Rapid and efficient synthesis of [¹¹C]ureas via the incorporation of [¹¹C]CO₂ into aliphatic and aromatic amines

Abstract

A rapid urea radiolabelling methodology has been developed. [¹¹C]CO₂ was activated by 1,8-diazabicycloundec-7-ene (DBU) in the presence of aliphatic and aromatic amines and reacted with Mitsunobu reagents to produce asymmetric ¹¹C radiolabelled ureas in high radiochemical yields.

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Positron emission tomography (PET) is a non-invasive molecular imaging technique that is used for medical diagnosis, drug development, and the understanding of normo- and pathophysiology.¹ Carbon-11 (t_1/2 = 20.4 min) is a commonly used radio-isotope for PET imaging, the ubiquity of carbon in all naturally occurring organic compounds making it an attractive radio-isotope for molecular imaging. Substituting carbon-12 (¹²C) in biologically active molecules with radioactive ¹¹C has no effect on the chemistry or the biological activity of the molecule.² Cyclotron-produced ¹¹C is commonly prepared in the form of [¹¹C]carbon dioxide ([¹¹C]CO₂) by the ¹⁴N(p,α)¹¹C nuclear reaction. Due to its poor reactivity, [¹¹C]CO₂ is typically converted into more reactive synthons such as [¹¹C]methyl iodide or triflate and subsequently used to radiolabel molecules of biological interest.³ Although these labelling synthons are useful, not all target molecules are accessible by these synthons and their preparation takes several minutes with a concomitant decrease in ¹¹C radioactivity due to decay. The development of methods to efficiently label compounds directly with [¹¹C]CO₂ is therefore of significant interest.

To overcome the low reactivity of CO₂, bases such as 1,8-diazabicycloundec-7-ene (DBU) or 2-tert-butylimino-2-diethylamino-1,3-dimethylperhydro-1,3,2-diazaphosphorine (BEMP) have recently been utilised as CO₂ activating agents in the synthesis of ¹¹C-labelled organic molecules.^4,5 These methods, however, produce very poor yields for unreactive aromatic amines, or the reactions are limited to a specific product.⁶

We report herein a rapid [¹¹C]CO₂ radiolabelling methodology which overcomes these limitations. DBU was used to trap the cyclotron-produced [¹¹C]CO₂ which was subsequently reacted with aliphatic, benzylic and aromatic amines (Scheme 1) to synthesise [¹¹C]ureas in a highly efficient manner.

Scheme 1 Synthesis of [¹¹C]urea with [¹¹C]CO₂.

Ureas are found in a plethora of biologically active molecules as has been extensively reported in the literature.⁷ The method reported herein provides a methodology to label this class of compounds with carbon-11.

Model reactions were initially conducted using nonradioactive CO₂.⁸ Compound 3 was subsequently chosen as the initial model reaction for optimisation with [¹¹C] CO₂.

[¹¹C]CO₂ from the cyclotron target was bubbled in a stream of helium gas at a flow rate of 1.4 ml min⁻¹ post target depressurisation directly into a solution containing a primary amine, a secondary amine and DBU in acetonitrile for one minute. The solution was stirred for one minute prior to the addition of Mitsunobu reagents di-tert-butyl azodicarboxylate (DBAD) and tributylphosphine (PBu₃).

Initially, experiments were performed in a number of different solvents at 40 °C (Table 1, entries 1–3) with the aim of identifying the best solvent for the reaction. Acetonitrile trapped cyclotron-produced [¹¹C]CO₂ very efficiently when bubbled directly into the reaction mixture (>95%), while DMSO and DMF were slightly less efficient (80–90%).

Table 1 Reaction optimisation

Entry^a	Solvent	T (°C)	Time (min)	RCY^b (%)
a Reaction conditions: primary amine (18.3 μmol), secondary amine (27.5 μmol), DBU (0.8 μmol), Mitsunobu reagents (36.6 μmol) in 400 μmol acetonitrile. n = 3. b Determined by radio-HPLC. c Reduced concentration.^9
1	MeCN	40	5	46 ± 7
2	DMF	40	5	13 ± 3
3	DMSO	40	5	18 ± 6
4^c	MeCN	25	5	0
5	MeCN	25	5	8 ± 1
6	MeCN	60	5	26 ± 12
7	MeCN	50	5	95 ± 3
8	MeCN	50	1	96 ± 2

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Moderate radiochemical yields (46%) of the desired [¹¹C]ureas were observed using acetonitrile as a solvent while DMF and DMSO gave yields of 13% and 18% respectively, despite good [¹¹C]CO₂ trapping (Table 1). Acetonitrile was therefore selected as the solvent of choice for subsequent reactions.

RCY was determined by radio-HPLC and defined as the amount of labelled [¹¹C]urea as a percentage of the cyclotron-produced [¹¹C]CO₂ trapped in solution obtained directly from the cyclotron and corrected for radioactive decay.

When the reactions were carried out at lower reagent concentrations (Table 1, entry 4), no [¹¹C]radiolabelled product was observed despite efficient [¹¹C]CO₂ trapping.⁹

The temperature dependency of the reaction was subsequently examined. Loss of [¹¹C]CO₂ from the reaction vial was observed when the reactions were performed at 60 °C. Reactions at 50 °C avoided these losses and resulted in over 95% incorporation of the [¹¹C]CO₂ into the target radiolabelled molecules (Table 1, entry 7). Reducing the reaction time from 5 to 1 minute still resulted in a RCY of 96% (Table 1, entry 8 and Fig. 1).

Fig. 1 HPLC chromotogram of the crude radiolabelled product (Table 1, entry 8). (A) Radioactivity (counts per second) target compound 3 at R_t 7.30 min. (B) UV absorption (254 nm) of compound 1 at R_t 3.25 min, compound 2R_t 3.45 min and by-products at 5.00 min, 5.30 min and 5.50 min.

The conditions for the model reaction were subsequently applied to the radiosynthesis of various asymmetric ureas using a range of aliphatic, benzylic and aromatic amines (Table 2).

Table 2 Radiolabelling various aliphatic, benzylic and aromatic amines with [¹¹C]CO₂

Entry^a	Product	RCY^b (%)
a n = 3. b Determined by radio-HPLC. Reaction conditions: [^11C]CO2, primary amine (18.3 μmol), secondary amine (27.5 μmol), DBU (0.8 μmol) in 400 μmol acetonitrile heated at 50 °C for 1 min. Mitsunobu reagents (36.6 μmol) added and stirred for 1 min.
1		74 ± 9
2		94 ± 2
3		69 ± 6
4		85 ± 6
5		83 ± 5
6		80 ± 10
7		19 ± 15

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The reactions between benzylic primary amines and the secondary amine, tetrahydroisoquinoline (Table 1, entry 8) resulted in high RCY while the reaction with N-methylbenzylamine produced slightly lower yields of the [¹¹C]urea (Table 2, entry 1). The high yields for tetrahydroisoquinoline can be explained by the rigidity of the molecule, having a locked planer confirmation and less steric hindrance.

Interestingly, RCYs of similar magnitudes were observed when a less reactive aromatic primary amine was used in place of a benzylic amine to form the target radiolabelled molecules (Table 2, entries 2 and 3).

The effect of functional groups on aromatic amines was also studied. Reactions with the electron rich aromatic amines m-toluidine, and p-anisidine resulted in over 80% RCY (Table 2, entries 5 and 6) and even poor nucleophiles such as 4-nitroaniline reacted efficiently, producing high RCY of 85% (Table 2, entry 4). The reaction favours the formation of asymmetric [¹¹C]ureas despite primary amines being present in excess of [¹¹C]CO₂. In the absence of secondary amines, various by-products are observed resulting in reduced RCY (Table 2, entry 7).

In conclusion, a rapid and robust methodology for the radiosynthesis of ureas has been developed. The method incorporates [¹¹C]CO₂ directly into aliphatic, benzylic and aromatic amines producing the target radiolabelled ureas in high RCY. Overcoming limitations of previous methods, even poorly reactive aromatic amines gave excellent RCY's of asymmetric [¹¹C]ureas within one minute after the addition of Mitsunobu reagents.

This novel radiolabelling methodology opens up new possibilities for ¹¹C radiolabelling molecules for in vivo molecular imaging applications.

Notes and references

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9. Reduced concentration conditions: primary amine (6.1 μmol), secondary amine (9.1 μmol), DBU (0.3 μmol), Mitsunobu reagents (12.2 μmol) and 400 μmol acetonitrile.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c3cc44046j

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