Highly efficient syntheses of [methyl-11C]thymidine and its analogue 4′-[methyl-11C]thiothymidine as nucleoside PET probes for cancer cell proliferation by Pd0-mediated rapid C-[ 11C]methylation

Article abstract of DOI:10.1039/c0ob01249a

Pd⁰-mediated rapid couplings of CH₃I (and then [ ¹¹C]CH₃I) with excess 5-tributylstannyl-2′- deoxyuridine and -4′-thio-2′-deoxyuridine were investigated for the syntheses of [methyl-¹¹C]thymidine and its stable analogue, 4′-[methyl-¹¹C]thiothymidine as PET probes for cancer diagnosis. The previously reported conditions were attempted using Pd ₂(dba)₃/P(o-CH₃C₆H₄) ₃ (1:4 in molar ratio) at 130°C for 5 min in DMF, giving desired products only in 32 and 30% yields. Therefore, we adapted the current reaction conditions developed in our laboratory for heteroaromatic compounds. The reaction using CH₃I/stannane/Pd₂(dba)₃/P(o- CH₃C₆H₄)₃/CuCl/K₂CO ₃ (1:25:1:32:2:5) at 80°C gave thymidine in 85% yield. Whereas, CH₃I/stannane/Pd₂(dba)₃/P(o-CH ₃C₆H₄)₃/CuBr/CsF (1:25:1:32:2:5) including another CuBr/CsF system promoted the reaction at a milder temperature (60°C), giving thymidine in 100% yield. Chemo-response of thiothymidine-precursor was different from thymidine system. Thus, the above optimized conditions including CuBr/CsF system gave 4′-thiothymidine only in 40% yield. The reaction using 5-fold amount of CuBr/CsF at 80°C gave much higher yield (83%), but unexpectedly, the reaction was accompanied by a considerable amount of undesired destannylated product. Such destannylation was greatly suppressed by changing to a CuCl/K₂CO₃ system using CH₃I/stannane/Pd₂(dba)₃/P(o-CH ₃C₆H₄)₃/CuCl/K₂CO ₃ (1:25:1:32:2:5) at 80°C, giving the 4′-thiothymidine in 98% yield. The each optimized conditions were successfully applied to the syntheses of the corresponding PET probes in 87 and 93% HPLC analytical yields. [¹¹C]Compounds were isolated by preparative HPLC after the reaction conducted under slightly improved conditions, exhibiting sufficient radioactivity of 3.7-3.8 GBq and specific radioactivity of 89-200 GBq mol ^-1 with radiochemical purity of ≥99.5% for animal and human PET studies.

Full text of DOI:10.1039/c0ob01249a

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PAPER
Highly efficient syntheses of [methyl-11C]thymidine and its analogue
4¢-[methyl-11C]thiothymidine as nucleoside PET probes for cancer cell
proliferation by Pd0-mediated rapid C-[11C]methylation†‡
Hiroko Koyama,a Siqin,b Zhouen Zhang,b Kengo Sumi,a Yuma Hatta,a Hiroko Nagata,b Hisashi Doib and
Masaaki Suzuki*b
Received 24th December 2010, Accepted 14th March 2011
DOI: 10.1039/c0ob01249a
Pd0-mediated rapid couplings of CH3I (and then [11C]CH3I) with excess 5-tributylstannyl-2¢-deoxy-
uridine and -4¢-thio-2¢-deoxyuridine were investigated for the syntheses of [methyl-11C]thymidine and its
stable analogue, 4¢-[methyl-11C]thiothymidine as PET probes for cancer diagnosis. The previously
reported conditions were attempted using Pd2(dba)3/P(o-CH3C6H4)3 (1 : 4 in molar ratio) at 130 ◦C for
5 min in DMF, giving desired products only in 32 and 30% yields. Therefore, we adapted the current
reaction conditions developed in our laboratory for heteroaromatic compounds. The reaction using
CH3I/stannane/Pd2(dba)3/P(o-CH3C6H4)3/CuCl/K2CO3 (1 : 25 : 1 : 32 : 2 : 5) at 80 ◦C gave thymidine
in 85% yield. Whereas, CH3I/stannane/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
thymidine in 100% yield. Chemo-response of thiothymidine-precursor was different from thymidine
system. Thus, the above optimized conditions including CuBr/CsF system gave 4¢-thiothymidine only
in 40% yield. The reaction using 5-fold amount of CuBr/CsF at 80 ◦C gave much higher yield (83%),
but unexpectedly, the reaction was accompanied by a considerable amount of undesired destannylated
product. Such destannylation was greatly suppressed by changing to a CuCl/K2CO3 system using
CH3I/stannane/Pd2(dba)3/P(o-CH3C6H4)3/CuCl/K2CO3 (1 : 25 : 1 : 32 : 2 : 5) at 80 ◦C, giving the
4¢-thiothymidine in 98% yield. The each optimized conditions were successfully applied to the syntheses
of the corresponding PET probes in 87 and 93% HPLC analytical yields. [11C]Compounds were isolated
by preparative HPLC after the reaction conducted under slightly improved conditions, exhibiting
sufficient radioactivity of 3.7–3.8 GBq and specific radioactivity of 89–200 GBq mmol-1 with
radiochemical purity of ≥99.5% for animal and human PET studies.
as PET probes4,5 for imaging tumor cell proliferation in living
Introduction
systems.3 Among the 11C-incorporation methods, particularly
Positron emission tomography (PET) is a non-invasive, in vivo
focusing on the methyl group in the structure of thymidine,
[11C]methylation could be ideal in view of the directness, ef-
ficiency, and practicability using the rapid cross-coupling of
[11C]CH3I and a soft metalloid thymidine precursor.5c In this
context, La˚ngstro¨m and coworkers synthesized 1-(2¢-deoxy-2¢-
fluoro-b-D-arabinofuranosyl)-[methyl-11C]thymine ([11C]FMAU,
[11C]2) in 28 5% decay-corrected radiochemical yield calculated
from [11C]methyl iodide using 5-trimethylstannyl precursors 3 by
a Stille-type cross-coupling reaction with [11C]methyl iodide.5c
However, this method possesses several problems to be solved
from practical points of view; (1) excess use of the potentially
toxic trimethylstannyl derivative; actually, highly toxic trimethyl-
stannyl iodide6 could be produced as a by-product after cross-
coupling reaction with [11C]methyl iodide; (2) suffering from
the production of undesired volatile [11C]ethane by scrambling
between [11C]CH3I and a (CH3)3Sn unit1a to cause safety and
environmental problems; (3) a decrease in the yield of the desired
molecular imaging method enabling the analysis of the dynamic
behavior of a radiotracer in living systems such as the brain, the
heart, and other active tissues and organs.2 Thymidine (1) is one
of the fundamental building blocks for deoxyribonucleic acid,
and hence, the labeling of thymidine and its derivatives attracts
much interest for cancer diagnosis.3 Actually, metabolically stable
11C or 18F-labeled thymidine derivatives have been synthesized
aDivision of Regeneration and Advanced Medical Science, Gifu University
Graduate School of Medicine, Yanagido 1-1, Gifu, 501-1193, Japan
bRIKEN Center for Molecular Imaging Science (CMIS),
Minatojima-minamimachi 6-7-3, Chuo-ku, Kobe, 650-0047, Japan.
E-mail: suzuki.masaaki@riken.jp
† Rapid methylation on carbon frameworks for PET tracer synthesis, Part
10; for parts 1–9, see refs. 1a–i.
‡ Electronic supplementary information (ESI) available: HPLC charts
after the rapid C-[11C]methylation and purification. See DOI:
10.1039/c0ob01249a
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product to a considerable extent in nature by such a scramble
reaction.7 The labeled compound of 4¢-thiothymidine ([11C]4),
which closely resembles the biological properties of thymidine and
is resistant to phosphorylase cleavage,8 has been first reported
by J. Toyohara and co-workers as an attractive PET probe for
tumor imaging.9 They synthesized [11C]4 by a reaction using the
tributylstannyl precursor 5 in order to avoid the strong toxicity
of trim◦ethylstannyl compound under the conditions of heating
at 130 C for 5 min using Pd2(dba)3/P(o-CH3C6H4)3 in DMF.9b
In this context, we have so far found that the tributylstannyl
precursor is much less reactive than the trimethyl one and
can be efficiently activated under mild conditions (60–80 ◦C)
using Pd0–bulky phosphine complexes mixed with the synergic
combinations of CuI/K2CO3 or CuI/CsF to realize the desired
rapid cross coupling (rapid C-methylations) in high yield without
scrambling.1a,f,7b The rapid C-methylations have covered the use of
aryl-,1a alkenyl-,1g alkynyl-,1e and hetero-aromatic tributylstannyl
precursors1i in addition to aryl- and alkenylboranes.1h Taking our
accumulated information on the Stille-type rapid cross-coupling
reactions into consideration, the reported reaction conditions in
both groups still seem insufficient. Thus, we have made an effort
to optimize the reaction conditions based on the introduction of
two kinds of synergic effects on the synthesis of thymidine and
4¢-thiothymidine. Described herein are truly efficient syntheses
of [methyl-11C]thymidine and 4¢-[methyl-11C]thiothymidine by the
Pd0-mediated rapid C-[11C]methylation.1j
synthesis in mind.10 To begin with, we evaluated the conditions
reported by B. La˚ngstro¨m and coworkers for the synthesis of
[11C]2.5c Thus, the reaction was conducted using Pd2(dba)3/P(o-
CH3C6H4)3 (1 : 4 in molar ratio) in DMF at 130 ◦C for 5 min
to give thymidine 1 only in 32% yield (HPLC analytical yield
based on the consumption of CH3I, HPLC: tR = 6.1 min,11
Table 1 entry 1). Continuously, we conducted the reaction
under our previously developed conditions for the sp3–sp2(phenyl)
rapid coupling1a using a combination of CH3I/6/Pd2(dba)3/P(o-
CH3C6H4)3/CuCl/K2CO3 (1 : 25 : 0.5 : 2 : 2 : 2 in molar ratio) at
60 ◦C for 5 min, giving 1 in 71% yield (Table 1 entry 2). The
reaction conducted at 100 ◦C did not improve the yield even at such
a high temperature (74%, Table 1 entry 2). Instead, the increasing
of the quantity of the added phosphine1g from 2 to 16 equiv was
not effective at 60 ◦C (60%, Table 1 entry 3), but the use of 2-fold
amount of Pd/phosphine (1 : 32 ratio) resulted in increasing the
yield at 80 ◦C to a considerable extent (85%, Table 1 entry 4). On
the other hand, the CuBr/CsF combination has also been found
to be an efficient synergic combination12 for the sp3–sp2(vinyl)
type rapid coupling reaction. Actually, though the reaction using
CH3I/6/Pd2(dba)3/P(o-CH3C6H4)3/CuBr/CsF (1 : 25 : 1 : 4 : 2 : 5
in molar ratio) at 60 ◦C in DMF for 5 min gave 1 in 53%
yield (in Table 1 ◦entry 5), the enhancement of the reaction
temperature to 80 C improved the yield to a great extent (97%,
Table 1 entry 5). In addition, the increase in the quantity of
CuBr/CsF enhanced the reaction even at a lower temperature
(60 ◦C, 87%, Table 1 entry 6). To our surprise, it was found
that the use of 8-fold quantity of phosphine (32 equiv) tremen-
dously promoted the reaction to give the desired methylated
product 6 quantitatively, even at lower temperature (60 ◦C)
without an increase in the quantity of CuI/CsF. Thus, the rapid
Results and discussion
The reaction using the 1 : 25 ratio of methyl iodide and stan-
nyl substrate 6 was set up by keeping the actual PET tracer
Table 1 Synthesis of a thymidine (1) by the rapid trapping of methyl iodide with 5-tributylstannyl-2¢-deoxyuridine (6)a
Yield of 1 (%)b
Temp. (◦C)
Entry
Pd2(dba)3(equiv)
P(o-CH3C6H4)3 (equiv)
CuI/base (molar ratio)
Solvent
60
80
100
130
1c,d
2e
3
4
5
6
7
8
9
1
4
2
16
32
4
none
DMF
DMF
DMF
DMF
DMF
DMF
DMF
DMF
DMF
NMP
0
—
—
—
85
97
—
—
89
—
100
—
74
—
—
—
93
97
—
—
—
32
—
—
—
—
—
—
—
—
—
0.5
0.5
1
1
1
CuCl/K2CO3 (2 : 2)
CuCl/K2CO3 (2 : 5)
CuCl/K2CO3 (2 : 5)
CuBr/CsF (2 : 5)
CuBr/CsF (10 : 25)
CuBr/CsF (2 : 5)
CuBr/CsF (2 : 5)
CuBr/CsF (2 : 5)
CuBr/CsF (2 : 5)
71
60
67
53
87
100
88
69
4
1
32
24
16
4
0.75
0.5
1
f
10
—
a Reaction was carried out with 25 equiv of 6 relative to methyl iodide (2.0 mmol). b The yields were determined by HPLC analysis based on the CH3I
consumption using acridine as an internal standard. c Reaction was carried out with 5 equiv of 6. d See reference 5c. e See reference 1a. f The mixture was
insoluble at this reaction temperature giving a product with low reproducibility.
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C-methylation was accomplished using CH3I/6/Pd2(dba)3/P(o-
CH3C6H4)3/CuBr/CsF (1 : 25 : 1 : 32 : 2 : 5 in molar ratio) at 60 ◦C
for 5 min in 100% yield (Table 1 entry 7). Such a combination
was tolerant of a wide range of the reaction temperature (60–
100 ◦C) to give 1 in high yield (Table 1 entry 7). The decrease of
the Pd0/CH3I ratio from 2 to 1.5 and 1 showed the lesser effect
in the order (Table 1 entries 8 and 9), indicating that the reaction
favors the use of an excess amount of Pd0 (Pd0/CH3I >2). This
tendency matches well with actual PET probe synthesis using an
extremely small amount of [11C]CH3I.1g,10 Here, the solvent was
changed from DMF to N-methyl-2-pyrrolidinone (NMP) known
as a better solvent for general rapid C-methylation of hetero-
aromatic frameworks of heteroaryl(tributyl)stannane.1i Because
the reaction mixture was not◦dissolved in NMP at 60 ◦C, the
reaction was conducted at 80 C to give the methylated product
quantitatively (Table 1 entry 10) similar to the result obtained in
DMF shown in entry 5 of Table 1.13 The effect of solvent compared
between entries 5 and 10 indicates that NMP is comparable to
DMF in the thymidine case,1i implying that the properties of 2,4-
pyrimidinedione structure 7 (keto form) in thymidine are different
from those of pyridine and its derivative as basic heteroaromatic
compounds and are rather regarded as phenol-type proton-donor
structures (enol forms) as illustrated by the equilibrium shown
in Scheme 1.14 A vinylstannyl moiety in the 2,4-pyrimidinedione
structure would also favor the reaction conditions including
the CuI/CsF synergic system over those including CuI/K2CO3
(Table 1 entries 7 vs. 4) as previously reported for the rapid
C-methylation of alkenylstannane precursors.1g,12 Although an
excess amount of phosphine is needed at low temperature to
obtain high yield (60 ◦C, Table 1 entry 7), such an excess
phosphine was not necessary at elevated temperature (80 ◦C, see
Table 1 entry 5). Overall, we concluded that the optimized con-
ditions for rapid C-methylation for 6 are CH3I/6/Pd2(dba)3/P(o-
CH3C6H4)3/CuBr/CsF (1 : 25 : 1 : 32 : 2 : 5 in molar ratio) at 60 ◦C
for 5 min.
CH3C6H4)3/CuBr/CsF (1 : 25 : 1 : 32 : 2 : 5 in molar ratio) at
60 ◦C for 5 min, unexpectedly giving the desired product 4 in
only 40% yield (Table 2 entry 2). Increasing the quantity of
CuBr/CsF at this temperature improved the yield considerably
but was still insufficient (64%, Table 2 entry 3). The use of
NMP as the solvent revealed a similar result (Table 2 entry
4).16 Here, we raised the temperature (80 ◦C) in DMF to
substantially accelerate the reaction, giving 4 in 83% yield
(Table 2 entry 3). However, HPLC analysis of the reaction
mixtures obtained in entries 2 and 3 showed that the reaction
generated the destannylated compound 8 (HPLC: tR = 6.8 min)11
as a byproduct to a considerable extent (20–49% for an initial
amount of 5).17 This observation prompted us to change the
use of the CuBr/CsF synergic system to CuCl/K2CO3 with
the aim of suppressing such destannylation. Thus, application
of our previously reported conditions1a for the synthesis of
toluene using the combination of CH3I/5/Pd2(dba)3/P(o-
CH3C6H4)3/CuCl/K2CO3 (1 : 25 : 0.5 : 2 : 2 in molar ratio) in
DMF at 60 and 80 ◦C for 5 min was attempted to give the
desired product 4 in 57 and 69% yields, respectively (Table 2
entry 5). Further, the reaction with increased Pd0/CH3I ratio
(2 : 1) using CH3I/5/Pd2(dba)3/P(o-CH3C6H4)3/CuCl/K2CO3
(1 : 25 : 1 : 4 : 2 : 5 in molar ratio) at 80 ◦C for 5 min in DMF gave
4 in 78% yield (Table 2 entry 6). Although the yield was lower
than that of the reaction shown as entry 3 in Table 2, it was
noted that the formation of the destannylated compound 8 was
much decreased (5–8%) compared with the CuBr/CsF system.17
Finally, the use of an increased amount of the phosphine (32
equiv) promoted the reaction markedly to give the desired product
4 in 83% yield at◦60 ◦C and then in 98% yield at a slightly raised
temperature (80 C, Table 2 entry 8). Decreasing the amount of
phosphine (16 equiv) gave slightly lower yield (Table 2 entry 7).
The promotion of destannylation in the CuBr/CsF system and
its suppression in the CuCl/K2CO3 system could be rationalized
by the following explanation as shown in Scheme 2. In the presence
of a CuI salt, the stannyl substrate 5 containing a sulfide ring could
establish an equilibrium with 5Cu1 and 5Cu2 formed by the first
coordination of a CuI salt with a sulfur atom in the sugar moiety
(5Cu1), and then by further coordination with the carbonyl of the
2,4-pyrimidinedione part (5Cu2, Scheme 2).18 By such copper(I)
behavior, the transmetallation of Sn/Cu could be slowed and the
N–H (or O–H in enol form, see Scheme 1) bond in pyrimidinedione
would become more acidic than that of non CuI-coordinated
substrate 5. As a result, the compound 5Cu2 could be a strong
proton donor capable of protonating a C–Cu bond in organo
copper compound 8, generated with one more CuI salt, giving
destannylated product 9 as a byproduct. On the contrary, in the
system of the CuCl/K2CO3, the stannyl substrate 5 was changed
to a proton acceptor 10 by deprotonation with a base, resulting
in suppressing the destannylation observed in the CuBr/CsF
system.19 We consider that excess phosphine serves to dissociate the
coordination of various heteroatoms in the structure of substrate
with Pd0 and/or CuI complexes to regenerate the reactivity of
native tin(IV) and copper(I) species to promote the reaction (see
also Table 1 entry 7).1i
Scheme 1 2,4-Pyrimidinedione structure 7 (keto form) is equilibrated
with two kinds of enol forms.
The
chemo-response
of
5-tributylstannyl-4¢-thio-2¢-
deoxyuridine (5) in the Pd0-mediated reaction was considerably
different from that of 6. The reaction was conducted similarly to
that with 6 by using methyl iodide and excess stannyl substrate 5
(1 : 25), which was synthesized from 2-deoxy-D-ribose via 12 steps
according to the previous paper.15 As seen in Table 2, we first
evaluated the reaction conditions reported by J. Toyohara and
co-workers using CH3I/5/Pd2(dba)3/P(o-CH3C6H4)3 (1 : 25 : 1 : 4
◦
in molar ratio) at 130 C for 5 min,9b giving the desired 4¢-
thiothymidine (4) in only 30% yield (Table 2 entry 1, HPLC:
tR = 12.0 min).11 Next, the optimized conditions elaborated in
the study on 6 (Table 1 entry 7) were applied to this stannyl
compound 5 using a combination of CH3I/5/Pd2(dba)3/P(o-
Thus, we found that appropriate selection of the CuBr/
CsF and CuCl/K2CO3 synergic systems is quite important to
realize the highly efficient rapid C-methylations of 6 and 5,
respectively.
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Table 2 Synthesis of a 4¢-thiothymidine (4) by the rapid trapping of methyl iodide with 5-tributylstannyl-4¢-thio-2¢-deoxyuridine (5)a
Yield of 4b (%)
Temp. (◦C)
Pd2(dba)3
(equiv)
P(o-CH3C6H4)3
(equiv)
CuI/base (molar
ratio)
Entry
Solvent
60
80
130
1
2
3
4
5
6
7
8
1
1
1
1
0.5
1
1
4
none
DMF
DMF
DMF
NMP
DMF
DMF
DMF
DMF
—
40
64
60
57
—
—
83
—
—
83
—
69
78
94
98
30
—
—
—
—
—
—
—
32
32
32
2
4
16
32
CuBr/CsF (2 : 5)
CuBr/CsF (10 : 25)
CuBr/CsF (10 : 25)
CuCl/K2CO3 (2 : 2)
CuCl/K2CO3 (2 : 5)
CuCl/K2CO3 (2 : 5)
CuCl/K2CO3 (2 : 5)
1
a Reaction was carried out with 25 equiv of 5 relative to methyl iodide (1.0 mmol). b The yields were determined by HPLC analysis based on the CH3I
consumption using acridine as an internal standard.
Scheme 2 Assumed equilibration between a stannyl thiothymidine 5 and a CuI salt.
The utility of the established reaction conditions was well
demonstrated by the syntheses of [methyl-11C]thymidine ([11C]1)
and 4¢-[11C-methyl]thiothymidine ([11C]4). In our experience with
actual PET studies, we had found that the continuous two-pot
operations to minimize the negative secondary effect of CuI
generated in situ1f gave a better result in terms of the repro-
ducibility. Therefore, the reaction was conducted according to such
two-pot operation using Pd2(dba)3/P(o-CH3C6H4)3/CuBr/CsF
(1 : 32 : 2 : 5 in molar ratio, see Table 1 entry 7)10 system. Thus, the
successive mixing of the Pd0/phosphine complex with [11C]methyl
iodide in DMF and then with the stannane 6 in the presence
of CuBr and CsF in DMF followed by heating at 65 ◦C for
5 min gave [11C]1 in 87% analytical yield20 as judged by HPLC
(Figs. 1a and b), indicating the high reaction efficiency.21,22
The 11C-labeled 4¢-thiothymidine ([11C]4) was also synthesized
using the corresponding tin substrate (5) under the similar
procedure as the synthesis of [11C]1 using the Pd2(dba)3/P(o-
CH3C6H4)3/CuCl/K2C◦O3 (1 : 32 : 2 : 5 in molar ratio, see Table 2
entry 8)10 system at 80 C for 5 min to give [11C]4 in 93% HPLC
analytical yield20 (Figs. 1c and d). The reactions for the isolation
of pure [11C]1 and [11C]4 were conducted by slightly improved
conditions using a half amount of phosphine (16 equiv) from
a practical point of view23 under the conditions Pd2(dba)3/P(o-
CH3C6H4)3/CuBr/CsF (1 : 16 : 2 : 10 in molar ratio)10 at 80 ◦C24
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Fig. 1 a) Synthetic scheme of [methyl-11C]thymidine ([11C]1), and b) the chart of high performance liquid chromatography in the analysis of [11C]1
(radioactivity and UV vs. time). c) Synthetic scheme of 4¢-[methyl-11C]thiothymidine ([11C]4), and d) the chart of high performance liquid chromatography
in the analysis of [11C]4 (radioactivity and UV vs. time). The peak at a retention time of 7.6 min shown in b) is [11C]1 and the peak at a retention time of
7.4 min shown in d) is [11C]4.
and Pd2(dba)3/P(o-CH3C6H4)3/CuCl/K2CO3 (1 : 16 : 3 : 8 in mo-
lar ratio)10 at 80 ◦C. Purifications by preparative HPLC gave pure
single products with high radiochemical purity (≥99.5% each, see
ESI‡). Total synthesis time was 42 min in each case until the
radiopharmaceutical formulation, exhibiting radioactivity of 3.7
and 3.8 GBq, respectively.
Decay-corrected radiochemical yields of [11C]1 and [11C]4 based
on the radioactivity of [11C]CH3I trapped in the Pd solution25
were 45 and 42–59%, respectively.26 Specific radioactivity of
[11C]1 and [11C]4 after the formulation was in the range of 89–
200 GBq mmol-1. Thus, the quantity of radioactivity and the
radiochemical quality of [11C]1 and [11C]4 obtained above are
sufficiently satisfactory for animal and human PET studies. We are
currently in the process of applying these synthetic procedures to
the synthesis under the guideline of Good Manufacturing Practice.
dine and its metabolically stable analog, 4¢-thiothymidine,
with the aim of incorporating a short-lived 11C in these
biologically significant compounds. The optimized reaction
conditions are CH3I/6/Pd2(dba)3/P(o-CH3C6H4)3/CuBr/CsF
(1 : 25 : 1 : 32 : 2 : 5 in molar ratio) at 60 ◦C for 5 min and CH3I/5/
Pd2(dba)3/P(o-CH3C6H4)3/CuCl/K2CO3 (1 : 25 : 1 : 32 : 2 : 5 in
molar ratio), giving 1 and 4 in 100 and 98% yields, respectively. The
utility of these rapid C-methylations was demonstrated by efficient
syntheses of the PET probes, [methyl-11C]thymidine and 4¢-
[methyl-11C]thiothymidine, giving [11C]1 and [11C]4 in 87 and 93%
(HPLC analytical yields), respectively. Pure [11C]1 and [11C]4 were
obtained under slightly improved reaction conditions followed by
preparative HPLC separation, and the radiopharmaceutical for-
mulation for administration, exhibiting sufficient radioactivity of
3.7–3.8 GBq and high radiochemical purities (≥99.5% each). These
radioactivities and radiochemical qualities are adequate for both
animal and human PET studies.27,28 These methods could readily
be applicable to 11C-labeling of the thymidine derivatives such as
[11C]FMAU3, [11C]FLT3 and their thiothymidine analogues29 as
well as thymidine-included artificial oligonucleotides with high
Conclusions
We elaborated the rapid C-methylations with high-yield and
practical reaction conditions for the syntheses of thymi-
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metabolic stability and binding affinity such as Bridged Nucleic
Acids as a bridged nucleic acid analogue possessing a fixed N-type
sugar conformation.30
1.0 mL min-1; detection, UV 265 nm; column temperature, 40 ◦C;
retention time, [11C]1, 7.6 min; 2¢-deoxyuridine, 5.8 min; mobil
phase, CH3CN/100 mM NH4HCO3 (pH 7.8) (8 : 92, v/v); flow
rate, 1.0 mL min-1; detection, UV 270 nm; column temperature,
30 ◦C; retention time, [11C]4, 7.4 min.
Experimental section
Rapid coupling of methyl iodide with 5-tributylstannyl-2¢-
General remarks
deoxyuridine (6) to afford thymidine (1; Table 1 entry 7)
All reactions were performed under argon (Ar) with Schlenk
techniques. Solvents and solutions were transformed by syringe–
septum and cannula techniques. Methyl iodide (Nacalai) was
distilled over P4O10 prior to use. Dehydrated N,N-dime-
thylformamide (DMF; Kanto), N-methyl-2-pyrrolidinone (NMP;
Kanto), tris(dibenzylideneacetone)dipalladium(0) (Aldrich), tri-
o-tolylphosphine (Aldrich), copper(I) chloride (Wako), copper(I)
bromide (Wako), potassium carbonate (Wako), and cesium fluo-
ride (Aldrich) were commercial grade. Stannane 6 was prepared
according to a literature procedure.31 Methyl iodide was distilled
over P4O10 prior to use. The [11C]methylation reactions in Fig. 1
were conducted in a lead-shielded hot cell with remote control of
all operations. [11C]Carbon dioxide was produced by a 14N(p,a)11C
reaction using a Sumitomo CYPRIS HM-12S cyclotron (Sumit-
omo Heavy Industries) and then converted into [11C]methyl iodide
by hydriodic acid using an original automated synthesis system for
11C-labeling in RIKEN CMIS. The [11C]methyl iodide obtained
was used for the palladium(0)-mediated rapid C-[11C]methylation
reaction shown in Fig. 1.
In a dry Schlenk tube (10 mL), Pd2(dba)3 (1.8 mg, 2.0 mmol), P(o-
CH3C6H4)3 (19.5 mg, 64.0 mmol), CuBr (0.6 mg, 4 mmol) and CsF
(1.5 mg, 10 mmol) were placed under Ar. After the addition of
DMF (250 mL), the mixture was stirred for 5 min at RT, followed
by successive additions of the solutions of stannane 6 (25.9 mg,
50.0 mmol) in DMF (150 mL) and methyl iodide (0.20 M DMF
solution, 10 mL, 2.0 mmol). After stirring at 60 ◦C for 5 min, the
mixture was rapidly cooled in an ice bath. The resulting mixture
was loaded onto a short column of silica-gel (0.5 g) and eluted with
DMF (ca. 1 mL), followed by the addition of acridine (0.10 M
DMF solution, 10 mL, 1.0 mmol) as an internal standard. The
resulting solution was analyzed HPLC under Conditions A. Yield
of 1: 100%. The product was identified by HPLC with an added
authentic reference (see ESI‡). The methylation reactions under
other conditions in Table 1 were conducted by the same procedure
as those in entry 7.
Rapid coupling of methyl iodide with 5-tributylstannyl-4¢-thio-2¢-
deoxyuridine (5) to afford 4¢-thiothymidine (4; Table 2 entry 8)
Product analysis
In a dry Schlenk tube (10 mL), Pd2(dba)3 (0.9 mg, 1 mmol), P(o-
CH3C6H4)3 (9.8 mg, 32 mmol), CuCl (0.2 mg, 2 mmol) and K2CO3
(0.7 mg, 5 mmol) were placed under Ar. After the addition of
DMF (150 mL), the mixture was stirred for 3 min at RT, followed
by successive additions of the solutions of stannane 5 (13 mg,
25 mmol) in DMF (100 mL) and methyl iodid◦e (40 mM DMF
solution, 25 mL, 1.0 mmol). After stirring at 80 C for 5 min, the
mixture was rapidly cooled in an ice bath. The resulting mixture
was loaded onto a short column of silica-gel (0.3 g) and eluted
with DMF (ca. 0.5 mL), followed by the addition of acridine
(0.10 M DMF solution, 20 mL, 2.0 mmol) as an internal standard.
The resulting solution was analyzed by HPLC under Conditions
B. Yield of 4: 98%. The product was identified by HPLC with an
added authentic reference (see ESI‡). The methylation reactions
under other conditions in Table 2 were conducted by the same
procedure as those in entry 8.
Yields of the reaction were determined by HPLC analysis using
following conditions.
Conditions A. Instrument: Shimadzu HPLC system with a
system controller (SCL-10AVP), a degasser (DGU-12A), a liquid
chromatograph (LC-10AT and LC-10ATVP), a column oven
(CTO-10AVP), an UV-vis detector (SPD-10A), and software
(CLASS-VP); column, SHIM-PACK CLC-ODS (6.0 mm I.D. ¥
150 mm, SHIMADZU); mobil phase, CH3CN:100 mM NaH2PO4
containing 200 mM NaClO4 (pH 2) (6 : 94, v/v); flow rate,
1.0 mL min-1; detection, UV 260 nm; column temperature, 40 ◦C;
retention time, thymidine (1), 6.1 min, 2¢-deoxyuridine, 4.7 min.
Conditions B. Instrument: JASCO HPLC system with a
chromatography interface (LC-Net II/ADC), a quaternary HPLC
pump (PU-2089Plus), a diode array detector (MD-2010Plus), a
column oven (CO-2065Plus), a recycling valve unit (RV-2080-02),
and software (chrom NAV chromatography data system); column,
SHIM-PACK CLC-ODS (6.0 mm I.D. ¥ 150 mm, SHIMADZU);
mobil phase, CH3CN/100 mM NaH2PO4 containing 200 mM
NaClO4 (pH 2) (6 : 94, v/v); flow rate, 1.0 mL min-1; detection,
UV 260 nm; column temperature, 40 ◦C; retention time, 4¢-
thiothymidine (4), 12.0 min; 2¢-deoxy-4¢-thiouridine (8), 6.8 min.
Rapid coupling of methyl iodide with 5-tributylstannyl-2¢-
deoxyuridine (6) to afford [methyl-11C]thymidine ([11C]1) in order
to determine the HPLC analytical yield
[11C]Methyl iodide was prepared from [11C]CO2 via reduction with
LiAlH4, followed by HI treatment according to the established
method.2 [11C]Methyl iodide was trapped in a solution of Pd2(dba)3
(0.9 mg, 1 mmol) and P(o-CH3C6H4)3 (9.7 mg, ◦32 mmol) in
DMF (300 mL) and the mixture was heated at 40 C for 1 min.
The resulting solution was added to a solution of stannane 6
(2.0 mg, 3.9 mmol), CuBr (0.3 mg, 2 mmol), and CsF (0.8 mg,
5 mmol) in DMF (130 mL). The combined mixture was heated
at 65 ◦C for 5 min and then diluted with water (1 mL). The
mixture was passed through cotton and then Syringe Filter (filter
Conditions C. Instrument: Shimadzu HPLC system with a
system controller (CBM-20A), an online degasser (DHU-20A3), a
solvent delivery unit (LC-20AD), a column oven (CTO-20AC), a
photodiode array detector (SPD-20A), and software (LC solution)
and an Aloka radioanalyzer (RLC-700); column, Luna 5 m
C18 (2) (4.6 mm I.D. ¥ 150 mm, Phenomenex); mobil phase,
CH3CN/100 mM NH4HCO3 (pH 7.8) (3 : 97, v/v); flow rate,
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media PVDF, pore size 0.2 mm, Whatman Inc.) and analyzed by
HPLC under Conditions C. HPLC analytical yield of [11C]1: 87%,
calculated by peak area ratio of the [11C]product distributions.
The identifications of [11C]1 was conducted by co-injecting with
the corresponding non-radioactive thymidine (see ESI‡).
mixture of stannane 5 (4.3 mg, 8.0 mmol), P(o-CH3C6H4)3 (3.7 mg,
12 mmol),32 CuCl (0.4 mg, 4 mmol), and K2CO3 (1.4 mg, 10
mmol) in DMF (80 mL) and then apparatus was rinsed with
DMF (250 mL). The radioactivity of [11C]CH3I trapped in the Pd
solution was measured as approximately 24 GBq. The combined
mixture was heated at 80 ◦C for 5 min, and then the salts
and palladium residue in the reaction mixture were removed by
passing through cotton and then Syringe Filter (pore size 0.2
mm) and washed with 10% aqueous acetonitrile (1.5 mL). The
combined elutes were injected into preparative HPLC {semi-
preparative column, Gemini 5 C18 (21.2 mm I.D. ¥ 250 mm,
Phenomenex); mobil phase, CH3CN/100 mM NH4HCO3 (pH 7.8)
(7 : 93, v/v); flow rate, 9.9 mL min-1; detection, UV 270 nm}
to give a radioactive fraction corresponding to [11C]4 (retention
time, 12.5 min, see ESI‡) with radioactivity of 6.9 GBq. The
decay-corrected radiochemical yield based on the radioactivity of
[11C]CH3I trapped in the Pd solution was calculated to be 59%. The
desired fraction was collected into a flask, and the organic solvent
was removed under reduced pressure. The desired [11C]product was
dissolved in a mixture of 25% ascorbic acid (200 mL) and saline
(4 mL). The total synthesis time including HPLC purification and
radiopharmaceutical formulation was 42 min. The radioactivity
of a formulated injection solution of saline (4–5 mL) was 3.8
GBq with the specific radioactivity in the range of 89–200 GBq
mmol-1. The chemical purity of the product was ≥98% and the
radiochemical purity of the product was ≥99.5%.
Synthesis of [methyl-11C]thymidine ([11C]1) for a PET study
[11C]Methyl iodide was trapped in a solution containing Pd2(dba)3
(1.2 mg, 1.3 mmol) and P(o-CH3C6H4)3 (2.5 mg, 8.2 mmol)23,32 in
DMF (300 mL) at RT. The solution was added to the mixture
of stannane 6 (5.5 mg, 11 mmol), P(o-CH3C6H4)3 (3.7 mg, 12
mmol),32 CuBr (0.4 mg, 3 mmol), and CsF (2.0 mg, 13 mmol) in
DMF (80 mL) and then apparatus was rinsed with DMF (250 mL).
The radioactivity of [11C]CH3I trapped in the Pd solution was
measured as approximately 24 GBq. The combined mixture was
heated at 80 ◦C for 5 min, and then salts and palladium residue in
the reaction mixture were removed by passing through cotton and
then Syringe Filter (pore size 0.2 mm) and washed with 8% aqueous
ascorbic acid solution (1.5 mL). The combined elutes were injected
into preparative HPLC {semi-preparative column, Gemini 5
C18 (21.2 mm I.D. ¥ 250 mm, Phenomenex); mobile phase,
CH3CN/100 mM NH4HCO3 (pH 7.8) (4 : 96, v/v); flow rate,
9.9 mL min-1; detection, UV 265 nm} to give a radioactive fraction
corresponding to [11C]1 (retention time, 12.5 min, see ESI‡) with
radioactivity of 5.4 GBq. The decay-corrected radiochemical yield
based on the radioactivity of [11C]CH3I trapped in the Pd solution
was calculated to be 45%. The desired fraction was corrected
into a flask containing 25% ascorbic acid solution (400 mL) and
evaporated under reduced pressure. The residue was diluted with
saline solution (4 mL). The total synthesis time including HPLC
purification and radiopharmaceutical formulation was 42 min.
The radioactivity of a formulated injection solution of saline
(4–5 mL) was 3.7 GBq with specific radioactivity of up to 89
GBq mmol-1. The chemical identity of [11C]1 was confirmed by
HPLC under Conditions C. The chemical purity was ≥98% and
the radiochemical purity was ≥99.5%.
Acknowledgements
This work was supported in part by the Research & Development
of Life Science Fields responding to the needs of society, Molecular
Imaging Research Program, of the Ministry of Education, Culture,
Sports, Science and Technology (MEXT) of Japan. We thank Mr.
Jeongwan Son for his technical assistance in part of the study for
preparation of the tin substrate 6.
Notes and references
1 Parts 1–10: (a) M. Suzuki, H. Doi, M. Bjo¨rkman, Y. Andersson, B.
Rapid coupling of methyl iodide with 5-tributylstannyl-4¢-thio-2¢-
deoxyuridine (5) to afford 4¢-[methyl-11C]thiothymidine ([11C]4) in
order to determine the HPLC analytical yield
Langstrom, Y. Watanabe and R. Noyori, Chem.–Eur. J., 1997, 3, 2039–
2042; (b) M. Suzuki, R. Noyori, B. La˚ngstro¨m and Y. Watanabe,
Bull. Chem. Soc. Jpn., 2000, 73, 1053–1070; (c) M. Suzuki, H. Doi,
K. Kato, M. Bjo¨rkman, B. La˚ngstro¨m, Y. Watanabe and R. Noyori,
Tetrahedron, 2000, 56, 8263–8273; (d) M. Bjo¨rkman, H. Doi, B. Resul,
˚
¨
[11C]Methyl iodide was trapped in a solution of Pd2(dba)3 (1.8 mg,
2.0 mmol) and P(o-CH3C6H4)3 (19 mg, 64 mmol) in DMF (300
mL) at RT. The solution was added to a solution of stannane 5
(4.3 mg, 8.0 mmol), CuCl (0.4 mg, 4 mmol), and K2CO3 (1.4 mg,
10 mmol) in DMF (100 mL). The resulting mixture was heated
at 80 ◦C for 5 min. The reaction mixture were passed through
cotton and then Syring Filter and were washed with water (1 mL).
The combined elutes were analyzed by HPLC under Conditions C.
HPLC analytical yield of [11C]4: 93%, calculated by peak area ratio
of the [11C]product distributions. The identifications of [11C]4 was
conducted by co-injecting with the corresponding non-radioactive
4¢-thiothymidine (see ESI‡).
M. Suzuki, R. Noyori, Y. Watanabe and B. Langstrom, J. Labelled
Compd. Radiopharm., 2000, 43, 1327–1334; (e) T. Hosoya, M. Wakao,
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(f) M. Suzuki, H. Doi, T. Hosoya, B. La˚ngstro¨m and Y. Watanabe,
TrAC, Trends Anal. Chem., 2004, 23, 595–607; (g) T. Hosoya, K.
Sumi, H. Doi, M. Wakao and M. Suzuki, Org. Biomol. Chem., 2006,
4, 410–415; Patent: M. Suzuki and T. Hosoya, JP 2005-306670, WO
02007/046258; (h) Patent: M. Suzuki, H. Doi and H. Tsukada, JP
2006-229169, WO/2008/023780; H. Doi, I. Ban, A. Nonoyama, K.
Sumi, C. Kuang, T. Hosoya, H. Tsukada and M. Suzuki, Chem.–
Eur. J., 2009, 15, 4165–4171; (i) M. Suzuki, K. Sumi, H. Koyama,
Siqin, T. Hosoya, M. Takashima-Horano and H. Doi, Chem.–Eur. J.,
2009, 15, 12489–12495; Patent: M. Suzuki, H. Doi and H. Koyama,
JP 2008-335250, WO/2010/074272; (j) For the preliminary reports
on this work, see: H. Koyama, Siqin, Z. Zhang, K. Sumi, Y. Hatta,
H. Nagata, H. Doi and M. Suzuki, the 2010 World Molecular Imaging
Congress, Kyoto, Sept. 8–11, 2010 (poster presentation), and the 2010
International Chemical Congress of Pacific Basin Societies, Honolulu,
Dec. 15–20, 2010 (oral presentation).
˚
¨
Synthesis of 4¢-[methyl-11C]thiothymidine ([11C]4) for a PET study
[11C]Methyl iodide was trapped in the solution of Pd2(dba)3
(1.1 mg, 1.2 mmol) and P(o-CH3C6H4)3 (2.1 mg, 6.9 mmol)23,32
in DMF (300 mL) at RT. The solution was transferred into the
2 (a) J. S. Fowler, A. P. Wolf, J. R. Barrio, J. C. Mazziotta and M. E.
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M. E. Phelps, J. C. Mazziotta and H. R. Schelbert, Raven Press, New
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18 V. W. Rosso, D. A. Lust, P. J. Bernot, J. A. Grosso, S. P. Modi, A.
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19 If 5Cu2 would have similar pKa as phenol (pKa = 10.0) by activation
with CuI, the formation of 10 by proton abstraction from 5Cu2 with
2-
CO3 would be calculated to be ca. 107 times larger than by F-, where
pKa = 10.3 and pKa = 3.2 were used for the corresponding conjugate
acids HCO3- and HF, respectively, supprting high superiority of K2CO3
to CsF for the ability to avoid the destannylation. For pKas, see; I.
Luyten, K. W. Pankiewicz, K. A. Watanabe and J. Chattopadhyaya,
J. Org. Chem., 1998, 63, 1033–1040; CRC Handbook of Chemistry
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6 M. Bragadin, D. Marton, G. Scutari and P. Dell’Antone, J. Inorg.
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(the cross-coupling reaction of methyl iodide and (p-methoxy)phenyl
tributylstannane using Pd(PPh3)4 as a catalyst, giving poor yield).
8 (a) J. A. Secrist III, K. N. Tiwari, J. M. Riordan and J. A. Montgomery,
J. Med. Chem., 1991, 34, 2361–2366; (b) W. B. Parker, S. C. Shaddix,
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synthesized using the corresponding trimethylstannyl precursor under
the same conditions; see also ref. 3c.
10 The synthesis of PET probes is rather different from the usual organic
syntheses. The reaction involves the trapping of an extremely small
amount of 11CH3I (approximately 100 nmol level containing 12CH3I)
with a large amount (mg level, >1000 equiv) of reacting substrate.
Therefore, we here set up the reaction using an excess amount of a
tributylstannyl substrate for CH3I ([11C]CH3I) to meet the demand.
11 For details, see the experimental section.
20 Yield was obtained according to the equation of 100% ¥ (radioactivity
of desired product)/(total radioactivity of the products) based on the
radioactivity observed in HPLC.
21 The same reaction was conducted using the conditions previously
reported5c for Pd2(dba)3/P(o-CH3C6H4)3 (1 : 4 in molar ratio) in DMF
at 130 ◦C for 5 min, giving [methyl-11C]thymidine [11C]1 in 17% (HPLC
analytical yield).
22 [11C]1 was obtained in 91% (HPLC analytical yield) under the
Pd2(dba)3/P(o-CH3C6H4)3/CuBr/CsF (1 : 32 : 10 : 25 in molar ratio)
system in DMF, 65 ◦C, 5 min. Thus the increase of the amount of
the CuBr/CsF tends to give the desired product in slightly higher yield.
23 We improved the reaction conditions to some extent under keeping
the specified smaller-sized reaction vial combined with a preparative
HPLC column for an actual PET probe synthesis in mind. Particularly,
the limited solubility of the phosphine was compelled to the tedious
preparative HPLC operations to purify small amounts of PET probes
from an insoluble heterogeneous mixture. See experimental section and
reference 1i.
24 See also Table 1 entry 5.
25 This study is focused on the rapid C-[11C]methylation. In this context,
11CH3PdI-phosphine complex is involved directly in the cross-coupling
reaction with a stannyl substrate, and therefore, the radioactivity of
non-volatile 11CH3PdI{P(o-CH3C6H4)3}n formed by trapping 11CH3I
with the Pd0 complex was selected as a first checking point of total
radioactivity.
26 Various factors such as radiolysis inducing radical reactions under high
concentration during the evaporation of the solvent and the separation
by preparative HPLC, the absorption on the HPLC solid support, time
elongation, etc. are considered to be reflected on lowering the isolated
yield. As a good example about the deviation of the isolated yield
and efforts to suppress such a yield decreasing by adding a radical
trapping agent to be sacrificed, see the synthesis of various 11C-labeled
2-arylpropionic acids and their esters, see: M. Takashima-Hirano, M.
Shukuri, T. Takashima, M. Goto, Y. Wada, Y. Watanabe, H. Onoe, H.
Doi and M. Suzuki, Chem.–Eur. J., 2010, 16, 4250–4258.
27 The radioactivities needed for animal and human PET imaging are
10-50 and 100-500 MBq, respectively.
12 S. P. H. Mee, V. Lee and J. E. Baldwin, Angew. Chem., Int. Ed., 2004,
43, 1132–1136.
13 NMP solvent was overlapped with thymidine on HPLC analysis and
therefore, NMP was removed by evaporation under reduced pressure
before analysis of the yield.
14 M. Maltese, S. Passerini, S. Nunzianta-Cesaro, S. Dobos and L.
Harsa´nyi, J. Mol. Struct., 1984, 116, 49–65; Yu. P. Blagoi, E. D.
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Chem. Phys. Lett., 1997, 269, 39–48.
28 We are also trying another approach for synthesis of 4¢-[methyl-
11C]thiothymidine using boronic acid ester.1h.
29 Y. Yoshimura, K. Kitano, K. Yamada, S. Sakata, S. Miura, N. Ashida
and H. Machida, Bioorg. Med. Chem., 2000, 8, 1545–1558; J. K. Watts,
K. Sadalapure, N. Choubdar, B. M. Pinto and M. J. Damha, J. Org.
Chem., 2006, 71, 921–925.
30 T. Imanishi and S. Obika, Chem. Commun., 2002, 1653–
1659.
15 P. Gunaga, H. R. Moon, W. J. Choi, D. H. Shin, J. G. Park and L. S.
Jeong, Curr. Med. Chem., 2004, 11, 2585–2637.
16 4¢-Thiothymidine was insoluble in NMP. Thus the resulting suspension
was dissolved in DMF before HPLC analysis.
17 In this context, the reaction of 6 using the CuBr/CsF synergic system
(Table 1 entry 7) gave only a small amount of destannylated product, 2¢-
deoxyuridine as byproduct (5% for initial amount of 6). The mechanism
of destannylation remains unclear. Heteroaromatic stannanes such
as neutral or pyridine-related basic compounds did not give such
destannylated compounds at all.1i.
31 C. F. Foulon, Y. Z. Zhang, S. J. Adelstein and A. I. Kassis, Appl. Radiat.
Isot., 1995, 46, 1039–1946.
32 In this two-pot procedure, the 6 equiv of phosphine for Pd2(dba)3 was
used to trap [11C]CH3I by a Pd0/phosphine complex, and 10 equiv of
phosphine was added in another flask to dissolve the copper salt as well
as to promote the coupling reaction efficiently.
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