Pd0�Mediated Rapid C�[11C]Methylation and C�[18F]Fluoromethylation: Revolutionary Advanced Methods for General Incorporation of Short�Lived Positron�Emitting 11C and 18F Radionuclides in an Organic Framework

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5
Pd0–Mediated Rapid C–[11C]Methylation and
C–[18F]Fluoromethylation: Revolutionary
Advanced Methods for General Incorporation
of Short–Lived Positron–Emitting 11C and
18F Radionuclides in an Organic Framework
Masaaki Suzuki1, Hiroko Koyama2,
Misato Takashima-Hirano1 and Hisashi Doi1
1RIKEN Center for Molecular Imaging Science (CMIS) and
2Gifu University Graduate School of Medicine
Japan
1. Introduction
The study of in vivo bioscience and medical treatment from molecular point of view requires
the precise evaluation of molecule behavior in living systems, especially involving the
human body. Positron emission tomography (PET) is a non–invasive imaging technology
with a good resolution, high sensitivity, and accurate quantification, which makes it
possible to timely and spatially analyze the dynamic behavior of molecules in in vivo
systems using a specific molecular probe labeled with positron–emitting radionuclides such
as 11C, 13N, 18F, and 76Br (Phelps, 2004). PET has been extensively used for the diagnosis of
diseases such as cancers, cerebral dysfunction, and etc., and recently, in medical checkups as
an early detection approach. In the current paradigm shift to drug discovery, PET molecular
imaging will provide an important new scientific platform to execute human microdosing
trials during the early stage of drug development, especially from the viewpoint of
promoting evidence–based medicine (Lappin & Garner, 2003; Bergström et al., 2003). A core
concept and the driving force of molecular imaging would truly be “Seeing is Believing”. It
is of significant value to unveil the vital functions and phenomena of living systems by
molecular imaging the in vivo behavior of a ligand and the localization of a biologically
significant target molecule. The potential of PET molecular imaging in an interdisciplinary
scientific area strongly depends on the availability of suitable radioactive molecular probes
with specific biological functions. The development of biologically significant novel PET
probes will be accomplished by the combination of an efficient synthetic strategy for
designed molecules and new advances in the field of labeling chemistry (Schubiger et al.,
2007).
Among the short–lived positron–emitting radionuclides, 11C and 18F with a half–life of 20.4
and 109.8 min, respectively, have often been used for radiolabeling as the most significant
radionuclides from both a chemical and biological perspective as well as from the viewpoint

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of radiation exposure safety. With respect to the 11C–incorporation on organic carbon
frameworks, we have been developing multiple–type Pd0–mediated rapid [11C]methylations
onto an arene including a heteroaromatic compound, and alkene, alkyne and alkane
structures by [11C]carbon–carbon bond forming reactions (rapid C–[11C]methylations) using
[11C]methyl iodide and an excess amount of an organostannane or organoboron within a
very short time span (5 min) (Suzuki et al., 1997; Hosoya et al., 2004; Hosoya et al., 2006; Doi
et al., 2009; Suzuki et al., 2009). These labeling reactions provide a high generality and
practicability as groundbreaking methods for introducing the [11C]methyl group into almost
any organic framework. Regarding the 18F radionuclide, a rather longer half–lived positron
emitter than 11C, the 18F–labeling can be mainly accomplished by ordinary methods
involving nucleophile substitution with the 18F anion, as exemplified by the synthesis of 2-
[18F]fluoro-2-deoxy-D-glucose ([18F]FDG) (Ido et al., 1978) and 3’-[18F]fluoro-3’-
deoxythymidine ([18F]FLT) (Grierson et al, 1997, as cited in Bading & Shields, 2008). In this
chapter, newly advanced methodologies for introducing the short–lived 11C radionuclide
into various carbon frameworks (rapid C–[11C]methylations) and the rather longer half–
lived 18F radionuclide into a benzene framework (C–[18F]fluoromethylation) are described in
detail in addition to their applications for radiolabeling biologically and clinically significant
organic molecules.
2. PET molecular imaging technology–principle, properties, and benefits
The short–lived positron emitting radionuclide 11C was first produced by Crane and
Lauritsen in 1934 (Lauritsen et al., 1934, as cited in Allard et al., 2008). They investigated the
physical properties of this radionuclide and demonstrated that 11C undergoes + decay with
a half–life of 20.4 min, yielding 11B as the stable nuclide (Figure 1). A positron (positively
charged electron, e+) ejected by this process collides with a nearby electron within a few
millimeters in tissue to produce two high–energy –ray photons of 511 keV each. These
photons travel in opposite directions at 180 degrees, penetrating the body, and can be
detected by a pair of opposing scintillation detectors. If the two opposite detectors are
simultaneously hit, it is assumed that the photons come from the same decay event. The
data are fed to a computer system that can reconstruct the three–dimensional tomographic
imaging and provide a highly accurate quantitative analysis of a radiolabeled drug in a
body over time, measured as becquerel (Bq) per pixel. Because of the really high specific
radioactivity of positron–emitter labeled compounds, PET enables in vivo imaging using an
extremely small mass of the compound (sub–femtomole), namely, at extremely low
concentrations (sub–picomolar) far below the critical concentration of pharmacological
effects. The other typical positron–emitting radionuclides for PET studies, along with their
half–lives (t1/2) are: 15O (t1/2 = 2.07 min); 13N (t1/2 = 9.96 min); 68Ga (t1/2 = 67.6 min); 18F (t1/2 =
109.7 min); 64Cu (t1/2 = 12.7 h). The benefits of the use of PET technology in scientific
research areas are as follows: (1) O, N, and C are included as ubiquitous elements
constituting a biologically active compound in nature, providing the diversity of the labeled
compounds without modifying the properties (or functions) of the molecule; (2) the
molecule including the positron emitting radionuclide can be externally and quantitatively
measured using a PET camera with a high resolution and sensitivity; (3) a short half–life is
very relevant to human PET studies in terms of the high required safety for radiation
exposure.

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Fig. 1. Principle of the brain imaging by PET as shown by 11C to 11B decay.
3. Rapid chemistry needed for 11C–labeling–working against time
The special aspects of PET radiochemistry such as short half–lives, extremely small amounts
of available radionuclides, and relatively high–energy radiation impose severe restrictions
on the synthesis of PET probes. In general, the synthesis of a pure, injectable 11C–labeled
probe must be accomplished within 2–half lives of ca. 40 min due to the quick decay of the
radioactivity. The synthesis process for the pharmaceutical formulation includes the
following steps: (1) derivatives of a 11C isotope produced by a cyclotoron to an appropriate
labeling precursor such as 11CH4, 11CH3I, 11CH3OTf, 11CO, and 11CO2; (2) evaluation of the
reaction efficiency (radiochemical yield) by analytical high performance liquid
chromatography (HPLC) after the 11C–labeling of the target probe; (3) work–up and
chromatographic purification of the desired 11C–labeled probe; and (4) preparation of an
injectable solution for an animal/human PET study (pharmaceutical formulation).
Therefore, the time allowed for a 11C–labeling reaction should be less than 5 min, inevitably
necessitating a rapid chemical reaction. Another difficulty encountered in the synthesis of a
11C–labeled PET probe is the availability of an extremely small amount (nano–mol level) of
the 11C–labeling precursor such as [11C]CH3I. Therefore, the labeling reaction is usually
carried out with a large amount (milli–gram level) of the reacting substrate to promote the
reaction. In addition, the efficient and secure purification of a small amount of the
synthesized 11C–labeled probe from a large amount of the remaining substrate must be
considered since a PET probe is usually intravenously injected into both living animals and
humans.
4. Attractive features of rapid C–[11C]methylation–four kinds of rapid
C–[11C]methylations
Thus far, in the field of PET chemistry, the [11C]methylation of the hetero atoms of N, O, and
S has mainly been explored and utilized because of its simple reaction conditions namely,
only by mixing 11CH3I and a large amount of the substrate (Allard et al., 2008). However, a
carbon–hetero atom bond tends to be readily metabolized to produce 11CH3OH, 11CH2O and
H11COOH, which are dispersed in whole organs, thus decreasing the credibility of a PET
image. It could be said that “the facts are the enemy of the truth.” We here considered that
the [11C]methylation by [11C]C–C bond formation (referred to as C–[11C]methylation)
+ decay
proton
neutron
11C
11B
180°
e–: electron
e+: positron
photon
511 keV
photon (ray)
511 keV
PET detector
computer-reconstructed
tomographic image

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Positron Emission Tomography – Current Clinical and Research Aspects
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(Suzuki et al., 1997) will have a number of benefits because of the following reasons: (1) The
[11C]methyl group introduced into a carbon will be metabolically stable, and therefore, such
a 11C–labeled probe will provide a highly credible PET image; (2) the methyl group is the
smallest nonpolar functional group, and therefore, the introduction of a methyl group has
the least influence on the biological activity of the parent compound; furthermore, the
methyl group is rather positively used in drug design as magic methyl to control the
lipophilicity as well as the fixation of the conformation of a molecule; (3) a short half–life of
the 11C–incorporated probe is favorable for the rapid screening involving optimization of
reaction conditions and the evaluation of the in vivo behavior, thus allowing several trials
per day. Accordingly, we have devised a plan to realize four types of rapid C–
[11C]methylations for arene, alkene, alkyne, and alkane frameworks (Figure 2), which allow
the 11C–labeling of almost any organic compound. The following pharmacokinetic
(PK)/pharmacodynamic (PD) studies in in vivo systems by PET provide a key methodology
to eventually promote “evidence–based medicine” at the molecular level. With regard to
such a [11C]C–C bond forming reaction, organometallic compounds comprised of the group
IA and IIA metals were previously used. For example, [methyl–11C]thymidine was prepared
in a radiochemical yield of 20% with radiochemical purities >99% by the reaction of
[11C]CH3I with the lithiated derivative obtained from the bromo precursor (Sundoro-Wu et
al., 1984). In such a reaction, however, the use of a moisture–sensitive organolithium
compound is difficult to justify the stoichiometry for an extremely small amount of
[11C]CH3I, resulting in the inevitable production of a large amount of an undesired
demethylated derivative due to the use of an excess amount of the lithiated substrate.
Furthermore, the undesired side reaction such as the rearrangement of the lithiation position
occurs under such drastic conditions. Consequently, the tedious separation of demethylated
side products and regioisomers is inevitably needed to purify the desired compound. Thus,
the reaction based on the use of “soft metalloids” as nucleophilic substrates was ideal for
this requirement, if realized, as described in detail in section 5.

Fig. 2. Attractive features of rapid C–[11C]methylations.

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119
5. Benefits of using of an organostannane as a trapping substrate for
[11C]methyl Iodide
A general protocol for the rapid C–methylation was established for the first time based on a
Stille–type reaction using phenyltributylstannane and CH3I, then [11C]CH3I, a frequently–
used 11C–labeling precursor (Suzuki et al., 1997). The Stille reaction is among the most
generally used C–C bond forming reactions in organic synthesis as a reaction of an
organometallic (–metaloid) reagent with an organic electrophile (Stille, 1986). The organotin
compounds can be prepared by a number of routes even if containing a variety of reactive
functional groups. Moreover, the reagent is not particularly oxygen or moisture sensitive. In
the palladium(0)–catalyzed coupling of an organic electrophile with an organotin reagent,
essentially only one of the groups on the tin atom selectively enters into the coupling
reaction, namely an unsymmetrical organotin reagent comprised of three simple alkyl
(except methyl) groups, and the fourth group, such as the arenyl, alkenyl, or alkynyl group.
The latter fourth group can selectively transfer. The Stille reaction was thought to be useful
for our purpose because of its favorable properties of the triorganostannane compounds,
such as (1) their high tolerance to various chemical reactions and chromatographic
purification conditions, enabling the incorporation of a radioisotope as the final step of the
PET–probe synthesis; and (2) the extremely low polarity of a trialkyltin(IV) derivative,
enabling an easy separation of the desired product from a large amount of the remaining tin
substrate. However, to the best of our knowledge, at that time, there was little information
on the Stille reaction using methyl iodide as an sp3–hybridized carbon partner in
comparison to its wide applicability to sp2–hybridized arenyl or sp3–hybridized allylic
halides; it seemed rather difficult to realize the methylation in high yield due to the
unavoidable scrambling between the methyl group in methyl iodide and phenyl groups in
the triphenylphosphine ligand, P(C6H5)3, by the reaction of methyl iodide with the less
reactive phenyltributylstannane in the presence of Pd{P(C6H5)3}4 (Morita et al., 1995). The
use of the higher reactive phenyltrimethylsytannane as a substrate also induces the
competition between 11CH3 in 11CH3I and CH3 groups in the stannane to produce
[11C]ethane as a byproduct (Suzuki et al., 1997, also see section 6). Furthermore, the labeled–
compound obtained from the trimethyltin derivative resulted in a much lower specific
activity than the tributyltin derivative (Samuelsson & Långström, 2003; Madsen et al., 2003).
It should be added that tributyltin derivative is practically non-toxic, while the trimethyl-
and triethyltins have a significant acute toxicity (Smith, 1998; Buck et al., 2003).
Consequently, we have been obliged to devise new reaction conditions capable of
promoting a rapid cross–coupling reaction using the less reactive tributyltin derivative as a
substrate for trapping [11C]CH3I.
6. Realization of Pd0–mediated rapid C–methylations by the reaction of
methyl iodide with an excess amount of arenyltributylstannanes (rapid
coupling between sp2(arenyl)– and sp3–hybridized carbons)
Keeping the 11C radiolabeling conditions of a PET–probe synthesis in mind, we set up a
model reaction using methyl iodide and an excess amount of phenyltributylstannane (1)
(CH3I/1 = 1:40 in molar ratio) to possibly restrict the reaction time to less than 5 min (Table
1) (Suzuki et al., 1997). The yield of the methylated product, toluene (2), was determined on
the basis of the CH3I consumption. As anticipated, the conventional Stille–reaction

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conditions with a reaction time of 30 min did not give the desired product at all (Table 1,
Entry 1), leading us to introduce the concept of coordinative unsaturation to activate the
palladium catalyst. Thus, we found that the use of a coordinatively unsaturated Pd0
complex, Pd{P(o-CH3C6H4)3}2 (Paul et al., 1995), generated in situ by mixing Pd2(dba)3 (dba:
dibenzylideneacetone) and the sterically bulky tri-o-tolylphosphine (P(o-CH3C6H4)3; cone
angle, 194°) (Tolman, 1977) instead of triphenylphosphine (P(C6H5)3; cone angle 145°)
(Tolman, 1977), significantly increased the coupling efficiency (76%, Table 1, Entry 2). Next,
we introduced an additional concept to shorten the reaction time (from 30 min to 5 min); the
simple heating (80 °C) was less effective for lowering the yield, but the stabilization of the
transiently formed palladium catalyst, strongly solvated by N,N-dimethylformamide (DMF),
effectively suppressed the decrease in the yield to a considerable extent. Furthermore, we
intended to enhance the reactivity by adding a CuI salt with the expectation of Sn to Cu
transmetallation, and K2CO3 in order to react with the (n-C4H9)3SnX (X = I and/or Cl)
generated during the reaction to neutralize the reaction system. Thus, the reaction using the
CH3I/1/Pd2(dba)3/P(o-CH3C6H4)3/CuCl/K2CO3 system (1:40:0.5:2:2:2) in DMF at 60 °C for 5
min gave the desired product in 91% yield (Table 1, Entry 6) (Suzuki et al., 1997). It should be
noted that when phenyltrimethylstannane was used instead of phenyltributylstannane, the
reaction produced toluene (2) in >100% yield (122–129%) together with ethane, indicating
the unexpected cross–coupling reactions (scrambling) between the methyl in methyl iodide
and the methyl on the tin atom. The reaction between the phenyl and methyl on the tin atom
was also contaminated to yield toluene (undesired product in actual PET probe synthesis) to
a significant extent (Suzuki et al., 1997), thereby decreasing the yield of the desired
[11C]toluene. The specific radioactivity of desired [11C]toluene would also be deduced by
the contamination of [12C]toluene formed by the reaction between the methyl


Entrya

1
Pd0 comlex
(μmol)
Pd{P(C6H5)3}4
(10)
Pd2(dba)3 (5)
Pd2(dba)3 (5)
Pd2(dba)3 (5)
Pd2(dba)3 (5)
Ligand (L) and/or
additive (μmol)
—
Pd0:L
(mol ratio)
—
Solvent Temp. Time Yield of 2
(°C) (min)
DMSO40
(%)a
0 30
2
3
4
5
P(o-CH3C6H4)3 (20)
P(o-CH3C6H4)3 (20)
P(o-CH3C6H4)3 (20)
P(o-CH3C6H4)3 (20),
CuI (20)
P(o-CH3C6H4)3 (20),
CuCl (20), K2CO3 (20)
1:2
1:2
1:2
1:2
DME
DME
DMF
DMF
40
80
80
60
30
5
5
5
76
41
63
3
6 Pd2(dba)3 (5) 1:2 DMF 60 5 91
aReaction was carried out with CH3I (10 mol), stannane 1 (400 mol), and Pd0 (10 mol). bYield was
determied by GLC analysis based on CH3I consumption. dba: dibenzylideneacetone; DMSO: dimethyl
sulfoxide; DME: 1,2-dimethoxyethane; DMF: N,N-dimethylformamide.
Table 1. Rapid cross–coupling of methyl iodide and phenyltributylstannane (1).
CH3I
+
Sn(n-C4H9)3
Pd0 catalyst
Additive
Solvent (1 mL)
CH3
12

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groups and phenyl groups on the tin atom (Madsen et al., 2003). It was assumed from these
results that the reaction of [11C]CH3I and phenyltrimethylstannane under PET radiolabeling
conditions would produce the undesired radioactive and volatile [11C]ethane. As described
later (see the section 8.2), these phenomena were observed during the palladium–mediated
reaction of 1-(2’-Deoxy-2’-fluoro--D-arabinofuranosyl)-5-(trimethylstannyl)uracil
synthesize 1-(2’-Deoxy-2’-fluoro--D-arabinofuranosyl)-[methyl–11C]thymidine (Samuelsson
& Långström, 2003). Therefore, we concluded that the arenyltributylstannane, though less
reactive, would be a much more suitable coupling partner than the arenyltrimethylstannane
in view of the increased efficiency of the reaction, relatively low toxicity, and the safety of
the radiation exposure.
to
The conditions of the reaction are significantly different from those of the originally
reported Stille coupling reaction. Thus, the coupling of methyl iodide and
phenyltributylstannane probably proceeds by the mechanism proposed in Equations 1–5
(Suzuki et al., 1997). In the first step, methyl iodide undergoes oxidative addition with

a Pd0 species to generate methyl–PdII iodide 3 (oxidative addition, Eq. (1)). The PdII complex
3 may directly react with the phenylstannane 1 to afford the (methyl)(phenyl)PdII complex 6
(substitution, Eq. (4)); however, the formation of the latter would be facilitated by the
phenyl–copper compound 4 formed by the preceding Sn/Cu transmetallation (Eq. (2)). The
effect of K2CO3 would be explained by the neutralization of (n-C4H9)3SnX to form the stable
bis(tributylstannyl)carbonate 5 (Eq. (3)). At the same time, K2CO3 serves to synergically
work with a CuI salt to promote the Sn/Cu transmetallation (Eqs. (2) and (3)) (Hosoya et al.,
2006). Finally, toluene is formed by reductive elimination from the PdII complex 6 (reductive
elimination, Eq. (5)). The significant ligand effect of tri-o-tolylphosphine is attributed to its
considerable bulkiness (cone angle = 194°, which is greater than that in tri-tert-
butylphosphine (182°)) (Tolman, 1997), which facilitates the generation of the coordinatively
unsaturated Pd0 and PdII intermediates (Louie & Hartwig, 1995). Transmetallation to give 6
and/or the reductive elimination of toluene requires the formation of the tricoordinate PdII
complex. DMF may stabilize such Pd intermediates even at high temperatures. It should be
noted that J. K. Stille et al. previously reported the reaction of methyl iodide and p-
methoxyphenyltributylstannane in the presence of Pd{P(C6H5)3}4 at 50 °C for 24 h, in which
the scrambling reaction between the methyl and the phenyl groups in the methyl iodide and
triphenylphosphines, respectively, preferably occurred to give the desired p-
methoxytoluene in only 3% together with 1-methoxy-4-phenylbenzene as the major
CH3I+[Pd{P(o-CH3C6H4)3}2]
+CuX
+
+ [Pd{P(o-CH3C6H4)3}]
[Pd(CH3)I{P(o-CH3C6H4)3}]
3
[Cu(C6H5){P(o-CH3C6H4)3}] (n-C4H9)3SnX
4
1
6
toluene (2)
[Pd(CH3)(C6H5){P(o-CH3C6H4)3}]
6
CH3–C6H5
P(o-CH3C6H4)3
C6H5Sn(n-C4H9)3
+ P(o-CH3C6H4)3
[(n-C4H9)3SnO]2C=O
5
K2CO3
+
X = Cl or Br; M = (n-C4H9)3Sn or Cu{P(o-CH3C6H4)3}n.
3 +C6H5M
+MI
2 (n-C4H9)3SnX+
+ 2 KX
(1)
(2)
(4)
(5)
(3)

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byproduct in 8% yield, suggesting that the promotion of the Stille reaction using methyl
iodide as an sp3–carbon partner could be difficult (Morita et al., 1995) until our successful
result was demonstrated (Suzuki et al., 1997).
7. Application for the synthesis of 15R-[11C]TIC methyl ester as specific probe
for prostaglandin receptor (IP2) in the central nervous system
In a preceding study of prostaglangin (PG), we succeeded in developing (15R)-16-m-tolyl-
17,18,19,20-tetranorisocarbacyclin (15R-TIC, 7), which was selectively responsive to a novel
prostacyclin receptor (IP2) in the central nervous system (Suzuki et al., 1996; Suzuki et al.,
2000b). The tolyl group in 7 was intended as a trigger component to create a PET molecular
probe. Therefore, we planned to apply the rapid C–methylation conditions to the synthesis
of a PET molecular probe, the 15R-[11C]TIC methyl ester using [11C]CH3I, prepared from
[11C]CO2 according to an established method (Fowler & Wolf, 1997), and the stannane 8
(Suzuki et al., 2000a). However, we found that the C–[11C]methylation under radiolabeling
conditions, even after using an excess amount of a CuI salt, lacked reproducibility for some
unknown reasons. During the course to overcome this difficulty along with the actual PET–
probe synthesis, we encountered some valuable information that led to a solution of the
problem by using CuI instead of CuCl that severely retarded the methylation of the
phenyltributylstannane (Table 1, Entry 5). In order to minimize this inhibitory effect of CuI,
we changed the one–pot operation to a two–pot stepwise procedure during the actual PET–
probe synthesis (Figure 3) (Suzuki et al., 2004). This procedure consists of independent
syntheses of a methylpalladium complex and a phenyl copper complex at room temperature
(25 °C), and then the mixing of these species in one portion at a higher temperature (65 °C, 5
min). As expected, the highly qualified PET probe, the 15R-TIC methyl ester ([11C]9), was
obtained by thus C–[11C]methylation procedure from 8 in an 85% isolated yield (decay–
corrected, based on the radioactivity of [11C]CH3I trapped in the Pd solution; it indicates the
production efficiency) with a purity of greater than 98%, which was applicable for a human
PET study with a sufficient radioactivity of 2–3 GBq and high reproducibility (Figure 3). The
specific radioactivity was 37–100 GBq mol–1. The total synthesis time was 35–40 min.
After the ethical committee gave its official approval for a human PET study, the principal
author, M. Suzuki, was nominated to be the first volunteer. Thus, the 15R-[11C]TIC methyl
ester ([11C]9) was injected into his right arm and it passed through the blood–brain barrier. It
was then hydrolyzed in the brain to a free carboxylic acid, which was eventually bound to
the IP2 receptor. PET images of horizontal slices indicated that a new receptor, IP2, was
distributed throughout various structures in the human brain (Figure 4) (Suzuki et al., 2004).
A PET study of the middle cerebral artery occlusion using a monkey model demonstrated
that 15R-TIC revealed a potent neuroprotective effect against focal cerebral ischemia as
judged by the [15O]O2 consumption and the uptake of [18F]FDG (Cui et al., 2006). Recently,
rat PET studies using the 15R-[11C]TIC methyl ester ([11C]9) showed that [11C]9 could be
useful for the in vivo analyses of the mrp2–mediated hepatobiliary transport (Takashima et
al., 2010). Furthermore, the PK/PD studies of [11C]9 in humans (submitted for publication)
as well as a translational study of Alzheimer’s disease patients to evaluate the progress of
such neurodegenerative diseases are now in progress. A PET probe of the 17-(3-
[11C]methylphenyl)-18,19,20-trinor-prostaglandin F2 isopropyl ester ([11C]10) (Björkman et
al., 2000) targeting the receptor of prostaglandin F2 (PGF2) was also synthesized using a
procedure similar to [11C]9.

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[11C]CH3I, formed from [11C]CO2 according to the established method, was trapped in a solution of
Pd2(dba)3 (1.3 mg, 1.4 µmol) and P(o-CH3C6H4)3 (1.7 mg, 5.6 µmol) in DMF (230 µL) at room
temperature. The solution was transferred to a vial containing stannyl precursor 8 (2.0 mg, 3.0 µmol),
CuCl (1.5 mg, 15µmol), and K2CO3 (2.1 mg, 15 µmol) in DMF (80 µL), and the resulting mixture was
heated at 65 °C for 5 min. Prior to the preparative HPLC, a solid phase extraction column was used to
remove the salts and palladium residue from the reaction mixture. The desired product, 15R-[11C]TIC
methyl ester [11C]9, after HPLC separation and intravenous formulation usually had a isolated
radioactivity of approximately 2.5 GBq, sufficient for an in vivo human PET study.
Fig. 3. Synthesis of 15R–[11C]TIC methyl ester ([11C]9) under PET radiolabeling conditions.
HO
HO H
COOR
X
7: R = H, X = CH3, 15R-TIC
8: R = CH3, X = (n-C4H9)3Sn
[11C]9: R = CH3, X = 11CH3
HO
HO
COOCH(CH3)2
OH
11CH3
[11C]10

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Positron Emission Tomography – Current Clinical and Research Aspects
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Fig. 4. PET imaging in human brain using [11C]9.
8. Rapid C–[11C]methylation of heteroaromatic compounds: importance of
using a large amount of bulky arenylphosphine, CuI/F– or CuI/K2CO3 synergic
effect, and the selection of an amide solvent
8.1 Pd0–mediated rapid coupling of methyl iodide and heteroarenylstannanes
applicable to 2- and 3-[11C]methylpyridines
There is a strong demand for the incorporation of a short–lived 11C–labeled methyl group
into the heteroaromatic carbon frameworks, because such structures often appear in major
drugs and their promising candidates. The Pd0–mediated rapid trapping of methyl iodide
with an excess amount of a hetero–aromatic ring–substituted tributylstannane 11a–i was
done (Suzuki et al., 2009) by first using our previously developed CH3I/11a–
i/Pd2(dba)3/P(o-CH3C6H4)3/CuCl/K2CO3 (1:40:0.5:2:2:2) combination system in DMF at 60
°C for 5 min (conditions A; Suzuki et al., 1997), but the reaction produced low yields of the
various kinds of heteroaromatic compounds (Table 2, Entries 1–9). An increase in the
phosphine ligand (conditions B) significantly improved the yield for the heteroarenyl
stannanes, 11b, 11c, and 11i, but the conditions were still insufficient in terms of the range of
adaptable heteroaromatic structures. Another CuBr/CsF combination system (conditions C)
also provided a result similar to conditions B using an increased amount of the phosphine.
Thus, pyridine and the related heteroaromatic compounds still remained as less reactive
substrates. Consequently, the problem was overcome by replacing the DMF solvent with N-
methyl-2-pyrolidinone (NMP). It is of interest that such a solvent effect was not observed for
the CuCl/K2CO3 combination system, but appeared for the CuBr/CsF reaction system
(Table 3, Entry 2), giving 2-methylpyridine (2-picoline, 12d) in 81% yield. The other solvents,
except for the amide–type solvent and amine additives, were not effective (Table 3, Entries
4–11). Thus, the reaction in NMP at 60–100°C for 5 min using the CH3I/11a–
i/Pd2(dba)3/P(o-CH3C6H4)3/CuBr/CsF (1:40:0.5:16:2:5) combination (conditions D) gave the
methylated products 12a–i in >80% yields (based on the reaction of CH3I) for all of the
heteroaromatic compounds listed in this study (Table 2, Entries 1–9). Thus the combined use
of NMP and increased amount of the bulky arenylphosphine is important to efficiently
promote the reaction. The conditions using a Pd{P(tert-C4H9)3}2/CsF system in NMP
reported by G. C. Fu et al. (Littke et al., 2002) were not effective by producing only a poor
yield (21%, Table 3, Entry 2) as judged by the methylation of 2-pyridyltributylstannane
(11d). The addition of CuBr to this system improved the yield to only a small extent (39%).

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Entrya Heteroarenyl
stannane
Methylated product Yield (%)b
Bc Ac Cc Dc
1
11a
12a
28 75 73 80
2
11b
12b
57 87 91 94
3
11c
12c
52 88 90 94
4
11d12d
16
(14)d
67 63 81
5
11e12e
25
(53)e
61 66 80
6
11f12f
79 60 68 87
7
11g 12g
3 50 48
62
(87)f
8
11h12h
25 72 70 90
9
11i12i
12 83 75 83
aReaction was carried out with CH3I (10 mol), stannane 11 (400 mol), and Pd0 (10 mol). bThe
products were identified by GLC analyses and comparison with authentic samples. Yields were
determined by GLC based on CH3I consumption using n-nonane and n-heptane as internal standards,
and are the average of 2 or 3 runs. cReaction conditions (molar ratio): A: CH3I/11/Pd2(dba)3/P(o-
CH3C6H4)3/CuCl/K2CO3 (1:40:0.5:2:2:2) in DMF at 60 °C for 5 min; B: CH3I/11/Pd2(dba)3/P(o-
CH3C6H4)3/CuCl/K2CO3 (1:40:0.5:16:2:5) in DMF at 60°C for 5 min; C: CH3I/11/Pd2(dba)3/P(o-
CH3C6H4)3/CuBr/CsF (1:40:0.5:16:2:5) in DMF at 60 °C for 5 min; D: CH3I/11/Pd2(dba)3/P(o-
CH3C6H4)3/CuBr/CsF (1:40:0.5:16:2:5) in NMP at 60 °C for 5 min. dCH3I/11d/Pd2(dba)3/P(o-CH3C6H4)3
(1:40:0.5:2) in DMF at 120 °C for 5 min (stepwise procedure) (Iida et al, 2004). eCH3I/11e/Pd2(dba)3/P(o-
CH3C6H4)3/CuCl/K2CO3 (1:40:0.5:2:2:2) in DMF at 80 °C for 3 min (Prabhakaran et al, 2005). fThe
reaction was conducted at 100 °C.
Table 2. General rapid C–methylation on various neutral and basic heteroaromatic rings.

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Entrya Solvent Additive (equiv) Yield (%)b
CuCl/K2CO3
synergic system
67
66
—
—
—
—
—
—
—
—
—
CuBr/CsF
synergic system
65
81 (21)c (39)d
69
18
20
38
23
34
19
20
6
1
2
3
4
5
6
7
8
9
10
11
DMF
NMP
DMA
DMI
toluene
THF
DMSO
HMPA
DMF
DMF
DMF
—
—
—
—
—
—
—
—
2,6-lutidine (17)
Triethylamine (14)
DABCO (18)
aReaction was carried out with CH3I (10 mol), stannane 11d (400 mol), and Pd0 (10 mol). Reaction
conditions (molar ratio): CH3I/11d/Pd2(dba)3/P(o-CH3C6H4)3/CuCl/K2CO3 (1:40:0.5:16:2:2) or
CH3I/11d/Pd2(dba)3/P(o-CH3C6H4)3/CuBr/CsF (1:40:0.5:16:2:5) at 60°C for 5 min. bYield (%) of 12d was
determined by GLC analyses based on CH3I consumption using n-heptane as the internal standard.
cFu’s original conditions (Littke et al., 2002) (molar ratio): CH3I/11d/Pd{P(tert-C4H9)3}2/CsF (1:40:1:2).
dFu’s original conditions + CuBr (molar ratio): CH3I/11d/Pd{P(tert-C4H9)3}2/CuBr/CsF (1:40:1:2:5).
NMP: N-methyl-2-pyrrolidinone; DMA: N,N-dimethylacetamide; DMI: 1,3-dimethylimidazolidin-2-one;
THF: tetrahydrofuran; HMPA: hexamethylphosphoric triamide; DABCO: 1,4-diazabicyclo[2.2.2]octane.
Table 3. Effect of a solvent and additives in increased phosphine and synergic systems on
the rapid trapping of methyl iodide with 2-pyridyltributylstannane (11d) to give 2-
methylpyridine (12d).
The utility of the general rapid methylation was well demonstrated by the syntheses of the
actual PET tracers, the 2- and 3-[11C]methylpyridines ([11C]12d and e), using Pd2(dba)3/P(o-
CH3C6H4)3/CuBr/CsF (1:16:2:5) in NMP at 60°C for 5 min, giving the desired products in 88
and 91% radio–HPLC analytical yields (definition: (radioactivity of desired product on
HPLC)/(total radioactivity of distributed materials on HPLC) x 100%; it means reaction
efficiency), respectively (Figure 5) (Suzuki et al., 2009).
(S)-(+)-2-Methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]homopiperazine (H-1152, or referred
to as H-1152P, 13) is known as the most potent, specific, and membrane–permeable inhibitor
of small G protein Rho-associated kinase (Rho–kinase). A 11C–labeled H-1152 as a novel PET
probe for imaging Rho-kinases was efficiently synthesized for the first time based on the
Pd0–mediated rapid C–[11C]methylation
heteroarenylstannane precursor using [11C]CH3I followed by rapid deprotection of the TFA
group (Suzuki et al., 2011a). Thus, the C-[11C]methylation on the isoquinoline derivative was
for trifluoroacetyl (TFA)–protected
CH3I
NSn(n-C4H9)3
NCH3
Pd2(dba)3
P(o-CH3C6H4)3
Additives
DMF or other solvents
(1 mL)
60 ?C, 5 min
11d
12d

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127
performed using Pd2(dba)3/P(o-CH3C6H4)3/CuBr/CsF (1:16:2:5 in molar ratio) in NMP at 80
°C for 5 min and the deprotection of TFA proceeded using 2 M NaOH at 25–50 °C for 1 min,
giving [11C]H-1152 ([11C]13) with 86 ± 4% (n = 3) radio–HPLC analytical yield. The isolated
total radioactivity was 3.8 ± 1.2 GBq (n = 3) with the radiochemical yield of 63 ± 14% (n = 3)
(decay–corrected, based on [11C]CH3I). Both chemical and radiochemical purities were
>99%. The total synthesis time was 38 min. The specific radioactivity at the end of the
formation was 97 ± 10 GBq mol–1 (n = 3). The use of [11C]13 for molecular imaging studies
of cardiovascular diseases is now in progress.

Red carbon in the structure means a radionuclide.
Fig. 5. Syntheses of 2- and 3-[11C]methylpyridines ([11C]12d, e).
8.2 Efficient syntheses of [methyl–11C]thymidine and 4’-[methyl–11C]thiothymidine
[18F]FLT has been developed as a more specific tumor imaging agent than [18F]FDG
(Grierson et al., 1997, as cited in Bading & Shields, 2008). This pyrimidine analogue lacking a
hydroxy group at C–3’ is phosphorylated by thymidine kinase 1 (TK1) and trapped in cancer
cell. The TK1 activity increases almost 10–fold during the DNA synthesis, and thus, the
imaging reflects the cell proliferation differentiating tumor from inflammation (Lee et al.,
2009). The first human imaging study was conducted with 1-(2’-deoxy-2’-[18F]fluoro-1--D-
arabinofuranosyl)thymine ([18F]FMAU) (Sun et al., 2005, as cited in Bading & Shields, 2008),
showing that the tumors in the brain, prostate, thorax, and bone could be clearly visualized.
However, there is the primary limitation in the use of [11C]- or [18F]FMAU which is being a
relatively poor substrate for TK1 and a relatively good substrate for TK2, probably

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accounting for its localization in the mitochondrion–rich human myocardium. On the other
hand, 4’-thiothymine (15b), which resembles the biological properties of thymidine (15a)
with a higher stability for the phosphorylase cleavage, underwent the 11CH3–labeling for the
tumor imaging using a rat, exhibited a higher potential as an attractive PET probe than
[18F]FLT (Toyohara et al., 2008). Although the PET imaging studies using various kinds of
11C– and 18F–labeled thymidne analogues have been extensively continued, it is still difficult
to synthesize the labeled compounds.
Thus, we applied the rapid C–[11C]methylation of a heteroarenylstannane (see the section
8.1) to the synthesis of the 11C–labeled thymidine and its thio–analogue (Koyama et al.,
2011). 1-(2’-Deoxy-2’-fluoro--D-arabinofuranosyl)-[methyl–11C]thymine ([11C]FMAU) and
4’-[methyl–11C]thiothymine ([11C]15b) have so far been labeled by 11C using 5-trimethyl
and/or tributylstannyl precursors via the Stille–type cross–coupling reaction with
[11C]methyl iodide (Samuelsson & Långström, 2003; Toyohara et al., 2008). However, as
anticipated, the previously reported conditions had fewer effects on the syntheses of 5-
tributylstannyl-2’-deoxyuridine (14a) and
Pd2(dba)3/P(o-CH3C6H4)3 (1:4 in molar ratio) at 130 °C for 5 min in DMF, giving the desired
products 15a and b in only 32 and 30% yields, respectively (Table 4, Entries 1 and 4).
Therefore, we tried to adapt the current reaction conditions, including the synergic systems
developed in our laboratory, for such heteroaromatic compounds. First, the reaction using
CH3I/14a/Pd2(dba)3/P(o-CH3C6H4)3/CuCl/K2CO3 (1:25:1:32:2:5) at 80 °C gave thymidine
(15a) in 85% yield (Entry 2). Whereas, CH3I/14a/Pd2(dba)3/P(o-CH3C6H4)3/CuBr/CsF
(1:25:1:32:2:5) including another CuBr/CsF system promoted the reaction at a milder
temperature (60 °C), giving 15a in quantitative yield (Entry 3). The chemo–response of the
thiothymidine–precursor 14b was different from the thymidine system 14a. The optimized
conditions obtained for 14a including the CuBr/CsF system gave 4’-thiothymidine (15b) in
only 40% yield (Table 4, Entry 5). The reaction using 5–fold amounts of CuBr/CsF at 80 °C
gave 15b in a much higher yield (83%, Entry 6), but unexpectedly, the reaction was
accompanied by a large amount of an undesired destannylated product. It was considered
that the destannylated product 17 would have been produced by proton transfer to the
transmetallated Cu intermediate 16 from 14bCu2 with the enhanced acidity by CuI
coordination of a sulfur atom in the thiothymidine structure (Figure 6). As expected, such a
destannylation was significantly suppressed by changing the medium to a much more basic
system, in which the stannyl substrate 14b would be changed to the deprotonated 18. Thus,
the conditions CH3I/14b/Pd2(dba)3/P(o-CH3C6H4)3/CuCl/K2CO3 (1:25:1:32:2:5) at 80 °C
gave 15b in nearly quantitative yield (98%, Table 4, Entry 7).
-4’-thio-2’-deoxyuridine (14b) using

N
R
SO2
N
HN
13: R = CH3, H-1152
[11C]13: R = [11C]CH3, [11C]H-1152

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Entrya X P(o-CH3C6H4)3
(equiv)
CuI, base (mol ratio) Yield (%)b
Temp. (°C)
60
0
67
100
—
40
64
83
80
—
85
—
—
—
83
98
100
—
—
97
—
—
—
—
130
32
—
—
30
—
—
—
1c
2
3
4
5
6
7
O
O
O
S
S
S
S
4
32
32
4
32
32
32
none
CuCl, K2CO3 (2:5)
CuBr, CsF (2:5)
none
CuBr, CsF (2:5)
CuBr, CsF (10:25)
CuCl, K2CO3 (2:5)
aX = O: reaction was carried out with CH3I (2.0 mol), stannane 14a (50 mol) and Pd0 (4.0 mol). X = S:
reaction was carried out with CH3I (1.0 mol), stannane 14b (25 mol) and Pd0 (2.0 mol). bThe yield
was determined by GLC based on CH3I consumption using acridine as the internal standard. cReaction
was carried out with 5 equiv. of 14a relative to methyl iodide.
Table 4. Synthesis of thymidine (15a) and 4’-thiothymidine (15b) by the rapid trapping of
methyl iodide with 5-tributylstannyl-2’-deoxyuridine (14a) and 5-tributylstannyl-4’-thio-2’-
deoxyuridine (14b).

Fig. 6. Assumed equilibration formed in the presence of a CuI salt.
Each optimized condition obtained for 14a and b was successfully used for the syntheses of the
corresponding PET probes with 87 and 93% radio–HPLC analytical yields (Figure 7) (Koyama
et al., 2011). The [11C]compounds were isolated by preparative HPLC after the reaction was
X
HO
OH
N
NH
CH3I
+
O
O
(n-C4H9)3Sn
Pd2(dba)3
P(o-CH3C6H4)3
CuI, base
DMF, 5 min
X
HO
OH
15a: X = O
15b: X = S
N
NH
O
O
H3C
14a: X = O
14b: X = S