Synthesis of (R,S)-[4-11C]baclofen via Michael addition of nitromethane labeled with short-lived 11C

Article abstract of DOI:10.1016/j.bmcl.2009.08.097

The synthesis of (R,S)-[4-¹¹C]baclofen, the first ¹¹C-labeled GABA_B agonist, was demonstrated via Michael addition of nitro[¹¹C]methane as a key step. A tetrabutylammonium fluoride promoted Michael addition of n

Full text of DOI:10.1016/j.bmcl.2009.08.097

Bioorganic & Medicinal Chemistry Letters 19 (2009) 6222–6224
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of (R,S)-[4-11C]baclofen via Michael addition of nitromethane
labeled with short-lived 11C
*
Koichi Kato , Ming-Rong Zhang, Kazutoshi Suzuki
Department of Molecular Probes, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan
a r t i c l e i n f o
a b s t r a c t
The synthesis of (R,S)-[4-11C]baclofen, the first 11C-labeled GABAB agonist, was demonstrated via Michael
addition of nitro[11C]methane as a key step. A tetrabutylammonium fluoride promoted Michael addition
of nitro[11C]methane to methyl p-chlorocinnamate, followed by the nitro-group reduction in the pres-
ence of NiCl2 and NaBH4 in aqueous MeOH and alkaline hydrolysis yielded (R,S)-[4-11C]baclofen in
36.4 1.8% radiochemical conversion in three steps within 20 min.
Article history:
Received 24 June 2009
Revised 6 August 2009
Accepted 31 August 2009
Available online 3 September 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Positron emission tomography
Nitro[11C]methane
Baclofen
GABAB
Positron emission tomography (PET) is a non-invasive in vivo
imaging technology using molecules labeled with short-lived pos-
itron-emitting radionuclides such as 11C and 18F (T1/2 = 20.3 and
109.7 min). There is an ongoing need for the development of new
versatile probe molecules for a variety of applications. Its success
as a tool for diagnosis and drug research and development depends
on employing a molecular probe with adequate pharmacokinetics
or high and specific uptake to target tissues.1 Many factors must
be considered when identifying potentially new PET tracers. Label-
ing position and type of isotope are important for the design of
tracers. However, the number of labeling reactions and the overall
time of the reaction sequence need to be limited because of the
short half-life of the radionuclide.2 Therefore, labeling methodolo-
gies and synthetic methods become vital to the success of the
probe.
tant targets of brain PET studies; however, almost all PET studies
of GABA receptors are focused on GABAA.4 There has been only
one report about the 11C-labeling synthesis of CGP62349, a GABAB
antagonist; however, significant results were not obtained because
of the negligible uptake into the brain.5 The importance of GABAB
PET for understanding seizures and epilepsy is suggested6 and re-
cently the tumor suppressive effect of a GABAB agonist for cancers
in peripheral organs has been reported.7 Therefore, the 11C-labeling
synthesis of baclofen should prove useful for PET studies of the GA-
BAB receptor.8 Moreover, development of the Michael addition
reaction of 1 can assist in the 11C-labeling synthesis of GABA deriv-
atives beyond baclofen, because it yields a common framework for
those compounds.
As outlined in Scheme 1, we planned the Michael addition med-
iated 11C-labeling synthesis of (R,S)-[4-11C]baclofen (2) by reaction
of 1 to methyl p-chlorocinnamate (3) followed by nitro-group
reduction and ester hydrolysis. Although Michael addition of nitro-
alkanes is one of the most important synthetic processes devoted
to CÀC bond formation and many reports have been published
on the topic, there are no applications using 1.9 Requirements of
rapid and appropriate conditions for a Michael addition reaction
We focused on the 11C-labeling synthesis of baclofen by the Mi-
chael addition reaction of nitro[11C]methane (1) that leads to the
construction of the
c
-amino acid framework as a key step (Scheme
1, R2 = p-Cl–C6H4). Baclofen is a
c
-amino butyric acid (GABA) ana-
log and is considered to be the only agonist of the GABAB receptors.
GABA is the major inhibitory transmitter in the central nervous
system and exerts its effect through three different receptor sub-
types: GABAA, GABAB, and GABAC.3 GABAA and GABAC are ligand-
gated ion channels permeable to anions and they convey fast syn-
aptic transmissions. GABAB is a G-protein coupled receptor; it is in-
volved in the presynaptic inhibition of transmitter release and
mediates the slow synaptic inhibition. GABA receptors are impor-
[
11C]H3NO2
NH2
R2
NO2
R2
O
*
1
O
*
O
R1O
HO
R1O
R2
2; R2 = p-Cl-C6H4
3; R1 = Me,
* = 11C
R
2 = p-Cl-C6H4
* Corresponding author.
E-mail address: katok@nirs.go.jp (K. Kato).
Scheme 1. Synthetic plan for 11C-labeled
c-amino acids via Michael addition.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.08.097

K. Kato et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6222–6224
6223
followed by a step such as nitro-group reduction hamper the
development of 11C-labeling synthesis. In this context, the Michael
addition of 1 to 3 was of interest to us.
is usually used as the base and alcohols are chosen as solvents for
increased solubility.16 However, treatment of 1 to 3 with KF did not
afford 4 in i-PrOH under 11C-labeling conditions. Further attempts
at the reaction with soluble fluoride led us to use TBAF in THF, and
it was found that TBAF promotes the Michael addition reaction of 1
to 3 efficiently in THF in 3 min.17 Thus, treatment of 1 and 3 with
AgNO2
1
(1)
[
11C]H3I
100 ºC
5
l
mol of TBAF in THF yielded the desired adduct 4 in 67.9 1.2%
30 mL/min
radiochemical conversion.18 When the TBAF was increased up to
20 lmol, conversion similarly increased to 77.1 1.1%. Although
O
a higher reaction temperature is frequently introduced for 11C-
labeling syntheses to increase radiochemical conversion, when
the reaction was carried out at 60 °C a decreased percent conver-
sion was obtained (entry 4). Thus, we decided to use the reaction
conditions shown in entry 3 for the synthesis of 2.
NO2
O
*
base
1
+
MeO
(2)
iPr
MeO
THF
Cl
rt, 3 min
Cl
N
3
4
It is critical that practical methods are developed and followed
for all steps in multi-step 11C-labeling syntheses. In this context,
we chose reduction by Ni2BÀNaBH4 and ester hydrolysis by aque-
ous alkaline solution in a one-pot system. The preparation of
Ni2BÀNaBH4 by the treatment of NiCl2 and NaBH4 in situ in MeOH
is simple enough for the synthesis of radio-labeled compounds.19
Moreover, the agent Ni2BÀNaBH4 does not seem to be affected
by the contamination of THF. Therefore, we carried out the reduc-
tion reaction of 4 without any purification of the Michael addition
mixture. However, we encountered a problem that might have
arisen from a competing 1,4-reduction of 3. Thus, a solution of
NiCl2 in MeOH was added to the mixture of the nitroaldol reaction
and then the resulting solution was transferred to the next reaction
vessel containing NaBH4. After alkaline hydrolysis, however, the
procedure did not yield 2. In contrast, the desired 2 was obtained
in about 15% conversion in three steps by adding MeOH to the
reaction mixture of the Michael addition and then placing NiCl2
and NaBH4 in the next reaction vessel. In the first case, low or no
yield of 2 resulted because of the incomplete nitro-group reduction
of 4 as the competing 1,4-reduction of 3 and rapid decomposition
of NaBH4 forming Ni2B in MeOH retarded formation of the corre-
sponding amine.19b In the second case, a measurable yield of 2
was obtained because of the gradual formation of Ni2B and the
remaining NaBH4. Further addition of NaBH4 can assist amine for-
mation under non-labeling conditions; however, because of issues
related to protection from radiation, this cannot be done. Several
attempts at a quick separation using a solid phase extraction car-
tridge were tried, but 3 and 4 were not separable.
iPr
iPr
base = t-BuOK,
N
P
N
N
N
DBU
N
5
The 11C-labeling agent 1 can be prepared by nitration of
iodo[11C]methane (Eq. 1),10 a routinely produced 11C-labeling
agent, and used for 11C-labeling reactions by us and other
groups.11,12 We carried out initial studies of the Michael addition
reaction yielding 4 in THF at room temperature in 3 min (Eq. 2).
The 11C-labeling agent 1 was prepared from iodo[11C]methane
via on-line synthesis by heating a plug of AgNO2.10b Our first at-
tempts of the reaction 1 and 3 using t-BuOK or DBU yielded no de-
sired product 4.13 Under non-labeling conditions, the reaction can
be carried out with an excess of nitroalkanes and a longer reaction
time to compensate for the slow reaction. In contrast, the 11C-
labeling reaction should be carried out under a sub-micromole
scale to realize the high specific activity and be terminated within
a few minutes due to competing decay of both 1 and 4. These big
differences between labeling and non-labeling reaction conditions
have been encountered frequently in other 11C-labeling syntheses.
Moreover, the proazaphosphatranes, P(RNCH2CH2)3N, developed
by Verkade, promote the Michael addition reaction efficiently
using a one-to-one ratio of nitroalkanes to
a,b-unsaturated com-
pounds under non-labeling conditions.14 However, under 11C-
labeling conditions, the desired 4 was not obtained by proazaphos-
phatrane 5 (R = i-Pr), although the 11C-labeling agent 1 was con-
verted completely by 5. Side reactions arising from the presence
of a large excess of 3 might complicate the reaction under the
11C-labeling conditions.
The constraints of short-lived and radioactive 11C prompted us
to investigate a milder condition for the decomposition of NaBH4
to increase amine formation. Since decomposition of NaBH4 is
slower in H2O than in MeOH, we used aqueous MeOH instead of
anhydrous MeOH. Thus, a 6:4 mixture of MeOH and H2O was
added to the nitroaldol reaction mixture and the resulting solution
was transferred to the next reaction vessel. After alkaline hydroly-
sis, this process yielded the desired 2 in 36.4 1.8% radiochemical
conversion in three steps (Scheme 2, Table 2).20
Thus, we developed an efficient method for the Michael
addition reaction of 1 to 3 promoted by TBAF under 11C-labeling
conditions. We also developed a practical method for the synthesis
of 2 based on the Michael addition reaction. Currently, automated
systems for the synthesis of 2 are under construction. Although the
Ionic fluoride can act as the base because of strong HÀF bond
energy, and the fact that its basicity against carbon acids like nitro-
alkanes is comparable to that of alkyl metals.15 Therefore, we
investigated the fluoride-assisted Michael addition reaction of 1
to 3 and the results are summarized in Table 1. Potassium fluoride
Table 1
Fluoride-assisted Michael addition reaction of 1–3a,b
Entry Base (l
mol) Solvent Temp Radiochemical conversion of 4c (%)
1
2
3
4
KF (10)
i-PrOH
THF
THF
rt
rt
rt
60 °C
0
TBAF (5)
TBAF (20)
TBAF (20)
67.9 1.2
77.1 1.1
<47d
NH2
NiCl2, NaBH4
MeOH—H2O
rt, 3 min
O
*
THF
TBAF
HO
1 + 3
4
a
NaOH aq.
THF
Reaction conditions: 1 (37–370 MBq); 3 (20
reaction time (3 min).
lmol); THF or i-PrOH (300 lL);
80 ºC, 3 min
rt, 3 min
Cl
* = 11C
2
b
Each reaction was carried out more than three times.
Determined by radiochromatogram of analytical HPLC after decay correction.
Roughly determined because of noise and peak arising from side reactions.
c
d
Scheme 2. Synthesis of 2.

6224
K. Kato et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6222–6224
Table 2
Summary of reaction conditions for the reduction of 4 and successive hydrolysisa
Entry
Reagent 1b
NiCl2 (10
MeOH (500
MeOH/H2O (6/4, 500
Reagent 2c
Reagent 3d
Radio-chemical conversion of 2 (three steps)e (%)
1
2
3
l
mol), MeOH (500
L)
lL)
NaBH4 (0.5 mmol)
5 N NaOH (300
5 N NaOH (300
5 N NaOH (300
lL)
lL)
lL)
0
<15
36.4 1.8
l
NiCl2 (10
NiCl2 (10
l
l
mol), NaBH4 (0.5 mmol)
mol), NaBH4 (0.5 mmol)
l
L)
a
b
c
Other conditions: for reduction, at room temperature and in 3 min; for hydrolysis, at 80 °C and in 3 min.
Reagents added to the mixture of Michael addition reaction.
Reagents containing the second reaction vessel.
Reagent added to the mixture of reduction reaction.
Determined by radiochromatogram of analytical HPLC after decay correction.
d
e
9. Ballini, R.; Bosica, G.; Fiorini, D.; Palmieri, A.; Petrini, M. Chem. Rev. 2005, 105,
933.
10. (a) Schoeps, K.-O.; Halldin, C.; Stone-Elander, S.; Långström, B.; Greitz, T. J.
Labelled Compd. Radiopharm. 1988, 25, 749; (b) Schoeps, K.-O.; Stone-Elander,
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11. (a) Kato, K.; Zhang, M. R.; Suzuki, K. Mol. BioSyst. 2008, 4, 53; (b) Kato, K.;
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12. (a) Schoeps, K.-O.; Långström, B.; Stone-Elander, S.; Halldin, C. Appl. Radiat. Isot.
1991, 42, 877; (b) Schoeps, K.-O.; Halldin, C. J. Labelled Compd. Radiopharm.
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synthetic method described here yields 2 as a racemic form, and a
chiral center is sometimes important for the PET image analysis,
11C-labeled 2 is the first GABAB agonist labeled with a positron-
emitting radionuclide. Moreover, the methods described in this
Letter will allow syntheses of 11C-labeled
c-amino acids and hence
promote the development of new PET tracers.
Acknowledgments
13. The reaction conditions of these attempts are similar to those of entry 2 in
Table 1.
14. Kisanga, P. B.; Ilankumaran, P.; Fetterly, B. M.; Verkade, J. G. J. Org. Chem. 2002,
67, 3555.
This work was supported in part by a consignment grant for the
Molecular Imaging Program on Research Base for PET Diagnosis
from the Ministry of Education, Culture, Sports, Science and Tech-
nology of the Japanese Government. We thank the staff of the
Cyclotron Operation and Radiochemistry sections for technical
support using radioisotopes.
15. Clark, J. H. Chem. Rev. 1980, 80, 429.
16. Kambe, S.; Yasuda, H. Bull. Chem. Soc. Jpn. 1966, 39, 2549.
17. Matsumoto, K. Angew. Chem., Int. Ed. Engl. 1981, 20, 770.
18. A ‘cold’ sample of 4 was prepared by the reaction of nitromethane and 3 using
TBAF. NMR spectra corresponded with those previously reported. Abbenante,
G.; Hughes, R.; Prager, R. H. Aust. J. Chem. 1994, 47, 1441. Analytical HPLC
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scintillation, retention time 5.8 min.
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20. Gaseous 1 was collected by a reaction vessel containing 3 (20
(300 L) with 30 mL/min flow rate at rt. To the mixture, a 1.0 M TBAF solution
in THF (20 mol) was added and stored at rt. After 3 min, a 60% MeOH aqueous
solution (500 L) was added and resulting mixture was transferred to the next
reaction vessel containing NiCl2 hexahydrate (2.4 mg) and NaBH4 (19 mg) at rt.
After 3 min, a 5 N NaOH aqueous solution (300 L) was added and the resulting
mixture was warmed to 80 °C. After 3 min at 80 °C, an aqueous buffer solution
was added (500 L) and the contents of the resulting mixture were analyzed by
m,
lmol) in THF
l
l
l
l
l
HPLC. HPLC conditions for 2: column J’sphere ODS-H80 (4.6 Â 150 mm, 4
l
YMC Co. Ltd), flow rate 0.7 mL/min, eluent 30/70 (MeOH/50 mM phosphoric
acid) detector NaI scintillation, retention time 5.1 min. The peak of
radioactivity of 2 corresponded with the UV absorption (254 nm) of non-
labeled baclofen purchased from Sigma–Aldrich Co. Ltd.