Synthesis of (+)-cis-N-(4-isothiocyanatobenzyl)-N-normetazocine, an isothiocyanate derivative of N-benzylnormetazocine as acylant agent for the σ1 receptor

Article abstract of DOI:10.1021/jm010501a

(+)-cis-N-(4-isothiocyanatobenzyl)-N-normetazocine (BNIT) (+)-(4) was designed and synthesized as a derivative of the potent and selective σ₁ receptor ligand (+)-cis-N-benzyl-Nnormetazocine for irreversibly blocking σ₁ binding sites.

Full text of DOI:10.1021/jm010501a

2662
J . Med. Chem. 2002, 45, 2662-2665
Syn th esis of (+)-cis-N-(4-Isoth iocya n a toben zyl)-N-n or m eta zocin e, a n
Isoth iocya n a te Der iva tive of N-Ben zyln or m eta zocin e a s Acyla n t Agen t for th e σ1
Recep tor
Giuseppe Ronsisvalle,*,† Orazio Prezzavento,† Agostino Marrazzo,† Franco Vittorio,† Michele Massimino,†
Giovanna Murari,‡ and Santi Spampinato‡
Department of Pharmaceutical Sciences, University of Catania, V.le A. Doria, 6, 95125 Catania, Italy,
and Department of Pharmacology, University of Bologna, Via Irnerio, 48, 40126 Bologna, Italy
Received October 29, 2001
(+)-cis-N-(4-Isothiocyanatobenzyl)-N-normetazocine (BNIT) (+)-(4) was designed and synthe-
sized as a derivative of the potent and selective σ1 receptor ligand (+)-cis-N-benzyl-N-
normetazocine for irreversibly blocking σ1 binding sites. Pretreatment of guinea pig brain
membranes with BNIT (0.1, 1, and 5 µM) caused a concentration-dependent loss of binding of
the selective σ1 ligand [3H]-(+)-pentazocine. Binding experiments with [3H]-1,3-di(2-tolyl)-
guanidine ([3H]-DTG), a ligand of σ1 and σ2 receptors, showed that pretreatment with BNIT
blocked only the σ1 component of [3H]-DTG binding.
In tr od u ction
In recent years, the σ receptor sites have been
extensively investigated and several compounds have
been synthesized and tested in radioligand binding
assays and in cellular and animal models.1 However,
F igu r e 1. Isothiocyanate probes for σ receptor.
although this effort has helped clarify numerous biologi-
cal and pharmacological properties unrelated to other
The design and synthesis of selective irreversible
known receptors, some fundamental points remain
ligands have proved very useful in the isolation, puri-
obscure; examples are the identification of an endog-
fication, and characterization of many receptor systems.
enous ligand and their neural function.
Portoghese et al.11 postulated that an irreversible ligand
The σ receptors can be divided into at least two
first recognizes a specific binding site and then forms a
covalent bond on or near that recognition site, resulting
in irreversible attachment. These compounds are gener-
ally derivatives of selected ligands that may be chemi-
cally modified to retain high receptor affinity and
selectivity while permitting covalent binding to the
receptor protein.
subtypes: σ1 and σ2.2-4 On the basis of this classifica-
tion, (+)-pentazocine and (+)-N-allylnormetazocine ((+)-
SK&F 10,047) are classified as specific σ1 receptor
ligands, and 1,3-di(2-tolyl)guanidine (DTG), a nonben-
zomorphan-type σ receptor ligand, has equally high
affinity for the σ1 and σ2 receptors. Thus, each receptor
can be identified solely by ligand binding experiments
using [3H]-(+)-pentazocine or [3H]-DTG with (+)-pen-
tazocine to mask the labeling of σ1 receptors.
σ1 sites have been cloned in various tissues of guinea
pig, mouse, rat, and human brain, but their structures
are unlike those of other known mammalian proteins.5-7
The σ1 protein has homology with ∆8,7-isomerase, an
enzyme of fungal sterol biosynthesis. This suggests a
role in sterol metabolism, and since progesterone showed
moderate affinity for these receptors, it has been as-
sumed that certain neurosteroids exert modulatory
effects through the σ receptors.8
Interest in the σ receptors is aroused by their involve-
ment in psychosis, neuroprotection, motor disorders,
cocaine abuse, learning processes, and tumors.9 σ ra-
dioligands might therefore be used for positron emission
tomography (PET) in cancer diagnosis, since several
human tumoral cell lines overexpress σ receptors.10
σ receptors have been examined with isothiocyanate
probes. Isothiocyanate derivatives were synthesized
starting from DTG (DIGIT) and phencyclidine (PCP)
(methaphit) (Figure 1). DIGIT inhibits the specific
binding of 3H-labeled ligands such as [3H]-DTG and
[3H]-(+)-3-PPP. This effect is selective for the σ site
because binding of ligands for PCP, dopamine D2,
benzodiazepine, and µ-opioid receptors is unaffected.
However there are no data for benzomorphan ligands.12
Metaphit had different effects on the binding of various
radiolabeled σ ligands.13 The order of sensitivity to
metaphit was [3H]-DTG > [3H]-(+)-3PPP . [3H]-(+)-
SK&F 10,047, half-maximal loss of binding occurring
at 2, 10, and 50 µM, respectively. This order of sensitiv-
ity seems to reflect the different modes of interaction
of benzomorphan and non-benzomorphan ligands with
σ receptors. SAR studies reported by Carroll et al.
showed that certain substitutions can be made at the
para position of the aromatic ring of the N-benzyl
substituent of the potent, selective σ1 ligand (+)-cis-N-
benzyl-N-normetazocine without any appreciable loss of
* To whom correspondence should be addressed. Phone/Fax: +39-
095-336722. E-mail address: ggrmedch@unict.it.
† University of Catania.
‡ University of Bologna.
10.1021/jm010501a CCC: $22.00 © 2002 American Chemical Society
Published on Web 05/02/2002

Brief Articles
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 12 2663
Sch em e 1a
Ta ble 1. Effect of BNIT on Binding Parameters (Kd and Bmax
of [3H]-(+)-Pentazocine to σ1 Sites in Guinea Pig Brain
Membranesa
)
treatment
Kd (nM)
Bmax (fmol/mg protein)
control
5.2 ( 0.3
987 ( 78.5
601 ( 139.8*
41.3 ( 11.3*
nd
0.1 µM BNIT
1 µM BNIT
5 µM BNIT
34.5 ( 18.6*
nd
nd
a
Scatchard analysis was carried out in guinea pig brain
membranes pretreated with BNIT. Control refers to membranes
not exposed to BNIT, as described in Materials and Methods.
Values are the mean ( SEM of three experiments, each carried
out in triplicate. nd ) not detectable (no accurate Kd could be
calculated). /represents P < 0.01 vs control (Dunnet’s test after
ANOVA).
a
(a) DMF, NaHCO3; (b) SnCl2, EtOH/EtOAc; (c) 2-pyridyl-thio-
carbonate, CH2Cl2.
All experiments were run in triplicate, and at least three
independent experiments were done. Incubations were termi-
nated by rapid filtration through glass-fiber filters (Schleicher
& Schuell, Dassel, Germany) presoaked in 0.5% polyethylen-
imine for at least 60 min before use. Radioactivity retained
on the filters was measured by liquid scintillation spectometry
using a 1414 Winspectral Perkin-Elmer Wallac (after over-
night incubation in 4 mL of Filter Count Cocktail (Packard))
with a counting efficiency of 60%. The apparent dissociation
constants (Kd), and the maximum number of binding sites
(Bmax) were calculated using the LIGAND or EBDA programs
(Elsevier-BIOSOFT, Cambridge, U.K.).
Data were fit to both a one-site and two-site model using
LIGAND program in order to determine which gave the best
fit. In all experiments, the best fits were determined by a run
test showing a nonserial correlation (P < 0.05) plus the
presence of a significant F test (P < 0.05) between the more
complex and the next simplest fit.
affinity.14,15 These results indicated that the relatively
small substituents retain nanomolar σ1 binding affinity.
Here, we describe the synthesis and receptor binding
profile of (+)-cis-N-(4-isothiocyanatobenzyl)-N-normeta-
zocine (+)-(4), (BNIT), a selective σ1 acylant.
Exp er im en ta l Section
Ch em istr y. Alkylation of (+)-(2S,6S,11S)-6,11-Dimethyl-
1,2,3,4,5,6-hexahydro-2,6-methano-3-benzazocin-8-ol (+)-1 with
1-(bromomethyl)-4-nitrobenzene in DMF using NaHCO3 as the
proton acceptor gave (+)-2 (Scheme 1). The (2S,6S,11S)-3-(4-
aminobenzyl)-6,11-dimethyl-1,2,3,4,5,6-hexahydro-2,6-methano-
3-benzazocin-8-ol (+)-3 was prepared by reduction of the N-4-
nitrobenzyl derivative (+)-2 with SnCl2 dihydrate in ethanol/
EtOAc (7:3). Compound (+)-3 was converted to (+)-(2S,6S,11S)-
3-(4-isothiocyanatobenzyl)-6,11-dimethyl-1,2,3,4,5,6-hexahydro-
2,6-methano-3-benzazocin-8-ol (+)-4 (BNIT) by reaction with
2-pyridyl-thio-carbonate in dichloromethane: mp 85 °C dec;
NMR (CDCl3) δ 0.80 (d, J ) 7.2 Hz, 3H), 1.20-1.26 (m, 2H),
1.30 (s, 3H), 1.67-2.00 (m, 2H), 2.02-2.20 (m, 1H), 2.34-2.48
(m, 1H), 2.56-2.74 (m, 2H), 2.80-3.04 (m, 2H), 3.57 (d, J )
13.8 Hz, 1H), 3.70 (d, J ) 13.8 Hz, 1H), 6.57-7.36 (m, 7H);
FTIR (KBr) 2101 (NdCdS) cm-1. Anal. (C22H24N2OS) C, H,
N.
P h a r m a cology. Ma ter ia ls a n d Meth od s. Membranes
from guinea pig brain were prepared according to Mach et al.16
For σ1 binding assay, membranes (1 mg/mL) were incubated
in Tris-HCl, pH 7.4, with different concentrations of BNIT (0.1,
1, and 5 µM) for 10 min at 4 °C, as reported by Bluth et al.13
The suspension was centrifuged (18 000 rpm, 6 min), the
supernatant was discarded, and the pellet was resuspended
in the same volume of buffer. This procedure was repeated
twice. After resuspension in the original buffer volume,
membranes were incubated for 60 min at 25 °C in order to
dissociate any noncovalently bound ligand. The suspension was
centrifuged at 18 000 rpm, the pellet was resuspended in half
the original volume, and the σ1 binding assay was done. For
σ2 binding assay, membranes (720 µg/mL) were incubated in
Tris-HCl, pH 8, and treated as above.
For σ1 saturation binding assays, guinea pig brain mem-
branes (500 µg of protein/assay tube) were incubated for 90
min at 25 °C in 1 mL of incubation buffer (50 mM Tris-HCl,
pH 7.4 at 37 °C) containing one of six concentrations of [3H]-
(+)-pentazocine (1-6 nM; specific activity (sa), 28 Ci/mmol).
Nonspecific binding was defined in the presence of 10 µM
haloperidol and accounted at most for ∼20% of the total
radioactivity retained in the filters. For σ2 saturation studies,
guinea pig brain membranes (360 µg of protein/assay tube)
were incubated for 90 min at 25 °C in 0.5 mL of incuba-
tion buffer (50 mM Tris-HCl, pH 8) containing one of six
concentrations of [3H]-DTG (1-6 nM; sa 31 Ci/mmol) and (+)-
pentazocine (200 nM) to mask σ1 sites.4 Nonspecific binding
was defined in the presence of 5 µM DTG and accounted
at most for ∼20% of the total radioactivity retained in the
filters.
Data are expressed as mean ( SEM. Statistical comparisons
were made by ANOVA for repeated measures and the post
hoc Dunnett’s multiple comparison test, with differences of P
less than 0.05 being considered significant (GraphPAD Soft-
ware, San Diego, CA).
Resu lts a n d Discu ssion
We first investigated the effect of BNIT on [3H]-(+)-
pentazocine binding in guinea pig brain membranes
exposed to various concentrations of BNIT for 10 min
at 4 °C. This was followed by extensive washing, as
described under Materials and Methods, and determi-
nation of the saturation binding of [3H]-(+)-pentazocine.
These values were compared to controls not exposed to
BNIT. After 90 min of incubation at 25 °C, [3H]-(+)-
pentazocine bound saturably to a single site with a Kd
of 5.2 ( 0.3 nM and a Bmax of 987 ( 78.5 fmol/mg
protein. BNIT (0.1, 1, and 5 µM) caused a concentration-
dependent loss of binding of the radioligand. Scatchard
analysis indicated that the maximum binding capacity
(Bmax) of [3H]-(+)-pentazocine was ∼40% lower in mem-
branes exposed to 0.1 µM BNIT and 96% lower in those
exposed to 1 µM BNIT (Table 1). The Kd value was
significantly higher in membranes pretreated with 0.1
µM BNIT (Table 1). After pretreatment with 1 µM
BNIT, there was extensive loss of receptor binding
labeled by [3H]-(+)-pentazocine such that we could not
calculate the Kd.
To determine the selectivity of inhibition by BNIT at
σ1 binding sites, saturation binding assays were carried
out in the presence of [3H]-DTG. In agreement with
previous studies,3,4 [3H]-DTG binding was best described
by a two-site model. Conceivably, the two sites may
correspond to σ1 and σ2 binding sites. The binding
parameters were Kd ) 7.5 and 15.6 nM for σ1 and σ2,

2664 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 12
Brief Articles
Ta ble 2. Kd (nM) and Bmax (fmol/mg protein) of [3H]-(+)DTG Binding to Guinea Pig Brain Membranes Exposed or Not Exposed
(Control) to BNIT As Described in Materials and Methodsa
treatment
Kd
Kd1 (σ1)
Kd2 (σ2)
Bmax
Bmax1(σ1)
Bmax2(σ2)
control
7.5 ( 1.4b
15.6 ( 3.5b
987 ( 78.5b
1280 ( 189.4b
0.1 µM BNIT
12.8 ( 1.6*b
21.4 ( 7.8b
674 ( 35.8**b
1330 ( 210.5b
1 µM BNIT
13.6 ( 0.3c
11.6 ( 0.5c
11.6 ( 2.8c
1013 ( 148.4c
957 ( 58.9c
5 µM BNIT
(+)-pentazocine (200 nM)
1120 ( 98.5c
a
Values are the mean ( SEM of three experiments, each carried out in triplicate. /represents P < 0.01. //represents P < 0.05 vs
control (Dunnet’s test after ANOVA). Two-site fit model was better than one-site fit. c One-site fit model was better than two-site fit.
b
respectively; the corresponding Bmax values were 987
and 1280 fmol/mg protein (n ) 3) (Table 2). The lower
concentration of BNIT (0.1 µM) caused a significant
elevation of Kd and a reduction of the Bmax values
limited to the σ1 component of the [3H]-DTG binding to
guinea pig brain membranes, whereas binding to σ2
sites was not modified (Table 2). The higher concentra-
tions of BNIT (1 and 5 µM) abolished [3H]-DTG binding
to σ1 sites; under these conditions, [3H]-DTG bound to
a single site (Table 2). BNIT 1 and 5 µM reduced the
maximum binding capacity of [3H]-DTG by approxi-
mately 45%, with no concentration-dependent effect,
whereas the Kd values related to σ2 binding sites of [3H]-
DTG were not modified by pretreatment with the higher
concentrations of this compound (Table 2). Exposure of
guinea pig brain membranes to BNIT irreversibly
blocked σ1 binding sites, whereas σ2 sites were not
sensitive to this compound, since it blocked only the σ1
component of [3H]-DTG binding. This is further sup-
ported by saturation binding experiments of [3H]-DTG
on guinea pig brain membranes in the presence of (+)-
pentazocine (200 nM). This latter is a selective σ1
ligand17 that blocks only σ1 sites and under these
conditions; thus, [3H]-DTG interacts only with σ2 sites.
In these experiments, the Bmax of [3H]-DTG was reduced
to 1120 ( 98.5 fmol/mg protein, similar to that in [3H]-
DTG binding assays on guinea pig membranes pre-
treated with BNIT (Table 2). Kd values of [3H]-DTG were
not modified by exposure to BNIT or the presence of
(+)-pentazocine (Table 2).
This study indicates that BNIT promises to be a
useful pharmacological tool for irreversibly blocking σ1
binding sites. This binding activity is not recoverable
even after extensive washing of guinea pig membranes
exposed to BNIT. However, BNIT did not cause any loss
of σ2 receptors. These findings add more evidence for
the existence of physically separate σ1 and σ2 binding
sites. Interestingly, at the lowest concentration em-
ployed (0.1 µM), BNIT drastically reduced the Bmax of
[3H]-(+)-pentazocine and the σ1 component of [3H]-DTG
binding to guinea pig brain membranes; moreover, it
lowered the affinity of these radioligands for σ1 sites.
BNIT may interact differently with these sites. First,
it may irreversibly bind to the protein receptor, and
second, it may affect the binding of [3H]-(+)-pentazocine
to the residual σ1 sites. Bluth et al.13 reported that
pretreatment of guinea pig brain membranes with 1-[1-
(3-isothiocyanatophenyl)cyclohexyl]piperidine (metaph-
it) caused irreversible loss of PCP receptors labeled by
[3H]-TCP while decreasing the affinity of [3H]-DTG to
σ sites. They suggested that metaphit may induce
acylation of PCP receptors. It is not able to acylate the
σ sites, but it may simply partition in the lipid environ-
ment, producing high local concentrations of ligand, or
it may acylate a nucleophile at a site adjacent to the σ1
receptor. Adams et al.12 found that di-o-tolylguanidine
isothiocyanate (DIGIT) was an irreversible inhibitor at
σ receptors. They suggested that DIGIT may acylate or
form covalent bonds with amino groups of the σ binding
protein. However, it could not discriminate between σ1
and σ2 receptors. We propose BNIT as a novel selective
agent capable of irreversibly blocking only the σ1 binding
sites. Further research efforts are necessary to decide
whether this compound acylates or forms covalent bonds
with the σ1 receptor or with any adjacent protein(s).
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