Novel synthesis of [18F]-fluorobenzene and pyridinecarbohydrazide-folates as potential PET radiopharmaceuticals

Article abstract of DOI:10.1002/jlcr.1022

In an attempt to visualize folate receptors that over-express on many cancers, [¹⁸F]-fluorobenzene and pyridine carbohydrazide-folates were synthesized using two different synthetic approaches starting from nucleophilic displacement reactions on ethyl-trimethylammonium-benzoate and pyridine carboxylate precursors. The intermediates ethyl [¹⁸F]-fluorinated benzene and pyridine esters were reacted with hydrazine to produce the [ ¹⁸F]-fluorobenzene and pyridine carbohydrazides followed by coupling with NHS-folate 11 in the first approach. Whereas hydrazide-folate 5 was reacted with 2,5-dioxoazolidinyl [¹⁸F]-fluorobenzenecarboxylate in the second approach. Based on starting [¹⁸F]-fluoride, radiochemical yields and synthesis times were found to be around 80% (45 min) and 35% (80 min) for the first and the second approaches, respectively. The first synthetic approach holds considerable promise as a rapid and simple method for the radiofluorination of folic acid with high radiochemical yield and short time. Copyright

Full text of DOI:10.1002/jlcr.1022

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2006; 49: 125–137.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.1022
Research Article
Novel synthesis of [18F]-fluorobenzene and
pyridinecarbohydrazide-folates as potential PET
radiopharmaceuticals
I. Al Jammaz*, B. Al-Otaibi, S. Okarvi and J. Amartey
Cyclotron and Radiopharmaceuticals Department, King Faisal Specialist Hospital and
Research Centre, P.O. Box 3354, Riyadh 11211, Kingdom of Saudi Arabia
Summary
In an attempt to visualize folate receptors that over-express on many cancers, [18F]-
fluorobenzene and pyridine carbohydrazide-folates were synthesized using two
different synthetic approaches starting from nucleophilic displacement reactions on
ethyl-trimethylammonium-benzoate and pyridine carboxylate precursors. The inter-
mediates ethyl [18F]-fluorinated benzene and pyridine esters were reacted with
hydrazine to produce the [18F]-fluorobenzene and pyridine carbohydrazides followed
by coupling with NHS-folate 11 in the first approach. Whereas hydrazide-folate 5 was
reacted with 2,5-dioxoazolidinyl [18F]-fluorobenzenecarboxylate in the second
approach. Based on starting [18F]-fluoride, radiochemical yields and synthesis times
were found to be around 80% (45 min) and 35% (80 min) for the first and the second
approaches, respectively. The first synthetic approach holds considerable promise as a
rapid and simple method for the radiofluorination of folic acid with high
radiochemical yield and short time. Copyright # 2005 John Wiley & Sons, Ltd.
Key Words: 18F-fluorination; 18F-fluorobenzene; 18F-fluoropyridine; 18F-fluorohy-
drazide; 18F-fluorofolic acid
Introduction
Advancement of scintigraphic tumor imaging is highly determined by
development of more tumor-specific radiotracers. Receptor-targeted radio-
tracers have shown promises in improvement of the specificity and sensitivity
*Correspondence to: Ibrahim Al Jammaz, Cyclotron and Radiopharmaceuticals Department, King Faisal
Specialist Hospital and Research Centre, P.O. Box 3354, Riyadh 11211, Kingdom of Saudi Arabia.
E-mail: jammaz@kfshrc.edu.sa
Contract/grant sponsor: Research Center of the King Faisal Specialist Hospital & Research Center;
contract/grant number: RAC # 2040027
Copyright # 2005 John Wiley & Sons, Ltd.
Received 15 March 2005
Revised 5 June 2005
Accepted 15 October 2005

126
I. AL JAMMAZ ET AL.
of nuclear medicine imaging procedures. Membrane-folic acid receptor is a
glycosylphospstidylinositol protein that overexpressed in approximately 100%
of serious ovarian adenocarcinomasa and various epithelial cancers
including cervical, colorectal and renal cancers.1,2 Meanwhile, this receptor
is highly restricted in most normal tissues which make these tumors as
excellent candidates for molecular targeting through the folate receptor
system. The vitamin folic acid has been mainly conjugated with macro-
molecules and low molecular weight chelates covalently via its gamma
carboxylate of the glutamic acid fragment.3–5 These conjugates have
considerable success for delivering various bioactive agents to folate-
receptor-positive tumor cells. In addition, folate-receptor has been successfully
targeted with folate–chelate conjugates through alpha carboxylate.6 In the last
decade considerable number of radiolabeled folate–chelate conjugates were
synthesized and evaluated for tumor imaging. These include 67Ga–deferox-
amine–folate (67Ga–DF–folate)5,7,8 and 111In–diethylenetriamine pentaacitic
acid (111In–DTPA–folate).9,10 These radiolabeled folate conjugates appeared
to be feasible in targeting tumor folate receptors in vivo. However, 111In–
DTPA–folate provided tumor-selective radionuclide delivery in vivo which has
led to the initial clinical study for imaging of ovarian malignancy and
differentiation between malignant and benign ovarian masses.11 Due to its
favorable physical properties and availability, numerous 99mTc–folate
conjugates have also been synthesized and biologically evaluated.12–15 For
example, the folate conjugates of 99mTc–6–hydrazinonicotinamidohydrazido
(HYNIC), 99mTc–ethylenedicysteine (EC), 99mTc–EC20 and 99mTc–DTPA
have shown capability of imaging folate receptor positive tumors. With the
increased use of positron emission tomography (PET), there has been great
interest in the development of positron emitters radiopharmaceuticals for
earlier detection and characterization of cancer, molecular assessment of
treatment effects and more fundamental understanding of the disease
process.16 Recently, Ga–DF–folate was radiolabeled with two positron-
emitting isotopes of gallium (66Ga 9.5 h half-life and 68Ga 68 min half-life) in
high radiochemical purity.17 Imaging of folate-receptor-positive tumor-
bearing mouse with 66Ga–DF–folate was feasible, however, high positron
(4.15 MeV) and gamma energies (1.04, 2.75 MeV) of this radionuclide may
hamper its application in humans. On the other hand 18F rather than 66Ga or
68Ga represent the ideal nuclear and chemical characteristics for PET
diagnostic imaging applications. These include relatively long half-life
(110 min), production in Curie quantities, low positron energy (0.64 MeV)
which result in low radiation doses and short range (2.3 mm) in tissues,
insignificant sterical changes and physiological properties when introduced to
metabolic substrates.16 As part of our ongoing research effort to develop
prosthetic precursors for radiofluorination of bioactive molecules, we here
Copyright # 2005 John Wiley & Sons, Ltd.
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NOVEL SYNTHESIS OF [18F]-FLUOROBENZENE
127
report the synthesis of [18F]-fluorobenzene and pyridine carbohydrazide-
folates using two different synthetic approaches.
Results and discussion
Chemistry
The reference 4-fluorobenzenecarbohydrazide 3 was synthesized as outlined
%
in Scheme 1. Scheme 2 was used to synthesize the reference compounds
2-[(4-{[(2-amino-4-oxohydropteridin-7yl)methyl]amino}phenyl)carbonylamino]-
4-{N-[(4-fluorophenyl)carbonylamino]carbamoyl}butanoic acid: (4-fluoroben-
zenecarbohydrazide-folate, 6) and 2-[(4-{[(2-amino-4-oxohydropteridin-7yl)-
%
CH3
OH
NH NH2
O
O
O
O
EtOH / H2SO4
Reflux
NH2NH2
EtOH/ reflux
F
F
F
1
2
3
Scheme 1. Synthesis of the reference 4-fluorobenzene-1-carbohydrazide ð3Þ
%
OH
O
O
5
N
TSTU/TEA
Folate-NHNH2 / TEA
O
DMSO / 50°C
90°C
HO
O
O
O
O
F
19
NH
7
F
O
21
22
NH
N
1
4
O
9
N
N
N
H
HN
OH
NH
H2N
O
O
O
R
N
TSTU/TEA
DMSO / 50°C
Folate-NHNH2 / TEA
5
O
90°C
O
N
6 , R=
9 , R =
O
F
N
F
N
F
F
7
8
Scheme 2. Synthesis of the reference fluorobenzene and pyridinecarbohydrazide-
folates ð6; 9Þ
% %
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128
I. AL JAMMAZ ET AL.
methyl]amino}phenyl)carbonylamino]-4-{N-[(2-fluoro (4-pyridyl))carbonyla-
mino]carbamoyl} butanoic acid: (2-fluoropyridine-4-carbohydrazide-folate,
9). These compounds were characterized by physical, chromatographic and
%
spectral data and were in agreement with the anticipated structures (see the
supporting information).
Compound 3 was obtained by converting the acid 1 to the ethyl ester 2
%
%
%
followed by addition of hydrazine hydrate. After subjection to preparative
thin-layer chromatography (TLC), yellowish crystals were separated in 57%
yield. This method was a modification of the previously published
procedure.18 The calculated molecular mass for C7H7FN2O was 154.14 which
was in agreement with the found ES-MS ½M þ 1ꢀþ ¼ 155.
Since, derivatization of the folate at the g-carboxyl moiety revealed that
its affinity for the folate receptors remains unaltered and comparable to
folic acid.5 The compound 2-[(4-{[(2-amino-4-oxohydropteridin-7yl)methyl]
amino}phenyl)carbonylamino]butanoic acid: (hydrazide-folate (g), 5) was
%
prepared to serve as a linkage between folic acid and fluorinated compounds.
This was confirmed by 1H NMR spectroscopy as protons on the
ethylene group of the g moiety shifted to lower field. The calculated
molecular mass for high-pressure liquid chromatography (HPLC)-purified
hydrazide-folate (g) 5 was 455.43. This was in agreement with the attained
ES-MS ½M þ 1ꢀþ ¼ 456, indicating the desired 1:1 conjugation ratio.
Chemical purity was found to be greater than 98% as determined by
analytical HPLC.
The amide-linked compound 6 was obtained as a yellow powder by
%
conjugation of 2,5-dioxoazolidinyl 4-fluorobenzoate 4 with the hydrazide-
%
folate (g) 5. The overall yield was greater than 90%. The calculated molecular
%
mass for C26H24FN9O6 was 577.52 and was in agreement with the found
ES-MS ½M þ 1ꢀþ ¼ 578 as well as the observed MS fragmentation pattern.
The base peak m=z ¼ 209, corresponding to the g moiety of the conjugated
folate (–CH2–CH2–CO–NH–NH–Ar–F).
The intermediate 2,5-dioxoazolidinyl 2-fluoropyridine-4-carboxylate 8 was
%
obtained as white crystals by the activation of the acid 7 using O-(N-
%
succinimidyl)-tetramethyluronium tetrafluoroborate (TSTU). Chemical yield
was 47% and the calculated molecular mass for C10H7FN2O4 was 238.17. This
was in agreement with the found ES-MS ½M þ 1ꢀþ ¼ 239.
Similarly, compound 9 was obtained as a yellow powder when compound 8
%
%
was conjugated with hydrazide-folate (g) 5 in basic condition. Chemical yield
%
was greater than 90% and the found ES-MS ½M þ 1ꢀþ ¼ 579 (calculated
578.51). The base peak m=z ¼ 211, correspond to the g moiety of the
conjugated folate (–CH2–CH2–CO–NH–NH–Ar–F). Chemical purity of
g-isomers of both reference compounds 6 and 9 as quantified by HPLC were
%
%
found to be greater than 98%.
Copyright # 2005 John Wiley & Sons, Ltd.
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NOVEL SYNTHESIS OF [18F]-FLUOROBENZENE
129
Radiochemistry
The synthetic approaches for preparation of [18F]-6 and [18F]-9 (Schemes 3 and
%
%
4) entailed several sequence of reactions. The key precursors 4-N,N,N-
trimethylammonium ethylbenzoate and pyridine carboxylate triflates 10 and
12 were treated using classical catalyzed nucleophilic no-carrier-added
radiofluoride described by Amartey et al.18 and Haka et al.19 The
intermediates ethyl 4-[18F]-fluorobenzoate and 2-[18F]-fluoro-4-pyridine
CH3
CH3
O
NH NH2
O
O
O
Kryptofix 222 / K2CO3
NH2NH2
EtOH/ 90oC
H18F / CH3CN / 90oC
CH3
O
N+
18F
18F
CH3
H3C
85%
85 %
10
11
NH-NH-Folate
Folate-NHS / TEA
80%
DMSO / 55oC
O
5
Folate-NHNH2 / TEA
40%
DMSO / 55oC
18F
CH3
O
O
N
Kryptofix 222 / K2CO3
O
H18F / CH3CN / 90oC
CH3
O
O
O
N+
18F
45 %
CH3
H3C
10
Scheme 3. Radiosynthetic approaches for preparation of 4–[18F]-fluorobenzene-
carbohydrazide-folate ([18F]-6)
%
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130
I. AL JAMMAZ ET AL.
CH3
CH3
NH NH2
O
O
O
Kryptofix222 / K2CO3
H18F / CH3CN / 90°C
O
NH2NH2
EtOH/ 90°C
13
O
N
N
N
18F
18F
90%
85%
N+ CH3
CH3
H3C
11
12
O
NH-NH-Folate
Folate-NHS / TEA
DMSO / 55°C
18F
80%
N
Scheme 4. Radiosynthesis of 2–[18F]-fluoropyridine-4-carbohydrazide-folate
([18F]-9)
%
carboxylate were extracted by ether and followed by passing through Sep-Pak
silica cartridge to remove polar impurities before conversion to either the
corresponding acids or hydrazides. In the first approach, the radiofluorinated
ethyl benzene and pyridine substrates were reacted with 5 ml of hydrazine
hydrate to give the corresponding 4-[18F]-fluorobenzenecarbohydrazide ([18F]-
3) and 2-[18F]-fluoropyridine-4-carbohydrazide ([18F]-13) in almost quantita-
%
tive yield (>98%) for the former and higher than 90% for the latter. These
results were determined by HPLC and confirmed by TLC silica gel (TLC-SG).
The Rf-values of [18F]-3 and [18F]-13 were 0.6 and 0.2, respectively, in TLC-SG
%
using ethyl acetate as a mobile phase, whereas retention times as determined
by HPLC found to be 9.7 and 3.5 min, respectively. A typical radio-
chromatograms are shown in Figure 1. The [18F]-3 and [18F]-13 intermediates
%
were reacted with 2-[(4-{[(2-amino-4-oxohydropteridin-7yl)methyl]amino}-4-
[(2,5-dioxoazolidinyl)oxycarbonyl]butanoic acid (NHS-folate 11) in DMSO at
pH 9 and reaction mixtures were then purified using Sep-Pak silica cartridge
followed by elution with saline to give the [18F]-6 and [18F]-9 in excellent yields
%
%
ready for biological studies. The overall radiochemical yields for both were
greater than 80% (based on starting [18F]-fluoride) with total synthesis time of
approximately 45 min. Radiochemical purities of the [18F]-6 and [18F]-9 were
%
%
always greater than 97% as determined by HPLC and confirmed by TLC-SG.
Copyright # 2005 John Wiley & Sons, Ltd.
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NOVEL SYNTHESIS OF [18F]-FLUOROBENZENE
131
Figure 1. HPLC chromatograms of [18F]-fluorobenzene and pyridine carbo-
hydrazide intermediates, [18F]-3 (A) and [18F]-13 (B)
%
Figure 2. HPLC chromatograms of [18F]-fluorobenzene and pyridine carbo-
hydrazide-folates, [18F]-6 (A) and [18F]-9 (B)
%
%
Retention times for both [18F]-6 and [18F]-9 were 9.5 and 10.6 min,
%
%
respectively, as shown in the radiochromatograms in Figure 2.
In the second approach, the resulted ethyl 4-[18F]-fluorobenzoate ([18F]-2)
%
was converted to the corresponding acid then activated with TSTU to form the
intermediate [18F]-4. This prosthetic intermediate was used to label the
%
hydrazide-folate (g) 5 in DMSO to give the folate conjugate [18F]-6. Work up
%
%
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I. AL JAMMAZ ET AL.
of this product by Sep-Pak silica column gave consistently [18F]-6 in
%
radiochemical purity greater than 97%. However, the overall yield was nearly
35% with total synthesis time of about 85 min.
Although, we have started with the purified hydrazide-folate (g) 5 which was
%
coupled with [18F]-4 in the second approach. Yet, the final product ([18F]-6) of
%
this approach was found to be identical to what have been obtained in the first
approach. In addition, the absence of a [18F]-folate in the radioactivity
chromatogram (7–8 min) implies that whether pure g folate or a mixture of
a and g folate conjugates are used for radiofluorination, radiochemical purity
of the [18F]-fluorofolates (g) were always better than 97% and other impurities
(53%) represent either 4-[18F]-fluorobenzoic acid or 2-[18F]-fluoropyridine-4-
carboxylic acid. That could be attributed to readily availability and less steric
hindrance of the gamma moiety compare to the alpha ones. In addition, the
measured specific activity for both [18F]-6 and [18F]-9 were >300 mCi/mmol.
%
%
Hence, the radiofluorinated folate conjugates would be suitable for
biochemical studies such as radioligand binding assays.
The first synthetic approach in comparison with the second one appears to
be advantageous and holds considerable promise in the synthesis of [18F]-
fluorofolate conjugates (g) in high radiochemical yield (>80%, based on
starting [18F]-fluoride), shorter synthesis time (45 min), less laborious way and
amenable for automation.
Moreover, the calculated partition coefficient for [18F]-6 and [18F]-9 were
%
%
found 0.38 ꢁ 0.02 and 0.14 ꢁ 0.01, respectively, representing a low lipophi-
licity of these compounds.
Experimental
The chemicals used in the study were all analytical reagent grade purchased
from Aldrich and were used without further purification unless stated.
Acetonitrile was kept over molecular sieves. Sep-Pak cartridges were
purchased from Waters-Millipore. TLC-SG sheets were purchased from
Gelman Sciences Inc. HPLC analysis was carried out on Econosil C-18
reversed phase columns (semipreparative, 250 mm ꢂ 10 mm or analytical,
250 mm ꢂ 4.6 mm). The solvent system used for the latter was non-linear
gradient (eluant A: water with 0.1 TFA; eluant B: acetonitrile/water, 3/1 v/v
with 0.1% TFA; gradient: 0–90% B, 90–90% B and 90–10% B over 10 min
each at flow rate of 1.5 ml/min). A Jasco chromatographic system equipped
with a variable wavelength ultraviolet monitor and in tandem with a Canberra
flow through radioactivity detector was used. Ultraviolet absorption was
monitored at 254 nm. Chromatograms were acquired and analyzed using
BORWIN software. Melting points were determined on a Thomas Hoover
Unimelt capillary melting point apparatus. Elemental analyses were performed
Copyright # 2005 John Wiley & Sons, Ltd.
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NOVEL SYNTHESIS OF [18F]-FLUOROBENZENE
133
by ResTech, Riyadh. Mass spectroscopy was run on Quattra electrospry mass
spectrometer (ES-MS). NMR was run on JEOL (400 MHz) Spectrometer.
4-Fluorobenzenecarbohydrazide 3
%
Compounds 2, 4, 10 and 12 in Schemes 1–4 were all synthesized utilizing the
% %
methods reported by Amartey et al.,18 Haka et al.19 and Wester et al.20
Compound 2, (230 mg, 1.37 mmol) was placed in 25 ml flask containing
%
ethanol (3 ml) followed by the addition of hydrazine mono-hydrate (100 ml,
2.05 mmol). Mixture was refluxed for over-night then excess of hydrazine and
volatile components were removed under reduced pressure. The yellowish
residue was re-solublilized in acetone (2 ml) and subjected to preparative TLC.
The desire product 3 was separated as yellow crystals (57% yield) using
%
ethylacetate/methanol (1/1) as eluant before drying under vacuum. Melting
point=121–1238C and the found ES-MS [M+1]+=155. Analysis calculated:
C, 54.90; H, 4.61; N, 18.29. Found: C, 54.44; H, 4.23; N, 17.95. H NMR
1
(CDCl3): d 8.06–8.12 (m, 2H, H1), d 7.10–7.17 (m, 2H, H2), d 1.25 (s, 2H,
NH2).
2-[(4-{[(2-Amino-4-oxohydropteridin-7yl)methyl]amino}phenyl)carbonylami-
no]butanoic acid: hydrazide-folate (g) 5
%
Starting from the folic acid and via the 2-[(4-{[(2-amino-4-oxohydropteridin-
7yl)methyl]amino}-4-[(2,5-dioxoazolidinyl)oxycarbonyl]butanoic acid (NHS-
folate 11) route, hydrazide-folate 5 was prepared and purified using methods
%
reported by Wang et al.9 and Guo et al.13 The g-isomer of hydrazide-folate was
separated from the a-isomer by HPLC on semipreparative C-18 column using
linear gradient (eluant A: water with 0.05% TFA; eluant B: acetonitrile;
gradient: 0–15% B over 40 min at flow rate of 5 ml/min). The elution time for
hydrazide-folate (g) and hydrazide-folate (a) were 26 and 34 min, respectively.
The fraction containing hydrazide-folate (g) was lyophilized and stored at
ꢃ158C. Melting point ꢄ2508C (decomposed) and the attained ES-MS
1
[M+1]+=456. H NMR (DMSO): d 8.64 (s, 1H, H7), d 7.59 (d, 2H, Ar),
d 6.63 (d, H2, Ar), d 4.47 (d, 2H, H9), d 4.33 (dd, 1H, H19), d 2.20 (m, 2H,
H22), d 1.90 (m, 2H, H21).
2-[(4-{[(2-Amino-4-oxohydropteridin-7yl)methyl]amino}phenyl)carbonylami-
no]-4-{N-[(4-fluorophenyl)carbonylamino]carbamoyl}butanoic acid 4-fluoro-
benzenecarbohydrazide-folates 6
%
The hydrazide-folate (g) (20 mg, 44 mmol) was dissolved in DMSO (2 ml)
followed by the addition of triethylamine (TEA) (11 ml, 66 mmol). 2,5-
dioxoazolidinyl 4-fluorobenzoate 4 (31 mg, 132 mmol) was then added and
%
mixture stirred while shielded from light at 508C for 3 h. The product was
precipitated by addition of acetonitrile (2 ml), centrifuged and then washed
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I. AL JAMMAZ ET AL.
several times with diethyl ether before drying into yellow powder under
vacuum and storing at ꢃ158C (83% yield). Melting point=ꢄ1998C
(decomposed) and the found ES-MS [M+1]+=578. Analysis calculated: C,
1
54.17; H, 4.20; N, 21.87. Found: C, 54.51; H, 4.32; N, 21.21. H NMR
(DMSO): d 8.65 (s, 1H, H7), d 8.09 (m, 2H, Ar–F), d 7.65 (d, 2H, Ar), d 6.65
(d, H2, Ar), d 4.47 (d, 2H, H9), d 4.30 (dd, 1H, H19), d 2.05 (m, 2H, H22), d
1.91 (m, 2H, H21).
2,5-Dioxoazolidinyl 2-fluoropyridine-4-carboxylate 8
%
To a mixture of 2-fluoropyridine-4-carboxylic acid 7 (50 mg, 0.35 mmol) and
%
TEA (100 ml, 0.71 mmol) dissolved in acetonitrile (300 ml), O-(N-succinimidyl)-
TSTU (128 mg, 0.43 mmol) was added and heated in a heating block for 1 h at
908C. The reaction mixture was diluted with hexane/ethyl acetate 8/2 v/v (1 ml)
and passed through a Sep-Pak silica cartridge. Then the product eluted by
hexane/ethyl acetate 8/2 v/v (10 ml). After solvent was evaporated to dryness,
white crystals separated and dried under vacuum (47% yield). Melting
point=121–1238C and the found ES-MS [M+1]+=239. Analysis calculated:
1
C, 50.64; H, 2.97; N, 11.81. Found: C, 50.11; H, 2.52; N, 11.99. H NMR
(CDCl3): d 10.2 (d, 1H, H6), d 9.34 (d, 1H, H5), d 9.23 (s, 1H, H3), d 3.0 (s, 4H,
NHS).
2-[(4-{[(2-Amino-4-oxohydropteridin-7yl)methyl]amino}phenyl)carbonylamino]-
4-{N-[(2-fluoro(4-pyridyl))carbonylamino]carbamoyl}butanoic acid: 2-fluoro-
pyridine-4-carbohydrazide-folates 9
%
The hydrazide-folate (g) (10 mg, 22 mmol) was dissolved in DMSO (1 ml)
followed by the addition of TEA (6 ml, 33 mmol). To this mixture, compound 8
%
(15 mg, 65 mmol) was added and stirred while shielded from light at 508C for
3 h. The product was precipitated by addition of acetonitrile (2 ml),
centrifuged, washed several times with diethyl ether and dried into yellow
powder under vacuum then stored at ꢃ158C (92% yield). Melting point
ꢄ1868C (decomposed) and the found ES-MS [M+1]+ = 579. Analysis
calculated: C, 51.99; H, 4.01; N, 24.25. Found: C, 51.33; H, 4.21; N, 24.01. 1H
NMR (DMSO): d 9.82 (d, 2H, Ar–F), d 8.94 (d, 2H, Ar–F), d 8.88 (s, 2H, Ar–
F), d 8.66 (s, 1H, H7), d 7.64 (d, 2H, Ar), d 6.66 (d, H2, Ar), d 4.47 (d, 2H, H9),
d 4.34 (dd, 1H, H19), d 2.06 (m, 2H, H22), d 1.90 (m, 2H, H21).
Radiosynthesis
2-[(4-{[(2-amino-4-oxohydropteridin-7yl)methyl]amino}phenyl)carbonylami-
no]-4-{N-[(4-[18F]-fluorophenyl)carbonylamino]carbamoyl}butanoic acid: 4-
[
18F]-fluorobenzenecarbohydrazide-folate ([ 18F]-6). First approach: Aqu-
eous [18F]-fluoride was produced by the 18O (p,n) 18F reaction. The flouride
activity (2–10 mCi, 74–370 MBq) was trapped in Kryptofix 2.2.2 (5 mg) and
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NOVEL SYNTHESIS OF [18F]-FLUOROBENZENE
135
potassium carbonate (1 mg) in acetonitrile/water solution (950/50 ml), dried by
azeotropic distillation with aliquots of acetonitrile. The solid residue was
resolubilized in CH3CN (0.2 ml) containing the precursor ethyl 4-(N,N,N-
trimethylammonium)-1-benzoate triflate 10 (5 mg). The reaction mixture was
heated in capped 2 ml reaction-vial at 908C for 5 min. The intermediate ethyl 4-
[18F]-fluorobenzoate was extracted with ether (2 ꢂ 1 ml) and passed through
Sep-Pak silica cartridge. Ether layer was dried with steady stream of nitrogen,
then residue resolubilized with absolute ethanol (500 ml) followed by the
addition of hydrazine hydrate (5 ml). Reaction mixture was heated in capped
2 ml reaction-vial at 908C for 5 min. The excess solvent and hydrazine were
evaporated with steady stream of nitrogen. To the dried activity was added
TEA (2 ml, 14 mmol) followed by addition of NHS-folate 11 (50 mg, 0.1 mmol) in
DMSO (100 ml). After 15 min at 558C, the reaction mixture was diluted with
diethyl ether (1 ml), passed through a Sep-Pak silica cartridge, washed several
times with diethyl ether and dried. The product [18F]-6 was then eluted by
%
saline (1 ml).
Second approach. The 2,5-dioxoazolidinyl 4-[18F]-fluorobenzoate ([18F]-4)
%
20
was prepared adopting procedures reported by Wester et al.
Briefly, the
precursor 10 was radiofluorinated and ethyl 4-[18F]-fluorobenzoate inter-
mediate was converted to corresponding acid ([18F]-1) by heating in the
%
presence of NaOH. This was followed by activation of the acid to the [18F]-4
%
using the TSTU. HPLC purified [18F]-4 was dried and resolubilized in DMSO
%
(100 ml) containing hydrazide-folate (g) 5 (50 mg, 0.11 mmol) and TEA (2 ml,
%
14 mmol). The reaction mixture was heated at 558C for 15 min then diluted
with diethyl ether (1 ml), passed through a Sep-Pak silica cartridge, washed
several times with diethyl ether and dried. Finally, the product [18F]-6 was
%
eluted by saline (1 ml).
2-[(4-{[(2-amino-4-oxohydropteridin-7yl)methyl]amino}phenyl)carbonylamino]-
acid
4-{N-[(2-[18F]-fluoro(4-pyridyl))carbonylamino]carbamoyl}butanoic
2-[18F]-fluoropyridine-4-carbohydrazide-folate([18F]-9). The desire com-
%
pound [18F]-9 was synthesized following the procedure mentioned above for
%
the preparation of [18F]-6. The prosthetic 2-[18F]-fluoropyridine-4-carbohy-
%
drazide 13 was synthesized adopting the method reported by Amartey et al.18
Briefly, the ethyl 2-(N,N,N-trimethylammonium)-4-pyridine carboxylate tri-
flate 12 was radiofluorinated ([18F]KF, Kryptofix, acetonitrile) at 908C for
2 min to obtain ethyl 2-[18F]-fluoro-4-pyridine carboxylate. This was converted
to the intermediate 13, by heating at 908C with hydrazine hydrate (5 ml) in
absolute ethanol (500 ml) for 5 min. After solvents evaporation, TEA (2 ml,
11 mmol) and NHS-folate 11 (50 mg, 0.1 mmol) in DMSO (100 ml) were added
and heated at 558C for 15 min. Finally, reaction mixture was diluted with
diethyl ether, passed through a Sep-Pak silica cartridge, washed several times
Copyright # 2005 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2006; 49: 125–137

136
I. AL JAMMAZ ET AL.
with diethyl ether and dried. The product [18F]-9 was then eluted by saline
%
(1 ml).
Partition coefficient
One hundred microliters each of [18F]-6 and [18F]-9 were added into test tubes
%
%
containing 1 ml of each n-octanol and buffered water (pH ¼ 7:3). The tubes
were shaken for 1 min. After partial separation of the phases by gravity, 0.7 ml
of each phase was transferred to separate tubes and centrifuged at 5000 rpm
for 5 min. Duplicate 0.2 ml aliquots of each phase will be taken for g-
radioactivity measurement and the partition coefficient will be determined by
the function: Partition coefficient = Log10 (counts in n-octanol layer/counts in
aqueous layer).
Conclusion
We have developed a synthetic schemes for the production of [18F]-
fluorobenzene and pyridine carbohydrazide-folates ([18F]-6, [18F]-9) as a new
%
%
PET radiopharmaceuticals. The overall radiochemical yields for both [18F]-6
%
and [18F]-9 were greater than 80% (based on starting [18F]-fluoride) with total
%
synthesis time of approximately 45 min. Radiochemical purities of both tracers
were always greater than 97% without HPLC purification which make this
method amenable for automation. In vitro and in vivo characterization of these
radiofluorinated folates are currently in progress.
Acknowledgements
The authors wish to thank Mr Mohamed Al-Amoudi for the MS analysis.
This project was supported by the Research Center of the King Faisal
Specialist Hospital & Research Center (RAC # 2040027).
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