Synthesis, σ1, σ2-receptors binding affinity and antiproliferative action of new C1-substituted adamantanes

Article abstract of DOI:10.1016/j.bmc.2012.03.038

The synthesis of N-{4-[a-(1-adamantyl)benzyl]phenyl}piperazines 2a-e is described. The in vitro antiproliferative activity of most compounds against main cancer cell lines is significant. The σ₁, σ₂-receptors and sodium channels binding affinity of compounds 2 were investigated. One of the most active analogs, 2a, had an interesting in vivo anticancer profile against the BxPC-3 and Mia-Paca-2 pancreas cancer cell lines with caspase-3 activation, which was associated with an anagelsic activity against the neuropathic pain.

Full text of DOI:10.1016/j.bmc.2012.03.038

Bioorganic & Medicinal Chemistry 20 (2012) 3323–3331
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, r1
,
r
2-receptors binding affinity and antiproliferative action of
new C1-substituted adamantanes
Stefanos Riganas a,e, Ioannis Papanastasiou a, George B. Foscolos a, , Andrew Tsotinis a,
Jean-Jacques Bourguignon b, Guillaume Serin c, Jean-François Mirjolet c, Kostas Dimas d,
Vassilios N. Kourafalos e, Andreas Eleutheriades e, Vassilios I. Moutsos e, Humaira Khan e,
Stavroula Georgakopoulou e, Angeliki Zaniou e, Margarita Prassa e, Maria Theodoropoulou e,
Stavroula Pondiki e, Alexandre Vamvakides e
⇑
a Faculty of Pharmacy, Department of Pharmaceutical Chemistry, National and Kapodistrian University of Athens, Panepistimioupoli-Zografou, 157 71 Athens, Greece
b Laboratoire de Pharmacochimie de la Communication Cellulaire, UMR 7175 du CNRS, Université Louis Pasteur, Faculté de Pharmacie, 74, route du Rhin, 67401 Illkirch Cedex, France
c Oncodesign S.A., 20 rue Mazen, F-21076, Dijon, France
d Division of Pharmacology, FBRAA, 11527 Athens, Greece
e Anavex Life Sciences, 27 Marathonos Avenue, 15351 Pallini, Athens, Greece
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of N-{4-[a-(1-adamantyl)benzyl]phenyl}piperazines 2a–e is described. The in vitro antipro-
Received 22 December 2011
Revised 14 March 2012
Accepted 18 March 2012
Available online 24 March 2012
liferative activity of most compounds against main cancer cell lines is significant. The r1, r2-receptors
and sodium channels binding affinity of compounds 2 were investigated. One of the most active analogs,
2a, had an interesting in vivo anticancer profile against the BxPC-3 and Mia-Paca-2 pancreas cancer cell
lines with caspase-3 activation, which was associated with an anagelsic activity against the neuropathic
pain.
Keywords:
C1-substituted adamantanes
Synthesis
Ó 2012 Elsevier Ltd. All rights reserved.
r1,r2-Binding affinity
In vitro antiproliferative
In vivo anticancer activity
1. Introduction
cytotoxic activity for
r
1-antagonists,15,16 r2 putative agonists,
r2 ligands15,19,21 and even one r1 agonist.22 More
specifically, Spruce et al.16 have shown that
1-antagonists induce
17–20
mixed r1
/
Sigma(
r
)-receptors are expressed to a greater degree in tumours
r
than they are in the surrounding normal tissue.1–3 Although they
have been considered as opioid receptors, the sigma receptors have
been classified as distinct pharmacological entity and their function
shown to be unrelated to the function of the opioid receptors.4–6
Based on the ligand selectivity in the binding assays, two subtypes,
caspase-dependent apoptosis, which is in accord with recent obser-
vations that r1 agonists prevent caspase-3 activation.9,10 On the
other hand,
r2-agonists have been shown to activate a caspase-
independent apoptotic pathway and have been proposed as puta-
tive anti-cancer drugs.17,18 Recent data have also suggested the
sigma-1 (
r
1) and sigma-2 (
r
2) receptors, were identified.5–7
importance of the
Clꢀ, Ca2+ 23–27
and
concerning the proliferation of cancer cells. The Na+ channels are, in
r
1-receptor modulated ion channels (Na+, K+,
Furthermore,
r
1-receptors have been shown to be involved in
)
r
1-receptor binding of cholesterol in lipid rafts28
programmed cell death (apoptosis), with
r1-agonists (activators
according to their molecular function)8 being antiapoptotic
and neuroprotective, with putative anti-neurodegenerative
particular, modulated by r1 receptors and implicated in the adhe-
sion, migration and apoptosis of cancer cells.25–27 These investiga-
tions provide new areas in which to elucidate the mechanism of
activity.6,9,10
r1-Antagonists (deactivators) or r2-agonists are proa-
poptotic and can, because of the high expression of r1 and r2
-
action of r-ligands and their putative role as therapeutic anticancer
receptors in the rapidly proliferating cancer cells11–14 act as puta-
tive anticancer drugs by inducing cell death via apoptosis.15–18
Presently, there is considerable evidence of antiproliferative and
agents. Classical SAR studies indicated that the presence of a cyclo-
alkyl or aryl group attached to the cationic amine center via a linker
of 3 to 5-membered chain (including a heteroatom), was essential
for affinity at the
r
-receptors.29,30
In the course of our program directed towards the design and
synthesis of new pharmacological active adamantane amino
⇑
Corresponding author. Tel.: +30 210 7274527; fax: +30 210 7274747.
E-mail address: gfosc@pharm.uoa.gr (G.B. Foscolos).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.03.038

3324
S. Riganas et al. / Bioorg. Med. Chem. 20 (2012) 3323–3331
derivatives, the above structural requirement for
r
-receptor affin-
7
R
8
N
6
5
ity prompted us to investigate the synthesis of the adamantane
piperazines 2 and 3. In the piperazine 2, one benzene ring is linked
to the second piperazine nitrogen by three atoms (N, 2C), while in
3, two benzene rings are linked to the second piperazine nitrogen
by a chain of 4 atoms (C, N, 2C). Our synthetic route gave mainly
compounds of type 2 with, in some cases, compounds of type 3
as by-products. Compounds 2 were investigated for their binding
10
1 α H
9
+
α
N
2
3
4
a
N
2a-d
3a,d
Cl
N
R
affinity at both r1 and
r2-receptors and we carried out an
O
in vitro study of their antiproliferative and cytotoxic activity. Final-
ly, the in vivo effect of 2a on pancreas cancer cells xenografts was
investigated in parallel with a prototypical study of its effect in the
formalin putative neuropathic pain amplified by the widely used
anticancer drug Paclitaxel.
b
1
N
H
+
4
N
5
2. Results and discussion
2.1. Chemistry
O
a: R=Me; b: R=Et; c: R=cyclohexyl; d: R=benzyl
Scheme 1. Reagents and conditions: (a) Monosubstituted piperazine, 130 °C, 12 h;
(b) morpholine, 110 °C, 14 h.
Reaction of excess 1-substituted piperazine with a-(1-adaman-
tyl)benzydryl chloride (1)31,32 gave 2 as the main product with
only minor amounts of 3. In the case of the reactions of 1-methyl
and 1-benzylpiperazine could these minor products be isolated
(3a,d). The main product 2 could be readily distinguished from
the minor product 3 in the 1H NMR spectrum as it had only nine
aromatic hydrogens compared with the ten required for 3, with
the first compound showing the characteristic methine proton on
Cl
H
1
Ca
. The structure of 2 was further confirmed by the 13C NMR
spectra.
For comparative purposes, derivative 4 (60%) was prepared by
N R
HN
H
the reaction of 1 with morpholine, the isomer 5 again being ob-
tained as the minor product (13%) (Scheme 1). The compounds
3a,d and 5 are unstable in the presence of acids and protic solvents.
A plausible pathway for the conversion of 1 to analogues 2, is
outlined in Scheme 2. According to the proposed mechanism, the
nucleophilic reagents (piperazines) attack preferentially the para-
position of one of the benzene rings, as the tertiary carbocation site
is sterically hindered by the bulky adamantane and phenyl
substituents.
N
H
N
N
2
R
N
R
Scheme 2. Plausible mechanism for the formation of piperazines 2a–d.
Reductive debenzylation of the benzyl derivative 2d with
ammonium formate and Pd on charcoal gave the adduct 2e
(Scheme 3).
H
H
2.2. Biology
a
N
N
The results for the in vitro antiproliferative activity of the deriv-
atives 2a–e and 4 are shown in Tables 1a, 1b. Also, their affinities
N
N
H
for both the r1 and
summarised in Table 2.
As is clear from Table 2, the piperazine analogues 2a–c and 2e
have a notable binding affinity for the r1 and 2-receptors and
also site 2 of the Na+ channels. The benzyl derivative 2d shows a
much lower affinity for
1-receptors and the Na+ channels. None
r
2-receptors and site 2 of Na+ channels are
PhH2C
2d
2e
Scheme 3. Reagents and conditions: (a) Pd/C, HCO2NH4, MeOH, reflux, 3 h.
r
selectivity and especially mode of action (agonistic or antagonistic)
are needed. However, it must be considered that, in fact, no vali-
dated in vitro isolated organ tests exist for the functional charac-
r
of these interactions are observed in the case of the morpholine
analogue 4. This difference in the activity of 2a–e and 4 may be
attributed to the presence of the second piperazine nitrogen atom
in 2a–e (Schemes 1 and 3). Analogue 2e seems to have moderate
terization of
r
ligands as agonists or antagonists.33 It is worth
noting that in the derivatives 2, the introduction of a bulky substi-
tuent on the second piperazine nitrogen (2c, d) causes a reduction
of cytotoxic and antiproliferative activity, which is accompanied by
binding activity at the
the 2-receptors.
These results, in conjunction with the in vitro antiproliferative
and cytotoxic action of the new adamantane piperazines
(Table 1a and 1b), imply that the new molecules act as mixed
r2 ligands, with a notable selectivity for 1- receptors. As the
r1-receptors, whilst it appears inactive at
r
a decrease of the
r1 affinity of 2d. However, the high affinity of 2c,
for the r1 receptors (more than 10-fold, compared to 2a), could
indicate that a decisive factor, in the expression of the cytotoxic
activity of these ligands may be also the intrinsic activity of these
2
r1/
r
ligands on
r1 and r2 receptors, that is, the type of activity (agonist
binding data do not classify the ligands as agonists or antagonists
and thus continuing attempts to define the characteristics of
adamantane ligands required for receptor activation, subtype
or antagonist), its efficacy (full or partial) and the interaction be-
tween r1 and r2 intrinsic activities. In conclusion, a rational SAR

S. Riganas et al. / Bioorg. Med. Chem. 20 (2012) 3323–3331
3325
Table 1a
Summary results from the in vitro screening of antiproliferative activity of tested derivatives on colon, prostate, breast, ovarian and central nervous system cancer cell lines
Compound
Values
Colon
Prostate
DU145
Breast
MCF7
Ovarian
OVCAR-5
CNS
HCT-116
HCT-15
PC3
IGROV-1
SF268
SF295
U251
2a
2b
2c
2d
2e
4
LC50
TGI
GI50
LC50
TGI
GI50
LC50
TGI
GI50
LC50
TGI
GI50
LC50
TGI
GI50
LC50
TGI
GI50
LC50
TGI
100
100
100
65.52
6.43
99.57
58.69
17.81
100
32.9
6.8
100
65.61
9.01
3.17
16.93
11.56
100
59
9.23
100
55
100
58.49
8.83
100
49
94.5
41.9
8
77.8
24.5
6
100
85.22
48.12
11.03
82.21
50.9
9.14
6.57
4
11.95
7.42
4.23
9.368
4.92
100
32.93
6.39
100
100
22.19
100
12.85
5.54
100
41.21
6.63
61.64
18.88
100
53.46
8.24
100
99.76
53.07
100
100
100
8.76
6.11
3.45
100
8
19.6
100
98.2
55.37
100
100
84.3
10.42
7.333
4.267
100
100
100
100
100
20
8.7
100
99.36
22.29
8.68
5.59
2.49
100
100
100
100
100
9.9
100
11.95
9.41
6.54
3.67
100
100
100
100
100
8.9
6.15
3.4
8.89
6.1
3.32
100
100
5FU
100
100
21
100
100
9.37
100
100
21.0
100
100
9.1
100
100
76.0
100
100
82
GI50
9.9
For experimental agent: LC50 is the concentration of drug resulting in 50% reduction in measured protein, TGI the concentration resulting in total growth inhibition, GI50 the
concentration resulting in growth inhibition of 50% (see also pharmacological protocol).
Table 1b
Summary results from the in vitro screening of antiproliferative activity of tested derivatives on lung, leukemia, pancreas, melanoma cancer cell lines and on normal cell lines
(HUVEC: Human Umbilical Vein Endothelial Cells, hMSC: Human Mesenchymal Stem Cells and NHDF: Normal Human Dermal Fibroblasts)
Compound
Values Non small cell lung
NCI-H460
Small lung
Leukemia
Pancreas
Liver
Melanoma
Normal cell lines
DMS 114 NCI-H69 H69AR HL-60 (TB) MiaPaca2 BX-PC3 SKHep1 LOX-IMVI
HUVEC hMSC NHDF
2a
2b
2c
2d
2e
4
LC50
TGI
GI50
LC50
TGI
GI50
LC50
TGI
GI50
LC50
TGI
GI50
LC50
TGI
GI50
LC50
TGI
GI50
LC50
TGI
100
7.66
4.41
100
9.44
5.77
100
100
47.3
100
100
54.74
9.7
6.72
3.74
100
100
100
100
100
6.5
100
100
100
7.09
100
100
5.54
90.47
18.43
6.04
100
7.82
4.75
78.51
9.63
5.04
100
100
6.39
16.61
6.96
3.84
>100
41.62
7.07
100
76.75
8.58
100
52.07
7.51
9.05
6.33
3.61
100
77.64
28.42
6.05
29.81
7.24
8.41
5.88
3.36
8.40
6.09
3.79
10.34
7.43
4.52
9.88
6.72
3.56
91.56 100
54.21 9.6
16.85 5.3
9.17
5.43
20.33
11.58
6.05
100
72
19.5
100
100
7.43
3.98
100
66.13
20.22
100
100
14.88
100
100
23.46
9.04
6.48
3.91
100
100
76.76
100
7.62
4.8
9.05
6.51
3.97
100
9.92
5.97
8.11
5.45
2.79
9.33
7.1
4.87
9.49
6.23
2.97
100
100
100
100
5FU
100
100
100
100
100
100
100
100
100
100
100
100
100
100
30
100
100
6.3
100
100
9.72
100
86.06
8.92
GI50
8.2
Gemcitabine LC50
100
100
35.83
8.71
9.05
3.64
TGI
GI50
For each experimental agent: LC50 is the concentration of drug resulting in 50% reduction in measured protein, TGI the concentration resulting in total growth inhibition, GI50
the concentration resulting in growth inhibition of 50% (see also pharmacological protocol).
study of sigma ligands could not be possible without validated
functional tests for the sigma receptors.
obtaining close to 30% of total apoptosis at 5
lM and more than
80% of total apoptosis at 50
l
M for 2a,40 in agreement with the
Compound 2a was initially screened in vitro on colon (colon
205), breast (MCF-7) and prostate (LNCap) cancer cells, by the try-
pan blue methodology, where significant apoptotic, anti prolifera-
tive and cytotoxic effects were observed. Then, apoptosis was more
extensively confirmed with the Annexin V (high throughput
cytometry) methodology34,35 on MCF-7 breast cancer cells, by
above (trypan blue) in vitro screening and with the sulforhoda-
mine B (SRB) staining36–38 (Tables 1a,b). Considering the in vitro
results (Tables 1a,b) of antiproliferative activity of derivatives 2
on cancer cell lines (colon, prostate, breast, ovarian, central ner-
vous system, lung, leukemia, pancreas, melanoma) and on normal
cell lines (HUVEC: Human Umbilical Vein Endothelial Cells, hMSC:

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S. Riganas et al. / Bioorg. Med. Chem. 20 (2012) 3323–3331
Table 2
important loss of weight in SCID mice during the xenografts exper-
iments. Concerning the mechanistic effects induced by 2a on the
BxPC-3, IGROV-1 and PC-3 cell cycle and apoptosis (Table 3), sig-
Affinities of the adamantane piperazines 2 for the r1 and
r
2-receptors
Compound
r
(nM)
1 IC50 SEM
IC50 SEM
(nM)
r1/r2
Na+ Channels
IC50 SEM (lM)
nificant increases of sub-G1 and, for BxPC-3 and PC-3 at 15 lM,
also of G1 populations were observed in cells treated with 2a, com-
pared to vehicle treated group, combined with a decrease of popu-
lations engaged in S phase, indicative of the apoptotic activity of
2a. Indeed, 2a exhibited apoptotic plasmatic membrane modifica-
2a
2b
2c
2d
2e
4
3.2 0.7
2.9 0.6
0.27 0.2
63.3 18.9
12.4 2.3
>100
37.6 9.3
4.0 2.8
97.0 36.0
119.0 44.0
>1000
11.8
1.4
360
1.9
1.2 0.15
0.7 0.1
0.7 0.1
1.6 0.5
1.2 0.2
>10.0
tions, from 5
lM for BxPC-3, associated, at 5 lM for IGROV-1 and
>1000
50 M for Bx-PC-3 or PC-3, with caspasse-3 activation (Table 3).
l
Results of in vitro assays concerning the effect of 2a on cell cycle
and apoptosis of BxPC3, IGROV-1 and PC3 cancer cells are summa-
rised in Table 3.
Finally, 2a was tested in a neuropathic pain model (described in
Section 4.740). Compound 2a exhibited a notable analgesic effect in
the formalin test41,42 operated on mice on which pain sensitiza-
tions was obtained by previous (2 weeks) administration of Paclit-
axel40 as shown in Fig 2.
Human Mesenchymal Stem Cells and NHDF: Normal Human
Dermal Fibroblasts) (Tables 1a,b), it appears that 2a exhibited the
most selective activity against melanoma and pancreas, given that
the cytotoxic effect of 2a on the HUVEC cells could, in vivo, also be
an antiangiogenic factor against the tumors.However, the in vivo
anti-tumor activity of 2a, in melanoma LOX-IMVI xenografts on
SCID mice, was not comparative to the reference drug Dacarbazine
(data not shown). In contrast, interesting results were obtained
with 2a in the BxPC-3 and Mia-Paca-2 pancreas cancer xenografts
(described in Section 4.738,39). Anti-tumor activity of 2a was in
most of the aforementioned xenografts, superior to those of 5-flu-
orouracile (5-FU) and similar to those of Gemcitabine, with 2a
exerting interesting synergies with both of these reference drugs
(Fig. 1).
The pharmacological profile of compound 2a was further ex-
tended by toxicological experiments. Toxicological studies were
performed on CD1 male and female mice and, for xenografts, on
mice with severe combined immune deficiency (SCID). Compound
2a was well tolerated at 40 mg/kg (ip) in chronic 3–4 weeks treat-
ments on CD1 and SCID mice. There were no dead animals or
3. Conclusion
The in vitro and in vivo toxicological and xenograft screening
studies showed that piperazine 2a, exhibited an acceptable toxico-
logical profile associated with a notable anti-tumor activity, in
good agreement with its in vitro results, was particularly promi-
nent in pancreas. It is noteworthy that this anti-tumor activity of
2a is associated with a putative antagonism of the neuropathic
pain induced by the clinically used anti-cancer drugs (particularly,
taxanes and platines). Given the fatal prognosis of pancreatic can-
cer, due to its high metastatic potential, compound 2a is currently
undergoing extensive investigations, with particular concern to its
putative anti-metastatic activity.
Tumor Growth Rate
Tumor Growth Rate
C
A
5T80
70 Gem
45 2a
45 2a + 70 Gem
ut
5T80
17 5FU
40 2a
1000
800
600
400
200
0
800.00
600.00
400.00
200.00
0.00
**
**
**
**
**
**
**
**
**
**
**
**
**
**
**
*
*
**
15
20
25
30
35
40
45
50
55
10
15
20
25
30
35
Days (post inoculation)
Days (post inoculation)
B
D
Figure 1. Growth of BX-PC3 (pancreas) and MiaPaca2 (pancreas) tumours in SCID mice treated with fluorouracil (5FU) (reference drug for pancreas) and Gemcitabine
(Gemzar) (reference drug for pancreas) and 2a. (A): tumour size (in mm3) of each mouse group (19-20 mice per group that is 38–40 tumours per group), (B) table showing %
reduction of tumours on day of termination of experiment.for BX-PC3 xenograft, (C) tumour size (in mm3) of each mouse group (12 mice per group that is 24 tumours per
group), (D) table showing % reduction of tumours on day of termination of experiment for MiaPaca2 xenograft. Ut, untreated animals; 5T80, animals treated with 5%Tween80;
17 5FU, animals treated with 17 mg/kg fluorouracil (dissolved in water for injection) twice a week, 40 2a, animals treated with 40 mg/Kg 2a (dissolved in 5% Tween 80) for 5
consecutive days (two cycles of treatment) and 2 administrations for one more week (for BX-PC3). 5T80, animals treated with 5%Tween80; 70 Gem, animals treated with
70 mg/kg Gemcitabine (dissolved in 5% Tween 80) twice a week; 45 2a, animals treated with 45 mg/Kg 2a (dissolved in 5% Tween 80) for 3 consecutive days (three cycles of
treatments); 45 2a + 70 Gem, co-administration of 45 mg/Kg 2a and 70 mg/kg Gemcitabine (three cycles of treatment) for MiaPaca2 xenograft. Statistical evaluation was
assessed with a two-tailed Students’ t-test. Points with p 60.05 are indicated by one asterisk (⁄), p 60.001 by two asterisks (⁄⁄).

S. Riganas et al. / Bioorg. Med. Chem. 20 (2012) 3323–3331
3327
Table 3
Summary results of the cell cycle and apoptotic in vitro assays performed with compound 2a at 0.5, 5, 15 and 50
lM on BxPC-3, PC-3 and IGROV-1 cancer cell lines
Cancer cell lines
Treatment (lM)
Cell cycle analysis (%)
<G1 G1 G2 >G2
Cleaved Caspase-3
containing cells (%)
Annexin V binding/7-AAD incorporation (%)
Early apoptosis Late apoptosis
Annexin Vꢀ 7-AADꢀ Annexin V+ 7-AADꢀ Annexin V+ 7-AAD+ death
S
Healthy cells
Total apoptotic
BxPC-3 (Pancreas)
IGR-OV-1 (Ovary)
PC-3 (Prostate)
0
0.5
5
15
50
0
0.5
5
15
50
0
0.5
5
9
11
8
8
25
4
3
5
11
41 33 12
37 34 12
43 30 12
51 19 16
33 24 13
27 29 19 21
26 28 20 20
25 29 19 21
21 27 21 20
6
8
8
5
5
10
3
5
11
16
4
5
7
30
77
84
65
60
26
86
89
88
62
31
92
95
92
89
61
11
10
17
12
15
9
8
8
18
46
5
9
6
17
16
33
4
3
3
15
16
2
20
16
33
28
48
13
10
11
33
62
7
NA NA NA NA NA 68
4
9
9
8
10
36 42 13
33 40 14
34 42 13
6
4
5
5
3
1
2
3
3
5
6
24
1
2
3
9
5
7
9
33
15
50
49 33
9
35 34 10 11
18
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 2a at 50 M in IGROV-1 cell line prevented correct cell
l
cycle analysis (NA, non applicable). In Caspase-3 assay, the percentage of cells showing cleaved/activated caspase-3 is indicated. In 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.
a range of concentrations of the compounds at 22 °C for 2 h in
5 nM Tris/HCl buffer, pH 7.4. The
r2 binding assay was performed,
according to Bowen et al. (1993),44 by incubating rat cerebral cor-
tex membranes (10–20 mg protein per tube) with [3H](+)1-DTG
(3-di-o-tolylguanidine; 5 nM) in the presence of (+)pentazocine
Control
60
50
40
30
20
10
0
#
#
GBP + Paclitaxel
2a+ Paclitaxel
Paclitaxel 6mgkg ip
**
(300 nM), to saturate
r1 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 de-
***
fined, in both assays, as that remaining in the presence of 10 lM
#
**
haloperidol. Affinities, for site 2 of Na+ channel were measured
by displacement of [3H]batrachotoxin benzoate (3H-BTX-B).45
Binding reactions were initiated by addition of 150
preparation containing 150–500 g protein to a solution in stan-
dard incubation buffer of 3H-BTX-B, 50
g Leiurus quinquestriatus
ll of vesicular
l
l
scorpion venom and various un labeled effectors as indicated.
The concentration of 3H-BTX-B was generally 20–25 nM, and the
Phase I
Phase II
total assay volume was 335 ll. Standard incubation buffer con-
tained 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 ice-cold wash buffer. The tissue was immediately collected
on Whatman GF/C glass fiber filters and washed 3 more times with
3 ml cold wash buffer. The wash buffer 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 deter-
mined by liquid-scintillation spectroscopy of the filters suspended
in 10 ml scintillation cocktail (3a70B;RPI). Nonspecific binding was
Figure 2. Effects of Orally Administered (po) 2a at the dose of100 mg/kg on
Paclitaxel treated mice in the Formalin test, compared to the reference drug
Gabapentin (GBP) at 100 mg/kg (ip). GBP was administered ip since oral admin-
istration of the compound exerts no analgesic effect in the formalin test, (data not
shown). One way Anova followed by LSD post hoc test confirmed the expected
hyperalgia of Paclitaxel treated animals compared to Vehicle treated animals in
both time intervals examined
(
⁄⁄p <0.01 and ⁄⁄⁄p <0.001, respectively). GBP
administration in Paclitaxel pretreated animals was found to exert a mild analgesic
effect in both time intervals, as indicated by their lower licking, of the administered
paw, times compared to Paclitaxel treated animals (# p<0.1 in both intervals).
Administration of 2a, however, exerted a stronger analgesic effect in Paclitaxel
treated animals. 2a Paclitaxel treated animals displayed lower licking times
compared to Paclitaxel treated animals both at 0–5 min (#p = 0.06) and 35–
40 min (⁄⁄p <0.01) after formalin injection.
determined from parallel incubations containing 250 lM veratri-
dine and has been subtracted from the data. Specific binding mea-
sured in this way was nominally 75% of the total binding.
4. Experimental
4.1. Binding studies
4.2. In vitro anti-proliferative and cytotoxic activity
Binding studies were carried out by CEREP (France) with the r1
binding assay performed in triplicate. Affinities of the adamantane
All human cancer cell lines were obtained from the National
Cancer Institute, NIH (Bethesda, MD, USA) with the exception of
BX-PC3 and the normal cells Hs888Lu, CCD18Co that were
obtained from ATCC, 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
piperazines 2 for the r1 and
r2-receptors measured by displace-
ment of [3H](+)pentazocine43 and [3H]1,3-di-o-tolylguanidine,44
respectively. The r1 binding assay was performed, according to
Ganapathy et al.,43 by incubating Jurkat cell membranes
(10–20 mg protein per tube) with [3H](+)pentazocine (8 nM) and
calf serum, 2 mM L-glutamine and antibiotics. The cultures were

3328
S. Riganas et al. / Bioorg. Med. Chem. 20 (2012) 3323–3331
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
by quantification of propidium iodide (PI) incorporation into geno-
mic DNA. After incubation, cells were detached from the well by
trypsinisation, transferred in tubes, washed and resuspended in
500 ll ice cold PBS before being fixed with 1.5 ml dropwise of
100
lL of medium at a the corresponding density (3500–30,000
100% cold ethanol for 3 h at 4 °C . Then, the cell suspensions were
cells per well) and subsequently, the plates were incubated at stan-
dard 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 sulforhodamine
B solution (SRB) staining, as described elsewhere.41–43 To deter-
mine the activity, each compound was dissolved in dimethyl sulf-
oxide (DMSO) and then was added at 10-fold dilutions (from 100
centrifuged at 1,500 rpm for 5 min and pellets were resuspended
in a mix of 100 ll of 200 lg/ml RNase and 100 ll of 1 mg/ml PI.
Cells were incubated for 45 min at room temperature in the dark.
The preparation was centrifuged 5 min at 1,500 rpm then the pel-
lets were resuspended in PBS for FACS analysis.
The apoptotic membrane modifications induced by compound
2a were evaluated by Annexin V binding at the end of the treat-
ment 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 labelled according to Annexin V-FITC/7-AAD Kit
(Beckman Coulter). Briefly, after being washed, cells were incu-
to 0.01 lM) 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.41–43
bated in ice-cold binding buffer. Then 10
solution and 20 l of 7-AAD viability dye were added to 100
cells suspension and incubated 15 min on ice in the dark. After
incubation, 800 l of binding buffer were added. Cell preparations
ll of Annexin V-FITC
l
l
l of
l
4.3. In vivo antitumor activity
were analyzed by FACS within 1 h.
The activation of caspase pathway by compound 2a was evalu-
ated by measuring the level of activated caspase-3 by FACS. After
incubation, cells were detached from the culture flask by trypsin-
isation, transferred to FACS tubes and labeled according to PE Ac-
tive Caspase-3 Apoptosis Kit (Pharmingen). Briefly, after being
washed, 106 cells were fixed and permeabilized in ice-cold BD
Cytofix/CytopermTM buffer for 20 min. Cells were then pelleted
and washed with BD Perm/WashTM buffer. Incubation with anti-
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 IIL-cages. Males or females
mice, 7–9 weeks old, were injected subcutaneously according to
the British practice of bilateral trocar implants at the axillary re-
gion. Each inoculum contained 106 cells exponentially growing
at the time of harvesting. The mice were subsequently randomly
divided into groups of 6 to 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 else-
where.43,44 All administrations were intraperitoneal. Treated ani-
body included in the kit was performed in 100
ll of same buffer
completed with 20 l of antibody for 30 min. Cell preparations
l
were analyzed by FACS within 1 h.
For all assays staining cells were analyzed with a CyFlowÒ space
flow cytometer (Partec) using a 488 nm wavelength laser excita-
tion. The acquisition was stopped after a total of 10,000 FSC/SSC-
gated cells were collected for each sample.
mals received
a single injection daily for 5 days per week
throughout the experiments. Tumor volume was measured with
a calliper twice per week. In addition to tumor volume, we calcu-
lated the parameter, %
D
T/
D
C, where
D
T = T ꢀ Do and
D
C = C ꢀ
4.5. Formalin test
Do (Do is the average tumor volume at the beginning of the treat-
ment; T and C are the volumes of treated and untreated tumors,
respectively, at a specified day). Concurrently, we scored the num-
ber of tumor-free animals, number of drug-related deaths, and
average number of days required to reach a defined tumor volume.
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 labo-
ratory diet and tap water. Paclitaxel (Brystol Myers Squibb Com-
pany) was diluted in saline and administered at one ip injection
(6 mg/kg) on day 0, as adapted from Matsumoto et al.40 On day
14, 1 h after oral administration (po) of the compound 2a
(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.41,42 The first phase con-
sists 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 sec-
ond phase reflects a state of central sensitization driven by the pre-
sumed first phase.41,42 The amount of time spent licking and biting
the injected paw and leg was recorded in 5-min intervals for 0–5
and 35–40 min after the formalin injection.
Optimal
DT/DC value 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 ani-
mals reached a volume of about 10–11% of animals’ weight.
4.4. In vitro cell cycle modifications and apoptotic activity
Supplying PC-3 (human prostatic adenocarcinoma), BxPC-3
(human pancreatic adenocarcinoma) and IGROV-1 (human ovarian
carcinoma) cancer cells, as well as, cell cycle and apoptosis assays
were carried out by Oncodesign S.A. (France). Cells were plated
24 h before treatment at the appropriate seeding density in 6-well
plates or 25 cm2 flasks according to the assay. Compound 2a was
dissolved at 10 mM in 100% DMSO then diluted in cascade to ob-
tain concentrations of 0.1, 1 and 3 mM in 100% DMSO. These dilu-
tions were further diluted at 1:10th with RPMI 1640 medium. The
last dilution was performed on cells at 1:20th to reach the appro-
4.6. 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., USA) software package. A difference was considered signifi-
cant if p <0.05.
priate concentrations of 0.5, 5, 15 or 50 lM. The final concentration
of DMSO was 0.5% in cell culture medium. The treatment period
was 24 h. The effect of compound 2a on cell cycle was evaluated

S. Riganas et al. / Bioorg. Med. Chem. 20 (2012) 3323–3331
3329
4.7. Materials and methods
3,5-H p), 3.32 (s, 1H,
a
-H), 6.75–6.77 (ꢂd, 2H, AA0BB0,
JAB = JA B = 8.5 Hz, 3,5-Har), 7.06–7.1 (ꢂt, 1H, 40-Har), 7.17–7.18
0
0
(ꢂt, 2H, 30,50-Har), 7.23–7.25 (ꢂd, 2H, AA0BB0, JAB = JA B = 8.5 Hz,
2,6-Har), 7.32–7.33 (ꢂd, 2H, 20,60-H ar). 13C NMR (100 MHz, CDCl3):
d 11.9 (CH3CH2), 28.8 (3,5,7 -C), 29.7 (1-C), 36.9 (2,8,9-C), 41.1
0
0
Melting points were determined using a Büchi capillary appara-
tus and are uncorrected. IR spectra were recorded on a Perkin-El-
mer 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 Ser-
vice Central de Microanalyse (CNRS) France, and the results ob-
tained had a maximum deviation of 0.4% from the theoretical
value.
(4,6,10-C), 48.9 (3,5-Cp), 52.4 (CH2CH3), 52.9 (2,6-Cp), 66.4 (a-C),
115.3 (3,5-Car), 125.6 (40-Car), 127.7 (30,50-Car), 129.9 (2,6-Car),
130.6 (20,60-Car), 133.3 (1-Car), 142.6 (40-Car), 149.2 (10-Car).
Monohydrochloride, mp 250 °C (EtOH–Et2O); IR (C29H39
ClN2 ꢁ ½H2O, nujol) mmax 3415 cm-1
;
Anal. Calcd for
C
29H39ClN2 ꢁ ½H2O (%): C, 75.70; H, 8.76; N, 6.09; found (%): C,
75.88; H, 8.70; N, 5.95.
4.7.1.4. 1-Cyclohexyl-4-{4-[
a
-(1-tricyclo[3.3.1.13,7]decyl) phen-
ylmethyl]phenyl}piperazine (2c). Piperazine 2c was ob-
4.7.1. General procedure for the preparation of analogues 2a–d
and 3a–d
tained as a waxy material in 31% yield from the reaction of
benzhydryl chloride 1 with 1-cyclohexylpiperazine following the
procedure used for 2a. 1H NMR (400 MHz, CDCl3): d 1.17–1.25
(m, 11H, 2,8,9-H, 2,3,4,5,6-Hax,cycl), 1.48–1.59 (m, 9H, 4,6,10-H,
3,4,5-Hax,cycl), 1.76 (br s, 2H, 2,6-Heq,cycl), 1.88 (br s, 3H, 3,5,7-
H), 2.28 (br s, 1H, 1-Hax,cycl), 2.68–2.70 (t, 4H, A2X2, JAX = 4.5 Hz,
2,6-Hp), 3.12–3.14 (t, 4H, A2X2, JAX = 4.5 Hz, 3,5-Hp), 3.36 (s, 1H,
A
stirred mixture of a-(1-adamantyl)benzhydryl chloride
(1)31,32 (1.7 g, 5 mmol) and 1-substituted piperazine (72 mmol)
was heated at 130 °C, under argon, for 12 h. After cooling, water
was added and the mixture was extracted with dichloromethane
(3 ꢁ 50 ml). The combined organic layers were washed with water,
dried over sodium sulfate and evaporated in vacuo. The residue
was purified by flash column chromatography.
a
-H), 6.78–6.80 (d, 2H, AA0BB0, JAB = 8 Hz, 3,5-Har), 7.07–7.13 (ꢂt,
1H, 40-Har), 7.18–7.22 (ꢂt, 2H, 30,50-Har), 7.25–7.27(d, 2H, AA0BB0,
-(1-tricyclo[3.3.1.13,7]decyl)phenyl me-
This compound was obtained in
JAB = JA B = 8 Hz, 2,6-Har), 7.35–7.37 (d, 2H, 20,60-Har). 13C NMR
(100 MHz, CDCl3): d 25.8 (3,5-Ccycl), 28.8 (2,6-Ccycl), 28.8 (3,5,7-
C), 29.7 (1-C), 36.9 (2,8,9-C), 41.1 (4,6,10-C), 49.0 (3,5-Cp), 49.3
0
0
4.7.1.1. 1-Methyl-4-{4-[
thyl]phenyl}piperazine (2a).
a
51% yield following the general method. Purification by flash
column chromatography using a mixture of Et2O/MeOH (97:3) as
eluent. Mp 171–173 °C (Et2O); 1H NMR (400 MHz, CDCl3): d
1.50–1.61 (m, 12H, 2,4,6,8,9,10-H), 1.91 (br s, 3H, 3,5,7-H), 2.30
(s, 3H, CH3), 2.50–2.53 (t, 4H, A2X2, JAX ꢂ4.8 Hz, 2,6-Hp),
(2,6-Cp), 63.5 (1-Ccycl), 65.4 (a
-C), 115.3 (3,5-Car), 125.7 (40-Car),
127.7 (30,50-Car), 129.9 (2,6-Car), 130.6 (20,60-Car), 133.2 (1-Car),
142.6 (4-Car), 149.3 (10-Car). Monohydrochloride, mp 178–181 °C
(EtOH–Et2O); IR (C33H45ClN2 ꢁ 1H2O, nujol) mmax 3430 cm-1; Anal.
Calcd for C33H45ClN2 ꢁ 1H2O (%): C, 75.76; H, 9.05; N, 5.35; found
(%): C, 75.65; H, 9.26; N, 5.08.
3.13–3.16 (t, 4H, A2X2, JAX ꢂ4.8 Hz, 3,5-Hp), 3.39 (s, 1H,
a-H),
6.81–6.83 (ꢂd, 2H, AA0BB0, JAB ꢂ8.6 Hz, 3,5-Har), 7.11–7.15 (ꢂt,
1H, 40-Har), 7.20–7.24 (ꢂt, 2H, 30,50-Har), 7.29–7.32 (ꢂd, 2H,
AA0BB0, JAB = JA B = 8.6 Hz, 2,6-Har), 7.38–7.40 (ꢂd, 2H, 20,60-Har).
13C NMR (50 MHz, CDCl3): d 28.7 (3,5,7-C), 29.5 (1-C), 36.8
(2,8,9-C), 41.0 (4,6,10-C), 46.0 (CH3), 48.8 (3,5-Cp), 55.0 (2,6-Cp),
65.4 (a-C), 115.2 (3,5-Car), 125.6 (40-Car), 127.6 (20,60-Car), 129.8
(30,50-Car), 130.5 (2,6-Car), 133.2 (1-Car), 142.5 (4-Car), 149.1
(1-Car). Anal. Calcd for C28H36N2 (%): C, 83.95; H, 9.06; found (%)
C, 83.75; H, 9.01. Monohydrochloride, mp 277–279 °C (dec) (ace-
tone–Et2O); IR (C28H37ClN2 ꢁ ½H2O, nujol) mmax 3420 cm-1; Anal.
Calcd for C28H37ClN2 ꢁ ½H2O (%): C, 75.39; H, 8.59; Cl, 7.95; N,
6.28; found (%): C, 75.65; H, 8.29; Cl, 8.03; N, 6.32.
4.7.1.5. 1-Phenylmethyl-4-{4-[
phenyl methyl]phenyl} piperazine (2d).
a
-(1-tricyclo[3.3.1.13,7]decyl)
Benzyl derivative
0
0
2d was prepared from benzhydryl chloride 1 and 1-benzylpipera-
zine following the procedure used for 2a. The residue obtained
was purified by flash column chromatography using a mixture of
ether/n-hexane (1:2) as eluent to give the title compound in 29%
yield as an off-white solid. Mp 78–80 °C (n-pentane); 1H NMR
(400 MHz, CDCl3): d 1.51–1.63 (m, 12H, 2,4,6,8,9,10-H), 1.91 (br
s, 3H, 3,5,7-H), 2.56–2.58 (t, 4H, A2X2, JAX ꢂ5 Hz, 2,6-Hp), 3.13–
3.15 (t, 4H, A2X2, JAX ꢂ5 Hz, 3,5-Hp), 3.38 (s, 1H, a-H), 3.54 (s, 2H,
PhCH2), 6.80–6.82 (d, 2H, AA0BB0, JAB = JA B = 8.5 Hz, 3,5-Har),
7.06–7.10 (t, 1H, 40-Har), 7.25–7.32 (complex m, 9H, 2,6,30,50, 200,
300, 400, 500, 600-Har), 7.38–7.40 (d, 2H, 20,60-Har). 13C NMR
(100 MHz, CDCl3): d 28.8 (3,5,7-C), 29.9 (1-C), 36.9 (2,8,9-C), 41.1
(4,6,10-C), 49.0 (3,5-Cp), 53.2 (2,6-Cp), 63.0 (PhCH2), 65.4 (a-C),
115.3 (3,5-C), 125.7 (40-C), 127.1 (400-Car), 128.20 (300,500-Car),
129.2 (200,600-Car), 129.9 (30,50-Car), 130.6 (2,6-Car), 133.2 (1-Car),
138.1 (100-Car), 142.6 (4-Car), 149.3 (10-Car). Monohydrochloride,
mp 241–242 °C (EtOH–Et2O); Anal. Calcd for C34H41ClN2 ꢁ H2O
(%): C, 75.76; H, 9.05; N, 5.35; found (%) C, 75.65; H, 9.26; N,
5.08. Monopicrate, mp 194–196 °C (acetone–Et2O); Anal. Calcd
for C40H43N5 O7 ꢁ CH3OH (%): C, 66.74; H, 6.42; N, 9.49; found
(%): C, 66.41, H, 6.06; N, 9.34.
0
0
4.7.1.2. 1-Methyl-4-[
methyl]piperazine (3a).
a
,a-diphenyl-(1-tricyclo[3.3.1.13,7] decyl)-
Compound 3a was isolated in 15%
yield as a less polar extract during the purification of 2a. Crystalline
solid. Mp 156–158 °C (Et2O/n-pentane); 1H NMR (400 MHz,
CDCl3): d 1.48–1.52 (m, 6H, 2,8,9-H), 1.87 (br s, 6H, 4,6,10-H),
1.90 (br s, 3H, 3,5,7-H) 2.42 (s, 3H, CH3), 2.43 (br s, 4H, 2,6-Hp),
2.80 (br s, 4H, 3,5-Hp), 7.16–7.26 (m, 6H, 2,4,6-Har), 7.54–7.56
(ꢂd, 4H, J ꢂ7.5 Hz, 3,5 -Har). 13C NMR (100 MHz, CDCl3): d 29.6
(3,5,7-C), 36.8 (2,8,9-C), 40.0 (4,6,10-C), 43.4 (1-C), 45.8 (CH3),
50.6 (3,5-Cp), 56.4 (2,6-Cp), 78.8 (a-C), 126.0 (4-Car), 126.3 (2,6-
Car), 131.7 (3,5-Car), 141.85 (1-Car); Anal. Calcd for C28H36N2
(%): C, 83.95; H, 9.06; N, 6.99; found (%): C, 83.65; H, 9.26; N, 5.04.
4.7.1.3. 1-Ethyl-4-{4-[
thyl]phenyl}piperazine (2b).
a
-(1-tricyclo[3.3.1.13,7]decyl)phenylme-
Piperazine 2b was obtained in
4.7.1.6. 1-Phenylmethyl-4-[
decyl)methyl]piperazine (3d).
a
,a-diphenyl-(1-tricyclo[3.3.1.13,7]-
Compound 3d was isolated
41% yield as a waxy material from the reaction of benzhydryl chlo-
ride 1 with 1-ethylpiperazine following the procedure used for 2a.
1H NMR (400 MHz, CDCl3): d 1.04–1.07 (t, 3H, A3X2, JAX = 7 Hz,
CH3CH2), 1.45–1.56 (m, 12H, 2,4,6, 8,9,10 -H), 1.85 (br s, 3H, 3,5,7
-H), 2.37–2.43 (q, 2H, A3X2, JAX = 7 Hz, CH2CH3), 2.51–2.54 (ꢂt,
4H, A2X2, JAX = 4.5 Hz, 2,6-Hp), 3.10–3.13 (ꢂt, 4H, A2X2, JAX = 4.5 Hz,
in 20% yield as a less polar extract during the purification of 2d.
Off-white solid. Mp 126–128 °C (ether/n-pentane); 1H NMR
(400 MHz, CDCl3): d 1.48–1.62 (m, 6H, 2,8,9-H), 1.88 (br s, 6H,
4,6,10-H), 1.91 (br s, 3H, 3,5,7 -H), 2.46 (br s, 4H, 2,6-Hp), 2.77
(br s, 4H, 3,5-Hp), 3.49 (s, 2H, PhCH2), 7.23–7.89 (m, 15H, Har).
13C NMR (100 MHz, CDCl3): d 29.6 (3,5,7-C), 36.9 (2,8,9-C), 40.0

3330
S. Riganas et al. / Bioorg. Med. Chem. 20 (2012) 3323–3331
(4,6,10-C), 43.4 (1-C), 50.8 (3,5-Cp), 54.6 (2,6-Cp), 63.4 (PhCH2),
71.1 (a-C), 125.9, 126.2, 128.1, 129.3, 131.8, 132.4
(2,3,4,5,6,20,30,40,50,60 -Car), 140.0 (10-Car), 141.8 (1-Car); Anal.
Calcd for C33H38N2 (%): C, 85.67; H, 8.28; N, 6.05; found (%): C,
85.65; H, 8.26; N, 6.04.
Acknowledgments
Dr Stefanos Riganas is idebted to National and Kapodistrian
University of Athens for the award of a scholarship and financial
support –Support of Universities Research Groups (2008–
2009)KA 70-|4|7835 and (2009–2012)KA70|4|10311.
4.7.2. 1-{4-[a
-(1-Tricyclo[3.3.1.13,7]decyl)phenylmethyl]-
phenyl}piperazine (2e)
References and notes
To a stirred suspension of N-benzyl derivative 2d (950 mg,
2 mmol) and 10% palladium on charcoal (950 mg) in methanol
(20 mL), ammonium formate (630 mg, 10 mmol) was added all
at once. The reaction mixture was refluxed under argon for 3 h
and after cooling 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 chroma-
tography using a mixture of DCM/MeOH (9:1) as eluent. Pipera-
zine derivative 2e was obtained in 74% yield as a white solid.
Mp 145–147 °C (Et2O); 1H NMR (400 MHz, CDCl3): d 1.48–1.56
(m, 12H, 2,4,6,8,9,10-H), 1.78 (br s, 1H, NH), 1.85 (br s, 3H,
3,5,7-H), 2.93–2.95 (m, 4H, 3,5-Hp), 3.02–3.04 (m, 4H, 2,6-Hp),
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3.33 (s, 1H,
a
-H), 6.75–6.77 (ꢂd, 2H, AA0BB0, JAB = JA B = 8.9 Hz,
0
0
3,5-Har), 7.06–7.10 (m, 1H, 40-Har), 7.15–7.19 (m, 2H, 30,50-
Har), 7.23–7.25 (ꢂd, 2H, AA0BB0, JAB = JA B =8.9 Hz, 2,6-Har),
7.32–7.34 (ꢂd, 2H, 20,60-Har). 13C NMR (100 MHz, CDCl3): d
28.8 (3,5,7-C), 36.9 (2,8,9-C), 41.1 (4,6,10-C), 46.2 (3,5-Cp), 50.3
0
0
(2,6-Cp), 52.8 (1-C), 65.5 (a
-C), 115.4 (3,5-Car), 125.8 (40-Car),
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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.;
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127.8 (30,50-Car), 129.9 (2,6-Car), 130.6 (20,60-Car), 133.4 (1-
Car), 142.6 (4-Car), 149.8 (10-Car). Dihydrochloride, mp 215–
216 °C (dec) (EtOH–Et2O); Anal. Calcd for C27H36Cl2N2 (%): C,
70.58; H, 7.90; found (%): C, 70.19; H, 8.09.
4.7.3. 4-{4-[a
-(1-Tricyclo[3.3.1.13,7]decyl)phenylmethyl]phenyl}
morpholine (4)
ꢀ
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Morpholine derivative 4 was prepared following the proce-
dure used for piperazine 2a. The residue obtained was purified
by flash column chromatography using a mixture of Et2O/n-hex-
ane (1:2) as eluent to give the title compound as a crystalline
solid in 60% yield. Mp 147–149 °C (Et2O); 1H NMR (400 MHz,
CDCl3): d 1.45–1.56 (m, 12H, 2,4,6,8,9,10-H), 1.85 (br s, 3H,
3,5,7-H), 3.02–3.05 (t, 4H, A2X2, JAX ꢂ5 Hz, 3,5-Hm), 3.33 (s,
1H,
a
-H), 3.75–3.77 (t, 4H, A2X2, JAX ꢂ5 Hz, 2,6-Hm), 6.73–6.75
0 0
(ꢂd, 2H, AA0BB0, JAB = JA B = 9.0 Hz, 3,5-Har), 7.07–7.09 (m, 1H,
40-Har), 7.10–7.19 (m, 2H, 30,50-Har), 7.24–7.27 (ꢂd, 2H, AA0BB0,
JAB = JA B = 9.0 Hz, 3,5- Har), 7.31–7.34 (ꢂd, 2H, 20,60-Har). 13C
NMR (100 MHz, CDCl3): d 28.8 (3,5,7-C), 36.9 (2,8,9-C), 38.8 (1-
C), 41.1 (4,6,10-C), 49.3 (3,5- Cm), 65.4 (a-C), 66.9 (2,6-Cm),
115.1 (3,5-Car), 125.8 (40-Car), 127.8 (30,50-Car), 129.9 (2,6-Car),
130.7 (20,60-Car), 133.8 (1-Car), 142.6 (4-Car), 149.2 (10-Car).
Fumarate, mp 178–181 °C (EtOH–Et2O); Anal. Calcd for
0
0
29. Ablordeppey, S. Y.; Fischer, J. B.; Law, H.; Glennon, R. A. Bioorg. Med. Chem.
2002, 10, 2759.
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4.7.4. 4-[a,
pholine (5)
a
-Diphenyl-(1-tricyclo[3.3.1.13,7]decyl)methyl]mor-
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Compound 6 was isolated in 13% yield as a less polar extract
during the purification of 5. Crystalline solid. Mp 137–139 °C
(Et2O/n-pentane); 1H NMR (400 MHz, CDCl3): d 1.45–1.54 (m, 6H,
2,8,9-H), 1.80–1.86 (m, 9H, 3,4,5,6,7,10-H), 2.66–2.68 (t, 4H, A2X2,
JAX = 4 Hz, 3,5-Hm), 3.61–3.64 (t, 4H, A2X2, JAX = 4 Hz, 2,6-Hm),
7.13–7.23 (m, 6H, 2,4,6-Har), 7.47–7.49 (ꢂd, 4H, J ꢂ9 Hz, 3,5-
Har). 13C NMR (100 MHz, CDCl3): d 29.3 (3,5,7-C), 36.6 (2,8,9-C),
39.9 (4,6,10-C), 42.9 (1-C), 51.9 (3,5-Cm), 68.1 (2,6-Cm), 70.9 (a-
C), 125.9 (4-Car), 126.1 (2,6-Car), 131.9 (3,5-Car), 140.82 (1-Car);
Anal. Calcd for C27H33NO (%): C, 83.68; H, 8.58; N, 3.61; found
(%): C, 83.65; H, 8.68; N, 3.64.
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