Synthesis of (4-[18F]fluorophenyl)triphenylphosphonium as a potential imaging agent for mitochondrial dysfunction

Article abstract of DOI:10.1002/jlcr.906

Tetraphenylphosphonium (TPP) cation is able to function as a molecular probe for monitoring mitochondrial disease. The F-18 labeled TPP, (4-[ ¹⁸F]fluorophenyl) triphenylphosphonium (¹⁸FTPP), was therefore developed as a PET radioligand for in vivo molecular imaging of mitochondrial dysfunction. ¹⁸FTPP was synthesized via direct nucleophilic substitution of no-carrier-added [¹⁸F]fluoride with the precursor 4-nitrophenyltriphenylphosphonium. After purification by HPLC, the average radiochemical yield was determined to be 10-15% and the specific activity was >500 Ci/ mmol at the end of synthesis. The total synthesis time was within 60min, and the radiochemical purity of the ¹⁸FTPP was above 95%. Copyright

Full text of DOI:10.1002/jlcr.906

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2005; 48: 131–137.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.906
Research Article
Synthesis of (4-[18F]fluorophenyl)triphenylphosphonium
as a potential imaging agent for mitochondrial
dysfunction
Zhen Cheng, Murugesan Subbarayan, Xiaoyuan Chen and Sanjiv Sam
Gambhir*
Department of Radiology and Bio-X Program, Molecular Imaging Program at Stanford
(MIPS), Stanford University, Stanford, CA, 94305, USA
Summary
Tetraphenylphosphonium (TPP) cation is able to function as a molecular probe for
monitoring mitochondrial disease. The F-18 labeled TPP, (4-[18F]fluorophenyl)
triphenylphosphonium (18FTPP), was therefore developed as a PET radioligand for
in vivo molecular imaging of mitochondrial dysfunction. 18FTPP was synthesized via
direct nucleophilic substitution of no-carrier-added [18F]fluoride with the precursor 4-
nitrophenyltriphenylphosphonium. After purification by HPLC, the average radio-
chemical yield was determined to be 10–15% and the specific activity was >500 Ci/
mmol at the end of synthesis. The total synthesis time was within 60 min, and the
radiochemical purity of the 18FTPP was above 95%. Copyright # 2005 John Wiley &
Sons, Ltd.
Key Words: phosphonium; F-18; mitochondria; imaging
Introduction
It has been recognized that a wide range of diseases, including cancers,
diabetes, heart failure, AIDS, degenerative diseases, and the pathophysiology
of aging are associated with mitochondria dysfunction.1–3 It has also become
widely accepted in the past few years that mitochondria play a key role during
apoptosis.4 Cumulative evidence reveals that dramatic changes of mitochon-
drial membrane potential (Dcm) are associated with these mitochondrial
diseases.1À4 For instance, the difference in Dcm between normal epithelial cells
*Correspondence to: Sanjiv Sam Gambhir, Molecular Imaging Program at Stanford, Department of
Radiology and Bio-X Program, Stanford University, 318 Campus Dr., Clark Center E-150, Stanford, CA
94305, USA. E-mail: sgambhir@stanford.edu
Contract/grant sponsor: Doris Duke Charitable Trust
Contract/grant sponsor: Molecular Imaging Program at Stanford (MIPS)
Copyright # 2005 John Wiley & Sons, Ltd.
Received 23 August 2004
Revised 26 October 2004
Accepted 26 October 2004

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Z. CHENG ET AL.
and many carcinoma cells is at least 60 mV,5 and the loss of Dcm is an early
event in cell apoptosis caused by pro-apoptotic agents.6 Therefore, to develop
drugs based on the changes in Dcm may serve as a novel effective approach for
disease treatment.7À10 Furthermore, the development of radiolabeled mito-
chondriotropic molecular probes may provide a powerful tool to investigate
the role of mitochondria in the pathophysiology and treatment of cancer using
a non-invasive imaging technology such as positron emission tomography
(PET).
It has been known for decades that lipophilic cations such as the rhodamine-
123 (Rh123) and tetraphenylphosphonium (TPP) salts, can penetrate the
plasma and mitochondrial membranes and selectively accumulate in mito-
chondria, because of the negative inner mitochondrial transmembrane
potential (À120 to À170 mV, negative inside).7À11 Many phosphonium and
other lipophilic cations have been evaluated as anticancer drugs, as well as
carriers to deliver passenger molecules selectively to mitochondria of cancer
cells, for manipulation of these cells.12À18 We and several other laboratories
have shown that 3H-labeled phosphonium analogs can function as a molecular
probe for selective accumulation in certain cancer cells.19À21. Several
radiolabeled phosphonium cations, [11C]methyltriphenylphosphonium,22 3-
[18F]fluoropropyl and 4-[18F]fluorobenzyltriarylphosphonium,23À24 have been
synthesized and evaluated as mitochondrial targeting agents.
Recently, the mitochondria targeting ability of a library of phosphonium
analogs were studied in our laboratory using matrix-assisted laser desorption/
ionization time of flight mass spectrometry (MALDI-TOF-MS). It was found
that (4-[19F]fluorophenyl)triphenylphosphonium exhibited 80% of the TPP
uptake in glioma C6 cells in vitro, which is much higher accumulation than
that of methyltriphenylphosphonium and butyltriphenylphosphonium (un-
published data). Having achieved good tumor accumulation and pharmaco-
kinetic properties of [3H]-TPP,21 we pursued further to label
tetraphenylphosphonium with Fluorine-18 which may be considered useful
as a novel molecular ‘voltage sensor’. We report in the present study the
radiosynthesis of (4-[18F]fluorophenyl)triphenylphosphonium.
Results and discussion
The radiosynthesis of (4-[18F]fluorophenyl)triphenylphosphonium (18FTPP)
via a one-step procedure is shown in Figure 1. The precursor for radio-
synthesis, 4-nitrophenyltriphenylphosphonium (1, NO2TPP) was first pre-
pared by following the procedure reported by Rieke et al.25 The diazonium
chloride intermediate was formed by slowly adding hydrochloric acid to 4-
nitroaniline at 08C. The formation of NO2TPP was obtained by the addition
of triphenylphosphine. Extraction with aqueous solution and precipitation
with sodium iodide (NaI) afforded pure precursor NO2TPP which was
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POTENTIAL IMAGING AGENT FOR MITOCHONDRIAL DYSFUNCTION
133
H2N
NO2
K18F, CH CN
18F
3
P
P
NO2
P
NaNO2, HCl, 0oC
Kryptofix222
3
1
F
I
P
F
Pd(OAc)2, p-Xylene
2
Figure 1. Synthetic scheme for (4-[18/19F]fluorophenyl)triphenylphosphonium
cation
confirmed by analytical HPLC, ESI-MS and NMR. 18FTPP was synthesized
by nucleophilic substitution reaction of NO2TPP and no-carrier-added
[18F]fluoride in the presence of potassium carbonate (K2CO3) and phase
transfer agent Kryptofix 222. After heating for 15 min at 1208C in the mineral
oil bath, the reaction solution was cooled and injected onto the semi-
preparative HPLC column to separate the labeled product. Under the HPLC
gradient described in the ‘Experimental’ section, the retention time of 18FTPP
and un-reacted NO2TPP were 26.0 and 24.5 min, respectively, which made the
separation of these two species feasible. Analytical HPLC analysis revealed
that the radiolabeled product exhibited identical retention time with a fully
characterized cold (4-[19F]fluorophenyl)triphenylphosphonium (FTPP) stan-
dard. The radiochemical purity of 18FTPP determined by analytical HPLC
was above 95%, and radiochemical yield for 18FTPP was 10–15% at end of
synthesis (EOS) with specific activity 576–715 Ci/mmol.
Experimental
No-carrier-added [18F]Fluoride was obtained from PETNET Pharmaceuticals,
Inc. (Palo Alto, CA). All other reagents were purchased from Sigma-Aldrich
Chemical Co. ESI-MS were performed by Vincent Coates Foundation Mass
Spectrometry Laboratory, Stanford University. NMR spectra were recorded
on a Varian 600 MHz instrument. Melting points were determined on an
Electrothermal’s TA 9100 digital melting point instrument (Barnstead/
Thermolyne, Dubuque, IA). HPLC was performed on a Dionex Summit1
HPLC system (Dionex Corporation, Sunnyvale, CA) equipped with a
170U 4-Channel UV-Vis absorbance detector and radioactive detector
(Carroll & Ramsey Associates, model 105S, Berkeley, CA). UV detection
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Z. CHENG ET AL.
wavelengths are 225 and 280 nm for all the experiments. Both semi-preparative
(Zorbax SB-C18, 9.4 mm  250 mm) and analytical (Dionex Acclaim1120
C18, 4.6 mm  250 mm) RP-HPLC columns were used. The mobile phase was
solvent A, 100 mM ammonium formate (HCO2NH4), and solvent B,
acetonitrile. CRC-15R PET dose calibrator (Capintec Inc., Ramsey, NJ)
was used for all radioactivity measurements.
Synthesis of 4-nitrophenyltriphenylphosphonium iodide
NO2TPP was prepared as a precursor for radiosynthesis of 18FTPP by the
method reported previously25. Briefly, 2.8 g (0.02 mol) 4-nitroaniline was
mixed with 10 ml concentrated hydrochloric acid at 08C. Then NaNO2
(0.02 mol, 1.38 g) dissolved in cold 10 ml H2O was added to the 4-nitroaniline
dropwise with stirring, followed by adding 5.6 g sodium acetate (NaOAc)
dissolved in water (20 ml). Triphenylphosphine (5.6 g) dissolved in 80 ml ethyl
acetate was then added dropwise to the reaction vial at 08C within 60 min.
After incubation at room temperature for 5 min, the reaction solution was
acidified by adding concentrated HCl (2 ml). The aqueous layer was then
washed twice using ether, and the ethyl acetate layer was extracted twice with
water. The aqueous solutions were then combined and NaI (2.2 g in 2 ml H2O)
was added to triturate the final product. The purity of 4-nitrophenyltriphe-
nylphosphonium iodide was over 95% as confirmed by analytic HPLC
(analytic column, flow rate: 1 ml/min, elution protocol starts with 95% A for
3 min, followed by a linear gradient to 15% A over 30 min, retention time:
23.4 min). Yield: 6.34 g (62%), pale brown solid; m.p. >2008C (lit25 2288C);
from the HMBC (multiple-bond CH correlation, 600 MHz, CDCl3) experi-
ment, we observe two aromatic hydrogens (8.57–8.58 ppm) coupled with C-2
and C-6 (127 ppm) carbons as doublet and two other aromatic hydrogens
(8.09–8.10 ppm) coupled with C-5, C-7 (137 ppm) as doublet of doublet with
integral values 1:1 accordingly (the carbon connected to NO2-group is
numbered as 1, and the other carbons in the same benzene ring are numbered
clockwise as 2–6, the same numbering system for carbon is applied for FTPP).
This indicates that NO2-group is in para position relative to phosphorus atom.
The rest of protons are combined in 7.6–8.1 ppm interval with integral value of
15 protons, thus it support the total integral ratio of 19 protons for compound
NO2TPP. ESI-MS (high-resolution): m/z calculated: 384.1153 (C24H19NO2P),
found: 384.1153.
Synthesis of (4-fluorophenyl)triphenylphosphonium formate
19FTPP was synthesized according to the method reported for synthesis of
(4-bromophenyl)triphenylphosphonium (Figure 1).26 Triphenylphosphine
(1.76 g, 6.72 mmol), 4-fluoro-iodobenzene (1.49 g, 6.72 mmol) and Pd(OAC)2
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(75 mg, 0.33 mmol, 5 mol%) in p-xylene (40 ml) were mixed together and
stirred at 130–1408C for 2 h. The color changed from yellow to brown and a
white precipitate formed. The solvent was removed in vacuo. 100 mg residue
was dissolved in CH3OH and purified by HPLC using semi-preparative
column (flow rate: 3 ml/min; elution protocol starts with 95% A for 3 min,
followed by a linear gradient to 15% A over 30 min, retention time: 23.8 min).
The purity of the product was analyzed by analytical HPLC (analytic column,
flow rate: 1 ml/min, same gradient as that for NO2TPP analysis, retention
time: 24.1 min, >95% purity). White solid, m.p. >2008C; three experiments,
1H NMR, HMBC and HSQC (heteronuclear single quantum correlation)
(600 MHz, CDCl3) were also performed for confirmation of the structure of
FTPP. From these experiments, we observe two protons (7.42–7.54, multiplet)
coupled with C-2 and C-6 (118 ppm) carbons and two other aromatic
hydrogens (7.68–7.75 ppm, multiplet) coupled with C-3 and C-5 (138 ppm),
which proves that fluorine-group is in para position relative to phosphorus
atom. The rest of protons are in the range of 7.58–7.67 ppm (multiplet, six
protons), 7.76–7.84 (multiplet, six protons), and 7.85–7.96 ppm (triplet, three
protons), supporting the total integral ratio of 19 protons for compound 2.
ESI-MS (high-resolution): m/z calculated: 357.1208 (C24H19FP), found:
357.1217.
Radiosynthesis and HPLC purification of (4-[18F]fluorophenyl)triphenylpho-
sphonium cation
[18F]fluoride was added to a pyrex glass reaction vessel containing 200 ml
25 mM potassium carbonate and Kryptofix 2.2.2. (3.0–4.0 mg) dissolved in
300 ml CH3CN. The solution was evaporated at 1208C by bubbling nitrogen
gas and the residue was dried by azeotropic distillation with acetonitrile
(3 Â 0.5 ml). To this anhydrous residue was added a solution of NO2TPP (1–
1.5 mg) in dry DMSO (0.32 ml). The reaction mixture was heated for 15 min at
1208C in an oil bath. The solution was cooled and injected onto a semi-
preparative HPLC column (the flow rate was 3 ml/min, with the mobile phase
starting from 28% solvent B (CH3CN) and 72% solvent A (0.1 M HCO2NH4
in water) (0–3 min) to 48% solvent B and 52% solvent A at 33 min, then going
to 85% solvent B and 15% solvent A (33–36 min), maintaining this solvent
composition for another 3 min (36–39 min) and returning to initial solvent
composition by 42 min). Pure [18F]FTPP, eluted off the column with a
retention time of 26 min, was collected in a small round bottle. The product
was dried in a rotary evaporator and was made isotonic with sodium chloride
and passed through a 0.22 mm membrane filter into a sterile multidose vial.
The product was found to be 495% radiochemically and chemically pure as
determined by analytical HPLC (same gradient as used for semi-preparative
HPLC; flow rate: 1.0 ml/min). The retention time for [18F]FTPP under
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analytical HPLC conditions was 25.5 min. Radiochemical yield ranged
between 10 and 15% (EOS; corrected for decay). Thus starting typically with
100 mCi of [18F]-fluoride ion, about 7 mCi of product, ready for injection, was
routinely obtained in 60 min.
Conclusion
In conclusion, radiosynthesis of 4-[18F]fluorophenyl)triphenylphosphonium in
high radiochemical purity was achieved through a one-step reaction. The yield
and specific radioactivity are sufficient for further biological evaluation of
18FTPP as a molecular probe for imaging mitochondrial dysfunction.
Acknowledgements
We would like to acknowledge funding support from the Doris Duke
Charitable Trust (S.S. Gambhir PI) and the Molecular Imaging Program at
Stanford (MIPS).
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