Synthesis of NCA [carbonyl-11C]amides by direct reaction of in situ generated [11C]carboxymagnesium halides with amines under microwave-enhanced conditions

Article abstract of DOI:10.1002/jlcr.786

No-carrier-added (NCA) aromatic and aliphatic [carbonyl- ¹¹C]amides were rapidly (< 5 min) synthesized in one pot in useful radiochemical yields (20-65%, decay-corrected) by directly coupling amines with NCA [¹¹C]carboxymagnesium halides generated in situ from Grignard reagents and cyclotronproduced [¹¹C]carbon dioxide. In this system cyclohexylcarboxymagnesium chloride (1b) is more reactive than 4-fluorophenylcarboxymagnesium bromide (2b) and primary amines (e.g. aniline, aminopyridines) far more reactive than secondary amines (e.g. 2-(methylamino)pyridine). The scope of the reaction was widened considerably by the application of microwaves, which allowed reactions to be carried out at much higher temperature than the boiling point of the solvent (i.e. tetrahydrofuran, b.p. 67°C). Copyright

Full text of DOI:10.1002/jlcr.786

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2003; 46: 1249–1259.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.786
Research Article
Synthesis of NCA [carbonyl-11C]amides by direct
reaction of in situ generated
[11C]carboxymagnesium halides with amines under
microwave-enhanced conditions
Shui-Yu Lu*, Jinsoo Hong and Victor W. Pike
PET Radiopharmaceutical Sciences, Molecular Imaging Branch, National
Institute of Mental Health, National Institutes of Health, Building 10, Room
B3C346, 10 Center Drive, Bethesda, MD 20892-1003, USA
Summary
No-carrier-added (NCA) aromatic and aliphatic [carbonyl-11C]amides were
rapidly (55 min) synthesized in one pot in useful radiochemical yields (20–
65%, decay-corrected) by directly coupling amines with NCA [11C]carboxy-
magnesium halides generated in situ from Grignard reagents and cyclotron-
produced [11C]carbon dioxide. In this system cyclohexylcarboxymagnesium
chloride (1b) is more reactive than 4-fluorophenylcarboxymagnesium bromide
(2b) and primary amines (e.g. aniline, aminopyridines) far more reactive than
secondary amines (e.g. 2-(methylamino)pyridine). The scope of the reaction
was widened considerably by the application of microwaves, which allowed
reactions to be carried out at much higher temperature than the boiling point
of the solvent (i.e. tetrahydrofuran, b.p. 678C). Copyright # 2003 John Wiley
& Sons, Ltd.
Key Words: carbon-11; amide; grignard reagent; microwave; [11C]carbon
dioxide
*Correspondence to: S.-Y. Lu, PET Radiopharmaceutical Sciences, Molecular Imaging Branch,
National Institute of Mental Health, National Institutes of Health, Building 10, Room B3C346, 10
Center Drive, Bethesda, MD 20892-1003, USA. E-mail: lus@intra.nimh.nih.gov
Copyright # 2003 John Wiley & Sons, Ltd.
Received 10 August 2003
Accepted 8 September 2003

1250
S.-Y. LU ET AL.
Introduction
Positron emission tomography (PET) imaging of animal or human
organs requires the development of new radiotracers labeled with
positron-emitters, such as carbon-11 (t1/2=20.4 min) and fluorine-18
(t1/2=109.8 min). Several methods have been developed to introduce
carbon-11 label into organic compounds from primary cyclotron-
produced radioactive precursors, such as no-carrier-added (NCA)
[11C]carbon dioxide and [11C]methane.1–3 Most of these involve multiple
steps. The fact that carbon-11 has a very short half-life besets
radiochemists with the considerable challenge of developing procedures
that are preferably single-step, rapid, isotope-efficient and easily
automated to produce radiotracers in high radiochemical purity and
high radiochemical yield.
Amides constitute a major functionality in a large proportion
of radiopharmaceuticals.4,5 Methods are known for introducing
carbon-11 into the carbonyl position. Traditionally, NCA
[carbonyl-11C]amides are prepared by reacting cyclotron-produced
[11C]carbon dioxide with organometallic reagents, such as Grignard
reagents, to obtain either the [11C]carboxymagnesium halides or
[11C]carboxylic acid followed by their conversion into a more reactive
species, such as a [11C]acid chloride,6–11 and reaction with the amine.
Other strategies to activate [11C]acids have also been reported.12–17
Nevertheless, the final step or reaction with amine is not only time
consuming, but also a part of the process that is not always reliable. A
labeling procedure that uses direct reaction of amine with [11C]carbox-
ymagnesium halide or [11C]carboxylic acid generated in situ would
provide a considerable reduction in time and efforts and would
therefore become highly attractive.
Lasne et al.18,19 reported the synthesis of [11C]aliphatic amides in
moderate decay-corrected radiochemical yields (RCYs) by treatment of
magnesium halide carboxylates with amines in tetrahydrofuran (THF)
in the presence of 2.5 equivalents of alkylmagnesium halides. The RCYs
were 15–60%, decay-corrected to the end of synthesis (EOS), and in
most cases reactions were complete in 5 min. However, success was
limited to the most reactive aliphatic Grignard reagents and amines. If
this single-pot procedure is to be generally useful its scope must be
expanded.
Microwave-enhanced radiochemistry20–22 can provide a faster,
cleaner, more selective and more highly atom-efficient approach than
Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 1249–1259

SYNTHESIS OF NCA [CARBONYL-11C]AMIDES
1251
conventional radiochemistry. The shorter reaction time coupled with
other benefits, such as higher product purity due to less reaction mixture
decomposition and the promotion of otherwise sluggish reactions, make
it an ideal tool to be explored in radiochemistry with short-lived
positron-emitters. Here we wish to report the synthesis of NCA
[carbonyl-11C]amides, both aliphatic and aromatic, by direct reaction of
amines with [11C]carboxymagnesium halides generated in situ from
Grignard reagents and [11C]carbon dioxide under microwave-enhanced
conditions.
Results and discussion
The two Grignard reagents used in this study, namely cyclohexylmag-
nesium chloride (1) and 4-fluorophenylmagnesium bromide (2), were
chosen not merely for their commercial availability, but more
importantly because they each represent the aliphatic and aromatic
moieties found in a large number of amide receptor ligands, especially
the 5-HT1A receptor ligands, WAY-10063523 and p-MPPF.24 A range of
commercially available primary and secondary aromatic amines of
interest to our radioligand development program was selected for this
study. However, the choice of Grignard reagent and amine need not be
limited to those listed in Scheme 1.
Scheme 1. Synthesis of NCA [carbonyl-11C]amides
Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 1249–1259

1252
S.-Y. LU ET AL.
Both Grignard reagents needed to be filtered through a polypropylene
filter (0.45 m) before use. Initial mixing of the Grignard reagent with
THF in the standard Pyrex glass reaction vessel was carried out in an
argon-protected glove box to minimize ingress of atmospheric carbon
dioxide, a potential source of carrier. Reactions were performed under
NCA conditions. Most of the amines have good solubility in THF,
except 4-aminopyridine, which has moderate solubility. After the
hydrolysis with sulfuric acid in THF, additional water was needed to
dissolve the salts. The reaction mixture separated into aqueous and
organic layers. The product [11C]amide and unreacted [11C]acid were
retained in the organic layer whilst the salts were kept in the aqueous
layer and easily removed. A representative chromatogram from the
analysis of crude organic phase containing [11C]13, showing absorbance
(254 nm) and radioactivity detector responses is illustrated in Figure 1.
The results for the synthesis of [carbonyl-11C]amides are summarized in
Table 1. Non-labeled compounds were also prepared and characterized
using LC-MS or GC-MS. Reaction conditions and properties are
summarized in Table 2. Authentic compounds were separated and used
for co-injection with the radioactive samples.
Both Grignard reagents 1 and 2 react with NCA [11C]carbon dioxide
at room temperature (RT) in THF. In the absence of amines, the
[11C]carboxymagnesium halides proceed to react with excess Grignard
reagent to form R1 11COR0 and R13 11COH at elevated temperature
(1008C). In the presence of amine, even non-reactive ones such as
Figure 1. HPLC analysis of the crude organic phase containing [11C]13, showing
absorbance at 254 nm (}) and radioactivity detector (- - -) channel outputs
Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 1249–1259

SYNTHESIS OF NCA [CARBONYL-11C]AMIDES
1253
Table 1. NCA [carbonyl-11C]amides from the direct coupling of NCA
[11C]carboxymagnesium halides with amines under microwave-enhanced
conditionsn
Entry Grignard Amine Reaction conditions
reagent
[11C]amide RCY(%)
(8C)
(min)
(W)
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
3
3
3
4
5
6
7
8
9
70
70
10
10
2
2
2
7
5
10
7
0
200
200
200
200
300
200
300
300
10
10
10
11
12
13
14
15
16
37
55
54
38
65
55
34
14
55
100
120
100
130
100
130
130
10
11
12
13
14
15
16
17
2
2
2
2
2
2
2
2
3
3
4
5
6
7
8
9
100
100
120
120
130
100
130
130
10
10
2
3
7
5
10
10
0
200
200
200
300
200
300
300
17
17
18
19
20
21
22
23
22
45
14
32
22
11
0
0
n Grignard reagent, 0.2 mmol from 2 M solution in ether (100 ml) diluted in THF (400 ml); amine,
0.04 mmol in THF (100 ml); sulfuric acid [0.2 mmol in THF (200 ml)]; water (200 ml).
Table 2. Synthesis and identification of amides from direct coupling of carboxylic
acids and amines in the presence of excess Grignard reagentn
Entry
Grignard
reagent
Acid
Amine
Amide
Retention time
in HPLC (min)
Mass by LC-MS
or GC-MS#
1
2
3
4
5
6
7
8
9
10
1
1
1
1
1
2
2
2
2
2
24
24
24
24
24
25
25
25
25
25
3
4
5
6
7
3
4
5
6
7
10
11
12
13
14
17
18
19
20
21
9.4
6.7
4.9
10.8
9.8
8.6
6.1
4.7
10.4
9.6
205 [M+1]+
205 [M+1]+
205 [M+1]+
203 [M]+#
217 [M]+#
217 [M+1]+
217 [M+1]+
217 [M+1]+
215 [M]+#
229 [M]+#
n Grignard reagent, [1 mmol from 2 M solution in ether (500 ml) diluted in THF (500 ml)]; acid
[0.33 mmol in THF (200 ml)]; amine [0.33 mmol in THF (200 ml)]; sulfuric acid [0.7 mmol in THF
(700 ml)]; water (700 ml).
pyridine, the formation of ketone and alcohol from 1 can be inhibited,
even at 1208C. It was noted that the intermediate, C6H1111COOMgCl
(1b), from the reaction of cyclohexylmagnesium chloride with [11C]
Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 1249–1259

1254
S.-Y. LU ET AL.
carbon dioxide, is much more reactive towards amine than the excess
Grignard reagent. The reverse is true for reactions involving 4-
fluorophenylmagnesium bromide; 4-F-C6H411COOMgBr (2b) is much
more reactive towards excess Grignard reagent than amine which leads
to the formation of ketone and tertiary alcohol. The addition of
pyridine did not prevent the formation of [11C]triarylmethanol in this
case, even at 1008C. This may be a reflection of the relative C–Mg bond
strength in these two Grignard reagents.25,26
Cyclohexylmagnesium chloride was more reactive and gave much
higher [11C]amide RCYs than 4-fluorophenylmagnesium bromide
(Table 1). Reactions starting with 4-flurophenylmagnesium bromide
require higher temperature. For example, reaction of 1b with 2-
aminopyridine (Table 1, entry 2) took place at 708C but no [11C]amide
was detected from the reaction of 2b with the same amine at 708C.
When the temperature was elevated to 1008C, the corresponding amide
was made in high RCY (Table 1, entry 11). However, further increase of
reaction temperature does not necessarily increase the yields; instead
more side reactions and decomposition take place.
Control of the reaction temperature is pivotal to the success
of the reaction. Microwave technology provides a fast, precise way to
control temperature. In this work a single mode microwave reactor
equipped with temperature and pressure sensors was used. At an early
stage, reactions using microwave reactor and heating block were
compared. The use of microwaves improved the reaction significantly;
for example RCYs increased 30–50% (e.g. from 37 to 55% and from 22
to 45%) for the two compared reactions (Table 1, entries 1 and 2, entries
10 and 11). It is also to our advantage that the microwave instrument
enables the reaction to be carried out at much higher temperature than
the boiling point of the solvent (here THF, b.p. 678C). With crimp
sealing of the septum no radioactivity was lost during the heating
process even when the reaction mixture in THF was heated to 1508C
and 80 p.s.i.
The carboxymagnesium halides are more reactive towards primary
amines than secondary amines. There are insufficient data to elucidate
the mechanism, but it appears that the reactivity of the amines is a
function of both their electronic and steric properties. Only 14% RCY
was achieved in the reaction of 1b with 2-(methylamino)pyridine (Table
1, entry 8). Variation of temperature, reaction time and concentration
did not improve the yield. The RCY for the reaction of 1b with
tetrahydroisoquinoline was even lower (Table 1, entry 9). Not
Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 1249–1259

SYNTHESIS OF NCA [CARBONYL-11C]AMIDES
1255
surprisingly, no amide was obtained from the aromatic Grignard
reagent 2 with secondary amines.
Non-labeled compounds were prepared using the reactions outlined
in Scheme 2. No optimization of yields was attempted. The compounds
served as standards for radio-HPLC analysis and were characterized by
LC-MS(ES) or GC-MS (EI) (Table 2).
Scheme 2. Synthesis of amides by direct coupling of carboxylic acids and amines
in the presence of excess Grignard reagent
In conclusion, both aromatic and aliphatic NCA [11C]amides were
successfully synthesized by the direct coupling of NCA [11C]carbox-
ymagnesium halides, generated in situ, with primary amines. The
procedure is less successful with secondary amines. The scope of the
reaction has been widened considerably by the application of
microwaves. Control of the reaction temperature at the right level is
pivotal to the success of the reaction; without the use of a microwave
reactor this would be difficult to achieve.
Experimental
Materials
Cyclohexylmagnesium chloride (2.0 M solution in diethyl ether pack-
aged under nitrogen in Sure/SealTM bottle, Aldrich), 4-fluorophenyl-
magnesium bromide (2.0 M solution in diethyl ether packaged under
nitrogen in Sure/SealTM bottle, Aldrich) were used as received. 2-
Aminopyridine (99+%, Aldrich), 3-aminopyridine (99+%, Aldrich),
4-aminopyridine (99+%, Aldrich), aniline (99%, Aldrich), benzylamine
(99%, Aldrich), 2-(methylamino)pyridine (98%, Aldrich), 1,2,3,4-
tetrahydroisoquinoline (98%, Aldrich) were used as received. THF
Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 1249–1259

1256
S.-Y. LU ET AL.
(99.9+%, inhibitor-free, Aldrich), sulfuric acid (95–98%, Aldrich),
phosphoric acid (85% in water, ACS grade, EM Science), ammonium
formate (96%, J.T. Baker), acetonitrile (high purity solvent, Burdick &
Jackson) were also used without further treatment.
General methods
LC-MS analysis was performed on a Thermo Finnigan Surveyor LC
system equipped with a Finnigan Quest LC QDECA ESI probe. GC-MS
analysis was performed on a Thermo Finnigan Trace GC equipped with
Polaris Q system. Microwave irradiation was performed with a CEM
DiscoverTM microwave reactor. HPLC analysis was performed on a
system comprising a Beckman Coulter Gold HPLC module [System
Gold 126 solvent module coupled with a 166 UV absorbance detector
(single wavelength)] plus a Bioscan Flow Count radioactivity detector
(diode or PMT). Radioactivity was measured using a calibrated Biodex
Medical Systems AtomlabTM 300 dose calibrator.
NCA [11C]Carbon dioxide was prepared by the 14N(p,a)11C nuclear
reaction on a cyclotron (PETtrace; GE) using a nitrogen gas target (1%
oxygen, pressure 150 p.s.i.) bombarded for 5 min with a 5 mA beam of
16 MeV protons. NCA[11C]carbon dioxide was first collected in a 5-loop
stainless steel tube cryogenic trap, equipped with a cartridge of mixtures
of chromium(III) oxide and copper(II) sulfate on silica gel to remove
nitrogen oxides and another cartridge of phosphorous(V) oxide to
remove moisture,27 before delivery to the reaction vessel by helium at
5 ml/min. Generally, this process gives [11C]carbon dioxide with a
specific radioactivity in the range 40–80 GBq/mmol within 5 min of the
end of irradiation.
Labeled compounds were identified by co-injection of the non-labeled
standards and co-elution from the column (Phenomenex Luna C18,
(
10 m, 100 A, 250 ꢀ 4.6 mm i.d.) with acetonitrile-0.1 M ammonium
formate solution containing 15.6 mM phosphoric acid, (pH=3.5) as
mobile phase at 2 ml/min.
General procedure for the synthesis of NCA ½carbonyl-11Cꢁamides; ½11Cꢁ
10–23
Within an argon-protected glove box, Grignard reagent (RMgX, 2 M
solution in ether; 100 ml, filtered through a 0.45 m filter) and THF
(400 ml) were added to a standard Pyrex glass microwave reaction vessel
Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 1249–1259

SYNTHESIS OF NCA [CARBONYL-11C]AMIDES
1257
(10 ml; CEM). The vessel was crimp-sealed with a PTFE-coated septum.
[11C]Carbon dioxide was bubbled into the vial at 5 ml/min at RT for
6 min via PEEK tubing (1/16 in) pierced through the septum. Amine
(0.04 mmol) in THF (100 ml) was added to the reaction mixture using an
epidermal needle also at RT. The mixture was then irradiated in the
microwave reactor at the pre-set temperature and for a set time. At the
end of microwave irradiation, stock sulfuric acid (1M in THF; 200 ml)
and water (200 ml) were added successively through epidermal needles.
The aqueous layer (ꢂ250 ml) was removed and the organic layer
(ꢂ700 ml) containing the 11C-labeled products analyzed by radio-HPLC
with UV absorbance (254 nm) and radioactivity detection.
General procedure for the synthesis of unlabeled amides, 10–23
Within an argon-protected glove box, Grignard reagent (RMgX; 2 M
solution in ether, 500 ml; filtered through 0.45 m filter) and THF (500 ml)
were added to a standard Pyrex glass microwave reaction vessel (10 ml;
CEM). The vessel was crimp-sealed with a PTFE-coated septum. Acid
(RCOOH; 0.3 mmol) in THF (200 ml) was added to the mixture at 08C,
followed 5 min later by amine (1.2 mmol) in THF (200ml), again at 08C.
The mixture was then irradiated in the microwave reactor at a pre-set
temperature and for a set time. At the end of microwave irradiation,
stock sulfuric acid (1 M in THF; 700 ml), and water (700 ml) were added
successively to destroy excess Grignard reagent and to dissolve the salt
formed again using epidermal needles. The aqueous layer (0.6 ml) was
removed and the organic layer (2.1 ml) containing the unlabeled
products was analyzed by HPLC as the crude reaction mixture. The
identity of compounds was confirmed by HPLC-MS or GC-MS analysis.
Acknowledgements
The authors wish to thank Dr Umesha Shetty (MIB, NIMH) for the
service of GC-MS and LC-MS analysis.
References
1. Welch MJ, Redvanly CS (eds). Handbook of Radiopharmaceuticals –
Radiochemistry and Applications. Wiley: Chichester, 2003.
Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 1249–1259

1258
S.-Y. LU ET AL.
.
2. Stocklin G, Pike VW (eds). Radiopharmaceuticals for Positron Emission
Tomography: Methodological Aspects. Kluwer Academic Publishers:
Dordrecht, 1993.
3. Fowler JS, Wolf AP. The Synthesis of Carbon-11, Fluorine-18 and
Nitrogen-13 Labeled Radiotracers for Biomedical Applications. Technical
Information Center, US Department of Energy: USA, 1982.
4. Iwata R. Reference Book for PET Radiopharmaceuticals. CYRIC,
Tohoku University, 2002. Available on the Internet at the following
address: http://kakuyaku.cyric.tohoku.ac.jp/public/Reference Book 2002.
pdf.
5. Passchier J, van Waarde A. Eur J Nucl Med 2001; 28: 113–129.
6. Luthra SK, Pike VW, Brady F. J Chem Soc Chem Commun 1985;
1423–1425.
7. Luthra SK, Pike VW, Brady F. Appl Radiat Isot 1990; 41: 471–476.
8. McCarron JA, Turton DR, Pike VW, Poole KG. J Label Compd
Radiopharm 1996; 38: 941–953.
!
9. Lasne M-C, Cairon Ph, Barre L. Appl Radiat Isot 1992; 43:
621–625.
10. Lang L, Jagoda E, Schmall B, Vuong BK, Adams HR, Nelson DL, Carson
RE, Eckelman WC. J Med Chem 1999; 42: 1576–1586.
11. Wilson AA, Inaba T, Fischer N, Dixon LM, Nobrega J, DaSilva JN,
Houle S. Nucl Med Biol 1998; 25: 769–776.
12. Mitchell JA, Reid EE. J Am Chem Soc 1931: 53: 1879–1883.
13. Jursic BS, Zdravkovski Z. Synth Commun 1993; 23: 2761–2770.
14. Rogers DA, Stone-Elander S, Ingvar M. J Label Compd Radiopharm 1994;
35: 327–328.
15. Lu SY, McCarron JA, Hong J, Musachio JL, Pike VW. J Label Compd
Radiopharm 2003; 46(S1): S229.
16. Lemoucheux L, Rouden J, Lasne M-C. Tetrahedron Lett 2000; 41:
9997–10001.
17. Azumaya I, Okamoto T, Imabeppu F, Takayanagi H. Tetrahedron 2003;
59: 2325–2331.
18. Aubert C, Huard-Perrio C, Lasne M-C. J Chem Soc, Perkin Trans I 1997;
2837–2842.
19. Perrio-Huard C, Aubert C, Lasne M-C. J Chem Soc, Perkin Trans I 2000;
311–316.
20. Elander N, Jones JR, Lu SY, Stone-Elander S. Chem Soc Rev 2000; 29:
239–249.
21. Jones JR, Lu SY. In Microwaves in Organic Synthesis, Loupy A (ed).
Wiley-VCH: Weinheim, 2002; 435–462.
22. Elander N, Stone-Elander S. J Label Compd Radiopharm 2002; 45:
715–746.
Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 1249–1259

SYNTHESIS OF NCA [CARBONYL-11C]AMIDES
1259
23. Pike VW, McCarron JA, Lammerstma AA, Osman S, Hume SP, Sargent
PA, Bench CJ, Cliffe IA, Fletcher A, Grasby PM. Eur J Pharmacol 1996;
301: R5–R7.
24. Shiue CY, Shiue GG, Mozley PD, Kung MP, Zhuang ZP, Kim HJ, Kung
HF. Synapse 1997; 25: 147–154.
25. Richey Jr HG (ed). Grignard Reagents – New Developments. Wiley:
Chichester, 2000.
26. Kharasch MS, Reinmuth O. Grignard Reactions of Nonmetallic Substances.
Prentice-Hall: New York, 1954.
27. Tewson TJ, Banks W, Franceschini M, Hoffpauir J. Appl Radiat Isot 1989;
40: 765–768.
Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 1249–1259