Radio labelling Method.
Field of the Invention.
The present invention relates to the field of radiopharmaceuticals for in vivo imaging,
in particular to automated methods for the preparation and purification of 1 F-labelled
tau imaging radiotracers. Also provided are interchangeable cassettes useful in the
methods, and the use of automated synthesizers and cassettes in the methods.
Background to the Invention.
Tau is a phosphoprotein having a physiological function of binding to tubulin to
stabilise microtubules. The degree of tau phosphorylation determines the binding
affinity to microtubules - tau hyperphosphorylation leads to weaker microtubule
binding. There is growing evidence that tau malfunction is implicated in, or triggers
neurodegeneration and dementia. There is therefore significant interest in the
molecular imaging of tau in vivo.
EP 1574500 A l (BF Research Institute Inc.) discloses diagnostic probes for Tau
proteins which comprise optionally radio labelled compounds of structure:
wherein:
Ri, R2, and R3 independently are H, Hal, OH, COOH, S0 3H, NH2, N0 2, CONH-
NH 2, Ci_4 alkyl or 0-Ci_ 4 alkyl, wherein two Ri groups together, may form
a benzene ring;
R 4 and R are independently H or Ci_4 alkyl; and
m and n are independently integers of value 0 to 4.
WO 2012/067863 discloses that quino lines can be radiolabelled with radioisotopes
suitable for PET or SPECT imaging to provide Tau imaging agents. WO
2012/067863 mentions that automated methods optionally including cassettes
used, but does not describe particular precursors, methods or cassettes.
WO 2012/057312 Al discloses Tau imaging radiotracers which are radiolabelled
compounds of Formula (I :
(I)
wherein
A is
R is Hal, a -C(=0)-lower alkyl group (said alkyl group may be each independently
substituted with one or more substituents selected from the group consisting of
NRaR , Hal, and OH), a lower alkyl group (said alkyl group may be each
independently substituted with one or more substituents selected from the group
consisting of Hal and OH), an -O-lower alkyl group (said alkyl group may be each
independently substituted with one or more substituents selected from the group
consisting of Hal and OH), or
, R4
wherein
R4 and R5 are each independently H, a lower alkyl group, or a cycloalkyl
group, or R4, R5, and the nitrogen atom to which they are attached are together form a
3- to 8-membered nitrogen-containing aliphatic ring (one or more carbon atoms
constituting said nitrogen-containing aliphatic ring may be replaced by a N, S or O
atom, and when the carbon atom is replaced by a N atom, said N atom may be
substituted with a lower alkyl group), or
R4 and the nitrogen atom to which it is attached, together with the ring A, form
an 8- to 16-membered nitrogen-containing fused bicyclic ring system (one or more
carbon atoms constituting said nitrogen-containing fused bicyclic ring system may be
replaced by a N, S or O atom, and when the carbon atom is replaced by a nitrogen
atom, said nitrogen atom may be substituted with a lower alkyl group), and R5 is H, a
lower alkyl group, or a cycloalkyl group,
where a solid line intersected with a broken line designates a linkage with
another structural portion in the general formulae above,
R2 or R3 is each independently Hal, OH, COOH, S0 3H, N0 2, SH, NRaR , a
lower alkyl group (said alkyl group may be each independently substituted with one
or more substituents selected from the group consisting of Hal and OH), or an -Olower
alkyl group (said alkyl group may be each independently substituted with one
or more substituents selected from the group consisting of Hal and OH),
the ring A is unsubstituted or substituted with R6 (wherein R6 is one or more
substituents independently selected from the group consisting of Hal, OH, COOH,
S0 H, N0 2, SH, NRaR , a lower alkyl group (said alkyl group may be each
independently substituted with one or more substituents selected from the group
consisting of Hal and OH), and an -O-lower alkyl group (said alkyl group may be
each independently substituted with one or more substituents selected from the group
consisting of Hal, OH, and an -O-lower alkyl group-O-lower alkyl group (said alkyl
group may be each independently substituted with Hal))),
Ra and R are independently H or a lower alkyl group (said alkyl group may be
each independently substituted with one or more substituents selected from the group
consisting of Hal and OH),
m is an integer from 0 to 4, and
n is an integer from 0 to 4.
WO 2012/057312 Al teaches that the 1 F-radiotracers are purified using a
combination of Sep-Pak cartridges followed by semi-preparative HPLC.
Okamura et al [J.Nucl.Med., 54(8), 1420-1427 (2013)] disclose that 1 Farylquinolines,
in particular 1 F-THK-5105 and 1 F-THK-51 17 are novel imaging
agents for imaging tau pathology in Alzheimer's disease. Okamura et al use the
following precursors and radiofluorination method:
precursor
R = R = CH3 THK-5105
R1 = H, R2 = CH3 THK-5 117.
Okamura et al use a manual radiolabelling reaction, plus semi-preparative HPLC for
purification of the radiotracer.
Blom et al [J.Radioanal.Nucl.Chem., 299, 265-270 (2014)] teach that the radiotracer
[1 F]-FMISO
[1 F]-FMISO, which also incorporates a fluorohydroxypropyl group, can be prepared
via an automated radiosynthesis. Blom et al studied various solid-phase extraction
(SPE) columns together with the chemical and radiochemical impurities, and
concluded that a hydrophilic-lipophilic balanced (HLB), polymer-based cartridge was
superior to both a mixed mode (MCX) cartridge and a Sep-Pak CI8 cartridge.
There is therefore still a need for alternative and/or improved methods of preparing
and purifying the tau imaging agents ofWO 2012/057312 Al and Okamura et al.
The Present Invention.
The precursor synthesis method of the present invention provides an automated
synthesis of quinoline-based [1 F]-labelled tau radiotracers. The automated method
includes an automated purification methodology, which uses only solid-phase
extraction (SPE) avoids the need for HPLC as taught by the prior art. The purification
method is thus fast (ensuring minimal loss of tracer due to radioactive decay), and
reproducible. The purification method has also been adapted to work effectively
across the wide range of operating temperatures (ca. 15-37 °C) that may be found in
practice in hot cells where radiosynthesizer apparatus is located.
The method comprises the use of an interchangeable, single-use cassette which is
adapted to make the radiosynthesis even more convenient for the operator, since
minimal operator intervention is required. The cassette approach also has the
advantages of: simplified set-up hence reduced risk of operator error; improved GMP
(Good Manufacturing Practice) compliance; multi-tracer capability; rapid change
between production runs; pre-run automated diagnostic checking of the cassette and
reagents; automated barcode cross-check of chemical reagents vs the synthesis to be
carried out; reagent traceability; single-use and hence no risk of cross-contamination,
as well as being tamper and abuse resistance.
Detailed Description of the Invention.
In a first aspect, the present invention provides an automated method of preparation of
an 1 F-labelled radiotracer of Formula (II), which comprises:
(i) provision of an automated synthesizer apparatus which comprises a
microprocessor, and an interchangeable, disposable cassette which comprises
a reaction vessel, a supply of solvents suitable for the preparation and
purification of said radiotracer, and a supply of the precursor of Formula (I):
(ii) microprocessor-controlled transfer of said precursor of Formula (I) from
step (i) to said reaction vessel, followed by reaction of said precursor with
[1 F]-fluoride in a suitable solvent, and removal of the Pg1 protecting group, to
give the 1 F-labelled radiotracer of Formula (II):
(II)
wherein:
A is chosen from:
X1 and X2 are independently an Xa or an Xb group;
X3 is an Xa or an X group;
Xa is -NR R2;
X is
R1 and R2 independently comprise H or Ci_4 alkyl, or R1 and R2 together with
the N atom and optionally the phenyl ring to which they are attached comprise
a 5- or 6- membered nitrogen-containing aliphatic or heteroaromatic ring,
optionally incorporating one further heteroatom chosen from -O- , -S- , =Nand
-NR a-, where Ra is H or Ci_4 alkyl;
R is Ci_4 alkyl, Ci_4 haloalkyl, C - aryl or C -i2 aralkyl;
Pg1 is an alcohol protecting group;
provided that in Formula (I), one X group is present, and in Formula (II), one X
group is present.
Thus, in the method of the first aspect, the X group of the precursor of Formula (I)
contains a reactive site (sulfonate ester group), which undergoes nucleophilic
radiofluorination with [1 F]-fluoride ion in step (ii) to give the corresponding X
substituent of the radiotracer product of Formula (II). The microprocessor control of
step (ii) is achieved via the microprocessor of said automated synthesizer apparatus.
The provisos of one X or X group being present imply that:
in Formula (I), one of X1 and X2 is an Xa group and the other is an X group;
in Formula (II), one of X1 and X3 is an Xa group and the other is an X group;
The term "radiotracer" has its' conventional meaning and refers to a
radiopharmaceutical used to trace a physiological or biological process without
affecting it. The term "radiopharmaceutical" has its' conventional meaning and refers
to a radiolabelled compound administered to the mammalian body in vivo for the
purpose of imaging or therapy.
By the term "automated synthesizer" is meant an automated module based on the
principle of unit operations as described by Satyamurthy et al [Clin.Positr.Imag., 2(5),
233-253 (1999)]. The term 'unit operations' means that complex processes are
reduced to a series of simple operations or reactions, which can be applied to a range
of materials. Such automated synthesizers are preferred for the method of the present
invention especially when a radiopharmaceutical composition is desired. They are
commercially available from a range of suppliers [Satyamurthy et al, above],
including: GE Healthcare; CTI Inc; Ion Beam Applications S.A. (Chemin du
Cyclotron 3, B-1348 Louvain-La-Neuve, Belgium); Raytest (Germany) and Bioscan
(USA).
Commercial automated synthesizers also provide suitable containers for the liquid
radioactive waste generated as a result of the radiopharmaceutical preparation.
Automated synthesizers are not typically provided with radiation shielding, since they
are designed to be employed in a suitably configured radioactive work cell. The
radioactive work cell provides suitable radiation shielding to protect the operator from
potential radiation dose, as well as ventilation to remove chemical and/or radioactive
vapours. The automated synthesizer preferably comprises a cassette. The automated
synthesizer comprises a microprocessor, which controls the operation of the
synthesizer apparatus, including the operation of any associated cassette.
By the term "cassette" is meant a unit piece of apparatus designed such that the whole
unit fits removably and interchangeably onto an automated synthesizer apparatus (as
defined above), in such a way that mechanical movement of moving parts of the
synthesizer controls the operation of the cassette from outside the cassette, i.e.
externally. Suitable cassettes comprise a linear array of valves, each linked to a port
where reagents or vials can be attached, by either needle puncture of an inverted
septum-sealed vial, or by gas-tight, marrying joints. Each valve has a male-female
joint which interfaces with a corresponding moving arm of the automated synthesizer.
External rotation of the arm thus controls the opening or closing of the valve when the
cassette is attached to the automated synthesizer. Additional moving parts of the
automated synthesizer are designed to clip onto syringe plunger tips, and thus raise or
depress syringe barrels.
The cassette is versatile, typically having several positions where reagents can be
attached, and several suitable for attachment of syringe vials of reagents or
chromatography cartridges (e.g. solid phase extraction or SPE). The cassette always
comprises a reaction vessel. Such reaction vessels are preferably 1 to 10 cm3, most
preferably 2 to 5 cm3 in volume and are configured such that 3 or more ports of the
cassette are connected thereto, to permit transfer of reagents or solvents from various
ports on the cassette. Preferably the cassette has 15 to 40 valves in a linear array,
most preferably 20 to 30, with 25 being especially preferred. The valves of the
cassette are preferably each identical, and most preferably are 3-way valves. The
cassettes are designed to be suitable for radiopharmaceutical manufacture and are
therefore manufactured from materials which are of pharmaceutical grade and ideally
also are resistant to radiolysis.
Preferred automated synthesizers of the present invention comprise a disposable or
single-use cassette which comprises all the reagents, reaction vessels and apparatus
necessary to carry out the preparation of a given batch of radiofluorinated
radiopharmaceutical. The cassette means that the automated synthesizer has the
flexibility to be capable of making a variety of different radiopharmaceuticals with
minimal risk of cross-contamination, by simply changing the cassette. The cassette
approach also has the advantages of: simplified set-up hence reduced risk of operator
error; improved GMP (Good Manufacturing Practice) compliance; multi-tracer
capability; rapid change between production runs; pre-run automated diagnostic
checking of the cassette and reagents; automated barcode cross-check of chemical
reagents vs the synthesis to be carried out; reagent traceability; single-use and hence
no risk of cross-contamination, as well as being tamper and abuse resistance.
By the term "precursor" refers to a 'radio labelling precursor' which means a non
radioactive compound suitable for reaction with a supply of a radioisotope in a
suitable solvent, to give the radiolabeled compound of interest in the minimum
numbers of steps. Thus, the precursor is designed such that the chemical and
radioactive yield is optimised, and the number of steps involving the handling of
radioactivity is minimized. The precursor is particularly suitable for radiolabelling
with 1 F.
By the term "protecting group" is meant a removable group which inhibits or
suppresses undesirable chemical reactions, and which is designed such that it can be
both attached and removed to/from the functional group in question under mild
enough conditions that do not modify or compromise the rest of the molecule. After
deprotection the desired product is obtained. The use of protecting groups is
described in Protective Groups in Organic Synthesis, 4th Edition, Theorodora W.
Greene and Peter G. M. Wuts, [Wiley Blackwell, (2006)]. The term "deprotection"
has its conventional meaning in the field of chemistry and/or radiochemistry, i.e. the
removal of a protecting group.
The alcohol protecting group (Pg1) of the first aspect protects the secondary alcohol
group of the X group. Suitable Pg1 groups include ethers (alkyl, aryl, aralkyl, or
silyl); esters or carbonates. Further details of alcohol protecting groups are provided
by Greene and Wuts (cited above).
When Pv 1 and R2 together with the N atom and optionally the phenyl ring to which
they are attached comprise a 5- or 6- membered nitrogen-containing aliphatic or
heteroaromatic ring, that means that the 5- or 6- membered ring incorporating one or
more of N , R 1 and R2 may either be a substituent on the phenyl ring, or be fused with
the phenyl ring bearing -NR'R . Examples of the former would be piperidine or
morpholine rings singly bonded to the phenyl ring. A preferred example of a fused
ring is when Xa is:
The X group incorporates a sulfonate ester group -OS0 2R . Such sulfonate esters
are important leaving groups in nucleophilic substitution, and the reactivity of the
sulfonate ester towards nucleophilic substitution can be adjusted depending on the
choice of R [M.B.Smith and J . March, March 's Advanced Organic Chemistry, Fifth
Edition, John Wiley & Sons Inc., (2001), pages 445-449].
The "suitable solvent" for step (ii), includes: acetonitrile, a Ci_4 alkyl alcohol,
dimethylformamide, tetrahydrofuran, or dimethylsulfoxide, or aqueous mixtures of
any thereof.
Preferred aspects.
In the method of the first aspect, step (ii) is preferably carried out by:
(a) reaction of the precursor of Formula (I) with [1 F]-fluoride in a suitable
solvent, to give an 1 F-labelled intermediate of Formula (III):
(III)
wherein
A is chosen from:
X4 and X5 are each independently an Xa or Xd group;
where Xd is:
provided that, in Formula (III) one Xd group is present;
then:-
(b) removal of the Pg1 protecting group from said intermediate to give the 1 Flabelled
radiotracer of Formula (II).
In the method of the first aspect, X2 is preferably X , such that the precursor is of
Formula (IA):
and the radiotracer product is of Formula IIA:
where A2 is chosen from:
In the method of the first aspect, the precursor is more preferably the ^-enantiomeric
form of Formula IB) :
(IIB).
The precursor may be enriched in said ^-enantiomeric form, to exceed the 50:50
content of the racemic mixture, and is preferably in substantially pure form.
In the method of the first aspect, A in Formulae (I), (IA), (IB), (II), (IIA), (IIB) and
(III) is preferably an A2 group of formula:
wherein - R is more preferably -NHCH 3 or -N(CH 3)2 and most preferably
-NHCH 3.
In the method of the first aspect, Pg1 is preferably a Pgla group, wherein Pgla
comprises:
(i) - c;
(i -Ar1;
(iii) -CH(Ar 1)2;
(v) tetrahydropyranyl optionally substituted with one or more substituents
chosen from Hal and OCH 3;
(vi) -CH 2OR ;
(vii) -SiRd
3;
(viii) -(C=0)R d;
(ix) -(C=0)OR wherein R is H, Rd, Ci_4 haloalkyl or vinyl; or
(x) -(C=0)NHR d;
wherein:
each R is independently Rd or C2_4 alkoxyalkyl optionally substituted with
one or more Hal;
each R is independently Ci_4 alkyl;
each Rd is independently R or Ar1; and
Ar 1 is independently benzyl or phenyl optionally substituted with one or more
substituents chosen from Hal, CH3, OCH3, N0 2 or -N(CH 3)2.
Pg1 is most preferably tetrahydropyranyl.
In the method of the first aspect, R is preferably chosen from: -CH3, -CF3, -C4F ,
-CH2CF3, -C6H4-CH3, -C6H4-N0 2 or -C6H4-Br. R3 is more preferably -C6H4-CH3.
In the method of the first aspect, the cassette preferably further comprises one to three
C18-reverse phase solid phase extraction (SPE) columns, and said method further
comprises step (iii):
(iii) microprocessor-controlled SPE purification of the 1 F-labelled radiotracer
of Formula (II) from step (ii) using said cassette SPE columns, and the
solvent(s) of said cassette.
The use of SPE avoids the need for HPLC purification, which is typically carried out
manually, and thus means that the radiosynthesis and purification of the radiotracer
can be carried out in a fully-automated manner using an appropriate cassette. Thus,
the purification method of the present invention preferably does not comprise HPLC.
In the SPE purification of the first aspect, the C18-reverse phase SPE column is
preferably silica-based, and is thus preferably a C18-silica SPE column and is more
preferably a tC18+ silica SPE column. Polymer-based SPE cartridges are less
preferred, since HLB type cartridges have been found to bind the radiotracers of the
present invention so strongly that elution becomes difficult. Reverse phase SPE
cartridges suitable for use in the present invention can be obtained from Waters
Limited (730-740 Centennial Court, Centennial Park, Elstree, Hertfordshire, UK). A
suitable size of SPE column for use in the present invention is 900 mg.
It is well-established that chromatography such as the SPE purification process is
subject to variations depending on the ambient temperature. So-called "hot cells" are
used for the production of PET radiotracers. These are enclosures with the necessary
facilities to carry out the radiosynthesis, but also having radiation-shielding and
suitable ventilation to protect the operator. Such hot cells range from large units that
are able to maintain room temperature (18 °C-22 °C) despite the large amount of
electrical equipment contained within them, to very small units that can reach
operating temperatures of 30 °C-40 °C. The present inventors have found that (see
Example 2), elevated temperatures affect the SPE purification such that the
radiotracer product elutes more quickly. As a result, satisfactory purification of the
radiotracer of Formula (II) can be achieved at temperatures ranges of 15-25 °C using
two 900 mg size SPE columns. At higher temperatures, however, of 15 to 40 °C three
SPE columns are necessary. Hence, the cassette and SPE method of the first aspect
preferably comprises the use of three 900 mg size SPE columns, since that permits
effective purification across the range of operating temperatures (ca. 15 to 40 °C)
likely to be found in radiosynthesis hot cells. Whilst it is possible that a smaller
number of larger SPE columns could be used, such larger columns are less likely to be
of a size compatible with automated synthesizer apparatus.
In the SPE purification of the first aspect, the C18-reverse phase SPE column is eluted
with an elution volume in the range 9 -12 mL, preferably 10.5 to 11.5 mL. In the SPE
purification method of the first aspect, the SPE columns are first eluted with an
aqueous, water-miscible organic solvent to remove impurities, and then eluted with
ethanol to elute the radiotracer of Formula (II). The "aqueous, water-miscible organic
solvent" refers to a mixture of water and the water-miscible organic solvent. Suitable
such organic solvents include acetonitrile, ethanol, THF, isopropanol and methanol,
and are preferably chosen from: acetonitrile, ethanol and THF, more preferably
acetonitrile and ethanol, most preferably acetonitrile. The aqueous acetonitrile
solvent, i.e. the acetonitrile/water solvent mixture is suitably in the range 20 to 50 %
v/v, and is preferably in the range 25 to 45%, more preferably in the range 35 to 40 %.
40% aqueous acetonitrile is most preferred.
By way of illustration of the SPE purification, the following discussion refers to
Compound 1 and Precursor 1 (see Scheme 1) - but the same principles apply for other
compounds within the scope of the first aspect.
Scheme 1.
Compound 1
Under the reaction conditions, there is a significant chemical excess of Precursor 1
over the chemical amount of [1 F]-fluoride present. Under the reaction conditions,
Precursor 1 also reacts and at least a portion thereof is converted primarily to the diol
(Impurity A; see structures below), and possibly some of Impurity B. The largest
impurity is Impurity A, which elutes and is removed when the SPE columns are eluted
with aqueous acetonitrile.
Precursor 1 is significantly more lipophilic than Compound 1, and remains bound to
SPE columns - when eluted with either aqueous acetonitrile or ethanol. Compound 1
does not elute when the SPE columns are washed with 10-12 mL of aqueous
acetonitrile, but is subsequently eluted when pure ethanol is used to elute the SPE
column(s). In this manner, Compound 1 is purified. Impurity B is observed less
frequently, but any present remains bound to the SPE column under the conditions of
the invention.
The method of the first aspect preferably further comprises, in addition to purification
step (iii), the following steps:
(iv) optionally diluting the purified [1 F]-radiotracer of Formula (II) from step (iii)
with a biocompatible carrier;
(v) aseptic filtration of the optionally diluted solution from step (iv) to give a
radiopharmaceutical composition comprising said radiotracer.
The "biocompatible carrier" is a fluid, especially a liquid, in which the radioconjugate
can be suspended or preferably dissolved, such that the composition is physiologically
tolerable, i.e. can be administered to the mammalian body without toxicity or undue
discomfort. The biocompatible carrier is suitably an injectable carrier liquid such as
sterile, pyrogen-free water for injection; an aqueous solution such as saline (which
may advantageously be balanced so that the final product for injection is isotonic); an
aqueous buffer solution comprising a biocompatible buffering agent (e.g. phosphate
buffer); an aqueous solution of one or more tonicity-adjusting substances (e.g. salts of
plasma cations with biocompatible counterions), sugars (e.g. glucose or sucrose),
sugar alcohols (e.g. sorbitol or mannitol), glycols (e.g. glycerol), or other non-ionic
polyol materials (e.g. polyethyleneglycols, propylene glycols and the like). Preferably
the biocompatible carrier is pyrogen-free water for injection, isotonic saline or
phosphate buffer.
The "radiopharmaceutical composition" is a pharmaceutical composition comprising
said radiotracer. Such compositions have their conventional meaning, and in
particular are in a form suitable for mammalian administration, especially via
parenteral injection. By the phrase "in a form suitable for mammalian administration"
is meant a composition which is sterile, pyrogen-free, lacks compounds which
produce toxic or adverse effects, and is formulated at a biocompatible pH
(approximately pH 4.0 to 10.5). Such compositions lack particulates which could risk
causing emboli in vivo, and are formulated so that precipitation does not occur on
contact with biological fluids (e.g. blood). Such compositions also contain only
biologically compatible excipients, and are preferably isotonic.
The production of [1 F]-fluoride suitable for radiopharmaceutical applications is wellknown
in the art, and has been reviewed by Hjelstuen et al [Eur.J.Pharm.Biopharm.,
78(3), 307-313 (201 1)], and Jacobson et al [Curr.Top.Med.Chem., 10(1 1), 1048-1059
(2010)].
A non-automated radiosynthesis of Compound 1 has been reported by Okamura et al
[J.Nucl.Med., 54(8) . 1420-1427 (2013)].
Substituted quinolones of Formula (I) can be synthesized by conventional quinoline
syntheses [Kouznetsov et al, Curr.Org.Chem., 9, 141-161 (2005)]. The syntheses of
several 2-arylquino lines has been provided by Tago et al [J.Lab.Comp.Radiopharm.,
57(1), 18-24 (2014)]. Further details of the precursor syntheses are provided in WO
2012/057312 Al. Thus, WO 2012/057312 Al discloses the following synthesis of the
1 F labelling precursors having alkoxy substituents at the 6-position functionalised
with hydroxy and 1 F groups:
where: Boc = fert-buryloxycarbonyl;
TBS = fert-butyldimethylsilyl
THP = tetrahydropyran;
Ts = 4-toluenesulfonyl.
The present supporting Examples provide further experimental details. The
corresponding enantiomers can be obtained by adapting the synthesis using chiral
starting materials, or resolution of the racemic mixture using e.g. chiral
chromatography or crystallisation of a chiral salt as is known in the art.
In a second aspect, the present invention provides a method of purification of the 1 Flabelled
radiotracer of Formula (II), (IIA) or (IIB) as defined in the first aspect, which
comprises the SPE purification method as described in a preferred embodiment of the
first aspect.
Preferred aspects of the radiotracer, precursor and purification method in the second
aspect, are as described in the first aspect (above).
In a third aspect, the present invention provides a cassette as described in the first
aspect (above). Preferred aspects of the cassette in the third aspect are as described in
the first aspect (above).
In a fourth aspect, the present invention provides the use of the automated synthesizer
apparatus as defined in the first aspect, to carry out the method of preparation of the
first aspect, or the method of purification of the second aspect. Preferred aspects of
the automated synthesizer apparatus and method in the fourth aspect, are as described
in the first aspect (above).
In a fifth aspect, the present invention provides the use of the cassette of the third
aspect, to carry out the method of preparation of the first aspect, or the method of
purification of the second aspect. Preferred aspects of the cassette in the fifth aspect
are as described in the third aspect (above).
Brief Description of the Figures.
Figure 1 and Figure 2 illustrate exemplary cassettes of the invention useful for
carrying out particular examples of the method of the invention.
Brief Description of the Examples.
The invention is illustrated by the non-limiting Examples detailed below. Example 1
provides the synthesis of a radiolabelling precursor of the invention ("Precursor 2").
Example 2 demonstrates the effect of elevated temperature on the radiosynthesis and
purification of Compound 1. Example 3 provides an improved synthesis and
purification of Compound 1, which is suitable for use at range of temperatures.
Compounds of the Invention.
Abbreviations.
Ac: Acetyl
Acm: Acetamidomethyl
ACN: Acetonitrile
AcOH: Acetic acid.
Boc: t rt-Butyloxycarbony1
tBu: t rt r -butyl
DCM: Dichloromethane
DIPEA: N,N -Diisopropylethyl amine
DMF: Dimethylformamide
EtOAc: ethyl acetate;
EtOH: ethanol
DMSO: Dimethylsulfoxide;
GMP: Good Manufacturing Practice;
HPLC: High performance liquid chromatography;
MeCH: acetonitrile
MW: molecular weight;
Ms: mesylate i.e. sulfonate ester of methanesulfonic acid.
RCP: radiochemical purity;
RCY: radiochemical yield;
RP-HPLC: reverse-phase high performance liquid chromatography;
SPE: solid phase extraction;
TBAF: tetrabutylammonium fluoride;
tBu: tert-butyl;
TFA: Trifiuoroacetic acid;
THF: Tetrahydrofuran;
THP: tetrahydropyranyl;
TLC: thin layer chromatography;
Trt: Trityl;
Tf: trifiate, i.e. sulfonate ester of trifluoromethanesulfonic acid.
Ts: tosylate, i.e. sulfonate ester of para-tolunesulfonic acid.
Example 1: Synthesis of Precursor 2.
Step (a): 2-(5-Fluoro-2-nitrophenyl)-l,3-dioxolane .
5-Fluoro-2-nitrobenzaldehyde (14.4 g, 85 mmol), ethane- 1,2-diol (14.48 mL, 260
mmol) and 4-toluenesulfonic acid monohydrate (0.826 g, 4.34 mmol) were added to
toluene (350 mL) and the mixture heated to reflux under nitrogen with a Dean & Stark
condenser. The reaction was allowed to cool after 4.5 h. After 30h, the solution was
decanted from the dark sticky residue at the bottom of the flask. Added EtOAc (275
mL) and washed with saturated aqueous sodium bicarbonate (70 mL), water (140
mL), brine (70 mL) and passed through a phase separator then evaporated to dryness
to give a dark brown oil (-18 g). This was dissolved in DCM:petrol (3:2) and purified
by chromatography on silica gel eluting with dichloromethane (A): Petroleum ether
(B) (60 %B, 340 g, 15 CV, 100 mL/min) to give the expected product as a yellow oil
(16.52 g, 91%).
1H NMR (400 MHz, ) d 8.10 - 7.95 (dd, J = 9.0, 4.9 Hz, 1H, Ar-H3), 7.58 - 7.44 (dd, J
= 9.1, 2.9 Hz, 1H, Ar-H4), 7.22 - 7.10 (ddd, J = 9.1, 7.2, 2.9 Hz, 1H, Ar-H6), 6.63 -
6.41 (s, 1H, OC(O)H) and 4.14 - 3.96 (dddd, J = 14.1, 8.6, 6.8, 3.3 Hz, 4H, 2 x CH2).
1 C NMR (101 MHz, ) d 164.8 (d, J = 259 Hz, C-F), 144.7 (C-NO2), 137.3 (d, J = 8
Hz, Ar-Cl), 127.7 (d, J = 9 Hz, Ar-C3), 116.5 (d, J = 25 Hz, Ar-C4/6), 115.1 (d, J =
25 Hz, Ar-C4/6), 99.1 (OCHO) and 65.5 (2 x CH2).
Step (b): 3-(l,3-Dioxolan-2-yl )-N-methyl-4-nitroaniline.
2-(5-Fluoro-2-nitrophenyl)-l,3-dioxolane [Step (a), 5.21 g, 24.44 mmol] was
dissolved in ethanol (37 ml) and methylamine (5.5 mL, 33wt% in ethanol, 46.9 mmol)
added. The yellow solution was stirred at ambient temperature for 10 minutes then
heated to reflux for 18 h, when LCMS and TLC (1:1 DCM:petrol) showed no
remaining starting material. The solution was allowed to cool and evaporated to
dryness, dissolved in DCM (100 mL) and washed with saturated aqueous sodium
bicarbonate (40 mL) then water (2 x 40 mL) and passed through a phase separator and
evaporated to a deep yellow-orange oil (5.45 g, 99%).
LCMS calcd for C10H12N2O4: 224.1; found 225.0 [M+H]+
1H NMR (400 MHz, CDCI3) d 8.05 (d, J = 9.0 Hz, 1H, Ar-5H), 6.92 (d, J = 2.7 Hz,
1H, Ar-2H), 6.62 (s, 1H, CH), 6.50 (dd, J = 9.1, 2.7 Hz, 1H, Ar-6H), 4.59 (br s, 1H,
NH), 4.06 (m, 4H, 2 x CH2) and 2.94 (d, J = 5.1 Hz, 3H, NCH3). 1 C NMR (101
MHz, CDCI3) d 153.4 (C-NH), 137.6 (C-NO2), 136.4 (Ar-3C), 128.7 (Ar-5C), 110.4
(Ar-6C), 109.8 (Ar-2C), 99.9 (CH), 65.3 (2 x CH2) and 30.2 (N-CH3).
Step c) 5-(Methylamino)-2-nitrobenzaldehyde.
3-(l,3-Dioxolan-2-yl )-N -methyl-4-nitroaniline [Step (b), 5.45 g, 24.31 mmol] was
dissolved in acetone (55 mL) and hydrochloric acid (IN) (2.00 g, 55 mmol) was
added and the yellow solution heated to 60 C for 3 h, when LCMS and TLC showed
no residual starting material. The solution was cooled and neutralised with aqueous
sodium bicarbonate and extracted into ethyl acetate (3 x 70 mL). The combined
organics were passed through a phase separator and evaporated to give a yellow solid
(4.28 g, 98%).
LCMS calcd for C H N20 3: 180.1; Found 180.92 [M+H]+
1H NMR (400 MHz, CDCI3) d 10.51 (s, 1H, HC=0), 8.10 (d, J = 9.1 Hz, 1H, Ar-3H),
6.85 (d, J = 2.8 Hz, 1H, Ar-6H), 6.68 (dd, J = 9.0, 2.8 Hz, 1H, Ar-4H), 4.83 (br s, 1H,
NH) and 2.98 (d, J = 5.1 Hz, 3H, N-CH3). 1 C NMR (101 MHz, CDCI3) d 190.2
(C=0), 153.8 (Ar-CNH), 137.9 (Ar-CN02), 135.6 (Ar-CCHO), 128.0 (Ar-3CH),
113.6 (Ar-4CH), 110.9 (Ar-6CH) and 30.3 (N-CH3).
Step d) 2-(4-Methoxyphenyl )-N -methylquinolin-6-amine.
5-(Methylamino)-2-nitrobenzaldehyde [Step (c), 1.39 g, 7.72 mmol] was dissolved in
ethanol (40 mL) in a 50 mL borosilicate tube and iron powder ( 1.72 g, 30.9 mmol)
and hydrochloric acid (3.86 mL, 0.1N, 0.386 mmol) added and the tube sealed with
PTFE/silicone screw-cap and heated in a pre-heated oil-bath at 100 C. After 2h, the
tube was removed and cooled in water and the pressure carefully released, when
LCMS showed no remaining starting material. Added l-(4-methoxyphenyl)ethanone
(1.16 g, 7.72 mmol) and powdered potassium hydroxide (0.52 g, 9.26 mmol) to the
mixture, resealed and heated at 100 C for 22h. Cooled, diluted with water (150 mL)
and extracted with DCM (4 x 50 mL), washed combined organics with water (50 mL)
and passed through a phase separator and evaporated to give a yellow-brown gum
( 1.94 g). This was purified by chromatography on silica gel eluting with petroleum
ether (A): ethyl acetate (B) (10-100 %B, 100 g, 15 CV, 85 mL/min) to give a pale
yellow solid (530 mg, 26% yield).
LCMS calcd for C17Hi6N20 264.1; found 265.0 [M+H]+
1H NMR (400 MHz, CDCI3) d 8.12 - 8.01 (m, 2H, Ph-H), 7.94 (d, J = 8.6 Hz, 1H, Ar-
H), 7.90 (d, J = 9.1 Hz, 1H, Ar-H) 7.68 (d, J = 8.6 Hz, 1H, Ar-H), 7.05 (dd, J = 9.0,
2.6 Hz, 1H, Ar-H), 7.01 (m, 2H, Ph-H), 6.66 (d, J = 2.5 Hz, 1H, Ar-H), 4.03 (br s, 1H,
NH), 3.58 (s, 3H, OCH3), 2.90 (s, 3H, NCH3). 1 C NMR (101 MHz, CDCI3) d 160.2
(C-OMe), 152.9 (Ar-C-N), 147.0 (C-NMe), 143.2 (Ar-CIO), 134.6 (Ar-C-4), 132.9
(Ph-Cl), 130.4 (Ar-C7), 128.8 (Ar-C9), 128.4 (Ph-C2&6), 121.4 (Ar-C8), 118.9 (Ar-
C3), 114.2 (Ph-C3&5), 102.5 (Ar-C5), 55.5 (O-CH3) and 30.8 (N-CH3).
Step e) 4-(6-(Methylamino)quinolin-2-yl)phenol.
2-(4-Methoxyphenyl)-N-methylquinolin-6-amine [Step (d), 680 mg, 2.57 mrnol] was
dissolved in DCM (35 mL) and boron tribromide (10.3 mL, 1M in DCM, 10.3 mmol)
was added and the mixture stirred for 18 h - some insoluble gum formed - when
LCMS showed mainly desired product with a little residual starting material. Added
methanol (2-3 mL dropwise) to destroy excess BBr3 and filtered off the yellow solid.
Stirred with saturated aqueous sodium bicarbonate and filtered. Allowed to dry on
filter paper to give the desired product as a yellow solid (602 mg, 93 %).
LCMS calcd for C1 H14N20 250.1; found 251.0 [M+H]+.
1H NMR (400 MHz, de-DMSO) d 7.96 (m, 3H), 7.77 (d, J = 8.7 Hz, 1H, C8-H), 7.65
(d, J = 9.0 Hz, 1H, C4-H), 7.1 1 (dd, J = 9.1, 2.0 Hz, 1H, C3-H), 6.81 (d, J = 8.5 Hz,
2H, C2'&6'-H), 6.59 (m, 1H, C5-H), 6.13 (d, J = 4.8 Hz, 1H, NH) and, 2.74(d, J =
4.8 Hz, 3H, N-CH3). 1 C NMR (101 MHz, d6-DMSO) d 159.3 (C-OH), 151.6 (C6-N),
148.0 (C9), 142.4 (C4'), 134.6 (C4-H), 129.9 (C7-H), 129.0 (CIO), 128.3 (C3-H'&
C5'-H), 122.1 (C8-H), 118.4 (C3-H), 116.1 (C2'-H & C6'-H), 101.2 (C5-H) and 30.3
Step (f) 3-(4-(6-(Methylamino)quinolin-2-yl)phenoxy)-2-((tetrahydro-2H-pyran-2-
yl)oxy)propyl 4-methylbenzenesulfonate .
4-(6-(Methylamino)quinolin-2-yl)phenol [Step (e), 300 mg, 1.2 mmol] and potassium
carbonate (215 mg, 1.56 mmol) were mixed in a 25 mL rb flask fitted with a rubber
septum and a nitrogen balloon. Dry DMF (10 mL) was added followed by 2-
((tetrahydro-2H-pyran-2-yl)oxy)propane-l,3-diyl z's(4-methylbenzenesulfonate) (581
mg, 1.2 mmol) [Oh et al, Nucl.Med.Biol, 32(8), 899-905 (2005)], and the mixture
stirred vigorously and heated at an external temp of 90 C. Cooled after 22 h, when
TLC showed incomplete reaction. Nevertheless, ice water (30 mL) was added and the
organic material extracted into ethyl acetate (3 x 15 mL). Washed the combined
organics with water ( x 15 mL), brine (15 mL) and passed through a phase separator
and evaporated. TLC (EtOAc:petrol 1:1) and LCMS showed the 2 main peaks as
starting material and product. Adsorbed onto silica from ethyl acetate and acetonitrile
mixture and purified by chromatography on silica gel eluting with petroleum ether
(A): ethyl acetate (B) (10-100 %B, 50 g, 20 CV, 40 mL/min) to give the major peak
being product but contaminated by starting material. Re-purified by chromatography
on silica gel eluting with dichloromethane (A): ethyl acetate (B) (20-60 %B with
initial isocratic at 21%, 25 g, 25 CV, 40 mL/min) to give pure product as a yellow
solid (65 mg, 10%).
LCMS calcd for C3iH34N 20 6S 562.2; found 563.0 [M+H]+.
1H NMR (400 MHz, CDC13) d 8.02 (m, 2H, C3'-H & C5'-H), 7.96 (d, J = 8.6 Hz, 1H,
C7-H), 7.90 (d, J = 9.1 Hz, 1H), 7.77 (m, 2H), 7.69 (d, J = 8.6 Hz, 1H), 7.26 (m, 2H),
7.08 (dd, J = 9.0, 2.6 Hz, 1H), 6.89 (m, 2H), 6.68 (d, J = 2.5 Hz, 1H), 4.81 (t, J = 3.3
Hz, 1H), 4.40 - 3.94 (m, 3H), 2.99 - 2.89 (s, 1H), 2.37 (s, 3H, Ar-CH 3), 1.86 - 1.63
(m, 2H), 1.61 - 1.44 (m, 3H). 1 C NMR (101 MHz, CDC13) d 158.9, 158.8, 152.6,
147.1, 145.0, 143.1, 134.6, 133.3, 132.6, 130.4, 130.0, 129.9, 128.9, 128.3, 128.1,
128.0, 121.5, 118.8, 114.8, 102.4, 99.1, 98.5, 72.7, 72.3, 69.4, 69.1, 66.9, 66.2, 62.9,
62.3, 60.5, 30.8, 30.6, 30.5, 25.4, 21.8, 21.2, 19.5, 19.1 and 14.3.
Example 2 : Effect of Temperature on the Automated Radiosynthesis of
Compound 1.
[1 F]-fluoride was produced using a GE PETtrace cyclotron with a silver target via the
[1 0](p,n) [1 F] nuclear reaction. Total target volumes of 3.2 - 4.8 mL were used.
The radio fluoride was trapped on a Waters QMA cartridge (pre-conditioned with
potassium carbonate), and the [1 F]-fluoride was eluted with a solution of TBAF
bicarbonate (0.75 M, 160 m ) in acetonitrile (640 m ) . Nitrogen was used to drive the
solution off the QMA cartridge to the reaction vessel. The [1 F]-fluoride was dried for
9 minutes at 120 °C under a steady stream of nitrogen and vacuum.
To investigate the impact of the anticipated PET cell temperature range on the
efficacy of the SPE process, radiosynthesis studies were conducted at the upper end of
the range (35 C) with a single Waters tC18+ SPE cartridge.
A cassette was fitted to a FASTlab synthesiser apparatus (GE Healthcare).
[1 F]Fluoride was transferred via the activity inlet of the FASTlab cassette using
vacuum. The activity was transferred from the activity inlet to the (pre-treated) QMA
cartridge where the [1 F] was trapped and the water passed through to the 1 0 water
recovery vial, using a combination of N2 to push and vacuum to pull. After the
transfer of the eluent containing the 1 F- activity into the reaction vessel, the solvents
were evaporated to dryness. The evaporation was carried out with heating under
nitrogen flow and under vacuum.
Precursor 1 (1.8 mL of a 1.5 mg/mL solution in DMSO) was added to the dry residue.
Nucleophilic substitution at 130 °C was carried out in the closed reaction vessel, in
which the tosylate group of the precursor was replaced by the 1 F- ions. After
labelling, the solution was cooled to 70°C. The tetrahydropyranylated intermediate
was converted into Compound 1 by removing the THP protecting group. This
deprotection was carried out in the reaction vessel by the addition of aqueous HC1
(0.35 mL of 4M HCL diluted with 0.82 mL of water), heating at 90°C for 35 seconds,
followed by quenching via the addition of 4% aqueous ammonia solution ( 1.4 mL).
The resulting Compound 1 was obtained in a DMSO/aqueous mixture, and was
adjusted to an 80:20 aqueous: organic mixture, prior to loading onto two Waters
tC18+ SPE cartridges in series.
Analysis of fractions of the 40% acetonitrile wash volume collected from the
FASTlab™ combined with reduced RCY showed significant loss of radiotracer. GE
FASTlab™ log files were used to determine that very low wash volumes were
sufficient to completely elute all the radiotracer at 35 C.
Example 3 : Automated Radiosynthesis of Compound 1.
The radiosynthesis of Example 2 was adapted using a third Waters tC18+ cartridge
added to the GE FASTlab™ cassette and this layout was studied over the temperature
range from 19.3 C- to 37.0 C. Figure 1 illustrates the cassette layout used wherein 1
indicates the activity inlet, 2 a buffer volume, 3 a supply of N2, each of 4a-j a valve, 5
effluent, 6 is the reaction vessel, 7-10 are reagent positions wherein 7 is precursor, 8 is
4M HC1, 9 is 4% ammonia, 10 is water and 11 is vacant. Reference number 12
indicates the three Waters tC18+ cartridges, 13 the product outlet, 14 the waste bottle,
15 40% MeCN and 16 100% EtOH.
The resulting Compound 1 was obtained in an acetonitrile/aqueous mixture, and was
adjusted to an 80:20 aqueous: organic mixture, prior to loading onto three Waters
tC18+ SPE cartridges in series. The SPE cartridges were then rinsed with water and
washed with 10.6 mL of 40% aqueous acetonitrile to remove Impurity A prior to
elution of Compound 1 with ethanol.
The Compound 1 obtained had a total chemical content of 5-10 mg/mL and
radiochemical purity (RCP) in the range 92 to 97% at a specific activity of 100-1000
GBq/ mihoI, for starting 1 F activities in the range 40-60 GBq. In addition, by studying
SPE wash fractions {via collecting the samples and analysing information provided by
the radio detectors in the GE FASTlab™ log file), it was noted that product losses
were negligible and hence good radiochemical yield (RCY) was achieved. Chemical
content, RCY and specific activity measurements were not observed to be affected by
the addition of a third SPE cartridge.
Example 4 : Automated Radiosynthesis of Compounds 2 & 3.
The cassette layout of Figure 2 was used to synthesise Compounds 2 & 3. In Figure
2 : 1 indicates the activity inlet, 2 a buffer volume, 3 a supply of N2, each of 4a-j a
valve, 5 effluent, 6 is the reaction vessel, 7-10 are reagent positions wherein 7 is
precursor (Precursor 3 and Precursor 4, respectively for Compound 2 and Compound
3), 8 is DMSO, 9 is 4M HC1, 10 is water and 11 is 4% ammonia. Reference number
12 indicates the three Waters tC18+ cartridges, 13 the product outlet, 14 the waste
bottle, 15 is MeCN (40%> and 28.5% for Compound 2 and Compound 3, respectively)
and 16 100% EtOH. Precursors 2 and 4 were obtained using methods similar to that
for Precursor 1 (i.e. as per methods described in Okamura et al J.Nucl.Med., 54(8),
1420-1427 (2013)).
For Compound 2, 11mL of 40% MeCN was required to give a chemical content of
0.1-1 .9 mg/mL over the temperature range from 2 1 C-39 C. A decay corrected yield of
42-57% was obtained when using 4 mg of precursor. The RCP was > 90%> when
starting with 60 GBq or less.
For Compound 3, 11.5 mL of ca. 28.5% MeCN was required to give a chemical
content of < 1.0 mg/mL over the temperature range 20-30°C. Decay corrected yields
of 20-25% were obtained with 3 mg precursor. For this compound, the starting
activity was increased to 100 GBq without any affect of RCP, showing RCP's of
>98%. However, the SPE purification works at a tighter temperature range as
compared to THK53 17. At around 25°C and below, the product is trapped on the 2nd
SPE cartridge and is eluted into the product vial, whereas at around 26-30°C the
product is trapped on the 3rd SPE cartridge before being eluted into the product vial.
Above 30°C some of the product is washed to waste and the resulting yield is thus
decreased. Therefore, the operating temperature for THK5351 is 20-30°C.
CLAIMS.
1. An automated method of preparation of an 1 F-labelled radiotracer of Formula
(II), which comprises:
(i) provision of an automated synthesizer apparatus which comprises a
microprocessor, and an interchangeable, disposable cassette which comprises
a reaction vessel, a supply of solvents suitable for the preparation and
purification of said radiotracer, and a supply of the precursor of Formula (I):
(ii) microprocessor-controlled transfer of said precursor of Formula (I) from
step (i) to said reaction vessel, followed by reaction of said precursor with
[1 F]-fluoride in a suitable solvent, and removal of the Pg1 protecting group, to
give the 1 F-labelled radiotra
A is chosen from:
X1 and X2 are independently an Xa or an Xb group;
X3 is an Xa or an X group;
Xa is -NR R2;
X is
R1 and R2 independently comprise H or Ci_4 alkyl, or R1 and R2 together with
the N atom and optionally the phenyl ring to which they are attached comprise
a 5- or 6- membered nitrogen-containing aliphatic or heteroaromatic ring,
optionally incorporating one further heteroatom chosen from -O- , -S- , =Nand
-NR a-, where Ra is H or Ci_4 alkyl;
R is Ci_4 alkyl, Ci_4 haloalkyl, C - aryl or C6-12 aralkyl;
Pg1 is an alcohol protecting group;
provided that in Formula (I), one X group is present, and in Formula (II) one X
group is present.
The method of claim 1, where step (ii) is carried out by:
(a) reaction of the precursor of Formula (I) with [1 F]-fluoride in a suitable
solvent, to give an 1 F-labelled intermediate of Formula (III):
(III)
wherein
A is chosen from:
X4 and X5 are each independently an Xa or X group;
where Xd is:
provided that, in Formula (III) one X group is present;
then:-
(b) removal of the Pg1 protecting group from said intermediate to give the 1 Flabelled
radiotracer of Formula (II).
3. The method of claim 1 or claim 2, where X2 is X , such that the precursor is of
Formula (IA):
and the radiotracer product is of Formula IIA:
(IIA)
where A2 is chosen from:
where Xa is as defined in claim 1.
4. The method of claim 3 where the precursor is the S-enantiomer of Formula (IB):
and the radiotracer product is the S-enantiomer of Formula (IIB)
(IIB).
5. The method of any one of claims 1 to 4, where A is an A2 group of formula:
6. The method of claim 5, where -NR R2 is -NHCH 3 or -N(CH 3)2.
7. The method of any one of claims 1 to 6, where the cassette further comprises one
to three C18-reverse phase solid phase extraction (SPE) columns, and said method
further comprises step (iii):
(iii) microprocessor-controlled SPE purification of the 1 F-labelled radiotracer
of Formula (II) from step (ii) using said cassette SPE columns, and the
solvent(s) of said cassette.
8. The method of claim 7, where the C18-reverse phase SPE column is C18-silica.
9. The method of claim 7 or claim 8, which is carried out at 15 to 40 °C with three
SPE columns.
10. The method of any one of claims 7 to 9, where the SPE columns are first eluted
with an aqueous, water-miscible organic solvent to remove impurities, and then eluted
with ethanol to elute the radiotracer of Formula (II).
11. The method of any one of claim 7 to 10, which further comprises:
(iv) optionally diluting the purified [1 F]-radiotracer of Formula (II) from step (iii)
with a biocompatible carrier;
(v) aseptic filtration of the optionally diluted solution from step (iv) to give a
radiopharmaceutical composition comprising said radiotracer.
12. A method of purification of the 1 F-labelled radiotracer of Formula (II), (IIA)
or (PB) as defined in any one of claims 1 to 6, which comprises the SPE purification
method as defined in any one of claims 7 to 10.
13. A cassette as defined in any one of claims 1 to 10.
14. Use of the automated synthesizer apparatus as defined in any one of claims 1
to 10, to carry out the method of preparation of any one of claims 1 to 11, or the
method of purification of claim 12.
15. Use of the cassette of claim 13, to carry out the method of preparation of any
one of claims 1 to 11, or the method of purification of claim 12.