Special Issue Review Received 30 April 2013,
Revised 26 June 2013,
Accepted 01 July 2013
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.3104
18F-labeled
folic acid derivatives for imaging of the folate receptor via positron emission tomography†
Hanno Schieferstein and Tobias L. Ross* The folate receptor (FR) is already known as a proven target in diagnostics and therapy of cancer. Furthermore, the FR is involved in inflammatory and autoimmune diseases. The major advantage as a valuable target is its strongly limited expression in healthy tissues. Over the past two decades, several folic acid-based radiopharmaceuticals addressing the FR have been developed, and some of them show great potential for applications in clinical routine. However, most of these radiofolates were developed for single photon emission computed tomography imaging, and only a few can be used for positron emission tomography (PET) imaging. The development of suitable 18F-labeled derivatives for PET imaging of the FR has aroused great interest and recent studies revealed very promising candidates for further development and translation into human applications. In this review, we focus on the development of 18F-labeled folic acid derivatives for PET imaging of the FR and discuss various radiochemical strategies and approaches towards 18F-folates. Besides radiochemistry and 18F-labeling, we briefly look into the crucial pharmacological parameters and the preclinical in vivo performance of those 18F-folates. Keywords: folic acid; PET imaging;
18
F-folates; folate receptor; radiofolates
Introduction
432
Folic acid (Figure 1), vitamin B9, is essential for the de novo DNA synthesis and amino acid homeostasis in eukaryotic cells. In the human body, folic acid itself is converted into the coenzyme 5,6,7,8-tetrahydrofolate, which acts as a cofactor and carries onecarbon building blocks such as methylene and formyl groups.1 The folate receptor (FR) is one of three mechanisms cells use for an active uptake of folic acid and folates. Normal cells favor the folate transport via membrane-spanning proteins, the reduced folate carrier (RFC) or the proton-coupled folate transporter (PCFT), both deliver folates directly into the cell cytosol. The PCFT is highly expressed in the duodenum and jejunum.2 The RFC is ubiquitous expressed with high levels in the placenta and liver. The RFC exhibits a high affinity to the reduced forms of folic acid, although it has only a low, micromolar affinity to folic acid (oxidized form).1 Proliferating cells such as cancer cells overexpress the FR, a membrane anchored 38 kDa glycoprotein receptor, which internalizes folic acid by endocytosis.3 The FR displays high affinity (Kd ~1 nM) for folic acid and folates in oxidized forms, but a much lower affinity for reduced folates.1 The FR is associated with various epithelial malignancies such as ovarian and endometrial carcinomas, breast cancer, and lung cancer.3 In addition, the FR plays an important role in autoimmune and inflammatory diseases.4 The positron emitter fluorine-18 is the most commonly used radionuclide in positron emission tomography (PET), which is due to its excellent characteristics for radiopharmaceutical and nuclear medical applications. In PET imaging, the high β-branching of 96.5% and the low β+-energy of 635 keV of 18 F support high-quality images with high sensitivity and resolution. Considering radiochemistry, the halogen allows covalent bonds to
J. Label Compd. Radiopharm 2013, 56 432–440
carbon as well as to silicon and boron with high thermodynamic stability. Additionally, the convenient half-life of 109.8 min offers the possibility of complex radiosyntheses as well as transporting of 18 F-radiopharmaceuticals and extended pharmacological in vivo studies. Consequently, 18F-labeled folate-based radiopharmaceuticals are of great interest for PET imaging of the folate receptor. Several folic acid-based radiopharmaceuticals and drug conjugates addressing the FR have been developed.5,6 Most of these radiofolates were developed for single photon emission computed tomography imaging and only a few for PET imaging.6,7 Recently, the development of 18F-labeled folic acid derivatives for PET imaging of FR-positive tumors has gained more and more interest. To date, some 18F-labeled folates have been developed and were evaluated in preclinical PET studies with promising results but none has been translated into humans, yet. A major issue in 18F-folate research has been to achieve the right balance between radiochemistry and pharmacokinetics. Recent developments have focused on highly efficient labeling and radiochemistry for
Institute of Nuclear Chemistry, Johannes Gutenberg-University, Fritz-Strassmann Weg 2, Mainz, Choose State, 55128, Germany *Correspondence to: Ross L. Tobias, Institute of Nuclear Chemistry, Johannes Gutenberg-University, Fritz-Strassmann Weg 2, Mainz, Choose State, 55128, Germany. Email: [email protected] † This article is published in Journal of Labelled Compounds and Radiopharmaceuticals as a special issue on IIS 2012 Heidelberg Conference, edited by Jens Atzrodt and Volker Derdau, Isotope Chemistry and Metabolite Synthesis, DSAR-DD, Sanofi-Aventis Deutschland GmbH, Industriepark Höchst G876, 65926 Frankfurt am Main, Germany.
Copyright © 2013 John Wiley & Sons, Ltd.
H. Schieferstein and T. L. Ross Biography
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Hanno Schieferstein was born in Bad Schwalbach, Germany, in 1983. He started the studies of Biomedical Chemistry at the Johannes GutenbergUniversity Mainz in 2004 and graduated (Diploma) in Chemistry in 2009. Subject of his diploma thesis was the development of 18F-labeling and evaluation of monoamine oxidase A inhibitors for positron emission tomography imaging, which he accomplished in the group of Professor Dr Frank Rösch. During his studies, he visited Professor Dr Joanna S. Fowler’s laboratories at the Medical Department, Brookhaven National Laboratory, Upton NY, in 2007. He started his PhD in the group of Professor Dr Tobias L. Ross in 2010, dealing with the labeling of folic acid derivatives for positron emission tomography imaging and therapy and the use of folic acid as targeting vector for macromolecular drug delivery systems. His work on radiolabeled folic acid and folates is published in different peer-reviewed articles and has been presented on several scientific meetings. Biography Tobias L. Ross was born in Frechen, Germany, in 1974. He studied Chemistry at the University of Cologne and graduated (Diploma) in Chemistry in 2002. He conducted his thesis “Synthesis of neuronal receptor ligands using no-carrier-added 4-[18F] fluorophenol” in the group of Professor Heinz H. Coenen at the Institute of Nuclear Chemistry at the Research Center Julich, Germany. In 2006, he received his PhD (University of Cologne) for his work on the “Synthesis of no-carrier-added [18F]fluoroarenes via the reaction of no carrier added [18F]fluoride and iodonium salts“ under the supervision of Professor Dr Heinz H. Coenen. He spent 4 years as a postdoctoral researcher and team leader in the laboratories of Professor Dr P. August Schubiger at the Center of Radiopharmaceutical Sciences of the Eidgenössische Technische Hochschule Zurich, Switzerland. Since 2010, he has been an associate professor for radiopharmaceutical chemistry at the Institute of Nuclear Chemistry of the Johannes Gutenberg-University Mainz, Germany. One of his major research interests is the use of folic acid and folates as radiotracers as well as targeting vectors for different applications in oncology.
J. Label Compd. Radiopharm 2013, 56 432–440
18
F-labeled folates via
18
F-prosthetic groups
The first approach toward 18F-labeled folic acid derivatives focused on the use of prosthetic groups and thus avoided major chemical changes in the molecule’s backbone. Previously developed (radio)conjugates of folic acid had already shown that chemical derivatization on the glutamate moiety in the molecule did not affect receptor affinity. Moreover, the folic acid molecule generally has a poor solubility in organic solvents and tends to decompose under harsh conditions, which makes difficulties in the preparation of direct 18F-labeling precursors and makes the direct 18F-radiolabeling somehow challenging. Because the first 18 F-labeled folates via prosthetic group labeling showed very promising in vivo data in preclinical PET-imaging,8 several further 18 F-folates based on 18F-labeling via prosthetic groups have been developed. Table 1 lists all 18F-folates labeled via prosthetic group strategies in chronological order. [18F]fluorobenzenecarbohydrazide and [18F]fluoropyridinecarbohydrazide folates The first report on 18F-folates focused on radiochemistry and labeling using three different prosthetic groups for 18F-labeling, 4-[18F]fluorobenzenecarbohydrazide, 2-[18F]fluoro-4-pyridinecarbohydrazide, and N-hydroxysuccinimide-4-[18F]fluorobenzoate ([18F] SFB, NHS-ester).9 The prosthetic groups were prepared in multi-step radiosyntheses starting from the ethyl esters of the N,N,Ntrimethylammonium triflate precursors. The 18F-versions were obtained in moderate to high radiochemical yields. The 18F-labeled intermediate 4-[18F]fluoro-benzoate ethyl ester was further transferred into the NHS-ester by using N,N,N′,N′-TSTU. Both 18 F-labeled carbohydrazides were obtained from reaction with hydrazine hydrate in very high radiochemical yields of 90%-98%. In a first approach, the 18F-labeled carbohydrazides were coupled with the activated NHS-ester of native folic acid to yield the final products [18F]fluorobenzenecarbohydrazide and [18F] fluoropyridinecarbohydrazide folates in high radiochemical yields of ~80% within very short radiosynthesis times of only 45 min (Figure 2). Interestingly, the final product was obtained in high radiochemical purities of ≥97% using only solid phase extraction (SPE) cartridges. In a second approach, the same coupling between activated NHS-esters and amines was used again, but vice versa, and the NHS-ester of 4-[18F]fluorobenzoic acid was reacted with γ-hydrazide-folate to yield the product, 4-[18F] fluorobenzenecarbohydrazide folate. However, this version gave much lower radiochemical yields of only ~35% and required longer radiosynthesis times of 85 min. In a following study, both 18F-hydrazide-folates of the first approach were employed for preclinical biological evaluation in vitro and in vivo.10 In tumor bearing mice (FR-positive human KB-tumors), both derivatives showed identical tumor uptake of ~6% ID/g, whereas the background in excretion organs was much lower for the [18F]fluoropyridinecarbohydrazide folate.
Copyright © 2013 John Wiley & Sons, Ltd.
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433
Figure 1. Molecule structure of folic acid. The molecule consists of two major building blocks: pteroic acid (pteridine and p-aminobenzoate) and L-glutamate.
F-folates with increased polarity for optimal pharmacokinetics. Most approaches towards 18F-folates are based on prosthetic group labeling using the free carboxylic acids on the glutamate moiety (Figure 1). Alternative strategies follow direct 18F-labeling methods, which require complex precursor synthesis and protection group chemistry.
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F-folate
4-[18F]fluorobenzyl-amine 6-[18F]fluoro-1-hexyne 2-[18F]fluoro-2-deoxy-glucopyranosyl azide 2-[18F]fluoro-2-deoxy-glucose 11-[18F]fluoro-triethylen-1-yne N-succinimidyl-4-[18F]fluorobenzoate
HOBt-folateb γ-azido-folate γ-alkyne-folate γ-hydrazide-oxime-folate γ-azido-OEG-folate γ-amino-PEG-folate
F-click-folate [ F]FDG-click-folate
[18F]FDG-folate [18F]fluoro-OEG-folate [18F]fluoro-PEG-folate
Copyright © 2013 John Wiley & Sons, Ltd.
b
X + Y equals number of radiochemical steps (X) + number of purification steps (Y). Folate-based compounds were employed as α/γ-isomeric mixtures. c The radiosynthesis time as well as the radiosynthetic steps do not include [18F]FDG synthesis. d Overall radiosynthesis time does not include all steps, rcy calculated starting from [18F]FDG.
a
18
18
2-[18F]fluoropyridine-4-carbohydrazide
4-[18F]fluorobenzene-carbohydrazide
NHS-folateb
18
F-prosthetic group
N-succinimidyl-4-[ F]fluorobenzoate
18
Oxime-formation Click-chemistry Amide formation (activated ester)
Amide formation (activated ester) Amide formation (activated ester) Amide formation (activated ester) Amide formation (activated ester) Click-chemistry Click-chemistry
Coupling chemistry
F-folates via 18F-prosthetic group labeling
γ-hydrazide-folate
Folate moiety
18
NHS-folateb
[ F]fluorobenzenecarboxyhydrazide folate [18F]fluorobenzenecarboxyhydrazide folate [18F]fluorobenzenecarboxyhydrazide folate α/γ-[18F]FBA-folates
18
18
Table 1. Details and results of the radiochemistry and labeling of different
80% (1 + 1)c 30% (2 + 2) ~30% (3 + 2)
35% (3 + 2) 25% (3 + 2)
~5% (3 + 3)
~80% (3 + 2)
~80% (3 + 2)
35% (3 + 2)
RCY (stepsa)
(20 min)c 90 min (73 min)d
90 min 180 min
135 minc
45 min
45 min
85 min
Time (EOB)
15 16 17
12 14
8
9,10
9,10
9
Ref.
H. Schieferstein and T. L. Ross
J. Label Compd. Radiopharm 2013, 56 432–440
H. Schieferstein and T. L. Ross
Figure 2.
18
18
F-labeling, conversion to 4-[ F]fluorobenzenecarbohydrazide and reaction to the final
18
18
F-folate.
18
Figure 3. F-labeling and reduction toward the prosthetic group 4-[ F]fluorobenzylamine and its subsequent reaction with the activated ester (HOBt, EDC) of native 18 folic acid. The resulting mixture of α- and γ-[ F]FBA-folate shows an isomeric ratio of 1:4, accordingly to the differences in reactivity of the two carboxylic acids.
Figure 4.
18
18
F-click-labeling of γ-(4-azido-butionyl)folic acid amide using the prosthetic group 6-[ F]fluorohex-1-yne.
α-/γ-[18F]FBA-folate 18
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18
F-Click-folate
To overcome the poor radiochemistry of the α/γ-[18F]FBA-folate, the Cu(I)-catalyzed 1,3-dipolar 18F-click-cycloaddition11 of
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Almost at the same time as the F-folates described earlier, the α/γ-[18F]FBA-folate was developed.8 The 18F-labeling was facilitated via the prosthetic group 4-[18F]fluorobenzylamine (4-[18F]FBA), which was obtained from the standard two-step procedure and a SPE cartridge purification. 4-[18F]FBA was reacted with the activated esters (EDC/HOBt) of native folic acid to yield the α/γ-[18F]FBA-folate as an isomeric mixture in radiochemical yields of ~5% (Figure 3). The use of native folic acid with both carboxylic acids free lead to a regioisomeric mixture of the α- and γ-isomers in a ratio of 1 : 4 in accordance to the relative reactivity of the carboxylic acids in the glutamate moiety. The isomeric mixture could be observed with a 1 : 4 peak ratio by means of analytical HPLC, but the 18F-labeled isomers
could not be isolated by HPLC leading over that the isomeric mixture was employed for subsequent biological studies. α/γ-[18F]FBA-folate was the first 18F-folate applied to preclinical PET imaging studies and showed very promising results in tumor bearing mice (human KB-tumors) with a good tumor visualization and a tumor uptake of ~7% ID/g. Although the α/γ-[18F]FBA-folate showed very promising results in preclinical in vivo PET studies, its unfavorable radiochemistry of a low-yielding multi-step radiosynthesis stopped further development of this tracer.
H. Schieferstein and T. L. Ross
Figure 5.
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18
F-click-labeling of γ-(4-azido-butionyl)folic acid amide using the clickable prosthetic group 6-[ F]fluorohex-1-yne.
alkynes and azides was chosen. The prosthetic group 6-[18F] fluorohex-1-yne was produced in a two-step procedure from the corresponding p-tosylate precursor and neatly purified by a co-distillation with acetonitrile while 18F-labeling took place (Figure 4).12 6-[18F]Fluorohex-1-yne was obtained in high radiochemical yields of ~80% and with radiochemical purities of ≥95% within 12 min. In a γ-selective build-up synthesis, a γ-azido-functionalized folic acid derivative, γ-(4-azido-butyl)-folic acid amide, was prepared and regiospecifically coupled to the 6-[18F]fluorohex-1-yne in the Cu(I)-catalyzed click reaction. For which, CuI as catalyst in acetonitrile/PBS (1 : 1) and a 1 : 2-mixture of DIPEA/2,6-lutidine, was found optimal. After 20 min at 80 °C, up to 85% conversion could be observed. Within the overall synthesis time of approximately 90 min, the product, 18F-click-
Figure 6.
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folate, could be isolated by HPLC in high radiochemical yields of 25–35% and high radiochemical purities of ≥99%. The 18F-click-folate derivative was applied for in vivo PET imaging studies and ex vivo biodistribution experiments by using tumor bearing mice (KB tumor xenografts). Ex vivo biodistribution data indicated a highly specific uptake of the 18 F-click folate in the FR-expressing tissues tumor and kidneys of ~3% ID/g and ~17% ID/g, respectively. The tracer exhibited a strong hepatobiliary excretion, which caused high radioactivity background in the abdominal region. A visualization of the KB tumors in whole body PET imaging could not be observed because of the unfavorably high background signal. 18F-click folate was obtained in high radiochemical yields in a convenient 18 F-click reaction, but shows limited in vivo properties.
18
F-labeling of a γ-aminoxy-hydrazide folate by the use of [ F]FDG.
18
18
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Figure 7. F-Click-labeling of γ-(11-azido-3,6,9-trioxaundecanyl)folic acid amide using the clickable prosthetic group 3-(2-(2-(2-[ F]fluoroethoxy)ethoxy)ethoxy) prop-1-yne.
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J. Label Compd. Radiopharm 2013, 56 432–440
H. Schieferstein and T. L. Ross [18F]FDG-Click-folate A clickable glucose-based 18F-prosthetic group, 2-[18F]fluoro-2deoxy-glucopyranosyl azide,13 was intended to combine highly efficient 18F-click chemistry and an enhanced polarity of the prosthetic group to circumvent the previously discussed strong hepatobliary excretion (Figure 5).14 The 2-[18F]fluoro-2-deoxy-glucopyranosyl azide was synthesized in a two-step procedure and an SPE cartridge purification. In a regioselective synthesis, a γ-alkynyl-folate was synthesized for Cu (I)-catalyzed 18F-click clycloaddition with 2-[18F]fluoro-2-deoxyglucopyranosyl azide. The highly efficient 18F-click reaction yielded the desired [18F]FDG-click-folate within 15 min (at 50 °C) quantitatively. After semipreparative HPLC, [18F]FDG-click-folate was obtained in high radiochemical yields of 25% after an overall radiosynthesis time of 3 h. The best conditions for the 18F-click cycloaddition were found to be a Cu(OAc)2/sodium ascorbatecatalyst in a mixture of water and ethanol. Although the radiosynthesis time of 3 h is very long, this was the first 18 F-folate labeled via a 18F-prosthetic group combining a highly efficient radiochemistry and an appropriate in vivo behavior for high-quality PET imaging. PET imaging studies using the [18F]FDG-click-folate in tumor bearing mice provided a clear tumor visualization with a reduced radioactivity background. The prosthetic group introduced an additional hydrophilicity into the 18F-folate and enhanced the pharmacokinetics for PET imaging with a high and specific tumor uptake of 10% ID/g. Beside the very favorable pharmacokinetics, the [18F]FDG-click-folate also showed a distinct liver uptake of 10% ID/g. [18F]FDG-folate In a similar approach, 2-[18F]fluoro-2-deoxy-glucose ([18F]FDG) was employed as prosthetic group for 18F-labeling of folic acid (Figure 6).15 [18F]FDG is reactive for oxime formation. As a result, the previously developed γ-hydrazide-folate9 was derivatized toward a γ- aminoxy-hydrazide-folate. [18F]FDG was synthesized according to literature procedures and applied without any changes. The γ- aminoxy-hydrazide-folate was mixed with
[18F]fluoro-OEG-folate This radiofolate is strongly linked to the original 18F-click-folate, only differing in the type of spacer, but retaining p-tosylate as a leaving group for radiolabeling.16 In this case, the alkyl spacer system has been replaced by an oligoethylene spacer system to increase polarity, which has previously been shown to enhance pharmacokinetics. The radiolabeling was accomplished by the 18F-click approach, which has proven to give high radiochemical yields, and the possibility to abandon protecting groups. The prosthetic group, 3-(2-(2-(2-[18F]fluoroethoxy)ethoxy) ethoxy)prop-1-yne, was labeled by using a tetrabutylammonium hydroxide/acetonitrile system at 100 °C, in this respect a strong temperature dependency has been observed, giving radiochemical yields greater than 75% (Figure 7). After purification by preparative HPLC, followed by fixation on a SPE cartridge, the 18 F-labeled prosthetic group was directly eluted in a vial equipped with azido-folic acid, which underwent a γ-regioselective built-up synthesis, the copper catalyst, and sodium ascorbate. The 18F-click reaction has been screened and optimized under conventional heating and microwave support, because the conditions described for the original
18
437
Figure 8.
[18F]FDG and heated to 60 °C for 10–15 min. The product [18F]FDG-folate was obtained in very high radiochemical yields of 80% within a 20 min radiosynthesis time starting from [18F]FDG. The [18F]FDG-folate was purified by SPE cartridge (C18) and was obtained in radiochemical purities of ≥ 98%. [18F]FDG-folate was evaluated in ex vivo biodistribution studies using KB-tumor bearing mice. Although the molecule shows an increased hydrophilicity and favorable pharmacokinetics were expected, only a moderate tumor uptake of 3% ID/g was found in tumor bearing mice. Surprisingly, the 18F-folate did not show the generally high kidney uptake of folates, which is because of a physiological expression site of the FR in the proxima tubuli. The authors assume an effect of the prosthetic group ([18F]FDG), which is negatively charged under physiological conditions, reducing the common kidney accumulation. However, this observation still remains unclear and needs further investigations.
F-labeling of the SFB precursor followed by the coupling to a folic acid-PEG amine via amide coupling.
J. Label Compd. Radiopharm 2013, 56 432–440
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H. Schieferstein and T. L. Ross 18
F-click-folate led to degradation of the tracer and precursor. Best radiochemical yields have been obtained during the microwave supported synthesis with yields over 90% and additionally a shorter reaction time of about 90 min. Conventional heating only gave radiochemical yields up to 75% and longer reaction times of 2.5 h were needed, but this enabled the transfer of the radiosynthesis into a hot cell. Additionally, the copper species had to be changed, meaning that for the microwave supported synthesis, Cu(I)I, and for the conventional labeling Cu(II)acetate was the catalyst of choice, whereas the solvent system has been the same as already described for the 18F-click-folate. Subsequently, biodistribution and small animal PET studies of the 18F-OEG-folate have been performed using KB tumor bearing mice, showing a high accumulation of the tracer in the kidneys, feces, and gall bladder. Tumor uptake has been determined to be ~3% ID/g, which is in the same range as the 18F-click-folate. However, blocking experiments indicated the high specificity of the tracer, meaning that 94% of the specific tracer uptake in the tumor could be blocked with native folic acid and even 99% in the kidneys. Interestingly the background signal decreased over time, which is contributed to the PEGylation. Nevertheless, small animal PET experiments have shown that the predominant accumulation of the 18F-OEG-folate in the gastro intestines led to a decrease of the tumor-to-background ratio and reflects the weak tumor signals. [18F]fluoro-PEG-folate
18
Almost at the same time as the 18F-OEG-folate was developed, another oligoethylene carrying radiofolate has been reported, but with the aim to target activated macrophages in a rat model of arthritis.17 The labeling approach was similar to the [18F] fluorobenzene-carboxyhydrazide folate, meaning that the folate derivative, which is carrying a tetraethyleneoxide spacer with a terminal amine function, is coupled through amide bond formation to the NHS-ester of 4-[18F]fluorobenzoate ([18F]SFB) (Figure 8). The [18F]fluoro-PEG-folate was synthesized in a three-step radiosynthesis, starting with 4-(tert-butylcarbonyl)-N, N,N-trimethylbenzenaminium salt, which has been 18F-labeled
Figure 9. Direct
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Figure 10. Direct
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by using the Kryptofix2.2.2/K2CO3-system in acetonitrile at 90°C for 10 min. Deprotection of the labeled precursor was accomplished with tetrapropylammonium hydroxide and subsequent treatment with TSTU has yielded the final prosthetic group, [18F]SFB, and a radiochemical yield of 30%-50%. The prosthetic group has been trapped on a SPE cartridge, eluted, and the solvent has been evaporated to a small volume (100 μL) before the coupling to the PEGylated folic acid derivative was performed. For 18F-labeling, the PEG-folate precursor has been dissolved in borate buffer (150 mM, pH 8.6) followed by the addition of the [18F]SFB in acetonitrile and a reaction time of 30 min at an ambient temperature giving the final [18F]fluoro-PEG-folate, after HPLC purification, in 70%-90% radiochemical yield. The tracer has then been applied to in vitro and in vivo testing, especially to visualize induced arthritis of a rat knee joints. Biodistribution studies showed tracer accumulation mainly in the kidney (~4% ID/g), small intestines (~1% ID/g), and the spleen (~1% ID/g), whereas ~0.4% ID/g accumulate in the arthritic knee. However, the tracer has shown high specificity, meaning that it has been possible to block the FR using glucosamine-folate. Small animal PET studies have also confirmed specific accumulation of the [18F]fluoro-PEG-folate in the arthritic knees of the rat. Interestingly, this tracer has also a high uptake in the gastro intestines as already seen by the [18F]fluoro-OEG-folate. F-labeled folates via direct
18
F-labeling
The synthesis of a suitable precursor for a direct 18F-labeling of folic acid requires a complete build-up synthesis of the folic acid backbone. Besides an obvious synthetic challenge, the choice of where to introduce the 18F without interfering receptor recognition is very limited. In general, modifications on the pteridine ring system as well as on the benzoyl ring have to be avoided, and the only safe site for derivatization is the glutamate part of the molecule. However, so far, three approaches toward a direct 18F-labeling precursor for 18F-labeled folic acid have been reported (Table 2).
18
F-labeling of 2’-[ F]fluorofolic acid.
18
18
F-labeling of γ-[ F]fluorofolic acid.
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J. Label Compd. Radiopharm 2013, 56 432–440
H. Schieferstein and T. L. Ross
Figure 11. Direct
18
18
2
F-labeling of 3’-aza-2’-[ F]fluorofolic acid using the precursor N -acetyl-3’-aza-2’-chlorofolic acid di-tert.-butylester.
Table 2. Details and results of the directly 18
F-folate 18
Leaving group
2’-[ F]fluorofolic acid
NO2
γ-[18F]fluorofolic acid
OMs
3’-aza-2’-[18F] fluoro-folic acid
Cl
18
F-labeled folic acid derivatives Precursor
Labeling chemistry
RCY (stepsa)
Time (EOB)
Ref.
aromatic labeling aliphatic labeling
4% (2 + 2)
80 min
18
~5% (2 + 2)
120 min
21
~9% (2 + 2)
110 min
22
2
N -(N,N-dimethylaminomethylene)2’-nitrofolic acid di-tert.-butylester N2-(N,N-dimethylaminomethylene)10-formyl-γ-mesyloxy-folic acid dimethylester N2-acetyl-3’-aza-2’-chloro-folic acid di-tert.-butylester
aromatic labeling
a
X + Y equals number of radiochemical steps (X) + number of purification steps (Y).
2’-[18F]fluorofolic acid
γ-[18F]fluorofolic acid
The first direct 18F-labeling of folic acid was based on an aromatic 18F-for-NO2-exchange.18 The precursor N2-(N,Ndimethylamino-methylene)-2’-nitrofolic acid di-tert.-butylester was synthesized via a complex built-up synthesis starting from 2-nitro-4-aminobenzoic acid (Figure 9).19 The direct aromatic 18 F-labeling was facilitated by the common Kryptofix2.2.2/ K2CO3-system in dimethylformamide. The aromatic 18F-for-NO2exchange requires quite harsh conditions and elevated temperatures, which is generally not suitable for folic acid structures. Furthermore, a cleavage of the protection groups is required to provide the final 2’-[18F]fluorofolic acid. Both steps, the radiolabeling and the deprotection, were intensively studied and optimized to increase radiolabeling yields as well as to avoid massive product degradation during deprotection. Finally, the best results were obtained after 20 min radiolabeling at 140 °C and a subsequent acidic hydrolysis using 4M HCl at moderate temperatures of 60 °C for 12 min. The radiolabeling yields were 4% after 80 min radiosynthesis. The relatively low radiochemical yields are mainly because of the previously mentioned harsh conditions and compound degradation as well as a poorly activated aromatic ring for a nucleophilic substitution. The amide functionality in ortho-position to the nitro leaving group acts as a moderate activating group (σp ( CONHMe) = 0.36), whereas the amine moiety in the meta-position is electron donating and increases the electron density (σm ( NHMe) = 0.21).20 2’-[18F]fluorofolic acid was evaluated in preclinical PET imaging studies in tumor bearing mice (human KB-tumors) and enabled a clear-cut visualization of tumors with almost no unspecific abdominal background. A high specific tumor uptake of 10% ID/g was obtained and the 2’-[18F]fluorofolic acid showed the most promising in vivo characteristics of 18F-labeled folates, so far.
Another direct 18F-labeling precursor focused on aliphatic 18 F-labeling in the glutamate backbone (Figure 10).21 Conditions for aliphatic 18F-labeling are generally milder than for aromatic approaches and were here intended to provide higher radiochemical yields and preserve the folic acid backbone from degradation. The precursor N2-(N,N-dimethylaminomethylene)10-formyl-γ-mesyloxy-folic acid dimethylester was synthesized in a multi-step build-up synthesis and employed for the radiolabeling. In the direct aliphatic 18F-labeling, tetrabutylammonium hydroxide was used as base in acetonitrile. The radiolabeling yielded the product after 35 min at 85 °C. For cleavage of the protecting groups, a basic hydrolysis using 1 M NaOH at 50 °C for 20 min was found sufficient. The final γ-[18F]fluorofolic acid was obtained from HPLC purification in radiochemical yields of 5% after 120 min overall radiosynthesis time. In in vivo, PET imaging studies of γ-[18F]fluorofolic acid in tumor bearing mice (human KB-tumors) were conducted. Excellent tumor visualization was observed and a low unspecific background. The specific tumor uptake was 8% ID/g and could be blocked with native folic acid by 90%. Besides the specific tumor and kidney uptake, elevated radioactivity levels were also found the in liver (14%ID/g).
In case of the 2’-[18F]fluorofolic acid, it was already discussed that the presented aromatic system is favorable for nucleophilic substitutions. With the idea to enhance the aromatic system for direct 18F-labeling, a pyridine-based system was introduced into the folic acid structure (Figure 11).22 For the direct aromatic 18 F-labeling, a chlorine leaving group in the ortho-position of the pyridine ring was employed. For radiolabeling, the precursor N2-acetyl-3’-aza-2’-chloro-folic acid di-tert.-butylester was used in a system of Kryptofix2.2.2/Cs2CO3 in dimethylsulfoxide and heated
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3’-aza-2’-[18F]fluorofolic acid
H. Schieferstein and T. L. Ross to 160 °C for 10 min. The protection groups could be cleaved in an acidic hydrolysis using 4M HCl solution at 60 °C for 10 min. 3’-aza-2’-[18F]fluorofolic acid was isolated from the radiolabeling mixture by means of HPLC and obtained in radiochemical yields of up to 9% after 110 min total radiosynthesis time. The 3’-aza-2’-[18F]fluorofolic acid exhibits a suitable radiochemistry which has, furthermore, the potential of an easy automation. 3’-aza-2’-[18F]fluorofolic acid has been evaluated in tumor bearing mice by using the human KB-tumor xenograft model. Ex vivo, biodistribution data revealed a high and specific tumor uptake of 12% ID/g as well as very limited abdominal background level. The in vivo PET imaging confirmed the biodistribution data with clear images of tumors and a fast renal clearance of the tracer. Unexpected high radioactivity levels were found in the liver, 14% ID/g, which could be assigned to radio-metabolites of the radiotracer.
Conclusions Several 18F-labeling strategies have been applied to folic acid derivatives. In this regard, the folic acid molecule is quite challenging because of its complex chemistry and its instability under harsh conditions. Therefore, most approaches for 18 F-labeling of folates use prosthetic groups. Especially, the 18 F-click chemistry has shown high potential and particular suitability for 18F-labeling of various folates. Although successful direct 18F-labeling strategies have been applied to 18F-folates, the laborious precursor syntheses, and the instability of the folic acid backbone during direct 18F-labeling and deprotection steps are major concerns. With respect to automation, the direct 18 F-labeling procedures are clearly favored. All developed 18F-folates, so far, show a strong dependency of their pharmacokinetics on derivatization for radiolabeling. Especially, the lipophilicity of the radiofolate is a crucial factor and strongly influences the balance between hepatobiliary and renal excretion. Particularly, very recent developments in 18F-labeled folates have overcome major issues in radiochemistry and unfavorable in vivo characteristics, thus they show great potential for a successful translation into human studies. Consequently, first human studies can be expected to confirm the high value of 18 F-labeled folates for PET imaging.
Acknowledgements The authors thank the research cluster SAMT of the Johannes Gutenberg-University Mainz for supporting Hanno Schieferstein and his folate-based research.
Conflict of Interest The authors did not report any conflict of interest.
References [1] A. C. Antony, Annu. Rev. Nutr. 1996, 16, 501. [2] H. Yuasa, K. Inoue, Y. Hayashi, J. Pharm. Sci. 2009, 98, 1608. [3] N. Parker, M. J. Turk, E. Westrick, J. D. Lewis, P. S. Low, C. P. Leamon, Anal. Biochem. 2005, 338, 284. [4] J. W. van der Heijden, R. Oerlemans, B. A. Dijkmans, H. Qi, C. J. van der Laken, W. F. Lems, A. L. Jackman, M. C. Kraan, P. P. Tak, M. Ratnam, G. Jansen, Arthritis Rheum. 2009, 60, 12. [5] P. S. Low, W. A. Henne, D. D. Doorneweerd, Acc. Chem. Res. 2008, 41, 120. [6] C. Y. Ke, C. J. Mathias, M. A. Green, Adv. Drug Delivery Rev. 2004, 56, 1143. [7] C. Müller, R. Schibli, J. Nucl. Med. 2011, 52, 1. [8] A. Bettio, M. Honer, C. Müller, M. Brühlmeier, U. Müller, R. Schibli, V. Groehn, A. P. Schubiger, S. M. Ametamey, J. Nucl. Med. 2006, 47, 1153. [9] I. Al Jammaz, B. Al-Otaibi, S. Okarvi, J. Amartey, J. Label. Compd. Radiopharm. 2006, 49, 125. [10] I. Al Jammaz, B. Al-Otaibi, S. Amer, S. M. Okarvi, J. Amartey, Nucl. Med. Biol. 2011, 38, 1019. [11] T. L. Ross, Curr. Radiopharm. 2010, 3, 202. [12] T. L. Ross, M. Honer, P. Y. H. Lam, T. L. Mindt, V. Groehn, R. Schibli, P. A. Schubiger, S. M. Ametamey, Bioconjugate Chem 2008, 19, 2462. [13] S. Maschauer, O. Prante, Carbohydr. Res. 2009, 344, 753. [14] C. R. Fischer, C. Müller, J. Reber, A. Müller, S. D. Krämer, S. M. Ametamey, R. Schibli, Bioconjugate Chem. 2012, 23, 805. [15] I. Al Jammaz, B. Al-Otaibi, S. Amer, N. Al-Hokbany, S. Okarvi, Nucl. Med. Biol. 2012, 39, 864. [16] H. Schieferstein, T. Betzel, C. R. Fischer, T. L. Ross, Eur. J. Nucl. Med. Mol. Imag. Res. 2013, submitted. [17] Y. Y. J. Gent, K. Weijers, C. F. M. Molthoff, A. D. Windhorst, M. C. Huisman, D. E. C. Smith, S. A. Kularatne, G. Jansen, P. S. Low, A. A. Lammertsma, C. J. van der Laken, Arthritis Res. Ther. 2013, 15, doi:10.1186/ar4191. [18] T. L. Ross, M. Honer, C. Müller, V. Groehn, R. Schibli, S. M. Ametamey, J. Nucl. Med. 2010, 51, 1756. [19] V. Groehn, R. Moser, T. L. Ross, T. Betzel, C. Müller, R. Schibli, S. M. Ametamey, Synthesis 2011, 3639. [20] C. Hansch, A. Leo, R. W. Taft, Chem. Rev. 1991, 91, 165. [21] R. Schibli, R. Moser, C. M. Müller, S. M. Ametamey, T. L. Ross, V. Groehn, Int. Pat. 2009. WO 2010/040854. [22] T. Betzel, C. Müller, V. Groehn, A. Müller, J. Reber, C. R. Fischer, S. D. Krämer, R. Schibli, S. M. Ametamey, Bioconjugate Chem. 2013, 24, 205.
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J. Label Compd. Radiopharm 2013, 56 432–440
Revised 26 June 2013,
Accepted 01 July 2013
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.3104
18F-labeled
folic acid derivatives for imaging of the folate receptor via positron emission tomography†
Hanno Schieferstein and Tobias L. Ross* The folate receptor (FR) is already known as a proven target in diagnostics and therapy of cancer. Furthermore, the FR is involved in inflammatory and autoimmune diseases. The major advantage as a valuable target is its strongly limited expression in healthy tissues. Over the past two decades, several folic acid-based radiopharmaceuticals addressing the FR have been developed, and some of them show great potential for applications in clinical routine. However, most of these radiofolates were developed for single photon emission computed tomography imaging, and only a few can be used for positron emission tomography (PET) imaging. The development of suitable 18F-labeled derivatives for PET imaging of the FR has aroused great interest and recent studies revealed very promising candidates for further development and translation into human applications. In this review, we focus on the development of 18F-labeled folic acid derivatives for PET imaging of the FR and discuss various radiochemical strategies and approaches towards 18F-folates. Besides radiochemistry and 18F-labeling, we briefly look into the crucial pharmacological parameters and the preclinical in vivo performance of those 18F-folates. Keywords: folic acid; PET imaging;
18
F-folates; folate receptor; radiofolates
Introduction
432
Folic acid (Figure 1), vitamin B9, is essential for the de novo DNA synthesis and amino acid homeostasis in eukaryotic cells. In the human body, folic acid itself is converted into the coenzyme 5,6,7,8-tetrahydrofolate, which acts as a cofactor and carries onecarbon building blocks such as methylene and formyl groups.1 The folate receptor (FR) is one of three mechanisms cells use for an active uptake of folic acid and folates. Normal cells favor the folate transport via membrane-spanning proteins, the reduced folate carrier (RFC) or the proton-coupled folate transporter (PCFT), both deliver folates directly into the cell cytosol. The PCFT is highly expressed in the duodenum and jejunum.2 The RFC is ubiquitous expressed with high levels in the placenta and liver. The RFC exhibits a high affinity to the reduced forms of folic acid, although it has only a low, micromolar affinity to folic acid (oxidized form).1 Proliferating cells such as cancer cells overexpress the FR, a membrane anchored 38 kDa glycoprotein receptor, which internalizes folic acid by endocytosis.3 The FR displays high affinity (Kd ~1 nM) for folic acid and folates in oxidized forms, but a much lower affinity for reduced folates.1 The FR is associated with various epithelial malignancies such as ovarian and endometrial carcinomas, breast cancer, and lung cancer.3 In addition, the FR plays an important role in autoimmune and inflammatory diseases.4 The positron emitter fluorine-18 is the most commonly used radionuclide in positron emission tomography (PET), which is due to its excellent characteristics for radiopharmaceutical and nuclear medical applications. In PET imaging, the high β-branching of 96.5% and the low β+-energy of 635 keV of 18 F support high-quality images with high sensitivity and resolution. Considering radiochemistry, the halogen allows covalent bonds to
J. Label Compd. Radiopharm 2013, 56 432–440
carbon as well as to silicon and boron with high thermodynamic stability. Additionally, the convenient half-life of 109.8 min offers the possibility of complex radiosyntheses as well as transporting of 18 F-radiopharmaceuticals and extended pharmacological in vivo studies. Consequently, 18F-labeled folate-based radiopharmaceuticals are of great interest for PET imaging of the folate receptor. Several folic acid-based radiopharmaceuticals and drug conjugates addressing the FR have been developed.5,6 Most of these radiofolates were developed for single photon emission computed tomography imaging and only a few for PET imaging.6,7 Recently, the development of 18F-labeled folic acid derivatives for PET imaging of FR-positive tumors has gained more and more interest. To date, some 18F-labeled folates have been developed and were evaluated in preclinical PET studies with promising results but none has been translated into humans, yet. A major issue in 18F-folate research has been to achieve the right balance between radiochemistry and pharmacokinetics. Recent developments have focused on highly efficient labeling and radiochemistry for
Institute of Nuclear Chemistry, Johannes Gutenberg-University, Fritz-Strassmann Weg 2, Mainz, Choose State, 55128, Germany *Correspondence to: Ross L. Tobias, Institute of Nuclear Chemistry, Johannes Gutenberg-University, Fritz-Strassmann Weg 2, Mainz, Choose State, 55128, Germany. Email: [email protected] † This article is published in Journal of Labelled Compounds and Radiopharmaceuticals as a special issue on IIS 2012 Heidelberg Conference, edited by Jens Atzrodt and Volker Derdau, Isotope Chemistry and Metabolite Synthesis, DSAR-DD, Sanofi-Aventis Deutschland GmbH, Industriepark Höchst G876, 65926 Frankfurt am Main, Germany.
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H. Schieferstein and T. L. Ross Biography
18
Hanno Schieferstein was born in Bad Schwalbach, Germany, in 1983. He started the studies of Biomedical Chemistry at the Johannes GutenbergUniversity Mainz in 2004 and graduated (Diploma) in Chemistry in 2009. Subject of his diploma thesis was the development of 18F-labeling and evaluation of monoamine oxidase A inhibitors for positron emission tomography imaging, which he accomplished in the group of Professor Dr Frank Rösch. During his studies, he visited Professor Dr Joanna S. Fowler’s laboratories at the Medical Department, Brookhaven National Laboratory, Upton NY, in 2007. He started his PhD in the group of Professor Dr Tobias L. Ross in 2010, dealing with the labeling of folic acid derivatives for positron emission tomography imaging and therapy and the use of folic acid as targeting vector for macromolecular drug delivery systems. His work on radiolabeled folic acid and folates is published in different peer-reviewed articles and has been presented on several scientific meetings. Biography Tobias L. Ross was born in Frechen, Germany, in 1974. He studied Chemistry at the University of Cologne and graduated (Diploma) in Chemistry in 2002. He conducted his thesis “Synthesis of neuronal receptor ligands using no-carrier-added 4-[18F] fluorophenol” in the group of Professor Heinz H. Coenen at the Institute of Nuclear Chemistry at the Research Center Julich, Germany. In 2006, he received his PhD (University of Cologne) for his work on the “Synthesis of no-carrier-added [18F]fluoroarenes via the reaction of no carrier added [18F]fluoride and iodonium salts“ under the supervision of Professor Dr Heinz H. Coenen. He spent 4 years as a postdoctoral researcher and team leader in the laboratories of Professor Dr P. August Schubiger at the Center of Radiopharmaceutical Sciences of the Eidgenössische Technische Hochschule Zurich, Switzerland. Since 2010, he has been an associate professor for radiopharmaceutical chemistry at the Institute of Nuclear Chemistry of the Johannes Gutenberg-University Mainz, Germany. One of his major research interests is the use of folic acid and folates as radiotracers as well as targeting vectors for different applications in oncology.
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18
F-labeled folates via
18
F-prosthetic groups
The first approach toward 18F-labeled folic acid derivatives focused on the use of prosthetic groups and thus avoided major chemical changes in the molecule’s backbone. Previously developed (radio)conjugates of folic acid had already shown that chemical derivatization on the glutamate moiety in the molecule did not affect receptor affinity. Moreover, the folic acid molecule generally has a poor solubility in organic solvents and tends to decompose under harsh conditions, which makes difficulties in the preparation of direct 18F-labeling precursors and makes the direct 18F-radiolabeling somehow challenging. Because the first 18 F-labeled folates via prosthetic group labeling showed very promising in vivo data in preclinical PET-imaging,8 several further 18 F-folates based on 18F-labeling via prosthetic groups have been developed. Table 1 lists all 18F-folates labeled via prosthetic group strategies in chronological order. [18F]fluorobenzenecarbohydrazide and [18F]fluoropyridinecarbohydrazide folates The first report on 18F-folates focused on radiochemistry and labeling using three different prosthetic groups for 18F-labeling, 4-[18F]fluorobenzenecarbohydrazide, 2-[18F]fluoro-4-pyridinecarbohydrazide, and N-hydroxysuccinimide-4-[18F]fluorobenzoate ([18F] SFB, NHS-ester).9 The prosthetic groups were prepared in multi-step radiosyntheses starting from the ethyl esters of the N,N,Ntrimethylammonium triflate precursors. The 18F-versions were obtained in moderate to high radiochemical yields. The 18F-labeled intermediate 4-[18F]fluoro-benzoate ethyl ester was further transferred into the NHS-ester by using N,N,N′,N′-TSTU. Both 18 F-labeled carbohydrazides were obtained from reaction with hydrazine hydrate in very high radiochemical yields of 90%-98%. In a first approach, the 18F-labeled carbohydrazides were coupled with the activated NHS-ester of native folic acid to yield the final products [18F]fluorobenzenecarbohydrazide and [18F] fluoropyridinecarbohydrazide folates in high radiochemical yields of ~80% within very short radiosynthesis times of only 45 min (Figure 2). Interestingly, the final product was obtained in high radiochemical purities of ≥97% using only solid phase extraction (SPE) cartridges. In a second approach, the same coupling between activated NHS-esters and amines was used again, but vice versa, and the NHS-ester of 4-[18F]fluorobenzoic acid was reacted with γ-hydrazide-folate to yield the product, 4-[18F] fluorobenzenecarbohydrazide folate. However, this version gave much lower radiochemical yields of only ~35% and required longer radiosynthesis times of 85 min. In a following study, both 18F-hydrazide-folates of the first approach were employed for preclinical biological evaluation in vitro and in vivo.10 In tumor bearing mice (FR-positive human KB-tumors), both derivatives showed identical tumor uptake of ~6% ID/g, whereas the background in excretion organs was much lower for the [18F]fluoropyridinecarbohydrazide folate.
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Figure 1. Molecule structure of folic acid. The molecule consists of two major building blocks: pteroic acid (pteridine and p-aminobenzoate) and L-glutamate.
F-folates with increased polarity for optimal pharmacokinetics. Most approaches towards 18F-folates are based on prosthetic group labeling using the free carboxylic acids on the glutamate moiety (Figure 1). Alternative strategies follow direct 18F-labeling methods, which require complex precursor synthesis and protection group chemistry.
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F-folate
4-[18F]fluorobenzyl-amine 6-[18F]fluoro-1-hexyne 2-[18F]fluoro-2-deoxy-glucopyranosyl azide 2-[18F]fluoro-2-deoxy-glucose 11-[18F]fluoro-triethylen-1-yne N-succinimidyl-4-[18F]fluorobenzoate
HOBt-folateb γ-azido-folate γ-alkyne-folate γ-hydrazide-oxime-folate γ-azido-OEG-folate γ-amino-PEG-folate
F-click-folate [ F]FDG-click-folate
[18F]FDG-folate [18F]fluoro-OEG-folate [18F]fluoro-PEG-folate
Copyright © 2013 John Wiley & Sons, Ltd.
b
X + Y equals number of radiochemical steps (X) + number of purification steps (Y). Folate-based compounds were employed as α/γ-isomeric mixtures. c The radiosynthesis time as well as the radiosynthetic steps do not include [18F]FDG synthesis. d Overall radiosynthesis time does not include all steps, rcy calculated starting from [18F]FDG.
a
18
18
2-[18F]fluoropyridine-4-carbohydrazide
4-[18F]fluorobenzene-carbohydrazide
NHS-folateb
18
F-prosthetic group
N-succinimidyl-4-[ F]fluorobenzoate
18
Oxime-formation Click-chemistry Amide formation (activated ester)
Amide formation (activated ester) Amide formation (activated ester) Amide formation (activated ester) Amide formation (activated ester) Click-chemistry Click-chemistry
Coupling chemistry
F-folates via 18F-prosthetic group labeling
γ-hydrazide-folate
Folate moiety
18
NHS-folateb
[ F]fluorobenzenecarboxyhydrazide folate [18F]fluorobenzenecarboxyhydrazide folate [18F]fluorobenzenecarboxyhydrazide folate α/γ-[18F]FBA-folates
18
18
Table 1. Details and results of the radiochemistry and labeling of different
80% (1 + 1)c 30% (2 + 2) ~30% (3 + 2)
35% (3 + 2) 25% (3 + 2)
~5% (3 + 3)
~80% (3 + 2)
~80% (3 + 2)
35% (3 + 2)
RCY (stepsa)
(20 min)c 90 min (73 min)d
90 min 180 min
135 minc
45 min
45 min
85 min
Time (EOB)
15 16 17
12 14
8
9,10
9,10
9
Ref.
H. Schieferstein and T. L. Ross
J. Label Compd. Radiopharm 2013, 56 432–440
H. Schieferstein and T. L. Ross
Figure 2.
18
18
F-labeling, conversion to 4-[ F]fluorobenzenecarbohydrazide and reaction to the final
18
18
F-folate.
18
Figure 3. F-labeling and reduction toward the prosthetic group 4-[ F]fluorobenzylamine and its subsequent reaction with the activated ester (HOBt, EDC) of native 18 folic acid. The resulting mixture of α- and γ-[ F]FBA-folate shows an isomeric ratio of 1:4, accordingly to the differences in reactivity of the two carboxylic acids.
Figure 4.
18
18
F-click-labeling of γ-(4-azido-butionyl)folic acid amide using the prosthetic group 6-[ F]fluorohex-1-yne.
α-/γ-[18F]FBA-folate 18
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18
F-Click-folate
To overcome the poor radiochemistry of the α/γ-[18F]FBA-folate, the Cu(I)-catalyzed 1,3-dipolar 18F-click-cycloaddition11 of
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Almost at the same time as the F-folates described earlier, the α/γ-[18F]FBA-folate was developed.8 The 18F-labeling was facilitated via the prosthetic group 4-[18F]fluorobenzylamine (4-[18F]FBA), which was obtained from the standard two-step procedure and a SPE cartridge purification. 4-[18F]FBA was reacted with the activated esters (EDC/HOBt) of native folic acid to yield the α/γ-[18F]FBA-folate as an isomeric mixture in radiochemical yields of ~5% (Figure 3). The use of native folic acid with both carboxylic acids free lead to a regioisomeric mixture of the α- and γ-isomers in a ratio of 1 : 4 in accordance to the relative reactivity of the carboxylic acids in the glutamate moiety. The isomeric mixture could be observed with a 1 : 4 peak ratio by means of analytical HPLC, but the 18F-labeled isomers
could not be isolated by HPLC leading over that the isomeric mixture was employed for subsequent biological studies. α/γ-[18F]FBA-folate was the first 18F-folate applied to preclinical PET imaging studies and showed very promising results in tumor bearing mice (human KB-tumors) with a good tumor visualization and a tumor uptake of ~7% ID/g. Although the α/γ-[18F]FBA-folate showed very promising results in preclinical in vivo PET studies, its unfavorable radiochemistry of a low-yielding multi-step radiosynthesis stopped further development of this tracer.
H. Schieferstein and T. L. Ross
Figure 5.
18
18
F-click-labeling of γ-(4-azido-butionyl)folic acid amide using the clickable prosthetic group 6-[ F]fluorohex-1-yne.
alkynes and azides was chosen. The prosthetic group 6-[18F] fluorohex-1-yne was produced in a two-step procedure from the corresponding p-tosylate precursor and neatly purified by a co-distillation with acetonitrile while 18F-labeling took place (Figure 4).12 6-[18F]Fluorohex-1-yne was obtained in high radiochemical yields of ~80% and with radiochemical purities of ≥95% within 12 min. In a γ-selective build-up synthesis, a γ-azido-functionalized folic acid derivative, γ-(4-azido-butyl)-folic acid amide, was prepared and regiospecifically coupled to the 6-[18F]fluorohex-1-yne in the Cu(I)-catalyzed click reaction. For which, CuI as catalyst in acetonitrile/PBS (1 : 1) and a 1 : 2-mixture of DIPEA/2,6-lutidine, was found optimal. After 20 min at 80 °C, up to 85% conversion could be observed. Within the overall synthesis time of approximately 90 min, the product, 18F-click-
Figure 6.
18
folate, could be isolated by HPLC in high radiochemical yields of 25–35% and high radiochemical purities of ≥99%. The 18F-click-folate derivative was applied for in vivo PET imaging studies and ex vivo biodistribution experiments by using tumor bearing mice (KB tumor xenografts). Ex vivo biodistribution data indicated a highly specific uptake of the 18 F-click folate in the FR-expressing tissues tumor and kidneys of ~3% ID/g and ~17% ID/g, respectively. The tracer exhibited a strong hepatobiliary excretion, which caused high radioactivity background in the abdominal region. A visualization of the KB tumors in whole body PET imaging could not be observed because of the unfavorably high background signal. 18F-click folate was obtained in high radiochemical yields in a convenient 18 F-click reaction, but shows limited in vivo properties.
18
F-labeling of a γ-aminoxy-hydrazide folate by the use of [ F]FDG.
18
18
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Figure 7. F-Click-labeling of γ-(11-azido-3,6,9-trioxaundecanyl)folic acid amide using the clickable prosthetic group 3-(2-(2-(2-[ F]fluoroethoxy)ethoxy)ethoxy) prop-1-yne.
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H. Schieferstein and T. L. Ross [18F]FDG-Click-folate A clickable glucose-based 18F-prosthetic group, 2-[18F]fluoro-2deoxy-glucopyranosyl azide,13 was intended to combine highly efficient 18F-click chemistry and an enhanced polarity of the prosthetic group to circumvent the previously discussed strong hepatobliary excretion (Figure 5).14 The 2-[18F]fluoro-2-deoxy-glucopyranosyl azide was synthesized in a two-step procedure and an SPE cartridge purification. In a regioselective synthesis, a γ-alkynyl-folate was synthesized for Cu (I)-catalyzed 18F-click clycloaddition with 2-[18F]fluoro-2-deoxyglucopyranosyl azide. The highly efficient 18F-click reaction yielded the desired [18F]FDG-click-folate within 15 min (at 50 °C) quantitatively. After semipreparative HPLC, [18F]FDG-click-folate was obtained in high radiochemical yields of 25% after an overall radiosynthesis time of 3 h. The best conditions for the 18F-click cycloaddition were found to be a Cu(OAc)2/sodium ascorbatecatalyst in a mixture of water and ethanol. Although the radiosynthesis time of 3 h is very long, this was the first 18 F-folate labeled via a 18F-prosthetic group combining a highly efficient radiochemistry and an appropriate in vivo behavior for high-quality PET imaging. PET imaging studies using the [18F]FDG-click-folate in tumor bearing mice provided a clear tumor visualization with a reduced radioactivity background. The prosthetic group introduced an additional hydrophilicity into the 18F-folate and enhanced the pharmacokinetics for PET imaging with a high and specific tumor uptake of 10% ID/g. Beside the very favorable pharmacokinetics, the [18F]FDG-click-folate also showed a distinct liver uptake of 10% ID/g. [18F]FDG-folate In a similar approach, 2-[18F]fluoro-2-deoxy-glucose ([18F]FDG) was employed as prosthetic group for 18F-labeling of folic acid (Figure 6).15 [18F]FDG is reactive for oxime formation. As a result, the previously developed γ-hydrazide-folate9 was derivatized toward a γ- aminoxy-hydrazide-folate. [18F]FDG was synthesized according to literature procedures and applied without any changes. The γ- aminoxy-hydrazide-folate was mixed with
[18F]fluoro-OEG-folate This radiofolate is strongly linked to the original 18F-click-folate, only differing in the type of spacer, but retaining p-tosylate as a leaving group for radiolabeling.16 In this case, the alkyl spacer system has been replaced by an oligoethylene spacer system to increase polarity, which has previously been shown to enhance pharmacokinetics. The radiolabeling was accomplished by the 18F-click approach, which has proven to give high radiochemical yields, and the possibility to abandon protecting groups. The prosthetic group, 3-(2-(2-(2-[18F]fluoroethoxy)ethoxy) ethoxy)prop-1-yne, was labeled by using a tetrabutylammonium hydroxide/acetonitrile system at 100 °C, in this respect a strong temperature dependency has been observed, giving radiochemical yields greater than 75% (Figure 7). After purification by preparative HPLC, followed by fixation on a SPE cartridge, the 18 F-labeled prosthetic group was directly eluted in a vial equipped with azido-folic acid, which underwent a γ-regioselective built-up synthesis, the copper catalyst, and sodium ascorbate. The 18F-click reaction has been screened and optimized under conventional heating and microwave support, because the conditions described for the original
18
437
Figure 8.
[18F]FDG and heated to 60 °C for 10–15 min. The product [18F]FDG-folate was obtained in very high radiochemical yields of 80% within a 20 min radiosynthesis time starting from [18F]FDG. The [18F]FDG-folate was purified by SPE cartridge (C18) and was obtained in radiochemical purities of ≥ 98%. [18F]FDG-folate was evaluated in ex vivo biodistribution studies using KB-tumor bearing mice. Although the molecule shows an increased hydrophilicity and favorable pharmacokinetics were expected, only a moderate tumor uptake of 3% ID/g was found in tumor bearing mice. Surprisingly, the 18F-folate did not show the generally high kidney uptake of folates, which is because of a physiological expression site of the FR in the proxima tubuli. The authors assume an effect of the prosthetic group ([18F]FDG), which is negatively charged under physiological conditions, reducing the common kidney accumulation. However, this observation still remains unclear and needs further investigations.
F-labeling of the SFB precursor followed by the coupling to a folic acid-PEG amine via amide coupling.
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H. Schieferstein and T. L. Ross 18
F-click-folate led to degradation of the tracer and precursor. Best radiochemical yields have been obtained during the microwave supported synthesis with yields over 90% and additionally a shorter reaction time of about 90 min. Conventional heating only gave radiochemical yields up to 75% and longer reaction times of 2.5 h were needed, but this enabled the transfer of the radiosynthesis into a hot cell. Additionally, the copper species had to be changed, meaning that for the microwave supported synthesis, Cu(I)I, and for the conventional labeling Cu(II)acetate was the catalyst of choice, whereas the solvent system has been the same as already described for the 18F-click-folate. Subsequently, biodistribution and small animal PET studies of the 18F-OEG-folate have been performed using KB tumor bearing mice, showing a high accumulation of the tracer in the kidneys, feces, and gall bladder. Tumor uptake has been determined to be ~3% ID/g, which is in the same range as the 18F-click-folate. However, blocking experiments indicated the high specificity of the tracer, meaning that 94% of the specific tracer uptake in the tumor could be blocked with native folic acid and even 99% in the kidneys. Interestingly the background signal decreased over time, which is contributed to the PEGylation. Nevertheless, small animal PET experiments have shown that the predominant accumulation of the 18F-OEG-folate in the gastro intestines led to a decrease of the tumor-to-background ratio and reflects the weak tumor signals. [18F]fluoro-PEG-folate
18
Almost at the same time as the 18F-OEG-folate was developed, another oligoethylene carrying radiofolate has been reported, but with the aim to target activated macrophages in a rat model of arthritis.17 The labeling approach was similar to the [18F] fluorobenzene-carboxyhydrazide folate, meaning that the folate derivative, which is carrying a tetraethyleneoxide spacer with a terminal amine function, is coupled through amide bond formation to the NHS-ester of 4-[18F]fluorobenzoate ([18F]SFB) (Figure 8). The [18F]fluoro-PEG-folate was synthesized in a three-step radiosynthesis, starting with 4-(tert-butylcarbonyl)-N, N,N-trimethylbenzenaminium salt, which has been 18F-labeled
Figure 9. Direct
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Figure 10. Direct
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by using the Kryptofix2.2.2/K2CO3-system in acetonitrile at 90°C for 10 min. Deprotection of the labeled precursor was accomplished with tetrapropylammonium hydroxide and subsequent treatment with TSTU has yielded the final prosthetic group, [18F]SFB, and a radiochemical yield of 30%-50%. The prosthetic group has been trapped on a SPE cartridge, eluted, and the solvent has been evaporated to a small volume (100 μL) before the coupling to the PEGylated folic acid derivative was performed. For 18F-labeling, the PEG-folate precursor has been dissolved in borate buffer (150 mM, pH 8.6) followed by the addition of the [18F]SFB in acetonitrile and a reaction time of 30 min at an ambient temperature giving the final [18F]fluoro-PEG-folate, after HPLC purification, in 70%-90% radiochemical yield. The tracer has then been applied to in vitro and in vivo testing, especially to visualize induced arthritis of a rat knee joints. Biodistribution studies showed tracer accumulation mainly in the kidney (~4% ID/g), small intestines (~1% ID/g), and the spleen (~1% ID/g), whereas ~0.4% ID/g accumulate in the arthritic knee. However, the tracer has shown high specificity, meaning that it has been possible to block the FR using glucosamine-folate. Small animal PET studies have also confirmed specific accumulation of the [18F]fluoro-PEG-folate in the arthritic knees of the rat. Interestingly, this tracer has also a high uptake in the gastro intestines as already seen by the [18F]fluoro-OEG-folate. F-labeled folates via direct
18
F-labeling
The synthesis of a suitable precursor for a direct 18F-labeling of folic acid requires a complete build-up synthesis of the folic acid backbone. Besides an obvious synthetic challenge, the choice of where to introduce the 18F without interfering receptor recognition is very limited. In general, modifications on the pteridine ring system as well as on the benzoyl ring have to be avoided, and the only safe site for derivatization is the glutamate part of the molecule. However, so far, three approaches toward a direct 18F-labeling precursor for 18F-labeled folic acid have been reported (Table 2).
18
F-labeling of 2’-[ F]fluorofolic acid.
18
18
F-labeling of γ-[ F]fluorofolic acid.
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J. Label Compd. Radiopharm 2013, 56 432–440
H. Schieferstein and T. L. Ross
Figure 11. Direct
18
18
2
F-labeling of 3’-aza-2’-[ F]fluorofolic acid using the precursor N -acetyl-3’-aza-2’-chlorofolic acid di-tert.-butylester.
Table 2. Details and results of the directly 18
F-folate 18
Leaving group
2’-[ F]fluorofolic acid
NO2
γ-[18F]fluorofolic acid
OMs
3’-aza-2’-[18F] fluoro-folic acid
Cl
18
F-labeled folic acid derivatives Precursor
Labeling chemistry
RCY (stepsa)
Time (EOB)
Ref.
aromatic labeling aliphatic labeling
4% (2 + 2)
80 min
18
~5% (2 + 2)
120 min
21
~9% (2 + 2)
110 min
22
2
N -(N,N-dimethylaminomethylene)2’-nitrofolic acid di-tert.-butylester N2-(N,N-dimethylaminomethylene)10-formyl-γ-mesyloxy-folic acid dimethylester N2-acetyl-3’-aza-2’-chloro-folic acid di-tert.-butylester
aromatic labeling
a
X + Y equals number of radiochemical steps (X) + number of purification steps (Y).
2’-[18F]fluorofolic acid
γ-[18F]fluorofolic acid
The first direct 18F-labeling of folic acid was based on an aromatic 18F-for-NO2-exchange.18 The precursor N2-(N,Ndimethylamino-methylene)-2’-nitrofolic acid di-tert.-butylester was synthesized via a complex built-up synthesis starting from 2-nitro-4-aminobenzoic acid (Figure 9).19 The direct aromatic 18 F-labeling was facilitated by the common Kryptofix2.2.2/ K2CO3-system in dimethylformamide. The aromatic 18F-for-NO2exchange requires quite harsh conditions and elevated temperatures, which is generally not suitable for folic acid structures. Furthermore, a cleavage of the protection groups is required to provide the final 2’-[18F]fluorofolic acid. Both steps, the radiolabeling and the deprotection, were intensively studied and optimized to increase radiolabeling yields as well as to avoid massive product degradation during deprotection. Finally, the best results were obtained after 20 min radiolabeling at 140 °C and a subsequent acidic hydrolysis using 4M HCl at moderate temperatures of 60 °C for 12 min. The radiolabeling yields were 4% after 80 min radiosynthesis. The relatively low radiochemical yields are mainly because of the previously mentioned harsh conditions and compound degradation as well as a poorly activated aromatic ring for a nucleophilic substitution. The amide functionality in ortho-position to the nitro leaving group acts as a moderate activating group (σp ( CONHMe) = 0.36), whereas the amine moiety in the meta-position is electron donating and increases the electron density (σm ( NHMe) = 0.21).20 2’-[18F]fluorofolic acid was evaluated in preclinical PET imaging studies in tumor bearing mice (human KB-tumors) and enabled a clear-cut visualization of tumors with almost no unspecific abdominal background. A high specific tumor uptake of 10% ID/g was obtained and the 2’-[18F]fluorofolic acid showed the most promising in vivo characteristics of 18F-labeled folates, so far.
Another direct 18F-labeling precursor focused on aliphatic 18 F-labeling in the glutamate backbone (Figure 10).21 Conditions for aliphatic 18F-labeling are generally milder than for aromatic approaches and were here intended to provide higher radiochemical yields and preserve the folic acid backbone from degradation. The precursor N2-(N,N-dimethylaminomethylene)10-formyl-γ-mesyloxy-folic acid dimethylester was synthesized in a multi-step build-up synthesis and employed for the radiolabeling. In the direct aliphatic 18F-labeling, tetrabutylammonium hydroxide was used as base in acetonitrile. The radiolabeling yielded the product after 35 min at 85 °C. For cleavage of the protecting groups, a basic hydrolysis using 1 M NaOH at 50 °C for 20 min was found sufficient. The final γ-[18F]fluorofolic acid was obtained from HPLC purification in radiochemical yields of 5% after 120 min overall radiosynthesis time. In in vivo, PET imaging studies of γ-[18F]fluorofolic acid in tumor bearing mice (human KB-tumors) were conducted. Excellent tumor visualization was observed and a low unspecific background. The specific tumor uptake was 8% ID/g and could be blocked with native folic acid by 90%. Besides the specific tumor and kidney uptake, elevated radioactivity levels were also found the in liver (14%ID/g).
In case of the 2’-[18F]fluorofolic acid, it was already discussed that the presented aromatic system is favorable for nucleophilic substitutions. With the idea to enhance the aromatic system for direct 18F-labeling, a pyridine-based system was introduced into the folic acid structure (Figure 11).22 For the direct aromatic 18 F-labeling, a chlorine leaving group in the ortho-position of the pyridine ring was employed. For radiolabeling, the precursor N2-acetyl-3’-aza-2’-chloro-folic acid di-tert.-butylester was used in a system of Kryptofix2.2.2/Cs2CO3 in dimethylsulfoxide and heated
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3’-aza-2’-[18F]fluorofolic acid
H. Schieferstein and T. L. Ross to 160 °C for 10 min. The protection groups could be cleaved in an acidic hydrolysis using 4M HCl solution at 60 °C for 10 min. 3’-aza-2’-[18F]fluorofolic acid was isolated from the radiolabeling mixture by means of HPLC and obtained in radiochemical yields of up to 9% after 110 min total radiosynthesis time. The 3’-aza-2’-[18F]fluorofolic acid exhibits a suitable radiochemistry which has, furthermore, the potential of an easy automation. 3’-aza-2’-[18F]fluorofolic acid has been evaluated in tumor bearing mice by using the human KB-tumor xenograft model. Ex vivo, biodistribution data revealed a high and specific tumor uptake of 12% ID/g as well as very limited abdominal background level. The in vivo PET imaging confirmed the biodistribution data with clear images of tumors and a fast renal clearance of the tracer. Unexpected high radioactivity levels were found in the liver, 14% ID/g, which could be assigned to radio-metabolites of the radiotracer.
Conclusions Several 18F-labeling strategies have been applied to folic acid derivatives. In this regard, the folic acid molecule is quite challenging because of its complex chemistry and its instability under harsh conditions. Therefore, most approaches for 18 F-labeling of folates use prosthetic groups. Especially, the 18 F-click chemistry has shown high potential and particular suitability for 18F-labeling of various folates. Although successful direct 18F-labeling strategies have been applied to 18F-folates, the laborious precursor syntheses, and the instability of the folic acid backbone during direct 18F-labeling and deprotection steps are major concerns. With respect to automation, the direct 18 F-labeling procedures are clearly favored. All developed 18F-folates, so far, show a strong dependency of their pharmacokinetics on derivatization for radiolabeling. Especially, the lipophilicity of the radiofolate is a crucial factor and strongly influences the balance between hepatobiliary and renal excretion. Particularly, very recent developments in 18F-labeled folates have overcome major issues in radiochemistry and unfavorable in vivo characteristics, thus they show great potential for a successful translation into human studies. Consequently, first human studies can be expected to confirm the high value of 18 F-labeled folates for PET imaging.
Acknowledgements The authors thank the research cluster SAMT of the Johannes Gutenberg-University Mainz for supporting Hanno Schieferstein and his folate-based research.
Conflict of Interest The authors did not report any conflict of interest.
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