Available via license: CC BY 4.0
Content may be subject to copyright.
Citation: Craig, A.; Kogler, J.; Laube,
M.; Ullrich, M.; Donat, C.K.; Wodtke,
R.; Kopka, K.; Stadlbauer, S.
Preparation of 18F-Labeled Tracers
Targeting Fibroblast Activation
Protein via Sulfur [18F]Fluoride
Exchange Reaction. Pharmaceutics
2023,15, 2749. https://doi.org/
10.3390/pharmaceutics15122749
Academic Editors: Ildiko Badea
and Leonard I. Wiebe
Received: 6 October 2023
Revised: 14 November 2023
Accepted: 2 December 2023
Published: 10 December 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
pharmaceutics
Article
Preparation of 18F-Labeled Tracers Targeting Fibroblast
Activation Protein via Sulfur [18F]Fluoride Exchange Reaction
Austin Craig 1, Jürgen Kogler 1,2 , Markus Laube 1, Martin Ullrich 1, Cornelius K. Donat 1,
Robert Wodtke 1, Klaus Kopka 1,2 and Sven Stadlbauer 1,2,*
1Helmholtz-Zentrum Dresden-Rossendorf, Institute of Radiopharmaceutical Cancer Research,
Bautzner Landstraße 400, D-01328 Dresden, Germany; a.craig@hzdr.de (A.C.)
2Faculty of Chemistry and Food Chemistry, School of Science, Technische Universität Dresden,
D-01062 Dresden, Germany
*Correspondence: s.stadlbauer@hzdr.de
Abstract:
Early detection and treatment of cancers can significantly increase patient prognosis and en-
hance the quality of life of affected patients. The emerging significance of the tumor microenvironment
(TME) as a new frontier for cancer diagnosis and therapy may be exploited by radiolabeled tracers for
diagnostic imaging techniques such as positron emission tomography (PET). Cancer-associated fibrob-
lasts (CAFs) within the TME are identified by biomarkers such as fibroblast activation protein alpha
(FAP
α
), which are expressed on their surfaces. Targeting FAP
α
using small-molecule
18
F-labeled in-
hibitors (FAPIs) has recently garnered significant attention for non-invasive tumor visualization using
PET. Herein, two potent aryl-fluorosulfate-based FAPIs,
12
and
13,
were synthetically prepared, and
their inhibition potency was determined using a fluorimetric FAP assay to be IC
50
9.63 and 4.
17 n
M,
respectively. Radiofluorination was performed via the sulfur [
18
F]fluoride exchange ([
18
F]SuFEx)
reaction to furnish [
18
F]
12
and [
18
F]
13
in high activity yields (AY) of 39–56% and molar activities
(A
m
) between 20–55 GBq/
µ
mol.
In vitro
experiments focused on the stability of the radiolabeled
FAPIs after incubation with human serum, liver microsomes and liver cytosol. Preliminary PET
studies of the radioligands were performed in healthy mice to investigate the
in vivo
biodistribution
and
18
F defluorination rate. Fast pharmacokinetics for the FAP-targeting tracers were retained and
considerable bone uptake, caused by either
18
F defluorination or radioligand accumulation, was
observed. In summary, our findings demonstrate the efficiency of [
18
F]SuFEx as a radiolabeling
method as well as its advantages and limitations with respect to PET tracer development.
Keywords:
automation; cancer-associated fibroblast; FAPI;
18
F fluorination; positron emission
tomography (PET); [18F]SuFEx
1. Introduction
Positron emission tomography (PET) is a highly sensitive non-invasive imaging tech-
nique routinely utilized in clinical practice in combination with MRI or CT for the diagnosis
of a plethora of human diseases [
1
–
6
]. PET imaging affords valuable and precise metabolic
information on a molecular level in real time, which aids clinicians to decide on effective
treatment plans for patients. Emerging biological targets for radiopharmaceuticals pro-
vide novel opportunities for enhanced tumor delineation using PET. Recently, the tumor
microenvironment (TME) has gained attention as a source of PET tracer targets, owing
to its inherent unique features such as the interconnection between cancer and stromal
ce
lls [7–10].
The overexpression of fibroblast activation protein alpha (FAP
α
) is a character-
istic of cancer-associated fibroblasts (CAFs), which are located in the stroma of epithelial
cells. In contrast, FAP expression in healthy tissue is relatively low [11].
To date, several examples of radiolabeled FAP inhibitors (FAPIs) have been de-
scribed [
11
–
14
]. The preparation of
68
Ga-labeled FAPIs has the advantage of utilizing
Pharmaceutics 2023,15, 2749. https://doi.org/10.3390/pharmaceutics15122749 https://www.mdpi.com/journal/pharmaceutics
Pharmaceutics 2023,15, 2749 2 of 18
a
68
Ge/
68
Ga generator for tracer production, thereby negating the need for an on-site
cyclotron and thus reducing tracer production costs. First-in-human experiments using
68
Ga-labeled FAPIs have provided high-contrast tumor PET images [
10
–
12
,
14
]. However,
the clinical applications of
68
Ga-labeled FAPIs are limited by the batch size of the PET
tracer due to generator-defined starting activities for the production and its short half-life
(6
8 mi
n) [
15
]. Moreover, the higher positron energy associated with gallium-68 affords PET
images of inferior quality compared to
18
F-labeled radiopharmaceuticals. To overcome
the limitations of 68Ga-based FAPIs, a series of 18F-labeled FAPIs have been developed. A
recent work by Linder et al. provides a useful summary of
18
F-labeled aluminum fluoride
complexes and 6-fluoronicotinamide FAPI derivatives (Scheme 1A,B) [
15
]. The advan-
tageous physicochemical properties of fluorine-18 are ideal for PET experiments. For
example, the half-life of fluorine-18 (109.7 min), the most predominant radionuclide used
in PET imaging, permits multi-step radiosynthetic protocols and tracer transport between
clinical facilities [
16
]. Moreover, the high positron (>97%
β+
) branching and low positron
energy (0.635 MeV) of fluorine-18 provide high-resolution PET images.
Pharmaceutics 2023, 15, x FOR PEER REVIEW 2 of 19
located in the stroma of epithelial cells. In contrast, FAP expression in healthy tissue is
relatively low [11].
To date, several examples of radiolabeled FAP inhibitors (FAPIs) have been described
[11–14]. The preparation of 68Ga-labeled FAPIs has the advantage of utilizing a 68Ge/68Ga
generator for tracer production, thereby negating the need for an on-site cyclotron and
thus reducing tracer production costs. First-in-human experiments using 68Ga-labeled
FAPIs have provided high-contrast tumor PET images [10–12,14]. However, the clinical
applications of 68Ga-labeled FAPIs are limited by the batch size of the PET tracer due to
generator-defined starting activities for the production and its short half-life (68 min) [15].
Moreover, the higher positron energy associated with gallium-68 affords PET images of
inferior quality compared to 18F-labeled radiopharmaceuticals. To overcome the
limitations of 68Ga-based FAPIs, a series of 18F-labeled FAPIs have been developed. A
recent work by Linder et al. provides a useful summary of 18F-labeled aluminum fluoride
complexes and 6-fluoronicotinamide FAPI derivatives (Scheme 1A,B) [15]. The
advantageous physicochemical properties of fluorine-18 are ideal for PET experiments.
For example, the half-life of fluorine-18 (109.7 min), the most predominant radionuclide
used in PET imaging, permits multi-step radiosynthetic protocols and tracer transport
between clinical facilities [16]. Moreover, the high positron (>97% β+) branching and low
positron energy (0.635 MeV) of fluorine-18 provide high-resolution PET images.
[18F]F−, AlCl3
0.5 M NaOAc buffer
DMSO
1) rt, 5min
2) 95 oC, 15 min
F3C O
O
N
OH
NN
OCN
R2
N
N
R1R1
MeCN, 40 oC, 5 min
O
NN
N
OH
NN
OCN
R2
N
N
R1R1
O
N
18F
B
N
OH
NN
OCN
R2
N
N
R1R1
O
N
N
N
[18F]8-10: [18F]FAPI-42/52/74
AY: 15–30%
RCP: >99%
[18F]3-4: [18F]FAPI-72/73
RCY: 50–70%
RCP: >99%
1: FAPI-72, R1= F, R2= NMe
2: FAPI-73, R1 = F, R2= O
5: FAPI-42, R1= F, R2= O
6: FAPI-52, R1= F, R2 = NMe
7: FAPI-74, R1= H, R2 = O
O
O
OO
Al
18F
N
OH
NN
OCN
R2
N
N
R1R1
O
N
N
N
O
HO
OOH
AY: 4 7%
Am: 4.7 GBq/µmol
RCP: >99%
Total synthesis time 40 min
N
OH
NN
OCN
O
FF
[18F]F−, BnEt3NCl
MeCN, 40 oC, 3 min
N
OH
NN
OCN
O
FF
C
DThis Work
A
[18F]F−
11 [18F]11
S
18F
OO
S
F
OO
N
OH
NN
OCN
ON
N
FF
O
[18F]F−, BnBu3NCl
MeCN, rt, 5 min
N
OH
NN
OCN
ON
N
FF
O
[18F]12: AY: 54 ± 1%
Am: 52 ± 2 GBq/µmol
RCP: >99%
OO
N
OH
NN
OCN
ON
N
FF
O
[18F]F−, BnBu3NCl
MeCN, rt, 5 min
N
OH
NN
OCN
ON
N
FF
O
[18F]13: AY: 43 ± 3%
Am: 22.58 GBq/µmol
RCP: >99%
OO
12
13
S
F
OO
S
18F
OO
S
S
O
O
O
O
F18F
Scheme 1.
(
A
–
C
) Previous preparations of
18
F-labeled FAPI derivatives. (
D
) This work:
18
F-labeled
FAPIs prepared via [18F]SuFEx reaction.
Pharmaceutics 2023,15, 2749 3 of 18
The most predominant
18
F-labeled tracer, 2-[
18
F]fluoro-2-deoxyglucose ([
18
F]FDG),
has been widely implemented in clinics for the detection of various tumors using PET [
17
].
Despite numerous other examples of promising radiofluorinated tracers being prevalent
in the literature, only ten
18
F-labeled PET tracers have been approved by the FDA to
date [
18
,
19
]. Therefore, emerging robust radiofluorination techniques that may facilitate
the development of PET tracers and their translation into clinics are still highly sought after.
The development of radiofluorination methods in recent years has primarily focused
on the formation of C–
18
F bonds [
20
–
34
]. Aside from several exceptions, radiofluorinations
involving C–
18
F bond formation typically necessitate harsh reaction conditions to ensure
fast
18
F incorporation rates, which may limit their substrate scopes and practicality for
clinical translation. Furthermore, fluorination strategies from larger-scale (e.g., millimolar)
concentrations in organic syntheses cannot be easily adapted towards radiofluorination
approaches due to the differing reactivity of fluorine-18 at low concentrations (micromolar).
The recently described sulfur [
18
F]fluoride exchange ([
18
F]SuFEx) chemistry has gained
attention for the rapid preparation of
18
F-labeled aryl-fluorosulfate (ArOSO
2
F) substrates
with ultra-fast reaction times, high radiochemical yields and rapid tracer isolation via
SPE purification, also demonstrated for the preparation of an
18
F-labeled FAPI derivative
(Scheme 1C) [
35
–
38
]. The
18
F-for-
19
F isotopic exchange reaction has also been shown
to produce
18
F-labeled compounds with high molar activities (A
m
) when a low starting
precursor concentration and high starting fluorine-18 activity concentration is utilized.
The
in vivo
stability of the [
18
F]ArOSO
2
F group was also demonstrated to be sufficient for
PET imaging in certain instances, yet a priori inferences regarding their stability have not
been established to date. This work reports the application of the [
18
F]SuFEx chemistry
towards two novel FAP-targeting radioligands and the evaluation of their suitability for
non-invasive imaging using PET (Scheme 1D).
2. Materials and Methods
2.1. Materials
Unless stated otherwise, all solvents and reagents were obtained from commercial
vendors and utilized without additional purification. All solvents used in experiments
were of HPLC or analytical grade, with the exception of water, which was ultrapure
(>18.2 MΩcm−1).
2.2. General Information
The
1
H,
13
C and
19
F NMR spectra provided were recorded on a Varian Inova-400 and
J-values are given in Hertz (Hz). All radiochemistry experiments were performed at the In-
stitute of Radiopharmaceutical Cancer Research, Helmholtz-Zentrum Dresden-Rossendorf
(HZDR). [
18
F]Fluoride was produced via the (p,n) reaction using a TR-FLEX (ACSI) cy-
clotron by irradiating [
18
O]H
2
O with 18–30 MeV protons [
39
]. Automated radiosynthesis
was carried out in a TRACERlab FX FDG (GE Healthcare) synthesis module and was
performed in a hot cell. Analytical radio-(U)HPLC was performed on the following system:
column Kinetex C-18 (Phenomenex Inc., Torrance, CA, USA; 50
×
2.1 mm, 1.7
µ
m, 1
00 Å
),
Shimadzu Nexera X2 UHPLC system (Shimadzu Corporation, Kyoto, Japan; degasser
DGU-20A
3R
and DGU-20A
5R
, pump LC-30AD, autosampler SIL-30AC, column oven CTO-
20AC with two column switching valves FCV-14AH, diode array detector SPD-M30A,
fluorescence detector RF-20A,
γ
detector Gabi Star (Raytest Isotopenmeßgeräte GmbH,
Straubenhardt, Germany), communication bus module CBM-20A), eluent: (A): 0.1% trifluo-
roacetic acid in H
2
O, (B): MeCN. Gradient A radio-UHPLC: flow rate 0.5 mL/min, gradient
(eluent A/B): t
0 min
95/5–t
0.3 min
95/5–t
4.5 min
5/95–t
5.5 min
5/95–t
6.0 min
95/5–t
7.5 min
95/5,
flow rate: 0.5 mL/min. Gradient B: radio-HPLC: flow rate 1.0 mL/min, gradient (eluent
A/B): t
0 min
95/5–t
3.0 min
95/5–t
28.0 min
5/95–t
34.0 min
5/95–t
35.0 min
95/5–t
40.0 min
95/5. The
radiochemical conversion (RCC) based on radio-UHPLC was determined by analyzing
an aliquot after dilution of the crude radiofluorination reaction mixture (MeCN) with
MeCN/H
2
O (1:1) and is based on the relative peak areas of the
γ
detector channel of
Pharmaceutics 2023,15, 2749 4 of 18
the chromatogram. The identity of the radiolabeled products was determined by asso-
ciating the UV-(U)HPLC traces of the suitable unlabeled reference compounds with the
radio-(U)HPLC traces of the radiolabeled products. Notably, as the concentrations of the
unlabeled precursor (which is also the appropriate reference compound due to the isotopic-
exchange radiolabeling mechanism of the [
18
F]SuFEx reaction) was typically sufficient for
detection in the UV chromatogram, co-injection with additional reference standard was
not performed. The radiochemical purity (RCP) of radiolabeled products was determined
based on the integration of the peaks in the chromatogram in the radio-(U)HPLC. The
isolated activity yield (AY) of a radiolabeled product is the non-decay-corrected (n.d.c.)
yield given as a percentage value, which is determined by dividing the product activity at
the end of synthesis (E.O.S.) by the initial starting activity and multiplying by one hundred.
The molar activity (A
m
) was determined with a (U)HPLC analysis based on the injection of
a known activity amount followed by determination of the injected amount of substance.
For that, the UV peak area corresponding to the radiolabeled product was determined
and the concentration of the non-radiolabeled “carrier” compound was calculated based
on a calibration curve (See Supplementary Materials Figure S21, 5.8913 pmol o.c., and
Figure S22, 22.521 pmol o.c.).
2.3. Preparation of Radiofluorinated FAPI Derivatives
The radiosynthetic procedure towards [
18
F]
12
and [
18
F]
13
commenced with the ad-
sorption of [
18
F]fluoride (50–10,000 MBq) on an anion-exchange resin (QMA light carbonate
cartridge, Waters Corp., Milford, MA, USA) followed by washing with dry MeOH (
0.7 mL
)
from the male side and elution of
18
F with a solution of either 3.0 mg (9.6
µ
mol) BnBu
3
NCl,
3.0 mg (13.1
µ
mol) BnEt
3
NCl or 1.0 mg (5.2
µ
mol) Et
4
NHCO
3
in 0.7 mL MeOH from
the female side (Scheme 1D). The methanolic solution was removed under vacuum at
70
◦
C for 5 min. The radiolabeling precursors (
12
or
13,
0.1 mg, 0.145
µ
mol) in MeCN
(0.5 mL) were added to the cooled reaction vessel and allowed to react for 5 min at room
temperature without stirring. The reaction mixture containing the crude radiofluorinated
product ([
18
F]
12
or [
18
F]
13
) was diluted with (5 mL) H
2
O and passed through an HLB SPE
cartridge. The HLB cartridge (Waters Corp.) was washed with water (10 mL) and eluted
with 2 mL EtOH to furnish either [
18
F]
12
at an activity yield (AY) of 55
±
1% (n= 3) or
[
18
F]
13
at an activity yield of 43
±
3% (n= 3) with a radiochemical purity of >95%. The
product solution was further diluted with 0.9% NaCl aqueous solution to obtain a final
product containing 10% of EtOH (v/v). The final products were analyzed using analytical
radio-HPLC for product identification. The RCC determination was carried out following
the quenching of the reaction mixture with (100
µ
L) H
2
O, taking an aliquot (5–15
µ
L) of the
crude reaction mixture and adding the mixture to a sample of (200
µ
L) H
2
O/MeCN (1:1,
v/v), and analyzed using radio-UHPLC. The RCC value was determined by integrating the
radiolabeled product peak area, dividing this value by the total integrated peak area values
and multiplying the value by one hundred. The AY was obtained by dividing the measured
activity of the product by the measured starting activity of [
18
F]F
−
prior to radiosynthesis
and multiplying by one hundred [
40
]. Unless noted otherwise, each experiment was per-
formed in triplicate. Calibration curves (see Supplementary Materials, Figures S21 and S22)
were constructed from the peak areas in the UV channel of the UHPLC chromatograms
of a series of non-radiolabeled products (e.g.,
12
) with known concentrations, in order to
determine exemplarily the carrier concentration in the final radiolabeled product mixture.
2.4. Radiosynthesis Automation
The automated radiosynthesis of [
18
F]
12
was carried out in a TRACERlab FX FDG
synthesis module (GE Healthcare, Waukesha, WI, USA). Approximately 6–60 GBq of
aqueous non-carrier-added (n.c.a.) [
18
F]fluoride was trapped on a QMA light carbonate
cartridge (Waters Corp., Position 1), washed with 2.5 mL MeOH (V1) and eluted from the
male side with Et4NHCO3(1.0 mg, 5.2 µmol, V2) in 700 µL MeOH.
Pharmaceutics 2023,15, 2749 5 of 18
Evaporation of the MeOH was carried out at 70
◦
C for 5 min under a vacuum. After
cooling the reaction vessel to 23
◦
C,
12
(0.1 mg, 0.145
µ
mol) in 0.5 mL MeCN (entry V3)
was transferred to the reactor. The reaction was performed for 5 min at 23
◦
C. Thereafter,
the reaction was quenched with the addition of 7 mL H
2
O into the reactor (V6), and the
resulting solvent mixture was loaded onto a preconditioned HLB cartridge (Waters Corp.,
position 1—HLB). Thereafter, the HLB cartridge was washed with 12 mL H
2
O (V7). Finally,
the elution of the trapped product [
18
F]
12
from the HLB cartridge was carried out using
2 mL
EtOH (V8) into the product vial. The purified radiotracer was diluted in isotonic
saline (0.9% NaCl) to obtain a final product containing 10% of EtOH (v/v). The final product
was analyzed using radio-HPLC for product identification. Overall, the implementation
of the radiosynthetic protocol into an automated radiosynthesizer furnished the desired
product [18F]12 within 60 min with an AY of 11 ±1% (n= 3).
2.5. Determination of Lipophilicity
The LogD
7.4
determination of [
18
F]
12
was determined in a similar manner as previ-
ously described and proceeded as follows [
41
]. [
18
F]
12
(1
µ
L, ca. 0.5 MBq) was pipetted into
an Eppendorf tube containing 600 µL of n-octanol (presaturated with phosphate-buffered
saline) and 600
µ
L of phosphate-buffered saline buffer (0.02 M, pH = 7.4). The tube was
vortexed for 15 min at room temperature, and the two phases were separated by centrifu-
gation at 5000 rpm for 3 min. An aliquot (400
µ
L) from the n-octanol layer was pipetted
in a vial containing phosphate-buffered saline (400
µ
L), the sample mixture was vortexed
for 15 min at room temperature and the two phases were separated by centrifugation at
5000 rpm for 3 min. Aliquots (250 µL) from each phase were taken and measured with an
automatic
γ
-counter after subtracting the background activity. The transfer of the aqueous
phase requires particular care, as described by Linclau et al. [
42
]. The partition coefficient
was calculated for the acquired samples using Equation (1).
logD7.4 =log counts per minute (octanol)
counts per minute (phosphate buffer)(1)
Equation (1) for lipophilicity (LogD) determination.
2.6. Serum Stability Assay
Either [
18
F]
12
or [
18
F]
13
(20–40 MBq) in 40
µ
L EtOH aliquots was incubated with
360
µ
L human serum (Sigma Aldrich, Darmstadt, Germany) at 37
◦
C. After specific time
intervals (5–120 min), 40
µ
L samples were taken from the serum incubation mixture, added
to 80
µ
L “Supersol” solubilizing agent (see Supplementary Materials Table S6) and cooled
over ice for 2 min. Thereafter, the cooled mixture was centrifuged at 4
◦
C (15,000 rpm) for
3 mi
n and 80
µ
L of the supernatant solution was monitored with radio-UHPLC. A gradient
of 5% to 45% acetonitrile in 7.5 min was used for enhanced separation.
2.7. Liver Microsome Assay
Microsome experiments with [
18
F]
12
in the presence of NADPH were performed
according to the procedure recently described by us with slight modifications [
43
,
44
]. Incu-
bations had a final volume of 250
µ
L. The radiotracer dissolved in EtOH (4
µ
L;
5 MBq/µL
)
was diluted with PBS (496
µ
L, 0.8% EtOH). This radiotracer solution (100
µ
L, 0.32% EtOH
and 16 MBq/mL or 1.6
µ
M final) was mixed with PBS (112.5
µ
L) and human liver micro-
somes (12.5
µ
L of 20 mg/mL stock; 1 mg/mL final; Gibco
™
Cat. No. HMMCPL, Lot. No.
PL050E-B) in a 1.5 mL Eppendorf tube and the mixture was warmed for 5 min at 37
◦
C.
Subsequently, NADPH (25
µ
L of freshly prepared 20 mM solution in PBS, 2 mM final)
was added and the mixture was incubated at 37
◦
C. As a control incubation, NADPH was
omitted and replaced by PBS. After distinct time points (10, 20, 30 and 60 min), an aliquot
(40
µ
L) was withdrawn and added to ice-cooled CH
3
CN (160
µ
L). The mixture was vor-
texed for 30 s, stored on ice for 4 min and centrifuged (5 min at 15,000 rpm). The resulting
supernatant was used for radio-HPLC analysis using the Shimadzu system described in
Pharmaceutics 2023,15, 2749 6 of 18
Section 2.2 with the following conditions. A C
18
column Kinetex
®
from Phenomenex (
5µm
,
100 Å, LC Column 250
×
4.6 mm) served as stationary phase. The eluent consisted of (A)
0.1% trifluoroacetic acid in H
2
O and (B) MeCN; flow rate 1 mL/min; elution profile A/B:
t0 min 70/30–t10.0 min 70/30–t11.0 min 5/95–t16.0 min 5/95–t17.0 min 70/30–t22.0 min 70/30.
Testosterone (40
µ
M final) was used as a positive control for the activity of the
HLM and was separately incubated under the conditions described above (0% EtOH
final). The metabolization was analyzed at distinct time points (10, 20, 30, 40 and 60 min)
with the HPLC system specified above (254 nm, elution profile A/B: t
0 min
55/45–t
10.0 min
55/45–t11.0 min 5/95–t16.0 min 5/95–t17.0 min 55/45–t25.0 min 55/45) and was completed after
>30 min.
2.8. Liver Cytosol Assay
Liver cytosol experiments with [
18
F]
12
were performed according to reported proce-
dures [
45
,
46
]. Incubations had a final volume of 250
µ
L. PBS (125
µ
L) and human (HLC,
Gibco
™
Cat. No. HMCYPL; Lot. No. PL028-J) or rat (RLC, Gibco
™
Cat. No. RTCYPL;
Lot. No. RT062-B) liver cytosol (25
µ
L of 20 mg/mL stock; 2 mg/mL final) were mixed in
a 1.5 mL Eppendorf tube and the mixture was warmed for 5 min at 37
◦
C. Subsequently,
the radiotracer solution (100
µ
L, 1% EtOH and 21 MBq/mL or 2.1
µ
M final) was added
and the mixture was incubated at 37
◦
C. After distinct time points (10, 30 and 60 min), an
aliquot (40
µ
L) was withdrawn and added to ice-cooled CH
3
CN (160
µ
L). The mixture
was vortexed for 30 s, stored on ice for 4 min and centrifuged (5 min at 15,000 rpm). The
resulting supernatant was used for radio-HPLC analysis using the conditions described in
Section 2.3.
Vanilin (40
µ
M final) was used as positive control for the activity of the liver cytosols
and was separately incubated under the conditions described above (1% EtOH final). Its
metabolization and the simultaneous formation of vanilic acid was analyzed at distinct
time points (10, 30 and 60 min) with the HPLC system specified above (280 nm, elution
profile A/B: t
0 min
78/22–t
10.0 min
78/22–t
11.0 min
5/95–t
16.0 min
5/95–t
17.0 min
78/22–t
22.0 min
78/22). More than 80% of the vanilin had been degraded after 60 min in the presence of
HLC, while around 60% of the vanilin had been degraded with RLC. There was no change
in the degradation rate with and without 1% EtOH in the incubation mixture.
2.9. FAP Fluorogenic Assay
To determine the inhibitory capacity, a commercial fluorogenic FAP assay was used
(BPS Bioscience, San Diego, CA, USA, #80210) according to the manufacturer’s instructions.
All tested compounds (
12
,
13
and FAPI-04) were diluted in DMSO. FAPI-04 was syntheti-
cally prepared in a similar manner to that previously reported [
12
] along with comparable
purity and characterization. On the day of testing, a 1:10 dilution in the supplied DPP
assay buffer was performed for all compounds. The final mix on the 96-well plate yielded
10 concentrations of each compound, ranging from 1
µ
M to 50 pM with 1% DMSO (v/v),
carried out in duplicates. The fluorogenic peptide substrate (Ala-Pro-AMC dipeptide) was
added and the reaction started with the addition of the human recombinant FAP (25 ng/
µ
L).
Immediately, the plate was loaded into a multilabel plate reader (Biotek Cytation 5, Agilent,
Bellevue, WA, USA) and each well was measured at an excitation wavelength window
of 360
±
20 nm and an emission wavelength window of 450
±
20 nm. The fluorescence
intensity was measured over 15 min in intervals of 15 s, resulting in 61 data points per curve.
Controls included blanks (omission of substrate, enzyme and inhibitor), negative (omission
of enzyme) and positive controls (omission of inhibitor). The recorded time courses of
the type (RFU
−
RFU0) = f(t) were analyzed with linear regression of the experimental
data over the entire measurement period. The IC
50
value, which is equal to the inhibitor
amount that causes 50% inhibition, and the Hill slope, nH, were calculated according to
Pharmaceutics 2023,15, 2749 7 of 18
E
quation (2)
, with “bottom” and “top” representing the lower and upper plateaus of the
sigmoid dose−response curve, respectively [44].
rate =Bottom +(Top −Bottom)×[I]nH
[I]nH +IC50nH (2)
Equation (2) for analyzing the sigmoid dose–response curve.
2.10. PET Imaging Experiments
All animal experiments were carried out according to the guidelines of the German
Regulation for Animal Welfare. The protocol was approved by the local Ethical Com-
mittee for Animal Experiments. Small-animal PET was performed using the nanoScan
®
PET/CT (Mediso Medical Imaging Systems, Budapest, Hungary). Each animal (BALB/cJRj
mice,
n= 2
) received an intravenous injection of 10 MBq [
18
F]
12
delivered in 0.15 mL of
0
.154 m
ol/L NaCl
aq
through a tail vein catheter. Emission of annihilation photons was
recorded continuously for 2 h. A corresponding X-ray CT image was recorded and used
for anatomical referencing and attenuation correction. List-mode data were sorted into
sinograms with 36 frames (15
×
10 s, 5
×
30 s, 5
×
60 s, 4
×
300 s, 3
×
600 s, 4
×
900 s) and
reconstructed using the Tera-Tomo
™
3D algorithm, applying corrections for decay, scatter
and attenuation. Images were post-processed and analyzed using ROVER (ABX, Radeberg,
Germany) and displayed as maximum-intensity projections (MIP) at indicated the time
points and scaling. Three-dimensional regions of interest (ROI) were created, applying fixed
contrast thresholds for delineation of blood (heart content, 80%), liver (80%), kidneys (50%),
bone (knee and shoulder joints, 50%), gall bladder and intestine (5%) and urinary bladder
(10%). Organ-specific activity concentrations were determined as ROI-averaged standard-
ized uptake values at mid-frame time (SUVmean). Time–activity curves were drawn using
Prism 9.0 (GraphPad Software, La Jolla, CA, USA) and the activity retention within each
organ was reported as the area under the curve (AUC). Excreted activity fractions were
determined as the percent of the initially administered activity dose (%ID).
3. Results
3.1. Organic Synthesis of Radiolabeling Precursors
The intermediate building blocks
18
and
28
were accessed via a previously described
synthetic route [
12
,
47
] and obtained in overall yields of 36% and 1%, respectively
(Scheme 2A,B). The fluorosulfonation of the commercially available phenols
29
and
31
was achieved using AISF (4-(acetylamino)phenyl]imidodisulfuryl difluoride) and DBU as a
base in THF to afford aryl-fluorosulfates
30
and
32
in 9% and 12% yields [
48
], respectively
(Scheme 2C) [
49
]. Thereafter, aryl-fluorosulfates
30
and
32
were coupled with
28
using
HATU and DIPEA in DMF to furnish the final compounds
12
and
13
in 46% and 43% yields,
respectively. Both
12
and
13
serve as radiolabeling precursors and reference compounds
for product identification in the radiosynthesis via [18F]SuFEx.
3.2. Fluorogenic FAP Assays
Employing a commercially available enzyme assay, compounds
12
and
13
along with
a reference (FAPI-04) were probed for their potency to inhibit recombinant human FAP
enzyme activity (Figure 1). Spanning a range of compound concentrations (
50 pM–1 µM
),
fluorescence intensity changes were followed over 15 min. For all compounds, a linear
response in relative fluorescence units (RFU) was observed, with the slope being determined
by the concentration of the inhibitor. This is exemplified by the response of FAPI-04,
provided in the supporting information.
Pharmaceutics 2023,15, 2749 8 of 18
Pharmaceutics 2023, 15, x FOR PEER REVIEW 8 of 19
Scheme 2. (A) Preparation of diuorinated proline building block 18; (B) synthetic route towards
FAPI intermediate 28 and (C) preparation of radiolabeling precursors 12 and 13.
3.2. Fluorogenic FAP Assays
Employing a commercially available enzyme assay, compounds 12 and 13 along with
a reference (FAPI-04) were probed for their potency to inhibit recombinant human FAP
enzyme activity (Figure 1). Spanning a range of compound concentrations (50 pM–1 µM),
uorescence intensity changes were followed over 15 min. For all compounds, a linear
response in relative uorescence units (RFU) was observed, with the slope being deter-
mined by the concentration of the inhibitor. This is exemplied by the response of FAPI-
04, provided in the supporting information.
By using this approach, compounds 12 and 13 were found to inhibit human FAP with
IC50 values of 9.63 and 4.17 nM, respectively (Table 1). These data are comparable to the
IC50 value for the reference compound FAPI-04 determined in the same assay (6.55 nM).
Scheme 2.
(
A
) Preparation of difluorinated proline building block
18
; (
B
) synthetic route towards
FAPI intermediate 28 and (C) preparation of radiolabeling precursors 12 and 13.
By using this approach, compounds
12
and
13
were found to inhibit human FAP with
IC
50
values of 9.63 and 4.17 nM, respectively (Table 1). These data are comparable to the
IC50 value for the reference compound FAPI-04 determined in the same assay (6.55 nM).
Table 1. IC50 values derived from FAPαenzyme inhibition by compounds 12,13 and FAPI-04.
Compound IC50 (nM) Hill Coefficient
12 9.63 (7.55–12.41) −1.56 (−2.43 to −1.059)
13 4.17 (3.01–5.56) −1.27 (−1.98 to −0.91)
FAPI-04 6.55 (4.58–9.35) −1.51 (−2.51 to −0.95)
IC
50
values and Hill coefficients were obtained by analyzing the sigmoid dose–response curves shown in Figure 1
with nonlinear regression using Equation (2) (see Section 2). IC
50
values and Hill coefficients are shown as mean
values with confidence intervals of 95% given in brackets.
Pharmaceutics 2023,15, 2749 9 of 18
Pharmaceutics 2023, 15, x FOR PEER REVIEW 9 of 19
Figure 1. Inhibition of human recombinant FAPα enzyme activity by compounds 12, 13 and FAPI-
04. Data shown are mean values (±SD) of one experiment for each compound, which was performed
in duplicate.
Table 1. IC50 values derived from FAPα enzyme inhibition by compounds 12, 13 and FAPI-04.
Compound IC50 (nM) Hill Coefficient
12 9.63 (7.55–12.41) −1.56 (−2.43 to −1.059)
13 4.17 (3.01–5.56) −1.27 (−1.98 to −0.91)
FAPI-04 6.55 (4.58–9.35) −1.51 (−2.51 to −0.95)
IC50 values and Hill coefficients were obtained by analyzing the sigmoid dose–response curves
shown in Figure 1 with nonlinear regression using Equation (2) (see Section 2). IC50 values and Hill
coefficients are shown as mean values with confidence intervals of 95% given in brackets.
3.3. Preparation of 18F-Labeled Tracers Targeting Fibroblast Activation Protein
The optimization of the radiofluorination was performed utilizing a selection of
phase transfer agents (PTA) in manual radiosyntheses. This covered 18F recovery as a
measure of the separation of [18F]fluoride from target water using the minimalist
approach, as well as the [18F]SuFEx reaction at room temperature by measuring the RCC
value at three time points (Table 2) [50]. Et4NHCO3 was chosen as a very common base in
[18F]fluorinations and as starting point for the developments, while BnBu3NCl and
BnEt3NCl were chosen as more novel PTAs, as introduced by Neumaier et al. for
[18F]SuFEx chemistry [36]. For all three PTAs, high 18F recovery (>90%) from the QMA was
found (Table 2). Radiofluorination was found to proceed fast and within 5 min with all
three PTAs. However, considerable differences were found for isolated activity yields (AY)
comparing Et4NHCO3, a more basic PTA, with the chloride salts BnBu3NCl and BnEt3NCl
(neutral PTAs). The basicity of Et4NHCO3 was found to lead to the decomposition of both
[18F]12 and [18F]13, as reflected by the significant RCC range between the radiolabeling
experiments and lower AY. In a direct comparison, BnBu3NCl was determined to be the
optimum PTA for both 18F recovery (97 ± 1%) and the final AY. Furthermore, the para-
derivative [18F]12 was found to provide a higher RCC compared to its meta-analog [18F]13
with all three PTAs. Under optimized conditions for manual radiosynthesis, [18F]12 and
[18F]13 could be obtained in 55 ± 1% and 43 ± 3% activity yields, respectively, using
BnBu3NCl. Under these conditions and a starting activity of ca. 10 GBq, molar activity
(Am) values of 19.7 GBq/µmol for [18F]12 and 22.6 GBq/µmol for [18F]13 were observed. The
Figure 1.
Inhibition of human recombinant FAP
α
enzyme activity by compounds
12
,
13
and FAPI-04.
Data shown are mean values (
±
SD) of one experiment for each compound, which was performed
in duplicate.
3.3. Preparation of 18F-Labeled Tracers Targeting Fibroblast Activation Protein
The optimization of the radiofluorination was performed utilizing a selection of phase
transfer agents (PTA) in manual radiosyntheses. This covered
18
F recovery as a measure of
the separation of [
18
F]fluoride from target water using the minimalist approach, as well as
the [
18
F]SuFEx reaction at room temperature by measuring the RCC value at three time
points (Table 2) [
50
]. Et
4
NHCO
3
was chosen as a very common base in [
18
F]fluorinations
and as starting point for the developments, while BnBu
3
NCl and BnEt
3
NCl were chosen as
more novel PTAs, as introduced by Neumaier et al. for [
18
F]SuFEx chemistry [
36
]. For all
three PTAs, high
18
F recovery (>90%) from the QMA was found (Table 2). Radiofluorination
was found to proceed fast and within 5 min with all three PTAs. However, considerable
differences were found for isolated activity yields (AY) comparing Et
4
NHCO
3
, a more
basic PTA, with the chloride salts BnBu
3
NCl and BnEt
3
NCl (neutral PTAs). The basicity of
Et
4
NHCO
3
was found to lead to the decomposition of both [
18
F]
12
and [
18
F]
13
, as reflected
by the significant RCC range between the radiolabeling experiments and lower AY. In a
direct comparison, BnBu
3
NCl was determined to be the optimum PTA for both
18
F recovery
(97
±
1%) and the final AY. Furthermore, the para-derivative [
18
F]
12
was found to provide
a higher RCC compared to its meta-analog [
18
F]
13
with all three PTAs. Under optimized
conditions for manual radiosynthesis, [
18
F]
12
and [
18
F]
13
could be obtained in 55
±
1%
and 43
±
3% activity yields, respectively, using BnBu
3
NCl. Under these conditions and a
starting activity of ca. 10 GBq, molar activity (A
m
) values of 19.7 GBq/
µ
mol for [
18
F]
12
and
22.6 GBq/
µ
mol for [
18
F]
13
were observed. The LogD
7.4
value for [
18
F]
12
was determined
by partitioning between phosphate-buffered saline at pH 7.4 (PBS) and octanol to be 1.81.
As part of the work process, the radiosynthesis using Et
4
NHCO
3
as base was trans-
ferred to an automated radiosynthesizer (Figure 2) including separation of [
18
F]fluoride
from target water, solvent evaporation, [
18
F]SuFEx and final SPE purification using an HLB
cartridge. Accordingly, [
18
F]
12
was obtained in 11
±
1% (n= 3) AY starting from 47.4 GBq
furnishing the radiotracer in Amof 53 ±2 GBq/µmol.
Pharmaceutics 2023,15, 2749 10 of 18
Table 2. 18
F Recovery,
18
F incorporation (RCC) and activity yield (AY) using different phase transfer
additive (PTA) salts.
PTA Salt 18F Recovery Compound RCC (%) 30 s RCC (%) 1 min RCC (%) 5 min AY (%)
Et4NHCO393 ±3% (n= 4) [18F]12 41 ±35 * 45 ±33 * 54 ±30 * 33 ±23 *
[18F]13 27 ±5 * 33 ±6 * 48 ±3 * 28 ±2 *
BnEt3NCl 96 ±2% (n= 6) [18F]12 60 ±13 65 ±9 71 ±9 52 ±2
[18F]13 44 ±2 48 ±2 61 ±2 36 ±1
BnBu3NCl 97 ±1% (n= 6) [18F]12 62 ±2 68 ±1 73 ±2 55 ±1
[18F]13 44 ±11 44 ±12 50 ±10 43 ±3
Reaction conditions as described in Section 2. RCC (radiochemical conversion) was analyzed using radio-HPLC.
The AY (activity yield) was obtained by dividing the measured activity of the product by the measured starting
activity of [
18
F]F
−
prior to radiosynthesis and multiplying by one hundred. Data shown are mean values (
±
SD)
and, unless noted otherwise, each experiment was performed in triplicate. * Experiments performed in duplicate.
Pharmaceutics 2023, 15, x FOR PEER REVIEW 10 of 19
LogD7.4 value for [18F]12 was determined by partitioning between phosphate-buffered
saline at pH 7.4 (PBS) and octanol to be 1.81.
Table 2. 18F Recovery, 18F incorporation (RCC) and activity yield (AY) using different phase transfer
additive (PTA) salts.
PTA Salt 18F Recovery Compound RCC (%) 30 s RCC (%) 1 min RCC (%) 5 min AY (%)
Et4NHCO3 93 ± 3% (n = 4) [18F]12 41 ± 35 * 45 ± 33 * 54 ± 30 * 33 ± 23 *
[18F]13 27 ± 5 * 33 ± 6 * 48 ± 3 * 28 ± 2 *
BnEt3NCl 96 ± 2% (n = 6) [18F]12 60 ± 13 65 ± 9 71 ± 9 52 ± 2
[18F]13 44 ± 2 48 ± 2 61 ± 2 36 ± 1
BnBu3NCl 97 ± 1% (n = 6) [18F]12 62 ± 2 68 ± 1 73 ± 2 55 ± 1
[18F]13 44 ± 11 44 ± 12 50 ± 10 43 ± 3
Reaction conditions as described in Section 2. RCC (radiochemical conversion) was analyzed using
radio-HPLC. The AY (activity yield) was obtained by dividing the measured activity of the product
by the measured starting activity of [18F]F- prior to radiosynthesis and multiplying by one hundred.
Data shown are mean values (±SD) and, unless noted otherwise, each experiment was performed in
triplicate. * Experiments performed in duplicate.
As part of the work process, the radiosynthesis using Et4NHCO3 as base was
transferred to an automated radiosynthesizer (Figure 2) including separation of
[18F]fluoride from target water, solvent evaporation, [18F]SuFEx and final SPE purification
using an HLB cartridge. Accordingly, [18F]12 was obtained in 11 ± 1% (n = 3) AY starting
from 47.4 GBq furnishing the radiotracer in Am of 53 ± 2 GBq/µmol.
Figure 2. Overview of the synthesis module TRACERlab FX FDG for the radiosynthesis of [18F]12.
(1, QMA) QMA light carbonate cartridge, (V1) 2.5 mL MeOH, (V2) BnEt3NCl (3 mg in 1.5 mL
MeOH), (V3) radiolabeling precursor (0.1 mg, 0.145 µmol of 12 in 500 µL MeCN), (V6) 7.0 mL H2O,
(V7) 7.0 mL H2O, (V8) 2.0 mL EtOH, (1, HLB) Sep-Pak® HLB, product vial was placed in neighboring
hot cell.
Figure 2.
Overview of the synthesis module TRACERlab FX FDG for the radiosynthesis of [
18
F]
12
. (1,
QMA) QMA light carbonate cartridge, (V1) 2.5 mL MeOH, (V2) BnEt
3
NCl (3 mg in 1.5 mL MeOH),
(
V3
) radiolabeling precursor (0.1 mg, 0.145
µ
mol of
12
in 500
µ
L MeCN), (V6) 7.0 mL H
2
O, (V7) 7.0
mL H
2
O, (V8) 2.0 mL EtOH, (1, HLB) Sep-Pak
®
HLB, product vial was placed in neighboring hot cell.
3.4. Stability Studies of Radiolabeled Compounds
The radiochemical purity and identity of [
18
F]
12
and [
18
F]
13
was analyzed with radio-
HPLC. The comparison to the non-radioactive references
12
and
13
confirmed their iden-
tities and revealed that a radiochemical purity (RCP) of >95% was obtained. Prior to a
biological evaluation, preliminary stability studies of [
18
F]
12
and [
18
F]
13
were performed
in human serum at 37
◦
C (Figure 3A,B). The stability studies were performed by taking
an aliquot of the radiolabeled product solution and incubating the product mixture in the
desired solvent or human serum at 37
◦
C for the given time points. In both cases, decom-
position of [
18
F]
12
and [
18
F]
13
was evident from the first peak in the radioactive channel
Pharmaceutics 2023,15, 2749 11 of 18
(t
R
: 0.6–1.0), most likely due to the release of [
18
F]fluoride, which could be detected using
UHPLC analysis from the first time point of 15 min and continued until 120 min. After
6
0 m
in, approximately 20% of [
18
F]
12
and 19% of [
18
F]
13
had been degraded; following a
120 min incubation, approximately 38% of both [
18
F]
12
and [
18
F]
13
had been degraded. For
comparison, both [
18
F]
12
and [
18
F]
13
have been shown to be stable after 120 min in PBS
(pH 7.4) (see Supplementary Materials Figures S19 and S20).
Pharmaceutics 2023, 15, x FOR PEER REVIEW 11 of 19
3.4. Stability Studies of Radiolabeled Compounds
The radiochemical purity and identity of [18F]12 and [18F]13 was analyzed with radio-
HPLC. The comparison to the non-radioactive references 12 and 13 confirmed their
identities and revealed that a radiochemical purity (RCP) of >95% was obtained. Prior to
a biological evaluation, preliminary stability studies of [18F]12 and [18F]13 were performed
in human serum at 37 °C (Figure 3A,B). The stability studies were performed by taking an
aliquot of the radiolabeled product solution and incubating the product mixture in the
desired solvent or human serum at 37 °C for the given time points. In both cases,
decomposition of [18F]12 and [18F]13 was evident from the first peak in the radioactive
channel (tR: 0.6–1.0), most likely due to the release of [18F]fluoride, which could be detected
using UHPLC analysis from the first time point of 15 min and continued until 120 min.
After 60 min, approximately 20% of [18F]12 and 19% of [18F]13 had been degraded;
following a 120 min incubation, approximately 38% of both [18F]12 and [18F]13 had been
degraded. For comparison, both [18F]12 and [18F]13 have been shown to be stable after 120
min in PBS (pH 7.4) (see Supplementary Materials Figures S19 and S20).
Figure 3. (A) Stability studies of [18F]12 in human serum at 37 °C. (B) Stability studies of [18F]13 in
human serum at 37 °C.
3.5. Liver Microsome Experiments
[18F]12 was furthermore subjected to incubation in the presence of human liver
microsomes (HLM) under oxidative conditions (NADPH) and human (HLC) and rat liver
cytosol (RLC) (Figure 4A). Liver microsome stability studies have shown that [18F]12
appeared to be largely stable toward HLC and RLC up to 60 min (see Supplementary
Materials Figure S23). However, a time-dependent degradation was observed toward
HLM, revealing a half-time of only 4.4 min (Figure 4B). At least three radiolabeled
metabolites could be detected using radio-RP-HPLC analysis, with two of them eluting
even at greater retention times than the parent radiotracer. The control experiment was
performed with the omission of NADPH, and it was found that [18F]12 was apparently
stable in the presence of HLM without oxidative conditions. Of note, liver microsome
experiments were analyzed with radio-HPLC, where [18F]fluoride and, hence, the release
of [18F]fluoride cannot be reliably detected.
Figure 3.
(
A
) Stability studies of [
18
F]
12
in human serum at 37
◦
C. (
B
) Stability studies of [
18
F]
13
in
human serum at 37 ◦C.
3.5. Liver Microsome Experiments
[
18
F]
12
was furthermore subjected to incubation in the presence of human liver micro-
somes (HLM) under oxidative conditions (NADPH) and human (HLC) and rat liver cytosol
(RLC) (Figure 4A). Liver microsome stability studies have shown that [
18
F]
12
appeared to
be largely stable toward HLC and RLC up to 60 min (see Supplementary Materials Figure
S23). However, a time-dependent degradation was observed toward HLM, revealing a
half-time of only 4.4 min (Figure 4B). At least three radiolabeled metabolites could be
detected using radio-RP-HPLC analysis, with two of them eluting even at greater reten-
tion times than the parent radiotracer. The control experiment was performed with the
omission of NADPH, and it was found that [
18
F]
12
was apparently stable in the presence of
HLM without oxidative conditions. Of note, liver microsome experiments were analyzed
with radio-HPLC, where [
18
F]fluoride and, hence, the release of [
18
F]fluoride cannot be
reliably detected.
Pharmaceutics 2023, 15, x FOR PEER REVIEW 12 of 19
normalized signal
fraction of intact [18F]12
Half Life 4.4
Figure 4. (A) Radio-HPLC chromatograms of [18F]12 after incubation with human liver microsomes
(HLM) for the indicated time periods. For “60 min control”, NADPH was omied in the incubations.
(B) Plot of fraction of intact [18F]12 determined with HPLC analysis vs. time including nonlinear
regression according to one-phase decay. The calculated half-time of [18F]12 toward HLM is given
in min in the box. Conditions: 10 mM PBS (pH 7.4), 1 mg/mL HLM, 2 mM NADPH, 16 MBq/mL or
1.6 µM [18F]12, 0.32% EtOH (v/v).
3.6. Distribution of [18F]12 in Mice
As exemplarily investigated for compound [18F]12, small-animal PET showed the in
vivo distribution of the radiofluorinated compound and its putative 18F-containing metab-
olites in mice within 2 h after injection (Figure 5A). Approximately 90% of the initial ac-
tivity concentration in blood (SUVmean of heart content) was cleared within 5 min after
compound injection (Figure 5B). Compound [18F]12 showed fast liver and kidney uptake
with maximum activity concentrations 2 min after injection followed by 90% clearance
from the liver within 82 min and from kidneys within 45 min (Figure 5C,D). Furthermore,
in vivo administration of [18F]12 in mice was followed by continuously increasing activity
concentrations in bones, most likely due to the release of [18F]fluoride during metabolic
turnover (Figure 5E).
Following [18F]12 injection, distribution and metabolic turnover in mice, the major
fraction of the initially delivered activity dose was excreted via the hepatobiliary pathway
(45%), as determined from its retention in gall bladder and intestine (Figure 5F). A con-
siderably smaller fraction was excreted via the renal pathway (10%), as determined from
its retention in urinary bladder (Figure 5G).
Figure 4.
(
A
) Radio-HPLC chromatograms of [
18
F
]12
after incubation with human liver microsomes
(HLM) for the indicated time periods. For “60 min control”, NADPH was omitted in the incubations.
(
B
) Plot of fraction of intact [
18
F]
12
determined with HPLC analysis vs. time including nonlinear
regression according to one-phase decay. The calculated half-time of [
18
F]
12
toward HLM is given in
min in the box. Conditions: 10 mM PBS (pH 7.4), 1 mg/mL HLM, 2 mM NADPH, 16 MBq/mL or
1.6 µM [18F]12, 0.32% EtOH (v/v).
Pharmaceutics 2023,15, 2749 12 of 18
3.6. Distribution of [18 F]12 in Mice
As exemplarily investigated for compound [
18
F]
12
, small-animal PET showed the
in vivo
distribution of the radiofluorinated compound and its putative
18
F-containing
metabolites in mice within 2 h after injection (Figure 5A). Approximately 90% of the initial
activity concentration in blood (SUV
mean
of heart content) was cleared within 5 min after
compound injection (Figure 5B). Compound [
18
F]
12
showed fast liver and kidney uptake
with maximum activity concentrations 2 min after injection followed by 90% clearance
from the liver within 82 min and from kidneys within 45 min
(
Figure 5C,D). Furthermore,
in vivo
administration of [
18
F]
12
in mice was followed by continuously increasing activity
concentrations in bones, most likely due to the release of [
18
F]fluoride during metabolic
turnover (Figure 5E).
Pharmaceutics 2023, 15, x FOR PEER REVIEW 13 of 19
Figure 5. Imaging of [
18
F]12 distribution in BALB/cJRj mice; (A) PET images presented as maximum
intensity projections (prone view) at different time points after compound injection; (B–E) kinetics
of organ-specific activity concentrations presented as region-averaged standardized uptake values
(SUV
mean
); areas under curve (AUC) indicate the overall activity retention within 2 h of observation;
(F,G) kinetics of hepatobiliary and renal excretion presented as percent of initially administered
activity dose.
4. Discussion
A novel class of
18
F-labeled tracers targeting FAP has been accessed via ultra-fast
[
18
F]SuFEx chemistry. Despite the introduction of an aryl-fluorosulfate in compounds 12
and 13, both compounds retained their excellent inhibitory capacity compared to previous
FAP inhibitors. The facile synthetic method towards radiolabeling precursors 12 and 13
and the highly efficient [
18
F]SuFEx radiofluorination approach allowed rapid access to the
radiolabeled compounds [
18
F]12 and [
18
F]13 under mild radiolabeling conditions. In the
context of existing
18
F-fluorination strategies, distinct advantages of the [
18
F]SuFEx radio-
labeling protocol include the omission of metal additives (e.g., Cu-mediator complexes),
the furnishing of structurally diverse radiofluorinated compounds in high AYs and the
simple radiosynthesis translation into commercially available radiosynthesizers. Addi-
tionally, the synthetic preparation of a cold reference compound for radiolabeled product
confirmation via analytical radio-HPLC co-injection is not required, as the radiolabeling
precursor may fulfil this purpose. Upon harnessing the [
18
F]SuFEx chemistry, to the best
of our knowledge, our work presents one of the highest-yielding radiosynthesis routes to
Figure 5.
Imaging of [
18
F]12 distribution in BALB/cJRj mice; (
A
) PET images presented as maximum
intensity projections (prone view) at different time points after compound injection; (
B
–
E
) kinetics
of organ-specific activity concentrations presented as region-averaged standardized uptake values
(SUV
mean
); areas under curve (AUC) indicate the overall activity retention within 2 h of observation;
(
F
,
G
) kinetics of hepatobiliary and renal excretion presented as percent of initially administered
activity dose.
Following [
18
F]
12
injection, distribution and metabolic turnover in mice, the major
fraction of the initially delivered activity dose was excreted via the hepatobiliary pathway
(45%), as determined from its retention in gall bladder and intestine (Figure 5F). A consid-
Pharmaceutics 2023,15, 2749 13 of 18
erably smaller fraction was excreted via the renal pathway (10%), as determined from its
retention in urinary bladder (Figure 5G).
4. Discussion
A novel class of
18
F-labeled tracers targeting FAP has been accessed via ultra-fast
[
18
F]SuFEx chemistry. Despite the introduction of an aryl-fluorosulfate in compounds
12
and
13,
both compounds retained their excellent inhibitory capacity compared to previous
FAP inhibitors. The facile synthetic method towards radiolabeling precursors
12
and
13
and the highly efficient [
18
F]SuFEx radiofluorination approach allowed rapid access to the
radiolabeled compounds [
18
F]
12
and [
18
F]
13
under mild radiolabeling conditions. In the
context of existing 18F-fluorination strategies, distinct advantages of the [18F]SuFEx radio-
labeling protocol include the omission of metal additives (e.g., Cu-mediator complexes),
the furnishing of structurally diverse radiofluorinated compounds in high AYs and the
simple radiosynthesis translation into commercially available radiosynthesizers. Addi-
tionally, the synthetic preparation of a cold reference compound for radiolabeled product
confirmation via analytical radio-HPLC co-injection is not required, as the radiolabeling
precursor may fulfil this purpose. Upon harnessing the [
18
F]SuFEx chemistry, to the best
of our knowledge, our work presents one of the highest-yielding radiosynthesis routes to
an
18
F-labeled FAPI ([
18
F]
12
, 55
±
1% AY) to date [
15
,
36
]. Manual radiosynthesis of both
[
18
F]
12
and [
18
F]
13
required 25 min preparation time, with both radioligands accessed in
>95% RCP. The preparation of [
18
F]
12
using the TRACERlab FX FDG synthesis module
required a preparation time of 50 min and it was obtained at an AY of 11
±
1% (n= 3) and
with >95% RCP.
The molar activity (A
m
) of radiotracers is an important value for PET imaging, as
target-site occupancy and unwanted toxic effects of injected “cold” products can be detri-
mental for furnishing high-resolution PET images and for ensuring patient safety. Thus,
radiofluorinated products with high A
m
values are advantageous, as they minimize the
concentration of unlabeled “carrier” compounds being introduced into a patient. As men-
tioned previously, the A
m
value for
18
F-for-
19
F isotopic-exchange reactions such as the
[
18
F]SuFEx reaction is of particular importance, as the impossibility of separating the ra-
diolabeling precursor (e.g.,
12
) from the radiolabeled product (e.g., [
18
F]
12
) can prove a
challenge for radiochemists [
51
]. Fortunately, high A
m
values for [
18
F]
12
were afforded
in both manual (A
m
: 19.7 GBq/
µ
mol from a starting activity of 10.4 GBq) and automated
(A
m
:
53 ±2 GBq/µmol
from a starting activity of with 47.4 GBq) radiosyntheses. The
relatively high A
m
values were achieved by employing a relatively low concentration
(0
.1 m
g, 0.1
45 µmo
l) of the radiolabeling precursors and commencing the radiosynthesis
with a high concentration of [18F]fluoride, as previously described [36].
In this work, we sought to examine whether the influence of the position of the flu-
orosulfate group on the aromatic ring of the radioligand plays a role in (i) the AY of the
final product and (ii) the overall stability of the radioligand. As of yet, a practical set of
guidelines providing information on the stability of the radiofluorinated aryl-fluorosulfate
([
18
F]ArOSO
2
F) motif has not been fully established. Studies have suggested that the
stability of the aryl-fluorosulfate motif may be dependent on the biovector bearing the
aryl-fluorosulfate [
35
,
36
] and on the presence of activating groups on the aromatic ring
bearing the fluorosulfate [
36
]. Neumaier et al. stated that “highly electron deficient radiola-
beled aryl-fluorosulfates are not sufficiently stable for
in vivo
applications” due to rapid
tracer defluorination (arising from nucleophilic displacement of the [
18
F]fluorosulfate) [
36
].
Moreover, the ability of the fluorosulfate (-OSO
2
F) group to potentially act as a chemical
warhead in a similar manner to a fluorosulfonate (-SO
2
F) motif may present additional
challenges [
52
,
53
], depending on the metabolic pathway of the radioligand. The exposure
of an
18
F-labeled compound bearing a fluorosulfate to proteins with a particular orienta-
tion will result in defluorination via nucleophilic displacement of the [
18
F]fluorosulfate,
reflected by the accumulation of fluorine-18 in the bones and joints in a resulting PET image.
Although the formation of a covalent bond of a radioligand to a target protein may provide
Pharmaceutics 2023,15, 2749 14 of 18
insight into the target binding, this phenomenon is largely detrimental for PET imaging
purposes using radiofluorinated aryl-fluorosulfates [
54
]. Therefore, information relating to
the defluorination rates of PET tracers prepared using [
18
F]SuFEx radiolabeling approaches
is of high significance for future radioligand design.
Extensive stability studies of the aryl-fluorosulfate group have been carried out in
the seminal work by the group of Wu et al. [
35
], where radiolabeled aryl-fluorosulfates
were incubated with nucleophilic amino acids at various pH ranges and in the presence of
oxidizing reagents. However, as the later work of Neumaier et al. discussed, the analytical
method initially reported by the group of Wu using a silica-based SunFire
®
C
18
HPLC
column with an acidic mobile phase (10–100% MeCN in 0.5% TFA) for the determination
of radiochemical conversion (RCC) and stability studies was not ideal, as [
18
F]HF formed
following defluorination and protonation from the acidic media may be adsorbed on the
HPLC column [
36
]. Ultimately, this would give the false impression of higher RCC values
of the radiolabeled products and would additionally overestimate the stability of the
18
F-
labeled products. Attempts to circumvent the instability of aryl-fluorosulfates may be
employed upon the utilization of aryl-sulfamoyl fluorides (Ar-N-SO
2
-F) as alternative
radiolabeling groups [
38
,
55
,
56
], although extensive stability studies of radiolabeled aryl-
sulfamoyl fluorides have not yet been reported.
The results displayed in Table 2show higher RCC and AY values for compound
[
18
F]
12
compared to [
18
F]
13
. Enhanced
18
F-fluorination of [
18
F]
12
was evident within 3
0 s
and consistent over 5 min with all PTAs evaluated. The higher
18
F fluorination rates for
[
18
F]
12
may be due to the electron-withdrawing effect of the amide group in the para-
position. Various PTAs were screened to evaluate their effect on RCC and on the stability
of the resulting radioligands. In contrast to the typically employed K
2
CO
3
/K
2.2.2.
elution
methods [
57
–
59
], Et
4
NHCO
3
, BnEt
3
NCl and BnBu
3
NCl were chosen as advantageous
alternatives in order to circumvent time-consuming azeotropic drying and to minimize
product losses to decomposition under basic conditions [
36
]. It was found that BnBu
3
NCl
afforded the highest
18
F recovery following elution (97
±
1%, n= 6) and furnished both
[18F]12 and [18F]13 in the highest AYs of 55 ±1% and 43 ±3%, respectively (Table 1).
In addition, we performed a series of stability studies of compounds [
18
F]
12
and
[
18
F]
13
in EtOH, phosphate-buffered saline (PBS, pH 7.4) and human serum (37
◦
C) to
provide an indication of the rates of
in vivo
defluorination. The stability studies indicated
that both [
18
F]
12
and [
18
F]
13
are stable in EtOH and PBS for 120 min, but show clear signs of
instability after only 15 min in human serum at 37
◦
C (Figure 3A,B). These findings correlate
with the unwanted accumulation of activity in the bones and joints obtained during PET
imaging experiments in a healthy mice (Figure 5A,B). Furthermore, the SUV
bone
uptake
was shown to be relatively higher for [
18
F]
12
compared to [
18
F]
13
(Figure 5), which would
support the theory that radiolabeled aryl-fluorosulfate PET tracers with electron-deficient
aromatic systems are disadvantageous for imaging applications [36].
However, the relatively higher lipophilicity of the
18
F-labeled compounds ([
18
F]
12
Log
D
7.4
: 1.81) may also be responsible for the high rate of
18
F defluorination, as the higher
lipophilicity of [
18
F]
12
and [
18
F]
13
is shown to promote excretion via the liver compared to
the kidney-excretion pattern observed for more hydrophilic FAPI derivatives (Scheme 1B,
[
18
F]
8
–
10
) [
15
]. Liver microsome stability studies have demonstrated that [
18
F]
12
is mostly
stable toward both HLC and RLC for up to 1 h, but is rapidly degraded upon treatment
with HML (t
1/2
of 4.4 min). Jansen et al. described the sufficient stability of FAPIs with a (4-
quinolinoyl)-glycyl-2-cyanopyrrolidine scaffold in microsome preparations, with half-lives
greater than 6 h (even 24 h) [
47
,
60
]. The signals of the radiometabolites in the radioactivity
channels provide evidence that the radiolabeled fluorosulfate motif is still intact, and would
therefore indicate that the radiometabolites observed herein originate from transformations
at the benzoyl-piperazine-alkyl moiety. These transformations might include hydroxylation
at the piperazine ring and probably also N-oxygenation at the tertiary amine by FMOs,
which are also present in microsome preparations [
61
]. As the quinoline system is a known
substrate motif for aldehyde oxidase (AO), which can lead to oxidation in ortho- and/or
Pharmaceutics 2023,15, 2749 15 of 18
para-position to the nitrogen, we studied stability in liver cytosols, which are a source of
AO [62]. Interestingly, [18F]12 was found not to be a substrate of AO.
Taken together, our findings suggest the unsuitability of the radiolabeled aryl-
fluorosulfates [
18
F]
12
and [
18
F]
13
for FAPI-PET imaging and indicate that alternating
the substitution pattern of the aryl-fluorosulfate has slight advantages regarding stability.
Notably, Toms et al. have shown that a high amount of activity concentration in the bones
and joints of mice was also evident following the radiofluorination of a FAPI derivative
via [
18
F]fluoroglycosylation [
63
], although this was shown to be reduced following ap-
propriate blocking ([
18
F]FGlc-FAPI: 6.0
±
2.5% ID/g vs. blocking: 0.36
±
0.06% ID/g)
with a non-radiolabeled FAP-targeting alkyne derivative. Due to the relatively strong
[
18
F]C-F bond present in the [
18
F]fluoroglycosylation radiolabeling approach, it is unlikely
that defluorination of the radiolabeled compound occurred, and this may indicate that
the intact radioligand is accumulating in the bones and joints through potential binding
to bone marrow fibroblasts. In the context of our findings, this could indicate that both
defluorination and radioligand accumulation in the bones and joints are occurring and
therefore, it is non-trivial to determine the extent of the fluorosulfate decomposition using
the FAPI derivative biovector using PET imaging experiments.
5. Conclusions
In conclusion, the rapid preparation of two
18
F-labeled tracers targeting FAP was
achieved by harnessing the ultra-fast reaction kinetics of the [18F]SuFEx reaction. Further-
more, the purification of the final radiofluorinated products with SPE, thereby avoiding
time-consuming semi-preparative HPLC purification, is a distinct advantage of this proto-
col compared to contemporary literature preparations and facilitated the implementation
of the radiosynthetic method into a commercially available automated radiosynthesizer.
As far as the authors are aware, our work presents one of the highest activity yields for a
radiofluorinated FAPI reported thus far. Notably, the work sheds light on the stability of the
[
18
F]aryl-fluorosulfate motif, with a comparison of our findings with the existing literature
being discussed. We believe our findings will aid and accelerate further applications of the
advantageous [18F]SuFEx chemistry towards PET tracer development.
Supplementary Materials:
The following supporting information can be downloaded at: https://
www.mdpi.com/article/10.3390/pharmaceutics15122749/s1, Experimental Procedures for Organic
Syntheses, Copies of HPLC and NMR data and FAP assay data are provided within the supporting
information. Detailed synthetic procedures and characterization data for the preparation of aryl-
fluorosulfate FAPIs; Figures S1–S6: NMR data of aryl-fluorosulfate FAPIs; Figures S7 and S8: HR-MS
chromatograms of aryl-fluorosulfate FAPIs; Figures S9 and S10: Analytical HPLC chromatograms
of aryl-fluorosulfate FAPIs. Tables S1–S5: Optimization data for preparation of radiolabeled aryl-
fluorosulfate FAPIs. Figures S11–S20: Radio-(U)HPLC chromatograms of radiolabeled aryl-fluorosulfate
FAPIs. Figures S21 and S22: Calibration curves for the molar activity determination of radiolabeled
aryl-fluorosulfate FAPIs. Figure S23: Radioactivity-detected HPLC chromatograms of a radiolabeled
aryl-fluorosulfate FAPI after incubation with human and rat liver cytosol. Table S6: Composition
of Supersol solubilizing agent. Figure S24: Linearity of fluorescence increase, catalyzed by human
recombinant FAPαprocessing of a peptide substrate.
Author Contributions:
Conceptualization, A.C., K.K. and S.S.; methodology, A.C., J.K., M.L., M.U.
and R.W.; investigation, A.C., J.K., M.U., R.W. and C.K.D.; resources, K.K. and S.S.; writing—original
draft preparation, A.C.; writing—review and editing, K.K., M.L. and S.S; visualization, A.C.; supervi-
sion, K.K. and S.S.; project administration, A.C., K.K. and S.S.; funding acquisition, K.K. and S.S. All
authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data are contained within the article and Supplementary Material.
Pharmaceutics 2023,15, 2749 16 of 18
Acknowledgments:
This work was presented at the 24th International Symposium on Radiopharma-
ceutical Sciences in Nantes, France. The authors are grateful for the support of Martin Kreller and the
cyclotron team for the provision of the fluorine-18 required for this work. Furthermore, the authors
wish to express their gratitude to the head and staff of the animal research facility, Birgit Belter, Katrin
Baumgart and Helge Gläser.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
Willmann, J.K.; Van Bruggen, N.; Dinkelborg, L.M.; Gambhir, S.S. Molecular Imaging in Drug Development. Nat. Rev. Drug
Discov. 2008,7, 591–607. [CrossRef] [PubMed]
2.
Ametamey, S.M.; Honer, M.; Schubiger, P.A. Molecular Imaging with PET. Chem. Rev.
2008
,108, 1501–1516. [CrossRef] [PubMed]
3. Tsien, R.Y. Imagining Imaging’s Future. Nat. Rev. Mol. Cell Biol. 2003,8, SS16–SS21.
4.
Fowler, J.S.; Wolf, A.P. Working against Time: Rapid Radiotracer Synthesis and Imaging the Human Brain. Acc. Chem. Res.
1997
,
30, 181–188. [CrossRef]
5.
Phelps, M.E. Positron Emission Tomography Provides Molecular Imaging of Biological Processes. Proc. Natl. Acad. Sci. USA
2000
,
97, 9226–9233. [CrossRef]
6. Gambhir, S.S. Molecular Imaging of Cancer with Positron Emission Tomography. Nat. Rev. Cancer 2002,2, 683–693. [CrossRef]
7.
Mori, Y.; Haberkorn, U.; Giesel, F.L.
68
Ga- or
18
F-FAPI PET/CT—What It Can and Cannot. Eur. Radiol.
2023
,33, 7877–7878.
[CrossRef]
8.
Gascard, P.; Tlsty, T.D. Carcinoma-Associated Fibroblasts: Orchestrating the Composition of Malignancy. Genes Dev.
2016
,30,
1002–1019. [CrossRef]
9.
Lamprecht, S.; Sigal-Batikoff, I.; Shany, S.; Abu-Freha, N.; Ling, E.; Delinasios, G.J.; Moyal-Atias, K.; Delinasios, J.G.; Fich, A.
Teaming Up for Trouble: Cancer Cells, Transforming Growth Factor-B1 Signaling and the Epigenetic Corruption of Stromal Naïve
Fibroblasts. Cancers 2018,10, 61. [CrossRef]
10.
Giesel, F.L.; Kratochwil, C.; Lindner, T.; Marschalek, M.M.; Loktev, A.; Lehnert, W.; Debus, J.; Jäger, D.; Flechsig, P.; Altmann, A.;
et al.
68
Ga-FAPI PET/CT: Biodistribution and Preliminary Dosimetry Estimate of 2 DOTA-Containing FAP-Targeting Agents in
Patients with Various Cancers. J. Nucl. Med. 2019,60, 386–392. [CrossRef]
11.
Kratochwil, C.; Flechsig, P.; Lindner, T.; Abderrahim, L.; Altmann, A.; Mier, W.; Adeberg, S.; Rathke, H.; Röhrich, M.; Winter, H.;
et al. 68Ga-FAPI PET/CT: Tracer Uptake in 28 Different Kinds of Cancer. J. Nucl. Med. 2019,60, 801–805. [CrossRef]
12.
Lindner, T.; Loktev, A.; Altmann, A.; Giesel, F.; Kratochwil, C.; Debus, J.; Jäger, D.; Mier, W.; Haberkorn, U. Development of
Quinoline-Based Theranostic Ligands for the Targeting of Fibroblast Activation Protein. J. Nucl. Med.
2018
,59, 1415–1422.
[CrossRef]
13.
Escudero-Castellanos, A.; Kurth, J.; Imlimthan, S.; Menéndez, E.; Pilatis, E.; Moon, E.S.; Läppchen, T.; Rathke, H.; Schwarzenböck,
S.M.; Krause, B.J.; et al. Translational Assessment of a DATA-Functionalized FAP Inhibitor with Facile
68
Ga-Labeling at Room
Temperature. Eur. J. Nucl. Med. Mol. Imaging 2023,50, 3202–3213. [CrossRef]
14.
Loktev, A.; Lindner, T.; Mier, W.; Debus, J.; Altmann, A.; Jäger, D.; Giesel, F.; Kratochwil, C.; Barthe, P.; Roumestand, C.; et al. A
Tumor-Imaging Method Targeting Cancer-Associated Fibroblasts. J. Nucl. Med. 2018,59, 1423–1429. [CrossRef]
15.
Lindner, T.; Altmann, A.; Giesel, F.; Kratochwil, C.; Kleist, C.; Krämer, S.; Mier, W.; Cardinale, J.; Kauczor, H.-U.; Jäger, D.; et al.
18F-Labeled Tracers Targeting Fibroblast Activation Protein. EJNMMI Radiopharm. Chem. 2021,6, 26. [CrossRef] [PubMed]
16.
Cunha, L.; Horvath, I.; Ferreira, S.; Lemos, J.; Costa, P.; Vieira, D.; Veres, D.S.; Szigeti, K.; Summavielle, T.; Máthé, D.; et al.
Preclinical Imaging: An Essential Ally in Modern Biosciences. Mol. Diagn. Ther. 2014,18, 153–173. [CrossRef] [PubMed]
17.
Ido, T.; Wan, C.-N.; Casella, V.; Fowler, J.S.; Wolf, A.P.; Reivich, M.; Kuhl, D.E. Labeled 2-Deoxy-D-Glucose Analogs.
18
F-Labeled
2-Deoxy-2-Fluoro-D-Glucose, 2-Deoxy-2-Fluoro-D-Mannose and 14C-2-Deoxy-2-Fluoro-D-Glucose. J. Label. Compd. Radiopharm.
1978,14, 175–183. [CrossRef]
18.
Halder, R.; Ritter, T.
18
F-Fluorination: Challenge and Opportunity for Organic Chemists. J. Org. Chem.
2021
,86, 13873–13884.
[CrossRef]
19.
Clarke, B.N. PET Radiopharmaceuticals: What’s New, What’s Reimbursed, and What’s Next? J. Nucl. Med. Technol.
2018
,46,
12–16. [CrossRef] [PubMed]
20.
Chen, W.; Huang, Z.; Tay, N.E.S.; Giglio, B.; Wang, M.; Wang, H.; Wu, Z.; Nicewicz, D.A.; Li, Z. Direct Arene C-H Fluorination
with 18F- via Organic Photoredox Catalysis. Science 2019,364, 1170–1174. [CrossRef]
21.
Craig, A.; Kolks, N.; Urusova, E.A.; Zischler, J.; Brugger, M.; Endepols, H.; Neumaier, B.; Zlatopolskiy, B.D. Preparation of
Labeled Aromatic Amino Acids via Late-Stage
18
F-Fluorination of Chiral Nickel and Copper Complexes. Chem. Commun.
2020
,
56, 9505–9508. [CrossRef]
22.
Graham, T.J.A.; Lambert, R.F.; Ploessl, K.; Kung, H.F.; Doyle, A.G. Enantioselective Radiosynthesis of Positron Emission
Tomography (PET) Tracers Containing [18F]Fluorohydrins. J. Am. Chem. Soc. 2014,136, 5291–5294. [CrossRef] [PubMed]
23.
Huiban, M.; Tredwell, M.; Mizuta, S.; Wan, Z.; Zhang, X.; Collier, T.L.; Gouverneur, V.; Passchier, J. A Broadly Applicable
[18F]Trifluoromethylation of Aryl and Heteroaryl Iodides for PET Imaging. Nat. Chem. 2013,5, 941–944. [CrossRef] [PubMed]
Pharmaceutics 2023,15, 2749 17 of 18
24.
Ichiishi, N.; Brooks, A.F.; Topczewski, J.J.; Rodnick, M.E.; Sanford, M.S.; Scott, P.J.H. Copper-Catalyzed [
18
F]Fluorination of
(Mesityl)(Aryl)Iodonium Salts. Org. Lett. 2014,16, 3224–3227. [CrossRef]
25.
Lee, E.; Kamlet, A.S.; Powers, D.C.; Neumann, C.N.; Boursalian, G.B.; Furuya, T.; Choi, D.C.; Hooker, J.M.; Ritter, T. A Fluoride-
Derived Electrophilic Late-Stage Fluorination Reagent for PET Imaging. Science 2011,334, 639–642. [CrossRef]
26.
Lee, S.J.; Makaravage, K.J.; Brooks, A.F.; Scott, P.J.H.; Sanford, M.S. Copper-Mediated Aminoquinoline-Directed Radiofluorination
of Aromatic C–H Bonds with K18F. Angew. Chem. Int. Ed. 2019,58, 3119–3122. [CrossRef] [PubMed]
27. Makaravage, K.J.; Brooks, A.F.; Mossine, A.V.; Sanford, M.S.; Scott, P.J.H. Copper-Mediated Radiofluorination of Arylstannanes
with [18F]KF. Org. Lett. 2016,18, 5440–5443. [CrossRef]
28.
Mossine, A.V.; Brooks, A.F.; Makaravage, K.J.; Miller, J.M.; Ichiishi, N.; Sanford, M.S.; Scott, P.J.H. Synthesis of [
18
F]Arenes via the
Copper-Mediated [18F]Fluorination of Boronic Acids. Org. Lett. 2015,17, 5780–5783. [CrossRef]
29.
Neumann, C.N.; Hooker, J.M.; Ritter, T. Concerted Nucleophilic Aromatic Substitution with
19
F
−
and
18
F
−
.Nature
2016
,534,
369–373. [CrossRef]
30.
Rotstein, B.H.; Stephenson, N.A.; Vasdev, N.; Liang, S.H. Spirocyclic Hypervalent Iodine(III)-Mediated Radiofluorination of
Non-Activated and Hindered Aromatics. Nat. Commun. 2014,5, 4365. [CrossRef]
31.
Levin, M.D.; Chen, T.Q.; Neubig, M.E.; Hong, C.M.; Theulier, C.A.; Kobylianskii, I.J.; Janabi, M.; O’Neil, J.P.; Toste, F.D. A Catalytic
Fluoride-Rebound Mechanism for C(Sp3)-CF3Bond Formation. Science 2017,356, 1272–1276. [CrossRef]
32.
Zischler, J.; Kolks, N.; Modemann, D.; Neumaier, B.; Zlatopolskiy, B.D. Alcohol-Enhanced Cu-Mediated Radiofluorination.
Chemistry 2017,23, 3251–3256. [CrossRef] [PubMed]
33.
Beyzavi, H.; Mandal, D.; Strebl, M.G.; Neumann, C.N.; D’Amato, E.M.; Chen, J.; Hooker, J.M.; Ritter, T.
18
F-Deoxyfluorination of
Phenols via Ru π-Complexes. ACS Cent. Sci. 2017,3, 944–948. [CrossRef] [PubMed]
34.
Bowden, G.D.; Chailanggar, N.; Pichler, B.J.; Maurer, A. Scalable
18
F Processing Conditions for Copper-Mediated Radiofluorination
Chemistry Facilitate DoE Optimization Studies and Afford an Improved Synthesis of [
18
F]Olaparib. Org. Biomol. Chem.
2021
,19,
6995–7000. [CrossRef]
35.
Zheng, Q.; Xu, H.; Wang, H.; Du, W.-G.H.; Wang, N.; Xiong, H.; Gu, Y.; Noodleman, L.; Sharpless, K.B.; Yang, G.; et al. Sulfur
[
18
F]Fluoride Exchange Click Chemistry Enabled Ultrafast Late-Stage Radiosynthesis. J. Am. Chem. Soc.
2021
,143, 3753–3763.
[CrossRef] [PubMed]
36.
Walter, N.; Bertram, J.; Drewes, B.; Bahutski, V.; Timmer, M.; Schütz, M.B.; Krämer, F.; Neumaier, F.; Endepols, H.; Neumaier, B.;
et al. Convenient PET-Tracer Production via SuFEx
18
F-Fluorination of Nanomolar Precursor Amounts. Eur. J. Med. Chem.
2022
,
237, 114383. [CrossRef] [PubMed]
37.
Craig, A.; Kogler, J.; Krutzek, F.; Brandt, F.; Laube, M.; Ullrich, M.; Donat, C.K.; Kopka, K.; Stadlbauer, S. Sulfur [
18
F]Fluoride
Exchange Reaction Enables Rapid Access to 18F-Labeled PET Tracers. Med. Sci. Forum 2022,14, 127. [CrossRef]
38.
Kim, M.P.; Cho, H.; Kayal, S.; Jeon, M.H.; Seo, J.K.; Son, J.; Jeong, J.; Hong, S.Y.; Chun, J.-H. Direct
18
F-Fluorosulfurylation of
Phenols and Amines Using an [18F]FSO2+Transfer Agent Generated In Situ. J. Org. Chem. 2023,88, 6263–6273. [CrossRef]
39.
Kreller, M.; Pietzsch, H.; Walther, M.; Tietze, H.; Kaever, P.; Knieß, T.; Füchtner, F.; Steinbach, J.; Preusche, S. Introduction of
the New Center for Radiopharmaceutical Cancer Research at Helmholtz-Zentrum Dresden-Rossendorf. Instruments
2019
,3, 9.
[CrossRef]
40.
Coenen, H.H.; Gee, A.D.; Adam, M.; Antoni, G.; Cutler, C.S.; Fujibayashi, Y.; Jeong, J.M.; Mach, R.H.; Mindt, T.L.; Pike, V.W.; et al.
Consensus Nomenclature Rules for Radiopharmaceutical Chemistry—Setting the Record Straight. Nucl. Med. Biol.
2017
,55, v–xi.
[CrossRef]
41.
Laube, M.; Frizler, M.; Wodtke, R.; Neuber, C.; Belter, B.; Kniess, T.; Bachmann, M.; Gütschow, M.; Pietzsch, J.; Löser, R. Synthesis
and Preliminary Radiopharmacological Characterisation of an
11
C-labelled Azadipeptide Nitrile as Potential PET Tracer for
Imaging of Cysteine Cathepsins. Label. Comp. Radiopharm. 2019,62, 448–459. [CrossRef] [PubMed]
42.
Linclau, B.; Wang, Z.; Compain, G.; Paumelle, V.; Fontenelle, C.Q.; Wells, N.; Weymouth-Wilson, A. Investigating the Influence
of (Deoxy)Fluorination on the Lipophilicity of Non-UV-Active Fluorinated Alkanols and Carbohydrates by a New Log P.
Determination Method. Angew. Chem. Int. Ed. 2016,55, 674–678. [CrossRef] [PubMed]
43.
Laube, M.; Gassner, C.; Neuber, C.; Wodtke, R.; Ullrich, M.; Haase-Kohn, C.; Löser, R.; Köckerling, M.; Kopka, K.; Kniess, T.; et al.
Deuteration versus Ethylation—Strategies to Improve the Metabolic Fate of an
18
F-Labeled Celecoxib Derivative. RSC Adv.
2020
,
10, 38601–38611. [CrossRef]
44.
Wodtke, R.; Hauser, C.; Ruiz-Gómez, G.; Jäckel, E.; Bauer, D.; Lohse, M.; Wong, A.; Pufe, J.; Ludwig, F.-A.; Fischer, S.; et al.
N
ε
-Acryloyllysine Piperazides as Irreversible Inhibitors of Transglutaminase 2: Synthesis, Structure–Activity Relationships, and
Pharmacokinetic Profiling. J. Med. Chem. 2018,61, 4528–4560. [CrossRef]
45.
Obach, R.S.; Huynh, P.; Allen, M.C.; Beedham, C. Human Liver Aldehyde Oxidase: Inhibition by 239 Drugs. J. Clin. Pharmacol.
2004,44, 7–19. [CrossRef] [PubMed]
46.
Sahi, J.; Khan, K.; Black, C. Aldehyde Oxidase Activity and Inhibition in Hepatocytes and Cytosolic Fraction from Mouse, Rat,
Monkey and Human. Drug Metab. Lett. 2008,2, 176–183. [CrossRef] [PubMed]
47.
Jansen, K.; Heirbaut, L.; Verkerk, R.; Cheng, J.D.; Joossens, J.; Cos, P.; Maes, L.; Lambeir, A.-M.; De Meester, I.; Augustyns, K.;
et al. Extended Structure–Activity Relationship and Pharmacokinetic Investigation of (4-Quinolinoyl)Glycyl-2-Cyanopyrrolidine
Inhibitors of Fibroblast Activation Protein (FAP). J. Med. Chem. 2014,57, 3053–3074. [CrossRef]