Synthesis of N-(2-chloro-3,4-dimethoxybenzylideneamino)guanidinium acetate [α-14C]

Article abstract of DOI:10.1002/jlcr.559

¹⁴C-Labelled N-(2-chloro-3,4-dimethoxybenzylideneamino)guanidinium acetate has been synthesized as a part of a four-step procedure which involved decarboxylation of 2-chloro-3,4-dimethoxybenzoic acid by Pb(OAc)₄ to give 2-chloro-3,4-dimethoxy-1-iodobenzene, followed by a selective lithiation at the iodine position and electrophilic substitution with N,N-dimethylformamide [α-¹⁴C] and final reaction with aminoguanidine bicarbonate. The specific activity was 59 mCi/mmol and the overall yield 49%. Copyright

Full text of DOI:10.1002/jlcr.559

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2002; 45: 371–377.
DOI: 10.1002/jlcr.559
Research Article
Synthesis of N-(2-chloro-3,4-
dimethoxybenzylideneamino)guanidinium
acetate [a-14C]
1,
Maria Almeida *, Arne Boman and Torbjorn Lundstedt
1
1,2
.
1
(
.
Melacure Therapeutics AB, Ullerakersvagen 38, SE-75643 Uppsala, Sweden
2 Department of Pharmaceutical Chemistry, Uppsala University, Box 574,
SE-741 23 Uppsala, Sweden
Summary
14C-Labelled N-(2-chloro-3,4-dimethoxybenzylideneamino)guanidinium acet-
ate has been synthesized as a part of a four-step procedure which involved
decarboxylation of 2-chloro-3,4-dimethoxybenzoic acid by Pb(OAc)4 to give
2-chloro-3,4-dimethoxy-1-iodobenzene, followed by a selective lithiation at the
iodine position and electrophilic substitution with N,N-dimethylformamide
[a-14C] and final reaction with aminoguanidine bicarbonate. The specific
activity was 59 mCi/mmol and the overall yield 49%. Copyright # 2002 John
Wiley & Sons, Ltd.
Key Words: N-(2-chloro-3,4-dimethoxybenzylideneamino)guanidinium acet-
ate; 2-chloro-3,4-dimethoxy-1-iodobenzene; oxidative decarboxylation
Introduction
The compound N-(2-chloro-3,4-dimethoxybenzylideneamino)guanidi-
nium acetate (1) has been shown to have a protective effect on tissue
following ischemia reperfusion injuries in several animal models. The
effect is partly due to an inhibitory effect on xanthine oxidase followed
(
*Correspondence to: M. Almeida, Melacure Therapeutics AB, Ullerakersvagen 38, SE-75643
.
Uppsala, Sweden. E-mail: maria.almeida@melacure.com
Copyright # 2002 John Wiley & Sons, Ltd.
Received 11 October 2001
Revised 15 November 2001
Accepted 21 November 2001

372
M. ALMEIDA ET AL.
by a decrease in the concentration of oxygen-free radicals, which results
in a tissue protective effect. Compound 1 decreased the mortality and
arrhythmia in a dose-dependent manner rat model after occlusion of
one cardiac artery.1 In further pharmacological evaluation such as
administration distribution metabolism elimination (ADME) and
metabolic studies, it became clear that a radioactive form of 1 was
required. The present work describes the methodology developed for the
synthesis of N-(2-chloro-3,4-dimethoxybenzylideneamino)guanidinium
acetate [a-14C].
Results and discussion
The synthesis of non-radioactive N-(2-chloro-3,4-dimethoxybenzylide-
neamino)-guanidinium acetate (1) was performed in good yield (75%)
by the reaction of aminoguanidinium bicarbonate with commercially
available 2-chloro-3,4-dimethoxy-benzaldehyde (2).2 This condensation
reaction would be the last step in the synthesis of the desired labelled
substrate. However, the synthesis of the [carbonyl-14C]-2 was not
straightforward and presented difficulties. The initial idea was to start
from the aldehyde 2, remove the functional group and then restore the
aldehyde function using a 14C-labelled reagent. Several possibilities were
tried for the removal of the carbonyl group (decarbonylation of
aldehydes,3 acyl halides4 and oxidative decarboxylation of carboxylic
acids5) and for the subsequent acylation step (Grignard and organo-
lithium reagents6).
The decarbonylation of the aldehyde 2 to 3-chloro-1,2-dimethox-
ybenzene (5), could be accomplished in a moderate yield (45%) using a
rhodium complex ((PPh3)3RhCl) in refluxing toluene or acetonitrile.3
The successful ortho-lithiation of chlorobenzenes using sec-BuLi in
THF at –1058C has been reported.7c This prompted us to try the same
reaction conditions for compound 5. Unfortunately, the lithiation was
extremely sluggish and only the starting material could be detected by
1H NMR.
Copyright # 2002 John Wiley & Sons, Ltd.
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SYNTHESIS OF N-(2-CHLORO-3,4-DIMETHOXY
373
Scheme 1.
The corresponding acyl halide, 2-chloro-3,4-dimethoxybenzoyl-bro-
mide (6), was easily prepared by radical-promoted oxidation of the
aldehyde with N-bromo-succinimide (NBS) in the presence of catalytic
amounts of 2,20-azobisisobutyronitrile (AIBN) as radical initiator.8
Again, the attempted decarbonylation of compound
6 using
(PPh3)3RhCl/I2 in refluxing toluene4 to afford compound 4 failed.
However, an alternative approach (Scheme 1) involving the oxidative
decarboxylation of the corresponding carboxylic acid 3 was successful.
The aldehyde 2 was readily oxidized using classical methods9 giving the
acid 3 in 76% yield and high purity, while the acid could be used directly
in the next key step. Thus, the decarboxylation was accomplished in an
easy and clean manner using Pb(OAc)4/I5d and catalytic amounts of
AIBN in CCl4 to afford 2-chloro-3,4-dimethoxy-1-iodobenzene (4) in
good yield (72%) after column chromatography. Having the key
intermediate 4, the acylation at the iodine position could, in principle,
be performed either by forming the Grignard or the organolithium
reagent.6 The Grignard reaction was found to be sluggish and a rather
complex mixture was obtained. On the other hand, the lithiation of 4 by
metal–iodine exchange followed by the introduction of the formyl group
using N,N-dimethyl-formamide in THF worked well and the aldehyde 2
was obtained in 65% yield after purification. It is worth mentioning that
the acylation of the lithiated compound was sensitive to the acylating
Copyright # 2002 John Wiley & Sons, Ltd.
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M. ALMEIDA ET AL.
reagent. When N-formyl-morpholine was used, no desired aldehyde was
1
obtained and the H NMR spectrum of the crude mixture showed 3-
chloro-1,2-dimethoxybenzene as the main product.
The last step in the synthetic procedure involved the condensation of the
aldehyde 2 with aminoguanidine to afford 1 in 75% yield. With the acqui-
sition of the labelled aldehyde by using DMF [a-14C] (59.6 mCi/mmol),
radioactive 1 could be easily obtained in 99.5% radiochemical purity.
Conclusion
We have developed a simple method for the labelling of the title
compound. Sisenwine et al.10 have reported the synthesis of N-(2,6-
dichlorobenzylidene-amino)guanidinium acetate [a-14C]. They used a
three-step synthesis in which the labelling of the desired carbon is
accomplished in the first step but only with an overall yield of 12%. In
our approach, the labelling of the aldehyde is performed one step before
the final reaction and, moreover in 49% overall yield.
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. 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. GC-MS analyses were performed on a Thermo-Quest Italia
S.p.A. GC 8000 Top Series gas chromatograph coupled to a Finnigan
Voyager mass spectrometer using a HP-5MS column (60 m ꢀ
0.25 mm ꢀ 0.25 mm) and helium as a carrier gas at an inlet pressure of
160 kPa. The samples were injected in a split mode at a ratio of 50:1 with
an injector temperature at 2208C and column oven temperature at
2808C. The mass spectra were recorded at an electron energy of 70 eV
and the ion source temperature was at 2008C.
Copyright # 2002 John Wiley & Sons, Ltd.
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SYNTHESIS OF N-(2-CHLORO-3,4-DIMETHOXY
375
2-chloro-3,4-dimethoxybenzoic acid (3)
A solution of potassium permanganate (3.40 g, 21.0 mmol) in water
(150 ml) was added dropwise to a stirred slurry of compound 2 (3.00 g,
15.0 mmol) in water (100 ml) at 758C. After addition (15–20 min),
stirring was continued for a further 2 h at 758C. The brownish reaction
mixture was made alkaline (10% NaOH solution) and the mixture was
filtered while hot. The manganese dioxide was washed with warm water
(3 ꢀ 100 ml). The combined filtrate and washings were once again
cooled and filtered to remove any starting material. The filtrate was
acidified with HCl until no further precipitate formed. The resulting
white precipitate was filtered, washed with cold water and dried to
afford 2.48 g (76%) of the product as a white solid identical with an
authentic sample (commercially available). The product was used
without further purification.
2-chloro-3,4-dimethoxy-1-iodobenzene (4)
The acid 3 (2.97 g, 13.7 mmol), lead tetraacetate (6.70 g, 15.0 mmol),
iodine (3.50 g, 15.0 mmol) and AIBN (66 mg, 0.40 mmol) were weighed
into a two-necked flask connected to a condenser under N2. Dry,
deoxygenated CCl4 (120 ml) was added via cannula and the resulting
reaction mixture refluxed with stirring for about 2 h. The reaction
mixture was cooled, diluted with CH2Cl2 (100 ml) and filtered. The
organic phase was then washed with 1 M Na2SO3 (4 ꢀ 50 ml), saturated
NaCl solution (4 ꢀ 50 ml), dried (anhydrous MgSO4), and evaporated to
afford 3.49 g of the crude product as a yellowish solid. Purification by
column chromatography (silica gel, n-hexane/diethyl ether 2:1) gave
2.96 g (72%) of the pure product as white crystals: mp 69.8–70.28C;
1H NMR d 7.56 (d, 1 H, J=8.8 Hz), 6.64 (d, 1 H, J=8.8 Hz), 3.88 (s,
3 H), 3.97 (s, 3 H); 13C NMR d 154.5, 146.5, 134.7, 133.5, 113.0, 87.7,
61.1, 56.6; MS m/z (relative intensity) 298 (M+, 100).
2-chloro-3,4-dimethoxybenzaldehyde (2)
n-Butyllithium (500 ml of a 2.5 M solution in hexane, 1.25 mmol) was
added dropwise via syringe under nitrogen to a solution of 2-chloro-3,4-
dimethoxy-1-iodobenzene (4) (300 mg, 1.00 mmol) in dry THF (25 ml)
cooled to –788C. The mixture was stirred for 2 h at –788C. Dry DMF
(116 ml, 1.5 mmol) was added dropwise via syringe. The mixture was
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M. ALMEIDA ET AL.
then brought to 08C and stirred for 1 h. The reaction was quenched with
dilute HCl (1 M) until the solution became acidic. The mixture was
extracted with diethyl ether (100 ml) and the organic layer washed in
turn with water (3 ꢀ 25 ml), 1 M NaHCO3 (3 ꢀ 25 ml), saturated NaCl
solution (3 ꢀ 25 ml), dried (anhydrous MgSO4), and finally the solvent
was evaporated. The crude product was then purified by silica gel
(hexane/diethyl ether 3:1) to afford 130 mg (65%) of the product as a
white solid identical with an authentic sample (commercially available).
N-(2-chloro-3,4-dimethoxybenzylideneamino)guanidinium acetate (1)
Amino-guanidinium bicarbonate (150 mg, 1.10 mmol) was suspended in
methanol (25 ml). The mixture was heated to 558C and acetic acid
(25 ml) was added. To the resulting clear solution, 2-chloro-3,4-
dimethoxybenzaldehyde (201 mg,1 mmol) was added in one portion.
The reaction mixture was refluxed until no aldehyde remained (51%
aldehyde (TLC), approximately 1 h). The methanol was removed and
the residue was crystallized from ethanol. Purification of the crude
product by HPLC (Lichrocart Select B (250 ꢀ 4.0 mm), 1.3 mM
ammonium acetate+0.08% formic acid: acetonitrile (1:1), isocratic,
flowrate: 0.7 ml/min) afforded the product in 75% yield as a white
powder: mp 2008C; 1H NMR d 8.32 (s, 1 H), 7.95 (d, 1 H, J=9 Hz), 7.07
(d, 1 H, J=9 Hz), 3.87 (s, 3 H), 3.75 (s, 3 H), 1.80 (s, 3 H); 13C NMR d
176.1, 158.0, 154.6, 144.8, 140.9, 127.4, 125.5, 122.8, 112.1, 60.5, 56.5,
24.0. Radioactive 1 was obtained in 99.4% radiochemical purity as
indicated by HPLC (conditions as above) with a specific activity of
59 mCi/mmol as determined by MS.
Acknowledgements
We thank Amersham Pharmacia Biotech, Cardiff Laboratories,
Whitchurch, Cardiff, CF14 7YT, for labelling of compound 1 using
the method described in this paper.
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SYNTHESIS OF N-(2-CHLORO-3,4-DIMETHOXY
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