SOLID PHASE EXTRACTION METHOD
Technical Field of the Invention
The present invention relates to radiochemistry and in particular to a method for the
preparation of a radiofluonnated compound. The method of the invention provides a
radiofluorination method that comprises purification by solid-phase extraction (SPE).
Description of Related Art
Radioflourinated tricyclic indole compounds are known from WO 2010/109007. These
compounds are useful as in vivo imaging agents that bind with high affinity to peripheral
benzodiazepine receptors (PBR). The compounds also have good uptake into the brain
following administration and good selective binding to PBR.
Abnormal PBR expression is known to be a feature of a variety of disease states, and in
particular disease states comprising neuroinflammation. The PBR selective ligand, (R)-
[ C]PKl 1195 provides a generic indicator of central nervous system (CNS)
inflammation. However, (R)-[ C]PKl 1195 is known to have high protein binding, and
low specific to non-specific binding. Furthermore, the role of its radiolabelled
metabolites is not known, and quantification of binding requires complex modelling. A
radiofluorinated tricyclic indole compound of the type disclosed by WO 2010/109007 is
therefore poised to provide an improved PBR selective in vivo imaging agent useful in
the diagnosis and monitoring of a variety of disease states.
In the experimental examples of WO 2010/109007 the preparation of radiolabelled
tricyclic indole compounds is described and includes purification of the compounds using
high-performance liquid chromatography (FIPLC). FIPLC requires a column, high
pressure pumps, and an ultraviolet detector which is a relatively complex system.
[1 F]-radiotracers in particular are now often conveniently prepared by means of an
automated radiosynthesis apparatus, e.g. Tracerlab™ and FASTlab™ from GE
Healthcare Ltd. For synthesisers like FASTlab™, a single-use disposable cassette in
which the radiochemistry is performed is fitted to the apparatus. The cassette normallyincludes fluid pathways, a reaction vessel, and ports for receiving reagent vials and
ideally solid phase extraction (SPE) cartridges for post-radiosynthetic clean up steps.
WO 2010/109007 discloses that a preferred method to obtain the radiolabelled tricyclic
indole compounds taught therein is by use of an automated synthesiser, wherein
purification is preferably carried out by solid phase extraction (SPE). However, no
particular methods are described.
It would be desirable to have an optimised method for the production of 1 F-labelled
tricyclic indole compounds wherein all the steps including purification are designed to be
carried out by means of an automated synthesiser.
Summary of the Invention
The present invention provides a method to prepare an 1 F-labelled tricyclic indole
compound wherein purification is carried out by solid-phase extraction (SPE) rather than
HPLC. This method is particularly suitable for carrying out the radiofluorination method
on a cassette suitable for use with an automated synthesiser. In addition to the
radiofluorination method, the present invention provides a cassette designed to carry out
the method on an automated synthesiser.
Detailed Description of the Invention
In one embodiment the present invention relates to a method to obtain a radiofluorinated
compound of Formula I :
wherein:R1 is hydrogen, halo or C1-3
alkoxy;
R andR are independently methyl, ethyl or benzyl, or together with the nitrogen to
which they are attached form a pyrrolidinyl, piperidinyl, azepanyl, or morpholinyl
ring;
CH2, CH2-CH2, CH(CH )-CH2, or CH2-CH2-CH2; and;
n is 1, 2 or 3 .
wherein said method comprises:
(i) providing a precursor compound of Formula la:
wherein la_3a y la and m are as defined for and are each the same as R1 ,
Y1 and n of Formula I, respectively, and LG is a sulfonate leaving group
having the formula -0-S0 2-R a wherein R a is a halogen, a straight-chain or
branched-chain C1-10
alkyl, a straight-chain or branched-chain C1-10
haloalkyl,
and a C -
io aryl;
reacting said precursor compound of Formula Ia with a suitable source
[ F]-fiuoride;
purifying the reaction mixture obtained in step (ii), wherein said purifying
step comprises:
(a) providing one or more solid-phase extraction (SPE) cartridges
wherein the sorbent comprises particles having a diameter between10-120µη and bonded hydrocarbons;
(b) conditioning said one or more SPE cartridges;
(c) loading the reaction mixture onto said one or more conditioned SPE
cartridges;
(d) washing said one or more SPE cartridges onto which said mixture is
loaded using a first solvent system comprising a ratio of
watenwater-miscible organic solvent in the range 100:0-0: 100; and,
(e) eluting said one or more SPE cartridges following said washing step
using a second solvent system comprising a ratio of watenwater-
miscible organic solvent in the range 70:30-0: 100.
The term "halogen " or "halo-" means a substituent selected from fluorine, chlorine, bromine
or iodine.
Unless otherwise specified, the term "alkoxy" means an alkyl radical comprising an ether
linkage. The term "alkyl" means a straight-chain or branched-chain radical having the
general formula CxH2x+i, e.g. methyl, ethyl, and propyl. The term "ether linkage " refers
to the group -C-0-C-. Examples of suitable alkyloxy radicals include methoxy, ethoxy,
ethoxyethyl, and propoxy.
The term "methyl" refers to the alkyl radical of formula CxH2x+i as defined above wherein x
is 1.
The term "ethyl" refers to the alkyl radical of formula C H2 +i as defined above wherein x is
2 .
The term "benzyl" refers to the monovalent aromatic radical C H5CH2- .
An "aromatic " radical is a conjugated hydrocarbon group with a number of π electrons that
equals (4z+2), wherein z is a positive integer or zero (Huckel's rule). The rule applies to
hydrocarbons compounds composed of only sp -hybridized carbon atoms.The term "pyrrolidinyl" refers to a five-membered aliphatic heterocycle containing four
carbon atoms and one nitrogen atom having the molecular formula C H N.
An "aliphatic" radical is either acyclic or cyclic and is not aromatic.
The term "piperidinyl" refers to a six-membered aliphatic heterocycle containing five carbon
atoms and one nitrogen atom having the molecular formula C5Hi
0N.
The term "azepanyl" refers to a seven-membered aliphatic heterocycle containing five
carbon atoms and one nitrogen atom having the molecular formula C Hi
2N.
The term "morpholinyl" refers to a six-membered aliphatic heterocycle containing four
carbon atoms, one nitrogen atom and one oxygen atom having the molecular formula
C4H NO.
A "precursor compound " comprises a non-radioactive derivative of a radiolabeled
compound, designed so that chemical reaction with a convenient chemical form of the
detectable label occurs site-specifically; can be conducted in the minimum number of
steps (ideally a single step); and without the need for significant purification (ideally no
further purification), to give the desired in vivo imaging agent. Such precursor
compounds are synthetic and can conveniently be obtained in good chemical purity.
The term "leaving group" generally refers to a moiety suitable for nucleophilic substitution
and is a molecular fragment that departs with a pair of electrons in heterolytic bond
cleavage. In the present invention, reaction of the precursor compound with [1 F]-fluoride
results in the nucleophilic displacement of the sulfonate leaving group from the precursor
compound.
The term "[1 F]-fluoride" refers to the anion 1 F .
The term "solid-phase extraction " (SPE) refers to the chemical separation technique that
uses the affinity of solutes dissolved or suspended in a liquid (known as the mobile phase)
for a solid through which the sample is passed (known as the stationary phase or sorbent) to
separate a mixture into desired and undesired components. The result is that either the
desired analytes of interest or undesired impurities in the sample are retained on the sorbent.The portion that passes through the sorbent is collected or discarded, depending on
whether it contains the desired analytes or undesired impurities. If the portion retained on
the sorbent includes the desired analytes, they can then be removed from the sorbent for
collection in an additional step, in which the sorbent is rinsed with an appropriate eluent.
The sorbent is typically packed between two porous media layers within an elongate
cartridge body to form a "solid-phase extraction (SPE) cartridge " wherein one ormore SPE
cartridges may be included in a cassette suitable for use with an automated synthesiser. A
typical SPE cartridge comprises a syringe barrel made from medical-grade plastic such as
polypropylene that is fitted with a luer tip, with frits holding the sorbent within the syringe
barrel.
The "sorbent " comprises particles, typically silica-based, to which have been bonded a
specific functional group. In the case of the present invention the sorbent suitably comprises
particles having a diameter between 10-120µπ . The functional groups bonded to the
sorbent particles are hydrocarbon chains of variable length. Typical hydrocarbon chain
lengths for SPE cartridge sorbents are C2, C8, C18 and C30.
The term "conditioning" refers to the step of rinsing the SPE sorbent with solvent prior to
loading the sample (in this case the reaction mixture). For the present invention, the
conditioning step typically comprises application of a water-miscible organic solvent
followed by water or an aqueous buffer.
The term "reaction mixture" refers to the crude product of the reaction between the
precursor compound of Formula la and the suitable source of [1 F]-fluoride. For example,
the reaction mixture is not subjected to any other purification steps such as HPLC prior to
loading onto the one or more conditioned SPE cartridges. The purifying step is therefore
the entire purification process for the reaction mixture.
The term "loading" as it applies to loading the reaction mixture onto the conditioned SPE
cartridges simply refers to the application of the reaction mixture to the cartridge, or in the
case of more than one cartridge to the first in the series.
The term "purifying" means the process of separating a desired chemical compound from a
mixture that comprises the desired chemical compound along with unwanted chemicalcompounds. In the context of the present invention the term purifying specifically refers to
SPE purifying wherein SPE is as defined above; HPLC is specifically excluded. The aim of
purifying is to remove as much as possible of the unwanted chemical compounds and as
little as possible of the desired chemical compound so that the desired chemical compound is
obtained in as high a proportion of the chemical composition of the purified product as
possible. In the specific context of the present invention, the purified product should
suitably have a ratio of compounds of Formula Ia:Formula I in the range 20:80 to 0 :100. In
reality a ratio of 0 :100 may not be achievable, therefore ratios of around 10:90 to 1:99 are
aimed for, with ratios in the range 5:95 to 1:99 being preferred. Most preferably, other
impurities are removed in addition to precursor compound of Formula la. As the
radiofiuorinated compound of Formula I is intended for in vivo use as a positron-emission
tomography (PET) tracer, it is necessary to remove any impurities that may have a toxic
effect on the mammalian body. Also, in order for the radiofiuorinated compound of
Formula I to bind most effectively to its biological target, it is desirable to remove as much
as possible of any impurities that have binding affinity to the same biological target. The
purifying step should result in the retention of as much radiofiuorinated compound of
Formula I as possible; suitably >75%, preferably >90%, and most preferably >95%.
The term "washing " refers to the step of the SPE procedure tailored for the removal of
unwanted impurities from the reaction mixture, i.e. in the case of the present invention any
chemical compounds in the reaction mixture other than the radiofiuorinated compound of
Formula I . In particular, it is desired to remove any unreacted compound of Formula la.
The term "solvent system" refers either to a single aliquot of solvent of a particular
concentration, or to multiple aliquots of solvent having different concentrations. Suitably,
said first solvent system comprises multiple aliquots of solvent wherein the concentration of
water-miscible organic solvent decreases with each successive aliquot. Suitably, said second
solvent system comprises one or more aliquots wherein the concentration of water-miscible
solvent is greater than that of any of the aliquots used in the first solvent system. The
volume of an aliquot in the context of the present invention can suitably be between 1-
50mL, typically between 5-30mL.
The term "water-miscible organic solvent" refers to a solvent other than water that readilyforms a homogenous solution with water at room temperature and at atmospheric pressure.
Examples of suitable water-miscible organic solvents include ethanol, methanol,
isopropanol, acetonitrile, dimethylformamide, dimethyl sulfoxide and formic acid. For
example, the solvent system could comprise one or more aliquots of 35% aqueous ethanol
as well as one or more aliquots of 40% aqueous ethanol and one or more aliquots of 55%
aqueous ethanol.
The term "eluting" refers to the step of the SPE procedure designed to remove the
compound of interest (the radiofluorinated compound of Formula I) from the SPE cartridge,
but to leave behind any impurities not removed by the washing step.
R1 of Formula I is preferably C1-3
alkoxy and is most preferably methoxy.
R2 and R of Formula I are preferably both methyl or both ethyl, and most preferably both
ethyl.
Y1 of Formula I is preferably CH2-CH2.
In Formula I n is preferably 2 .
Rla of Formula la is preferably C1-3
alkoxy and is most preferably is methoxy.
R a and R a of Formula la are preferably both methyl or both ethyl, and most preferably both
ethyl.
Yla of Formula la is preferably CH2-CH2.
In Formula la, m is preferably 2 .
LG of Formula la is preferably selected from toluenesulfonic acid (tosylate),
nitrobenzenesulfonic acid, benzenesulfonic acid, trifluoromethanesulfonic acid (triflate),
fluoro sulfonic acid, methanesulfonic acid (mesylate) and perfluoroalkylsulfonic acid. In a
most preferred embodiment LG is tosylate, triflate or mesylate and is especially preferably
mesylate.
In an especially preferred radiofluorinated compound of Formula I :R1 is Ci-3 alkoxy and preferably is methoxy;
R2 and R are either both methyl or both ethyl, and preferably both ethyl;
Y1 is CH2-CH2; and
n is 2 .
In an especially preferred precursor compound of Formula la:
Rla is Ci-3 alkoxy and preferably is methoxy;
R a and R a are either both methyl or both ethyl, and preferably both ethyl;
Yla is CH2-CH2;
m is 2; and,
LG is selected from toluenesulfonic acid (tosylate), nitrobenzenesulfonic acid,
benzenesulfonic acid, trifluoromethanesulfonic acid (triflate), fluorosulfonic acid,
methanesulfonic acid (mesylate) and perfluoroalkyl sulfonic acid; preferably tosylate, triflate
or mesylate and most preferably mesylate.
Said radiofluorinated compound of Formula I is preferably Compound 1:
and said precursor compound of Formula l a is preferably Compound la:wherein OMs is mesylate.
The compounds of Formula I and Formula la have a chiral centre and been illustrated
above as their racemates. In a particularly preferred embodiment, the compounds of
Formula I and Formula la are provided in an enantiomerically pure form, preferably the
S-enantiomer. The S-enantiomer of Compound I is as follows:
and the S-enantiomer of Compound la is as follows:A preferred particle diameter distribution for the sorbent of said one or more SPE
cartridges is between 35-120µιη, more preferably between 35-60µιη and most especially
preferably between 35-55 µιη. Preferably, within this size distribution, the sorbent of the
one or more SPE cartridges includes at least some particles having a diameter of
between 35-40µιη, with more preferred sorbents comprising a greater proportion of
particles having a diameter between 35-40µιη. Furthermore, it is preferred that the
bonded hydrocarbons of said sorbent have a chain length of C18 or C30. It is also
preferred that said one or more SPE cartridges used in step (iii) of the purifying step
comprise between 300mg and 3.0g of sorbent, and most preferably between 1.5-2.0g of
sorbent. The amount of sorbent can generally be provided as 1-3 SPE cartridges,
typically two SPE cartridges. For example, in a particularly preferred embodiment, 2
SPE cartridges having 900mg of sorbent each are provided. Non-limiting examples of
commercially-available SPE cartridges that are suitable for use in the purifying step of
the method of the invention include e.g. Waters tC18 Sep Pak Plus 900mg, Waters CI8
Sep Pak Plus 360mg, Varian Bond Elute 500mg, Macherey Nagel C18 ec 530mg,
Princeton C30 950mg. Preferred of these commercially-available SPE cartridges are the
Waters tC18 Sep Pak Plus 900mg, Varian Bond Elute 500mg and Princeton C30
950mg, with the Waters tC18 Sep Pak Plus 900mg being most preferred.
The preferred embodiments of the SPE cartridges as described in the previous paragraph
are particularly preferred where the method of the invention relates to obtaining
Compound 1 by radiofluorination of Compound la.
Preferably, in the purifying step of the method of the invention, said water-miscible organic
solvent of said first and second water-miscible organic solvent systems is selected from
ethanol (EtOH), acetonitrile (MeCN), methanol and isopropanol. Preferably, the first
solvent system comprises one or more aliquots having watenwater-miscible organic solvent
in a ratio of between 65:35-60:40, i.e. 35-40% aqueous water-miscible organic solvent,
wherein each successive aliquot used in the first solvent system has a lower concentration of
water-miscible organic solvent, e.g. a first aliquot of 40% aqueous water-miscible organic
solvent followed by a second aliquot of 35% aqueous water-miscible organic solvent.
Preferably, the volume of said first aliquot is greater than that of said second aliquot, e.g.said first aliquot is 20-30mL and said second aliquot is 5-15mL. Preferably, said second
solvent system comprises one or more aliquots of aqueous water-miscible organic solvent
each having water: water-miscible organic solvent in a ratio of between 60:40 to 0 :100, i.e.
40-100% aqueous water-miscible organic solvent. Most preferably, said second solvent
system comprises one or more aliquots wherein the concentration of water-miscible organic
solvent is greater than that of any of the aliquots in the first solvent system. For example,
said second solvent system preferably comprises one or more aliquots having a
concentration of water-miscible organic solvent in the range 50-80%, most preferably 50-
70% and most especially preferably 50-60%>. Said first and second solvent systems may also
comprise an aliquot of water as a final aliquot. The most preferred water-miscible organic
solvent for said first and second water-miscible organic solvent systems is EtOH. Most
preferably when EtOH is said water-miscible organic solvent, in said first solvent system a
first aliquot is 40% aqueous water-miscible organic solvent and a second aliquot is 35%
aqueous water-miscible organic solvent, optionally followed by a third aliquot of water; and,
in said second solvent system a first aliquot is 50-60%> aqueous EtOH, optionally followed
by subsequent aliquots having EtOH concentration greater than said first aliquot.
Non-limiting examples of particularly preferred solvent systems for use in the purifying step
of the method of the invention are tabulated below (% values are % >water-miscible organic
solvent in water, where said organic solvent is preferably EtOH):
The preferred embodiments described in the above paragraph relating to solvent systems areparticularly preferred where the method of the invention relates to obtaining Compound 1
by radiofluorination of Compound la, and in particular the S-enantiomers thereof.
The method of the invention primarily aims to remove as much unreacted precursor
compound of Formula la from the reaction mixture as possible. In preferred
embodiments, the method of the invention also removes additional impurities. Notably,
where the method of the invention relates to obtaining Compound 1 by radiofluorination
of Compound la, the experimental examples demonstrated that the method of the
invention removes 90-98% of the precursor compound and 85-90% of a hydroxy
impurity and only traces of a vinyl impurity are left. The hydroxy and vinyl impurities
are, respectively, as follows:
A further notable impurity is the acetyl impurity, which has the following structure:
Methods suitable for the preparation of precursor compound of Formula la are described in
detail in WO 2010/109007. For example, a precursor compound wherein LG is mesylate
can be prepared from commercially-available starting materials according to the general
method illustrated in Scheme 1below:1. 60°C
Scheme 1 2. IPA, ZnC
In Scheme 1 above and in Scheme l a below, the variables R a~ a and Y a are as suitably and
preferably provided herein in respect of Formula la. R5a in Scheme 1 represents
CH2CwaterBn wherein Bn is benzyl, Et is ethyl, OTs represents a tosylate leaving group,
IPA stands for isopropyl alcohol, and OMs represents a mesylate leaving group.
Alternatively, where Rla of the precursor compound of Formula la is at the top position on
the ring, the general synthetic route illustrated in Scheme la below can be used:LiAlH
THF
ZnCl 3eq Diethyl ether
Reflux 5days
Silica chromatography Scheme l a
10-50% ethyl acetyate/petrol
In Scheme la, Bn is benzyl, THF is tetrahydrofuran, KFDVIDS is potassium
hexamethyldisilazane, eq stands for equivalent(s), and EtAc is ethyl acetate. The resultant
hydroxyl compound can be readily converted into a precursor compound of Formula la, e.g.
by reaction with methane sulfonyl chloride for addition of a methane sulfonate leaving
group.[ F]-fluoride is normally obtained as an aqueous solution from the nuclear reaction
1 0(p,n) 1 F. In order to increase the reactivity of fluoride and to avoid hydroxylated by
products resulting from the presence of water, water is typically removed from [1 F]-
fluoride prior to the reaction, and fluorination reactions are carried out using anhydrous
reaction solvents (Aigbirhio et al 1995 J Fluor Chem; 70: 279-87). The removal of water
from [1 F]-fluoride is referred to as making "naked" [1 F]-fluoride. A further step that is
used to improve the reactivity of [1 F]-fluoride for radiofluorination reactions is to add a
cationic counterion prior to the removal of water. Suitably, the counterion should possess
sufficient solubility within the anhydrous reaction solvent to maintain the solubility of the
[1 F]-fluoride. Therefore, counterions that are typically used include large but soft metal
ions such as rubidium or caesium, potassium complexed with a cryptand such as
Kryptofix™, or tetraalkylammonium salts, wherein potassium complexed with a cryptand
such asKryptofix™, or tetraalkylammonium salts are preferred. [1 F]-fluoride that has been
made reactive according to these steps is what is meant in the context of the present
invention as a "suitable source of [1 F1-fluoride .
[1 F]-radiotracers in particular are now often conveniently prepared on an automated
radiosynthesis apparatus. There are several commercially-available examples of such
apparatus, including Tracerlab™ and FASTlab™ (GE Healthcare Ltd). Such apparatus
commonly comprises 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 solid-phase extraction cartridges used in post-radiosynthetic clean up
steps. In a preferred embodiment therefore, the method of the invention is automated.
Additionally, in a further aspect, the present invention provides a cassette for carrying out
the method of the invention on an automated synthesis apparatus, wherein said cassette
comprises:
(i) a vessel containing a precursor compound of Formula la as defined herein
for the method of the invention;
(ii) means for eluting the vessel with a suitable source of [1 F]-fluoride; and,(iii) one or more SPE cartridges as defined herein for the method of the
invention.
All the suitable, preferred, most preferred, especially preferred and most especially preferred
embodiments of the precursor compound of Formula la, [1 F]-fluoride and the SPE
cartridges that are presented herein in respect of the method of the invention also apply to
the cassette of the invention.
The cassette of the invention may furthermore comprise:
(iv) an ion-exchange cartridge for removal of excess [1 F]-fluoride.
The invention is now illustrated by the following non-limiting examples:
Brief Description of the Examples
Example 1 describes the preparation of spiked samples for SPE screening experiments.
Example 2 describes the SPE screening experiments.
Example 3 describes preparation of decayed FASTLab crude samples for SPE
purification experiments.
Example 4 describes the SPE purification of Crude 1.
Example 5 describes the SPE purification of Crude 2 .
Example 6 describes the SPE purification of Crude 3 .
Example 7 describes the SPE purification of Crudes 4 & 5 .
Example 8 describes the SPE purification of Crude 6 .
Example 9 describes a number of FASTlab runs that were carried out including SPE
purification on the FASTlab cassette.List of Abbreviations used in the Examples
aq aqueous
DAD diode array detector
ESI electrospray ionisation
EtOH ethanol
HPLC high performance liquid chromatography
LC-MS liquid chromatography mass spectrometry
MeCN acetonitrile
MS mass spectrometry
SPE solid phase extraction
UV ultraviolet
Examples
Example 1: Preparation of Spiked Samples for SPE Screening Experiments
Non-radioactive Compound 1 and Compound l a were prepared in accordance with the
methods described in Examples 2 and 1, respectively, of WO 2010/109007.
To prepare each spiked sample, lmg of Compound l a was weighed in and dissolved in
ImL of MeCN. Then ΙΟΟµΙ of Compound 1 stock solution (lmg/mL in 50:50
H20:MeCN) was added. The sample was then diluted with ImL of water before use in
the experiments described in Example 2 below.
Example 2: SPE Screening Experiments
Samples containing -25 g of Compound 1 (a compound of Formula I) and lmg ofCompound l a (a compound of Formula la) were prepared.
2mL of sample in 50% aq MeCN was used in each experiment. Before application of
sample, the cartridge(s) were activated using 3mL EtOH, equilibrated using lOmL
water, and dried by application of a vacuum. Following the washing steps, the
cartridge(s) were dried by application of a vacuum, then eluted using 3mL EtOH and
dried again by application of a vacuum.
Analysis of the various fractions was carried out by HPLC with an Agilent 1100 Series,
OSLC016 with UV detection at 230nm, 270nm, DAD detection and MS detection. The
column used was a Zorbax Stable Bond C18 1.8 µιη 4.6 x 50 mm and the mobile phases
were A: 0.1% HCOOH in water, B : 0 .1% HCOOH in 80% MeCN. The flow rate was
1mL/min and the column oven was set to 40 C. The following gradient was used:
Amounts of Compound 1 and Compound l a were estimated based on standard curves
generated for Compound 1.
Analysis was also carried out by LC-MS using an Agilent single TOF (LC-UV/MS) in
ESI+ ionization mode and a fragmentor voltage of 70V. Detection was carried out by
UV at 230nm, 270nm, DAD detection and MS detection. The column was a Zorbax
Stable Bond C18 1.8 µιη 4.6 x 50 mm and the mobile phases were A: 0 .1% HCOOH in
water, B : 0.1 % HCOOH in 80% MeCN. The flow rate was 1.5 mL/min and the column
oven was set to 40 C. The following gradient was used:6.30 90
9.20 90
9.30 40
12.00 40
The table below summarises the experiments carried out and the results obtained:
Example 3: Preparation of FASTLab Crude Samples for SPE Purification
Experiments
Generally for the preparation of each FASTLab crude sample, a FASTLab cassette was
assembled with an eluent vial, a QMA cartridge (preconditioned, Waters), a precursor
vial and an MeCN vial. The FASTLab samples were prepared by carrying out the
FASTLab process up to and including the labelling step, followed by transfer of the
crude (approximately 1.3mL MeCN) to a vial for storage in until analysis. For the non-
radioactive runs, the labelling step was carried out without any fluoride. More detail in
respect of each sample is now provided.
Crude 1
120.6MBq of [1 F]fluoride obtained from a GE PETrace cyclotron was made up to
1.5mL with water, introduced into the FASTLab synthesiser (GE Healthcare), and
trapped on the QMA cartridge. 825 eluent solution (KHCO3+ kryptofix inMeCN/water (80/20, v/v)) was used to elute the [ F]fluoride off the QMA cartridge
into the reaction vessel. The material in the reaction vessel was then dried at 120°C for
10 minutes followed by transfer of 4.0mg of Compound l a dissolved in 1.6mL MeCN to
the reaction vessel. Labelling was carried out at 100°C for 15 minutes. The contents of
the reaction vessel following labelling (in 1.3 mL MeCN) were transferred to a vial and
allowed to decay at room temperature for 1 day prior to storage in the freezer until
analysis.
500 L decayed crude in 500µΙ MeCN was spiked with 40µΙ 1.lmg/mL Compound 1
and then diluted with ImL water; 2mL of this was used in the experiment described in
Example 4 .
Crudes 2 & 3
The process as described for Crude 1was carried out except that (i) instead of trapping
1 F-fluoride on the QMA cartridge, 1.5mL water was passed through the QMA
cartridge, (ii) 1200µΙ of eluent solution was used rather than 825 eluent solution, (iii)
drying was at 100°C for 20 minutes, and (iv) 3.2mg of Compound l a in 1.6mL MeCN
was transferred to the reaction vessel for the labelling reaction step.
Samples were prepared for 2 experiments. For each sample, 500 crude (in MeCN)
was spiked with 20µΙ Compound 1 solution (1.24mg/mL in 50:50 H20:MeCN) and
then diluted with ImL water. 2mL of each solution was loaded onto the SPE column
for the experiments described in Examples 5 and 6 .
Crude 4
The process as described for the preparation of Crudes 2 and 3 was carried out except
that 3.1mg of Compound l a in 1.6mL MeCN was used for the labelling reaction step.
500µ crude (in MeCN) was spiked with 20µΙ Compound 1 solution (1.24mg/mL in
50:50 H20:MeCN) and then diluted with ImL water. 2mL of this solution was loaded
onto the SPE column for the experiment described in Example 7 .
Crude 5The process as described for the preparation of Crudes 2 and 3 was carried out except
that 4.8mg of Compound l a in 1.6mL MeCN was used for the labelling reaction step.
500 L crude (in MeCN) was spiked with 20µΙ Compound 1 solution (1.24mg/mL in
50:50 H20:MeCN) and then diluted with ImL water. 2mL of this solution was loaded
onto the SPE column for the experiment described in Example 7 .
Crude 6
The process as described for the preparation of Crudes 2 and 3 was carried out except
that 3.5mg of Compound l a in 1.6mL MeCN was used for the labelling reaction step.
500 L crude (in MeCN) was spiked with 20 Compound 1 solution (1.24mg/mL in
50:50 H20:MeCN) and then diluted with ImL water. 2mL of this solution was loaded
onto the SPE column for the experiment described in Example 8.
The table below details the amounts in g of the main components in the FASTLab
crude samples prepared according to this example as applied to the SPE cartridges in
Examples 4-8:
Example 4: SPE Purification of Crude 1
2 x 900mg Waters tC18 SPE cartridges were used in series. The cartridges were
activated with 3mL EtOH, equilibrated with lOmL water and dried by application of a
vacuum. Then, 2mL Crude 1 (prepared as described in Example 3) was applied to the
cartridges. The cartridges were washed firstly with 27mL 40% aq EtOH, then lOmL35% aq EtOH, and then 5mL water. The cartridges were then dried by application of a
vacuum, followed by elution using 3mL EtOH and a further drying step.
Analysis of the various fractions was carried out by HPLC as described in Example 2
above.
The table below details the amounts of each component in g coming off the cartridg
following each step:
Approximately 80% of Compound l a and 30% of the hydroxy are removed during the
wash with 27mL 40% aq EtOH. Another 20% of Compound l a and 30% of the
hydroxy are removed during the wash with 35% EtOH (total of lOmL). Only small
amounts of Compound l a and hydroxy are removed during the wash with 5mL water.
Left in the eluate is hydroxy/Compound la/Compound 1/vinyl to a ratio 18/3/36/43. As
expected, no vinyl is removed as it elutes later than Compound 1.
Example 5: SPE Purification of Crude 2
The method as described in Example 4 was used for 2mL of Crude 2 (prepared as
described in Example 3) except that elution was carried out using 3mL of 50% aq EtOH,
3mL of 60% aq EtOH, 3mL 70% aq EtOH and 3mL of 80% aq EtOH were used.
The table below details the amounts of each component in g coming off the cartridges
following each step:
Wash Hydroxy Compound l a Compound 1 Vinyl
27mL 40% aq EtOH 5 261 0 0Compound 1 eluted mainly during the 3mL of 60%> aq EtOH, but some Compound 1
was also observed in the 3mL of 50% aq EtOH and 3mL 70% aq EtOH. 85% of the
vinyl eluted during the 3mL of 70% aq EtOH and the last 15% during 3mL of the 80%
aq EtOH. The wash with 50% aq EtOH before elution and after the wash with 5mL
water was shown to be effective for the removal of the hydroxy.
Example 6: SPE Purification of Crude 3
The method as described in Example 5 was used to purify 2mL of Crude 3 (prepared as
described in Example 3) except that the 3mL 50% aq EtOH step was changed to a 3mL
40% aq EtOH step, and followed by 3mL of 65% aq EtOH and 3mL of 100% EtOH.
The table below details the amounts of each component in g coming off the cartridges
following each step:
The removal of hydroxy decreases as compared to the method described in Example 5,
but less loss of Compound 1was observed. Compound 1mainly eluted in the 3mL of
65% aq EtOH wash.Example 7: SPE Purification of Crudes 4 & 5
Crudes 4 and 5 were purified using the method as described in Example 4, except that
the 3mL EtOH step was replaced with 3mL 50% aq EtOH, then 3mL 65%> EtOH, and
then 3mL 100% EtOH, with each of these steps followed by drying by application of a
vacuum.
The table below details the amounts of each component in g coming off the cartridges
following each step in respect of Crude 4 :
The table below details the amounts of each component in g coming off the cartridges
following each step in respect of Crude 5:
The experiments for Crude 4 and Crude 5 showed similar trends. After the wash with
27mL of 4 0% aq EtOH approximately 60-70% of Compound l a was removed together
with approximately 10% of the hydroxy impurity. The lOmL wash with 35% aq EtOHremoved nearly the rest of Compound la. A total of 90% was removed for Crude 5 (the
total amount Compound l a in the injected sample was 1.7 mg) and 98% Crude 4 (the
total amount of Compound l a in the injected sample was 0.9mg). A total of 40-50% of
the hydroxy was removed after this wash. 5mL water washed out further amounts of
both hydroxy and Compound la. The wash with 3mL of 50% aq EtOH removed
another 35% of the hydroxy and small amounts of Compound la. The 3mL of 65% aq
EtOH, contained 50/50 Compound 1/hydroxy and traces of Compound l a and vinyl.
This means that approximately 85-90% of the hydroxy was removed during the
procedure. The vinyl impurity is mainly trapped on the cartridge and eluted out with
100% EtOH.
Crude 5 contains almost double gs of Compound l a compared to Crude 4, but the
results are comparable. The method was able to remove nearly all Compound l a in both
crudes.
Example 8: SPE Purification of Crude 6
An experiment was performed to examine the composition in the eluate when the sample
injected was not spiked with product. SPE purification was performed on Crude 6
(prepared as described in Example 3) with the method as described in Example 7 for the
purification of Crudes 4 and 5 .
The table below details the amounts of each component in g coming off the cartridges
following each step:
The table shows that the composition (based on estimated amounts [ of the eluate ishydroxyl/precursor/product/vinyl = 90/6/4/0. The ratio given in the table is based on the
area under the peak at 230nm. The lowest values included in the standard curve were
0.1 15µ .
Example 9: FASTlab Runs
A FASTlab process was carried out for the production of a number of batches of the S-
enantiomer of Compound 1. Up to 80GBq of [1 F]fluoride obtained from a GE PETrace
cyclotron (from H2
1 0 ) was introduced into the FASTLab synthesiser (GE Healthcare),
and trapped on the QMA cartridge. Approximately 475 µΐ eluent solution (KHCO3+
kryptofix in MeCN/water (80/20, v/v) was used to elute the [1 F]fluoride off the QMA
cartridge into the reaction vessel. The material in the reaction vessel was then dried at
120°C for 9 minutes followed by transfer of 4.0mg of Compound l a dissolved in 1.6mL
MeCN to the reaction vessel. Labelling was carried out at 100°C for 6 minutes.
In each case, following labelling, the reaction mixture was applied to the first in a series
of 2 conditioned 900mg Waters tC18 SPE cartridges in situ on the FASTlab cassette
and the SPE purification process was carried out as follows: a first solvent system
comprising 22mL 40% EtOH followed by lOmL 35% EtOH and a second solvent
system comprising 3.5mL 55% EtOH and 3.5mL water.
Figure 1 provides details of the runs that were carried out, including initial activity,
uncorrected end of synthesis (UEOS) yield, radioactive concentration (RAC),
radiochemical purity (RCP), as well as the amounts of each compound (all S-enantiomer
compounds) separated in the SPE process. RCP values in excess of 95% were routinely
achieved.Claims
A method to obtain a radiofluorinated compound of Formula I :
wherein:
R1 is hydrogen, halo or C1-3
alkoxy;
R andR are independently methyl, ethyl or benzyl, or together with the nitrogen to
which they are attached form a pyrrolidinyl, piperidinyl, azepanyl, or morpholinyl
ring;
CH2, CH2-CH2, CH(CH )-CH2, or CH2-CH2-CH2; and;
n is 1, 2 or 3 .
wherein said method comprises:
(i) providing a precursor compound of Formula la:wherein la_3a y la and m are as defined for and are each the same as R1 3 ,
Y1 and n of Formula I, respectively, and LG is a sulfonate leaving group;
(ii) reacting said precursor compound of Formula la with a suitable source of
[1 F]-fluoride;
(iii) purifying the reaction mixture obtained in step (ii), wherein said purifying
step comprises:
(a) providing one or more solid-phase extraction (SPE) cartridges
wherein the sorbent comprises particles having a diameter between
10-120µπ and bonded hydrocarbons;
(b) conditioning said one or more SPE cartridges;
(c) loading the reaction mixture onto said one or more conditioned SPE
cartridges;
(d) washing said one or more SPE cartridges onto which said mixture is
loaded using a first solvent system comprising a ratio of
watenwater-miscible organic solvent in the range 100:0-0:100; and,
(e) eluting said one or more SPE cartridges following said washing step
using a second solvent system comprising a ratio of watenwater-
miscible organic solvent in the range 70:30-0: 100.
(2) The method as defined in Claim 1wherein each of R1 and Rla is C1-3
alkoxy.
(3) The method as defined in Claim 2 wherein each of R1 and Rla is methoxy.
(4) The method as defined in any one of Claims 1-3 wherein each of R2, R , R a and R a
is either methyl or ethyl.
(5) The method as defined in Claim 4 wherein each of R2, R3, R a and R a is ethyl.
(6) The method as defined in any one of Claims 1-5 wherein each of Y1 and Yla is CH2-CH2
The method as defined in any one of Claims 1-6 wherein each of n and m is 2 .
The method as defined in any one of Claims 1-7 wherein LG is selected from
tosylate, triflate and mesylate.
The method as defined in Claim 8 wherein LG is mesylate.
The method as defined in any one of Claims 1-9 wherein said radiofluorinated
compound of Formula I is:
and said precursor compound of Formula la is:
wherein OMs is mesylate.
The method as defined in any one of Claims 1-10 wherein said one or more SPEcartridges used in step (iii) comprise between 900mg and 2.0g of sorbent.
(12) The method as defined in Claim 11 wherein said one or more SPE cartridges
comprise between 1.5-2.0g of sorbent.
(13) The method as defined in any one of Claims 1-12 wherein the sorbent of said SPE
cartridges used in step (ii) comprises particles having a diameter distribution of
between 35-60 µπ .
(14) The method as defined in any one of Claims 1-13 wherein for said SPE cartidges
used in step (ii) said bonded hydrocarbons of said sorbent have a carbon chain
length of 18 or 30.
(15) The method as defined in any one of Claims 1-14 wherein said water-miscible
organic solvent of said first and second water-miscible organic solvent systems used
in step (ii) of said method is selected from ethanol (EtOH) and acetonitrile (MeCN).
(16) The method as defined in Claim 15 wherein said water-miscible organic solvent is
EtOH.
(17) The method as defined in Claim 16 wherein said first solvent system comprises one
or more aliquots of aqueous EtOH each having water :EtOH in a ratio of between
65:35-60:40, and one or more aliquots of water.
(18) The method as defined in Claim 17 wherein said second solvent system comprises
one or more aliquots of aqueous EtOH each having watenEtOH in a ratio of
between 60:40-0:100.
(19) The method as defined in either Claim 17 or Claim 18 wherein said first solvent
system consists of a first aliquot of 27mL 40% EtOH, a second aliquot of lOmL
35% EtOH, and a third aliquot of 5mL water; and wherein said second solvent
system consists of a first aliquot of 3mL 50% EtOH, a second aliquot of 3mL 65%
EtOH and a third aliquot of 3mL 100% EtOH.
(20) The method as defined in either Claim 17 or Claim 18 wherein said first solventsystem consists of a first aliquot of 22mL 40% EtOH and a second aliquot of lOmL
35% EtOH; and wherein said second solvent system consists of a first aliquot of
3.5mL 55% EtOH, a second aliquot of 3.5mL water.
(21) The method as defined in any one of Claims 1-20 which is automated.
(22) A cassette for carrying out the method as defined in Claim 2 1which comprises:
(i) a vessel containing a precursor compound of Formula la as defined in the
method of any one of Claims 1-10;
(ii) means for eluting the vessel with a suitable source of [1 F]-fluoride; and,
(iii) one or more SPE cartridges as defined in the method of any one of Claims 1
and 11-14.
(23) The cassette as defined in Claim 22 which further comprises:
(iv) an ion-exchange cartridge for removal of excess [1 F]-fluoride.