ACS Medicinal Chemistry Letters
Letter
labeled analogue of norepinephrine developed by Wieland and
co-workers at our institution,11 is the best known example and
has been used as a radiotracer for imaging and treatment of
neuroendocrine tumors as well as noninvasive imaging of
cardiac sympathetic nerves. The latter application stems from
the fact that, in heart disease, cardiac autonomic dysfunction
contributes to morbidity and mortality due to disease-induced
alterations in the nervous control of the heart.12 Such
alterations are the result of 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 regional degeneration of postganglionic para-
sympathetic and/or sympathetic nerve fibers in the heart.
Benzylguanidines have proven to be very useful for scinti-
graphic imaging studies of cardiac sympathetic innervation, but
their uptake rates into cardiac sympathetic neurons are too
rapid to allow for robust and reliable compartmental modeling
of their kinetics. This, in combination with limitations of
scintigraphy, has created much interest in developing
guanidine analogues labeled with carbon-11 for PET imaging.
Thus, [11C]phenethylguanidine as well as analogues such as N-
[11C]guanyl-(−)-m-octopamine ([11C]GMO),13,14 ([11C]-m-
hydroxyphenethylguanidine (MHPG), and ([11C]-p-fluoro-m-
hydroxyphenethylguanidine (4F-MHPG) (Figure 1), have
been evaluated as radiotracers with improved kinetics for
quantifying cardiac nerve density with PET.13 Building on
these early radiotracers, after bioevaluation of 12 phenenthyl-
guanidines, Raffel and co-workers developed meta- and para-
substituted structural isomers of [18F]fluoro-hydroxyphene-
thylguanidine ([18F]4F-MHPG and [18F]3F-PHPG) for
imaging cardiac sympathetic denervation (Figure 1).15 First-
in-human studies provided robust regional metrics of cardiac
sympathetic nerve density.16
In an effort to investigate further tuning of the myocardial
kinetics of guanidine PET radiotracers to improve quantitative
analysis and imaging of neuroendocrine tumors, we were
interested in synthesizing hydroxyphenoxyethylguanidine
analogues ([11C]-3-fluoro-4-hydroxyphenoxyethylguanidine
([1 1C]3F-PHPOG, [11C]3i), [11C]-1-(3-fluoro-5-
hydroxyphenethyl)guanidine ([11C]3c), and [11C]-1-(3,4-
dihydroxyphenethyl)guanidine ([11C]3d)). Since its initial
discovery, guanidine has been found in a variety of natural
products,17,18 and many guanidine synthesis methods have
been investigated. However, while there has been extensive
research devoted to studying guanidino compounds and their
syntheses,17,18 methods for labeling guanidines with carbon-11
remain scarce. Almost 30 years ago, Westerberg and
compound, meta-fluorobenzylamine (1a) (Scheme 1 and
Table 1). A GE TracerLab FXM automated synthesis module
Scheme 1. Initial [11C]Cyanation of 1a
was used with simple modifications (see the Supporting
Information). To accomplish the transformation, [11C]hydro-
gen cyanide ([11C]HCN) was produced from [11C]CO2, and
then converted to [11C]BrCN using pyridinium perbromide
and Sb metal, per previous reports.5 The tube containing
pyridinium perbromide and plug of Sb metal was connected
inline between the process panel (where [11C]HCN is
generated) and the FXM synthesis module. The flow of
[11C]BrCN was then directly sparged into a solution
containing amine precursor 1a in the reactor of the synthesis
module. Initial generation of cyanamide intermediate [11C]2a
proceeded in 42% RCY (based on HPLC analysis of the crude
1). Encouraged by this initial result, we proceeded to
investigate the subsequent generation of guanidine product
[11C]3a (Table 1). However, initial efforts to treat cyanamide
intermediate [11C]2a with ammonium chloride and generate
guanidine product [11C]3a resulted in low yield (3%) (Table
1, entry 1). We hypothesized that the second step was slow
and inefficient, and that increased temperature, pressure and/
or reaction time could improve radiochemical yield. The
automated module allows increasing the pressure of the
reaction chamber by over pressurizing with nitrogen gas.
Overpressurizing for 15 s was insufficient to notably improve
yield of [11C]3a from [11C]2a (Table 1, entry 2). Switching to
a saturated ammonium chloride solution and further increasing
the time to over pressurize the rector resulted in an improved
radiochemical yield of 36−49%, n = 2 (Table 1, entry 3).
Encouraged by these results, we next automated the entire
synthesis, including purification of [11C]3a by semipreparative
HPLC. This resulted in a total synthesis time of 46 min and a
noncorrected activity yield (AY) of 31% (Table 1, entry 4). In
a previously reported carbon-11 guanylation synthesis by
Raffel,13 NH4Br was used to form the guanidine instead of
NH4Cl because of the higher solubility of NH4Br in NH4OH.
However, using our automated method, the use of NH4Cl
(Table 1, entry 4) provided higher yields of [11C]3a than
NH4Br (Table 1, entry 5). While developing the semi-
preparative HPLC method we noted that adjusting pH with
acetic acid (Table 1, entry 5) compared to phosphoric acid
(Table 1, entry 4) shortened the purification process (and
therefore total synthesis time) without impairing separation
which is advantageous when working with short-lived carbon-
11. Lastly, during the optimization process, we observed that
yields of guanidine [11C]3 improved when freshly prepared
NH4Cl solutions were used.
19
Långstrom synthesized [ C]MIBG, 1,3-di(2-tolyl)-[11C]-
guanidine,20 and [11C]GG16721 using [11C]cyanogen bromide
([11C]BrCN).22 Building on this literature precedent, Raffel
recently synthesized a variety of [11C]phenethylguanidines
using similar approaches.13 However, the syntheses of all these
radiolabeled guanidines were performed manually using
homemade equipment that is no longer particularly amenable
to routine clinical production demands in cGMP-compliant
PET radiochemistry laboratories. To address this gap in
radiochemistry, herein we report a new automated method for
radiolabeling guanidines with carbon-11 and use it to prepare a
series of [11C]guanidines. Proof-of-concept is demonstrated
through preclinical cardiac PET in rabbits with [11C]3F-
PHPOG
11
̈
Our investigation began with optimization of the reaction
conditions for the carbon-11 guanylation of a model
We next applied the fully automated and optimized carbon-
11 guanylation conditions to a series of primary amines (Figure
B
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX