Novel imidazoline compounds as partial or full agonists of D 2-like dopamine receptors inspired by I2-imidazoline binding sites ligand 2-BFI

Article abstract of DOI:10.1016/j.bmc.2010.08.005

Based on the well known biological versatility of the imidazoline nucleus, we prepared the novel derivatives 3a-k inspired by 2-BFI scaffold to assess imidazoline molecules as D₂-like dopamine receptor ligands. Conservative chemical modificatio

Full text of DOI:10.1016/j.bmc.2010.08.005

Bioorganic & Medicinal Chemistry 18 (2010) 7085–7091
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel imidazoline compounds as partial or full agonists of D2-like
dopamine receptors inspired by I2-imidazoline binding sites ligand 2-BFI
Gianfabio Giorgioni a, , Dario Ambrosini a, Cristian Vesprini a, Alan Hudson b, Cinzia Nasuti c,
⇑
Antonio Di Stefano d, Piera Sozio d, Osele Ciampi e, Barbara Costa f, Claudia Martini f, Antonio Carrieri g,
Giuseppe Carbonara g, Christoph Enzensperger h, Maria Pigini a
a Scuola di Scienze del Farmaco e dei Prodotti della Salute, Università degli Studi di Camerino, via Sant’Agostino 1, 62032 Camerino, Italy
b Department of Pharmacology, 9-70 Medical Sciences Building, University of Alberta, Edmonton, Alberta, Canada T6G 2H7
c Scuola di Scienze del Farmaco e dei Prodotti della Salute, Università degli Studi di Camerino, via Madonna delle Carceri, 62032 Camerino, Italy
d Dipartimento di Scienze del Farmaco, Università ‘G. D’Annunzio’, via dei Vestini, 66100 Chieti, Italy
e Department of Human Morphology and Applied Biology, University of Pisa, via Volta 4, 56126 Pisa, Italy
f Department of Psychiatry, Neurobiology, Pharmacology and Biotechnology, University of Pisa, via Bonanno, 6-56126 Pisa, Italy
g Dipartimento Farmaco-Chimico, Università degli Studi di Bari, via E. Orabona 4, 70125 Bari, Italy
h Friedrich-Schiller Universiät Jena, Institut für Pharmazie, Philosophenweg 14 D-07745 Jena, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 16 February 2010
Revised 26 July 2010
Accepted 2 August 2010
Available online 6 August 2010
Based on the well known biological versatility of the imidazoline nucleus, we prepared the novel deriv-
atives 3a–k inspired by 2-BFI scaffold to assess imidazoline molecules as D2-like dopamine receptor
ligands. Conservative chemical modifications of the lead structure, such as the introduction of an hydroxy
group in the aromatic ring alone or associated with N-benzyl substitution, provided partial (3f) or nearly
full (3e and 3h) agonists, all endowed with D2-like potency comparable to that of dopamine.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Dopamine receptors
2-BFI
Imidazoline binding sites
I2-IBS
D2-like receptors
Imidazoline
1. Introduction
positive and negative symptoms of schizophrenia. Indeed, the nor-
malization of dopaminergic activity induced by these agents does
Dopamine (DA), a neurotransmitter distributed both centrally
and peripherally, plays important neuroendocrine, cognitive, emo-
tional and locomotor functions. These effects are mediated by five
distinct receptor subtypes belonging to the superfamily of G-pro-
tein-coupled receptors (GPCRs). Based on their ability to activate
or inhibit the enzyme adenylate cyclase, DA receptors are divided
into two classes, D1-like (D1 and D5) and D2-like (D2–D4).1 Selective
drugs targeting these receptors show broad utility in the treatment
of the diseases where the DA system is altered.2 In particular, the
D2-like receptor family is considered the primary target of antipar-
kinson and antipsychotic drugs. In addition to L-DOPA, the imme-
diate precursor of DA, D2-like full agonists proved to be able to
reduce bradykinesia, rigidity, and tremor typical of Parkinson’s dis-
ease (PD).2 In recent years, attention has been turned to D2-like
partial agonists as potential tools for the treatment of both the
not seem to be associated with the extrapyramidal side effects
(EPS), generally observed in the therapy with D2 antagonists.3
Several studies of some of us have demonstrated the biological
versatility of the imidazoline ring and the crucial role played by the
bridge (X) and the aromatic area (Ar) forming the substituent in
position 2 (Chart 1).4 In particular, the peculiar chemical nature
of the bridge determined preferential recognition by a specific
biological system, whereas that of the aromatic region was
responsible for the ligand functional behaviour. Based on these
observations, the consequent rational design allowed us to obtain
several novel imidazoline molecules, preferentially targeting
imidazoline binding sites (I1- and I2-IBS) or
a2-adrenoreceptors
(a
2-ARs).4 Although the I2-IBS identity still remains unknown,
these binding proteins are widely distributed both centrally and
peripherally and they are involved in psychiatric disorders, opiate
withdrawal, PD and Alzheimer’s diseases (AD).5 In addition, brain
dialysis revealed that I2-IBS ligands induced an increase of extra-
cellular monoamine concentration.6 The imidazoline derivative
⇑
Corresponding author. Tel.: +39 0737402360; fax: +39 0737637345.
E-mail address: gianfabio.giorgioni@unicam.it (G. Giorgioni).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.08.005

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G. Giorgioni et al. / Bioorg. Med. Chem. 18 (2010) 7085–7091
N
N
5
6
2
Ar
X
N
H
O
N
H
HO
N
O
Me
1
2
Chart 1. Common structural features of 2-BFI (1) and tetrahydrochromenoisoquinolines (2).
2-(2-benzofuranyl)-2-imidazoline (2-BFI, 1, Chart 1), where Ar and
X groups are represented by the benzofurane moiety, is currently
considered a ligand of choice for the I2-IBS study.7
the D2-like receptor family (EC50 = 37.7; ia = 0.57), and conserva-
tive chemical modifications of its base structure modulated its I2-
IBS and dopaminergic properties. In particular, we observed that
the introduction of one hydroxy group in position 6 of the aromatic
ring (compound 3f) enhanced the D2-like profile of the lead. In-
deed, 3f showed a better affinity value (Ki = 14.45) compared to 1
(Ki = 47.0),8 behaved as a partial agonist (ia = 0.48), and displayed
a potency value (EC50 = 5.7) comparable to that of DA (EC50 = 4.8,
Nevertheless, it also proved to be able to bind with some affinity
(47 l
M) to D2-like receptors. and to display central DA releasing/
depleting property.8 Moreover, our preliminary studies on porcine
striatal membranes demonstrated that 1 was lacking of D1-like
affinity (Ki >100 lM). 2-BFI (1) shares a molecular framework with
the tetrahydrochromenoisoquinolines (2, Chart 1), which we dis-
covered to be endowed with D3 affinity in micromolar range.9
Based on the above considerations, we prepared the novel deriva-
tives 3a–k inspired by 1 scaffold (Chart 2) with the aim of assessing
imidazoline molecules as DA receptor ligands.
The designed chemical modifications were performed both in
the aromatic region and to the imidazoline nitrogen without
changing the core structure of the lead, also to preserve a possible
favourable synergism between I2-IBS and DA systems. The choice
of the alternative or simultaneous insertion of hydroxy groups in
position 5 or 6 of the phenyl ring was suggested by the presence
of these functions in the aromatic area of DA and numerous known
D2 agonists. Similarly, the presence of N-benzyl or N-propyl pen-
dant groups in efficacious D2-ligands,2 suggested the N-substitu-
tion. The D2-like affinities of compounds 3a–k and the potencies
of the most interesting derivatives (3e–3h and 3k) and 1 were
Ki = 3.90). Moreover, 3f retained
(Ki = 0.076) and high I2-IBS/ 2-ARs selectivity. In contrast, the 5-
hydroxy isomer 3a, endowed with a similar good I2-IBS affinity
(Ki = 0.088) and I2-IBS/ 2-ARs selectivity, failed to target the D2-
like receptors. The peculiar structure of the imidazoline nucleus al-
lows the supposed dopaminergic pharmacophoric functions (basic
amino and phenolic moieties) of both 5- and 6-hydroxy derivatives
3a and 3f be located at similar distances (7.96 Å and 7.82 Å, respec-
tively).11 Therefore, since only the 6-hydroxy derivative 3f dis-
played significant D2-like affinity, it appeared that the OH
function triggered a productive hydrogen bond only if its spatial
relationship with the oxygen bridge was comparable to that of
the meta hydroxy function of DA.
a significant I2-IBS affinity
a
a
As expected, the good I2-IBS properties of 3a and 3f agreed with
previous results obtained with ligands structurally related to 1.12
A
negative influence of the 5,6-dihydroxy substitution was observed
in the catechol derivatives 3i, which reduced the I2-IBS affinity and
failed to interact with D2-like receptors. A sharp, but profitable
modulation of the biological profile of the lead was instead ob-
tained with the N-substitution. Interestingly, the observed general
decrease of the I2-IBS affinity was associated, in some cases, with a
significant D2-like potency enhancement. In particular, the N-ben-
zyl derivatives 3e and 3h behaved as a nearly D2-like full agonists
(for 3e, ia = 0.85; for 3h, ia = 0.82), with affinity (for 3e, Ki = 5.0; for
3h, Ki = 5.66), and potency values (for 3e, EC50 = 6.3; for 3h,
EC50 = 5.8) comparable to those of DA. The presence of the N-ben-
zyl substituent proved to be so beneficial that it was able to coun-
terbalance the aforementioned negative effect of both hydroxy
group in position 5 (compare 3e vs 3a) and catechol moiety (com-
pare 3k vs 3i). This result might be conferred to a favourable
hydrophobic interactions of the highly lipophilic N-benzyl group
with the cluster of aromatic residues located in the sixth trans-
membrane helix, namely Phe6.44, Trp6.48, Phe6.51 and Phe6.52
according to the numbering proposed by Ballesteros and
Weinstein.13 Such a cluster, is involved in the ligand binding as
evaluated. In addition, I2-IBS and
derivatives were determined on rat whole brain membranes.
a2-AR affinities of all the novel
2. Chemistry
The imidazolines 5a and 5f–k were obtained by reaction of the
esters 4a–c10 with the proper ethylendiamines (Scheme 1). The
imidazolines 5b–e were obtained by treatment of 5a with the suit-
able alkyl iodide. The target compounds 3a–k could be promptly
obtained by O-demethylation carried out with a solution of BBr3
in dichloromethane or with a solution of 48% HBr and acetic acid
under reflux temperature.
3. Results and discussion
Affinity (Ki,
lM), potency (EC50, lM) and intrinsic activity (ia)
values, of compounds 3a–k are reported in Table 1 along with
those of 1. From our study it emerged that 1 partially activated
R1
R2
N
O
N
R
3a
3b
3c
3d
3e
3f
R=H
R1=OH
R1=OH
R1=OH
R1=OH
R1=OH
R1=H
R2=H
R2=H
R2=H
R2=H
R2=H
R2=OH
3g
3h
3i
3j
3k
R=n-C3H7 R1=H
R=CH2Ph R1=H
R2=OH
R2=OH
R2=OH
R2=OH
R2=OH
R=CH3
R=C2H5
R=n-C3H7
R=CH2Ph
R=H
R=H
R1=OH
R=n-C3H7 R1=OH
R=CH2Ph R1=OH
Chart 2. Novel imidazoline molecules 3a–k inspired by 2-BFI (1).

G. Giorgioni et al. / Bioorg. Med. Chem. 18 (2010) 7085–7091
7087
R1
R1
R2
N
N
iii
O
i
3a-k
O
O
OEt
R2
R
5a
R = H
4a
4b
R1 = OCH3 R2 = H
R1 = OCH3 R2 = H
R1 = OCH3 R2 = H
R1 = OCH3 R2 = H
R1 = OCH3 R2 = H
R1 = OCH3 R2 = H
R1 = H R2 = OCH3
5bii R = CH3
4c R1 = OCH3 R2 = OCH3
5cii R = C2H5
5dii R = n-C3H7
5eii R = CH2Ph
5f
R = H
R1 = H
R1 = H
R1 = H
R2 = OCH3
R2 = OCH3
R2 = OCH3
5g R = n-C3H7
5h R = CH2Ph
5i
5j
R = H
R = n-C3H7
R = CH2Ph
R1 = OCH3 R2 = OCH3
R1 = OCH3 R2 = OCH3
R1 = OCH3 R2 = OCH3
5k
Scheme 1. Reagents: (i) RNHCH2CH2NH2; Me3Al; (ii) from 5a: NaH, R-X; (iii) 48% HBr/CH3COOH or BBr3.
Table 1
Binding affinity, efficacy and intrinsic activity of compounds 3a–ka
Asp3.32
Compd
D2-like receptorsb I2-IBSc
M) EC50 M) ia Ki ( M)
0.57 0.0034 0.0012 3.98 0.60
a
2-ARsc
Ki (
l
(
l
l
Ki ( M)
l
1 (2-BFI) 47d
37.7 5.1
Phe6.51
3a
3b
3c
3d
3e
3f
3g
3h
3i
>100
>100
>100
15.81 1.01 nt
5.0 0.23
14.45 1.10 5.7 0.7
13.84 1.30 13.3 1.6
5.66 1.00
>100
>100
18.49 1.3
3.90 0.59
nt
nt
nt
nt
nt
nt
nt
0.0881 0.005
nt
>100
2.95 0.53
nt
25.5 5.82
3.64 0.50
4.39 0.51
11.0 2.31
1.92 0.32
2.08 0.20
24.21 6.71
10.16 3.92
4.43 2.80
nt
0.70 0.20
6.3 0.8
0.85 1.10 0.31
0.48 0.076 0.02
0.64 2.10 0.51
0.82 5.78 0.48
nt
nt
Ser5.42
Ser5.46
Trp6.48
Phe6.44
5.8 0.8
nt
nt
11.9 1.2
4.8 0.6
5.69 0.52
28.02 2.33
3j
3k
DA
0.52 10.39 1.80
1.00 nt
Phe6.52
a
The values are means SEM of at least three experiments; nt = not tested.
Inhibition of [3H] YM-09-151-2 (D2-like) binding to porcine striatal membranes.
b
EC50 and ia were measured by the [35S]GTP
cS binding experiments in striatal
Figure 1. Docking of 3h into the D2 receptor binding. Side chains of Asp3.32,
Ser5.42, Ser5.46, Phe6.44, Trp6.48, Phe6.51 and Phe6.52 are displayed to help
interpretation of the proposed pose that was scored with a favourable free energy of
binding of ꢀ3.93 kcal/mol.
membranes.
c
I2-IBS and
a2-ARs binding were determined on rat whole brain membranes.
d
Ref.8.
well as in the structural rearrangements that participate in recep-
tor activation.14 It is also reported to be highly conserved among
the whole family A of GPCRs.15 However, taking also into account
that the catechol derivatives 3i and 3j lack of D2-like affinity, the
possibility that the imidazoline molecules bind the receptor in an
alternative mode compared to that of catecholamines can not be
ruled out. On the other hand, Waugh et al. already proposed that
imidazolines bind differently in the agonist pocket than phenethyl-
amine agonists.16,17
To investigate this hypothesis, in the absence of any experimen-
tal structural data related to the biological target considered in this
study, a theoretical model of the human D2 receptor, based on the
rhodopsine X-ray structure was considered.18 On the basis of the
obtained biological experimental data, we considered determinant
for the binding to the receptor the hydrophobic interaction of the
pendant benzyl group with the aforementioned cluster of aromatic
residues. In our simulation (Fig. 1), a plausible correspondence of
critical fragments of the ligand with the aminoacids crucial for
D2-like activity, can be achieved when 3h interacts with the bind-
ing pocket in an almost flipped out way compared to that of the
natural ligand DA. Such an unexpected observation, might be pos-
sible only if the key interaction, such as the ionic bridge towards
Asp3.32 of TM3,19 characterizing the docking of DA, might be
played by a charge reinforced hydrogen bond given by the 6-hy-
droxy group of 3h with the same aminoacid. Beside this polar
interaction, the benzofuran ring might make hydrophobic interac-
tion with Trp6.48 and Phe6.51, while the aromatic tail of N-benzyl
group is packed against the benzene rings of Phe6.44 and Phe6.52,
perhaps stabilizing, at the same time, the positive charge of the
imidazoline ring through a charge transfer complex. Moreover,
the docking of 5h, the 3h intermediate bearing a methoxy group
in 6 position, did not produce any valuable pose in the receptor
binding site. The proposed binding mode, might better rationalize
the increased intrinsic activity observed for 3h compared to 3f.
Deeper investigations are under progress to verify such
hypothesis.
a
Anyway, the degree of receptor activation, favoured by the N-
substitution was affected by the feature of the pendant group
(i.e., lipophilicity) and, sometimes, also by the structural character-
istics of the parent compound. The 6-hydroxy-N-propyl derivative
3g, for example, displayed affinity, potency and intrinsic activity
values (Ki = 13.84; EC50 = 13.3, ia = 0.64) slightly lower than those
of the corresponding N-benzyl derivative 3h. Comparable D2-like
affinity was observed for the 5-hydroxy derivative 3d
(Ki = 15.81), whereas no D2-like binding was shown by the 5,6-
diydroxy-N-propyl (3j), 5-hydroxy-N-methyl (3b) and N-ethyl
(3c) derivatives. Generally, the new compounds displayed
a2-AR
affinity similar to that of the lead.

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G. Giorgioni et al. / Bioorg. Med. Chem. 18 (2010) 7085–7091
stirred for 5 min then evaporated to dryness. The residue was
4. Conclusion
recrystallized to give the product as oxalate salt (Table 3).
Similarly 3b–e were prepared (Table 3). 3f was characterized as
hydrobromide after the first solvent evaporation.
In conclusion, the present study: (i) demonstrated the validity
of our design, directed to obtain novel imidazoline molecules tar-
geting D2-like receptors; (ii) confirmed the biological versatility
of the imidazoline nucleus and the role of its substituent in posi-
tion 2; (iii) highlighted that the N-substitution induced a decrease
of I2-IBS affinity, but might positively modulate the D2-like activ-
ity; (iv) identified the D2-like nearly full agonists 3h and 3e, and
the partial agonist 3f, all endowed with a potency value compara-
ble to that of DA. 3f, in addition, displayed a significant I2-IBS affin-
ity. To evaluate in vivo the behaviour of our compounds, the
agonist 3h was, preliminarily, assessed on open field test in rats.
It is known that the performance in open field task may provide
a good measure of the stimulant effects induced by DA agonists
and L-DOPA.20 We observed that intracerebroventricular injection
5.1.3. 2-(1-Propyl-4,5-dihydro-1H-imidazol-2-yl)benzofuran-6-
ol hydrobromide (3g)
To a solution of 5g (1.16 mmol) in CH2Cl2 (7 mL) at ꢀ78 °C, a
2 M solution of BBr3 (10.45 mmol) in CH2Cl2 was added dropwise.
The mixture was left to warm to rt and stirred overnight. It was
cooled again to ꢀ78 °C, and MeOH (3 mL) was carefully added.
After stirring at rt for 15 min, the solvent was removed and the res-
idue was recrystallized (Table 3).
Similarly 3h–k were prepared. (Table 3).
5.1.4. 2-(5-Methoxybenzofuran-2-yl)-4,5-dihydro-1H-imidazole
(5a)
with equimolar doses (0.0149 lM/kg body weight) of 3h or
Ethylendiamine (7.27 mmol) was added to a 2 M solution of
Me3Al (7.27 mmol) in toluene at a temperature below 10 °C (ice
bath). When the gas formation was finished, ethyl 5-meth-
oxybenzofuran-2-carboxylate (4a)10 (4.54 mmol) was added por-
tionwise at rt. The solution was refluxed for 3 h; after cooling,
water (2 mL) was added and the mixture diluted with a 50% solu-
tion of CH2Cl2 in MeOH (15 mL), then refluxed for 15 min. After fil-
tration and evaporation to dryness, ethyl acetate (25 mL) was
added and the solution refluxed for 15 min. Evaporation of the
dried mixture gave a residue that was recrystallized (Table 3).
Similarly 5f–k were prepared (Table 3).
L-DOPA induced a similar trend with increased locomotion and
rearing in the exploratory behaviour (Table 2). The overall results
justify the interest for this novel class of compounds and encour-
ages the continuation of our study.
5. Experimental section
5.1. Chemistry
5.1.1. General procedures
Melting points were taken in glass capillary tubes on a Büchi
SMP-20 apparatus and are uncorrected. IR and NMR spectra were
recorded on Perkin–Elmer 297 and Varian EM-390 instruments,
respectively. Chemical shifts are reported in parts per million
(ppm) relative to tetramethylsilane (TMS), and spin multiplicities
are given as s (singlet), d (doublet), t (triplet), q (quartet), or m
(multiplet). Mass spectra were recorded on a HPLC-DAD-RID-MS
(ion trap) 1100—LC MSD trap sl Agilent Technologies. IR spectra
data, not shown because of the lack of unusual features, were ob-
tained for all compounds reported and are consistent with the as-
signed structures. The microanalyses were performed by the
Microanalytical Laboratory of the Department of Chemical Sci-
ences. The elemental composition of the compounds agreed to
within 0.4% of the calculated value. Chromatographic separations
were performed on silica gel columns (Kieselgel 40, 0.040–
0.063 mm, Merck) by flash chromatography. The term ‘dried’ refers
to the use of anhydrous sodium sulphate. Compounds were named
following the IUPAC rules proposed by CSC ChemDraw (version
9.0).
5.1.5. 2-(5-Methoxybenzofuran-2-yl)-1-methyl-4,5-dihydro-1H-
imidazole (5b)
To a solution of 5a (2.59 mmol) in dry THF (20 mL) a 60% sus-
pension of NaH in mineral oil was added portionwise. The mixture
was refluxed until the gas bubbling ceased, then cooled to rt.
Methyl iodide (2.59 mmol) was added and the solution stirred at
rt for 12 h, then poured onto ice and extracted with ethyl acetate.
Evaporation of the dried solvent gave the crude product that was
used in the next step without further purification (Table 3).
Similarly 5c–e were prepared (Table 3).
5.2. Biological experiments
5.2.1. General procedures to estimate Ki values
The tested compounds were dissolved in DMSO. The level of
DMSO did not exceed 1% and was maintained constant in all tubes.
At least six different concentrations of each compound were used.
The IC50 values, computer-generated using a nonlinear regression
formula on the computer program GraphPad Prism, version 4.0
for Windows (San Diego, California, USA), were converted to Ki
values according to the equation of Cheng and Prusoff.21 Data
represent the mean SEM of 3–5 separate experiments performed
in triplicate. Protein concentration was assayed by the method of
Bradford.22
5.1.2. 2-(4,5-Dihydro-1H-imidazol-2-yl)benzofuran-5-ol oxalate
(3a)
The imidazoline 5a (1.85 mmol) was dissolved in a mixture of
acetic acid (3 mL) and 48% HBr solution (3 mL) and refluxed
overnight. The solvent was removed and the solid residue was dis-
solved in a small amount of NaHCO3 solution and extracted with
ethyl acetate. The evaporation of the dried organic fraction gave
a residue that was dissolved in a solution of oxalic acid in ethanol,
5.2.1.1. I2-IBS and 2-ARs binding assays. Rat whole brain mem-
branes were prepared as previously described.23 Briefly, brains
were homogenized in 10 volumes (w/v) of Tris–sucrose buffer
(50 mM Tris–HCl, 320 mM sucrose and 1 mM MgCl2, pH 7.4) using
a motor driven Teflon glass homogeniser. The homogenate was
centrifuged at 1000g for 10 min at 4 °C and the resulting superna-
tant was recentrifuged at 32,000g for 20 min at 4 °C. The resulting
pellet was washed twice by resuspension in 10 volumes of assay
buffer (50 mM Tris–HCl and 1 mM MgCl2, pH 7.4) and centrifuga-
tion at 32,000g for 20 minutes at 4 °C. The membrane pellets were
stored at ꢀ70 °C until use. Prior to binding studies, pellets were
thawed and washed twice more by centrifugation as described
Table 2
Effects of icv injection of vehicle (control group), L-Dopa and 3h on ambulatory and
rearing episodes in rats
Groups
% Ambulatory episodes
% Rearing episodes
Control
L-Dopa
3h
3.2 0.4
5.7 0.9*
6.3 1.2*
0.5 0.3
2.0 0.3*
2.4 0.7*
Data are expressed as mean SE of 8 rats per group.
*
P <0.05 versus control.

G. Giorgioni et al. / Bioorg. Med. Chem. 18 (2010) 7085–7091
7089
Table 3
Physicochemical characteristics of compounds 3a–k and 5a–k
Compd
Yield (%)
Anal. C, H, N
mp (°C)
ESIMS
NMR, d# (DMSO-d6), d& (CDCl3)
m/z (MH+)
3aa
70
C11H10N2O2ꢁH2C2O4ꢁH2O
203
[1H]NMR d# 10.81 (br s, 2H, NH2þ); 9.65 (br s, 1H, OH); 7.88 (s, 1H, ArH); 7.55 (d,
J = 8.96 Hz, 1H, ArH); 7.08–7.02 (m, 2H, ArH); 3.98 (s, 4H, 2CH2). [13C]NMR d# 164.2
(2 ꢂ C@O); 155.6 (C imid); 154.6 (C, Ar); 149.7 (C, Ar); 139.4 (C, Ar), 127.2 (C, Ar);
118.8 (CH, Ar); 115.6 (CH, Ar); 112.4 (CH, Ar); 106.8 (CH, Ar); 44.1 (2 ꢂ CH2)
>300
3ba
3ca
3da
3ea
68
72
58
62
C12H12N2O2ꢁH2C2O4
217
[1H]NMR d# 10.46 (br s, 1H, NH+); 9.70 (br s, 1H, OH); 8.00 (s, 1H, ArH); 7.60 (d,
J = 8.89 Hz, 1H, ArH); 7.15–7.04 (m, 2H, ArH); 4.10–3.86 (m, 4H, 2CH2); 3.40 (s, 3H,
CH3). [13C]NMR d# 162.3 (2 ꢂ C@O); 155.0 (C imid); 154.6 (C, Ar); 150.2 (C, Ar); 138.8
(C, Ar), 127.6 (C, Ar); 119.7 (CH, Ar); 117.4 (CH, Ar); 113.3 (CH, Ar); 107.1 (CH, Ar);
50.7 (CH2); 42.7 (CH2); 42.2 (CH3)
>300
C13H14N2O2ꢁH2C2O4
231
245
293
[1H]NMR d# 10.52 (br s, 1H, NH+); 9.55 (br s, 1H, OH); 7.73 (s, 1H, ArH); 7.48 (d,
J = 8.96 Hz, 1H, ArH); 7.11–7.02 (m, 2H, ArH); 3.88 (s, 4H, 2CH2); 3.69 (q, 2H, CH2);
1.23 (t, J = 7.10 Hz, 3H, CH3). [13C]NMR d# 162.3 (2 ꢂ C@O); 155.0 (C imid); 154.5 (C,
Ar); 149.9 (C, Ar); 138.7 (C, Ar), 127.4 (C, Ar); 119.3 (CH, Ar); 117.2 (CH, Ar); 112.9
(CH, Ar); 106.9 (CH, Ar); 50.6 (CH2); 43.0 (CH2); 42.7 (CH2); 12.3 (CH3)
[1H]NMR d# 10.58 (br s, 1H, NH+); 9.45 (br s, 1H, OH); 7.96 (s, 1H, ArH); 7.57 (d,
J = 9.00 Hz, 1H, ArH); 7.16–7.05 (m, 2H, ArH); 4.05–3.71 (m, 6H, 3CH2); 1.78–1.67 (m,
2H, CH2); 0.95 (t, J = 7.46 Hz, 3H, CH3). [13C]NMR d# 162.3 (2 ꢂ C@O); 154.6 (C imid);
154.5 (C, Ar); 149.5 (C, Ar); 138.4 (C, Ar), 127.0 (C, Ar); 118.9 (CH, Ar); 116.8 (CH, Ar);
112.5 (CH, Ar); 106.5 (CH, Ar); 50.8 (CH2); 48.7 (CH2); 42.7 (CH2); 20.3 (CH2); 10.8 (CH3)
[1H]NMR d# 10.82 (br s, 1H, NH+); 9.61 (br s, 1H, OH); 7.96 (s, 1H, ArH); 7.60 (d,
J = 8.94 Hz, 1H, ArH); 7.46–7.40 (m, 5H, ArH); 7.16–7.05 (m, 2H, ArH); 5.08 (s, 2H,
CH2); 3.99 (s, 4H, 2CH2). [13C]NMR d# 162.3 (2 ꢂ C@O); 154.6 (2 ꢂ C, Ar); 149.5 (C,
Ar); 138.4 (C, Ar), 134.3 (C, Ar); 128.9 (2 ꢂ CH, Ar); 128.2 (CH, Ar); 127.7 (2 ꢂ CH, Ar);
126.8 (C, Ar); 119.1 (CH, Ar); 117.2 (CH, Ar); 112.5 (CH, Ar); 106.5 (CH, Ar); 50.9
(CH2); 50.7 (CH2); 42.7 (CH2)
>300
C14H16N2O2ꢁH2C2O4ꢁ4H2O
235–238
C18H16N2O2ꢁH2C2O4ꢁ2H2O
260–263
3fb
76
53
C11H10N2O2ꢁHBr
202
245
[1H]NMR d# 10.61 (br s, 2H, NH2þ); 10.35 (s, 1H, OH); 7.97 (s, 1H, ArH); 7.72 (d,
J = 8.65 Hz,1H, ArH); 7.01–6.92 (m, 2H, ArH); 3.97 (s, 4H, 2CH2). [13C]NMR d# 159.7 (C
imid); 157.1 (C, Ar); 155.35 (C, Ar); 137.2 (C, Ar), 124.3 (CH, Ar); 118.4 (CH, Ar); 116.7
(C, Ar); 1115.1 (CH, Ar); 97.2 (CH, Ar); 44.4 (2 ꢂ CH2)
[1H]NMR d# 10.36 (br s, 2H, NH+ and OH); 8.04 (s, 1H, ArH); 7.72 (d, J = 8.60 Hz, 1H,
ArH); 7.04–6.93 (m, 2H, ArH); 4.10–3.71 (m, 6H, 3CH2); 1.75–1.71 (m, 2H, CH2); 0.95
(t, J = 7.41 Hz, 3H, CH3). [13C]NMR d# 159.8 (C imid); 156.8 (C, Ar); 154.3 (C, Ar);
136.44 (C, Ar), 124.2 (C, Ar); 118.3 (CH, Ar); 117.9 (CH, Ar); 115.3 (CH, Ar); 97.3 (CH,
Ar); 50.7 (CH2); 48.7 (CH2); 42.5 (CH2); 20.3 (CH2); 10.8 (CH3)
>300
3gb
C14H16N2O2ꢁHBr
273–275
3hb
69
C18H16N2O2ꢁHBrꢁH2O
293
[1H]NMR d# 10.40 (br s, 2H, NH+ and OH); 8.02 (s, 1H, ArH); 7.70 (d, J = 8.66 Hz, 1H,
ArH); 7.44–7.35 (m, 5H, ArH); 6.97–6.92 (m, 2H, ArH); 5.09 (s, 2H, CH2); 3.97 (s, 4H,
2CH2). [13C]NMR d# 159.9 (C imid); 156.9 (C, Ar); 154.5 (C, Ar); 136.4 (C, Ar), 134.4 (C,
Ar); 128.9 (2 ꢂ CH, Ar); 128.1 (CH, Ar); 127.6 (2 ꢂ CH, Ar); 124.2 (C, Ar); 118.2 (C, Ar);
118.3 (CH, Ar); 115.3 (CH, Ar); 97.3 (CH, Ar); 50.9 (CH2); 50.7 (CH2); 42.7 (CH3)
>300
3ib
3jb
62
42
C11H10N2O3ꢁHBrꢁH2O
219
261
[1H]NMR d# 10.49 (br s, 2H, NH2þ); 10.00 (br s, 1H, OH); 9.48 (br s, 1H, OH); 7.84 (m,
1H, ArH); 7.13–7.02 (m, 2H, ArH); 3.96 (s, 4H, 2CH2). [13C]NMR d# 155.3 (C, Ar); 150.9
(C, Imid); 149.6 (C, Ar); 144.76 (C, Ar); 136.8 (C, Ar), 117.9 (C, Ar); 116.5 (CH, Ar);
106.2 (CH, Ar); 97.4 (CH, Ar); 44.31 (2 ꢂ CH2)
[1H]NMR d# 10.24 (s, 1H, NH+); 10.02 (br s, 1H, OH); 9.56 (br s, 1H, OH); 7.94 (s, 1H,
ArH); 7.14 and 7.06 (2s, 2H, ArH); 4.07–3.89 (m, 4H, 2CH2); 3.73 (t, J = 7.45, 2H, CH2);
1.74–1.70 (m, 2H, CH2); 0.95 (t, J = 7.47 Hz, 3H, CH3). [13C]NMR d# 154.1 (C, Ar); 150.6
(C imid); 149.7 (C, Ar); 144.9 (C, Ar), 135.9 (C, Ar); 117.8 (C, Ar); 117.7 (CH, Ar); 105.9
(CH, Ar); 97.4 (CH, Ar); 50.7 (CH2); 48.6 (CH2); 42.4 (CH2); 20.3 (CH2); 10.8 (CH3)
>300
C14H16N2O3ꢁHBrꢁH2O
>300
3kb
38
C18H16N2O3ꢁHBr
308
[1H]NMR d# 10.46 (s, 1H, NH+); 10.02 (br s, 1H, OH); 9.06 (br s, 1H, OH); 7.90 (s, 1H,
ArH); 7.43–7.39 (m, 5H, ArH); 7.09–6.95 (m, 2H, ArH); 5.06 (s, 2H, CH2); 3.95 (s, 4H,
2CH2). [13C]NMR d# 154.4 (C, Ar); 150.8 (C imid); 149.9 (C, Ar); 144.9 (C, Ar); 135.9 (C,
Ar), 134.5 (C, Ar); 128.9 (2 ꢂ CH, Ar); 128.1 (CH, Ar); 127.6 (2 ꢂ CH, Ar); 118.1 (C, Ar);
117.8 (CH, Ar); 106.0 (CH, Ar); 97.4 (CH, Ar); 50.2 (CH2); 50.6 (CH2); 42.6 (CH2)
258–260
5ac
5b
5c
45
58
51
40
140–144
217
231
245
259
[1H]NMR d& 7.38 (d, J = 8.98 Hz, 1H, ArH); 7.27 (s, 1H, ArH); 7.06–6.94 (m, 2H, ArH);
3.86 (s, 3H, OCH3); 3.82 (br s, 4H, 2CH2)
[1H]NMR d& 7.48 (d, J = 9.18 Hz, 1H, ArH); 7.20 (s, 1H, ArH); 7.10–6.85 (m, 2H, ArH);
4.00–3.80 (m, 5H, OCH3 and CH2); 3.45 (t, J = 10.15 Hz, 2H, CH2); 3.18 (s, 3H, CH3)
[1H]NMR d& 7.42 (d, 1H, ArH); 7.27 (s, 1H, ArH); 7.18–7.10 (m, 2H, ArH); 4.20–3.95
(m, 6H, 3CH2); 3.80 (s, 3H, OCH3); 1.40 (t, J = 7.14, 3H, CH3)
—
—
—
5d
[1H]NMR d# 8.00 (s, 1H, ArH); 7.71 (d, J = 9.12 Hz, 1H, ArH); 7.38–7.18 (m, 2H, ArH);
4.13–3.66 (m, 9H, OCH3 and 3CH2); 1.83–1.66 (m, 2H, CH2); 0.95 (t, J = 7.43 Hz, 3H,
CH3)
5e
58
307
217
[1H]NMR d# 7.95 (s, 1H, ArH); 7.68 (d, 1H, ArH); 7.42–7.35 (m, 6H, ArH); 7.20 (m, 1H,
ArH); 5.10 (s, 2H, CH2); 4.02 (s, 4H, 2CH2); 3.80 (s, 3H, OCH3)
5fc
43.5
139–141
[1H]NMR d# 7.61–7.56 (d, J = 8.62 Hz, 1H, ArH); 7.26–7.20 (m, 2H, ArH); 6.95–6.89
(m, 1H, ArH); 3.79 (s, 3H, OCH3); 3.59 (br s, 4H, 2CH2)
(continued on next page)

7090
G. Giorgioni et al. / Bioorg. Med. Chem. 18 (2010) 7085–7091
Table 3 (continued)
Compd
Yield (%)
Anal. C, H, N
mp (°C)
ESIMS
NMR, d# (DMSO-d6), d& (CDCl3)
m/z (MH+)
5g
51
—
259
[1H]NMR d& 7.50 (d, J = 8.65 Hz, 1H, ArH); 7.27 (s, 1H, ArH); 7.04–6.90 (m, 2H, ArH);
4.02–3.81 (m, 5H, OCH3, CH2); 3.61–3.39 (m, 4H, 2CH2); 1.73–1.58 (m, 2H, CH2);
1.03–0.91 (t, J = 7.44 Hz, 3H, CH3)
5h
5ic
5j
50
50
46
43
—
307
247
289
337
[1H]NMR d&7.48–6.87 (m, 9H, ArH); 4.69 (s, 2H, CH2); 4.02–3.81 (m, 5H, OCH3 and
CH2); 3.51–3.40 (m, 2H, CH2)
140–142
[1H]NMR d# 7.41–7.36 (d, J = 8.93 Hz, 1H, ArH); 7.27 (s, 1H, ArH); 6.99–6.94 (m, 1H,
ArH); 3.84–3.82 (m, 10H, 5,6-OCH3 and 2CH2)
[1H]NMR d& 7.27–7.01 (m, 3H, ArH); 3.99–3.90 (m, 8H, 2OCH3 and CH2); 3.57–3.60
(m, 4H, 2CH2); 1.73–1.64 (m, 2H, CH2); 1.03–0.95 (t, J = 7.46, 3H, CH3)
[1H]NMR d& 7.37–7.26 (m, 6H, ArH); 7.13–7.00 (m, 2H, ArH); 4.68 (s, 2H, CH2); 3.97–
—
—
5k
3.91 (m, 8H, 5,6-OCH3, CH2); 3.45 (s, 2H, CH2)
a
b
c
Recrystallized from ethanol/diethyl ether.
Recrystallized from methanol.
Recrystallized from ethyl acetate.
above. Rat brain membranes (about 300
l
g of proteins) were
parameters of [3H]SCH23390 were: Kd = 1.33 0.12 nM and
Bmax = 320 36 fmol/mg of proteins.
incubated to equilibrium (30 min, room temperature) with
increasing concentrations of the tested ligands and fixed concen-
tration (1 nM) of [3H]2-BFI for I2-IBS or [3H]RX821002 for
a2-ARs.
5.2.2. [35S]GTP
35S]GTP
cS binding experiments in striatal membranes were
cS binding assays
Incubations were terminated by rapid filtration through 0.5% poly-
ethyleneimine pre-soaked Whatman GF/B filters using a Brandel
M-24 cell harvester. Filters were washed twice with 5 mL of ice-
cold assay buffer and the amount of radioactivity retained on them
was determined by liquid scintillation counting. Nonspecific bind-
ing of [3H]2-BFI or [3H]RX821002 was defined in the presence,
[
performed as previously described by Odagaki and Toyoshima.25
This method allows to study G protein activation through D2-like
receptors. Briefly, aliquots of striatal membranes equivalent to
20
50 mM Tris–HCl buffer (pH 7.4) containing 100
MgCl2, 0.1 mM EDTA, 0.2 mM EGTA, 0.2 mM DTT and 150 mM NaCl
in the presence of 0.2 nM [35S]GTP
S and six different concentra-
tions of newly synthesized compounds. Non specific binding was
determined in the presence of 100 M unlabeled GTP S. The reac-
l
g of protein were incubate at 30 °C for 60 min in 500
ll of
lM GDP, 5 mM
respectively of 10
2-ARs.
lM BU224 for I2-IBS or 10 lM rauwolscine for
a
c
5.2.1.2. D2-like receptors binding assays. Porcine striatal mem-
branes were prepared as previously described.24 In brief, tissue
was homogenized in 20 volumes of ice-cold 50 mM Tris–HCl buffer
l
c
tion was terminated by rapid filtration through GF/C glass fiber fil-
ter washing twice with 4 ml of ice-cold 50 mM Tris/HCl buffer (pH
7.4). The bound radioactivity was measured by liquid scintillation
spectrometry. The EC50 values were obtained by non linear regres-
sion method using the computer program GraphPad Prism, version
4.0 for Windows and are presented as mean SEM of three inde-
pendent experiments. Intrinsic activity is relative to the full agonist
dopamine where the percent maximal of increase above the basal
binding (%Emax) was set to 1.
at pH 7.4 (buffer T) containing protease inhibitors (20
lg/mL soy-
bean trypsin inhibitor, 200 g/mL, and 160 g/mL benzamidine),
l
l
using an Ultra-Turrax TP-1810. The homogenate was centrifuged
at 50,000g for 10 min at 4 °C. The resulting pellet was then washed
once by resuspension in buffer T and recentrifuged. The final pellet
was frozen at ꢀ20 °C until the time of assay. Striatal membranes
(about 200 lg of proteins) were incubated to equilibrium (60 min,
at 30 °C) with increasing concentrations of the tested ligands and
fixed concentration (0.3 nM) of [3H]YM-09-151-2. Incubation was
terminated by dilution to 5 mL with ice cold buffer T, followed
immediately by rapid filtration through glass fiber Whatman GF/C
filters. The filters were then washed (3 ꢂ 5 mL) with buffer T and
the amount of radioactivity retained on them was determined by li-
quid scintillation counting. Nonspecific binding was defined in the
presence of 2.5 mM dopamine. In porcine striatal membranes, the
equilibrium binding parameters of [3H]YM-09-151-2 were:
Kd = 0.40 0.03 nM and Bmax = 350 39 fmol/mg of proteins.
5.2.3. In vivo studies
5.2.3.1. Animals. Male Wistar rats (n = 24) (Harlan, UD, Italy) that
weighed 200–225 g at the beginning of the experiments were used.
The animals were individually housed in a room on a 12 h light/
dark cycle (lights off at 7:00 A.M.) at constant temperature (20–
22 °C) and humidity (45–55%). Rats were offered food pellets
(4RF; Mucedola, Settimo Milanese, Italy) and tap water ad libitum.
All procedures were conducted in adherence to the European Com-
munity Council Directive for Care and Use of Laboratory Animals.
5.2.1.3. D1-like receptors binding assays. Porcine striatal mem-
branes were prepared as previously described for D2-like receptors
binding assays. [3H]SCH23390 binding to D1-like receptors was as-
sayed in a final incubation volume of 0.5 mL, which contained
crude membranes (ꢃ0.2 mg of protein), radioligand (ꢃ0.5 nM),
and the tested compound in the range 10ꢀ8–10ꢀ4 M concentrations
at 30 °C for 60 min. Incubation was terminated by dilution to 5 mL
with ice cold buffer T, followed immediately by rapid filtration
through glass fiber Whatman GF/C filters. The filters were then
washed (3 ꢂ 5 mL) with buffer T and the amount of radioactivity
retained on them was determined by liquid scintillation counting.
Nonspecific binding was defined in the presence of 2.5 mM dopa-
mine. In porcine striatal membranes, the equilibrium binding
5.2.3.2. Surgical procedure and treatments. After anesthesia
with 10 mg/kg of a mixture of zolazepam and tiletamine (Zoletil
100, Italmed, Italy) by intraperitoneally injection, a stainless steel
cannula was implanted in the animal’s lateral cerebroventricle
using a stereotaxic instrument into the right ventricle (1.0 mm
posterior to the bregma, 1.8 mm lateral to the midline, and
3.5 mm ventral to the surface of the skull, according to the brain
atlas of Paxinos and Watson.26 Rats were divided in three groups:
the control group received vehicle (physiological solution) into the
right ventricle; the second group was administered by icv with
0.0149
lM/kg body weight of L-Dopa and the third group was trea-
ted by icv with equimolar dose of 3h.

G. Giorgioni et al. / Bioorg. Med. Chem. 18 (2010) 7085–7091
7091
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a
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Acknowledgement
The authors kindly acknowledge Dr. Henri Xhaard for the sup-
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