Synthesis of two novel oxocyclam-binding technetium complexes containing an analogue of cocaine

Article abstract of DOI:10.1002/jlcr.560

In order to visualize and quantify dopamine transporters, the synthesis of two novel ligands labelled with technetium-99m (^99mTc) has been investigated. A multi-step synthesis afforded two target ligands with a tropane skeleton and a macrocycli

Full text of DOI:10.1002/jlcr.560

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2002; 45: 379–393.
DOI: 10.1002/jlcr.560
Research Article
Synthesis of two novel oxocyclam-binding
technetium complexes containing an
analogue of cocaine
F. Turpin1, F. Masri2, F. Riche2,*, E. Berthommier1, P. Emond3,
´
M. Vidal4, P. Auzeloux5, C. Loc’h6, N. Neuquelman1 and
L. Mauclaire1
1 CIS bio international, subsidiary of Schering,
BP 32 – 91192 Gif-sur-Yvette, France
2
!
Universite Joseph Fourier, LEDSS–UMR CNRS 5616,
BP 53 – 38041 Grenoble Cedex 9, France
3
!
INSERM U 316, Laboratoire de Biophysique Medicale et Pharmaceutique,
31 avenue Monge – 37200 Tours, France
4 ERAS Labo – 38330 St Nazaire les Eymes, France
5 INSERM U484, rue Montalembert, 63005 Clermont-Ferrand Cedex 05, France
6
! !
Service Hospitalier Frederic Joliot, DRM, CEA, 91406 Orsay, France
Summary
In order to visualize and quantify dopamine transporters, the synthesis of two
novel ligands labelled with technetium-99 m (99mTc) has been investigated.
A multi-step synthesis afforded two target ligands with a tropane skeleton and
a macrocyclic complexing moiety. The choice and the position of substituents
are in adequation with dopamine transporter structure. The radiolabelling of
these ligands with 99mTc has been studied and the results make them good
candidates for SPECT imaging. Copyright # 2002 John Wiley & Sons, Ltd.
Key Words: synthesis; cocaine analogues; tropanes; dopamine transporter;
SPECT; oxocyclam; technetium
!
*Correspondence to: Dr. F. Riche, Universite Joseph Fourier, LEDSS–UMR CNRS 5616,
BP 53 – 38041 Grenoble Cedex 9, France. E-mail: francoise.riche@ujf-grenoble.fr
Contract/grant sponsor: EUREKA-DOPIMAG program; contract/grant number: EU1836
Copyright # 2002 John Wiley & Sons, Ltd.
Received 11 October 2001
Revised 20 November 2001
Accepted 21 November 2001

380
F. TURPIN ET AL.
Introduction
The presynaptic dopamine transporter (DAT) is an essential part of
dopaminergic transmission regulation.l,2 In vitro and in vivo studies have
shown that DAT is depleted in Parkinson’s disease and that the extent
of depletion correlates with the loss of dopamine essentially in some
brain areas (striatum and substantia nigra) where dopaminergic neurons
are located.3,4 A quantitative measure of the DAT density may be
achieved by selective binding of ligands containing a radioactive label.
A decreased specific striatal uptake of such a radiolabelled DAT ligand
may therefore indicate pathological states and allow an early diagnosis.
Among drugs which bind DAT selectively, the most studied belong to
the family of bicyclo[3.2.l]octanes modified at C2, C3 or N (WIN-
analogs). All molecules with the basic R-cocaine skeleton are known to
inhibit DAT (Figure 1).
Recently, many compounds have been proposed as PET or SPECT
imaging agents. Several ligands have been reported for use in PET
imaging, including [11C]cocaine,5 [11C]-2b-carbomethoxy-3b-(4-iodo-
phenyl)tropane (b-CIT6) or N-fluoropropyl-2b-carbomethoxy-3b-(4-
iodophenyl)tropane (FPbCIT7). Positron-emitting nuclides have short
half-lives (e.g., 11C, 20 min; 18F, 109 min), so they require an on-site
cyclotron for their production. More recently, SPECT imaging agents
have been developed, labelled with 123I (g, 160 keV, t1/2=13 h), like
[
123I]-b-CIT,8 [123I]-N-(fluoropropyl)-b-CIT,7 [123I]-N-(3-iodoprop-(2E)-
enyl)-2b-carbomethoxy-3b-(4-methylphenyl)nortropane (PE2I9). All
these derivatives showed a very good affinity for DAT but their
specificity was insufficient. Other SPECT imaging agents are labelled
with 99mTc. This metal is the most widely used radionuclide in
diagnostic nuclear medicine. 99mTc (t1/2=6 h) is readily produced by
99Mo/99mTc generator and its g-ray energy emission (140 keV) is suitable
for g-camera detection. But, unlike halogenated compounds, labelling
Figure 1.
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 379–393

SYNTHESIS OF TWO NOVEL OXOCYCLAM-BINDING
381
with technetium needs a chelating structure of this transition metal. All
the examples of Tc-99m labelled tropane derivatives have been modified
at the N-position (R1) or at C2 (R2) to introduce a technetium-carrying
moiety. The best candidates are technepine,10 ‘‘3+1’’-mixed-ligand11
and TRODAT-112 which showed a good fixation on striatum neurons.
Nevertheless, they are still not perfect for the development of a SPECT
imaging agent of dopaminergic neuronal function.
We propose new compounds with a WIN-analogue structure. We
chose to keep the N-methyl group by analogy to cocaine and a tolyl
group at C3 by analogy to the good results of PE2I. The chelating
structure chosen was oxocyclam for its remarkable properties in terms
of complexation of the [TcO+2 ] core leading to a very stable complex,
neutral at physiological pH and thus potentially able to cross the blood
brain barrier.13 This chelating structure was conjugated to the tropane
ring at the C2 position with a linker via an ester or an amide group as
shown in Figure 2.
The choice of an ester or amide function at C2 was controlled by SAR
studies suggesting the presence in DAT of one or two hydrogen bond
acceptor sites localized in the vicinity of the 2b-carbomethoxy group of
cocaine.14
Results and discussion
Chemistry
The synthesis of ligands 9 and 17, leading by complexation of the
[TcO+2 ] core to target compounds 10 and 18, requires the building of the
macrocyclic structure on one side and functionalised enantiomerically
pure tropane on the other side. The key-step in this synthesis is the
reaction of 8-methyl-3S-p-tolyl-8-azabicyclo[3.2.l]octane-2S-carboxylic
acid 8 with 13-[5-hydroxypentyl]-1,4,8,11-tetraazacyclotetradecan-5-one
Figure 2. Target complexes
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 379–393

382
F. TURPIN ET AL.
Scheme 1. Synthesis of 8-methyl-3S-p-tolyl-8-azabicyclo[3.2.1]octane-2S-car-
boxylic acid 5-(12-oxo-1,4,8,11-tetraazacyclotetradec-6-yl)pentyl ester 9
to give 9 or with 13-(5-aminopentyl)-1,4,8,11-tetraazacyclotetradecan-5-
one affording 17. The two total syntheses are shown in Schemes 1 and 2.
The starting material was 5-chloropentan-1-ol, a bifunctional
compound used as a linker. After protection of the hydroxyl function
by a tetrahydropyranyl group, a three-carbon bridge was introduced by
a classic malonic synthesis, resulting in the condensation of mono-
sodium salt of diethylmalonate on the chloro-compound 1 with a yield
of 60%. The four nitrogens required for the complexation are easily
introduced through the action of excess ethylenediamine on the diester
leading to diamido/diamino compound 3 (85%). It was necessary to
deprotect the alcohol before the reduction of the amide groups by
BH3–Me2S complex because it is known that ketals undergo reductive
cleavage with borane15 affording a 4-hydroxybutyloxy group in place of
tetrahydropyran-2-oxy group. The alcohol 4 was quantitatively
obtained in acidic conditions. The reduction of amide groups was
realised via Brown’s method, BH3/Me2S complex in THF,16 with a
modification: the four amine functions resulting from reduction were
protected in situ to make the purification easier. Thus, 5 was obtained
with a global yield of 65%. Deprotection of amines was achieved with
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 379–393

SYNTHESIS OF TWO NOVEL OXOCYCLAM-BINDING
383
Scheme 2. Synthesis of 8-methyl-3S-p-tolyl-8-azabicyclo[3.2.1]octane-2S-car-
boxylic acid 5-(12-oxo-1,4,8,11-tetraazacyclotetradec-6-yl)pentyl amid 17
concentrated aqueous HCl in MeOH to give 6 which was used without
further purification. Cyclisation of 6 on methacrylate was realised as
described17 after the addition of 4 equiv. of Et3N or MeONa to assure
deprotonation of the amine groups, which is essential before cyclisation.
Cyclisation occurred with a yield of 35%, which is a good value in the
absence of a template effect or high dilution.
The synthesis of 8-methyl-3S-p-tolyl-8-azabicyclo[3.2.1]octane-2S-
carboxylic acid 8 starting from natural R(+)-cocaine is described
elsewhere.18 The esterification reaction between 7 and 8 was carried out
in the presence of dicyclohexylcarbodiimide in DMF, catalysed by
dimethylaminopyridine. Compound 9 was isolated with a yield of 18%
after purification. This low yield results from the low reactivity of the
alcohol function but also from a very difficult purification.
The synthesis used to prepare 17 is described in Scheme 2.
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 379–393

384
F. TURPIN ET AL.
The transformation of the alcohol in amine function is classically
realised by a Gabriel synthesis via a phthalimide. A Mitsunobu reaction
using phthalimide, DEAD and PPh3 gave 11. But with various
conditions of temperature and solvent, we never exceeded a yield of
40%. Compound 11 was not isolated. We also chose to introduce the
phthalimide group by another route which first required amine
protection. Then, treatment of 12 with methanesulfonyl chloride
afforded 13 which, when heated in THF containing a phase transfer
catalyst and potassium phthalimide, yielded 14. Compound 15 was
obtained by hydrazinolysis (95%), then treated in acidic conditions
to afford 16. The amide 17 was synthesized under the same conditions
as the esterification, leading to 9; the yield was also relatively low
(26%).
Labelling
The radiolabelling of the two ligands 9 and 17 with sodium
[
99mTc]pertechnetate was achieved using stannous (II) tartrate as the
reducing agent as described in the literature.13 Ligands (200 mg) were
dissolved in 4 ml NaOH 0.1 M in water. Then 4 ml of a saturated aqueous
solution of SnC4H4O6 were added followed by introduction of 18 ml
(0.8 MBq) TcOꢀ4 in physiological serum. Reaction occurred at 70–808C
over 45 min.
The labelling yields were measured by chromatography on silica gel
using a ternary system, dichloromethane 3, ethanol 2 and ammonia
22% 0.5. After counting the radiochromatogram by electronic auto-
radiography, silica gel plates were treated with a phosphomolybdic acid
ethanolic solution to visualize the ligand position. These yields varied
between 46 and 72% depending on the compounds and experimental
conditions. Typical chromatogram are given in Figure 3 with the free
ligand encircled. Rf values were the same for 3 and 17.
This profile leads to the following comments:
*
absence of pertechnetate,
presence of Tc-tartrate, intermediary complex which leads to Tc–L by
*
exchange with the ligand.
good separation of L and Tc–L, allowing a purification of Tc–L by liquid
chromatography to obtain a pure complex free of ligand.
*
The [99mTc-oxocyclam] structure has been reported previously.13,19
These studies, conducted on the moeity oxocyclam without
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 379–393

SYNTHESIS OF TWO NOVEL OXOCYCLAM-BINDING
385
Figure 3. Chromatograms of labelling solutions of 9 or 17 (99mTc–L+L),
99mTc-tartrate and 99mTcO4ꢀ
any substituent at tracer level and macroscopic level, showed that
the macrocyclic oxocyclam complexed with the [TcO+2 ] core and
that the resulting complex established an acid–basic equilibrium in
solution by protonation/deprotonation of the amide function as
described in Scheme 3. The preponderant form at pH 7.4 is neutral so
we hope that the target molecules will be able to cross the blood brain
barrier.
Scheme 3. Structure of the complex 99mTcO2-oxocyclam
Experimental
The compound 8-methyl-3S-p-tolyl-8-azabicyclo[3.2.l]octane-2S-car-
boxylic acid 8 was made available by the ERAS Laboratory.
1H NMR spectra were run at 300 MHz and 13C NMR at 75 MHz.
Et2O and THF were dried over and distilled from sodium metal with
benzophenone as the indicator. DMF and CH2Cl2 were dried over and
distilled from CaH2. MeOH and EtOH were dried over and distilled
from Mg. Analytical TLC was carried out on Merck Kieselgel 60F254-
0,20 mm plates, and column chromatography on Merck Kieselgel
Geduran Si 60 (0.06–0.20 mm). Radioactivity was measured by
autoradiography (Instant Imager, Packard). Commercially available
reagents were used without further purification.
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 379–393

386
F. TURPIN ET AL.
2-(5-chloropentyloxy)tetrahydropyran (1)
5-Chloropentanol (30 g, 0.245 mol) was dissolved in 70 ml of anhydrous
THF and the resulting solution was cooled to 08C. After addition of a
catalytic amount of p-toluenesulfonic acid, dihydropyran (20.6 g,
0.245 mol) was introduced dropwise. The resulting mixture was warmed
to room temperature and stirred overnight. THF was evaporated off,
then the crude mixture was dissolved in Et2O. The ether layer was
washed with NaHCO3 saturated aqueous solution, then water. After
evaporation in vacuo of ether, the crude product was purified by liquid
chromatography (silica gel, Et2O:cyclohexane, 50:50) to afford 47.51 g
1
of 1 (94%) as an oil. H NMR (300 MHz, CDCl3) d 1.5–1.8 (m, 12 H,
3
CH2CH2CH(2)), 1.84 (qt, 2 H, CH2CH2Cl, JðCH 2CH Þ ¼ 6:7 HzÞ, 3.5–
2
2
3
3.9 (m, 4 H, CH2O), 3.55 (t, 2 H, CH2Cl, JðCH 2CH Þ ¼ 6:7 HzÞ, 4.6 (t,
2
2
lH, CH(O)2, JðCH2CH Þ ¼ 3:1 Hz). 13C NMR (75 MHz, CDCl3) d 20.0,
3
2
24.1, 25.8, 29.4 (CH2CH2CH(2)), 31.1 (CH2CH(O)2), 32.8 (CH2CH2Cl),
45.3 (CH2Cl), 62.7, 67.6 (CH2O), 99.3 (CH2CH(O)2).
2-[5-ðtetrahydropyran-2-oxy)pentyl]malonic acid ethyl ester (2)
An ethanolate solution was prepared by dissolving Na (6.2 g, 0.269 mol)
in anhydrous ethanol (250 ml). After addition of diethylmalonate
(36.9 g, 0.23 mol), the solution was heated and stirred at 608C for l h.
Then, 2-(5-chloropentyloxy)tetrahydropyran (47.5 g, 0.23 mol) was
added and the resulting mixture was heated to 808C and stirred for
24 h. Ethanol was evaporated off, the residue was dissolved in water and
the product was extracted with ether. 2 was purified by liquid
chromatography (silica gel, pentane:ether:ethyl acetate, 20:2:1) and
1
obtained as an oil (51.74 g, 68%). H NMR (300 MHz, CDCl3) d
3
1.25 (t, 6 H, CH3CH2O, JðCH 2CH Þ ¼ 7:2 Hz), 1.3–2 (m, 14 H,
2
3
CH2CH2CH(2)), 3.3 (t, lH, CH2CH(CO)2), 3.3–3.9 (m, 4 H, CH2CH2O),
4.2 (q, 4 H, CH3CH2O, 3JðCH 2CH Þ ¼ 7:2 Hz), 4.6 (m, lH, CH(O)2). 13
C
2
3
NMR (75 MHz, CDCl3) d 13.8 (CH3CH2O), 19.3, 25.2, 25.6, 26.6, 28.3,
29.1, 30.4 (CH2CH2CH(2)), 51.6 (CH(CO)2), 60.9 (CH3CH2O), 61.9,
67.0 (CH2CH2O), 98.4 (CH(O)2), 169.1 (C¼O).
N,N0-bis(2-aminoethyl)-2-[5-(tetrahydropyran-2-yloxy)pentyl]malona-
mide (3)
The diester 2 (51.74 g, 0.156 mol) was dissolved in ethylenediamine
(93.8 g, 1.56 mol) and the resulting mixture was heated at 558C for 2
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 379–393

SYNTHESIS OF TWO NOVEL OXOCYCLAM-BINDING
387
days. Ethanol and ethylenediamine were evaporated off in vacuo.
Purification by liquid chromatography (silica gel, CHCl3:CH3OH:
NH4OH 22%, 3:2:0.5) afforded 46.2 g of 3 (orange wax, 83%).
1H NMR (300 MHz, D2O, pH=9–10)
d
1.5–1.8 (m, 14 H,
3
CH2CH2CH(2)), 2.64 (t, 4 H, CH2NH2, JðCH 2CH Þ ¼ 6:3 Hz), 3.2 (t,
2
2
4 H, CH2NH, 3JðCH 2CH Þ ¼ 6:3 Hz), 3–3.2 (m, lH, CH2CH(CO)2), 3.4–
2
2
3.85 (m, 4 H, CH2O), 4.5 (m, 1 H, CH(O)2, JðCH2CH Þ ¼ 3:l Hz). 13C
3
2
NMR (75 MHz, D2O, pH=9–10) d 19.7, 25, 25.4, 26.7, 28.9, 29.8, 30.6
(CH2CH2CH(2)), 40.2, 42.2 (CH2NH2, CH2NH), 54.3 (CH(CO)2), 63.7,
68.4 (CH2CH2O), 99.9 (CH(O)2), 172.8 (C¼O).
N; N0-bis(2-aminoethyl)-2-[5-hydroxypentyl]malonamide (4)
To a solution containing 28.78 g, 0.08 mol of 3 in 500 ml of absolute
ethanol, p-toluenesulfonic acid monohydrate (38.8 g, 0.206 mol) was
added and the resulting mixture was heated at 608C for 18 h. NH4OH
22% was added until pH=9. A white precipitate appeared, which was
removed by filtration. Ethanol was evaporated off in vacuo and the
compound 4 was purified by liquid chromatography (silica gel,
CHCl3:CH3OH:NH4OH 22%, 3:2:0.5) to afford 21.8 g (viscous oil,
1
99%) of pure product. H NMR (300 MHz, D2O, pH=9–10) d 1–1.25
3
(m, 4 H, CH2CH2CH(2)), 1.3 (m, 2 H, CH2CH2OH, JðCH 2CH Þ
¼
2
2
6:5 HzÞ, 1.5–1.7 (m, 2 H, (CO)2CHCH2), 2.55 (t, 4 H, CH2NH,
3
3JðCH 2CH Þ ¼ 6:3 Hz), 3.1 (t, 4 H, CH2NH2, JðCH -CH Þ ¼ 6:3 Hz), 3.2
2
2
2
2
(s, lH, (CO)2CHCH2), 3.4 (t, 2 H, CH2OH, JðCH 2CH Þ ¼ 6:5 Hz). 13C
3
2
2
NMR (75 MHz, D2O, pH=9–10) d 25, 26.7 (CH2CH2CH(2)), 29.7
((CO)2CHCH2), 31.4 (CH2CH2OH), 40.1 (CH2NH), 42.0 (CH2NH2),
49.2 (CH(CO)2), 61.9 (CH2OH), 172.8 (C¼O).
(2-tert-butoxycarbonylaminoethyl)-(2-{[tert-butoxycarbonyl)-(2-tert-but-
oxycarbonylaminoethyl)-amino]methyl}-7-hydroxyheptyl)carbamic
acid tert-butyl ester (5)
In a Schlenk tube containing 250 ml of anhydrous THF were dissolved
13.3 g, 0.048 mol of diamide 4 and 33 ml of boran-dimethyl sulphide
complex. The resulting mixture was gently refluxed for 5 days. THF was
evaporated off, then 100 ml of methanol was introduced dropwise and
refluxed for 3 h. A methanolic solution of HCl (30 ml aqueous HCl 32%
in 250 ml MeOH) was added and then heated to reflux over 12 h. After
evaporation of the solvents, 100 ml of methanol were added and
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 379–393

388
F. TURPIN ET AL.
immediately evaporated to eliminate methyl borate. This last operation
was repeated three times. The crude product was dissolved in 350 ml of
methanol and treated with NaHCO3 (60 g) and di-tert-butyldicarbonate
(42.67 g, 0.195 mol) over 48 h at room temperature. Methanol was
evaporated off, the residue dissolved in water and extracted with ether.
Evaporation of ether afforded 5 which was purified by liquid
chromatography (silica gel, CHCl3:CH3OH, 4:0.2) as a white wax
(19.76 g, 65%). 1H NMR (300 MHz, CDCl3) d 1.2–2 (m, 9 H,
CH2CH2CH(2), CH), 1.3 and 1.4 (s, 36 H, (CH3)3CO), 3–3.2 (m, 12 H,
NHCH2CH2N), 3.6 (m, 2 H, CH2OH), 5 (broad s, 2 H, NH). 13C NMR
(75 MHz, CDCl3) d 26.0, 29.8, 32.5, 39.4 (CH2CH2CH(2)), 28.4
((CH3)3CO), ꢁ 37 (broad, CH), 47.1, 49.7 (broad, CHCH2N), 63.2
(CH2OH), 79.2, 80 ((CH3)3CO), 156.1 (CONH).
7-(2-aminoethylamino)-6-[(2-aminoethylamino)methyl]-heptan-1-ol (6)
The compound 5 (19.76 g, 0.03 mol) was dissolved in 300 ml of methanol
and 32 ml of aqueous HCl 32%. After 36 h at room temperature, the
solvents were evaporated off and 6 was isolated as its tetrachlorohydrate
1
salt (11.5 g, 97%). H NMR (300 MHz, D2O, pH=1) d 1–1.4 (m, 8 H,
CH2CH2CH(2)), 2.2 (m, 1 H, CH), 3.1 (m, 4 H, NHCH2CH), 3.15–3.25
(m, 8 H, NH2CH2CH2NH), 3.4 (t, 2 H, CH2OH, 3JðCH 2CH Þ ¼ 6:3 Hz).
2
2
13C NMR (75 MHz, D2O, pH=1)
d
25, 25.3, 28.9, 31.4
(CH2CH2CH(2)), 34.2 (CH), 35.9 (NHCH2CH), 45.6, 49.7
(NH2CH2CH2NH), 61.9 (CH2OH).
13-(5-hydroxypentyl)-1,4,8,11-tetraazacyclotetradecan-5-one (7)
The amine salt 6 (11.5 g, 29.3 mmol) was dissolved in dry methanol
(600 ml) and triethylamine (6 equiv.) was added. Then methacrylate
(2.71 ml, 29.6 mmol) was introduced and the mixture was stirred under
an inert atmosphere over 2 days at room temperature and 15 days under
reflux. After evaporation of the solvent, purification on silica gel
(CHCl3:CH3OH:NH4OH 22%, 10:4.5:0.75) afforded 7 (1.95 g, orange
1
oil, 22%). H NMR (300 MHz, D2O, pH=9) d 1.05–1.25 (m, 6 H,
3
CH2CH2CH(2)), 1.4 (qt, 2 H, CH2CH2OH, JðCH 2CH Þ ¼ 6:6 Hz), 1.6–
2
2
3
1.75 (m, 1 H, CH), 2.25 (t, 2 H, CH2CO, JðCH 2CH Þ ¼ 5:8 Hz), 2.4–2.9
(m, 12 H, CH2NHCH2), 3.1–3.3 (m, 2 H, CH2NHCO), 3.4 (t, 2 H,
CH2OH, JðCH 2CH Þ ¼ 6:6 Hz). 13C NMR (75 MHz, D2O, pH=9) d
2
2
3
2
2
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 379–393

SYNTHESIS OF TWO NOVEL OXOCYCLAM-BINDING
389
25.4, 26, 30.4, 31.5 (CH2CH2CH(2)), 34.0 (CH2CO), 35.1 (CH), 37.9,
43.7, 45.2, 46.9, 48.8, 54.3, 54.4 (CH2NH), 62.1 (CH2OH), 175.4 (C¼O).
8-methyl-3S-p-tolyl-8-azabicyclo[3:2:1] octane-2S-carboxylic acid
5-(12-oxo-1,4,8,11-tetraazacyclotetradec-6-yl)pentyl ester (9)
The acid 8 (97 mg, 0.345 mmol), the alcohol 7 (210 mg, 0,69 mmol),
dicyclohexylcarbodiimide (78 mg, 0.38 mmol) and dimethylaminopyr-
idine (46 mg, 0.38 mmol) were dissolved in freshly distilled DMF
(20 ml). After 3 days at room temperature, the solvent was evaporated
off and the product purified by liquid chromatography (silica gel,
1
(CHCl3:CH3OH:NH4OH 22%, 10:4.5:0.75) to give 9 as an oil. H
NMR (300 MHz, CDCl3) d 0.9–1 (m, 2 H, 2H12), 1–1.28 (m, 4H, 2H11,
0
2H13), 1.3–1.5 (m, 2H, 2H10), 1.51–1.7 (m, 4H, H4a, H6 , H7 , H14), 1.95–
0
2.3 (m, 2H, H6, H7), 2.21 (s, 3H, N–CH3), 2.28 (s, 3H, ArCH3), 2.3–3
0
0
0
(m, 13H, 2H16, 2H20, 2H21, 2H22, H3, H4b, H15 , H17 , H23 ), 3.05–3.27
(m, 2H, H15, H23), 3.35 (m, 1H, H5), 3.55 (m, 2H, H1, H17), 3.85 (dt, 1H,
H9), 4.05 (dt, 1H, H9), 7–7.2 (m, 4H, CArH). 13C NMR (75 MHz,
CDCl3) d 21 (ArCH3), 25.5 (C6, C7), 26.1, 26.2, 32.2 (C11, C12, C13), 28.5
(C10), 33.2 (C3), 34.1 (C4), 36.1, 37.7 (C17, C19), 39.7 (C14), 42 (NCH3),
44.4, 46.2 (C20, C21), 52.9 (C2), 53.1, 53.8 (C16, C22), 56.7 (C15, C23), 62.3
0
(C5), 63.5 (C9), 65.5 (C1), 127.1, 128.6 (C5 , C6 , C8 , C9 ), 135, 140.1 (C4 ,
0
0
0
0
C7), 171.7, 172.5 (C8, C18).
6-(5-hydroxypentyl)-12-oxo-1,4,8,11-tetraazacyclotetradecane-1,
4,8-tricarboxylic acid tri-tert-butyl ester (12)
To a solution of 7 (317 mg, 1.05 mmol) and NaHCO3 (500 mg) in 25 ml
of methanol was added di-tert-butyldicarbonate (1.38 g, 6.33 mmol).
The mixture was stirred at room temperature for 2 days. The solvent
was evaporated off and the residue dissolved in ether. This phase was
washed with water, stored over anhydrous Na2SO4, filtered and
evaporated. A purification on silica gel (ether:methanol, 10:l) afforded
12 (595 mg, 94%) as a white oil. 1H NMR (300 MHz, CDCl3) d 1.2–1.65
(m, 8H, CH2CH2CH(2)), 1.44, 1.48 (s, 27H, (CH3)3CO), 1.65–1.95 (m,
1H, CH), 2.2–3.9 (m, 16H, CH2N, CH2CO), 3.6 (t, 2H, CH2OH,
3JðCH 2CH Þ ¼ 6:3 Hz), 6.85–7.15 (broad s, 1H, NHCO). 13C NMR
2
2
(75 MHz, CDCl3) d 26.4, 30.2, 32.7, 37.1 (CH2CH2CH(2)), 28.8
((CH3)3CO), 38.2 (CH), 41.1 (CH2CO), 47, 47.9, 52.7 (CH2,N), 62.7
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 379–393

390
F. TURPIN ET AL.
(CH2OH), 79.9, 80.3, 81.1 ((CH3)3CO), 156.3 (OCONH), 172.8
(NHCOCH2).
6-(5-methanesulfonyloxypentyl)-12-oxo-1,4,8,11-tetraazacyclotetrade-
cane-1,4,8-tricarboxylic acid tri-tert-butyl ester (13)
The alcohol 12 (412 mg, 0.686 mmol) and triethylamine (69 mg,
0.689 mmol) were dissolved in 20 ml CH2Cl2. The methanesulpho-
nylchloride (78 mg, 0.686 mmol) was added dropwise and the resulting
solution was stirred at room temperature for 12 h, washed with water
then brine. The dichloromethane was evaporated off and 13 (395 mg,
85%) was isolated as a white wax by liquid chromatography (silica gel,
1
CHCl3:methanol, 6:l). H NMR (300 MHz, CDCl3) d 1–1.6 (m, 6H,
CH2CH2CH(2)), 1.45, 1.48 (s, 27H, (CH3)3,C–O), 1.65–1.8 (m, 1H, CH),
3
1.73 (qt, 2H, CH2CH2OH, JðCH 2CH Þ ¼ 6:6 Hz), 2.3–3.9 (m, 16H,
CH2N, CH2CO), 3.0 (s, 3H, CH3S), 4.2 (t, 2H, CH2O,
2
2
3JðCH 2CH Þ ¼ 6; 6 Hz), 6.7–7 (broad s, 1H, NHCO). 13C NMR
2
2
(75 MHz, CDCl3) d 26.2, 29.2, 30.2, 37.2 (CH2CH2CH(2)), 28.8
((CH3)3CO), 37.7 (CH3S), 38.8 (CH), 41.2 (CH2CO), 47.2, 49.6, 50.5,
52.9 (CH2N), 70.2 (CH2–O), 79.8, 80.3, 81.1 ((CH3)3CO), 156.3
(OCONH), 172.6 (NHCOCH2).
6-[5-(1,3-dioxo-1,3-dihydroisoindol-2-yl)-pentyl]-12-oxo-1,4,8,11-tetra-
azacyclotetradecane-1,4,8-tricarboxylic acid tri-tert-butyl ester (14)
A solution of 13 (288 mg, 0.424 mmol), potassium phthalimide (116 mg,
0.636 mmol) in 30 ml of toluene was refluxed for 10 h. After evaporation
in vacuo, the residue was dissolved in ether, washed with water, then
brine, and finally evaporated. The product was purified by liquid
chromatography on silica gel (ether:methanol, 10:1) to give compound
14 (263 mg, 85%) as a white wax. 1H NMR (300 MHz, CDCl3) d 1–1.55
(m, 6H, CH2CH2CH(2)), 1.43, 1.45, 1.47 (s, 27H, (CH3)3CO), 1.66 (qt,
3
2H, CH2CH2N(CO)2, JðCH 2CH Þ ¼ 7 Hz), 1.75–1.85 (m, 1H, CH),
2.35–3.95 (m, 16H, CH2N, CH2CO), 3.66 (t, 2H, CH2N(CO)2,
2
2
3JðCH 2CH Þ ¼ 7 Hz), 6.75–6.95 (broad s, 1H, NHCO), 7.65–7.75 (m,
2
2
2H, CHAr), 7.77–7.89 (m, 2H, CHAr). 13C NMR (75 MHz, CDCl3) d
26.6, 27.6, 30.4, 37.2 (CH2CH2CH(2)), 30.2 ((CH3)3C–O), 38.1
(CH2N(CO)2), 38.6 (CH), 41.3 (CH2CO), 47.1, 47.6, 49.7, 52.8
(CH2N), 79.8, 80.3, 81.1 ((CH3)3CO), 123.5 (CArH), 132.5 (CAr),
ꢀ
134.2 (CArH), 156.3 (OCO–NH), 168.7(NCOCAr ), 172.6 (NHCOCH2).
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 379–393

SYNTHESIS OF TWO NOVEL OXOCYCLAM-BINDING
391
6-(5-aminopentyl)-12-oxo-1,4,8,11-tetraazacyclotetradecane-1,4,8-tri-
carboxylic acid tri-tert-butyl ester (15)
To a solution of 14 (240 mg, 0.329 mmol) in absolute ethanol (25 ml)
was added a large excess of hydrazine hydrate (1 ml). The mixture was
refluxed for 8 h. After cooling, a white precipitate appeared. The solvent
was evaporated off and the residue dissolved in ether and aqueous KOH
30%. The organic layer was stored over anhydrous Na2SO4 and then
evaporated. Purification on a silica gel column (CHCl3:MeOH, 5:1)
1
afforded 15 (187 mg, 95%) as an oil). H NMR (300 MHz, CDCl3) d
0.9–1.55 (m, 8H, CH2CH2CH(2)), 1.36, 1.40 (s, 27H, (CH3)3CO), 1.6–1.8
(m, 1H, CH), 2.2–3.9 (m, 16H, CH2N, CH2CO), 2.64 (t, 2H, CH2NH2,
3JðCH 2CH Þ ¼ 6:8 Hz), 6.45–6.9 (broad s, 1H, NHCO). 13C NMR
2
2
(75 MHz, CDCl3) d 26.9, 27.6, 30.4, 37.2 (CH2CH2CH(2)), 28.8
((CH3)3CO), 38.6 (CH), 41.2 (CH2CO), 42.4 (CH2NH2), 47.1, 47.9,
50.0, 52.9 (CH2N), 79.8, 80.3, 81.0 ((CH3)3CO), 156.3 (OCONH), 172.5
(NHCOCH2).
13-(5-aminopentyl)-1,4,8,11-tetraazacyclotetradecan-5-one,
tetrachlorhydrate (16)
Deprotection of 15 (96 mg, 0.16 mmol) was realised in methanol (25 ml)
and aqueous HCl 32% (3 ml) during 24 h at room temperature. Solvents
were evaporated off and the residue was dissolved in water and washed
with ether. The water was evaporated off in vacuo to give 16 (71 mg,
99%). 1H NMR (300 MHz, D2O, pH=l) d 1.32–1.55 (m, 6H,
3
CH2CH2CH(2)), 1.4 (qt, 2H, CH2CH2NH2, JðCH 2CH Þ ¼ 7:2 Hz),
2
2
3
2.35 (m, 1H, CH), 2.97 (t, 2H, CH2CO, JðCH 2CH Þ ¼ 5:7 Hz), 3.02 (t,
2
2
2H, CH2NH2, JðCH 2CH Þ ¼ 7:6 Hz), 3.3–3.95 (m, 14H, CH2NH).13C
3
2
2
NMR (75 MHz, D2O, pH=1) d 25.6, 25.7, 26.8, 29.4 (CH2–CH2–
CH(2)), 29.9 (CH2CO), 34 (CH), 37.3, 42.2, 43.5, 44.4, 47, 48.1, 49
(CH2–NH), 39.7 (CH2–NH2), 176.4 (C¼O).
8-methyl-3S-p-tolyl-8-azabicyclo[3.2.1]octane-2S-carboxylic acid [5-
(12-oxo-1,4,8, 11-tetraazacyclo tetradec-6-yl)-pentyl]amide (17)
The work-up was the same as for the synthesis of 9. The chromato-
graphic conditions were silica gel, CHCl3:MeOH:NH4OH 22%,
1
5:1:0.2). Yield was 26% of 17, as a yellow oil. H NMR (300 MHz,
CDCl3) d 0.9–1 (m, 2H, 2H12), 1–1.28 (m, 4H, 2H11, 2H13), 1.3–1.5 (m,
0
2H, 2H10), 1.51–1.7 (m, 4H, H4a, H6 , H7 , H14), 1.95–2.3 (m, 2H, H6,
0
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 379–393

392
F. TURPIN ET AL.
H7), 2.21 (s, 3H, NCH3), 2.28 (s, 3H, ArCH3), 2.3–3 (m, 13 H, 2H16,
0
2H20, 2H21, 2H22, H3, H4b, H15 , H17 , H23 ), 3.05–3.27 (m, 2H, H15,
0
0
H23), 3.35 (m, 1H, H5), 3.55 (m, 2H, H1, H17), 3.85 (dt, 1H, H9), 4.05
(dt, 1H, H9 ), 7–7.2 (m, 4H, CAr–H). 13C NMR (75 MHz, CDCl3) d 21
0
(ArCH3), 25.5 (C6, C7), 26.1, 26.2, 32.2 (C11, C12, C13), 28.5 (C10), 33.2
(C3), 34.1 (C4), 36.1, 37.7 (C17, C19), 39.7 (C14), 42 (NCH3), 44.4, 46.2
(C20, C21), 52.9 (C2), 53.1, 53.8 (C16, C22), 56.7 (C15, C23), 62.3 (C5), 63.5
0
0
0
0
0
(C9), 65.5 (C1), 127.1, 128.6 (C5 , C6 , C8 , C9 ), 135, 140, 1 (C4 , C7),
171.7, 172.5 (C8, C18).
Labelling of 9 and 17 (10 and 18)
Compounds 9 or 17 (0.2 mg) were labelled by incubating with stannous
tartrate saturated solution (4 ml), NaOH 0.1 M (4 ml) and 99mTc-
pertechnetate (ꢂ0.8 MBq, 18 ml) for 45 min at 808C. The solutions
were chromatographed on silica gel plates (CH2Cl2:EtOH:NH4OH
22%, 3:2:0.5). The plates were autoradiographed, then a phosphomo-
lybdic acid ethanolic solution (5%) was used to visualize the ligands.
Conclusion
In order to visualize and quantify dopamine transporters by SPECT, we
synthesized two ligands with a tropane skeleton and a macrocyclic
moiety, allowing the complexation of 99mTc. The radiolabelling of these
ligands led to two novel complexes, good candidates for imaging the
dopamine transporters. Biological studies are in progress and the results
will be published in due course.
Acknowledgements
This work was post of the EUREKA-DOPIMAG program: Project
EU1836.
References
1. Ritz MC, Lamb RJ, Goldberg SR, Kuhar MJ. Science 1987; 237: 1219–
1223.
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 379–393

SYNTHESIS OF TWO NOVEL OXOCYCLAM-BINDING
393
2. Koob GF, Bloom FE. Science 1988; 242: 715–723.
3. Hantraye P, Brownell AL, Elmaleh D, et al. NeuroReport 1992; 3: 265–268.
4. Kaufman MJ, Madras BK. Synapse 1991; 9: 43–49.
5. Fowler JS, Volkow ND, Wolf AP, et al. Synapse 1989; 4: 371–377.
.
6. Muller L, Halldin C, Farde L, Karlsson P, Hall H, Swahn CG, Neumeyer
JL, Gao Y, Milius RA. Nucl Med Biol 1993; 20: 249–255.
7. Neumeyer JL, Wang S, Gao Y, et al. J Med Chem 1994; 37: 1558–1561.
8. Neumeyer JL, Wang S, Milius RA, Baldwin RM, Zea-Ponce Y, Hoffer PB,
Sybirska EH, Al-Tikriti MS, Charney DS, Malison RT, Laruelle M, Innis
RB. J Med Chem 1991; 34: 3144–3146.
9. Emond P, Garreau L, Chalon S, Boazi M, Caillet M, Bricard J, Frangin Y,
Mauclaire L, Besnard JC, Guilloteau D. J Med Chem 1997; 40: 1366–1372.
10. Madras BK, Jones AG, Mahmood A, Zimmerman RE, Garada B,
Holman BL, Davison A, Blundell P, Meltzer PC. Synapse 1996; 22:
239–246.
11. Meegalla SK, Plossl K, Kung MP, Stevenson DA, Liable Sands LM,
Rheingold AL, Kung HF. J Am Chem Soc 1995; 117: 11 037–11 038.
12. Meegalla S, Plossl K, Kung MP, Chumpradit S, Stevenson DA, Kushner
SA, McElgin WT, Mozley PD, Kung HF. J Med Chem 1997; 40: 9–17.
!
13. Riche F, Trimcev I, Godard J, Benabed A, Vidal M, Pasqualini R. Appl
Radiat Isot 1993; 44: 907–910.
14. Carroll FI, Lewin AH, Boja JW, Kuhar MJ. J Med Chem 1992; 35:
969–981.
15. Lane CF. Chem Rev 1976; 76: 773–799.
16. Brown HC, Choi YM, Narasimhan S. J Org Chem 1982; 47: 3153–3163.
17. Hay RW, Pujari MS, Perroti A. Trans Met Chem 1985; 10: 396–399.
18. (a) Xu L, Trudell ML. J Heterocyclic Chem 1996; 33: 2037–2039.
(b) Carroll FI, Kotian P, Dehghani A, Gray JL, Kuzemko MA, Parkam
KA, Abraham P, Lewin AH, Boja SW, Kuhar MJ. J Med Chem 1995; 38;
379–388.
!
19. Riche F, Vidal M, Bolzati C, Uccelli L, Marchi A, Duatti A. Inorg Chim
Acta 1995; 231: 147–151.
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 379–393