Targeting Colorectal Cancer with Conjugates of a Glucose Transporter Inhibitor and 5-Fluorouracil

Transporter Inhibitor and 5 ‑Fluorouracil

Article

Targeting Colorectal Cancer with Conjugates of a Glucose

Chun-Kai Chang, Pei-Fang Chiu, Hui-Yi Yang, Yu-Pu Juang, Yen-Hsun Lai, Tzung-Sheng Lin,

Lih-Ching Hsu, Linda Chia-Hui Yu, and Pi-Hui Liang*

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sı Supporting Information

ABSTRACT: Overexpression of glucose transporters (GLUTs) in

colorectal cancer cells is associated with 5-ﬂuorouracil (1, 5-FU)

resistance and poor clinical outcomes. We designed and

synthesized a novel GLUT-targeting drug conjugate, triggered by

glutathione in the tumor microenvironment, that releases 5-FU

and GLUTs inhibitor (phlorizin (2) and phloretin (3)). Using an

orthotopic colorectal cancer mice model, we showed that the

conjugate exhibited better antitumor eﬃcacy than 5-FU, with

much lower exposure of 5-FU during treatment and without

signiﬁcant side eﬀects. Our study establishes a GLUT-targeting

theranostic incorporating a disulﬁde linker between the targeting

module and cytotoxic payload as a potential antitumor therapy.

INTRODUCTION

cells through pyruvate scavenging of free radicals involved in 5-

FU-induced cytotoxicity.

■

Colorectal cancer (CRC) is the fourth most common and

second most lethal cancer in the world, with 19.7 million cases

and almost 880,000 deaths in 2018. First line chemotherapy

consists of 5-ﬂuorouracil (1, 5-FU), in combination with other

reagents such as FOLFOX4 (leucovorin, 5-FU, and oxaliplatin),

FOLFIRI (leucovorin, 5-FU, and irinotecan), and FOLFOXIRI

Targeting of glucose transporters has attracted a great deal of

18,19

interest as an anticancer strategy in recent years.

Phloretin

(

3, Figure 1), a well-known and naturally abundant GLUT1

inhibitor, has been investigated for its anticancer activities and

demonstrated the ability to overcome therapeutic resistance of

anticancer agents when administered in combination with them

−4

(

5-FU, leucovorin, oxaliplatin, and irinotecan).

The 5-FU-

20−23

in preclinical studies.

Phlorizin (2, Figure 1) is a SGLT-1

based chemotherapy is eﬃcacious in patients with metastatic

CRC but poorly selective for malignant vs healthy cells, causing

marrow suppression and polyneuropathy, both major dose-

inhibitor that diﬀers from phloretin in that it bears an additional

glucose conjugate at the ortho-hydroxyl of a phenyl moiety.

Treatment of mice bearing tumor xenografts with SGLT

limiting toxicities. Chemotherapy resistance is also a signiﬁcant

inhibitors resulted in increased necrosis within the tumors.

Since these glucose transporters are physiologically expressed in

problem and the main reason for the high mortality rate of

25−27

CRC.

a wide array of cells and tissues,

selective targeting of

Cancer cells rely on anaerobic glycolysis pathways to a greater

SGLT-1 or GLUT1 on cancer cells would be necessary. One

extent than mitochondrial oxidative phosphorylation compared

to noncancer cells, the so-called Warburg eﬀect. There are two

possible approach is through a small molecule drug conjugation

28,29

(SMDC) strategy,

but to the best of our knowledge, SMDC

categories of glucose transporters that enable cancer cells to

using glucose transporter inhibitors as a targeting ligand has not

been attempted.

Such a strategy is predicated on the ability to selectively

release the conjugated drug in the tumor tissue. Given that the

concentration of GSH is about 10-fold higher in tumor cells

uptake glucose: (1) facilitative GLUT1 to GLUT4, and (2)

secondary active sodium-glucose cotransporters (SGLT-1 and

,10

SGLT-2).

Tumor cells also overexpress glycolysis enzymes

and glucose transporters, both of which were correlated with the

1−14

invasiveness and metastatic potential of cancers,

and

enhanced glucose uptake is known to diminish the cytotoxicity

Received: May 27, 2020

Published: April 5, 2021

15,16

of 5-FU in colorectal cancer.

We analyzed specimens from

CRC patients previously treated with 5-FU and found

signiﬁcantly higher transcript levels of SGLT-1and GLUT1 in

those refractory to the therapy. Overexpression of GLUTs is

known to be associated with 5-FU resistance in colon cancer

2021 American Chemical Society

Journal of Medicinal Chemistry

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J. Med. Chem. 2021, 64, 4450−4461

Article

as a base gave compound 15 (54%) as a major product. The

regioselective nature of this alkylation was presumably due to the

electro-withdrawing eﬀect of the carbonyl group at the para-

position of a benzyl group and the reduced steric hindrance at

this position. Deprotection of a t-butythiol group by 4.0 N

NaOH and TCEP gave phlorizin derivative 16 in 85% yield,

which was reﬂuxed with 1.0 N HCl at 90 °C for 3 h quenching

with NaHCO (aq) to give phloretin derivative 17 (73% yield).

Phlorizin- and phloretin azide-containing analogs 20 and 21

were similarly prepared. MsCl was added slowly to a solution of

triethylene glycol and triethylamine in DCM at 0 °C, with

stirring. After 20 min, the reaction mixture was concentrated and

the residue was dissolved in EtOH, to which was added NaN₃.

After reﬂuxing for 24 h, azide linker 18 was obtained. This was

mesylated, and then the mesyl group exchanged with bromine

using lithium bromide to provide compound 19, which was used

to derivatize phlorizin to give the desired intermediate 20. After

hydrolysis, phloretin derivative 21 was obtained.

N-1 alkyl-substituted 5-FU derivatives are poorly cytotoxic

and not converted to 5-FU in vivo. In anticipation of this

problem, ester functionality was designed into the linker; it was

hypothesized that this ester would be cleavable in the tumor

microenvironment by endogenous esterases. The reaction of 5-

FU with formaldehyde followed by 6-maleimidohexanoic acid,

-(pyridin-2-yldisulfanyl)-butanoic acid, or 4-pentynoic acid

and the coupling reagents DCC and DMAP gave compounds

2−24, respectively (Scheme 2). Thiol-containing phlorizin

derivative 16 and phloretin derivative 17 were conjugated with

maleimide of 5-FU (22) to give 4 and 5, respectively, and the

carboxylic acid of 23 to give 6 and 7, respectively. Azide-

containing phlorizin derivative 20 and phloretin derivative 21

were conjugated with alkyne-containing 5-FU (24) under the

(Cu(I)-catalyzed azide-alkyne cycloaddition) click condition to

give compounds 8 and 9.

Figure 1. Structures of 5-FU (1), phlorizin (2), and phloretin (3) and

proposed design of glucose transporter-targeting drug conjugates 4−9.

compared to the normal cells (except hepatocytes) and 10−100

0−32

times higher intracellularly,

we designed compounds 4−7,

wherein phloretin 3 (or phlorizin 2) are connected to 5-FU

through a redox-sensitive linker, as depicted in Figure 1. In

conjugates 4 and 5, a succinimidyl thioether (a moderately

Plasma Stability and Release Proﬁles in Diﬀerent GSH

Concentrations. The in vitro human plasma stability and

release proﬁles of compounds 5, 7, and 9 are depicted in Figure

2. The stability tests comprised incubation of compounds 5, 7,

and 9 in human plasma at 37 °C at diﬀerent time intervals 0, 1, 2,

4, 8, 12, 24, and 48 h. Samples were collected and analyzed by

RP-HPLC (Figure 2a for 7 and Figure S49 for 5 and 9). As

shown in Figure 2b, the half-lives of compounds 5, 7, and 9 were

0.8, 13, and 3.6 h, respectively. The reason for the inferior

plasma stability of compounds 5 and 9 compared to 7 is

unknown but can be attributed to the high pKa of the linkers

(the pKa values of both succinimide and triazole are around

8.5−9.5) increasing the susceptibility of the ester portion of the

sensitive linker) was used, whereas conjugates 6 and 7

incorporate a disulﬁde linker (a highly sensitive linker) instead.

We also designed compounds 8 and 9, incorporating triazole

conjugates as a stable linker-bearing group (Figure 1). The

stability of these conjugates in human plasma and in solutions

containing varying concentrations of GSH was studied as well as

their in vitro cytotoxicity across two tumor cell lines (HCT-116

and HT-29). The antitumor eﬀect of the compounds was

evaluated in the orthotopic CRC mice model, and pharmaco-

kinetic evaluation was performed to elucidate tumor-targeting

eﬃcacy.

linker to degradation. Therefore, pH stability analysis of

compound 9 was conducted, and it was found that compound 9

was stable at pH 4−5 but was prone to fully degrade when the

pH was ≥7. In order to predict the fate of those conjugates in the

body, we collected the metabolites of 7 from the plasma treating

assay and identiﬁed two major metabolites: metabolite 1, which

arises from hydrolysis of the ester bond of 7, and metabolite 2,

the disulﬁde exchange intermediate (Figure 2a and Figure S48).

Since the concentration of GSH is about 5−10 mM in the tumor

RESULTS AND DISCUSSION

■

Chemistry. Conjugates 4−9 were constructed by sequential

derivatization of the central linker (Scheme 1 , detailed synthesis

procedures can be found in the Supporting Information).

Addition of triethylene glycol and TsCl to ethanol/H O gave the

tosylated intermediate, to which was added potassium

thioacetate in acetone. After this mixture was heated at reﬂux

for 1 h, thiol ester 11 was obtained in 85% yield. Hydrolysis of

thiol ester 11 was accomplished using potassium carbonate

cells and 1−10 μM in plasma, we then evaluated the release of

5-FU from conjugates 5, 7, and 9 using GSH. The concentration

of GSH used was set to 5 mM, mimicking the tumor

microenvironment. Less 5-FU was released from compounds

5 and 9 after 48 h; however, the release of 5-FU from compound

7 was found to be dependent on the concentration of GSH used.

The cleavage percentage of compound 7 after 4 h exposure to 5

(

0.05 M) to give compound 12 in 85% yield, the free thiol of

which was protected using 2-methyl-2-propanethiol to give

compound 13 in 70% yield. After mesylation and bromide

substitution, brominated compound 14 was obtained in 57%

yield. Alkylation of phlorizin with 14 using potassium carbonate

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Scheme 1. Synthetic Route for Phlorizin and Phloretin Conjugates

mM GSH was up to 60% but only ∼10% in the presence of 1

mM GSH, indicating that compound 7 is cleaved much slower in

similar inhibition activity to phloretin, despite bearing

substitution at 4-OH.

mM GSH than 5 mM GSH (Figure 2d). The half-lives of

Cytotoxicity to Human CRC Cells. Compounds 4−9, 16−

17, and 5-FU were evaluated for their cytotoxicity against the

CRC cell lines HCT-116 and HT-29 (both of which overexpress

GLUT1, Figure S50) using the sulforhodamine B (SRB) assay

compound 7 at 5 and 1 mM GSH were 4.0 and 10.3 h,

respectively. In the presence of 5 μM GSH, only 30%

degradation of compound 7 was observed after 48 h (Figure

38−40

d). The stability and release proﬁles of three phlorizin-bearing

(Table 1).

5-FU exhibited an IC of approximately 15 μM

derivatives 4, 6, and 8 were also evaluated; their results were all

similar to their corresponding congeners 5, 7, and 9,

respectively. Disulﬁde bonds in compounds 6 and 7 have

relatively short half-lives (<5 h) in highly reductive environ-

ments while maintaining a degree of stability in circulation.

GLUT1 Inhibitory Activity. A previous crystallography

study established that the benzene-1,3,5-triol ring of phloretin is

in CRC cells (entry 1, Table 1). To mimic the tumor

microenvironment, 10 mM GSH was ﬁrst attempted to add to

the medium; however, we found that 10 mM GSH interfered

CRC cell growth. Thus, 5 mM GSH was added to the culture

media of CRC cell lines. Phlorizin derivative 16 did not exhibit

any inhibitory activity. Derivative 17 exhibited some inhibitory

activity, with an IC₅₀of 15−30 μM. Derivatives 4 and 5 were

poorly cytotoxic, with or without the addition of GSH. The

cytotoxicity of the disulﬁde-containing compounds 6 and 7 was

sensitive to GSH, for example, the IC of 6 to HCT-116 cells

stabilized in a pocket of GLUT1 by forming three H-bonds,

but the extent to which this would be perturbed by a substituent

at the 4-position (such as in our phloretin derivatives 17) was

unknown. We examined the inhibitory activity of 16 and 17

toward GLUT1 using COS-7 cells that overexpress GLUT1 in a

was 23.8 μM with GSH and 54.9 μM without GSH. These

cytotoxicites of 6 and 7 in these cancer cells might be due to the

sub-millimolar level of intracellular GSH that gradually cleaved

the disulﬁde bonds of 6 and 7 to release 5-FU. The IC of 7 in

-NBDG ((2-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-

deoxy-D-glucose) uptake assay. Since phlorizin could not

inhibit GLUT1, it was not surprising that compound 16 (a

phlorizin derivative) was not able to inhibit GLUT1 at either 50

or 100 μM (Figure 3). On the contrary, compound 17 had

the presence of GSH was 10.5 μM for HCT-116 and 3.8 μM for

HT-29, which were close to the IC values of 5-FU in both cell

lines (13.9 and 5.2 μM, respectively). Compounds 8 and 9 were

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Scheme 2. Synthetic Route for the Conjugates 4−9

Figure 2. In vitro plasma stability and release proﬁles of compounds under diﬀerent conditions. (a) Analysis of compound 7 with human plasma by

using RP-HPLC. (b) Stability of compounds 5, 7, and 9 in human plasma at 37 °C. (c) Analysis of compound 7 with GSH (5 mM) by using RP-HPLC.

(d) In vitro release proﬁle of compound 7 under diﬀerent GSH concentrations (5 μM, 1 mM, and 5 mM) and formations of 5-FU under 1 and 5 mM

GSH. Data are shown as mean ± S.E.M. (n = 3).

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analysis of GLUT1 in Figure S50 ) were also examined; it was

Article

found that both 5-FU and 7 had no obvious inhibition eﬀect

IC₅₀values of 70.4 ± 7.2 and 90.0 ± 2.6 μM, respectively),

(

suggesting that the conjugate 7 was less toxic in normal cells.

Evaluation of the In Vivo Activity of the Synthetic

Compounds in an Orthotopic CRC Mice Model. Com-

pound 7 was selected for in vivo evaluation based on its release

proﬁle and the results of the cytotoxicity experiments. An

orthotopic CRC mice model was established by intraperitoneal

(

i.p.) injection of AOM (10 mg/kg body weight) to BALB/c

mice (6 weeks, male) and supplementation of their drinking

water with 2% dextran sodium sulfate (DSS) (Figure 4a).

39,42

Figure 3. Inhibition of GLUT1 activity of compounds 16 and 17 by the

-NBDG uptake assay. Data represent mean ± S.E.M. (n = 4; n.s.,

After 2 months, solutions of 5-FU (50 mg/kg), compound 7 (50

mg/kg), and 5-FU + 17 (10 mg/kg of 5-FU and 30 mg/kg of 17,

dosing is based on compound 7) in 0.2 mL of PBS were injected

i.p. once every 3 days for 3 weeks. Based on calculations of tumor

volume, compound 7 showed better tumor suppression than 5-

FU at a dose of 50 mg/kg (0.07 mmol, when only 19% of 5-FU

was given (Figure 4b,c). Since GLUT1/3/4 is generally

nonsigniﬁcant diﬀerence; ****P < 0.0001 (two-way ANOVA)).

Table 1. Cytotoxicity of Compounds 4−9, 16−17, and 5-FU

in HCT-116 and HT-29 Cell Lines

HCT-116 (μM)

HT-29 (μM)

compounds

w/o GSH

5 mM GSH

w/o GSH

5 mM GSH

overexpressed in CRC patients and phloretin is known to

-FU

14.9 ± 2.24

>100

13.9 ± 0.60

>100

15.9 ± 0.89

>100

14.9 ± 1.86

>100

29.1 ± 6.14

>100

43.7 ± 5.0

59.8 ± 7.93

19.6 ± 3.86

>100

85 ± 9.1

21.0 ± 9.38

10.3 ± 4.33

5.20 ± 0.12

>100

43,44

inhibit GLUT1/3/4 inhibition,

compound 7 might display

targeting through GLUT1/3/4, leading to signiﬁcant tumor

inhibition. Mice treated with free 5-FU were noted to lose about

15.5 ± 0.72

97.4 ± 20.3

75.0 ± 13.7

54.9 ± 3.36

21.2 ± 1.69

>100

21.9 ± 2.98

10.4 ± 1.25

24.2 ± 0.40

93.6 ± 9.4

86.9 ± 2.9

8.00 ± 0.52

3.80 ± 0.65

0% of their body weight rapidly after injection (Figure 4d),

82.8 ± 4.18

23.8 ± 1.15

10.5 ± 1.15

presumably a reﬂection of the systemic toxicity of 5-FU; there

was no signiﬁcant change in the body weight of the mice who

received compound 7. The combination of 5-FU (with 19% of

free 5-FU dose) and compound 17 also reduced tumor size in

the mice model, exhibiting synergistic eﬀects, which was also

observed in the cell assay.

-FU + 16

-FU + 17

13.6 ± 0.73

8.73 ± 0.64

8.58 ± 0.09

3.03 ± 0.03

Evaluation of Pharmacokinetic and Biodistribution

Proﬁles. Plasma concentration and organ distribution of

compound 7 were evaluated in BALB/c mice (n = 3) with

single dose injection of compounds. To analyze the 5-FU release

proﬁle of compound 7, blood samples were collected at 1, 3, 5,

10, 15, 30, and 45 min and 1, 2, 4, and 8 h after intravenous

injection (i.v.) (50 mg/kg), and the concentration of compound

7 was determined with UPLC-MS/MS. The concentration of

compound 7 dropped rapidly in 10 min (Figure 5a), presumably

related to the expression of carboxylesterase and dihydropyr-

imidine dehydrogenase (DPD) and the high tissue penetration

Cell lines were incubated with compounds for 48 h. IC₅₀values were

determined by SRB assay. ND = not determined.

poorly cytotoxic. The cytotoxicities of 16 and 17 were also

assayed in combination with 5-FU: 5-FU combined with 17 was

more eﬀective at inhibiting cell viability than the other

compounds when given alone, reﬂecting a synergistic inhibitory

eﬀect. Additionally, the cytotoxicities of 5-FU and 7 in the low

expression GLUT1 normal cell line NHDF (western blot

Figure 4. (a) Schematics of the mouse model for CRC. (b) Macroscopic pictures of the mice colonic tumors. (c) Tumor area and (d) body weight

change of the mice treated with control (PBS alone), 5-FU (50 mg/kg), 7 (50 mg/kg), and 5-FU + 17 (10 mg/kg of 5-FU and 30 mg/kg of 17, dosing is

based on compound 7). Data represent mean ± S.E.M. (n = 5, ****P < 0.0001 or *P < 0.05 (one-way ANOVA)).

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Figure 5. Pharmacokinetic proﬁle of compound 7 and 5-FU in mice following i.v. and i.p. injection. (a, b) Plot shows plasma concentration as the

function of time after i.v. injection of compound 7 (50 mg/kg) and i.p. injection of compound 7 and 5-FU (50 mg/kg). The data points exhibiting

concentrations lower than the detection limit of the LC−MS/MS method used were arbitrarily placed just below 0.003 μg/mL. (c) Distributions of 5-

FU at 1 h after i.p. injection of free 5-FU (9.5 mg/kg) and compound 7 (50 mg/kg, 5-FU equivalent dose) in BALB/c mice. Data represent mean ±

S.E.M. (n = 3, *P < 0.05).

of 7 in mouse blood. ³⁻⁴⁵5-FU released from compound 7 was

detected within 2 h and persisted in blood longer than

compound 7 (Figure 5a). Compound 7 exhibited an unusual

plasma pharmacokinetic (PK) proﬁle in that its concentration

was undetectable (lower detection limit = 0.003 μg/mL) after

distributes to the cytosol of CRC cell, where GSH is 10-fold

more than the normal cells. The high tissue penetration of 7 led

to the delayed release of 5-FU, prolonging exposure to 5-FU and

enhancing its tumor eradicating eﬀects. Organ distribution

analysis revealed the colon-targeting eﬀect of 5-FU, leading to

promising antitumor eﬃcacy with low toxicity.

0 min but detectable again after 45 min and 4 h, suggesting it to

be rapidly absorbed by mouse cells and tissues and reach an

equilibrium during elimination. To investigate the distribu-

tion-related antitumor eﬃcacy of compound 7, blood samples

were collected after intraperitoneal injection (i.p.) (50 mg/kg)

of compound 7 and 5-FU in BALB/c mice (n = 3). The

concentration of compound 7 was below the detection limit

after 5 min due to lipophilicity-directed high tissue penetra-

CONCLUSIONS

■

A series of drug conjugates comprising 5-FU and either phlorizin

or phloretin joined with a succinimidyl thioether, disulﬁde, or

triazole linker have been designed, synthesized, and biologically

evaluated. Most of these compounds were less potent against

CRC cell lines than 5-FU; however, compound 7, incorporating

5-FU, phloretin, and a disulﬁde bond, showed good stability (t₁_/₂

tion. The T of 5-FU from compound 7 was delayed from 10

max

to 45 min compared to the 5-FU group, and the concentration of

13 h) in human plasma and lability in the presence of 5 mM

-FU in both groups was undetectable after 2 h, indicating that

the antitumor eﬃcacy might be related to high tissue penetration

Figure 5b). Consequently, the distributions of compound 7 (50

GSH (t₁_/₂= 4.0 h), and its cytotoxicity toward HCT-116 and

HT-29 cell lines was similar to that of 5-FU. In an orthotopic

mice CRC model, compound 7 exhibited excellent antitumor

eﬃcacy and low toxicity, reducing tumor volume by 67% (only a

(

mg/kg, 5-FU equivalent dose) and 5-FU (9.5 mg/kg) in the

organs of BALB/c mice were evaluated at 1 h post i.p. injection;

compound 7 was found to be distributed in the stomach, colon,

heart, and liver (Figure S56). Since the amounts of compound 7

in each organ were low, further analysis of 5-FU released from

compound 7 was performed, showing that 5-FU was detected

mainly in colon, with small amounts in the kidney and stomach,

while free 5-FU was distributed in stomach, spleen, and kidney

3% reduction was seen with 5-FU) without body weight loss.

The colon-targeting eﬀect was a consequence of high tissue

penetration and GLUT-targeting eﬃcacy, leading to consid-

erable tumor inhibition in comparison to 5-FU. Targeting

GLUT with cytotoxic small molecule drugs conjugated with the

glutathione-sensitive linker is therefore proposed as a novel

strategy for the treatment of CRC. Further work to expand this

conjugation strategy to other anticancer agents with a view to

the development of novel chemotherapeutic agents in the future

is ongoing in our laboratory.

(

Figure 5c), similar to the previous report. These results

indicate that our conjugation of compound 7 is an appropriate

strategy to eﬀect the delivery of 5-FU into the colon. This

delivery is posited to be mediated by GLUT1, which is mostly

expressed in the colon. As the phloretin moiety binds to the

glucose transporter, two pathways by which cleavage of the

disulﬁde bond might be accomplished: extracellularly, after

binding of GLUT, or intracellularly, after the conjugate

EXPERIMENTAL SECTION

■

General Chemicals. Reagents and solvents for synthesis were of

reagent grade and used without further puriﬁcation. HPLC analysis was

performed on a HITACHI D-2000 Elite system equipped with a BDS

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HYPERSIL C18 250-4.6 column. The mobile phase was a mixture of

phosphate buﬀer (pH = 8.0). The mixture was stirred for 2 h at ambient

temperature. The solvent was removed in vacuo. The residue was

puriﬁed by column chromatography (silica gel; DCM/MeOH = 15/1

ACN and dd H O, which were ﬁltered through a 0.45 mm membrane

ﬁlter before use. The column was eluted with the mobile phase at a ﬂow

rate of 1.0 mL/min. The eluate was monitored by measuring the

absorption at 254 nm at 25 °C. The purities of all ﬁnal products were

conﬁrmed by HPLC to be >95% prior to their in vitro and in vivo use.

Thin-layer chromatography (0.25 mm, E. Merck silica gel 60 F₂₅₄) was

used to monitor reaction progress; plates were visualized by UV (254

to 10/1) to give compound 6 (107 mg, 0.13 mmol, >99%) as oil; H

NMR (400 MHz, CD OD) δ 7.89 (t, J = 6.1 Hz, 1H), 7.07 (t, J = 8.4

Hz, 2H), 6.68 (t, J = 8.4 Hz, 2H), 6.32 (d, J = 2.2 Hz, 1H), 6.13 (d, J =

2.2 Hz, 1H), 5.62 (s, 2H), 5.10−5.03 (m, 1H), 4.64 (t, J = 7.2 Hz, 1H),

4.17−4.05 (m, 2H), 3.90 (dd, J = 12.4 Hz, 2.0 Hz, 1H), 3.83−3.95 (m,

2H), 3.59−3.73 (m, 7H), 3.44−3.54 (m, 6H), 3.34−3.39 (m, 1H), 2.87

(m, 4H), 2.72 (t, J = 7.0 Hz, 2H), 2.49 (t, J = 7.0 Hz, 2H), 1.98 (t, J = 7.0

nm) or by staining with ninhydrin and heating. Acquisition of H and

C nuclear magnetic resonance (NMR) spectra was performed on a

Bruker-AV-400 (400 MHz) (See the Supporting Information).

Chemical shifts are referenced to residual solvent peaks in parts per

Hz, 2H); C NMR (100 MHz, CD OD) δ 205.5, 172.9, 166.0, 165.0,

160.6, 159.8 (d, JCCF = 27 Hz), 155.0, 149.8, 140.0 (d, JCF = 233 Hz),

132.4, 129.3 (d, JCCF = 35 Hz), 129.1 (2C), 114.7 (2C), 106.3, 100.8,

95.8, 93.8, 77.1, 77.1, 73.4, 70.0 (2C), 69.8, 69.5 (2C), 67.6, 66.3, 61.1,

45.8, 38.0, 37.3, 31.7, 29.3, 23.6 ppm; HRMS (ESI TOF-MS)

million (δ): H δ = 2.50, C δ = 39.52 for d -DMSO; H δ = 3.31, C δ

49.00 for CD OD; H δ = 2.05, C δ = 29.84, 206.26 for d -acetone;

H δ = 7.26, C δ = 77.16 for CDCl . Coupling constants (J) are given

C H FN O S [M + H] calc. 845.2267, found 845.2279.

in Hertz (Hz). Splitting patterns are denoted as s (singlet), d (doublet),

dd (double doublet), t (triplet), q (quartet), and m (multiplet). Mass

spectra were acquired using a Burker bioTOF III and are reported in m/

36 45

16 2

(

5-Fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl

-((2-(2-(2-(3,5-dihydroxy-4-(3-(4-hydroxyphenyl)propanoyl)-

phenoxy)ethoxy)ethoxy)ethyl)disulfanyl)butanoate (7). To a

solution of compound 17 (55 mg, 0.13 mmol) in THF were added

compound 23 (50 mg, 0.13 mmol) and phosphate buﬀer (pH 8.0). The

mixture was stirred for 2 h at ambient temperature. The solvent was

removed in vacuo. The residue was puriﬁed by column chromatography

(

5-Fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl

-(3-((2-(2-(2-(3-hydroxy-4-(3-(4-hydroxyphenyl)propanoyl)-

-(((2S,3R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)-

tetrahydro-2H-pyran-2-yl)oxy)phenoxy)ethoxy)ethoxy)ethyl)-

thio)-2,5-dioxopyrrolidin-1-yl)hexanoate (4). To a solution of 16

(silica gel; DCM/MeOH = 20/1) to give compound 7 (85 mg, 0,12

(

35 mg, 0.06 mmol) in dry methanol (2 mL) was added compound 22

21 mg, 0.06 mmol). The reaction mixture was stirred for 10 min and

mmol, 92%) as a solid; H NMR (600 MHz, d -acetone) δ 7.93 (d, J =

6.4 Hz, 1H), 7.09 (d, J = 8.4 Hz, 2H), 6.75 (d, J = 9.4 Hz, 2H), 5.69 (s,

2H), 6.01 (s, 2H), 4.15 (t, J = 4.6 Hz, 2H), 3.81 (t, J = 4.3 Hz, 2H), 3.70

(t, J = 6.5 Hz, 2H), 3.66−3.60 (m, 4H), 3.35 (t, J = 8.0 Hz, 2H), 2.89−

2.77 (m, 4H), 2.78 (t, J = 7.2 Hz, 2H), 2.52 (t, J = 7.2 Hz, 2H), 2.07−

then was concentrated in vacuo. The residue was puriﬁed by column

chromatography (silica gel; DCM/MeOH = 12/1) to give 4 (53 mg,

.06 mmol, >99%) as foam. H NMR (600 MHz, CD OD) δ 7.88 (d, J

6 Hz, 1H, H-29), 7.07 (d, J = 8.2 Hz, 2H, H-2, H-6), 6.67 (d, J = 8.2

2.04 (m, 2H); C NMR (150 MHz, d -acetone) δ 205.8, 173.3, 166.1,

Hz, 2H, H-3, H-5), 6.31−6.29 (m, 1H, H-3′), 6.11−6.09 (m, 1H, H-

165.3, 157.9 (d, J_C_C_F= 27 Hz), 156.4, 150.1, 140.9 (d, J_C_F= 231 Hz),

133.4, 130.2 (2C), 129.0 (d, J_C_C_F= 34.5 Hz), 115.2 (2C), 105.7, 94.0

(2C), 71.4, 71.3, 71.0, 70.1, 70.0, 68.5, 47.0, 39.3, 38.1, 32.7, 30.5, 30.3,

′), 5.61 (s, 2H, H-23), 5.11 (d, J = 7.2 Hz, 1H, H-1″), 4.15 (t, J = 4.8

Hz, 2H), 4.03−4.00 (m, 1H), 3.90 (d, J = 4.2 Hz, 1H), 3.81 (t, J = 4.2

Hz, 2H), 3.77−3.70 (m, 3H), 3.67 (t, J = 4.2 Hz, 2H), 3.63 (t, J = 5.4

Hz, 2H), 3.50−3.45 (m, 5H), 3.42−3.30 (m, 3H), 3.17−3.11 (m, 1H),

24.7 ppm. HRMS (ESI TOF-MS) C H FN O S [M + H] calc.

11 2

683.1739, found 683.1763.

.09−3.05 (m,1H), 2.89−2.81 (m,3H), 2.50 (dd, J = 15.6, 3 Hz, 1H),

(5-Fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl

3-(1-(2-(2-(2-(3-hydroxy-4-(3-(4-hydroxyphenyl)propanoyl)-5-

(((2S,3R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)-

tetrahydro-2H-pyran-2-yl)oxy)phenoxy)ethoxy)ethoxy)ethyl)-

.33 (t, J = 7.2 Hz, 2H, H-21), 1.60−1.58 (m, 2H, H-20), 1.52−1.50

(

m, 2H, H-18), 1.28−1.26 (m, 2H, H-19) ppm; C NMR (150 MHz,

CD OD) 206.8, 178.9, 177.2, 174.6, 167.2, 166.2, 161.9, 159.6 (d, J

CCF

H-1,2,3-triazol-4-yl)propanoate (8). To a solution of 20 (95 mg,

26.1 Hz), 156.3, 151.0, 141.4 (d, J_C_F= 232.5 Hz), 133.7, 130.5 (d,

.16 mmol) in EtOH/water (3/1, 2 mL) were added TBTA (18 mg,

.03 mmol), sodium ascorbic acid (20.8 mg, 0.10 mmol), copper(II)

J_C_C_F= 33.1 Hz), 130.4 (2C), 116.0 (2C), 107.6, 102.1, 97.1, 95.2, 78.4,

4.7, 72.1, 71.7, 71.6, 71.2, 71.1, 70.4, 69.0, 62.5, 49.8, 47.0, 40.8, 39.4,

7.3, 34.3, 32.0, 30.6, 28.0, 26.9, 25.0 ppm; HRMS (ESI TOF-MS)

sulfate pentahydrate (3.3 mg, 0.01 mmol), and 24 (38 mg, 0.16 mmol).

The reaction mixture was stirred for 12 h and then concentrated in

vacuo. The mixture was dissolved in EtOAc and was extracted with

water. The organic layers were concentrated in vacuo and puriﬁed by

C H FN O SNa [M + Na] calc. 960.2843, found 960.2847.

(

5-Fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl

-(3-((2-(2-(2-(3,5-dihydroxy-4-(3-(4-hydroxyphenyl)-

propanoyl)phenoxy)ethoxy)ethoxy)ethyl)thio)-2,5-dioxopyr-

rolidin-1-yl)hexanoate (5). To a solution of 17 (22 mg, 0.05 mmol)

in dry MeOH (1 mL) was added 22 (19 mg, 0.05 mmol). The reaction

mixture was stirred for 10 min and then was concentrated in vacuo. The

residue was puriﬁed by column chromatography (silica gel; DCM/

column chromatography (silica gel; DCM/MeOH = 10/1) to give 8

(117 mg, 0.14 mmol, 88%) as foam. H NMR (600 MHz, CD OD) δ

7.81 (d, J = 6 Hz, 1H, H-24), 7.77 (s, 1H, H-13), 7.06 (d, J = 8.4 Hz, 2H,

H-2, H-6), 6.67 (d, J = 8.4 Hz, 2H, H-3, H-5), 6.30 (d, J = 2.4 Hz, 1H,

H-3′), 6.12 (d, J = 2.4 Hz, 1H, H-5′), 5.59 (s, 2H, H-18), 5.07 (d, J = 7.2

Hz, 1H, H-1″), 4.50 (t, J = 4.8 Hz, 2H, H-12), 4.15 (t, J = 4.2 Hz, 2H, H-

7), 3.88 (dd, J = 10, 2 Hz, 1H), 3.80 (t, J = 4.8 Hz, 2H), 3.79−3.77 (m,

2H), 3.68 (q, J = 6 Hz, 1H), 3.66−3.61 (m, 4H), 3.5−3.4 (m, 5H),

3.38−3.34 (m, 1H), 2.94 (t, J = 7.8 Hz, 2H, H-16), 2.88 (t, J = 7.2 Hz,

MeOH = 12/1) to give 5 (39 mg, 0.05 mmol, >99%) as foam. H NMR

(

400 MHz, CD OD) δ 7.89 (d, J = 6 Hz, 1H, H-29), 7.05 (d, J = 8.4 Hz,

H, H-2, H-6), 6.68 (d, J = 8.4 Hz, 2H, H-3, H-5), 5.92 (s, 2H, H-3′, H-

′), 5.61 (s, 2H), 4.09 (t, J = 4.4 Hz, 2H), 4.09−3.99 (m, 1H), 3.81 (t, J

4.4 Hz, 2H), 3.76−3.72 (m, 2H), 3.69−3.64 (m, 4H), 3.42 (t, J = 7

2H, H-β), 2.72 (t, J = 7.2 Hz, 2H, H-17) ppm; C NMR (150 MHz,

CD OD) δ 206.8, 173.5, 167.2, 166.2, 161.8, 159.5 (d, J = 26 Hz),

Hz, 2H), 3.21−3.20 (m, 1H), 3.11−3.07 (m, 2H), 2.87−2.83 (m, 3H),

CCF

.47 (dd, J = 14.8, 3.6 Hz, 1H), 2.34 (t, J = 7.3 Hz, 2H), 1.62−1.50 (m,

156.4, 150.9, 146.9, 141.3 (d, J_C_F= 232 Hz), 133.7, 130.5 (d, J_C_C_F= 34

Hz), 130.3 (2C), 124.4, 116.0 (2C), 107.6, 102.1, 97.1, 95.1, 78.5, 78.4,

74.7, 71.6, 71.5, 71.4, 71.1, 70.4, 70.3, 68.9, 62.4, 51.3, 47.0, 34.0, 30.6,

21.4 ppm; HRMS (ESI TOF-MS) C H FN O [M + H] ⁺calc.

H), 1.29−1.27 (m, 2H) ppm; C NMR(100 MHz, CD OD) δ 206.7,

78.9, 177.1, 174.5, 166.5, 165.5 (2C), 159.7 (d, J = 27.4 Hz), 156.4,

CCF

51.3, 141.6 (d, J_C_F= 227 Hz), 133.8, 130.5 (d, J_C_C_F= 32.2 Hz), 130.3

(

2C), 116.1 (2C), 106.1, 94.8 (2C), 72.1, 71.7, 71.6, 71.2, 70.4, 68.7,

834.2840, found 834.2855.

7.4, 40.7, 39.4, 37.2, 34.3, 32.0, 31.2, 28.0, 26.9, 25.0 ppm; HRMS (ESI

(5-Fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl

TOF-MS) C H FN O SNa [M + Na]⁺calc. 798.2315, found

3-(1-(2-(2-(2-(3-hydroxy-4-(3-(4-hydroxyphenyl)propanoyl)-5-

(

((2S,3R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)-

98.2319.

5-Fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl

-((2-(2-(2-(3-hydroxy-4-(3-(4-hydroxyphenyl)propanoyl)-5-

tetrahydro-2H-pyran-2-yl)oxy)phenoxy)ethoxy)ethoxy)ethyl)-

H-1,2,3-triazol-4-yl)propanoate (9). To a solution of 21 (62.3 mg,

.14 mmol) in EtOH/water (3: 1, 2 mL) were added TBTA (7.9 mg,

(

((2S,3R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)-

tetrahydro-2H-pyran-2-yl)oxy)phenoxy)ethoxy)ethoxy)ethyl)-

disulfanyl)butanoate (6). To a solution of compound 16 (76 mg,

0.01 mmol), sodium ascorbic acid (45 mg, 0.22 mmol), copper(II)

sulfate pentahydrate (5.6 mg, 0.02 mmol), and 24 ( 35.7 mg, 0.15

mmol). The reaction mixture was stirred for 12 h and then concentrated

.13 mmol) in THF were added compound 23 (50 mg, 0.13 mmol) and

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J. Med. Chem. 2021, 64, 4450−4461

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Article

in vacuo. The mixture was then dissolved in EtOAc and was extracted

with water. The organic layers were concentrated in vacuo and puriﬁed

2H), 3.54 (t, J = 6.4 Hz, 2H), 3.27 (t, J = 7.6 Hz, 2H), 2.83 (t, J = 7.6 Hz,

2H), 2.62−2.57 (m, 2H); C NMR(100 MHz, CDCl ) δ 205.2, 164.5

by column chromatography (silica gel: DCM/MeOH = 14/1) to give 9

(2C), 153.7, 133.4, 129.5 (2C), 115.3 (2C), 94.5 (2C), 77.2, 72.8, 70.4,

70.4, 69.9, 69.4, 67.0, 45.8, 29.8, 23.9; HRMS (ESI TOF-MS)

(

62.9 mg, 0.09 mmol, 64%) as foam; H NMR (400 MHz, d -DMSO) δ

.11 (d, J = 6.4 Hz, 1H, H-24), 7.85 (s, 1H, H-13), 7.05 (d, J = 8.4 Hz,

H, H-2, H-6), 6.69 (d, J = 8.4 Hz, 2H, H-3, H-5), 5.96−5.96 (m, 2H,

C H O SNa [M + Na] calc. 445.1291, found 445.1328.

21 26 7

1-(4-(2-(2-(2-Azidoethoxy)ethoxy)ethoxy)-2-hydroxy-6-

H-3′, H-5′), 5.59 (s, 2H, H-18), 4.48 (t, J = 5 Hz, 2H, H-12), 4.12−4.05

(((2S,3R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)-

tetrahydro-2H-pyran-2-yl)oxy)phenyl)-3-(4-hydroxyphenyl)-

propan-1-one (20). Phlorizin (2.31 g, 5.30 mmol) in dry DMF (15

mL) was added K CO (1.11 g, 8.03 mmol) at ambient temperature

(

m, 2H), 3.82 (t, J = 5 Hz, 2H), 3.75−3.65 (m, 2H), 3.59−3.51 (m,

H), 3.27 (t, J = 7.6 Hz, 2H), 2.89 (t, J = 7.2 Hz, 2H, H-16), 2.80 (t, J =

.4 Hz, 2H, H-β)), 2.7 (t, J = 7.2 Hz, 2H, H-17) ppm; C NMR (100

and stirred for 10 min. Compound 19 (1.66 g, 7.00 mmol, see the

Supporting Information for its preparation) was added, and the reaction

mixture was heated at 55 °C for 12 h. The reaction mixture was

concentrated in vacuo. The residue was extracted with water/EtOAc

three times. The organic layers were combined and concentrated in

vacuo. The residue was puriﬁed by column chromatography (silica gel;

MHz, CD OD) δ 206.7, 173.6, 166.5, 165.6 (2C), 159.7 (d, J

= 26

CCF

Hz), 156.5, 151.1, 147.0, 141.3 (d, J_C_F= 232 Hz), 133.9, 130.4 (d, J_C_C_F

34 Hz), 130.3 (2C), 124.4, 116.1 (2C), 106.1, 94.8 (2C), 71.6, 71.6,

1.4, 70.5, 70.3, 68.7, 51.3, 47.4, 34.0, 31.2, 21.5 ppm; HRMS (ESI

TOF-MS) C H FN O [M + H] calc. 672.2312, found 672.2318.

-(4-(2-(2-(2-(tert-Butyldisulfanyl)ethoxy)ethoxy)ethoxy)-2-

DCM/MeOH = 10/1) to give compound 20 (2.13 g, 3.59 mmol, 68%)

h y d r o x y - 6 - ( ( ( 2 S , 3 R , 5 S , 6 R ) - 3 , 4 , 5 - t r i h y d r o x y - 6 -

as foam; H NMR (600 MHz, CD OD) δ 7.08 (d, J = 8.4 Hz, 2H, H-2,

(

hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)phenyl)-3-(4-

hydroxyphenyl)propan-1-one (15). Phlorizin (1.0 g, 2.29 mmol) in

H-6), 6.70 (d, J = 8.4, 2H, H-3, H-5), 6.34 (d, J = 2.4 Hz, 1H, H-3′),

6.15 (d, J = 2.4 Hz, 1H, H-5′), 5.10 (d, J = 7.2 Hz, 1H, C-1″), 4.16 (t, J =

4.4 Hz, 2H, H-9), 3.91 (d, J = 2 Hz, 1H), 3.83 (t, J = 4.4 Hz, 1H), 3.73−

3.70 (m, 5H), 3.69−3.67 (m, 1H), 3.52−3.50 (m, 4H), 3.49−3.40 (m,

dry DMF (10 mL) was added K CO (436 mg, 3.15 mmol) at ambient

temperature and stirred for 10 min. Compound 14 (1.0 g, 3.16 mmol,

see the Supporting Information ) was added, and the reaction mixture

was heated at 55 °C for 12 h. The reaction mixture was concentrated in

vacuo. The residue was extracted with water/EtOAc three times. The

organic layers were combined and concentrated in vacuo. The residue

5H), 2.90 (t, J = 7.8 Hz, 2H, H-12) ppm; C NMR (150 MHz,

CD OD) δ 206.8, 167.2, 166.2, 161.8, 156.3, 133.7, 130.3 (2C), 116.0

(2C), 107.6, 102.1, 97.1, 95.1, 78.4, 74.7, 71.7, 71.3, 71.1, 70.7, 70.5,

was puriﬁed by column chromatography (silica gel; DCM/MeOH =

68.9, 62.4, 51.7, 47.0, 30.6 ppm; HRMS (ESI TOF-MS)

0/1) to give compound 15 (837 mg, 1.25 mmol, 54%) as foam; H

12Na [M + Na] calc. 616.2113, found 616.2115.

NMR (400 MHz, CD OD) δ 7.06 (d, J = 8.4 Hz, 2H, H-2, H-6), 6.68

1-(4-(2-(2-(2-Azidoethoxy)ethoxy)ethoxy)-2,6-dihydroxy-

phenyl)-3-(4-hydroxyphenyl)propan-1-one (21). To a solution of

compound 20 (195 mg, 0.33 mmol) in EtOH (5 mL) was added 1.0 N

HCl (3 mL). The mixture was reﬂuxed at 90 °C for 3 h. The reaction

mixture was cooled, and the solvent was evaporated in vacuo. The

residue was extracted with water /EtOAc and dried with MgSO4. The

organic layers were combined and concentrated in vacuo. The residue

was puriﬁed by column chromatography (silica gel; Hex/EtOAc = 1/1)

(

d, J = 8.4 Hz, 2H, H-3, H-5), 6.33 (d, J = 2.4 Hz, 1H, H-3′), 6.14 (d, J =

(

.4 Hz, 1H, H-5′), 5.08 (d, J = 7.6 Hz, 1H, H-1″), 4.15 (t, J = 4.4 Hz,

H), 3.91−3.88 (m, 1H), 3.82 (t, J = 4.4 Hz, 2H), 3.71−3.62 (m, 5H),

.61−3.60 (m, 2H), 3.50−3.44 (m, 5H), 3.34 (d, J = 9.2 Hz, 1H),

.90−2.84 (m, 4H), 1.30 (s, 9H, H-14, H-15, H-16) ppm; C NMR

150 MHz, CD OD) δ 206.8, 167.2, 166.2, 161.8, 156.3, 133.7, 130.3

2C), 116.0 (2C), 107.6, 102.1, 97.1, 95.1, 78.4, 74.7, 71.6, 71.3, 71.1,

to give compound 21 (110 mg, 0.26 mmol, 79%) as foam; H NMR

0.7, 70.5, 68.9, 62.4, 48.4, 47.0, 40.9, 30.6, 30.2 (3C) ppm; HRMS

(400 MHz, CDCl ) δ 7.04 (d, J = 8.4 Hz, 2H, C-2, C-6), 6.71 (d, J = 8.4

(

ESI TOF-MS) C H NaO S Na [M + Na] calc. 695.2166, found

12 2

Hz, 2H, C-3, C-5), 5.95−5.85 (m, 2H, C-3′, C-5′), 4.06−4.04 (m, 2H,

95.2174.

-(2-Hydroxy-4-(2-(2-(2-mercaptoethoxy)ethoxy)ethoxy)-6-

((2S,3R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)-

C-7), 3.82 (t, J = 4.4 Hz, 2H), 3.73−3.70 (m, 2H), 3.67−3.65 (m, 2H),

.60 (t, J = 5 Hz, 2H), 3.33−3.28 (m, 4H, C-α, C-12), 2.89 (t, J = 7.7

(

tetrahydro-2H-pyran-2-yl)oxy)phenyl)-3-(4-hydroxyphenyl)-

propan-1-one (16). To a stirred solution of 15 (156 mg, 0.23 mmol)

in dry THF (5 mL) were added TCEP (133 mg, 0.46 mmol) and 4.0 N

NaOH solution (1 mL). The reaction mixture was stirred for 5 h and

then was concentrated in vacuo. The residue was dissolved in EtOAc,

and the precipitate was ﬁltered. The ﬁltrate was concentrated in vacuo

and was puriﬁed by column (silica gel; DCM/MeOH = 10/1) to give

Hz, 2 Hz, C-β) ppm; C NMR (100 MHz, CDCl ) δ 204.9, 164.5

(2C), 153.7, 133.7, 129.5 (2C), 115.2 (2C), 105.0, 94.6 (2C), 70.6,

70.4, 69.9, 69.8, 67.1, 50.5, 45.8, 29.7 ppm; HRMS (ESI TOF-MS)

C H N O Na [M + Na] calc. 454.1585, found 454.1583.

1-(6-Maleimidohexanoyloxymethyl)-5-ﬂuorouracil (22). To a

solution of 5-FU (100 mg, 0.77 mmol) in water (0.5 mL) was added

formaldehyde (37%, 0.5 mL) at 60 °C and stirred for another 3 h after

the solids completely disappeared. Then, the solvent was removed in

vacuo to obtain colorless and viscous oil. In another ﬂask, to 6-

maleimidohexanoic acid (195 mg, 0.92 mmol) in dry ACN (2 mL) were

added DCC (190 mg, 0.92 mmol) and DMAP (9.5 mg, 0.08 mmol) at 0

°C and stirred for 10 min. Then, the abovementioned oil in dry ACN

(0.5 mL) was added to the reaction ﬂask. The reaction mixture was

stirred at 0 °C for 30 min and then stirred at room temperature for 12 h.

The precipitates formed in the reaction were ﬁltered, and the ﬁltrate was

evaporated in vacuo. The residue in EtOAc was extracted with 1.0 N

compound 16 (115 mg, 0.20 mmol, 86%) as foam; H NMR (400

MHz, CD OD) δ 7.06 (d, J = 8.4 Hz, 2H, H-2, H-6), 6.68 (d, J = 8.4 Hz,

H, H-3, H-5), 6.33 (d, J = 2.4 Hz, 1H, H-3′), 6.14 (d, J = 2.4 Hz, 1H,

H-5′), 5.08 (d, J = 7.2 Hz, 1H, H-1″), 4.15 (t, J = 4.4 Hz, 1H, H-7), 3.91

dd, J = 10, 2 Hz, 1H), 3.84−3.81 (m, 2H), 3.71−3.68 (m, 3H), 3.64−

.62 (m, 2H), 3.59 (t, J = 6.4 Hz, 2H), 3.50−3.43 (m, 5H), 3.39−3.37

m, 1H), 2.88 (t, J = 7.5 Hz, 2H), 2.64 (t, J = 6.4 Hz, 2H) ppm; ¹³

NMR(100 MHz, CD OD) δ 206.8, 167.3, 166.3, 161.8, 156.3, 133.7,

(

30.3 (2C), 116.0 (2C), 107.6, 102.1, 97.1, 95.1, 78.5, 78.4, 74.7, 74.1,

1.6, 71.1 (2C), 70.5, 68.9, 62.4, 47.0, 30.7, 24.6 ppm; HRMS (ESI

HCl, saturated NaHCO

, and water. The organic layers were dried over

TOF-MS) C H O SNa [M + Na] calc. 607.1820, found 607.1821.

MgSO and concentrated in vacuo. The residue was puriﬁed by column

36 12

-(2,6-Dihydroxy-4-(2-(2-(2-mercaptoethoxy)ethoxy)-

chromatography (silica gel; Hex/EtOAc = 1/1) to give compound 22

ethoxy)phenyl)-3-(4-hydroxyphenyl)propan-1-one (17). To a

solution of compound 16 (210 mg, 0.36 mmol) in EtOH (2 mL) was

added 1.0 N HCl (1 mL). The mixture was heated at reﬂux for 3 h. The

reaction mixture was cooled, and the solvent was evaporated in vacuo.

The residue was extracted with water and EtOAc and dried over

(117 mg, 0.33 mmol, 43%) as foam. H NMR (600 MHz, CD OD) δ

7.92 (d, J = 6 Hz, 1H, H-6), 6.79 (s, 2H, H-16, H-17), 5.63 (s, 2H, H-7),

3.48 (t, J = 6.9 Hz, 2H, H-13), 2.38 (t, J = 7.3 Hz, 2H, H-9), 1.68−1.63

(m, 2H, H-10), 1.59−1.54 (m, 2H, H-12), 1.32−1.27 (m, 2H, H-11);

C NMR (150 MHz, CD OD) δ 174.6, 172.5 (2C), 159.7 (d, J = 25

CCF

MgSO . The organic layers were combined and concentrated in vacuo.

The residue was puriﬁed by column chromatography (silica gel; Hex/

EtOAc = 1/1) to give compound 17 (111 mg, 0.26 mmol, 73%) as

foam; H NMR (400 MHz, CDCl ) δ 7.00 (d, J = 8.4 Hz, 2H, H-2, H-

), 6.70 (d, J = 8.4 Hz, 2H, H-3, H-5), 5.87 (s, 2H, H-3′, H-5′), 4.00 (t, J

3.2 Hz, 2H), 3.79−3.77 (m, 2H), 3.71−3.69 (m, 2H), 3.63−3.61 (m,

H), 151.1, 141.4 (d, J_C_F= 233 Hz), 135.3 (2C), 130.5 (d, J_C_C_F= 34 Hz),

71.6, 38.2, 34.3, 29.1, 27.0, 25.1; HRMS (ESI TOF-MS)

C H FN O C Na [M + Na] calc. 376.0915, found 376.0915.

15 16 3 6 21

1-(4-(Pyridin-2-yldisulfanyl) butyroyloxymethyl)-5-ﬂuorour-

acil (23). A solution of 5-FU (100 mg, 0.77 mmol) in 37%

formaldehyde solution (0.5 mL) was stirred at 60 °C until the solids

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J. Med. Chem. 2021, 64, 4450−4461

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Article

completely disappeared and was stirred at 60 °C for another 3 h. The

solvent was removed in vacuo to obtain a colorless oil. In another ﬂask

were added 4-(2-pyridyldithio)butanoic acid (206 mg, 0.90 mmol),

DCC(190 mg, 0.92 mmol), and DMAP (11 mg, 0.09 mmol) in ACN (5

mL) at 0 °C, and the reaction was stirred for 10 min. Then, the solution

was added to the abovementioned colorless oil. The reaction mixture

was stirred at 0 °C for 30 min and then stirred at room temperature for

temperature. The bound protein stain was dissolved with 100 μL of 10

mM Tris base (pH 10.5) and shaken for 10 min. Optical density (O.D.)

was measured at 510 nm with a multimode microplate reader

(SpectraMax Paradigm, Beckman Coulter, U.S.). The fraction of cell

survival was calculated as follows: survival fraction = (OD treated −

blank)/(OD control − blank). IC values (the concentrations that

produce 50% inhibition of cell growth) were calculated using nonlinear

regression curve-ﬁtting models (GraphPad Prism 7, U.S.). Each

experiment was repeated three times.

2 h. The precipitates formed in the reaction were ﬁltered, and the

ﬁltrate was evaporated in vacuo. The residue in EtOAc was extracted

with 1.0 N HCl, saturated NaHCO , and water. The organic layers were

2-NBDG Uptake Assay. COS-7 cells were seeded in 96-well culture

plates and grown in low-glucose Dulbecco’s Modiﬁed Eagle’s Medium

supplemented with 10% fetal bovine serum and 2 mM L-glutamine for

24 h. After washing with Krebs Ringer bicarbonate buﬀer (KRB) (10

dried over MgSO and concentrated in vacuo. The residue was puriﬁed

by column chromatography (silica gel; CHCl /MeOH = 25/1) to give

compound 23 (95.5 mg, 0.26 mmol, 34%, two steps) as a solid; H

NMR (400 MHz, CDCl ) δ 8.41 (d, J = 4.8 Hz, 1H), 7.62 (t, J = 7.3 Hz,

mM HEPES, 129 mM NaCl, 4.7 mM KCl, 2 mM CaCl , 1.2 mM

H), 7.58 (d, J = 5.4 Hz, 1H), 7.11−7.01 (m, 1H), 5.60 (s, 2H), 2.78 (t,

MgSO , 1.2 mM KH PO , and 5 mM NaHCO , pH 7.4), cells were

J = 7 Hz, 2 H), 2.50 (t, J = 7.2 Hz, 2H), 1.21−1.91 (m, 2H); C NMR

incubated with the compounds to be tested in KRB containing 2-

(100 MHz, CDCl ) δ 173.1, 159.8 (d, J = 27 Hz), 156.9, 149.8,

NBDG (200 μM) for 90 min at 37 °C in a 5% CO atmosphere, then

CCF

49.3, 140.3 (d, J_C_F= 238 Hz), 137.2, 128.5 (d, J_C_C_F= 24 Hz), 120.9,

20.0, 69.8, 37.6, 32.2, 23.5 ppm; HRMS (ESI TOF-MS)

washed twice with KRB, and lysed with lysis buﬀer (1% Nonidet P-40,

1% sodium deoxycholate, 40 mM KCl, and 20 mM Tris, pH 7.4).

Finally, the lysates were transferred to black 96-well plates and

ﬂuorescence intensity was detected with a multimode microplate reader

(SpectraMax Paradigm, Beckman Coulter, USA) using an excitation

C H F N O S [M + H] calc. 372.0485, found 372.0483.

4 2

1-(Pent-4-ynoyloxymethyl)-5-ﬂuorouracil (24). A solution of

-FU (100 mg, 0.77 mmol) in 37% formaldehyde solution (0.5 mL)

was stirred at 60 °C until the solids completely disappeared and was

stirred at 60 °C for another 3 h. The solvent was removed in vacuo to

obtain a colorless oil. In another ﬂask was added pent-4-ynoic acid (105

mg, 1.07 mmol), DCC (285 mg, 1.38 mmol), and DMAP (11 mg, 0.09

mmol) in ACN (5 mL) at 0 °C and the reaction was stirred for 10 min

to give pre-activated pent-4-ynoic acid. Then, pre-activated pent-4-

ynoic acid in ACN was added to the abovementioned colorless oil. The

reaction mixture was stirred at 0 °C for 30 min and then stirred at room

temperature for 12 h. The solids were ﬁltered, and the ﬁltrate was

evaporated in vacuo. The residue in EtOAc was extracted with 1.0 N

wavelength of 475 nm and an emission wavelength of 550 nm.

In Vivo Studies. Speciﬁc pathogen-free BALB/c mice (6 weeks of

age, male) were purchased from the National Laboratory Animal

Center. The animals were housed in clean plastic microisolator cages

(ﬁve mice/cage), maintained on standard laboratory pellet diet and

water ad libitum. Animal rooms were kept at constant temperature,

humidity, and 12 h dark/light cycle. All animal procedures were in

accordance with the recommendations of the Committee for the

Laboratory Animal Care Committee in National Taiwan University

College of Medicine [IACUC 20180076].

HCl, saturated NaHCO solution, and water sequentially. The organic

layers were dried over MgSO4, concentrated in vacuo, and puriﬁed by

The azoxymethane (AOM)-induced murine CRC model was

applied to model human CRC. BALB/c mice (6 weeks of age, male)

were intraperitoneally (i.p.) injected once every 2 weeks with AOM (10

mg/kg body weight) or the PBS vehicle. Seven days after each AOM

injection, 2% dextran sulfate sodium (DSS) was given in the drinking

water for 4 days followed by 3 days of regular water. This cycle was

repeated three times. The mice were randomly divided into four groups

(n = 5) on day 60 after the ﬁrst AOM treatment. 5-FU (50 mg/kg body

weight), compound 7 (50 mg/kg body weight each), and 5-FU + 17 (5-

FU (10 mg/kg) and 17 (30 mg/kg)) in 0.2 mL of PBS were injected i.p.

once every 3 days for 3 weeks. Body weight was measured every 3 days.

The mice were sacriﬁced on day 21 after the ﬁrst treatment. Tumor

volumes and body weights were recorded at sacriﬁce. The numbers of

tumors were determined under a dissecting microscope, and the area

covered by tumors was measured by imaging software (AxioVision LE

4.8.2.0).

column chromatography (silica gel; Hex/EtOAc = 2/1) to give

compound 24 (118 mg, 0.49 mmol, 63%) as amorphous solids; H

NMR (400 MHz, CDCl ) δ 7.59 (d, J = 5.4 Hz, 1H, H-6), 5.65 (s, 2H,

H-7), 2.67−2.55 (m, 2H), 2.54−2.46 (m, 2H), 1.97 (t, J = 2.5 Hz, 1H,

H-12) ppm; ¹³C NMR (150 MHz, CDCl ) δ 171.9, 156.4 (d, J = 28

CCF

Hz), 148.8, 140.1 (d, J_C_F= 237 Hz), 128.3 (d, J_C_C_F= 33 Hz), 81.5, 69.7,

9.5, 32.9, 14.1 ppm; HRMS (ESI TOF-MS) C H FN O Na [M +

10 9 2 4

Na] calc. 263.0439, found 263.0443.

Cell Culture and Cytotoxic Assay. Cancer cell lines were

obtained from ATCC unless otherwise noted. HT-29 and HCT-116

cells were cultured in Dulbecco’s Modiﬁed Eagle Medium (DMEM,

Thermo Fisher Scientiﬁc) with 10% fetal bovine serum (FBS, Thermo

Fisher Scientiﬁc) and 1% antibiotic−antimycotic (Thermo Fisher

Scientiﬁc). Normal cell line NHDF cells were obtained from

PromoCell unless otherwise noted. NHDF cells were cultured in

Dulbecco’s Modiﬁed Eagle Medium (DMEM, Sigma-Aldrich, Product

In the following PK experiments, BALB/c mice (6 weeks of age,

male) were randomly divided into two groups (n = 3). The two groups

of mice were treated with 5-FU solution (9.5 mg/kg) and compound 7

(50 mg/kg, equivalent as 5-FU), respectively, by intraperitoneal and

intravenous injection. Blood samples were collected via femoral vein by

syringes at 0, 1, 3, 5, 10, 15, 30, and 45 min and 1, 2, 4, and 24 h after

drug administration. A total of 50 μL of blood samples was collected

into 100 μL of extraction solvent (EtOAc/MeOH, 1:1) and vortexed

for 30 s. The supernatants were separated from the mixture by

centrifugation at 10,000 rpm for 10 min and frozen at −30 °C, pending

UPLC-MS/MS analysis. For biodistribution experiments, the entire

dissected organs were placed into weight-known 2 mL microcentrifuge

tubes with 1 mL of extraction solvent (EtOAc/MeOH, 1:1) and 7 mm

stainless steel beads and then homogenized for 30 min. All of the

containers were maintained at 4 °C throughout the processes. The

supernatants were separated from the mixture by centrifugation at

10,000 rpm for 10 min and frozen at −30 °C. The 200 μL solvent from

crude organ suspensions was removed using a dryer, and then they were

dissolved in 50 μL of extraction solvent. All samples were stored at −30

°C and analyzed within 24 h. All of the samples were ﬁltered through

0.22 μm PTFE membranes into 12 × 32 mm vials for UPLC-MS/MS

6429) with 10% fetal bovine serum (FBS, Thermo Fisher Scientiﬁc)

and 1% antibiotic−antimycotic (Thermo Fisher Scientiﬁc). The cells

were incubated at 37 °C in a humidiﬁed incubator with 5% CO and

95% air.

Cytotoxicity of the compounds against HT-29, HCT-116, and

NHDF cells was determined by SRB assay. Brieﬂy, CRC cells were

seeded at a density of 5 × 10 cells/well in 96-well microtiter plates (100

μL/well), separated into two groups with or without GSH. After

incubation for 24 h, the culture medium was replaced by fresh medium

(100 μL). For non-GSH control groups, a series of concentrations of

compounds (100 μL, diluted by medium) were added to cells directly;

for GSH groups, cells were transiently treated with 30 mM GSH (20

μL/well, diluted by medium) for 1 h, and then a series of concentration

of compounds (40 μL/well) and 10 mM GSH (40 μL/well) were

added, making the ﬁnal GSH concentration 5 mM, and incubated for 48

h. After incubation, 10% cold trichloroacetic acid was gently added to

each well and incubated at 4 °C for 1 h. The medium was removed,

carefully washed with water, and dried at room temperature. Next,

plates were stained with 100 μL of 0.057% SRB for 30 min, rinsed with

1% acetic acid to remove the unbound dye, and dried at room

analyses using 5-FU- N as a marker. Pharmacokinetic parameters

458

J. Med. Chem. 2021, 64, 4450−4461

Journal of Medicinal Chemistry

Complete contact information is available at:

Article

were calculated using the WinNonlin Software (version 5.2, Pharsight,

MO, USA.)

Pei-Fang Chiu − School of Pharmacy, College of Medicine,

National Taiwan University, Taipei 100, Taiwan

UPLC-MS/MS Analyses. Analyses were performed on a ACQUITY

UPLC I-Class/Xevo TQ-XS IVD System (Waters, Milford, MA, USA)

from College of Public Health, National Taiwan University. The reverse

phase BEH C18 (100 mm × 2.1 mm, 1.7 μm, Waters) and VanGuard

BEH C18 (5 mm × 2.1 mm, 1.7 μm, Waters) precolumns were used to

separate the analytes. All data were acquired by MassLynx V4.2. The

mobile phase consisted of a 10 mM aqueous solution of ammonium

acetate (A) and acetonitrile (B), set as follows: 0.00 min 98% A → 1.00

min 98% A → 3.00 min 10% A → 4.50 min 10% A → 4.60 min 98% A →

Hui-Yi Yang − School of Pharmacy, College of Medicine,

National Taiwan University, Taipei 100, Taiwan

Yu-Pu Juang − School of Pharmacy, College of Medicine,

National Taiwan University, Taipei 100, Taiwan

Yen-Hsun Lai − School of Pharmacy, College of Medicine,

National Taiwan University, Taipei 100, Taiwan

Tzung-Sheng Lin − School of Pharmacy, College of Medicine,

National Taiwan University, Taipei 100, Taiwan

Lih-Ching Hsu − School of Pharmacy, College of Medicine,

National Taiwan University, Taipei 100, Taiwan

6.00 min 98% A, at a rate of 0.5 mL/min for 6.00 min with 4 μL per

injection. The column oven was maintained at 60 °C. A multiple

reaction monitoring (MRM) method was applied in quantiﬁcation.

Mass spectrometer parameters were as follows: capillary voltage, 3.0

kV/3.0 kV, respectively, for positive/negative ion mode; ion source

temperature, 120 °C; desolvation temperature, 450 °C; cone gas ﬂow

Linda Chia-Hui Yu − Graduate Institute of Physiology, College

of Medicine, National Taiwan University, Taipei 100, Taiwan

https://pubs.acs.org/10.1021/acs.jmedchem.0c00897

(

N ), 50 L/h; desolvation gas ﬂow (N ), 700 L/h; multiplier, 650 V;

−

and collision gas pressure (Ar), 3−4 × 10 mbar. Other parameters are

listed in Table S1 .

Author Contributions

Plasma Stability and Drug Releasing Assay. To access human

plasma stability, synthesized conjugates were dissolved in DMSO, and

the analysis solutions were prepared by diluting 1 μL of stock with 4 μL

of PBS (pH 7.4) and 95 μL of human plasma to a ﬁnal concentration 2.5

mM. After incubation for the indicated time, proteins were denatured

by the addition of ACN, and samples were centrifuged to collect the

clear supernatant and stored at −30 °C until analysis; for releasing

analysis, synthesized conjugates were dissolved in DMSO, and the

analysis solutions were prepared by diluting 50 μL of stock in 750 μL of

PBS (pH 7.4, containing 5 μM, 1 mM, or 5 mM GSH) to a ﬁnal

concentration 2.5 mM. After incubation for the indicated time, samples

were centrifuged to collect the clear supernatant and stored at −30 °C

until analysis. RP-HPLC injections were carried out under the speciﬁed

conditions, and the area of peak was integrated for further calculation.

Statistical Analysis. All data were obtained at least in triplicate, and

results are reported as mean ± mean of standard deviation (S.E.M.).

Comparisons among groups were analyzed via t tests, one-way

ANOVA, and two-way ANOVA analysis using SAS Version 9.2 (SAS

Institute, Cary, NC). The statistical signiﬁcance was determined: n.s.,

nonsigniﬁcant diﬀerence; ****P < 0.0001; *P < 0.05.

P.-H.L. designed the research, and C.-K.C., H.-Y.Y., P.-F.C., and

Y.-H.L. synthesized the compounds. C.-K.C. performed in vitro

cytotoxicity assay and in vivo assay. C.-K.C., H.-Y.Y., and T.-S.L.

did the stability assay. P.-F.C. and Y.-P.J. performed western

analysis. L.-C.H. performed GLUT1 inhibition assay and

provided NHDF cells. L.C.-H.Y. provided HT-29 and HCT-

11 cell lines and designed animal experiments. C.-K.C. and P.-

H.L. wrote the manuscript, which was contributed by all authors,

who have approved the submitted version of the manuscript.

Funding

This research was ﬁnancially supported by the Ministry of

Science and Technology (MOST 108-3114-Y-001-002; 107-

2320-B-002-019-MY3; 106-2320-B-002-017; and 104-2320-B-

02-008-MY3) and Academia Sinica [grant no. AS-SUMMIT-

09].

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

ASSOCIATED CONTENT

sı Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c00897.

■

C.-K.C. and P.-F.C. are supported by a scholarship from the

XinChen Medical Research Foundation, Taiwan. Y.-P.J. is

supported by the Program of Research Performance Enhance-

ment via Students Entering Ph.D. Programs Straight from an

Undergraduate/Master’s Program from National Taiwan

University.

Synthesis of linkers 14 and 19, NMR spectra of all new

structures, and H NMR and C NMR spectra of

compounds; serum stability of compounds 5 and 9 and

HPLC purities of ﬁnal compounds; Western analysis of

CRC and NHDF cell lines; and detailed UPLC-MS/MS

analyses to quantify serum and organ concentrations of 5-

FU and compound 7 (PDF )

ABBREVIATIONS

■

ACN, acetonitrile; AOM, azoxymethane; CRC, colorectal

cancer; DCC, dicyclohexylcarbodiimide; DCM, dichlorome-

thane; DMAP, 4-dimethylaminopyridine; DMEM, Dulbecco’s

Modiﬁed Eagle Medium; DMF, dimethylformamide; DPD,

dihydropyrimidine dehydrogenase; DSS, dextran sulfate so-

dium; 5-FU, 5-ﬂuorouracil; GLUT, glucose transporter; GSH,

glutathione; Hex, hexane; HRMS, high-resolution mass

spectrometry; i.v., intravenous; i.p., intraperitoneal; KRB,

Krebs Ringer bicarbonate buﬀer; 2-NBDG, (2-N-(7-nitrobenz-

Molecular formula strings (CSV )

AUTHOR INFORMATION

■

Corresponding Author

Pi-Hui Liang − School of Pharmacy, College of Medicine,

National Taiwan University, Taipei 100, Taiwan; The

Genomics Research Center, Academia Sinica, Taipei 128,

Taiwan; orcid.org/0000-0001-6668-4642;

Email: phliang@ntu.edu.tw

-oxa-1,3-diazol-4-yl)amino)-2-deoxy-D-glucose; NHDF, nor-

mal human dermal ﬁbroblasts; NMR, nuclear magnetic

resonance; PBS, phosphate buﬀered saline; PK, pharmacoki-

netic; RP-HPLC, reversed phase high-performance liquid

chromatography; SGLT, sodium-glucose cotransporters;

SMDC, small molecule drug conjugation; SRB, sulforhodamine

B; TBTA, tris1-benzyl-1H-1,2,3-triazol; TCEP, tris(2-

carboxyethyl)phosphine; THF, tetrahydrofuran

Authors

Chun-Kai Chang − School of Pharmacy, College of Medicine,

National Taiwan University, Taipei 100, Taiwan

459

J. Med. Chem. 2021, 64, 4450−4461

Journal of Medicinal Chemistry

Article

19) Shi, Y.; Liu, S.; Ahmad, S.; Gao, Q. Targeting key transporters in

tumor glycolysis as a novel anticancer strategy. Curr. Top. Med. Chem.

018, 18, 454−466.

20) Shao, X.; Bai, N.; He, K.; Ho, C. T.; Yang, C. S.; Sang, S. Apple

REFERENCES

■

(

1) Bray, F.; Ferlay, J.; Soerjomataram, I.; Siegel, R. L.; Torre, L. A.;

Jemal, A. Global cancer statistics 2018: GLOBOCAN estimates of

incidence and mortality worldwide for 36 cancers in 185 countries. CA:

Cancer J. Clin. 2018, 68, 394−424.

polyphenols, phloretin and phloridzin: new trapping agents of reactive

dicarbonyl species. Chem. Res. Toxicol. 2008, 21, 2042−2050.

21) Cao, X.; Fang, L.; Gibbs, S.; Huang, Y.; Dai, Z.; Wen, P.; Zheng,

(

2) Loupakis, F.; Cremolini, C.; Masi, G.; Lonardi, S.; Zagonel, V.;

(

Salvatore, L.; Cortesi, E.; Tomasello, G.; Ronzoni, M.; Spadi, R.;

Zaniboni, A.; Tonini, G.; Buonadonna, A.; Amoroso, D.; Chiara, S.;

Carlomagno, C.; Boni, C.; Allegrini, G.; Boni, L.; Falcone, A. Initial

therapy with FOLFOXIRI and bevacizumab for metastatic colorectal

cancer. N. Engl. J. Med. 2014, 371, 1609−1618.

X.; Sadee, W.; Sun, D. Glucose uptake inhibitor sensitizes cancer cells to

daunorubicin and overcomes drug resistance in hypoxia. Cancer

Chemother. Pharmacol. 2007, 59, 495−505.

(

22) Chang, H. C.; Yang, S. F.; Huang, C. C.; Lin, T. S.; Liang, P. H.;

Lin, C. J.; Hsu, L. C. Development of a novel non-radioactive cell-based

method for the screening of SGLT1 and SGLT2 inhibitors using 1-

NBDG. Mol. Biosyst. 2013, 9, 2010−2020.

(

3) Boige, V.; Mendiboure, J.; Pignon, J. P.; Loriot, M. A.; Castaing,

M.; Barrois, M.; Malka, D.; Trégouët, D. A.; Bouché, O.; Le Corre, D.;

Miran, I.; Mulot, C.; Ducreux, M.; Beaune, P.; Laurent-Puig, P.

Pharmacogenetic assessment of toxicity and outcome in patients with

metastatic colorectal cancer treated with LV5FU2, FOLFOX, and

FOLFIRI: FFCD 2000-05. J. Clin. Oncol. 2010, 28 , 2556 −2564.

(23) Abdel-Wahab, A. F.; Mahmoud, W.; Al-Harizy, R. M. Targeting

glucose metabolism to suppress cancer progression: prospective of anti-

glycolytic cancer therapy. Pharmacol. Res. 2019, 150, 104511.

(24) Scafoglio, C.; Hirayama, B. A.; Kepe, V.; Liu, J.; Ghezzi, C.;

4) Modest, D. P.; Pant, S.; Sartore-Bianchi, A. Treatment sequencing

in metastatic colorectal cancer. Eur. J. Cancer 2019, 109, 70−83.

5) Munker, S.; Gerken, M.; Fest, P.; Ott, C.; Schnoy, E.; Fichtner-

Satyamurthy, N.; Moatamed, N. A.; Huang, J.; Koepsell, H.; Barrio, J.

R.; Wright, E. M. Functional expression of sodium-glucose transporters

in cancer. Proc. Natl. Acad. Sci. U. S. A. 2015 , 112 , E4111 −E4119.

(

Feigl, S.; Wiggermann, P.; Vogelhuber, M.; Herr, W.; Stroszczynski, C.;

Schlitt, H. J.; Evert, M.; Reng, M.; Klinkhammer-Schalke, M.; Teufel, A.

Chemotherapy for metastatic colon cancer: No effect on survival when

the dose is reduced due to side effects. BMC Cancer 2018, 18, 455.

(25) Pessin, J. E.; Bell, G. I. Mammalian facilitative glucose transporter

family: structure and molecular regulation. Annu. Rev. Physiol. 1992, 54,

11−930.

26) Huang, C. Y.; Yu, L. C. Distinct patterns of interleukin-12/23 and

tumor necrosis factor alpha synthesis by activated macrophages are

(6) Cuezva, J. M.; Krajewska, M.; de Heredia, M. L.; Krajewski, S.;

modulated by glucose and colon cancer metabolites. Chin. J. Physiol.

Santamaria, G.; Kim, H.; Zapata, J. M.; Marusawa, H.; Chamorro, M.;

Reed, J. C. The bioenergetic signature of cancer: a marker of tumor

progression. Cancer Res. 2002, 62, 6674 −6681.

(

020, 63, 7−14.

27) Huang, C.-Y.; Kuo, W.-T.; Huang, C.-Y.; Lee, T.-C.; Chen, C.-T.;

(7) Medina, R. A.; Owen, G. I. Glucose transporters: expression,

Peng, W.-H.; Lu, K.-S.; Yang, C.-Y.; Yu, L. C.-H. Distinct cytoprotective

roles of pyruvate and ATP by glucose metabolism on epithelial

necroptosis and crypt proliferation in ischaemic gut. J. Physiol. 2017,

regulation and cancer. Biol. Res. 2002, 35, 9−26.

8) Bhattacharya, B.; Mohd Omar, M. F.; Soong, R. The Warburg

effect and drug resistance. Br. J. Pharmacol. 2016, 173, 970−979.

9) Guo, G. F.; Cai, Y. C.; Zhang, B.; Xu, R. H.; Qiu, H. J.; Xia, L. P.;

29) Srinivasarao, M.; Galliford, C. V.; Low, P. S. Principles in the

95, 505−521.

28) Srinivasarao, M.; Low, P. S. Ligand-targeted drug delivery. Chem.

Rev. 2017, 117, 12133−12164.

(

Jiang, W. Q.; Hu, P. L.; Chen, X. X.; Zhou, F. F.; Wang, F.

Overexpression of SGLT1 and EGFR in colorectal cancer showing a

correlation with the prognosis. Med. Oncol. 2011, 197 −203.

design of ligand-targeted cancer therapeutics and imaging agents. Nat.

(10) Wright, E. M.; Loo, D. D. F.; Hirayama, B. A. Biology of human

Rev. Drug Discovery 2015, 14, 203 −219.

sodium glucose transporters. Physiol. Rev. 2011, 91, 733−794.

(30) Bansal, A.; Simon, M. C. Glutathione metabolism in cancer

(11) Villani, L. A.; Smith, B. K.; Marcinko, K.; Ford, R. J.; Broadfield,

progression and treatment resistance. J. Cell Biol. 2018, 217, 2291−

298.

31) Zhao, M.; Liu, Q.; Gong, Y.; Xu, X.; Zhang, C.; Liu, X.; Zhang, C.;

L. A.; Green, A. E.; Houde, V. P.; Muti, P.; Tsakiridis, T.; Steinberg, G.

R. The diabetes medication Canagliflozin reduces cancer cell

proliferation by inhibiting mitochondrial complex-I supported

respiration. Mol. Metab. 2016, 5, 1048−1056.

(

Guo, H.; Zhang, X.; Gong, Y.; Shao, C. GSH-dependent antioxidant

defense contributes to the acclimation of colon cancer cells to acidic

microenvironment. Cell Cycle 2016, 15, 1125 −1133.

(12) Younes, M.; Lechago, L. V.; Somoano, J. R.; Mosharaf, M.;

Lechago, J. Wide expression of the human erythrocyte glucose

(32) Forman, H. J.; Zhang, H.; Rinna, A. Glutathione: overview of its

transporter Glut1 in human cancers. Cancer Res. 1996, 56, 1164−1167.

protective roles, measurement, and biosynthesis. Mol. Aspects Med.

009, 30, 1−12.

33) Fishkin, N.; Maloney, E. K.; Chari, R. V. J.; Singh, R. A novel

(13) Haber, R. S.; Rathan, A.; Weiser, K. R.; Pritsker, A.; Itzkowitz, S.

H.; Bodian, C.; Slater, G.; Weiss, A.; Burstein, D. E. GLUT1 glucose

transporter expression in colorectal carcinoma: a marker for poor

prognosis. Cancer 1998, 83, 34−40.

pathway for maytansinoid release from thioether linked antibody −drug

conjugates (ADCs) under oxidative conditions. Chem. Commun. 2011,

(14) Brown, R. S.; Wahl, R. L. Overexpression of Glut-1 glucose

transporter in human breast cancer An immunohistochemical study.

7, 10752−10754.

34) Pan, X.; Wang, C.; Wang, F.; Li, P.; Hu, Z.; Shan, Y.; Zhang, J.

Cancer 1993, 72, 2979−2985.

(15) Law, A. A.; Collie-Duguid, E. S.; Smith, T. A. Influence of

Development of 5-Fluorouracil derivatives as anticancer agents. Curr.

Med. Chem. 2011, 18, 4538−4556.

resistance to 5-fluorouracil and tomudex on [18F]FDG incorporation,

glucose transport and hexokinase activity. Int. J. Oncol. 2012, 41, 378−

(35) Ponte, J. F.; Sun, X.; Yoder, N. C.; Fishkin, N.; Laleau, R.; Coccia,

J.; Lanieri, L.; Bogalhas, M.; Wang, L.; Wilhelm, S.; Widdison, W.;

Pinkas, J.; Keating, T. A.; Chari, R.; Erickson, H. K.; Lambert, J. M.

Understanding how the stability of the thiol-maleimide linkage impacts

the pharmacokinetics of lysine-linked antibody-maytansinoid con-

jugates. Bioconjugate Chem. 2016, 27, 1588−1598.

(36) Michelet, F.; Gueguen, R.; Leroy, P.; Wellman, M.; Nicolas, A.;

Siest, G. Blood and plasma glutathione measured in healthy subjects by

HPLC: relation to sex, aging, biological variables, and life habits. Clin.

Chem. 1995, 41, 1509−1517.

382.

(16) Ma, Y.-S.; Yang, I.-P.; Tsai, H.-L.; Huang, C.-W.; Juo, S.-H. H.;

Wang, J.-Y. High glucose modulates antiproliferative effect and

cytotoxicity of 5-fluorouracil in human colon cancer cells. DNA Cell

Biol. 2014, 33, 64−72.

(17) Huang, C.-Y.; Huang, C.-Y.; Pai, Y.-C.; Lin, B.-R.; Lee, T.-C.;

Liang, P.-H.; Yu, L.-C. Glucose metabolites exert opposing roles in

tumor chemoresistance. Front. Oncol. 2019, 9, 1282.

(18) Ma, P.; Chen, J.; Bi, X.; Li, Z.; Gao, X.; Li, H.; Zhu, H.; Huang, Y.;

Qi, J.; Zhang, Y. Overcoming multidrug resistance through the GLUT1-

mediated and enzyme-triggered mitochondrial targeting conjugate with

redox-sensitive Paclitaxel release. ACS Appl. Mater. Interfaces 2018, 10,

(37) Salas-Burgos, A.; Iserovich, P.; Zuniga, F.; Vera, J. C.; Fischbarg,

J. Predicting the three-dimensional structure of the human facilitative

glucose transporter glut1 by a novel evolutionary homology strategy:

insights on the molecular mechanism of substrate migration, and

12351−12363.

460

J. Med. Chem. 2021, 64, 4450−4461

Journal of Medicinal Chemistry

M.; Hasegawa, S.; Togashi, K.; Sakai, Y. Regulation of 18F-FDG

Article

binding sites for glucose and inhibitory molecules. Biophys. J. 2004, 87,

(

990−2999.

38) Iwamoto, M.; Kawada, K.; Nakamoto, Y.; Itatani, Y.; Inamoto, S.;

Toda, K.; Kimura, H.; Sasazuki, T.; Shirasawa, S.; Okuyama, H.; Inoue,

accumulation in colorectal cancer cells with mutated KRAS. J. Nucl.

Med. 2014, 55, 2038−2044.

(39) Kuo, W.-T.; Lee, T.-C.; Yu, L. C.-H. Eritoran suppresses colon

cancer by altering a functional balance in Toll-like receptors that bind

lipopolysaccharide. Cancer Res. 2016, 76, 4684−4695.

(40) Juang, Y. P.; Lin, Y. Y.; Chan, S. H.; Chang, C. K.; Shie, J. J.;

Hsieh, Y. S. Y.; Guh, J. H.; Liang, P. H. Synthesis, distribution analysis

and mechanism studies of N-acyl glucosamine-bearing oleanolic

saponins. Bioorg. Chem. 2020, 99, 103835.

(41) Pei, Q.; Hu, X.; Li, Z.; Xie, Z.; Jing, X. Small molecular

nanomedicines made from a camptothecin dimer containing a disulfide

bond. RSC Adv. 2015, 5, 81499−81501.

(42) Kuo, W.-T.; Lee, T.-C.; Yang, H.-Y.; Chen, C.-Y.; Au, Y.-C.; Lu,

Y.-Z.; Wu, L.-L.; Wei, S.-C.; Ni, Y.-H.; Lin, B.-R.; Chen, Y.; Tsai, Y.-H.;

Kung, J. T.; Sheu, F.; Lin, L.-W.; Yu, L. C.-H. LPS receptor subunits

have antagonistic roles in epithelial apoptosis and colonic carcino-

genesis. Cell Death Differ. 2015, 22 , 1590 −1604.

(43) Kasahara, T.; Kasahara, M. Characterization of rat Glut4 glucose

transporter expressed in the yeast Saccharomyces cerevisiae: compar-

ison with Glut1 glucose transporter. Biochim. Biophys. Acta 1997, 1324,

44) Augustin, R. The protein family of glucose transport facilitators:

11−119.

It ’s not only about glucose after all. IUBMB Life 2010, 62 , 315 −333.

45) Rudakova, E. V.; Boltneva, N. P.; Makhaeva, G. F. Comparative

H. W.; Christman, J. W.; Pei, D. A peptidyl inhibitor that blocks

analysis of esterase activities of human, mouse, and rat blood. Bull. Exp.

Biol. Med. 2011, 152, 73−75.

(46) Dougherty, P. G.; Karpurapu, M.; Koley, A.; Lukowski, J. K.;

Qian, Z.; Srinivas Nirujogi, T.; Rusu, L.; Chung, S.; Hummon, A. B.; Li,

calcineurin-NFAT interaction and prevents acute lung injury. J. Med.

Chem. 2020, 63, 12853−12872.

(47) Geary, R. S.; Norris, D.; Yu, R.; Bennett, C. F. Pharmacokinetics,

biodistribution and cell uptake of antisense oligonucleotides. Adv. Drug

Delivery Rev. 2015, 87, 46−51.

(48) Visser, G. W. M.; Gorree, G. C. M.; Peters, G. J.; Herscheid, J. D.

M. Tissue distribution of [18F]-5-fluorouracil in mice: effects of route

of administration, strain, tumour and dose. Cancer Chemother.

Pharmacol. 1990, 26, 205−209.

461