Mapping Aldehyde Dehydrogenase 1A1 Activity using an [18F]Substrate-Based Approach

Article abstract of DOI:10.1002/chem.201805473

Aldehyde dehydrogenases (ALDHs) catalyze the oxidation of aldehydes to carboxylic acids. Elevated ALDH expression in human cancers is linked to metastases and poor overall survival. Despite ALDH being a poor prognostic factor, the non-invasive assessment of ALDH activity in vivo has not been possible due to a lack of sensitive and translational imaging agents. Presented in this report are the synthesis and biological evaluation of ALDH1A1-selective chemical probes composed of an aromatic aldehyde derived from N,N-diethylamino benzaldehyde (DEAB) linked to a fluorinated pyridine ring either via an amide or amine linkage. Of the focused library of compounds evaluated, N-ethyl-6-(fluoro)-N-(4-formylbenzyl)nicotinamide 4 b was found to have excellent affinity and isozyme selectivity for ALDH1A1 in vitro. Following ¹⁸F-fluorination, [¹⁸F]4 b was taken up by colorectal tumor cells and trapped through the conversion to its ¹⁸F-labeled carboxylate product under the action of ALDH. In vivo positron emission tomography revealed high uptake of [¹⁸F]4 b in the lungs and liver, with radioactivity cleared through the urinary tract. Oxidation of [¹⁸F]4 b, however, was observed in vivo, which may limit the tissue penetration of this first-in-class radiotracer.

Full text of DOI:10.1002/chem.201805473

DOI: 10.1002/chem.201805473
Full Paper
&
Biochemistry
Mapping Aldehyde Dehydrogenase 1A1 Activity using an
[18F]Substrate-Based Approach
Raul Pereira,[a] Thibault Gendron,[b] Chandan Sanghera,[a] Hannah E. Greenwood,[a]
Joseph Newcombe,[b, c] Patrick N. McCormick,[a] Kerstin Sander,[b] Maya Topf,[c] Erik ꢀrstad,[b]
and Timothy H. Witney*[a]
Abstract: Aldehyde dehydrogenases (ALDHs) catalyze the
oxidation of aldehydes to carboxylic acids. Elevated ALDH
expression in human cancers is linked to metastases and
poor overall survival. Despite ALDH being a poor prognostic
factor, the non-invasive assessment of ALDH activity in vivo
has not been possible due to a lack of sensitive and transla-
tional imaging agents. Presented in this report are the syn-
thesis and biological evaluation of ALDH1A1-selective chemi-
cal probes composed of an aromatic aldehyde derived from
N,N-diethylamino benzaldehyde (DEAB) linked to a fluorinat-
ed pyridine ring either via an amide or amine linkage. Of the
focused library of compounds evaluated, N-ethyl-6-(fluoro)-
N-(4-formylbenzyl)nicotinamide 4b was found to have excel-
lent affinity and isozyme selectivity for ALDH1A1 in vitro.
Following 18F-fluorination, [18F]4b was taken up by colorectal
tumor cells and trapped through the conversion to its 18F-la-
beled carboxylate product under the action of ALDH. In vivo
positron emission tomography revealed high uptake of
[18F]4b in the lungs and liver, with radioactivity cleared
through the urinary tract. Oxidation of [18F]4b, however, was
observed in vivo, which may limit the tissue penetration of
this first-in-class radiotracer.
Introduction
tion, ALDHs control the detoxification of exogenous reactive
aldehydes and therapeutic drugs such as cyclophosphamide.[6]
Aberrant expression of ALDH is associated with many diseases,
including cancer, with increased expression and activity of
ALDH shown to be a predictor of metastatic potential and
poor overall survival.[7] In particular, the ALDH1A1 isozyme is a
well-characterized marker of cancer stem cells, which are
known for their tumor-initiating properties and resistance to
conventional therapy.[8] Studies have shown resistance to che-
motherapy and poor prognosis is associated with high
ALDH1A1 activity in breast,[9] ovarian,[10] prostate,[11] colon[12]
and lung[13] cancer. As a consequence, ALDH1A1 has been se-
lected as a target for anti-cancer therapy, with ALDH inhibitors
shown to reverse chemoresistance in a range of preclinical
tumor models.[14]
Aldehyde dehydrogenases (ALDHs) are a family of enzymes
that catalyze the NAD(P)+-dependent oxidation of a wide vari-
ety of aldehydes to their corresponding carboxylic acids.[1]
There are currently 20 known functional human ALDHs[2] that
mediate the metabolism of aldehydes generated during oxida-
tive stress,[3] amino acid and biogenic amine metabolism,[4] reti-
noic acid biosynthesis,[5] and ethanol metabolism.[3a] In addi-
[a] Dr. R. Pereira, C. Sanghera, H. E. Greenwood, Dr. P. N. McCormick,
Dr. T. H. Witney
Centre for Advanced Biomedical Imaging
University College London
Paul O’Gorman Building, 72 Huntley Street
London WC1E 6DD (UK),
and
Given the causal link between ALDH expression and cancer
drug resistance, the non-invasive identification of ALDH-ex-
pressing tumors is of great clinical importance. The measure-
ment of chemoresistance through ALDH imaging could poten-
tially enable the clinician to select the most suitable therapeu-
tic intervention for the individual patient (e.g. chemotherapy
versus immunotherapy) with the possibility to improve out-
comes and reduce unnecessary treatment. Currently, the in
vitro assessment of ALDH activity has been restricted to fluo-
rescence-based assays.[15] Despite these commercially available
imaging agents being widely-adopted for the isolation of
ALDH-positive cells in cell culture, the poor tissue penetration
of the fluorescent signal currently limits their in vivo utility. In
order to circumvent these inherent limitations, we propose the
use of positron emission tomography (PET) as an alternative to
Current address: Department of Imaging Chemistry & Biology
King’s College London, St. Thomas’ Hospital
London SE1 7EH (UK)
E-mail: tim.witney@kcl.ac.uk
[b] Dr. T. Gendron, J. Newcombe, Dr. K. Sander, Prof. Dr. E. ꢀrstad
Department of Chemistry
University College London
20 Gordon Street
London WC1H 0AJ (UK)
[c] J. Newcombe, Prof. Dr. M. Topf
Department of Biological Sciences
Birkbeck, University of London
Malet Street
London WC1E 7HX (UK)
Supporting information and the ORCID identification number(s) for the au-
thor(s) of this article can be found under:
https://doi.org/10.1002/chem.201805473.
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fluorescence-based assays.[16] Previous attempts to develop
ALDH1A1-specific radiotracers have so far failed due to the
poor cellular retention of the carboxylate product, presumed
to be a consequence of its high hydrophobicity.[17] Here, we
report the synthesis and biological evaluation of 18F-fluorinated
aldehyde-based probes for the non-invasive detection of
ALDH1A1 activity in tumor cell models.
using recombinant human ALDH1A1, ALDH2 and ALDH3A1 en-
zymes, which are commonly expressed in human cancer.
ALDH2 is expressed in the mitochondrial matrix and plays a
critical role in alcohol metabolism,[19] whereas ALDH3A1 is lo-
calized in both the nucleus and cytosol and functions to detox-
ify aldehydes formed during UV-induced lipid peroxidation.[20]
Furthermore, these three isoforms exhibit three different rate-
limiting steps: ALDH1A1’s being the cofactor dissociation,[21]
the deacylation-step for ALDH2,[22] and hydride transfer for
ALDH3A1.[23]
Results and Discussion
We made a single-point modification to 2, with the addition
of a fluorine to the aromatic ring—an essential requirement
for 18F-radiofluorine-based radiotracers—to give compounds
3a and 3b. The lithium/bromine exchange on 11 followed by
quenching with DMF afforded aldehyde 12. Stirring 12 with
NaBH(OAc)3 and diethylamine in DCE afforded the crude re-
ductive amination product which was directly treated with
aqueous hydrochloric acid in THF to furnish 3a (Scheme 1A).
Reduction of nitrile 13 with diisobutylaluminium hydride clean-
ly produced aldehyde 14 which was thereafter stirred with
excess diethylamine in THF to yield aldehyde 3b (Scheme 1B).
Amide 16 was prepared by reacting the acid chloride of 5-fluo-
ronicotinic acid 15 with amine B (Scheme 1C). The acid-cata-
lyzed cleavage of the acetal furnished compound 4a. LiAlH4 re-
duction of the amide bond in 16 followed by the acid-cata-
lyzed acetal cleavage afforded amine 5 (Scheme 1C). In a simi-
lar manner, the acid chloride of 6-fluronicotinic acid 16 was re-
acted with amine A to yield amide 18 which following an acid-
catalyzed acetal cleavage furnished aldehyde 4b. The sulfona-
mide 6 was accessed by reacting the commercially available 5-
fluoropyridine-3-sulfonyl chloride 19 with amine A following
an acid-catalyzed acetal cleavage (Scheme 1E).
ALDH1A1 chemical probes were designed to have a) an alde-
hyde that can serve as a substrate for ALDH1A1; b) contain a
(radio)fluorine atom that would allow for detection via gamma
counting/PET imaging; c) a suitable hydrophobic-hydrophilic
balance which would allow for passive diffusion in and out of
cells, and importantly; d) subsequent trapping of the in situ
generated carboxylic acid product within the cytosol as a
result of the acquired negative charge (Figure 1A). We took a
substrate-based approach for the imaging of ALDH1A1 to pro-
vide a functional readout of enzymatic activity. Substrate-
based radiotracers provide an advantage over radiolabeled in-
hibitors which only report on enzyme expression. Moreover,
multiple substrate molecules can be turned over by a single
enzyme, thereby increasing the sensitivity of detection when
compared to radiolabeled inhibitors.
The benzylic amine 2 showed a higher affinity (lower KM)
and catalytic efficiency (Vmax/KM) for ALDH1A1 over both
ALDH2 and ALDH3A1 (Table 1, entry 1 and Figure 2B, respec-
tively), indicating that the isozyme selectivity of DEAB was
maintained. Interestingly, 3a and 3b exhibited lower affinity
for ALDH1A1 than the non-fluorinated analogue 2, with a KM
of 0.28Æ0.12 mm, 0.26Æ0.08 mm and 0.16Æ0.03 mm, respec-
tively (Table 1, entries 1–3).
Figure 1. A) Schematic illustrating ALDH-mediated trapping of 18F-labeled al-
dehydes by conversion to their corresponding acid. B) Chemical structures
of DEAB (1), an ALDH1A1 inhibitor, and 2, an ALDH1A1 substrate.
Given that fluorination proximal to the aldehyde moiety de-
creased affinity for ALDH1A1, we next explored compounds
with fluorine atoms that were remote from the aldehyde. Com-
pounds 4a and 4b exhibited a five-fold increase in ALDH1A1
affinity over 2 (Table 1, entries 1, 4 and 5). Furthermore, the
enzyme efficiency for 4a was 7-fold higher for ALDH1A1 than
ALDH2, with the enzyme efficiency for ALDH1A1 over 20-fold
higher with respect to ALDH3A1; that is, the linking of the pyri-
dine via an amide bond resulted in improved selectivity for
ALDH1A1 (Figure 2B; Table 1, entry 4). The position of the fluo-
rine on the pyridine ring crucially did not play a key role in
substrate kinetics, as seen with amide 4b which exhibited
analogous behavior to 4a, albeit with marginally decreased
ALDH1A1 enzyme selectivity (Table 1, entry 5). In the absence
of the amide linkage, the tertiary amine 5, whilst exhibiting a
similar ALDH1A1 binding profile as compounds 4a and 4b,
was readily oxidized by ALDH2 when compared to the other
The starting point for our small molecule probe develop-
ment was N,N-diethylaminobenzaldehyde (DEAB) 1, which is a
well-known inhibitor of ALDH1A1 (Ki values of 10–40 nm).[18]
Given the advantages of substrate-based radiotracers, our ini-
tial goal was to convert 1 in to a substrate whilst maintaining
suitable ALDH1A1 selectivity over other commonly expressed
ALDH isozymes. 1 is thought to form a stalled acyl-enzyme in-
termediate as a result of the delocalization of the electron lone
pair of the para-substituted amine into the aromatic ring.[18a,c]
To convert 1 from an inhibitor to a substrate, we uncoupled
the amine nitrogen from the aromatic p-system through the
introduction of a methylene linker to give 2 (Figure 1B), which
was rapidly converted to the carboxylate product under the
action of ALDH1A1 (Figure S1). In order to assess the enzyme
kinetics of the compounds in this study, we examined the
effect of substrate concentration on the initial enzyme velocity
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Scheme 1. Synthetic route to 3a-b,4a-b, 5, 6, 7, 8 and 9. All yields are isolated yields. See Supporting Information for further details and for the synthesis of
A and B.
compounds tested (Figure 2B; Table 1, entry 6). Replacing the
amide of compound 4a with a sulfonamide gave us com-
pound 6 with diminished ALDH1A1 enzyme efficiency when
compared to 4a (Figure 2B; Table 1, entries 5 and 7). In sum-
mary, we have shown 4a, 4b and 5 to be excellent substrates
for ALDH1A1, with 4a and 4b to have good ALDH1A1 selectiv-
ity over the other isoforms tested (Figure 2B).
pounds 2–6, and the natural ALDH1A1 substrates 9-cis-retinal,
13-cis-retinal, and all-trans-retinal, were docked into protein
structures for ALDH1A1, ALDH2, and ALDH3A1 (Protein data
bank ID: 4WB9, 1O01 and 4L2O respectively). In comparison to
ALDH2 and ALDH3A1, ALDH1A1 has the largest access tunnel
to the active site residue (Figure S3), which suggests that bulki-
er and more rigid substrates would be preferentially turned
over by ALDH1A1. Tunnel topography may also explain why
reducing the amide in compound 4a to a flexible tertiary
amine in compound 5, resulted in increased affinity for ALDH2
To understand why compounds 4a and 4b exhibited en-
hanced selectivity for ALDH1A1 over the other isozymes
tested, we carried out an in silico docking study wherein com-
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Table 1. Kinetic properties of human ALDH1A1, ALDH2 and ALDH3A1 towards oxidation of aldehydes in this study.
Entry
Com-
ALDH1A1
ALDH2
ALDH3A1
pound
Vmax
KM
[mm]
Vmax/KM
Vmax
KM
[mm]
Vmax/KM
Vmax
KM
[mm]
Vmax/KM
[nmolminÀ1 mgÀ1
]
[nmolminÀ1 mgÀ1
]
[nmolminÀ1 mgÀ1
]
1
2
3
4
5
6
7
2
1.22Æ0.09
1.10Æ0.18
1.42Æ0.17
1.08Æ0.07
0.98Æ0.02
1.36Æ0.08
1.5Æ2.68
0.16Æ0.03
0.28Æ0.12
0.26Æ0.08
0.03Æ0.01
0.03Æ0.01
0.04Æ0.01
0.10Æ0.24
7.9
3.9
5.5
33.9
28.7
33.3
14.6
0.09Æ0.01
0.30Æ0.02
0.19Æ0.01
0.67Æ0.01
0.53Æ0.01
0.32Æ0.02
0.27Æ0.01
0.03Æ0.01
0.64Æ0.12
0.12Æ0.01
0.13Æ0.01
0.07Æ0.01
0.02Æ0.01
0.03Æ0.01
2.7
0.47
1.5
4.9
6.8
2.81Æ0.03
0.89Æ0.03
1.51Æ0.10
1.87Æ0.03
2.22Æ0.18
1.6Æ0.13
1.87Æ0.06
1.78Æ0.11
0.99Æ0.13
1.19Æ0.06
1.10Æ0.18
0.18Æ0.03
0.02Æ0.01
1.5
0.5
1.5
1.5
2.0
8.7
1.7
3a
3b
4a
4b
5
19.6
7.4
6
0.03Æ0.01
presumably via p-stacking between the pyridine and the tyro-
sine-Y296 of ALDH1A1 (Figure 3). This putative p-stacking in-
teraction may explain the increase in binding affinity for com-
pounds 4a, 4b, 5, and 6 when compared to compounds that
lack the pyridine functional group. The low binding affinity of
compounds 4a, 4b, 5, and 6 for ALDH2 (Figure 2 and Table 1)
might be ascribed to the equivalent phenylalanine residue
within the active site, which being less electron-rich than tyro-
sine, results in a lowered p-stacking efficiency.[24] In ALDH3A1
the active site tyrosine is replaced by a methionine, which is
incapable of p-stacking. Consequently, no pattern in binding
affinity for ALDH3A1 was observed for substrates with the pyr-
idyl substituent.
Figure 2. Structure-activity relationship. A) Chemical structures for the com-
pounds in this study. B) Enzyme efficiency of human recombinant ALDH1A1,
ALDH2 and ALDH3A1 for compounds 2–6. The “enzyme efficiency” (Vmax/KM)
provides a measure of how efficiently an enzyme converts a substrate into
product. Note: see Scheme 1 and Supporting Information for chemical syn-
thesis of compounds 2–6 and Figure S2 for Michaelis Menten plots used to
derive enzyme kinetic data.
Compound 4b was further considered as a potential radio-
chemical probe to assess cellular ALDH activity as it not only
had excellent enzyme efficiencies and selectivity for ALDH1A1,
but also potential radiochemical accessibility. Radiolabeling via
nucleophilic substitution with [18F]fluoride allows for substitu-
tion at the 2-position on a pyridine ring, whilst the 3-position
4a exhibits poor reactivity.[25] Consequently, the 18F-radiola-
beled analogue of 4b, N-ethyl-6-(fluoro-18F)-N-(4-formylbenzyl)-
nicotinamide was chosen as our lead candidate radiotracer. To
access [18F]4b, a nucleophilic aromatic substitution (SNAr) was
performed on the 6-chloronicotinamide precursor 7, which
was prepared from the acid chloride of 6-chloronicotinic acid
20 and amine A (Scheme 1F). Stirring 7 with [18F]KF/K222 in
DMSO at 1508C for 25 min followed by an acid-catalyzed
acetal cleavage step furnished [18F]4b in 35Æ1% (n=3)
decay-corrected radiochemical yield (RCY) after reverse phase
semi-preparative HPLC purification following manual radiola-
beling (Scheme 2). Automation of this procedure improved the
RCY to 43Æ1% (decay-corrected; n=3). Starting the synthesis
and ALDH3A1. Compounds 4a and 4b displayed similar bind-
ing modes as 2 to ALDH1A1 in our in silico model, as shown in
Figure 3. The benzaldehyde portions of compounds 4a, 4b, 5,
and 6 occupied a similar site to that of compound 2; however,
the pyridyl-ring was predicted to make additional contacts,
Figure 3. In silico modeling. Predicted binding modes of ALDH1A1 sub-
strates from in silico docking studies. Predicted binding modes in ALDH1A1
of 9-cis-retinal (purple), 2 (magenta), 4b (green) and 5 (cyan), shown in ball
and stick representation. Catalytic residue C302, and Y296 which was identi-
fied in p-stacking interactions with the substrates are shown as dark grey
sticks. Ribbons are shown in grey and faded for clarity, the cofactor NAD(H)
is shown in pastel orange, green, red and blue ball and stick representation.
Scheme 2. Radiofluorination of [18F]4b. See Supporting Information for fur-
ther details. RCY, radiochemical yield; d.c. =decay-corrected.
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with ꢀ1.0 GBq of [18F]fluoride, the radiotracer was obtained
with a radiochemical purity of 99% (see Supporting Informa-
tion) and a molar activity of up to 4.4 GBqmmolÀ1 (manual)
and up to 7.2 GBqmmolÀ1 (automated).
intracellular trapping of either [18F]4b or its products (Fig-
ure 4B). To confirm the identity of the intracellular radioactive
species present, we performed radio-HPLC analysis of the re-
sulting cell lysates. 20 min after the addition of the radiotracer,
near-complete conversion of the [18F]4b aldehyde to the corre-
sponding carboxylic acid [18F]8 was observed (Figure 4C; see
Scheme 1G for structures of 8 and 9), confirmed through co-
injection with non-radioactive [19F]8 (See Supporting Informa-
tion for details). Incubation of cells with DEAB resulted in a
substantial reduction in the production of [18F]8, with >90%
of radioactivity present as the parent compound (Figure 4C),
suggesting that in the absence of ALDH1A1 activity the alde-
hyde does not undergo intracellular oxidation and therefore is
not retained.
Whilst the levels of [18F]4b in DEAB-treated cells did not sig-
nificantly change over the 60 min time course of the experi-
ment (P>0.05; Figure 4B, red circles), the amount of intracellu-
lar radioactivity decreased in a time-dependent fashion in vehi-
cle (DMSO)-treated cells following addition of [18F]4b (Fig-
ure 4B, blue circles). Despite washout of radioactivity from ve-
hicle-treated cells, cell-associated radioactivity at 60 minutes
remained 2.4-fold higher than cells treated with DEAB, at 2.8Æ
0.3% radiotracer dosemgÀ1 and 1.2Æ0.1% radiotracer
dosemgÀ1 protein, respectively (P=0.0005; n=3). Analysis of
media samples by radio-HPLC following cell incubation
showed a progressive increase in the levels of [18F]8 and the
subsequent appearance of the alcohol [18F]9 (see Scheme 1G
for structures), indicating imperfect intracellular trapping of
[18F]8 following its production by ALDH (Figure 4D). Appear-
ance of [18F]8 in the media therefore accounts for the time-de-
pendent reduction in cell-associated radioactivity observed in
vehicle-treated cells following incubation with [18F]4b, poten-
tially as the result of efflux pump-mediated excretion of the
carboxylate. Media incubation of [18F]4b at 378C for 60 min in
the absence of cells did not result in the production of [18F]8
(Supporting Information; chromatogram S3), confirming that
the conversion to the carboxylate was cell-mediated. Together,
these data show [18F]4b to be specific and sensitive marker of
ALDH activity in tumor cells.
Furthermore, reference compounds for cellular metabolite
analysis, carboxylic acid 8 and alcohol 9, were prepared by the
KMnO4 mediated oxidation of 4b, and the NaBH4 mediated re-
duction of aldehdye 4b, respectively (Scheme 1G). With a
tracer candidate in hand we assessed whether [18F]4b could
provide a readout of ALDH activity in tumor cells grown in cul-
ture. We used the HCT116 KrasG13D/À mutant (HCT116 mut)
human colorectal cancer cell line as a model of aggressive,
therapy-resistant cancer. HCT116 mut lines displayed high
ALDH activity, which was specifically inhibited through the in-
cubation of cells with the ALDH inhibitor DEAB (30 mm; Fig-
ure 4A). Incubation of HCT116 mut cells with [18F]4b resulted
in rapid cell uptake and intracellular retention of radioactivity,
reaching 5.7Æ0.2% radiotracer dosemgÀ1 protein at 20 min
(n=3). Treatment of cells with DEAB resulted in an 83% reduc-
tion in cell-associated radioactivity to 1.0Æ0.1% radiotracer
dosemgÀ1 protein (n=3; P<0.0001), indicating ALDH-specific
Given that [18F]4b can measure ALDH activity in HCT116
mut cells with high sensitivity and specificity, we next explored
[18F]4b’s in vivo biodistribution. Dynamic microPET imaging
following intravenous injection of [18F]4b into healthy balb/c
mice (Figure 5) revealed rapid and extensive uptake in the
lungs, known to express high levels of ALDH1A1. Liver uptake
peaked at 5 min post injection (p.i.) at 20.0Æ2.6% injected
dose (ID)gÀ1 tissue (n=3 mice). [18F]4b clearance was initially
via the kidneys and bladder to afford high contrast images
with low uptake in background tissue (2.2Æ0.3%IDgÀ1 in the
muscle at 5 min p.i.; n=3 mice). However, hepatobiliary excre-
tion was evident by 30 min p.i., as shown by radiotracer
uptake in the gallbladder and gastrointestinal (GI) tract, indi-
cating possible metabolism of the parent compound at this
time point. [18F]4b was further cleared from all other organs
other than gallbladder and GI over the remainder of the imag-
ing time course. For time activity curves for organs with high
[18F]4b uptake, see Figure S4.
Figure 4. Detection of ALDH activity in HCT116 mut cells. A) ALDH activity in
vehicle (DMSO) and DEAB-treated cells (30 mm, 45 min), as measured by
ALDH-mediated trapping of Aldefluor and detection by flow cytometry. Data
are means ÆSD (n=4). B) Intracellular radioactivity levels in HCT116 mut
cells treated with either vehicle or DEAB (30 mm, 45 min) following incuba-
tion with [18F]4b. Data are means ÆSD (n=3). C) Radio-HPLC chromato-
grams from cell lysates following 20 min incubation of [18F]4b (red peak)
with (bottom), or without DEAB treatment (top). The green peak corre-
sponds to the carboxylate [18F]8. D) Composition of radioactive metabolites
in the media following 20, 40 and 60 min incubation of [18F]4b (red) with
HCT116 mut cells. The presence of [18F]8 (green peak) along with the alcohol
[18F]9 (blue peak) were identified via co-injection with [19F]4b, [19F]8, and
[19F]9 and their detection at 254 nm (see Supporting Information for further
information). ***, P<0.001 in vehicle vs. DEAB-treated cells.
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Experimental Section
See Supporting Information for detailed synthetic, radiochemical
methods, enzyme assays, cellular and studies in mice.
Acknowledgements
This study was funded through a Wellcome Trust and Royal So-
ciety Sir Henry Dale Fellowship (107610/Z/15/Z), the CRUK &
EPSRC Comprehensive Cancer Imaging Centre at KCL & UCL
(C1519/A16463). UCL radiochemistry is funded in-part by the
Department of Health’s NIHR Biomedical Research Centres
funding Scheme. We would like to thank Dr. Adam Shuhendler
for his thoughtful comments during the preparation of this
manuscript.
Figure 5. PET/CT maximum intensity projections of [18F]4b in a healthy balb/
c mouse. Representative time course images are shown to illustrate [18F]4b
pharmacokinetics. Gray scale is CT image, and color scale is PET image.
Uptake in key organs are illustrated. GI, gastrointestinal tract. Images are
representative of three separate mice.
Conflict of interest
The authors declare no conflict of interest.
The combined renal and hepatobiliary excretion observed at
30 min p.i., prompted us to assess the in vivo stability of
[18F]4b. Radio-HPLC analysis of plasma taken by terminal ex-
sanguination from anesthetized mice following tail vein intra-
venous (i.v.) injection of [18F]4b revealed complete conversion
to [18F]8 by 2 min (Supporting Information; chromatogram S7).
The appearance of a second, unknown peak of similar reten-
tion time to [18F]8 (Supporting Information chromatogram S7)
was evident by 5 min, increasing to ꢀ40% total radioactivity
in the blood by 60 min, which may account for the mixed
routes of excretion observed at later imaging time points. Im-
portantly, the rapid metabolism of the free aldehyde may limit
the in vivo tissue penetration of [18F]4b.
Keywords: [18F]fluorination
cancer · radiochemistry · radiolabeling
·
aldehyde dehydrogenase
·
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Manuscript received: November 1, 2018
Revised manuscript received: December 5, 2018
Accepted manuscript online: December 6, 2018
Version of record online: && &&, 0000
Chem. Eur. J. 2019, 25, 1 – 8
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ꢁ 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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These are not the final page numbers! ÞÞ

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FULL PAPER
Presented in this report are the syn-
thesis and biological evaluation of
ALDH1A1-selective chemical probes
composed of an aromatic aldehyde de-
rived from N,N-diethylamino benzalde-
hyde (DEAB) linked to a fluorinated pyri-
dine ring either via an amide or amine
linkage.
&
Biochemistry
R. Pereira, T. Gendron, C. Sanghera,
H. E. Greenwood, J. Newcombe,
P. N. McCormick, K. Sander, M. Topf,
E. ꢀrstad, T. H. Witney*
&& – &&
Mapping Aldehyde Dehydrogenase
1A1 Activity using an [18F]Substrate-
Based Approach
&
&
Chem. Eur. J. 2019, 25, 1 – 8
www.chemeurj.org
8
ꢁ 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!