Synthesis, dopamine D2 receptor binding studies and docking analysis of 5-[3-(4-arylpiperazin-1-yl)propyl]-1H-benzimidazole, 5-[2-(4-arylpiperazin-1-yl)ethoxy]-1H-benzimidazole and their analogs

European Journal of Medicinal Chemistry 40 (2005) 481–493

www.elsevier.com/locate/ejmech

Original article

Synthesis, dopamine D₂receptor binding studies and docking analysis

of 5-[3-(4-arylpiperazin-1-yl)propyl]-1H-benzimidazole,

5-[2-(4-arylpiperazin-1-yl)ethoxy]-1H-benzimidazole and their analogs

V. Šukalovic´ ^a, Deana Andric´ ^b, G. Roglic´ ^b, Sladjana Kostic´-Rajacˇic´ ^a,

A. Schrattenholz ^c, V. Šoškic´ ^a,c,

*

^aInstitute for Chemistry, Technology and Metallurgy, Center for Chemistry, Njegoševa 12, 11000 Belgrade, Serbia and Montenegro

^bFaculty of Chemistry, University of Belgrade, Studentski trg 16, 11000 Belgrade, Serbia and Montenegro

^cProteoSys AG, Carl Zeiss Street 51, 55129 Mainz, Germany

Received 13 May 2004; received in revised form 18 October 2004; accepted 21 October 2004

Available online 01 April 2005

Abstract

5-[3-(4-Arylpiperazin-1-yl)propyl]-1H-benzimidazoles and 5-[2-(4-arylpiperazin-1-yl)ethoxy]-1H-benzimidazoles were synthesized and

their afﬁnity for the D₁, D₂and 5-HT_1Areceptors examined. They expressed a rather high afﬁnity for the D₂dopamine receptor. The main

features of ligand–D₂receptor interactions revealed by docking analyses were: salt bridge between piperazine ring protonated N₁and Asp 86,

hydrogen bonds of ligand bezimidazole part with Ser 141, Ser 122 and His 189, edge-to-face interactions of arylpiperazine aromatic ring with

Phe 178, Tyr 216 and Trp 182 and hydrogen bond between ethereal oxygen in ethylenoxy ligands and hydrogen of Phe 185 or Trp 115. The

most active 5-{2-[4-(2-methoxyphenyl)-piperazin-1-yl]ethoxy}-1,3-dihydro-2H-benzimidazole-2-thione (27) has a maximal number of attrac-

tive interactions. A satisfactory correlation between docking of the compounds into the D₂receptor and competition binding results was

observed.

Keywords: Arylpiperazines; D₂receptor; 5-HT_1Areceptor; Binding pocket; Docking analysis

1. Introduction

imidazoles that can be considered as non-catechol bio-

isosteres of catecholamines [3]. The most active compounds

were obtained by connecting benzimidazole ring through a

ﬂexible spacer with N-arylpiperazines, structure of one in such

a way obtained ligand is presented in Chart 1. It was observed

that the afﬁnity of the obtained ligands for the binding to the

D₂DAR depends on both the structure of benzimidazole and

arylpiperazine part of the molecule and the spacer itself [4,5].

In order to obtain a better structure/DA-ergic activity rela-

tionship and to evaluate the inﬂuence of the spacer in this

type of ligands on their binding afﬁnity, we have synthesized,

Since the introduction of chlorpromazine, a known dopam-

ine (DA) antagonist, DA-ergic drugs have been widely used

in medicinal practice. During the last years, different DA ago-

nists and antagonists have been applied in human medicine

for the treatment of numerous disorders, e.g. Parkinson’s dis-

ease, schizophrenia, some neurohumoral disturbances, etc.

Since a great number of these compounds expressed undesir-

able side effects, the need for new drugs with side effects

reduced to a minimum is increasing. Scientiﬁc literature in

this ﬁeld of interest is numerous and can be found summa-

rized in recent review articles [1,2].

Within the scope of the program aimed at the discovery of

new DA-ergic ligands, we have synthesized a series of benz-

*

Corresponding author. Tel.: +49 613 1501 9246; fax: +49 613 1501 9211.

Chart 1.

doi:10.1016/j.ejmech.2004.10.006

482

V. Šukalovic´ et al. / European Journal of Medicinal Chemistry 40 (2005) 481–493

evaluated and performed docking analyses of a series of new

compounds, derivatives of 5-[3-(4-arylpiperazin-1-yl)propyl]-

1H-benzimidazole and 5-[2-(4-arylpiperazin-1-yl)ethoxy]-

1H-benzimidazole. On the other hand oxygen/methylene isos-

teric replacement that introduce an aryl–O bond has signiﬁcant

effect on both electronic and conformational properties stud-

ied compounds [6,7] and therefore can effect ligand–recep-

tor interaction on several levels. Our previous ﬁnding shows

that interactions between benzoimidazole type ligand and D₂

DAR are sensitive to above mentioned molecular properties

[5,8] what initiated investigations presented in this paper.

Binding pocket of the D₂DAR was deﬁned according to

Teeter and DuRand [10], while docking analysis was done

using Insight II software packet from Accelerys.

2. Chemistry

The derivatives 1–13 and 20–43 (Table 1) were synthe-

sized as outlined in Scheme 1. Compounds with propylene

spacer were prepared according to previously described strat-

egy [4] starting from 4-(3-chloropropyl)-2-nitroaniline (1).

Ethylenoxy compounds were obtained starting from 4-(2-

Table 1

Chemical structure and binding afﬁnity of the examined arylpiperazine ligands for the D₁and D₂dopamine and 5-HT_1Aserotonin receptors

Number

X

Y

R

K_i(nM) S.E.M.

D₂

D₁

5-HT_1A

71.1 8.2

>1000

47 4.0

177 29

>1000

8.6 1.1

>1000

284 35

330 42

>1000

308 29

>1000

4

5

6

7

8

9

CH

CH₂

O

H

>1000

446 32

>1000

405 30

>1000

129 19

>1000

CH

3-CF₃

4-Cl

CH

>1000

CH

4-OMe

2-OMe

H

>1000

CH

97 21

C=S

CH

3.0 0.2

5.8 0.4

20 2.8

33 4.2

0.4 0.05

274 18

>1000

10

11

12

13

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

3-CF₃

4-Cl

4-OMe

2-OMe

H

CH

O

3-CF₃

2-MeO

3-MeO

4-MeO

H

CH

O

12.6 0.9

>1000

CH

O

CH

O

>1000

C=S

C=O

N

O

0.67 0.06

5.94 0.3

0.19 0.06

13.2 1.2

105 8.1

2.75 0.4

7.06 0.9

>1000

O

3-CF₃

2-MeO

3-MeO

4-MeO

2-Cl

O

3-Cl

O

4-Cl

O

H

7.53 1.0

26.1 4.2

1.30 0.1

44.8 6.2

>1000

O

3-CF₃

2-MeO

3-MeO

4-MeO

2-Cl

O

>1000

O

3-Cl

45.5 3.0

>1000

O

4-Cl

O

H

>1000

N

O

3-CF₃

2-MeO

>1000

N

O

1.51 0.4

Structure of 5-[3-(4-arylpiperazine-1-yl)propyl]-1H-benzimidazoles and 5-[2-(4-arylpiperazine-1-yl)ethoxy]-1H-benzimidazoles and their analogs tested for

the binding to the D₁and D₂dopamine and 5-HT_1Aserotonin receptors is shown. K_ivalues are the means of three independent experiments done in triplicate

performed at eight to 10 competing ligand concentrations.

V. Šukalovic´ et al. / European Journal of Medicinal Chemistry 40 (2005) 481–493

483

Scheme 1. Synthesis of 5-[3-(4-arylpiperazine-1-yl)propyl]-1H-benzimidazole, 5-[2-(4-arylpiperazine-1-yl)ethoxy]-1H-benzimidazole and their analogs. (a)

Arylpiperazine, K₂CO₃, DMF; (b) Ra–Ni, N₂H₄, EtOH, DCE; (c) HCOOH, reﬂux; (d) CS₂, KOH, EtOH; (e) 1,1′-carbonyldiimidazole and (f) NaNO₂, AcOH.

Obtained yields are clamed in parentheses.

chloroethoxy)-2-nitroaniline (17) as a parent compound

(Scheme 2). Brieﬂy, 1-(2-chloroethoxy)-4-nitrobenzene (14)

was prepared by alkylating 4-nitrophenol with 1,2-

dichloroethane.A simultaneous reduction and acetylation with

zinc dust in acetanhydride/acetic acid mixture afforded aceta-

nilide (15). Nitration of 15 in boiling 20% nitric acid gave

o-nitroacetanilide (16) that was further hydrolyzed to o-aniline

17 with boiling HCl. Compound 17 readily alkylates

N-phenyl-piperazine in DMF in the presence of K₂CO₃and

KI at elevated temperature affording 2-nitro-4-[2-(4-

arylpiperazin-1-yl)ethoxy]-anilines (18a–h). Further reduc-

tion with Ra–Ni/hydrazine afforded o-phenylenediamines

19a–h.

were calculated from the displacement curves obtained in

three independent experiments done in triplicate and per-

formed at eight to 10 competing ligand concentrations. The

pharmacological results are listed in Table 1 .

4. Results

None of the compounds synthesized and evaluated for the

DA-ergic/serotonergic activity throughout the present study

expressed the afﬁnity for the binding to the D₁DAR. Ligands

5–7, 24, 37, 38 and 40–42 were completely inactive competi-

tors in both D₂and 5-HT_1Abinding assays. Besides, com-

pounds 10–12, 20, 25, 26, 28–31, 33–36, 39 and 43 acted

only as [³H]spiperone displacers.Also, only one of the ligands

(23) acted as an active displacer of [³H]8-OH-DPAT. The

remaining novel compounds (4, 8, 9, 13, 22 and 27) behaved

as efﬁcient displacers of [³H]spiperone and [³H]8-OH-

DPAT, expressing the binding afﬁnity for the corresponding

receptor in a nanomolar range of concentrations.

Target benzimidazoles (4–8 and 20–24), benzimidazole-

2-thiones (9–13 and 25–32), benzimidazole-2-ones (33–40)

and benzotriazoles (41–43) were prepared as described ear-

lier [3].

3. Pharmacology

Among all tested ligands, compound 27 with a K_ivalue of

0.19 nmol was the most potent displacer of [³H]spiperone,

while in [³H]-OH-DPAT binding assay compound 13 with a

K_iof 8.6 nmol was the most active (Table 1 ).

Binding pocket of the D₂DAR was deﬁned according to

Teeter and DuRand [10]. The main features of the D₂DAR

model shown in Fig. 1 using compound 27 as a ligand were:

(a) salt bridge between protonated N1 of the piperazine ring

All compounds were evaluated for the binding afﬁnity to

the D₁and D₂dopamine and serotonin 5-HT_1Areceptors by

in vitro competition displacement of the speciﬁc radioli-

gands ([³H]SCH 23390, [3H]spiperone and 8-OH-[³H]DPAT,

respectively) from synaptosomal membranes prepared from

fresh bovine caudate nuclei (D₁and D₂receptor) and hippoc-

ampi (5-HT_1Areceptor). K_ivalues of individual compounds

Scheme 2

yields are clamed in parentheses.

. Synthesis of 4-(2-chloroethoxy)-2-nitrophenylamine (17). (a) DCE, K₂CO₃, MEK; (b) Zn, Ac₂O/AcOH; (c) 20% HNO₃, D; (d) HCl, H₂O. Obtained

484

V. Šukalovic´ et al. / European Journal of Medicinal Chemistry 40 (2005) 481–493

Fig. 1

.

Schematic representation of ligand 27 interaction with the D₂dopamine receptor. Schematic model of the proposed interaction of the studied compound

27 with the D₂DAR. 3D model describes a possible interaction of compound 27 and theoretical dopamine D₂receptor model.

and negatively charged Asp 86 (calculated distance 1.63 Å);

(b) hydrogen bonds between benzimidazole part of the ligand

and Ser 141, Ser 122 and His 189; (c) edge-to-face interac-

tions of the aromatic ring or arylpiperazine part of the ligand

with Phe 178, Tyr 216 and Trp 182 of the receptor. Similar

results were obtained with other substituents in piperazine

ring; (d) in addition, 2-methoxypiperazine derivatives 8, 13,

22, 27, 35 and 43 could build one more hydrogen bond with

Trp 182 and (e) interaction of ethereal oxygen of ethylenoxy

ligands (20–43) with hydrogen of Phe 185 or Trp 115. To

calculate free energy (DG) of this particular interaction, the

structures obtained in docking analysis were used. Coordi-

nates of a ligand and Phe 185 of the receptor were frozen,

while all other elements of the receptor were removed. Single

point calculation was employed to deﬁne total energy of

thereof deﬁned system. In the following step, the ligand was

translated along X-axis by 10 Å and the calculation was

repeated. The difference in the obtained energies was taken

as a measure of the system stabilization. For this calculation,

B3LYP method was used with basis set 6–31 g* in a Gauss-

ian 98W software [11,12]. The obtained free energy value

ranged from 2.5 to 3.8 kcal mol^–1.

5. Discussion

Arylpiperazines have been known to have the activity pro-

ﬁles similar to those of atypical antipsychotics [13–15]. In

V. Šukalovic´ et al. / European Journal of Medicinal Chemistry 40 (2005) 481–493

485

order to design new drugs with desired pharmacological pro-

ﬁles, we have investigated the effects of various structural

modiﬁcations of these molecules on their DA-ergic and sero-

tonergic activity [3–5]. Based on these results, we have found

of interest to prepare derivatives with ethylenoxy and propy-

lene bridge between arylpiperazine and heteroaryl part of the

molecule and to examine their DA-ergic and serotonergic

activity. In contrast to a high D₂DAR binding afﬁnity of a

number of tested compounds, only a few were active in the

5-HT_1Acompetition binding assay, while none of them acted

as a displacer of [³H]SCH 23390. Therefore, our efforts were

directed toward a better understanding of the D₂DAR–

ligand interactions at molecular level using docking of newly

designed ligands in the binding pocket of this receptor.

Docking was performed by considering all receptor amino

acids that could interact after initial positioning of the ligands

against Asp 86, Ser 141 and Ser 122 residues. The binding

pocket designed in this way provided the data that matched

the experimental results obtained in competition binding

assays.

Generally, uncaring points of a ligand docked in the bind-

ing pocket of the D₂DAR are localized around benzimida-

zole and arylpiperazine part of the ligands separated by poly-

methylene spacer. Benzimidazole structural motif interacts

with the receptor through hydrogen bonds that involve amino

acids Ser 122 and Ser 141 in TM II and TM III, that are a part

of catechol binding site of the D₂DAR [10,16]. In addition,

docking analysis revealed that His 189 in TM VI could be

also involved in hydrogen bond formation.Arylpiperazine part

of the ligands interacts with the receptor through hydropho-

bic (edge-to-face) type of interactions [17,18]. A cluster of

aromatic amino acid residues that are highly conserved in

GPCR family of classA are responsible for this kind of inter-

actions. Bondensgaard et al. [19] postulated that this amino

acid cluster builds an ancillary pocket in GPCRs that is

involved in docking of so-called “privileged structures”. This

aspect of ligand–receptor interaction that is more elaborated

in our previous paper [9] can be summarized as follows: ary-

plpiperazine structural motif can be considered as a privi-

leged structure ﬁtting within the ancillary pocket of the D₂

DAR that is preserved in most of the GPRCs. The aryplpip-

erazine moiety is positioned in the ancillary pocket spanned

by the three conserved aromatic residues, i.e. Phe 178, Trp

182 and Tyr 216, providing favorable aromatic–aromatic inter-

actions. The results of docking studies revealed that close

interaction of protonated N1 of the piperazine ring with Asp

86, and edge-to-face interactions of the aromatic ring or

arylpiperazine part of the ligand with Phe 178, Trp 182 and

Tyr 216 of the receptor, represent the main stabilizing forces.

In addition, 2-methoxy derivative (8, 13, 22, 27, 35, 43) could

build one additional hydrogen bond with Trp 182. Bulky sub-

stituents in position 4 of the aromatic part of phenylpipera-

zine ring are not tolerated because of the unfavorable steric

interactions with Phe 178. Substituents in position 2 and 3 of

phenylpiperazine are sterically well tolerated. Electron attrac-

tive groups decreased the binding afﬁnity, while electron

donors like—OMe increased the afﬁnity for the binding at

the D₂DAR in comparison with the unsubstituted phenylpip-

erazine. These effects can be explained by stronger edge-to-

face interactions of negative ESP in the center of aromatic

residues of the ligands and positive ESP of the protons of the

receptor aromatic residues. Methoxy group, increase the

afﬁnities of ligands if attached to position 2, since one addi-

tional hydrogen bond could be formed with Trp 182.

Methylene groups connecting benzimidazole part of the

ligands to arylpiperazine part can be deﬁned as spacers that

determine the distance between two clusters of ligand uncar-

ing points. Therefore, the number of methylene groups in the

spacer critically inﬂuenced the binding afﬁnity of the ligands.

Compounds with a single methylene group had the lowest

afﬁnity that originates from partial occupation of receptor

binding site. Ligands with ethylene and propylene (or ethyl-

enoxy) spacer can completely occupy the binding site of the

receptor. The main difference between these two groups of

ligands comes from the involvement of His 189 in binding of

propylene or ethylenoxy ligands. This increases the number

of hydrogen bonds and consequently, the binding afﬁnity to

the D₂DAR (Fig. 2 ).

Ethylenoxy ligands expressed a higher binding afﬁnity

compared to propylene counterparts because of one addi-

tional attractive interaction with the receptor molecule. This

stabilizing interaction may originate in the formation of an

atypical hydrogen bond between aromatic hydrogens in Phe

185 or Trp 115 and ethereal oxygen of the ligands. This type

of hydrogen bonds was postulated and studied in some other

biological systems [20]. It was shown that these weak inter-

actions, typically 3–4 Å long, represent an important stabi-

lizing factor in protein folding [21]. In docking analyses per-

formed in the present study, distances between the aromatic

hydrogen and ethereal oxygen ranging from 2.55 to 3.25 Å

were found, what makes the formation of this bond quite likely

(Fig. 3). Calculated free energy (DG) of this particular inter-

action was 2.14 kcal mol^–1. This value is slightly exceeding

the value of 0.3–1.9 kcal mol^–1claimed by Gu et al. [22] but

this can be explained by limitations of the available model of

the D₂DAR binding site. In addition to this, some recent pub-

lished data [6,7] shows signiﬁcant difference in torsional

potential between aryl–CH₂and aryl–O. In studied anisol like

systems, the global minimum corresponds to the eclipsed

structure in which the methoxy group is coplanar with the

aromatic ring. A signiﬁcant decrease of the calculated rota-

tional barrier at the orthogonal structure by 1.5–3.0 kcal mol^–1

was observed. This is an additional constrain that itself can

inﬂuence overall stability of receptor–ligand complex.

Differences in binding afﬁnity among the ligands that dif-

fer only in benzimidazole part of the molecule can be ascribed

to the number of possible hydrogen bonds formed with Ser

122 (TM IV), Ser 141 (TM V) and His 189 (TM VI). Benz-

imidazole derivatives 4–8 and 20–24 are capable of building

only one hydrogen bond with Ser 141 (Fig. 2a), while ben-

zotriazoles 41–43 are interacting by forming one or two

hydrogen bonds with Ser 141 and His 189 (Fig. 2b).

486

V. Šukalovic´ et al. / European Journal of Medicinal Chemistry 40 (2005) 481–493

Fig. 2

.

Interaction of benzimidazole part of the ligands with the D₂dopamine receptor. Schematic representation of the interaction of (a) benzimidazole, (b)

benzotriazole, (c) benzimidazole-2-one and (d) benzimidazole-2-thione derivatives with Ser 141, Ser 122 and His 189 of the D₂DAR.

Benzimidazole-2-ones 33–40 are building two hydrogen

bonds, one with Ser 141 and one with His 189 (Fig. 2c), while

benzimidazole-2-thiones 9–13 and 25–32 are capable of build-

ing one additional hydrogen bond with Ser 122 via long C=S

bond (Fig. 2 d). Accordingly, benzimidazole-2-thione deriva-

tives 9–13 and 25–32 should have the highest binding afﬁn-

ity. They are followed by benzimidazole-2-ones 33–40, ben-

zotriazoles 41–43 and benzimidazoles 4–8 and 20–24, as

conﬁrmed by experimental results obtained in competition

binding assays listed in Table 1.

The effects of substituents introduced in arylpiperazines

on DA-ergic activity could be summarized as follows: (a) sub-

Fig. 3. Interaction of ethereal oxygen of 5-[2-(4-phenylpiperazine-1-yl)ethoxy]-1H-benzimidazole type of ligands with the D₂DAR. Interaction of ethereal

oxygen of 5-[2-(4-phenylpiperazine-1-yl)ethoxy]-1H-benzimidazole (20) with. Phe 185 and Trp 115. Figures a and b are showing docked ligand from two

different angles.

V. Šukalovic´ et al. / European Journal of Medicinal Chemistry 40 (2005) 481–493

487

stituents in position 2 and 3 of piperazine phenyl ring are

sterically well tolerated; (b) substituents with electron with-

drawal effect in this position, such as 3-triﬂuoromethyl (5,

10, 21, 26, 34 and 42) and 3-chloro (31 and 39) are affecting

the afﬁnity by decreasing electron density in the benzene ring

of these ligands that results in the reduction of the energy of

edge-to-face interactions; (c) electron donor groups, e.g. meth-

oxy (8, 13, 22, 27, 28, 35, 36 and 43), expressed a beneﬁcial

effect on ligand binding by facilitating edge-to-face interac-

tions; (d) in the case of ligands with methoxy group in posi-

tion 2 (8, 13, 22, 27, 35 and 43), one additional hydrogen

bond can be built with Trp 182, and as a consequence, these

ligands were the most active among all analogs; (e) ligands

with bulky substituents in position 4 of arylpiperazine ring

(6, 7, 11, 12, 24, 29, 32, 37 and 40) are involved in unfavor-

able steric interaction of substituents with Phe 178 in the

receptor binding pocket what reduced the afﬁnity of these

ligands.

For analytical thin-layer chromatography E. Merck (Darm-

stadt, Germany) F-256 plastic-backed thin-layer silica gel

plates were used. Chromatographic puriﬁcations were per-

formed on Merck-60 silica gel columns, 230–400 mesh

ASTM, under medium pressure (MPLC). Solutions were rou-

tinely dried over anhydrous Na₂SO₄prior to evaporation.

7.2. Molecular modeling

Ligand models were constructed using a Hyperchem v.

7.0 software (Hypercube Inc., Gainesville, USA) and inbuilt

PM3 routine for molecular geometry optimization. It was pos-

tulated that the ligands are bound to the receptors in proto-

nated form [23,24], therefore, a formal charge of +1 was added

to piperazine nitrogen (1N). The results obtained were fur-

ther optimized in a Gaussian 98W, Rev. A.9 (Gaussian Inc.,

Pittsburgh, USA) using the DFT B3LYP method and a 6–31 g

basis set [11,12].

Modeling of the ligand–D₂DAR complexes was done

using the Docking module within an INSIGHT II software

(Accelerys Inc., Cambridge, UK) on a SGI Octane 2 work-

station (Silicon Graphics Inc., Mountain View, USA). Dock-

ing of the ligands described here was performed initially, using

SA docking algorithm and up to 100 structures were gener-

ated applying a Monte Carlo method. Each structure was fur-

ther minimized for 4000 cycles or until 0.01 kcal mol^–1Å^–1

was reached. Minimization was performed by ﬁxing all the

protein backbone atoms and keeping ligand and amino acid

residues in the binding site ﬂexible. In this way, the relax-

ation of Van der Waals interactions was permitted. Subse-

quently, all structures were ﬁltered using the general rule that

the best structure is the one with the shortest salt bridge

between the ligand andAsp 86, and with a maximum number

of hydrogen bonds with the D₂DAR. The obtained results

were visualized using a DS View software (Accelerys Inc.,

Cambridge, UK).

6. Conclusions

The results of docking studies on newly synthesized

compounds—D₂DAR complexes revealed that these ligands

interact through protonated N1 of the piperazine ring with

Asp 86 (salt bridge), hydrogen bonds between bezimidazole

part of a ligand and Ser 141, Ser 122 and His 189, edge-to-

face interactions of the aromatic ring or arylpiperazine part

of a ligand with Phe 178, Tyr 216 and Trp 182 of the receptor.

In addition, 2-methoxy derivatives could build one additional

hydrogen bond with Trp 182. Similarly, ethereal oxygen of

ethylenoxy derivatives interacts with hydrogen of Phe 185 or

Trp 115. Compound of the highest afﬁnity 5-{2-[4-(2-

methoxyphenyl)piperazin-1-yl]ethoxy}-1,3-dihydro-2H-

benzimidazole-2-thione (27) is the one with the maximal num-

ber of the above listed attractive interactions. The results

presented here provide a basis for further rational design of

DA-ergic compounds.

7.3. Chemistry

7.3.1. Syntheses

7.3.1.1. Preparation of 1-(2-chloroethoxy)-4-nitrobenzene

(14). To 0.5 mol 4-nitrophenol solution in 350 ml of ethyl–

methyl–ketone, 60 g (0.5 mol) of anhydrous potassium car-

bonate and 150 ml 1,2-dichloroethane were added with stir-

ring. The mixture was reﬂuxed (36 h) with a constant stirring.

After the reaction was completed, the reaction mixture was

cooled to ambient temperature and poured into 1 l water. The

product was extracted three times with 300 ml methylene

chloride. After evaporation, crude product was crystallized

from 90% ethanol. Yield: 72%; m.p. 48 °C; IR (cm^–1): 1258,

1339, 1507, 1596; ¹H NMR: d 3.87 (t, 2H, J = 5.8 Hz), 4.33

(t, 2H, J = 5.6 Hz), 7.00 (d, 2H, J = 9.2 Hz,ArH), 8.22 (d, 2H,

J = 9.4 Hz, ArH).

7. Experimental protocols

7.1. General

A Boetius PHMK apparatus (VEBAnalytic, Dresden, Ger-

many) was used to determine melting points (m.p.), pre-

sented here as uncorrected. H NMR spectra recorded on a

1

Gemini 2000 spectrometer (Varian, PaloAlto, CA, USA) with

CDCl₃as a solvent unless otherwise stated are reported in

ppm downﬁeld from the internal standard tetramethylsilane.

The IR spectra were run on a Perkin–Elmer 457 Grating

Infrared Spectrophotometer (Perkin–Elmer, Beaconsﬁeld,

UK). High resolution mass spectra were acquired on a Bruker

Biﬂex MALDI TOF (Bruker, Bremen, Germany). Elemental

analyses were performed by a Perkin–Elmer 240B microana-

lyzer.Analyses indicated by the symbols of the elements were

within 0.4% of the theoretical values.

7.3.1.2. Preparation of N-[4-(2-(chloroethoxy)phenyl]aceta-

mide (15). 1-(2-Chloroethoxy)-4-nitrobenzene (14) (0.25 mol)

was dissolved in the mixture of 300 ml acetic acid and 250 ml

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V. Šukalovic´ et al. / European Journal of Medicinal Chemistry 40 (2005) 481–493

acetanhydride. The mixture was stirred and upon adding 195 g

zinc dust in three portions, heated in an oil bath (80 °C) with

constant stirring.After the reaction was completed (about 6 h),

the mixture was ﬁltered through sinter glass and the ﬁltrate

evaporated under reduced pressure. Crude product was crys-

tallized from ethanol. Yield: 88%; m.p. 110 °C; IR (cm^–1):

1256, 1530, 1663, 3313; ¹H NMR: d 2.16 (s, 3H, CH₃), 3.80

(t, 2H, J = 6 Hz), 4.21 (t, 2H, J = 6 Hz), 6.89 (d, 2H, J = 9 Hz,

ArH), 7.18 (s, 1H, NH), 7.41 (d, 2H, J = 9 Hz, ArH).

J = 8.6 Hz, ArH), 7.05–7.35 (m, 5H, ArH), 7.96 (d, 1H,

J = 2 Hz, ArH).

7.3.2.3. 2-Nitro-4-{3-[4-(4-chlorophenyl)piperazin-1-yl]-

propyl}aniline (2c). Yield: 70%; m.p. 118 °C; IR (cm^–1):

1451, 1499, 1640, 2825, 3045; ¹H NMR: d 1.81 (quint, 2H,

J = 8 Hz), 2.39 (t, 2H, J = 7.8 Hz), 2.59 (m, 6H), 3.17 (t, 4H,

J = 5 Hz), 5.98 (s, 2H, NH₂), 6.75 (d, 1H, J = 8.4 Hz, ArH),

6.84 (dd, 1H, J = 4.6 Hz, J = 2.2 Hz, ArH), 7.21 (m, 4H,

ArH), 7.95 (d, 1H, J = 2.2 Hz, ArH).

7.3.1.3. Preparation of N-[4-(2-chloroethoxy)-2-nitrophe-

nyl]acetamide (16). Finely powdered N-[4-(2-(chloro-

ethoxy)phenyl]acetamide (15) (0.20 mol) was added in por-

tions into 300 ml of boiling 20% nitric acid with stirring.After

1 h, the reaction mixture was cooled and poured onto 500 g

crushed ice. The precipitate was ﬁltered and washed with cold

water. Crude product was crystallized from 90% ethanol.

Yield: 68%; m.p. 95 °C; IR (cm^–1): 1242, 1277, 1508, 1697,

3366; ¹H NMR: d 2.28 (s, 3H, CH₃), 3.84 (t, 2H, J = 5.6 Hz),

4.28 (t, 2H, J = 5.6 Hz), 7.27 (d, 1H, J = 3 Hz, J = 6.1 Hz,

ArH), 7.70 (d, 1H, J = 3 Hz, ArH), 8.68 (d, 1H, J = 9.4 Hz,

ArH), 10.09 (s, 1H, NH).

7.3.2.4. 2-Nitro-4-{3-[4-(4-methoxyphenyl)piperazin-1-yl]-

propyl}aniline (2d). Yield: 68%; m.p. 109 °C; IR (cm^–1):

1242, 1513, 1635, 2822, 3320; ¹H NMR: d 1.81 (quint, 2H,

J = 8 Hz), 2.39 (t, 2H, J = 7.8 Hz), 2.59 (m, 6H), 3.17 (t, 4H,

J = 5 Hz), 5.98 (s, 2H, NH₂), 6.75 (d, 1H, J = 8.4 Hz, ArH),

6.84 (dd, 1H, J = 4.6 Hz, J = 2.2 Hz, ArH), 7.21 (m, 4H,

ArH), 7.95 (d, 1H, J = 2.2 Hz, ArH).

7.3.2.5. 2-Nitro-4-{3-[4-(2-methoxyphenyl)piperazin-1-yl]-

propyl}aniline (2e). Yield: 65%; m.p. 110 °C; IR (cm^–1):

1248, 1512, 1641, 2818, 3060; ¹H NMR: d 1.80 (quint, 2H,

J = 8 Hz), 2.41 (t, 2H, J = 7.8 Hz), 2.62 (m, 6H), 3.11 (m,

4H), 3.82 (s, 3H, OCH₃), 5.99 (s, 2H, NH₂), 6.73 (d, 1H,

J = 8.4 Hz, ArH), 6.82–7.00 (m, 4H, ArH), 7.11 (dd, 1H,

J = 2 Hz, J = 4.8 Hz, ArH), 7.97 (d, 1H, J = 2.2 Hz, ArH).

7.3.1.4. Preparation of 4-(2-chloroethoxy)-2-nitroaniline

(17). N-[4-(2-Chloroethoxy)-2-nitrophenyl]acetamide (16)

(0.15 mol) was resuspended in 120 ml 4 N HCl and reﬂuxed

for 4 h. The solution was cooled to ambient temperature and

transferred in a refrigerator overnight. The crystals were sepa-

rated by ﬁltration and recrystallized from 90% EtOH. Yield:

75%; m.p. 77 °C; IR (cm^–1): 1114, 1453, 1510, 1669, 2356,

7.3.2.6. 2-Nitro-4-[2-(4-phenylpiperazin-1-yl)ethoxy]ani-

line (18a). Yield: 70%; m.p. 127 °C; IR (cm^–1): 1217, 1246,

1

1417, 1509, 1596, 2944, 3372; H NMR: d 2.74 (m, 4H),

2.83 (t, 2H, J = 6 Hz), 3.21 (m, 4H), 4.12 (t, 2H, J = 5.8 Hz),

5.93 (s, 2H, NH₂), 6.81–6.97 (m, 4H, ArH), 7.23–7.34 (m,

3H, ArH), 8.11 (s, 1H, ArH).

1

3057; H NMR: d 3.81 (t, 2H, J = 5.8 Hz), 4.20 (t, 2H,

J = 5.6 Hz), 6.02 (s, 2H, NH₂), 6.80 (d, 1H, J = 8.8 Hz, ArH),

7.70 (d, 1H, J = 2.8 Hz, J = 6.2 Hz,ArH), 7.53 (d, 1H, J = 3 Hz,

ArH).

7.3.2.7. 2-Nitro-4-(2-{4-[3-(triﬂuoromethyl)phenyl]piperazin-

1-yl}ethoxy)aniline (18b). Yield: 72%; m.p. 85 °C; IR (cm^–

1

): 1215, 1315, 1416, 1513, 2828, 3171; ¹H NMR: d 2.75 (t,

7.3.2. General alkylation procedure

Solution of 1 or 17 (20 mmol) was mixed with 22 mmol

arylpiperazines, 25 ml dimethylformamide (DMF) and the

mixture of 3.18 g K₂CO₃and 1.0 g KI. The resulting mixture

was stirred (24 h, 80 °C). After cooling, the precipitate was

discarded and the ﬁltrate evaporated in vacuo. The residue

was chromatographed on silica gel and recrystallized from

hot isopropanol.

4H, J = 5 Hz), 2.87 (t, 2H, J = 5.4 Hz), 3.38 (t, 4H, J = 5 Hz),

4.12 (t, 2H, J = 4.8 Hz), 5.92 (s, 2H, NH₂), 6.77 (d, 1H,

J = 9.2 Hz), 7.05–7.39 (m, 5H,ArH), 7.59 (d, 1H, J = 2.8 Hz).

7.3.2.8. 2-Nitro-4-{2-[4-(2-methoxyphenyl)piperazin-1-yl]-

ethoxy}aniline (18c). Yield: 62%; m.p. 120 °C; IR (cm^–1):

1

1254, 1517, 1596, 2945; H NMR (d₆DMSO): d 2.62 (m,

4H), 2.73 (t, 2H, J = 5.6 Hz), 2.96 (m, 4H), 3.77 (s, 3H,

OCH₃), 4.06 (t, 2H, J = 5.4 Hz), 6.87–6.95 (m, 4H, ArH,

NH₂), 7.00 (d, 1H, J = 9.4 Hz,ArH), 7.19 (dd, 1H, J = 2.8 Hz,

J = 6.2 Hz, ArH), 7.27 (s, 2H, ArH), 7.42 (d, 1H, J = 3 Hz,

ArH).

7.3.2.1. 2-Nitro-4-[3-(4-phenylpiperazin-1-yl)propyl]ani-

line (2a). Yield: 60%; m.p. 98 °C; IR (cm^–1): 1343, 1520,

3475; ¹H NMR: d 1.83 (m, 2H), 2.45 (t, 2H, J = 7 Hz), 2.60

(m, 6H), 3.24 (m, 4H), 6.05 (s, 2H, NH₂), 6.75 (d, 1H,

J = 8 Hz, ArH), 6.84 (t, 2H, J = 8 Hz, ArH), 6.92 (d, 1H,

J = 8 Hz, ArH), 7.24 (m, 3H, ArH), 7.98 (s, 1H, ArH).

7.3.2.9. 2-Nitro-4-{2-[4-(3-methoxyphenyl)piperazin-1-yl]-

ethoxy}aniline (18d). Yield: 66%; m.p. 119 °C; IR (cm^–1):

1138, 1256, 1419, 1515, 1596, 2944; ¹H NMR (d₆DMSO): d

3.23 (m, 4H), 3.32 (m, 2H), 3.63 (m, 4H), 3.73 (s, 3H, OCH₃),

4.36 (t, 2H, J = 4 Hz), 4.51 (s, 2H, NH₂), 6.43–6.61 (m, 4H,

ArH), 7.05 (d, 1H, J = 9.2 Hz, ArH), 7.26 (dd, 1H, J = 3 Hz,

J = 9.2 Hz, ArH), 7.52 (d, 1H, J = 3 Hz, ArH).

7.3.2.2. 2-Nitro-4-(3-{4-[3-(triﬂuoromethyl)phenyl]piperazin-

1-yl}propyl)aniline (2b). Yield: 62%; m.p. 99 °C; IR (KBr)

(cm^–1): 1408, 1449, 1566, 1642, 2950, 3083; ¹H NMR: d 1.82

(quint, 2H, J = 8 Hz), 2.41 (t, 2H, J = 8 Hz), 2.60 (m, 6H),

3.25 (t, 4H, J = 5 Hz), 5.98 (s, 2H, NH₂), 6.76 (d, 1H,

V. Šukalovic´ et al. / European Journal of Medicinal Chemistry 40 (2005) 481–493

489

7.3.2.10. 2-Nitro-4-{2-[4-(4-methoxyphenyl)piperazin-1-yl]-

ethoxy}aniline (18e). Yield: 65%; m.p. 132 °C; IR (cm^–1):

1210, 1417, 1512, 2825; ¹H NMR: d 2.76 (t, 4H, J = 2.8 Hz),

2.86 (t, 2H, J = 5.6 Hz), 3.13 (t, 4H, J = 4.6 Hz), 3.77 (s, 3H,

OCH₃), 4.11 (t, 2H, J = 5.6 Hz), 6.02 (s, 2H, NH₂), 6.75–6.94

(m, 5H, ArH), 7.00 (dd, 1H, J = 3 Hz, J = 6.2 Hz, ArH), 7.58

(d, 1H, J = 3 Hz, ArH).

J = 8 Hz, ArH), 7.09 (d, 1H, J = 8 Hz, ArH), 7.25 (t, 2H,

J = 7.8 Hz, ArH), 7.45 (s, 1H, ArH), 7.53 (d, 1H, J = 8 Hz,

ArH), 8.23 (s, 1H, CH). MS: m/e 320.198. Anal.: C₂₀H₂₄N₄

(C, H, N).

7.3.4.2. 5-(3-{4-[3-(Triﬂuoromethyl)phenyl]-piperazin-1-

yl}propyl)-1H-benzimidazole (5). Yield: 60%; m.p. oil; IR

(cm^–1): 1451, 1495, 1612, 2947, 3029; ¹H NMR: d 1.92 (quint,

2H, J = 8 Hz), 2.47 (t, 2H, J = 8.2 Hz), 2.62 (t, 4H J = 4.8 Hz),

2.79 (t, 2H, J = 7.6 Hz), 3.25 (t, 4H, J = 7 Hz), 7.03–7.16 (m,

4H, ArH), 7.34 (d, 1H, J = 7.6 Hz, ArH), 7.47 (s, 1H, ArH),

7.58 (d, 1H, J = 8.2 Hz, ArH), 8.05 (s, 1H, CH). MS: m/e

388.181. Anal.: C₂₁H₂₃F₃N₄(C, H, N).

7.3.2.11. 2-Nitro-4-{2-[4-(2-chlorophenyl)piperazin-1-yl]-

ethoxy}aniline (18f). Yield: 72%; m.p. 139 °C; IR (cm^–1):

1

1417, 1456, 1594, 2825, 3312; H NMR: d 2.78 (t, 4H,

J = 4.6 Hz), 2.88 (t, 2H, J = 5.8 Hz), 3.12 (t, 4H, J = 4.6 Hz),

4.12 (t, 2H, J = 5.8 Hz), 6.22 (s, 2H, NH₂), 6.83 (d, 1H,

J = 9.2 Hz, ArH), 6.93–6.14 (m, 3H, ArH), 7.12–7.26 (m,

1H, ArH), 7.36 (dd, 1H, J = 1.6 Hz, J = 6.4 Hz, ArH), 7.57 (d,

1H, J = 2.8 Hz, ArH).

7.3.4.3. 5-{3-[4-(4-Chlorophenyl)piperazin-1-yl]propyl}-1H-

benzimidazole (6). Yield: 66%; m.p. 177 °C; IR (cm^–1): 1451,

1

1495, 1627, 2939, 3122; H NMR: d 1.80 (quint, 2H,

J = 7.6 Hz), 2.33 (t, 2H, J = 6.8 Hz), 2.51 (m, 4H), 2.71 (t,

2H, J = 7.6 Hz), 3.18 (t, 4H, J = 4.8 Hz), 6.93 (d, 2H,

J = 7.2 Hz, ArH), 7.05 (d, 1H, J = 8.2 Hz, ArH), 7.22 (d, 2H,

J = 9 Hz, ArH), 7.45 (m, 2H, ArH), 8.14 (s, 1H, CH), 12.31

(s, 1H, NH). MS: m/e 354.160. Anal.: C₂₀H₂₃ClN₄(C, H, N).

7.3.2.12. 2-Nitro-4-{2-[4-(3-chlorophenyl)piperazin-1-yl]-

ethoxy}aniline (18g). Yield: 68%; m.p. 110 °C; IR (cm^–1):

1

1216, 1255, 1513, 1595, 2830; H NMR: d 2.72 (m, 4H),

2.83 (t, 2H, J = 5.4 Hz), 3.23 (t, 4H, J = 5 Hz), 4.11 (t, 2H,

J = 5.4 Hz), 5.91 (s, 2H, NH₂), 6.74–6.82 (m, 3H,ArH), 6.87–

6.89 (m, 1H, ArH), 7.08–7.21 (m, 2H, ArH), 7.59 (d, 1H,

J = 2.8 Hz, ArH).

7.3.4.4. 5-{3-[4-(4-Methoxyphenyl)piperazin-1-yl]propyl}-

1H-benzimidazole (7). Yield: 70%; m.p. 189 °C; IR (cm^–1):

1246, 1512, 1630, 2951, 3060, 3411; ¹H NMR: d 1.88 (quint,

2H, J = 7.6 Hz), 2.43 (t, 2H, J = 6.8 Hz), 2.60 (m, 4H), 280 (t,

2H, J = 7.6 Hz), 3.15 (t, 4H, J = 4.8 Hz), 3.79 (s, 3H, OCH₃),

6.80–6.93 (m, 4H, ArH), 7.13 (dd, 1H, J = 1.4 Hz, J = 7 Hz,

ArH), 7.45 (m, 1H, ArH), 7.57 (d, 1H, J = 8 Hz, ArH), 8.05

(s, 1H, CH), 12.31 (s, 1H, NH). MS: m/e 350.209. Anal.:

C₂₁H₂₆N₄O (C, H, N).

7.3.2.13. 2-Nitro-4-{2-[4-(4-chlorophenyl)piperazin-1-yl]-

ethoxy}aniline (18h). Yield: 65%; m.p. 106 °C; IR (cm^–1):

1

1207, 1416, 1509, 1642, 2819; H NMR: d 2.74 (m, 4H),

2.86 (t, 2H, J = 5.6 Hz), 3.19 (t, 4H, J = 5 Hz), 4.11 (t, 2H,

J = 5.8 Hz), 5.91 (s, 2H, NH₂), 6.73–6.88 (m, 3H,ArH), 7.07–

7.23 (m, 3H, ArH), 7.58 (d, 1H, J = 2.8 Hz, ArH).

7.3.3. General procedure for reduction

7.3.4.5. 5-{3-[4-(2-Methoxyphenyl)piperazin-1-yl]propyl}-

Ra–Ni (0.4–0.5 g) was added in small portions to a stir-

ring solution of 6.5 mmol of either nitro compound (2a–j,

18a–h) in 12 ml EtOH, 12 ml 1,2-dichloro-ethane and 2 ml

(20 mmol) hydrazine hydrate at 30 °C. After the addition of

Ra–Ni was completed, the mixture was heated in a water bath

(50 °C, 60 min) and ﬁltered through celite. The ﬁltrate was

evaporated in vacuo and crude product used for further syn-

theses.

1H-benzimidazole (8). Yield: 66%; m.p. oil; IR (cm^–1): 1242,

1

1513, 1638, 2942, 3083; H NMR: d 1.92 (quint, 2H,

J = 8 Hz), 2.49 (t, 2H, J = 8.2 Hz), 2.68 (m, 4H), 2.79 (t, 2H,

J = 7.6 Hz), 3.12 (m, 4H), 3.86 (s, 3H, OCH₃), 6.85–6.97 (m,

4H, ArH), 3.13 (d, 1H, J = 8.2 Hz, ArH), 7.44 (s, 1H, ArH),

7.57 (d, 1H, J = 8.6 Hz, ArH), 8.01 (s, 1H, CH). MS: m/e

350.212. Anal.: C₂₁H₂₆N₄O (C, H, N).

7.3.4.6. 5-[2-(4-Phenylpiperazin-1-yl)ethoxy]-1H-benzimi-

dazole (20). Yield: 62%; m.p. 74 °C; IR (cm^–1): 1165, 1238,

1452, 1598, 2823, 3149; ¹H NMR: d 2.77 (t, 4H, J = 4.8 Hz),

2.90 (t, 2H, J = 5.6 Hz), 3.23 (t, 4H, J = 5.2 Hz), 4.18 (t, 2H,

J = 5.6 Hz), 6.83–6.97 (m, 3H, ArH), 7.10 (d, 1H, J = 2 Hz,

ArH), 7.23–7.29 (m, 3H, ArH), 7.54 (d, 1H, J = 9 Hz, AH),

7.98 (s, 1H, CH), 12.23 (s, 1H, NH). MS: m/e 322.183.Anal.:

C₁₉H₂₂N₄O (C, H, N).

7.3.4. General procedure for the synthesis of 1H-benzimi-

dazoles

Two milimoles of diamine 3 or 19 and 5.6 mmol 96% for-

mic acid were heated (100 °C, 2 h). After cooling to ambient

temperature, 5 ml of 10% NaHCO₃were added, the product

extracted with CH₂Cl₂and concentrated in vacuo. The result-

ing 1H-benzimidazoles were puriﬁed by chromatography or

recrystallized from hot EtOH.

7.3.4.1. 5-[3-(4-Phenylpiperazin-1-yl)propyl]-1H-benzimi-

dazole (4). Yield: 70%; m.p. 199–200 °C; IR (cm^–1): 1408,

1677, 2948, 3503; ¹H NMR of (d₆DMSO): d 2.02 (m, 2H),

2.75 (t, 2H, J = 7.6 Hz), 3.04 (t, 2H, J = 7.6 Hz), 3.23 (m,

4H), 3.29 (m, 4H), 6.84 (t, 1H, J = 7.2 Hz, ArH), 6.98 (d, 2H,

7.3.4.7. 5-(2-{4-[3-(Triﬂuoromethyl)phenyl]piperazin-1-yl}-

ethoxy)-1H-benzimidazole (21). Yield: 64%; m.p. oil; IR (cm^–

1

): 1121, 1314, 1453, 1615, 2952; H NMR: d 2.71 (t, 4H,

J = 4.8 Hz), 2.85 (t, 2H, J = 5 Hz), 3.22 (t, 4H, J = 5 Hz), 4.11

(t, 2H, J = 5.4 Hz), 6.89–7.13 (m, 4H, ArH), 7.30 (t, 2H,

490

V. Šukalovic´ et al. / European Journal of Medicinal Chemistry 40 (2005) 481–493

J = 7.8 Hz, ArH), 7.54 (d, 1H, J = 8.8 Hz, ArH), 8.08 (s, 1H,

CH), 9.69 (s, 1H, NH). MS: m/e 390.166.Anal.: C₂₀H₂₁F₃N₄O

(C, H, N).

2H, J = 3.6 Hz), 2.52 (m, 4H), 2.65 (t, 2H, J = 7.4 Hz), 3.22

(m, 4H), 6.97–7.08 (m, 4H, ArH), 7.19 (s, 1H, ArH), 7.23 (d,

1H, J = 8.2 Hz, ArH), 7.40 (d, 1H, J = 8.2 Hz, ArH), 12.45 (s,

2H, NH). MS: m/e 420.161. Anal.: C₂₁H₂₃F₃N₄S (C, H, N).

7.3.4.8. 5-{2-[4-(2-Methoxyphenyl)piperazin-1-yl]ethoxy}-

1H-benzimidazole (22). Yield: 57%, m.p. oil; IR (cm^–1): 1242,

1528, 1598, 2943; H NMR: d 2.83 (m, 6H), 3.12 (m, 4H,

J = 5.2 Hz), 3.81 (s, 3H, OCH₃), 4.10 (t, 2H, J = 5.4 Hz), 6.81

(s, 1H, ArH), 6.85–6.89 (m, 3H, ArH), 6.95–7.04 (m, 2H,

ArH), 7.46 (d, 1H, J = 8.8 Hz, AH), 8.05 (s, 1H, CH). MS:

m/e 352.184. Anal.: C₂₀H₂₄N₄O₂(C, H, N).

7.3.5.3. 5-{3-[4-(4-Chlorophenyl)piperazin-1-yl]propyl}-1,3-

dihydro-2H-benzimidazole-2-thione (11). Yield: 55%; m.p.

>250 °C; IR (cm^–1): 1241, 1497, 1596, 2829, 3034; ¹H NMR

(d₆DMSO): d 1.75 (quint, 2H, J = 7 Hz), 2.32 (t, 2H,

J = 7.4 Hz), 2.50 (m, 4H), 2.64 (t, 2H, J = 7.2 Hz), 3.12 (m,

4H), 6.91–7.08 (m, 5H, ArH), 7.23 (s, 1H, ArH), 7.24 (s, 1H,

ArH), 12.40 (s, 2H, NH). MS: m/e 386.133. Anal.:

C₂₀H₂₃ClN₄S (C, H, N).

1

7.3.4.9. 5-{2-[4-(3-Methoxyphenyl)piperazin-1-yl]ethoxy}-

1H-benzimidazole (23). Yield: 60%; m.p. oil; IR (cm^–1): 1166,

1208, 1499, 1604, 2652; ¹H NMR: d 2.76 (t, 4H, J = 5 Hz),

2.91 (t, 2H, J = 5.6 Hz), 3.24 (t, 4H, J = 4.8 Hz), 3.78 (s, 3H,

OCH₃), 4.19 (t, 2H, J = 5.8 Hz), 6.40–6.47 (m, 2H, ArH),

6.55 (dd, 1H, J = 1.8 Hz, J = 6.6 Hz, ArH), 6.96 (dd, 1H,

J = 2.4 Hz, J = 6.4 Hz, ArH), 7.13 (t, 1H, J = 2 Hz, ArH), 7.20

(d, 1H, J = 8.2 Hz, ArH), 7.55 (d, 1H, J = 8.8 Hz, AH), 7.98

(s, 1H, CH). MS: m/e 352.182.Anal.: C₂₀H₂₄N₄O₂(C, H, N).

7.3.5.4. 5-{3-[4-(4-Methoxyphenyl)piperazin-1-yl]propyl}-

1,3-dihydro-2H-benzimidazole-2-thione (12). Yield: 61%;

m.p. >250 °C; IR (cm^–1): 1243, 1513, 1623, 2827, 3089, 3418;

¹H NMR (d₆DMSO): d 1.74 (quint, 2H, J = 7 Hz), 2.30 (t,

2H, J = 6.8 Hz), 2.50 (m, 4H), 2.64 (t, 2H, J = 7.4 Hz), 3.00

(m, 4H), 3.68 (s, 3H, OCH₃), 6.78–6.91 (m, 4H, ArH), 6.91–

7.07 (m, 3H, ArH), 12.45 (s, 2H, NH). MS: m/e 382.184.

Anal.: C₂₁H₂₆N₄OS (C, H, N).

7.3.4.10. 5-{2-[4-(4-Methoxyphenyl)piperazin-1-yl]ethoxy}-

1H-benzimidazole (24). Yield: 57%; m.p. 115 °C; IR (cm^–1):

1151, 1251, 1458, 1512, 1632, 2827; ¹H NMR (d₆DMSO): d

2.66 (t, 4H, J = 4.6 Hz), 2.79 (t, 2H, J = 5.6 Hz), 3.03 (t, 4H,

J = 4 Hz), 3.68 (s, 3H, OCH₃), 4.14 (t, 2H, J = 5.6 Hz), 6.78–

6.92 (m, 5H,ArH), 7.11 (s, 1H,ArH), 7.47 (d, 1H, J = 8.8 Hz,

ArH), 8.09 (s, 1H, CH), 12.20 (s, 1H, NH). MS: m/e 352.191.

Anal.: C₂₀H₂₄N₄O₂(C, H, N).

7.3.5.5. 5-{2-[4-(2-Methoxyphenyl)piperazin-1-yl]ethoxy}-

1,3-dihydro-2H-benzimidazole-2-thione (13). Yield: 55%;

m.p. 127 °C; IR (cm^–1): 1241, 1511, 1625, 2831, 3092, 3418;

¹H NMR (d₆DMSO): d 1.80 (m, 2H), 2.49 (t, 2H, J = 7.4 Hz),

2.65 (m, 6H), 3.01 (m, 4H), 3.77 (s, 3H, OCH₃), 6.87–7.09

(m, 7H, ArH), 12.48 (s, 2H, NH). MS: m/e 382.181. Anal.:

C₂₁H₂₆N₄OS (C, H, N).

7.3.5. General procedure for the synthesis

7.3.5.6. 5-[2-(4-Phenylpiperazin-1-yl)ethoxy]-1,3-dihydro-

of benzimidazole-2-thiones

2H-benzimidazole-2-thione (25). Yield: 45%; m.p. 262 °C;

1

Carbon disulﬁde (0.28 ml, 5.8 mmol) and KOH (0.28 g in

0.6 ml water) were added to 2.0 mmol of diamine 3 or 19

previously dissolved in 5 ml EtOH. After reﬂuxing for 3 h,

activated charcoal was added and the suspension ﬁltered

through celite. The solvent was removed in vacuo and the

residue resuspended in 10% NaHCO₃, extracted with CH₂Cl₂

and concentrated in vacuo. The resulting benzimidazole-2-

thiones were puriﬁed by chromatography or recrystallized

from hot EtOH.

IR (cm^–1): 1170, 1226, 1463, 1498, 1598, 2819, 3165; H

NMR (d₆DMSO): d 2.63 (t, 4H, J = 4.6 Hz), 2.75 (t, 2H,

J = 5.8 Hz), 3.13 (t, 4H, J = 5 Hz), 4.10 (t, 2H, J = 5.4 Hz),

6.73–6.80 (m, 3H, ArH), 6.91–7.05 (m, 3H, ArH), 7.17–7.25

(m, 2H, ArH), 12.40 (d, 2H, J = 9.4 Hz, NH). MS: m/e

354.153. Anal.: C₁₉H₂₂N₄OS (C, H, N).

7.3.5.7. 5-(2-{4-[3-(Triﬂuoromethyl)phenyl]piperazin-1-yl}-

ethoxy)-1,3-dihydro-2H-benzimidazole-2-thione (26). Yield:

55%; m.p. 76 °C; IR (cm^–1): 1122, 1316, 1456, 1629, 2948;

¹H NMR (d₆DMSO): d 2.69 (t, 4H, J = 4.8 Hz), 2.80 (t, 2H,

J = 5.2 Hz), 3.25 (t, 4H, J = 4.8 Hz), 4.12 (t, 2H, J = 4.8 Hz),

6.73 (s, 1H, ArH), 6.76 (d, 1H, J = 8 Hz, ArH), 7.06 (t, 2H,

J = 5.8 Hz, ArH), 7.17–7.24 (m, 2H, ArH), 7.42 (t, 1H,

J = 7.8 Hz, ArH), 12.39 (s, 1H, NH), 12.45 (s, 1H, NH). MS:

m/e 422.140. Anal.: C₂₀H₂₁F₃N₄OS (C, H, N).

7.3.5.1. 5-[3-(4-(Phenylpiperazin-1-yl)propyl]-1,3-dihydro-

2H-benzimidazole-2-thione (9). Yield: 62%; m.p. >250 °C;

IR (cm^–1): 1183, 1465, 1493, 1620, 2927; ¹H NMR

(d₆DMSO): d 1.75 (m, 2H), 2.31 (t, 2H, J = 6.8 Hz), 2.48 (m,

4H), 2.64 (t, 2H, J = 6.8 Hz), 3.12 (m, 4H), 6.76 (t, 1H,

J = 7.4 Hz, ArH), 6.89–7.07 (m, 5H, ArH), 7.20 (t, 2H,

J = 8 Hz, ArH). MS: m/e 352.172. Anal.: C₂₀H₂₄N₄S (C, H,

N).

7.3.5.8. 5-{2-[4-(2-Methoxyphenyl)piperazin-1-yl]ethoxy}-

1,3-dihydro-2H-benzimidazole-2-thione (27). Yield: 48%;

m.p. 99 °C; IR (cm^–1): 1170, 1242, 1462, 1499, 1635, 2829;

¹H NMR (d₆DMSO): d 2.68 (m, 4H), 2.86–2.90 (m, 2H), 2.97

(m, 4H), 3.78 (s, 3H, OCH₃), 4.10 (t, 2H, J = 5.6 Hz), 6.73 (s,

1H, ArH), 6.78 (d, 1H, J = 2.2 Hz), 6.88–6.93 (m, 4H, ArH),

7.3.5.2. 5-(3-{4-[3-(Triﬂuoromethyl)phenyl]piperazin-1-yl}-

propyl)-1,3-dihydro-2H-benzimidazole-2-thione (10). Yield:

58%; m.p. >250 °C; IR (cm^–1): 1162, 1460, 1614, 2836, 3081;

¹H NMR (d₆DMSO): d 1.76 (quint, 2H, J = 7 Hz), 2.32 (t,

V. Šukalovic´ et al. / European Journal of Medicinal Chemistry 40 (2005) 481–493

491

7.03 (d, 1H, J = 8.4 Hz, ArH), 12.39 (s, 1H, NH), 12.44 (s,

1H, NH). MS: m/e 384.162. Anal.: C₂₀H₂₄N₄O₂S (C, H, N).

When the addition was completed, the mixture was stirred

for additional 30 min and transferred in a refrigerator over-

night. The solvent was removed in vacuo and the residue

recrystallized in CHCl₃/EtOH mixture.

7.3.5.9. 5-{2-[4-(3-Methoxyphenyl)piperazin-1-yl]ethoxy}-

1,3-dihydro-2H-benzimidazole-2-thione (28). Yield: 52%;

1

m.p. 201 °C; IR (cm^–1): 1168, 1208, 1461, 1607, 2826; H

7.3.6.1. 5-[2-(4-Phenylpiperazin-1-yl)ethoxy]-1,3-dihydro-

2H-benzimidazole-2-one (33). Yield: 58%; m.p. 243 °C; IR

(cm^–1): 1170, 1499, 1701, 1754, 2824; ¹H NMR (d₆DMSO):

d 2.63 (t, 4H, J = 5 Hz), 2.73 (t, 2H, J = 5.8 Hz), 3.13 (t, 4H,

J = 5 Hz), 4.05 (t, 2H, J = 5.6 Hz), 6.51–6.55 (m, 2H, ArH),

6.75–6.82 (m, 2H, ArH), 6.90–6.95 (m, 2H, ArH), 7.17–7.25

(m, 2H,ArH), 10.38 (s, 1H, NH), 10.52 (s, 1H, NH). MS: m/e

338.176. Anal.: C₁₉H₂₂N₄O₂(C, H, N).

NMR (d₆DMSO): d 2.62 (t, 4H, J = 4.8 Hz), 2.74 (t, 2H,

J = 5.4 Hz), 3.13 (t, 4H, J = 4.4 Hz), 3.71 (s, 3H, OCH₃), 4.09

(t, 2H, J = 5.6 Hz), 6.36 (dd, 1H, J = 2 Hz, J = 6 Hz, ArH),

6.44 (t, 1H, J = 2 Hz, ArH), 6.52 (dd, 1H, J = 1.8 Hz,

J = 6.4 Hz, ArH), 6.73 (s, 1H, ArH), 6.77 (d, 1H, J = 2.2 Hz,

ArH), 7.02–7.14 (m, 2H, ArH), 12.40 (s, 2H, NH). MS: m/e

384.160. Anal.: C₂₀H₂₄N₄O₂S (C, H, N).

7.3.6.2. 5-(2-{4-[3-(Triﬂuoromethyl)phenyl]piperazin-1-yl}-

7.3.5.10. 5-{2-[4-(4-Methoxyphenyl)piperazin-1-yl]ethoxy}-

1,3-dihydro-2H-benzimidazole-2-thione (29). Yield: 49%;

m.p. 255 °C; IR (cm^–1): 1168, 1253, 1462, 1512, 1629, 2825;

¹H NMR (d₆DMSO): d 2.67 (t, 4H, J = 4.6 Hz), 2.76 (t, 2H,

J = 5.4 Hz), 3.02 (t, 4H, J = 4.2 Hz), 3.68 (s, 3H, OCH₃), 4.10

(t, 2H, J = 5.6 Hz), 6.72–6.91 (m, 6H, ArH), 7.03 (d, 1H,

J = 8.4 Hz, ArH), 12.39 (s, 1H, NH), 12.44 (s, 1H, NH). MS:

m/e 384.164. Anal.: C₂₀H₂₄N₄O₂S (C, H, N).

ethoxy)-1,3-dihydro-2H-benzimidazole-2-one (34). Yield:

1

53%; m.p. 226 °C; IR (cm^–1): 1166, 1503, 1696, 2972; H

NMR (d₆DMSO): d 2.63 (t, 4H, J = 5 Hz), 2.73 (t, 2H,

J = 5.6 Hz), 3.23 (t, 4H, J = 5.2 Hz), 4.06 (t, 2H, J = 6 Hz),

6.51 (d, 1H, J = 2.4 Hz, ArH), 6.55 (s, 1H, ArH), 6.80 (d, 1H,

J = 9.2 Hz, ArH), 7.06 (d, 1H, J = 7.2 Hz, ArH), 7.16 (s, 1H,

ArH), 7.21 (d, 1H, J = 8.2 Hz, ArH), 7.41 (t, 1H, J = 8.2 Hz,

ArH), 10.38 (s, 1H, NH), 10.52 (s, 1H, NH). MS: m/e 406.163.

Anal.: C₂₀H₂₁F₃N₄O₂(C, H, N).

7.3.5.11. 5-{2-[4-(2-Chlorophenyl)piperazin-1-yl]ethoxy}-

1,3-dihydro-2H-benzimidazole-2-thione (30). Yield: 62%,

7.3.6.3. 5-{2-[4-(2-Methoxyphenyl)piperazin-1-yl]ethoxy}-

1,3-dihydro-2H-benzimidazol-2-one (35). Yield: 56%; m.p.

182 °C; IR (cm^–1): 1168, 1242, 1502, 1643, 1703, 2927; ¹H

NMR: d 2.85 (m, 6H), 3.15 (m, 4H), 3.86 (s, 3H, OCH₃),

4.11 (t, 2H, J = 5.4 Hz), 6.58 (d, 1H, J = 2.4 Hz, ArH), 6.62

(s, 1H, ArH), 6.84–7.05 (m, 5H, ArH), 9.22 (s, 1H, NH), 9.48

(s, 1H, NH). MS: m/e 368.184. Anal.: C₂₀H₂₄N₄O₃(C, H,

N).

1

m.p. 232 °C; IR (cm^–1): 1170, 1464, 1632, 3131; H NMR

(d₆DMSO): d 2.73 (m, 4H), 2.85 (m, 2H), 3.00 (m, 4H), 4.12

(t, 2H, J = 5.4 Hz), 6.73 (s, 1H, ArH), 7.00–7.08 (m, 2H,

ArH), 7.11–7.18 (m, 2H,ArH), 7.26–7.34 (m, 1H,ArH), 7.39–

7.46(m, 1H, ArH), 12.42 (d, 2H, J = 9.6 Hz, NH). MS: m/e

388.112. Anal.: C₁₉H₂₁ClN₄OS (C, H, N).

7.3.5.12. 5-{2-[4-(3-Chlorophenyl)piperazin-1-yl]ethoxy}-

1,3-dihydro-2H-benzimidazole-2-thione (31). Yield: 58%;

7.3.6.4. 5-{2-[4-(3-Methoxyphenyl)piperazin-1-yl]ethoxy}-

1,3-dihydro-2H-benzimidazol-2-one (36). Yield: 54%; m.p.

238 °C; IR (cm^–1): 1171, 1580, 1608, 1703, 2835; ¹H NMR

(d₆DMSO): d 2.61 (t, 4H, J = 5 Hz), 2.72 (t, 2H, J = 5.6 Hz),

3.13 (t, 4H, J = 4.6 Hz), 3.71 (s, 3H, OCH₃), 4.05 (t, 2H,

J = 5.8 Hz), 6.36 (dd, 1H, J = 2.4 Hz, J = 5.8 Hz, ArH), 6.44

(s, 1H,ArH), 6.49–6.55 (m, 3H,ArH), 6.80 (d, 1H, J = 9.2 Hz,

ArH), 7.10 (t, 1H, J = 8.2 Hz, ArH), 10.38 (s, 1H, NH), 10.52

(s, 1H, NH). MS: m/e 368.182. Anal.: C₂₀H₂₄N₄O₃(C, H,

N).

1

m.p. 170 °C; IR (cm^–1):1171, 1463, 1498, 1596, 2829; H

NMR (d₆DMSO): d 2.62 (t, 4H, J = 4.8 Hz), 2.74 (t, 2H,

J = 5.4 Hz), 3.18 (t, 4H, J = 4.6 Hz), 4.10 (t, 2H, J = 5.8 Hz),

6.72–6.79 (m, 3H, ArH), 6.88–6.94 (m, 2H, ArH), 7.01 (d,

1H, J = 8.8 Hz, ArH), 7.20 (t, 1H, J = 8 Hz, ArH), 12.38

(broad s, 2H, NH). MS: m/e 388.114. Anal.: C₁₉H₂₁ClN₄OS

(C, H, N).

7.3.5.13. 5-{2-[4-(4-Chlorophenyl)piperazin-1-yl]ethoxy}-

1,3-dihydro-2H-benzimidazole-2-thione (32). Yield: 50%;

m.p. 194 °C; IR (cm^–1): 1168, 1463, 1498, 1596, 1632, 2825;

¹H NMR (d₆DMSO): d 2.62 (t, 4H, J = 5.2 Hz), 2.74 (t, 2H,

J = 5.2 Hz), 3.13 (t, 4H, J = 4.4 Hz), 4.09 (t, 2H, J = 5.8 Hz),

6.72–6.76 (d, 2H, J = 8.2 Hz, ArH), 6.91–7.05 (m, 3H, ArH),

7.22 (d, 2H, J = 8.8 Hz, ArH), 12.36 (broad s, 2H, NH). MS:

m/e 388.110. Anal.: C₁₉H₂₁ClN₄OS (C, H, N).

7.3.6.5. 5-{2-[4-(4-Methoxyphenyl)piperazin-1-yl]ethoxy}-

1,3-dihydro-2H-benzimidazole-2-one (37). Yield: 55%; m.p.

185 °C; IR (cm^–1): 1172, 1253, 1511, 1755, 2830; ¹H NMR

(d₆DMSO): d 2.63 (t, 4H, J = 4.8 Hz), 2.72 (t, 2H, J = 5.6 Hz),

3.01 (t, 4H, J = 5.2 Hz), 3.68 (s, 3H, OCH₃), 4.05 (t, 2H,

J = 5.8 Hz), 6.51 (d, 1H, J = 2.4 Hz, ArH), 6.55 (s, 1H, ArH),

6.56–6.91 (m, 5H, ArH), 10.38 (s, 1H, NH), 10.51 (s, 1H,

NH). MS: m/e 368.186. Anal.: C₂₀H₂₄N₄O₃(C, H, N).

7.3.6. General procedure for the synthesis

of benzimidazole-2-ones

1,1′-Carbonyldiimidazole (1.4 g, 8.6 mmol) was added

step-wise to the stirred solution of 12 ml dried acetonitrile

and diamine 19 (0.72 g, 2 mmol) at ambient temperature.

7.3.6.6. 5-{2-[4-(2-Chlorophenyl)piperazin-1-yl]ethoxy}-1,3-

dihydro-2H-benzimidazol-2-one (38). Yield: 65%; m.p.

1

217 °C; IR (cm^–1): 1170, 1479, 1713, 2943; H NMR

492

V. Šukalovic´ et al. / European Journal of Medicinal Chemistry 40 (2005) 481–493

(d₆DMSO): d 2.67 (m, 4H), 2.75 (t, 2H, J = 5.6 Hz), 2.99 (m,

4H), 4.05 (t, 2H, J = 5.6 Hz), 6.51–6.56 (m, 2H, ArH), 6.80

(d, 1H, J = 9 Hz, ArH), 6.91–7.04 (m, 1H, ArH), 7.18 (dd,

1H, J = 1.4 Hz, J = 6.6 Hz, ArH), 7.26–7.33 (m, 1H, ArH),

7.40 (dd, 1H, J = 1.4 Hz, J = 6.2 Hz,ArH), 10.39 (s, 1H, NH),

10.53 (s, 1H, NH). MS: m/e 372.137. Anal.: C₁₉H₂₁ClN₄O₂

(C, H, N).

7.3.7.3. 5-{2-[4-(2-Methoxyphenyl)piperazin-1-yl]ethoxy}-

1H-benzotriazole (43). Yield: 59%; m.p. 82 °C; IR (cm^–1):

1217, 1242, 1455, 1500, 1628, 2831; ¹H NMR (d₆DMSO): d

2.89 (m, 6H), 3.18 (m, 4H), 3.82 (s, 3H, OCH₃), 4.13 (t, 2H,

J = 4.8 Hz), 6.84–7.04 (m, 5H, ArH), 7.28 (s, 1H, ArH), 7.74

(d, 1H, J = 8.8 Hz,ArH), 12.27 (s, 1H, NH). MS: m/e 353.187.

Anal.: C₁₉H₂₃N₅O₂(C, H, N).

7.3.6.7. 5-{2-[4-(3-Chlorophenyl)piperazin-1-yl]ethoxy}-1,3-

dihydro-2H-benzimidazole-2-one (39). Yield: 61%, m.p.

183 °C; IR (cm^–1): 1169, 1594, 1698, 1751, 2836; ¹H NMR

(d₆DMSO): d 2.62 (t, 4H, J = 4.8 Hz), 2.73 (t, 2H, J = 5.6 Hz),

3.18 (t, 4H, J = 4.6 Hz), 4.05 (t, 2H, J = 5.6 Hz), 6.51–6.55

(m, 2H, ArH), 6.75–6.79 (m, 2H, ArH), 6.87–6.94 (m, 2H,

ArH), 7.20 (t, 1H, J = 7.8 Hz, ArH), 10.39 (s, 1H, NH), 10.52

(s, 1H, NH). MS: m/e 372.134. Anal.: C₁₉H₂₁ClN₄O₂(C, H,

N).

7.4. Pharmacology

7.4.1. Membrane preparation, radioligand binding assays

and data analysis

Synaptosomal membranes from fresh bovine caudate

nuclei and hippocampi for radioligand binding assays were

prepared as previously described [25]. [³H]SCH 23390 (spec.

act. 80 Ci mmol^–1), [³H]spiperone (spec. act. 70.5 Ci mmol^–1)

and 8-OH-[³H]DPAT (spec. act. 223 Ci mmol^–1) used to label

D₁, D₂and 5-HT_1Areceptors, respectively, were purchased

from Amersham Buchler GmbH (Braunschweig, Germany).

7.3.6.8. 5-{2-[4-(4-Chlorophenyl)piperazin-1-yl]ethoxy}-1,3-

dihydro-2H-benzimidazole-2-one (40). Yield: 60%; m.p.

181 °C; IR (cm^–1): 1168, 1233, 1699, 1751, 2819; ¹H NMR

(d₆DMSO): d 2.63 (t, 4H, J = 4.8 Hz), 2.74 (t, 2H, J = 5.6 Hz),

3.14 (t, 4H, J = 4.6 Hz), 4.05 (t, 2H, J = 5.6 Hz), 6.51–6.55

(m, 2H, ArH), 6.80 (d, 1H, J = 9.4 Hz, ArH), 6.93 (d, 2H,

J = 9 Hz, ArH), 7.22 (d, 2H, J = 8.8 Hz, ArH), 10.39 (s, 1H,

NH), 10.52 (s, 1H, NH). MS: m/e 372.134. Anal.:

C₁₉H₂₁ClN₄O₂(C, H, N).

7.4.1.1. [³H]Spiperone receptor binding assay. [³H]Spiper-

one binding was assayed in 1.0 mM EDTA, 4 mmol MgCl₂,

1.5 mM CaCl₂, 5 mmol KCl, 120 mM NaCl, 25 mM Tris–

HCl solution, pH 7.4, at membrane protein concentration of

0.7 mg ml^–1at 37 °C for 20 min in a total volume of incuba-

tion mixture of 1.0 ml. Binding of the radioligand to 5-HT₂

receptors was prevented by 50 mM ketanserin. The K_ivalues

of the tested compounds were determined by competition

binding at 0.2 nmol of the radioligand and eight to 10 differ-

ent concentrations of each compound (10^–4–10^–10M). Non-

speciﬁc binding was measured in the presence of 1.0 mmol

(+)-butaclamol. The reaction was terminated by rapid ﬁltra-

tion through Whatman GF/C ﬁlters, which were further

washed three times with 5.0 ml of ice-cold incubation buffer.

Each point was determined in triplicate. Retained radioactiv-

ity was measured by introducing dry ﬁlters into 10 ml of

toluene-based scintillation liquid and counting in a 1219 Rack-

beta Wallac scintillation counter at an efﬁciency of 51–55%

for tritium.

7.3.7. General procedure for the synthesis

of 1H-benzotriazoles

Cold sodium nitrite solution (0.24 g, 3.47 mmol) and

0.36 ml water were poured in a cooled mixture (0 °C) of

diamine 19 (0.8 g, 3.1 mmol), 0.7 ml acetic acid and 1.4 ml

water. Upon the addition was completed, the mixture was

heated at 70 °C for additional 10–15 min, cooled to ambient

temperature, neutralized with 10% sodium carbonate solu-

tion, extracted with CH₂Cl₂and concentrated in vacuo. The

resulting benzotriazoles were puriﬁed by chromatography or

recrystallized from hot EtOH.

7.3.7.1. 5-[2-(4-Phenylpiperazin-1-yl)ethoxy]-1H-benzo-

triazole (41). Yield: 58%; m.p. 91 °C; IR (cm^–1): 1209, 1452,

1598, 1627, 2780, 2952; ¹H NMR (d₆DMSO): d 2.66 (t, 4H,

J = 4.6 Hz), 2.82 (t, 2H, J = 5.6 Hz), 3.14 (t, 4H, J = 4.8 Hz),

4.23 (t, 2H, J = 5.6 Hz), 6.77 (t, 1H, J = 7.2 Hz,ArH), 6.92 (d,

2H, J = 7.8 Hz, ArH), 7.04 (dd, 1H, J = 2.2 Hz, J = 6.8 Hz,

ArH), 7.17–7.26 (m, 3H,ArH), 7.84 (d, 1H, J = 9.2 Hz,ArH).

MS: m/e 323.175. Anal.: C₁₈H₂₁N₅O (C, H, N).

7.4.1.2. [³H]SCH 23390 receptor binding assay. Binding of

[³H]SCH 23390 was examined by the same rapid ﬁltration

assay discussed for [³H]spiperone, in the absence of ket-

anserin.

7.4.1.3. 8-OH-[³H]DPAT receptor binding assay. Each tube

contained 1.0 mM EDTA, 4 mM MgCl₂, 1.5 mM CaCl₂,

5 mM KCl, 120 mM NaCl, 25 mM Tris–HCl, 10 µmol niala-

mide and 0.1% ascorbic acid, pH 7.4, membrane protein con-

centration of 0.7 mg ml^–1, 0.6 nmol 8-OH-[³H]DPAT and vari-

ous concentrations (10^–4–10^–10M) of the tested compounds

in a ﬁnal volume of 0.5 ml. The tubes were incubated (20 min,

37 °C) and the reaction terminated by vacuum ﬁltration

through Whatman GF/B ﬁlters. The ﬁlters were washed three

times with 5 ml of ice-cold 25 mM Tris–HCl buffer, pH 7.4,

and bound radioactivity measured by liquid scintillation spec-

7.3.7.2. 5-(2-{4-[3-(Triﬂuoromethyl)phenyl]piperazin-1-yl}-

ethoxy)-1H-benzotriazole (42). Yield: 59%; m.p. oil; IR (cm^–

1

): 1121, 1165, 1452, 1621, 2831; H NMR: d 2.81 (t, 4H,

J = 4.6 Hz), 2.93 (t, 2H, J = 5.2 Hz), 3.28 (t, 4H, J = 5 Hz),

4.16 (t, 2H, J = 5 Hz), 6.93 (dd, 1H, J = 2 Hz, J = 7.2 Hz,

ArH), 7.01–7.09 (m, 4H,ArH), 7.78 (d, 1H, J = 9.2 Hz,ArH),

11.63 (s, 1H, NH). MS: m/e 391.163. Anal.: C₁₉H₂₀F₃N₅O

(C, H, N).

V. Šukalovic´ et al. / European Journal of Medicinal Chemistry 40 (2005) 481–493

493

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ence between the binding in the absence and in the presence

of 10 µM 5-hydroxytryptamine.

Competition binding data were analyzed by the iterative

non-linear least-squares curve-ﬁtting program LIGAND [26].

Acknowledgements

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This work was supported by the Ministry for Science, Tech-

nology and Development of Serbia, Grant #1698 (V.

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Article Doi

Synthesis, dopamine D2 receptor binding studies and docking analysis of 5-[3-(4-arylpiperazin-1-yl)propyl]-1H-benzimidazole, 5-[2-(4-arylpiperazin-1-yl)ethoxy]-1H-benzimidazole and their analogs

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