Rapid C-carboxylation of nitro[¹¹C]methane for the synthesis of ethyl nitro[2-¹¹C]acetate

Abstract

The labelling synthesis of ethyl nitro[2-¹¹C]acetate, a synthetic intermediate feasible for ¹¹C-labelled PET tracers, by C-carboxylation of [¹¹C]MeNO₂ with 1-ethoxycarbonylbenzotriazole, and its simple application are presented.

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Positron emission tomography (PET) is a non-invasive imaging technology that can visualise molecular distribution in humans and animals in vivo. Recently, the importance of PET has developed because PET can be utilised for diagnosis and the acceleration of drug development.^1,2Carbon-11 (¹¹C) and fluorine-18 are the most frequently used positron emitters, with half-lives of 20 and 110 min, respectively. Rapid labelling of “short-lived” radionuclides on a sub-microscale is critical for PET tracer synthesis, and remote-controlled synthesis in the lead-shielded hot-cell is required for radiation protection. Such different situations from organic synthesis cause chemical and technical problems and restrictions,³ resulting in obstacles for the development of new labelling methods.

New ¹¹C-labelling methodology is inevitable for the development of wide-ranging PET tracer synthesis. ¹¹C is prepared by a cyclotron-induced nuclear reaction in the form of [¹¹C]O₂ and [¹¹C]H₄, giving access to a limited number of ¹¹C-labelling precursors (e.g., [¹¹C]MeI, [¹¹C]O, and in this paper [¹¹C]MeNO₂ ([¹¹C]1)) (Fig. 1). The labelling precursor [¹¹C]1 is synthesised from [¹¹C]MeI, widely used for ¹¹C-labelling syntheses;^4,5 however, applications using [¹¹C]1 have not been sufficient so far.⁶ We focused on the C-carboxylation of [¹¹C]1 which was a useful ¹¹C–C bond-forming method affording ¹¹C-labelled nitroacetate. Nitroacetic acid ester is an important synthetic intermediate for amino acids, amino alcohols and heterocyclics,⁷ which are interesting small molecules as PET tracers. In addition, nitroacetic acid is an effective inhibitor of the oxidation of succinate.⁸

Fig. 1 Structures of [¹¹C]1, [¹¹C]2, [¹¹C]3, and 4.

Proštenik and Butula reported that ethyl nitroacetate (2) was obtained by the C-carboxylation of the sodium salt of methyl nitronate (3) with 1-ethoxycarbonylbenzotriazole (4) in dimethyl sulfoxide (DMSO) or dimethylformamide (DMF).⁹ In their synthesis, excess 1 and a longer reaction time were required. On the other hand, both the amount of [¹¹C]1 and reaction time were restricted in ¹¹C-labelling. Excess reactants and ¹¹C-labelling precursor have been used for the labelling reaction. The ¹¹C-labelling reaction has been terminated within 5 min owing to the competing decay both of precursor and product. Although the reaction conditions are different from radiolabelling synthesis, the benzotriazole method is straightforward for the development of C-carboxylation of [¹¹C]1. Herein we describe the labelling synthesis of [¹¹C]2 by C-carboxylation of [¹¹C]1 with 4.

As generally reported, cyclotron-produced [¹¹C]O₂ was converted to [¹¹C]MeIvia reduction with lithium aluminium hydride (LAH) and subsequent iodination by hydroiodic acid (HI). [¹¹C]MeI was distilled and gaseous [¹¹C]MeI was converted to [¹¹C]1 by passing through a heating column comprising a plug of AgNO₂ (Scheme 1) in 3 min. The gaseous [¹¹C]1 was collected by the next reaction vessel.

Scheme 1 Preparation of [¹¹C]MeNO₂ ([¹¹C]1).

First we employed a procedure collecting [¹¹C]1 in the presence of the base (procedure A). We assumed that [¹¹C]3 was prepared during the collection of [¹¹C]1 and would react immediately with 4 by this procedure, resulting in a shorter reaction time. Sodium hydride (NaH) and DMSO was the first choice as a base and solvent, and a C-carboxylation reaction was performed for 5 min at 30 °C. Thus, [¹¹C]1 was collected in DMSO in the presence of NaH and then a DMSO solution of 4 was added (Table 1). Little or no yield of ethyl nitro[2-¹¹C]acetate ([¹¹C]2) was obtained (less than 8% radiochemical yield). Most [¹¹C]1 was consumed and radioactive polar compounds were obtained (entry 1). DMF was another good solvent for C-carboxylation; however, again, radioactive polar compounds were obtained as major products (entry 2). Katritzky and co-workers reported that C-acylation of 1 was achieved using the dipotassium salt of dianion of 1 and acylbenzotriazoles in DMSO.¹⁰ We therefore examined K(O-t-Bu) as another base for the carboxylation reaction of [¹¹C]1 under the labelling conditions. By this reaction, radioactive polar compounds were major products and the desired [¹¹C]2 was obtained; however, the radiochemical yield of [¹¹C]2 was low (17 ± 16%) and the labelling reactions were not reproducible (entry 3). For the reaction using K(O-t-Bu), we employed a higher reaction temperature (60 °C); however, more complicated radioactive mixtures were obtained and the radiochemical yield of [¹¹C]2 did not improve (data not shown).

Table 1 Radiochemical yield of [¹¹C]1, [¹¹C]2, and polar products by procedure A^a,b,c

Entry	Base	Solvent	[^11C]2 (% yield)	[^11C] Polar products (% yield of mixture)	[^11C]1 (% yield)
a All reactions were performed for 5 min at 30 °C. b [^11C]1 (37–370 MBq), base (22 µmol), 4 (18 µmol), solvent (700 µL). c Decay corrected radiochemical yields were determined by radio chromatogram of HPLC. d Base (13 µmol).
1	NaH	DMSO	<8	>75	14 ± 2
2	NaH	DMF	0	69	31
3^d	K(O-t-Bu)	DMSO	17 ± 16	47 ± 5	33 ± 13

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Reducing the radioactive polar compound formation was an issue to improve the radiochemical yield and reproducibility of [¹¹C]2. When we analysed a DMSO solution of [¹¹C]1 in the absence of a base, polar compounds were not observed.¹¹ No detailed study on polar compound formation has been undertaken, but they are supposed to be formed by the radiolytic decomposition of [¹¹C]3 in the reaction mixture. Alkyl nitronates are good spin trapping agents and have been used to trap radical species.¹² Under radiolabelling conditions, radical species could be formed and reacted with [¹¹C]3. Radiolysis of radioactive compounds was suppressed under low specific radioactivity. Indeed, the formation of radioactive polar compounds was almost suppressed by the addition of 22 µg (0.36 µmol) of non-radioactive 1 to a DMSO solution of [¹¹C]1 in the presence of base (22 µmol).

Thus, we had to employ other procedures in which [¹¹C]3 would react with 4 before radiolysis. For this purpose, [¹¹C]1 was collected in DMSO in the presence of 4 and then a suspension of base in solvent was added (Table 2, procedure B). The C-carboxylation reaction of [¹¹C]1 by NaH as a base in DMSO was sufficiently fast and was completed in 5 min at 30 °C. The desired [¹¹C]2 was obtained at 75 ± 6% radiochemical yield and the radiochemical yield of polar compounds was 19 ± 6% (entry 1). The radiochemical yields of [¹¹C]2 and radioactive polar compounds changed with the amount of 4 (entry 2). Although DMF gave similar results for the C-carboxylation reaction of 1 in DMSO,⁹C-carboxylation of [¹¹C]1 did not proceed well in DMF by procedure B. The radiochemical yield of [¹¹C]2 was 6%, and 84% yield of [¹¹C]1 was recovered (entry 3). Moreover, the C-carboxylation of [¹¹C]1 by K(O-t-Bu) in DMSO was examined. Compound [¹¹C]2 was obtained in 41 ± 11% radiochemical yield but the results were not as good as by NaH (entry 4).

Table 2 Radiochemical yield of [¹¹C]1, [¹¹C]2, and polar products by procedure B^a,b,c

Entry	Base	Solvent	[^11C]2 (% yield)	[^11C] Polar products (% yield of mixture)	[^11C]1 (% yield)
a All reactions were performed for 5 min at 30 °C. b [^11C]1 (37–370 MBq), base (22 µmol), 4 (18 µmol), solvent (700 µL). c Decay-corrected radiochemical yields were determined by radio chromatogram of HPLC. d 4 (10 µmol). e Base (13 µmol).
1	NaH	DMSO	75 ± 6	19 ± 6	6 ± 1
2^d	NaH	DMSO	59	35	6
3	NaH	DMF	6	10	84
4^e	K(O-t-Bu)	DMSO	41 ± 11	34 ± 2	25 ± 9

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Quick and efficient isolation of [¹¹C]2 was required for further transformation. Solid phase extraction (SPE) is a suitable technology for this purpose. A large excess of NaH was used for the C-carboxylation of [¹¹C]1. Compound 2 is a relatively strong carbon acid (pK_a(DMSO) = 9.08, pK_a(H₂O) = 5.82);¹³ therefore, [¹¹C]2 must be in anion form in the reaction mixture. Anion-exchange SPE and successive acid elution are advantageous because the eluted solution is directly used to reduce the nitro group of [¹¹C]2 by metal, such as Zn and In.^7c,7d A Sep-Pak^® QMA Light cartridge was employed for the separation of [¹¹C]2. The reaction mixture solution of the synthesis of [¹¹C]2 was directly subjected to SPE, and the filtrate was collected as waste. The radioactivity of [¹¹C]2 detected in the waste solution was less than 1% for total radioactivity; therefore, most of [¹¹C]2 was extracted by SPE. A 1 : 4 mixture of 1 N HCl and EtOH was employed for the elution of [¹¹C]2 and more than 90% of the total radioactivity was eluted from SPE. Acetic acid was another appropriate acidic solvent for the reduction of the nitro group by Zn powder, but elution efficiency from SPE was not as good as the HCl–EtOH mixture (50–90%). The eluted solution was directly used for reduction of the nitro group of [¹¹C]2, and [2-¹¹C]glycine ethyl ester ([¹¹C]5) was obtained quantitatively using Zn powder within 3 min at 100 °C (Fig. 2). Compound [¹¹C]5 can be used as an in vivo tracer without ester hydrolysis because the ethyl ester group is hydrolysed under physiological conditions.

Fig. 2 Reduction of the nitro group of [¹¹C]2.

Thus, we have shown a rapid and efficient synthesis of [¹¹C]2 and its simple application for the synthesis of [¹¹C]5. Radiolysis of [¹¹C]3 seems to be a common problem to develop [¹¹C]1 as a reliable labelling precursor in DMSO or DMF, the most frequently used solvents in radiolabelling synthesis. Moreover, ¹¹C–C bond forming between [¹¹C]3 and 4 is sufficiently rapid for ¹¹C-labelling. We think that our results will prompt other labelling methods using [¹¹C]1.

Notes and references

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