[18F]PRIMATX, a New Positron Emission Tomography Tracer for Imaging of Autotaxin in Lung Tissue and Tumor-Bearing Mice

Article abstract of DOI:10.1002/cmdc.201900297

Autotaxin (ATX) is a secreted enzyme with tissue levels associated with tissue injury, which increase during wound healing and chronic fibrotic diseases. We selected [¹⁸F](R,E)-3-(4-chloro-2-((5-methyl-2H-tetrazol-2-yl)methyl)phenyl)-1-(4-((5-(2-fluoroethoxy)pyridin-2-yl)methyl)-2-methylpiperazin-1-yl)prop-2-en-1-one ([¹⁸F]PRIMATX, [¹⁸F]2), a tracer for positron emission tomography, to image ATX expression in vivo. It successfully differentiates expression levels in lung tissue samples from idiopathic pulmonary fibrosis patients, and allows the detection of ATX-expressing tumors in living mice, confirming its potential for development as a clinical imaging agent.

Full text of DOI:10.1002/cmdc.201900297

Accepted Article
Title: [18F]PRIMATX, a new positron emission tomography tracer for in
vivo imaging of autotaxin in lung and tumour tissues
Authors: Emmanuelle Briard, Aniket Joshi, Shiva Shanmukhappa,
Ohad Ilovich, and Yves P. Auberson
This manuscript has been accepted after peer review and appears as an
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the VoR from the journal website shown below when it is published
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To be cited as: ChemMedChem 10.1002/cmdc.201900297
Link to VoR: http://dx.doi.org/10.1002/cmdc.201900297
A Journal of
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[18F]PRIMATX, a new positron emission tomography tracer for
imaging of autotaxin in lung tissue and tumour-bearing mice.
Dr Emmanuelle Briard,[a] Dr. Aniket D. Joshi,[b] Dr. Shiva Shanmukhappa,[c] Dr. Ohad Ilovich,[d] Dr. Yves
P. Auberson*[a]
[a]
Dr. Y. P. Auberson (ORCID 0000-0001-5800-4811), Dr. E. Briard (ORCID 0000-0001-9496-1395)
Global Discovery Chemistry
Novartis Institutes for BioMedical Research
Novartis Pharma AG, Klybeckstrasse 141, 4057 Basel, Switzerland
E-mail: yves.auberson@novartis.com
[b]
[c]
[c]
Dr. A. Joshi, ORCID(s) of Author(s)
Clinical and Translational Imaging
Novartis Pharmaceuticals
45 Sidney Street, 1203K, Cambridge, MA 01239
Dr. S. Shanmukhappa, ORCID 0000-0001-7781-7586)
Discovery and Investigative safety, Preclincial safety
Novartis Institutes for BioMedical Research
250 Massachusetts avenue Cambridge, MA 01239
Dr. Ohad Ilovich
inviCRO, LLC
27 Dry Dock Avenue, 7th floor west
Boston, MA 02210
carcinoma is correlated with tumour grade as well as with the risk
of death.[10] Autotaxin quantification by imaging in fibrosis or
cancer patients, therefore, has the potential to become an
important diagnostic and prognostic tool.
Abstract: Autotaxin (ATX) is a secreted enzyme with tissue levels
associated with tissue injury, which increase during wound
healing and chronic fibrotic diseases. We selected [18F]-(R,E)-3-
(4-chloro-2-((5-methyl-2H-tetrazol-2-yl)methyl) phenyl)-1-(4-((5-
(2-fluoroethoxy)pyridin-2-yl)methyl)-2-methylpiperazin-1-yl)prop-
2-en-1-one ([18F]PRIMATX, [18F]2), a tracer for positron emission
tomography, to image ATX expression in vivo. It successfully
differentiates expression levels in lung tissue samples from
idiopathic pulmonary fibrosis patients, and allows the detection of
ATX-expressing tumours in living mice, confirming its potential for
development as a clinical imaging agent.
Non-invasive nuclear imaging techniques provide information
about the physiology and biochemistry of experimental animals,
patients and volunteers. They rely on the use of imaging
instruments that can detect radiation emitted from a radioisotope
bound to a physiologically relevant molecule (a radiotracer).
Quantification of the emitted radiation allows the reconstruction
of images revealing the distribution and concentration of the
radiotracer as
a
function of time.[11] Positron emission
tomography (PET) is a preferred technique as it offers a
comparatively high spatial and temporal resolution. It involves the
use of radiotracers labelled with positron emitting radionuclides
such as carbon-11 (11C) or fluorine-18 (18F), both of which have
short physical half-lives, allowing for same-day imaging. The
system detects pairs of 511 keV gamma rays resulting from the
annihilation of the emitted positron with a local electron.
Introduction
Autotaxin
(ATX),
also
known
as
ectonucleotide
pyrophosphatase/phosphodiesterase (ENPP2), is a secreted
phosphodiesterase possessing lysophospholipase D activity.[1] It
produces the bioactive lipid mediator lysophosphatidic acid (LPA),
by hydrolysis of lysophosphatidylcholine (LPC) in serum.[2]
Enpp2+/- heterozygotes have plasma LPA levels that are half that
of wild-type mice.[2b] LPA is involved in the pathogenesis of a
number of diseases, including cancer,[3] pulmonary fibrosis,[4] liver
fibrosis,[5] and possibly neuropathic pain.[6]
A number of PET tracers have already been developed to image,
quantify and monitor diseases. The most frequently used is 2-
deoxy-2-[18F]fluoroglucose ([18F]FDG), which is useful to monitor
glucose metabolism and identify hyper-metabolic cancerous
lesions.[11] Further examples are tracers binding to the
translocator protein (TSPO) on mitochondrial membranes for
imaging inflammation,[12] and tracers for imaging Tau
aggregates[13] or amyloid plaques,[14] the hallmarks of Alzheimer’s
disease.
LPA and ATX levels markedly increase following tissue injury,
during wound healing and in chronic fibrotic diseases. In lung
fibrosis elevated local LPA levels have been shown to play a role
in the migration, differentiation and secretion profile of fibroblasts
acting primarily via the G-protein coupled lysophosphatidic acid
receptor LPA1. Samples of lung tissue from idiopathic pulmonary
fibrosis (IPF) patients show increased levels of ATX
expression.[4b] Likewise, ATX is over-expressed in multiple
human cancer cell lines.[7] It was shown that ATX is associated
with the initiation and progression of breast cancer in mice[8] and
human,[9] and that ATX expression in human hepatocellular
Another important application of PET imaging studies is that,
when conducted with an appropriate radiotracer, it facilitates the
in vivo, non-invasive determination of the level of target
engagement of a drug candidate without having to label the drug
itself. This is particularly important when it comes to optimizing
dose and regimen of a new molecular entity, and to precisely
quantify the duration of its pharmacodynamic effects. While blood
1
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sampling makes it easy to determine the duration of exposure in
plasma, it only gives a very rough approximation of exposure in
the organ of interest and ignores binding kinetics. In particular,
multiple parameters such as the rate of tissue permeation, P-
glycoprotein mediated efflux, and koff influence target
engagement in tumours. These parameters are complex to
measure precisely, making predictions of clinical efficacy difficult
and imprecise. Target occupancy studies allows addressing
questions such as whether the drug has sufficiently penetrated
the tissue of interest, and whether the selected dose achieves
enough receptor occupancy for the desired effects, with an
adequate safety margin. Selecting this approach to dose
selection in drug development will help reduce the risk of drug
failure due to lack of sufficient target engagement.
N
N
N
N
N
N
N
Br
N
N
N
N
R
N
R
O
a
b, c
Br
Br
Br
+
OH
R
R
R = Cl, CF3
3a: R = Cl
3b: R= CF3
4a: R = Cl
4b: R= CF3
5a: R = Cl
5b: R= CF3
N
N
N
N
N
N
N
N
R
O
O
O
N
N
d, e
f
N
N
NH
R
6a: R = Cl
1:
7:
R = Cl
6b: R= CF3
R= CF3
The lead structure for PET tracer optimization (1, Figure 1)
originates in the modification of a hit from a high-throughput
screen.[15] Optimized as a drug candidate, 1 displays a high
affinity for ATX, and PET tracer-like properties. It however
displays an elimination half-life that is too long for a potential PET
g
h
N
N
N
N
N
N
N
N
R
O
N
O
N
N
F
O
N
N
N
R
8a: R = Cl
8b: R= CF3
9a: R = Cl
9b: R= CF3
tracer (t = 1.1h in male Sprague-Dawley rats after 1 mg/kg i.v,),
½
especially if labelled with 11C (t = 20.3 min). To address this
½
issue, we sought to identify a fluorine-containing derivative with a
Scheme 1. Experimental condfitions: a) K2CO3, DMF, RT, 2 h, 4a: 37%, 4b:
37%; b) P(o-tol)3, NEt3, DMF, Pd(OAc)2, 100°C, 18 h; c) 2M NaOH, EtOH, RT,
18 h, quant.; d) HATU, DIPEA, NMP, RT, 2h; e) TFA, CH2Cl2, RT, 4 h, 6a: 95%,
6b: 84%; f) 5-methoxypicolinaldehyde, 2-picoline-borane, AcOH, MeOH, RT,
16h, 7: 55%; g) 2-fluoro-5-formylpyridine, 2-picoline-borane, AcOH, MeOH, RT,
16h, 8a: 13%, 8b: 16%; h) 9a: Pt/C, EtOH, H2, RT, 21 h, 21%; 9b: Pd/C, EtOH,
H2, RT, 17 h, 84%.
shorter half-life, for labelling with 18F (t = 109.8 min). We also
½
explored structural modifications to attempt increasing affinity
further. Several suitable candidates were identified. Compound
2 (PRIMATX) was selected for characterization of its imaging
potential.
N
N
N
N
N
N
Scheme 2 shows the preparation of picolinaldehydes 12a,b, and
their coupling to 6a,b to provide the two pairs of analogues 2, 13
and 14a,b. Treatment of 6-methylpyridin-3-ol with 2-fluoroethyl 4-
methylbenzene sulfonate[16] in the presence of potassium
carbonate and sodium iodide led to 10a. The methyl group of 10a
was oxidized following a published synthetic sequence[17] to yield
aldehyde 12a, which was condensed with 6a,b in the presence of
acetic acid and 2-picoline-borane to give target compounds 2 and
13. Alternatively, alkylation of 6-methylpyridin-3-ol with 2-(2-
chloroethoxy)ethan-1-ol and subsequent treatment with
tetrabutylammonium fluoride gave 10b. Oxidation of the methyl
group led to 12b, which was condensed with 6a or 6b to provide
target compounds 14a,b. Finally, Pd/C catalyzed hydrogenation
of 13 yielded the saturated analogue 15.
N
N
O
O
O
O
N
N
N
N
18F
N
N
Cl
Cl
18
1
2
F]
[
Figure 1. Structure of lead 1 and radiolabelled derivative [18F]2.
Results and Discussion
Synthesis
Scheme 1 shows the synthetic route toward compounds 1, its CF3
analogue (7), as well as variations of the pyridine ring (8a,b) and
saturation of the double bond (9a,b). Alkylation of 1-bromo-2-
(bromomethyl)-4-chlorobenzene or 1-bromo-2-(bromomethyl)-4-
(trifluoromethyl)-benzene with 5-methyl-2H-tetrazole led to a
mixture of regioisomers. Subsequent isolation by filtration gave
the desired compounds 4a,b, which were reacted with ethyl
acrylate and hydrolyzed to provide acids 5a,b. Carboxylic
activation by HATU and coupling with a partially protected
piperazine, followed by deprotection, furnished intermediates
6a,b. Condensation with picolinaldehyde in the presence of
acetic acid and 2-picoline-borane as reducing agent gave target
compounds 1 and 7. The 6-fluoropyridinyl analogues 8a,b were
prepared by condensation of 6a,b with 6-fluoronicotinaldehyde,
while a Pd/C catalyzed hydrogenation of 1 and 7 yielded 9a and
9b, respectively.
2
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toluenesulfonate), Cs2CO3, DMF, RT, 2 h, 62%; c) [18F]KF, crypt-222,
DMSO, 140°C, 10 min.
O
O
OH
N
F
a (n=1)
N
F
e, f
N
n
n
O
or b-d (n=2)
O
10a
10b
11a
11b
, n=1
, n=2
, n=1
, n=2
Optimization and pharmacological characterization
N
N
N
O
N
N
O
F
n
Starting from structure 1, we initially explored the exchange of the
aromatic chlorine by a trifluoromethyl group. This modification
was tolerated, only leading to a slight decrease in affinity (7).
Replacing the 5-methoxypyridin-2-yl group by a 6-fluoropyridin-3-
yl (8a), which would allow an easy radiolabelling strategy, was
less favorable. Interestingly, the combination of these two
modifications led to a partial recovery of affinity (8b). Reduction
of the double bond (9a, 9b) did not bring any advantage, with both
derivatives being less potent that the lead structure.
O
i
g, h
O
N
N
F
n
N
R
: R = Cl, n = 1
: R = CF
: R = Cl, n = 2
: R = CF
2
13
14a
14b
12a
12b
, n=1
, n=2
3, n = 1
3, n = 2
N
N
N
N
O
O
j
N
N
F
2
N
CF3
15
To introduce another option for radiolabelling, we extended the
methoxy group to a fluorine-containing side-chain, envisaging the
introduction of 18F from the corresponding tosylate precursor. We
first prepared the fluoroethyl analogues 2 and 13, which proved
to have affinities equivalent to the lead structure. Extending the
side-chain to a fluoroethoxyethoxy group had a minimal effect on
affinity (14a,b). Further variations, as exemplified by 15 and 18,
identified additional derivatives with high affinity and similar
overall properties. The good permeability and low efflux ratio of
these molecules in the MDCK-MDR1 assay hints at a good organ
distribution, including a high likelihood of brain penetration. Their
properties appeared promising and only differed minimally, so we
selected the lowest molecular-weight compound (2, MW = 514)
for further evaluation. Furthermore, the radiosynthesis of the
radiolabelled [18F]2 was expected to be straightforward.
Scheme 2. Experimental conditions: a) FEtOTs, K2CO3, NaI, DMF,80°C, 16 h,
10a: 83%, 10b: 85%; b) 2-(2-chloroethoxy)ethan-1-ol, K2CO3, NaI, DMF, 85°C,
16 h; c) p-tolylsulfonyl chloride, CH2Cl2, RT, 16 h; d) TBAF, THF, 65°C, 30 min;
e) m-CPBA, CHCl3, 0°C, 3 h; f) Ac2O, 130°C, 30 min; g) NaOH, H2O, EtOH,
reflux, 1 h; h) MnO2, CH2Cl2, RT, 2 days; i) AcOH, MeOH, 2-picoline borane,
RT, 16 h, 2: 23%, 13: 58%, 14a: 50%, 14b: 49%; j) Pd/C, H2, EtOH, 18 h, 47%.
The oxazole-carbaldehyde 17 was prepared according to
scheme 3. Fluorination of methyl 2-(chloromethyl)oxazole-4-
carboxylate led to compound 16. Consecutive reduction and
oxidation led to aldehyde 17, whose condensation with 6b
afforded compound 18.
O
O
O
F
F
Cl
a
b, c
O
O
O
N
N
N
Table 1. Compound list and in vitro properties.
O
O
17
16
IC50 (nM)
CHI(IAM)[a]
logD[b]
%HSA[c]
flux[d]
efflux
ratio[]
1.0
1.9
1.0
1.1
1.1
1.2
0.9
1.6
3.4
-
Cmpd
N
N
N
1
2
7
3
4
-
-
-
12.5
8.1
14.1
7.4
N
O
d
35
35
36
36
34
35
35
34
33
-
3.0
3.2
3.3
3.5
2.9
3.2
3.2
3.2
3.0
3.1
2.8
91
91
93
94
88
89
91
88
87
87
86
O
F
N
10
51
13
22
15
3
6
5
3
2
N
N
8a
8b
9a
9b
13
14a
14b
15
18
F3C
8.3
18
12.9
26.3
7.7
10.4
-
Scheme 3. Experimental conditions: a) TBAF, CH3CN, 21 hours, RT, 32%; b)
DIBAL-H, THF, -78°C, time, 41%; c) MnO2, CH2Cl2, RT, 18 hours; d) 6b, AcOH,
MeOH, 2-picoline borane, RT, 16 h, 24%.
10.3
13.8
1.3
1.8
33
The precursor for the radiosynthesis of [18F]2 was prepared
according to Scheme 4. Condensation of 6a with 5-
hydroxypicolinaldehyde led to compound 19. Further treatment
with ethane-1,2-diyl bis(4-methylbenzenesulfonate) and cesium
carbonate provided 20, the precursor for the radiosynthesis of
[18F]2. The latter was obtained after treatment with [18F]KF in
presence of Kryptofix-222.
[a] Chromatographic Hydrophilicity Index (CHI) measured by HPLC on
Immobilized Artificial Membranes (IAM)[18]; [b] logD measured in octanol/buffer
pH 7.4; [c] ; [d] measured in the Madin Darby Canine Kidney (MDCK) cell line
transfected with the human multidrug resistance (MDR1) gene.
The selectivity of 2 was tested at a concentration of 30 µM in a
panel of 63 pharmacological targets from the central nervous
system and periphery; no activity was found (results not shown).
Its CHI(IAM) value of 35, similar to all compounds in this series,
indicates a very low propensity to bind to cell membranes,
predicting minimal non-specific binding in vivo.[19] The rat plasma
protein free fraction was determined to be 5%. Finally, the
pharmacokinetic properties of the tracer candidate (Table 2)
indicate a faster elimination than the lead compound, with a
plasma half-life of 0.7 h. It also shows a good exposure in the
lung, an organ of interest for pulmonary fibrosis. A separate
N
N
N
N
N
N
N
N
O
O
OH
O
a
b
N
N
N
N
OTs
c
18F]2
6a
[
N
N
Cl
Cl
19
20
Scheme 4. Radiosynthesis of [18F]2: a) 5-hydroxypyridine-2-carboxaldehyde,
NaBH(OAc)3, CH3COOH, (ClCH2)2, RT, 17h; b) ethylene di(p-
3
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pharmacokinetics study showed that in rats, brain penetration
was modest, with a brain/blood ratio of 0.24, five minutes after
intravenous administration (1 mg/kg). In combination with a high
affinity for ATX and
a low non-specific binding, this is
nevertheless expected to be sufficient for PET imaging of the
central nervous system.
Table 2. Pharmacokinetic properties of 2, one hour after intravenous
administration (1 mg/kg) in Sprague Dawley male rats (n=3). Formulation as a
solution in 1% 1M HCl, 10% PEG300, 25% (20% solutol), 64% PBS.
Blood
(nM)[a]
55±13
Lung
(nM)[a]
241±80
Lung/blood
ratio[a]
t½ (h)
CL[a]
(ml/min/kg)
23±11
Vss[b]
(L/kg)
Figure 3. In vitro autoradiography with [18F]2 on human fibrotic lung sections,
logarithmically scaled. Upper sections: total binding; lower sections: binding
after blocking with 10 µM 2; a) section with moderate ATX expression; b) and
d) sections with high ATX expression as determined via IHC.
4.6
0.7±0.1
1.0±0.3
[a] CL = clearance; [b] Vss = Distribution volume at steady state
Encouraged by this profile, we moved to the characterization of
the radiolabelled tracer, [18F]2. We first performed immunohisto-
chemistry for ATX in human IPF lung samples, to qualitatively
estimate the level of expression of ATX in diseased tissues
(Figure 2). One sample showed a low level of ATX expression,
while the other two showed a robust signal, visible under the
microscope as a diffuse brown staining, as expected for a
secreted protein.
After failing to reliably and reproducibly induce lung fibrosis using
the bleomycin model in mice,[20] and in view of the scarcity of
alternative fibrosis animal models, we decided to pursued the in
vivo evaluation of [18F]2 in mice bearing autotaxin over-
expressing tumours. Our aim was to confirm that the tracer is able
to image its target in vivo, and that its signal can be blocked by
pre-treatment with a structurally different autotaxin inhibitor.
A
B
C
Four B6D2F1 mice inoculated with subcutaneous B16F10
melanoma tumours (140 – 391 mm3) were injected with [18F]2
(1.09 ± 0.33 MBq, < 200 μL). The animals underwent a 60-minute
whole body dynamic PET scan, followed by computer
tomography (CT) for anatomical reference. The following day, the
same four animals were administered 100 mg/kg 21[11] as
blocking agent, via oral gavage. Thirty minutes later, animals
were injected with [18F]2 (10.5 ± 0.22 MBq, < 200 μL) and
underwent a second 60-minute whole body dynamic PET scan,
followed by a CT scan for anatomical reference.
E
F
D
Figure 2. Immunohistochemistry with anti-ATX antibody on human fibrotic lung
sections. Upper panel: A, lung with low expression of ATX; B and C, lungs with
high expression of ATX. Lower panel, higher magnifications of image from
upper panel showing brown staining of secreted ATX.
A subsequent in vitro immersion autoradiography on adjacent
lung sections, both under control and self-blocking conditions
(Figure 3), confirmed the ability of [18F]2 to detect the increase in
autotaxin expression associated with fibrosis.[4] The first section
(A) was shown to have a moderate autotaxin expression by
immuno-histochemistry (IHC), while sections B and C have a high
level of expression. Self-blocking decreased signal intensity
significantly, providing a first indication of high selectivity of
uptake and that [18F]2 might be suitable for the intended purpose.
Figure 4. Structure of blocker 21 and time course of percent injected dose per
gram (%ID/g, mean +/- SEM) in a) tumour; b) brain. Black lines represent
baseline signals, red lines represent signal after blocking with 100 mg/kg 21.
[18F]2 shows an average uptake of 4.4 %ID/g in B16F10 tumours
(Figure 4). The uptake was decreased to 2.1 %ID/g after blocking,
indicating that approximately 60% of uptake is antigen specific.
In the brain, average uptake was 3.4 %ID/g, and decreased by
4
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isocratic; flow rate: 1.0 mL.min-1. Preparative high-pressure liquid
chromatography (HPLC) purifications were run as follows (% = percent
per volume): Method C: Gilson Trilution LC, column: SunFire C18OBD 5
µm, 30 x 100 mm; mobile phase: H2O + 0.1% TFA) (A) / CH3CN + 0.1%
TFA (B); gradient: from 5 to 50% B in 20 min; flow rate: 40 mL min-1.
39% to 2.1 %ID/g upon blocking, confirming sufficient brain
penetration for imaging purposes. The distribution of radioactivity
suggests that [18F]2 and its metabolites are mostly excreted by a
combination of renal and hepatic clearance (Figure 5).
2-(2-Bromo-5-chlorobenzyl)-5-methyl-2H-tetrazole (4a): 5-Methyl-2H-
tetrazole (77 g, 913 mmol) was placed in a flask with dry DMF (400 mL)
at 0°C using an ice bath. Potassium carbonate (168 g, 1217 mmol) was
added in portions followed by dropwise addition of 1-bromo-2-
(bromomethyl)-4-chlorobenzene (173 g, 608 mmol) in DMF (400 mL). The
resulting mixture was stirred at room temperature for 2 h. The mixture was
poured into water and the resulting suspension collected by filtration. The
solid was triturated with iso-hexane and the undissolved solid was
removed by filtration. The filtrate was concentrated under reduced
pressure giving a white solid which was suspended in water and stirred
overnight. The product was filtered and washed with water to afford the
desired product (64.9 g, 37 %), which was used in the next step without
further purification. LC/MS: tRA=1.15 min, m/z: 289.0 [M+H].
Figure
5 Representative averaged PET image, scaled to 0-35 %ID/g
(measured between 20 and 55 minutes after [18F]2 injection), tumour centred,
60 min.; a) baseline; b) blocked; white arrows point to tumour.
2-(2-Bromo-5-(trifluoromethyl)benzyl)-5-methyl-2H-tetrazole (4b): To
a stirred solution of 5-methyl-2H-tetrazole (19.44 g, 231 mmol) in DMF
(154 mL) at 10°C under N2 was added K2CO3 (42.6 g, 308 mmol). The
resulting suspension was cooled to - 2°C (ice salt bath) and a solution of
1-bromo-2-(bromomethyl)-4-(trifluoromethyl)benzene (49 g, 154 mmol) in
DMF (66 mL) was added dropwise over 30 min keeping the internal
temperature below 5 °C. The mixture was then allowed to warm to room
temperature and the resulting white suspension stirred overnight. Water
(400 mL) was added slowly to the mixture which was then extracted with
EtOAc (2 x 500 mL). The combined organic extracts were washed with
brine (500 mL), dried (MgSO4) and concentrated in vacuo to yield a
colourless oil. Iso-hexane (150 mL) was added and the resulting slurry
was filtered and the solid washed with iso-hexane (2 x 50 mL). The filtrate
was concentrated in vacuo to yield a colourless oil. Purification by
chromatography on silica gel eluting with 0 to 50% EtOAc in iso-hexane
afforded the title compound (18.4 g, 37%). LC/MS: tRA =1.30 min, m/z:
Conclusions
The optimization of lead compound 1 led to the selection of its
fluorinated derivative 2, a very selective, high-affinity and
permeable autotaxin ligand, as a promising PET tracer candidate
with low non-specific binding. Pharmacokinetic studies
demonstrated good penetration into lung and a shortened half-
life of 0.7h, compatible with the requirements for a PET imaging
agent radiolabelled with fluorine-18. Immersion autoradiographic
studies in human fibrotic lung tissue confirm the ability of [18F]2
to differentiate areas of low and high autotaxin expression. A
subsequent in vivo PET study in mice bearing autotaxin-
expressing B16F10 tumours demonstrated the potential of this
tracer for tumour imaging. A clear imaging signal associated with
the tumour was blocked by a structurally different autotaxin ligand.
It is, to our knowledge, the first time an autotaxin tracer
successfully demonstrate the possibility to image melanoma
cancer cells in vivo, confirming the relevance of this target for
diagnostic purposes. Taken together, these results confirm that
[18F]2 fulfils the requirement of a PET tracer candidate for further
clinical development.
1
321.3 [M+H]. H NMR (400 MHz, CDCl3) δ= 2.58 (3 H, s), 5.83 (2 H, s),
7.11 (d, J = 2.46 Hz, 1H), 7.23 (dd, J = 8.5 Hz, 11.9 Hz, 1H), 7.57 ppm (d,
J = 8 Hz, 1H).
(E)-Ethyl
3-(4-chloro-2-((5-methyl-2H-tetrazol-2-yl)methyl)phenyl)
acrylate: Compound 4a (15 g, 52.2 mmol), tri-o-tolylphosphine (0.79 g,
2.61 mmol) and triethylamine (10.56 g, 104 mmol) were placed in a flask
with dry, degassed DMF (80 mL). Ethyl acrylate (7.83 g, 78 mmol) was
added, followed by palladium diacetate (0.59 g, 2.61 mmol) and the
reaction mixture was stirred at 100°C overnight. The mixture was allowed
to cool to room temperature, diluted with EtOAc (150 mL) and filtered to
remove solids, then partitioned between EtOAc and water. The organic
phase was washed with water and brine, dried over MgSO4, filtered, and
the solvent was removed in vacuo. When ~75% of the solvent was
removed, a solid precipitated out which was collected by filtration and
dried to afford the title compound as a white solid (8.5 g, 53 %). LC/MS:
Experimental Section
t
RA =1.33 min, m/z: 307.5 [M+H]. 1H NMR (400 MHz, DMSO-d6): δ 7.92 (1
General methods: All chemicals, reagents, and solvents for the synthesis
of the compounds were analytical grade, purchased from commercial
sources and used without purification, unless otherwise specified. 1H NMR
spectra were acquired on a Bruker 400 MHz instrument. Chemical shift (δ)
values are reported in parts per million (ppm) relative to the residual
solvent peak.
H, d), 7.89 (1 H, d), 7.59 (1 H, d), 7.51 (1 H, d of d), 6.59 (1 H, d), 6.09 (2
H, s), 4.20 (2 H, q), 2.41 (3 H, s), 1.26 ppm (3 H, t).
(E)-3-(4-Chloro-2-((5-methyl-2H-tetrazol-2-yl)methyl)phenyl)acrylic
acid (5a): (E)-Ethyl 3-(4-chloro-2-((5-methyl-2H-tetrazol-2-yl)methyl)
phenyl)acrylate (8.75 g, 28.5 mmol) was placed in a flask with EtOH (100
mL). 2M aq. NaOH (57.1 mL, 11 mmol) was added and the reaction
mixture stirred at room temperature overnight. The ethanol was removed
in vacuo and the reaction mixture acidified with 2M aq. HCl. The resulting
precipitate was collected by filtration, washed with water and dried to
afford the title compound as a white solid. It was used in the next step
without further purification. LC/MS: tRA =0.99 min, m/z: 279.2 [M+H].
Liquid chromatography/mass spectrometry (LC/MS) analyses were run as
follows (% = percent per volume): Method A (tRA = retention time A):
UPLC-ZQ2000, column: Acquity HSS-T3 1.8 µm, 2.1 x 50 mm, 60°C;
mobile phase: H2O + 0.05% H2CO2H + 3.75 mM CH3CO2NH4 (A) / CH3CN
+ 0.04% HCO2H (B); gradient: from 5 to 98% B in 1.4 min, then 0.7 min
isocratic; flow rate: 1.0 mL.min-1. Method B (tRB = retention time B): UPLC-
ZQ2000, column: Acquity HSS-T3 1.8 µm, 2.1 x 50 mm, 60°C; mobile
phase: H2O + 0.05% H2CO2H + 3.75 mM CH3CO2NH4 (A) / CH3CN +
0.04% HCO2H (B); gradient: from 5 to 98% B in 9.4 min, then 0.7 min
(E)-Ethyl 3-(2-((5-methyl-2H-tetrazol-2-yl)methyl)-4- (trifluoromethyl)
phenyl)acrylate: To a stirred solution of 4b (17 g, 52.9 mmol) in DMF (76
mL) was added tri-o-tolylphosphine (0.81 g, 2.65 mmol) and triethylamine
5
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900297
ChemMedChem
FULL PAPER
(14.76 mL, 106 mmol). The solution was de-gassed by bubbling N2
through it for 20 min. Pd(OAc)2 (0.59 g, 2.65 mmol) and ethyl acrylate
(8.66 mL, 79 mmol) were added and the reaction mixture heated to 90 °C
under N2. After cooling to room temperature, the mixture was partitioned
between water (150 mL) and EtOAc (250 mL). The phases were
separated and the aqueous phase extracted with more EtOAc (250 mL).
The combined organic layers were washed with brine (2 x 250 mL), dried
over anhyd. MgSO4 and concentrated in vacuo to yield the title compound
as orange oil (5.2 g, 28.7%), and used as such in the next step.LC/MS:
sodium sulphate, filtered and concentrated in vacuo. Purification was
performed by silica gel column chromatography eluting with a gradient of
iso-hexane to ethyl acetate. The product fractions were combined and
evaporated in vacuo to give a white solid. LC/MS: tRA=1.39 min, m/z: 395.3
[M-100+H] which was directly used in the next step.
(R,E)-3-(2-((5-Methyl-2H-tetrazol-2-yl)methyl)-4-
(trifluoromethyl)phenyl)-1-(2-methylpiperazin-1-yl)prop-2-en-1-one
(6b): TFA (10 mL) was added to a solution of (R,E)-tert-butyl 3-methyl-4-
(3-(2-((5-methyl-2H-tetrazol-2-yl)methyl)-4-(trifluoromethyl)phenyl)
acryloyl)piperazine-1-carboxylate (2.7 g, 5.46 mmol) in CH2Cl2 (10 mL)
and the resulting mixture stirred for 1 h. Toluene (100 mL) was added and
the reaction concentrated in vacuo. The resulting gum was stirred in
diethyl ether (250 mL), water (1mL) was added and the resulting solid was
collected by filtration, washed with ether and dried under vacuum to give
the title compound as a trifluoroacetate salt (1.8 g, 84%). LC/MS: tRA=0.74
min, m/z: 395.0 and 397.5 [M+H]. 1H NMR (400 MHz, CDCl3) δ= 1.40 (d,
J = 7.09 Hz, 3 H), 2.45 (s, 3 H), 2.76 - 2.95 (m, 1 H), 3.09 (d, J = 2.69 Hz,
2 H), 3.17 - 3.53 (m, 2 H), 4.05 - 4.82 (m, 2 H), 5.72 (s, 2 H), 6.70 (d, J =
15.28 Hz, 1 H), 7.25 (d, J = 2.08 Hz, 1 H), 7.30 (dd, J = 8.38, 2.02 Hz, 1
H), 7.43 (d, J = 8.31 Hz, 1 H), 7.93 ppm (d, J = 15.28 Hz, 1 H).
t
RA=1.36 min, m/z: 341.5 [M+H].
(E)-3-(2-((5-Methyl-2H-tetrazol-2-yl)methyl)-4-(trifluoromethyl)
phenyl)acrylic acid (5b): To a stirred solution of crude (E)-ethyl 3-(2-((5-
methyl-2H-tetrazol-2-yl)methyl)-4-(trifluoromethyl)phenyl)acrylate (18.02
g, approx. 53 mmol) in EtOH (212 mL) was slowly added 2M aq. NaOH
(79 mL, 159 mmol). The resulting orange solution was stirred at room
temperature overnight, then concentrated in vacuo to a volume of 100 ml,
and then filtered. 5M HCl (38 mL) was added slowly to adjust the pH to 2
whereupon a solid started to crystallize out of solution. The mixture was
stirred at room temperature for 2 h to allow full crystallization. The resulting
slurry was filtered, and the filter cake washed with 50% aq. EtOH (2 x 20
mL). The solid was dried in vacuo at 40 °C overnight to afford the title
compound, which was directly used in the next step. LC/MS: tRA=1.14 min,
m/z: 313.4 [M+H].
(R,E)-1-(4-((5-methoxypyridin-2-yl)methyl)-2-methylpiperazin-1-yl)-3-
(2-((5-methyl-2H-tetrazol-2-yl)methyl)-4-(trifluoromethyl)phenyl)
prop- 2-en-1-one (7): To a solution of compound 6b (62 mg, 0.16 mmol)
in MeOH (1.4 mL) were added AcOH (0.14 mL) and 5-methoxy-2-
pyridinecarboxaldehyde (32.3 mg, 0.24 mmol). After stirring for 5 min, 2-
picoline borane (26.6 mg, 0.25 mmol) was added. The reaction mixture
was stirred at RT for 16 h. The volatiles were evaporated and the residue
purified by flash chromatography (mobile phase: CH2Cl2 (A) / MeOH (B);
gradient: 100% A to 10% B in 15 min). The desired fractions were
concentrated to give the product as an orange oil (46 mg, 55%). LC/MS:
(R,E)-tert-Butyl
4-(3-(4-chloro-2-((5-methyl-2H-tetrazol-2-
yl)methyl)phenyl)acryloyl)-3-methylpiperazine-1-carboxylate: HATU
(2.11 g, 5.55 mmol) was added to a solution of compound 5a in NMP (15
mL) and the mixture stirred at room temperature for 5 minutes. (R)-tert-
butyl-3-methylpiperazine-1-carboxylate (0.93 g, 4.63 mmol) was added
followed by DIPEA (1.62 mL, 9.26 mmol). The reaction mixture was stirred
at room temperature for 2 h, then was poured into water and extracted
with EtOAc. The organic portion was washed with water, sat. aq. sodium
bicarbonate, water, brine, and dried over a phase separator. The solvent
was removed under reduced pressure. Purification of the crude product
by chromatography on silica using a gradient from 0 to 100% EtOAc in
iso-hexane afforded the title compound (2.0 g, 95 %). LC/MS: tRA =1.23
min, m/z: 461.3 [M+H]. 1H NMR (400 MHz, CDCl3) δ= 1.20 (d, J = 7.09 Hz,
3 H), 1.42 (s, 9 H), 1.61 (s, 1 H), 1.90 - 2.00 (m, 2 H), 2.23 - 2.34 (m, 2 H),
2.45 (s, 3 H), 2.65 (s, 1 H), 2.77 (s, 3 H), 2.94 - 3.10 (m, 1 H), 3.31 (t, J =
7.03 Hz, 2 H), 5.72 (s, 2 H), 6.73 (d, J = 15.28 Hz, 1 H), 7.18 (d, J = 1.96
Hz, 1 H), 7.28 (dd, J = 8.44, 2.08 Hz, 1 H), 7.43 (d, J = 8.44 Hz, 1 H), 7.91
ppm (d, J = 15.28 Hz, 1 H).
t
RA= 0.9 min, m/z: 517.2 [M+H]. 1H NMR (400 MHz, DMSO) δ= 1.16 - 1.30
(m, 4 H), 1.93 - 2.22 (m, 2 H), 2.41 (s, 3 H), 2.64 - 2.73 (m, 1 H), 2.85 (d,
J = 10.51 Hz, 1 H), 3.45 - 3.67 (m, 2H), 3.83 (s, 3 H), 3.93 - 4.29 (m, 1 H),
4.34 - 4.76 (m, 1 H), 6.12 (d, J = 2.08 Hz, 2 H), 7.22 (d, J = 15.28 Hz, 1
H), 7.32 - 7.52 (m, 2 H), 7.67 - 7.92 (m, 3 H), 8.06 (d, J = 8.07 Hz, 1 H),
8.14 - 8.30 ppm (m, 1 H).
(R,E)-3-(4-chloro-2-((5-methyl-2H-tetrazol-2-yl)methyl)phenyl)-1-(4-
((6-fluoropyridin-3-yl)methyl)-2-methylpiperazin-1-yl)prop-2-en-1-
one (8a): To a solution of compound 6a (120 mg, 0.33 mmol) in MeOH
(3.0 mL) were added AcOH (0.3 mL) and 2-fluoro-5-formylpyridine (65.0
mg, 0.49 mmol). After stirring for 5 min, 2-picoline borane (56.4 mg, 0.53
mmol) was added. The reaction mixture was stirred at RT for 16 h and
was concentrated. The crude product was purified by reversed-phase
preparative HPLC (method C) to give 25 mg of a white powder (13%).
LC/MS: tRA=1.01 min, m/z: 470 [M+H]. 1H NMR (400 MHz) δ= 1.28 (dd, J
= 6.78, 2.26 Hz, 4 H), 2.07 - 2.36 (m, 2 H), 2.41 - 2.47 (m, 3 H), 2.64 - 2.81
(m, 1 H), 2.88 (br. s., 1 H), 3.65 (br. s., 2 H), 4.15 (d, J = 13.18 Hz, 1 H),
4.55 (br. s., 1 H), 5.96 (s, 2 H), 7.03 (d, J = 15.31 Hz, 1 H,) 7.14 (d, J =
8.41 Hz, 1 H), 7.45 - 7.54 (m, 2 H), 7.71 (d, J = 15.31 Hz, 1 H), 7.83 (s, 1
H), 7.95 (br. s., 1 H), 8.20 ppm (br. s., 1 H).
(R,E)-3-(4-Chloro-2-((5-methyl-2H-tetrazol-2-yl)methyl)phenyl)-1-(2-
methylpiperazin-1-yl)prop-2-en-1-one (6a): To (R,E)-tert-butyl 4-(3-(4-
chloro-2-((5-methyl-2H-tetrazol-2-yl)methyl)phenyl)acryloyl)-3-
methylpiperazine-1-carboxylate (2.1 g, 4.56 mmol) in CH2Cl2 (22 mL) was
added TFA (4.21 mL, 54.7 mmol) and the mixture was stirred at room
temperature for 4 h. The solvent was removed under reduced pressure.
The resulting residue was loaded onto an Isolute® SCX-2 cartridge eluting
with MeOH followed by
2 M NH3 in MeOH. The fractions were
concentrated under reduced pressure to afford the title compound (1.2 g,
95%). LC/MS: tRA = 0.64 min, m/z = 361.6 [M+H]. 1H NMR (400 MHz,
CDCl3) δ= 1.40 (d, J = 7.09 Hz, 3 H), 2.45 (s, 3 H), 2.76 - 2.95 (m, 1 H),
3.09 (d, J = 2.69 Hz, 2 H), 3.17 - 3.53 (m, 2 H), 4.05 - 4.82 (m, 2 H), 5.72
(s, 2 H), 6.70 (d, J = 15.28 Hz, 1 H), 7.25 (d, J = 2.08 Hz, 1 H), 7.30 (dd,
J = 8.38, 2.02 Hz, 1 H), 7.43 (d, J = 8.31 Hz, 1 H), 7.93 ppm (d, J = 15.28
Hz, 1 H).
(R,E)-1-(4-((6-fluoropyridin-3-yl)methyl)-2-methylpiperazin-1-yl)-3-(2-
((5-methyl-2H-tetrazol-2-yl)methyl)-4-(trifluoromethyl)phenyl)prop-2-
en-1-one (8b): AcOH (0.28 mL) and 2-fluoro-5-formylpyridine (59.5 mg,
0.46 mmol) were added to a solution of compound 6b (120 mg, 0.30
mmol) in MeOH (2.8 mL). After stirring for 5 min, 2-picoline borane (51.6
mg, 0.49 mmol) was added. The reaction mixture was stirred at RT for 16
(R,E)-tert-Butyl 3-methyl-4-(3-(2-((5-methyl-2H-tetrazol-2-yl)methyl)-
4-(trifluoromethyl)phenyl)acryloyl)piperazine-1-carboxylate: T3P®
50% in ethyl acetate (4.5 mL, 7.7 mmol) was added to a solution of 5b (2
g, 6.41 mmol), (R)-tert-butyl 3-methylpiperazine-1-carboxylate (2.0 g, 6.4
mmol) and NEt3 (3.6 mL, 25.6 mmol) in CH2Cl2 (20 mL) and the resulting
mixture stirred for 1 h at room temperature. The reaction mixture was
diluted with sat. aq. sodium bicarbonate (100 mL) The aqueous solution
was extracted with ethyl acetate (3x100 mL). The combined organic
solutions were washed with water (50 mL), brine (50 mL), dried over
h
and concentrated. The crude product was purified by flash
chromatography: mobile phase: cyclohexane (A) / EtOAc (B); gradient: A
to B in 20min, to provide 26 mg as a white solid (16%). LC/MS: tRA=1.06
min, m/z: 504 [M+H]. 1H NMR (400 MHz, DMSO) δ= 1.23 (d, J = 5.50 Hz,
3 H), 1.90 - 2.24 (m, 2 H), 2.42 (s, 3 H), 2.62 - 2.74 (m, 1 H), 2.84 (d, J =
10.27 Hz, 1 H), 3.44 - 3.69 (m, 2 H), 3.95 - 4.32 (m, 1 H), 4.37 - 4.71 (m,
1 H), 6.12 (d, J = 1.96 Hz, 2 H), 7.12 - 7.28 (m, 2 H), 7.72 - 7.89 (m, 3 H),
6
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900297
ChemMedChem
FULL PAPER
7.96 (td, J = 8.22, 2.38 Hz, 1 H), 8.07 (d, J = 8.07 Hz, 1 H) 8.19 ppm (s, 1
H).
flash chromatography (mobile phase: cyclohexane (A) / EtOAc (B);
gradient: 100% A to 100% B) to afford 1.02 g of the desired product as
yellow oil, and which was directly used in the next step. LC/MS: tRA=0.85
min, m/z: 352.4 [M+H].
(R)-3-(4-chloro-2-((5-methyl-2H-tetrazol-2-yl)methyl)phenyl)-1-(4-((5-
methoxypyridin-2-yl)methyl)-2-methylpiperazin-1-yl)propan-1-one
(9a): A solution of compound 7 (90 mg, 0.19 mmol) and Pt/C (5%) (32 mg,
8.20 µmol) in EtOH (4.7 mL) was stirred at RT under hydrogen for 21
h. The reaction mixture was filtered over Celite and concentrated to give
78 mg as a yellow oil. The crude product was purified by reversed-phase
preparative HPLC (method C) to give 29 mg of pale yellow oil. The residue
was diluted with CH2Cl2 and passed through a phase separator,
concentrated and dried on high vacuum, at 50°C overnight to provide 24
mg of pale yellow oil (21%). LC/MS: tRB=2.84 min, m/z: 485.0 [M+H]. 1H
NMR (400 MHz, DMSO) δ= 1.24 (d, J = 6.78 Hz, 4 H), 2.46 (d, J = 1.76
Hz, 3 H), 2.55 - 2.69 (m, 3 H), 2.90 - 3.05 (m, 3 H), 3.12 - 3.27 (m, 2 H),
3.89 (s, 3 H), 3.93 - 4.17 (m, 3 H), 4.47 (br. s., 1 H), 5.92 (s, 2 H), 7.20 -
7.42 (m, 3 H), 7.46 (s, 2 H), 8.32 ppm (s, 1 H).
5-(2-(2-fluoroethoxy)ethoxy)-2-methylpyridine (10b): To a solution of
2-(2-((6-methylpyridin-3-yl)oxy)ethoxy)ethyl 4-methylbenzenesulfonate
(1.02 g, 2.48 mmol) in THF (15.7 mL) was added TBAF (1M in THF, 12.40
mL, 12.40 mmol) and the resulting solution was stirred at 65°C for 30 min.
The solvent was evaporated, and the residue taken up in EtOAc and
washed with water. The aqueous layer was extracted twice with EtOAc.
The combined organic layers were washed with brine, dried over a phase
separator and concentrated to the give crude product as brown oil, which
was purified by flash chromatography (cyclohexane (A) / EtOAc (B);
gradient: 100% A to 100% B) to afford the title compound as orange oil
(465 mg, 85%). LC/MS: tRA=0.44 min, m/z: 200.1 [M+H]. 1H NMR (400
MHz, DMSO): δ=2.41 (s, 3H), 3.64 - 3.72 (m, 1H), 3.76 - 3.83 (m, 3H),
4.16 (dd, J = 5.44, 3.73 Hz, 2H), 4.45 - 4.53 (m, 1H), 4.61 - 4.64 (m, 1H),
7.18 (d, J = 8.56 Hz, 1H), 7.31 (dd, J = 8.50, 3.00 Hz, 1H), 8.17 ppm (d, J
= 2.93 Hz, 1H).
(R)-1-(4-((5-methoxypyridin-2-yl)methyl)-2-methylpiperazin-1-yl)-3-
(2-((5-methyl-2H-tetrazol-2-yl)methyl)-4-(trifluoromethyl)phenyl)
propan-1-one (9b):
A
solution of compound
7
(24 mg, 0.05
5-(2-fluoroethoxy)-2-methylpyridine 1-oxide: A solution of compound
10a (591 mg, 3.81 mmol) in CHCl3 (17.3 mL) at 0°C was added m-
chloroperbenzoic acid (789 mg, 4.57 mmol). The resulting mixture was
stirred at 0°C for 2 h. The reaction mixture was quenched with a saturated
solution of Na2CO3 and extracted twice with CH2Cl2. The combined
organic layers were washed with brine, dried over a phase separator and
concentrated to give 145 mg of crude product as white solid, and which
was used in the next step without further purification. LC/MS: tRA=0.43 min,
m/z: 172.0 [M+H]..
mmol) and Pd/C (10%) (2.8 mg, 2.61 µmol) in EtOH (1.2 mL) was stirred
at RT under hydrogen for 17h. The reaction mixture was filtered over
Celite and concentrated to give 20.5 mg of a pale yellow oil (84%). LC/MS:
t
RB=3.12 min, m/z: 518.0 [M+H]. 1H NMR (400 MHz, DMSO) δ= 1.10 - 1.25
(m, 4 H), 1.78 - 2.08 (m, 2 H), 2.37 - 2.45 (m, 4 H), 2.56 - 3.20 (m, 6 H),
3.40 - 3.62 (m, 3 H), 3.82 (s, 3 H), 3.89 - 4.60 (m, 2 H), 6.06 (br. s., 2 H),
7.38 (s, 2 H), 7.55 (br. s., 1 H), 7.68 (br. s., 2 H), 8.19 ppm (s, 1 H).
5-(2-fluoroethoxy)-2-methylpyridine (10a):
methylpyridin-3-ol (0.5 g, 4.58
A
suspension of 6-
mmol), 2-fluoroethyl-4-
(5-(2-fluoroethoxy)pyridin-2-yl)methyl acetate (11a): To acetic
anhydride (748 µl, 7.93 mmol) was added at 80°C 5-(2-fluoroethoxy)-2-
methylpyridine 1-oxide (145 mg, 0.61 mmol). The reaction mixture was
heated up to 130°C and stirred for 30 min., poured into ice water and then
stirred at room temperature for 15 min. The product was extracted with
EtOAc. The organic layer was washed with sat. aq. NaHCO3 and brine.
The aqueous layer was extracted with EtOAc. The combined organic
layers were dried over a phase separator and concentrated to give the
crude product as brown oil, and which was purified by flash
chromatography (mobile phase: cyclohexane (A) / EtOAc (B); gradient:
100% A to 40% B) to afford 73 mg of the title compound as yellow oil, and
which was used in the next step without further purification. LC/MS:
methylbenzenesulfonate (1.5 g, 6.87 mmol), K2CO3 (0.82 g, 5.96
mmol) and NaI (0.04 g, 0.26 mmol) was stirred at 80°C for 16 h under
argon. The reaction mixture was allowed to cool to room temperature,
diluted with EtOAc and washed with water three times. The combined
aqueous layers were extracted with EtOAc. The combined organic layers
were washed with brine, dried over
a phase separator and
concentrated. The crude product was purified by flash chromatography on
silica gel (mobile phase: cyclohexane (A) / EtOAc (B); gradient: 100% A
to 100% B in 15min), to afford 591 mg of an orange solid (83%). LC/MS:
t
RA=0.37 min, m/z: 156.0 [M+H]. 1H NMR (400 MHz, CDCl3) δ= 2.43 (s, 3
H), 4.07 - 4.16 (m, 1 H), 4.17 - 4.26 (m, 1 H), 4.56 - 4.68 (m, 1 H), 4.71 -
4.80 (m, 1 H), 6.97 - 7.03 (m, 1 H), 7.05 - 7.14 (m, 1 H), 8.15 ppm (d, J =
2.81 Hz, 1 H).
t
RA=0.66 min, m/z: 214.0 [M+H].
5-(2-(2-fluoroethoxy)ethoxy)-2-methylpyridine 1-oxide: To a solution
of compound 10b (465 mg, 2.1 mmol) in CHCl3 (10.6 mL) at 0°C was
added m-chloroperbenzoic acid (435 mg, 2.52 mmol). The resulting
mixture was stirred at 0°C for 3 h, then quenched with sat. aq. Na2CO3
and extracted twice with CH2Cl2. The combined organic layers were
washed with brine, dried over a phase separator and concentrated to give
562 mg of crude product as yellow oil, and which was used in the next
step without further purification. LC/MS: tRA=0.48 min, m/z: 216.1 [M+H].
2-(2-((6-methylpyridin-3-yl)oxy)ethoxy)ethanol: A suspension of 6-
methylpyridin-3-ol
(1.5
g,
13.75
mmol),
diethylene
glycol
monochlorohydrine (5.83 mL, 55.0 mmol), K2CO3 (2.85 g, 20.62 mmol)
and NaI (0.11 g, 0.76 mmol) in DMF (18.6 mL) was stirred at 85°C for 16
h under an argon atmosphere. The reaction mixture was allowed to cool
to room temperature, diluted with EtOAc and washed twice with water.
The aqueous layer was extracted with CH2Cl2/i-PrOH 4:1. The combined
organic layers were washed with brine, dried over a phase separator and
concentrated. The combined organic layers were dried and concentrated
to give the crude product as purple oil, which was purified by flash
chromatography (mobile phase: CH2Cl2 (A) /MeOH (B); gradient: 0 to
10% B) to afford 5.0 g of brown oil, which was directly used in the next
step. LC/MS: tRA=0.32 min, m/z: 198.1 [M+H].
(5-(2-(2-fluoroethoxy)ethoxy)pyridin-2-yl)methyl acetate (11b): To
acetic anhydride (2018 µl, 21.39 mmol) was added at 80°C 5-(2-(2-
fluoroethoxy)ethoxy)-2-methylpyridine 1-oxide (562 mg, 1.64 mmol). The
reaction mixture was heated up to 130°C and stirred for 30 min., then
poured into ice water and stirred at RT for 15 min. The product was
extracted with EtOAc. The organic layer was washed with sat. aq.
NaHCO3 and brine. The aqueous layer was extracted with EtOAc. The
combined organic layers were dried over a phase separator and
concentrated to give the crude product as a brown oil which was purified
by flash chromatography (mobile phase: cyclohexane (A) / EtOAc (B);
gradient: 100% A to 40% B), to afford 352 mg of title compound as yellow
oil, and which was used in the next step without further purification.
LC/MS: tRA=0.71 min, m/z: 258.1 [M+H].
2-(2-((6-methylpyridin-3-yl)oxy)ethoxy)ethyl
4-methylbenzene
sulfonate: To stirred solution of 2-(2-((6-methylpyridin-3-
a
yl)oxy)ethoxy)ethanol (2.35 g, 4.05 mmol) in CH2Cl2 (10 mL) was slowly
added p-tolylsulfonyl chloride (1.87 g, 9.72 mmol) followed by
triethylamine (2.82 mL, 20.26 mmol) at 0°C, under argon. The reaction
mixture was stirred at RT for 16 h, then diluted with CH2Cl2 and water. The
organic layer was washed with brine, dried over a phase separator and
concentrated to give the crude product as brown oil, which was purified by
7
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900297
ChemMedChem
FULL PAPER
(5-(2-fluoroethoxy)pyridin-2-yl)methanol: To a solution of compound
11a (430 mg, 1.81 mmol) in EtOH (4.7 mL) and water (0.82 mL) was
added NaOH (145 mg, 3.63 mmol). The resulting reaction mixture was
stirred at reflux for 1h. The solvents were evaporated and the residue
extracted twice with EtOAc. The combined organic layers were dried over
a phase separator and concentrated to give 340 mg of crude product as
yellow oil, which was directly used in the next step. LC/MS: tRA=0.44 min,
m/z: 215.8 [M+H].
(R,E)-3-(4-chloro-2-((5-methyl-2H-tetrazol-2-yl)methyl)phenyl)-1-(4-
((5-(2-(2-fluoroethoxy)ethoxy)pyridin-2-yl)methyl)-2-
methylpiperazin-1-yl)prop-2-en-1-one (14a): To a solution of 6a (80 mg,
0.22 mmol) in MeOH (2.0 mL) were added AcOH (0.2 mL) and 12b (93
mg, 0.33 mmol). After stirring for 5 min, 2-picoline borane (44.2 mg, 0.35
mmol) was added. The reaction mixture was stirred at RT for 20h. The
volatiles were evaporated and the residue purified by preparative HPLC
(method C). The desired fractions were concentrated. The residue was
taken up in EtOAc and washed with sat. aq. NaHCO3. The aqueous layer
was extracted with EtOAc. The combined organic layers were washed
with brine, dried over a phase separator and concentrated to give 63 mg
5-(2-fluoroethoxy)picolinaldehyde (12a): To
a solution of (5-(2-
fluoroethoxy)pyridin-2-yl)methanol (340 mg, 1.63 mmol) in CH2Cl2 (6.5
mL) was added MnO2 (1416 mg, 16.29 mmol). The resulting reaction
mixture was stirred at RT for 2 days. The reaction mixture was filtered over
Celite and washed three times with EtOAc. The filtrate was evaporated to
dryness to give 196 mg of crude product as yellow oil which was directly
used in the next step. LC/MS: tRA=0.61 min, m/z: 170.0 [M+H].
1
yellow oil (50%). LC/MS: tRA=0.87 min, m/z: 558.2 [M+H]. H NMR (400
MHz, DMSO, 100°C) δ=1.28 (d, J = 6.78 Hz, 3H), 2.01 - 2.15 (m, 1H), 2.25
(dd, J = 11.48, 3.07 Hz, 1H), 2.44 (s, 3H), 2.73 (d, J = 11.42 Hz, 1H), 2.88
(d, J = 11.29 Hz, 1H), 3.17 (br. s., 1H), 3.52 - 3.66 (m, 2H), 3.70 - 3.76 (m,
1H), 3.78 - 3.88 (m, 3H), 4.12 (d, J = 12.55 Hz, 1H), 4.18 - 4.28 (m, 2H),
4.44 - 4.56 (m, 2H), 4.57 - 4.65 (m, 1H), 5.96 (s, 2H), 7.03 (d, J = 15.31
Hz, 1H), 7.40 (d, J = 1.76 Hz, 2H), 7.44 - 7.51 (m, 2H), 7.71 (d, J = 15.31
Hz, 1H), 7.81 (d, J = 8.41 Hz, 1H), 8.24 ppm (t, J = 1.69 Hz, 1H).
(5-(2-(2-fluoroethoxy)ethoxy)pyridin-2-yl)methanol: To a solution of
compound 11b (352 mg, 1.08 mmol) in EtOH (4.7 mL) and water (0.82
mL) was added NaOH (86 mg, 2.16 mmol). The resulting reaction mixture
was stirred at reflux for 1h. The solvents were evaporated and the residue
was extracted twice with EtOAc. The combined organic layers were dried
over a phase separator and concentrated to give 282 mg of crude product
as orange oil, which was directly used in the next step. LC/MS: tRA=0.43
min, m/z: 216.1 [M+H].
(R,E)-1-(4-((5-(2-(2-fluoroethoxy)ethoxy)pyridin-2-yl)methyl)-2-
methylpiperazin-1-yl)-3-(2-((5-methyl-2H-tetrazol-2-yl)methyl)-4-
(trifluoromethyl)phenyl)prop-2-en-1-one (14b): The title compound
was prepared by a similar method to compound 14a, from compounds 6b
and 12b (85 mg, 0.30 mmol) to give 60 mg (49%) of title compound as
yellow oil. LC/MS: tRA=0.92 min, m/z: 592.2 [M+H]. 1H NMR (400 MHz,
DMSO) δ=1.29 (d, J = 6.65 Hz, 3 H) 2.09 (td, J = 11.67, 3.14 Hz, 1 H) 2.24
(dd, J = 11.36, 3.70 Hz, 1 H) 2.40 - 2.46 (m, 3 H) 2.72 (d, J = 11.29 Hz, 1
H) 2.88 (d, J = 11.17 Hz, 1 H) 3.12 - 3.27 (m, 1 H) 3.50 - 3.64 (m, 2 H)
3.70 - 3.75 (m, 1 H) 3.78 - 3.87 (m, 3 H) 4.12 (d, J = 13.80 Hz, 1 H) 4.19 -
4.26 (m, 2 H) 4.43 - 4.54 (m, 2 H) 4.57 - 4.65 (m, 1 H) 6.06 (s, 2 H) 7.11
(d, J = 15.43 Hz, 1 H) 7.40 (d, J = 1.13 Hz, 2 H) 7.69 - 7.84 (m, 3 H) 7.99
(d, J = 8.53 Hz, 1 H) 8.24 ppm (s, 1 H).
5-(2-(2-fluoroethoxy)ethoxy)picolinaldehyde (12b): To a solution of (5-
(2-(2-fluoroethoxy)ethoxy)pyridin-2-yl)methanol (282 mg, 1.05 mmol) in
CH2Cl2 (4.0 mL) was added MnO2 (911 mg, 10.5 mmol). The resulting
reaction mixture was stirred at RT for 2 days, then filtered over Celite and
washed three times with EtOAc. The filtrate was evaporated to dryness to
give 185 mg of crude product as yellow oil, which was directly used in the
next step. LC/MS: tRA=0.65 min, m/z: 214.0 [M+H].
(R)-1-(4-((5-(2-fluoroethoxy)pyridin-2-yl)methyl)-2-methylpiperazin-
1-yl)-3-(2-((5-methyl-2H-tetrazol-2-yl)methyl)-4-
(R,E)-3-(4-chloro-2-((5-methyl-2H-tetrazol-2-yl)methyl)phenyl)-1-(4-
((5-(2-fluoroethoxy)pyridin-2-yl)methyl)-2-methylpiperazin-1-yl)prop-
2-en-1-one (2): To a solution of 6a (100 mg, 0.28 mmol) in MeOH (2.5
mL) were added AcOH (0.25 mL) and 12a (70.3 mg, 0.42 mmol). After
stirring for 5 min, 2-picoline borane (55.3 mg, 0.44 mmol) was added. The
reaction mixture was stirred at room temperature for 16 h. The volatiles
were evaporated and the residue purified by reversed-phase preparative
HPLC (method C) to provide a white solid (33 mg, 23%). LC/MS: tRA=0.92
min, m/z: 548.0 [M+H]. 1H NMR (400 MHz, DMSO, 100°C) δ=1.29 (d, J =
6.78 Hz, 3H) 2.00 - 2.14 (m, 1H) 2.21 - 2.29 (m, 1H) 2.44 (s, 3H) 2.66 -
2.76 (m, 1H) 2.84 - 2.92 (m, 1H) 3.12 - 3.25 (m, 1H) 3.60 (d, J = 10.67 Hz,
2H) 4.06 - 4.17 (m, 1H) 4.28 - 4.34 (m, 1H) 4.35 - 4.42 (m, 1H) 4.45 - 4.58
(m, 1H) 4.66 - 4.74 (m, 1H) 4.78 - 4.87 (m, 1H) 6.06 (s, 2H) 7.11 (d, J =
15.43 Hz, 1H) 7.42 (d, J = 1.88 Hz, 2H) 7.77 (d, J = 5.65 Hz, 3H) 7.92 -
8.04 (m, 1H) 8.26 ppm (s, 1H).
(trifluoromethyl)phenyl)propan-1-one (15): A solution of compound 13
(46 mg, 0.084 mmol) and Pd/C (10%) (5.72 mg, 5.38 µmol) in EtOH (4.00
mL) was stirred at room temperature under hydrogen for 18 h. The
reaction mixture was filtered over Celite and concentrated. The residue
was taken up in 1mL of MeOH and purified by preparative HPLC (method
C). The fractions containing the product were combined and lyophilized
overnight. The resulting white powder was diluted with EtOAc and washed
with a saturated solution of NaHCO3. The aqueous layer was extracted
with EtOAc and the combined organic layers washed with brine, dried over
a phase separator, concentrated and dried under vacuum to give the title
compound as yellow oil (22 mg, 47%). LC/MS: tRA=0.89 min, m/z: 550.1
[M+H]. 2 min Final Analysis. 1H NMR (400 MHz, DMSO) δ=1.16 - 1.22 (m,
3H), 1.27 - 1.31 (m, 1H), 1.92 - 2.13 (m, 2H), 2.46 (s, 3H), 2.55 - 2.85 (m,
4H), 3.04 - 3.07 (m, 3H), 3.48 - 3.62 (m, 2H), 3.78 - 4.03 (m, 1H), 4.28 -
4.41 (m, 2H), 4.66 - 4.85 (m, 2H), 5.96 - 6.08 (m, 2H), 7.35 - 7.44 (m, 2H),
7.53 - 7.71 (m, 3H), 8.21 - 8.29 ppm (m, 1H).
(R,E)-1-(4-((5-(2-fluoroethoxy)pyridin-2-yl)methyl)-2-
methylpiperazin-1-yl)-3-(2-((5-methyl-2H-tetrazol-2-yl)methyl)-4-
(trifluoromethyl)phenyl)prop-2-en-1-one (13): To a solution of 6b ( 100
mg, 0.25 mmol) in MeOH (2.3 mL) were added AcOH (0.23 mL) and 12a
(64.3 mg, 0.38 mmol). After stirring for 5 min, 2-picoline borane (50.6 mg,
0.41 mmol) was added. The reaction mixture was stirred at room
temperature for 16 h. The volatiles were evaporated and the residue
purified by flash chromatography to provide a white solid (81 mg, 58%).
LC/MS: tRB= 3.4 min, m/z: 548.1 [M+H]. 1H NMR (400 MHz, DMSO,
100°C) δ=1.29 (d, J = 6.78 Hz, 3 H), 2.00 - 2.14 (m, 1 H), 2.21 - 2.29 (m,
1 H), 2.44 (s, 3 H), 2.66 - 2.76 (m, 1 H), 2.84 - 2.92 (m, 1H), 3.12 - 3.25
(m, 1 H), 3.60 (d, J = 10.67 Hz, 2 H), 4.06 - 4.17 (m, 1 H), 4.28 - 4.34 (m,
1 H), 4.35 - 4.42 (m, 1 H), 4.45 - 4.58 (m, 1 H), 4.66 - 4.74 (m, 1 H), 4.78
- 4.87 (m, 1 H), 6.06 (s, 2 H), 7.11 (d, J = 15.43 Hz, 1 H), 7.42 (d, J = 1.88
Hz, 2 H), 7.77 (d, J = 5.65 Hz, 3 H), 7.92 - 8.04 (m, 1 H), 8.26 ppm (s, 1
H).
Methyl 2-(fluoromethyl)oxazole-4-carboxylate (16): To a solution of
methyl 2-(chloromethyl)oxazole-4-carboxylate (1.0 g, 5.70 mmol) in
CH3CN (28.5 mL) was added TBAF (1M in THF, 17.09 mL, 17.09 mmol)
at room temperature. The resulting green solution was stirred for 21 h
under argon at RT. The reaction mixture was poured in water and
extracted twice with EtOAc. The combined organic layers were washed
with brine, dried over a phase separator and concentrated to give crude
product as orange oil. The crude product was purified by preparative
HPLC (method C). The fractions containing the desired product were
combined, diluted with EtOAc and washed with sat. aq. NaHCO3. The
aqueous layer was extracted with EtOAc. The combined organic layers
were washed with brine, dried over a phase separator and concentrated
to give 291 mg of a white solid (32%). LC/MS: tRA=0.49 min, m/z: 160.0
[M+H]. 1H NMR (400 MHz, DMSO): δ=3.76 (s, 3 H) 5.43 (s, 1 H) 5.55 (s,
1 H) 8.91 ppm (d, J = 1.34 Hz, 1 H).
8
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900297
ChemMedChem
FULL PAPER
(2-(fluoromethyl)oxazol-4-yl)methanol: To a solution of compound 16
(291 mg, 1.46 mmol) in THF (3.6 mL) at -78°C under argon, was added
dropwise DIBAL-H (1M in THF, 3.22 mL, 3.22 mmol). The reaction mixture
was then stirred at -78°C for 3 h and at room temperature for 16 h.
UPLC/MS showed remaining starting material therefore the reaction
mixture was cooled down to -78°C and DIBAL-H (1M in THF, 3.22 mL,
3.22 mmol) was added and the reaction mixture stirred at -78°C for 3 h,
then diluted with CH2Cl2 at -78°C, quenched with MeOH/water/2M aq.
NaOH (1 mL) and stirred for 15 min. Solid Na2SO4 was added at room
temperature and the suspension stirred for 15 min. The salts were
removed by filtration and the filtrate concentrated under vacuum. The
crude product was purified by flash chromatography (mobile phase:
CH2Cl2 (A) / MeOH (B); gradient: : from 100% A to 5% B), to afford the
title compound as yellow oil (79 mg, 41%). LC/MS: tRA=0.32 min, m/z:
silica gel (mobile phase: cyclohexane (A) / EtOAc (B); gradient: 100% A
to 100% B), to obtain the product as colourless oil (180 mg, 62%). LC/MS:
t
δ=1.12-1.27 (m, 3H), 1.97 (s, 1H), 2.11 (s, 1H), 2.40 (d, J = 4.0 Hz, 6H),
2.64 (d, J = 11.3 Hz, 1H) 2.80 (d, J = 11.0 Hz, 1H), 3.27 (s, 1H), 3.44-3.59
(m, 2H), 4.01 (q, J = 7.1 Hz, 1H), 4.22 (dd, J = 5.3, 2.8 Hz, 2H), 4.34 (dd,
J = 5.3, 2.8 Hz, 2H), 4.4 (s, 1H), 6.03-5.91 (m, 2H), 7.11 (d, J = 15.2 Hz,
1H), 7.29 (dd, J = 8.6, 2.9 Hz, 1H), 7.35 (d, J = 8.6 Hz, 1H), 7.43-7.51 (m,
4H), 7.70 (d, J = 15.2 Hz, 1H), 7.78 (d, J = 8.3 Hz, 2H), 7.87 (d, J = 9.1
Hz, 1H), 8.07 ppm (d, J = 2.8 Hz, 1H).
RA=1.06 min, m/z: 666.5 [M+H]. 1H NMR (400 MHz, 300°C, DMSO)
[18F]-(R,E)-3-(4-chloro-2-((5-methyl-2H-tetrazol-2-yl)methyl) phenyl)-
1
1-(4-((5-(2-fluoroethoxy)pyridin-2-yl)methyl)-2-methylpiperazin-1-
132.0 [M+H]. H NMR (400 MHz, DMSO) δ=4.31 - 4.34 (m, 2H) 5.32 (s,
1H) 5.44 (s, 1H) 7.84 - 8.03 ppm (m, 1H).
yl)prop-2-en-1-one ([18F]2):
[
18F]fluoride produced via the 18O(p,n)18F
nuclear reaction was purchased from PETNET. The [18F]fluoride in 1.5 mL
of [18O]H2O was trapped on a QMA-Light Sep-Pak (Waters Accell Plus
QMA cartridge, 46 mg, PN 1860045-40). It was then eluted into the reactor
with a solution of K2CO3 (0.75 mg) and Kryptofix-2.2.2 (7.5 mg) in
deionized water (0.4 mL) and MeCN (0.4 mL). The [18F]fluoride was dried
by heating under vacuum (5 min. at 70 °C and 5 min. at 100 °C) in a stream
of nitrogen. A solution of 20 (2 mg, 3 µmol) in anhydrous. DMSO (0.7 mL)
was added and heated to 140 °C under stirring for 10 min., then cooled to
50 °C, diluted with 4.3 mL of HPLC water, and the mixture passed through
an Alumina N Light cartridge. The filtrate was loaded onto a semi-
preparative HPLC system for purification (column: Phenomenex Luna
C18(2), 5 μm, 250 x 10 mm, CAT# 00G-4252-N0; mobile phase: 50%
MeCN /50% water/0.1% v/v TEA; flow: 4 mL-min). The product peak (rt =
12.2 min.) was collected into a flask containing 0.5% w/v NaAsc in water
2-(fluoromethyl)oxazole-4-carbaldehyde (17): To a solution of (2-
(fluoromethyl)oxazol-4-yl)methanol (78 mg, 0.6 mmol) in CH2Cl2 (6 mL)
was added MnO2 (517 mg, 5.95 mmol). The resulting mixture was stirred
at RT for 2 days. The reaction mixture was filtered over Celite and washed
three times with CH2Cl2. The filtrate was evaporated to dryness to give 32
mg of crude product, which was used in the next step without further
purification. LC/MS: tRA=0.34 min, m/z: 130.0 [M+H].
(R,E)-1-(4-((2-(fluoromethyl)oxazol-4-yl)methyl)-2-methylpiperazin-1-
yl)-3-(2-((5-methyl-2H-tetrazol-2-yl)methyl)-4-
(trifluoromethyl)phenyl)prop-2-en-1-one (18): The title compound was
prepared by a similar method to compound 14a, from compounds 6b (80
mg, 0.20 mmol) and 17 (26.2 mg, 0.20 mmol), to give a of colourless oil
(25 mg, 24%). LC/MS: tRA=0.89 min, m/z: 508.3 [M+H]. 1H NMR (400 MHz,
DMSO) δ=1.27 (d, J = 6.97 Hz, 3H), 2.05-2.11 (m, 1H), 2.23-2.27 (m, 1H),
2.44 (s, 3H), 2.76-2.80 (m, 1H), 2.90-2.95 (m, 1H), 3.10-3.20 (m, 1H), 3.5
(s, 2H), 4.1 (d, 1H), 4.5 (s, 1H), 4.45 (d, 2H), 6.06 (s, 2H), 7.1 (d, 1H), 7.70
- 7.80 (m, 4H), 7.98-8.01 (m, 1H).
(45 mL). The purified
[
18F]2 was trapped on
a C18-E cartridge
(Phenomenex, C18-E-50 mg cartridge, PN 8B-S001-DAK), washed with
0.5% w/v NaAsc in water (5 mL), eluted with EtOH (0.5 mL), then diluted
with 0.33% w/v NaAsc, 0.33% v/v Tween-80 in 0.9% w/v NaCl (4.5 mL).
The [18F]2 formulation was transferred to a sterile dose vial and submitted
for quality control. The radiochemical yield was ~15% (decay corrected)
and produced a tracer with <99% radiochemical purity, >93% chemical
purity and a specific activity of 304±136 GBq/µmole.
(R,E)-3-(4-chloro-2-((5-methyl-2H-tetrazol-2-yl)methyl)phenyl)-1-(4-
((5-hydroxypyridin-2-yl)methyl)-2-methylpiperazin-1-yl)prop-2-en-1-
one (19): A stirred solution of compound 6a ( 3.0 g, 8.3 mmol) in (ClCH2)2
(60 mL) was treated with 5-hydroxypyridine-2-carboxaldehyde (2.0 g, 16.6
mmol), sodium triacetoxyborohydride (3.5 g, 16.62 mmol) and acetic acid
(0.95 mL) at 0°C. The resulting mixture was stirred at room temperature
for 17 h. The solvent was evaporated, water was added to the residue,
and the aqueous phase extracted with EtOAc. The organic layer was
washed with sat. aq. NaHCO3, brine, dried over solid Na2SO4, filtered and
concentrated under vacuum. The residue was purified by flash
chromatography on silica gel (mobile phase: CH2Cl2 (A) / MeOH (B);
gradient: from 100% A to 5% B), to afford the title compound as a white
Acknowledgements
The authors would like to acknowledge Caroline Radoch for the CHI(IAM)
assay, Dr Berndt Oberhauser, Rrezarta Ramqaj and Emanuele Mauro for skilful
synthetic support, Clare Thomas for immunohistochemistry, Ildiko Polyak
(Invicro) for autoradiographic measurements, and Dr. David Alagille (Invicro)
for radiochemistry.
1
powder (2.8 g, 72%). LC/MS: tRA= 0.73 min, m/z: 468.2 [M+H]. H NMR
(600 MHz, DMSO-d6) δ=1.24 (s, 3H), 2.19-2.89 (m, 2H), 2.39 (s, 1H), 2.41
(s, 3H), 2.62 (s, 1H), 2.66 (d, J = 11.3 Hz, 1H), 2.82 (s, 1H), 3.45 (d, J =
13.7 Hz, 1H), 3.53 (d, J = 13.7 Hz, 1H), 3.98-4.28 (m, 1H), 4.36-4.65 (m,
1H), 5.96-6.05 (m, 2H), 7.11-7.19 (m, 1H), 7.28 (d, J = 8.4 Hz, 1H), 7.51-
7.53 (m, 1H), 7.72 (d, J = 15.2 Hz, 1H), 7.90 (d, J = 8.6 Hz, 1H), 8.04 (d,
J = 2.8 Hz, 1H), 9.78 (s, 1H).
Keywords: Enzymes • Imaging agents • Oncology • Fibrosis •
Fluorinated ligands
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10.1002/cmdc.201900297
ChemMedChem
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PRIMATX is a novel PET tracer for imaging the secreted enzyme autotaxin (ATX). It proved sensitive to fibrosis-associated ATX
expression in fibrotic lung tissue from idiopathic pulmonary fibrosis patients, and allows the detection of autotaxin-expressing
melanoma tumors in living mice with a good signal-to-noise ratio. This study provides a potential diagnostic tool for clinical
development, and confirms the relevance of ATX for this purpose.
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