Effect of ring-constrained phenylpropyloxyethylamines on sigma receptors

Article abstract of DOI:10.1016/j.bmc.2013.06.068

A series of ring-constrained phenylpropyloxyethylamines, partial opioid structure analogs and derivatives of a previously studied sigma (σ) receptor ligand, was synthesized and evaluated at σ and opioid receptors for receptor selectivity. The results of this study identified several compounds with nanomolar affinity at both σ receptor subtypes. Compounds 6 and 9 had the highest selectivity for both σ receptor subtypes, compared to μ opioid receptors. In addition, compounds 6 and 9 significantly reduced the convulsive effects of cocaine in mice, which would be consistent with antagonism of σ receptors.

Full text of DOI:10.1016/j.bmc.2013.06.068

Bioorganic & Medicinal Chemistry 21 (2013) 4923–4927
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Effect of ring-constrained phenylpropyloxyethylamines on sigma
receptors
Lidiya Stavitskaya a, , Michael J. Seminerio b, , Jason R. Healy b, Bahar Noorbakhsh b, Rae R. Matsumoto b,
,
⇑
Andrew Coop a,
⇑
a Department of Pharmaceutical Sciences, University of Maryland, School of Pharmacy, 20 N. Pine Street, Baltimore, MD 21201, USA
b Department of Basic Pharmaceutical Sciences, West Virginia University, PO Box 9500, Morgantown, WV 26506, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of ring-constrained phenylpropyloxyethylamines, partial opioid structure analogs and deriva-
tives of a previously studied sigma ( ) receptor ligand, was synthesized and evaluated at and opioid
receptors for receptor selectivity. The results of this study identified several compounds with nanomolar
affinity at both receptor subtypes. Compounds 6 and 9 had the highest selectivity for both receptor
subtypes, compared to opioid receptors. In addition, compounds 6 and 9 significantly reduced the
convulsive effects of cocaine in mice, which would be consistent with antagonism of receptors.
Received 18 April 2013
Revised 19 June 2013
Accepted 28 June 2013
Available online 11 July 2013
r
r
r
r
l
r
Keywords:
Sigma
Ó 2013 Published by Elsevier Ltd.
Opioid
Phenylpropyloxyethylaminesl
Antagonists
25–28
1. Introduction
compared to r1
.
A recent study identified the r2 binding site
as the progesterone receptor membrane component 1 (PGRMC-
1).29 In addition, r2 receptors are believed to be associated with
the inhibition of cell proliferation and induction of apoptosis, pro-
ducing transient and sustained release of calcium ions.30
Finding effective pharmacotherapies to treat cocaine abuse and
addiction remains a major challenge.1 Considerable efforts have
been put forth towards the development of potential anti-cocaine
therapeutics that attenuate the toxic and addictive effects.2 Our ap-
Prior to the discovery of the two subtypes, initial
r receptor–
proach utilized the fact that cocaine interacts with sigma (
r
)
ligand structure–activity relationship (SAR) studies were
performed on a range of opioid-related compounds and it was
determined that (a) phenylpiperidine containing analogs had rel-
receptors3–5 and
r
antagonists attenuate acute (convulsions,
lethality, locomotor activity) and subchronic (sensitization, place
conditioning) effects of cocaine, making these receptors a promis-
ing target for developing treatments for cocaine abuse.3,6–9 In addi-
tion, recent data from cocaine self administration studies suggests
sigma receptor activation may have a role in stimulant abuse.10–13
atively high binding affinity at the
N-alkyl lipophilic substituents produce greater affinity for both
subtypes, and (c) there is no predetermined set of rigid con-
straints for the intramolecular distances required for receptor
binding.15 Though these initial
ligands helped gain insight into
the SAR, their interaction with other biological systems such as
r receptor binding sites, (b)
r
r
r
Receptors were initially proposed by Martin and co-workers
r
as a subtype of opioid receptor to account for some benzomorphan
r
activity.14 However, due to the inability of naloxone to antagonize
opioid receptors, dopamine transporters, or N-methyl-D-aspartate
r
-induced effects,
ique class of receptors.15
types, r1 and
2, with cocaine interacting with both subtypes.16
To date, r1 receptors are the only cloned
receptor.17–20 Studies
r
receptors were later considered to be a un-
(NMDA) receptors14 impeded the understanding of theirtrue bio-
logical function. Subsequent studies included a partial opioid,
N-phenylpropyl derivative of a ring opened benzomorphinan
r
Receptors are comprised of two sub-
r
r
(PPAP), which had high selectivity for the r receptor versus the
have shown that r1 receptors are involved in intracellular signal-
ing, synaptic transmission, modulation of inositol phosphates, pro-
tein kinases, and calcium.17,21–24 Though not yet cloned, r2
receptors appear to exist as heterodimers and are smaller in size
phencyclidine (PCP) sites and dopamine D1 and D2 receptors31
and thus served as the lead compound for many years in detailed
20,32,33
structure activity investigation.
Specifically, the effect of
longer-chain, aryl substituents, as well as conformational con-
straint on PPAP derivates were examined.33 These studies resulted
in agents which were selective for
r over other biological systems
⇑
Corresponding authors. Tel.: +1 4107062029.
E-mail address: acoop@rx.umaryland.edu (A. Coop).
These authors contributed equally to the work.
while displaying equivalent or higher affinity for
r1 and r2 recep-
tor subtypes.33
0968-0896/$ - see front matter Ó 2013 Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.bmc.2013.06.068

4924
L. Stavitskaya et al. / Bioorg. Med. Chem. 21 (2013) 4923–4927
Earlier studies from our laboratory had shown that AC927 (N-
phenethylpiperidine), a mixed 1 and 2 antagonist, demonstrated
high selectivity for the
receptors.34 Additional studies showed
that AC927 attenuated the behavioral and neurotoxic effects of
cocaine in mice.35 However, AC927 has a relatively narrow thera-
peutic window, which can result in deaths in mice at suprathera-
peutic doses (unpublished finding; R.R. Matsumoto, Morgantown,
WV). Accordingly, analogs of AC927 and structurally similar
compounds are required to determine the optimal substituents
targets were converted to oxalate salts and characterized using
NMR and MS. All elemental analyses of salts were within 0.4%.
r
r
r
2.2. Opioid and
r receptor binding
All of the tested compounds exhibited low to negligible affinity
(1000 to >10,000 nM) for the three opioid receptors subtypes
delta (d) and kappa ( ). Among the tested compounds, compound
l,
j
3 displayed the highest affinity for the r1 receptor (4.6 nM), with
the greatest selectivity for the r1 receptor when compared to the
and structural backbone needed to improve selectivity for each
r
r2 receptor (r2
/r
1 = 240). Reduction of a double bond on the cin-
subtype and to increase the therapeutic window for cocaine treat-
ment.
namyl group to give 4 decreased affinity at
r
1 and 2 receptors (59
r
and 3800 nM, respectively). Introduction of a phenylpropyl group
on the oxygen position of compound 6 led to compound 7, which
displayed higher selectivity for r1 (compared to 6) with 84 nM
affinity. In contrast, introduction of a phenylpropyl group to give
compound 10 resulted in dramatic decreases in both r1 and r2
receptor affinities, exhibiting low affinity at r1 receptors
(790 nM) and negligible affinity at r2 receptors (>10,000 nM). In
agreement with previous reports,34 increasing the chain length
from phenethyl (6) to phenylpropyl (9) gave rise to higher r1
and r2 affinities as indicated by 9. These results suggest that the
phenylpropylamines are required for r2 activity in the cyclohexa-
nol series. Compounds 6 and 9 were selected, as they produced no
N
AC927
The current series of compounds were initially synthesized to
determine the minimal structural requirements for high affinity
and efficacy at mu (
low to negligible affinity for the
l
) opioid receptors,36 however they displayed
l
opioid receptor. Their close
resemblance to AC927 and their inability to bind to the opioid
receptors prompted our decision to further investigate the partial
opioid structures at the two established
affinity for the
l opioid receptor and had a similar r binding pro-
r receptors subtypes
file to AC927, to undergo further in vivo testing in order to deter-
mine the ability of the compounds to block cocaine-induced
convulsions (see Table 1).
(
r1, r
2). The structural differences of the novel compounds will
aid in the design of r1 and r2 ligands and ultimately provide
insight into future drug development.
2.3. Cocaine-induced convulsions
2. Results and discussion
2.1. Chemistry
Based on their binding affinity for
r receptors, compounds 6
and 9 were investigated in vivo for anticonvulsant actions in co-
caine-treated mice (Fig. 1). Results indicate that pretreatment of
mice with compound 6 led to significant attenuation of cocaine-in-
duced convulsions at the highest dose tested (30 mg/kg, ip;
p < 0.05, Fisher’s exact tests). Pretreatment with compound 9 sig-
nificantly and dose-dependently attenuated cocaine-induced con-
vulsions at lower doses (1 and 10 mg/kg, ip), likely due to its
The synthetic sequences used to prepare compounds 3, 4, 6, 7, 9,
and 10 are displayed in Scheme 1. In brief, analogs 3 and 4
(Scheme 1A) were synthesized via N-methylation of trans-2-
aminocyclohexanol hydrochloride (1) using the Eschweiler–Clark
methylation37 followed by alkylation with the corresponding
bromide in the presence of NaH.38 Compound 6 and the intermedi-
ate of 9 (Scheme 1B) were synthesized by addition of the appropri-
ate N-alkylamine to an epoxide under reflux conditions.39
Eschweiler–Clark methylation reaction37 was utilized again to
obtain target 9. Analogs 7 and 10 were achieved by alkylation with
the appropriate phenylalkyl bromide in presence of NaH.38 All
higher affinity for
r receptors when compared to 6. Moreover,
the additional methylene group in 9 causes the expected increase
in cLogP of 0.3 units (ChemBioDraw Ultra 12.0, CambridgeSoft;
Cambridge, MA), which indicates that potency may be related to
lipophilicity due to greater brain penetration. Since cocaine
interacts with
r
receptors8 and our compounds display significant
A
NH2
OH
N
N
O
a
83%
b
1
2
7%; 10%
OH
1
2
3
4
1,2 = CH2-CH2
1,2 = CH=CH
B
N
N
c
d
6%
n
O
70%
O
OH
g
5
6
8
7: n = 1
e
33%
10:
n = 2
H
N
89%
N
f
52%
OH
OH
9
Scheme 1. Synthesis of 3, 4, 6, 7, 9 and 10. Reagents and conditions: A, (a) HCHO, HCOOH, reflux; (b) cinnamyl bromide or 1-bromo-3-phenylpropane, NaH, DMF, 50 °C, 3 h. B,
(c) N-methyl-phenethylamine, EtOH, reflux; (d) 1-bromo-3-phenylpropane, NaH, DMF, 50 °C, 3 h; (e) 3-phenylpropylamine, EtOH, reflux; (f) HCHO, HCOOH, reflux; (g)
1-bromo-3-phenylpropane, NaH, DMF, 50 °C.

L. Stavitskaya et al. / Bioorg. Med. Chem. 21 (2013) 4923–4927
4925
Table 1
Opioid and
r
receptor binding affinities
Compd
Opioid bindinga Ki SEM (nM)
Sigma bindingb Ki SEM (nM)
Selectivity
a
b
l
d
j
r1
r2
r2/r1
3
4
6
7
2300 74
4200 140
>10,000
1600 41
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
6200 770
>10,000
>10,000
>10,000
21,000 93
>10,000
4.6 0.20
59 2.0
120 8.0
84 15
19 0.90
790 76
30 2.0
1100 29
3800 800
220 6.0
570 31
6.7 0.30
>10,000
240
65
2.0
7.0
0.40
9
10
3100 62
2200 280
>13
5.0
AC927c
140 18
a
Displacement of [3H](+)-pentazocine.
b
c
Displacement of [3H]di-o-tolylguanidine (DTG) in presence of (+)-pentazocine.
Citations reference previously known compounds and results Ref.34
.
100
100
75
50
25
0
A
B
75
50
25
0
*
**
**
0
.1
1
10
0
1
10
30
Compound 6 Dose (mg/kg, i.p.)
Compound 9 Dose (mg/kg, i.p.)
Figure 1. Cocaine-induced convulsions. (A) Pretreatment of Swiss Webster mice with compound 6 (0–30 mg/kg ip), followed by a convulsive dose of cocaine (70 mg/kg ip)
produced a reduction in the convulsive effects of cocaine. (B) Pretreatment of Swiss Webster mice with compound 9 (0–10 mg/kg ip), followed by a convulsive dose of cocaine
(70 mg/kg ip) produced a dose-dependent reduction in the convulsive effects of cocaine. ⁄p < 0.05; ⁄⁄p < 0.01; Fisher’s exact test.
affinities for
r
receptors, compounds 6 and 9 may in part produce
(Analtech, Inc., Newark, DE). All compounds were purified using
standard techniques (crystallization, etc.) and characterized using
standard spectroscopic methods such as 1H NMR (Varian Inova
500 MHz) and MS (ThermoFinnigan LCQ Classic, Waltham, MA).
All optically active compounds were prepared as racemates and
evaluated for their biological activities. Elemental analysis was
performed by Atlantic Microlabs (Norcross, GA).
their protective effects through these receptors. Recent evidence
has suggested both r1 and r2 receptor antagonists possess anti-
cocaine activity.2,3,15,21
3. Conclusions
In the present study, we found that ring-constrained phenylpro-
pyloxyethylamines exhibit low to negligible affinity for opioid
4.1.1. trans-2-(Dimethylamino)cyclohexanol (2)
A solution of trans-2-aminocyclohexanol (1, 1 g, 8.68 mmol,
1.0 equiv) 37% formaldehyde (HCHO) (14 mL, 471 mmol,
54.2 equiv) and formic acid (HCOOH) (14 mL, 365 mmol,
42.0 equiv) was refluxed overnight. The resulting crude mixture
was concentrated and dissolved with ether and washed with 5 N
NaOH. The organic extracts were combined, dried with K2CO3,
and evaporated. MS ESI m/z 144 (M+H+); yield 83% (1.03 g).
receptors while displaying significant binding affinity for
r recep-
tors. Binding studies showed that introduction of a cinnamyl group
(compound 3) resulted in an increase in selectivity for the r1
receptor when compared to the r2 receptor. Compounds 6 and 9
bind to
with previous studies, increasing the chain length from phenethyl
(6) to phenylpropyl (9) increased affinity at both receptor
r receptors at nanomolar concentrations and in agreement
r
subtypes. Moreover, compounds 6 and 9 produced significant
4.1.2. trans-N,N-Dimethyl-2-(3-phenylpropoxy)cyclohexan-
amine (3)
reductions in cocaine-induced convulsions, providing further evi-
dence of
r involvement in the actions of cocaine and thus identi-
Compound 3 was prepared through alkylation of trans-2-
(dimethylamino)cyclohexanol (2, 0.72 g, 5.03 mmol, 1.0 equiv)
with 1-bromo-3-phenylpropane, (2.29 mL, 15.1 mmol, 3.0 equiv)
in the presence of NaH (0.84 g, 35.2 mmol, 7.0 equiv) following
the method described above. Yield 7% (0.092 g); 1H NMR
(500 MHz, D2O) d 7.41 (t, 7.67 Hz, 2H), d 7.26–7.33 (m, 3H), d
3.69–3.76 (m, 1H), d 3.47–3.59 (m, 2H), d 3.11–3.19 (m, 1H), d
2.68–2.94 (m, 8H), d 2.28–2.35 (m, 1H), d 2.04–2.11 (m, 1H), d
1.91–1.99 (m, 2H), d 1.89 (d, 6.79 Hz, 1H), d 1.75–1.81 (m, 1H);
MS ESI m/z 262 (M+H+); Anal. (C17H27NO(C2H2O4)2) C, H, N.
fies compound 9 as a viable lead compound for the development
of treatments for cocaine abuse.
4. Experimental methods
All reagents, solvents and compounds were purchased from Sig-
ma Aldrich Inc. (St. Louis, MO) and used without further purifica-
tion, unless stated otherwise. The radioligands were purchased
from Perkin Elmer (Boston, MA).
4.1. Synthesis
4.1.3. trans-2-(Cinnamyloxy)-N,N-dimethylcyclohexanamine (4)
To a solution of DMF and NaH (1.21 g, 50.3 mmol, 7.0 equiv)
was added trans-2-(dimethylamino)cyclohexanol (2, 1.03 g,
All reactions were carried out under an atmosphere of nitrogen.
Thin layer chromatography was performed on silica 60 F254 plated

4926
L. Stavitskaya et al. / Bioorg. Med. Chem. 21 (2013) 4923–4927
7.19 mmol, 1.0 equiv) dropwise and allowed to stir at room tem-
perature for 30 min prior to adding cinnamyl bromide (4.25 g,
21.6 mmol, 3.0 equiv). The reaction was heated for 3 h at 50 °C.
After the reaction reached completion, it was quenched with
20 mL of ethanol and the solvent was reduced under pressure.
The crude product was dissolved in H2O and extracted with Et2O.
The reaction mixture was then extracted into 6 M HCl and basified
to pH 12–13 with 5 M NaOH (aq) and extracted with Et2O. The
combined organic layers were washed with brine solution and
dried over Na2SO4. After removal of the solvent under reduced
pressure, the crude product was purified by column chromatogra-
phy (silica gel, CHCl3/5% MeOH/1% NH4OH) followed by formation
of an oxalate salt from ether. Yield 10% (0.18 g); 1H NMR (500 MHz,
D2O) d 7.52 (d, 3.56 Hz, 2H), d 7.42 (t, 7.33 Hz, 2H), d 7.33–7.38 (m,
1H), d 6.73–6.79 (m, 1H), d 6.36–6.44 (m, 1H), d 4.36–4.41 (m, 1H),
d 4.20–4.26 (m, 1H), d 3.65–3.72 (m, 1H), d 3.15–3.22 (m, 1H), d
2.80 (s, 6H), d 2.38–2.44 (m, 1H), d 2.07 (d, 5.86 Hz, 1H) d 1.87
(d, 6.07 Hz, 1H), d 1.76–1.81 (m, 1H), d 1.41–1.51 (m, 1H), d
1.19–1.38 (m, 4H); MS ESI m/z 260 (M+H+); Anal. (C17H25NO(C2H2-
O4)1) C, H, N.
1H), d 3.05–3.34 (m, 3H), d 2.91–3.00 (m, 1H), d 2.88 (s, 1H), d
2.70–2.83 (m, 4H), d 2.00–2.20 (m, 3H), d 1.89–1.96 (m, 1H), d
1.79–1.88 (m, 1H), d 1.70–1.78 (m, 1H), d 1.21–1.48 (m, 4H); MS
ESI m/z 248 (M+H+); Anal. (C16H25NO(C2H2O4)1(H2O)0.25) C, H, N.
4.1.8. trans-N-Methyl-2-(3-phenylpropoxy)-N-(3-
phenylpropyl)cyclohexanamine (10)
Compound 10 was prepared through alkylation of trans-2-
(methyl(3-phenylpropyl)amino)cyclohexanol (9, 0.6 g, 2.43 mmol,
1.0 equiv) with 1-bromo-3-phenylpropane (1.84 mL, 12.13 mmol,
5.0 equiv) in the presence of NaH (0.41 g, 17.0 mmol, 7.0 equiv) fol-
lowing the method described previously.38 Yield 33% (0.29 g); 1H
NMR (500 MHz, D2O) d 7.23–7.32 (m, 5H), d 7.14–7.24 (m, 5H), d
3.54–3.62 (m, 1H), d 3.40–3.47 (m, 1H), d 3.18–3.26 (m, 1H), d
2.54–2.75 (m, 5H), d 2.42–2.51 (m, 1H), d 2.28–2.38 (m, 3H), d
2.06–2.14 (m, 1H), d 1.84–1.93 (m, 2H), d 1.72–1.83 (m, 3H), d
1.63–1.72 (m, 2H), d 1.53–1.60 (m, 1H), d 1.04–1.30 (m, 4H); MS
ESI m/z 366 (M+H+); Anal. (C25H35NO(C2H2O4)1) C, H, N.
4.2. Receptor binding assays
4.1.4. trans-2-(Methyl(phenethyl)amino)cyclohexanol (6)
N-Methylphenethylamine (2.70 mL, 18.8 mmol, 1.0 equiv) and
cyclohexene oxide (5, 3.70 mL, 36.6 mmol, 2.1 equiv) were dis-
solved in 50 mL of ethanol and refluxed overnight. The ethanol
was evaporated under reduced pressure and purified by column
chromatography (silica gel, CHCl3/5% MeOH/1% NH4OH). Yield
70% (3.04 g); 1H NMR (500 MHz, D2O) d 7.44 (t, 7.23 Hz, 2H), d
7.35–7.40 (m, 3H), d 3.74–3.86 (m, 1H), d 3.51–3.64 (m, 1H), d
3.38–3.46 (m, 1H), d 3.01–3.31 (m, 3H), d 2.98 (s, 1H), d 2.83 (s,
2H), d 2.01–2.15 (m, 2H), d 1.82–1.90 (m, 1H), d 1.71–1.79 (m,
1H), d 1.21–1.55 (m, 4H); MS ESI m/z 234 (M+H+); Anal. (C15H23-
NO(C2H2O4)1) C, H, N.
The opioid receptor binding affinities of compounds were deter-
mined in membranes prepared from CHO cells stably transfected
with and expressing each of the three human opioid receptor sub-
types (l, d, and j l
) using standard in vitro methods.36,40 Briefly,
receptors were labeled with 1.3 nM [3H]DAMGO (53.4 Ci/mmol),
d receptors were labeled with 1.2 nM [3H]DPDPE (45 Ci/mmol),
and
mmol). Nonspecific binding for the opioid receptor assays was
determined in the presence of 1 M unlabelled DAMGO, DPDPE,
or U69,593 for the respective subtypes.
The radioligand binding assays for
j
receptors were labeled with 1.7 nM [3H]U69,593 (42.7 Ci/
l
r
receptors were performed
in rat brain homogenates using methods previously described in
detail.41,42 Briefly, r1 receptors were labeled with 5 nM [3H](+)-
4.1.5. N-Methyl-N-phenethyl-2-(3-
phenylpropoxy)cyclohexanamine (7)
pentazocine and
ylguanidine (DTG) in the presence of 300 nM (+)-pentazocine to
block r1 receptors. Nonspecific binding for the receptor assays
was determined in the presence of 10 M haloperidol.
r
2 receptors were labeled with 3 nM [3H]di-o-tol-
Compound 7 was prepared through alkylation of trans-2-
(methyl(phenethyl)amino)cyclohexanol (6) (1.5 g, 6.43 mmol,
1.0 equiv) with 1-bromo-3-phenylpropane (2.93 mL, 19.3 mmol,
3.0 equiv) in the presence of NaH (1.08 g, 45.0 mmol, 7 equiv) fol-
lowing the method described previously.38 Yield 6% (0.14 g); 1H
NMR (500 MHz, D2O) d 7.33–7.47 (m, 6H), d 7.26–7.33 (m, 4H), d
3.59–3.69 (m, 3H), d 3.39–3.48 (m, 1H), d 3.25–3.32 (m, 1H), d
3.16–3.24 (m, 1H), d 3.09–3.15 (m, 1H), d 2.94–3.00 (m, 2H), d
2.80–2.84 (m, 1H), d 2.59–2.68 (m, 2H), d 2.27–2.33 (m, 1H), d
2.01–2.14 (m, 1H), d 1.73–1.90 (m, 4H), d 1.44–1.56 (m, 1H), d
1.11–1.39 (m, 4H); MS ESI m/z 352 (M+H+); Anal. (C24H33NO(C2H2-
O4)1) C, H, N.
r
l
The competition binding studies were performed in the pres-
ence of up to 12 concentrations of each test compound and were
incubated for 120 min at 25 °C. Reactions were terminated by rapid
vacuum filtration through Perkin Elmer UnifilterÒ-96, GF/B filters
(Fisher Scientific, Hanover Park, IL) previously soaked in 0.5% poly-
ethyleneimine (PEI). Bound radioactivity was quantified by liquid
scintillation counting. Affinities (Ki) were calculated using the
Cheng–Prusoff equation.43
4.3. Cocaine-induced convulsions
4.1.6. trans-2-(3-Phenylpropylamino)cyclohexanol (8)
Phenylpropylamine (1.58 mL, 11.1 mmol, 1.0 equiv) and cyclo-
hexene oxide (5, 2.36 mL, 23.3 mmol, 2.1 equiv) were dissolved
in 50 mL of ethanol and refluxed overnight. The ethanol was evap-
orated under reduced pressure and purified by column chromatog-
raphy (silica gel, 94% CHCl3/5% MeOH/1% NH4OH). Yield 89%
(2.30 g); MS ESI m/z 234 (M+H+).
To probe for anticonvulsant actions of compounds 6 and 9
against cocaine, male, Swiss Webster mice (n = 10/group) were
pretreated (ip) with compounds 6 (0, 1, 10, or 30 mg/kg) or 9 (0,
0.1, 1, or 10 mg/kg) 15 min prior to administration of a convulsive
dose of cocaine (70 mg/kg, ip), similar to previously described.21
The mice were observed for the occurrence of convulsions for
30 min following the cocaine injection and results were recorded.
Convulsions were operationally defined as clonic or tonic limb
movements, which were accompanied by the loss of righting re-
flexes for at least 5 s, and/or popcorn jumping. Fisher’s exact test
was utilized to determine significant differences between each
group.
4.1.7. trans-2-(Methyl(3-phenylpropyl)amino)cyclohexanol (9)
A solution of trans-2-(3-phenylpropylamino)cyclohexanol (8,
2.27 g, 9.73 mmol, 1.0 equiv) 37% formaldehyde (HCHO) (21 mL,
706 mmol, 72.6 equiv) and formic acid (HCOOH) (21 mL,
548 mmol, 56.3 equiv) was refluxed overnight. The resulting crude
mixture was concentrated and dissolved with ether and washed
with 5 N NaOH. The organic extracts were combined, dried with
K2CO3, and evaporated. Yield 52.4% (1.26 g); 1H NMR (500 MHz,
D2O) d 7.41 (t, 7.41 Hz, 2H), d 7.30–7.30 (m, 3H), d 3.71–3,79 (m,
Acknowledgments
This work was supported in part by the National Institute on
Drug Abuse, National Institutes of Health (NIDA, NIH)

L. Stavitskaya et al. / Bioorg. Med. Chem. 21 (2013) 4923–4927
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.06.068.
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