Synthesis and characterization of N,N-dialkyl and N-alkyl-N-aralkyl fenpropimorph-derived compounds as high affinity ligands for sigma receptors

Article abstract of DOI:10.1016/j.bmc.2010.04.078

The sigma-1 receptor is a unique non-opioid, non-PCP binding site that has been implicated in many different pathophysiological conditions including psychosis, drug addiction, retinal degeneration and cancer. Based on the structure of fenpropimorph, a hig

Full text of DOI:10.1016/j.bmc.2010.04.078

Bioorganic & Medicinal Chemistry 18 (2010) 4397–4404
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and characterization of N,N-dialkyl and N-alkyl-N-aralkyl
fenpropimorph-derived compounds as high affinity ligands for sigma receptors
Abdol R. Hajipour a,b, Dominique Fontanilla a, Uyen B. Chu a, Marty Arbabian a, Arnold E. Ruoho a,
*
a Department of Pharmacology, University of Wisconsin School of Medicine and Public Health, Madison, WI 53706, United States
b Pharmaceutical Research Laboratory, Department of Chemistry, Isfahan University of Technology, Isfahan 84156, IR, Iran
a r t i c l e i n f o
a b s t r a c t
Article history:
The sigma-1 receptor is a unique non-opioid, non-PCP binding site that has been implicated in many dif-
ferent pathophysiological conditions including psychosis, drug addiction, retinal degeneration and can-
cer. Based on the structure of fenpropimorph, a high affinity (Ki = 0.005 nM)1 sigma-1 receptor ligand
and strong inhibitor of the yeast sterol isomerase (ERG2), we previously deduced a basic sigma-1 receptor
pharmacophore or chemical backbone composed of a phenyl ring attached to a di-substituted nitrogen
atom via an alkyl chain.2 Here, we report the design and synthesis of various N,N-dialkyl or N-alkyl-N-
aralkyl derivatives based on this pharmacophore as well as their binding affinities to the sigma-1
receptor. We introduce three high affinity sigma-1 receptor compounds, N,N-dibutyl-3-(4-fluoro-
phenyl)propylamine (9), N,N-dibutyl-3-(4-nitrophenyl)propylamine (3), and N-propyl-N0-4-aminophenyl-
ethyl-3-(4-nitrophenyl)propylamine (20) with Ki values of 17.7 nM, 0.36 nM, and 6 nM, respectively. In
addition to sigma receptor affinity, we show through cytotoxicity assays that growth inhibition of various
tumor cell lines occurs with our high affinity N,N-dialkyl or N-alkyl-N-aralkyl derivatives.
Received 19 November 2009
Revised 23 April 2010
Accepted 25 April 2010
Available online 29 April 2010
Keywords:
Sigma receptor
Cancer
Tumor cell lines
Fenpropimorphy
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
receptor. Sigma-1 receptor function, however, has proven to be rel-
atively unclear because unlike the yeast or mammalian sterol
To date, two subtypes of the sigma receptor have been identi-
fied, the sigma-1 receptor and the sigma-2 receptor, which are dis-
tinguishable by their pharmacology, function, and molecular
weight. The sigma-1 receptor was first cloned from guinea pig liver
in 19963 and subsequently from other sources including human
placental choriocarcinoma cells,4 human brain,5 rat brain,6,7 and
mouse brain.8 The sigma-2 receptor, however, has yet to be cloned.
For over a decade, the sigma-1 receptor, has been known to exclu-
sively share significant amino acid sequence similarity with the
yeast sterol C8–C7 isomerase (ERG2 protein) as demonstrated by
the Glossman group in 1996.3 A fundamental enzyme in ergosterol
biosynthesis, which is the fungal counterpart of cholesterol in
mammalians, the ERG2 protein is 30.3% identical and 66.4% similar
to the sigma-1 receptor.3 These amino acid sequence similarities
isomerases, it lacks sterol isomerase activity.3 Recently, however,
the sigma-1 receptor was discovered to possess chaperone activity
as a Ca2+-sensitive and ligand-operated chaperone complexed with
another chaperone protein known as BiP.9 The C-terminus of the
sigma-1 receptor has also been implicated in the activation of IP3
receptors by inducing its dissociation from ankyrin B 220.10 In
Chinese Hamster Ovary (CHO-K1) cells, ligand-activated sigma-1
receptors target to focal adhesion contacts (FAC) and colocalize
with Talin and Kv1.4 potassium channels.11 We have purified the
recombinant guinea pig sigma-1 receptor to homogeneity12 and
shown that ligand binding sites on the sigma-1 receptors include
regions of the receptor that have been identified as steroid binding
domains (SBDLI and SBDLII) in the yeast sterol isomerase.13,14
In 1997, the Glossman lab investigated the ability of sterol
C8-C7 isomerase inhibitors to compete with (+)-[3H]-pentazocine
labeled sigma-1 receptors.1 Interestingly, they discovered that of
all the inhibitors tested, an agricultural fungicide, fenpropimorph,
bound with exceptionally high affinity to the guinea pig hepatic
(Ki 0.011 nM),1 cerebral (Ki 0.005 nM),1 and yeast-expressed sig-
ma-1 receptor (Ki 0.08 nM).1 Other pharmacological studies have
indicated that this receptor also binds a wide range of compounds
including opiates, antipsychotics, antidepressants, anti-histamines,
PCP-like compounds, beta-adrenergic receptor ligands, serotoner-
gic compounds, cocaine and cocaine analogs, neurosteroids, and
neuropeptides. Previously, we observed that these drugs have a
were thought to provide
a pharmacological and structural
correlation between the yeast sterol isomerase and the sigma-1
Abbreviations: ERG2, yeast sterol C8–C7 isomerase; CHO-K1, Chinese hamster
ovary cells; FAC, focal adhesion contacts; SBDLI, steroid binding domain-like I;
SBDLII, steroid binding domain-like II; BD1047, N-[2-(3,4-dichlorophenyl)ethyl]-N-
methyl-2-(dimethylamino)ethylamine; BD1063, 1-[2-(3,4-dichlorophenyl)ethyl-4-
methylpiperazine; DTG, ditolyl guanidine.
* Corresponding author. Address: Department of Pharmacology, University of
Wisconsin Medical School, 1300 University Ave., Madison, WI 53706, United States.
Tel.: +1 608 263 5382; fax: +1 608 262 1257.
E-mail address: aeruoho@wisc.edu (A.E. Ruoho).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.04.078

4398
A. R. Hajipour et al. / Bioorg. Med. Chem. 18 (2010) 4397–4404
common pharmacophore, which can also be generated from the
chemical structure of fenpropimorph.2 This chemical backbone is
composed of a phenyl ring attached to a di-substituted nitrogen
atom by an alkyl chain. Further examination led to the observation
that similar chemical backbones could be derived from other high
affinity sigma-1 ligands such as haloperidol and cocaine,2 resulting
in a common N,N-dialkyl or N-alkyl-N-aralkyl product. A number of
other sigma-1 ligands reported in the literature support this struc-
tural pharmacophore such as N-[2-(3,4-dichlorophenyl)ethyl]-
N-methyl-2-(dimethylamino)ethylamine (BD1047),15 1-[2-(3,4-
dichlorophenyl)ethyl-4-methylpiperazine (BD1063),15 metham-
phetamine,16 FN/C-1 to FN/C-4,17 (piperazin-2-yl)methanol deriva-
tives,18 1-aralkyl-4-benzylpiperazine derivatives,19 dimemorfan,20
(R/S)-4-(dimethylamino)-2-(napthalen-2-yl)butan-2-ol,21 1-methoxy-
carbonyl-1-phenyl-2-cyclopropylmethylamines22 and [11C]SA4503.23
One of the striking features of the sigma receptor is the discov-
ery that sigma-1 and sigma-2 receptors are overexpressed in many
human and non-human tumors24,25 such as rodent C6 glioma,
rodent N1E-115 neuroblastoma, human t47D breast ductal carci-
noma, human MCF7 breast adenocarcinoma, human NCI-H727
lung carcinoid, human A375 melanoma, rodent PC12 pheochromo-
cytoma cells,26 and NCB-20 cells.27,28 Consequently, the pharmaco-
logical study of small molecule sigma receptor ligands for potential
clinical treatment and imaging applications has been a developing
area of cancer research. In 2004, for example, it was demonstrated
that the small molecule sigma-1 receptor ligands rimcazole, BD-
1047, and BD-1063, inhibited tumor cell survival while SKF-
10047 and pentazocine repressed these effects. In another study,25
the sigma-1 receptor was found to be expressed in most neoplastic
breast epithelial cells and cell lines. Furthermore, the sigma recep-
tor ligands haloperidol and progesterone were found to inhibit
growth of several breast cancer cell lines in a dose-dependent
manner.25 Sigma-1 antagonists have previously been shown to ini-
tiate tumor-selective and caspase-dependent apoptosis, which
could be rescued by sigma-1 agonists.29 In addition, the sigma-2
receptor is emerging as an important player in tumor imaging
efforts since sigma-2 receptors are highly concentrated in tumor
cells.30 Sigma ligands inhibit proliferation and induce apoptosis
in mammary and colon carcinoma cell lines,31–33 which in some in-
stances are attributed to their actions on the sigma-2 receptor.33,34
It has also been shown that sigma-2 receptor activation by selec-
tive and non-selective ligands triggers cell death in various tumors
through a pathway involving reactive oxygen species and lyso-
somal membrane leakage.35 In addition to the sigma-2 selective
compound, ibogaine, several high affinity sigma-2 ligands have
been synthesized and generally contain N-alkylated piperazine or
piperidine rings.36–40
Currently, we report the design, synthesis, and evaluation of the
relative affinities of several N,N-dialkyl or N-alkyl-N-aralkyl com-
pounds to the sigma-1 receptor by competition assays against
(+)-[3H]-pentazocine and to the sigma-2 receptor using [3H]-ditolyl
guanidine ([3H]-DTG).14 In addition we test our high affinity
N,N-dialkyl or N-alkyl-N-aralkyl derivatives in cytotoxicity assays
for their ability to inhibit the growth of various tumor cell lines.
2. Results and discussion
3-(4-Nitrophenyl)propylbromide was prepared by reaction of
3-propylbromide with HNO3 in the presence of P2O5/silica gel un-
der solvent-free conditions.36 The N,N-dialkyl derivatives 1–3 were
prepared by reaction 2-(4-nitrophenyl)propylbromide or 3-(4-
nitrophenyl)propylbromideref with amines as demonstrated in
Scheme 1. Amides 4–8 were synthesized employing 4-flurophenyl-
propionic acid and appropriate amines in the presence of DCC and
the isolated amide without further purification were reduced with
LiAlH4 in THF to the corresponding amines 9–13 in excellent yields
(Scheme 2). As shown in Scheme 3 compounds 11 and 12 were re-
duced to corresponding amines 14 and 15 using H2/Pd–C in meth-
anol in quantitative yields and then the isolated amine 14 and 15
were converted to compounds 16–17 in high yields. Compounds
(CH2)n-Br
NHR1R2
Et3N/ether
(CH2)n-NR1R2
1, n = 1, R1 = H, R2 = n-Propyl
, n = 2, R1 = H, R2 = n-Propy
2
3, n = 3, R1 = R2 = n-Butyl
O2
N
O2N
Scheme 1.
O
O
1. DCC
NR1R2
OH
2. HNR1R2
F
F
4, R1 = R2 = n-Butyl
5, R1 = R2 = n-Octyl
6, R1 = n-Propyl, R2 = (4-nitrophenyl)methyl
7, R1 = n-Propyl, R2 = (4-nitrophenyl)ethyle
8, R1 =H, R2 = n-Propyl
NR1R2
LiAlH4/THF
9, R1 = R2 = n-Butyl
1
, R = R2 = n-Octyl
10
F
11, R1 = n-Propyl R2 = (4-nitrophenyl)methyl
12, R1 = n-Propyl R2 = (4-nitrophenyl)ethyle
13, R1 =H, R2 = n-Propyl
Scheme 2.

A. R. Hajipour et al. / Bioorg. Med. Chem. 18 (2010) 4397–4404
4399
-
(CH2)3
(CH2)3
Me4NICl2
(CH2)n
(CH2)n
H2/Pd-c
N
Solvent-free
N
CH3OH
11 or 12
F
F
NH2
14 or 15
NO2
(CH)3
I
(CH2)n
(CH)3
I
NaNO2/HCl
(CH2)n
N
N
F
NH2
F
16 or 17
N3
18 or 19
11,14, 16, 18, n = 1, 12, 15, 17, 19, n = 2
N
O2
N
20
H2
N
Scheme 3.
20 was synthesis by reaction of 3-(4-nitrophenyl)propylbromide
and N-propyl-N-4-aminophenylethylmine in the presence of Et3N
in Et2O in 94% yields.
adding 5 mM Haloperidol as a control condition. Curve fitting using
‘GraphPad Prism version 4.0C’ indicated that all the compounds fit to
a single binding site for the sigma-1 receptor with regression values
(R2) between 0.94 and 0.99 (Table 1). Selectivity ratios between the
sigma-1 receptor and the sigma-2 receptor were also calculated to
determine relative specificity and are summarized in Table 1.
With the exception of compound 16, the compounds which
were synthesized based on our proposed sigma-1 receptor ligand
pharmacophore2 generally had a higher affinity and specificity
for the sigma-1 receptor than for the sigma-2 receptor. Specific
binding of compounds 4, 5, and 6, either could not be detected to
bind to the sigma-1 receptor (Ki >100,000 nM) or possessed very
low affinity, presumably because the amide group present in these
compounds traps the nitrogen’s lone pair, which is needed for opti-
mal sigma receptor binding as previously reported by the Glennon
group.41 The importance of the nitrogen’s lone pair is further illus-
trated by comparing the Ki values of compounds 4 (32,063 nM)
with 9 (17.7 nM) and 5 (53,579 nM) with 10 (665.3 nM), showing
a 2000-fold and 100-fold affinity difference, respectively. In con-
trast, compounds 9, 3, and 20 were found to be exceptionally high
affinity compounds for the sigma-1 receptor with Ki values of
17.7 nM, 0.3 nM, and 6 nM, respectively. The nitro substituent on
the phenyl ring of compound 20 likely enhances binding to the sig-
ma-1 receptor due to its greater electron withdrawing character13
as demonstrated by the 400-fold difference in affinity between com-
pound 20(6 nM) and N-propyl-N-(4-aminophenyl-ethyl)-3-(4-fluo-
rophenyl)propylamine (compound 15, sigma-1 Ki = 2590 nM),42
which contains a fluorine atom replacing the nitro group. In a similar
manner, addition of a nitro group on the phenylring of the high affin-
ity compound 9 (sigma-1 Ki = 17.7 nM), further improves binding to
the sigma-1 receptor as demonstrated by compound 3 (sigma-1
Ki = 0.3 nM; sigma-1 vs sigma-2 selectivity is 1347-fold). Interest-
ingly, 6 clearly demonstrates that compounds with amide groups,
even in the presence of a nitro substituent on the phenyl ring,
effectively prevented sigma-1 receptor binding, providing further
The synthesized N,N-dialkyl (1–3,) or N-alkyl-N-aralkyl com-
pounds (compounds 4–18) were tested for their binding affinities
to sigma-1 receptors in guinea pig liver membranes, and to sigma-
2 receptors in rat liver membranes, as summarized in Table 1. The
binding affinities of these compounds were determined by compet-
itive displacement of [3H]-(+)-pentazocine (10 nM) and showed
high affinity and selectivity to the sigma-1 receptor. For determina-
tion of binding to the sigma-2 receptor, 3 nM [3H]-ditolyl guanidine
(DTG) was utilized in the presence of non-radioactive (+)-pentazo-
cine (100 nM), which masked the sigma-1 receptor population from
binding to [3H]-DTG. Non-specific binding was determined by
Table 1
Binding affinities of N,N0-dialkyl and N-alkyl-N0-aralkyl derivatives
Ligand
Sigma 1 Ki values (nM)
( SEM, n = 3), R2 value
Sigma 2 Ki values (nM)
( SEM, n = 3), R2 value
Ratio r2/r1
2
3
4
5
6
2254 ( 1.2, 0.95)
0.3 ( 1.29, 0.96)
32,063 ( 2.16, 0.94)
53,617
404 ( 1.21, 0.97)
23.8
1347
3.94
ndb
126,333
a
53,579 ( 3.70, 0.98)
a
236,000
ndb
9
17.7 ( 1.07, 0.99)
665.3 ( 1.13, 0.98)
91 ( 1.16, 0.97)
164 ( 1.16, 0.97)
2590 ( 0.63, 0.96)c
393,000
89,000 ( 3.94, 0.96)
7240 ( 2.03, 0.98)c
6 ( 1.21, 0.96)
685 ( 1.17, 0.98)
1653 ( 1.69, 0.96)
230 ( 1.75, 0.90)
2150 ( 1.79, 0.93)
120 ( 0.045, 0.91)c
133,200
38.7
2.49
2.53
13.11
10
13
14
15c
16
18
19c
20
0.046c
0.3389
570.8
0.178c
13.93
>500,00,000
1290 ( 3.4, 0.96)c
83.6 ( 1.68, 0.85)
a
b
c
Does not compete with [3H]-pentazocine or [3H]-DTG.
nd—not determined.
Fontanilla, D. et al. Biochemistry 2008, 47, 7205–7217.

4400
A. R. Hajipour et al. / Bioorg. Med. Chem. 18 (2010) 4397–4404
Figure 1. Growth inhibition of tumor cell lines. Compounds 3, 9, 14, 15, 19, and 20 were used in cytotoxicity assays to measure their ability to inhibit growth of various tumor
cells. IC50 values of the compounds are reported in tabular form. Also depicted is the graphical representation of 1/IC50 of the compounds plotted against the various tumor
cell lines.
evidence that the nitrogen’s lone pair is vital for optimal sigma-1
receptor binding.2,41,42
3 has a higher affinity and higher selectively for the sigma-1 recep-
tor than compound 9 (Table 1), and robustly inhibits growth in
MB-MDA231, MCF7, Du145, and NCI-H460 cell lines in addition
to SKOV-3 cells, the only cell line whose growth is inhibited by
compound 9. Compounds 15 and 20 are also structurally similar
to one another, but do not follow the same trend as compounds
9 and 3. Compound 15 has a higher binding affinity for the sig-
ma-2 receptor than the sigma-1 receptor (Table 1) and is cytotoxic
to more tumor cell lines than the structurally similar compound
20, which has high affinity for sigma-1 (Fig. 1, Table 1). As previ-
ously mentioned, sigma-2 receptor ligands have been demon-
strated to inhibit cell proliferation in mammary and colon
carcinoma cells33,34 and furthermore, cell death initiated by sig-
ma-2 ligands seems to occur through pathways involving reactive
oxygen species and lysosomal membrane leakage.35 Interestingly,
the observations reported here suggests that the cytotoxicity pro-
duced by compound 15 might be due to actions on sigma-2 recep-
tors, while the reponses produced by compound 3 seem to occur
through sigma-1 receptors. Although compounds 15 and 3 have a
substantial amount of selectivity for their respective sigma recep-
tor subtypes, it is unclear at this time whether these cytotoxicity
responses are due to simultaneous actions on both sigma-1 and
sigma-2 receptors.
Since sigma-1 and sigma-2 receptors are overexpressed in
numerous tumor cell lines, which include breast cancer, lung car-
cinoma, renal carcinoma, colon carcinoma, sarcoma, brain tumors,
melanoma, glioblastoma, neuroblastoma, and prostate cancer, we
tested the ability of our compounds to inhibit the growth of vari-
ous tumor cell lines. Cytotoxicity assays revealed that our N,N-dial-
kyl or N-alkyl-N-aralkyl derivatives are cytotoxic against a number
of cancer cells lines (Fig. 1) including breast, lung, prostate, ovar-
ian, colorectal, and CNS, indicating their utility as potential anti-
cancer or diagnostic agents. Specifically, we tested compounds 9,
3, 14, 15, 19, and 20 (Schemes 2 and 3), based on their high
affinities and specificities for the sigma-1 receptor. Except for com-
pound 14, the selected N,N-dialkyl or N-alkyl-N-aralkyl derivatives
could inhibit cell growth in vitro (Fig. 1). Interestingly, as illus-
trated in Figure 1, compound 19 was non-specifically cytotoxic in
almost all the cell lines tested whereas compound 14 lacked any
cytotoxic properties. Furthermore, only specific cell lines were sus-
ceptible to compounds 3, 9, 15, and 20 (Fig. 1). Cell lines that
seemed to have the greatest susceptibility to the N,N-dialkyl or
N-alkyl-N-aralkyl derivatives were NCI-H460 (human lung adeno-
carcinoma), SKOV-3 (human ovarian adenocarcinoma), MCF7 (hu-
man breast adenocarcinoma), and MB-MDA-231 (human breast
adenocarcinoma). Correlation between the cytotoxicity growth
inhibition levels and the sigma receptor binding affinities is less
clear due to unknown involvement of the sigma-1 versus sigma-
2 receptor with regard to these novel compounds. Though struc-
turally similar except for a nitro substituent (Scheme 1), compound
In conclusion, the findings from this study show thatN,N-dialkyl
and N-alkyl-N-aralkyl fenpropimorph derivatives are sigma ligands
that exhibit increased sigma-1 receptor affinity with the addition
of electron withdrawing nitro substituents. Alternatively, sigma-1
receptor affinity is abolished when an amide group is introduced
into the compound structure. Furthermore, these fenpropimorph

A. R. Hajipour et al. / Bioorg. Med. Chem. 18 (2010) 4397–4404
4401
derivatives exhibit specific cytotoxic activity against numerous tu-
mor cell lines, demonstrating their potential use as clinical anti-
cancer, imaging, or diagnostic agents.
(t, 6H, J = 7.8). Anal. Calcd for C16H26N2O2: C, 69.03; H, 9.41; N,
10.06. Found: C, 69.80; H, 9.20; N, 10.10.
3.4. General procedure for preparation of amides (4–8)
3. Methods
A mixture of DCC (1 mmol, 2.1 g) and 4-flurophenylpropionic
acid (0.17 g, 1 mmol) was ground with a pestle in a mortar for
30 s and then the amine (1 mmol) was added to the reaction mix-
ture. The reaction was ground with a pestle until TLC showed no
remaining 4-flurophenylpropionic acid (n-hexane/EtOAc, 75:25)
(20 min). Then to the reaction mixture was added a mixture of
ether (20 mL) and H2O (5 mL). The etheral layer was washed with
saturated NaHCO3, HCl 5% and water and the organic phase dried
(MgSO4), and evaporated by a rotary evaporator to give a residue.
The residue was used without further purification for the next step.
3.1. Chemistry
Yields refer to isolated pure products after column chromatog-
raphy. The products were characterized by their spectral (IR, 1H
and 13C NMR and CHN Analysis). All 1H NMR spectra were recorded
at 300 MHz in CDCl3 relative to TMS (0.00 ppm) and IR spectra were
recorded on a Shimadzu 435 IR spectrometer. Melting points were
determined with a Thomas-Hoover capillary melting point appara-
tus and are reported uncorrected. Chemicals were obtained from Al-
drich Chemical Co. and utilized without further purification.
3.4.1. N.N-Dibutyl-3-(4-fluorophenyl)propionamide (4)
Yield: (0.26 g, 93%), mp 162–164 °C. IR (KBr): 1658 cmꢁ1 1H
.
3.2. Preparation of 3-(4-nitrophenyl)-propylbromine
NMR (CDCl3): d 7.18 (m, 2H), 6.98 (m, 2H), 3.17 (t, 4H, J = 7.8),
2.8 (t, 2H, J = 7.8), 2.59 (t, 2H, J = 7.8), 1.1–4-1.0 (m, 8H), 0.96
(t, 6H, J = 7.8). Anal. Calcd for C17H26FNO: C, 73.08; H, 9.38; N,
5.01. Actual: C, 72.90; H, 9.40; N, 5.20.
2 g of P2O5/silica gel (65% w/w) (10 mmol)36 and 3-phenylpro-
pylbromine (10 mmol, 1.98 g) was ground for 30 seconds, and then
5 ml of HNO3 65% was added. The mixture was ground with a
pestle at rt until a deep-yellow color appeared (2 min). When
TLC (n-hexane/EtOAc 90:10) showed complete disappearance of
3-phenylpropylbromide (10 min), ether (100 ml) was added to
the reaction mixture and the solid was separated through a short
pad of silica gel and washed with ether (3 ꢀ 20 ml). The filtrate
was washed with 10% NaHCO3 (3x20 ml) and dried (MgSO4). The
solvent was evaporated under reduced pressure and the residue
was purified by short column chromatography (n-hexane/EtOAc,
90:10). 3-(4-Nitrophenyl)-propylbromide was obtained (8.3 mmol,
2.02 g 83%) as a yellow oil. 1H NMR (CDCl3): d 8.2 (d, J = 6.3), 7.38
(d, 2H, 6.3), 3.4 (t, 2H, J = 7.8), 2.90 (m, 2H, J = 7.8), 2.2 (m, 2H).
Anal. Calcd for C9H10BrNO2: C, 44.29: H, 4.13; N, 5.74. Found: C,
44.50; H, 48.30; N, 5.80.
3.4.2. N,N-Dioctyl-3-(4-fluorophenyl)propionamide (5)
Yield: (0.37 g, 95%), mp 190–193 °C. IR (KBr): 1663 cmꢁ1 1H
.
NMR (CDCl3): d 7.18 (m, 2H), 6.97 (m, 2H), 3.18 (t, 2H, J = 7.8),
2.80 (t, 2H, J = 7.8), 2.60 (t, 2H, J = 7.8), 1.50 (m, 4H), 1.26 (m,
22H), 0.90 (t, 6H, J = 7.8). Anal. Calcd for C25H42FNO: C, 76.68; H,
10.81; N, 3.58. Actual: C, 76.40; H, 10.90; N, 3.40.
3.4.3. 3-(4-Fluorophenyl)-N-(3-nitrobenzyl)-N-propylpropana-
mide (6)
Yield: (0.31 g, 90%), Yellow oil. IR (KBr): 1661 cmꢁ1 1H NMR
.
(CDCl3): d 8.18 (d, 2H, J = 6.8), 7.46 (d, 2H, J = 6.8), 7.15 (m, 2H),
6.94 (m, 2H), 4.85 (s, 2H), 3.22 (t, 2H, J = 7.8), 2.79 (t, 2H, J = 7.8),
2.30 (m, 2H), 1.42 (m, 2H), 0.96 (t, 3H, J = 7.8). Anal. Calcd for
C19H21FN2O3: C, 66.26; H, 6.15; N, 8.13. Actual: C, 66.10; H, 6.30;
N, 8.00.
3.3. General procedure for preparation of amines 1–3
To
a stirring mixture of 3-(4-nitrophenyl)ethylbromide or
3.4.4. 3-(4-Fluorophenyl)-N-(3-nitrophenethyl)-N-propylpropana-
4-nitrobenzyl bromide (1 mmol), Et3N (1.1 mmol, 0.11 g) in Et2O
(10 ml) was added the appropriate amines (1.0 mmol). The reac-
tion mixture was stirred at room temperature for 10 h. After filtra-
tion, the solvent was removed to give a yellow residue. The crude
products were purified by column chromatography (silica gel,
toluene/Et2NH, 20:1) to afford pure product.
mide (7)
Yield: (0.32 g, 88%), Yellow oil. IR (KBr): 1663 cmꢁ1 1H NMR
.
(CDCl3): d 8.18 (d, 2H, J = 6.8), 7.46 (d, 2H, J = 6.8), 7.15 (m, 2H),
6.94 (m, 2H), 3.22 (t, 2H, J = 7.8), 2.82 (m, 2H), 2.35 (m, 2H), 1.42
(m, 2H), 0.96 (t, 3H, J = 7.8). Anal. Calcd for C20H23FN2O3: C,
67.02; H, 6.47; N, 7.82. Actual: C, 67.13; H, 6.30; N, 7.70.
3.3.1. N-(4-Nitrobenzyl)propan-1-amine (1)
3.4.5. 3-(4-Fluorophenyl)-N-propylpropanamide (8)
Pale yellow oil, bp 120–122 °C (15 mm Hg). Yield 80% (0.15 g,
Yield: (0.19 g, 90%), Yellow oil. IR (KBr): 1660 cmꢁ1 1H NMR
.
0.80 mmol). IR (KBr): 3268 cmꢁ1 1H NMR (CDCl3): d 8.14 (d, 2H,
.
(CDCl3): d 7.28 (s, 1H,), 7.18 (m, 2H), 6.94 (m, 2H), 3.60 (t, 2H,
J = 7.8), 2.78 (t, 2H, J = 7.8), 2.39 (t, 2H, J = 7.8), 1.45 (m, 2H), 0.96
(t, 3H, J = 7.8). Anal. Calcd for C12H16FNO: C, 68.88; H, 7.71; N,
6.69. Actual: C, 68.90; H, 7.50; N, 6.80.
J = 6.8), 7.38 (d, 2H, J = 6.8), 3.99 (s, 2H), 2.60 (t, 2H, J = 7.8), 1.55
(m, 2H), 1.40 (s, 1H, NH), 0.96 (t, 3H, J = 7.8). Anal. Calcd for
C10H14N2O2: C, 61.84; H, 7.27; N, 14.42. Actual: C, 61.50; H, 7.40;
N, 14.20.
3.5. General procedure for reduction of amides (4–8) to the corre-
sponding amines (9–13)
3.3.2. N-propyl-N-3-(4-nitrophenyl)-ethylamine (2)
Mp 142–144 °C. Yield 86% (0.18 g, 0.86 mmol). IR (KBr):
3261 cmꢁ1 1H NMR (CDCl3): d 8.18 (d, 2H, J = 6.8), 7.58 (d, 2H,
.
In a double-necked round bottomed flask equipped with sep-
tum and condenser, a solution of amides (1 mmol) in anhydrous
THF (5 ml) was added via a syringe dropwise to a stirred solution
of LiAlH4 (0.74 g, 2 mmol) in anhydrous THF (5 ml) under argon.
TLC indicated the reaction to be almost completed after 15 min
at room temperature. The reaction mixture was driven to comple-
tion by brief refluxing (15 min) and when it was cooled to rt, it was
diluted by adding 5 ml THF. The excess LiAlH4 was destroyed by
dropwise addition of water 1 ml. The reaction mixture was stirred
J = 6.8), 2.60 (t, 2H, J = 7.8), 2.46 (m, 2H), 2.16 (s, 1H, NH), 1.86
(m, 2H), 1.45 (m, 2H), 0.96 (t, 3H, J = 7.8). Anal. Calcd for
C11H16N2O2: C, 63.44; H, 7.74; N, 13.45. Actual: C, 63.30; H, 7.90;
N, 13.20.
3.3.3. N-Dibutyl-3-(4-nitrophenyl)ethylamine (3)
1H NMR (CDCl3): d 8.16 (d, 2H), 7.24 (d, 2H), 3.04 (t, 2H, J = 7.8),
2.8 (m, 4H), 1.55 (m, 2H), 1.72 (m, 2H), 1.2–0.98 (m, 8H), 0.80

4402
A. R. Hajipour et al. / Bioorg. Med. Chem. 18 (2010) 4397–4404
for 30 min at rt and then the solids were removed by filtration. The
filtrate was dried (MgSO4), and the solvent evaporated by a rotary
evaporator to give pure products as yellow oils in quantitative
yield.
3.7. General method for iodination of amine 14 and 15 to the
corresponding amino iodoamino derivative 16 and 17
A mixture of amines 14 or 15 (0.5 mmol) and tetramethylam-
monium dichloroiodate (0.5 mmol, 0.14 g)43 in a mortar was
ground with a pestle to produce a homogenous paste and the mix-
ture was left at room temperature until TLC (toluene/Et2NH, 20:1)
showed complete disappearance of amines. To the brown solid was
added 5 ml sodium bisulfate (5%) and the reaction mixture was ex-
tracted with dichloromethane (3 ꢀ 5 ml). The combined extracts
were dried with MgSO4. Evaporation of the solvent gave the corre-
sponding iodo derivatives (16 or 17). The product was purified by
column chromatography (silica gel, toluene/Et2NH, 20:1).
3.5.1. N.N-Dibutyl-3-(4-fluorophenyl)propylamine (9)
1H NMR (CDCl3): d 7.18 (m, 2H), 6.92 (m, 2H), 3.03 (t, 2H,
J = 7.8), 2.40 (m, 6H), 1.72 (m, 2H), 1.33 (m, 8H), 0.95 (t, 6H,
J = 7.8). Anal. Calcd for C17H28FN: C, 76.93; H, 10.63; N, 5.28. Ac-
tual: C, 77.10; H, 10.70; N, 5.20.
3.5.2. N,N-Dioctyl-3-(4-fluorophenyl)propylamine (10)
1H NMR (CDCl3): d 7.18 (m, 2H), 3.64 (t, 2H, J = 7.8), 3.13 (t, 2H,
J = 7.8), 2.67 (t, 2H, J = 7.8), 2.40 (m, 4H), 1.85 (m, 2H), 1.50–1.26
(m, 22H), 0.90 (t, 6H, J = 7.8). Anal. Calcd for C25H44N: C, 79.52;
H, 11.74; N, 3.71. Actual: C, 79.60; H, 11.50; N, 3.80.
3.7.1. N-Propyl-N-(4-amino-3-iodo-benzyl)-3-(4-fluorophenyl)-
propylamine (16)
Oil, 84% yield. IR (KBr): 3245 cmꢁ1 1H NMR (CDCl3): d 8.18 (m,
.
3.5.3. 3-(4-Fluorophenyl)-N-(4-nitrobenzyl)-N-propylpropan-1-
amine (11)
2H, J = 6.8), 7.98 (m, 2H, J = 6.8), 6.70–6.45 (m, 3H, J = 6.8), 4.00 (s,
2H), 3.45 (s, 2H, NH2), 2.78 (t, 2H, J = 7.8), 2.45 (m, 4H), 1.66 (m,
2H), 1.30 (m, 2H), 0.96 (t, 2H, J = 7.8). Anal. Calcd for C19H24IFN2:
C, 53.53; H, 5.67; N, 6.57. Actual: C, 53.40; H, 6.70; N, 6.40.
IR (KBr): 3258 cmꢁ1. 1H NMR (CDCl3): d 8.20 (d, 2H, J = 6.8), 7.58
(d, 2H, J = 6.8), d 7.18 (m, 2H), 6.98 (m, 2H), 3.64 (s, 2), 2.62 (t, 2H,
J = 7.8), 2.42 (m, 4H), 1.80 (m, 2H), 1.42 (m, 2H), 0.96 (t, 3H, J = 7.8).
Anal. Calcd for C19H23FN2O2: C, 69.07; H, 7.02; N, 8.48. Actual: C,
69.21; H, 7.30; N, 8.30.
3.7.2. N-Propyl-N-(4-amino-3-iodo-phenylethyl)-3-(4-fluoro-
phenyl)propylamine (17)
Oil, 81% yield. IR (KBr): 3245 cmꢁ1. 1H NMR (CDCl3): d 6.99–6.60
(m, 7H), 3.22 (s, 2H, NH2), 2.62 (t, 6H, J = 7.8), 2.40 (m, 4H), 1.80 (m,
2H), 1.38 (m, 2H), 1.01 (t, 2H, J = 7.8). Anal. Calcd for C20H26IFN2: C,
54.55; H, 5.95; N, 6.36. Actual: C, 54.40; H, 6.10; N, 6.20.
3.5.4. 3-(4-Fluorophenyl)-N-(4-nitrophenethyl)-N-propylpropan-
1-amine (12)
1H NMR (CDCl3): d 8.18 (d, 2H, J = 6.8), 7.55 (d, 2H, J = 6.8), d
7.10 (m, 2H), 6.95 (m, 2H), 2.60 (t, 6H, J = 7.8), 2.42 (m, 4H), 1.80
(m, 2H), 1.42 (m, 2H), 0.96 (t, 3H, J = 7.8). Anal. Calcd for
C20H25FN2O2: C, 69.74; H, 7.32; N, 8.13. Actual: C, 69.60; H, 7.50;
N, 8.10.
3.8. General procedure for conversion of amino iodo derivative
16–17 to the corresponding azido iodo derivative 18–19
To a cold mixture (0 °C) of 16 or 17 (0.1 mmol) in H2O (2 ml),
concentrated HCl (0.4 ml) was added an aqueous solution of
NaNO2 (0.30 mmol, 21 mg, in 0.5 ml H20) in 5 min in a round bot-
tomed flask. The reaction mixture stirred at room temperature for
30 min. Then to the reaction mixture at rt and darkness was added
an aqueous solution of NaN3 (0.36 mmol, 23 mg, in 0.5 ml H20)
dropwise. The reaction mixture was stirred at rt and darkness for
30 min and then extracted with EtOAc (3 ꢀ 3 ml). The combined
EtOAc solution was dried with MgSO4 and the solvent was evapo-
rated with rotary evaporator to afford orange oil. The crude prod-
ucts were purified by column chromatography (silica gel, first
toluene/Et2NH, 20:1 and then toluene/Et2NH, 4:1) to give the prod-
uct as a yellow liquid.
3.5.5. 3-(4-Fluorophenyl)-N-propylpropan-1-amine (13)
IR (KBr): 3323 cmꢁ1 1H NMR (CDCl3): d 7.18 (m, 2H), 6.94 (m,
.
2H), 2.60 (m, 6H), 1.80 (m, 3H), 1.45 (m, 2H), 0.96 (t, 3H, J = 7.8).
Anal. Calcd for C12H18FN: C, 73.81; H, 9.29; N, 7.17. Actual: C,
73.90; H, 9.50; N, 7.10.
3.6. General procedure for reduction of nitro groups of compounds
11 and 12 to the corresponding amino group 14 and 15
A mixture of nitro compounds (1 mmol) and 10 mg of Pd/C
(10%) in methanol (10 ml) was reduced with H2 at normal pres-
sure. The mixture was stirred at room temperature over night.
After filtration, solvent was removed to give a yellow residue.
The crude products were purified by column chromatography
(silica gel, toluene/Et2NH, 20:1) to afford pure amine.
3.8.1. N-Propyl-N-(3-iodo-4-azido-benzyl)-3-(4-fluorophenyl)-
propylamine (18)
Oil, 98% yield. 1H NMR: d 6.99–6.80 (m, 7H), 4.10 (s, 2H, NH2),
2.78 (t, 2H, J = 7.8), 2.49 (m, 4H), 1.82 (m, 2H), 1.48 (m, 2H), 0.99
(t, 2H, J = 7.8). Anal. Calcd for C19H22IFN4: C, 50.45; H, 4.90; N,
12.39. Actual: C, 50.40; H, 5.00; N, 12.40.
3.6.1. N-Propyl-N-(4-amino-benzyl)-3-(4-fluorophenyl)propyl-
amine (14)
Yield: (0.28 g, 94%), Semisolid. IR (KBr): 3258, 1661 cmꢁ1 1H
.
NMR (CDCl3): d 8.14 (d, 2H, J = 6.8), 7.38 (d, 2H, J = 6.8), 6.96 (d,
2H, J = 6.8), 6.62 (d, 2H, J = 6.8), 4.00 (s, 2H), 3.60 (s, 2H, NH2),
2.78 (t, 4H, J = 7.8), 2.43 (m, 6H), 1.65 (m, 2H), 1.34 (m, 2H), 0.94
(t, 3H, J = 7.8). Anal. Calcd for C19H25FN2: C, 75.96; H, 8.39; N,
9.32. Actual: C, 75.80; H, 8.50; N, 9.10.
3.8.2. N-Propyl-N-(3-iodo-4-azido-phenyethyl)-3-(4-fluoro-
phenyl)propylamine (18)
Oil, 91% yield. 1H NMR (CDCl3): d 6.99–6.80 (m, 7H), 2.60 (t, 6H,
J = 7.8), 2.41 (m, 4H), 1.80 (m, 2H), 1.47 (m, 2H), 1.01 (t, 2H, J = 7.8).
Anal. Calcd for C20H24IFN4: C, 51.51; H, 5.19; N, 12.01. Actual: C,
51.40; H, 5.40; N, 12.10.
3.6.2. N-Propyl-N-(4-amino-phenylethyl)-3-(4-fluorophenyl)pro-
pylamine (15)
Yield: (0.29 g, 94%), yellow oil. IR (KBr): 3258, 1661 cmꢁ1
.
1H
3.8.3. Synthesis of N-propyl-N-4-aminophenylethyl-3-(4-nitro-
NMR (CDCl3): d 8.14 (d, 2H, J = 6.8), 7.38 (d, 2H, J = 6.8), 6.93 (d,
2H, J = 6.8), 6.65 (d, 2H, J = 6.8), 3.60 (s, 2H, NH2), 2.70 (t, 6H,
J = 7.8), 2.45 (m, 6H), 1.79 (m, 2H), 1.43 (m, 2H), 0.94 (t, 3H,
J = 7.8). Anal. Calcd for C20H27FN2O: C, 76.39; H, 8.65; N, 8.91.
Actual: C, 76.50; H, 8.80; N, 8.70.
phenyl)propylamine (20)
To
a stirring mixture of 3-(4-nitrophenyl)propylbromide
(1 mmol) , Et3N (1.1 mmol, 0.11 g) in Et2O (10 ml) was added N-pro-
pyl-N-4-aminophenylethylmine (3 mmol). The reaction mixture
was stirred at room temperature for 10 h. After filtration, solvent

A. R. Hajipour et al. / Bioorg. Med. Chem. 18 (2010) 4397–4404
4403
was removed to give a yellow residue. The crude products were puri-
fied by column chromatography (silica gel, toluene/Et2NH, 20:1) to
afford pure product. Semisolid. Yield 94% (0.32 g, 0.94 mmol). IR
(KBr): 3245 cmꢁ1. 1H NMR (CDCl3): d 8.14 (d, 2H, J = 6.8), 7.38 (d,
2H, J = 6.8), 6.93 (d, 2H, J = 6.8), 6.65 (d, 2H, J = 6.8), 3.60 (s, 2H,
NH2), 2.80 (t, 6H, J = 7.8), 2.49 (m, 4H), 1.98 (m, 2H), 1.63 (m, 2H),
1.10 (t, 3H, J = 7.8). Anal. Calcd for C20H27N3O2: C, 70.35; H, 7.97;
N, 12.31. Actual: C, 70.40; H, 8.10; N, 12.40.
ported in part by the Center of Excellence in Sensor and Green
Chemistry Research to A.R.H. and by NIH Grant R01 MH065503
to A.E.R.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.04.078.
3.9. Preparation of rat liver and guinea pig liver membranes
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Acknowledgements
Member of the Molecular and Cellular Pharmacology Graduate
Program (D.F. and U.B.C). Supported in part by the MCP training
grant from the National Institute of Health (NIH) #T32 GM08688
and by the National Institutes of Health (NIH) under Ruth L. Kirsch-
stein National Research Service Award (#F31 DA022932) from the
National Institute on Drug Abuse (NIDA) to D.F. This work was sup-

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A. R. Hajipour et al. / Bioorg. Med. Chem. 18 (2010) 4397–4404
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