induced activation of ﬂoxuridine

Figure 2. Properties of ﬂoxuridine oligomer prodrugs. (A) Nuclease

degradation of ﬂoxuridine oligomer F6 and its prodrugs in 10% FBS

within 48 h incubation time course. (B) Eﬀects of ﬂoxuridine

oligomer prodrugs (0.1 μM) on the viability of A549 cells under

NO2

oligomers in living cells, F6, F6 , and F6 were incubated

with A549 cells under normoxia (O = 20%) or hypoxia (O =

normoxia (O = 20%) and hypoxia conditions (O = 1%). Six

NO2

independent experiments were averaged, and the error bars represent

standard deviations. * p ≤ 0.05 and ** p ≤ 0.01 by an unpaired

Student’s t test.

%) conditions and cell viability assays were conducted. F6

and F6 showed a much lower cytotoxicity than F6 at 0.1 μM

concentrations, indicating that the caging groups eﬀectively

masked the anticancer ability of the ﬂoxuridine oligomer F6

under normoxia conditions (Figure 2B). In contrast, in hypoxia

and F6 both showed anticancer eﬀects

similar to that of F6. A better activation of F6

To investigate whether our hypoxia-activated ﬂoxuridine is

eﬀective in vivo, we administered ﬂoxuridine oligomer

prodrugs in A549-tumor-bearing BALB/cAJcl nude mice;

solid tumors consist of hypoxic tumor microenvironments,

which, in turn, were expected to activate the hypoxia-activated

ﬂoxuridine within the tumor in vivo. We evaluated the

NO2

environments, F6

NO2

than its

monomeric form might be explained by the previous study

showing that nitro groups in oligonucleotides are eﬃciently

33−35

reduced by reductases.

Again, importantly, the parent F6

was less cytotoxic under hypoxia than under normoxia

therapeutic potential of F6 , which was the most potent F6

NO2

conditions, while the cytotoxicities of F6

and F6 were

prodrug in vitro and in the cell viability experiments. A549 cells

NO2

the opposite. The clear recovery in the drug eﬃcacy of F6

and F6 validates our design of hypoxia-responsive groups

were implanted in BALB/cAJcl nude mice (n = 6 in each

group) and grown until tumor sizes were around 100 mm

that are reduced by reductases and generate the active form of

before drug treatment. PBS, F6, or F6 was then intra-

F6 only in hypoxic cells. Indeed, F6^N^O²and F6 were 3.9 and

venously administered (on days 0, 3, and 6). The changes in

the tumor growth and the body weight were recorded during

.3 times less toxic than F6 under normoxia conditions,

respectively. Meanwhile, the diﬀerence in anticancer activity

between F6 and both F6 and F6

the treatment. As shown in Figure 3A−C, F6 was found to

NO2

was negligible under

eﬃciently inhibit the tumor growth at the same levels as F6,

hypoxia conditions (2.0 and 1.3 times less toxic than F6,

respectively). The similar therapeutic eﬀects as ﬂoxuridine

monomer prodrugs were observed even if the drug

concentrations were 1/10, proving the high therapeutic eﬃcacy

of oligomerized ﬂoxuridine prodrugs. The concentration-

dependent anticancer activities of both oligomer prodrugs

were conﬁrmed (Figure S7 ). The above results demonstrate

the ability to conditionally activate a highly toxic ﬂoxuridine

oligomer on the basis of a nucleobase caging strategy.

approximately 2.5-fold reduction in tumor growth, suggesting

that F6 recognized the hypoxic tumor microenvironment

and was activated within the region. We also want to

emphasize the reliability of F6 eﬃcacy because there was

little variance in tumor size of F6 -treated mice compared

with those of F6-treated mice (Figure 3A,C). The changes of

body weight in the F6 -treated group were similar to those of

the PBS control group (Figure S8 ), suggesting the F6 has

minor toxicity to the mice. We also measured the alanine

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functionalities were reduced by intracellular reductases into

amino groups, generating highly cytotoxic ﬂoxuridine oligomer

in situ. We also evaluated the anticancer activity of the

ﬂoxuridine oligomer prodrug in vivo. Collectively, the results

imply that our approach has great potential for an eﬃcient

targeted therapy of tumors.

EXPERIMENTAL SECTION

■

General. All chemicals were purchased from Sigma-Aldrich, Wako

Chemicals, Tokyo Chemical Industry, and Glen Research. Reagents

were used as received from commercial suppliers unless otherwise

speciﬁed. Air- and/or moisture-sensitive experiments were carried out

under a N or Ar atmosphere. TLC was run on silica gel 60 F

254

aluminum sheets (Merck). Column chromatography was carried out

with silica gel (Kanto Chemical; Silica Gel 60 (spherical), 63-210 μm)

or reverse phase silica gel (Merck; LiChroprep RP-18, 40−63 μm).

H, C, and P NMR spectra were measured with an Avance 600

(

Bruker Biospin), operating at 600, 150, and 240 MHz, respectively.

Chemical shifts are reported in parts per million referenced to internal

tetramethylsilane (0.00), residual CHCl (7.26), methanol (3.31), or

DMSO-d (2.50) for H NMR and chloroform-d (77.16), methanol-

d₄(49.00), or DMSO-d₆(39.52) for C NMR. Electrospray

ionization mass (ESI-MS) spectrometry was recorded by a microTOF

II-NAC (Bruker Daltonics). Matrix-assisted laser desorption ioniza-

tion time-of-ﬂight (MALDI-TOF) mass spectrometry was performed

by a microﬂex-NAC (Bruker Daltonics). Modiﬁed oligonucleotides

were synthesized on an NTS H-8 DNA/RNA synthesizer (Nihon

Techno Service) and then puriﬁed by a HPLC system composed by

GILSON and JASCO modules. The pH was measured by LAQUA F-

Figure 3. In vivo evaluation of F6^N^Aas an anticancer agent. (A)

Tumor volumes of each group (n = 6). (B) Representative images of

mice on day 21. (C) Photograph of tumors dissected from nude mice

on day 21. Error bars represent standard errors. * p ≤ 0.05 and ** p ≤

2 (Horiba).

0.01 by an unpaired Student’s t test.

Synthesis of 2. To a solution of 1,2,4-triazole (4.33 g, 62.7 mmol)

in MeCN (150 mL) were added POCl (2.5 mL, 26.8 mmol) and

Et N (10.2 mL, 73.2 mmol), and the mixture was stirred for 1 h in an

aminotransaminase (ALT) levels to evaluate the detailed

ice bath. Then, compound 1 (1.87 g, 3.95 mmol) in MeCN (25

mL) was added, and the mixture was stirred for 16 h at room

temperature. The reaction was quenched with saturated aqueous

adverse eﬀects because some synthetic oligonucleotides are

known to have severe hepatotoxicity. F6 did not result in

elevated ALT levels compared to a positive control (Figure

NaHCO and evaporated. The mixture was extracted with DCM and

S9 ), which means the adverse eﬀect of F6 is very low.

Taken together, these results indicate that F6

applied to silica gel column (hexanes/EtOAc = 3/4 (v/v)) to yield

is a

compound 2 as a white solid (1.83 g, 88%). H NMR (600 MHz,

promising anticancer prodrug targeting hypoxic tumor micro-

environments. Our approach enables the speciﬁc activation of

oligomerized nucleoside drugs, which has attracted great

attention as a new antitumor drug platform because of their

higher therapeutic eﬃcacy. Furthermore, we can propose a

new hypoxia-responsive 4-nitroazobenzyl group, which shows

excellent bioreductive properties compared to traditional

caging groups. This can be applied to develop hypoxia-

sensitive prodrugs based on not only nucleoside molecules but

also other modalities including peptides. For developing future

drug therapies, the molecular design of the oligomerized

prodrugs can be adapted to other nucleic acid therapeutics,

including antisense oligonucleotides (ASOs), small interfering

CDCl ): δ 9.25 (1H, s), 8.85 (1H, d, J = 6 Hz), 8.22 (1H, s), 6.22

(1H, s), 4.42 (1H, dd, J = 12 and 6 Hz), 4.06−4.02 (2H, m), 3.82

(

1H, d, J = 12 Hz), 2.63−2.58 (1H, m), 2.25−2.21 (1H, m), 0.94

9H, s), 0.89 (9H, s), 0.16−0.07 (12H, m). C NMR (151 MHz,

CDCl ): δ 154.8, 154.6, 151.7, 148.9, 148.8, 145.4, 145.2, 144.3,

36.9, 135.2, 88.3, 88.2, 87.8, 87.7, 87.3, 69.8, 69.7, 61.7, 42.4, 25.9,

5.7, 18.5, 17.9, −4.1, −4.5, −4.9. ESI-MS calculated for

C H FN O Si m/z [M + Na] : 548.2501; found 548.2504.

Synthesis of 3. To a solution of compound 2 (320 mg, 0.61

mmol) and 4-nitrobenzyl alcohol (100 mg, 0.65 mmol) in THF (6.2

mL) was added DBU (0.14 mL, 0.938 mmol), and the mixture was

stirred at room temperature for 1.5 h. The mixture was evaporated

and extracted with DCM and applied to silica gel column (hexanes/

EtOAc = 2/1 (v/v)) to yield compound 3 as a yellow solid (595 mg,

92%). H NMR (600 MHz, CDCl ): δ 8.35 (1H, d, J = 6 Hz), 7.59

RNAs (siRNAs), and aptamers. On the contrary, the tumor

suppression eﬀect of our prodrug in mice was not so dramatic.

Thus, our future work will include the optimization of the

length of ﬂoxuridine oligomers and the pattern of nucleobase

protections.

(

2H, d, J = 6 Hz), 6.18 (1H, s), 5.55 (2H, s), 4.36 (1H, d, J = 6 Hz),

.96−3.91 (2H, m), 3.76 (1H, d, J = 12 Hz), 2.47−2.43 (1H, m),

.11−2.07 (1H, m), 0.89−0.84 (18H, s), 0.10−0.03 (12H, s).

NMR (151 MHz, DMSO-d ): δ 161.9, 161.8, 153.4, 148.2, 142.6,

137.4, 135.8, 129.2, 128.6, 124.2, 124.1, 88.3, 87.5, 87.2, 87.1, 86.7,

0.6, 70.5, 67.9, 62.3, 26.3, 26.1, 18.8, 18.3, −3.8, −4.2, −4.6, −5.0.

CONCLUSIONS

ESI-MS calculated for C H FN O Si m/z [M + Na] : 632.2600;

found 632.2609.

■

28 44 3 7 2

We have developed an oligomerized ﬂoxuridine that is

activated in hypoxia environments by the installation of nitro

and azo functionalities onto the 5-ﬂuorouracil nucleobases.

The introduction of hypoxia-responsive groups abolished the

cytotoxicity of ﬂoxuridine oligomer in normoxia conditions

O = 20%) through the enhancement of nuclease resistance.

In contrast, under hypoxia conditions (O = 1%), nitro and azo

Synthesis of 4. To a solution of compound 2 (736 mg, 1.03

mmol) and 4-[(4-dimethylaminophenyl)azo]benzyl alcohol (446 mg,

.75 mmol) in MeCN (14 mL) was added DBU (0.46 mL, 3.08

mmol), and the mixture was stirred in ice bath for 5 min and then

room temperature for overnight . The mixture was evaporated and

extracted with DCM and applied to silica gel column (hexanes/

EtOAc = 3/2 (v/v)) to yield compound 4 as an orange solid (780 mg,

(

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(

8%). H NMR (600 MHz, CDCl ): δ 8.32 (1H, d, J = 6 Hz), 7.88

4.29 (1H, s), 3.89 (1H, s), 3.71 (2H, d, J = 12 Hz), 3.06 (6H, s), 2.31

(1H, t, J = 6 Hz), 2.12 (1H, dd, J = 12 and 6 Hz). ¹C NMR (151

2H, d, J = 12 Hz), 7.85 (2H, d, J = 6 Hz), 7.56 (2H, d, J = 6 Hz),

.76 (2H, d, J = 12 Hz), 6.25 (1H, s), 5.57 (1H, d, J = 12 Hz), 5.52

1H, d, J = 12 Hz), 4.40 (1H, d, J = 6 Hz), 3.98 (1H, d, J = 12 Hz),

.94 (1H, s), 3.79 (1H, d, J = 12 Hz), 3.10 (6H, s), 2.50−2.47 (1H,

m), 2.17−2.12 (1H, m), 0.92 (9H, s), 0.88 (9H, s), 0.13−0.06 (12H,

MHz, DMSO-d ): δ 61.1, 161.0, 152.6, 152.4, 142.6, 136.6, 134.9,

129.9, 129.7, 129.3, 129.0, 128.9, 128.8, 125.5, 125.4, 125.0, 124.9,

124.6, 124.4, 122.5, 122.4, 122.1, 121.6, 121.5, 111.6, 87.8, 86.7, 85.7,

69,6, 69.5, 68.3. ESI-MS calculated for C H FN O m/z [M + Na] :

s). C NMR (151 MHz, CDCl ): δ 161.9, 161.8, 153.2, 153.1, 152.4,

506.1810; found 506.1839.

43.4, 137.2, 135.7, 135.6, 129.6, 129.4, 128.7, 128.5, 128.1, 127.9,

25.5, 125.4, 124.7, 124.5, 122.8, 122.7, 121.8, 111.32, 87.7(3),

7.6(6), 86.0, 70.0, 69.9, 61.8, 40.6, 39.8, 26.1, 25.9, 25.8, 25.7, 25.5,

8.4, 17.9, −5.2, −5.3, −5.9. ESI-MS calculated for C H FN O Si

Synthesis of 9. To a solution of compound 5 (1.46 g, 2.05 mmol)

in THF (20 mL) was added AcOH (0.26 mL, 4.55 mmol) and TBAF

(1.0 M THF solution, 4.6 mL, 4.60 mmol), and the mixture was

stirred at room temperature for 4 h. The mixture was evaporated, and

m/z [M + Na] : 734.3540; found 734.3548.

H O was added to the resulting orange solid. The liquid was ﬁltered

Synthesis of 5. To a solution of compound 2 (1.50 g, 2.86 mmol)

and 4-[(4-nitrophenyl)azo]benzyl alcohol (868 mg, 3.38 mmol) in

THF (29 mL) was added DBU (0.65 mL, 4.35 mmol), and the

mixture was stirred at room temperature for 1.5 h. The mixture was

evaporated and extracted with DCM and applied to silica gel column

oﬀ, and then, the remaining solid was applied to a silica gel column

(MeOH/DCM= 1/20 (v/v)) to yield compound 9 as an orange solid

(645 mg, 65%). H NMR (600 MHz, DMSO-d ): δ 8.57 (1H, d, J = 6

Hz), 8.46 (2H, d, J = 6 Hz), 8.11 (2H, d, J = 12 Hz), 8.02 (2H, d, J =

6 Hz), 7.73 (2H, d, J = 6 Hz), 6.09 (1H, t, J = 6 Hz), 5.56 (2H, s),

5.29 (1H, d, J = 6 Hz), 5.22 (1H, t, J = 6 Hz), 4.25 (1H, s), 3.86 (1H,

d, J = 6 Hz), 3.69−3.67 (1H, m), 3.61−3.59 (1H, m), 2.29−2.25 (1H,

(

hexanes/EtOAc = 3/1 to 2/1 to 1/1 (v/v)) to yield compound 5 as

an orange solid (482 mg, 24%). H NMR (600 MHz, CDCl ): δ 8.31

(

2H, d, J = 6 Hz), 8.29 (1H, d, J = 6 Hz), 7.97 (2H, d, J = 6 Hz), 7.91

2H, d, J = 12 Hz), 7.57 (2H, d, J = 6 Hz), 6.19 (1H, d, J = 6 Hz),

m), 2.11−2.07 (1H, m). C NMR (151 MHz, DMSO-d ): δ 161.8,

161.7, 155.9, 153.1, 152.4, 149.4, 140.9, 137.2, 135.6, 130.5, 130.2,

129.5, 125.9, 124.4, 124.1, 123.8, 123.6, 88.7, 86.5, 70.4, 70.3, 68.6,

61.4. ESI-MS calculated for C H FN O m/z [M + Na] : 508.1239;

.53 (2H, t, J = 12 Hz), 4.34 (1H, t, J = 6 Hz), 3.93−3.88 (2H, m),

.73 (1H, d, J = 12 Hz), 2.45−2.41 (1H, m), 2.09−2.05 (1H, m),

.86 (9H, s), 0.82 (9H, s), 0.07−0.00 (12H, m). C NMR (151

found 508.1211.

MHz, CDCl ): δ 162.2, 162.1, 156.0, 153.6, 152.6, 149.2, 139.8,

Synthesis of 10. To a solution of compound 6 (53 mg, 74.7

μmol) in THF (1 mL) was added TBAF (1.0 M THF solution, 164

μL, 164 μmol), and the mixture was stirred at room temperature for 1

h. The mixture was evaporated and applied to a silica gel column

(MeOH/EtOAc = 1/18 (v/v) and MeOH/DCM = 1/184 (v/v)) to

37.6, 136.0, 129.9, 129.6, 129.1, 128.8, 128.6, 125.1, 124.3, 123.7,

8.3, 70.7, 70.6, 68.8, 62.4, 26.3, 26.1, 25.9, 18.8, 18.3, −4.8, −4.9,

5.4. ESI-MS calculated for C H FN O Si m/z [M + Na] :

Synthesis of 6. To a solution of compound 2 (228 mg, 0.380

yield compound 10 as a yellow solid (32 mg, 89%). H NMR (600

MHz, DMSO-d ): δ 8.36 (1H, d, J = 6 Hz), 7.47 (2H, d, J = 6 Hz),

7.42 (2H, d, J = 6 Hz), 7.26 (2H, d, J = 6 Hz), 7.09 (1H, d, J = 12

Hz), 6.94 (1H, d, J = 18 Hz), 6.71 (2H, d, J = 12 Hz), 6.19 (1H, t, J =

6 Hz), 5.30 (1H, s), 5.22 (1H, d, J = 6 H), 4.97 (2H, d, J = 6 H), 4.25

(1H, s), 3.80 (1H, s), 3.63−3.52 (2H, m), 2.92 (6H, s), 2.16 (2H, d, J

= 6 Hz). C NMR (151 MHz, DMSO-d ): δ 157.5, 157.3, 150.8,

149.9, 139.6, 138.0, 135.6, 129.4, 128.8, 127.9, 127.1, 126.1, 125.7,

113.1, 88.5, 70.8, 70.7. ESI-MS calculated for C H FN O m/z [M

+ H] : 482.2087; found 482.2089.

Hypoxia Activation with Rat Liver Microsomes in Vitro. The

hypoxic condition was prepared by bubbling argon gas into the

reaction solution (0.1 M potassium phosphate buﬀer, pH 7.4) for 30

min. Rat liver microsomes (140 μg/mL) purchased from BioIVT were

preincubated at 37 °C for 5 min, and then, a 1 μM prodrug containing

1% DMSO as a cosolvent was added. As a cofactor for reductases, 100

μM nicotinamide adenine dinucleotide phosphate (NADPH) was

added at 5 min.

found 732.3663.

Oligonucleotide Synthesis. All modiﬁed oligonucleotides were

synthesized using a standard DNA synthesis method. Each

phosphoramidite was dissolved in dry acetonitrile and ﬁltrated to

prepare a 0.1 M solution. Each phosphoramidite solution was reacted

on a DNA synthesizer with 0.25 or 0.5 M 5-ethylthio-1H-tetrazole

solution as an activator for 9 min. Glen UnySupport FC (Glen

Research) was used for the synthesis. For the deprotection of DMTr

protecting group, 3% DCA solution was used instead of 3% TCA

solution. For every sequence, a 5′-end DMTr protecting group was

eliminated on the synthesizer. Synthesized DNA was cleaved from the

support by 28% ammonium solution at room temperature for 5 min.

Immediately after the cleavage, the solution was puriﬁed through

NAP-10 or -5 columns (GE Healthcare) to eliminate ammonium

solution. All the products were puriﬁed by a reverse-phased HPLC.

Cell Viability Assay. The monolayer cell culture was trypsinized

and counted. To each well of the 96-well plate was 180 μL of the

diluted cell suspension (10 000 cells/mL). After 24 h, when a partial

monolayer was formed, the medium in each well was removed by

aspiration and 180 μL of fresh medium with diﬀerent compound

concentrations (0.001, 0.01, 0.1, or 1.0 μM) was added to each well.

Synthesis of 7. To a solution of compound 3 (2.22 g, 3.65 mmol)

in THF (37 mL) was added TBAF (1.0 M THF solution, 8.0 mL,

.00 mmol), and the mixture was stirred at room temperature for 8 h.

The mixture was evaporated, and H O was added. The liquid was

ﬁltered oﬀ, and the resultant solid was applied to silica gel column

(

MeOH/DCM= 22/1 (v/v)) to yield compound 7 as a white solid

1.20 g, 86%). H NMR (600 MHz, DMSO-d ): δ 8.68 (1H, d, J = 12

Hz), 8.38 (2H, d, J = 12 Hz), 7.84 (2H, d, J = 12 Hz), 6.18 (1H, t, J =

Hz), 5.68 (2H, s), 5.38 (1H, d, J = 6 Hz), 5.32 (1H, d, J = 6 Hz),

.35 (1H, s), 3.95 (1H, s), 3.79−3.76 (1H, m), 3.72−3.69 (1H, m),

.38−2.34 (1H, m), 2.20−2.16 (1H, m). C NMR (151 MHz,

DMSO-d ): δ 153.0, 148.1, 144.0, 135.6, 130.2, 129.2, 124.6, 88.7,

0.4, 70.3, 68.0. ESI-MS calculated for C H FN O m/z [M + Na] :

04.0870; found 404.0889.

Synthesis of 8. To a solution of compound 4 (780 mg, 1.61

22 20 5 7

mmol) in THF (11 mL) was added TBAF (1.0 M THF solution, 2.5

mL, 2.50 mmol), and the mixture was stirred at room temperature for

h. The mixture was evaporated and applied to a silica gel column

(MeOH/EtOAc = 1/18 (v/v)) to yield compound 8 as an orange

solid (596 mg, quant). H NMR (600 MHz, DMSO-d ): δ 8.60 (1H,

An hypoxia environment (O concentration of 1%) was generated

d, J = 6 Hz), 7.83 (4H, d, J = 6 Hz), 7.62 (2H, d, J = 12 Hz), 6.82

with an nBIONIX (Sugiyama-Gen). After 72 h, 20 μL of Prestoblue

(Invitrogen) was added to each well, and the plate was incubated for 1

(2H, d, J = 12 Hz), 6.13 (1H, s), 5.49 (2H, s), 5.35−5.29 (2H, m),

345

J. Am. Chem. Soc. 2021, 143, 3340−3347

Journal of the American Chemical Society

https://pubs.acs.org/10.1021/jacs.0c10732

Article

h. The ﬂuorescence of each well was measured spectrophotometrically

in a multiwell Cytation 5 plate reader (BioTek Instruments) at a

wavelength of 590 nm, excited by a wavelength of 560 nm. The

relative viability was calculated according to the following formula

with the ﬂuorescence intensity obtained:

Masako Takatsu − Research Center for Advanced Science and

Technology (RCAST), The University of Tokyo, Tokyo 153-

8904, Japan

Hiraki Osumi − Department of Chemistry and Biotechnology,

Graduate School of Engineering, The University of Tokyo,

Tokyo 113-8656, Japan

Tsuyoshi Osawa − Department of Chemistry and

Biotechnology, Graduate School of Engineering and Research

Center for Advanced Science and Technology (RCAST), The

University of Tokyo, Tokyo 113-8656, Japan

average (x μM) − average (DMEM)

relative cell viability (x μM) =

average (0 μM) − average (DMEM)

Nuclease Degradation Assay. In a 0.6 mL plastic tube, 72 μL of

0% FBS in 1 × DMEM (+Pe/St), 5 μL of 100 μM Rhodamine B

dissolved in MQ, and 3 μL of 10 μM F6 derivatives in MQ were

mixed. The mixture was reacted for several hours at 37 °C. The

reaction mixture was ﬁltered and analyzed by reverse phased HPLC.

The relative peak area of the F6 derivatives was calculated by the

dividing peak area of the F6 derivatives (260 nm) by that of

Rhodamine B (550 nm).

Complete contact information is available at:

Notes

The authors declare no competing ﬁnancial interest.

In Vivo Studies. F6 (10 nmol) and F6 (10 nmol) were

dissolved in 100 μL of PBS solution to a ﬁnal concentration of 100

μM. Seven week old BALB/cAJcl nude mice (purchased from CLEA)

were subcutaneously implanted with tumors by the injection of A549

cells. Nine nude mice were randomly assigned to three groups. After

ACKNOWLEDGMENTS

■

This work was supported by the Tokyo Biochemical Research

Foundation (TBRF) (to K.M.), the Japan Science and

Technology Agency (JST) ACT-X (JPMJAX191I to K.M.),

and the Japan Society for the Promotion of Science (JSPS)

KAKENHI (18H03931, 18H05504, and 19K22245 to A.O.).

The Table of Contents graphic was created with BioRender.-

com.

the implanted tumors were grown to about 100 mm , drug injections

(

intravenous) were performed over a period of 3 days, and the length

and width of the tumors were recorded. The tumor volume was

calculated as V = (a·b )/2, where V, a, and b indicate the tumor

volume, length of tumor, and width of tumor, respectively. The

experiments were repeated twice. Blood was collected from the

inferior vena cava following sacriﬁce and centrifuged for 20 min at

300g to obtain the serum. The ALT was measured using an alanine

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