An interactive SAR approach to discover novel hybrid thieno probes as ligands for D2-like receptors with affinities in the subnanomolar range

Article abstract of DOI:10.1002/cbdv.201300204

A series of [(phenylpiperazinyl)alkyl]-isoindole-1,3-dione derivatives was synthesized to serve as probes for dopaminergic receptors. Among this series, compound 6a showed the highest affinity towards D4 and D3 receptors with K _i values in the low nanomolar range, and D2/D4- and D2/D3-selectivity indices of 72 and 20, respectively. Optimization rounds were adopted and led to the D4-selective ligand thiophene-2-carboxamide 9a with a K_i(D4) value of 0.62 nM, and to its butyl analog, 10a, with K_i(D4) and K_i(D3) values of 0.03 and 0.26 nM, respectively. Docking experiments revealed the importance of the unique D4 residue Arg186 in manipulating the ligands' D4-subtype-receptor selectivity. Copyright

Full text of DOI:10.1002/cbdv.201300204

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An Interactive SAR Approach to Discover Novel Hybrid Thieno Probes as
Ligands for D2-Like Receptors with Affinities in the Subnanomolar Range
by Mohamed A. O. Abdel-Fattaha)b), Jochen Lehmannb), and Ashraf H. Abadi*a)
a) Department of Pharmaceutical Chemistry, Faculty of Pharmacy and Biotechnology, German
University in Cairo, Cairo 11835, Egypt
(phone: þ202-27590716; fax: þ202-27581041; e-mail: ashraf.abadi@guc.edu.eg)
b) Institut fꢀr Pharmazie, Lehrstuhl fꢀr Pharmazeutische/Medizinische Chemie, Friedrich Schiller
Universitꢁt Jena, Philosophenweg 14, DE-07743 Jena
A series of [(phenylpiperazinyl)alkyl]-isoindole-1,3-dione derivatives was synthesized to serve as
probes for dopaminergic receptors. Among this series, compound 6a showed the highest affinity towards
D4 and D3 receptors with Ki values in the low nanomolar range, and D2/D4- and D2/D3-selectivity
indices of 72 and 20, respectively. Optimization rounds were adopted and led to the D4-selective ligand
thiophene-2-carboxamide 9a with a Ki(D4) value of 0.62 nm, and to its butyl analog, 10a, with Ki(D4) and
Ki(D3) values of 0.03 and 0.26 nm, respectively. Docking experiments revealed the importance of the
unique D4 residue Arg186 in manipulating the ligandsꢂ D4-subtype-receptor selectivity.
1. Introduction. – Psychosis is a mental illness referred to as the disease of mind and
soul, and featured by radical changes, impairment in personality and functioning, as
well as a state of nonexistent sense of objective reality [1]. Abnormalities in brain
chemistry manifested as singular rise in dopamine brain levels have been shown to be
linked to this disorder [2][3]. This can be counteracted through blocking the dopamine
neurotransmission via antagonizing its action mainly at the different D2-like receptor
subtypes [4]. This family of receptors includes D2, D3, and D4 receptor subtypes, and
differs from the D1-like family receptors in their molecular structure and the signaling
cascade occurring upon stimulation [5–7]. The stimulation of D1-like receptors
including D1 and D5 receptor subtypes leads to activation of Adenylate Cyclase (AC)
that provokes the production of cAMP and hence Protein Kinase A (PKA) activation,
while the stimulation of D2-like family members leads to negative regulation of the
production of cAMP and accordingly to a decrease in PKA activity [8][9].
Among the well-known D2-like family antagonists, the butyrophenone derivative
Haloperidol (1) is one of the most commonly marketed typical antipsychotic agents
[10]. These typical agents are characterized by their ability to block the D2-like
receptor subtypes unselectively, and, though being useful in curing the positive
symptoms of psychosis, they possess marked Extra Pyramidal Parkinsonꢂs like adverse
effects [11]. This undesired propensity is thought to be a result of blocking D2 receptor
subtype that is mainly concentrated in striatal areas of the brain [12]. On the contrary,
atypical antipsychotic agents relieve both positive and negative signs of psychosis
showing much lower incidence of the undesired Parkinsonꢂs like symptoms [13]. This
special behavior of atypical antipsychotic agents is believed to be either due to their
ꢃ 2013 Verlag Helvetica Chimica Acta AG, Zꢀrich

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ability to block D3/D4 receptor subtypes selectively over D2 ones [14], or due to their
loose binding and their rapid dissociation off D2 receptor subtype [15]. Recent studies
have linked this special pharmacological behavior of atypical agents to their cross-
interaction with 5-HT2A receptors [16].
Among these atypical agents, the benzamide derivative sulpiride (2) and the
thienobenzodiazepine derivative olanzapine (3) are commonly used. Ketanserine (4) is
another ligand characterized by its moderate interaction with D2 receptor subtype,
while exhibiting much higher binding affinity to D4 and 5-HT2A receptors.
Unfortunately, the use of atypical antipsychotic agents is still limited because of their
many adverse effects, including increased risk of brain stroke, cardiovascular diseases,
metabolic and diabetic complications, weight gain, and impaired sexual function,
leaving the field in need of newer probes [17].
Trying to develop novel selective D3 and/or D4 ligands, some probes were designed
to serve as hybrids bearing combined structural features from both the typical and
atypical lead compounds haloperidole, sulpiride, olanzapine, and ketanserine (1–4,
resp.; Scheme 1). The designed probes bear a scaffold in which the aromatic ring
appendage and the basic N-atom of the phenylpiperazine are separated with an
amidoalkyl linker and thus would keep the primary recognition elements that have
been proved to be necessary for fitting into the binding pockets of the target
dopaminergic receptors [18][19].
2. Results and Discussion. – 2.1. Chemistry. The synthesis of the [(phenylpiper-
azinyl)propyl]-isoindole-1,3-dione derivatives as outlined in Scheme 2 started with a
reaction of phthalic anhydride (11) with 3-chloropropylamine hydrochloride salt to
afford 2-(3-chloropropyl)isoindole-1,3-dione (12), which in turn was subjected to a
nucleophilic substitution reaction with the corresponding 1-phenylpiperazine to furnish
the desired derivatives [20]. Due to the non-availability of the 4-chlorobutylamine
hydrochloride salt, the synthesis of the [(phenylpiperazinyl)butyl]-isoindole-1,3-dione
derivatives was conducted by adopting Gabriel synthesis [21] in which the phthalimide
potassium salt (13) was N-alkylated with 1,4-dibromobutane to give 2-(4-bromobutyl)-
isoindole-1,3-dione (14), which was again subjected to a nucleophilic substitution
reaction with the corresponding 1-phenylpiperazine to yield the desired compounds
(Scheme 3).
The syntheses of the benzamides and thienoamides as depicted in Scheme 4 were
carried out starting from the corresponding previously synthesized [(phenylpiperazi-
nyl)alkyl]-isoindole-1,3-dione derivatives by adopting IngꢀMansk reaction [22] that
involves refluxing the [(phenylpiperazinyl)alkyl]-isoindole-1,3-dione derivative with
aqueous hydrazine in 95% EtOH to afford the corresponding [(phenylpiperazinyl)al-
kyl]amine derivatives, 15a–15j and 16a–16j, which in turn were immediately reacted
with either benzoyl chloride (17) or thiophene-2-carbonyl chloride (19) in presence of
Et3N (TEA) to furnish the corresponding amide derivatives [22]. The commercially
non-available thiophene-2-carbonyl chloride (19) was prepared by reaction of
thiophene-2-carboxylic acid (18) with SOCl2 [23].
2.2. Pharmacology. The final target compounds were screened for their binding
affinities towards human cloned dopamine receptor subtypes D1, D2, D3, D4, and D5
utilizing radioligand binding assay according to our previously published protocol [24].

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Scheme 1. Design of Hybrid Dopaminergic Probes Based on Marketed Typical and Atypical
Antipsychotic Agents
Scheme 2. Synthesis of [(Phenylpiperazinyl)propyl]-isoindole-1,3-dione Derivatives
[3H]SCH23390 was used as radioligand for the D1-like family receptors, while
[3H]spiperone was the radioligand used for the D2-like family receptors. Incubations at
278 were terminated after 90 min by rapid filtration with a Perkin-Elmer Mach III

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Scheme 3. Synthesis of [(Phenylpiperazinyl)butyl]-isoindole-1,3-dione Derivatives
Scheme 4. Synthesis of Benzamide and Thienoamide Derivatives
harvester. At least two independent experiments were carried out, each in triplicate.
The designed compounds have exhibited functional activities towards D2-like family
receptor subtypes ranging from partial agonists to full antagonists in fluorescent Ca2þ
assay [24].
2.3. StructureꢀActivity Relatioship. The Ki affinity binding data compiled in Table 1
show that our designed probes lack the affinity towards D1-like family receptor

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subtypes, while exhibiting appreciable binding affinity towards D2-like family
receptors showing diverse affinity and selectivity patterns.
Starting with the propyl linker-bearing compounds, it is obvious that the isoindole-
1,3-dione derivatives 5a and 5b have shown the lowest affinity to all the D2-like
receptors. The benzamide analogs 7a and 7b and the thienoamide analogs 9a and 9b
have displayed improvement in affinity towards D2 receptor subtypes, marked
selectivity to D4 receptor subtypes (view selectivity indices in Table 2), but still with
low affinity to D3 subtypes.
Table 2. D2/D3 and D2/D4 Data of Isoindole-1,3-dione, Benzamide, and Thienoamide Derivatives
Compound
D2/D3
D2/D4
Compound
D2/D3
D2/D4
5a
5b
6a
6b
7a
7b
8a
1.3
1.7
20
9
11
72
72
247
37
8b
9a
9b
10a
10b
1
23
0.2
0.2
110
54
50
222
23
950
50
20
0.23
0.35
32
1.5
0.08
46
Moving to the butyl-linker-bearing compounds, benzamide derivatives 8a and 8b
and thienoamide derivatives 10a and 10b have exhibited great improvement in affinity
to D2, D3, D4 subtypes with marked selectivities to D3 and D4 over D2 subtypes (see
the selectivity indices in Table 2). The isoindole-1,3-dione analogs 6a and 6b exhibited
the least binding affinities within this set of compounds.
From the obtained binding data, we can discuss the effect of three major factors on
the affinity and selectivity pattern of the synthesized compounds.
2.3.1. Effect of the Spacerꢀs Length. In the whole series, compounds bearing the
butyl linker have shown better affinities to the D2-like receptors than their propyl
analogs. In terms of affinity, the length of the spacer could have prominent effect on the
pKa value of the basic N-atom of the 1-phenylpiperazine unit that is reported to be
involved in a key salt-bridge interaction with Asp 3.32 residue of the target receptors
[18][25][26]. The calculated pKa values of the basic piperazin N-atom range from 6.8 to
7.8 in the compounds with the propyl linker, while they range from 7.3 to 8.3 in their
butyl-linker-bearing analogs. This could enable the compounds bearing the butyl linker
to afford better salt-bridge interaction with Asp 3.32 residue explaining their better
affinity to the target receptors compared to their propyl linker-bearing counterparts.
In terms of selectivity, the length of the spacer could play a crucial role in enabling
the ligandꢂs aromatic appendage to come into contact and then afford hydrophobic
interaction with certain amino acid residues reported to figure the D2/D3 subtype
receptor selectivity. These amino acid residues are located in the extracellular side EL2
of the binding pockets of D2 and D3 receptor subtypes, and include Glu 181 and Ile 183
in D2 that face Val 180 and Ser 182, respectively, in D3 [18]. This will be discussed
thoroughly later in the docking section.
2.3.2. Effect of the Ligandꢀs Aromatic Appendage. It is worth considering the nature
of the aromatic appendage responsible for the hydrophobic interaction with the target

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receptors. Among the whole series, compounds with thiophene system have shown the
highest binding affinities at all the D2-like receptor subtypes, followed by those with
the benzene ring, and finally came the largest-in-size isoindole-1,3-dione system with
the least binding affinity. The superiority of the thiophene system relative to the
benzene may be a function of better hydrophobic interaction at the binding pocket.
This is mainly due to the fact that the thiophene system is richer in electrons, as the lone
pair of electrons in the p orbital of the S-atom contributes to the Hꢁckel aromatic sextet
and pushes high electron density toward the ring C-atoms that accordingly acquire
partial negative charge. Thus, it was suggested that the large atomic polarizability of the
S-atom and the electron-rich thiophene system would provide higher dispersion forces
compared to benzene, leading to better pꢀp stacking and/or Van der Waals interaction
[27] with the hydrophobic residues lining the hydrophobic pocket of the target
receptors.
2.3.3. Effect of the Nature of the Substituent at the Phenylpiperazine Moiety. To
investigate the impact of changing the nature and the position of the substituent in the
phenylpiperazine moiety on the affinity and selectivity, compounds 9c–9j and 10c–10j
were synthesized and biologically evaluated. In Table 1, the binding affinity data of
these compounds towards the human cloned dopamine receptor subtypes are collected.
It is noticeable that the propyl-linker-bearing ligands 9c–9j exhibited appreciable
affinities to D4 receptor subtypes. The best affinity was observed with compounds with
ortho-oxygenated and dihalogenated substituents, followed by those with ortho-
monohalogenated substituents. The affinity of the synthesized ligands displayed four-
to ten-fold decrease, when the substituent was shifted to the para position, as deduced
from the comparison of the Ki values of compounds 9b, 9c, 9d, and 9e with those of their
para-substituted analogs 9h, 9f, 9g, and 9i. This indicated the importance of an ortho-
substituted phenylpiperazine moiety with a specific electrostatic potential to regulate
affinity towards D4 receptor subtypes.
All of the butyl-linker-bearing ligands, 10c–10j, have shown superior affinities to
both D3 and D4 receptor subtypes, relative to D2 ones. Again the nature and position
of the substituent in the phenylpiperazine moiety determined the affinity of these
derivatives in a similar fashion exhibited by their propyl counterparts.
2.4. In silico Docking Experiments. Docking experiments were carried out to
configure the binding fashion of the synthesized compounds to the target receptor
subtypes. All compounds have been docked to the human D3 model (PDB ID: 3PBL),
and the validated D2 and D4 homology models developed before [28]. MOE Software
[29] has been used for this purpose.
Compound 9a with the highest selectivity to D2 over D3 among all derivatives
bearing the propyl linker was docked to the D2 receptor homology model and showed
the contact between the ligandꢂs arene and Ile 183 residue. Docking the same
compound to D3 receptor model revealed the contact between the ligandꢂs arene and
the Ser 182 in the EL2 of the binding site of the D3 receptors that is occupied with the
more hydrophobic Ile 183 in the D2 receptor subtype, ensuring better hydrophobic
interaction with the target receptors. This confirms that these amino acid residues
manipulate the ligandsꢂ selectivity towards D2/D3. The other propyl-linker-bearing
compounds, 7a, 7b, and 9b, have been also docked to D2 and D3 receptor models, and
over-relayed compound 9a in the binding site of the target receptors (Fig. 1).

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Compound 10a with the highest selectivity to D3 over D2 among all the derivatives
bearing the butyl linker has been docked to both D2 and D3 models. The docking
revealed that the butyl spacer enables the ligandsꢂ arene contact with the Glu 181 that is
conserved in the binding site of the D2 subtype and faces the more hydrophobic Val 180
in D3. Although Val 180 is conserved in the binding site of D3 receptor subtypes, the 2D
interactions of compound 10a with D3 receptor model did not show this residue in the
binding site. The other butyl linker-bearing compounds 8a, 8b, 10b have been also
docked to D2 and D3 models and over-relayed compound 10b in the binding site of the
target receptors (Fig. 2).
As for D4 receptor subtypes, docking both compounds 9a and 10a that were among
the compounds with the highest D4 affinity in the whole series have emphasized that
the unique D4 amino acid residue Arg 186 turned out to be involved in the H-bond
interaction with the C¼O moiety of the synthesized ligands. The other propyl-linker-
bearing compounds 7a, 7b, and 9b, and the butyl-linker-bearing compounds 8a, 8b, and
10b have been also docked to D4 model and showed to over-relay compounds 9a and
10a, respectively, in the binding site of the target receptor (Fig. 3).
It is remarkable that the generally enhanced binding affinity of all the butyl-linker-
bearing candidates towards all the D2-like members emphasizes the role of the butyl
linker in enabling the compound to afford a specific folded conformation stabilized by
an intramolecular H-bond between the amide C¼O and the protonated piperazin N-
atom. This conformation is assumed to possess the optimum distance between the
pharmacophore elements leading to optimum binding affinity to the target receptor
subtypes [30].
3. Conclusions. – Novel chemical probes bearing three different chemical scaffolds
have been designed and synthesized, and their binding affinities to the five subtypes of
dopamine receptors were determined. It turned out that the C4 linker separating the
two pharmacophore elements (aromatic appendage and phenylpiperazine unit) of the
synthesized probes is crucial to enhance the affinity to all D2-like receptors with
marked selectivity towards D3 and D4 over D2 subtypes, giving rise to potential
atypical antipsychotic agents. Docking experiments confirmed the rule of certain amino
acid residues in the second extra-cellular loop of the target receptors in manipulating
subtype-receptor selectivity. These residues include Glu 181 and Ile 183 in D2
receptors, Val 180 and Ser 182 in D3 receptors, and finally Arg 186 in D4 receptors.
These findings are important for understanding the interactions with such G-protein-
coupled receptors (GPCRs) and for the discovery of highly specific ligands.
Experimental Part
1. General. All reactions were performed with commercially available reagents, and they were used
without further purification. Solvents were dried by standard methods and stored over molecular sieves.
TLC: silica gel F254 plates (Merck); detection of compounds was made by short UV light. Column
chromatography (CC): mainly silica gel 60 (SiO2; 63–200 mm (Baker). M.p.: in open cap. tubes, with a
Gallenkamp melting-point apparatus; uncorrected. FT-IR Spectra: Nicolet Avatar 380 spectrometer.
1H-NMR spectra: Bruker Advance 250 spectrometer (250 MHz). MS Data: determined by GC/MS, using
a Hewlett-Packard GCD-Plus (G1800C) apparatus (HP-5 MS column; J&W Scientific). Elemental
analyses were performed on a Heraeus Vario EL apparatus in Organic chemistry institute, Friedrich

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Schiller University, Jena; found values were within ꢁ0.4% of the theoretical ones. The pKa values were
calculated using Marvin Sketch software.
Preparation of 2-(3-Chloropropyl)-1H-isoindole-1,3(2H)-dione (12). Phthalic anhydride (11; 1.48 g,
10 mmol) and 3-chloropropylamine hydrochloric salt (1.43 g, 11 mmol) were heated in an oil bath at 1608.
The reaction mixture was maintained at the same temp. for 15 min. Then, it was cooled to r.t., and 30 ml
H2O were added just before formation of a slurry. The product was filtered, washed twice with H2O, and
1
purified by CC (SiO2; CH2Cl2) to afford pure 12 (1.7 g, 75%). White crystals. M.p. 69–718. H-NMR
(CDCl3): 2.11–2.22 (m, CH2(2’)); 3.54–3.59 (t, CH2(3’)); 3.82–3.87 (t, CH2(1’)); 7.70–7.91 (m, 4 arom.
H). GC/MS: 63 (25), 76 (95), 104 (75), 133 (25), 160 (100), 188 (20), 223 (5, Mþ ).
General Procedure for the Preparation of 2-[3-(4-Arylpiperazin-1-yl)propyl]isoindole-1,3-diones. To
a soln. of 12 (1.0 g, 4.5 mmol) in 40 ml of dry MeCN was added the corresponding 1-phenylpiperazine
(4.5 mmol) and Et3N (TEA; 1.5 g, 15 mmol). The mixture was allowed to reflux under inert atmosphere
for 48 h, and then left to cool to r.t. The org. solvent was removed under reduced pressure, and the residue
was subjected to CC (SiO2; CH2Cl2/MeOH 100 :2).
2-{3-[4-(2-Ethoxyphenyl)piperazin-1-yl]propyl}-1H-isoindole-1,3(2H)-dione (5a). Yield: 1.3 g
(71%). Yellow crystals. M.p. 109–1118. IR: 1708, 1737 (2 C¼O). 1H-NMR (CDCl3): 1.42 (t, J ¼ 7,
Me); 1.89–1.95 (m, CH2(2’)); 2.5 (t, J ¼ 6.9, CH2(3’)); 2.59 (br. s, 2 CH2); 2.96 (br. s, 2 CH2); 3.78 (t, J ¼
6.9, CH2(1’)); 4.03 (q, J ¼ 7, MeCH2); 6.78–6.96 (m, 4 arom. H); 7.68–7.82 (m, 4 arom. H). GC/MS: 77
(10), 104 (5), 130 (10), 191 (60), 219 (90), 378 (40), 393 (100, Mþ ). Anal. calc. for C23H27N3O3 (393.48): C
70.21, H 6.92, N 10.68; found: C 69.84, H 7.08, N 10.89.
2-{3-[4-(2,3-Dichlorophenyl)piperazin-1-yl]propyl}-1H-isoindole-1,3(2H)-dione (5b) [31]. Yield:
1.3 g (70%). Yellowish white crystals. M.p. 103–1058. IR: 1711, 1766 (2 C¼O). 1H-NMR (CDCl3):
1.88–1.96 (m, CH2(2’)); 2.50 (t, J ¼ 6.9, CH2(3’)); 2.57 (br. s, 2 CH2); 2.90 (br. s, 2 CH2); 3.79 (t, J ¼ 6.9,
CH2(1’)); 6.80–7.15 (m, 3 arom. H); 7.70 (dd, J ¼ 3, 5.5, 2 arom. H); 7.84 (dd, J ¼ 3, 5.5, 2 arom. H). GC/
MS: 77 (20), 104 (100), 130 (40), 174 (10), 243 (80), 269 (20), 417 (90, Mþ ), 419 (55, [Mþ2]þ ), 421 (20,
[Mþ4]þ ). Anal. calc. for C21H21Cl2N3O2 (418.32): C 60.30, H 5.06, N 10.05; found: C 60.44, H 4.90, N
9.98.
2-{3-[4-(2-Fluorophenyl)piperazin-1-yl]propyl}-1H-isoindole-1,3(2H)-dione (5c). Yield: 1.2 g
(71%). Yellow crystals. M.p. 96–988. IR: 1709, 1765 (2 C¼O). 1H-NMR (CDCl3): 1.84–1.95 (m,
CH2(2’)); 2.48 (t, J ¼ 6.8, CH2(3’)); 2.56 (t, J ¼ 4.8, 2 CH2); 2.94 (t, J ¼ 4.8, 2 CH2); 3.79 (t, J ¼ 6.8,
CH2(1’)); 6.79–7.05 (m, 4 arom. H); 7.67–7.72 (m, 4 arom. H); 7.67–7.72 (m, 4 arom. H). GC/MS: 77
(80), 104 (100), 130 (20), 193 (60), 352 (10), 368 (30, Mþ ). Anal. calc. for C21H22FN3O2 (367.42): C 68.65,
H 6.04, N 11.44; found: C 69.02, H 6.11, N 11.41.
2-{3-[4-(2-Chlorophenyl)piperazin-1-yl]propyl}-1H-isoindole-1,3(2H)-dione (5d). Yield: 1.1 g
(66%). Yellow crystals. M.p. 127–1298. IR: 1714, 1779 (2 C¼O). 1H-NMR (CDCl3): 1.84–1.95 (m,
CH2(2’)); 2.49 (t, J ¼ 6.85, CH2(3’)); 2.56 (br. s, 2 CH2); 2.89 (br. s, 2 CH2); 3.79 (t, J ¼ 6.85, CH2(1’));
6.87–7.20 (m, 4 arom. H); 7.26–7.72 (m, 4 arom. H). GC/MS: 77 (35), 104 (100), 130 (20), 209 (85), 348
(20), 383 (35, Mþ ), 385 (10, [Mþ2]þ ). Anal. calc. for C21H22ClN3O2 (383.87): C 65.71, H 5.78, N 10.95;
found: C 65.32, H 5.41, N 10.76.
2-{3-[4-(2-Hydroxyphenyl)piperazin-1-yl]propyl}-1H-isoindole-1,3(2H)-dione (5e). Yield: 1.0 g
(60%). Yellow crystals. M.p. 135–1378. IR: 3220 (ꢀOH), 1712, 1769 (2 C¼O). 1H-NMR (CDCl3):
1.88–1.93 (m, CH2(2’)); 2.49 (t, J ¼ 6.7, CH2(3’)); 2.53 (br. s, 2 CH2); 2.70 (br. s, 2 CH2); 3.81 (t, J ¼ 6.7,
CH2(1’)); 6.81–7.04 (m, 4 arom. H); 7.74 (dd, J ¼ 3, 5.4, 2 arom. H); 7.87 (dd, J ¼ 3, 5.4, 2 arom. H). GC/
MS: 77 (20), 104 (10), 163 (100), 217 (95), 230 (40), 350 (30), 365 (20, Mþ ). Anal. calc. for C21H23N3O3
(365.43): C 69.02, H 6.34, N 11.50; found: C 68.78, H 6.32, N 11.24.
2-{3-[4-(4-Fluorophenyl)piperazin-1-yl]propyl}-1H-isoindole-1,3(2H)-dione (5f) [32]. Yield: 1.2 g
(72%). Brown crystals. M.p. 103–1058. IR: 1712, 1769 (2 C¼O). 1H-NMR (CDCl3): 1.76–1.95 (m,
CH2(2’)); 2.47 (t, J ¼ 6.9, CH2(3’)); 2.53 (t, J ¼ 4.8, 2 CH2); 2.95 (t, J ¼ 4.8, 2 CH2); 3.79 (t, J ¼ 6.9,
CH2(1’)); 6.76–6.96 (m, 4 arom. H); 7.68 (dd, J ¼ 3, 5.5, 2 arom. H); 7.83 (dd, J ¼ 3, 5.5 2 arom. H). GC/
MS: 77 (90), 104 (100), 130 (50), 193 (30), 352 (15), 368 (35, Mþ ). Anal. calc. for C21H22FN3O2 (367.42):
C 68.65, H 6.04, N 11.44; found: C 68.70, H 5.80, N 11.07.
2-{3-[4-(4-Chlorophenyl)piperazin-1-yl]propyl}-1H-isoindole-1,3(2H)-dione (5g). Yield: 1.2 g
(72%). Pale-yellow crystals. M.p. 119–1218. IR: 1701, 1774 (2 C¼O). 1H-NMR (CDCl3): 1.84–1.95

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(m, CH2(2’)); 2.46 (t, J ¼ 7, CH2(3’)); 2.52 (t, J ¼ 5.1, 2 CH2); 2.98 (t, J ¼ 5.1, 2 CH2); 3.79 (t, J ¼ 7,
CH2(1’)); 6.75 (d, J ¼ 4.7, 2 arom. H); 7.16 (d, J ¼ 4.7, 2 arom. H); 7.66–7.84 (m, 4 arom. H). GC/MS: 77
(40), 104 (100), 130 (10), 209 (90), 348 (15), 383 (50, Mþ ), 385 (15, [Mþ2]þ ). Anal. calc. for
C21H22ClN3O2 (383.87): C 65.71, H 5.78, N 10.95; found: C 65.83, H 5.68, N 11.37.
2-{3-[4-(3,4-Dichlorophenyl)piperazin-1-yl]propyl}-1H-isoindole-1,3(2H)-dione (5h). Yield: 1.3 g
(67%). Yellow crystals. M.p. 88–908. IR: 1713, 1760 (2 C¼O). 1H-NMR (CDCl3): 1.83–1.94 (m,
CH2(2’)); 2.46 (t, J ¼ 6.8, CH2(3’)); 2.51 (t, J ¼ 5.2, 2 CH2); 2.99 (t, J ¼ 5.2, 2 CH2); 3.79 (t, J ¼ 6.8,
CH2(1’)); 6.67 (dd, J ¼ 2.85, 9, 1 arom. H); 6.85 (d, J ¼ 2.85, 1 arom. H); 7.23 (d, J ¼ 9, 1 arom. H); 7.67–
7.83 (m, 4 arom. H). GC/MS: 77 (50), 104 (100), 130 (10), 174 (20), 243 (90), 269 (15), 417 (90, Mþ ), 419
(55, [Mþ2]þ ), 421 (20, [Mþ4]þ ). Anal. calc. for C21H21Cl2N3O2 (418.32): C 60.30, H 5.06, N 10.05;
found: C 60.20, H 4.95, N 9.99.
2-{3-[4-(4-Hydroxyphenyl)piperazin-1-yl]propyl}-1H-isoindole-1,3(2H)-dione (5i). Yield: 1.0 g
(62%). Brown crystals. M.p. 145–1478. IR: 3209 (ꢀOH), 1704, 1759 (2 C¼O). 1H-NMR (CDCl3):
1.85–1.96 (m, CH2(2’)); 2.51 (t, J ¼ 6.9, CH2(3)); 2.55 (t, J ¼ 4.7, 2 CH2); 2.90 (t, J ¼ 6.7, 2 CH2); 3.78 (t,
J ¼ 6.9, CH2(1’)); 6.69–7.77 (m, 4 arom. H); 7.69 (dd, J ¼ 3, 5.5, 2 arom. H); 7.83 (dd, J ¼ 3, 5.5, 2 arom.
H). GC/MS: 77 (20), 104 (10), 163 (100), 217 (80), 230 (50), 350 (40), 365 (25, Mþ ). Anal. calc. for
C21H23N3O3 (365.43): C 69.02, H 6.34, N 11.50; found: C 68.71, H 6.24, N 11.62.
2-[3-(4-Phenylpiperazin-1-yl)propyl]-1H-isoindole-1,3(2H)-dione (5j) [33]. Yield: 1.2 g (76%).
Yellow crystals. M.p. 105–1078. IR: 1703, 1760 (2 C¼O). 1H-NMR (CDCl3): 1.86–1.97 (m, CH2(2’)); 2.50
(t, J ¼ 7, CH2(3’)); 2.56 (t, J ¼ 5, 2 CH2); 3.06 (t, J ¼ 5, 2 CH2); 3.80 (t, J ¼ 7, CH2(1’)); 6.80–7.26 (m, 5
arom. H); 7.68 (dd, J ¼ 3, 5.4, 2 arom. H); 7.84 (dd, J ¼ 3, 5.4, 2 arom. H). GC/MS: 77 (95), 104 (100), 130
(50), 160 (60), 175 (30), 349 (10, Mþ ). Anal. calc. for C21H23N3O2: C 72.18, H 6.63, N 12.03; found: C
72.09, H 6.67, N 11.98.
Preparation of 2-(4-Bromobutyl)-1H-isoindole-1,3(2H)-dione (14). Phthalimide potassium salt (13;
1.85 g, 10 mmol) was added slowly to a soln. of 1,4-dibromobutane (2.40 g, 11 mmol) in 60 ml acetone.
The mixture was refluxed for 24 h, and the precipitate was filtered. The filtrate was evaporated under
reduced pressure, and the resulting pale-yellow oil was subjected to CC (SiO2; CH2Cl2) to afford pure
creamy white crystals of 14 (2.0 g, 75%). M.p. 67–688. 1H-NMR (CDCl3): 1.75–1.99 (m, CH2(2’),
CH2(3’)); 3.2 (t, J ¼ 6.9, CH2(4’)); 3.71 (t, J ¼ 6.9, CH2(1’)); 7.32–7.91 (m, 4 arom. H). GC/MS: 77 (60),
105 (50), 133 (100), 160 (95), 202 (98), 336 (10), 281 (20, [Mꢀ2]þ ), 283 (20, Mþ ).
General Procedure for the Preparation of 2-[4-(4-Arylpiperazin-1-yl)butyl]isoindole-1,3-diones. To a
soln. of (14; 1.2 g, 4.5 mmol) in 40 ml of dry MeCN, were added the corresponding 1-phenylpiperazine
(4.5 mmol) and TEA (1.5 g, 15 mmol). The mixture was allowed to reflux under inert atmosphere for
48 h and then left to cool to r.t. The org. solvent was removed under reduced pressure, and the residue
was subjected to CC (SiO2; CH2Cl2/MeOH 100 :2).
2-{4-[4-(2-Ethoxyphenyl)piperazin-1-yl]butyl}-1H-isoindole-1,3(2H)-dione (6a). Yield: 1.2 g (68%).
1
Orange resin. IR: 1705, 1741 (2 C¼O). H-NMR (CDCl3): 1.44 (t, J ¼ 7, Me); 1.64–1.74 (m, CH2(2’),
CH2(3’)); 2.44 (t, J ¼ 7.2, CH2(4’)); 2.64 (br. s, 2 CH2); 3.11 (br. s, 2 CH2); 3.73 (t, J ¼ 7.2, CH2(1’)); 4.05
(q, J ¼ 7, MeCH2); 6.82–6.92 (m, 4 arom. H), 7.70 (dd, J ¼ 3, 5.5, 2 arom. H); 7.84 (dd, J ¼ 3, 5.5, 2 arom.
H). GC/MS: 70 (40), 104 (20), 130 (70), 160 (100), 172 (30), 407 (15, Mþ ). Anal. calc. for C24H29N3O3
(407.51): C 70.74, H 7.17, N 10.31; found: C 71.03, H 7.42, N 9.96.
2-{4-[4-(2,3-Dichlorophenyl)piperazin-1-yl]butyl}-1H-isoindole-1,3(2H)-dione (6b) [31]. Yield:
1.3 g (69%). Yellow crystals. M.p. 119–1218. IR: 1710, 1744 (2 C¼O). 1H-NMR (CDCl3): 1.57–1.77
(m, CH2(2’), CH2(3’)); 2.44 (t, J ¼ 7, CH2(4’)); 2.61 (br. s, 2 CH2); 3.04 (br. s, 2 CH2); 3.73 (t, J ¼ 7,
CH2(1’)); 6.92–6.14 (m, 3 arom. H); 7.69–7.83 (m, 4 arom. H). GC/MS: 70 (60), 104 (30), 130 (50), 160
(100), 172 (30), 243 (50), 432 (5, Mþ ). Anal. calc. for C22H23Cl2N3O2 (432.34): C 61.12, H 5.36, N 9.72;
found: C 60.73, H 5.42, N 9.93.
2-{4-[4-(2-Fluorophenyl)piperazin-1-yl]butyl}-1H-isoindole-1,3(2H)-dione (6c). Yield: 1.2 g (72%).
Yellowish-white crystals. M.p. 120–1228. IR: 1703, 1746 (2 C¼O). 1H-NMR (CDCl3): 1.51–1.79 (m,
CH2(2’), CH2(3’)); 2.43 (t, J ¼ 7, CH2(4’)); 2.61 (t, J ¼ 4.7, 2 CH2); 3.09 (t, J ¼ 4.7, 2 CH2); 3.73 (t, J ¼ 7,
CH2(1’)); 6.87–6.07 (m, 4 arom. H); 7.70 (dd, J¼3, 5.5, 2 arom. H); 7.81 (d, J ¼ 4, 2 arom. H). GC/MS: 70
(90), 104 (30), 122 (90), 160 (100), 193 (80), 366 (5), 381 (10, Mþ ). Anal. calc. for C22H24FN3O2 (381.44):
C 69.27, H 6.34, N 11.02; found: C 69.43, H 6.03, N 10.95.

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
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2-{4-[4-(2-Chlorophenyl)piperazin-1-yl]butyl}-1H-isoindole-1,3(2H)-dione (6d). Yield: 1.3 g (74%).
Pale-yellow crystals. M.p. 141–1438. IR: 1704, 1745 (2 C¼O). 1H-NMR (CDCl3): 1.57–1.77 (m, CH2(2’),
CH2(3’)); 2.45 (t, J ¼ 7.1, CH2(4’)); 2.62 (br. s, 2 CH2); 3.06 (br. s, 2 CH2); 3.73 (t, J ¼ 7.1, CH2(1’)); 6.92–
6.35 (m, 4 arom. H); 7.71 (dd, J ¼ 3, 5.5, 2 arom. H); 7.83 (d, J ¼ 3, 2 arom. H). GC/MS: 70 (50), 104 (30),
138 (50), 160 (100), 209 (70), 382 (2), 397 (5, Mþ ). Anal. calc. for C22H24ClN3O2 (397.90): C 66.41, H 6.08,
N 10.56; found: C 66.20, H 6.02, N 10.43.
2-{4-[4-(2-Hydroxyphenyl)piperazin-1-yl]butyl}-1H-isoindole-1,3(2H)-dione (6e). Yield: 1.0 g
(63%). Yellowish-white crystals. M.p. 118–1208. IR: 3215 (ꢀOH), 1716, 1741 (2 C¼O). 1H-NMR
(CDCl3): 1.57–1.75 (m, CH2(2’), CH2(3’)); 2.45 (t, J ¼ 7.3, CH2(4’)); 2.61 (br. s, 2 CH2); 2.91 (br. s,
2 CH2); 3.73 (t, J ¼ 7.3, CH2(1’)); 6.81–6.17 (m, 4 arom. H); 7.70–7.86 (m, 4 arom. H). GC/MS: 70 (50),
104 (35), 120 (100), 160 (90), 191 (20), 364 (5), 379 (5, Mþ ). Anal. calc. for C22H25N3O3 (379.45): C
69.64, H 6.64, N 11.07; found: C 69.31, H 6.83, N 10.89.
2-{4-[4-(4-Fluorophenyl)piperazin-1-yl]butyl}-1H-isoindole-1,3(2H)-dione (6f) [32]. Yield: 1.3 g
(76%). Pale-yellow crystals. M.p. 116–1188. IR: 1717, 1776 (2 C¼O). 1H-NMR (CDCl3): 1.53–1.80 (m,
CH2(2’), CH2(3’)); 2.43 (t, J ¼ 7.1, CH2(4’)); 2.58 (t, J ¼ 5.1, 2 CH2); 3.10 (t, J ¼ 5.1, 2 CH2); 3.73 (t, J ¼
7.3, CH2(1’)); 6.82–6.98 (m, 4 arom. H); 7.71 (dd, J ¼ 3, 5.5 2 arom. H); 7.81 (d, J ¼ 4, 2 arom. H). GC/
MS: 70 (80), 104 (30), 122 (80), 160 (100), 193 (70), 366 (5), 381 (10, Mþ ). Anal. calc. for C22H24FN3O2
(381.44): C 69.27, H 6.34, N 11.02; found: C 69.48, H 6.01, N 10.92.
2-{4-[4-(4-Chlorophenyl)piperazin-1-yl]butyl}-1H-isoindole-1,3(2H)-dione (6g). Yield: 1.4 g (77%).
Yellow crystals. M.p. 149–1518. IR: 1707, 1755 (2 C¼O). 1H-NMR (CDCl3): 1.56–1.77 (m, CH2(2’),
CH2(3’)); 2.42 (t, J ¼ 7.3, CH2(4’)); 2.57 (t, J ¼ 4.8, 2 CH2); 3.14 (t, J ¼ 4.8, 2 CH2); 3.73 (t, J ¼ 7.3,
CH2(1’)); 6.82 (d, J ¼ 9, 2 arom. H); 7.19 (d, J ¼ 9, 2 arom. H); 7.71 (dd, J ¼ 3, 5.5, 2 arom. H); 7.84 (dd,
J ¼ 3, 5.5, 2 arom. H). GC/MS: 70 (60), 104 (30), 138 (40), 160 (100), 209 (50), 382 (5), 397 (15, Mþ ).
Anal. calc. for C22H24ClN3O2 (397.90): C 66.41, H 6.08, N 10.56; found: C 66.14, H 6.04, N 10.43.
2-{4-[4-(3,4-Dichlorophenyl)piperazin-1-yl]butyl}-1H-isoindole-1,3(2H)-dione (6h). Yield: 1.4 g
(72%). Yellow crystals. M.p. 125–1278. IR: 1711, 1742 (2 C¼O). 1H-NMR (CDCl3): 1.56–1.77 (m,
CH2(2’), CH2(3’)); 2.41 (t, J ¼ 7.3, CH2(4’)); 2.55 (t, J ¼ 5.25, 2 CH2); 3.14 (t, J ¼ 5.25, 2 CH2); 3.73 (t, J ¼
7.3, CH2(1’)); 6.72 (dd, J ¼ 2.75, 9, 1 arom. H); 6.93 (d, J ¼ 2.75, 1 arom. H); 7.25 (d, J ¼ 9, 1 arom. H);
7.71 (dd, J ¼ 3, 5, 2 arom. H); 7.84 (dd, J ¼ 3, 5, 2 arom. H). GC/MS: 70 (50), 104 (35), 130 (60), 160
(100), 172 (60), 243 (50), 432 (15, Mþ ). Anal. calc. for C22H23Cl2N3O2 (432.34): C 61.12, H 5.36, N 9.72;
found: C 61.16, H 5.49, N 10.17.
2-{4-[4-(4-Hydroxyphenyl)piperazin-1-yl]butyl}-1H-isoindole-1,3(2H)-dione (6i). Yield: 1.0 g
1
(62%). Pale-yellow crystals. M.p. 151–1538. IR: 3217 (ꢀOH), 1712, 1748 (2 C¼O). H-NMR (CDCl3):
1.65–1.78 (m, CH2(2’), CH2(3’)); 2.40 (t, J ¼ 7, CH2(4’)); 2.57 (br. s, 2 CH2); 3.01 (br. s, 2 CH2); 3.64 (t,
J ¼ 7, CH2(1’)); 6.68 (d, J ¼ 9, 2 arom. H); 6.78 (d, J ¼ 9, 2 arom. H); 7.68–7.76 (m, 4 arom. H). GC/MS:
70 (15), 104 (5), 120 (20), 160 (25), 191 (100), 364 (20), 379 (90, Mþ ). Anal. calc. for C22H25N3O3: C
69.64, H 6.64, N 11.07; found: C 69.84, H 7.03, N 10.79.
2-[4-(4-Phenylpiperazin-1-yl)butyl]-1H-isoindole-1,3(2H)-dione (6j) [32]. Yield: 1.2 g (73%).
Yellow crystals. M.p. 137–1398. IR: 1710, 1756 (2 C¼O). 1H-NMR (CDCl3): 1.54–1.80 (m, CH2(2’),
CH2(3’)); 2.43 (t, J ¼ 7, CH2(4’)); 2.59 (t, J ¼ 5, 2 CH2); 3.18 (t, J ¼ 5, 2 CH2); 3.73 (t, J ¼ 7, CH2(1’));
6.81–6.93 (m, 3 arom. H); 7.22–7.28 (m, 2 arom. H); 7.69–7.84 (m, 4 arom. H). GC/MS: 77 (95), 104
(100), 130 (40), 160 (80), 175 (70), 348 (5), 363 (15, Mþ ). Anal. calc. for C22H25N3O2 (363.45): C 72.70, H
6.93, N 11.56; found: C 72.41, H 6.96, N 11.53.
Procedure for the Preparation of Thiophene-2-carbonyl Chloride (19) [23]. To a 100-ml round-
bottom flask containing thiophene-2-carboxylic acid (18; 0.5 g, 4 mmol) was added slowly and with
continuous stirring 10 ml of SOCl2. After complete addition, the mixture was allowed to reflux for 6 h.
The flask was then cooled to r.t., 50 ml of H2O was added, and the desired org. product was extracted with
CH2Cl2 (2ꢂ50 ml). The org. layers were collected, dried (Na2SO4), and evaporated under reduced
pressure. The final product was retrieved in form of brownish-black oil (0.42 g, 72%) and was used for the
further reaction without additional purification.
General Procedure for the Preparation of the Amide Derivatives. A soln. of a isoindole-1,3-dione
derivative (2 mmol) and NH2NH2 ·H2O (80%, 0.25 g, 6 mmol) in 20 ml of EtOH (95%) was heated to
reflux for 5 h. After cooling to r.t., any insoluble material was filtered off, washed with EtOH 95% (2ꢂ

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CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
20 ml), and the filtrate was evaporated under reduced pressure. The product was extracted with CHCl3
(2ꢂ30 ml), and the desired amines obtained, 15a–15j and 16a–16j were introduced to the following
reaction without further purification, where a soln. of benzoyl chloride (17; 0.28 g, 2 mmol) or 19 (0.30 g,
2 mmol) in 10 ml of dry THF was added slowly to the soln. of the previously obtained corresponding
amine derivative (2.3 mmol) and TEA (0.5 g, 5 mmol) in dry THF (30 ml) at 08. The mixture was then
stirred at r.t. for 5 h. The mixture was then poured into 30 ml of H2O and extracted with CH2Cl2 (2ꢂ
40 ml). The org. layers were collected, dried (Na2SO4), and evaporated to yield a residue of the desired
product that was purified by CC (SiO2; CH2Cl2/MeOH 200 :3).
N-{3-[4-(2-Ethoxyphenyl)piperazin-1-yl]propyl}benzamide (7a). Yield: 0.53 g (72%). Yellow crys-
tals. M.p. 121–1238. IR: 1637 (C¼O). 1H-NMR (CDCl3): 1.44 (t, J ¼ 7, Me), 1.80–1.87 (m, CH2(2’)); 2.64
(t, J ¼ 5.75, CH2(3’)); 2.71 (br. s, 2 CH2); 3.11 (br. s, 2 CH2); 3.59 (q, J ¼ 5.5, CH2(1’)); 4.05 (q, J ¼ 7,
MeCH2); 6.84–6.99 (m, 4 arom. H); 7.35–7.50 (m, 3 arom. H); 7.74–7.83 (m, 2 arom. H); 8.31 (br. s, NH).
GC/MS: 77 (100), 105 (95), 120 (95), 219 (20), 352 (15), 367 (10, Mþ ). Anal. calc. for C22H29N3O2
(367.48): C 71.90, H 7.95, N 11.43; found: C 71.58, H 7.74, N 10.84.
N-{3-[4-(2,3-Dichlorophenyl)piperazin-1-yl]propyl}benzamide (7b). Yield: 0.60 g (78%); Pale-
yellow crystals. M.p. 112–1148. IR: 1640 (C¼O). 1H-NMR (CDCl3): 1.81–1.86 (m, CH2(2’)); 2.65 (t,
J ¼ 6.25, CH2(3’)); 2.70 (br. s, 2 CH2); 3.04 (br. s, 2 CH2); 3.59 (q, J ¼ 5.75, CH2(1’)); 6.86–6.90 (m, 1
arom. H); 7.12–7.20 (m, 2 arom. H); 7.37–7.51 (m, 3 arom. H); 7.81–7.85 (m, 2 arom. H); 8.11 (br. s, NH).
GC/MS: 77 (80), 105 (100), 219 (30), 375 (15), 392 (10, Mþ ). Anal. calc. for C20H23Cl2N3O (392.32): C
61.23, H 5.91, N 10.71; found: C 60.96, H 5.84, N 10.47.
N-{4-[4-(2-Ethoxyphenyl)piperazin-1-yl]butyl}benzamide (8a). Yield: 0.50 g (66%). Pale-yellow
crystals. M.p. 106–1088. IR: 1641 (C¼O). 1H-NMR (CDCl3): 1.44 (t, J ¼ 7, Me); 1.60–1.71 (m, CH2(2’),
CH2(3’)); 2.30 (t, J ¼ 6.75, CH2(4’)); 2.70 (br. s, 2 CH2); 3.12 (br. s, 2 CH2); 3.48 (q, J ¼ 6.2, CH2(1’)); 4.06
(q, J ¼ 7, MeCH2); 6.82–6.97 (m, 4 arom. H, NH); 7.38–7.48 (m, 3 arom. H); 7.76–7.87 (m, 2 arom. H).
GC/MS: 77 (60), 105 (100), 120 (30), 219 (20), 297 (10), 366 (10), 382 (5, Mþ ). Anal. calc. for
C23H31N3O2 (381.51): C 72.41, H 8.19, N 11.01; found: C 71.87, H 7.98, N 10.87.
N-{4-[4-(2,3-Dichlorophenyl)piperazin-1-yl]butyl}benzamide (8b) [34]. Yield: 0.64 g (80%). Yel-
lowish-white crystals. M.p. 128–1308. IR: 1643 (C¼O). 1H-NMR (CDCl3): 1.62–1.72 (m, CH2(2’),
CH2(3’)); 2.46 (t, J ¼ 6.9, CH2(4’)); 2.62 (br. s, 2 CH2); 3.02 (br. s, 2 CH2); 3.48 (q, J ¼ 6.25, CH2(1’)); 6.69
(br. s, NH), 6.87–6.91 (m, 1 arom. H); 7.09–7.17 (m, 2 arom. H); 7.38–7.51 (m, 3 arom. H); 7.74–7.82 (m,
2 arom. H). GC/MS: 77 (100), 105 (95), 172 (80), 205 (70), 243 (60), 410 (5, [Mþ4]þ ). Anal. calc. for
C21H25Cl2N3O (406.35): C 62.07, H 6.20, N 10.34; found: C 61.84, H 6.66, N 10.34.
N-{3-[4-(2-Ethoxyphenyl)piperazin-1-yl]propyl}thiophene-2-carboxamide (9a). Yield: 0.50 g (68%).
Yellow resin. IR: 1631 (C¼O). 1H-NMR (CDCl3): 1.45 (t, J ¼ 7, Me); 1.76–1.86 (m, CH2(2’)); 2.62 (t, J ¼
6, CH2(3’)); 2.71 (br. s, 2 CH2); 3.17 (br. s, 2 CH2); 3.57 (q, J ¼ 5.6, CH2(1’)); 4.07 (q, J ¼ 7, MeOH2);
6.84–7.04 (m, 5 arom. H); 7.40 (dd, J ¼ 1.05, 5, 1 arom. H); 7.56–7.58 (m, 1 arom. H); 7.79 (br. s, NH).
GC/MS: 77 (20), 111 (35), 168 (30), 219 (70), 358 (100), 372 (50, Mþ ). Anal. calc. for C20H27N3O2S
(373.51): C 64.31, H 7.29, N 11.25; found: C 64.72, H 7.25, N 11.25.
N-{3-[4-(2,3-Dichlorophenyl)piperazin-1-yl]propyl}thiophene-2-carboxamide (9b). Yield: 0.52 g
(65%). Yellow resin. IR: 1639 (C¼O). 1H-NMR (CDCl3): 1.80–1.87 (m, CH2(2’)); 2.64 (t, J ¼ 6,
CH2(3’)); 2.71 (br. s, 2 CH2); 3.11 (t, J ¼ 4.8, 2 CH2); 3.57 (q, J ¼ 5.6, CH2(1’)); 6.91–6.95 (m, 1 arom. H);
7.05 (dd, J ¼ 3.6, 5, 1 arom. H); 7.12–7.20 (m, 2 arom. H); 7.43 (dd, J ¼ 1.15, 5, 1 arom. H); 7.57 (dd, J ¼
1.15, 3.6, 1 arom. H); 7.85 (br. s, NH). GC/MS: 77 (20), 111 (80), 168 (80), 197 (100), 381 (20), 398 (10,
Mþ ). Anal. calc. for C18H21Cl2N3OS (398.35): C 54.27, H 5.31, N 10.55; found: C 54.11, H 5.36, N 10.52.
N-{3-[4-(2-Fluorophenyl)piperazin-1-yl]propyl}thiophene-2-carboxamide (9c). Yield: 0.48 g (69%).
Brown resin. IR: 1643 (C¼O). 1H-NMR (CDCl3): 1.79–1.84 (m, CH2(2’)); 2.63 (t, J ¼ 6, CH2(3’)); 2.70 (t,
J ¼ 4.8, 2 CH2); 3.16 (t, J ¼ 4.8, 2 CH2); 3.57 (q, J ¼ 5.5, CH2(1’)); 6.90–7.06 (m, 5 arom. H); 7.40–7.56
(m, 2 arom. H); 7.98 (br. s, NH). GC/MS: 77 (15), 111 (100), 122 (80), 193 (40), 331 (40), 347 (10, Mþ ).
Anal. calc. for C18H22FN3OS (347.45): C 62.22, H 6.38, N 12.09; found: C 62.47, H 6.29, N 12.07.
N-{3-[4-(2-Chlorophenyl)piperazin-1-yl]propyl}thiophene-2-carboxamide (9d). Yield: 0.45 g (63%).
Yellow resin. IR: 1634 (C¼O). 1H-NMR (CDCl3): 1.78–1.96 (m, CH2(2’)); 2.60 (t, J ¼ 6, CH2(3’)); 2.91
(br. s, 2 CH2); 3.25 (br. s, 2 CH2); 3.61 (q, J ¼ 5.5, CH2(1’)); 6.99–7.28 (m, 4 arom. H); 7.35–7.68 (m, 3

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
2263
arom. H); 8.09 (br. s, NH). GC/MS: 77 (25), 111 (85), 168 (100), 197 (95), 347 (40), 364 (10, [Mþ2]þ ).
Anal. calc. for C18H22ClN3OS (363.90): C 59.41, H 6.09, N 11.55; found: C 59.56, H 6.04, N 11.52.
N-{3-[4-(2-Hydroxyphenyl)piperazin-1-yl]propyl}thiophene-2-carboxamide (9e). Yield: 0.47 g
1
(68%). Yellow resin. IR: 3208 (ꢀOH), 1647 (C¼O). H-NMR (CDCl3): 1.78–1.85 (m, CH2(2’)); 2.63
(t, J ¼ 6, CH2(3’)); 2.66 (br. s, 2 CH2); 2.97 (br. s, 2 CH2); 3.57 (q, J ¼ 5.5, CH2(1’)); 6.88–7.61 (m, 7 arom.
H); 7.95 (br. s, NH). GC/MS: 77 (15), 111 (75), 168 (80), 197 (100), 330 (10), 345 (15, Mþ ). Anal. calc.
for C18H23N3O2S (345.46): C 62.58, H 6.71, N 12.16; found: C 62.51, H 6.78, N 12.24.
N-{3-[4-(4-Fluorophenyl)piperazin-1-yl]propyl}thiophene-2-carboxamide (9f). Yield: 0.48 g (70%).
1
Yellowish-white crystals. M.p. 103–1058. IR: 1648 (C¼O). H-NMR (CDCl3): 1.79–1.86 (m, CH2(2’));
2.61 (t, J ¼ 6, CH2(3’)); 2.66 (t, J ¼ 5.1, 2 CH2); 3.16 (t, J ¼ 5.1, 2 CH2); 3.58 (q, J ¼ 5.6, CH2(1’)); 6.84–
7.03 (m, 5 arom. H); 7.38–7.53 (m, 2 arom. H); 7.82 (br. s, NH). GC/MS: 77 (20), 111 (100), 122 (50), 197
(90), 331 (60), 347 (20, Mþ ). Anal. calc. for C18H22FN3OS (347.45): C 62.22, H 6.38, N 12.09; found: C
62.65, H 6.52, N 12.30.
N-{3-[4-(4-Chlorophenyl)piperazin-1-yl]propyl}thiophene-2-carboxamide (9g). Yield: 0.54 g (74%).
Pale-yellow crystals. M.p. 144–1468. IR: 1641 (C¼O). 1H-NMR (CDCl3): 1.79–1.86 (m, CH2(2’)); 2.60 (t,
J ¼ 6, CH2(3’)); 2.64–2.68 (t, J ¼ 5, 2 CH2); 3.20 (t, J ¼ 5, 2 CH2); 3.58 (q, J ¼ 5.7, CH2(1’)); 6.83 (d, J ¼ 9,
2 arom. H); 6.0 (dd, J ¼ 3.7, 5, 1 arom. H); 7.13 (d, J ¼ 9, 2 arom. H); 7.38–7.52 (m, 2 arom. H); 7.78 (br. s,
NH). GC/MS: 77 (20), 111 (55), 168 (70), 197 (100), 347 (35), 362 (20, Mþ ). Anal. calc. for
C18H22ClN3OS (363.90): C 59.41, H 6.09, N 11.55; found: C 59.45, H 6.21, N 11.62.
N-{3-[4-(3,4-Dichlorophenyl)piperazin-1-yl]propyl}thiophene-2-carboxamide (9h). Yield: 0.58 g
(73%). Pale-yellow crystals. M.p. 107–1098. IR: 1649 (C¼O). 1H-NMR (CDCl3): 1.76–1.86 (m,
CH2(2’)); 2.58 (t, J ¼ 6, CH2(3’)); 2.64 (t, J ¼ 5.2, 2 CH2); 3.20 (t, J ¼ 5.2, 2 CH2); 3.56 (q, J ¼ 5.7,
CH2(1’)); 6.72 (dd, J ¼ 2.8, 8.8, 1 arom. H); 6.94 (d, J ¼ 2.8, 1 arom. H); 7.02 (dd, J ¼ 3.6, 5, 1 arom. H);
7.27 (d, J ¼ 8.8, 1 arom. H); 7.40 (dd, J ¼ 1, 5, 1 arom. H); 7.51 (d, J ¼ 3.6, 1 arom. H); 7.78 (br. s, NH).
GC/MS: 77 (20), 111 (55), 168 (70), 197 (100), 381 (30), 398 (15, Mþ ). Anal. calc. for C18H21Cl2N3OS
(398.35): C 54.27, H 5.31, N 10.55; found: C 54.20, H 5.29, N 10.57.
N-{3-[4-(4-Hydroxyphenyl)piperazin-1-yl]propyl}thiophene-2-carboxamide (9i). Yield: 0.51 g
(74%). Creamy-white crystals. M.p. 156–1588. IR: 3217 (ꢀOH), 1639 (C¼O). 1H-NMR (CDCl3):
1.77–1.87 (m, CH2(2’)); 2.70 (t, J ¼ 6, CH2(3’)); 2.75 (t, J ¼ 4.9, 2 CH2); 3.23 (t, J ¼ 4.9, 2 CH2); 3.58 (q,
J ¼ 5.5, CH2(1’)); 6.97–7.38 (m, 7 arom. H); 8.00 (br. s, NH). GC/MS: 77 (20), 111 (35), 168 (40), 197
(100), 330 (70), 345 (80, Mþ ). Anal. calc. for C18H23N3O2S (345.46): C 62.58, H 6.71, N 12.16; found: C
62.46, H 6.80, N 12.17.
N-[3-(4-Phenylpiperazin-1-yl)propyl]thiophene-2-carboxamide (9j). Yield: 0.50 g (76%). Creamy-
white crystals. M.p. 162–1648. IR: 1635 (C¼O). 1H-NMR (CDCl3): 1.77–1.87 (m, CH2(2’)); 2.61 (t, J ¼ 6,
CH2(3’)); 2.67 (t, J ¼ 5, 2 CH2); 3.25 (t, J ¼ 5, 2 CH2); 3.57 (q, J ¼ 5.6, CH2(1’)); 6.85–7.02 (m, 4 arom.
H); 7.25–7.40 (m, 3 arom. H); 7.55–7.56 (m, 1 arom. H); 7.97 (br. s, NH). GC/MS: 77 (95), 111 (100), 132
(40), 175 (30), 197 (25), 314 (15), 329 (10, Mþ ). Anal. calc. for C18H23N3OS (329.46): C 65.62, H 7.04, N
12.75; found: C 65.61, H 6.89, N 12.57.
N-{4-[4-(2-Ethoxyphenyl)piperazin-1-yl]butyl}thiophene-2-carboxamide (10a). Yield: 0.57 g (74%).
Orange resin. IR: 1641 (C¼O). 1H-NMR (CDCl3): 1.45 (t, J ¼ 7, Me); 1.67–1.85 (m, CH2(2’), CH2(3’));
2.47 (t, J ¼ 6.7, CH2(4’)); 2.65 (br. s, 2 CH2); 3.12 (br. s, 2 CH2); 3.46 (q, J ¼ 5.8, CH2(1’)); 4.05 (q, J ¼ 7,
MeCH2 ); 6.46 (br. s, NH); 6.82–7.07 (m, 5 arom. H); 7.43–7.49 (m, 2 arom. H). GC/MS: 70 (30), 111
(100), 121 (40), 134 (20), 219 (20), 372 (5), 387 (5, Mþ ). Anal. calc. for C21H29N3O2S (387.54): C 65.08, H
7.54, N 10.84; found: C 64.78, H 7.28, N 10.47.
N-{4-[4-(2,3-Dichlorophenyl)piperazin-1-yl]butyl}thiophene-2-carboxamide (10b). Yield: 0.58 g
1
(71%). Yellowish-brown resin. IR: 1637 (C¼O). H-NMR (CDCl3): 1.77–1.94 (m, CH2(2’), CH2(3’));
2.95 (t, J ¼ 6.8, CH2(4’)); 3.12 (br. s, 2 CH2); 3.36 (br. s, 2 CH2); 3.52 (q, J ¼ 6.2, CH2(1’)); 6.96 (br. s, H);
7.13–7.43 (m, 4 arom. H); 7.46 (d, J ¼ 3, 1 arom. H); 7.50 (d, J ¼ 2.3, 1 arom. H). GC/MS: 70 (50), 130
(60), 160 (100), 174 (40), 243 (40), 397 (5), 412 (5, Mþ ). Anal. calc. for C19H23Cl2N3OS (412.38): C 55.34,
H 5.62, N 10.19; found: C 55.44, H 5.62, N 9.94.
N-{4-[4-(2-Fluorophenyl)piperazin-1-yl]butyl}thiophene-2-carboxamide (10c). Yield: 0.51 g (71%).
Creamy-white crystals. M.p. 135–1378. IR: 1634 (C¼O). 1H-NMR (CDCl3): 1.60–1.65 (m, CH2(2’),
CH2(3’)); 2.45 (t, J ¼ 6.8, CH2(4’)); 2.62 (t, J ¼ 4.9, 2 CH2); 3.10 (t, J ¼ 4.9, 2 CH2); 3.47 (q, J ¼ 6.3,

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CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
CH2(1’)); 6.44 (br. s, NH); 6.89–7.07 (m, 5 arom. H); 7.44 (d, J ¼ 4.3, 1 arom. H); 7.50 (d, J ¼ 3.6, 1 arom.
H). GC/MS: 70 (50), 111 (100), 122 (60), 193 (40), 211 (20), 346 (10), 361 (5, Mþ ). Anal. calc. for
C19H24FN3OS (361.48): C 63.13, H 6.69, N 11.62; found: C 62.84, H 6.72, N 12.03.
N-{4-[4-(2-Chlorophenyl)piperazin-1-yl]butyl}thiophene-2-carboxamide (10d). Yield: 0.53 g (71%).
Yellowish-brown resin. IR: 1649 (C¼O). 1H-NMR (CDCl3): 1.67–1.80 (m, CH2(2’), CH2(3’)); 2.47 (t, J ¼
6.8, CH2(4’)); 2.65 (br. s, 2 CH2); 3.08 (br. s, 2 CH2); 3.44 (q, J ¼ 6, CH2(1’)); 6.39 (br. s, NH); 6.93–7.46
(m, 7 arom. H). GC/MS: 70 (70), 111 (100), 138 (50), 194 (10), 211 (40), 362 (5), 377 (5, Mþ ). Anal. calc.
for C19H24ClN3OS (377.93): C 60.38, H 6.40, N 11.12; found: C 60.01, H 6.39, N 10.79.
N-{4-[4-(2-Hydroxyphenyl)piperazin-1-yl]butyl}thiophene-2-carboxamide (10e). Yield: 0.46 g
1
(64%). Reddish-brown resin. IR: 3225 (OH), 1635 (C¼O). H-NMR (CDCl3): 1.66–1.69 (m, CH2(2’),
CH2(3’)); 2.51 (t, J ¼ 6.7, CH2(4’)); 2.68 (br. s, 2 CH2); 2.93 (t, J ¼ 4.75, 2 CH2); 3.44 (q, J ¼ 5.8, CH2(1’));
6.56 (br. s, NH); 6.82–6.95 (m, 2 arom. H); 7.04–7.14 (m, 3 arom. H); 7.45 (d, J ¼ 4.8, 1 arom. H); 7.54 (d,
J ¼ 3.8, 1 arom. H). GC/MS: 70 (40), 111 (50), 148 (25), 199 (40), 211 (100), 355 (10), 359 (5, Mþ ). Anal.
calc. for C19H25N3O2S (359.49): C 63.48, H 7.01, N 11.69; found: C 63.11, H 6.95, N 11.72.
N-{4-[4-(4-Fluorophenyl)piperazin-1-yl]butyl}thiophene-2-carboxamide (10f). Yield: 0.53 g (74%).
Greysh-white crystals. M.p. 143–1458. IR: 1644 (C¼O). 1H-NMR (CDCl3): 1.61–1.67 (m, CH2(2’),
CH2(3’)); 2.46 (t, J ¼ 6.7, CH2(4’)); 2.61 (t, J ¼ 5, 2 CH2); 3.12 (t, J ¼ 5, 2 CH2); 3.44 (q, J ¼ 6.3, CH2(1’));
6.40 (br. s, NH); 6.83–7.06 (m, 5 arom. H); 7.44 (d, J ¼ 4.9, 1 arom. H); 7.50 (d, J ¼ 3.8, 1 arom. H). GC/
MS: 70 (35), 111 (100), 122 (35), 193 (20), 211 (15), 346 (5), 361 (5, Mþ ). Anal. calc. for C19H24FN3OS
(361.48): C 63.13, H 6.69, N 11.62; found: C 62.94, H 6.83, N 11.35.
N-{4-[4-(4-Chlorophenyl)piperazin-1-yl]butyl}thiophene-2-carboxamide (10g). Yield: 0.57 g (76%).
Creamy-white crystals. M.p. 158–1608. IR: 1649 (C¼O). 1H-NMR (CDCl3): 1.66–1.74 (m, CH2(2’),
CH2(3’)); 2.46 (t, J ¼ 6.8, CH2(4’)); 2.60 (t, J ¼ 4.8, 2 CH2); 3.16 (t, J ¼ 4.8, 2 CH2); 3.44 (q, J ¼ 6,
CH2(1’)); 6.29 (br. s, NH); 6.82 (d, J ¼ 9, 2 arom. H); 7.05 (m, 1 arom. H); 7.19 (d, J ¼ 9, 2 arom. H); 7.44–
7.48 (m, 2 arom. H). GC/MS: 70 (70), 111 (100), 140 (35), 196 (10), 211 (45), 362 (5), 377 (5, Mþ ). Anal.
calc. for C19H24ClN3OS (377.93): C 60.38, H 6.40, N 11.12; found: C 60.72, H 6.55, N 10.82.
N-{4-[4-(3,4-Dichlorophenyl)piperazin-1-yl]butyl}thiophene-2-carboxamide (10h). Yield: 0.62 g
(76%). Pale-yellow crystals. M.p. 132–1348. IR: 1641 (C¼O). 1H-NMR (CDCl3): 1.65–1.74 (m,
CH2(2’), CH2(3’)); 2.43 (t, J ¼ 6.7, CH2(4’)); 2.57 (t, J ¼ 5, 2 CH2); 3.16 (t, J ¼ 5, 2 CH2); 3.47 (q, J ¼ 6.2,
CH2(1’)); 6.28 (br. s, NH); 6.72 (dd, J ¼ 3, 8.8, 1 arom. H); 6.93 (d, J ¼ 3, 1 arom. H); 7.06 (d, J ¼ 8.8, 1
arom. H); 7.24–7.47 (m, 3 arom. H). GC/MS: 70 (80), 130 (40), 160 (100), 174 (30), 243 (50), 397 (5),
416 (5, [Mþ4]þ ). Anal. calc. for C19H23Cl2N3OS (412.38): C 55.34, H 5.62, N 10.19; found: C 55.54, H
5.67, N 10.15.
N-{4-[4-(4-Hydroxyphenyl)piperazin-1-yl]butyl}thiophene-2-carboxamide (10i). Yield: 0.46 g
1
(64%). Buff crystals. M.p. 139–1418. IR: 3236 (OH), 1633 (C¼O). H-NMR (CDCl3): 1.66–1.69 (m,
CH2(2’)), CH2(3’)); 2.52 (t, J ¼ 6.7, CH2(4’)); 2.68 (br. s, 2 CH2); 2.95 (br. s, 2 CH2); 3.47 (q, J ¼ 5.8,
CH2(1’)); 6.56 (br. s, NH); 6.82–7.55 (m, 7 arom. H). GC/MS: 70 (60), 111 (90), 148 (30), 199 (45), 211
(100), 355 (20), 359 (10, Mþ ). Anal. calc. for C19H25N3O2S (359.49): C 63.48, H 7.01, N 11.69; found: C
63.87, H 6.65, N 11.75.
N-[4-(4-Phenylpiperazin-1-yl)butyl]thiophene-2-carboxamide (10j). Yield: 0.50 g (73%). Creamy-
white crystals. M.p. 115–1178. IR: 1641 (C¼O). 1H-NMR (CDCl3): 1.65–1.68 (m, CH2(2’), CH2(3’)); 2.47
(t, J ¼ 6.8, CH2(4’)); 2.63 (t, J ¼ 5, 2 CH2); 3.21 (t, J ¼ 5, 2 CH2); 3.46 (q, J ¼ 6.2, CH2(1’)); 6.42 (br. s,
NH); 6.83–7.45 (m, 8 arom. H). GC/MS: 70 (30), 104 (40), 111 (50), 132 (60), 211 (100), 328 (50), 343
(30, Mþ ). Anal. calc. for C19H25N3OS (343.49): C 66.44, H 7.34, N 12.23; found: C 66.56, H 7.36, N 12.07.
2. Molecular Modeling. 2.1. Energy Minimization and Conformational Search Procedure. The
compounds were drawn using ChemDraw with the basic piperazin N-atom protonated and saved as mol
file. The latter were subjected to energy minimization using Force Field MMFF94x by Molecular
Operating Environment (MOE) software, MOE, Chemical Computing Group Inc. http://www.chem-
comp/com.
2.2. Source of Target Proteins. The crystal structure of human D3 receptors complexed with its
antagonist eticlopride (PDB ID code: 3PBL) was downloaded from the Protein Data Bank and opened
with MOE software. The homology model of human D4 receptor was downloaded from the Supporting
Information of the article published in American Chemical Society Publications [29].

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
2265
2.3. Docking Procedure of D3 Receptors. The co-crystallized compound was selected, and the binding
site was identified according to residues in 4-ꢄ proximity to those interacted with the co-crystallized
antagonist eticlopride. Ligand interactions were computed for the X-ray co-crystallized compound,
eticlopride, to reveal the different types of interaction as a validation for the docking procedure. Default
settings of MOE-Dock were used, including ꢅRotate Bondsꢂ option in order to allow flexible ligandꢀrigid
receptor docking. The scoring function was London dG with a replacement of Triangle Matcher. Thirty
poses of each ligand docked to the identified binding site were retained and ranked in order of increasing
scoring function. The 2D ligandꢀreceptor interactions of these poses were viewed using the ꢅcompute
ligand interactionꢂ option of MOE.
2.4. Docking Procedure of D4 Receptors. The binding site was isolated off the validated homology
model using the default settings of the site finder panel option of MOE. Again, the default settings of
MOE-Dock were used, including ꢅRotate Bondsꢂ option in order to allow flexible ligandꢀrigid receptor
docking. The scoring function was London dG with a replacement of Triangle Matcher. Thirty poses of
each ligand docked to the identified binding site were retained and ranked in order of increasing scoring
function. The 2D ligandꢀreceptor interactions of these poses were viewed using the ꢅcompute ligand
interactionꢂ option of MOE.
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Received June 24, 2013