Substituted benzylaminoalkylindoles with preference for the σ2 binding site

Article abstract of DOI:10.1016/j.ejmech.2007.09.012

In the attempt to develop new σ ligands we synthesized a series of N-benzyl-3-[1-(4-fluorophenyl)-1H-indol-3-yl]-N-methylpropan-1-amines and N-benzyl-4-[1-(4-fluorophenyl)-1H-indol-3-yl]-N-methylbutan-1-amines variously substituted on the phenyl ring. The displacement percentages of [³H]-DTG and [³H]-(+)-pentazocine determined in rat liver homogenates by these compounds at the fixed 100 nM concentration have been determined as a preliminary evaluation of their σ₁ and σ₂ affinity, respectively. The results suggested that the phenyl substituents may positively modulate, in comparison with the unsubstituted compound, the ability to displace [³H]-DTG from σ₂ sites, whereas the same phenyl substituents reduced the displacement percentages of [³H]-(+)-pentazocine from σ₁ sites. Some of these compounds were selected for radioligand binding assays. Compounds with a butylene intermediate chain displayed the greatest binding affinity for σ₂ over σ₁ receptors. The butylene derivative with 2,4-dimethyl substitution on the phenyl ring showed the greatest σ₂ affinity (σ₂K_i = 5.9 nM) and an appreciable σ₂ over σ₁ selectivity (σ₁K_i/σ₂K_i = 22). The obtained results suggest that a butylene chain separating the indole moiety from variously substituted benzylamino groups may be required to their interaction with a hypothetical secondary σ₂ binding site.

Full text of DOI:10.1016/j.ejmech.2007.09.012

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 43 (2008) 2073e2081
http://www.elsevier.com/locate/ejmech
Original article
Substituted benzylaminoalkylindoles with preference
for the s2 binding site*
a
b
b
Maria Grazia Mamolo a, , Daniele Zampieri , Caterina Zanette , Chiara Florio ,
*
Simona Collina c, Mariangela Urbano c, Ornella Azzolina c, Luciano Vio a
a Department of Pharmaceutical Sciences, University of Trieste, P.ale Europa 1, 34127 Trieste, Italy
b Department of Biomedical Sciences (Pharmacology Section), University of Trieste, Via A. Fleming 32, 34127 Trieste, Italy
c Department of Pharmaceutical Chemistry, University of Pavia, V.le Taramelli 12, 27100 Pavia, Italy
Received 14 February 2007; received in revised form 25 June 2007; accepted 17 September 2007
Available online 26 September 2007
Abstract
In the attempt to develop new s ligands we synthesized a series of N-benzyl-3-[1-(4-fluorophenyl)-1H-indol-3-yl]-N-methylpropan-1-amines
and N-benzyl-4-[1-(4-fluorophenyl)-1H-indol-3-yl]-N-methylbutan-1-amines variously substituted on the phenyl ring. The displacement percent-
ages of [3H]-DTG and [3H]-(þ)-pentazocine determined in rat liver homogenates by these compounds at the fixed 100 nM concentration have
been determined as a preliminary evaluation of their s1 and s2 affinity, respectively.
The results suggested that the phenyl substituents may positively modulate, in comparison with the unsubstituted compound, the ability to
displace [3H]-DTG from s2 sites, whereas the same phenyl substituents reduced the displacement percentages of [3H]-(þ)-pentazocine from s1
sites. Some of these compounds were selected for radioligand binding assays. Compounds with a butylene intermediate chain displayed the
greatest binding affinity for s2 over s1 receptors. The butylene derivative with 2,4-dimethyl substitution on the phenyl ring showed the greatest
s2 affinity (s2Ki ¼ 5.9 nM) and an appreciable s2 over s1 selectivity (s1Ki/s2Ki ¼ 22). The obtained results suggest that a butylene chain sep-
arating the indole moiety from variously substituted benzylamino groups may be required to their interaction with a hypothetical secondary s2
binding site.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: s Receptors; Benzylaminoalkyl indole derivatives; Radioligand binding assays
1. Introduction
affinity for (þ)-benzomorphans such as (þ)-pentazocine and
(þ)-N-allylnormetazocine (NANM, SKF-10,047) and reduced
affinity for the corresponding (ꢀ)-enantiomers. (þ)-Pentazocine
shows a very low affinity for s2 receptors and represents a typ-
ical selective agonist used as tritiated ligand to label s1 recep-
tors. Several compounds selectively binding the s1 receptors
and s1 pharmacophoric models have been proposed by Glen-
non et al. [5e7] and Gund et al. [8]. However, known s2
receptor ligands generally display a poor selectivity profile.
Selective s2 over s1 ligands such as ibogaine and the phenyl-
morphans CB-64D and CB-184 also show affinity for NMDA
(ibogaine) and m-opioid (CB-64D and CB-184) receptors
[9,10]. The antipsychotic haloperidol and 1,3-di(2-tolyl)guani-
dine (DTG) possess high affinity for both s subtypes [11].
DTG is the most used s2 radioligand but it needs a s1 masking
The s receptor was first described as a subtype of opioid
receptors [1]. Now the existence of s sites has been estab-
lished and the s1 and s2 subtypes are universally recognized
[2]. The s1 protein has been purified and cloned from several
animal species and humans [3,4]. This receptor has been iden-
tified to be a mammalian homologue of the yeast sterol C8eC7
isomerase. The molecular identity of the s2 receptor has not
been fully determined [3,4]. The s1 subtype exhibits high
*
A preliminary account of this work was presented at the XVII National
Meeting on Medicinal Chemistry, Pisa, September 6e10, 2004.
* Corresponding author. Tel.: þ39 40 5583719; fax: þ39 40 52572.
E-mail address: mamolo@units.it (M.G. Mamolo).
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2007.09.012

2074
M.G. Mamolo et al. / European Journal of Medicinal Chemistry 43 (2008) 2073e2081
Table 1
Yields and physical data of the synthesized compounds
agent. However, a number of benzamide derivatives, that have
higher affinity and selectivity for s2 versus s1 receptors, have
been recently reported [12].
(CH2)n
N
The s1 receptors have several modulatory roles in neuro-
transmitter systems such as dopaminergic, serotoninergic,
muscarinic systems [2,13,14] and in NMDA-stimulated neuro-
transmitter release [15]. Moreover, s1 receptors are involved
in neuroprotective and antiamnesic activities [16], modulation
of opioid analgesia [17] and attenuation of cocaine-induced lo-
comotor activity and toxicity [18]. Besides, s1 antagonists
have shown to be effective against negative symptoms of
schizophrenia without producing extrapyramidal side effects
[19,20].
R
CH3
N
1 a-l n = 3
2 a-l n = 4
F
On the contrary, s2 receptors may contribute to the acute
side effects of typical neuroleptic drugs and s2 antagonists at-
tenuate the extrapyramidal effects, dystonic reactions and tar-
dive dyskinesia [2,13,21e23] suggesting their potential use in
the treatment of psychoses [19,20]. Furthermore, s2 receptors
are involved in regulation of cell proliferation and mainte-
nance of cell viability. They are highly expressed in several
tumoral cell lines [24,25], where s2 agonists produce morpho-
logical changes and apoptosis. The s2 receptor agonists pro-
mote Ca2þ release from endoplasmic reticulum and
mitochondrial stores [26] with subsequent cell death by cas-
pase-independent apoptosis [25]. Apoptosis may also be in-
duced in tumoral cells by regulation of the sphingolipid
pathway [27]. Therefore, s2 agonists may be useful as novel
anticancer agents. Moreover, s2 selective ligands may be use-
ful as imaging agents in cancer diagnosis by Positron Emission
Tomography (PET) [28] and Single Photon Emission Com-
puted Tomography (SPECT) [29,30].
Thus, selective s1 and s2 agonists and antagonists may be
potentially useful drugs for treatment of several pathologic
conditions such as psychiatric disorders, cocaine abuse, mem-
ory and learning disorders, dyskinesia and dystonic reactions
induced by classical antipsychotic drugs, cancer and tumor di-
agnosis. Now several compounds binding s1 receptors with
high affinity and selectivity have been discovered, whereas
s2 receptor ligands generally have poor selectivity over s1
receptors and new s2 ligands are needed in order to define
the structural features that may improve their affinity and
selectivity.
With the aim to develop new ligands with high affinity and
selectivity for s receptors, we synthesized the series of
compounds 1ael and 2ael (Table 1).
Compounds 2ael are structurally related to the indole de-
rivative 3 (Lu 28-179) (Fig. 1) and analogs, characterized by
high affinity and selectivity for s2 subtype over s1 subtype re-
ceptors [31].
Lu 28-179 is a member of a series of 3-(u-aminobutyl)-1H-
indoles variously substituted on the 1-position of the indole
group and linked by the butylene chain to a spiro[isobenzo-
furan-1(3H ),40-piperidine] moiety [31]. All these spiropiperi-
dines showed selectivity for s2 versus s1 binding sites and
their IC50 values were below 1 nM for a majority of
compounds. However, introduction of the 4-fluorophenyl
substituent at the indole nitrogen atom produced compound 3
R
Comp., Yield Anal.
n ¼ 3
Comp., Yield Anal.
(%)
C, H, N
n ¼ 4
2a
(%)
C, H, N
H
2-Cl
1a
1b
1c
1d
1e
1f
20
44
48
52
38
46
52
46
46
50
52
58
C25H25N2F
37
59
49
48
49
59
35
38
50
57
57
46
C26H27N2F
C25H24N2FCl 2b
C25H24N2FCl 2c
C25H24N2FCl 2d
C25H23N2FCl2 2e
C25H24N2FBr 2f
C25H24N2FBr 2g
C25H24N2FBr 2h
C26H26N2FCl
C26H26N2FCl
C26H26N2FCl
C26H25N2FCl2
C26H26N2FBr
C26H26N2FBr
C26H26N2FBr
C26H26N2F2
C27H29N2F
3-Cl
4-Cl
3,4-(Cl)2
2-Br
3-Br
4-Br
2-F
1g
1h
1i
C25H24N2F2
C26H27N2F
C26H27N2F
C27H29N2F
2i
3-CH3
4-CH3
2,4-(CH3)2 1l
1j
2j
2k
2l
1k
C27H29N2F
C28H31N2F
with the highest s2 binding affinity (s1IC50 ¼ 17 nM;
s2IC50 ¼ 0.12 nM) and s2 over s1 selectivity (s1IC50/
s2IC50 ¼ 140) in the series. Furthermore, shorter interspacing
alkylene chains seem to reduce potency and selectivity. The
introduction of substituents on the benzene moiety of the spiro-
piperidine ring system of Lu 28-179 produced effects on s1 and
O
N
N
F
3
(CH2)n
N
N
R
R = H, OCH3, OH
n = 0-6
8
Fig. 1. Structures of the s2 selective ligands 3 and 8.

M.G. Mamolo et al. / European Journal of Medicinal Chemistry 43 (2008) 2073e2081
2075
OH
s2 affinity and selectivity, indicating that this benzene ring may
COOH
(CH2)n
be involved in binding to s1 and s2 sites [32]. Furthermore,
modifications of the structure of the spiropiperidine ring system
producing a different spatial localization of the benzene ring
relative to the basic nitrogen atom determined to a large extent
the affinity for both s1 and s2 receptors [32]. The results of
this study [32] indicated that both substituents on the benzene
ring and their position relative to the basic nitrogen atom might
influence the binding characteristics of the spiropiperidine de-
rivatives under examination. The series of new indole deriva-
tives 1ael and 2ael have been synthesized by replacing the
spiro[isobenzofuran-1(3H ),40-piperidine] moiety of Lu 28-
179 and analogous compounds with a variously substituted ben-
zylamino group linked to the indole moiety by an alkylene
chain, in order to verify if this simple modification may produce
compounds with affinity for s receptorial sites.
(CH2)n
LiAlH4
Et2O
5a (n = 3)
5b (n = 4)
N
N
H
H
4a (n = 2)
4b (n =3)
K2CO3, CuI, ZnO
NMP, 155°C, 72h
Br
F
OSO2CH3
(CH2)n
OH
(CH2)n
N
N
6a (n = 3)
6b (n = 4)
CH3SO2Cl
CH2Cl2 , triethylamine, RT
7a (n = 3)
7b (n = 4)
2. Chemistry
F
F
CH3
N
H
The substituted N-benzyl-3-[1-(4-fluorophenyl)-1H-indol-
3-yl]-N-methylpropan-1-amines 1ael and N-benzyl-4-[1-(4-
fluorophenyl)-1H-indol-3-yl]-N-methylbutan-1-amines 2ael
(Table 1) have been synthesized (Scheme 1) starting from 3-
(1H-indol-3-yl)propanoic acid 4a and 4-(1H-indol-3-yl)buta-
noic acid 4b, which were reduced with LiAlH4 to 3-(1H-in-
dol-3-yl)propan-1-ol 5a and 4-(1H-indol-3-yl)butan-1-ol 5b
following, with minimal modifications, a literature procedure
[33]. By heating compounds 5a and 5b with 4-bromofluoro-
benzene in the presence of K2CO3, CuI and ZnO in
1-methyl-2-pyrrolidinone, the corresponding compounds
3-[1-(4-fluorophenyl)-1H-indol-3-yl]propan-1-ol 6a and 4-[1-
(4-fluorophenyl)-1H-indol-3-yl]butan-1-ol 6b [31] were ob-
tained. By treatment of 6a and 6b with methanesulfonyl
chloride the methanesulfonate esters 7a and 7b were prepared,
from which compounds 1ael and 2ael were obtained by reac-
tion with substituted benzylamines.
R
MIBK, K2CO3
150°C, 16h
H3C
N
R
(CH2)n
R = H,2-Cl, 3-Cl, 4-Cl, 3,4-(Cl)2,
2-Br, 3-Br, 4-Br,2-F,3-CH3,
4-CH3, 2,4-(CH3)2
N
1 a-l (n = 3)
2 a-l (n = 4)
F
Scheme 1. Synthesis of compounds 1ael and 2ael.
Actually, in a series of 4-(tetralin-1-yl)alkyl derivatives of
1-cyclohexylpiperazine 8 (Fig. 1) the little structural differ-
ences preferentially between s1 and s2 ligands allowed to hy-
pothesize the existence of different secondary sites in the
corresponding receptorial subtypes [34] with the intermediate
chain length exerting a significant influence on s2 binding
affinity.
The tetralin derivative 8 (R ¼ 5-OCH3, n ¼ 3) was charac-
terized by the greatest s2 affinity (s2Ki ¼ 0.34 nM). If only
the aromatic portion of this tetralin derivative is considered,
the spacer corresponding to a three-methylene chain (n ¼ 3)
produced a butyl bridge connecting the aromatic portion
with the piperazine ring [34].
On the basis of these considerations we maintained in the
new compounds 2ael (n ¼ 4) the butylene spacer present in
the high s2 affinity compounds 3 and 8 (R ¼ 5-OCH3,
n ¼ 3) in order to verify if this spacer, together with the termi-
nal substituted benzyl group, may produce receptorial interac-
tions with the proposed s2 binding site [34].
3. Results and discussion
Since a four atom spacer appeared to be important for the
s2 affinity of the spiropiperidine derivative 3 (Fig. 1) and anal-
ogous compounds [31], we retained in compounds 2ael
(n ¼ 4) (Table 1) the 1-(4-fluorophenyl)-indole moiety and
the four carbon atoms alkylene chain, replacing the spiropiper-
idine moiety with the N-benzylamino group variously
substituted on the aromatic ring.
The new indole derivatives 2ael have been designed, ac-
cording to the s1-receptorial model proposed by Glennon
et al. [5e7], with the assumption that the indole moiety may
interact with a primary hydrophobic site corresponding to
the phenyl ‘‘B’’ region [5e7], the basic N-atom linked by
the alkylene chain (n ¼ 4) to the indole moiety may interact
with a receptorial proton-donor site and the substituted N-ben-
zyl moiety may bind a secondary hydrophobic region similar
to the phenyl ‘‘A’’ region of the s1-receptorial model [5e7]
and modulate the binding affinity of the compounds for s1
or s2 receptors.
Another series of compounds 1ael (n ¼ 3) (Table 1) with
a propylene chain and the same aromatic substitution pattern

2076
M.G. Mamolo et al. / European Journal of Medicinal Chemistry 43 (2008) 2073e2081
Table 3
Displacement percentages of [3H]-(þ)-pentazocine from s1 sites
was synthesized to evaluate the effect of this simple modifica-
tion on s receptor affinity and selectivity.
R
Comp.,
n ¼ 3
% Meana,b
Comp.,
n ¼ 4
% Meana,b
In a preliminary assay the displacement percentages of
[3H]-DTG from s2 receptors produced by compounds 1ael
(n ¼ 3) and 2ael (n ¼ 4) were evaluated on rat liver homoge-
nates at the fixed 100 nM concentration in the presence of
(þ)-pentazocine to mask s1 receptors (Table 2). The displace-
ment percentages produced by compounds 1ael were in the
range 27e85%, whereas all 2ael compounds were found to
produce >50% inhibition of binding at s2 receptors (range
68e93%).
From these preliminary data it appeared that both the series
of compounds produced displacement percentages of [3H]-
DTG from s2 receptors modulated by the substituents on the
phenyl ring, but the butylene derivatives 2ael produced dis-
placement percentages higher than those of the corresponding
propylene derivatives 1ael.
ꢁ SD
ꢁ SD
H
2-Cl
1a
1b
1c
1d
1e
1f
78 ꢁ 5
2a
2b
2c
2d
2e
2f
76 ꢁ 1
58 ꢁ 5
22 ꢁ 5
72 ꢁ 11
10 ꢁ 8
46 ꢁ 14
28 ꢁ 10
70 ꢁ 9
49 ꢁ 7
51 ꢁ 11
71 ꢁ 19
37 ꢁ 8
56 ꢁ 7
3-Cl
4-Cl
42 ꢁ 17
68 ꢁ 9
3,4-(Cl)2
2-Br
3-Br
28 ꢁ 12
41 ꢁ 18
40 ꢁ 5
1g
1h
1i
2g
2h
2i
4-Br
2-F
71 ꢁ 10
59 ꢁ 3
3-CH3
4-CH3
2,4-(CH3)2
1j
63 ꢁ 27
69 ꢁ 14
38 ꢁ 11
2j
1k
1l
2k
2l
Pentazocine, 1 nM
Pentazocine, 10 nM
Pentazocine, 100 nM
24 ꢁ 12
60 ꢁ 2
95 ꢁ 2
Moreover, all the [3H]-DTG displacement values from s2
sites produced by substituted compounds 1bel and 2bel
were higher than those of the corresponding unsubstituted
compounds 1a and 2a.
a
Mean displacement percentages produced on rat liver homogenates by
compounds 1ael (n ¼ 3) and 2ael (n ¼ 4) at the fixed 100 nM concentration.
b
Data are expressed as mean values ꢁ SD of 3 experiments performed in
duplicate.
With the same approach the displacement percentages of
[3H]-(þ)-pentazocine from s1 sites by the compounds 1ael
and 2ael at the fixed 100 nM concentration were determined
(Table 3). The displacement percentages were in the range
28e78% for compounds 1ael and 10e76% for compounds
2ael (n ¼ 4).
displacement percentages of [3H]-DTG from s2 sites were se-
lected, together with the corresponding compounds 1c and 1l,
for determination of their Ki values at both receptor types (Table
4).
From the obtained results it appears that the substituents on
the phenyl ring can modulate, according to the previously de-
termined displacement percentage data, the s1 and s2 binding
affinity of these compounds. As regards s1 receptors, the un-
substituted compounds 1a (n ¼ 3) and 2a (n ¼ 4) displayed s1
binding affinity higher than that of the corresponding
substituted derivatives 1c, 1l and 2c, 2l, respectively. The
most potent s1 ligand was the propylene derivative 1a, whose
s1Ki value was 20.8 nM with a selectivity ratio s1Ki/
s2Ki ¼ 0.05. Conversely, the s2 affinity of the substituted
compounds 1c, 1l (n ¼ 3) and 2c, 2l (n ¼ 4) was higher than
that of the unsubstituted compounds 1a and 2a, respectively,
and dependent on the alkylene chain, with the butylene deriv-
atives having higher affinity than the corresponding propylene
compounds. The highest s2 binding affinity was attained by
the butylene derivative 2l, whose s2Ki value was 5.9 nM, sug-
gesting an appreciable s2 affinity and selectivity of this com-
pound over s1 sites (s1Ki/s2Ki ¼ 22). On the other hand, the
corresponding propylene derivative 1l (n ¼ 3), even if it is
showing some preference for s2 sites, displayed reduced s2
Percentage reductions of [3H]-(þ)-pentazocine binding to
s1 site, even if varied along with the substitution on the aro-
matic ring, were less than the percentage inhibition of the un-
substituded compounds, suggesting that aromatic substitution
may be detrimental for the s1 binding of these compounds.
The unsubstituted compounds 1a (n ¼ 3) and 2a (n ¼ 4) and
compounds 2c and 2l, which produced the greatest
Table 2
Displacement percentages of [3H]-DTG from s2 sites
R
Comp.,
n ¼ 3
% Meana,b
Comp.,
n ¼ 4
% Meana,b
ꢁ SD
ꢁ SD
H
2-Cl
1a
1b
1c
1d
1e
1f
27 ꢁ 8
40 ꢁ 10
59 ꢁ 8
59 ꢁ 8
39 ꢁ 5
43 ꢁ 1
62 ꢁ 4
54 ꢁ 9
46 ꢁ 9
64 ꢁ 9
78 ꢁ 5
85 ꢁ 5
2a
2b
2c
2d
2e
2f
68 ꢁ 8
87 ꢁ 2
89 ꢁ 3
85 ꢁ 10
69 ꢁ 3
86 ꢁ 7
78 ꢁ 22
84 ꢁ 10
88 ꢁ 8
85 ꢁ 12
87 ꢁ 8
93 ꢁ 9
3-Cl
4-Cl
3,4-(Cl)2
2-Br
3-Br
1g
1h
1i
2g
2h
2i
4-Br
2-F
3-CH3
4-CH3
2,4-(CH3)2
1j
2j
Table 4
Binding affinity (Ki ꢁ SEM nM) and selectivity
1k
1l
2k
2l
Comp.
n
R
s1Ki, nM
s2Ki, nM
s1Ki/s2Ki
ratio
DTG, 3 nM
DTG, 30 nM
DTG, 300 nM
a
9 ꢁ 5
47 ꢁ 10
82 ꢁ 2
1a
1c
1l
3
3
3
4
4
4
H
3-Cl
20.8 ꢁ 1
98.9 ꢁ 10
118 ꢁ 16
43 ꢁ 7
444.3 ꢁ 21
123.5 ꢁ 23
46.9 ꢁ 12
57.7 ꢁ 16
25.6 ꢁ 9
0.05
0.80
2.5
Mean displacement percentages produced on rat liver homogenates by
compounds 1ael (n ¼ 3) and 2ael (n ¼ 4) at the fixed 100 nM concentration
in the presence of 100 nM (þ)-pentazocine to mask s1-receptors.
2,4-(CH3)2
H
3-Cl
2a
2c
2l
0.76
7.9
201 ꢁ 21
129 ꢁ 22
b
Data are expressed as mean values ꢁ SD of 3 experiments performed in
2,4-(CH3)2
5.9 ꢁ 2
22
duplicate.

M.G. Mamolo et al. / European Journal of Medicinal Chemistry 43 (2008) 2073e2081
2077
affinity (s2Ki ¼ 46.9 nM) and selectivity (s1Ki/s2Ki ¼ 2.5) in
4.1.2. 4-(1H-Indol-3-yl)butan-1-ol 5b
In an analogous way 4-(1H-indol-3-yl)butan-1-ol 5b [33]
was prepared. Yield: 8.88 g (93%).
comparison with compound 2l. The obtained results suggest
that the new compound 2l (n ¼ 4) and other butylene spaced
compounds 2, variously substituted on the phenyl ring, may
interact with a secondary s2 site similar to the hydrophobic
binding site ‘‘A’’ of the Glennon’s s1 [5e7] model.
IR (nujol, cmꢀ1): 3350, 2927. 1H NMR (CDCl3eTMS) ppm
(d): 1.24 (t, 2H, eCH2eCH2eCH2eCH2eOH), 1.73 (br s, 1H,
OH, disappearing on deuteration), 2.82 (m, 2H, eCH2eCH2e
CH2eCH2eOH), 3.52 (m, 2H, eCH2eCH2eCH2eCH2e
OH), 3.71 (t, 2H, eCH2eCH2eCH2eCH2eOH), 6.99 (s, 1H,
CH ind.), 7.08e8.02 (m, 4H arom.), 8.02 (s, 1H, NH, disappear-
ing on deuteration). Anal. Calcd for C12H15NO (MW 189.25):
C, 76.16; H, 7.99; N, 7.40%. Found:C, 75.96; H, 7.69; N, 8.18%.
The indole moiety may establish a favorable interaction
with a hydrophobic binding site resembling the s1 primary
binding site proposed by Glennon and the butylene chain sep-
arating the N-basic atom seems to produce the most favorable
distance between the primary and secondary hydrophobic cen-
ters. The positive and variable contribution to the s2 affinity of
the substituents on the phenyl ring in compounds 1c, 1l and 2c,
2l and their negative effect on s1 affinity may encourage the
design of structural modifications useful for the development
of new ligands with higher s2 affinity and selectivity.
4.1.3. 3-[1-(4-Fluorophenyl)-1H-indol-3-yl]propan-1-ol 6a
A mixture of 2.5 g (14.3 mmol) of 3-(1H-indol-3-yl)pro-
pan-1-ol 5a, 4.24 g (24.25 mmol) of 4-bromofluorobenzene,
2.56 g (18.5 mmol) of potassium carbonate, 0.68 g
(3.56 mmol) of CuI and 0.18 g (2.28 mmol) of ZnO in
100 ml of 1-methyl-2-pyrrolidinone (NMP) was heated at
155 ꢂC for 72 h. After cooling the precipitated inorganic salts
were filtered off and 20 ml of ethyl ether and 80 ml of 10%
NaOH were added to the solution. The organic phase was sep-
arated, washed with NaCl solution and dried over anhydrous
CaCl2. The filtered solution was concentrated under reduced
pressure and the remaining crude product was purified as an
oil by column chromatography on silica gel (eluted with
a 3:2 mixture of CH3Cl and toluene). Yield: 1.54 g (40%).
4. Experimental section
4.1. Chemistry
Melting points were determined with a Buchi 510 capillary
¨
apparatus, and are uncorrected. Infrared spectra in nujol mulls
were recorded on a Jasko FT 200 spectrophotometer. Proton
nuclear magnetic resonance (1H NMR) spectra were deter-
mined on a Varian Gemini 200 spectrometer, chemical shifts
are reported as d (ppm) in CDCl3 solution. Reaction courses
and product mixtures were routinely monitored by thin-layer
chromatography (TLC) on silica gel precoated F254 Merck
plates. Column chromatographies were performed using
Backer 70e230 mesh silica gel60. ESI-MS spectra were ob-
tained on a PE-API I spectrometer by infusion of a solution
of the sample in MeOH. Elemental analyses (C, H, N) were
performed on a Carlo Erba analyzer and were within ꢁ0.3
of the theoretical value.
1
IR (nujol, cmꢀ1): 3348. H NMR (CDCl3eTMS) ppm (d):
1.52 (s, 1H, OH, disappearing on deuteration), 2.06 (m, 2H, e
CH2eCH2eCH2eOH), 2.95 (t, 2H, eCH2eCH2eCH2eOH),
3.8 (t, 2H, eCH2eCH2eCH2eOH), 7.14e7.7 (m, 8H arom.,
1H ind.). Anal. Calcd for C17H16FNO (MW 269.31): C,
75.82; H, 5.99; N, 5.20%. Found: C, 75.66; H, 5.72; N, 5.08%.
4.1.4. 4-[1-(4-Fluorophenyl)-1H-indol-3-yl]butan-1-ol 6b
In an analogous way 4-[1-(4-fluorophenyl)-1H-indol-3-yl]-
butan-1-ol 6b [31] was obtained. Yield: 1.6 g (40%).
1
4.1.1. 3-(1H-Indol-3-yl)propan-1-ol 5a
IR (nujol, cmꢀ1): 3352. H NMR (CDCl3eTMS) ppm (d):
3-(1H-Indol-3-yl)propanoic acid 4a (3 g, 15.8 mmol) in
50 ml of ethyl ether was stirred under reflux and 1.5 g
(39.6 mmol) of LiAlH4 in 50 ml of ethyl ether was added
dropwise to the solution. After heating for 3 h, the solution
was stirred at room temperature for additional 20 h. The solu-
tion was filtered and the excess of LiAlH4 was eliminated by
treating with water and then with a 1:3 mixture of concen-
trated H2SO4 and water. The aqueous phase was extracted
with ethyl ether and the collected organic phases were dried
on anhydrous sodium sulphate, filtered and concentrated under
reduced pressure. The obtained yellow oil was chromatograph-
ically pure and was used without further purification. Yield:
2.71 g (98%).
IR (nujol, cmꢀ1): 3345, 2935. 1H NMR (CDCl3eTMS)
ppm (d): 1.9 (m, 2H, eCH2eCH2eCH2eOH), 2.5 (br s, 1H,
OH, disappearing on deuteration), 2.85 (t, 2H, eCH2eCH2e
CH2eOH), 3.75 (t, 2H, eCH2eCH2eCH2eOH), 7.0 (s, 1H,
CH ind.), 7.1e7.7 (m, 4H arom.), 8.1 (s, 1H, NH, disappearing
on deuteration). Anal. Calcd for C11H13NO (MW 175.23): C,
75.40; H, 7.48; N, 7.99%. Found: C, 75.26; H, 7.52; N, 8.08%.
1.27 (t, 2H, eCH2eCH2eCH2eCH2eOH), 1.32 (br s, 1H,
OH, disappearing on deuteration), 1.78 (m, 2H, eCH2e
CH2eCH2eCH2eOH), 2.87 (m, 2H, eCH2eCH2eCH2e
CH2eOH), 3.73 (t, 2H, eCH2eCH2eCH2eCH2eOH),
7.05e7.82 (m, 8H arom., 1H ind.). Anal. Calcd for
C18H18FNO (MW 283.34): C, 76.30; H, 6.40; N, 4.94%.
Found: C, 76.55; H, 6.68; N, 5.12%.
4.1.5. 3-[1-(4-Fluorophenyl)-1H-indol-3-yl]propyl-1-
methanesulfonate 7a
To a solution of 1.54 g (5.72 mmol) of 3-[1-(4-fluoro-
phenyl)-1H-indol-3-yl]propan-1-ol 6a in 50 ml of CH2Cl2,
0.64 g (6.29 mmol) of triethylamine was added. The solution
was stirred and cooled in an ice bath and 0.72 g
(6.29 mmol) of methanesulfonyl chloride in 20 ml of di-
chloromethane was added dropwise by keeping the temper-
ature below 5 ꢂC. The temperature was allowed to reach
room temperature and the mixture was stirred for additional
1.5 h. Water was added and the organic phase was separated
and evaporated under reduced pressure. The solid residue

2078
M.G. Mamolo et al. / European Journal of Medicinal Chemistry 43 (2008) 2073e2081
was crystallized from ethanol. Yield: 1.59 g (80%); melting
point 65 ꢂC.
4.1.9. N-(3-Chlorobenzyl)-3-[1-(4-fluorophenyl)-
1H-indol-3-yl]-N-methylpropan-1-amine 1c
1H NMR (CDCl3eTMS) ppm (d): 2.19 (m, 2H, eCH2e
CH2eCH2eOH), 2.99 (t, 2H, eCH2eCH2eCH2eOH), 3.03
(s, 3H, CH3), 4.34 (t, 2H, eCH2eCH2eCH2eOH), 7.1e7.7
(m, 8H arom., 1H ind.). Anal. Calcd for C18H18FNO3S (MW
347.1): C, 62.23; H, 5.22; N, 4.03%. Found: C, 62.46; H,
5.31; N, 4.18%.
1H NMR (CDCl3eTMS) ppm (d): 2.1 (m, 2H, eCH2e
CH2eCH2eNH), 2.36 (s, 3H, CH3), 2.65 (t, 2H, eCH2e
CH2eCH2eNH), 2.86 (t, 2H, eCH2eCH2eCH2eNH), 3.55
(s, 2H, CH2), 7.05 (s, 1H ind.), 7.1e7.65 (m, 12H arom.).
MS: m/z 407 [MHþ], 409 [MHþ þ 2]. Anal. Calcd for
C25H24ClFN2 (MW 406.92): C, 73.79; H, 5.94; N, 6.88%.
Found: C, 73.92; H, 5.81; N, 6.63%.
4.1.6. 4-[1-(4-Fluorophenyl)-1H-indol-3-yl]
butyl-1-methanesulfonate 7b
4.1.10. N-(4-Chlorobenzyl)-3-[1-(4-fluorophenyl)-
1H-indol-3-yl]-N-methylpropan-1-amine 1d
1H NMR (CDCl3eTMS) ppm (d): 1.99 (m, 2H, eCH2e
CH2eCH2eNH), 2.25 (s, 3H, CH3), 2.51 (t, 2H, eCH2e
CH2eCH2eNH), 2.9 (t, 2H, eCH2eCH2eCH2eNH), 3.2 (s,
2H, CH2), 7.04 (s, 1H ind.), 7.16e7.7 (m, 12H arom.). MS:
m/z 407 [MHþ], 409 [MHþ þ 2]. Anal. Calcd for
C25H24ClFN2 (MW 406.92): C, 73.79; H, 5.94; N, 6.88%.
Found: C, 73.56; H, 5.72; N, 6.99%.
With the same procedure, 4-[1-(4-fluorophenyl)-1H-indol-
3-yl]butyl-1-methanesulfonate (7b) was obtained as an oil
and purified by column chromatography on silica gel (eluted
with a 9:1 mixture of dichloromethane and toluene). Yield:
1.3 g (80%).
1H NMR (CDCl3eTMS) ppm (d): 2.19 (t, 2H, eCH2eCH2e
CH2eCH2eOH), 2.99 (m, 2H, eCH2eCH2eCH2eCH2eOH),
3.03 (s, 3H, CH3), 3.73 (m, 2H, eCH2eCH2eCH2eCH2eOH),
4.34 (t, 2H, eCH2eCH2eCH2eCH2eOH), 7.1e7.7 (m, 8H
arom., 1H ind.). Anal. Calcd for C19H20FNO3S (MW 361.43):
C, 63.14; H, 5.58; N, 3.88%. Found: C, 62.96; H, 5.34; N, 3.62%.
4.1.11. N-(3,4-Dichlorobenzyl)-3-[1-(4-fluorophenyl)-
1H-indol-3-yl]-N-methylpropan-1-amine 1e
1H NMR (CDCl3eTMS) ppm (d): 2.02 (m, 2H, eCH2e
CH2eCH2eNH), 2.25 (s, 3H, CH3), 2.51 (t, 2H, eCH2e
CH2eCH2eNH), 2.85 (t, 2H, eCH2eCH2eCH2eNH), 3.47
(s, 2H, CH2), 7.05 (s, 1H ind.), 7.15e7.69 (m, 11H arom.).
MS: m/z 441 [MHþ], 443 [MHþ þ 2]. Anal. Calcd for
C25H23Cl2FN2 (MW 441.37): C, 68.03; H, 5.25; N, 6.35%.
Found: C, 68.25; H, 5.52; N, 6.19%.
4.1.7. N-Benzyl-3-[1-(4-fluorophenyl)-1H-indol-3-yl]
-N-methyl-propan-1-amine 1a
To 3-[1-(4-fluorophenyl)-1H-indol-3-yl]propy1 methane-
sulfonate 7a (0.5 g, 1.4 mmol) in 50 ml of 4-methyl-2-penta-
none (MIBK), 0.17 g (1.4 mmol) of N-methylbenzylamine
and 0.4 g (2.7 mmol) of potassium carbonate were added.
The mixture was heated under reflux at 150 ꢂC for 16 h. After
cooling, inorganic salts were filtered off and 4-methyl-2-pen-
tanone was evaporated under reduced pressure. The remaining
mixture was treated with ethyl acetate and water and the or-
ganic phase was separated, dried over anhydrous MgSO4
and concentrated in vacuo. The remaining crude oil was puri-
fied by column chromatography on silica gel, eluting with
a 9:1 mixture of CHCl3 and toluene. Yield: 0.20 g (40%).
1H NMR (CDCl3eTMS) ppm (d): 2.05 (m, 2H, eCH2e
CH2eCH2eN), 2.36 (s, 3H, CH3), 2.5 (t, 2H, eCH2eCH2e
CH2eN), 2.91 (t, 2H, eCH2eCH2eCH2eN), 3.65 (s, 2H,
CH2), 7.0 (s, 1H ind.), 7.1e7.3 (m, 13H arom.). MS: m/z
373 [MHþ]. Anal. Calcd for C25H25FN2 (MW 372.48): C,
80.61; H, 6.77; N, 7.52%. Found: C, 80.48; H, 6.92; N, 7.68%.
The following compounds 1bel and 2ael were similarly
obtained. Yields are reported in Table 1.
4.1.12. N-(2-Bromobenzyl)-3-[1-(4-fluorophenyl)-
1H-indol-3-yl]-N-methylpropan-1-amine 1f
1H NMR (CDCl3eTMS) ppm (d): 2.03 (m, 2H, eCH2e
CH2eCH2eNH), 2.33 (s, 3H, CH3), 2.62 (t, 2H, eCH2e
CH2eCH2eNH), 2.89 (t, 2H, eCH2eCH2eCH2eNH), 3.61
(s, 2H, CH2), 7.02 (s, 1H ind.), 7.2e7.7 (m, 12H arom.).
MS: m/z 451 [MHþ], 453 [MHþ þ 2]. Anal. Calcd for
C25H24BrFN2 (MW 451.37): C, 66.52; H, 5.36; N, 6.21%.
Found: C, 66.25; H, 5.53; N, 6.09%.
4.1.13. N-(3-Bromobenzyl)-3-[1-(4-fluorophenyl)-
1H-indol-3-yl]-N-methylpropan-1-amine 1g
1H NMR (CDCl3eTMS) ppm (d): 1.99 (m, 2H, eCH2e
CH2eCH2eNH), 2.26 (s, 3H, CH3), 2.52 (t, 2H, eCH2e
CH2eCH2eNH), 2.87 (t, 2H, eCH2eCH2eCH2eNH), 3.5
(s, 2H, CH2), 7.03 (s, 1H ind.), 7.1e7.66 (m, 12H arom.).
MS: m/z 451 [MHþ], 453 [MHþ þ 2]. Anal. Calcd for
C25H24BrFN2 (MW 451.37): C, 66.52; H, 5.36; N, 6.21%.
Found: C, 66.73; H, 5.22; N, 6.40%.
4.1.8. N-(2-Chlorobenzyl)-3-[1-(4-fluorophenyl)-
1H-indol-3-yl]-N-methylpropan-1-amine 1b
1H NMR (CDCl3eTMS) ppm (d): 2.03 (m, 2H, eCH2e
CH2eCH2eNH), 2.32 (s, 3H, CH3), 2.61 (t, 2H, eCH2e
CH2eCH2eNH), 2.89 (t, 2H, eCH2eCH2eCH2eNH), 3.66
(s, 2H, CH2), 7.02 (s, 1H ind.), 7.2e7.65 (m, 12H arom.).
MS: m/z 407 [MHþ], 409 [MHþ þ 2]. Anal. Calcd for
C25H24ClFN2 (MW 406.92): C, 73.79; H, 5.94; N, 6.88%.
Found: C, 73.58; H, 6.12; N, 7.03%.
4.1.14. N-(4-Bromobenzyl)-3-[1-(4-fluorophenyl)-
1H-indol-3-yl]-N-methylpropan-1-amine 1h
1H NMR (CDCl3eTMS) ppm (d): 1.98 (m, 2H, eCH2e
CH2eCH2eNH), 2.22 (s, 3H, CH3), 2.5 (t, 2H, eCH2e
CH2eCH2eNH), 2.86 (t, 2H, eCH2eCH2eCH2eNH), 3.48
(s, 2H, CH2), 7.03 (s, 1H ind.), 7.19e7.69 (m, 12H arom.).
MS: m/z 451 [MHþ], 453 [MHþ þ 2]. Anal. Calcd for

M.G. Mamolo et al. / European Journal of Medicinal Chemistry 43 (2008) 2073e2081
2079
C25H24BrFN2 (MW 451.37): C, 66.52; H, 5.36; N, 6.21%.
Found: C, 66.64; H, 5.49; N, 6.37%.
eCH2eCH2eCH2eCH2eN), 2.84 (t, 2H, eCH2eCH2e
CH2eCH2eN), 3.56 (s, 2H, CH2), 7.09 (s, 1H ind.), 7.14e
7.93 (m, 12H arom.). MS: m/z 421 [MHþ], 423 [MHþ þ 2].
Anal. Calcd for C26H26ClFN2 (MW 420.95): C, 74.18; H,
6.23; N, 6.65%. Found: C, 74.32; H, 6.36; N, 6.43%.
4.1.15. N-(2-Fluorobenzyl)-3-[1-(4-fluorophenyl)-
1H-indol-3-yl]-N-methylpropan-1-amine 1i
1H NMR (CDCl3eTMS) ppm (d): 2.02 (m, 2H, eCH2e
CH2eCH2eNH), 2.3 (s, 3H, CH3), 2.57 (t, 2H, eCH2e
CH2eCH2eNH), 2.88 (t, 2H, eCH2eCH2eCH2eNH), 3.62
(s, 2H, CH2), 7.06 (s, 1H ind.), 7.1e7.7 (m, 12H arom.).
MS: m/z 391 [MHþ]. Anal. Calcd for C25H24F2N2 (MW
390.37): C, 76.90; H, 5.25; N, 6.35%. Found: C, 76.62; H,
5.40; N, 6.47%.
4.1.21. N-(3-Chlorobenzyl)-4-[1-(4-fluorophenyl)-
1H-indol-3-yl]-N-methylbutan-1-amine 2c
1H NMR (CDCl3eTMS) ppm (d): 1.58e1.91 (m, 4H, e
CH2eCH2eCH2eCH2eNH), 2.21 (s, 3H, CH3), 2.45 (t, 2H,
eCH2eCH2eCH2eCH2eNH), 2.84 (t, 2H, eCH2eCH2e
CH2eCH2eNH), 3.48 (s, 2H, CH2), 7.12 (s, 1H ind.), 7.14e
7.76 (m, 12H arom.). MS: m/z 421 [MHþ], 423 [MHþ þ 2].
Anal. Calcd for C26H26ClFN2 (MW 420.95): C, 74.18; H,
6.23; N, 6.65%. Found: C, 74.01; H, 6.12; N, 6.74%.
4.1.16. N-(3-Methylbenzyl)-3-[1-(4-fluorophenyl)-
1H-indol-3-yl]-N-methylpropan-1-amine 1j
1H NMR (CDCl3eTMS) ppm (d): 2.05 (m, 2H, eCH2e
CH2eCH2eNH), 2.27 (s, 3H, CH3), 2.36 (s, 3H, CH3), 2.54
(t, 2H, eCH2eCH2eCH2eNH), 2.87 (t, 2H, eCH2eCH2e
CH2eNH), 3.51 (s, 2H, CH2), 7.03 (s, 1H ind.), 7.05e7.71
(m, 12H arom.). MS: m/z 387 [MHþ]. Anal. Calcd for
C26H27FN2 (MW 386.50): C, 80.80; H, 7.04; N, 7.25%.
Found: C, 80.92; H, 7.22; N, 7.03%.
4.1.22. N-(4-Chlorobenzyl)-4-[1-(4-fluorophenyl)-
1H-indol-3-yl]-N-methylbutan-1-amine 2d
1H NMR (CDCl3eTMS) ppm (d): 1.60e1.89 (m, 4H, e
CH2eCH2eCH2eCH2eNH), 2.26 (s, 3H, CH3), 2.53 (t, 2H,
eCH2eCH2eCH2eCH2eNH; J ¼ 6.71), 2.84 (t, 2H, e
CH2eCH2eCH2eCH2eNH; J ¼ 7.32), 3.57 (s, 2H, CH2),
7.09 (s, 1H ind.), 7.13e7.73 (m, 12H arom.). MS: m/z 421
[MHþ], 423 [MHþ þ 2]. Anal. Calcd for C26H26ClFN2 (MW
420.95): C, 74.18; H, 6.23; N, 6.65%. Found: C, 74.33; H,
6.41; N, 6.60%.
4.1.17. N-(4-Methylbenzyl)-3-[1-(4-fluorophenyl)-
1H-indol-3-yl]-N-methylpropan-1-amine 1k
1H NMR (CDCl3eTMS) ppm (d): 2.12 (m, 2H, eCH2e
CH2eCH2eNH), 2.3 (s, 3H, CH3), 2.4 (s, 3H, CH3), 2.65 (t,
2H, eCH2eCH2eCH2eNH), 2.85 (t, 2H, eCH2eCH2e
CH2eNH), 3.65 (s, 2H, CH2), 7.06 (s, 1H ind.), 7.1e7.7 (m,
12H arom.). MS: m/z 387 [MHþ]. Anal. Calcd for
C26H27FN2 (MW 386.50): C, 80.80; H, 7.04; N, 7.25%.
Found: C, 80.66; H, 6.90; N, 7.38%.
4.1.23. N-(3,4-Dichlorobenzyl)-4-[1-(4-fluorophenyl)-
1H-indol-3-yl]-N-methylbutan-1-amine 2e
1H NMR (CDCl3eTMS) ppm (d): 1.56e1.93 (m, 4H, e
CH2eCH2eCH2eCH2eNH), 2.20 (s, 3H, CH3), 2.42 (t, 2H,
eCH2eCH2eCH2eCH2eNH), 2.83 (t, 2H, eCH2eCH2e
CH2eCH2eNH), 3.42 (s, 2H, CH2), 7.04e7.76 (m, 11H
arom., 1H ind.). MS: m/z 455 [MHþ], 457 [MHþ þ 2]. Anal.
Calcd for C26H25Cl2FN2 (MW 455.39): C, 68.57; H, 5.53;
N, 6.15%. Found: C, 68.72; H, 5.40; N, 6.02%.
4.1.18. N-(2,4-Dimethylbenzyl)-3-[1-(4-fluorophenyl)-
1H-indol-3-yl]-N-methylpropan-1-amine 1l
1H NMR (CDCl3eTMS) ppm (d): 2.07 (m, 2H, eCH2e
CH2eCH2eNH), 2.29 (s, 3H, CH3), 2.34 (s, 3H, CH3), 2.42
(s, 3H, CH3), 2.58 (t, 2H, eCH2eCH2eCH2eNH), 2.89 (t,
2H, CH2eCH2eCH2eNH), 3.54 (s, 2H, CH2), 6.98 (s, 1H
ind.), 7.04e7.7 (m, 11H arom.). MS: m/z 401 [MHþ]. Anal.
Calcd for C27H29FN2 (MW 400.53): C, 80.96; H, 7.30; N,
6.99%. Found: C, 80.71; H, 7.04; N, 7.14%.
4.1.24. N-(2-Bromobenzyl)-4-[1-(4-fluorophenyl)-
1H-indol-3-yl]-N-methylbutan-1-amine 2f
1H NMR (CDCl3eTMS) ppm (d): 1.60e1.93 (m, 4H, e
CH2eCH2eCH2eCH2eNH), 2.24 (s, 3H, CH3), 2.54 (t, 2H,
eCH2eCH2eCH2eCH2eNH), 2.85 (t, 2H, eCH2eCH2e
CH2eCH2eNH), 3.61 (s, 2H, CH2), 7.04e7.78 (m, 12H
arom., 1H ind.). MS: m/z 465 [MHþ], 467 [MHþ þ 2]. Anal.
Calcd for C26H26BrFN2 (MW 465.40): C, 67.10; H, 5.63; N,
6.02%. Found: C, 67.38; H, 5.42; N, 5.89%.
4.1.19. N-Benzyl-4-[1-(4-fluorophenyl)-
1H-indol-3-yl]-N-methyl-butan-1-amine 2a
1H NMR (CDCl3eTMS) ppm (d): 1.55e1.89 (m, 4H, e
CH2eCH2eCH2eCH2eN), 2.17 (s, 3H, CH3), 2.46 (t, 2H,
eCH2eCH2eCH2eCH2eN), 2.77 (t, 2H, eCH2eCH2e
CH2eCH2eN), 3.51 (s, 2H, CH2), 7.09 (s, 1H ind.), 7.11e
7.77 (m, 13H arom.). MS: m/z 387 [MHþ]. Anal. Calcd for
C26H27FN2 (MW 386.50): C, 80.80; H, 7.04; N, 7.25%.
Found: C, 80.61; H, 7.26; N, 7.10%.
4.1.25. N-(3-Bromobenzyl)-4-[1-(4-fluorophenyl)-
1H-indol-3-yl]-N-methylbutan-1-amine 2g
1H NMR (CDCl3eTMS) ppm (d): 1.56e1.91 (m, 4H, e
CH2eCH2eCH2eCH2eNH), 2.22 (s, 3H, CH3), 2.47 (t, 2H,
eCH2eCH2eCH2eCH2eNH; J ¼ 6.71), 2.84 (t, 2H, e
CH2eCH2eCH2eCH2eNH; J ¼ 7.32), 3.49 (s, 2H, CH2),
7.10 (s, 1H ind.), 7.16e7.83 (m, 12H arom.). MS: m/z 465
[MHþ], 467 [MHþ þ 2]. Anal. Calcd for C26H26BrFN2 (MW
465.40): C, 67.10; H, 5.63; N, 6.02%. Found: C, 67.01; H,
5.73; N, 6.13%.
4.1.20. N-(2-Chlorobenzyl)-4-[1-(4-fluorophenyl)-
1H-indol-3-yl]-N-methylbutan-1-amine 2b
1H NMR (CDCl3eTMS) ppm (d): 1.56e1.90 (m, 4H, e
CH2eCH2eCH2eCH2eN), 2.20 (s, 3H, CH3), 2.53 (t, 2H,

2080
M.G. Mamolo et al. / European Journal of Medicinal Chemistry 43 (2008) 2073e2081
4.1.26. N-(4-Bromobenzyl)-4-[1-(4-fluorophenyl)-
1H-indol-3-yl]-N-methylbutan-1-amine 2h
stock solutions of the compounds were prepared in 100%
DMSO and diluted with 50 mM TriseHCl buffer on the day
of the experiment. The final DMSO concentration in the incu-
bation tubes was 0.1%.
1H NMR (CDCl3eTMS) ppm (d): 1.53e1.87 (m, 4H, e
CH2eCH2eCH2eCH2eNH), 2.19 (s, 3H, CH3), 2.43 (t, 2H,
eCH2eCH2eCH2eCH2eNH), 2.81 (t, 2H, eCH2eCH2e
CH2eCH2eNH), 3.43 (s, 2H, CH2), 7.07 (s, 1H ind.), 7.12e
7.89 (m, 12H arom.). MS: m/z 465 [MHþ], 467 [MHþ þ 2].
Anal. Calcd for C26H26BrFN2 (MW 465.40): C, 67.10; H,
5.63; N, 6.02%. Found: C, 67.22; H, 5.54; N, 6.26%.
For s2 receptor assays, 150 mg of rat liver homogenate was in-
cubated in 50 mM TriseHCl, pH 8.0, in the presence of 100 nM
(þ)-pentazocine, 0.5 ml final volume. After 120 min at room
temperature,incubationwasstoppedbyrapidfiltrationunder vac-
uum on GF/B filters pre-soaked in 0.5% polyethylenimine. For
saturation analysis, increasing concentrations (0.3e300 nM) of
[3H]-DTG (PerkineElmer, specific activity 58.1 Ci/mmol)
were used in the absence (total binding) and in the presence (non-
specific binding) of 10 mM haloperidol. Kd and Bmax values were
calculated by nonlinear regression using the SigmaPlot software
and corresponded to 19.7 ꢁ 3 nM and 8263 ꢁ 420 fmol/mg pro-
tein, respectively (mean ꢁ SEM of 3 experiments performed in
duplicate). For competition experiments, a final concentration
of 3 nM [3H]-DTG was used and unlabelled DTG (3, 30 and
300 nM) was assayed as internal standard. When estimated,
IC50 values were obtained using 11 increasing concentrations of
the tested compound (0.1e10.000 nM). The corresponding Ki
values were calculated by means of the ChengePrusoff equation,
and utilizing the experimental one as the Kd value. The Ki values
for the test compounds represent the mean ꢁ SE of three separate
determinations performed in duplicate.
For s1 receptor assays, 250 mg of rat liver homogenate was
incubated in 50 mM TriseHCl, pH 8.0, in 0.5 ml final volume.
After 120 min at 37 ꢂC the reactions were terminated as de-
scribed above. For saturation analysis, increasing concentra-
tions (0.3e50 nM) of [3H]-(þ)-pentazocine (PerkineElmer,
specific activity 34.9 Ci/mmol) were used in the absence (total
binding) and in the presence (nonspecific binding) of 10 mM
haloperidol. The calculated Kd and Bmax values were
16.7 ꢁ 4 nM and 4842 ꢁ 409 fmol/mg protein, respectively
(mean ꢁ SEM of 3 experiments performed in duplicate). For
competition experiments, a final concentration of 1 nM [3H]-
(þ)-pentazocine was used and unlabelled (þ)-pentazocine
(1, 10 and 100 nM) was assayed as internal standard. The Ki
values of selected compounds were obtained as described
above.
4.1.27. N-(2-Fluorobenzyl)-4-[1-(4-fluorophenyl)-
1H-indol-3-yl]-N-methylbutan-1-amine 2i
1H NMR (CDCl3eTMS) ppm (d): 1.60e1.95 (m, 4H, e
CH2eCH2eCH2eCH2eNH), 2.24 (s, 3H, CH3), 2.49 (t, 2H,
eCH2eCH2eCH2eCH2eNH), 2.85 (t, 2H, eCH2eCH2e
CH2eCH2eNH), 3.60 (s, 2H, CH2), 6.95e7.79 (m, 12H
arom., 1H ind.). MS: m/z 405 [MHþ]. Anal. Calcd for
C26H26BrFN2 (MW 404.49): C, 77.20; H, 6.48; N, 6.93%.
Found: C, 77.41; H, 6.33; N, 7.08%.
4.1.28. N-(3-Methylbenzyl)-4-[1-(4-fluorophenyl)-
1H-indol-3-yl]-N-methylbutan-1-amine 2j
1H NMR (CDCl3eTMS) ppm (d): 1.61e1.91 (m, 4H, e
CH2eCH2eCH2eCH2eNH), 2.17 (s, 3H, CH3), 2.37 (s, 3H,
CH3), 2.47 (t, 2H, eCH2eCH2eCH2eCH2eNH), 2.85 (t,
2H, eCH2eCH2eCH2eCH2eNH), 3.49 (s, 2H, CH2), 7.09
(s, 1H ind.), 7.13e7.75 (m, 12H arom.). MS: m/z 401
[MHþ]. Anal. Calcd for C27H29FN2 (MW 400.53): C, 80.96;
H, 7.30; N, 6.99%. Found: C, 81.12; H, 7.06; N, 7.15%.
4.1.29. N-(4-Methylbenzyl)-4-[1-(4-fluorophenyl)-
1H-indol-3-yl]-N-methylbutan-1-amine 2k
1H NMR (CDCl3eTMS) ppm (d): 1.69e1.99 (m, 4H, e
CH2eCH2eCH2eCH2eNH), 2.35 (s, 3H, CH3), 2.46 (s, 3H,
CH3), 2.70 (t, 2H, eCH2eCH2eCH2eCH2eNH; J ¼ 6.71),
2.85 (t, 2H, eCH2eCH2eCH2eCH2eNH; J ¼ 7.32), 3.82
(s, 2H, CH2), 7.05e7.72 (m, 12H arom., 1H ind.). MS: m/z
401 [MHþ]. Anal. Calcd for C27H29FN2 (MW 401.53): C,
80.96; H, 7.30; N, 6.99%. Found: C, 80.81; H, 7.41; N, 6.80%.
4.1.30. N-(2,4-Dimethylbenzyl)-4-[1-(4-fluorophenyl)-
1H-indol-3-yl]-N-methylbutan-1-amine 2l
1H NMR (CDCl3eTMS) ppm (d): 1.56e1.99 (m, 4H, e
CH2eCH2eCH2eCH2eNH), 2.20 (s, 3H, CH3), 2.31 (s, 3H,
CH3), 2.35 (s, 3H, CH3), 2.47 (t, 2H, eCH2eCH2eCH2e
CH2eNH), 2.82 (t, 2H, eCH2eCH2eCH2eCH2eNH), 3.44
(s, 2H, CH2), 6.93e7.84 (m, 11H arom., 1H ind.). MS: m/z
401 [MHþ]. Anal. Calcd for C28H31FN2 (MW 414.56): C,
81.12; H, 7.54; N, 6.76%. Found: C, 80.93; H, 7.32; N, 6.91%.
Acknowledgment
This research was carried out with the financial support
from Italian Ministry for University and Research (MIUR),
Rome, Italy (PRIN 2003).
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