Electron-donating para-methoxy converts a benzamide-isoquinoline derivative into a highly Sigma-2 receptor selective ligand

Article abstract of DOI:10.1016/j.bmc.2011.10.046

The sigma-2 (σ2) receptor has been suggested to be a promising target for pharmacological interventions to curb tumor progression. Development of σ2-specific ligands, however, has been hindered by lack of understanding of molecular determinants that under

Full text of DOI:10.1016/j.bmc.2011.10.046

Bioorganic & Medicinal Chemistry 19 (2011) 7435–7440
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Bioorganic & Medicinal Chemistry
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Electron-donating para-methoxy converts a benzamide-isoquinoline derivative
into a highly Sigma-2 receptor selective ligand
Abdol R. Hajipour a,b, Lian-Wang Guo a, , Arindam Pal a, Timur Mavlyutov a, Arnold E. Ruoho a,
⇑
⇑
a Department of Neuroscience, University of Wisconsin School of Medicine and Public Health, 1300 University Ave., Madison, WI 53706, USA
b Pharmaceutical Research Laboratory, College of Chemistry, Isfahan University of Technology, Isfahan 84156, Islamic Republic of Iran
a r t i c l e i n f o
a b s t r a c t
Article history:
The sigma-2 (
to curb tumor progression. Development of
understanding of molecular determinants that underlie selective ligand-
explored effects of electron donating and withdrawing groups on ligand selectivity for the
r
2) receptor has been suggested to be a promising target for pharmacological interventions
2-specific ligands, however, has been hindered by lack of
2 interactions. Here we have
2 versus
1 receptor using new benzamide-isoquinoline derivatives. The electron-donating methoxy group
Received 8 August 2011
Revised 7 October 2011
Accepted 15 October 2011
Available online 20 October 2011
r
r
r
r
increased but the electron-withdrawing nitro group decreased
r2 affinity. In particular, an extra meth-
Keywords:
Sigma-2 receptor
Methoxy
Electron-donating
para-Position
Selectivity
oxy added to the para-position (5e) of the benzamide phenyl ring of 5f dramatically improved (631 fold)
the 2 selectivity relative to the 1 receptor. This para-position provided a sensitive site for effective
manipulation of the sigma receptor subtype selectivity using either the methoxy or nitro substituent.
Our study provides a useful guide for further improving the 2-over- 1 selectivity of new ligands.
Ó 2011 Elsevier Ltd. All rights reserved.
r
r
r
r
Isoquinoline
1. Introduction
remarkably, proliferating tumor cells express nearly 10-fold higher
amounts of the
2 receptor than quiescent tumor cells.11 Impor-
tantly, 2 receptor agonists have been found to induce apoptosis
in different tumor cell lines.12–16 These findings strongly support
use of the 2 receptor as a biomarker for solid proliferating tumors
and thereby selective 2 agonists as potential anti-cancer drugs.
r
The
r
1 and
r
2 receptors are unique, non-opioid binding sites
r
ubiquitously distributed in various tissues (see review1). These
two subtypes are pharmacologically distinguishable.2 With no
r
homology to any known mammalian protein,3 the
r
1 receptor
r
has been identified as a unique ligand-operated chaperone residing
However, only a very few highly
r2 selective drugs are now under
in the endoplasmic reticulum membrane.4 In contrast, however,
development (see review17).
little had been known about the molecular properties of the
receptor until most recently the progesterone receptor membrane
r
2
It is interesting to note that the putative
r
2 receptor (PGRMC1)
2 binding sites
has a molecular size (25 kDa)5 distinct from the
r
component 1 (PGRMC1) protein complex was identified as a puta-
previously observed through photoaffinity labeling (21.5 and/or
tive
Sigma receptor ligands have been reported to have various ther-
apeutic potentials. Numerous studies have proposed selective
r
2 receptor binding site.5
18 kDa).18–21 It remains to be clarified whether they are different
splice variants. Therefore it is important to develop
ligands for understanding the enigmatic 2 receptor binding
site(s). Ligand binding has been the main approach for detecting
the 2 receptor. The commonly used ligands including DTG
(1,3-di(2-tolyl)guanidine) and haloperidol, however, are not selec-
tive over the 1 receptor. Considerable effort has been paid to cre-
ate 2 ligands with improved affinity and selectivity, some of
r2 selective
r
1
r
agonists as therapeutic agents for anxiety, depression, psychosis,
and learning and memory improvement, and recently, neuropro-
r
tective effects of
(see reviews6–9). Thus far, the most prominent function of the
receptor has been linked to tumor progression. It is known that
the
2 receptor is highly expressed in various tumor cells,10 and
r1 agonists have garnered increasing attention
r
2
r
r
r
which have been developed into imaging agents for proliferating
tumor detection as well as for tissue and cellular distribution of
this receptor (see review17). In spite of these advances, only a
few compounds, such as CB-184,22 PB2814, siramesine,15 WC-26
and tetrahydroisoquinoline derivatives,5,23 have shown excellent
Abbreviations: RT, room temperature; DCC, dicyclohexylcarbodiimide; THF,
tetrahydrofuran; Ar, aromatic ring; OMe, methoxy; EtOH, ethanol; DTG,
1,3-di(2-tolyl)guanidine;
125I]-IAF, 1-N-(20,60-dimethyl-morpholino)-3-(4-azido-
[
3-[125I]iodo-phenyl propane.
r
2 over
A major barrier for discovery of
of understanding of the molecular determinants for ligand/
r1 selectivity.
⇑
Corresponding authors. Tel.: +1 608 263 3980; fax: 608 262 1257 (L.-W.G.).
E-mail addresses: lianwangguo@wisc.edu (L.-W. Guo), aeruoho@wisc.edu (A.E.
r
2-selective ligands is the lack
r
2
Ruoho).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.10.046

7436
A. R. Hajipour et al. / Bioorg. Med. Chem. 19 (2011) 7435–7440
interactions. Development of
r
2-selective ligands has not been
dramatic difference of affinity for the
electron-donating methoxy was added to the para-position (5e),
whereas 1 affinity was slightly reduced, 2 affinity was increased
by 500 fold, and compound 5e had thus acquired an ꢀ400 fold
selectivity for the 2 over the 1 receptor. Also supporting the
2 affinity-enhancing effect of electron-donating, iodine at the
para-position (5g), which in this case is relatively electron-donat-
ing compared to the hydrogen (5h), increased 2 affinity by 4.5
r2 receptor. When an extra
straightforward, and in some cases, identification of ligands with
high affinity for the 2 receptor has been a serendipitous result
of drug screening intended for other targets.17 The purpose of the
current study was to identify 2-favoring molecular determinants
in ligands. We have explored the contribution of electron-donating
and electron-withdrawing groups to receptor selectivity,
r
r
r
r
r
r
r
r
2
through the synthesis and characterization of a series of new com-
pounds featuring a benzamide moiety and an isoquinoline moiety
linked by an alkyl chain. We found that adding an extra methoxy
group (electron-donating) to the para-position (5e) of the benzam-
r
fold in comparison to compound 5h. On the contrary, however,
the electron-withdrawing nitro group on the benzamide phenyl
ring reduced
para-position (5d) or ortho-position (5a) led to a great decrease
of affinity for the 2 receptor in comparison to compound 5c.
Interestingly, the nitro group on the isoquinoline side of compound
5h also reduced 2 affinity as compared to 5I. While the nitro
group on the benzamide reduced 2 affinity, it significantly
improved the 1 binding (see comparison of 5c to 5d and 5a), con-
sistent with our previous findings.24,25 Moreover, the
1-affinity
r2 affinity. For example, a nitro group placed at the
ide phenyl ring of 5f dramatically improved the
r2 selectivity.
r
2. Chemistry
r
r
A commercial N-(4-bromoalkyl) phthalimide (1) was treated
with 1,2,3,4 tetrahydroisoquinoline (2) to yield an imide derivative
(3). Hydrazinolysis of 3 afforded N-(3-aminoalkyl)-1,2,3,4-tetrahy-
droisoquinolines (4a and 4b) which, by reaction with appropriate
carboxylic acid in the presence of DCC in THF, were converted to
(5) (Scheme 1).
r
r
enhancing effect was more profound when the nitro group was
placed at the para-position (5d) than at the ortho-position (5a).
Thus, it appeared that the electron-donating methoxy group
was favorable but the electron-withdrawing nitro group was unfa-
vorable for
or on the isoquinoline side. The para-position of the benzamide
phenyl ring was a sensitive location for changing the 2/ 1 selec-
r2 affinity, when placed either on the benzamide side
3. Results and discussion
r
r
We have previously discovered that the electron-withdrawing
tivity by adding either an electron-donating group or an electron-
withdrawing group.
It is also noteworthy that all the tested isoquinoline derivatives
in Table 1 that contain an amide group generally showed impaired
nitro group greatly improved ligand affinity for the
This prompted us to test the effects of electron-withdrawing, or
conversely, electron-donating groups on ligand affinity for the
receptor. A class of isoquinoline derivatives, which have previously
shown good
2 selectivity,23 provided an ideal template for our
r
1 receptor.24,25
r
2
r
1 affinity. This data is consistent with a previously reported
observation that an amide group abrogates
1 binding.26 In our
current study, including an amide group together with electron-
donating groups likely facilitated higher 2 selectivity over
r
r
experiments. The molecular structures of benzamide and isoquino-
line groups linked by a butyl chain provided us the flexibility to
modify the phenyl ring either on the isoquinoline side or on the
benzamide side. We thus synthesized a series of new derivatives
with electron withdrawing or donating substituents, and deter-
r
r1
in some of the isoquinoline derivatives, such as compound 5e.
Additionally, compared to 5e, replacing benzamide with phthali-
mide (3b) achieved a similar
mide may provide another good template for generating high
affinity compounds for 2.
To further confirm the
r2 affinity, suggesting that phthali-
mined their affinities for the
The results are presented in Table 1.
The new finding from our data is that the electron-donating
methoxy group favored 2 affinity, but conversely, the electron-
withdrawing nitro group on the phenyl ring mitigated against
ligand binding to the 2 receptor. The most interesting evidence
was revealed by the comparison of 5e to 5f, which showed a
r2 receptor as well as the r1 receptor.
r
r
2 selectivity of 5e shown by the
r
binding assays (Table 1), this compound and two other new
compounds (3b and 5f) were used as competitors to block sigma
receptor photoaffinity labeling by [125I]-IAF (1-N-(20,60-dimethyl-
morpholino)-3-(4-azido-3-[125I]iodo-phenyl propane) (Fig. 1). As
r
Scheme 1.

A. R. Hajipour et al. / Bioorg. Med. Chem. 19 (2011) 7435–7440
7437
Table 1
Characterization of new isoquinoline derivatives
Compounds
r
2 Ki (nM)
r
1 Ki (nM)
r1/r2
5e
26.78 ( 2.92)
10,320 ( 363)
385
5f
12,930 ( 55.84)
7,870 (264)
0.61
5g
5h
5i
866.70 ( 138.6)
4,000 ( 177.9)
1,400 ( 286)
5,290 ( 408)
74,680 ( 305)
11,200 ( 469)
67,800 ( 4,155)
>107
86.17
2.80
48.40
5c
5d
5a
152,000 ( 4,106)
14,690 ( 1,121)
0.096
>107
1.21 ꢁ 106
3b
21.26 ( 2.41)
87.5 ( 3.07)
4.12
Ki is presented as a mean ( SEM).
also shown in our previous studies20,21, [125I]-IAF labeled both
r1
4. Conclusion
and
peridol blocked photolabeling of both
2). Consistent with the binding data (Table 1), compound 5e elim-
inated 2 labeling but left 1 labeling essentially unaltered (lane
4). In contrast, compound 5f which showed low affinities for both
1 and 2, did not block the labeling of either receptor (lane 5).
Compound 3b, which was determined to have relative high affini-
ties for both subtypes, however, diminished both bands (lane 3).
Thus, the photolabeling data (Fig. 1) agreed well with the compet-
itive radioligand binding data (Table 1).
r
2 in the absence of competitors (lane 1). As a control, halo-
r
1 and 2 receptors (lane
r
Our data indicate that in a class of new benzamide-isoquinoline
derivatives, the electron-donating methoxy group favored
affinity but not 1 binding, thus improving 2 selectivity. Most
remarkably, an extra methoxy group at the para-position of the
benzamide phenyl ring (5e) dramatically increased 2 selectivity.
This para-position appeared to be highly sensitive to molecular
manipulations for the purpose of altering 2/ 1 selectivity. The
new information from our study offers a useful guide for designing
2 specific compounds.
r
2
r
r
r
r
r
r
r
r
r
r

7438
A. R. Hajipour et al. / Bioorg. Med. Chem. 19 (2011) 7435–7440
121 °C. 1H NMR, d: 8.05 (m, 2H), 7.60 (m, 1H,), 3.82 (s, 2H), 3.38
(t, 2H, J = 7.4 Hz), 3.12 (t, 2H, J = 7.4 Hz), 2.83 (s, 1H). 13C NMR, d:
150.6, 145.0, 140.3, 129.6, 122.6, 121.4, 46.9, 44.1, 28.1. EMS
[M+H+] for C9H10N2O2, Calcd 179.0740. Found, 179.1121.
5.1.2. Preparation of compounds 3a and 3b
A mixture of N-(4-bromobutyl)-phthalimide (10 mmol) and iso-
quinoline derivative (10 mmol) and K2CO3 (20 mmol) in ethanol
(100 ml) was refluxed for 12 h. The reaction mixture was then fil-
tered off, and the solvent was evaporated to give a crude product,
that was purified by column chromatography (silica gel, tolu-
ene:diethylamine, 20:1).
5.1.3. 2-(4-(6,7-Dimethoxy-3,4-dihydroisoquinoline-2(1H)-yl)-
butyl)isoindoline-1,3-dione (3a)
Oil, yield 75%; 1H NMR, d: 7.8 (m, 6 H), 7.43 (d, 2H, J = 8.6 Hz),
3.72 (s, 2H), 3.61 (t, 2H, J = 7.4 Hz), 2.90 (t, 2H, J = 7.4 Hz), 2.58
(t, 2H, J = 7.6 Hz), 1.59 (m, 4H). 13C NMR, d: 171.0, 149.8, 149.3,
132.2, 126.6, 123.7, 111.4, 108.3, 57.2, 56.1, 53.8, 37.2, 27.3, 25.6,
25.0. EMS [M+H+] for C23H26N2O4, Calcd 395.1863. Found,
395.1839.
Figure 1. Sigma-2 receptor-specific protection of
[
125I]-IAF photolabeling by
compound 5e photoaffinity labeling of RT4 cell membranes with 1 nM [125I]-IAF
was performed in the absence (lane 1) or presence of 10 lM competing
compounds: haloperidol (lane 2), 3b (lane 3), 5e (lane 4), or 5f (lane 5).
5.1.4. 2-(4-(7-Nitro-3,4-dihydroisoquinoline-2(1H)-yl)butyl)-
isoindoline-1,3-dione (3b)
Oil, yield 72%; 1H NMR, d: 8.3–7.8 (m, 3H), 6.84 (s, 2H,), 3.83 (s,
6 H), 3.7 (s, 2H), 3.61 (t, 2H, J = 7.4 Hz), 2.90 (t, 2H, J = 7.4 Hz), 2.58
(t, 2H, J = 7.6 Hz), 1.59 (m, 4H). 13C NMR, d: 171.0, 151.0, 144.6,
134.3, 129.4, 127.5, 122.4, 57.9, 56.8, 53.8, 37.2, 27.3, 25.6, 25.0.
EMS [M+H+] for C21H21N3O4, Calcd 380.1503. Found, 380.1529.
5. Experimental section
5.1. Chemistry
All yields refer to isolated products after purification. All prod-
ucts were characterized by their spectral (IR, 1H NMR, CHN, TLC
and GC) and physical data (melting and boiling points). The melt-
ing point for compound 2b was measured on a Gallenkamp melt-
ing apparatus. The remainder compounds are oil. All the amine
derivatives are free bases. 1H-NMR spectra were recorded using
400 MHz in CDCl3 solutions at room temperature (TMS was used
as an internal standard) on a Bruker Avance 500 MHz instrument
(Rheinstetten, Germany) or Varian 400 MHz NMR spectrometer.
FT-IR spectra were recorded on a spectrophotometer (Jasco-680,
Japan). Spectra of solids were carried out using KBr pellets.
Vibrational transition frequencies are reported in the wave number
(cmꢂ1). Furthermore, we used GC (BEIFIN 3420 Gas Chromato-
graph equipped a Varian CP SIL 5CB column-30 m, 0.32 mm,
5.1.5. Preparation of compounds 4a and 4b
A solution of 3a or 3b (free bases, 1.2 mmol) and hydrazine
monohydrate (0.05 ml, 15 mmol) in ethanol (95%, 15 ml) was re-
fluxed for 1 h. The reaction mixture was cooled and treated with
an additional amount of ethanol (95%, 15 m) and concentrated
HCl (1.3 ml). The reaction mixture was then refluxed for 4 h and
left overnight in a refrigerator. The precipitate was filtered off,
and the solvent was evaporated. The residue was treated with n-
hexane (20 ml) and NH3 (aqueous, 15 ml). The solution was ex-
tracted with CHCl3 (3 ꢁ 15 ml), the organic layer was dried over
anhydrous K2CO3, and the solvents were evaporated to give 4a or
4b which were used without further purification.
5.1.6. 4-(6,7-Dimethoxy-3,4-dihydroisoquinoline-2(1H)-yl)-
butan-1-amine (4a)
0.25 lm) for examination of reaction completion and yields. CHN
analyses were measured by Isfahan University of Technology using
a Vario EL III Element Analyzer (Germany). All of the starting
materials were purchased from Merck or Sigma–Aldrich. TLC plates
were from Merck.
Oil, yield 85%; 1H NMR, d: 6.92 (s, 2H, J = 8.6 Hz), 3.83 (s, 6H), 3.7
(s, 2H), 3.70 (t, 2H, J = 7.4 Hz), 2.95 (t, 2H, J = 7.4 Hz), 2.78 (t, 2H,
J = 7.6 Hz), 2.63 (t, 2H, J = 7.4 Hz), 2.46 (t, 2H), 2.32 (s, 2H), 1.72
(m, 2H), 1.48 (m, 2H). 13C NMR, d:148.2, 146.7, 126.9, 126.4,
111.4, 108.3, 57.2, 56.1, 53.7, 41.7, 27.3, 26.2, 25.4. EMS [M+H+]
for C15H24N2O2, Calcd 265.1838. Found, 265.3521.
5.1.1. Preparation of 7-nitro-1,2,3,4-tetrahydroisoquinoline
(2b)
In a mortar 2 g of P2O5/silica gel (65% w/w)1 (10 mmol) and
1,2,3,4-tetrahydroisoquinoline (10 mmol, 1.33 g) (Scheme 1) was
triturated for 30 s, and then 5 ml of HNO3 65% was added drop-wise
and the mixture was further triturated with a pestle at room tem-
perature for 20 min until a deep-yellow color appeared, at which
point TLC (n-hexane:EtOAc 70:30) showed complete disappearance
of 1,2,3,4-tetrahydroisoquinoline (30 min). To the reaction mixture
was added diethyl ether (50 ml) and the solid was separated
through a short pad of silica gel and washed with diethyl ether
(2 ꢁ 15 ml). The filtrate was washed with NaHCO3 10% (20 ml)
and dried (MgSO4). The solvent was evaporated under reduced
pressure and the residue was purified by column chromatography
(n-Hexane:EtOAc, 2:1), 7-nitro-1,2,3,4-tetrahydroisoquinoline
(2b) was obtained (8 mmol, 1.4 g 80%) as a yellow solid, mp
5.1.7. 4-(7-Nitro-3,4-dihydroisoquinoline-2(1H)-yl)butan-1-
amine (4b)
Yellow oil, yield 79%; 1H NMR, d: 8.07 (s, 1H), 7.02 (m, 1H), 7.43
(m, 1H,), 3.75 (s, 2H), 3.61 (t, 2H, J = 7.4 Hz), 2.95 (t, 2H, J = 7.4 Hz),
2.68 (t, 2H, J = 7.6 Hz), 2.65 (t, 2H, J = 7.6 Hz), 2.54 (s, 2H), 2.46 (t,
2H, J = 7.6 Hz), 1.74 (m, 2H),1.54 (m, 2H). 13C NMR, d: 150.8,
142.3, 135.4, 129.7, 122.8, 122.3, 53.9, 53.5, 53.2, 41.7, 27.0, 26.4,
25.0. EMS [M+H+] for C13H19N3O2, Calcd 250.3089, Found,
250.1841.
5.1.8. Preparation of compounds 5a–5i
To a solution of benzoic acid derivatives (1.1 mmol) in anhy-
drous THF (50 ml) was added dicyclohexylcarbodiimide (DCC,
1.1 mmol) in anhydrous THF (5 ml) and the reaction mixture was

A. R. Hajipour et al. / Bioorg. Med. Chem. 19 (2011) 7435–7440
7439
stirred at room temperature. After 5 min, a solution of the amine
4a or 4b in THF (5 ml) was added to this reaction mixture, and then
was stirred at room temperature overnight. The precipitate was fil-
tered off, and the solvent was evaporated. The organic layer was
dried over anhydrous MgSO4 and the solvents were evaporated
to give 5a-5i, which were purified using column chromatography
(silica gel, CHCl3: MeOH, 95:5).
J = 7.6 Hz), 2.40 (t, 2H, J = 7.5 Hz), 1.70 (t, 2H, J = 7.5 Hz), 1.50 (t,
2H, J = 7.5 Hz). 13C NMR, d: 172.4, 155.2, 150.6, 141.1, 134.2,
129.7, 122.9, 122.4, 120.3, 111.3, 104.4, 91.03, 60.3, 56.1, 53.9,
53.6, 39.4, 27.8, 27.4, 25.3. EMS [M+H+] for C21H24IN3O5, Calcd
526.3368. Found, 526.3984.
5.1.15. 2-Hydroxy-3-methoxy-N-(4-(7-nitro-3,4-
dihydroisoquinoline-2(1H)-yl)butyl)benzamide (5h)
Light yellow oil, yield 79%; 1H NMR, d: 13.78 (s, 1H), 8.50 (s, 1H),
8.02 (m, 2H), 7.42 (m, 3H), 7.08 (m, 1H,), 3.86 (s, 3H), 3.70 (s, 2H),
3.34 (t, 2H, J = 7.4 Hz), 2.98 (t, 2H, J = 7.4 Hz), 2.78 (t, 2H, J = 7.6 Hz),
2.40 (t, 2H, J = 7.6 Hz), 1.70 (t, 2H, J = 7.5 Hz), 1.50 (t, 2H, J = 7.4 Hz).
13C NMR, d: 171.4, 149.0, 148.4, 141.1, 134.2, 129.7, 122.9, 122.4,
120.3, 111.3, 104.4, 91.03, 60.3, 56.1, 53.9, 53.6, 39.4, 27.8, 27.4,
25.3. EMS [M+H+] for C21H25N3O5, Calcd 400.4403. Found,
400.4913.
5.1.9. N-(4-(6,7-Dimethoxy-3,4-dihydroisoquinoline-2(1H)-yl)-
butyl)-3-nitrobenzamide (5a)
Yellow oil, yield 78%; 1H NMR, d: 8.76 (s, 1H), 8.48 (m, 3H), 7.92
(m, 1H), 6.92 (s, 2H,), 3.88 (s, 6 H), 3.68 (s, 2H, J = J = 7.4 Hz), 3.34 (t,
2H, J = 7.4 Hz), 2.98 (t, 2H, J = 7.6 Hz), 2.78 (t, 2H, J = 7.6 Hz,
J = 7.6 Hz), 2.40 (t, 2H, J = 7.6 Hz), 1.70 (t, 2H, J = 7.8 Hz), 1.50 (t,
2H, J = 7.8 Hz). 13C NMR, d: 169.4, 148.2, 148.0, 135.4,129.7,
126.9, 126.4, 111.3, 108.4, 57.2, 56.1, 53.9, 53.6, 39.4, 27.8, 27.4,
25.3. EMS [M+H+] for C22H27N3O5, Calcd 414.4669, Found,
414.2243.
5.1.16. N-(4-(6,7-dimethoxy-3,4-dihydroisoquinoline-2(1H)-yl)-
butyl)-2-hydroxy-3-methoxybenzamide (5I)
5.1.10. N-(4-(6,7-Dimethoxy-3,4-dihydroisoquinoline-2(1H)-yl)-
butyl)benzamide (5c)
Oil, yield 83%; 1H NMR, d: 13.81 (s, 1H), 8.44 (s, 1H), 7.40 (m,
2H), 7.04 (m, 2H), 6.85 (m, 2H,), 3.88 (s, 6 H), 3.83 (m, 3H), 3.70
(s, 2H), 3.34 (t, 2H, J = 7.4 Hz), 2.98 (t, 2H, J = 7.4 Hz), 2.78 (t, 2H,
J = 7.6 Hz), 2.40 (t, 2H, J = 7.6 Hz), 1.70 (t, 2H, J = 7.5 Hz), 1.50 (t,
2H, J = 7.6 Hz). 13C NMR, dd: 171.6, 149.0, 148.4, 146.7, 126.8,
126.5, 122.9, 122.4, 120.3, 111.3, 104.4, 91.03, 60.3, 56.6, 56.2,
53.9, 53.6, 39.4, 27.8, 27.4, 25.3. EMS [M+H+] for C23H30N2O5, Calcd
415.44947. Found, 415.5231.
Oil, yield 75%; 1H NMR, d: 8.40 (s, 1H), 8.03 (m, 2H), 7.75 (m,
3H), 6.85 (m, 2H,), 3.80 (s, 6 H), 3.68 (s, 2H, J = 7.4 Hz), 3.34 (t,
2H, J = 7.4 Hz), 2.98 (t, 2H, J = 7.5 Hz), 2.78 (t, 2H, J = 7.6 Hz), 2.40
(t, 2H, J = 7.6 Hz), 1.70 (t, 2H, J = 7.8, J = 7.6 Hz Hz), 1.50 (t, 2H).
13C NMR, d: 167.5, 148.2, 146.5, 134.4, 132.1, 128.9, 127.4, 111.3,
108.4, 56.1, 53.9, 53.6, 39.4, 27.8, 27.4, 25.3. EMS [M+H+] for
C22H28N2O3, Calcd 369.4693. Found, 369.2639.
5.2. Preparation of rat liver membranes and RT4 cell
membranes
5.1.11. N-(4-(6,7-Dimethoxy-3,4-dihydroisoquinoline-2(1H)-yl)-
butyl)-4-nitrobenzamide (5d)
Oil, yield 69%; 1H NMR, d: 8.44 (s, 1H), 8.40 (m, 3H), 8.11 (d, 2H,
J = 8.6 Hz), 6.85 (m, 2H), 3.80 (s, 6 H), 3.68 (s, 2H), 3.34 (t, 2H,
J = 7.4 Hz), 2.98 (t, 2H, J = 7.4 Hz), 2.78 (t, 2H, J = 7.6 Hz), 2.40 (t,
2H, J = 7.6 Hz), 1.70 (t, 2H, J = 7.5 Hz), 1.50 (t, 2H, J = 7.6 Hz). 13C
NMR, d: 167.5, 151.3, 148.2, 146.5, 133.2, 124.0, 128.9, 127.4,
111.3, 108.4, 56.1, 53.9, 53.6, 39.4, 27.8, 27.4, 25.3. EMS [M+H+]
for C22H27N3O5, Calcd 414.4669. Found, 414.2436.
Rat livers (65 g, Pel-Freez Biologicals) were minced in 100 ml
homogenization buffer (20 mM Tris–HCl pH 8.0, 0.32 M sucrose)
that contains the Protease Inhibitor Cocktail (Sigma–Aldrich
P8340-5ML, for use with mammalian cell and tissue extracts),
and then homogenized on ice using a Brinkman Polytron Homoge-
nizer (setting 6, 4 bursts of 10 s each) followed by a glass homog-
enizer (Teflon pestle by
6 slow passes at 3000 rpm). The
homogenized tissues were first centrifuged at 1,000 g for 10 min,
and the supernatant was then centrifuged at 100,000ꢁg for 1 h at
4 °C. The membrane pellets were resuspended in the homogeniza-
tion buffer, and used for competitive sigma receptor binding
assays.
Human urinary bladder transitional papilloma RT4 cells (HTB-2,
ATCC) were harvested from the cell culture, and lysed using a soni-
cator (Branson Sonifier, output 50%, duty cycle 50%, 6 ꢁ 10 s) in the
homogenization buffer. The cell homogenates were centrifuged at
1,000ꢁg for 10 min, and the supernatant was then centrifuged at
100,000ꢁg for 1 h at 4 °C. The pelleted membranes were resus-
pended by brief sonication and used fresh for the photolabeling
assays.
5.1.12. 2,3,4-Trimethoxy-N-(4-(7-nitro-3,4-
dihydroisoquinoline-2(1H)-yl)butyl)benzamide (5e)
Oil, yield 82%; 1H NMR, d: 8.44 (s, 1H), 8.05 (m, 2H), 7.37 (m,
1H), 6.73 (m, 2H,), 3.88 (s, 3H), 3.83 (s, 6 H), 3.76 (s, 2H), 3.34 (t,
2H, J = 7.4 Hz), 2.98 (t, 2H, J = 7.4 Hz), 2.78 (t, 2H, J = 7.6 Hz), 2.40
(t, 2H, J = 7.6 Hz), 1.70 (t, 2H, J = 7.5 Hz), 1.50 (t, 2H, J = 7.5 Hz).
13C NMR, d: 168.6, 160.2, 151.3, 143.2, 141.5, 134.2, 129.7, 122.9,
122.4, 120.3, 111.3, 104.4, 60.9, 60,8, 56.1, 53.9, 53.6, 39.4, 27.8,
27.4, 25.3. EMS [M+H+] for C23H29N3O6, Calcd 444.4929. Found,
444.2573.
5.1.13. 2,3-Dimethoxy-N-(4-(7-nitro-3,4-dihydroisoquinoline-
2(1H)-yl)butyl)benzamide (5f)
Oil, yield 82%; 1H NMR, d: 8.44 (s, 1H), 8.05 (m, 2H), 7.41 (m,
1H), 7.13 (m, 1H,), 3.88 (s, 3H), 3.83 (s, 3H), 3.76 (s, 2H), 3.34 (t,
2H, J = 7.4 Hz, J = 7.4 Hz), 2.98 (t, 2H, J = 7.6 Hz), 2.78 (t, 2H,
J = 7.6 Hz), 2.40 (t, 2H, J = 7.4 Hz), 1.70 (t, 2H, J = 7.5 Hz), 1.50 (t,
2H, J = 7.5 Hz). 13C NMR, d: 168.6, 153.5, 150.3, 149.4, 141.5,
134.2, 129.7, 122.9, 122.4, 120.3, 111.3, 104.4, 60.9, 60,8, 56.1,
53.9, 53.6, 39.4, 27.8, 27.4, 25.3. EMS [M+H+] for C22H27N3O5, Calcd
414.4669. Found, 414.2247.
5.3. Sigma receptor radioligand binding assays
Competitive binding assays were performed to determine bind-
ing affinities of the new compounds for the
r
r
2 and
2 binding were per-
lg of total proteins per
r1 receptors as
previously described.24,27 Briefly, assays for
formed using rat liver membranes (ꢀ50
reaction) and 10 nM [3H]-DTG (PerkinElmer, 58.1 Ci/mmol) in
50 mM Tris–HCl pH 8.0, and in the presence of 100 nM cold
(+)-pentazocine to block
r1 binding sites. r1 binding in rat liver
5.1.14. 2-Hydroxy-4-iodo-3-methoxy-N-(4-(7-nitro-3,4-
dihydroisoquinoline-2(1H)-yl)butyl)benzamide (5g)
membranes was assayed by using 10 nM [3H]-(+)-pentazocine
(PerkinElmer, 34.8 Ci/mmol). Haloperidol (Sigma–Aldrich) of
Oil, Yield 75%; 1H NMR, d: 13.42 (s, 1H), 8.28 (s, 1H), 8.03 (m,
2H), 7.43 (m, 1H), 7.19 (m, 1H,), 3.84 (s, 3H), 3.70 (s, 2H), 3.34 (t,
2H, J = 7.4 Hz), 2.98 (t, 2H, J = 7.4 Hz, J = 7.6 Hz), 2.78 (t, 2H,
10 lM was used to determine non-specific binding. Serial dilutions
of the stocks (in DMSO) of compounds listed in Table 1 were added
to the reactions and incubated for 1.5 h at 32 °C. To assess the

7440
A. R. Hajipour et al. / Bioorg. Med. Chem. 19 (2011) 7435–7440
ability of the new compounds to displace radioligands from the
sigma receptor subtypes, following the incubation, the samples
were filtered through Brandel GF/B Fired Membranes (which were
pretreated with 0.5% polyethyleneimine) using a Brandel Cell Har-
vester (M-48T). Radioactivity on the filters was counted using a
Beckman Scintillation Counter (LS-6500) in an NEN formula 989
scintillation cocktail (Ultima Gold MV, PerkinElmer). Values were
fit to a non-linear regression curve (one-site competition) using
Graphpad Prism Version 4.0c. Ki was calculated using the Cheng-
Prusoff equation.28 For the Ki calculation, a Kd of 50 nM was used
for [3H]-DTG and a Kd of 25 nM was used for (+)-pentazocine in
rat liver membranes.
References and notes
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5.4. Photoaffinity labeling of sigma receptors
Radiochemical synthesis of the sigma receptor photolabel [125I]-
IAF
(1-N-(20,60-dimethyl-morpholino)-3-(4-azido-3-[125I]iodo-
phenyl propane) was performed as previously described.20 Fresh
RT4 cell membranes were used for [125I]-IAF photoaffinity labeling
of sigma receptors since this cell line is highly enriched in the
receptor. To test the 2 or 1 binding specificity of the new com-
pounds, compounds 3b, 5e, and 5f were first preincubated with
RT4 membranes (200 g total proteins per reaction) in 50 mM Tris
r2
r
r
l
(pH 7.4) for 30 min at 32 °C. [125I]-IAF was then added to the mem-
branes to a final concentration of 1 nM (final 1% ethyl acetate) and
incubated for another 30 min at 32 °C. Following the incubation,
the [125I]-IAF photoreactive label was activated by exposure for
6 s to a high-pressure AH-6 mercury lamp (10 cm distance). The
photolysis reactions were then quenched immediately with the
sample buffer containing 200 mM b-mercaptoethanol and 1%
SDS. Proteins were separated on a SDS–Tricine gel, and radiola-
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Dynamics).
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Acknowledgements
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This work was supported by NIH Grants MH065503 and
DA027191, and a Retina Research Foundation Edwin & Dorothy
Gamewell Professorship (to A.E.R). We thank Dr. Uyen B. Chu for
assistance in the radiochemical synthesis of [125I]-IAF.
28. Cheng, Y.; Prusoff, W. H. Biochem. Pharmacol. 1973, 22, 3099.