Ether modifications to 1-[2-(3,4-dimethoxyphenyl)ethyl]-4-(3-phenylpropyl)piperazine (SA4503): Effects on binding affinity and selectivity for sigma receptors and monoamine transporters

Article abstract of DOI:10.1016/j.bmc.2014.11.007

Two series of novel ether analogs of the sigma (σ) receptor ligand 1-[2-(3,4-dimethoxyphenyl)ethyl]-4-(3-phenylpropyl)piperazine (SA4503) have been prepared. In one series, the alkyl portion of the 4-methoxy group was replaced with allyl, propyl, bromoethyl, benzyl, phenethyl, and phenylpropyl moieties. In the second series, the 3,4-dimethoxy was replaced with cyclic methylenedioxy, ethylenedioxy and propylenedioxy groups. These ligands, along with 4-O-des-methyl SA4503, were evaluated for σ₁ and σ₂ receptor affinity, and compared to SA4503 and several known ether analogs. SA4503 and a subset of ether analogs were also evaluated for dopamine transporter (DAT) and serotonin transporter (SERT) affinity. The highest σ₁ receptor affinities, K_i values of 1.75-4.63 nM, were observed for 4-O-des-methyl SA4503, SA4503 and the methylenedioxy analog. As steric bulk increased, σ₁ receptor affinity decreased, but only to a point. Allyl, propyl and bromoethyl substitutions gave σ₁ receptor K_i values in the 20-30 nM range, while bulkier analogs having phenylalkyl, and Z- and E-iodoallyl, ether substitutions showed higher σ₁ affinities, with K_i values in the 13-21 nM range. Most ligands studied exhibited comparable σ₁ and σ₂ affinities, resulting in little to no subtype selectivity. SA4503, the fluoroethyl analog and the methylenedioxy congener showed modest six- to fourteen-fold selectivity for σ₁ sites. DAT and SERT interactions proved much more sensitive than σ receptor interactions to these structural modifications. For example, the benzyl congener (σ₁ K_i = 20.8 nM; σ₂ K_i = 16.4 nM) showed over 100-fold higher DAT affinity (K_i = 121 nM) and 6-fold higher SERT affinity (K_i = 128 nM) than the parent SA4503 (DAT K_i = 12650 nM; SERT K_i = 760 nM). Thus, ether modifications to the SA4503 scaffold can provide polyfunctional ligands having a broader spectrum of possible pharmacological actions.

Full text of DOI:10.1016/j.bmc.2014.11.007

Bioorganic & Medicinal Chemistry 23 (2015) 222–230
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Ether modifications to 1-[2-(3,4-dimethoxyphenyl)ethyl]-
4-(3-phenylpropyl)piperazine (SA4503): Effects on binding affinity
and selectivity for sigma receptors and monoamine transporters
Rong Xu a, Sarah A. Lord b,d, Ryan M. Peterson a, Emily A. Fergason-Cantrell b,d, John R. Lever b,d, ,
,
⇑
Susan Z. Lever a,c,
⇑
,à
a Department of Chemistry, University of Missouri, Columbia, MO 65211, USA
b Department of Radiology, University of Missouri, Columbia, MO 65212, USA
c Department of MU Research Reactor Center, University of Missouri, Columbia, MO 65211, USA
d Harry S. Truman Memorial Veterans’ Hospital, Columbia, MO, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Two series of novel ether analogs of the sigma (
r
) receptor ligand 1-[2-(3,4-dimethoxyphenyl)ethyl]-
Received 13 August 2014
Revised 25 October 2014
Accepted 4 November 2014
Available online 11 November 2014
4-(3-phenylpropyl)piperazine (SA4503) have been prepared. In one series, the alkyl portion of the
4-methoxy group was replaced with allyl, propyl, bromoethyl, benzyl, phenethyl, and phenylpropyl moi-
eties. In the second series, the 3,4-dimethoxy was replaced with cyclic methylenedioxy, ethylenedioxy
and propylenedioxy groups. These ligands, along with 4-O-des-methyl SA4503, were evaluated for r1
and
r2 receptor affinity, and compared to SA4503 and several known ether analogs. SA4503 and a subset
Keywords:
of ether analogs were also evaluated for dopamine transporter (DAT) and serotonin transporter (SERT)
affinity. The highest r1 receptor affinities, Ki values of 1.75–4.63 nM, were observed for 4-O-des-methyl
SA4503, SA4503 and the methylenedioxy analog. As steric bulk increased,
but only to a point. Allyl, propyl and bromoethyl substitutions gave 1 receptor Ki values in the 20–30 nM
Sigma receptors
Dopamine transporter
Serotonin transporter
In vitro binding
r1 receptor affinity decreased,
r
range, while bulkier analogs having phenylalkyl, and Z- and E-iodoallyl, ether substitutions showed
higher r1 affinities, with Ki values in the 13–21 nM range. Most ligands studied exhibited comparable
r1 and r2 affinities, resulting in little to no subtype selectivity. SA4503, the fluoroethyl analog and the
methylenedioxy congener showed modest six- to fourteen-fold selectivity for r1 sites. DAT and SERT
interactions proved much more sensitive than
r receptor interactions to these structural modifications.
For example, the benzyl congener (r1 Ki = 20.8 nM; r2 Ki = 16.4 nM) showed over 100-fold higher DAT
affinity (Ki = 121 nM) and 6-fold higher SERT affinity (Ki = 128 nM) than the parent SA4503 (DAT
Ki = 12650 nM; SERT Ki = 760 nM). Thus, ether modifications to the SA4503 scaffold can provide polyfunc-
tional ligands having a broader spectrum of possible pharmacological actions.
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
normal tissue, the r2 receptors serve as markers of cell prolifera-
tion, and
r1 antagonists/r2 agonists have potential for cancer ther-
Sigma (
r
) receptors can be classified into distinct r1 and r2
apy.1–3 In the CNS realm, r1 receptor agonists improve cognition,
subtypes that play prominent roles in certain cancers and in
central nervous system (CNS) disorders. Malignancies often
express high levels of r1 and r2 receptors with respect to the
show antidepressant and anxiolytic properties, and promote
neuroregeneration.4–6 Moreover, selective
r1, and perhaps r2,
receptor antagonists show potential as medications for treatment
of psychostimulant abuse.7–10 Compounds from many different
chemical classes show varying degrees of affinity and selectivity
⇑
Corresponding authors. Tel.: +1 (573) 882 8395; fax: +1 (573) 882 2754 (S.Z.L.);
for
r receptors, and structurally diverse ligands including proges-
tel.: +1 (573) 814 6000x53686; fax: +1 (573) 814 6551 (J.R.L.).
E-mail addresses: leverj@health.missouri.edu (J.R. Lever), levers@missouri.edu
(S.Z. Lever).
terone, N,N0-dimethyltryptamine and sphingolipids have been sug-
gested as endogenous ligands.11,12
The r1 receptor has been cloned,13,14 while the primary struc-
ture of the r2 receptor remains elusive.3 Protein biochemistry
studies and homology modeling efforts are now revealing specific
Present address: Department of Radiology, University of Missouri—Columbia,
M292 Medical Sciences Building, One Hospital Drive, Columbia, Missouri 65212, USA.
à
Present address: Room 125 Chemistry Building, University of Missouri—Colum-
bia, 601 South College Avenue, Columbia, MO 65211, USA.
http://dx.doi.org/10.1016/j.bmc.2014.11.007
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

R. Xu et al. / Bioorg. Med. Chem. 23 (2015) 222–230
223
features of the r1 receptor binding site at the amino acid level.15
Less is known about r2 receptors, but recent studies show the
orthosteric binding site to be located within the progesterone
receptor membrane component 1 (PGRMC1) protein complex.3,16
Nonetheless, medicinal chemistry studies show a degree of com-
monality, with both r1 and r2 receptor pharmacophores simply
described by an amine binding site flanked by two hydrophobic
binding pockets.17,18 These basic models are fit well by N,N0-disub-
stituted piperazines having phenethyl and phenylpropyl groups to
occupy the hydrophobic regions.
receptor binding results. In work aimed toward radioiodinated
SA4503 analogs, the 4-methoxy group was replaced by (Z)- and
(E)-iodoallyloxy substituents (IA-SA4503, Fig. 1).39 These modifica-
tions reduced
r1 affinity and increased r2 affinity, leading to a pair
of non-selective isomeric ligands having Ki values of 11–20 nM for
both subtypes.
To gain a better understanding of the qualitative structure–
affinity relationships associated with ether modifications of
SA4503, we prepared nine novel analogs (Fig. 2) for a more system-
atic investigation. Two types of structural modifications were
investigated. The first involved replacements of the 4-methoxy
group with ethers of increasing steric bulk and hydrophobicity,
Two active series from this structural class of
r receptor ligands
have attracted attention as potential pharmaceuticals. These are
denoted as either ‘SA’ compounds19,20 or ‘YZ’ compounds
(Fig. 1).21,22 They differ in the patterns and types of substituents
on the phenethyl and phenylpropyl moieties, and typically display
relatively high r1 receptor binding affinity accompanied by
modest selectivity against r2 sites. Subtle structural differences
influence their r1 and r2 receptor binding, as well as the
behavioral pharmacology observed in animal models.22–27
SA4503 (Fig. 1), the most widely studied ligand from this class, is
also known as cutamesine and is regarded as a r1 receptor ago-
nist.4–6,19 SA4503 causes dissociation of immunoglobulin binding
as well as differing potential for
p and other interactions (1–6).
The second involved replacement of the 3,4-dimethoxy moiety
with cyclic ethers of different ring sizes (7–9). Each compound
was tested in vitro for affinity and selectivity of binding to r1
and r2 receptors using standard techniques. The
r receptor bind-
ing affinities of the known 4-O-des-methyl-SA450336b (Fig. 1) have
not been reported, and this material was tested as well.
In previous work, SA4503 showed low affinity for about 40 dif-
ferent receptors, ion channels and second messenger systems,19
although a significant secondary interaction with the vesicular ace-
tylcholine transporter, Ki = 50 nM, has been observed.40 Interac-
tions of SA4503 with dopamine transporters (DAT) and serotonin
transporters (SERT) have not been reported to our knowledge. Sev-
eral of the novel SA4503 ether analogs were designed to bear a
structural resemblance to ligands such as GBR 12909 (vanoxerine)
and certain rimcazole analogs that show high DAT and SERT affin-
protein from the
r1 receptor, a definitive biochemical test of stim-
ulation.28 SA4503 has been evaluated in Phase II clinical trials for
treatment of major depressive disorder29 and to aid in functional
recovery after stroke.30,31 In the stroke trial,30 SA4503 proved well
tolerated, and the data indicated improvements in individuals who
received a 3 mg/d dosage for 28 days. Interestingly, SA4503 shows
mixed properties in recent behavioral studies, and can either
potentiate or attenuate certain effects of cocaine and methamphet-
amine in mice as a function of dose.23,24 By contrast, the YZ analogs
ities accompanied by weaker interactions with the
r receptor sub-
types.41,42 Considering the importance of
r
receptors and the DAT
in mediating the effects of cocaine, compounds having actions at
both sites are of interest from the viewpoint of psychostimulant
medications development.41–43 As exemplified by fluvoxamine,4
profile as
r1 receptor antagonists based upon their ability to block
cocaine-induced convulsions in mice.22
Although a number of different SA/YZ compounds have been
studied, relatively little information is available regarding the
ligands with actions at both r receptors and the SERT are of inter-
est as medications for treatment of depression and anxiety.4–6
Accordingly, we determined the affinities of SA4503 and a subset
of the ether analogs for DAT and SERT with the aim of identifying
structural modifications that would provide polyfunctional ligands
having a broader spectrum of pharmacological actions.
effects of ether modifications on
r receptor binding. Findings come
primarily from the development of radiolabeled SA4503 analogs
for molecular imaging applications.32,33 The 4-methoxy position
of SA4503 has been isotopically radiolabeled with carbon-11 for
positron emission tomography (PET) studies of central and periph-
eral r1 receptors in human beings and animal models of dis-
ease.34,35 The fluorine-18 labeled 4-fluoroethoxy36 (FE-SA4503,
Fig. 1) and fluoromethoxy37 (FM-SA4503, Fig. 1) analogs have also
2. Results and discussion
2.1. Chemistry
been prepared, and used for PET studies of cerebral
r1 receptors in
conscious monkeys.36,37 In our hands, SA4503 displays a 4.6 nM
Target compounds 1–9 were prepared by the routes shown in
Scheme 1. Direct reaction of the phenolate anion generated
in situ from 4-O-des-methyl SA450336b with primary alkyl, allyl
or benzyl bromides provided piperazines 1–6 (Scheme 1). Isolated
yields from the Williamson ether synthesis ranged from 44–90%
for 1, 2, 5 and 6 using phase transfer conditions, 40% KOH/tetra-
n-butylammonium hydroxide (TBAH), at 50 °C. This set of reaction
conditions emerged from studies on the synthesis of compound 1
apparent affinity (Ki) for r1 receptors with 14-fold selectivity for
r
1 over
ethyl modification caused a two-fold reduction in affinity for both
1 and 2 receptors while preserving the
1 subtype selectivity.38
As previously discussed in detail,38 these
receptor subtype
r
2 sites.38 In side-by-side binding comparisons, the fluoro-
r
r
r
r
selectivities are quite different from those initially reported for
SA450319 and FE-SA4503,36 primarily as a consequence of the r2
SA4503, R = CH3
FM-SA4503, R = -CH2F
FE-SA4503, R = -CH2CH2F
(Z)-IA-SA4503, R = (Z)-CH2CH=CH2I
(E)-IA-SA4503, R = (E)-CH2CH=CH2I
4-O-des-methyl-SA4503, R = H
1, R = -CH2CH2CH3
2, R = -CH2CH2Br
3, R = -CH2CH=CH2
4, R = -CH2Ph
5, R = -CH2CH2Ph
6, R = -CH2CH2CH2Ph
N
N
N
N
OCH3
O-R
N
OCH3
O-R
N
7, n = 1
8, n = 2
9, n = 3
YZ-184, R = 2-OCH3
YZ-185, R = 3-OCH3
YZ-067, R = 4-OCH3
YZ-069, R = 3,4-di-Cl
N
O
N
(CH2)n
R
O
Figure 1. Representative ‘SA’ and ‘YZ’ sigma receptor ligands.
Figure 2. Novel SA4503 ether analogs 1–9.

224
R. Xu et al. / Bioorg. Med. Chem. 23 (2015) 222–230
calls for KOH at reflux for 17 h. Coupling reactions were then per-
N
N
N
OCH3
O-H
formed using 1-hydroxybenzotriazole monohydrate (HOBtꢀH2O),
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
(EDCꢀHCl) and 4-methylmorpholine in CH2Cl2 at 0 °C under N2 to
give 13–15. These amides were isolated, characterized and then
reduced with LiAlH4 in THF to give 7–9. Coupling reactions were
accomplished in >90% yields, while the reduction steps provided
33–85% isolated yields of the final targets.
(a)
1, R = -CH2CH2CH3
2, R = -CH2CH2Br
3, R = -CH2CH=CH2
4, R = -CH2Ph
5, R = -CH2CH2Ph
6, R = -CH2CH2CH2Ph
N
OCH3
O-R
2.2. Sigma receptor binding
Scheme 1. Synthesis of SA4503 ether analogs 1–6. Reagents and reaction condi-
tions: (a) 1, 2, 5 and 6: corresponding alkyl bromide, KOH/TBAH, 50 °C, 50 min; 3:
allyl bromide, KOH/TBAH, 0 °C to ambient temperature, 50 min; 4: benzyl bromide,
EtOH/K2CO3, 25 °C, N2 overnight.
Table 1 shows binding affinities of 4-O-des-methyl SA4503 and
novel ether analogs 1–9 for
r1 and r2 receptors in guinea pig brain
membranes. Published data, obtained using the same assay condi-
tions, for SA4503 and FE-SA4503,38 as well as (Z)- and (E)-IA-
SA4503,39 are included for comparison. Literature binding data
for several YZ analogs and other reference ligands are also
included. In addition, Table 1 shows a spot test of interactions with
the DAT using a single high concentration (10,000 nM) of SA4503
and analogs. This test was performed to prioritize the selection
of compounds for more detailed studies (see Section 2.3).
by varying the type of base (NaOH, KOH) and the number of molar
equivalents of base and alkylating agent. For the more reactive
electrophile, allyl bromide, the reaction was performed from 0 °C
to ambient temperature to give 3 in 57% yield. The phase transfer
method was not amenable to use with benzyl bromide, and a num-
ber of products were formed that were not characterized. However,
treatment of 4-O-des-methyl SA4503 with benzyl bromide and
K2CO3 in absolute EtOH at ambient temperature overnight pro-
vided 4 in 35% yield. These reactions were not optimized further.
Cyclic ethers 7–9 were synthesized by coupling substituted
phenylacetic acids to 1-(3-phenylpropyl)piperazine to yield amide
intermediates that were subsequently reduced with LiAlH4
(Scheme 2). The 3,4-(methylenedioxy)phenylacetic acid 10
required for preparation of 7 was commercially available. The
known 3,4-(ethylenedioxy)phenylacetic acid 1144 and 3,4-(propy-
lenedioxy)phenylacetic acid 1245 required for preparation of 8
and 9, respectively, were synthesized in 52% and 42% yields by
treatment of 3,4-dihydroxyphenylacetic acid with K2CO3 and the
appropriate alkyl dibromide in ethylene glycol at 120 °C for 4.5 h.
Kashima and colleagues46 reported these conditions for the
synthesis of cyclic ethers from catechols. This one-step route is a
convenient alternative to the multi-step method of Sasamoto,44
and uses milder conditions than the method of Alker et al.45 that
Of the fourteen compounds studied, 4-O-des-methyl SA4503
showed the highest binding affinity for both r1 and r2 receptors
(Table 1). The
r2 receptor binding is sensitive to this modification,
with a ten-fold increase in affinity for 4-O-des-methyl SA4503
compared to SA4503. These findings are consistent with our recent
report of a structurally similar phenol that showed much higher r2
receptor affinity than the corresponding ligand bearing a methoxy
group.47 4-O-des-methyl SA4503 is the only ligand tested that can
be a hydrogen bond donor. By contrast, the hydrogen bond accep-
tors FE-SA4503 and FM-SA4503 showed the poorest r2 receptor
affinities of the series (Table 1). YZ-184 and YZ-185 have only
hydrogen at the 4-position, but still display high
r2 receptor affin-
ity, while YZ-067 and YZ-069 that have a chlorine or methoxy at
the 4-position show a somewhat lower r2 receptor affinity
(Table 1). The data indicate that either having a hydroxyl group
or no substituent at the 4-position promotes r2 binding. Rhoades
et al.48 have developed a three-dimensional model for the r2
receptor pharmacophore, based upon ligands from seven different
HO
OH
OH
O
(b)
O
(a)
O
O
HO
HO
O
O
HO
O
O
O
O
10
11
12
(c)
(c)
(c)
N
N
N
O
O
N
O
O
N
O
O
N
O
O
O
15
13
14
(d)
(d)
(d)
N
N
N
O
O
N
O
O
N
O
O
N
9
8
7
Scheme 2. Synthesis of SA4503 ether analogs 7–9. Reagents and reaction conditions: (a) BrCH2CH2Br, K2CO3/HOCH2CH2OH, 120 °C, 4.5 h; (b) Br(CH2)3Br, K2CO3/
HOCH2CH2OH, 120 °C, 4.5 h; (c) 1-(3-phenylpropyl)piperazine, HOBtꢀH2O/EDCꢀHCl/4-methylmorpholine, 0 °C, N2 overnight; (d) LiAlH4/THF, 0 °C–ambient temperature, 7.5 h.

R. Xu et al. / Bioorg. Med. Chem. 23 (2015) 222–230
225
Table 1
r
hydrophobic substituents reduced r1 but increased r2 receptor
affinity with respect to SA4503. The presence of an aromatic ring
modestly increased the affinities of 4–6 for both r1 and r2 recep-
tors as compared to alkyl and allyl analogs 1–3 (Table 1). Phenethyl
ether analog 5 gave the highest overall r1 and r2 affinity of this
series, but the chain length variations had small effects.
The previously reported (Z)- and (E)-iodoallyl ether analogs
show r1 and r2 receptor affinities comparable to those of novel
phenylalkyl ether analogs 4–6 (Table 1). Notably, the presence of
iodine confers 2- to 3-fold higher r1 and r2 binding affinities for
the iodoallyl analogs over the simple allyl analog 3. The similarity
receptor affinity of SA4503 and novel ether analogsa,b, along with literature data for
selected YZ and reference compounds
R
Sigma receptor binding
DATc
r1 Ki (nM)
r2 Ki (nM)
r2/
r1 % Inh.
–H (des-methyl SA4503)
–CH3(SA4503)d
1.75 0.17
6.72 0.21
4.63 0.21 63.09 4.33
8.03 0.41 113.2 11.7
3.8
13.6
14.1
39.0
1.1
1.6
1.3
1.1
1.4
0.8
1.2
1.3
5.8
3.4
2.7
1.9
7.3
22
20
23
19
—
–CH2CH2F (FE-SA4503)d
–CH2F (FM-SA4503)e
(E)–CH2CH@CHIf
(Z)–CH2CH@CHIf
–CH2CH2CH3
–CH2CH2Br
–CH2CH@CH2
–CH2Ph
–CH2CH2Ph
6.4 1.3
250 16
16.91 1.28 18.20 1.99
10.67 1.32 16.71 1.20
25.60 2.56 33.40 1.11
25.37 2.02 27.12 0.96
30.13 1.24 42.10 0.66
20.79 1.10 16.43 1.13
13.26 0.82 16.57 0.64
18.45 0.48 23.31 3.34
2.55 0.19 14.83 0.43
10.20 0.52 35.20 1.69
19.10 3.11 52.10 4.91
69
94
43
38
44
93
89
97
42
33
43
—
1
2
3
4
5
6
7
8
9
of
r receptor affinities for the (Z)- and (E)-configurations indicates
little sensitivity to these particular geometric constraints. Thus, the
extended ether analogs having bulky, hydrophobic and polarizable
aromatic ring or iodine substituents are readily accommodated by
–CH2CH2CH2Ph
the
r receptor binding pockets. While discrete p–p stacking inter-
–Methylenedioxy–
–Ethylenedioxy–
–Propylenedioxy–
YZ-184 (2-OCH3)g
YZ-185 (3-OCH3)g
YZ-067 (4-OCH3)g
YZ-069 (3,4-di-Cl)g
(+)-Pentazocined
Ditolylguanidine (DTG)d
actions for the phenylalkyl ether moieties of 4–6 may contribute to
binding, the overall data suggest that substituent bulk and hydro-
phobicity are main factors. These properties confer higher r1 and
r2 receptor binding affinities for 4–6, as well as (Z)- and (E)-IA-
SA4503, than observed for analogs 1–3 that have smaller molecular
volumes.
3.9 0.6
1.4 0.2
1.3 0.3
2.2 0.2
7.5 0.2
10.2 0.5
28.6 1.9
38.8 1.6
—
—
—
24
1.62 0.15 728.4 48.8 450
35.45 1.20 39.87 2.61 1.1
—
—
Cyclic ether 7, with its compact methylenedioxy group, showed
2- to 4-fold increases in both r1 and r2 receptor affinity with
respect to SA4503 (Table 1). Compared to each other, cyclic ethers
7–9 showed progressive 7.5- to 3.5-fold decreases in both r1 and
r2 receptor affinities when moving from methylenedioxy (7) to
the bulkier ethylenedioxy (8) and propylenedioxy (9) congeners.
Thus, these three constrained ether analogs of SA4503 follow the
pattern discussed earlier where a smaller substituent volume leads
to relatively higher r1 and r2 receptor affinities accompanied by
modest r1 selectivity.
a
Ki values are means SEM for 3–6 trials, each performed in duplicate.
1.0 nM [3H](+)-pentazocine 1), 3.0 nM [3H]ditolylguanidine/200 nM (+)-
b
(r
pentazocine (r2); guinea pig brain membranes.
c
% Inhibition for a 10,000 nM concentration against [125I]RTI-121 specific bind-
ing in mouse striatal membranes.
d
Binding data reported in Ref. 38.
Ref. 37.
Ref. 39.
Ref. 22.
e
f
g
structural classes, that includes at least four hydrogen bond accep-
tor groups located at different positions of the binding pocket.
Thus, the hydrogen bond donor properties of the phenol may con-
tribute to high affinity r2 binding.
2.3. DAT and SERT binding
SA4503 and analogs varied widely in their potential to interact
with the DAT (Table 1). Using a 10,000 nM concentration, their
ability to inhibit [125I]-RTI-121 binding ranged from 19% to 97%.
SA4503, FE-SA4503 and 4-O-des-methyl SA4503 exhibited the
lowest inhibition (19%–23%), while the alkyl/allyl ethers 1–3 and
the cyclic ethers 7–9 showed a moderate level of inhibition
(38%–44%). Phenylalkyl ethers 4–6 and the (Z)- and (E)-iodoallyl
ethers analogs gave the highest degree of inhibition (69%–97%).
Thus, the presence of large, hydrophobic ether substituents at the
4-O-position of the SA4503 phenethyl leads to stronger DAT
binding.
Based upon these results, SA4503, as well as selected ether
analogs displaying moderate (1, 3, 7) or high (4, 6, (Z)- and (E)-
IA-SA4503) potential for DAT binding, were studied in more detail.
Over a 100-fold range of DAT affinities was observed, while SERT
affinities varied by only 7-fold (Table 2). SA4503 displayed the
weakest interactions with both the DAT and SERT, with a decided
16-fold higher affinity for SERT over DAT. The DAT Ki of
12,650 nM proved about 10-fold lower than the DAT Ki values pre-
viously reported for monomethoxy analogs YZ-185 and YZ-067
(Table 2).22 By contrast, SERT affinities for SA4503 (Ki = 760 nM)
and YZ-185 (Ki = 985 nM)22 were quite similar. Somewhat surpris-
ingly, the DAT affinity for SA4503 proved close to that previously
reported by Matsumoto et al.22 for dichloro analog YZ-069
(Table 2). The DAT and SERT binding data for SA4503 were repli-
cated, with no significant differences (n = 4; t-test, P >0.05), using
fresh serial dilutions prepared from powder.
For the SA4503 analogs described thus far, a smaller substituent
volume leads to higher affinity binding to both
r1 and r2 receptors
that is accompanied by modest selectivities for the r1 subtype. In
this regard, oxygen substituents at both the 3- and 4-positions of
the phenethyl ring are not essential for high affinity
r receptor
binding. For example, monomethoxy analog YZ-185 binds to r1
and r2 receptors with Ki values of 1.4 nM and 10.2 nM, respec-
tively, (Table 1).21,22 The bulkier and more hydrophobic propyl,
bromoethyl and allyl ether substitutions of analogs 1–3 proved
detrimental to r1 receptor binding, but slightly advantageous for
r
2 receptor binding as compared to SA4503 (Table 1). These three
compounds showed the poorest r1 receptor affinities of the com-
pounds tested, and displayed Ki values of 25–30 nM (Table 1). Their
r
2 receptor affinities of 27–42 nM were also nearly equivalent, and
engendered no appreciable subtype selectivity. The similarity of
binding data across analogs 1–3 suggests that neither halogen
bonding49 of bromine, nor
p effects of the ethylene system, con-
tribute significantly to the interactions. Further, these data indicate
that the high electronegativity of fluorine, rather than a steric con-
straint, is responsible for the low
r2 receptor affinity of FE-SA4503
and FM-SA4503. Fluorine participation in hydrogen bonding
acceptor or other dipole–dipole interactions50 that inhibit r2
receptor binding fits with the notion that hydrogen bonding donor
properties help promote the high affinity r2 receptor binding of
4-O-des-methyl SA4503.
The homologous series of phenylalkyl ethers 4–6 had no appre-
ciable subtype selectivity, and displayed a tight range of Ki values,
13–23 nM, for binding to both r1 and r2 sites (Table 1). As
observed for analogs 1–3, incorporation of these bulkier and more
All novel ether analogs tested proved more potent than SA4503
for binding to both DAT and SERT (Table 2). Ligands 1, 3 and 7 dis-
played nearly equal affinities, approximately 2000 nM, for DAT
sites, while SERT binding showed more sensitivity to structure.

226
R. Xu et al. / Bioorg. Med. Chem. 23 (2015) 222–230
Table 2
DAT and SERT affinity of SA4503 and novel ether analogsa,b,c, along with literature data for selected YZ and reference compounds
b
f
c
f
R
DAT Ki (nM)
DAT/r1
SERT Ki (nM)
SERT/r1
–CH3 (SA4503)
(E)-CH2CH@CHI
(Z)–CH2CH@CHI
–CH2CH2CH3
–CH2CH@CH2
–CH2Ph
–CH2CH2CH2Ph
–Methylenedioxy–
YZ-185 (3-OCH3)d
YZ-067 (4-OCH3)d
YZ-069 (3,4-di-Cl)d
GBR 12909e
12650 832
560 57
237 13
1821 174
2083 156
121 16
2732
33
22
71
69
760 31
174 11
ND
124 13
295 19
164
10.3
ND
4.8
9.8
1
3
4
6
7
6
5
128
ND
6
6.2
ND
87
5
1840 311
1408 164
1466 19
8196 1005
12.0 1.9
721
1006
1128
3725
0.04f
468 41
184
703
1328
1302
0.78f
985 126
1726 57
2865 484
24.1 2.3
a
b
c
d
e
f
Ki values are means SEM for n = 3–6 trials, each performed in duplicate, except for DAT assays of compounds 6 and (Z)—CH2CH@CHI, where n = 2. ND = not determined.
15 pM [125I]RTI, mouse striatal membranes.
0.3 nM [3H]paroxetine, mouse whole brain membranes.
Binding data reported in Ref. 22.
Ref. 41.
Based upon the r1 Ki values reported in Table 1, and the r1 Ki value of 318 nM reported for GBR 12909 in Ref. 41.
Their SERT Ki values of 124 nM to 468 nM varied by 4-fold. Benzyl
analog 4 and the (E)-iodoallyl congener displayed SERT Ki values of
128 nM and 174 nM, respectively, (Table 2). Thus, further increases
in ether substituent size and hydrophobicity preserved, but did not
enhance, SERT affinity. By contrast, DAT affinities of the SA4503
ether analogs increased substantially in conjunction with increas-
ing substituent size and hydrophobicity. Phenylalkyl ethers 4 and 6
gave the highest DAT affinities of the ligands tested, with Ki values
of 121 nM and 87 nM (Table 2), and exhibited over 100-fold higher
DAT affinity than SA4503. Considering the lower DAT affinity of the
(Z)- and (E)-iodoallyl ether analogs (Table 2), the terminal aromatic
rings on the ether substituents of 4 and 6 play an important role in
this interaction.
Poses shown have the phenylpropyl piperazine moieties aligned,
and were minimized using the MMFF94 force field routine of pro-
gram Avogadro.52 Qualitatively, the overall molecular dimensions
of the two compounds are comparable, and there is substantial
spatial overlap between the terminal aromatic ring of the ether
substituent of 4 and the diphenylmethoxy ring system of GBR
12909. GBR 12909,41 as well as the YZ-series of analogs,22 typically
display low affinities, >1000 nM, for the norepinephrine trans-
porter. Consequently, the affinities of SA4503 and analogs were
not determined for this site.
3. Conclusions
We anticipated the possibility of finding moderately high DAT
and SERT affinities for the phenylalkyl ether analogs of SA4503
based upon their structural resemblance to GBR 12909 (Fig. 3), a
ligand that shows high DAT (Ki = 12 nM) and SERT (Ki = 24 nM)
affinities accompanied by weaker interactions with both r1
SA4503, 4-O-des-methyl-SA4503 and two series of ether ana-
logs have been prepared and evaluated for r1 and r2 receptor
binding. The 14 compounds tested could be divided into two
groups having different structure affinity relationships. The first
group included 6 compounds, SA4503, 4-O-des-methyl SA4503,
FE-SA4503 and cyclic ether analogs 7–9. These displayed relatively
high r1 receptor affinities, Ki values 1.75–19 nM, and variable r2
receptor affinities, Ki values 6.7–113 nM, leading to modest r1
receptor selectivities. The other group of eight SA4503 analogs
had bulkier, hydrophobic ether substituents, and included alkyl/
allyl ethers 1–3, phenylalkyl ethers 4–6 and the (Z)- and (E)-iodo-
allyl ether analogs. These compounds showed moderately high,
nearly equal affinities for both r1 and r2 receptors, leading to
ligands with little to no subtype selectivity. Considering there are
(Ki = 318 nM) and
r
2 (Ki = 116 nM) sites.41 A more recent determi-
nation puts the apparent affinity of GBR 12909 for r1 receptors at
50.8 nM.42 SA4503 and GBR 12909 are flexible molecules that can
adopt hundreds of closely related low energy conformations.20,51 In
both cases, the piperazine rings are thought to take a chair confor-
mation, and the phenyl alkyl chains are fully extended. These fea-
tures have been observed in crystal structures of SA4503 and
analogs.20 One possible structural superimposition of GBR 12909
with 4, the benzyl ether analog of SA4503, is given in Figure 3.
F
N
N
N
O
N
F
O
GBR 12909
4
OCH3
Figure 3. Comparison of GBR 12909 with 4, the benzyl ether analog of SA4503, and superimposition of one of many possible low energy conformers from each using program
Avogadro.

R. Xu et al. / Bioorg. Med. Chem. 23 (2015) 222–230
227
two piperazine nitrogens in each ligand and two main hydrophobic
binding pockets for each receptor subtype, at least eight different
diluted with H2O (30 mL) and extracted with CH2Cl2 (3 ꢁ 20 mL).
The organic extracts were washed with saturated NaCl solution,
dried over MgSO4, and concentrated under reduced pressure. The
crude oily residues, pale yellow to dark brown in color, were puri-
fied by silica gel column chromatography (CHCl3/CH3OH = 20:1).
4.1.1.1.1. 1-(3-Methoxy-4-propoxyphenethyl)-4-(3-phenylpropyl)-
piperazine (1). Treatment of 4-O-des-methyl-SA4503 (200 mg,
0.56 mmol) with 40% KOH (1.1 mL), tetra-n-butylammonium
hydroxide (0.1 mL) and 1-bromopropane (1200 mg, 9.74 mmol)
according to the general procedure gave 1 (201 mg, 90%) 1H
NMR: (250 MHz, CDCl3, d) 1.03 (t, 3H, J = 7.5 Hz, CH3), 1.77–1.89
(m, 4H, CH2), 2.35–2.78 (m, 16H, CH2), 3.84 (s, 3H, OCH3), 3.94 (t,
2H, J = 7.5 Hz, OCH2), 6.70–6.80 (m, 3H, CH), 7.13–7.29 (m, 5H,
CH). 13C NMR: (62.5 MHz, CDCl3, d) 10.4, 22.5, 28.6, 33.2, 33.7,
53.2, 55.9, 57.97, 60.6, 70.6, 112.6, 113.2, 120.5, 125.7, 128.2,
128.3, 133.0, 142.1, 146.8, 149.3. Anal. Calcd for the di-HCl salt
(C25H38Cl2N2O2): C, 63.96; H, 8.16; N, 5.97. Found: C, 63.91; H,
8.13; N, 5.91; mp 212 °C.
4.1.1.1.2. 1-(4-(2-Bromoethoxy)-3-methoxyphenethyl)-4-(3-phen-
ylpropyl)piperazine (2). Treatment of 4-O-des-methyl-SA4503
(400 mg, 1.13 mmol) with 40% KOH (2.2 mL), tetra-n-butylammo-
nium hydroxide (0.2 mL) and 1,2-dibromoethane (690 mg,
3.66 mmol) according to the general procedure gave 2 (430 mg,
83%). 1H NMR: (250 MHz, CDCl3, d) 1.77–1.89 (m, 2H, CH2), 2.35–
2.78 (m, 16H, CH2), 3.62 (t, 2H, J = 7.5 Hz, CH2), 3.84 (s, 3H, CH3),
4.28 (t, 2H, J = 7.5 Hz, CH2), 6.70–6.84 (m, 3H, CH), 7.14–7.30 (m,
5H, CH). 13C NMR: (62.5 MHz, CDCl3, d) 28.6, 29.0, 33.2, 33.7,
53.2, 56.0, 58.0, 60.5, 69.5, 113.0, 115.2, 120.7, 125.7, 128.2,
128.3, 134.7, 142.1, 145.7, 149.8; Anal. Calcd for the di-HCl salt
(C24H35BrCl2N2O2): C, 53.94; H, 6.60; N, 5.24. Found: C, 53.94; H,
6.70; N, 5.24; mp 248–251 °C.
4.1.1.1.3. 1-(4-Allyloxy)-3-methoxyphenethyl)-4-(3-phenylpropyl)-
piperazine (3). 4-O-des-Methyl-SA4503 (250 mg, 0.070 mmol)
was treated with 40% KOH (1.4 mL) and tetra-n-butylammonium
hydroxide (0.12 mL), followed by dropwise addition of allyl bromide
(1500 mg, 12.40 mmol) at 0 °C. The mixture was then stirred at
room temperature for 50 min, and worked up according to the gen-
eral procedure to give 3 (160 mg, 57%). 1H NMR: (300 MHz, CDCl3, d)
1.81–1.86 (app p, 2H, J = 7.5 Hz, CH2), 2.36–2.76 (m, 16H, CH2), 3.85
(s, 3H, OCH3), 4.55–4.58 (ddd, 2H, CH2, J = 5.4, 1.5, 1.5 Hz), 5.23–5.28
(ddt, 1H, J = 10.5, 1.5, 1.5 Hz), 5.35–5.42 (ddt, 1H, J = 17.1, 1.5,
1.5 Hz), 6.00–6.13 (ddt, 1H, J = 17.4, 10.5, 5.4 Hz), 6.70–6.81 (m,
3H, CH), 7.17–7.29 (m, 5H, CH). 13C NMR: (75 MHz, CDCl3, d) 28.6,
33.2, 33.7, 53.2, 55.8, 58.0, 60.6, 69.9, 112.4, 113.6, 117.7, 120.4,
125.7, 128.3, 128.4, 133.4, 133.5, 142.1, 146.3, 149.3; Anal. Calcd
for the di-HCl salt (C25H36Cl2N2O2): C, 64.23; H, 7.76; N, 5.99. Found:
C, 64.33; H, 7.71; N, 5.96. mp 222 °C.
r
binding motifs are possible. Thus, one set of motifs may be favored
for the binding of SA4503 and the compact analogs described
above, while different motifs may be preferred as the ether substit-
uents become larger and more hydrophobic. In this regard, some
effects observed are likely to be unique to the exact position and
type of substituent on the SA4503 scaffold. Diverse substituent
pattern effects on
r receptor binding affinity and subtype selectiv-
ity are often found between studies of piperazines that have differ-
ing arylalkyl systems.17,18,20–22,53–58
SA4503 and a subset of ether analogs were also evaluated for
DAT and SERT interactions. SA4503 displayed very weak binding
to the DAT, Ki = 12650 nM. Affinity increased with progressively
larger ether substitutions, and the phenylpropyl ether analog 6
showed a 145-fold higher DAT affinity of 87 nM. SERT binding var-
ied 7-fold between SA4503 and analogs. Overall, monoamine
transporter binding proved more sensitive than
to these structural modifications of SA4503.
r receptor binding
Together, the data provide some insight into how manipula-
tions of the substituents at the 4-position of the phenethyl side
chain of SA4503 modulate
ity. None of the novel ether analogs displayed higher
r
receptor subtype affinity and selectiv-
receptor
r
subtype affinity or selectivity than SA4503 or known congeners.
On the other hand, ether substituents can be chosen that greatly
increase DAT and SERT binding affinities, and the work identifies
SA4503 as a scaffold for the preparation of ligands that can target
multiple receptors. For instance, benzyl ether 4 shows moderately
high affinities for r1 and r2 receptors, as well as for the DAT and
SERT. Thus, certain ether analogs of SA4503 may well be polyfunc-
tional in vivo, and would be of interest for behavioral testing in
animal models of psychostimulant abuse, depression and anxiety.
4. Experimental section
4.1. Chemistry
4.1.1. General methods
Chemicals and solvents were reagent grade, and used as
received from commercial sources. 4-O-des-methyl-SA4503 was
prepared according to the literature procedure.36b 1H NMR spectra
were obtained on Bruker ARX-250 (250 MHz), DRX-300 (300 MHz)
or DRX-500 (500 MHz) spectrometers. Chemical shifts (1H) are
reported for the free base in ppm (d) relative to internal Me4Si in
CDCl3. Splitting patterns are identified as singlet (s), broad singlet
(br s), doublet (d), triplet (t), apparent pentet (app p), multiplet,
(m), doublet of doublets of doublets, (ddd), doublet of doublets
of triplets, (ddt). Elemental analyses were determined by Atlantic
Microlab, Inc., Norcross GA. Analytical TLC was conducted on Poly-
gram SIL G/UV254 plates, with additional visualization by staining
with cerium molybdate. Preparative TLC was conducted on silica
4.1.1.1.4. 1-(4-(Benzyloxy)-3-methoxyphenethyl)-4-(3-phenylpro-
pyl)piperazine (4). To
a stirred solution of 4-O-des-methyl-
SA4503 (250 mg, 0.70 mmol) in absolute EtOH (2.5 mL) was added
K2CO3 (193 mg, 1.40 mmol) and benzyl bromide (192 mg,
1.12 mmol). The mixture was left under N2 overnight, and then fil-
tered through Celite. Concentration under reduced pressure fol-
lowed by preparative TLC (CHCl3/CH3OH = 10:1) gave 4 as a pale
yellow solid (110 mg, 35%). 1H NMR: (250 MHz, CDCl3, d) 1.82–
1.88 (m, 2H, CH2), 2.38–2.77 (m, 16H, CH2), 3.89 (s, 3H, CH3),
5.13 (s, 2H, CH2), 6.68–6.83 (m, 3H, CH), 7.19–7.46 (m, 10H, CH).
13C NMR: (62.5 MHz, CDCl3, d) 28.6, 33.2, 33.7, 53.2, 55.9, 58.0,
60.6, 71.2, 112.6, 114.2, 120.5, 125.70 127.2, 127.7, 128.2, 128.3,
128.4, 133.6, 137.4, 142.1, 146.5, 149.5. Anal. Calcd for the di-HCl
salt (C29H38Cl2N2O2ꢀ0.5 H2O): C, 66.15; H, 7.47; N, 5.32. Found: C,
66.00; H, 7.34; N, 5.38. mp 213 °C.
gel plates (20 ꢁ 20 cm, 1000
lm) from Analtech Inc., Newark DE.
Column chromatography was performed using SilicycleÒultra pure
silica gel (230–400 mesh) under N2 pressure. For storage and ease
of handling, final targets were converted into the di-HCl salts. The
free bases were dissolved in EtOH, concentrated HCl was added,
the mixtures were evaporated to dryness, and the solid white
products were recrystallized. Compounds 1–9 showed >95% purity
by reversed-phase HPLC analysis.
4.1.1.1. General procedure for alkylation of 4-O-des-methyl-
SA4503 (1, 2, 3, 5 and 6).
Mixtures of 4-O-des-methyl-SA4503
4.1.1.1.5. 1-(3-Methoxy-4-phenylethoxyphenethyl)-4-(3-phenyl-
propyl)piperazine (5). Treatment of 4-O-des-methyl-SA4503
(200 mg, 0.56 mmol) with 40% KOH (1.1 mL), tetra-n-butylammo-
nium hydroxide (0.1 mL) and (2-bromoethyl)benzene (1800 mg,
(200–400 mg, 0.56–1.12 mmol), 40% KOH (1.1–2.2 mL), tetra-
n-butylammonium hydroxide (1 M, CH3OH; 0.1–0.2 mL), and
excess alkylating agent were heated at 50 °C for 50 min, and then

228
R. Xu et al. / Bioorg. Med. Chem. 23 (2015) 222–230
9.733 mmol) according to the general procedure gave 5 (113 mg,
44%). 1H NMR: (250 MHz, CDCl3, d) 1.82–1.89 (m, 2H, CH2), 2.38–
2.78 (m, 16H, CH2), 3.17 (t, 2H, J = 7.5 Hz, CH2), 3.86 (s, 3H, CH3),
4.22 (t, 2H, J = 7.5 Hz, CH2), 6.74–6.83 (m, 3H, CH), 7.19–7.31 (m,
10H, CH). 13C NMR: (62.5 MHz, CDCl3, d) 28.6, 33.2, 33.7, 35.8,
53.2, 56.0, 58.0, 60.6, 70.0, 112.8, 113.6, 120.7, 125.7, 125.8,
126.4, 127.3, 128.3, 128.4, 128.4, 129.0, 133.4, 138.1, 142.1,
146.6, 149.4. Anal. Calcd for the di-HCl salt (C30H40Cl2N2O2): C,
67.79; H, 7.58; N, 5.27. Found: C, 67.53; H, 7.64; N, 5.24. mp 252 °C.
4.1.1.1.6. 1-(3-Methoxy-4-(3-phenylpropoxy)phenethyl)-4-(3-phen-
ylpropyl)piperazine (6). Treatment of 4-O-des-methyl-SA4503
(200 mg, 0.56 mmol) with 40% KOH (1.1 mL), tetra-n-butylammo-
using 3-phenylpropylpiperazine (316 mg, 1.55 mmol) and 3,4-
(ethylenedioxy)phenylacetic acid (11, 301 mg, 1.55 mmol) to yield
14 (577 mg, 98%). 1H NMR: (250 MHz, CDCl3, d) 1.78 (app p, 2H,
J = 7.5 Hz, CH2), 2.24–2.39 (m, 6H, CH2), 2.62 (t, 2H, J = 7.5 Hz,
CH2), 3.43 (t, 2H, J = 5.0 Hz, CH2), 3.63 (t, 2H, J = 5.0 Hz, CH2), 3.59
(s, 2H, CH2), 4.20 (s, 4H, OCH2CH2O), 6.66–6.81 (m, 3H, CH),
7.15–7.30 (m, 5H, CH). 13C NMR: (62.5 MHz, CDCl3, d) 28.4, 33.4,
40.0, 41.7, 46.0, 52.7, 53.1, 57.5, 64.2, 64.3, 117.3, 121.5, 125.8,
128.2, 128.3, 128.3, 141.9, 142.3, 143.5, 169.4. Anal. Calcd for C23-
H28N2O3ꢀ0.5H2O: C, 70.93; H, 7.50; N, 7.19. Found: C, 70.73; H,
7.31; N, 7.20.
4.1.1.2.3. 2-(3,4-Propylenedioxy)phenyl-1-(4-(3-phenylpropyl)pip-
erazin-1-yl)ethanone (15). The general procedure was followed
using 3-phenylpropylpiperazine (255 mg, 1.25 mmol) and 3,4-
(ethylenedioxy)phenylacetic acid (12, 260 mg, 1.25 mmol) to yield
15 (443 mg, 90%) 1H NMR: (500 MHz, CDCl3, d) 1.63 (app p, 2H,
J = 7.5 Hz, CH2), 1.98 (app p, 2H, J = 6.0 Hz, CH2), 2.09 (t, 2H,
J = 5.0 Hz, CH2), 2.16 (t, 2H, J = 7.5 Hz, CH2), 2.21 (t, 2H, J = 5.0 Hz,
CH2), 2.47 (t, 2H, J = 7.5 Hz, CH2), 3.27 (t, 2H, J = 5.0 Hz, CH2), 3.45
(s, 2H, CH2), 3.48 (t, 2H, J = 5.0 Hz, CH2), 3.98–4.01 (m, 4H, CH2),
6.62–6.77 (m, 3H, CH), 7.00–7.15 (m, 5H, CH). 13C NMR:
(125 MHz, CDCl3, d) 28.3, 31.8, 33.4, 39.8, 41.7, 46.0, 52.6, 53.0,
57.5, 70.4, 121.6, 121.7, 123.4, 125.7, 128.2, 128.3, 130.2, 141.9,
149.9, 151.1, 169.2. Anal. Calcd for C23H28N2O3ꢀ0.5H2O: C, 68.64;
H, 7.19; N, 6.27. Found: C, 69.09; H, 7.31; N, 6.66.
nium
hydroxide
(0.1 mL)
and
1-bromo-3-phenylpropane
(1460 mg, 7.31 mmol) according to the general procedure gave 6
(214 mg, 81%). 1H NMR: (300 MHz, CDCl3, d) 1.87 (app p, 2H,
J = 6.0 Hz, CH2), 2.15 (app p, 2H, J = 6.0 Hz, CH2), 2.43–2.85 (m,
18H, CH2), 3.86 (s, 3H, CH3), 3.99 (t, 2H, J = 6.0 Hz, CH2), 6.71–6.80
(m, 3H, CH), 7.16–7.31 (m, 10H, CH). 13C NMR: (75 MHz, CDCl3, d)
28.2, 30.8, 31.3, 32.1, 32.2, 32.3, 33.0, 33.6, 34.4, 52.8, 56.0, 58.5,
61.5, 68.2, 112.7, 113.6, 120.6, 125.8, 125.9, 125.9, 128.4, 128.4,
128.4, 128.5, 132.8, 141.5, 141.8, 146.9, 149.5; Anal. Calcd for the
di-HCl salt (C31H42Cl2N2O2): C, 68.24; H, 7.76; N, 5.13. Found: C,
67.94; H, 7.77; N, 5.12. mp 227 °C.
4.1.1.1.7. 3,4-Ethylenedioxyphenylacetic acid (11) and 3,4-propylen-
edioxyphenylacetic acid (12). To a solution of 3,4-dihydroxyphen-
ylacetic acid (500 mg, 3.0 mmol) and either 1,2-dibromoethane
(1.13 g, 6.0 mmol) or 1,3-dibromopropane (1210 mg, 6.0 mmol)
in ethylene glycol (5 mL) was added anhydrous K2CO3 (1240 mg,
9.0 mmol). After heating at 120 °C for 4.5 h, the mixtures were
cooled, diluted with H2O (50 mL), acidified to pH <1 (2 N HCl),
and extracted with EtOAc (3 ꢁ 20 mL). The organic layers were
concentrated under reduced pressure, and residues were purified
by column chromatography (EtOAc/Hexane = 50:50 for 11; 40:60
for 12) to give 1144 (301 mg, 52%) or 1245 (260 mg, 42%). 11 1H
NMR: (300 MHz, CDCl3, d) 3.56 (s, 2H, CH2), 4.23 (s, 4H, CH2),
6.75–6.87 (m, 3H, CH), 11.64 (br s, 1H, ACOOH). 12 1H NMR:
(250 MHz, CDCl3, d) 2.19 (app p, 2H, J = 7.5 Hz, CH2), 3.55 (s, 2H,
CH2), 4.21 (m, 4H, CH2), 6.81–6.95 (m, 3H, CH), 10.50 (br s, 1H,
ACOOH).
4.1.1.3. General procedure for amide reduction (7–9).
Solu-
tions of substituted amides (13–15, 0.79–1.27 mmol) in THF
(7–12 mL) were added to a suspension of 3 equivalents of LiAlH4
in THF (7–12 mL) under N2 at 0 °C. Mixtures were then stirred at
ambient temperature for 7.5 h, cooled in an ice bath, and quenched
by sequential addition of EtOAc (3 mL) and HCl (2 N, 1 mL), fol-
lowed by addition of solid NaHCO3 until CO2 evolution ceased.
Mixtures were filtered, diluted with saturated NaHCO3 and
extracted with EtOAc. Organic extracts were dried over MgSO4, fil-
tered and evaporated to dryness under reduced pressure. Cyclic
ether 7 was isolated by preparative TLC (CHCl3/CH3OH = 10:1)
while 8 and 9 were purified on silica gel columns (CHCl3/CH3-
OH = 30: 1).
4.1.1.3.1. 1-(3,4-Methylenedioxyphenethyl)-4-(3-phenylpropyl)-
piperazine (7). The general procedure was followed using amide
13 (288 mg, 0.79 mmol) to give 7 as a yellow oil (90 mg, 33%). 1H
NMR: (250 MHz, CDCl3, d) 1.75–1.87 (m, 2H, CH2), 2.33–2.73 (m,
16H, CH2), 5.85 (s, 2H, OCH2O), 6.60–6.71 (m, 3H, CH), 7.15–7.28
(m, 5H, CH). 13C NMR: (75 MHz, CDCl3, d) 28.6, 33.3, 33.6, 53.2,
57.9, 60.7, 100.7, 108.1, 109.1, 121.3, 125.7, 128.2, 128.3, 134.0,
142.1, 145.7, 147.5; Anal. Calcd for the di-HCl salt (C22H30Cl2N2O2):
C, 62.12; H, 7.11; N, 6.59. Found: C, 61.85; H, 7.04; N, 6.55. mp
230 °C.
4.1.1.2. General procedure for phenylacetic acid coupling to
3-phenylpropylpiperazine (13–15).
Equimolar amounts of
3-phenylpropylpiperazine (0.83–1.55 mmol), HOBtꢀH2O, EDCꢀHCl
and the substituted phenylacetic acid (10–12) were treated with
3 equiv of 4-methylmorpholine in CH2Cl2 (7–15 mL) under N2 at
0 °C. Mixtures were stirred at ambient temperature overnight,
and then evaporated to dryness under reduced pressure. Residues
were diluted with saturated NaHCO3 and extracted with EtOAc.
Organic layers were washed with saturated NaCl solution, dried
over MgSO4, filtered and evaporated to dryness under reduced
pressure. Residues were purified by column chromatography
(CHCl3/CH3OH = 20:1).
4.1.1.2.1. 2-(3,4-Methylenedioxy)phenyl-1-(4-(3-phenylpropyl)-
piperazin-1-yl)ethanone (13). The general procedure was fol-
lowed with 3-phenylpropylpiperazine (170 mg, 0.83 mmol) and
3,4-(methylenedioxy)phenylacetic acid (10, 150 mg, 0.83 mmol)
to provide 13 (288 mg, 94%) 1H NMR: (300 MHz, CDCl3, d) 1.76–
1.81 (m, 2H, CH2), 2.25–2.39 (m, 6H, CH2), 2.62 (t, 2H, J = 6.0 Hz,
CH2), 3.45 (t, 2H, J = 6.0 Hz, CH2), 3.63 (t, 4H, J = 6.0 Hz, CH2), 5.92
(s, 2H, OCH2O), 6.66–6.75 (m, 3H, CH), 7.15–7.27 (m, 5H, CH). 13C
NMR: (75 MHz, CDCl3, d) 28.3, 33.4, 40.4, 41.7, 46.0, 52.6, 53.0,
57.5, 100.9, 108.3, 109.0, 121.5, 125.7, 128.2, 128.3, 128.6, 141.8,
146.31, 147.8, 169.3. Anal. Calcd for C22H26N2O3ꢀ0.25 H2O: C,
71.23; H, 7.20; N, 7.55. Found: C, 71.41; H, 7.18; N, 7.54.
4.1.1.3.2.
1-(3,4-Ethylenedioxyphenethyl)-4-(3-phenylpropyl)-
piperazine (8). The general procedure was followed using amide
14 (484 mg, 1.27 mmol) to give 8 as a yellow oil (340 mg, 73%).
1H NMR: (250 MHz, CDCl3, d) 1.84 (app p, 2H, J = 7.5 Hz, CH2),
2.36–2.74 (m, 16H, CH2), 4.19 (s, 4H, OCH2CH2O), 6.65–6.80 (m,
3H, CH), 7.15–7.31 (m, 5H, CH). 13C NMR: (62.5 MHz, CDCl3, d)
28.6, 33.3, 33.6, 53.2, 57.9, 60.7, 100.7, 108.1, 109.1, 121.3, 125.7,
128.2, 128.3, 134.0, 142.1, 145.7, 147.5; Anal. Calcd for the di-
HCl salt (C23H32Cl2N2O2): C, 62.87; H, 7.34; N, 6.38. Found: C,
63.14; H, 7.49; N, 6.39. mp 230 °C.
4.1.1.3.3.
1-(3,4-Propylenedioxyphenethyl)-4-(3-phenylpropyl)-
piperazine (9). The general procedure was followed using amide
15 (390 mg, 0.99 mmol) to give 9 as a yellow oil (321 mg, 85%).
1H NMR: (300 MHz, CDCl3, 1.84 (app p, 2H, J = 6.0 Hz, CH2), 2.15
(app p, 2H, J = 6.0 Hz, CH2), 2.37–2.75 (m, 16H, CH2), 4.16 (t, 4H,
J = 6.0 Hz, -CH2O), 6.74–6.91 (m, 3H, CH), 7.15–7.30 (m, 5H, CH).
13C NMR: (75 MHz, CDCl3, d) 28.6, 32.0, 32.7, 33.7, 53.2, 58.0,
4.1.1.2.2. 2-(3,4-Ethylenedioxy)phenyl-1-(4-(3-phenylpropyl)pip-
erazin-1-yl)ethanone (14). The general procedure was followed

R. Xu et al. / Bioorg. Med. Chem. 23 (2015) 222–230
229
3. Mach, R. H.; Zeng, C.; Hawkins, W. G. J. Med. Chem. 2013, 56, 7137.
60.5, 70.4, 121.3, 121.6, 123.4, 125.7, 128.3, 128.4, 135.6, 142.1,
149.4, 151.0; Anal. Calcd for the di-HCl salt (C24H34Cl2N2O2): C,
63.57; H, 7.56; N, 6.18. Found: C, 63.44; H, 7.71; N, 6.16. mp 212 °C.
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r
receptor assays. Fresh male CD-1Ò mouse brains (Charles River Lab-
oratories International, Inc.; Wilmington, MA) were harvested, after
animal euthanasia by cervical dislocation, as needed for the DAT
and SERT assays. Studies involving living animals were performed
humanely in compliance with the Guide for the Care and Use of Lab-
oratory Animals promulgated by the U.S. National Institutes of
Health, and with prior approvals from the Institutional Animal Care
and Use Committees of the University of Missouri and the Harry S.
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Assays for
[3H](+)-pentazocine (
200 nM (+)-pentazocine (
guinea pig brains as previously described in detail.38 Non-specific
binding was defined by haloperidol (1.0 M; 1) or by DTG
(100 M; 2). Binding assays for DAT and SERT were performed
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receptor binding were performed using 1.0 nM
1), 3.0 nM [3H]ditolylguanidine ([3H]DTG)/
2), and membranes from whole male
r
r
l
r
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using literature procedures with minor modifications. In brief,
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with non-specific binding defined by 0.5
assays used [3H]paroxetine at 0.3 nM and mouse whole brain mem-
branes, with non-specific binding defined by 10
M fluoxetine.61
l
M GBR 12909.60 SERT
l
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sigmoidal four-parameter logistic fit (Prism 6.0c; GraphPad Soft-
ware, Inc.; La Jolla, CA). Ki values were derived from inhibition
IC50 data by the Cheng–Prusoff relationship,62 using reported Kd
values of 2.3 nM for [3H](+)-pentazocine,38 23.9 nM for [3H]DTG,38
0.12 nM for [125I]RTI,60 and 0.13 nM for [3H]paroxetine.61
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We thank the National Cancer Institute (P50 CA103130: Center
for Single Photon-Emitting Cancer Imaging Agents) and the
National Institute for Drug Abuse (1RC1DA028477: Development
of Anti-Cocaine Medications) for partial support of this research.
We thank the National Science Foundation (NSF CHE-0353891:
Research Experience for Undergraduates (REU)—Site for an Intro-
duction to Radiochemistry Research) for supporting Dr. Ryan M.
Peterson, and the University of Missouri Life Sciences Undergradu-
ate Research Opportunity Scholars program for supporting Dr.
Sarah A. Lord (née Violand). We thank Mr. Michael W. Lever for
assistance with Avogadro. We thank NSF CHE-95-31247 for NMR
instrumentation support. We also acknowledge resources and
facilities provided by the Harry S. Truman Memorial Veterans’
Hospital. This work does not represent the views of the U.S.
Department of Veterans Affairs or the United States Government.
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