WO2006133732A1

WO2006133732A1 - Process for synthesizing labelled compounds

Info

Publication number: WO2006133732A1
Application number: PCT/EP2005/010051
Authority: WO
Inventors: Vincent Tadino; Marco Müller
Original assignee: Advanced Accelerator Applications; Abx Advanced Biochemical Compounds - Biomedizinische Forschungsreagenzien Gmbh
Priority date: 2005-06-17
Filing date: 2005-06-17
Publication date: 2006-12-21

Abstract

The main objectives of the invention are notably: the shrinkage of the process time because of the low half-life of [18F]FLT, as well as the increase of the yield, especially without HPLC purification which is a complex and a time consuming step. Other intended improvements are the reduction of the duration and the complexity of the synthesis and of the device. The purpose is to facilitate the methods of synthesis used by the Positron Emission Tomography (TEP) laboratories. The invention concerns a process for synthesizing labelled compounds with an isotopic element, comprising the steps of: a. preparing at least one labelling agent; b. labelling at least one precursor with the labelling agent, wherein the precursor is a protected substrate; c. possibly pre-purifying the labelled precursor; d. retaining the labelled precursor on at least one solid support; e. deprotecting the labelled precursor by hydrolysis so as to obtain the labelled compound, directly on at least one solid support which is contained in at least one column or cartridge; f. recovering the labelled compound by elution g. and purifying the eluate, wherein the labelled compound is 3’-deoxy-3’-[18F]fluorothymidine ([18F]FLT) wherein the deprotectionhydrolysis is carried out by means of at least one deprotectionhydrolysis is carried out by means of at least one deprotecting/hydrolyzing agent selected in the organic acids groups comprising e.g.: trifluoroacetic acid (TFA) and/or the purifying step (g) comprises transferring the eluate containing the labelled compound on at least one hydrophilic-lipophilic water-wettable reversed-phase sorbent for solid-phase extraction.

Description

PROCESS FOR SYNTHESIZING LABELLED COMPOUNDS

FIELD OF THE INVENTION

The present invention a new fully automated method for the synthesis of labelled compounds such as 3'-deoxy-3'-[¹⁸F]fluorothymidine ([¹⁸F]FLT), by modifying a commercial FDG synthesizer (for instance the "TRACERlab MX_FDG ®, marketed by the GE Medical Systems company) and its disposable fluid pathway.

Other synonyms Of [¹⁸F]FLT are : (l-(3'-Deoxy-3'-fluoro-β-D-pentofuranosyl)thymine, 3'- Deoxy-3'-fluorothymidine, 3'-Fluorodeoxythymidine, 3'-Fluorothymidine, Alovudine, CL 184824.

The invention also relates to a device for synthesizing said labelled compounds which relies on this method, making an automatic processing possible, including possibly a single use kit of materials.

The process according to the invention relates to synthesis methods for labelled compounds with any isotopic element and used particularly in the medical field (NMR, therapy, medical imaging), based upon the labelling of an organic substrate the functional groups of which are protected beforehand by protecting groups which, after the labelling step, can be easily removed by hydrolysis.

The term "functional groups" means functions such as alcohol, thiol, phenol, thiophenol, amines, ketones, aldehydes, carboxylic acids, etc. The term "protecting groups" means groups (according to the function to be protected) such as acetyl, ethers, esters, thioesters, thioethers, imines, enamines, amides, carbamates, N-alkyles, N-aryles, N-hetero derivates, cetals, acetals, etc.

BACKGROUND OF THE INVENTION

[¹⁸F]FLT is a tracer increasingly used in nuclear medical imaging. This molecule labelled with the radionucleide ¹⁸F, appears to be the one of the most promising radiopharmaceuticals because of the lack of in vivo degradation, metabolic trapping in proliferating cells, and the favorable half life for Positron Emission Tomography (PET) imaging. This molecule, labelled with the radionucleide ¹⁸F, allows the mapping and the quantification of this fundamental mechanism. Recent clinical studies have also demonstrated that it would be a promising tumor therapy response marker for lung and other kinds of cancer.

Several methods have been developed and are presently used in various (TEP) laboratories to produce [¹⁸F]FLT for immediate use closely from the place of synthesis.

Before mentioning the closest prior art relating to [¹⁸F]FLT, it is referred hereinafter to a technological background regarding 2-[¹⁸F]fluoro-2-deoxy-D-glucose, more commonly called fluoro-deoxy glucose or FDG. FDG is also a tracer used in nuclear medical imaging. This molecule labelled with the radionucleide ¹⁸F, behaves in a way similar to glucose in the first step of its metabolization in the human body and allows to map and quantify this fundamental mechanism. It is indicated for diagnosis of numerous diseases. The most widely spread FDG synthesis method is the so-called Hamacher method, described by Hamacher K., Coenen H. and Stocklin G. in "Efficient Stereospecific Synthesis of No-carrier-added-2- [¹⁸F] fluoro-2- deoxy-D-glucoso Using Aminopolyether Supported Nucleo-philic Substitution", Journal of Nuclear Medicine 27, 235 (1986).

US-B-6,172,207 discloses a method for synthesizing [¹⁸F]fluoro-2-deoxy-D-glucose using multiple stopcock manifolds and disposible sterile syringes. This corresponds to the commercial "TRACERlab MXFDG ®", marketed by the GE Medical Systems company The synthesis reaction according to US-B-6,172,207 occurs as follows:

I. recovery of the [¹⁸O] enriched water using an anionic resin namely the 4, 7, 13, 16, 21, 24-hexaoxo-l,10-diazabicyclo-[8.8.8]-hexacosane [KRYPTOFIX®. (also called K2.2.2)] which is a ¹⁸F "activating" agent; 2. recovery, by elution of the anionic resin, of the activity in the form of [K/222]¹⁸⁺, F^" in a solution in a mixture CH3CN/H₂O,

3. evaporation of the solvent by IR heating (105.⁰C.) under nitrogen flow (2 min. 30 sec),

4. addition of 1 ml CH₃CN, evaporation (2 min. 30 sec),

5. addition of 1 ml CH₃CN, evaporation to siccity (determination of the evaporation end through a temperature probe),

6. cooling of the reactor to 7O.°C,

7. addition of a solution of a labelling precursor (15 mg) in CH₃CN (1.7 ml),

8. heating at 95. degree. C. during 3 min. (labelling step),

9. dilution of the resulting solution in 25 ml water, 10. transfer of the diluted solution through a Cl 8 cartridge (conditioned beforehand with 5 ml ethanol followed with 10 ml water) to the waste,

II. rinsing of the cartridge with 10 ml 0.1 N HCl and 10 ml water which are sent to the waste, 12. drying of the cartridge under nitrogen flow,

13. addition of 0.7 ml 1.5M NaOH on the C18 cartridge,

14. deprotection (hydrolysis) 1.5 min. at room temperature,

15. elution of the FDG with 5 ml water into a syringe containing 0.8 ml 1.5M HCl and 5 ml of citrate buffer, and

16. transfer of the resulting solution through a Cl 8 cartridge, a neutral alumina cartridge and a 0.22 μm filter; the solution is collected in a sterile vial.

[¹⁸F]FLT is a labelled compound different from FDG and constitutes an interesting alternative to FDG. [¹⁸F]FLT offers a better precision than FDG in PET imaging for cancer diagnosis.

Furthermore, to use [ F]FLT in clinical studies, automation is necessary to reduce unnecessary radiation exposure for the operators and to obtain high radiochemical yield with reproducibility. In this context, there is an obvious interest to improve and to simplify the [¹⁸F]FLT synthesis. In particular, the search of higher yield of synthesis is an important issue.

These were the aims of the authors of the following publication: "Fully automated synthesis system of 3'-deoxy-3'-^lsF fluorothymidine : Oh, S. J.; Mosdzianowski, C; Chi, D. Y; Kim, J. Y; Kang, S. K; Ryu, J. S.; Yeo, J. S.; Moon, D. H., Nucl Med Biol, VOLUME 31, N° 6, 2004 Aug. PP 803-9".

Said publication described a [¹⁸F]FLT synthesis with an automated production system based on the commercial [¹⁸F]FDG synthesis module, namely the "TRACERlab MX_FDG ®, marketed by the GE Medical Systems company" as described in the US-B-6,172,207. This synthesis is substantially based upon the following operating steps: Preparation of the fluorinatine asent

In a first step, the ¹⁸F is activated through "activating" agents such as an anionic resin namely the 4, 7, 13, 16, 21, 24-hexaoxo-l,10-diazabicyclo-[8.8.8]-hexacosane [KRYPTOFIX®. (also called K2.2.2)], so as to make it more reactive. In some publications, they are called "phase transfer agents". The radionucleide is produced beforehand, generally by irradiation of ¹⁸O enriched water with a proton beam originating from a particle accelerator, as F^" (for instance H¹⁸F, in an aqueous solution). The labelling of the precursor The fluorinating agent, made totally anhydrous by additions of acetonitrile (CH₃CN) and dry evaporations, is put in presence of a labelling substrate (precursor), generally the precursor 1 -(2'-Deoxy-3 '-O-(4-nitrobenzenesulfonyl)-5'-O-(4,4'-dimethoxytrityl)-beta-D- threo-pentafuranosyl)-3-(ter^butyloxycarbonyl)thymine also called 3-N-Boc-l-[5-O-(4,4'- dimethoxytrityl)-3-C>-nitrophenylsulfonyl-2-deoxy-β-D-lyxofuranosyl]thymidine (5'-O- DMTrdimethoxytrityl-2'-deoxy-3'-0-nosyl-β-D-threo-pentofuranosyl)-3-N-BOC-thymine, solubilized in acetonitrile. A substitution reaction then occurs, where the nosyl group of the substrate is replaced by the ¹⁸F atom, resulting in the formation of BOC- DMTr- undeprotected product.

Hydrolysis

The fluorination solvent is partially evaporated and the hydrochloric acid is added in the reactor, after heating the solution, hydrolysis is completed, to give a bulk solution of.

[¹⁸F]FLT

The global chemical mechanism is the following:

Purification

The solution is then neutralized and injected in HPLC for purification.

The final product is collected after HPLC purification and is formulated in a directly injectable solution of water and ethylic alcohol.

The obtained radiochemical yield is 42±5.4% after HPLC.

This known procedure has, however, a number of drawbacks, the main ones of which are: The duration of such a procedure is about fifty minutes, particularly because of the important number of the successive heating and evaporating steps as well as liquid transfers, resulting in a loss of activity just because of the 110 minute half- life of ¹⁸F. Moreover, the purification with HPLC is inevitable and the automation of said HPLC purification is almost impossible, and requests addition of hardware.

AIMS OF THE INVENTION

Faced to this prior art, the inventors aimed to improve the method for the synthesis of 3^l- deoxy-3418F]fluorothymidme ([18F]FLT), as disclosed in Oh, S. Jet al. Nucl Med Biol, VOLUME 31, N° 6, 2004 Aug. So, some of the main objectives of the present invention are notably: the shrinkage of the process time because of the low half-life of [¹⁸F]FLT, as well as the increase of the yield using a new hydrolysis method, and, preferably to simplify the process, the possibility to remove (if need and/or wish be) the final HPLC purification which is a complex and a time consuming step.

Other intended improvements are the reduction of the duration and the complexity of the synthesis and of the device.

The purpose is to facilitate the methods of synthesis used by the Positron Emission Tomography (TEP) laboratories.

BRIEF DESCRIPTION OF THE INVENTION

These objectives, among others, have been reached by the present invention, which relates to a process for synthesizing labelled compounds with an isotopic element, comprising the steps of: a. preparing at least one labelling agent; b. labelling at least one precursor with the labelling agent, wherein the precursor is a protected substrate; c. possibly pre-purifying the labelled precursor; e. deprotecting the labelled precursor by hydrolysis so as to obtain the labelled compound; f. recovering the labelled compound; g. and purifying the recovered labelled compound,

wherein the deprotectionhydrolysis is carried out by means of at least one deprotecting/hydrolyzing agent consisting in at least one acid and/or at least one base, with the exception of HCl and NaOH, preferably in at least one organic acid, said organic acid(s) being more preferably selected in the group comprising the acetic acid and its derivates, notably the halogenated acetic acid, and even more preferably in the sub-group comprising: trifluoroacetic acid (TFA), acetic acid, formic acid, the analogs thereof and mixtures thereof.

Preferably, the labelled compound is 3'-deoxy-34¹⁸F]fluorothymidine ([¹⁸F]FLT).

The process according to the invention makes it possible to raise significantly the [¹⁸F]FLT yield, even without HPLC. The hydrolysis allows to increase the yield, that is why purification without HPLC is possible. In summary, better yield are got for the synthesis, which enables to carry out a purification different from HPLC purification (namely, for example Solid Phase Extraction (SPE) purification), in order to enhance easily the final purity of the labelled compound. In such case priority is given to the purity. So, the overall yield could be a little bit lower, but it's to the credit of the invention to offer this possibilty to give priority to the purity (possibly without HPLC) or to the yield.

The deprotection/hydrolysis step (e) of the process of the invention can occurs in a reactor which is a container (vial, vat, etc.) and/or on and/or into a solid support capable to adsorb/trap the precursor (cartridges, columns, wells, plates and so on...).

This latter type of reactor is in the core of a preferred way of implementation for the process according to the invention. Said preferred way comprises the steps of: a. preparing at least one labelling agent; b. labelling at least one precursor with the labelling agent, wherein the precursor is a protected substrate; c. possibly pre-purifying the labelled precursor; d. retaining the labelled precursor on at least one solid support; e. deprotecting the labelled precursor by hydrolysis so as to obtain the labelled compound, directly on at least one solid support which is contained in at least one column or cartridge; f. recovering the labelled compound by elution g. and purifying the eluate.

This embodiment correspond to an automated synthesis which is easy, rapid, reliable and reproductible. Thus, it can be carried out where the injection Of[¹⁸F]FLT to the patients are done, given the limited half-life of 110 min of this radiolabelling compound.

So, the users of this labelled compound who are not equipped to make chemical synthesis, could implement this process according to the invention, which facilitates the marketing of this promising labelling compound.

One of the keys of the invention consists in the selection of a specific group of acidic hydrolysis agent, which acts, possibly in conjunction with the solid support on which the undeprotected labelled precursor is fixed (preferred embodiment), as a deprotective agent enabling in the case of [¹⁸F]FLT, the lysis of the bonds between BOC- & DMTr- groups and FLT molecule. For the [¹⁸F]FLT, the undeprotected labelled precursor is, for example, selected in the group of precursors including those corresponding to the formula in the right of the following reaction schema:

deprotection/hydrolysis agent with

Rl = H, BOC, tBOC, C₂ to C₁₀ alkyloxy carbonyl

R2 = Nos (O-nitrophenylsulfonyl), R-SO₂ (with R= C₁ to C₅ alkyl or unsubstitued phenyl group) R3=, triphenylmethyl, substituted on the phenyl group, trialkylmetyl, triphenylsilyl substituted on the phenyl group, trialkylsilyl,Trityl, DMTrityl, ,....

In practice, the undeprotected labelled precursor can be l-(2'-Deoxy-3'-O-(4- nitrobenzenesulfonyl)-5'-0-(4,4'-dimethoxytrityl)-beta-D-threo-pentafuranosyl)-3-(teλ^- butyloxycarbonyl)thymine, or

(5'-O-DMTr-2'-deoxy-3'-O-nosyl-β-D-tlireo-pentofuranosyl)-3-N-BOC-thymine

The worthwhile amount of precursor used in the process can be comprised —for example- between 1 mg and 100 mg of a solid support. The preferred quantities are from 5 to 50 mg. More preferably, it can be for example 25 mg.

AU the numerical values given in the present exposure are given with a tolerance of +/- 10% for example, with or without the adverb "about".

Advantageously, the deprotecting/hydrolyzing agent includes an acid aqueous solution, preferably of TFA.

Practically, the deprotecting/hydrolyzing agent includes, for example, an acid aqueous solution of TFA, which [TFA] concentration is greater or equal to 40% by weight, preferably greater or equal to 50% by weight, and more preferably comprised between 55 and 65% by weight. According to an interestering feature of the invention, the step (e) of deprotecting/hydrolyzing lasts between 0.1s to 500s (s = seconds), preferably, between Is and 120s.

The elimination of said protecting groups in the deprotection step (e) is obtained directly on a solid support comprised in a column, a cartridge, in plates or in wells, exhibiting a high affinity to the protected molecule and a low affinity to the deprotected molecule. The term "column" "cartridge" "plates" or "wells" means equally any kind of stationary phase conditioning which may be used in chromatography, possibly including plastic or glass containers, columns, etc. These products are commercially available and are in particular used in SPE (Solid Phase Extraction) applications and in solid phase chromatography. The column or cartridge containing the solid support which adsorbs the labelled precursor (step -d-) and where the deprotection (e) is directly performed, can be also used for the previous step (d) of pre-purifϊcation of the labelled precursor (elimination of the residual reagents and elimination of the used organic solvent(s) e.g. acetonitrile).

The possible cartridge used may be for example of the types Cl 8, C8, tC18, NH2, diol, polystyrene divinylbenzene (SDB) or other polymeric phases, as for example available under the following trademarks: Maxi-clean®. cartridges from Alltech®: Cl 8, 300 mg cartridge (Alltech®No. 20922) C8, 300 mg cartridge (Alltech®No. 20946) NH2, 300 mg cartridge (Alltech®No. 210040) These cartridges also exist in 600 and 900 mg versions. Waters® cartridges, from 50 mg to 10 g, in particular: Cl 8 cartridges of Sep-Pak short body type (Waters®No. WAT 020 515) tC18 cartridges (trifunctional) of Sep-Pak® short body type 400 mg (Waters®No. WAT 036 810) Waters® OASIS® HLB extraction cartridge. Varian® cartridges: Microbond Elut ® C18 (re No. 1214-4002) Microbond Elut® C8 (re No. 1214-4405) Microbond Elut ® PS-SDB (re No. 1214-4011) Macherey- Nagel ® cartridges: Chromabond ® Cl 8 500 mg (re No. 730 003) Chromabond ® Phenyl 500 mg (re No. 730 084).

The cartridges and columns that are used, contain -for example- between 50 mg and 1O g of a solid support. The preferred quantities are from 200 to 800 mg. Other quantities are also possible.

According to an interesting feature of the invention, the solid-support of step (e) is selected in the group comprising: normal phase, reverse phase, intermediate polarity phase, low polarity phase, ion exchange phase and mixtures thereof.

The more preferred supports are, for instance: low polarity phase, ion exchange phase mixtures of one or several ion exchange phase(s) with one or several normal or reverse phases. Practically, it is suitable that the deprotection step (e) be performed on a column or cartridge selected from the group including the SEP PAK® cartridges marketed by the WATERS® company under the names Cl 8, C8, tC18, NH2, diol, and polystyrene divinylbenzene (SDB).

According to a preferred embodiment of the invention, the purifying step (g) comprises tranferring the eluate containing the labelled compound on one or several —preferably at least two- hydrophilic-lipophilic water-wettable reversed-phase sorbents for solid-phase extraction (SPE).

Advantageously, these sorbents are assimilable to solid supports as described above for the trapping step (d) and the deprotection step (e).

These two or more sorbents are advantageously in serial. That means that the eluate containing the labelled compound is flushed through to a first sorbent and then through a second sorbent (or more) successively.

Actually, the number of sorbents (columns or cartridges) is not the only parameter to take into consideration. Indeed, the mass of the material constituting the sorbent(s). Regarding the the flow and the volume of liquid involved in the process, the man skilled in the art could easily determine this mass. For instance, said mass could be comprised between 900 and 1100 mg.

It is beneficial that the purifying step (g) be carried out at the ambient temperature.

Advantageously, the flow of the eluate through the sorbent(s) is chosen between about 2 to about 5 rriL per minute.

Profitably, the hydrophilic-lipophilic water-wettable reversed-phase sorbents can be any of those hereabove described. In particular, the hydrophilic-lipophilic water-wettable reversed-phase sorbent for solid-phase extraction of step (g), can belong to the group of those which contain at least one copolymer comprising at least one hydrophilic comonomer (e.g. N-vinylpyrrolidone) and one lipophilic comonomer (e.g. divinylbenzene). More particularly, the hydrophilic-lipophilic water-wettable reversed- phase sorbents can be selected in the sub-group of the WATERS® OASIS® HLB family of sample extraction products, e.g. cartridges, columns, wells, or plates, the OASIS® HLB cartridges being especially preferred (e.g. OASIS® HLB plus cartridges, 225mg of sorbent, 60μm particle size). The hydrophilic-lipophilic water-wettable reversed-phase sorbent for solid-phase extraction of step (g) and/or the solid-support of step (e) is (are) contained in a cartridge or column in a amount between 50 mg and 10 g of a solid support/sorbent.

In an remarkable variant, the step (g) is followed by a step (gl) comprising at least one rinsing of the solid sorbent(s) with a liquid selected in the group comprising aqueous solutions, physiological solutions, water, alcohols (preferably ethanol), and mixtures water/alcohol(s).

The step (gl) includes a rinsing (gl 1) of the solid sorbent(s) with water and a rinsing (gl2) of the solid sorbent(s) with a mixture water/alcohol(s), preferably a mixture water/alcohol(s) 99/1 - more than 70/less than 30 by volume, for example about 9/1 by volume. The rinsing step (gl2) can be assimilated to an elution of impureties, which affinity for the sorbent(s) is less than the affinity of the labelled compound for said sorbent(s).

The purifying step (g) comprises at least one elution (g2) of the solid sorbent(s) with an eluent selected in the group comprising alcohols (preferably ethanol) and mixtures water/alcohol(s), preferably a mixture water/alcohol(s) 70/30 -0/100 by volume, for example about 70/30 by volume.

According to a worthwhile way of implementation, at least the elution (g2) is e.g. a countercurrent elution of the labelled compound(s)

The hydrophilic-lipophilic water-wettable reversed-phase sorbent for solid-phase extraction of step (g) and/or the solid-support of step (e) is (are) in the form of grains, membranes, sheets or capillaries.

The conditions of injection through the solid support/sorbent, of rinsing and/or of elution (e.g. flow, volume of injection rinsing liquid/eluent, temperature....) are for example those given by the manufacturer and providers of SPE solid support or sorbent, notably of hydrophilic-lipophilic water-wettable reversed-phase sorbents for solid-phase extraction.

For example, these conditions can be as follows:

The uses of syringe drivers and vacuum allows to control exactly the flow through the cartridges.

■ To load the solid support of step(s) (d/e), e.g.C-18 cartridge, before the hydrolysis: about 7mL/min ^■ To rinse the solid support of step(s) (d/e), e.g. C-18 cartridge, before hydrolysis: about 7mL/min

^■ To elute the bulk solution out of the solid support of step(s) (d/e), e.g.C-18 cartridge: about 5mL/min

^■ To load the bulk on the hydrophilic-lipophilic water-wettable reversed-phase sorbent for solid-phase extraction (SPE), e.g.OASIS® HLB -step (g)-: about 7mL/min

■ To rinse the SPE, e.g. OASIS ® HLB -step (g)-: about 7mL/min

^■ To elute the impurities e.g. with the 9-1 water-ethanol mixture —step (g)~: about 3-5mL/min

■ To elute the final product e.g with the 7-3 water-ethanol mixture -step (g)-: about 5mL/mm

The process temperature is e.g. the room temperature

Advantageously, the eluate recovered at step (f) comprises organic solvent and, if need be, is diluted in water so as to insure an organic solvent concentration lower or equal to 10% by volume, in order to insure the best trapping efficiency.

According to a possible way of implementation, the purification g) of the eluate containing the labelled compound(s) comprises a HPLC purification, instead of or in addition to a purification on SPE.

DETAILED DESCRIPTION OF THE INVENTION

The steps (a) to (g) are detailed hereinafter:

The preparation (a) of the labelling agent is well known in the art.

The radionucleide is produced beforehand, generally by irradiation of ¹⁸O enriched water with a proton beam originating from a particle accelerator, as F^" (for instance H¹⁸F, in an aqueous solution). Then, the ¹⁸F is recovered under an activated form through "activating" agents such as KRYPTOFIX® (also called K2.2.2), a trademark used in connection with the compound 4, 7, 13, 16, 21, 24-hexaoxo-l, 10-diazabicyclo-[8.8.8]-hexacosane, so as to make it more reactive. In some publications, they are called "phase transfer agents". Practically, the [¹⁸O] enriched water containing [¹⁸F^"] is passed through an anionic resin that can be contained in a Strong Anionic Exchange cartridge such as the one marketed under the trademark QMA SepPak® Light Waters®.

Advantageously, the [ rl8 O] enriched water is removed to the O water collection vial in the module.

Collected and activated radioactivity is recovered by washing with K222/K₂Cθ₃ CH₃CN/H₂O mixture solution

After this activation, the fluorinating agent, is made totally anhydrous by additions of at least one organic solvent, and by dry evaporations.

Preferably, the organic solvent is selected in the group comprising: acetonitrile (CH₃CN), dimethylsulfoxyde, dimethylformamide, acetone, and mixtures thereof. For example, acetonitrile (CH₃CN) is particularly suitable.

In the labelling step (b), the dried fluorinating agent is put in presence of a labelling substrate (precursor), generally the precursor Nosyl-Boc-DMTr-FLT for the [¹⁸F]FLT synthesis said precursor being solubilized in at least one organic solvent, for example: acetonitrile (CH₃CN). A substitution reaction then occurs at 160⁰C. In the [¹⁸F]FLT synthesis, the Nosyl-Boc- groups of the substrate are replaced by the ¹⁸F atom and H, resulting in the formation of the undeprotected labelled precursor DMTr-FLT.

With :

Rl = H, BOC, tBOC, C₂ to C₁₀ alkyloxy carbonyl

R2 = Nos (O-nitrophenylsulfonyl), R-SO₂ (with R= C₁ to C₅ alkyl or unsubstitued phenyl group)

R3=, triphenylmethyl, substituted on the phenyl group, trialkylmetyl, triphenylsilyl substituted on the phenyl group, trialkylsilyl,Trityl, DMTrityl, , ....

The possible step (c) of prepurification can be carried out as follows The reaction mixture recovered at step (b) comprising a solution of the undeprotected labelled precursor in the organic solvent(s) can be diluted in water so as to insure an organic solvent (for example: acetonitrile (CH₃CN)), concentration lower or equal to 10% by volume. Advantageously, once the dilution is completed the diluted reaction mixture recovered at step (b), the prepurification (c) can consists in flushing said diluted reaction mixture through a SPE solid support, preferably the SPE solid support used in the steps (d) and (e), for example those hereinabove described such as a WATERS® C- 18 SepPak® cartridge. The prepurification (c) makes it possible to remove the reagent residues, particularly the Kryptofϊx® K2.2.2. and the unreacted ¹⁸F fluorides.

In the preferred way of implementation consisting in using the same solid support for the prepurification (c), for the selective adsorption (d) and for the deprotection (e), the adsorption or the trapping on the solid support (e.g. Cl 8 SepPak®) of the undeprotected labelled precursor (step (d)), occurs during the migration of the diluted reaction mixture through the solid support (e.g. Cl 8 SepPak®) for the purpose of prepurification (c).

Then, it is preferable that the solid support (e.g. Cl 8 Sep-Pak®) be then rinsed e.g. with water.

The following step is the deprotection step (e), a mixture of water/TFA -e.g.40/60- (deprotecting/hydrolyzing agent) is pushed on the solid support (e.g. Cl 8 Sep-Pak® cartridge), and kept in contact with the solid support for a time of Is to 120s.

The recovery of [¹⁸F]FLT (step (f)) essentially consists in rinsing e.g. with water to elute the bulk solution including the [¹⁸F]FLT.

The purifying step (g) which follows, comprises, for instance, tranferring the eluate containing the labelled compound on two hydrophilic-lipophilic water-wettable reversed- phase sorbents for solid-phase extraction. Practically, the aqueous solution is flushed through two OASIS® HLB cartridges, the [¹⁸F]FLT and impurities is so trapped on the OASIS® HLB cartridges.

The preferred purification (g) advantageously comprises a a rinsing step (gl 1) with water and a rinsing step (gl2), in which a first mixture of water and at least one rinsing liquid selected from aqueous solutions, physiological solutions, water, alcohols (preferably ethanol) and mixtures water/alcohol(s). organic solvent (e.g. ethylic alcohol), is pushed through the OASIS® HLB cartridges to elute all the impurities and by products. Preferably the first mixture is water/alcohol(s) 9/1 by volume. After that, the purifying step (g) comprises at least one elution (g2) with a second mixture of water and at least one rinsing liquid selected from aqueous solutions, physiological solutions, water, alcohols (preferably ethanol) and mixtures water/alcohol(s). organic solvent (e.g. ethylic alcohol), is pushed through the OASIS® HLB cartridges to elute all the pure final product [¹⁸F]FLT or [¹⁸F]FDG of the solid sorbent(s). Preferably the second mixture is water/alcohol(s) 7/3 by volume. Advantageously, the elution (g2) is a countercurrent elution of the labelled compound(s).

Possibly, but not necessarily, the purification (g) of the eluate containing the labelled compound(s) comprises a HPLC purification using, for example a Breeze HPLC pump, a UV 2457 (Waters, Milford, USA) and a NaI pin detector system (Bioscan, Washington DC, USA). The UV and NaI detector were installed in the hot-cell but HPLC pump and control PC were installed outside of hot-cells. For sample injection, we used a 10 mL HPLC loop and an automatic injector (Rheodyne, Rohnert Park, USA). To collect purified [¹⁸F]FLT, we used one three-way valve between detector outlet point, a waste bottle and a [¹⁸F]FLT collection vial, which was operated by a compressed air supply. The reaction mixture was injected to HPLC loop by syringe pressure of the module. Air in the tubing and impurities in reaction mixture were removed by 0.22 μm vented filter before injection. Purification conditions were ethanol:water (10:90), 5 mL/min, and 267 nm (UV detector). A Econosil C18 (Alltech, 10 μm, 10x250 mm) HPLC column is used. Purified [¹⁸F]FLT was collected on a 20 mL vial and passed via a sterile 0.22 μm filter.

A last aspect of the present invention concerns the device for the synthesis of 3'-deoxy-3'- [¹⁸F]fluorothymidine ([¹⁸F]FLT) through the process of the invention, in which a solid support is used in the deprotection step, preferably included in a single use kit of materials. Said device is advantageously automated.

DESCRIPTION OF A PREFERRED EMBODIMENT OF THE INVENTION

The invention will be described more in details, in reference to an example of a specific embodiment which is illustrated schematically in the enclosed of the single figure which is a schematic drawing illustrating the synthesis of 3'-deoxy-3'-[¹⁸F]fluorothymidine ([¹⁸F]FLT).

EXAMPLES

Materials:

The method to prepare the ¹⁸F-FLT according to the examples, consists in using an adapted form of a commercial single use kit which trademark is "TRACERlab MXFD_G ®'\ and which is marketed by the GE Medical Systems company. The module (kit) includes multiple stopcock manifolds and disposable sterile syringes. This adapted material is described in the single enclosed schematic figure comprising the following single use components mentioned in Table 1 :

TABLE 1

And to insure the connexions, different caps, lueur and needles

Description of the single figure:

TABLE 2

Chemicals

1 -(2'-Deoxy-3 '-O-(4-nitrobenzenesulfonyl)-5 '-O-(4,4'-dimethoxytrityl)-beta-D-threo- pentafuranosyl)-3-(ter^-butyloxycarbonyl)thymine (5'-O-DMTr-2'-deoxy~3'-O-nosyl-β-D- threo-pentofuranosyl)-3-N-BOC-thymine was used as a precursor, which was prepared according to the previous methods Solvents and reagents were purchased from Sigma- Aldrich and used as supplied.

COMPARATIVE EXAMPLE 1 (according to the synthesis disclosed in the hereinabove cited prior reference Oh et al)

Preparation of the kit:

Valve 2: QMA + reservoir

Valve 3 : 7mL of acetonitrile Valve 4: disposable 3OmL syringe

Valve 5: 40mg of precursor in 3mL of acetonitrile

Valve 6: connexion to reactor in

Valve 7: 25OmL of Water For Injection (WFI)

Valve 8: NaOH 2M Valve 9: HCl IM

Valve 10: nothing

Valve 11: nothing

Valve 12: HPLC out

Valve 13: Nothing Valve 14: disposable 3OmL syringe

Valve 15: reactor out

In that system, there is no pre-purification cartridge between valve 10 and 11.

Step fa) al. recovery of the [¹⁸O] enriched water using an anionic resin [KRYPTOFIX®.

(also called K2.2.2)] which is a ¹⁸F "activating" agent. a2. recovery, by elution of the anionic resin, of the activity in the form of

[K/222].¹⁸⁺, F^" in a solution in a mixture CH₃CN/H₂O. a3. evaporation of the solvent by IR heating (95.⁰C.) under nitrogen flow (2 min. 30 sec). a4. addition of 0.4 ml CH₃CN, evaporation (2 min. 30 sec). a5. addition of 0.4 ml CH₃CN, evaporation to siccity.

Step fb)

Labelling of the precursor. bl . addition of a solution of a labelling precursor (1-50 mg) in CH₃CN (3 ml). b2. heating at 160. degree. C. during 6 min.

Step (c):

Partial evaporation of the fluorination solvent. Step fd):

Deprotection (hydrolysis), dl addition of 3mL HCl into the reactor. d2. heating for 300sec at 105 degrees Celcius.

Step (e):

Neutralization of the hydrolysis mixture. e. addition of NaOH 2M into the reactor.

Step (e):

HPLC injection.

Results :

Yield: average is 50% corrected. Total synthesis time: 60 minutes including HPLC purification.

In this example, it is emphasized that there is no pre-purification cartridge and that all the process happen in the reactor.

EXAMPLE 2 : Process according to the invention with a HPLC purification

Preparation of the kit:

Valve 2: QMA + reservoir

Valve 3: 7mL of acetonitrile Valve 4: disposable 3OmL syringe

Valve 5: 1 to 50mg of precursor in 3mL of acetonitrile

Valve 6: connexion to reactor in

Valve 7: 25OmL of Water For Injection (WFI)

Valve 8: 7mL of ethanol Valve 9: NaOH ethanolic solution

Valve 10 TFA 60% (v/v) in water ImL

Valve 11 : outlet to HPLC

Valve 12: Nothing

Valve 13: Nothing Valve 14: disposable 3OmL syringe

Valve 15: reactor out Step (a) al. recovery of the [¹⁸O] enriched water using an anionic resin [KRYPTOFIX®. (also called K2.2.2)] which is a ¹⁸F "activating" agent. a2. recovery, by elution of the anionic resin, of the activity in the form of [K/222].¹⁸⁺, F^" in a solution in a mixture CH₃CNZH₂O. a3. evaporation of the solvent by IR heating (95. °C.) under nitrogen flow (2 min. 30 sec). a4. addition of 0.4 ml CH₃CN, evaporation (2 min. 30 sec). a5. addition of 0.4 ml CH₃CN, evaporation to sicciiy.

Step (V)

Labelling of the precursor. bl. addition of a solution of a labelling precursor (1-50 mg) in CH₃CN (3 ml). b2. heating at 16O.degree. C. during 6 min.

Step (c) & (d):

Pre-purification with adsorption of the labelled compound on the solid support. between stopcocks 10 & 11. cl. dilution of the resulting solution in 25 ml water. c2dl. transfer of the diluted solution through a Cl 8 cartridge (conditioned beforehand with 5 ml ethanol followed with 10 ml water) to the waste. d2. rinsing of the cartridge with 3 times 10 ml of water which are sent to the waste. d3. drying of the cartridge under nitrogen flow.

Step (e): Deprotection (hydrolysis) on solid support in acidic media. el addition of 0.7 ml a 60% (v/v) aqueous solution of trifluoroacetic acid on the Cl 8 cartridge. e2. deprotection (hydrolysis) 2 min. at room temperature.

Step (f):

Recovery of the labelled compound. f. elution of the bulk solution with 3 ml of an aqueous solution of NaOH into a syringe. fl. rinse of the C- 18 cartridge with 3mL of water which are sent also to the syringe containing the bulk.

Step (a):

HPLC injection. Results :

Yield: average is 50% uncorrected 63% corrected to be compared to the 42 % of Oh publication.

Total synthesis time: 38 minutes including HPLC purification.

EXAMPLE 3 : Process according to the invention without a HPLC purification

Preparation of the kit: Method with cartridges:

Valve 2: QMA + reservoir

Valve 3: 7mL of acetonitrile

Valve 4: disposable 3OmL syringe

Valve 5: 5-50mg of precursor in 3mL of acetonitrile Valve 6: connexion to reactor in

Valve 7: 25OmL of WFI

Valve 8: Solutionl

Valve 9: Solution 2

Valve 10 TFA water ImL Valve 11 : To HPLC outlet

Valve 12: Purification cartridge

Valve 13: Purification cartridge

Valve 14: disposable 3OmL syringe

Valve 15: reactor out

Step fa) al. recovery of the [¹⁸O] enriched water using an anionic resin [KRYPTOFIX®. (also called K2.2.2)] which is a ¹⁸F "activating". a2. recovery, by elution of the anionic resin, of the activity in the form of [K/222].¹⁸⁺, F^" in a solution in a mixture CH₃CN/H₂O. a3 evaporation of the solvent by IR heating (95.⁰C.) under nitrogen flow (2 min. 30 sec). a4. addition of 0.4 ml CH₃CN, evaporation (2 min. 30 sec). a5. addition of 0.4 ml CH₃CN, evaporation to siccity.

Step fb)

Labelling of the precursor. bl. addition of a solution of a labelling precursor (25 mg) in CH₃CN (3 ml). b2. heating at lόO.degree. C. during 6 min. (labelling step). Step To) & (d):

Pre-purification with adsorption of the labelled compound on the solid support between stopcocks 10 & 11. cldilution of the resulting solution in 25 ml water. c2dl transfer of the diluted solution through a Cl 8 cartridge to the waste. d2 rinsing of the cartridge with 3 times 10 ml of water which are sent to the waste. d3 drying of the cartridge under nitrogen flow.

Step Te): Deprotection (hydrolysis) on solid support in acidic media. el addition of 0.7 ml a 60% (v/v) aqueous solution of trifluoroacetic acid on the Cl 8 cartridge. e2. deprotection (hydrolysis) 2 min. at room temperature.

Step (f):

Recovery of the labelled compound. elution of the bulk solution with 10 ml of an water into a syringe containing 15mL of water.

Step (g):

Final purification on solid support. g. loading the purification cartridges_12;13, the product remains on the cartridge and the liquid go to waste. gl 1. rinse of the purification cartridge with 3mL of water which are sent also to the syringe containing the bulk.

Results :

Yield: 18%

Total synthesis time: 42 minutes The non use of HPLC makes it possible to gain significant time preparation, e.g. at least 2h with regard to the comparative example 1. Therefore, it induces economy for the process, and that even more HPLC is expensive.

Claims

1) A process for synthesizing labelled compounds with an isotopic element, comprising the steps of:

a. preparing at least one labelling agent; b. labelling at least one precursor with the labelling agent, wherein the precursor is a protected substrate; c. possibly pre-purifying the labelled precursor; e. deprotecting the labelled precursor by hydrolysis so as to obtain the labelled compound; f. recovering the labelled compound; g. and purifying the recovered labelled compound,

wherein the deprotection/hydrolysis is carried out by means of at least one deprotecting/hydrolyzing agent consisting in at least one acid and/or at least one base, with the exception of HCl and NaOH, preferably in at least one organic acid, said organic acid(s) being more preferably selected in the group comprising the acetic acid and its derivates, notably the halogenated acetic acid, and even more preferably in the sub-group comprising: trifluoroacetic acid (TFA), acetic acid, formic acid, the analogs thereof and mixtures thereof.

2) A process according to claim 1, comprising the steps of: a. preparing at least one labelling agent; b. labelling at least one precursor with the labelling agent, wherein the precursor is a protected substrate; c. possibly pre-purifying the labelled precursor; d. retaining the labelled precursor on at least one solid support; e. deprotecting the labelled precursor by hydrolysis so as to obtain the labelled compound, directly on at least one solid support which is contained in at least one column or cartridge; f. recovering the labelled compound by elution g. and purifying the eluate.

3) A process according to claim 1 or 2 wherein the labelled compound is 3'-deoxy-3'- [¹⁸F]fluorothymidine ([¹⁸F]FLT). 4) A process according to claim 1 or 2 wherein the deprotecting/hydrolyzing agent includes an acid aqueous solution.

5) A process according to claim 3 wherein the deprotecting/hydrolyzing agent includes an acid aqueous solution, which [acid] concentration is greater or equal to 40% by weight, preferably 50% by weight, and more preferably comprised between 55 an 65% by weight.

6) A process according to any of the preceding claims wherein the step (e) of deprotecting/hydrolyzing lasts between 0.1s to 500s, preferably, between Is and 120s.

7) A process according to any of the preceding claims wherein the purifying step (g) comprises tranferring the labelled compound on at least one hydrophilic-lipophilic water- wettable reversed-phase sorbent for solid-phase extraction (SPE).

8) A process according to claim 3 wherein the solid-support of step (e) the solid- support of step (e) is selected in the group comprising: normal phase, reverse phase, intermediate polarity phase, low polarity phase, ion exchange phase and mixtures thereof.

9) A process according to claim 3 wherein the deprotection step (e) is performed on a column or cartridge selected from the group including those marketed by the WATERS® company under the names C18, C8, tC18, NH2, diol, and polystyrene divinylbenzene (SDB).

10) A process according to claim 3 wherein the purifying step (g) comprises tranferring the eluate containing the labelled compound on one or several -preferably at least two- hydrophilic-lipophilic water-wettable reversed-phase sorbents for solid-phase extraction (SPE).

11) A process according to claim 7 wherein the step (g) is followed by a step (gl) comprising at least one rinsing of the solid sorbent(s) with a liquid selected in the group comprising aqueous solutions, physiological solutions, water, alcohols (preferably ethanol) and mixtures water/alcohol(s).

12) A process according to claim 11 wherein the step (gl) includes a rinsing (gll) of the solid sorbent(s) with water and a rinsing (gl2) of the solid sorbent(s) with a mixture water/alcohol(s), preferably a mixture water/alcohol(s) 99/1 - more than 70/less than 30 by volume, for example about 9/1 by volume. 13) A process according to claim 7 wherein the purifying step (g) comprises at least one elution (g2) of the solid sorbent(s) with an eluent selected in the group comprising alcohols (preferably ethanol) and mixtures water/alcohol(s), preferably a mixture water/alcohol(s) 70/30 -0/100 by volume, for example about 70/30 by volume.

14) A process according to claim 13 wherein at least the elution (g2) is a countercurrent elution of the labelled compound(s).

15) A process according to claim 7 wherein the hydrophilic-lipophilic water- wettable reversed-phase sorbent for solid-phase extraction of step (g), belong to the group of those which contain at least one copolymer comprising at least one hydrophilic comonomer and one lipophilic comonomer, the preferred hydrophilic-lipophilic water-wettable reversed- phase sorbents being selected in the sub-group of the WATERS® OASIS® HLB family of sample extraction products, the OASIS® HLB cartridges being especially preferred.

16) A process according any of the preceding claims, wherein the hydrophilic- lipophilic water-wettable reversed-phase sorbent for solid-phase extraction of step (g) and/or the solid-support of step (e) is (are) contained in a cartridge or column in a amount between 50 mg and 10 g of a solid support/sorbent.

17) The process according any of the preceding claims, wherein the hydrophilic- lipophilic water-wettable reversed-phase sorbent for solid-phase extraction of step (g) and/or the solid-support of step (e) is (are) in the form of grains, membranes, sheets or capillaries.

18) The process according any of the preceding claims, wherein the eluate recovered at step (b) comprises organic solvent and, if need be, is diluted in water so as to insure an organic solvent concentration lower or equal to 10% by volume.

19) The process according to claim 18, wherein the organic solvent is selected in the group comprising: acetonitrile (CH₃CN),, dimethylsulfoxyde, dimethylformamide, acetone, and mixtures thereof.

20) The process according any of the preceding claims, wherein the purification of the eluate containing the labelled compound(s) comprises a HPLC purification.

21) The device for implementing the process according to any of the claims 1 to 20.