Characterization of novel N,N'-disubstituted piperazines as σ receptor ligands

Article abstract of DOI:10.1021/jm980143k

σ Receptors have been the focus of extensive studies because of their potential functional role in several important physiological and biochemical processes. To further evaluate the properties of σ receptors, especially σ- 1 and σ-2 subtypes, we have synt

Full text of DOI:10.1021/jm980143k

4950
J . Med. Chem. 1998, 41, 4950-4957
Ch a r a cter iza tion of Novel N,N′-Disu bstitu ted P ip er a zin es a s σ Recep tor Liga n d s
Ying Zhang, Wanda Williams, Cheryl Torrence-Campbell, Wayne D. Bowen, and Kenner C. Rice*
Laboratory of Medicinal Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases,
National Institutes of Health, 8 Center Drive, MSC 0815, Bethesda, Maryland 20892-0815
Received March 9, 1998
σ Receptors have been the focus of extensive studies because of their potential functional role
in several important physiological and biochemical processes. To further evaluate the properties
of σ receptors, especially σ-1 and σ-2 subtypes, we have synthesized a series of N,N′-disubstituted
piperazine compounds (1-32). The design of these compounds was based upon the early
structure-activity relationship (SAR) studies of the minimum structural requirements of a
molecule necessary to elicit σ receptor binding activity. In the N-(3-phenylpropyl)piperazine
series, compounds with the ethylenediamine moiety (8-11, 15-17) showed 6-20-fold higher
affinity for σ-1 and 2-40-fold higher affinity for σ-2 relative to their corresponding amides
(1-7). The (m-nitrophenethyl)piperazine 10 exhibits a subnanomolar affinity for the σ-1 site,
whereas the corresponding o-nitro compound 9 shows the highest affinity for the σ-2 site (Ki )
4.9 nM). Compounds with a free amino terminus were designed as precursors for use as
bioconjugated affinity compounds. Some of these compounds displayed high affinity for σ-1
and moderate affinity for σ-2 sites and are currently used for the purification and characteriza-
tion of the receptor subtypes.
Ch a r t 1
In tr od u ction
σ Receptors are involved in many important biological
and physiological processes. Some of these include
regulation of motor behavior and postural tone,1-3
negative modulation of the phosphoinositide response
to muscarinic cholinergic agonists,4,5 neuroprotective6,7
and neurodegenerative8-10 effects, and modulation of
glutamatergic and dopaminergic neurotransmission.11,12
A wide array of structurally diverse compounds have
been reported to bind to σ receptors. These include
benzomorphans,13 disubstituted guanidines,14,15 substi-
tuted ethylenediamines,16 substituted piperidines,17 and
substituted piperazines.18,19 However, most of those
compounds showed cross-reactivity with other receptors,
such as dopamine D2, PCP, or opioid receptors.20 For
example, the 6,7-benzomorphan (+)-pentazocine is one
of the compounds that binds selectively to σ receptors
with high affinity,21 whereas the 6,7-benzomorphans as
a class show cross-reactivity with PCP receptors.13
Haloperidol, a neuroleptic agent, binds with comparable
affinity to dopamine D2 receptors and σ sites.22 Thus,
it has been a challenge to develop selective σ ligands
that do not cross-react with the other receptor systems.
Furthermore, the study of σ receptors has been compli-
cated by the discovery of subtypes based partly on the
differential binding properties of structurally diverse σ
ligands.23,24 The existence of at least two σ subtypes:
σ-1 and σ-2 sites, has been proposed.25 The σ-1 site has
been implicated to regulate gastrointestinal effects,26
mediate the inhibition by σ ligands of the muscarinic
acetylcholine receptor phosphoinositide response,5 modu-
late NMDA-stimulated neurotransmitter release,11,12
and mediate neuroprotective and antiamnesic effects of
σ ligands.27 The σ-2 site has shown the ability to
mediate the motor effects of σ ligands,3,28 mediate the
effects of certain σ drugs on K+ channels,29,30 modulate
intracellular calcium levels,31,32 and mediate σ ligand-
induced morphological changes and cell death.33 How-
ever the functional roles of σ-1 and σ-2 receptor sites
are far from fully understood at the present time and
have yet to be characterized using more selective and
high-affinity σ ligands. Therefore, identifying novel
classes of high-affinity, subtype-selective σ ligands has
been an ongoing task.
It has been reported that the cis isomers (-)- and (+)-I
(Chart 1) of the κ-selective agonist U50,488 displayed
moderate affinity for the σ sites (labeled with [3H]-(+)-
PPP), showing Ki values of 81 and 211 nM,34 whereas
their reduction products cyclohexanediamines (-)- and
(+)-II exhibited high affinity against displacement of
10.1021/jm980143k This article not subject to U.S. Copyright. Published 1998 by the American Chemical Society
Published on Web 11/12/1998

Disubstituted Piperazines as σ Receptor Ligands
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 25 4951
Ch a r t 2
Sch em e 1a
[3H]-(+)-PPP (Ki values of 1.3 and 6.0 nM, respec-
tively).16 The higher-affinity isomer (-)-II shows higher
selectivity for the σ sites and does not cross-react with
κ, dopamine D2, or PCP receptors. Further manipula-
tion of the cyclohexyl moiety of II yielded a novel class
of highly specific σ ligand III, N-[2-(3,4-dichlorophenyl)-
ethyl]-N-methyl-2-(1-pyrrolidinyl)ethylamine, which binds
to both σ-1 and σ-2 sites with nanomolar affinity (Ki-
(σ1) ) 2.1 nM, Ki(σ2) ) 8.1 nM).16 Since then, much
effort has been exerted in the studies of structure-
activity relationships (SAR) and in seeking novel classes
of σ ligands to further delineate the biological functions
of the receptors. The earlier SAR studies on III yielded
a series of analogues of III derived from incorporating
III into more conformationally restricted structures
such as piperazines, 2-aminotetralines, and rigid bicyclic
bridgehead ring systems or from modifying the ethylene
spacer and pyrrolidinyl moiety.16,18 These studies in-
dicated that an ethylenediamine moiety is the minimal
structural requirement to achieve high affinity for σ
receptor sites. To extend the SAR studies on the
ethylenediamine series, we prepared a series of novel
N, N′-disubstituted piperazines (Chart 2) and investi-
gated their binding properties on both σ-1 and σ-2 sites.
Portions of this work have been previously reported in
abstract form.35
a
(a) 3-Phenylpropyl chloride, K2CO3, DMF; (b) mono- or disub-
stituted phenylacetic acid, DCC, CH2Cl2, rt; (c) 1 or 0.5 M AlH3,
THF.
Sch em e 2a
Ch em istr y
a
The preparation of compounds 1-17 and 24-32
proceeded from a monoalkylation of commercial pipera-
zine with 1-chloro-3-phenylpropane (Scheme 1).36 The
resulting N-(3-phenylpropyl)piperazine was purified as
the oxalate from methanol in 79% yield. The free base
of N-(3-phenylpropyl)piperazine was then acylated with
mono- or disubstituted phenylacetic acid in the presence
of dicyclohexylcarbodiimide (DCC) in methylene chloride
at room temperature to give compounds 1-7, followed
by AlH3 reduction16,37 to give compounds 8-17. Com-
pounds 1-17 were purified from methanol or acetone
as their oxalates.
(a) o-, m-, or p-Methoxyphenylacetic acid, DCC, CH2Cl2, rt;
(b) 1 M AlH3 in THF, rt.
acid in chloroform to afford the amides 27-29. Alane
reduction of these compounds in THF provided the
corresponding triamines 30-32. Compounds 24-32
were purified as their oxalate salts from methanol or
acetone.
The symmetrical disubstituted piperazine derivatives
18-23 was prepared from a coupling reaction between
o-, m-, or p-methoxyphenylacetic acid and piperazine to
give the diamides 18-20, followed by amide reduction
to afford 21-24 (Scheme 2). The purification of the
diamines 21-24 was achieved by salt formation with
maleic acid in ethanol or methanol. The structure and
purity of these compounds were confirmed by CI-MS,
N-(3-Phenylpropyl)piperazine was acylated with N-
Boc-protected amino acids, Boc-glycine, Boc-â-alanine,
or Boc-GABA, to give 24-26 (Scheme 3), followed by
the deprotection of amino group in 30% trifluoroacetic

4952 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 25
Zhang et al.
Sch em e 3a
σ-1 and σ-2 receptors tolerate the amide functional
group in the disubstituted piperazine series far better
than the N-pyrrolidinylethylamine series. In the N-
pyrrolidinylethylamine series, the σ-1 affinity decreased
75-350-fold simply by replacing the arylethylamine
moiety with an arylacetamide, although affinity for the
σ-2 subtype dropped only 4.8-22-fold.38 In comparison,
only 6-20-fold reduction for σ-1 affinity and 2-40-fold
reduction for σ-2 affinity were observed in the piperazine
series. De Costa et al. reported earlier that in the
piperazine series containing the N′-3,4-dichlorophen-
ethyl substituent,18 increasing the size of the N-sub-
stituent from methyl to pentyl resulted in an increase
in affinity with optimal binding occurring at butyl. In
the current piperazine series, the σ-1 subtype well-
tolerates further increase in size of the N-substituent
to a phenylpropyl (8, Ki(σ-1) ) 2.2 nM), showing an
affinity comparable to the N-pentylpiperazine derivative
(Ki(σ-1) ) 1.7 nM). Considering the fact that the N-nor
compound in the de Costa series showed a Ki (σ-1) value
of 119 nM, a 70-fold decrease relative to the N-pen-
tylpiperazine derivative in σ-1 binding, we conclude that
a hydrophobic substituent is necessary on this nitrogen
for strong binding interaction for the σ-1 site.
In the symmetrical piperazine series (18-23), the
pattern is evident that the diamine compounds possess
higher affinity for the σ receptors than their corre-
sponding amide derivatives, showing as high as 1200-
fold improvement in σ-1 binding. It can be concluded
that at least one basic nitrogen is necessary to maintain
the high-affinity binding for the σ receptors in the
piperazine series. In this series, it also follows that the
ethylenediamine compounds show higher affinity for the
σ-1 subtype than the σ-2, with the only exception being
that compound 21, bis(o-methoxyphenethyl)piperazine,
showed a Ki value of 16.7 nM for the σ-2 site, 8.7-fold
selective for σ-2 relative to σ-1. This compound is by
far the most selective compound for the σ-2 binding site
among the ethylenediamine series. Considering the fact
that ditoluylguanidine (DTG) possesses similar affinity
for σ-1 and σ-2 sites, the structural similarity of this
compound to DTG probably plays an important role.
a
(a) Boc-protected amino acid, DCC, CH2Cl2, rt; (b) 30% TFA
in CHCl3, rt; (c) 1 M AlH3 in THF, rt.
1H NMR (300 MHz), and elemental analysis. The
physical and chemical data of compounds 1-32 are
reported in Tables 1-3.
Resu lts a n d Discu ssion
As a continuation of the SAR study on the ethylene-
diamine σ ligands, we have synthesized a series of N,N′-
disubstituted piperazine compounds and evaluated their
binding properties for σ-1 and σ-2 receptors. In the
N-(3-phenylpropyl)piperazine series (1-17), the com-
pounds with the ethylenediamine moiety (8-11, 15-
17) showed 6-20-fold higher affinity for σ-1 and 2-40-
fold higher affinity for σ-2 relative to their corresponding
amides (1-7). This notion coincides with our observa-
tion in the N-(arylethyl)-N-methyl-2-(1-pyrrolidinyl)-
ethylamine series38 that compounds with the ethylene-
diamine moiety possess an overall higher affinity for
both σ-1 and σ-2 subtypes than their corresponding
arylacetamides.
As demonstrated in our prior study,38 the nitro
compounds, once again, showed the highest affinity for
both σ-1 and σ-2 among the compounds with other
aromatic substitutions. Compound 9, with a nitro
substitution at the ortho position, showed an affinity of
4.9 nM at σ-2, the highest binding affinity for the σ-2
site among the known ethylenediamine compounds,
whereas 10, a m-nitro compound showed a subnano-
molar affinity for the σ-1 site. However, unlike the N-(1-
pyrrolidinyl)ethylamine series,38 the presence of an
electron-donating group, such as a methoxy or an amino
function in the aromatic region, shows only minimal
affects on the binding affinity for σ receptors. As shown
in Table 4, compounds 12-17 containing electron-
donating groups showed 1.3-4.8 nM affinity for σ-1 and
7.5-37 nM affinity for σ-2, while compounds with an
electron-withdrawing group (9-11) showed affinities of
0.72-2.6 nM for σ-1 and 4.9-24.9 nM for σ-2.
Compounds 24-32 were designed as precursors for
use as bioconjugated affinity compounds. By coupling
the high-affinity disubstituted piperazine derivatives
with a column matrix or affinity probe such as biotin,
the macromolecules could be useful in the purification
and identification of σ receptor subtypes. All the
compounds of this series have a fixed alkyl group on
one of the piperazine nitrogens, but with various lengths
of the carbon chain on the other nitrogen. Compounds
with a shorter chain seem to favor σ-1 binding as both
the triamine and diamine amide compounds 30 and 27
showed high binding affinity (2.6 and 4.1 nM, respec-
tively). These two compounds also showed relatively
higher affinity for the σ-2 site compared to the rest of
the triamines with a longer carbon chain. Of the nine
compounds in this series, 26 showed an exceptionally
high affinity for the σ-1 site, with Ki(σ-1) ) 0.32 nM.
This appears to be due to derivatization of the basic
alkyl nitrogen when the σ-1 affinity of the free amine
compound 29 is compared. The same pattern is ob-
served in a comparison of compound 25 to 28. However,
at this point, more structurally related compounds are
Similar to observations in the N-(1-pyrrolidinyl)-
ethylamine series,38 no clear pattern was observed
between the orientation of aromatic substituents and
the σ binding activity in both diamine compounds (8-
17) and amine amide compounds (1-7). However, both

Disubstituted Piperazines as σ Receptor Ligands
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 25 4953
Ta ble 1. Physical and Chemical Data of Compounds 1-17
no.
method
X
R
salt
solvent
mp (°C)
analysisa,c
1b
2b
A
A
A
A
A
A
A
D
D
D
D
C
C
C
C
C
C
CdO
CdO
CdO
CdO
CdO
CdO
CdO
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
3,4-di-Cl
o-NO2
m-NO2
p-NO2
o-OCH3
m-OCH3
p-OCH3
3,4-di-Cl
o-NO2
m-NO2
p-NO2
o-NH2
m-NH2
p-NH2
o-OCH3
m-OCH3
p-OCH3
oxalate
oxalate
oxalate
oxalate
oxalate
oxalate
oxalate
oxalate
oxalate
oxalate
oxalate
oxalate
oxalate
oxalate
oxalate
oxalate
oxalate
acetone
acetone
acetone
acetone
acetone
acetone
acetone
methanol
methanol
methanol
methanol
methanol
methanol
methanol
methanol
methanol
methanol
183-4
C21H24N2Cl2O‚C2H2O4
C21H25N3O3‚C2H2O4
C21H25N3O3‚C2H2O4
C21H25N3O3‚C2H2O4
C22H28N2O2‚C2H2O4
C22H28N2O2‚C2H2O4
C22H28N2O2‚C2H2O4
C21H26N2Cl2‚2C2H2O4
C21H27N3O2‚2C2H2O4
C21H27N3O2‚2C2H2O4
C21H27N3O2‚2C2H2O4
C21H29N3‚2C2H2O4
153-4
3b
180-1
4b
174-5
5b
171-2
6b
150-1
7b
128-30
219-21
221 dec
220 dec
220 dec
214 dec
200 dec
209 dec
227 dec
226 dec
223 dec
8b
9b
10b
11b
12b
13b
14b
15b
16b
17b
C21H29N3‚2C2H2O4
C21H29N3‚2C2H2O4‚H2O
C22H30N2O‚2C2H2O4
C22H30N2O‚2C2H2O4
C22H30N2O‚2C2H2O4
a
b
Elemental compositions (%) were found to be within (0.4% of the theoretical values of C, H and N. The 1H NMR data for the free
base of this compound are shown in the Experimental Section. c All compounds gave CI-MS consistent with the expected structure.
Ta ble 2. Physical and Chemical Data of Compounds 18-23
no.
method
X
R
salt
solvent
mp (°C)
analysisa,c
C22H26N2O4
C22H26N2O4
C22H26N2O4
C22H30N2O2‚2C4H4O4
C22H30N2O2‚2C4H4O4
C22H30N2O2‚2C4H4O4
18b
19b
20b
21b
22b
23b
B
B
B
C
C
C
CdO
CdO
CdO
CH2
CH2
CH2
o-OCH3
m-OCH3
p-OCH3
o-OCH3
m-OCH3
p-OCH3
ethanol/water
ethanol
ethanol
ethanol
ethanol
173-4
132-3
155-6
197-8
193-5
202-3
maleate
maleate
maleate
methanol
a
b
Elemental compositions (%) were found to be within (0.4% of the theoretical values of C, H, and N. The 1H NMR data for the free
base of this compound are shown in the Experimental Section. c All compounds gave CI-MS consistent with the expected structure.
Ta ble 3. Physical and Chemical Data of Compounds 24-32
no.
method
X
n
R
salt
solvent
acetone
acetone
acetone
acetone
acetone
acetone/methanol
methanol
acetone
mp (°C)
analysisa,c
24b
25b
26b
27b
28b
29b
30b
31b
32b
A
A
A
E
E
E
C
C
C
CdO
CdO
CdO
CdO
CdO
CdO
CH2
1
2
3
1
2
3
1
2
3
t-Boc
t-Boc
t-Boc
H
oxalate
oxalate
oxalate
oxalate
oxalate
oxalate
oxalate
oxalate
oxalate
145-6
64-6
C20H31N3O3‚C2H2O4
C21H33N3O3‚C2H2O4
143-4
180-1
110-2
145-6
215-6
209-10
202-3
C22H35N3O3‚C2H2O4‚0.25H2O
C15H23N3O‚2C2H2O4‚0.5 acetone
C16H25N3O‚2C2H2O4‚H2O
C17H27N3O‚2C2H2O4
H
H
H
H
C15H25N3‚3C2H2O4
CH2
CH2
C16H27N3‚2C2H2O4
H
methanol
C17H29N3‚2C4H4O4
a
b
Elemental compositions (%) were found to be within (0.4% of the theoretical values of C, H, and N. The 1H NMR data for the free
base of this compound are shown in the Experimental Section. c All compounds gave CI-MS consistent with the expected structure.
needed for the further SAR study to delineate the cause
of the high binding affinity of this compound.
In conclusion, we have demonstrated a novel class of
high-affinity ligands for σ-1 and σ-2 receptors. Some
of these compounds may prove useful in functional
studies and purification of the receptors.
reported uncorrected. Elemental analyses were obtained from
Atlantic Microlabs, Atlanta, GA, or Galbraith Laboratories,
Inc., Knoxville, TN, and were determined to be within (0.4%
of the theoretical values for carbon, hydrogen, and nitrogen.
CI-MS (chemical ionization mass spectra) were performed
using a Finnigan 1015 mass spectrometer. 1H NMR spectra
were obtained on a Varian XL-300 spectrometer using CDCl3
solutions of free bases. All the chemical shifts reported are
relative to a tetramethylsilane internal reference in ppm on
the δ scale. Thin-layer chromatography (TLC) was performed
on Analtech GHLF silica gel plates (250 µm) with a solvent
system of 90:9:1 CHCl3/MeOH/concentrated NH4OH.
Exp er im en ta l Section
Ch em ica l Ma ter ia ls a n d Meth od s. Melting points were
determined on a Mel-Temp II capillary apparatus and are

4954 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 25
Ta ble 4. Biological Data for 1-32a
Zhang et al.
Ki([3H]-(+)-pent)
in guinea pig
brain (σ1) (nM)
Ki([3H]-DTG +
dextrallorphan) in
rat liver (σ2) (nM)
no.
R1
R2
n
X
σ1/σ2
1
2
3
4
5
6
7
8
9
Ph(CH2)3
Ph(CH2)3
Ph(CH2)3
Ph(CH2)3
Ph(CH2)3
Ph(CH2)3
Ph(CH2)3
Ph(CH2)3
Ph(CH2)3
Ph(CH2)3
Ph(CH2)3
Ph(CH2)3
Ph(CH2)3
Ph(CH2)3
Ph(CH2)3
Ph(CH2)3
Ph(CH2)3
o-methoxyphenethyl
m-methoxyphenethyl
p-methoxyphenethyl
o-methoxyphenethyl
m-methoxyphenethyl
p-methoxyphenethyl
Ph(CH2)3
3,4-dichlorophenyl
o-nitrophenyl
m-nitrophenyl
p-nitrophenyl
o-methoxyphenyl
m-methoxyphenyl
p-methoxyphenyl
3,4-dichlorophenyl
o-nitrophenyl
m-nitrophenyl
p-nitrophenyl
o-aminophenyl
m-aminophenyl
p-aminophenyl
o-methoxyphenyl
m-methoxyphenyl
p-methoxyphenyl
o-methoxyphenyl
m-methoxyphenyl
p-methoxyphenyl
o-methoxyphenyl
m-methoxyphenyl
p-methoxyphenyl
NHBoc
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
3
1
2
3
1
2
3
CdO
CdO
CdO
CdO
CdO
CdO
CdO
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CdO
CdO
CdO
CH2
CH2
CH2
CdO
CdO
CdO
CdO
CdO
CdO
CH2
CH2
CH2
28 ( 1
7.9 ( 1.2
7.6 ( 0.6
14 ( 1
78 ( 3
76 ( 11
65 ( 0.2
167 ( 4
294 ( 10
206 ( 2
464 ( 34
39 ( 2
4.9 ( 0.01
9.4 ( 1.5
25 ( 2
18 ( 0.2
37 ( 1
14 ( 1
7.5 ( 0.2
10 ( 1
29 ( 2
>10 µM
11.8 ( 1.5
744 ( 35
17 ( 2
29 ( 3
99 ( 4
0.4
0.1
0.1
0.08
0.2
0.1
0.03
0.06
0.2
0.08
0.1
0.3
0.05
0.3
56 ( 8
26 ( 0.01
13 ( 2
2.2 ( 0.2
1.2 ( 0.04
0.72 ( 0.14
2.6 ( 0.8
4.8 ( 0.1
2.0 ( 0.3
4.1 ( 0.02
3.9 ( 0.6
1.4 ( 0.2
1.3 ( 0.3
1465 ( 220
NAb
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
0.5
0.1
0.05
<0.15
NAb
>14
8.7
>10 µM
145 ( 8
5.3 ( 0.7
8.2 ( 0.9
NAb
0.2
0.08
NAb
0.02
0.003
0.01
0.08
0.05
0.01
0.09
0.2
175 ( 38
4561 ( 537
126 ( 14
303 ( 62
1783 ( 103
3839 ( 72
188 ( 13
2000 ( 334
999 ( 19
Ph(CH2)3
Ph(CH2)3
Ph(CH2)3
Ph(CH2)3
Ph(CH2)3
Ph(CH2)3
Ph(CH2)3
Ph(CH2)3
NHBoc
NHBoc
NH2
NH2
NH2
NH2
NH2
NH2
86 ( 5
0.32 ( 0.07
4.1 ( 1.2
148 ( 6
202 ( 41
2.6 ( 0.09
181 ( 16
177 ( 16
a
Twelve concentrations of unlabeled test ligand ranging from 0.05 to 10 000 nM or from 0.5 to 100 000 nM were incubated with guinea
pig brain membranes and [3H]-(+)-pentazocine (σ-1 receptors) or with rat liver membranes and [3H]DTG in the presence of 1 µM
dextrallorphan (σ-2 receptors) as described in the Experimental Section. IC50 values were determined using the iterative curve-fitting
program GraphPAD InPlot (San Diego, CA). IC50 values were then converted to apparent Ki values using the Cheng-Prusoff equation and
b
radioligand Kd values. Values are the averages of 2-3 experiments, ( SEM. Each experiment was carried out in duplicate. Data not
available.
N-(3-P h en ylp r op yl)p ip er a zin e. This compound was pre-
pared according to the reported method with modification.36
To a suspension of 50 g (0.58 mol, 8 equiv) of piperazine in
100 mL of DMF was added 10.0 mL (70 mmol) of 1-chloro-3-
phenylpropane dropwise. The mixture was stirred at room
temperature for 2 days, and a yellow-colored solution with
precipitate was obtained. The solvent was then evaporated
on a rotavapor, and the obtained residue was dissolved in 150
mL of 10% aqueous NaOH solution. The aqueous layer was
extracted with methylene chloride (50 mL) four times; then
the combined organic layer was washed with water twice and
saturated aqueous NaCl once, dried over Na2SO4, and evapo-
rated to give 13.2 g (64.7 mmol) of crude product as a light-
yellow-colored liquid (92%). This compound was purified
through the oxalate from methanol to provide 21.1 g (78.5%)
of salt as a white solid.
Gen er a l Meth od A. To a stirred solution of DCC in CH2-
Cl2 (5 mmol of DCC in 20 mL of CH2Cl2) was added a solution
of mono- or disubstituted phenylacetic acid or N-Boc-protected
amino acid (5 mmol of carboxylic acid to 20 mL of CH2Cl2).
The mixture was allowed to stir at room temperature for 10
min before a solution of N-(3-phenylpropyl)piperazine in CH2-
Cl2 (5 mmol of the free base in 10 mL of CH2Cl2) was added.
The molar ratio of DCC/carboxylic acid:amine was 2:1.3-1.5:
1. The reaction mixture was allowed to stir at room temper-
ature until TLC indicated the completion of the reaction. The
white precipitate of DCU was then filtered, and the filter cake
was washed with CH2Cl2 twice. The combined organic layer
was extracted with 15% aqueous citric acid solution three
times or until TLC indicated that all product was extracted
into the aqueous layer. The aqueous layer was back-washed
with CH2Cl2 once, basified with concentrated NH4OH to pH
9-11, and then extracted with CH2Cl2 three times. The
obtained organic layer was washed with water and brine, dried
over Na2SO4, and evaporated to give the desired amides. The
crude product was purified through salt formation (see Tables
1-3), or, if necessary, purified on a silica gel column with 150:
10:1 CH2Cl2/MeOH/concentrated NH4OH before salt formation.
The yield range for this method was 85-95%.
Meth od B. To a stirred solution of DCC in CH2Cl2 (5 mmol
of DCC to 20 mL of solvent) was added a solution of o-, m-, or
p-methoxyphenylacetic acid in CH2Cl2 (5 mmol of carboxylic
acid to 20 mL of solvent). The mixture was allowed to stir at
room temperature for 10 min before piperazine was added
slowly. The molar ratio of DCC/carboxylic acid:piperazine was
2.5:2.0-2.5:1, and the reaction was terminated when TLC
indicated the disappearance of piperazine. The white precipi-
tate of DCU was then filtered, and the filtrate was washed
with water once, 3 N aqueous HCl twice, and brine once. The
resulting organic layer was dried over Na2SO4 and evaporated
to give the desired diamide. The crude product mixed with a
small amount of DCU was recrystallized from ethanol to give
the pure diamide as white crystalline solid. The yield range
for this method was 80-95%.
Meth od C. A solution of amide in a minimum amount of
THF was added dropwise to a stirred, freshly prepared solution
of 1 M AlH3 in THF.16,37 The molar ratio of amide:AlH3 was
about 1:5. The reaction mixture was stirred at room temper-

Disubstituted Piperazines as σ Receptor Ligands
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 25 4955
ature for 10 min when TLC showed the disappearance of the
amide. The mixture was then poured slowly into a 15%
aqueous NaOH solution in an ice-water bath. The layers
were separated, and the aqueous layer was extracted with CH2-
Cl2 three times. The combined organic layer was then washed
with water and brine, dried over Na2SO4, and evaporated to
give the corresponding amine. The base was then purified
through crystallization of the salt in the appropriate solvent
(see Tables 1-3). The yield range for this method was 80-
95%.
Meth od D. This method was the same as method C except
that 0.5 M AlH3 was utilized. The obtained mixture was
worked up according to method C, and the crude product was
purified on a silica gel column or on a preparative TLC plate
with 200-400:10:1 CH2Cl2/MeOH/concentrated NH4OH. The
purified amine compounds were then crystallized as their salt
from appropriate solvents (see Tables 1-3). The yield range
for this method was 50-70%.
Meth od E. To a mixture of 40 mL of trifluoroacetic acid in
80 mL of chloroform was added 10 mmol of the N-Boc-protected
compound 24-26. The solution turned dark-brown and was
allowed to stir at room temperature for 2 h when TLC showed
the completion of the reaction. The solution was then evapo-
rated to dryness on a rotavapor, and the residue was dissolved
in 1:1 15% aqueous NaOH/CH2Cl2. The layers were separated,
and the aqueous layer was extracted with CH2Cl2 twice. The
combined organic layer was then washed with water and brine,
dried over Na2SO4, and evaporated to give the deprotected
compound with a terminal amino function (27-29) as a light-
yellow oil. The crude product was purified through salt
formation or purified on a silica gel column prior to the salt
formation if necessary (see Table 3). The yield range for this
method was 60-75%.
2: 8.10 (d, J ) 7.5 Hz, 1H, ArH), 7.57 (t, J ) 7.2 Hz, 1H,
ArH), 7.44 (t, J ) 7.8 Hz, 1H, ArH), 7.18-7.35 (m, 6H, ArH),
4.05 (s, 2H, NCOCH2), 3.58-3.67 (m, 4H, CONCH2), 2.66 (t,
J ) 7.7 Hz, 2H, ArCH2), 2.35-2.52 (m, 6H, NCH2), 1.84 (tt, J
) 7.3 Hz, 2H).
3: 8.14 (d, J ) 8.7 Hz, 1H, ArH), 8.11 (s, 1H, ArH), 7.61 (d,
J ) 7.8 Hz, 1H, ArH), 7.51 (t, J ) 7.8 Hz, 1H, ArH), 7.16-
7.31 (m, 5H, ArH), 3.81 (s, 2H, NCOCH2), 3.67 (t, J ) 5.1 Hz,
2H, CONCH2), 3.51 (t, J ) 5.1 Hz, 2H, CONCH2), 2.64 (t, J )
7.6 Hz, 2H, ArCH2), 2.37 (m, 6H, NCH2), 1.81 (tt, J ) 7.5 Hz,
2H).
4: 8.20 (d, J ) 8.7 Hz, 2H, ArH), 7.42 (d, J ) 8.7 Hz, 2H,
ArH), 7.16-7.43 (m, 5H, ArH), 3.81 (s, 2H, NCOCH2), 3.66 (t,
J ) 5.1 Hz, 2H, CONCH2), 3.48 (t, J ) 5.1 Hz, 2H, CONCH2),
2.64 (t, J ) 7.7 Hz, 2H, ArCH2), 2.31-2.42 (m, 6H, NCH2),
1.80 (tt, J ) 7.8 Hz, 2H).
5: 7.15-7.30 (m, 7H, ArH), 6.85-6.94 (m, 2H, ArH), 3.82
(s, 3H, OCH3), 3.69 (s, 2H, NCOCH2), 3.66 (t, J ) 5.1 Hz, 2H,
CONCH2), 3.46 (t, J ) 5.0 Hz, 2H, CONCH2), 2.63 (t, J ) 7.7
Hz, 2H, ArCH2), 2.39 (t, J ) 5.1 Hz, 2H, NCH2), 2.33 (t, J )
7.5 Hz, 2H, NCH2), 2.27 (t, J ) 5.0 Hz, 2H, NCH2), 1.79 (tt, J
) 8.0 Hz, 2H).
6: 7.15-7.25 (m, 7H, ArH), 6.78-6.83 (m, 2H, ArH), 3.79
(s, 3H, OCH3), 3.70 (s, 2H, NCOCH2), 3.65 (t, J ) 5.1 Hz, 2H,
CONCH2), 3.44 (t, J ) 5.1 Hz, 2H, CONCH2), 2.62 (t, J ) 7.7
Hz, 2H, ArCH2), 2.38 (t, J ) 5.1 Hz, 2H, NCH2), 2.32 (t, J )
7.4 Hz, 2H, NCH2), 2.24 (t, J ) 4.9 Hz, 2H, NCH2), 1.78 (tt, J
) 7.6 Hz, 2H).
7: 7.13-7.25 (m, 7H, ArH), 6.85 (d, J ) 8.7 Hz, 2H, ArH),
3.79 (s, 3H, OCH3), 3.64 (m, 4H, NCOCH2 + CONCH2), 3.44
(t, J ) 5.1 Hz, 2H, CONCH2), 2.62 (t, J ) 7.7 Hz, 2H, ArCH2),
2.37 (t, J ) 5.1 Hz, 2H, NCH2), 2.32 (t, J ) 7.6 Hz, 2H, NCH2),
2.24 (t, J ) 5.0 Hz, 2H, NCH2), 1.78 (tt, J ) 7.5 Hz, 2H).
8: 7.18 (m, 7H, ArH), 7.04 (dd, J 1 ) 7.8 Hz, J 2 ) 2.0 Hz,
1H, ArH), 2.75 (m, 2H, ArCH2), 2.64 (t, J ) 7.8 Hz, 2H, ArCH2),
2.55-2.60 (m, 10H, NCH2), 2.40 (t, J ) 7.8 Hz, 2H, NCH2),
1.86 (tt, J ) 7.8 Hz, 2H).
Biologica l Ma ter ia ls a n d Meth od s. [3H]-(+)-Pentazocine
(51.7 Ci/mmol) was synthesized as described previously.21,39
[3H]DTG (39.1 Ci/mmol) was purchased from DuPont/New
England Nuclear (Boston, MA). Dextrallorphan was provided
by Dr. F. I. Carroll (Research Trangle Institute, Research
Triangle Park, NC). Haloperidol, Tris-HCl, and poly(ethyl-
enimine) were purchased from Sigma Chemicals (St. Louis,
MO).
σ Recep tor Bin d in g Meth od . σ-1 Receptors were labeled
as described previously, using the σ-1-selective probe [3H]-(+)-
pentazocine and guinea pig brain membranes.39 Guinea pig
brain membranes (300-350 µg of membrane protein) were
incubated with 3 nM [3H]-(+)-pentazocine in a total volume
of 0.5 mL of 50 mM Tris-HCl, pH 8.0. Incubations were carried
out for 120 min at 25 °C. Nonspecific binding was determined
in the presence of 10 µM unlabeled haloperidol. Assays were
terminated by dilution with 5 mL of ice-cold 10 mM Tris-HCl,
pH 8.0, and vacuum filtration through glass fiber filters using
a Brandel cell harvester (Gaithersburg, MD). Filters were
soaked in 0.5% poly(ethylenimine) for at least 30 min at 25
°C prior to use. Filters were counted in CytoScint cocktail
(ICN, Costa Mesa, CA) after an overnight extraction of counts.
Membranes were prepared from frozen guinea pig brains
(minus cerebella) as previously described.39
σ-2 Receptors were labeled as previously described using
rat liver membranes, a rich source of σ-2 sites, and [3H]-1,3-
di-o-tolylguanidine ([3H]DTG) in the presence of 1 µM dex-
trallorphan to mask σ-1 receptors.40 Assays were performed
in 50 nM Tris-HCl, pH 8.0, for 120 min at 25 °C in a volume
of 0.5 mL with 160 µg of membrane protein and 5 nM
radioligand. Assays included 1 µM dextrallorphan to mask
σ-1 binding. Nonspecific binding was determined in the
presence of 10 µM haloperidol. All other manipulations were
prepared from the liver of male Sprague-Dawley rats as
previously described.40
1H NMR (CDCl3) data for compounds 1-32 are as follows.
1: 7.16-7.41 (m, 7H, ArH), 7.09 (dd, J 1 ) 8.8 Hz, J 2 ) 2.0
Hz, 1H, ArH), 3.66 (s, 2H, NCOCH2), 3.65 (t, J ) 4.8 Hz, 2H,
CONCH2), 3.45 (t, J ) 4.9 Hz, 2H, CONCH2), 2.64 (t, J ) 7.8
Hz, 2H, ArCH2), 2.31-2.42 (m, 6H, NCH2), 1.83 (tt, J ) 7.8
Hz, 2H).
9: 7.90 (d, J ) 7.6 Hz, 1H, ArH), 7.51 (dd, J 1 ) 8.1 Hz, J 2
)
1.9 Hz, 1H, ArH), 7.37 (d, J ) 7.7 Hz, 2H, ArH), 7.18-7.32
(m, 5H, ArH), 3.09 (dd, J 1 ) 9.0 Hz, J 2 ) 2.5 Hz, 2H, CH2-
ArNO2), 2.53-2.69 (m, 12H, NCH2), 2.41 (t, J ) 7.7 Hz, 2H,
ArCH2), 1.84 (tt, J ) 7.7 Hz, 2H).
10: 8.09 (m, 2H, ArH), 7.54 (d, J ) 7.6 Hz, 1H, ArH), 7.44
(t, J ) 7.8 Hz, 1H, ArH), 7.18-7.31 (m, 5H, ArH), 2.90 (dd, J 1
) 9.9 Hz, J 2 ) 3.0 Hz, 2H, CH2ArNO2), 2.35-2.70 (m, 14H,
ArCH2 + NCH2), 1.83 (m, 2H).
11: 8.13 (d, J ) 8.5 Hz, 2H, ArH), 7.35 (d, J ) 8.5 Hz, 2H,
ArH), 7.17-7.37 (m, 5H, ArH), 2.90 (m, 2H, CH2ArNO2), 2.34-
2.64 (m, 14H, ArCH2 + NCH2), 1.82 (m, 2H).
12: 7.18-7.30 (m, 5H, ArH), 7.02 (m, 2H, ArH), 6.68 (m,
2H, ArH), 4.50 (bs, 2H, NH2), 2.35-2.73 (m, 16H, ArCH2
NCH2), 1.82 (m, 2H).
+
13: 7.15-7.30 (m, 5H, ArH), 7.07 (t, J ) 7.9 Hz, 1H, ArH),
6.61 (d, J ) 7.6 Hz, 1H, ArH), 6.54 (s, 1H, ArH), 6.52 (d, J )
6.9 Hz, 1H, ArH), 2.36-2.74 (m, 16H, ArCH2 + NCH2), 1.83
(tt, J ) 7.7 Hz, 2H).
14: 7.15-7.30 (m, 5H, ArH), 6.99 (d, J ) 8.2 Hz, 2H, ArH),
6.62 (d, J ) 8.5 Hz, 2H, ArH), 2.36-2.72 (m, 16H, ArCH2
NCH2), 1.83 (tt, J ) 7.8 Hz, 2H).
+
15: 7.13-7.30 (m, 7H, ArH), 6.85 (m, 2H, ArH), 3.80 (s, 3H,
OCH3), 2.83 (m, 2H, ArCH2), 2.54-2.66 (m, 12H, NCH2), 2.40
(m, 2H, ArCH2), 1.83 (tt, J ) 7.7 Hz, 2H).
16: 7.15-7.30 (m, 6H, ArH), 6.76 (m, 3H, ArH), 3.79 (s, 3H,
OCH3), 2.79 (m, 2H, ArCH2), 2.53-2.66 (m, 12H, NCH2), 2.38
(m, 2H, ArCH2), 1.83 (tt, J ) 7.7 Hz, 2H).
17: 7.15-7.30 (m, 5H, ArH), 7.12 (d, J ) 8.6 Hz, 2H, ArH),
6.82 (d, J ) 8.6 Hz, 2H, ArH), 3.77 (s, 3H, OCH3), 2.75 (m,
2H, ArCH2), 2.53-2.66 (m, 12H, NCH2), 2.39 (m, 2H, ArCH2),
1.83 (tt, J ) 7.8 Hz, 2H).
18: 7.22 (d, J ) 7.7 Hz, 4H, ArH), 6.92 (t, J ) 7.6 Hz, 2H,
ArH), 6.86 (d, J ) 8.0 Hz, 2H, ArH), 3.82 (s, 6H, OCH3), 3.70
(s, 4H, CONCH2), 3.61 (s, 2H, ArCH2), 3.45 (s, 4H, CONCH2),
3.28 (s, 2H, ArCH2).

4956 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 25
Zhang et al.
(5) Inhibition of the Cholinergic Phosphoinositide Response by σ
Ligands: Distinguishing a σ Receptor-Mediated Mechanism from
a Mechanism Involving Direct Cholinergic Antagonism; Bowen,
W. D., Tolentino, P. J ., Hsu, K. K., Cutts, J . M., Naidu, S. S.,
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19: 7.23 (t, J ) 7.8 Hz, 2H, ArH), 6.79 (s + d, J ) 7.8 Hz,
6H, ArH), 3.79 (s, 6H, OCH3), 3.70 (s, 4H, CONCH2), 3.60 (s,
2H, ArCH2), 3.42 (d, J ) 3.9 Hz, 4H, CONCH2), 3.19 (s, 2H,
ArCH2).
20: 7.13 (d, J ) 7.8 Hz, 4H, ArH), 6.85 (d, J ) 8.7 Hz, 4H,
ArH), 3.79 (s, 6H, OCH3), 3.66 (s, 4H, CONCH2), 3.59 (s, 2H,
ArCH2), 3.41 (s, 4H, CONCH2), 3.19 (s, 2H, ArCH2).
21: 7.15-7.19 (m, 4H, ArH), 6.89 (t, J ) 6.8 Hz, 2H, ArH),
6.85 (d, J ) 7.8 Hz, 2H, ArH), 3.82 (s, 6H, OCH3), 2.57-2.87
(m, 16H, ArCH2 + NCH2).
22: 7.21 (m, 2H, ArH), 6.77 (m, 6H, ArH), 3.80 (s, 6H, OCH3),
2.62-2.84 (m, 16H, ArCH2 + NCH2).
23: 7.13 (d, J ) 8.5 Hz, 4H, ArH), 6.83 (d, J ) 8.5 Hz, 4H,
ArH), 3.79 (s, 6H, OCH3), 2.76 (m, 4H, ArCH2), 2.58 (m, 12H,
NCH2).
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24: 7.17-7.29 (m, 5H, ArH), 3.96 (s, 50%, COCH2), 3.94 (s,
50%, COCH2), 3.64 (t, J ) 4.9 Hz, 2H, CONCH2), 3.39 (t, J )
4.9 Hz, 2H, CONCH2), 2.65 (t, J ) 7.8 Hz, 2H, ArCH2), 2.35-
2.44 (m, 6H, NCH2), 1.82 (tt, J ) 7.8 Hz, 2H), 1.45 (s, 9H,
CH3).
25: 7.17-7.28 (m, 5H, ArH), 3.62 (t, J ) 5.4 Hz, 2H,
CONCH2), 3.42 (m, 4H, COCH2 + CONCH2), 2.65 (t, J ) 7.8
Hz, 2H, ArCH2), 2.50 (t, J ) 4.9 Hz, 2H, CH2NHBoc), 2.40 (m,
6H, NCH2), 1.82 (tt, J ) 7.6 Hz, 2H), 1.43 (s, 9H, CH3).
26: 7.17-7.29 (m, 5H, ArH), 5.30 (bs, 1H, NHBoc), 3.62 (t,
J ) 4.9 Hz, 2H, CONCH2), 3.46 (t, J ) 4.9 Hz, 2H, CONCH2),
3.18 (t, J ) 5.9 Hz, 50%, COCH2), 3.16 (t, J ) 5.9 Hz, 50%,
COCH2), 2.65 (t, J ) 7.3 Hz, 2H, ArCH2), 2.33-2.43 (m, 8H,
NCH2 + CH2NHBoc), 1.80-1.87 (m, 4H), 1.44 (s, 9H, CH3).
27: 7.16-7.30 (m, 5H, ArH), 3.64 (t, J ) 5.1 Hz, 2H,
CONCH2), 3.43 (s, 2H, COCH2), 3.36 (t, J ) 5.0 Hz, 2H,
CONCH2), 2.64 (t, J ) 7.7 Hz, 2H, ArCH2), 2.34-2.60 (m, 6H,
NCH2), 1.81 (tt, J ) 7.6 Hz, 2H).
28: 7.17-7.31 (m, 5H, ArH), 3.63 (t, J ) 5.1 Hz, 2H,
CONCH2), 3.47 (m, 2H, CONCH2), 3.00 (t, J ) 6.0 Hz, 2H,
COCH2), 2.65 (t, J ) 7.7 Hz, 2H, ArCH2), 2.46 (t, J ) 6.1 Hz,
2H, CH2NH2), 2.40 (m, 6H, NCH2), 1.82 (tt, J ) 7.6 Hz, 2H).
29: 7.17-7.31 (m, 5H, ArH), 3.63 (t, J ) 5.0 Hz, 2H,
CONCH2), 3.48 (t, J ) 5.0 Hz, 2H, CONCH2), 2.75 (t, J ) 6.9
Hz, 2H, COCH2), 2.65 (t, J ) 7.7 Hz, 2H, ArCH2), 2.35-2.43
(m, 8H, NCH2), 1.73-1.87 (m, 4H).
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NH2), 2.63 (t, J ) 7.8 Hz, 2H, ArCH2), 2.34-2.48 (m, 12H,
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31: 7.17-7.30 (m, 5H, ArH), 2.74 (t, J ) 6.9 Hz, 2H, CH2-
NH2), 2.63 (t, J ) 7.8 Hz, 2H, ArCH2), 2.34-2.48 (m, 12H,
NCH2), 1.82 (m, 2H), 1.66 (m, 2H).
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Metabolites of Haloperidol Display Preferential Activity at σ
Receptors Compared to Dopamine D-2 Receptors. Eur. J . Phar-
macol. 1990, 177, 111-118.
32: 7.19-7.30 (m, 5H, ArH), 2.71 (t, J ) 6.9 Hz, 2H, CH2-
NH2), 2.63 (t, J ) 7.8 Hz, 2H, ArCH2), 2.32-2.48 (m, 12H,
NCH2), 1.82 (tt, J ) 7.6 Hz, 2H), 1.49 (m, 4H).
Ack n ow led gm en t. The authors offer their sincere
appreciation to Noel Whittaker and Wesley White, LAC,
NIDDK, for performing mass spectral analyses of all the
compounds in this paper. Y. Zhang acknowledges the
NIDDK Intramural Research Training Award Program
for financial support.
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