Reduced 18F-Folate Conjugates as a New Class of PET Tracers for

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Reduced 18F-Folate Conjugates as a New Class of PET Tracers for Folate Receptor Imaging Silvan D Boss, Cristina Müller, Klaudia Siwowska, Josephine Büchel, Rafaella Schmid, Viola Groehn, Roger Schibli, and Simon Mensah Ametamey Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00775 • Publication Date (Web): 07 Feb 2018 Downloaded from http://pubs.acs.org on February 17, 2018

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Bioconjugate Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Bioconjugate Chemistry

Reduced

18

F-Folate Conjugates as a New Class of PET Tracers for

Folate Receptor Imaging Silvan D. Boss†, Cristina Müller‡, Klaudia Siwowska‡, Josephine I. Büchel†, Raffaella M. Schmid‡, Viola Groehn§, Roger Schibli†,‡ and Simon M. Ametamey*,† †Department of Chemistry and Applied Biosciences, Institute of Pharmaceutical Sciences, ETH Zurich, Zurich, Switzerland ‡Center for Radiopharmaceutical Sciences ETH-PSI-USZ, Paul Scherrer Institut, Villigen-PSI, Switzerland §Merck & Cie, Schaffhausen, Switzerland

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ABSTRACT 5-Methyltetrahydrofolate (5-MTHF), a reduced folate form, is the biologically active folate involved in many different metabolic processes. To date, there are no studies available in the literature on

18

F-labeled 6S- and 6R-5-MTHF radiotracers for imaging FR-α-positive tissues. 18

Therefore, the goal of this study was to synthesize four

F-labeled 5-MTHF derivatives

conjugated at either the α- or γ-carboxylic functionality of glutamate and to assess their suitability for folate receptor (FR)-targeting. Organic syntheses of the precursors and the four reference compounds, namely 6S-α, 6S-γ, 6R-α and 6R-γ-click-fluoroethyl-5-MTHF, were carried out in low to moderate overall chemical yields. The radiosyntheses of the α- and γ-conjugated

18

F-labeled folate derivatives were

accomplished in approximately 100 min, low radiochemical yields (1-7% d.c.) and high molar activities (139-245 GBq/µmol). Radiochemically pure tracers were obtained after the addition of a mixture of antioxidants consisting of sodium ascorbate and L-cysteine. In vitro, all four 5MTHF conjugates showed similar binding affinities to FR-α (IC50 = 17.7-24.0 nM), whereas folic acid showed a significantly higher binding affinity to the FR-α. Cell uptake and internalization experiments with KB cells demonstrated specific uptake and internalization of the radiofolate conjugates. Metabolite studies in mice revealed high in vivo stability of the radiotracers in mice. Biodistribution and positron emission tomography (PET) imaging studies in FR-positive KB tumor-bearing mice demonstrated that the 6S- and 6R-5-MTHF conjugates exhibited a different accumulation pattern in various organs including the kidneys and the liver, whereas no significant differences in radioactivity accumulation in the kidneys and the liver were found for both the α- and γ-conjugated diastereoisomers. Despite the considerably lower binding 2


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affinities of the 5-MTHF derivatives compared to the corresponding folic acid conjugates similar high KB tumor uptake was observed for all the folate conjugates investigated (8-11% IA/g). Based on these results, we conclude that

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F-labeled 5-MTHF conjugates are a promising new

class of radiotracers for targeting FR-positive tumor tissues.

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INTRODUCTION The concept of modifying folic acid (Figure 1) at the glutamic acid moiety is a promising approach for the selective delivery of attached payloads to folate receptor (FR)-positive tumors. The FR is a promising tumor target as it is overexpressed in many epithelial cancer types but present in only few healthy tissues.1,2 Low molecular weight chemotherapeutics or immunotherapeutics have been conjugated to folic acid and used for FR-targeted therapy in ovarian and lung cancer patients demonstrating promising antitumor activity.3,4 Diagnostic agents were prepared for magnetic resonance and near-infrared imaging, the latter being particularly promising for intraoperative imaging of FR-positive cancer lesions.5,6 Radiolabeled folate conjugates have proven to be useful tools for non-invasive imaging using single photon emission computed tomography (SPECT) or positron emission tomography (PET).7 However, varying the chemical moieties attached to folic acid did not result in a major increase in FRpositive tumor uptake of the folic acid conjugates.8 Almost all folic acid (pteroylglutamic acid) radioconjugates developed to date were obtained via derivatization at the γ-carboxylic functionality of the glutamate entity due to easier synthetic accessibility compared to the carboxylic group at the α-position.8 In the last decade, our group has focused on the development of 18F-based radiofolates.9,10,11 Recently, we have reported on an extensive comparative study of three pairs of

18

F-labeled α- and γ-conjugated folic acid

derivatives.12 The study showed that the site of conjugation (α or γ) has a considerable impact on the in vivo characteristics of the 18F-labeled folic acid conjugates. The α-conjugated derivatives exhibited a significantly lower liver uptake compared to the corresponding γ-conjugated derivatives, whereas kidney retention was higher for the α-regioisomers. In contrast, the FR4


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positive KB tumor uptake was similar for both regioisomers. This previous study was performed to investigate the influence of different chemical moieties linked to the α/γ carboxyl group folic acid. As a next logical step, we set out to investigate modifications of the targeting molecule (folic acid) itself. 5-Methyltetrahydrofolate (5-MTHF) is a reduced form of folic acid and the biologically active and predominant folate present in blood and represents therefore an interesting alternative to folic acid as FR-targeting molecule. 5-MTHF is, however, less stable in solution than folic acid and can easily be oxidized by oxygen, free radicals or UV light.13,14 5-MTHF exists either as the physiological 6S- or as the naturally not existing 6R-diastereoisomer (Figure 1). Both isomers have an approximately 5-10-fold lower affinity to the FR compared to folic acid as reported by Wang et al..15 This group further showed that the natural 6S-5-MTHF exhibits selectivity to the FR-α expressed on tumor cells over FR-β, which is overexpressed on activated macrophages involved in inflammations.15 The unnatural 6R-5-MTHF has similar high binding affinities to both FR isoforms.15,16 Besides binding to the FR, it was reported in the literature that 6S-5MTHF is predominantly transported through the reduced folate carrier (RFC), an anionic exchanger ubiquitously expressed in eukaryotic cells.17 The lack of FR-selectivity over RFCmediated transport, the lower affinity of 6S-5-MTHF to the FR compared to folic acid and its low chemical stability are potential reasons why 5-MTHF was probably not taken into consideration as a targeting molecule for FR-positive tumors. So far, only two reduced folates have been radiolabeled and reported in the literature. Saeed et al. developed the radiosynthesis of a N5,N10-methylenetetrahydrofolate radiotracer labeled with carbon-11, whereas Vaitilingam et al. reported on a

99m

Tc-labeled N5,N10-dimethyltetrahydrofolate derivative.18,19 Both groups 5



reported on the synthesis of these radiofolates, but only the

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99m

Tc-labeled N5,N10-

dimethyltetrahydrofolate radiotracer existing as 1:1 mixture of the 6R- and 6S-diastereoisomers, was biologically evaluated. Although the organic synthesis of 5-MTHF was reported by Zhou et al. in 2013, there are no studies available in the literature to date about the biological behavior of diastereomerically pure 6S- and 6R-5-MTHF radiotracers and whether such radiofolates can be used to image FR-positive tissues using PET.20 The aim of the present study was, therefore, to prepare diastereomerically pure

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F-labeled 6S-

and 6R-5-MTHF derivatives, namely 6S-α-, 6R-α-, 6S-γ- and 6R-γ-click-[18F]fluoroethyl-5MTHF conjugates (6S-α-, 6R-α-, 6S-γ- and 6R-γ-1, Figure 1) and to assess their utility as imaging agents for FR-positive tumor tissues. These four radiofolates were synthesized, biologically evaluated in vitro and investigated in in vivo experiments using tumor-bearing mice. Conclusions were drawn on the suitability of this new class of radiofolates as imaging agents for FR-positive tumor tissues. Furthermore, the influence of the site of conjugation (α vs. γ) on the glutamate of 5-MTHF was investigated.

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Figure 1: Chemical structures of 6S- and 6R-5-MTHF, 6S-α-[18F]1 and 6R-α-[18F]1, 6S-γ-[18F]1 and 6R-γ-[18F]1.

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RESULTS Syntheses of the α-Conjugated Precursor Folate Alkynes. The syntheses of the α-conjugated folate alkynes 6S-α-8 and 6R-α-8 (Scheme 1), which served as precursors, were accomplished in analogy to a procedure previously reported by our group.12 The first step involved a coupling reaction of Boc-Glu(OMe)-OH (2) and H-Pra-OMe (3) to afford intermediate 4, which was deprotected under acidic conditions to yield amine 5. Coupling of compound 5 with either 6S- or 6R-10-formyl-5-methyltetrahdropteroic acid (6S-6 or 6R-6) using HBTU as coupling reagent gave 6S-α-7 or 6R-α-7 in 30% and 18% chemical yield, respectively. After cleavage of the protecting groups of 6S-α-7 or 6R-α-7 using NaOH followed by semipreparative HPLC purification, 6S-α-8 or 6R-α-8 was obtained in a chemical purity of >95% and overall yields of 18% and 8%, respectively.

8


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Scheme 1: Synthesis of 6S-α- and 6R-α-folate alkynes (6S-α-8 and 6R-α-8)a

a

(i) HBTU, DIPEA, DMF, 0 °C to rt, 3 h, 86%; (ii) CH2Cl2/TFA (10:1), rt, 2 h, quant.; (iii) 5,

HBTU, Et3N, DMF, 0 °C to rt, 5 h: 6S-α-7: 30%, 6R-α-7: 18%; (iv) 1M NaOH, rt, 3 h: 6S-α-8: 68%, 6R-α-8: 58%.

Syntheses of Reference Compounds. The syntheses of the 6S-α- and 6R-α-click-fluoroethyl-5-MTHFs (6S-α-1 and 6R-α-1) were accomplished following a similar synthetic route as previously described (Scheme 2).12 Click reaction of fluoroethyl azide 9 with alkyne 4 gave click product 10, which was Boc-deprotected using TFA to afford intermediate 11. Coupling of amine 11 with either 6S- or 6R-10-formyl-59



methyltetrahdropteroic acid (MTHP) gave the protected reference compounds 6S-α-12 or 6R-α12, respectively. Basic deprotection using NaOH followed by semipreparative HPLC purification yielded reference compounds 6S-α-1 or 6R-α-1 in high chemical purity of >95% and overall yields of 7% and 6%, respectively. Scheme 2: Synthesis of reference compounds 6S-α-1 and 6R-α-1a

a

(i) Cu(OAc)2∙H2O, Na-(+)-L-ascorbate, MeCN/H2O, 60 °C, 78%; (ii) CH2Cl2/TFA (10:1), rt, 2

h, quant.; (iii) 11, HBTU, Et3N, DMF, 0 °C to rt, 5 h: 6S-α-12: 38%; 6R-α-12: 17%; (iv) 1M NaOH, rt, 4 h, 6S-α-1: 22%, 6R-α-1: 42%. Reference compounds 6S-γ- and 6R-γ-click-fluoroethyl-5-MTHFs (6S-γ-1 and 6R-γ-1, Scheme 3) were obtained after a deprotection reaction of intermediates 6R-γ-12 or 6S-γ-12 (provided by 10


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Merck & Cie, Schaffhausen, Switzerland) using NaOH and subsequent purification by semipreparative HPLC in chemical yields of 52% and 50%, respectively and high chemical purity of >95%.

Scheme 3: Deprotection reaction to afford reference compounds 6S-γ-1 and 6R-γ-1a

a

(i) 1M LiOH, rt, 3 h, 6S-γ-1: 52%, 6R-γ-1: 50%.

Radiochemistry. The radiosyntheses of 6S- and 6R-α-click-[18F]fluoroethyl-5-MTHF (6S-α- and 6R-α-[18F]1) were accomplished in a two-step reaction sequence involving either 6S-α-8 or 6R-α-8 with [18F]fluoroethyl azide (Scheme 4 A). 6S-α- and 6R-α-[18F]1 were obtained after approximately 100 min of total radiosynthesis time in radiochemical yields of 1-4% and 2-3% (d.c.), respectively and a radiochemical purity of >95% with molar activity in the range of 160-245 GBq/µmol. The identity of 6S-α- and 6R-α-[18F]1 was confirmed by co-injection of the 11



nonradioactive reference compounds. A similar radiosynthetic procedure was followed for the radiosyntheses of the corresponding γ-conjugated 6R- and 6S-5-MTHF radiotracers (6S-γ- and 6R-γ-[18F]1) by reacting [18F]9 with either 6S-γ-8 or 6R-γ-8 (Scheme 4, B). At the end of the syntheses, 6S-γ- and 6R-γ-[18F]1 were obtained in radiochemical yields of 5-7% and 2-4% (d.c.), respectively and high chemical purity of >95%. Molar activity for both radiotracers was in the range of 139-233 GBq/µmol. Co-injection of the reference compounds in the analytical HPLC confirmed the identities of 6S-γ- and 6R-γ-[18F]1. 6S-α- and 6R-α-[18F]1 as well as 6S-γ- and 6R-γ-[18F]1 were purified by semipreparative HPLC using a solvent system of EtOH in phosphate buffer pH 7.4 containing sodium ascorbate (50 g/L). The product peak was directly collected in the product vial which contained 1 mL of a Lcysteine solution (40 mg/mL). The product solution was directly used for further experiments. The distribution coefficients (logD7.4) of the four reduced radiofolate conjugates were not significantly different ranging from -2.2 to -2.6, whereas the 6S-derivatives (P = 0.0006), but not the 6R-conjugates (P > 0.01) exhibited significant differences compared to the corresponding folic acid derivatives (LogD7.4 = -3.0, Table S2).12 Scheme 4: Two-step radiosynthesis of 6S- and 6R-α-click-[18F]fluoroethyl-5-MTHFs (A) and 6S- and 6R-γ-click-[18F]fluoroethyl-5-MTHFs (B)a

12


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a

(i) [18F]KF-K222, MeCN, 80 °C, 15 min; (ii) [18F]9, Cu(OAc)2∙H2O, Na-(+)-L-ascorbate,

EtOH/H2O/MeCN, 85 °C, 20 min.

In Vitro Characterization. The FR-binding affinities of the four nonradioactive reference compounds 6S-α-1, 6R-α-1, 6S-γ1 and 6R-γ-1, the physiological 6S-5-MTHF, the non-physiological 6R-5-MTHF and folic acid were determined using KB tumor cells in a displacement assay with [3H]-folic acid (Table 1). Similar affinities in the range of 17.7 to 25.8 nM were found for all four synthesized folate conjugates as well as for non-derivatized 6S- and 6R-5-MTHF. All reduced folate conjugates showed considerably lower FR-binding affinities compared to folic acid and the previously reported corresponding oxidized forms α/γ-click-fluoroethyl-folic acid derivatives (IC50 = ~1.5 nM).12

13



Table 1: Comparison of in vitro binding affinities to the FR-α of the four 5-MTHF conjugates, 6S- and 6R-5-MTHF and folic acid. (n = 3) compound

IC50 [nM]

6S-α-1

24.0 േ 4.6

6R-α-1

22.3 േ 3.0

6S-γ-1

17.7 േ 7.2

6R-γ-1

22.2 േ 6.9

6S-5-MTHF

20.6 േ 0.4

6R-5-MTHF

25.8 േ 5.8

folic acid

0.61 േ 0.13

Cell Uptake and Internalization Studies. Cell uptake was in the range of 50-60% for all four folate radioconjugates after an incubation period of 3 h (Figure 2). In all cases, cell internalization increased over time resulting in about 15% after 3 h of incubation. FR-specific binding was confirmed by co-incubating cells with an excess of folic acid which resulted in an almost complete blockade of the uptake (95% demonstrating the sensitivity of 5-MTHF derivatives to decomposition in the presence of ionizing radiation. L-Cysteine was found to be the optimal thiol-bearing antioxidant for stabilizing the radiofolates since it is well tolerated by mammals and is highly effective in stabilizing reduced folates determined in a non-radioactive stability test (Supporting Information, Table S1). However, L-cysteine could not be used as antioxidant for the semipreparative HPLC purification of the radiotracers due to solubility issues and its effect on the pH of the HPLC eluent. The in vitro binding affinity experiments with FR-expressing KB cells demonstrated that neither the configuration at position 6 of 5-MTHF, nor conjugation at either the α- or γ-position of the glutamate had an influence on the binding to FR-α since all the four reference compounds exhibited binding affinity values of around 20 nM similar to the values of the non-derivatized 6S- and 6R-5-MTHF. As expected, the previously reported corresponding α/γ-click-fluoroethylfolic acid derivatives exhibited higher affinities (~1.5 nM) confirming the results of Wang et al., who showed that folic acid exhibits a higher affinity to FR-α compared to 6S- and 6R-5MTHF.12,15 Surprisingly, in vitro cell uptake and internalization experiments performed with the four 5-MTHF radioconjugates showed high and FR-specific binding to the FR-α similar to folic acid-based radiotracers revealing that the affinity of the 5-MTHF derivatives is high enough for binding the receptor. Low background activity such as in the muscle, heart, lung and skin throughout the whole animal for all four radiofolate conjguates was found in the biodistribution and PET studies suggesting that the 5-MTHF conjugates are not transported by the ubiquitously expressed RFC. These results support the data reported by Westerhof et al. who showed that the glutamic acid group, 20


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which is twice negatively charged under physiological conditions, is crucial for efficient transport of reduced folates by the RFC.22 Furthermore, the increased molecular size of the folate conjugates may also prevent transport by the RFC. Therefore, it is likely that 5-MTHF conjugated at either the α- or γ-carboxylic group of the glutamate exhibit potential selectivity to FR-α over RFC. Similar uptake values were found for the two pairs of regioisomers, 6S-α- and 6S-γ-[18F]1, as well as for 6R-α- and 6R-γ-[18F]1 in the most important tissues including the liver and the kidneys (Figure 3) indicating that the site of conjugation at either the α- or γ-carboxylic functionality of the glutamate of either 6S- or 6R-5-MTHF has no major impact on the in vivo distribution pattern of

18

F-labeled folate conjugates. Interestingly, a considerable differences in

the in vivo behavior of the 6S- and the 6R-5-MTHF conjugates were found in both the biodistribution and the PET studies. A significantly lower amount of the 6S-isomers (6S-α- and 6S-γ-[18F]1) accumulated in the kidneys compared to the corresponding 6R-isomers (6R-α- and 6R-γ-[18F]1) and the previously reported corresponding folic acid derivatives (α-click-[18F]FEfolic acid: 54.0 േ 10.4% IA/g, γ-click-[18F]FE-folic acid: 24.0 േ 3.27% IA/g), which may be advantegous for radiotherapeutic applications in future as kidney damage represents a critical issue in this field.12,23 Possibly the binding affinities of the R- and S-form of the reduced folate conjugates to the murine FR-α may be different than to the human FR expressed on the KB tumor cells, however, this will have to be verified in future investigations. In contrast and despite the 10-20-fold reduced FR-binding affinities of the 5-MTHF conjugates compared to the folic acid derivatives, similar high KB tumor uptake was observed for the folate conjugates demonstrating that the binding affinities of 6S- and 6R-5-MTHF are sufficiently high for targeting FR-α-positive tissues in vivo. The 6S-5-MTHF conjugates exhibited improved tumor21



to-kidney ratios compared to the ratios of the 6R-conjugates and the corresponding folic acid derivatives (Table 2). With respect to imaging purposes, a major drawback of the 6S-5-MTHF conjugates is, however, the increased uptake into the liver compared to the 6R-5-MTHF derivatives resulting in considerably lower tumor-to-liver ratios. These results cannot be explained by the hydrophobicity of the derivatives as similar hydrophobicities were found for all four reduced folate conjugates. In order to find explanations for the different pharmacokinetics of the 6S- and 6R-diastereoisomers, experiments investigating the transport of the α- and γconjuagted 6S- and 6R-5-MTHF conjugates by different transport and carrier systems (e.g. proton-coupled folate transporter) are currently ongoing in our laboratories. Furthermore, investigations on the selectivity of the 6S- and 6R-5-MTHF conjugates to the FR-α over the FRβ, which is overexpressed on activated macrophages involved in inflammations, will be carried out in order to evaluate the possibility of selective tumor imaging with these reduced folate conjugates.

CONCLUSION In this study, we synthesized a set of

18

F-labeled 5-MTHF conjugates for the imaging of FR-

positive tumors. Whereas 6S- and 6R-diastereoisomers of 5-MTHF conjugates exhibited different biological characteristics in mice, the conjugation sites (α vs γ) did not influence the in vivo behavior of the derivatives. The 6R-5-MTHF conjugates seem to be more favorable for tumor PET imaging due to the lower liver uptake compared to the 6S-conjugates. Clearly, our results demonstrate that using 5-MTHF as FR targeting molecule represents an alternative and promising concept for imaging of FR-positive tumors. 22


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EXPERIMENTAL PROCEDURES General. Reagents and solvents were purchased from Sigma-Aldrich Chemie GmbH, Acros Organics and used without purification. The building blocks were bought from ABCRChemicals, Apollo Scientific, Fluka-Chemie and Merck & Cie (Schaffhausen, Switzerland). 6Rand 6S-10-formyl-5-MTHP (6R-6 and 6S-6) and 6R-γ-8, 6S-γ-8, 6R-γ-12 and 6S-γ-12 were provided by Merck & Cie (Schaffhausen, Switzerland). Quality control of these building blocks was performed with 1H-NMR, 13C-NMR, COSY NMR and HR-MS. The solvents for conducting the flash chromatography, transferring reaction mixtures, extraction and washing processes were directly purchased from the fuel depot from ETH Zurich. 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 following abbreviations are used in this section for the description of the 1H NMR: singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m), doublet of doublets (dd), doublet of doublets of doublets (ddd) and broad signal (bs). The chemical shifts of complex multiplets are given as the range of their occurrence. High-resolution mass spectra (HR-MS) were recorded by the MS service of the Laboratorium für Organische Chemie at ETH Zurich on a Varian IonSpec Ultima MALDI-FT-ICR or a Bruker Daltonics Ultra-Flex II MALDI-TOF. Trans-2-[3-(4-tertButylphenyl)-2-methyl-2-propenyli-dene]malononitrile

(DCTB)

and

3-hydroxypyridine-2-

carboxylic acid (3-HPA) served as matrices for MALDI mass spectrometry. ESI mass spectra were recorded with a Bruker FTMS 4.7 T BioAPEXII (ESI). Analytical and semipreparative HPLC was performed with a Merck-Hitachi system, equipped with a D-7000 interface, L-7400 24


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UV detector, and a L-7100 pump, using a sunfire C18 column (4.6 x 150 mm, 5 µm) or a sunfire C18 column (10 x 150 mm, 5 µm), respectively. Preparative HPLC was performed with a Merck-Hitachi system, equipped with a D7000 interface, L-7400 UV detector, and a L-7150 pump, using an Ultimate XB-C18 column (150 x 21.2 mm, 5 µm) (Ultisil, Welch Materials). Analytical radio-HPLC was performed on an Agilent 1100 series HPLC system, equipped with a 100 µL-lop and a GabiStar radiodetector (Raytest) using the analytical Phenomenex Gemini C18 column (4.6 x 250 mm, 5µm, 110 Å). Semipreparative radio-HPLC system was equipped with smartline pump 1000, smartline manager 5000, smartline UV detector 2500 (Knauer), 5 mLloop, and a GabiStar radiodetector (Raytest) using a Phenomenex Gemini C18 column (10 x 250 mm, 5 µm, 110 Å).

Organic Syntheses Synthesis of 6S-α-5-MTHF Alkyne. The synthesis of the 6S-α-5-MTHF alkyne 6S-α-7 was performed in analogy to the synthesis of the α-alkyne folic acid derivative published previously (Scheme 1).12 Boc-Glu(OMe)-OH∙DCHA 2 (406 mg, 0.92 mmol) was dissolved in anhydrous DMF (11 mL) at 0 °C and DIPEA (234 µL, 1.38 mmol) and HBTU (383 mg, 1.0 mmol) were added. A solution of H-Pra-OMe∙HCl 3 (150 mg, 0.92 mmol) in anhydrous DMF (3 mL and DIPEA (234 µL, 1.38 mmol) was added to the solution containing 2 and the resulting reaction mixture was stirred for 1 h at 0 °C, allowed to warm up to room temperature and stirred for 1 h. Afterwards, the reaction mixture was diluted with H2O (50 mL) and extracted with EtOAc (3 x 50 mL). The combined organic phases were washed with brine (3 x 50 mL), dried over Na2SO4 and concentrated under reduced pressure. The crude product was purified by flash 25



chromatography on silica gel with hexane/EtOAc (60:40→40:60). Evaporation of the organic solvents of the product fractions afforded product 4 as a white solid (292 mg, 86 %). 1H-NMR (400 MHz, DMSO) δ 8.30 (d, J = 8.0 Hz, 1H), 6.93 (d, J = 8.0 Hz, 1H), 4.47 – 4.38 (m, 1H), 4.06 – 3.98 (m, 1H), 3.63 (s, 3H), 3.58 (s, 3H), 2.91 (t, J = 2.4 Hz, 1H), 2.62 (dd, J = 6.6, 2.6 Hz, 2H), 2.38 – 2.31 (m, 2H), 1.94 – 1.84 (m, 1H), 1.81 – 1.70 (m, 1H), 1.38 (s, 9H). HR-MS (ESI): calculated for C17H26N2NaO7: 393.1632, m/z found was 393.1628. For the removal of the Boc protecting group, intermediate 4 was dissolved in CH2Cl2/TFA (10:1, 4.5 mL) and the reaction mixture was stirred for 2 h at room temperature. After completion of the reaction, the solution was concentrated under reduced pressure to yield the TFA salt of amine 5 quantitatively (73.0 mg), which was directly used for the coupling with 6R-10-formyl-5-MTHP (6R-6). For the coupling, 6R-6 (116 mg, 0.32 mmol) was dissolved in anhydrous DMF (2 mL) at 0 °C and DIPEA (138 µL, 0.81 mmol) and HBTU (154 mg, 0.41 mmol) were added. The TFA salt of amine 5 (104 mg, 0.27 mmol) was dissolved in anhydrous DMF (2 mL) and DIPEA (138 µL, 0.81 mmol, 3 eq.) was added at 0 °C and the resulting solution was added to the reaction mixture containing pteroic acid 6R-6. The resulting solution was stirred for 1 h at 0 °C, allowed to warm up to room temperature and stirred for 3 h. Reaction monitoring was done by analytical HPLC and after completion of the reaction, the solvent was evaporated under reduced pressure and the crude product was purified via preparative HPLC. After lyophilization of the product fractions intermediate 6R-α-7 was obtained as a white solid (49.0 mg, 30%). 1H-NMR (400 MHz, DMSO) δ 9.85 (s, 1H), 8.52 (s, 1H), 8.48 (d, J = 7.2 Hz, 2H), 7.88 (d, J = 8.4 Hz, 2H), 7.53 (d, J = 8.4 Hz, 2H), 6.50 (d, J = 3.6 Hz, 1H), 6.38 (d, J = 3.6 Hz, 1H), 5.86 (d, J = 12.0 Hz, 2H), 4.58 – 4.51 (m, 1H), 4.47 – 4.40 (m, 1H), 3.64 (s, 3H), 3.58 (s, 3H), 3.15 – 3.10 (m, 1H), 26


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3.05 – 2.98 (m, 2H), 2.94 – 2.91 (m, 1H), 2.65 (d, J = 2.4 Hz, 1H), 2.63 (d, J = 2.4 Hz, 1H), 2.42 (s, 4H), 2.11 – 2.02 (m, 1H), 2.01 – 1.92 (m, 1H). HR-MS (MALDI/ESI) calculated for C28H34N8O8; 610.2494, m/z found was: 610.2494. Cleavage of the protecting groups was carried out by dissolving intermediate 6R-α-7 (48.8 mg, 0.08 mmol) in 1M NaOH (1 mL) and stirring for 3 h at room temperature. Reaction monitoring was done by analytical HPLC and after completion of the reaction, the pH was adjusted to pH 8 using 1M HCl. Purification of the product was performed via preparative HPLC and after lypophilization of the product peak, 6S-α-8 was obtained as a white solid (30.2 mg, 68%) with a chemical purity of >95%. 1H NMR (500 MHz, DMSO) δ 8.52 (d, J = 4.0 Hz, 1H), 7.66 (d, J = 8.7 Hz, 2H), 7.61 (d, J = 6.3 Hz, 1H), 6.56 (d, J = 8.8 Hz, 2H), 6.49 (d, J = 2.5 Hz, 1H), 6.17 (s, 1H), 6.08 (d, J = 0.6 Hz, 1H), 4.31 (dd, J = 13.5, 7.7 Hz, 1H), 3.87 – 3.82 (m, 2H), 3.22 – 3.11 (m, 2H), 2.92 (d, J = 6.7 Hz, 1H), 2.89 – 2.83 (m, 1H), 2.83 – 2.76 (m, 1H), 2.70 – 2.63 (m, 1H), 2.54 (d, J = 2.6 Hz, 1H), 2.48 (s, 2H), 2.26 (dd, J = 13.4, 7.0 Hz, 2H), 2.03 – 1.84 (m, 2H). 13C NMR (125 MHz, DMSO) δ 175.10, 172.16, 171.26, 166.51, 159.13, 153.18, 151.47, 129.12, 120.68, 110.83, 99.54, 81.84, 71.96, 55.22, 53.59, 52.45, 43.48, 42.71, 35.49, 31.55, 27.30, 21.74. HR-MS (MALDI/ESI): calculated for C25H31N8O7; 555.2310, m/z found was: 555.2312.

Synthesis of 6R-α-5-MTHF Alkyne. The synthesis of 6R-α-5-MTHF alkyne (6R-α-8) was performed in analogy to the synthesis of the corresponding 6S-α-5-MTHF alkyne. Intermediate 5 was coupled to 6S-10-formyl-5-MTHP (6S-6, 519 mg, 1.45 mmol) was dissolved in anhydrous DMF (8 mL) at 0 °C and DIPEA (620 µL, 3.63 mmol) and HBTU (690 mg, 1.82 mmol) were added. The TFA salt of amine 5 (327 mg, 1.21 mmol) was dissolved in anhydrous DMF (4 mL) 27



and DIPEA (620 µL, 3.63 mmol) was added at 0 °C and the resulting solution was added to the reaction mixture containing 6S-6. The resulting solution was stirred for 1 h at 0 °C, allowed to warm up to room temperature and stirred for 3 h. Reaction monitoring was done by analytical HPLC and after completion of the reaction, the solvent was evaporated under reduced pressure and the crude product was purified via preparative HPLC. After lyophilization of the product fractions, intermediate 6S-α-7 was obtained as a white solid (132 mg, 18%). 1H NMR (400 MHz, DMSO) δ 9.83 (s, 1H), 8.53 (s, 1H), 8.49 (d, J = 8.0 Hz, 2H), 7.88 (d, J = 8.6 Hz, 2H), 7.53 (d, J = 8.6 Hz, 2H), 6.38 (d, J = 4.0 Hz, 1H), 5.84 (s, 2H), 4.59 – 4.51 (m, 1H), 4.47 – 4.40 (m, 1H), 3.64 (s, 3H), 3.58 (s, 3H), 3.14 – 3.08 (m, 1H), 3.05 – 2.97 (m, 2H), 2.94 – 2.91 (m, 1H), 2.64 (dd, J = 6.5, 2.6 Hz, 2H), 2.42 (s, 3H), 2.11 – 2.03 (m, 1H), 2.01 – 1.92 (m, 1H). HR-MS (MALDI/ESI) calculated for C28H34N8O8; 610.2494, m/z found was: 610.2495. Cleavage of the protecting groups was carried out by dissolving intermediate 6S-α-7 (45.0 mg, 0.07 mmol) in 1M NaOH (1 mL) and stirring for 2 h at room temperature. Reaction monitoring was done by analytical HPLC. After completion of the reaction the pH was adjusted to pH 8 using 1M HCl. Purification of the product was performed via preparative HPLC and after lyophilization of the product fractions, 6R-α-8 was obtained as a white solid (23.9 mg, 58%) with a chemical purity of >95%. 1H NMR (500 MHz, DMSO) δ 8.33 (s, 1H), 7.66 (d, J = 8.6 Hz, 2H), 6.56 (d, J = 8.6 Hz, 2H), 6.48 (s, 1H), 6.15 (s, 2H), 6.08 (s, 1H), 4.37 – 4.32 (m, 1H), 3.91 – 3.87 (m, 1H), 3.20 – 3.13 (m, 2H), 2.96 – 2.90 (m, 1H), 2.89 – 2.83 (m, 1H), 2.83 – 2.76 (m, 1H), 2.70 – 2.64 (m, 1H), 2.60 – 2.54 (m, 2H), 2.49 (s, 3H), 2.43 (s, 1H), 2.28 (t, J = 7.4 Hz, 2H), 2.01 (dd, J = 13.6, 5.8 Hz, 1H), 1.89 (dd, J = 13.6, 8.6 Hz, 1H). 13C NMR (125 MHz, DMSO) δ 175.13, 175.10, 172.57, 171.68, 166.90, 159.48, 153.55, 151.86, 129.55, 121.09, 111.22, 99.99, 28


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82.13, 72.48, 55.70, 53.73, 52.75, 43.96, 43.15, 35.89, 31.56, 27.67, 22.14. HR-MS (MALDI/ESI): calculated for C25H31N8O7; 555.2310, m/z found was: 555.2316.

Synthesis of 6S-α-Click-FE-5-MTHF. The synthesis of the 6S-α-click-FE-5-MTHF (6S-α-1, Scheme 2) was performed in analogy to the synthesis of the α-click-FE folic acid derivative as previously reported.12 Intermediate 4 (100 mg, 0.27 mmol) was dissolved in anhydrous DMF (1 mL) and aqueous solutions of Cu(OAc)2∙H2O (100 µL, 0.3 mM) and Na-(+)-L-ascorbate (100 µL, 1.2 mM) were added. In situ generated fluoroethyl azide 9 (48.1 mg, 0.54 mmol) was added and the reaction mixture was stirred for 2.5 h and heated at 60 °C. Then, additional fluoroethyl azide 9 (24.0 mg, 0.27 mmol, 1 eq.) was added to the reaction solution. Reaction monitoring was performed via analytical HPLC using 10 mM NH4HCO3 solution (solvent A) and MeCN (solvent B) and a gradient as follows: 0–30 min: 90 – 45% A, 30–31 min: 45 – 20% A, 31–35 min: 20% A, 35–36 min: 20 – 90% A, 36–40 min: 90% A. The absorption was measured at 210 nm. After completion of the reaction, the solution was allowed to cool down to room temperature. H2O (20 mL) was added to the reaction mixture and an extraction with EtOAc (3 x 20 mL) was carried out. The combined organic phases were washed with brine, dried over Na2SO4, and concentrated to give the crude mixture of the product. Purification was achieved by preparative HPLC using 10 mM NH4HCO3 solution (solvent A) and MeCN (solvent B) and a gradient as follows: 0–30 min: 90 – 45% A, 30–31min: 45 – 20% A, 31–35 min: 20% A, 35–36 min: 20 – 90% A, 36–40 min: 90% A. Absorption was measured at 210 nm. After lyophilization of the product fractions, intermediate 10 was obtained as a white solid (97.2 mg, 78%). 1H-NMR (400 MHz, DMSO) δ 8.32 (d, J = 7.6 Hz, 1H), 7.90 (s, 1H), 6.94 (d, J = 8.4 Hz, 1H), 4.85 (t, J = 29



4.4 Hz, 1H), 4.75 – 4.71 (m, 1H), 4.70 – 4.67 (m, 1H), 4.64 – 4.60 (m, 1H), 4.57 – 4.50 (m, 1H), 3.98 – 3.90 (m, 1H), 3.60 (s, 3H), 3.59 (s, 3H), 3.11 (d, J = 5.4 Hz, 1H), 3.05 (d, J = 8.4 Hz, 1H), 2.35 – 2.28 (m, 2H), 1.88 – 1.79 (m, 1H), 1.78 – 1.68 (m, 1H), 1.37 (s, 9H). HR-MS (ESI) calculated for: C19H30FN5NaO7: 482.2021, m/z found was: 482.2019. Boc deprotection was achieved by dissolving 10 (97.2 mg, 0.21 mmol) in CH2Cl2/TFA (10:1, 5.5 mL) and stirring at room temperature until TLC showed completion of the reaction. The organic solvents were directly evaporated under reduced pressure to afford the TFA salt of amine 11 (99.1 mg, quant.), which was directly used for the coupling with 6R-α-6. For the coupling, 6R-α6 (91.0 mg, 0.25 mmol) was dissolved in anhydrous DMF (2 mL) at 0 °C. Then, DIPEA (108 µL, 0.63 mmol) and HBTU (120 mg, 0.32 mmol) were added and the reaction mixture was stirred for 1 h at 0 °C. The TFA salt of amine 11 (100 mg, 0.21 mmol) was dissolved in anhydrous DMF (2 mL) at 0 °C and DIPEA (108 µL, 0.63 mmol, 3 eq) was added. The solution was transferred to the solution containing 6R-α-6 and the resulting reaction mixture was stirred for 1 h at 0 °C. The transparent reaction mixture was allowed to warm up to room temperature and stirring was continued for 1 h. After completion of the reaction, the organic solvents were evaporated and purification of the crude product was carried out by preparative HPLC. After lyophilization of the product fractions, 6R-α-12 was obtained after lyophilization as a white solid (56.3 mg, 38%). 1H NMR (400 MHz, DMSO) δ 9.84 (s, 1H), 8.52 (d, J = 5.6 Hz, 2H), 7.95 (s, 1H), 7.88 (d, J = 8.4 Hz, 1H), 7.53 (d, J = 8.8 Hz, 1H), 6.38 (d, J = 3.6 Hz, 1H), 5.84 (s, 1H), 4.87 – 4.82 (m, 1H), 4.75 – 4.70 (m, 1H), 4.69 – 4.63 (m, 1H), 4.62 – 4.57 (m, 1H), 4.55 – 4.48 (m, 1H), 4.47 – 4.41 (m, 1H), 3.62 (s, 3H), 3.59 (s, 3H), 3.29 (s, 2H), 3.18 (d, J = 5.6 Hz, 1H), 3.14 (d, J = 5.2 Hz, 1H), 3.10 (d, J = 3.4 Hz, 1H), 3.09-3.05 (m, 1H), 3.05-3.01 (m, 1H), 3.01 – 30


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2.98 (m, 1H), 2.67 (s, 1H), 2.42 (s, 3H), 2.04 – 1.91 (m, 2H). HR-MS (MALDI/ESI): calculated for: C30H38FN11NaO8: 722.2781, m/z found was: 722.2780. For the deprotection, 6R-α-12 (10.0 mg, 0.01 mmol) was dissolved in 1M NaOH (300 µL) and the solution was stirred for 4 h at room temperature. Reaction monitoring was done by analytical HPLC. After completion of the reaction, the pH was adjusted to pH 8 using 1 M HCl and purification of the product was achieved via semipreparative HPLC. The reference compound 6S-α-1 was obtained after lyophilization of the product fractions in 22% yield (1.91 mg) and high chemical purity of >99 %. 1H NMR (500 MHz, DMSO) δ 8.16 (d, J = 4.4 Hz, 1H), 7.90 (s, 1H), 7.87 (s, 1H), 7.66 (d, J = 8.6 Hz, 2H), 6.55 (d, J = 8.6 Hz, 2H), 6.47 (s, 1H), 6.18 – 5.96 (m, 3H), 4.84 – 4.67 (m, 2H), 4.61 – 4.51 (m, 2H), 4.33 (dd, J = 13.7, 7.7 Hz, 1H), 4.22 – 4.15 (m, 1H), 3.17 (d, J = 10.3 Hz, 2H), 2.97 (d, J = 7.8 Hz, 1H), 2.96 – 2.88 (m, 2H), 2.88 – 2.76 (m, 2H), 2.49 (s, 3H), 2.27 (t, J = 7.6 Hz, 2H), 2.00 – 1.81 (m, 2H).

13

C NMR (125 MHz, DMSO) δ

174.97, 171.81, 166.89, 159.43, 153.54, 151.89, 144.29, 129.53, 123.67, 120.94, 111.24, 100.02, 82.92, 81.59, 55.65, 53.49, 53.28, 50.31, 50.15, 43.94, 43.11, 35.94, 31.32, 28.45, 27.41. HR-MS (MALDI/ESI): calculated for: C27H35FN11O7: 644.2699, m/z found was: 644.2711.

Synthesis of 6R-α-Click-FE-5-MTHF. The synthesis of 6R-α-click-FE-5-MTHF (6R-α-1, Scheme 2) was performed in analogy to the synthesis of the corresponding 6S-α-1. For the coupling, 6S-α-6 (104 mg, 0.29 mmol) was dissolved in anhydrous DMF (2 mL) at 0 °C. Then, DIPEA (120 µL, 0.72 mmol) and HBTU (137 mg, 0.36 mmol) were added and the reaction mixture was stirred for 1 h at 0 °C. The TFA salt of amine 11 (115 mg, 0.24 mmol) was dissolved in anhydrous DMF (2 mL) at 0 °C and DIPEA (108 µL, 0.63 mmol) was added. The 31



solution was transferred to the solution containing 6S-α-6 and the resulting reaction mixture was stirred for 1 h at 0 °C. The transparent reaction mixture was allowed to warm up to room temperature and stirring was continued for 1 h. After completion of the reaction, the organic solvents were evaporated and purification of the crude product was carried out by preparative HPLC. 6S-α-12 was obtained after lyophilization of the product fractions as a white solid (29.0 mg, 17%). 1H NMR (400 MHz, DMSO) δ 9.82 (s, 1H), 8.53 (s, 1H), 8.51 (s, 1H), 7.95 (s, 1H), 7.89 (d, J = 8.6 Hz, 2H), 7.53 (d, J = 8.6 Hz, 2H), 6.38 (d, J = 3.4 Hz, 1H), 5.84 (s, 2H), 4.84 (dd, J = 8.8, 4.4 Hz, 1H), 4.72 (dd, J = 8.8, 4.4 Hz, 1H), 4.68 – 4.65 (m, 1H), 4.61 – 4.58 (m, 1H), 4.54 – 4.50 (m, 1H), 4.47 – 4.42 (m, 1H), 3.62 (s, 3H), 3.59 (s, 3H), 3.18 – 2.98 (m, 6H), 2.42 (s, 3H), 2.04 – 1.93 (m, 2H). HR-MS (MALDI/ESI): calculated for: C30H39FN11O8: 700.2962, m/z found was: 700.2962. For the deprotection, 6S-α-12 (10.0 mg, 0.01 mmol) was dissolved in 1M NaOH (300 µL) and the solution was stirred for 2 h at room temperature. Reaction monitoring was done by analytical HPLC. After completion of the reaction, the pH was adjusted to pH 8 using 1 M HCl and purification of the product was achieved via semipreparative HPLC. The reference compound 6R-α-1 was obtained after lyophilization of the product fractions in 42% yield (3.9 mg) and high chemical purity of >95 %. 1H NMR (500 MHz, DMSO) δ 9.96 (s, 1H), 8.21 (d, J = 8.0 Hz, 1H), 8.04 (d, J = 7.6 Hz, 1H), 7.93 (s, 1H), 7.67 (d, J = 8.6 Hz, 2H), 6.56 (d, J = 8.6 Hz, 2H), 6.47 (s, 1H), 6.08 (s, 1H), 5.93 (s, 2H), 4.87 – 4.77 (m, 1H), 4.77 – 4.69 (m, 1H), 4.66 – 4.54 (m, 2H), 4.43 – 4.34 (m, 2H), 3.23 – 3.11 (m, 4H), 3.04 – 2.95 (m, 1H), 2.95 – 2.84 (m, 2H), 2.82 – 2.76 (m, 1H), 2.49 (s, 3H), 2.33 – 2.27 (m, 2H), 2.02 – 1.93 (m, 1H), 1.92 – 1.84 (m, 1H). 13C NMR (125 MHz, DMSO) δ 174.68, 172.24, 166.91, 159.27, 153.46, 151.90, 143.67, 129.56, 123.84, 32


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120.88, 111.22, 100.04, 82.92, 81.58, 55.66, 53.20, 52.41, 50.38, 50.22, 43.97, 43.11, 35.94, 30.98, 27.91, 27.29. HR-MS (ESI): calculated for: C27H35FN11O7: 644.2699, m/z found was: 644.2701.

Synthesis of 2-Fluoroethyl Azide. Compound 9 was synthesized in analogy to procedures published in the literature.24,25

6S-γ-Click-Fluoroethyl-5-Methyltetrahydrofolate. 6R-γ-12 (10.1 mg, 14.4 µmol) was dissolved in 1M LiOH (300 µL) and the solution was stirred for 3 h at room temperature (Scheme 3). After completion of the reaction, the solution was neutralized with 1M HCl and the product was purified by semipreparative HPLC. Lyophilization of the product fractions afforded the reference compound 6S-γ-1 as a white solid in 50% yield (4.81 mg) and high chemical purity of >95%. 1H NMR (500 MHz, DMSO) δ 7.89 (d, J = 6.2 Hz, 1H), 7.86 – 7.82 (m, 1H), 7.80 (s, 1H), 7.60 (d, J = 8.2 Hz, 2H), 6.55 (d, J = 8.2 Hz, 2H), 6.43 – 6.18 (m, 2H), 6.06 (s, 1H), 4.79 (t, J = 4.6 Hz, 1H), 4.69 (t, J = 4.6 Hz, 1H), 4.64 (t, J = 4.6 Hz, 1H), 4.59 (t, J = 4.6 Hz, 1H), 4.30 – 4.23 (m, 1H), 4.19 – 4.12 (m, 1H), 3.24 – 3.09 (m, 3H), 2.96 – 2.78 (m, 4H), 2.49 (s, 3H), 2.21 – 2.10 (m, 2H), 2.04 – 1.94 (m, 1H), 1.90 – 1.80 (m, 1H). 13C NMR (125 MHz, DMSO) δ 174.46, 173.85, 171.66, 165.99, 153.36, 151.31, 144.11, 128.74, 123.07, 121.00, 110.89, 99.46, 82.64, 81.31, 55.22, 53.16, 49.87, 49.71, 43.55, 42.70, 39.52, 35.48, 32.19, 28.27, 27.72. HR-MS (MALDI/ESI) calculated for C27H34FN11O7: 643.2621; found: 643.2621.

33



6R-γ-Click-Fluoroethyl-5-Methyltetrahydrofolate. 6S-γ-12 (10.0 mg, 14.3 µmol) was dissolved in 1M LiOH (300 µL) and the solution was stirred for 3 h at room temperature (Scheme 3). After completion of the reaction, the solution was neutralized with 1M HCl and the product was purified by semipreparative HPLC. Lyophilization of the product fractions afforded the reference compound 6R-γ-1 as a white solid in 52% yield (4.54 mg) and high chemical purity of >95%. 1H NMR (500 MHz, DMSO) δ 10.22 (s, 1H), 7.99 (d, J = 7.0 Hz, 1H), 7.94 (d, J = 7.8 Hz, 1H), 7.80 (s, 1H), 7.62 (d, J = 8.6 Hz, 2H), 6.55 (d, J = 8.6 Hz, 2H), 6.48 (s, 1H), 6.19 – 6.00 (m, 3H), 4.79 (t, J = 4.6 Hz, 1H), 4.70 (t, J = 4.6 Hz, 1H), 4.65 (t, J = 4.6 Hz, 1H), 4.59 (t, J = 4.6 Hz, 1H), 4.33 – 4.27 (m, 1H), 4.20 – 4.15 (m, 1H), 3.22 – 3.08 (m, 3H), 2.95 – 2.84 (m, 3H), 2.81 – 2.75 (m, 1H), 2.49 (s, 3H), 2.23 – 2.12 (m, 2H), 2.04 – 1.95 (m, 1H), 1.91 – 1.82 (m, 1H). 13

C NMR (125 MHz, DMSO) δ 172.09, 166.58, 159.49, 153.53, 151.78, 144.27, 129.26, 123.55,

121.31, 111.31, 99.97, 83.06, 81.72, 55.65, 53.24, 50.30, 50.15, 43.97, 43.12, 35.95, 32.54, 28.45, 27.75. HR-MS (MALDI/ESI) calculated for C27H34FN11O7: 643.2621; found: 643.2621.

Chemical Stability Test. A nonradioactive chemical stability test with the precursor folate alkyne 6S-γ-8 was performed in order to find an appropriate antioxidant, which is efficient to stabilize the 18F-labeled 5-MTHF conjugates. 6S-γ-8 (1 mg, 1.8 µmol) was dissolved in a 10 mM phosphate buffer pH 7.4 (0.5 mL) and the stability of the folate was determined by analytical HPLC over time. The area of the folate peak directly after dissolving was used as a control. Ten vials were prepared containing the folate and one certain antioxidant and the stability of the folate was determined by analytical HPLC at several time points (Supporting Information, Table S1 and Figure S1). 34


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Radiochemistry. Production of Dried [18F]Fluoride. The procedure for the production of dried [18F]fluoride is available in the Supporting Information.

Preparation of 6R- and 6S-α-Click-[18F]Fluoroethyl-5-Methyltetrahydrofolates. 6S- and 6Rα-click-FE-5-MTHF (6S-α- and 6R-α-[18F]1) were prepared in a two-step radiosyntheses similar to a procedure previously reported (Scheme 4).12 6S-α- or 6R-α-8 (2 mg, 3.6 µmol) was dissolved in H2O (500 µL) and the ethanolic solution containing [18F]fluoroethyl azide [18F]9 was added to the dissolved folate. Solutions of Cu(OAc)2∙H2O in H2O (100 mM, 100 µL) and Na-(+)-Lascorbate in H2O (500 mM, 200 µL) were added and the resulting reaction mixture was stirred for 20 min at 80 °C. After that, the solution was allowed to cool down for 2 min and sodium phosphate buffer (10 mM, pH 7.4) containing 20 mg/mL of Na-(+)-L-ascorbate (1.4 mL) was added. Then, the product was purified by semipreparative radio-HPLC and the product fraction containing either 6S-α-[18F]1 or 6R-α-[18F]1 was collected directly through a sterile filter into the vial containing a filtrated L-cysteine solution (40 mg/mL). Quality control of the tracer was performed on an analytical radio-HPLC. At the end of synthesis, 300-800 MBq (1-4% d.c.) of the final radiotracers were obtained. Molar activity ranged from 160-245 GBq/µmol and radiochemical purity was greater 95%.

Preparation

of

6R-

and

6S-γ-Click-[18F]Fluoroethyl-5-Methyltetrahydrofolate.

The

radiosyntheses of 6R- and 6S-γ-click-[18F]FE-5-MTHF (6S-γ- and 6R-γ-[18F]1, Scheme 4) was 35



performed in analogy to the radiosyntheses of the corresponding 6S-α- and 6R-α-[18F]1, but clicking [18F]9 with either 6S-γ- or 6R-γ-8 (2 mg, 3.6 µmol). At the end of synthesis, 700-1800 MBq (2-7% d.c.) of the final radiotracers were obtained. Molar activity was in the range of 139233 GBq/µmol and radiochemical purity was greater 95%.

Determination of Distribution Coefficient. Determination of the distribution coefficients (logD7.4) of 6S-α- and 6R-α-[18F]1, as well as 6S-γ- and 6R-γ-[18F]1 was done using the shakeflask method as previously described.26

In Vitro Binding Affinity. The binding affinities of reference compounds 6S-α-1, 6R-α-1, 6S-γ1, 6R-γ-1, 6R- and 6S-5-MTHF and folic acid to the FR-α were determined in a competitive in vitro binding assay on FR-positive KB cells according to a previously published procedure.11

Cell Uptake and Internalization. Cell uptake and internalization experiments with KB cells were performed with all four radiofolate conjugates 6S-α-[18F]1, 6R-α-[18F]1, 6S-γ-[18F]1 and 6R-γ-[18F]1 as previously described.27

Preparation of Tumor-Bearing Mice. Animal experiments were performed in compliance with Swiss and local laws on animal protection and approved by the Veterinary Office of Switzerland. Female CD-1 nude mice were purchased from Charles River (Germany) and kept on a folatedeficient rodent diet (ssniff Spezialdiäten GmbH, Soest, Germany) starting one week prior to the tumor cell inoculation. Experiments were performed 2 weeks after KB tumor cell inoculation. 36


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Cell suspension (5×106 cells in 100 µL of PBS pH 7.4) was inoculated into the subcutis of each shoulder.

Biodistribution Studies. Animals were injected with ~5 MBq (~0.03 nmol, 100 µL) of the corresponding radiotracer via a lateral tail vein (n = 4). Blocking studies (n = 3) were performed with excess folic acid dissolved in PBS pH 7.4 (100 µg; 100 µL per mouse) which was intravenously injected 2-3 min before injection of the radiotracer. Animals were sacrificed 60 min after injection of the radiotracer. Organs and tissues were collected and measured in a γcounter. The incorporated radioactivity was expressed as percentage of injected activity per gram of tissue [% IA/g]. Significances were calculated using a t-test (GraphPad Prism software, version 6.05) and a P-value of

Reduced 18F-Folate Conjugates as a New Class of PET Tracers for

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