New chemical and radiochemical routes to [18F]Rho6G-DEG-F, a delocalized lipophilic cation for myocardial perfusion imaging with PET

Article abstract of DOI:10.1039/c7md00326a

New chemical and radiochemical syntheses are described for the preparation of [¹⁸F]Rho6G-DEG-F, an ¹⁸F-labeled analogue of the fluorescent dye rhodamine 6G, which has shown promise as myocardidal perfusion imaging agent. Tosylated precursors of [¹⁸F]Rho6G-DEG-F amenable to ¹⁸F-labeling were obtained either through a two-step synthesis from rhodamine 6G lactone (33% yield), or in one step from rhodamine 575 (64% yield), then purified by preparative C₁₈ chromatography. Manual synthesis of [¹⁸F]Rho6G-DEG-F was achieved in a single radiochemical step from either the tosylate salt or the tosylate/formate double salt in DMSO under standard nucleophillic aliphatic ¹⁸F-fluorination conditions (K[¹⁸F]F/K₂CO₃/Kryptofix 2.2.2.). Incorporation of the [¹⁸F]F^- was found to be satisfactory (≥34% by TLC), despite the protic character of the precursor molecules. [¹⁸F]Rho6G-DEG-F was manually synthesized in final decay-corrected radiochemical yields of 11-26% (tosylate salt) and 9-21% (tosylate/formate double salt). The protocol was transferred to an automated synthesis unit, where the product was obtained in 3-9% radiochemical yield (n = 3) decay corrected to start-of-synthesis, >99% radiochemical purity, and a molar activity of 122-267 GBq μmol^-1 (3.3-7.2 Ci μmol^-1).

Full text of DOI:10.1039/c7md00326a

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RESEARCH ARTICLE
New chemical and radiochemical routes to
[18F]Rho6G-DEG-F, a delocalized lipophilic cation
for myocardial perfusion imaging with PET
Cite this: DOI: 10.1039/c7md00326a
ab
ab
c
J. A. H. Inkster,
S. Dubey,
†
S. Zhang, †‡ab V. Akurathi,
§
A. Belanger,
c
bc
ab
*
S. T. Treves
and A. B. Packard
New chemical and radiochemical syntheses are described for the preparation of [18F]Rho6G-DEG-F, an
18F-labeled analogue of the fluorescent dye rhodamine 6G, which has shown promise as myocardidal per-
fusion imaging agent. Tosylated precursors of [18F]Rho6G-DEG-F amenable to 18F-labeling were obtained
either through a two-step synthesis from rhodamine 6G lactone (33% yield), or in one step from rhodamine
575 (64% yield), then purified by preparative C18 chromatography. Manual synthesis of [18F]Rho6G-DEG-F
was achieved in a single radiochemical step from either the tosylate salt or the tosylate/formate double salt
in DMSO under standard nucleophillic aliphatic 18F-fluorination conditions (Kij18F]F/K2CO3/Kryptofix 2.2.2.).
Incorporation of the [18F]F− was found to be satisfactory (≥34% by TLC), despite the protic character of the
precursor molecules. [18F]Rho6G-DEG-F was manually synthesized in final decay-corrected radiochemical
yields of 11–26% (tosylate salt) and 9–21% (tosylate/formate double salt). The protocol was transferred to an
automated synthesis unit, where the product was obtained in 3–9% radiochemical yield (n = 3) decay
corrected to start-of-synthesis, >99% radiochemical purity, and a molar activity of 122–267 GBq μmol−1
(3.3–7.2 Ci μmol−1).
Received 27th June 2017,
Accepted 24th August 2017
DOI: 10.1039/c7md00326a
rsc.li/medchemcomm
of the attractive nuclear properties of this radionuclide [i.e.,
Introduction
high positron yield (97%), low positron energy (β+
= 250
avg
Myocardial perfusion imaging (MPI) is an important compo-
nent in the diagnosis and risk-stratification of coronary artery
disease. Owing to its cost-effectiveness, readily available radio-
pharmaceuticals, and clinical utility, single-photon emission
computed tomography (SPECT) MPI using 99mTc radiophar-
maceuticals (e.g., 99mTc-sestamibi, 99mTc-tetrofosmin) remains
a popular imaging technique.1 However, there is currently
considerable interest in the development of positron emission
tomography (PET) radiopharmaceuticals for MPI because of
the inherent advantages of PET in terms of spatial resolution,
mitigation of attenuation artifacts, and the ability to accu-
rately measure myocardial perfusion.2 In this context, an 18F-
labeled MPI radiopharmaceutical is highly desirable because
keV), and intermediate half-life (t1/2 = 109.8 min)] relative to
other PET radionuclides used for MPI. For example, 82Rb (t1/2
= 1.3 min) is available from the 82Sr/82Rb decay pathway, but
the generator is very expensive. Thus, high patient throughput
is required to make this system cost-effective. [13N]NH3 is an
effective MPI radiopharmaceutical, but the short half-life of
13N (t1/2 = 9.97 min) limits it use to facilities with an on-site
cyclotron. To date, the most promising 18F-labeled MPI candi-
date is [18F]flurpiridaz, a pyridaben derivative that shows su-
perior extraction fraction relative to all other MPI agents save
[18O]H2O and is currently in a phase 3 clinical trial.3–5 Other
18F-labeled pyridaben derivatives are also being investigated
for MPI,6,7 as are a number of [18F]fluoroalkyl8–12 and
[18F]fluoroaryl13 triphenylphosphonium salts.
Rhodamines are delocalized lipophilic cations (DLCs) that
accumulate in mitochondria in proportion to mitochondrial
membrane potential14 and thus have been utilized extensively
as potentiometric dyes15 for the ex vivo study of
cardiomyocytes.16 With regard to 18F-labeled rhodamines, we
first synthesized rhodamine B derivatives bearing 18F-alkyl17
and 18F-polyoxyethylene18,19 esters and evaluated them for
chemical and pharmacological parameters important to PET-
MPI. Subsequently, AlJammaz et al. synthesized and
a Boston Children's Hospital, Division of Nuclear Medicine and Molecular
Imaging, Boston, MA, 02115, USA. E-mail: alan.packard@childrens.harvard.edu;
Tel: +(617) 355 7539
b Harvard Medical School, Boston, MA 02115, USA
c Brigham & Women's Hospital, Boston, MA 02115, USA
† These authors contributed equally to this work.
‡ Current address: Nemours/Alfred I. DuPont Hospital for Children, 1600
Rockland Road, Wilmington, DE 19803.
§ Current address: University of Iowa Hospitals and Clinics, 200 Hawkins Drive,
Iowa City, IA 52242.
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evaluated an [18F]FDG-modified amide analogue of rho- Results and discussion
damine
12320
and
Trencsényi
et
al.
prepared
2-[18F]fluoroethylrhodamine B for the in vitro measurement
P-glycoprotein function.21 Building on our earlier work with
rhodamine B, we later compared the pharmacological proper-
ties of several 2-(2-[18F]fluoroethoxy)ethyl ester derivatives
bearing different rhodamine cores.22 These 18F-labeled rho-
damine esters were prepared by condensing a rhodamine in
the lactone form with the requisite 18F/tosyl-bearing bifunc-
tional prosthetic molecule. An example synthesis is shown in
Scheme 1. On the basis of these experiments, rhodamine 6G
2-(2-[18F]fluoroethoxy)ethyl ester, chloride salt ([18F]Rho6G-
DEG-F, 18F-[1]ijCl]; Scheme 1) was identified as the most
promising MPI candidate, exhibiting effective localization in
isolated rat cardiomyocytes (4.6% 0.7% ID g−1 at 1 min),
rapid and sustained uptake into rat hearts (1.3% 0.3% ID
g−1 at 60 min), and excellent contrast between the heart and
nearby liver tissues.22 This two-step method for the prepara-
tion of 18F-[1]ijCl] is not, however, optimal for routine produc-
tion using automated synthesis modules primarily because
the lactone precursor is sparingly soluble in many organic
solvents, including MeCN. An additional limitation is that it
requires two radiochemical steps. Thus clinical studies of
[18F]Rho6G-DEG-F are predicated on the development of an
improved synthesis for this potential PET-MPI radiopharma-
ceutical, one that is amenable to use in automated synthesis
systems. To this end, we now report the one-step synthesis of
18F-[1]ijCl] by radiofluorination of the tosylate salt [2]ijOTs]
and the double salt, [2]ijHCOO]ijOTs], both of which can be
Two routes were developed for the synthesis of lipophilic cat-
ion [2]. The first was a two-step method, starting from the
chloride salt of rhodamine 6G ([3]ijCl]; Scheme 2). In this syn-
thesis,
a
nucleophilic carboxylate anion is generated
in situ by way of base-mediated hydrolysis of the ester,
and the resultant zwitterion is converted to non-fluorescent
lactone 4. Formation of neutral 4 eliminates the chloride an-
ion from the final product, thus removing it from potential
competition with [18F]F− during subsequent radio-
fluorination reactions. In the second synthetic step, 4 was
suspended in acetonitrile and heated in the presence of N,N-
diisopropylethylamine (DIPEA) and diethylene glycol bisIJp-
toluenesulfonate) (5). The solvent and nitrogen base are grad-
ually distilled off over time. The reaction presumably pro-
ceeds by way of zwitterion formation (6), followed by nucleo-
philic attack on 5 by the carboxylate anion. This general
approach was first used for the preparation of modified rho-
damine esters bearing carboxylic acid and benzyl chloride
functionalities for conjugation to peptides,23 and later for the
synthesis of 19F reference material 19F-[1]ijCl].22 Cation [2] was
purified on a glass column packed with C18 sorbent (50 : 50
MeCN : H2O with 0.5% acetic acid), followed by solid-phase
extraction (SPE) from the liquid chromatography (LC) eluent.
The final product was determined to be the singly charged
tosylate salt [2]ijOTs] (Scheme 2). The overall chemical yield of
[2]ijOTs] using this two-step approach was 33%.
In the second route to precursor cation [2], commercially
available inner salt rhodamine 575 (6) was esterified directly
(Scheme 3). The product was purified on a preparative col-
umn of C18 sorbent using 45 : 55 MeCN : H2O containing
0.1% formic acid as the mobile phase. In this method, how-
ever, the product salt was not extracted from the acidic LC el-
uent prior to concentration, which resulted in the formation
of doubly charged [2]ijHCOO]ijOTs] as suggested by 1H NMR
and elemental analysis. Conventional understanding of
prepared in
a single step from commercially available
starting materials.
K2CO3/Kryptofix 2.2.2. (K2.2.2.)
18F-fluorination chemistry sug-
gests that this precursor may be inferior to [2]ijOTs] owing
to the presence of an additional acidic proton that could
interfere with fluoride nucleophilicity through the generation
of H[18F]F.24 However, [2]ijHCOO]ijOTs] proved a serviceable
precursor for standard nucleophilic aliphatic 18F-fluorina-
tions (vide infra). The isolated chemical yield of
[2]ijHCOO]ijOTs] was 64%, approximately double the yield of
[2] that was obtained using the two-step approach.
The utility of [2]ijOTs] and [2]ijHCOO]ijOTs] for the manual
radiosynthesis of [18F]Rho6G-DEG-F was verified using stan-
dard nucleophilic aliphatic 18F-fluorination methods
(Scheme 4). When the precursor salt was heated in anhy-
drous DMSO in the presence of the dried Kij18F]F/K2.2.2. com-
plex and K2CO3, incorporation of [18F]F− was found to be 34–
47% (n = 3) and 34–69% (n = 7) by TLC for [2]ijOTs] and
[2]ijHCOO]ijOTs], respectively. These incorporation yields are
somewhat surprising given the deleterious effect of Brønsted-
Lowry acidic functionalities observed on many nucleophilic
Scheme 1 Two-step radiosynthesis of [18F]Rho6G-DEG-F.
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Scheme 2 Two-step synthesis of precursor [2]ijOTs].
18F-fluorination reactions. After reverse-phase semi-prepara-
tive HPLC, the product was isolated from the HPLC solvent
by solid-phase extraction using a tC18 cartridge, concen-
trated to dryness, and formulated in 10% EtOH in isotonic
saline.¶
MBq) of [18F]fluoride ion obtained from transfer line flushes
after clinical production of other radiotracers. The fastest
manual synthesis time was 97 min from SOS.
Finally, [18F]Rho6G-DEG-F was prepared in an automated
synthesis module (GE Tracerlab FXFN) using conditions simi-
lar to those employed for the manual syntheses. As the yield
and molar activity obtained using the two precursors were
similar, [2]ijOTs] was employed as precursor for the auto-
mated synthesis. Decay-corrected radiochemical yields of 3, 7
and 9% (6 3%) were achieved. The relatively low yields of
18F-[1]ijCl] during automated syntheses are in part attributable
to the inefficient extraction of [18F]F− from Sep-Pak® Light
QMA anion exchange cartridges (130 mg) using an elution
matrix containing 0.9 mg mL−1 K2CO3 as the anion exchange
reagent. Under these elution conditions, the non-decay-
corrected (NDC) elution efficiency is only 50–65%.
Manual radiochemical yields of 18F-[1]ijCl] from start-of-
synthesis (SOS) were 11–26% (n = 3) decay-corrected (DC) and
9–21% (n = 6) DC for [2]ijOTs] and [2]ijHCOO]ijOTs] respec-
tively. Molar activities were calculated based on a calibration
curve prepared from an 19F standard22 ([2]ijOTs] = 3–9 GBq
μmol−1; [2]ijHCOO]ijOTs] = 7–35 GBq μmol−1). These molar ac-
tivity values are similar to those typically observed for manual
18F syntheses carried out using small quantities (370–740
Scheme 3 One-step synthesis of precursor [2]ijHCOO]ijOTs].
¶ After preparative HPLC purification the radiotracer is assumed to be present
as a TFA salt ([1]ijTFA]), which converts to the chloride salt ([1]ijCl]) upon formu-
lation in 10% EtOH in isotonic saline.
Scheme 4 One-step radiosynthesis of [18F]Rho6G-DEG-F.
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Furthermore, we observed that that the lipophilic 18F-[1]ijCl]
product was partially retained by the PTFE tubing and the
sterile filter used in the automated synthesis system. Despite
these inefficiencies, the use of a remote synthesis apparatus
allowed for the current “good manufacturing practices”
(cGMP) production of 41, 56 and 81 GBq of [18F]Rho6G-DEG-
F and molar activities of 122, 180 and 267 (190 73) GBq
μmol−1, respectively. The fastest automated synthesis time
was 83 min from SOS.
were obtained from Cambridge Isotope Laboratories (Ando-
ver, MA, USA). Chemical shifts (δ) are reported in ppm rela-
tive to the hydrogenated residue of the deuterated solvents.
Elemental microanalysis (EA) was carried out by Atlantic
Microlab, Inc. (Norcross, GA, USA).
HPLC systems described below, including data acquisition
modules, are Shimadzu Prominence. The gamma detectors
were optimized for 511 keV photons.
HPLC A. For manual preparations. Pump: LC-20AT. UV/vis
detector: SPD-20A. Radiation detection: Carroll & Ramsey
Model 105S.
HPLC B. Analytical. Pump: LC-20 AD. Diode array detector
(DAD): SPD-M20A. Radiation detection: Harshaw NaI(Tl) de-
tector with Canberra NIM electronics. For low resolution liq-
uid chromatography-mass spectroscopy, an Advion expres-
sionS CMS apparatus was added in-line.
Conclusions
Two synthetic routes were developed to the tosylated precur-
sor of [18F]Rho6G-DEG-F (18F-[1]ijCl]),
a
promising
mitochondria-targeting 18F tracer for PET-MPI. Despite being
delocalized lipophilic salts, [2]ijOTs] and [2]ijHCOO]ijOTs] can
be labeled with 18F using conventional nucleophilic aliphatic
18F-fluorination chemistry in a single radiochemical step.
This facilitated the easy translation of this protocol to an au-
tomated synthesis system that will be used for the cGMP pro-
duction of this radiopharmaceutical in upcoming human tri-
als. Starting from precursor [2]ijOTs], 18F-[1]ijCl] was prepared
in an automated synthesis unit in 6 3% decay-corrected ra-
diochemical yield from start-of-synthesis, high radiochemical
HPLC C. For automated preparations. Pump: Sykam
S1122. UV/vis detector: WellChrom Filter-Photometer K-2001.
Radiation detection: GE FXFN internal detector.
HPLC D. Analytical. Pump: LC-20AB. UV/Vis detector: SPD-
20A Radiation Detection: Carroll & Ramsey Model 105S.
Program A. Column: EMD Millipore Purosphere® RP-18
endcapped, 5 μm, 4 mm × 125 mm. Solvent system: gradient
elution, 10% MeCN in H2O containing 0.1% formic acid for 2
min, raised to 90% MeCN in H2O containing 0.1% formic
acid over 13 min, hold for 5 min, flow rate = 1 mL min−1. De-
tector: 190–800 nm (DAD).
purity (>99%), and high molar activity (190
μmol−1).
73 GBq
Experimental
General information
Program B. Column: EMD Millipore Purosphere® RP-18
endcapped, 5 μm, 4 mm × 125 mm. Solvent system: gradient
elution, 10% MeCN in H2O containing 0.1% trifluoroacetic
acid (TFA) for 2 min, raised to 90% MeCN in H2O containing
0.1% TFA over 13 min, hold for 5 min, flow rate = 1 mL
min−1. Detector: 190–800 nm (DAD).
Program C. Column: ES Industries Chromegabond WR
C18, 5 μm, 120 Å, 9.6 mm × 250 mm. Solvent system: iso-
cratic elution, 50 : 50 MeCN : H2O containing 0.1% TFA, flow
rate = 3 mL min−1. Detector: 254 nm.
Program D. Column: EMD Millipore Purosphere® RP-18
endcapped, 5 μm, 4 mm × 125 mm. Solvent system: isocratic
elution, 50 : 50 MeCN : H2O containing 0.1% TFA, flow rate =
1 mL min−1. Detector: 190–800 nm (DAD).
Program E. Column: Phenomenex Luna C18, 10 μm, 100
Å, 10 mm × 250 mm. Solvent system: isocratic elution, 45:55
MeCN: citrate buffer (0.1 M, pH = 2.7), flow rate = 5 mL
min−1. Detector: 254 nm.
Chemicals and media. Unless otherwise noted, reagents
and solvents were purchased from Acros Organics or Alfa Aesar
and were used without further purification. Rhodamine 575 (6;
laser grade) was obtained from Organica (Bitterfeld-Wolfen,
Germany). Diethylene glycol bisIJp-toluenesulfonate) (5) was
purchased from TCI America (Portland, OR, USA). Silica gel
(40–63 μm) for flash chromatography was obtained from
Silicycle, Inc. (Quebec City, Canada). LiChroprep® RP-18 sor-
bent (40–63 μm) was obtained from Merck KGaA. Quaternary
methyl ammonium (QMA) anion-exchange cartridges (carbon-
ate form) for manual [18F]F− trapping and release were
obtained from MedChem Imaging (Boston, USA). Sep-Pak®
Light QMA cartridges (carbonate form) for automated synthesis
were obtained from ABX (Radeberg, Germany). tC18 Light and
C18 Plus Sep-Pak® solid phase extraction cartridges were
obtained from Waters (Milford, MA, USA). Thin-layer chroma-
tography (TLC) was performed on aluminum oxide coated poly-
ethylene terephthalate sheets from Fluka (Ronkonkoma, NY,
USA). Oxygen-18-enriched water (98% isotopic enrichment) was
purchased from Rotem Industries Ltd. (Mishor Yamin, Israel).
Program F. Column: Phenomenex Luna C18, 5 μm, 100 Å,
4.6 mm × 250 mm. Solvent system: isocratic elution, 50 : 50
MeCN : H2O containing 0.1% TFA, flow rate = 1 mL min−1.
Detector: 254 nm.
Equipment
Non-radioactive chemistry
A Fujifilm BAS-5000 Phosphor Imager with Multi Gauge v3.0
software was used to assay TLC plates.
Synthesis of rhodamine 6G lactone (4). Rhodamine 6G
([3]ijCl]; 4.4 g, 9.2 mmol) was dissolved in MeCN (30 mL). To
this solution 6 M NaOH (30 mL) was added with stirring, and
the reaction mixture was refluxed overnight. After cooling to
1H and 13C NMR spectra were recorded with a Varian 400-
MR Spectrometer with a 400 MHz magnet. NMR solvents
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room temperature, the reaction mixture was extracted with 3 ×
50 mL chloroform. The organic layers were combined and
dried over anhydrous sodium sulfate. After filtration, the sol-
vent was evaporated in vacuo. The crude product was further
purified on a flash column of silica gel. The column was eluted
first with 3% triethylamine (TEA) in 6 : 4 CH2Cl2 : hexanes,
followed by a step-wise decrease in hexanes content until all
visible impurities were eluted. The product was then eluted
from the column with 8 : 2 CH2Cl2 : EtOH containing 3% TEA.
The fractions containing the product were pooled and concen-
trated in vacuo, with the addition of 3 × 10 mL of EtOH added
to facilitate the removal of TEA. After drying under high vac-
uum, 2.5 g (66%) of 4 was obtained as a pink powder. The
identity and purity of lactone 4 was verified by 1H NMR23 and
LC-MS (HPLC B, Program A, tR = 10.3 min, m/z 415 [M+H]+).
Synthesis of rhodamine precursor [2]ijOTs]. To a round-
bottom flask containing rhodamine 6G lactone (4) (1.66 g,
4.0 mmol) in MeCN (30 mL), diethylene glycol bisIJp-
toluenesulfonate) (5; 3.32 g, 8.0 mmol) and DIPEA (3 mL, 18
mmol) were added. The flask was capped with a rubber stop-
per punctured with a vent needle and the reaction mixture
was heated to 90 °C overnight with stirring, during which
time the solvent partially evaporated. The next morning, the
reaction was heated to 130 °C and held there until all
remaining solvent was removed. The flask was then cooled to
room temperature, and the crude product (a purple oil) was
ditional MeCN (20 mL) and centrifuged for 10 min (13 000
rpm). The solvent was decanted off and concentrated in vacuo
to form a viscous maroon oil which crystallized upon stand-
ing. The product was purified by preparative C18 chromatog-
raphy. Elution of the column with 45 : 55 MeCN : H2O
containing 0.1% formic acid followed by concentration of the
collected fractions at reduced pressure yielded tosylate/for-
mate double salt [2]ijHCOO]ijOTs] (619 mg, 64%) as a glassy
red solid. 1H NMR (400 MHz, CD2Cl2): δ 1.37 (t, J = 7.2 Hz,
6H), 2.15 (s, 6H), 2.32 (s, 3H), 2.41 (s, 3H), 3.31–3.37 (m, 2H),
3.43–3.54 (m, 6H), 3.98–4.05 (m, 4H), 6.49 (br s, 2H), 6.71 (s,
2H), 6.82 (d, J = 0.9 Hz, 2H), 7.13 (d, J = 7.9 Hz, 2H), 7.29–
7.37 (m, 3H), 7.67–7.75 (m, 3H), 7.75–7.85 (m, 2H), 8.25 (s,
1H), 8.34 (dd, J = 1.4, 7.8 Hz, 1H). 13C NMR (100 MHz,
CD2Cl2): δ 14.22, 17.85, 17.86, 21.55, 21.57, 21.94, 21.96,
39.29, 64.81, 69.00, 69.28, 69.74, 94.42, 94.44, 114.37, 126.17,
126.45, 128.34, 129.06, 129.41, 129.42, 130.42, 130.48, 130.71,
130.77, 132.06, 133.37, 133.52, 134.52, 139.95, 139.97, 145.84,
156.61, 157.88, 158.48, 162.85, 165.47. LC-MS (HPLC B, Program
A, tR = 12.0 min, m/z 657 [M]+). Anal. calcd. for C45H50N2O12S2:
C, 61.77; H, 5.76; N, 3.20%. Found: C, 61.96; H, 5.94; N, 3.40%.
Radiochemistry
Preparation of [18F]fluoride ion. No-carrier-added [18F]fluo-
ride ion was produced by proton bombardment of 3.5 mL of
[18O]water [18OIJp,n)18F reaction] on the GE 16.5 MeV PETtrace
cyclotron at Brigham and Woman's Hospital BICOR facility.
An estimated 96 GBq activity was produced at EOB for auto-
mated syntheses. For manual experiments, 740–1850 MBq of
[18F]fluoride ion from an earlier production run was used.
purified on
a glass column packed with C18 sorbent
(LiChroprep® RP-18). The column was eluted by gravity with
50 : 50 MeCN : H2O containing 0.5% acetic acid as the mobile
phase. The collected fractions were pooled and diluted with
water (200 mL), then a fraction of this solution (∼1/3) was
passed through two solid-phase extraction cartridges placed
in-line [Sep-Pak® C18 Plus, activated previously with EtOH (5
mL) and water (20 mL)]. This step was repeated twice more
with the remaining fractions. The product was eluted from
the three sets of cartridges with EtOH (5 mL each) and con-
centrated under reduced pressure to afford [2]ijOTs] as a pur-
Example radiosyntheses of [18F]Rho6G-DEG-F (18F-[1]ijCl])
Manual radiosynthesis. [18F]Fluoride ion was extracted
from [18O]H2O via immobilization on a QMA anion-exchange
cartridge (carbonate form; 10–12 mg; MedChem Imaging),
which was previously activated with water (1 mL). The activity
was eluted from the sorbent into a glass conical vial (2 mL)
with a solution of K2CO3 (0.5 mg) in water (0.2 mL), followed
by K2.2.2. (2.5 mg) in dry MeCN (0.8 mL). The extraction effi-
ciency of this step was 90% NDC. Solvent was removed by
azeotropic distillation at 110 °C under an argon stream. An
additional portion of dry MeCN (1 mL) was added, and a sec-
ond evaporation step was carried out at 110 °C. In the same
fashion a third evaporation step was carried out. The evapo-
ration steps took 15 minutes total. The reaction vessel was
cooled by partially submerging it in tap water, then the pre-
cursor compound [2]ijHCOO]ijOTs] (0.5 mg) in anhydrous
DMSO (0.5 mL) was added. The vial was sealed and heated to
110 °C for 15 min. After cooling, a portion was removed for
radio-TLC (neutral alumina, 10% MeOH in CH2Cl2) and ana-
lytical HPLC (HPLC B, Program B, tR = 12.6 min), then the
bulk mixture was quenched with an aqueous solution of
0.1% TFA (500 μL). After semi-preparative HPLC purification
(HPLC A, Program C, tR = 22.5 min), the collected product¶
was diluted with water (50 mL) and trapped on a Sep-Pak®
1
ple solid (1.65 g, 50%). H NMR (400 MHz, CDCl3): δ 1.32 (t, J
= 8.0, 6H), 2.19 (s, 6H), 2.31 (s, 3H), 2.41 (s, 3H), 3.32–3.50
(m, 8H), 4.05 (m, 4H), 6.50 (s, 2H), 6.68 (s, 2H), 7.12 (d, J =
8.0, 2H), 7.27–7.40 (m, 5H), 7.73–7.82 (m, 6H), 8.35 (dd, J =
1.2, 7.8, 1H). 13C NMR (100 MHz, CDCl3): δ 13.65, 17.95,
21.27, 21.62, 38.47, 64.20, 68.43, 68.73, 68.99, 93.48, 113.18,
126.05, 126.22, 127.84, 128.23, 128.40, 129.75, 129.90, 131.60,
132.73, 133.01, 134.22, 138.86, 143.91, 145.04, 156.03, 156.27,
156.94, 157.69, 164.98. LC-MS (HPLC B, Program A, tR = 12.0
min, m/z 657 [M]+. Anal. calcd. for C44H48N2O10S2: C, 63.75;
H, 5.84; N, 3.38%. Found: C, 63.95; H, 5.83; N, 3.51%.
Synthesis of rhodamine precursor [2]ijHCOO]ijOTs]. Into a
round-bottomed flask containing diethylene glycol bisIJp-
toluenesulfonate) (5; 2.50 g, 6.0 mmol) in dry MeCN (20 mL)
was added rhodamine 575 (6; 0.50 g, 1.2 mmol), in portions,
with stirring. The reaction was heated to reflux for 21 h. Re-
action monitoring was carried out using neutral alumina TLC
(10% EtOH in CHCl3). Upon cooling to room temperature, a
precipitate formed. The reaction mixture was diluted with ad-
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Light tC18 SPE cartridge that was previously activated with
EtOH (10 mL) and water (10 mL). 18F-[1]ijTFA] was eluted from
the cartridge with EtOH (1 mL), and the eluate was evapo-
rated at 110 °C. The final product was formulated in 10%
EtOH in 0.9% saline (1 mL) and assayed by analytical HPLC
(HPLC B, Program D, tR = 6.7 min). Non-decay corrected pre-
parative yield of 18F-[1]ijCl] was 11% (21% DC) from SOS. To-
tal synthesis time was 97 min from SOS.
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Automated radiosynthesis. [18F]Fluoride ion in [18O]H2O was
transferred to a GE Tracerlab FXFN automated radiosynthesis
module where the [18F]fluoride ion was immobilized on a Sep-
Pak® Light QMA cartridge (ABX). [18F]Fluoride ion was partially
eluted into a reaction vessel using a mixture of K2.2.2. (5 mg) in
dry MeCN (0.9 mL) and K2CO3 (0.9 mg) in H2O (0.1 mL) provid-
ing approximately 81 GBq of activity (65% NDC efficiency). The
extraction solvent was heated to 60 °C under a continuous
stream of helium, and the temperature was held there for 7
min. Next, the temperature was increased to 120 °C to complete
the azeotropic distillation process, followed by immediate
cooling of the reaction vessel to 50 °C. Precursor [2]ijOTs] (1 mg)
in DMSO (1 mL) was added and the reaction mixture was heated
to 100 °C for 30 min. Upon completion of the reaction, the
crude product was diluted with 3.5 mL of HPLC mobile phase
and injected onto a semi-preparative HPLC column (HPLC C,
Program E, tR = ∼18 min). 18F-[1]ijTFA] was collected and diluted
with water (50 mL), followed by trapping on a Sep-Pak® C18
Plus SPE cartridge (previously conditioned with 5 mL EtOH and
10 mL water). The tracer was eluted from the cartridge using
EtOH (1 mL), followed by 0.9% saline (5.5 mL). This solution
was then passed through a 0.2 μm sterile filter and collected in
a vented vial. An additional aliquot of saline (3.5 mL) was passed
through the filter giving 6.6 GBq (5.3% NDC, 9.0% DC yield
from SOS) of the injectable final product, 18F-[1]ijCl], in 10%
EtOH in saline. The identity of the formulated compound was
confirmed by analytical HPLC (HPLC D, Program F, tR = 11.3
min). Total automated synthesis time was 86 min from SOS.
17 T. K. Heinrich, V. Gottumukkala, E. Snay, P. Dunning, F. H.
Fahey, S. Ted Treves and A. B. Packard, Appl. Radiat. Isot.,
2010, 68, 96–100.
Conflict of interest
18 M. D. Bartholoma, V. Gottumukkala, S. Zhang, A. Baker, P.
Dunning, F. H. Fahey, S. T. Treves and A. B. Packard, J. Med.
Chem., 2012, 55, 11004–11012.
The authors declare no competing interests.
19 M. D. Bartholoma, H. He, C. A. Pacak, P. Dunning, F. H.
Fahey, F. X. McGowan, D. B. Cowan, S. T. Treves and A. B.
Packard, Nucl. Med. Biol., 2013, 40, 1043–1048.
20 I. AlJammaz, B. Al-Otaibi, H. AlHindas and S. M. Okarvi,
Nucl. Med. Biol., 2015, 42, 804–808.
21 G. Trencsényi, I. Kertesz, Z. T. Krasznai, G. Mate, G. Szaloki,
P. Szabo Judit, L. Karpati, Z. Krasznai, T. Marian and K.
Goda, Eur. J. Pharm. Sci., 2015, 74, 27–35.
Acknowledgements
The authors would like to thank Organica (Bitterfeld-Wolfen,
Germany) and Charkit Chemical Corporation (Norwalk, CT,
USA) for the gift of rhodamine 575. This work was supported
by the Children's Hospital Radiology Foundation and NIH
Grant 5R01HL108107 (PI: A. B. Packard).
22 M. D. Bartholoma, S. Zhang, V. Akurathi, C. A. Pacak, P.
Dunning, F. H. Fahey, D. B. Cowan, S. T. Treves and A. B.
Packard, Nucl. Med. Biol., 2015, 42, 796–803.
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Med. Chem. Commun.
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