Reevaluation of fenpropimorph as a σ receptor ligand: Structure-affinity relationship studies at human σ1 receptors

Article abstract of DOI:10.1016/j.bmcl.2017.04.088

Fenpropimorph (1) is considered a “super high-affinity” σ₁ receptor ligand (K_i?=?0.005?nM for guinea pig σ₁ receptors). Here, we examine the binding of 1 and several of its deconstructed analogs at human σ₁ (hσ<

Full text of DOI:10.1016/j.bmcl.2017.04.088

Accepted Manuscript
Reevaluation of Fenpropimorph as a σ Receptor Ligand: Structure-Affinity Re-
lationship Studies at Human σ Receptors
1
Elena Sguazzini, Hayden R. Schmidt, Kavita A. Iyer, Andrew C. Kruse,
Małgorzata Dukat
PII:
S0960-894X(17)30474-2
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.04.088
BMCL 24941
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Accepted Date:
10 April 2017
27 April 2017
Please cite this article as: Sguazzini, E., Schmidt, H.R., Iyer, K.A., Kruse, A.C., Dukat, M., Reevaluation of
Fenpropimorph as a σ Receptor Ligand: Structure-Affinity Relationship Studies at Human σ Receptors, Bioorganic
1
&
Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.04.088
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Reevaluation of Fenpropimorph as a  Receptor Ligand: Structure-Affinity
Relationship Studies at Human  Receptors
1
a
b
a
b
Elena Sguazzini, Hayden R. Schmidt, Kavita A. Iyer, Andrew C. Kruse, Małgorzata
a*
Dukat
a
Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond,
Virginia 23298, USA,
b
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical
School, Boston, Massachusetts 02115, USA.
*
Corresponding author. Tel.: 8048285256; fax: 8048287625.
E-mail address: mdukat@vcu.edu (M. Dukat)
Address: Department of Medicinal Chemistry, School of Pharmacy, Virginia Commonwealth
University, PO Box 980540, Richmond, VA 23298-540, USA
Bioorg. Med. Chem. Lett._submitted April 10, 2017

ABSTRACT
Fenpropimorph (1) is considered a “super high-affinity”  receptor ligand (K = 0.005 nM
1
i
for guinea pig  receptors). Here, we examine the binding of 1 and several of its
1
deconstructed analogs at human  (h ) receptors. We monitored their subtype selectivity by
1
1
determining the binding affinity at  receptors. In addition, we validated an existing
2
pharmacophore model at the molecular level by conducting 3D molecular modeling studies,
using the crystal structure of h receptors, and Hydrophatic INTeractions (HINT) analysis. Our
1
structure affinity relationship studies showed that 1 binds with lower affinity at h receptors (Ki
1
=
17.3 nM) compared to guinea pig; moreover, we found that none of the fenpropimorph
methyl groups is important for its binding at h receptors, nor is stereochemistry. For
1
example, removal of all methyl groups as seen in 4 resulted in an almost 5-fold higher
affinity at h receptors compared to 1 and 350-fold selectivity versus  receptors. In
1
2
addition, although the O atom of the morpholine ring does not contribute to affinity at h1
receptors (and might even detract from it), it plays role in subtype ( versus  receptor)
1
2
selectivity.
KEY WORDS
SAFiR, Radioligand binding, 3D Molecular modeling, HINT analysis
1
. Introduction

1
The sigma1 ( ) receptor experienced a resurgence in interest beginning in 2011 when a
1
2
familial mutation in the receptor was shown to cause amyotrophic lateral sclerosis (ALS).
Since then, the  receptor has emerged as a potential therapeutic target for a variety of
1
neurological disorders including pain, Parkinson’s, Alzheimer’s and Huntington’s diseases,
3
, 4
depression, amnesia, and schizophrenia.
Originally,  receptors were classified as a subtype of the opioid receptor superfamily
5
based on radioligand-binding studies, but further pharmacological studies and the molecular
cloning of the  receptor confirmed that both it and the pharmacologically similar but
1
6
genetically distinct σ receptor are unrelated to opioid receptors. The σ receptor has a
2
1
particularly diverse pharmacological profile as it can bind a remarkable variety of small
molecules with high (<100 nM) affinity. This spurred the creation and identification of a
myriad of molecules that can target the receptor. Combined with its emerging disease
relevance, the pharmacological tractability of the σ receptor makes it an ideal target for
1
therapeutic intervention.
Recently, the Kruse laboratory reported the 3D crystal structure of the human  (h )
1
1
receptor in complex with two structurally and functionally diverse ligands, an antagonist,
PD144418 (Figure 1), and the yet to be defined agonist/inverse agonist, N-(1-
benzylpiperidin-4-
yl)-4-iodobenzamide (4-IBP), revealing a trimeric structure with a single transmembrane
7
domain for each protomer.

Figure 1. Structures of the  receptor ligands fenpropimorph (1) and PD144418.
Fenpropimorph (1) is, perhaps, the highest-affinity  receptor ligand ever reported. It
1
binds with extremely high affinity at guinea pig whole-brain (K = 0.005 nM) and liver (K =
i
i
3
0
.011 nM), and yeast-expressed (K = 0.08 nM) homogenate  receptors labeled by (+)-[ H]-
i
1
8
, 9
pentazocine (PTZ) as radioligand. It might be considered as a “super high-affinity” 1
receptor ligand. Not surprisingly, the Ruoho laboratory used this agent as a scaffold to
1
0
develop newer, high-affinity  receptor ligands. Unfortunately, it is not known if any of
1
the modifications introduced to the fenpropimorph structure resulted in a higher-affinity
ligand than the parent compound because 1 was not used as a positive control in their guinea-
1
0
pig liver assay.
The picomolar affinity of fenpropimorph makes it a very attractive starting point for the
development of novel  receptor agents. But, the question arises: what structural features
1
make fenpropimorph such a high-affinity ligand? Moreover, does fenpropimorph retain
comparable picomolar affinity at human  receptors? Here we address these questions,
1
leveraging both synthetic medicinal chemistry, the hσ receptor crystal structure, and
1
homology modeling studies, to provide a complete structural analysis of fenpropimorph
binding.
We began by examining the binding of fenpropimorph at h receptors. As an indication
1
of selectivity, we also examined  receptor binding. To understand which structural aspects
2

of fenpropimorph account for binding to human  receptors we used the “deconstruction-
1
1
1
elaboration” approach.
Chemical entities with similar structure had already been synthesized by Glennon’s
1
1
4, 11
group, and a  receptor pharmacophore has been proposed.
Here, we synthesized a
1
new series of fenpropimorph-related compounds to determine if they fit the pharmacophore
4
, 7, 11
model and, especially, the recently crystalized structure of the h receptor.
That is, the
1
proposed pharmacophore model suggested that the branched chain between the phenyl ring
and amine moiety was not required and that the morpholine oxygen atom should, if anything,
slightly detract from binding. The pharmacophore model also implicated a “primary
4
, 11
hydrophobic site” at a certain distance from the amine.
Hence, the hydrophobic tert-butyl
group of 1 should be a contributor to binding. We prepared a series of analogs to test these
hypotheses that we present here in one of the first structure-guided analyses of hσ receptor
1
pharmacology.
2
2
. Results and Discussion
.1. Synthesis
The commercially available free base of fenpropimorph (1) was converted to its more
water-soluble hydrochloride salt, which had not been previously reported. A series of
fenpropimorph analogs was synthesized using a common pathway (Scheme 1). Classical
Knoevenagel condensation with the Doebner modification was applied to obtain known 4-
1
2
tert-butyl-phenylacrylic acid (9a), using the corresponding commercially available
aldehyde 8a and malonic acid. Catalytic reduction of the olefinic double bond of 9a with 10%
13
Pd/C under a H atmosphere yielded carboxylic acids 10a. The acid chlorides 11a and 11b
2
were generated in situ from the carboxylic acid 10a and commercially available

dihydrocinnamic acid (10b), respectively, in reaction with thionyl chloride. The acid
chlorides were then reacted with the corresponding amine using a literature procedure for a
1
4
similar compounds to obtain the amides 12-16, followed by reduction with diborane or a
borane dimethyl sulfide complex (in preparation of 7) to afford the target compounds 2, and
4
-7. The trans isomer 3 was obtained by separation of an isomeric mixture of 2 using flash
chromatography. All target compounds were prepared as water-soluble salts. This pathway
gave satisfactory yields.
Scheme 1. Synthetic pathway for fenpropimorph analogs 2-7.

a
Reagents and conditions: (i) CH (COOH) , piperidine, pyridine; (ii) H , 10% Pd/C, EtOAc;
2
2
2
(
iii) SOCl ; (iv) a.) CH Cl , Et N, stirring at room temperature; b.)10% HCl, Et O; (v)
2 2 2 3 2
BH THF or BH S(CH ) (for 7), THF, reflux.
3
3
3 2
1
The structures of 1-7 were confirmed by H NMR, IR, mp (where appropriate) and C, H, N
analysis. The hydrochloride salt of 5 was previously synthesized by a different synthetic
1
5
route, but was not fully characterized. Compound 6 was not known at the time we prepared
1
6
it (Scheme 1) but was subsequently reported by Khan and Bhanage using a different method
of preparation.
2
.2. Structure-Affinity Relationship (SAfiR) Studies

1
receptor binding data and SAfiR are described here in the context of an existing
pharmacophore model. The proposed pharmacophore model suggests three major features are
important for high binding affinity at  receptors: i) a “primary hydrophobic region”
1
(constituted of aryl ring or similar hydrophobic moiety) situated at 6-10 Å (optimum = 8.3
Å) distant from an amine, ii) a secondary or tertiary amine (the amine can be a part of cyclic
structure), and iii) a small “secondary hydrophobic region” distant at 2.4-3.9 Å from the
4
, 11
amine (Figure S1).
Fenpropimorph (1) seems to meet these structural requirements (Figure S1). That is, the
basic tertiary amine of 1 is surrounded by the “primary hydrophobic region” (corresponding
to tert-butylphenyl; the distance from N atom to the t-butyl tertiary C atom = 8.71 Å) and the
“
secondary hydrophobic region” (the distance from N atom to CH = 3.3 Å).
3

3
We assessed the binding affinity of the analogs 1-7 for the h receptor using [ H](+)-
1
PTZ as a radioligand and we generated the corresponding binding curves (Figure 2).

3
Figure 2. Radioligand binding curves for the σ receptor. A) A [ H](+)-PTZ saturation curve
1
depicting total (red circles) and non-specific (blue squares) binding to Sf9 membranes. K ±
d
3
SEM = 20.0 ± 1.2 nM. B) Competition binding curve against [ H](+)-PTZ in Sf9 membranes
for compounds 1 (orange diamonds), 2 (brown hexagons), 3 (green triangles pointed up), 4
(red squares), 5 (blue circles), 6 (purple triangles pointed down), and 7 (pink half-filled
circles). K values are listed in Table 1. Error bars represent SEM. Curves depicted in this
i
figure are representative curves from at least two independent experiments each performed in
triplicate.
Compounds 2 and 3 (Table 1), with nearly comparable  receptor affinity, and an affinity at
1
least twice that of 1, show that the side chain methyl group does not contribute to binding,
that the stereochemistry of the morpholine methyl groups are relatively unimportant, and that
removal of all the methyl groups (i.e., 4, Table 1) is tolerated. However, removing the tert-
butyl group results in a dramatic (i.e., 265-fold) decrease in affinity (i.e., 5).

Table 1. Binding affinity (K ± SEM nM) of fenpropimorph (1) and its analogs 2-7 at human
i
1
and rat  receptors.
2
a
i
K ± SEM (nM)
1 selectivity
h1
r2
1
2
3
4
5
6
7
(fenpropimorph) 17.3 ± 1.2
8.4 ± 0.6
854 ± 6.8
77.9 ± 0.9
66.1 ± 1.1
930 ± 14
2019 ± 41
31.5 ± 0.7
13.8 ± 0.3
50
9
2.3 ± 0.1
29
360
3
2.6 ± 0.1
706 ± 19
1.5 ± 0.04
21
3
5.4 ± 0.4
a
Values presented as the mean ± SEM (n = 3)
Removal of the morpholine oxygen atom from 4 (i.e., 6, Table 1) reveals it to be unimportant
for  receptor affinity and, indeed, slightly improves binding affinity. Piperidine analog 6
1
displays ten times higher affinity than 1. Elaborated compound 7 (Table 1) possesses
structural features required for binding and binds with at least thrice the affinity of 1.
In addition we monitored the subtype selectivity of analogs 1-7 for  vs  receptors.
1
2

3
Thus, all analogs were examined for their binding affinity at  receptors with [ H] DTG as a
2
radioligand and binding curves were generated (Figure 3).
3
Figure 3. Radioligand binding curves for the σ receptor. A) A [ H] DTG saturation curve
2
depicting total (red circles) and non-specific (blue squares) binding to PC-12 membranes. K
d
3
±
SEM = 23.0 ± 3.9 nM. B) Competition binding curve against [ H] DTG in PC-12
membranes for compounds 1 (orange diamonds), 2 (brown hexagons), 3 (green triangles
pointed up), 4 (red squares), 5 (blue circles), 6 (purple triangles pointed down), and 7 (pink
half-filled circles). K values are listed in Table 1. Error bars represent SEM. Curves depicted
i
in this figure are representative curves from two independent experiments performed in
triplicate.
We found that although the morpholine O atom is not important for binding affinity of
fenpropimorph analogs at  receptors, its removal resulted in a dramatic decrease in subtype
1
selectivity. For example 4 showed 360-fold selectivity at  receptors compared to 
1
2
receptors, whereas its piperidine counterpart 6 is only 21-fold selective for  receptors
1
compared to  receptors.
2
2
.3. Molecular Modeling
The two high-resolution crystal structures of the σ receptor (PDB ID: 5HK1 and
1
7
5
HK2) served as excellent starting points for our docking studies. Since the conformation of
the receptor was highly similar for the antagonist and the agonist/inverse agonist-bound
forms, we conducted our docking studies on the higher resolution antagonist-bound crystal
structure (PDB ID: 5HK1) of the hσ receptor. The binding site, as identified by Schmidt et
1
7
al., consists primarily of hydrophobic residues with the exception of two acidic residues,
Glu172 and Asp126. We utilized the glutamate residue (Glu172), previously shown to be
1
7
important for binding by mutagenesis data, to dock our compounds in a spherical region of
6
Å radius. To quantify the interactions observed between the ligands and the receptor, we

performed a Hydrophatic INTeractions (HINT) analysis that takes into account all non-
1
8
covalent interactions between two molecules. A higher HINT score implies a more
1
8
favorable interaction between the two (i.e., the receptor and a ligand in this case).

The SAfiR studies indicated that none of the methyl substituents on fenpropimorph
(
1) contribute to its affinity at the h1 receptor. Thus, we docked analog 4 (K = 2.6 nM) and
i
Tyr103
Leu105 Leu95
Tyr103
A
B
Ala98
Ile178
Tyr206
Ala98
Ile178
Leu105
Tyr206
Glu172
Glu172
Leu182
Leu182
Ala185
Ala185
Figure 4. Two views of analog 4 (yellow capped sticks) docked in the binding site of the hσ1
receptor (PDB ID: 5HK1). A) The amino acid residues of the crystal structure are shown as
pale blue-capped sticks. The N atom of the morpholine ring of analog 4 appears to be
involved in a bidendate ionic interaction (dashed red lines) with the O atoms of the
carboxylate group of Glu172. B) The 4-tert-butyl group is surrounded by and interacting with
hydrophobic residues – Leu95, Ala98, Tyr103, Leu105, Ile178, Leu182, Ala185 and Tyr206 –
at the binding site.
found that the 4-tert-butylphenyl group occupied a pocket consisting of hydrophobic residues
–
Leu95, Ala98, Tyr103, Leu105, Ile178, Leu182, Ala185 and Tyr206, whereas the
morpholine nitrogen atom formed a key bidentate ionic interaction with the carboxylate O
atoms of Glu172 (Figures 4 A and B). The lower-affinity des 4-tert-butyl analog 5 (K = 706
i
nM) lacks these extensive hydrophobic interactions of 4 and appears to be oriented in the
opposite direction (Figure S2). Analog 5 did show weak hydrophobic interactions with
Val152 and Ala185 while retaining the key bidentate interaction with Glu172 (Figure S2).
HINT analysis of the solution representing its binding mode might potentially explain the
~
265-fold difference in the affinity of 5 as compared to 4 (Table 1) at h receptors.
1

In our biological studies we examined racemic fenpropimorph (1) and because
radioligand binding affinity data for (R)- and (S)-fenpropimorph are lacking we docked both
isomers. Both, the (R)- and (S)-isomer of 1 mimicked the binding mode of 4. In both cases,
the bidentate interactions of the N atom with the O atoms of the carboxylate group of Glu172
as well as the hydrophobic interactions of the 4-tert-butylphenyl group were retained both by
the (R)- and (S)-isomer of 1 (Figure S3). The HINT scores for the two isomers, (R)- and (S)-
1
, were comparable (Table 2) suggesting that the stereocenter might not be significantly
important for binding. This is supported by our biological data.
Table 2. Summary of HINT scores for the deconstructed analogs 4, 5 and the (R)- and (S)-
fenpropimorph docked in the binding site of the hσ receptor.
1
d
Ligand
Total HINT
Total
hydrophobic
Totalc
polar
Hydrophobic
a
b
score
4
2041
394
905
-756
149
1136
1150
1197
1269
1828
488
5
(
R)-Fenpropimorph (1)
1346
1592
1968
2111
(
S)-Fenpropimorph (1)
323
a
Sum of all noncovalent interactions including hydrophobic and polar interactions between two
1
8
b
entities,
sum of hydrophobic and hydrophobic/polar (which represent desolvation energy)
18 c
interactions, sum of attractive acid/base and hydrogen bond interactions and repulsive acid/acid and
base/base interactions, sum of hydrophobic interactions between individual atoms of the ligand and
the receptor
18 d
1
8
To understand the >3500-fold difference in the binding affinity of fenpropimorph (1)
at guinea pig vs human orthologues of the  receptor, we aligned amino acid sequences of
1
both orthologues and examined their sequence identity in the orthosteric binding site (Figure

S4). It appears that the orthosteric binding sites in both species are highly conserved and there
are no differences in the amino acid sequences that might explain the extremely high affinity
of fenpropimorph (1) at guinea pig relative to human  receptors. Differences in results
1
might be related to experimental differences in the radioligand binding procedures.
Our docking studies and HINT analysis are in agreement with our biological data
which suggests that the chiral center on the 2-methylpropyl linker, the 3,5-dimethyl groups,
and the oxygen atom of the morpholine ring might not be important for binding affinity of
fenpropimorph at human σ receptors.
1
3
. Conclusions
Fenpropimorph (1) does not bind at human  receptors with nearly the same affinity it
1
display for  receptors of other species. Nevertheless, even with lower affinity for h
1
1
receptors, it is still 50-fold selective for  vs  receptors. The present findings demonstrate
1
2
that: i) phenpropimorph binds at human  receptors with much lower affinity than its
1
reported affinity for guinea pig  receptors, ii) the side chain methyl group of 1 is not
1
required for  binding, iii) the lipophilic character on the “left hand” portion of the molecule
1
(
as drawn here) contributes to binding, iv) the stereochemistry about the morpholine methyl
substituents is unimportant, and that v) the morpholine oxygen atom does not contribute to
and might even detract from) σ receptor binding.
(
1
In addition, given the availability of the human  receptor crystal structure we, for the first
1
time, are able to provide support for Glennon’s pharmacophore model at an atomic level.
Alignment of amino acid sequences for the human versus guinea pig orthologs of the σ1
receptor could not explain the differences in affinities observed for fenpropimorph. Docking

studies and HINT analysis indicated that the high-scoring ionic interactions with Glu172,
retained by all analogs, might be a contributing factor for their high affinity for the hσ1
receptor. HINT analysis further indicated a high degree of contribution by hydrophobic
interactions with the amino acid residues of the binding site and might point towards the
necessity of the 4-t-butylphenyl group for the binding of fenpropimorph. Our current findings
also show that fenpropimorph (1) does not bind with nearly the high affinity for human σ1
receptors that it displays for other species (e.g. guinea pig), and that this must be taken in
consideration for future studies with fenpropimorph analogs. Nevertheless, simplified
fenpropimorph analogs such as 4 and 5 are worthy of continued studies. Most interesting,
perhaps, is that 4, is >350-fold selective for σ vs σ receptor binding.
1
2
In summary, our findings support and confirm a previously proposed  receptor
1
1
1
pharmacophore; and, with respect to compound 4, suggest it might be a suitable template
for exploitation to develop compounds with significant  versus  receptor selectivity.
1
2
4
4
. Experimental section
.1. Synthesis
All solvents and reagents used were purchased from Sigma-Aldrich. Routine thin layer
chromatography (TLC) was performed on silica gel GHLF plates (250 , 2.5 x 10 cm;
Analtech Inc. Newark, DE) and examined by UV (model UVGL-25 mineralight lamp,
multiband UV –  = 254/365 nm (UVP, Upland, CA). TLC plates were also developed using
an iodine chamber containing a few crystals of iodine mixed with silica. Chromatographic
separations were performed on silica gel columns (Silica Gel 62, 60-200 mesh, 150 Å,
Sigma-Aldrich). Flash chromatography was performed on a CombiFlash Companion/TS
Teledyne Isco Inc. (Lincoln, NE) instrument using normal phase silica flash chromatography

columns (35-70 , mesh 230-400, 60 Å, RediSep). Melting points were determined in glass
®
capillary tubes on a Thomas-Hoover melting point apparatus and are uncorrected.
Hydrogenation reactions were performed using an hydrogenation apparatus (4833/3900
1
apparatus, Parr Instrument Company, Moline, IL). H NMR spectra were recorded with a
Varian EM-390 300 MHz with tetramethylsilane (TMS) as an internal standard. Peak
positions are reported in parts per million (). Elemental analyses were performed by Atlantic
Microlab Inc. (Norcross, GA) for the indicated elements and results are within 0.4 % of
calculated values.
12
13
Compounds 9a and 10a were prepared according to literature procedures.
4
.1.1. Fenpropimorph Hydrochloride (1). Fenpropimorph was purchased from Sigma-
Aldrich and converted to its HCl salt. The resulting solid was recrystallized from Et O/MeOH
2
1
yielding a white crystalline product: mp 220-222 °C. H NMR (DMSO-d )  0.91 (d, J = 6
6
Hz, 3H, CH ), 1.11 (m, 6H, CH ), 1.27 (s, 9H, CH ), 2.3 (m, 2H, CH ), 2.53 (m, 1H, CH),
3
3
3
2
2
2
1
.58 (m, 2H, CH ), 3 (m, 2H, CH ), 3.45 (m, 2H, CH ), 4.08 (m, 2H, CH), 7.16 (d, J = 12 Hz,
2 2 2
H, CH), 7.32 (d, J = 12 Hz, 2H, CH). Anal. Calcd for (C H NO·HCl): C = 70.66%, H =
2
0
33
0.08%, N = 4.12%. Actual: C = 70.50%, H = 10.23%, N = 4.11%.
4
.1.2. 4-[3-[(4-tert-Butylphenyl)propyl]-2,6-dimethylmorpholine Hydrochloride (2).
Diborane-THF complex (11.5 mL) was added in a dropwise manner to a solution of 12 (2 g,
.01 mol) in dry THF (10 mL) at 0 °C under a N atmophere. The reaction mixture was
0
2
heated at reflux for 3 h. A solution of 10% HCl (20 mL) was added in a dropwise manner at 0
C to the reaction mixture, which was then heated at reflux for additional 15 h. The solvent
was removed under reduced pressure. The reaction was basified to pH 10 by the addition of
% NaOH solution and the mixture was extracted with Et O (3 x 20 mL). The Et O solution
°
2
2
2

was dried (Na SO ), filtered, and the solvent evaporated under reduced pressure. An Et O
2
4
2
solution of the residue was treated with a solution of Et O/HCl and the resultant precipitate
2
was collected by filtration, washed well with Et O, and recrystallized from an anhydrous
2
Et O/MeOH mixture to yield 1.86 g (93%) of 2 as white crystals as a mixture of cis and trans
2
1
isomers: mp 230 – 244 °C. H NMR (DMSO-d )  1.11 (d, J = 6 Hz, 6H, CH ), 1.27 (s, 9H,
6
3
CH ), 1.43 (m, 2H, CH ), 2.1 (m, 2H, CH ), 2.51 (m, 2H, CH ), 2.6 (m, 2H, CH ), 3.1 (m,
3
2
2
2
2
2
H, CH ), 3.4 (m, 2H, CH), 7.16 (d, J = 6 Hz, 2H, CH), 7.32 (d, J = 6 Hz, 2H, CH). Anal.
2
Calcd for (C H NO∙HCl): C = 70.02%, H = 9.90%, N = 4.30%. Actual: C = 70.14%, H =
1
9
31
1
0.04%, N = 4.30%.
4
.1.3. Trans-4-[3-[(4-tert-Butylphenyl)propyl]-2,6-dimethylmorpholine Hydrochloride
(3). A part of the isomeric mixture (free base) 2 (0.2 g, 0.001 mol) was separated using silica
gel flash chromatography (hexane-EtOAc = 9:1) to give a yellow oil which was treated with a
solution of Et O/HCl. The resultant precipitate was collected by filtration, washed well with
2
anhydrous Et O and recrystallized from an Et O/MeOH mixture to yield 0.11 g (56%) of 3 as
2
2
1
white crystals: mp 248-249 °C. H NMR (DMSO-d )  1.10 (d, J = 6 Hz, 6H, CH ), 1.25 (s,
6
3
9
H, CH ), 1.99 (m, 3H, CH ), 2.57 (m, 4H, CH ), 2.99 (m, 3H, CH ), 3.89 (m, 2H, CH), 7.13
3 2 2 2
(
d, J = 9 Hz, 2H, CH), 7.29 (d, J = 9 Hz, 2H, CH). Anal. Calcd for (C H NO∙HCl): C =
19 31
7
0.02%, H = 9.90, N = 4.30%. Actual: C = 70.27%, H = 9.91%, N = 4.29%.
Analogs 4-6 were obtained following the procedure for the preparation of 2 from amides 13-
1
5, respectively.
4
.1.4. 4-[3-(4-tert-Butylphenyl)propyl]-morpholine Oxalate (4).

1
White crystals: mp 176-178 °C (Et O/MeOH). Yield 0.07 g (14%). H NMR (DMSO-d ) 
2
6
1
.25 (s, 9H, CH ), 1.89 (m, 2H, CH ), 2.55 (m, 2H, CH ), 2.7-3.1 (m, 6H, CH ), 3.74 (m, 4H,
3 2 2 2
CH ), 7.12 (d, J = 9 Hz, 2H, CH), 7.28 (d, J = 6, 2H, CH). Anal. Calcd for
2
(
C H NO∙C H O ): C = 64.93%, H = 8.32%, N = 3.99. Actual: C = 64.77%, H = 8.17%, N
17 27 2 2 4
=
3.94%.
4
.1.5. 4-(3-Phenylpropyl)-morpholine Hydrochloride (5).
1
White crystals: mp 137-138 °C (i-PrOH/Et O). Yield 0.05 g (7%). H NMR (DMSO-d ) 
2
6
2
3
.02 (m, 2H, CH ), 2.49 (m, 2H, CH ), 2.62 (t, J= 6 Hz, 2H, CH ), 3.03 (m, 4H, CH ), 3.89-
2 2 2 2
+
.80 (m, 4H, CH ), 7.24 (m, 5H, CH), 11.12 (s, 1H, NH ). Anal. Calcd for (C H NO·HCl):
2
3
13 19
C = 64.59%, H = 8.34%, N = 5.79%. Actual: C = 64.46%, H = 8.37%, N = 5.78%.
4
.1.6. 4-[3-(4-tert-Butylphenyl)propyl]-piperidine Oxalate (6).
1
White crystals: mp 177-179 °C (Et O/MeOH). Yield 0.16 g (4.5%). H NMR (DMSO-d ) 
2
6
1
.26 (s, 9H, CH ), 1.5 (m, 2H, CH ), 1.71 (m, 4H, CH ), 1.93 (m, 2H, CH ), 2.51 (t, 2H,
3
2
2
2
CH ), 2.57 (t, 2H, CH ), 2.97 (t, 2H, CH ), 3.08 (m, 2H, CH ), 7.15 (d, J = 6 Hz, 2H, CH),
2
2
2
2
7
.31 (d, J = 6 Hz, 2H, CH). Anal. Calcd for (C H NO∙C H O ): C = 68.74%, H = 8.94%, N
18 29 2 2 4
=
4.01%. Actual: C = 68.45%, H = 9.08%, N = 3.94%.
4
.1.7. 4-[3-(4-tert-Butylphenyl)propyl]-4-methylpiperidine Oxalate (7).
Borane dimethyl sulfide complex (0.9 mL) was added in a dropwise manner to a solution of
6 (1.65 g, 0.006 mol) in dry THF (25 mL) at 0 °C under a N atmosphere. The reaction
1
2
mixture was heated at reflux for 3 h. An aqueous solution of 10% HCl (20 mL) was added in
a dropwise manner at 0 °C to the reaction mixture which was then heated at reflux for
additional 15 h. The solvent was removed under reduced pressure. The reaction mixture was

basified to pH 10 by adding 2% aqueous NaOH solution and the mixture was extracted with
Et O (3 x 40mL). The Et O solution was dried (Na SO ) and evaporated under reduced
2
2
2
4
pressure. The free base of 7 was dissolved in anhydrous Et O and treated with a solution of
2
oxalic acid. The resultant precipitate was collected by filtration, washed well with anhydrous
Et O, and recrystallized from a Et O/MeOH mixture to yield 0.22 g (13%) of 7 as white
2
2
1
crystals: mp 168-176 °C. H NMR (DMSO-d )  0.92 (d, J = 6.44 Hz, 3H, CH ), 1.26 (s, 9H,
6
3
CH ), 1.6 (m, 1H, CH), 1.77 (m, 2H, CH ),1.91 (m, 2H, CH ), 2.5 (m, 2H, CH ), 2.58 (t, 2H,
3
2
2
2
CH ), 2.97 (t, 2H, CH ), 2.98 (m, 2H, CH ), 3.38 (m, 2H, CH ), 7.15 (d, J = 8 Hz, 2H, CH),
2
2
2
2
7
3
.32 (d, J = 8 Hz, 2H, CH). Anal. Calcd for (C H N∙C H O ): C = 69.39%, H = 9.15%, N =
19 31 2 2 4
.81%. Actual: C = 69.13%, H = 9.04%, N = 3.81%.
4
.1.8. 3-(4-tert-Butyl)phenyl propanoyl chloride (11a).
Compound 10a (3.81 g, 0.02 mol) was dissolved in dry benzene (5 mL, 0.06 mol) at room
temperature then SOCl (10 mL, 0.14 mol) was added in a dropwise manner. The reaction
2
mixture was heated at reflux for 15 h. The solvent was evaporated under reduced pressure to
give 11a (4.49 g, 95%) as a yellow oil. The crude product was used in the next step without
further purification or characterization.
4
.1.9. 3-Phenylpropanoyl chloride (11b).
Dihydrocinnamic acid (10b) (3.0 g, 0.02 mol) was dissolved in dry benzene (5 mL, 0.06 mol)
at room temperature then SOCl (10 mL, 0.14 mol) was added in a dropwise manner. The
2
reaction mixture was allowed to heat at reflux for 4 h. The solvent was evaporated under
reduced pressure to give (3.4 g, 95%) of 11b as yellow oil. The crude product was used in the
next step without further purification or characterization.

4
.1.10. 3-(4-tert-Butyl)-phenyl-1-(2,6-dimethylmorpholinyl)propan-1-one (12).
A solution of 3-(4-tert-butyl)-3-phenylpropanoyl chloride (11a) (2.83 g, 0.01 mol) in dry
CH Cl (10 mL) was added in a dropwise manner to a stirred solution of 2,6-
2
2
dimethylmorpholine (1.86 mL, 0.02 mol), TEA (3.45 mL, 0.03 mol), and dry CH Cl (20
2
2
mL). The reaction mixture was allowed to stir at room temperature for 15 h. The solvent was
evaporated under reduced pressure. A solution of 10% HCl (20 mL) was added to the oily
residue. The mixture was washed with Et O (3 x 20 mL). The Et O portion was dried
2
2
(
Na SO ) and the solvent was removed under reduced pressure to give (2.00 g, 56%) of 12 as
2 4
a yellow oil. The crude product was used in the next step without further purification or
characterization.
Amides 13-16 were obatined using a procedure similar to that used for 12.
4
.2. Biological studies
Recombinant receptor expression
7
Human σ receptors were expressed in Sf9 insect cells as described previously. In
1
short, the human σ receptor was cloned into the pFastBac vector and expressed using the
1
baculovirus expression system. Cells were grown in a shaker at 27° C, and infected when
6
cells reached a density of 4 x 10 cells/mL. At 48 hours post-infection, cells were harvested
by centrifugation and used for membrane preparation.
Preparation of cell membranes for radioligand binding experiments
Membranes were prepared from PC-12 (for σ binding) or Sf9 (for σ binding) cells
2
1
1
9
with a protocol based on that of Vilner et al. In summary, PC-12 cells were washed with ice
cold PBS and harvested with a cell scraper and Sf9 cells were pelleted by centrifugation. Cell
pellets were suspended in 20 mM HEPES pH 7.5 supplemented with cOmplete Mini, EDTA-

free Protease Inhibitor Cocktail Tablets‎(Roche, 1 tablet per 50 mL buffer). The cells were
homogenized using a needle and syringe, and then centrifuged at 50,000 x g for 20 min. The
supernatant was discarded and the membranes were resuspended in cold 50 mM Tris pH 8.0
containing cOmplete Mini, EDTA-free Protease Inhibitor Cocktail Tablets (Roche, 1 tablet
per 50 mL buffer). The membranes were again centrifuged at 50,000 x g for 20 min, and then
resuspended in1-5 mL of the same Tris/protease inhibitor buffer. Total protein content was
measured using the Biorad DC protein assay according to the manufacturer’s instructions.
Membranes divided into 200 μL aliquots, flash frozen, and stored at -80° C until use.
Radioligand saturation binding
Both σ and σ receptor radioligand saturation binding to membranes was determined
1
2
20
using an assay similar to that described by Chu and Ruoho, with minor differences. Briefly,
membranes from infected Sf9 insect cells (2.5 μg total protein per reaction) and 0 - 300 nM
3
H(+)-PTZ for σ binding assays, or PC-12 cells (15-30 μg total protein per reaction) 0 - 30
1
3
nM H-ditolylguanidine (DTG) for σ binding assays, were incubated in a 100 μL reaction
2
buffered with 50 mM Tris pH 8.0. In the σ binding assays, concentrations of 100 and 300
2
3
nM DTG were assayed by isotopic dilution to minimize use of H-DTG, and σ receptor sites
1
were blocked by the addition of 1.8 μM (+)-SKF-10,047. For each saturation curve, a second
curve that was otherwise identical save for the addition of 2 μM haloperidol was measured in
parallel to determine nonspecific binding. Reactions were incubated on a shaker at 37 °C for
9
0 min and then terminated via filtration through glass fiber filters using a Brandel cell
harvester. Glass fiber filters were soaked in 0.3% polyethylenimine for at least 20 min at
room temperature prior to harvesting. After washing, filters were soaked in 5 mL Cytoscint
scintillation fluid overnight and measured on a Beckman Coulter LS 6500 scintillation
counter the next morning. K values were calculated using non-linear regression tools from
d

Graphpad Prism version 7.0b.
Competition binding assays
Radioligand competition curves testing the binding of compounds 1-7 to both σ and
1
2
0
σ receptors were performed similarly to what has been described by Chu and Ruoho, with
2
slight modifications. For σ receptor competition curves, Sf9 insect membranes
1
overexpressing σ receptor (2.5 μg of total protein per reaction) were incubated in a 100 μL
1
3
reaction buffered with 50 mM Tris pH 8.0, with 10 nM H(+)-PTZ and 8 concentrations
ranging from 10 pM – 100 μM of the competing cold ligand. For σ receptor competition
2
curves, PC-12 membranes (12-30 μg total protein per reaction) were incubated in the same
3
volume with the same buffer and 30 nM H DTG and 1.8 μM (+)SKF-10,047 to block σ
1
receptor binding sites. As in the saturation curves, reactions were incubated for 90 min at 37
°
C, and were then terminated by filtration through a glass fiber filter using a Brandel cell
harvester. Glass fiber filters were soaked in 0.3% polyethylenimine for at least 20 min at
room temperature prior to harvesting. All curves were assessed by two or three independent
experiments performed in triplicate. After the filters were washed, they were soaked in 5 mL
of Cytoscint scintillation fluid overnight and radioactivity was measured using a Beckman
Coulter LS 6500 scintillation counter. K values were computed directly without the use of
i
the Cheng-Prusoff correction using Graphpad Prism version 7.0b software.
4
.3. Molecular modeling
The X-ray crystal structure of the σ receptor co-crystallized with PD144418 was retrieved
1
7
from the Protein DataBank (PDB ID: 5HK1). Only a single monomer of the receptor (chain
B) was retained for the docking studies. The ligands, including PD144418, were sketched and
energy minimized using Tripos Force Field and Gasteiger-Hückel charges in SYBYL X-2.1

2
1
(
Tripos International). For the purpose of the docking studies, we utilized GOLD suite 5.2
to define a spherical region of radius 6Å around the amino acid Glu172 with each ligand
being docked 30 times in the binding site and early termination switched off. The solutions
generated were analyzed on the basis of GOLD score and selected ligand-receptor complexes
1
8
were energy minimized in SYBYL X-2.1 (Tripos International). HINT analysis was
performed in SYBYL 8.1 (Tripos International) with all parameters set at default and the
output examined to arrive at HINT scores.
Acknowledgements
We thank Prof. Richard A. Glennon for discussions and proof-reading the manuscript. We
thank Prof. Simona Collina for sponsoring Elena Sguazzini to conduct her research at VCU
as an exchange student from the University of Pavia, Italy. Kavita A. Iyer is the recipient of
a Lowenthal Award (2013-2015). We acknowledge support to A.C.K. from the Vallee
Foundation, the Esther A. and Joseph Klingenstein Fund, and the Harvard Brain Initiative
Winthrop Fund.
Supplementary data
Supplementary data associated with this article can be found, in the online version, at…
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Graphical abstract
Glu172
Analog 4
h K = 2.6 ± 0.1 nM
1
i