One-pot synthesis of high molar activity 6-[18F]fluoro-l-DOPA by Cu-mediated fluorination of a BPin precursor

Article abstract of DOI:10.1039/c9ob01758e

A one-pot two-step synthesis of 6-[¹⁸F]fluoro-l-DOPA ([¹⁸F]FDOPA) has been developed involving Cu-mediated radiofluorination of a pinacol boronate ester precursor. The method is fully automated, provides [¹⁸F]FDOPA in good

Full text of DOI:10.1039/c9ob01758e

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Tanzey, A. F. Brooks, K. J. Makaravage, N. Ichiishi, J. M. Miller, B. D. Henderson, M. Skaddan, M. S. Sanford
and P. J. H. Scott, Org. Biomol. Chem., 2019, DOI: 10.1039/C9OB01758E.
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DOI: 10.1039/C9OB01758E
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COMMUNICATION
One-pot Synthesis of High Molar Activity 6-[18F]Fluoro-L-DOPA by
Cu-Mediated Fluorination of a BPin Precursor†
a
c
c
Andrew V. Mossine, a Sean S. Tanzey,a,b Allen F. Brooks, Katarina J. Makaravage, Naoko Ichiishi,
‡
‡
‡
Received 00th January 20xx,
Accepted 00th January 20xx
Jason M. Miller, a,b Bradford D. Henderson,a Marc B. Skaddan,d Melanie S. Sanford*c and Peter J. H.
‡
Scott*a,b
DOI: 10.1039/x0xx00000x
www.rsc.org/
A one-pot two-step synthesis of 6-[18F]fluoro-L-DOPA ([18F]FDOPA)
Due to the inherent limitations of electrophilic
has been developed involving Cu-mediated radiofluorination of a radiofluorination reactions, a synthesis of [18F]FDOPA that uses
pinacol boronate ester precursor. The method is fully automated, nucleophilic [18F]fluoride has long been in demand. In contrast
provides [18F]FDOPA in good activity yield (104 ± 16 mCi, 6 ± 1%), to the electrophilic reagents described above, [18F]fluoride is
excellent radiochemical purity (>99%) and high molar activity (3799 readily available in high molar activity and is routinely handled
± 2087 Ci/mmol), n=3, and has been validated to produce the in radiochemistry production facilities. However, the electronic
radiotracer for human use.
mismatch between the nucleophilic 18F- and the electron rich
catechol ring has hampered efforts to develop an operationally
simple nucleophilic synthesis of high molar activity [18F]FDOPA.
The typical approach involves nucleophilic radiofluorination of
a benzaldehyde precursor with an appropriate leaving group
(e.g. -F, -NO2, -N+Me3).1b,7 The 18F-labelled aldehyde
intermediate then converted to the ester via a Dakin oxidation.
Finally, hydrolysis of the ester with concentrated HI or HBr
generates [18F]FDOPA (Figure 1b). While this approach yields
[18F]FDOPA in good yields and molar activity, it is confined to
certain synthesis modules (or manual syntheses) because of the
requirements for automation of multiple steps after the
introduction of 18F and the use of corrosive reagents during the
deprotection step. Finally, the complexity of this process results
in multiple potential fail points (both chemical and mechanical)
during automated radiosynthesis.
6-[18F]Fluoro-L-DOPA ([18F]FDOPA, 118F
) is a diagnostic
radiopharmaceutical for positron emission tomography (PET)
imaging.1 The first [18F]FDOPA PET study of the human brain was
reported in 19832 and, since its introduction, [18F]FDOPA PET
imaging has been used to image Parkinson’s disease,3 brain
tumors,4 and focal hyperinsulinism of infancy.5
Despite the numerous important applications in molecular
imaging, [18F]FDOPA PET remains underutilized because of
synthetic challenges associated with accessing the radiotracer
for clinical use.1b Chief amongst these is the need to
radiofluorinate a highly electron rich catechol ring in the
presence of an amino acid. Historically this has been
accomplished with an organostannane or organomercury
precursor via electrophilic aromatic substitution (SEAr) with
[18F]F2 or [18F]acetyl hypofluorite (Figure 1a).6 However, the
production and handling of these reagents requires specialized
equipment that is not widely accessible. Furthermore, the site-
and chemoselectivities of SEAr reactions are typically modest,
and the [18F]FDOPA produced using electrophilic methods
generally has low molar activity.1b
There thus remains a need for a one-pot, two-step
(fluorination + deprotection) synthesis of [18F]FDOPA from
nucleophilic [18F]fluoride that is high yielding, uses milder
reagents, and is easily automated. While such a method has
eluded radiochemists to date, fluorine-18 radiochemistry has
undergone a renaissance in recent years.8 For instance,
hypervalent
iodine
reagents,9
organoborons,10
organostannanes,11 Ni/Pd complexes,12 and phenols13 have
recently been introduced as precursors for nucleophilic
radiofluorination of electron rich arenes. While we and others
have used a number of these approaches to synthesize
[18F]FDOPA in proof-of-concept studies,9,10d,11,12b,14 a method
that is compliant with current Good Manufacturing Practice
(cGMP) and validated for production of human doses has yet to
be reported. For example, the full-scale automated synthesis of
a number of radiotracers from BPin precursors, including
a. Department of Radiology, University of Michigan, Ann Arbor, MI 48109, USA. E-
mail: pjhscott@umich.edu
b. Department of Medicinal Chemistry, University of Michigan, Ann Arbor, MI
48109, USA.
c. Department of Chemistry, University of Michigan, Ann Arbor, MI 48109, USA. E-
mail: mssanfor@umich.edu
d. AbbVie Translational Imaging, North Chicago, IL 60030, USA.
† Electronic Supplementary Information (ESI) available: Experimental procedures,
optimization details, radio-HPLC/TLC traces and spectral data. See
DOI: 10.1039/x0xx00000x.
‡ Current affiliations: AVM: Zevacor; KJM: Gordon Center for Medical Imaging,
Massachusetts General Hospital, Harvard Med. School; NI: Takeda; JMM: US Army.
This journal is © The Royal Society of Chemistry 20xx
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[18F]FDOPA, was reported by Gouverneur (Figure 1c).10d selected because it is commercially available (ABX Advanced
View Article Online
However, since [18F]FDOPA was not the main focus of that Biochemicals), and has MOM and Boc protecting groups that
DOI: 10.1039/C9OB01758E
paper, extensive development work was not done and in its enable mild deprotection with HCl. Our radiofluorination
published form the method gives doses of [18F]FDOPA methodology does not require the introduction of air,10b
contaminated with a chemical impurity that disqualify it from simplifying automation. Lastly, we have also developed a new
clinical use at the University of Michigan. Moreover, the approach for purification and reformulation of [18F]FDOPA that
requirement to introduce air into the radiofluorination reaction utilizes hydrophilic interaction liquid chromatography (HILIC).
is difficult to automate given that radiochemistry synthesis HILIC is an alternative technique to HPLC for separating
modules are typically kept under an inert atmosphere and particularly polar compounds (for an overview of the method,
closed to the environment. The use of 57% HI in the see: 15). HILIC employs traditional polar stationary phases (e.g.
deprotection step is also problematic as it is highly corrosive to silica, amino or cyano), but mobile phases used are similar to
the valves and lines employed in automated synthesis modules. reversed-phase HPLC and, in this case, it provided [18F]FDOPA in
high chemical, radiochemical and enantiomeric purity.
a) Traditional electrophilic synthesis of [18F]FDOPA
To develop a synthesis of [18F]FDOPA (
3), we elected to use
O
O
our recently developed Cu-mediated radiofluorination of
organoboron precursors,10b which was expected to simplify
automation as, unlike the method described above it does not
require the and began by conducting the automated
NHOCH
NH2
EtO
i) [18F]F2 (or [18F]AcOF)
ii) HBr (or HI) Deprotection
HO
tBuOCOO
tBuOCOO
HO
HO
Sn
18F
X Requirement to handle F2
X Uses corrosive HI/HBr
X Low specific activty
e.g. Luurtsema: EJNMMI Radiopharm. Chem., 2016, 1, 7.
[Non-decay-corrected yield: 23 ± 4%a]
radiofluorination of BPin
1 using a TRACERLab FXFN synthesis
module (Table 1). [18F]Fluoride from the cyclotron was trapped
on a bicarbonate-preconditioned QMA cartridge, eluted into
the reactor with an aqueous solution of 10 mg/mL KOTf / 0.1
mg/mL K2CO3 (0.5 mL) and azeotropically dried with MeCN (1
mL). For initial proof-of-concept, manual radiofluorination was
b) Multi-step nucleophilic syntheses of [18F]FDOPA
O
i) [18F]KF
MeO
ii) NaBH4
iii) HBr
-OTf
O
MeO
N
iv) (Ph)2CNCH2COOtBu
v) HI
NH2
HO
Lemaire: Eur. J. Org. Chem., 2004, 2899.
[Non-decay-corrected yield: 13-16%]
conducted using our standard labelling protocol (
1 (4 µmol),
HO
Cu(OTf)2 (20 µmol) and pyridine (500 µmol) in 1 mL DMF for 20
HO
O
H
18F
min at 110 ⁰C). This provided protected [18F]FDOPA (
2) in 49 ± 7
N
tBuO
Trt
√ Nucleophilic [18F]F-
i) [18F]TBAF
ii) mCPBA
iii) HCl
% radiochemical yield (RCY§) (entry 1). This process was readily
translated to an automated process on the synthesis module to
provide 2 in 38 ± 4 % RCY (entry 2).
√ High specific activty
X Difficult automation
O
MOMO
NO2
X Multiple potential fail points
√ HCl can replace HBr/HI
Mueller: J. Labelled Comp. Radiopharm., 2015, 58 (Sup. 1), S164.
[Non-decay-corrected yield: >10%]
Table 1: Optimization of the Labeling of 1
c) One-pot Cu-mediated nucleophilic synthesis of [18F]FDOPA
O
O
O
O
i) [18F]KF/K2.2.2
CuPy4OTf2, DMF
ii) 57% HI Deprotection
NBoc2
NH2
EtO
HO
[
18F]XF, Cu(OTf)2
NBoc2
NBoc2
tBuO
tBuO
MeO
MeO
HO
HO
Pyridine, DMF
110 oC, 20 min
MOMO
MOMO
MOMO
MOMO
BPin
18F
BPin
18F
Gouverneur: Chem. Commun., 2016, 52, 8361.
[Non-decay-corrected yield: 22 ± 3%b]
√ Nucleophilic [18F]F-
√ High specific activty
X Simple automation
X HI deprotection
1
2
Entrya
18F]XF
§
[
RCY
1b
2c
[
[
18F]KF
49 ± 7%
38 ± 4%
55 ± 13%
18F]KF
X Chemical impurity
3 c
[
18F]TBAF
d) One-pot Cu-mediated nucleophilic synthesis of [18F]FDOPA
a Conditions: 1BPin (4 µmol), Cu(OTf)2 (20 µmol), and pyridine (500 µmol) in DMF at 4
O
O
mM concentration of the BPin precursor in DMF, [18F]XF, 110 °C, 20 min; manual
b
i) [18F]TBAF, CuPy4OTf2
Pyridine, DMF
ii) HCl Deprotection
NBoc2
NH2
syntheses; c automated syntheses.
tBuO
HO
MOMO
MOMO
HO
HO
We next focused on optimizing the radiofluorination step.
Our prior work has shown that revealed that both the 18F-
processing technique and the order/temperature of reagent
addition were both key to reaction outcome in related
systems.10e,f Thus, we used these as a starting points for
BPin
18F
Scott, Sanford: This Work
[Non-decay-corrected yield: 6 ± 1%]
√ Nucleophilic [18F]F-
√ High specific activty
√ Simple automation
√ HCl deprotection
Fig. 1 Radiosyntheses of [18F]FDOPA and motivation for this optimizing the [18F]FDOPA synthesis. In our previous work, the
work. a based on [18F]F2 in reactor; b doses not reformulated for
clinical use.
dissolution of 18F- before heating the fluorination reaction
proved critical to avoid competing reactions (e.g.
protodeborylation
and/or
hydroxydeborylation)
To address this issue, we
that
competitively consume 1 10e,f
.
To address the outstanding need in the PET radiochemistry
community for ready access to [18F]FDOPA, in this
communication we describe a new one-pot, two-step synthesis
of the radiotracer from a BPin precursor, and validate it for
developed an alternate eluent in order to facilitate rapid
dissolution of 18F-. Given the greater solubility of
tetrabutylammonium (TBA+) and Cs+ cations relative to K+ in
DMF, without loss of anion exchange properties, we settled on
production of clinical doses (Figure 1d). Precursor
1 was
an
aqueous
eluent
consisting
of
15
mg/mL
2 | J. Name., 2012, 00, 1-3
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tetrabutylammonium triflate (TBAOTf) and 0.2 mg/mL Cs2CO3
(0.5 mL), as a replacement for KOTf and K2CO3, respectively. This
eluent gave good recovery of 18F- from the QMA, and improved
reformulation of small molecule radiopharmaceuticals using,
View Article Online
for example, C18 or Oasis HLB cartridges,DbOuIt: t1h0.i1s0i3s9n/Co9tOpBo0s1s7i5b8leE
with [18F]FDOPA due to its hydrophilicity. We thus employed a
HILIC Strata NH2 cartridge for reformulation. We found
trapping/release efficiency for [18F]FDOPA of 70% and 75% for
the 100 mg and 200 mg cartridges, respectively, and selected
the 200 mg cartridges for routine use.
the RCY of 2 to 55 ± 13% (entry 3).
With an optimized fluorination in hand, we next investigated
the deprotection step. Historically, deprotection steps to
generate [18F]FDOPA have most commonly utilized
concentrated HI or HBr to remove methoxy protecting
groups.1b While such reagents can be used with automated
synthesis modules, they are highly corrosive and greatly reduce
the lifetime of lines and valves in the synthesis module. We
therefore sought to employ a milder acid for deprotection, and
reasoned that HCl should be both compatible with our synthesis
module and adequate to deprotect the methoxymethyl ether
Finally, we automated the one-pot, two-step synthesis of
[18F]FDOPA using a TRACERLab FXFN synthesis module and
validated the synthesis for cGMP production of doses for clinical
use. To simplify routine automation, we changed the Cu source
from Cu(OTf)2 to the less hygroscopic Cu(pyridine)4(OTf)2. This
Cu source has been used to radiofluorinate BPin esters by
Gouverneur but, as stated above, that method requires the
introduction of air into the radiofluorination reaction which is
difficult to automate.10a,d To negate this issue, we adapted
Cu(Py)4(OTf)2 for use in our chemistry, which is compatible with
the inert atmosphere of the TRACERLab synthesis module,10b by
maintaining the same relative ratio of substrate: copper:
pyridine (1BPin (4 µmol), Cu (20 µmol), and pyridine (420
µmol)). Radiofluorination and deprotection then proceeded as
described above. The reaction mixture was diluted with MeCN
(3 mL) and purified by semi-preparative HILIC. The peak
corresponding to [18F]FDOPA (tR ~22–23 min, see Figure S2 in
Supporting Information for a typical trace) was collected in 100
mL MeCN and this solution was passed through the HILIC Strata
NH2 cartridge to trap the radiotracer. Following trapping and
rinsing with US Pharmacopeia (USP) grade ethanol (2-3 mL) to
remove residual MeCN, [18F]FDOPA was eluted from the
cartridge with 0.9% saline, USP (10 mL) to produce doses
formulated for injection. The final drug product was dispensed
into a septum-sealed, sterile, pyrogen-free glass vial through a
0.22 µm sterile filter (Millex GV) to afford formulated doses of
[18F]FDOPA (104 ± 16 mCi, n = 3). The total synthesis time was
approximately 110 min from end-of-bombardment, and the
activity yield (AY) was 6 ± 1%, based upon 1.8 Ci of [18F]fluoride.
Radiochemical purity (RCP) was >99% and molar activity was
3799 ± 2087 Ci/mmol. Doses were submitted for full quality
control (QC) testing to validate the method, and all doses met
or exceeded release criteria for clinical application at the
University of Michigan, including purity, sterility, residual TBA
levels, and residual solvent analysis (Table 3). Notably,
enantiomeric purity was found to be >99% using chiral HPLC,
confirming that the stereochemistry of the precursor was
retained throughout the entire manufacturing process. Doses
produced using Cu-mediated reactions also need to be free of
residual Cu if they are to be applied in the clinic, since the
permitted daily exposure limit for Cu is ≤340 µg/day for
parenteral administration.17 Samples from each of the
qualification runs were submitted for inductively coupled
plasma mass spectrometry (ICP-MS) analysis and were found to
contain residual Cu below the limit of quantification (0.11 ± 0.02
ppm), well under the established limit for Cu.
(MOM) and tert-butyl ester groups of
attempts to treat in the fluorination reaction mixture with 12N
HCl resulted in significant decomposition and minimal (<1%)
[18F]FDOPA
(entry 1). We hypothesized that the
2 (Table 2). Initial
2
(3)
decomposition could be due to the presence of Cu(II) salts,
which could promote numerous potential side reactions.16 As
such, we examined the addition of ascorbic acid during the
deprotection, as this is known to reduce the Cu(II) to Cu(I).
Gratifyingly, this resulted in a dramatic enhancement in the
yield of the deprotection step, providing [18F]FDOPA in 84±8%
RCY (entry 2). Intermediate 2 could also be purified by SPE prior
to deprotection using a modified synthesis module. This
resulted in an even cleaner deprotection that proceeded in
>99% RCY (entry 3).
Table 2: Optimization of the Deprotection of [18F]2
O
O
NBoc2
NH2
tBuO
HO
Deprotection
110 oC, 10 min
MOMO
MOMO
HO
HO
18F
18F
2
3
Entrya
§
RCYb
Deprotection
1
2
12 M HCl
12 M HCl + 0.25 M Ascorbic Acid
12 M HCl + 0.25 M Ascorbic Acid
Decomp.
84 ± 8%
>99%
3c
a Conditions: HCl ± ascorbic acid, 110 °C, 10 min; b RCY represents transformation of 2
à
3; c2 purified by SPE prior to deprotection.
We next sought to develop a robust semi-preparative
chromatography system that would enable purification of
[18F]FDOPA from reactants and potential by-products (e.g. OH-
DOPA and H-DOPA). Prior reports utilized reverse-phase HPLC
with C18 columns, but we have found these to be unsatisfactory
due to the close retention times of [18F]FDOPA and both OH-
DOPA and H-DOPA by-products which result from competing
hydroxy- and proto-deborylation, respectively. We therefore
switched to HILIC purification and evaluated several different
columns (see Supporting Information). The best results were
achieved using a Phenomenex Luna NH2 5 µ column and an
eluent with a high organic content: 75% MeCN incl. 10mM KOAc
buffered with acetic acid to pH: 5.0-5.5 (near the hypothetical
isoelectric point of FDOPA).¶ This system enables adequate
separation of FDOPA, OH-DOPA and H-DOPA using both semi-
preparative and analytical columns (see Supporting
Information). PET radiotracers purified using MeCN-based HILIC
eluents require reformulation into an injectable vehicle such as
ethanolic saline. Reverse phase SPE is typically used for
In summary, we report the validation of our Cu-mediated
radiofluorination of BPin esters for the cGMP synthesis of
[18F]FDOPA for clinical use. The synthesis was fully automated
using a commercial radiochemistry synthesis module, and doses
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and F. Wüst, Nuklearmedizin, 2008, 47, 62; c) G. Luurtsema,
met all QC criteria for human use. We expect to initiate clinical
imaging studies with [18F]FDOPA in the near future.
View Article Online
H. H. Boersma, M. Schepers, A. M. T. de Vries, B. Maas, R.
Zijlma, E. F. J. de Vries and P. H. ElsingaD, EOJIN: 1M0.1M03I 9R/aCd9iOoBp0h1a7r5m8E.
Chem., 2017, 1, article 7.
All hazardous laboratory chemicals were used by trained
personnel under the supervision of University of Michigan (UM)
Environmental Health and Safety. Radioactivity was used by
trained personnel under the approval of the UM Radiation
Policy Committee (Protocol 12-029) and supervision of the UM
Radiation Safety Service. We acknowledge NIH (R01EB021155
to MSS and PJHS) and US DOE/NIBIB (DE-SC0012484 to PJHS) for
financial support.
7
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Radiopharm., 2001, 44 (Suppl. 1), S146; b) F. M. Wagner, J.
Ermert and H. H. Coenen, J. Nucl. Med., 2009, 50, 1724; c) C.
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Luxen, Eur. J. Org. Chem., 2004, 2899; d) B. Shen, W.
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M. Mueller and D. Goblet, J. Labelled Comp. Radiopharm.,
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For reviews of new late-stage fluorination approaches, see: a)
A. F. Brooks, J. J. Topczewski, N. Ichiishi, M. S. Sanford and P.
J. H. Scott, Chem. Sci., 2014, 5, 4545–4553; b) M. G. Campbell
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2580–2605.
Table 3: Validation data for cGMP Synthesis of [18F]FDOPA (3)
i) [18F]TBAF, CuPy4OTf2
Pyridine, DMF
O
O
8
9
NH2
NBoc2
HO
tBuO
110 oC, 20 min
HO
HO
MOMO
MOMO
ii) 12 M HCl
0.25 M Ascorbic Acid
110 oC, 10 min
18F
BPin
1
3
QC Test
Radioactivity Conc.
FDOPA Conc.
Specifications
≥10mCi/batch
≤5µg/mL
Result (n = 3)
104 ± 16 mCi
0.69 ± 0.47 µg/mL
a) N. Ichiishi, A. F. Brooks, J. J. Topczewski, M. E. Rodnick, M.
S. Sanford and P. J. H. Scott, Org. Lett. 2014, 16, 3224; b) B. H.
Rotstein, N. A. Stephenson, N. Vasdev and S. H. Liang. Nature
Commun., 2014, 5, 4365.
Molar activity
≥ 500 Ci/mmol
>90%
3799 ± 2087 Ci/mmol
99.7 ± 0.3
1.02 ± 0.002
>99%
Radiochemical Purity
Radiochemical Identity
Enantiomeric Purity
Visual Inspection
pH
RRTa = 0.9-1.1
≥ 95% L-FDOPA
Clear, colorless, no ppt
4.5-7.5
Pass
5.5 ± 0
10 a) M. Tredwell, S. M. Preshlock, N. J. Taylor, S. Gruber, M.
Huiban, J. Passchier, J. Mercier, C. Génicot, V. Gouverneur,
Angew. Chem., Int. Ed. 2014, 53, 7751; b) A. V. Mossine, A. F.
Brooks, K. J. Makaravage, J. M. Miller, N. Ichiishi, M. S.
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Zlatopolskiy, J. Zischler, P. Krapf, F. Zarrad, E. A. Urusova, E.
Kordys, H. Endepols, B. Neumaier, Chem. Eur. J. 2015, 21,
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Cailly, M. Schedler, T. L. Collier, J. Passchier, R. Smits, J.
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Radionuclidic Identity
T1/2 = 105-115 min
≤260 µg/mL by
Dragendorff reagent
≤880 ppm
112 ± 2 min
< 260 µg/mL
Residual TBA+
Residual DMF
Residual MeCN
Residual Cu
106 ± 56 ppm
179 ± 78 ppm
0.11 ± 0.02 ppm
56 ± 1 psi
≤410 ppm
≤34 ppm
Filter membrane integrity
Bacterial endotoxins
Sterility
≥50 psi
≤ 2.00 EUb/mL
No microbial growth
<2.00 EUb/mL
Pass
a Relative retention time (RRT) = [HPLC retention time of [18F]FDOPA / HPLC retention
time of FDOPA reference standard]; EU = endotoxin units.
b
Conflicts of interest
There are no conflicts to declare.
Notes and references
§
Radiochemical yields (RCY) are non-isolated and were
calculated by % integrated area of the appropriate 18F product
peak versus total 18F- peaks in a radio-TLC trace.
¶
The HPLC employs a dual eluent (0-13 min: 90% MeCN (10mM
KOAc pH: 7.0-7.5); 13-30 min: 75% MeCN (10mM KOAc pH:
5.0-5.5) to separate FDOPA from ascorbic acid used during
deprotection).
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COMMUNICATION
16 a) S. D. McCann and S. S. Stahl, Acc. Chem. Res., 2015, 48, 1756
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View Article Online
DOI: 10.1039/C9OB01758E
17 Guidelines
for
Elemental
Impurities.
http://www.ich.org/fileadmin/Public_Web_Site/ICH_Product
s/Guidelines/Quality/Q3D/Q3D_Step_4.pdf; accessed 17-
May-2019.
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