13C- and 14C-labelling of N-[1-(4-chlorophenyl)-1H- pyrrol-2-yl-methyleneamino] guanidinium acetate

Article abstract of DOI:10.1002/jlcr.957

N-[1-(4-chlorophenyl)-1H-pyrrol-2-yl-¹³C₄- methyleneamino]guanidinium acetate has been synthesized by a four-step procedure. This involved reduction of the Weinreb amide N,N′-dimethyl-N, N′-dimethyloxybutane-1,4-diamide-1,2,3,4-

Full text of DOI:10.1002/jlcr.957

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2005; 48: 621–627.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.957
Research Article
13C- and C-labelling of N-[1-(4-chlorophenyl)-1H-
14
pyrrol-2-yl-methyleneamino] guanidinium acetate
Maria Almeida1,*, Petra Johannesson2, Arne Boman3 and Torbjorn
¨
Lundstedt3,4
1 Dirigentvagen 160, SE-75654, Uppsala, Sweden
¨
2 AstraZeneca R&D Molndal, Molndal, Sweden
¨
¨
3 AcurePharma Consulting AB, Uppsala, Sweden
4 Department of Pharmaceutical Chemistry, Uppsala University, Box 574,
SE-75123 Uppsala, Sweden
Summary
N-[1-(4-chlorophenyl)-1H-pyrrol-2-yl-13C4-methyleneamino]guanidinium acetate has
been synthesized by a four-step procedure. This involved reduction of the Weinreb
amide N,N0-dimethyl-N,N0-dimethyloxybutane-1,4-diamide-1,2,3,4-13C4 by Dibal-H
to give the corresponding unstable dialdehyde which is reacted in situ with 4-
chloroaniline to form 1-(4-chlorophenyl)-1H-pyrrole-13C4. This pyrrole analogue
underwent a Vilsmeyer acylation with POCl3/DMF followed by final reaction with
aminoguanidine bicarbonate to produce the desired labelled compound with 99%
atom 13C. By using DMF [a-14C] a radio-labelled analogue was synthesized with a
specific activity of 60 mCi/mmol. Copyright # 2005 John Wiley & Sons, Ltd.
Key Words: N-[1-(4-chlorophenyl)-1H-pyrrol-2-yl-13C4-methyleneamino]guanidinium
acetate; 1-(4-chlorophenyl)-1H-pyrrole-13C4; succinic acid-13C4; Paal–Knorr reaction
Introduction
Compounds labelled with stable isotopes are routinely used as internal
standards in LC–MS assays.1 When a chlorine atom is present in the molecule,
a mass increase of at least M+4 is normally required so the parent ion cluster
is well separated from that of the non-labelled compound. In the development
of a bioanalysis method, the stable labelled analogue 5a was needed. The
present work describes the method developed for the preparation of N-[1-(4-
chlorophenyl)-1H-pyrrol-2-yl-13C4-methyleneamino] guanidinium acetate (5a)
using commercially available succinic acid-13C4, suitable for the synthesis of a
*Correspondence to: Maria Almeida, Dirigentvagen 160, SE-75654 Uppsala, Sweden. E-mail: mlalmeida
¨
@comhem.se
Copyright # 2005 John Wiley & Sons, Ltd.
Received 18 March 2005
Revised 28 March 2005
Accepted 2 April 2005

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M. ALMEIDA ET AL.
multiply and specifically labelled pyrrole ring. For pharmacological
and metabolic studies, the radioactive form 5b was synthesized starting
from the commercially available 1-(4-chlorophenyl)-1H-pyrrole (3b) and
DMF [a-14C].
Results and discussion
The synthesis of non-labelled N-1[(4-chlorophenyl)-1H-pyrrol-2-yl-methyle-
neamino]-guanidinium acetate is easily prepared from commercially available
1-(4-chlorophenyl)-1H-pyrrole by a Vilsmeyer acylation,2 yielding 1-(4-
chlorophenyl)-1H-pyrrole-2-carbaldehyde followed by the reaction with the
aminoguanidinium bicarbonate.3 For the synthesis of the labelled analogue 5a
the key step was the labelling of the pyrrole ring.
Our first attempt was to N-arylate the commercially available pyrrole-d5
with 1-chloro-4-iodobenzene. Several reported procedures4–6 were tried
for the N-arylation of pyrrole. No reaction occurred either using the
NaH4 or the Cu catalysed5 methods. The microwave assisted reaction using
K2CO3/KOH and catalytic amounts of tetrabutylammonium bromide6 turned
out to be sluggish and only 10% of a mixture of the correct compound 1-(4-
chlorophenyl)-1H-pyrrole and 1-(4-iodophenyl)-1H-pyrrole was detected by
1H NMR.
The next approach was to take advantage of the Paal–Knorr reaction,7
which is the most important preparative method for pyrroles. A variety of N-
substituted pyrroles can be prepared using 2,5-dimethoxytetrahydrofuran as a
succinaldehyde equivalent.8 In our case the idea was to generate in situ the
labelled dialdehyde starting from the commercially available 1,4-butane-
diol-13C4. First tried was the oxidation of 1,4-butanediol to the corresponding
dialdehyde by using several oxidation procedures (Swern,9 Dess–Martin,10
PCC/Al2O131) followed by the addition of 4-Cl-aniline. While the Swern9 and
Dess–Martin10 oxidations failed, the PCC/Al2O311 method gave the correct
product in 30% yield. However, a side product was detected to some extent,
which was difficult to separate, and thus no attempts were made to optimize
the reaction further.
Finally, an alternative approach starting from succinic acid proved to be
successful (Scheme 1). The labelled succinic acid-13C4 (1a) was readily
transformed to the Weinreb12 diamide 2a by using standard coupling reagents
in 79% yield after purification. The diamide 2a was then reduced by Dibal-H
followed by in situ addition of 4-Cl-aniline. The labelled pyrrole derivative 3a
was obtained in 40% yield after purification. The Vilsmeyer acylation of 3a
with POCl3/DMF was then easily performed yielding compound 4a in 65%
yield after column chromatography. The last step involved the condensation of
the aldehyde 4a with aminoguanidine to afford 5a in good yield (80%) with
99.9% 13C-atom purity.
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13C- AND 14C-LABELLING
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Scheme 1.
The synthesis of the radioactive analogue 5b could be easily accomplished in
a two-step reaction. Formylation of the commercially available 3b with
labelled DMF [a-14C] formed the aldehyde 4b in 84% yield. The standard
condensation of 4b with aminoguanidine gave the desired radio-labelled 5b in
74% yield with 99% radiochemical purity.
Conclusion
A convenient method was developed for the synthesis of a multiply and
specifically labelled N-substituted 13C-pyrrole ring starting from commercially
available succinic acid-13C. Using this method, the 13C4-labelled compound 5a
was efficiently synthesized with 99.9% atom purity. The Vilsmeyer reaction
using labelled DMF [a-14C] is the method of choice for the introduction of the
radioactive acyl group at position 1 in a pyrrole ring. Thus, the radio-labelled
5b was easily prepared in 62% overall yield with 99% radiochemical purity.
Copyright # 2005 John Wiley & Sons, Ltd.
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M. ALMEIDA ET AL.
Experimental
Melting points were recorded on an Electrothermal IA9200 apparatus and are
uncorrected. All solvents applied as reaction media were of analytical grade
and dried for several days over molecular sieves (4 A). Major chemicals were
purchased from Maybridge and Aldrich and used as received. Succinic
acid-13C4 (99.9% 13C) was purchased from Aldrich. TLC analyses were
performed on Merck silica gel (60 F254) plates (0.25 mm), precoated with a
fluorescent indicator. Visualization was effected with UV light, I2 atmosphere,
or phosphomolybdic acid reagent (PMA) 10% solution in ethanol. Column
chromatography was carried out on Aldrich silica gel 60 (70–230 mesh). NMR
spectra were routinely recorded in CDCl3 on a Bruker Avance-300 instrument
at 300 MHz (1H) and 75 MHz (13C). Chemical shifts are measured in parts per
million (ppm) relative to chloroform (7.25 and 77.0 ppm) as internal reference.
MS analyses were performed on a Micromass Quattro Ultima mass
spectrometer.
Synthesis of N-[1-(4-chlorophenyl)-1H-pyrrol-2-yl-13C4-methyleneamino]gua-
nidinium acetate
N,N0-dimethyl-N,N0-dimethyloxysuccinimide-13C4 (2a). To a suspension of 1a
(1.00 g, 8.20 mmol) and methylmethyloxyamine hydrochloride (2.40 g, 24.6 mmol)
in dry methylene chloride (80 ml) was added triethylamine (4.15 g, 41.0 mmol). To
this mixture was added 1-hydroxybenzotriazole (2.44 g, 18.0 mmol) and the
resulting solution stirred at room temperature. N,N0-dicyclohexylcarbodiimide
(4.06 g, 19.7 mmol) in dry methylene chloride (20 ml) was then added to the
reaction mixture and left with stirring overnight at room temperature. The solvent
was evaporated and the residue treated with diethyl ether (200 ml). The white
precipitate was filtered off and washed several times with diethyl ether. The
ethereal solution was evaporated and the crude colourless oil was chromato-
graphed (silica gel, dichloromethane/methanol 20:1) to afford 1.35 g (79%) of the
product as a white solid: 1H NMR d 3.75 (s, 6 H), 3.20 (br d, 6 H, J=1.6 Hz), 2.79
(dm, 4 H, JC–H=125.6 Hz).
1-(4-chlorophenyl)-1-H-pyrrole-13C4 (3a). Dibal-H (14.1 ml of a 1 M solution
in THF, 14.1 mmol) was added dropwise via syringe to a solution of diamide
2a (1.33, 6.39 mmol) and cooled to ꢀ788C under nitrogen. The mixture was
stirred for about 3 h at ꢀ788C under nitrogen. 4-Chloroaniline (3.26 g,
25.6 mmol) in dry tetrahydrofuran (10 ml) was added dropwise via syringe
followed by addition of water (5 ml). The reaction mixture was left to warm to
room temperature and stirred overnight. The mixture was extracted with
diethyl ether (200 ml) and the organic layer washed in turn with saturated citric
acid solution (3 ꢁ 50 ml), saturated sodium bicarbonate solution (3 ꢁ 50 ml),
saturated sodium chloride solution (3 ꢁ 50 ml), dried (anhydrous sodium
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sulphate), and finally the solvent evaporated. The crude product was then
purified by silica gel chromatography (petroleum ether/diethyl ether 20:1) to
afford 540 mg (46%) of the product as a white solid: 1H NMR d 7.49–7.30 (m,
4H), 7.06 (dm, 2H, JC–H=177 Hz), 6.42 (dm, 2H, JC–H=201 Hz).
1-(4-chlorophenyl)-1H- pyrrole-13C4 –2-carbaldehyde (4a). To a stirred and
ice-bath cooled solution of N,N-dimethylformamide (256 mg, 3.50 mmol) in
1,2-dichloroethane (5 ml) was added phosphorus oxychloride (537 mg,
3.50 mmol) via syringe under nitrogen. The ice-bath was removed and the
mixture stirred at room temperature under nitrogen for 15 min. The reaction
mixture was again ice-bath cooled and additional 1,2-dichloroethane (10 ml)
was added. A solution of 3a (530 mg, 2.92 mmol) in 1,2-dichloroethane (5 ml)
was added via syringe to the cooled and stirred reaction mixture under
nitrogen. The resulting clear solution was refluxed with stirring for 30 min and
then cooled to room temperature. A solution of sodium acetate (1.20 g,
14.6 mmol) in water (25 ml) was added and the resulting mixture refluxed with
stirring for about 20 min. After cooling to room temperature, the organic layer
was separated and the aqueous phase extracted with diethyl ether (3 ꢁ 20 ml).
The combined organic layer was washed in turn with saturated sodium
carbonate solution (2 ꢁ 20 ml), saturated sodium chloride (3 ꢁ 20 ml), dried
(anhydrous sodium sulphate), and finally the solvent was evaporated. The
crude product was then purified by silica gel chromatography (petroleum
ether/diethyl ether 2:1) to afford 400 mg (65%) of the product 4a as a white
1
solid: H NMR d 9.59 (d, 1H, JC–H=28.1 Hz), 7.54–7.41 (m, 2H), 7.35–7.25
(m, 2H), 7.12 (dm, 1H, JC–H=147 Hz), 7.06 (dm, 1H, JC–H=189 Hz), 6.44
(dm, 1H, JC–H=180 Hz).
N-[1-(4-chlorophenyl)-1H-pyrrol-2-yl-13C4-methyleneamino]guanidinium acet-
ate (5a). Aminoguanidinium bicarbonate (286 mg, 2.10mmol) and aldehyde
4a (400 mg, 1.91 mmol) were suspended in methanol (30 ml). Acetic acid (2 ml)
was added and the resulting mixture refluxed for about 5 h. The solvent was
evaporated and diethyl ether was added to the resulting yellow oil. After
cooling, the product crystallized as an off-white powder (500 mg, 80%): mp165–
1
1668C; H NMR (DMSO-d6) d 7.79 (d of unresolved d, 1H, JC–H=9.4,
J=2.8Hz), 7.62–7.52 (m, 2H), 7.44–7.30 (m, 2H), 7.04 (dm, 1H, JC–H=190Hz),
6.69 (dm, 1H, JC–H=174 Hz), 6.30 (dm, 1H, JC–H=158 Hz), 6.15–5.70 (br
signal, 4H), 1.81 (s, 3H); MS m/z (relative intensity) 267 (M+, 100).
Synthesis of N-[1-(4-chlorophenyl)-1H-pyrrol-2-yl–methylene-14C-amino]gua-
nidinium acetate
1-(4-chlorophenyl)-1H-pyrrole-2-carbaldehyde-14C (4b). The pyrrole 3b
(500 mg, 2.81 mmol), DMF [a-14C] (253 mg, 3.37 mmol) and phosphorus
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M. ALMEIDA ET AL.
oxychloride (517 mg, 3.37 mmol) underwent the Vilsmeyer reaction as
described for 4a to yield 480 mg (84%) of the pure product as white crystals
identical with an authentical sample.
N-[1-(4-chlorophenyl)-1H-pyrrol-2-yl-methylene-14C-amino]guanidinium acet-
ate (5b). The aldehyde 4b (480 mg, 2.11 mmol) and aminoguanidinium
bicarbonate (287 mg, 2.11 mmol) were reacted as described for 5a. Purification
of the crude product by HPLC (Hypersil BDS C8 3m (100 ꢁ 3.0 mm), 10 mM
ammonium acetate +1% formic acid:acetonitrile (65:35), isocratic, flow rate:
0.3 ml/min) afforded 532 mg (74%) of the product as an off-white powder: mp
1
166.0–166.58C; H NMR (DMSO-d6) d 7.80 (s, 1H), 7.62–7.56 (m, 2H),
7.44–7.33 (m, 2H), 7.06 (dd, 1H, J=2.4 and 1.6 Hz), 6.75 (dd, 1H, J=3.6 and
1.6 Hz), 6.29 (t, 1H, J=3.2 Hz), 6.90–6.10 (br s, 5H), 1.78 (s, 3H). Radioactive
5b was obtained in 99% radiochemical purity as indicated by HPLC
(conditions as above) with a specific activity of 60 mCi/mmol as determined
by MS.
Acknowledgements
We thank Amersham Pharmacia Biotech, Cardiff Laboratories, Whitchurch,
Cardiff, CF14 7YT, for preparing the radioactive compound 5b using the
method described in this paper, and Action Pharma A/S, Denmark for
permitting to publish the content of this paper. We also thank Ms. Johanna
Tjarnstrom and Ms. Louise Johansson for their help in carrying out the mass
¨
spectroscopy analyses.
¨
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