New approaches for the synthesis of isotopically labelled guanidine-derived amino acids and noradrenaline reuptake inhibitors

Article abstract of DOI:10.1002/jlcr.1242

A new approach for the stereoselective synthesis of guanidine-derived amino acids, L-arginine and (+)-blastidic acid, has been devised which allows the selective incorporation of isotopic labels in both the side chain of the amino acid as well as the guanidine unit. A new asymmetric synthesis of the (S,R)-diastereomer of reboxetine, an antidepressant, has also been completed which allows the specific incorporation of radiolabelled iodine for SPECT imaging. Copyright

Full text of DOI:10.1002/jlcr.1242

Journal of Labelled Compounds and Radiopharmaceuticals
J Label Compd Radiopharm 2007; 50: 323–326.
Published online in Wiley InterScience
JLCR
(www.interscience.wiley.com). DOI: 10.1002/jlcr.1242
Short Research Article
New approaches for the synthesis of isotopically labelled
guanidine-derived amino acids and noradrenaline reuptake
inhibitorsy
ROLAND BISCHOFF, DEBORAH J. HAMILTON, NICOLA K. JOBSON and ANDREW SUTHERLAND*
WestCHEM, Department of Chemistry, Joseph Black Building, University of Glasgow, Glasgow G12 8QQ, UK
Received 21 July 2006; Revised 20 December 2006; Accepted 21 December 2006
Abstract: A new approach for the stereoselective synthesis of guanidine-derived amino acids, l-arginine and
(+)-blastidic acid, has been devised which allows the selective incorporation of isotopic labels in both the side chain
of the amino acid as well as the guanidine unit. A new asymmetric synthesis of the (S,R)-diastereomer of reboxetine,
an antidepressant, has also been completed which allows the specific incorporation of radiolabelled iodine for SPECT
imaging. Copyright # 2007 John Wiley & Sons, Ltd.
Keywords: asymmetric synthesis; guanylation; isotope labelling; amino acids and reboxetine
Introduction
Results and discussion
The efficient synthesis of selectively labelled biologi-
cally active compounds is important for a range of
studies including metabolic investigations as well as
the elucidation of protein structure by NMR spectro-
scopy.1 Ideally, incorporation of the isotope should
occur at a late stage of the synthesis thereby maximiz-
ing the utility of the relatively expensive isotopically
labelled reagent. Herein we describe our work on new
routes for the stereoselective synthesis of guanidine-
containing amino acids for use in biosynthetic studies.
These new routes allow the selective incorporation of
stable isotopes at various positions during the later
stages of the synthesis. We also describe our initial
investigations in identifying a molecular imaging agent
for the noradrenaline reuptake transporter (NAT) with
the first synthesis of an iodinated (S,R)-diastereomer of
reboxetine, a known inhibitor of NAT.2
Our first objective was to develop a short and efficient
synthesis of l-arginine which would also allow the
selective incorporation of stable isotopes. This was
achieved by initially synthesizing a suitably protected
ornithine derivative and coupling this with a commer-
cially available, 1-guanyl-pyrazole reagent to achieve
the key step. The ornithine derivative was prepared as
shown in Scheme 1. l-Aspartic acid 1 was converted in
two steps to N,N-di-tert-butoxycarbonyl l-aspartic acid
dimethyl ester 2. Regioselective reduction of the b-ester,
first with DIBAL-H and then with sodium borohydride
gave primary alcohol 3 in good yield. The alcohol
functional group was activated in two steps to give
iodide 4. Incorporation of a stable isotope was achieved
by nucleophilic displacement of the iodide with potas-
sium [13C]cyanide in DMF. To prevent racemization of
the stereogenic centre or any cyclization reactions from
taking place, one of the Boc-protecting groups was
removed using TFA and the ester functional group was
hydrolysed using lithium hydroxide. Finally, hydroge-
nation of nitrile 5 using platinum oxide as a catalyst
gave the desired ornithine derivative 6 in excellent
yield.
*Correspondence to: Andrew Sutherland, WestCHEM, Department of
Chemistry, Joseph Black Building, University of Glasgow, Glasgow
G12 8QQ, UK. E-mail: andrews@chem.gla.ac.uk
Contract/grant sponsor: University of Glasgow
Contract/grant sponsor: EPSRC
Contract/grant sponsor: SHERT
The key step was carried out involving the reaction of
ornithine derivative 6 with commercially available N,N-
bis(tert-butoxycarbonyl)-1H-pyrazole-1-carboxamidine
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.

324 R. BISCHOFF ET AL.
i. TMSCl, MeOH then
NEt3, Boc2O, 85%
i. DIBAL-H, -78 oC
CO2H
HO2C
CO2Me
NBoc2
HO
CO2Me
NBoc2
MeO2C
ii. NaBH4, 91% over
two steps
ii. Boc2O, DMAP, 96%
NH2
1
2
3
i. MsCl, NEt3,
DMAP, 85%
ii. NaI, ∆,
89%
K13CN, 87%
NC13
CO2H
NHBoc
I
CO2Me
NBoc2
NC13
CO2Me
i. TFA, 88%
ii. LiOH, 99%
NBoc2
4
5
H2, PtO2, 87%
13
CO2H
NHBoc
+H3N
Cl-
6
Scheme 1
NBoc
NH
EtN(iPr)2,
75%
TFA,
87%
NBoc
13
13
13
CO2H
NHBoc
CO2H
NHBoc
CO2H
NH2
N
+
+H3N
Cl-
BocN
N
H
H2N
N
H
BocHN
N
7
6
8
9
Scheme 2
7 in the presence of N-ethyldiisopropylamine which gave
the tri-Boc protected arginine analogue 8 in 75% yield
(Scheme 2). Finally, cleavage of the three Boc-protecting
groups using TFA followed by ion exchange chromato-
graphy gave [5-13C]-l-arginine 9 in 12-steps and in 24%
overall yield.3 We utilized this approach for the synthesis
of [5-13C]-l-arginine only. However, the use of other
stable isotopes of potassium cyanide or isotopically
Having achieved a simple and efficient synthesis of
isotopically labelled l-arginine we proposed that a
similar approach involving the coupling of an ornithine
derivative with N,N-bis(tert-butoxycarbonyl)-1H-pyra-
zole-1-carboxamidine should lead to an improved
synthesis of blastidic acid 11 and also allow the
selective incorporation of stable isotopes for further
biosynthetic studies. The ornithine derivative was
prepared as outlined in Scheme 3. A one-pot reaction
for the protection of b-alanine 12 followed by N-
methylation gave 13 in excellent yield. Reduction of
the ester 13 followed by a one-pot Swern/Wadsworth–
Emmons reaction gave exclusively the E-alkene 14. The
b-amino functionality was then introduced using an
asymmetric conjugate addition,7 which gave major
diastereomer 15 in 85% yield. After exchange of the
benzyl groups for the Cbz-protecting group, the tert-
butyl ester and Boc-protecting groups were cleaved
using TFA to give the b-ornithine derivative 17.
labelled
N,N-bis(tert-butoxycarbonyl)-1H-pyrazole-1-
carboxamidine 7 (prepared from commercially available
isotopically labelled cyanamide)4 would allow the pre-
paration of further analogues of labelled l-arginine.
l-Arginine has been used to investigate the biosyn-
thetic pathway of blasticidin S 10, a peptidyl-nucleoside
antibiotic first isolated from Streptomyces griseochro-
mogenes.5 Gould and co-workers demonstrated that
the b-amino acid component of blasticidin S, blastidic
acid 11, is biosynthesized from arginine via an intramo-
lecular migration of the a-nitrogen to the b-position.6
HO2C
O
N
NH
NH2
O
NH
NH2
O
O
N
H2N
N
N
H
NH2
H2N
N
OH
CH3
CH3
10
11
Copyright # 2007 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2007; 50: 323–326
DOI: 10.1002.jlcr

SYNTHESIS OF ISOTOPICALLY LABELLED GUANIDINE-DERIVED AMINO ACIDS 325
NaH, MeI,
93%
DIBAL-H,
79%
TMSCl, MeOH
CO2Me
CO2H
CO2Me
BocHN
H2N
BocN
CH3
BocN
CH3
OH
then Boc2O
NEt3, 99%
12
13
i. DMSO, NEt3
(COCl)2
97% over
two steps
ii.
CO Bu
Ph P
i. NH4HCO2
NHCbz
Ph
N
Ph
Ph
N
Ph
5% Pd/C, ∆
t
H
CO2 Bu
t
t
CO2 Bu
CO2 Bu
BocN
CH3
BocN
CH3
BocN
CH3
nBuLi, 85%
ii. BnOCOCl,
NaHCO3,
74% over two steps
16
15
14
TFA
NHCbz
NH
NH2
NBoc
N
NHCbz
TMSI, 82%
7, EtN(iPr)2
CO2H
CO2H
CO2H
HN
CH3
H2N
N
BocHN
82% from 16
CH3
CH3
11
17
18
Scheme 3
CHO
CO2Et
LiCl, DBU
DIBAL-H
72%
OH
(EtO)2POCH2CO2Et
Quant.
Br
Br
Br
19
20
(+)-DIPT
Ti(OiPr)4
tBuOOH
74%,
>98% e.e.
Br
Br
O Na
Br
O
TsCl, NEt3
OEt
H
OH
OH
DMAP
65%
H
OH
67%
O
O
OEt
OEt
OH
OTs
23
21
22
NH4OH
49%
Br
Br
Br
O
i.
Cl
Cl
BH3.THF
62%
NEt3, 83%
OH
O
O
ii. tBuO-Na+
79%
O
O
O
OEt
OEt
OEt
NH2
N
H
O
N
H
25
24
Scheme 4
As described for the synthesis of l-arginine, the
guanidine unit was introduced by coupling of 17 with 7
in the presence of N-ethyldiisopropylamine which gave
18 in excellent yield. Finally, deprotection of 18 with
trimethylsilyl iodide gave blastidic acid 11 in 11 steps
and 30% overall yield.8 Again, the use of isotopically
Copyright # 2007 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2007; 50: 323–326
DOI: 10.1002.jlcr

326 R. BISCHOFF ET AL.
Br
I
I
i. Boc2O, NEt3
cat. DMAP,
75%
TFA
57%
O
O
O
ii. NaI, CuI, 120 oC
NH2(CH2)3NH2
O
O
O
OEt
OEt
OEt
N
H
N
H
N
49%
Boc
25
26
Scheme 5
labelled N,N-bis(tert-butoxycarbonyl)-1H-pyrazole-1-
carboxamidine would allow the selective incorporation
of stable isotopes at a late stage of this synthesis of
blastidic acid.
first synthesis of an (S,R)-diastereomer of reboxetine
which after binding studies will be radioiodinated for
SPECT imaging.
Acknowledgement
Financial support from the University of Glasgow,
EPSRC and SHERT is gratefully acknowledged.
More recently our research programme for the
synthesis of isotopically labelled, biologically active
compounds has focused on identifying a new imaging
tracer for the NAT. Our aim has been to synthesize
radioiodinated analogues of reboxetine, a known in-
hibitor of NAT which could be used for SPECT imaging.
The synthesis of an iodinated (S,R)-diastereomer of
reboxetine has been completed using a Wadsworth–
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Copyright # 2007 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2007; 50: 323–326
DOI: 10.1002.jlcr