TROTEC-1: A new high-affinity ligand for labeling of the dopamine transporter

Full text of DOI:10.1021/jm981008a

© Copyright 1998 by the American Chemical Society
Volume 41, Number 23
November 5, 1998
Communications to the Editor
radentate N2S2 chelating unit. This chelating unit
forms a very stable complex with the oxometal(V) core.
TROTEC-1: A New High -Affin ity Liga n d
for La belin g of th e Dop a m in e
Tr a n sp or ter
Alexander Hoepping,*,† Martina Reisgys,†
Peter Brust,† Sepp Seifert,† Hartmut Spies,†
Roger Alberto,‡ and Bernd J ohannsen†
Institut fu¨r Bioanorganische und Radiopharmazeutische
Chemie, Forschungszentrum Rossendorf, POB 51 01 19,
D-01314 Dresden, Germany, and Abteilung fu¨r
Radiopharmazie, Paul Scherrer Institut,
CH-5232 Villigen, Switzerland
Received J uly 27, 1998
Parkinson’s disease is characterized by a significant
loss of dopaminergic neurons in the basal ganglia. A
reduction in the dopamine transporter (DAT) located at
the presynaptic nerve terminals in the striatum (ST)
can be measured by selective binding of DAT ligands
containing a radioactive label. A decreased specific
striatal uptake of such a radiolabeled DAT ligand may
therefore indicate early pathological states and allow a
diagnosis.1 Due to its favorable properties, technetium-
99m is the preferred nuclide for routine diagnosis by
means of single photon computed tomography (SPECT)
in nuclear medicine. Considerable efforts have been
undertaken toward its incorporation into a variety of
central nervous system (CNS) receptor ligands.2,3
The first technetium complexes derived from a 2â-
carbomethoxy-3â-(halophenyl)tropane useful for in vivo
imaging of dopaminergic neuronal function were re-
cently reported.4,5 Technepine was the first reported
technetium tracer crossing the blood-brain barrier and
accumulating selectively in the striatum.4 Also [99mTc]-
Trodat with the chelate moiety in the 2-position of the
tropane ring shows high affinity to the dopamine
transporter in vivo.5 Both compounds contain a tet-
One of the disadvantages of the tetradentate N2S2
ligands is the occurrence of additional stereoisomers due
to the complexation reaction. These stereoisomers can
exhibit different binding affinities and therefore require
separation by chromatographic methods, complicating
their clinical use. Another problem is the low brain
uptake of the complexes known so far, due in part to
the polar coordination sphere6 and its large size. To
enable metal complexes to cross the blood-brain barrier
by nonspecific transport, the complexes should be non-
polar and lipophilic.
To improve the brain uptake, we are investigating
new ligand/metal systems which are nonpolar, neutral,
and small in size. One of the most promising ap-
proaches seemed to be the use of a dithioether/metal
carbonyl(I) unit: [TcClS2(CO)3]. This unit is available
by reduction of [TcO4]- under a low-pressure carbon
monoxide atmosphere.7 In solution the (NEt4)2[TcCl3-
(CO)3] obtained undergoes a rapid exchange of two of
the chloro ligands by the solvent. At this point, the
solvent in the coordination sphere can be exchanged
with a dithioether chelate. The remaining chlorine
atom is necessary for the formation of neutral octahe-
dral complexes of the common formula [TcCl“S2”(CO)3]
(“S2” ) RS(CH2)2SR′).8 While this system does not
overcome the stereoisomer formation upon complex-
* Address correspondence to this author at Forschungszentrum
Rossendorf. Phone: +49 351 2603694. Fax: +49 351 2603232. E-
mail: hoepping@fz-rossendorf.de.
† Forschungszentrum Rossendorf.
‡ Paul Scherrer Institut.
10.1021/jm981008a CCC: $15.00 © 1998 American Chemical Society
Published on Web 10/20/1998

4430 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 23
Communications to the Editor
Sch em e 1a
tion by flash chromatography, 2â-(hydroxymethyl)-3â-
(4-fluorophenyl)tropane (2) was obtained in a yield of
85-95%. Esterification of 2 with 4,7-dithiaoctanoic acid
using N′-(3-dimethylaminopropyl)-N-ethylcarbodiimide
(EDCI) and 4-(N,N-dimethylamino)pyridine (DMAP)
was performed in methylene chloride at room temper-
ature. The desired product 3 was obtained in excellent
1
yield. In the H NMR spectrum of 3, the chemical shift
of the N-methyl group is consistent with 2â,3â-stereo-
chemistry.11
The synthesis of the rhenium complex was performed
by reaction of 3 with (NEt4)2[ReBr3(CO)3] in methanol.
After workup, the complex 4 was obtained in 83% yield.
Elemental analysis, mass spectra, and IR spectra
clearly confirm the proposed structure. The tricarbon-
ylrhenium core gives rise to the strong vibration bands
at 2032, 1940, and 1904 cm-1. An additional CdO band
at 1728 cm-1 is due to the ester group. The mass
spectrum (FAB+) shows the molecular peak at m/z 762.
Three routes of fragmentation are observed. First, there
is a loss of all three carbonyl ligands and the methyl
group (m/z 663). The second route is the cleavage of
the bond between the aromatic unit and the tropane
ring followed by loss of one carbonyl ligand (m/z 531,
503). Last, the sulfur-carbon bond connecting the
chelate unit with the tropane ester unit is cleaved (m/z
457). The fragment m/z 443 can be assigned to a
following loss of the S-methyl group.
a
Reagents: (a) DIBAL-H, toluene, 0 °C, 85-95%; (b) EDCI,
DMAP, CH2Cl2, 25 °C, quant; (c) (NEt4)2[MX3(CO)3] (M ) Re, X
) Br; M ) 99mTc or 99Tc, X ) Cl), MeOH, 25 °C, 83%.
The 99mTc complex (TROTEC-1) was synthesized by
a modification of the literature procedure7 and purified
by HPLC (tR ) 6.2 min).
In Vitr o Bin d in g Stu d ies. The binding affinities
of the compounds 3 and 4 for the DAT, NET, and 5-HTT
were determined, using competitive binding assays.15,16
ation, it is nevertheless promising because of its small
size and high lipophilicity. While it has recently been
shown that different stereoisomers of SPECT ligands
for the DAT indeed displayed different properties,9 it is
also worth mentioning that in this special case the
mixture of the diastereomers gave the same image in a
baboon brain as the better one of the separated diaster-
eomers. For different receptors, a different behavior
cannot be ruled out, but for the first we decided to place
back the problem of diastereoisomerism.
Recent structure-activity relationship (SAR) studies
revealed the possibility of accommodating bulky sub-
stituents in the 2â-position of the tropane ring.10
Introduction of the nonbasic thioether carbonyl moiety
into this position of â-CFT should afford a SPECT ligand
exhibiting high affinity at the DAT with improved
biodistribution properties. Originally, the existence of
one or two hydrogen-bonding sites for the 2â-substituent
had been postulated.11 More recently, it has been shown
that the DAT tolerates a large variety of 2â-substitu-
ents, including weakly polar or nonpolar12 and also
rather polar substituents.13 By connecting the dithio-
ether via an ester group, one potential H-bonding
interaction was retained, and the two sulfur atoms could
potentially increase the affinity by additional hydro-
phobic interactions.
Resu lts a n d Discu ssion . Starting from â-CFT, a
new chelating ligand (3) was prepared by introduction
of a dithioether unit into the 2â-position. Binding data
to cloned human DAT revealed a high binding affinity.
For both compounds 3 and 4 a significantly improved
fit in the estimation of the IC50 values was obtained, if
the existence of both a low- and a high-affinity binding
site was postulated. Interestingly, the rhenium complex
4 binds as strongly to the high-affinity site as the metal-
free precursor 3 but, on the other hand, exhibits
decreased binding to the low-affinity site. The calcu-
lated affinity with assumption of a single binding site
is better for 4 (IC50 ) 1.5 ( 0.4 nM) than it is for 3 (IC50
) 3.2 ( 0.3 nM). High- and low-affinity binding of
â-CFT has previously been described on membranes of
rat15 and monkey18 striatum, as well as for the cloned
human19 DAT. Our IC50 values obtained on cloned
human DAT expressed in CHO cells are about 4-fold
lower than those found by Pristupa et al.19 using COS-7
cells. Cell type differences may explain this discrepan-
cy, but differences in the assay procedures cannot be
excluded as a possible source. Thus, Gracz and Madras
found 10-fold differences in the high- and low-affinity
binding between fresh or frozen and soluble striatal
membrane preparations of monkeys.18
In the present study, we report the synthesis of the
DAT ligand 2â-[(4,7-dithiaoctanoyloxy)methyl]-3â-(4-
fluorophenyl)tropane (3) as well as its complexation to
the tricarbonylmetal(I) centers of Re and 99mTc (TRO-
TEC-1). We also report in vitro competition binding
studies at the DAT, serotonin transporter (5-HTT), and
norepinephrine transporter (NET).
Ch em istr y. The synthesis was carried out according
to Scheme 1. â-CFT obtained by common methods14
was reduced with DIBAL-H in toluene.12 After purifica-
It is known that â-CFT also exhibits a high selectivity
over the 5-HTT and the NET (Table 1). Introduction
of our chelate moiety increases the affinity toward the
5-HTT and NET and consequently decreases the selec-
tivity.

Communications to the Editor
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 23 4431
(4) Madras, B. K.; J ones, A. G.; Mahmood, A.; Zimmerman, R. E.;
Garada, B.; Holman, B. L.; Davison, A.; Blundell, P.; Meltzer,
P. C. Technepine: a high-affinity 99mTechnetium probe to label
the dopamine transporter in brain by SPECT imaging. Synapse
(N. Y.) 1996, 22, 239-246.
(5) (a) Kung, M. P.; Stevenson, D. A.; Plo¨ssl, K.; Meegalla, S. K.;
Beckwith, A.; Essman, W. D.; Mu, M.; Lucki, I.; Kung, H. F.
[Tc-99m]TRODAT-1: A novel technetium-99m complex as a
dopamine transporter imaging agent. Eur. J . Nucl. Med. 1996,
24, 372-380. (b) Dresel, S. H. J .; Kung, M. P.; Plo¨ssl, K.;
Meegalla, S. K.; Kung, H. F. Pharmacological effectes of dopa-
minergic drugs on in vivo binding of [99mTc]TRODAT-1 to the
central dopamine transporters in rats. Eur. J . Nucl. Med. 1998,
25, 31-39.
Ta ble 1. Binding Data (IC50 Values) of â-Cft, 3, and 4 to the
Monoamine Transportersa
DAT high
affinity (nM) affinity (nM)
DAT low
NET
(nM)
5-HTT
(nM)
â-CFT 2.62 ( 1.06
139 ( 72
5.69 ( 1.20
20.3 ( 16.1
834 ( 4517 759 ( 4717
3
4
0.227 ( 0.202
0.146 ( 0.042
16.3 ( 0.6
7.3 ( 1.1
28.5 ( 4.6
71.8 ( 1.4
a
Data are means ( SEM (n ) 3-4).
The most important result is the markedly enhanced
affinity of the tricarbonylrhenium(I) complex 4 in
comparison with â-CFT. Although other metal com-
plexes exhibit a very high affinity to the DAT, for
(6) Meegalla, S. K.; Plo¨ssl, K.; Kung, M. P.; Chumpradit, S.;
Stevenson, D. A.; Kushner, S. A.; McElgin, W. T.; Mozley, P. D.;
Kung, H. F. Synthesis and characterization of technetium-99m-
labeled tropanes as dopamine transporter-imaging agents. J .
Med. Chem. 1997, 40, 9-17.
example, a “3 + 1” complex derived from IPT,20
a
cyclopentadienyl carbonyl complex derived from â-CIT,21
and technepine again derived from â-CFT,4 the affinity
was more or less retained in the same order of magni-
tude compared to the starting ligand. With complex 4,
we achieved an affinity of about 1 order of magnitude
higher than that of â-CFT. This can only be explained
by participation of the complex unit in the binding to
the transporter in the sense that the assumed hydrogen
bonding between the C-2 ester group and the DAT is
complemented by an additional lipophilic interaction
exerted by the sulfur atoms of the dithioether. This
lipophilic interaction can slightly be enhanced by com-
plexation. This interpretation is consistent with previ-
ously reported results from Kozikowski et al. regarding
a 2-cocaine analogue bearing a 2-vinyl substituent22 and
of Carroll et al. regarding cocaine analogues with
heterocyclic substituents in position 2.23 Both reports
suggest a weak hydrophobic contribution to binding at
the DAT.
(7) European Patent Application 972012322.
(8) Reisgys, M.; Wu¨st, F.; Alberto, R.; Schibli, R.; Schubiger, P. A.;
Pietzsch, H. J .; Spies, H.; J ohannsen, B. Synthesis of rhenium-
(I) and technetium(I) carbonyl/dithioether ligand complexes
bearing 3,17â-estradiol. Bioorg. Med. Chem. Lett. 1997, 7, 2243-
2246.
(9) Meegalla, S. K.; Plo¨ssl, K.; Kung, M. P.; Stevenson, D. A.; Mu,
M.; Kushner, S. A.; Liable-Sands, L. M.; Rheingold, A. L.; Kung,
H. F. Specifity of diastereomers of [99mTc]TRODAT-1 as dopa-
mine transporter imaging agents. J . Med. Chem. 1998, 41, 428-
436.
(10) Meltzer, P. C.; Blundell, P.; Madras, B. K. Structure activity
relationships of inhibition of the dopamine transporter by
3-arylbicyclo[3.2.1]octanes. Med. Chem. Res. 1998, 8, 12-34.
(11) Lewin, A. H.; Gao, Y.; Abraham, P.; Boja, J . W.; Kuhar, M. J .;
Carroll, F. I. 2â-Substituted analogues of cocaine. Synthesis and
inhibition of binding to the cocaine receptor. J . Med. Chem. 1992,
35, 135-140.
(12) Kozikowski, A. P.; Saiah, M. K. E.; J ohnson, K. M.; Bergmann,
J . S. Chemistry and Biology of the 2â-Alkyl-3â-phenyl Analogues
of Cocaine: Subnanomolar Affinity Ligands that Suggest a New
Pharmacophore Model at the C-2 Position. J . Med. Chem. 1995,
38, 3086-3093.
(13) Carroll, F. I.; Kotian, P.; Gray, J . L.; Abraham, P.; Kuzemko,
M. A.; Lewin, A. H.; Boja, J . W.; Kuhar, M. J . 3â-(4′-chlorophen-
yl)tropan-2â-carboxamides and cocaine amide analogues: new
high affinity and selective compounds for the dopamine trans-
porter. Med. Chem. Res. 1993, 7, 468-472.
(14) Xu, L. F.; Trudell, M. L. Stereoselective synthesis of 2â-
carbomethoxy-3â-phenyltropane derivatives. Enhanced stereo-
selectivity observed for the conjugate addition reaction of
phenylmagnesium bromide derivatives with anhydrous dichlo-
romethane. J . Heterocycl. Chem. 1996, 33, 2037-2039.
(15) Madras, B. K.; Spealman, R. D.; Fahey, M. A.; Neumeyer, J . L.;
Saha, J . K.; Milius, R. A. Cocaine receptors labeled by [3H]2â-
carbomethoxy-3â-(4-fluorophenyl)tropane. Mol. Pharmacol. 1989,
36, 518-524.
(16) Reith, M. E. A.; Xu, C.; Coffey, L. L. Binding domains for blockers
and substrates on the cloned human dopamine transporter
studied by protection against n-ethylmaleimide-induced reduc-
tion of [3H]2â-carbomethoxy-3â-(4-fluorophenyl) tropane ([3H]-
WIN35,428) binding. Biochem. Pharmacol. 1996, 52, 1435-1446.
(17) Boja, J . W.; Kuhar, M. J .; Kopajtic, T.; Yang, E.; Abraham, P.;
Lewin, A. H.; Carroll, F. I. Secondary amine analogues of 3â-
(4′-substituted phenyl)tropane-2â-carboxylic acid esters and
N-norcocaine exhibit enhanced affinity for serotonin and nore-
pinephrine transporters. J . Med. Chem. 1994, 37, 1220-1223.
(18) Gracz, L. M.; Madras, B. K. [3H]WIN 35,428 ([3H]CFT) binds to
multiple charge-states of the solubilized dopamine transporter
in primate striatum. J . Pharmacol. Exp. Ther. 1995, 273, 1224-
1234.
Con clu sion s. TROTEC-1 (4) represents the first
example of a dithioether/carbonyl complex with excep-
tional high affinity to the DAT. We have thus demon-
strated that the Tc-complexed chelator moiety, rather
than necessarily representing an obstacle to binding as
a consequence of its steric bulk, may in favorable cases
even improve binding through additional interactions
if its position within the ligand molecule is chosen
judiciously.
Additional studies focused on the biodistribution
behavior of the 99mTc complex and the question of which
structural features influence the pharmacological activ-
ity of further dithioether/carbonyl complexes are cur-
rently in progress.
Su p p or tin g In for m a tion Ava ila ble: Experimental de-
scriptions of the chemistry and radioligand binding assays (4
pages). Ordering information is given on any current mast-
head page.
Refer en ces
(1) Tedroff, J .; Aquilonius, S. M.; Hartvig, P. G.; Langstrom, B.
Functional positron emission tomographic studies of striatal
dopaminergic activity - changes induced by drugs and nigro-
striatal degeneration. In Parkinson’s Disease; Battistin, L.,
Scarlato, G., Caraceni, T., Ruggieri, S., Eds.; Lippincott-Raven
Publ.: New York, 1996; Vol. 69, pp 443-448.
(2) J ohannsen, B.; Scheunemann, M.; Spies, H.; Brust, P.; Wober,
J .; Syhre, R.; Pietzsch, H. J . Technetium(V) and rhenium(V)
complexes for 5-HT2A serotonin receptor binding: Structure-
affinity considerations. Nucl. Med. Biol. 1996, 23, 429-438.
(3) Samnick, S.; Brandau, W.; Sciuk, J .; Steinstra¨âer, A.; Schober,
O. Synthesis, Characterization and Biodistribution of Neutral
and Lipid-soluble 99mTc-Bisaminoethanethiol Spiperone. Nucl.
Med. Biol. 1995, 22, 573-583.
(19) Pristupa, Z. B.; Wilson, J . M.; Hoffman, B. J .; Kish, S. J .; Niznik,
H. B. Pharmacological heterogeneity of the cloned and native
human dopamine transporter: disassociation of [3H]WIN 35,-
428 and [3H]GBR 12,935 binding. Mol. Pharmacol. 1994, 45,
125-135.
(20) Meegalla, S.; Plo¨ssl, K.; Kung, M.-P.; Stevenson, D. A.; Liable-
Sands, L. M.; Rheingold, A. L.; Kung, H. F. First Example of a
99mTc Complex as a Dopamine Transporter Imaging Agent. J .
Am. Chem. Soc. 1995, 117, 11037-11038.
(21) Zoghbi, S. S.; Tamagnan, R. M.; Baldwin, R. M.; Gao, Y.;
Neumeyer, J . L.; Baldessarini, R.; Charney, D. S.; Seibyl, J . S.;
Innis, R. B. Synthesis of a dopamine transporter binding
cyclopentadiene phenyltropane conjugate complexed with rhe-

4432 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 23
Communications to the Editor
nium and Tc-99m. Abstracts of the 44th Annual Meeting of the
Society of Nuclear Medicine, J une 1-5, 1997; No. 368.
(22) Kozikowski, A. P.; Roberti, M.; Xiang, L.; Bergmann, J . S.;
Callahan, P. M.; Cunningham, K. A.; J ohnson, K. M. Structure-
activity relationship studies of cocaine: replacement of the C-2
ester group by vinyl argues against hydrogen-bonding and
provides an esterase-resistant, high-affinity cocaine analogue.
J . Med. Chem. 1992, 35, 4764-4766.
(23) Kotian, P.; Mascarella, S. W.; Abraham, P.; Lewin, A. H.; Boja,
J . W.; Kuhar, M. J .; Carroll, F. I. Synthesis, Ligand Binding,
and Quantitative Structure-Activity Relationship Study of
3â-(4′-Substituted phenyl)-2â-heterocyclic Tropanes: Evidence
for an Electrostatic Interaction at the 2â-Position. J . Med. Chem.
1996, 39, 2753-2763.
J M981008A