Synthesis and radiosynthesis of a novel PET fluorobenzyl piperazine for melanoma tumour imaging; [18F]MEL054

Article abstract of DOI:10.1071/CH12489

2-{2-[4-(4-[18F]-Fluorobenzyl)piperazin-1-yl]-2-oxoethyl}isoindolin-1-one ([18F]MEL054), is a new potent indolinone-based melanin binder designed to target melanotic tumours. [18F]MEL054 was prepared by an automated two-step radiosynthesis, comprising of

Full text of DOI:10.1071/CH12489

CSIRO PUBLISHING
Aust. J. Chem. 2013, 66, 491–499
Full Paper
http://dx.doi.org/10.1071/CH12489
Synthesis and Radiosynthesis of a Novel PET
Fluorobenzyl Piperazine for Melanoma Tumour
Imaging; [18F]MEL054
A C
,
A F
,
Maxine P. Roberts, Naomi A. Wyatt,
A
Stephen R. Taylor,
A
A D
,
A E
,
Thomas Bourdier, Peter Roselt,
B
Tien Q. Pham, Daniela Stark,
A E
,
A
Andrew Katsifis, and Ivan Greguric
A
LifeSciences Division, Australian Nuclear Science and Technology Organisation,
Locked Bag 2001, Kirrawee DC, NSW 2232, Australia.
B
Centre for Molecular Imaging, The Peter MacCallum Cancer Centre,
12 St Andrew’s Place, East Melbourne, Vic. 3002, Australia.
C
Current address: Department of Nuclear Medicine, Level 3, Ned Hanlon Building,
Royal Brisbane and Women’s Hospital, Metro North Health Service District,
Herston, Qld 4029, Australia.
D
Current address: Cyclopet Pty Ltd Macquarie University Hospital, 3 Technology Place,
Macquarie University, Lower Northern Sydney, NSW 2341, Australia.
Current address: Department of PET & Nuclear Medicine, Building 63, Level A7,
Royal Prince Alfred Hospital, Missenden Road, Camperdown, NSW 2050, Australia.
Corresponding author. Email: maxine.roberts@ansto.gov.au
E
F
2-{2-[4-(4-[18F]-Fluorobenzyl)piperazin-1-yl]-2-oxoethyl}isoindolin-1-one ([18F]MEL054), is a new potent indolinone-
based melanin binder designed to target melanotic tumours. [18F]MEL054 was prepared by an automated two-step
radiosynthesis, comprising of the preparation of 4-[18F]fluorobenzaldehyde from 4-formyl-N,N,N-trimethylanilinium
triflate, followed by reductive alkylation with 2-(2-oxo-2-piperazin-1-ylethyl)isoindolin-1-one. 4-[18F]Fluorobenzaldehyde
was prepared on a GE TRACERlab FXFN module in 68 ꢀ 8 % radiochemical yield (RCY, non-decay corrected), purified
by a Sep-Pak Plus C18 cartridge and eluted into the reactor of an in-house modified Nuclear Interface [18F]FDG synthesis
module for the subsequent reductive alkylation reaction. HPLC purification produced [18F]MEL054 in a collected RCY of
34 ꢀ 9 % (non-decay corrected), the total preparation time (including Sep-Pak Plus C18 and HPLC purification) did not
exceed 105 min. The radiochemical purity of [18F]MEL054 was greater than 99 % with a specific radioactivity of
71–119 GBq mmolꢁ1 and [18F]MEL054 remained stable in saline solution (.98 %) after 3 h.
Manuscript received: 29 October 2012.
Manuscript accepted: 12 December 2012.
Published online: 18 February 2013.
Introduction
with high selectivity for melanoma tissue offer the potential for
treatment using internal radionuclide therapy. Consequently,
intense interest has resulted in the search for melanoma-specific
diagnostic imaging probes and radiopharmaceuticals during the
past two decades.[3–5]
Several biochemical systems have been described as a means
of delivering radiolabelled chemical entities to melanoma cells
and tissues, including: immunoconjugates,[6,7] specific upregu-
lated receptors,[8] and melanin binding compounds,[9,10] many
of which have shown promising results in animal studies and
preliminary clinical investigations. One of the key features of
melanoma tumours is the presence of excess melanin pigment
which has consequently become an attractive and precise target
for the development of diagnostic and therapeutic melanin-
targeting radiopharmaceuticals.[9,10] Therefore, monoclonal
antibodies,[11] iodothiouracils,[9] melanocortin-1 receptor
targeting peptides,[12–14] indoquinolines,[15] methylene blue
Melanoma is an aggressive cancer that accounts for over 75 %
of all skin cancer related deaths and in recent decades there
has been a marked increase in the incidence of melanoma.
Melanoma-related mortality has become a significant public
health problem worldwide. Early detection and accurate staging
of melanoma has shown that the 5-year survival rates are better
than 99 % if the disease is detected before it has metastasised;
however, this figure falls to below 10 % in patients with distant
metastases. The lack of an effective treatment for melanoma has
therefore focussed research on improved methods for early
detection, detection of disease progression, and treatment
monitoring.[1,2]
The molecular imaging modalities, positron emission
tomography (PET) and single-photon emission computer
tomography (SPECT), offer an excellent opportunity for imag-
ing and staging melanoma. Furthermore, radiopharmaceuticals
Journal compilation Ó CSIRO 2013
www.publish.csiro.au/journals/ajc

492
S. R. Taylor et al.
O
O
O
123I
N
N
N
N
H
I123
N
H
N
H
123I
N
[
123I]BZA
[
123I]BZA2
[
123I]MEL008
1
2
3
O
O
O
N
N
N
N
N
H
18F
N
O
123I
[
18F]MEL050
[
123I]MEL037
5
4
Fig. 1. Previously described radiolabelled melanin binders for the single-photon emission computer tomography and positron
emission tomography imaging of melanoma.
dye,[16,17] iodobenzamides[10,18–20] and more recently [18F]
fluorine labelled benzamides and nicotinamides have been
investigated as melanin targeting radiolabelled agents.[21,22]
It is, however, the [123I]iodinated and [18F]fluorinated
benzamides that have shown most promise as melanin binding
radiopharmaceuticals for targeting melanoma tumours.
The uptake of the first radioiodinated benzamides, N-(2-
Preliminary biodistribution and imaging results for [18F]9[33]
indicates it is a superior agent for clinical staging, compared
with 5; possessing better lesion contrast, more sensitive detec-
tion of small metastases, and greater potential for monitoring
melanotic melanoma. In addition, the chemistry of [18F]9 can be
simply translated to an iodine based [131I]radiopharmaceutical
for future radiotherapeutic treatment (publication in progress).[34]
In this study we describe the synthesis and automation of [18F]9,
our most potent [18F]radiopharmaceutical, for imaging of
melanoma tumours using PET.
diethylaminoethyl)-4-[123I]iodobenzamide
[
123I]BZA (1,
Fig. 1) and [123I]BZA2 (2, Fig. 1), in melanoma cells was
shown to be dependent on melanin binding and melanin content
rather than a receptor-mediated mechanism. Evaluation of
[
123I]iodobenzamides 1 and 2 in patients revealed high affinity
Results and Discussion
binding to melanin-containing melanocytes and confirmed their
in vivo properties as selective SPECT imaging agents for
cutaneous and ocular melanoma.[23–25] In addition, extensive
structure–activity studies revealed significant structural diver-
sity in melanin targeting moieties, which not only offered
targeting entities suitable for diagnostic imaging but also
provided an opportunity for internal radionuclide therapy
employing [131I]iodine analogues.[26,27]
More recently, several benzamide and nicotinamide
derivatives bearing the PET radionuclide [18F]fluoride have
been developed. The 4-[18F]fluorophenyl derivative [18F]N-(2-
diethylaminoethyl)-4-fluorobenzoate, prepared by a three-step
radiosynthetic procedure, demonstrated good tumour uptake in
melanoma tumour bearing mice.[4,21,22] Even more interestingly,
the corresponding [18F]fluoronicotinamide analogue [18F]N-(2-
diethylaminoethyl)-6-fluoronicotinamide ([18F]MEL050, 5,
Fig. 1), not only displayed excellent tumour-to-background
ratios, favourable pharmacokinetics for imaging, good in vivo
stability, and predominantly renal clearance from the rest of the
body, but was also prepared in one easily automated radio-
synthetic step.[28–30]
Chemistry
The synthesis of the non-radioactive labelling standard 2-{2-[4-
(4-fluorobenzyl)piperazin-1-yl]-2-oxoethyl}isoindolin-1-one
(MEL054, 9) was achieved in five steps in an overall yield
of 45 % according to Scheme 1. 2-Carboxybenzaldehyde was
condensed with glycine methyl ester hydrochloride in the
presence of sodium triacetoxyborohydride (STAB) in
acetonitrile (CH3CN) at room temperature (RT) to yield methyl
(1-oxo-1,3-dihydro-2H-isoindol-2-yl)acetate (6) in 88 % yield.
Methyl ester 6 was subsequently hydrolysed by K2CO3 in
aqueous methanol, and the resulting acid coupled with
tert-butyl-piperazine-1-carboxylate in the presence of
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide)
(EDC)/
hydroxybenzotriazole (HOBt) to give tert-butyl-4-(2-(1-
oxoisoindolin-2-yl)acetyl)piperazine-1-carboxylate (7) in 73 %
over the two steps. The Boc-deprotection of 7 by TFA in
dichloromethane (DCM) afforded the TFA salt precursor, 2-(2-
oxo-2-(piperazin-1-yl)ethyl)isoindolin-1-one (8), which was
reductively alkylated with 4-fluorobenzaldehyde in 1,2-
dichloroethane using STAB producing the labelling standard 9
in 70 % yield over two steps.[35,36]
Our radiopharmaceutical research program has delivered a
variety of structurally diverse compounds including [123I]N-(2-
diethylaminoethyl-5-iodonicotinamide (MEL008, 3, Fig. 1)
and 2-(2-(4-(4-[123I]-iodobenzyl)piperazine-1-yl)-2-oxoethyl)
isoindoline-1,3-dione (MEL037, 4, Fig. 1); which demonstrate
high and prolonged tumour uptake, rapid whole body clearance,
and potential therapeutic application.[20,31,32] The favourable
pharmacokinetic and tumour uptake profiles of these melanin-
specific radiopharmaceuticals provided the basis for further
development. Our aim was to combine the attractive biological
properties of the SPECT analogues 3 and 4 with an 18F label for
PET imaging to provide the novel tracer [18F]MEL054 ([18F]9).
Radiosynthesis Development
[18F]9 was prepared in a two-step radiochemical synthesis;
4-[18F]fluorobenzaldehyde (11) was 1) prepared, then purified
on a Sep-Pak Plus C18 cartridge, and 2) reductively alkylated
with piperazine (8) in the presence of NaCNBH3 (Scheme 2).
In comparison to the synthesis of the labelling standard 9, STAB
could not be used as the reducing agent in the [18F]radioactive
work due to instability at high temperatures; undertaking the
reaction at a lower temperature over a long period of time would
not be feasible due to [18F]fluoride decay. For this reason a more

Radiosynthesis of [18F]MEL054
493
O
O
COOH
CHO
OMe
OMe
(a)
HClH2N
ϩ
N
88 %
O
6
(b)
73 %
O
O
O
O
(c)
quantative
N
N
Boc
N
NH TFA
N
N
7
8
(d)
70 %
O
O
N
N
N
9
F
Scheme 1. Synthesis of non-radioactive radiolabelling standard 9. Reagents and conditions: (a) Na(OAc)3BH,
CH3CN, room temperature (RT), 12 h; (b) (i) K2CO3, CH3OH, H2O, 408C, 3 h, (ii) tert-butyl-piperazine-1-
carboxylate, hydroxybenzotriazole, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide), N,N-diisopropylethylamine,
DMF, 16 h; (c) TFA, dichloromethane (DCM), RT, 2 h; (d) 4-fluorobenzaldehyde, Na(OAc)3BH, 1,2-dichloroethane
(1,2-DCE), RT, 16 h.
CHO
CHO
(a)
CHO
(b)
ϩ
N
45 %
70 % manual,
68 % automated
N
18F
10
11
(c)
33 % manual,
34 % automated
O
N
O
N
N
18F
[
18F
]9
Scheme 2. Synthesis of [18F]9. Reagents and conditions: (a) MeOTf, DCM, N2, 12 h; (b) K18F.Kryptofix 2.2.2
complex, DMF, 1008C, 15 min, (70 ꢀ 5 % for manual synthesis and 68 ꢀ 8 % for automated); (c) 8, NaCNBH3,
CH3OH, acetic acid, 1208C, 25 min (33 ꢀ 10 % for manual synthesis and 34 ꢀ 9 % for automated).
heat stable reducing agent, NaCNBH3, was chosen. NaCNBH3
was also considered a suitable reducing agent due to its pH
selectivity; by carrying out reductive alkylation reactions under
weakly acidic to neutral conditions the reactants are able to form
imines without consumption of aldehydes or ketones through
reduction. The reaction pH in this case was 3.5, hence suitable
for the reductive alkylation reaction.[36]
Radiosynthesis of 11 was performed with no-carrier-added
[18F]fluoride (6–7 GBq) through the activated K18F.K222
complex generated by the evaporation of an aqueous
solution of H[18F], K2CO3, and K222 (Kryptofix 2.2.2), followed
by azeotropic drying twice more with anhydrous CH3CN.
The optimisation of the radiosynthesis of 11 was first undertaken
by manually reacting 4-formyl-N,N,N-trimethylanilium triflate
(10) (prepared from commercially available 4-(dimethylamino)
benzaldehyde and methyl triflate, in 45 % yield)[37,38] with
anhydrous K18F.K222 complex in DMF at 80, 100, and 1508C
for 5, 15, and 30 min. The optimum [18F] incorporation yield
was obtained at 1008C for 15 min, and the results displayed in
Fig. 2. The reaction solvent and precursor mass were not

494
S. R. Taylor et al.
70
60
50
40
30
20
10
0
time and temperature were optimised by investigating the
reductive alkylation in methanol at 100 and 1208C for 5, 15,
and 30 min (Fig. 3). The optimal temperature was found to be
1208C and the yield between the 15 and 30 min time points did
not differ greatly, hence taking into account the short half-life of
[18F] a reaction time of 25 min was chosen to balance increased
yield versus radiochemical decay.
The reductive alkylation of 11 with piperazine 8 and
NaCNBH3 using methanol and acetic acid as the solvent, in a
tightly sealed vessel at 1208C for 25 min, afforded [18F]9
isolated by preparative HPLC in 33 ꢀ 10 % (RCY non-decay
corrected, n ¼ 12). The total preparation time of [18F]9 did not
exceed 105 min with specific radioactivity in the range of
71–119 GBq mmolꢁ1. In comparison to our results, literature
describing the preparation of 11 followed by a reductive alkyl-
ation with a secondary amine reports a RCY of 17 % using
methanol and acetic acid at 1358C for 15 min;[41] and a RCY of
7–13 % with the use of a piperazine derivative in DMF at 1208C
for 10 min.[42]
80ЊC
100ЊC
150ЊC
0
5
10
15
20
25
30
35
Time [min]
Fig. 2. Non-decay corrected radiochemical yield of 11 from precursor
10 in DMF with K18F.Kryptofix 2.2.2 complex as a function of time and
temperature.
35
100ЊC
30
25
20
15
10
5
120ЊC
Automated Radiosynthesis
The manual radiochemical optimisation of the aldehyde
synthon 11, and the following reductive alkylation reaction
(with 8) to produce [18F]9 provided a good starting point for
the automated radiosynthesis development. Although the
manual preparation of [18F]9 was successful, our anticipated
pre-clinical evaluation studies required an automated radio-
synthesis. Automated synthesis of [18F]radiotracers signifi-
cantly reduces the dose to the operator, enabling higher
amounts of radioactivity to be used and also increases the
reproducibility of the radiosynthesis. Radiolabelled synthons
such as 11 are often used to introduce radioisotopes into
molecules not amenable to direct labelling. The translation of a
radiosynthesis from manual to automated can be very difficult
and often requires substantial additional optimisation. Typical
modifications for a successful translation often involve the
reaction time, solvent type and volume, precursor mass,
radioactivity scale, and purification method. Precursor mass
and reagents are commonly the limiting constraint due to a
fixed number of reagent vessels on the synthesis module, and
the need to remove excess reagents by preparative HPLC.
0
0
5
10
15
20
25
30
35
Time [min]
Fig. 3. Non-decay corrected radiochemical yield of [18F]9 from 11 with
NaCNBH3 in methanol/acetic acid as a function of time and temperature.
modified on account of the good yield and literature precedent
for the use of DMF in this reaction.[39,40] After cooling, the
reaction mixture was diluted with aqueous HCl and passed
through a Sep-Pak Plus C18 cartridge to trap the labelled
benzaldehyde and remove the water soluble precursor and
reagents. Subsequent elution of the Sep-Pak with methanol
afforded 11 in 68 ꢀ 8 % non-decay corrected radiochemical
yield (RCY). In comparison, Speranza et al.[40] reported a
similar RCY of 67 %, using DMSO at 608C for 15 min (auto-
mated synthesis), and Berndt et al.[39] reported a RCY of 80 %,
using similar reaction conditions of DMF at 1208C for 15 min.
Optimisation of the reductive alkylation included modifica-
tion of the reaction solvent, the amine precursor mass (8),
reaction time, and temperature. Considering the anticipated
high temperature of the reaction (1208C) we initially selected
a solvent with a high boiling point (DMF), however we observed
reduced elution of 11 from the Sep-Pak Plus C18 cartridge, low
solubility of the reducing agent (NaCNBH3), in addition to
difficult HPLC purification and low yield (10 % RCY, non-
decay corrected) of [18F]9. Methanol was found to be an
improved solvent as it easily eluted benzaldehyde 11 from the
cartridge and also readily solubilised NaCNBH3 for the subse-
quent reductive alkylation reaction. Using a precursor (8) mass
of 4.5 mg for the reductive alkylation gave a favourable balance
between sufficient radiochemical yield and acceptable HPLC
loading and separation on a preparative column. The reaction
Two-Step, One-Pot Automated Synthesis
The radiosynthesis method of [18F]9, by the formation of 11,
was transferred to the GE TRACERlab FXFN automated
synthesis module (Supplementary Material 1.7, a), using an
in-house programmed reaction sequence. [18F]Fluoride was
separated from H218O by trapping onto an anion exchange
cartridge (Sep-Pak Light QMA cartridge) followed by elution
with K2CO3 and K222 in CH3CN/H2O, into a reaction vessel that
was then dried under vacuum for 7 min at 758C and for 5 min at
1208C. Triflate 10 in DMF was added to the activated K18F.K222
complex and the mixture heated at 1208C for 15 min. Following
the formation of 11 the reactor was cooled to 308C. Piperazine 8
in methanol and acetic acid, along with NaCNBH3 in methanol,
were then added to the reactor and heated to 1208C for 25 min.
Following preparative HPLC purification of the reaction mix-
ture, [18F]9 was collected in a poor radiochemical yield of
5–10 %, which is explained by the reductive alkylation of
piperazine 8 by unlabelled 10 in a competitive side reaction.[42]
The competitive reaction did not occur in the initial optimisation
reactions as 11 was purified by a Sep-Pak Plus C18 cartridge
before proceeding with the alkylation reaction, however, this

Radiosynthesis of [18F]MEL054
495
HPLC gamma detector
250
200
150
[
18F]9
100
50
0
HPLC UV detector [AU]
600
500
400
300
200
100
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
Time [min]
Fig. 4. The radio-HPLC chromatogram (top) of [18F]9 radiosynthesis, with the corresponding UV absorbance at 254 nm (bottom). [18F]9 was
collected at a retention time between 12.4 and 13.4 min. Injection sample contained entire reaction contents and 400 mL CH3OH/H2O (50 : 50, v/v).
purification step was not possible with the use of a single reactor
module.
observation of solvent condensation up the transfer lines of the
reactor vessel; this was not observed during the manual
development reactions due to the absence of transfer lines
and the smaller reaction surface area. Despite this difference,
the radiochemical yields from both approaches were compara-
ble, however, the possibility of the solvent partially being in
the vapour phase in combination with a relatively long radio-
chemistry reaction time may have prevented a higher yield of
[18F]9 being obtained.
Two-Step, Two-Pot Automated Synthesis
Multistep radiosyntheses performed on commercial synthe-
sisers with a single reactor can be problematic as described in the
previous sentence, hence in order to overcome the poor yield of
the second step the automated synthesis of 11 and subsequently
[18F]9 were modified. This was achieved by creating a two-pot
dual-type reactor by linking a GE TRACERlab FXFN module to
an in-house modified Nuclear Interface [18F]FDG synthesis
module (Supplementary Material 1.7, b) with a programmed
reaction sequence (Supplementary Table 1.8). Benzaldehyde 11
was prepared on the GE TRACERlab FXFN reactor as described,
diluted with aqueous HCl, and transferred to a Sep-Pak Plus C18
cartridge on the Nuclear Interface module (position D,
Supplementary Material 1.7, b, where the QMA cartridge is
usually located). The synthon 11 was eluted from the cartridge
with methanol into the reaction vial of the Nuclear Interface
module for the subsequent reductive alkylation with piperazine
precursor 8 in methanol/acetic acid and NaCNBH3 in methanol
for 25 min at 1208C. The tracer was purified by preparative
HPLC and [18F]9 was isolated in a non-decay corrected
collected yield of 34 ꢀ 9 % (n ¼ 10).
Formulation of [18F]9 for In Vivo Studies
The [18F]9 HPLC collection (Fig. 4) (of the two-step, two-pot
automated radiosynthesis) was diluted with 12 mL of H2O and
the resulting solution passed through a Sep-Pak Light C18
cartridge. Trapped [18F]9 was eluted from the cartridge with
0.5 mL of ethanol, evaporated, and diluted with saline to a final
concentration containing less than 2 % ethanol for use in in vivo
studies. The total preparation time including HPLC purification
and formulation did not exceed 105 min.
Purity Specific Activity and Stability of [18F]9
The radiochemical purity of [18F]9 was found to be 99.1 %. UV
analysis of the final solution of [18F]9 revealed no contamination
from the starting material or reaction breakdown products
(Fig. 5). The specific activity of [18F]9 ranged from 71 to
119 GBq mmolꢁ1. The stability of [18F]9 in a 2 % ethanol in
saline formulation was analysed using an analytical reversed-
phase HPLC column (Luna Peek 5 mm C18 column
150 ꢂ 4.6 mm; mobile phase acetonitrile/0.1 M ammonium
acetate 35 : 65, v/v; flow rate 1 mL minꢁ1; l 254 nm) at room
temperature. Time points were taken at t ¼ 0 and 180 min, and
Thorough consideration of the numerous automated trans-
lation issues discussed has enabled the technical transfer to
work very well in this case. In terms of the chemistry,
additional reaction(s) optimisation was not required, fluid
transfers were efficient and comparable yields were consis-
tently obtained over a wide range of [18F]activities (1–74 GBq).
One issue with the automated reductive alkylation was the

496
S. R. Taylor et al.
Chromatogram:
cps
Radioactivity
6500.0
6000.0
5500.0
5000.0
4500.0
4000.0
3500.0
3000.0
2500.0
2000.0
1500.0
1000.0
500.0
0
0:00
10:00
20:00
30:00mm:ss
Chromatogram:
UV 254 nm
mV
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0
0:00
10:00
20:00
30:00mm:ss
Fig. 5. HPLC QC radiochromatogram of [18F]9 (5.8 MBq in 25 mL sample in saline, ,2 % ethanol, solution). Radioactivity is shown on
the top and UV 254 nm on the bottom.
the HPLC QC (quality control) radiochemical analysis indicated
that there was no significant decrease in radiochemical purity
(99.1 to 98.6 %) over 180 min.
was greater than 99 % with a specific radioactivity range of
71–119 GBq mmolꢁ1. The tracer was stable in saline (.98 %)
after 3 h.
The automated development resulted in [18F]9 being pro-
duced in sufficient quantities and purity that enabled the in vivo
assessment of this radiotracer (publication in progress)[33] and
the promising results have encouraged us to further investigate
the use of automation for multi-step radiochemical synthesis.
Our use of two combined reactor modules was not ideal due to
obvious inefficiencies involving duplication of all associated
equipment; preparing two modules to run together smoothly,
multiple software programs, hot cell space, and numerous power
supplies. These obstacles, however, have motivated our research
group to continue towards the development of more efficient
dual reaction technology, such as the iPHASE Technologies
FlexLAB Dual Reactor, for easier synthesis and translation for
future clinical studies (publication in progress).[43] The core
Conclusion
[18F]9 was specifically designed to detect and image melanotic
tumours through melanin uptake. Preliminary in vivo results
suggest it is our most promising melanin binding [18F]tracer.[33]
[18F]9 was prepared by a two-step automated process on a GE
TRACERlab FXFN automated synthesis module in combination
with an in-house modified Nuclear Interface [18F]FDG
synthesis module. The radiochemical synthesis consisted of the
automated preparation of 11 (RCY 68 ꢀ 8 %, n ¼ 29, non-decay
corrected) followed by the reductive alkylation with the piper-
azine precursor,
8
(RCY 34 ꢀ 9 %, n ¼ 10, non-decay
corrected). The radiochemical purity of the final product [18F]9

Radiosynthesis of [18F]MEL054
497
structure of [18F]9 is also amenable to iodine chemistry for
radiotherapeutic applications and so we are currently deve-
loping new iodinated analogues as radiotherapeutic agents,
anticipated to clinically treat secondary metastases, in combina-
tion with the imaging agent, [18F]9 (publication in progress).[34]
4.39 (s, 2H, –CH2–COOMe), 3.73 (s, 3H, –OCH3). dC (CDCl3,
100 MHz) 169.4, 168.7, 141.4, 131.7, 131.6, 127.9, 123.8,
122.7, 52.2, 50.4, 43.6; m/z (LRMS ESI, þve) 205 (38 %,
Mþ), 146 (100). m/z (HRMS ESI, þve) Calc. for C11H11NO3:
205.0738. Found: 205.0733.
Experimental
tert-Butyl-4-[(1-oxo-1,3-dihydro-2H-isoindol-2-yl)
acetyl]piperazine-1-carboxylate (7)
General
All reagents were purchased from Sigma–Aldrich, and used
1
Potassium carbonate (538 mg, 3.89 mmol) and 6 (400 mg,
1.94 mmol) were dissolved in a solution of CH3OH (5 mL) and
H2O (5 mL) and stirred at 408C for 3 h. The CH3OH was
evaporated under reduced pressure and the aqueous solution
acidified with 1 M HCl, diluted with H2O (20 mL), and extracted
into EtOAc (2 ꢂ 20 mL). The EtOAc washings were combined,
dried over anhydrous Na2SO4, filtered, and evaporated to give
(1-oxo-1,3-dihydro-2H-isoindol-2-yl)carboxylic acid (283 mg),
which (250 mg, 1.31 mmol) was subsequently treated with
1-tert-butyl-piperazine (268 mg, 1.43 mmol), HOBt (264 mg,
1.96 mmol), and N,N-diisopropylethylamine (0.57 mL,
3.27 mmol) in dry DMF (7 mL). EDC (376 mg, 1.96 mmol)
was added and the resulting solution stirred overnight at RT.
The reaction was diluted with H2O (20 mL) and extracted with
DCM (3 ꢂ 20 mL). The organic layers were combined, washed
with saturated NaHCO3 solution (2 ꢂ 20 mL) and water
(4 ꢂ 20 mL), dried over anhydrous MgSO4, filtered, and the
solvent evaporated. Purification by column chromatography
using EtOAc/petroleum ether (66 : 33) as the mobile phase gave
7 as a white powder (345 mg, 73 %). dH (CDCl3, 400 MHz) 7.85
(d, J 7.6, 1H, ArH-7), 7.55 (t, J 8.0, 1H, ArH-5), 7.46 (t, J 8.0,
2H, ArH-4, 6), 4.55 (s, 2H, Ar–CH2–N), 4.45 (s, 2H, –CH2–
CON), 3.57–3.54 (m, 4H, N–(CH2–CH2)2–N), 3.45–3.38 (m,
4H, N–(CH2–CH2)2–N), 1.45 (s, 3H, –C(CH3)3). dC (CDCl3,
100 MHz) 167.6, 166.2, 153.7, 142.0, 131.7, 131.1, 127.5,
123.0, 122.6, 79.1, 50.2, 43.2, 42.9, 27.8. m/z (LRMS ESI,
þve) 359 (1 %, Mþ), 259 (75), 146 (100). m/z (HRMS ESI, þve)
Calc. for C19H25N3O4: 359.1845. Found: 359.1832.
without further purification. H and 13C NMR spectra were
measured on a Bruker DPX 400, at 400 (1H) and 100 MHz (13C)
in CDCl3 or d6-DMSO. Chemical shifts are reported in parts per
million (d) relative to TMS (0.00 ppm), which was used as an
internal standard. Coupling constants are given in Hz and cou-
pling patterns are abbreviated as: s (singlet), d (doublet),
t (triplet), m (mutiplet), and dt (doublet of triplets). Low-
resolution mass spectrometry (LRMS) was performed on a
Waters Micromass ZQ Quadrupole Mass Spectrometer and
high-resolution mass spectrometry (HRMS) was performed at
the University of Wollongong using a Micromass QT Ultima or
AutoSpec ToF. TLC were run on pre-coated plastic-backed
plates of silica gel 60F254 (Merck) and Rf values were estab-
lished using a UV lamp at 254 nm. Column chromatography was
undertaken on Merck 60 silica gel (40–63 mm) columns. All
Sep-Pak cartridges were purchased from Waters; Sep-Pak Light
AccellTM QMA cartridges were activated with potassium car-
bonate (0.1 M aq, 5 mL), and then washed with water (15 mL)
before use and Sep-Pak Plus and Light C18 cartridges were
activated with ethanol (2 mL), and then washed with water
(15 mL) before use.
H[18F] was produced on a GE PETtrace Cyclotron by the
18O(p, n)18F nuclear reaction (Cyclotek, Australia). Compound
11 was analysed by HPLC, consisting of a Waters 510 pump, a
Linear UV-Vis detector (l 254 nm) in series with a Carroll and
Ramsey model 105S gamma detector, on a Phenomenex Bond-
clone C18 column (300 ꢂ 7.8 mm, 10 mm) at 2.0 mL minꢁ1 with
CH3CN/H2O/TFA (50 : 50 : 0.1, v/v) as the mobile phase. [18F]9
was purified by HPLC (as described above) using CH3CN/H2O/
TFA (25 : 75 : 0.1, v/v) as the mobile phase. HPLC QC analysis
of [18F]9 was performed on an analytical HPLC system consist-
ing of a Waters 600W pump, a Waters Linear UV-Vis detector
(l 231 nm) in series with an Ortec ACE Mate bþ-flow detector
on a Luna PEEK C18 column (150 ꢂ 4.6 mm, 5 mm) at
1.0 mL minꢁ1 with CH3CN/0.1 M ammonium acetate (35 : 65,
v/v) as the mobile phase. The syntheses of 11 and [18F]9 were
confirmed by co-injection with the non-radioactive standard by
HPLC. Radioactivity was measured with a Capintec R15C dose
calibrator.
2-(2-Oxo-2-(piperazin-1-yl)ethyl)isoindolin-1-one (8)
Compound 7 (280 mg, 0.78 mmol) in DCM (2 mL) was
treated with TFA (2 mL) and stirred for 2 h at RT. After
evaporation of the TFA to dryness, the residue was dissolved
in toluene (10 mL) and evaporated to dryness to give 8 (quanti-
tative recovery). dH (d6-DMSO) 7.70 (m, 1H), 7.61 (m, 2H),
7.50 (m, 1H), 4.51 (s, 2H), 4.47 (s, 2H), 3.73 (m, 2H), 3.65 (m,
2H), 3.20 (m, 2H), 3.11 (m, 2H). dC (d6-DMSO) 167.9, 166.5,
142.3, 131.9, 131.5, 127.9, 123.5, 122.9, 50.4, 43.3, 42.7, 42.6,
41.2, 38.3.
Chemistry
2-{2-[4-(4-Fluorobenzyl)piperazin-1-yl]-2-oxoethyl}
Methyl (1-oxo-1,3-dihydro-2H-isoindol-2-yl)acetate (6)
isoindolin-1-one (9)
2-Carboxybenzaldehyde (1.0 g, 6.66 mmol), glycine methyl
ester hydrochloride (875 mg, 6.96 mmol), and STAB (1.69 g,
7.99 mmol) were dissolved in anhydrous CH3CN (20 mL) and
stirred overnight at RT under a N2 atmosphere. The resulting
suspension was diluted with H2O (20 mL) and extracted into
DCM (2 ꢂ 20 mL). The organics were dried over anhydrous
MgSO4, filtered, and evaporated to dryness. The residue crystal-
lised on standing to give 6 as white needles which were used
without any further purification (1.21 g, 88 %). dH (CDCl3,
400 MHz) 7.84 (d, J 7.2, 1H, ArH-7), 7.53 (t, J 7.6, 1H,
ArH-5), 7.44 (t, J 7.2, 2H, ArH-4, 6), 4.49 (s, 2H, Ar–CH2–N),
The evaporated residue, 8, was dissolved in dry 1,2-
dichloroethane (5 mL) and stirred overnight at RT with 4-
fluorobenzaldehyde (175 mL, 1.64 mmol) and STAB (363 mg,
1.71 mmol). The resulting suspension was diluted with H2O
(20 mL) and washed with DCM (2 ꢂ 20 mL). The organic layers
were dried over anhydrous MgSO4, filtered, and evaporated to
dryness. The product was purified by column chromatography
using EtOAc/CH3OH (95 : 5) as the mobile phase and isolated as
a yellow film (201 mg, 70 %). dH (CDCl3, 400 MHz) 7.96 (d, J
7.2, 1H, ArH-7), 7.65 (dt, J 7.6, 1.2, 1H, ArH-5), 7.56 (t, J 7.8,
2H, ArH-4, 6), 7.39–7.35 (m, 2H, (ArH)–F) 7.13–7.08 (m, 2H,

498
S. R. Taylor et al.
(ArH)–F), 4.66 (s, 2H, –CH2–CON), 4.55 (s, 2H, Ar–CH2–N),
3.70 (dt, J 20.0, 5.2, 4H, N–(CH2–CH2)2–N), 3.58 (s, 3H, –N–
CH2–ArF), 2.56–2.51 (m, 4H, N–(CH2–CH2)2–N). dC (d6-
DMSO, 100 MHz) 167.6, 165.9, 161.2 (J 241.7), 142.0, 133.7
(J 3.0), 131.7, 131.1, 130.4 (J 7.8), 127.5, 123.0, 122.6, 114.6
(J 20.8), 60.6, 52.0, 50.2, 43.1. m/z (LRMS ESI, þve) 368
(100 % [M þ H]þ). m/z (HRMS ESI, þve) Calc. for
C21H22FN3O2: 367.1696. Found: 367.1698.
of K2CO3 (2 mg) and K222 (10 mg) in CH3CN/H2O (1 mL,
80 : 20 v/v). The eluate was dried by vacuum at 758C for 7 min
and 1208C for 5 min. To the activated K18F.K222 complex was
added triflate 10 (2 mg, 6.4 mmol) in DMF (1 mL) and the
mixture heated at 1208C for 15 min. The solution was diluted
with HCl solution (0.1 M, 12 mL) and transferred onto a Sep-Pak
Plus C18 cartridge on the in-house modified Nuclear Interface
[18F]FDG automated synthesis module.
The cartridge was washed with H2O (5 mL) before CH3OH
(1 mL) eluted 11 from the C18 cartridge (68 þ 8 %, non-decay
corrected, n ¼ 29) into the second reactor, after which pipera-
zine 8 (4.5 mg, 12.6 mmol) in CH3OH (0.4 mL) and acetic acid
(20 mL), plus NaCNBH3 (2 mg, 31.8 mmol) in CH3OH (0.4 mL)
were added and heated at 1208C for 25 min.
The reaction mixture was diluted with H2O (2 mL) and
purified by preparative HPLC (Phenomenex Bondclone C18
column; 300 ꢂ 7.8 mm, 10 mm, CH3CN/H2O/TFA, 25 : 75 : 0.1,
v/v; 2.5 mL minꢁ1) eluting [18F]9 at 12 min, in a collected yield
of 34 % ꢀ 9 % (non-decay corrected, n ¼ 10).
Radiochemistry
Manual Radiosynthesis of 2-{2-[4-(4-[18F]-Fluorobenzyl)
piperazin-1-yl]-2-oxoethyl}isoindolin-1-one ([18F]9)
An aqueous H[18F] solution (6–7 GBq) was added to a 10 mL
vial containing a solution of K2CO3 (2 mg, 20 mL in H2O) and
K222 (10 mg, 100 mL in anhydrous CH3CN). The solvent was
evaporated under a stream of nitrogen at 1008C under vacuum
and the residue azeotropically dried twice more by further
addition and evaporation of anhydrous CH3CN (2 ꢂ 1 mL). To
the activated K18F.K222 complex was added 10 (2 mg, 6.4 mmol)
in DMF (1 mL) and the mixture heated at 1008C for 15 min.
After cooling the mixture was diluted with 0.1 M HCl solution
(10 mL) and passed through a reverse phase C18 Sep-Pak Plus
cartridge. The cartridge was washed with H2O (5 mL) and then
CH3OH (1 mL) eluted 11 from the cartridge (RCY 70 ꢀ 5 %,
non-decay corrected n ¼ 13). To this solution of 11 was added
the piperazine precursor 8 (4.5 mg, 5.6 mmol) in methanol
(0.4 mL) with acetic acid (20 mL) and NaCNBH3 (2 mg,
31.8 mmol) in methanol (0.4 mL). The reaction mixture was
heated at 1208C for 25 min in a sealed vial. Purification of the
reaction mixture by preparative HPLC (Phenomenex Bondclone
C18 column; 300 ꢂ 7.8 mm, 10 mm; CH3CN/H2O/TFA,
25 : 75 : 0.1, v/v; 2.5 mL minꢁ1) eluted [18F]9 at 12 min in a
collected radiochemical yield of 33 ꢀ 10 % (non-decay cor-
rected, n ¼ 12).
Purity, Specific Activity, and Stability Measurement
For radiochemical purity, stability, and specific activity
measurements, [18F]9 was ascertained on an analytical HPLC
system (Luna PEEK C18 column 150 ꢂ 4.6 mm, 5 mm at
1.0 mL minꢁ1 with CH3CN/0.1 M ammonium acetate, 35 : 65,
v/v). Specific activity measurements were assessed by HPLC.
The specific activity value for the final compound was
obtained by measurement of the radioactivity injected and
the UV absorbance associated with the radioactive [18F]9 peak.
The concentration was then obtained by comparison of the
UV area under the curve to the standard concentration curve
plot of 9.
Supplementary Material
Two-Step, One-Pot Automated Radiosynthesis of [18F]9
The Supplementary Material contains characterisation data of 9
and a schematic of the instrumentation used to synthesise [18F]9
and are available on the Journal’s website.
An aqueous [18F]fluoride solution (1–74 GBq) was added to
a GE TRACERlab FXFN automated synthesis module and the
reaction program started. [18F]Fluoride was separated from the
H218O by an anion exchange cartridge (Sep-Pak Light QMA)
and eluted from the cartridge into the reaction vessel with a
solution of K2CO3 (2 mg) and K222 (10 mg) in CH3CN/H2O
(1 mL, 80 : 20 v/v). The eluate was dried by vacuum at 758C for
7 min and 1208C for 5 min. To the activated K18F.K222 complex
was added triflate 10 (2 mg, 6.4 mmol) in DMF (1 mL) and the
mixture heated at 1208C for 15 min. The reactor was cooled to
308C, and then the piperazine precursor 8 (4.5 mg, 12.6 mmol) in
methanol (0.4 mL) and acetic acid (20 mL), along with
NaCNBH3 (2 mg, 31.8 mmol) in methanol (0.4 mL), were added
to the reactor that was then heated to 1208C for 25 min. The
reaction mixture was purified by preparative HPLC (Phenom-
enex Bondclone C18 column; 300 ꢂ 7.8 mm, 10 mm, CH3CN/
H2O/TFA, 25 : 75 : 0.1, v/v; 2.5 mL minꢁ1) eluting [18F]9 at
12 min in a collected radiochemical yield of 5–10 %.
Acknowledgements
The authors gratefully acknowledge Tim Jackson for technical assistance
and Christian Wichmann for his contribution. They thank the
Commonwealth Government of Australia for their financial support to the
Collaborative Research Centre for Biomedical Imaging Development
(CRC-BID).
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