Synthesis, dopaminergic profile, and molecular dynamics calculations of N-aralkyl substituted 2-aminoindans

Article abstract of DOI:10.1016/j.bmc.2007.12.027

Brain dopaminergic system has a crucial role in the etiology of several neuropsychiatric disorders, including Parkinson's disease, depression, and schizophrenia. Several dopaminergic drugs are used to treat these pathologies, but many problems are attributed to these therapies. Within this context, the search for new more efficient dopaminergic agents with less adverse effects represents a vast research field. The aim of the present study was to synthesize N-[2-(4,5-dihydroxyphenyl)-methyl-ethyl]-4,5-dihydroxy-2-aminoindan hydrobromide (3), planned to be a dopamine ligand, and to evaluate its dopaminergic action profile. This compound was assayed as a diastereoisomeric mixture in two experimental models: stereotyped behavior (gnaw) and renal urinary response, after central administration. The pharmacological results showed that compound 3 significantly blocked the apomorphine-induced stereotypy and dopamine-induced diuresis and natriuresis in rats. Thus, compound 3 demonstrated an inhibitory effect on dopaminergic-induced behavior and renal action. N-[2-(-Methyl-ethyl)]-4,5-dihydroxy-2-aminoindan hydrobromide (4) was previously reported as an inotropic agent, and in the present work it was also re-evaluated as a diastereoisomeric mixture for its possible central action on the behavior parameters such as stereotypy and dopamine-induced diuresis and natriuresis in rats. Our results indicate that compound 4 produces an agonistic response, possibly through dopaminergic mechanisms. To better understand the experimental results we performed molecular dynamics simulations of two complexes: compound 3/D₂DAR (dopamine receptor) and compound 4/D₂DAR. The differential binding mode obtained for these complexes could explain the antagonist and agonist activity obtained for compounds 3 and 4, respectively.

Full text of DOI:10.1016/j.bmc.2007.12.027

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 3233–3244
Synthesis, dopaminergic profile, and molecular
dynamics calculations of N-aralkyl substituted 2-aminoindans
Sebastian A. Andujar,a Biagina Migliore de Angel,b Jaime E. Charris,c Anita Israel,d
b
e
d
f
´
´
Heberto Suarez-Roca, Simon E. Lopez, Maria R. Garrido, Elvia Victoria Cabrera,
Gonzalo Visbal,g Cecire Rosales,f Fernando D. Suvire,a
Ricardo D. Enriza, and Jorge E. Angel-Guıo
f,
*
*
´
aDepartamento de Quı´mica, Universidad Nacional de San Luı´s, Chacabuco 915-5700, San Luis, Argentina
bSeccio´n de Farmacologı´a, Instituto de Investigaciones Clı´nicas, Dr. Ame´rico Negrette, Facultad de Medicina,
Universidad del Zulia, Apartado # 526, Maracaibo, Venezuela
cLaboratorio de Sı´ntesis Orga´nica, Facultad de Farmacia, Universidad Central de Venezuela, Venezuela
dLaboratorio de Neurope´ptidos, Facultad de Farmacia, Universidad Central de Venezuela, Venezuela
eLaboratorio de Quı´mica Medicinal y Heterociclos, Dpto. de Quı´mica, Universidad Simo´n Bolı´var, Venezuela
fLaboratorio de Sı´ntesis Orga´nica y Disen˜o de Fa´rmacos, Departamento de Quı´mica,
Facultad Experimental de Ciencias Universidad del Zulia, Apartado # 526, Maracaibo, Venezuela
gLaboratorio de Sı´ntesis Orga´nica y Productos Naturales, Centro de Quı´mica,
Instituto Venezolano de Investigaciones Cientı´ficas (IVIC), Alto de Pipe, Venezuela
Received 28 August 2007; revised 7 December 2007; accepted 11 December 2007
Available online 31 December 2007
Abstract—Brain dopaminergic system has a crucial role in the etiology of several neuropsychiatric disorders, including Parkinson’s
disease, depression, and schizophrenia. Several dopaminergic drugs are used to treat these pathologies, but many problems are
attributed to these therapies. Within this context, the search for new more efficient dopaminergic agents with less adverse effects
represents a vast research field. The aim of the present study was to synthesize N-[2-(4,5-dihydroxyphenyl)-methyl-ethyl]-4,5-dihy-
droxy-2-aminoindan hydrobromide (3), planned to be a dopamine ligand, and to evaluate its dopaminergic action profile. This com-
pound was assayed as a diastereoisomeric mixture in two experimental models: stereotyped behavior (gnaw) and renal urinary
response, after central administration. The pharmacological results showed that compound 3 significantly blocked the apomor-
phine-induced stereotypy and dopamine-induced diuresis and natriuresis in rats. Thus, compound 3 demonstrated an inhibitory
effect on dopaminergic-induced behavior and renal action. N-[2-(-Methyl-ethyl)]-4,5-dihydroxy-2-aminoindan hydrobromide (4)
was previously reported as an inotropic agent, and in the present work it was also re-evaluated as a diastereoisomeric mixture
for its possible central action on the behavior parameters such as stereotypy and dopamine-induced diuresis and natriuresis in rats.
Our results indicate that compound 4 produces an agonistic response, possibly through dopaminergic mechanisms. To better under-
stand the experimental results we performed molecular dynamics simulations of two complexes: compound 3/D2DAR (dopamine
receptor) and compound 4/D2DAR. The differential binding mode obtained for these complexes could explain the antagonist
and agonist activity obtained for compounds 3 and 4, respectively.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
of these substances in the brain. The dopamine system is
involved in the regulation of brain regions that subserve
motor, cognitive, and motivational behaviors, and dis-
ruptions of dopamine function have been implicated in
neurological and psychiatric disorders, including Par-
kinson’s disease, depression and, mainly, schizophre-
nia.1,2 In addition, brain dopamine (DA) is now
recognized as an important modulator of systemic blood
pressure through the regulation of fluid and sodium
metabolism and vasopressin release.3,4
Dopamine belongs to the monoaminergic group of neu-
rotransmitters and constitutes about 80% of the content
Keywords: Dopamine; N-Aralkyl 2-aminoindan derivatives; Dopa-
mine; Apomorphine-stereotypy; Molecular dynamics simulations.
*
Corresponding authors. Tel.: +54 2652 424689; fax: +54 2652 431301
(R.D.E.); e-mail addresses: denriz@unsl.edu.ar; jangel63@yahoo.
com; jangel@luz.edu.ve
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.12.027

3234
S. A. Andujar et al. / Bioorg. Med. Chem. 16 (2008) 3233–3244
Schizophrenia is a chronic psychiatric illness that affects
approximately 1% of the world population. Usually, a
schizophrenic patient presents two main kinds of symp-
toms: positive (delusions, hallucinations) and negative
(avolition, anhedonia, attentional impairment) symp-
toms. Most of the drugs used for treating this disorder
are D2-like receptor antagonists and are called typical
antipsychotic. These drugs are effective only for treating
the positive symptoms and present an important profile
of adverse effects, mainly the extrapyramidal distur-
bances such as tardive dyskinesia.1,2 For these reasons
the search for new more efficient dopaminergic agents
with lower adverse effects is still an extremely active re-
search field.
possible action on the dopaminergic system, we deter-
mined the behavioral parameters such as the stereotypy
and dopamine-induced diuresis and natriuresis after
central administration (ICV) in rats. In addition, for
the better understanding of the mechanism of action
of compounds 3 and 4 at molecular level, we simulated
the molecular interactions of both compounds with the
D2 dopamine receptor (D2DAR). These simulations
were carried out using molecular dynamics (MD) calcu-
lations. Binding pocket of the D2DAR was defined
according to Teeter et al.,14 while docking analysis was
done using GROMACS software package.15,16
2. Results and discussion
2.1. Biological activity
During the last decades, a large amount of N-substituted
2-aminoindan (1) and 2-aminotetraline (2) analogs with
peripheral and central nervous system action have been
reported.5–8 According to the well-established structure–
biological relationship for the dopaminergic activity9–12
as well as from our conformational studies,13 we predict
that compounds N-[2-(4,5-dihydroxyphenyl)-methyl-
ethyl]-4,5-dihydroxy-2-aminoindan hydrobromide (3)
and N-[2-(-methyl-ethyl)]-4,5-dihydroxy-2-aminoindan
hydrobromide (4) could be ligands with dopaminergic
activity. In particular since in their structures they have
the fragment of the pharmacophore needed for the inter-
action with the dopaminergic receptor (m-hydroxyphe-
nylethylamino fragment). Compound 3 represents a
rigid analog of the dopamine a rotamer, while com-
pound 4 has the same rotamer lacking the hydroxylic
substituent on the aromatic ring in the aralkyl group
(Fig. 1). Compound 4 was previously reported as an ino-
tropic agent. In the present study we were promoted to
synthesize compound 3 as a diastereoisomeric mixture
and evaluated the pharmacological profiles of both,
compounds 3 and 4. Thus, taking in consideration their
Stereotypy is a major component of several psychiatric
disorders, including childhood autism17 or schizophre-
nia.18 It is well-established that stereotypy (including
sniffing or gnawing) is a dopamine-dependent behav-
ior19 and the neural substrate of apomorphine-induced
stereotyped behavior in animals has been shown to in-
clude the central dopaminergic projections to the cau-
date-putamen region.20 Apomorphine is known to be a
mixed D1/D2 dopamine receptor agonist.19,21 The acti-
vation of the D1/D2 dopamine receptors on striatum is
expressed as the response of an excessive and repetitive
behavior (stereotypy). Furthermore, it has been demon-
strated that in adult and neonatal rats, lesioned with 6-
hydroxydopamine (6OH-DA), the stereotypy behavior
is the product of the activation of both receptor sub-
types. Recently, receptor D1 has been postulated as
responsible for the initiation of the stereotypy behavior,
while the participation of receptor D2 involved the
maintenance of such action, but not the initiation pro-
cess. That is why the combined action of the D1/D2
antagonists blocks the stereotypy in a more effective
way than the selective block through a selective antago-
nist of D1.22
The results of the experiments reported in the present
study demonstrated that compound 3 (0.6 lg/ll and
6 lg/ll), by itself, did not induce the stereotyped behav-
ior (gnaw). On the other hand, the central administra-
tion of compound 3 significantly reduced (6 lg/ll) or
inhibited (0.6 lg/ll) the stereotypy-induced by apomor-
phine (Fig. 3), indicating that in the central nervous sys-
tem compound 3 acts as a dopaminergic antagonist.
However, the selectivity as antagonist of the dopaminer-
gic receptors involved in the stereotypy can be reached
with the use of low doses.
The renal response to central administration of com-
pound 3 was also evaluated23–26 and is shown in Figure
4. Intracerebroventricular administration of dopamine
increased urinary volume at 6 h and sodium excretion
at 3 and 6 h periods of collection. Central pretreatment
with compound 3 did not alter basal urinary volume or
the sodium excretion, while completely blocked the
diuresis and natriuresis-induced by DA (Fig. 4). Several
neurotransmitters centrally applied are able to modulate
Figure 1. Structures of compounds (1–4). Showing the four torsion
angles h1–h4.

S. A. Andujar et al. / Bioorg. Med. Chem. 16 (2008) 3233–3244
3235
OMe
OMe
MeO
NaBH3CN
MeOH
OMe
NH2HCl
+
O
(5)
(6)
OMe
OMe
MeO
HBr 48%
OMe
NH
(7)
OH
OH
OH
HO
HBr
NH
(3)
Figure 2. Synthetic route of N-[2-(3,4-dihydroxyphenyl)-methyl-ethyl]-4,5-dihydroxy-2-aminoindan bromohydride 4.
500
300
200
100
0
6
5
4
3
2
1
0
*
Control
DA
**
Control
DA
*
**
*
**
*
400
300
200
100
0
**
*
**
*
**
**
*
**
*
**
*
*
**
2
**
6
**
8
**
9
**
4
**
10 11
**
3
**
5
**
7
*
0
1
OBSERVATION INTERVALS
C3 (6µg/µl)
SALINE
APOMORPHINE
C3 (0.6µg/µl)
C3 (6µg/µl) + APO
V
C3
V
C3
3 hr
V
C3
6 hr
V
C3
3 hr
C3 (0.6µg/µl) + APO
6 hr
Figure 3. Effect of intracerebroventricular administration of com-
pound 3 (0.6–6 lg/ll) on the stereotyped behavior (gnawing) in rats.
On the ordinate are the numbers of gnawing for interval. On the
abscise, the intervals of measurements of the behavior, observed during
4.5 min. The controls were injected with NaCl 0.9% and apomorphine
(1 mg/kg, sc). The results are expressed as means SEM of four
independent experiments. *p < 0.0001 compared with the vehicle and
**p < 0.0001 compared with apomorphine. One- or two-way ANOVA
was used, followed by the Duncan test. The results of compound 3 and
compound 3 + apomorphine are overlapped with the saline control.
Figure 4. Effect of compound 3 on the DA-induced diuresis and
natriuresis. Urinary response to ICV-DA in rats pre-treated with
compound 3. Groups of rats received an intracerebroventricular
injection of compound 3 (C3) (50 lg/5 ll) (N = 6–8) or vehicle (V)
(5 ll) (N = 6–8) 15 min before ICV-DA (100 lg/5 ll) or saline (5 ll)
followed by water po (20 ml/kg). Significant F; ratios (*p < 0.05 and
**p < 0.01) from two-way ANOVA were as follows: volume effect-DA
at 6 h and C8 at 3 h; and Na+ effect, dopamine at 3 and 6 h.
compared to the saline control. These behaviors were
blocked by haloperidol, a known antagonist of the
dopaminergic receptors, suggesting an effect through
dopaminergic mechanisms. It is important to mention
that compound 4-induced licking behavior was signifi-
cantly higher than that of apomorphine. In regard to
the renal effect observed in Figure 6, we demonstrate
that intracerebroventricular administration of com-
pound 4 significantly increased urinary volume and so-
dium excretion at the 6 h periods of collection
(p < 0.05), while compound 3 was ineffective.
the excretion of water and electrolytes.23–26 DA has been
postulated to function as a neurotransmitter, a neuro-
hormone, or both and it has been involved in the regu-
lation of fluid and electrolyte balance.23,24 In effect, DA
ICV-administered increases urinary volume and sodium
excretion (Fig. 4), suggesting a role for the dopaminergic
neurons besides the regulation of blood pressure and
thirst, possibly through the control of vasopressin
release.4
As shown in Figure 5, evaluation of compound 4 in the
stereotypy assays demonstrated a significant increment
in the licking, sniffing, and grooming behavior when
Our results of the inhibitory action of compound 3 on
the stereotypy behavior (gnaw) induced by apomor-
phine and the diuresis and natriuresis-induced by

3236
S. A. Andujar et al. / Bioorg. Med. Chem. 16 (2008) 3233–3244
º
SALINE
APOMORPHINE
HALO
C 4
*
2500
*
80
70
60
50
40
30
20
10
0
º
2000
*
C 4/HAL
1500
**
NS
1000
500
0
NS
NS
*
**
SALINE APO HALO
C4 C4/HAL
SALINE APO HALO
C4
C4/HAL
2250
1750
1250
750
*
1200
950
700
450
200
25
º
*
NS
250
10
*
**
0
0
SALINE APO HALO C4 C4/HAL
SALINE APO HALO
C4
C4/HAL
Figure 5. Effect of compound 4 (50 lg/5 ll) on the stereotyped behavior in rats blocked with haloperidol. On the ordinate, the sum of the behavior
measurements. On the abscise, the tested compounds. The observations were carried out during 1 h. The results are expressed as means SEM of
four independent experiments. The data were analyzed using one-way analysis of variance (ANOVA) and the test of Newman–Keul’s. *p < 0.001
compared to saline; **p < 0.001 compared to compound 4, ꢁp < 0.05 significant compared to apomorphine.
300
the opposite response on the dopaminergic system. This
Control
Control
*
Compound 4
4
3
2
1
0
would be in agreement with the reported results related
to the antagonist responses when a hydroxyl group is
present on the aromatic ring of the aralkyl fragment.13
There are several explanations for the different activities
obtained for compounds 3 and 4. Probably the reason
for the differential pharmacological response shown by
the two compounds, 3 and 4, could be due to the dispo-
sition of the hydroxyl groups on the aromatic ring of
compound 4, which could attribute a change in the con-
formational disposition of the latter in respect to the for-
mer, a fact that would not allow the adequate
orientation and interaction with the dopamine receptor
to promote the biological action of these compounds.13
Compound 4
*
250
200
150
100
50
0
3 hr
6 hr
3 hr
6 hr
Figure 6. Effect of
a
single intracerebroventricular injection of
To better understand the above experimental results, we
conducted a computer-assisted molecular dynamics sim-
ulation of the molecular interactions of both compounds
(3 and 4) with the D2 dopamine receptor.
compound 4 (50 lg/10 ll) on urine volume and sodium excretion in
conscious hydrated rats. Significant differences (two-way ANOVA) are
indicated. *p < 0.05, compared with control-saline. N = 6–8 animals
per group.
2.2. Molecular dynamics simulations
dopamine are in agreement with the predicted confor-
mational theoretical study and strongly suggest that
compound 3 represents a potential dopaminergic antag-
onist. The fact that compound 4 showed an opposite re-
sponse to that shown by compound 3 with respect to the
stereotypy behavior induced by apomorphine and the
diuresis and natriuresis-induced by dopamine suggests
that the incorporation of hydroxyl groups on the aro-
matic ring of the aralkyl fragment is responsible for
It should be noted that compounds 3 and 4 possess two
chiral centers and therefore they are diastereomeric with
the possibility of up to 4 isomers. However, we did not
perform a diastereomeric resolution for the biological as-
says; thus, only one isomer of each compound was evalu-
ated in the MD simulations. To choose one of the isomeric
forms of 3 and 4, we consider on one hand previously
reported results27 and on the other hand calculations

S. A. Andujar et al. / Bioorg. Med. Chem. 16 (2008) 3233–3244
3237
specially performed here determining the energetically
preferred form of these compounds. Our previous results
suggest that RR or RS forms would be the preferred iso-
mers for these compounds.27 We first optimize both iso-
mers RR and RS of compounds 3 and 4 using DFT
calculations. Our results indicate that RS isomeric forms
are energetically preferred with respect to the RR isomers
by 2.26 and 2.36 kcal/mol, for compounds 3 and 4, respec-
tively. Thus, on the basis of the DFT results we decide to
use RS isomers of 3 and 4 for the MD simulations. In addi-
tion, to better characterize these isomeric forms we calcu-
lated their respective specific rotations [a]D.
the ligand–receptor complexes reached stable average
values around 0.5 ns, whereas others takes longer time
periods. For such reason to ensure full equilibration,
only the last 9.5 ns were taken into account for the anal-
ysis. After discarding the first 0.5 ns of the trajectory, we
follow the changes of spatial ordering of the ligand–
receptor complexes (Fig. 7).
A highly conserved aspartic acid (Asp 86) in TMH3 is
important for the binding of both agonists and antago-
nists to the D2 receptor,36–38 and its terminal carboxyl
group may function as an anchoring point for ligands
with a protonated amino group.39 This structural
requirement was also observed for the D1 receptors.40
In the present study, both ligands compounds 3 and 4
were docked into the receptor with the protonated ami-
no group near Asp 86. After 10 ns of MD simulations,
the ligands had moved slightly but in a different form
compared with the initial position. Anyhow, the strong
interaction with Asp 86 was maintained for both com-
plexes (see Fig. 8), supporting the suggestion that Asp
86 functions as an anchoring point for ligands with a
protonated amino group. From Figure 8a and b it is
clear that a strong salt bridge forms for both compounds
between the protonated amino groups and the carboxyl
group of Asp 86.
Optical rotation calculations have been previously re-
ported for different molecules.28,29,32,35 Polavarapu28
and Polavarapu and Chakraborty29 reported calcula-
tions of [a]D for several compounds using the Amos
methodology30 in the CADPAC program.31 Polavarapu
and Zhao32 reported calculations of [a]D using the Hel-
gaker et al. methodology33 using the DALTON pro-
gram.34 However, the accuracies of optical rotations
calculated using the methodologies of Amos and Helga-
ker et al. are limited by their use of the HF/SCF meth-
odology, in which electron correlation is neglected.
More recently Stephens et al.35 reported that DFT
(B3LYP) provides substantially more accurate rotations
than HF/SCF methodologies and therefore constitutes
the current method of choice for stereochemical applica-
tions. They also showed that for these calculations, [a]D
values are strongly basis set dependent. In this sense
large basis sets containing diffuse functions give very
consistent results. On the basis of those results, we calcu-
late the [a]D using both RHF/6-31G(d) and B3LYP/6-
311++G(2d,2p) calculations. Our calculations predict
[a]D values of 106.32 and 103.5 for compound 3 using
RHF and DFT calculations, respectively. Whereas for
compound 4, these calculations predict [a]D values of
31.37 and 28.96, respectively. However caution is re-
quired for these results. It should be noted that the
above techniques apply to the calculations of [a]D values
of rigid molecules. In the case of conformationally flex-
ible molecules (like compounds 3 and 4), calculated [a]D
values are less accurate for two reasons. First, the [a]D
values of different conformations often differ in sign,
reducing the magnitude of predicted [a]D values. Second,
errors due to uncertainties in conformational popula-
tions are introduced. In this case the [a]D were calculated
for the significantly populated conformations obtained
from DFT calculations for compounds 3 and 4.
The equilibrium state of the complexes was observed from
onset of simulation until 10 ns. Temperature stabilized at
310 4 K for both complexes. Whereas the Potential En-
ergy stabilized at ꢀ462,000
2000 kJ/mol and
ꢀ467,500 2000 kJ/mol for compound 3/D2DAR com-
plex and compound 4/D2DAR complex, respectively.
These parameters stabilized in a short time period
(around 0.5 ns) suggesting that the system is well equili-
brated (see Figs. S1, S2, S3, and S4 in supporting
information).
Figure 7. Spatial view of the dopamine D2 receptor model from
reference 15 using the chimera program as graphic interface. In this
figure the binding pocket is denoted with a circle (dote line) including
compound 3 (in yellow), Asp 86 (in light blue), Ser 122, Ser 141, and
Ser 144 (in red).
Considering the 10 ns of MD simulation and from the
time profiles it was concluded that some properties of

3238
S. A. Andujar et al. / Bioorg. Med. Chem. 16 (2008) 3233–3244
0.60
0.55
0.50
0.45
0.40
0.35
0.30
0.25
0.20
0.15
0.10
0.60
compound 3
0.55
compound 4
0.50
0.45
0.40
0.35
0.30
0.25
0.20
0.15
0.10
0
1000 2000 3000 4000 5000 6000 7000 8000 9000 10000
time (ps)
0
1000 2000 3000 4000 5000 6000 7000 8000 9000 10000
time (ps)
Figure 8. The length of salt bridge between the Asp 86 and the protonated amine group of compounds 3 (a) and 4 (b). A geometric average of
interatomic distances between the three atoms of COOꢀ group with the nitrogen atom of amine was considered in this figure.
Pharmacological data with dopaminergic ligands36,41
indicate that the meta-hydroxyl group of dopaminergic
agonists is primarily important in stabilizing agonist
binding, suggesting that the serine residues (141 and
144) of the D2 receptor may not be equally important
for binding affinity. Individual mutation of serines 141
and 144 in TMH5 to alanine produced asymmetrical ef-
fects on dopamine receptor binding. These results indi-
cated that Ser 141 might be differentially important for
dopamine binding. In addition site-directed mutagenesis
studies have indicated that a cluster of serine residues in
TMH5 (Ser 141, Ser 144) and in TMH4 (Ser 122 and Ser
118) is important for agonist binding and receptor acti-
vation.38,39,42,43 It was suggested that the serine cluster
and dopamine form a hydrogen-bonding network. Such
a hydrogen-bonding network was reproduced by the
MD simulation of the compound 4/D2DAR complex
(Fig. 9). In this complex the strongest contributor to
˚
the network was Ser 141 (atomic distance 1.8 A) which
is consistent with the experimental observation that a
Ser 141 Ala mutated receptor completely lost dopa-
mine-induced activation.14 It should be noted that the
meta-hydroxyl group (X in Fig. 1) of compound 4 dis-
played another significant hydrogen bond interaction
with Ser 118, however this interaction is weaker (atomic
distance 2.0 A) with respect to the hydrogen bond with
Ser 141. In contrast to the above results, compound 3/
D2DAR complex does not show hydrogen bond be-
Figure 9. Interactions of compound 4 (ligand) with the D2 dopamine
receptor. Spatial view of two interactions: salt bridge (Asp 86 with
˚
protonated amino group) to the right and hydrogen bond between the
meta-hydroxyl group (X in Fig. 1) with Ser 141. The rest of the
tween meta-hydroxyl group and Ser 141 (Fig. 10). Only
the starting conformation of compound 3 displayed this
interaction due to the fact that we start the simulation
using a spatial ordering possessing this interaction.
However, during the rest of the 9.5 ns of simulation this
interaction just disappears. In fact, none of the hydroxyl
groups of compound 3 displayed any significant hydro-
gen bond with Ser 141. This is a striking difference with
respect to the results obtained for compound 4/D2DAR
complex. In the average complex after 10 ns MD, only
the hydroxyl R1 of compound 3 formed a strong hydro-
aminoacids were deleted to better appreciate the molecular interactions
and the spatial ordering of compound 4.
The conformational behavior of the four torsional an-
gles (h1–h4) of compounds 3 and 4 during the simulation
is shown in Figures 11 and 12. From these figures it is
clear that the conformational behavior of torsional an-
gles h1, h2, and h3 are very similar for both compounds
(Figs. 11 and 12a–c). Each of these three torsional angles
displayed a highly preferred conformation; gauche + -
form for h1 and h3 and anti form for h2. However it is
interesting to note that the conformational behavior
˚
gen bond with Ser 122 (atomic distance of 2.0 A)
(Fig. 10).

S. A. Andujar et al. / Bioorg. Med. Chem. 16 (2008) 3233–3244
3239
direction of Ser 122. The different conformation
adopted by compounds 3 and 4 in their respective com-
plexes might be well appreciated comparing Figures 9
and 10. Thus, it is reasonable to think that these differ-
ent interactions with serine cluster would be the princi-
pal factor responsible for the different conformational
behavior obtained for these complexes. Also it is reason-
able to think that this different binding mode could be
responsible for the antagonist and agonist effects dis-
played by compounds 3 and 4, respectively.
In conclusion, compound 3 seems to have a profile of ac-
tion coherent to dopaminergic function. On the basis of
the results obtained on the apomorphine-induced stereo-
typy and centrally mediated renal actions, we can con-
clude that compound 3 has demonstrated to be a
potential antagonist of the centrally mediated actions in-
duced by dopamine. The different binding mode ob-
tained from MD simulations for compound 3/D2DAR
and compound 4/D2DAR complexes could explain, at
least in part, the antagonist and agonist effect obtained
for compounds 3 and 4, respectively.
3. Experimental
3.1. Chemistry
Figure 10. Interactions of compound 3 (ligand) with the D2 dopamine
receptor. Spatial view of two interactions: salt bridge (Asp 86 with
protonated amino group) to the right and hydrogen bond between the
R1-hydroxyl group with Ser 122. The rest of the aminoacids were
deleted to better appreciate the molecular interactions and the spatial
ordering of compound 3.
Compound 3 was prepared through a reductive amina-
tion, combining a mixture of the derivative (5) and the
ketone (6) as shown in Figure 2. Thus, the 4,5-dime-
thoxy-2-aminoindan hydrochloride (5) was obtained
according to the procedure described in Refs. 6, 7, and
44 Subsequently, derivative (7) was hydrolyzed to pro-
duce compound 3 in good yield.9
obtained for torsional angle h4 of compound 4 (Fig. 12d)
is different from that of compound 3 (Fig. 11d). While h4
of compound 3 displays only one conformation (a per-
pendicular form with h4 ffi 90ꢁ); h4 of compound 4
shows a conformational change from perpendicular
(h4 ffi 90ꢁ) to anti-perpendicular (h4 ffi ꢀ90ꢁ) (see
Fig. 12d). This conformational change occurs at ffi7 ns
of simulation. This result is in a complete agreement
with the different positional RMSD (root mean square
deviation) profiles obtained for compounds 3 and 4,
respectively (Fig. 13). It appears that the strong hydro-
gen bond between the OH group at R1 of compound 3
and Ser 122 maintains this ring in the same conforma-
tion during all the simulation. In contrast, the phenyl
ring of compound 4 is allowed to perform an almost free
rotation during the simulation, and therefore a confor-
mational change takes place in this portion of the
molecule.
Uncorrected melting points were determined using a
Thomas Hoover Capillary Melting Point Apparatus.
NMR spectra were recorded using a Jeol Eclipse
270 MHz spectrometer using CDCl3 or DMSO-d6 and
are reported in ppm downfield from CHCl3 or DMSO
residual. Infrared spectra were determined as KBr pel-
lets on a Shimadzu model 470 spectrophotometer. Mass
spectra were recorded on a Hewlett Packard 5995 mass
spectrometer. The purity of all compounds was accessed
by thin layer chromatography using different polarity
solvents. Elemental analyses were performed on a Per-
kin-Elmer 2400CHN analyser, results were within
0.4% of predicted values. All solvents were distilled
and dried in the usual manner.
3.1.1.
N-[2-(4,5-Dimethoxyphenyl)-methyl-ethyl]-4,5-
Comparing the results obtained for both complexes
(compound 3/D2DAR and compound 4/D2DAR), inter-
esting conclusions might be obtained. Consistent with
previous experimental35,27 and theoretical37,29 results,
our simulations indicate the importance of the nega-
tively charged aspartate 86 for the binding of com-
pounds 3 (antagonist) and 4 (agonist). The main
difference in binding mode between compounds 3 and
4 was that the agonist bound the receptor with the
meta-hydroxyl in the direction of Ser 141 (in TMH5),
while the antagonist has the hydroxyl group R1 in the
dimethoxy-2-aminoindan hydrochloride (7). 4,5-Dime-
thoxy-2-aminoindan hydrochloride (5) (2.0 g, 10 mmol),
the corresponding 4,5-dimethoxy-phenyl acetone (6)
(1.94 g, 10 mmol), and sodium cyanoborohydride
(0.94 g, 15 mmol) in CH3OH 80 ml were stirred under
a nitrogen atmosphere for 48 h. The resulting mixture
was treated with an excess of 2 N HCl. The solvent
was removed under reduced pressure, the remaining so-
lid was dissolved in H2O, and the aqueous solution was
extracted with ether. The aqueous layer was made basic
using 20% NaOH solution and extracted with CHCl3.

3240
S. A. Andujar et al. / Bioorg. Med. Chem. 16 (2008) 3233–3244
400
350
300
250
200
150
100
50
400
θ1
θ2
350
300
250
200
150
100
50
0
0
0
2000
4000
6000
time (ps)
8000
10000
0
2000
4000
6000
time (ps)
8000
10000
400
350
300
250
200
150
100
50
400
350
300
250
200
150
100
50
c
d
θ3
θ4
0
0
0
2000
4000
6000
time (ps)
8000
10000
0
2000
4000
6000
8000
10000
time (ps)
Figure 11. Evolution of the angles h1 (a), h2 (b), h3 (c), and h4 (d) of compound 3 with time.
The organic layer was dried over anhydrous magnesium
sulfate; a steam of dry HCl was passed through the solu-
tion. Resulting crystals were collected by filtration.
Recrystallization was carried out from isopropanol–
ether (70%); mp 167–168 ꢁC. IR (KBr) cmꢀ1: 2950
(CH3); 2900 (CH2); 2.800–2750, 1625, 1612, 1525
ðR2 NH2þClꢀÞ; 1488, 1463 (C@C); 1275, 1263, 1075,
Indan); 121 (C2-Aralkyl); 122 (C7-Indan). MS m/e: the
molecular ion did not show up, 220 (100%); 177
(50%); 146 (10%); 91 (6%), 77 (4%). C22H30ClNO4:
407.94. Anal. Calcd C, 64.77; H, 7.41; N, 3.43. Found:
C, 65.03; H, 7.17; N, 3.26.
3.1.2. N-[2-(4,5-Dihydroxyphenyl)-methyl-ethyl]-4,5-
dihydroxy-2-aminoindan hydrobromide (3). The hydro-
chloride amine (7) (1.0 mmol) in 5 ml of 48% HBr was
heated at reflux under a nitrogen atmosphere for 5 h.
The solvent was removed under reduced pressure and
the solid residue was recrystallized from isopropanol–
ether (70%), mp 149–150 ꢁC. IR (KBr) cmꢀ1: 3625–
3050 (OH); 2975 (CH3); 1625, 1512 ðR2 NH2þÞ; 1506
1
1028 (C–O–C); 800 (ArH). H NMR (CDCl3): d 1.06
(d, 3H, J = 10.8 Hz, CH3); 1.72 (bb, R2NH); 2.36–2.48
(m, 2H, CH2Ar); 2.55 (dd, 1H, J = 5.4 Hz, Hax (C1-In-
dan)); 2.65 (dd, 1H, J = 5.4 Hz, Hax (C3-Indan)); 3.06
(dd, 1H, J = 5.4 Hz, Heq (C1-Indan)); 3.16 (dd, 1H,
J = 5.4 Hz, Heq (C3-Indan)); 2.93–3.26 (m, 1H, –
CH(CH3)CH2Ar); 3.86 (m, 13H (–OCH3)4, CH(C2-In-
dan)); 6.64 (dd, 1H, J = 8.1 and 2.97 Hz, ArH (C6-aral-
kyl)); 6.67 (d, 1H, J = 8.1 Hz, ArH (C5-Aralkyl)); 6.70
(d, 1H, J = 8.1 Hz, ArH, (C6-Indan)); 6.75 (d, 1H,
J = 2.97 Hz, ArH (C2-Aralkyl)); 6.81 (d, 1H,
J = 8.1 Hz, ArH (C7-Indan)). 13C NMR: 15 (CH3); 35
(CH2Ar); 37 (C1-C3 Indan); 41 (CH(CH3)CH2Ar); 51
(C2-Indan); 56, 57, 60 (OCH3)4; 112, 113, 114, 121,
122, 130, 133, 134, 145, 148, 149, 152 (C, ArH). DEPT:
15(CH3); inverted signals: 35 (CH2Ar); 37 (C1–C3-In-
dan); 41 (CH(CH3)CH2Ar); 51 (C2-Indan); 56, 57, 60
(OCH3)4; 112 (C6-Aralkyl); 113 (C5-Aralkyl); 114 (C6-
1
(C@C); 1300–1225 (C–O–H). H NMR (DMSO-d6): d
1.07 (d, 3H, J = 6.43 Hz, CH3); 2.5–3.47 (m, 7H, CH2Ar,
CH2(C1-Indan); CH2(C3-Indan); CH(CH3)CH2Ar); 3.95
(m, 1H, CH(C2-Indan)); 6.51 (d, 1H, J = 7.91 Hz, ArH
(C6-Aralkyl)); 6.53 (d, 1H, J = 7.91 Hz, ArH (C5-Aral-
kyl)); 6.61 (d, 1H, J = 7.91 Hz, ArH (C6-Indan)); 6.64
(s, 1H, ArH, (C2-Aralkyl)); 6.69 (d, 1H, J = 7.91 Hz,
(C7-Indan)); 8.04–8.60 (bb, (OH)4); 8.90 (bb,
NH2þBrꢀ). 13C NMR: d 15.70 (CH3); 18.66 (CH2Ar);
34.83 (C1,C3-Indan); 37.29 (CH(CH3)CH2Ar); 51.12
(C2-Indan); 115.30 (C6-Aralkyl); 116.28 (C5-Aralkyl);

S. A. Andujar et al. / Bioorg. Med. Chem. 16 (2008) 3233–3244
3241
400
400
350
θ2
θ1
350
300
250
200
150
100
50
300
250
200
150
100
50
0
0
0
2000
4000
6000
time (ps)
8000
10000
0
2000
4000
6000
8000
10000
time (ps)
400
350
300
250
200
150
100
50
400
350
300
250
200
150
100
50
θ3
θ4
0
0
0
2000
4000
6000
time (ps)
8000
10000
0
2000
4000
6000
8000
10000
time (ps)
Figure 12. Evolution of the angles h1 (a), h2 (b), h3 (c), and h4 (d) of compound 4 with time.
117.21 (C6-Indan); 121 (C2-Aralkyl); 126.8 (C7-Indan);
127, 128, 131, 142, 143, 145, 146 (qc). C18H22BrNO4:
396.28. Anal. Calcd C, 54.56; H, 5.59; N, 3.54. Found:
C, 54.93; H, 5.27; N, 3.59.
(32 · 28 · 28 cm). Computer-assisted recording of the
stereotyped (repetitive and purposeless) gnawing was
carried out at 4.5 min interval for 45 min. Analysis of
variance followed by Duncan’s multiple range test was
performed to determine a significant difference between
means of each treatment group. Four rats were use for
each measurement.
3.2. Biological activity evaluation
3.2.1. Behavioral study. Adult Sprague–Dawley rats
´
(Centro Tecnologico, IVIC, Caracas), weighting 200–
3.2.2. Renal action evaluation. Adult male Sprague–
Dawley rats (220–280 g) were housed under controlled
conditions of temperature and photoperiod (lights on
from 06:00 to 18:00 h) and were provided with free ac-
cess to laboratory chow and water. A cannula was im-
planted in the left lateral cerebroventricle, 1 mm
caudal to the coronal suture and 1.5 mm lateral to the
midsagittal suture, with the aid of a stereotaxic instru-
ment and under pentobarbital anesthesia (40 mg/kg,
ip). The cannula was secured to the skull with acrylic ce-
ment. A minimum of three days was allowed for recov-
ery. Single ICV injections were made with a Hamilton
syringe fitted with a stop to prevent needle penetration
past the cannula tip.
250 g, were housed under controlled conditions of tem-
perature and light with free access to laboratory chow
and water. An indwelling cannula was stereotaxically
implanted, under pentobarbital anesthesia (40 mg/kg,
ip), in the right lateral ventricle of each rat according
to the following coordinates: 0.40 mm bregma, 1.1 mm
lateral, 2.2 mm ventral. After three days of recovery
from the surgical procedure, compounds 3 (0.6, 6.0 lg/
5 ll) and 4 (50 lg/5 ll) were slowly infused into the lat-
eral ventricle at a rate of 0.5 ll/min. Control rats were
infused with vehicle, 0.9% NaCl. After 5 min of ICV
infusion, animal behavior was monitored for 45 min.
A group of rats pre-treated with compound 3 or vehicle
were injected with apomorphine HCl (APO) (Sandoz
S.A., Basel, Switzerland) (1 mg/kg, ip) one minute be-
fore the initiation of the behavioral observation. Ani-
mals were observed in transparent Plexiglas chambers
The animals were randomly distributed into the fol-
lowing groups: V: control vehicle, receiving NaCl
0.9% (5 or 10 ll); DA: dopamine (100 lg/5 ll, ICV);

3242
S. A. Andujar et al. / Bioorg. Med. Chem. 16 (2008) 3233–3244
Compound 3
0.16
Compound 4
0.16
0.14
0.12
0.10
0.08
0.06
0.04
0.02
0.00
0.14
0.12
0.10
0.08
0.06
0.04
0.02
0.00
0
2000
4000
6000
time (ps)
8000
10000
0
2000
4000
6000
time (ps)
8000
10000
Figure 13. The root mean square deviation (rmsd) of both complexes during the 10 ns of MD simulation. (a) Compound 3/D2 DAR complex and (b)
compound 4/D2DAR complex; in this case a conformational change might be observed around 7 ns of simulation.
C3: compound 3 (50 lg/5 ll, ICV); DA + C3 (15 min
before DA), and C4: compound 4 (50 lg/10 ll, ICV).
N = 6–8 animals per group. All the rats were weighed,
orally loaded with 20 ml/kg of water, and than placed
in individual metabolic cages. Urine was collected at 3
and 6 h; the bladder was emptied at 6 h by gentle
suprapubic massage. Food and water were not avail-
able during the experiment. Ventricular cannula place-
ment was confirmed postmortem by examining the
distribution of an ICV injection of 5 ll of fast green
dye, given before animal sacrifice. Data were used
only if the dye was distributed in the lateral, third,
and forth ventricles. Urine samples were assayed for
sodium and potassium content by flame photometry.
All data are presented as means SEM. Statistical dif-
ferences between groups were analyzed using two-way
analysis of variance (ANOVA) and by the Newman–
Keul’s Student range statistics.
ligand complexes were prepared in order to obtain the
input files for MD runs. Several docking positions were
considered and the strongest receptor interactions were
examined in detail.
The MD simulations and analysis are performed using
the GROMACS 3.2.1 simulation package15,16 and the
GROMACS46–50 united-atoms force field (FF) and
the rigid SPC water model51,52 in a cubic box with peri-
odic boundary conditions. The receptor–ligand com-
plexes were embedded in a box containing the SPC
water model that extended to at least 1 nm between
the receptor and the edge of the box resulting in a
box of 7.17 nm of side lengths. The total number of
water molecules was 11,030 for both simulations. Then
three Na+ ions were added to the systems by replacing
water in random positions, thus making the whole sys-
tem neutral. The time step for the simulations was
0.001 ps. for a complete simulation time of 10 ns. For
long-ranged interactions the article-mesh Ewald
(PME)53–55 method was used with a 1 nm cut-off and
a Fourier spacing of 0.12 nm. The MD protocol con-
sisted of several preparatory steps; energy minimization
using the conjugate gradient model,56,57 density stabil-
ization (NPT conditions), and finally production of
the MD simulation trajectory. All production simula-
tions are performed under NVT conditions at 310 K,
using Berendsen’s coupling algorithm58 for keeping
the temperature constant. The compressibility was
4.8 · 10ꢀ5 barꢀ1. All coordinates are saved every 5 ps.
The SETTLE49 algorithm is used to keep water mole-
cules rigid. Also the LINCS59 algorithm was used to
constrain all Ca atom position for the receptor in order
to avoid desfolding problems.
The procedures used in these experiments were reviewed
and approved by the Animal Care and Use Committee
of The Central University of Venezuela, School of Phar-
´
macy, Caracas, and Instituto de Investigaciones Clıni-
cas. Dr. Americo Negrette, Facultad de Medicina.
´
Universidad del Zulia.
3.3. Molecular dynamics simulations
A 3D model of the human dopamine D2 receptor was
used for the Molecular Dynamics (MD) simulations,
based on the X-ray structure of bacteriorhodopsin14
(PDB Acquisition Code: 1I15). The ligands’ topologies
were built using the Dundee PRODRG45 server. For
this purpose we used the previously optimized geometry
at RHF/6-31G(d) level of theory of the global minimum
of each ligand. In the present study, we have used an ap-
proach where manual docking was guided by informa-
tion from site-directed mutagenesis and short docking
simulations with both the receptor and the ligand free
to move. Structurally similar parts of the ligands were
oriented in similar positions in the receptor model which
was described by Mansour et al.36 Thus, these receptor–
3.4. Quantum mechanicals calculations
All calculations reported here were carried out using the
Gaussian 03 program.60 Geometrical optimizations for
the low-energy forms of RR and RS isomeric forms of
3 and 4 were performed using B3LYP/6-31G(d)61
calculations.

S. A. Andujar et al. / Bioorg. Med. Chem. 16 (2008) 3233–3244
3243
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D. M.; Palmiter, R. D. Proc. Natl. Acad. Sci. U.S.A. 2001,
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The author (Angel-Guıo, JE) wishes to dedicate this
manuscript to memory of his mother, Mrs. Ligia Guıo
de Angel ( ).
´
We are indebted to the Condes-LUZ Grant Nos. 0158-
06 and 0681-04, also to the Fonacit-Venezuela, Grant
No. S1 97000394. This work was partially supported
by Grants of UNSL. Sebastian A. Andujar thanks a fel-
lowship of CONICET-Argentina. Ricardo D. Enriz is
member of the CONICET staff.
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