Synthesis of carbon-14 and stable isotope labelled NN414: A potent potassium channel opener

Article abstract of DOI:10.1002/jlcr.805

Currently, NN414, a potent β-cell selective potassium channel opener, is undergoing clinical trials for the treatment of type 2 diabetes. Here, we report the synthesis of carbon-14 and stable isotope labelled NN414 for use in metabolic studies and as an internal standard in pharmacokinetic assays, respectively. The carbon-14 labelling was performed in two steps starting from an advanced intermediate. This provided [¹⁴C]NN414 in 60% overall radiochemical yield with a specific activity of 58mCi/ mmol. The stable isotope labelling was accomplished from benzyl tert-butyl malonate in eight steps using [¹³C,²H₃]iodomethane and [²H ₂]dibromomethane as the source of carbon-13/deuterium. The synthetic sequence, which included a Mannich reaction followed by deamination, a Simmons-Smith-type cyclopropanation and a modified Curtius reaction, provided [¹³C,²H₅]NN414 in 8.6% overall yield. Copyright

Full text of DOI:10.1002/jlcr.805

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2004; 47: 127–138.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.805
Research Article
Synthesis of carbon-14 and stable isotope labelled
NN414: a potent potassium channel opener
.
Steen K. Johansen*, Jesper B. Kristensen, Lars K. Muller, Claus U. Jessen
and Christian Foged
(
Novo Nordisk A/S, Novo Nordisk Park, Malꢀv DK-2760, Denmark
Summary
Currently, NN414, a potent b-cell selective potassium channel opener, is undergoing
clinical trials for the treatment of type 2 diabetes. Here, we report the synthesis of
carbon-14 and stable isotope labelled NN414 for use in metabolic studies and as an
internal standard in pharmacokinetic assays, respectively. The carbon-14 labelling was
performed in two steps starting from an advanced intermediate. This provided
[14C]NN414 in 60% overall radiochemical yield with a specific activity of 58 mCi/
mmol. The stable isotope labelling was accomplished from benzyl tert-butyl malonate
in eight steps using [13C,2H3]iodomethane and [2H2]dibromomethane as the source of
carbon-13/deuterium. The synthetic sequence, which included a Mannich reaction
followed by deamination, a Simmons–Smith-type cyclopropanation and a modified
Curtius reaction, provided [13C,2H5]NN414 in 8.6% overall yield. Copyright # 2004
John Wiley & Sons, Ltd.
Key Words: carbon-13; carbon-14; deuterium; Mannich reaction; Simmons–Smith
cyclopropanation; Curtius reaction
Introduction
Type 2 diabetes is the most prevalent type of diabetes mellitus, a complex
metabolic disease affecting a significant proportion of people worldwide.1
Among other things, type 2 diabetes is characterized by insulin resistance in
the peripheral tissue. Owing to the lowered response to insulin, the pancreatic
b-cells responsible for production of insulin are stressed in order to produce
more of the hormone. This can ultimately lead to the death of the b-cells and
the onset of type 1 diabetes.
It has been shown that potassium channel openers2 such as diazoxide
(Figure 1) induce a resting state in the b-cells, thereby prolonging the life of
(
*Correspondence to: S. K. Johansen, Novo Nordisk A/S, Novo Nordisk Park, DK-2760 Malꢀv, Denmark.
E-mail: skjo@novonordisk.com
Copyright # 2004 John Wiley & Sons, Ltd.
Received 2 October 2003
Revised 11 November 2003
Accepted 13 November 2003

128
S. K. JOHANSEN ET AL.
H
N
H
N
S
N
Cl
NH
N
S
Cl
S
O
O
O
O
Diazoxide
NN414
Figure 1. Structure of diazoxide and NN414
these cells and postponing the time by which treatment with insulin is
necessary.3
NN4144 (Figure 1) is a potent b-cell selective potassium channel opener,
which is currently undergoing clinical trials for the treatment of type 2
diabetes. In order to study the metabolic properties of NN414, a carbon-14
radiolabelled form of the compound was required for use in absorption,
distribution, metabolism and excretion (ADME) studies. A stable isotope
labelled form of the compound was also desirable for use as an internal
analytical standard in pharmacokinetic assays. Here we report the labelling of
NN414 with carbon-14 and various stable isotopes.5
Results and discussion
Carbon-14 labelling
From a metabolic point of view, it is advantageous to have the carbon-14 label
located in the heterocyclic part of NN414, as the methylcyclopropyl moiety is
susceptible to elimination in vivo. Therefore, we started the synthesis of
carbon-14 labelled NN414 from the thiadiazine derivate 1, which can be
prepared by coupling of 3-amino-5-chlorothiophene-2-sulfonamide and ethyl
[14C]chloroformate with triethylamine in DMSO. This was performed by the
authors for the first preparation of [14C]NN414 with low specific activity and
subsequently performed at Amersham Biosciences, Cardiff, UK.
Direct amination of thiadiazine derivate 1 with alkylamines is possible but
was not viewed as a suitable approach, as the yields are low.4 Instead, we
decided to introduce a more suitable leaving group at C-3 in order to facilitate
coupling with the amine. This was accomplished by chlorination of 1 with
triethylamine hydrochloride in activated phosphoryl chloride,6 which provided
the chloride 2 in quantitative yield with a radiochemical purity of 90%
(Scheme 1).
Generally, the nucleophilic aromatic substitution of the chloride 2 gives the
best yields with simple unbranched amines, whereas bulky amines give low
yields due to incomplete conversion.4 This was initially also true in our hands,
as the reaction with 1-methylcyclopropyl-1-amine (3) consistently stopped at
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SYNTHESIS OF CARBON-14 AND STABLE
129
H
N
NH2
O
14C
NH
O
14C
Et3N
Cl
Cl
+
NH2
S
Cl
O
S
S
S
DMSO
O
O
O
O
1
HCl
H2N
H
H
N
H
N
3
Et3N·HCl
H3PO4
N
Cl
14C
N
14C
Et3N
THF
Cl
Cl
N
POCl3
S
S
S
S
O
O
O
O
2
4
Scheme 1. Synthesis of [14C]NN414 (4)
30–40% conversion of starting material when running the reaction with
triethylamine in ethanol even when using extended reaction times and high
temperatures.
The reaction can be improved by switching to a better leaving group such as
the fluoride. Thus, the reported yield of NN414 is increased from 26 to 54%
using the fluoride instead of the chloride.4 However, this would introduce an
additional step to the synthesis and was not viewed as optimal.
Instead, we screened a number of solvents and bases hoping to optimize the
reaction conditions. This proved successful, and we found that optimal
conditions could be achieved by switching the solvent from ethanol to THF
and keeping triethylamine as the base. These conditions gave complete
conversion of starting material although extended reaction times and high
temperatures were still necessary, and [14C]NN414 (4) was obtained in 60%
overall radiochemical yield after purification by HPLC. The chemical and
radiochemical purities were found to be >98% and the specific activity was
found to be 58 mCi/mmol by mass spectroscopy.
Stable isotope labelling
The use of an internal standard is important in the determination and
quantification of analytical samples in pharmacokinetic studies, as it provides
a simple and accurate analytical method. Consequently, the choice of the
internal standard is equally important as properties such as solubility,
extraction and chromatographic behaviour and stability can affect the assay.
Generally, it is acknowledged that a stable isotope labelled version of the drug
candidate provides the ideal standard for such an assay. This is due to the
standard having identical properties in all these aspects, while at the same
time, the difference in molecular weight makes it distinguishable in LC/MS
assays. Normally, a three mass unit difference between the parent compound
and the isotope labelled standard is sufficient to obtain good separation
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130
S. K. JOHANSEN ET AL.
between drug candidate and standard in the assay. However, in the case of
NN414, a five mass unit difference is required due to the presence of a chlorine
atom in the NN414 and the fact that naturally occurring chlorine is a mixture
of chlorine-35 and chlorine-37.
Looking at the structure of NN414, it quickly became clear that
introduction of five extra mass units in the heterocyclic part of the molecule
would not be feasible. Therefore, we were left with the task of introducing
these into the methylcyclopropylamine moiety. While alkylation of cyclopro-
panecarboxylic acid with carbon-13/deuterium labelled iodomethane could
give access to 1-methyl-1-cyclopropylcarboxylic acid labelled with four extra
mass units we dismissed this approach as no obvious way exists of introducing
the last extra mass unit.
Instead, we decided to go back one step and synthesize the cyclopropyl
moiety according to the retrosynthetic sequence shown in Figure 2. Starting
from a suitable protected malonic acid A, the first four extra masses would be
introduced by alkylation with [13C,2H3]iodomethane. After removal of one of
the protecting groups, a Mannich reaction with diethylamine and formalde-
hyde followed by elimination in situ would give access to the acrylic ester B. A
standard copper/zinc catalysed Simmons–Smith reaction would then produce
the cyclopropyl moiety and simultaneously introduce an additional two mass
units (from [2H2]dibromomethane) to give C. Finally, removal of the second
protecting group followed by a modified Curtius reaction would lead to the
desired product, 1-methyl-1-cyclopropylamine D.
A number of reports exist in the literature on the preparation of deuterium
labelled methacrylate esters starting from malonic esters.7 However, we felt
that there was still room for improvement concerning three important aspects.
First of all, the reported yields for the three steps leading to the methacrylate
ester could be improved (40–42%). Also, due to the lack of functional groups,
these compounds are very difficult to visualize either by using UV-detection or
by various TLC reagents, and therefore the introduction of a UV-absorbing
D
D
D
D
H2N
POOC
13CD3
13CD3
D
C
O
O
O
PO
OP
P = Protecting group
PO
13CD3
A
B
Figure 2. Retrosynthesis of stable isotope labelled NN414
Copyright # 2004 John Wiley & Sons, Ltd.
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SYNTHESIS OF CARBON-14 AND STABLE
131
functionality could be beneficial. Finally, by introducing an orthogonal
protection scheme, sequential elimination of the protecting groups would be
possible, thus avoiding the need to carefully monitor the partial hydrolysis of a
symmetric diester.
With these aspects in mind, benzyl tert-butyl malonate (5) was selected as
the starting material for the synthesis of stable isotope labelled 1-methyl-1-
cyclopropylamine (Scheme 2). Alkylation with [13C,2H3]iodomethane and
NaH was carried out with a slight excess of 5 (1.1 eq.) in order to minimize
formation of the dialkylated product. This provided the pure monomethylated
malonate 6 in excellent yield (82% based on methyl iodide) after flash
chromatography. The use of flash chromatography was instrumental in
removing unreacted 5 as well as the small quantities of dimethyl malonate
formed, something which has posed a problem for some of the reported
syntheses.7b,c Removal of the tert-butyl protecting group was accomplished by
treatment with trifluoroacetic acid in dichloromethane to give 7 in quantitative
yield. Then compound 7 was reacted in a Mannich-type reaction with
formaldehyde and diethylamine to give the methacrylate 8, after an in situ
deamination, in excellent purity and yield (82%) after flash chromatography.
The overall yield for the three steps was 67%, which is a distinct improvement
over the reported syntheses.
O
O
O
O
O
13CD3I, NaH
TFA
O
BnO
BnO
BnO
O
F
TH
CH2Cl2
5
13CD3
6
O
O
Zn, CuCl
CD2Br2, TiCl4
CH2O, Et2NH
THF, H2O
OH
BnO
Et2O
13CD3
13CD3
7
8
O
D
D
D
D
Pd/C, H2
DPPA, Et3N
HCl
HOOC
BnO
EtOH
t-BuOH
EtOAc
13CD3
13CD3
H
N
9
10
Cl
H
N
H
N
Cl
D
D
12
N
S
S
H2N
HCl
13CD3
Cl
Et3N
O
O
D
N
S
S
D
13CD3
THF
O
O
11
13
Scheme 2. Synthesis of stable isotope labelled NN414 (13)
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S. K. JOHANSEN ET AL.
Friedrich and co-workers have reported on a convenient version of the
Simmons–Smith cyclopropanation reaction,8 which avoids the use of specially
prepared zinc–copper couples by using titanium(IV) chloride catalysis
(the use of acetyl chloride as catalyst in the reaction has also been reported.9
Reaction of methacrylate 8 with [2H2]dibromomethane using activated
zinc dust, copper(I) chloride and titanium(IV) chloride in refluxing anhy-
drous diethyl ether provided the carboxylic ester 9 in moderate yield (49%)
and excellent purity after flash chromatography. We found the use of pro-
perly activated zinc dust important for this reaction, as the use of par-
tially or un-activated zinc dust gave incomplete conversion of the starting
material.10
The benzyl protecting group was removed by hydrogenolysis to give the acid
10 in excellent yield (97%). Then, this was subjected to a modified Curtius
reaction using diphenyl phosphorazidate (DPPA).11 Reaction of 10 with Et3N
and DPPA in anhydrous tert-butanol provided the BOC-protected amine,
which then was deprotected by treatment with acidic ethylacetate to provide 1-
[13C,2H3]methyl-[2,2-2H2]cyclopropyl-1-amine hydrochloride (11) in moderate
yield (48%) after recrystallization. The use of anhydrous conditions in the
Curtius reaction proved paramount to the success of the reaction. Initially, the
reaction was run under normal conditions using standard analytical grade tert-
butanol. However, this gave a mixture of 11 and a closely related unknown
compound as seen by 1H NMR. LC–MS revealed the unknown compound to
have a MW of 181.3 (M+1), which (combined with the 1H NMR data, which
suggested a symmetrical structure) lead us to believe that the compound is the
sym-urea derivative of 11 (MW 180.2). The formation of this derivative is
almost certainly caused by the presence of water in the solvent, something
which is widely supported by the literature.12 This was confirmed by the use of
anhydrous conditions and freshly distilled tert-butanol, which avoided the
formation of this derivative altogether.
It should be noted that the use of [2H2]formaldehyde in the Mannich-type
reaction would replace the last two hydrogen atoms present and thereby give
access to 1-methylcyclopropyl-1-amine labelled with deuterium at all
positions. Furthermore, the use of carbon-13 labelled formaldehyde and/or
malonic acid (which is available labelled in various positions) would give
access to 1-methylcyclopropyl-1-amine labelled with carbon-13 in any
combination of positions.
The final step in the synthesis of stable isotope labelled NN414 was the
coupling of the amine 11 with the heterocyclic moiety 12. This was performed
using the optimized conditions developed for the carbon-14 labelled synthesis
but employing 0.88 equivalents of 11 instead of the 3.2 equivalents used in the
former synthesis. This provided [13C,2H5]NN414 in 56% yield and >99%
purity after purification by flash chromatography and recrystallization giving
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SYNTHESIS OF CARBON-14 AND STABLE
133
an overall yield of 8.6% for the eight-step synthesis. The isotopic purity was
confirmed to be >98% by mass spectroscopy.
Conclusion
In summary, we have developed synthetic routes for the labelling of the potent
b-cell selective potassium channel opener NN414 with carbon-14 as well as
carbon-13/deuterium. NN414 was labelled with carbon-14 in the thiadiazine
moiety in 60% overall starting from the thiadiazine derivative 2. Following
HPLC purification, [14C]NN414 was obtained in high radiochemical purity
(>98%) and with high specific radioactivity (58 mCi/mmol).
Stable isotope labelled NN414 was synthesized from benzyl tert-butyl
malonate (5) using [13C,2H3]iodomethane and [2H2]dibromomethane as the
source of carbon-13/deuterium. This provided [13C,2H5]NN414 in an overall
yield of 8.6% for the eight-step synthesis, which included a Mannich reaction,
a Simmons–Smith-type cyclopropanation and a modified Curtius reaction.
The methodology presented here can be applied to the synthesis of 1-
methylcyclopropyl-1-amine labelled with deuterium in many different combi-
nations including complete labelling. Likewise, the methodology can be
applied to the synthesis of 1-methylcyclopropyl-1-amine labelled with carbon-
13 in any combination of positions.
Experimental
General
[3-14C]-6-Chloro-2,3-dihydro-3-oxo-4H-thieno[3,2-e]-1,2,4-thiadiazine 1,1-di-
oxide (specific activity: 554 mCi/mmol) was supplied by Amersham
Biosciences, UK. [13C,2H3]iodomethane and [2H2]dibromomethane were
supplied by Aldrich. Benzyl tert-butyl malonate and diphenyl-phosphonic
azide (DPPA) were supplied by Lancaster. 1-Methylcyclopropyl-1-amine
hydrochloride and 3,6-dichloro-4H-thieno[3,2-e]-1,2,4-thiadiazine 1,1-dioxide
were supplied Chemical Development, Novo Nordisk A/S. THF was distilled
from sodium/benzophenone and t-BuOH from calcium hydride under
.
nitrogen. Dry diethyl ether was obtained from Riedel–deHaen and stored
under nitrogen. All other reagents and solvents were of analytical grade and
used without further purification. Reactions performed under a nitrogen
atmosphere were performed in flame dried glassware.
HPLC was performed using a Merck Hitachi Intelligent Pump L6200A
equipped with a Merck Hitachi Column Thermostat T5025 (set at 408C) with a
Rheodyne injector, and a Merck Hitachi UV Detector L4000A (detection at
220 nm). Detection of carbon-14 was performed on a Canbarra Packard flow
detector A500.
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S. K. JOHANSEN ET AL.
Analytical HPLC was performed on an RP C18 column (4.6 ꢀ 250 mm,
(
5 mm, OdDMeSi 120 A, Novo Nordisk) with a flow of 1.0 ml/min. using one of
the following systems. System 1: 85% ! 60% A 0–20 min (A: 10%
acetonitrile in 0.1% aq. TFA; B: 90% acetonitrile in 0.1% aq. TFA). System
2: 100% A 0–40 min followed by 0% A for 10 min (A: 25% acetonitrile in
0.25% aq. TEA, pH 7; B: 90% acetonitrile in 0.1% aq. TFA). Purifications were
performed out using system 2 employing a 10 ꢀ 250 mm column (5.0 ml/min.).
Radioactivity measurements were performed on a Packard Tri-Carb 1000
liquid scintillation analyser using Ultima FloTM M (Packard Bioscience) as
liquid scintillation cocktail. Specific activity was determined on a Sciex API
300 mass spectrometer equipped with an ionspray interface. 1H and 13C NMR
spectra were recorded on a Bruker DRX 300 spectrometer. Flash chromato-
(
graphy was performed with silica gel 60 A (Merck, 40–63 mm). TLC was
performed on Alugram SIL G/UV254 pre-coated silica plates and spots were
detected by UV-detection and/or by dipping in a solution of potassium
permanganate (1.5 g KMnO4, 10 g K2CO3, 2.5 ml 5% aq. NaOH and 150 ml
water). Concentrations were performed on a rotary evaporator at a
temperature below 408C. Melting points are uncorrected. Elemental analyses
were performed by the Department of Structural Chemistry, Novo Nordisk.
[3-14C]-3,6-Dichloro-4H-thieno[3,2-e]-1,2,4-thiadiazine 1,1-dioxide (2)
Et3N ꢁ HCl (382 mg, 2.77 mmol) and H3PO4 (0.094 ml, 85% aq., 1.39 mmol)
were added to a solution of [3-14C]-6-chloro-2,3-dihydro-3-oxo-4H-thieno[3,2-
e]-1,2,4-thiadiazine 1,1-dioxide (1) (49.5 mCi, 0.85 mmol, 554 mCi/mmol) in
POCl3 (5 ml, 54.6 mmol) in a glass flask. A glass stopper was securely fastened,
and the mixture was stirred at 1008C for 4 days, at which time HPLC analysis
(System 1) showed 89% radiochemical conversion. The resulting black
solution was carefully quenched by adding it to water (10 ml) (Caution–
exothermic reaction!) followed by addition of acetonitrile (5 ml) and additional
water (40 ml). This solution was applied to a pre-activated Sep-Pak1 cartridge
(Waters, RP-C18, 10 g) followed by washing with water (20 ml). Then the
product was eluted with aqueous acetonitrile (90%, 40 ml) to give 2 (49.5 mCi,
100%) with a radiochemical purity of 90% (System 1).
[3-14C]-6-Chloro-3-(1-methylcyclopropyl)amino-4H-thieno[3,2-e]-1,2,4-thia-
diazine 1,1-dioxide (NN414) (4)
1-Methylcyclopropyl-1-amine hydrochloride (3) (242 mg, 2.25 mmol) and
Et3N (0.53 ml, 3.80 mmol) were added to a solution of [3-14C]-3,6-dichloro-
4H-thieno[3,2-e]-1,2,4-thiadiazine 1,1-dioxide (2) (40.6 mCi, 0.70 mmol) in dry
THF (4 ml) in a pressure vial. The screw cap was securely fastened, and the
mixture was stirred at 808C for 6 days, at which time HPLC analysis (System
2) showed 82% radiochemical conversion. The reaction mixture was purified
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SYNTHESIS OF CARBON-14 AND STABLE
135
by HPLC (System 2) and the fractions containing 4 were concentrated
followed by addition of 10% aq. EtOH (50 ml). This mixture was applied to a
pre-activated Sep-Pak1 cartridge (Waters, RP-C18, 10 g) followed by washing
with water (20 ml). Then the product was eluted with aqueous EtOH (90%,
40 ml), concentrated and re-dissolved in EtOH (100 ml). This provided 3 (24.2
mCi, 60%) with chemical and radiochemical purities >98% (System 2). The
specific activity was calculated to be 58 mCi/mmol (MS).
Benzyl tert-butyl [13C,2H3]methylmalonate (6)
A solution of benzyl tert-butyl malonate (5) (7.55 g, 30.2 mmol) in THF
(7.5 ml) was slowly added over 5 min to a suspension of pentane-washed NaH
(964 mg, 40.2 mmol) in THF (20 ml) under a nitrogen atmosphere. The
mixture was stirred for 0.5 h resulting in a clear solution; to this was added a
solution of [13C,2H3]iodomethane (3.91 g, 26.8 mmol) in THF (3 ml). The
solution was stirred for a further 0.75 h and then quenched with aqueous acetic
acid (10%, 10 ml) followed by evaporation of THF. The residual aqueous
phase was extracted with dichloromethane (3 ꢀ 40 ml) and the combined
organic phases were washed with brine (10 ml), dried (MgSO4), and
concentrated. Purification by flash chromatography (EtOAc/heptane 1:19)
gave the title compound 6 as a colourless oil (5.86 g, 82% based on
[13C,2H3]iodomethane). RF 0.42 (EtOAc/heptane 2:8).
1H NMR (300 MHz, CDCl3): d 1.37 (9 H, s), 3.35 (1 H, d, J 4.5), 5.09–5.19
(2 H, m,), 7.24–7.35 (5 H, m). 13C NMR (75 MHz, CDCl3): d 12.4 (septet,
13CD3), 27.4, 46.6, 66.4, 81.2, 128.0, 128.2, 135.4, 169.7, 169.9.
C1413CH17D3O4 (268.34): calculated C + 13C as C 67.14, H + D as H 7.51;
found C + 13C as C 66.90, H + D as H 7.85.
Benzyl [13C,2H3]methylmalonate (7)
TFA (4 ml) was added to a solution of benzyl tert-butyl [13C,2H3]methylma-
lonate (6) (2.71 g, 10.1 mmol) in dichloromethane (20 ml), and the mixture was
stirred for 6 h. Concentration gave the title compound 7 as a clear oil (2.28 g,
100%). RF 0.14 (EtOAc/heptane 2:8).
1H NMR (300 MHz, CDCl3): d 3.55 (1 H, d, J 5.0), 5.18 (2 H, s), 7.25–7.35
(5 H, m), 11.30 (1 H, s). 13C NMR (75 MHz, CDCl3): d 12.5 (septet, 13CD3),
45.7, 67.7, 128.0, 128.5, 134.8, 170.5, 175.9.
Benzyl [13C,2H3]methacrylate (8)
Formaldehyde (0.97 ml, 37% aq., 12.9 mmol) and Et2NH (1.35 ml, 13.0 mmol)
were added to a solution of benzyl [13C,2H3]methylmalonate (7) (2.71 g,
12.8 mmol) in THF/water (10 ml/10 ml), and the reaction mixture was stirred
at 508C for 16 h. The mixture was concentrated and the residue dissolved in
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S. K. JOHANSEN ET AL.
water (10 ml) and extracted with dichloromethane (3 ꢀ 40 ml). The combined
organic phases were washed with brine (10 ml), dried (MgSO4), and
concentrated. Purification by flash chromatography (EtOAc/heptane 1:19)
gave the title compound 8 as a colourless oil (1.89 g, 82%). RF 0.52 (EtOAc/
heptane 2:8).
1H NMR (300 MHz, CDCl3): d 5.17 (2 H, s), 5.54 (1 H, d, J 1.5, J 6.0), 6.14
(1 H, dd, J 2.0, J 10.0), 7.24–7.36 (5 H, m). 13C NMR (75 MHz, CDCl3): d 17.3
(septet, 13CD3), 66.1, 125.8, 127.8, 127.9, 128.3, 136.0, 166.9.
C1013CH9D3O2 (180.23): calculated C + 13C as C 73.31, H + D as H 6.71;
found C + 13C as C 73.11, H + D as H 6.99.
Benzyl 1-[13C,2H3]methyl-[2,2-2H2]cyclopropane-1-carboxylate (9)
A solution of benzyl [13C,2H3]methacrylate (8) (1.68 g, 9.32 mmol) in Et2O
(2 ml) as well as [2H2]dibromomethane (2.00 ml, 5.00 g, 28.4 mmol) were added
to a suspension of activated zinc dusty (2.20 g, 33.7 mmol) and CuCl (0.336 g,
3.39 mmol) in Et2O (20 ml) under a nitrogen atmosphere. After stirring at
room temperature for 0.5 h, TiCl4 (0.08 ml, 0.73 mmol) was added, and the
mixture was refluxed for 21 h, at which time further [2H2]dibromomethane
(0.42 ml, 1.05 g, 5.97 mmol) was added and the mixture refluxed for an
additional 2.5 h. After cooling to room temperature, the mixture was carefully
quenched with aqueous HCl (2 N, 5 ml) followed by filtration and addition of
water (5 ml). The phases were separated, and the aqueous phase was extracted
with Et2O (2 ꢀ 20 ml). The combined organic phases were washed with brine
(10 ml), dried (MgSO4) and concentrated. Purification by flash chromato-
graphy (EtOAc/heptane 1:19) gave the title compound 9 as a colourless oil
(0.886 g, 49%). RF 0.52 (EtOAc/heptane 2:8).
1H NMR (300 MHz, CDCl3): d 0.63 (1 H, t), 1.22 (1 H, s), 5.07 (2 H, s),
7.23–7.35 (5 H, m). 13C NMR (75 MHz, CDCl3): d 16.3, 18.3 (septet, 13CD3),
65.9, 127.6, 127.8, 128.3, 136.2, 175.4.
C1113CH9D5O2 (196.28): calculated C + 13C as C 73.43, H + D as H 7.19;
found C + 13C as C 73.36, H + D as H 7.37.
1-[13C,2H3]Methyl-[2,2-2H2]cyclopropane-1-carboxylic acid (10)
Pd/C (40 mg) was added to a solution of benzyl 1-[13C,2H3]methyl-
[2,2-2H2]cyclopropane-1-carboxylate (9) (0.853 g, 4.35 mmol) in EtOH
(15 ml). The mixture was stirred under an H2 atmosphere for 16 h and then
filtered and concentrated to give the title compound 10 as a colourless oil
(0.448 g, 97%).
y
Zinc dust was activated by sequential washing with 1N NaOH (10 min), water, 1N NC1 (10 min), water,
ethanol, acetone and diethyl ether followed by drying in in vacuo for 2 h.
Copyright # 2004 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2004; 47: 127–138

SYNTHESIS OF CARBON-14 AND STABLE
137
1H NMR (300 MHz, CDCl3): d 0.72 (1H, t, J 4.0), 1.27 (1H, s), 11.89 (1H, s).
13C NMR (75 MHz, CDCl3): d 17.2, 17.9 (septet, 13CD3), 183.3.
1-[13C,2H3]Methyl-[2,2-2H2]cyclopropyl-1-amine hydrochloride (11)
Et3N (0.65 ml, 4.66 mmol) and diphenylphosphonic azide (0.96 ml, 4.45 mmol)
were added to a solution of 1-[13C,2H3]methyl-[2,2-2H2]cyclopropane-1-
carboxylic acid (10) (0.448 g, 4.22 mmol) in dry t-BuOH (10 ml) under a
nitrogen atmosphere, and the reaction mixture was stirred at 508C for 16 h.
The mixture was concentrated to an oil, which was extracted with Et2O (4 ꢀ
50 ml). The combined organic phases were washed with aqueous NaOH (1N, 2
ꢀ 20 ml), aq. H2SO4 (0.1N, 2 ꢀ 20 ml) and brine (20 ml), dried (MgSO4), and
concentrated to give the BOC protected amine as a yellow oil (0.591 g, 79%).
1H NMR (300 MHz, MeOH-d4): d 0.50 (1 H, t, J 4.0), 0.65 (1 H, d, J 4.5),
1.42 (9 H, s). 13C NMR (75 MHz, CDCl3): d 14.5, 22.4 (septet, 13CD3), 23.1,
28.8, 79.6, 158.2.
A solution of the BOC protected amine (0.591 g) in EtOAc (50 ml) was
cooled to 08C and HCl (g) was passed through the solution for 0.5 h followed
by stirring at room temperature for 16 h. Concentration gave a crystalline
product, which was purified by recrystallization (EtOAc/MeOH) to give the
title compound 11 as colourless crystals (0.230 g, 48% for the two steps); Mp
206–2098C (an unlabelled sample had mp 196–1978C).
1H NMR (300 MHz, MeOH-d4): d 0.73 (1 H, dd, J 4.0), 0.92 (1 H, dd, J 5.5).
13C NMR (75 MHz, MeOH-d4): d 11.2, 20.2 (septet, 13CD3), 30.3.
C3 13C CH5 D5 NCl (113.62): calculated C + 13C as C 42.28, H + D as H
8.87, N 12.33, Cl 31.20; found C + 13C as C 42.37, H + D as H 9.20, N 12.28,
Cl 30.93.
6-Chloro-3-(1-[13C,2H3]methyl-[2,2-2H2]cyclopropyl)amino-4H-thieno[3,2-
e]-1,2,4-thiadiazine 1,1-dioxide (NN414) (13)
A suspension of 1-[13C,2H3]methyl-[2,2-2H2]cyclopropyl-1-amine hydrochlor-
ide (11) (0.100 g, 0.88 mmol) and 3,6-dichloro-4H-thieno[3,2-e]-1,2,4thiadia-
zine 1,1-dioxide (12) (0.257 g, 1.00 mmol) in dry THF (20 ml) was heated to
658C. Then, Et3N (0.20 ml, 1.44 mmol) was added and the mixture stirred at
658C for 5 days. Concentration gave a dark oil, which was purified by flash
chromatography (dichloromethane/MeOH, 19:1) to give a crude product
(0.173 g, 66%). Final purification was performed by recrystallization (EtOH)
to give the title compound 13 as off-white crystals (0.147 g, 56%), Mp 263–
2648C (an unlabelled sample had mp 251–2528C). Purity >99% (HPLC
System 2). RF 0.17 (dichloromethane/MeOH 19:1).
Copyright # 2004 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2004; 47: 127–138

138
S. K. JOHANSEN ET AL.
1H NMR (300 MHz, MeOH-d4): d 0.72 (1 H, t, J 4.5), 0.83 (1 H, d, J 5.0),
6.92 (1 H, s). 13C NMR (75 MHz, MeOH-d4): d 15.1, 21.8 (septet, 13CD3),
29.7, 113.3, 118.7, 137.1, 140.1, 152.0.
MS (ionspray): 298.2 (M+H) shows less than 1.1% of the unlabelled
compound present (292.2).
Acknowledgements
We are grateful to Mr Ole Wassmann for mass spectra analyses, Mr Flemming
Gundertofte for elemental analyses and Dr Flemming E. Nielsen for helpful
discussions.
References
.
1. Johansen SK, Kristensen JB, Muller LK, Jessen CU, Foged C. Presented at the
8th International Symposium on the Synthesis and Applications of Isotopes and
Isotopically Labelled Compounds, Boston, MA, USA, 1–5 June 2003.
2. WHO fact sheet No. 236 September 2002 (http://www.who.int/mediacentre/
factsheets/fs236/en).
3. Coghlan MJ, Carroll WA, Gopalakrishnan. J Med Chem 2001; 44: 1627–1653.
4. Tagmose TM, Mogensen JP, Agerholm PC, Arkhammar POG, Wahl P, Worsaae
A, Hansen JB. Bioorg Med Chem Lett 2001; 11: 1749–1752.
5. Nielsen FE, Bodvarsdottir TB, Worsaae A, MacKay P, Stidsen CE, Boonen
HCM, Pridal L, Arkhammar POG, Wahl P, Ynddal L, Junager F, Dragsted N,
Tagmose TM, Mogensen JP, Koch A, Treppendahl SP, Hansen JB. J Med Chem
2002; 45: 4171–4187.
6. Andersen K, Begtrup M. Acta Chem Scand 1992; 46: 1130–1132.
7. (a) Hosokawa T, Yamanaka T, Itotani M, Murahashi S. J Org Chem 1995; 60:
6159–6167. (b) Keah HH, Rae ID, Hawthorne DG. Aust J Chem 1992; 45:
!
# !
659–669. (c) Nikoletic M, Borcic S, Sunko DE. Tetrahedron 1967; 23: 649–660.
8. Friedrich EC, Lunetta SE, Lewis EJ. J Org Chem 1989; 55: 2388–2390.
9. Friedrich EC, Lewis EJ. J Org Chem 1990; 56: 2491–2494.
10. For a review on the preparation of activated zinc metal, see Erdik E. Tetrahedron
1987; 43: 2203–2212.
11. Ninomiya K, Shioiri T, Yamada S. Tetrahedron 1974; 30: 2151–2157.
12. Smith PAS. Org Reactions 1946; 3: 337–449.
Copyright # 2004 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2004; 47: 127–138