Norepinephrine-Transporter-Targeted and DNA-Co-Targeted Theranostic Guanidines

Article

Cite This: J. Med. Chem. XXXX, XXX, XXX −XXX

pubs.acs.org/jmc

Norepinephrine-Transporter-Targeted and DNA-Co-Targeted

Theranostic Guanidines

Zbigniew P. Kortylewicz, Donald W. Coulter, Guang Han, and Janina Baranowska-Kortylewicz*^,^†

†

‡

†,§

^†Department of Radiation Oncology, J. Bruce Henriksen Cancer Research Laboratories, University of Nebraska Medical Center,

Omaha, Nebraska 68132-6850, United States

‡

Department of Pediatrics, University of Nebraska Medical Center, Omaha, Nebraska 68132-2168, United States

§

Department of Radiation Oncology, Hubei Cancer Hospital, Tongji Medical College, Huazhong University of Science and

Technology, Wuhan, Hubei 430074, China

*

S Supporting Information

ABSTRACT: High risk neuroblastoma often recurs, even with aggressive treatments. Clinical evidence suggests that

proliferative activities are predictive of poor outcomes. This report describes syntheses, characterization, and biological

properties of theranostic guanidines that target norepinephrine transporter and undergo intracellular processing, and

subsequently their catabolites are eﬃciently incorporated into DNA of proliferating neuroblastoma cells. Radioactive guanidines

are synthesized from 5-radioiodo-2′-deoxyuridine, a molecular radiotherapy platform with clinically proven minimal toxicities

and DNA-targeting properties. The transport of radioactive guanidines into neuroblastoma cells is active as indicated by the

competitive suppression of cellular uptake by meta-iodobenzylguanidine. The rate of intracellular processing and DNA uptake is

inﬂuenced by the agent’s catabolic stability and cell population doubling times. The radiotoxicity is directly proportional to

1

25

DNA uptake and duration of exposure. Biodistribution of 5-[ I]iodo-3′-O-(ε-guanidinohexanoyl)-2′-deoxyuridine in a mouse

neuroblastoma model shows signiﬁcant tumor retention of radioactivity. Neuroblastoma xenografts regress in response to the

clinically achievable doses of this agent.

meta-[¹³¹I]iodobenzylguanidine (¹³¹I-MIBG), a functional

INTRODUCTION

■

analog of norepinephrine. Originally developed as the

Survival of patients diagnosed with neuroblastoma (NB)

depends on the biological characteristics of their tumor.

Current treatment strategies take into account many of these

characteristics, resulting in substantial outcome improvements

9

1

,2

adrenomedullary imaging agent, MIBG is taken up by the

adrenergic tissues via NET. This radioactive drug has been

10

introduced to the management of NB over 30 years ago. In

3

,4

131

the intervening years, many clinical studies of I-MIBG were

in cases of the low- and intermediate-risk disease. However,

more than half of patients have metastatic disease at diagnosis.

Others develop refractory disease or relapse after the initial

treatment. The 5-year survival in this group is only ∼30%

conducted. Published reports indicate that the treatment is

comparatively safe and provides good disease control.

However, tumor responses are transient. Even at the maximum

5

11

despite multimodal therapies. Still lower survivals of <10% are

tolerated dose of >600 MBq/kg, remissions are of short

1

2−14

131

reported for patients who do not achieve partial response with

induction chemotherapy or have residual bone marrow

duration.

Failure of

I-MIBG to produce durable

responses can be attributed to the dose limiting radiotoxicities

6

disease.

NB is unique among pediatric cancers for its expression of

the norepinephrine transporter (NET). Because NB is one of

the most radioresponsive of all human tumors, the image-

guided NET-targeted molecular radiotherapy is possible with

Special Issue: Women in Medicinal Chemistry

7

,8

Received: March 18, 2019

Published: June 24, 2019

©

XXXX American Chemical Society

A

DOI: 10.1021/acs.jmedchem.9b00437

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Scheme 1. Synthesis of Guanidinoalkyl Esters of 5-[¹²⁵I]Iodo-2′-deoxyuridine^a

a

Reagents and conditions: (a) TsCl, pyridine, 5 °C overnight; (b) DMTrCl in pyridine, DMAP (0.05 equiv), 0 °C → rt, 10 h; (c) levulinic acid,

DCC/DMAP (0.05 equiv), DCM/diethyl ether, rt; (d) ZrCl (1 equiv), CH CN, 15 min, rt; (e) TBDMSCl, pyridine, rt, 14 h. (f) Method A: (i)

4

3

+

from 5′-O-p-tolylsulfonyl-2′-IUdR (5), LiN , DMF, 70 °C; (ii) Dowex 50 H in 50% EtOH; (iii) triphenylphosphine, 1 N NH OH, 35 °C. Method

3

4

B: (i) from 5′-O-p-tolylsulfonyl-2′-deoxyuridine, NaN , DMF, 90 °C; (ii) hydrogenation (30 psi), 10% Pd/C, rt, 2 h; (iii) Hg(OAc) in 0.5 M

3

2

+

CH COONa, pH 6, 50 °C; (iv) I /EtOH, Dowex 50 H column; (g) N,N′-bis(t-butoxycarbonyl)-N″-triﬂuoromethanesulfonyl guanidine (1.1

3

2

equiv), TEA (2.2 equiv), dioxane/water, 6 h, rt; (h) Sn (CH ) , (1.25−1.70 equiv) in EtOAc or dioxane, TEA (2−4 equiv), (Ph P)Pd(II)Cl

2

3

6

3

2

1

25

(

0.06−0.10 equiv), reﬂuxed under nitrogen (1−3 h); (i) Na I in NaOH (22.2−148 MBq), 30% H O (5 μL), TFA/CH CN (0.1% v/v), 1 min

2

3

sonication, 6−15 min reaction time, HPLC puriﬁcation; (j) (i) TFA (neat, 100 μL), sealed vial at 55−65 °C, 20−35 min; (ii) HPLC separation

with solvents containing 0.07% TFA (v/v); (iii) ﬁnal HPLC separation, eluting with a gradient of EtOH in potassium phosphate buﬀer (10 mM

PB, pH 6.1); (k) TBDMS-IUdR (4), 6-N-Boc-aminohexanoic acid, DCC/DMAP in DCM, rt, 6 h; (l) (i) TBAF (1 M) in THF, rt, 5 h; (ii) TFA,

CH CN, rt 1 h; (m) 5-iodo-3′-O-levulinyl-2′-deoxyuridine (3), N-Boc-β-alanine (for 14a) or 6-N-Boc-aminohexanoic acid (for 14b), DCC/

3

DMAP activation, 4−6 h; (n) TFA/MeCN, rt, 3 h; (o) N H ·H O/pyridine/AcOH (0.3:9.5:2.4, v/v); (p) (i) 5-iodo-3′-O-levulinyl-2′-

2

4

2

deoxyuridine (3), cyclo-3,5-di(tert-butyl)-6-ﬂuorochlorophosphite, CH CN, DIPEA, −40 °C → rt, 2 h; (ii) t-BuOOH, −20 °C → rt, 1 h; (r), (i) 6-

3

N-Boc-aminohexanoic acid, DCC/DMAP, in DCM, rt, 4 h; (ii) TFA/MeCN, rt, 4 h; (s) (i) 5-iodo-3′-O-levulinyl-2′-deoxyuridine (3), 4-(Boc-

aminomethyl)benzoic acid, DCC/DMAP activation, rt, 3 h; (t) 5-iodo-5′-O-DMTr-IUdR (2), 6-(Boc-aminohexanoyl)-N-phenylglycine, DCC/

DMAP in DCM, rt, 5 h.

of ¹³¹I and the mean range of β-particles that leaves small

transplant. Some patients also develop secondary malignan-

19−22

131

tumors underdosed while the decay energy is deposited in the

cies.

High-dose therapy with I-MIBG is reported to

1

31

23

adjacent normal tissues. Additionally, the washout of

I-

damage ovaries. Thyroid disorders including hypothyroidism

and thyroid nodules are common after I-MIBG therapy.

Over 80% of the long-term survivors treated with I-MIBG

1

31

MIBG from NB cells is rapid with the half-life t < 60

1

/2

1

5−17

131

min.

This relatively poor retention of MIBG in NB cells

24

reduces the cumulative radiation doses delivered to tumor

cells.

develop hypothyroidism. Nonetheless, the risk of these

complications is lower than the risk of disease progression. It is

evident that targeted molecular radiotherapy has a curative

potential in NB; however, this approach must be optimized.

The success of many therapeutic approaches is eclipsed by the

fact that children develop severe acute and delayed toxicities.

¹³¹I-MIBG is radiotoxic to normal tissues and suppresses the

bone marrow function. Hematologic toxicities are the main

1

1−14,18

short-term adverse event.

Patients treated with high

131

doses, ≥400 MBq/kg, of

I-MIBG require bone marrow

B

DOI: 10.1021/acs.jmedchem.9b00437

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

the subsequent non-carrier-added electrophilic ²⁵I-iododes-

1

New therapies must attain a rational balance between survival

and morbidity from these late adverse eﬀects. An optimal

therapy would selectively deliver cytotoxic doses to NB cells

while causing as little injury as possible to the healthy tissues

and organs.

1

25

tannylation of organotin precursors to the target I-iodinated

products. This well-proven radioiodination sequence based on

27

the modiﬁed Stille coupling and originally applied in our

1

25

laboratories to produce no-carrier-added 5-[ I]iodo-2′-

28

29−32

We developed a series of new compounds that can be

radiolabeled with Auger electron-emitting radioiodines, which

are expected to be superior as the therapeutic radionuclides in

NB. Auger electrons are extraordinarily radiotoxic only when

either within the structure of DNA or in its immediate vicinity

and practically harmless when located in the cytoplasm or

extracellular spaces. Nearly all electron energy associated with

deoxyuridine and other radiolabeled compounds

served

just as eﬀectively in this project.

To prepare nonradioactive iodo analogues, after the

esteriﬁcation of selected N-Boc-amino acid (using DCC/

DMPA or BDDC activation) by a suitably 5′-O- or 3′-O-

protected derivative of IUdR (1), the N-Boc group of resulting

amino ester was cleaved and the guanidinylation carried out

1

25

the I decay is deposited within a sphere smaller than the cell

with N,N′-bis(tert-butyloxycarbonyl)-N″-triﬂuoromethanesul-

2

5,26

33,34

nucleus.

New radioactive guanidines are transported into

fonyl guanidine.

In the preparation of 6, the required 5-

NB cells via NET, much like MIBG. Subsequently, these

agents are also processed intracellularly into a thymidine

analogue and their mode of action includes targeting of DNA

in proliferating NB cells. The incorporation of the radioactive

catabolite into the structure of DNA ensures retention of the

radionuclide within the NB cell. Decaying Auger electron

emitting radionuclides deposit high linear energy transfer

iodo-5′-amino-2′,5′-dideoxyuridine was obtained in the overall

yield of ∼56%, as follows: 5-iodo-5′-O-p-tolylsulfonyl-2′-

deoxyuridine (5) was reacted with lithium azide, attained 5-

iodo-5′-azido-2′,5′-dideoxyuridine reacted with triphenylphos-

phine, and a crude product hydrolyzed with concentrated

ammonium hydroxide. The yield of 5-iodo-5′-amino-2′,5′-

dideoxyuridine was later improved to ∼78% by replacing 5

with5′-O-p-tolylsulfonyl-2′-deoxyuridine in the initial reaction

with lithium azide. The resultant 5′-azido- 2′,5′-dideoxyuridine

was hydrogenated to 5′-amino-2′,5′-dideoxyuridine, reacted

with mercuric acetate to produce 5-mercuriacetate of 5′-

amino-2′,5′-dideoxyuridine, which was iodinated with ele-

mental iodine. To acquire guanidinoalkyl ester 10, IUdR was

selectively 5′-O-silylated with TBDMSCl in pyridine to yield 4

in 79% and reacted with 6-N-Boc-aminohexanoic acid. The 5′-

O-position of N-Boc-aminohexanoyl ester was deprotected

with a 2% solution of TBAF/THF and the N-Boc-group

cleaved in a 20% TFA/MeCN mixture. The subsequent

guanidinylation gave 10 in the overall yield of 67%.

Preparation of guanidino esters 14a, 14b, and 22 started

with 5-iodo-3′-O-levulinyl-2′-deoxyuridine (3) obtained from

(

LET) radiation within this most radiosensitive structure of

the NB cell and remain there until the cell’s death. The unused

agent is rapidly degraded to free radioiodide and eliminated.

With this approach, we expect to deliver therapeutic radiation

doses to the NB cells while sparing normal tissues.

RESULTS

■

Chemistry. A common radioiodination method allowed

preparation of all reported guanidinoalkyl esters of 5-

1

25

[

I]iodo-2′-deoxyuridine: 13 (IDG), 17a, 17b, 21, 25 along

125

with 5-[ I]iodo-5′-N-guanidino-2′,5′-dideoxyuridine (9) and

1

25

125

3

-[ I]iodobenzylguanidine ( I-MIBG, 29). The processes

shown in Schemes 1 and 2 involve three consecutive steps: the

initial preparation of nonradioactive iodo analogs followed by

the stannylation of iodides to the corresponding stannanes and

5-iodo-5′-O-dimethoxytrityl-2′-deoxyuridine (2) in the reac-

tion with 4-oxopentanoic acid (DCC/DMAP activation, ∼78%

yield) and the subsequent cleavage of dimethoxytrityl group-

Scheme 2. Synthesis of ¹²⁵I-MIBG^a

35

with ZrCl in CH CN. The 3′-O-Lev protected 3 was used in

4

3

the similarly carried out esteriﬁcation of Boc-β-alanine, 6-(Boc-

amino)caproic acid, and 4-(Boc-aminomethyl)benzoic acid to

yield correspondingly 5-iodo-5′-O-(3-N-Boc-aminopropionyl),

5

(

-iodo-5′-O-(6-N-Boc-aminohexanonyl), and 5-iodo-5′-O-[4-

N-Boc-aminomethyl)benzoyl] derivatives of 5-iodo-3′-levu-

linyl-2′-deoxyuridine. These, after the removal of N-Boc amino

protection, the guanidinylation, and the ﬁnal cleavage of 3′-O-

3

6

Lev group, gave the target guanidinoalkyl esters in

satisfactory yields, 14a (73%), 14b (69%), and 22 (67%).

The synthesis of guanidino ester 18 started with 5-iodo-5′-O-

a

Reagents and conditions: (a) N,N′-bis(tert-butoxycarbonyl)-N″-

[

cyclo-3,5-di(tert-butyl)-6-ﬂuorosaligenyl]-2′-deoxyuridine

triﬂuoromethanesulfonylguanidine (1.1 equiv), TEA (1.1 equiv) in

DCM, 0 °C → rt, ∼4 h; (b) (i) Sn (CH ) , (1.9 equiv) in EtOAc,

TEA (4 equiv), (Ph P)Pd(II)Cl (0.1 equiv), reﬂuxed under argon

37

monophosphate attained via phosphorus(III) route, as we

2

3 6

30

previously reported. In two steps, 5-iodo-3′-O-levulinyl-2′-

deoxyuridine 3 was coupled with cyclo-3,5-di(tert-butyl)-6-

ﬂuorochlorophosphite in the presence of N,N-diisopropylethyl-

amine and the generated phosphite, oxidized directly with tert-

butyl hydroperoxide, produced the 5′-cyclo-saligenyl derivative

of 3, which again, after cleavage of 3′-O-Lev group and the ﬁnal

reaction with 6-(Boc-amino)caproic acid, provided 5-iodo-5′-

O-[cyclo-3,5-di(tert-butyl)-6-ﬂuorosaligenyl]-3′-O-(6-N-Boc-

aminohexanonyl)-2′-deoxyuridine monophosphate. The elim-

ination of N-Boc protecting group followed by the

guanidinylation led to the guanidinoalkyl ester 18 with the

overall yield of 79%.

3

2

(

1−3 h); (ii) a silica gel column using EtOAc/n-hexanes (1:4), ∼91%

pure by HPLC analysis (at 220/280 nm); (iii) further HPLC

puriﬁcation, C18 100 Å, (5 μm, 10 mm × 250 mm), semipreparative

1

25

column (60 mg, ∼5 mg per injection); (c) (i) Na I in NaOH, done

1

25

twice with 59.2 and 58.1MBq of Na I/NaOH, to give overall 104.4

MBq (89% average yield), 30% H O (5 μL), TFA/CH CN (0.1% v/

2

3

v), 1 min sonication, 6−15 min at rt; (ii) HPLC puriﬁcation; (d) (i)

carried out with 31.8, 44.4, and 50.3 MBq of 28, TFA (neat, 100 μL),

sealed vial at 55−65 °C, 20−35 min, 86% yield; (ii) HPLC separation

with solvents containing 0.07% TFA (v/v); (iii) ﬁnal HPLC

separation, eluting with a gradient of EtOH in potassium phosphate

buﬀer (10 mM PB, pH 6.1).

C

DOI: 10.1021/acs.jmedchem.9b00437

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Organotin precursors were obtained through the standard

stannylations of above-described iodides with hexamethylditin

catalyzed by bis(triphenylphosphine)palladium(II) dichloride

in the presence of triethylamine. Reactions were carried out

under nitrogen in reﬂuxed dioxane or ethyl acetate, depending

on the solubility of starting iodide. In order to achieve ≥98%

purity of separated stannanes, successive HPLC puriﬁcations

were often necessary. However, once puriﬁed, anhydrous

samples of stannanes stored with the exclusion of light under

nitrogen at −20 °C did not decompose excessively, ≤7% by

the HPLC analysis, and even after lengthy storage of up to six

sera having comparable levels of nonspeciﬁc esterases but

lacking carboxylesterase EC 3.1.1.1. And second, we intend to

use juvenile pigs as the animal model in preclinical safety

40−42

studies.

humans.

Pigs have many physiological similarities to

Their tissue volumes are comparable to humans,

43,44

and so the assessment of radiation eﬀects in clinically relevant

volumes and more realistic estimates of the radionuclide

biodistribution and absorbed radiation doses to normal organs

4

3,45,46

are possible.

Compound 21 is slowly processed in

1

25

human serum to IUdR monophosphate with a half-life of

∼15 h (Supporting Information pp S78, S79). A similar

degradation pathway is observed during the cellular processing

of 21. The doubly negative charge of this catabolite provides a

lock-in mechanism; i.e., this product is unable to eﬄux across

the cellular membrane. Consequently, it is trapped intra-

cellularly and available at any time the cell commences its

DNA synthesis. Compound 9 , similar to MIBG, is stable in

human and mouse sera (Supporting Information p S14). The

estimated half-lives of new compounds span a wide range from

∼0.4 to 15 h. As expected, 5′-substituted derivatives appear to

be more susceptible to hydrolysis than 3′-regioisomers.

Shifting the substitution from the 5′- to 3′-position, 17b

versus IDG, almost triples the half-life in human serum. The

lengthening of the 5′-side chain to caproic acid also prolongs

the half-life. The addition of a bulky substituent at the 5′-

position as in 21 extends the serum half-life to 15 h. This

ﬁnding suggests potential new directions for the design of

additional derivatives if the preclinical performance of the

current candidate is found inadequate.

1

25

months were suitable for the immediate

I-radioiodo-

destannylation. Higher hydrophobicity of stannanes compared

to the matching iodides allowed for a complete separation of

the trimethyltin precursor from ¹I-iodinated products in a

single HPLC puriﬁcation even when large volumes of the

crude reaction mixtures were loaded onto the column.

25

Biological Evaluation. Stability. The ex vivo stability of

radioiodinated guanidines was assessed in PBS, cell culture

medium without NB cells and in the presence of NB cells after

various times of incubation and in human, mouse, and porcine

sera. The analytical details are provided in the Supporting

Information (pp S29−S32, S47−S48, S61−S64, S77−S79,

S92−S94). Half-lives of new compounds in serum were

calculated from the HPLC analysis of the area under the peak

of recovered intact compounds and their catabolite(s) in the

processed incubation mixtures (Table 1). The degradation

Table 1. Half-Lives of Radiolabeled Compounds in Human

Serum

In advance of the in vitro studies, we also measured the

stability of new agents in PBS and in cell culture media

longitudinally to ensure that the data acquired in the in vitro

experiments represent properties of the intact compound. The

tested compounds are stable in PBS and in cell culture in the

absence of NB cells (Supporting Information pp S19, S35, S54,

S69, S86, S102). Figure 1D shows the analyses of IDG

incubated in cell culture medium for 24 h at 37 °C in 5% CO₂

atmosphere. After 24 h at these conditions, 86% of IDG

remains intact. The estimated half-life of IDG in the cell

culture medium is ∼110 h. When radioactive guanidines are

added to the exponentially growing monolayers of NB cell

compd

half-life (std err), h

a

9

nd

IDG

2.28 (0.08)

0.39 (0.04)

0.89 (0.09)

15.00 (0.62)

0.76 (0.22)

1

2

7a

7b

1

5

a

Stable for days.

rates followed ﬁrst order kinetics, and data were ﬁtted into a

monoexponential equation with the intercept set at 100%

(

Figures 1E and 5F) and incubated with cells for 24 h, the

recovered media contain ∼30%−44% of the intact compounds

(

Figure 1C). Data in Figure 1 illustrate the HPLC method

1

25

and ∼40%−45% of IUdR. Since these compounds are stable

used to establish diﬀerences in the stability of new compounds

in serum. In a typical experiment, the radioactive compound

was added to serum to produce a ﬁnal concentration

in media without cells, the considerable loss of the intact

1

25

compound and appearance of I-1 in the extracellular spaces

are indicative of the uptake and intracellular processing of

radioactive guanidines. Nucleosides can cross the plasma

membrane of mammalian cells by a facilitated diﬀusion process

131

approximating the levels of₁I-MIBG in blood of children

1,38

receiving therapeutic doses.

Compounds IDG, 17a, 17b, and 25, irrespective of their

47,48

125

overall stability, produce a common radioactive catabolite in

that is bidirectional.

We propose therefore that IUdR is

1

25

the form of IUdR, ensuring that the DNA targeting can be

realized. After 5 min incubation in human serum, 93% of IDG

remains intact (Figure 1A). In comparison, >20% of 17b is

produced intracellularly, and if it is not taken up into the DNA,

1

25

IUdR diﬀuses freely through the cellular membrane and

1

25

emerges in the cell culture media. The production of IUdR

as a common catabolite of new radioactive guanidines is

considered a positive outcome. Preclinical and clinical studies

established that radioiodinated IUdR undergoes rapid hepatic

degradation and has a half-life in the systemic circulation of

∼10 min, ensuring minimal normal tissue exposure to

1

25

converted into

IUdR (Figure 1B) under the same

conditions. None of the tested compounds are stable in

mouse serum, which contains four esterases including

carboxylesterase. Human and porcine sera do not contain

39

any carboxylesterase activity. Half-lives of IDG, the agent

43,49

selected for the in vivo testing, were estimated at 0.059 h ±

radiation, especially important in pediatric subjects.

0

.021 h, 2.28 h ± 0.08 h, and 3.59 h ± 0.12 h (average ± std

Cellular Uptake. All new guanidines have pK ∼ 12 (Table

a

err) in mouse, human, and pig serum, respectively. The

evaluation of new compounds in porcine serum served a dual

purpose. First, we were able to compare the stability in two

2) and are protonated at physiological pH similar to MIBG,

which exists as a monocation over a broad pH range resulting

in a poor cell membrane permeability that hinders cellular

D

DOI: 10.1021/acs.jmedchem.9b00437

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Figure 1. Determination of the stability of radioactive guanidines in human serum. HPLC analyses of regioisomers IDG and 17b. (A) HPLC

analysis of IDG incubated in human serum. (B) HPLC analysis of 17b incubated in human serum. (C) Comparison of the monoexponential

degradation curves for tested compounds in human serum. Dotted line indicates 50%. (D) HPLC analyses of IDG incubated for 24 h in cell culture

media without cells. (E) HPLC analyses of IDG incubated for 24 h in cell culture media with neuroblastoma cells SK-N-SH. (F) HPLC analyses of

1

7b incubated for 24 h in cell culture media with neuroblastoma cells SK-N-SH. Peaks highlighted in red indicate the intact compound. Blue peaks

1

25

correspond to the catabolic product IUdR. HPLC conditions: column Columbus C18 100 Å (5 μm; 4.6 mm × 250 mm); ﬂow 0.8 mL/min;

linear gradient from 0% to 95% CH CN over 50 min followed by isocratic 95% CH CN for 20 min; both solvents with 0.07% TFA.

3

Table 2. pK , pI, and log P of Radioactive Guanidines

the passive diﬀusion across the cell membrane, whereas the

active transport and binding to NET appear to be sterically

hindered by the deoxyribose residue. While this outcome is

a

compd

pK_a

pI

log P

9

IDG

1

2

11.5

12.1

11.9

12.2

12.1

11.9

11.8

9.99

−1.30

−0.20

−1.48

−0.20

4.58

disappointing (9 was produced with the expectation that it

10.29

10.18

10.31

10.30

10.16

nd

52,53

might mimic the in vivo biological behavior of MIBG

and

7a

7b

1

25

additionally allow the incorporation and retention of I into

54

DNA of proliferating cells ), the lack of uptake of 9 by NB

cells conﬁrms that our radioactive guanidines require active,

NET-facilitated transport across the plasma membrane.

The remaining target compounds IDG, 17a, 17b, 21, and 25

are taken up by NB in a time- and concentration-dependent

manner (Figure 2A and Figure 2B). Uptake increases linearly

within the tested range of extracellular concentrations, up to

5

0.26

1.63

MIBG

50,51

uptake by passive diﬀusion.

Cellular uptake of radioactive

guanidines was evaluated in two human NB cells lines SK-N-

SH (SK) and BE(2)-C (BE) and in one murine NB cell line

N1E-115 (N1E). Compound 9 is not taken up by NB cells.

After 1 h with 9 at the extracellular concentration of 38 kBq/

−

9

2

00 kBq/mL (2.45 × 10 M), resembling MIBG uptake,

−

7

which is linear up to ∼1 × 10 M and begins to saturate at

−

7

55

1

25

concentrations >5 × 10 M. Uptake of new radioactive

mL, amounts of I barely detectable in a γ-counter were

−

5

−5

guanidines is also dependent on the cell population doubling

recovered with SK cells (1.0 × 10 ± 1.4 × 10 mBq/cell). A

time (T ). When cells are incubated with IDG for 1 h, washed

continuous exposure of SK cells to 9 for 24 h produced only a

D

1

25

−4

marginal increase in the cellular I levels (5.4 × 10 ± 1.8 ×

with nonradioactive medium and PBS, and processed

immediately, cellular uptake is nearly twice as high in BE

cells (T = 19 h) as it is in SK cells (T = 40 h), indicating

−

5

1

0

mBq/cell). This lack of cellular uptake can be attributed

to the positive charge of the guanidine residue, which prevents

D

E

DOI: 10.1021/acs.jmedchem.9b00437

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Journal of Medicinal Chemistry

Article

Figure 2. Cellular uptake and retention of radioactive guanidines in neuroblastoma cell lines in vitro. (A) Time dependent uptake of 17b in SK-N-

SH cells. (B) Concentration dependent uptake of IDG in SK-N-SH cells. (C) Comparison of cellular uptake of IDG by BE(2)-C (orange) and SK-

N-SH (blue) cells. Cells were treated with IDG for 1 h and harvested and immediately processed (1 h) or were washed with nonradioactive

medium and PBS, and returned to the incubator for an additional 23 h (24 h). (D) Subcellular distribution of IDG in SK-N-SH cells. (E)

Subcellular distribution of IDG in BE(2)-C cells. Cytoplasm = black; DNA in SK-N-SH = blue; DNA in BE(2)-C cells = orange. (F) Comparison

1

25

of subcellular distribution of I-MIBG (blue) and 17b (orange) in N1E-115 cells. (G) Cellular uptake of 21 in SK-N-SH cells after 1 and 24 h as

described above. (H) Surviving fractions of SK-N-SH cells treated with 21 for 1 h, washed, and incubated for additional 23 h. (J) Subcellular

distribution of 21 in SK-N-SH cells; cytoplasm = black; DNA = blue. (K) Comparison of cellular uptake of several radioactive guanidines in SK-N-

SH cells normalized to the extracellular concentration of 37 kBq/mL. All data are shown as average (n ≥ 3) and standard deviation unless otherwise

indicated.

greater levels of ¹²⁵I incorporation into the DNA of rapidly

proliferating BE cells. Allowing cells to grow for additional 23 h

after the removal of the radioactive media equalizes per cell

uptake because the number of BE cells more than doubles in

subcellular distribution of ¹²⁵I-MIBG and 17b in N1E cells is

1

25

shown in Figure 2F. The majority of I-MIBG after 24 h

incubation was recovered in the cytoplasmic fraction. Only

1

25

∼19% of I was recovered in the nuclear fraction and 0% in

1

25

2

4 h and now each daughter cell has approximately one-half of

DNA. In contrast, in cells exposed to 17b, practically all

I

the initial radioactivity (Figure 2C). These experiments also

was recovered in the DNA pellet (>99%). The overall

comparison of all radioactive guanidines in terms of the total

cellular uptake is shown in Figure 2K. Compound 25,

structurally closest to MIBG, is retained by SK cells at levels

conﬁrmed the intracellular processing of these agent with

1

25

production of IUdR, which can diﬀuse from cells reducing

its intracellular pool. Subcellular distribution studies corrobo-

rate this notion. After 1 h with IDG, SK cells retain ∼80% of

the total recovered ¹I in their DNA compared to 93% in

DNA of BE cells (Figure 2D, Figure 2E). At 24 h, practically all

1

25

marginally lower than I-MIBG. Because of their short half-

lives in human serum, 25 (t₁_/₂= 45 min), 17a (t₁_/₂= 23 min),

and 17b (t₁_/₂= 53 min) were not evaluated further. On the

25

1

25

125

I is associated with DNA in both cells lines. The intracellular

basis of the high cellular uptake, >4× greater compared to I-

processing of radioactive guanidines and DNA co-targeting is

also evident in studies of compound 21. The subcellular

distribution of 21, which is slowly catabolized to mono-

phosphate of IUdR, reveals the intracellular trapping of this

negatively charged catabolite and consequently a higher

cellular and cytoplasmic retention of radioactivity at 1 and

MIBG, and good stability in human serum (t1/2 = 137 min),

IDG appeared to be the candidate drug most likely to succeed

in the in vivo studies.

1

25

Competition with MIBG. SK and BE cells express high

levels of endogenous NET and represent a well-established

53,56−59

robust model for the NET-speciﬁc MIBG uptake.

Two

2

4 h, even after repeated washes (Figure 2G, Figure 2J). In the

variations of the binding assay were employed to determine the

NET-directed uptake of new radioactive guanidines: the cell

suspension and the cell monolayer method. Both assays

conﬁrmed that the active uptake of radioactive guanidines is

competitively inhibited by nonradioactive MIBG indicating the

same speciﬁc transport mechanism. MIBG inhibits uptake of

IDG, 17b, and 21, three compounds evaluated in this assay.

Exponentially growing monolayers of BE cells exposed to 21

case of 21, which is more stable compared to IDG, the fraction

of

corroboration of the intracellular trapping and processing of

radioactive guanidines. Cell survival is proportional to the

levels of I incorporation (Figures 2H and 4). Similar results

were observed in N1E cells, an NB cell line of murine origin

with T_D= 36 h similar to SK. The comparison of the

1

25

I associated with DNA is less, providing further

1

25

F

DOI: 10.1021/acs.jmedchem.9b00437

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Journal of Medicinal Chemistry

Article

Figure 3. Dependence of the cellular uptake of new agents on the availability of norepinephrine transporters in neuroblastoma cell lines. (A)

Competition of 21 with nonradioactive MIBG in BE(2)-C cells grown as a monolayer. Cells were exposed to 37 kBq/mL 21 without (blue) and

with (orange) 100 μM nonradioactive MIBG. (B) Temperature-dependent uptake of IDG (300 kBq/mL) in BE(2)-C cells and competition with

nonradioactive MIBG analyzed in a ﬁltration assay. (C) Cell cycle analysis of quiescent SK-N-SH cells. (D) Lack of competition of IDG with

nonradioactive MIBG in quiescent SK-N-SH cells. (E) Western blot of norepinephrine transporter expression in cell lysates from SK-N-SH cell in

the exponential (exp) growth or in G0. (F) Western blot of β-actin to verify uniform protein loads. (G) Cellular uptake of IDG in SK-N-SH cells at

various levels of conﬂuency. All data are shown as average and standard deviation (n ≥ 3).

after 1 h incubation with 100 μM nonradioactive MIBG had

uptake of 21 reduced by ∼28% compared to cells treated with

of 0.1 mM and in some instances up to 1 mM are

63,64

employed.

MIBG can disrupt normal cellular functions at

2

1 alone. The extended 3 h incubation with nonradioactive

such high concentrations. Moreover, MIBG is far more

lipophilic than our new compounds (Table 2) and can cross

the plasma membrane unaided by NET. At low concentrations

comparable to the physiological levels of norepinephrine, ∼1 ×

MIBG reduced the uptake by 40% (Figure 3A). Cellular

uptake of radioactive guanidines is also energy dependent

conﬁrming their active transport into the NB cell. When cells

are cooled on ice to 4 °C and the MIBG inhibition assay is

conducted at this temperature, signiﬁcantly reduced uptake of

IDG is observed compared to experiments run at 37 °C

−

9

−7

10 to 1 × 10 M, MIBG uptake is predominantly via the

NET-facilitated active transport process, whereas at non-

physiological levels typically used in the competitive binding

65,66

(Figures 3B). At low temperatures, precipitation of MIBG was

assays, MIBG uptake occurs through the passive diﬀusion.

Concentrations of our radioactive guanidines do not exceed 1

noted at concentrations of >80 μM. Similar to BE cells, uptake

in SK cells depends on the temperature. SK cells incubated

with 37 kBq/mL IDG at 37 °C take up 1.85 ± 0.01 mBq/cell.

In a parallel uptake study conducted at 4 °C, SK cells retained

^oⁿ^l^y⁰^.⁰⁵^±⁰^.⁰²^m^B^q^/^c^e^l^l^.^T^h^e^e^s^tⁱ^m^a^t^e^d^I^C⁵0 values are

^lⁱ^s^t^e^dⁱⁿ^T^a^b^l^e³^.^W^e^c^oⁿ^sⁱ^d^e^r^t^h^e^I^C⁵0 estimates from

−

8

× 10 M, i.e., are well within the active transport limits.

We have also undertaken studies intended to uncouple

MIBG-competed uptake from the DNA targeting. We

hypothesized that quiescent SK cells produced by the cell

density-dependent inhibition of proliferation will not be able to

1

25

incorporate IUdR, the catabolic product of our compounds,

into their DNA but will retain their ability to transport

guanidines via the NET function. BE and N1E cells cannot be

used in this experiment because their growth is not inhibited at

high cell densities. Density-inhibited SK cells were maintained

in culture with daily additions of fresh media for 3 days after

reaching 100% conﬂuency. Cell cycle analyses indicated nearly

Table 3. Half Maximal Inhibitory Concentrations (IC ) of

Nonradioactive MIBG in Competition with IDG in BE(2)-C

and SK-N-SH Human Neuroblastoma Cell Lines

50

average IC₅₀(std err), μM

conditions

BE(2)-C

8.2 (2.1)

SK-N-SH

76.2 (9.3)

monolayer (1 h)

cell suspension

9

0% of cells in G0 phase with no discernible S phase cells

(

Figure 3C). However, we unexpectedly found that uptake of

3

4

7 °C

°C

28.7 (4.4)

23.9 (1.5)

35.2 (16.6)

22.0 (6.6)

IDG by quiescent SK cells is insensitive to MIBG inhibition.

Only ∼15% reduction of uptake was detected at the highest

tested MIBG concentration (Figure 3D). The Western blot of

proteins derived from lysates of exponentially growing SK cells

indicates the presence of a single protein band, MW ∼ 68 kDa,

detectable with anti-NET antibodies. In the same Western

blot, protein lysates from quiescent SK cells show two protein

bands, the minor 68 kDa band and a more prominent 47 kDa

band (Figure 3E). It appears that allowing SK cells to arrest in

G0 not only aﬀects proliferation but also renders a signiﬁcant

fraction of NET inactive. In follow-up experiments, we found

that cellular uptake of radioactive guanidines by SK cells is

inversely proportional to the cell density (Figure 3G).

Consequently, it was not possible to clearly separate MIBG-

inhibited uptake of new compounds from the DNA co-

targeting. An exhaustive literature search did not uncover any

information regarding the expression and functional activity of

NET in quiescent NB cells. This aspect of NET expression and

experiments conducted in a monolayer a better representation

^o^f^t^h^e^c^o^m^p^e^tⁱ^tⁱ^v^eⁱⁿ^hⁱ^bⁱ^t^o^r^y^e^ﬀ^e^c^t^o^f^M^I^B^G^.^T^h^e^hⁱ^g^h^e^r^I^C⁵0

in SK cells is a manifestation of the fact that the SK cell line

consists of three distinct subpopulations, N-, S- and I-types of

60

which N-type cells express ∼7× higher NET than the I-type.

In contrast, BE cell line is a homogeneous I-subtype. The cell

suspension assay has several shortcomings in the evaluation of

compounds that co-target DNA. SK and BE cells in suspension

fail to maintain their normal functions after ∼1−2 h and are

especially vulnerable at 4 °C. SK cells seem to be a little more

resilient. Further complications arise from the cytotoxicity of

nonradioactive MIBG when used at high concentrations.

MIBG is cytotoxic in a large panel of diverse cancer cell lines

61,62

without preference against tumor cells of neural origin.

In a

typical competitive inhibition assay, high MIBG concentrations

G

DOI: 10.1021/acs.jmedchem.9b00437

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Figure 4. Radiotoxicity of guanidines in SK-N-SH and BE(2)-C cells. (A) Surviving fractions of SK-N-SH cells 24 h after treatment with various

concentration of IDG. (B) Concentration-dependent clonogenic survival of SK-N-SH cells treated with IDG. (C) Colonies formed by SK-N-SH

cells 14 days after plating: (a) untreated controls cells; (b) cells treated with 18 kBq/mL IDG; (c) cells treated with 35 kBq/mL IDG. (D) Number

125

of I accumulated in DNA of BE(2)-C cells exposed to the increasing concentrations of IDG for 1 h, extensively washed and harvested with

1

25

trypsin. (E) Evaluation of clonogenic survival of BE(2)-C as a function of the intracellular content of I. (F) Colonies formed by BE(2)-C

1

25

untreated control cells. (G) Colonies formed by BE(2)-C with the intracellular I accumulation of 0.3 mBq/cell after 1 h treatment with IDG.

(

H) ImageJ analyses of colonies in (F). (J) ImageJ analyses of colonies in (G). All data are shown as an average (n ≥ 3) and standard deviation

unless otherwise indicated.

function in NB cells will require comprehensive studies beyond

the scope of this report.

important to determine if the radioactivity retained by BE cells

after short treatment of 1 h is suﬃcient to produce measurable

radiotoxicity.

Radiotoxicity. Radiotherapeutic potential of radioactive

guanidines was ﬁrst evaluated in vitro. The survival of NB

cells was assessed at 24 h after treatment using the trypan blue

exclusion method followed by the clonogenic assay to measure

the reproductive integrity of treated cells. Both end points were

dependent on the compound concentration and duration of

exposure (Figure 4). Twenty-four hours after the exposure to

IDG, only ∼60% of SK cells survive at the extracellular

concentration of 180 kBq/mL (Figure 4A). The clonogenic

assay of cells harvested in this experiment, replated at 500 live

cells/ﬂask and grown in nonradioactive medium for 14 days,

indicates that after the exposure to the extracellular

concentration of 35 kBq/mL IDG, the majority of SK cells,

To answer this question and relate the cell survival to the

duration of exposure, BE cells were treated for 1 h with several

concentrations of IDG, washed, harvested and replated for the

clonogenic assay. Cellular uptake was measured and the

1

25

number of I atoms per cell in DNA estimated (Figure 4D).

A brief 1-h exposure to IDG resulted in the incorporation of

1

25

>6,000

I/cell. Extracellular concentrations required to

1

25

achieve this level of I in DNA are expected to be easily

attainable in a clinical setting. At the intracellular retention

levels of 0.3 mBq/cell and 0.8 mBq/cell, only a fraction of BE

cells, 0.6 and 0.4, respectively, survive, retain their reproductive

integrity, and form colonies (Figure 4E), which are

considerably smaller compared to the untreated cells (Figure

4F, Figure 4G). Image analyses also revealed the signiﬁcant

decrease in the crystal violet intensity in colonies. This

parameter is proportional to the number of cells in each colony

(Figure 4H, Figure 4J).

>

90%, lose their reproductive integrity (Figure 4B). Cells

treated with concentrations IDG > 100 kBq/mL did not

produce any colonies even after 21 days in culture. Typical

colony forming assay results are shown in Figure 4C for the SK

cells, untreated (a) and after treatment with IDG at the

concentration of 18 kBq/mL (b) and 35 kBq/mL (c). BE cells

respond to treatment with radioactive guanidines much like SK

cells. Given the ∼2.3 h half-life of IDG in human serum, it was

The estimated extracellular concentration of IDG required

to kill 63% of cells (D ) is approximately 25× greater for the 1

3

7

h treatment as compared to the 24 h treatment (Table 4).

H

DOI: 10.1021/acs.jmedchem.9b00437

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Journal of Medicinal Chemistry

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Table 4. Estimated Intracellular and Extracellular ¹²⁵I

Concentrations, Average (std error), Needed To Kill 63%

escapes into the extracellular spaces, reducing the cellular pool

1

25

of IUdR available for DNA uptake. The cell survival is

inversely proportional to the radioactivity located in the cell

(

D ) of BE(2)-C cells treated for 1 h and SK-N-SH cells

37

1

25

treated for 24 h with IDG

nucleus. This comparison of I-MIBG and IDG conﬁrms a

1

25

remarkable dependence of I radiotoxicity on the subcellular

location and highly localized energy deposition around the

BE(2)-C

SK-N-SH

24 h

1

h

125

decay site. This property of I also indicates that compared to

1

31

extracellular (kBq/mL)

intracellular (mBq/cell)

398 (48)

0.91 (0.29)

15.6 (2.3)

1.20 (0.27)

I-MIBG, a signiﬁcant reduction of absorbed radiation doses

by normal tissues is achievable, a property of great

consequence in pediatric patients.

In Vivo Evaluation. On the basis of its favorable stability in

human serum, in vitro uptake, and radiotoxicity in human NB

cells, IDG was selected for biodistribution, whole body

clearance, and therapy studies in immunodeﬁcient female

and male mice bearing subcutaneous (SQ) NB xenografts of

BE cells. Intratumor injections (ITs) were used as the

preferred route of administration to avoid rapid degradation

of IDG in blood by carboxylesterases. One group of mice was

However, predictably, the estimated intracellular D doses are

very similar for BE and SK cells. The radiotoxicity of I,

which produces a cascade of monoenergetic electrons known

37

1

25

as conversion and Auger electrons, is dependent on the

_i_n_t_r_a_n_u_c_l_e_a_r_l_o_c_a_l_i_z_a_t_i_o_n_;_i_._e_._,125I is radiotoxic when within

2

6,67−69

DNA or in the immediate vicinity of DNA.

The cell

_s_u_r_v_i_v_a_l_i_s_p_r_o_p_o_r_t_i_o_n_a_l_t_o_l_e_v_e_l_s_o_f1 I incorporation into

25

DNA. After the intracellular processing of radioactive

1

25

_g_u_a_n_i_d_i_n_e_s_,_b_o_t_h_c_e_l_l_l_i_n_e_s_i_n_c_o_r_p_o_r_a_t_e125IUdR into their

given IT injections of

I-MIBG to compare its tissue

distribution with IDG. Figure 6A and Table 1S (Supporting

Information p S118) summarize tissue and tumor uptake of

these two radioactive agents at 24 h after administration. The

radioactive content of tumors extirpated from mice treated

with IDG is on average 3.93% ± 0.23 %ID/g, twice the content

DNA (Figure 2), and accordingly, the intracellular doses D₃₇

are similar.

_T_h_e_r_e_q_u_i_r_e_m_e_n_t_f_o_r_t_h_e_i_n_t_r_a_n_u_c_l_e_a_r_l_o_c_a_l_i_z_a_t_i_o_n_o_f₁25I was

further conﬁrmed in studies comparing the radiotoxicity of our

125

radioactive guanidine to I-MIBG (Figure 5). Cells exposed

1

25

1

25

of tumors injected with I-MIBG, which retained 1.85% ±

.35 %ID/g (P = 0.02). The gender of mice did not inﬂuence

to I-MIBG store this drug predominantly in the cytoplasmic

compartments, 87% and 74% of total cell-associated radio-

activity after 1 and 24 h, respectively, whereas IDG

accumulates predominantly in DNA, ∼70%−80% and >97%

after 1 and 24 h, respectively (Figures 2D, 5B, 5C). Number of

colonies formed after 1 h and 24 h treatments is signiﬁcantly

0

tumor retention of IDG (Figure 5B). The corresponding

tumor-to-blood ratios were estimated at 161 ± 30 for IDG and

1

25

1

9 ± 7 for I-MIBG (Table 1S, Supporting Information p

1

25

18). As expected, I-MIBG is also taken up by adrenal

70

125

glands at 4.64% ± 1.40 %ID/g 24 h after injection. Many

lower mammals including mice have high levels of carbox-

ylesterases in blood, while little or no expression of this

smaller in SK cells treated with IDG as compared to I-MIBG

25

(

Figure 5D, Figure 5E). Cells exposed to ¹I-MIBG for 1 h

produced 229 ± 13.4 colonies compared to SK cells exposed to

IDG, which produced 163 ± 8.3 colonies. The 24 h exposure

resulted in 250 ± 18.7 colonies in ¹I-MIBG group and 97 ±

3

9

enzyme is observed in higher primates and humans.

25

Consequently in mice studies, all traces of IDG that leak out

from the tumor site are rapidly degraded by carboxylases in the

blood, preventing any uptake in tissues other than the tumor.

Human NB cells have high levels of carboxylesterase

4

.3 colonies in cells treated with IDG. More colonies were

observed in cells in the 1 h group treatment with IDG because

when the radioactive medium is removed and cells are washed

with PBS, harvested with trypsin, and replated for the

clonogenic assay, a substantial amount of ¹IUdR already

produced and present in the cytoplasm diﬀuses from cells and

71−73

activities.

We attribute greater tumor retention of IDG

25

as compared to MIBG to the intracellular processing of IDG

and co-targeting of DNA in proliferating NB cells. To

Figure 5. Comparison of cellular uptake and subcellular distribution eﬀects on the reproductive integrity in SK-N-SH cell treated with ¹²⁵I-MIBG

1

25

(

extracellular concentration 39.3 ± 0.3 kBq/mL) and IDG (extracellular concentration 40 ± 0.5 kBq/mL). (A) Cellular uptake of I-MIBG and

125 125

IDG in SK-N-SH cells. (B) Cytoplasmic distribution of I-MIBG and IDG after 1 h and 24 h treatments. (C) Distribution of I-MIBG and IDG

1

25

in the cell nucleus after 1 h and 24 h treatments. I activity in the nuclei of cells treated with IDG was recovered in DNA. All cells were exposed to

radioactive compounds for 1 h. Cells in the 1 h group were washed with nonradioactive medium, PBS, harvested and replated for the clonogenic

assay. Cells in 24 h group were washed with nonradioactive medium, PBS, given fresh nonradioactive medium, and returned to the incubator for 23

1

25

125

h. (D) Colony forming assay results after treatment with I-MIBG and IDG. Blue bars represent averages for I-MIBG; orange bars represent

1

25

averages for IDG; standard deviations of n = 4. (E) Colonies formed by SK-N-SH treated with I-MIBG and IDG.

I

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Figure 6. In vivo evaluation of IDG in mice. (A) Biodistribution of IDG (orange) and ¹²⁵I-MIBG (blue) in athymic Ncr nu/nu female and male

mice bearing subcutaneous BE(2)-C xenografts at 24 h after the intratumor administration. (B) Comparison of IDG uptake by BE(2)-C xenografts

in female and male mice. (C) Eﬀects of carboxylesterase inhibition with bis-para-nitrophenylphosphate (BNPP) on the retention of IDG in BE(2)-

C xenografts. (D) Volume of BE(2)-C xenografts in vehicle-injected control mice (blue bar) and in mice treated with 8.4 ± 0.3 MBq IDG (orange

bar); average ± std err. (∗) Control mice were killed 7 days after the vehicle injection. (E) Whole body clearance of IDG in mice with BE(2)-C

xenografts after injection of therapeutic doses of IDG. (F) Radioactivity retained in tumor, blood, and the whole body at necropsy 14 days after the

administration. (G) Hemoglobin levels in blood of control (NT) mice and mice treated with IDG. (∗∗) Normal range of Hb values in adult mice.

substantiate this notion, we conducted the in vivo tumor

uptake studies in BE xenograft-bearing mice injected with bis-

para-nitrophenylphosphate (BNPP). BNPP is an irreversible

weight in the NT group was 3.69 ± 1.48 g. Treated mice were

terminated 14 days after the injection of IDG when the average

3

tumor volume was 2.55 ± 1.08 cm . This tumor volume

74

carboxylase inhibitor. It has been used in vitro and in vivo to

inhibit carboxylase activity and alter the pharmacokinetic

corresponded to the average tumor weight of 2.71 ± 1.77 g at

necropsy. The diﬀerence between treatment and control

groups is statistically signiﬁcant (P = 0.01). It is evident that

a single dose of 8.4 ± 0.3 MBq/mouse (227 ± 9 μCi/mouse)

produced a signiﬁcant tumor growth delay. The estimated

75,76

proﬁles of drugs hydrolyzed by this enzyme.

Groups of

mice were injected with IDG alone or alongside BNPP at two

doses: 9 μmol and 18 μmol. In this experiment, 3.31 ± 0.85 %

ID/g was found in tumors extirpated from mice receiving only

IDG (Figure 6C). Tumors from mice preinjected with 9 and

tumor doubling times (T ) from day 0 to day 7 were 2.4 days

D

in the NT group and 14.9 days in mice treated with IDG. The

1

8 μmol of BNPP retained far less of the radioactive guanidine,

.63% ± 0.13 %ID/g and 1.66% ± 0.16 %ID/g, respectively.

overall T , calculated from the time 0 to day 14, for tumors

treated with IDG was 5.3 ± 0.4 days.

D

These diﬀerences are statistically signiﬁcant as compared to the

compound alone uptake (P = 0.0014). BNPP-inhibited tumor

uptake of IDG is nearly identical to tumor uptake of ¹I-MIBG

The whole body clearance after the injection of therapeutic

doses of IDG is biphasic (Figure 6D). The initial rapid decline

of radioactivity has the biological half-life of 1.35 ± 0.07 h and

is followed by a slow elimination with the biological half-life of

76.2 ± 12.6 h. Over 50% of whole-body radioactivity is

eliminated by 2 h postinjection. The remaining activity is

primarily tumor-associated. The slow phase of the whole-body

25

(

1.85 ± 0.35 %ID/g), conﬁrming the intracellular processing

125

with subsequent incorporation and retention of I-1 in DNA.

Therapeutic potential of radioactive guanidines was tested in

athymic Ncr nu/nu female and male mice bearing SQ

xenografts of BE cells randomized via a lottery into two

groups: treatment with IDG and control (NT). On the day of

1

25

elimination parallels the clearance of I from the tumor site.

On day 14, the tumor radioactive content evaluated in terms of

%ID/g was >70× greater compared to blood (Figure 6E). The

estimated tumor absorbed radiation dose is 1200 ± 147 mGy/

MBq (Table 5). We also estimated whole body absorbed

radiation doses for 1-year-old and 5-year-old pediatric patients.

3

treatment, initial tumor volumes were 0.39 ± 0.15 cm and

0

3

.42 ± 0.14 cm in NT and IDG groups, respectively (P =

0

.85). One week after the administration of IDG, tumor

3

volume in mice treated with IDG was 0.54 ± 0.12 cm (Figure

1

31

6

D). All control mice had to be terminated at this time because

The reported I-MIBG whole-body radiation is estimated at

3

77

their tumor volume at 3.26 ± 1.20 cm was reaching the 10%

0.23 mGy/MBq, approximately 16× higher than doses

body weight limit. At necropsy on day 7, the average tumor

estimated for new radioactive guanidines. Hb levels in mice

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Table 5. Estimated Tumor and Whole Body Absorbed

Radiation Doses

IDG suﬃcient to kill >90% of NB cells is ∼37 kBq/mL. This

corresponds to <100 MBq needed to achieve the same levels in

blood of a 3-year old child. Similarly, in our in vivo studies,

tumor mean residence time (MRT)

absorbed radiation dose, 1 g tumor

whole body dose estimate, 1-year old

whole body dose estimate, 5-years old

11.6 (1.4) h

IDG given as a single dose of ∼8 MBq increases tumor T

D

1200 ± 147 mGy/MBq

0.014 mSv/MBq

0.017 mSv/MBq

nearly 7 times, from a couple of days to over 2 weeks, and

signiﬁcantly arrests tumor growth, notwithstanding the rapid

degradation by mouse carboxylesterases. Biodistribution and

therapy studies were conducted in a SQ mouse model of NB

using human BE cells. We were faced with two major

challenges related to this in vivo model. A mouse is an

treated with IDG are within the normal range values (Figure

F) and support the postulated low normal tissue toxicity of

6

1

25

91

I-labeled compounds.

imperfect model for xenografts of human NB and for the

evaluation of drugs that are susceptible to circulating

39,92,93

DISCUSSION AND CONCLUSIONS

carboxylases.

Nonetheless, mice are always used as the

■

initial in vivo animal model in the preclinical evaluation. To

enhance the function of this model in our studies, we chose an

intratumor route of the administration to avoid rapid

catabolism of radioactive guanidines by circulating carbox-

ylesterases. We also used BNPP, a powerful carboxylase

inhibitor, to assess tumor uptake in the absence of this enzyme.

And ﬁnally, we selected a human NB cell line that is radio- and

chemoresistant. BE cells were derived from a bone marrow

biopsy taken from a 2-year-old boy with disseminated,

relapsed, and refractory NB after multiple courses of

NB is a heterogeneous disease. Some tumors regress or

diﬀerentiate spontaneously. Others exhibit extremely malig-

nant behavior with life-threatening progression, very low cure

2

,78−80

rates, and very few therapeutic options.

year of age or older are frequently unresectable or metastatic

and are fatal in ∼60% of cases despite intensive multimodal

NBs in children

1

31

therapies. I-MIBG has been introduced to the management

1

0

of NB nearly 30 years ago and became an essential

component of treatment strategies for relapsed and refractory

1

1,12,18,22,81

disease with overall response rates of ∼30%.

9

4

However, the therapeutic applications of 31_I_-_M_I_B_G_a_r_e

1

chemotherapy and radiotherapy. Tumor responses to

clinically manageable doses of IDG are signiﬁcant in this

model. Moreover, the absorbed radiation dose estimates

indicate that therapeutically relevant tumor doses are

achievable. It is also reasonable to assume that the higher

doses of IDG may be delivered to tumors as a bolus or

fractionated administration without ampliﬁcation of the

limited. Tumor responses are transient. Myelosuppression

and other adverse eﬀects related to the radiation exposure of

normal tissues prevent administration of higher, more eﬀective

doses. New therapies must attain a balance between improved

survival rates and the morbidity of side eﬀects. Our radioactive

guanidines are radiolabeled with Auger electron emitters and

are expected to have a more favorable toxicity proﬁle that will

permit the administration of curative doses. Low energy

1

31

detrimental side eﬀects associated with

I-MIBG. The

1

25

radiation dose from I falls oﬀ rapidly outside the tumor

volume, ensuring that doses to tissues and organs remote from

the tumor site are greatly reduced. For comparison,

hematological or normal tissue toxicities were not observed

1

25

electrons from I decaying in DNA produce nonrepairable

8

2,83

strand breaks and lesions

high LET radiations. Each decay of DNA-incorporated

produces double DNA strand breaks with the probability of

analogous to those produced by

1

25

I

1

25

after administration of up to ∼30 GBq (>800 mCi) I-A33

95

8

4−87

antibody, which has a far longer half-life in circulation. The

whole body doses that might be expected in pediatric patients

treated with IDG are also reassuring since the estimates are

nearly one and several single DNA strand breaks.

Approximately 90% of these DNA breaks are located within

0 nm of the I decay site; i.e., nearly all of the electron

energy associated with the I decay is deposited within a

sphere smaller than the cell nucleus,

1

25

1

31

1

25

∼16× lower compared to I-MIBG. Additionally, we should

125

25,26

note that radionuclides such as I, which emit only low

energy photons, are easily shielded, greatly simplifying the

radiation protection of medical and nursing staﬀ.

ensuring minimal

irradiation of the surrounding healthy tissues and organs.

Synthetic methods and the design of new radioactive

guanidines can be altered to produce derivatives with distinct

stabilities under physiologic conditions and varied cellular

uptakes. The chemical structure accommodates therapeutic as

well as diagnostic radionuclides. For these reasons, the clinical

potential of radioactive guanidines is high. New compounds

mimic MIBG in their mechanism of NB targeting; however,

unlike MIBG they subsequently undergo intracellular process-

ing and their catabolites are eﬃciently incorporated into DNA

of NB cells with high proliferation activities, i.e., cells in the

high risk and aggressive disease. Moreover, the structure can

In conclusion, radioactive guanidines can be eﬃciently

synthesized from 5-radioiodo-2′-deoxyuridine, a molecular

radiotherapy platform with clinically proven minimal toxicities

and DNA targeting properties. In vitro and in vivo properties

of new compounds indicate their signiﬁcant potential as novel

theranostics for the management of NB.

EXPERIMENTAL SECTION

■

Reagents and Instrumentation. Chemicals and reagents

purchased from commercial suppliers were used without further

puriﬁcation, unless explicitly indicated. 5-Iodo-2′-deoxyuridine 1

(IUdR) was purchased from Sigma-Aldrich (St. Louis, MO). Reagents

88

comprise groups that allow the lock in mechanism, such as in

compound 21, to trap the catabolites within the cancer cells,

ensuring their sustained availability throughout the cell cycle.

This feature is important in eradicating slowly proliferating

4

and 5 were prepared using routine procedures, and these are

described brieﬂy in the Supporting Information (pp S5, S20, S39).

1

25

−5

8

9,90

Solutions of sodium [ I]iodide in 1 × 10 NaOH (pH 8−11), with

speciﬁc activities of ∼78 000 GBq/mmol (carrier-free) were obtained

from PerkinElmer (Billerica, MA). Radioactivity was measured with

Minaxi γ-counter (Packard, Waltham, MA) and a dose calibrator

tumor cells

that can be expected to occur in a

heterogeneous cell population of NB.

Biological properties of radioactive guanidines support their

potentially signiﬁcant role as theranostics for diagnostic and

eﬀective nontoxic therapeutic strategies in pediatric patients

diagnosed with high risk NB. For example, a concentration of

(

CapIntec Inc., Ramsey, NJ). Analytical TLC was carried out on

plastic plates coated with a 0.2 mm layer of silica (normal phase

Merck 60 F₂₅₄), and spots were visualized with either short wave UV

K

DOI: 10.1021/acs.jmedchem.9b00437

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

or iodine vapors. Flash column chromatography was performed using

Merck silica gel 60 (40−60 μM) as a stationary phase. Compounds

were resolved, and their purity was evaluated by the HPLC analyses

conducted on Gilson (Middleton, WI) and ISCO (Lincoln, NE)

systems, with 5 μm, 250 mm × 4.6 mm analytical columns, either

Columbus C8 or C18 (Phenomenex, Torrance, CA) and ACE C18

the resulting solid was directly treated with a solution of hydrazine

hydrate (0.5 M, ∼5 equiv) in a pyridine/AcOH mixture (4:1, v/v, 5

mL), cooled on an ice−water bath. The stirring continued for ∼10

min at room temperature, and water (40 mL) and EtOAc (50 mL)

were added. The organic layer was separated, washed with 10%

aqueous solution of NaHCO (20 mL), water (20 mL), dried over

3

(

Advanced Chromatography Technologies, www.ace-hplc.com). All

MgSO , and evaporated.

4

compounds were determined to be ≥95% pure by this method.

Columns protected by guard ﬁlters were eluted at a rate of 0.8 mL/

min with various gradients of acetonitrile (CH CN; 10−95%) in

General Procedure B: Preparation of Trimethyltin Precur-

sors 7, 11, 15a,b, 19, 23, and 27. Stirred solutions of 5-iodo-3′-O-

or 5-iodo-5′-O-[(N,N′-bis(tert-butyloxycarbonyl)-N″-alkylcarboxy)-

guanidino]-2′-deoxyuridine 10, 14a,b, 18, 22, 5-iodo-5′-N-[N′,N″-

bis(tert-butyloxycarbonyl)guanidino]-2′,5′-dideoxyuridine 6, or N,N′-

bis(tert-butyloxycarbonyl)-3-iodobenzylguanidine 26 (1.0 equiv), in

ethyl acetate or dioxane (depending on solubility), containing

hexamethylditin (1.25−1.70 equiv), triethylamine (2−4 equiv), and

dichloro-bis(triphenylphosphine)palladium II catalyst (0.10 equiv),

were gently reﬂuxed under nitrogen until the starting iodide could be

detected (1−3 h). The reaction progress was checked often by TLC.

After cooling to ambient temperature, the mixture was ﬁltered

through a thin pad of silica (washed with EtOAc/hexanes, 2:1) to

separate the remaining catalyst and the solvent was evaporated. Two

major products were always present: the corresponding trimethyl-

stannane (with high TLC mobility), isolated in 30−62% yield, and the

protodestannylated form of starting iodide (slower on TLC). Crude

products were separated and puriﬁed by repeating a silica gel ﬂash

column chromatography, using mixtures of EtOAc/hexanes (2:1−2,

v/v) and/or with various gradients of MeOH in DCM (0.3−0.7:10).

In order to reach ≥98% purity of stannylated products, the successive

preparative HPLC puriﬁcations were usually required. Puriﬁed,

anhydrous samples of stannanes (100 μg/vial) were stored with the

exclusion of light under nitrogen at −20 °C.

3

water, with or without triﬂuoroacetic acid (TFA; 0.07%, w/v). Two

variable wavelength UV detectors: UVIS-205 (Linear, Irvine, CA) and

UV116 (Gilson) were used jointly with the sodium iodide crystal

Flow-count radioactivity detector (Bioscan, Washington, DC)

connected to the outlet of UV detector. Both signals were monitored

and analyzed simultaneously. All target nonradioactive compounds

were found to be ≥98% pure by the rigorous HPLC analysis, with the

integration of a peak area (detected at 220 and/or 280 nm).

Radioiodinated products were identiﬁed and evaluated through the

independently prepared nonradioactive reference compounds by

comparing UV signals of the nonradioactive standards with signals

from radio-TLC (R ) and radio-HPLC (t ) of the radioactive

f

R

products. Before testing the biological activities and stability, each

ﬁnal target compound was once again puriﬁed by HPLC using linear

gradient of ethanol (EtOH; 0−70%) in phosphate buﬀer (10 mM, pH

6

.1) to eliminate TFA and CH CN in tested samples. Solutions

3

containing the product were evaporated with a stream of sterile

nitrogen and reconstituted in a preferred solvent at the required

concentration and then ﬁltered through a sterile 0.2 μm ﬁlter

1

13

31

(

Millipore) into a sterile evacuated vial. H, C, and P NMR

spectra were recorded in (CD ) SO or CDCl at ambient temperature

3

2

3

General Procedure C: Preparation of ¹I-Radioiodinated 3′-

25

on Bruker Avance III HD 600 MHz spectrometer. All NMR analyses

were performed by the University of Nebraska Medical Center Fred &

Pamela Buﬀett Cancer Center Structural Biology Facility Shared

Resource (Omaha, NE). Chemical shifts are given as δ (ppm) relative

to TMS as internal standard with J in hertz. Deuterium exchange and

decoupling experiments were performed to further assist signals

assignments of protons. High resolution electrospray ionization (ESI-

HR) mass spectra (in positive mode) were acquired on a Waters

Synapt G2 HDMS mass spectrometer at the Washington University

Resource for Biomedical and Bio-Organic Mass Spectrometry (St.

Louis, MO).

125

and 5′-Guanidino-N-alkylcarboxy Esters of 5-[ I]Iodo-2′-

1

25

deoxyuridine 13 (IDG), 17a,b, 21, 25, 5-[ I]Iodo-5′-N-

1

25

guanidino-2′,5′-dideoxyuridine 9, and 3-[ I]Iodobenzyl-

guanidine 29. Into a glass tube containing the selected tin

precursor, 7, 11, 15a,b, 19, 23, or 27 (∼100 μg, 0.1−0.2 μmol) in

1

25

MeCN (50 μL), a solution of Na I/NaOH (10−50 μL, 22.2−148

MBq) was added, followed by a solution of 30% H O in water (5 μL)

2

and 2 min later, a solution of TFA (50 μL, 0.1% in MeCN). The

resulting mixture, brieﬂy vortexed and/or sonicated, was left for 15

min at room temperature and then quenched with a solution of

Na S O (90−100 μg) in water (60 μL). At this point, one of the two

2

3

General Procedure A: Preparation of 5-Iodo-5′-N-[N′,N″-

bis(tert-butyloxycarbonyl)guanidino]-2′,5′-dideoxyuridine (6)

and 3′-O -and 5′-O-[(N,N′-bis(tert-butyloxycarbonyl)-N″-

alkylcarboxy)guanidino] Esters of 5-Iodo-2′-deoxyuridine:

pathways was implemented.

Pathway 1: The reaction mixture was taken up into a syringe and

the reaction tube washed twice with a solution of MeCN/H O (50

2

1

0, 14a,b, 18, 22. The Boc group of each starting 5-iodo-3′-O- or

μL, 1:1−3, depending on the stannane solubility). The reaction

mixture and washes were combined, injected onto the HPLC system,

and separated on the C8 or C18 reverse phase column with a linear

gradient of MeCN in water. Eluted fractions with one of the

radioiodinated products, 8, 12, 16a,b, 20, 24, 28, were pooled,

evaporated with a stream of dried nitrogen, or else dissolved in dry

MeCN (∼18.5 MBq/mL) for further analysis or storage. To remove

the guanidine Boc-protective groups, neat TFA (100 μL) was added

−

5′-O-(N-tert-butyloxycarbonyl)aminoalkylcarboxy)-2′-deoxyuridine

was cleaved with ∼15% TFA in dichloromethane (DCM) or MeCN.

When reaction was completed (monitoring by TLC), the mixture was

evaporated under a vacuum at room temperature, the resulting oily

residue treated with 15 mL of EtOAc/hexane (1:1) and sonicated

brieﬂy a few times. The solvent was carefully decanted from a formed

solid, a crude product was washed with Et O, and a solvent was drawn

2

1

25

oﬀ again. This washing procedure was repeated, and then the

remaining TFA salt was kept under a vacuum to dry. A resulting TFA

salt of 5-iodo-3′-O- or 5-iodo-5′-O-aminoalkylcarboxy-2′-deoxyur-

idine (1.0 molar equiv) was suspended in DCM, placed on an ice

bath, and to a stirred mixture TEA (1.05 molar equiv) was added,

immediately followed by N,N′-bis(tert-butoxycarbonyl)-N″-

triﬂuoromethanesulfonylguanidine (1.1 molar equiv). The mixture

was stirred for 5 min, and to a resulting clear solution a second

portion of TEA (1.0 molar equiv) added. The stirring continued for

to a dried residue of I-iodinated product, and the resulting mixture

was vortexed and then heated in a sealed vial at 55−65 °C for 20−35

min. After cooling, the mixture diluted with CH CN (200 μL) was

3

evaporated repetitively with a stream of nitrogen, each time leaving

∼20 μL of the liquid in the reaction vial, to prevent the adhesion of

products to walls of a vial. This process was repeated at least three

times to ensure that an excess of TFA was eliminated. The residue

was then dissolved in a solution of 50% MeCN in water, injected onto

the HPLC system, and separated on the C18 reverse phase column

with a gradient of MeCN in water, with both solvents containing

0.07% TFA (v/v), as the eluant.

2

−6 h (TLC monitoring) at room temperature. Upon the

completion, an excess of amines and triﬂic amide were removed by

aqueous workup with 5% citric acid and saturated brine. Organic

Pathway 2. Alternatively, if the initial separation of Boc-protected

radioiodinated product was not essential, a crude radioiodination

mixture was evaporated with a stream of nitrogen and/or kept under a

high vacuum, then it was directly treated with neat TFA at elevated

temperature and separated using HPLC, as described previously. To

phase was dried over MgSO , ﬁltered, and evaporated. Crude

4

products were puriﬁed using a silica gel column with gradients of

MeOH in DCMor EtOAc in hexanes. In preparations conducted with

3′-O-levulinyl-protected deoxyuridines, to remove the O-Lev group,

L

DOI: 10.1021/acs.jmedchem.9b00437

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

fully eliminate the presence of TFA and CH CN, the combined

same column and the elution rate; eluent, solvent A water, solvent B

3

HPLC fractions containing a ﬁnal separated product were evaporated

with a stream of nitrogen and to the residue, ethanol (80 μL) was

added, followed by potassium phosphate buﬀer (100 μL, 10 mM PB,

pH ∼ 6.1). The resulting mixture was injected for a second time on

HPLC reverse phase column and eluted with a linear gradient of

EtOH in potassium phosphate buﬀer. In every HPLC separation or

analysis of radiolabeled products, the eluate from a column was

monitored with a radioactivity detector connected to an outlet of UV

detector (detection at 220 and 280 nm). Solutions containing the

product were reconstituted in a preferred solvent to the required

concentration and then ﬁltered through a sterile ﬁlter (Millipore, 0.22

μm) into a sterile evacuated vial. Identities of the radiolabeled

products were conﬁrmed by the evaluation of the UV signals of

nonradioactive iodo analogs with both signals of radiolabeled

CH CN; 45% B for 30 min, then a linear gradient of B to 95% over 30

3

1

min, continued for 30 min. H NMR (CDCl , 600 MHz) δ: 10.62 (bs,

3

1H, NH-uridine), 9.05 (bs, 1H, NH), 8.31 (t, 1H, NH, J = 5.72 Hz),

3

7.49 (s, 1H, H6-uridine, J

= 19.5 Hz), 5.98 (dd, 1H, H1′, J₁_′_,₂_′=

Sn,H

3

6.2 Hz, J

= 4.6 Hz), 4.21−4.15 (m, 1H, H4′), 3.78−3.73 (m, 2H,

1

′,2″

H3′, OH), 3.63−3.59 (m, 1H, H5″), 2.34−2.28 (m, 1H, H2′), 2.19−

2.04 (m, 1H, H2″), 1.44 (s, 9H, 3 × CH -t-Bu), 1.41 (s, 9H, 3 ×

3

2

13

CH -t-Bu), 0.32 (s, 9H, 3 × CH , J = 29.1 Hz) ppm. C NMR

Sn,H

3

(CDCl , 100 MHz) δ: 165.6 (C4), 160.46 (C-guanidine), 158.4 (C1-

3

Boc-N′), 156.2 (C1-Boc-N″), 150.9 (C2), 143.4 (C6), 112.7 (C5),

86.8 (C1′), 85.2 (C4′,), 79.7 (C2-Boc-N′), 75.3 (C2-Boc-N″), 71.6

(C3′), 67.0 (C5′), 38.9 (C2′), 28.1 (C3-Boc-N′), 28.1 (C3-Boc-N″),

+

−7.15 (3 × CH3-Sn) ppm. HDMS (m/z): [M + H] calcd for

1

12

112

C H N O Sn, 626.1919; found 626.1909 using the Sn isotope

2

3

39

5

8

compounds and by comparing R obtained from the radio-TLC and

signal.₁

f

25

t_Rfrom the radio-HPLC analysis. All radiolabeled products, if kept in

a solution overnight at ambient temperature, were puriﬁed one more

time shortly before conducting further experiments, although the

HPLC analysis rarely indicated less than 95% of the radiochemical

purity.

5-[ I]Iodo-5′-N-[N′,N″-bis(tert-butyloxycarbonyl)-

guanidino]-2′,5′-dideoxyuridine (8). A total of four radio-

iodinations was carried out according to general procedure C, with

1

25

∼100 μg of stannane 7 and Na I/NaOH, within the 18.5−77.7 MBq

range, to give overall 140.6 MBq of 8. An average yield of the puriﬁed

product was 84%. The HPLC puriﬁcation of a crude reaction mixture

proceeded on ACE C8 100 Å (5 μm, 4.6 mm × 250 mm), eluted at

0.8 mL/min initially with 45% MeCN in water for 30 min, then with a

linear gradient of MeCN from 45% to 95% MeCN over 30 min, and

95% MeCN was held for 15 min longer. The product 8 eluted within

19.5−22.0 min after the injection and was collected in three fractions

(>98% pure). An excess of unreacted stannane 7, eluting between

46.5 and 49.0 min, was completely separated from the product. Two

HPLC co-injections of puriﬁed 8 and nonradioactive analog 6

(Bioscan NaI(T) detector and UV signal at 220/280 nm) conﬁrmed

5

-Iodo-5′-N-[N′,N″-bis(tert-butyloxycarbonyl)guanidino]-

′,5′-dideoxyuridine (6). To a stirred solution of 5-iodo-5′-amino-

′,5′-dideoxyuridine (1.91 g, 5.39 mmol) in 30 mL of dioxane/water

2

mixture (12:1, v/v), N,N′-bis(tert-butyloxycarbonyl)-N″-

triﬂuoromethanesulfonylguanidine (2.11 g, 5.39 mmol) was added,

followed by TEA (750 μL, 5.4 mmol). The stirring continued for 6 h,

dioxane was evaporated, water (60 mL) added, and the mixture

extracted with DCM (2 × 40 mL). The organic phase was washed

with brine, then dried over MgSO and evaporated. The residue of a

4

crude product was puriﬁed on a silica gel column (DCM/MeOH,

1

0:0.4), followed by a second silica gel puriﬁcation (EtOAc/n-

the same mobility of both compounds: (1) t = 20.1 min, ACE C8 100

R

hexanes, 2:1) to yield 6 (2.57 g, 79%) as colorless foam. HPLC

analysis: (1) t = 19.89 min (98.2% pure at 254 nm) on Columbus C8

column, eluted at the rate of 0.8 mL/min, with 45% MeCN in water

for 30 min and then with a linear gradient of MeCN from 45% to 95%

over 30 min. (2) t = 34.56 min (98.2% pure at 280 nm) on ACE C18

Å (5 μm, 4.6 mm × 250 mm), eluted at 0.8 mL/min at ﬁrst with 45%

MeCN in water for 30 min, then a linear gradient of MeCN from 45%

to 95% MeCN over 30 min and kept at 95% MeCN for 15 min. (2) t_R

= 37.4 min, ACE C18 100 Å (5 μm, 4.6 mm × 250 mm), eluted at 0.8

mL/min of the eluant: a linear gradient of MeCN in water from 5% to

95% over 60 min, then kept at 95% MeCN for 30 min.

R

1

00 Å, (5 μm, 4.6 mm × 250 mm) column eluted at the rate of 0.8

125

mL/min with a linear gradient of MeCN in water from 10% to 95%

over 35 min, then 95% MeCN for 25 min. H NMR (CDCl , 600

MHz) δ:11.40 (s, 1H, NH-uridine), 9.13 (bs, 1H, NH), 8.07 (t, 1H,

NH, J = 5.85 Hz), 7.81 (s, 1H, H6-uridine), 6.08 (dd, 1H, H1′, J₁_′_,₂_’=

5-[ I]Iodo-5′-N-guanidino-2′,5′-dideoxyuridine (9). Apply-

ing general procedure C, the radioiodination reaction mixture of 7 or

dried residue of puriﬁed 8 was treated with TFA (neat) and kept at 75

°C for 15 min to completely cleave Boc-protection of guanidine. The

deprotection progress of 8 using a solution of 40% TFA in MeCN was

also monitored by HPLC at room temperature and was completed

within 70 min. An average yield of the puriﬁed 9 was 91%. Product

was separated on ACE C18 100 Å (5 μm, 4.6 mm × 250 mm),

starting with 5% MeCN in water for 10 min and eluted at 0.8 mL/

min, followed by a linear gradient of MeCN from 5% to 95% over 30

min and 95% MeCN was kept constant for 15 min longer. Both

1

3

6

.4 Hz, J = 4.8 Hz), 4.31−4.25 (m, 2H, H3′, C3′-OH), 3.78−3.73

1

′,2″

(

m, 2H, H4′, H5′), 3.63−3.59 (m, 1H, H5″), 2.24−2.18 (m, 1H,

H2’), 2.13−2.06 (m, 1H, H2″), 1.51 (s, 9H, 3 × CH -BocN′), 1.47

3

1

3

(

s, 9H, 3 × CH -BocN″) ppm. C NMR (CDCl , 100 MHz) δ:

3

1

62.4 (C4), 159.8 (C1-Boc-N′), 157.7 (C1-Boc-N″), 153.0 (C2),

49.66 (C-guanidine), 144.2 (C6), 118.3 (C5), 84.7 (C1′), 84.3

1

(

C4′,), 77.3 (C2-Boc-N′), 77.0 (C2-Boc-N″), 73.5 (C3′), 67.0 (C5′),

3

8.9 (C2′), 28.2 (C3-Boc-N′), 28.1 (C3-Boc-N″) ppm. HDMS (m/

solvents contained 0.07% TFA (v/v). The product (t = 10.4 min,

R

+

z): [M + H] calcd for C H IN O , 596.1212; found, 596.1219.

≥98% purity, Bioscan/UV 280 nm) eluting within 9.5−12.0 min after

injection was collected in three fractions, which were combined,

evaporated, and to the tube containing the product residue 80 μL of

ethanol was added, followed by potassium phosphate buﬀer (100 μL,

10 mM PB, pH 6.1). The resulting solution of 9 was injected again on

HPLC system equipped with Luna CN (5 μm, 4.5 mm × 250 mm), a

reverse phase column eluted at 0.8 mL/min with the two consecutive

gradients of EtOH in potassium phosphate buﬀer (10 mM PB, pH

6.1): 0−5% in 10 min and 10−60% over the next 30 min. The

product was collected within 21.5−23.0 min after the injection.

2

0

30

5

8

5

-Trimethylstannyl-5′-N-[N′,N″-bis(tert-butyloxycarbonyl)-

guanidino]-2′,5′-dideoxyuridine (7). General procedure B was

carried out twice with (520 mg, 0.873 mmol and 712 mg, 1.196

mmol) 5-iodo-5′-N-[(N′,N″-bis-Boc)guanidinyl]-2′,5′-dideoxyuridine

6

in EtOAc containing hexamethylditin 486 mg (1.48 mmol) and 670

mg (2.05 mmol) respectively, TEA (300 μL, 2.15 mmol), and the

palladium catalyst (30 mg, 0.043 mmol). Combined crude products

were puriﬁed on a silica gel column using a mixture of EtOAc/hexanes

(

2:1, v/v) to give 7 as a pale foam (680 mg) in 52% yield. R = 0.51

DCM/MeOH, 10:0.3). The ﬁnal HPLC puriﬁcation (160 mg, ∼11

f

HPLC analysis: t = 22.5 min (≥98% purity, Bioscan/UV 280 nm).

R

mg per injection) was done on a semipreparative Columbus C18, 100

Å (10 mm × 250 mm) column, eluted at 2.2 mL/min with 55%

MeCN in water for 20 min, then with a linear gradient of MeCN from

5-Iodo-3′-O-[ε-(N,N′-bis(tert-(butyloxycarbonyl)-

guanidino))hexanoyl]-2′-deoxyuridine (10). General procedure

A was carried out with 5-iodo-3′-O-(6-N-Boc-aminohexanoyl)-2′-

deoxyuridine (1.12 g, 1.97 mmol). The Boc protecting group was

cleaved, and the separated dried TFA salt after addition of TEA (279

μL, 2.0 mml) reacted immediately with 0.82 g (2.10 mmol) of N,N′-

bis(tert-butoxycarbonyl)-N″-triﬂuoromethanesulfonylguanidine in

DCM (23 mL) in the presence of TEA (280 μL, 2.0 mmol). A

crude product was puriﬁed on a silica gel column using a gradient of

5

5% to 95% over 15 min and 95% MeCN kept further 30 min. HPLC

analysis: (1) t = 30.02 min (97.3% pure at 280 nm) on ACE C18

R

1

00 Å, (5 μm, 4.6 mm × 250 mm) column, eluted at 0.8 mL/min

with 50% MeCN in water for a 20 min, then with a linear gradient

from 50% to 95% of MeCN over 15 min and 95% MeCN continued

for next 30 min. (2) t = 47.06 min (98.4% pure at 280 nm), using the

R

M

DOI: 10.1021/acs.jmedchem.9b00437

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

MeOH in DCM (0.3−0.5:10) to give 938 mg of 10 in 67% yield. R =

protection and was completed in 20 min. An average yield from four

preparations of the puriﬁed IDG was 89%. The product (19.37 MBq,

ﬁrst preparation) was separated on ACE C18 100 Å (5 μm, 4.6 mm ×

250 mm) column, eluted at 0.8 mL/min with a linear gradient of

MeCN from 0% to 95% over 50 min and 95% MeCN kept for an

additional period of 20 min, and both solvents contained 0.07% TFA

(v/v); t_R= 23.67 min (≥98% radiochemical purity). The product

eluting within 23.2−24.5 min after the injection was collected in three

fractions, which were combined and evaporated. To eliminate the

presence of TFA and MeCN in the sample, ethanol (80 μL) was

added to a portion of the product residue (9.1 MBq), followed by

potassium phosphate buﬀer (100 μL, 10 mM PB, pH 6.1). The

resulting solution was injected again on HPLC system equipped with

Luna CN (5 μm, 4.5 mm × 250 mm) column and eluted at the rate of

0.8 mL/min with 15% EtOH in potassium phosphate buﬀer (10 mM

PB, pH 6.1) for a period of 10 min, then with a linear gradient of 15−

50% EtOH in PB over a 20 min period. The product (8.2 MBq, 90%

f

0

.42 (DCM/MeOH, 10:0.4). HPLC analysis: t = 29.9 min (≥96.6%

R

pure at 254/280 nm) on ACE C18 100 Å, (5 μm, 4.6 mm × 250 mm)

column, at the elution rate of 0.8 mL/min with a linear gradient of

MeCN in water from 40% to 95% over 45 min, then 95% MeCN kept

1

for 15 min. H NMR (DMSO-d , 600 MHz) δ: 11.61 (s,1H, NH-

6

uridine), 8.32 (bs, 1H, NH), 8.16 (t, 1H, NH, J = 5.52 Hz), 8.06 (s,

3

1

H, H6-uridine), 6.28 (dd, 1H, H1′, J

= 6.40 Hz, J

= 4.85

1

′,2′

1′,2″

Hz), 5.15−4.87 (m, 2H, H3′, OH), 4.19−4.02 (m, 1H′, H4′), 3.77−

3

(

.56 (m, 4H, H5′, H2-Ac), 2.89−2.76 (m, 2H, H6-Hex)′ 2.58−2.46

m, 1H, H2′), 2.38−2.29 (m, 1H, H2″), 1.72−1,64 (m, 2H, H3-

Hex), 1.61−1.54 (m, 2H, H5-Hex), 1.52 (s, 9H, H3-BocN′, 1.46 (s,

13

9

(

H, H3-BocN″), 1.36−1.29 (m, 2H, H4-Hex) ppm. C NMR

DMSO-d , 100 MHz) δ:172.8 (C1-Hex), 162.8 (C4), 158.8 (C1-

6

Boc-N′), 156.7 (C1-Boc-N″), 153.5 (C2), 149.52 (C-guanidine),

1

42.5 (C6), 110.3 (C5), 85.19 (C1′), 84.4 (C4′), 80.8 (C2-Boc-N′),

9.7 (C2-Boc-N″), 74.7 (C3′), 61.4 (C5′), 41.3 (C2-hex), 40.5 (C6-

7

Hex), 37.7 (C2′), 31.8 (C5-Hex), 28.4 (C3-Boc-N′), 28.3 (C3-Boc-

N″), 26.5 (C4-Hex), 24.7 (C3-Hex) ppm. HDMS (m/z): [M + H]

yield) was collected within 19.3−22.5 min after the injection (t =

R

+

19.9 min, ≥97% purity Bioscan/UV 280 nm).

calcd for C H IN O , 710.1892; found, 710.1906.

5-Iodo-5′-O-[(N,N′-bis(tert-(butyloxycarbonyl)-N″-

propionyl)guanidino]-2′-deoxyuridine (14a). As described in

general procedure A, the Boc protecting group of 5-iodo-5′-O-(6-N-

Boc-aminopropionyl)-3′-O-levulinyl-2′-deoxyuridine (1.51 g, 2.42

mmol) was cleaved in MeCN (22 mL) containing 3.5 mL of TFA

and the separated TFA salt was suspended in DCM (27 mL) reacted

directly with 0.95 g (2.45 mmol) of N,N′-bis(tert-butoxycarbonyl)-

N″-triﬂuoromethanesulfonylguanidine in the presence of TEA (720

μL, 5.16 mmol) added in two portions. The residue was puriﬁed by

chromatography on a silica gel column using a gradient of MeOH in

26

40

5

10

5

- T r i m e t h y l s t a n n y l - 3 ′ - O - [ ε - ( N , N ′ - b i s ( t e r t -

(

butyloxycarbonyl)guanidino))hexanoyl)]-2′-deoxyuridine

11). Stannylation of 5-iodo-3′-O-[ε-(N,N′-bis(tert-

butyloxycarbonyl)guanidino))hexanoyl)]-2′-deoxyuridine (10) was

conducted two times, with 230 mg (0.324 mmol) and repeated with

16 mg (0.445 mmol) of 10, and hexamethylditin 160 mg (0.488

3

mmol) and 219 mg (0.668 mmol), respectively, with the palladium

catalyst (25 mg, 0.036 mmol) and TEA (200 μL, ∼1.4 mmol). Crude

products were puriﬁed on a silica gel column using a gradient of

MeOH in DCM (0.3−0.5:10) to give 11 (89 mg and 136 mg) in 37%

DCM (0.3−0.5:10) to give 1.57g (74%) of the product (R = 0.67 in

f

and 41% yield, respectively. R = 0.47 (DCM/MeOH, 10:0.4). HPLC

DCM/MeOH 10:0.3). The 3′-O-Lev group was cleaved with a

f

analyses: t = 36.8 min (95.9% pure at 220/280 nm) on ACE C18

mixture of N H ·H O/pyridine/AcOH (0.3/9.5/2.4 mL) and a crude

R

2

4

2

1

00 Å, (5 μm, 4.6 mm × 250 mm) column, eluted at the rate of 0.8

product puriﬁed again on a silica gel column (DCM/MeOH,

mL/min with a linear gradient of MeCN in water, from 40% to 95%

over 50 min, then 95% MeCN was kept further 20 min. H NMR

10:0.35), to give 1.01g (73% yield) of 14a as colorless foam; R =

f

1

0.60 (DCM/MeOH, 10:0.5). HPLC analysis: (1) t_R= 35.6 min

(≥98.81% pure at 280 nm) on Columbus C18 100 Å, (5 μm, 4.6 mm

(

DMSO-d , 600 MHz) δ: 11.66 (s,1H, NH-uridine), 8.72 (bs, 1H,

6

3

NH), 8.37 (t, 1H, NH, J = 5.48 Hz), 7.76 (s, 1H, H6-uridine, J

=

× 250 mm) column, eluent solvent A water, solvent B CH CN; eluted

Sn,H

3

1

4

3

8.42 Hz), 6.32 (dd, 1H, H1′, J

= 6.45 Hz, J = 4.80 Hz),

.95−4.81 (m, 1H, H4′), 4.39−4.22 (m, 1H′ H3′), 3.77−3.64 (m,

H, H5′,OH), 2.89−2.77 (m, 2H, H6-Hex), 2.59−2.42 (m, 3H, H2′,

at the rate of 0.8 mL/min with a linear gradient of B from 10% to 90%

1′,2′

1′,2″

over 40 min, 90−95% within 40−45 min and 95% B kept over 15

min. On ACE C18 100 Å, (5 μm, 4.6 mm × 250 mm) column. (2) t

R

H2-Hex), 2.38−2.29 (m, 1H, H2″), 1.70−1,63 (m, 2H, H3-Hex),

= 34.5 min, (≥98.35% pure at 220 nm); eluent solvent A water,

1

.58−1.50 (m, 2H, H5-Hex), 1.41 (s, 9H, H3-BocN′, 1.37 (s, 9H,

solvent B CH CN; eluted at the rate of 0.8 mL/min with a linear

3

H3-BocN″), 1.30−1.27 (m, 2H, H4-Hex), 0.27 (s, 9H, 3 × SnCH ,

gradient of B from 40% to 80% over 65 min, then 95% of B kept over

3

2

13

1

J

= 29.2 Hz) ppm. C NMR (DMSO-d , 100 MHz) δ:173.2 (C1-

25 min. H NMR (CDCl , 600 MHz) δ: 11.46 (s,1H, NH-uridine),

Sn,H

6

3

Hex), 163.37 (C4), 160.1 (C1-Boc-N′), 158.3 (C1-Boc-N″), 153.5

8.71 (bs, 1H, NH), 8.66 (t, 1H, NH, J = 4.52 Hz), 7.96 (s, 1H, H6-

uridine), 6.21 (t, 1H, H1′, J = 5.55 Hz), 4.48−4.42 (m, 2H, H3′,

OH), 4.37−4.31 (m, 1H, H4′), 3.75−3.53 (m, 4H, H5′, H2-Prop),

2.85−2.74 (m, 2H, H3-Prop), 2.58−2.48 (m, 1H, H2′), 2.24−2.19

(

C2), 149.6 (C-guanidine), 142.3 (C6), 110.2 (C5), 85.9 (C1′), 84.3

C4′), 80.9 (C2-Boc-N′), 79.9 (C2-Boc-N″), 74.5 (C3′), 61.5 (C5′),

(

4

1.2 (C6-hex), 40.5 (C2-Hex), 37.7 (C2′), 31.9 (C5-Hex), 28.4 (C3-

Boc-N′), 27.4 (C3-Boc-N″), 26.5 (C4-Hex), 24.7 (C3-Hex), −7.9 (3

×

(m, 1H, H2″), 1.49 (s, 9H, H3-BocN′), 1.46 (s, 9H, H3-BocN″)

+

13

CH -Sn) ppm. HDMS (m/z): [M + H] calcd for

ppm. C NMR (CDCl

3

, 100 MHz) δ:171.7 (C1-Prop), 163.3 (C4),

3

C H N O Sn, 740.2600; found 740.2589 using the ¹¹²Sn isotope

112

160.0 (C1-Boc-N′), 156.2 (C1-Boc-N″), 153.9 (C2), 149.9 (C-

guanidine), 143.4 (C6), 111.7 (C5), 85.7 (C1′), 84.3 (C4′), 77.2

(C2-Boc-N′), 77.1 (C2-Boc-N″), 74.3 (C3′), 67.5 (C5′), 44.2 (C2-

Prop), 42.4 (C3-Prop), 38.7 (C2′), 28.3 (C3-Boc-N′), 28.0 (C3-Boc-

29

49

5

8

1

25

+

N″) ppm. HDMS (m/z): [M + H] calcd for C H IN O ,

23

34

5

10

1

25

carried out two times, with 40.7 and 57.4 MBq of Na I/NaOH, to

give overall 82 MBq of 12. An average yield of the isolated product

was 85%. A crude reaction mixture was separated and puriﬁed by

HPLC on Jupiter C18 100 Å (5 μm, 4.6 mm × 250 mm) column

eluted at the rate of 0.8 mL/min, using a linear gradient of MeCN in

water from 40% to 95% over 45 min, then 95% MeCN was held for

further 25 min. The product was collected within 29.3−30.8 min after

the injection, and an excess of unreacted tin precursor 11 was fully

668.1423; found, 668.1398.

5-Iodo-5′-O-[ε-(N,N′-bis(tert-(butyloxycarbonyl)-

guanidino))hexanoyl]-2′-deoxyuridine (14b). General procedure

A was implemented, starting with 5-iodo-5′-O-(6-N-Boc-amino-

hexanoyl)-3′-levulinyl-2′-deoxyuridine (2.25 g, 3.38 mmol). The

Boc protecting group was cleaved, and the separated TFA salt was

reacted without delay with 1.36 g (3.47 mmol) of N,N′-bis(tert-

butoxycarbonyl)-N″-triﬂuoromethanesulfonylguanidine in the pres-

ence of TEA (970 μL, 6.95 mmol) added in two portions. The

product residue dissolved in 60 mL of EtOAc was washed with

separated, eluting ∼7 min later. HPLC analysis: t = 29.90 min

R

(

≥98% purity, Bioscan/UV 280 nm).

125

5

-[ I]Iodo-3′-O-(ε-guanidinohexanoyl)-2′-deoxyuridine

solution of citric acid and brine, organic phase was dried over MgSO ,

4

(13, IDG). The deprotection (HPLC monitoring) of 12 to IDG in a

evaporated and the residue puriﬁed on silica gel column (EtOAc/n-

hexane, 2:1). The 3′-O-Lev group was cleaved with a mixture of

N H ·H O (0.26 mL, 5.36 mmol) in pyridine/AcOH (4:1, 5 mL) and

solution of 40% TFA in MeCN at room temperature required ∼250

min. A treatment of 12 ( dried residue, 21.8 MBq) with neat TFA (30

μL) at 75 °C accelerated the elimination of the guanidine Boc-

2

4

2

a crude product was puriﬁed again on a silica gel column (DCM/

N

DOI: 10.1021/acs.jmedchem.9b00437

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

MeOH, 10:0.6) to give 1.65 g (69% overall yield) of 14b as pale foam,

R_f= 0.35 (DCM/MeOH, 10:0.6), and was 93.7% pure by HPLC

analysis (at 254/280 nm). Further HPLC puriﬁcation of this product

Columbus C18, 100 Å, (5 μm, 10 mm × 250 mm) semipreparative

column, eluted with a solution of 65% MeCN in water for 25 min,

followed by a linear gradient of MeCN from 65% to 95% over 15 min

and ﬁnally, 95% MeCN for the last 15 min, at the rate of 2.5 mL/

(

1

106 mg, ∼5 mg per injection) was performed on a Columbus C18,

00 Å, (5 μm, 10 mm × 250 mm) column eluted with a linear

min.HPLC analysis conﬁrmed its high purity: t = 31.1 min (≥98.2%

R

gradient of MeCN in water from 50% to 95% over 30 min at the rate

of 2.7 mL/min. Subsequent HPLC analysison ACE C18 100 Å, (5

μm, 4.6 mm × 250 mm) column, conﬁrmed an acceptable purity

pure, UV at 220/280 nm); ACE 100 Å, 5 μm, 4.6 mm × 250 mm),

eluted at the rate of 0.8 mL/min with a linear gradient of MeCN in

1

water, from 40% to 95% over 40 min and 95% for 15 min. H NMR

(

≥95.4% at 280 nm) of a ﬁnal product: t = 40.6 min, eluent solvent

(DMSO-d , 600 MHz) δ: 11.16 (s,1H, NH-uridine), 8.44 (bs, 1H,

R

6

3

A 10% CH CN in water, solvent B CH CN; run with a linear gradient

NH), 8.14 (t, 1H, NH, J = 5.50 Hz), 7.81 (s, 1H, H6-uridine, J

=

3

Sn,H

3

of B from 10% to 95% over 45 min, then 95% B for 25 min, elution

19.4 Hz), 6.32 (dd, 1H, H1′, J = 6.45 Hz, J

= 4.64 Hz), 4.92−

1′,2′

1′,2″

1

rate 0.8 mL/min. H NMR (CDCl , 600 MHz) δ: 11.48 (s,1H, NH-

4.84 (m, 1H, H4′), 4.35−4.12 (m, 2H, H5′), 3.59−3.54 (m, 3H, H3′,

OH), 2.87−2.74 (m, 2H, H6-Hex), 2.39−2.32 (m, 3H, H2′, H2-

Hex), 2.18−2.09 (m, 1H, H2″), 1.74−1,66 (m, 2H, H3-Hex), 1.57−

1.51 (m, 2H, H5-Hex), 1.40 (s, 9H, H3-BocN′, 1.38 (s, 9H, H3-

3

uridine), 8.45 (bs, 1H, NH), 8.29 (t, 1H, NH, J = 4.54 Hz), 7.95 (s,

1

H, H6-uridine), 6.23 (t, 1H, H1′, J = 5.50 Hz), 4.47−4.39 (m, 2H,

H3′, OH), 4.30−4.18 (m, 1H, H4′), 3.42−3.38 (m, 2H, H5′), 2.85−

.74 (m, 2H, H2-Hex), 2.54−2.47 (m, 3H, H2′, H6-Hex), 2.20−2.15

m, 1H, H2″), 1.75−1.69 (m, 2H, H3-Hex), 1.63−1.58 (m, 2H, H5-

Hex), 1.51 (s, 9H, H3-BocN′), 1.49 (s, 9H, H3-BocN″), 1.44−1.36

2

(

BocN″), 1.33−1.26 (m, 2H, H4-Hex), 0.29 (s, 9H, 3 × SnCH , J

3

Sn,H

1

3

= 29.8 Hz) ppm. C NMR (DMSO-d , 100 MHz) δ: 173.4 (C1-

6

Hex), 163.5 (C4), 158.9 (C1-Boc-N′), 156.5 (C1-Boc-N″), 152.7

(C2), 149.8 (C-guanidine), 143.2 (C6), 110.4 (C5), 87.3 (C1′), 85.2

(C4′), 83.5 (C2-Boc-N′), 79.7 (C2-Boc-N″), 71.3 (C3′), 62.3 (C5′),

41.9 (C6-hex), 40.5 (C2-Hex), 38.6 (C2′), 30.3 (C5-Hex), 28.6 (C3-

1

3

(

m, 2H, H4-Hex) ppm. C NMR (CDCl , 100 MHz) δ: 173.1 (C1-

3

Hex), 163.5 (C4), 159.6 (C1-Boc-N′), 156.2 (C1-Boc-N″), 153.3

(

C2), 149.6 (C-guanidine), 144.2 (C6), 111.7 (C5), 85.8 (C1′), 84.5

C4′), 81.0 (C2-Boc-N′), 79.5 (C2-Boc-N″), 70.9 (C3′), 68.4 (C5′),

Boc-N′), 27.3 (C3-Boc-N″), 26.1 (C4-Hex), 24.3 (C3-Hex), −7.3 (3

+

4

1.1 (C2-hex), 40.6 (C6-Hex), 38.7 (C2′), 31.2 (C5-Hex), 28.3 (C3-

× CH -Sn) ppm. HDMS (m/z): [M + H] calcd for

3

1

12

112

Boc-N′), 28.1 (C3-Boc-N″), 26.3 (C4-Hex), 24.5 (C3-Hex) ppm.

C H N O

Sn, 740.2600; found 740.2601 using the Sn isotope

2

9

49

5

10

+

HDMS (m/z): [M + H] calcd for C H IN O , 710.1892; found,

signal.₁

2

6

40

5

10

25

7

10.1906.

5-[ I]Iodo-5′-O-[(N,N′-bis(tert-(butyloxycarbonyl)-N″-

propionyl)guanidino]-2′-deoxyuridine (16a). Radioiodination

(general procedure C) of stannane 15a (∼100 μg) was done twice,

5

-Trimethylstannyl-5′-O-[(N,N′-bis(tert-(butyloxycarbonyl)-

N″-propionyl)guanidino]-2′-deoxyuridine (15a). General proce-

1

25

dure B was carried out with 336 mg (0.504 mmol) of 5-iodo-5′-O-

with 40.1 and 34.4 MBq of Na I/NaOH, to give overall 67.8 MBq of

16a. An average yield of the isolated product was 91%. The reaction

mixture was separated and puriﬁed by HPLC using Columbus C18

100 Å (5 μm, 4.6 mm × 250 mm) column, eluted at the rate of 0.8

mL/min with a linear gradient of MeCN in water from 0% to 95%

over 60 min, then 95% MeCN was held further 30 min. The product

was collected in three fractions (within 39−41 min after the injection)

and an excess of unreacted stannane 15a was suﬃciently separated

[

(N,N′-bis(tert-(butyloxycarbonyl)-N″-propionyl)guanidino]-2′-de-

oxyuridine (14a) in EtOAc containing TEA (300 μL, 2.15 mmol) in

the presence of the palladium catalyst (35 mg, 0.05 mmol). A crude

product was initially puriﬁed on a silica gel column (EtOAc/n-

hexanes, 3:1) to give this stannane as colorless foam in 41% yield, R =

f

0

.69 (DCM/MeOH, 10:0.5). The product was only ∼91.3% pure by

HPLC analysis (at 254/280 nm). Further HPLC puriﬁcation (177

mg, ∼8 mg per injection) was performed on a Columbus C18, 100 Å,

eluting ∼4.5 min later. HPLC analysis: t = 39.9 min, (≥98% purity).

R

1

25

(

5 μm, 10 mm × 250 mm) column eluted with a solution of 65%

5-[ I]Iodo-5′-O-[ε-(N,N′-bis(tert-(butyloxycarbonyl)-

guanidino))hexanoyl]-2′-deoxyuridine (16b). Radioiodination

(general procedure C) of stannane 15b (∼100 μg) was completed

MeCN in water for 28 min, then with a linear gradient of MeCN from

5% to 95% over 7 min and 95% MeCN for a further 25 min, at the

rate of 2.4 mL/min. Subsequent HPLC analysis (ACE 100 Å, 5 μm,

6

1

25

with ∼34.1 MBq of Na I/NaOH giving 28.3 MBq of the isolated

product in 83% yield. The reaction mixture was separated and puriﬁed

by HPLC on Jupiter C18 100 Å (5 μm, 4.6 mm × 250 mm) column,

eluted at the rate of 0.8 mL/min with a linear gradient of MeCN in

water from 40% to 95% over 40 min, then 95% MeCN held for

additional 20 min. The product was collected in two fractions (within

25.1−26.5 min after the injection), and an excess of unreacted tin

precursor was fully separated, eluting ∼4.5 min later. Further HPLC

4

.6 mm × 250 mm) conﬁrmed high purity of the ﬁnal product (99.6%

at 220/280 nm): t = 40.85 min, eluent solvent A water, solvent B

R

CH CN; eluted with a linear gradient of B from 5% to 95% over 45

3

1

min and then 95% B for 15 min, at the rate of 0.8 mL/min. H NMR

(

8

CDCl , 600 MHz) δ: 11.51 (s,1H, NH-uridine), 8.64 (bs, 1H, NH),

.49 (t, 1H, NH, J = 5.50 Hz), 7.16 (s, 1H, H6-uridine, J_S_n_,_H= 18.6

3

Hz), 6.19 (t, 1H, H1′, J = 6.56 Hz), 4.49−4.38 (m, 2H, H3′, OH),

4

2

.34−4.29 (m, 1H, H4′), 4.07−3.78 (m, 2H, H5′), 3.26−3.14 (m,

H, H3-Prop), 2.68−2.57 (m, 2H, H2-Prop), 2.46−2.40 (m, 1H,

analysis of the product conﬁrmed its high purity: t

(≥98% purity, Bioscan/UV 280 nm).

= 36.4 min,

R

1

25

H2′), 2.27−2.18 (m, 1H, H2″), 1.50 (s, 9H, H3-BocN′), 1.49 (s, 9H,

5-[ I]Iodo-5′-O-propionylguanidino-2′-deoxyuridine

2

13

(17a). The cleavage process of N-Boc guanidine protecting groups of

H3-BocN″), 0.28 (s, 9H, 3 × SnCH , J

= 27.9 Hz) pm. C NMR

3

Sn,H

1

6a was conducted initially (monitored by HPLC) in a solution of

(

CDCl , 100 MHz) δ: 171.7 (C1-Prop), 166.1 (C4), 163.4 (C-

3

40% TFA in MeCN at room temperature and required ∼190 min. To

guanidine), 156.1 (C1-Boc-N′), 153.2 (C1-Boc-N″), 150.9 (C2),

complete the N-Boc cleavage faster (general procedure C), to a dried

residue of 16a (31.5 MBq) under nitrogen, TFA (40 μL) was added

and the solution was kept in a tightly covered vial at 75 °C for 20 min.

An average yield of the puriﬁed 17a was 83%. The product (26.1

MBq) was separated on Columbus C18 100 Å (5 μm, 4.6 mm × 250

mm) column, eluted at 0.8 mL/min with a linear gradient of MeCN

in water from 0% to 95% over 60 min and 95% MeCN held 10 min

1

43.4 (C6), 113.3 (C5), 86.2 (C1′), 84.0 (C4′), 83.4 (C2-Boc-N′),

9.5 (C2-Boc-N″), 71.6 (C3′), 64.0 (C5′), 39.9 (C2′), 36.0 (C3-

7

Prop), 33.9 (C2-Prop), 28.3 (C3-Boc-N′), 28.1 (C3-Boc-N″), −9.28

+

(

3 × CH3-Sn) ppm. HDMS (m/z): [M + H] calcd for

112_S_n_,₆₉₈_.₂₁₃₁_;_f_o_u_n_d₆₉₈_.₂₁₁₅_u_s_i_n_g_t_h_e112Sn isotope

C H N O

26

43

5

10

signal.

5

- T r i m e t h y l s t a n n y l - 5 ′ - O - [ ε - ( N , N ′ - b i s ( t e r t -

(

butyloxycarbonyl)guanidino))hexanoyl]-2′-deoxyuridine

longer. Both solvents contained 0.07% TFA (v/v); t = 21.2 min

R

15b). Stannylation of the iodide 14b (0.386 g, 0.54 mmol) was

(≥98% purity, Bioscan/UV 280 nm). The product eluting within

20.8−22.0 min after the injection was collected in three fractions,

which were combined and evaporated. To the portion of the product

dry residue (17.95 MBq) ethanol (80 μL) was added, followed by

potassium phosphate buﬀer (100 μL, 10 mM, pH 6.1). The resulting

solution was injected again on HPLC system equipped with Luna CN

(5 μm, 4.5 mm × 250 mm) column eluted at the rate of 0.8 mL/min

with 15% EtOH in potassium phosphate buﬀer (10 mM PB, pH 6.1)

conducted with hexamethylditin 267 mg (0.82 mmol) with the

palladium catalyst (38 mg, 0.05 mmol) and TEA (150 μL, ∼1.0

mmol) in EtOAc (15 mL). A crude product was puriﬁed on a silica

gel column using EtOAc/n-hexanes (2:1) to give 134 mg of 15b in

2

9

9% yield as yellow foam, R = 0.56 (DCM/MeOH, 10:0.4) and was

1.4% pure by HPLC analysis (at 220/280 nm). Further HPLC

f

puriﬁcation (96 mg, ∼4 mg per injection) was performed on a

O

DOI: 10.1021/acs.jmedchem.9b00437

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

for a period of 10 min and then with a linear gradient of EtOH in PB,

from 15% to 70% over 30 min. The product (16.1 MBq, 89%) was

twice on a silica gel column EtOAc/n-hexanes (1:1) was separated as

yellow foam (390 mg, 30% yield), R = 0.71 (DCM/MeOH, 10:0.5)

f

collected within 16.5−20.5 min after the injection (t = 16.8 min,

and was ≥93.7% pure by HPLC analysis (at 220/280 nm). The

HPLC puriﬁcation (168 mg, ∼11 mg per injection) was performed on

a Columbus C18, 100 Å (5 μm, 10 mm × 250 mm) semipreparative

column, eluted with a linear gradient of MeCN in water, from 65% to

95% over 60 min at the rate of 2.4 mL/min. HPLC analysis conﬁrmed

R

≥

97% purity Bioscan/UV 280 nm).

1

25

5

-[ I]Iodo-5′-O-(ε-guanidinohexanoyl)-2′-deoxyuridine

(17b). The standard radioiodination (general procedure C) of

stannane 15b (∼100 μg) was carried out three times with 30.7, 47.5,

and 50.5 MBq of Na¹I/NaOH and gave overall 87.5 MBq of 17b in

an average yield of 68%. A crude reaction mixture, quenched with a

solution of Na S O , was evaporated with a stream of nitrogen and

25

acceptable purity of the product, a mixture of diastereomers: t_R₁

=

57.8 and t_R₂= 58.1 min (≥98% pure, UV at 220/280 nm); ACE C18

column 100 Å (5 μm, 4.6 mm × 250 mm); eluted with a linear

gradient of MeCN in water, from 70% to 95% MeCN over 60 min,

2

3

brieﬂy kept under a high vacuum. The intermediate separation of Boc-

protected product 16b was omitted, and to the resulting dried residue

TFA (100 μL) was added and the mixture heated in a sealed vial at 70

1

then 95% B for 30 min, at the rate of 0.8 mL/min. H NMR (DMSO-

d₆, 600 MHz) δ: 11.27, 11.19 (2s,1H, HN-uridine), 8.23 (bs, 1H,

NH), 8.12 (t, 1H, NH, J = 4.35 Hz), 7.79, 7.74 (2s, 1H, H6-uridine,

°

C for ∼25 min. After cooling, the mixture was diluted with CH CN

3

(

200 μL) and evaporated with a stream of nitrogen, leaving ∼20 μL of

J_S_n_,_H= 19.47 Hz), 7.35−7.29 (m, 1H, aryl-H4), 5.91−5.84 (m, 1H,

the liquid in the reaction vial. This process was repeated three times.

To the tube containing the reaction mixture residue, ethanol (80 μL)

was added, followed by potassium phosphate buﬀer (100 μL, 10 mM

PB, pH 6.1). The resulting solution was injected on HPLC reverse

phase column eluted with a linear gradient of EtOH in potassium

phosphate buﬀer and was separated and puriﬁed by HPLC on Luna

CN 100 Å (5 μm, 4.6 mm × 250 mm) column eluted at the rate of 0.8

mL/min with a linear gradient of EtOH in potassium phosphate

buﬀer (10 mM PB, pH 6.1) from 15% to 70% over 60 min, then 70%

EtOH in PB was kept for further 10 min. The product was collected

within 17.5−20.5 min after the injection. The HPLC analysis of the

H1′), 5.44−5.39 (m, 2H, benzyl), 4.95−4.81 (m, 1H, H4′), 4.29−

4

3

.15 (m, 3H, H5′, H3′), 2.90−2.84 (m, 2H, H6-Hex), 2.49−2.30 (m,

H, H2′, H2-Hex), 2.26−2.10 (m, 1H, H2″), 1.72−1.63 (m, 2H, H3-

Hex), 1.61−1.55 (m, 2H, H5-Hex), 1.48 (s, 9H, H3-BocN′), 1.40 (s,

9

H, H3-BocN″), 1.39 (s, 9H, 3 × CH -t-Bu), 1.37−1.28 (s, 9H, 3 ×

3

2

CH -t-Bu; m, 2H, H4-Hex), 0.29 (s, 9H, 3 × CH −Sn, J

ppm. C NMR (DMSO-d , 100 MHz) δ:173.5 (C1-Hex), 163.6

= 29.7

Sn,H

3

1

3

6

(

1

C4), 160.3 (C1-Boc-N′), 158.6 (C1-Boc-N″), 155.8 (C2-aryl),

53.42 (C2), 149.8 (C-guanidine), 143.9 (C6-aryl), 143.3 (C6),

37.3 (C5-aryl), 130.5 (C3-aryl), 127.4 (C4-aryl), 111.2 (C5), 110.2

(

C1-aryl), 89.5 (C1′), 85.2, (C4′), 82.6 (C2-Boc-N′), 79.8 (C2-Boc-

N″), 73.7 (C3′), 68.4 (C-benzyl), 67.8 (C5′), 41.5 (C2-Hex), 40.7

C6-Hex), 38.0 (C2′), 34.7, 34.6 (2 × C1 t-Bu), 30.8 (C5-Hex), 29.9,

product 17b (t = 20.1 min) showed its satisfactory ≥98% purity

R

(

Bioscan/UV 280 nm).

-Iodo-5′-O-[cyclo-3,5-di(tert-butyl)-6-ﬂuorosaligenyl]-3′-O-

(

5

2

9.8 (C2 t-Bu), 28.9 (C3-Boc-N′), 28.5 (C3-Boc-N″), 26.6 (C4-

[

ε-(N,N′-bis(tert-(butyloxycarbonyl)guanidino))hexanoyl)]-2′-

31

Hex), 24.6 (C3-Hex), −7.62 (3 × CH3-Sn) ppm. P NMR (DMSO-

deoxyuridine Monophosphate (18). General procedure A was

carried out with 5-iodo-5′-O-[cyclo-3,5-di(tert-butyl)-6-ﬂuorosaligen-

yl]-3′-O-(6-N-Boc-aminohexanoyl)-2′-deoxyuridine monophosphate

d ) δ: −8.73, −8.81 (2s, diastereomers) ppm. HDMS (m/z): [M +

6

+

112

H] calcd for C H N O

Sn, 1038.3735; found 1038.3723 using

4

69

5

13

112

the Sn isotope signal.

(

1.20 g, 1.39 mmol), the Boc protecting group was cleaved, and the

125

5

-[ I]Iodo-5′-O-[cyclo-3,5-di(tert-butyl)-6-ﬂuorosaligenyl]-

separated TFA salt reacted directly with 0.60 g (1.53 mmol) of N,N′-

bis(tert-butoxycarbonyl)-N″-triﬂuoromethanesulfonylguanidine in

DCM (35 mL) in the presence of TEA (390 μL, 2.80 mmol)

added in two portions. A crude product puriﬁed twice on a silica gel

column (DCM/MeOH, 10:0.3−0.5) was obtained as colorless rigid

3

′-O-[ε-(N,N′-bis(tert-(butyloxycarbonyl)guanidino))-

hexanoyl)]-2′-deoxyuridine Monophosphate (20). Radioiodina-

tion (general procedure C) of stannane 19 (∼100 μg) was conducted

1

25

twice with 59.2 and 42.6 MBq of Na I/NaOH and gave 89.6 MBq

of 20 with an average yield of 88%. The reaction mixture was

separated and puriﬁed by HPLC using ACE-100 C18 100 Å (5 μm,

4.6 mm × 250 mm) column eluted at the rate of 0.8 mL/min with a

linear gradient of MeCN in water, from 80%−95% over 60 min, and

95% MeCN held constant for additional 30 min. The product was

collected in three fractions (within 35−38 min after the injection). An

excess of unreacted tin precursor 19 was separated suﬃciently, eluting

∼14.5 min later. The HPLC analysis of the product showed the

diastereomeric mixture: t = 34.62 min, t = 35.35 min (≥98%

foam: 1.11 g (79% yield), R = 0.52 (DCM/MeOH, 10:0.5). The

f

HPLC analysis showed a mixture of diastereomers: t_R₁= 42.74 and t_R₂

=

43.40 min (≥96% pure, UV at 254/280 nm); ACE C18 column 100

Å (5 μm, 4.6 mm × 250 mm); eluted at the rate of 0.8 mL/min with a

linear gradient of MeCN in water, from 70% to 95% MeCN over 60

min, then 95% B kept for 30 min. H NMR (DMSO-d , 600 MHz) δ:

1

6

1

1.39, 11.31 (2s,1H, NH-uridine), 8.56 (bs, 1H, NH), 8.23 (t, 1H,

NH, J = 4.4 Hz), 7.96, 7.94 (2s, 1H, H6-uridine), 7.30−7.24 (m, 1H,

R1

R2

aryl-H4), 6.26−6.20 (m, 1H, H1′), 5.54−5.37 (m, 2H, benzyl), 4.45−

purity, Bioscan/UV 280 nm).

125

4

.21 (m, 2H, H3′, H4′), 3.55−3.41 (m, 2H, H5′), 2.90−2.82 (m, 2H,

5-[ I]Iodo-5′-O-[cyclo-3,5-di(tert-butyl)-6-ﬂuorosaligenyl]-

3′-O-(ε-guanidinohexanoyl]-2′-deoxyuridine Monophosphate

(21). The removal of guanidine N-Boc protecting groups of

compound 20 was tested initially in a solution of 50% TFA in

MeCN at room temperature and required ∼140 min. To complete the

N-Boc cleavage faster (general procedure C), to a dried residue of 20

H2-Hex), 2.44−2.34 (m, 3H, H2′, H6-Hex), 2.22−2.16 (m, 1H,

H2″), 1.76−1.68 (m, 2H, H3-Hex), 1.64−1.57 (m, 2H, H5-Hex),

1

.54 (s, 9H, H3-BocN′), 1.49 (s, 9H, H3-BocN″), 1.46 (s, 9H, 3 ×

CH -t-Bu), 1.39−1.36 (s, 9H, 3 × CH -t-Bu; m, 2H, H4-Hex) ppm.

3

13

C NMR (DMSO-d , 100 MHz) δ: 173.2 (C1-Hex), 163.4 (C4),

6

(

three preparations conducted with 24.15, 31.5, and 16.65 MBq)

1

60.15 (C1-Boc-N′), 158.3 (C1-Boc-N″), 155.3 (C6-aryl), 154.2

under nitrogen, TFA (40 μL) was added and the solution was kept in

a tightly covered vial, at 75 °C for 20 min. An excess of TFA was

evaporated with a stream of dry nitrogen. The product was separated

on ACE-100 C18 100 Å (5 μm, 4.6 mm × 250 mm) column eluted at

(

C2), 149.62 (C-guanidine), 148.6 (C2-aryl), 144.2 (C6), 133.4 (C5-

aryl), 130.5 (C3-aryl), 127.2 (C4-aryl), 111.7 (C5), 110.6 (C1-aryl),

8.7 (C1′), 84.6, (C4′), 81.6 (C2-Boc-N′), 80.1 (C2-Boc-N″), 71.2

C3′), 69.3 (C-benzyl), 68.4 (C5′), 41.3 (C2-Hex), 40.8 (C6-Hex),

8.9 (C2′), 34.6, 34.6 (2 × C2 t-Bu), 31.2 (C5-Hex), 29.9, 29.9

CH -t-Bu), 28.3 (C3-Boc-N′), 28.1 (C3-Boc-N″), 26.3 (C4-Hex),

8

(

3

0

.8 mL/min with a linear gradient of MeCN in water from 0% to 95%

over 60 min and 95% MeCN was kept for an additional 10 min; both

solvents contained 0.07% TFA (v/v). The product eluted within

(

2

3

1

4.5 (C3-Hex) ppm. P NMR (DMSO-d ) δ: −8.69, −8.89 (2s,

6

+

4

3.2−44.5 min after the injection. An average yield of the puriﬁed 21

diastereomers) ppm. HDMS (m/z): [M + H] calcd for

was 83%; diastereomeric mixture: t = 42.1, t = 42.4 min (≥98%

C H IN O , 1008.3027; found, 1008.3049.

R‑1

R‑2

41

60

5

13

purity, Bioscan/UV 280 nm). To eliminate an excess of TFA, the

solution containing the product was evaporated again and to the dry

residue of 21 (64.4 MBq) 80 μL of ethanol was added, followed by

potassium phosphate buﬀer (100 μL, 10 mM, pH 6.1). The resulting

solution was injected on HPLC system equipped with Luna CN (5

μm, 4.5 mm × 250 mm) column and was eluted with phosphate

5

-Trimethylstannyl-5′-O-[cyclo-3,5-di(tert-butyl)-6-ﬂuorosa-

ligenyl]-3′-O-[ε-(N,N′-bis(tert-(butyloxycarbonyl)guanidine))-

hexanoyl)]-2′-deoxyuridine Monophosphate (19). The iodide

1

2

(

8 (1.26 g, 1.25 mmol) was reacted with hexamethylditin (0.69 g,

.19 mmol) with the palladium catalyst (90 mg, 0.13 mmol) and TEA

950 μL, 6.82 mmol) in C H (32 mL). A crude product puriﬁed

6 6

P

DOI: 10.1021/acs.jmedchem.9b00437

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

+

buﬀer (10 mM PB, pH 6.1) and EtOH at the rate of 0.8 mL/min with

a linear gradient of EtOH from 50% to 80% over 30 min, then 80%

EtOH held for 10 min. The product (57.3 MBq, 89%) collected

(C3-Boc-N″), −7.74 (3 × CH3-Sn) ppm. HDMS (m/z): [M + H]

1

12

calcd for C H N O

10

Sn, 760.2287; found 760.2293 using the

3

1

45

5

1

12

Sn isotope signal.

1

25

within 23.3−24.6 min after the injection was analyzed on HPLC: t =

5-[ I]Iodo-5′-O-[(N,N′-bis(tert-(butyloxycarbonyl)-N″-

R

2

3.5 min, ≥97% purity Bioscan/UV 280 nm.

methyl-4-benzoyl)guanidino]-2′-deoxyuridine (24). Stannane

23 (∼100 μg) was radioiodinated twice (general procedure C) with

5

-Iodo-5′-O-[(N,N′-bis(tert-(butyloxycarbonyl)-N″-methyl-4-

benzoyl)guanidino]-2′-deoxyuridine (22). Starting with 5-iodo-

′-O-[4-(N-Boc-aminomethyl)benzoyl]-3′-levulinyl-2′-deoxyuridine

2.76 g, 4.03 mmol), general procedure A was carried out as follows:

1

25

2

0.3 and 37.6 MBq of Na I/NaOH and gave ∼47 MBq of the

5

(

product 24 with an average yield 81%. The reaction mixture was

separated on HPLC using ACE-100 C18 100 Å (5 μm, 4.6 mm × 250

mm) column, eluted at the rate of 0.8 mL/min with a linear gradient

of MeCN in water from 50% to 85% over 50 min, then from 85% to

the Boc protecting group was cleaved, and the separated TFA salt was

reacted right away with 1.23 g (4.89 mmol) of N,N′-bis(tert-

butoxycarbonyl)-N″-triﬂuoromethanesulfonylguanidine in the pres-

ence of TEA added in two portions (2 × 680 μL, 9.75 mmol). The

crude product dissolved in 60 mL of EtOAc was washed with a

^s^o^l^u^tⁱ^oⁿ^o^f^cⁱ^t^rⁱ^c^a^cⁱ^d^aⁿ^d^b^rⁱⁿ^e^,^o^r^g^aⁿⁱ^c^p^h^a^s^e^w^a^s^d^rⁱ^e^d^o^v^e^r^M^g^S^O4^,

evaporated and the remaining residue puriﬁed on a silica gel column

9

5% in 10 min and 95% MeCN held for further 10 min. The product

was collected in three fractions within 19−21 min after the injection.

An excess of unreacted stannane 23 was well separated, eluting ∼10

min later. The HPLC analysis of the separated product (t = 18.8

R

min) conﬁrmed its adequate radiochemical purity (≥98%, Bioscan/

(

EtOAc/n-hexane, 2:1) to give 2.57 g of white foam. This was treated

UV 280 nm).

with a mixture of N H ·H O (0.5 M, 0.26 mL) in pyridine/AcOH

(

a silica gel column (DCM/MeOH, 10:0.4) was obtained as a white

125

2

4

2

5

-[ I]Iodo-5′-O-methyl-4-benzoylguanidino-2′-deoxyuri-

4:1, 5 mL) to cleave the 3′-O-Lev group. A crude product puriﬁed on

dine (25). Cleavage of guanidine N-Boc protecting groups of

compound 24 carried out in a solution of 40% TFA in MeCN at room

temperature was completed within ∼220 min. Faster cleavage rate

(general procedure C) was achieved when a dried residue of 24

amorphous solid: 1.52 g, 67% yield, R = 0.54 (DCM/MeOH, 10:0.5).

f

HPLC analysis: t = 41.39 (≥96% pure, UV at 280 nm) on ACE C18

R

column 100 Å (5 μm, 4.6 mm × 250 mm); eluted at the rate of 0.8

(

conducted twice with 13.2 and 30.7 MBq) was treated with neat

TFA (40 μL) under nitrogen and kept in a tightly covered vial at 75

C. After 30 min, the reaction was completed (checked by HPLC).

An excess of TFA was evaporated with a stream of dry nitrogen and

the product was separated on HPLC using ACE-100 C18 100 Å (5

μm, 4.6 mm × 250 mm) column, eluted at 0.8 mL/min with a linear

gradient of MeCN in water from 0% to 95% over 45 min and with

5% MeCN held for an additional 25 min; both solvents contained

.07% TFA (v/v). The product eluted within 18−24 min after the

injection, and an average yield of separated 25 was 76%. The HPLC

mL/min with a linear gradient of MeCN in water, from 10% to 95%

1

MeCN over 45 min, then 95% B kept for 15 min. H NMR (DMSO-

°

d₆, 600 MHz) δ: 11.46 (s,1H, NH-uridine), 8.63 (bs, 1H, NH), 8.21

(

t, 1H, NH, J = 4.5 Hz), 7.97 (s, 1H, H6-uridine), 7.87−7.74 (m, 2H,

aryl-H2, aryl-H6), 7.53−7.48 (m, 2H, aryl-H3, aryl-H5), 6.19−6.12

m, 1H, H1′), 4.34−4.27 (m, 1H, H4′), 4.24−4.19 (m, 1H, H3′,

OH), 3.94−3.87 (m, 2H, benzyl), 3.76−3.68 (m, 2H, H5′), 2.34−

(

9

0

2

.26 (m, 1H, H2′), 2.20−2.15 (m, 1H, H2″), 1.50 (s, 9H, H3-

1

3

BocN′), 1.46 (s, 9H, H3-BocN″) ppm. C NMR (DMSO-d , 100

6

MHz) δ: 171.4 (C1-benzoyl), 163.4 (C4), 160.3 (C1-Boc-N′), 158.4

analysis: t = 22.3 min, ≥98% purity, Bioscan/UV 280 nm). Puriﬁed

5 was evaporated again with a stream of nitrogen, and to the dry

R

(

C1-Boc-N″), 154.4 (C2), 149.7 (C-guanidine), 144.2 (C4-aryl),

2

1

39.3 (C2-aryl), 134. (C6), 127.3 (C3-aryl), 126.9 (C5-aryl), 111.6

C1-aryl), 110.2 (C5), 89.8 (C1′), 86.5, (C4′), 82.3 (C2-Boc-N′),

1.3 (C2-Boc-N″), 71.7 (C3′), 69.3 (C-benzyl), 68.4 (C5′), 38.9

C2′), 28.3 (C3-Boc-N′), 28.1 (C3-Boc-N″) ppm. HDMS (m/z): [M

residue (∼33.0 MBq) ethanol (80 μL) was added, followed by

potassium phosphate buﬀer (100 μL, 10 mM, pH 6.1). The resulting

solution was injected on HPLC system equipped with Luna CN (5

μm, 4.5 mm × 250 mm) column and was eluted at the rate of 0.8 mL/

min with a linear gradient of EtOH in potassium phosphate buﬀer (10

mM PB, pH 6.1); from 10% to 15% over 10 min, next from 15% to

0% over a period of 30 min and 60% EtOH for further 30 min. The

product freed from an excess of TFA (28.4 MBq, 86% yield) was

collected within 28.5−30.0 min after the injection and analyzed on

(

8

(

+

H] calcd for C H IN O , 730.1580; found, 730.1610.

28 36 5 10

5

-Trimethylstannyl-5′-O-[(N,N′-bis(tert-(butyloxycarbonyl)-

N″-methyl-4-benzoyl)guanidino]-2′-deoxyuridine (23). Stanny-

lation of the iodide 22 (0.748 g, 1.025 mmol) was done with

hexamethylditin 605 mg (1.846 mmol), the palladium catalyst (72

mg, 0.01 mmol), and TEA (700 μL, 5.0 mmol) in EtOAc (20 mL). A

crude product 23 (286 mg, 36% yield) separated on a silica gel

6

HPLC: t = 28.1 min (≥98% purity, Bioscan/UV 280 nm).

R

N,N′-Bis(tert-butyloxycarbonyl)-3-iodobenzylguanidine

column using EtOAc/n-hexanes (2:0.5−1), R = 0.62 (DCM/MeOH,

f

(

26). To a stirred solution of 3-iodobenzylamine (0.81g, 3.47 mmol)

1

0:0.5), and was ∼92% pure by HPLC analysis (at 220/280 nm) and

in DCM (40 mL), placed in an ice bath, TEA (490 μL, 3.51 mmol)

and N,N′-bis(tert-butoxycarbonyl)-N″-triﬂuoromethanesulfonyl-

guanidine (1.37g, 3.50 mmol) were added, and the mixture was

stirred at room temperature for ∼4 h (TLC monitoring), ﬁltered and

the solvent evaporated under reduced pressure. To the remaining

residue, 100 mL of EtOAc/n-hexane (4:1) was added, and an excess

of amines and triﬂic amide were removed by aqueous workup with 5%

citric acid and saturated brine. The organic phase was dried over

was further puriﬁed on a Columbus C18, 100 Å (5 μm, 10 mm × 250

mm) semipreparative column (166 mg, ∼7 mg per injection), eluted

at the rate of 2.4 mL/min with a linear gradient of MeCN from 50%

to 67% over 30 min, then from 67% to 95% over 10 min and 95%

MeCN kept for 20 min. Following HPLC, analysis conﬁrmed high

purity of the product: t = 24.6 min (97.7% pure at 220/280 nm) on

R

Columbus C18 100 Å (5 μm, 4.6 mm × 250 mm) column, eluent

solvent A water, solvent B CH CN; eluted with a linear gradient of B

3

MgSO , ﬁltered, and evaporated. A crude product was separated and

puriﬁed twice by column chromatography on a silica gel (DCM/

from 50% to 95% over 30 min and then 95% B for 30 min and the

4

1

elution rate 0.8 mL/min. H NMR (DMSO-d , 600 MHz) δ: 11.14

6

hexanes, 2:0.5−1) to yield 1.26 g (76%) of the guanidine 26; R =

(

s,1H, NH-uridine), 8.24 (bs, 1H, NH), 8.12 (t, 1H, NH, J = 4.46

f

3

0

.62 in DCM/MeOH 10:0.5. HPLC analysis: t = 23.39 (≥97% pure,

Hz), 7.94 (s, 1H, H6-uridine, J

= 19.42 Hz), 7.84−7.72 (m, 2H,

R

Sn,H

UV at 280 nm) on ACE C18 column 100 Å (5 μm, 4.6 mm × 250

aryl-H2, aryl-H6), 7.48−7.36 (m, 2H, aryl-H3, aryl-H5), 6.10−5.89

m, 1H, H1′), 4.87−4.62 (m, 1H, H4′), 4.49−4.27 (m, 1H, H3′,

OH), 3.97−3.87 (m, 2H, benzyl), 3.68−3.53 (m, 2H, H5′), 2.38−

.29 (m, 1H, H2′), 2.16−2.08 (m, 1H, H2″), 1.47 (s, 9H, H3-

(

mm); eluted at the rate of 0.8 mL/min with a linear gradient of

MeCN in water, from 70% to 95% MeCN over 45 min, then 95%

1

2

MeCN was kept for 15 min. H NMR (CDCl

3

, 600 MHz) δ: 11.47

2

BocN′), 1.44 (s, 9H, H3-BocN″), 0.29 (s, 9H, 3 × CH -Sn, J

=

Sn,H

(bs, 1H, NH-Boc), 8.58 (bs, 1H, NH-benzyl), 7.69−7.61 (m, 2H,

3

2

1

9.5 ppm. ¹³C NMR (DMSO-d , 100 MHz) δ:170.2 (C1-benzoyl),

aryl-H4, aryl-H2), 7.29−7.09 (m, aryl-H5, aryl-H6), 4.58 (s, 2H,

6

1

3

63.4 (C4), 159.8 (C1-Boc-N′), 158.5 (C1-Boc-N″), 153.7 (C2),

benzyl), 1.53 (s, 9H, H3-BocN), 1.48 (s, 9H, H3-BocN′) ppm. C

49.6 (C-guanidine), 144.6 (C4-aryl), 143.8 (C6), 139.7 (C2-aryl),

27.5 (C5-aryl), 126.7 (C1-aryl), 111.9 (C3-aryl), 110.3 (C5), 90.37

NMR (DMSO-d , 100 MHz) δ: 163.5 (C1-BocN), 156.2 (C1-

6

BocNH), 153.1 (C-guanidine), 140.4 (C1-aryl), 139.8 (C2-aryl),

136.9 (C4-aryl), 130.4 (C5-aryl), 127.1 (C6-aryl), 94.5 (C3-aryl),

83.4 (C2-BocNH), 79.5 (C2-BocN), 44.1 (C-benzyl), 28.3 (C3-

(

6

C1′), 86.2, (C4′), 84.2 (C2-Boc-N′), 81.8 (C2-Boc-N″), 70.7 (C3′),

9.12 (C-benzyl), 68.8 (C5′), 39.5 (C2′), 28.5 (C3-Boc-N′), 28.1

Q

DOI: 10.1021/acs.jmedchem.9b00437

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

+

BocNH), 28.1 (C3-BocN) ppm. HDMS (m/z): [M + H] calcd for

vacuum. To a resulting dried residue, neat TFA (100 μL) was added,

and the mixture remained in a sealed vial at 65 °C for ∼35 min. After

C H IN O , 476.1041; found, 476.1033.

18

26

3

4

N,N′-Bis(tert-butyloxycarbonyl)-3-trimethylstannyl-

cooling, it was diluted with CH CN (200 μL) and evaporated with a

3

benzylguanidine (27). A mixture of N,N′-bis(tert-butyloxycarbon-

yl)-3-iodobenzylguanidine 26 (0.206 g, 0.433 mmol), hexamethylditin

stream of nitrogen leaving ∼20 μL of the liquid. This process was

repeated twice. To the ﬁnal residue ethanol (80 μL) was added

followed by potassium phosphate buﬀer (100 μL, 10 mM PB, pH 6.1)

and the solution injected on HPLC equipped with Luna CN 100 Å (5

μm, 4.6 mm × 250 mm) column eluted with a linear gradient of

EtOH in potassium phosphate buﬀer as applied in method A. Using

this direct preparation three times, starting with 40.7, 38.9, and 24.1

2

95 mg (0.90 mmol), palladium catalyst (30 mg, 0.043 mmol), and

TEA (150 μL, 1.8 mmol) was reﬂuxed in EtOAc (17 mL) under

argon atmosphere for as long as the starting iodide was detectable

∼3 h, checked often by TLC). After cooling to room temperature

and evaporation of the mixture, a crude product 27 was separated (98

mg, 44% yield) on a silica gel column using EtOAc/n-hexanes (1:4),

R = 0.65 (DCM/MeOH, 10:0.4), ∼91% pure by HPLC analysis (at

20/280 nm) and was further puriﬁed on the Columbus C18, 100 Å

(

1

25

MBq of Na I/NaOH, the guanidine 29 was attained with an average

yield of 87%. HPLC analysis: t_R= 24.7 min, ≥98% radiochemical

purity (Bioscan/UV 280 nm.

f

2

(

5 μm, 10 mm × 250 mm) semipreparative column: (60 mg, ∼5 mg

per injection), eluted at the rate of 2.4 mL/min with a linear gradient

of MeCN from 50% to 85% over 45 min, then from 85% to 95% over

Cells. Two human NB cell lines SK-N-SH (SK) and BE(2)-C (BE)

and one murine NB cell line N1E-115 (N1E) were purchased from

the American Type Culture Collection (ATCC; Manassas, VA). Cells

were maintained according to the vendor’s instructions. The

subcultivation ratio of 1:3 was routinely applied. For animal studies,

cells were harvested when ∼70% conﬂuent and cryopreserved. When

suﬃcient cell numbers were available, cells were thawed, washed twice

with full medium, resuspended in the appropriate growth medium

without fetal bovine serum, cell viability was determined using the

trypan blue assay and cell suspension distributed into sterile 1 mL

syringes equipped with 22 gauge needle.

Proteins, Sera, Buﬀers, and Cell Culture Reagents. ATCC-

formulated Eagle’s minimum essential medium and DMEM:F-12

medium were from the American Type Culture Collection (ATCC;

Manassas, VA). Fetal bovine serum (FBS) was purchased from

Rockland Immunochemicals Inc. (Limerick, PA) and ThermoFisher

Scientiﬁc (Waltham, MA). L-Glutamine, penicillin−streptomycin, and

sodium pyruvate were from GE Healthcare Life Sciences (Marl-

borough, MA) and ThermoFisher Scientiﬁc. Pierce NE-PER nuclear

and cytoplasmic extraction reagents and Micro BCA protein assay kit

were from ThermoFisher Scientiﬁc.

Mice. Research involving animals was performed in accordance

with the UNMC institutional guidelines and protocols as deﬁned by

the Institutional Animal Care and Use Committee for U.S.

institutions. Athymic NCr nude mice (spontaneous mutant) were

purchased from Taconic (Rensselaer, NY). The intratumoral route

(IT) was used for the dose administration. All experiments were

conducted in mice of both genders 4- to 8 weeks-old. Biodistribution

studies were conducted in groups of 14 male and 12 female mice.

Twenty mice received the radioactive compounds alone, and six mice

also received BNPP (3 male, 3 female). The experiment was

conducted using two independent production lots of IDG. The

evaluation of the therapeutic potential was conducted in groups of six

female and six male mice.

1

5 min and 95% MeCN kept for another 10 min. The HPLC analysis

conﬁrmed higher purity of the product: t = 35.4 min (≥98.5% pure

R

at 220/280 nm) on a Columbus C18 100 Å (5 μm, 4.6 mm × 250

mm) column eluted at the rate of 0.8 mL/min with a linear gradient

of MeCN in water from 70% to 95% over 45 min and then 95%

MeCN kept for another 15 min. H NMR (CDCl , 600 MHz) δ:

1.53 (bs, 1H, NH-Boc), 8.62 (bs, 1H, NH-benzyl), 7.40−7.19 (m,

H, aryl), 5.12 (s, 2H, benzyl), 1.52 (s, 9H, H3-BocN), 1.49 (s, 9H,

1

3

1

4

+

H3-BocN′), 0.31 (s, 9H, 3 × CH -Sn) ppm. HDMS (m/z): [M + H]

3

1

4

12

112

calcd for C H N O Sn, 506.1749; found 506.1728 using the Sn

21

35

3

isotope signal.

N,N′-Bis(tert-butyloxycarbonyl)-3-[¹²⁵I]iodobenzyl-

guanidine (28). Radioiodination (general procedure C) of stannane

1

25

2

7 (∼100 μg) was done twice with 59.2 and 58.1 MBq of Na I/

NaOH to give overall 104.4 MBq (89% average yield) of the isolated

product. The reaction mixture was separated and puriﬁed by HPLC

on Columbus C8 100 Å (5 μm, 4.6 mm × 250 mm) column, eluted at

the rate of 0.8 mL/min with a linear gradient of MeCN in water from

5

0% to 95% over 45 min, then 95% MeCN was held for further 35

min. The product was collected in three fractions (within 40.5−42.5

min after the injection), and an excess of unreacted tin precursor was

fully separated, eluting ∼7.5 min later. Performed HPLC analysis of

the product conﬁrmed its high purity: t = 40.7 min (≥98% purity,

R

Bioscan/UV 280 nm).

3

-[¹²⁵I]Iodobenzylguanidine (29). Method A. A dried residue of

8, treated with neat TFA (60 μL) under nitrogen, was kept in a

tightly covered vial at ∼65 °C. After 30 min the reaction (monitored

by HPLC) was completed. An excess of TFA was evaporated with a

stream of dry nitrogen, and 100 μL of 50% MeCN in water added.

The mixture was injected on HPLC ﬁtted with ACE-100 C18 100 Å

2

(

1

5 μm, 4.6 mm × 250 mm) column and eluted at 0.8 mL/min with a

0% MeCN in water for 10 min, then by a linear gradient of MeCN in

Gel Electrophoresis and Western Immunoblotting. All

analyses were conducted on 4−20% or Any kD Mini-PROTEAN

TGX precast protein gels (10-well, 30 μL) using the Mini-PROTEAN

electrophoresis cell (Bio-Rad, Hercules, CA). Protein concentration

in cell lysates was measured using Micro BCA protein assay kit

(ThermoFisher Scientiﬁc). Cell lysate aliquots were added to the 2×

sample buﬀer. Protein samples were denatured at 95 °C for 5 min,

cooled, and loaded onto gels at 100 μg total protein per well. Gels

were run at the constant voltage (150 or 190 V) for ∼1 h. Proteins

were transferred onto a Hybond-P 0.45 μm PVDF membrane

(Amersham Biosciences, Piscataway NJ) in a cold room using a

constant current of 30 mA for 18 h. Kaleidoscope prestained

standards (Bio-Rad Laboratories, Hercules, CA) were used as

molecular weight markers and to monitor the eﬃciency of protein

transfer. The membrane was incubated for 1 h in the blocking buﬀer

containing 5% w/v nonfat dry milk. The blocked membrane was

incubated in the same buﬀer containing primary antibodies overnight

at 4 °C. Norepinephrine transporter antibody PA5-42080 (1:2,000

dilution, v/v) and Pierce goat anti-rabbit poly-HRP 32260 (1:5000

dilution, v/v) were purchased from ThermoFisher Scientiﬁc (Rock-

ford, Il). Rabbit anti-β-actin mAb 13E5 (Cell Signaling Technology,

Danvers, MA; 1:2000 dilution, v/v) was used as a marker of the

protein load. Antigens were detected using the chemiluminescence

water from 10% to 95% over 40 min and 95% MeCN held for an

additional 50 min; both solvents contained 0.07% TFA (v/v). The

product was collected within 26−31 min after the injection. An

average yield of 29 (from three preparations carried out with 31.8,

4

4.4, and 50.3 MBq of 28) was 86%. The subsequent HPLC analysis

showed t = 28.7 min and ≥97% purity (Bioscan/UV 280 nm). To

eliminate the presence of TFA, the solution of separated product 29

R

(

∼20.7 MBq) was evaporated and ethanol (80 μL) added, followed

by potassium phosphate buﬀer (100 μL, 10 mM, pH 6.1). The

resulting solution was again injected on HPLC system equipped with

Luna CN (5 μm, 4.5 mm × 250 mm) column and eluted at the rate of

0

.8 mL/min with a linear gradient of EtOH in potassium phosphate

buﬀer (10 mM PB, pH 6.1); from 30% to 70% over 60 min and 70%

EtOH kept for further 10 min. The product freed from an excess of

TFA (18.3 MBq, 88% yield) was collected within 24.0−33.0 min after

the injection and was analyzed on HPLC: t = 24.8 min, ≥98%

R

radiochemical purity (Bioscan/UV 280 nm).

Method B. Alternatively, the typical radioiodination (general

procedure C) of stannane 27 (∼100 μg) was carried out, but the

separation of Boc-protected guanidine 28 was omitted. A crude

radioiodination mixture, quenched with a solution of Na S O , was

2

3

evaporated with a stream of dried nitrogen and kept under a high

R

DOI: 10.1021/acs.jmedchem.9b00437

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

substrate ChemiGlow according to the manufacturer’s instructions

undisturbed for an additional 23 h, at which time cells were washed

with PBS, trypsinized, and counted and their viability was determined.

Cells were replated in T25 ﬂasks at plating densities of 200 and 500

live cells/ﬂask. Each cell density was tested in quadruplicate.

Radioactive medium from cells in a 24 h group was removed after

24 h of incubations. Cells were washed, harvested, and replated as

described above. The clonogenic assay cells were allowed to grow

undisturbed for 2−3 weeks. The medium was changed once a week.

At 14−21 days later, colonies were washed with 5 mL of ice-cold PBS

followed by 5 mL of PBS/methanol (1:1; v/v) and ﬁxed in 5 mL of

methanol for 10 min. Methanol was removed, and ﬂasks were left

open to dry for a few hours. Crystal violet (5 mL; 0.25% in 1:1 PBS/

methanol) was added to each ﬂask to stain colonies. After

approximately 10 min, the dye was removed, and ﬂasks were rinsed

thoroughly with tap water followed by distilled water and were left to

dry. Colonies were counted by two or three independent readers with

the aid of ImageJ software (http://rsb.info.nih.gov/ij/).

(

Alpha Innotech Corporation, San Leandro, CA).

Stability Studies. Into 1980 μL of 1:1 diluted serum or cells

culture medium, 370−740 kBq of the radioactive compound in 20 μL

of 25% EtOH/PB (100 mM, pH 6.1) was added. This mixture was

brieﬂy vortexed and kept at ambient temperature. At each time point,

a volume of 0.5 mL was withdrawn and processed as follows. Each

aliquot of the incubated mixture was mixed with 0.5 mL of CH CN,

3

vortexed, and centrifuged (2000 rpm, 15 min). Aliquots of

supernatant (500 μL) were acidiﬁed to pH ∼ 6 with 0.05 N TFA

(

2−8 μL). The excess of CH CN was evaporated with a stream of

3

nitrogen and 100 μL water added. A mixture was passed through a 0.2

μm ﬁlter and each sample (∼100 μL, 62.5−130 kBq) injected onto

the HPLC system.

Time-Dependent Cellular Uptake. For each compound tested,

cells were plated in TPP T25 ﬂasks (Midwest Scientiﬁc, Valley Park,

5

MO) at 5 × 10 cells/well in 5 mL of growth medium. Radioactive

compounds were diluted in fresh medium to produce the required

concentration, and 5 mL of this solution was added to each ﬂask after

cells were maintained in culture for 24−48 h, depending on the

doubling time of the cell line. Aliquots of the radioactive medium

were counted in a γ counter to conﬁrm the concentration. Cells were

incubated with radioactive compounds for 15 min, 30 min, 1 h, 2 h, 3

h, 4 h, 5 h, 24 h. Each point in time was tested in triplicate. At the end

of incubation, aliquots of media (0.5 mL) were removed from each

ﬂask for γ counting. The remaining radioactive medium was aspirated

and disposed as a radioactive material. Cells were washed twice with 3

mL of ice-cold PBS. Aliquots of wash PBS (0.5 mL) were also taken

for γ counting. Cells were trypsinized with trypsin/EDTA,

enumerated using a Cellometer disposable cell counting chambers

Subcellular Fractionation. NB cells were plated into six T75 or

T150 ﬂasks and allowed to attach overnight. Cells were grown as a

monolayer to approximately 60−70% conﬂuency. The nonradioactive

growth medium was removed and replaced with 10 mL of fresh

medium containing tested radioactive compounds. Cells were exposed

for 1 h after which time the radioactive medium was removed and

replaced with fresh medium. Aliquots of all radioactive growth media

were counted in the γ counter before and after the cell culture. Cells

in three ﬂasks were processed immediately. Cells in the remaining

three ﬂasks were cultured in fresh nonradioactive medium for 24 h

and then processed. Cell monolayers were rinsed with 5 mL of PBS,

trypsinized, cells were enumerated, and their viability was determined.

By use of NE-PER nuclear and cytoplasmic extraction reagents or

Mem-PER Plus membrane protein extraction kit (ThermoFisher

Scientiﬁc), the cell content was fractionated and counted in a γ

counter to determine the compound distribution in various

compartments of the cancer cell.

(

Nexcelom Bioscience, Lawrence, MA), and centrifuged at 1200 rpm

for 10 min, and cell pellets were washed with PBS. All washes and

spent medium were counted in a γ counter alongside the cell pellet to

determine the cell-associated radioactivity.

Uptake in Exponential and Conﬂuent SK Cells. SK cells were

plated into cell culture ﬂasks (n = 14), allowed to attach for 24 h,

given fresh media, and grown until ∼60%, ∼80%, >90%, and 100%

conﬂuent. Cells in 100% conﬂuent ﬂasks (G0) were given fresh

medium and incubated for additional 3 days with daily media

replacement. Cell in the remaining ﬂasks were given media containing

IDG, incubated for 1 h at 37 °C in the radioactive medium, and

harvested by scraping to avoid losses of the membrane-bound

radioactivity. Cells were counted, centrifuged at 1500 rpm at 4 °C for

Competition with MIBG. Monolayer Method. Cells were plated

into T25 ﬂasks and allowed to grow until ∼60−70% conﬂuent. Fresh

medium containing variable concentrations of nonradioactive MIBG

was added in duplicate to ﬂasks and incubated for up to 3 h. A

constant amount of radioactive guanidines was added to each ﬂask

and cells were returned into the incubator for 1−3 h at which time

media were removed, monolayer washed 1× with full medium, 2×

with PBS, and cells were harvested with the nonenzymatic cell

dissociation solution (Sigma-Aldrich). N1E cells were harvested by

ﬂushing the monolayer with spent media. Cell numbers were

determined, and their radioactive content was measured in a γ

counter.

Filtration Method. Cells in the exponential growth phase were

harvested with the nonenzymatic cell dissociation solution, enum-

erated, centrifuged, washed, and resuspended in PBS. 1 mL aliquots

were dispensed into glass tubes and various concentrations of

nonradioactive MIBG added, and cells were incubated on a Labquake

shaker (Barnstead Thermolyne) at either 37 or 4 °C. To determine

nonspeciﬁc binding, a parallel set of tubes was prepared without cells.

Radioactive guanidines diluted in PBS were added, and the tubes were

gently vortexed. Aliquots (0.1 mL) were withdrawn to determine the

total radioactivity. Both sets of tubes were incubated on a Labquake

shaker for 1 h. Using the vacuum manifold, cells were ﬁltered, washed

with PBS, and the radioactive content was measured in a γ counter.

The content of tubes without cells was also ﬁltered to measure

nonspeciﬁc binding of radioactivity to ﬁlters.

1

0 min, media were collected, and 1 mL aliquot was counted in a γ

counter. Cell pellets were washed with 5 mL of ice-cold PBS,

centrifuged at 1500 rpm at 4 °C for 10 min, PBS was collected, and 1

mL was counted in a γ counter. This procedure was repeated twice.

Cell pellets were resuspended in 1 mL of PBS, transferred into γ

counter tubes, and the cellular radioactivity was measured. Cells from

three 100% conﬂuent ﬂasks and two ∼60% conﬂuent ﬂasks were also

harvested. These cells were enumerated and aliquoted. One aliquot of

6

3

× 10 cells was processed for the cell cycle analyses. The remaining

cells were lysed for Western immunoblotting. The protein content of

lysates was determined using the Micro BCA method.

Clonogenic Assay (Reproductive Integrity). BE and SK cells

were plated in T75 ﬂasks. After ∼18−48 h in culture, the growth

medium was removed from all ﬂasks and replaced with either 15 mL

of fresh medium containing various concentrations of radioactive

guanidines or 15 mL of fresh nonradioactive medium containing PBS

in amounts identical to these added with the radioactive compounds.

Triplicate 0.1 mL aliquots of medium were withdrawn from each ﬂask

and counted in a γ counter to determine the extracellular

concentration of radiolabeled compounds. Cells were processed

after 1 or 24 h in the incubator at 37 °C. Medium was removed from

all ﬂasks in 1 h and 1 h + 23 h groups. Monolayers were washed once

with full nonradioactive medium and PBS. Cells in a 1 h group were

harvested, enumerated, and replated in T25 ﬂasks for clonogenic assay

at 200 and 500 live cells/ﬂask. Each cell density was tested in

quadruplicate. To cells in the 1 h + 23 h group, 15 mL of fresh

nonradioactive medium was added and cells were allowed to grow

Biodistribution. Groups of athymic NCr nu/nu male and female

mice bearing SQ BE xenografts received an intratumor average dose

of 0.33 ± 0.05 MBq of IDG in PBS containing 0.1% HSA. Control

1

25

mice were injected with I-MIBG (0.44 ± 0.1 MBq per tumor). For

the carboxylase inhibitions studies, mice bearing BE xenografts were

given IT injection of BNPP (8.8 and 17.6 μmol) followed by IDG, an

average dose 0.83 ± 0.09 MBq. Control mice in the BNPP study

received only IDG at the same dose. Weights of the syringe, with the

dose and empty after the injection, were determined. Aliquots of the

injected dose were counted in a γ-counter before the dose

S

DOI: 10.1021/acs.jmedchem.9b00437

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

administration and later alongside all extirpated tissues. Mice were

euthanized 24 h after the administration. Lungs, heart, liver, spleen,

pancreas, stomach, small intestine, cecum, large intestine, kidneys,

adrenals, urinary bladder, genitourinary tract (male, en bloc), uterus

and ovaries (female), brain, skin, ears, muscle, thyroid, bone, and

tumor were dissected, rinsed in PBS, and patted dry, and their wet

weight was determined using the analytical balance. Blood, dissected

tissues, and tumors were placed in γ-counter tubes and the radioactive

content was measured using 15−80 keV channel of the Packard

Cobra II model 5003 or Packard Minaxi A5530 γ-counter. Tissue

uptake values were calculated in terms of the percent injected dose

per gram tissue (%ID/gram) for each mouse.

HPLC analyses of all compounds from 3 through 29

(

PDF)

Molecular formula strings and some data (CSV)

AUTHOR INFORMATION

Corresponding Author

E-mail: jbaranow@unmc.edu.

■

*

ORCID

Janina Baranowska-Kortylewicz: 0000-0002-2254-0791

Author Contributions

All authors contributed to the data acquisition, analyses, and

manuscript and have given approval to the ﬁnal version of the

manuscript.

Therapy. Four-week-old NCr nu/nu mice, male (n = 6) and

6

female (n = 5), received SQ implant of 5 × 10 BE cells in BD

Matrigel basement membrane matrix (BD Biosciences, San Jose, CA).

Three weeks later, mice were randomly assigned via a lottery to either

control group or treatment group. Control mice were given IT

injection of the vehicle. Mice in the therapy group received a bolus IT

injection of 8.41 ± 0.32 MBq IDGper tumor. Whole body

radioactivity was measured at 15 min, 2 h, 20 h, 90 h, 142 h, and

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

The authors thank Ed Ezell of the Fred & Pamela Buﬀett

Cancer Center Structural Biology Facility Shared Resource

National Cancer Institute Award P30 CA036727) for his help

■

1

66 h after injection. Necropsy was performed as described in the

biodistribution protocol. Blood was collected via cardiac puncture,

and aliquots were reserved for determination of Hb (HemoCue,

Angelholm, Sweden). The whole body radioactivity was used for the

(

̈

with the NMR analyses. Mass spectrometry analyses were

conducted by the NIH/NIGMS Mass Spectrometry Resource,

Washington University, in St. Louis School of Medicine, St.

Louis, MO. Authors also acknowledge Dr. S. Tian’s technical

assistance in the uptake experiments. These studies were

funded in part by the National Cancer Institute Grant

R21CA187548 (J.B.-K., PI), the National Institutes of

Health/National Institute of General Medical Sciences Grant

P41GM103422 (Gross, PI), and the State of Nebraska

Pediatric Cancer Research Grant LB905 (D.W.C., PI).

construction of the clearance curve and the area under the curve

(

AUC) and mean residence time (MRT) determinations. AUC was

determined using GraphPad Prism 7 for Windows, version 7.04

(

GraphPad Software, La Jolla, CA).

Absorbed Radiation Dose Estimates. The AUCs for the

radioactivity−time curves were integrated from the time of injection

of IDG to 166 h after injection, not corrected for radioactive decay,

31

using previously described by us methods. AUC from 166 h after

injection to inﬁnity was estimated, assuming elimination only by the

radioactive decay, by setting t_f_i_n_a_lto at least 2 times the half-life. MRT

of radioactivity in tumor and whole body was calculated by dividing

the total AUC₀_h_→_∞by the average injected dose. The radiation-

absorbed doses (mGy/MBq) in 1-year-old (9.8 kg) and 5-year0-old

ABBREVIATIONS USED

■

(

19 kg) phantoms were estimated on the basis of the whole-body

BDDC, bis[[4-(2,2-dimethyl-1,3-dioxolyl)]methyl]-

carbodiimide; Boc, tert-butyloxycarbonyl; DCC, 1,3-dicyclo-

hexylcarbodiimide; DCM, dichloromethane; DIPEA, diisopro-

pylethylamine; DMF, dimethylformamide; DMPA, 4-

residence times with the aid of the MIRDOSE 3.0 computer program

(

96

copyright by Oak Ridge Associated Universities).

Statistical Analyses. All variables are expressed as average ±

standard deviation or ±standard error. The cellular uptake, cell

survival and other biological properties of new compounds were

analyzed using two-sided Student’s t test. Two-sided P values of <0.05

were considered statistically signiﬁcant. The least-squares ﬁt of

clearance curves was performed with either Prism or SigmaPlot. A

(

dimethylamino)pyridine; DMSO, dimethyl sulfoxide;

DMTrCl, 4,4′-dimethoxytrityl (triphenylmethyl) chloride;

EtOAc, ethyl acetate; EtOH, ethanol; HPLC, high perform-

1

25

ance liquid chromatography; IDG, 5-[ I]iodo-3′-O-(ε-

guanidinohexanoyl)-2′-deoxyuridine; IUdR, 5-iodo-2′-deoxy-

uridine; Lev, levulinyl (4-oxopentanoyl); MeCN, acetonitrile;

MIBG, 3-iodobenzylguanidine; PB, potassium phosphate

buﬀer; TBAF, tetrabutylammonium ﬂuoride; TBDMSCl, tert-

butyldimethylsilyl chloride; TEA, triethylamine; TFA, tri-

ﬂuoroacetic acid; THF, tetrahydrofuran; TLC, thin layer

chromatography; TMS, tetramethylsilane

2

correlation coeﬃcient R > 0.8 was used as the lowest acceptable

goodness-of-ﬁt criterion. AUC was integrated by the trapezoidal

^m^e^t^h^o^d⁽^P^rⁱ^s^m⁾^.^T^u^m^o^r^g^r^o^w^t^h^c^u^r^v^e^s^w^e^r^e^u^s^e^d^t^o^d^e^t^e^r^mⁱⁿ^e^TD

values. Tumor weights at the necropsy were used as the ﬁnal measure

of the tumor response. These data were analyzed using ANOVA with

the Tukey−Kramer multiple comparisons. All diﬀerences were

considered signiﬁcant when P < 0.05. SigmaPlot/SigmaStat from

Systat Software, Inc., Point Richmond, CA, and GraphPad InStat

computer software from GraphPad Software, Inc., La Jolla, CA, were

used for these analyses.

REFERENCES

■

(

1) Schmidt, M. L.; Lukens, J. N.; Seeger, R. C.; Brodeur, G. M.;

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Supporting Information

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ACS Publications website at DOI: 10.1021/acs.jmed-

chem.9b00437.

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DOI: 10.1021/acs.jmedchem.9b00437

J. Med. Chem. XXXX, XXX, XXX−XXX

Article Doi

Norepinephrine-Transporter-Targeted and DNA-Co-Targeted Theranostic Guanidines

DOI: 10.1021/acs.jmedchem.9b00437

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Full text of DOI:10.1021/acs.jmedchem.9b00437

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