Synthesis of novel lactam derivatives and their evaluation as ligands for the dopamine receptors, leading to a D4-selective ligand

Article abstract of DOI:10.1016/j.bmc.2007.06.002

The preparation of some lactam (cyclic amide) derivatives bearing various phenylpiperazinylbutyl side chains attached to the amide nitrogen together with their dopamine receptor affinity study is described. The synthesis of the target compounds involved t

Full text of DOI:10.1016/j.bmc.2007.06.002

Bioorganic & Medicinal Chemistry 15 (2007) 5811–5818
Synthesis of novel lactam derivatives and their evaluation as
ligands for the dopamine receptors, leading to a D4-selective ligand
Fadi M. Awadallah,c Franziska Muller,b Jochen Lehmannb and Ashraf H. Abadia,*
¨
aDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy and Biotechnology, The German University in Cairo, Cairo, Egypt
bInstitute of Pharmacy, Pharmaceutical/Medicinal Chemistry, Friedrich-Schiller University, Jena, Germany
cDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, Cairo, Egypt
Received 17 April 2007; revised 29 May 2007; accepted 3 June 2007
Available online 7 June 2007
Abstract—The preparation of some lactam (cyclic amide) derivatives bearing various phenylpiperazinylbutyl side chains attached to
the amide nitrogen together with their dopamine receptor affinity study is described. The synthesis of the target compounds involved
the preparation of the intermediate bromobutyl derivatives of the appropriate lactam followed by N-alkylation of the appropriate
phenylpiperazines with these intermediates. Radioligand binding studies at D2–D5 receptor subtypes and a functional calcium assay
of the target compounds at D2 and D5 receptor subtypes were performed. All compounds, except 12a and 12b, showed selectivity
towards the D2-like receptor subtypes. Selectivity of the indolinone derivatives 11a–d at the D4 receptors was observed. Compound
11b exhibited a remarkable affinity to hD4 receptors with Ki value of 0.04 0.02 nm and was >43,000-fold selective over the hD2
receptor. In the functional assay, all the active compounds were of antagonistic activity.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
macological target for the development of antipsychotic
agents with low risk of extrapyramidal side effects.
Disturbances of the dopaminergic system account for
many neurological and neuropsychiatric disorders
including Parkinson’s disease, Tourette’s syndrome,
schizophrenia, tardive dyskinesia and addiction to psy-
chostimulants.1,2 Five subtypes of dopamine receptors
have been identified: D1, D2, D3, D4 and D5. These sub-
types are generally grouped into two families of dopa-
mine receptors, D1-like (D1 and D5) and D2-like (D2,
D3 and D4) receptors, based on similar pharmacologic
properties and primary structure homology.2,3 Recent
studies have revealed that the dopamine D3 receptor
subtype may be an important biological target for phar-
macotherapeutic agents used in treatment of schizophre-
nia.4 Essentially, all clinically useful neuroleptics are
antagonists at the D2 and D3 receptors. Therefore, it
has been concluded that the blockade of D2 receptor
in the caudate putamen region of the brain may be
responsible for extrapyramidal side effects, while block-
ade of D2/D3 receptors in the limbic regions of the brain
may be associated with antipsychotic effects.5,6 This sug-
gests that the D3 receptor subtype may be a good phar-
Several reports had shown molecules with a butylamide
linking chain, an extended aromatic terminus on one
end and an arylpiperazine on the other end are selective
D3 ligands with the type of intrinsic activity almost
dependent on the phenyl substituent, for example, BP
897, NGB 2904 and GR 103691.7–9 Structurally similar
compounds made up of cyclic benzamides linked
through an alkyl spacer to arylpiperazines were evalu-
ated as mixed D2/5-HT2 receptor antagonists and atyp-
ical antipsychotic agents. In most of successful cases, the
cyclic benzamide building blocks were linked to various
arylpiperazines through a 4-carbon spacer that has been
proved related to optimum activity.10–12 As pointed out
by Norman et al., the butyl spacer enabled the com-
pound to adopt a folded conformation stabilized by an
intramolecular hydrogen bond between the protonated
piperazine and the amide carbonyl. This flexible butyl
chain, in turn, allowed an optimum conformation and
distance between the terminal pharmacophores resulting
in optimum binding to the D2 receptors11 (Fig. 1).
Moreover, the dopaminergic activities of phenylpipera-
zines are not limited to D3 receptors, thus phenylpiper-
azine derivatives linked to various heteroaryls by a
Keywords: Dopamine ligands; Arylpiperazines; Cyclic amides; Lac-
tams; Receptor selectivity.
*
Corresponding author. E-mail: ashraf.abadi@guc.edu.eg
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.06.002

5812
F. M. Awadallah et al. / Bioorg. Med. Chem. 15 (2007) 5811–5818
O
3. Results and discussion
N
N
Table 1 shows binding data for the target compounds on
hD2L, hD3, hD4.4 and hD5 receptors, stably expressed in
CHO or HEK293 cells. [3H]-SCH 23390 and [3H]-Spip-
erone were used as radioligands for experiments at the
D1-like and D2-like receptor family, respectively. The
functional assay has been made upon hD2 and hD5
receptors.
H
BP 897
N
MeO
NGB 2904
N
O
Cl
N
Cl
Binding data reveal general affinity of the target com-
pounds towards the D2-like family of dopamine recep-
tors with the exception of the isoindolinone derivatives
12a,b that show D5 dopamine receptor affinity. Affinities
of compounds 9,10 (a–d) are almost distributed between
the D2 and D4 receptors with compounds 9a and 9d
being the most potent at D2 receptors and 10d being
the most potent at D4 antagonist as revealed by the
functional assay, Ki = 1.2 nm. All the indolinone deriva-
tives 11(a–d) were selective to the D4 receptors, this is
regardless of the nature of the phenyl substituent. Com-
pound 11b is the most potent, with Ki = 0.04 nm for the
D4 receptors, thus it is >43,000-fold selective over the
hD2 receptor, >30,000-fold selective over the D3 and
>5000-fold more selective over the D5 receptors,
respectively.
N
H
GR 103691
N
O
N
OMe
N
H
H3COC
Figure 1. D3 selective ligands with the general skeleton of an exocyclic
amide butyl spacer, linking an extended aromatic terminus on one end
and an arylpiperazine on the other.
methylene bridge looks like template for D4
selectivity.13,14
Interestingly, compounds 12(a–d), which are the posi-
tional isomers of 11(a–d), exhibit a different affinity pro-
file compared to all of the previous series. The affinity of
these compounds is equally distributed between the two
dopamine receptor families with compounds 12a,b
showing selectivity at D5 receptors and 12c,d showing
selectivity at D3 receptors.
It is deem of interest to test if the D3 selectivity will be
retained for structurally similar compounds with the
amide in an endocyclic rather than an exocyclic form.
Thus, the goal of the current study is to explore the
structure–activity/affinity relationships of various aryl-
piperazinylbutyllactams as candidates for dopamine
receptors’ ligands. Several heterocycles possessing cyclic
amides particularly, saccharin, 4-quinazolinone, indoli-
none and isoindolinone linked through a butyl chain
to various phenylpiperazines were prepared and evalu-
ated in vitro for their binding affinities by radioligand-
binding experiments and functional activities using a
calcium fluorescence assay at different dopamine recep-
tors. The protocol of these experiments has been de-
scribed by us previously.15–17
The impact of the phenylpiperazine substituent viz. H,
2-OCH3, 3-CF3 and 4-Cl function upon activity is not
absolute. However, compounds with the H substituent
9d, 10d, 11d, 12d were more biased towards the D4 sub-
type selectivity; meanwhile compounds with the 3-CF3
substituent 9c, 10c, 11c, 12c are more biased towards
imparting D3 subtype selectivity.
All the active compounds in the functional assay were of
antagonistic activity and they were more active on the
D2 rather than D5. The functional assay is dependent
on the fact that stimulation of the dopamine receptors
by agonists, for example, quinpirole for D2-like recep-
tors and SKF 38393 for hD5 will cause an increase in
intracellular Ca2+ which appears to represent a universal
second messenger signal for a majority of recombinant
GPCRs. Assay of the produced Ca2+ can be performed
using Oregon Green 488 BAPTA-1/AM and a micro-
plate reader. Dopamine agonists or antagonists are ex-
pected to alter the response to the standard agonists.
The microplate reader based calcium assay is a very use-
ful method for simply and quickly selecting the active
compounds out of a considerable number of com-
pounds. Comparing radioligand and calcium data, it
can be seen that there are differences in the Ki values ob-
tained, as the calcium-assay monitors a fast calcium-sig-
nal, these results represent non-equilibrium data,
whereas radioligand binding studies were performed
2. Chemistry
Sixteen lactam derivatives 9–12(a–d) bearing the butyl
piperazinyl un- or substituted-phenyl moiety were pre-
pared as outlined in Scheme 1. The general approach
for the preparation of the target compounds involved
alkylation of the appropriate phenylpiperazine with
the appropriate bromobutyl lactam derivative. The re-
quired intermediate alkylating agents 5–8 were synthe-
sized according to published procedures by the
reaction of the precursor lactams 1–4 with 1,4-dibromo-
butane. The intermediate bromo derivatives 5–8 were
then reacted with the appropriate phenylpiperazine in
refluxing acetonitrile in the presence of triethylamine un-
der nitrogen atmosphere to give the final compounds.

F. M. Awadallah et al. / Bioorg. Med. Chem. 15 (2007) 5811–5818
5813
R
Br
O
O
N
N
N
O
HN
R
N
N
Br
Br
NH
S
S
O
S
O
O
O
O
O
R
9 a-d
5
Br
1
O
N
N
N
O
N
O
HN
Br
R
N
N
Br
NH
N
N
10 a-d
6
R
2
Br
O
O
N
N
N
N
Br
HN
R
N
Br
O
N
H
11 a-d
3
7
R
Br
O
N
N
N
R
HN
N
N
Br
Br
NH
O
O
12 a-d
8
4
a: R = H; b: R= 4-Cl; c: R = 3-CF3; d: R = 2-OCH3
Scheme 1.
under equilibrium conditions. The assay is fast, simple,
and avoids the use of radioactivity.16
CDCl3 on Varian spectrometer at 300 MHz using tet-
ramethylsilane (TMS) as internal reference. Chemical
shift values are given in ppm. High resolution mass
spectra (GC-HRMS) were recorded on an Agilent
Mass spectrometer using positive ion electrospray ion-
ization(ESI, purity >95%). MS data were determined
by GC/MS, using a Hewlett Packard GCD-Plus
(G1800C) apparatus (HP-5MS column; J&W Scien-
tific). Elemental analysis of compound 11a was made
by the Microanalytical Unit, Faculty of Science, Cairo
University, Egypt. IR spectra were recorded on Bru-
ker FT-IR spectrophotometer as thin film for oils or
as potassium bromide disc for solids. Compounds 2,
4, 5–8 were prepared according to the published pro-
cedures.18–20
From the obtained results it seems that the D3 selectivity
is in great part dependent on the presence of the amide
of the butylamido spacer in an exocyclic form, while its
presence in an endocyclic form will lead to switch of
affinities to other dopamine receptor subtypes.
4. Experimental
4.1. Chemistry
Unless otherwise noted, all materials were obtained
from commercial suppliers and used without further
purification. Dimethylformamide (DMF) and acetoni-
4.2. General procedure for the preparation of the substi-
tutedphenylpiperazine derivatives 9–12(a–d)
´
˚
trile were dried over molecular sieve 4A and anhy-
drous
sodium
sulfate,
respectively.
Column
chromatography was performed using silica gel (s.d.
fine-chem. limited MUMBAI 400025, mesh size 200–
400) using chloroform/ethanol (9:1) as eluent. TLC
was performed on FLUKA silica gel TLC aluminium
cards (0.2 mm thickness) with fluorescent indicator
254 nm. Melting points were determined in open cap-
illaries on Electrothermal melting-point apparatus and
are uncorrected. 1H-NMR spectra were recorded in
A mixture of 5–8 (0.5 mmol), the appropriate phenylpip-
erazine (0.55 mmol) and triethylamine (2 mL) in dry
DMF (5 mL) was heated at 120 ꢁC under nitrogen.
The course of the reaction was followed by TLC. At
the end of the reaction time, the reaction mixture was
poured on to ice-water and extracted with chloroform.
The extracts were dried over anhydrous sodium sulfate,
filtered and evaporated untill dryness under reduced

5814
F. M. Awadallah et al. / Bioorg. Med. Chem. 15 (2007) 5811–5818
Table 1. Radioligand binding study and functional calcium assay of compoundsa 9–12
Compound
Radioligand binding study
Calcium assay
Change % of receptor
Ki-values [nM]
Change % of the agonist
Ki-values [nM]
bound radioactivity
by a 10 lM solution
of the compound
Average SD or SEM
(number of experiments
in triplicate)b
induced fluorescence by
a 10 lM solution of
the compound
Average SD or SEM
(number of experiments
each including six values)b
9a
D2 ꢀ100
D3 ꢀ99
D4 ꢀ101
D5 ꢀ75
D2 38 2 (2)
D3 66 4 (2)
D2 ꢀ62
D5 ꢀ12
D2 4.1 3.1 (4 · 6)
D5 386 131 (2 · 6)
D4 136 75 (3)
D5 1629 216 (2)
9b
D2 ꢀ99
D3 ꢀ80
D4 ꢀ101
D5 ꢀ87
D2 177 58 (2)
D3 287 43 (2)
D4 36 13 (3)
D5 230 15 (2)
D2 ꢀ24
D2 >10,000
D5 inactive
D5 +4
9c
D2 ꢀ100
D3 ꢀ98
D4 ꢀ54
D5 ꢀ89
D2 113 11 (2)
D3 107 72 (3)
D4 >10,000
D2 ꢀ44
D2 >10,000
D5 inactive
D5 +8
D5 1756 636 (2)
9d
D2 ꢀ100
D3 ꢀ94
D4 ꢀ83
D5 ꢀ90
D2 360 31 (2)
D3 187 33 (2)
D4 79 18 (2)
D5 1132 30 (2)
D2 ꢀ56
D2 6.2 4.2 (3 · 6)
D5 inactive
D5 +5
10a
10b
10c
10d
11a
11b
11c
11d
12a
12b
D2 ꢀ100
D3 ꢀ89
D4 ꢀ96
D5 ꢀ5
D2 ꢀ100
D3 ꢀ81
D4 ꢀ102
D5 ꢀ76
D2 ꢀ107
D3 ꢀ98
D4 ꢀ98
D5 ꢀ76
D2 ꢀ105
D3 ꢀ86
D4 ꢀ91
D5 ꢀ56
D2 ꢀ106
D3 ꢀ99
D4 ꢀ100
D5 ꢀ94
D2 ꢀ106
D3 ꢀ83
D4 ꢀ101
D5 ꢀ93
D2 ꢀ105
D3 ꢀ96
D4 ꢀ96
D5 ꢀ93
D2 ꢀ108
D3 ꢀ97
D4 ꢀ97
D5 ꢀ95
D2 46 7 (2)
D3 527 295 (3)
D4 81 26 (2)
D5 >10,000
D2 ꢀ37
D2 >10,000
D5 inactive
D5 +7
D2 140 92 (4)
D3 1780 161 (2)
D4 158 42 (3)
D5 507 78 (3)
D2 ꢀ44
D2 >10,000
D5 inactive
D5 +5
D2 73 9 (2)
D2 ꢀ38
D2 >10,000
D5 inactive
D3 102 13 (2)
D4 185 79 (3)
D5 1703 44 (2)
D5 +9
D2 184 14 (2)
D3 306 8.5 (2)
D4 123 66 (3)
D5 >10,000
D2 ꢀ58
D2 1.2 0.66 (2 · 6)
D5 inactive
D5 +2
D2 162 14 (2)
D3 80 27 (2)
D4 4.2 1.8 (2)
D5 313 47 (2)
D2 ꢀ58
D5 ꢀ2
D2 153 14 (2 · 6)
D5 >10,000
D2 1747 118 (2)
D3 1204 512 (3)
D4 0.04 0.02 (3)
D5 202 95 (2)
D2 ꢀ28
D2 375 23 (2 · 6)
D5 inactive
D5 +8
D2 323 20 (2)
D3 247 64 (3)
D4 28 13 (2)
D5 190 5 (5)
D2 ꢀ55
D2 416 108 (2 · 6)
D5 inactive
D5 +26
D2 405 91 (2)
D3 195 30 (2)
D4 9.6 0.9 (2)
D5 330 36 (2)
D2 ꢀ54
D2 286 3 (2 · 6)
D5 inactive
D5 +14
D2 ꢀ109
D3 ꢀ100
D4 ꢀ96
D5 ꢀ80
D2 189 62 (3)
D3 35.8 5.0 (2)
D4 216 56 (2)
D5 80 7.2 (2)
D2 ꢀ54
D5 ꢀ2
D2 60 42 (2 · 6)
D5 >10,000
D2 ꢀ105
D2 760 118 (2)
D3 660 206 (3)
D2 ꢀ7
D2 >10,000
D5 inactive
D3 ꢀ75
D5 +21

F. M. Awadallah et al. / Bioorg. Med. Chem. 15 (2007) 5811–5818
5815
Table 1 (continued)
Compound
Radioligand binding study
Calcium assay
Change % of receptor
bound radioactivity
by a 10 lM solution
of the compound
Ki-values [nM]
Change % of the agonist
Ki-values [nM]
Average SD or SEM
(number of experiments
each including six values)b
Average SD or SEM
(number of experiments
in triplicate)b
induced fluorescence by
a 10 lM solution of
the compound
D4 ꢀ91
D4 142 18 (2)
D5 33.6 2.8 (2)
D5 ꢀ100
12c
12d
D2 ꢀ106
D3 ꢀ99
D4 ꢀ95
D5 ꢀ99
D2 ꢀ107
D3 ꢀ98
D4 ꢀ94
D5 ꢀ89
D2 387 2 (2)
D3 14 4.2 (3)
D4 368 176 (3)
D5 80.8 5 (2)
D2 ꢀ53
D2 26 3.9 (2 · 6)
D5 inactive
D5 +41
D2 319 (1)
D2 ꢀ52
D5 ꢀ12
D2 34 19 (2 · 6)
D5 137 59 (2 · 6)
D3 30 23 (3)
D4 134 22 (2)
D5 75.4 21 (2)
a Binding assays using [3H]-Spiperone for D2, D3 and D4 and [3H]-SCH 23390 for D5.
b SD, standard deviation; SEM, standard error of the mean; the SEM is used when the number of values is less than three.
pressure. The crude compounds were purified using col-
umn chromatography.
J = 6 Hz, aromatic H). IR, Cmꢀ1: 3091 (aromatic
CH), 2924 (aliphatic CH), 1734 (CO), 1337 and 1162
(SO2). HRMS calculated for C22H25F3N3O3S (M+H+)
468.1569, found 468.1510.
4.3. 2-(4-(4-(2-Methoxyphenyl)-1-piperazinyl)butyl)-1,2-
benzisothiazole-3(2H)-one 1,1-dioxide (9a)
4.6. 2-(4-(4-Phenyl-1-piperazinyl)butyl)-1,2-benzisothia-
zole-3(2H)-one 1,1-dioxide (9d)
1
Yield 45%; mp 107–109 ꢁC; H NMR d 1.69 (m, 2H,
J = 7.2 Hz, ACONCH2CH2CH2CH2NA), 1.916 (m, 2H,
J = 7.5 Hz, ACONCH2CH2CH2CH2NA), 2.48 (t, 2H,
J = 7.5 Hz, ACONCH2CH2CH2CH2NA), 2.664 (br s,
4H, (CH2)2N1 piperazinyl), 3.09 (br s, 4H, (CH2)2N4 pip-
erazinyl), 3.80 (t, 2H, J = 7.2 Hz, ACONCH2CH2CH2
CH2NA), 3.85 (s, 3H, AOCH3), 6.83–6.98 (m, 4H, aro-
matic H), 7.813–7.903 (m, 3H, aromatic H), 8.043 (d,
1H, J = 5 Hz, aromatic H). HRMS calculated for
C22H28N3O4S (M+H+) 430.1801, found 430.1792.
1
Yield 53%; mp 122–124 ꢁC; H NMR d 1.66 (m, 2H,
J = 7.5 Hz, ACONCH2CH2CH2CH2NA), 1.92 (m, 2H,
J = 7.5 Hz, ACONCH2CH2CH2CH2NA), 2.46 (t, 2H,
J = 7.5 Hz, ACONCH2CH2CH2CH2NA), 2.61 (br s,
4H, (CH2)2N1 piperazinyl), 3.20 (br s, 4H, (CH2)2N4 pip-
erazinyl),
3.83
(t,
2H,
J = 7.5 Hz,
ACONCH2CH2CH2CH2NA), 6.82–7.73 (m, 5H), 7.80–
7.94 (m, 3H), 8.06 (d, 1H, J = 6 Hz). MS m/z (%): M+
400 (1.6), 399 (6.5), 175 (100.0). HRMS calculated for
C21H26N3O3S (M+H+) 400.1695, found 400.1682.
4.4. 2-(4-(4-(4-Chlorophenyl)-1-piperazinyl)butyl)-1,2-
benzisothiazole-3(2H)-one 1,1-dioxide (9b)
4.7. 3-(4-(4-(2-Methoxyphenyl)-1-piperazinyl)butyl)qui-
nazolin-4(3H)-one (10a)18
1
Yield 52%; mp 119–121 ꢁC; H NMR d 1.66 (m, 2H,
J = 7.5 Hz, ACONCH2CH2CH2CH2NA), 1.92 (m, 2H,
J = 7.5 Hz, ACONCH2CH2CH2CH2NA), 2.48 (t, 2H,
J = 7.2 Hz, ACONCH2CH2CH2CH2NA), 2.61 (br s,
4H, (CH2)2N1 piperazinyl), 3.17 (br s, 4H, (CH2)2N4
1
Yield 62%; Oil; H NMR d 1.67 (m, 2H, J = 7.2 Hz,
ACONCH2CH2CH2CH2NA), 1.89 (m, 2H, J = 7.2 Hz,
ACONCH2CH2CH2CH2NA), 2.52 (t, 2H, J = 7.5 Hz,
ACONCH2CH2CH2CH2NA), 2.70 (br s, 4H,
(CH2)2N1 piperazinyl), 3.13 (br s, 4H, (CH2)2N4 piper-
azinyl), 3.87 (s, 3H, OCH3), 4.07 (t, 2H, J = 7.2 Hz,
ACONCH2CH2CH2CH2NA), 6.86–7.77 (m, 7H, aro-
matic H), 8.061 (s, 1H, ANACH@NA), 8.34 (d, 1H,
J = 7 Hz, aromatic H). HRMS calculated for
C23H29N4O2 (M+H+) 393.2291, found 393.2260.
piperazinyl),
3.83
(t,
2H,
J = 7.2 Hz,
ACONCH2CH2CH2CH2NA), 6.84 (d, 2H, J = 9 Hz,
aromatic H), 7.20 (d, 2H, J = 9 Hz, aromatic H), 7.82–
7.90 (m, 3H, aromatic H), 8.07 (d, 1H, J = 5 Hz, aro-
matic H). HRMS calculated for C21H25ClN3O3S
(M+H+) 434.1305, found 434.1284.
4.5. 2-(4-(4-(3-a,a,a-Trifluorotolyl)-1-piperazinyl)butyl)-
1, 2-benzisothiazole-3(2H)-one 1,1-dioxide (9c)
4.8. 3-(4-(4-(4-Chlorophenyl)-1-piperazinyl)butyl)quinazo-
lin-4(3H)-one (10b)
1
Yield 40%; mp 111–113 ꢁC; H NMR d 1.69 (m, 2H,
1
J = 7.2 Hz, ACONCH2CH2CH2CH2NA), 1.93 (m, 2H,
J = 7.2 Hz, ACONCH2CH2CH2CH2NA), 2.51 (t, 2H,
J = 7.2 Hz, ACONCH2CH2CH2CH2NA), 2.62 (br s,
4H, (CH2)2N1 piperazinyl), 3.25 (br s, 4H, (CH2)2N4
Yield 58%; mp 163–165 ꢁC; H NMR d 1.66 (m, 2H,
J = 7.5 Hz, ACONCH2CH2CH2CH2NA), 1.86 (m, 2H,
J = 7.5 Hz, ACONCH2CH2CH2CH2NA), 2.47 (t, 2H,
J = 7.5 Hz, ACONCH2CH2CH2CH2NA), 2.62 (br s,
4H, (CH2)2N1 piperazinyl), 3.17 (br s, 4H, (CH2)2N4 pip-
erazinyl), 4.06 (t, 2H, J = 7.5 Hz, ACONCH2CH2
CH2CH2NA), 6.82–7.78 (m, 7H, aromatic H), 8.05 (s,
piperazinyl),
3.83
(t,
2H,
J = 7.2 Hz,
ACONCH2CH2CH2CH2NA), 7.05–7.33 (m, 4H, aro-
matic H), 7.83–7.91 (m, 3H, aromatic H), 8.05 (d, 1H,

5816
F. M. Awadallah et al. / Bioorg. Med. Chem. 15 (2007) 5811–5818
1H, ANACH@NA), 8.339 (d, 1H, J = 7 Hz, aromatic H).
IR, Cmꢀ1: 3057 (aromatic CH), 2940 (aliphatic CH), 1660
(CO), 1612 (C@N). MS m/z (%): M+ 397 (8.0), 396 (30.6),
209 (100.0). HRMS calculated for C22H26ClN4O (M+H+)
397.1795, found 397.1791.
4.13. 1-(4-(4-(3-a,a,a-Trifluorotolyl)-1-piperazinyl)butyl)-
indolin-2-one (11c)
Yield 37%; Oil; 1H NMR d 1.65 (m, 2H, J = 7 Hz,
ACONCH2CH2CH2CH2NA), 1.76 (m, 2H, J = 7 Hz,
ACONCH2CH2CH2CH2NA), 2.46 (t, 2H, J = 7 Hz,
ACONCH2CH2CH2CH2NA), 2.62 (br s, 4H,
(CH2)2N1 piperazinyl), 3.26 (br s, 4H, (CH2)2N4 piper-
azinyl), 3.54 (s, 2H, CH2CON indoline), 3.77 (t, 2H,
J = 7.2 Hz, ACONCH2CH2CH2CH2NA), 6.86–7.35
(m, 8H, aromatic H). MS m/z (%): M+ 417 (5.0), 243
(100.0). HRMS calculated for C23H27F3N3O (M+H+)
418.2106, found 418.2119.
4.9. 3-(4-(4-(3-a,a,a-Trifluorotolyl)-1-piperazinyl)butyl)-
quinazolin-4(3H)-one (10c)
Yield 46%; mp 77–79 ꢁC; 1H NMR d 1.64 (m, 2H,
J = 7.5 Hz, ACONCH2CH2CH2CH2NA), 1.89 (m, 2H,
J = 7.5 Hz, ACONCH2CH2CH2CH2NA), 2.47 (t, 2H,
J = 7.5 Hz, ACONCH2CH2CH2CH2NA), 2.61 (br s,
4H, (CH2)2N1 piperazinyl), 3.24 (br s, 4H, (CH2)2N4
piperazinyl),
4.07
(t,
2H,
J = 7.5 Hz,
4.14. 1-(4-(4-Phenyl-1-piperazinyl)butyl)indolin-2-one (11d)
ACONCH2CH2CH2CH2NA), 7.04–7.78 (m, 7H, aro-
matic H), 8.06 (s, 1H, ANACH@NA), 8.34 (d, 1H,
J = 7 Hz, aromatic H). HRMS calculated for
C23H26F3N4O (M+H+) 431.2059, found 431.2048.
1
Yield 30%; Oil; H NMR d 1.649 (m, 2H, J = 7 Hz,
ACONCH2CH2CH2CH2NA), 1.758 (m, 2H, J = 7 Hz,
ACONCH2CH2CH2CH2NA), 2.456 (t, 2H, J = 7 Hz,
ACONCH2CH2CH2CH2NA), 2.611 (br s, 4H,
(CH2)2N1 piperazinyl), 3.209 (br s, 4H, (CH2)2N4 piperaz-
inyl), 3.532 (s, 2H, CH2CON indoline), 3.786 (t, 2H,
J = 7.2 Hz, ACONCH2CH2CH2CH2NA), 6.858–7.292
(m, 9H, aromatic H). MS m/z (%): M+ 349 (14.2), 175
(100.0). HRMS calculated for C22H28N3O (M+H+)
350.2232, found 350.2249.
4.10. 3-(4-(4-Phenyl-1-piperazinyl)butyl)quinazolin-
4(3H)-one (10d)19
1
Yield 61%; mp 136–138 ꢁC; H NMR d 1.61 (m, 2H,
J = 7.5 Hz, ACONCH2CH2CH2CH2NA), 1.89 (m, 2H,
J = 7.5 Hz, ACONCH2CH2CH2CH2NA), 2.47 (t, 2H,
J = 7.5 Hz, ACONCH2CH2CH2CH2NA), 2.61 (br s,
4H, (CH2)2N1 piperazinyl), 3.23 (br s, 4H, (CH2)2N4
4.15. 2-(4-(4-(2-Methoxyphenyl)-1-piperazinyl)butyl)-1-
isoindolinone (12a)
piperazinyl),
4.07
(t,
2H,
J = 7.5 Hz,
ACONCH2CH2CH2CH2NA), 6.87–7.78 (m, 8H, aro-
matic H), 8.06 (s, 1H, ANACH@NA), 8.35 (d, 1H,
J = 7 Hz, aromatic H). MS m/z (%): M+ 362 (44.2),
175 (100.0). HRMS calculated for C22H27N4O
(M+H+) 363.2185, found 363.2198.
Yield 34%; Oil; 1H NMR d 1.65 (m, 2H, J = 7 Hz,
ACONCH2CH2CH2CH2NA), 1.76 (m, 2H, J =
7 Hz, ACONCH2CH2CH2CH2NA), 2.51 (t, 2H,
J = 7 Hz, ACONCH2CH2CH2CH2NA), 2.70 (br s, 4H,
(CH2)2N1 piperazinyl), 3.09 (br s, 4H, (CH2)2N4
4.11. 1-(4-(4-(2-Methoxyphenyl)-1-piperazinyl)butyl)-
indolin-2-one (11a)
piperazinyl),
3.67
(t,
2H,
J = 7 Hz,
ACONCH2CH2CH2CH2NA), 3.86 (s, 3H, OCH3),
4.40 (s, 2H, CH2NCO isoindoline), 6.87–7.80 (m, 8H,
aromatic H). MS m/z (%): M+ 379 (0.87), 216 (100.0).
Analysis calculated for C23H29N3O2: C, 72.79; H, 7.70;
N, 11.07. Found C, 72.39; H, 7.19; N, 11.20.
1
Yield 41%; Oil; H NMR d 1.65 (m, 2H, J = 7.2 Hz,
ACONCH2CH2CH2CH2NA), 1.76 (m, 2H, J =
7.2 Hz, ACONCH2CH2CH2CH2NA), 2.465 (t, 2H,
J = 7.2 Hz, ACONCH2CH2CH2CH2NA), 2.654 (br s,
4H, (CH2)2N1 piperazinyl), 3.101 (br s, 4H, (CH2)2N4 pip-
erazinyl), 3.53 (s, 2H, CH2CON indoline), 3.761 (t, 2H,
J = 7.2 Hz, ACONCH2CH2CH2CH2NA), 3.869 (s, 3H,
4.16. 2-(4-(4-(4-Chlorophenyl)-1-piperazinyl)butyl)-1-iso-
indolinone (12b)
OCH3), 6.878–7.272 (m, 8H, aromatic H). IR, Cmꢀ1
:
Yield 42%; Oil; 1H NMR d 1.64 (m, 2H, J = 7 Hz,
ACONCH2CH2CH2CH2NA), 1.76 (m, 2H, J =
7 Hz, ACONCH2CH2CH2CH2NA), 2.49 (t, 2H,
J = 7 Hz, ACONCH2CH2CH2CH2NA), 2.55 (br s, 4H,
(CH2)2N1 piperazinyl), 3.109 (br s, 4H, (CH2)2N4 piper-
3056 (aromatic CH), 2924 (aliphatic CH), 1707 (CO).
MS m/z(%): M+ 379 (15.8), 205 (100.0). HRMScalculated
for C23H30N3O2 (M+H+) 380.2338, found. 380.2303.
4.12. 1-(4-(4-(4-Chlorophenyl)-1-piperazinyl)butyl)indo-
lin-2-one (11b)
azinyl),
3.675
(t,
2H,
J = 7 Hz,
ACONCH2CH2CH2CH2N), 4.392 (s, 2H, CH2NCO
isoindoline), 6.788–7.577 (m, 8H, aromatic H). HRMS
calculated for C22H27ClN3O 384.1843, found 384.1849.
Yield 35%; Oil; 1H NMR d 1.65 (m, 2H, J = 7 Hz,
ACONCH2CH2CH2CH2NA), 1.74 (m, 2H, J =
7 Hz, ACONCH2CH2CH2CH2NA), 2.46 (t, 2H,
J = 7 Hz, ACONCH2CH2CH2CH2NA), 2.60 (br s, 4H,
(CH2)2N1 piperazinyl), 3.16 (br s, 4H, (CH2)2N4 piper-
azinyl), 3.53 (s, 2H, CH2CON indoline), 3.76 (t, 2H,
J = 7.2 Hz, ACONCH2CH2CH2CH2NA), 6.82–7.27
(m, 8H, aromatic H). MS m/z (%): M+ 384 (3.1), 383
(11.7), 209 (100.0). HRMS calculated for C22H27N3O
(M+H+) 384.1843, found 384.1810.
4.17. 2-(4-(4-(3-a,a,a-Trifluorotolyl)-1-piperazinyl)butyl)-
1-isoindolinone (12c)
Yield 40%; Oil; 1H NMR d 1.65 (m, 2H, J = 7 Hz,
ACONCH2CH2CH2CH2NA), 1.75 (m, 2H, J =
7 Hz, ACONCH2CH2CH2CH2NA), 2.489 (t, 2H,
J = 7 Hz, ACONCH2CH2CH2CH2NA), 2.614 (br s,
4H, (CH2)2N1 piperazinyl), 3.146 (br s, 4H, (CH2)2N4

F. M. Awadallah et al. / Bioorg. Med. Chem. 15 (2007) 5811–5818
5817
piperazinyl),
3.665
(t,
2H,
J = 7 Hz,
whole-cell-suspension were carried out in triplicate in a
volume of 550 lL (final concentration): Tris–Mg2+-buf-
fer (345 lL), [3H]-ligand (50 lL), whole-cell-suspension
(100 lL) and appropriate drugs (55 lL). Non-specific
binding was determined using fluphenazine (100 lM)
in D5 test and haloperidol (10 lM) in D2, D3 and D4
tests. For a fast screening the drugs were used in a con-
centration of 100 lM, and the percentage of removed
radioligand determined. The incubation was initiated
by addition of the radioligand. It was carried out in
96-deep well plates (Greiner bio-one Frickenhausen)
using a Thermocycler (Thermocycler comfort, Eppen-
dorf, Wessling) at 27 ꢁC for 11/2 h, and stopped by rapid
filtration with a Perkin-Elmer Mach III Harvester using
a Perkin-Elmer Filtermat A., previously treated with
0.25% polyethyleneimine solution (Sigma–Aldrich),
which was washed once with ice-cold water. The filter-
mat was dried for 3 min with 400 W microwave
(WM21, Clatronic, Kemoen). The dry filtermat was
placed on a filter paper (Omni filter plates, Perkin-Elmer
Life Sciences) and each field of the filtermat moistened
with 50 lL Microscint 20 scintillation cocktail. The
radioactivity retained on the filters was counted using
a Top Count NXT microplate scintillation counter
(Packard, Ct, USA). For determining the Ki values at
least two independent experiments in triplicate were per-
formed. The competition binding data were analysed by
the software GraphPad PrismTM using non-linear squares
fit. For calculating the mean, SD and SEM the software
Microsoft Excel was used. Ki values were calculated
from IC50 values applying the equation of Cheng and
Prusoff.21
ACONCH2CH2CH2 CH2N), 4.4 (s, 2H, CH2NCO iso-
indoline), 7.041–7.81 (m, 8H, aromatic H). MS m/z
(%): M+ 417 (1.6), 217 (100.0). HRMS calculated for
C23H27F3N3O (M+H+) 418.2116, found 418.2106.
4.18. 2-(4-(4-Phenyl-1-piperazinyl)butyl)-1-isoindolinone
(12d)20
1
Yield 40%; Oil; H NMR d 1.645 (m, 2H, J = 7 Hz,
ACONCH2CH2CH2CH2NA), 1.782 (m, 2H, J =
7 Hz,
ACONCH2CH2CH2CH2NA),
2.496
(t,
2H, J = 7 Hz, ACONCH2CH2CH2CH2NA), 2.634 (br
s, 4H, (CH2)2N1 piperazinyl), 3.245 (br s, 4H,
(CH2)2N4 piperazinyl), 3.679 (t, 2H, J = 7 Hz,
ACONCH2CH2CH2CH2N), 4.403 (s, 2H, CH2NCO
isoindoline), 6.859–7.8 (m, 9H, aromatic H). MS m/z
(%): M+ 349 (6.6), 217 (100.0). HRMS calculated for
C22H28N3O (M+H+) 350.2232, found 350.2243.
5. Pharmacology15–17
5.1. Dopamine receptor ligand activity
5.1.1. Cell culture. Human D2L, D3, D4.4 and D5 recep-
tors were stably expressed in Chinese hamster ovary
(CHO) cells and human embryonic kidney cells
(HEK293), respectively. The density of receptors mea-
sured with [3H]-SCH 23390 was 679.44 fmol/mg protein
for D5 receptor in HEK cells. The densities of receptors
measured with [3H]-Spiperone were 186.53 fmol/mg pro-
tein for D2 receptor expressed in HEK cells; 6043 fmol/
mg for the D4 receptor and 14,474 fmol/mg for D3
receptor, both expressed in CHO cells. Cells were grown
at 37 ꢁC under a humidified atmosphere of 5% CO2:
95% air in HAM/F12-medium (Sigma–Aldrich) for
CHO cells and Dulbecco‘s modified Eagle’s medium
Nutient mixture F-12 Ham for HEK293 cells, each sup-
plemented with 10% foetal bovine serum, 1 mM L-gluta-
mine, 20 U/mL penicillin G, 20 lg/L streptomycin and
0.2 lg/mL G 418 (all from Sigma–Aldrich).
6. Functional assay measuring intracellular Ca2+ with a
fluorescence microplate reader16,22
6.1. Cell culture
Human D2L and D5 receptors were stably expressed in
human embryonic kidney cells (HEK293) and cultured
as above-mentioned.
5.1.2. Preparation of whole-cell-suspension.17 Human
D2, D3, D4 and D5 receptor cell lines (CHO) were grown
on T 175 culture dishes (Greiner bio-one, Frickenhau-
sen) to 85% confluency, the medium was removed and
the cells were incubated with 6 mL trypsin-EDTA-solu-
tion (Sigma–Aldrich) to remove the cells from the cul-
ture dish. After incubation, cells were suspended in 3–
6 mL added medium in order to stop the effect of tryp-
sin-EDTA solution.The resulting suspension was centri-
fuged (1800–2400 rpm/min, 4 ꢁC, 4 min), the pellet
resuspended in 10 mL PBS (ice-cooled, calcium- and
magnesium-free), pelleted, and this procedure repeated.
The resulting pellet was then resuspended in 12 mL buf-
fer (5 mM magnesium chloride, 50 mM Tris–HCl, pH
7.4) and the resulting suspension was directly used for
the radioligand binding assay.
6.2. Preparation of whole-cell-suspension
Human D2 and D5 receptor cell lines were grown on
T 175 culture dishes (Greiner bio-one, Frickenhausen)
to 85–90% confluency. The medium was removed and
cells rinsed twice with 6 mL Krebs-Hepes buffer
(118 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4,
1.2 mM KH2PO4, 4.2 mM NaHCO3, 11.7 mM D-Glu-
cose, 1.3 mM CaCl2, 10 mM Hepes, pH 7.4) each
time. After two washes, cells were loaded with 3 lL
of a 0,5 M Oregon Green 488 BAPTA-1/AM-solution
(Molecular Probes, Eugene, OR) (in DMSO) in 6 mL
of the same buffer containing 3 lL of a 20% Pluronic
F-127-solution (Sigma–Aldrich) (in DMSO) for 45 min
at 37 ꢁC. After 35-min incubation, the culture dish
was rapped slightly in order to remove all cells from
the dish for further incubation. Then 5 mL of
Krebs-Hepes buffer was added and cells were sus-
pended. The resulting suspension was separated in
10 vials (1.5 mL) and centrifuged (10,000 rot/min,
5.1.3. Radioligand binding assay17. For the binding stud-
ies a procedure according to the protocol previously
published but in 96-well format.15 The assays with the

5818
F. M. Awadallah et al. / Bioorg. Med. Chem. 15 (2007) 5811–5818
10 s), the pellets were resuspended in 1 mL Krebs-
Hepes buffer twice per five pellets and centrifuged
again. The pellets were resuspended in 16 mL (for
screening of antagonistic activity) or 18 mL (for
screening of agonistic activity) Krebs-Hepes buffer
and plated into 96-well plates (OptiPlate HTRF-96,
Packard, Meriden, CT; Cellstar, Tissue Culture Plate,
96W, Greiner bio-one, Frickenhausen). Microplates
were kept at 37 ꢁC under a humidified atmosphere
of 5% CO2: 95% air for 30 min.
instruments and equipments used in this research to
Cairo University.
References and notes
1. Mach, R. H.; Huang, Y.; Freeman, R. A.; Wu, L.; Blair,
S.; Luedtke, R. R. Bioorg. Med. Chem. 2003, 17, 225.
2. Missale, C.; Nash, S. R.; Robinson, S. W.; Jaber, M.;
Caron, M. G. Physiol. Rev. 1998, 78, 189.
3. Civelli, O.; Bunzow, J. R.; Grandy, D. K. Annu. Rev.
Pharmacol. Toxicol. 1993, 32, 281.
6.3. Calcium assay16
4. De Oliveira, I. R.; Juruena, M. F. J. Clin. Pharm. Ther.
2006, 31, 523.
Screening for agonistic and antagonistic activity was
performed using
5. Dikeos, D. G.; Papadimitriou, G. N.; Avramopoulos, D.;
Karadima, G.; Daskalopoulou, E. G.; Souery, D.; Mend-
lewicz, J.; Vassilopoulos, D.; Stefanis, C. N. Psychiatr.
Genet. 1999, 9, 189.
6. Newman, A. H.; Grundt, P.; Nader, M. A. J. Med. Chem.
2005, 48, 3663.
7. Gilbert, J. G.; Newman, A. H.; Gardner, E. L.; Ashby, C.
R., Jr.; Heidbreder, C. A.; Pak, A. C.; Peng, X. Q.; Xi, Z.
X. Synapse 2005, 57, 17.
8. Salama, I.; Schlotter, K.; Utz, W.; Hubner, H.; Gmeiner,
P.; Boeckler, F. Bioorg. Med. Chem. 2006, 14, 5898.
9. Audinot, V.; Newman-Tancredi, A.; Gobert, A.; Rivet, J.
M.; Brocco, M.; Lejeune, F.; Gluck, L.; Desposte, I.;
Bervoets, K.; Dekeyne, A.; Millan, M. J. J. Pharmacol.
Exp. Ther. 1998, 287, 187.
10. Norman, M. H.; Navas, F., 3rd; Thompson, J. B.; Rigdon,
G. C. J. Med. Chem. 1996, 39, 4692.
11. Norman, M. H.; Minick, D. J.; Rigdon, G. C. J. Med.
Chem. 1996, 39, 149.
12. Norman, M. H.; Rigdon, G. C.; Navas, F., 3rd; Cooper,
B. R. J. Med. Chem. 1994, 37, 2552.
13. Abadi, A. H. Arch. Pharm. Pharm. Med. Chem. 2004, 337,
383.
a NOVOstar microplate reader
(BMG LabTechnologies) with a pipettor system. Ago-
nistic activity was tested after 30-min incubation of
the plated cell suspension by injecting 20 lL buffer
alone, standard agonist, or test compounds, respec-
tively, dissolved in buffer sequentially into separate
wells. Screening of compounds for antagonist activity
or dose–response curves in presence of an antagonist
was performed by preincubating the cells with 20 lL
of the solutions of compounds (final concentrations:
100 lM, 50 lM, 10 lM, 5 lM, 1 lM, 500 nM,
100 nM, 50 nM, 10 nM, 1 nM, 0,1 nM) at 37 ꢁC for
30 min prior to injection of 20 lL standard agonist.
Final concentration of test compounds for screening
of agonist or antagonist activity was 10 lM, respec-
tively. Quinpirole was used as standard agonist for
hD2 receptors and SKF 38393 for hD5 receptors (final
concentration: 1 lM). Fluorescence intensity was mea-
sured at 520 nm (bandwidth 25 nm) for 30 s at 0.4 s
intervals. Excitation wavelength was 485 nm (band-
width 20 nm). IC50 values were obtained by determi-
nation of the maximum fluorescence intensity of
each data set and non-linear regression with sigmoidal
dose–response equation using a four-parameter logistic
equation on GraphPadPrismTM 3.0. Ki values were then
calculated to account for different agonist concentra-
tions and EC50 values applying a modified Cheng–
Prusoff equation21:
14. Lober, S.; Hubner, H.; Gmeiner, P. Bioorg. Med. Chem.
Lett. 1999, 9, 97.
15. Kassack, M. U.; Hofgen, B.; Decker, M.; Eckstein, N.;
Lehmann, J. Naunyn-Schmiedeberg’s Arch. Pharmacol.
2002, 366, 543.
16. Hoefgen, B.; Decker, M.; Mohr, P.; Schramm, A. M.;
Rostom, S. A.; El Subbagh, H.; Schweikert, P. M.;
Rudolf, D. R.; Kassack, M. U.; Lehmann, J. J. Med.
Chem. 2006, 49, 760.
17. Decker, M.; Lehmann, J. Arch. Pharm. Pharm. Med.
Chem. 2003, 336, 466.
18. Bojarski, A. J.; Kowalski, P.; Kowalska, T.; Duszynska,
B.; Charakchieva-Minol, S.; Tatarczynska, E.; Klodzins-
ka, A.; Chojnacka-Wojcik, E. Bioorg. Med. Chem. 2002,
10, 3817.
19. Kowalski, P.; Kowalska, T.; Mokrosz, M. J.; Bojarski, A.
J.; Charakchieva-Minol, S. Molecules 2001, 6, 784.
20. Bojarski, A. J.; Paluchowska, M. H.; Duszynska, B.;
Bugno, R.; Klodzinska, A.; Tatarczynska, E.; Chojnacka-
Wojcik, E. Bioorg. Med. Chem. 2006, 14, 1391.
21. Cheng, Y.; Prusoff, W. H. Biochem. Pharmacol. 1973, 22,
3099.
IC50
Ki ¼
L
1 þ
EC50
L: concentration of standard agonist (M); EC50: effective
concentration 50% of the standard agonists (M); IC50:
inhibitory concentration 50% of test compounds at the
given experimental conditions, that is, standard agonist
concentration.
Acknowledgment
The authors are grateful to the Alexander von Hum-
boldt foundation, Germany, for donating some of the
22. Kassack, M. U.; Ho¨fgen, B.; Lehmann, J.; Eckstein, N.;
Quillan, J. M.; Sadee, W. J. Biomol. Screening 2002, 8, 233.