Novel ergopeptides as dual ligands for adenosine and dopamine receptors

Article abstract of DOI:10.1021/jm060947x

Multivalent ligands are promising pharmacological tools that may be more efficacious for several diseases than highly selective single-target drugs. A combined therapy using dopaminergic agonists and adenosinergic antagonists is currently being evaluated for the treatment of Parkinson's disease. [(a) Kanda, T.; et al. Exp. Neurol. 2000, 162, 321-327. (b) Jenner, P. Expert Opin. Invest. Drugs 2005, 14, 729-738. (c) Kase, H.; et al. Neurology 2003, 61 (Suppl 6), S97-S100.] Here we prepared dual ligands acting on adenosine and dopamine receptors by applying a combinatorial approach based on the ergolene privileged structure. The potency and efficacy of these novel compounds were determined by radioligand binding studies and intracellular cAMP production assays in cells expressing adenosine and dopamine receptors. Selected compounds displayed dual dopamine agonist and adenosine antagonist activity. Molecules with this pharmacological profile are potentially useful for studying dopamine-adenosine cross-talk in the central nervous system and for testing the therapeutic potential of multivalent drugs for Parkinson's disease.

Full text of DOI:10.1021/jm060947x

3
062
J. Med. Chem. 2007, 50, 3062-3069
Novel Ergopeptides as Dual Ligands for Adenosine and Dopamine Receptors
Marc Vendrell, Ester Angulo, Vicent Casad o´ , Carme Lluis, Rafael Franco, Fernando Albericio,* and Miriam Royo*,†
†
‡
‡
‡
‡
,§,||
Combinatorial Chemistry Unit, Barcelona Science Park, Department of Biochemistry and Molecular Biology, Molecular Neurobiology Unit,
IDIBAPS, Institut d’InVestigacions Biom e` diques August Pi i Sunyer, IRB Barcelona, Barcelona Science Park, and Department of Organic
Chemistry, UniVersity of Barcelona, 08028 Barcelona, Spain
ReceiVed August 4, 2006
Multivalent ligands are promising pharmacological tools that may be more efficacious for several diseases
than highly selective single-target drugs. A combined therapy using dopaminergic agonists and adenosinergic
antagonists is currently being evaluated for the treatment of Parkinson’s disease. [(a) Kanda, T.; et al. Exp.
Neurol. 2000, 162, 321-327. (b) Jenner, P. Expert Opin. InVest. Drugs 2005, 14, 729-738. (c) Kase, H.;
et al. Neurology 2003, 61 (Suppl 6), S97-S100.] Here we prepared dual ligands acting on adenosine and
dopamine receptors by applying a combinatorial approach based on the ergolene privileged structure. The
potency and efficacy of these novel compounds were determined by radioligand binding studies and
intracellular cAMP production assays in cells expressing adenosine and dopamine receptors. Selected
compounds displayed dual dopamine agonist and adenosine antagonist activity. Molecules with this
pharmacological profile are potentially useful for studying dopamine-adenosine cross-talk in the central
nervous system and for testing the therapeutic potential of multivalent drugs for Parkinson’s disease.
Introduction
in the first case, or complex pharmacokinetics and pharmaco-
dynamics, as in the second.
Recent studies indicate that highly selective drugs alone may
not be sufficient to modulate complex in vivo systems, in spite
of binding to a single given target. This observation may help
to explain the scarcity of successful new single-target drugs
during the past decade, despite extensive drug-development
efforts. Network models support the notion that partial inhibition
of a minimal number of targets is more efficient than the
The potential of multivalent ligands to relieve the symptoms
and halt the progress of complex neurodegenerative diseases
has been partially explored. The limited efficacy of some single-
target drugs for certain disorderssmainly those related to
cognition, memory, and movementshas been reported.11 Thus,
although patients suffering from neurodegenerative diseases such
as Parkinson’s disease (PD) or Alzheimer’s disease (AD) are
currently benefiting from single-target drugs, current pharma-
cological approaches are limited in their capacity to significantly
alter the course of these diseases, and they offer partial or
2
3
complete inhibition of a single target. The application of a
multitarget ligand approach is a new trend in medicinal
chemistry, and in some cases has superseded the traditional “one
target-one disease” philosophy. This is especially true for
disorders in which the alteration of a single receptor is
1
2
transient benefits at best. This is particularly relevant to PD,
where patients receiving traditional treatment, based on dopam-
ine replacement therapy using L-DOPA or other dopamine
agonists, experience response fluctuation, often accompanied
4
therapeutically insufficient. For these cases, balanced modula-
tion of a small number of targets has been shown to have
superior efficacy and fewer side effects than single-target
13,14
by dyskinesias.
Recently several nondopaminergic receptors
5
treatments.
expressed in the basal ganglia have been reported as alternative
The growth of this trend in pharmacological research is
reflected in the numerous relevant compounds that are currently
in late stages of clinical development. For instance, in the field
of antipsychotic drugs, the first selective dopamine antagonists
targets for PD treatment.15 For instance, a combination of A2ARa
antagonists with selective D1R or D2R agonists has exhibited
antiparkinsonian activity in rodent as well as nonhuman primate
1a
models of the disease. Functional interactions between ad-
6
described, such as haloperidol, have been gradually replaced
enosine and dopamine receptors occur at both molecular and
by the so-called atypical antipsychotics, which have a broader
receptor affinity profile. Recent research has focused on the
synthesis of ligands with an optimum pKi for 5-HT2A/D2
receptors, as this multireceptor affinity profile provides broader
efficacy against both the negative and positive symptoms of
16,17
functional levels.
Molecular cross-talk between A1R and D1R
has been reported, which demonstrates the adenosinergic
negative modulation of dopaminergic activation of striatonigral-
18,19
striatopenduncular neurons.
complexes.
A2AR and D2R form heteromeric
20-23
These receptors are colocalized in striatal
7
-9
schizophrenia.
medium spiny neurons, and it has been proposed that adenosine
binding to A2AR lowers the affinity of dopamine for D2R, thus
Multiple pharmacological targets can be affected through the
use of drug cocktails, multicomponent drugs, or multivalent
ligands.1 However, the latter have become increasingly popular
as they do not have drawbacks such as patient compliance, as
24
modulating the function of the latter receptor. A2AR selective
0
antagonists have antiparkinsonian activities in animal models
25
of PD, and in addition, some have exhibited neuroprotective
properties, a benefit rarely observed in most currently used
2
6
*
To whom correspondence should be addressed. F.A.: phone, 0034
antiparkinsonian drugs. Thus, the synthesis of multivalent
9
34037088; fax, 0034 934037126; e-mail, albericio@pcb.ub.es. M.R.:
a
phone, 0034 934037122; fax, 0034 934037126; e-mail, mroyo@pcb.ub.es.
Abbreviations: A1R, adenosine A1 receptor; A2AR, adenosine A2A
†
Combinatorial Chemistry Unit.
Department of Biochemistry and Molecular Biology.
IRB Barcelona.
Department of Organic Chemistry.
receptor; cAMP, cyclic adenosine monophosphate; D1R, dopamine D1
receptor; D2R, dopamine D2 receptor. Abbreviations used for amino acids
follow the IUPAC-IUB Comission of Biochemical Nomenclature in Jones,
J.H. J. Pept. Sci. 2003, 9, 1-8.
‡
§
||
10.1021/jm060947x CCC: $37.00 © 2007 American Chemical Society
Published on Web 06/01/2007

Ergopeptides as Dual Ligands for A1,2AR and D1,2
R
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 13 3063
Figure 1. (a) General structure of natural ergopeptines; (b) scheme of the library of ergot linear tripeptide alkaloids synthesized.
ligands that combine D1 and D2 dopamine agonism to A1 and
A2A adenosine antagonism in a single molecule may lead to
novel treatments for PD.
L-proline, nipecotic acid and L-indoline-2-carboxylic acid were
selected as proline-like amino acids to determine the role of
the size of the C-terminal cyclic amino acid. In the other two
positions, non-natural aromatic amino acids and hydrophobic
amino acids, such as valine or homocyclohexylalanine, were
used. The 36-compound library, resulting from the combination
of the three C-terminal amino acids with 12 possible combina-
tions of R1 and R2 (in Figure 1b, R1 and R2 were always distinct
amino acids), was synthesized on solid-phase using Rink-PS-
resin as a polymeric support. Tripeptides were synthesized using
standard peptide coupling conditions (DIPCDI and HOBt as an
additive). D-Lysergic acid was attached via an amide bond to
the tripeptides using DIPCDI as the coupling reagent, HOAt as
an additive, and N-methylpyrrolidinone (NMP) as solvent,
because D-lysergic acid has low solubility in the other common
solvents used for peptide synthesis (DMF, DCM, toluene, THF).
Finally, cleavage with TFA-H2O (95:5) released the final
ergopeptides [1-36, Table 1 in the Supporting Information (SI)],
which were then analyzed by RP-HPLC-MS. To check the
binding properties of the ergolene scaffold itself, the corre-
sponding primary amide of D-lysergic acid was also synthesized.
In this case, direct coupling of D-lysergic acid to the Rink-PS
resin (DIPCDI, HOAt in NMP) and subsequent cleavage with
TFA-H2O (95:5) yielded D-lysergilamide (37) (Table 1 in SI).
Privileged structures,27 with their inherent capacity to interact
with diverse biological receptors, are an ideal source of scaffolds
for the design and synthesis of combinatorial libraries targeted
at multiple receptors.2 Ergot alkaloids contain the tetracyclic
ergolene or ergoline system and are privileged structures because
of their interactions at more than one receptor.30 Here we
synthesized a 36-member library of ergolene analogs containing
distinct linear peptidic moieties in order to study the interaction
and pharmacology of novel ergopeptides at dopamine and
adenosine receptors.
8,29
Results
Library Design and Synthesis. A number of ergolene
derivatives interact selectively with some GPCRs. The affinity
of these interactions varies with alkaloid structure. Selective
modification of the ergolene system may therefore provide
molecules that interact with a desired set of receptors. Among
the many families of natural products containing the ergolene
system, the ergot peptide alkaloids family, also known as
ergopeptines, displays a broad spectrum of pharmacological
activity. Ergopeptines comprise a D-lysergic acid skeleton linked
to a tricyclic peptide moiety via an amide bond. Naturally
occurring ergopeptines have a double bond at position 9,10
Biological Assays: Binding Properties of Ergopeptides.
The binding properties of 1-37 were assayed via competitive
radioligand binding experiments at 50 µM in D1R, D2R, A1R,
and A2AR (Figure 1 in SI)
(Figure 1a). In this figure, Ra and Rb correspond to side chains
of hydrophobic amino acids such as alanine, â-alanine, leucine,
valine, isoleucine, or phenylalanine3
0,31
(Figure 1a).
For all the receptors except A2AR, some ergopeptides were
more potent than D-lysergilamide (37). Further screening at
lower ligand concentrations was performed for compounds with
the capacity to displace more than 50% of the specific
radioligand binding (Figure 2 in SI). After this second screening,
nine ergopeptides (4, 6, 9, 10, 14, 15, 17, 27, and 36), with
relatively high affinity for either dopamine or adenosine
receptors, were selected, resynthesized, and purified to confirm
their binding properties and to characterize their pharmacological
profiles. It should be noted that, to date, these molecules are
the first derivatives of the ergolene system described to interact
with adenosine receptors.
In addition to natural ergopeptines, the biosyntheses of several
of these compounds have revealed the presence of intermediates
3
2
in which the peptidic moiety is partially or completely linear.
Linear intermediates can be synthesized more easily than the
corresponding cyclic compounds, thus accelerating the evalu-
ation of the effects that peptide substitution has on the
pharmacological properties of novel ergopeptides. On the basis
of this premise, we synthesized a set of novel ergopeptides by
attaching 36 distinct linear tripeptides to D-lysergic acid (Figure
1
b).
The library of ergopeptides was designed so that the ergolene
system was attached to the N-terminal position of distinct linear
tripeptides with a carboxamide group at the C-terminus. Peptidic
moieties of natural ergopeptines always consist of an L-proline
plus two other hydrophobic amino acids. Therefore, to mimic
the diversity space defined by the natural products, we
introduced three proline-like amino acids at the C-terminal
position of the tripeptides while four different L-amino acids
were introduced at the other two positions. In addition to
Resyntheses of Compounds with Higher Affinity (4, 6, 9,
10, 14, 15, 17, 27, and 36). Nine ergopeptides were resynthe-
sized as previously described with the exception that D-lysergic
acid was coupled in solution. The tripeptides were thus cleaved
from the resin using TFA-H2O (95:5) prior to coupling and
D-lysergic acid was attached using DIPCDI, HOAt, and DIEA.
Coupling of the acid in solution instead of on solid-phase
afforded higher global yields and required a much smaller

3
064 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 13
Vendrell et al.
Table 1. Chemical Structures of the Selected Ergopeptides and Their Corresponding KD Valuesa
a
KDh correspond to KD in the receptor high-affinity state.
amount of the costly D-lysergic acid. However, coupling between
tripeptides and D-lysergic acid proceeded with moderate epimer-
ization (around 20% in most cases) at the C8 carbon of the
ergolene scaffold (Figure 1a), thereby leading to diastereoiso-
meric mixtures. As the chromatographic separation of both
diastereoisomers was complex and there was no evidence of a
preferred C8 configuration to interact with dopamine and
adenosine receptors, the crude mixtures were purified by
semipreparative RP-HPLC, thereby collecting the two diaste-
reoisomers for each sample. However, to evaluate the effects
of the C8 configuration on ergopeptide pharmacology, 9 was
further purified so that the two corresponding diastereoisomers
dopamine receptors, the corresponding KD values were deter-
mined from competition experiments (see Table 1).
The results shown in Table 1 indicate that incorporation of
some linear peptidic moieties onto the ergolene structure leads
to improved affinities for several subtypes of adenosine and
dopamine receptors when compared to the ergolene scaffold
alone. This improvement is dependent on the sequence of the
peptidic moiety, with some amino acids exhibiting moderate
selectivity at certain receptors. Regarding the contribution of
the C-terminal amino acid, ergopeptides containing L-proline
or nipecotic acid (6, 9, 10, 14, and 17) showed higher affinities
for dopamine receptors, while those containing L-indolin-2-
carboxylic acid (27 and 36) showed greater affinities for
adenosine receptors. Concerning the amino acid in the inter-
mediate position, stronger interactions at adenosine receptors
were obtained with homocyclohexylalanine or valine (15, 27,
and 36), while this specificity was not detected in the case of
ergopeptides binding to dopamine receptors. Finally, in terms
of the amino acid directly attached to the ergolene scaffold,
p-nitrophenylalanine and valine conferred more affinity for
adenosine and dopamine binding (6, 9, 14, 15, 17, 27, and 36).
In addition, not only the amino acids but their relative position
in the global structure is relevant to ergopeptide binding
properties. Consequently, it is not surprising that the binding
(
9a and 9b) were isolated. The chemical structures of 9a
1
13
and 9b were determined by H and C NMR spectroscopy
Experimental Section), which indicate that 9b is a D-lysergic
(
acid derivative, whereas 9a is an L-lysergic acid derivative.
In addition, the corresponding tripeptides of the nine
selected ergopeptides were synthesized using the same
R
procedure described above but including an N -acetylation step
R
at the N -terminus prior to cleavage. Purification by semi-
preparative RP-HPLC afforded the final tripeptides (38-46)
(Table 2 in SI).
Biological Assays: KD Determination. To determine the
binding affinity of selected compounds to adenosine and

Ergopeptides as Dual Ligands for A1,2AR and D1,2
R
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 13 3065
Figure 2. Effects of ergopeptides (50 µM) on intracellular cAMP levels
in SH-SY5Y neuroblastoma cells. CGS (150 nM) is a selective A2A
agonist. *Significantly different (p < 0.05) compared to basal.
*Significantly different (p < 0.05) compared to CGS-treated cells.
R
Figure 3. Effects of ergopeptides (50 µM) on intracellular cAMP levels
in SH-SY5Y neuroblastoma cells. Raclopride (50 µM) is a selective
*
2 2
D R antagonist and quinpirole (150 nM) is a selective D R agonist.
§
Cells were preincubated (5-10 min, 22 °C) with ZM (5 µM) to block
properties of ergopeptides with the same composition but with
amino acids placed in a different order vary greatly. For instance,
matching pairs of ergopeptides (6 and 11, 9 and 12, 36 and 33,
and 4 and 1) exhibited very different affinities in D1R, D2R,
A1R, and A2AR, respectively. Thus, whereas 6, 9, 36, and 4
were selected from the nine ergopeptides with higher affinity,
binding of endogenous adenosine to A2AR. *Significantly different (p
<
0.05) compared to cells activated with Fk and pretreated with ZM.
**Significantly different (p < 0.05) compared to cells activated with
Fk, pretreated with ZM and after addition of raclopride and quinpirole.
whether they acted as A2AR antagonists, cAMP levels were
determined in SH-SY5Y cells pretreated with the A2AR selective
1
1, 12, 33, and 1 were discarded after the competition
3
5
experiments.
agonist CGS (Figure 2b). All compounds antagonized CGS-
induced cAMP production and therefore were A2A antagonists,
taking into consideration that 9a, 9b, 10, 15, and 17 may behave
as inverse agonists.
To determine the contribution of the peptidic moiety to the
affinity of ergopeptides for the receptors under study, radioligand
binding competition studies were performed using 38-46 for
D1R, D2R, A1R, and A2AR under the same experimental
conditions as those used for the ergopeptide library. None of
the tripeptides at 50 µM significantly (more than 40%) displaced
the specific radioligand binding at any receptor (see Figure 3
in SI); however, as previously mentioned, the peptidic moiety
appeared to be crucial in determining the binding and selectivity
properties of ergopeptides. This phenomenon may be explained
by the fact that the ergolene scaffold interacts at the binding
site itself, whereas tripeptides interact with adjacent amino acids.
Therefore, the peptidic interaction would determine a better or
a worse accommodation of the ergolene scaffolds in the binding
site and thus establishes several selectivity criteria.
A similar procedure was used to distinguish between agonism
and antagonism in ergopeptides at D2R. D2R activation results
in coupling to GRi-proteins, which leads to the inhibition of
adenylate cyclase and therefore to a decrease in cAMP
36,37
levels.
Thus, D2R agonism causes a decrease in the increased
cAMP levels induced by forskolin. Given that activation of
adenosine-binding A2AR decreases the affinity of dopamine for
2
4a,38
D2R,
SH-SY5Y cells were preincubated with the selective
A2AR antagonist ZM [4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo-
[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol], thereby preventing
D2R binding caused by endogenous adenosine. Experiments
were performed in the presence of 10 µM forskolin to detect
decreases in cAMP levels. Compounds 4, 9a, 9b, 10, 14, 17,
and 36 provoked a significant decrease in the cAMP levels of
forskolin-activated cells (Figure 3a). This effect was antagonized
by the selective D2R antagonist raclopride (3,5-dichloro-N-(1-
ethylpyrrolidin-2-ylmethyl)-2-hydroxy-6-methoxybenzamide),
thereby indicating that compounds 4, 9a, 9b, 10, 14, 17, and
36 behaved as D2R agonists. Concerning the effect of C8
configuration in D2R pharmacology, the diastereoisomer 9b was
a slightly more potent agonist than its corresponding isomer
9a. In contrast, ligands 6, 15, and 27 acted as D2R antagonists,
since they reverted the decrease in the cAMP levels of cells
activated by the selective D2R agonist quinpirole [(4aR,8aR)-
5-propyl-4,4a,5,6,7,8,8a,9-octahydro-1H-pyrazolo[3,4-g]quino-
line] (Figure 3b).
Biological Assays: Identification of Agonist/Antagonist
Behavior. To characterize the pharmacological profile of 4, 6,
9a, 9b, 10, 14, 15, 17, 27, and 36 at D1R, D2R, A1R, and A2AR,
the ligands were tested by evaluating intracellular cAMP levels
derived from the activation or inhibition of adenylate cyclase
in two cell lines: SH-SY5Y neuroblastoma cells, which
endogenously express A2AR and were transfected with dopamine
D2LR, and fibroblasts cotransfected with A1R and D1R.
We first evaluated the effects of the compounds on A2AR
activation by analyzing cAMP levels in SH-SY5Y cells
separately treated with each of the nine ergopeptides or the A2AR
agonist CGS [2-p-(2-carboxyethyl)phenethylamino-5′-N-ethyl-
carboxamido adenosine hydrochloride] (Figure 2a). Activated
A2AR couples to GRs-protein, which leads to the activation of
adenylate cyclase and therefore to an increase in cAMP
Analogously to A2AR, D1R couples to GRs-protein; thus,
activation of the receptors also causes an increase in cAMP
3
3,34
levels.
As none of the ergopeptides increased basal cAMP
3
6,37
levels, they were not considered to be A2AR agonists. To test
levels.
The nine ergopeptides tested proved to be D1R

3
066 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 13
Vendrell et al.
Activated A1R couples to GRi-protein, which leads to the
inhibition of adenylate cyclase. This observation implies that
33,34
A1R agonists decrease cAMP levels.
A1R-D1R transfected
fibroblasts were preincubated with SCH (50 µM) to prevent
any agonist interaction of the ergopeptides with D1R during
pharmacological evaluation of A1R. Experiments were per-
formed in the presence of 10 µM forskolin to detect decreases
in cAMP levels. In contrast to the effect of the selective A1R
6
agonist R-PIA [N -(R)-(2-phenylisopropyl)adenosine], none of
the ligands caused a decrease in the forskolin-induced cAMP
levels (Figure 5a). Furthermore, all compounds acted as A1R
antagonists as they reverted the cAMP decrease induced by
R-PIA, similarly to DPCPX (1,3-dipropyl-8-cyclopentylxan-
thine), a selective A1R antagonist (Figure 5b).
Conclusions
The incorporation of linear peptidic moieties into the ergolene
privileged structure has led to molecules with improved affinity
for dopamine and adenosine receptors compared to the ergolene
scaffold alone. This improvement is dependent on the sequence
of the peptidic moiety, which therefore determines the binding
and selectivity properties of ergopeptides. However, none of
the tripeptides alone interacted significantly in the binding site
of any receptor. A possible explanation for this binding mode
considers the interaction of the ergolene scaffold at the binding
site while tripeptides interact with adjacent amino acids. In
addition, our pharmacological assays revealed that the agonist/
antagonist behavior of ergopeptides relies on the ergolene
system. This observation is also consistent with our hypothesis
of ergopeptides binding mode, which assumes that the peptidic
moiety does not directly interact at receptor binding sites.
Concerning the effect of ergolene system chirality, the C8
configuration is not relevant in ergopeptides pharmacology, as
the diastereoisomers 9a and 9b showed similar behavior in the
four receptors evaluated, observing only slight differences in
their agonist potency at D2R. All the selected ergopeptides
behaved as A1R antagonists, A2AR inverse agonists or antago-
nists, and D1R agonists. Regarding D2R, 4, 9a, 9b, 10, 14, 17,
and 36 acted as agonists, whereas 6, 15, and 27 were antagonists.
Therefore, these novel ergopeptides can be described as mul-
tifunctional compounds, as they induce distinct pharmacological
responses for receptors. Given their affinities in the low
micromolar range and complementary pharmacological profiles
Figure 4. Effects of ergopeptides (50 µM) on intracellular cAMP levels
in A R and D R transfected fibroblasts. SCH (50 µM) is a selective
R antagonist and SKF (20 nM) is a selective D R agonist.
Significantly different (p < 0.05) compared to basal.
1
1
D
*
1
1
(D1-D2 agonism and A1-A2A antagonism), these ergopeptides
provide a good starting point to generate new ligands with dual
activity in dopamine and adenosine receptors. In addition, these
molecules are useful tools to evaluate adenosine-dopamine
cross-talk mechanisms and to test the potential of multivalent
ligands in the treatment of PD and other neurological disorders.
The incorporation of novel amino acids with several functional
groups may lead to more potent and selective compounds. Using
the above-mentioned ergopeptides, we are currently working
on the design and synthesis of new molecules that show
improved affinity for a number of subtypes of dopamine and
adenosine receptors.
Figure 5. Effects of ergopeptides (50 µM) on intracellular cAMP levels
in A R and D R transfected fibroblasts. DPCPX (50 µM) is a selective
R antagonist and R-PIA (20 nM) is a selective A
were preincubated (5-10 min, 22 °C) with SCH (50 µM) to block
binding of ergopeptides as D R agonists. *Significantly different (p <
.05) compared to cells activated with Fk, pretreated with SCH and
after addition of R-PIA.
1
1
§
A
1
1
R agonist. Cells
1
0
agonists since they increased the cAMP levels in A1R-D1R
transfected fibroblasts to a similar extent as the selective D1R
agonist SKF [(()-1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benza-
zepine-7,8-diol]. This increase was also antagonized by the
selective D1R antagonist SCH [(R)-7-chloro-8-hydroxy-3-
methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine] (Figure
Experimental Section
Materials and Equipment. All Fmoc-amino acids were pur-
chased from Neosystem (Strasbourg, France), and Fmoc-Rink-PS
resin was supplied by Calbiochem-Novabiochem AG. DIPCDI was
obtained from Fluka Chemika (Buchs, Switzerland) and HOBt from
Albatross Chem, Inc. (Montreal, Canada). Solvents for peptide
synthesis and RP-HPLC equipment were obtained from Scharlau
(Barcelona, Spain). Trifluoroacetic acid was supplied by KaliChe-
mie (Bad Wimpfen, Germany). Other chemicals were purchased
4
a), thus confirming D1R agonism. None of the compounds
decreased the cAMP levels in SKF-activated fibroblasts (Figure
b), thereby indicating that they were not D1R antagonists.
4

Ergopeptides as Dual Ligands for A1,2AR and D1,2
R
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 13 3067
from Aldrich (Milwaukee, WI) and were of the highest com-
mercially available purity. All commercial reagents and solvents
were used as received.
H
2
O (95:5, v/v) in 1 h afforded 3.0 mg of d-lysergilamide (yield:
32%), which was dried under vacuum and lyophilized before
biological assays. The product was characterized using the same
conditions as those used for the ergopeptide library (HPLC data
included in Table 1 in SI).
Adenosine deaminase (EC 3.5.4.4) was purchased from Roche
3
(
Basel, Switzerland). Geneticine G-418, and [ H]-R-PIA were
supplied by Amersham Biosciences (Buckinghamshire, UK). For-
skolin, quinpirole, raclopride, dopamine, MgCl , CGS 21680,
R-PIA, and DPCPX were purchased from Sigma (St. Louis, MO).
Resyntheses of Compounds 4, 6, 9a, 9b, 10, 14, 15, 17, 27,
r
2
and 36 and the Corresponding N -Acetylated Tripeptides (38-
46). Each synthesis was carried out using Fmoc-Rink-PS resin (400
R
ZM 241385 and SCH 23390 were supplied by Tocris Biosciences
mg). Upon synthesis of the free N -terminus tripeptides, the resins
(
Avonmouth, UK). SKF 38393 was purchased from Research
were split equally into two syringes to eventually provide both the
3
3
R
Biochemicals (Natick, MA) and [ H]SCH 23390, [ H]YM 09151-
2
ergopeptides and the corresponding N -acetylated tripeptides.
3
3
r
, [ H]ZM 241385, and [ H]CGS 21680 were supplied by Perkin-
N -Acetylated Tripeptides. The resins were treated with Ac
2
O
Elmer (Boston, MA). Ecoscint H scintillation cocktail was pur-
chased from National Diagnostics (Atlanta, GA) and cAMP Biotrak
Enzyme Immunoassay (EIA) kits from Amersham Biosciences
(135 µL, 10 equiv) and DIEA (240 µL, 10 equiv) in DCM (2 × 15
min). Reaction completion was checked by the Kaiser test. Cleavage
for 1 h with a solution of TFA-H
2
O (95:5, v/v) led to the crude
R
(Buckinghamshire, UK). Penicilin, streptomycin, hygromycine, and
N -acetylated tripeptides.
R
all other supplements were purchased from Invitrogen (Paisley,
UK).
Ergopeptides. Free N -terminus tripeptides were cleaved from
2
the resin with a solution of TFA-H O (95:5, v/v). After evapora-
Analytical RP-HPLC-MS were performed using a 2795 Waters
Milford, MA) Alliance with a Micromass ZQ Mass Spectrometer
tion, the crude products were solved in DMF and treated with DIEA
(24 µL, 1 equiv). D-Lysergic acid (38 mg, 1 equiv) was activated
in DMF by HOAt (19 mg, 1 equiv). The resulting suspension and
DIPCDI (22 µL, 1 equiv) were added to each crude peptide.
Couplings were performed overnight at room temperature and the
crude products were evaporated under vacuum. The compounds
were purified by semipreparative RP-HPLC using a reverse-phase
(
and 996 PDA detector. Semipreparative RP-HPLC was performed
on a 2767 Waters chromatography system with a Micromass ZQ
mass spectrometer. Multiple sample evaporation was carried out
1
in a Discovery SpeedVac ThermoSavant (Waltham, MA). H and
1
3
C NMR spectra were recorded on a Mercury 400 spectrometer
Unitat de RMN, Serveis Cientifico-T e` cnics, University of Barce-
2
(
Symmetry C18 (3 × 10 cm ) 5 µm column with elution systems
lona); chemical shifts (δ scale) are reported in parts per million
2
(A) H O-TFA 99.9:0.1 and (B) ACN-TFA 99.9:0.1 at 25 mL/
(ppm) and coupling constants are given in hertz (Hz). Radioligand
min flow rate and a purification gradient of 0% B to 100% B in 30
min.
binding experiments were performed using a Brandel (Gaithersburg,
MD) cell harvester and a Packard 1600 TRI-CARB scintillation
counter. Absorbance measures were carried out in a Merck Elisa
System Mios plate reader.
Sample Characterization. The purified compounds were char-
acterized by analytical RP-HPLC-MS with a reverse-phase Sym-
2
metry C18 (3.9 × 150 mm ) 5 µm column and flow rate of 1 mL/
Synthesis. Solid-Phase General Procedure. Peptide syntheses
were performed manually in a polypropylene syringe fitted with a
polyethylene porous disk. Solvents and soluble reagents were
removed by suction. Washings between deprotection, coupling, and
subsequent deprotection steps were carried out with DMF (5 × 1
min) and DCM (5 × 1 min) using 10 mL of solvent/g of resin
each time. Peptide syntheses were carried out with Fmoc-Rink-PS
resin using an Fmoc/tBu solid-phase strategy. Fmoc-AA-OH (3
equiv) was coupled using DIPCDI (3 equiv) and HOBt (3 equiv)
in DCM-DMF (1:1) for 2-4 h at room temperature. After each
coupling, the resin was washed with DMF (5 × 1 min) and DCM
min in two separate elution systems. The purities obtained using
each of the two elution systems are specified in Table 3 (SI).
r
N -Acetylated Tripeptides. HPLC data and yields are included
in Table 2 (SI).
Ergopeptides. 4 (yield 31%): 37.6 mg, Mexp 697.4 (Mcalc 696.4),
93%.
6 (yield 1%): 3.2 mg, Mexp 629.3 (Mcalc 628.3), 94%.
9a (yield 5%): 4.4 mg, Mexp 656.3 (Mcalc 655.3), 98%; H NMR
1
6
(400 MHz, DMSO-d ) 11.03 (s, 1H), 10.37 (bs, 2H), 8.52 (d, 1H,
J ) 8.9 Hz), 8.41 (d, 1H, J ) 7.9 Hz), 8,14 (d, 2H, J ) 8.5 Hz),
7,67 (d, 2H, J ) 8.6 Hz), 7.35 (d, 1H, J ) 8.0 Hz), 7.30 (bs, 1H),
7.20 (t, 1H, J ) 7.7 Hz), 7.10 (d, 1H, J ) 7.3 Hz), 6.50 (bs, 1H),
4.92 (m, 1H), 4.38 (m, 1H), 4.33 (dd, 1H, J ) 3.8-8.4 Hz), 4.27
(m, 1H), 3.98 (m, 1H), 3.72 (m, 1H), 3.70 (m, 1H), 3.60 (m, 2H),
3.50 (m, 1H), 3.35 (m, 1H), 3.19 (s, 3H), 3.05 (d, 1H, J ) 9.3 Hz),
2.94 (m, 1H), 2.11 (m, 2H), 1.99 (m, 1H), 1.91 (m, 2H), 0.89 (d,
(
5 × 1 min). Reaction completion was checked by the Kaiser or
chloranil tests.
Fmoc group removal involved the following sequence: (i) DMF
(
5 × 1 min), (ii) piperidine-DMF (2:8) (1 × 1 min + 2 × 15
min), (iii) DMF (5 × 1 min).
13
Synthesis of Ergopeptides Library (1-36). Each compound
was synthesized starting from Fmoc-Rink-PS resin (85 mg, loading
3H, J ) 6.9 Hz), 0.87 (d, 3H, J ) 6.9 Hz); C NMR (400 MHz,
DMSO-d ) 174.1, 171.3, 169.6, 169.4, 146.7, 134.4, 131.4, 125.8,
6
0
.7 mmol/g). Upon synthesis of the tripeptides as described above,
D-lysergic acid (47 mg, 3 equiv) was activated in NMP by HOAt
25 mg, 3 equiv). The resulting solution and DIPCDI (28 µL, 3
125.4, 125.1, 123.4, 121.0, 120.1, 116.1, 112.4, 111.6, 110.0, 106.3,
61.9, 60.4, 58.6, 53.4, 52.0, 47.5, 41.9, 40.2, 37.1, 31.0, 30.0, 25.1,
24.5, 19.9, 18.9.
9b (yield: 3%): 2.9 mg, Mexp 656.3 (Mcalc 655.3), 99%; H NMR
6
(400 MHz, DMSO-d ) 10.94 (s, 1H), 9.15 (bs, 2H), 8.46 (d, 1H, J
(
1
equiv) were added to the resin, which had previously been swollen
in NMP. The coupling was carried out overnight at room temper-
ature. After coupling, the resins were washed with NMP, DMF,
DCM, and MeOH (5 × 1 min with every solvent). Cleavages were
) 8.8 Hz), 8.43 (d, 1H, J ) 7.9 Hz), 8,09 (d, 2H, J ) 8.4 Hz),
7.56 (d, 2H, J ) 8.4 Hz), 7.21 (bs, 1H), 7.16 (bs, 1H), 7.11 (bs,
1H), 7.10 (bs, 1H), 6.68 (d, 1H, J ) 5.4 Hz), 4.76 (m, 1H), 4.30
(m, 1H), 4.18 (dd, 1H, J ) 4.0-8.4 Hz), 4.14 (dd, 1H, J ) 1.5-
7.0 Hz), 3.72 (m, 1H), 3.62 (m, 4H), 3.51 (m, 1H), 3.22 (dd, 1H,
J ) 4.0-14.0 Hz), 3.11 (dd, 1H, J ) 4.1-13.9 Hz), 3.10 (s, 3H),
2.01 (m, 1H), 1.90 (m, 1H), 1.89 (m, 2H), 1.80 (m, 1H), 0.73 (d,
2
performed at room temperature with a solution of TFA-H O (95:
5
, v/v). The products were evaporated under pressure and lyoph-
ilized before biological assays. All compounds were characterized
by analytical RP-HPLC-MS using a reverse-phase Symmetry C18
2
(
3.9 × 150 mm ) 5 µm column, flow 1 mL/min (HPLC data
13
included in Table 1 in SI).
3H, J ) 6.4 Hz), 0.72 (d, 3H, J ) 6.4 Hz); C NMR (400 MHz,
Synthesis of D-Lysergilamide (37). Fmoc-Rink-PS resin (50 mg)
was swollen in DMF and the Fmoc group was removed as above-
mentioned. D-Lysergic acid (28 mg, 3 equiv) and HOAt (15 mg, 3
equiv) were dissolved in NMP. The solution obtained and DIPCDI
DMSO-d ) 174.0, 171.2, 171.0, 169.6, 146.9, 134.5, 131.5, 125.9,
6
125.4, 123.9, 123.8, 123.7, 123.4, 121.2, 118.6, 112.1, 111.7, 106.3,
62.1, 60.4, 58.4, 53.4, 52.0, 47.5, 43.1, 39.3, 37.0, 31.4, 30.0, 25.1,
24.8, 19.9, 18.9.
(
17 µL, 3 equiv) were added to the resin, and the coupling was
10 (yield 27%): 30.0 mg, Mexp 631.4 (Mcalc 630.4), 99%.
14 (yield 9%): 23.9 mg, Mexp 738.2 (Mcalc 737.5), 87%.
15 (yield 6%): 10.6 mg, Mexp 645.4 (Mcalc 644.5), 97%.
17 (yield 20%): 20 mg, Mexp 736.3 (Mcalc 735.3), 99%.
carried out overnight at room temperature. Afterward, the resin was
washed with NMP, DMF, DCM, and MeOH (5 × 1 min with every
solvent). Cleavage at room temperature with a solution of TFA-

3
068 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 13
Vendrell et al.
2
3
7 (yield 13%): 22.7 mg, Mexp 679.4 (Mcalc 678.4), 94%.
6 (yield 4%): 10.7 mg, Mexp 704.4 (Mcalc 703.3), 97%.
mL geneticine G-418. Both cell lines were grown at 37 °C in
saturation humidity in a 5% CO -95% air atmosphere.
2
cAMP Determination. The accumulation of cAMP was mea-
sured with a competitive enzyme immunoassay of the cAMP
Biotrak enzyme immunoassay system. Cells showing 80% conflu-
Biological Assays. Radioligand Binding Experiments. General
Procedure. Membrane suspensions of lamb striatum were obtained
following the method described in refs 39 and 40. Radioligand
binding assays of membrane suspensions (0.5 mg of protein/mL
determined with bicinchoninic acid kits) were carried out at 22 °C
in 50 mM Tris-HCl buffer, pH 7.4 (see conditions below used for
each receptor). After radioligand incubation, free and membrane-
bound ligand were separated by rapid filtration of 500 mL aliquots
in a cell harvester through Whatman GF/C filters embedded in
1 1
ence (NBt or A D cells) were serum-starved for 12-16 h, and the
medium was replaced by fresh HBSS. The phosphodiesterase
inhibitor zardaverine (50 µM) was added, and after 10 min of
incubation, the ligands were added at the concentrations indicated
in the legends of Figures 2-5. After 35 min overall incubation,
the reactions were stopped by treatment with the lysis buffer
provided by the kit and samples were processed following the
manufacturer’s instructions. Data are means ( SD of three to six
determinations, and the differences with respect to controls were
tested for significance (two-tailed; p < 0.05) using the Student’s t
test for unpaired samples.
3
polyethylenimine (except for [ H]CGS). Nonspecific binding was
determined with unlabeled ligands at the concentration indicated
below. In all cases, the filters were incubated with 10 mL of
Ecoscint H scintillation cocktail overnight at room temperature.
Radioactivity counts in the vials were determined in the counter
with an efficiency of 60%.
Screening of Ergopeptides Library. Binding experiments of
the whole library were performed at a concentration of 50 µM
ergopeptide.
Acknowledgment. This work was partially supported by
funds from CICYT (CTQ 2005-0315 and BQU 2003-00089)
and the Fundaci o´ La Marat o´ de TV3 Grant Marat o´ /2001/012710
(R.F.). M.V. thanks the MECD (Spain) for a predoctoral
Dopamine D
1
Receptor. Membranes were incubated with 1.2
fellowship.
3
nM [ H]SCH 23390 (85 Ci/mmol) in 50 mM Tris-HCl buffer (pH
7
1
.4) for 1 h. Nonspecific binding was measured in the presence of
mM dopamine.
Supporting Information Available: Tables containing chemi-
cal structures, HPLC data, and yields for the whole library of
R
ergopeptides, N -acetylated tripeptides, and resynthesized ergopep-
Dopamine D
2
Receptor. Membranes were incubated with 0.9
3
tides as well as results for preliminary radioligand binding experi-
ments. This material is available free of charge via the Internet at
http://pubs.acs.org.
nM [ H]YM 09151-2 (83 Ci/mmol) in 50 mM Tris-HCl buffer (pH
7
1
.4) for 2 h. Nonspecific binding was measured in the presence of
00 µM raclopride.
Adenosine A
1
Receptor. Membranes were incubated with 2.2
nM [ H]-R-PIA (46 Ci/mmol) in 50 mM Tris-HCl buffer (pH 7.4)
containing 10 mM MgCl and 0.2 U/mL ADA for 1 h. Nonspecific
References
3
(
1) (a) Kanda, T.; Jackson, M. J.; Smith, L. A.; Pearce, R. K. B.;
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2
binding was measured in the presence of 50 µM R-PIA.
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3
nM [ H]CGS 21680 (40.5 Ci/mmol) in 50 mM Tris-HCl buffer
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(
pH 7.4) containing 10 mM MgCl
Nonspecific binding was measured in the presence of 1 mM CGS
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2
and 2 U/mL ADA for 2 h.
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2
Compounds with the capacity to displace more than 50% of the
specific radioligand binding were then tested at lower concentrations
using the same procedure. Each ergopeptide concentration is
indicated in the corresponding legend of Figure 2a-d (SI).
D
Competition Experiments and K Determination. Competition
experiments were performed by incubating membranes under the
same conditions as described above, in the absence or presence of
increasing concentrations of ergopeptides. Nonspecific binding was
determined as previously outlined. Radioligand displacement curves
were analyzed by nonlinear regression using the commercial
program GRAFIT (Erithacus Software, Surrey, UK) by fitting the
2
003, 61 (Suppl 6), S97-S100.
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total binding data to the displacement models with one or two
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modified F test was used to analyze whether the fit to the two-site
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35a
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(
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cells were grown under the same conditions as NBt cells but
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1
(
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1 1
A D

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