Labelling of a potent glucagon receptor antagonist with tritium, carbon-14 and stable isotopes

Full text of DOI:10.1002/jlcr.1197

Journal of Labelled Compounds and Radiopharmaceuticals
J Label Compd Radiopharm 2007; 50: 466–467.
Published online in Wiley InterScience
JLCR
(www.interscience.wiley.com). DOI: 10.1002/jlcr.1197
Short Research Article
Labelling of a potent glucagon receptor antagonist with
tritium, carbon-14 and stable isotopesy
STEEN K. JOHANSEN*, SUSSI H. WAGNER, CLAUS U. JESSEN, JACOB S. VALSBORG and LARS MARTINY
Isotope Chemistry Group, Novo Nordisk A/S, Novo Nordisk Park, DK-2760 Maaloev, Denmark
Received 13 July 2006; Revised 3 November 2006; Accepted 22 November 2006
Keywords: glucagon receptor antagonist; carbon-13; carbon-14; deuterium; tritium
Introduction
additional masses were required for the internal mass
spectroscopy standard with six of the additional
masses introduced from [U-13C]benzene and another
two from [2H5]aniline.
3-{4-[1-(4-Cyclohex-1-enylphenyl)-3-(3,5-dichlorophenyl)
ureidomethyl]-benzoylamino}propionic acid (1) is
a
potent glucagon receptor antagonist, which has been
investigated in pre-clinical trials for the treatment of
type 2 diabetes (Scheme 1). Here, we present the
syntheses of tritium, carbon-14 and stable isotope
labelled 1 for use in a range of preclinical studies.
For the first intermediate, [U-13C]benzene was con-
verted to 1,4-bis(chloromethyl)benzene (3) by reaction
with paraformaldehyde, ZnCl2 and thionyl chloride
(Scheme 2). Oxidation with HNO3 then gave 4-formyl-
benzoic acid (4) along with several oxidative by-
products, which were removed after protection of the
acid moiety to provide 4-formylbenzoic acid methyl
ester (5) in 5.1% overall yield (3 steps). For the second
intermediate, [2H5]aniline was reacted with cyclohex-
anone to give the adduct 6. This was then treated with
concentrated HCl and cracked under distillation to
provide 4-cyclohex-1-enylphenylamine (7) in 30% over-
all yield (2 steps).
Results and discussion
Tritium labelling was achieved using Crabtree’s catalyst
in good yield (36%) and high specific activity (95 Ci/
mmol) (Scheme 1). Tritium NMR determined the
labelling positions to be in the ortho-positions of the
benzoic acid moiety as well as in the b-positions of the
b-alanine moiety.
The convergent step in the synthesis was the
reductive amination of aldehyde 5 with amine 7 to give
the benzoic acid derivative 8 (85%). This was then
reacted with 3,5-dichlorophenyl isocyanate followed by
hydrolysis to give the benzoic acid 10 (68%). Finally,
reaction of 10 with b-alanine methyl ester using
standard peptide coupling chemistry followed by hy-
drolysis provided [13C6,2H2]1 (79%). The overall yield
was 2.4% based on the longest linear sequence (eight
steps) with the isotopic purity found to be >99% by
LCMS analysis.
Carbon-14 labelling was achieved in two steps
starting from [1-14C]b-alanine ethyl ester (Scheme 1).
First, [1-14C]b-alanine ethyl ester was reacted with the
benzoic acid 2 using standard peptide coupling chem-
istry followed by hydrolysis of the ester group to give
[
14C]L in 41% overall yield and with a specific activity of
52 mCi/mmol.
Stable isotope labelling was achieved using a con-
vergent route employing a total of ten steps with eight
steps in the longest linear sequence. At least seven
*Correspondence to: Steen K. Johansen, Isotope Chemistry Group,
Novo Nordisk A/S, Novo Nordisk Park, DK-2760 Maaloev, Denmark.
E-mail: skjo@novonordisk.com
yProceedings of the Ninth International Symposium on the Synthesis
and Applications of Isotopically Labelled Compounds, Edinburgh,
16–20 July 2006.
Copyright # 2007 John Wiley & Sons, Ltd.

LABELLING OF A POTENT GLUCAGON RECEPTOR ANTAGONIST 467
3H2, Crabtree
CH2Cl2
3H
O
O
O
O
3H
3H
36%
HO
HO
N
N
H
H
H
H
Cl
N
Cl
N
N
N
3H
[3H]-1
1
O
O
Cl
Cl
EDAC, HOBt
DIPEA, CH2Cl2
LiOH
THF/H2O
O
O
O
14C
41%
HO
HO
N
H
H
H
N
N
Cl
N
Cl
N
2
[14C]-1
O
14C
O
O
+
NH3Cl
O
Cl
Cl
Scheme 1
MeOH, SOCl,
then acetone
(CH2O)n
ZnCl2, SOCl2
HNO3
Cl
O
O
U-13
C
U-13
C
U-13
C
U-13
C
Cl
HO
O
27%
19%
2 steps
3
4
5
O
O
2H
2H
2H
2H
2H
2H
2H
2H
HCl
EtOH
HCl, ∆
H2N
+
30%
2 steps
H2N
NH2
2H
O
2H 2
H
NH2
6
7
2H
OCN
Cl
NaBH3CN
MeOH
O
2H
2H
U-13
C
CH3CN
H2N
MeO
+
O
Cl
85%
5
7
2H
O
79%
O
U-13
C
NH
O
8
LiOH
THF/H2O
O
NH3Cl
EDAC, HOBt
DIPEA, CH2Cl2
LiOH
THF/H 2O
2H
2H
86%
O
2H
2H
O
O
98%
81%
HO
HO
N
U-13
C
U-13
C
H
H
H
N
N
N
Cl
N
Cl
[2H2,13C6]-1
10
O
O
Cl
Cl
Scheme 2
Copyright # 2007 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2007; 50: 466–467
DOI: 10.1002.jlcr