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).
1 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 18F into organic molecules can be broadly divided into two categories,
namely nucleophilic and electrophilic reactions. For nucleophilic fluorination, [1 F]-
fluoride ion ( F ) is used as the source of 1 F. It is normally obtained as an aqueous
solution from the nuclear reaction 0(p,n) 8F. Once it is made reactive by the addition
of a cationic counterion and the removal of water F can be reacted with a compound
comprising a suitable leaving group so that 1 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 inflate (OTf). The F-labelled
compound obtained can either be the final product, or is an 1 F-labelled synthon that s
used as a labelling reagent to obtain the final product. An example of such a synthon is
18F-(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.
8F-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 v al is
required for each reagent and t 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.
One example of an F-fluoroalkylation reaction to obtain a PET tracer is the following
reaction used to obtain F-labelled S-fluoroalkyl diarylguanidines as reported by
Robins et al (2010 Bioorg Med Chem Letts; 20: 1749-51):
The F-fluoroalkyl tosylate synthons were prepared by reaction in step (i) of the
ditosylate starting material with K F/Kryptofix 2.2.2 in acetonitrile at 90°C for 15
minutes. Although not explicitly stated in the paper, the F-fluoroalkyl tosylate
synthons were purified by PLC prior to use in the next step. 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 CS2CO3. Since CS2CO3 used in this alkylation reaction is not
soluble in acetonitrile, the method for cannot be readily automated.
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-!abcled amino acid 0-(2-[ SF]fluoroethy! )-L-tyrosine ([i F]FET):
[ F]Fluoroethyl tosylate was prepared in step (i) by displacement of a tosyl group from
1,2-bistosyloxyethane by reaction with 18F/Kryptofix 2.2.2 in acetonitrile at 90°C for
10 minutes. The purified [1 F]fluoroethyl 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 1 F-labelled S-fluoroalkyl diarylguanidines as reported by Robins et al
{supra), this method for preparation of [18F]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
[1 F]fluoropropyl-p-CIT (b-CIT· (-)-2p-Carbomethoxy-3p-(4-iodophenyl)tropane) using
the [l F]fluoropropyl bromide as the labelling reagent. In step (i) [1 F]fluoropropyl
bromide was prepared by a nucleophilic fluonnation of 1,3-dibromopropane with [I F]
potassium Kryptofix complex. [\ 8FjFluoropropyl 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-CIT:
The 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 novel radiofluorination methods that comprise Ffluoroalkylation
that overcome the problems associated with the known methods in
order to be readily automated h particular it would be desirable to reduce the number
of process steps and to minimise the number of reagents used.
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, 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 relates to a method for the synthesis of a compound
of Formula I :
or a salt or 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,
C _ alkyl, or C5-1 2 aryl; and,
n is an integer of 1-6;
wherein said method comprises:
providing [ F]Fluoride trapped on an ion-exchange cartridge;
eluting the ion-exchange cartridge of step (i) with an aqueous solution
comprising a first aliquot of an eluent, wherein said ekient comprises a
catiomc counterion a suitable solvent, to obtain a [18FJFluoride eluent;
reacting a compound of Formula II
wherein LG and LG2 are the same or different and each represent a leaving
group, and n is as defined for Formula I;
in a first solvent with the [ F]Fluoride eluent obtained in step (ii) to obtain
a compound of Formula III:
wherein LG2 and n are as defined for Formula II;
(iv) deprotonating a compound of Formula IV:
R
A [IV]
or a protected version thereof, wherein A and R are as defined for Formula
I ;
by addition of a second aliquot of the eluent as defined in step (ii);
(v) reacting the compound of Formula III obtained in step (iii) with said
deprotonated compound obtained in step (iv), or a protected version
thereof, in a second solvent to obtain said compound of Formula I, or a
protected version thereof, wherein said second solvent is an alkanol or an
aqueous alkanol,
(vi) removing any protecting groups.
A suitable "salt" according to the invention maybe selected from: (i) physiologically
acceptable acid addition salts such as those derived from mineral acids, for example
hydrochloric, hydrobromic, phosphoric, metaphosphonc, 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 "alkyl" used either alone or as part of another group is defined as any straight,
branched or cyclic, saturated or unsaturated C H2 +i group.
The term "aryl" used either alone or as part of another group is defined as any C -i4
molecular fragment or group which is derived from a monocyclic or polycyclic aromatic
hydrocarbon, or a monocyclic or polycyclic heteroaromatic hydrocarbon.
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 ammo 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.
A suitable "ion-exchange cartridge" in the context of the present invention is a solidphase
extraction (SPE) cartridge that retains 1 and allows 1 0 to pass through when
an aqueous solution from the nuclear reaction 0(p,n) 1 F is passed through. Preferably,
said ion-exchange cartridge is an anion exchange cartridge, most preferably a quaternary
methylammonium (QMA) cartridge.
A "cationic counterion " in the context of the present invention is a positively-charged
counterion that acts to improve the reactivity of [1 F]Fluonde when combined
therewith. A suitable cationic countenon for use in the method of the present invention
may be a large but soft metal ion such as rubidium or caesium, a metal complex of a
cryptand or a tetraalkylammonium salt. A preferred cationic counterion is a metal
complex of a cryptand or a tetraalkylammonium salt. A preferred metal in a metal
complex of a cryptand is potassium. A preferred cryptand in a metal complex of a
cryptand is Kryptofix 222. A preferred tetraalkylammonium salt is selected from P N +
wherein R is ethyl, methyl or butyl. The "suitable solvent " for the eluent is an alkanol,
and is preferably ethanol or methanol, most preferably ethanol.
The "aqueous solution comprising a first aliquot of an eluent comprising a cationic
counterion " refers to a solution comprising an aliquot of said eluent made up with
water. This aqueous solution is used as a phase transfer catalyst to improve solubility
and nucleophilicity of [1 F]fluoride. In the eluting step said aqueous solution is passed
through the ion-exchange cartridge, bringing with it the [1 F]fluoride to result in an
" F eluent" comprising [ F]fluoride in said aqueous solution.
Said [, F]Fluoride eluent may be dried before subsequent use, suitably by evaporation
of water to result in anhydrous [ F]Fluoride eluent. This drying step is for example
carried out by application of heat and use of a solvent such as acetonitrile to provide a
lower boiling azeotrope.
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. A preferred suitable leaving group can be an aryl
or alkyl sulphonate, for example, tosylate, triflate, nosylate or mesylate.
The "first solvent " used in step (iii) of the method of the invention is suitably one in
which both the compound of Formula II and the dried [ F]fluoride eluent are soluble.
Generally, a dipolar aprotic solvent is suitable, preferably an alky] nitrile, most
preferably acetonitrile.
As in the case of the [ FJFluoride eluent, the compound of Formula III may be dried
before subsequent use to remove the solvent, which can be particularly important when
the solvent is an alkyl nitrile such as acetonitrile. The present inventors have observed
that the presence of such a solvent n the alkylation reaction mixture can lead to the
generation of acetyl impurities that are difficult to remove from the final product. For
the compound of Formula III, drying is suitably carried out by application of heat and/or
vacuum and/or use of gas flow(typically nitrogen).
The term "deprotonating " refers to the removal of a proton (H+) from a molecule. The
step of deprotonating the compound of Formula IV is carried out using a second aliquot
of the eluent as defined in step (ii) where in this part of the process the eluent acts as
soluble base.
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 second solvent used in step (v) of the method of the invention 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. Suitably said second 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.
In Formulae II and III, n is preferably 1-4, most preferably 1-3 and most especially
preferably 1-2.
Reacting step (v) which is the alkylation step may be carried out either at room
temperature or at higher temperatures (typically 90-1 30°C). In a prefen-ed embodiment,
the compound of Formula III from step (iii) is used directly in the alkylation step (v).
That is, no purification step is carried out on the crude reaction product of step (iii)
before carrying out step (v), which makes the method relatively simple and therefore
even more amenable to automation. It is also envisaged that reacting step (v) can be
followed by a purification step to obtain substantially pure compound of Formula I .
Examples of suitable purification methods are solid-phase extraction (SPE) and highperformance
liquid chromatography (HPLC).
An additional advantage of the present method over known methods is that purification
of the compound of Formula I can be made easier by avoiding generation in the
presence of acetonitrile of acetyl impurities. For example, the present inventors found
that in the synthesis of 3-(2-chloro-5-((2-[ 1 F]fluoroethyl)thio)phenyl)-l-methyl-l-(3-
(methylthio)phenyl)guanidine from 3-(2-chloro-5-((2-hydroxyethyl)thio)phenyl)-l -
methyl- 1-(3-(methyl thio)phenyl)guanidine, an acetyl impurity was formed that proved
difficult to separate. The scheme below illustrates the proposed mechanism by which
this impurity is formed:
Use of an alkanoi i place o acetonitriie i the [ F] fluoroalkylation step avoids the
production of this acetyl impurity.
The method reported by Robins et al (2010 Bioorg Med Chem Letts; 20: 1749-51) for
the synthesis of 18F-labelled .V-fluoroalkyl diarylguanidines comprises
[, F]fluoroalkylation of a thiol group. The method can be readily adapted to be a
method of the present invention.
Firstly, the [ F] fluoroalkylation step (ii) is carried out in an aqueous alkanoi rather
than acetonitrile. Also, there is no requirement to purify the [1 F]fluoroethyltosylate in
order to avoid the acetyl impurity. In addition, an aliquot of the solution the eluent of
2CO3 and Kryptofix 222 where the complex K(Kryptofix)2C0 3 is used to make
"reactive" [1 F][K(Kryptofix)]F for use in step (i) is used in place of CS2CO3 in step (ii)
where the complex K(Kryptofix)2C0 3 is used as a base.
Accordingly, an example of a preferred BTM of formula R'-A-H in the method of the
present invention is a compound of Formula la:
wherein A is as defined in Claim 1, and.
Ra is selected from hydrogen or C alkyl;
R is halo;
R is selected from halo, C alkylthio, or C 1-4 alkyl; and,
P and P are independently hydrogen or an amine protecting group, preferably
hydrogen.
Preferably, said BTM of Formula a is a compound of Formula lb:
wherein A, Ra , Pa and P are as defined for Formula la.
Ra is preferably C alkyl and most preferably methyl.
R is preferably chloro.
R is preferably alkylthio, and most preferably methylthio.
A is preferably S.
For any particular compound of Formula la or Formula lb:
R is C1-4 alkyl and is most preferably methyl;
R group is chloro;
R group is alkylthio, and is most preferably methylthio;
A is S.
A particularly preferred compound of Formula lb is the following compound:
The above-defined compounds of Formulae la and lb 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 skilled person will appreciate that the method of the present invention may be
applied to the preparation of a range of F-labelled compounds. For instance, a nonlimiting
example of a known method that can be adapted in a straightforward manner to
result in a method of the present invention is the method comprising
[ 1 F]fluoroalkylation of a phenol described by Wang et al (2006 J Radioanalyt Nuc
Chem; 270(2): 439-43) to obtain the 1 F-labeled amino acid 0-(2-[ F]fluoroethyl)-Ltyrosine
([ 1 F]FET):
[ 1 F]Fluoroethyl 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 2C O and Kryptofix
222 (rather than NaOH) previously used in the method to make reactive
[, F][K(Kryptofix)]F for use in step (i).
Accordingly, another example of a preferred BTM of formula R ' -A-H in the method of
the present invention is the following compound.
Another non-limiting example of a known method that may be easily adapted is the
method described by Lundkvist et al (1997 Nuc Med Biol; 24: 621-7) for the synthesis
of [ F]fluoropropyl-p-CIT (b-CIT: (-)-2p-Carbomethoxy -3P-(4-iodophenyl)tropane) a
secondary amine is alkylated using [ FJfluoropropyl bromide:
This method can be readily converted to be a method of the present invention by
carrying out step (ii) in an aqueous alkanol solution using an aliquot of K2CO3 and
Kryptofix 222 (with acetonitrile removed) used to make reactive [ 1 F][K(Kryptofix)]F
for use in step (i).
Accordingly, another example of a preferred BTM of formula R'-A-H in the method of
the present invention 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 [ F]fluoroalkyl labelling
reagent using [1 F]fluoride as the source of 1 F, and (ii) [ F]fluoroalkylation of a thiol,
hydroxy or amine functionality in a precursor compound.
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, and also that
it minimises the number of reagent vials used since the eluent reagent vial would be
used twice once as phase transfer catalyst and once as a base.
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® (GBA) . 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 solidphase
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 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 eluent as defined in step (ii) of Claim 1;
(ii) a second vessel containing a compound of Formula II as defined in step (iii)
of Claim 1;
(iii) a third vessel containing a compound of Formula rV as defined in step (iv)
of Claim 1;
(iv) a fourth vessel in which reacting steps (iii) and (v) as defined in Claim 1are
carried out; and,
(v) an ion-exchange cartridge for trapping [18F]fluoride.
Any indications for the cassette of the present invention that have been defined above
for the method of the present invention are as suitably and preferably defined herein for
the method of the invention.
The term "vessel" is taken to mean a reagent vial suitable for placing in a position on a
cassette to be used with an automated synthesis cartridge.
Depending on the stability of the compound of Formula II and of the compound of
Formula IV, either of the vials containing these compounds may optionally be provided
separately to the cassette in order to be stored, e.g. under refrigeration or frozen, until
use for carrying out the method of the invention when the vial is brought to room
temperature and then included in the cassette. The compounds of Formulae II and IV
may each be provided in their respective vial either in solution or in dried, e.g.
lyophilised, form to be reconstituted before use with the appropriate solvent set out
above for the method of the invention.
Additional vessels may be present specific to the chemistry/BTM 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 if HPLC 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.
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-
[ F]fluoroethyl)thio)phenyl)-l-methyl-l-(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
K222 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 -rF]fluoroethyl)thio)phenyl)-
l-methyl-l-(3-(methylthio)phenyl)guanidine
A cassette for use with a FASTlab synthesiser comprised the following vials:
*3-(2-chloro-5-((2-hydroxyethyl)thio)phenyl)-l -methyl- l -(3-(methylthio)phenyl)guanidine
The cassette is also illustrated in Figure 1.
Hi) 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 [' 8F]fluoride off the QMA
70m of the eluent vial (K222, K2CO3) was removed from the eluent vial using the lmL
syringe. 550m of water was then withdrawn from the water bag and added to the eluent
in the lmL syringe. The [ F]fluoride trapped on the QMA cartridge was then eluted
into the reaction vessel using the eluent/water solution in the 1mL syringe and a vacuum
applied to the reaction vessel to draw the solution through the QMA cartridge.
l(iv) Drying f Flfluoride
The [1 F]fluoride and eluent solution was dried for 20 minutes by heating (100°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 18F]-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 [1 F] fluoride/K222/K 2C0 3 (reactive [1 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 f F]'-fluoroethyltosylate
The crude [I 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 500uL of eluent to precursor vial
500m of eluent vial (K222, 2C0 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 (26miho1) of precursor (3-(2-chloro-5-((2-hydroxyethyl)thio)phenyl)-l -methyl- -
(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) Loop flush 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 0.1M 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 transferred 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:
0-60 mins 40%(B)
Column ACE CI 100c 10 h h 5m
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 ImL ethanol from the vial using
the right hand 5mL syringe into a vial containing 4mL saline containing 1.5mg
ascorbic acid.
In summary, the following were observed.
Example 2: Comparison of FASTlab Synthesis of 3-(2-chloro-5-((2-
f' Flfluoroethyl)thio)phenyl)-l-methyl-l-(3-(methylthio)phenyl)guanidine using
Ethanol or Acetonitrile as the solvent
The process described in Example 1 was carried out up to step l(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.ImL)
Mobile phase B: MeCN
0-1 mm 40%B; 1-25 min 40 -95%B
HPLC column: Luna CI8 (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
A method for the synthesis of a compound of Formula I :
R 1
[I]
or a salt or 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 NR 2 wherein R 2 is hydrogen,
C alkyl, or C5-i2 aryl; and,
n is an integer of 1-6;
wherein said method comprises:
(i) providing [ F]Fluoride trapped on an ion-exchange cartridge;
(ii) eluting the ion-exchange cartridge of step (i) with an aqueous solution
comprising a first aliquot of an eluent, wherein said eluent comprises a
cationic counterion in a suitable solvent, to obtain a [18F]Fluoride eluent;
(iii) reacting a compound of Formula II:
[II]
wherein LG1 and LG2 are the same or different and each represent a leaving
group, and n is as defined for Formula I ;
in a first solvent with the [ F]Fluoride eluent obtained in step (ii) to obtain
a compound of Formula III:
F LG2 [HI]
wherein LG2 and n are as defined for Formula II;
(iv) deprotonating a compound of Formula IVH
R
[IV]
or a protected version thereof, wherein A and R1 are as defined for Formula
I;
by addition of a second aliquot of the eluent as defined in step (ii);
(v) reacting the compound of Formula III obtained in step (iii) with said
deprotonated compound obtained in step (iv), or a protected version
thereof, in a second solvent to obtain said compound of Formula I, or a
protected version thereof, wherein said second solvent is an alkanol or an
aqueous alkanol;
(vi) removing any protecting groups.
(2) The method as defined in Claim 1 wherein said ion-exchange cartridge is an anion
exchange cartridge.
(3) The method as defined in Claim 2 wherein said anion exchange cartridge is a
quaternary methylammonium (QMA) cartridge.
(4) The method as defined any one of Claims 1-3 wherein said cationic counterion is
a metal complex of a cryptand.
(5) The method as defined in Claim 5 wherein said metal complex of a cryptand is a
potassium salt of Kxyptofix 222.
(6) The method as defined in any one of Claims 1-5 wherein LG1 and LG2 of Formula
II are independently selected from halo or an aryl or alkyl sulphonate.
(7) The method as defined in Claim 6 wherein LG and LG are independently a halo
selected from chloro, iodo and bromo.
(8) The method as defined in Claim 6 wherein LG1 and LG2 are independently an aryl
or alkyl sulphonate selected from tosylate, triflate and mesylate.
(9) The method as defined in any one of Claims 1-8 wherein said first solvent is an
alkyl nitrile.
(10) The method as defined in Claim 9 wherein said alkyl nitrile is acetonitrile.
( 1 ) The method as defined in any one of Claims 1-10 wherein said alkanol is ethanol.
(12) The method as defined in any one of Claims 1-1 1 which is automated.
( ) The method as defined in any one of claims 1-12 wherein said BTM is an enzyme
substrate, enzyme antagonist, enzyme agonist, enzyme inhibitor or receptor-binding
compound.
(14) The method as defined in Claim 13 wherein said BTM is a receptor-binding
compound.
(15) The method as defined in either Claim 3 or Claim 14 wherem said BTM is a nonpeptide.
(16) The method as defined in any one of Claims 13-15 wherein said BTM is synthetic.
(17) The method as defined in any one of Claims 1-16 wherein said BTM is a compound
of Formula la:
wherein A is as defined Claim 1, and:
Ra is selected from hydrogen or C alkyl;
R is halo;
R is selected from halo, C -4 alkylthio, or C alkyl; and,
P and P are independently hydrogen or an amine protecting group.
(18) The method as defined in Claim 17 wherein BTM is a compound of Formula lb:
wherein A, R , P and P are as defined for Formula la.
The method as defined in any one of Claims 1-16 wherein said BTM
following compound:
(20) The method as defined in any one of Claims 1-16 wherein said BTM
following compound:
A cassette for carrying out the method as defined in Claim 1 comprising:
(i) a first vessel containing eluent as defined in step (ii) of Claim 1;
(ii) a second vessel containing a compound of Formula II as defined in step (
of Claim 1:
(iii) a third vessel containing a compound of Formula IV as defined in step (iv)
of Claim 1;
(iv) a fourth vessel in which reacting steps (iii) and (v) as defined in Claim 1are
carried out; and,
(v) an ion-exchange cartridge for trapping [ F]fluoride.