Nucleophilic 18F-fluorination of phosphorofluoridates and phosphonofluoridic acids via imidazole-activated precursors

Article abstract of DOI:10.1016/j.tetlet.2021.152917

¹⁸F-Labeled organofluorophosphates are important radiosynthons that have only been previously accessible via ^18/19F-isotope-exchange with limited molar activities. Herein, a novel ¹⁸F-fluorination methodology has been developed to prepare ¹⁸F-labeled phosphorofluoridates and phosphonofluoridic acids via the [¹⁸F]F^? nucleophilic substitution of imidazole-activated precursors. The efficient one-step ¹⁸F-fluorination affords stable products in the presence of Zn(Ⅱ) with high radiochemical yields and high molar activities. This ¹⁸F-fluorination method could be used to prepare various phosphorofluoridate and phosphonofluoridic acid analogs for use as ¹⁸F-radiosynthons and potential positron emission tomography tracers.

Full text of DOI:10.1016/j.tetlet.2021.152917

Tetrahedron Letters 68 (2021) 152917
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Nucleophilic 18F-fluorination of phosphorofluoridates and
phosphonofluoridic acids via imidazole-activated precursors
⇑
Zhaobiao Mou, Xueyuan Chen, Chao Wang, Tao Wang, Hongzhang Yang, Zijing Li
Center for Molecular Imaging and Translational Medicine, State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, School of Public Health, Xiamen
University, Xiamen 361102, China
a r t i c l e i n f o
a b s t r a c t
18F-Labeled organofluorophosphates are important radiosynthons that have only been previously acces-
sible via 18/19F-isotope-exchange with limited molar activities. Herein, a novel 18F-fluorination method-
ology has been developed to prepare 18F-labeled phosphorofluoridates and phosphonofluoridic acids
via the [18F]Fꢀ nucleophilic substitution of imidazole-activated precursors. The efficient one-step 18F-flu-
orination affords stable products in the presence of Zn(Ⅱ) with high radiochemical yields and high molar
activities. This 18F-fluorination method could be used to prepare various phosphorofluoridate and phos-
phonofluoridic acid analogs for use as 18F-radiosynthons and potential positron emission tomography
tracers.
Article history:
Received 19 December 2020
Revised 1 February 2021
Accepted 8 February 2021
Available online 16 February 2021
Keywords:
18F-Fluorination
Phosphorofluoridate
Phosphonofluoridic acid
Imidazole
Ó 2021 Elsevier Ltd. All rights reserved.
Nucleophilic substitution
Introduction
phosphorus moieties may involve individual synthesis of the pre-
cursors, multistep radiosynthesis [8], and potential change of
Phosphate and phosphonic acid groups are privileged scaffolds
that are widespread in biologically and medicinally active com-
pounds [1]. Additionally, radiolabeled phosphates and phosphonic
acids are irreplaceable as radiosynthons and radiotracers for the
investigation of phosphate-related physiological processes and dis-
eases [2]. In prior studies, only 32P-labeled phosphates were avail-
able to monitor their physiological pathways in vitro [3], which
could not provide real-time dynamic images in vivo due to the
nuclide property of 32P (100% b– decay). Instead, 18F (97% b+ decay,
635 keV, t1/2 ꢁ 109.8 min), the most widely used positron emitting
isotope, allows wide bioisosteric replacement and in vivo imaging
with positron emission tomography (PET) [4]. However, 18F-
labeled organofluorophosphates were only previously accessible
via 18/19F-isotope-exchange with limited molar activities (Am, the
measured radioactivity per mole of compound, measured in
bioactivity of the substrate [9]. Therefore, the ideal fluorination
sites are the terminal positions of the phosphate and phosphonic
acid groups. Imidazole-activated phosphates prepared via a one-
step coupling reaction from phosphates [10] can be fluorinated
using aqueous fluoride [11]. This process can be accelerated by
the presence of Lewis acids [12], making these promising candi-
dates for precursors. It was hypothesized that the same fluorina-
tion and coupling reaction may also be applicable to phosphonic
acids.
Herein, a method for the 18F-fluorination of phosphorofluori-
dates and phosphonofluoridic acids via imidazole-activated pre-
cursors is described. Zn2+ species were determined as the best
Lewis acid catalysts among the tested metal cations (e.g. Mg2+
and Mn2+) to facilitate the elimination of imidazole via activation
of the P-N bond [13]. In addition, the in vitro and in vivo stabilities
of the 18F-labeled phosphorofluoridates and phosphonofluoridic
acids were evaluated for potential applications as radiosynthons
and PET tracers.
Bq/mol or GBq/lmol) [5,6].
Due to analogous van der Waals radius and valence electron
numbers between fluoro and hydroxyl groups, fluorination of the
phosphate or phosphonic acid moiety can produce a chemically
stable phosphorofluoridate or phosphonofluoridic acid derivative
which is isostructural and isoelectronic to the unmodified
substrate [7]. Additionally, nucleophilic fluorination on non-
Results and discussion
Five imidazole-activated precursors, sodium (1H-imidazol-1-yl)
phosphonates (1a–4a) and sodium benzyl (1H-imidazol-1-yl)phos-
phinate (5a), were synthesized in 35–82% yield from the corre-
sponding phosphates or phosphonic acid via the one-step
⇑
Corresponding author.
E-mail address: zijing.li@xmu.edu.cn (Z. Li).
https://doi.org/10.1016/j.tetlet.2021.152917
0040-4039/Ó 2021 Elsevier Ltd. All rights reserved.

Z. Mou, X. Chen, C. Wang et al.
Tetrahedron Letters 68 (2021) 152917
coupling with imidazole in the presence of triphenylphosphine and
2,20-dithiodipyridine [10] (Scheme 1). Phosphorofluoridates (1–4)
and benzyl phosphonofluoridic acid (5) were synthesized as refer-
ence compounds in 58–78% yield from 1a-5a by ZnCl2-mediated
nucleophilic fluorination [12] (Scheme 2).
A solution of 1a and various Zn(II) salts (Zn(OAc)2, ZnCl2, or Zn
(NO3)2) dissolved in anhydrous DMSO were shaken with an
azeotropically dried [18F]KF/K222 complex, which was prepared
by mixing [18F]F–, K222 ([2.2.2]-cryptand), and K2CO3, in order to
determine the optimized reaction conditions. A time-based study
(Fig. 1b) showed that the 18F-fluorination reaction was almost
complete after 20 min and afforded 67.5 1.2% radiochemical yield
(RCY). The RCY increased with an increase in temperature, and the
effect of increased temperature on RCY became negligible beyond
70 °C (Fig. 1c). In addition, the optimum precursor amount was
0.2 mg (Fig. 1d), and the optimal amount of the Zn(II) salt
was ꢂ 5.0 equiv. with respect to the precursor (Fig. 1e). Although
86.9 0.9% and 82.0 0.9% RCYs were respectively obtained in
the presence of Zn(NO3)2 and ZnCl2 for [18F]1, it is safer to use
ZnCl2 as a catalyst because Zn(NO3)2 is a hazardous chemical
which can easily explode [14]. The Am of [18F]1 was determined
Scheme 1. General synthetic route for imidazole-activated precursors 1a–5a.
Reagents and conditions: (i) (a) phosphate or phosphonic acid (1.0 equiv.),
imidazole (8.0 equiv.), PPh3 (4.0 equiv.), 2,20-dithiodipyridine (4.0 equiv.), DMF,
RT, 24–30 h; (b) NaI (8.0 equiv.), acetone, 0 °C, 2 h.
as 7.5 GBq/l
mol and the characterization of [18F]1 was performed
Table 1
Substrate scope for the synthesis of 18F-labeled phosphorofluoridates.a
Compound
Additives (mg)
RCY (%)b
K222 (0.8 mg)
K2CO3 (0.1 mg)
K222 (4.0 mg)
K2CO3 (0.5 mg)
57.1 2.5
1.9 0.4
K222 (0.8 mg)
K2CO3 (0.1 mg)
K222 (4.0 mg)
K2CO3 (0.5 mg)
69.1 11.4
10.2 2.3
K222 (4.0 mg)
90.7 4.7
K2CO3 (0.5 mg)
Scheme 2. General synthetic route for phosphorofluoridates and phosphonofluo-
ridic acids 1–5 and the radiosynthetic route for
[
18F]1–[18F]5. Reagents and
a
Reagents and conditions: 2a–4a (0.2 mg), ZnCl2 (5.0 equiv.), DMSO, 70 °C,
20 min.
conditions: (i) (a) 1a–5a (1.0 equiv.), ZnCl2 (8.0 equiv.), TBAF (3.0 equiv.), THF,
60 °C, 12 h; (b) 732 strong acid cation exchange resin, H2O, RT, 0.5 h; (ii) 1a–5a,
b
RCYs were determined by radio-TLC (n = 2–3).
[
18F]KF/K222, Zn(Ⅱ) salts (Zn(OAc)2, ZnCl2, or Zn(NO3)2), DMSO.
Fig. 1. Optimization of the 18F-fluorination conditions for [18F]1. (a) Radio-HPLC characterization of [18F]1 via co-injection of [18F]1 and 1; (b) effect of the reaction time: 1a
(0.2 mg, 0.77 mol), Zn(OAc)2 (5.0 equiv.), 70 °C; (c) effect of the temperature: 1a (0.2 mg, 0.77 mol), Zn(OAc)2 (5.0 equiv.), 20 min; (d) effect of the precursor amount: Zn
(OAc)2 (5.0 equiv.), 70 °C, 20 min; (e) effect of the equivalents of Zn(Ⅱ): 1a (0.2 mg, 0.77 mol), Zn(OAc)2, 70 °C, 20 min; (f) effect of the utilized Zn(Ⅱ) salt: 1a (0.2 mg,
0.77
l
l
l
l
mol), Zn(Ⅱ) (5.0 equiv.), 70 °C, 20 min. HPLC conditions: column: Nacalai Tesque Cosmosil 5C18-MS-Ⅱ column (4.4 mm, 4.6 mm ꢃ 250 mm); elution: isocratic, 70%
phosphate buffered saline (0.01 mol/L, pH = 7.40) and 30% CH3OH; flow rate: 1.0 mL/min. RCYs were determined by radio-TLC (n = 2–3).
2

Z. Mou, X. Chen, C. Wang et al.
Tetrahedron Letters 68 (2021) 152917
Fig. 2. Optimization for the 18F-fluorination conditions for [18F]5. (a) Radio-HPLC characterization of [18F]5 via co-injection of [18F]5 and 5; (b) effect of the reaction time: 5a
(0.2 mg, 0.82 mol), ZnCl2 (5.0 equiv.), 70 °C; (c) effect of the temperature: 5a (0.2 mg, 0.82 mol), ZnCl2 (5.0 equiv.), 5 min; (d) effect of the precursor amount: ZnCl2 (5.0
equiv.), 70 °C, 5 min; (e) effect of the equivalents of Zn(Ⅱ): 5a (0.2 mg, 0.82 mol), ZnCl2, 70 °C, 5 min; (f) effect of the utilized Zn(Ⅱ) salts: 5a (0.2 mg, 0.82 mol), Zn(Ⅱ) (5.0
l
l
l
l
equiv.), 70 °C, 5 min. HPLC conditions: column: Nacalai Tesque Cosmosil 5C18-MS-Ⅱ column (4.4 mm, 4.6 mm ꢃ 250 mm); elution: isocratic, 80% phosphate buffered saline
(0.01 mol/L, pH = 7.40) and 20% CH3OH; flow rate: 1.0 mL/min. RCYs were determined by radio-TLC (n = 2–3).
Fig. 3. Stabilities of [18F]1 and [18F]5 in vitro and in vivo. (a) Radio-HPLC analysis of [18F]1 after 90 min incubation in saline and mouse serum; (b) radio-HPLC analysis of [18F]1
in mouse blood at 30 min post-injection; (c) radio-HPLC analysis of [18F]1 in mouse urine at 30 min post-injection; (d) radio-HPLC analysis of [18F]5 after 90 min incubation in
saline and mouse serum; (e) radio-HPLC analysis of [18F]5 in mouse blood at 30 min post-injection; (f) radio-HPLC analysis of [18F]5 in mouse urine at 30 min post-injection.
HPLC conditions for the analysis of [18F]1 and [18F]5 are consistent with those used in Fig. 1 and Fig. 2, respectively.
using radio-high-performance liquid chromatography (radio-
HPLC) (Fig. 1a).
excess K2CO3 can result in a weakly alkaline pH (8.0–8.5), leading
to low RCYs.
As shown in Table 1, precursor 2a was 18F-fluorinated in
57.1 2.5% RCY. Compound 3a with chlorine substitution at the
para-position of the benzene ring afforded 69.1 11.4% RCY. Hete-
rocyclic substituted precursor 4a was also suitable for this trans-
formation and afforded 90.7 4.7% RCY. However, the RCYs for
The 18F-fluorination of sodium benzyl (1H-imidazol-1-yl)phos-
phinate (5a) was also explored. A RCY of 53.5 5.5% was observed
after only one minute at 70 °C in the presence of ZnCl2 (5.0 equiv.),
and the 18F-fluorination reaction was almost complete after 20 min
(Fig. 2b). A RCY of 63.1 1.2% was obtained after 5 min at room
temperature (RT), which increased by only 15.8% when the tem-
perature was increased to 110 °C (Fig. 2c). Additionally, a RCY
higher than 69.4 4.4% was observed for [18F]5 when ꢂ 0.2 mg
of 5a and ꢂ 5.0 equiv. (with respect to 5a) of Zn(Cl)2 were used.
(Fig. 2e). [18F]5 was synthesized from 5a (0.2 mg) in the presence
of Zn(NO3)2 (5.0 equiv.) at 70 °C for 5 min with 87.0 1.6% RCY
[
18F]1–[18F]3 decreased significantly when the amounts of K222
and K2CO3 were increased five fold (Table 1 and Table S1) due to
a change in the pH caused by K2CO3. Excess ZnCl2 is used to ensure
weakly acidic pH conditions, which are important for this nucle-
ophilic fluorination. The necessary addition of 0.1 mg K2CO3 for
[
18F]F– activation affords a favorable pH (6.0–6.5). Nevertheless,
3

Z. Mou, X. Chen, C. Wang et al.
Tetrahedron Letters 68 (2021) 152917
(Fig. 2f) and 2.2 GBq/l
mol Am. The characterization of [18F]5 was
Acknowledgments
performed using radio-HPLC (Fig. 2a).
The stabilities of 18F-labeled phosphorofluoridates and phos-
phonofluoridic acids were also evaluated in vitro and in vivo. Model
compounds [18F]1 and [18F]5 (radiochemical purity (RCP) ꢂ 95.0%
after purification) were incubated with saline and mouse serum
at 37 °C for 90 min, and the stabilities were analyzed using
radio-HPLC. All the stability studies were repeated 3 times, and
the utilized mice were ICR female mice. No defluorination was
observed after 90 min in saline or serum (Fig. 3a and Fig. 3d), indi-
cating the strong in vitro stabilities of [18F]1 and [18F]5. The in vivo
metabolic stabilities of [18F]1 and [18F]5 were measured by analyz-
ing the radio-metabolites in mice blood and urine at 30 min post-
injection. For [18F]1, the radioactivity in the blood could be barely
detected and no parent compound could be detected in mice urine
(Fig. 3b and Fig. 3c). This indicated that [18F]1 was cleared from the
blood after 30 min, presumably due to its good water-solubility,
and the radiolabeled product was hydrolyzed during circulation.
After 30 min circulation of [18F]5, the radioactivity in the blood
could barely be detected. However, a maximum 85.0% and an aver-
age 65.6 16.2% (n = 3) of the radioactive compound detected in
the urine was [18F]5 (Fig. 3e and Fig. 3f), confirming less defluori-
nation than that of [18F]1. It is hypothesized that the replacement
of the OAP bond by the CAP bond in [18F]5 can reduce the recog-
nition of phosphonofluoridic acid by phosphatases and therefore
strengthen the in vivo metabolic stabilities [15].
This study was financially supported by the National Natural
Science Foundation of China (81971674), Science Fund for Distin-
guished Young Scholars of Fujian Province (2019J06006), Funda-
mental Research Funds for the Central Universities
(20720180050), Scientific Research Foundation of State Key Labo-
ratory of Molecular Vaccinology and Molecular Diagnostics
(2016ZY002).
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.tetlet.2021.152917.
References
[1] (a) W.W. Cleland, A.C. Hengge, Chem. Rev. 106 (2006) 3252–3278;
(b) P.J. Thornton, H. Kadri, A. Miccoli, Y. Mehellou, J. Med. Chem. 59 (2016)
10400–10410;
(c) A. Mucha, P. Kafarski, L. Berlicki, J. Med. Chem. 54 (2011) 5955–5980;
(d) P. Clézardin, F.H. Ebetino, P.G.J. Fournier, Cancer Res. 65 (2005) 4971–4974.
[2] (a) O.N. Witte, A. Dasgupta, D. Baltimore, Nature 283 (1980) 826–831;
(b) P. Mackie, J. Fisher, H. Zhou, P. Choong, Br. J. Cancer 84 (2001) 951–958.
[3] (a) M. Galiè, F. Boschi, I. Scambi, F. Mergo, P. Marzola, L. Altabella, U.
Lavagnolo, A. Sbarbati, A.E. Spinelli, Theranostics 7 (2018) 4399–4409;
(b) A.J. Rosenberg, H. Liu, Z. Tu, Appl. Radiat. Isot. 102 (2015) 5–9;
(c) A.J. Taylor, P.D. Gorman, A. Farb, T.G. Hoopes, R. Virmani, Circulation 100
(1999) 2366–2372.
[4] (a) S. Preshlock, M. Tredwell, V. Gouverneur, Chem. Rev. 116 (2015) 719–766;
(b) X. Deng, J. Rong, L. Wang, N. Vasdev, L. Zhang, L. Josephson, S.H. Liang,
Angew. Chem. Int. Ed. 58 (2019) 2580–2605.
Conclusion
[5] H. Hong, L. Zhang, F. Xie, R. Zhuang, D. Jiang, H. Liu, J. Li, H. Yang, X. Zhang, L.
Nie, Z. Li, Nat. Commum. 10 (2019) 1–7.
[6] (a) N.H. Coenen, A.D. Gee, M. Adam, G. Antoni, C.S. Cutler, Y. Fujibayashi, J.M.
Jeong, R.H. Mach, T.L. Mindt, V.W. Pike, A.D. Windhorst, Nucl. Med. Biol. 55
(2017) v-xi.;
In summary, an efficient method for the 18F-fluorination of
phosphorofluoridates and phosphonofluoridic acids has been
developed via imidazole-activated phosphate and phosphonic acid
precursors in the presence of Zn(Ⅱ). This method affords >50% RCYs
for the phosphorofluoridate analogs and >80% RCYs for thebenzyl
phosphonofluoridic acid as well as high Am. The 18F-labeled benzyl
phosphorofluoridate and benzyl phosphonofluoridic acid show
sufficient in vitro stabilities, and the latter exhibits better in vivo
metabolic stability. This nucleophilic 18F-fluorination method
could be applied to prepare various phosphorofluoridate and phos-
phonofluoridic acid analogs as 18F-radiosynthons and potential PET
tracers.
(b) N. Savisto, J. Bergman, J. Aromaa, S. Forsback, O. Eskola, T. Viljanen, J.
Rajander, S. Johansson, J. Grigg, S. Luthra, O. Solin, Nucl. Med. Biol. 64–65
(2018) 8–15.
[7] (a) N. Brown, Bioisosteres in Medicinal Chemistry, Wiley, 2012, pp. 6–8;
(b) T. Hiyama, K. Kanie, T. Kusumoto, Y. Morizawa, M. Shimizu, Organofluorine
Compounds: Chemistry and Applications, Springer, 2000, pp. 2–11.
[8] C.E. McKenna, B.A. Kashemirov, A. Negahbani, K. Chen, Patent
US20200206370A1 (2020).
[9] (a) D.B. Berkowitz, M. Bose, J. Fluor. Chem. 11 (2001) 213–233;
(b) D. Liu, Y. Gao, J.H. Voigt, K. Lee, M.C. Nicklaus, L. Wu, Z. Zhang, T.R. Burke,
Bioorg. Med. Chem. Lett. 13 (2003) 3005–3007.
[10] (a) M. Strenkowska, P. Wanat, M. Ziemniak, J. Jemielity, J. Kowalska, Org. Lett.
14 (2012) 4782–4785;
(b) A.M. Marmelstein, L.M. Yates, J.H. Conway, D. Fiedler, J. Am. Chem. Soc. 136
(2014) 108–111.
Ethical statement
[11] (a) M.F. Aldersley, P.C. Joshi, H.M. Schwartz, A.J. Kirby, Tetrahedron Lett. 55
(2014) 1464–1466;
(b) M.F. Aldersley, P.C. Joshi, J. Mol. Evol. 78 (2014) 275–278.
[12] M.R. Baranowski, A. Nowicka, A.M. Rydzik, M. Warminski, R. Kasprzyk, B.A.
Wojtczak, J. Wojcik, T.D.W. Claridge, J. Kowalska, J. Jemielity, J. Org. Chem. 80
(2015) 3982–3997.
All experimental animal procedures were performed in accor-
dance with the National Institutes of Health Guidelines for the Care
and Use of Laboratory Animals and were approved by the Institu-
tional Animal Care and Utilization Committee of Xiamen
University.
[13] (a) M. Kadokura, T. Wada, C. Urashima, M. Sekine, Tetrahedron Lett. 38 (1997)
8359–8362;
(b) J. Jemielity, J. Kowalska, A.M. Rydzik, E. Darzynkiewicz, New J. Chem. 34
(2010) 829–844.
Declaration of Competing Interest
[14] H. Fan, G. Li, Environ. Protect. Xinjiang 36 (2014) 29–33.
[15] (a) H. Fleisch, Bisphosphonates in Bone Disease: From the Laboratory to the
Patient, Academic Press (2000) 27–30;
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared
to influence the work reported in this paper.
(b) K. Ogawa, H. Saji, Int. J. Mol. Imaging. (2011) 537687.
4