Synthesis and in vitro evaluation of tetrahydroisoquinolinyl benzamides as ligands for σ receptors

Article abstract of DOI:10.1016/j.bmcl.2007.02.005

5-Bromo-N-[4-(6,7-dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-butyl)]-2,3-dimethoxy-benzamide (1) is one of the most potent and selective σ₂ receptor ligands reported to date. Previous structure-activity relationship studies of such tetrahydroisoquinolinyl benzamides have focused on the linker that connects the ring systems and the effects of benzamide ring substituents. The present study explores the effects of fusing methylene-, ethylene-, and propylenedioxy rings onto the tetrahydroisoquinoline in place of the two methoxy groups. These modifications decreased σ₂ affinity by 8- to 12-fold, with no major differences noted with ring size. By contrast, the methylenedioxy analog showed a 10-fold greater σ₁ affinity than 1, and progressively lower σ₁ affinities were then noted with increasing ring size. We also opened the tetrahydroisoquinoline ring of 1 to study the effects of greater conformational fluidity on σ receptor binding. The σ₂ affinity of the open-ring compound decreased by 1700-fold, while σ₁ affinity was not changed. Thus, a constrained tetrahydroisoquinoline ring system is key to the exceptional σ₂ receptor binding affinity and selectivity of this active series.

Full text of DOI:10.1016/j.bmcl.2007.02.005

Bioorganic & Medicinal Chemistry Letters 17 (2007) 2594–2597
Synthesis and in vitro evaluation of tetrahydroisoquinolinyl
benzamides as ligands for r receptors
Rong Xu,a John R. Leverb,c,d and Susan Z. Levera,e,
*
aDepartment of Chemistry, University of Missouri-Columbia, Columbia, MO, USA
bDepartment of Radiology, University of Missouri-Columbia, Columbia, MO, USA
cDepartment of Medical Pharmacology and Physiology, University of Missouri-Columbia, Columbia, MO, USA
dHarry S. Truman Veterans Administration Medical Center, Columbia, MO, USA
eResearch Reactor Center, University of Missouri–Columbia, Columbia, MO, USA
Received 26 December 2006; revised 30 January 2007; accepted 1 February 2007
Available online 4 February 2007
Abstract—5-Bromo-N-[4-(6,7-dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-butyl)]-2,3-dimethoxy-benzamide (1) is one of the most
potent and selective r2 receptor ligands reported to date. Previous structure-activity relationship studies of such tetrahydroisoquin-
olinyl benzamides have focused on the linker that connects the ring systems and the effects of benzamide ring substituents. The pres-
ent study explores the effects of fusing methylene-, ethylene-, and propylenedioxy rings onto the tetrahydroisoquinoline in place of
the two methoxy groups. These modifications decreased r2 affinity by 8- to 12-fold, with no major differences noted with ring size.
By contrast, the methylenedioxy analog showed a 10-fold greater r1 affinity than 1, and progressively lower r1 affinities were then
noted with increasing ring size. We also opened the tetrahydroisoquinoline ring of 1 to study the effects of greater conformational
fluidity on r receptor binding. The r2 affinity of the open-ring compound decreased by 1700-fold, while r1 affinity was not changed.
Thus, a constrained tetrahydroisoquinoline ring system is key to the exceptional r2 receptor binding affinity and selectivity of this
active series.
Ó 2007 Elsevier Ltd. All rights reserved.
Functional sigma (r) receptors are located throughout
the brain and periphery, and can be differentiated into
r1 and r2 subtypes.1–4 These subtypes play distinct func-
tional roles and have different pharmacological charac-
teristics. Both r1 and r2 subtypes are involved in
central nervous system disorders such as schizophrenia,
depression, and dementia.1,2 Certain r receptor antago-
nists can ameliorate the effects of cocaine and other psy-
chostimulant drugs of abuse, and have potential as
medications.1–3 Moreover, r receptors are over-ex-
pressed by many cancers.4 Some r receptor ligands in-
duce apoptosis in cancer cells,5–7 and one is in a
clinical trial for prostate cancer treatment.8 Thus, there
is much interest in subtype selective r receptor ligands
as molecular probes and as therapeutic agents.
of selective binding models.9,10 Although a number of
studies have investigated the effects of structure on rela-
tive r1/r2 receptor binding affinity and selectivity, few
truly selective compounds are known. Recently, Mach
and colleagues11 identified a series of tetrahydroisoquin-
olinyl benzamides that rank among the most selective r2
receptor ligands known to date. For example, 1 displays
high apparent affinity, Ki = 8.2 nM, for r2 sites in vitro
accompanied by 1573-fold selectivity over r1 sites.
OCH3
H3CO
H3CO
O
N
N
OCH3
H
Br
1
A variety of structural classes are avid binders to both r
receptor subtypes which has hampered the development
Published structural modifications have concentrated on
the length of the alkyl spacer connecting the two differ-
ent ring systems, and the effects of various benzamide
substituents.11–13 To extend the structure-activity rela-
tionships for this active series, we report on the effects
Keywords: r Receptors; Tetrahydroisoquinoline.
*
Corresponding author at present address: 125 Chemistry, 601 S.
College Ave., Columbia, MO 65211, USA. Tel.: +1 573 882 8395;
fax: +1 573 882 2754; e-mail: levers@missouri.edu
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.02.005

R. Xu et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2594–2597
2595
of fusing methylene-, ethylene-, and propylenedioxy
OMe
OMe
OH
OH
a
rings onto the tetrahydroisoquinoline. This stems from
work on 1,4-disubstituted piperazines, where we found
that r receptor subtype affinity and selectivity can be
modulated by similar manipulations of dimethoxyben-
zene moieties.14 In addition, we noticed that C–N bond
rotation in 1 is limited by the tetrahydroisoquinoline
ring. Thus, we wished to open this ring to gain insight
into the contributions of conformational fluidity to r
receptor binding.
HN
HCl
HN
HBr
b
OH
OH
O
O
c, d
O
N
HN
•HCl
( )
n
Compound 1 was obtained for reference using the
reported methods.11 The novel congeners were prepared
as shown in Schemes 1–3. For methylenedioxy analog 2,
the corresponding tetrahydroisoquinoline was synthe-
sized from piperonal using an established route that cul-
minates with the Pictet–Spengler reaction.15–17
Alkylation with 4-bromobutanenitrile, followed by
reduction and amidation with 5-bromo-2,3-dim-
ethoxybenzoyl chloride, afforded 2 which was character-
ized as the oxalate salt (Scheme 1).18
O
e, f, g
O
O
H3CO
O
( )
H3CO
N
n
N
H
Ethylenedioxy (3) and propylenedioxy (4) analogs were
synthesized in parallel fashion from their corresponding
tetrahydroisoquinolines (Scheme 2). In turn, these three-
ring heterocycles were obtained from N-Boc protected
tetrahydroisoquinoline diol by base-promoted cyclo-
alkylation with the appropriate dibromoalkane cata-
lyzed by tetrabutylammonium bromide (Scheme 2).
Br
3, n=1
4, n=2
Scheme 2. Reagents and conditions: (a) 48% HBr, 120 °C, 2 h; (b)
(Boc)2O, MeOH, Et3N; (c) Br-(CH2)n-Br: n = 2, 3, TBAB; (d) 4 N HCl;
(e) 4-bromobutanenitrile, K2CO3, NaI, DMF; (f) LiAlH4; (g) 5-
bromo-2,3-dimethoxybenzoyl chloride.
As shown in Scheme 3, open-ring compound 5
was prepared by alkylation of the commercially
OCH3
OCH3
a, b
NC
N
O
NH2
OCH3
O
OCH3
O
O2N
O
O
O
O
c, d
a
H
OCH3
H3CO
H3CO
O
b
N
N
H
OCH3
O
O
Br
c
H2N
O
O
O
O
e
HN
OCH3
OCH3
d, e, f
H3CO
H3CO
O
NH
N
H
5
O
O
Br
H3CO
H3CO
O
N
Scheme 3. Reagents: (a) 4-bromobutanenitrile; (b) (Boc)2O, MeOH,
Et3N; (c) LiAlH4; (d) 5-bromo-2,3-dimethoxybenzoyl chloride; (e) 10%
TFA, CH2Cl2.
N
H
2
Br
available 2-(3,4-dimethoxyphenyl)ethanamine, followed
by N-Boc protection, reduction, amidation, and
deprotection.
Scheme 1. Reagents: (a) CH3NO2, MeOH, NaOH; (b) LiAlH4; (c)
paraformaldehyde; (d) 4-bromobutanenitrile, K2CO3, NaI, DMF; (e)
LiAlH4; (f) 5-bromo-2,3-dimethoxybenzoyl chloride.

2596
R. Xu et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2594–2597
Table 1. Binding properties of compounds 1–5 at r1 and r2 receptors19
compound having 5-fold selectivity for binding to r1
receptors. Thus, the constrained tetrahydroisoquinoline
ring is critically important to high r2 receptor binding
affinity and selectivity.
Compound
Ki (nM)
r2
2.7 0.1
Ratio r1/r2
r1
881 15
1
2
3
4
5
326
4
82.2 5.6
338 8.4
1430 36
880 60
20.7 2.0
21.7 1.2
32.6 1.5
4616 247
In conclusion, we determined that modifications of the
two methoxy groups of the tetrahydroisoquinolinyl ben-
zamides can be used to modulate the relative affinities
and selectivities of ligand binding to r1 and r2 receptor
subtypes. We also demonstrated that a constrained tet-
rahydroisoquinoline ring system is key to the exception-
al r2 receptor binding affinity and selectivity observed
for this active series.
16
44
0.2
Values are means SEM (n = 3–5) from competition assays against
[3H](+)-pentazocine (r1) and [3H]DTG/(+)-pentazocine (r2) in mem-
branes from male guinea pig brains.
As expected, compound 1 displayed very high affinity
and selectivity for r2 sites in vitro (Table 1). The degree
of r2 selectivity, based upon Ki ratios, was somewhat
less than previously found11 as a consequence of a high-
er apparent affinity for r1 sites. The r1 receptor assay in
guinea pig brain membranes is susceptible to slight
changes in conditions. So, we also tested 1 using the pre-
viously reported regimen (pH 8.0 vs pH 7.4 buffer,
3.0 nM vs 1.0 nM [3H](+)-pentazocine, 25 °C vs 37 °C,
120 min vs 150 min, and 10 lM (+)-pentazocine vs
1.0 lM haloperidol to define nonspecific binding). The
r1 receptor IC50 value of 1273 22 nM found for 1 un-
der the present conditions increased substantially, about
50%, to 1895 110 nM. Comparing this lower affinity
r1 receptor IC50 with the r2 receptor IC50 of 3.0 0.11
for 1 under the present conditions would double the
selectivity assigned. Also, the r2 receptor binding was
assessed using rat liver membranes in the previous work,
while guinea pig brain membranes were employed in the
present study. In such ways, experimental factors can
impact the r1/r2 subtype selectivity determinations from
various laboratories.
Acknowledgments
We thank the National Cancer Institute (P50 CA
103130: Center for Single Photon-Emitting Cancer
Imaging Agents) for partial support of this research.
We also acknowledge facilities provided by Truman
Memorial Veterans’ Hospital, and NSF CHE-95-31247
and NIH 1S10RR11962-01 grant awards for NMR
instrumentation.
References and notes
1. Guitart, X.; Codony, X.; Monroy, X. Psychopharmacol-
ogy 2004, 174, 301.
2. Hayashi, T.; Su, T.-P. CNS Drugs 2004, 18, 269.
3. Matsumoto, R. R.; Liu, Y.; Lerner, M.; Howard, E. W.;
Brackett, D. J. Eur. J. Pharmacol. 2003, 469, 1.
4. Aydar, E.; Palmer, C. P.; Djamgoz, M. B. Cancer Res.
2004, 64, 5029.
5. Colabufo, N. A.; Berardi, F.; Contino, M.; Niso, M.;
Abate, C.; Perrone, R.; Tortorella, V. Naunyn. Schmiede-
bergs Arch. Pharmacol. 2004, 370, 106.
Replacement of the two methoxy groups by a methy-
lene-, ethylene- or propylenedioxy ring decreased r2
affinity by 8- to 12-fold, with no major effects attribut-
able to the specific sizes of the rings (Table 1). By con-
trast, methylenedioxy analog 2 showed a 10-fold
greater r1 affinity than the parent scaffold 1. Further
effects of ring size were well defined, with progressively
4-fold lower r1 affinities noted for the ethylenedioxy (2)
and propylenedioxy (3) analogs. Thus, r1 binding
exhibits the most sensitivity to these perturbations.
Together, the data indicate that r1/r2 receptor binding
affinity and selectivity can be modulated by subtle
changes in molecular volumes, ring conformations,
and the precise orientations of the oxygen atoms in this
region.
6. Azzariti, A.; Colabufo, N. A.; Berardi, F.; Porcelli, L.;
Niso, M.; Simone, G. M.; Perrone, R.; Paradiso, A. Mol.
Cancer Ther. 2006, 5, 1807.
7. Rothfuss, J.; Zeng, C.; Vangveravong, S.; Chu, W.; Tu, Z.;
Hotchkiss, R.; Chang, K.C.; Mach, R.H. Abstracts of
Papers, 232nd National Meeting of the American Chem-
ical Society, San Francisco, CA, September 10-14, 2006;
American Chemical Society: Washington, DC, 2006;
MEDI 040.
8. Casellas, P.; Galiegue, S.; Bourrie, B.; Ferrini, J.-B.; Jbilo,
O.; Vidal, H. Anticancer Drugs 2004, 15, 113.
9. Glennon, R. A. Mini-Rev. Med. Chem. 2005, 5, 927.
10. Glennon, R. A. Braz. J. Pharm. Sci. 2005, 41, 1.
11. Mach, R. H.; Huang, Y.; Freeman, R. A.; Wu, L.;
Vangveravong, S.; Luedtke, R. R. Bioorg. Med. Chem.
Lett. 2004, 14, 195.
12. Xu, J.; Tu, Z.; Jones, L. A.; Vangveravong, S.; Wheeler,
K. T.; Mach, R. H. Eur. J. Pharmacol. 2005, 525, 8.
13. Rowland, D. J.; Tu, Z.; Xu, J.; Ponde, D.; Mach, R. H.;
Welch, M. J. J. Nucl. Med. 2006, 47, 1041.
14. Xu, R.; Lever, J.R.; Lever, S.Z. Abstracts of Papers, 233rd
National Meeting of the American Chemical Society,
Chicago, IL, March 25-29, 2007; American Chemical
Society: Washington, DC, 2007; MEDI 462.
15. Bowman, R. K.; Johnson, J. S. J. Org. Chem. 2004, 69,
8537.
16. Koseki, Y.; Katsura, S.; Kusano, S.; Sakata, H.; Sato, H.;
Monzene, Y.; Nagasaka, T. Heterocycles 2003, 59, 527.
Remarkably, the r2 affinity of open-ring compound 5
decreased by 1700-fold, while the r1 affinity was not
changed (Table 1). It is difficult to provide a molecular
explanation for such an interesting result. Nevertheless,
this observation may aid in developing r receptor bind-
ing models for tetrahydroisoquinolinyl benzamides.
Clearly, the greater conformational freedom of 5 with
respect to 1 is detrimental to r2 receptor binding but
has no influence on binding interactions with r1 recep-
tors. The effect is pronounced and leads to a low affinity

R. Xu et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2594–2597
2597
17. Ruchirawat, S.; Chaisupakitsin, M.; Patranuwatana, N.;
Cashaw, J. L.; Davis, V. E. Synth. Commun. 1984, 14,
1221.
C23H31BrN2O5 Æ C2H2O4: C, 51.29; H, 5.68; N, 4.79.
Found: C, 51.07; H, 5.72; N, 4.75.
19. The r receptor binding assays were performed as previ-
ously described in detail,20 except membranes were
prepared exclusively from male guinea pig brains. In brief,
r1 assays used [3H](+)-pentazocine (1.0 nM) in 50 mM
Tris–HCl buffer (pH 7.4, 25 °C) with nonspecific binding
defined by haloperidol (1.0 lM). Assay tubes were incu-
bated for 150 min at 37 °C using 0.25 mg protein in a final
volume of 1.0 mL. The r2 assays used [3H]ditolylguanidine
([3H]DTG, 3.0 nM) with 200 nM (+)-pentazocine added as
a r1 receptor mask. Incubations were performed using
50 mM Tris–HCl buffer (pH 8.0, 25 °C) with nonspecific
binding defined by DTG (100 lM). Assay tubes were
incubated for 120 min at 25 °C using 0.25 mg protein in a
final volume of 0.5 mL. Test compounds were dissolved in
water containing 0.1% HOAc and 1.0% EtOH, and
comprised 10% of the final assay volumes. Ten concen-
trations were used that were centered on the IC50 and
spaced equally on log scale. Assays were terminated by
addition of ice-cold incubation buffer followed by rapid
filtration through Whatman GF/B glass fiber filters
presoaked in 0.5% polyethylenimine using a Brandel cell
harvester. Filters were washed three times with 3–4 mL of
ice-cold buffer, dried, and extracted with Hi-Safe 2
scintillation cocktail. Radioactivity was measured using
a Wallac 1409 liquid scintillation counter at a tritium
efficiency of 44%. Binding data were analyzed with curve-
fitting programs Prism 4.0b and Radlig 6.0. Ki values were
computed from IC50’s using the Cheng-Prusoff relation-
ship, with Kd input values of 2.3 nM for [3H](+)-
pentazocine and 23.9 nM for [3H]DTG.20
1
18. Data for 2. H NMR (free base, CDCl3, d) 1.67 (m, 4H,
CH2); 2.51 (t, 2H, CH2); 2.65 (t, 2H, CH2); 2.75 (t, 2H,
CH2); 3.44–3.48 (m, 4H, CH2); 3.84–3.86 (2s, 6H,
OCH3); 5.86 (s, 2H, OCH2O); 6.44 (s, 1H, aromatic
CH); 6.52 (s, 1H, aromatic CH); 7.08 (d, 1H, CH); 7.74
(d, 1H, aromatic CH); 8.01 (t, 1H, amide NH). Mp.
174–175 °C (mono-oxalate salt); Anal. Calcd for
C23H27BrN2O5 Æ C2H2O4: C, 51.64; H, 5.03; N, 4.82.
Found: C, 51.80; H, 5.15; N, 4.80.
1
Data for 3. H NMR (free base, CDCl3, d) 1.68 (p, 4H,
CH2); 2.52 (t, 2H, CH2); 2.66 (t, 2H, CH2); 2.75 (t, 2H,
CH2); 3.49 (m, 4H, CH2). 3.86 (2s, 6H, OCH3); 4.20 (s,
4H, OCH2CH2O); 6.49 (s, 1H, CH); 6.57 (s, 1H, CH);
7.10 (d, 1H, CH); 7.75 (d, 1H, CH); 7.99 (t, 1H, amide
NH). Mp. 146–147 °C (mono-oxalate salt); Anal. Calcd
for C24H29BrN2O5 Æ C2H2O4: C, 52.45; H, 5.25; N, 4.70.
Found: C, 52.66; H, 5.30; N, 4.61.
1
Data for 4. H NMR (free base, CDCl3, d) 1.66 (m, 4H,
CH2); 2.13 (m, 2H, CH2); 2.50 (t, 2H, CH2); 2.65 (t, 2H,
CH2); 2.77 (t, 2H, CH2); 3.40–3.48 (m, 4H, CH2). 3.84–3.86
(2s, 6H, OCH3); 4.12 (s, 4H, CH2O); 6.61 (s, 1H, CH); 6.69
(s, 1H, CH); 7.10 (d, 1H, CH); 7.75 (d, 1H, CH); 7.98 (t, 1H,
amide NH). Mp. 163–164 °C (mono-oxalate salt); Anal.
Calcd for C25H31BrN2O5 Æ C2H2O4: C, 53.21; H, 5.46; N,
4.60. Found: C, 53.26; H, 5.48; N, 4.60.
Data for 5. 1H NMR: (free base, CDCl3, d) 1.66 (br m, 4H,
CH2); 2.85 (t, 2H, CH2); 2.96 (t, 2H, CH2); 3.43 (t, 2H, CH2);
3.83–3.86 (4s, 12H, OCH3); 5.75 (br s, 1H, NH); 6.70–6.80
(m, 3H, aromatic CH); 7.11 (d, 1H, aromatic CH); 7.74 (d,
1H, aromatic CH); 7.99 (t, 1H, amide NH). Mp.
176–177 °C (mono-oxalate salt); Anal. Calcd for
20. Lever, J. R.; Gustafson, J. L.; Xu, R.; Allmon, R. L.;
Lever, S. Z. Synapse 2006, 59, 350.