METHOD FOR THE SYNTHESIS OF 18F-LABELLED BIOMOLECULES
Technical Field of the Invention
The present invention relates to the field of radiopharmaceuticals, and in particular to
the preparation of compounds suitable for use in positron emission tomography (PET).
A method for the synthesis of compounds labelled with F is provided, which is
preferably an automated method. Also provided by the present invention is a cassette
suitable for carrying out the automated method of the invention.
Description of Related Art
Due to its physical and chemical properties, 1 F is radionuclide a preferred radionuclide
for use in positron emission tomography (PET) tracers. The chemical reactions used to
incorporate F into organic molecules can be broadly divided into two categories,
namely nucleophilic and electrophilic reactions. For nucleophilic fluorination, [, SF]-
fluoride ion ( 1 F ) is used as the source of 1 F. It is normally obtained as an aqueous
solution from the nuclear reaction 1 0(p,n) F. Once it is made reactive by the addition
of a cationic counterion and the removal of water 1 F can be reacted with a compound
comprising a suitable leaving group so that F becomes incorporated into the
compound in place of the leaving group. Suitable leaving groups include CI, Br, I,
tosylate (OTs), mesylate (OMs), nosylate (ONs) and triflate (OTf). The 1 F-labelled
compound obtained can either be the final product, or is an 1 F-labelled synthon that is
used as a labelling reagent to obtain the final product. An example of such a synthon is
' F-(CH2) -LG wherein LG represents a leaving group, which can be used to alkylate
thiol, hydroxy, or amine groups in a precursor compound to result in an F-labelled
product. In order for the alkylation reaction to proceed successfully, deprotonation of
the thiol, hydroxy, or amine group is necessary and as such the reaction is typically
carried out in the presence of a base.
1 , F-labelled radiotracers are at present conveniently prepared by means of an automated
radiosynthesis apparatus. There are several commercially-available examples of such
apparatus. An apparatus such as FASTlab™ (GE Healthcare) comprises a disposable
cassette in which the radiochemistry is performed, which is fitted to the apparatus to
perform the radiosynthesis. In order for a radiofluorination reaction to be carried out on
such an automated synthesis apparatus, it is necessary for each of the reagents to be
soluble in order to be transported around the device. In addition, a separate vial is
required for each reagent and it is desirable for there to be as few vials as possible in
order to simplify the chemistry process, reduce the cost of goods and simplify or
minimise the burden of validation and documentation of reagents required for GMP
clinical production.
Radiolabeled alkylthiophenyl-guanidine compounds and their potential applications in
imaging central nervous system receptors have been reported in WO 2006/136846 and
by Zhao et al (J Label Compd Radiopharm, 2006; 49: 163-70). It has been
demonstrated that these compounds have high affinity for N-methyl-D-aspartate
(NMDA) receptors (<5nM) and have potential utility for the diagnosis of NMDAmediated
disorders such as epilepsy, stroke, neuropathic pain and schizophrenia.
The manual synthesis of two particular radiolabeled alkylthiophenyl-guanidine
compounds was recently reported by Robins et al (Bioorganic and Medicinal Chemistry
Letters, 2010; 20 (5): 1749-51):
The F-fluoroalkyl tosylate synthons were prepared by reaction in step (i) of the
ditosylate starting material with K1 F/Kryptofix 2.2.2 in acetonitrile at 90°C for 15
minutes. The labelled guanidine compounds were obtained in step (ii) by alkylation of
the associated thiol precursor compound with the relevant F fluoroalkyl tosylate
synthon in acetonitrile in the presence of the base Cs2C0 3. The present inventors have
observed an acetyl impurity generated on carrying out the above step (ii):
This acetyl impurity was found to be difficult to remove from the crude reaction
mixture by HPLC, resulting in an overly-long and complicated radiosynthesis.
Another example of an F-fluoroalkylation reaction to obtain a PET tracer is the
reaction described by Wang et al (2006 J Radioanalyt Nuc Chem; 270(2): 439-43) used
to obtain the , F-labeled amino acid 0-(2-[ 1 F]fluoroethyl)-L-tyrosme ([' F]FET):
[18FjFluoroethyl tosylate was prepared in step (i) by displacement of a tosyl group from
1,2-bistosyloxyethane by reaction with K F/Kryptofix 2.2.2 in acetonitrile at 90°C for
10 minutes. The purified [ Fjfluoroethyl tosylate was then reacted in step (ii) with a
solution of L-tyrosine and 10% aqueous NaOH in DMSO (or di-Na-salt of L-tyrosine in
DMSO) 20 minutes at 90°C to obtain [1 F]FET. In contrast to the method for
preparation of F-labelled S-fluoroalkyl diarylguanidines as reported by Robins et al
(supra), this method for preparati *on of [ 8F]FET uses a soluble base in the alkylation
reaction. However, the reaction is still not ideal for carrying out on an automated
synthesis device that uses a cassette due to the fact that and additional vial is required
for the base used for the subsequent fluoroalkylation step.
Lundkvist et al (1997 Nuc Med Biol; 24: 621-7) describe the synthesis of
[ F]fluoropropyl -P-CIT (b-CIT: (-)-2p-Carbomethoxy-3P-(4-iodophenyl)tropane) using
the [, F]fluoropropyl bromide as the labelling reagent. In step (i) [ F]fluoropropyl
bromide was prepared by a nucleophilic fluonnation of 1,3-dibromopropane with [ F]
potassium Kryptofix complex. [ F]Fluoropropyl bromide in dimethyl formamide
(DMF) was then used in step (ii) to alkylate nor-b- T at 130°C for 25 minutes to form
[ F]fluoropropyl-p-CITThe
above method is not ideal for automation since it requires the purification of the
synthon via distialltion and an additional reagent vial for the base.
There is therefore a need for a method to obtain the above-described and similar , Flabelled
compounds that overcomes the various problems and renders the methods more
amenable to automation.
Summary of the Invention
The present invention provides a method for the synthesis of F-labelled biomolecules,
which is amenable to automation. The present invention also provides a cassette for
automating the method of the invention. The method of the present invention provides
numerous advantages over the prior art methods. It requires one less purification step as
compared with known methods. Furthermore, in a preferred embodiment it makes use
of a particular reagent in two steps thereby minimises the number of reagent vials
required. The chemistry process is thereby simplified, the cost of goods is reduced and
the burden of validation and documentation of reagents required for GMP clinical
production is minimised.
Detailed Description of the Invention
In one aspect, the present invention provides a method to prepare a compound of
Formula I :
or a salt of a solvate thereof, wherein:
R'-A- is a deprotonated radical of a biological targeting molecule (BTM) of
formula R'-A-H wherein A is selected from S, O or NR2 wherein R2 is hydrogen,
Ci_6 alkyl, or C - 2 aryl, and,
n is an integer of 1-6;
wherein said method comprises:
(i) reacting in a suitable solvent a compound of Formula II:
wherein:
LG and LG are the same or different and each represents a leaving
group LG; and,
and m is an integer of between 1-4;
with a suitable source of [ F]fluoride to obtain a first crude reaction
product comprising said compound of Formula II and a compound of
Formula III:
wherein LG is a leaving group LG and p is as defined for m of
Formula II;
(ii) deprotonating a compound of Formula IV·
F I
A [IV]
or a protected version thereof, wherein -A -R" is as defined for -A
R1 of Formula I;
(iii) reacting in an alkanol solvent said first crude reaction product obtained in
step (i) with said deprotonated compound obtained in step (ii) to obtain a
second crude reaction product comprising said compound of Formula , or a
protected version thereof; and,
(iv) removing any protecting groups.
A suitable "salt." according to the invention may be selected from (i) physiologically
acceptable acid addition salts such as those derived from mineral acids, for example
hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric and sulphuric acids, and
those derived from organic acids, for example tartaric, trifluoroacetic, citric, malic,
lactic, fumaric, benzoic, glycollic, gluconic, succinic, methanesulphonic, and paratoluenesulphonic
acids; and (ii) physiologically acceptable base salts such as
ammonium salts, alkali metal salts (for example those of sodium and potassium),
alkaline earth metal salts (for example those of calcium and magnesium), salts with
organic bases such as triethanolamine, N-methyl-D-glucamine, piperidine, pyridine,
piperazine, and morpholine, and salts with amino acids such as arginine and lysine.
A suitable "solvate" according to the invention may be formed with ethanol, water,
saline, physiological buffer and glycol.
The term "biological targeting moiety" (BTM) is meant a compound which, after
administration, is taken up selectively or localises at a particular site of the mammalian
body in vivo. Such sites may for example be implicated in a particular disease state or
be indicative of how an organ or metabolic process is functioning. The BTM may be of
synthetic or natural origin, but is preferably synthetic.
The term "synthetic" has its conventional meaning, i.e. man-made as opposed to being
isolated from natural sources e.g. from the mammalian body. Such compounds have the
advantage that their manufacture and impurity profile can be fully controlled. The
molecular weight of the BTM is preferably up to 3,000 Daltons, more preferably 200 to
2,500 Daltons, most preferably 300 to 2,000 Daltons, with 400 to 1,500 Daltons being
especially preferred.
Preferably the BTM is an enzyme substrate, enzyme antagonist, enzyme agonist,
enzyme inhibitor or receptor-binding compound, in particular a non-peptide, and
preferably is synthetic. By the term "non-peptide " is meant a compound which does not
comprise any peptide bonds, i.e. an amide bond between two amino acid residues.
When the BTM is an enzyme substrate, enzyme antagonist, enzyme agonist or enzyme
inhibitor, preferred such biological targeting molecules of the present invention are
synthetic, drug-like small molecules i.e. pharmaceutical molecules. Non-limiting
examples of particular such biological targeting molecules are described in more detail
hereunder.
The term "alkyj" used either alone or as part of another group is defined as any straight,
branched or cyclic, saturated or unsaturated CnH2n+i group.
The term "aryj" used either alone or as part of another group is defined as any C -i
molecular fragment or group which is derived from a monocyclic or polycyclic aromatic
hydrocarbon, or a monocyclic or polycyclic heteroaromatic hydrocarbon.
The "suitable solvent" for use in said reacting step (i) is one in which the reactants are
readily soluble and readily react to result in the desired product. Examples include N,Ndimethylformamide
(DMF), acetone, dichloromethane (DCM), chloroform,
dimethylsulphoxide (DMS), methanol, ethanol, propanol, isopropanol, tetrahydrofuran
(THF), or acetonitrile, and aqueous solutions thereof. An "aqueous solution" in the
context of the suitable solvent for step (i) preferably means 5-20% water, most
preferably 10-15% water. Either aqueous ethanol or aqueous acetonitrile are preferred
for reacting step (i), but where aqueous acetonitrile is used it is necessary following said
reacting step to remove acetonitrile before using the first crude reaction product in
reacting step (iii). Aqueous ethanol is preferred.
The term "leaving group" refers to a molecular fragment that departs with a pair of
electrons in heterolytic bond cleavage. A suitable leaving group can be a halo, e.g.
selected from chloro, iodo, or bromo, or an aryl or alkyl sulphonate. A preferred
leaving group is selected from CI, Br, I, tosylate (OTs), mesylate (OMs) and triflate
(OTf). Preferably LG1 and LG2 are the same.
A "suitable source of [1 F fluoride" is [1 F]fluoride that has been made reactive,
typically by drying and addition of a cationic counterion. The step of "drying" said
[ 8F]fluoride comprises evaporation of water to result in anhydrous [ 8F]fluoride. This
drying step are suitably carried out by application of heat and use of a solvent such as
acetonitrile to provide a lower boiling azeotrope. A "cationic counterion" is a
positively-charged counterion examples of which include large but soft metal ions such
as rubidium or caesium, potassium complexed with a cryptand, or tetraalkylammonium
salts. A preferred cationic counterion is a metal complex of a cryptand, most preferably
wherein said metal is potassium and wherein said cryptand is Kryptofix 222.
The term "crude reaction product" as used in the context of both the first crude reaction
product and the second crude reaction product is taken to mean the product of the
reaction that has not been subjected to any purification. The term "purification " refers
to any method used to isolate from the crude reaction product that chemical compound
which is the desired reaction product. The other chemical compounds are generally
referred to as "impurities " Typically, this is done by separating the various chemical
compounds present in the crude reaction product from each other by means of
techniques well-known to those skilled in the art such as chromatography and solidphase
extraction.
The term "deprotonating " refers to the removal of a proton (H+) from the compound of
Formula IV and is carried out using a base. This step facilitates the subsequent
alkylation reaction. The "base" can be an inorganic base such as potassium or caesium
carbonate, potassium hydroxide, or sodium hydride, or an organic base such as a
trialkylamine, for example triethylamine, diisopropylethylamine, or
dimethylaminopyridine. In a one preferred embodiment, rather than being a separate
reagent, the base used for the deprotonating step is the cationic counterion used in
preparing reactive [, F]fluoride. This preferred embodiment is particularly suitable for
automation because less reagent vials are required.
Suitable "protecting groups" and methods for "removing protecting groups" are well
known to those skilled in the art. The use of protecting groups is described in
'Protective Groups in Organic Synthesis', by Greene and Wuts (Fourth Edition, John
Wiley & Sons, 2007). The step of removing these protecting groups, if present, is
preferably carried out after the alkylation step.
The "alkanol solvent " for reacting step (iii) may be an alkanol or an aqueous alkanol,
wherein the term "alkanol " is taken to mean a simple aliphatic alcohol. An "aqueous
alkanol" consists of water and an alkanol and in the context of this step (iii) means a
solution comprising water. Suitably said alkanol solvent does not comprise any
solvents apart from water and alkanol, and in particular does not comprise acetonitrile.
Suitable alkanols in the context of the present invention include methanol, ethanol and
propanol, with ethanol being most preferred.
Compounds of Formula II can be readily obtained by use or straightforward adaptation
of methods described by Block et al (J Label Comp Radiopharm, 1988; 25: 201) or by
Neal et al (J Label Comp Radiopharm, 2005; 48 557).
Compounds of Formula IV may be prepared by use or straightforward adaptation of the
methods described variously in WO 94/27591, WO 2004/007440, WO 2006/136846,
Hu et al (J Med Chem, 1997; 40: 4281-9), Zhao et al (J Label Compd Radiopharm,
2006; 49: 163-70) and Robins et al (Bioorganic and Medicinal Chemistry Letters, 2010;
20 (5): 1749-51).
The indications n, m, p and q are in each instance preferably 1-4, most preferably 1-3
and most especially preferably 1-2.
The alkylation step (iii) may be carried out either at room temperature or at higher
temperatures (typically 90-1 30°C), and following the removal of any protecting groups,
the method can comprise the additional step (v) of purifying said second crude reaction
product to obtain purified compound of Formula I . Suitably said purifying is carried out
by chromatography or solid-phase extraction (SPE), wherein said chromatography is
preferably high-performance liquid chromatography (HPLC).
The method of the present invention has the advantage that it does not require
purification of the compound of Formula III for use in the alkylation step.
In a preferred embodiment of the method of the invention said compound of Formula I
is a compound of Formula la:
or a salt or solvate thereof, wherein:
Ala is an A group as defined for Formula 1,
R, is an Ra group selected from hydrogen or C alkyl;
R a is an R group which is halo; and
R4 is an Rd group selected from halo, C alkylthio, or C alkyl;
said compound of Formula IV is a compound of Formula IVa:
wherein R, , , R, and R,4a are respectively an R , R and Rd group as
defined for Formula la, A is an A group as defined for Formula I, and P
and P2 are each a P group selected from hydrogen or a protecting group,
preferably hydrogen.
The term "halogen " or "halo" means a substituent selected from fluorine, chlorine,
bromine or iodine.
The term "alkylthio" refers to an alkyl group as defined above comprising a sulphur
the chain, preferably at the proximal end, i.e. -S-alkyl.
Most preferably said compound of Formula la is a compound of Formula lb:
wherein Rl , R , and R4 are respectively an Ra, R and Rd group as defined for
Formula la, A is an A group as defined for Formula I, and n is as defined for n of
Formula I ;
said compound of Formula IV is a compound of Formula IVb:
wherein R,lb , R1 , and R, 1
4b are respectively an R , R and R group as defined for
Formula la, A2 is an A group as defined for Formula I, and Pl and P are each a P
group as defined for Formula IVa.
Each Ra group is preferably C alkyl and most preferably methyl.
Each R group is preferably chloro.
Each Rd group is preferably alkylthio, and most preferably methylthio.
Each A group is preferably S and R12 is preferably SH.
For particular compounds of Formulae la and b as defined hereinabove it is preferred
that:
each R group is C alkyl and is most preferably methyl;
each R group is chloro;
each R group is alkylthio, and is most preferably methylthio; and,
each A group is S and R12 is preferably SH; and,
Where the method of the invention is for the synthesis of a compound of Formula la, the
method reported by Robins et al (2010 Bioorg Med Chem Letts; 20: 1749-51) is easily
adapted to result in a method of the present invention. This method of Robins et al for
the synthesis of F-labelled .S-fluoroalkyl diarylguani dines comprises
[1 F]fluoroalkylation of a thiol group using the following method:
In the above reaction scheme (i) represents 1 F Kryptofix 2.2.2, MeCN, 90°C, 15
minutes and (ii) represents the thiol derivative of the final product, CS2CO , MeCN,
10°C, 5 minutes. The [, F]fluoroalkyltosylate labelling reagent is purified before use
in this method of Robins et al. In the method of the present invention the [ F]
fluoroalkylation step is instead carried out in an aqueous alkanol rather than acetonitrile
(MeCN), and the [ F]fluoroalkyltosylate labelling reagent is used without having been
purified.
A particular advantage of the present method over known methods where the compound
of Formula I is a compound of Formula la is that purification is made easier by avoiding
generation of acetyl impurities in the alkylation step, a problem which was found by the
present inventors. The scheme below illustrates the mechanism by which an acetyl
impurity is believed to be formed in the synthesis of 3-(2-chloro-5-((2-
[ F]fluoroethyl)thio)phenyl)-l -methyl- 1-(3-(methylthio)phenyl)guanidine from 3-(2-
chloro-5-((2-hydroxyethyl)thio)phenyl)-l -methyl- l-(3-(methylthio)phenyl)guanidine:
It is proposed that use of alkanol solvent in place of acetonitrile in the
[ F]fluoroalkylation step (iii) avoids the production of this acetyl impurity.
Furthermore, use of the alkanol solvent in the [ F]fluoroalkylation step means that the
first crude reaction product comprising the compounds of Formulae II and III can be
used directly the alkylation without having to purify to remove unreacted compound
of Formula II, which would be necessary if acetonitrile were to be used in the
[1 F]fluoroalkylation step to avoid the reaction illustrated above. When an alkanol
solvent is used, any unreacted compound of Formula II can still react with the
deprotonated compound of Formula IV, but the impurity generated as a consequence
will be a hydroxyl impurity which is straightforward to separate in any subsequent
purification step.
Another known method that can be adapted in a straightforward manner to be a method
of the present invention is the method comprising [ F]fluoroalkylation of a phenol
described by Wang et al (2006 J Radioanalyt Nuc Chem; 270(2): 439-43) to obtain the
, F-labeled amino acid 0-(2-[ 1 F]fluoroethyl)-L-tyrosine ([ F]FET):
[ FjFluoroethyl tosylate obtained in step (i) can be reacted in step (ii) (without needing
to first be purified) with a solution of L-tyrosine in an aqueous alkanol (rather than
DMSO) which has been treated with an aliquot of the solution of K2C0 3 and Kryptofix
222 (rather than NaOH) previously used in the method to make reactive
[1 F][K(Kryptofix)]F for use in step (i).
Accordingly, another example of a prefen-ed compound of Formula IV is the following
compound:
In the method described by Lundkvist et al (1997 Nuc Med Biol; 24: 621-7) for the
synthesis of [1 F]fiuoropropyl -P-CIT (b-CIT: (-)-2p-Carbomethoxy-3P-(4-
iodophenyl)tropane) a secondary amine is alkylated using [ FJfluoropropyl bromide:
This method can be readily adapted to be a method of the present invention by carrying
out step (ii) in an aqueous alkanol solution. Preferably an aliquot of K2C0 3 and
Kryptofix 222 (with acetonitrile removed), which is used to make reactive
[1 F][K(Kryptofix)]F in step (i), is used in step (ii) as a base.
Accordingly, another example of a preferred compound of Formula IV is the following
compound:
The above-described compounds merely provide illustrations of how the method of the
present invention may be applied. It will be clearly appreciated by the skilled person
that the method of the present invention can also be applied to achieve similar
advantages to any reaction that comprises (i) synthesis of an [1 F]fluoroalkyl labelling
reagent using [, F]fluoride as the source of 1 F, and (ii) [1 F]fluoroalkylation of a thiol,
hydroxy or amine functionality in a precursor compound.
The method of the present invention is particularly amenable to automation as
compared to known methods. Automation may be carried out on an automated
radiosynthesis apparatus. There are several commercially-available examples of such
apparatus, including Tracerlab MX ™ and FASTlab™ (GE Healthcare), FDGPlus
Synthesizer (Bioscan) and Synthera® (IBA). Such apparatus may comprise a "cassette",
often disposable, in which the radiochemistry is performed, which is fitted to the
apparatus in order to perform a radiosynthesis. The cassette normally includes fluid
pathways, a reaction vessel, and ports for receiving reagent vials as well as any sohdphase
extraction cartridges used in post-radiosynthetic clean up steps. As the method of
the present invention does not require purification of the first crude reaction product,
and as the second crude reaction product is relatively easy to purify, the method of the
present invention is amenable to automation. Therefore, in a preferred embodiment, the
method of the present invention is automated, most preferably by means of a cassette on
an automated radiosynthesis apparatus. The present invention therefore provides in
another aspect a cassette for the automated synthesis of the compound of Formula I as
defined herein wherein said cassette comprises:
(i) a first vessel containing a compound of Formula II as defined herein;
(ii) means for eluting said first vessel with a suitable source of [18F]-fluoride;
and,
(iii) a second vessel containing a compound of Formula IV as defined herein.
The suitable and preferred embodiments of the compounds of Formulae II and IV, and
the suitable source of [18F]fluoride as defined hereinabove for the method of the present
invention are also applicable to the cassette of the present invention.
The term "vessel" is taken to mean a reagent vial suitable for placing in a position on a
cassette suitable for use with an automated radiosynthesis apparatus.
Additional vessels maybe present specific to the chemistry/biomolecule synthesis e.g. vials
for solvents for deprotection, purification, formulation, reformulation. Additional
cartridges (SPE) may also be present for purification and/or re-formulation. There may
also be a connection line from the cassette to a HPLC unit ifHPLC purification is required,
and there may be a connection line from the "HPLC cut vial" to the cassette if there is a
requirement for solvent reformulation post purification.
Therefore in another embodiment, the cassette of the present invention may additionally
comprise either or both of:
(iv) an ion-exchange cartridge for removal of excess [ F]fluoride; and,
(v) a cartridge for carrying out the step of removing any protecting groups.
The reagents, solvents and other consumables required for the automated synthesis may
also be included together with a data medium, such as a compact disc carrying software,
which allows the automated synthesiser to be operated in a way to meet the end user's
requirements for concentration, volumes, time of delivery etc.
Brief Description of the Examples
Example 1 describes the automated synthesis of 3-(2-chloro-5-((2-
[1 F]fluoroethyl)thio)phenyl)- 1-methyl- -(3-(methylthio)phenyl)guanidine using the
method of the present invention.
Example 2 describes an experiment comparing FASTlab™ synthesis of 3-(2-chloro-5-
((2-[ F]fluoroethyl)thio)phenyl)- 1-methyl- 1-(3-(methylthio)phenyl)guanidine using
ethanol or acetonitrile as the solvent.
List of Abbreviations used in the Examples
EtOH ethanol
HPLC high performance liquid chromatography
222 Kryptofix 2.2.2
MeCN acetonitrile
QMA quaternary methylammonium
SPE solid phase extraction
TsO tosylate
Examples
Example 1: FASTlab™ Synthesis of 3-(2-chloro-5-((2-f l sF]fluoroethyl)thio)phenyl)-
1-methyl-l-(3-(m ethylthio)phen guanidine
A cassette for use with a FASTlab™ synthesiser comprised the following vials:
*3-(2-chloro-5-((2-hydioxyethyl)thio)p enyl)- l -methyl- l -(3-(methylthio)phenyl)guanidine
The cassette is also illustrated in Figure 1.
l(i) Transfer of F fluoride to cassette
[ F]Fluoride was supplied from GE Healthcare on a GE PETrace cylcotron. The initial
activity was transferred via the activity inlet of the FASTlab cassette using vacuum.
l(ii) Trapping [ F]fluoride on the QMA
The activity was transferred from the activity inlet to the (pre-treated) QMA cartridge
where the [ F] was trapped and the water passed through to the O water recovery vial,
using a combination of N2 to push and vacuum to pull.
l(iii) Elution of f '8Flfluoride off the QMA
70m of the eluent vial (K222, 2C0 3) was removed from the eluent vial using the ImL
syringe. 550m of water was then withdrawn from the water bag and added to the eluent
in the I L syringe. The [I F]fluoride trapped on the QMA cartridge was then eluted
into the reaction vessel using the eluent/water solution in the ImL syringe and a vacuum
applied to the reaction vessel to draw the solution through the QMA cartridge.
1(iv) Drying [' F fluoride
The [18F]fluoride and eluent soluti'on was dried for 20 minutes by heating ( 00°C) and a
combination of nitrogen and vacuum were used to remove the evaporated solvent and
water from the reaction vessel to a waste collection vessel.
l(v) Radiosynthesis of [ F] -fluoroethyltosylate
lmL of the ethylene ditosylate solution (2.5mg per mL of MeCN) was removed from
the vial using the centre (5ml) syringe and dispensed into the reaction vessel containing
the dried [, F] fluoride/K222/K 2C0 3 (reactive [, F][K(Kryptofix)]F). The reaction
vessel was then sealed and the reaction carried out by heating for 15 minutes at 86°C.
l(vi) Removal of solvent from the ['8F]-fluoroethyltosylate
The crude [, F]-fluoroethyltosylate /ethylene ditosylate solution was dried for 10
minutes by heating (80°C) and a combination of nitrogen and vacuum was used to
remove the evaporated solvent from the reaction vessel to a waste collection vessel.
l(vii) Introduction of 500må of eluent to precursor vial
500m of eluent vial (K222 , K2C0 3) was removed from the eluent vial and added into the
precursor vial using the lmL syringe. The solution was held for 1minute.
l(viii) Introduction of precursor to reaction vessel
lOmg (26mh o1) of precursor (3-(2-chloro-5-((2-hydroxyethyl)thio)phenyl)-l -methyl- 1-
(3-(methylthio)phenyl)guanidine) in 1.5mL of ethanol was removed from the vial by
creating a vacuum in the reaction vessel.
l(ix) Alkylation of precursor
The reaction vessel was then sealed and the alkylation carried out by initially heating for
2 minutes at 80°C, then 13 minutes at 100°C.
l(x) Loopflush out with water
A total of lOmL water was removed from the water bag using the centre (5ml) syringe
and sent through the HPLC loop in two syringe movements.
l(xi) Quench reaction, and transfer out of FASTlab to HPLC loop
2mL water was added to the reaction vessel from the water bag using the centre 5mL
syringe. lmL 0. M HC1 was added to the reaction vessel from the vial using the centre
5mL syringe. This was then withdrawn from the reaction vessel using the same syringe
and transfen-ed from the cassette to the HPLC loop, followed by a purge of the line and
cassette fluid path with nitrogen to clear any residual solution to the HPLC loop.
l(xii) HPLC purification and SPE formulation
The following HPLC method was used:
0-60mins 40%(B)
Column ACE C18 1OOx 10mm 5m h
Mobile phase Mobile phase A (pump A): Acetonitrile (pump B)
Loop Size 10ml
Pump speed 3ml/min
Wavelength 254nm
Mobile Phase A: 0.8%TEA [TEA (8ml) and H20 (992ml)], pH adj to ca. 7.5
with 85%H3P04 (ca. 2.1ml)
The HPLC run was controlled from the HPLC software until the cut was performed.
The HPLC cut was transferred back to the FASTlab using the right hand (5ml) syringe
to draw the cut back on to the cassette then add to the dilution water bag. The diluted
HPLC cut (>100mL) was loaded on to a tC18+ SPE cartridge by applying a vacuum for
1 minutes to draw the full content of the water bag through the cartridge to a waste
collection vessel. The SPE cartridge was eluted with lmL ethanol from the vial using
the right hand 5mL syringe into a vial containing 14mL saline containing .5mg
ascorbic acid.
In summary, the following were observed:
Example 2: Comparison of FASTlab Synthesis of 3-(2-chloro-5-((2-
f18F]fluoroethylHhio)phenyl)-l-methyl-l-(3-(methylthio)phenyl)suanidme usins
Ethanol or Acetonitrile as the solvent
The process described in Example 1 was carried out up to step 1(xi) but wherein the
following step was analytical HPLC using the following method:
Mobile Phase A: 0.8%TEA (8mL TEA and 992mL H20 ), pH adj. to ca. 7.5 with
85%H3P0 4 (ca. 2.1mL)
Mobile phase B: MeCN
0-1 in 40%B; 1-25 min 40 -95%B
HPLC column: Luna C 8 (150 x 4.6mm)
Flow rate: lmL/min
In addition, the same process was carried out wherein acetonitrile was used as the
solvent in place of ethanol. Figure 2 compares the synthesis wherein acetonitrile (top)
was used in place of ethanol (bottom) as the solvent. It can be clearly seen that the
acetyl chemical impurity that elutes around 12 minutes (with product eluting just
afterwards) is not formed when acetonitrile has been removed from the alkylation step.

Claims
(1) A method to prepare a compound of Formula I :
or a salt of a solvate thereof, wherein.
R -A- is a deprotonated radical of a biological targeting molecule (BTM) of
2 2 fomiula R -A-Hwherein A is selected from S, O or NR wherein R is hydrogen,
C i - alkyl, or C5-1 2 aryl; and,
n is an integer of 1-6;
wherein said method comprises:
(i) reacting in a suitable solvent a compound of Formula II:
wherein:
LG1and LG2 are the same or different and each represents a leaving
group LG; and,
and m is an integer of between 1-4;
with a suitable source of [ FJfluoride to obtain a first crude reaction
product comprising said compound of Formula II and a compound of
Formula III:
wherein LG 2 is a leaving group LG and p is as defined for m of
Formula II;
(ii) deprotonating a compound of Formula IV:
H 1
A2 [IV]
or a protected version thereof, wherein -A2-R is as defined for -A1-
R 1 of Formula I;
(iii) reacting in an alkanol solvent said first crude reaction product obtained in
step (i) with said deprotonated compound obtained in step (ii) to obtain a
second crude reaction product comprising said compound of Formula I, or a
protected version thereof; and,
(iv) removing any protecting groups.
(2) The method as defined in Claim 1 wherein:
said compound of Formula I is a compound of Formula la:
or a salt or solvate thereof, wherein:
Al is an A group as defined for Formula 1,
R is an R group selected from hydrogen or C alkyl;
R A is an R group which is halo; and
R4 is an R group selected from halo, C -4 alkylthio, or C alkyl;
said compound of Formula IV is a compound of Formula FVa:
wherein R,la , R and R14a are respectively an R , R and R group as
defined for Formula la, A is an A group as defined for Formula I, and P1
and P are each a P group selected from hydrogen or a protecting group.
The method as defined in Claim 1 wherein:
said compound of Formula I is a compound of Formula lb:
wherein R , R b, and R4 are respectively an R , R and Rd group as defined in
Claim 2 for Formula la, A is an A group as defined for Formula I, and n is as
defined for n of Formula I;
said compound of Formula IV is a compound of Formula b:
[rvb]
wherein Rllb , R , and R1l4b are respectively an R , R and R group as defined
Claim 2 for Formula la, A2 is an A group as defined for Formula I, and P l and P
are each a P group as defined in Claim 2 for Formula IVa.
(4) The method as defined in either Claim 2 or Claim 3 wherein each R group is C 4
alkyl.
(5) The method as defined in Claim 4 wherein each R group is methyl.
(6) The method as defined in any one of Claims 2-5 wherein each Rb group is -18F-C1-2
fluoroalkyl.
(7) The method as defined in any one of Claims 2-6 wherein each R group is chloro.
(8) The method as defined in any one of Claims 2-7 wherein each R group is alkylthio.
(9) The method as defined in Claim 8 wherein each Rd group is methylthio.
(10) The method as defined in any one of Claims 2-9 wherein said A group is S.
( 11) The method as defined in Claim 1 wherein said compound of Formula IV is the
following compound:
The compound as defined in any one of Claim 1 wherein said compound
Formula IV is the following compound:
(13) The method as defined in any one of Claims 1-12 wherein said leaving group LG is
selected from CI, Br, I, tosylate (OTs), mesylate (OMs) and triflate (OTf).
(14) The method as defined in any one of Claims 1-13 wherein LG1 and LG2 are the
same.
(15) The method as defined in any one of Claims 1-14 which comprises the additional
step (v) of purifying said second crude reaction product to obtain purified
compound of Formula I .
(16) The method as defined in any one of Claims 1-15 which is automated.
(17) A cassette for carrying out the method as defined in Claim 16 comprising:
(i) a first vessel containing a compound of Formula II as defined in either
Claim 1 or Claim 14;
(ii) means for elutmg said first vessel with a suitable source of [18F]-fluoride;
and,
(iii) a second vessel containing a compound of Formula IV as defined in any onr
of Claims 1-12.
(18) The cassette as defined in Claim 7 which comprises one or both of:
(iv) an ion-exchange cartridge for removal of excess [ 8F]fluoride; and,
(v) a cartridge for carrying out step (iv) as defined in Claim 1 of removing
any protecting groups.