CC BY ND NC 4.0 · SynOpen 2017; 01(01): 0173-0179
DOI: 10.1055/s-0036-1591863
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Copyright with the author

Synthesis and Radiosynthesis of Prospective 2-Nitroimidazole Hypoxia­ PET Tracers via Thiazolidine Ligation with 5-Fluorodeoxyribose (FDR)

a  Institute of Medical Sciences and Kosterlitz Centre for Therapeutics, School of Medicine, Medical Sciences and Nutrition, University of Aberdeen, Foresterhill, Aberdeen, AB25 2ZD, Scotland, UK   Email: m.zanda@abdn.ac.uk   Email: s.dallangelo@abdn.ac.uk
,
a  Institute of Medical Sciences and Kosterlitz Centre for Therapeutics, School of Medicine, Medical Sciences and Nutrition, University of Aberdeen, Foresterhill, Aberdeen, AB25 2ZD, Scotland, UK   Email: m.zanda@abdn.ac.uk   Email: s.dallangelo@abdn.ac.uk
,
M. Zanda*
a  Institute of Medical Sciences and Kosterlitz Centre for Therapeutics, School of Medicine, Medical Sciences and Nutrition, University of Aberdeen, Foresterhill, Aberdeen, AB25 2ZD, Scotland, UK   Email: m.zanda@abdn.ac.uk   Email: s.dallangelo@abdn.ac.uk
b  C.N.R. – I.C.R.M., via Mancinelli 7, 20131 Milan, Italy
› Author Affiliations
M.M. gratefully acknowledges SULSA (http://www.sulsa.ac.uk/) for a PhD studentship
Further Information

Publication History

Received: 19 October 2017

Accepted: 21 November 2017

Publication Date:
12 December 2017 (online)

 

Abstract

The first prospective fluorinated PET tracers for imaging hypoxia obtained via thiazolidine-ligation are reported. Three 1,2-thiol-amine linkers were combined with four different 2-nitroimidazole spacers via amide or urea bond formation. The resulting compounds were submitted to thiazolidine-ring-forming ligation reaction with the fluorinated carbohydrate l-5-fluoro-5-deoxy-ribose (FDR), affording the desired candidate PET tracers in variable yields. The same ligation reactions performed on l-ribose – a by-product of [18F]FDR radiosynthesis – under conditions mimicking a radiochemical production showed that the fluorinated adducts can be efficiently purified and isolated by HPLC. Finally, one of the prospective hypoxia tracers was successfully produced in radiolabelled form in 29.2% radiochemical yield from [18F]FDR.


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Hypoxia occurs in cells and tissues when oxygen demand exceeds supply.[1] [2] The irregular vasculature typical of solid tumours does not sufficiently support cellular oxygen demand, leading to the development and progression of heterogeneous hypoxic cancer areas, which are generally poorly responsive to chemo- and radio-therapies.[3,4] Accurate imaging of hypoxic regions could allow clinicians to stratify patients and develop more efficient treatment strategies to improve therapeutic outcomes.[5,6] Identification and quantification of hypoxic areas in tumours – and in other pathologies – is therefore important for planning the most appropriate and personalised therapeutic approach.[7] [8] Owing to its high sensitivity and non-invasive nature, PET imaging is emerging as the method of choice for in vivo identification, characterization and discrimination of hypoxic areas.[6] [9] In the last two decades, several PET tracers for hypoxia have been described, but all of them are affected by significant drawbacks, such as low signal-to-noise ratios, slow accumulation in hypoxic regions and poor brain uptake, therefore the development of new hypoxia-targeted PET tracers remains a very active area of research.[10] [11] l-5-Fluoro-5-deoxy-ribose ([18F]FDR) 2 (Figure [1]) has recently emerged as a promising prosthetic group for rapid, indirect radiolabelling of bioactive molecules via oxime bond formation.[12] [13] [14] [15]

As an alternative to the oxime bond, thiazolidine ring formation could be used as a site-specific ligation method via reaction of a 1,2-thiol-amine function with a carbonyl group – including masked carbonyls of carbohydrates and hemiacetals – in mildly acidic or basic conditions (pH 4 to 8).[16] [17] [18] Importantly, the thiazolidine ring is generally stable in a wide pH range (from 4 to 10), thus representing an attractive linkage option. In order to further investigate the efficiency of [18F]FDR as a radiolabelling agent and expand the library of prospective PET tracers for hypoxia imaging, we designed a novel class of candidate tracers [18F]1 (Figure [1]) taking advantage of the last-step formation of a thia­zolidine ring linkage between [18F]FDR 2 and terminal 2-amino-thiols 3 carrying a hypoxia-reactive 2-nitroimidazole group.

Zoom Image
Figure 1 Novel hypoxia PET tracers via thiazolidine ligation with [18F]FDR

Three different 2-aminoethanethiol linkers 4ac (Scheme [1]) were selected to modulate the steric constraints and lipophilicity of the final candidate tracers. The synthesis was based on the conditions described by Duthaler et al.[19] A mixture of racemic cysteine 5 and conc. HCl in acetone was heated at reflux for 6 h, affording the thiazolidine intermediate 6. Samples of 6 were invariably found (by 1H NMR spectroscopy) to contain 5–10% of cysteine hydrochloride 7. The mixture of 6 and 7 was allowed to react with (Boc)2O in pyridine for 3 days to give the N-Boc-derivative 4a.[19] NMR spectroscopy showed that this compound exists as a mixture of rotamers, the signals of which did not show coalescence at 60 °C either in CDCl3 or in CD3OD. Compound 4a was converted into the Weinreb amide 8 by reaction with HATU and DIPEA, followed by addition of N,O-di­methylhydroxylamine hydrochloride. Reduction of 8 using LiAlH4 at 0 °C provided in good yield the aldehyde 9, which was submitted to Wittig reaction with the phosphonium ylide Ph3P=CHCO2Me to give exclusively the trans isomer of the α,β-unsaturated ester 10.[20] Hydrogenation reaction of 10 using H2 over Pd/C catalyst gave in quantitative yield the saturated intermediate 11, which afforded the free carboxylic acid 4b [21] by basic hydrolysis of the ester function. The carbinol 12 was obtained upon treatment of 11 with LiAlH4 at 0 °C, whereas the amine derivative 4c [22] was obtained via Mitsunobu reaction of phthalimide with 12 to give compound 13, followed by phthalimide-ring cleavage with hydrazine monohydrate.

Zoom Image
Scheme 1 Synthesis of thiazolidine linkers 4ac. Reagents and conditions: (a) acetone, conc. HCl, reflux, 6 h; (b) Boc2O, pyridine, N2 atm., r.t., 72 h; (c) N,O-Dimethylhydroxylamine hydrochloride, DIPEA, CH2Cl2, HATU, from 0 °C to r.t., 18 h; (d) LiAlH4, Et2O, N2 atm., 0 °C, 15 min; (e) Ph3P=CHCO2Me, THF, reflux, 18 h; (f) Pd/C 10wt. %, H2 atm., MeOH, r.t., 24 h; (g) LiAlH4, THF, N2 atm., 0 °C, 1 h; (h) Phthalimide, PPh3, diisopropyl azodicarboxylate (DIAD), THF, r.t., 16 h; (i) NH2NH2 .H2O, reflux, 3 h; (j) LiOH, THF, r.t., 18 h.

The 2-nitro-imidazole spacers 14ad (Scheme [2] and Scheme [3]) were selected with the aim of introducing structural diversity within the series. The structure of the spacer was expected to have an important effect on lipophilicity, metabolic stability and ultimately on the imaging potential of the candidate tracers 1.

Amines 14a,b were synthesised via Gabriel reaction (Scheme [2]) starting respectively from commercial 1,3-dibromopropane (15a) and 1,5-dibromopentane (15b). The resulting phthalimides 16a,b [23] were reacted with 2-nitroimidazole and K2CO3 in DMF upon heating to 115 °C to give compounds 17a,b [24] in good yields. The desired amines 14a,b were obtained by quantitative cleavage of the phthalimido group with hydrazine monohydrate. The 1,2,3-triazole-amine 14c was prepared via Huisgen cycloaddition reaction between the azide 18, which was obtained by bromine displacement reaction of 16a with sodium azide,[25] and 1-propargyl-2-nitroimidazole 19, which was prepared according to the literature,[26] to afford phthalimide derivative 20. Removal of the phthalimido group with hydrazine gave compound 14c in good overall yield.

Zoom Image
Scheme 2 Synthesis of 2-nitroimidazole spacers 14ac. Reagents and conditions: (a) Phthalimide, TEA, DMF, r.t., 48 h; (b) 2-Nitroimidazole, K2CO3, DMF, 110–120 °C, 3–5 h; (c) NH2NH2·H2O, EtOH, 60–100 °C, 1–4 h; (d) NaN3, DMF, 120 °C, 4 h; (e) 1-Propargyl-2-nitroimidazole 19, CuSO4, sodium ascorbate, t-BuOH/H2O, r.t., 20 h.

2-Nitro-imidazolyl-acetic acid 14d [27] (Scheme [3]) was prepared in four steps starting from 21, which provided compound 22 after protection of the hydroxy group as tetrahydropyranyl acetal (THP) followed by introduction of the 2-nitroimidazole function in K2CO3 and DMF upon heating to 115 °C. The resulting intermediate 23 was then dissolved in a 6 M aq. HCl solution in MeOH to cleave the THP group, followed by treatment of the resulting carbinol 24 with Jones reagent (CrO3/H2SO4/acetone) in acetone to give the desired compound 14d in 41% yield over the three steps.

Zoom Image
Scheme 3 Synthesis of 2-nitroimidazole spacer 14d. Reagents and conditions: (a) DHP, PPTS, CH2Cl2, r.t., 18 h; (b) 2-Nitroimidazole, K2CO3, DMF, 115 °C, 5 h; (c) 6 M HCl, MeOH, r.t., 18 h; (d) CrO3/H2SO4/acetone, r.t., 12 h.

Assembling of linkers 4ac and spacers 14ad to give the tracers’ precursors 3af is shown in Scheme [4]. Treatment of carboxylic acid derivatives 4ac with HATU and DIPEA gave the corresponding activated esters, which were reacted in situ with the amines 14ad to afford the amides 25ae.[28] Different conditions were used to prepare the urea derivative 25f.[29] In this case, the amine 4c was added dropwise to a solution of carbonyldiimidazole (CDI) in CH2Cl2 at 0 °C to give the intermediate imidazocarboxyamide, which gave the desired urea 25f upon in situ treatment with the amine 14a.

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Scheme 4 Synthesis of the 2-amino-thiol tracer precursors 3af. Reagents and conditions: (a) HATU, DIPEA, CH2Cl2, r.t., 18 h; (b) CDI, CH2Cl2, from 0 °C to r.t., 18 h; (c) TFA/H2O/MeOH 3:2:1, from r.t. to 65 °C, 2–4 h.

The final unprotected 2-aminoethanethiol derivatives 3af [30] (Scheme [4]) were obtained by treatment of 25af with a TFA/H2O/MeOH 3:2:1 mixture upon heating to 65 °C for 2–4 h, followed by solvents removal under reduced pressure at 60 °C. Then the crude compounds were dissolved in ethanol (except compound 3c, which is only soluble in aqueous solutions) and eluted through a SiliaBond ® carbonate pad (silica bound equivalent of tetramethylammonium carbonate), which trapped residual TFA, acid by-products and free-based 2-nitroimidazolium trifluoroacetate salts formed during the thiazolidine hydrolysis.

In all cases, variable amounts of disulphide dimers were obtained in mixture with the desired thiol monomers 3af, as evidenced by both HPLC/MS analysis and NMR spectroscopy. However, we did not attempt to purify further the samples, as the disulphide dimers could be readily reduced back to the monomeric thiols by treatment with 1,4-dithiothreitol (DTT) before the following ligation reaction with FDR 2 (Scheme [5]).

The thiazolidine ring formation was performed by reaction of 3af with cold [19F]FDR 2 using 1 M acetate buffer as reaction medium in the presence of DTT. Acetate buffers with different molarity (from 0.1 to 4.0) and pH (from 3 to 6) were tested at different temperatures (from r.t. to 50 °C) in order to optimise the thiazolidine ring formation rate. The optimised conditions were 2.5 equiv of 3af reacted with 1 equiv of [19F]FDR(2) in the presence of 2.5 equiv of DTT, using 1 M acetate buffer at pH 4.5 as reaction medium, for 20 min at 30 °C. The purification step was performed by gradient RP-HPLC using a mixture of H2O/ACN + 0.05% (v/v) of TFA as eluent. 1,2-Aminothiol derivatives 3af showed markedly different reactivity towards [19F]FDR 2, showing that the spacers’ structure plays an important role in the cyclisation reaction (see Table [1] for yields). Only the 1,2-thiol-amine derivative 3c, incorporating a triazole ring, failed to react under all the conditions explored, affording in very low yields (<5%) the corresponding thiazolidine 1c, which could not be isolated in pure form by RP-HPLC purification.

The radiosynthesis of [18F]FDR 2 is known to produce an excess of l-ribose 26 as by-product,[12] which, although less reactive than 2, will compete with it in the thiazolidine ring formation, affording the corresponding non-fluorinated thiazolidines 27af (Scheme [5]) and decreasing the chemical purity of the tracer.

Zoom Image
Scheme 5 (a) 1 M acetate buffer CH3COOH/CH3COONa, pH 4.5, r.t., 20 min

To simulate the radiosynthesis conditions, the thiazolidine ring formation reaction was carried out in the presence of 10 equiv of 26 along with 1 equiv of [19F]FDR 2 and 1,2-aminothiol derivatives 3af. This experiment was performed with the aim of assessing the formation of the desired FDR thiazolidines in the presence of l-ribose 26 and the possibility of performing an HPLC purification for separating the [18F]FDR-derived tracers 1af from the non-radioactive l-ribose-derived thiazolidines 27af. As shown in Table [1], as well as in the HPLC profiles (see the Supporting Information), the retention times of the target FDR-thiazolidines 1af are indeed significantly different to those of the ribose-derived thiazolidines 27af.

Table 1 Synthesis of Cold Tracers [19F]1af

Compound

X

Y

Yield (%)

tR –F (min)

tR –OH (min)a

log P (±SED)

1a

CO

NH(CH2)2

61.3

12.5

8.3

0.33 (±0.04)

1b

CO

NH(CH2)4

67.9

27.3, 28.4

20.0

0.64 (±0.04)

1d

(CH2)2CO

NH(CH2)2

11.2

22.4–23.4

20.3

0.68 (±0.04)

1e

(CH2)3NH

CO

31.8

15.6–18.4

10.0–11.5

0.26 (±0.04)

1f

(CH2)3NH

CONH(CH2)3

42.3

27.7–29.4

22.0–23.5

0.50 (±0.02)

a Retention time of the corresponding l-ribose analogue 26.

Therefore, the final cold tracers [19F]1af could be isolated and characterised by LC-MS. Their Log P values were determined by RP-HPLC (isocratic phase H2O/EtOH 90:10). Considering that the gold standard hypoxia tracer [18F]FMISO has a Log P = 0.42, candidate tracers 1 appear to have suitable lipophilicity for use in vivo. Thiazolidines 1af presented very complex NMR spectra owing to the presence of four diastereomers, originated by the two (R/S) thiazolidine stereogenic centres, plus different rotamers and trifluoroacetate salts. Only compound 1a [31] was isolated in sufficient quantity for being satisfactorily characterised by NMR spectroscopy, after treatment with SiliaBond ® carbonate in order to freebase the trifluoroacetate salts.

Radiolabelling tests for producing [18F]1a [32] were conducted on the 1,2-aminothiol derivative 3a, which was treated with [18F]FDR (2)[12] [13] using a sodium acetate buffer solution (Scheme [6]). Also in this case, different reaction conditions were tested with the aim of achieving the maximum radiochemical conversion within 40 minutes (see Table [1]S, Supporting Information).

Zoom Image
Scheme 6 Radiosynthesis of [18F]1a. Reagents and conditions: (a) 6 M acetate buffer, pH 4.5, r.t., 30 min.

Eventually, we found that the use of a 6 M acetate buffer solution (70% v/v concentration) in the reaction mixture containing [18F]FDR (2), 3a and DTT (1:1) (in the range 2–4 M) at pH 4.5, provided the highest RCY (29.2%, decay corrected). No further improvements could be achieved by changing buffer concentration, pH or extending further the reaction time.

The tracer identity was confirmed by superimposition of the UV-HPLC profile of the cold reference [19F]1a with the semi-preparative RP-HPLC radio-chromatogram of [18F]1a, acquired before purification of the radiotracer (Figure [2]).

Zoom Image
Figure 2 In black: semi-prep RP-HPLC Radio-analysis of radiotracer [18F]1a formation using 70% v/v of a 6 M acetate buffer solution in the aqueous solution of [18F]FDR (2). In red: superimposed UV chromatogram of the cold reference [19F]1a obtained using the same RP-HPLC conditions.

In conclusion, we have designed and synthesised the first candidate PET tracers (1) for hypoxia imaging based on the use of [18F]FDR 2 as radiolabelling agent. The synthesis is based on the formation of a thiazolidine-ring-linkage between [18F]FDR 2 and 1,2-thiol-amines 3, which occurs with moderate to good efficiency depending on the structure of spacer and linker featured in 3. The method was successfully tested for the radiosynthesis of [18F]1a, which was produced in 29.2% radiochemical yield and successfully purified by RP-HPLC.


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Acknowledgment

Mr Federico Toson is gratefully acknowledged for conducting preliminary experiments.

Supporting Information

  • References and Notes

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  • 20 O’Connell CE. Ackermann K. Rowell CA. Garcia AM. Lewis MD. Schwartz CE. Bioorg. Med. Chem. Lett. 1999; 9: 2095
  • 21 Synthesis of 4b: An aqueous 1 M LiOH solution (1.64 mL, 1.64 mmol) was added at r.t. to a solution of 11 (200 mg, 0.66 mmol) in THF (1.7 mL). The reaction mixture was stirred for 18 h at r.t. and then neutralised with a 1 M aq. HCl solution, then extracted with EtOAc (3 × 2 mL), dried and concentrated under reduced pressure to give 4b (186 mg, 97.4%) as an oil. 1H NMR (CDCl3, 400 MHz): δ = 9.07 (br, 1 H), 4.35 (br, 1 H), 3.12 (dd, J = 11.9, 5.9 Hz, 1 H), 2.58 (d, J = 11.9 Hz, 1 H), 2.39–2.19 (m, 2 H), 2.12–1.93 (m, 2 H), 1.72 (s, 6 H), 1.45 (s, 9 H). 13C NMR (CDCl3, 100 MHz): δ = 178.9, 152.7, 80.7, 69.6, 63.7, 51.2, 32.3, 31.3, 29.6 (2C), 28.4 (3C) MS (ESI): m/z calcd for C13H23NO4S: 290.2 [M+H]+, 312.1 [M+Na]+; found: 290.2 [M+H]+, 312.1 [M+Na]+
  • 22 Synthesis of 4c: Hydrazine monohydrate (124 μL, 2.52 mmol) was added to a solution of 13 (340 mg, 0.84 mmol) in EtOH (5 mL) and the reaction mixture was heated at reflux for 3 h. After cooling to 0 °C the resulting white precipitate was filtered off and the filtrate was concentrated under reduced pressure. The residue was dissolved with Et2O (5 mL) and the resulting white precipitate was filtered, then the filtrate was concentrated under reduced pressure to afford 4c (448 mg, 98.4%) as a yellow oil. 1H NMR (CDCl3, 400 MHz): δ = 4.16 (br, 1 H), 3.00 (dd, J = 11.6, 6.1 Hz, 1 H), 2.65–2.54 (m, 2 H), 2.47 (d, J = 11.6 Hz, 1 H), 1.73–1.49 (m, 4 H), 1.60 (s, 6 H), 1.44–1.41 (m, 2 H), 1.33 (s, 9 H). 13C NMR (CDCl3, 100 MHz): δ = 152.3, 79.8, 69.4, 64.1, 41.8, 31.3, 30.9, 30.2, 29.6 (2C), 28.4 (3C). MS (ESI): m/z calcd for C13H26N2O2S: 275.1 [M+H]+, 297.2 [M+Na]+ 303.2 [M+K]+; found: 275.2 [M+H]+, 297.2 [M+Na]+, 303.1 [M+K]+
  • 23 Böhmer V. Dozol J.-F. Grüttner C. Liger K. Matthews SE. Rudershausen S. Saadioui M. Wang P. Org. Biomol. Chem. 2004; 2: 2327
  • 24 Hay MP. Wilson WR. Moselen JW. Palmer BD. Denny WA. J. Med. Chem. 1994; 37: 381
  • 25 Ebran J.-P. Dendane N. Melnyk O. Org. Lett. 2011; 13: 4336
  • 26 Bejot R. Carroll L. Bhakoo K. Declerck J. Gouverneur V. Bioorg. Med. Chem. 2012; 20: 324
  • 27 A shorter synthesis of 14d has been reported, see: Joyard Y., Azzouz R., Bischoff L., Papamicaël C., Labar D., Bol A., Bol V., Vera P., Grégoire V., Levacher V., Bohn P.; Bioorg. Med. Chem.; 2013, 21: 3680; and references therein. However, imidazolyl carbinol 24 was already being used in our labs for a related project, therefore it was used as an intermediate for 14d
  • 28 Synthesis of 25a: DIPEA (156 μL, 0.92 mmol) and HATU (350 mg, 0.92 mmol) were added to a solution of 4a (200 mg, 0.77 mmol) in anhydrous CH2Cl2 (10 mL) at 0 °C and the mixture was allowed to react at r.t. for 1 h. Then the amino derivative 14a (260 mg, 1.53 mmol) dissolved in CH2Cl2 (2 mL) was added to the mixture. After 16 h under stirring, the mixture was washed with a 0.5 M aq. NaOH solution (3 × 6 mL) and then with a 0.1 M aq. HCl solution (3 × 6 mL), dried over Na2SO4 and concentrated under reduced pressure. The crude material was purified by flash chromatography (Hex/EtOAc, from 8:2 to 7:3) to afford 25a (229 mg, 72.3%) as a yellow oil. 1H NMR (CDCl3, 400 MHz, mixture of rotamers): δ = 7.32 (s, 1 H), 7.08 (s, 1 H), 6.55 (br, 1 H), 4.72 (br, 1 H), 3.42–3.11 (m, 4 H), 1.82 (s, 3 H), 1.73 (s, 3 H), 1.42 (s, 9 H). 13C NMR (CDCl3, 100 MHz): δ = 171.7, 153.3, 144.7, 128.4, 127.0, 81.8, 71.4, 67.5, 47.4, 36.0, 31.0, 29.3, 28.9, 28.4 (3C). MS (ESI): m/z calcd for C17H27N5O5S: 436.2 [M+Na]+, 452.0 [M+K]+; found: 436.1 [M+Na]+, 452.0 [M+K]+
  • 29 Synthesis of 25f: A solution of 4c (152 mg, 0.56 mmol) in CH2Cl2 (2 mL) was added dropwise to a solution of CDI (90 mg, 0.56 mmol) in anhydrous CH2Cl2 (3 mL), at 0 °C under N2 atmosphere, then the mixture was allowed to react at r.t. for 1 h. After 16 h under stirring, the mixture was added via syringe to a solution of 14a (226 mg, 1.33 mmol) in CH2Cl2 (3 mL) under N2 atmosphere. After 16 h under stirring the mixture was concentrated under recued pressure. Purification by FC on silica gel (Hex/EtOAc, from 3:7 to 7:3) gave 25f (148 mg, 56.7%) as a yellow oil. 1H NMR (CDCl3, 400 MHz, 2 rotamers): δ = 7.35 (br, 1 H), 7.02 (br, 1 H), 5.55 (br, 2 H), 4.40 (t, J = 6.9 Hz, 2 H), 4.19 (br, 1 H), 3.24–2.94 (m, 5 H), 2.49 (d, J = 11.8 Hz, 1 H), 2.05–1.86 (m, 2 H), 1.81–1.66 (m, 2 H), 1.63 (s, 3 H), 1.61 (s, 3 H), 1.48–1.26 (m, 11 H). 13C NMR (CDCl3, 100 MHz, 2 rotamers): δ = 159.0, 152.7, 144.6, 128.2, 127.1, 80.3, 69.4, 64.0, 47.8, 39.8, 36.6, 31.6, 31.4, 30.6, 30.1, 29.6, 28.4 (3C), 27.3. MS (ESI): m/z calcd for C20H34N6O5S: 471.2 [M+H]+, 493.2 [M+Na]+; found: 471.2 [M+H]+, 493.2 [M+Na]+
  • 30 Synthesis of 3a: Compound 25a was dissolved in a TFA/H2O/MeOH 3:2:1 mixture and heated to 65 °C for 2 h. Solvents were then concentrated under reduced pressure at 60 °C, then the residue was dissolved in ethanol and passed through a SiliaBond ® carbonate pad to give the crude 3a as trifluoroacetate salt (55.7 mg, 74.8%). The compound was used in the next reaction without any further purification. 1H NMR (CD3OD, 400 MHz, in mixture with the dimer): δ = 7.62–7.54 (m, 1 H), 7.20–7.15 (m, 1 H), 4.60–4.42 (m, 2 H), 4.31–4.19 (m, 1 H), 3.52–3.33 (m, 2 H), 3.24–3.09 (m, 2 H), 2.19–2.00 (m, 2 H). 13C NMR (CD3OD, 100 MHz, in mixture with the dimer): δ = 167.4, 144.6, 127.4, 127.2, 51.7, 47.4, 37.8, 36.3, 29.7. MS (ESI): m/z calcd for C9H15N5O3S: 274.1 [M+H]+, 296.1 [M+Na]+; found: 274.0 [M+H]+, 296.0 [M+Na]+
  • 31 Synthesis of 1a: [19F]FDR ([19F]2) (5 mg, 0.033 mmol) was added to a solution of 3a (32.0 mg, 0.083 mmol) and DTT (12.8 mg, 0.083 mmol) in a 1 M sodium acetate buffer solution (pH 4.5), then the mixture was allowed to react at 30 °C for 20 min. Purification by RP-HPLC (Column: Phenomenex Luna C18 250 × 10.00 mm, 5 μm; mobile phase: A (H2O + 0.05% TFA), B (ACN + 0.05% TFA); gradient: from 5% B to 6% B in 15 min; flow: 5 mL min−1; tR : 12.5 min) gave 1a as trifluoroacetate salt (10.6 mg, 61.3 %). NMR analyses were performed after treatment of 1a with SiliaBond ® carbonate (10% w/w) in EtOH, under gentle stirring for 1 h in order to freebase trifluoroacetate salt. 1H NMR (CD3OD, 400 MHz, – four diastereoisomers – two major isomer in ~3:2 ratio were identified): δ = 7.55 (d, J = 1.2 Hz, 1 H), 7.16 (d, J = 1.2 Hz, 1 H), 4.89–4.83 (m, 1 H), 4.62–4.42 (m, 4 H), 4.25 (dd, J = 7.0, 6.8 Hz, 1 H), 4.09–4.04 (m, 1 H), 3.91 (dd, J = 7.4, 4.6 Hz, 1 H), 3.66 (dd, J = 7.4, 5.8 Hz, 1 H), 3.39–3.23 (m, 3 H), 3.02–2.91 (m, 1 H), 2.16–2.05 (m, 2 H); δ (second isomer) = 7.57 (d, J = 1.2 Hz, 1 H), 7.17 (d, J = 1.2 Hz, 1 H), 4.92 (d, J = 2.6 Hz, 1 H), 4.67 (dd, J = 9.8, 3.0 Hz, 1 H), 4.63–4.42 (m, 3 H), 4.25 (dd, J = 7.0, 6.8 Hz, 1 H), 4.04–3.95 (m, 1 H), 3.85–3.75 (m, 2 H), 3.39–3.14 (m, 3 H), 3.02–2.91 (m, 1 H), 2.16–2.05 (m, 2 H). 13C NMR (CD3OD, 100 MHz, – four diastereoisomers – two major isomers in ~3:2 ratio were identified): δ (first isomer) = 172.5, 144.7, 127.2, 127.1, 84.1 (d, J CF = 167 Hz), 73.6 (d, J CF = 7 Hz), 72.3, 72.1, 71.8 (d, J CF = 18 Hz), 71.4, 65.8, 35.8, 34.9, 30.0; δ (second isomer) = 172.4, 144.7, 127.2, 127.1, 84.3 (d, J CF = 167 Hz), 73.6 (d, J CF = 7 Hz), 72.3, 72.0, 71.9, 71.9 (d, J CF = 18 Hz), 70.2, 66.2, 36.6, 34.9. 29.9. 19F NMR (376 MHz, CD3OD): δ (first isomer) = –233.0 (dt, J = 48.0, 22.7 Hz); δ (second isomer) = –233.6 (dt, J 1 = 48.0 Hz, J 2 = 22.3 Hz); MS (ESI): m/z calcd for: C14H22FN5O6S: 408.1 [M+H]+, 430.1 [M+Na]+; found: 408.0 [M+H]+, 430.0 [M+Na]+
  • 32 Optimised radiosynthesis of [18F]1a: A solution of sodium acetate buffer (6 M, pH 4.5) was added to a solution of 3a (2.5 mg, 6.4 μmol), DTT (1 mg, 6.4 μmol) and [18F]FDR ([18F]2) (2.5–10 MBq) in 0.5–1.0 mL of H2O to form a 70% v/v sodium acetate buffer solution (final concentration 4.2 M). After ~20 min the mixture was purified by RP-HPLC (Column: Phenomenex Luna C18 250 × 10.00 mm, 5 μm) to give [18F]1a in 29% RCY (decay corrected)

  • References and Notes

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  • 2 Höckel M. Vaupel P. JNCI J. Natl. Cancer Inst. 2001; 93: 266
  • 3 Ke Q. Costa M. Mol. Pharmacol. 2006; 70: 1469
  • 4 Muz B. de la Puente P. Azab F. Azab AK. Hypoxia 2015; 83
  • 5 Harada H. J. Radiat. Res. (Tokyo) 2011; 52: 545
  • 6 Padhani AR. Krohn KA. Lewis JS. Alber M. Eur. Radiol. 2007; 17: 861
  • 7 Wigerup C. Påhlman S. Bexell D. Pharmacol. Ther. (Supplement C) 2016; 164: 152
  • 8 Horsman MR. Mortensen LS. Petersen JB. Busk M. Overgaard J. Nat. Rev. Clin. Oncol. 2012; 9: 674
  • 9 Carlin S. Humm JL. J. Nucl. Med. 2012; 53: 1171
  • 10 Lopci E. Grassi I. Chiti A. Nanni C. Cicoria G. Toschi L. Fonti C. Lodi F. Mattioli S. Fanti S. Am. J. Nucl. Med. Mol. Imaging 2014; 4: 365
  • 11 Peeters SG. J. A. Zegers CM. L. Lieuwes NG. van Elmpt W. Eriksson J. van Dongen GA. M. S. Dubois L. Lambin P. Int. J. Radiat. Oncol. 2015; 91: 351
  • 12 Li X.-G. Dall’Angelo S. Schweiger LF. Zanda M. O’Hagan D. Chem. Commun. 2012; 48: 5247
  • 13 Dall’Angelo S. Zhang Q. Fleming IN. Piras M. Schweiger LF. O’Hagan D. Zanda M. Org. Biomol. Chem. 2013; 11: 4551
  • 14 Keinänen O. Li X.-G. Chenna NK. Lumen D. Ott J. Molthoff CF. Sarparanta M. Helariutta K. Vuorinen T. Windhorst AD. Airaksinen AJ. ACS Med. Chem. Lett. 2015; 7: 62
  • 15 Li X.-G. Helariutta K. Roivainen A. Jalkanen S. Knuuti J. Airaksinen AJ. Nat. Protoc. 2013; 9: 138
  • 16 Forget D. Boturyn D. Defrancq E. Lhomme J. Dumy P. Chem. Eur. J. 2001; 7: 3976
  • 17 Zhang L. Tam JP. Anal. Biochem. 1996; 233: 87
  • 18 Liu C.-F. Tam JP. J. Am. Chem. Soc. 1994; 116: 4149
  • 19 Duthaler RO. Wyss B. Eur. J. Org. Chem. 2011; 24: 7419
  • 20 O’Connell CE. Ackermann K. Rowell CA. Garcia AM. Lewis MD. Schwartz CE. Bioorg. Med. Chem. Lett. 1999; 9: 2095
  • 21 Synthesis of 4b: An aqueous 1 M LiOH solution (1.64 mL, 1.64 mmol) was added at r.t. to a solution of 11 (200 mg, 0.66 mmol) in THF (1.7 mL). The reaction mixture was stirred for 18 h at r.t. and then neutralised with a 1 M aq. HCl solution, then extracted with EtOAc (3 × 2 mL), dried and concentrated under reduced pressure to give 4b (186 mg, 97.4%) as an oil. 1H NMR (CDCl3, 400 MHz): δ = 9.07 (br, 1 H), 4.35 (br, 1 H), 3.12 (dd, J = 11.9, 5.9 Hz, 1 H), 2.58 (d, J = 11.9 Hz, 1 H), 2.39–2.19 (m, 2 H), 2.12–1.93 (m, 2 H), 1.72 (s, 6 H), 1.45 (s, 9 H). 13C NMR (CDCl3, 100 MHz): δ = 178.9, 152.7, 80.7, 69.6, 63.7, 51.2, 32.3, 31.3, 29.6 (2C), 28.4 (3C) MS (ESI): m/z calcd for C13H23NO4S: 290.2 [M+H]+, 312.1 [M+Na]+; found: 290.2 [M+H]+, 312.1 [M+Na]+
  • 22 Synthesis of 4c: Hydrazine monohydrate (124 μL, 2.52 mmol) was added to a solution of 13 (340 mg, 0.84 mmol) in EtOH (5 mL) and the reaction mixture was heated at reflux for 3 h. After cooling to 0 °C the resulting white precipitate was filtered off and the filtrate was concentrated under reduced pressure. The residue was dissolved with Et2O (5 mL) and the resulting white precipitate was filtered, then the filtrate was concentrated under reduced pressure to afford 4c (448 mg, 98.4%) as a yellow oil. 1H NMR (CDCl3, 400 MHz): δ = 4.16 (br, 1 H), 3.00 (dd, J = 11.6, 6.1 Hz, 1 H), 2.65–2.54 (m, 2 H), 2.47 (d, J = 11.6 Hz, 1 H), 1.73–1.49 (m, 4 H), 1.60 (s, 6 H), 1.44–1.41 (m, 2 H), 1.33 (s, 9 H). 13C NMR (CDCl3, 100 MHz): δ = 152.3, 79.8, 69.4, 64.1, 41.8, 31.3, 30.9, 30.2, 29.6 (2C), 28.4 (3C). MS (ESI): m/z calcd for C13H26N2O2S: 275.1 [M+H]+, 297.2 [M+Na]+ 303.2 [M+K]+; found: 275.2 [M+H]+, 297.2 [M+Na]+, 303.1 [M+K]+
  • 23 Böhmer V. Dozol J.-F. Grüttner C. Liger K. Matthews SE. Rudershausen S. Saadioui M. Wang P. Org. Biomol. Chem. 2004; 2: 2327
  • 24 Hay MP. Wilson WR. Moselen JW. Palmer BD. Denny WA. J. Med. Chem. 1994; 37: 381
  • 25 Ebran J.-P. Dendane N. Melnyk O. Org. Lett. 2011; 13: 4336
  • 26 Bejot R. Carroll L. Bhakoo K. Declerck J. Gouverneur V. Bioorg. Med. Chem. 2012; 20: 324
  • 27 A shorter synthesis of 14d has been reported, see: Joyard Y., Azzouz R., Bischoff L., Papamicaël C., Labar D., Bol A., Bol V., Vera P., Grégoire V., Levacher V., Bohn P.; Bioorg. Med. Chem.; 2013, 21: 3680; and references therein. However, imidazolyl carbinol 24 was already being used in our labs for a related project, therefore it was used as an intermediate for 14d
  • 28 Synthesis of 25a: DIPEA (156 μL, 0.92 mmol) and HATU (350 mg, 0.92 mmol) were added to a solution of 4a (200 mg, 0.77 mmol) in anhydrous CH2Cl2 (10 mL) at 0 °C and the mixture was allowed to react at r.t. for 1 h. Then the amino derivative 14a (260 mg, 1.53 mmol) dissolved in CH2Cl2 (2 mL) was added to the mixture. After 16 h under stirring, the mixture was washed with a 0.5 M aq. NaOH solution (3 × 6 mL) and then with a 0.1 M aq. HCl solution (3 × 6 mL), dried over Na2SO4 and concentrated under reduced pressure. The crude material was purified by flash chromatography (Hex/EtOAc, from 8:2 to 7:3) to afford 25a (229 mg, 72.3%) as a yellow oil. 1H NMR (CDCl3, 400 MHz, mixture of rotamers): δ = 7.32 (s, 1 H), 7.08 (s, 1 H), 6.55 (br, 1 H), 4.72 (br, 1 H), 3.42–3.11 (m, 4 H), 1.82 (s, 3 H), 1.73 (s, 3 H), 1.42 (s, 9 H). 13C NMR (CDCl3, 100 MHz): δ = 171.7, 153.3, 144.7, 128.4, 127.0, 81.8, 71.4, 67.5, 47.4, 36.0, 31.0, 29.3, 28.9, 28.4 (3C). MS (ESI): m/z calcd for C17H27N5O5S: 436.2 [M+Na]+, 452.0 [M+K]+; found: 436.1 [M+Na]+, 452.0 [M+K]+
  • 29 Synthesis of 25f: A solution of 4c (152 mg, 0.56 mmol) in CH2Cl2 (2 mL) was added dropwise to a solution of CDI (90 mg, 0.56 mmol) in anhydrous CH2Cl2 (3 mL), at 0 °C under N2 atmosphere, then the mixture was allowed to react at r.t. for 1 h. After 16 h under stirring, the mixture was added via syringe to a solution of 14a (226 mg, 1.33 mmol) in CH2Cl2 (3 mL) under N2 atmosphere. After 16 h under stirring the mixture was concentrated under recued pressure. Purification by FC on silica gel (Hex/EtOAc, from 3:7 to 7:3) gave 25f (148 mg, 56.7%) as a yellow oil. 1H NMR (CDCl3, 400 MHz, 2 rotamers): δ = 7.35 (br, 1 H), 7.02 (br, 1 H), 5.55 (br, 2 H), 4.40 (t, J = 6.9 Hz, 2 H), 4.19 (br, 1 H), 3.24–2.94 (m, 5 H), 2.49 (d, J = 11.8 Hz, 1 H), 2.05–1.86 (m, 2 H), 1.81–1.66 (m, 2 H), 1.63 (s, 3 H), 1.61 (s, 3 H), 1.48–1.26 (m, 11 H). 13C NMR (CDCl3, 100 MHz, 2 rotamers): δ = 159.0, 152.7, 144.6, 128.2, 127.1, 80.3, 69.4, 64.0, 47.8, 39.8, 36.6, 31.6, 31.4, 30.6, 30.1, 29.6, 28.4 (3C), 27.3. MS (ESI): m/z calcd for C20H34N6O5S: 471.2 [M+H]+, 493.2 [M+Na]+; found: 471.2 [M+H]+, 493.2 [M+Na]+
  • 30 Synthesis of 3a: Compound 25a was dissolved in a TFA/H2O/MeOH 3:2:1 mixture and heated to 65 °C for 2 h. Solvents were then concentrated under reduced pressure at 60 °C, then the residue was dissolved in ethanol and passed through a SiliaBond ® carbonate pad to give the crude 3a as trifluoroacetate salt (55.7 mg, 74.8%). The compound was used in the next reaction without any further purification. 1H NMR (CD3OD, 400 MHz, in mixture with the dimer): δ = 7.62–7.54 (m, 1 H), 7.20–7.15 (m, 1 H), 4.60–4.42 (m, 2 H), 4.31–4.19 (m, 1 H), 3.52–3.33 (m, 2 H), 3.24–3.09 (m, 2 H), 2.19–2.00 (m, 2 H). 13C NMR (CD3OD, 100 MHz, in mixture with the dimer): δ = 167.4, 144.6, 127.4, 127.2, 51.7, 47.4, 37.8, 36.3, 29.7. MS (ESI): m/z calcd for C9H15N5O3S: 274.1 [M+H]+, 296.1 [M+Na]+; found: 274.0 [M+H]+, 296.0 [M+Na]+
  • 31 Synthesis of 1a: [19F]FDR ([19F]2) (5 mg, 0.033 mmol) was added to a solution of 3a (32.0 mg, 0.083 mmol) and DTT (12.8 mg, 0.083 mmol) in a 1 M sodium acetate buffer solution (pH 4.5), then the mixture was allowed to react at 30 °C for 20 min. Purification by RP-HPLC (Column: Phenomenex Luna C18 250 × 10.00 mm, 5 μm; mobile phase: A (H2O + 0.05% TFA), B (ACN + 0.05% TFA); gradient: from 5% B to 6% B in 15 min; flow: 5 mL min−1; tR : 12.5 min) gave 1a as trifluoroacetate salt (10.6 mg, 61.3 %). NMR analyses were performed after treatment of 1a with SiliaBond ® carbonate (10% w/w) in EtOH, under gentle stirring for 1 h in order to freebase trifluoroacetate salt. 1H NMR (CD3OD, 400 MHz, – four diastereoisomers – two major isomer in ~3:2 ratio were identified): δ = 7.55 (d, J = 1.2 Hz, 1 H), 7.16 (d, J = 1.2 Hz, 1 H), 4.89–4.83 (m, 1 H), 4.62–4.42 (m, 4 H), 4.25 (dd, J = 7.0, 6.8 Hz, 1 H), 4.09–4.04 (m, 1 H), 3.91 (dd, J = 7.4, 4.6 Hz, 1 H), 3.66 (dd, J = 7.4, 5.8 Hz, 1 H), 3.39–3.23 (m, 3 H), 3.02–2.91 (m, 1 H), 2.16–2.05 (m, 2 H); δ (second isomer) = 7.57 (d, J = 1.2 Hz, 1 H), 7.17 (d, J = 1.2 Hz, 1 H), 4.92 (d, J = 2.6 Hz, 1 H), 4.67 (dd, J = 9.8, 3.0 Hz, 1 H), 4.63–4.42 (m, 3 H), 4.25 (dd, J = 7.0, 6.8 Hz, 1 H), 4.04–3.95 (m, 1 H), 3.85–3.75 (m, 2 H), 3.39–3.14 (m, 3 H), 3.02–2.91 (m, 1 H), 2.16–2.05 (m, 2 H). 13C NMR (CD3OD, 100 MHz, – four diastereoisomers – two major isomers in ~3:2 ratio were identified): δ (first isomer) = 172.5, 144.7, 127.2, 127.1, 84.1 (d, J CF = 167 Hz), 73.6 (d, J CF = 7 Hz), 72.3, 72.1, 71.8 (d, J CF = 18 Hz), 71.4, 65.8, 35.8, 34.9, 30.0; δ (second isomer) = 172.4, 144.7, 127.2, 127.1, 84.3 (d, J CF = 167 Hz), 73.6 (d, J CF = 7 Hz), 72.3, 72.0, 71.9, 71.9 (d, J CF = 18 Hz), 70.2, 66.2, 36.6, 34.9. 29.9. 19F NMR (376 MHz, CD3OD): δ (first isomer) = –233.0 (dt, J = 48.0, 22.7 Hz); δ (second isomer) = –233.6 (dt, J 1 = 48.0 Hz, J 2 = 22.3 Hz); MS (ESI): m/z calcd for: C14H22FN5O6S: 408.1 [M+H]+, 430.1 [M+Na]+; found: 408.0 [M+H]+, 430.0 [M+Na]+
  • 32 Optimised radiosynthesis of [18F]1a: A solution of sodium acetate buffer (6 M, pH 4.5) was added to a solution of 3a (2.5 mg, 6.4 μmol), DTT (1 mg, 6.4 μmol) and [18F]FDR ([18F]2) (2.5–10 MBq) in 0.5–1.0 mL of H2O to form a 70% v/v sodium acetate buffer solution (final concentration 4.2 M). After ~20 min the mixture was purified by RP-HPLC (Column: Phenomenex Luna C18 250 × 10.00 mm, 5 μm) to give [18F]1a in 29% RCY (decay corrected)

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Figure 1 Novel hypoxia PET tracers via thiazolidine ligation with [18F]FDR
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Scheme 1 Synthesis of thiazolidine linkers 4ac. Reagents and conditions: (a) acetone, conc. HCl, reflux, 6 h; (b) Boc2O, pyridine, N2 atm., r.t., 72 h; (c) N,O-Dimethylhydroxylamine hydrochloride, DIPEA, CH2Cl2, HATU, from 0 °C to r.t., 18 h; (d) LiAlH4, Et2O, N2 atm., 0 °C, 15 min; (e) Ph3P=CHCO2Me, THF, reflux, 18 h; (f) Pd/C 10wt. %, H2 atm., MeOH, r.t., 24 h; (g) LiAlH4, THF, N2 atm., 0 °C, 1 h; (h) Phthalimide, PPh3, diisopropyl azodicarboxylate (DIAD), THF, r.t., 16 h; (i) NH2NH2 .H2O, reflux, 3 h; (j) LiOH, THF, r.t., 18 h.
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Scheme 2 Synthesis of 2-nitroimidazole spacers 14ac. Reagents and conditions: (a) Phthalimide, TEA, DMF, r.t., 48 h; (b) 2-Nitroimidazole, K2CO3, DMF, 110–120 °C, 3–5 h; (c) NH2NH2·H2O, EtOH, 60–100 °C, 1–4 h; (d) NaN3, DMF, 120 °C, 4 h; (e) 1-Propargyl-2-nitroimidazole 19, CuSO4, sodium ascorbate, t-BuOH/H2O, r.t., 20 h.
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Scheme 3 Synthesis of 2-nitroimidazole spacer 14d. Reagents and conditions: (a) DHP, PPTS, CH2Cl2, r.t., 18 h; (b) 2-Nitroimidazole, K2CO3, DMF, 115 °C, 5 h; (c) 6 M HCl, MeOH, r.t., 18 h; (d) CrO3/H2SO4/acetone, r.t., 12 h.
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Scheme 4 Synthesis of the 2-amino-thiol tracer precursors 3af. Reagents and conditions: (a) HATU, DIPEA, CH2Cl2, r.t., 18 h; (b) CDI, CH2Cl2, from 0 °C to r.t., 18 h; (c) TFA/H2O/MeOH 3:2:1, from r.t. to 65 °C, 2–4 h.
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Scheme 5 (a) 1 M acetate buffer CH3COOH/CH3COONa, pH 4.5, r.t., 20 min
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Scheme 6 Radiosynthesis of [18F]1a. Reagents and conditions: (a) 6 M acetate buffer, pH 4.5, r.t., 30 min.
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Figure 2 In black: semi-prep RP-HPLC Radio-analysis of radiotracer [18F]1a formation using 70% v/v of a 6 M acetate buffer solution in the aqueous solution of [18F]FDR (2). In red: superimposed UV chromatogram of the cold reference [19F]1a obtained using the same RP-HPLC conditions.