Tc and Re chelates of 8α-amino-6-methyl-ergoline: Synthesis and affinity to the dopamine D2 receptor

Article abstract of DOI:10.1016/S0968-0896(02)00214-6

The influence of structural changes at the 8α-amino position of 8α-amino-6-methyl-ergoline on the lipophilicity and affinity to the D₂ receptor was studied. 8α-amino-6-methyl-ergoline (1) was converted into the derivatives (2a-f) by mercaptoacetylation of the amino group to make it possible to prepare the rhenium and technetium complexes (3, 4a,b). Binding tests on cloned human dopamine D₂ receptors show that the affinities of the coordination compounds (IC₅₀ values between 50 and 240 nM) are less than those of the derivatives 2a-f (IC₅₀=3-50 nM) but more than those of the parent compound 1. Biodistribution studies of the Tc complexes 4a,b performed on Wistar rats show a slow blood clearance with substantial accumulation and retention in the liver and kidneys and low brain uptake.

Full text of DOI:10.1016/S0968-0896(02)00214-6

Bioorganic & Medicinal Chemistry 10 (2002) 3523–3528
Tc and Re Chelates of 8ꢀ-Amino-6-methyl-ergoline: Synthesis and
Affinity to the Dopamine D2 Receptor
H. Spies,* B. Noll, St. Noll, M. Findeisen, P. Brust, R. Syhre and R. Berger
Forschungszentrum Rossendorf e.V., Institut fur Bioanorganische und Radiopharmazeutische Chemie,
¨
POB 510119, D-01314 Dresden, Germany
Received 16 April 2002; accepted 7 June 2002
Abstract—The influence of structural changes at the 8a-amino position of 8a-amino-6-methyl-ergoline on the lipophilicity and
affinity to the D2 receptor was studied. 8a-amino-6-methyl-ergoline (1) was converted into the derivatives (2a–f) by mercaptoace-
tylation of the amino group to make it possible to prepare the rhenium and technetium complexes (3, 4a,b). Binding tests on cloned
human dopamine D2 receptors show that the affinities of the coordination compounds (IC50 values between 50 and 240 nM) are less
than those of the derivatives 2a–f (IC50=3–50 nM) but more than those of the parent compound 1. Biodistribution studies of the Tc
complexes 4a,b performed on Wistar rats show a slow blood clearance with substantial accumulation and retention in the liver and
kidneys and low brain uptake.
# 2002 Elsevier Science Ltd. All rights reserved.
Introduction
baboons as a potential tool for quantitative in-vivo
imaging of D2 receptors, using positron emission tomo-
graphy.12
The ergot alkaloids,1,2 derivatives of lysergic acid or
ergoline structures exhibit a broad range of pharmaco-
logical acitivities. Many of them are dopamine D2
receptor agonists,3,4 but some ergoline derivatives also
interact with serotonin receptors5ꢀ7 and adrenocep-
tors8,9 with very high affinities. The tetracyclic structure
of the ergolines contains the essential features of the
monoamine neurotransmitters norepinephrine, dopa-
mine and serotonin. This structural relationship may
contribute to the ability of many naturally occuring and
synthetic ergot alkaloids to act as agonists or antago-
nists at receptors for these neurotransmitters.
Many efforts were and are being made to design recep-
tor binding technetium tracers in view of the excellent
nuclide properties and wide availability of the isotope
99mTc. To gain access to such tracers, a receptor-binding
organic structure is combined with a small Tc chelate
unit.13 However, the introduction of a technetium che-
late causes drastic alterations in the molecular and bio-
logical properties of the lead structure having the
required receptor affinity. Studying the influence that
structural modifications required for binding techne-
tium may have on the binding to the receptor in ques-
tion is therefore of basic interest.
This observation stimulated the search for partial
structures and derivatives with the aim to improve
pharmacological selectivity. Numerous modifications
were carried out at the 8-position of ergoline. They
produced, for example, the alpha-ureido derivative ter-
guride10 with a high D2 activity. The high receptor affi-
nities of the ergolines were also examined for the design
of potential receptor-binding radiotracers, for example
123I]2-iodolisuride, which was successfully used as a
SPECT imaging agent in the study of healthy volunteers
and patients.11 [76Br]bromolisuride was evaluated in
The general pharmacological activity observed for many
derivatives favours the ergoline moiety as a subject of
such investigations. As deduced from terguride (Fig. 1)
and a series of other N-substituted derivatives, the free
amino group should be blocked without drastically
alterating the receptor-binding ability. The 8-amino
group of 8a-amino-6-methylergoline was therefore cho-
sen as a suitable position in which to perform structural
modifications. After reporting on the preparation and
molecular structure of the oxorhenium(V) complex,14
we now describe the functionalization of the ergoline
moiety with a 2-mercaptoacyl group and the subsequent
[
*Corresponding author. Tel.:+49-351-260-3678; e-mail: spies@
fz-rossendorf.de
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00214-6

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H. Spies et al. / Bioorg. Med. Chem. 10 (2002) 3523–3528
introduce the required SH-functionality, the parent 8a-
amino-6-methylergoline (1) was converted into the
S-benzoylated derivatives 2a by condensation of the
amino group with benzoylmercaptoacetic acid.
Removal of the protective group by sodium methylate
yielded the free mercaptane 2c, from which the methy-
lated derivative 2e resulted. The derivatives with
R=methyl (2b, 2d, 2f) were obtained, using 2-benzoyl-
mercaptopropionic acid instead of benzoylmercapto-
acetic acid (Fig. 2).
Figure 1. Terguride.
For preparation of the complexes, the benzoyl group
was removed immediately before complexation. The
resulting mercaptanes 2c,d were used directly without
purification. The complexes were prepared in solid form
from the precursor molecules [ReO(SSS)Cl]16 and
[TcO(SSS)Cl].17 These species contain the preformed
oxometal-chelate unit, from which the ergoline-deriva-
tized complexes are obtained as solids after simply
replacing the chlorine by the mercaptide ligand 2c and
2d. The synthesis of the rhenium complex 3 was descri-
bed before.14 The corresponding technetium complexes
4a,b were obtained in mg amounts and their composi-
tion was confirmed by 1H NMR spectroscopy. The
technetium complexes 4a,b were also prepared in solu-
tion by a two-step procedure, starting with the reaction
of the ergolines 2c,d as the monodentate ligand with
Tc(V) gluconate, and subsequent addition of the tri-
dentate thiolate ligand HS–CH2CH2–S–CH2CH2–SH
(HSSSH). The complexes were separated by HPLC
and identified by comparison of the Rt values and
TLC data with those of the isolated 99Tc complexes
(Fig. 3).
synthesis of neutral mixed-ligand oxotechnetium(V) and
oxorhenium(V) complexes containing 8a-amino-6-
methylergoline as a pendant group. Using terguride
as a reference molecule, we investigated the effects
on lipophilicity and affinities to the D2 receptor of
the derivatives that the introduction of the chelate
unit in the parent 8a-amino-6-methylergoline would
produce.
Results and Discussion
Chemistry
For coupling the metal to the ergoline moiety the ‘3+1’
mixed-ligand concept was used.15 In the ‘3+1’
approach, a mercapto group has to be introduced into
the ergoline for binding to the oxometal(V) core, while
the remaining free coordination sites at the metal should
have an additional tridentate chelate ligand, thus form-
ing a neutral MO/monodentate/tridentate complex. To
Figure 2. Reaction scheme for the preparation of derivatives 2a–f of 8a-amino-6-methyl-ergoline.

H. Spies et al. / Bioorg. Med. Chem. 10 (2002) 3523–3528
3525
Figure 3. Reaction scheme of the preparation of Re complex (3) and Tc complexes (4a,b) derived from 1.
Lipophilicity Data
Table 1. Lipophilicity values (PHPLC) and affinity to the dopamine
D2 receptor (IC50 values) of ergoline derivatives
The lipophilicity data PHPLC were determined for the
derivatives 2a–f as well as for the chelate-bearing species
3,4a,b. While the PHPLC value of the dimethylcarbamoyl
derivative, terguride, was comparable to that of
8a-amino-6-methylergoline 1, the derived compounds
(2a–f) showed PHPLC values exceeding those of 1 by
about one order of magnitude. The highest values were
found for compounds containing the S-protecting ben-
zoyl group 2a,b, while the chelate group containing ergo-
lines show relatively moderate lipophilicity. A remarkable
enhancement of the PHPLC values was due to the influence
of the methyl group located at the mercapto acyl bridge.
Compound
PHPLC
IC50 (nM)
Terguride
1
5
4
80
252
n.d.
n.d.
8
15
33
4.8
1730
10.7
2.7
42.9
45.0
22.1
17.2
139
2a
2b
2c
2d
2e
2f
3
4a
4b
45
70
235
54
species 2a–f have moderate to high affinities to cloned
human dopamine D2 receptors (Table 1). Functionali-
zation at the amino group in the 8a-amino-6-methy-
lergoline drastically increases the affinity, resulting in
IC50 values between 3 and 45 nM. The benzoyl deriva-
tive 2b has an even better affinity than the reference
molecule terguride. The introduction of a chelate into
this molecule (complexes 3, 4a,b) results in a decrease of
affinity. The steric requirements of the chelate unit are
obviously not suited to matching the ergoline moiety
and producing highly affine species, although the affi-
nities of this coordination species are higher (IC50 values
between 54 and 240 nM) than those of the parent com-
pound 1.
Affinity to the D2 Receptor
The main point of interest was the question as to how
the introduction of the chelate unit influences the affi-
nity to the dopamine D2 receptor. From the litera-
ture,1,2,18 it is obvious, that bulky substituents at the
8-amino position increase the affinity. While the affinity
of the parent 8a-amino-6-methylergoline is in the mmo-
lar level (Table 1), many bulky group-bearing ergolines
show high affinities.18ꢀ20 For instance, the ureido deri-
vative terguride, which serves as a reference molecule in
our studies, shows an affinity to the D2 receptor at the
nanomolar level, as previously shown.6,20
Our experiments with 8a-amino-6-methylergoline mod-
ified by Tc and Re chelates reveal that the non-chelate
Another remarkable observation is the fact that both the
lipophilicity and the receptor affinity are very sensitive to

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H. Spies et al. / Bioorg. Med. Chem. 10 (2002) 3523–3528
Table 2. Biodistribution (% injected dose/organ) of Tc complexes 4a and 4b in male Wistar rats (means; n=3)
Complex
Min pi
Brain
Lung
Kidney
Liver
Blood
4a
10
60
0.17
0.16
2.5
2.4
7.7
21.2
24.9
24.7
38.7
27.9
4b
10
60
0.13
0.13
1.8
1.210.9 1.9
10.1
2
23.2
25.2
16.2
structural changes at the mercaptoacyl bridge. This can
be clearly seen in the influence of a methyl group on the
pairs 2a,b, 2c,d, 2e,f, and 4a,b.
the derived complexes of rhenium (3) and technetium
(4) are nevertheless higher than those of the parent
compound 1.
In general, as the ergot alkaloids and their derivatives
display a broad spectrum of pharmacological action
and as there is some tolerance in substitution at least at
the 8a-N position, the ergoline moiety is well suited as a
leading structure in the development of receptor-bind-
ing Tc complexes.
Biodistribution Studies
Biodistribution studies of the technetium complexes in
rats (Table 2) show a high blood pool activity and a
slow blood clearance with substantial accumulation and
retention in liver and kidneys. A long plasma half-life
was also described for other ergoline derivatives such as
transdihydrolisuride, bromerguride, terguride and
cabergoline.21ꢀ24 It was observed that the elimination of
terguride occurs mainly in the urine,23 and lisuride is
also eliminated via the kidneys and liver.25 The brain
uptake of the Tc complexes 4a,b is low. The brain
uptake as well as the slow brain clearance of the com-
plexes seem to indicate their capability of crossing the
blood–brain barrier of rats and being trapped in the
brain.
Experimental
General
1
All reagents are of commercial grade. H NMR spectra
were recorded on a Varian Inova-400 spectrophoto-
meter. Elemental analyses were carried out on a LECO
Elemental Analyzer CHNS-932.
Chemical synthesis
8ꢀ-Amino-6-methyl-ergoline (1). 5.15 g (0.015 mol) ter-
guride was suspended in 500 mL of 1 N hydrochloric
acid and refluxed for 20 h. The solvent was removed by
rotary evaporation until a volume of 100 mL. While
stirring sodium hydrogencarbonate was added to a pH
of 8.5 and stirring was continued for 40 min. The pre-
cipitate was separated and dried in the vacuum. This
product was dissolved in 1000 mL of hot methanol and
filtered on a short column filled with silica gel. The
methanol was removed until the product began to crys-
tallize and standing overnight deliver 1. Yield: 2.2 g
(61%); mp: 292 ꢁC, TLC (Silufol//methanol/25%
ammonia: 95:5): Rf 0.4 (UV, ninhydrine). Anal. calcd
for C15H19N3: C, 74.65; H, 7.94; N, 17.41. Found: C,
74.40; H, 7.90; N, 17.21.
Conclusions
The experiments were aimed at studying, at a pharma-
cologically potent class of compounds, the influence of
the structure of ergoline conjugates on their lipophilicity
and their binding to the D2 receptor. The general phar-
macological activity observed for many derivatives
favours the ergoline moiety for such investigations. The
experience gained from studying many derivatives
shows that a room-filling group is a necessary condition
for dopamine D2 affinity. It is therefore reasonable to
bind the chelate to the external amino group. Step-by-
step binding, first of the mercaptoacetyl groups and
then of the Tc/Re chelates to amino ergoline produced
derivatives whose molecular weight and steric require-
ments increased in this order. Compared with references
1 and terguride, the lipophilicity of all derived species
was enhanced by one to two orders of magnitude. The
observations concerning affinity made in our experi-
ments with derivatives (2a–f) were consistent with our
expectations. The affinity (C50 values) was much higher
for 8a-amino-6-methylergolines functionalized with
small substituents than for ergoline with the free amino
group (1) and seemed to increase with increasing size of
the derivatizing group. Surprisingly, one derivative (2b)
showed a higher affinity than the reference molecule
terguride.
N-(2-(Benzoylmercapto)acetyl-8-ꢀ-amino-6-methylergo-
line (2a). Prepared by reaction of a mixture of 98 mg
(0.5 mmol) S-benzoylmercaptoacetic acid, 165 mg (0.5
mmol) TBTU and 170 mL (1 mmol) DIPEA in 1 mL
N-methylpyrrolidone with a solution of 96 mg (0.4
mmol) 8a-amino-6-methyl-ergoline (1) in
1
mL
N-methylpyrolidone, stirring for 4 h, and purification of
the reaction product by MPLC (RP8, eluent 0.1%
TFA/acetonitrile). Yield: 33.5 mg (20%); mp: 175 ꢁC.
1H NMR (400 MHz, d DMSO-d6=2.51): d 1.87–1.90
(2H, CH2), 2.70–2.73 (2H, 2ꢂCH), 2.99–3.18 (3H, N–
CH3), 3.57–3.60 (2H, CH2), 3.88–4.02(4H, 2 ꢂCH2),
4.27 (1H, CH), 6.88–6.90 (1H, CH), 7.09–7.20 (2H,
2ꢂCH), 7.23–7.25 (1H, CH), 7.56–7.94 (5H, CHarom),
8.67–8.68 (1H, NHchain), 10.93 (1H, NHring). Anal.
The steric tolerance of the ergoline molecule seems,
however, to be overtaxed by the chelate, since its intro-
duction results in a loss of activity. The binding data of

H. Spies et al. / Bioorg. Med. Chem. 10 (2002) 3523–3528
3527
calcd for C24H25N3O2S: C, 68.71; H, 6.01; N, 10.02, S,
7.64. Found: C, 68.57; H, 5.95; N, 9.94; S, 7.61.
N-CH3), 3.11–3.14 (2H, CH2), 3.22–3.26 (2H, CH2),
3.60–3.71 (2H, CH2), 4.30 (1H, CH–N), 6.86–6.88 (1H,
CHring), 7.08–7.12(H2, 2
ꢂCHring), 7.22–7.24 (1H,
N-(2(Benzoylmercapto)propionyl-8-ꢀ-amino-6-methyler-
golin (2b). A mixture of 105 mg (0.5 mmol) S-benzoyl-
mercapto propionic acid, 165 mg (0.5 mmol) TBTU and
170 mL (1 mmol) DIPEA in 1 mL N-methylpyrrolidone
is stirred for 3 min and dropped into a solution of 96 mg
(0.4 mmol) 8a-amino-6-methyl-ergoline (1) in 1 mL
N-methylpyrrolidone. After stirring for another 4 h, the
reaction product is purified by MPLC (RP8, eluent
0.1% TFA/acetonitrile). Evaporation of the solvent
yields a light brown solid of 2b. Yield: 43.3 mg (25%);
mp: 123 ꢁC. 1H NMR: d 1.54–1.58 (3H, CH3), 1.84–1.90
(2H, CH2), 2.67–2.74 (1H, CH), 2.88–2.94 (1H, CH),
3.00 (3H, N–CH3), 3.88–4.02(4H, 2 ꢂCH2), 4.28 (1H,
CH), 4.41–4.46 (1H, CH), 6.87–6.89 (1H, CH), 7.07–
7.13 (2H, 2ꢂCH), 7.23–7.25 (1H, CH), 7.55–7.93 (5H,
CHarom), 8.65 (1H, NHchain), 10.91 (1H, NHring). Anal.
calcd for C25H27N3O2S: C, 69.26; H, 6.28; N, 9.69; S,
7.39: Found: C, 68.99; H, 6.14; N, 9.43; S, 7.27.
CHring), 8.39–8.40 (1H, NHchain), 10.92(1H, NH ring).
Anal. calcd for C18H23N3OS: C, 65.62; H, 7.04; N,
12.75; S, 9.73; Found C, 65.43; H, 7.02; N, 12.61; S,
9.68.
N-(2-Methylmercapto)propionyl-8-ꢀ-amino-6-methyler-
goline (2f). As described for 2e, starting from 2b. Yield:
23.5 mg (23%); mp: 215 ꢁC, C19H25N3OS. 1NMR: 1.37–
1.39 (3H, CH3), 1.84–1.87 (2H, CH2), 2.09–9.10 (3H, S–
CH3), 2.66–2.69 (2H, 2ꢂCH), 3.02(3H, N–CH ), 3.57–
3
3.65 (4H, 2CH2), 4.28 (1H, N–CH), 4.37 (1H, CH), 6.84–
6.89 (1H, CHring), 7.07–7.17 (2H, 2ꢂCHring), 7.17–7.24
(1H, CHring), 8.31 (1H, NHchain), 10.91 (1H, NHring).
Rhenium complex 3. This is reported in ref 14.
Chloro(3 - thiapentane - 1.5 - dithiolato)oxotechnetium(V).
This was prepared by the reaction of the tridentate ligand
HS–CH2CH2—S–CH2CH2–SH and teraphenylarsonium
tetrachlorotechnetate(V) AsPh4[TcOCl4] in acetonitrile as
described in ref 17.
N-(2-Mercapto)acetyl-8-ꢀ-amino-6-methylergoline (2c).
Sodium methylate (1 mL, 0.15 M,) was added to a stir-
red solution of 2a (0.1 mmol) in 1 mL methanol. The
mixture is neutralized by methanolic HCl after 1 h.
After filtration and evaporation a white powder was
obtained. Purification was arried out by MPLC (RP8,
eluent 0.1% TFA/acetonitrile). Yield: 9.5 mg (30%);
Tc complex 4a. The methanolic reaction mixture of the
preparation of 2c is given to a solution of 30.3 mg, (0.1
mmol) [TcO(SSS)Cl] in 5 mL acetonitrile. The stirred
mixture is kept under nitrogen. After continued stirring
for 2h and standing overnight, the precipitating sodium
chloride was filtered off and the solvent removed by a
nitrogen stream. The residue was dissolved in DMS and
purified by column chromatography [silica gel 60
(Merck) eluted with methanol (95%)–NH3(conc)(5%)].
The brown fraction delivers after reducing the volume at
0.5 mL a brown solid substance. Yield: 12.8 mg (22%).
mp: >200 ꢁC; TLC (silica gel/95% methanol) Rf: 0.52.
UV–vis: lmax; 280 nm. 1NMR: 1.40 (1H, C–H), 1.52(1H,
CH), 1.68 (1H, CH), 1.93 (1H, CH), 2.29 (3H, N–CH3),
2.32 (1H, CH), 2.44 (1H, CH), 2.75 (m, 2H, CO–CH2–S),
3.13–3.26 (3H, CH), 3.50–4.08 (6H, CH), 4.29 (2H, CH),
6.61 (1H, CHarom), 6.95–7.10 (3H, CHarom and olefin), 7.75
(1H, NH chain), 10.63 (s, 1H, NHring).
mp: 152 ꢁC. H NMR: d 1.87 (t, 1H, J=12.5 Hz, CH2),
1
2.69 (d, 1H, J=13.5 Hz, CH2), 2.79 (t, 1H, J=7.7 Hz,
SH), 2.88–2.94 (m, 1H, CH2), 2.98 (s, 3H, N–CH3),
3.16–3.30 (m, 2H, CO–CH2–S), 3.39–3.59 (m,
6H+H2O, CH– and CH2), 3.73 (t, 1H, J=11.6 Hz,
CH– or CH2), 4.24 (s, 1H, >CH–N), 6.88 (d, 1H,
J=6.8 Hz, arom), 7.08–7.12(m, 2H, arom and olefin),
7.23 (d, 1H, J=8.2Hz arom), 8.39 (s, 1H, NH–chain),
10.91 (s, 1H, NH–ring). Anal. calcd for C17H21N3OS:
C, 64.73; H, 6.71; N, 13.32; S, 10.16; Found C, 64.51; H,
6.42; N, 13.18; S, 10.25.
N-(2-Mercapto)propionyl-8-ꢀ-amino-6-methylergoline
(2d). As described for 2c, starting from 2b. Yield: 15.5
mg (47%); mp: 137 ꢁC. NMR d 1.44 (1H, SH), 1.47
1
(3H, CH3), 1.87–1.91 (1H, CH2), 2.69 (1H, CH2), 2.70
(2H, 2ꢂCH), 2.96–2.97 (3H, N–CH3), 3.00 (2H, CH2),
3.50 (1H, CH–CH3), 3.65–3.69 (2H, CH2), 4.22 (1H,
CH–N), 6.86–6.90 (1H, CHring), 7.08–7.13 (2H,
2ꢂCHring), 7.22–7.24 (1H, CHring), 8.31 (1H, NHchain),
10.91 (1H, NHring). Anal. calcd for C18H23N3OS: C,
65.62; H, 7.04; N, 12.75; S, 9.73; Found C, 65.46, H,
6.99, N, 12.66: S. 9.71.
Tc complex 4b. This was prepared in the same manner
using 2d instead of 2c. Yield: 9.5 mg (16%).
Mp:>200 ꢁC; TLC Rf: 0.58. UV–vis: lmax; 280 nm.
99m/99Tc-complexes 4a,b in solution
Tc gluconate was prepared by gradual addition of 100
mL 0.01 M stannous chloride solution in 0.1 M HCl to
an aqueous solution of 100 mL 0.01 M KTcO4 and 0.5
mL 99mTc generator eluate in 1 mL 0.1 M sodium glu-
conate. 4 mmol of the S-benzoylic protected mercap-
tanes 2a resp. 2b were dissolved in 200 mL MeOH and
saponified by adding 50 mL 1.1 M sodium methylate.
After 20 min, the alkaline solution was given to Tc glu-
conate. After another 20 min, 2 mmol of the tridentate
dithiolate ligand HSSSH, dissolved in 100 mL acetone
was added. The solution was neutralized by 1 M HCl
and applied to HPLC-separation [RP18 column,4ꢂ250
mm, 0.01 M phosphate buffer pH 5.8 (A) and acetonitrile
N-(2-Methylmercapto)acetyl-8-ꢀ-amino-6-methylergoline
(2e). A mixture of 2a (0.3 mmol), 5% Pd/CaCO3 (0.7
mg) and sodium methylate (0.42mmol) in 5 mL
methanol was stirred under hydrogen atmosphere at 67
kPa for 90 min. The filtered mixture was refluxed with
methyl iodide (0.3 mmol) for 1 h. The volume of the
solution was reduced to about 1 mL by evaporation and
the product was purified by MPLC. Yield: 36.5 mg
ꢁ
1
(37%); mp: 163 C; NMR: 1.83–1.89 (2H, CH2), 2.15
(3H, S–CH3), 2.66–2.69 (2H, 2ꢂCH), 2.95–2.98 (3H,

3528
H. Spies et al. / Bioorg. Med. Chem. 10 (2002) 3523–3528
(B). 3 min 95% A/5% B; 15 min from 75% A/25% B to
10% A/90% B; 5 min 10% A/90% B; Flow rate: 1 mL/
min]. 4a: Rt 21,5 min, 4b: Rt 22.5 min. TLC [silica gel
(Merck Kieselgel60), 95% ethanol]. 4a: Rf 0.52, 4b: Rf 0.58.
Acknowledgements
This work was supported by the Deutsche For-
schungsgemeinschaft and the Fonds der Chemischen
Industrie. The authors thank Miss B. Große and Mr. A.
Rother for technical assistance.
The eluted fractions (about 40% each) were separated,
the solvent removed by evaporation in a nitrogen
stream and the residue dissolved in propylene glycol/
water (radioactive concentration about 5–10 MBq per
mL) for biodistribution studies.
References and Notes
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¨
of Ergot Alkaloids and Related Compounds as a Basis of
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Pharmacology, Vol 49: Ergot Alkaloids and Related Com-
pounds; Berde, B., Schild, H. O., Eds.; Springer: Berlin,
Heidelberg, New York, 1978; p 1.
2. Mantegani, S.; Brambilla, E.; Varasi, M. Farmaco 1999, 54,
288.
3. Rabey, J. M.; Nissipeanu, P.; Inzelberg, R.; Korczyn, A. D.
Clin. Neuropharmacol. 1994, 17, 286.
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Determination of lipophilicity data
Lipophilicity data lgPHPLC of all species were deter-
mined by reversed-phase HPLC using a Hamilton PRP-
1 column (250ꢂ4.1 mm, 10 mm) and the Perkin–Elmer
HPLC Controller System Model 1022 equipped with a
UV–vis spectrometer detector at 254 nm. An isocratic
eluent [acetonitrile and 0.01 M phosphate buffer (ph 7.4)
3:1,v/v] was applied with a flow rate of 1.5 mL/min. The
pH value of the eluent was measured by a glass elec-
trode, which was calibrated with standard buffer daily.
PHPLC values were derived from the retention times
according to the equation log P=a log k0+b, where
k0=(tR–t0)/t0 is the capacity factor, tR is the retention
time of the sample and t0 the retention time of methanol
as an unretained solute. Aniline (0.9), benzene (2.13)
and bromobenzene (2.99) served as log P references.
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Blood Flow Metab. 1991, 11, 914.
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347.
Radioligand binding assay
The IC50 values of all species were determined for their
ability in D2receptor binding. [ 3H]Spiperone (from
NEN) was used as the radioligand. The binding assay was
carried out in a final volume of 5 mL tris HCl buffer, pH
7.4, containing 10 nM MgCl2, 1 nM EDTA, 0.27 nM
[3H]Spiperone, 1 unit of cloned D2 receptor and various
concentrations of the ergoline derivatives, dissolved in a
small volume of DMSO. The samples were incubated in
triplicates at 20ꢁC for 60 min. The incubation was termi-
nated by rapid filtration through GF/B glass fiber filters
(Whatman) using a 30-port Brandel Cell Harvester. The
filters were rapidly washed with four 4-mL portions of ice-
cold buffer, transferred into 10 mL scintillation fluid
(Ultima-Gold, Packard) and analysed for radioactivity. In
case of 99Tc complexes interference by 99Tc radioactivity
in 3H counting was eliminated by appropriate adjustment
of the energy channel, except at the highest 99Tc con-
centration used, which required correction.
Biodistribution studies
Animal experiments were carried out according to the
relevant national regulations. The studies were per-
formed on male Wistar rats (5–6 weeks old). 0.5 mL of
the complex solution were injected in the tail vein and
the animals were sacrified by heart puncture (2–60 min
pi). The selected organs were isolated for weighing and
counting and the percentage of injected dose per organ
was calculated. The blood pool activity was calculated
from the respective blood concentration under the
assumption that the total blood volume represents 6%
of the body weight of rats.