Sigma-1 ligands: Tic-hydantoin as a key pharmacophore

Article abstract of DOI:10.1016/j.ejmech.2009.10.004

Sigma-1 receptors are involved in numerous pathological dysfunctions and the synthesis of selective ligands is of interest. We identified a fused tetrahydroisoquinoline-hydantoin (Tic-hydantoin) structure with high affinity and selectivity for these receptors. We report here our efforts towards the pharmacomodulation of this substructure, the synthesis of 9 analogs with stereochemistry inversion, opening of isoquinoline ring, removal of isoquinoline nitrogen, replacement of isoquinoline by pyridine, of Tic-hydantoin moiety by quinazolinedione heterocycle. All these analogs provided a loss in the affinity for the sigma-1 receptor. The present work underlines the real importance of the Tic-hydantoin moiety for the obtainment of high affinity ligands.

Full text of DOI:10.1016/j.ejmech.2009.10.004

European Journal of Medicinal Chemistry 45 (2010) 256–263
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Sigma-1 ligands: Tic-hydantoin as a key pharmacophore
Marion Toussaint a, Deborah Mousset a, Catherine Foulon b, Ulrich Jacquemard a, Claude Vaccher b,
,
Patricia Melnyk a
*
a UMR CNRS 8161 - Universite´ Lille Nord de France - Institut Pasteur de Lille 1 rue du Professeur Calmette, B.P. 447, 59021 Lille cedex, France
b EA4034, Faculte´ de Pharmacie de Lille, Universite´ Lille Nord de France, 3 rue du Professeur Laguesse, 59006 Lille, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Sigma-1 receptors are involved in numerous pathological dysfunctions and the synthesis of selective
ligands is of interest. We identified a fused tetrahydroisoquinoline-hydantoin (Tic-hydantoin) structure
with high affinity and selectivity for these receptors. We report here our efforts towards the pharma-
comodulation of this substructure, the synthesis of 9 analogs with stereochemistry inversion, opening of
isoquinoline ring, removal of isoquinoline nitrogen, replacement of isoquinoline by pyridine, of Tic-
hydantoin moiety by quinazolinedione heterocycle. All these analogs provided a loss in the affinity for
the sigma-1 receptor. The present work underlines the real importance of the Tic-hydantoin moiety for
the obtainment of high affinity ligands.
Received 26 May 2009
Received in revised form
10 August 2009
Accepted 1 October 2009
Available online 9 October 2009
Keywords:
Sigma receptor
Hydantoin
Ó 2009 Elsevier Masson SAS. All rights reserved.
Tetrahydroisoquinoline
1. Introduction
such as norepinephrine, dopamine, serotonin, acetylcholine and
glutamate as well as the activity of opiate receptors [6]. Conse-
Sigma receptors were first described by Martin et al. in 1976 [1].
They are now classified into two distinct subtypes denoted s1 and
s2 [2]. These subtypes display a different tissue distribution and
a distinct physiological and pharmacological profile in the central
and peripheral nervous system. Based on the predicted 223 amino
acid sequence, sigma-1 receptor shares no homology with any
mammalian proteins. Homology analysis of the amino acid
sequence has suggested that the sigma-1 receptor has two trans-
membrane segments, resulting in an extracellular loop of approx-
imately 50 amino acids and an intracellular C-terminus of
approximately 125 amino acids [3,4]. According to this model, the
N-terminus is very short and also localised intracellularly. The
ligand binding region together with the second hydrophobic region
has been suggested to be important for the binding of ligands [5]
but the exact binding site has still to be elucidated. s1 receptors
were described to modulate the transmission of neurotransmitters
quently, they are associated with some functions or disorders such
as nociception [7], cocaine addiction [8–10], mnesic disorders [11],
depression [12], anxiety, epilepsy [13] and are implicated in neu-
roprotection [14,15]. Furthermore, s1 proteins are over expressed in
tumor cells, which make them a possible target for cancer treat-
ment [16].
Previous studies in our laboratory evidenced the affinity of
compound
1 (Fig. 1) containing the tetrahydroisoquinoline-
hydantoin structure towards sigma-1 receptors [17] and a chemical
approach to this structure was developed [18]. This compound was
first optimized modifying in parallel the Tic-hydantoin structure
and the amino side chain to provide a first lead 2 (Fig. 1). The
modification of the Tic-hydantoin part consisted in substitution of
the aromatic nucleus, aromatic deletion, reduction of the size of the
central quinoline ring, reduction of the hydantoin ring and
formation of a thiohydantoin [17]. The modification of the amino
side chain took advantage of Glennon and Ablorpeddey model
[19,20]. The optimal chain was elected as n ¼ 3 and m ¼ 1, regarding
several criteria such as sigma-1 affinity (IC50 guinea pig ¼ 3.9 nM),
selectivity towards sigma-2 receptor (IC50 > 500 nM), a limited
number of free rotation bond and a very low cytotoxicity providing
a high selectivity index (ratio CC50/IC50) greater than 50,000 [21].
In this paper, we report our continuing efforts towards the
modulation of Tic-hydantoin core of the lead compound 2 (n ¼ 3,
m ¼ 1) and complete our previous study. Obviously, we decided to
evaluate different changes in the substructure (Fig. 2). We focused
Abbreviations: PHPLC, Purity determined by HPLC; TLC, Thin-layer chromatog-
raphy; tR, HPLC retention time; CDI, 1,10-Carbonyldiimidazole; tCDI, 1,10-Thio-
carbonyldiimidazole; EP, Petroleum ether; AcOEt, Ethyl acetate; Hex, n-Hexane;
Cyh, Cyclohexane; DCM, Dichloromethane; CAN, Acetonitrile; THF, Tetrahydro-
furan; IsoQ, Isoquinoline; Pyr, Pyridine; DIEA, Diisopropylethylamine; TFA, Tri-
fluoroacetic acid; PTSA, Paratoluenesulfonic acid; HBTU, 2-(1H-benzotriazol-1-yl)-
1,1,3,3-tetramethyluronium hexafluorophosphate; HOBt, 1-Hydroxybenzotriazole;
aro, Aromatic.
* Corresponding author. Tel.: þ33 3 20 87 12 19; fax: þ33 3 20 87 12 33.
E-mail address: patricia.melnyk@ibl.fr (P. Melnyk).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.10.004

M. Toussaint et al. / European Journal of Medicinal Chemistry 45 (2010) 256–263
257
O
O
N
p
N
N
n N
R
m
N
N
O
O
1
2
Fig. 1. Structure of compounds 1 and 2.
our work on the importance of the stereochemistry, the synthesis
of thiohydantoin, moderate modifications that were shown to be
effective in our last work. To evaluate the importance of the con-
strained structure, we designed an open quinoline ring. The major
changes were introduced with the deletion of the nitrogen atom,
the deletion of the central ring with the dance of the nitrogen atom
and the extension of the hydantoin ring.
orthoquinodimethane was envisaged. Though modest, the higher
yields were obtained according to the synthetic pathway described
in Scheme 4. Maleimide 13 was synthesized from amine 3 and
maleic anhydride in two steps. Intermediate orthoquinodimethane
was obtained from sultine 14 easily prepared from a,a
0-dichloro-o-
xylene [24]. Pyridine analogs 16 and 17 were synthesized according
to the same methodology starting from pyridine-2,3-dicarboxylic
anhydride for 16 and pyridine-3,4-dicarboxylic anhydride for 17
(Scheme 5). The poor yields were due to the instability of the final
compounds towards nucleophiles (methanol for instance) and the
difficulty to take them out of silica.
2. Chemistry
N-methyl-N-benzyl-1,3-diamine 3 was synthesized according to
our previously described procedure [21,22]. Slightly unstable and
difficult to purify, it is conserved as its Boc protected form 4. The
synthesis of enantiomer compound 5 follows the same synthetic
strategy as compound 2 and is described in Scheme 1. The starting
material was commercial Boc-1,2,3,4-tetrahydroisoquinoline-3-
carboxylic acid 6 (Boc-D-Tic-OH) whose secondary amine function
was protected using Boc2O. Amine 3 was preliminary deprotected
by a TFA/CH2Cl2 1:1 treatment and followed by a large excess of
DIEA (15 equiv), then coupled with acid 6 using HOBt/EDCI acti-
vation and an excess of DIEA to yield intermediate 7. After depro-
tection of the secondary amino group, evaporation, and addition of
THF and DIEA, 1,10-carbonyldiimidazole (CDI) was added to yield
hydantoin 5. Both enantiomers are chemically and enantiomeri-
cally stable. The enantiomeric purity of both enantiomers was
evaluated higher than 98% (chiral HPLC). The optical rotation of the
Quinazolinedione analogs 23–25 were synthesized in two steps
starting from isatoic anhydrides (Scheme 6). These latter
compounds were either commercially available or synthesized
according to Carter et al. [25] (compounds 18, 19).
3. Biological results and discussion
All the compounds were evaluated in binding assays on human
cerebral cortex s1 receptor using haloperidol as reference
compound [26]. For compounds showing high s1 affinity, binding
assays were also performed on rat s2 receptor [27]. The specific
ligand binding to the receptors is defined as the difference between
the total binding and the non-specific binding determined in the
presence of an excess of unlabelled ligand. The biochemical results
are presented as IC50 value, concentration causing a half-maximal
inhibition of control specific binding (Table 1) [28].
two compounds are [
a
] ¼ þ132.0 at 28 ꢀC for compound 2 and
[
a
] ¼ ꢁ132.1 at 28 ꢀC for 5. Preliminary study showed that those
The lead compound 2 has a high affinity for
s receptors and
compounds were stable under acidic or neutral solution for more
than 48 h.
good s1 versus s2 selectivity. The configuration of the asymmet-
rically substituted carbon seems to have an interesting influence
on the affinity for s1 receptor resulting in a slight increase for
the (R)-enantiomer 5, while the selectivity versus s2 receptor is
preserved.
Replacement of urea by thiourea in compound 8 increased the
affinity, as we already described [17] but in this case the results
have to take into account the chemical and enantiomeric unsta-
bility of the compound. Moreover, thiourea-containing compounds
often show many adverse reactions, while in many cases the cor-
responding urea compounds do not cause similar toxicity [29].
Concerning the modifications of the Tic core, the opening of the
isoquinoline ring in amine 11 causes a dramatic loss of affinity,
highlightening the importance of a constrained structure whereas
the elimination of the isoquinoline nitrogen in compound 15
results in a 6.5-fold decrease in affinity.
Synthesis of thiohydantoin analog 8 was similar, starting from L-
Tic-OH 9. The cyclisation step was performed using thio-
carbonyldiimidazole (Scheme 2). This thiohydantoin revealed a low
chemical stability. Furthermore, HPLC experiments showed
a complete racemisation of the compound. After preparative
separation of both enantiomers, each compound showed
a complete racemisation in ethanol after 30 min at 50 ꢀC. Further
details of the racemisation process will be published elsewhere.
The synthesis of analog 11 (Scheme 3) necessitates the prepa-
ration of N-Boc-N-methylphenylalanine 12 according to published
method [23].
In order to obtain the analog compound 15 without the quino-
line nitrogen, a Diels Alder cycloaddition of maleimide 13 and
stereochemistry
quinoline
The replacement of the Tic core by a more polar pyridine
nucleus core provides complete loss of s1 affinity for compounds 16
and 17, which is consistent with Glennon’s model.
O
replacement
As our lead compound 2 does not fit exactly with this model
because of a too big distance between hydrophobic Tic substructure
and central nitrogen atom, the replacement of Tic-hydantoin
structure by quinazolinedione heterocycles should be of interest. In
our case, non polar substituents were introduced on the aromatic
ring providing in all examples an important decrease of affinity at
least 10-fold.
N
N
N
nitrogen dance
O
ring opening
hydantoin
modification
Fig. 2. Pharmacomodulations around the Tic-hydantoin core.

258
M. Toussaint et al. / European Journal of Medicinal Chemistry 45 (2010) 256–263
RHN
N
4 R=Boc
3 R=H
a
O
O
b
N
H
N
a, c
O
N
N
NBoc
N
OH
5
O
7
NBoc
6
Reagents: (a) i) TFA/CH2Cl2 1:1, rt, 30 min, (ii) DIEA 15 eq., THF, rt, 15 min; (b) HOBt 1.2
eq., HBTU 1.2 eq., CH2Cl2, rt, 12 h; (c) CDI 3 eq., THF, reflux, 12 h
Scheme 1. Synthesis of enantiomer 5.
4. Conclusion
equipped with a UV detector set at 254 nm. Compounds were
dissolved in Tampon B or MeOH and injected through a 50 L loop.
m
Sigma-1 receptors are associated with
a
lot of neuro-
The following eluent systems were used: Tampon A (H2O/TFA,
100:0.05) and Tampon B (CH3CN/H2O/TFA, 80:20:0.05). HPLC
retention times (HPLC tR) were obtained, at a flow rate of 1 mL/min,
using the following conditions: for the 10 min method: a gradient
run from 100% eluent A during 30 s, then to 100% eluent B over the
next 8 min and for the 40 min method: a gradient run from 100%
eluent A during 1 min, then to 100% eluent B over the next 30 min.
Chiral chromatography was carried out on a Chiralpak AD (tris-2,5-
psychiological disorders and the identification of potent ligands is
of great interest. In the past, we identified a high affinity sigma-1
ligands related to the Tic-hydantoin structure and optimized the
amino side chain. In this work, we described our efforts to modu-
late this substructure and the synthesis of about 9 new compounds.
All of them showed a lower affinity or even no affinity for this
receptor. Our work underlines the importance of the Tic-hydantoin
core for affinity.
dimethylphenylcarbamate, 250 ꢂ 4.6 mm I.D.; 10
mm; Daicel
Chemical Industries, Baker, France) using a gradient Waters 600E
metering pump model equipped with a Waters 996 photodiode
array spectrophotometer (Waters, St Quentin-en-Yvelines, France).
Chromatographic data were collected and processed on a computer
5. Materials and methods
5.1. Chemistry
running Millennium 2010. The sample loop was 20
7125 injector). The column eluate was monitored at 206 nm and
¨ns 2 and 5, chiral separation was performed at
mL (Rheodyne
All reactions were monitored by thin-layer chromatography
carried out on 0.2 mm E. Merck silica gel plates (60F-254) using UV
light as a visualizing agent. Chromatography was undertaken using
silica gel 60 (230–400 mesh ASTM) from Macherey-Nagel. Thin
layer chromatography (TLC) was performed using silica gel from
Merck, from which the compounds were extracted by the following
solvent system: CH2Cl2/MeOH/NH4OH, 80:20:1. NMR spectra were
266 nm. For hydantoı
40 ꢀC using an isocratic mobile phase of n-hexane/ethanol (40:60,
¨n 8, chiral sepa-
v/v) at a flow rate of 1 mL/min. For thiohydantoı
ration was performed at 20 ꢀC using an isocratic mobile phase of n-
hexane/2-propanol (90:10, v/v) at a flow rate of 1 mL/min. The peak
of the solvent front was considered to be equal to the dead time (t0)
and was about 4.50 min. Compounds were chromatographed by
dissolving them in ethanol to a concentration of 0.5 mM and passed
obtained using a Bruker 300 MHz spectrometer, chemical shifts (d)
were expressed in ppm relative toTMS used as an internal standard.
The attributions of the carbons are deduced after 2D experiments
have been performed. Mass spectra were recorded on a MALDI-TOF
Voyager-DE STR (Applied Biosystems, Palo Alto CA) with a trihy-
droxy-acetophenone matrix. The purity of final compounds was
verified by two types of high pressure liquid chromatography
(HPLC) columns: C18 Deltapak (C18N) and C4 Interchrom UP5WC4-
25QS (C4). Analytical HPLC was performed on a Shimadzu system
through a 0.45 mm membrane filter prior to loading the column.
The optical rotation of methanolic solutions (concentration 1 mg/
mL) using Na D line (589 nm) was obtained using a polarimeter
(Perkin Elmer Life and Analytical Systems, Boston, MA). The volume
of the cell and the optical path were 1 mL and 10 cm, respectively.
Measurements were performed at 28 ꢀC. Reagents were obtained
from Acros, Aldrich, Lancaster, Novabiochem and Avocado.
a
4
3
O
O
b
N
H
N
a, c
O
N
N
NBoc
N
OH
8
S
10
NBoc
9
Reagents: (a) i) TFA/CH2Cl2 1:1, rt, 30 min, (ii) DIEA 15 eq., THF, rt, 15 min; (b) HOBt 1.2
eq., HBTU 1.2 eq., CH2Cl2, rt, 12 h; (c) tCDI 3 eq., THF, reflux, 12 h.
Scheme 2. Synthesis of thiohydantoin 8.

M. Toussaint et al. / European Journal of Medicinal Chemistry 45 (2010) 256–263
259
a
4
3
O
O
b
N
H
N
a, c
O
N
N
NBoc
N
OH
11
O
13
NBoc
12
Reagents: (a) i) TFA/CH2Cl2 1:1, rt, 30 min, (ii) DIEA 15 eq., THF, rt, 15 min; (b) HOBt 1.2
eq., HBTU 1.2 eq., CH2Cl2, rt, 12 h; (c) CDI 3 eq., THF, reflux, 24 h.
Scheme 3. Synthesis of compound 11.
5.1.1. (R)-3-[3-(Benzyl-methyl-amino)-propylcarbamoyl]-3,4-
dihydro-1H-isoquinoline-2-carboxylic acid tert-butyl ester 7
A solution of compound 4 (500 mg, 1.79 mmol) in DCM/TFA (10/
10 mL), was stirred at room temperature during 30 min. The
solvent was then evaporated to provide free amine 3. A solution of
solvent was evaporated in vacuo and the residue taken up in THF
(20 mL). DIEA (3.15 mL, 15 equiv) was added. After stirring the
mixture at room temperature for 10 min, CDI (588 mg, 3 equiv) was
added and stirring was continued for 12 h at reflux. The solvent was
evaporated in vacuo and the residue taken up in ethyl acetate
(30 mL). The organic layer was washed with a solution of NaHCO3
5% (2 ꢂ 15 mL), brine (1 ꢂ15 mL) and then dried over MgSO4. The
solvent was evaporated. The residue was purified by TLC (DCM/
methanol/NH4OH, 9/1/0.2) to yield expected compound 5 as yellow
oil (379 mg, 87% yield). TLC: Rf 0.5 (DCM/methanol/NH4OH, 9/1/
0.2). HPLC (C18, 10 min) PHPLC 99%, tR 4.5 min; HPLC (C4, 40 min)
PHPLC 99%, tR 14.4 min. 1H NMR (CDCl3): 1.89 (2H, quint, CH2,
J ¼ 7 Hz), 2.21 (3H, s, NCH3), 2.45 (2H, t, NCH2, J ¼ 7 Hz), 2.78 (1H,
Boc-D-1,2,3,4-tetrahydroisoquinoleine-3-carboxylic acid
6
(R)
(Boc-D-TicOH; 496 mg, 1 equiv), HOBt (290 mg, 1.2 equiv), HBTU
(814 mg, 1.2 equiv) in DCM (30 mL) was stirred at room tempera-
ture during 15 min. Then, a solution of free amine 3 and DIEA
(4.7 mL, 15 equiv) in DCM (15 mL) was added and the reaction was
stirred at room temperature overnight. The organic layer was
washed with
a
solution of NaHCO3 5% (1 ꢂ 20 mL), brine
(1 ꢂ 20 mL) and then dried over MgSO4. The residue was purified by
TLC (DCM/methanol, 9/1) to yield expected compound 7 as yellow
oil (485 mg, 62% yield). TLC: Rf 0.6 (DCM/methanol, 9/1). HPLC
(C18-10 min) PHPLC 90%, tR 5.3 min. 1H NMR (CDCl3): 1.4–1.5 (11H,
m, C(CH3)3 and H2), 1.9–2.0 (3H, m, NCH3), 2.2–2.3 (2H, m, H1), 2.9–
3.0 (2H, m, H3), 3.3–3.4 (4H, m, isoQ-H4 and NCH2Ph), 4.3–4.6 (3H,
m, isoQ-H1 and isoQ-H3), 7.2–7.3 (9H, m, Haro); 13C NMR (CDCl3)
25.9 (CH2), 28.5 (C(CH3)3), 31.8 (isoQ-C4), 37.3 (C1), 45.0 (isoQ-C1),
50.5 (NCH3), 54.9 (C3), 56.8 (isoQ-C3), 62.1 (NCH2Ph), 126.3–126.9–
127.6–128.6–129.4 (Caro); Formula C26H36N3O3, molecular weight
437.6, m/z 438.3 [M þ H]þ.
dd, H10
,
2J ¼ 16 Hz, J10ꢁ10a ¼ 12 Hz), 3.25 (1H, dd, H10
,
2J ¼ 16 Hz,
J10ꢁ10a ¼ 5 Hz), 3.49 (2H, s, NCH2Ph), 3.64 (2H, t, NCH2, J ¼ 7 Hz),
4.05 (1H, dd, H10a, J10aꢁ10 ¼ 12 Hz, J10aꢁ10a ¼ 5 Hz), 4.42 (1H, d, H5,
2J ¼ 17 Hz), 5.03 (1H, d, H5, 2J ¼ 17 Hz), 7.1–7.3 (m, 9H, Haro); 13C
NMR (CDCl3) 25.8 (CH2), 30.9 (C10), 37.1 (NCH2), 41.4 (C5), 41.8
(NCH3), 54.3 (NCH2), 54.3 (C10a), 62.1 (CH2Ph), 126.6–128.5–129.3
(Caro); Formula C22H25N3O2, molecular weight 363.4, m/z 364.1
[M þ H]þ; ½a 2D8
¼ þ132.0 (MeOH, 1.0 mg/mL).
ꢃ
5.1.3. (S)-3-[3-(Benzyl(methyl)amino)propylcarbamoyl]-3,4-
dihydro-1H-isoquinoline-2-carboxylic acid tert-butyl ester 10
A solution of compound 4 (411 mg, 1.48 mmol) in DCM/TFA
(10/10 mL) was stirred at room temperature during 30 min. The
solvent was then evaporated to provide free amine 3. A solution
of Boc-L-1,2,3,4-tetrahydroisoquinoleine-3-carboxylic acid 9 (S)
5.1.2. (R)-2-[3-(Benzyl(methyl)amino)propyl]-10,10a-dihydro-5H-
imidazo[1,5-b]isoquinoline-1,3-dione 5
A solution of compound 7 (528 mg, 1.2 mmol) in DCM/TFA
(2/2 mL) was stirred at room temperature during 30 min. The
O
O
O
N
3
b
N
H
O
N
O
a
N
O
O
OH
12
13
O
O
Cl
13
c
d
S
N
O
N
Cl
O
14
15
Reagents: (a) DIEA (2 eq.), THF, rt, 12h; (b) (acetic anhydride/acetic acid) (2/1), 55°C,
overnight; (c) rongalite (2 eq.), TBAB (0.2 eq.), DMF, rt overnight; (d) 13 (1.2 eq.), toluene,
reflux, 24h.
Scheme 4. Synthesis of compound 15.

260
M. Toussaint et al. / European Journal of Medicinal Chemistry 45 (2010) 256–263
Table 1
O
O
Binding affinities for target compounds on s1 and s2 sites.
3
N
O
Compound
IC50 s1 (nM)
IC50 s2 (nM)
Ratio s2/s1
X
X
a
Y
Y
Haloperidol
2
5
8
11
15
16
17
23
24
25
23.0
8.7
13.7
4.5
>200
57
>200
>200
116
230
650
1000
10
75
76
–
–
–
–
–
–
–
N
O
O
–
–
–
–
–
–
–
–
16 X = CH, Y = N
17 X = N, Y = CH
Reagents: (a) PTSA (0.05 eq.), toluene, reflux, overnight.
>200
103
Scheme 5. Synthesis of compounds 16 and 17.
–
Mean IC50 values for 2–3 independent experiments are shown with less than 10%
deviation
(Boc-L-TicOH; 411 mg, 1 equiv), HOBt (240 mg, 1.2 equiv), HBTU
(673 mg, 1.2 equiv) in DCM (30 mL) was stirred at room tempera-
ture during 15 min. Then, a solution of free amine 3 and DIEA
(3.9 mL, 15 equiv) in DCM (15 mL) was added and the reaction was
stirred overnight at room temperature. The organic layer was
90%, tR 19.4 min. 1H NMR (CDCl3): 1.94 (2H, quint, CH2, J ¼ 7 Hz),
2.19 (3H, s, NCH3), 2.46 (2H, t, NCH2, J ¼ 7 Hz), 2.81 (1H, dd, H10
,
2J ¼ 15 Hz, J10ꢁ10a ¼ 12 Hz), 3.26 (1H, dd, H10
,
2J ¼ 15 Hz, J10–
washed with
a
solution of NaHCO3 5% (1 ꢂ 20 mL), brine
¼ 5 Hz), 3.48 (2H, s, NCH2Ph), 3.96 (2H, t, NCH2, J ¼ 7 Hz), 4.12
10a
(1 ꢂ20 mL) and then dried over MgSO4. The residue was purified by
TLC (DCM/methanol, 9/1) to yield expected compound 10 as yellow
oil (576 mg, 89% yield). TLC: Rf 0.6 (DCM/methanol, 9/1). HPLC (C18-
10 min) PHPLC 90%, tR 5.3 min. 1H NMR (CDCl3): 1.4–1.5 (11H, m,
C(CH3)3 and H2), 1.9–2.0 (3H, m, NCH3), 2.2–2.3 (2H, m, H1), 2.9–3.0
(2H, m, H3), 3.3–3.4 (4H, m, isoQ-H4 and NCH2Ph), 4.3–4.6 (3H, m,
isoQ-H1 and isoQ-H3), 7.2–7.3 (9H, m, Haro); 13C NMR (CDCl3) 25.9
(CH2), 28.5 (C(CH3)3), 31.8 (isoQ-C4), 37.3 (C1), 45.0 (isoQ-C1), 50.5
(NCH3), 54.9 (C3), 56.8 (isoQ-C3), 62.1 (NCH2Ph), 126.3–126.9–
127.6–128.6–129.4 (Caro); Formula C26H36N3O3, molecular weight
437.6, m/z 438.3 [M þ H]þ.
(1H, dd, H10a, J10aꢁ10 ¼ 12 Hz, J10aꢁ10a ¼ 5 Hz), 4.60 (1H, d, H5,
2J ¼ 17 Hz), 5.48 (1H, d, H5, 2J ¼ 17 Hz), 7.1–7.3 (m, 9H, Haro); 13C
NMR (CDCl3) 25.5 (CH2), 30.9 (C10), 40.2 (NCH2), 42.2 (NCH3); 46.1
(C5); 54.8 (NCH2); 57.5 (C10a); 62.4 (CH2Ph); 125.3–126.9–127.0–
127.5–127.7–128.3–129.2–129.4 (Caro); Formula C22H25N3OS,
molecular weight 379.5, m/z 380.2 [M þ H]þ.
5.1.5. {1-[3-(Benzyl(methyl)amino)propylcarbamoyl]-2-
phenylethyl}methylcarbamic acid tert-butyl ester 13
A solution of compound 4 (114 mg, 0.41 mmol) in DCM/TFA (2/
2 mL), was stirred at room temperature during 30 min. The solvent
was then evaporated to provide free amine 3. A solution of N-tert-
butoxycarbonyl-N-methylphenylalanine 12 (S) (114 mg, 1 equiv),
HOBt (66 mg, 1.2 equiv), HBTU (186 mg, 1.2 equiv) in DCM (8 mL)
was stirred during 15 min. Then, a solution of free amine 3 and DIEA
(1.1 mL, 15 equiv) in DCM (8 mL) was added and the reaction was
stirred overnight at room temperature. The organic layer was
washed with a solution of NaHCO3 5% (2 ꢂ10 mL), brine (1 ꢂ10 mL)
and then dried over MgSO4. The residue was purified by TLC (DCM/
methanol, 95/5) to yield expected compound 13 as white powder
(50 mg, 28% yield). TLC: Rf 0.3 (DCM/methanol, 95/5). HPLC (C18-
10 min) PHPLC 96%, tR 2.5 min. 1H NMR (CDCl3): 1.30 (9H, s, C(CH3)3),
1.60 (2H, m, CH2), 2.16 (3H, s, NCH3), 2.35 (2H, t, NCH2, J ¼ 6 Hz),
2.68 (3H, s, NBocCH3), 2.72 (2H, m, CH2Ph), 3.22 (2H, m, NHCH2),
4.43 (2H, s, NCH2Ph), 4.67 (1H, m, CO–CH), 7.20–7.30 (m, 10H, Haro);
13C NMR (CDCl3) 27.4 (CH2), 29.0 (C(CH3)3), 31.7 (CH2Ph), 35.2
(NHCH2), 39.8 (NCH3), 54.5 (NCH2), 62.2 (NCH2Ph), 62.5 (COCH),
5.1.4. 2-[3-(Benzyl(methyl)amino)propyl]-3-thioxo-2,3,10,10a-
tetrahydro-5H-imidazo[1,5-b] isoquinoline-1-one 8
A solution of compound 10 (366 mg, 0.84 mmol) in DCM/TFA
(4/4 mL) was stirred at room temperature during 30 min. The
solvent was evaporated in vacuo and the residue taken up in THF
(20 mL). DIEA (2.18 mL, 15 equiv) was added. After stirring the
mixture at room temperature for 10 min, tCDI (447 mg, 3 equiv)
was added and stirring was continued overnight at reflux. The
solvent was evaporated in vacuo and the residue taken up in ethyl
acetate (30 mL). The organic layer was washed with a solution of
NaHCO3 5% (2 ꢂ15 mL), brine (1 ꢂ15 mL) and then dried over
MgSO4. The solvent was evaporated. The residue was purified by
TLC (DCM/methanol/NH4OH, 9/1/0.1) to yield expected compound
8 as yellow oil (83 mg, 26% yield). TLC: Rf 0.4 (DCM/methanol, 95/5).
HPLC (C18, 10 min) PHPLC 90%, tR 5.3 min; HPLC (C4, 40 min) PHPLC
H
H
N
O
O
NH2
N
O
a
b
H
N
N
O
R
R
R
O
O
N
N
20 R = H
21 R = 5-Cl
22 R = 5-I
23
24
25
18 R = 5-Cl
19 R = 5-I
Reagents: (a) 3 (1.3 eq.), DIEA (5 eq.), DCM, rt, overnight; (b) DIEA (5 eq.), DCM, rt, 10
min, then CDI (3 eq.), THF, reflux, overnight.
Scheme 6. Synthesis of compounds 23–25.

M. Toussaint et al. / European Journal of Medicinal Chemistry 45 (2010) 256–263
261
127.2–127.5–128.4–128.8–129.3–129.7 (Caro); Formula C26H37N3O3,
colorless oil (132 mg, 62% yield). TLC: Rf 0.5 (DCM/methanol, 95/5).
HPLC (C18, 10 min) PHPLC 98%, tR 5.0 min; HPLC (C4, 40 min) PHPLC
98%, tR 11.2 min. 1H NMR (CDCl3): 1.33 (2H, quint, CH2, J ¼ 7 Hz),
1.95 (3H, s, NCH3), 1.96 (2H, t, NCH2, J ¼ 7 Hz), 2.88 (2H, m, CH,
2J ¼ 14 Hz), 3.13 (2H, m, CH, 2J ¼ 14 Hz), 3.1–3.2 (2H, m, CHCO),
3.24 (2H, s, NCH2Ph), 3.34 (2H, t, NCH2, J ¼ 7 Hz), 7.1–7.3 (9H, m,
Haro); 13C NMR (CDCl3) 24.8 (CH2), 29.6 (CH), 36.8 (NCH2), 39.8
(CHCO), 41.4 (NCH3), 54.0 (NCH2), 61.2 (NCH2Ph), 126.8–127.2–
127.7–128.0–128.9 (Caro); Formula C23H26N2O2, molecular weight
362.4, m/z 363.2 [M þ H]þ.
molecular weight 439.6, m/z 440.5 [M þ H]þ.
5.1.6. (S)-5-Benzyl-3-[3-(benzyl(methyl)amino)propyl]-1-
methylimidazolidine-2,4-dione 11
A solution of compound 13 (324 mg, 0.96 mmol) in DCM/TFA
(4/4 mL) was stirred at room temperature during 30 min. The
solvent was evaporated in vacuo and the residue taken up in THF
(15 mL). DIEA (2.49 mL, 15 equiv) was added. After stirring the
mixture at room temperature for 10 min, CDI (156 mg, 3 equiv)
was added and stirring was continued overnight at reflux. The
solvent was evaporated in vacuo and the residue taken up in ethyl
acetate (40 mL). The organic layer was washed with a solution of
NaHCO3 5% (20 mL), brine (20 mL) and then dried over MgSO4. The
solvent was evaporated. The residue was purified by TLC (AcOEt/
methanol/NH4OH, 9/1/0.05) to yield expected compound 11 as
yellow oil (126 mg, 36% yield). TLC: Rf 0.4 (AcOEt/methanol, 96/4).
HPLC (C18, 10 min) PHPLC 90%, tR 4.3 min; HPLC (C4, 40 min) PHPLC
97%, tR 21.8 min. 1H NMR (CDCl3): 1.50 (2H, quint, CH2, J ¼ 7 Hz),
2.11 (3H, s, NCH3), 2.24 (2H, t, NCH2, J ¼ 7 Hz), 2.94 (3H, s, NCH3),
3.15 (2H, m, CH2Ph), 3.42 (m, 4H, NCH2, NCH2Ph), 4.07 (1H, t,
COCH, J ¼ 4 Hz), 7.22 (m, 5H, Haro); 13C NMR (CDCl3) 26.5 (CH2),
29.8 (NCH3), 35.1 (CH2Ph), 37.1 (NCH2), 41.8 (NCH3), 54.5 (NCH2),
62.2 (NCH2Ph), 62.5 (CHCO), 127.2–127.5–128.4–128.8–129.3–
129.7 (Caro); Formula C22H27N3O2, molecular weight 365.4, m/z
366.3 [M þ H]þ.
5.1.10. 6-[3-(Benzyl(methyl)amino)propyl]pyrrolo[3,4-b]pyridine-
5,7-dione 16
To a solution of amine 3 (135 mg, 0.76 mmol) in toluene
(5 mL) in a Dean–Stark apparatus was added PTSA (9.5 mg,
0.05 equiv) and pyridine-2,3-dicarboxylic anhydride (113 mg,
1 equiv). Stirring was continued at 110 ꢀC overnight. The organic
layer was washed with a solution of NaHCO3 5% (10 mL), brine
(10 mL) and then dried over MgSO4. The solvent was evaporated.
The residue was purified by TLC (AcOEt/Cyh, 9/1) to yield
expected compound 16 as colorless oil (68 mg, 29% yield). TLC: Rf
0.5 (AcOEt/Cyh, 9/1). HPLC (C18, 10 min) PHPLC 99%, tR 3.3 min;
HPLC (C4, 40 min) PHPLC 99%, tR 9.8 min. 1H NMR (CDCl3): 1.99
(2H, quint, CH2, J ¼ 7 Hz), 2.27 (3H, s, NCH3), 2.60 (2H, t, NCH2,
J ¼ 7 Hz), 3.59 (2H, s, NCH2Ph), 3.83 (2H, t, NCH2, J ¼ 7 Hz), 7.10–
7.30 (9H, m, Haro), 7.61 (1H, dd, Pyr-H3, J ¼ 7 Hz, J ¼ 5 Hz), 8.15
(1H, dd, Pyr-H4, J ¼ 7 Hz, J ¼ 1 Hz), 8.97 (1H, dd, Pyr-H2, J ¼ 5 Hz,
J ¼ 1 Hz); 13C NMR (CDCl3) 25.8 (CH2), 36.6 (NCH2), 41.5 (NCH3),
54.4 (NCH2), 61.9 (NCH2Ph), 127.4 (Pyr-C3), 128.6–128.8–129.6
(Caro), 131.3 (Pyr-C4), 155.3 (Pyr-C2); Formula C18H19N3O2,
molecular weight 309.3, m/z 310.1 [M þ H]þ.
5.1.7. (Z)-3-[3-(Benzyl(methyl)amino)propylcarbamoyl]acrylic
acid 12
A solution of amine 3 (373 mg, 0.96 mmol) and DIEA (0,22 mL,
2 equiv) in THF (2 mL) was stirred during 10 min. Maleic anhydride
(113 mg, 1,2 equiv) was added and stirring was continued overnight
at room temperature. The solvent was evaporated. The residue was
purified by flash chromatography (Acetone/NH4OH, 9/1) to yield
expected compound 12 as yellow oil (228 mg, 86% yield). TLC: Rf 0.3
(Acetone/NH4OH, 9/1). HPLC (C18, 10 min) PHPLC 97%, tR 2.9 min. 1H
NMR (CDCl3): 1.86 (2H, quint, CH2, J ¼ 7 Hz), 2.17 (3H, s, NCH3), 3.35
(2H, t, NCH2, J ¼ 7 Hz), 3.75 (2H, s, NCH2Ph), 5.88 (2H, d, CH–CONH,
J ¼ 12 Hz), 6.25 (2H, d, CH–COOH, J ¼ 12 Hz), 7.3–7.4 (5H, m, Haro),
10.11 (1H, s, OH); 13C NMR (CDCl3) 27.2 (CH2), 32.2 (NCH3), 40.6
(NCH2), 56.9 (NCH2), 63.5 (NCH2Ph), 130.9–131.3–132.4 (CH, Caro),
138.2 (CH); Formula C15H20N2O3, molecular weight 276.3, m/z 277.1
[M þ H]þ.
5.1.11. 2-[3-(Benzyl(methyl)amino)propyl]pyrrolo[3,4-c]pyridine-
1,3-dione 17
To a solution of amine 3 (129 mg, 0.73 mmol) in toluene (5 mL)
in a Dean–Stark apparatus was added PTSA (6.9 mg, 0.05 equiv) and
pyridine-3,4-dicarboxylic anhydride (108 mg, 1 equiv). Stirring was
continued at 110 ꢀC overnight. The organic layer was washed with
a solution of NaHCO3 5% (10 mL), brine (10 mL) and then dried over
MgSO4. The solvent was evaporated. The residue was purified by
TLC (AcOEt/Cyh, 9/1) to yield expected compound 16 as colorless oil
(7 mg, 3% yield). TLC: Rf 0.7 (DCM/methanol, 95/5). HPLC (C18,
10 min) PHPLC 95%, tR 3.3 min; HPLC (C4, 40 min) PHPLC 96%, tR
9.4 min. 1H NMR (CDCl3): 1.89 (2H, quint, CH2, J ¼ 7 Hz), 2.16 (3H, s,
NCH3), 2.45 (2H, t, NCH2, J ¼ 7 Hz), 3.46 (2H, s, NCH2Ph), 3.79 (2H, t,
NCH2, J ¼ 7 Hz), 7.10–7.30 (9H, m, Haro), 7.74 (1H, dd, Pyr-H4,
J ¼ 5 Hz, J ¼ 1 Hz), 9.06 (1H, d, Pyr-H3, J ¼ 5 Hz), 9.13 (1H, d, Pyr-H1,
J ¼ 1 Hz); 13C NMR (CDCl3) 26.4 (CH2), 37.1 (NCH2), 42.2 (NCH3),
54.9 (NCH2), 62.6 (NCH2Ph), 117.3 (Pyr-C4), 127.3–128.5–129.3
(Caro), 144.9 (Pyr-C1), 155.9 (Pyr-C3); Formula C18H19N3O2, molec-
ular weight 309.3, m/z 310.1 [M þ H]þ.
5.1.8. 1-[3-(Benzyl(methyl)amino)propyl]pyrrole-2,5-dione 13
A solution of acid 13 (218 mg, 0.79 mmol) in glacial acetic acid/
acetic anhydride (2/1 mL) was stirred under nitrogen atmosphere
at 50 ꢀC for 24 h. The solvent was evaporated. The residue was
purified by TLC (DCM/methanol, 95/5) to yield expected compound
13 as yellow oil (82 mg, 40% yield). TLC: Rf 0.3 (DCM/methanol, 95/
5). HPLC (C18, 10 min) PHPLC 95%, tR 3.4 min. 1H NMR (CDCl3): 1.79
(2H, quint, CH2, J ¼ 7 Hz), 2.16 (3H, s, NCH3), 2.39 (2H, t, NCH2,
J ¼ 7 Hz), 3.46 (2H, s, NCH2Ph), 3.58 (2H, t, NCH2, J ¼ 7 Hz), 6.65 (2H,
s, CH), 7.2–7.3 (5H, m, Haro); 13C NMR (CDCl3) 26.4 (CH2), 36.4
(NCH2), 42.1 (NCH3), 54.7 (NCH2), 62.5 (NCH2Ph), 127.2–128.4–
129.3 (Caro), 134,3 (CH); Formula C15H18N2O2, molecular weight
258.3, m/z 259.2 [M þ H]þ.
5.1.12. Opening of isatoic anhydrides, general protocol A
A solution of isatoic anhydride (0.83 mmol), amine 3 (1.3 equiv)
and DIEA (5 equiv) in DCM (5 mL) was stirred overnight at room
temperature under inert atmosphere. The solvent was evaporated.
The residue was purified by flash chromatography (DCM/methanol,
98/2) to yield the desired compound.
5.1.9. 2-[3-(Benzyl(methyl)amino)propyl]-3a,4,9,9a-
tetrahydrobenzo[f]isoindole-1,3-dione 15
5.1.13. 2-Amino-N-[3-(benzyl(methyl)amino)propyl]benzamide 20
White solid (84% yield). TLC: Rf 0.4 (DCM/methanol, 9/1). HPLC
(C18, 10 min) PHPLC 96%, tR 3.5 min. 1H NMR (CDCl3): 1.79 (2H,
quint, CH2, J ¼ 7 Hz), 2.24 (3H, s, NCH3), 2.57 (2H, t, NCH2, J ¼ 7 Hz),
3.40–3.50 (4H, m, NHCH2, NCH2Ph), 5.62 (2H, large s, NH2), 6.50
(1H, td, H5, J ¼ 8 Hz, J ¼ 1.5 Hz), 6.66 (1H, dd, H3, J ¼ 8 Hz,
To a solution of sultin 14 (100 mg, 0.59 mmol) in toluene
(10 mL) was added maleimide 13 (183 mg, 1.2 equiv). Stirring was
continued under nitrogen atmosphere at 50 ꢀC for 24 h. The
solvent was evaporated. The residue was purified by TLC (DCM/
AcOEt/methanol, 6/4.5/0.5) to yield expected compound 15 as

262
M. Toussaint et al. / European Journal of Medicinal Chemistry 45 (2010) 256–263
J ¼ 1.5 Hz), 7.12 (1H, dd, H6, J ¼ 8 Hz, J ¼ 1.5 Hz), 7.17 (1H, td, H2,
J ¼ 8 Hz, J ¼ 1.5 Hz), 7.2–7.3 (5H, m, Haro); 13C NMR (CDCl3) 27.6
(CH2), 42.1 (NHCH2), 44.0 (NCH3), 59.3 (NCH2), 65.5 (NCH2Ph),
118.7 (C5), 119.4 (C3), 129.4 (Caro), 129.5 (C6), 130.6–131.5 (Caro),
134.1 (C2); Formula C18H23N3O, molecular weight 297.4, m/z 298.2
[M þ H]þ.
(3H, s, NCH3), 2.32 (2H, t, NCH2, J ¼ 7 Hz), 3.36 (2H, s, NCH2Ph), 3.86
(2H, t, NCH2, J ¼ 7 Hz), 7.00–7.30 (6H, m, H8, Haro); 7.62 (1H, dd, H7,
J ¼ 8.5 Hz, J ¼ 2.5 Hz), 7.78 (1H, d, H5, J ¼ 2.5 Hz), 11.47 (1H, large s,
NH); 13C NMR (DMSO-d6) 25.6 (CH2), 39.4 (NCH2), 42.7 (NCH3), 55.1
(NCH2), 62.1 (NCH2Ph), 118.0 (C8), 127.0 (C5), 127.5–128.8–129.4
(Caro), 135.5 (C7); Formula C19H20ClN3O2, molecular weight 357.1–
359.1, m/z 358.2–360.2 [M þ H]þ.
5.1.14. 2-Amino-N-[3-(benzyl(methyl)amino)propyl]-5-
chlorobenzamide 21
5.1.19. 3-[3-(Benzyl(methyl)amino)propyl]-6-iodo-1H-quinazoline-
2,4-dione 25
Colorless oil (89% yield). TLC: Rf 0.5 (DCM/methanol, 9/1).
HPLC (C18, 10 min) PHPLC 98%, tR 4.2 min. 1H NMR (CDCl3): 1.68
(2H, quint, CH2, J ¼ 7 Hz), 2.19 (3H, s, NCH3), 2.46 (2H, t, NCH2,
J ¼ 7 Hz), 3.35 (2H, q, NHCH2, J ¼ 7 Hz), 3.43 (2H, s, NCH2Ph), 5.54
(2H, large s, NH2), 6.50 (1H, d, H3, J ¼ 8.5 Hz), 7.00 (1H, dd, H4,
J ¼ 8.5 Hz, J ¼ 2.5 Hz), 7.08 (1H, d, H6, J ¼ 2.5 Hz), 7.10–7.20 (5H, m,
Haro), 7.98 (1H, large t, NH); 13C NMR (CDCl3) 23.9 (CH2), 38.8
(NHCH2), 40.8 (NCH3), 55.2 (NCH2), 61.7 (NCH2Ph), 117.3 (C3),
125.9 (C6), 126.3–127.3–128.0 (Caro), 130.6 (C4); Formula
C18H22ClN3O, molecular weight 331.1–333.1, m/z 332.3–334.3
[M þ H]þ.
White solid (46% yield). TLC: Rf 0.5 (DCM/methanol, 9/1). HPLC
(C18, 10 min) PHPLC 95%, tR 5.3 min; HPLC (C4, 40 min) PHPLC 93%, tR
10.6 min. 1H NMR (CDCl3): 2.08 (2H, quint, CH2, J ¼ 7 Hz), 2.36 (3H,
s, NCH3), 2.69 (2H, t, NCH2, J ¼ 7 Hz), 3.68 (2H, s, NCH2Ph), 4.28 (2H,
t, NCH2, J ¼ 7 Hz), 6.95 (1H, d, H8, J ¼ 8.5 Hz), 7.20–7.50 (5H, m,
Haro), 7.87 (1H, dd, H7, J ¼ 8.5 Hz, J ¼ 2 Hz), 8.55 (1H, d, H5, J ¼ 2 Hz),
10.45 (1H, large s, NH); 13C NMR (CDCl3) 25.5 (CH2), 39.7 (NCH2),
42.0 (NCH3), 54.9 (NCH2), 62.0 (NCH2Ph), 116.9 (C8), 126.9–128.2–
129.0 (Caro), 136.9 (C5), 143.4 (C7); Formula C19H20IN3O2, molecular
weight 449.2, m/z 450.1 [M þ H]þ.
5.1.15. 2-Amino-N-[3-(benzyl(methyl)amino)propyl]-5-
iodobenzamide 22
5.2. Binding assays to
s receptors
Brown oil (58% yield). TLC: Rf 0.5 (DCM/methanol, 9/1). HPLC
(TSK gel, 10 min) PHPLC 91%, tR 6.9 min. 1H NMR (DMSO-d6): 1.69
(2H, quint, CH2, J ¼ 7 Hz), 2.11 (3H, s, NCH3), 2.34 (2H, t, NCH2,
J ¼ 7 Hz), 3.20 (2H, q, NHCH2, J ¼ 7 Hz), 3.45 (2H, s, NCH2Ph), 6.52
(2H, large s, NH2), 6.54 (1H, d, H3, J ¼ 8.5 Hz), 7.20–7.29 (5H, m,
Haro), 7.37 (1H, dd, H4, J ¼ 8.4 Hz, J ¼ 2 Hz), 7.69 (1H, d, H6, J ¼ 2 Hz),
8.31 (1H, large t, NH); 13C NMR (DMSO-d6) 27.1 (CH2), 38.0
(NHCH2), 42.2 (NCH3), 55.1 (NCH2), 62.1 (NCH2Ph),119.6 (C3),127.5–
128.9–129.4 (Caro), 136.5 (C6), 140.2 (C4); Formula C18H22IN3O,
molecular weight 423.3, m/z 424.1 [M þ H]þ.
The s1 binding assays were performed by CEREP (Paris, France),
according to Ganapathy et al. [26] for s1 binding and Bowen et al.
[27] for s2 binding. In brief, the s1 binding assay was performed by
incubating Jurkat cell membranes (10–20 mg protein per tube)
with [3H](þ)-pentazocine (8 nM) and a range of concentrations of
test compounds, at 22 ꢀC for 2 h, in 5 mM Tris/HCl buffer, pH ¼ 7.4.
The s2 binding assay was performed by incubating rat cerebral
cortex membranes (10–20 mg protein per tube) with [3H](þ)-DTG
(5 nM), in the presence of (þ)-pentazocine (300 nM) to saturate s1
sites, and a range of concentrations of test compounds, at 22 ꢀC for
2 h, in 5 mM Tris/HCl buffer, pH ¼ 7.4. The final assay volume was
0.5 mL. Non-specific binding was defined, in both assays, as that
5.1.16. Formation of quinazolinediones, general protocol B
A
solution of 2-amidobenzamide (0.90 mmol) and DIEA
remaining in the presence of 10 mM haloperidol. The reaction was
(5 equiv) in DCM (30 mL) was stirred for 10 min at room
temperature under inert atmosphere. CDI (3 equiv) was added
and the reaction mixture was heated overnight at reflux. The
solvent was evaporated in vacuo and the residue taken up in ethyl
acetate (40 mL). The organic layer was washed with a solution of
NaHCO3 5% (20 mL), brine (20 mL) and then dried over MgSO4.
The solvent was evaporated. The residue was purified by flash
chromatography (DCM/methanol, 95/5) to yield expected
compound.
terminated by rapid filtration through Whatman GF/B filters, which
were then washed with 5 ꢂ1 mL ice-cold 0.9% NaCl (saline) solu-
tion and allowed to dry before bound radioactivity was measured
using liquid scintillation counting. The protein concentration in the
homogenates was determined using the method of Bradford [30].
Acknowledgments
´
We express our thanks to Gerard Montagne for NMR experi-
5.1.17. 3-[3-(Benzyl(methyl)amino)propyl]-1H-quinazoline-2,4-
dione 23
ments, Marie-Ange Debreu-Fontaine for her contribution in organic
´
synthesis and Herve Drobecq for MS spectra. This work was sup-
White solid (99% yield). TLC: Rf 0.3 (DCM/methanol, 9/1).
HPLC (C18, 10 min) PHPLC 98%, tR 4.0 min; HPLC (C4, 40 min) PHPLC
99%, tR 9.9 min. 1H NMR (CDCl3): 1.95 (2H, quint, CH2, J ¼ 7 Hz),
2.21 (3H, s, NCH3), 2.54 (2H, t, NCH2, J ¼ 7 Hz), 3.51 (2H, s,
NCH2Ph), 4.18 (2H, t, NCH2, J ¼ 7 Hz), 7.03 (1H, d, H8, J ¼ 8 Hz),
7.22 (1H, d, H6, J ¼ 8 Hz), 7.20–7.30 (5H, m, Haro), 7.54 (1H, d, H7,
J ¼ 8 Hz), 8.13 (1H, d, H5, J ¼ 8 Hz); 13C NMR (CDCl3) 28.0 (CH2),
42.1 (NCH2), 44.5 (NCH3), 57.4 (NCH2), 64.6 (NCH2Ph), 117.3 (C8),
125.8 (C6), 129.3–130.7–130.9 (Caro), 131.5 (C5), 137.4 (C7);
Formula C19H21N3O2, molecular weight 323.3, m/z 324.1
[M þ H]þ.
´
ported by Universite de Lille II and MILDt. M.T. was a recipient of
´
fellowships from the Region Nord-Pas de Calais and CNRS.
References
[1] W.R. Martin, C.E. Eades, J.A. Thompsom, R.E. Huppler, The effects of morphine-
and nalorphine- like drugs in the nondependent and morphine-dependent
chronic spinal dog. J. Pharmacol. Exp. Ther. 197 (1976) 517–532.
[2] R. Quirion, W.D. Bowen, Y. Itzhak, J.L. Junien, J.M. Musacchio, R.B. Rothman,
T.P. Su, S.W. Tam, D.P. Taylor, A proposal for the classification of sigma binding
sites. Trends Pharmacol. Sci. 13 (1992) 85–86.
[3] E. Aydar, C.P. Palmer, V.A. Klyachko, M.B. Jackson, The sigma receptor as
a ligand-regulated auxiliary potassium channel subunit. Neuron 34 (2002)
399–410.
[4] G. Duncan, L. Wang, Focus on molecules: the sigma-1 receptor. Exp. Eye Res.
81 (2005) 121–122.
[5] H. Yamamoto, R. Miura, T. Yamamoto, K. Shinihara, M. Watanabe, S. Okuyama,
A. Nakasato, T. Nukada, Amino acid residues in the transmembrane domain of
the type 1 sigma receptor critical for ligand binding. FEBS Lett. 445 (1999)
19–22.
5.1.18. 3-[3-(Benzyl(methyl)amino)propyl]-6-chloro-1H-
quinazoline-2,4-dione 24
White solid (73% yield). TLC: Rf 0.5 (DCM/methanol, 9/1). HPLC
(C18, 10 min) PHPLC 98%, tR 5.1 min; HPLC (C4, 40 min) PHPLC 92%, tR
10.5 min. 1H NMR (DMSO-d6): 1.69 (2H, quint, CH2, J ¼ 7 Hz), 2.01

M. Toussaint et al. / European Journal of Medicinal Chemistry 45 (2010) 256–263
263
[6] D.L. DeHaven-Hudkins, L.C. Fleissner, Competitive interactions at [3H]1,3-di(2-
tolyl)guanidine (DTG)-defined sigma recognition sites in guinea pig brain. Life
Sci. 50 (1992) 65–70.
[19] R.A. Glennon, Pharmacophore identification for sigma-1 (s1) receptor binding:
application of the "deconstruction–reconstruction–elaboration" approach.
Mini-Rev. Medic. Chem. 5 (2005) 927–940.
[7] D.H. Roh, H.W. Kim, S.Y. Yoon, H.S. Seo, Y.B. Kwon, K.W. Kim, H.J. Han, A.J. Beitz,
J.H. Lee, Intrathecal administration of sigma-1 receptor agonists facilitates
nociception: involvement of protein kinase C-dependant pathway. J. Neurosci.
Res. 86 (2008) 3644–3654.
[8] T. Maurice, R. Martin-Fardon, P. Romieu, R.R. Matsumoto, Sigma(1) (sigma(1))
receptor antagonists represent a new strategy against cocaine addiction and
toxicity. Neurosci. Biobehav. Rev. 26 (2002) 499–527.
[20] S.Y. Ablordeppey, J. Fischer, H. Law, R.A. Glennon, Probing the proposed
phenyl-A region of the sigma-1 receptor. Bioorg. Med. Chem. 10 (2002) 2759–
2765.
[21] A. Cazenave Gassiot, J. Charton, S. Girault-Mizzi, M.-A. Debreu-Fontaine,
P. Melnyk, C. Sergheraert, Synthesis and pharmacological evaluation of Tic-
hydantoin derivatives as selective s1. Part 2. Bioorg. Med. Chem. Lett. 15 (2005)
4828–4832.
[9] X. Guitart, X. Codony, X. Monroy, Sigma receptors: biology and therapeutic
potential. Psychopharmacology 174 (2004) 301–319.
[10] P. Romieu, R. Martin-Fardon, W.B. Bowen, T. Maurice, Sigma-1 receptor-related
neuroactive steroids modulate cocaine-induced reward. J. Neurosci. 23 (2003)
3572–3576.
[11] K. Matsuno, T. Senda, T. Kobayashi, S. Mita, Involvement of sigma 1 receptor in
(þ)-N-allylnormetazocine-stimulated hippocampal cholinergic functions in
rats. Brain Res. 690 (1995) 200–206.
[12] H.C. Akunne, K.T. Zoski, S.Z. Whetzel, J.J. Cordon, R.M. Brandon, F. Roman,
T.A. Pugsley, Neuropharmacological profile of a selective sigma ligand, igme-
sine: a potential antidepressant. Neuropsychopharmacology 41 (2001) 138–149.
[13] A. Meurs, R. Clinckers, G. Ebinger, Y. Michotte, I. Smolders, Sigma 1 receptor-
mediated increase in hippocampal extracellular dopamine contributes to the
mechanism of the anticonvulsant action of neuropeptide Y. Eur. J. Neurosci. 11
(2007) 3079–3092.
[22] M. Toussaint, B. Delair, C. Foulon, N. Lempereur, C. Vaccher, T. Maurice,
P. Melnyk, Tic hydantoin sigma-1 agonist: pharmacological characterization
on cocaine-induced stimulant and appetitive effects. Eur. Neuropsychopharm.
19 (2009) 504–515.
[23] L. Rivas, L. Quintero, J.L. Fourrey, R. Benhida, A short synthesis of aspergilla-
mide B. The marine natural product from Aspergillus sp. Tetrahedron Lett. 43
(2002) 7639–7642.
[24] M.D. Hoey, D.C. Dittmer, A convenient synthesis of 1,4-dihydro-2,3-benzox-
athiin 3-oxide, a useful precursor of o-quinodimethane. J. Org. Chem. 56
(1991) 1947–1948.
[25] M.C. Carter, D.G. Alber, R.C. Baxter, S.K. Bithell, J. Budworth, A. Chubb,
G. Stuart Cockerill, V.C.L. Dowdell, E.A. Henderson, S.J. Keegan, R.D. Kelsey,
M.J. Lockyer, J.N. Stables, L.J. Wilson, K.L. Powell, 1,4-Benzodiazepines as
inhibitors of respiratory syncytial virus. J. Med. Chem. 49 (2006) 2311–
2319.
[14] T. Hayashi, T.P. Su, An update on the development of drugs for neuropsychi-
atric disorders: focusing on the sigma 1 ligand. Expert Opin. Ther. Targets 12
(2008) 45–58.
[15] C. Katnik, W.R. Guerrero, K.R. Pennypacker, Y. Herrera, J.J. Cuevas, Sigma-1
receptor activation prevents intracellular calcium dysregulation in cortical
neurons during in vitro ischemia. Pharmacol. Exp. Ther. 319 (2006) 1355–
1365.
[16] E. Aydar, P. Onganer, R. Perrett, M.B. Djamgoz, C.P. Palmer, The expression and
functional characterization of sigma (sigma) 1 receptors in breast cancer cell
lines. Cancer Lett. 242 (2006) 245–257.
[26] M.E. Ganapathy, P.D. Prasad, W. Huang, P. Seth, F.H. Leibach, V.J. Ganapathy,
Molecular and ligand-binding characterization of the sigma-receptor in the
Jurkat human T lymphocyte cell line. Pharmacol. Exp. Ther. 289 (1999) 251–
260.
[27] W.D. Bowen, B.R. de Costa, S.B. Hellewell, M. Walker, K.C. Rice, [3H](þ)-
pentazocine: a potent and highly selective benzomorphan-based probe for
sigma1 receptors. Mol. Neuropharmacol. 3 (1993) 117–126.
[28] IC50 values from competitive inhibition experiments were determined using
the Marquardt-Levenberg nonlinear curve fitting procedure of Xfit macro
(Microsoft Excel).
[17] J. Charton, A. Cazenave Gassiot, S. Girault-Mizzi, P. Gilleron, M.-A. Debreu-
Fontaine, C. Sergheraert, P. Melnyk, Synthesis and pharmacological evaluation
of Tic-hydantoin derivatives as selective s1 ligands. Part 1. Bioorg. Med. Chem.
Lett. 15 (2005) 4833–4837.
[29] R.C.A. Onderwater, J.N.M. Commandeur, E.J. Groot, A. Sitters, W.M.P.B. Menge,
N.P.E. Vermeulen, Cytotoxicity of a series of mono- and di-substituted thiourea
in freshly isolated rat hepatocytes: a preliminary structure–toxicity relation-
ship study. Toxicology 125 (1998) 117–129.
[18] J. Charton, A. Cazenave Gassiot, P. Melnyk, S. Girault-Mizzi, C. Sergheraert,
Optimized Synthesis of tetrahydroisoquinoline-hydantoins. Tetrahedron Lett.
45 (2004) 7081–7085.
[30] M.M. Bradford, A rapid and sensitive method for the quantitation of micro-
gram quantities of protein utilizing the principle of protein-dye binding. Anal.
Biochem. 72 (1976) 248–254.