Fluoro-Hydroxyphenethylguanidines: Efficient Synthesis and Comparison of Two Structural Isomers as Radiotracers of Cardiac Sympathetic Innervation

Article abstract of DOI:10.1021/acschemneuro.7b00051

Fluorine-18 labeled phenethylguanidines are currently under development in our laboratory as radiotracers for quantifying regional cardiac sympathetic nerve density using PET imaging techniques. In this study, we report an efficient synthesis of ¹⁸

Full text of DOI:10.1021/acschemneuro.7b00051

Research Article
pubs.acs.org/chemneuro
[18F]Fluoro-Hydroxyphenethylguanidines: Efficient Synthesis and
Comparison of Two Structural Isomers as Radiotracers of Cardiac
Sympathetic Innervation
Yong-Woon Jung, Keun Sam Jang, Guie Gu, Robert A. Koeppe, Phillip S. Sherman, Carole A. Quesada,
and David M. Raffel*
Division of Nuclear Medicine, Department of Radiology, 2276 Medical Sciences I Building, University of Michigan Medical School,
Ann Arbor, Michigan 48109, United States
ABSTRACT: Fluorine-18 labeled phenethylguanidines are currently under development in our laboratory as radiotracers for
quantifying regional cardiac sympathetic nerve density using PET imaging techniques. In this study, we report an efficient
synthesis of 18F-hydroxyphenethylguanidines consisting of nucleophilic aromatic [18F]fluorination of a protected diaryliodonium
salt precursor followed by a single deprotection step to afford the desired radiolabeled compound. This approach has been shown
to reliably produce 4-[18F]fluoro-m-hydroxyphenethylguanidine ([18F]4F-MHPG, [18F]1) and its structural isomer 3-[18F]fluoro-
p-hydroxyphenethylguanidine ([18F]3F-PHPG, [18F]2) with good radiochemical yields. Preclinical evaluations of [18F]2 in
nonhuman primates were performed to compare its imaging properties, metabolism, and myocardial kinetics with those obtained
previously with [18F]1. The results of these studies have demonstrated that [18F]2 exhibits imaging properties comparable to
those of [18F]1. Myocardial tracer kinetic analysis of each tracer provides quantitative metrics of cardiac sympathetic nerve
density. Based on these findings, first-in-human PET studies with [18F]1 and [18F]2 are currently in progress to assess their
ability to accurately measure regional cardiac sympathetic denervation in patients with heart disease, with the ultimate goal of
selecting a lead compound for further clinical development.
KEYWORDS: 3-[18F]Fluoro-p-hydroxyphenethylguanidine, [18F]3F-PHPG, 4-[18F]fluoro-m-hydroxyphenethylguanidine,
[18F]4F-MHPG, norepinephrine transporter, sympathetic nervous system
referred to as “parasympathetic withdrawal”.4 The chronically
elevated levels of the neurotransmitter norepinephrine that
result from this imbalance of parasympathetic and sympathetic
influences are cardiotoxic and contribute to the progression of
INTRODUCTION
Cardiac autonomic dysfunction contributes to morbidity and
mortality in many diseases that damage the heart, including
congestive heart failure, myocardial ischemia, myocardial
■
infarction, and diabetic autonomic neuropathy.1−3 Disease-
heart failure, including development of left ventricular
dilation.5,6 Throughout the time course of heart failure,
induced alterations in the nervous control of the heart can be
caused by changes in the outflow of nervous impulses to the
parasympathetic and sympathetic branches of the autonomic
nervous system arising from central sites in the brain, and by
autonomic dysfunction can promote malignant arrhythmias,
leading to sudden cardiac death.7,8
Clinical imaging studies of cardiac sympathetic denervation
using nuclear scintigraphy techniques have provided important
regional degeneration of postganglionic parasympathetic and/
insights into how different diseases affect this nerve
or sympathetic nerve fibers in the heart. The complex
population.9,10 Our laboratory has previously developed several
interaction between aberrant parasympathetic and sympathetic
influences on the heart often evolves with the progression of
disease. For example, early heart failure is characterized by
hyperactivity of sympathetic nerve pathways, with a concom-
itant reduction in parasympathetic activation, a process that
Received: February 1, 2017
Accepted: March 21, 2017
Published: March 21, 2017
© XXXX American Chemical Society
A
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Figure 1. Structures of norepinephrine and some radiotracers for imaging cardiac sympathetic nerve terminals.
radiotracers for noninvasive imaging studies of cardiac
sympathetic nerves, including [123I]metaiodobenzylguanidine
([123I]MIBG) for planar scintigraphy and SPECT imaging, and
[11C]-(−)-m-hydroxyephedrine ([11C]HED) for PET imaging
(Figure 1).11,12 These radiolabeled analogs of the endogenous
neurotransmitter norepinephrine are transported into presy-
naptic sympathetic nerve terminals by the norepinephrine
transporter (NET). Since NET expression in the heart is only
associated with sympathetic nerve varicosities, the cardiac
retention levels of these tracers can be used as metrics of
regional sympathetic nerve density. Clinical trials with
nerve density.17 In control studies and pharmacological
blocking studies with the NET inhibitor desipramine (DMI)
in rhesus macaque monkeys, the Ki values from compartmental
modeling analysis were found to decrease with increasing DMI
doses along a sigmoidal dose−response curve, with an IC50 of
0.087 mg/kg and a Hill slope nH = −0.70. Similar results were
obtained using the Patlak slopes Kp (mL/min/g) from Patlak
graphical analysis of [11C]GMO kinetics, as an alternative
estimate of the Ki values (IC50 = 0.068 mg/kg, nH = −0.54).
These results strongly suggest that regional estimates of Ki from
tracer kinetic analysis of [11C]GMO kinetics in human hearts
would serve as a reproducible metric of regional cardiac
sympathetic nerve density.
[
123I]MIBG and [11C]HED tracers in heart failure patients
have demonstrated that cardiac sympathetic denervation is
associated with a significantly increased risk of sudden cardiac
death. For example, the multicenter ADMIRE-HF study of 961
heart failure patients showed that a low heart-to-mediastinum
ratio (H/M) of [123I]MIBG obtained from planar gamma
camera images, a global measure of cardiac sympathetic
denervation, was a predictor of mortality and fatal arrhythmic
events in patients eligible for implantable cardioverter
defibrillator (ICD) therapy.13 More recently, the prospective
PAREPET trial used [11C]HED to assess regional sympathetic
denervation, [13N]ammonia to assess myocardial perfusion, and
[18F]FDG to assess myocardial viability in 204 heart failure
patients staged for an ICD.14 The PAREPET results showed
that the regional extent of sympathetic denervation in the left
ventricle (as defined by [11C]HED retention deficits) was the
strongest predictor of sudden cardiac arrest among all imaging
parameters measured. Patients with regional sympathetic
denervation encompassing more than 38% of their left ventricle
defined the upper tertile of patients with the highest risk of
sudden cardiac arrest (p < 0.001). The results of the ADMIRE-
HF and PAREPET trials suggest that noninvasive imaging
studies of cardiac sympathetic denervation may find a clinical
role in improved risk stratification of heart failure patients for
ICD placement.15
We have recently been investigating radiolabeled phenethyl-
guanidines as next generation sympathetic nerve tracers with
improved myocardial kinetics for accurate and sensitive
quantification of regional sympathetic nerve density using
PET and tracer kinetic analysis methods.16 Our initial work
focused on the carbon-11 labeled hydroxyphenethylguanidine
N-[11C]guanyl-(−)-m-octopamine ([11C]GMO, Figure 1).
PET studies with [11C]GMO in nonhuman primates showed
that the myocardial kinetics of this tracer could be successfully
analyzed using compartmental modeling methods or Patlak
graphical analysis to obtain an estimates of the “net uptake rate
constant” Ki (mL/min/g) as a measure of regional sympathetic
While the preclinical PET studies with [11C]GMO were
highly encouraging, the short half-life of carbon-11 (20.4 min)
limits its use to PET centers with an on-site cyclotron.
Conversely, fluorine-18 has a sufficiently long half-life (1.83 h)
that the production and distribution of 18F-labeled radiophar-
maceuticals from a central production facility to stand-alone
PET imaging centers is feasible. To develop a fluorine-18
labeled phenethylguanidine, we initially chose to prepare 4-
[18F]fluoro-m-hydroxyphenethylguanidine ([18F]4F-MHPG,
[18F]1, Figure 1). In previous studies in an isolated rat heart
model, the kinetics of [11C]4F-MHPG were comparable to
those of [11C]GMO.16
A previous report described several preclinical evaluations of
[18F]1, including PET imaging studies in nonhuman
primates.18 The results of the PET studies of [18F]1 closely
paralleled those obtained with [11C]GMO,17 supporting further
development of this agent for clinical translation. For these
pilot studies, our initial approach to the preparation of [18F]1
was a multistep automated radiosynthesis involving production
of a 18F-labeled intermediate using a diaryliodonium salt
precursor followed by a deprotection step, an N-guanylation
step, a second deprotection step and finally HPLC purification
to provide [18F]1.18 Because of the multiple steps required, this
initial method relied on the use of two automated radiosyn-
thesis modules in adjacent hot-cells, linked in series, to provide
the final product. While this approach was adequate for
preclinical evaluations of [18F]1, it was poorly suited to routine
production of the radiotracer for clinical studies in human
subjects. Thus, one goal of this work was to develop an efficient
automated radiosynthesis of [18F]1 capable of providing
sufficiently high radiochemical yields for clinical PET studies.
We report here a new two-step approach to the radiosyn-
thesis of [18F]1 that utilizes a novel N,N′,N″,N″-tetrakis-Boc
protected guanidinyliodoium salt precursor for nucleophilic
aromatic [18F]fluorination, followed by a single deprotection
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Scheme 1. Ineffective Initial Approach to an Improved Radiosynthesis of [18F]1
Scheme 2. 18F-Fluorination of a Test Compound with a Fully Protected Guanidinyl Moiety
Table 1. [18F]Fluorination of Diaryliodonium Salts (5, 11 and 21) under Various Conditions
a
b
b
c
entry
I+ salt
18F-fluoride [18F]F−X+
method
T (°C)
t (min)
solvent
TEMPO and H2O
product
yield (%)
NR
n
1
5
[18F]F−K+
[18F]F−K+
[18F]F−TBA+
[18F]F−Cs+
[18F]F−Cs+
[18F]F−Cs+
[18F]F−Cs+
[18F]F−Cs+
[18F]F−Cs+
[18F]F−Cs+
[18F]F−Cs+
[18F]F−Cs+
[18F]F−Cs+
manual
manual
manual
manual
manual
manual
manual
manual
manual
manual
manual
automated
automated
150
150
150
150
150
130
120
150
150
150
150
150
10
10
10
10
5
MeCN
DMF
DMF
DMF
DMF
DMF
DMF
DMF
DMF
DMF
DMF
DMF
DMF
−
−
−
−
+
+
+
+
+
+
+
+
+
[18F]6
[18F]6
[18F]6
[18F]6
[18F]6
[18F]6
[18F]6
[18F]6
[18F]6
[18F]22
[18F]22
[18F]1
[18F]2
2
2
5
3
2
2
2
3
5
trace
9
4
5
2
2
5
5
51
31
28
43
30
16
11
5
5
3
5
5
5
4
3
6
5
5
3
7
5
5
3
8
5
15
25
5
2
9
5
2
10
11
12
13
11
11
11
21
5
15
5
3
d
e
7.0 3.5
8.0 3.5
13
12
150
5
a
b
Amount of diaryliodonium salt precursor used was 4−6 mg. Reaction solvent (MeCN or DMF; 500 μL) prepared without (−) or with (+) 1 mg
c
TEMPO and 10 μL of H2O. Progress of the reaction and yields for manual reactions were analyzed by radio-TLC (developing solvent: ethyl
d
acetate/hexane = 30:70, vol/vol). Yield of the isolated pure product [18F]1 by semipreparative column (Phenomenex Synergi 10 μ Hydro-RP 80A,
e
250 × 10 mm) using HPLC (5% EtOH in 40 mM NH4OAc buffer, λ = 254 nm, 4.0 mL/min). Yield of the isolated pure product [18F]2 by
semipreparative column (Phenomenex Synergi 10 μ Hydro-RP 80A, 250 × 10 mm) using HPLC (3.5% EtOH in 40 mM NH4OAc buffer, λ = 254
nm, 4.0 mL/min).
step, to provide [18F]1 in good yields and high specific activities
for clinical PET studies. This new approach is performed using
a single automated radiosynthesis module and has proven to be
very reliable.
step automated radiosynthesis (Scheme 1). However, many
attempts at 18F-radiofluorination of the N,N′-bis-Boc-guanidi-
nyliodonium salt precursor 3 with different [18F]fluoride
sources under a variety of reaction conditions were unsuccessful
in providing the desired 18F-labeled intermediate [18F]4. We
hypothesized that this disappointing outcome was related to the
two unprotected protons of the guanidinyl group being acidic,
causing a disturbance in the 18F-radiofluorination reaction. To
confirm this hypothesis, we explored the 18F-radiofluorination
of a test compound with a fully protected guanidinyl moiety to
eliminate the unprotected protons (Scheme 2). The
N,N′,N″,N″-tetrakis-Boc-guanidinyliodonium salt precursor 5
was synthesized, and radiolabeling tests were carried out under
various conditions, including different [18F]fluoride sources,
reaction solvents (e.g., DMF and MeCN), reaction times and
reaction temperatures to produce the 18F-labeled compound
[18F]6 (Table 1).19 The reaction of K[18F]F−K222 or
[18F]TBAF with precursor 5 afforded [18F]6 in low yields
(0−3%) at 150 °C for 10 min. When precursor 5 was reacted
with Cs[18F]F under general anhydrous radiofluorination
conditions, the yield of [18F]6 was improved (∼9% at 150
°C for 10 min). However, when 5 was reacted with Cs[18F]F
for a longer reaction time (150 °C for 25 min), the yield of the
With the establishment of this new synthetic approach to
18F-hydroxyphenethylguanidines, the second objective of this
study was to prepare 3-[18F]fluoro-p-hydroxyphenethylguani-
dine ([18F]3F-PHPG, [18F]2, Figure 1), the structural isomer of
[18F]1. Preclinical evaluations of this compound were
performed to compare its characteristics as a cardiac
innervation tracer with those of [18F]1. Studies in nonhuman
primates showed that [18F]2 is as good or better than [18F]1 as
a cardiac PET imaging agent. Based on the positive preclinical
testing results with [18F]1 and [18F]2, these new cardiac
sympathetic innervation radiotracers are currently being
evaluated in first-in-human PET studies under an exploratory
investigational new drug (IND) clearance from the FDA.
RESULTS AND DISCUSSION
■
Chemistry and Radiochemistry. Our initial strategy for
improving the radiosynthesis of [18F]1 was to utilize a N,N′-bis-
Boc-protected diaryliodonium salt precursor for direct no-
carrier-added nucleophilic aromatic [18F]fluorination via a two-
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a
Scheme 3. Synthetic Route to the Diaryliodonium Salt Precursor 11
a
(a) (Boc)2CO, DMAP, Et3N, THF, rt, 48h, 92%; (b) Sn2Me6, Pd(PPh3)4, toluene, reflux, N2, 30 min, 86%; (c) (i) 2-(diacetoxyiodo)thiophene, p-
TsOH·H2O, MeCN, CH2Cl2, N2, rt, 1 h; (ii) 9, MeCN, CH2Cl2, N2, rt, 20 h, 87%; (d) KBr, MeCN, H2O, 65 °C-rt, 1 h, 86%.
a
Scheme 4. Synthetic Route to the Diaryliodonium Salt Precursor 21
a
(a) (Boc)2CO, Et3N, THF, rt, 24 h, 98%; (b) NaI, NaOCl, KOH, MeOH, 0 °C, 43%; (c) BnBr, K2CO3, acetone, 4 h, 78%; (d) HCl, 65 °C, 1 h,
97%; (e) 1,3-N,N’-bis(tert-butoxycarbonyl)-2-methyl-2-thiopseudourea, Et3N, DMF, 0 °C-rt, 24 h, 58%; (f) (Boc)2CO, DMAP, Et3N, THF, rt, 20 h,
63%; (g) Sn2Me6, Pd(PPh3)4, toluene, reflux, N2, 30 min, 98%; (h) (i) 2-(diacetoxyiodo)thiophene, p-TsOH·H2O, MeCN, CH2Cl2, N2, rt, 1 h; (ii)
19, MeCN, CH2Cl2, N2, rt, 44 h, 86%; (i) KBr, MeCN, H2O, 65 °C to rt, 1 h, 96%.
desired product decreased to ∼4%. We hypothesized that the
reduced radiochemical yields at longer reaction times might be
due to the decomposition of the iodonium salt precursor 5 and
the radiofluorinated product [18F]6 at the 150 °C reaction
temperature. In tests, 5 started slow thermal decomposition at
150 °C in open capillary tubes in a melting point apparatus,
supporting this hypothesis. The addition of TEMPO (1.0 mg)
as a radical scavenger and a small amount of water (10 μL) in
the reaction solvent (DMF, 0.5 mL) to increase the solubility of
Cs[18F]F greatly increased the radiochemical yields, up to
∼51% for manual syntheses at 150 °C for 5 min. Additional
tests using a reduced temperature of 130 °C for 5 min,
decreased the yield of [18F]6 to ∼31%. Tests at temperatures
greater than 150 °C did not produce any improvements in the
yields.
This encouraging result led us to prepare the required
N,N′,N″,N″-tetrakis-Boc-protected guanidinyliodonium salt
precursor for a simplified automated production of [18F]1 for
clinical studies (Scheme 3). The N′,N″-bis-Boc-protected
iodophenethylguanidine 7 was previously synthesized in our
laboratory.18 This compound was converted to the
N,N′,N″,N″-tetrakis-Boc-protected guanidine 8 by treatment
with di-t-butyl dicarbonate in the presence of dimethylamino-
pyridine and triethylamine in THF. Reaction of 8 with
bis(trimethyl)tin in the presence of tetrakis-
(triphenylphosphine)palladium provided the trimethylstannane
compound 9. [2-hydroxy(tosyloxy)iodo]thiophene,20 which
was generated in situ by a mixture of 2-(diacetoxyiodo)-
thiophene21 with p-toluenesulfonic acid under nitrogen
atmosphere, was reacted with trimethylstannane 9 to provide
(2-thienyl)iodonium tosylate 10 in 87% yield as a yellow solid.
Among various counteranions, bromide was found to be
especially reactive, increasing the radiochemical yield.22 There-
fore, (2-thienyl)iodonium tosylate 10 was converted into the
corresponding bromide 11 as a gray powder in 86% yield using
KBr in a solution of CH3CN and H2O.
A similar approach was used to prepare the guanidinyl-
iodonium salt precursor 21 for production of [18F]2 (Scheme
4). The N-Boc-protected tyramine 13 was iodinated in the
presence of sodium iodide and sodium hypochlorite to afford
14 in low yields (43%). The iodophenol 14 was protected by
benzylation with benzyl bromide followed by subsequent
deprotection of the Boc group to afford the benzylated 3-
iodotyramine intermediate 16. Condensation of 16 with 1,3-
D
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a
Scheme 5. Efficient Radiosynthetic Route to [18F]1 and [18F]2
a
(a) Cs[18F]F, TEMPO, H2O, DMF, 150 °C, 5 min; (b) 3.0 N HBr, 120 °C, 15 min.
bis(t-butoxycarbonyl)-2-methyl-2-thiopseudourea afforded the
N′,N″-bis-Boc-protected iodophenethylguanidine 17. This
compound was converted to the N,N′,N″,N″-tetrakis-Boc-
protected guanidininyliodonium salt 21 using the same
sequence of steps used to prepare 11 as shown in Scheme 3.
Briefly, reaction of the trimethylstannane 19 with [2-hydroxy-
(tosyloxy)iodo]-thiophene provided the (2-thienyl)iodonium
tosylate 20 in 86% yield as a yellow solid. Conversion of 20 to
the corresponding bromide 21 yielded a gray powder in 96%
yield. The structures of 11 and 21 were confirmed by 1H NMR,
13C NMR, high-resolution mass spectrometry (HRMS) spectra,
and C, H and N elemental analysis.
([11C]MIBG) and [11C]HED.16 Extraneuronal uptake (“up-
take-2”), which transports some norepinephrine analogs into
myocytes, was blocked pharmacologically by adding 54 μM
corticosterone to all heart perfusates.23 At the beginning of the
study, a 10 min constant infusion of tracer in the heart
perfusate is performed to measure a neuronal uptake rate, Kup
(mL perfusate/min/g wet). After this, the heart is switched to
normal heart perfusate to measure clearance rates from
neuronal spaces, expressed as a major clearance half-time,
T1/2 (h). As shown in Figure 2, [18F]2 had almost identical
A TRACERlab FXFN radiosynthesis module (GE Healthcare)
was used to achieve a fully automated radiosynthesis of [18F]1
and [18F]2 using a two-step reaction as shown in Scheme 5.
The first step incorporates [18F]F− into the aromatic ring of the
protected diaryliodonium salt precursor to yield an 18F-labeled
intermediate ([18F]22 or [18F]23) using Cs[18F]F in DMF
(150 °C, 5 min, 1 mg TEMPO, 10 μL H2O). The second step
is the simultaneous cleavage of the benzyl ether and the
N,N′,N″,N″-tetrakis-Boc protecting groups by treatment with
3.0 N HBr at 120 °C for 15 min followed by HPLC purification
to provide the desired radiotracer, [18F]1 or [18F]2. The
structure of the 18F-labeled product was confirmed by
comparing its retention time against the corresponding
nonradioactive fluorine-19 standard using reverse-phase
HPLC. The total synthesis time of the fully automated process
is 90 min. For [18F]1, the purified product was obtained in 7.0
3.5% yield at end of synthesis (EOS), (n = 13, decay-
corrected based on starting activity) with >98% radiochemical
purities. [18F]2 was obtained in 8.0 3.5% yield (n = 12) with
>99% radiochemical purities (Table 1). Activity levels in the
final product ranged from 0.6 to 2.0 GBq for [18F]1 and 1.0−
2.4 GBq for [18F]2. Specific activities were 56 19 GBq/μmol
for [18F]1 and 104 26 GBq/μmol for [18F]2 at EOS. Other
than the radiolabeled product, no other nonradioactive
compounds were observed in the HPLC/UV trace during
quality control of the final product. Mass concentrations
averaged 0.48 0.29 μg/mL for [18F]1 and 0.27 0.14 μg/mL
for [18F]2. Stability tests of 3 batches of each compound
showed that both compounds are completely stable in the
ammonium acetate buffer at room temperature for at least 6 h.
Isolated Rat Heart Studies. The kinetics of [18F]2 were
measured in the isolated working rat heart model, which has
been used previously to measure the neuronal uptake and
retention kinetics of [18F]1,18 the carbon-11 analog of MIBG
Figure 2. Kinetics of [18F]2 in isolated rat heart. Data for [18F]1,
[11C]MIBG, and [11C]HED are shown for comparison. The tracer is
infused for 10 min to measure its neuronal uptake rate (Kup, mL/min/
g wet), then the heart is switched to normal perfusate to measure
tracer clearance rates (T1/2, h). [18F]1 and [18F]2 have very long
neuronal retention times due to efficient storage inside norepinephrine
storage vesicles. ADV: apparent distribution volume.
kinetics as [18F]1 in this model, with neuronal uptake rates Kup
= 0.79 mL/min/g wet for [18F]2 and 0.77 mL/min/g wet for
[18F]1. Major clearance halftimes were T1/2 > 50 h for [18F]2
and T1/2 > 24 h for [18F]1. The long neuronal retention times
of these compounds are due to their efficient uptake and
storage in norepinephrine storage vesicles. This shows that
[18F]1 and [18F]2 are good substrates of the vesicular
monoamine transporter, isoform 2 (VMAT2), which is
localized in cell membranes of storage vesicles in peripheral
sympathetic nerve terminals.24 In comparison, [11C]MIBG and
[11C]HED have much faster neuronal uptake rates, with Kup
=
3.65 mL/min/g wet for [11C]MIBG and Kup = 2.35 mL
perfusate/min/g wet for [11C]HED. They also have much
faster clearance rates in this experimental model. [11C]MIBG
has a major clearance half-time T1/2 = 2.1 h and [11C]HED has
T1/2 = 1.6 h. These clearance rates are faster than are seen in
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human imaging studies with [123I]MIBG and [11C]HED
because the isolated rat heart has coronary flow rates that are
about 10 times higher than resting blood flow in a human
heart.25 If tracer molecules diffuse out of sympathetic neurons
in the isolated rat heart they are more likely to be cleared from
interstitial spaces into the capillary bed rather than re-entering
nerve terminals by NET transport. In contrast, several studies
have shown that [11C]HED continuously diffuses out of nerve
terminals only to be taken back up into the nerves during PET
studies in normal human hearts.25−27 The slower neuronal
uptake rates and long neuronal retention times of [18F]1 and
[18F]2 are favorable for tracer kinetic analysis,17 including
Patlak graphical analysis, which requires irreversible trapping of
a tracer in a tissue compartment.28
PET Studies in Nonhuman Primates. Dynamic PET
studies with [18F]2 were performed in rhesus macaque
monkeys to characterize the imaging properties, metabolism
and myocardial kinetics of the tracer (n = 4). Representative
cardiac PET images of [18F]2 show that the tracer provides
high quality images of the distribution of sympathetic nerve
terminals in the heart (Figure 3). Corresponding PET images
Figure 4. Time course of the metabolic breakdown of [18F]1 and [18F]
2 in the plasma of rhesus macaque monkeys.
showed that the main metabolic pathway for this compound in
rhesus macaques was sulfate conjugation at its m-hydroxyl
group.18 Incubations of [18F]2 under identical conditions were
unable to produce any radiometabolite, suggesting that this
compound is not a substrate for sulfate conjugation in rhesus
monkeys. Additional in vitro tests with [18F]2 using a monkey
liver microsomal fraction and the glucuronidation cofactor
uridine 5′-diphospho-glucuronic acid (UDPGA) were also
negative. Further tests are needed to identify the unknown
polar metabolite of [18F]2 produced in rhesus macaque
monkeys.
Blood Activity Partitioning. Using aliquots of plasma and
whole blood, the ratio of the activity concentration in plasma
(Cp) over that in whole blood (Cwb) was determined for each
venous blood sample. For [18F]2, the mean ratio Cp/Cwb
measured was 1.23 0.05 (n = 4). The Cp/Cwb ratio tended
to be constant throughout the PET study. This is similar to
previous results for [18F]1 which found Cp/Cwb = 1.25 0.06
(n = 4).18
Figure 3. Representative PET images of [18F]1 and [18F]2 in rhesus
macaque monkeys.
Tracer Kinetic Analysis. The myocardial tissue kinetics
Ct(t) of [18F]2 and its estimated kinetics in plasma Cp(t) were
analyzed using compartmental modeling and Patlak graphical
analysis as previously described.17,18 For compartmental
modeling analysis, the model used has two tissue compart-
ments, one for extraction into and clearance from extracellular
spaces, with rate constants K1 (mL/min/g) and k2 (min−1),
respectively, and the second for irreversible uptake into
sympathetic neurons, with rate constant k3 (min−1). A blood
volume fraction term BV (dimensionless) was also included to
account for activity in myocardial blood. The estimates of K1,
k2, and k3 from compartmental modeling were used to calculate
a “net uptake rate constant” Ki (mL/min/g) = (K1k3)/(k2 + k3),
which reflects the net rate of tracer influx into tissue
compartments. K1 estimates were fairly consistent for [18F]2,
while k2 and k3 were more variable, similar to previous findings
with [18F]1. The mean values of K1 for [18F]2 were K1 = 0.46
0.05 mL/min/g, compared with K1 = 0.56 0.07 mL/min/g
for [18F]1. Estimates of k3 (the rate constant related to
neuronal uptake of the tracer) were too variable to be useful as
a quantitative measure of nerve density, but the values of the
net uptake rate constant Ki calculated from the rate constant
estimates were very consistent. For [18F]2, the mean net uptake
rate constant was Ki = 0.221 0.029 mL/min/g, which can be
compared with Ki = 0.341 0.041 mL/min/g for [18F]1. Patlak
graphical analysis also gave consistent results. This analysis
approach uses a mathematical transformation of Cp(t) and Ct(t)
to construct a “Patlak plot” which has a distinct linear phase.
The slope of the linear phase, the Patlak slope Kp (mL/min/g),
is theoretically equal to the calculated net uptake rate constant
Ki derived from the compartmental modeling rate constants.
For [18F]2, the measured Patlak slopes averaged Kp = 0.169
for [18F]1 are also provided. There is little retention of either
tracer in the lungs, providing very high heart-to-lung contrast.
[18F]2 exhibited good heart-to-blood (H/B) contrast in the
final PET images (t = 85 min), with H/B = 4.0 0.3. This was
better heart-to-blood contrast than the ratio measured for [18F]
1, which was H/B = 3.0 0.5.17,18 Final heart-to-liver (H/L)
0.8. Due to faster clearance of
activity from the liver, contrast between heart and liver was a
little better for [18F]1, with final H/L = 2.5
0.3. No
significant uptake of free fluorine-18 was observed in the
vertebral bones of the spine for either compound, indicating
that these tracers are not susceptible to defluorination in
nonhuman primates.
ratios for [18F]2 were 2.2
Metabolism of [18F]2. To assess the metabolism of [18F]2,
six venous blood samples were drawn during the dynamic PET
studies. Plasma was separated from blood and processed for
analysis on a reverse-phase HPLC system with an in-line
radiation detector optimized for detection of 511 keV positron
annihilation photons to determine the fraction of plasma
activity in the form of radiolabeled metabolites.18 Representa-
tive data showing the metabolic breakdown of [18F]2 in plasma
are shown in Figure 4. Data on the metabolic breakdown of
[18F]1 in rhesus macaques are shown for comparison. For both
compounds, the metabolism time course was biphasic, with an
initially rapid phase of metabolism followed by a slower phase.
[18F]2 was metabolized more slowly than [18F]1. The mean
time at which 50% of parent radiotracer was still intact was 6.7
2.4 min for [18F]2 vs 2.3 0.9 min for [18F]1 (n = 4 each).
Using in vitro incubations of [18F]1 with a monkey liver
cytosol fraction and the cofactor for sulfur conjugation, 3′-
phospho-adenosine-5′-phosphosulfate (PAPS), we previously
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0.021 mL/min/g, comparied with Kp = 0.302
0.031 mL/
the targeted properties were (a) slower NET transport rates
than [11C]HED and [123I]MIBG; (b) rapid vesicular uptake
due to efficient VMAT2 transport; (c) resistance to intra-
neuronal metabolism; and (d) low lipophilicity to promote long
neuronal retention times. The slower neuronal uptake rate
makes the rate constant associated with this process more
identifiable from the cardiac PET kinetics, and the rapid uptake
and long retention in storage vesicles eliminates one rate
constant from the kinetic model, leading to more reliable and
consistent parameter estimates.17
min/g for [18F]1. For any given study, relative to their
corresponding Ki values, the Patlak slopes are biased a little
lower in magnitude because Patlak analysis does not account
for myocardial blood activity in the tissue kinetics Ct(t), while
the blood volume term BV used in compartmental modeling
does account for the blood activity. Representative examples of
the results of compartmental modeling and Patlak analysis of
[18F]2 are shown in Figure 5. In previous PET studies in
To achieve this goal, radiolabeled phenethylguanidines were
investigated because several phenethylguanidines are potent
neuron blocking agents due to their prolonged retention inside
norepinephrine storage vesicles.29−31 The isolated rat heart
studies with [18F]1 and [18F]2 presented above (Figure 2)
demonstrate that these compounds satisfy the targeted
properties (a), (b), and (d). PET studies showing very long
neuronal retention times for [18F]1 and [18F]2 in nonhuman
primate myocardium are consistent with (b) rapid vesicular
uptake and (d) very little diffusion of the tracers from storage
vesicles. For (c), the guanidine group of the side chain of [18F]1
and [18F]2 confers stability against neuronal enzymes such as
tyrosine hydroxylase, MAO, dopamine-β-hydroxylase, or
DOPA decarboxylase.32 Previous studies with the carbon-11
analog of [18F]1 in rats showed that 100% of the activity in the
myocardium at t = 30 min after tracer administration was in the
form of the parent tracer, consistent with no intraneuronal
metabolism of the tracer.33 Thus, [18F]1 and [18F]2 have been
found to satisfy all of our targeted neuronal tracer properties.
Structure−activity studies of a series of 11C-labeled
phenethylguanidines in the isolated rat heart showed that
several hydroxyphenethylguanidines had extremely long
retention times in this model. This was particularly true for
ring hydroxylation at the m- and p- positions, the beta-position
of the side chain, or combinations of these hydroxyl
substitutions.16 In contrast, none of the 11C-labeled benzylgua-
nidine compounds tested, including [11C]-p-hydroxybenzylgua-
nidine, were found to have long neuronal retention times in the
isolated rat heart. These results suggest that hydroxyphene-
thylguanidines are better VMAT2 substrates than benzylguani-
dines, or are retained in storage vesicles much more efficiently,
leading to high uptake and prolonged retention in storage
vesicles. The highly acidic interior of vesicles has been
described as an “amine trap”, since the low pH inside vesicles
further encourages high pKa molecules to exist in their ionized
form.34 The very high pKa of the guanidine group of
hydroxyphenethylguanidines (pKa = 10 to 13) likely causes
them to be highly protonated inside vesicles, further enhancing
their vesicular retention.
Figure 5. Myocardial tracer kinetic analyses for [18F]2 kinetics in the
same monkey. Parameter estimates from compartment modeling (A)
were used to calculate a net uptake rate constant Ki (mL/min/g).
Patlak graphical analysis (B) provided a Patlak slopes Kp (mL/min/g).
nonhuman primates with [11C]GMO and [18F]1, using the
NET inhibitor desipramine (DMI) to pharmacologically induce
different degrees of NET transporter occlusion showed that net
uptake rate constants Ki and Patlak slopes Kp each tracked
sensitively and reproducibly with DMI-induced declines in
available NET transporters.17,18 Taken together, the results of
these studies suggest that values of Ki or Kp obtained from
tracer kinetic analysis of the kinetics of [18F]1 or [18F]2 can be
used as quantitative metrics of regional sympathetic nerve
density in the heart.
Advantages of 18F-Hydroxyphenethylguanidines.
[18F]1 and [18F]2 offer some advantages over existing cardiac
sympathetic innervation radiotracers. The longer half-life of a
fluorine-18 (1.83 h) compared with the 20.4 min half-life of
carbon-11 in compounds such as [11C]HED and [11C]GMO
was one advantage discussed previously. In terms of structure−
activity relationships, there are four major factors that govern
the neuronal uptake and retention kinetics of sympathetic nerve
radiotracers: (a) rate of neuronal uptake by NET (equal to
Two 18F-labeled analogs of [123I]MIBG, m-[18F]-
fluorobenzylguanidine ([18F]MFBG) and p-[18F]-
fluorobenzylguanidine ([18F]PFBG) were synthesized previ-
ously for PET studies of cardiac sympathetic innervation and
neuroendocrine tumors.35,36 Studies of [18F]PFBG in the
isolated rat heart model showed it had a neuronal uptake rate
Kup = 2.80 mL/min/g wet, ∼77% of the rate of 3.65 mL/min/g
wet measured for [123I]MIBG.16,37 The major neuronal
clearance halftime for [18F]PFBG was T1/2 = 0.59 h, compared
with T1/2 = 2.1 h for [123I]MIBG. Thus, [18F]PFBG has rapid
NET transport rate into sympathetic neurons and clears from
the neurons faster than [123I]MIBG. We are not aware of any
human cardiac imaging studies with [18F]PFBG or [18F]MFBG,
although one report used [18F]PFBG to assess cardiac nerve
Vmax/Km for NET transport); (b) rate of vesicular uptake
(equal to Vmax/Km for VMAT2 transport); (c) vulnerability to
intraneuronal metabolism by enzymes such as monoamine
oxidase (MAO); and (d) membrane diffusion rates, which
influence vesicular storage and neuronal retention, and are
partly determined by a tracer’s lipophilicity (log P). [18F]1 and
[18F]2 were developed in an effort to design a tracer with
optimal properties in each of these four categories. We
hypothesized that such a tracer would possess myocardial
kinetics that could be successfully analyzed using established
tracer kinetic analysis techniques to provide quantitative
measures of regional sympathetic nerve density. Specifically,
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Research Article
damage in a canine model of infarction.38 While these tracers
are certainly capable of imaging cardiac sympathetic nerves,
their kinetics would not satisfy the four tracer properties we had
targeted in the development of [18F]1 and [18F]2. Thus,
another advantage of the 18F-hydroxyphenethylguanidines [18F]
1 and [18F]2 over radiolabeled benzylguanidines is the ability to
analyze their irreversible myocardial kinetics using standard
tracer kinetic methods, such as Patlak analysis, to obtain
quantitative regional estimates of sympathetic nerve density.
Reagents and solvents were purchased from commercial sources
and used without further purification unless otherwise noted. [19F]4F-
MHPG and [19F]3F-PHPG (standards for HPLC analysis) and N,N′-
bis(tert-butoxycarbonyl)-N-3-benzyloxy-4-iodophenethylguanidine (7)
were prepared in our laboratory using previously reported
methods.18,39
N,N′,N″,N″-Tetrakis(tert-butoxycarbonyl)-N-3-benzyloxy-4-iodo-
phenethylguanidine (8). A solution of di-tert-butyl dicarbonate (6.1
mmol, 6.1 mL of 1.0 M solution in THF) was added to a solution of
N′,N″-bis(tert-butoxycarbonyl)-N-3-benzyloxy-4-iodophenethylguani-
dine 7 (600 mg, 1.0 mmol), dimethylaminopyridine (74 mg, 0.61
mmol) and triethylamine (0.85 mL, 6.1 mmol) in anhydrous THF (12
mL) at room temperature. The mixture was stirred for 48 h and then
poured over water (50 mL). The mixture was diluted with ethyl
acetate (50 mL) and extracted with ethyl acetate (2 × 50 mL). The
combined extracts were washed with brine, dried over Na2SO4 and
concentrated under reduced pressure. The residue was purified by
flash column chromatography (silica gel, 15% ethyl acetate in hexane)
CONCLUSION
■
Our previous multistep radiosynthesis of [18F]1 was not a
practical approach for routine production of [18F]fluoro-
hydroxyphenethylguanidines in a clinical PET radiochemistry
facility. A more efficient method involving 18F-labeling of a
N,N′,N″,N″-tetrakis-Boc protected guanidinyliodoium salt
precursor followed by a single deprotection step and HPLC
purification has been developed. This new radiosynthetic
approach has proven to be very reliable and provides sufficient
radiochemical yields and specific activities for clinical studies in
human subjects. This new method was used to synthesize [18F]
2, a structural isomer of [18F]1. Preclinical tests of [18F]2
demonstrated that it has imaging properties and kinetics that
are very similar to those observed with [18F]1, and that
quantitative metrics of regional cardiac sympathetic nerve
density can be obtained from tracer kinetic analyses of its
myocardial kinetics.
1
to afford the product 8 (730 mg, 92%) as a white oil; H NMR (500
MHz, CDCl3) δ 7.69 (d, J = 7.9 Hz, 1H), 7.53 (d, J = 7.4 Hz, 2H),
7.40 (t, J = 7.4 Hz, 2H), 7.32 (t, J = 7.4 Hz, 1H), 6.83 (d, J = 1.7 Hz,
1H), 6.66 (dd, J = 7.9, 1.7 Hz, 1H), 5.15 (s, 2H), 3.94 (td, J = 8.0, 4.9
Hz, 2H), 2.90 (td, J = 8.0, 4.9 Hz, 2H), 1.52−1.46 (m, 36H); 13C
NMR (125 MHz, CDCl3) δ 157.95, 157.48, 151.39, 147.63, 143.88,
141.05,139.51,136.74, 131.09, 128.72, 128.05, 127.40, 123.71, 113.89,
84.30, 83.92, 83.90, 82.29, 71.05, 48.78, 33.36, 28.25, 28.17, 28.13; MS
(ESI) m/z 796 (M+H)+, HRMS (ESI) calcd for C36H50IN3O9
818.2484 (M + Na)+, found 818.2491; Anal. Calcd For
C36H50IN3O9: C, 54.34; H, 6.34; N, 5.28. Found: C, 54.54; H, 6.41;
N, 5.14.
Due to the different metabolic pathways of [18F]1 and [18F]
2, both agents are currently being evaluated in first-in-human
studies under an exploratory IND clearance from the FDA
(ClinicalTrials.gov, NCT02385877). The results of these
studies will be used to select a lead radiotracer for further
clinical development. If PET studies with one of these
radiotracers can provide accurate and sensitive regional
measures of cardiac sympathetic nerve density in human
hearts, it could be used to investigate the contribution of
cardiac sympathetic denervation to mechanisms that lead to
sudden cardiac death from heart diseases. Based on previous
clinical studies with [123I]MIBG and [11C]HED, a potential
clinical role for [18F]1 or [18F]2 would be in improved risk
stratification of heart failure patients being staged for
implantable cardioverter defibrillator (ICD) therapy.
N,N′,N″,N″-Tetrakis(tert-butoxycarbonyl)-N-3-benzyloxy-4-trime-
thylstannylphenethyl Guanidine (9). Hexamethylditin (0.36 mL, 1.73
mmol) was added to a solution of compound 8 (688 mg, 0.86 mmol)
and tetrakis(triphenylphosphine)palladium (50 mg, 0.04 mmol) in
anhydrous toluene (8.0 mL) at room temperature under nitrogen
atmosphere. The resulting mixture was heated to 130 °C for 30 min,
cooled down to room temperature and filtered through a Celite pad.
Celite pad was washed with ethyl acetate and the solvent was removed
under reduced pressure. The residue was purified by flash column
chromatography (silica gel, 100% hexane to 10% ethyl acetate in
1
hexane) to afford the product 9 (621 mg, 86%) as a yellow oil; H
NMR (500 MHz, CDCl3) δ 7.40−7.34 (m, 4H), 7.31−7.29 (m, 2H),
6.88 (d, J = 7.2 Hz, 1H), 6.82 (s, 1H), 5.02 (s, 2H), 3.96 (t, J = 8.1 Hz,
2H), 2.92 (t, J = 8.1 Hz, 2H), 1.53−1.31 (m, 36H), 0.17 (s, 9H); 13C
NMR (126 MHz, CDCl3) δ 158.0, 157.5, 151.4, 147.6, 143.9, 141.1
139.5, 136.7, 128.7, 128.5, 127.4, 123.7, 113.9, 84.3, 83.9, 83.9, 82.3,
71.1, 48.8, 33.4, 28.3, 28.2, 28.1, 0.2; MS (ESI) m/z 834 (M + H)+;
HRMS (ESI) calcd for C39H59N3O9Sn 856.3166 (M + Na)+, found
856.3183. Anal. Calcd For C39H59N3O9Sn: C, 56.26; H, 7.14; N, 5.05.
Found: C, 56.00; H, 7.14; N, 4.88.
2-Benzyloxy-4-{2′-(N,N′,N″,N″-tetrakis(tert-butoxycarbonyl)-
guanidinyl)ethyl}phenyl (2-thienyl)iodonium Tosylate (10). A
solution of 2-(diacetoxy)iodothiophene (92 mg, 0.278 mmol) in
CH2Cl2 (1.0 mL) was added to a solution of p-toluenesulfonic acid
hydrate (53 mg, 0.278 mmol) in MeCN (1.0 mL) at room
temperature under nitrogen atmosphere. The white precipitate was
immediately generated, and the mixture was stirred for 1 h. A solution
of compound 9 (232 mg, 0.278 mmol) in CH2Cl2 (1.0 mL) and
MeCN (1.0 mL) was added slowly to the reaction mixture. After the
white precipitate had disappeared, the mixture was stirred at room
temperature for 20 h under nitrogen atmosphere. The solvent was
removed under reduced pressure and the residue was purified by flash
column chromatography (silica gel, 100% CH2Cl2 to 20:1 = CH2Cl2/
MeOH) to afford the tosilate salt 10 (255 mg, 87%) as a yellow solid.
mp 79−84 °C; 1H NMR (500 MHz, DMSO-d6) δ 8.26 (d, J = 8.1 Hz,
1H), 7.89 (dd, J = 5.3, 1.3 Hz, 1H), 7.76 (dd, J = 3.8, 1.3 Hz, 1H),
7.52−7.37 (m, 7H), 7.24 (d, J = 1.6, 1H), 7.11−7.08 (m, 3H), 6.95
(dd, J = 8.2, 1.6 Hz, 1H), 5.34 (s, 2H), 3.91 (t, J = 7.4 Hz, 2H), 2.91 (t,
J = 7.4 Hz, 2H), 2.28 (s, 3H), 1.43−1.39 (m, 27H), 1.29 (s, 9H); 13C
NMR (126 MHz, DMSO-d6) δ 157.05, 155.10, 150.54, 146.676,
METHODS
■
Chemicals and General Instrumentation. NMR spectra were
obtained on a Varian Inova 500 (499.90 MHz for 1H; 125.70 MHz for
13C) spectrometer. H and 13C NMR chemical shifts (δ) are reported
1
in parts per million (ppm) relative to internal standard TMS and
coupling constants (J) are in Hz. High-resolution mass spectra were
obtained on a VG (Micromass) 70-250S spectrometer using
electrospray ionization (ESI) in positive ion mode, direct chemical
ionization (DCI) or electron impact (EI) at 70 eV. Melting points
were determined on a Mel-Temp capillary melting point apparatus in
open capillary tubes. Flash column chromatography was performed
with E. Merck 230−400 mesh silica gel. Analytical TLC was performed
with Analtech 0.25 mm glass-backed plates with fluorescent back-
ground. Visualization of TLC plates was achieved by UV illumination
or treatment with phosphomolybdic acid (PMA). High pressure liquid
chromatography (HPLC) was performed on a Hitachi pump L-7100
instrument equipped with Hitachi D-7500 integrator and Hitachi L-
4000 UV detector. Radioactivity detection was done with Bioscan
coincidence (model B-FC-4000) detector. Reverse-phase HPLC
analysis of the formation of radiometabolites in plasma samples was
performed on a PerkinElmer Series 410 LC instrument equipped with
an Ortec model 905-4 NaI(Tl) radiodetector (Oak Ridge, TN).
H
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146.23, 145.82, 143.64, 139.44, 137.51, 136.87, 136.33, 135.67, 129.23,
128.63, 128.40, 128.01, 127.96, 127.28, 125.48, 123.77, 114.20, 108.08,
101.56, 83.43, 83.21, 81.51, 70.90, 48.86, 47.40, 46.17, 40.01, 32.64,
27.49, 27.41, 27.19, 20.77; HRMS (EI) calcd for C40H53IN3O9S
878.2542 (M − OTs)+, found 878.2546.
1H), 6.79 (d, J = 8.3 Hz, 1H), 5.13 (s, 2H), 4.52 (br. s, NH), 3.32 (t, J
= 6.5 Hz, 2H), 2.70 (t, J = 6.5 Hz, 2H), 1.44 (S, 9H); 13C NMR (126
MHz, CDCl3) δ 155.90, 155.80, 139.67, 136.56, 133.56, 130.87,
128.79, 128.54, 127.86, 126.98, 112.72, 86.92, 79.33, 70.97, 41.78,
34.80, 28.45, 28.41. CAS Registry Number: 788824-73-7.
2-Benzyloxy-4-{2′-(N,N′,N″,N″-tetrakis(tert-butoxycarbonyl)-
guanidinyl)ethyl}phenyl (2-thienyl)iodonium Bromide (11). A
solution of KBr (98 mg, 0.82 mmol) in H2O (1.0 mL) was added
to a solution of compound 10 (200 mg, 0.19 mmol) in MeCN (1.0
mL) at 60 °C for 5 min. The reaction mixture was stirred at room
temperature for 1 h. The precipitate was washed with ice H2O (10
mL), filtered, washed further with hexane several times, and dried in
vacuo to afford the bromide salt 11 (156 mg, 86%) as a light yellow
solid. mp 116−119 °C; 1H NMR (500 MHz, DMSO-d6) δ 8.25 (d, J =
8.1 Hz, 1H), 7.86 (d, J = 5.3 Hz, 1H), 7.73 (d, J = 3.7 Hz, 1H), 7.52−
7.38 (m, 5H), 7.22 (s, 1H), 7.08 (dd, J = 5.3, 3.7 Hz, 1H), 6.94 (d, J =
8.1 Hz, 1H), 5.33 (s, 2H), 3.91 (t, J = 7.4 Hz, 2H), 2.90 (t, J = 7.4 Hz,
2H), 1.43−1.39 (m, 27H), 1.30 (s, 9H); 13C NMR (126 MHz,
DMSO-d6) δ 157.05, 155.06, 150.53, 146.73, 146.00, 143.63, 139.03,
136.90, 135.96, 135.70, 129.10, 128.61, 128.35, 127.92, 123.72, 114.17,
109.53, 108.87, 102.82, 83.43, 83.19, 81.49, 70.87, 47.38, 32.64, 27.48,
27.40, 27.20; HRMS (ESI) calcd for C40H53IN3O9S 878.2542 (M −
Br)+, found 878.2555. Anal. Calcd For C40H53BrIN3O9S: C, 50.11; H,
5.57; N, 4.38. Found: C, 50.04; H, 5.69; N, 4.37.
2-(4-Hydroxyphenyl)ethylamine tert-Butylcarbamate (13). To a
solution of 2-(4-hydroxy phenyl)ethylamine 12 (4.00 g, 29.16 mmol)
in THF (48 mL) was added triethylamine (4.3 mL, 30.82 mmol). Di-
tert-butyl-dicarbonate (6.70 g, 30.70 mmol) was added to the resulting
solution and the reaction mixture was stirred at room temperature
overnight. The solvent was evaporated under reduced pressure and the
residue was purified by flash column chromatography (silica gel, 20%
ethyl acetate in hexane) to afford the desired compound 13 (6.78 g,
98%) as a colorless oil; 1H NMR (500 MHz, CDCl3) δ 7.03 (d, J = 8.1
Hz, 2H), 6.77 (d, J = 8.1 Hz, 2H), 4.57 (br. s, NH), 3.33 (t, J = 6.5 Hz,
2H), 2.71 (t, J = 6.5 Hz, 2H), 1.47 (s, 9H); 13C NMR (126 MHz,
CDCl3) δ 154.43, 129.88, 115.43, 98.46, 67.47, 42.01, 33.25, 28.46.
CAS Registry Number: 64318-28-1.
2-(4-Benzyloxy-3-iodophenyl)ethylamine Hydrochloride (16).
Hydrochloric acid (45 mL of 4.0 M solution in 1,4-dioxane, 180
mmol) was added to a solution of compound 15 (0.80 g, 17.6 mmol)
in ethyl acetate (5 mL). The resulting solution was stirred at 65 °C for
1 h. The solvent was evaporated under reduced pressure. The crude
product was dissolved again in ethyl acetate and concentrated under
reduced pressure. The residue was purified by flash column
chromatography (silica gel, 5% methanol, and 0.3% NH4OH in
methylene chloride) to afford the compound 16 (0.61 g, 97%) as a
yellow powder; mp 162−167 °C; 1H NMR (500 MHz, CDCl3) δ 7.70
(s, 1H), 7.46 (d, J = 7.4 Hz, 2H), 7.37 (t, J = 7.4 Hz, 2H), 7.30 (t, J =
7.4 Hz, 1H), 7.15 (d, J = 8.4 Hz, 1H), 6.76 (d, J = 8.4 Hz, 1H), 5.07 (s,
2H), 3.20 (t, J = 7.8 Hz, 2H), 3.00 (t, J = 7.8 Hz, 2H); 13C NMR (126
MHz, CDCl3) δ 156.50, 139.75, 136.35, 130.52, 129.83, 128.55,
127.89, 126.97, 112.91, 110.01, 87.48, 87.22, 70.92, 45.82, 41.12. CAS
Registry Number: 794507-50-9.
N′,N″-Bis(tert-butoxycarbonyl)-N-4-benzyloxy-3-iodophenethyl-
guanidine (17). To a cooled (0 °C) solution of compound 16 (0.37 g,
1.06 mmol) and triethylamine (0.75 mL, 5.38 mmol) in anhydrous
DMF (3.5 mL) was added in portion 1,3-bis(tert-butoxycarbonyl)-2-
methyl-2-thiopseudourea (0.34 g, 1.16 mmol). The resulting mixture
was stirred at 0 °C for 1 h, warmed to room temperature and stirred
overnight. The mixture was diluted with ethyl acetate (50 mL), washed
with saturated NH4Cl solution (200 mL), and extracted with ethyl
acetate (2 × 150 mL). The combined extracts were washed with brine,
dried over Na2SO4 and concentrated under reduced pressure. The
residue was purified by flash column chromatography (silica gel, 10%
ethyl acetate in hexane) to afford the product 17 (0.37 g, 58%) as a
1
white solid; mp 115−117 °C; H NMR (500 MHz, CDCl3) δ 11.46
(br. s, 1NH), 8.38 (br. s, 1NH), 7.67 (s, 1H), 7.49 (d, J = 7.6 Hz, 2H),
7.39 (t, J = 7.6 Hz, 2H), 7.32 (t, J = 7.6 Hz, 1H), 7.11 (d, J = 9.0 Hz,
1H), 6.78 (d, J = 9.0 Hz, 1H), 5.13 (s, 2H), 3.62 (q, J = 6.7 Hz, 2H),
2.77 (t, J = 6.7 Hz, 2H), 1.50 (s, 18H); 13C NMR (126 MHz, CDCl3)
δ 156.11, 155.99, 139.76, 136.57, 133.15, 129.69, 128.53, 127.84,
126.98, 112.74, 86.91, 83.12, 79.26, 70.96, 42.15, 33.93, 28.31, 28.09;
HRMS calcd for C26H34IN3O5 596.1616, found 596.1615.
2-(3-Iodo-4-hydoxyphenyl)ethylamine tert-Butylcarbamate (14).
Sodium iodide (7.41 g, 49.45 mmol) and NaOH (1.54 g, 38.50 mmol)
were added to a solution of compound 13 (7.82 g, 32.98 mmol) in
MeOH (60 mL). The resulting solution was cooled to 0 °C. Sodium
hypochlorite (4.0−4.9% in water, 81.8 mL, 49.45 mmol) was added
slowly to the solution by dropping funnel. The reaction temperature
was kept at 0−3 °C. After adding NaOCl, the reaction mixture was
stirred for one more hour at 0−5 °C. A solution of sodium thiosulfate
(10% in H2O, 70 mL) was added, and the pH was adjusted to 6.5 by
addition of HCl (2.0 N solution). The product was extracted by ethyl
acetate (3 × 100 mL), and the combined extracts were washed with
brine, dried over Na2SO4, and concentrated under reduced pressure.
The residue was purified by flash column chromatography (silica gel,
20% ethyl acetate in hexane) to afford the compound 14 (5.15 g, 43%)
N,N′,N″,N″-Tetrakis(tert-butoxycarbonyl)-N-4-benzyloxy-3-iodo-
phenethylguanidine (18). A solution of di-tert-butyl dicarbonate (40.7
mmol, 40.7 mL of 1.0 M solution in THF) was added to a solution of
compound 17 (4.04 g, 6.78 mmol), N,N-dimethylaminopyridine (497
mg, 4.07 mmol), and triethylamine (5.67 mL, 40.1 mmol) in
anhydrous THF (82 mL) at room temperature. The mixture was
stirred for 48 h and then poured over water (200 mL). The mixture
was diluted with ethyl acetate (200 mL). After decantation, the
aqueous layer was extracted with ethyl acetate (2 × 200 mL). The
combined extracts were washed with brine, dried over Na2SO4, and
concentrated under reduced pressure. The residue was purified by
flash column chromatography (silica gel, 10% ethyl acetate in hexane)
1
as a white powder; mp 112−115 °C; H NMR (500 MHz, CDCl3) δ
7.49 (s, 1H), 7.06 (d, J = 8.0 Hz, 1H), 6.92 (d, J = 8.0 Hz, 1H), 4.53
(br. s, NH), 3.31 (t, J = 6.5 Hz, 2H), 2.70 (t, J = 6.5 Hz, 2H), 1.47 (s,
9H); 13C NMR (126 MHz, CDCl3) δ 155.86, 153.51, 138.23, 133.11,
130.64, 115.02, 85.67, 79.40, 44.56, 34.78, 28.54. CAS Registry
Number: 788824-50-0.
1
to afford the product 18 (3.41 g, 63%) as a white oil; H NMR (500
MHz, CDCl3) δ 7.71 (s, 1H), 7.49 (d, J = 7.6 Hz, 2H), 7.39 (t, J = 7.6
Hz, 2H), 7.32 (t, J = 7.6 Hz, 1H), 7.17 (d, J = 8.4 Hz, 1H), 6.78 (d, J =
8.4 Hz, 1H), 5.13 (s, 2H), 3.93 (t, J = 8.0 Hz, 2H), 2.85 (t, J = 8.0 Hz,
2H), 1.50 (s, 36H); 13C NMR (126 MHz, CDCl3) δ 157.68, 155.85,
147.34, 139.77, 136.58, 133.51, 129.92, 128.54, 127.85, 126.98, 112.71,
86.84, 83.66, 82.03, 70.96, 48.73, 31.87, 28.04, 27.98, 27.91; HRMS
calcd for C36H50IN3O9 818.2484, found 818.2479.
N,N′,N″,N″-Tetrakis(tert-butoxycarbonyl)-N-4-benzyloxy-3-trime-
thylstannylphenethylguanidine (19). Hexamethylditin (2.0 mL, 9.60
mmol) was added to a solution of compound 18 (2.79 g, 3.50 mmol)
and tetrakis(triphenylphosphine)palladium (200 mg, 0.16 mmol) in
anhydrous toluene (30 mL) at room temperature under nitrogen
atmosphere. The resulting mixture was heated to 130 °C for 30 min,
cooled down to room temperature, and filtered through a Celite pad.
Celite pad was washed with ethyl acetate and the solvent was removed
under reduced pressure. The residue was purified by flash column
2-(4-Benzyloxy-3-iodophenyl)ethylamine tert-Butylcarbamate
(15). Benzyl bromide (0.52 g, 3.03 mmol) and K2CO3 (0.63 g, 4.55
mmol) were added to a solution of compound 14 (1.10 g, 3.03 mmol)
in acetone (17 mL). The resulting solution was stirred at 70 °C for 4
h. The reaction mixture was filtered and the solvent was evaporated
under reduced pressure. Water was added to the residue, the product
was extracted by ethyl acetate (3 × 100 mL), and the combined
extracts were washed with brine, dried over Na2SO4, and concentrated
under reduced pressure. The residue was purified by flash column
chromatography (silica gel, 10% ethyl acetate in hexane) to afford the
1
compound 15 (1.07 g, 78%) as a white powder; mp 70−72 °C; H
NMR (500 MHz, CDCl3) δ 7.63 (s, 1H), 7.49 (d, J = 7.4 Hz, 2H),
7.39 (t, J = 7.4 Hz, 2H), 7.32 (t, J = 7.4 Hz, 1H), 7.09 (d, J = 8.3 Hz,
I
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chromatography (silica gel, 100% hexane to 10% ethyl acetate in
hexane) to afford the product 19 (2.87, 98%) as a yellow oil; 1H NMR
(500 MHz, CDCl3) δ 7.42−7.37 (m, 4H), 7.32 (t, J = 6.5 Hz, 2H),
6.91 (d, J = 7.1 Hz, 1H), 6.84 (s, 1H), 5.04 (s, 2H), 3.98 (t, J = 8.1 Hz,
2H), 2.94 (t, J = 8.1 Hz, 2H), 1.51−1.49 (m, 36H), 0.19 (s, 9H); 13C
NMR (126 MHz, CDCl3) δ 163.2, 157.8, 151.2, 147.4, 143.6, 141.2,
137.1, 136.5, 128.4, 128.1, 127.8, 127.7, 121.8, 110.9, 83.6, 83.4, 82.0,
70.0, 48.9, 33.6, 28.1, 28.0, 27.9, 14.1; HRMS (ESI) calcd for
C39H59N3O9Sn 856.3166 (M+Na)+, found 856.3172. Anal. Calcd For
C39H59N3O9Sn: C, 56.26; H, 7.14; N, 5.05. Found: C, 56.00; H, 7.23;
N, 4.90.
solution was heated at 120 °C for 15 min and then cooled to 50 °C.
Next, a mixture solution of NaOH solution (1.0 mL, 4.0 M in H2O)
and buffer solution (1.8 mL, 5% EtOH in 40 mM NH4OAc) was
added into the reactor vessel. This mixture was injected onto a reverse-
phase HPLC column (Phenomenex Synergi 10 μ Hydro-RP 80A, 250
× 10 mm, 5% EtOH in 40 mM NH4OAc buffer, flow rate 4.0 mL/min,
λ = 254 nm) and [18F]1 was collected at Rt = 30−32 min. The
collected [18F]1 fraction was passed through a 0.22 μm sterilizing filter
directly into a 10 mL septum-sealed sterile pyrogen-free glass vial.
Specific activity (SA) was determined by injecting a sample of [18F]
1 with known activity (kBq) onto an HPLC system used for quality
control. The area under the UV absorbance peak associated with the
[18F]1 radioactivity peak was compared against a predetermined
standard curve to estimate the total mass ([18F]4F-MHPG + [19F]4F-
MHPG) in micrograms. The ratio of 18F-activity to total mass
(converted from μg to μmol using the molecular weight of [19F]4F-
MHPG) gave the specific activity.
2-Benzyloxy-5-{2′-(N,N′,N″,N″-tetrakis(tert-butoxycarbonyl)-
guanidinyl)ethyl}phenyl(2-thienyl)iodonium Tosylate (20). A sol-
ution of 2-(diacetoxy)iodothiophene (209 mg, 0.637 mmol) in
CH2Cl2 (5.0 mL) was added to a solution of p-toluenesulfonic acid
hydrate (121 mg, 0.637 mmol) in MeCN (5.0 mL) at room
temperature under nitrogen atmosphere. The white precipitate was
immediately generated and the mixture was stirred for 1 h. A solution
of compound 19 (530 mg, 0.637 mmol) in CH2Cl2 (3.0 mL) and
MeCN (3.0 mL) was added slowly to the reaction mixture. After the
white precipitate disappeared, the mixture was stirred at room
temperature for 20 h under nitrogen atmosphere. The solvent was
removed under reduced pressure, and the residue was purified by flash
column chromatography (silica gel, 100% CH2Cl2 to 20:1 = CH2Cl2/
MeOH) to afford the tosylate salt 20 (482 mg, 86%) as a yellow solid;
Radiosynthesis of [18F]2. The same methods described for the
synthesis of [18F]1 were used to prepare [18F]2, except that the
appropriate precursor 21 was used instead of precursor 11. HPLC
purification conditions were slightly different: Phenomenex Synergi 10
μ Hydro-RP 80A, 250 × 10 mm, 3.5% EtOH in 40 mM NH4OAc
buffer, flow rate 4.0 mL/min, λ = 254 nm. In this system, [18F]2 was
collected at Rt = 31−33 min.
Isolated Rat Heart Studies. Hearts from male Sprague−Dawley
rats (225−500 g) were perfused under moderate workload conditions
(7.3 mmHg preload, 73 mmHg afterload) using a working heart
preparation.40 Two parallel perfusion circuits were connected to the
left atrial cannula with a three-way connector to allow for rapid
switching from one circuit to the other. The heart perfusate was Krebs-
Henseleit (KH) bicarbonate buffer (118 nM NaCl, 4.7 mM KCl, 2.55
mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, and 25 mM NaHCO3)
containing 5 mM glucose, oxygenated with a 95% O2/5% CO2 gas
mixture and held at 37 °C. Corticosterone (54 μM) was added to the
perfusate to block extraneuronal uptake (uptake-2) of the radiotracer
into the rat myocardium.23
Fluorine-18 activity in the heart was measured externally using a
pair of cesium fluoride (CsF) scintillation detectors with crystal size 51
cm diameter and 51 cm thick (Crismatec 51Y51; Saint-Gobain,
Nemours, France). The front faces of the two CsF detectors were
positioned directly opposite each other, ∼4 cm apart, with the heart
centered between them. Each detector was enclosed in a large
cylindrical lead collimator (2 cm wall thickness, 25 cm long) to shield
against background counts from radioactive sources outside the heart.
Two coincidence detection circuits were established between the
detectors using standard Nuclear Instrumentation Module (NIM)
electronic modules. One circuit measured total coincident events
between the two detectors (true + random coincident events), and the
second measured only random coincident events. A computerized data
acquisition system interfaced to the NIM-module coincidence circuits
was used to acquire and record the whole-heart radioactivity data
throughout the study.41
Hearts were initially perfused for a 30 min stabilization period using
KH buffer in the first perfusion circuit. During this time, [18F]2 was
added to 1.0 L of KH buffer circulating in the second perfusion circuit
and allowed to equilibrate over several minutes. Three 1.0 mL aliquots
were drawn from the second perfusion circuit for counting in a gamma
counter (Cobra II Auto-Gamma, PerkinElmer, Waltham, MA) to
determine the radioactivity concentration in the perfusate (Cp), which
was ∼74 kBq/mL perfusate. After initiating data acquisition from the
CsF detectors, the heart was rapidly switched to the second perfusion
circuit to begin a constant infusion of [18F]2 for 10 min. Then the
heart was switched back to the first perfusion circuit for 120 min to
measure clearance rates of the tracer from the heart.
1
mp 140−141 °C; H NMR (500 MHz, DMSO-d6) δ 8.21 (s, 1H),
7.90 (t, J = 5.3 Hz, 1H), 7.77 (t, J = 5.3 Hz, 1H), 7.49−7.37 (m, 7H),
7.32 (d, J = 8.5 Hz, 2H), 7.12−7.08 (m,3H), 5.33 (s, 2H), 3.88 (t, J =
7.8 Hz, 2H), 2.85 (t, J = 7.8 Hz, 2H), 2.28 (s, 3H), 1.43−1.36 (m,
36H); 13C NMR (126 MHz, DMSO-d6) δ 157.1, 153.7, 150.6, 146.7,
145.8, 143.6, 139.5, 137.5, 136.5, 136.3, 135.8, 134.7, 133.7, 129.2,
128.6, 128.3, 128.0, 127.7, 125.5, 113.9, 110.3, 101.3, 83.6, 83.2, 81.6,
70.9, 47.5, 31.1, 27.9, 27.52, 27.4, 27.3, 27.2, 20.9.
2-Benzyloxy-5-{2′-(N,N′,N″,N″-tetrakis(tert-butoxycarbonyl)-
guanidinyl)ethyl}phenyl(2-thienyl)iodonium Bromide (21). A sol-
ution of KBr (583 mg, 4.90 mmol) in H2O (5.0 mL) was added to a
solution of compound 20 (430 mg, 0.49 mmol) in MeCN (7.0 mL) at
60 °C for 5 min. The reaction mixture was stirred at room temperature
for 1 h. The precipitate was washed with ice H2O (10 mL), filtered,
washed further with hexane several times, and dried in vacuo to afford
the bromide salt 21 (411 mg, 96%) as a light yellow solid; mp 140−
141 °C; 1H NMR (500 MHz, CDCl3) δ 7.97 (s, 1H), 7.53 (d, J = 5.2
Hz, 1H), 7.46 (d, J = 5.2 Hz, 1H), 7.41−7.35 (m, 6H), 6.96−6.91 (m,
2H), 5.21 (s, 2H), 3.92 (t, J = 8.1 Hz, 2H), 2.90 (t, J = 8.1 Hz, 2H),
1.52−1.48 (m, 36H); 13C NMR (126 MHz, CDCl3) δ 157.6, 153.6,
151.1, 147.3, 143.5, 137.9, 136.5, 135.2, 134.7, 134.6, 133.8, 128.8,
128.7, 128.5, 127.5, 114.4, 113.8, 83.89, 83.8, 82.2, 71.9, 48.4, 33.8,
32.0, 29.7, 28.1, 28.0, 27.9; HRMS (ESI) calcd for C40H53IN3O9S
878.2542 (M-Br)+, found 878.2540. Anal. Calcd For C40H53BrIN3O9S:
C, 50.11; H, 5.57; N, 4.38. Found: C, 51.86; H, 5.95; N, 4.24.
Radiosynthesis of [18F]1. A TRACERlab FXFN computer-
controlled radiosynthesis module (GE Healthcare) was used to
achieve fully automated radiosyntheses of [18F]1 and [18F]2. [18F]F−
was prepared by the 18O(p,n)18F reaction using H218O as the target
material in a GE PETrace cyclotron. [18F]F− was isolated from the
enriched water by trapping on a Waters Sep-Pak Light QMA cartridge
(preactivated with 10 mL of ethanol and 10 mL of H2O) and eluted
from the cartridge into the glassy-carbon reactor vial of the
TRACERlab FXFN system with a solution of 0.5 mL Cs2CO3 (0.05
M in H2O). MeCN (1.0 mL) was added to the reactor vessel and then
water/acetonitrile was evaporated at 80 °C under vacuum with a
nitrogen stream to yield dried Cs[18F]F. After cooling to 60 °C, a
mixed solution of 0.5 mL of DMF and 20 μL of Milli-Q H2O,
containing 5.5−6.0 mg of the diaryliodonium salt precursor 11 and 1.0
mg of TEMPO (2,2,6,6-tetramethylpiperidine-N-oxyl) were added to
the reactor vessel containing Cs[18F]F. The sealed reaction mixture
was heated at 150 °C for 5 min to produce 3-benzyloxy-4-
[18F]fluorophenethyl-N,N′,N″,N″-tetrakis-BOC-guanidine [18F]22 as
intermediate. After cooling to 70 °C, a solution of 48% HBr (0.5 mL)
and MeCN (0.5 mL) was added to the reaction mixture. The reaction
The acquired whole-heart radioactivity data (counts per second;
cps) at each time point were converted to an “apparent distribution
volume” (ADV; mL perfusate/g wet), by dividing by the perfusate
radioactivity concentration Cp (kBq/mL perfusate), the detector
system calibration factor Zcalib (cps/kBq), and the measured wet mass
of the heart Mw (g wet). Neuronal uptake rates of the radiotracers
J
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(Kup; mL perfusate/min/g wet) were calculated as the slope of a linear
regression of the ADV data between t = 1 min and t = 4 min of the 10
min infusion study. Clearance rates were estimated by fitting the ADV
data during the clearance phase of the study to multiple exponential
decay processes. The exponential clearance rate constants (λi) were
used to calculate corresponding clearance half-times: T1/2 = ln(2)/λi.
The slowest rate, associated with clearance from sympathetic neurons,
is reported for each compound.
Plasma Time−Activity Curve. Region-of-interest analysis of the
dynamic PET images was used to determine a time-activity curve of
the activity concentration in whole blood, Cwb(t). The whole blood
time-activity curve Cwb(t) was then multiplied by the measured ratio of
activity in plasma over whole blood (Cp/Cwb) and by the percentage of
intact parent tracer fraction in plasma data, fintact(t), to estimate the
kinetics of the plasma concentration of intact tracer, Cp(t). The
estimated plasma curve Cp(t) was used as the input function for tracer
kinetic analyses.
Tracer Kinetic Analysis. For each PET study, the final four
dynamic image frames were summed and used to draw a region-of-
interest in the left ventricular wall, encompassing three to four
transaxial slices, to extract a time-activity curve for myocardial tissue
Ct(t). For compartmental modeling analysis, Ct(t) and the plasma
kinetics Cp(t) for [18F]2 were analyzed using a two-tissue compart-
ment model with irreversible trapping to estimate the rate constants K1
(mL/min/g), k2 (min−1), k3 (min−1) and a blood volume fraction BV
(dimensionless). The Ct(t) and Cp(t) data were also analyzed using
Patlak analysis to estimate a Patlak slope, Kp (mL/min/g).
PET Imaging Studies with [18F]2. Cardiac PET studies (n = 4)
were performed in rhesus macaque monkeys using a microPET P4
primate scanner (Siemens/CTI Concorde Microsystems, Knoxville,
TN). After anesthetizing the animal, a percutaneous angiocather was
placed in the saphenous vein of each leg, one for tracer injection, the
other for blood sampling. Vital signs, including heart rate (bpm),
blood oxygen saturation levels (SpO2) and body temperature were
monitored continuously (model V3404P, SurgiVet, Norwell, MA).
Dynamic PET data were acquired in list-mode for 90 min after
intravenous injection of 155−230 MBq of [18F]2. List-mode emission
data were rebinned into a 27-frame dynamic sequence (12 × 10 s, 2 ×
30 s, 2 × 60 s, 2 × 150 s, 2 × 300 s, 7 × 600 s). Rebinned emission
data were corrected for attenuation and scatter, and transaxial images
reconstructed using maximum a posteriori (MAP) reconstruction,42 an
iterative method that accounts for the detector point spread function
in the model of the system.
Animal Care. The care of all animals used in this study was done in
accordance with the Animal Welfare Act and the National Institute of
Health’s Guide for the Care and use of Laboratory Animals.44 Animal
protocols were approved by the Institutional Animal Care and Use
Committee (IACUC) at the University of Michigan.
Blood Partitioning and Radiometabolite Analysis. Venous
blood samples (1.5−2.0 mL) were centrifuged for 1 min at 12 000g to
separate plasma and red blood cells. Plasma was deproteinized by
adding perchloric acid (HClO4; final concentration 0.4 N) and
centrifuging for 5 min at 12 000g. The supernatant was neutralized
with KOH (pH 7.0−7.5) and filtered twice (Millex GS 0.22 μm,
Millipore, Billerica, MA). Aliquots (0.1 mL) of whole blood, plasma,
and the final supernatant were counted in a gamma counter. Count
data for plasma and whole blood aliquots were decay corrected and
used to calculate the relative concentrations of [18F]2 in plasma and
whole blood (Cp/Cwb). The final supernatant was analyzed by HPLC
(Synergi 10 μm Hydro-RP column, 4.6 × 250 mm, 60 mM sodium
phosphate buffer, pH 5.4 with 8% ethanol, flow rate 1.0 mL/min) with
an in-line radiation detector. Under the HPLC conditions used, [18F]2
had a retention time Rt = 12.7 min, while the main polar
radiometabolite formed had Rt = 9.3 min. Peak area analysis of the
radiation detection curve was used to estimate the percentage intact
parent tracer fraction (fintact) for each plasma sample.
In Vitro Metabolism Tests. To test for sulfate conjugation of
[18F]2, a 20 μL aliquot of monkey liver cytosol (#452461, BD
Biosciences, San Jose, CA) was added to a 10 μL aliquot of 10 mM
sulfotransferase cofactor PAPS (adenosine-3′-phosphate-5′-phospho-
sulfate lithium salt hydrate; #A1651, Sigma-Aldrich, Milwaukee, WI)
dissolved in 50 μL of 1.0 mM Tris-HCl buffer (pH 7.4) and 170 μL of
ultrapure water (18 MΩ·cm Milli-Q, Millipore, Billerica, MA) and
incubated at 37 °C for 5 min.43 Next, 740 kBq of [18F]2 in 250 μL of
ultrapure water was added to the reaction mixture (final volume 500
μL) and incubated at 37 °C for 20 min. The reaction was terminated
by centrifugation at 16 000g for 5 min at 4 °C. The supernatant was
filtered (Millex GS 0.22 μm, Millipore, Billerica, MA) and analyzed
using HPLC with radiation detection as described above for the rhesus
macaque plasma samples. To test for glucuronidation of [18F]2, a 25
μL aliquot containing 0.5 mg of monkey liver microsomes (#452413,
BD Biosciences, San Jose, CA) was added to a reaction mixture of 50
μL of 20 mM glucuronidation cofactor UDPGA (uridine 5′-
diphosphoglucuronic acid; #U5625 Sigma-Adrich, Milwaukee, WI)
and 50 μL of 30 mM DTT (dithiothreitol, #D0632, Sigma-Aldrich,
Milwaukee, WI) dissolved in 1.0 M glycine/NaOH buffer containing
50 mM MgCl2 (pH 9.2). An additional 125 μL of the glycine/NaOH
buffer was added and the mixture incubated at 37 °C for 5 min. Next,
740 kBq of [18F]2 in 250 μL of ultrapure water was added to the
reaction mixture (final volume 500 μL) and incubated at 37 °C for 20
min. Using the same procedures for the sulfate conjugation test, the
reaction mixture was centrifuged and the supernatant filtered for
analysis using radio-HPLC.
AUTHOR INFORMATION
■
Corresponding Author
*Mailing address: Division of Nuclear Medicine, Department of
Radiology, 2276 Medical Science I Bldg, SPC-5610, University
of Michigan Medical School, Ann Arbor, MI 48109, USA.
Telephone: 734-936-0725. Fax: 734-764-0288. E-mail: raffel@
umich.edu.
ORCID
David M. Raffel: 0000-0002-7188-9463
Author Contributions
D.M.R. and Y.-W.J. designed the tracers; Y.-W.J., K.S.J., and
D.M.R. developed the radiosynthetic approach; Y.-W.J and
K.S.J. prepared the radiosynthetic precursors and the non-
radioactive standards for HPLC, and performed all radio-
syntheses; D.M.R. performed the isolated rat heart studies;
P.S.S., C.A.Q., G.G., Y.-W.J., and D.M.R. performed the PET
imaging studies; D.M.R. performed the PET image analysis and
kinetic data analysis with contributions from R.A.K.; G.G.,
D.M.R., and Y.-W.J. performed all metabolism studies.
Funding
We gratefully acknowledge the support of this work through
PHS Grant R01-HL079540 from the National Heart Lung and
Blood Institute, National Institutes of Health, Bethesda, MD.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors thank the staff of the University of Michigan
Cyclotron Facility for their many contributions to this study.
ABBREVIATIONS USED
■
DMI, desipramine; [11C]HED, [11C]-(−)-m-hydroxyephedrine;
[11C]GMO, N-[11C]guanyl-(−)-m-octopamine; [123I]MIBG,
[
123I]m-iodobenzylguanidine; NET, norepinephrine transport-
er; TEMPO, 2,2,6,6-tetramethylpiperidine-N-oxyl; VMAT2,
vesicular monoamine transporter, isoform 2
K
DOI: 10.1021/acschemneuro.7b00051
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dine ([18F]4F-MHPG): a novel imaging agent for cardiac sympathetic
innervation. Mol. Imaging Biol. 17 (Suppl 1), S997.
REFERENCES
■
(1) Fukuda, K., Kanazawa, H., Aizawa, Y., Ardell, J. L., and
Shivkumar, K. (2015) Cardiac innervation and sudden cardiac death.
Circ. Res. 116, 2005−2019.
(2) Shen, M. J., and Zipes, D. P. (2014) Role of the autonomic
nervous system in modulating cardiac arrhythmias. Circ. Res. 114,
1004−1021.
(3) Kuehl, M., and Stevens, M. J. (2012) Cardiovascular autonomic
neuropathies as complications of diabetes mellitus. Nat. Rev.
Endocrinol. 8, 405−416.
(4) Floras, J. S. (1993) Clinical aspects of sympathetic activation and
parasympathetic withdrawal in heart failure. J. Am. Coll. Cardiol. 22 (4
Suppl. A), 72A−82A.
(20) Kazmierczak, P., Skulski, L., and Kraszkiewicz, L. (2001)
Synthesis of (diacetoxyiodo)arenes or iodylarenes from iodoarenes,
with sodium periodate as the oxidant. Molecules 6, 881−891.
(21) Chun, J.-H., and Pike, V. W. (2012) Regiospecific syntheses of
functionalized diaryliodonium tosylates via [hydroxy(tosyloxy)iodo]-
arenes generated in site from (diacetoxyiodo)arenes. J. Org. Chem. 77,
1931−1938.
(22) Ross, T. L., Ermert, J., Hocke, C., and Coenen, H. H. (2007)
Nucleophilic 18F-fluorination of heteroaromatic iodonium salts with
no-carrier-added [18F]fluoride. J. Am. Chem. Soc. 129, 8018−8025.
(23) Salt, P. J. (1972) Inhibition of noradrenaline uptake2 in the
isolated rat heart by steroids, clonidine and methoxylated phenyl-
ethylamines. Eur. J. Pharmacol. 20, 329−340.
(5) Baker, A. J. (2014) Adrenergic signaling in heart failure: a balance
of toxic and protective effects. Pfluegers Arch. 466, 1139−1150.
(6) Gardner, R. T., Ripplinger, C. M., Myles, R. C., and Habecker, B.
A. (2016) Molecular mechanisms of sympathetic remodeling and
arrhythmias. Circ.: Arrhythmia Electrophysiol. 9, e001359.
(7) Vaseghi, M., and Shivkumar, K. (2008) The role of the
autonomic nervous system in sudden cardiac death. Prog. Cardiovasc.
Dis. 50, 404−419.
(8) Zipes, D. P., and Rubart, M. (2006) Neural modulation of cardiac
arrhythmias and sudden cardiac death. Heart Rhythm 3, 108−113.
(9) Henneman, M. M., Bengel, F. M., van der Wall, E. E., Knuuti, J.,
and Bax, J. J. (2008) Cardiac neuronal imaging: application in the
evaluation of cardiac disease. J. Nucl. Cardiol. 15, 442−455.
(10) Wollenweber, T., and Bengel, F. M. (2014) Molecular imaging
to predict ventricular arrhythmia in heart failure. J. Nucl. Cardiol. 21,
1096−1109.
(11) Wieland, D. M., Brown, L. E., Rogers, W. L., Worthington, K.
C., Wu, J.-L., Clinthorne, N. H., Otto, C. A., Swanson, D. P., and
Beierwaltes, W. H. (1981) Myocardial imaging with a radioiodinated
norepinephrine storage analog. J. Nucl. Med. 22, 22−31.
(12) Rosenspire, K. C., Haka, M. S., Van Dort, M. E., Jewett, D. M.,
Gildersleeve, D. L., Schwaiger, M., and Wieland, D. M. (1990)
Synthesis and preliminary evaluation of carbon-11-meta-hydroxyephe-
drine: a false transmitter agent for heart neuronal imaging. J. Nucl.
Med. 31, 1328−1334.
(13) Jacobson, A. F., Senior, R., Cerqueira, M. D., Wong, N. D.,
Thomas, G. S., Lopez, V. A., Agostini, D., Weiland, F., Chandna, H.,
and Narula, J. (2010) Myocardial iodine-123 meta-iodobenzylguani-
dine imaging and cardiac events in heart failure: results of the
prospective ADMIRE-HF (AdreView Myocardial Imaging for Risk
Evaluation in Heart Failure) study. J. Am. Coll. Cardiol. 55, 2212−
2221.
(14) Fallavollita, J. A., Heavey, B. M., Luisi, A. J., Michalek, S. M.,
Baldwa, S., Mashtare, T. L., Hutson, A. D., DeKemp, R. A., Haka, M.
S., Sajjad, M., Cimato, T. R., Curtis, A. B., Cain, M. E., and Canty, J. M.
(2014) Regional myocardial sympathetic denervation predicts the risk
of sudden cardiac arrest in ischemic cardiomyopathy. J. Am. Coll.
Cardiol. 63, 141−149.
(15) Deyell, M. W., Krahn, A. D., and Goldberger, J. J. (2015)
Sudden cardiac death risk stratification. Circ. Res. 116, 1907−1918.
(16) Raffel, D. M., Jung, Y. W., Gildersleeve, D. L., Sherman, P. S.,
Moskwa, J. J., Tluczek, L. J., and Chen, W. (2007) Radiolabeled
phenethylguanidines: novel imaging agents for cardiac sympathetic
neurons and adrenergic tumors. J. Med. Chem. 50, 2078−2088.
(17) Raffel, D. M., Koeppe, R. A., Jung, Y. W., Gu, G., Jang, K. S.,
Sherman, P. S., and Quesada, C. A. (2013) Quantification of cardiac
sympathetic nerve density of N-11C-guanyl-meta-octopamine and
tracer kinetic analysis. J. Nucl. Med. 54, 1645−1652.
(24) Schafer, M. K. H., Weihe, E., and Eiden, L. E. (2013)
̈
Localization and expression of VMAT2 across mammalian species: a
translational guide for its visualization and targeting in health and
disease. Adv. Pharmacol. 68, 319−334.
(25) DeGrado, T. R., Hutchins, G. D., Toorongian, S. A., Wieland, D.
M., and Schwaiger, M. (1993) Myocardial kinetics of carbon-11-meta-
hydroxyephedrine: retention mechanisms and effects of norepinephr-
ine. J. Nucl. Med. 34, 1287−1293.
(26) Raffel, D. M., and Wieland, D. M. (2001) Assessment of cardiac
sympathetic nerve integrity with positron emission tomography. Nucl.
Med. Biol. 28, 541−559.
(27) Raffel, D. M. (2012) Targeting norepinephrine transporters in
cardiac sympathetic nerve terminals. In Targeted Molecular Imaging
(Welch, M. J., and Eckelman, W. C., Eds.), pp 305−320, CRC Press,
Boca Raton, FL.
(28) Patlak, C. S., and Blasberg, R. G. (1985) Graphical evaluation of
blood-to-brain transfer constants from multiple-time uptake data.
Generalizations. J. Cereb. Blood Flow Metab. 5, 584−590.
(29) Costa, E., Kuntzman, R., Gessa, G. L., and Brodie, B. B. (1962)
Structural requirements for bretylium and guanethidine-like activity in
a series of guanidine derivatives. Life Sci. 1, 75−80.
(30) Fielden, R., and Green, A. L. (1965) The effects of some
aralkylguanidines in mice. Br. J. Pharmacol. Chemother. 24, 408−417.
(31) Green, A. L., Fielden, R., Bartlett, D. C., Cozens, M. J., Eden, R.
J., and Hills, D. W. (1967) New norepinephrine-depleting agents. β-
hydroxyphenethylguanidine and related compounds. J. Med. Chem. 10,
1006−1008.
(32) Maxwell, R. A., and Wastila, W. B. (1977) Adrenergic Neuron
Blocking Drugs. In Antihypertensive Agents. Handbook of Pharmacology
XXXIX (Gross, F., Ed.), pp 161−211, Springer-Verlag, New York.
(33) Raffel, D. M. (2015) Preclinical evaluations of cardiac
sympathetic innervation radiotracers. In Autonomic Innervation of the
Heart: Role of Molecular Imaging (Slart, R. H. J. A., Tio, R. A., Elsinga,
P. H., and Schwaiger, M., Eds.), pp 201−234, Springer, Berlin.
(34) Graefe, K.-H., and Bonisch, H. (1988) The transport of amines
̈
across axonal membranes of noradrenergic and dopaminergic
neurones. In Catecholamines I, Handbook of Experimental Pharmacology
(Trendelenburg, U., and Werner, N., Eds.), pp 192−245, Springer-
Verlag, Berlin.
(35) Garg, P. K., Garg, S., and Zalutsky, M. R. (1994) Synthesis and
preliminary evaluation of para- and meta-[18F]fluorobenzylguanidine.
Nucl. Med. Biol. 21, 97−103.
(36) Zhang, H., Huang, R., Pillarsetty, N., Thorek, D. L. J.,
Vaidyanathan, G., Serganova, I., Blasberg, R. G., and Lewis, J. S. (2014)
Synthesis and evaluation of 18F-labeled benzylguanidine analogs for
targeting the human norepinephrine transporter. Eur. J. Nucl. Med.
Mol. Imaging 41, 322−332.
(37) Berry, C. R., Garg, P. K., Zalutsky, M. R., Coleman, R. E., and
DeGrado, T. R. (1996) Uptake and retention kinetics of para-fluorine-
18-fluorobenzylguanidine in isolated rat heart. J. Nucl. Med. 37, 2011−
2016.
(18) Jang, K. S., Jung, Y. W., Gu, G., Koeppe, R. A., Sherman, P. S.,
Quesada, C. A., and Raffel, D. M. (2013) 4-[18F]fluoro-m-
hydroxyphenethylguanidine: a radiopharmaceutical for quantifying
regional cardiac sympathetic nerve density with positron emission
tomography. J. Med. Chem. 56, 7312−7323.
(19) Jang, K. S., Jung, Y. W., Song, H. C., and Raffel, D. M. (2015)
Strategies for radiolabeling of 4-[18F]fluoro-m-hydroxyphenethylguan-
(38) Berry, C. R., Garg, P. K., DeGrado, T. R., Hellyer, P., Weber, W.,
Garg, S., Hansen, B., Zalutsky, M. R., and Coleman, R. E. (1996) Para-
L
DOI: 10.1021/acschemneuro.7b00051
ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

ACS Chemical Neuroscience
Research Article
[18F]fluorobenzylguanidine kinetics in a canine coronary artery
occlusion model. J. Nucl. Cardiol. 3, 119−129.
(39) Jang, K. S., Jung, Y. W., Sherman, P. S., Quesada, C. A., Gu, G.,
and Raffel, D. M. (2013) Synthesis and bioevaluation of [18F]4-fluoro-
m-hydroxyphenethylguanidine ([18F]4F-MHPG): a novel radiotracer
for quantitative PET studies of cardiac sympathetic innervation. Bioorg.
Med. Chem. Lett. 23, 1612−1616.
(40) Taegtmeyer, H., Hems, R., and Krebs, H. A. (1980) Utilization
of energy providing substrates in the isolated working rat heart.
Biochem. J. 186, 701−711.
̀
(41) Raffel, D., Loc’h, C., Mardon, K., Maziere, B., and Syrota, A.
(1998) Kinetics of the norepinephrine analog [Br-76]-meta-
bromobenzylguanidine in isolated working rat heart. Nucl. Med. Biol.
25, 1−16.
(42) Qi, J., and Leahy, R. M. (2000) Resolution and noise properties
of MAP reconstruction for fully 3D-PET. IEEE Trans. Med. Imaging
19, 493−506.
(43) Narimatsu, S., Kobayashi, N., Asaoka, K., Masubuchi, Y., Horie,
T., Hosokawa, M., Ishikawa, T., Ohmori, S., Kitada, M., Miyano, J.,
Kataoka, H., and Yamamoto, S. (2001) High-performance liquid
chromatographic analysis of the sulfation of 4-hydroxypropranolol
enantiomers by monkey liver cytosol. Chirality 13, 140−147.
(44) National Research Council (1985) Guide for the Care and Use of
Laboratory Animals, U.S. Department of Health and Human Services,
National Institutes of Health, Bethesda, MD.
M
DOI: 10.1021/acschemneuro.7b00051
ACS Chem. Neurosci. XXXX, XXX, XXX−XXX