Comparative Studies of Three Pairs of α- And γ-Conjugated Folic Acid Derivatives Labeled with Fluorine-18

Article abstract of DOI:10.1021/acs.bioconjchem.5b00644

The folate receptor (FR) is upregulated in various epithelial cancer types (FR α-isoform), while healthy tissues show only restricted expression. FR-targeted imaging using folate radiopharmaceuticals is therefore a promising approach for the detection of FR-positive cancer tissue. Almost all folate-based radiopharmaceuticals have been prepared by conjugation at the γ-carboxylic functionality of the glutamate moiety of folic acid. In this work, three pairs of fluorinated α- and γ-conjugated folate derivatives were synthesized and their in vitro and in vivo properties compared. The syntheses of all six regioisomers were obtained in good chemical yields using a multistep synthetic approach including the highly selective Cu(I)-catalyzed 1,3-dipolar cycloaddition. The radiosyntheses of the α- and γ-conjugated ¹⁸F-labeled folate derivatives were accomplished in moderate to good radiochemical yields, high radiochemical purities (>95%), and specific activities ranging from 25 to 196 GBq/μmol. In vitro, all folate derivatives showed high binding affinity to the FR-α (IC₅₀ = 1.4-2.2 nM). In vivo PET imaging and biodistribution studies in FR-positive KB tumor-bearing mice demonstrated similar FR-specific tumor uptake for both regioisomers of each pair of compounds. However, FR-unspecific liver uptake was significantly lower for the α-regioisomers compared to the corresponding γ-regioisomers. In contrast, kidney uptake was up to 50% lower for the γ-regioisomers than for the α-regioisomers. These results show that the site of conjugation in the glutamyl moiety of folic acid has a significant impact on the in vivo behavior of ¹⁸F-based radiofolates, but not on their in vitro FR-binding affinity. These findings may potentially stimulate new directions for the design of novel ¹⁸F-labeled folate-based radiotracers.

Full text of DOI:10.1021/acs.bioconjchem.5b00644

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Article
Comparative Studies of Three Pairs of #- and #-
Conjugated Folic Acid Derivatives Labeled with Fluorine-18
Silvan D Boss, Thomas Betzel, Cristina Müller, Cindy R Fischer, Stephanie
Haller, Josefine Reber, Viola Groehn, Roger Schibli, and Simon M. Ametamey
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/
acs.bioconjchem.5b00644 • Publication Date (Web): 04 Dec 2015
Downloaded from http://pubs.acs.org on December 6, 2015
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Bioconjugate Chemistry is published by the American Chemical Society. 1155
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Comparative Studies of Three Pairs of αꢀ and γꢀConjugated Folic Acid
Derivatives Labeled with Fluorineꢀ18
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Silvan D. Boss†, Thomas Betzel†, Cristina Müller‡, Cindy R. Fischer†, Stephanie Haller‡,
Josefine Reber‡, Viola Groehn§, Roger Schibli†,‡ and Simon M. Ametamey*,†
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†
Department of Chemistry and Applied Biosciences, Institute of Pharmaceutical Sciences, ETH Zurich, Zurich, Switzerland
‡
Center for Radiopharmaceutical Sciences ETHꢀPSIꢀUSZ, Paul Scherrer Institute, VilligenꢀPSI, Switzerland
§
Merck & Cie, Schaffhausen, Switzerland
Abstract
The folate receptor (FR) is upregulated in various epithelial cancer types (FR αꢀisoform), while
healthy tissues show only restricted expression. FRꢀtargeted imaging using folate
radiopharmaceuticals is therefore a promising approach for the detection of FRꢀpositive cancer
tissue. Almost all folateꢀbased radiopharmaceuticals have been prepared by conjugation at the γꢀ
carboxylic functionality of the glutamate moiety of folic acid. In this work, three pairs of
fluorinated αꢀ and γꢀconjugated folate derivatives were synthesized and their in vitro and in vivo
properties compared. The syntheses of all the six regioisomers were obtained in good chemical
yields using a multiꢀstep synthetic approach including the highly selective Cu(I)ꢀcatalyzed 1,3ꢀ
dipolar cycloaddition. The radiosyntheses of the αꢀ and γꢀconjugated 18Fꢀlabeled folate
derivatives were accomplished in moderate to good radiochemical yields, high radiochemical
purities (> 95%) and specific activities ranging from 25ꢀ196 GBq/µmol. In vitro, all folate
derivatives showed high binding affinity to the FRꢀα (IC50 = 1.4ꢀ2.2 nM). In vivo PET imaging
and biodistribution studies in FRꢀpositive KB tumorꢀbearing mice demonstrated similar FRꢀ
specific tumor uptake for both regioisomers of each pair of compounds. However, FRꢀunspecific
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liver uptake was significantly lower for the αꢀregioisomers compared to the corresponding γꢀ
regioisomers. In contrast, kidney uptake was up to 50% lower for the γꢀregioisomers than for the
αꢀregioisomers. These results show that the site of conjugation in the glutamyl moiety of folic
acid has a significant impact on the in vivo behavior of 18Fꢀbased radiofolates but not on their in
vitro FRꢀbinding affinity. These findings may potentially stimulate new directions for the design
of novel 18Fꢀlabeled folateꢀbased radiotracers.
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Introduction
The folate receptor (FR) has emerged as an interesting target for imaging FRꢀpositive tumors due
to the overexpression of FRꢀα on a variety of epithelial cancer types including cancer of the
ovaries, uterus, lung, breast, kidneys and colonꢀrectum.1,2 The expression of FR in normal
healthy tissue is limited to a few sites including the kidneys, lung, choroid plexus, salivary
glands and the placenta.1,3,4
Structurally, folic acid consists of two chemical entities, namely pteroate and glutamate.
Together, they form pteroylglutamic acid which is used as a synonym for folic acid (Figure 1).
Figure 1. Chemical structure of folic acid consisting of pteroic acid (blue) and glutamic acid
(black). The two carboxylic functionalities are accessible for derivatization (black arrows).
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Chemical modification of folic acid by covalent attachment of chemotherapeutics or prosthetic
groups using the pendant approach can in general be accomplished via any of the two carboxylic
functionalities at the alpha (α) and gamma (γ) positions of the glutamate moiety.5 However,
almost all folateꢀbased chemotherapeutic drugs and radiopharmaceuticals have been prepared via
derivatization at the γꢀposition probably because the αꢀcarboxylic acid group in the glutamate
part of folic acid is synthetically less easily accessible for conjugation due to steric hindrance.6,7
The first biological investigations of αꢀ and γꢀregioisomers of folate derivatives were performed
by Wang et al. who conjugated a deferoxamine chelator to the αꢀ or γꢀcarboxylic functionalities
in the glutamic acid moiety.8 The authors observed very low or virtually no binding affinity to
the FR of the αꢀregioisomer of deferoxamine folate, whereas the γꢀregioisomer exhibited an
affinity similar to that of folic acid. One year later, the same group reported on an
ethylendiamineꢀfolate derivative, which was labeled with fluorescein isothiocyanate.9 Similar
results with regard to binding affinities were obtained for the αꢀ and γꢀconjugated derivatives.
The αꢀregioisomer exhibited virtually no affinity to FR, whereas the γꢀregioisomer showed
nanomolar affinity similar to that of folic acid.
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In contrast to the results reported by Wang et al., Leamon et al. observed similar binding
affinities to FRꢀpositive cells for proteins conjugated to folic acid either at the αꢀ or γꢀcarboxylic
acid group of the glutamic acid moiety.10 This observation was also confirmed by Müller et al.
who showed equal in vitro binding affinities to the FR of both αꢀ and γꢀregioisomers of 99mTcꢀ
labeled folate derivatives (99mTc(CO)3ꢀPAMAꢀfolates).11
Wedeking et al. observed comparable in vitro binding affinities of αꢀ and γꢀconjugated folate
derivatives labeled with 153Gd or 99mTc to FRꢀpositive tumor cells.5 In vivo studies of the αꢀ and
γꢀfolate conjugates in tumorꢀbearing animals showed equal or greater tumor accumulation of the
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αꢀisomers compared to the corresponding γꢀregioisomers. Renal clearance of the αꢀisomers was
significantly higher when compared to the γꢀregioisomers. It is, however, not clear whether the
results obtained from the radiometallated folates can be generalized to include fluorineꢀ18
labeled folateꢀbased radiopharmaceuticals. In order to gain more insight and a better
understanding of the biological behavior of the alpha and gamma regioisomers, we designed and
synthesized three pairs of fluorinated αꢀ and γꢀconjugated folic acid derivatives. The structures of
these six different folate congeners are depicted in Figure 2. Our main goal was to evaluate the in
vitro and in vivo properties of these regioisomers, and to assess whether differences exist in their
in vitro binding affinities and in vivo characteristics.
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Figure 2. Three pairs of fluorinated αꢀ and γꢀfolate conjugates; clickꢀfluoroꢀdeoxyꢀglucose
folates α/γꢀ1 (clickꢀFDG folates), clickꢀfluoroethyl folates α/γꢀ2 (clickꢀFE folates) and clickꢀ
fluorobutyl folates α/γꢀ3 (clickꢀFB folates).
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RESULTS
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Synthesis of NonꢀRadioactive Compounds. The synthesis of the αꢀfolate alkyne precursor (αꢀ
10, Scheme 1) was accomplished following the procedure previously described for the γꢀisomer
(γꢀ10).12 Coupling of the two commercially available amino acids BocꢀGlu(OMe)ꢀOH (4) and Hꢀ
PraꢀOMe (5) gave glutamate derivative 6 in 80% yield. Acidic Bocꢀdeprotection of intermediate
6 using TFA afforded quantitatively the free amine 7, which was conjugated in the next step to
protected pteroic acid 8 using HBTU as the coupling reagent. Compound 9 was deprotected
using NaOH and after preparative HPLC purification of the reaction crude, αꢀ10 was obtained in
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26% yield and purity of > 98%. Compound αꢀ10 was prepared in four steps in an overall
chemical yield of 21%. For the other regioisomer γꢀ10, an overall chemical yield of 31% was
obtained as previously reported. The structure of αꢀ10 was confirmed by NMR spectroscopy and
mass spectrometry.
Compounds αꢀ1 and γꢀ1 were synthesized by a Cu(I)ꢀcatalyzed cycloaddition between folate
alkyne 10 and the azido derivative 11, which was prepared in 83% chemical yield following a
previously reported procedure (Scheme 2).13 Preparative HPLC purification gave the nonꢀ
radioactive reference compounds αꢀ1 and γꢀ1 in yields of 62% and 52%, respectively, and
chemical purities of both regioisomers were >98%. In Scheme 3 is shown the synthetic route to
αꢀ and γꢀclickꢀFE folates (αꢀ2 and γꢀ2). Both regioisomers were obtained from the corresponding
folate alkyne building blocks α/γꢀ10 and 2ꢀfluoroethylazido compound 12. 2ꢀFluoroethyl 4ꢀ
methylbenzenesulfonate 24 was obtained in 74% yield by treatment of 2ꢀfluoroethanol 23 with
tosyl chloride (Scheme S2). Replacing the tosyl leaving group with azide functionality afforded
2ꢀfluoroethyl azide 12 which was directly used for the clickꢀreaction with either precursor αꢀ10
or γꢀ10 to give reference compounds αꢀ2 and γꢀ2. After semipreparative HPLC purification, the
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αꢀ and γꢀclickꢀFE folates (αꢀ2, γꢀ2) were obtained in 29% and 34% yield, respectively. For
radiolabeling, 2ꢀazidoethyl 4ꢀmethylbenzenesulfonate 22 (Scheme S1) was synthesized using a
oneꢀpot/twoꢀstep synthesis in 67% yield after column chromatography.
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Nonꢀradioactive αꢀclickꢀFB folate (αꢀ3) was synthesized following the procedure described
previously for the γꢀisomer (γꢀ3).14 Treatment of the commercially available Nꢀ1ꢀBocꢀ1,4ꢀ
diamine 13 with in situ generated triflyl azide gave the azide intermediate 14 in 96% yield
(Scheme 4). Bocꢀdeprotection of intermediate 14 afforded the free amine 15, which was coupled
to protected amino acid BocGlu(OMe)ꢀOH (4) to give glutamate derivative 16 in 64% yield. The
Bocꢀprotecting group in compound 16 was cleaved using TFA to yield free amine 17
quantitatively, which was coupled to protected pteroic acid 8 using HBTU as a coupling reagent
to give compound 18 in 55% chemical yield after semipreparative HPLC purification.
Deprotection of 18 using NaOH, followed by semipreparative HPLC purification afforded αꢀ19
in 63% yield and high purity of >99%. NMR spectroscopy and mass spectrometry confirmed the
structure of αꢀ19. The synthesis of 6ꢀFluorohexꢀ1ꢀyne 20 (Scheme S3) was accomplished in a
twoꢀstep reaction sequence by first reacting hexꢀ5ꢀyneꢀ1ꢀol 25 with tosyl chloride to give
tosylate 26 in 81% yield. Nucleophilic substitution of tosylate 26 with fluoride in a second step
delivered alkyne 20 in 25% yield. Compound 20 was directly used for the Cu(I)ꢀcatalyzed
cycloaddition with folate azides α/γꢀ19 in aqueous solution. Nonꢀradioactive reference
compounds αꢀ3 and γꢀ3 (Scheme 5) were obtained in 65% and 81% yield, respectively, and a
chemical purity of >98% after semipreparative HPLC purification.
Radiochemistry. αꢀ and γꢀclickꢀ[18F]FDG folates (α/γꢀ[18F]1, Scheme 6) were obtained in a
twoꢀstep reaction sequence by reacting γꢀfolate alkynes αꢀ10 and γꢀ10 with
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[18F]fluoroglucopyranosyl azide ([18F]11).12 [18F]11 was synthesized in a twoꢀstep reaction
sequence (Scheme S4, Supporting Information) and used directly for the Cu(I)ꢀcatalyzed
cycloaddition to afford αꢀ or γꢀ[18F]1. After semipreparative HPLC, both radiolabeled
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regioisomers αꢀ[18F]1 and γꢀ[18F]1 were obtained in radiochemical yields of 3ꢀ10% and 5ꢀ25%,
respectively. The radiochemical purity of both αꢀ and γꢀ[18F]1 was higher ≥ 95% with SA
ranging from 30ꢀ170 GBq/µmol.12 Coꢀinjection of the nonꢀradioactive reference compounds α/γꢀ
1 confirmed the identity of α/γꢀ[18F]1.
The radiosyntheses of αꢀ and γꢀclickꢀ[18F]FE folates (α/γꢀ[18F]2, Scheme 7) were accomplished
in a two stepꢀreaction procedure similar to the radiosyntheses of α/γꢀ[18F]1. The first step
involved the nucleophilic substitution of the tosylate precursor 21 with [18F]fluoride.15 18Fꢀ
incorporation was greater than 80% after heating at 80 °C for 15 min. Solid phase extraction
using a silica cartridge was employed for the removal of nonꢀreacted [18F]fluoride and
cryptofix/carbonate salts which potentially could interfere with the following click reaction. The
second step involving the Cu(I)ꢀcatalyzed reaction between radiolabeled azido compound [18F]12
and alkynes α/γꢀ10 was carried out at 80 °C for 20 min to afford αꢀ and γꢀ[18F]2 in 2ꢀ4% decay
corrected radiochemical yield. At the end of synthesis, 400ꢀ800 MBq of the final radioproducts
were obtained in up to 2 mL injectable solution, which was sufficient for in vitro and in vivo
experiments. SA ranged from 25–122 GBq/µmol and radiochemical purity was greater 98%.
The radiosyntheses of αꢀ and γꢀclickꢀ[18F]FB folates (α/γꢀ[18F]3) were accomplished as shown in
Scheme 8. Using hexꢀ5ꢀynꢀ1ꢀyl 4ꢀmethylbenzenesulfonate 26 as the precursor, 6ꢀ[18F]fluorohexꢀ
18
1ꢀyne [18F]20 was obtained with an Fꢀincorporation yield ranging from 50ꢀ70%. In order to
remove unreacted [18F]fluoride and cryptofix/carbonate salts from the reaction mixture, [18F]20
was distilled at 110 °C into a DMF/DMSO solution containing the folate azide α/γꢀ19. The
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subsequent click reaction was carried out in the presence of aqueous solutions of CuSO4×5H2O
and Naꢀ(+)ꢀLꢀascorbate at 90 °C for 20 min. After semipreparative HPLC purification and SPE,
a physiological product solution of αꢀ or γꢀ[18F]3 was obtained. Radiochemical purity of αꢀ and
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γꢀ[18F]3 was >95% and the SA ranged from 63–196 GBq/µmol. At the end of the synthesis,
2.35–4.17 GBq of radiotracer with a decay corrected yield of 19ꢀ23% was obtained.
In Vitro Characterization. To analyze the influence of αꢀ and γꢀconjugation on FRꢀbinding
affinity, in vitro affinity studies were performed with all the three pairs of synthesized reference
compounds (α/γꢀ1, α/γꢀ2, α/γꢀ3) using KB cells in a displacement experiment with 3Hꢀfolic acid.
The binding affinities of the compounds were in the same range, independent of the site of
conjugation. The obtained affinities were similar to that of folic acid (Table 1).
Table 1. Comparison of in vitro affinity data of αꢀ and γꢀ folate regioisomers in comparison to
folic acid. (n = 3)
Compound
IC50 [nM]
folic acid
1.1 ± 0.4
αꢀisomer
γꢀisomer
1.5 ± 0.3
1.4 ± 0.2
2.1 ± 0.2
clickꢀFDG folates
clickꢀFE folates
clickꢀFB folates
1.6 ± 0.2
1.6 ± 0.2
2.2 ± 0.1
The distribution coefficient (logD7.4) was determined using the shakeꢀflask method.16 As
expected, both regioisomers of all the three pairs of clickꢀfolates, α/γꢀ[18F]1, α/γꢀ[18F]2 and α/γꢀ
[18F]3, exhibited identical logD7.4 values (Table 2).
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Table 2. Experimentally determined LogD7.4 values using the shakeꢀflask method. LogD7.4 value
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of γꢀclickꢀFDG folate was taken from the literature.12
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Compound
LogD7.4
α/γꢀisomer
ꢀ4.2 ± 0.1
ꢀ3.0 ± 0.1
ꢀ2.7 ± 0.1
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clickꢀFDG folates
clickꢀFE folates
clickꢀFB folates
Biodistribution. The results of the biodistribution studies are summarized in Figure 3, Table S1
and Table S2 (Supporting Information). Biodistribution data for γꢀ[18F]1 were taken from the
literature12, whereas for γꢀ[18F]3 new biodistribution experiments were performed. In each
experiment, mice were sacrificed at 30, 60 or 90 min p.i.. Mice which received a preꢀinjection of
folic acid for blockage experiments were sacrificed 60 min p.i..
FRꢀpositive tumor uptake of the αꢀ and γꢀclickꢀ[18F]FDG folate (Figure 3, A) after 30 min p.i.
was already 9.22 ± 0.30% IA/g and 9.61 ± 1.73% IA/g, respectively. Tumor uptake slightly
increased for αꢀ[18F]1 over time to 10.9 ± 0.52% IA/g at 90 min p.i.. In contrast, tumor
accumulation of γꢀ[18F]1 remained at similar values during the time course of the investigation
(9.05 ± 2.12% IA/g at 90 min p.i.). Preꢀinjection of folic acid prior to αꢀ[18F]1 or γꢀ[18F]1
application efficiently blocked tumor uptake. Liver uptake of αꢀ[18F]1 was 7.84 ± 1.05% IA/g at
30 min, p.i. and decreased to a value of 3.01 ± 0.48% IA/g at 90 min p.i. A significantly higher
liver uptake was observed for γꢀ[18F]1 already at 30 min p.i.(10.8 ± 1.68% IA/g), and remained
high over the time course of the study (8.37 ± 1.19% IA/g at 90 min p.i.). High uptake of αꢀ
[18F]1 was found in the FRꢀexpressing organs such as kidneys (85.8 ± 8.06% IA/g, 30 min p.i.)
and salivary glands (10.1 ± 2.13% IA/g, 30 min p.i.). These values were significantly higher
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compared to the values obtained for γꢀ[18F]1 in the kidneys (32.4 ± 1.84% IA/g, 30 min p.i.) and
salivary glands (4.61 ± 0.44% IA/g, 30 min p.i.).
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A similar biodistribution pattern was observed for the α/γꢀclickꢀ[18F]FE folates (Figure 3, B).
Already 30 min after injection of the radiotracer, a significant difference in tumor uptake was
found for αꢀ[18F]2 (8.31 ± 0.63% IA/g) and γꢀ[18F]2 (5.61 ± 1.26% IA/g). Tracer accumulation in
the FRꢀpositive tumors increased for both isomers to values of 12.5 ± 1.04% IA/g for αꢀ[18F]2
and 7.24 ± 0.99% IA/g for γꢀ[18F]2 at 90 min p.i.. Preꢀinjection of folic acid blocked the tumor
uptake of αꢀ[18F]2 and γꢀ[18F]2 efficiently. Under baseline conditions, liver uptake of αꢀ[18F]2
(7.03 ± 1.02% IA/g) was more than twoꢀfold lower compared to the corresponding γꢀregioisomer
(16.0 ± 2.69% IA/g) already 30 min p.i.. Radioactivity uptake for both isomers in the liver
decreased over time, however, the liver uptake for γꢀ[18F]2 (8.66 ± 1.19% IA/g) was still
significantly higher compared to the corresponding αꢀisomer (2.77 ± 0.57% IA/g) at 90 min p.i..
As expected, liver uptake of both radiotracers was not affected under blocking conditions, since
the FR is not expressed in the liver. Kidney uptake was more than twoꢀfold higher for αꢀ[18F]2
(32.7 ± 3.52% IA/g) compared to γꢀ[18F]2 (16.9 ± 1.19% IA/g) at 90 min p.i..
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Biodistribution data of α/γꢀclickꢀ[18F]FB folates (Figure 3, C) showed low tumor uptake of the αꢀ
isomer at all different time points (3.68 ± 0.43% IA/g at 30 min p.i.; 3.31 ± 0.33% IA/g at 60 min
p.i.; 3.73 ± 0.62% IA/g at 90 min p.i.). Similar observations were found for the γꢀisomer,
wherein tumor uptake was also low (2.90 ± 0.63% IA/g at 30 min p.i.; 3.10 ± 1.03% IA/g at 60
min p.i.; 3.22 ± 1.05% IA/g at 90 min p.i.). Under blocking conditions, radioactivity in the tumor
xenografts was efficiently reduced. Nonspecific accumulation of α/γꢀ[18F]3 was observed in the
liver, whereby the uptake for the αꢀ[18F]3 (30 min p.i.: 2.44 ± 0.12% IA/g; 60 min p.i.: 1.50 ±
0.34% IA/g; 90 min p.i.: 0.90 ± 0.22% IA/g) was significantly lower compared to γꢀ[18F]3 (30
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min p.i.: 5.12 ± 1.07% IA/g; 60 min p.i.: 3.38 ± 0.63% IA/g; 90 min p.i.: 2.25 ± 0.15% IA/g) at
all the investigated three time points. Salivary gland uptake was significantly lower for αꢀ[18F]3
and decreased over time to 2.27 ± 0.76% IA/g at 90 min p.i,, whereas uptake for γꢀ[18F]3
increased to 9.24 ± 0.73% IA/g at 90 min p.i.. High kidney uptake was found for both
regioisomers, whereas the γꢀregioismer (32.9 ± 1.67% IA/g) showed a significantly lower uptake
compared to the αꢀregioisomer (44.2 ± 4.03% IA/g;) at 90 min p.i.. High radiotracer
accumulation was also found in the gall bladder, urine, and feces, suggesting biliary and renal
extraction of αꢀ[18F]3 and γꢀ[18F]3.
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Tumorꢀtoꢀliver ratio was significantly higher for all the αꢀregioisomers when compared to the
corresponding γꢀregioisomers due to the low liver uptake of the αꢀconjugated derivatives (Table
S2, Supporting Information).
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Figure 3. Results of biodistribution study of some selected tissues at 90 min p.i. of α/γꢀclickꢀ
[18F]FDG folates (A), α/γꢀclickꢀ[18F]FE folates (B) and of α/γꢀclickꢀ[18F]FB folates (C). *P ꢀ
0.01 and **P ꢀ 0.001.
A summary of some important characteristics of the six regioisomers is presented in Table 3.
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Table 3. Summary of some important values of the six regioisomers. Biodistribution data are
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given at 90 min p.i.. The indicated RCY is decay corrected.
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Compound
ꢀ4.2±0.1 1.6±0.2 3ꢀ10 30ꢀ170 10.9±0.52 52.9±4.20 3.01±0.48
αꢀ[18F]1
ꢀ4.2±0.1 1.5±0.3 5ꢀ25 52ꢀ128 9.05±2.12 27.1±1.53 8.37±1.19
γꢀ[18F]1
ꢀ3.0±0.1 1.6±0.2 2ꢀ4 25ꢀ122 12.5±1.04 32.7±3.52 2.77±0.57
αꢀ[18F]2
ꢀ3.0±0.1 1.4±0.2 2ꢀ4 39ꢀ112 7.24±0.99 16.9±1.19 8.66±1.19
γꢀ[18F]2
ꢀ2.7±0.1 2.2±0.1 19ꢀ23 63ꢀ71 3.73±0.62 44.2±4.03 0.90±0.22
αꢀ[18F]3
ꢀ2.7±0.1 2.1±0.2 19ꢀ21 92ꢀ196 3.22±1.05 32.9±1.67 2.25±0.15
γꢀ[18F]3
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In Vivo PET Imaging. Whole body PET scans were performed with all the Fꢀlabeled folates
for 30 min starting from 75 min p.i.. In Figure 4 PET/CT images of KB tumorꢀbearing mice after
i.v. injection of the radioligands are shown. High and similar uptake of radiotracer was found in
KB xenografts for both αꢀ and γꢀclickꢀ[18F]FDG folates, αꢀ[18F]1 and γꢀ[18F]1 (Figure 4, panel
A). The FRꢀpositive kidneys were also clearly visualized in both cases, whereby the αꢀ
regioisomer showed a lower accumulation in the kidneys. Liver uptake was considerably lower
for the αꢀisomer compared to the γꢀisomer. For the α/γꢀclickꢀ[18F]FE folates, αꢀ[18F]2 and γꢀ
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[18F]2 (Figure 4, panel B) high tumor uptake was evident. Kidney accumulation was lower for
the γꢀisomer compared to the αꢀisomer similar to the results obtained in the biodistribution
studies. For α/γꢀclickꢀ[18F]FB folates, αꢀ[18F]3 and γꢀ[18F]3 (Figure 4, panel C), most of the
injected radioactivity was localized in the gastrointestinal tract, the kidneys and the gall bladder,
whereas the tumors were not visualized at SUV=14. Similar to regioisomers αꢀ[18F]2 and γꢀ
[18F]2, lower kidney accumulation was observed for the γꢀregioisomer compared to the αꢀisomer.
Preꢀinjection of folic acid resulted in a remarkably reduced uptake in FRꢀpositive tissues
(xenografts, kidneys, salivary glands) for all the six radioligands (data not shown).
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Figure 4. Maximum intensity projections of PET/CT scans (averaged from 75–105 min p.i.)
performed with KB tumorꢀbearing mice. A: αꢀclickꢀ[18F]FDG folate (left) and γꢀclickꢀ[18F]FDG
folate (right), SUVmax = 5; B: αꢀclickꢀ[18F]FE folate (left) and γꢀclickꢀ[18F]FE folate (right),
SUVmax = 6; C: αꢀclickꢀ[18F]FB folate (left) and γꢀclickꢀ[18F]FB folate (right), SUVmax = 14; Tu
= KB tumor, Li = liver, Ki = kidney, GB = gall bladder, Bl = urinary bladder, Int =
intestines/feces.
Metabolite Studies. Metabolite studies were carried out with αꢀ[18F]1, αꢀ[18F]2 and γꢀ[18F]2 in
mice in order to determine the in vivo stability of the tracers. For all the three radioprobes only
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intact parent compounds were found 30 min after injection of the tracers (Figures S1, S2 and S3,
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Supporting Information) similar to results previously reported for γꢀ[18F]1.12
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DISCUSSION
In this work, we designed and prepared three [18F]fluorinated prosthetic groups which were
coupled to modified folic acid by chemical linkage to either the αꢀ or γꢀglutamyl carboxylate site
using clickꢀchemistry to afford three pairs of αꢀ and γꢀconjugated folate conjugates with different
overall lipophilicities. The synthetic pathways towards αꢀ and γꢀfolate alkyne precursors, αꢀ
10 and γꢀ10, which were key intermediates in the syntheses of αꢀ1, αꢀ2, γꢀ1 and γꢀ2 are shown in
Schemes 1, 2 and 3. The overall chemical yields for both alkynes αꢀ10 and γꢀ10 were 21% and
31%, respectively. A similar trend was observed for the αꢀfolate azide αꢀ19 (21%), which was
obtained in a lower chemical yield compared to its corresponding regioisomer, γꢀ19 (25%)
(Scheme 4). The lower yields of the regioisomers αꢀ10, αꢀ19, compared to γꢀ10 and γꢀ19 may
be explained by the steric hindrance around the αꢀposition. The highly selective Cu(I)ꢀcatalyzed
1,3ꢀdipolar cycloadditions afforded the target compounds αꢀ1, γꢀ1, αꢀ2, γꢀ2, αꢀ3, γꢀ3 after
HPLC purification in chemical yields ranging from 29ꢀ81%. The IC50 values of all six
compounds were in the range of 1.4 to 2.2 nM, and very similar to that of folic acid (1.1 nM).
These results suggest that the site of conjugation in the glutamyl moiety has no dramatic effects
on the binding affinities of the resulting αꢀ and γꢀfolic acid conjugates. These results are in
agreement with the recently published crystal structure of FRꢀα in complex with folic acid which
showed that the pteroate moiety in folic acid is buried in the FR, whereas the glutamate
functionality is solventꢀexposed and sticks out of the pocket entrance allowing it to be
conjugated to drugs without adversely affecting binding to the FRꢀα.17 These results also confirm
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data from Wedeking and coworkers5, Leamon et al.10, Müller et al.11 and Bettio et al.18 who
showed that αꢀ and γꢀregioisomers of folic acid conjugates exhibit similar in vitro binding
affinities to FR.
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18
The radiosyntheses of the Fꢀlabeled folate conjugates followed a highly reliable and efficient
procedure.15,19 The first step involved the nucleophilic substitution of a tosylate or triflate leaving
group in the prosthetic group with 18Fꢀfluoride followed by a Cu(I)ꢀcatalyzed 1,3ꢀdipolar
cycloaddition. αꢀ[18F]1 and γꢀ[18F]1 were obtained in moderate to good decay corrected
radiochemical yields of 3ꢀ10% and 5ꢀ25%, respectively. High radiochemical yields were also
achieved for αꢀ[18F]3 (20ꢀ23%, decay corrected) and γꢀ[18F]3 (19ꢀ21%, decay corrected),
whereas αꢀ[18F]2 and γꢀ[18F]2 were obtained in only 2ꢀ4% decay corrected radiochemical yields.
These low radiochemical yields of α/γꢀ[18F]2 were not due to the inefficiency of the click
reaction but rather due to loss of radioactivity during the semiꢀpreparative HPLC purification of
the two tracers. No attempts were made to optimize the semiꢀpreparative HPLC purification
since enough radioactivity was obtained for the in vivo evaluation of αꢀ[18F]2 and γꢀ[18F]2. All
the six radiotracers were isolated in high radiochemical purity (> 95%) after HPLC purification
and SA ranged from 25ꢀ196 GBq/µmol.
In metabolite studies, αꢀ[18F]1, γꢀ[18F]1, αꢀ[18F]2 and γꢀ[18F]2 exhibited high in vivo stability,
since only intact parent compounds were found in samples taken 30 min after injection of the
radiotracers (Figures S1, S2 and S3, Supporting Information). Previous metabolite studies with γꢀ
[18F]1 and γꢀ[18F]3 showed no radioactive metabolites or defluorination12,14 therefore in this
study no metabolite studies were performed for these two radioligands.
PET imaging experiments and biodistribution data showed similar tumor uptake values for the
respective αꢀ and γꢀregiosiomers whereby the αꢀregioisomers showed slightly higher tumor
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uptake values than the corresponding γꢀregioisomers. A possible explanation might be the lower
hepatobiliary clearance of the αꢀregioisomers compared to the γꢀregiosiomers since a lower
accumulation of the αꢀregioisomers in the liver was observed. Regioisomer αꢀ[18F]2 exhibited
the highest tumor uptake value of 12.5 ± 1.04% IA/g at 90 min p.i.. Of all the regioisomers
investigated αꢀ[18F]3 and γꢀ[18F]3 showed the lowest tumor uptake. We attribute this low uptake
to the lower hydrophilicity of these regiosiomers (LogD7.4 = ꢀ2.7 ± 0.1) compared to α/γꢀ[18F]1
(LogD7.4 = ꢀ4.2 ± 0.1) and α/γꢀ[18F]2 (LogD7.4 = ꢀ3.0 ± 0.1).
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An interesting finding of this study is that radioactivity accumulation in a nonꢀtargeted tissue
such as the liver was generally lower for all the αꢀregioisomers when compared to their
corresponding γꢀisomers. As a consequence, the tumorꢀtoꢀliver ratios were considerably higher
for the αꢀregioisomers than the corresponding γꢀregioisomers for all the three pairs of the folate
conjugates. Since FR is not expressed in liver cells, we speculate unspecific carrierꢀmediated
uptake via organic anion transporters or protonꢀcoupled folate transporter (PCFT) might be
operating. The PCFT is mainly expressed in the liver and the intestinal tract.20,21 We presume
that the steric constraints imposed on the αꢀregioisomers through conjugation to the αꢀglutamyl
moiety render the αꢀregioisomers less accessible to the PCFT and other unspecific carriers. This
assumption is purely a speculation and needs to be verified in future in vitro studies. The low
uptake of the αꢀisomers in the liver has an advantage from an imaging point of view because it
would allow delineating FRꢀpositive tumors that are located close to the liver. Furthermore, the
assessment of liver metastases would be feasible due to a reduced background activity or spillꢀ
over from the liver.
The detection of liver metastasis with the clinically used γꢀconjugated folate radiopharmaceutical
99mTcꢀEC20 is not possible due to substantial liver uptake in humans, although mice did not
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show liver uptake.22,23 These results suggest that the pharmacokinetics of folate
radiopharmaceuticals can differ considerably in mice and humans. However, the use of αꢀ
conjugated folate radiopharmaceuticals could be a possible way to overcome the problem of
unspecific liver uptake. Clearly, the results of this study have shown that the site of conjugation
in folic acid of folateꢀbased radiopharmaceuticals has an impact on the in vivo characteristics of
the 18Fꢀlabeled folic acid conjugates.
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CONCLUSION
It could be shown for the first time that the site of conjugation in folic acid has an impact on the
in vivo distribution pattern of 18Fꢀlabeled folate conjugates but not on the in vitro binding affinity
towards FR. It is expected that these new findings may stimulate new directions for the design of
novel fluorinated folateꢀbased PET radiotracers but may also have an impact on the synthesis of
folateꢀbased ligands for other applications.
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EXPERIMENTAL PROCEDURES
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General. Reagents and solvents were purchased from Bachem, SigmaꢀAldrich Chemie GmbH,
Acros Organics and VWR International AG and used without further purification. The N2ꢀN,Nꢀ
dimethylaminomethyleneꢀ10ꢀformylpteroic acid (protected pteroic acid, 8) was generously
provided by Merck & Cie (Schaffhausen, Switzerland). Nuclear magnetic resonance spectra
were recorded on a Bruker 400 or 500 MHz spectrometer with the corresponding solvent signals
as an internal standard. Chemical shifts are reported in parts per million (ppm) relative to
tetramethylsilane (0.00 ppm). Values of the coupling constant (J) are given in hertz (Hz); the
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following abbreviations are used in this section for the description of the H NMR: singlet (s),
doublet (d), triplet (t), quartet (q), multiplet (m), doublet of doublets (dd), and bs (broad signal).
The chemical shifts of complex multiplets are given as the range of their occurrence. Highꢀ
resolution mass spectra (HRꢀMS) were recorded with a Bruker FTMS 4.7 T BioAPEXII (ESI).
Preparative HPLC was performed with a MerckꢀHitachi system, equipped with a D7000
interface, Lꢀ7400 UV detector and a Lꢀ7150 pump, using a reversedꢀphase column (UltisilTM,
former named Ultimate, C18 5 µm, 21.2×150 mm, Welch Materials) at a flow rate of either 15
mL/min (α/γꢀ19) or 18 mL/min (α/γꢀ1 and α/γꢀ10).
Analytical and semipreparative HPLC was performed with a MerckꢀHitachi system, equipped
with a Dꢀ7000 interface, Lꢀ7400 UV detector and a Lꢀ7100 pump, using a reversedꢀphase
column (analytical columns: α/γꢀ1: Gemini C18, 5 ꢁm, 4.6×250 mm, Phenomenex; α/γꢀ2:
XBridge C18, 5 ꢁm, 4.6×150 mm, Waters; α/γꢀ3: Sunfire C18, 5 ꢁm, 4.6×150 mm, Waters;
semipreparative columns: α/γꢀ1: Gemini C18, 5 ꢁm, 10×250 mm, Phenomenex; α/γꢀ2: XBridge
C18, 5 um, 10×150 mm; α/γꢀ3: Sunfire C18, 5 µm, 10×150 mm). Analytical HPLC was
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performed at a flow rate of 1 mL/min, whereas the flow rate for the semipreparative HPLC was
either 3 mL/min (α/γꢀ[18F]1) or 4 mL/min (α/γꢀ[18F]2 and α/γꢀ[18F]3).
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Analytical radioꢀHPLC was performed on an Agilent 1100 series HPLC system, equipped with a
100 µLꢀloop and a GabiStar radiodetector (Raytest). An analytical column (Gemini C18, 5 ꢁm,
4.6×250 mm, Phenomenex) was used at a flow rate of 1 mL/min.
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Purification of the radiolabeled products were performed by using a semipreparative radioꢀHPLC
system equipped with a Smartline Pump 1000, Smartline Manager 5000, Smartline UV detector
2500 (Knauer), 5 mLꢀloop and a GabiStar radiodetector (Raytest). A semipreparative HPLC
column (Gemini, C18, 5 µm, 250×10 mm, Phenomenex) was used at a flow rate of either 3
mL/min (α/γꢀ[18F]1) or 4 mL/min (α/γꢀ[18F]2 and α/γꢀ[18F]3). All the semipreparative and
analytical HPLC methods are available in the Supporting Information.
For the radiometabolite study, an ultraperformance liquid chromatography (UPLC, Waters)
system with an Acquity UPLC BEH C18 column (2.1×50 mm, 1.7 ꢁm, Waters) and an attached
coincidence detector (FlowStar LB513, Berthold) was used with 50 mM NH4HCO3 solution
(solvent A) and acetonitrile (solvent B) as solvents and a gradient from 0ꢀ0.5 min 100% A, 0.5ꢀ
3.5 min 100ꢀ30% A, 3.5ꢀ3.9 min 30% A at a flow rate of 0.5 mL/min for αꢀ[18F]1. For the
analysis of α/γꢀ[18F]2, 10 mM NH4HCO3 solution (solvent A) and acetonitrile (solvent B) as
solvents were used and a gradient from 0ꢀ0.3 min 100% A, 0.3ꢀ1.0 min 100ꢀ85% A, 1.0ꢀ1.6 min
85% A, 1.6ꢀ2.8 min 85ꢀ40% A, 2.8ꢀ3.5 min 40% A, 3.5ꢀ3.7 min 40ꢀ100% A and 3.7ꢀ4.0 min
100% A at a flow rate of 0.6 mL/min.
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Synthesis of αꢀ and γꢀAlkyne Folate Isomers. The synthesis of γꢀalkyne folate was performed
as previously described.12 The αꢀisomer was synthesized following the same synthetic procedure
(Scheme 1). BocꢀGlu(OMe)ꢀOH×DCHA 4 (487 mg, 1.10 mmol) was dissolved in anhydrous
DMF (10 mL) and NEt3 (612 µL, 4 eq.) and HBTU (492 mg, 1.30 mmol) were added at 0 °C.
This mixture was added to a solution of HꢀPraꢀOMe×HCl 5 (147 mg, 1.16 mmol) in anhydrous
DMF (4 mL) and NEt3 (612 µL, 4 eq.) at 0 °C and stirred for 1 h at 0 °C. The solution was then
allowed to warm up to room temperature and stirring was continued overnight. The reaction
mixture was diluted with 1 M citric acid (50 mL) and extracted with EtOAc (50 mL). Thereafter
the organic phase was rinsed with brine, dried over Na2SO4, filtered and concentrated under
reduced pressure. Purification was achieved by flash chromatography on silica gel with
CH2Cl2/MeOH (50:1), providing the desired product 6 as a clear oil (326 mg, 80%). For removal
of the Boc group, compound 6 (270 mg, 0.73 mmol) was dissolved in CH2Cl2 (4.5 mL) and
trifluoroacetic acid (0.5 mL) was added. The mixture was stirred at room temperature for 5 h and
was then concentrated under reduced pressure to yield the TFA salt of amine 7 as a yellow oil
(198 mg, quantitative). At 0 °C HBTU (314 mg, 0.83 mmol) was added to a suspension of N2ꢀ
N,Nꢀdimethylaminomethylenꢀ10ꢀformylꢀpteroic acid 8 (246 mg, 0.62 mmol) in anhydrous DMF
(2 mL) and NEt3 (165 µL, 2 eq.) and the reaction mixture was stirred for 5 min. The resulting
orange solution was added at 0 °C to a solution of compound 7 (160 mg, 0.59 mmol) in
anhydrous DMF (3 mL), containing NEt3 (165 µL, 2 eq.). Thereafter, the clear yellow solution
was stirred at 0 °C for 4 h and then allowed to warm up to room temperature and stirred
overnight. Removal of volatile compounds under reduced pressure and purification of the residue
by flash chromatography on silica gel with CH2Cl2/MeOH (10:1) provided compound 9 as a
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yellow oil, which was directly deprotected, using 1 M NaOH (1 mL). The solution was stirred
over night at room temperature and thereafter extracted with small amounts of EtOAc. The pH of
the aqueous phase was adjusted to pH 8 with 2 M HCl. The solution was diluted with 50 mM
NH4HCO3 and αꢀ10 was purified via preparative HPLC using 50 mM NH4HCO3 solution
(solvent A) and MeCN (solvent B) as solvent system and a gradient as follows: 0ꢀ20 min: 100ꢀ
90% A, 20ꢀ22 min 90% A, 22ꢀ28 min 90ꢀ40% A, 28ꢀ34 min 90% A. After preparative HPLC,
the αꢀalkyne folate precursor αꢀ10 was obtained in an overall yield of 16% yield, whereas the
yield of γꢀ10 was 19%. Both precursor isomers (αꢀ10 and γꢀ10) were used for the preparation of
radioactive labeled αꢀ and γꢀclickꢀ[18F]FDG folate and αꢀ and γꢀclickꢀ[18F]FE folate and their
nonꢀradioactive reference compounds.
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Scheme 1. Synthesis of αꢀfolate alkynea
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a(i) HBTU, Et3N, DMF, 0 °C to rt, 20 h, 80%; (ii) TFA/CH2Cl2 (1:9), rt, 5 h, quant.; (iii) HBTU,
Et3N, DMF, 0 °C to rt, 18 h; (iv) aq. NaOH (1 M), rt, 15 h, 21% (over four steps).
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Synthesis of 2ꢀDeoxyꢀ2ꢀfluoroglucopyranosyl azide. Compound 11 was prepared according to
the procedure reported by Maschauer et al..13
Synthesis of αꢀ and γꢀClickꢀFDG Folates. The synthesis of γꢀclickꢀFDG folate (γꢀ1) was
performed as previously described.12 Preparation of αꢀclickꢀFDG folate (αꢀ1) followed the same
procedure (Scheme 2). 2ꢀdeoxyꢀ2ꢀfluoroglucopyranosyl azide 11 (18 mg, 86 µmol) was reacted
with αꢀfolate alkyne αꢀ10 (22 mg, 40 µmol) in tꢀBuOH/H2O (1:1, 1.8 mL), a 100 mM aqueous
solution of Cu(OAc)2×H2O (0.1 eq., 40 µL) and a 100 mM aqueous solution of Naꢀ(+)ꢀLꢀ
ascorbate (0.2 eq., 80 µL). The solution was shaken at 500 rpm at room temperature for 90 min
until complete conversion. For isolation of αꢀ1, the mixture was injected into a preparative
HPLC, applying the same method as for the purification of compound αꢀ10.
After preparative HPLC, αꢀ1 and γꢀ1 were obtained in 62% (18 mg) and 52% (7.2 mg) yield,
respectively, and purities were determined to be > 95%. αꢀ1: 1H NMR (D2O/NaOD) δ/ppm 8.63
(s, 1H), 7.98 (s, 1H), 7.67 (d, 3J = 8.4 Hz, 2H), 6.86 (d, 3J = 8.8 Hz, 2H), 5.69 (dd, 3J1 = 8.8 Hz,
4J2 = 2.4 Hz, 1H), 4.66 (t, 3J = 9.2 Hz, 1H), 4.62 (s, 2H), 4.49 (q, 3J = 4.4 Hz, 1H), 4.43 (dd, 3J1 =
5.2 Hz, 3J2 = 4.4 Hz, 1H), 3.94 – 3.83 (m, 1H), 3.71 (dd, 3J1 = 12.4 Hz, 4J2 = 2.2 Hz, 1H), 3.65 –
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3.53 (m, 2H), 3.52 – 3.45 (m, 1H), 3.34 (dd, J1 = 14.8 Hz, J2 = 4.4 Hz, 1H), 3.11 (dd, J1 =
14.8 Hz, 3J2 = 9.2 Hz, 1H), 2.30 – 2.24 (m, 2H), 2.12 – 2.02 (m, 1H), 2.02 – 1.90 (m, 1H); HRꢀ
MS (ES+) calculated for C30H35FN11O11: 744.2496; found: 744.2497.
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Scheme 2. Synthesis of αꢀ and γꢀclickꢀFDG folate reference compoundsa
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a(i) tꢀBuOH/H2O, Cu(OAc)2×H2O, Naꢀ(+)ꢀLꢀascorbate, 90 min, rt; αꢀ1: 62%; γꢀ1: 52%
Synthesis of 2ꢀAzidoethyl 4ꢀmethylbenzenesulfonate. Compound 22 was prepared according
to a published procedure (Scheme S1, Supporting Information).24
Synthesis of 2ꢀFluoroethyl azide. Compound 12 was prepared in analogy to procedures
published in literature (Scheme S2, Supporting Information). 25,26
Synthesis of αꢀ and γꢀClickꢀFE Folates. The syntheses of αꢀ and γꢀclickꢀFE folate (α/γꢀ2) are
shown in Scheme 3. αꢀ or γꢀFolate alkyne α/γꢀ10 (5.4 mg, 0.01 mmol) was dissolved in H2O
(400 µL) and a 100 mM aqueous solution of Cu(OAc)2×H2O (0.1 eq., 10 µL) and a 100 mM
aqueous solution of Naꢀ(+)ꢀLꢀascorbate (0.2 eq., 20 µL) were added, followed by 100 µL of 2ꢀ
fluoroethyl azide solution (12). The reaction mixture was shaken at 900 rpm on an Eppendorf
shaker at room temperature. After two hours, additional 50 µL of the 2ꢀfluoroethyl azide solution
were added and reacted for additional 60 min. The progress of the reaction was followed by
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analytical HPLC using 50 mM NH4HCO3 solution (solvent A) and MeCN (solvent B) as a
solvent system with a gradient from 0ꢀ4 min 100% A, 4ꢀ25 min 100ꢀ80% A, 25ꢀ26 min 80ꢀ40%
A, 26ꢀ35 min 40% A, 35ꢀ36 min 40ꢀ100% A, 36ꢀ45 min 100% A. After completion of the
reaction, semiꢀpreparative HPLC was performed applying the same gradient and solvent system
used for the analytical HPLC to yield αꢀ2 (1.8 mg, 29%, purity >98%) or γꢀ2 (2.1 mg, 34%,
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purity >99%). αꢀ2: 1H NMR (DMSOꢀd6/D2O) δ/ppm 8.64 (s, 1H), 7.82 (s, 1H), 7.65 (d, 3J = 8.8
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Hz, 2H), 6.64 (d, J = 8.8 Hz, 2H), 4.83 – 4.74 (m, 1H), 4.71 – 4.62 (m, 1H), 4.59 – 4.54 (m,
1H), 4.48 (s, 2H), 4.31 (dd, 3J1 = 8.8, 3J2 = 5.6 Hz, 1H), 4.17 – 4.09 (m, 1H), 3.16 (dd, 2J1 = 14.8,
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3J2 = 4.8 Hz, 1H), 2.95 (dd, J1 = 14.8, J2 = 7.6 Hz, 1H), 2.26 (t, J = 7.6 Hz, 2H), 2.01 – 1.90
(m, 1H), 1.89 – 1.78 (m, 1H); HRꢀMS (ES+) calculated for C26H29FN11O7: 626.2230; found:
626.2231; γꢀ2: 1H NMR (DMSOꢀd6/D2O) δ/ppm 8.64 (s, 1H), 7.77 (s, 1H), 7.61 (d, 3J = 8.8 Hz,
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2H), 6.64 (d, J = 8.8 Hz, 2H), 4.79 (t, J = 4.8 Hz, 1H), 4.65 (dt, J1 = 9.2, J2 = 4.4 Hz, 2H),
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3J2 = 4.4 Hz, 1H), 3.10 (dd, 2J1 = 14.8, 3J2 = 4.8 Hz, 1H), 2.91 (dd, 2J1 = 14.8, 3J2 = 8.4 Hz, 1H),
2.21 – 2.11 (m, 2H), 2.02 – 1.91 (m, 1H), 1.91 – 1.78 (m, 1H); HRꢀMS (ES+) calculated for
C26H29FN11O7: 626.2230; found: 626.2230.
Scheme 3. Synthesis of reference compounds αꢀ and γꢀclickꢀFE folatea
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a(i) 2ꢀFluoroethyl azide 12, Cu(OAc)2×H2O, Naꢀ(+)ꢀLꢀascorbate, MeCN/H2O/EtOH, 60 min, rt;
αꢀ2: 29%, γꢀ2: 34%.
Synthesis of αꢀ and γꢀAzide Folate Isomers. The synthesis of the αꢀazide folate (αꢀ19, Scheme
4) was accomplished following the procedure described previously for the γꢀregioisomer (γꢀ
19).14 A solution of NaN3 in CH2Cl2/H2O (2:1, 15 mL) was slowly treated with Tf2O at 0 °C
under vigorous stirring. The solution was allowed to warm to room temperature and stirred for
2h. Extraction of the TfN3 was performed with CH2Cl2 (2×5 mL). Thereafter, the combined
organic phases were washed with saturated Na2CO3 (15 mL) and then slowly added to a solution
of Nꢀ1ꢀBocꢀ1,4ꢀdiaminobutane∙HCl (500 mg, 2.22 mmol), K2CO3 (460 mg, 3.33 mmol) and
CuSO4×5H2O (5 mg, 0.02 mmol) in MeOH/H2O (3:2, 25 mL) and stirred at room temperature
overnight. The organic solvents were evaporated and the pH of the solution was adjusted to pH 6
by the addition of concentrated HCl. Dilution of the reaction solution was performed with 0.2 M
phosphate buffer pH 6.2 (15 mL) and an extraction with CH2Cl2 (4×40 mL) was performed. The
combined organic phases were washed with brine, dried over MgSO4 and the concentrated under
reduced pressure to give compound 14 as a colorless oil (339 mg, 96 %). Boc deprotection was
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performed by dissolving intermediate 14 (214 mg, 1.0 mmol) in CH2Cl2/TFA (9:1, 5 mL) and
stirring at room temperature overnight. The organic solvent was evaporated and the obtained oily
product was directly used for the following coupling with the protected glutamate. For the
coupling, a solution of BocꢀGlu(OMe)ꢀOH×DCHA 4 (443 mg, 1.0 mmol) in DMF (5 mL) was
treated with DIPEA (510 µL, 3.0 mmol) at 0 °C under stirring. HBTU (380 mg, 1.0 mmol) was
added and the solution was stirred at 0 °C for 30 min. Intermediate 15 was dissolved in DMF (5
mL) and DIPEA (255 µL, 1.5 mmol) and thereafter the solution was slowly added to the other
solution containing the glutamate and stirred at 0 °C for 2 h. Stirring was continued overnight at
room temperature. Citric acid (1M, 75 mL) was added to the brownish transparent solution and
an extraction with EtOAc (4×40 mL) was carried out. The combined organic phases were
washed with brine, dried over MgSO4 and concentrated to give the crude mixture of the product.
Purification was achieved by flash chromatography on silica gel with hexane/EtOAc (1:2),
providing the desired product 16 as a transparent oil (230 mg, 64 %). Boc deprotection was
achieved by dissolving 16 in CH2Cl2/TFA (9:1, 5 mL) and stirring at room temperature until
TLC showed completion of the reaction. The organic solvents were evaporated and the obtained
product 17 was directly used for the coupling reaction without any further purification. For the
coupling, a solution of N2ꢀN,Nꢀdimethylaminomethylenꢀ10ꢀformylꢀpteroic acid 8 (233 mg, 0.59
mmol) in DMF (10 mL) at 0 °C was treated with TEA (124 µL, 0.9 mmol). HBTU (224, 0.59
mmol) was added and the solution was stirred at 0 °C for 1 h. Afterwards, a solution containing
17 (220 mg, 0.59 mmol) in DMF (9 mL) and TEA (295 µL, 2.1 mmol) was slowly added and
stirred at 0 °C for 1 h. Stirring was continued overnight at room temperature. H2O (100 mL) was
added to the solution and an extraction with EtOAc (2×50 mL) and CH2Cl2 (2×50 mL) was
performed. The combined organic phases were washed with brine, dried over MgSO4 and
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concentrated. Flash chromatography on silica gel with CH2Cl2/MeOH (7 → 9% of MeOH) and
evaporation of the solvent gave 18 in 55% yield (204 mg). For deprotection, 18 (108 mg, 0.17
mmol) was dissolved in 1M NaOH (2.5 mL) and the solution was stirred overnight at room
temperature. Precipitation of product αꢀ19 was achieved by acidification to pH 2 using
concentrated HCl. The solid could be separated by centrifugation (3500 rpm, 10 min), after
which the precipitation was dissolved in 10 mM NH4HCO3 and purified via preparative HPLC
using 10 mM NH4HCO3 solution (solvent A) and MeCN (solvent B) as solvent system and a
gradient as follows: 0ꢀ50 min: 100ꢀ75% A, 50ꢀ80 min 75% A, 80ꢀ90 min 75ꢀ50% A, 90ꢀ91 min
50ꢀ100% A, 91ꢀ100 min: 100% A. After preparative HPLC, the αꢀ and γꢀazide folates αꢀ19 and
γꢀ19 were obtained in an overall chemical yield of 21% and 25%, respectively. Chemical purity
was greater than 95%.
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Scheme 4. Synthesis of αꢀfolate azide isomera
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