This invention relates to methods for allowing very polar radiopharmaceuticals to be rapidly converted to solutions ready for injection. This method is suitable for radiopharmaceuticals containing multiple acidic and/or phosphonic acid functional groups.

Background

The invention relates to the subject matter referred to in the claims, i.e. rapid robust formulation of very polar radiopharmaceuticals containing multiple acidic functional groups.

Molecular imaging has the potential to detect disease progression or therapeutic effectiveness earlier than most conventional methods in the fields of oncology, neurology and cardiology. Of the several promising molecular imaging technologies having been developed as optical imaging and MR!, PET is of particular interest for drug development because of its high sensitivity and ability to provide quantitative and kinetic data.

Positron emitting isotopes include carbon, nitrogen, and oxygen. These isotopes can replace their non-radioactive counterparts in target compounds to produce tracers that function biologically and are chemically identical to the original molecules for PET imaging. On the other hand, ' ⁸ F is the most convenient labelling isotope due to its relatively long half-life (109.6 min) which permits the preparation of diagnostic tracers and subsequent study of biochemical processes. In addition, its high β+ yield and low β+ energy (635 keV) are also advantageous.

In the preparation of radiopharmaceuticals the final step of the process is to ensure that the said radiopharmaceutical is suitable for injecting into mammals, e.g. have a suitable pH, osmolality, etc. Typically in the radiosyntheses of radiopharmaceuticals a purification step using high pressure liquid chromatography (HPLC) is used. This HPLC purification step uses toxic or potentially toxic substances, e.g. acetonifriie, methanol, trifluoroacetic acid, formic acid etc., and steps have to be taken to ensure these toxic or potentially toxic substances are removed. The process of taking a HPLC purified solution of the said radiopharmaceutical which contains toxic or potentially toxic substance and converting it into a solution suitable for injecting into mammals is typically referred to as a "reformulation step". This reformulation step is well known for lipophilic compounds as the HPLC purified solution of the said radiopharmaceutical containing the toxic or potentially toxic substances can be diluted with water passed through a siica or polymer based resin functionalized with carbon chains, e.g. C-18 (ociadecyi) solid phase extraction (SPE) cartridge where the said radiopharmaceutical is retained due to the lipophilic character of the said radiopharmaceutical. The toxic or potentially toxic substances are then washed from the SPE cartridge by various washing steps and the desired radiopharmaceutical is eiuted from the SPE using a solution which upon dilution is suitable for injecting into mammals, typically ethanoi is used and then diluted with saline or phosphate buffered saline (PBS).

For polar compounds (log D <1 ) is reformulation step by taken the HPLC purified fraction of the said radiopharmaceutical and concentrating it under reduced pressure or blowing dry under a gas stream at elevated temperatures. This procedure has been successfully used for amino acid imaging agent, D-fluoromethyi tyrosine (DFMT, Tsukada et a!., Eur, J. Nuc. Med. Mol. I mag. 2006, 33, 1017-1024), where the final product is concentrated under reduced pressure, which is a time-consuming step, and then redissolved in saline to give the formulated product. Other examples for polar compounds containing multiple carboxylic acid functional groups are glutamate-hetero urea dimers which are imaging agents that target Prostate Specific Membrane Antigen (PSMA). The glutamate-hetero urea N- [N-[(S)-1 ,3- dicarboxypropyl]carbamoyl]-4-[ ¹⁸ F]fluoroben2yl-L-cysteine (DCFBC, Mease et al., Clin Cancer Res. 2008, 14, 3036-3043) where the HPLC purified radiopharmaceutical was concentrated under reduced pressure and then redissolved in saline to give the formulated product. Another glutamate-hetero urea is 2-[3-[1-Carboxy-5-(4-[ ⁱ⁸ F]fluoro-benzoylamino)- pentyi]-ureidoj-pentanedioic Acid (Chen et a!., J. Med. Chem. , 2008, 51 , 7933-7943) where the HPLC purified radiopharmaceutical was also purified under reduced pressure. The major drawback with this concentration step is whether ail traces of the toxic or potentially toxic additives, i.e. acetonitrile. Trifiuoroacetic acid, are really fully removed.

Problem to be solved by the invention and its so ution

Despite the aforementioned advances in reformulation methods, there is relatively little known for the reformulation of very polar compounds (log D <0), especially those very polar radiopharmaceuticals that contain multiple acidic functional groups, for example carboxylic acid or phosphonic acid moieties. For the said radiopharmaceuticals containing multiple acidic functional groups, the reformulation step is typically carried out by concentration either under vacuum or under a stream of nitrogen or helium. This step can be relatively time consuming as one must ensure that all the traces of the toxic or potentially toxic substances are removed. Time consuming steps are to be avoided in the syntheses of radiopharmaceuticals especially those containing positron emitting (PET) radioisotopes as they are not compatible with the half-life of short-lived radioisotopes, e.g. C-1 1 (20 mins), F- 18 (1 10 mins), Tc-99m (6 h), 1-123 (13.2 h), etc.

The reformulations methods for obtaining reformulated radiopharmaceutical solution as disclosed in the present invention allow for a surprisingly rapid and simple reformulation of very polar radiopharmaceuticals containing multiple acidic functional groups wherein the obtained solution is suitable for injecting into mammals. Summary

The invention relates to the subject matter referred to in the claims, i.e. surprisingly rapid and simple reformulation of very polar radiopharmaceuticals containing multiple acidic functional groups to solutions suitable for injecting into mammals.

In a first aspect, the invention is directed to a method for reformulation of a radiopharmaceutical, wherein the radiopharmaceutical comprises two or more carboxylic acid groups and/or one or more phosphonic acid. In embodiments of the first aspect, the radiopharmaceutical is a compound of formula (I), {11} or mixture thereof.

In a second aspect, the invention is directed to a reformulated radiopharmaceutical solution. In a third aspect, the invention is directed to a kit comprising

- an anionic exchange resin cartridge, and

- a vial containing an Elution solvent comprising sodium chloride (NaCi) characterised in that the kit is useful for conducting the method of the first aspect. Description

In a first aspect, the invention is directed to a method for reformulation of a radiopharmaceutical, wherein the radiopharmaceutical comprises two or more carboxylic acid groups and/or one or more phosphonic acid, comprising the step of:

Eluting the radiopharmaceutical from an anionic exchange resin cartridge with an elution solvent comprising sodium chloride (NaCI).

Preferred features:

Preferably, the radiopharmaceutical comprises one (1 ) to ten (10) carboxylic acid groups and/or one (1 ) to five (5) phosphonic acid. More preferably, the radiopharmaceutical comprises two (2) to five (5) carboxylic acid groups and/or one (1 ) or two (2) phosphonic acid groups. Even more preferably, the radiopharmaceutical comprises three (3) or four (4) carboxylic acid groups and/or one (1 ) or two (2) phosphonic acid groups. Even more preferably, the radiopharmaceutical comprises four (4) carboxylic acid groups and one (1 ) phosphonic acid.

Preferably, the radiopharmaceutical comprises one (1 ) to ten (10) carboxylic acid groups. More preferably, the radiopharmaceutical comprises one (1 ) to five (5) carboxylic acid groups. Even more preferably, the radiopharmaceutical comprises three (3) or four (4) carboxyiic acid groups. Even more preferably, the radiopharmaceutical comprises three (3) carboxyiic acid groups. Preferably the anionic exchange resin is a siiica based or polymer based weak anionic exchange resin, a medium anionic exchange resin or a strong anionic exchange resin or the anionic exchange resin is a siiica based or polymer based mixed mode weak anionic exchange resin or strong anionic exchange resin. More preferably the anionic exchange resin is a silica based or polymer based strong anionic exchange resin (SAX - e.g. Sep-Pak Accell Pius Q A, Cieanert SAX, LC-SAX, AccuBOND SAX, Bond E!ut SAX etc.) or a mixed mode silica based or polymer based strong anionic exchange resin (MAX - Bond E!ut Certify !!, Chormabond Drug II, Screen-A, Chromabond HR-XA, Cieanert PAX, Oasis MAX etc.). Even more preferably, the anionic exchange resin is a siiica based or polymer based strong anionic resin. Even more preferably the anionic exchange resin is a quaternary alkylated ammonium resin. Even more preferably the anionic exchange resin is a quaternary trimethy!ated ammonium exchange resin. Even more preferably the anionic exchange resin is a quaternary trimethy!ammonium exchange resin, wherein the trimethylammonium moiety is connected via a propyl linker to an acrylamide copolymer on diol silica that is commercially available as Sep-Pak Accell Plus QMA Plus Short cartridges.

Preferably, the eiution solvent comprises pharmaceutically acceptable sodium salt at the concentration of 2 M to 0.3 M of the said pharmaceutically acceptable sodium salt. More preferably, the eiution solvent comprises pharmaceutically acceptable sodium salt at the concentration of 1 M to 0.3 M of the said pharmaceutically acceptable sodium salt. Even more preferably, the eiution solvent comprises pharmaceutically acceptable sodium salt at the concentration of 0.8 M to 0.3 M of the said pharmaceutically acceptable sodium salt. Even more preferably, the Eiution solvent comprises pharmaceutically acceptable sodium salt at the concentration of 0.5 M of the said pharmaceutically acceptable sodium salt. As a preferred feature, the invention method for reformulation of a radiopharmaceutical comprises additionally the following steps before eluting:

Diluting the fraction obtained from the HPLC that contains the purified radiopharmaceutical with an aqueous solution comprising of a base, and

Trapping the purified radiopharmaceutical on an anionic exchange resin cartridge.

In a first embodiment, the invention is directed to a method for reformulation of a radiopharmaceutical, comprising the step of: Eiuting the radiopharmaceutical from an anionic exchange resin cartridge with an elution solvent comprising sodium chloride (NaCI),

wherein the radiopharmaceutica! is a compound of formula (I),

wherein

R is a radiolabeled pendant group,

each R-i is independently from each other hydrogen or a pharmaceutically acceptable salt, and

In an embodiment of the method, the method for reformulation of a radiopharmaceutical comprises additionally the following steps before eiuting:

Diluting the fraction obtained from the HPLC that contains the purified radiopharmaceutical with an aqueous solution comprising of a base, and

Trapping the purified radiopharmaceutical on an anionic exchange resin cartridge, wherein the radiopharmaceutical is a compound of formula (Ί).

In an embodiment of formula (I), X is CH ₂ .

In an embodiment of formula (I), X is CH ₂ -CH ₂ .

In an embodiment of formula (I), R is phenyl or pyridyl, each substituted with r one [ ¹⁸ F]~fluoro group and optionally substituted with a second group selected from the group consisting of chioro and cyano. embodiment of formula (I), R ^:

wherein R ³ is - ¹⁸ F; and R ² is chioro or cyano. In an embodiment of formula (I R is

wherein R ^J is - ^1a F; and R is chloro or cyano.

In an embodiment of formula (I R is

wherein R ^J is - ^' . and R ² is chloro or cyano.

In an embodiment of

wherein R

In an embodiment of form is

wherei .X? or

In another embodiment of formula (i), R is

In another embodiment of formula (I), R is

In another embodiment of formula (I), R is

In another embodiment of formula (I), R is ¹⁸ F ^{' "} N

In an embodiment of formula (I), R ¹ is hydrogen. mbodiment of formula (I), R is a pharmaceutically acceptable salt.

In another embodiment, the compound of formula (!) is

In a second embodiment the invention is directed to a method for reformulation of a radiopharmaceutical, comprising the step of:

Eiuting the radiopharmaceutical from an anionic exchange resin cartridge with an elution solvent comprising sodium chloride (NaCI),

wherein the radiopharmaceutical is a compound of formula (II),

wherein

Rc is the cold counter-part of a radiolabeled pendant group,

each R-i is independently from each other hydrogen or a pharmaceutically acceptable salt, and

X is CH ₂ or CH ₂ -CH ₂ .

In an embodiment of the method, the method for reformulation of a radiopharmaceutical comprises additionally the following steps before eluting:

Diluting the fraction obtained from the HPLC that contains the purified radiopharmaceutical with an aqueous solution comprising of a base, and

Trapping the purified radiopharmaceutical on an anionic exchange resin cartridge, wherein the radiopharmaceutical is a compound of formula (Π),

In an embodiment of formula (11), X is CH ₂ .

In an embodiment of formula (II), X is CH ₂ -CH ₂ .

In an embodiment of formula (Π), R is phenyl or pyridyl, each substituted with one [Fj-fluoro group and optionally substituted with a second group selected from the group consisting of chioro and cyano.

In an embodiment of formula II), R is

wherein R ^J is -F; and R ² is chioro or cyano

In an embodiment of formula (II), R is

herein R ^J is -F; and R ² is chloro or cyano.

in an embodiment of formula (11 R is

wherein R~ ^! is -F ; and R is chloro or cyano.

In an embodiment of formula (II), R is

wherein R ^J is -F

in an embodiment of formula (11), R

herein R ^J is -F.

In another embodiment of formula (M), R is F

In another embodiment of formula (Π), R is

In an embodiment of formula (11), R is hydrogen.

In an embodiment of formula (II), R ¹ is pharmaceutically acceptable salt.

In another embodiment, the compound of formula (II) is

or a pharmaceutically acceptable salts thereof.

In a third embodiment the invention is directed to a method for reformulation of a radiopharmaceutical, wherein the radiopharmaceutical comprises two or more carboxylic acid groups and/or one or more phosphonic acid, comprising the steps of

Diluting the fraction obtained from the HPLC that contains the purified radiopharmaceutical with an aqueous solution comprising of a base,

Trapping the purified radiopharmaceutical on an anionic exchange resin cartridge, and

- E!uting the radiopharmaceutical from an anionic exchange resin cartridge with an e!ution solvent comprising sodium chloride (NaCI). Preferably, the eiution solvent comprises pharmaceutically acceptable sodium salt at the concentration of 2 to 0.3 of the said pharmaceutically acceptable sodium salt. More preferably the eiution solvent comprises pharmaceutically acceptable sodium salt at the concentration of 1 to 0.3 of the said pharmaceutically acceptable sodium salt. Even more preferably, the eiution solvent comprises pharmaceutically acceptable sodium salt at the concentration of 0.8 M to 0.3 M of the said pharmaceutically acceptable sodium salt. Even more preferably, the Eiution solvent comprises pharmaceutically acceptable sodium salt at the concentration of 0.5 IV! of the said pharmaceutically acceptable sodium salt. Preferred features as disclosed in first aspect are included the first, second and third embodiment thereof.

In a second aspect, the invention is directed to a reformulated radiopharmaceutical solution, wherein the radiopharmaceutical comprises two or more carboxyiic acid groups and/or one or more phosphonic acid, comprising

- a radiopharmaceutical, wherein the radiopharmaceutical comprises two or more carboxyiic acid groups and/or one or more phosphonic acid, and

- an eiution solvent comprising sodium chloride (NaCI). Preferably, the radiopharmaceutical comprising two or more carboxyiic acid groups and/or one or more phosphonic acid is a compound formula (I), (II) or mixture thereof.

Preferably, the reformulated radiopharmaceutical solution is obtained by the method as described in first aspect.

In a first embodiment the invention is directed to a reformulated radiopharmaceutical solution, wherein the radiopharmaceutical comprises two or more carboxyiic acid groups and/or one or more phosphonic acid, comprising

- a reformulated radiopharmaceutical, and

- an eiution solvent comprising sodium chloride (NaCI).

Preferably, the radiopharmaceutical comprising two or more carboxyiic acid groups and/or one or more phosphonic acid is a compound formula (I), (II) or mixture thereof. Preferably, the reformulated radiopharmaceutical solution is obtained by the method as described in first aspect. Preferred features and embodiments as disclosed in first aspect are included here thereof.

In a third aspect, the invention is directed to a kit comprising

- an anionic exchange resin cartridge, and

- a vial containing an Elution solvent comprising sodium chloride (NaCI) characterised in that the kit is useful for conducting the method of the first aspect.

The preferred features and sub-embodiments disclosed in the first aspect are herein incorporated in the second and third aspects.

Definitions

The terms used in the present invention are defined below but are not limiting the invention scope.

An anionic exchange resin is a resin containing a cation group, typically amino groups that are protonated to give ammonium salt or quaternary alkylated amino groups, which attract and retain anions present in the solution surrounding the said resin.

A resin is organic polymer or functionaiized silica that is insoluble in most organic solvents, aqueous solutions and mixtures thereof.

A quaternary alkylated amino resin is a resin that it functionaiized with one or more amino groups and these amino groups are substituted independently with three alkyi or alkylaryl groups or mixture thereof to give an ammonium salt (N ^÷ R ^! R ² R ³ R ⁴ ) where are R ¹ is the resin. R ² , R ³ and R ⁴ is methyl, ethyl, propyl, butyl, benzyl, or ethylphenyl.

If chiral centers or other forms of isomeric centers are present in a compound according to the present invention, ail forms of such stereoisomers, including enantiomers and diastereoisomers, are intended to be covered herein. Compounds containing chiral centers may be used as racemic mixture or as an enantiomerically enriched mixture or as a diastereomeric mixture or as a diastereomericaily enriched mixture, or these isomeric mixtures may be separated using well-known techniques, and an individual stereoisomer maybe used alone. In cases wherein compounds may exist in tautomeric forms, such as kefo-enoi taufomers, each tautomeric form is contemplated as being included within this invention whether existing in equilibrium or predominantly in one form. In the context of the present invention, preferred salts are pharmaceutically acceptable salts of the compounds according to the invention. The invention also comprises salts which for their part are not suitable for pharmaceutical applications, but which can be used, for example, for isolating or purifying the compounds according to the invention.

Pharmaceutically acceptable salts of the compounds according to the invention include acid addition salts of mineral acids, carboxylic acids and sulphonic acids, for example salts of hydrochloric acid, hydrobromic acid, sulphuric acid, phosphoric acid, methanesulphonic acid, ethanesulphonic acid, to!uenesulphonic acid, benzenesulphonic acid, naphthalene disulphonic acid, formic acid, acetic acid, trifluoroacetic acid, propionic acid, lactic acid, tartaric acid, malic acid, citric acid, fu marie acid, maieic acid and benzoic acid.

Pharmaceutically acceptable salts of the compounds according to the invention also include salts of customary bases, such as, by way of example and by way of preference, alkali metal salts (for example sodium salts and potassium salts), alkaline earth metal salts (for example calcium salts and magnesium salts) and ammonium salts, derived from ammonia or organic amines having 1 to 16 carbon atoms, such as, by way of example and by way of preference, ethyiamine, diethylamine, triethylamine, ethyldiisopropylamine, monoethanolamine, diethano!amine, triethano!amine. dicyclohexyiamine. dimethy!aminoethano!, procaine, dibenzy!amine, N-methy!morpholine, arginine, lysine, ethy!enediamine and N- methylpiperidine.

Those skilled in the art will further recognise that acid addition salts of the claimed compounds may be prepared by reaction of the compounds with the appropriate inorganic or organic acid via any of a number of known methods. Alternatively, alkali and alkaline earth metal salts of acidic compounds of the invention are prepared by reacting the compounds of the invention with the appropriate base via a variety of known methods.

The present invention includes ail possible salts of the compounds of the present invention as single salts, or as any mixture of said salts, in any ratio.

The term "buffering agents" as employed herein include but are not limited to potassium metaphosphate, dipotassium phosphate, sodium acetate, sodium citrate anhydrous and sodium citrate dehydrate.

A compound is polar when an electric charge is not symmetrically distributed, so that there is a separation of charge or partial charge and formation of definite positive and negative poles. Polar compounds are defined as having a log D (partition coefficient determined with octanol and water at pH 7.4) in the range of -2 to 0, and very polar compounds are defined as having a log D <-3. The term "haiide" as employed herein by itself or as part of another group is known or obvious to someone skilled in the art, and means fiuoro, chloro, bromo, and iodo.

The term "Radiopharmaceutical" as employed herein refers to a drug that contains radioactive atom(s). Radiopharmaceuticals are administered to patient as diagnostic tracer for the diagnosis and/or treatment of diseases. Radioactive atom(s) are ⁸ F- fluorine, ' ²⁴ \~, ¹²⁴ l-, ¹ "!- and ^1S6 I~, ^{13 '} !-iodine, ^b!5 Ga-Gaiiiurn and ^99m Tc-Technetiurn (list not exhaustive).

Present invention is directed to any radiopharmaceuticals falling within the scope of the invention. Preferably the radiopharmaceutical contains fluorine or iodine radioisotope.

For the purpose of the invention, radiopharmaceutical includes also drug comprising the nonradioactive counterpart (cold isotope).

Well known radiopharmaceuticals are

[F-18]-giutamic acid.

Where the plural form of the word compounds, salts, polymorphs, hydrates, solvates and the like, is used herein, this is taken to mean also a single compound, salt, polymorph, isomer, hydrate, solvate or the like.

Unless otherwise specified, when referring to the compounds of formula the present invention per se as well as to any pharmaceutical composition thereof the present invention includes all of the hydrates, salts, and complexes. General synthesis of F-18 compounds All solvents and chemicals were obtained from commercial sources and used without further purification. Anhydrous solvents and inert atmosphere (nitrogen or argon) were used if not stated otherwise. The preceding table lists the abbreviations used in this paragraph and in the Intermediates and Examples sections as far as they are not explained within the text body.

The radiofluorination reaction can be carried out, for example in a typical reaction vessel (e.g. Wheaton vial) which is known to someone skilled in the art or in a microreactor. The reaction can be heated by typical methods, e.g. oil bath, heating block or microwave. The radiofluorination reactions are carried out in dimethylformamide with potassium carbonate as base and "kryptofix" as crown-ether. But also other solvents can be used which are well known to experts. These possible conditions include, but are not limited to: dimethylsu!foxide and acetonitrile as solvent and tetraalkyl ammonium and tetraaikyl phosphonium carbonate as base. Water and/or alcohol can be involved in such a reaction as co-solvent. The radiofluorination reactions are conducted for one to 60 minutes. Preferred reaction times are five to 50 minutes. Further preferred reaction times are 10 to 40 rnin. This and other conditions for such radiofluorination are known to experts (Coenen, FIuorine-18 Labeling Methods: Features and Possibilities of Basic Reactions, (2008), in: Schubiger PA, Friebe ., Lehmann L, (eds), PET-Chemistry - The Driving Force in Molecular Imaging. Springer, Berlin Heidelberg, pp.15-50). The radiofluorination can be carried out in a "hot-cell" and/or by use of a module (review: Krasikowa, Synthesis Modules and Automation in F-18 labeling (2006), in: Schubiger P.A., Friebe M., Lehmann L, (eds), PET-Chemistry - The Driving Force in Molecular Imaging. Springer, Berlin Heidelberg, pp. 289-316) which allows an automated or semi-automated synthesis.

The compounds produced may require purification. Purification of organic compounds is well known to the person skilled in the art and there may be several ways of purifying the same compound. In some cases, no purification may be necessary. in some cases, the compounds may be purified by preparative HPLC according to the preparative HPLC methods listed below.

Abbreviations

AcCi Acetyl Chloride Cs COs Cesium carbonate

DMF W.W-Dimethyiformamide

DMF-DMA Dimethylformamide dimethyl acetai

D SO Dimethyisulfoxide

EtOAc Ethyl Acetate

EtOH Ethanol

GBq GigaBequerei

h Hour

HBF ₄ Tetrafiuoroboric acid

HPLC High Pressure Liquid Chromatography

K ₂ C0 ₃ Potassium carbonate

K ₂₂₂ Kryptofix 2.2.2

M Molar concentration "1 molar" (1 M).

1 mol/L

Min(s) Minute(s)

MBq MegaBequerei

TB Methyl t-butyl ether

MeCN Acetonitrile

MeOH Methanol

MS Mass Spectrometry

N ₂ Nitrogen

nd Not determined

NMR Nuclear Magnetic Resonance

PET Positron Emission Technology

PMPA 2-{Phosphonomethyi) pentanedioic acid

RT Room Temperature

SPE Solid Phase Extraction

TEA Triethy!amine

TFA Trif!uoroacetic acid

THF Tetrahydrofuran

TLC Thin Layer Chromatography

1. Radiolabelsrscj and HPLC purification

Exampie 1 N-(4~[ ⁱ⁸ F]flL3orobenzoyi)-L~gamma-glL3tarnyl-0-[{[(1 S)-1 ,3 dicarboxypropyl]amino}(hydroxy) phosphorylj-L-serine (SFB-CTT)

8F] Fluoride was produced by an ¹⁸ 0 (p, n) ^B F nuclear reaction by bombardment of a 98% 8Q-enriched water target with an 1 1 MeVproton beam at the RDS1 1 1 cyclotron. The aqueous [ ¹⁸ Fjf!uoride solution was trapped in a small anion exchange Sep-Pak™ Plus Q A cartridge (Waters) {preconditioned with 5 mi 0.5 M K ₂ CQ ₃ solution and 10 mL water). The radioactivity was eiuted with a solution mixture (1 .0 mg K CQ ₃ in 0.5 ml water and 5.27 rng K ₂₂₂ in 1 .5 ml MeCN) from the QMA cartridge into a 5 mL conic Wheaton vial. The solvent was evaporated under a stream of nitrogen at 1 10°C. Azeotropic drying was repeated three times with 1 .0 mL portions of acetonitriie. Ethyl 4-(trimethy!ammonium trifiate)benzoate (5.0 mg, 20 mmo!) in anhydrous MeCN (1 mL) was added to the dried K222/K[ ¹⁸ F]F and the mixture heated at 100°C for 10 min to produce ethyl 4-[18F]fluorobenzoate. The ethyl ester was subsequently hydrolyzed with 20 ί of tetrapropy!ammonium hydroxide (40%) in acetonitril (1 mL) at 120°C for 3 min, and then the mixture azeotropical!y dried using MeCN (1 mL). Subsequently, a solution of N,N,N',N'-tetramethyi-0-(N-succinimidyi) uranium hexaf!uorophosphate (H8TLJ) (12 mg, 33 mmoi) in MeCN (1 mL) was added and the solution heated at 100°C for 6 min. After cooling, the reaction mixture was diluted with water (2.5 mL) and acetonitriie (1 mL) and purified by prep. HPLC (ACE 5μ C18, 250 x 10mm, isocratic 65% MeCN in 35% water + 0.1 %TFA, flow: 4m!/min). The SFB product peak was collected and diluted with 40ml water and passed through a preconditioned Sep-Pak ^{1 M} Light C18 cartridge (Waters) (preconditioned with 5ml acetonitriie and with 10ml water). The SPE was washed with water (5 mL) and was eiuted with acetonitriie (1.5 mi). The acetonitriie solution was dried under gentle N ₂ -stream at 60°C. To the dried SFB was added a solution of CTT54 (2 mg) in 50 μΙ water and 100 μ! 0.1 M Na2C03. The reaction vessel was sealed and heated at 50°C for 10 mins. The reaction mixture was diluted with water (4 ml) and purified by prep. HPLC (Synergi Hydro RP 4μ, 250 x 9.4mm, isocratic 5% MeCN in 95% water + 0.1 %TFA, flow: 3ml/min). The product peak was collected and diluted with 15ml Q.02IVI K CQ ₃ aqueous solution and passed through a preconditioned small anion exchange Sep-Pak ⁱ Pius QMA cartridge

(Waters) (preconditioned by washing the cartridge with 5mi methanol and 10 ml 0.02M K ₂ C0 ₃ aqueous solution). The QMA was washed with water (2 mi) and eiuted with 0.5M NaCI (500 μΙ) into PBS buffer (1 ml, pH ~ 8) to give the desired product in a radiochemical yield 3.88±2.70% in a synthesis time of 215±46 min. The radiochemical purity was 94±3.3%. Radiochemical purity was analyzed on a ZIC HILiC column (4.6mm x 100mm; 5μ: SeQuant) and radioactivity detection was performed on a GABi Star from Raytest. The eiution solution was 0.1 M ammonium formiate {ρΗ 3.2)/actonitri!e (3:7), and the flow rate was 0.5 mL/min. A Corona charged aerosol detector (CAD) from ESA was used to check the separation of the final hot tracer from excess of non-UV-active excess of biological vector CTT54. The residual amount of CTT54 was beiow detection limit of 0.5 g/mL.

E am e 2

N-[(6-[ ^1a F]fluoropyridin-3-yi)carbonyl]-L-gamma-glutamyi-0-[{[{ 1 S)-1 ,3-dicarboxypropy1j amino}(hydroxy)phosphoryi]-L-homoserine (SFN-hCTT)

[ ¹⁸ F] Fluoride was produced by an ⁸ 0 (p, n) ¹⁸ F nuclear reaction by bombardment of a 98% ¹⁸ 0-enriched water target with an 1 1 eVproton beam at the RDS1 1 1 cyclotron. The aqueous [ ¹⁸ F]fiuoride solution was trapped in a small anion exchange Sep-Pak ^{1 M} Plus Q A cartridge (Waters) (preconditioned with 5 mi 0.5 M K2CO3 solution and 10 mL water). The radioactivity was eiufed with a solution mixture (1 .0 mg K ₂ C0 ₃ in 0.5 ml water and 5.27 mg K222 in 1 .5 ml eCN) from the QMA cartridge into a 5 mL conic Wheaton vial. The solvent was evaporated under a stream of nitrogen at 1 10°C. Azeotropic drying was repeated three times with 1 .0 mL portions of acetonitrile. Ethyl 6-chioronicotinate (15.0 mg, 8.1 mmo!) in anhydrous MeCN (1 mL) was added to the dried K222/K[ ^B F]F and the mixture heated at 100"C for 15 min to produce ethyl 6-[ ¹⁸ F]f!uoronicotinate. The ethyl ester was subsequently hydrolyzed with 65 μί of tetrapropylammonium hydroxide (40%) in acetonitril (1 mL) at 35°C for 3 min, and then the mixture azeotropically dried using MeCN (1 mL). Subsequently, a solution of Ν,Ν.Ν',Ν'- tetramethyl-O-(N-succinimidyl) uronium hexafluorophosphate (HSTU) (40 mg, 1 10 mmol) in MeCN (1 mL) was added and the solution heated at 90°C for 6 min. After cooling, the reaction mixture was diluted with water (3.0 mL) and acetonitrile (0.5 mL) and purified by prep. HPLC {ACE 5μ C18, 250 x 10mm, isocratic 75% acetonitrile in 25% water + Q.1 %TFA, flow: 4ml/min). The N-succinimidyl 4-[ ' ⁸ F]fiuoronicotinate ([ ' ⁸ F]SFN) peak was collected and diluted with 30mi water and passed through a preconditioned Sep-Pak™ Light C18 cartridge

(Waters) (preconditioned with 5m! acetonitrile and with 10ml water). The SPE was washed with water (5 mL) and was eluted with acetonitrile (1.5 ml). The acetonitrile solution was dried under gentle N ₂ -stream at 60°C. To the dried SFB was added a solution of hCTT54 (2 mg) in 10 μΙ water and 50 μΙ 1 M Na ₂ C0 ₃ buffer. The reaction vessel was sealed and heated at 50 ^" C for 10 min. The reaction mixture was diluted with water (4 ml) and purified by prep. HPLC (Zorbax Bonus RP 4μ, 250 x 9.4mm, flow: 3ml/min) using the following gradient: (the eluent components were A: water + 0.1 %TFA; B: acetonitriie + 0.1 %TFA): 0 min— 95%A''5%B; 20 min— 50%A''50%B. The product peak was collected and diluted with 20 ml 0.02M K ₂ C0 ₃ aqueous solution and passed through a preconditioned small anion exchange Sep-Pak™ Pius QMA cartridge (Waters) (preconditioned by washing the cartridge with 5ml methanol and 10 mi 0.02M K ₂ C0 ₃ aqueous solution). The QMA was washed with water (2 mi) and eluted with 0.5M NaCI (500 μ!) into PBS buffer (1 ml, pH ~ 8) to give the desired product in a radiochemical yield 2.32±1.54 % in a synthesis time of 201 ±74 min. The radiochemical purify was 98±0.5%. Radiochemical purity was analyzed on a ZIC HILIC column (4.6mm x 100mm: 5μ; SeQuant) and radioactivity detection was performed on a GABI Star from Raytest. The eiution solution was 0.1 M ammonium formiate (pH 3.2)/actonitrile (3:7), and the flow rate was 0.5 mL/min. A Corona charged aerosol detector (CAD) from ESA was used to check the separation of the final hot tracer from excess of non-UV-active excess of biological vector hCTT54. The residual amount of hCTT54 was below detection limit of 0.5 pg/mL.

N-[(5-chloro-6-[ ¹⁸ F]fiuoropyridin-3-yl)carbonyl]-L-gamma-g!utamy!-0-[{[( 1 S)-1 ,3- dicarboxypropy!]amino}(hydroxy)phosphoryI]-L-homoserine (SC!FN-hCTT)

[ ¹⁸ F] Fluoride was produced by an ^t3 0 (p, n) ¹ⁱ⁵ F nuclear reaction by bombardment of a 98% ^,8 0~enriched water target with an 1 1 MeVproton beam at the RDS1 1 1 cyclotron. The aqueous [ ' ⁸ Fjfluoride solution was trapped in a small anion exchange Sep-Pak ^Tiv1 Plus QMA cartridge (Waters) (preconditioned with 5 mi 0.5 M K ₂ C0 ₃ solution and 10 mL water). The radioactivity was eluted with a solution mixture (1 .0 mg K ₂ C0 ₃ in 0.5 ml water and 5.27 mg K ₂₂₂ in 1 .5 ml eCN) from the QMA cartridge into a 5 mL conic Wheaton vial. The solvent was evaporated under a stream of nitrogen at 1 10°C. Azeofropic drying was repeated three times with 1 .0 mL portions of acetonitriie. Ethyl 5,6-dichloronicotinate (15.0 mg, 6.8 mmol) in anhydrous MeCN (1 mL) was added to the dried K222/K[ ^!8 F]F and the mixture heated at 100°C for 15 min to produce ethyl 5-chloro-6-[ ' ⁸ F]fiuoronscotinate. The ethyl ester was subsequently hydrolyzed with 65 μί of tetrapropylammonium hydroxide (40%) in acetonitril (1 mL) at 35°C for 3 min, and then the mixture azeotropicaily dried using MeCM (1 mL). Subsequently, a solution of rvl,N,N',N'-tetramethyi-Q-(N-succinimidyi) uronium hexafiuorophosphate (HSTU) (40 mg, 1 10 mmol) in eCN (1 mL) was added and the solution heated at 90°C for 6 min. After cooling, the reaction mixture was diluted with water (3.0 mL) and acetonitri!e (0.5 ml) and purified by prep. HPLC (ACE 5μ C18, 250 x 10mm, isocratic 70% acetonitrile in 30% water + 0.1 %TFA, flow: 4m!/min). The N-succinimidyl 5-chloro-8-fluoro-[ ^s Fjfluoronicotinate ([ ^t3 FJSC!FN) peak was collected and diluted with 30ml water and passed through a preconditioned Sep~Pak ^{] M} Light C18 cartridge (Waters) (preconditioned with 5ml acetonitrile and with 10ml water). The SPE was washed with water (5 mL) and was eiuted with acetonitrile (1 .0 ml). To the acetonitrile solution S was added a solution of hCTT54 (2 mg) in 10 μΙ water and 50 μΙ 1 M NaHCQS buffer. The reaction vessel was left open and heated at 80°C for 10 min. The reaction mixture was diluted with water (4 mi) and purified by prep. HPLC (Zorbax Bonus RP 4μ, 250 x 9.4mm, flow: 3m!/min) using the following gradient (the e!uent components were A: water + 0.1 %TFA; B: acetonitrile + 0.1 %TFA): 0 min— 95%A/5%B; 20 min— 50%A/5Q%B, The product peak was collected and diluted with 20 ml 0.02 M K2CO3 aqueous solution and passed through a preconditioned small anion exchange Sep-Pak™ Pius QMA cartridge (Waters) (preconditioned by washing the cartridge with 5ml methanol and 10 mi 0.02 M K2CO3 aqueous solution). The QMA was washed with water (2 mi) and eiuted with 0.5 M NaCI (500 μΙ) into PBS buffer (1 mi, pH ~ 8) to give the desired product in a radiochemical yield 2.02±0.86 % in a synthesis time of 147±6 min. The radiochemical purity was 99±0.5%. Radiochemical purity was analyzed on a ZIC H!LIC column (4,6mm x 100mm; 5μ; SeQuant) and radioactivity detection was performed on a GABI Star from Raytest. The eiution solution was 0.1 M ammonium formiate (pH 3.2)/actonitrile (3:7), and the flow rate was 0.5 mL/min. A Corona charged aerosol detector (CAD) from ESA was used to check the separation of the final hot tracer from excess of non-UV-active excess of biological vector hCTT54. The residual amount of hCTT54 was below detection limit of 0.5 9;Υηί.

Reformulation of N-(4-[ ^B F]fluorobenzoyl)-L-gamma-glutamyi-0-[{[(1 S)-1 ,3

dicarboxypropyl]amino}(hydroxy)phosphoryl]-L-serine(SFB-C TT) on C18 plus, ZiC-HILIC and QMA plus cartridges

Tab!e 1 : Tested solid-phase extraction cartridges for reformulation after final preparative HPLC in the synthesis of N-(4-[ ¹⁸ F]fluorobenzoy!)-L-gamma-glutamyi-0-[{[(1 S)-1 ,3 dicarboxypropy!]amino}(hydroxy)phosphoryl]-L-serine (SFB-CTT)

Cartridge Lo acting Solvent ssfoing Solvent Efsjti ng Solv< snt

[%]: acitivty sticking [%]: a citivty sticking on £%/; aciti vty stici dng on on SPE SP E SPE

1 SPE from Waters (preconditioned with 5 ml ethanoi and 10 ml water); 2 SPE from Sequant (Product P/N: 2942-051- 500 mg solid phase material in 3 ml polypropylene cartridge) (preconditioned with 5 ml ethanoi and 0 ml water); 3 SPE from Waters (preconditioned with 5ml methanol and 10 ml 0.02M K ₂ C ⁽ ¾ aqueous solution)

Example 2

Reformulation of N-[(6-[ ^1a F]fluoropyridin-3~yl)carbonylj-L-gamma-giutamyl-0-[{[( 1 S)-1 ,3- dicarboxypropyl] amino}(hydroxy)phosphory!]-L-homoserine (SFN-hCTT) on QMA plus cartridge

Table 2: Tested solid-phase extraction cartridges for reformulation after final preparative HPLC in the synthesis of N~[(8-[' ⁸ F]fluo! pyridin~3-yl)carbonyl]~L-gamma~giutamyl~0-[{[(1 S)- 1 ,3-dicarboxypropyl] amino}(hydroxy)phosphoryl]-L-homoserine (SFN-hCTT)

1 SPE from Waters (preconditioned with 5ml methanol and 10 mi 0.02M K _s C0 ₃ aqueous solution)