Radiosynthesis, in vitro and preliminary biological evaluation of [18F]2-amino-4-((2-((3-fluorobenzyl)oxy)benzyl)(2-((3-(fluoromethyl)benzyl)oxy)benzyl)amino)butanoic acid, a novel alanine serine cysteine transporter 2 inhibitor-based positron

Baguet Tristan (Orcid ID: 0000-0002-1067-9374)

Descamps Benedicte (Orcid ID: 0000-0001-8879-1772)

18

Radiosynthesis, in vitro and preliminary biological evaluation of [ F]FABABA, a novel ASCT-2

inhibitor based PET tracer.

1

2

1

Tristan Baguet *, Jakob Bouton , Jonas Janssens , Glenn Pauwelyn , Jeroen

1

3

2

3

Verhoeven , Benedicte Descamps , Serge Van Calenbergh , Christian Vanhove , Filip

De Vos¹.

¹Laboratory of Radiopharmacy, Ghent University, Ghent, Belgium

²Laboratory for Medicinal Chemistry, Ghent University, Ghent, Belgium

³IBiTech-MEDISIP, Department of Electronics and Information Systems, Ghent University,

Ghent, Belgium

*

Correspondence:

Tristan Baguet

Email: Tristan.baguet@ugent.be

Number of words: 5962

1

8

Keywords: Positron emission tomography, oncology, glutamine, [ F]2-amino-4-((2-((3-

fluorobenzyl)oxy)benzyl)(2-((3-(fluoromethyl)benzyl)oxy)benzyl)amino)butanoic acid,

[¹⁸

F]FABABA

This article has been accepted for publication and undergone full peer review but has not been

through the copyediting, typesetting, pagination and proofreading process which may lead to

differences between this version and the Version of Record. Please cite this article as doi:

10.1002/jlcr.3863

Abstract

Introduction: The metabolic alterations in tumors make it possible to visualize the latter by

means of positron emission tomography, enabling diagnosis and providing metabolic

information. The alanine serine cysteine transporter-2 (ASCT-2) is the main transporter of

glutamine and is upregulated in several tumors. Therefore, a good positron emission tracer

targeting this transport protein would have substantial value. Hence, the aim of this study is to

develop a fluorine-18 labelled version of a V-9302 analogue, one of the most potent inhibitors

of ASCT-2.

Methods: The precursor was labelled with fluorine-18 via a nucleophilic substitution of the

corresponding benzylic bromide. The cold reference product was subjected to in vitro assays

3

with [ H]glutamine in a PC-3 and F98 cell line to determine the affinity for both the human

and rat alanine serine cysteine transporter-2. To evaluate the tracer potential dynamic µPET

images were acquired in a mouse xenograft model for prostate cancer.

Results: The tracer could be synthesized with an overall non-decay corrected yield of 3.66 ±

1

.90 %. In vitro experiments show inhibitor constants K of 90 µM and 125 µM for the PC-3

i

and F98 cells, respectively. The experiments in the PC-3 xenograft demonstrate a low uptake

in the tumor tissue.

1

8

Conclusions: We have successfully synthesized the radiotracer [ F]2-amino-4-((2-((3-

fluorobenzyl)oxy)benzyl)(2-((3-(fluoromethyl)benzyl)oxy)benzyl)amino)butanoic acid. In

vitro experiments show a good affinity for both the human and rat ASCT-2. However, the tracer

suffers from poor in vivo tumor uptake in the PC-3 model. Briefly, we present the first fluorine-

1

8 labelled derivative of compound V-9302, a promising novel ASCT-2 blocker used for

inhibition of tumor growth.



Introduction

Cancer is characterized by genetic and epigenetic changes of individual cells leading to

1

,2

metabolic divergencies and unrestrained cell division . Tumor can be classified based on their

specific metabolic deviations. Attempts to exploit these changes has led to the development of

anticancer drugs and imaging probes directed towards these altered molecular targets. Positron

emission tomography (PET) is an imaging modality that uses probes labelled with radioactive

isotopes. In oncology, PET tracers target the altered metabolic pathway, enabling the

visualization of the tumor and providing metabolic information of the type of targeted

metabolic pathway³.

The alanine serine cysteine transporter-2 (ASCT-2) is the predominant transporter of glutamine

in the human body. Considering the importance of glutamine in several types of tumor, research

has been conducted towards the role of ASCT-2 in tumors. Upregulated levels of the transporter

have been found in numerous tumors (e.g. colorectal cancer, pancreatic ductal carcinoma,

4

–6

7,8

breast cancer) . In addition, it also appears to be a prognostic factor in some malignancies .

Developing radioactive probes to visualize ASCT-2 could therefore provide valuable

information. Several attempts have been made to develop probes targeting ASCT-2, however

18

none of them are selective or display good tumor uptake (see Scheme 1). [ F]fluciclovine is a

radiotracer binding to ASCT-2 showing promising results with regard to prostate cancer

imaging. However, this compound is not ASCT-2 selective as it also binds to large amino acid

9

18

transporter-1 (LAT-1) . (2S,4R)4-[ F]fluoroglutamine, a glutamine-based PET tracer, targets

ASCT-2 but suffers from in vivo defluorination¹⁰^,¹¹. A new ASCT-2 tracer, [ F]ala-BF

18

, shows

3

1

2

potential but has only been preliminary tested .

Scheme 1: Overview of ASCT-2 directed, fluorine-18 labelled, PET-radiotracers.

In 2016, Schulte et al. reported a class of compounds, the aryloxybenzyl aminobutanoic acids,

which display the highest affinity for ASCT-2 reported to date, with IC50-values as low as 3

13

µM . One of these compounds, V-9302, was further evaluated for its antitumor activity both

in vitro and in vivo: V-9302 reduced cellular viability and increased cell death in vitro, and was

1

4

capable of inhibiting tumor growth in vivo . Given these promising results as an antitumor

agent, we aimed at developing a fluorine-18 labelled version of a V-9302 analogue (see Scheme

2

). In this analogue, both meta-methyl groups of the double 2-((3-methylbenzyl)oxy)-benzyl

substituents on the nitrogen are replaced by a fluorine and fluoromethyl substituent,

respectively, the latter containing the fluorine-18 label. A bromomethyl precursor would allow

3

straightforward labelling. Cold reference product was subjected to in vitro [ H]glutamine

uptake experiments to test affinity. The radioactive tracer was evaluated in vivo using a PC-3

xenograft mouse model to evaluate its imaging potential.

Scheme 2: Left: Chemical structure of V-9302, the lead molecule on which the new radiotracer

1

8

is based upon. Right: Chemical structure of [ F]FABABA, modified on both meta-methyl

1

8

groups, which are replaced with a fluorine and [ F]fluoromethyl group, respectively.



Results

Chemistry

A five-step synthesis was developed to obtain cold reference product 7 (Scheme 4). The same

synthesis was used to synthesize the precursor for radiolabeling. Fluorination of [3-

(bromomethyl)phenyl]methanol with DAST afforded the requested 1-(bromomethyl)-3-

(fluoromethyl)benzene (1) for the synthesis of intermediate 3 (Scheme 3). A Williamson ether

synthesis was used to convert 2-hydroxybenzaldehyde to the ether products 2, 3 and 4. The

yield of 4 was significantly lower than for 2 and 3, due to formation of the double ether side-

product with 1,3-bis(bromomethyl)benzene as a major side-product. Next, reductive amination

of aldehyde 2 with (2S)-tert-butyl 4-amino-2-((tert-butoxycarbonyl)amino)butanoate

1

3

hydrochloride according to Schulte et al. (2016) afforded 5 . A small excess of N-Boc-L-2,4-

diaminobutyric acid tert-butylester hydrochloride proved critical to avoid over-amination. A

second reductive amination with benzaldehydes 3 and 4 resulted in compounds 6 and 8.

Compound 8 was then used as precursor for labelling with fluoride-18. Alternatively,

deprotection of 6 with anhydrous HCl (4M in 1,4-dioxane) gave cold reference product 7.

Scheme 3: Reagents and conditions: a) DAST, CH

2

Cl , RT, 1 h, 61%.

2

Scheme 4: Reagents and conditions: a) appropriate benzylbromide, K

00%; b) Boc-dab-OtBu, NaBH(OAc) , CH Cl , RT, overnight, 53%; c) Aldehyde,

NaBH(OAc) , CH Cl , RT, overnight, 44-73%; d) 4M HCl in 1,4-dioxane , 40°C, 4 h, 100%.

2

CO , DMF, RT, 3 h, 28-

3

1

3

2

3

2

Radiochemistry

18

Synthesis

of

[ F]2-amino-4-((2-((3-fluorobenzyl)oxy)benzyl)(2-((3-

1

8

(

fluoromethyl)benzyl)oxy)benzyl)amino)butanoic acid ([ F]FABABA) (Scheme 5) was

achieved with an overall non-decay corrected yield of 3.66 ± 1.90 % and a total synthesis time

of 122 ± 19 minutes (n=3). Although high yields (>80%) of radiofluorination were observed,

the total yield dropped significantly due to the challenging deprotection step which could not

be improved. The combination of strong acidic conditions and high temperature is essential for

the removal of protecting groups, but also results in partial degradation of the radiotracer. Co-

injection with cold reference product on analytical HPLC confirmed the chemical purity (t

[¹⁸

F]FABABA = 18.5 min) (See Fig. S1 and Fig. S2). Quality control by means of analytical

HPLC showed a radiochemical purity of >99%. The logD7.4 was measured to be 1.42 ± 0.28

n=6).

r

(

1

8

18

-

Scheme 5: Radiochemical synthesis of [ F]FABABA. Reagents and conditions: a) [ F]F ,

ACN, 120°C, 5 min; b) HCl (4 M in 1,4-dioxane), 90°C, 3.5 min, 3.66%.

In vitro experiments

In vitro stability

1

8

Stability testing showed no degradation of [ F]FABABA in its formulation. After 180 minutes

only the mother compound was detected on analytical HPLC.

Optimization of parameters for radiochemical labelling

Optimization of the different reaction parameters showed inflection points for all three

variables. Changing the solvent to acetonitrile increased the labelling yield significantly. Rising

the temperature over 100°C turned out to be necessary for high yields. A total amount of 2 mg

precursor is sufficient for good yields, while further increasing the precursor amount does not

provide any additional gain in product. The ideal labeling parameters are acetonitrile as solvent,

a reaction temperature of 120°C and at least 2 mg mass precursor. Graphical visualization of

the influence of different parameters can be found in Figure 1.

1

8

Figure 1: Optimization of the radiochemical synthesis yield of [ F]FABABA. The

optimization was done stepwise (one-parameter at a time, n=3). A: The labelling yield in

function of reaction solvent (DMSO, DMF and acetonitrile). B: The labelling yield in function

of the temperature. The reaction temperatures tested were 60°C, 80°C, 100°C and 120°C. C:

The labelling yield in function of the precursor mass.

ASCT-2 expression assay

Expression of ASCT-2 was confirmed on both the PC-3 and F98 cell lines (Figure 2Error!

Reference source not found.). The PC-3 and F-98 cell line showed a median population of

1

2,056 (Specimen_001-PC3 intra) and 28,585 (Specimen_001-F98 intra), respectively. The

median population of the negative control was 576 (Specimen_001-PC3 surface) and 919

Specimen_001-F98 surface) for the surface staining of PC-3 and F98 cell line, respectively.

(

Figure 2: FACS measurement of ASCT-2 expression on PC-3 and F98 cells. Specimen_001-

PC3 intra and specimen_001-F98 intra represents the intracellular staining of the PC-3 and

F98 cells, respectively. Specimen_001-PC3 surface figure and specimen_001-F98 surface

figure acts as a negative control and is the surface staining of the PC-3 and F98 cells,

respectively.

Figure 3 represents the FACS analyses evaluating the ASCT-2 expression on the HEK-293 WT

and HEK-293 KO cell lines as performed by the Creative Biogene company. The results show

a positive expression population 66.3% and 0.454% for ASCT-2 on the HEK-293 WT and

HEK-293 KO cell lines, respectively. The HEK-293 cell-iso and HEK-293 KO cell-iso, which

act as a negative control for the experiment, have a positive expression population of 0.643%

and 0.151%, respectively.

Figure 3: Top left: HEK-293 WT cell-iso; Top righ: HEK-293 WT cell; Bottom left: HEK-293

KO cell Iso; Bottom Right: HEK-293 KO cell.

Concentration dependency

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9

The affinity of [ F]FABABA for ASCT-2 was determined in both rat F98 cells and human

PC-3 cells. As a comparison, the affinity of V-9302 was also determined in the human PC-3

1

9

cell line. [ F]FABABA shows a K value of 90 µM towards the glutamine transporter and

i

inhibits glutamine uptake in a non-competitive manner (see Table 1). Compound V-9302

inhibits the glutamine transport in a comparative manner with a Ki value of 150µM. In Figure

the Michaelis-Menten plots for the glutamine uptake can be found in both absence and

presence of the inhibitor.

4

Glutamine (A)

FABABA (B)

1

50000

150000

100000

50000

0

100000

5

0000

0

500

1000

1500

0

500

1000

1500

[

S] (µM)

[S] (µM)

Glutamine (C)

V-9302 (D)

1

50000

150000

100000

50000

0

100000

5

0000

0

500

1000

0

500

1000

[

S] (µM)

[S] (µM)

Glutamine (E)

FABABA (F)

8

0000

80000

60000

40000

20000

0

6

0000

4

0000

2

0000

0

500

1000

0

500

1000

[

S] (µM)

[S] (µM)

3

Figure 4: A: Concentration dependent uptake of [ H]glutamine in PC-3 cells. B: Concentration

3

dependent uptake of [ H]glutamine in presence of FABABA in PC-3 cells. C: Concentration

3

dependent uptake of [ H]glutamine in PC-3 cells. D: Concentration dependent uptake of

[³

H]glutamine in presence of V-9302 in PC-3 cells. E: Concentration dependent uptake of

3

H]glutamine in F98 cells. F: Concentration dependent uptake of [ H]glutamine in presence

of FABABA in F98 cells.

3

Inhibition [ H]-

L

-glutamine uptake in knock-out cell line:

19

The selectivity of [ F]FABABA towards the ASCT-2 cell line was assessed using a knock-out

1

9

cell line. [ F]FABABA shows a 32 ± 16% and 29 ± 10% inhibition of glutamine transport in

ASCT-2 expressing F98 and HEK-293 WT cell lines, while in the ASCT-2 knock out cell line

[¹⁹

F]FABABA inhibits transport with 71 ± 10%. The inhibitor is therefore not selective for

ASCT-2.

In vivo experiments

18

In vivo experiments in PC-3 xenografts show that [ F]FABABA has only limited uptake in

tumor tissue (see Figure 5). In both mice the tracer has a low standardized uptake value (SUV)

and T/M ratio (see Table 2).

[¹⁸

F]FABABA PET (A)

[¹⁸F]FABABA CT (B)

18

Figure 5: µPET acquisition of [ F]FABABA (A) and CT image (B) of the PC-3 xenograft

tumor model (tumors delineated with a white circle).

Biodistribution study

18

The results of the biodistribution of [ F]FABABA are shown in Table 3. Five minutes post-

injection, the majority of radioactivity is found inside the liver tissue (28.87 ± 2.82 %ID/ g)

and the blood pool (8.96 ± 3.20 %ID/ g). After twenty minutes, a large fraction remains in the

liver (19.65 ± 1.10 %ID/ g) and the blood pool (6.11 ± 1.03 %ID/ g). Tumor uptake of the

radiotracer is neglectable with only 0.74 ± 0.15 %ID/ g found in tumor tissue. Fractions of the

radioactivity was found in the muscle (0.62 ± 0.19 %ID/ g), brain (0.27 ± 0.35 %ID/ g), small

intestine (1.07 ± 0.46 %ID/ g), bladder (1.71 ± 0.73 %ID/ g) and bone tissue (1.47 ± 0.27 %ID/

g).



Discussion

The unique tumor principle states every tumor possesses unique characteristics. Not only are

there genetic and epigenetic differences between tumors, exogenous factors such as diet and

15

lifestyle also contribute to uniqueness of neoplasms . This heterogeneity makes it hard to

estimate therapy effectiveness as each tumor will respond differently to the therapy. Grouping

tumors to molecular (sub)classifications helps in predicting tumor behavior¹⁶^,¹⁷. Collecting

molecular information is therefore an important step in multimodality oncologic diagnosis and

therapy. Positron emission tomography can provide such molecular information. Moreover, the

more different kinds of tracers that are developed, the more information this modality can

provide. In this study, we synthesized a novel PET-tracer targeting the alanine-serine-cysteine

transporter-2. As the major transporter of the amino acid glutamine, we believe that imaging

this transporter could provide valuable information in the metabolic mapping of the tumor.

A five-step synthesis was successfully used to gain access to the cold reference product. The

precursor for fluorine-18 tagging was synthesized following the same scheme. To obtain an

1

8-fluorine derivative of compound V-9302, chemical adjustments to the structure had to be

made. While doing this, the possibility to obtain a less liphophilic compound without impairing

the pharmacological characteristics of V-9302 was explored. Changing the methyl-group to a

fluorine was a non-invasive switch and was supported by the following two arguments. First,

the LogP was computed in Chemdraw for V-9302 (6.52) and FABABA (5.84), which was

1

3

advantageous for FABABA. Secondly, in the previous paper described by Schulte (2016) ,

the incorporation of a fluorine directly on the arene altered the affinity in only a small manner.

This trade-off seemed positive and therefore the methyl arm was chosen over the fluor arm.

Radiochemical synthesis was successful, however only low non-decay corrected yields were

obtained. Despite good labelling with fluorine-18, the deprotection step deteriorated the overall

yield. Optimization of this step was not successful. The in vitro K experiments showed that the

i

FABABA compound has a moderate affinity for both the human and rat ASCT-2. The observed

affinity in the PC-3 cell line (90 µM) is comparable to that of compound V-9302 (150 µM), on

which FABABA was based upon. Moreover, Schulte et al. (2016) showed that comparable

compounds also inhibited ASCT2-mediated glutamine uptake in human cells in a

concentration-dependent fashion and reported IC50 values ranging from 1.3 - 68.2 µM and from

1

3

7

.2 - 141.7 µM, in rat and human cell lines, repectively . A comparison of V-9302 and

FABABA with regard to selectivity was not pursued in this manuscript. However, experiments

using FABABA in the ASCT-2 knock-out cell line suggests the latter not being selective to

ASCT-2 whereas literature on V-9302 does suggest ASCT-2 selectivity. This can be explained

to the above mentioned substitutions of methylgroups with fluorine and methylfluorine on the

structure of V-9302. Despite the good affinities we observed in vitro, the in vivo experiments

showed almost no uptake in the tumor tissue. Both the dynamic PET acquisitions and the

biodistribution study demonstrate a slightly higher uptake in tumor tissue compared to muscle

tissue. However, this is better explained by a very low uptake in muscle tissue rather than an

elevated tumor uptake. As organs such as liver and intestines are immediately visible and

dominate the uptake of the fluorine-18, we presume this radiotracer undergoes heavy

metabolization (e.g., O-dealkylation, N-dealkylation, oxidation). This is supported by the

results in the biodistribution study, where the radiotracer is primarily found in the liver tissue

five minutes post-injection. In contrast, compound V-9302 that was described by Schulte et al.

1

4

(

2018) showed good inhibition of tumor growth , which indicates tumor uptake. However,

administration was at much higher dosage (75 mg/ kg) compared to the tracer amount we used.

Moreover, kinetics of metabolization are hard to compare between the radiotracer and cold

product used for treatment purposes. The functional groups, where metabolization is possible,

are inherent to the compound and are necessary for good affinity, indicating that metabolic

optimization for molecular imaging will be very challenging.



Conclusion

[¹⁸

F]FABABA could be synthesized, albeit with low overall yields. In vitro experiments show

good affinity towards ASCT-2, comparable to the affinity of lead compound V-9302. However,

the in vivo experiments show neglectable uptake in the tumor and demonstrate that the

[¹⁸

F]FABABA compound is not able in properly visualizing the PC-3 xenograft tumors.

18

Further research should clarify the potential of [ F]FABABA in visualizing other tumor types.

We hereby reported the first fluorine-18 labelled derivative of compound V-9302, a promising

novel ASCT-2 blocker used for inhibition of tumor growth.



Material and methods

General

All reactions were performed under N atmosphere. The chemicals used in these reactions were

2

at least reagent grade, obtained from Sigma Aldrich (Bornem, Belgium), TCI Europe

Zwijndrecht, Belgium), Activate Scientific (Prien, Germany), Acros (Geel, Belgium) or

(

ChemCollect (Wuppertal, Germany) and used as received. Solvents were at least HPLC-grade

and were obtained from Chemlab (Zedelgem, Belgium). Chemical reactions were monitored

on pre-coated TLC sheets (ALUGRAM® Xtra SIL G/UV254) under a wave length of 254 nm.

Spots were visualized by spraying with either basic aqueous solution of KMnO

4

(1.0 g KMnO

O); 2,4dinitrophenylhydrazine (12 g 2,4-DNP, 60 mL H SO , 80 mL

O, 200 mL 95% EtOH) or ninhydrin (1.5g ninhydrin, 3.0 mL acetic acid, 100 mL EtOH)

4

,

2

.0 g K

2

CO

3

, 100 mL H

2

4

H

2

followed by subsequent charring. Purifications were done either by manual (Machery-Nagel

Kieselgel 60 (63-200 µm)) or automated (Reveleris X2 (Grace)) flash chromatography. A mass

spectrometer (Waters LCT Premier XE Time of Flight (TOF)), equipped with electronspray

ionization (ESI) and modular Lockspray^T^Mwas used to obtain high resolution mass spectra.

NMR spectra were recorded on a Varian Mercury-300BB (300 MHz) spectrometer.

Chemistry

General Procedure A (Williamson ether synthesis)

Benzylic bromide (19.7 mmol, 1.2 eq) was dissolved in anhydrous DMF (85 mL; 5 mL/ mmol)

in a flame-dried flask with stir bar under N

and K CO (5.65 g; 41.0 mmol; 2.5 eq) were added and the mixture was allowed to stir

overnight at room temperature. The next day, the solvent was removed in vacuo and the residue

2

atmosphere. Salicylaldehyde (1.71 mL, 16.4 mmol)

2

3

was partitioned between CH

2

Cl

2

and H

2

O. The phases were separated, and the aqueous phase

SO and

extracted twice more with CH

2

Cl

2

. The combined organic phases were dried over Na

2

4

concentrated in vacuo. The residue was purified by flash column chromatography (FCC).

1

-(bromomethyl)-3-(fluoromethyl)benzene (1)

(3-(bromomethyl)phenyl)methanol (1.00 g; 5.00 mmol) (Scheme 3) was dissolved in CH

2

Cl

2

(10 mL; 2 mL/ mmol) in a flame-dried flask and stirred for 10 min at 0°C. Diethylaminosulfur

trifluoride (DAST, 0.66 mL, 5.00 mmol, 1 equivalent) was added dropwise, the reaction

mixture was removed from the ice bath and was allowed to stir for 1 hour at room temperature.

Upon disappearance of all starting material on TLC, the mixture was neutralized via dropwise

addition of H

phases were separated, and the aqueous layer further extracted with CH

organic phases were dried over Na SO and concentrated in vacuo. The residue was adsorbed

on celite® and purified by automated column chromatography (0% EtOAc → 5% EtOAc in

2

O (10 mL). Next, the compound was partitioned between H

2

O and CH

2

Cl . The

2

Cl

2

(2x). The combined

2

4

hexane) to afford 1 (0.613 g; 3.05 mmol) as a transparent liquid in 61% yield. R 0.66 (10%

f

1

EtOAc/ 90% Hex). H NMR (300 MHz, CDCl

3

): δ 4.50 (s, 2H, CH

): δ 32.98, 82.93, 85.17, 127.28,

27.35, 127.84, 127.93, 129.06, 129.12, 129.29, 129.34, 129.57, 136.80, 137.03, 138.25,

2

Br), 5.38 (d, J= 48 Hz, 2H,

1

3

CH

2

F), 7.28-7.43 (m, 4H, HPhe). C NMR (75 MHz, CDCl

3

1

9

38.40. F NMR (282 MHz, CDCl

3

): δ -208,75 (t, J= 47.9 Hz). Exact Mass: (ESI-MS) for

C

8

H

8

BrF [M+H] found, non; calcd, 202.9872

2

-((3-fluorobenzyl)oxy)benzaldehyde (2)

Salicylaldehyde (1.71 mL; 16.4 mmol) and 1-(bromomethyl)-3)fluorobenzene (2.45 mL; 19.7

mmol) were coupled using procedure A. The product was purified by automated flash

chromatography (4% EtOAc →14% EtOAc in hexane) to afford 2 (3.51 g; 15.3 mmol) as a

1

white powder in 93% yield. R

f

0.36 (10% EtOAc/ 90% Hex). H NMR (300 MHz, CDCl

3

): δ

5

7

6

1

.19 (s, 2H, CH

.85-7.89 (m, 1H, HPhe), 10.57(d, J= 0.7, 1H, CHO). C NMR (75 MHz, CDCl

9.64, 112.88, 113.94, 114.23, 115.03, 115.31, 121.26, 122.56, 122.60, 125.18, 128.66, 130.30,

2

-O), 7.00-7.24 (m, 5H, HPhe), 7.33-7.42 (m, 1H, HPhe), 7.50-7.57(m, 1H, HPhe),

1

3

): δ 69.60,

1

9

30.40, 135.92, 138.61, 160.68, 189.51. F NMR (282 MHz, CDCl

3

): δ [-112.40; -112.30]

(m). Exact Mass: (ESI-MS) for C14

H11FO

2

[M+H] found 231.0810, calcd, 231.0821

2

-((3-(fluoromethyl)benzyl)oxy)benzaldehyde (3)

Salicylaldehyde (0.258 mL; 2.48 mmol) and compound 1 (0.441 mL, 2.97 mmol) were coupled

using procedure A. The product was purified by automated flash chromatography (5% EtOAc

→

20% EtOAc in hexane) to afford 3 (0,606 g; 2.48 mmol) as a transparent liquid in

1

quantitative yield. R

f

0.67 (25% EtOAC/ 75% Hex). H NMR (300 MHz, CDCl

3

): δ 5.21 (s,

2

7

8

1

4

H, CH

.85-7.89 (m, 1H, HPhe), 10.56 (d, J=0.73Hz, 1H, CHO). C NMR (75 MHz, CDCl

3.14, 85.37, 112.94, 121.15, 125.18, 126.07, 126.13, 127.16, 127.28, 127.54, 127.57, 128.57,

2

O), 5.42 (d, J=51 Hz, 2H, CH F), 7.02-7.09 (m, 2H, HPhe), 7.33-7.58 (m, 5H, HPhe),

2

1

3

): δ 70.17,

1

9

29.06, 135.93, 136.62, 136.97, 160.88, 189.63. F NMR (282 MHz, CDCl

3

): δ -208.15 (t, J=

7.1 Hz). Exact Mass: (ESI-MS) for C15 [M+H] found, 245.0984; calcd, 245.0978

H14FO

2

-((3-(bromomethyl)benzyl)oxy)benzaldehyde (4)

Salicylaldehyde (1.71 mL; 16.4 mmol) and 1,3-bis(bromomethyl)benzene (5.20 g; 19.7 mmol)

were coupled using procedure A. The product was purified by manual flash chromatography

(5% EtOAc → 15% EtOAc in hexane) to afford 4 (1.40 g; 4.59 mmol) as a white powder in

1

2

8% yield. R

CH Br), 5.19 (s, 2H, CH

H, HPhe), 10.56 (d, J=0.7 Hz, 1H, CHO). C NMR (75 MHz, CDCl

21.17, 125.18, 127.26, 127.79, 128.59, 128.92, 129.24, 135.93, 136.83, 138.39, 160.78,

89.62. Exact Mass: (ESI-MS) for C15 [M+H] found, 305.0327; calcd, 305.0177

f

0.38 (10% EtOAc/ 90% Hex). H NMR (300 MHz, CDCl

3

): δ 4.51 (s, 2H,

2

O), 7.02-7.09 (m,2H, HPhe), 7.36-7.57 (m, 5H, HPhe), 7.84-7.89 (m,

1

3

1

3

): δ 33.06, 70.08, 112.94,

H14BrO

2

Tert-butyl 2-((tert-butoxycarbonyl)amino)-4-((2-((3 fluorobenzyl)oxy)benzyl)amino)butanoate

5)

(

To a vial equipped with a magnetic stir bar, NBoc-L-2,4 diaminobutyric acid tert-butyl ester

hydrochloride (2.98 g; 9.58 mmol) was dissolved in dry CH Cl (80 mL; 10 mL/ mmol).

2

Compound 2 (2.10 g; 9.13 mmol) was added together with sodium triacetoxyborohydride (4.50

g; 21.2 mmol). The reaction was stirred at room temperature overnight. Upon completion, the

reaction mixture is partitioned between H

2

O and CH

2

Cl

2

, the phases separated, and the aqueous

phase extracted with additional CH Cl (2X) and dried over anhydrous sodium sulfate. After

2

concentration in vacuo the residue was purified by automated flash chromatography (40%

EtOAc → 70% EtOAc in hexane). Compound 5 (2.36 g; 4.84 mmol) was obtained as a yellow

1

oil in 53% yield. Rf 0.19 (100% EtOAc). H NMR (300 MHz, CDCl

3

): δ 1.40 (s, 9H, tBu),

-CH), 2.71-2.83 (m, 2H, NH-CH -CH ), 3.94

-NH), 4.09-4.22 (m, 1H, CH -CH-NH), 5.12 (s, 2H,

1

.42 (s, 9H, tBu), 1.72-1.89 (m, 2H, CH

2

-CH

2

(

dd, J = 18,5 Hz, J = 13,1 Hz, 2H, CH

1

2

CH

2

O), 5.51-5.62 (m, 1H, NHBoc), 5.99 (s, 1H, CH

2

-NH-CH

2

), 7.86-7.25 (m, 6H, HPhe), 7.29-

1

3

7

6

1

.40 (m, 2H, HPhe). C NMR (75 MHz, CDCl

3

): δ 27.90, 28.27, 31.41, 44.54, 47.84, 52.47,

9.29, 79.73, 82.05, 111.72, 113.92, 114.21, 114.77, 115.03, 121.18, 122.57, 122.62, 129.15,

30.21, 130.32, 130.72, 130.39, 139.51, 156.46, 161.33, 164.60, 171.39. ¹⁹F (282 MHz,

CDCl

3

): δ [-112.60; -112.90] (m). Exact Mass: (ESI-MS) for C27

H38FN

2

5

O [M+H] found

4

89.2766, calcd, 489.2765.

Tert-butyl2-((tert-butoxycarbonyl)amino)-4-((2-((3-fluorobenzyl)oxy)benzyl)(2-((3-

fluoromethyl)benzyl)oxy)benzyl)amino)butanoate (6)

(

To a flame-dried flask, with stir bar, was added compound 5 (400 mg; 0.82 mmol) dissolved

in CH Cl (10 mL; 10 mL/mmol), compound 3 (0.209 mL; 0.86 mmol) and NaBH(OAc) (608

mg; 2.87 mmol). The mixture was stirred overnight at room temperature under N atmosphere.

Upon completion of the reaction, the mixture was transferred to a separation funnel followed

by addition of 10 mL H O. The phases were separated, and the aqueous phase was extracted

twice more with CH Cl . The combined organic layers were dried over Na SO and

concentrated in vacuo. The residue was purified with flash chromatography (5% EtOAc in

2

3

2

4

hexane +1% Et

3

N, isocratic). Compound 6 (0.429 g; 0.599 mmol) was obtained as a white oil

1

with 73% yield. Rf = 0,28 (10% EtOAC in hexane + 1%Et

.32 (s, 9H, tBu), 1.42 (s, 9H, tBu), 1.74-1.90 (m, 1H, CH

CH -CH), 2.42-2.74 (m, 2H, N-CH -CH ), 3.72 (dd, J = 15 Hz, J

.18 (m, 1H, CH -CH-NH), 5.06 (s, 2H, CH O), 5.09 (s, 2H, CH O), 5,37 (d, J= 47.7 Hz, 2H,

F), 5.72 (d, J= 6Hz, 1H, NH), 6.81-7.21 (m, 9Hz, HPhe), 7.27-7.53 (m, 7H, HPhe). C NMR

): δ 27.85, 28.39, 29.32, 50.68, 52.28, 53.41, 69.16, 69.73, 79.13, 81.27, 83.30,

3

N). H NMR (300 MHz, CDCl

3

): δ

-CH), 1.93-2.09 (m, 1H, CH

= 9Hz, 4H, CH N), 4.04-

1

2

-CH

2

-

2

1

2

4

2

1

3

CH

2

(75 MHz, CDCl

3

8

1

5.50, 111.63, 111.72, 113.82, 114.09, 114.44, 114.73, 120.89, 121.00, 122.47, 122.50, 126.12,

26.19, 126.74, 126.82, 127.50, 127.54, 127.79, 127.81, 127.87, 128.85, 129.99, 130.10,

30.32, 136.42, 136.65, 137.88, 139.95, 140.05, 155.44, 156.49, 156.71, 161.30, 164.57,

19

71.70. F NMR (282 MHz, CDCl

3

): δ [-112.90; -112.72] (m), -207.60 (t, J= 27.9 Hz). Exact

Mass: (ESI-MS) for C42

H

51

F

2

N

2

O

6

[M+H] found 717.3606, calcd, 717.3715.

2

-amino-4-((2-((3-fluorobenzyl)oxy)benzyl)(2-((3-

(fluoromethyl)benzyl)oxy)benzyl)amino)butanoic acid (7)

Compound 6 (230 mg; 0.32 mmol) was dissolved in HCl (4M in 1,4-dioxane, 5.0 mL) in a

flame-dried flask. The mixture was stirred for 1 hour at 40°C under N -atmosphere after which

2

it was concentrated in vacuo. Pure compound 7 (0.32 mmol) was obtained as a white powder

1

in quantitative yield. R

f

0,37 (10% MeOH in CH

2

Cl

2

). H NMR (300 MHz, DMSO-d

6

, 80°C):

δ 4.31 (s, 4H, CH N), 5.15 (d, 6.9 Hz, 4H, CH

2

O), 5.37 (d, J= 47.4 Hz, 2H, CH2F), 6.86-7.72

1

3

(

m, 16H, HPhe). C NMR (75 MHz, CDCl3) 24.83, 29.66, 50.38, 51.08, 52.80, 59.42, 61.66,

6

1

7.09, 69.62, 70.22, 83.23, 85.51, 112.33 114.13, 114.85, 117.72, 121.64, 123.07, 126.57,

27.27, 127.97, 129.14, 130.47, 131.64, 133.78, 136.66, 139.11, 156.74, 156.98, 161.06,

1

9

64.32, 170.08. F NMR (282 MHz, DMSO-D6, 80°C) δ [-113.06; -112.78] (m), -206.06 (t,

[M+H] found 561.2553, calcd,

J = 47.8 Hz). Exact Mass: (ESI-MS) for C33

61.2565.

H

35

F

2

N

2

O

4

5

tert-butyl 4-((2-((3-(bromomethyl)benzyl)oxy)benzyl)(2-((3-fluorobenzyl)oxy)benzyl)amino)-

-((tert-butoxycarbonyl)amino)butanoate (8)

2

In a flame-dried flask with stirbar, compound 5 (2.18g; 4.44 mmol) was dissolved in anhydrous

CH Cl (80 mL; 20 mL/mmol). Compound 4 (1427 mg; 4.69 mmol) was added, followed by

sodium triacetoxyborohydride (3.32 g; 15.7 mmol). The reaction was stirred for 24 h, followed

by the addition of H O and extraction with CH Cl (3x). The combined organic layers were

washed with brine and dried over Na SO . The reaction mixture was concentrated in vacuo and

purified by automated flash chromatography (10% EtOAc → 18 % EtOAc in hexane + 1%

2

4

Et

3

N). Compound 8 (1.52 g; 1.95 mmol) was obtained as a transparent wax in 44% yield. R

f

1

0

1

2

.38 (10% EtOac in hexane + 1% Et

.41(s, 9H, tBu), 1.75-1.91 (m, 1H, CH

.70 (m, 2H, N-CH -CH ), 3.72 (dd, J =15Hz, J

Br), 5.06 (s, 4H, CH O), 5.66-5.77 (m, 1H, NH), 6.80-7.03 (m, 5H,

Phe), 7.07-7.22 (m, 4H, HPhe), 7.27-7.54 (m, 7H, HPhe). C NMR (75 MHz, CDCl3): δ 27.87,

3

N). H NMR (300 MHz, CDCl

3

): δ 1.32 (s, 9H, tBu),

-CH), 1.93-2.06 (m, 1H, CH -CH -CH), 2.43-

= 9Hz, 4H, CH N), 4.03-4.15 (m, 1H, CH2-

2

-CH

2

1

2

CH-NH), 4.47(s, 2H, CH

2

1

3

H

2

1

8.39, 29.31, 33.32, 50.66, 52.28, 53.05, 53.39, 69.16, 69.67, 111.64, 111.75, 113.81, 114.07,

14.45, 114.74, 121.00, 122.51, 127.24, 127.79, 128.41, 129.02, 130.02, 130.11, 130.32,

1

9

38.05, 155.42, 156.42, 156.71,161.30, 164.54, 171.69. F NMR (300 MHz, CDCl

3

): δ -112.77

(s). Exact Mass: (ESI-MS) for C42

H

51BrFN

2

O

6

[M+H] found 777.2947, calcd, 777.2915

18

Radiosynthesis of [ F]2-amino-4-((2-((3-fluorobenzyl)oxy)benzyl)(2-((3-

1

8

(

fluoromethyl)benzyl)oxy)benzyl)amino)butanoic acid ([ F]FABABA)

Fluoride-18 (1.11 GBq) derived from cyclotron was trapped on a QMA light Sep-Pak cartridge

Water, Zellik, Belgium; preconditioned with 0.05 M K CO (10 mL) and ultrapure water (10

mL)) and eluted with cryptand-2.2.2/ K CO (1 mL of the following mixture: 9.4 mg cryptand-

.2.2. and 1.75 mg K CO dissolved in respectively acetonitrile (960 µL) and ultrapure water

40 µL)). The solvent was evaporated in a warm oil bath (100°C) under a gentle flow of N -

(

2

3

2

3

2

3

(

2

gas followed by two azeotropic drying steps after adding extra dry acetonitrile (1.0 mL). After

placing the vial in an ice bath for 3 minutes, the reaction mixture (4.0 mg of compound 8 in

2

00 µL extra dry acetonitrile) was added and the vial was heated to 120°C for 5 minutes.

Subsequently, the vial was placed in the ice bath for 3 minutes again, HCl (200 µL, 4.0 M in

,4-dioxane) was added and the mixture was heated to 90°C for 3.5 minutes. PBS (100 µL)

1

was added and the mixture was injected onto HPLC for purification. HPLC was done by using

a C18 prep column (Atlantis T3 OBD prep column, 100Å, 10µM, 10mm x 250mm, 1/pkg,

18

Waters), [ F]FABABA eluted after 15 minutes (mobile phase: acetonitrile/ammonium acetate

10 mM) in ultrapure water 45:55 (V:V); flowrate 6 mL/min). After elution, the collected

fraction was diluted with PBS and trapped on a C18 Sep-Pak (preconditioned with acetonitrile

10 mL) and ultrapure water (10 mL)), washed with PBS (10 mL) and subsequently eluted with

EtOH (1.0 mL). The EtOH was evaporated under a gentle flow of N -gas (90°C) and the

(

2

radiotracer was solubilized in an ethanol:PBS mixture (400 µL,10:90 (V:V)) to obtain the

desired formulation.

The obtained radiotracer was subjected to quality control by means of analytical HPLC with

radio and UV detection (205 nm; Waters) using a 1.0 mL/min flow and a Grace Alltima C18

(4.6 x 250 mm; 5 µm) column. Identification of the radiotracer was done by co-injection with

compound 7 and comparing the retention time of the radiopeak to the UV-peak (18 min; mobile

phase: gradient from 10 percent acetonitrile in ammonium acetate (10 mM) to 100 percent

acetonitrile). logD7.4 determination was done by adding 1 MBq radiotracer to a mixture of PBS

water and n-octanol (1:1; V:V). Samples were vortexed and centrifuged (10 min; 1100 g) where

after aliquots were taken from each layer which were measured in a NaI (Tl) scintillation

counter (Capintec; Ramsey, NJ, USA). logD7.4 was calculated as the logarithm of the

proportion of counts in the octanol and PBS layers.

In vitro experiments

General information

The cell lines were purchased from ATCC (PC-3, F98 and HEK-293 wild-type) and Creative

Biogene (HEK-293 SLC1A5 knock-out cell line). RPMI medium enriched with 10% fetal calf

serum, 1% -glutamine and penicillin/streptomycin (50 U/ mL) was used to cultivate the PC-3

L

cells. The culture medium for the F98 cell line, HEK-293 wild-type (WT) and SLC1A5 knock-

out (KO) cell line was Dulbecco’s modified Eagle’s medium (DMEM) enriched with 10% fetal

calf serum, 1%

stored in an incubator set to 37°C and 5% CO

FACS) was used to evaluate the presence of ASCT-2 expression in PC-3 and F98 cell lines

L

-glutamine and penicillin/streptomycin (50 U/ mL). All culture flasks were

2

environment. Fluorescence-activated cell sorting

(

1

8

following the protocol described by De Munter et al. . Both cell lines (100,000 cells/ 100 µL)

were stained at their surface with a primary [rabbit ab; ASCT-2 (V501)] and secondary

antibody [Fab2 (PE Conjugate); anti-rabbit IgG] against ASCT-2 as a negative control. The

cells were fixed and permeabilized, followed by intracellular binding with the primary

antibody. After binding of the primary antibody, the secondary antibody was added followed

by an incubation period. All samples were analyzed at the end of the incubation period using

the LSR II (BD Biosciences). The ASCT-2 KO cell line was engineered with CRISPR-CAS 9

using a HEK-293 WT cell line. Expression of ASCT-2 on both the HEK-203 WT and HEK-

2

93 KO cell lines was also verified using fluorescence-activated cell sorting by Creative

Biogene.

One day prior to the test, PC-3, F98, HEK-293 WT and ASCT-2 KO cells were seeded in 24-

well plates (VWR, US) with a concentration of 175,000; 125,000; 175,000 and 175,000 cells

per milliliter, respectively. To achieve good attachment of the HEK-293 WT and HEK-293 KO

cells, the 24-well plates were coated prior to seeding: 300 µL of poly-D-lysine stock solution

(0.1 mg/ mL) was added to each well and the plates were dried for 30 minutes until all solvent

was removed. Upon removal of the solvent the plates were ready for cell attachment. A

HEPES+ buffer (pH 7.4; 100 mM NaCl (Sigma Aldrich, Belgium), 2 mM KCl (Sigma Aldrich,

Belgium), 1 mM MgCl

2

(VWR, US), 1 mM CaCl (VWR, US), 10 mM Hepes (Sigma Aldrich,

2

Belgium), 5 mM Tris (VWR, US), 1 g/ L glucose (VWR, US) and 1 g/ L Bovine Serum

3

Albumin (Sigma Aldrich, Belgium)) was used to perform the [ H]glutamine uptake studies.

The desired concentration series were made from a glutamine stock concentration (43.8 mg/

3

0mL HEPES+). To each stock solution a total of 111 kBq/ mL [ H]-

L

-glutamine (Perkin

19

Elmer, Massachusetts) was added. Concentration series containing [ F]FABABA were made

next to the reference glutamine concentration series. The experiments were started by aspiring

the medium and washing all wells with HEPES+ twice. After removal of HEPES+, test solution

(

250 µL) was added, and the plates were incubated for 5 minutes at 37°C. After 5 minutes

uptake was stopped by placing the plates on ice and adding ice cold PBS + BSA (1 mL, 1 g/

00 mL). After removal of PBS +BSA, the wells were washed twice with ice cold PBS (2 mL)

1

and cells were lysed by adding 0.1 M NaOH (250 µL) (VWR, US) and placing the plates on a

shaker. Aliquots of 150 µL were pipetted out of each well into a scintillation bottle (Perkin

Elmer, Massachusetts, USA) and 5 mL of scintillation cocktail (Ultima Gold, Perking Elmer,

Massachusetts, USA) was added. Samples were measured with an automated scintillation

counter (TriCarb 2900 TR; Perkin Elmer, Massachusetts, USA). Aliquots of 25 µL cell lysis

were subjected to the bicinchoninic acid assay (BCA) (ThermoFisher Scientific) to correct for

protein density.

3

Concentration dependency: The uptake of [ H]-

L

-glutamine was tested in PC-3 human cells

and F98 rat cells in absence of cold reference product and was compared to the uptake in the

3

presence of cold reference products. Additionally, the uptake of [ H]-

L

-glutamine in PC-3 cells

was also evaluated in the presence of compound V-9302. The glutamine stock solution used

ranged from 10 µM to 1200 µM. Michaelis-Menten parameters were obtained by plotting the

data of the liquid scintillation counter via non-linear regression in GraphPad prism v5.01

(

GraphPad software, San Diego, CA, USA). K

m

(values in absence of cold product) and Km,app

values

(apparent values in presence of cold product) data were used to calculate the K

i

ꢀꢁꢂꢃ

according to the formula: 푉푚푎푥, 푎푝푝 = ₁, with [I] being the concentration of inhibitor.

[

퐼]

+

퐾푖

3

Inhibition [ H]-

L

-glutamine uptake in knock-out cell line: To evaluate the selectivity of the

3

radiotracer to ASCT-2, the inhibition of the [ H]-

L

-glutamine uptake in absence of ASCT-2

3

was measured in a knock-out cell line. The cells were incubated with 250 µL 50 µM [ H]-

L

-

glutamine/glutamine in absence and presence of compound 8 for 5 minutes at 37°C. The

inhibition of glutamine uptake in HEK-293 KO was compared to glutamine inhibition in

ASCT-2 expressing HEK-293 WT and F98 cells which was evaluated using the same protocol.

In vitro evaluation of stability in formulation

18

The stability of [ F]FABABA was evaluated in its formulation. After radiosynthesis the

radiotracer formulation was incubated at 37°C for 30, 70, 120 and 180 minutes. At the indicated

time points aliquots were taken (7 MBq) and analyzed by analytical HPLC (method cfr QC of

[¹⁸

F]FABABA).

Optimization of parameters for radiochemical labelling

The reaction parameters of the radiofluorination were optimized. Solvent, reaction temperature

and mass precursor were altered one variable at a time. DMF, DMSO and acetonitrile were

tested as reaction solvents. The different reaction temperatures were 60°C, 80°C, 100°C and

1

20°C. The concentration of precursor solution tested were 2 mg/ 200 µL; 4 mg/ 200 µL; 6

mg/ 200 µL; 8 mg/ 200µL extra dry acetonitrile. Radiochemical labelling yields were

determined by TLC analysis.

18

In vivo evaluation of [ F]2-amino-4-((2-((3-fluorobenzyl)oxy)benzyl)(2-((3-

fluoromethyl)benzyl)oxy)benzyl)amino)butanoic acid

(

The Ghent University Ethical Committee on Animal Experiments approved the in vivo

evaluation of the tracer in a PC-3 xenograft model prior to the start of the study, which was

performed according to the appropriate guidelines and regulations (ECD 17/08). The

xenografts were obtained by inoculating male swiss nu/nu mice with PC-3 tumor cells. At the

day of inoculation, the PC-3 cells were harvested and suspended in non-enriched RPMI

7

medium to obtain a 5 x 10 cells/mL cell suspension. The mice (n=2) were injected with 5 x

1

0⁶cells (+ 0.1 mL Matrigel®) in the right flank at the age of 8 weeks; one week later, the left

6

flank was inoculated with 5 x 10 cells (+ 0.1 mL Matrigel®). Tumor growth was followed

with manual caliper measurements. During animal experiments, food and water were provided

ad libitum. Dynamic µPET scans were acquired seven weeks post-inoculation and animals

were fasted minimal six hours prior to imaging. Anesthesia was induced with 5.0% isoflurane

(

V:V in O ) and maintained with 2.0% until the end of the experiment. After intravenous

2

injection of 18.5 MBq radiotracer in the lateral tail vein, dynamic PET acquisitions of 1 hour

were performed on a FLEX Triumph II small animal PET/CT-scanner (PET field of view: 7.5

cm axial; 1.2 mm spatial resolution; TriFoil Imaging). Upon completion of the PET-scan an

additional computed tomography (CT) scan was performed for anatomical correlation

purposes. The resulting PET data were obtained in list mode and reconstructed iteratively (50

iterations) in frames of one minute the first twenty-five minutes and frames of five minutes for

the following thirty-five minutes. PET and CT images were aligned using the AMIDE (Source

1

9

Forge Media, La Jolla, CA, USA) software . Region of interests (ROI) were drawn manually

around the tumor and leg muscle as comparison tissue. Tumor-to-muscle (T/M) ratios were

calculated and standard uptake values (SUV) were determined according to the following

formula:

SUV = Radioactivity in ROI (MBq/ml) / injected activity (MBq) x body weight (g)

Biodistribution study

1

8

The distribution of the [ F]FABABA to the different organs of PC-3 inoculated mice was

evaluated by means of a biodistribution study. The PC-3 xenograft model was set up as

discussed above. Seven weeks post-inoculation, 5.0 MBq of radiotracer was injected

intravenously via the lateral tail vain. At the indicated time points (5 min and 20 min post-

injection) the mice were euthanized by cervical dislocation (n=3 for each time point). Blood

was extracted via cardiac puncture and the organs were removed during dissection. To avoid

any interference of adhering blood, all organs were rinsed with water. Subsequent weighing of

the samples and counting of the radioactive signal provided the uptake in the organs expressed

as percentage injected dose per gram of tissue (%ID/ g) ± SD.



List of abbreviations

ASCT-2: Alanine serine cysteine transporter-2

CT: Computed tomography

DAST: Diethylaminosulfur trifluoride

DMEM: Dulbecco’s modified Eagle’s medium

DMF: Dimethylformamide

DMSO: Dimethylsulfoxide

[¹⁸

F]FABABA: [ F]2-amino-4-((2-((3-fluorobenzyl)oxy)benzyl)(2-((3-

18

(fluoromethyl)benzyl)oxy)benzyl)amino)butanoic acid

KO: Knock-out

LAT1: Large amino acid transporter-1

PBS: Phosphate buffered saline

PET: Positron emission tomography

ROI: Region of interest

SUV: Standardized uptake value

T/M: Tumor-to-muscle



Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or

financial relationships that could be construed as a potential conflict of interest.



Authors’ contribution

Conceptualization: TB JB JJ GP JV BD CV FDV

Data curation: TB JB JJGP JV BD CV

Formal analysis: TB JB JJ GP

Investigation: TB JB JJ GP JV

Methodology: TB BD SVC CV FDV

Project administration: SVC CV FDV

Resources: CV FDV

Software: TB JB JJ GP JV

Supervision: SCV CV FDV

Validation: TB GP

Visualization: TB

Writing - original draft: TB GP JV

Writing - review & editing: TB JB JJ GP JV BD SVC CV FDV

Tristan Baguet: TB; Jakob Bouton: JB, Jonas Janssens: JJ; Glenn Pauwelyn: GP; Jeroen

Verhoeven: JV; Benedicte Descamps: BD; Serge Van Calenbergh: SVC, Christian Vanhove:

CV; Filip De Vos: FDV



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Table 1: Affinity constant K for FABABA in human PC-3 cells and rat F98 cells.

i

Compound

FABABA

K

i

value PC-3 (µM)

K

i

value F98 (µM)

9

0

125

V-9302

1

50

Not determined.

Table 2: Semiquantitative values of [18F]FABABA uptake in PC-3 xenograft. Mean tumor-to-

muscle (T/Mmean) ratios and mean standardized uptake values (SUVmean) are given for two

mice.

Mouse 1

1.23

Mouse 2

1.20

T/Mmean

SUVmean

0.10

0.07

18

Table 3: Tissue uptake of [ F]FABABA in various organs and the blood of Swiss nu/nu male

mice at 5- and 20-minutes post-injection. Values are expressed as % ID/ g of tissue ± SD (n=3).

[¹⁸

F]FABABA

Organ

%ID/ g 5 min

post-injection

%ID/ g 20 min

post-injection

Blood

8.96 ± 3.20

0.091 ± 0.070

0.15 ± 0.17

-0.01 ± 0.23

0.55 ± 0.24

0.08 ± 0.14

28.87 ± 2.82

0.98 ± 0.34

2.48 ± 0.91

3.80 ± 0.86

0.74 ± 0.32

0.53 ± 0.35

0.22 ± 0.19

-0.16 ± 0.47

0.52 ± 0.33

2.17 ± 0.53

6.11 ± 1.03

0.74 ± 0.15

0.62 ± 0.19

0.27 ± 0.35

1.38 ± 0.18

0.69 ± 0.36

19.65 ± 1.10

1.13 ± 0.10

2.50 ± 0.20

4.00 ± 0.29

0.63 ± 0.41

1.07 ± 0.46

0.19 ± 0.08

1.71 ± 0.73

1.47 ± 0.27

1.87 ± 0.13

Tumor

Muscle

Brain

Skin

Fat

Liver

Spleen

Lung

Kidney

Stomach

Small intestine

Large intestine

Bladder

Bone

Heart

Article Doi

Radiosynthesis, in vitro and preliminary biological evaluation of [18F]2-amino-4-((2-((3-fluorobenzyl)oxy)benzyl)(2-((3-(fluoromethyl)benzyl)oxy)benzyl)amino)butanoic acid, a novel alanine serine cysteine transporter 2 inhibitor-based positron

DOI: 10.1002/jlcr.3863

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Authors:

Article abstract of DOI:10.1002/jlcr.3863

Full text of DOI:10.1002/jlcr.3863

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