C h a l l e n g e s f o r
`D e v e l o p i n g P E T
`Tr a c e r s : I s o t o p e s ,
`C h e m i s t r y, a n d
`R e g u l a t o r y A s p e c t s
`
`Robert H. Mach, PhD*, Sally W. Schwarz, MS, BCNP
`
`KEYWORDS
`
` Carbon 11  Fluorine 18  PET regulatory  Chemistry
`
`PET continues to be the primary functional
`imaging technique for conducting translational
`research studies aimed at identifying the molec-
`ular basis of human disease. Although PET
`imaging studies of the central nervous system
`(CNS)
`initially focused on global or
`regional
`changes in brain function such as glucose utiliza-
`tion, cerebral blood flow, and oxygen metabolism,
`a major effort in PET research in the past 20 years
`has focused on the development of specific
`probes that are capable of studying the change
`in neurotransmitter receptors, second messenger
`systems, and neuronal networks in the brain. As
`our knowledge of the diversity of neurotransmitter
`systems continues to grow, so will the need to
`develop molecular imaging probes with an even
`higher target specificity than first-generation PET
`radiotracers. For example, before the cloning of
`dopamine receptors, when PET imaging studies
`of the dopamine D2 receptor began in the mid-
`1980s, it was believed that there were only 2 types
`of dopamine receptors: D1 and D2. It is now known
`that there are 2 different families of dopamine
`receptors: D1-like (consisting of the D1 and D5
`receptors) and D2-like (consisting of the D2, D3,
`and D4 receptors).1 There is a growing body of
`evidence that within the D2-like family of receptors,
`D2 and D3 receptors are regulated in an opposing
`manner under
`conditions of
`increased or
`decreased dopaminergic tone. For example, auto-
`radiography studies have shown that
`there is
`
`a 15% increase in D2 receptors in the caudate
`and putamen and a 45% decrease in D3 receptors
`in the ventral striatum of postmortem brain
`samples of Parkinson disease.2 Similar results
`have been reported in the study of chronic expo-
`sure to cocaine on D2 and D3 receptor function.
`That is, D2 receptors are reduced in autoradiog-
`raphy3 and PET imaging studies of
`rhesus
`monkeys that have self-administered cocaine,4
`and in chronic cocaine abusers.5,6 However, the
`autoradiography studies conducted by Staley
`and Mash7 reported an upregulation of D3 recep-
`tors in human cocaine overdose victims compared
`with age-matched controls. Because PET radio-
`tracers such as [11C]raclopride,
`[18F]fallypride,
`and [11C]PHNO have a high affinity for D2 and D3
`receptors, newer
`radiotracers having a higher
`affinity and selectivity for D3 versus D2 receptors
`are needed to study the role of the D3 receptor in
`CNS disorders. This same argument can be
`applied to many other CNS receptor systems,
`including the serotonergic system (7 different fami-
`lies for receptors and at least 13 different receptor
`subtypes),8 glutamate receptors (2 different
`classes, ionic and metabotropic, with at least 11
`different subtypes),9 and the adrenergic receptors
`(6 different subtypes of a adrenergic receptors and
`3 different subtypes of b receptors).10 As the need
`for more subtype-selective radiotracers grows, the
`success criteria for these newer radiotracers are
`expected to be higher than PET chemists have
`
`pet.theclinics.com
`
`Department of Radiology, Mallinckrodt Institute of Radiology, Washington University School of Medicine, 510
`South Kingshighway Boulevard, St Louis, MO 63110, USA
`* Corresponding author.
`E-mail address: rhmach@mir.wustl.edu
`
`PET Clin 5 (2010) 131–153
`doi:10.1016/j.cpet.2010.02.002
`1556-8598/10/$ – see front matter ª 2010 Elsevier Inc. All rights reserved.
`
`Petitioner GE Healthcare – Ex. 1033, p. 131
`
`

`

`132
`
`Mach & Schwarz
`
`striven to achieve in the past. For example, as
`a PET radiotracer becomes more subtype selec-
`tive, the target density to be imaged by the tracer
`within the CNS (ie, receptor density or Bmax) invari-
`ably decreases. The development of high-affinity,
`high-selectivity,
`and
`low-nonspecific-binding
`radiotracers will likely be required to provide a suit-
`able signal-to-noise ratio in PET imaging studies.
`Factors such as improved specific activity also
`play a key role in the ability to image receptors,
`and other protein targets that have a low expres-
`sion in the CNS.
`This article does not review the advances in
`radiotracer design with respect to subtype-selec-
`tive PET imaging probes; that topic is covered in
`other articles in this and other issues of PET
`Clinics. This article provides an overview of the
`basic principles of PET radiochemistry and
`describes recent advances in PET radiosynthesis
`to assist the development of subtype-selective
`PET radiotracers needed to address the next level
`of scientific inquiry within clinical neuroscience
`research. The general principles of radiotracer
`design used by many chemists in the development
`of a PET radiotracer are also described, especially
`when this involves the incorporation of fluorine 18
`(18F) into a molecule that does not possess a fluo-
`rine atom. A description of the current regulatory
`guidelines relevant to the production of PET radio-
`tracers for human use studies is also provided.
`
`RADIONUCLIDES USED IN PET
`
`The 4 positron-emitting radionuclides most
`frequently used in PET are oxygen 15 (15O),
`nitrogen 13 (13N), carbon 11 (11C), and 18F. There
`are several reasons why these radionuclides are
`routinely used in PET imaging applications. The
`first is that most of these radionuclides can be
`substituted into biologically active molecules with
`a hot-for-cold substitution. That is, 11C can be
`substituted for nonradioactive 12C in a biologically
`active molecule without altering the biologic
`
`Table 1
`Radionuclides commonly used in PET
`
`properties of the molecule. In this example, 11C
`is a true radiotracer because the structure of the
`biologically active molecule has not been altered
`to introduce the PET radiolabel. The same concept
`applies to 15O and 13N; however, the short half-
`lives of 15O and 13N have limited their use in PET
`radiotracer development. Consequently,
`these
`radionuclides are largely used as perfusion
`[15O]water
`tracers:
`for brain perfusion studies
`and [13N]ammonia for heart perfusion studies.
`An exception to the principle of hot-for-cold
`substitution is 18F; although fluorine is the most
`abundant halogen in soil samples, the abundance
`of naturally occurring organofluorine compounds
`is low and limited to fluorinated carboxylic acids
`such as fluoroacetate,
`fluorocitrate, and fluori-
`nated fatty acids produced in numerous plant
`species indigenous to Africa, Australia, and South
`America.11 There are no naturally occurring orga-
`nofluorine compounds in animals. However, fluo-
`rine is a frequently used substituent
`in drug
`development, and there are several fluorine-con-
`taining drugs targeting neurotransmitter receptors
`in the CNS.12 These include the antidepressants
`fluoxetine and paroxetine, the tricyclic antipsy-
`chotic fluphenazine, the butyrophenones haloper-
`idol
`and
`spiperone,
`and
`the
`atypical
`antipsychotics risperidone and setoperone. These
`fluorine-containing drugs have served as a direct
`source of leads for PET radiotracers involving the
`principle of an 18F for 19F substitution.13–19 In addi-
`tion to these examples, it is often possible to incor-
`porate fluorine into a nonfluorine-containing lead
`compound without losing activity at the target
`protein; 1 example is the dopamine D2 imaging
`agent fluoroclebopride, an analogue of the dopa-
`mine antagonist clepopride.20
`A second reason for the prominent use of these
`radionuclides in PET imaging studies is that each
`can be produced in high yield and high specific
`activity with a low energy (ie, 11–17 MeV), medical
`cyclotron using either the p,n (15O, 13N, 18F) or p,a
`(11C) nuclear reaction (Table 1). Because the
`
`Radionuclide
`
`Half-life (min)
`
`Oxygen 15
`Nitrogen 13
`Carbon 11
`Fluorine 18
`
`2
`10
`20
`110
`
`Nuclear
`Reaction
`
`15N(p,n)15O
`13C(p,n)13N
`14N(p,a)11C
`18O(p,n)18F
`
`Average b D
`Energy (keV)
`
`b D Range
`mm (H2O)
`
`Maximum
`Specific Activity
`(Ci/mmol)
`
`735
`491
`385
`242
`
`8.2
`5.39
`4.1
`2.39
`
`91730
`18900
`9220
`1710
`
`Routine specific activity: 11C, ~10 Ci/mmol; [18F]fluoride, 2–10 Ci/mmol.
`
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`
`

`

`Challenges for Developing PET Tracers
`
`133
`
`target material is either gas (15O, 11C) or liquid (13N,
`18F), the radionuclides are readily transferred from
`the cyclotron target to a hot cell in the radiochem-
`istry laboratory, where the radionuclide can be
`incorporated into a biologically active molecule.
`There are also several commercially available
`automated chemistry systems that are capable
`of converting these PET radionuclides, obtained
`as low molecular weight species from the cyclo-
`tron target,
`into either a chemically reactive
`species or a radiolabeled prosthetic group. Auto-
`mated chemistry systems are typically designed
`to conduct 2 to 3 organic reactions in series, fol-
`lowed by either a resin-based or high-performance
`liquid chromatography (HPLC) purification step,
`leading to the synthesis of a PET radiotracer that
`is suitable for
`imaging studies in preclinical
`imaging studies in animal models or in clinical
`research studies. Although simplistic in theory,
`the adaptation of a commercially available auto-
`mated PET chemistry system to enable the
`synthesis of structurally diverse PET radiotracers,
`and the routine review and optimization of the
`synthesis to ensure reproducible radiochemical
`yields and high specific activity, requires consider-
`able expertise in organic chemistry and chemical
`engineering. Quality assurance testing to confirm
`that the radiotracer meets criteria for radiochem-
`ical and chemical purity, specific activity, sterility,
`pyrogen levels, and presence of residual solvents
`for use in humans also requires expertise in analyt-
`ical chemistry and radiopharmacy. Therefore,
`most PET centers actively engaged in clinical
`research consist of a collaboration between
`organic/medicinal chemists, chemical engineers,
`analytical
`chemists,
`and
`radiopharmacists
`working in concert to meet the significant time
`constraints imposed by the short-lived radioiso-
`topes routinely used in PET.
`Because 11C and 18F are the 2 main radionu-
`clides used in the development of PET radio-
`tracers for
`imaging brain function,
`this article
`focuses on a review of the basic principles and
`recent developments in the radiosynthesis of
`11C- and 18F-labeled compounds.
`
`11C Radiochemistry
`
`11C is produced in the cyclotron target using the
`14N(p,a)11C nuclear reaction. The target material
`is nitrogen (N2) containing trace quantities of
`the 11C into
`oxygen (0.5% O2)
`to convert
`[11C]CO2.21,22 The simplest reaction using 11C
`involves the transfer of [11C]CO2 from the target
`to a chemistry module or a reaction vessel con-
`taining a Grignard reagent
`that
`reacts with
`[11C]CO2 to give the corresponding 11C-labeled
`
`carboxylic acid. The most prominent examples of
`this process are the metabolic tracers [11C]acetate
`and [11C]palmitate (Fig. 1), which are prepared by
`the direct venting of [11C]CO2 into a solution of
`either methylmagnesium bromide or 1-pentade-
`cylmagnesium bromide in an anhydrous solvent
`such as tetrahydrofuran or ether.23 There are 2
`examples in which [11C]CO2 has been used in
`the synthesis of a PET radiotracer for imaging
`CNS receptors: (1) the serotonin 5-hydroxytrypta-
`mine1A (5-HT1A) antagonist [11C]Way-1006524,25;
`and (2) the radiolabeled dopamine D2/D3 agonist,
`[11C](1)-PHNO, which is primarily used to image
`the high affinity state of the dopamine D2 and D3
`receptors.26–28 In each case, [11C]CO2 is vented
`through a solution of the Grignard reagent (cyclo-
`hexyl magnesium chloride for Way-10065 and
`ethylmagnesium bromide for (1)-PHNO) to give
`11C-labeled carboxylic acid; conversion to the cor-
`responding acid chloride results in the formation of
`11C-labeled acid chloride, which is reacted with
`the secondary amine (see Fig. 1) to give the corre-
`[11C](1)-PHNO
`sponding amide. Synthesis of
`requires the additional reduction of the amide
`with lithium aluminum hydride resulting in the
`formation of [11C](1)-PHNO.26
`The most common chemical transformation of
`[11C]CO2
`is into [11C]methyliodide ([11C]CH3I)
`(Fig. 2), a highly reactive species that can be
`used to introduce 11C into biologically active mole-
`cules via alkylation of N-, O-, or S-nucleophiles
`(Fig. 3). The initial method for preparing
`[11C]CH3I
`involved passing [11C]CO2
`through
`a solution of lithium aluminum hydride (LiAlH4)
`followed by
`and tetrahydrofuran (see Fig. 3),
`quenching with hydrioidic acid. This method has
`been largely replaced with the gas phase method
`of synthesizing [11C]methyl iodide, which involves
`[11C]CO2
`the
`catalytic
`reduction
`of
`to
`[11C]methane ([11C]CH4), followed by iodination
`with molecular iodine (I2) to produce [11C]CH3I.
`An alternative method for preparing [11C]CH3I
`involves the direct production of [11C]CH4 by using
`a target gas composition of 10% H2/N2 in the
`target, followed by iodination with molecular I2
`(see Fig. 2). The rationale for using the in-target
`production of [11C]CH4 is the lower amount of
`methane in the atmosphere (~1.6 ppm) relative to
`CO2 (~300 ppm); in theory, this should result in
`improved specific activity of [11C]CH3I. However,
`the inexplicable low recovery for the in-target
`production of [11C]CH4 has prevented the wide-
`spread use of this method for the synthesis of
`[11C]CH3I. Average specific activities of 11C-
`labeled radiotracers using [11C]CH3I have been re-
`ported in the range of 2 to 10 Ci/mmol (74–370
`GBq/mmol). This specific activity is lower than the
`
`Petitioner GE Healthcare – Ex. 1033, p. 133
`
`

`

`134
`
`Mach & Schwarz
`
`CH3(CH2)14MgBr
`
`11COOH
`CH3(CH2)14
`
`[11C]Palmitate
`
`11CO2
`
`CH3MgBr
`
`CH3
`
`11COOH
`
`[11C]Acetate
`
`1. CH3CH2MgBr
`
`2. Phthaloyl chloride
`
`CH3CH2
`
`11COCl
`
`HO
`
`NH
`
`HO
`
`O
`
`N
`
`*
`
`11COCl
`
`LiAlH4
`
`1. cyclohexylMgCl
`
`2. Thionyl chloride
`
`N
`
`NH
`
`OCH3
`
`N
`
`N
`
`OCH3
`
`N
`
`N
`
`N
`
`N
`
`*
`
`O
`
`[11C]Way-100635
`
`HO
`
`N
`
`*
`
`[11C](+)-PHNO
`
`Fig. 1. Synthesis of [11C]palmitate, [11C]acetate, [11C](1)-PHNO and [11C]Way-100635 using [11C]CO2.
`
`maximum theoretic specific activity of 11C (see
`Table 1). Therefore, for an 11C-labeled radiotracer
`having a specific activity of 10 Ci/mmol, only 1 in
`1000 tracer molecules contain 11C, with the re-
`maining containing 12C.
`Another highly reactive form of 11C is [11C]meth-
`yltriflate ([11C]CH3OTf). [11C]CH3OTf is formed by
`passing gaseous [11C]CH3I over a column contain-
`ing silver triflate (AgOTf) at 200C (see Fig. 2).21
`The advantage of [11C]CH3OTf is that this method
`generally requires lower reaction temperatures,
`shorter reaction times, and lower amounts of the
`des-methyl precursor for labeling than is usually
`required to label a compound using [11C]CH3I.
`Because HPLC retention times of the des-methyl
`precursor are similar to the 11C-labeled N-methyl
`
`product, reducing the amount of precursor for
`the labeling reaction can simplify the HPLC purifi-
`cation of the radiotracer as a result of a reduced
`tendency of peak broadening by the des-methyl
`precursor. Although this time saving may seem
`trivial, reducing the synthesis time from 30 minutes
`to 25 minutes leads to an approximately 20%
`increase in specific activity of a 11C-labeled radio-
`tracer simply by reducing the loss of activity by
`radioactive decay. Examples comparing radiolab-
`eling with [11C]CH3I and [11C]CH3OTf are shown in
`Fig. 4.22,29–31 Perhaps the best example of the
`advantage of [11C]CH3OTf over [11C]CH3I is the
`synthesis of
`the b amyloid imaging agent
`[11C]PiB.
`[11C]PiB from
`The
`synthesis
`of
`[11C]CH3I requires the addition of a base such as
`
`11CO2
`
`LiAlH4
`
`11CH2OH
`
`HI
`
`11CH3I
`
`I2
`
`11CH4
`
`Ni/H2
`
`11CO2
`
`N2
`
`H2
`"In Target"
`
`Fig. 2. Synthesis of [11C]CH3I and [11C]CH3OTf.
`
`AgOSO2CF3
`
`11CH3OSO2CF3
`
`Petitioner GE Healthcare – Ex. 1033, p. 134
`
`

`

`Challenges for Developing PET Tracers
`
`135
`
`COOH
`
`H2N
`
`S-11CH3
`
`[11C](S)-Methionine
`
`11CH3
`
`N
`
`O
`
`OC
`
`H3O
`
`O
`
`H
`
`[11C]cocaine
`
`[11C]CH3OTf or [11C]CH3I
`
`HO
`
`S
`
`N
`
`11CH3
`N
`
`H
`
`OH
`
`O
`
`Cl
`
`H
`
`N
`
`NH
`
`O11CH3
`
`Cl
`
`[11C]Raclopride
`
`O
`
`N
`
`11CH3
`
`N
`
`O
`
`N
`
`F
`
`[11C]NMSP
`
`[11C]PiB
`Fig. 3. Examples of 11C-labeled radiotracers synthesized using [11C]CH3I or [11C]CH3OTf.
`
`Yield:
`[11C]CH3I: ~50%
`[11C]CH3OTf: ~75%
`
`F
`
`COOCH3
`
`11C
`H3
`[11C]CH3I/1 mg/110oC/3 min
`
`N
`
`COOCH3
`
`HN
`
`[11C]CH3OTf/0.15 mg/60oC/1 min
`
`F
`
`nor-CFT
`
`[11C]CFT
`
`Nucl. Med. Biol. 22: 335; 1995
`
`Yield:
`[11C]CH3I: ~45%
`[11C]CH3OTf: ~80%
`
`O
`
`N
`
`NH
`
`H
`O11CH3
`
`OCH3
`
`[11C]FLB 457
`
`[11C]CH3I/2.2 mg/110oC/3 min
`
`Br
`
`H
`
`N
`
`[11C]CH3OTf/0.3 mg/60oC/1 min
`
`J. Labelled Cpd.Radiopharm. 41: 545; 1998
`
`O
`
`NH
`
`OH
`
`OCH3
`
`FLB 604
`
`Br
`
`11CH3
`N
`H
`
`11CH3
`N
`H
`
`15% Yield EOS
`
`25% Yield EOS
`
`NS
`
`[11C]PIB
`
`1) 11CH3I/KOH
`DMSO
`
`HO
`
`NH2
`
`2) HCl/MeOH
`
`NS
`
`CH3OCH2O
`
`MOM-nor-PIB
`
`NS
`
`[11C]CH3OTf
`
`HO
`
`MEK
`
`H H
`
`N
`
`NS
`
`HO
`
`nor-PIB
`Fig. 4. Comparison of 11C-radiolabeling using [11C]CH3I and [11C]CH3OTf.
`
`[11C]PIB
`
`Petitioner GE Healthcare – Ex. 1033, p. 135
`
`

`

`136
`
`Mach & Schwarz
`
`sodium hydroxide because of the low reactivity of
`the amine nitrogen (see Fig. 4). This process
`requires the protection of the hydroxyl group of
`the precursor as the corresponding methoxymethyl
`(MOM) ether to prevent the unwanted reaction of
`[11C]CH3I at
`the oxygen atom. Consequently,
`a separate acid-catalyzed deprotection step is
`required to prepare [11C]PiB following N-alkylation
`of the amine group with [11C]CH3I.32,33 In compar-
`ison, because [11C]CH3OTf has a higher chemical
`reactivity than [11C]CH3I, the reaction at the aniline
`nitrogen occurs without the addition of a base
`catalyst, thereby eliminating the need to protect
`the phenol group as the corresponding MOM
`ether. This 1-step synthesis using [11C]CH3OTf is
`the preferred method for making [11C]PiB because
`of its shorter synthesis time and higher radiochem-
`ical yield.34–36
`
`18F Radiochemistry
`
`Two forms of 18F are used in the synthesis of PET
`radiotracers: nucleophilic and electrophilic. Nucle-
`ophilic fluoride is produced via the 18O(p,n)18F
`nuclear reaction using [18O]water as the target
`material. This process results in the formation of
`[18F]HF, which is transferred from the target to
`either a reaction vessel containing a base such
`as potassium carbonate (to make [18F]KF) or tetra-
`butylammonium hydroxide (to give [18F]TBAF).
`Alternatively,
`the radioactivity can be passed
`through an anion exchange resin, which traps the
`activity as [18F]fluoride. The activity is then
`removed from the anion exchange resin by elution
`
`with an aqueous solution of base (usually potas-
`sium
`carbonate
`or
`tetrabutylammonium
`to give [18F]fluoride as a salt
`hydroxide)
`(ie,
`[18F]KF or [18F]TBAF). An advantage of [18F]TBAF
`is that it can be directly solubilized into organic
`solvents such as acetonitrile and dimethylsulfox-
`ide, whereas [18F]KF requires the addition of the
`crown ether
`[2.2.2]kryptofix to solubilize the
`reactivity.
`[18F]Fluoride is incorporated into biologically
`active molecules via 2 types of reaction mecha-
`nisms: SN2 and SNAr2. The SN2 mechanism
`occurs when an appropriate leaving group is dis-
`placed from an aliphatic (ie, sp3) carbon atom
`(Fig. 5). The SNAr2 mechanism occurs when the
`leaving group is displaced from an aromatic (ie,
`sp2) carbon atom such as a benzene ring.
`Although fluoride is a relatively unreactive nucleo-
`phile, high yields of an 18F-labeled radiotracer can
`be obtained with the SN2 mechanism if
`the
`precursor contains a good leaving group such as
`a triflate (R 5 CF3) or mesylate (R 5 CH3) group.
`In the SNAr2 mechanism, the para position must
`be activated by an electron-withdrawing group to
`increase the rate of reaction so that an acceptable
`yield of the labeled compound can be obtained
`within the time constraints imposed by 18F. Exam-
`ples of activating groups are the cyano (CN), nitro
`(NO2), keto (RC 5 O) and aldehyde (HC 5 O)
`groups (see Fig. 5). Although the nitro group was
`historically the first leaving group used in radio-
`fluorinations using the SNAr2 mechanism, the tri-
`methylammonium group is more commonly used
`today because of its rapid rate of reaction and
`
`O
`
`O
`
`O
`
`S
`
`R
`
`[18F]MF
`
`∆ or
`microwave
`
`O
`
`R = CH3, CF3
`
`X
`
`LG
`
`[18F]MF
`
`∆ or
`microwave
`
`X
`
`18F
`
`18F
`
`O
`
`SN2 Mechamism
`
`SNAr2 Mechanism
`
`LG = Cl. Br, NO2, NMe3
`
`+
`
`X = CN, CHO, ketone, NO2, SO2R, COOR
`
`R = CH3, p-CH3Ph, CF3
`Fig. 5. Reaction mechanism for introducing 18F into organic molecules. M refers to a positive cation such as K1,
`Cs1, or (Bu)4N1.
`
`Petitioner GE Healthcare – Ex. 1033, p. 136
`
`

`

`Challenges for Developing PET Tracers
`
`137
`
`the ease of eliminating unreacted precursor result-
`ing from the high polarity of the charged trimethy-
`lammonium group.37 There are 2 methods used for
`increasing the rate of reactivity of [18F]fluoride:
`heating the reaction mixture (from 90 to 160 C)
`and microwave irradiation. Microwave irradiation
`is rapidly becoming the preferred method of nucle-
`ophilic [18F]fluoride incorporation because the
`reaction time needed to achieve a high radiochem-
`ical yield of the product is typically faster than the
`simple thermal method of incorporation.
`Although activation of the paraposition within 1 of
`these groups facilitates the incorporation of
`[18F]fluoride, it does not guarantee a high radio-
`chemical yield. An example of this is shown in
`Fig. 6. Whereas [18F]fluoride is readily incorporated
`into the nitroprecursor, resulting in a high radio-
`chemical yield of [18F]setoperone, similar reaction
`conditions result in only a low yield of [18F]N-methyl-
`spiperone even although both substrates have
`a ketone as the activating group in the paraposi-
`tion.19,38 The low yield of [18F]N-methylspiperone
`is likely caused by keto-enol tautomerization, which
`leads to the formation of the nonreactive enol (see
`Fig. 6). Because of the long half-life of 18F (110
`minutes), a multistep synthesis of [18F]NMSP was
`developed39 (Fig. 7), which afforded yields suitable
`for human imaging studies.40
`
`A second example of the SNAr2 reaction mech-
`anism for incorporating 18F into biologically active
`molecules involves the displacement of a leaving
`group in either the 2 or 6 position of a pyridine
`ring. Because pyridine is a p-deficient heteroaro-
`matic ring system, it is prone to nucleophilic attack
`at the 2, 4, and 6 positions. Therefore, a pyridine
`ring does not require the presence of an elec-
`tron-withdrawing group to activate the ring system
`to nucleophilic attack by [18F]fluoride. This
`strategy has been used extensively in the
`synthesis of
`ligands for imaging nicotinic a4b2
`receptors (Fig. 8).41–44
`introducing high
`An alternative strategy for
`specific activity, nucleophilic [18F]fluoride into
`ligands is to use an 18F-labeled prosthetic group.
`This method uses the same basic principle as
`labeling a molecule with [11C]CH3I, which is essen-
`tially an 11C-labeled prosthetic group.
`In this
`approach, 18F is incorporated into an organic
`molecule, which is
`then used to alkylate
`leading to an 18F-labeled radio-
`a precursor
`tracer.17 As with [11C]CH3I, the most common
`reaction is the alkylation of a nitrogen atom. The
`following different 18F-labeled prosthetic groups
`have been used to date: [18F]2-fluoroethoxytriflate
`[18F]2-fluoroethyltosylate45,46;
`[18F]4-
`or
`and,
`iodide (or bromide).47–49 Examples
`fluorobenzyl
`
`S
`
`N
`
`N
`
`O
`
`O
`
`N
`
`S
`
`N
`
`N
`
`O
`
`O
`
`N
`
`[18F]KF
`Kryptofix
`
`180oC
`30 min
`
`NO2
`
`[18F]Setoperone
`
`18F
`
`Yield: 55%
`
`O2N
`
`Keto
`
`O
`
`OH
`
`N
`
`N
`
`O
`
`N
`
`O
`
`N
`
`O2N
`
`Enol
`
`CH3
`
`N
`
`[18F]KF
`Kryptofix
`
`O
`
`N
`
`CH3
`
`N
`
`O
`
`N
`
`18F
`
`[18F]NMSP
`
`Yield: 1 - 3%
`
`CH3
`
`N
`
`Fig. 6. Examples of nucleophilic incorporation of [18F]fluoride. Note the difference in radiochemical yield even
`although both substrates are activated by a ketone group in the para position.
`
`Petitioner GE Healthcare – Ex. 1033, p. 137
`
`

`

`138
`
`Mach & Schwarz
`
`O2N
`
`18F
`
`O
`
`O
`
`O
`
`O
`
`[18F]CsF
`
`aq. DMSO
`160oC
`
`18F
`
`Cl
`
`amine/KI
`
`HCl/CH3OH
`
`CH3
`
`N
`
`O
`
`N
`
`N
`
`18F
`
`[18F]NMSP
`
`Overall Yield: 7-15%
`
`Fig. 7. Synthesis of [18F]NMSP by the PET group at Brookhaven National Laboratory.
`
`of the use of these prosthetic groups in radiotracer
`synthesis are shown in Figs. 9 and 10.
`A recent development in PET radiochemistry is
`the use of copper-assisted 1,3-dipolar cycloaddi-
`tion reactions to prepare 18F-labeled compounds.
`The method, often referred to as click chemistry,
`uses either an 18F-labeled fluorinated acetylene
`or an 18F-labeled organic azide as the prosthetic
`group (Fig. 11). The first click radiolabeling reac-
`tions used simple [18F]fluoroalkynes50 and [18F]2-
`fluoroethylazide.51 Reported radiochemical yield
`using this strategy has been high, often in excess
`of 80%.50,51 This labeling strategy has been used
`
`most often in the radiolabeling of peptides
`because it avoids the need to protect the multiple
`functional groups in a polypeptide that could react
`with the prosthetic groups currently used in
`producing 18F-labeled peptides (Fig. 12). Several
`second-generation 18F-labeled click synthons
`have been introduced recently52–54 and are shown
`in Fig. 11. A review of the use of click chemistry in
`PET radiotracer design was recently published by
`Glaser and Robbins.55
`A second method for introducing 18F into biolog-
`ically active molecules is through the use of an
`electrophilic fluorination reaction with [18F]F2.
`
`NMe3
`
`+
`
`K18F/K222
`
`DMSO
`
`70% yield
`
`Boc
`
`N
`
`18F
`
`N
`
`Ding et al.,
`Synapse 1996
`
`18F
`
`N
`
`Horti et al.,
`JLCR 1996
`
`HN
`
`Br
`
`K18F/K222
`
`DMSO
`
`50 min
`10% yield
`>2000 mCi/mmol
`
`N
`
`N
`
`Boc
`
`N
`
`HN
`
`Dolle et al,
`J. Med. Chem. 1999
`
`70 - 90%
`
`18F
`
`N
`
`NH
`
`TFA
`
`CH2CH2
`RT/1 min
`
`K18F/K222
`DMSO/150oC/2 min
`or
`microwave 1 min
`
`N
`
`+Me3N
`
`N B
`
`oc
`
`Fig. 8. Synthesis of 18F-labeled radiotracers involving the direct introduction of 18F into the 2-position of a pyri-
`dine ring.
`
`Petitioner GE Healthcare – Ex. 1033, p. 138
`
`

`

`Challenges for Developing PET Tracers
`
`139
`
`O
`
`N
`
`18F
`
`O
`
`N
`
`N
`
`spiperone
`
`18F
`
`[18F]Fluoroethyspiperone
`
`CF3SO2O
`
`[18F]KF
`
`CF3SO2O
`
`OSO2CF3
`
`18F
`
`Bis-triflate
`
`des-methyl-
`flumazenil
`
`F
`
`N
`
`O
`
`N
`
`O
`
`N
`
`OCH2CH3
`
`18F
`
`[18F]Fluoroethyl-flumazenil
`Fig. 9. Radiosynthesis using the [18F]2-fluoroethoxytriflate as a prosthetic group.
`
`There are 2 different ways to produce electrophilic
`[18F]F2. The first method, which requires a cyclo-
`tron designed to accelerate deuterons,
`is the
`20Ne(d,a)18F nuclear reaction (Fig. 13). The neon
`target gas contains ~2% F2 to complete the
`
`[18F]F2. The second
`in-target production of
`method for producing [18F]F2
`involves
`the
`18O(p,n)18F nuclear reaction. This method, which
`is generally used on proton-only cyclotrons,
`requires
`a multistep process
`to produce
`
`O
`
`OCH3
`
`NH
`
`N
`
`NH2
`
`[18F]FCP
`
`Cl
`
`18F
`
`N
`
`COCH2CH3
`
`Cl
`
`18F
`
`[18F]FCT
`
`CHO
`
`CHO
`
`[18F]CsF
`
`aq. DMSO
`110oC
`
`1. LiBH4
`
`2. HI
`
`+
`NMe3
`
`18F
`
`CH2I
`
`18F
`
`[18F]4-FBI
`
`N
`
`N
`
`OH
`
`18F
`
`+
`
`P
`
`18F
`
`[18F]FBT
`
`[18F]Fluorobenzyl Triphenylphosphonium
`Fig. 10. Examples of 18F radiolabeling using the prosthetic group, [18F]4-fluorobenzyl iodide. (Data from Refs.47–49)
`
`Petitioner GE Healthcare – Ex. 1033, p. 139
`
`

`

`140
`
`Mach & Schwarz
`
`18F
`
`n
`
`18F
`
`N
`
`N+
`
`N–
`
`Marik and Sutcliffe Tet. Lett. 47: 6681-84; 2006
`
`Glaser and Arstad Bioconjugate Chem. 18: 989-993; 2007
`
`N–
`
`N+
`
`N
`
`Thonon et al., Bioconjugate Chem. 20: 817-8236; 2009
`
`O
`
`N
`
`18F
`
`18F
`
`O
`
`N
`
`18F
`
`[18F]FPyKYNE
`Kuhhast et al., J. Labelled. Cmpd.
`Radiopharm. 51: 336-342; 2008
`
`[18F]FPy5yne
`Inksteret al., J. Labelled. Cmpd.
`Radiopharm. 51:444-452; 2008
`Fig. 11. Prosthetic groups used in click labeling with 18F.
`
`[18F]F2.56 The first step involves irradiation of 2%
`F2/Ar mixture to passivate the target. The target
`is then filled with [18O]O2, and a second irradiation
`is conducted to produce 18F-labeled species that
`are deposited on to the interior surface of the
`target. This deposition of the 18F on to the target
`surface enables the recovery of the [18F]O2 target
`gas. Once the [18O]O2 has been recovered, the
`target is filled with 2%F2/Ar and a third irradiation
`is conducted to recover the [18F]F2. Electrophilic
`fluorination with [18F]F2 is the preferred method
`for making [18F]fluoro-L-dihydroxyphenylalanine
`([18F]FDOPA)57 and [18F]fluoro-meta-tyrosine,58
`radiotracers that measure presynaptic dopami-
`nergic terminal density (see Fig. 13). Other PET
`radiotracer synthesized using [18F]F2 include 2b-
`carbomethoxy-3b-(4-[18F]-fluorophenyl)tropane,
`[18F]CFT,59 which labels the dopamine transporter
`(another marker of dopamine terminal density) and
`2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentaflu-
`oropropyl)-acetamide (EF-5) labeled with 18F-fluo-
`rine, [18F]EF-5, a radiotracer for imaging hypoxia.60
`The main drawback to using electrophilic fluorina-
`tion for introducing 18F is low specific activity that
`is achieved using [18F]F2, because of the require-
`ment of adding unlabeled F2 to recover the radio-
`activity from the target. However, a method for
`producing electrophilic [18F]F2 from nucleophilic
`
`[18F]fluoride was reported by Bergman and Solin
`in 1997.61 Although the addition of unlabeled F2
`required to form [18F]F2, a significant
`is still
`increase in specific activity of [18F]FDOPA and
`[18F]CFT have been reported using this method
`of electrophilic radiofluorination.61
`
`FACTORS DETERMINING THE ABILITY
`OF PET RADIOTRACERS TO CROSS
`THE BLOOD-BRAIN BARRIER
`
`There are several factors to contend with in the
`design of a radiotracer for imaging targets within
`the CNS. A description of these factors of prereq-
`uisites for CNS PET radiotracers can be found in
`the review by Ametamey and colleagues.62 A brief
`summary is provided in the next section.
`
`Affinity and Selectivity for the Target
`Receptor or Enzyme
`
`Ideally, a PET probe should possess a high (ie,
`nanomolar) affinity and high selectivity (>100-
`fold) for the target protein versus other proteins
`in the CNS. However, the exact affinity of a suitable
`PET ligand for
`its target protein depends on
`a variety of factors, including the density of the
`receptor or protein target, the presence of endog-
`enous ligands that could compete with the
`
`Petitioner GE Healthcare – Ex. 1033, p. 140
`
`

`

`Challenges for Developing PET Tracers
`
`141
`
`NH
`
`O
`
`HN
`
`NH
`
`O
`
`N3
`
`"Click Chemistry"
`
`Cu2+/ascorbate
`15 min/room temperature
`
`NH2
`
`O
`
`O
`
`HN
`
`O
`
`NH
`
`TsO
`
`[18F]KF
`
`18F
`
`N3
`
`CH3CN, 15 min
`
`NH2
`
`NH
`
`O
`
`O
`
`O
`
`HN
`
`O
`
`NH
`
`HN
`
`O
`
`NH
`
`N
`
`N
`
`N
`
`18F
`
`Yield: 92%
`Fig. 12. Click chemistry and an example of its use in 18F-labeled peptide chemistry. (Data from Glaser M and E
`‘‘Click labeling’’ with 2-[18F]fluoroethylazide for positron emission tomography. Bioconjug Chem
`Arstad.
`2007;18(3):989–93.)
`
`radiotracer for ligand binding site on the protein,
`and the level of nonspecific binding of the tracer.
`Tracers having a high affinity (ie picomolar) for
`the target receptor often bind irreversibly to the
`receptor. That is, there is no washout of radio-
`tracer from regions of the brain expressing the
`receptor during the data acquisition period of
`PET. The uptake of tracers displaying irreversible
`kinetics in vivo can be dependent on and influ-
`enced by differences in cerebral blood flow.
`Therefore, kinetically irreversible tracers often
`require correction for differences in cerebral blood
`[15O]water study or K1 estimates by
`flow (ie,
`analyzing the early part of the tissue-time activity
`curves) to obtain accurate measures of the density
`or binding potential of the receptor or protein
`under
`investigation. A prime example of
`this
`concept is the development of PET radiotracers
`for
`imaging striatal versus extrastriatal D2/D3
`[11C]Raclopride, which has ~5 nM
`receptors.
`affinity for D2 and D3 receptors,
`is a suitable
`
`radiotracer for imaging striatal D2/D3 receptors.
`There is a high density of D2/D3 receptors in the
`caudate and putamen and the tracer binds revers-
`ibly to D2/D3 receptors. However, it is not generally
`useful for imaging extrastriatal D2/D3 receptors,
`which are expressed in lower density in dopamine
`receptor–enriched extrastriatal regions versus the
`caudate and putamen. The short half-life of 11C
`[11C]raclopride from
`and rapid dissociation of
`dopamine receptors results in a low signal-to-
`noise ratio in regions with a low density of D2/D3
`receptors. The best radiotracers for imaging extra-
`striatal D2/D3 receptors are [18F]fallypride and
`[18F]FLB-457, which have a picomolar affinity for
`D2 and D3 receptors, and a low level of nonspecific
`binding. These properties (picomolar affinity and
`low nonspecific binding) make it possible to obtain
`stable measures of extrastriatal
`receptors ex-
`pressing in brain regions containing a low density
`of D2 and D3 receptors. The irreversible binding
`[18F]fallypride and [18F]FLB-457 to D2/D3
`of
`
`NH2
`
`OH
`
`O
`
`NH2
`
`OH
`
`O
`
`Petitioner GE Healthcare – Ex. 1033, p. 141
`
`

`

`142
`
`Mach & Schwarz
`
`BocO
`
`BocO
`
`BocO
`
`COOCH2CH3
`[18F]F2
`
`NHCHO
`)
`
`3
`
`Sn(CH
`
`3
`
`CHCl3
`
`HCl
`140oC for 5 min
`
`HO
`
`HO
`
`COOH
`
`NH2
`
`18F
`
`[18F]F-DOPA
`
`COOCH2CH3
`[18F]F2
`
`NHBoc
`
`Sn(CH
`
`)
`
`3
`
`3
`
`CHCl3
`
`HBr
`140oC for 5 min
`
`HO
`
`COOH
`
`NH2
`
`18F
`
`[18F]fluoro-meta-tyrosine
`
`H