Synthesis and biological evaluation of [18F](2S,4S)4-(3-fluoropropyl) arginine as a tumor imaging agent

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Synthesis and biological evaluation of [ F](2S,4S)4-(3-fluoropropyl) arginine as a

tumor imaging agent

Renbo Wu, Song Liu, Yajing Liu, Yuli Sun, Xuebo Cheng, Yong Huang, Zequn Yang,

Zehui Wu

PII:

S0223-5234(19)30882-7

DOI:

https://doi.org/10.1016/j.ejmech.2019.111730

EJMECH 111730

Reference:

To appear in: European Journal of Medicinal Chemistry

Received Date: 9 August 2019

Revised Date: 21 September 2019

Accepted Date: 21 September 2019

Please cite this article as: R. Wu, S. Liu, Y. Liu, Y. Sun, X. Cheng, Y. Huang, Z. Yang, Z. Wu, Synthesis

18

and biological evaluation of [ F](2S,4S)4-(3-fluoropropyl) arginine as a tumor imaging agent, European

Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.111730.

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Synthesis and Biological Evaluation of [¹⁸F](2S,4S)4-(3-fluoropropyl) Arginine as a Tumor Imaging Agent

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Renbo Wu, ^aSong Liu, ^aYajing Liu, ^bYuli Sun, ^aYong Huang, ^aZequn Yang, ^aand Zehui Wu ^a*

1

journal homepage: www.elsevier.com

Synthesis and Biological Evaluation of [¹⁸F](2S,4S)4-(3-fluoropropyl) Arginine as

a Tumor Imaging Agent

Renbo Wu, ^aSong Liu, ^aYajing Liu, ^bYuli Sun, ^aXuebo Cheng, ^aYong Huang, ^aZequn Yang ^aand Zehui Wu

a*

^aBeijing Institute of Brain Disorders, Capital Medical University, Beijing, 100069, China;

b

School of Pharmaceutical Science, Capital Medical University, Beijing 100069, China.

ARTICLE INFO

ABSTRACT

Article history:

Received

Revised

Accepted

Available online

Designing novel ¹⁸F-labeled amino acid derivatives for targeted amino acid

transporters is an attractive strategy for the development of therapeutic and diagnostic

agents for cancer therapy. In this work, we have developed a novel 3-fluoropropyl

analog of arginine, namely, (2S,4S)4-[¹⁸F]FPArg, [¹⁸F]1, to be used as a probe for

studying arginine metabolism. Optically pure and labeled with ¹⁸F and ¹⁹F, (2S,4S)4-

(3-fluoropropyl)arginine was synthesized and isolated in high radiochemical purity

(>95%). In vitro uptake assays in human MCF-7 cells revealed that [¹⁸F]1 enters cells

mainly via sodium-independent cationic amino acid transporters and was

inhibited >62% by arginine. [¹⁸F]1 showed a high cellular uptake of 7.3 ± 0.24% and

6.07 ± 0.3% uptake/100 mg protein after incubation in MCF-7 and MDA-MB-231

cells for 120 min, respectively. In vivo biodistribution studies demonstrated that [¹⁸F]1

provided high tumor uptake and high tumor to muscle ratios (5:1 at the 30 and 60 min

time points). In vivo PET imaging studies demonstrated tumor-specific uptake in nude

mice bearing MCF-7 breast tumors with an excellent tumor-to-muscle ratio. These

results suggest that [¹⁸F]1 is a promising tracer for clinical breast cancer imaging and

may be used to diagnose and monitor diseases that are associated with arginine

metabolism.

Keywords:

Arginine

Breast Cancer

Imaging agent

Cationic Amino Acid Transporter,

Positron Emission Tomography

1. Introduction

diagnosis of clinical gliomas because they can cross the blood-

brain barrier.[16, 31, 32] [¹⁸F](2S,4R)4-FGln has been

Radiolabeled amino acids are structurally and functionally

diverse tumor imaging agents suitable for positron emission

tomography (PET) and single photon emission computed

tomography (SPECT), which have been used extensively for

oncologic imaging in both preclinical and human clinical

studies.[1-4] Imaging agents determined by their amino acid

transporters can potentially provide uniquely and clinically

biological tumor information.[1] Traditionally, the amino acid

transporter family has been functionally characterized as a

transport system, such as system L, system A, system N,

system ASC, system xCT and cationic amino acid transporter

(CAT), many of which contain multiple family members.[5-10]

The research and identification of new radiolabeled amino

acids with novel features has focused on targeting system L

and ASC because they are upregulated in various human

cancers.[3, 10-14] Many imaging agents targeting system L

(e.g., [¹¹C] MET,[15, 16] [¹⁸F] FET,[17-19] [¹⁸F] FDOPA,[20,

21] [¹²³I] IMT,[22]) and system ASC ([¹⁸F] FMA,[23] [¹¹C]

Gln,[24] [¹⁸F] (2S, 4R)4-FGln,[25, 26] [¹⁸F] (2S,4R)4-

FPGln,[27] anti-3-[¹⁸F] FACBC[28]) have been reported in the

past few decades (Figure 1). Imaging agent targeting system A

(e.g., S-[¹⁸F] FAMP and S-[¹⁸F] MeFAMP[29]) and system

xCT (e.g., [¹⁸F] (2S,4R)4F-Glu[30] and [¹⁸F] (2S,4S) FSPG[30])

have also been gaining interest (Figure 1). Among these,

targeting system L imaging agents are widely used in the

demonstrated to be useful in detecting breast tumors in

women.[33] ¹⁸F(2S, 4S)FSPG has a high cancer detection rate

in patients with hepatocellular carcinoma.[34] Anti-3-[¹⁸F]

FACBC (Axumin) has been approved by the FDA for routine

clinical applications of prostate cancer. Recently, amino acid

imaging agent development has explored cationic amino acid

transport systems for oncologic imaging, such as the cationic

tyrosine

analog

O-2((2-[¹⁸F]fluoroethyl)methyl-

amino)ethyltyrosine ([¹⁸F]FEMAET) selective for target

system ATB^0,+.[5] The 1H-[1,2,3] triazole substituted amino

acid ((S)-[¹⁸F]AFETP) is mediated by a combination of

cationic amino acid transport and system L transport.[35] [¹⁸F]

AFETP is expected to be used for the early detection of the

tumor response to arginine depletion due to ADI-PEG20

treatment.[36] Though studies have shown that cationic amino

acid transport is increasingly recognized as important targets

for oncology imaging and therapy,[3, 36-38] imaging agents of

natural cationic amino acid derivatives, such as L-arginine and

L-lysine, have not been reported yet. Therefore, designing and

synthesizing natural cationic amino acid derivative imaging

agents are particularly urgent.

Corresponding authors.

E-mail addresses: wzhhuey2012@ccmu.edu.cn

1

carcinogenesis and the biological behavior of tumors through

various biological metabolic pathways.[40, 41] Some normal

cells are able to synthesize arginine from citrulline by

argininosuccinate synthetases (ASS). However, the expression of

ASS is lacking in some tumor cells such as melanoma and

hepatocellular carcinoma, which renders the arginine auxotrophy

of the tumor. The requirement of exogenous arginine for cellular

growth, proliferation and survival in ASS lacking tumor provides

an important strategy for the development of therapeutic and

diagnostic agents for cancer.[3, 42] Therefore, L-arginine and its

analogs could serve as biomarkers for oncologic imaging in

terms of detection, prognosis and response to therapeutic

monitoring of arginine metabolism-related tumors. Herein, we

report the first efficient organic and radiosynthetic routes to

obtain the L-arginine derivative (2S,4S)4-FPArg, 1. This novel

tracer was evaluated both in vitro cell uptake assays and in vivo

biodistribution studies and PET/CT imaging with nude mice

transplanted with human MCF-7 tumor.

Fig. 1. Chemical structures of [¹¹C]Gln, [¹⁸F](2S,4R)4-FGln, [¹⁸F](2S,4S)4-

FPGln, anti-3-[¹⁸F]FACBC, [¹⁸F](2S,4S)FSPG, (S)-[¹⁸F]AFETP and

[¹⁸F]FEMAET

Arginine is a cationic amino acid which can be synthesized

from citrulline in two steps by the urea cycle enzymes

argininosuccinate synthetase and argininosuccinate lyase[37, 39].

Arginine is closely related to the tumor suppression,

Reagents and conditions: (a) Pd/C, methanol, rt, 3h; (b) N,N′-Di-Boc-1H-pyrazole -1-carboxamidine, N,N-Diisopropylethylamine, ACN, 50 ^oC, 3h; (c)

Methanesulfonyl chloride, Et₃N, DCM, rt, 4h; (d) TASF, Et₃N^.(HF)₃, CH₂Cl₂/THF, 55 ^oC, overnight.

Scheme 1. An attempt to synthesis of 4-FArg

arginine attacked the activated ester at position 4 of arginine to

2. Results and discussion

form a stable five-membered ring. Therefore, at least one of the

NH groups of guanidine of arginine must be protected, but the

protected arginine cannot introduce an activated ester at carbon

4 due to the large steric hindrance, and the corresponding 4-

FArg cannot be obtained. 4-FArg may be obtained by

introducing a fluorine atom into the starting material and then

performing a multistep reaction. However, radiosynthesis is

complicated to operate and has low yields, which is not

conducive to clinical application.

2.1. Synthesis

Our strategy was to modify the carbon at position 4 of

arginine based on the analysis of the arginine structure and the

experience of synthesizing glutamine derivatives.[25, 27] 4-

FArg was first considered to be synthesized because the

structure of arginine was minimally altered (Scheme 1). The

synthesis of compound 2 was obtained by the guanidinylation

of the amino group with N, N'-Di-Boc-1H-pyrazole-1-

carboxamidine according to the reported procedure.[43]

Surprisingly, the tosylation reaction of 2 did not work,

probably because the steric hindrance from the carbon at

position 4 in arginine was too large. The activating group was

replaced by a less sterically hindered methanesulfonyl chloride,

and 3 was obtained in low yield (32%). In the reaction solution,

3 easily converted to 4 (checked by LC-MS). Unfortunately,

the fluorination reactions cannot provide any of the desired

product 5 regardless of whether the fluorinating reagent is

tetrabutylammonium fluoride, potassium fluoride or

To obtain a radioactive arginine derivative with simple

labeling conditions, we tried to introduce a fluoropropane at the

carbon at position 4 of the arginine (Scheme 2, (2S,4S)4-(3-

fluoropropyl)arginine, (2S,4S)4-FPArg, 1), which will make

the fluorine SN2 substitution reaction on the primary carbon

easier and will not racemize during the labeling reaction.

According to the experience from the synthesis of 4-FArg, at

least one NH at carbon position 2 or 5 of the precursor of 1

must be protected, and a six-membered ring can be formed

easily by the NH at carbon position 5, which reacts with the

sulfonyl ester; therefore, the NH at carbon position 5 was

selected for protection. Following our design (Scheme 2), 7

tris(dimethylamino)

(TASF). We reasoned that the NH groups of guanidine of

sulfonium

difluorotrimethylsilicate

2

was obtained by our reported synthetic method.[27] Compound

7 was mixed with an anhydride, followed by reduction with

sodium borohydride to give 8 in a total yield of 85%. Notably,

guanidinylation of 8 is a critical step in the whole scheme. A

Mitsunobu reaction was carried out by using 8, N, N'-Di-Boc-

1H-pyrazole-1-carboxamidine, triphenylphosphine and diethyl

azodicarboxylate to give 9 in 89% yield (Scheme 2). This step

enabled the introduction of the NH (carbon at position 5 of

arginine protected) with a tert-butoxycarbonyl (Boc) protecting

group. The next step involved coupling of a suitable amino

protecting group that could be easily removed after

radiolabeling. 4-Methoxybenzyl was chosen as the amino

protecting group because it is easily removed in the presence of

TFA. Substituting 4-methoxybenzylamine for the activated

ester (pyrazole) of 9 gave 10 in 75% yield. Removal of the OH

protecting group in 10 by pyridinium p-toluenesulfonate gave

11 in 84% yield. Tosylation of 11 gave tosylate 12 (the

precursor for fluorination) in 86% yield. The O-tosylated 12

was treated with TASF and Et₃N(HF)₃to give the desired

compound 13 in 55% yield. Optimization of the fluorination

reaction conditions using TASF and Et₃N(HF)₃as the reagents

that were reported previously for the preparation of 4-

fluoroglutamine.[27] Deprotection using TFA at room

temperature produced the final products, (2S,4S)4-FPArg, 1 in

93% yield. To better distinguish the peaks of the isomers

produced during the labeling reaction and to investigate the

ability of tumors to take up D and L arginine, we synthesized

the corresponding isomer of 1, (2R,4R)4-FPArg, 25, using the

same synthesis method as 1 (Scheme 2 and S1).

.

Reagents and conditions: (a) tert-butyl 2,2,2-trichloroacetimidate (TBTA), BF₃·Et₂O, DCM, cyclohexane, rt, overnight; (b) LiHMDS, Allyl bromide, THF, -78

^oC, 4 h; (c) 9-BBN, H₂O₂, NaOH, 0 ^oC - rt, 48 h; (d) DHP, PPTS, DCM, rt, 3 h; (e) Pd/C, H₂, EtOH, rt, 2 h; (f) Ethyl chloroformate, NaBH₄, THF, H₂O, 0 ^oC - rt,

4 h; (g) N,N′-Di-Boc-1H-pyrazole-1-carboxamidine, triphenyl phosphine, diethyl azodicarboxylate, THF, 0 ^oC - rt, overnight; (h) 4-Methoxybenzylamine, N,N-

Diisopropylethylamine, ACN, 3h; (i) PPTS, ethanol, 2h; (j) p-toluenesulfonyl chloride, Et₃N, DCM, rt, overnight; (k) TASF, Et₃N^.(HF)₃, CH₂Cl₂/THF, 55 ^oC,

overnight; (l) TFA, rt, overnight.

Scheme 2. Synthesis of (2S,4S)4-FPArg, 1 and structure of (2S,4S)4-FPArg, 25

indicates that the elimination reaction was faster than the

nucleophilic substitution reaction during the reaction. This

problem may be solved by protecting the NH at the carbon at

position 2. To our delight, 18-crown-6 replaced Kryptofix 222 as

the phase transfer catalyst, and the labeling yield was greatly

improved when the reaction solution was acetonitrile/tert-amyl

alcohol (1/9) (Table 1, entry 5). A large amount of the labeled

precursor was unreacted as the fluorinated reactant was detected

by LC-MS, therefore, the labeling conditions were further

optimized by increasing the amount of the phase transfer catalyst.

When the amount of the phase transfer catalyst was doubled, the

labeling yield increased from 3.52% to 5.93% (Table 1, entry 6).

Further increasing the temperature of the reaction, the labeling

yield continued to improve (Table 1, entry 7). In summary, the

labeling reaction was carried out in two steps. The first step,

nucleophilic [¹⁸F] fluoride substitution, was successfully

2.2. Radiolabeling and Stability

The radiosynthesis was conducted in two steps (Scheme 3).

Initially, the radiosynthesis was conducted in two steps following

the (2S,4R)4-[¹⁸F]FPGln labeling method,[27] but the

radiolabeling failed despite trying to increase the temperature or

change the solvent (entries 1-3). According to Kim's method,[44,

45] the tert-amyl alcohol solvent system may have some

beneficial effects on the mechanism of nucleophilic substitution;

therefore, tert-amyl alcohol was selected as the reaction solvent

for the labeling reaction. Kryptofix 222 was first chosen as the

phase transfer catalyst for the labeling reaction; surprisingly, the

yield was still very low, and the product was racemic (Figure S4a

and entry 4). The precursor, 12, was converted into 26 or 27 as

determined by the LC-MS analysis of the first step reaction

solution (Figures 2a, S1 and Table 1, entries 1-4), which

3

performed in tert-amyl alcohol/acetonitrile (9/1) at 100 ^oC to

afford the intermediate [¹⁸F]13 from pure precursor 12. Using

solid phase extraction with a C18 cartridge, the [¹⁸F]13 was

retained while the unreacted [¹⁸F] fluoride was removed. Then,

the [¹⁸F]13 was eluted from the cartridge with ethanol. The

radiochemical purity of [¹⁸F]13 was determined by radiometric

thin-layer chromatography (ethyl acetate/petroleum ether = 2/3,

Rf = 0.6) and analytical HPLC (Figure S2). In the second step,

deprotection was achieved with TFA, followed by removal of the

6

7

(1) 18-crown-6(320 mg), KHCO₃(58 mg),

90 ^oC, 15 min, tert-amyl alcohol and

acetonitrile (9/1)^{d b}

(1) 18-crown-6(320 mg), KHCO₃(58 mg),

100 ^oC, 15 min, tert-amyl alcohol and

acetonitrile (9/1)^{d b}

5.93 ±

1.01%

NO

6.95 ±

1.51%

(a) 1 mL 160 mg 18-Crown-6 in 18.6 mL acetonitrile/29 mg KHCO₃in

3.4 mL water; (b) TFA/anisole (495/5), 60 C, 5 min; (c) 1 mL 220 mg

K₂₂₂in 18.6 mL acetonitrile/40 mg K₂CO₃in 3.4 mL water; (d) 1 mL 18-

Crown-6 in 18.6 mL acetonitrile/ KHCO₃in 3.4 mL water. (f) yield

uncorrected, n = 3.

o

reaction solution.

A solution containing 10% ethanol in

physiological saline was added and passed through a 0.22 µm

nylon filter to provide the final product (2S,4S)4-[¹⁸F]FPArg,

[¹⁸F]1(log P = -0.72). Radiochemical purity was determined by

chiral analytic HPLC (Figure 2b and S4). Notably, first step was

purified by a solid phase column, and occasionally two

radioactive peaks were observed (ethyl acetate/petroleum ether =

2/3, Rf = 0.4 and 0.6, Figure S3). We speculate the other

radioactive peak may due to the removal of one or more Boc

groups from intermediate [¹⁸F]13. This is because only the target

compound [¹⁸F]1 was obtained when the mixture of the first

reaction was hydrolyzed by TFA. [¹⁸F]1 displayed high stability

in PBS buffer (10 mΜ, pΗ = 7.4) at room temperature and plasm

at 37 °C for 2 h, as confirmed by radio-HPLC (Figure 3a). The

radiolabeling of [¹⁸F]25 was consistent with the radiolabeling

conditions of [¹⁸F]1 (Scheme S2).

2.3. Cell Uptake Assays and Inhibition Studies

MCF-7 (ER+) and MAD-MB-231 (ER-), the two typical types

of breast cancer are chosen for uptake capacity study. One

reason for the selection is based on estrogen receptor (ER)

expression level, which determines the following treatment

strategy optimization. The other is the membrane presentation of

transporters, which is crucial for our interested biological

function. Previous study has showed that argininosuccinate

synthetase 1 (ASS1) expression level is highly related to the

subcellular localization of CAT-1[36] and the mRNA level of

ASS1 is high in MCF-7 (log2(RPKM) = 7.99) and low in MAD-

MB-231

(log2(RPKM)

=1.89)

[CCLE,

https://portals.broadinstitute.org/ccle]. However, the cation

transporters present in both cell types are primarily CAT-1

subtypes.[46] Both cells can uptake and accumulate [¹⁸F]1 from 5

min to 120 min, which suggests [¹⁸F]1 can be used as a tracer for

the diagnosis of ER+ and ER- breast cancers (Figure 3b).

However, the corresponding isomer [¹⁸F]25 showed a lower

uptake in both cells, which is consistent with the high uptake of

the L-type natural amino acid reported in the literature.[25]

Additionally, whether for [¹⁸F]1 or [¹⁸F]25, the uptake by MCF-7

cells is higher than that by MDA-MB-231 cells at the late stage.

The result is accordance with the membrane presentation level of

CAT-1. [¹⁸F]25 was not further subjected to biological evaluation

due to the low uptake of [¹⁸F]25 by the tumor cells. The in vitro

cell uptake of [¹⁸F]1, [¹⁸F]FDG and [¹⁸F](2S, 4R)4-FGln in

MCF-7 cell was further compared. As Figure 3c exhibited, in

vitro cell uptake of [¹⁸F]1 showed lower uptake than [¹⁸F](2S,

4R)4-FGln and [¹⁸F]FDG in MCF-7 cell culture for 5 min to 120

min.

Reagents and conditions: (a) 1 mL of 18-crown-6/KHCO₃(320 mg of 18-

crown-6 in 18.6 mL of ACN/58 mg of KHCO₃in 3.4 mL of water), 100 °C,

15 min, tert-amyl alcohol and acetonitrile (9/1); (b) TFA, anisole, 60 °C, 5

min.

Scheme 3. Radiosynthesis of (2S,4S)4-[¹⁸F]FPArg, [¹⁸F]1

Fig. 2. (a) Structures of two byproducts in labeling reaction; (b) HPLC

profiles of (2S,4S)4-FPArg, 1, (2R,4R)4-FPArg, 25 and (2S,4S)4-[¹⁸F]FPArg,

[¹⁸F]1, using HPLC system (Column: Chirex 3126 (D)-penicillamine 250 ×

4.6 mm, 4.6 µm). Mobile phase (isocratic): Methanol/1 mM CuSO₄= 5/95.

Table 1. The optimal conditions for (2S,4S)4-[¹⁸F]FPArg, [¹⁸F]1

Ent

ry

1

Reaction conditions

Yield^f

Epimeri

zation

NO

(1) 18-crown-6, KHCO₃, 80 ^oC, 15 min,

0.57 ±

0.15%

ACN ^{a b}

2

3

(1) 18-crown-6, KHCO₃, 100 ^oC, 15 min,

ACN ^{a b}

(1) 18-crown-6, KHCO₃, 100 ^oC, 15 min,

NO

1.04 ±

0.21%

1.08

DMSO ^{a b}

±0.19%

1.42 ±

0.23%

4

5

(1) K₂₂₂, K₂CO₃, 100 ^oC, 15 min, tert-amyl

alcohol ^{c b}

(1) 18-crown-6(160 mg), KHCO₃(28 mg),

90 ^oC, 15 min, tert-amyl alcohol and

acetonitrile (9/1)^{d b}

Yes

NO

3.52 ±

0.56%

4

conditions inhibited 62% of the uptake relative to the sodium-

free control (p < 0.01), indicating that the cell uptake of [¹⁸F]1

was mainly mediated by sodium-independent cationic amino acid

transport (Figure 3d). From the above experimental results, it can

be seen that arginine has a stronger inhibitory effect on [¹⁸F]1

than lysine, but the usual transport proteins of the CAT family

have similar Km values to arginine and lysine. To investigate the

differential inhibition of arginine compared with lysine, the half-

maximal inhibitory concentrations (IC₅₀, mM) of arginine and

lysine were measured (Figure 3e-f). The results indicate that

arginine (IC₅₀= 1.76 mM) exhibits a higher affinity than lysine

(IC₅₀= 2.36 mM) for the CAT family of transport proteins. It has

been reported that cation transporter proteins in MCF-7 cells

mainly include CAT-1, CAT-2A and CAT-2B, and CAT-1 plays

an important role in the cellular uptake of arginine.[46] The

CAT-1 protein shows a higher affinity for amino acids, while

CAT-2A and CAT-2B show a lower affinity.[47, 48] The CAT

proteins seem to have a higher affinity for cationic amino acids

with a long carbon backbone.[49] Combined with the above

reports and the results of this experiment, we can conclude that

the order of affinity for CAT proteins is [¹⁸F]1 > arginine >

lysine, the inhibition rate of arginine for [¹⁸F]1 should be higher

than lysine, and [¹⁸F]1 may be a high-affinity CAT-1 transporter

substrate. Taken together, the cationic amino acid transport

substrate L-arginine was the most effective inhibitor of [¹⁸F]1

uptake. These findings suggest that a significant proportion of the

in vitro uptake of [¹⁸F]1 is mediated by cationic amino acid

transporters, although other amino acid transporters that

recognize neutral amino acids, including systems L and ASC also

contribute. More extensive amino acid transport assays will be

needed to determine which of these transport systems mediate the

uptake of [¹⁸F]1 by MCF-7 cells.

Fig. 3. (a) The stability in PBS and Plasm of (2S,4S)4-[¹⁸F]FPArg, [¹⁸F]1

(n = 3) (b) In vitro cell uptakes of [¹⁸F]1 in MCF-7 and MDA-MB-231 cell

lines, (n = 5) (c) In vitro cell uptakes of [¹⁸F]FDG, [¹⁸F](2S, 4R)4-FGln and

[¹⁸F]1 in MCF-7, (n = 5). At the same time point, the cell uptake of [¹⁸F] 1

was compared with [¹⁸F]FDG (significantly different at 4 time points are less

than 0.001), [¹⁸F](2S, 4R)4-FGln (significantly different at 4 time points are

less than 0.001), respectively. (d) In vitro cell uptake inhibition studies of

[¹⁸F]1 conducted in MCF-7 cells, incubation 30 min. Na solution= PBS buffer,

Na free solution = K or choline replacement Na PBS buffer, MeAIB = 10

mM N-methyl a aminoisobutyric acid (system A inhibitor), BCH = 10 mM

aminobicyclo(2,2,1)-heptane-2-carboxylic acid (system L inhibitor), Arg = 10

mM arginine, His = 10 mM histidine, Lys = 10 mM lysine, ASC = a mixture

of L-Ala, L-Ser, L-Cys (3.3 mM of each amino acid), RKH = L-Arg, L-Lys,

L-His mixture (3.3 mM of each amino acid). The sodium PBS control

condition was compared to the PBS BCH*, ASC*, His*, Arg**, Lys** and

RKH** (*, p < 0.05 and **, p < 0.01) condition using two-tailed t tests. The

choline PBS control condition was compared to the choline PBS Arg (***, p

< 0.01) condition using two-tailed t tests. IC₅₀assay: inhibition of [¹⁸F]1

uptake by increasing different concentrations of L-Arg (e) or L-Lys (f),

incubation 120 min. Each data point is n = 5.

2.4. Biodistribution Studies

The results of the biodistribution studies with nude mice with

subcutaneous MCF-7 tumors at 5, 30, 60 and 120 min

postinjection (p.i.) are shown in Table 2 and are expressed as the

percent of the total injected dose per gram of tissue (% ID/g). The

tumor uptake of [¹⁸F]1 was rapid with 1.6 ± 0.21 % ID/g at 5 min

and remained relatively constant over the course of the study to

1.78 ± 0.25 % ID/g at 30 min and 1.68 ± 0.13 % ID/g at 60 min

(Table 2). A slow wash out was observed for the tumor uptake

and retention in the MCF-7 tumor to 1.14 ± 0.07 % ID/g at 120

min p.i. At 30 min, the tumor-to-background (tumor-to-muscle

and tumor-to-blood) ratios of [¹⁸F]1 were 5.34 ± 0.83 and 4.61 ±

0.81, respectively. At each time point, the brain uptake of [¹⁸F]1

was relatively low. As shown in Table S1, maximal blood, liver

and kidney uptake of [¹⁸F]1 in BALB/c mice were rapid with

3.48 ± 0.79 % ID/g, 22.88 ± 3.68 % ID/g and 24.98 ± 5.33 %

ID/g respectively, 5 min after the injection with progressive

washout at 30, 60 and 120 min, which is consistent with the

biodistribution of nude mice bearing MCF-7 tumors (Table 2).

The highest levels of uptake for [¹⁸F]1 in nude mice bearing

MCF-7 tumors were observed in the kidneys and livers at the 5,

30 and 60 min time points, but pancreatic uptake was relatively

moderate and constant at the same time points (Table 2), which is

different from the common pattern seen with radiolabeled amino

acids. The uptake of [¹⁸F]1 was prominent in the kidney, liver

and urinary bladder, indicating that the radiotracer was mainly

excreted by the kidney and liver. In other organs including the

muscle, spleen, heart and stomach, the uptake was low to

moderate (Table 2). The bone uptake of [¹⁸F]1 was relatively

constant, indicating that no in vivo defluorination occurred over

the time course of the study (Table 2). [¹⁸F]1 showed moderate

uptake in the large and small intestines uptake in nude mice

bearing MCF-7 tumors with a relatively slow washout rate (Table

The mechanisms of transport of [¹⁸F]1 were assessed through

in vitro uptake assays with MCF-7 cells in the absence and

presence of amino acid transport inhibitors. A variety of inhibitor

conditions were used to determine which amino acid transport

system or systems were responsible for the cell uptake of [¹⁸F]1.

As shown in Figure 3d, there was no uptake inhibition of [¹⁸F]1

in the presence of MeAIB, indicating that [¹⁸F]1 did not use

system A to enter MCF-7 cells. In contrast, the uptake of [¹⁸F]1

under Na BCH and Na ASC conditions was 76 ± 5.5% (p < 0.05)

and 67 ± 4.1% (p < 0.05) relative to the control, respectively

(Figure 3d). These results indicate a component of systems L and

ASC transport [¹⁸F]1. Because [¹⁸F]1 is a derivative of L-arginine,

the basic side chain amino acids L-arginine, L-histidine and L-

lysine individually and together (RKH) were selected as

competitive inhibitors for inhibition assays. In the presence of 10

mM L-lysine, approximately 48% of the [¹⁸F]1 uptake by MCF-7

cells was blocked relative to the sodium control (p < 0.01). The

uptake of [¹⁸F]1 in the Na histidine and Na RKH conditions was

60 ± 7.2% (p < 0.05) and 53 ± 6.8% (p < 0.01) relative to the

control, respectively (Figure 3d). The uptake of [¹⁸F]1 was

partially blocked by arginine with 40 ± 5.2% uptake relative to

the sodium control (p < 0.01). As expected, the arginine

5

2), which was consistent with the reported better uptake of

arginine by the intestine.[50]

Further differences in the biodistribution of [¹⁸F]FDG,

[¹⁸F](2S,4R)4-FGln and [¹⁸F]1 in nude mice bearing MCF-7

tumors were compared. Unlike [¹⁸F]1, at 60 min and 120 min p.i.,

the heart uptake was the highest in all organs in the [¹⁸F]FDG

biodistribution study (Table S2), and the pancreas uptake was the

highest for the [¹⁸F](2S, 4R)4-FGln study.[33] [¹⁸F]1 (Table 2)

exhibited a faster clearance compared to [¹⁸F]FDG (Table S2)

and [¹⁸F](2S, 4R)4-FGln (T/B, 6.37 ± 0.45 and 1.52 ± 0.16) at 60

min and 120 min p.i..[33] The tumor to muscle ratio observed

with [¹⁸F]1 (Table 2) and [¹⁸F](2S, 4R)4-FGln (T/M, 3.91 ± 0.42)

were much higher than with [¹⁸F]FDG (Table S2, p < 0.01) at

60 min p.i. due to the much higher uptake of [¹⁸F]FDG in

muscle,[33] indicating that [¹⁸F]1 and [¹⁸F](2S, 4R)4-FGln

would be useful for imaging breast cancer. At 120 min p.i., the

T/M of [¹⁸F]1 was still significantly higher than [¹⁸F]FDG

(Table S2, p < 0.01) and [¹⁸F](2S, 4R)4-FGln (1.52 ± 0.16, p <

0.05)

imaging times.

,

[33] indicating that [¹⁸F]1 has a wider range of optimal

Table 2. In Vivo Biodistribution of [¹⁸F]1 in nude mice bearing MCF-7

organ

5 min

30 min

60 min

120 min

blood

2.29 ± 0.54

1.60 ± 0.21

0.14 ± 0.04

1.13 ± 0.25

10.73 ± 1.32

0.79 ± 0.13

2.25 ± 0.29

16.2 ± 2.13

0.53 ± 0.13

1.28 ± 0.24

1.88 ± 0.06

3.01 ± 0.54

1.83 ± 0.25

2.95 ± 0.37

6.37 ± 1.29

1.60 ± 0.69

0.72 ± 0.15

3.19 ± 0.78

0.37 ± 0.08

1.78 ± 0.25

0.07 ± 0.01

0.34 ± 0.04

6.36 ± 0.60

0.63 ± 0.10

0.92 ± 0.09

6.01 ± 0.63

0.4 ± 0.16

1.17 ± 0.19

0.71 ± 0.05

3.91 ± 0.46

1.58 ± 0.38

2.81 ± 0.33

1.52 ± 0.31

0.89 ± 0.14

4.61 ± 0.81

5.34 ± 0.83

0.22 ± 0.05

1.68 ± 0.13

0.09 ± 0.01

0.34 ± 0.05

4.34 ± 0.51

0.53 ± 0.06

0.84 ± 0.12

2.94 ± 0.38

0.39 ± 0.19

2.01 ± 0.18

0.77 ± 0.12

3.26 ± 0.36

1.47 ± 0.07

2.93 ± 0.53

1.65 ± 0.46

0.93 ± 0.11

7.68 ± 1.41

4.31 ± 1.12

0.11 ± 0.01

1.14 ± 0.07

0.06 ± 0.01

0.25 ± 0.03

1.67 ± 0.28

0.34 ± 0.02

0.57 ± 0.10

1.21 ± 0.10

0.39 ± 0.06

1.78 ± 0.22

0.77 ± 0.29

1.79 ± 0.22

1.31 ± 0.06

2.33 ± 0.48

1.29 ± 0.21

0.64 ± 0.11

10.05 ± 0.91

2.89 ± 0.81

tumor*

brain

heart

liver

spleen

lung

kidney

muscle

bone

skin

pancreas

large intestine

small intestine

bladder

stomach

tumor/blood(T/B)

tumor/muscle(T/M)

^a1.29 MBq of [¹⁸F]1 was administrated via tail vein injection without anesthesia. The animals were euthanized at 5, 30, 60,

120 min (n = 5) after injection. The data are expressed as mean % ID/g with standard deviation. There was significant

difference in tumor and muscle uptake values after 5 min (p < 0.01), 30 min (p < 0.01), 60 min (p < 0.001), and 120 min (p <

0.001) of injection.

low normal brain uptake of [¹⁸F]1 may be due to the exclusion of

these tracers by the BBB. High liver, kidney and bladder uptake

2.5. Small Animal PET/CT Imaging in nude Mice Bearing

MCF-7 Tumors and blocking study

were also observed. The relatively low levels of activity in the

bone indicated that no substantial in vivo defluorination occurred

during the 180 min time course of the study. To assess the in vivo

kinetics, a region of interest analysis was performed to generate a

time-activity curve (Figure 4b). The kinetic curves confirmed that

the tracers exhibited higher uptake in the tumor compared to the

muscle (background) regions. In addition, the tumor uptake still

To further investigate tumor uptake, tumor imaging of [¹⁸F]1

was studied using a dynamic small animal PET study of a nude

mice with MCF-7 tumors. As demonstrated by Figure 4a, the

MCF-7 tumors could be visualized with [¹⁸F]1. The relatively

6

increased at 3 h, indicating that the tumor has high uptake and

high retention of [¹⁸F]1 (Figure 4b). The SUV of tumor uptake

continued to increase, which was different than the

biodistribution result. The reason for this difference is not clear

but may be due to the effects of anesthesia during the small

animal PET studies. Within 3 h, the liver still exhibited high

uptake, which is consistent with the literature reports that the

liver has a higher uptake of arginine.[51] These results clearly

indicate that [¹⁸F]1 is suitable as a breast cancer imaging tracer.

To further confirm that [¹⁸F]1 is specific for the CAT protein,

microPET imaging with [¹⁸F]1 uptake blocked by arginine in the

same mice was performed. As shown in Figure 4c, the SUV of

tumor uptake was reduced from 3.6 ± 0.24 to 2.9 ± 0.15

(relative to no arginine inhibition, p < 0.05) when the amount

of pretreated arginine was 0.5 mg. As the amount of arginine was

further increased to 2 mg, the SUV of tumor uptake was further

reduced to 2.4 ± 0.21 (relative to no arginine inhibition, p <

0.05, Figure 4c), suggesting potent in vivo competitive binding

of arginine with [¹⁸F]1 for the CAT protein.

Fig. 4. Representative PET images of MCF-7 tumor-bearing nude mice after intravenous injection of [¹⁸F]1 into nude mice (n = 3). The images of the views (a)

are from a summed 3 h scan. (b) Time-activity curves of [¹⁸F]1 uptake in tumor and muscle. (c) Small-animal PET images of MCF-7 tumor-bearing nude mice at

45 - 65 min p.i. of [¹⁸F]1 with (left) 0 mg, 0.5 mg (middle) and 2 mg (right) injection of arginine. The red arrows represent the location of tumors.

the previous reported PET imaging data of [¹⁸F](2S, 4R)4-

The targeted CAT tracers [¹⁸F]AFETP and [¹⁸F]FEMAET have

FGln,[33] the T/M value of [¹⁸F]1 is higher than that of [¹⁸F](2S,

been reported for many years, but neither have been clinically

4R)4-FGln at 60 min (2.06 ± 0.04) and 120 min (2.49 ± 0.08)

,

used. [¹⁸F]FEMAET is most likely to selectively targets ATB^0,+

,

so tumors delineated by [¹⁸F]1 PET imaging are easier. To the

conclusion that [¹⁸F]1 and [¹⁸F](2S, 4R)4-FGln are superior in the

imaging of breast cancer, a more detailed comparison of

biodistribution and PET imaging of nude mice bearing MCF-7

tumor is still required.

which can reflect the ATB^0,+activity in vivo through PET

imaging.[5, 52] However, [¹⁸F]FEMAET still needs to be further

developed because it is not an optically pure compound, and the

effect of the proportion of its racemate on the imaging has not

been considered.[5, 52] [¹⁸F]AFETP is mainly mediated by LAT

and CAT, its synthesis is simple and the labeling yield is high. It

is expected to be used for the diagnosis of high-grade gliomas

and to monitor the response of tumors to arginine depletion

treatment. Compared with [¹⁸F]AFETP and [¹⁸F]FEMAET, the

radiolabeling conditions of [¹⁸F]1 are simple and easy to handle

their labeling success rate is high, and the yield is stable.

However, the labeling yield of [¹⁸F]1 is low, and it is still

necessary to further optimize the reaction conditions and the

structure of the radiolabeling precursor. The unique advantage of

[¹⁸F]1 is that it can reflect the physiological function of arginine

to a great extent because it possesses all of the functional groups

of arginine and its uptake may be mediated by the same

transporter as arginine.

In summary, a new arginine analog [¹⁸F]1 was designed and

prepared, which has simple synthesis conditions and a high yield.

The labeling conditions of [¹⁸F]1 are mild and easy to handle, and

their stability in vivo is high. [¹⁸F]1 showed the high cellular

uptake of 7.3 ± 0.24% and 6.07 ± 0.3% uptake/100 mg protein

after incubation in MCF-7 and MDA-MB-231 cells for 120 min,

respectively. Inhibition experiments found that L-arginine was a

more effective inhibitor than L-lysine and L-histidine, blocking

62% of the [¹⁸F]1 uptake. These properties allow the tracer to be

a specific probe for cationic transporters in vivo. In vivo

biodistribution studies have shown that [¹⁸F]1 provides high

tumor uptake and high tumor to muscle ratio (approximately 5:1

at the 30 and 60 min time points). Small animal PET studies with

[¹⁸F]1 demonstrated good tumor visualization of MCF-7 tumors

up to 3 h p.i. These results support the conclusion that [¹⁸F]1 may

be a promising candidate for the PET imaging of breast cancer.

The specific role of arginine and its derivatives in the process of

tumorigenesis and development is very complicated. Therefore,

the use of [¹⁸F]1 is expected to reveal the function of arginine and

nitric oxide, thereby revealing new intersections between

metabolism and disease and finding new opportunities for

therapeutic intervention.

Preliminary in vitro cell uptake experiments showed that

MCF-7 cells uptake of [¹⁸F]FDG and [¹⁸F](2S, 4R)4-FGln were

much higher than [¹⁸F]1, which may be because glucose and

glutamine are the two main nutrients used by cancer cells for

proliferation and survival. Comparing the biodistribution data, it

can be seen that the tumor uptake of [¹⁸F]FDG, [¹⁸F](2S, 4R)4-

FGln and [¹⁸F]1 were high at 60 min p.i., but due to the higher

background of [¹⁸F]FDG, the T/M ratio is lower and the image

contrast is not high enough. [¹⁸F]1 has a longer washout time in

tumor than [¹⁸F](2S, 4R)4-FGln (more than 1 hour), and the T/M

ratio is higher and the optimal imaging time is wider. Comparing

3. Experimental section

7

3.1. General information

159.40, 155.60, 129.16, 128.28, 114.36, 84.88, 83.24, 82.80,

81.85, 79.28, 55.31, 52.15, 50.95, 47.41, 35.58, 34.66, 28.31,

28.20. HRMS calcd for C22H40NO8+, 446.2748[M+H]⁺; found,

446.2762.

All reagents were commercial products used without further

purification unless otherwise indicated. Deionized water used in

our experiments was obtained from a Milli-Q water system.

MCF-7 cells and MDA-MB-231 cells were obtained from the

American, ATCC, Manassas, VA. Fetal bovine serums were

purchased from Beijing YuanHeng ShenMa Biology Technology

Research Institute. NMR spectra were recorded at 400 MHz at

ambient temperature. Chemical shifts are reported in parts per

million downfield from TMS (tetramethylsilane). Coupling

tert-butyl

(2S,4S)-2-((tert-butoxycarbonyl)

amino)-4-

(hydroxymethyl)-7-((tetrahydro-2H-pyran-2 -yl)oxy)heptanoate

(8) Acid 7 (1 g, 2.24 mmol) was dissolved in 5 mL THF in a 50

o

mL round bottom flask and the solution was cooled to 0 C. To

this solution Et₃N (0.24 mL, 2.24 mmol) and ethyl chloroformate

(0.21 mL, 2.79 mmol) were added dropwise. After stirring at 0 ºC

for 30 min, the reaction mixture was filtered off. To a mixture of

NaBH₄(0.17 g, 4.48 mmol) with 2 mL H₂O in a 100 mL round

bottom flask cooled with an ice bath the above filtrate was added

slowly. The mixture was stirred at room temperature for a further

1 h and was then acidified with 1 M HCl until the pH = 7 under

cooling with ice bath. The organic phase was collected and water

phase was extracted with ethyl acetate (20 mL × 3). The organic

phases were combined, washed with Sat. NaHCO₃(20 mL) and

brine (20 mL), and dried with MgSO₄. The filtrate was

evaporated in vacuo and the residue was purified by FC (ethyl

1

constants in H NMR are expressed in Hertz. High-resolution

mass spectrometry (HRMS) data was obtained with an Agilent

(Santa Clara, CA) G3250AA LC/MSD TOF system. Thin-layer

chromatography (TLC) analyses were performed using Merck

(Darmstadt, Germany) silica gel 60 F254 plates. Crude

compounds were generally purified by flash column

chromatography (FC) packed with Teledyne ISCO. Small animal

PET imaging data were recorded on a microPET (Inveon,

Siemens, Germany).

3.2. Synthesis

1

acetate/hexane 30/70) to give an oil 8 (0.82 g, 85.2%). HNMR

tert-butyl (2S)-5-((E)-2,3-bis (tert-butoxycarbonyl)guanidino)-

2- ((tert-butoxycarbonyl) amino)-4-hydroxypentanoate (2) A

solution of tert-butyl (2S)-5-amino-2-((tert- butoxycarbonyl)

amino)-4-hydroxypentanoate (0.5 g, 1.64 mmol), N,N′-Di-Boc-

1H-pyrazole -1-carboxamidine (0.28 g, 2 mmol) and N,N-

(400 MHz, CDCl₃) δ: 5.21 (s, 1H), 4.58 (d, J = 4.8 Hz, 1H), 4.22

(s, 1H), 3.90 - 3.85 (m, 1H), 3.78 - 3.73 (m, 2H), 3.58 - 3.51 (m,

1H), 3.43 - 3.38 (m, 1H), 1.85 - 1.78 (m, 1H), 1.72 - 1.55 (m,

12H), 1.48 (s, 9H), 1.45 (s, 9H). ¹³C NMR (100 MHz, CDCl₃) δ:

172.20, 155.54, 99.01, 67.77, 67.72, 64.87, 62.51, 62.47, 52.25,

37.43, 30.75, 28.32, 28.00, 27.96, 26.95, 25.45, 19.75, 19.73.

HRMS calcd for C22H41NNaO7+, 454.2775[M+Na]⁺;

found,454.2740.

o

Diisopropylethylamine (1 mL) at 50 C in 30 mL acetonitrile for

3 h. The solvent was removed under vacuum, and purified by FC

(ethyl acetate/hexane 30/70) to get little yellow oil 2 (0.77 g,

86.3%).¹H NMR (400 MHz, CDCl₃) δ 11.44 (s, 1H), 8.71 (s, 1H),

5.45 (s, 1H), 5.25 (s, 1H), 4.25 (s, 1H), 3.93 (s, 1H), 3.61 - 3.58

(m, 1H), 3.43-3.33 (m, 1H), 2.11 - 1.80 (m, 2H), 1.51 (s, 36H).

¹³C NMR (101 MHz, CDCl₃) δ 171.60, 162.73, 157.54, 152.94,

83.49, 81.99, 79.53, 77.34, 77.02, 76.70, 69.06, 65.73, 51.96,

47.31, 37.48, 28.33, 28.18, 28.02, 27.98, 27.96. HRMS calcd for

C25H47N4O9+, 547.3338 [M+H]⁺; found, 547.3339.

tert-butyl (2S,4S)-4-(((E)-N,N'-bis(tert- butoxycarbonyl)-1H-

pyrazole-1-carboximidamido)

butoxycarbonyl)amino)

methyl)-2-((tert-

-7-((tetrahydro-2H-pyran-2-

yl)oxy)heptanoate (9) To a solution of N,N′-Di-Boc-1H-pyrazole-

1-carboxamidine (0.58 g, 1.86 mmol), 8 (0.8 g, 1.86 mmol) and

o

triphenylphosphine (0.49 g, 1.86 mmol) at 0 C in anhydrous

THF, diethyl azodicarboxylate (0.34 mL, 1.86 mmol) was added

dropwise, after 10 min, the reaction was warmed at room

temperature and stirred overnight. The solvent was removed

under vacuum, and purified by FC (ethyl acetate/hexane 20/80)

to get colorless oil 9 (1.2g, 89.1%). ¹HNMR (400 MHz, CDCl₃) δ:

7.95 (s, 1H), 7.68 (s, 1H), 6.37 - 6.36 (m, 1H), 6.07 (s, 1H), 4.48

(d, J = 1.2 Hz, 1H), 3.93 - 3.88 (m, 1H), 3.77 - 3.72 (m, 1H), 3.65

- 3.60 (m, 2H), 3.40 - 3.37 (m, 1H), 3.32 - 3.26 (m, 1H), 1.94 -

1.69 (m, 3H), 1.64 - 1.58 (m, 3H), 1.48 - 1.40 (m, 16H), 1.34 -

1.32 (m, 18H), 1.17 (s, 9H). ¹³C NMR (100 MHz, CDCl₃)

δ:172.40, 157.20, 156.14, 152.13, 143.16, 130.60, 109.07, 98.69,

98.53, 82.68, 82.56, 80.72, 18.69, 77.47, 77.15,, 76.38, 67.28,

67.17, 62.06, 60.20, 53.10, 52.15, 35.04, 34.90, 34.05, 30.06,

28.28, 28.19, 27.84, 27.80, 27.60, 26.48, 25.40, 19.47. HRMS

calcd for C36H62N5O10+ 724.4491[M+H]⁺; found, 724.4504.

tert-butyl (2S)-5-((E)-2,3-bis(tert-butoxy carbonyl)guanidino)-

2-((tert-butoxycarbonyl)amino)-4-

((methylsulfonyl)oxy)pentanoate (3) To a stirred solution of 2

(0.5 g, 0.91 mmol) in CH₂Cl₂(25 mL) at 0 ^oC, Et₃N (0.3 mL, 2.7

mmol), Methanesulfonyl chloride (MsCl, 0.31 g, 2.7 mmol) and

catalytic amount 4-dimethylaminopyridine (DMAP, 0.012 g, 0.1

o

mmol) were added sequentially. After maintaining at 0 C for 15

min., the ice bath was removed, the reaction kept at room

temperature for overnight. The solution was washed by H₂O, and

dried by Na₂SO₄. The crude product was purified by FC (ethyl

acetate /hexanes, 20/80) to provide a light yellow oil 3 (0.18 g,

1

32.6%). H NMR (400 MHz, CDCl₃) δ 11.41 (s, 1H), 8.65 (s,

1H), 5.44 - 5.27 (m, 1H), 5.04 - 4.84 (m, 1H), 4.24 (m, 1H), 3.85

- 3.74 (m, 1H), 3.16 (s, 3H), 2.35 - 2.17 (m, 1H), 2.15 - 2.04 (m,

1H), 1.68 - 1.34 (m, 36H).¹³C NMR (101 MHz, CDCl₃) δ 171.11,

170.64, 162.82, 156.57, 155.47, 152.88, 83.54, 82.27, 79.96,

77.92, 77.35, 77.04, 76.72, 60.36, 50.99, 44.35, 39.35, 38.29,

34.53, 28.29, 28.19, 28.01, 27.97, 27.93, 21.02, 14.18. HRMS

calcd for C26H49N4O11S+, 625.3113[M+H]⁺; found, 625.3114.

tert-butyl (2S,4S)-4-(((Z)-1,3-bis(tert- butoxycarbonyl)-2-(4-

methoxybenzyl)guanidino)methyl)-

butoxycarbonyl)amino)-7-

2-((tert-

((tetrahydro-2H-pyran-2-

yl)oxy)heptanoate (10) A solution of 9 (1 g, 1.38 mmol), 4-

Methoxybenzylamine (0.28 g, mmol) and N,N-

2

o

(2S,4S)-5-(tert-butoxy)-4-((tert-butoxycarbonyl)amino)-5-oxo-

2-(3-((tetrahydro-2H-pyran-2-yl)oxy)propyl)pentanoic acid (7) A

mixture of the ester 6 (1.5 g, 2.8 mmol) and 10% Pd/C (0.2 g) in

absolute EtOH (20 mL) was stirred under H₂for 3 h. This

mixture was then filtered and the filtrate was concentrated under

Diisopropylethylamine (1 mL) at 50 C in 30 mL acetonitrile for

3 h. The solvent was removed under vacuum, and purified by FC

(ethyl acetate/hexane 30/70) to get little yellow oil 10 (0.71 g,

1

75.1%). HNMR (400 MHz, CDCl₃) δ: 7.26 (d, J = 8.8Hz, 2H),

6.90 (d, J = 8.8Hz, 2H), 5.10 - 4.96 (m, 1H), 4.56 (s, 1H), 4.40 (s,

1H), 4.16 (s, 1H), 3.82 (s, 4H), 3.74 - 3.64 (m, 2H), 3.51 - 3.47

(m, 1H), 3.39 - 3.31 (m, 1H), 1.90 - 1.80 (m, 2H), 1.68 - 1.65 (m,

4H), 1.61 - 1.54 (m, 7H), 1.49 (s, 18H), 1.46 (s, 9H). ¹³C NMR

(100 MHz, CDCl₃) δ:172.27, 159.37, 129.22, 114.33, 98.73,

82.62, 81.67, 79.50, 79.24, 67.57, 67.50, 62.23, 60.39, 55.30,

1

vacuum to give a white solid 7 (1.24 g, 100%). HNMR (400

MHz, CDCl₃) δ: 5.09 (s, 1H), 4.58 (d, J = 4.0 Hz, 1H), 4.23 (d, J

= 5.6 Hz, 1H), 4.0 (s, 1H), 3.86 (d, J = 6.0 Hz, 2H), 2.59-2.44 (m,

1H), 1.91 - 1.79 (m, 2H), 1.71 - 1.69 (m, 6H), 1.55-1.53 (m, 4H),

1.47 (s, 9H), 1.45 (s, 9H). ¹³C NMR (100 MHz, CDCl₃) δ: 172.14,

8

52.36, 51.31, 47.37, 34.93, 30.70, 28.32, 28.22, 28.19, 27.99,

27.56, 26.06, 25.49, 21.95, 21.72, 21.05, 19.61, 14.20. HRMS

calcd for C41H69N4O11+, 793.4957[M+H]⁺; found, 793.4955.

28.20, 28.17, 27.98. HRMS calcd for C36H60FN4O9+,

711.4339[M+H]⁺; found, 711.4335.

(2S, 4S)4-(3-fluoropropyl)Arginine (2S,4S)4-FPArg,

1 A

tert-butyl (2S,4S)-4-(((Z)-1,3-bis(tert-butoxy carbonyl)-2-(4-

solution of 13 (0.1 g, 0.14 mmol) and trifluoroacetic acid (TFA, 5

mL) was stirred at room temperature for overnight. The solvent

was removed under vacuum, diethyl ether was added, washed by

diethyl ether, the sticky colorless oil in flask was collected, and

methoxybenzyl)guanidino)methyl)

-2-((tert-

butoxycarbonyl)amino)-7- hydroxyheptanoate (11) A solution of

10 (1 g, 1.26 mmol) and Pyridinium p-toluenesulfonate (0.31 g,

1.26 mmol) at 50 ^oC in 30 mL ethanol for 2 h. Saturated NaHCO₃

(0.13 g, 1.26 mmol) was added, filtered. The solvent was

removed under vacuum, and purified by FC (ethyl acetate/

purified by HPLC (30 mg, 93.7%). [α]²⁵= + 25.9 (c = 1.0),

D

MeOH. ¹HNMR (400 MHz, D₂O) δ: 4.51 (t, J = 5.6Hz, 1H), 4.39

(t, J = 5.6Hz, 1H), 3.96 (t, J = 2.8Hz, 1H), 3.24 - 3.19 (m, 1H),

3.14 - 3.08 (m, 1H), 1.97 - 1.88 (m, 2H), 1.78 - 1.59 (m, 3H),

1.49 - 1.41 (m, 2H). ¹³C NMR (100 MHz, D₂O) δ: 172.70, 157.02,

85.95, 84.37, 71.25, 51.37, 43.77, 33.37, 32.37, 26.14, 25.95,

21.07. ¹⁹F NMR (376.5 MHz, CD₃OD): -220.34 (H-F decoupled).

HRMS calcd for C9H20FN4O2+, 235.1565[M+H]⁺; found,

235.1567.

1

hexane 40/60) to get white solid 11 (0.67 g, 84.2%). HNMR

(400 MHz, CDCl₃) δ: 9.38 (s, 1H), 7.26 (d, J = 8.8Hz, 2H), 6.90

(d, J = 8.8Hz, 2H), 5.05 - 5.01 (m, 1H), 4.40 - 4.30 (m, 2H), 4.20

(t, J = 8.0Hz, 1H), 3.87 - 3.82 (m, 1H), 3.79 (s, 1H), 3.61 - 3.49

(m, 3H), 2.81 (s, 1H), 1.79 (s, 1H), 1.65 - 1.60 (m, 6H), 1.54 -

1.52 (m, 18H), 1.48 (s, 9H), 1.46 (s, 9H). ¹³C NMR (100 MHz,

CDCl₃) δ: 172.15, 171.78, 159.39, 155.87, 153.65, 129.14,

128.21, 114.35, 114.22, 98.94, 98.88, 82.82, 81.92, 79.91, 79.25,

77.39, 77.07,76.76, 62.40, 62.37, 61.22, 60.34, 55.26, 55.22,

51.79, 50.68, 47.43, 44.75, 36.55, 33.31, 30.71, 28.47, 28.39,

28.29, 28.16, 28.13, 27.95, 25.77, 25.43, 20.99, 19.68. HRMS

calcd for C36H61N4O10+, 709.4382[M+H]⁺; found, 709.4381.

3.3. Radiosynthesis

[¹⁸F] Fluoride and ¹⁸F-FDG was produced from department of

Nuclear Medicine in Peking University Cancer Hospital &

Institute with a HM-20 medical cyclotron (Sumitomo, Kyoto,

Japan) as an [¹⁸O] enriched aqueous solution of [¹⁸F]fluoride.

Solid-phase extraction (SPE) cartridges such as Sep-Pak QMA

Light and Oasis HLB cartridges were purchased from Waters

(Milford, MA). High performance liquid chromatography (HPLC)

was performed on Agilent 1100 series system with different

HPLC columns.

tert-butyl (2S,4S)-4-(((Z)-1,3-bis(tert-butoxy carbonyl)-2-(4-

methoxybenzyl)guanidino)methyl)

-2-((tert-

butoxycarbonyl)amino)-7- (tosyloxy)heptanoate (12) To a stirred

solution of 11 (0.8 g, 1.1 mmol) in CH₂Cl₂(25 mL) at 0 ^oC, Et₃N

(0.33 mL, 3.3 mmol), p-toluenesulfonyl chloride (TsCl, 0.418 g,

2.2 mmol) and catalytic amount 4-dimethylaminopyridine

(DMAP, 0.11 mmol, 0.013 g) were added sequentially. After

Typical radiosyntheses began with approximately 740 to 1480

MBq (20 to 40 mCi) of [¹⁸F] fluoride. An activated SepPak Light

QMA Carb was loaded with [¹⁸F] fluoride and eluted with 1 mL

of 18-crown-6/KHCO₃(320 mg of 18-crown-6 in 18.6 mL of

ACN/58 mg of KHCO₃in 3.4 mL of water). The solution was

blown with argon until dry and dried thrice azeotropically with 1

mL of acetonitrile at 100 ^oC under a flow of argon. The dried [¹⁸F]

fluoride was cooled in an ice bath and 5 mg of tosylate precursor

12 was dissolved in 1 mL of tert-amyl alcohol and acetonitrile

(9/1), and added to the dried [¹⁸F] fluoride. The mixture was

o

maintaining at 0 C for 15 min., the ice bath was removed, the

reaction kept at room temperature for overnight. The solution

was washed by H₂O, and dried by Na₂SO₄. The crude product

was purified by FC (ethyl acetate /hexanes, 30/70) to provide a

light yellow oil 12 (0.7 g, 86.3%). [α]²⁵= - 41.3 (c = 1.0),

D

1

MeOH. HNMR (400 MHz, CDCl₃) δ: 9.46 (s, 1H), 7.76 (d, J =

8.8Hz, 2H), 7.32 (d, J = 8.8Hz, 2H), 7.23 (d, J = 8.8Hz, 2H), 6.89

(d, J = 8.8Hz, 2H), 5.04 (s, 1H), 4.36 (s, 2H), 4.09 (s, 1H), 4.00 -

3.97 (m, 2H), 1.47 - 1.46 (m, 18H), 1.43 (s, 9H), 1.40 (s, 9H). ¹³C

NMR (100 MHz, CDCl₃) δ: 171.97, 159.39, 155.59, 153.40,

144.59, 133.15, 129.81, 127.81, 114.36, 82.82, 81.87, 79.58,

79.28, 77.37, 77.25, 77.05, 76.74, 70.68, 60.36, 55.28, 55.12,

50.68, 47.35, 35.50, 34.65, 28.18, 28.15, 27.96, 26.76, 25.50.

21.59. HRMS calcd for C43H67N4O12S+, 863.4471[M+H]⁺;

found, 863.4475.

o

heated for 15 min at 100 C. The mixture was then cooled in an

ice bath and added to 9 mL of water. The mixture was loaded

onto an activated Oasis HLB 3 cm³cartridge, pushed through,

and washed with 10 mL of water. The desired radiolabeled

intermediate [¹⁸F]13 was eluted with 1 mL of ethanol. The

radiochemical purity of the intermediate [¹⁸F]13 was assessed by

coinjection of the nonradioactive cold 13, onto an analytical

column (Phenomenex Gemini-Nx C18 110A (250 × 4.6 mm × 5

µm) using a solution of 15/85 0.1% formic acid aqueous

solution/acetonitrile as mobile phase with a flow rate of 1

mL/min and λ = 254 nm. [¹⁸F]13 had retention times of 16.5 min

(Figure S2). The radiochemical purity was > 95%.

tert-butyl (2S,4S)-4-(((Z)-1,3-bis(tert-butoxy carbonyl)-2-(4-

methoxybenzyl)guanidino)

butoxycarbonyl)amino)-7- fluoroheptanoate (13) To a stirred

solution of tris(dimethylamino) sulfonium

methyl)-

2-((tert-

difluorotrimethylsilicate (TASF, 1.38 g, 5.0 mmol) in

CH₂Cl₂/THF (1.5 mL/1.5 mL) was added Et₃N^.(HF)₃(0.25 mL)

dropwise. The above solution was added to tosylate 12 (0.5 g,

The ethanol solution was blown until dry. A mixture of TFA

(0.495 mL) and anisole (5 µL) was added and heated for 5 min at

o

60 C in a capped 10 mL vial. TFA was removed under argon

0.58 mmol) in 5 mL THF, the reaction was stirred at 55 C for

while still warm. The reaction tube was then cooled in an ice bath.

1 mL of 10% ethanol physiological saline solution was slowly

added into the mixture, vortexed and mixed, filtered by sterile

membrane, yielded the desired radioactive (2S,4S)4-[¹⁸F]FPArg,

[¹⁸F]1, (pH = 5 - 7). The radiochemical purity was > 95% (Figure

2b and S4b).

overnight. The reaction mixture was diluted with ethyl acetate

and washed with saturated NaHCO₃, water and brine

subsequently. The ethyl acetate phase was collected, dried by

MgSO₄, filtered, concentrated in vacuo. The left residue was

purified by FC (ethyl acetate /hexanes, 20/80) to provide a light

yellow oil 13 (0.23 g, 55.6%). [α]²⁵_D= - 30.2 (c = 1.0), MeOH.

¹HNMR (400 MHz, CDCl₃) δ: 7.27 (d, J = 8.8Hz, 2H), 6.91 (d, J

= 8.8Hz, 2H), 5.01 (s, 1H), 4.48 (t, J = 6.0Hz, 1H), 4.39 (s, 1H),

4.19 - 4.14 (m, 1H), 3.82 (s, 1H), 3.80 - 3.75 (m, 1H), 3.66 - 3.61

(m, 1H), 1.86 (s, 1H), 1.82 - 1.54 (m, 6H), 1.49 (s, 18H), 1.45 (s,

9H), 1.42 (s, 9H). ¹³C NMR (100 MHz, CDCl₃) δ: 172.14, 159.40,

155.60, 153.49, 129.16, 128.28, 114.36, 84.88, 83.24, 82.80,

81.85, 79.28, 55.31, 52.15, 50.95, 47.41, 35.58, 34.66, 28.31,

The radiochemical and stereochemical purities of [¹⁸F]1 were

determined by two different HPLC systems. System 1. Column:

Chirex 3126 (D)-penicillamine 250 × 4.6 mm, 4.6 µm. Mobile

phase (isocratic): Methanol /1 mM CuSO₄solution=5/95, 1

o

mL/min, column temperature at 30 C. The retention times of

[¹⁸F]1 are 11.6 min (Figure 2b). System 2. Methanol /1 mM

o

CuSO₄solution=10/90, 1 mL/min, column temperature at 30 C.

9

The retention times of [¹⁸F]1 are 8.2 min (Figure S4b). The

specific activity [¹⁸F]1 was > 11 GBq/µmol at the end of

synthesis.

cold PBS (without Ca²⁺and Mg²⁺). After washing with ice-cold

PBS, 350 µL of 1M NaOH was used to lyse the cells. The lysed

cells were collected onto filter paper and counted together with

samples of the incubation dose using a gamma counter. A total of

100 µL of the cell lysate was used to determine the protein

concentration (Modified Lowry Protein Assay). The data was

normalized as percentage uptake of initial dose (ID) relative to

100 µg of protein content (% ID/100 µg of protein).

The labeling of [¹⁸F]25 follows the labeling method of [¹⁸F]1.

The radiochemical purity of the intermediate [¹⁸F]24 was

assessed by coinjection of the nonradioactive standard 24, onto

an analytical column (Phenomenex Gemini-Nx C18 110A (250 ×

4.6 mm × 5 µm) using a solution of 15/85 0.1% formic acid

aqueous solution/acetonitrile as mobile phase with a flow rate of

1 mL/min and λ = 254 nm. [¹⁸F]24 had retention times of 16.2

min (Figure S5). The radiochemical purity was > 95%. The

radiochemical and stereochemical purities of [¹⁸F]25 were

determined by two different HPLC systems. System 1. Column:

Chirex 3126 (D)-penicillamine 250 × 4.6 mm, 4.6 µm. Mobile

phase (isocratic): Methanol /1 mM CuSO₄solution=5/95, 1

To characterize the transport of [¹⁸F]1, competitive inhibition

studies were conducted using the MCF-7 cell line. Various

inhibitors were then added to the cells in sodium PBS solution or

sodium free PBS solution at a concentration of 10 mM. Selected

inhibitors included synthetic amino acid transport inhibitors such

as N-methyl-a-aminoisobutyric acid (MeAIB, 10 mM), a mixture

of L-Ala/L-Ser/L-Cys (ASC, 3.3 mM of each amino acid),

aminobicyclo(2,2,1)-heptane -2-carboxylic acid (BCH, 10 mM),

L-arginine (Arg, 10 mM), L-lysine (Lys, 10 mM), L-histidine

(His, 10 mM), and a mixture of L-Arg/L-Lys/L-His (RKH, 3.3

mM of each amino acid).[35] Natural amino acid arginine, lysine,

histidine was also used as inhibitors. Two buffer conditions with

and without sodium were used for the assays. In sodium free

studies, the phosphate buffered saline solution contained 105 mM

sodium chloride, 3.8 mM potassium chloride, 1.2 mM potassium

bicarbonate, 25 mM sodium phosphate dibasic, 0.5 mM calcium

chloride dehyrate, 1.2 mM magnesiumsulfate, and 5.6 mM D-

glucose. In sodium free studies, PBS buffer was replaced with

Na⁺free solution (143 mM choline chloride, 2.68 mM KCl and

1.47 mM KH₂PO₄).[53] The assays were performed as at pH 7.40

with each condition performed in 5 replicates. Briefly, cells were

washed twice with 37 °C assay buffer (2 mL) and then incubated

with different inhibitors (10 mM) for 30 min before starting the

assay. The above cells are then incubated with [¹⁸F]1 (37

kBq/mL/well) in assay buffer for 30 min at 37 °C under the

control or inhibitor conditions. The cells are then lysed and the

protein concentration is calculated, which is consistent with cell

uptake assays.

o

mL/min, column temperature at 30 C. The retention times of

[¹⁸F]25 are 20.1 min (Figure S6). System 2. Methanol/1 mM

o

CuSO₄solution=10/90, 1 mL/min, column temperature at 30 C.

The retention times of [¹⁸F]25 are 14.6 min (Figure S6). The

specific activity [¹⁸F]25 was > 11 GBq/µmol at the end of

synthesis.

Partition Coefficient (Log P): The partition coefficients

were measured by mixing [¹⁸F]1 (37 kBq) with 3 g each of 1-

octanol and buffer (pH 7.4, 0.1 M phosphate) in a test tube.

The test tube was then vortexed for 2 min and centrifuged for

10 min at room temperature. Two samples (2 g) from the 1-

octanol and buffer layers were weighed and counted in a

gamma counter. The partition coefficient was determined by

calculating the ratio of counts per min/gram in octanol to that

of the buffer. Samples of the 1-octanol layer were

repartitioned until consistent partition coefficient values were

obtained. The measurement was repeated three times

Stability in plasm: Fresh blood was collected into 1.5 mL

heparin-coated EP tube from anesthetized C57BL/6 mice. Cells

were removed by centrifugation for 10 min at 2000 g. The

resulting supernatant was the plasma for test. Incubations were

carried out at 37 °C in a water bath. Then the sample was

taken and centrifuged for 5 min at 14 000 rpm, 10 µL of the

supernatant layer was measured by radio-HPLC at 30, 60 and

The IC₅₀assay was similar to inhibition studies. Different

concentrations of arginine (0, 10^-4, 10^-3, 10^-2, 10^-1, 1, 2, 4, 5, 10,

20 mmol/L) or lysine (0, 2×10^-4, 2×10^-3, 2×10^-2, 2×10^-1, 2, 5, 10,

20, 40 mmol/L) were incubated with the above pretreated MCF-7

cells for 30 min, after which [¹⁸F]1 (74 kBq/mL/well) was added

to the above cells and incubation was continued for 2 h. The cells

are then lysed and the protein concentration is calculated, which

is consistent with cell uptake assays. Competition experiments

were analyzed using the GraphPad prism 7.00 nonlinear curve-

fitting program to obtain half-maximal inhibitory concentrations

(IC₅₀) values.

120 min

.

Stability in PBS: [¹⁸F]1 (370 kBq) was added to 1 mL PBS (pH

= 7.4, 0.1 M) solution. The solutions were incubated at room

temperature for PBS. Then the sample was taken to radio-HPLC

analysis to measure the stability in PBS at 30, 60 and 120 min.

3.4. Cell Uptake Assays and Inhibition Studies

3.5. Biodistribution Studies.

MCF-7 cells were cultured in RPMI 1640 (SIGMA)

supplemented with 10% fetal bovine serum (YHSM) and 1%

penicillin/ streptomycin (Gibco). The cells were maintained in T-

75 culture flasks under humidified incubator conditions (37 ^oC, 5%

CO₂) and were routinely passaged at confluence. MDA-MB-231

cells were maintained in L-15 medium supplemented with 10%

fetal calf serum and 1% penicillin / streptomycin (Gibco) at 37 ^oC

in a humidified atmosphere of 5% CO₂with the change of fluid

2-3 times per week. Tumor cells were plated (2.0 × 10⁵cells/well)

24 h in the media prior to ligand incubation. On the day of the

experiment, the culture media was aspirated and the cells were

washed three times with warm PBS (containing 0.90 mM of Ca²⁺

and 1.05 mM of Mg²⁺). [¹⁸F]1, [¹⁸F]FDG or [¹⁸F](2S, 4R)4-FGln

(37 kBq/mL/well) were mixed in PBS, respectively(with Ca²⁺

and Mg²⁺) solution and then added to each well. The cells were

incubated at 37 °C for 5, 30, 60, and 120 min. At the end of the

incubation period, the PBS solution containing the ligands was

aspirated and the cells were washed three times with 1 mL of ice

Nude mice (female, weight, 12 - 16 g) bearing MCF-7 tumors

(purchased from Cancer Hospital Chinese Academy of Medical

Sciences, Beijing, China), MCF-7 cells (∼10⁶) in PBS (0.1 mL)

were injected subcutaneously into the lower right flank of the

nude mice. The tumors took 12-15 days to reach appropriate size

(0.5 cm diameter). BALB/c mice (18-22 g) were purchase from

Beijing Charles river Laboratories. All the animals were

maintained according as the Chinese government guidelines for

care and use of laboratory animals. Studies of the in vivo

distribution of [¹⁸F]1 was performed in nude mice bearing MCF-

7 tumors. Approximately 1.29 MBq [¹⁸F]1 or [¹⁸F]FDG was

administrated via tail vein injection in conscious animals. Groups

of five animals were euthanized at 5, 30, 60, and 120 min p.i. All

animals were not fasted prior to the study. The organs of interest

were removed, weighed, and the radioactivity was counted with a

gamma counter (Packard Cobra). The results were expressed as

10

the percent uptake of injected dose per gram of tissue (% ID/g)

and presented as mean ± SD.

iodo-a-methyl tyrosine; [¹⁸F] FDOPA, L-3,4-dihydroxy-6-

[¹⁸F]fluorophenyl alanine; [¹⁸F] FMA, [¹⁸F]-2-Amino-4-

fluorobutanoic acid; S-[¹⁸F] FAMP, (S)-2-amino-3-[¹⁸F]fluoro-2-

methylpropanoic acid S-[¹⁸F] MeFAMP, [¹⁸F] (2S,4R)4F-Glu,

(2S,4R)-2-amino-4-(fluoro-¹⁸F)pentanedioic acid; L-Ser, L-

Serine; L-Cys, L-Cysteine; L-Ala, L-Alanine; LC-MS, Liquid

3.6. Small Animal PET/CT Imaging in nude Mice Bearing

MCF-7 Tumors.

Dynamic small animal PET imaging studies were conducted

with [¹⁸F]1 similar to that reported previously.[27] All scans were

performed on a dedicated animal PET scanner (Inveon, Siemens,

Germany). Nude mice with MCF-7 tumors were used for the

imaging studies. A total of 8-11 MBq of activity was injected

intravenously via the lateral tail vein. For blocking study, 0.5 mg

or 2 mg of arginine was injected into nude mice 30 min before

administration of [¹⁸F]1, and PET images were collected for 45 -

65 min after administration. All animals were sedated with

isoflurane anesthesia (2-3%, 1 L/min oxygen) and were then

placed on a heating pad in order to maintain body temperature

throughout the procedure. The animals were visually monitored

for breathing and any other signs of distress throughout the entire

imaging period. The data acquisition began after an intravenous

injection of the tracer. All scans were conducted over a period of

180 min (dynamic, 5 min/frame). Regions of interest (ROIs)

were drawn over tumor guided by CT images using Amira 3.1

image visualization and analysis software.

Chromatography-Mass Spectrometrys;

LiHMDS, Lithium

bis(trimethylsilyl)amide; PPTS, Pyridinium p-toluenesulfonate;

DHP, 3,4-Dihydro-2H-pyran; PBS, phosphate buffer saline;

HPLC, high performance liquid chromatography; HRMS, High-

resolution mass spectrometry; SUV, standardized uptake value;

SD, standard deviation

trifluoroacetic acid

; TLC, thin-layer chromatography; TFA,

.

Supplementary Material

Supplementary data (detailed procedure for the synthesis of

(2R,4R)4-FPArg, 25, analytical data, radiolabeling condition)

associated with this article can be found in the online version, at.

at doi:………

Reference

3.7. Statistical Analysis

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Financial support

This research was supported by the National Natural Science

Foundation of China (81701753) and Scientific Research

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Author contributions

Zehui Wu conceived and planned the experiments. Renbo Wu

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13

Highlights

Arginine derivative [¹⁸F]1 and [¹⁸F]25 were prepared;

[¹⁸F]1 may be a high-affinity CAT-1 transporter substrate;

[¹⁸F]1 showed high tumor uptake and high tumor to muscle ratios;

[¹⁸F]1 is a promising tracer for clinical breast cancer imaging.

Article Doi

Synthesis and biological evaluation of [18F](2S,4S)4-(3-fluoropropyl) arginine as a tumor imaging agent

DOI: 10.1016/j.ejmech.2019.111730

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Article abstract of DOI:10.1016/j.ejmech.2019.111730

Full text of DOI:10.1016/j.ejmech.2019.111730

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