Electrostatic surface potential calculation on several new halogenated benzimidazole-like dopaminergic ligands

Article abstract of DOI:10.1002/ardp.200300846

We examined the effects of the electron density distribution (electrostatic surface potential; ESP) of several new benzimidazole-type ligands on their binding affinity for the D₁ and D₂ dopamine receptors (DAR). Receptors were prepared from synaptosomal membranes of bovine caudate nuclei. [³H]SCH 23390 and [³H]spiperone were used as specific radiolabels for the D₁ and D₂ receptors, respectively. The ESP of these compounds was calculated using Gaussian 98 W software. Calculations performed with known dopaminergic ligands showed that the electron density charge in the aromatic ring of these compounds favors a higher binding affinity for the D₂ DAR. This was confirmed by the synthesis of halogenated analogues of several known dopaminergic ligands. Halogenation resulted in an increase in the positive charge of the aromatic part of the molecule. None of the newly synthesized compounds was efficient in displacing [³H]SCH 23390 from the D₁ DAR. The introduction of chlorine into the molecule led to a higher binding affinity for the D ₂ DAR of the new ligands in comparison to both parent compounds and brominated ligands. This difference probably originates from the difference in the sizes of chlorine and bromine atoms, which could influence the interaction of a ligand with the receptor binding site. However, among the new ligands with bromine as a substituent, two compounds (8b and 10b) expressed a higher binding affinity and two of them (9b and 11b) a lower binding affinity for the D ₂ DAR, when compared to unsubstituted parent compounds. These results indicate that the electrostatic surface potential of a ligand is an important factor in its interaction with the D₂ DAR and that this should be taken into account during design and synthesis of dopaminergic compounds.

Full text of DOI:10.1002/ardp.200300846

´
376 Andric et al.
Arch. Pharm. Pharm. Med. Chem. 2004, 337, 376−382
a
ˇ
´
V. Sukalovic ,
Electrostatic Surface Potential Calculation on
Several New Halogenated Benzimidazole-like
Dopaminergic Ligands
b
´
Deana Andric ,
b
´
G. Roglic ,
Sladjana Kostic-Rajacic ,
´
ˇ ´a
a,c
ˇ
ˇ
´
V. Soskic
We examined the effects of the electron density distribution (electrostatic surface
potential; ESP) of several new benzimidazole-type ligands on their binding affin-
ity for the D1 and D2 dopamine receptors (DAR). Receptors were prepared from
synaptosomal membranes of bovine caudate nuclei. [3H]SCH 23390 and
[3H]spiperone were used as specific radiolabels for the D1 and D2 receptors,
respectively. The ESP of these compounds was calculated using Gaussian
98 W software. Calculations performed with known dopaminergic ligands
showed that the electron density charge in the aromatic ring of these com-
pounds favors a higher binding affinity for the D2 DAR. This was confirmed by
the synthesis of halogenated analogues of several known dopaminergic ligands.
Halogenation resulted in an increase in the positive charge of the aromatic part
of the molecule. None of the newly synthesized compounds was efficient in
displacing [3H]SCH 23390 from the D1 DAR. The introduction of chlorine into
the molecule led to a higher binding affinity for the D2 DAR of the new ligands in
comparison to both parent compounds and brominated ligands. This difference
probably originates from the difference in the sizes of chlorine and bromine
atoms, which could influence the interaction of a ligand with the receptor binding
site. However, among the new ligands with bromine as a substituent, two com-
pounds (8b and 10b) expressed a higher binding affinity and two of them (9b
and 11b) a lower binding affinity for the D2 DAR, when compared to unsubsti-
tuted parent compounds. These results indicate that the electrostatic surface
potential of a ligand is an important factor in its interaction with the D2 DAR and
that this should be taken into account during design and synthesis of dopa-
minergic compounds.
a Centre for Chemistry, Institute
for Chemistry, Technology
and Metallurgy,
11000 Belgrade,
Serbia Montenegro
b Faculty of Chemistry,
University of Belgrade,
11000 Belgrade,
Serbia Montenegro
c ProteoSys AG,
D-55129 Mainz, Germany
Keywords: Benzimidazole; Electrostatic surface potential; Electron density
distribution; Dopaminergic; Dopamine receptors; D2 receptor
Received: October 1, 2003; Accepted: December 1, 2003 [FP846]
DOI 10.1002/ardp.200300846
binding affinity for the D1 and D2 dopamine receptors
(DAR). The influence of ESP on the biological activity
of different classes of compounds has been well docu-
mented [5Ϫ7]. Wilcox and Teeter [8] provided a theo-
retical model of the ESP distribution of various DA-
ergic ligands. We have also observed previously that
electrostatic potential in the benzene part of several
benzimidazole type ligands affects D2 DAR affinity [4].
Introduction
Benzimidazoles and their structural analogues can be
considered as non-catechol bioisosteres of cat-
echolamines. As a result, numerous new compounds
with varying binding affinity for catecholamine recep-
tors have been designed and synthesized [1, 2]. How-
ever, the physicochemical basis of the above bioisos-
terism is still far from being fully understood. This
prompted us to study the effects of the electron density
distribution (molecular electrostatic surface potential;
ESP) [3, 4] around the heterocyclic nuclei of several
known dopaminergic (DA-ergic) ligands of benzimida-
zole type and their halogenated derivatives on their
In the present work, we have further evaluated this
observation. For this purpose, starting from four known
DA-ergic benzimidazole type ligands as parent com-
pounds [3], we prepared halogenated derivatives with
the halogen atom directly attached to the benzene ring
(Figure 1). Due to their high electron withdrawal effect,
it was expected that the halogen atoms would in-
crease positive ESP in the aromatic part of the ligands.
Upon ESP mapping, i.e. ESP calculation of parent and
halogenated ligands, the halogenated compounds
´
Correspondence: Deana Andric, Faculty of Chemistry,
University of Belgrade, Studentski trg 16, 11000 Belgrade,
Serbia Montenegro. Phone: +38 111636061, Fax: +38
111636061; e-mail: deanad@helix.chem.bg.ac.yu
 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Arch. Pharm. Pharm. Med. Chem. 2004, 337, 376−382
New Halogenated Benzimidazole-like Dopaminergic Ligands 377
Figure 2. ESP values were mapped on electron den-
sity surface for simpler comparisons. Values in dark
gray indicate strong, negative ESP, whereas those in
light grey correspond to a strong, positive ESP.
Figure 1. Structure of benzimidazole-like dopamin-
ergic bioisosteres.
were synthesized and their affinities for binding to the
D1 and D2 DAR were estimated. Correlations between
ESP maps in the heterocyclic part of the molecule and
the D2 DAR binding properties are discussed.
in acetic acid, respectively. Nitration of N-[2-halogen-
4-(2-chloro-ethyl)-phenyl]-acetamides 3a and 3b in
acetanhydride with sulfuric acid/100% nitric acid af-
forded the corresponding N-[2-halogen-4-(2-chloro-
ethyl)-6-nitro-phenyl]-acetamides 4a and 4b. After hy-
drolysis in 4 N HCl, the resulting 2-halogen-4-(2-
chloro-ethyl)-6-nitro-phenylamines 5a and 5b readily
alkylated dipropylamine in the presence of sodium car-
bonate and potassium iodide in dimethylformamide.
The obtained 2-halogen-4-(2-dipropylamino-ethyl)-6-
nitro-phenylamines 6a and 6b were reduced with
Ra-Ni/hydrazine to produce 3-halogen-5-(2-dipropyl-
amino-ethyl)-benzene-1,2-diamines 7a and 7b. 4-
Chloro-6-(2-dipropylamino-ethyl)-1,3-dihydro-benz-
imidazole-2-thione (8a), 4-bromo-6-(2-dipropylamino-
ethyl)-1,3-dihydro-benzimidazole-2-thione (8b), [2-(7-
chloro-1H-benzimidazol-5-yl)-ethyl]-dipropyl-amine
(9a), [2-(7-bromo-1H-benzimidazol-5-yl)-ethyl]-dipro-
pyl-amine (9b), [2-(7-chloro-1H-benzotriazol-5-yl)-
ethyl]-dipropyl-amine (10a), [2-(7-bromo-1H-benzotri-
azol-5-yl)-ethyl]-dipropyl-amine (10b), 5-chloro-7-(2-
dipropyl-amino-ethyl)-1,4-dihydro-quinoxaline-2,3-
dione (11a), and 5-bromo-7-(2-dipropylamino-ethyl)-
1,4-dihydro-quinoxaline-2,3-dione (11b), used as tar-
get ligands, were prepared analogously to the corre-
sponding phenylethylamines described previously
[17].
Calculations
Ligand models were constructed using the Hyperchem
program [9]. Initial geometry optimization was under-
taken using Vega software [10] with the PM3 method
[11].
The results obtained were further optimized in a Gaus-
sian G 98W [11]. ESP were calculated in G 98W using
the DFT B3LYP method and a 6Ϫ31 g basis set [12,
13]. The ESP cube output from Gaussian G 98W was
visualized in a gOpenMol program [14] following the
recommended Gaussian procedure to display the cal-
culated properties. The data from the available litera-
ture pointed to the fact that DA-ergic ligands bind to
the corresponding receptors in a protonated state [15].
We performed ESP calculations using 5-[2-(N,N-di-n-
propylamino)ethyl]benzimidazol-2-thione (compound
12) in both the protonated and the unprotonated form
(Figure 2). In the protonated form, formiate anion was
used to mimic ASP 86 present in the D2 DAR binding
site.
Chemistry
The chemical structures of the compounds synthe-
sized in the present study are shown in Scheme 1.
1-(2-Chloro-ethyl)-4-nitro-benzene (1) was reduced
with stannous chloride in absolute ethanol and the re-
sulting amine was acylated without purification with
acetanhydride to produce N-[4-(2-chloro-ethyl)-phen-
yl]-acetamide (2) [16]. Compound 2 was converted
Results and discussion
All compounds were evaluated for their binding affinity
for the D1 and D2 dopamine receptor subtypes from
the synaptosomal membranes, prepared from bovine
caudate nuclei, by the in vitro competitive displace-
ment of specific radioligands [18]. The binding param-
either
into
N-[2-chloro-4-(2-chloro-ethyl)-phenyl]-
acetamide (3a) or N-[2-bromo-4-(2-chloro-ethyl)-phen-
yl]-acetamide (3b) using sulfuryl chloride or bromine
 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

´
378 Andric et al.
Arch. Pharm. Pharm. Med. Chem. 2004, 337, 376−382
Scheme 1. Pathways for the synthesis of the ligands.
eters of the compounds are listed in Table 1. The dis-
tribution of electrostatic charges in these compounds
was calculated and the results for parent compounds
in protonated and unprotonated form are shown in Fig-
ure 2. In the heterocyclic part of the ligands no signifi-
cant differences were observed between the ESP of
protonated and unprotonated forms. Therefore, we de-
cided to rely on the ESP calculations of the unpro-
tonated form of the novel ligands considered in the
present study.
ligands 8a-11a had a higher affinity for binding to the
D2 DAR in comparison with both parent compounds
and their brominated analogues. Among the bromine-
substituted ligands, two compounds (8b and 10b) had
a higher binding affinity, while the other two (9b and
11b) had a lower binding affinity for the D2 DAR when
compared to the parent compounds. This can be as-
cribed to the size of the bromine atom, which could
prevent sterically favorable interactions of the bromin-
ated ligand with the receptor ligand binding pocket. On
the other hand, the introduction of halogen atoms into
the benzene ring did not significantly affect the relative
order of magnitude of DA-ergic activity of the different
types of ligands, i.e. benzimidazolethiones Ͼ quinoxa-
line-2,3-diones ~ benzotriazoles Ͼ benzimidazoles.
As seen from Table 1, none of the compounds exam-
ined were active displacers of [3H]SCH 23390 from the
D1 DAR. D2 DAR binding studies revealed an interest-
ing profile of activities of the novel ligands. Chlorinated
 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Arch. Pharm. Pharm. Med. Chem. 2004, 337, 376−382
New Halogenated Benzimidazole-like Dopaminergic Ligands 379
Table 1. Affinity of the new ligands for binding to the
D1 and D2 dopamine receptors. Values are the means
of three independent experiments done in triplicate
performed at eight competing ligand concentrations
(10Ϫ4Ϫ10Ϫ9 M) and 0.2 mM [3H]SCH23390 or
[3H]spiperone.
No
X
Hal
Ki S.E.M. (µM)
D1
D2
8a
8b
9a
C=S
C=S
CH
CH
N
Cl
Br
Cl
Br
Cl
Br
Cl
Br
H
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
0.05 0.003
0.2 0.04
4.0 0.1
>100
0.3 0.01
0.7 0.02
0.9 0.04
>100
9b
10a
10b
11a
11b
12
13
14
15
N
CO-CO
CO-CO
C=S
CH
1.0 0.2
Figure 3. ESP values of benzimidazoles, benzimida-
zolethiones, benzotriazoles, quinoxalinediones and
their halogenated derivatives. ESP values were map-
ped on the electron density surface for a simpler com-
parison. Values in blue indicate a strong, negative
ESP, whereas values in red correspond to a strong,
positive ESP.
H
H
H
50
9
N
5.0 2.0
3.0 0.1
CO-CO
For the G protein-coupled receptors, whose native li-
gands are catecholamines, it seems that the catechol
moiety of the ligands interacts with the receptor via
hydrogen bonds to serine residues in the fifth trans-
membrane spanning region (TMV) of the receptors
[19]. For the D2 dopamine receptor, that contains three
conserved serines in the TMV [Ser193 (V8), Ser194
(V9), and Ser197 (V12)] [20, 21], mutation of these resi-
dues to alanines affects the binding of certain antagon-
ists and agonists. These studies indicated that the
conserved serine residues clearly act together to alter
ligand binding and effector coupling. Results of the
ESP calculations for the novel ligands are shown in
Figure 3. Examination of the ESP maps suggested
that high electron density areas could be necessary
for hydrogen bond formation between the central front
end of a ligand and the serines in the binding pocket
of the D2 DAR.
located negative ESP, which might favor hydrogen
bonding with Ser193 (V8), Ser194 (V9), and Ser197
(V12) of the D2 receptor. Benzotriazoles 10a, 10b and
14 and quinoxaline-2,3-diones 11a, 11b and 15 were
less active displacers of [3H]spiperone than the above
benzimidazolethiones. Benzimidazoles 9a, 9b and 13
were found to have a laterally located negative ESP
on only one side of the molecule (Figure 3), which
could render the formation of the hydrogen bond be-
tween the ligand and the receptor binding site difficult.
Comparison of the ESP maps of parent compounds
with the halogenated ligands (Figure 3) clearly demon-
strated that the introduction of a halogen atom in the
benzene part of the former molecules resulted in a de-
crease in electron density in the benzene ring, just as
expected. The increasing ESP in the benzene part of
the heterocyclic ring was accompanied by an in-
creased affinity for binding to the D2 DAR, and 8a,b,
Benzimidazolethiones 8a, 8b and 12 are the most po-
tent DA-ergic agents among the ligands tested. We
believe this is because they have the highest centrally
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380 Andric et al.
Arch. Pharm. Pharm. Med. Chem. 2004, 337, 376−382
in ppm downfield from the internal standard tetramethylsil-
ane. The IR spectra were run on a Perkin Elmer 457 Grating
Infrared Spectrophotometer (Perkin Elmer, Beaconsfield,
GB). The mass spectra were determined by a Finnigan Mat
8230 mass spectrometer (Finnigan, Bremen, Germany). For
analytical thin-layer chromatography Merck (Darmstadt, Ger-
many) F-256 plastic-backed thin-layer silica gel plates were
used. Chromatographic purifications were performed on
Merck-60 silica gel columns, 230-400 mesh ASTM, under
medium pressure (MPLC). Solutions were routinely dried
over anhydrous Na2SO4 prior to evaporation.
10a,b and 11a were the most active DA-ergic ligands
among the newly synthesized compounds. Since the
decrease in electron density in the benzene ring of
chlorinated (8aϪ11a) and brominated (8bϪ11b) li-
gands was similar, the differences in the DA-ergic ac-
tivity between these two groups of compounds can be
ascribed to the difference in size between the chlorine
and bromine atoms. At present, the details of the inter-
action between the part of ligand characterized by low
electron density and the D2 receptor are insufficiently
known, although the presence of an electron-rich
counterpart in the receptor ligand binding pocket could
be postulated. Generally, a compound with the lowest
electron density in the aromatic part and the highest
negative ESP in the central front end of the molecule
should express the highest DA-ergic activity.
Chemistry
Synthesis of N-[2-chloro-4-(2-chloro-ethyl)-phenyl]-acet-
amide (3a)
The mixture containing 0.56 g (3 mmol) N-[4-(2-chloro-ethyl)-
phenyl]-acetamide (2), 5 mL CHCl3 and 0.4 mL (5 mmol) sulf-
uryl chloride was stirred (2 h, 70°C). The reaction mixture
was cooled to ambient temperature, washed with diluted
NaHCO3 solution, dried and evaporated in vacuo. The resi-
due was chromatographed on silica gel. Yield: 75%; 1H-NMR
(ppm): 2.23 (s, 3H, CH3), 3.01 (t, 2H, J = 7.2 Hz, CH2), 3.68
(t, 2H, J = 6.8 Hz, CH2), 7.13 (dd, 1H, J = 8.4 Hz, ArH), 7.25
(d, 1H, J = 2 Hz, ArH), 7.60 (s, 1H, NH), 8.29 (d, 1H, J = 8.4
Hz, ArH).
As alternative explanation for the results obtained, the
observed differences may be due to the fact that not
all of the compounds interact in the same way with
the binding site or the receptor. This would influence
hydrogen bond geometry and, therefore, the energy of
binding in general. Without exact 3D crystal data it is
difficult to distinguish between the two models of re-
ceptor-ligand interaction presented above.
Synthesis of N-[2-bromo-4-(2-chloro-ethyl)-phenyl]-acet-
amide (3b)
The mixture of 1.0 g (5 mmol) N-[4-(2-chloro-ethyl)-phenyl]-
acetamide (2) and 3.0 mL CH3CO2H was heated to 45°C,
then 0.26 mL (0.82 g, 5.05 mmol) bromine were added drop-
wise at a rate that maintained the temperature of the well-
stirred mixture at 50Ϫ55°C. After all bromine was added, stir-
ring was continued for a further 30 min, then the reaction
mixture was poured into a well-stirred mixture of ice and solid
sodium metabisulphite, and extracted with CH2Cl2. The or-
ganic layer was dried and evaporated in vacuo. The residue
was chromatographed on silica gel. Yield: 60%; 1H-NMR
(ppm): 2.23 (s, 3H, CH3), 3.00 (t, 2H, J = 7 Hz, CH2), 3.68 (t,
2H, J = 7.2 Hz, CH2), 7.17 (dd, 1H, J = 7.4 Hz, ArH), 7.41 (d,
1H, J = 2 Hz, ArH), 7.57 (s, 1H, NH), 8.27 (d, 1H, J = 8.4
Hz, ArH).
Our current hypothesis is that the overall affinity of the
compounds synthesized during the present study and
evaluated for the DA-ergic activity represents a sum of
electronic and bulk interactions as well as of hydrogen
bonding. However, the influence of some other factors
such as ESP cannot be excluded. A full understanding
of the way in which ESP influences the overall DA-
ergic activity of a ligand might be an important step
in the direction of design and synthesis of new DA-
ergic ligands.
Synthesis of N-[2-chloro-4-(2-chloro-ethyl)-6-nitro-phenyl]-
acetamide (4a) and N-[2-bromo-4-(2-chloro-ethyl)-6-nitro-
phenyl]-acetamide (4b)
Acknowledgments
This work was supported by the Ministry for Science,
Technology and Development of the Republic of
Conc. H2SO4 (0.6 mL) was added to the cooled solution
(0°C) of 40 mmol of either acetamide 3a or 3b in 60 mL acet-
anhydride. The reaction mixture was cooled to Ϫ5°C and 2
mL 100% HNO3 were introduced dropwise. After overnight
stirring, the reaction mixture was poured into ice, made alka-
line with sodium hydroxide solution and extracted with
CH2Cl2. The organic layer was dried and evaporated in vac-
uo. The residue was chromatographed on silica gel.
ˇ
´
´
Serbia, Project No. 1698 (V. Sukalovic, G. Roglic, S.
ˇ ´
´
´
Kostic-Rajacic and D. Andric), and ProteoSys AG,
ˇ
ˇ
´
Mainz, Germany (V. Soskic).
(4a): Yield: 70%; 1H-NMR (ppm): 2.23 (s, 3H, CH3), 3.20 (t,
2H, J = 6.8 Hz, CH2), 3.81 (t, 2H, J = 6.8 Hz, CH2), 7.10 (s,
1H, NH), 7.72 (d, 1H, J = 2 Hz, ArH), 7.90 (d, 1H, J = 2
Hz, ArH).
Experimental
General
A Boetius PHMK apparatus (VEB Analytic, Dresden, Ger-
many) was used to determine melting points, presented here
as uncorrected. 1H-NMR and 13C-NMR spectra recorded on
a Gemini 2000 spectrometer (Varian, Palo Alto, CA, USA)
with CDCl3 as a solvent unless otherwise stated are reported
(4b): Yield: 65%; 1H-NMR (ppm): 2.82 (s, 3H, CH3), 3.22 (t,
2H, J = 6.6 Hz, CH2), 3.82 (t, 2H, J = 6.6 Hz, CH2), 7.27 (s,
1H, NH), 7.93 (d, 1H, J = 2 Hz, ArH), 8.10 (d, 1H, J = 2
Hz, ArH).
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Arch. Pharm. Pharm. Med. Chem. 2004, 337, 376−382
New Halogenated Benzimidazole-like Dopaminergic Ligands 381
Synthesis of 2-chloro-4-(2-chloro-ethyl)-6-nitro-phenylamine
(5a) and 2-bromo-4-(2-chloro-ethyl)-6-nitro-phenylamine (5b)
113.16, 122.87, 128.47, 133.76, 136.87, 169.40; MS: m/z
114(100), 311(M+).
(8b): Yield: 430 mg, 60%; m.p. >250°C; IR (KBr) (cmϪ1):
3055, 2963, 1570, 1485, 1403; 1H-NMR (DMSO) (ppm): 0.80
(t, 6H, J = 7.2 Hz, CH3), 1.42 (m, 4H, CH2), 2.66 (m, 4H,
CH2), 3.45 (m, 4H, CH2CH2), 6.98 (s, 1H, ArH), 7.20 (s, 1H,
ArH), 8.33 (s, 1H, NH); 13C-NMR: 11.94, 20.09, 32.44, 55.43,
55,76, 100.73, 108.92, 125.74, 130.20, 133.39, 137.41,
169.29; MS: m/z 114(100), 356(M+1).
Of either acetamide 4a or 4b, 0.5 mmol were resuspended in
50 mL of 4 N HCl and refluxed for 4 h. The solution was
cooled to ambient temperature and diluted with 150 mL of an
ice-water mixture. The excess acid was neutralized with 10%
NaOH solution keeping the temperature below 30°C, and the
product was extracted with CH2Cl2. After drying and evapor-
ation in vacuo, the residue was chromatographed on silica
gel.
Synthesis of [2-(7-chloro-1H-benzimidazol-5-yl)-ethyl]-dipro-
pyl-amine (9a) and [2-(7-bromo-1H-benzimidazol-5-yl)-ethyl]-
dipropyl-amine (9b)
(5a): Yield: 90%; 1H-NMR (ppm): 2.98 (t, 2H, J = 6.8 Hz,
CH2), 3.70 (t, 2H, J = 6.8 Hz, CH2), 6.51 (s, 2H, NH2), 7.52
(d, 1H, J = 2 Hz, ArH), 7.96 (d, 1H, J = 2 Hz, ArH).
Two mmol of either diamine 7a or 7b and 0.44 mL (7.3 mmol)
98% formic acid were heated in an oil bath at 100°C for 2 h.
After cooling to ambient temperature, 15 mL 10% NaHCO3
were added and the product was extracted with CH2Cl2. The
solvent was removed in vacuo and the residue chromato-
graphed on silica gel.
(5b): Yield: 93%; 1H-NMR (ppm): 2.93 (t, 2H, J = 6.6 Hz,
CH2), 3.71 (t, 2H, J = 6.6 Hz, CH2), 6.60 (s, 2H, NH2), 7.61
(d, 1H, J = 2 Hz, ArH), 8.06 (d, 1H, J = 2 Hz, ArH).
Synthesis of 2-chloro-4-(2-dipropylamino-ethyl)-6-nitro-phen-
ylamine (6a) and 2-bromo-4-(2-dipropylamino-ethyl)-6-nitro-
phenylamine (6b)
(9a): Yield: 335 mg, 60%; m.p. 113°C; IR (KBr) (cmϪ1): 3029,
1
2932, 1578, 1477, 1433; H-NMR (ppm): 0.89 (t, 6H, J = 7.4
The stirred mixture of 2.28 mmol of either 5a or 5b, 0.7 mL
(0.5 g, 5 mmol) dipropylamine, finely powdered sodium car-
bonate (0.5 g) and potassium iodide (0.04 g) in 10 mL dimeth-
ylformamide was heated overnight at 80°C. The reaction mix-
ture was cooled to ambient temperature, filtered, washed with
toluene and evaporated in vacuo. The residue was chromato-
graphed on silica gel.
Hz, CH3), 1.50 (m, 4H, CH2), 2.51 (m, 4H, CH2), 2.81 (m, 4H,
CH2CH2), 7.16 (s, 1H, ArH), 7.27 (s, 1H, NH), 7.35 (s, 1H,
ArH), 8.08 (s, 1H, CH); 13C-NMR: 11.95, 20.13, 32.80, 55.50,
56.07, 118.90, 122.56, 125.63, 131.24, 134.83, 136.30,
142.77; MS: m/z 114(100), 280(M+1).
(9b): Yield: 421 mg, 65%; m.p. 141°C; IR (KBr) (cmϪ1): 3025,
(6a): Yield: 54%; 1H-NMR (ppm): 1.03 (t, 6H, J = 7.6 Hz,
CH3), 1.86 (m, 4H, CH2), 3.03 (m, 4H, CH2), 3.17 (s, 4H,
CH2CH2), 6.58 (s, 2H, NH2), 7.56 (d, 1H, J = 2 Hz, ArH), 7.95
(d, 1H, J = 2 Hz, ArH).
1
2958, 1570, 1463, 1434; H-NMR (ppm): 0.89 (t, 6H, J = 7.2
Hz, CH3), 1.49 (m, 4H, CH2), 2.52 (m, 4H, CH2), 2.84 (m, 4H,
CH2CH2), 7.32 (s, 1H, ArH), 7.39 (s, 1H, ArH), 8.09 (s, 1H,
CH); 13C-NMR: 11.97, 20.03, 32.69, 55.50, 56.50, 117.81,
119.32, 125.56, 135.46, 139.21, 140.52, 142.62; MS: m/z
114(100), 324(M+1).
(6b): Yield: 60%; 1H-NMR (ppm): 0.88 (t, 6H, J = 7.4 Hz,
CH3), 1.46 (m, 4H, CH2), 2.46 (m, 4H, CH2), 2.67 (s, 4H,
CH2CH2), 6.50 (s, 2H, NH2), 7.61 (d, 1H, J = 2 Hz, ArH), 7.98
(d, 1H, J = 2 Hz, ArH).
Synthesis of [2-(7-chloro-1H-benzotriazol-5-yl)-ethyl]-dipro-
pyl-amine (10a) and [2-(7-bromo-1H-benzotriazol-5-yl)-ethyl]-
dipropyl-amine (10b)
Synthesis of 3-chloro-5-(2-dipropylamino-ethyl)-benzene-1,2-
diamine (7a) and 3-bromo-5-(2-dipropylamino-ethyl)-ben-
zene-1,2-diamine (7b)
Of either diamine 7a or 7b, 2 mmol were dissolved in a mix-
ture of 0.5 mL acetic acid and 0.9 mL water. After that, 0.16
g (2.35 mmol) NaNO2 dissolved in 0.25 mL water were added
at 0°C. The solution was heated (70°C, 10 min.) and, after
cooling to ambient temperature, the solvent was removed in
vacuo. The residue was resuspended in 10 mL of 5%
NaHCO3 and the product was extracted with CH2Cl2. The sol-
vent was removed in vacuo and the residue chromato-
graphed on silica gel.
Raney-Ni (0.06Ϫ0.08 g) was added in small portions to a
stirring solution of 2 mmol of either nitro compound (6a or 6b)
in 5 mL EtOH, 10 mL 1,2-dichloro-ethane and 0.9 mL hydra-
zine hydrate at 30°C. After the addition of Ra-Ni was com-
pleted, the mixture was heated in a water bath (50°C, 60
min.) and filtered through celite. The filtrate was evaporated
in vacuo and crude products were used for further syntheses.
(10a): Yield: 343 mg, 61%; oil; IR (KBr) (cmϪ1): 3055, 2965,
1554, 1467, 1417; 1H-NMR (ppm): 0.92 (t, 6H, J = 7.6 Hz,
CH3), 1.64 (m, 4H, CH2), 2.81 (m, 4H, CH2), 3.05 (d, 4H, J =
3.6 Hz, CH2CH2), 7.12 (s, 1H, NH), 7.13 (s, 1H, ArH), 7.62
(s, 1H, ArH); 13C-NMR: 11.88, 19.93, 32.56, 55.34, 56.04,
111.64, 120.54, 125.82, 138.29, 138.65, 140.69; MS: m/z
114(100), 280(M+).
Synthesis of 4-chloro-6-(2-dipropylamino-ethyl)-1,3-dihydro-
benzimidazole-2-thione (8a) and 4-bromo-6-(2-dipropyl-
amino-ethyl)-1,3-dihydro-benzimidazole-2-thione (8b)
Carbon disulfide (0.24 mL, 4 mmol) and KOH (0.25 g in 0.6
mL water) were added to 2 mmol of either diamine 7a or 7b
in 10 mL EtOH. After refluxing for 3 h, 0.3 mL of acetic acid
in 3.3 mL water were added. The solvent was removed in
vacuo and the residue chromatographed on silica gel.
(10b): Yield: 423 mg, 65%; oil; IR (KBr) (cmϪ1): 3100, 2970,
1548, 1466, 1413; 1H-NMR (ppm): 0.92 (t, 6H, J = 7.2 Hz,
CH3), 1.60 (m, 4H, CH2), 2.79 (m, 4H, CH2), 3.03 (s, 4H,
CH2CH2), 7.28 (s, 1H, ArH), 7.63 (s, 1H, ArH), 10.47 (s, 1H,
NH); 13C-NMR: 11.83, 19.76, 32.33, 55.45, 56.11, 108.68,
112.16, 122.17, 129.00, 137.80, 140.86; MS: m/z 114(100),
324(M+).
(8a): Yield: 340 mg, 52%; m.p. >250°C; IR (KBr) (cmϪ1):
1
3065, 2962, 1567, 1491, 1403; H-NMR (DMSO)(ppm): 0.81
(t, 6H, J = 7.4 Hz, CH3), 1.40 (m, 4H, CH2), 2.45 (m, 4H,
CH2), 2.69 (m, 4H, CH2CH2), 6.96 (s, 1H, ArH), 7.09 (s, 1H,
ArH); 13C-NMR: 11.92, 20.66, 32.29, 55.34, 55.59, 108.57,
 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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382 Andric et al.
Arch. Pharm. Pharm. Med. Chem. 2004, 337, 376−382
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´
Synthesis of 5-chloro-7-(2-dipropylamino-ethyl)-1,4-dihydro-
quinoxaline-2,3-dione (11a) and 5-bromo-7-(2-dipropylamino-
ethyl)-1,4-dihydro-quinoxaline-2,3-dione (11b)
[4] V. Sukalovic, Structural aspects of dopaminergic benzi-
midazole type ligands and D2 dopamine receptor Ϫ
Master thesis, Faculty of Chemistry, University of Bel-
grade, Belgrade 2001.
Two mmol of either diamine 7a or 7b, 0.55 g (4.4 mmol) oxalic
acid and 2.5 mL 4 N HCl were refluxed for 60 min. After cool-
ing to ambient temperature, the solvent was removed in va-
cuo. The residue was resuspended in 20 mL 10% NaHCO3
and the product was extracted with CH2Cl2. The solvent was
removed in vacuo and the residue chromatographed on sil-
ica gel.
[5] S. Mecozzi, A. West, D. Dougherty, Proc. Natl. Acad.
Sci. 1996, 93, 10566Ϫ10571.
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Pearlman, M. Teter, C. DuRand, B. Wiens, K. Neve, J.
Med. Chem. 2000, 43, 3005Ϫ3019.
[7] H. S. Parihar, K. S. Kirschbaum, Bioorg. Med. Chem.
Lett. 2002, 12, 2743Ϫ2747.
(11a): Yield: 280 mg, 43%; m.p. 230°C; IR (KBr) (cmϪ1):
3046, 2965, 1699, 1601, 1392; 1H-NMR (DMSO) (ppm): 0.92
(t, 6H, J = 7.2 Hz, CH3), 1.69 (m, 4H, CH2), 3.05 (m, 4H,
CH2), 3.13 (m, 2H, CH2CH2), 3.46 (m, 2H, CH2N), 7.01 (s,
1H, ArH), 7.25 (s, 1H, ArH), 11.45 (s, 1H, NH), 12.18 (s, 1H,
NH); 13C-NMR: 11.14, 16.60, 28.46, 52.81, 53.34, 114.66,
118.79, 121.94, 123.82, 127.29, 133.17, 155.04, 155.49; MS:
m/z 114(100), 322(MϪ1).
[8] R. Wilcox, T. Tseng, M. Kim, B. Ginsburg, R. Pearlman,
M. Teter, C. DuRand, S. Starr, K. Neve, J. Med. Chem.
1998, 41, 4385Ϫ4391.
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47Ϫ49.
(11b): Yield: 295 mg, 40%; m.p. 152°C; IR (KBr) (cmϪ1):
3146, 2967, 1695, 1620, 1395; 1H-NMR (DMSO) (ppm): 0.92
(t, 6H, J = 7 Hz, CH3), 1.69 (m, 4H, CH2), 3.04 (m, 4H, CH2),
3.24 (m, 4H, CH2CH2), 7.03 (s, 1H, ArH), 7.40 (s, 1H, ArH),
11.06 (s, 1H, NH), 12.15 (s, 1H, NH); 13C-NMR: 11.84, 16.60,
28.38, 52.83, 53.30, 108.16, 115.33, 123.11, 127.06, 129.72,
133.65, 155.02, 155.57; MS: m/z 114(100), 367(M+).
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Synaptosomal membrane preparation, binding assays and
data analysis
The synaptosomal membranes of the bovine caudate nuclei
that were used as a source of the dopamine D1 and D2 receptor
subtypes were prepared exactly as described previously [22].
[3H]SCH 23390 (80 Ci mmolϪ1) and [3H]spiperone (70 Ci
mmolϪ1) used to label D1 and D2 receptor subtypes, respec-
tively, were purchased from Amersham Buchler GmbH
(Braunschweig, Germany). Briefly, [3H]spiperone binding was
assayed in binding buffer at 37°C for 20 min in a total volume
of 0.5 mL. The binding of the radioligand to 5-HT2 receptors
was prevented by 50 nM ketanserin. Ki values were deter-
mined by competition binding at 0.2 nM of the radioligand,
and eight to ten concentrations of each ligand were tested
(0.1 µMϪ0.1 mM). Nonspecific binding was measured in the
presence of 1.0 mM (+)-butaclamol. The reaction was termin-
ated by a rapid filtration through Whatman GF/C filters, before
washing three times with 5.0 mL ice-cold incubation buffer.
Radioligand binding for each compound concentration was
determined in triplicate. Retained radioactivity was measured
by introducing dry filters into 10 mL toluene-based scintillation
liquid and by counting in a 1219 Rackbeta Wallac scintillation
counter. Binding of [3H]SCH 23390 was examined by the
same rapid filtration assay discussed for [3H]spiperone but in
the absence of ketanserin.
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Mol. Structure 1999, 484, 181Ϫ196.
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[16] S. Dukic, D. Dragovic, V. Soskic, J. Joksimovic, Arz-
neim.-Forsch./Drug Res. 1996, 46(II), 741Ϫ746.
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[17] S. Kostic-Rajacic, V. Soskic, J. Joksimovic, Arch. Pharm.
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ˇ
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[18] S. Kostic, V. Soskic, J. Joksimovic, Arzneim.-Forsch./
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17979Ϫ17982.
Competition binding data were analyzed by the non-linear
least-squares curve-fitting program LIGAND [23].
[20] R. Woodward, C. Coley, S. Daniell, L. H. Naylor, P. G.
Strange, J. Neurochem. 1996, 66, 394Ϫ402.
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 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim