Synthesis of I-131 labelled 4-iodophenylacetic acid

Article abstract of DOI:10.1002/jlcr.1812

Phenylacetate has been reported to have a potent anti-proliferative and anti-differentiating effect in haematological malignancies and in solid tumours at non-toxic concentrations. This study is a preliminary investigation into the potential of 4-iodophen

Full text of DOI:10.1002/jlcr.1812

Research Article
Received 19 January 2010,
Revised 6 May 2010,
Accepted 3 June 2010
Published online 16 September 2010 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.1812
Synthesis of I-131 labelled 4-iodophenylacetic
acid
Zoltan Szucs,a Mike Sathekge,b Biljana Marjanovic-Painter,a
Ã
Judith Wagener,a Thato Sello,a Carl Wagener,a and Jan Rijn Zeevaartc
Phenylacetate has been reported to have a potent anti-proliferative and anti-differentiating effect in haematological
malignancies and in solid tumours at non-toxic concentrations. This study is a preliminary investigation into the potential
of 4-iodophenylacetic acid radiolabelled by 131I as a radiopharmaceutical equivalent. The radiolabelling by isotope
exchange gave a radiochemical yield of 5376%, and a radiochemical purity of 97.871.2% as qualified by HPLC. The
product contained 4% ester by-product and is suitable for studies in animals.
Keywords: biotracer; phenylacetic acid metabolism; prostate carcinoma; 131I
In patients with hormone-refractory prostate cancer and high-
grade glioma, Phase I trials of phenylacetate showed minimal
Introduction
Phenylacetic acid (Figure 1) and its deprotonated form at
physiological pH (phenylacetate) is an aromatic molecule and is
present in low concentration in human serum as a product of
phenylalanine metabolism. It is conjugated with glutamine in
the liver by phenyl-acetyl coenzyme A to form phenyl-acetyl-
glutamine, which is excreted in the urine.1
toxicity, and a partial response was observed at a serum
concentration of 2–10 Â 10À3 M.12,13
Increasing evidence points to phenylacetate having multiple
effects on gene expression and on regulatory proteins
responsible for its antineoplastic effects.14 Thus, phenylacetate
has been shown to down-regulate Bcl-2 and up-regulate bax/
p21 apoptosis-related genes in ovarian carcinoma cells and up-
regulate p21 in K-ras mutant MCF-7 ras breast cancer cell
lines.9,15 Phenylacetate also activates the human peroxisome
proliferator-activated receptors (PPAR) which belong to the
superfamily of nuclear steroid receptors such as retinoids,
vitamin D, and thyroid hormone receptors – all important
regulators of cell growth and differentiation.16,17 In all these
studies phenylacetate exerts its effects at clinically acceptable
serum concentration levels (2.5–10 Â 10À3 M).14
In recent years, the quest for an agent that can be used for
both the diagnosis and therapy of a disease is being addressed
world-wide. Most notable is the application of the synergism
between the chemistry of the isotopes of technetium (99mTc) and
rhenium (186Re and 188Re), in which the former isotope is used for
diagnosis and the latter for therapy of the disease.18,19
131I-labelled compounds fulfil the criteria of being both a diagnostic
and/or therapeutic agent. Although the g-energy of 131I is not ideal
(t1/2 =8.02 d; g-energy = 364 keV; b-energy = 0.610.8 MeV,19 it is
adequate for use in diagnostic studies using a high-energy
Phenylacetate has been reported to have a potent anti-
proliferative and anti-differentiating effect in haematological
malignancies and in solid tumours at non-toxic concentrations.
It has been used safely to treat children with inborn errors of
urea synthesis and also in patients with hyperammonaemia.1–3
Furthermore, it has been shown to inhibit tumor growth while
sparing normal tissue and to induce phenotypic reversion and
differentiation of malignant cells with some of the antiprolifera-
tive effect of phenyl-acetate in prostate carcinoma,4 melanoma,5
rhabdomyosarcoma,6 breast cancer,7 pancreatic adenocarcino-
10
ma,8,9 ovarian carcinoma,9 B-chronic lymphocytic leukemia
and medullablastoma11 cell lines.
H
CH
COOH
C
COOH
2
NH
2
Phenylacetic acid
Phenylalanine
aNecsa, Radiochemistry, Pretoria, South Africa
bUniversity of Pretoria/Pretoria Academic Hospital Pretoria, Pretoria, South Africa
I
CH
COOEt
I
CH
COOH
2
2
cCARST, North West University, Mafikeng Campus, PO Box 582, Pretoria 0001,
Mmabatho, South Africa
4-iodophenylacetic acid
Ethyl 2-(4-iodophenyl)acetate
*Correspondence to: Jan Rijn Zeevaart, CARST, PO Box 582, Pretoria 0001,
South Africa.
E-mail: janrijn.zeevaart@necsa.co.za
Figure 1. The structures of 4-iodophenylacetic acid, phenylacetic acid, phenyla-
lanine and ethyl 2-(4-iodophenyl)acetate.
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Z. Szucs et al.
collimator. The b-energy is sufficient to deliver an internal
The Agilent HPLC-MS instrument was equipped with a 1200
therapeutic radiation dose to the target organ or carcinoma. A series quaternary and an isocratic pump, a single quad mass
further advantage of 131I is that the biodistribution of the spectrometer, a 1200 series diode array and multiple wave-
labelled phenylacetic acid and radiation dose of a therapeutic length detector and connected to gamma and beta detectors
dose to the target organ or carcinoma can be evaluated by (Raytest, Germany). The column was the same as for the Varian
imaging the emitted g-radiation. These diagnostic and ther- instrument.
apeutic effects are obtained by using extremely small amounts
of the agent, as the effects observed are determined by the
Radiosynthesis of 4-[131I] iodophenylacetic acid
radioactivity of the compound and not by the mass of material
used. Radiolabelling with iodine also affords the options of 123I
About 0.3 mg (1 mmol) cold 4-iodophenylacetic acid was
(SPECT imaging with t1/2 = 13.3 h; g-energy = 159 keV), 124I (PET
dissolved in 10 ml ethanol in a 1 ml vial. Freshly prepared
imaging with t1/2 = 4.2 d; b1-energy = 1.5 MeV) and therapeutic
ascorbic acid (0.5 mg, 3 mmol) in 20 ml water, 0.6 mmol of CuCO3
catalyst and 2.1 mg (11 mmol) citric acid was dissolved in 20 ml
125I (t1/2 = 59.4 d; Auger electrons).
To our knowledge radioiodinated 4-iodophenylacetic acid has
never been synthesized before and the biodistribution of 131I
labelled phenylacetic acid has never been evaluated in
experimental animals or humans. This novel synthesis attempt
was based on the general reaction parameters for Cu(I) assisted
radiohalogenation as proposed in the literature.20 Because of
the closeness of the structure of 4-iodophenylacetic to that
of phenylacetic acid, as well as the reported biological activity
of phenylacetic acid, it is hypothesized that useful infor-
mation of the biodistribution of the 131I labelled agent can be
obtained from experimental animals, especially with regards to
its uptake in neoplastic versus normal tissue. The results
presented here describe the synthesis of 4-[131I] iodophenyl-
acetic acid as a first step in assessing its potential as diagnostic
imaging and/or therapeutic agent for the treatment of
neoplastic conditions.
water and transferred to the 1 ml vial. The reaction mixture was
diluted with 50 ml water and 40 ml ethanol. The reaction contents
were purged with argon gas, a micro-magnetic stirrer bar was
introduced and 100 ml (4150 MBq) of the radioactive 131I was
added. The reaction vial was hermetically closed and the isotope
exchange reaction was carried out at 1501C for one hour with
vigorous stirring. The reaction mixture was left to cool to room
temperature and the reaction contents were transferred to the
AG-MP-1M column. The reaction vial was washed with 2 Â 100 ml
ethanol, which was also pushed through the AG-MP-1M column.
The column was washed additionally with 1 ml of a 1:1 mixture
of saline and ethanol. The eluates were collected and gently
evaporated under argon gas flow down to 1 ml. The residue was
diluted by saline to 3 ml and filtered through a 0.22 mm Millex GV
(Millipore) sterile filter into a sterile, pyrogen-free bottle with a
silicon-based septum. A polyethylene film was placed between
the septum and the solution. Quality control was carried out by
the Varian HPLC equipped with the Agilent Eclipse XDB-C18
column. Separation was carried out by a gradient method, using
ethanol and 0.01 M ortho-phosphoric acid in water as solvents.
The ratio of the solvents was 70/30% (ethanol/water) for the first
4 min, then it was decreased to 40/60% over a 5 min period, kept
at 40/60% for another 5 min, after which it was increased back to
70/30% over a 1 min period and kept at this level until the end
of the chromatographic elution. The flow rate was 1.0 ml/min.
About 20 ml of the final product solution was injected. The
analysis was done at room temperature. The total time of
analysis was 25 min. The radioactivity of the sample was
detected by a 30 ml flow-cell and the chemical composition by
the UV-detector at 210 nm.
Experimental
General
All chemicals and solvents used were of AR or high performance
liquid chromatography (HPLC) grade purity (Merck, Darmstad,
Germany, Sigma-Aldrich). 4-Iodophenylacetic acid was pur-
chased from Alfa Aesar L13345 with a certified purity of 97%.
The 131I solution was supplied by NTP Radioisotopes Pty. Ltd,
Pretoria, South Africa, in a 0.05 M sodium hydroxide solution
without thiosulphate or buffer solution. The fission 131I had a
radiochemical purity of 495%, a radionuclidic purity of 99.9%
and a specific activity of 294 TBq 131I/mmol I (62 Ci/mg). The AG-
MP-1M Biorad anion exchanger resin was treated with 1 M
sodium hydroxide, washed with water and packed in the
column one day before the synthesis. The solution of ascorbic
acid was freshly prepared just before the synthesis.
Two HPLC instruments (a Varian and an Agilent coupled to an
MS detector) were used for separation and identification of
components. The Varian HPLC system consisted of a Zorbax
Eclipse XDB C-18 4.6 Â 150 mm, 5 mm particle size column,
Varian ProStar model 230 dual pumps, a synchronized Rheodine
7725i injector, an in-line Varian ProStar model 325 UV and VIS
detector, and a single sodium iodide crystal flow radioactivity
detector (Raytest Gabi.). Varian HPLC chromatograms were
recorded by a Varian Star 800 channel control/interface module
connected to a computer running Galaxie Chromatographic
Data System software. The radioactive chromatograms were
obtained using the Gina Star 4.07 version of software. The
radioactivity was measured by a Capintec CRC-15 bETA Radio-
isotope Dose calibrator meter and by g-spectrometry with the
Genie-2000 3.1 Gamma Analyzer Software, Canberra.
Radiosynthesis of ethyl 2-(4-[131I] iodophenyl)acetate
The possibility exists that the ester of 4-iodophenylacetic acid
will form as a by-product during the above synthesis. To
establish the radiochemical purity of the 4-[131I] iodophenyla-
cetic acid by HPLC analysis, the retention time of the ester
(ethyl 2-(4-[131I] iodophenylacetate) had to be determined.
Therefore, ethyl 2-(4-[131] iodophenyl)acetate was synthesized as
follows.
About 100 ml of 131I labelled 4-iodophenylacetic acid
solution was introduced into a solution of ethanol (400 ml)
and concentrated sulphuric acid (10 ml) in a reaction vial.
The reaction mixture was heated in a pre-heated (1001C)
lead pot for 1.5 h. The reaction mixture was left to cool to
room temperature and injected in the HPLC-MS for
separation and identification. The same HPLC separation
method and conditions as described in the earlier section were
used.
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Z. Szucs et al.
drops, while a too high temperature causes decomposition of the
4-iodophenylacetic acid. A freshly prepared solution of ascorbic
acid is also essential. When the ascorbic acid was prepared the day
before, a lower yield was recorded. The reaction time could be
reduced from the 12 h initially used to 1 h, without changing the
yield. The ratio of substrate to catalyst was investigated, but no
detrimental effect was found when a different ratio is used than
reported herein. The precursor 4-iodophenylacetic acid was added
to the reaction mixture in solid form taken directly from the
Results
Radiosynthesis of 4-[131I] iodophenylacetic acid
During the development phase of the labelling process, it was
found that an inert atmosphere is key to the success of the
synthesis. When oxygen is allowed in the reaction vial, the yield
drops to less than 20%. Furthermore, the temperature should be
kept between 145 and 1551C. If the temperature is lower, the yield
Figure 2. UV chromatograms of 4-iodophenylacetic acid (retention time ꢀ12 min, top), ethyl 2-(4-iodophenyl)acetate (retention time ꢀ16 min, middle) and
radiochromatogram of radiolabelled 4-iodophenylacetic acid and ethyl 2-(4-Iodophenyl)acetate.
www.jlcr.org
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J. Label Compd. Radiopharm 2011, 54 54–58

Z. Szucs et al.
100
80
60
40
20
0
100
200
300
m/z
Figure 3. Mass spectrum of ethyl 2-(4-iodophenyl)acetate.
refrigerator. The ratio of the reaction constituents was chosen 4-iodophenylacetic acid will not adversely interfere with the
according to Gysemans et al.20 The reaction mixture was pushed biological processes in the rat. The amount of phenylacetic acid
through an AG-MP-1M anion exchanger column to separate the in normal tissue is 16.8 mg/ml,22 which is also an order of
un-reacted anionic 131I. Six labelling procedures were performed magnitude higher than the amount of 4-iodophenylacetic acid
with an average radiochemical purity of 97.871.2% and yield of that will be injected. This implies that the biodistribution of the
5376%. The radiochemical purity of the run in which the HPLC- tracer will not be influenced by its metabolic product, due to
MS analysis was used (using only 150 MBq of 131I) was 96% equilibrium with phenylacetic acid already present in the body.
(Figure 2). The results presented herein further refer to this For therapeutic doses, the preparation method will have to be
particular run, which had a lower yield and radiochemical purity, adapted to make use of a direct labelling method, such as that
probably due to the lower activity of 131I used. The activity of the described by Machulla et al.,23 to increase the specific activity.
product was 64 MBq. The radiochemical yield was 43% (decay For the preparations used for animal studies, the percentage
corrected). The calculated specific activity (from HPLC analysis) ethanol (that is higher than 10% in the current labelling
was 63.7 MBq/mmol (6.6Ci/g).
procedure) will be lowered by reducing the volume further
during the evaporation step using Ar. This has been demon-
strated in one of the runs, where the volume was reduced to
100 ml without causing a lower radiochemical purity or yield.
Radiosynthesis of ethyl 2-(4-[131I] iodophenyl) acetate
After the synthesis step, 20 ml of product solution was injected
into the HPLC-MS. The separation conditions as described in the
previous section were used, except for orthophosphoric acid,
which was exchanged with the same concentration of formic
acid to avoid undue suppression of the MS signal. The retention
time of ethyl 2-(4-[131I] iodophenyl) acetate was approximately
16 min, compared to the retention time of the 4-[131I]
iodophenylacetic acid of approximately 12 min (Figure 2). (Please
note the different total analysis times for the three chromato-
grams.) The positive ES mass spectrum of the cold compounds
shows ions at [M1H]1 m/z 290.1, [M1Na]1 m/z 312.8, [M1H-
COOCH2CH3]1 m/z 216.9 and [COOCH2CH3]1 m/z 74.1. The MS
spectrum (Figure 3) confirmed that the product was the ester.
Conclusions
4-[131I]-Iodophenylacetic acid was successfully prepared,
although 4% of the ester by-product was present. These data
indicate that 4-[131I] iodophenylacetic acid shows promise for
in vivo investigations and therapy.
Acknowledgements
The authors thank Prof. B. J. Meyer for suggesting this study
before his untimely death and dedicate this paper to his
memory. Thanks to Dr Knoesen for the initial exploratory work.
The authors thank Necsa for permission to publish this work.
Discussion
References
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of magnitude higher than the amount that will be injected and
one can therefore assume that the 0,075 mg/kg injected
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