Benzimidazolone-based selective σ2 receptor ligands: Synthesis and pharmacological evaluation

Article abstract of DOI:10.1016/j.ejmech.2019.01.019

Sigma receptors (σRs) are considered to be a significant and valid target for developing new medications to address several diseases. Their potential involvement in numerous central nervous system disorders, neuropathic pain, addiction, and cancer has bee

Full text of DOI:10.1016/j.ejmech.2019.01.019

Accepted Manuscript
Benzimidazolone-based selective σ receptor ligands: Synthesis and
2
pharmacological evaluation
Sebastiano Intagliata, Walid F. Alsharif, Christophe Mesangeau, Nicola Fazio,
Michael Seminerio, Yan-Tong Xu, Rae R. Matsumoto, Christopher R. McCurdy
PII:
S0223-5234(19)30029-7
DOI:
https://doi.org/10.1016/j.ejmech.2019.01.019
EJMECH 11026
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 4 October 2018
Revised Date: 1 January 2019
Accepted Date: 8 January 2019
Please cite this article as: S. Intagliata, W.F. Alsharif, C. Mesangeau, N. Fazio, M. Seminerio, Y.-T.
Xu, R.R. Matsumoto, C.R. McCurdy, Benzimidazolone-based selective σ receptor ligands: Synthesis
2
and pharmacological evaluation, European Journal of Medicinal Chemistry (2019), doi: https://
doi.org/10.1016/j.ejmech.2019.01.019.
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ACCEPTED MANUSCRIPT
Benzimidazolone-based selective σ2 receptor ligands: synthesis and pharmacological
evaluation
a
b
b
b
Sebastiano Intagliata , Walid F. Alsharif , Christophe Mesangeau , Nicola Fazio , Michael
Seminerio c, Yan-Tong Xu c, Rae R. Matsumoto c,d, Christopher R. McCurdy a, b
a Department of Medicinal Chemistry, College of Pharmacy, University of Florida, Gainesville,
Florida 32610, USA
b
Department of Medicinal Chemistry, School of Pharmacy, The University of Mississippi,
University, Mississippi 38677, USA
c Department of Basic Pharmaceutical Sciences, West Virginia University, Morgantown, West
Virginia 26506, USA
d College of Pharmacy, Touro University California, Vallejo, CA 94592, USA
Abstract
Sigma receptors (σRs) are considered to be a significant and valid target for developing new
medications to address several diseases. Their potential involvement in numerous central nervous
system disorders, neuropathic pain, addiction, and cancer has been extensively reported. In
particular, the σ2R has been identified as potential target for the development of pharmaceutical
agents intended to treat the negative effects associated with drugs of abuse. As a continuation of
our previous efforts to develop new selective σ2R ligands, a series of benzimidazolone
derivatives were designed, synthesized, and characterized. The newly synthesized ligands were
evaluated through in vitro radioligand binding assays to determine their affinity and selectivity
towards both σ1 and σ2 receptors. Several derivatives displayed high affinity for the σ2R (Ki =
0.66-68.5 nM) and varied from preferring to selective, compared to σ1R (σ1/σ2 = 5.8-1139).
Among
them,
compound
1-{4-[4-(4-fluorophenyl)piperazin-1-yl]butyl}-3-propyl-1,3-
dihydrobenzimidazol-2-one dihydrochloride (14) displayed the ability to produce a dose-
dependent reduction in the convulsive effects of cocaine in a rodent model after injecting 10
Corresponding author. Tel.: +1 (352) 294-8691; fax: +1 (352) 392-9455. E-mail address: CMccurdy@cop.ufl.edu (C.R.
McCurdy).

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mg/kg (i.p.). These preliminary results support the use of selective σ2R ligands in the
development of useful pharmacological tools or potential pharmacotherapies for cocaine toxicity.
Keywords: Sigma receptors, Sigma-2 receptors, Sigma-2 ligands, Benzimidazolone,
Structure-affinity relationships, Cocaine-induced convulsions, Cocaine’s toxicity.
1. Introduction
Sigma receptors (σRs) were originally classified as members of the opioid receptor family.
This was based on the behavioral actions observed with the opiate benzomorphan, (±)-N-allyl-
normetazocine, and its analogs [1]. The effects of (±)-N-allylnormetazocine were reported to be
partially antagonized by the opioid antagonist naloxone, thus resulting in σRs being classified as
a new opioid receptor subtype. Through subsequent studies using purified enantiomers, it was
shown that the “psychotomimetic” effect produced by (-)-SKF-10047 was instigated through
kappa opioid receptors and the “psychotomimetic” effect of (+)-SKF-10047 was not blocked by
naltrexone, a potent analog of naloxone, and must be mediated through non-opioid receptors.
(+)-SKF-10047 was eventually shown to interact with at least two distinct binding sites: 1) the
phencyclidine (PCP) site on N-methyl-D-aspartate (NMDA) receptors, and it was due to this
interaction that the term sigma-PCP was prevalent in the early 1980s, and 2) other binding sites,
distinct from opioid and NMDA receptors, which today retains the designation of σRs [2, 3].
Later, several studies proved that σRs are unique proteins highly preserved across species,
cell types, and organelles [4-7]. Additional studies identified two subtypes of the receptor, σ1R
and σ2R. Although historical sigma ligands interacted with both σ1R and σ2R, the subtypes are
distinguishable from each other by their amino acid sequences and pharmacological preferences
[8-10]. The σ1R was first cloned in 1996 from guinea pig liver and later from other sources,
representing different species and tissues such as human placental choriocarcinoma cells [4],
human brain [5], rat brain [6], and mouse brain [8]. The σ1R is highly conserved across species
with an amino acid sequence that is distinct from other known mammalian receptors [4]. The
σ2R has been more difficult to understand and clone. In 2011, the σ2R was suggested to be part
of the progesterone receptor membrane component 1 (PGRMC1) protein complex [11].

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However, further investigations showed no correlation between PGRMC1 expression and σ2R
binding levels in knockout or over-expression studies [12]. More recently, the σ2R was purified
from calf liver tissue by affinity chromatography and, subsequently, the gene that codes for the
σ2R was identified as identical to Transmembrane Protein 97 (TMEM97), which is an
endoplasmic reticulum-resident transmembrane protein that acts as a binding partner of the
lysosomal cholesterol transporter NPC1. Indeed, this latest finding provided further evidence that
the σ2R and PGRMC1 are two distinct proteins [13]. Consensus in the field now accepts the
TMEM97 protein as the σ2R. Over-expression of σ2R in proliferative cancer cells has been
reported, suggesting that selective radiolabeled σ2R ligands may be useful to pinpoint the
malignant tissues in SPECT (single-photon emission computed tomography) and PET (positron
emission tomography) imaging [14-16]. Moreover, literature-based reports have shown that σ2R
agonists are able to induce apoptosis and reduce cancer cell proliferation, thus, there is a rising
interest in the development of σ2R agents as therapeutics or adjuvants to treat cancer [17-19].
The involvement of σRs in several CNS disorders such as depression, schizophrenia, and
dementia, as well as their potential role in other neurological and psychological disorders, drove
much of the early interest in these receptors [20-24]. This interest was due to the majority of
typical antipsychotics, such as haloperidol, having affinity for σRs. Also, agents that instigate
psychotomimetic activity, such as (±)-N-allylnormetazocine and its analogs, shared affinity for
σRs [25]. Moreover, evidence-based literature demonstrated that some σR antagonists were able
to attenuate the effects of psychostimulant drugs such as cocaine [26-28]. Conversely, σR
agonists aggravated some of these effects, including the convulsive effects of cocaine, resulting
in a leftward shift in the cocaine convulsion dose–response curve [28, 29]. There still remains a
need to develop new medications to attenuate the convulsions associated with an overdose of
cocaine, which cannot be controlled with existing antiepileptic drugs. However, unlike the σ1R,
the involvement of the σ2R in the toxicity of cocaine is still unclear, and improved, selective σ2R
ligands are needed [30].
Previously, we reported a series of 1,3-benzothiazole-2(3H)-thione with high mixed affinity
for σ1Rs and σ2Rs [31]. Among them, 3-[4-(4-cyclohexylpiperazin-1-yl)butyl]-2(3H)-
benzothiazolethione compound 1 (CM156, Fig. 1) produced both acute and subchronic effects
against cocaine in behavioral models in rodents [32]. Further studies showed the ability of 1 to
mitigate the effects of methamphetamine in mice [33]. Furthermore, we developed 6‐acetyl‐3‐{4‐

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[4‐(4‐fluorophenyl)piperazin‐1‐yl]butyl}‐2(3H)-benzoxazolone compound 2 (SN79, Fig. 1)
which reduced the toxic and stimulant effects of cocaine, and also caused sedation and
incoordination with acute treatment [34]. More studies have suggested that pretreatment with
compound 2 afforded protection against methamphetamine-induced hyperthermia, and striatal
dopaminergic and serotonergic neurotoxicity in mice [35].
Within the frame of our efforts to develop novel selective σ2R ligands [36], we have
developed several σ ligands with high affinity for σ2Rs and preference over σ1Rs. Among them,
the most selective σ2R ligand was selected in order to study its effect against cocaine-induced
convulsions in a preclinical rodent model, and to assess the selectivity requirement of both
receptor subtypes. As a strategy in the current study, we replaced the 1,3-benzothiazole-2(3H)-
thione, and the 1,3-benzoxazol-2(3H)-one of 1 and 2 with the bioisostere 1,3-
dihydrobenzimidazol-2-one. In particular, the new versatile core allowed us to perform 1,3-bis-
functionalization along with the introduction of an acetyl group in the 5-position of the
heterocyclic ring. Similarly to 1 and 2, the cyclic amine fragment of the new ligands were both
1-cyclohexylpiperazine and 1-(4-fluorophenyl)piperazine. Finally, based on our previous data, a
four-carbon chain was selected as a linker as it results in greater σ2R preference [31].

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Fig. 1. Chemical structures and affinity values of compounds 1 and 2.
2. Results and discussion
2.1. Chemistry
The synthesis of final compounds 5 and 6, was very straightforward as outlined in Scheme
1. Treatment of commercially available compound 3 with 1,4-dibromobutane in the presence of
potassium carbonate in DMF, gave the 4-bromobutyl derivative (4) which was then coupled with
1-cyclohexylpiperazine, or 1-(4-fluorophenyl)piperazine in the presence of potassium carbonate
in DMF to afford target compounds 5 and 6, respectively (Scheme 1).
Scheme 1. Reagents and conditions: (a) 1,4-dibromobutane, K2CO3, DMF, 60 °C, 70%; (b) cyclic amine, K2CO3,
DMF, 60 °C, 56-62%.
The synthesis of final compounds 12-20 is depicted in Scheme 2. N-alkylation of
commercially
available
2-(4-bromobutyl)isoindole-1,3-dione
(7)
with
1-(4-
fluorophenyl)piperazine provided compound 8. The desired primary amine was then liberated
from the phtalimide by hydrazinolysis to afford compound 9 which was subsequently reacted,
through a nucleophilic aromatic substitution, with 1-fluoro-2-nitrobenzene to give 10 or, with 1-
(4-chloro-3-nitrophenyl)ethanone to give 11. The nitro groups of 10 and 11 were reduced in the
presence of Pd/C and the corresponding 1,2-diamine derivatives which were reacted with 1,1′-

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carbonyldiimidazole to give benzimidazolones 12 and 13. The final compounds 14-20, were
obtained by N-alkylation with the appropriate alkyl halides.
F
F
F
N
N
N
N
Br
O
N
N
(a)
(b)
(c)
N
N
HN
O
O
O
O2N
R1
NH2
7
8
9
10 R1 = H
11 R1 = COCH3
F
F
N
N
N
N
(d)
(e)
14 R1 = H, R2 = nC3H7
N
N
15 R1 = H, R2 = nC5H11
O
O
16 R1 = H, R2 = nC10H21
17 R1 = COCH3, R2 = CH3
18 R1 = COCH3, R2 = nC3H7
19 R1 = COCH3, R2 = nC5H11
20 R1 = COCH3, R2 = nC10H21
R1
N
R2
R1
N
H
12 R1 = H
13 R1 = COCH3
Scheme 2. Reagents and conditions: (a) 1-(4-fluorophenyl)piperazine, K2CO3, DMF, 60 °C, 60%; (b) NH2-NH2,
EtOH, reflux, 83%; (c) 1-fluoro-2-nitrobenzene or 1-(4-chloro-3-nitrophenyl)ethanone, DIPEA, DMF, 100-120 °C,
83-93%; (d) H2 (40 psi), 10% Pd/C, EtOH then CDI, THF, 20 °C, 51-68%; (e) NaH, TBAI, alkyl halide, DMF, 20
°C, 33-80% or K2CO3, TBAI, alkyl halide, DMF, 60 °C, 84-89%.

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2.2. Binding studiesand extended non-σ binding profile
All final compounds were tested in radioligand binding studies at the σ1R and σ2R in rat
brain membranes using well-established and previously described assay conditions [31]. The
σ1Rs were labeled with [3H](+)-pentazocine. The σ2Rs were labeled with [3H]di-o-tolylguanidine
(DTG) in the presence of (+)-pentazocine to block σ1Rs. Nonspecific binding was determined in
the presence of haloperidol. The binding affinities (Ki in nM), and selectivity ratios (Ki σ1/ Ki σ2)
of the new compounds at σRs are summarized in Table 1.
Initially, we synthesized compounds 5 and 6 to investigate the effect of replacement of the
heterocyclic scaffolds in 1 and 2 with a benzimidazolone moiety. This structural modification led
to an increase of the σ1/σ2 selectivity ratio for both analogs (5 vs 1, 6 vs 2). In particular,
compound 6 resulted in 46-fold more selective preference than 2 for the σ2R over the σ1R. This
result prompted us to maintain the 1-(4-fluorophenyl)piperazine ring as a fixed portion of the
new series. Subsequently, the introduction of different alkyl substituents on the N-3 (such as
nC3H7, nC5H11, and nC10H21), as well as an acetyl group at the 5-position of the benzimidazolone
scaffold, gave compounds 12-20 which resulted in affinity values in the low or subnanomolar
range for the σ2R (Ki = 0.66-32.5 nM).
Based on the binding data, reported in Table 1, the main structure-affinity relationships
(SARs) were as follows. In general, the new benzimidazolone derivatives retained high σ2R
affinity and showed increased selectivity over the σ1R (selectivity 5.8-1139 fold). Replacement
of the 1-cyclohexylpiperazine with 1-(4-fluorophenyl)piperazine, greatly improved the
selectivity for the σ2R subtype (5 vs 6). Functionalization at the N-3 of the benzimidazolone ring
generally increased affinity for the σ2R. Introduction of an acetyl group at the 5-position of the
heterocyclic ring led to a loss of the σ1/σ2 selectivity. Thus, the presence of both a bulky
substituent as R2 and an acetyl group as R1 was detrimental for σ2R affinity. Regarding the set of
compounds with no substituent on the benzo-fused ring (6, 12, and 14-16), the introduction of R2
groups were ranked based on their affinity (Ki values) at σ2R as follows: nC3H7 > CH3 > nC5H11
> nC10H21 > H. A different ranking was observed for the set of 5-acetyl-benzoimidazolone
analogs 13, and 17-20: nC3H7 > nC5H11 > CH3 > H > nC10H21. Among both sets, n-propyl and n-
pentyl analogs (14, 18, and 15, 19) displayed better results in terms of affinity and selectivity for
the σ2R than other alkyl-substituted derivatives.

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Based on these results, we selected compounds 14 and 15 which had the highest ratios of
σ1/σ2 ( >1000 fold and >500 fold, respectively) for further evaluation in extended non-σRs
binding experiments (Table 2), using previously established and published methods [28].
Substantially, compound 14 did not exhibit significant affinities for the non-σ proteins tested,
except for serotonin (5-HT2) receptors (Ki = 54.70 nM, 5-HT2/σ2R = 83 fold preference for
σ2Rs). Compound 15 showed similar results at serotonin (5-HT2) receptors (Ki = 249.6 nM, 5-
HT2/σ2R = 58 fold selectivity for σ2Rs), although possessed very low affinity for serotonin
transporters (Ki = 445.9 nM, 5-HTT/σ2R = 104 fold selectivity for σ2Rs).
Table 1. σRs binding affinities and selectivity ratios of compounds 5, 6, 12-20, and haloperidol.
Compd.
R1
R2
Ki (nM ± SEM)a
σ2R
K
i σ1/ Ki σ2
σ1
R
H
CH3
CH3
H
50.22 ± 7.59
366.23 ± 101
144 ± 6.6
2.57 ± 0.47
2.05 ± 0.33
18.4 ± 1.4
28.5 ± 8.6
0.66 ± 0.01
4.27 ± 0.29
19.5
178.6
7.8
5
H
6
H
12
13
14
15
COCH3
H
166 ± 12
5.8
H
H
nC3H7
nC5H11
752 ± 63
1139
532.5
2274 ± 187

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H
nC10H21
724 ± 13.6
72.3 ± 2.6
273.5 ± 1.33
299 ± 32
4.55 ± 0.21
159.1
9.5
16
COCH3
COCH3
COCH3
COCH3
CH3
7.6 ± 0.31
1.81 ± 0.02
2.15 ± 0.94
32.5 ± 5.3
17
nC3H7
nC5H11
nC10H21
151.1
139
18
19
988 ± 53
30.4
0.042
20
Haloperidol
3.35 ± 0.80
80.60 ± 14.10
aThe values represent the mean ± SEM from triplicate assays.
Table 2. Binding profile of 14 and 15 for other non-σ proteins.
Compd.
Ki (nM ± SEM)a
Monoamine transporters
Dopamine Serotonin Norepinephrine Dopamine
Other receptors
Serotonin
(5-HT2)
NMDA
Opioid
(D2)
>1000
>1000
>1000
>10000
>10000
>10000
54.70 ± 0.27 >10000 >10000
14
15
445.9 ± 3.2
>1000
249.6 ± 4.8 >10000 >10000
a Affinities (Ki values in nanomolar) were determined in rat brain homogenates. The values represent the mean
± SEM from triplicate assays. Values of >10000 nM represent less than 30% displacement of the radioligand
at that concentration.
2.3. In vivo convulsion studies
Seizure is a common result of cocaine overdose and earlier σR antagonists attenuated the
convulsive effects of cocaine in mice [27, 28]. Therefore, we tested whether pretreatment of
mice with 1-{4-[4-(4-fluorophenyl)piperazin-1-yl]butyl}-3-propyl-1,3-dihydrobenzimidazol-2-
one dihydrochloride (14), similarly attenuated cocaine-induced convulsions. Compound 14 was
selected because it possessed a very good extended non-σ binding profile, thus acting as a
relatively selective σ2R ligand. As can be seen from Fig. 2, compound 14 produced a dose-
dependent reduction in the convulsive effects of cocaine, with the protective effects being
statistically significant at the 10 mg/kg pretreatment dose (P<0.005). These results suggest that

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σ2R-selective ligands can mitigate cocaine-induced convulsions, and support the hypothesis of an
active role for this target in cocaine’s toxicity.
Fig. 2. In vivo cocaine-induced convulsion studies on compound 14. ** P<0.005 vs vehicle response; n = 6-8 per
group.
3. Conclusion
We successfully synthesized a series of benzimidazolone derivatives, and evaluated their
binding affinity for σRs. In this work, a versatile 1,3-dihydrobenzimidazol-2-one scaffold was
used, along with a 1-(4-fluorophenyl)piperazine as a cyclic amine moiety. Based on the SARs,
both structural elements were identified as preferential for optimizing affinity and selectivity for
the σ2R. Derivatives 14, and 15, which gave the best results in terms of affinity for the σ2R (Ki =
0.66 and 4.27 nM) and selectivity over the σ1R (σ1/σ2 =1139 and 532), were selected for
additional binding evaluation versus different non-σ sites. In particular, only moderate affinity
for serotonin (5-HT2) receptors (14 and 15, Ki = 54.70 and 249.60 nM respectively) and very low
affinity for serotonin transporters (15, Ki = 445.9 nM) were observed. Noteworthy, in vivo
convulsion studies in mice showed that compound 14 was able to dose-dependently attenuate
cocaine-induced convulsions. Thus, the data reported in this paper are indicative of the potential
utility of targeting σ2Rs to mitigate the convulsive effects of cocaine. The synthesis of new

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derivatives and further in vivo studies are currently under development and will be reported
elsewhere.
4. Experimental protocols
4.1. Chemistry
Reagents and starting materials were obtained from commercial suppliers and were used
without purification. Precoated silica gel GF Uniplates from Analtech were used for thin-layer
chromatography (TLC). Column chromatography was performed on silica gel 60 (Sorbent
1
Technologies). H and 13C NMR spectra were recorded with a Bruker APX400 in CDCl3 and
DMSO-d6 solution. Chemical shift (δ) values are given in parts per million (ppm) downfield to
tetramethylsilane (TMS, δ = 0.00 ppm); coupling constants (J values) are given in hertz (Hz).
For signal multiplicities, the following abbreviations are used as follows: s (singlet), d (doublet),
dd (doublets of doublet), t (triplet), q (quartet), quint (quintet), br s (broad singlet) and m
(multiplet). Except where otherwise noted, 1H and 13C NMR data for final compounds are given
for materials in their salt form. The high resolution mass spectra (HRMS) were recorded on a
Waters Micromass Q-Tof Micro mass spectrometer with a lock spray source. The mass spectra
(MS) were recorded on a WATERS ACQUITY Ultra Performance LC with ZQ detector in ESI
mode. Elemental analysis (C, H, N) was recorded on an elemental analyzer, Perkin-Elmer
CHN/SO Series II Analyzer.
4.1.1. 1-(4-bromobutyl)-3-methyl-1,3-dihydrobenzimidazol-2-one (
K2CO3 (0.56 g, 4.05 mmol) and 1,4-dibromobutane (1.12 mL, 9.45 mmol) were added,
under mechanical stirring, to a solution of 3-methyl-1H-benzimidazol-2-one ( ) (0.20 g, 1.35
mmol) in anhydrous DMF (8 mL). The reaction mixture was heated at 60 C for 3 h. After
cooling, the mixture was poured into 100 mL of water, extracted with ethyl acetate (3 40 mL),
4).
3
°
×
washed with saturated aqueous NaCl and dried. The solvent was removed in vacuo, and the
residue was purified by chromatography on a silica gel column using petroleum ether/ethyl
1
acetate (7:3) as the eluent to give
CDCl3):
3H), 1.89-1.85 (m, 4H). 13C NMR (100 MHz, CDCl3):
4
(0.27 g, 70%), as a colorless oil. H NMR (400 MHz,
δ
7.06-7.03 (m, 2H), 6.96-6.91 (m, 2H), 3.88-3.86 (m, 2H), 3.41-3.38 (m, 2H), 3.63 (s,
154.41, 130.10, 129.17, 121.26, 121.23,
δ

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107.53, 107.50, 40.08, 33.13, 29.66, 27.15, 26.98. MS (ESI) m/z 305 [M + Na]+ for 79Br, m/z 307
[M + Na]+ for 81Br.
4.1.2.
dihydrochloride (
K2CO3 (0.06 g, 0.44 mmol) and 1-cyclohexylpiperazine (0.04 g, 0.21 mmol) were added,
under mechanical stirring, to a solution of (0.05 g, 0.18 mmol) in anhydrous DMF (3 mL). The
reaction mixture was heated at 60 C for 2 h. After cooling, the mixture was poured into 20 mL
of water, extracted with ethyl acetate (3 30 mL), and the combined organic layers were washed
1
‐[4‐(4‐cyclohexylpiperazin
‐1
‐yl)butyl]
‐3‐
methyl-1,3-dihydrobenzimidazol-2-one
5
).
4
°
×
with saturated aqueous NaCl and dried. The solvent was removed in vacuo, and the residue was
chromatographed on a silica gel column using methylene chloride/methanol (95:5) as the eluent.
The free base was converted into the hydrochloride salt by addition of HCl/dioxane to give
5
1
(0.05 g, 62%), as a white solid. H NMR (400 MHz, DMSO-d6):
δ 12.01-11.80 (m, 2H), 7.21-
7.19 (m, 1H), 7.15-7.12 (m, 1H), 7.08-7.06 (m, 2H), 3.84-3.02 (m, 17H), 1.80-1.77 (m, 2H),
1.66-1.05 (m, 11H). 13C NMR (100 MHz, DMSO-d6):
154.01, 129.80, 128.89, 121.33, 121.30,
δ
108.09, 64.32, 55.37, 48.60, 45.39, 39.96, 27.13, 26.37, 25.28, 24.75, 24.55, 21.14. HRMS (ESI)
calcd for C22H35N4O [M + H]+ 371.2811, found 371.2828. Anal. calcd for C22H36Cl2N4O. H2O:
C, 57.26; H, 8.30; N, 12.14. Found: C, 57.46; H, 7.92; N, 12.11.
4.1.3.
dihydrochloride (
This compound was prepared from
Yield 56%, as a white solid. 1H NMR (400 MHz, DMSO-d6):
1
‐
{4
‐
[4
‐
(4
‐
fluorophenyl)piperazin
‐
1
‐
yl]butyl}
‐
3
‐
methyl-1,3-dihydrobenzimidazol-2-one
6
).
4
and 1-(4-fluorophenyl)piperazine as described for
5.
δ
11.40 (br s, 1H), 7.30-7.00 (m,
9H), 3.88 (t, J = 6.8 Hz, 2H), 3.70-3.67 (m, 2H), 3.50-3.47 (m, 2H), 3.32 (s, 3H), 3.23-3.08 (m,
13
6H), 1.81-1.68 (m, 4H). C NMR (100 MHz, DMSO-d6):
δ 154.81 (d, J = 258.0 Hz), 149.09,
129.59, 128.69, 120.78, 120.75, 117.84 (d, J = 7.0 Hz), 115.25 (d, J = 22.0 Hz), 107.65, 107.63,
54.68, 50.33, 45.95, 39.64, 26.80, 25.16, 20.18. HRMS (ESI) calcd for C22H28N4OF [M + H]+
383.2247, found 383.2237. Anal. calcd for C22H29Cl2FN4O: C, 58.02; H, 6.42; N, 12.30. Found:
C, 58.31; H, 6.14; N, 12.23.
4.1.4. 2‐{4‐[4‐(4‐fluorophenyl)piperazin‐1‐yl]butyl}-1H-isoindole-1,3(2H)-dione (8).

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K2CO3 (8.45 g, 61.2 mmol) and 1-(4-fluorophenyl)piperazine (4.77 g, 26.50 mmol) were
added, under mechanical stirring, to a solution of 2-(4-bromobutyl)isoindole-1,3-dione (
g, 20.00 mmol) in anhydrous DMF (30 mL). The reaction mixture was stirred at 60 C for 1 h
and at room temperature for 15 h. The reaction mixture was then poured into 40 mL of water,
extracted with ethyl acetate (3 40 mL), and the combined organic layers were washed with
7) (3.75
°
×
saturated aqueous NaCl and dried. The solvent was removed in vacuo, and the residue was
purified by chromatography on a silica gel column using a gradient of methylene chloride and
methanol (10:0 to 9.5:0.5) as the eluent to give
(400 MHz, DMSO-d6): 7.86-7.80 (m, 4H), 7.00 (t, J = 9.0 Hz, 2H), 6.90-6.87 (m, 2H), 3.58 (t,
8
(4.70 g, 60%), as a pale yellow solid. 1H NMR
δ
J = 7.0 Hz, 2H), 3.01-2.99 (m, 4H), 2.43-2.41 (m, 4H), 2.30 (t, J = 7.0 Hz, 2H), 1.63-1.57 (m,
13
2H), 1.48-1.42 (m, 2H). C NMR (100 MHz, DMSO-d6):
δ 167.88, 155.87 (d, J = 234.0 Hz),
147.89 (d, J = 1.8 Hz), 134.27, 131.56, 122.91, 116.91 (d, J = 7.5 Hz), 115.13 (d, J = 21.6 Hz),
57.14, 52.65, 48.92, 37.31, 25.90, 23.58.
4.1.5. 4-[4-(4-fluorophenyl)piperazin-1-yl]butan-1-amine (
9).
A solution of (4.25 g, 11.10 mmol), and hydrazine monohydrate (2.7 mL, 55.70 mmol) in
8
ethanol (220 mL) was heated at reflux for 16 h. The reaction mixture was then cooled with an ice
bath. The precipitate was eliminated by filtration, and the filtrate was evaporated. The residue
was purified by chromatography on a silica gel column using a gradient of methylene chloride
and methanol saturated with ammonia (10:0 to 8:2) as the eluent to give
9
(2.33 g, 83%), as a
1
low melting point pale yellow solid. H NMR (400 MHz, CDCl3):
δ 6.91-6.89 (m, 2H), 6.83-
6.79 (m, 2H), 3.06 (t, J = 4.8 Hz, 4H), 2.67 (t, J = 6.8 Hz, 2H), 2.54 (t, J = 5.0 Hz, 4H), 2.34 (t, J
= 7.0 Hz, 2H), 2.00 (br s, 2H), 1.55-1.40 (m, 4H). 13C NMR (100 MHz, CDCl3):
157.24 (d, J =
δ
237.1 Hz), 148.11 (d, J = 2.1 Hz), 117.88 (d, J = 7.5 Hz), 115.58 (d, J = 21.9 Hz), 58.52, 53.35,
50.23, 42.07, 31.57, 24.42.
4.1.6. N
A mixture of 1-fluoro-2-nitrobenzene (2.51 g, 17.80 mmol), compound
mmol), N,N-diisopropylethylamine (7.8 mL, 44.56 mmol) in DMF (30 mL) was heated at 120
for 2 h. After cooling, the mixture was poured into 50 mL of water, extracted with ethyl acetate
(3 50 mL), and the combined organic layers were washed with brine and dried. The solvent
‐{4‐[4‐(4‐fluorophenyl)piperazin‐1‐yl]butyl}‐2‐nitroaniline (10).
9
(2.24 g, 8.91
°C
×

ACCEPTED MANUSCRIPT
was removed in vacuo, and the residue was purified by chromatography on a silica gel column
1
using ethyl acetate/hexanes (6:4) as the eluent to give 10 (3.09 g, 93%), as an orange solid. H
NMR (400 MHz, DMSO-d6):
δ 8.14-8.11 (m, 1H), 8.04 (d, J = 8.6 Hz, 1H), 7.50 (t, J = 7.6 Hz,
1H), 7.06 (d, J = 8.8 Hz, 1H), 7.01 (t, J = 8.8 Hz, 2H), 6.92-6.88 (m, 2H), 6.65 (t, J = 7.6 Hz,
1H), 3.36 (q, J = 6.3 Hz, 2H), 3.04-3.02 (m, 4H), 2.47-2.45 (m, 4H), 2.34 (t, J = 6.9 Hz, 2H),
13
1.68-1.61 (m, 2H), 1.57-1.50 (m, 2H). C NMR (100 MHz, DMSO-d6):
δ 155.90 (d, J = 234.0
Hz), 147.91 (d, J = 1.8 Hz), 145.21, 136.52, 130.79, 126.19, 116.93 (d, J = 7.5 Hz), 115.16 (d, J
= 21.6 Hz), 115.00, 114.51, 57.09, 52.65, 48.96, 42.00, 26.07, 23.50.
4.1.7. 1
A mixture of 1-(4-chloro-3-nitrophenyl)ethanone (3.89 g, 19.50 mmol), compound
g, 9.75 mmol), N,N-diisopropylethylamine (6.32 g, 48.74 mmol) in DMF (50 mL) was heated at
100 C for 6 h. After cooling, the mixture was poured into 80 mL of water, extracted with ethyl
acetate (3 80 mL), and the combined organic layers were washed with brine and dried. The
‐[4
‐({4
‐[4‐(4‐fluorophenyl)piperazin
‐1
‐
yl]butyl}amino)
‐3
‐
nitrophenyl]ethan
‐1
‐one (11).
9
(2.45
°
×
solvent was removed in vacuo, and the residue was purified by chromatography on a silica gel
column using a gradient of ethyl acetate and hexanes (6:4 to 10:0) as the eluent to give 11 (3.35
g, 83%), as a yellow solid. 1H NMR (400 MHz, DMSO-d6): δ 8.61-8.60 (m, 2H), 7.99 (d, J = 5.2
Hz, 1H), 7.16 (d, J = 9.1 Hz, 1H), 7.02 (t, J = 9.0 Hz, 2H), 6.93-6.90 (m, 2H), 3.45 (q, J = 2.5
Hz, 2H), 3.37 (s, 3H), 3.05 (t, J = 4.0 Hz, 4H), 2.48 (s, 4H), 2.37 (t, J = 6.9 Hz, 2H), 1.66 (quint,
J = 7.0 Hz, 2H), 1.54 (quint, J = 6.9 Hz, 2H).
4.1.8. 1‐{4‐[4‐(4‐fluorophenyl)piperazin‐1‐yl]butyl}‐1,3-dihydrobenzimidazol-2-one (12).
A solution of 10 (3.00 g, 8.06 mmol) in methanol (220 mL) was hydrogenated in the
presence of 10% Pd/C (0.30 g) under pressure (40 psi) for 1 h. The catalyst was removed by
filtration and the solvent was evaporated. The residue was dissolved in THF (80 mL), and 1,1′-
carbonyldiimidazole (2.61 g, 16.10 mmol) was added. The reaction mixture was stirred under
argon at room temperature. After 2 h, brine was added and the mixture was extracted with ethyl
acetate (3 x 40 mL). The combined organic layers were washed with brine, dried and evaporated.
The residue was purified by chromatography on a silica gel column using a gradient of
methylene chloride and methanol (10:0 to 9.5:0.5) as the eluent to give 12 (2.03 g, 68%), as a
white solid. A small portion was converted to the corresponding dihydrochloride salt for the

ACCEPTED MANUSCRIPT
1
biological testing. H NMR (free amine, 400 MHz, DMSO-d6):
δ 10.81 (s, 1H), 7.12-7.10 (m,
1H), 7.03-6.97 (m, 5H), 6.90-6.87 (m, 2H), 3.79 (t, J = 6.9 Hz, 2H), 3.01-2.99 (m, 4H), 2.44-2.42
13
(m, 4H), 2.30 (t, J = 7.0 Hz, 2H), 1.68-1.62 (m, 2H), 1.48-1.41 (m, 2H). C NMR (free amine,
100 MHz, DMSO-d6):
δ 155.90 (d, J = 234.0 Hz), 154.73, 147.91 (d, J = 1.8 Hz), 130.19,
128.22, 120.62, 120.39, 116.93 (d, J = 7.5 Hz), 115.15 (d, J = 21.7 Hz), 108.64, 107.67, 57.04,
52.60, 48.93, 39.59, 25.61, 23.33. Anal. calcd for C21H27Cl2FN4O.1/2 H2O: C, 56.00; H, 6.27; N,
12.44. Found: C, 56.44; H, 6.02; N, 12.44.
4.1.9.
13).
This compound was prepared from 11 as described for 12. Yield 51%, as a white solid. A
5‐acetyl‐1‐{4‐[4‐(4‐fluorophenyl)piperazin‐1‐yl]butyl}‐1,3-dihydrobenzimidazol-2-one
(
small portion was converted to the corresponding dihydrochloride salt for the biological testing.
1H NMR (free amine, 400 MHz, DMSO-d6): δ 11.16 (br s, 1H), 7.71 (d, J = 6.5 Hz, 1H), 7.51 (s,
1H), 7.24 (d, J = 6.5 Hz, 1H), 7.00 (t, J = 7.0 Hz, 2H), 6.89-6.87 (m, 2H), 3.84 (t, J = 5.4 Hz,
2H), 2.99 (s, 4H), 2.53 (s, 3H), 2.41 (s, 4H), 2.30 (t, J = 5.6 Hz, 2H), 1.66 (quint, J = 5.3 Hz,
2H), 1.44 (quint, J = 5.4 Hz, 2H). 13C NMR (free amine, 100 MHz, DMSO-d6): δ 196.59, 155.93
(d, J = 237.3 Hz), 154.54, 147.93, 134.34, 130.18, 128.26, 122.59, 116.95 (d, J = 5.9 Hz), 115.19
(d, J = 17.3 Hz), 108.03, 107.22, 57.05, 52.64, 48.96, 40.01, 26.51, 25.66, 23.32. Anal. calcd for
C23H29Cl2FN4O2. H2O: C, 55.09; H, 6.23; N, 11.17. Found: C, 55.25; H, 5.81; N, 10.67.
4.1.10. 1-{4-[4-(4-fluorophenyl)piperazin-1-yl]butyl}-3-propyl-1,3-dihydrobenzimidazol-2-one
dihydrochloride (14).
Sodium hydride (60% in mineral oil, 0.02 g, 0.38 mmol) and tetrabutylammonium iodide
(0.01 g, 0.03 mmol) were added to a solution of 12 (0.10 g, 0.27 mmol) in anhydrous DMF (4
mL) at room temperature under argon. After stirring for 15 min, iodopropane (0.03 mL, 0.32
mmol) in DMF (2 mL) was added. The reaction mixture was stirred for 4 h at room temperature,
poured into brine (20 mL) and extracted with ethyl acetate (3 x 20 mL). The organic layers were
combined, washed with water, dried and evaporated. The residue was purified by
chromatography on a silica gel column using ethyl acetate as the eluent. The free base was
converted into the hydrochloride salt by the addition of HCl/dioxane to give 14 (0.10 g, 80%), as
a white solid. 1H NMR (400 MHz, DMSO-d6):
δ 11.41 (s, 1H), 7.23-7.22 (m, 1H), 7.19-7.18 (m,

ACCEPTED MANUSCRIPT
1H), 7.11-7.01 (m, 6H), 6.39 (br s, 1H), 3.86 (t, J = 6.6 Hz, 2H), 3.79 (t, J = 6.9 Hz, 2H), 3.69 (d,
J = 12.3 Hz, 2H), 3.48 (d, J = 11.3 Hz, 2H), 3.23-3.08 (m, 6H), 1.78-1.62 (m, 6H), 0.85 (t, J =
7.0 Hz, 3H). 13C NMR (100 MHz, DMSO-d6):
δ 156.75 (d, J = 235.8 Hz), 153.50, 146.20,
129.09, 128.78, 120.89, 120.80, 118.03 (d, J = 7.7 Hz), 115.56 (d, J = 21.9 Hz), 107.96, 107.91,
54.76, 50.42, 46.10, 41.90, 39.69, 25.26, 21.22, 20.30, 11.08. Anal. calcd for C24H33Cl2FN4O: C,
59.63; H, 6.88; N, 11.59. Found: C, 59.38; H, 6.66; N, 11.10.
4.1.11. 1-{4-[4-(4-fluorophenyl)piperazin-1-yl]butyl}-3-pentyl-1,3-dihydrobenzimidazol-2-one
dihydrochloride (15).
1
This compound was prepared from 12 as described for 14. Yield 68%, as a white solid. H
NMR (400 MHz, DMSO-d6):
δ 11.42 (s, 1H), 7.24-7.21 (m, 1H), 7.18-7.16 (m, 1H), 7.11-7.01
(m, 6H), 3.86 (t, J = 6.8 Hz, 2H), 3.81 (t, J = 7.0 Hz, 2H), 3.68 (d, J = 12.7 Hz, 2H), 3.49 (d, J =
11.2 Hz, 2H), 3.23-3.08 (m, 6H), 1.78-1.62 (m, 6H), 1.31-1.21 (m, 4H), 0.82 (t, J = 6.6 Hz, 3H).
13C NMR (100 MHz, DMSO-d6):
δ 156.93 (d, J = 236.0 Hz), 153.61, 146.23, 129.11, 128.88,
121.11, 121.00, 118.23 (d, J = 7.6 Hz), 115.70 (d, J = 21.9 Hz), 108.09, 108.08, 54.92, 50.60,
46.32, 40.44, 39.81, 28.42, 27.63, 25.31, 21.86, 20.47, 13.97. Anal. calcd for C26H37Cl2FN4O: C,
61.05; H, 7.29; N, 10.95. Found: C, 60.74; H, 7.25; N, 10.65.
4.1.12.
dihydrochloride (16).
This compound was prepared from 12 as described for 14. Yield 33%, as a white solid. 1H
NMR (400 MHz, DMSO-d6): 11.15 (s, 1H), 7.24-7.22 (m, 1H), 7.19-7.16 (m, 1H), 7.09-7.01
1‐decyl‐3‐{4‐[4‐(4‐fluorophenyl)piperazin‐1‐yl]butyl}-1,3-dihydrobenzimidazol-2-one
δ
(m, 6H), 5.85 (br s, 1H), 3.86 (t, J = 6.7 Hz, 2H), 3.81 (t, J = 7.0 Hz, 2H), 3.68 (d, J = 11.9 Hz,
2H), 3.48 (d, J = 11.2 Hz, 2H), 3.17-3.06 (m, 6H), 1.76-1.62 (m, 6H), 1.15-1.20 (m, 14H), 0.83
(t, J = 6.2 Hz, 3H). 13C NMR (125 MHz, DMSO-d6):
δ 156.61 (d, J = 235.7 Hz), 153.41, 146.37,
129.00, 128.77, 120.86, 120.76, 117.87 (d, J = 7.7 Hz), 115.48 (d, J = 21.9 Hz), 107.88, 107.87,
54.77, 50.49, 46.02, 40.30, 39.75, 31.23, 28.88, 28.87, 28.61, 28.59, 27.76, 26.09, 25.20, 22.03,
20.29, 13.91. Anal. calcd for C31H47Cl2FN4O: C, 64.01; H, 8.14; N, 9.63. Found: C, 63.94; H,
8.04; N, 9.59.

ACCEPTED MANUSCRIPT
4.1.13. 5-acetyl-1-(4-(4-(4-fluorophenyl)piperazin-1-yl)butyl)-3-methyl-1H-benzo[d]imidazol-
2(3H)-one dihydrochloride (17
)
A solution of 13 (0.11 g, 0.27 mmol), potassium carbonate (0.11 g, 0.80 mmol),
tetrabutylammonium iodide (0.30 g, 0.08 mmol) in anhydrous DMF (10 mL) was heated at 60
°C under argon. After 30 min, a solution of iodomethane (16.7 µl, 0.27 mmol) in anhydrous
DMF (8 mL) was added dropwise over 1 h. After 3 h, a solution of iodomethane (5 µl, 0.08
mmol) in anhydrous DMF (3 mL) was added dropwise and the reaction was stirred for another 3
h. The mixture was then poured into 80 mL of water, extracted with ethyl acetate (3 x 50 mL)
and the solvent was evaporated. The residue was purified by chromatography on a silica gel
column using a gradient of ethyl acetate and methanol saturated with ammonia (10:0 to 9:1) as
the eluent. The free base was converted into the hydrochloride salt by addition of HCl/dioxane to
give 17 (0.11 g, 89%), as a white solid. 1H NMR (400 MHz, DMSO-d6): δ 11.17 (br s, 1H), 7.78
(d, J = 8.2 Hz, 1H), 7.73 (s, 1H), 7.36 (d, J = 8.2 Hz, 1H), 7.11-6.99 (m, 4H), 5.20 (br s, 1H),
3.92 (t, J = 6.7 Hz, 2H), 3.69 (d, J = 11.7 Hz, 2H), 3.49 (d, J = 11.0 Hz, 2H), 3.40 (s, 3H), 3.18-
3.06 (m, 6H), 2.58 (s, 3H), 1.74-1.71 (m, 4H). 13C NMR (100 MHz, DMSO-d6): δ 196.80,
156.77 (d, J = 236.0 Hz), 154.01, 146.17, 132.92, 130.42, 129.76, 122.84, 118.05 (d, J = 8.5 Hz),
115.56 (d, J = 21.8 Hz), 107.42, 107.39, 54.82, 50.48, 46.15, 40.10, 27.14, 26.50, 20.29. Anal.
calcd for C24H31Cl2FN4O2. H2O: C, 55.92; H, 6.45; N, 10.87. Found: C, 54.88; H, 6.05; N, 10.58.
4.1.14.
5‐acetyl‐1
‐{4
‐[4‐(4‐fluorophenyl)piperazin
‐1‐
yl]butyl}
‐3
‐propyl-1,3-
dihydrobenzimidazol-2-one dihydrochloride (18).
1
This compound was prepared from 13 as described for 17. Yield 89%, as a white solid. H
NMR (400 MHz, DMSO-d6): δ 11.48 (br s, 1H), 9.69 (br s, 1H), 7.76 (d, J = 9.0 Hz, 1H), 7.74
(s, 1H), 7.36 (d, J = 8.1 Hz, 1H), 7.11-7.04 (m, 4H), 3.93-3.84 (m, 4H), 3.69 (d, J = 12.1 Hz,
2H), 3.49 (d, J = 11.3 Hz, 2H), 3.22 (t, J = 12.1 Hz, 2H), 3.19-3.09 (m, 4H), 2.57 (s, 3H), 1.79-
1.64 (m, 6H), 0.86 (t, J = 7.3 Hz, 3H). 13C NMR (100 MHz, DMSO-d6): δ 196.83, 156.79 (d, J =
235.7 Hz), 153.85, 146.12, 132.90, 130.47, 129.13, 122.78, 118.07 (d, J = 7.6 Hz), 115.57 (d, J =
21.9 Hz), 107.52, 107.44, 54.74, 50.41, 46.13, 42.04, 40.45, 26.69, 25.28, 21.23, 20.26, 11.06.
Anal. calcd for C26H35Cl2FN4O2. 1/2H2O: C, 58.43; H, 6.79; N, 10.48. Found: C, 58.85; H, 6.46;
N, 10.46.

ACCEPTED MANUSCRIPT
4.1.15.
5
‐acetyl
‐1‐{4
‐[4
‐(4
‐
fluorophenyl)piperazin
‐1
‐
yl]butyl}
‐3
‐pentyl-1,3-
dihydrobenzimidazol-2-one dihydrochloride (19).
1
This compound was prepared from 13 as described for 17. Yield 84%, as a white solid. H
NMR (400 MHz, DMSO-d6): δ 11.53 (br s, 1H), 8.75 (br s, 1H), 7.76 (d, J = 8.2 Hz, 1H), 7.71
(s, 1H), 7.36 (d, J = 8.2 Hz, 1H), 7.11-7.02 (m, 4H), 3.93-3.86 (m, 4H), 3.69 (d, J = 12.4 Hz,
2H), 3.51 (d, J = 11.4 Hz, 2H), 3.23 (t, J = 11.8 Hz, 2H), 3.20-3.09 (m, 4H), 2.57 (s, 3H), 1.79-
1.62 (m, 6H), 1.31-1.22 (m, 4H), 0.81 (t, J = 6.7 Hz, 3H). 13C NMR (100 MHz, DMSO-d6): δ
196.82, 156.85 (d, J = 235.7 Hz), 153.79, 146.07, 132.93, 130.47, 129.08, 122.84, 118.13 (d, J =
7.7 Hz), 115.59 (d, J = 21.9 Hz), 107.53, 107.32, 54.74, 50.39, 46.19, 40.87, 40.45, 28.28, 27.53,
26.69, 25.28, 21.75, 20.27, 13.86. Anal. calcd for C28H39Cl2FN4O2.1/2 H2O: C, 59.78; H, 7.17;
N, 9.96. Found: C, 59.71; H, 7.04; N, 9.93.
4.1.16. 5‐acetyl‐3
‐
decyl
‐
1
‐{4
‐[4‐
(4‐fluorophenyl)piperazin
‐1
‐yl]butyl}-1,3-dihydrobenzimidazol-
2-one dihydrochloride (20).
1
This compound was prepared from 13 as described for 17. Yield 87%, as a white solid. H
NMR (free amine, 400 MHz, CDCl3): δ 7.71 (dd, J = 6.7, 1.5 Hz, 1H), 7.61 (d, J = 1.3 Hz, 1H),
7.00 (d, J = 8.2 Hz, 1H), 6.93-6.89 (m, 2H), 6.84-6.81 (m, 2H), 3.92 (t, J = 7.2 Hz, 2H), 3.87 (t, J
= 7.4 Hz, 2H), 3.09 (t, J = 5.0 Hz, 4H), 2.61 (t, J = 4.9 Hz, 4H), 2.59 (s, 3H), 2.45 (t, J = 7.4 Hz,
2H), 1.79 (quint, J = 7.3 Hz, 2H), 1.72 (quint, J = 7.2 Hz, 2H), 1.59 (quint, J = 7.8, 2H), 1.30-
1.21 (m, 16H), 0.83 (t, J = 7.0 Hz, 3H). 13C NMR (free amine, 100 MHz, CDCl3): δ 197.22,
157.44 (d, J = 237.6 Hz), 154.64, 147.98, 133.67, 131.13, 129.91, 123.47, 118.08 (d, J = 8.0 Hz),
115.74 (d, J = 22.0 Hz), 107.32, 106.86, 57.73, 53.12, 50.01, 41.57, 41.23, 32.04, 29.70, 29.45,
29.43, 28.60, 27.00, 26.65, 26.41, 23.73, 22.83, 21.67, 14.28. Anal. calcd for C33H49Cl2FN4O2:
C, 63.55; H, 7.92; N, 8.98. Found: C, 63.49; H, 7.67; N, 8.81.
4.2. Competition binding assays for theσ1R and σ2
R
Preparation of rat brain membranes and in vitro binding assays for the σ1R and σ2R were
performed using methods published previously in detail [31]. In brief, homogenates of whole rat
brain excluding the cerebellum (400-500 µg) were incubated with 5 nM [3H](+)-pentazocine to
label σ1Rs, or 3 nM [3H]-DTG in the presence of 300 nM (+)-pentazocine to label σ2Rs. Non-

ACCEPTED MANUSCRIPT
specific binding was determined in the presence of 10 µM haloperidol. Ten concentrations (0.1-
o
1000 nM) of each test compound were incubated for 120 min at 25 C in 50 mM Tris-HCl, pH
8.0 to measure their ability to displace each radioligand from its binding sites. The total reaction
volume was 500 µl. All of the assays were terminated with the addition of ice-cold buffer and
rapid vacuum filtration over glass fiber filters. Counts were extracted from the filters using
Ecoscint (National Diagnostics, Manville, NJ) for at least 8 h prior to counting. Ki values were
calculated using the Cheng-Prusoff equation [37] and Kd values determined in separate saturation
assays.
4.3. Extended non-σ binding profile
The two compounds with the highest σ1/σ2 ratios (14 and 15) were tested for interactions
with other binding sites known to interact with many historic σ compounds and
psychostimulants: opioid receptors, PCP binding site on NMDA receptors, dopamine (D2)
receptors, serotonin (5-HT2) receptors, monoamine transporters (dopamine, serotonin,
norepinephrine). Well-established, previously published methods were used [28]. Briefly,
dopamine transporters were labeled using 0.5 nM [3H]WIN 35,428 in membranes prepared from
rat striata; nonspecific binding was determined in the presence of 50
transporters were labeled using 0.2 nM [3H]paroxetine in membranes prepared from rat
brainstem; nonspecific binding was determined in the presence of 1.5 M imipramine.
Norepinephrine transporters were labeled using 0.5 nM [3H]nisoxetine in membranes prepared
from rat cerebral cortex; nonspecific binding was determined in the presence of 4
desipramine. All of the receptor assays were conducted using rat brain membranes minus
cerebellum, similarly to those for
Rs. Dopamine (D2) receptors were labeled using 5 nM [3H](-
)-sulpiride and nonspecific binding was determined with 1 M haloperidol. Serotonin (5-HT2)
receptors were labeled using 2 nM [3H]ketanserin and nonspecific binding was determined with
M mianserin. The PCP site on NMDA receptors were labeled using 5 nM [3H]1-[1-(2-
thienyl)cyclohexyl]piperidine (TCP) and nonspecific binding was determined with 10
cyclazocine. Opioid receptors were labeled using 0.5 nM [3H]bremazocine and nonspecific
binding was determined with 10 M levallorphan. All of the assays were terminated with the
µM cocaine. Serotonin
µ
µM
σ
µ
1
µ
µ
M
µ

ACCEPTED MANUSCRIPT
addition of ice-cold buffer and vacuum filtration through glass fiber filters. Counts were
extracted and Ki values determined as described for the σR assays.
4.4. In vivo convulsion studies
Male, Swiss Webster mice (21-30 g; Harlan, USA) were pretreated (i.p.) with either saline
or compound 14 (0.1, 1 or 10 mg/kg), then challenged 15 min later with a convulsive dose of
cocaine (70 mg/kg, i.p.). Mice were observed for the next 30 min for convulsions, which were
defined as a loss of righting reflexes for at least 5 s, combined with the presence of clonic limb
movements or popcorn jumping. The data were analyzed using Fisher’s exact tests. Animals
were housed 1-5 per cage with a 12:12H light/dark cycle (lights on 06:00 hours) and ad libitum
food and water. They were allowed to climatize following their arrival for one week prior to
being utilized in any experiment. All procedures were approved by the Institutional Animal Care
and Use Committee at West Virginia University.
Acknowledgments
We appreciate the technical assistance of Matthew Robson, Colleen Beatty, and Bahar
Noorbakhsh. This project was supported by grants from NIDA R01 DA013978 and R01
DA023205.
References
[1] W.R. Martin, C.G. Eades, J.A. Thompson, R.E. Huppler, P.E. Gilbert, The effects of
morphine- and nalorphine- like drugs in the nondependent and morphine-dependent chronic
spinal dog, The Journal of pharmacology and experimental therapeutics, 197 (1976) 517-532.
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Benzimidazolone-based selective σ2 receptor ligands: synthesis and pharmacological
evaluation
a
b
b
b
Sebastiano Intagliata , Walid F. Alsharif , Christophe Mesangeau , Nicola Fazio , Michael
Seminerio c, Yan-Tong Xu c, Rae R. Matsumoto c,d, Christopher R. McCurdy a, b
a
Department of Medicinal Chemistry, College of Pharmacy, University of Florida, Gainesville,
Florida 32610, USA
b
Department of Medicinal Chemistry, School of Pharmacy, The University of Mississippi,
University, Mississippi 38677, USA
c
Department of Basic Pharmaceutical Sciences, West Virginia University, Morgantown, West
Virginia 26506, USA
d College of Pharmacy, Touro University California, Vallejo, CA 94592, USA
Highlights
•
A series of benzimidazolone derivatives as highly selective σ2R ligands were prepared and
characterized.
•
Replacement of the heterocyclic scaffolds of the lead compounds with a benzimidazolone
moiety increased the σ2 selectivity.
•
The cyclic amine fragment 1-(4-fluorophenyl)piperazine is preferred rather than 1-
cyclohexylpiperazine for the σ2 selectivity.
•
Introduction of n-propyl and n-pentyl group at N-3 position of the benzimidazolone, resulted
in better affinity and selectivity for the σ2R.
•
Selective σ2R ligands might attenuate cocaine-induced convulsions.
Corresponding author. Tel.: +1 (352) 294-8691; fax: +1 (352) 392-9455. E-mail address: CMccurdy@cop.ufl.edu (C.R.
McCurdy).