François Liger

Dr Ture Eijsbouts

Thierry Billard
email: thierry.billard@univ-lyon1.frwww.fmi-lyon.fr

Dr D Le Bars

F Cadarossanesaib

Dr C Tourvieille

Direct 11 C-Methylation of Amines from [ 11 C]CO 2 for the Synthesis of PET Radiotracers

C-Methylation (t1/2 = 20.4 min) is a main labeling strategy in the development of PET radiotracers. A straight radiomethylation of amines by use of cyclotron-produced 11 CO2 was developed to obtain various radiolabeled compounds with good radiochemical yields. This strategy constitutes a new approach to simplify the radiolabeling processes and transpositions onto automatic synthesizers. This method was applied to the syntheses of radiolabeled drugs and a PET radiotracer used in Alzheimer's disease studies.

Introduction

PET (positron emission tomography) imaging is a nuclear medicine technology increasingly used not only in routine clinical diagnosis [1] but also in biomedical research and drug development. [2] These growing applications of PET imaging require the development of more and more various radiotracers. However, the short-lived positron-emitters impose specific synthetic methods, easily transferable onto automatic synthesizers. [1c, 3] In general, [ 18 F] labeled molecules are preferred for production and use in routine clinical imaging due to the higher half-life of fluorine-18, as illustrated by the recent development of various methods for radiofluorination. [4] Nevertheless, despite the 11 C short half-life (t1/2 = 20.4 min), [ 11 C] labeled molecules still represent valuable targets for research studies. Indeed, easy-to-introduce carbon-substituents are present in a large majority of drugs and bioactive compounds. Consequently, a direct radiolabeled equivalent of molecule is easier to envisage.

The radioisotope carbon-11 is usually produced from a cyclotron by the 14 N(p,a) 11 C nuclear reaction in the form of either of two precursors: 11 CH4 or 11 CO2. Direct use of these two compounds is quite limited and they are, usually, converted into more reactive species. Some direct synthetic applications of 11 CO2 have been previously described [5] to obtain [ 11 C] labeled carboxylic acids by reaction with Grignard and organolithium reagents [3a, 6] or, more recently, by cross-coupling reactions with boronic acids. [7] The syntheses of [ 11 C] labeled ureas and carbamates have been also described. [8] Methylation reactions of heteroatoms, in particular nitrogen, constitute one of the more classic approaches for 11 C-labeling of molecules, [3a, 6] as illustrated by the radiosynthesis of the bamyloid imaging radioligand [ 11 C]PIB. [9] These radiomethylations require preliminary transformation of 11 CO2 into very reactive species, 11 CH3I or 11 CH3OTf. [3a, 6, 10] These reagents are often used under basic conditions in a strictly anhydrous environment. Such conditions can require additional protection and deprotection steps of incompatible functional groups that substantially prolong and complicate the radiolabeling process. Furthermore, from a technical point of view the simplest radiosynthesis, with a minimum of steps, will lead to an easier automation. [10][11] Consequently, a direct radiomethylation of amines using 11 CO2, generated from cyclotron, appears very attractive. Some previous works have been described, [12] however, these strategies require a multi-steps procedure: 11 CO2 is first fixed with silylated amines (preliminary prepared) to give O-silyl carbamates, which are subsequently reduced in situ by LiAlH4 (a harsh reducer). Therefore, a more direct and soft procedure could be beneficial.

Results and Discussion

Recently, some groups have described the application of CO2 as a C1-building block for catalytic methylation of amines. [13] Such elegant works appeared in perfect adequacy with our effort to develop a simpler and more direct way to perform 11 C-methylation of amines. This approach would eliminate the preliminary time-consuming, and sometimes sources of failure during the automatic radiolabeling, steps of preparation of reactive methylating reagents. We have focused our interest onto the strategy developed by Cantat et al. using standard and simple catalytic system (ZnCl2, IPr), without use of additional hydrogen gas. [13b] As within this article, N-methyl aniline (1a) has been selected as model substrate (Table 1).

With a similar catalytic system, in the same solvent and at the same temperature than the optimal described conditions, only 3% of expected methylation was observed in 20 min (entry 1). Such disappointing results can be rationalized by the short time of reaction, imposed by 11 C half-life, compared to the 20 hours required in original publication. To increase the reaction kinetic, a solvent with similar properties and polarity but with a higher boiling point was selected to increase reaction temperature. Thus, in diglyme, at 150°C, a satisfactory radiochemical yield of 40% has been obtained (entry 2).

Because of the small molar amount of 11 CO2 generated by cyclotron (e.g. 1 Ci, i.e. 37 GBq, correspond to 108 pmol), the precursor (1a) quantity was decreased to match standard amount (around 1-2 mg) used in classic radiolabeling without any change in yield (entry 3). It should be noticed that, for practical reasons (weighing), the same amount of catalyst was preserved, bringing the catalyst amount to stoichiometry (related to 1a). With a shortened reaction time of 10 min, the radiochemical yield was halved (entry 4), even with a doubled catalyst amount (entry 5). The presence of both ZnCl2 and IPr is proving to be essential (entries 6-8). Preliminary complex formation [ZnCl2;IPr] [14] appears to be deleterious for radiolabeling, in contrast with the work of Cantat et al. (entry 9). [13b] In order to increase 11 CO2 concentration in solution to potentially accelerate the reaction, TMEDA (N,N,N',N'tetramethylethylenediamine) as CO2 trap was added, as described by Pike et al. [7] However, no reaction was then observed: TMEDA certainly competes with 1a to catch CO2 (entry 10). Finally, microwave irradiation appeared to be also detrimental for the reaction; the increased molecular agitation contributes to break the amine-CO2 interaction, which seems to be essential to initiate the methylation (entry 11). Furthermore, Dyson et al. have described similar methylation in metal-free conditions with another carbene (NHC, 1,3-bis(2,4,6trimethylphenyl)-1,3-dihydro-2H-imidazol-2-ylidene). [13e] Unfortunately, in our conditions, no expected radiolabeling was observed (entry 12).

Moreover, the trapping efficiency of cyclotron-produced 11 CO2 was measured using the optimal conditions (Table 1, entry 3) at a trapping flow rate of 10 mL/min. Untrapped 11 CO2 was confined within an ascarite ® cartridge attached to the reactor outlet. Up to 80% of the delivered 11 CO2 was kept inside the reactor at the end of entrapment (trapping time = 60s). More precisely, the trapping efficiency depends on the amines basicity: 80% with 1d, 70% with 1a and 65% with 1e. 5 [c] 0.023 Diglyme 150 10 27

6 [d] 0.023 Diglyme 150 20 0

7 [e] 0 With the optimal reaction conditions in hand (Table 1, entry 3), the scope of this new radiolabeling strategy was investigated using various amines (scheme 1). These 11 C-methylations gave generally satisfactory radiochemical yields with aromatic or aliphatic amines. Primary amines are selectively mono-methylated and, generally, only a small amount of bis-methylation was observed. With anilines, the presence of electron-withdrawing or electron-donor substituents seems to have no relevant influence (3b-3h). In accordance with the supposed mechanism (see scheme 2), electron-withdrawing groups should favor the first step of CO2 trapping by amine whereas electron-donor substituents should more favor the second and third reduction steps. This reaction is also compatible with ester function (3f). In the case of cyano group, a more moderate yield is observed, maybe due to partial chelation of ZnCl2 by the nitrogen atom partially quenching the reaction. Starting from p-bromoaniline (1h), a satisfactory yield of expected product was obtained, despite the formation of 3b, arising from the partial reduction of the C-Br bond. This radiolabeling appears also to be chemoselective since in case of amino-alcohol, only N-methylation is observed (3k). However, to achieve similar radiochemical yields compared to other amines, an excess of amino-alcohol (vs ZnCl2) was required. This could be explained by chelate formation between amino-alcohol and zinc which could deactivate the nitrogen atom and thus would disfavor the CO2 trapping. Finally, this methodology could be applied to the synthesis of radiolabeled drugs, such as ephedrine (sympathomimetic amine, 3k), imipramine (tricyclic antidepressant, 3j) and the b-amyloid radiotracer [ 11 C]PIB (Alzheimer disease diagnostic, 3l).

From a mechanistic point of view, Cantat et al. have proposed the over reduction of an intermediate formamide (Scheme 2, steps 2-3). [13b] Furthermore, radiomethylation by reduction of a formamide has been previously reported. [15] In our conditions the CO2-amine interaction appeared to be determining. Indeed, an increase of molecular agitation by micro-wave [16] disfavored the reaction by breaking this interaction. In addition, starting from imipramine hydrochloride (3g), no labeling was observed due to the impossible CO2-amine interaction. During the radiomethylation of 1a, the corresponding labeled formamide, could be identified by HPLC. These observations led us to proposed the following pathway (Scheme 2): 1) formation of CO2-amine complex; 2) 1 st reduction in formamide; 3) 2 nd reduction in expected methyl amine. This direct 11 C-labeling strategy was applied to effective production of the b-amyloid radiotracer [ 11 C]PIB [17] (3i) with HPLC purification and final formulation step (Scheme 3).

[ 11 C]PIB was produced in sufficient radioactive amount (57 mCi -RCY = 38%), with a high radiochemical and chemical purity. Such result is very encouraging and demonstrates the possibility to extrapolate this new strategy to a "productive scale" of radiotracers. Specific radioactivity remains however weak (SA = 15 GBq.µmol -1 ), certainly due to excess of environmental CO2 which dilutes 11 CO2. Nevertheless, in a classical production of [ 11 C]PIB ([ 11 C]methyl triflate method) with the same synthesizer and a similar production of 11 CO2 by the same cyclotron, [ 11 C]PIB is obtained with a similar radiochemical yield of 45% and a specific activity of around 50 GBq.µmol -1 .

R 1 N R 2 11 CO 2 H R 1 H N R 2 11 CO 2 [ZnCl 2 /IPr] PhSiH 3 R 1 N R 2 11 C H O [ZnCl 2 /IPr] PhSiH 3 R 1 N R 2 11 CH

Conclusions

In conclusion, we have brought the proof of concepts of direct and selective amine 11 C-methylation by direct use of cyclotronproduced 11 CO2. Even if some improvements are still required, these preliminary data should open the way for new developments of direct and simple radiolabeling methods.

Experimental Section

Synthesis of [ 11 C]PIB (3i). Cyclotron-produced 11 CO2, trapped (953 mCi) on a column of molecular sieves (4 Å), was released by purging the heated column (350°C) with He gas and bubbled, through Teflon lines, into the reactor containing ZnCl2 (1.3mg), IPr (3.5 mg), 1i (1.7 mg) in 400 µL of diglyme and PhSiH3 (23µL) cooled at 0°C ( 11 CO2 traps: 711 mCi). Teflon lines were removed when radioactivity content reached a maximum and the reacting mixture was heated at 150°C for 20 min. After cooling, 2.2 mL of HPLC mobile phase were added to the mixture and the resulting solution was purified by HPLC on a Waters Symmetry-Prep C18 column (7 µm, 7.8 x 300 mm) at a flow rate of 4 mL/min (H2O/MeCN 60:40 v/v). [ 11 C]PIB fraction (tR = 11 min) was collected, diluted in 40 mL of water and formulated by SPE. After rinsing with 10 mL of water, the purified product was released from the Sep-Pak (Waters Plus tC18) with 1 mL of ethanol and 2 mL of water in a sterile vial (57 mCi).

Scheme 1 .

 1 Scheme 1. Direct 11 C-methylation of amines with 11 CO2. RCY values are mean (n=3). They are estimated from trapped 11 CO2 within the reactor and are decay-corrected from the end of 11 CO2 trapping inside reactor. [a] ratio 3h/3b = 60:40.[b] with 2 equiv. of 1h vs ZnCl2.

Scheme 2 .

 2 Scheme 2. Hypothesized mechanism for the 11 C-methylation with 11 CO2.

Table 1 .

 1 Direct 11 C-methylation of 1a with 11 CO2.[a]

	Entry	[1a] (M)	Solvent	T (°C)	t (min)	RCY [b] (%)
	1	0.231	THF	80	20	3
	2	0.231	Diglyme	150	20	40
	3	0.023	Diglyme	150	20	43
	4	0.023	Diglyme	150	10	23

  Scheme 3. Production of b-amyloid radiotracer [ 11 C]PIB. RCY value is estimated from trapped 11 CO2 within the reactor and are decay-corrected from the end of 11 CO2 trapping inside reactor

								iPr
							iPr
							N	N
							iPr	iPr
	R 1	H N R 2 1	+	2 (20 equiv.) PhSiH 3	+ 11 CO 2	ZnCl 2 (1 equiv.) IPr (1 equiv.)	3 R 1 N R 2 11 CH 3
	11 CH 3		11 CH 3			11 CH 3	11 CH 3
		N			NH			NH	NH
	3a 41 %		3b 48 % (mono/bis = 95:5)		MeO (mono/bis = 96:4) 3c 60 %	OMe (mono/bis = 98:2) 3d 75 %
		NH 11 CH 3			NH 11 CH 3		NH 11 CH 3	NH 11 CH 3
	F 3 C (mono/bis = 92:8) 3e 53 %		EtO 2 C (mono/bis = 95:5) 3f 55 %	NC	3g 24 %	Br (mono/bis = 92:8) 3h 48 % [a]
				NH				H
		11 CH 3 (mono/bis > 95:5) 3i 47 %	N [ 11 C]Imipramine 3j	N 11 CH 3	NH HO H 11 CH 3 3k [ 11 C]Ephedrine
							26 %	29 %
								42 % [b]
	HO	S		11 CH 3	3l			(mono/bis > 95:5)
		N		NH	[ 11 C]PIB 45 %

Acknowledgements

This work has been supported by the RADIOMI project (EU FP7-PEOPLE-2012-ITN-RADIOMI). Dr Anis Tlili (ICBMS -UMR CNRS 5246) is thanked for fruitful discussions. Frédéric Bonnefoi and Thibaut Iecker (CERMEP -in vivo imaging) are acknowledged for their technical assistance. The authors wish to thank CNRS for financial supports.

Keywords: alkylation • carbon-11 • positron emission tomography • radiochemistry • isotopic labeling