Synthesis and evaluation of the novel 2-[18F]fluoro-3-propoxy- triazole-pyridine-substituted losartan for imaging AT1 receptors

Article abstract of DOI:10.1016/j.bmc.2014.06.011

The 2-[¹⁸F]fluoro-3-pent-4-yn-1-yloxypyridine ([ ¹⁸F]FPyKYNE) analog of the potent non-peptide angiotensin II type 1 receptor (AT₁R) blocker losartan was produced via click chemistry linking [¹⁸F]FPyKYNE to azide-modified tetrazole-protected losartan followed by TFA deprotection. Preliminary small animal imaging with positron emission tomography (PET) in rats displayed high uptake in the kidneys with good contrast to surrounding tissue. Rat metabolism displayed the presence of 23% unchanged tracer in plasma at 30 min. Upon co-administration with AT ₁R blocker candesartan (2.5, 5 and 10 mg/kg), a dose-dependent reduction (47-65%) in tracer uptake was observed in the kidney, while no difference was observed following AT₂R blocker PD123,319 (5 mg/kg), indicating binding selectivity for AT₁R over AT₂R and potential for imaging AT₁R using PET.

Full text of DOI:10.1016/j.bmc.2014.06.011

Bioorganic & Medicinal Chemistry 22 (2014) 3931–3937
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
18
Synthesis and evaluation of the novel 2-[ F]fluoro
3-propoxy-triazole-pyridine-substituted losartan
for imaging AT receptors
-
1
Natasha Arksey a,b, Tayebeh Hadizad , Basma Ismail , Maryam Hachem a,b, Ana C. Valdivia a,b
a
a,b
,
a,b
a
a,b,c,
⇑
Rob S. Beanlands , Robert A. deKemp , Jean N. DaSilva
a
Cardiac PET Centre, University of Ottawa Heart Institute, 40 Ruskin St., Ottawa, ON, Canada
Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, ON, Canada
Department of Radiology, Radio-Oncology and Nuclear Medicine, University of Montreal, University of Montreal Hospital Research Centre (CRCHUM), Montréal, Québec, Canada
b
c
a r t i c l e i n f o
a b s t r a c t
The 2-[18F]fluoro-3-pent-4-yn-1-yloxypyridine ([18F]FPyKYNE) analog of the potent non-peptide angio-
tensin II type 1 receptor (AT R) blocker losartan was produced via click chemistry linking [ F]FPyKYNE
1
to azide-modified tetrazole-protected losartan followed by TFA deprotection. Preliminary small animal
imaging with positron emission tomography (PET) in rats displayed high uptake in the kidneys with good
contrast to surrounding tissue. Rat metabolism displayed the presence of 23% unchanged tracer in plasma
Article history:
Received 26 February 2014
Revised 28 May 2014
Accepted 6 June 2014
Available online 17 June 2014
18
at 30 min. Upon co-administration with AT
dent reduction (47–65%) in tracer uptake was observed in the kidney, while no difference was observed
following AT R blocker PD123,319 (5 mg/kg), indicating binding selectivity for AT R over AT R and poten-
tial for imaging AT R using PET.
1
R blocker candesartan (2.5, 5 and 10 mg/kg), a dose-depen-
Keywords:
Fluorine-18
Click chemistry
Losartan
2
1
2
1
Ó 2014 Elsevier Ltd. All rights reserved.
FPyKYNE
AT
PET
1
1
. Introduction
Previous AT
1
R PET tracers have been labeled with C-11,2–4 how-
ever F-18 offers some key advantages: namely, a longer half-life
5
The renin-angiotensin-system (RAS) is an important regulator
of blood pressure and exerts its effects mainly through activation
of the AT R and AT R subtypes by angiotensin II. Activation of
the AT R induces most of the known pathophysiologic effects of
(109.6 vs 20.4 min) allowing for multiple patient scans from a sin-
gle formulation and shipment to other sites without radiochemis-
try or cyclotron capabilities, and a shorter RMS (Root Mean
Squared) positron range (0.23 vs 0.39 mm in soft tissue) enabling
1
2
6
1
angiotensin II, including vasoconstriction, sodium and water reten-
higher resolution images. Structure–activity relationship studies
and recent work reported that large prosthetic groups can be intro-
duced at the imidazole 5-position with minimal changes both in
binding properties and antagonistic efficacy compared to the par-
tion, hypertrophy, cell proliferation and fibrosis.1 In addition to
1
angiotensin-converting enzyme inhibitors, AT R blockers such as
losartan, are well established therapies for the treatment of hyper-
tension and cardiovascular diseases. Several diseases including
renal hypertension, diabetes and myocardial infarction exhibit
2
,7
ent compound.
8
Click chemistry is a popular method of conjugation, owing to
the regiospecific nature of the reactions, mild reaction conditions,
high yields and ease of purification. The mainstay of click chemis-
try is the copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC).9
This ligation is one of only a few truly chemoselective and regiose-
lective reactions. The resulting linkage, a 1,4-disubstituted triazole,
acts as amide bond isostere, but is more resistant to hydrolysis,
1
AT R dysregulation. The ability to non-invasively detect this
abnormal regulation by molecular imaging modalities such as
PET provides the potential to monitor disease progression and
guide therapy accordingly.
10
oxidation and reduction in physiological conditions. Several
1
8
⇑
Corresponding author. Addresses: Department of Radiology, Radio-Oncology
and Nuclear Medicine, University of Montreal. CRCHUM - Tour Viger, 900 rue
St-Denis, Room R11.440, Montréal, QC H2X 0A9, Canada. Tel.: +1 (514)890
F-labeled alkynes and their subsequent use in click chemistry
11,12
have been described in the literature.
Herein we describe the conjugation of [18F]FPyKYNE, following
8
000x30653.
1
3
the procedure already published, to azide-modified trityl losartan
E-mail address: jean.dasilva@umontreal.ca (J.N. DaSilva).
http://dx.doi.org/10.1016/j.bmc.2014.06.011
968-0896/Ó 2014 Elsevier Ltd. All rights reserved.
0

3
932
N. Arksey et al. / Bioorg. Med. Chem. 22 (2014) 3931–3937
via click chemistry using an automated dual reactor module
TRACERlab FX N Pro) and provide a preliminary in vivo evaluation
of the novel F-labeled losartan derivative in rats using micro-PET
imaging. To the best of our knowledge, there are currently no publi-
10 min. Purified 2b was delivered to the second reaction vessel
in a 15–20% yield (decay-corrected) by passing the reaction mix-
ture through a series of three silica cartridges with an eluent of
50:50 ether/pentane. Following evaporation of the solvent, the
labeled prosthetic group 2b was then conjugated to the azide-
modified tetrazole-protected losartan precursor 4 for 30 min at
(
1
8
1
6
1
cations describing the synthesis of an F-18 labeled AT R PET tracer.
9
5 °C in DMSO. The reaction mixture was then cooled, treated with
2
2
. Results and discussion
.1. Chemistry
TFA, and heated to 80 °C for 2 min prior to loading onto HPLC. The
fraction containing 5b was collected and loaded onto a precondi-
tioned C18 cartridge (SPE) to remove the HPLC solvents. Pure 5b
was eluted with minimal EtOH and diluted in saline to provide
the final product in 44–70% yield (decay-corrected from 2b), or
an overall yield of 7–14% (decay-corrected from end-of-beam
The synthesis of 2-butyl-4-chloro-5-[[3-{[1-methyl-4-yl](1,2,3)
0
triazol}propoxy]-2-fluoro-pyridine]-1-[[2 -[tetrazole-5-yl]biphenyl-
4
-yl]methyl]imidazol, 5a, hereafter named FPyKYNE-losartan, is
(
EOB)). The yield is much lower than that of the non-radioactive
described in Scheme 1. It’s precursor, 2-butyl-4-chloro-5-(azido
methyl)-1-[[2 -[(triphenylmethyl)tetrazole-5-yl]biphenyl-4-yl]
methyl]imidazol 4, was prepared according to the method previ-
ously reported, a nucleophilic substitution reaction that converts
the 5 hydroxymethyl group in 3 to the corresponding azide moiety
in 87% yield. Compound 1, NO
0
synthesis likely due to shorter reaction times, the amount of F-18
fluoride available for radiofluorination compared to the stoichiom-
etric quantities used for the synthesis of the standard, and product
loss in the components of the module (i.e., reaction vessels, tubing,
SPE cartridges).
1
4
0
2
PyKYNE (2-nitro-3-pent-4-yn-1-
The identity of the product was confirmed by a single peak on
analytical HPLC following a co-injection of the formulated product
with cold standard (Fig. 1). The radiochemical purity was consis-
tently greater than 98% with specific activity from 200–
yloxypyridine), 2a FPyKYNE (2-fluoro-3-pent-4-yn-1-yloxypyri-
1
8
dine) and 2b [ F]FPyKYNE were synthesized according to the
1
5,16
literature.
Replacement of the nitro group on 1 by fluorine
using potassium fluoride (KF) readily afforded the cold prosthetic
1
4
200 mCi/lmol (7.4–155 GBq/lmol). The entire synthesis, includ-
group 2a. Both compounds 1 and 2a were characterized by
H
1
3
ing reformulation, took less than 2 h from EOB. In a representative
and C NMR and high-resolution mass spectrometry (HRMS).
19
synthesis, 40 mCi (1.48 GBq) of labeled product 5b could be
Compound 2a was further characterized by F NMR. The results
proved consistent with those reported.15 Cyclocondensation of 2a
and 4 was achieved through the copper(I)-catalyzed cycloaddition
1
8
obtained from 850 mCi (31.45 GBq) of [ F]fluoride.
The level of copper in the final formulations measured by induc-
tively coupled plasma (ICP) MS was 23.3 ± 5.4 ppb, 26.3 ± 6.5 ppb
in blanks, 23.6 ppb in pure saline, and 18 ppb in pure ethanol, all
well below the pharmaceutically maximum acceptable exposure
to residual metals on a chronic basis. These results indicate no
meaningful contamination of copper in the final formulation.
4
reaction using an aqueous solution of copper(II) sulfate (CuSO )
and sodium ascorbate as the source of Cu(I). The reaction was
amenable to a range of temperatures (room temperature to
1
2 2
20 °C) and a variety of co-solvents (e.g., DMSO, DMF, CH Cl ,
MeCN). A quick acid hydrolysis of the protected tetrazole with
TFA at 80 °C followed by flash column chromatography purification
afforded the desired product 5a, FPyKYNE-losartan, in 44% yield.
Compound 5a was fully characterized by accurate mass measure-
2
.3. Biological evaluation in rats
1
13
19
Preliminary in vivo evaluation of the binding properties of
F]FPyKYNE-losartan was performed using micro-PET imaging.
ment, H NMR, C NMR, and F NMR.
[18
Normal untreated rats showed the greatest accumulation of activ-
ity in the liver and kidneys, respectively (Fig. 2A). The time-activity
curves (TAC) derived from the left kidney displayed a sharp
increase in activity uptake in the first few minutes, following the
arterial blood input functions, which then washed out slowly to
background levels around 55 min (Fig. 2B). The renal activity had
2
.2. Radiochemistry
The first step in the production of [18F]FPyKYNE-losartan, 5b
was radiofluorination of the nitro precursor 1 (Scheme 1). This step
occurred in the first reaction vessel of the TRACERlab FX N Pro
using
1
8
2 3
[ F]KF/K222/K CO in anhydrous DMSO at 120 °C for
Scheme 1. Synthesis of [18F]FPyKYNE-losartan and standard via the Cu(I)-catalyzed [2 + 3] cycloaddition reaction between azide-modified losartan and [18F]FPyKYNE,
performed in the TRACERlab FXF-N automated module (GE Healthcare).

N. Arksey et al. / Bioorg. Med. Chem. 22 (2014) 3931–3937
3933
(
(
(
A)
B)
C)
2.3.1. In vivo competition studies
In rats treated with the AT R antagonist PD 123,319 (IC50 for rat
R = 34 nM), kidney TAC followed similar time course
to normal rats (Fig. 2). No effect on tracer binding to AT R was
2
1
7
adrenal AT
2
1
observed (DV values were 2.76 ± 0.68 and 3.02 ± 0.20 (p = 0.28)
for normal and PD 123,319 blocking scans, respectively) confirm-
1
8
1 2
ing [ F]FPyKYNE-losartan binding selectivity for AT R over AT R.
A significant 47–65% (p <0.02) dose-dependent reduction in
renal uptake of the tracer was observed after administration of
1
8
candesartan (IC50 for AT
specificity. Doses of 2.5, 5.0, and 10 mg/kg reduced the SUV at
0 min to 1.25 ± 0.17, 0.99 ± 0.38 and 0.61 ± 0.15, respectively.
1 1
R = 110 nM), suggesting AT R binding
1
Similarly, the DV values were reduced in a dose-dependent manner
3
to 1.46 ± 0.30, 1.45 ± 0.21 and 0.96 ± 0.06 mL/cm with doses of
2
.5, 5 and 10 mg/kg, respectively (Fig. 3).
2
.3.2. Radiolabeled metabolite analysis of [18F]FPyKYNE-
losartan in rat plasma
Column-switch High Performance Liquid Chromatography
1
8
(
HPLC) metabolite analysis of [ F]FPyKYNE-losartan exhibited
three distinct radioactive peaks in rat plasma, with retention times
of approximately 0.5–1 min (peak 1), 6.8 min (peak 2) and 10 min
Figure 1. Quality control for [18F]FPyKYNE-losartan formulated product by analyt-
ical HPLC. (A) UV chromatogram of cold standard; (B) radiation and UV chromato-
gram of product formulation; (C) UV chromatogram from co-injection of product
and standard overlaid on UV chromatogram of product from B. UV spectra were
(
peak 3) post-switch, respectively (Fig. 4). Peak 1 corresponds to
hydrophilic labeled metabolite(s) eluted from the capture column,
that is unlikely to bind to AT R. Peak 2 is a hydrophobic labeled
metabolite that can potentially bind to AT R, and peak 3 represents
1
recorded at 254 nm. Column: Phenomenex Luna C18(2) (10
lM, 250 ꢀ 4.6 mm)
1
with 40:60 MeCN/AF (0.1 M) at 2 mL/min; PeakSimple 3.93 Analysis Software.
unchanged tracer. At 30 min after injection, 78 ± 10% of total radio-
activity (noise- and decay-corrected) was from peak 1, 1 ± 1% peak
1
8
2
, and 23 ± 10% was unchanged [ F]FPyKYNE-losartan (Fig. 5). In
control plasma samples, radioactivity present was solely represen-
tative of unchanged [ F]FPyKYNE-losartan (data not shown).
(
A)
1
8
3
. Conclusions
Radiosynthesis of [18F]FPyKYNE-losartan has been achieved in
high radiochemical purity and acceptable specific activity for renal
imaging. The production method was reliable, providing sufficient
amount of tracer, with no meaningful copper contamination, for
1
8
multiple PET scans. [ F]FPyKYNE-losartan PET images obtained
in rats displayed high tissue contrast and binding selectivity for
1
8
renal AT
1 2
R over AT R. [ F]FPyKYNE-losartan metabolism to mostly
hydrophilic labeled compounds in plasma suggests minimal inter-
1
8
ference of F-labeled metabolites to AT
1
Rs. These findings support
(
B)
the use of this tracer for renal AT
imaging in rats.
1
R evaluation with non-invasive
4
4
. Experimental section
.1. Materials and methods
Reaction solvents, including MeCN and DMSO as well as purifi-
cation solvents (pentane and diethyl ether) were anhydrous (>99%)
ACS grade by Sigma–Aldrich. HPLC grade MeCN was purchased
from Fisher Scientific, Trifluoroacetic acid (TFA, 99%) from Alfa
Aesar and losartan potassium (98%) from LKT Laboratories, Inc
Figure 2. Representative microPET images (coronal view) of [18F]FPyKYNE-losartan
showing liver and kidney activity (specific uptake values) at 5-10 min post-
(
Medicorp, Montreal, QC). All other reagents, including deuterated
solvents for NMR, were purchased from Sigma–Aldrich and classi-
fied as 98% purity or greater. EtOH (99.9%) and saline solution
(0.9%) for reformulation were obtained from sterile capped bottles.
No further manipulation or purification was made to purchased
solvents and/or reagents.
injection in (A) normal animals (n = 7) and AT
23,319 (n = 3). (B) Tracer time-activity curves for average blood input (left atrium)
and kidney are presented as specific uptake values normalized to body weight
SUVBW) from 0 to 60 min.
2
R blocked animals with 5 mg/kg PD
1
(
a specific uptake value (SUV) of 1.52 ± 0.58 at 10 min post-injec-
tion. Using Logan graphical analysis of image-derived renal versus
left atrium blood TAC, the average distribution volume (DV) of
Analytical TLC was performed on silica gel-coated aluminum
sheets (Sigma–Aldrich). Unless specified otherwise, TLC was run
with a solvent of 30:70 EtOAc/Hexanes. Preparative TLC was per-
1
8
3
[
F]FPyKYNE-losartan in the left kidney was 2.76 ± 0.68 mL/cm .
formed on Analtech TLC Uniplates (silica gel matrix, 1000 lM). A

3
934
N. Arksey et al. / Bioorg. Med. Chem. 22 (2014) 3931–3937
(
A)
(
B)
(C)
Figure 3. (A) MicroPET images (coronal view) of [18F]FPyKYNE-losartan showing liver and kidney activity (specific uptake values) at 10–15 min post-injection. Effect of AT
R
1
blocking with increasing candesartan doses (2.5 mg/kg, n = 3; 5 mg/kg, n = 5; and 10 mg/kg, n = 3) on (B) specific uptake value (SUV) time-activity curves from 0 to 60 min,
and (C) distribution volume (DV) of [ F]FPyKYNE-losartan determined by Logan analysis in the kidney of rats. P <0.02 compared to baseline; #P <0.02 compared to previous
1
8
⁄
dose.
(
A)
(B)
(
C)
(D)
Figure 4. Representative HPLC chromatograms displaying presence of unchanged [18F]FPyKYNE-losartan and its labeled metabolites, in rat plasma at respective time points.
1
8
[
F]FPyKYNE-losartan (peak 3) is metabolized into a hydrophilic metabolite (peak 1) and a hydrophobic metabolite (peak 2). Plasma samples were analyzed at 5 min (A),
0 min (B), 20 min (C) and 30 min (D) post tracer injection.
1
technical grade silica gel (DavisilÒ grade 633, 60 Å pore size,
00–425 mesh) purchased from Sigma–Aldrich was used for flash
column chromatography. A Phenomenex Luna C18(2) column
250 ꢀ 4.6 mm, 10 M) was used for all analytical HPLC and a
Phenomenex Luna C18(2) column (250 ꢀ 10 mm, 10 M) was
installed in the module for HPLC purification. PeakSimple Chroma-
tography Data System (6-port) was used in combination with a
Waters 486 Tunable Absorbance Detector and Water 515 HPLC
Pump and data analysed by PeakSimple (v 3.93). Sep-Pak Light
Waters Accell Plus QMA cartridges and Sep-Pak Light C18
2
(
l
Ò
l

N. Arksey et al. / Bioorg. Med. Chem. 22 (2014) 3931–3937
3935
min]: 4.6 min. 1H NMR (300 MHz, CDCl
) d
8.08 (dd, J = 3.2,
3
H
2
.6 Hz, 1H); 7.51–7.50 (m, 2H); 4.23 (t, J = 6.0 Hz, 2H); 2.45 (td,
J = 6.8, 2.6 Hz, 2H); 2.03 (qn, J = 6.1 Hz, 2H); 1.95 (t, J = 2.6 Hz,
1
3
1
H). C NMR (75 MHz, CDCl
CH); 128.53 (CH); 123.43 (CH); 82.68 (C); 69.43 (CH); 67.88
CH ); 27.58 (CH ); 14.87 (CH ). HRMS: calcd for C10
M+H] , 207.0677, found 207.0323. The H and C NMR as well
3 C
) d 150 (C); 147.14 (C); 139.20
(
(
[
2
2
2
10 2 3
H N O
+
1
13
as HRMS data confirmed product identity and were in agreement
with the reported values.
4
.2.2. 2-Fluoro-3-pent-4-yn-1-yloxypyridine (FPyKYNE, 2a)
1
5
As described previously, compound 2a was synthesized by
reacting NO PyKYNE 1 with potassium fluoride and Kryptofix2.2.2
2
in DMSO at 145–165 °C for 90 min. The reaction was monitored for
completion by TLC. After cooling to room temperature, the reaction
mixture was diluted with EtOAc and extracted with water and
2 4
brine then dried over Na SO before removing the solvent under
reduced pressure. The residue was purified by flash column chro-
matography on silica gel (85:15 Hexanes/EtOAc) to afford 2a as
Figure 5. Proportions of [18F]FPyKYNE-losartan and its labeled metabolites in rat
plasma over time. In the control sample, 100% of radioactivity represents
1
8
unchanged tracer. From 5 to 30 min unchanged [ F]FPyKYNE-losartan is rapidly
metabolized and reduced from 78% to 23%, respectively.
clear crystalline oil. HPLC [50:50 MeCN/H
2
O (0.1 M AF), 2 mL/
) d 7.75 (dd, J = 4.8,
.6 Hz, 1H); 7.32 (m, 1H); 7.12 (dd, J = 7.8, 4.9 Hz, 1H); 4.17 (t,
J = 6.1 Hz, 2H); 2.45 (td, J = 6.9, 2.6 Hz, 2H); 2.05 (qn, J = 6.4 Hz,
1
min]: 4.6 min. H NMR (300 MHz, CDCl
3
H
1
cartridges for SPE were purchased from Waters. The QMA car-
tridges were conditioned by passing through 7–8 mL of a 0.55 M
13
2
J
1
H); 2.0 (t, J = 2.6 Hz, 1H). C NMR (75 MHz, CDCl
3
) d
C
153.85 (C,
1
2
3
C–F = 237.3 Hz); d: 142.23 (C, J C–F = 25.4 Hz); 137.37 (C, J C–F
3.2 Hz); d: 122.81 (C, J C–F = 4.4 Hz); d: 121.66 (C, J C–F = 4.3 Hz);
=
solution of potassium carbonate (7.60 g or 0.055 mol K
2
CO
3
in
3
4
1
00 mL water), followed by water (7–8 mL) and drying under a
19
8
2
2.93 (C); 69.16 (CH); 67.50 (CH ); 27.83 (CH
2
); 14.96 (CH
2
).
F
stream of argon. The C18 cartridges were pre-conditioned by pass-
ing through 5 mL of EtOH followed by 10 mL of water. Silica car-
tridges were used as received. F-18 eluent was prepared by
NMR d
F
ꢁ84.79 (referenced with respect to TFA). HRMS: calcd for
+
10
C H10FNO [M+H] , 180.0732, found 180.0443.
dissolving 27.5 mg (0.20 mmol) K
Kryptofix2.2.2 (K222) in a 5 mL solution of MeCN (4.75 mL) and
water (250 l). Melting points (Mp) were measured by a Fisher Sci-
2
CO
3
and 150 mg (0.40 mmol)
0
4
.2.3. 2-Butyl-4-chloro-5-(hydroxymethyl)-1-[[2 -
[(triphenylmethyl)tetrazole-5-yl]biphenyl-4-
l
yl]methyl]imidazol (tetrazole-protected losartan, 3)
Compound 3 was prepared from commercially available losar-
tan potassium as previously described. Briefly, to a solution of
losartan in DMF was added trityl chloride and Et N. The solution
3
was stirred at room temperature overnight after which it was
diluted with EtOAc, washed with water and brine, dried over Na2-
4
and solvent removed. The residue was purified by flash column
chromatography on silica gel to obtain a white powder. HPLC
35:65 MeCN/AF (0.1 M), 2 mL/min]: 3.6 min. H NMR (400 MHz,
DMSO-d ) d 7.79 (d, J = 6.0 Hz, 1H); 7.61 (t, J = 6.0 Hz, 1H); 7.54
t, J = 6.0 Hz, 1H); 7.31–7.44 (m, 10H); 7.05 (d, J = 6.4 Hz, 2H);
.90 (d, J = 6.4 Hz, 2H); 6.86 (d, J = 6.0 Hz, 6H); 5.24 (t, J = 4.0 Hz,
H); 5.19 (s, 2H); 4.21 (d, J = 4.0 Hz, 2H); 2.37 (t, J = 6.0 Hz, 2H);
.41 (q, J = 6.0 Hz, 2H); 1.15 (sx, J = 6.0 Hz, 2H); 0.73 (t, J = 6.0 Hz,
entific melting point apparatus.
NMR, HRMS, Accurate Mass Measurement and ICP MS were per-
formed at the University of Ottawa. 1H NMR was carried out in
CDCl3, unless otherwise indicated either by a Bruker AVANCE 300
2
1
3
or 400 MHz, or a Varian Inova 500 MHz spectrometer. C NMR
1
9
and
F NMR spectra were obtained on a Bruker AVANCE
SO
3
00 MHz spectrometer. Proton chemical shifts (d) are reported in
parts per million (ppm) relative to tetramethylsilane (TMS) inter-
nal standard. Coupling constants (J) are reported in Hz. HRMS were
done on either a Concept HRes, EI Mass Spectrometer or a Micro-
mass QToF, ESI HRes Mass Spectrometer (for accurate mass mea-
surement). ICP MS was performed on a Varian (Agilent) Vista-Pro
Inductively coupled plasma emission spectrometer, equipped with
a charge-coupled device (CCD) detector.
1
[
6
H
(
6
1
1
3
6
H). HRMS: calcd for C41
87.2638.
H37ClN
6
O [M+Na], 687.2615, found
1
8
ꢁ
[
F]F was produced in a CTI/Siemens RDS III cyclotron by pro-
1
8
18
18
2
ton irradiation of [ O]H O (>97%) via the O(p,n) F nuclear reac-
tion. The TRACERlab FX N Pro automated synthesizer, including UV
detector, HPLC pump, and software, were purchased from GE
0
4
.2.4. 2-Butyl-4-chloro-5-(azidomethyl)-1-[[2 -
[(triphenylmethyl)tetrazole-5-yl]biphenyl-4-
Healthcare. Radioactivity was measured in
calibrator.
a
Capintec dose
yl]methyl]imidazol (tetrazole-protected azido-losartan, 4)
To a solution of 3 (300 mg, 0.44 mmol) in THF (4 mL) were
added
0
0
18-diazabicyclo[5.4.0]-7-undecene
.48 mmol) and diphenylphosphoryl azide (DPPA, 105
.49 mmol). The reaction mixture was stirred at ambient tempera-
(DBU,
74 mg,
4
.2. Chemistry
ll,
4
.2.1. 2-Nitro-3-pent-4-yn-1-yloxypyridine (NO
2
PyKYNE, 1)
ture under argon for overnight (16 h) and monitored by TLC for
completion. The reaction was then quenched with water (2 mL).
The organic phase was separated, and the aqueous phase extracted
with EtOAc. The organic phases were combined and dried over
Na SO . The solvent was removed under reduced pressure and
2 4
the residue purified by flash column chromatography (EtOAc/
1
5
Compound 1 was synthesized according to the literature.
solution containing 2-nitro-3-hydroxypyridine, potassium carbon-
ate (K CO ), sodium iodide (NaI) and 5-chloropent-1-yne in DMF
was stirred overnight at 80 °C. The reaction mixture was cooled
and diluted with EtOAc for extraction with water and brine. After
drying over Na SO , the solvent was removed under reduced pres-
2 4
sure and the residue purified by flash column chromatography on
silica gel (85:15 Hexane/EtOAc) to afford 1 as a white crystalline
powder. Mp 56–58 °C. HPLC [50:50 MeCN/H O (0.1 M AF), 2 mL/
2
A
2
3
Hexanes 5:95–30:70) to afford 4 (275 mg, 87%) as a white crystal-
line powder. Mp 130–132 °C. H NMR (300 MHz, CDCl
1
3
) d
H
7.96 (m,
1
H); 7.41-7.51 (m, 2H); 7.30-7.36 (m, 10H); 7.11 (d, J = 8.2 Hz, 2H);
6
.90 (d, J = 7.7 Hz, 2H); 6.70 (d, J = 8.2 Hz, 6H); 4.98 (s, 2H); 3.94

3
936
N. Arksey et al. / Bioorg. Med. Chem. 22 (2014) 3931–3937
ꢁ
3
ꢁ3
(
s, 2H); 2.51 (t, J = 7.8 Hz, 2H); 1.66 (qn, J = 7.7 Hz, 2H); 1.29 (sx,
9.4 ꢀ 10 ꢁ 14.1 ꢀ 10 mmol) and sodium ascorbate (3.25–
ꢁ3 ꢁ3
13
J = 7.4 Hz, 2H); 0.86 (t, J = 7.3 Hz, 3H). C NMR (75 MHz, CDCl
3
)
4.9 mg, 16.4 ꢀ 10 ꢁ 24.7 ꢀ 10 mmol). The level of copper in
the final formulations measured by ICP MS, was above the limit
of detection (0.06 ppb), and the results were compared with blanks
containing 10% ethanol in saline (prepared in different types of
vials, n = 3), pure ethanol (n = 2) or pure saline (n = 1).
d
C
149.42 (C); 146.80 (C); 145.20 (3 ꢀ C); 141.23 (C); 130.61
3 ꢀ C); 129.64 (C); 128.40 (7 ꢀ CH); 127.90 (9 ꢀ CH); 127.6
4 ꢀ CH); 126.99 (3 ꢀ CH); 125.31 (C); 77.24 (C); 42.14 (CH );
9.91 (CH ); 29.67 (CH ); 22.29 (CH ); 22.23 (CH ); 13.46 (CH ).
HRMS: calcd for C41 36ClN [M+Na], 712.2680, found 712.2664.
(
(
2
2
2
2
2
2
3
H
9
4
.4. Animal studies
4
.2.5. 2-Butyl-4-chloro-5-[[3-{[1-methyl-4-
yl](1,2,3)triazol}propoxy]-2-fluoro-pyridine]-1-[[2 -[tetrazole-
-yl]biphenyl-4-yl]methyl]imidazol (FPyKYNE-losartan, 5a)
To a stirred solution of tetrzol-protected azido-losartan 4
50 mg, 0.072 mmol) in CH Cl (1.5 mL) was added a solution of
FPyKYNE, 2a (14.28 mg, 0.080 mmol) in CH Cl (400 l) followed
by aqueous solutions of copper(II) sulfate (CuSO , 50 l, 0.06 M)
and sodium ascorbate (120 l, 0.05 M). The biphasic solution was
0
All experiments were performed with male Sprague Dawley
5
rats weighing 200–660 g (Charles River Laboratories, Montreal,
Canada) and were conducted in accordance with the guidelines
of the Canadian Council of Animal Care (CCAC) and with approval
from the Animal Care Committee (ACC) at the University of
Ottawa. Rats were housed in pairs, kept on a 12 h light-dark cycle,
and followed a standard diet.
(
2
2
2
2
l
l
4
l
stirred at ambient temperature overnight (16 h). The reaction
was monitored by TLC for completion after which it was diluted
with EtOAc and extracted with water (2 ꢀ 30 mL) and brine
4.4.1. In vivo microPET
Dynamic PET images were acquired with a Siemens Inveon
small animal dedicated micro-PET scanner (LSO scintillation crys-
tals, 1.4 mm spatial resolution, 12.7 cm axial FOV). Rats (n = 7)
were anaesthetized with isoflurane (2–2.5%), weighed, and placed
on the scanning bed in supine position with movement restricted
by light taping. Anesthesia was maintained throughout the scan-
ning process by a continuous flow of isoflurane (1–2%) through a
(
2 4
30 mL) then dried over Na SO before removing the solvent under
reduced pressure. The residue was diluted with a solution of TFA in
DMF (400 l, 0.13 mM) and heated at 80–85 °C for 1–2 min. The
l
product was purified by semi-prep TLC (silica on glass, EtOAc/Hex-
anes/MeOH 80:20:5) to obtain 5a (20 mg, 44%) as a clear oil which
was then crystallized with chloroform to yield a white solid. Mp
1
18
1
62–165 °C. H NMR (300 MHz, CDCl
3
) d
H
7.86 (dd, J = 7.51,
nose cone. A slow-bolus of 0.4–1.0 mCi of [ F]FPyKYNE-losartan
1
.4 Hz, 1H); 7.65-7.68 (m, 1H); 7.52 (td, J = 7.47, 1.6 Hz, 1H); 7.46
was injected via a catheter in the lateral tail vein. List-mode data
were acquired for 60 min and subsequently binned into 26 frames
of 12 ꢀ 10 s, 3 ꢀ 60 s, and 11 ꢀ 300 s. Dynamic data were recon-
structed on a 128 ꢀ 128 pixel image matrix (0.345 mm pixel size)
using OSEM3D/MAP (b = 1.0, OSEM3D iterations = 2, MAP itera-
tions = 18) with corrections for dead-time, isotope decay, detector
efficiencies, attenuation, scatter and random events. A 10 min
transmission scan with two rotating Co (122 keV) point sources
was performed either prior to, or following the dynamic F-18 emis-
sion scan for attenuation correction.
(
td, J = 7.53, 1.7 Hz, 1H); 7.24–7.36 (m, 1H); 6.98-7.07 (m,4H);
6
4
.81 (d, J = 8.4 Hz, 2H); 6.39 (d, J = 8.3 Hz, 2H); 5.40–5.24 (m,
H); 3.81 (t, J = 6.3 Hz, 2H); 2.60 (t, J = 7.8 Hz, 4H); 1.86 (qn,
J = 6.7 Hz, 2H); 1.67 (qn, J = 7.7 Hz, 2H); 1.34 (sx, J = 7.5 Hz, 2H);
1
9
0
.86 (t, J = 7.3 Hz, 3H). F NMR (referenced with respect to TFA)
13
d
(
1
F
ꢁ84.88 (d, J = 8.78 Hz). C NMR (75 MHz, CDCl
C); 152.17 (C); 150.15 (C); 147.29 (C); 142.03 (C); 139.97 (C);
38.30 (C); 137.56 (C); 135.16 (C); 131.89 (C); 131.55 (C); 130.47
3 C
) d 153.75
5
7
(
(
2 ꢀ CH); 130.06 (2 ꢀ CH); 129.46 (CH); 128.49 (CH); 124.65
CH); 123.15 (CH); 122.85 (CH); 121.93 (CH); 120.64 (CH);
In order to assess binding specificity for the AT
(n = 3–5 per group) were co-injected with AT R blocker candesar-
tan (2.5 mg/kg, 5 mg/kg and 10 mg/kg). Binding specificity for
1
R, subsets of rats
1
2
20.24 (CH); 67.83 (CH
9.93 (CH ); 28.33 (CH
2
); 53.62 (CH
); 26.89 (CH
2
); 47.34 (CH
); 22.55 (CH
2
); 42.55 (CH
); 13.85 (CH
2
);
).
1
2
2
2
2
3
3
,19
HRMS: calcd for C32
H32ClFN10O [MꢁH], 625.2355, found 625.2357.
AT
2
R
was examined in another rat group (n = 3) by injection
of PD123,319 (5 mg/kg) 5 min prior to tracer injection.
4
4
.3. Radiochemistry
4
.4.2. Image analysis
Reconstructed dynamic images were analyzed using Siemens
.3.1. 2-Butyl-4-chloro-5-[[3-{[1-methyl-4-
18
0
yl](1,2,3)triazol}propoxy]-2-[ F]fluoro-pyridine]-1-[[2 -
Inveon Research Workplace (IRW) software. TACs were generated
for the arterial blood input function (derived from left atrium
[
tetrazole-5-yl]biphenyl-4-yl]methyl]imidazol ([18F]FPyKYNE-
1
8
losartan, 5b)
region of interest) and left kidney tissue uptake. [ F]FPyKYNE-
losartan renal activity was calculated as SUV (g/mL) at 5–10 min
post-injection (frame 16). The SUV is a relative quantification of
activity in a single or summed frame, normalized to the injected
activity and the body weight of the rat, to allow comparison
between subjects. Preliminary tracer binding was assessed by
Logan graphical analysis. Logan analysis is a graphical method
used to quantify the tracer DV, which is the expected ratio of radi-
otracer in tissue relative to plasma at equilibrium for reversibly
The F-18 enriched target water was delivered to the module and
loaded onto a QMA cartridge whereby F-18 was then eluted with
an aqueous solution of potassium carbonate and Kryptofix2.2.2
(K222) into the first reaction vessel. Azeotropic drying by evapora-
tion of acetonitrile provided active un-solvated K /K222
+
18 ꢁ
/ F .
2
0
Nucleophilic substitution of the nitro group on NO PyKYNE 1 with
2
1
8
F-18 produced [ F]FPyKYNE 2b which was then transferred to a
series of three silica cartridges. The labeled product was eluted into
the second reaction vessel and combined with tetrazole-protected
azido-losartan 4 for conjugation. The product was deprotected
with acid and sent to the semi-prep HPLC for purification. The col-
lected fraction containing the product was then diluted with water
and passed through a C-18 cartridge. After washing with water it
was then eluted with EtOH (minimum value 0.4 mL) and reformu-
lated in saline (maximum 10% EtOH solution).
3
binding receptor ligands. The tracer DV (mL/cm ) is an index of
receptor density (Bmax) and ligand affinity (1/K
Physiologically, the DV can be used as an indicator of protein
expression and/or receptor-ligand binding potential (Bmax/K ) for
reversibly binding ligands, that is, higher protein expression or
binding affinity will result in a higher DV value.
d
) for the receptor.
d
4
.4.3. Radiolabeled metabolite analysis in plasma
4
.3.2. Level of copper in the final formulation
Restrained animals were injected with 2-4 mCi (74–148 MBq)
1
8
[18F]FPyKYNE-losartan via the lateral tail vein and sacrificed at
The [ F]FPyKYNE-losartan 5b, was produced several times
n = 11) using different amounts of copper sulfate (1.5–2.25 mg,
(
ꢁ5 (control), 5, 10, 20 and 30 min post-injection (n = 3). Plasma

N. Arksey et al. / Bioorg. Med. Chem. 22 (2014) 3931–3937
3937
samples were prepared following centrifugation (4000ꢀg, 5 min),
and Stroke Foundation of Ontario Program Grant (PRG6242), and
the Canadian Institutes of Health Research (MOP-287694).
by mixing with 1 g urea, to break binding with macromolecules,
filtered through 0.2 lm syringe filters (Nylon Membrane, Acro-
Ò
disc ), and injected onto the HPLC system. A modification of the
Supplementary data
HPLC column-switch method was used to quantify plasma metab-
2
1
olites.
Waters): one eluting solvent A (1:99 MeCN/water [v/v]) at
.5 mL/min across the capture cartridge (Alltech Direct-Connect
refillable guard column, 2 ꢀ 20 mm) packed with sorbent (HLB
VAC RC, Waters Oasis) and fitted with 2.5 m frits (Alltech, 2-
mm filter elements); and the other eluting solvent B (35:65 MeCN/
.1 M ammonium formate [v/v]) at 3 mL/min across the analytical
m; Phenome-
Briefly, the HPLC apparatus consisted of 2 pumps
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2014.06.011.
(
1
References and notes
l
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3
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0
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l
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(
2004, 31, 571.
were integrated using the PeakSimple Six-Port Chromatography
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vent B across the capture cartridge, eluting retained compounds
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1
1
1
1
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2
2
Acknowledgments
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The authors wish to thank the radiochemistry personnel, Yanick
Lee and the Department of Chemistry of University of Ottawa for
the chemistry. We would also like to extend our sincerest gratitude
to Dr. Stephanie Thorn and Dr. James Thackeray for the help with
the PET/bio-testing portion of this project. This work was sup-
ported in part by, the Ontario Preclinical Imaging Consortium
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1
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2
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(
OPIC) Grant# RE03-51 (Ontario Research Foundation), the Heart