New adamantane phenylalkylamines with σ-receptor binding affinity and anticancer activity, associated with putative antagonism of neuropathic pain

Article abstract of DOI:10.1021/jm3013008

The synthesis of the adamantane phenylalkylamines 2a-d, 3a-c, and 4a-e is described. These compounds exhibited significant antiproliferative activity, in vitro, against eight cancer cell lines tested. The σ₁, σ₂, and sodium channel binding affinities of compounds 2a, 3a, 4a, and 4c-e were investigated. The most interesting analogue, 4a, exhibited significant in vivo anticancer profile on pancreas, prostate, leukemia, and ovarian cancer cell line xenografts together with apoptosis and caspase-3 activation. Inhibition of the cancer cells cycle at the sub-G1 level was also obtained with 4a. Finally, encouraging results were observed with 4a in vivo on mice, suggesting putative antimetastatic and analgesic activities of this compound.

Full text of DOI:10.1021/jm3013008

Article
pubs.acs.org/jmc
New Adamantane Phenylalkylamines with σ‑Receptor Binding
Affinity and Anticancer Activity, Associated with Putative
Antagonism of Neuropathic Pain
Stefanos Riganas,†,∥ Ioannis Papanastasiou,† George B. Foscolos,*,† Andrew Tsotinis,† Guillaume Serin,‡
Jean-Franco̧
is Mirjolet,‡ Kostas Dimas,§ Vassilios N. Kourafalos,∥ Andreas Eleutheriades,∥
Vassilios I. Moutsos,∥ Humaira Khan,∥ Stavroula Georgakopoulou,∥ Angeliki Zaniou,∥ Margarita Prassa,∥
Maria Theodoropoulou,∥ Athanasios Mantelas,∥ Stavroula Pondiki,∥ and Alexandre Vamvakides∥
†Department of Pharmaceutical Chemistry, Faculty of Pharmacy, National and Kapodistrian University of Athens,
Panepistimioupoli-Zografou, 15771 Athens, Greece
‡Oncodesign S.A., 20 Rue Mazen, F-21076 Dijon, France
§Division of Pharmacology, Foundation for Biomedical Research of the Academy of Athens (FBRAA), 11527 Athens, Greece
∥Anavex Life Sciences, 27 Marathonos Avenue, 15351 Pallini, Athens, Greece
S
* Supporting Information
ABSTRACT: The synthesis of the adamantane phenylalkylamines 2a−d, 3a−c,
and 4a−e is described. These compounds exhibited significant antiproliferative
activity, in vitro, against eight cancer cell lines tested. The σ1, σ2, and sodium
channel binding affinities of compounds 2a, 3a, 4a, and 4c−e were investigated. The
most interesting analogue, 4a, exhibited significant in vivo anticancer profile on
pancreas, prostate, leukemia, and ovarian cancer cell line xenografts together with
apoptosis and caspase-3 activation. Inhibition of the cancer cells cycle at the sub-G1
level was also obtained with 4a. Finally, encouraging results were observed with 4a
in vivo on mice, suggesting putative antimetastatic and analgesic activities of this compound.
apoptotic pathway and were proposed as putative anticancer
drugs,22,25 but caspase-3 activation was also described for σ2
anticancer ligands.28,30,31 Recent data have also suggested the
importance of the σ1-receptor modulated ion channels (Na+,
K+, Cl−, Ca2+)2,32−35 and σ1-receptor binding of cholesterol in
lipid rafts concerning the proliferation of cancer cells.36,37 The
Na+ channels, in particular, are modulated by σ1-receptors and
are implicated in the adhesion, migration, and apoptosis of
cancer cells.34,35,38−40 Important advances were recently made
on the mechanism of action of σ-ligands and their putative role
as therapeutic anticancer agents. In particular, the σ1-receptor
was cloned2,41 allowing more accurate pharmacological
evaluation42 of its specific role in the endoplasmic reticulum
(ER), in the apoptosis of cancer cells,1,2,4,16 and in its
connection with the impairing action on the G0/G1 cell cycle
phases.31,32 Classical SAR studies indicated that the presence of
a cycloalkyl or aryl group attached to the cationic amine center
via a linker of a three- to five-membered chain (including a
heteroatom) was essential for affinity at the σ-receptors.43,44 In
previous work, we reported 1-[p-[α-(1-adamantyl)benzyl]-
phenyl]piperazines 1 as antiproliferative agents. Piperazine 1a
(R = CH3) presented appreciable anticancer activity, which was
related to its affinity for σ-receptors and binding to site 2 of the
INTRODUCTION
■
Cancer and neurogenerative diseases, two of the most
important areas for medical research, both implicate σ-
receptors. σ-Ligands can therefore be expected to be putative
anticancer drugs.1−5 Indeed, σ-receptors are expressed to a
greater degree in tumors than they are in the surrounding
normal tissue.6−13 σ-Receptors have been classified as a distinct
pharmacological entity, and their function was shown to be
unrelated to the function of opioid receptors.2,5,14 On the basis
of the ligand selectivity in the binding assays, two subtypes, σ1
and σ2 receptors, were identified.2,5,15 Further, σ1-receptors
have been shown to be involved in programmed cell death
(apoptosis), with σ1-agonists being antiapoptotic and neuro-
protective, with putative antineurodegenerative activity.2,16−20
σ1-Antagonists or σ2-agonists are proapoptotic and can, because
of the high expression of σ1 and σ2 receptors in the rapidly
proliferating cancer cells,6−13 act as putative anticancer drugs by
inducing cell death via apoptosis.21−32 There is considerable
evidence of antiproliferative and cytotoxic activity for σ1-
antagonists,21,23,24,26,29,31 σ2-putative agonists,22,23,25,28,30 mixed
σ1/σ2-ligands,21,23,26 and even one σ1-agonist.27 More specifi-
cally, it has been shown that σ1-ligands (putative antagonists)
induce caspase-dependent apoptosis,24,31 which is in accord
with recent observations that σ1-agonists prevent caspase-3
activation.2,17,19 On the other hand, σ2-ligands (putative
agonists) have been shown to activate a caspase-independent
Received: September 10, 2012
© XXXX American Chemical Society
A
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Na+ channels.31 The structures of derivatives 1 involve a
scaffold of a benzene ring A linked to the second piperazine
nitrogen via a chain of three atoms (N, 2C), the first piperazine
nitrogen, and the following two piperazine carbons (Figure 1).
The length of the above linker satisfies the structural
requirement for σ-receptor binding affinity.
a
Scheme 1
a
Reagents and conditions: (a) (i) p-tolylmagnesium bromide/Et2O,
reflux, 2 h, (ii) HCl 10% at 0 °C; (b) NBS-dibenzoyl peroxide/CCl4,
reflux, 8 h; (c) R2NH/THF, reflux, 10 h; (d) (i) TFA/DCM, Ar, rt, 15
min, (ii) Et3SiH, Ar, rt, 1 h, (iii) H2O at 0 °C, then sat. solution of
Na2CO3.
Figure 1. Adamantane phenylpiperazines 1.
In this work, we describe the design, synthesis, and σ-
receptor affinity and antiproliferative potency of 1-[p-[α-(1-
adamantyl)benzyl]phenyl]alkylpiperazines 2a−c, 3a,b, and 4a−
d (Figure 2). In the new derivatives, the benzene ring A is
Reduction of 8 with triethylsilane in trifluoroacetic acid gave
the desired benzylamines 2a−d. The second pathway was
accomplished in five steps (Scheme 2). Reaction of p-
bromophenylmagnesium bromide with ketone 5 gave bromo
alcohol 9, which was reduced by triethylsilane in trifluoroacetic
acid to the aryl bromide 10. Conversion of bromide 10 to the
corresponding Grignard reagent and reaction of the latter with
dry gaseous carbon dioxide gave benzoic acid 11. Trans-
formation of the acid 11 to the acid chloride and coupling of
this with the appropriate secondary amine gave the benzamides
12a,d, which were reduced by LiAlH4 to the benzylamines 2a,d.
Phenethylamines 3a−c were prepared by two synthetic
pathways (Schemes 4 and 5). In the first synthetic route p-
bromophenethylamines 17 were used as starting materials,
which were synthesized by the reaction sequence shown in
Scheme 3.
Bromophenethyl alcohol 13 was esterified to tosylate 14,
which was converted to amines 17 by reacting with the
appropriate secondary amine. Alternatively,47 p-bromophene-
thylamines 17 can be prepared by reacting paraformaldehyde
with the trifluoroacetate of 1-methyl- or 1-ethylpiperazine or
piperidine hydrochloride in N-methyl-2-pyrrolidinone and
addition of p-bromobenzylzinc bromide to the intermediate
cations 16 (Scheme 3). The p-bromophenethylamines 17 were
then lithiated with tert-butyllithium at −80 °C to give the
corresponding aryllithium intermediates 18, which were then
reacted with ketone 5 to give the amino alcohols 19. Reduction
of 19 with triethylsilane in trifluoroacetic acid gave the desired
phenethylamines 3a−c (Scheme 4).
Figure 2. Adamantane phenylalkylamines 2, 3, and 4 with
antiproliferative activity.
linked to the first piperazine nitrogen by one, two, and three
methylene carbons. We also synthesized piperidine adducts 2d,
3c, and 4e in order to evaluate the contribution of the first
piperazine nitrogen to antiproliferative activity. Derivative 4a,
having shown the most interesting antiproliferative activity in
vitro, was also evaluated in vivo.
In the second route to the amines 3a−c, aryl bromide 10 was
converted to the intermediate aryllithium with n-butyllithium at
−80 °C and then reacted with DMF to give the benzaldehyde
20. Reduction of 20 gave the benzyl alcohol 21, which was then
treated with thionyl chloride to give the benzyl chloride 22.
The latter was converted to phenylacetonitrile 23 with sodium
cyanide in DMSO. Carbonitrile 23 could also be obtained by
the reaction of aldehyde 20 with tosylmethyl isocyanide
(TosMIC) in the presence of potassium tert-butoxide, followed
by addition of methanol.48,49 Alcoholysis of nitrile 23 led to
ethyl phenylacetate 24, and this was saponified to the
corresponding phenylacetic acid, which was converted to the
intermediate acid chloride and then to the phenylacetamides
25. Reduction of amides 25 with LiAlH4 then gave the desired
phenethylamines 3a−c (Scheme 5).
CHEMISTRY
■
For the synthesis of benzylamines 2, 1-adamantyl phenyl
ketone (5) was used as starting material and was prepared
either by reacting diphenylcadmium with 1-adamantanecarbon-
yl chloride in boiling benzene or by reacting phenyllithium with
1-adamantanecarboxylic acid in di-n-butyl ether at −80 °C.45,46
Two different synthetic pathways were followed for the
preparation of benzylamines 2, as illustrated in Schemes 1 and
2. In the first synthetic route, p-tolylmagnesium bromide was
added to ketone 5 to give α-phenyl-α-(p-tolyl)-1-adamantane-
methanol (6). Carbinol 6 was then brominated by NBS in the
presence of a catalytic amount of dibenzoyl peroxide to afford
bromomethylcarbinol 7, which was coupled with the
appropriate secondary amine to give amino alcohols 8.
B
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a
Scheme 2
a
Reagents and conditions: (a) p-bromophenylmagnesium bromide/Et2O, reflux, 2 h, (ii) sat. solution of NH4Cl at 0 °C; (b) (i) TFA/DCM, Ar, rt,
15 min, (ii) Et3SiH, rt, 1 h, (iii) H2O at 0 °C, then sat. solution of Na2CO3; (c) (i) Mg turnings/THF, reflux, 3 h, (ii) dry CO2 gas, 3 h, (iii) HCl
(10%) at 0 °C; (d) (i) SOCl2, reflux, 1 h, (ii) R2NH/THF, reflux, 3 h; (e) LiAlH4/THF, reflux, 3 h, then NaOH 10% at 0 °C.
a
a
Scheme 3
Scheme 5
a
Reagents and conditions: (a) TsCl/Py−DCM, at 0 °C, 30 min, then
at 4 °C, 12 h; (b) R2NH/THF, reflux, 6 h; (c) paraformaldehyde/
NMP, rt, 20 min, then at 60 °C, 12 h; (d) p-bromobenzylzinc
bromide/THF, rt, 30 min.
a
Scheme 4
a
Reagents and conditions: (a) (i) n-BuLi/THF, 2 h at −80 °C, (ii)
DMF, rt, (iii) HCl 10%, at 0 °C; (b) LiAlH4/THF, reflux, 1 h, then
NaOH 10% at 0 °C; (c) SOCl2 and CaCO3/Et2O, rt, 12 h; (d)
NaCN/DMSO, 12 h; (e) (i) t-BuOK and TosMIC/DME, 2 h at −60
°C, (ii) MeOH, rt, then reflux, 30 min; (f) gas HCl/ethanol, ∼20%,
reflux, 2 h, then water drops, reflux, 1 h; (g) (i) NaOH/EtOH−H2O,
reflux, 2 h, then HCl 10% at 0 °C, (ii) SOCl2 at 60 °C, 1 h, (iii)
R2NH/THF, reflux, 12 h; (h) LiAlH4/THF, reflux, 3 h, then NaOH
10% at 0 °C.
a
a
Scheme 6
Reagents and conditions: (a) t-BuLi/THF, 2 h at −80 °C; (b) (i)
ketone 5/THF, 30 min at −80 °C, then rt, 2 h, (ii) sat. solution of
NH4Cl at 0 °C; (c) (i) TFA/DCM, rt, 15 min, (ii) Et3SiH, rt, 1 h, (iii)
H2O at 0 °C, then sat. solution of Na2CO3.
The synthesis of propylamines 4a−c,e was accomplished by
the reaction sequence shown in Scheme 6. γ-[p-[α-(1-
Adamantyl)benzyl]phenyl]propanol (26) was tosylated to the
intermediate ester, which was then reacted with the requisite
secondary amines to give the desired amines 4a−c,e. This
substitution was accompanied by the formation of the p-
toluenesulfonamides byproducts 27.
a
Reagents and conditions: (a) TsCl/Py/DCM; (b) requisite amine/
EtOH, Δ.
a
Scheme 7
Piperazine 4d was prepared from the benzyl derivative 4c by
reductive debenzylation in the presence of palladium on
charcoal, with ammonium formate as hydrogen donor (Scheme
7).
a
Reagents and conditions: (a) HCO2NH4/10% Pd−C/MeOH, Δ.
The preparation of propyl alcohol 12 is described in our
previous paper as a low yield byproduct.50 In this work, 12 was
prepared by three different synthetic routes. In the first route,
addition of allylmagnesium chloride to aryl bromide 10 in
presence of a catalytic amount of copper(I) iodide gave a
C
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a
mixture of olefin 28 and 1-(α-benzhydryl)adamantane (29) via
a free radical mechanism. Hydroboration of the above mixture
led to propyl alcohol 26, which was separated from hydro-
carbon 29 by column chromatography (Scheme 8).
Scheme 10
a
Scheme 8
a
Reagents and conditions: (a) p-allylphenylmagnesium bromide/THF,
NH4Cl/H2O at 0 °C; (b) BH3 (>2 equiv)/THF and then NaOH/
H2O2 at 0 °C; (c) (i) TFA/DCM and then Et3SiH; (ii) H2O at 0 °C;
(iii) NaOH/H2O/THF at rt.
a
Reagents and conditions: (a) allylmagnesium chloride/THF, CuI;
(b) (i) BH3·THF; (ii) NaOH/H2O2.
BIOLOGY
■
The results for the affinities of the derivatives for both the σ1
and σ2 receptors and site 2 of Na+ channels are summarized in
Table 1. The results for the in vitro antiproliferative activity of
the derivatives 2, 3, and 4 are shown in Table 2.
In the second route, the application of Heck reaction
conditions,51,52 treatment of aryl bromide 10 with ethyl acrylate
in presence of palladium(II) acetate and triphenylphosphine,
gave the ethyl cinnamate 30. The corresponding cinnamic acid
is a mixture of diastereomers (R,E) and (S,E) (Figure 3)
Table 1. Affinities of Some Adamantane Phenylalkylamines
for the σ1 and σ2 Receptors
IC50 SEM (nM) (n = 3)
IC50 SEM (nM)
(n = 3)
compd
σ1
σ2
σ1/σ2
Na+ channels
a
2a
3a
4a
4c
4d
4e
5.2 1.3
110.4 13.1
80.1 9.4
21.2
27.6
1.77
0.78
11.0
1.06
nd
a
2.9 0.7
nd
48.1 17.7
36.1 8.3
42.0 21.0
12.3 4.2
85.0 18.3
28.3 7.2
>1000
11.5 7.0
>1000
Figure 3. Mixture of corresponding cinnamic acid diastereomers.
461.4 141.7
13.0 3.9
>100
according to the 1H NMR spectra for this compound. Catalytic
hydrogenation of the unsaturated ester 30 and sequential
reduction of the intermediate propionic ester 31 with LiAlH4
gave the desired alcohol 26 (Scheme 9).
a
nd: not determined.
It is clear from Table 1 that adamantane analogues 4a−e
have a significant binding affinity for the σ1 and σ2 receptors
and, except for the 4c, a much lower or insignificant affinity for
site 2 of the Na+ channels. Analogue 4d seems to have
moderate binding affinity at the σ2-receptors, while derivatives
2a and 3a seem to act as selective σ1-ligands.
a
Scheme 9
These results, in conjunction with the in vitro antiprolifer-
ative action of the new adamantane derivatives (Table 2), imply
that the new molecules act as mixed σ1/σ2 ligands. Since
binding data do not classify the ligands as agonists or
antagonists, further work is required to define the character-
istics of adamantane ligands that are needed for receptor
activation, subtype selectivity, and particularly the nature of the
action (agonistic or antagonistic). No validated in vitro isolated
organ test exists for the functional characterization of σ-ligands
as agonists or antagonists.53 However, the antiproliferative
activity of the new compounds is possibly linked with their
affinity for the σ-receptors, while other mechanisms of action
cannot be ruled out. Indeed, we argue that in contrast to
analogues 1,31 where the antiproliferative activity and the σ
affinities are clearly related to the second piperazine nitrogen
(benzyl and morpholino derivatives exhibited diminution of or
no σ affinities and antiproliferative activities), the antiprolifer-
ative action and σ binding affinities of analogues 2a, 3a, and
4a−d are due to both piperazine atoms. The relative
a
Reagents and conditions: (a) ethyl acrylate, palladium(II) acetate,
triphenylphosphine, triethylamine at 95−100 °C; (b) H2/PtO2, EtOH
at 50−60 psi; (c) (i) LiAlH4/THF; (ii) H2O/OH− at 0 °C.
In the third synthetic route (Scheme 10), addition of p-
allylphenylmagnesium bromide to ketone 5 gave the unsatu-
rated alcohol 32, which was transformed to diol 33 by
hydroboration. The latter was reduced to propyl alcohol 26
with triethylsilane in trifluoroacetic acid. Of the three synthetic
pathways, the third route gave the best yield of 26.
D
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conservation of the antiproliferative activity of the piperidino
analogue 4e reinforces this argument. However, the high σ
binding affinity of the benzyl-substituted compound 4c is not
accompanied by any significant antiproliferative activity. This,
which has also been observed in analogous benzyl group
bearing adamantanes,50 is possibly due to the σ2-receptor
antagonistic profile of 4c induced by the benzyl substitution.
Considering the in vitro results of antiproliferative activity of
derivatives 4 on cancer cell lines (colon, prostate, breast,
ovarian, central nervous system, leukemia, pancreas, liver) and
on normal cell lines (HUVEC, human umbilical vein
endothelial cell; hMSC, human mesenchymal stem cell;
NHDF, normal human dermal fibroblast) (Table 2), it appears
that 4a exhibited the most selective activity against cancer cells,
given that the cytotoxic effect of 4a on the HUVECs could, in
vivo, also be an antiangiogenic factor against the tumors.
Interesting results were also obtained with 4a in the BxPC-3
(pancreas), OVCAR-5 (ovarian), DU-145 and PC3 (prostate),
and HL-60 (leukemia) cancer xenografts (described in
Experimental Section38,39). Antitumor activity of 4a was, in
most of these xenografts, superior to those of 5-fluorouracil
(5FU) and aracytin and similar to those of gemcitabine,
paclitaxel (Taxol), and cis-Pt, with 4a exerting interesting
synergies with both the reference drugs 5FU and gemcitabine
(Figures 4−8). Finally, some encouraging results were obtained
with 4a on the metastasis of PC3 and OVCAR-5 cancer cells
xenografts (Figures 6 and 8)
The pharmacological profile of compound 4a was further
extended by toxicological experiments. These studies were
performed on CD-1 male and female mice and, for xenografts,
on mice with severe combined immune deficiency (SCID).
Compound 4a was well tolerated at 40−55 mg/kg (ip) in
chronic 2−3 weeks of treatments on CD-1 and SCID mice.
There were no more dead animals than in Tween-80 (5%) or
Cremophor or reference drugs used in the above protocols and
no important loss of weight in SCID mice during the xenograft
experiments. Concerning the mechanistic effects induced by 4a
on the BxPC-3, IGROV-1, and PC-3 cell cycle and apoptosis
(Table 3), significant increases of sub-G1 for BxPC-3, IGROV-
1, and PC-3 at 15 μM (for PC3 even from 5 μM) and also of
G1 populations of BxPC3 at 5 μM were observed in cells
treated with 4a, compared to the vehicle treated group.
Combined with a decrease of populations engaged in the S
phase, these alterations of the cell cycle are indicative of the
apoptotic activity of 4a. Indeed, 4a exhibited apoptotic
plasmatic membrane modifications at 15 and 50 μM (for Bx-
PC-3 at 15 μM and for IGROV-1 at 15 and 50 μM) with
caspase-3 activation. It is noteworthy that the antiproliferative
activity of 4a on BxPC-3 (pancreas), IGROV-1 (ovarian), and
PC3 (prostate) cancer cells is conjugated with apoptotic
activity (in the order IGROV-1 > BxPC-3 > PC3), an inhibition
of these cancer cells cycle at the sub-G1 level (in the order PC3
> IGROV-1 ≥ BxPC-3), and caspase-3 activation only for
IGROV-1 and BxPC-3. The above results could indicate that
the mixed σ1/σ2 ligand 4a, in agreement with previous
data,28,30−33,54 exhibited cell cycle inhibition and caspase-3
activation in BxPC-3 and IGROV-1 but also cell cycle
inhibition and caspase-3 independent apoptosis in PC3 cancer
cells, in agreement with results concerning σ2-ligands.22,25,54
Results of in vitro assays concerning the effect of 4a on the cell
cycle and apoptosis of BxPC3, IGROV-1, and PC3 cancer cells
are summarized in Table 3.
F
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Figure 4. Growth of BXPC3 (pancreas) tumors in SCID mice treated with fluorouracil (5FU) (reference drug for pancreatic cancer) and 4a. (Aa)
Tumor size (in mm3) of each mouse group (10 mice per group, that is, 20 tumors per group). (Bb) Table showing reduction of tumors on day of
termination of experiment for BXPC3 xenograft: Ut, untreated animals; 5T80, animals treated with 5% Tween 80; 17 5FU, animals treated with 17
mg/kg 5FU (dissolved in water for injection) administered twice a week (three cycles of treatment); 40 4a, animals treated with 40 mg/kg 4a
(dissolved in 5% Tween 80) administered for 3 consecutive days per week (three cycles of treatment)l 17 5FU + 40 4a, coadministration of 17 mg/
kg 5FU and 40 mg/kg 4a. Statistical evaluation was carried out using a two-tailed Student's t test. Points with p < 0.05 are indicated by one asterisk
(∗), and points with p < 0.001 are indicated by two asterisks (∗∗). Shown is the growth of BXPC3 (pancreas) tumors in SCID mice treated with
gemcitabine (Gem) (reference drug for pancreatic cancer) and 4a. (Cc) Tumor size (in mm3) of each mouse group (10 mice per group, that is, 20
tumors per group). (Dd) Table showing reduction of tumors on day of termination of experiment for BXPC3 xenograft: Ut, untreated animals;
5T80, animals treated with 5% Tween 80; 70Gem, animals treated with 70 mg/kg Gem (dissolved in water for injection) administered twice a week
(three cycles of treatment); 45 4a, animals treated with 45 mg/kg 4a (dissolved in 5% Tween 80) administered for 3 consecutive days per week
(three cycles of treatment); 40 4a, animals treated with 40 mg/kg 4a (dissolved in 5%Tween 80) administered for 3 consecutive days per week
(three cycles of treatment; 70Gem + 40 4a, coadministration of 70 mg/kg Gem and 40 mg/kg 4a. Statistical evaluation was carried out using a two-
tailed Student's t test. Points with p < 0.05 are indicated by one asterisk (∗) and those with p < 0.001 by two asterisks (∗∗).
Finally, in relation to the role of σ1-receptors in the central
sensitization processes of neuropathic pain,2,55−58 4a was tested
in a neuropathic pain model (described in Experimental
Section31,59). 4a exhibited a notable analgesic effect in the
formalin test60,61 operated on mice on which pain sensitization
was obtained by previous (2 weeks) administration of
paclitaxel,31,59 as shown in Figure 9.
that, in good agreement with its in vitro cell line results, was
particularly prominent in pancreas, prostate, ovarian, and
leukemia xenografts on SCID mice (Figures 4−8). It is
noteworthy that this antitumor activity of 4a is associated with
a putative antagonism of the neuropathic pain induced by the
clinically used anticancer drugs (particularly, taxanes). En-
couraging results were also obtained with 4a on the metastasis
of prostate and ovarian xenografts (Figures 7 and 8).
Consequently, compound 4a is currently under investigation
with particular interest in its putative antimetastatic activity.
CONCLUSION
■
The in vitro and in vivo toxicological and xenograft screening
studies showed that piperazine 4a exhibited an acceptable
toxicological profile associated with a notable antitumor activity
G
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Figure 5. Growth of BXPC3 (pancreas) tumors in SCID mice treated with fluorouracil (5FU), gemcitabine (Gem) (reference drugs for pancreatic
cancer) and 4a. (Aa) Tumor size (in mm3) of each mouse group (10 mice per group, that is, 20 tumors per group). (Bb) Table showing reduction of
tumors on day of termination of experiment for BXPC3 xenograft: 5T80, animals treated with 5% Tween 80; 35 5FU, animals treated with 35 mg/kg
5FU (dissolved in water for injection) administered twice a week (three cycles of treatment); 70Gem, animals treated with 70 mg/kg gemcitabine
(dissolved in water for injection) administered twice a week (three cycles of treatment); 40 4a, animals treated with 40 mg/kg 4a (dissolved in 5%
Tween 80) administered for 3 consecutive days per week (three cycles of treatment); 35 5FU + 40 4a, coadministration of 35 mg/kg 5FU and 40
mg/kg 4a; 70 Gem + 40 4a, coadministration of 70 mg/kg Gem and 40 mg/kg 4a. Shown is the growth of HL-60(TB) (leucaemias) tumors in SCID
mice treated with aracytin (Ara) (reference drug for leucaemias) and 4a. (Cc) Tumor size (in mm3) of each mouse group (9 mice per group, that is,
20 tumors per group). (Dd) Table showing reduction of tumors on day of termination of experiment for HL-60 (TB) xenograft: 5T80, animals
treated with 5% Tween 80; 10Aracytin, animals treated with 10 mg/kg aracytin (dissolved in water for injection) administered 5 times a week (one
cycle of treatment); 50 4a, animals treated with 50 mg/kg 4a (dissolved in 5% Tween 80) administered for 3 consecutive days per week (one cycle of
treatment). Statistical evaluation was carried out using a two-tailed Student's t test. Points with p < 0.05 are indicated by one asterisk (∗) and those
with p < 0.001 by two asterisks (∗∗).
both assays, as that remaining in the presence of 10 μM haloperidol.
EXPERIMENTAL SECTION
■
Affinities for site 2 of Na+ channel were measured by displacement of
Binding Studies. Binding studies were carried out by CEREP
[3H]batrachotoxin benzoate (3H-BTX-B).64 Binding reactions were
(France) with the σ1 binding assay performed in triplicate. Affinities of
initiated by addition of 150 μL of vesicular preparation containing
the new adamantane alkylamines for the σ1 and σ2 receptors were
150−500 μg of protein to a solution in standard incubation buffer of
3H-BTX-B, 50 μg of Leiurius quinquestriatus scorpion venom, and
various unlabeled effectors as indicated. The concentration of 3H-
BTX-B was generally 20−25 nM, and the total assay volume was 335
μL. Standard incubation buffer contained 130 mM choline chloride, 50
mM HEPES buffer adjusted to pH 7.4 with Tris base, 5.5 mM glucose,
0.8 mM MgSO4, 5.4 mM KCl, and 1 mg/mL BSA. Incubations were
carried out for 60 min at the indicated temperature and were then
terminated by addition of 3 mL of ice-cold wash buffer. The tissue was
immediately collected on Whatman GF/C glass fiber filters and
washed 3 more times with 3 mL of cold wash buffer. The wash buffer
measured by displacement of [3H](+)-pentazocine and [3H]1,3-di-o-
tolylguanidine, respectively. The σ1 binding assay62 was performed by
incubating Jurkat cell membranes (10−20 mg of protein per tube)
with [3H](+)-pentazocine (8 nM) and a range of concentrations of the
compounds at 22 °C for 2 h in 5 nM Tris-HCl buffer, pH 7.4. The σ2
binding assay63 was performed by incubating rat cerebral cortex
membranes (10−20 mg of protein per tube) with [3H](+)-1-DTG (3-
di-o-tolylguanidine, 5 nM) in the presence of (+)-pentazocine (300
nM) to saturate σ1 site binding and a range of concentrations of the
compounds at 22 °C for 2 h in 5 nM Tris-HCl buffer, pH 7.4. The
final assay volume was 0.5 mL. Nonspecific binding was defined, in
H
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Figure 6. Growth of PC3 (prostate) tumors in SCID mice treated with paclitaxel (reference drug for prostate cancer) and 4a. (Aa) Tumor size (in
mm3) of each mouse group (14 mice per group, that is, 28 tumors per group). (Bb) Table showing reduction of tumors on day of termination of
experiment for PC3 xenograft. (Cc) Occurrence of primary (n = 7) and secondary metastatic tumors (n = 7) at the axillary (AM) and bone (BM)
sites in the control and drug-treated groups: 5 T80, animals treated with 5% Tween 80; Crem, animals treated with a (1:1 ethanol and Cremophor
stock solution diluted 1:3 in saline; 20 Tax, animals treated with 20 mg/kg paclitaxel (dissolved in Cremophor and diluted in saline) administered
once a week (two cycles of treatment); 55 4a, animals treated with 55 mg/kg 4a (dissolved in 5% Tween 80) administered for 5 consecutive days
(two cycles of treatment). Statistical evaluation was carried out using a two-tailed Student's t test. Points with p < 0.05 are indicated by one asterisk
(∗) and those with p < 0.001 by two asterisks (∗∗).
contained 163 mM choline chloride, 5 mM HEPES (pH 7.4), 1.8 mM
CaCl2, 0.8 mM MgSO4, and 1 mg/mL BSA. Radioactivity associated
with the tissue was determined by liquid scintillation spectroscopy of
the filters suspended in 10 mL of scintillation cocktail (3a70B;RPI).
Nonspecific binding was determined from parallel incubations
containing 250 μM veratridine and has been subtracted from the
data. Specific binding measured in this way was nominally 75% of the
total binding.
In Vitro Antiproliferative and Cytotoxic Activity. All human
cancer cell lines were obtained from the National Cancer Institute,
NIH (Bethesda, MD, U.S.) with the exception of BX-PC3 and the
normal cells Hs888Lu and CCD18Co that were obtained from ATCC
and the hMSCs, NHF, and HUVECs that were purchased from Lonza.
All cell lines were adapted to propagate in RPMI 1640 medium
supplemented with 5% heat-inactivated fetal calf serum, 2 mM L-
glutamine, and antibiotics. The cultures were grown in a humidified 37
°C incubator with 5% CO2 atmosphere. Cell viability was assessed at
the beginning of each experiment by the trypan blue dye exclusion
method and was always greater than 95%. Cells were seeded into 96-
well microtiter plates in 100 μL of medium at the corresponding
density (3500−30000 cells per well), and subsequently, the plates
were incubated at standard conditions for 24 h to allow the cells to
resume exponential growth prior to addition of the agents to be tested.
Then in order to measure the cell population, cells in one plate were
fixed in situ with trichloroacetic acid (TCA) followed by sulforhod-
amine B solution (SRB) staining, as described elsewhere.65−67 To
determine the activity, each compound was dissolved in dimethylsulf-
oxide (DMSO) and then was added at 10-fold dilutions (from 100 to
0.01 μM), and incubation continued for an additional period of 48 h.
The assay was terminated by addition of cold TCA followed by SRB
staining and absorbance measurement at 540 nm in an DAS plate
reader to determine the GI50, that is, the concentration required in the
cell culture to inhibit cell growth by 50%; TGI, the concentration that
is required to completely inhibit cell growth; and the LC50, the
concentration that is needed in culture to kill 50% of the cellular
population as described.65−67
In Vivo Antitumor Activity. SCID (NOD.CB17 Prkdcscid) mice
were purchased from Jackson Laboratories/Charles River Laboratories
(L'Arbresle, France). The mouse colony was maintained under
restricted flora conditions in a pathogen-free environment in type
I
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Figure 7. Growth of DU145 (prostate) tumors in SCID mice treated with paclitaxel (reference drug for prostate cancer) and 4a. (Aa) Tumor size (in
mm3) of each mouse group (minimum number of animals used n = 30). (Bb) Table showing reduction of tumors on day of termination of
experiment for DU145 xenograft: 5 T80, animals treated with 5% Tween 80; Crem, animals treated with a 1:1 ethanol and Cremophor stock
solution diluted 1:3 in saline; 20 Tax, animals treated with 20 mg/kg paclitaxel (dissolved in Cremophor and diluted in saline) administered once a
week (two cycles of treatment); 55 4a, animals treated with 55 mg/kg 4a (dissolved in 5% Tween 80) administered for 5 consecutive days (two
cycles of treatment). Statistical evaluation was carried out using a two-tailed Student’s t test. Points with p < 0.05 are indicated by one asterisk (∗)
and those with p < 0.001 by two asterisks (∗∗).
IIL cages. Male or female mice, 7−9 weeks old, were subjected to
subcutaneous injections according to the British practice of bilateral
trocar implants at the axillary region. Each inoculum contained 106
cells exponentially growing at the time of harvesting. The mice were
subsequently randomly divided into groups of 6−20 animals per group
when the average tumor volume had reached about 100 mm3.
Treatments started at that point. Tumor volume was calculated as
described elsewhere.67,68 All administrations were intraperitoneal.
Treated animals received a single injection daily for 5 days per week
throughout the experiments. Tumor volume was measured with a
caliper twice per week. In addition to tumor volume, we calculated the
parameter, % ΔT/ΔC, where ΔT = T − Do and ΔC = C − Do (Do is
the average tumor volume at the beginning of the treatment; T and C
are the volumes of treated and untreated tumors, respectively, at a
specified day). Concurrently, we scored the number of tumor-free
animals, number of drug-related deaths, and average number of days
required to reach a defined tumor volume. Optimal ΔT/ΔC was used
as a measure of drug activity. Losses of weight, neurological disorders,
and behavioral and dietary changes were also recorded as indicators of
toxicity (side effects). The experiment was terminated when tumor
size in untreated animals reached a volume of about 10−11% of the
animals’ weight.
Evaluation of Metastasis in the OVCAR5 Xenograft. The
OVCAR5 ovarian cancer xenograft was observed for the presence of
metastasis. On day 54 (days postinoculation), animals from all drug
groups were sacrificed and observed for the occurrence of metastasis.
Metastatic tumors were observed at two main sites: the auxiliary region
(named “auxillary metastasis”, AM) and the abdominal region attached
to the bone (named “bone metastasis”, BM). Figure 8, Table C
represents the occurrence of the AM and BM tumors for the different
drug groups as observed on day 54 of the experiment. Isolation,
subculture and in vitro analysis of the metastatic-derived tissue
indicated that the three types of cultures (primary, AM-derived, and
BM-derived) were indeed of similar morphological arigin, confirming
that the metastatic tissue was OVCAR5 ovarian-cancer-cell derived.
In Vitro Cell Cycle Modifications and Apoptotic Activity.
Supplying PC-3 (human prostatic adenocarcinoma), BxPC-3 (human
pancreatic adenocarcinoma), and IGROV-1 (human ovarian carcino-
ma) cancer cells and carrying out cell cycle and apoptosis assays were
done by Oncodesign S.A. (France). Cells were plated 24 h before
treatment at the appropriate seeding density in six-well plates or 25
cm2 flasks according to the assay. Compound 4a was dissolved at 10
mM in 100% DMSO and then diluted in cascade to obtain
concentrations of 0.1, 1, and 3 mM in 100% DMSO. These dilutions
were further diluted at 1:10 with RPMI 1640 medium. The last
dilution was performed on cells at 1:20 to reach the appropriate
concentrations of 0.5, 5, 15, and 50 μM. The final concentration of
DMSO was 0.5% in cell culture medium. The treatment period was 24
h. The effect of compound 4a on cell cycle was evaluated by
quantification of propidium iodide (PI) incorporation into genomic
DNA. After incubation, cells were detached from the well by
trypsinisation, transferred into tubes, washed, and resuspended in
500 μL of ice cold PBS before being fixed with 1.5 mL dropwise of
100% cold ethanol for 3 h at 4 °C . Then the cell suspensions were
centrifuged at 1500 rpm for 5 min, and pellets were resuspended in a
mix of 100 μL of 200 μg/mL RNase and 100 μL of 1 mg/mL PI. Cells
were incubated for 45 min at room temperature in the dark. The
preparation was centrifuged 5 min at 1500 rpm. Then the pellets were
resuspended in PBS for FACS analysis.
The apoptotic membrane modifications induced by compound 4a
were evaluated by annexin V binding at the end of the treatment
period. 7-AAD, a fluorescent agent incorporated into DNA, was also
used to differentiate early (no membrane disruption) and late
(membrane disruption) apoptosis. After incubation, cells were
detached from the well by gentle scraping, transferred to FACS
tubes, and labeled according to the annexin V-FITC/7-AAD kit
(Beckman Coulter). Briefly, after being washed, cells were incubated in
ice-cold binding buffer. Then 10 μL of annexin V-FITC solution and
20 μL of 7-AAD viability dye were added to 100 μL of cell suspension
and incubated 15 min on ice in the dark. After incubation, an amount
of 800 μL of binding buffer was added. Cell preparations were
analyzed by FACS within 1 h.
The activation of caspase pathway by compound 4a was evaluated
by measuring the level of activated caspase-3 by FACS. After
incubation, cells were detached from the culture flask by trypsinization,
transferred to FACS tubes, and labeled according to PE active caspase-
3 apoptosis kit (Pharmingen). Briefly, after being washed, 106 cells
were fixed and permeabilized in ice-cold BD Cytofix/Cytoperm buffer
J
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Figure 8. Growth of OVCAR5 (ovarian) tumors in SCID mice treated with cisplatin (reference drug for ovarian cancer) and 4a. (Aa) Tumor size (in
mm3) of each mouse group (16 mice per group, that is, 32 tumors per group). (Bb) Table showing reduction of tumors on day of termination of
experiment for OVCAR5 xenograft. (Cc) Average weight of primary tumors (n = 7−8) with % reduction and occurrence of secondary metastatic
tumors (n = 7−8) at the axillary (AM) and bone (BM) sites in the control and drug-treated groups: 5 T80, animals treated with 5% Tween 80; 2.5
Cis, animals treated with 2.5 mg/kg cisplatin administered twice a week (three cycles of treatment); 55 4a, animals treated with 55 mg/kg 4a
(dissolved in 5% Tween 80) administered for 5 consecutive days (two cycles of treatment). Statistical evaluation was carried out using a two-tailed
Student’s t test. Points with p < 0.05 are indicated by one asterisk (∗) and those with p < 0.001 by two asterisks (∗∗).
for 20 min. Cells were then pelleted and washed with BD Perm/Wash
buffer. Incubation with antibody included in the kit was performed in
100 μL of the same buffer complete with 20 μL of antibody for 30 min.
Cell preparations were analyzed by FACS within 1 h.
For all assays staining cells were analyzed with a CyFlow space flow
cytometer (Partec) using a 488 nm wavelength laser excitation. The
acquisition was stopped after a total of 10 000 FSC/SSC gated cells
were collected for each sample.
Formalin Test. CD1 male mice weighing 34−40 g were used. They
were kept in a room maintained at 21−22 °C with free access to
standard laboratory diet and tap water. Paclitaxel (Brystol Myers
Squibb Company) was diluted in saline and administered at one ip
injection59 (6 mg/kg) on day 0. On day 14, 1 h after oral
administration (po) of compound 4a (100 mg/kg) the formalin test,
as a tonic and persistent pain model of nociception, was performed.
Injection of formalin into the hind paw is followed by two phases of
behavior.60,61 The first phase consists of intense licking and biting of
the injected paw for the first 5 min followed by a period of little
activity. The second phase spans from 15 to 40 min after the formalin
injection and involves periods of licking and biting of the injected paw.
The first phase is thought to be a model of acute chemical pain,
whereas the second phase reflects a state of central sensitization driven
by the presumed first phase.60,61 The amount of time spent licking and
biting the injected paw and leg was recorded at 5 min intervals for 0−5
and 35−40 min after the formalin injection.
Statistical Analysis. Significant difference in tumor volume was
determined by the Student’s t test using the SPSS for Windows
(release 11.0.0, SPSS Inc., U.S.) software package. A difference was
considered significant if p < 0.05.
K
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Table 3. Summary Results of the Cell Cycle and Apoptotic in Vitro Assays Performed with Compound 4a at 0.5, 5, 15, and 50
a
μM on BxPC-3, PC-3, and IGROV-1 Cancer Cell Lines
cell cycle analysis (%)
annexin V binding/7-AAD incorporation (%)
healthy cells early apoptosis late apoptosis
cleaved caspase-3
containing cells
(%)
total
cancer cell
line
treatment
(μM)
apoptotic
<G1 G1
S
G2 >G2
annexin V−/7-AAD− annexin V+/7-AAD− annexin V+/7-AAD+ death (%)
BxPC-3
(pancreas)
0
9
41
33
12
6
8.2
79
9
9
18
0.5
5
11
8
41
53
32
31
27
31
23
27
33
29
11
10
12
9
7
4
82
77
35
12
86
10
11
19
2
7
17
21
44
46
13
5
4
10
26
44
5
15
50
0
24
23
4
5
17.5
4.8
3.9
4
IGR-OV-1
(ovary)
19
21
9
0.5
5
4
25
29
23
30
36
26
27
25
28
42
23
21
27
10
12
20
20
17
15
6
4.2
89
83
35
26
91
8
3
11
15
57
64
8
4
12.6
61.4
15.8
3.2
10
37
40
5
5
15
50
0
12
18
4
20
25
3
PC-3
(prostate)
0.5
5
4
34
36
25
29
38
36
31
31
15
10
11
11
7
1.7
2
95
92
71
43
3
1
2
7
9
5
11
25
12
6
5
7
15
50
7
2.5
4
9
16
23
17
15
a
In cell cycle analysis, the percentage of cells was estimated for each phase of cell cycle (G0/G1, S, and G2/M) according to their DNA content.
Cells having less than n chromosomes (<G0/G1) or more than 2n chromosomes (>G2/M) were also reported. The high toxicity of compound 4a at
50 μM in the IGROV-1 cell line prevented correct cell cycle analysis. In the caspase-3 assay, the percentage of cells showing cleaved/activated
caspase-3 is indicated. In the annexin V/7-AAD assay, the percentage of healthy, early apoptotic, and late apoptotic cells was estimated according to
their affinity to annexin V-FITC and 7-AAD intercalating agent.
sium bromide, which was prepared from magnesium turnings
(0.8 g, 0.032 g at) and 4-bromotoluene (6 g, 0.035 mol) in
anhydrous diethyl ether (50 mL), was added dropwise a
solution of 1-adamantyl phenyl ketone (5) (3.4 g, 0.014 mol) in
anhydrous diethyl ether (50 mL) under an argon atmosphere.
The mixture was heated to mild reflux for 1 h and then
quenched by adding water and aqueous HCl (10%) in an ice
bath. The aqueous layer was separated and extracted with
diethyl ether. The combined organics were washed with water,
saturated solution of Na2CO3 (10%), dried over Na2SO4, and
concentrated in vacuo. The residue obtained was crystallized
and triturated with n-pentane to give 2.8 g of a solid product.
Yield 60%. Mp 146−148 °C (ether−n-pentane).
Figure 9. Effects of orally administered (po) 4a at doses of 50 and 100
mg/kg po on paclitaxel treated mice in the formalin test compared to
α-(4-Bromomethylphenyl)-α-phenyl-1-tricyclo-
[3.3.1.13,7]decanemethanol (7). To a stirred solution of
carbinol 6 (1.35 g, 4 mmol) in dry CCl4 (20 mL) were added
NBS (0.9 g, 5 mmol) and a catalytic amount of dibenzoylper-
oxide (50 mg). The reaction mixture was gently refluxed for 8
h, then cooled to ambient temperature and filtered. The
insoluble material was washed with CCl4 and the combined
filtrates were evaporared in vacuo to give a residue, which was
used as such in the next step.
α-[4-(4-Methyl-1-piperazinylmethyl)phenyl]-α-phe-
nyl-1-tricyclo[3.3.1.13,7]decanemethanol (8a). To a sol-
ution of the crude benzyl bromide 7 in anhydrous THF (10
mL) was added 1-methylpiperazine (1.8 g, 18 mmol), and the
reaction mixture was heated to reflux for 10 h. The solvent was
then removed in vacuo. Water was poured into the residue, and
the resulting mixture was extracted with DCM. The organic
layer was thoroughly washed with water, dried over Na2SO4,
and evaporated to give a residue, which was purified by flash
column chromatography, using as eluent a mixture of CHCl3/
the reference drug gabapentin (GBP) at 100 mg/kg (ip).
Administration of 4a exerted a significant analgesic effect in paclitaxel
treated animals. 4a, 50 and 100 mg/kg (po), in paclitaxel treated
animals resulted in lower licking times compared to paclitaxel treated
animals at 0−5 min ((∗∗) p = 0.0013 and (∗) p = 0.041, respectively)
and 35−40 min ((∗∗) p = 0.012 and (∗) p = 0.015) after formalin
injection.
Methods and Materials. Melting points were determined using a
Buchi capillary apparatus and are uncorrected. IR spectra were
̈
recorded on a Perkin-Elmer 833 spectrometer. 1H and 13C NMR
spectra were recorded on a Bruker MSL 400 spectrometer using
CDCl3 as solvent and TMS as internal standard. Carbon multiplicities
were established by DEPT experiments. The 2D NMR experiments
(HMQC, COSY, and NOESY) were performed for the elucidation of
the structures of the new compounds. Microanalyses were carried out
by the Service Central de Microanalyse (CNRS), France, and the
results obtained had a maximum deviation of 0.4% from the
theoretical value.
α-(4-Methylphenyl)-α-phenyl-1-tricyclo[3.3.1.13,7]-
decanemethanol (6). To a stirred solution of p-tolylmagne-
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MeOH (9:1) to afford 1.02 g of foamy product (yield 59% from
alcohol 6).
1-Methyl-4-{4-[α-(1-tricyclo[3.3.1.13, 7]decyl)-
phenylmethyl]phenylcarbonyl}pirerazine (12a). Thionyl
chloride (12 mL) was added to the carboxylic acid 11, and the
mixture was gently refluxed for 1 h. The excess of thionyl
chloride was evaporated in vacuo, and the last traces were
removed azeotropically with dry benzene. The residue was
dissolved in anhydrous THF (20 mL), and the resulting
solution was added dropwise to a solution of 1-methylpiper-
azine (2.6 g, 25.6 mmol) in anhydrous THF (20 mL) at 0 °C.
The reaction mixture was refluxed for 3 h, and then the solvent
was evaporated in vacuo. Water was added to the residue, and
the mixture was extracted with DCM. The combined organic
phases were washed with water and dried over Na2SO4. The
residue was purified by flash column chromatography, using as
eluent a mixture of DCM/MeOH (9:1) to give 1.32 g of a
viscous oil, which was crystallized after cooling (yield 37%). Mp
137−138 °C (Et2O).
α-[4-(4-Ethyl-1-piperazinylmethyl)phenyl]-α-phenyl-
1-tricyclo[3.3.1.13,7]decanemethanol (8b). Amino alcohol
8b was prepared from carbinol 6 in a similar way to amino
alcohol 8a. The product was purified by flash column
chromatography, using as eluent a mixture of DCM/MeOH
(9:1) to afford amino alcohol 8b as a foamy product (yield
57.5% from alcohol 6).
α-[4-(1-Piperazinylmethyl)phenyl]-α-phenyl-1-
tricyclo[3.3.1.13,7]decanemethanol (8c). Amino alcohol 8c
was prepared from carbinol 6 in a similar way to amino alcohol
8a. The product was purified by flash column chromatography,
using as eluent a mixture of DCM/MeOH (7:3) to afford
amino alcohol 8c as a foamy product (yield 38% from alcohol
6).
α-[4-(1-Piperidinylmethyl)phenyl]-α-phenyl-1-
tricyclo[3.3.1.13,7]decanemethanol (8d). Amino alcohol 8d
was prepared from carbinol 6 in a similar way to amino alcohol
8a. The product was purified by flash column chromatography,
using as eluent a mixture of DCM/MeOH (9:1) to afford
amino alcohol 8d as a sticky product (yield 67.5% from alcohol
6).
1-{4-[α-(1-Tricyclo[3.3.1.13,7]decyl)phenylmethyl]-
phenylcarbonyl}pireridine (12d). Carboxamide 12d was
prepared from carboxylic acid 11 in a similar way to benzamide
12a. Flash column chromatography gave a solid product, using
as eluent a mixture of DCM/MeOH (9:1). Yield 42% from aryl
bromide 12. Mp 196−198 °C (Et2O).
1-Methyl-4-{4-[α-(1-tricyclo[3.3.1.13, 7]decyl)-
phenylmethyl]phenylmethyl}pirerazine (2a). Method A.
To a solution of amino alcohol 8a (1 g, 2.3 mmol) in
anhydrous DCM (10 mL) was added trifluoroacetic acid (3.6 g,
31.2 mmol) under an argon atmosphere, and the mixture was
stirred at room temperature for 15 min. Triethylsilane (350 mg,
3 mmol) was added dropwise to the reaction mixture under
mild external cooling, and the resulting mixture was stirred at
room temperature for 1 h. The reaction mixture was quenched
with chilled water, made alkaline on treatment with solid
Na2CO3, and then extracted with DCM. The combined organic
layers were washed with water, dried over Na2SO4, and
concentrated in vacuo. To a solution of the residue obtained in
THF was added KOH (1 g) in a minimum amount of water,
and the resulting mixture was stirred for 2 h. Then the solvent
was evaporated in vacuo. Water was added to the residue, and
the mixture was extracted with DCM. The combined organic
phases were washed with water and dried over Na2SO4. The
residue was purified by flash column chromatography, using as
eluent a mixture of CHCl3/MeOH (9:1) to afford 630 mg of a
α-(4-Bromophenyl)-α-phenyl-1-tricyclo[3.3.1.13,7]-
decanemethanol (9). Carbinol 9 was synthesized by adding
p-bromophenylmagnesium bromide to 1-adamantyl phenyl
ketone (5) in a similar way as for carbinol 6. Yield 81%. Mp
128−129 °C (ether−n-pentane).
4-Bromo-α-phenyl-α-(1-tricyclo[3.3.1.13,7]decyl)-
methylbenzene (10). To a solution of carbinol 9 (4 g, 10
mmol) in anhydrous DCM (10 mL) was added trifluoroacetic
acid (7.6 g, 67 mmol) under an argon atmosphere, and the
mixture stirred at room temperature for 15 min. Triethylsilane
(1.28 g, 1.8 mL, 11 mmol) was added dropwise to the reaction
mixture under mild external cooling, and the resulting mixture
was stirred at room temperature for 1 h. Then the reaction was
quenched with chilled water and the mixture extracted with
DCM. The combined organic layers were washed with water,
saturated solution of Na2CO3 (10%), dried over Na2SO4, and
concentrated in vacuo. To a solution of the residue obtained in
THF, KOH (2 g) in a minimum amount of water was added
and the resulting mixture stirred for 2 h. Then the solvent was
evaporated in vacuo. Water was added to the residue, and the
mixture was extracted with ether. The combined ethereal
phases were washed with water, dried over Na2SO4, and
concentrated in vacuo. The residue was purified by flash
column chromatography, using hexane as eluent to afford 2.1 g
of a crystalline product (yield 54%). Mp 144−145 °C (Et2O).
4-[α-(1-Tricyclo[3.3.1.13,7]decyl)phenylmethyl]-
benzene Carboxylic Acid (11). A solution of aryl bromide
10 (3.2 g, 8.4 mmol) in anhydrous THF (16 mL) containing
1,2-dibromoethane (10 drops) was added dropwise to
magnesium turnings (0.44 g, 18.4 mmol) activated with iodine,
under an argon atmosphere. The reaction mixture was heated
to reflux for 3 h and then cooled to room temperature. Dry
carbon dioxide gas bubbled into the mixture for 3 h. The
reaction was quenched by adding an aqueous solution of HCl
(10%) at 0 °C. The mixture was extracted with DCM, and the
combined organic layers were washed with water, dried over
Na2SO4, and concentrated in vacuo to give 2.24 g of a sticky
product, which was used as such in the next step without
further purification.
1
solid product (yield 60%). Mp 196−198 °C. H NMR (400
MHz, CDCl3) δ (ppm): 1.45−1.55 (complex m, 12H, 2,4,6,
8,9,10-H), 1.85 (br s, 3H, 3,5,7-H), 2.18−2.62 (very br s, 8H,
2,3,5,6-Hp), 2.20 (s, 3H, CH3), 3.38 (br s, 2H, α-H), 3.39 (s,
1H, β-H), 7.10−7.13 (m, 3H, 2,6,4′-Har), 7.16−7.20 (m, 2H,
3′, 5′-Har), 7.28−7.30 (m, 2H, 3,5-Har), 7.33−7.35 (m, 2H, 2,
6-H). 13C NMR (100 MHz, CDCl3) δ (ppm): 28.8 (3,5,7-C),
36.8 (2,8,9-C), 41.0 (1,4,6,10-C), 46.0 (CH3), 53.0 (2,6-Cp),
55.1 (3,5-Cp), 62.7 (α-C), 66.1 (β-C), 125.9 (4′-Car), 127.8
(2,6-Car), 128.7 (3′,5′-Car), 129.8 (3,5-Car), 130.0 (2′,6′-Car),
135.6 (1-Car), 140.9 (4-Car), 142.2 (1′-Car). Dihydrochloride,
mp > 260 °C.
Method B. To a stirred suspension of LiAlH4 (1.0 g, 26
mmol) in anhydrous THF (20 mL) was added dropwise a
solution of benzamide 12a (800 mL, 1.92 mmol) in anhydrous
THF (10 mL). The reaction mixture was refluxed for 3 h, then
hydrolyzed by adding ethanol, water, and a solution of NaOH
(10%) at 0 °C. The inorganic material was filtered, and the
filtrate was evaporated. Water was added to the residue, and the
resulting mixture was extracted with DCM. The combined
M
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organic phases were washed with water, dried over Na2SO4, and
concentrated in vacuo. The residue was purified by flash
column chromatography, using as eluent a mixture of CHCl3/
MeOH (9:1) to afford 677 mg of a solid product (yield 85%).
1-Ethyl-4-{4-[α-(1-tricyclo[3.3.1.13 , 7 ]decyl)-
phenylmethyl]phenylmethyl}pirerazine (2b). Benzyl-
amine 2b was prepared from amino alcohol 8b in a similar
way to benzylamine 2a. The product was purified by flash
column chromatography, using as eluent a mixture of DCM/
MeOH (9:1) to afford amino alcohol 2b as a solid product
mmol) in anhydrous DCM (3 mL) at 0 °C. The reaction
mixture was stirred at the same temperature for 30 min and
then at 4 °C for 12 h. Water was added to the mixture, then
acidified with a solution of aqueous HCl (10%) and extracted
with DCM. The combined organic phases were washed with
water, dried over Na2SO4, and concentrated in vacuo. The
residue was purified by flash column chromatography, using
DCM as eluent to afford 1.65 g of a solid product (yield 90%).
Mp 86−88 °C.
1-Methyl-4-(4-bromophenylethyl)piperazine (17a).
Method A. To a solution of tosylate 14 (1.5 g, 4.2 mmol) in
anhydrous THF (15 mL) was added 1-methylpiperazine (2.1 g,
2.1 mmol), and the reaction mixture was refluxed for 8 h. Then
the solvent was removed in vacuo, water was added into the
residue, and the mixture was extracted with DCM. The
combined organic phases were washed with water, dried over
Na2SO4, and concentrated in vacuo. The residue was purified
by flash column chromatography, using as eluent a mixture of
DCM/MeOH (9:1) to afford 750 mg of a solid product (yield
63%). Mp 52−53 °C.
Method B. To a stirred solution of 1-methylpiperazine (1g, 10
mmol) in NMP (4 mL) was added dropwise a solution of
trifluoroacetic acid (1.14 g, 10 mmol) in NMP (4 mL) at 0 °C.
Paraformaldehyde (445 mg, 15 mmol) was added to the
reaction mixture, and the resulting suspension was stirred at
room temperature for 10 min, then heated to 60 °C for 12 h
under an argon atmosphere. After the mixture was cooled to
room temperature, a solution of p-bromobenzylzinc bromide (8
mL, 0.5M in THF, 4 mmol) was added into the reaction
mixture in one portion. The reaction mixture was stirred at
room temperature for 20 min. Then ethyl acetate (40 mL) and
a saturated solution of Na2CO3 were added. The resulting
suspension was stirred at room temperature for 30 min. Then
the white solid was filtered through a pad of Celite and washed
with ethyl acetate. The combined filtrates were washed with a
saturated solution of Na2CO3 and extracted with 1 N HCl. The
acidic layer was washed with ethyl acetate and basified to pH >
10 with a solution of NaOH (50%) in the presence of ethyl
acetate. The organic layer was washed with a saturated solution
of NaHCO3, dried over Na2SO4, and concentrated in vacuo.
The residue was purified by flash column chromatography,
using as eluent a mixture of DCM/MeOH (9:1) to afford 510
mg of amine 17a (yield 45% based on p-bromobenzylzinc
bromide).
1-Ethyl-4-(4-bromophenylethyl)piperazine (17b).
Method A. Phenethylamine 17b was prepared from tosylate
14 in a similar way to amine 17a. Yield 77%. Mp 51−53 °C.
Method B. Phenethylamine 17b was also prepared from 1-
ethylpiperazine trifluoroacetate in a similar way to amine 17a.
Yield 47%.
1-(4-Bromophenylethyl)piperidine (17c). Method A.
Phenylethylpiperidine 17c was prepared from tosylate 14 in a
similar way to piperazine 17a. Oily product, yield 79%.
Method B. Phenylethylpiperidine 17c was also prepared from
piperidine hydrochloride in a similar way to amine 17a. Yield
70%.
α-{4-[2-(4-Methyl-1-piperazinyl)ethyl]phenyl}-α-phe-
nyl-1-tricyclo[3.3.1.13,7]decanemethanol (19a). To a
stirred solution of aryl bromide 17a (1.2 g, 4.2 mmol) in
anhydrous THF (10 mL) was added tert-butyllithium (3 mL,
1.7 M solution in hexane, 5 mmol) at −80 °C under an argon
atmosphere. The reaction mixture was stirred at the same
temperature for 2 h. Then a solution of ketone 5 (1.2 g, 5
1
(yield 60%). Mp 45−47 °C. H NMR (400 MHz, CDCl3) δ
(ppm): 0.98−1.02 (t, 3H, A3X2, JAX ≈ 7.2 Hz, CH3), 1.45−1.57
(m, 12H, 2,4,6,8,9,10-H), 1.86 (br s, 3H, 3,5,7-H), 2.22−2.62
(very br s, 8H, 2,3,5,6-Hp), 2.32−2.37 (q, 2H, A3X2, JAX ≈ 7.2
Hz, CH2CH3), 3.39 (s, 3H, α, β-H), 7.08−7.13 (m, 3H, 2,6,4′-
Har), 7.28−7.30 (m, 2H, 3′,5′-H), 7.33−7.35 (m, 2H, 2′,6′-
Har). 13C NMR (100 MHz, CDCl3) δ (ppm): 11.9 (CH3), 28.8
(3,5,7-C), 36.8 (2,8,9-C), 41.1 (1,4,6,10-C), 52.3 (CH2CH3),
52.7 (2,6-Cp), 52.9 (3,5-Cp), 62.7 (α-C), 66.1 (β-C), 125.9
(4′-Car), 127.8 (2′,6′-Car), 128.7 (3′,5′-Car), 130.0 (3,5,2′,6′-
Car), 135.5 (1-Car), 141.0 (4-Car), 142.2 (1′-Car). Dihydro-
chloride, mp > 260 °C (dec) (EtOH−Et2O).
1-{4-[α-(1-Tricyclo[3.3.1.13,7]decyl)phenylmethyl]-
phenylmethyl}pirerazine (2c). Benzylamine 2c was pre-
pared from amino alcohol 8c in a similar way to benzylamine
2a. The product was purified by flash column chromatography,
using as eluent a mixture of DCM/MeOH (7:3) to afford
amino alcohol 2c as a solid product (yield 63%). Mp 63−65 °C.
1H NMR (400 MHz, CDCl3) δ (ppm): 1.45−1.57 (m, 12H,
2,4,6,8,9,10-H), 1.86 (br s, 4H, 3,5,7-H, 4-Hp), 2.32 (br s, 4H,
2,6-Hp), 2.78−2.80 (t, 4H, J ≈ 4.8 Hz, 3,5-H), 3.36 (s, 2H, α-
H), 3.39 (s, 1H, β-H), 7.10−7.13 (m, 3H, 2, 6, 4′-Har), 7.17−
7.20 (m, 2H, 3′,5′-Har), 7.28−7.30 (m, 2H, 3,5-Har), 7.34−
7.35 (m, 2H, 2′,6′-Har). 13C NMR (100 MHz, CDCl3) δ
(ppm): 28.8 (3,5,7-C), 36.8 (2,8,9-C), 41.1 (1,4,6,10-C), 48.0
(3,5-Cp), 54.3 (2,6-Cp), 63.3 (α-C), 66.1 (β-C), 125.9 (4′-
Car), 127.8 (2,6-Car), 128.7 (3′,5′-Car), 129.8 (3,5-Car), 130.0
(2′,6′-Car), 135.5 (1-Car), 140.9 (4-Car), 142.2 (1′-Car).
Dihydrochloride, mp 240 °C (dec) (EtOH−Et2O).
1-{4-[α-(1-Tricyclo[3.3.1.13,7]decyl)phenylmethyl]-
phenylmethyl}piperidine (2d). Method A. Benzylpiperidine
2d was prepared from amino alcohol 8d in a similar way to
benzylpiperazine 2a. The product was purified by flash column
chromatography, using as eluent a mixture of DCM/MeOH
(9:1) to afford amine 2d as a solid product (yield 87%). Mp
1
56−57 °C. H NMR (400 MHz, CDCl3) δ (ppm): 1.35 (br s,
2H, 4-Hp), 1.45−1.56 (complex m, 16H, 2,4,6,8,9,10-H, 3,5-
Hp), 1.86 (br s, 4H, 3,5,7-H, 4-Hp), 2.30 (br s, 4H, 2,6-Hp),
3.36 (s, 2H, β-H), 3.41 (s, 1H, α-H), 7.09−7.14 (m, 3H, 2,6,4′-
Har), 7.17−7.21 (m, 2H, 3′,5′-H), 7.29−7.31 (m, 2H, 3,5-Har),
7.34−7.37 (2,6-Har). 13C NMR (100 MHz, CDCl3) δ (ppm):
24.3 (4-Cp), 25.8 (3,5-Cp), 28.8 (3,5,7-C), 36.8 (2,8,9-C), 41.1
(1,4,6,10-C), 54.4 (2,6-Cp), 63.4 (β-C), 66.1 (α-C), 125.9 (4′-
Car), 127.8 (2,6-Car), 128.8 (3′,5′-Car), 129.8 (3,5-Car), 130.1
(2′,6′-Car), 131.9 (1-Car), 140.8 (4-Car), 142.3 (1′-Car),
Hydrochloride, mp 244−246 °C (dec) (EtOH−Et2O).
Method B. Benzylpiperidine 2d was also prepared from
benzamide 12d in a similar way to benzylpiperazine 2a. Yield
87%.
4-Bromobenzenethanol 4-Methylbenzenesulfonate
(14). To a stirred solution of p-bromophenethyl alcohol (13)
in a mixture of anhydrous DCM (5 mL) and pyridine (4.7 mL)
was added dropwise a solution of tosyl chloride (1.9 g, 10
N
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mmol) in anhydrous THF (10 mL) was added dropwise into
the mixture, which was stirred for 1 more hour at the same
temperature. The reaction mixture was allowed to gradually
reach ambient temperature and was stirred at the same
temperature for 2 h. The reaction was quenched by adding a
saturated solution of NH4Cl at 0 °C. The organic solvents were
removed in vacuo, and water was added to the residue. The
mixture was extracted with DCM. The combined organic
phases were washed with water, dried over Na2SO4, and
concentrated in vacuo. The residue was purified by flash
column chromatography, using as eluent a mixture of DCM/
MeOH (9:1) to give 870 mg of a foamy solid (yield 47%).
α-{4-[2-(4-Ethyl-1-piperazinyl)ethyl]phenyl}-α-phenyl-
1-tricyclo[3.3.1.13,7]decanemethanol (19b). Amino alcohol
19b was prepared from ketone 5 and aryl bromide 17b in a
similar way as for amino alcohol 19a. Viscous oil. Yield 45%.
α-{4-[2-(1-Piperidinyl)ethyl]phenyl}-α-phenyl-1-
tricyclo[3.3.1.13,7]decanemethanol (19c). Amino alcohol
19c was prepared from ketone 5 and aryl bromide 17c in a
similar way as for amino alcohol 19a. Viscous oil. Yield 48%.
4-[α-(1-Tricyclo[3.3.1.13,7]decyl)phenylmethyl]-
benzenecarboxaldehyde (20). To a stirred solution of aryl
bromide 10 (1.6 g, 4.23 mmol) in anhydrous THF (15 mL)
was added n-butyllithium (2.1 mL, 2.5 M solution in hexane,
5.21 mmol) at −80 °C, under an argon atmosphere. The
reaction mixture was stirred at the same temperature for 2 h,
and then DMF (1.7 mL) was added to the mixture, which was
allowed to gradually reach ambient temperature. The reaction
was quenched by adding a solution of HCl (10%) at 0 °C. The
organic solvents were removed in vacuo, and water was added
to the residue. The mixture was extracted with DCM. The
combined organic phases were washed with water, dried over
Na2SO4, and concentrated in vacuo. The residue was purified
by flash column chromatography, using as eluent a mixture of
n-hexane/Et2O (5:1) to give 1.07 g of a crystalline product
(yield 76.5%). Mp 141−143 °C (ether−n-pentane).
was added sodium cyanide (430 mg, 8.8 mmol). The reaction
mixture was stirred at room temperature for 12 h under an
argon atmosphere. The reaction was quenched with chilled
water, and the mixture was extracted with DCM. The combined
organic phases were washed with water, dried over Na2SO4, and
concentrated in vacuo. The residue was purified by flash
column chromatography, using as eluent a mixture of n-hexane/
Et2O (4:1) to give 650 mg of a crystalline product (yield
86.5%). Mp 168−169 °C (ether).
Method B. To a stirred suspension of potassium tert-butoxide
(943 mg, 8.4 mmol) in dry DME (5 mL) was added a solution
of tosylmethyl isocyanide (TosMIC) (860 mg, 4.4 mmol) in
dry DME (5 mL) at −30 °C under an argon atmosphere. The
reaction mixture was cooled at −60 °C, and then a solution of
aldehyde 20 (1.32 g, 4 mmol) in dry DME (10 mL) was added
dropwise into the mixture, which was stirred at the same
temperature for 1 more hour. The reaction was controlled with
TLC (n-pentane/ether, 4:1), and an excess of TosMIC (100
mg) was added in one portion into the mixture, which stirred
for 30 more minutes. Absolute methanol (12 mL) was added,
and the reaction mixture was allowed to reach room
temperature and then heated at 75−80 °C for 30 min. The
solvents were removed in vacuo. Water and acetic acid (1 mL)
were added into the residue, and the resulting mixture was
extracted with DCM. The combined organic phases were
washed with water and a saturated solution of NaHCO3, dried
over Na2SO4, and concentrated in vacuo. The residue was
purified by flash column chromatography, using as eluent a
mixture of n-hexane/Et2O, 4:1, to give 755 mg of a crystalline
product (yield 55%).
Ethyl 4-[α-(1-Tricyclo[3.3.1.13,7]decyl)phenylmethyl]-
benzeneacetate (24). Phenylacetonitrile 23 (1.4 g, 4.1
mmol) was added to a mixture of saturated ethanolic solution
of hydrogen chloride (12 mL) and absolute ethanol (8 mL).
The reaction mixture was refluxed for 2 h. Then water (8
drops) was added to the mixture, which was heated for 1 more
hour. Ethanol was removed in vacuo. Water was added to the
residue, and the mixture was extracted with ether. The
combined organic phases were washed with water and a
saturated solution of Na2CO3, dried over Na2SO4, and
concentrated in vacuo. The residue was purified by flash
column chromatography, using as eluent a mixture of n-hexane/
Et2O (2:1) to give 1.31 g of a solid product (yield 82%). Mp
81−82 °C.
4-[α-(1-Tricyclo[3.3.1.13,7]decyl)phenylmethyl]-
benzenemethanol (21). To a stirred suspension of LiAlH4
(700 mg, 18.5 mmol) in anhydrous THF (10 mL) was added
dropwise a solution of benzaldehyde 20 (970 mg, 2.94 mmol)
in anhydrous THF (10 mL). The reaction mixture was gently
refluxed for 2 h, then hydrolyzed by adding ethanol, water, and
a solution of NaOH (10%) at 0 °C. The inorganic material was
filtered, and the filtrate was evaporated. Water was added to the
residue, and the resulting mixture was extracted with ether. The
combined organic phases were washed with water, dried over
Na2SO4, and concentrated in vacuo to give 880 mg of a solid
product (yield 90%). Mp 150−151 °C (ether).
1-Methyl-4{4-[α-(1-tricyclo[3.3.1.13 , 7 ]decyl)-
phenylmethyl]phenylacetyl}pirerazine (25a). To a stirred
solution of ethyl ester 24 (670 mg, 1.72 mmol) in ethanol (10
mL), a solution of NaOH (2 g) in a minimum amount of water
was added, and the reaction mixture was refluxed for 2 h.
Ethanol was removed in vacuo. Water was added to the residue,
and the resulting mixture was acidified with an aqueous
solution of HCl (10%) at 0 °C. The mixture was extracted with
ether, and the organic phase was washed with water, dried over
Na2SO4, and concentrated in vacuo. Then thionyl chloride (3
mL) was added and the reaction mixture was gently refluxed for
1 h. The excess of thionyl chloride was removed in vacuo with
the aid of dry benzene. The residue was dissolved in anhydrous
THF (10 mL), and the resulting solution was added dropwise
to a solution of 1-methylpiperazine (2 mL) in anhydrous THF
(10 mL) at 0 °C. The reaction mixture was stirred at room
temperature for 12 h. Then the solvent was removed in vacuo
and water was added to the residue. The mixture was extracted
with DCM. The combined organic phases were washed with
4-[α-(1-Tricyclo[3.3.1.13,7]decyl)phenylmethyl]-
phenylmethyl Chloride (22). To a stirred solution of benzyl
alcohol 21 (2 g, 6 mmol) in dry ether (10 mL) were added a
small amount of anhydrous calcium chloride and then a
solution of thionyl chloride (0.8 g, 6.7 mmol) in dry ether (3
mL). Then, calcium carbonate (0.6 g) was added into the
reaction mixture, which was stirred at room temperature for 12
h. The inorganic material was filtered out and the combined
filtrates were evaporated in vacuo to give a residue (yield almost
quantitative), which was used as such in the next step without
any further purification. TLC (ether−n-pentane) shows one
spot.
4-[α-(1-Tricyclo[3.3.1.13,7]decyl)phenylmethyl]-
benzeneacetonitrile (23). Method A. To a stirred solution of
crude benzyl chloride 22 (1 g, 2.2 mmol) in DMSO (10 mL)
O
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water, dried over Na2SO4, and concentrated in vacuo. The
residue was purified by flash column chromatography, using as
eluent a mixture of DCM/MeOH (97:3) to give 480 mg of a
sticky product (yield 63%).
3c was prepared from amino alcohol 19c in a similar way to
phenethylamine 3a. Flash column chromatography, using as
eluent a mixture of DCM/MeOH (9:1), gave a solid product
(yield 58%). Mp 127−129 °C. 1H NMR (400 MHz, CDCl3) δ
(ppm): 1.35−1.30 (m, 2H, 4-Hp), 1.46−1.56 (complex m,
10H, 2,8,9-H, 3,5-Hp), 1.55 (br s, 6H, 4,6,10-H), 1.86 (br s,
3H, 3,5,7-H), 2.35−2.47 (complex m, 4H, α-H, 2,6-Hp), 2.67−
2.71 (m, 2H, β-H), 3.37 (s, 1H, γ-H), 7.01−7.03 (d, 2H, 3, 5-
Har), 7.07−7.12 (m, 1H, 4′-Har), 7.14−7.21 (m, 2H, 3′, 5′-
Har), 7.25−7.27 (∼d, 2H, 3,5-Har), 7.33−7.34 (∼d, 2H, 2′,6′-
Har). 13C NMR (100 MHz, CDCl3) δ (ppm): 24.4 (4-Cp),
25.9 (3,5-Cp), 28.8 (3,5,7-C), 33.1 (β-C), 36.5 (2,8,9-C), 41.1
(1,4,6,10-C), 54.5 (2,6-Cp), 61.3 (α-C), 66.0 (γ-C), 125.9 (4′-
Car), 127.8 (2,6-Car), 128.2 (3′,5′-Car), 130.0 (3,5,2′,6′-Car),
138.0 (1-Car), 139.8 (4-Car), 142.3 (1′-Car). Hydrochloride,
mp > 250 °C (EtOH−Et2O).
1-Ethyl-4{4-[α-(1-tricyclo[3.3.1.13 , 7 ]decyl)-
phenylmethyl]phenylacetyl}pirerazine (25b). Amide 25b
was prepared in a similar way as for amide 25a using ester 24 as
starting material. Flash column chromatography, using as eluent
a mixture of DCM/MeOH (97:3), gave a sticky solid (yield
60%).
1-{4-[α-(1-Tricyclo[3.3.1.13,7]decyl)phenylmethyl]-
benzeneacetyl}pireridine (25c). Phenylacetylpiperidine 25c
was prepared in a similar way as for amide 25a, using ester 24
as starting material. Flash column chromatography, using as
eluent a mixture of DCM/MeOH (97:3), gave a sticky solid
(yield 71%).
1-Methyl-4-{4-[α-(1-tricyclo[3.3.1.13,7]decyl)-
phenylmethyl]phenylethyl}pirerazine (3a). Method A.
Phenethylamine 3a was synthesized by reduction of amino
alcohol 19a with triethylsilane/trifluoracetic acid in a similar
way to benzylamine 2a from amino alcohol 8a. Flash column
chromatography, using as eluent a mixture of DCM/MeOH
Method B. Phenethylamine 3c was also prepared by reduction
amide 25c with the aid of LiAlH4 in a similar way to
benzylamine 2a. Yield 97%.
4-[α-(1-Tricyclo[3.3.1.13,7]decyl)phenylmethyl]-
benzenepropanol (26). Method A. To a stirred solution of
allylmagnesium chloride (2 M) in THF (8 mL, 16 mmol) was
added dropwise, under an argon atmosphere, a solution of aryl
bromide 10 (1.9 g, 5 mmol) in anhydrous THF (20 mL) in the
presence of catalytic amount of copper(I) iodide. The reaction
mixture was heated to reflux for 4 h, then quenched by adding a
saturated solution of NH4Cl at 0 °C. The mixture was filtered,
and the filtrate was evaporated to remove the solvent. Water
was added to the residue, and the mixture was extracted with
ether. The combined organic layers were washed with water,
dried over Na2SO4, and concentrated in vacuo. The residue was
purified by flash column chromatography, using cyclohexane as
1
(9:1), gave a glassy solid (yield 63%). H NMR (400 MHz,
CDCl3) δ (ppm): 1.46−1.57 (m, 6H, 2,8,9-H), 1.54 (s, 6H,
4,6,10-H), 1.85 (br s, 3H, 3,5, 7-H), 2.23 (s, 3H, CH3), 2.30−
2.75 (very br s, 8H, 2,3,5,6-Hp), 2.49−2.51 (m, 2H, α-H),
2.66−2.68 (m, 2H, β-H), 3.37 (s, 1H, γ-H), 7.01−7.03 (∼d,
2H, 3,5-Har), 7.07−7.11 (m, 1H, 4′-Har), 7.16−7.19 (m, 2H,
3′,5′-Har), 7.25−7.27 (∼d, 2H, 3,5-Har), 7.32−7.34 (∼d, 2H,
2′,6′-Har). 13C NMR (100 MHz, CDCl3) δ (ppm): 28.8 (3,5,7-
C), 33.0 (β-C), 36.8 (2,8,9-C), 41.1 (1,4,6,10-C), 45.9 (CH3),
53.0 (3,5-Cp), 55.0 (2,6-Cp), 60.4 (α-C), 66.0 (γ-C), 126.0 (4′-
Car), 127.8 (2,6-Car), 128.2 (3′,5′-Car), 130.0 (3,5-Car), 130.1
(2′, 6′Car), 137.6 (1-Car), 139.9 (4-Car), 142.3 (1′-Car).
Dihydrochloride, mp > 260 °C (EtOH−Et2O).
1
an eluent, to give 1.1 g of a viscous product. H NMR analysis
showed a mixture of alkene 28 and 1-(α-benzhydryl)-
adamantane (29) (45:55). To a solution of the above mixture
in THF (15 mL) was added a solution of borane (7 mL, 1 M in
THF, 7 mmol) slowly at 0 °C under an argon atmosphere. The
reaction mixture was stirred at room temperature for 3 h, then
quenched by adding chilled water until no foaming was further
formed. A solution of NaOH, 10% (1.5 mL), and H2O2, 30%
(1.5 mL), was added dropwise into the previous mixture. The
resulting mixture was heated to 50−60 °C for 1 h. Then the
solvent was evaporated and water was added to the residue.
The resulting mixture was extracted with ether. The combined
organic layers were washed with water, dried over Na2SO4, and
concentrated in vacuo to give a residue, which was further
purified by flash column chromatography, using cyclohexane as
an eluent, to give 550 mg of 1-(α-benzhydryl)adamantane (29)
Method B. Phenethylamine 3a was also prepared by reduction
of phenylacetamide 25a with the aid of LiAlH4 in a similar way
to benzylamine 2a from benzamide 12a. Yield almost
quantitative.
1-Ethyl-4-{4-[α-(1-tricyclo[3.3.1.13 , 7 ]decyl)-
phenylmethyl]phenylethyl}pirerazine (3b). Method A.
Phenethylamine 3b was prepared from amino alcohol 19b in
a similar way to phenethylamine 3a. Flash column chromatog-
raphy, using as eluent a mixture of DCM/MeOH (8:2), gave a
viscous oil (yield 48%). 1H NMR (400 MHz, CDCl3) δ (ppm):
1.00−1.04 (t, 3H, A3X2, JAX ≈ 7.2 Hz, CH3), 1.44−1.55 (m, 6H,
2,8,9-H), 1.54 (br s, 6H, 4,6,10-H), 1.85 (br s, 3H, 3,5,7-H),
2.30−2.72 (very br s, 8H, 2, 3,5,6-Hp), 2.33−2.39 (q, 2H, A3X2,
JAX ≈ 7.2 Hz, CH2CH3), 2.48−2.52 (m, 2H, α-H), 2.67−2.71
(m, 2H, β-H), 3.37 (s, 1H, γ-H), 7.01−7.03 (∼d, 2H, 2,6-Har),
7.07−7.11 (m, 1H, 4′-Har), 7.15−7.19 (m, 2H, 3′,5′-Har),
7.25−7.27 (∼d, 2H, 3,5-Har), 7.32−7.34 (∼d, 2H, 2′,6′-Har).
13C NMR (100 MHz, CDCl3) δ (ppm): 11.9(CH3), 28.8
(3,5,7-C), 33.1 (β-C), 36.8 (2,8,9-C), 41.1 (1,4,6,10-C), 52.3
(CH2CH3), 52.8 (3,5-Cp), 53.1 (2,6-Cp), 60.5 (α-C), 66.0 (γ-
C), 125.9 (4′-Car), 127.8 (2,6-Car), 128.2(3′,5′-Car), 130.0
(3,5,2′,6′-Car), 137.8 (1-Car), 139.9 (4-Car), 142.3 (1′-Car).
Dihydrochloride, mp > 250 °C (EtOH−Et2O).
1
(yield 36%). Mp 81−83 °C. H NMR (400 MHz, CDCl3) δ
(ppm): 1.48−1.56 (complex m, 12H, 2,4,6, 8,9,10-H), 2.20 (br
s, 3H, 3,5,7-H), 3.40 (s, 1H, α-H), 7.08−7.11 (m, 2H, 4-Har),
7.16−7.20 (m, 4H, 3, 5-Har), 7.34−7.36 (∼d, 4H, 2,6-Har).
13C NMR (100 MHz, CDCl3) δ (ppm): 28.8 (3,5,7-C), 30.3
(1-C), 36.8 (2,8,9-C), 41.1 (4,6,10-C), 66.4 (α-C), 126.0 (4-
Car), 127.8 (3,5-Car), 130.1 (2,6-Car), 142.2 (1-Car). An
amount of 640 mg of crystalline alcohol 26 was obtained (yield
35%) by using a mixture of cyclohexane/ethyl acetate (8:2) as
an eluent. Mp 71−73 °C (n-pentane).
Method B. Phenethylamine 3b was also prepared by
reduction amide 25b with the aid of LiAlH4 in a similar way
to benzylamine 2a. Yield almost quantitative.
Ethyl 4-[α-(1-Tricyclo[3.3.1.13,7]decyl)phenylmethyl]-
benzenepropanoate (31). To a stirred solution of aryl
bromide 10 (1.92 g, 5 mmol) in triethylamine (10 mL) were
added triphenylphosphine (2.62 g, 10 mmol), palladium(II)
acetate (112 mg, 0.5 mmol), and ethyl acrylate (1.2 g, 12
1-{4-[α-(1-Tricyclo[3.3.1.13,7]decyl)phenylmethyl]-
phenylethyl}pireridine (3c). Method A. Phenethylpiperidine
P
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Journal of Medicinal Chemistry
Article
mmol). The reaction mixture was heated to 95−100 °C for 15
h under an argon atmosphere. After cooling, the mixture was
filtered through a Celite pad and the precipitate was washed
well with water and ethyl acetate. The organic layer was
separated, and the inorganic phase was extracted with ethyl
acetate. The combined organic layers were washed with water,
dried over Na2SO4, decolorized with norit, and evaporated. The
residue was purified by flash column chromatography to give
the unreacted aryl bromide 10 (less polar extract), using n-
hexane as eluent, and ethyl cinnamate 30, using a mixture of n-
hexane/Et2O (1:1) as eluent. Ethyl cinnamate 30 was obtained
in 1.54 g yield (77%) as crystalline product. Mp 109−111 °C.
1H NMR analysis of the corresponding cinnamic acid from
ester saponification showed a mixture of (R,E) and (S,E)
diastereomers. 1H NMR δ (ppm): 1.45−1.55 (very br m, 12H,
2,4,6,8,9,10-H), 1.87 (br s, 3H, 3H, 3,5,7-H), 3.44 (s, 1H, γ-H),
6.30−6.34 and 6.36−6.40 (dd, 2H, J ≃ 12 Hz, α-H), 7.12−7.48
(complex dm, 9H, Har), 7.65−7.69 and 7.70−7.74 (dd, 1H, J ≃
12 Hz, β-H). To a solution of ethyl cinnamate 30 in a small
portion of ethyl acetate and ethanol (30 mL) was added
platinum oxide (200 mg), and the reaction mixture was
hydrogenated for 7−8 h under a pressure of 55−60 psi. Then
the catalyst was filtered out and the filtrate evaporated in vacuo.
The residue was purified by flash column chromatograph, using
a mixture of n-hexane/Et2O (4:1) as eluent, to afford 1.4 g of
ethyl propionate 31 as a viscous oil, which was crystallized on
standing (total yield from aryl bromide 10 70%). Mp 43−45
°C.
(method A). Flash column chromatography, using as eluent a
mixture of cyclohexane/ethyl acetate (3:2), gave 3.18 g of diol
33 as a viscous oil (yield 65%).
4-[α-(1-Tricyclo[3.3.1.13,7]decyl)phenylmethyl]-
benzenepropanol (26). Method C. Reduction of diol 33 with
triethylsilane in a similar way as for aryl bromide 10 gave
propanol 26. Flash column chromatography, using as eluent a
mixture of cyclohexane/ethyl acetate (2:1), afforded propanol
26. Yield 71%.
1-Methyl-4-{3-[4-[α-(1-tricyclo[3.3.1.13,7]decyl)-
phenylmethyl]phenyl]propyl}piperazine (4a). A solution
of propanol 26 (1.19 g, 3.29 mmol) in dry DCM (5 mL) was
added dropwise to a stirring mixture of p-toluenesulfonyl
chloride (1.26 g, 5.29 mmol) in DCM/Py (6 mL, 1:1) at 0 °C
under an argon atmosphere. The reaction mixture was stirred at
0 °C for 4 h and then at 10 °C overnight. The above mixture
was acidified with a solution of aqueous HCl (1:4) and
extracted with DCM. The combined organic phases were
washed with water and an aqueous solution of Na2CO3, dried
over Na2SO4, and concentrated in vacuo to afford 1.69 g of a
viscous tosyl ester (yield 97%). TLC analysis with a mixture of
n-hexane/ether (2:1) showed a single spot. To a solution of the
intermediate tosyl ester (1.03 g, 2 mmol) in EtOH absolute (10
mL) was added 1-methylpiperazine (2 mL), and the reaction
mixture was refluxed for 30 min. Then the solvent was
evaporated, water was added to the residue, and the resulting
mixture was extracted with DCM. The combined organic
phases were washed with water and dried over Na2SO4. The
residue was purified by flash column chromatography using a
mixture of DCM/MeOH (98:2) to afford 204 mg of
sulfonamide 27a as a less polar product. Mp 155−157 °C.
1H NMR (400 MHz, CDCl3) δ (ppm): 2.19 (s, 3H, CH3N),
2.35 (s, 3H, 4-CH3C6H4), 2.39−2.42 (t, 2H, J ≃ 5 Hz, 2,6-Hp),
2.95 (br. s, 2H, 3, 5-Hp), 7.24−7.26 (d, 2H, AA′BB′, JAB = JA′B′
≃ 8.2 Hz, JAA′ = JBB′ ≃ 0 Hz, 3,5-Har), 7.55−7.57 (d, 2H,
AA′BB′, JAB = JA′B′ ≃ 8.2 Hz, JAA′ = JBB′ ≃ 0 Hz, 2, 6-Har).
Piperazine 4a was eluted as the more polar extract. Yield 490
4-[α-(1-Tricyclo[3.3.1.13,7]decyl)phenylmethyl]-
benzenepropanol (26). Method B. To a stirred suspension of
LiAlH4 (1.08 g, 28.4 mmol) in anhydrous THF (30 mL) was
added dropwise a solution of ethyl propanoate 31 (1.14 g, 2.84
mmol) in anhydrous THF (20 mL). The reaction mixture was
stirred at room temperature overnight, then hydrolyzed by
adding ethanol, water, and a solution of NaOH (10%) at 0 °C.
The inorganic material was filtered, and the filtrate was
evaporated. Water was added to the residue, and the resulting
mixture was extracted with ether. The combined organic phases
were washed with water, dried over Na2SO4, and concentrated
in vacuo to give 1.02 g of alcohol 26 (yield almost quantitative).
α-Phenyl-α-[4-(2-propenyl)pheny]-1-tricyclo-
[3.3.1.13,7]decanemethanol (32). To a stirred solution of p-
allylphenylmagnesium bromide, which was prepared from
magnesium turnings (0.8 g, 0.032 g at) and p-allylbromoben-
zene (5.9 g, 0.03 mol) in anhydrous THF (40 mL) was added
dropwise a solution of 1-adamantyl phenyl ketone (5) (2.4 g,
0.01 mol) in anhydrous THF (15 mL) under an argon
atmosphere. The mixture was heated at 40 °C for 3 h and then
quenched by adding a saturated solution of NH4Cl in an ice
bath. The inorganic material was filtered off and washed with
THF. The filtrate was evaporated in vacuo, and water was
added to the residue. The resulting mixture was extracted with
ether. The ethereal extracts were washed with water, dried over
Na2SO4, and concentrated. The residue was heated at 70 °C
under high vacuum to remove the volatiles and then further
purified by flash column chromatography using a gradient
eluent from n-hexane to a mixture of n-hexane/Et2O (1:1) and
gave 3.28 g of a waxy solid (yield 91%).
1
mg (55%) of a viscous oil. H NMR (400 MHz, CDCl3) δ
(ppm): 1.45−1.48 (m, 3H, 2,8,9-Hax), 1.54−1.57 (complex m,
9H, 2,8,9-Heq, 4,6,10-H), 1.66−1.75 (m, 2H, β-H), 1.85 (br s,
3H, 3, 5, 7-H), 2.21 (s, 3H, CH3), 2.27−2.31 (t, 2H, J ≃ 7.6 Hz,
α-H), 2.48−2.52 (t, 2H, J ≃ 7.8 Hz, γ-H), 2.25−2.65 (very br s,
8H, 2, 3, 5, 6-Hp), 3.37 (s, 1H, δ-H), 6.99−7.01 (d, 2H, J ≃ 8.1
Hz, 2, 6-Har), 7.08−7.11 (m, 1H, 4′-Har), 7.16−7.20 (m, 2H,
3′, 5′-Har), 7.24−7.26 (d, 2H, J ≃ 8.1 Hz, 3,5-Har), 7.33−7.35
(m, 2H, 2′,6′-Har). 13C NMR (100 MHz, CDCl3) δ (ppm):
28.5 (β-C), 28.7 (3,5,7-C), 33.2 (γ-C), 36.8 (2,8,9-C), 41.1
(1,4,6,10-C), 46.0 (CH3), 53.1 (3,5-Cp), 55.1 (2,6-Cp), 58.1
(α-C), 67.0 (δ-C), 125.9 (4′-Car), 127.8 (2,6,3′,5′-Car), 129.9
(3, 5-Car), 130.0 (2′, 6′-Car), 139.5 (1-Car), 139.6 (4-Car),
142.3 (1′-Car). Dihydrochloride, mp 276 °C (EtOH−Et2O).
1-Ethyl-4-{3-[4-[α-(1-tricyclo[3.3.1.13,7]decyl)-
phenylmethyl]phenyl]propyl}piperazine (4b). Ethylpiper-
azine 4b was prepared from alcohol 26 in a similar way as for
piperazine 4a. The product was purified by flash column
chromatography using a mixture of DCM/MeOH (98:2) to
afford sulfonamide 27b (yield 50%) as a less polar product. Mp
1
4-[α-Hydroxy-α-(1-tricyclo[3.3.1.13 , 7 ]decyl)-
phenylmethyl]benzenepropanol (33). Diol 33 was synthe-
sized by hydroboration of unsaturated alcohol 32 (4.66 g, 0.013
mol) in anhydrous THF (30 mL) with a solution of borane (30
mL, 1 M in THF, 0.030 mol) in a similar way as for alcohol 26
43−44 °C. H NMR (400 MHz, CDCl3) δ (ppm): 0.94−0.97
(t, 3H, A3X2, JAX ≃ 7.2 Hz, CH3CH2), 2.31−2.35 (q, 2H, A3X2,
JAX ≃ 7.2 Hz, CH3CH2), 2.35 (s, 3H, 4-CH3C6H4), 2.44 (br s,
2H, 2, 6-Hp), 2.96 (br s, 2H, 3,5-Hp), 7.23−7.24 (d, 2H,
AA′BB′, JAB = JA′B′ ≃ 7.8 Hz, JAA′ = JBB′ ≃ 0 Hz, 3,5-Har), 7.55−
Q
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Journal of Medicinal Chemistry
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7.57 (d, 2H, AA′BB′, JAB = JA′B′ ≃ 7.8 Hz, JAA′ = JBB′ ≃ 0 Hz,
7.07−7.10 (m, 1H, 4′-Har), 7.14−7.18 (m, 2H, 3′,5′-Har),
7.22−7.24 (d, 2H, J ≃ 8.1 Hz, 3,5-Har), 7.32−7.33 (∼d, 2H, J
≃ 7.6 Hz, 2′,6′-Har). 13C NMR (100 MHz, CDCl3) δ (ppm):
24.4 (4-Cp), 25.8 (3, 5-Cp), 28.3 (β-C), 28.8 (3,5,7-C), 33.3
(γ-C), 36.8 (2, 8,9-C), 41.1 (1,4,6,10-C), 54.5 (2, 6-Cp), 58.9
(α-C), 66.0 (δ-C), 125.9 (4′-Car), 127.8 (2,6,3′,5′-Car), 129.9
(3,5-Car), 130.0 (2′,6′-Car), 139.5 (1-Car), 139.7 (4-Car),
142.4 (1′-Car). Hydrochloride, mp 230−232 °C (dec)
(EtOH−Et2O).
2,6-Har).
1
Piperazine 4b was eluted as an oil (yield 45%). H NMR δ
(ppm): 0.99−1.02 (t, 3H, A3X2, JAX ≃ 7.2 Hz, CH3CH2), 1.46−
1.48 (m, 3H, 2,8,9-Hax), 1.55−1.57 (complex m, 9H, 2,8,9-
Heq, 4,6,10-H), 1.69−1.74 (m, 2H, β-H), 1.85 (br s, 3H, 3, 5,
7-H), 2.28−2.30 (t, 2H, J ≃ 8 Hz, α-H), 2.32−2.35 (q, 2H,
A3X2, JAX ≃ 7.2 Hz, CH3CH2), 2.49−2.51 (t, 2H, J ≃ 7.7 Hz, γ-
H), 2.23−2.55 (very br s, 8H, 2,3,5,6-Hp), 3.37 (s, 1H, δ-H),
6.98−7.00 (d, 2H, J ≃ 8.1 Hz, 2,6-Har), 7.07−7.10 (m, 1H, 4′-
Har), 7.16−7.18 (m, 2H, 3′,5′-Har), 7.23−7.25 (d, 2H, J ≃ 8.1
Hz, 3,5-Har), 7.32−7.34 (m, 2H, 2′, 6′-Har). 13C NMR (100
MHz, CDCl3) δ (ppm): 11.9 (CH3CH2), 28.5 (β-C), 28.8
(3,5,7-C), 33.2 (γ-C), 36.8 (2,8,9-C), 41.1 (1,4,6,10-C), 52.3
(CH3CH2), 52.7 (3,5-Cp), 53.1 (2,6-Cp), 58.1 (α-C), 66.0 (δ-
C), 125.8 (4′-Car), 127.7 (2,6-Car), 127.8 (3′,5′-Car), 129.9
(3,5-Car), 130.0 (2′,6′-Car), 139.5 (1-Car), 139.7 (4-Car),
142.3 (1′-Car). Dihydrochloride, mp 250 °C (EtOH−Et2O).
1-Phenylmethyl-4-{3-[4-[α-(1-tricyclo[3.3.1.13,7]decyl)-
phenylmethyl]phenyl]propyl}piperazine (4c). Ethylpiper-
azine 4c was prepared from alcohol 26 in a similar way as for
piperazine 4a. The product was purified by flash column
chromatography using a mixture of n-hexane/ether (2:1) to
afford sulfonamide 27c (yield 40%) as a less polar product. Mp
1-{3-[4-[α-(1-Tricyclo[3.3.1.13,7]decyl)phenylmethyl]-
phenyl]propyl}piperazine (4d). To a stirred suspension of
N-benzyl derivative 4c (800 mg, 1.5 mmol) and 10% palladium
on charcoal (800 mg) in methanol (20 mL) was added
ammonium formate (480 mg, 7.5 mmol) all at once. The
reaction mixture was refluxed under argon for 1.5 h. The
reaction was monitored by TLC analysis. After consumption of
the starting material, the mixture was cooled to room
temperature and the catalyst was removed by filtration and
washed with chloroform (20 mL). The filtrate was concentrated
in vacuo and the residue was purified by flash column
chromatography using a mixture of DCM/MeOH (5:1) as
eluent to afford 420 mg of piperazine derivative 4d in 64% yield
1
as a viscous oil. H NMR (400 MHz, CDCl3) δ (ppm): 1.44−
1.47 (m, 3H, 2,8,9-Hax), 1.53−1.56 (complex m, 9H, 2,8,9-
Heq, 4,6,10-H), 1.65−1.73 (m, 2H, β-H), 1.84 (br s, 3H, 3,5,7-
H), 2.25−2.28 (t, 2H, J ≃ 7.7 Hz, α-H), 2.35 (br s, 4H, 2,6-
Hp), 2.47−2.51 (t, 2H, J ≃ 7.8 Hz, γ-H), 2.82−2.84 (t, 4H, J ≃
5 Hz, 3,5-Hp), 2.88 (br s, 1H, NH), 3.37 (s, 1H, δ-H), 6.97−
7.00 (d, 2H, J ≃ 8.1 Hz, 2,6-Har), 7.06−7.10 (m, 1H, 4′-Har),
7.15−7.19 (m, 2H, 3′,5′-Har), 7.23−7.25 (d, 2H, J ≃ 8.1 Hz,
3,5-Har), 7.32−7.34 (m, 2H, 2′,6′-Har). 13C NMR (100 MHz,
CDCl3) δ (ppm): 28.2 (β-C), 28.8 (3,5,7-C), 33.2 (γ-C), 36.8
(2,8,9-C), 41.1 (1,4,6,10-C), 45.7 (3,5-Cp), 53.9 (2,6-Cp), 58.5
(α-C), 66.0 (δ-C), 125.9 (4′-Car), 127.8 (2,6,3′,5′-Car), 129.9
(3,5-Car), 130.0 (2′,6′-Car), 139.5 (1-Car), 139.6 (4-Car),
142.3 (1′-Car). Monofumarate, mp 225−227 °C (dec)
(EtOH−Et2O). Dipicrate, mp 230−233 °C (dec) (acetone).
1
48−50 °C. H NMR, δ (ppm): 2.35 (s, 3H, CH3), 2.43−2.45
(t, 4H, J ≃ 4.7 Hz, 2,6-Hp), 2.93 (br s, 4H, 3,5-Hp), 3.40 (s,
2H, α-H),7.15−7.18 (m, 5H, 2′,3′,4′,5′,6′-Har), 7.22−7.23 (d,
2H, AA′BB′, JAB = JA′B′ ≃ 8.1 Hz, JAA′ = JBB′ ≃ 0 Hz, 3, 5-Har),
7.54−7.56 (d, 2H, AA′BB′, JAB = JA′B′ ≃ 8.1 Hz, JAA′ = JBB′ ≃ 0
Hz, 2,6-Har). Benzylpiperazine 4c as a semisolid product (yield
1
55%), H NMR, (400 MHz, CDCl3) δ (ppm): 1.45−1.48 (m,
3H, 2,8,9-Hax), 1.53−1.55 (complex m, 9H, 2,8,9-Heq, 4,6,10-
H), 1.67−1.75 (m, 2H, β-H), 1.85 (br s, 3H, 3,5,7-H), 2.27−
2.31 (t, 2H, J ≃ 7.7 Hz, α-H), 2.47−2.51 (t, 2H, J ≃ 7.8 Hz, γ-
H), 2.22−2.55 (very br s, 8H, 2,3,5,6-Hp), 3.37 (s, 1H, δ-H),
3.43 (s, 2H, ε-H), 6.98−7.00 (d, 2H, J ≃ 8.1 Hz, 2,6-Har),
7.07−7.11 (m, 1H, 4′-Har), 7.16−7.19 (m, 3H, 3′,5′,4″-Har),
7.23−7.26 (m, 6H, 3,5,2″,3″,5″,6″-Har), 7.33−7.35 (m, 2H,
2′,6′-Har). 13C NMR (100 MHz, CDCl3) δ (ppm): 28.4 (β-C),
28.8 (3,5,7-C), 33.2 (γ-C), 36.8, 36.8 (2,8,9-C), 41.1 (1,4,6,10-
C), 52.5 (2,6-Cp), 53.1 (3,5-Cp), 58.1 (α-C), 63.0 (ε-C), 66.0
(δ-C), 125.9 (4′-Car), 127.0 (4″-Car), 127.8 (2,6,3′,5′-Car),
128.2 (3″,5″-Car), 129.2 (2″,6″-Car), 129.9 (3,5-Car), 130.0
(2′,6′-Car), 138.0 (1″-Car),139.5 (1-Car), 139.6 (4-Car), 142.3
(1′-Car). Dihydrochloride·H2O, mp > 250 °C (EtOH−Et2O).
Dipicrate, mp 237−238 °C (dec) (acetone).
ASSOCIATED CONTENT
* Supporting Information
■
S
IR and NMR characterization data of all compounds but finals
and elemental analysis data. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: +30 210 7274527. Fax: +30 210 7274747. E-mail:
gfosc@pharm.uoa.gr.
■
1-{3-[4-[α-(1-Tricyclo[3.3.1.13,7]decyl)phenylmethyl]-
phenyl]propyl}piperidine (4e). Piperidine 4e was prepared
from alcohol 26 in a similar way as for piperazine 4a. The
product was purified by flash column chromatography using
DCM as an eluent to afford sulfonamide 27e (yield 40%) as a
less polar product. Mp 51−53 °C. 1H NMR (400 MHz,
CDCl3) δ (ppm): 1.31−1.35 (m, 2H, 4-Hp), 1.53−1.57(m, 4H,
3, 5-Hp), 2.35 (s, 3H, CH3), 2.87−2.89 (t, 4H, J ≃ 5.5 Hz, 2, 6-
Hp), 7.23−7.25 (d, 2H, AA′BB′, JAB = JA′B′ ≃ 8.3 Hz, JAA′ = JBB′
≃ 0 Hz, 3,5-Har), 7.54−7.56 (d, 2H, AA′BB′, JAB = JA′B′ ≃ 8.3
Hz, JAA′ = JBB′ ≃ 0 Hz, 2,6-Har). Piperidine 4e was eluted as an
oil (yield 55%) using a mixture of DCM/MeOH (98:2) as
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
S.R. is indebted to National and Kapodistrian University of
Athens, Greece, for the award of a scholarship and financial
support: Support of Universities Research Groups Grant KA70|
4|7835 (2008−2009) and Grant KA70|4|10311 (2009−2012).
1
ABBREVIATIONS USED
eluent. H NMR (400 MHz, CDCl3) δ (ppm): 1.33−1.34 (m,
■
2H, 4-Hp), 1.43−1.62 (very br m, 16H, 2, 4, 6, 8,9, 10-H, 3, 5-
Hp), 1.69−1.74 (m, 2H, β-H), 1.84 (br s, 3H, 3, 5, 7-H), 2.23−
2.35 (m, 6H, α-H, 2,6-Hp), 2.45−2.49 (t, 2H, J ≃ 7.7 Hz, γ-H),
3.36 (s, 1H, δ-H), 6.97−6.99 (d, 2H, J ≃ 8.1 Hz, 2, 6-Har),
DCM, dichloromethane; EtOH, ethanol; MeOH, methanol;
Et2O, diethyl ether; DMSO, dimethylsulfoxide; Py, pyridine;
THF, tetrahydrofuran; TFA, trifluoroacetic acid; TosMIC,
tosylmethyl isocyanide; NMR, nuclear magnetic resonance;
R
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neyrodegeneration by sigma receptor 1 ligand (+)-pentazocine. Invest.
Ophthalmol. Visual Sci. 2008, 49 (9), 4154−4161.
(19) Villard, V.; Espallergues, J.; Keller, E.; Alkam, T.; Nitta, A.;
Yamada, K.; Nabeshima, T.; Vamvakides, A.; Maurice, T. Antiamnesic
and neuroprotective effects of the aminotetrahydrofuran derivative
ANAVEX1-41 against amyloid beta(25-35)-induced toxicity in ice.
Neuropsychopharmacology 2009, 34, 1552−1566.
(20) Villard, V.; Espallergues, J.; Keller, E.; Vamvakides, A.; Maurice,
T. Anti-amnesic and neuroprotective potentials of the mixed
muscarinic receptor/sigma 1 (σ1) ligand ANAVEX2-73, a novel
aminotetrahydrofuran derivative. J. Psychopharmacol. 2011, 25 (8),
1101−1117.
(21) Vilner, B. J.; De Costa, B. R.; Bowen, W. D. Cytotoxic effects of
sigma ligands: sigma receptor-mediated alterations in cellular
morphology and viability. J. Neurosci. 1995, 15, 117−134.
(22) Crawford, K. W.; Bowen, W. D. Sigma-2 receptor agonists
activate a novel apoptotic pathway and potentiate antineoplastic drugs
in breast tumor cell lines. Cancer Res. 2002, 62, 313−322.
(23) Colabufo, N. A.; Berardi, F.; Contino, M.; Niso, M.; Abate, C.;
Perrone, R.; Tortorella, V. Antiproliferative and cytotoxic effects of
some σ2 agonists and σ1 antagonists in tumour cell lines. Naunyn-
Schmiedeberg’s Arch. Pharmacol. 2004, 370, 106−113.
(24) Spruce, B. A.; Campbell, L. A.; McTavish, N.; Cooper, A.;
Appleyard, M. V. L; O’Neil, M.; Howie, J.; Samson, J.; Watt, S.;
Murray, K.; McLean., D.; Leslie, N. R.; Safrany, S. T.; Ferguson, M. J.;
Peters, J. A.; Prescott, A. R.; Box, G.; Hayes, A.; Nutley, B.; Raynand,
F.; Downes, C. P.; Lambert, J. J.; Thompson, A. M.; Eccles, S. Small
molecule antagonists of the σ-1 receptor cause selective release of the
death program in tumor and self-reliant cells and inhibit tumor growth
in vitro and in vivo. Cancer Res. 2004, No. 64, 4875−4886.
NMP, N-methyl-2-pyrrolidone; NBS, N-bromosuccinimide;
Et3SiH, triethylsilane; TCA, trichloroacetic acid; HEPES, 4-
(2-hydroxyethyl)-1-piperazineethanesulfonic acid; BCA, bicin-
choninic acid; SRB, sulforhodamine B; HUVEC, human
umbilical vein endothelial cell; hMSC, human mesenchymal
stem cell; NHDF, normal human dermal fibroblast; SCID,
severe combined immune deficiency; 5FU, 5-fluorouracil; GBP,
gabapentin; Gem, gemcitabine; Ara, aracytin
REFERENCES
■
(1) Megalizzi, V.; Le Mercier, M.; Decaetsecker, C. Sigma receptors
and their ligands in cancer biology: overview and new perspectives for
cancer therapy. Med. Res. Rev. 2012, 32 (2), 410−427.
(2) Maurice, T.; Su, T. P. The pharmacology of sigma-1 receptors.
Pharmacol. Ther. 2009, 124, 195−206.
(3) Van Waarde, A.; Rybczynska, A. A.; Ramakrishnan, N.; Ishiwata,
K.; Elsinga, P. H.; Dierckx, R. A. Sigma receptors in oncology:
therapeutic and diagnostic applications of sigma ligands. Curr. Pharm.
Des. 2010, 16 (31), 3519−3537.
(4) Tsai, S. Y.; Hayashi, T.; Mori, T.; Su, T. P. Sigma-1 receptor
charepones and diseases. Cent. Nerv. Syst. Agents Med. Chem. 2009, 9
(3), 184−189.
(5) Bowen, D. W. Sigma receptors: recent advances and new clinical
potentials. Pharm. Acta Helv. 2000, 74, 211−218.
(6) Bem, T. W.; Thomas, E. G.; Mamone, Y. J.; Homan, M. S.; Levy,
K. B.; Johnson, E. F.; Coscia, J. C. Overexpression of sigma receptors
in nonneural human tumors. Cancer Res. 1991, 51, 6558−6562.
(7) Vilner, B. J.; John, C. S.; Bowen, W. D. Sigma-1 and sigma-2
receptors are expressed in a wide variety of human and rodent tumor
cell lines. Cancer Res. 1995, 55, 408−413.
(8) John, C. S.; Vilner, B. J.; Schwartz, A. M.; Bowen, W. D.
Characterization of sigma receptor binding sites in human biopsied
solid breast tumors. J. Nucl. Med. 1996, 37, 267P.
(9) Zamora, P. O.; Moody, T. W.; John, C. S. Increased binding to
sigma sites of N-[1′(2-piperidinyl)ethyl-4-[I-125]-iodobenzamide (I-
125-PAB) with onset of tumor cell proliferation. Life Sci. 1998, 63,
1611−1618.
(10) Wang, B.; Rouzier, R.; Albarracin, C. T.; Sahin, A.; Wagner, P.;
Yang, Y.; Smith, T. L.; Meric-Bernstam, F.; Aldaz, C. M.; Hortobagyi,
G. N.; Puszta, L. Expression of sigma-1 receptors in human breast
cancer. Breast Cancer Res. Treat. 2004, 87 (3), 205−214.
(11) Aydar, E.; Onganer, P.; Perrett, R.; Djamgoz, M. B.; Palmer, C.
P. The expression and functional characterization of sigma (sigma) 1
receptors in breast cancer cell lines. Cancer Lett. 2006, 242, 245−257.
(12) Colabufo, N. A.; Berardi, F.; Contino, M.; Ferorelli, S.; Niso, M.;
Perrone, R; Paqliarulo, A.; Saponaro, P.; Paqliarulo, V. Correlation
between σ2 receptor protein expression and histopathologic grade in
human bladder cancer. Cancer Lett. 2006, 237, 83−88.
(13) Tucci, M.; Quatraro, C.; Dammacco, F.; Silvestris, F. Role of
active drug transporters in refractory multiple myeloma. Curr. Top.
Med. Chem. 2009, 9, 218−224.
(14) Martin, R. W.; Eades, G. C.; Thompson, E. R.; Huppler, E. P.;
Gilbert, E. P. The effects of morphine- and nalorphine-like drugs in the
nondependent and morphine-dependent chronic spinal dog. J.
Pharmacol. Exp. Ther. 1976, 197, 517−532.
(15) Hellewell, S. B.; Bruce, A.; Feinstein, J.; Orringer, J.; Williams,
W.; Bowen, W. D. Rat liver and kidney contain high densities of sigma-
1 and sigma-2 receptors: characterization by ligand binding and
photoaffinity labeling. Eur. J. Pharmacol. 1994, 268, 9−18.
(16) Hayashi, T.; Su, T. P. Sigma-1 receptor chaperones at the ER-
mitochondrion interface regulate Ca(2+) signaling and cell survival.
Cell 2007, 131, 596−610.
(17) Yang, S.; Bhardwaj, A.; Cheng, J.; Alkayed, N. J.; Hurn, P. D.;
Kirsch, J. R. Sigma receptor agonists provide neuroprotection in vitro
by preserving bcl-2. Anesth. Analg. 2007, 104, 1179−1184.
(18) Smith, S. B.; Duplantier, J.; Dun, Y.; Mysona, B.; Roon, P.;
Martin, P. M.; Ganapathy, V. In vivo protection against retinal
(25) Ostenfeld, M. S.; Fehrenbacher, N.; Høy
Thomsen, C.; Farkas, T.; Jaatela, M. Effective tumor cell death by
́
er-Hansen, M.;
̈
̈
̈
sigma-2 receptor ligand siramesine involves lysosomal leakage and
oxidative stress. Cancer Res. 2005, 65, 8975−8983.
(26) Azzariti, A.; Colabufo, N. A.; Berardi, F.; Porcelli, L.; Niso, M.;
Simone, G. M.; Perrone, R.; Paradiso, A. Cyclohexylpiperazine
derivative PB28, a sigma2 agonist and sigma1 antagonist receptor,
inhibits cell growth, modulates P-glycoprotein, and synergizes with
anthracyclines in breast cancer. Mol. Cancer Ther. 2006, 5, 1807−1816.
(27) Megalizzi, V.; Mathieu, V.; Mijatovic, T.; Gailly, P.; Debeir, O.;
De Neve, N.; Van Damme, M.; Bontempi, G.; Haibe-Kains, B.;
Decaestecker, C.; Kondo, Y.; Kiss, R.; Lefranc, F. 4-IBP, a sigma1
receptor agonist, decreases the migration of human cancer cells,
including glioblastoma cells, in vitro and sensitises them in vitro and in
vivo to cytotoxic insults of proapoptotic and proautophagic drugs.
Neoplasia 2007, 9, 358−369.
(28) Kashiwagi, H.; McDunn, J. E.; Simon, P. O., Jr; Goedegebuure,
P. S.; Xu, J.; Jones, L.; Chang, K.; Johnston, F.; Trinkaus, K.;
Hotchkiss, R. S.; Mach, R. H.; Hawkins, W. G. Selective sigma-2
ligands preferentially bind to pancreatic adenocarcinomas: applications
in diagnostic imaging and therapy. Mol. Cancer 2007, 6, 48−60.
(29) Piergentili, A.; Amantini, C.; Del Bello, F.; Giannella, M.;
Mattioli, L.; Palmery, M.; Perfurmi, M.; Pigini, M.; Santoni, G.; Tucci,
P.; Zotti, M.; Quaglia, W. Novel highly potent and selective sigma 1
receptor antagonists related to spipethiane. J. Med. Chem. 2010, 53 (3),
1261−1269.
(30) Hornick, J. R.; Xu, J.; Vangveravong, S.; Tu, Z.; Mitchem, J. B.;
Spitzer, D.; Goedegebuure, P.; Mach, R. H.; Hawkins, W. G. The novel
sigma-2 receptor ligand SW43 stabilizes pancreas cancer progression in
combination with gemcitabine. Mol. Cancer 2010, 22 (9), 298.
(31) Riganas, S.; Papanastasiou, I.; Foscolos, G. B.; Tsotinis, A.;
Bourguignon, J. J.; Serin, G.; Mirjolet, J. F.; Dimas, K.; Kourafalos, V.
N.; Eleutheriades, A.; Moutsos, V. I.; Khan, H.; Georgakopoulou, S.;
Zaniou, A.; Prassa, M.; Theodoropoulou, M.; Pondiki, S.; Vamvakides,
A. Synthesis, σ(1), σ(2)-receptors binding affinity and antiproliferative
action of new C1-substituted adamantanes. Bioorg. Med. Chem. 2012,
20 (10), 3323−3331.
(32) Renaudo, A.; Watry, V.; Chassot, A. A.; Ponzio, G.; Ehrenfeld,
J.; Soriani, O. Inhibition of tumor cell proliferation by σ ligands is
S
dx.doi.org/10.1021/jm3013008 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
associated with K+ channel inhibition and p27kip1 accumulation. J.
Pharmacol. Exp. Ther. 2004, 311, 1105−1114.
(33) Renaudo, A.; L’ Hoste, S.; Guizouarn, H.; Borgese, F.; Soriani,
O. Cancer cell cycle modulated by a functional coupling between
sigma-1 receptors and Cl− channels. J. Biol. Chem. 2007, 282, 2259−
2267.
(34) Johannessen, M.; Ramachandran, S.; Riemer, L.; Ramos-
Serrano, A.; Ruoho, A. E.; Jackson, M. B. Voltage-gated sodium
channel modulation by sigma-receptors in cardiac myocytes and
heterologous systems. Am. J. Physiol.: Cell Physiol. 2009, 296, 1049−
1057.
(35) Johannessen, M.; Fontanilla, D.; Mavlyutov, T.; Ruoho, A. E.;
Jackson, M. B. Antagonist action of progesterone at σ-receptors in the
modulation of voltage-gated sodium channels. Am. J. Physiol.: Cell
Physiol. 2011, 300 (2), C328−37.
(36) Palmer, C. P.; Mahen, R.; Schnell, E.; Djamgoz, M. B. A.; Aydar,
E. Sigma-1 receptors bind cholesterol and remodel lipid rafts in breast
cancer cell lines. Cancer Res. 2007, 67, 11166−11175.
(37) Hayashi, T.; Su, T. P. Cholesterol at the endoplasmic reticulum:
roles of the sigma-1 receptor charepone and implications thereof in
human diseases. Subcell. Biochem. 2010, 51, 381−398.
(38) Ding, Y.; Brackenbury, W. J.; Onganer, P. U.; Montano, X.;
Porter, L. M.; Bates, L. F.; Djangoz, M. B. Epidermal growth factor
upregulates motility of Mat-LyLu rat prostate cancer cells partially via
voltage-gated Na+ channel activity. J. Cell. Physiol. 2008, 215, 77−81.
(39) Onkal, R.; Djamgoz, M. B. Molecular pharmacology of voltage-
gated sodium channel expression in metastatic disease: clinical
potential of neonatal Nav1.5 in breast cancer. Eur. J. Pharmacol.
2009, 625, 206−219.
(40) Jansson, K. H.; Lynch, J. E.; Lepori-Bui, N.; Czymmek, K. J.;
Duncan, R. L.; Sikes, R. A. Overexpression of the Vssc-associated
CAM, beta-2, enhances LNCaP cell metastasis associated behavior.
Prostate 2012, 72 (10), 1080−1092.
(41) Hanner, M.; Moebius, F. F.; Flandorfer, A.; Knaus, H. G.;
Striessnig, J.; Kempner, E.; Glossmann, H. Purification, molecular
cloning, and expression of the mammalian sigma1 binding site. Proc.
Natl. Acad. Sci. U.S.A. 1996, 93 (15), 8072−8077.
(42) Cobos, E. J.; Entrena, J. M.; Nieto, F. R.; Cendan, C. M.; Del
Pozo, E. Pharmacology and therapeutic potential of sigma(1) receptor
ligands. Curr. Neuropharmacol. 2008, 6 (4), 344−366.
(43) Ablordeppey, S. Y.; Fischer, J. B.; Law, H.; Glennon, R. A.
Probing the proposed phenyl-A region of the sigma-1 receptor. Bioorg.
Med. Chem. 2002, 10, 2759−2765.
(44) Ganapathy, V.; Ganapathy, M. E.; Inoue, K. In Sigma Receptors:
Chemistry in Cell Biology and Clinical Implications; Matsumoto, R. R.,
Bowen, W. D., Su, T.-P., Eds.; Springer: New York, 2007; pp 99−112.
(45) Papaconstantinou-Garoufalias, S.; Foscolos, G. B.; Costakis, E.
Adamantane analogs of diphenhydramine. Chim. Chron., New Ser.
1984, 13, 225−237.
(46) Stetter, H.; Rauscher, E. Uber verbindungen mit urotropin-
struktur XVII. Zur kenntnkder adamantan-carbosaure-(I). Chem. Ber.
1960, 93, 1161−1166.
(47) Ku, Y.-Y.; Grieme, T.; Pu, Y. M.; Bhatia, A. V.; King, S. A. A
simple one-pot procedure for the imminium salt formation: an
efficient route to [beta]-arylethylamines. Tetrahedron Lett. 2005, 46,
1471−1474.
(48) Oldenziel, O. H.; Van Leusen, D.; Van Leusen, A. M. Chemistry
of sulfonylmethyl isocyanides. A general one-step synthesis of nitriles
from ketones using tosylmethyl isocyanide. Introduction of a one
carbon unit. J. Org. Chem. 1977, 42, 3114−3118.
(51) Heck, R. F.; Nolley, J. P., Jr. Palladium-catalyzed vinylic
hydrogen substitution reactions with aryl, benzyl and styryl halides. J.
Org. Chem. 1972, 37, 2320−2322.
(52) Mizoroki, T.; Mori, K.; Bulll, O. A. Arylation of olefin with
aryliodide catalyzed by palladium. Chem. Soc. Jpn. 1971, 44, 581−583.
(53) Colabufo, N. A.; Contino, M.; Inglese, C.; Niso, M.; Perrone, R.;
Roperto, S.; Roperto, F. In vitro and ex vivo characterization of sigma-
1 and sigma-2 receptors: agonists and antagonists in biological assays.
Cent. Nerv. Syst. Agents Med. Chem. 2009, 9, 161−171.
(54) Zeng, C.; Rothfuss, J.; Zhang, J.; Chu, W.; Vangveravong, S.; Tu,
Z.; Pan, F.; Chang, K. C.; Hotchkiss, R.; Mach, R. H. Sigma-2 ligands
induce tumour cell death by miltiple signalling pathways. Br. J. Cancer
2012, 106 (4), 693−701.
(55) De la Puente, B.; Nadal, X.; Portillo-Salido, E.; San
R.; Ovalle, S.; Palacios, G.; Muro, A.; Romero, L.; Entrena, J. M.;
Baeyens, J. M.; Lopez-García, J. A.; Maldonado, R.; Zamanillo, D.;
́
chez-Arroyos,
́
Vela, J. M. Sigma-1 receptors regulate activity-induced spinal
sensitization and neuropathic pain after peripheral nerve injury. Pain
2009, 145 (3), 294−303.
(56) Díaz, J. L.; Zamanillo, D.; Corbera, J.; Baeyens, J. M.; Aldonado,
R.; Pericas, M. A.; Vela, J. M.; Torrens, A Selective sigma-1 (sigma1)
̀
receptor antagonists: emerging target for the treatment of neuropathic
pain. Cent. Nerv. Syst. Agents Med. Chem. 2009, 9 (3), 172−183.
́
(57) Cendan, C. M.; Pujalte, J. M.; Portillo-Salido, E.; Montoliu, L.;
Baeyens, J. M. Formalin-induced pain is reduced in sigma(1) receptor
knockout mice. Eur. J. Pharmacol. 2005, 511 (1), 73−74.
(58) Roh, D. H.; Kim, H. W.; Yoon, S. Y.; Seo, H. S.; Kwon, Y. B.;
Kim, K. W.; Han, H. J.; Beitz, A. J.; Na, H. S.; Lee, J. H. Intrathecal
injection of the sigma(1) receptor antagonist BD1047 blocks both
mechanical allodynia and increases in spinal NR1 expression during
the induction phase of rodent neuropathic pain. Anesthesiology 2008,
109 (5), 879−889.
(59) Matsumoto, M.; Inoue, M.; Hald, A.; Xie, W.; Ueda, H.
Inhibition of paclitaxel-induced A-fiber hypersensitization by gaba-
pentin. J. Pharmacol. Exp. Ther. 2006, 318 (2), 735−740.
(60) Coderre, T. J.; Vaccarino, A. L.; Melzack, R. Central nervous
system plasticity in the tonic pain response to subcutaneous formalin
injection. Brain Res. 1990, 535 (1), 155−158.
(61) Laughlin, T. M.; Tram, K. V.; Wilcox, G. L.; Birnbaum, A. K.
Comparison of antiepileptic drugs tiagabine, lamotrigine and
gabapentin in mouse models of acute, prolonged and chronic
nociception. J. Pharmacol. Exp. Ther. 2002, 302 (3), 1168−1175.
(62) Ganapathy, M. E.; Prasad, P. D.; Huang, W.; Seth, P.; Leibach,
F. H.; Ganapathy, V. Molecular and ligand-binding characterization of
the sigma -receptor in the Jurkat human T lymphocyte cell line. J.
Pharmacol. Exp. Ther. 1999, 289, 251−260.
(63) Bowen, W. D.; De Costa, B. R.; Hellewell, S. B.; Walker, M.;
Rice, K. C. [3H](+)-Pentazocine: a potent and highly selective
benzomorphan-based probe for sigma-1 receptors. Mol. Neuro-
pharmacol. 1993, 3, 117−126.
(64) Brown, G. B. 3H-Batrachotoxinin-A benzoate binding to voltage-
sensitive sodium channels: inhibition by the channel blockers
tetrodotoxin and saxitoxin. J. Neurosci. 1986, 6, 2064−2070.
(65) Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.;
Vistica, D.; Warren, J. T.; Bokesch, H.; Kenney, S.; Boyd, M. R. New
coloremetric cytotoxicity assay for anticancer-drug screening. J. Natl.
Cancer Inst. 1990, 82 (13), 1107−1112.
(66) Keepers, Y. P.; Pizao, P. E.; Peters, G. J.; Van Ark-Otte, J.;
Winograd, B.; Pinedo, H. M. Comparison of the sulforhodamine B
protein and tetrazolium (MMT) assays for in vitro chemosensitivity
testing. Eur. J. Cancer 1991, 27 (7), 897−900.
(49) Van Leusen, A. M.; Oomkes, P. G. One-step conversion of
aldehydes to nitriles. Introduction of a one-carbon unit. Synth.
Commun. 1980, 10, 399−403.
(50) Riganas, S.; Papanastasiou, I.; Foscolos, G. B.; Tsotinis, A.;
Dimas, K.; Kourafalos, V. N.; Eleutheriades, A.; Moutsos, V. I.; Khan,
H.; Prassa, M.; Georgakopoulou, S.; Zaniou, A.; Theodoropoulou, M.;
Mantelas, A.; Pondiki, S.; Vamvakides, A. New adamantane derivatives
with sigma affinity and antiproliferative activity. Med. Chem. 2012, 8,
569−586.
(67) Dimas, K.; Hatziantoniou, S.; Tseleni, S.; Khan, H.;
Georgopoulos, A.; Alevizopoulos, K.; Wyche, J. H.; Pantazis, P.;
Demetzos, C. Sclareol induces apoptosis in human HCT116 colon
cancer cells in vitro and suppression of HCT116 tumor growth in
immunodeficient mice. Apoptosis 2007, 12 (4), 685−694.
(68) Overholser, J. P.; Prewett, M. C.; Hooper, A. T.; Waksal, H. W.;
Hicklin, D. J. Epidermal growth factor receptor blockade by antibody
T
dx.doi.org/10.1021/jm3013008 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
IMC-C225 inhibits growth of a human pancreatic carcinoma xenograft
in nude mice. Cancer 2000, 89 (1), 74−82.
U
dx.doi.org/10.1021/jm3013008 | J. Med. Chem. XXXX, XXX, XXX−XXX