Synthesis and antiproliferative properties of N3/8-disubstituted 3,8-diazabicyclo[3.2.1]octane analogues of 3,8-bis[2-(3,4,5-trimethoxyphenyl)pyridin-4-yl]methyl-piperazine

Article abstract of DOI:10.1016/j.ejmech.2006.11.013

A series of novel N_3/8-disubstituted-3,8-diazabicyclo[3.2.1]octanes in order to improve the in vitro activity of the prototype 3,8-bis[2-(3,4,5-trimethoxyphenyl)pyridyl-4-yl)methylpiperazine (1) were synthesized and evaluated by assays of growth inhibition against several tumor cell lines. Compounds 2a,b,f and m demonstrated not only growth-inhibitory activities against leukemia cancer cells, but also fairly good activities against the growth of certain solid tumors. Among them, 2a is the most potent one with IC₅₀ values in the low micromolar range. Moreover, compound 2a has been selected for in vitro testing on MCF-7 cell to evaluate the mode of action of this lead compound.

Full text of DOI:10.1016/j.ejmech.2006.11.013

European Journal of Medicinal Chemistry 42 (2007) 293e306
http://www.elsevier.com/locate/ejmech
Original article
Synthesis and antiproliferative properties of N3/8-disubstituted
3,8-diazabicyclo[3.2.1]octane analogues of 3,8-bis[2-(3,4,5-
trimethoxyphenyl)pyridin-4-yl]methyl-piperazine
a
a
Rosanna Filosa a, , Antonella Peduto , Paolo de Caprariis ,
*
Carmela Saturnino a, Michela Festa a, Antonello Petrella a, Amedeo Pau b,
b
Gerard Aime Pinna , Paolo La Colla , Bernardetta Busonera , Roberta Loddo
c
c
c
´
´
a Dipartimento di Scienze Farmaceutiche, Universita` di Salerno, Via ponte don Melillo, 84084 Fisciano (SA), Italy
b Dipartimento Farmaco Chimico Tossicologico, Universita` di Sassari, Via Muroni 23/A, 07100 Sassari, Italy
c Dipartimento di Scienze e Tecnologie Biomediche, Sezione di Microbiologia e Virologia Generale e Biotecnologie Microbiche,
Universita` di Cagliari, Cittadella Universitaria, SS 554-Km 4.500, 09042 Monserrato (CA), Italy
Received 6 July 2006; received in revised form 29 September 2006; accepted 6 November 2006
Available online 5 December 2006
Abstract
A series of novel N3/8-disubstituted-3,8-diazabicyclo[3.2.1]octanes in order to improve the in vitro activity of the prototype 3,8-bis[2-(3,4,
5-trimethoxyphenyl)pyridyl-4-yl)methylpiperazine (1) were synthesized and evaluated by assays of growth inhibition against several tumor
cell lines. Compounds 2a,b,f and m demonstrated not only growth-inhibitory activities against leukemia cancer cells, but also fairly good
activities against the growth of certain solid tumors. Among them, 2a is the most potent one with IC50 values in the low micromolar range.
Moreover, compound 2a has been selected for in vitro testing on MCF-7 cell to evaluate the mode of action of this lead compound.
Ó 2006 Elsevier Masson SAS. All rights reserved.
Keywords: Piperazine; 3,8-Diazabicyclo[3.2.1]octane
1. Introduction
Clinical strategies have been developed to address such is-
sues, especially cycles of therapy, but unfortunately this ap-
proach may result, in the tumor cell population and in an
increased selection pressure which would induce dangerous
drug resistance. It is interesting to note that this general profile
applies, to a different extent, to all cytotoxic agents even if
they act with different mechanisms.
Because of this critical situation, the search for new
drugs for antitumour chemotherapy continues at a steady
pace [1].
The piperazine nucleus is often found embedded in chemo-
therapic agents exhibiting a wide range of biological activities
(anthelmintic, antiprotozoal, bactericidal, fungicidal, antiviral,
and antitumour properties [2,3]). A series of simple N,N0-
bisarylpyridinylmethyl piperazines such as 1 have been shown
to exhibit in vitro growth-inhibitory properties against MCF-7
and HCT-15 cells with IC50 values in the micromolar
The global burden of cancer at the beginning of the 21st
century is immense. Cancer is a collection of over 100 devas-
tating diseases characterized by malignant cells that are clearly
distinguished from normal cells by an out-of-control growth.
Clinically, weapons in the fight against cancer are surgery,
radiotherapy and chemotherapy. Current chemotherapy con-
sists of cytotoxic (cell-killing) agents and antihormonal drugs
which reduce the chaotic proliferation of cancerous aberrant
cells. Significant side effects such as nausea, vomiting, diar-
rhea, hair loss and serious infections are often encountered
during chemotherapy.
* Corresponding author. Tel.: þ39 089962922; fax: þ39 089962828.
E-mail address: rfilosa@unisa.it (R. Filosa).
0223-5234/$ - see front matter Ó 2006 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2006.11.013

294
R. Filosa et al. / European Journal of Medicinal Chemistry 42 (2007) 293e306
Conformational restriction approach
OMe
MeO
OMe
OMe
N
OMe
N
MeO
MeO
MeO
N
OMe
OMe
OMe
OMe
N
N
N
N
N
1
2a
Chart 1. Structure modification of piperazine derivative 1.
concentration range (Chart 1). HTC cell IC50 6 mM, MCF-7
cell IC50 6 mM [4].
Synthesis of the intermediate 3 in Scheme 1 was accomplished
in three steps from the known 3,8-dibenzyl-3,8-diazabyci-
clo[3.2.1]octane-2,4-dione [5,6] 4 as chloridrate by converting
it to the N8-dibenzyl-derivative 6 by a reaction with hydrogen
and catalyst (Pd/C) in ethanol. This imide on reduction with so-
dium bis(2-methoxyethoxy)aluminium hydride (SMEAH) in
toluene at room temperature produced diamine 6 which was
then N3-debenzylated by catalytic hydrogenation to afford 3
as dihydrochloride salt in 87% yield. Amine salt was then
alkylated with various halides in the presence of a base under
microwave irradiation to produce different title compounds.
The substituted 4-(chloromethyl)-2-phenylpyridine 10a, den
was readily prepared from 2-chloro-isonicotinic ethyl ester 7.
Suzuki reaction of 7 with designed arylboronic esters in the
presence of catalytic amount of [1,10-bis(diphenylphosphino)-
ferrocene] dichloro palladium (II) (PdCl2(dppf)) afforded aryl-
pyridinyl esters 8 in moderate to good yield. Esters 8 were
transformed into the required halides 10a, den using standard
procedure, that is, by the reaction with DIBALH to give the cor-
responding alcohols 9 which were treated with thionyl chloride
(Scheme 2).
In our effort to search for novel antitumour compounds we
decided to assume 1 as a lead structure designing new ana-
logue 2a in which the cis-dimethyl piperazine moiety of 1
was replaced with a 3,8-diazabicyclo[3.2.1]octanyl ring sys-
tem. Here, we wish to report on the synthesis of our target
compounds and on the results of an initial in vitro testing
for antitumour activity.
2. Chemistry
Compounds 2aer (Table 1) were retrosynthetically discon-
nected at CeN3/8 bonds to give 3,8-diazabicyclo[3.2.1]octane
(3) and appropriate halides (Fig. 1).
Table 1
3,8-Bisdiaza-bicyclo[3.2.1]octane derivatives
Compound
X
R
The analogues 10b and 10c were prepared using different
starting material as outlined in Schemes 3 and 4.
N
2
R
N
X
Catalytic Grignard cross-coupling of commercially avail-
able 2-bromo-4-methyl pyridine 11 proceeded with high yields
in the presence of nickel catalyst coordinated with 1,2-bis(di-
phenylphosphino) ethane (dppe). The methyl group in the
cross-coupling product 12 was oxidized with potassium per-
manganate to afford compound 13. Reduction of the corre-
sponding methyl ester 14 furnished alcohol 15 and subsequent
reaction with thionyl chloride yielded the desired intermediate
4-(chloromethyl)-2-(4-methoxyphenyl)pyridine 10b.
Compound 10c was prepared starting from (2-bromopyri-
din-4-yl)methanol 16 which was protected as silyl ether and
under catalytic Grignard cross-coupling conditions gave 17
in 65% yield.
X
N
N
N
N
N
N
N
N
N
N
N
N
N
C
C
C
R
a
b
c
d
e
f
3-(30,40,50 Trimethoxyphenyl)
3-para-Methoxy phenyl
3-meta-Methoxy phenyl
3-para-Hydroxy phenyl
3-meta-Hydroxy phenyl
3-para-Methyl phenyl
3-meta-Methyl phenyl
3-para-Acetamido phenyl
3-meta-Acetamido phenyl
3-para-Chloro phenyl
3-para-Nitro phenyl
3-meta-Nitro phenyl
3-Phenyl
g
h
i
j
k
l
m
n
o
p
3-Phenyl
4-Phenyl
After removal of the protecting group, halogeno-substitu-
tion of hydroxyl function proceeded efficiently to give
4-(chloromethyl)-2-(3-methoxyphenyl)pyridine 10c.
H
2q
2r
N
N
N
A
HN
N
A
X
+
A
NH
N
X = Cl or Br
N
2
3
Fig. 1. Antithesis of title derivatives 2.

R. Filosa et al. / European Journal of Medicinal Chemistry 42 (2007) 293e306
295
Cl
N
CH2Cl
Me
R
N
Me
N
O
N
O
N
O
a
b
O
a
b
e
HN
H
N
N
Br
4
5
OMe
OMe
OMe
Cl
H2N
R=COOH13
11
12
10 b
c,d
Cl
HN
R=CH2OH15
c
N
NH2
Scheme 3. Reagents and conditions: (a) 4-methoxyphenyl magnesium bro-
mide, NiCl2(dppe), THF, rt, 18 h; (b) KMnO4, tert-BuOH/H2O, reflux, 24 h;
(c) i: SOCl2, 60 ꢀC, 4 h; ii: MeOH, rt, 2 h; (d) DIBALH, toluene, rt, 1 h; (e)
SOCl2, CH2Cl2, rt, 4 h.
6
3
Scheme 1. Reagents and conditions: (a) i: Pd/C 10%, ethanol, rt, 3 h, 3 atm; ii:
10% Na2CO3; (b) SMEAH, toluene, rt, 2 h; (c) Pd/C 10%, ethanol, 6 N HCl,
rt, 24 h.
WIL-2NS and CCRF-SB) (Table 2) or solid (SKMEL28, MCF-
7, SKMES-1, HepG2, and DU145) human tumors (Table 3).
Initial substitution of the cis-dimethyl piperazine nucleus of
1 with 3,8-diazabicyclo[3.2.1]octane ring system provided 2a
as an active compound against leukemia cancer cells with IC50
in the low micromolar range. Moreover, 2a showed a wide
spectrum of inhibitory effect on cancer cell growth of solid
tumors ranging from 4 to 14 mM.
In the case of analogues 2q and 2r the deviation from
derivative 2a was more pronounced. On the aromatic units,
were carried out respectively, naphthyl and anthranyl moieties.
The final alkylation was performed using microwave pro-
gram, which was composed by appropriate ramping and hold-
ing steps [7]. Identification of the optimum profile was
reported.
The IC50 obtained for N3/8-methoxypyridylmethyl-3,8-dia-
zabicyclo[3.2.1]octane derivative 2b showed that simple
para-methoxy substitution of the side chain appendages favor-
ably affected the antiproliferative activity being more potent
than counterpart 2a against certain solid tumor cell lines
(SKMEL28, SKMES-1, HepG2, DU145). Moreover com-
pound 2b did have respectable antiproliferative activity
against leukemia cell lines with IC50 values up to 3 mM. The
lower IC50 values for meta-methoxy derivative 2c clearly
showed that substitution at this position was less tolerated.
The para-hydroxy analogue 2d displayed respectable cytotox-
icity but it was less active than the ground term 2a. Compound
2e showed weak selective cytotoxicity against CCRF-CEM
and CCRJ-Sb with IC50 values of 14 and 16 mM, respectively;
however, 2e was non-cytotoxic against almost all other tumor
cells (Table 4).
3. Biological results
The antiproliferative activity of test compounds was evalu-
ated against a panel of cell lines derived from haematological
(CCRF-CEM, WIL-2NS and CCRF-SB) (Table 2) or solid
(SKMEL28, MCF-7, SKMES-1, HepG2, and DU145) human
tumors (Table 3).
They were also evaluated against cell lines derived from
normal human tissues (CRL 7065).
4. Results and discussion
The antiproliferative activity of test compounds was evalu-
ated against a panel of cell lines derived from normal human
tissues (CRL 7065) either haematological (CCRF-CEM,
These results suggest that the methoxy group at phenyl
para position is important for anticancer response.
R1
R
R1
R
O
Cl
CO2Et
R2
R2
O
Cl
b,c
a
X
X
X
7
8a, d-n
10a, d-n
a R,R1,R2=OMe
d R=OH
X=N
R1,R2=H
R1,R2=H
X=N
X=N
e R1=OH
g R1=Me
R,R2=H
R,R2=H
R,R2=H
X=N
X=N
X=N
f
R=Me
h R=NHCOMe
R=Cl
R1,R2=H X=N
i
R1=NHCOMe
j
R1,R2=H
R1,R2=H
R1,R2=H
X=N
X=N
X=N
k R=NO2
m R=H
l R1=NO2
n R=H
R,R2=H
X=N
X=C
R1,R2=H
Scheme 2. Reagents and conditions: (a) arylboronic acid or ester, K2CO3, PdCl2(dppf), EtOH, 60 W, 110 ꢀC, 10 min; (b) DIBALH, toluene, rt, 15 min or THF, rt
18 h; (c) SOCl2, benzene, reflux, 10 min.

296
R. Filosa et al. / European Journal of Medicinal Chemistry 42 (2007) 293e306
Table 2
Microwave program applied for final alkylation
Cl
HO
O
Si
a,b
c,d
Time (min)
Power (W)
Temperature (ꢀC)
OCH3
OCH3
N
N
N
5
80
300
200
80
120
110
Br
60
5
16
17
10c
Scheme 4. Reagents and conditions: (a) TBDSCl, imidazole, DMF, 25 ꢀC,
13 h; (b) 3-methoxyphenyl magnesium bromide, NiCl2(dppe), THF, rt, 18 h;
(c) TBAF, THF, 0 ꢀC, 25 ꢀC, 3 h; (d) SOCl2, CH2Cl2, rt, 4 h.
When the nitrogen atom of 2m was replaced with a carbon
atom, as seen in 2n, cytotoxicity against solid tumor cell lines
decreased, but 2n still exhibited weak activity against leuke-
mia cell lines.
Compound 2f containing a para-methyl group demon-
strated good selective cytotoxicity against the solid tumor
cell lines while the meta-methyl derivative exhibited marginal
activity. A direct comparison of 2b,c and 2h,i allowed a deter-
mination of the effect of replacing the OCH3 with NHCOCH3,
since the two pairs of structure were otherwise identical. The
decreased IC50 values for acetylamino derivatives 2h,i clearly
showed that this type of substitution was less tolerated.
Turning to the analogues containing an electron withdraw-
ing group such as Cl or NO2, compounds 2jek, only the
chloro derivative 2j displayed significant cytotoxicity espe-
cially against CCRF-CEM and solid tumor cell lines.
Compound 2m, the unsubstituted bisphenyl pyridylmethyl-
3,8-diazabicyclo[3.2.1]octane derivative, showed moderate cy-
totoxicity toward all of the tested cell lines, except against
MT-4 and WIL-2NS.
Compound 2o, having a phenyl group at C-4 pyridylmethyl
segment showed moderate cytotoxicity against leukemia cell
lines as well as against SH-MEL-28 and DU145. Furthermore,
replacing the phenyl moiety of 2o with a hydrogen atom yielded
2p which was non-toxic against all tumor cell lines, suggesting
that the phenyl pyridylmethyl side chain appendages are struc-
tural features that bestow antiproliferative activity. However, it
is interesting to note that compounds 2q and 2r bearing a naph-
thylmethyl or an anthracenylmethyl moiety at N3/8-diazabicy-
cloalkane core displayed moderate to good anticellular activity.
In conclusion, we have designed and prepared a new series
of N3/8-disubstituted-diazabicyclo[3.2.1]octane analogues 2 of
3,8-bis [2(3,4,5-trimethoxyphenyl)pyridin-4yl)methylpipera-
zine (1) for evaluation in an in vitro preclinical screening
against some human tumor cell lines. The results showed
Table 3
Cytotoxicity against leukemia/lymphoma
Compound
IC50 (mM)a
MT-4b
CCRF-CEMc
WIL-2NSd
CCRF-SBe
CRL 7065f
2a
2b
2c
2d
2e
2f
2
2 ꢁ 0.2
3 ꢁ 0.05
11 ꢁ 0.05
15 ꢁ 2
3 ꢁ 0.05
6 ꢁ 0.6
13 ꢁ 1
18 ꢁ 3
28 ꢁ 8
26 ꢁ 7
25 ꢁ 3
30 ꢁ 2
25 ꢁ 0.7
29 ꢁ 2
>100
3 ꢁ 1
>100
>100
>100
80 ꢁ 0.1
>100
>100
>100
>100
>100
>100
>100
>100
>100
41 ꢁ 2
>100
>100
20 ꢁ 0.9
69 ꢁ 0.1
>100
>100
4
5 ꢁ 1
N.T.
N.T.
N.T.
N.T.
>100
18
12 ꢁ 1
12 ꢁ 1
16 ꢁ 3
12 ꢁ 2
18 ꢁ 4
25 ꢁ 5
17 ꢁ 0.2
13 ꢁ 2
>100
14 ꢁ 0.2
8 ꢁ 0.5
18 ꢁ 5
2g
2h
2i
19 ꢁ 2
>100
N.T.
N.T.
N.T.
19
16 ꢁ 0.2
7 ꢁ 0.3
91 ꢁ 10
36 ꢁ 2
2j
2k
2l
63 ꢁ 19
15 ꢁ 0.7
12 ꢁ 0.5
11 ꢁ 2
60 ꢁ 6
7 ꢁ 0.01
6 ꢁ 0.1
>100
27 ꢁ 6
11 ꢁ 0.05
10 ꢁ 2
6 ꢁ 0.4
81 ꢁ 19
7 ꢁ 0.7
9 ꢁ 0.1
88 ꢁ 12
>100
2m
2n
2o
2p
2q
2r
5
8 ꢁ 2
N.T.
>100
>100
N.T.
N.T.
>100
>100
12 ꢁ 0.5
7 ꢁ 1
46 ꢁ 0.2
8 ꢁ 0.3
6 ꢁ 0.1
85 ꢁ 12
>100
6
>100
6MP
CPT
a
0.1 ꢁ 0.02
1 ꢁ 0.05
3 ꢁ 0.05
1 ꢁ 0.1
>100
0.5 ꢁ 0.005
0.004 ꢁ 0.0004
0.003 ꢁ 0.0005
0.005 ꢁ 0.0005
0.003 ꢁ 0.0005
Compound concentration required to reduce cell proliferation by 50%, as determined by the MTT method, under conditions allowing untreated controls to
undergo at least three consecutive rounds of multiplication. Data represent mean values (ꢁSD) for three independent determinations.
b
CD4þ human T-cells containing an integrated HTLV-1 genome.
c
CD4þ human acute T-lymphoblastic leukemia.
Human splenic B-lymphoblastoid cells.
d
e
Human acute B-lymphoblastic leukemia.
Human foreskin fibroblasts, 6MP: 6-mercapto purine, CPT: camptothecin.
f

R. Filosa et al. / European Journal of Medicinal Chemistry 42 (2007) 293e306
297
Table 4
Cytotoxicity against solid tumor-derived cell lines
a
Compound
IC50 (mM)
SK-MEL-28b
MCF-7c
SKMES-1d
HepG2e
DU145f
2a
2b
2c
2d
2e
2f
4 ꢁ 0.1
2 ꢁ 0.3
8 ꢁ 1
5 ꢁ 0.9
6 ꢁ 2
10 ꢁ 0.5
3 ꢁ 0.6
8 ꢁ 0.7
23 ꢁ 2
>100
8 ꢁ 0.2
3 ꢁ 0.2
15 ꢁ 1
13 ꢁ 2
>100
14 ꢁ 3
2 ꢁ 0.2
9 ꢁ 0.7
15 ꢁ 3
>100
15 ꢁ 2
25 ꢁ 0.3
>100
19 ꢁ 2
>100
7 ꢁ 2
7 ꢁ 0.6
24 ꢁ 7
43 ꢁ 2
23 ꢁ 5
10 ꢁ 0.5
>100
8 ꢁ 0.3
18 ꢁ 0.9
22 ꢁ 0.7
33 ꢁ 3
12 ꢁ 0.5
>100
7 ꢁ 0.3
41 ꢁ 7
30 ꢁ 1
43 ꢁ 5
9 ꢁ 0.1
>100
7 ꢁ 0.3
34 ꢁ 3
42 ꢁ 0.5
24 ꢁ 2
9 ꢁ 0.1
>100
2g
2h
2i
15 ꢁ 4
20 ꢁ 0.8
34 ꢁ 0.1
10 ꢁ 0.4
>100
2j
2k
2l
31 ꢁ 0.4
8 ꢁ 0.4
15 ꢁ 0.3
6 ꢁ 2
68 ꢁ 0.1
9 ꢁ 3
30 ꢁ 0.1
11 ꢁ 0.5
10 ꢁ 2
43 ꢁ 7
>100
41 ꢁ 1
11 ꢁ 0.3
38 ꢁ 2
45 ꢁ 14
>100
85 ꢁ 1.5
8 ꢁ 0.9
14 ꢁ 3
7 ꢁ 0.5
>100
2m
2n
2o
2p
2q
2r
5
25 ꢁ 3
84 ꢁ 17
>100
>100
13 ꢁ 2
30 ꢁ 10
96 ꢁ 4
>100
11 ꢁ 3
54 ꢁ 1
86 ꢁ 13
>100
17 ꢁ 0.1
40 ꢁ 1
81 ꢁ 20
>100
18 ꢁ 0.1
50 ꢁ 0.2
92 ꢁ 8
>100
80 ꢁ 0.1
41 ꢁ 1.4
87 ꢁ 13
>100
6
6MP
CPT
a
15 ꢁ 1
3 ꢁ 0.5
58 ꢁ 2
8 ꢁ 0.5
2 ꢁ 0.05
0.04 ꢁ 0.005
0.04 ꢁ 0.01
0.01 ꢁ 0.004
0.03 ꢁ 0.005
0.01 ꢁ 0.005
Compound concentration required to reduce cell proliferation by 50%, as determined by the MTT method, under conditions allowing untreated controls to
undergo at least three consecutive rounds of multiplication. Data represent mean values (ꢁSD) for three independent determinations.
b
Human skin melanoma.
Human breast adenocarcinoma.
c
d
Human lung squamous carcinoma.
Human hepatocellular carcinoma.
Human prostate carcinoma.
e
f
that the antitumour activities of these derivatives were strongly
related to the nature of the side chain appendages on 3,8-dia-
zabicyclo[3.2.1]octane core with compounds 2a,b showing
significant activity against leukemia cell lines and some solid
tumor cell lines.
This cellular line was selected to compare the analysis with
the derivative 1 described in the literature.
First, we have checked the activity of 2a on apoptosis using
propidium iodide (PI) staining by flow cytometry.
MCF-7 cells were incubated with 2a (10e50 mM) for 24 h.
Results in Fig. 2 showed that there was no significant increase
in apoptotic cells after treatment with 2a.
Generally, they were less active against normal cells and
cells derived from solid tumors.
Compound 2a, bearing a trimethoxy group resulted as be-
ing the most potent, showing an IC50 against the various cell
lines ranging between 2 and 14.0 mM, thus resulting as being
equally potent as 6-MP against the haematological tumors. It
is interesting to note that the above compound resulted as be-
ing six-fold more potent than 6-MP against lung squamous
carcinoma, and equally potent as 6-MP against breast adeno-
carcinoma, hepatocellular and prostate carcinoma.
Despite the enormous variation in tumor types, a common
theme in neoplasia is that normal cell proliferation controls
are lost at the level of either cell signalling, cell-cycle arrest,
differentiation, or apoptotic cell death [8].
Recently, increased attention has been focused on the role
of apoptosis in mediating the cytotoxic effects of anticancer
agents. The induction of apoptosis has been shown to be an
important determinant of the response of most tumors to cyto-
toxic therapy.
6
4
2
0
C
10 µM
50 µM
2a
Fig. 2. Apoptosis detection by propidium iodide (PI) staining of hypodiploid
nuclei. MCF-7 cells were incubated with 2a (10e50 mM) for 24 h. Compound
2a had no effects on MCF-7 apoptosis. Data shown are representative of three
experiments performed in triplicate.
For these reasons we investigate primarily the effects of 2a
on apoptosis and cellular cycle of MCF-7 human breast cancer
cells.

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R. Filosa et al. / European Journal of Medicinal Chemistry 42 (2007) 293e306
This allows us to speculate this derivative, bearing favourable
R substituents, which could act with a cytostatic mechanism.
Furthermore we have studied effects of 2a on cell cycle of
MCF-7 cells.
In particular, cells were treated with 2a (10 mM) for 24 h.
At the end the incubation time cell cycle was analysed as
DNA content of individual cells by flow cytometry. As shown
in Fig. 3 treatment of cells with 2a induces an accumulation of
MCF-7 cells in G0/G1 phase of the cell cycle. Under control
condition 58.2% of MCF-7 cells are in G0/G1 phase of cell cy-
cle, and after 2a treatment for 24 h the percentage of cells is
increased to 87.
During this phase kinases developed a central role in the
control of the cell cycle. For the kinases to be active it must
be associated with another type of protein called cyclins.
Hence they are called cyclin-dependent kinases or ‘‘Cdks’’.
Cdks control the various checkpoints, they are the gatekeepers
that decide whether to pass or fail cells as they go through the
various quality control checkpoints in the cell cycle.
It has been shown that the cyclin-dependent kinase inhibi-
tor p21CIP/WAF-1 is an important regulator gene of cell-cycle
progression [9].
Fig. 4. Expression of p21CIP1/Waf-1 in MCF-7 cells after 2a treatment at dif-
ferent times measured by western blotting analysis. Compound 2a (10 mM)
induced a time dependent increase of p21/Waf-1. Results are representative
of three experiments.
with normal CRL 7065 line will be taken into account even
though comparable structures in the literature have the ability
to bind tightly but reversible to DNA by intercalation between
the base pairs of the double helix [10,11].
Optimisation of the design of these compounds from
a structureeactivity relationship point of view, as well as the
elucidation of their mechanisms of action, could very well
lead to the development of novel types of antineoplastic drug.
6. Experimental
6.1. General
For this reason we analysed the expression of this protein
after 2a treatment. MCF-7 cells were treated with 2a
(10 mM) at different times and then total intracellular protein
extracts were analysed by Western blotting using p21CIP-1/
Waf-1 monoclonal antibody. Results in Fig. 4 show a signifi-
cant increase of p21CIP1/Waf-1 protein after 2a treatment
for 24 h.
Cellular arrest in G0/G1 phase of cell cycle of MCF-7 cells
was accompanied by an increase in p21CIP1/Waf-1 protein
levels suggesting that antiproliferative effects of 2a are medi-
ated by p21CIP1/Waf-1 expression.
Microwave experiments were performed in a CEM Dis-
cover monomode reactor (CEM Corp., Matthews, NC). All re-
actions were conducted in a specially adapted cylindrical
Pyrex vessel. All reagents were of analytical grade and pur-
chased from SigmaeAldrich (Milan, Italy). Flash chromatog-
raphy was performed on Carlo Erba silica gel 60 (230e400
mesh; Carlo Erba, Milan, Italy). TLC was carried out using
plates coated with silica gel 60F254 purchased from Merck
(Darmstadt, Germany). Melting points were determined in
open capillary tubes on an Electrothermal 9100 apparatus
and are uncorrected. 1H and 13C NMR spectra were registered
on a Bruker AC 300. Chemical shifts are reported in parts per
million. The abbreviations used are as follows: s, singlet; d,
doublet; dd, double doublet; br s, broad singlet. The analytical
HPLC analyses were carried out on Beckman Coulter 125 S,
equipped with two high pressure binary gradient delivery
5. Conclusion
The precise mechanism of action of some of these deriva-
tives is currently under investigation in our laboratory. The
ability of 2a to cause accumulation of p21 and their inactivity
2a (10 µM) 24 h
G0/G1 58.2
G0/G1 87.0
S
34.8
S
4.6
400
300
200
100
0
G2/M 6.9
G2/M 7.5
0
30
60
90
120
0
20
40
60
80
100
120
DNA content
DNA content
Fig. 3. Cell cycle analysis. Treatment of cells with 2a (10 mM) for 24 h induces an accumulation of MCF-7 cells in the G0/G1 phase of the cell cycle. Data shown
are representative of three experiments.

R. Filosa et al. / European Journal of Medicinal Chemistry 42 (2007) 293e306
299
systems, a System Gold 166 variable-wavelength UVevis
detector and a Rheodyne 7725i injector (Rheodyne, Inc., Co-
tati, CA, USA) with a 20-mL stainless steel loop.
6.2. General procedures for 8a,den
Methyl 2-chloropyridine-4-carboxylate (1.7 mmol), phenyl-
boronic acid or ester (1.5 eq), K2CO3 (2 eq), PdCl2(dppf)
(5 mol%), 6 mL of 95% ethanol were taken in glass tube with
a magnetic stir bar. The vessel was sealed with a septum and
placed into the microwave cavity. Microwave irradiation of
60 W was used, the temperature being ramped from room tem-
perature to 110 ꢀC.
The reaction mixture was held at this temperature for
10 min. After the mixture was allowed to cool to room temper-
ature, the reaction vessel was opened and the contents were
poured in a flask.
For the analytical tests, compounds were prepared dissolv-
ing in methanol (0.5 mg/mL). Each solution (20 mL) was in-
jected in a Jupiter Phenomenex RP 18 (4.5 ꢂ 250 mm)
analytical column. The mobile phase was a combination mix-
ture of H2O þ 0.1% TFA (solvent A) and CH3CN þ 0.1% TFA
(solvent B). The elution was made in gradient from 5% of B to
71% in 22 min, the flow rate was 1.0 mL/min. UV detection
wavelength was set at 210 nm. Mass spectrometry analysis
ESI-MS was carried out on a Finnigan LCQ Deca ion trap
instrument.
The solvent was removed and saturated NaHCO3 were
added. After further extractions of the aqueous layer with
CH2Cl2, the organic washings were combined, dried over
Na2SO4 and concentrated in vacuo, left a residue which was
then subjected to flash chromatography.
6.1.1. 3-Benzyl-3,8-diazabicyclo[3.2.1]octan-2,4-dione (5)
A solution of 4 (3 g, 8.4 mmol) in absolute ethanol (70 mL)
was hydrogenated in the presence of 10% palladium charcoal
(0.3 g, 0.3 mmol), in Parr apparatus, at room temperature and
at 3 atm for 3 h. The catalyst was filtered off and ethanol was
removed under reduced pressure to give 1.98 g of 5 as hydro-
chloric salt, which was dissolved in a solution of 10% Na2CO3
(8 mL) and the aqueous solution was extracted with Et2O. The
organic layer was dried over Na2SO4 and evaporated affording
6.2.1. Ethyl 2-(3,4,5-trimethoxyphenyl)pyridine-4-
carboxylate (8a)
Elution with n-hexane/EtOAc (50:10e45:15) afforded 8a
(85%) as a pale oil. 1H NMR (CDCl3): 1.48 (t, 3H,
J ¼ 7.23 Hz); 3.95 (s, 3H); 4.01 (s, 6H); 4.49 (q, 2H,
J ¼ 7.23 Hz); 7.34 (s, 2H); 7.79 (d, 1H, J ¼ 4.82 Hz); 8.27
(s, 1H); 8.84 (d, 1H, J ¼ 4.82 Hz).
1
5 (85%) as free base. H NMR (CDCl3): 1.65e1.97 (m, 4H);
2.2 (d, 2H, J ¼ 12 Hz); 6.45 (d, 2H, J ¼ 9.2 Hz); 3.42 (m, 2H);
3.46 (s, 4H); 7.18e7.38 (m, 10H).
6.2.2. Ethyl-(4-hydroxyphenyl)-pyridine-4-carboxylate (8d)
Elution with light petroleum/EtOAc (95:5) afforded 8d
6.1.2. 3-Benzyl-3,8-diazabicyclo[3.2.1]octane (6)
1
A solution of SMEAH in toluene (5.74 mL, 19.1 mmol)
was added dropwise to a, magnetically stirred, cold (0 ꢀC) so-
lution of 5 (1 g, 4.34 mmol) in dry toluene (50 mL) and kept
under argon atmosphere. The reaction mixture was stirred at
room temperature for 2 h. The solution was cooled to 0 ꢀC
and quenched with 20% Na2CO3. The aqueous layer was sep-
arated and extracted with Et2O. The organic layer was dried
over Na2SO4 and evaporated to give a residue which was
then subjected to flash chromatography. Elution with CHCl3/
MeOH/NH3 (95:5:0.05) afforded 0.92 g of 6 (70%) as a white
solid. 1H NMR (CDCl3): 1.65e1.97 (m, 4H); 2.2 (d, 2H,
J ¼ 12 Hz); 6.45 (d, 2H, J ¼ 9.2 Hz); 3.42 (m, 2H); 3.46 (s,
2H); 7.18e7.38 (m, 5H).
(72%). H NMR (CDCl3): 1.47 (t, 3H, J ¼ 7.02 Hz); 4.48 (q,
2H, J ¼ 7.02 Hz); 6.97 (d, 2H, J ¼ 8.55 Hz); 7.45 (d, 1H,
J ¼ 5.04 Hz); 7.99 (d, 1H, J ¼ 58.55 Hz); 8.26 (s, 1H); 8.81
(d, 1H, J ¼ 5.04 Hz).
6.2.3. Ethyl-2(3-hydroxyphenyl)-pyridine-4-carboxylate (8e)
Elution with light petroleum/EtOAc (9:1) afforded 8e
1
(62%). H NMR (CDCl3): 1.45 (t, 3H, J ¼ 7.23 Hz); 4.46 (q,
2H, J ¼ 7.23 Hz); 7.41 (d, 2H, J ¼ 5.26); 7.81 (d, 1H,
J ¼ 5.26 Hz); 7.93 (t, 1H, J ¼ 3.95 Hz); 8.07 (s, 1H); 8.26 (s,
1H); 8.82 (d, 1H, J ¼ 5.26 Hz).
6.2.4. Ethyl-(2-p-tolyl)-pyridine-4-carboxylate (8f)
Elution with light petroleum/EtOAc (95:5) afforded 8f
6.1.3. 3,8-Diazabicyclo[3.2.1]octane (DBO) (3)
1
(44%). H NMR (CDCl3): 1.45 (t, 3H, J ¼ 7.02 Hz); 2.45 (s,
A solution of 7 (1.31 g, 6.5 mmol) in absolute ethanol
(55 mL) and 6 N HCl (3 mL) was hydrogenated in the pres-
ence of 10% palladium charcoal (0.3 g, 0.3 mmol), in Parr
apparatus, at room temperature and at 3 atm for 12 h. The
catalyst was filtered off and ethanol was removed under re-
duced pressure. The residue was dissolved in Et2O and etha-
nol was added dropwise until the formation of a precipitate.
The precipitate was collected affording 1 g of 3 (83%) as
3H); 4.50 (q, 2H, J ¼ 7.02 Hz); 7.33 (d, 2H, J ¼ 8.55 Hz);
7.77 (d, 1H, J ¼ 5.04 Hz); 7.98 (d, 2H, J ¼ 8.55 Hz); 8.30 (s,
1H); 8.83 (d, 1H, J ¼ 5.04 Hz).
6.2.5. Ethyl-2(3-tolyl)-pyridine-4-carboxylate (8g)
Elution with n-hexane/EtOAc (95:5) afforded 8g (65%). 1H
NMR (CDCl3): 1.45 (t, 3H, J ¼ 7.02 Hz); 2.5 (s, 3H); 4.49 (q,
2H, J ¼ 7.02 Hz); 7.31 (d, 1H, J ¼ 7.67 Hz); 7.81 (d, 1H,
J ¼ 5.04 Hz); 7.87 (d, 1H, J ¼ 7.67 Hz); 7.92 (s, 1H); 8.32
(s, 1H); 8.86 (d, 1H, J ¼ 5.04 Hz).
1
a white solid. H NMR (CD3OD): 2.35e2.45 (m, 4H); 3.55
(d, 2H, J ¼ 13.59 Hz); 3.68 (d, 2H, J ¼ 13.59 Hz); 4.30
(s, 2H).

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R. Filosa et al. / European Journal of Medicinal Chemistry 42 (2007) 293e306
6.2.6. Ethyl-2(4-acetamidophenyl)-pyridine-4-carboxylate (8h)
Elution with light petroleum/EtOAc (8:2e4:6) afforded 8h
quenched with a saturated solution of NaHCO3. The mixture
was filtered and the solvent was removed in vacuo.
1
(60%). H NMR (CDCl3): 1.47 (t, 3H, J ¼ 7.02 Hz); 2.25 (s,
3H); 4.48 (q, 2H, J ¼ 7.02 Hz); 7.68 (d, 2H, J ¼ 8.55 Hz);
7.78 (d, 1H, J ¼ 5.04 Hz); 8.08 (d, 2H, J ¼ 8.55 Hz); 8.29 (s,
1H); 8.83 (d, 2H, J ¼ 5.04 Hz).
6.3.1. 2-(3,4,5-(Trimethoxyphenyl)pyridin-4-yl)methanol
(9a)
The residue was diluted with water and extracted with
CH2Cl2. The organic layer was dried over anhydrous
Na2SO4, filtered and the solvent evaporated to give a residue
which was then subjected to flash chromatography. Elution
with hexane/EtOAc (7:3e4:6) afforded 9a (58%) as a white
6.2.7. Ethyl-2(3-acetamidophenyl)-pyridine-4-carboxylate (8i)
Elution with light petroleum/EtOAc (8:2e4:6) afforded 8i
1
(60%). H NMR (CDCl3): 1.47 (t, 3H, J ¼ 7.02 Hz); 2.25 (s,
1
3H); 4.48 (q, 2H, J ¼ 7.02 Hz); 7.74 (t, 1H, J ¼ 7.89 Hz);
7.9 (d, 1H, J ¼ 5.04 Hz); 8.33 (d, 1H, J ¼ 7.89 Hz); 8.39 (s,
1H); 8.46 (d, 1H, J ¼ 7.89 Hz); 8.91 (d, 1H, J ¼ 5.04 Hz);
8.98 (s, 1H).
powder. H NMR (CDCl3): 3.93 (s, 3H); 3.98 (s, 6H); 4.79
(s, 2H); 7.24 (d, 1H, J ¼ 4.82); 7.25 (s, 2H); 7.72 (s, 1H);
8.66 (d, 1H, J ¼ 4.82 Hz).
6.3.2. 4-(4-(Hydroxymethyl)pyridine-2-yl)phenol (9d)
The residue was chromatographed on silica gel using light
petroleum/EtOAc (6:4) as an eluent yielding 9d (71%) as
a pale oil. 1H NMR (CDCl3): 4.80 (s, 2H); 6.95 (d, 2H,
J ¼ 8.77 Hz); 7.19 (d, 1H, J ¼ 5.04 Hz); 7.68 (s, 1H); 7.88
(d, 2H, J ¼ 8.77 Hz); 8.61 (d, 1H, J ¼ 5.04 Hz).
6.2.8. Ethyl-(4-chlorophenyl)-pyridine-4-carboxylate (8j)
Elution with n-hexane/EtOAc (95:5) afforded 8j (50%). 1H
NMR (CDCl3): 1.43 (t, 3H, J ¼ 7.1 Hz); 4.44 (q, 2H,
J ¼ 7.1 Hz); 7.44 (dd, 2H, J ¼ 7.2e1.7 Hz); 7.77 (dd, 1H,
J ¼ 5.1e1.4 Hz); 7.98 (dd, 1H, J ¼ 6.7e1.7 Hz); 8.23 (s,
1H); 8.79 (d, 1H, J ¼ 4.9 Hz).
6.3.3. 3-(4-(Hydroxymethyl)pyridine-2-yl)phenol (9e)
The residue was chromatographed on silica gel using light
petroleum/Et2O (3:7) as an eluent yielding 9e (68%) as a pale
oil. 1H NMR (CDCl3): 4.85 (s, 2H); 6.93 (d, 1H, J ¼ 4.82 Hz);
7.27 (d, 1H, J ¼ 7.89 Hz); 7.37 (t, 1H, J ¼ 7.89 Hz); 7.54 (s,
1H); 7.55 (d, 1H, J ¼ 7.89 Hz); 7.75 (s, 1H); 8.67 (d, 1H,
J ¼ 4.82 Hz).
6.2.9. Ethyl-2(4-nitrophenyl)-pyridine-4-carboxylate (8k)
Elution with light petroleum/EtOAc (95:5) afforded 8k
1
(66%). H NMR (CDCl3): 1.47 (t, 3H, J ¼ 7.02 Hz); 4.46 (q,
2H, J ¼ 7.02 Hz); 7.97 (d, 1H, J ¼ 5.04 Hz); 8.28 (d, 2H,
J ¼ 8.11 Hz); 8.38 (d, 3H, J ¼ 8.11 Hz); 8.93 (d, 1H,
J ¼ 5.04 Hz).
6.2.10. Ethyl-2(3-nitrophenyl)-pyridine-4-carboxylate (8l)
Elution with hexane/EtOAc (85:15) afforded 8l (87%). H
NMR (CDCl3): 1.49 (t, 3H, J ¼ 7.02 Hz); 4.51 (q, 2H,
J ¼ 7.02 Hz); 7.74 (t, 1H, J ¼ 7.89 Hz); 7.9 (d, 1H,
J ¼ 5.04 Hz); 8.33 (d, 1H, J ¼ 7.89 Hz); 8.39 (s, 1H); 8.46
(d, 1H, J ¼ 7.89 Hz); 8.91 (d, 1H, J ¼ 5.04 Hz); 8.98 (s, 1H).
1
6.3.4. (2-p-Tolylpyiridin-4-yl)methanol (9f)
The residue was diluted with water and extracted with Et2O.
The organic layer was dried over anhydrous Na2SO4, filtered
and the solvent evaporated to give a residue which was then
subjected to flash chromatography. Elution with hexane/EtOAc
(8:2) afforded 9f (68%), as a pale oil. 1H NMR (CDCl3): 2.43
(s, 3H); 4.69 (s, 2H); 7.33 (d, 2H, J ¼ 8.11 Hz); 7.37 (d, 2H,
J ¼ 5.04 Hz); 7.82 (s, 1H); 7.84 (d, 2H, J ¼ 8.11 Hz).
6.2.11. Ethyl-(2-phenyl)-pyridine-4-carboxylate (8m)
Elution with light petroleum/EtOAc (95:5) afforded 8m
1
(92%). H NMR (CDCl3): 1.48 (t, 3H, J ¼ 7.23 Hz); 4.49 (q,
6.3.5. (2-m-Tolylpyridin-4-yl)methanol (9g)
2H, J ¼ 7.23 Hz); 7.43e7.59 (m, 3H); 7.82 (dd, 1H,
J ¼ 1.32e4.82 Hz); 8.1 (dd, 1H, J ¼ 8.55e1.10 Hz); 8.34 (t,
1H, J ¼ 1.10 Hz); 8.87 (dd, 1H, J ¼ 5.04e0.66 Hz).
The residue was diluted with water and extracted with
EtOAc. The organic layer was dried over anhydrous Na2SO4,
filtered and the solvent evaporated to give a residue which
was then subjected to flash chromatography. Elution with light
petroleum/EtOAc (9:1) afforded 9g (78%), as a pale yellow oil.
1H NMR (CDCl3): 2.0 (br s, OH); 2.47 (s, 3H); 4.84 (s, 2H);
7.24e7.38 (m, 2H); 7.39 (t, 1H, J ¼ 7.89); 7.63 (s, 1H); 7.80
(d, 2H, J ¼ 7.87 Hz); 8.68 (d, 1H, J ¼ 5.04 Hz).
6.2.12. Ethyl-3-phenylbenzoate (8n)
The residue was chromatographed on silica gel using n-hex-
ane/Et2O (95:5) as an eluent yielding 8n (77%) as a pale oil.
1H NMR (CDCl3): 1.47 (t, 3H, J ¼ 7.02 Hz); 4.48 (q, 2H,
J ¼ 7.02 Hz); 7.29e7.58 (m, 4H); 7.68 (d, 1H, J ¼ 5.04 Hz);
7.82(d, 1H, J ¼ 4.82 Hz);8.09(d, 1H, J ¼ 4.82 Hz); 8.36(s, 1H).
6.3.6. N-4-(4-(Hydroxymethyl)pyridin-2-yl)phenyl-
acetamide (9h)
6.3. General procedures for 9a,den
The residue was chromatographed on silica gel using light
petroleum/Et2O (6:4) as an eluent yielding 9h (55%) as a pale
oil. 1H NMR (CDCl3): 4.85 (s, 2H); 6.93 (d, 1H, J ¼ 4.82 Hz);
7.27 (d, 1H, J ¼ 7.89 Hz); 7.37 (t, 1H, J ¼ 7.89 Hz); 7.54 (s,
1H); 7.55 (d, 1H, J ¼ 7.89 Hz); 7.75 (s, 1H); 8.67 (d, 1H,
J ¼ 4.82 Hz).
A solution of 1.5 M DIBALH in toluene or THF (3 eq) was
added dropwise to a magnetically stirred solution of 10
(1.535 mmol) in toluene or THF (15 mL) and cooled at
0 ꢀC. The reaction mixture was stirred at room temperature
until disappearance of starting material. The reaction was

R. Filosa et al. / European Journal of Medicinal Chemistry 42 (2007) 293e306
301
6.3.7. N-(3-(4-Hydroxymethyl)pyridine-2-yl)phenyl)
acetamide (9i)
6.4. General procedures for 10a,den
The residue was chromatographed on silica gel using light
petroleum/EtOAc (6:4) as an eluent yielding 9i (57%) as
a pale oil. 1H NMR (CDCl3): 2.19 (s, 3H); 4.88 (s, 2H);
7.34 (d, 1H, J ¼ 5.04 Hz); 7.68 (t, 1H, J ¼ 7.89); 7.87 (s,
1H); 8.3 (d, 1H, J ¼ 7.89 Hz); 8.42 (d, 1H, J ¼ 7.89 Hz);
8.72 (d, 1H, J ¼ 5.04 Hz); 8.91 (s, 1H).
A solution of thionyl chloride (1.77 eq) in dry benzene
(3 mL) was added dropwise to a cold (0 ꢀC) solution of
9a,den (120 mg) in dry benzene (6 mL).
The reaction mixture was refluxed for 10 min and then
cooled at 0 ꢀC. The solvent was removed and the residue
was diluted with saturated NaHCO3 and extracted with
CH2Cl2 (3 ꢂ 15 mL). The combined organic phases were dried
over anhydrous Na2SO4, filtered and the solvent evaporated in
vacuo.
6.3.8. (2-(4-Chlorophenyl)pyridine-4-yl)methanol (9j)
The residue was diluted with water and extracted with
Et2O. The organic layer was dried over anhydrous Na2SO4, fil-
tered and the solvent evaporated to give a residue which was
then subjected to flash chromatography. Elution with light pe-
troleum/EtOAc (8:2e1:1) afforded 9j (84%), as a pale yellow
6.4.1. 4-(Chloromethyl)-2-(3,4,5-trimethoxyphenyl)
pyridine (10a)
The residue was crystallized from diethyl ether affording
1
10a (99%) as a white powder. H NMR (CDCl3): 3.93 (s,
3H); 3.98 (s, 6H); 4.65 (s, 2H); 6.88 (d, 1H, J ¼ 4.82 Hz);
1
oil. H NMR (CDCl3): 1.93 (t, 1H, J ¼ 5.7 Hz); 4.85 (d, 2H,
J ¼ 5.7 Hz); 7.27 (d, 2H, J ¼ 5.04 Hz); 7.48 (d, 2H,
J ¼ 8.55 Hz); 7.75 (s, 1H); 7.99 (d, 2H, J ¼ 8.55 Hz); 8.68
(d, 1H, J ¼ 5.04 Hz).
6.95 (s, 2H); 7.44 (s, 1H); 8.57 (s, 1H).
6.4.2. 4-(4-Chloromethyl)pyridin-2-ylphenol (10d)
The residue was chromatographed on silica gel using light
petroleum/EtOAc (6:4) as an eluent yielding 10d (72%) as
6.3.9. (2-(4-Nitrophenyl)pyridine-4-yl)methanol (9k)
1
a pale yellow oil. H NMR (CDCl3): 4.63 (s, 2H); 6.96 (d,
The residue was diluted with water and extracted with
EtOAc. The organic layer was dried over anhydrous
Na2SO4, filtered and the solvent evaporated to give a residue
which was then subjected to flash chromatography. Elution
with light petroleum/EtOAc (7:3) afforded 9k (77%), as
2H, J ¼ 8.77 Hz); 7.24 (d, 1H, J ¼ 5.04 Hz); 7.71 (s, 1H);
7.94 (d, 2H, J ¼ 8.77 Hz); 8.67 (d, 1H, J ¼ 5.04 Hz).
6.4.3. 3-(4-Chloromethyl)pyridin-2-ylphenol (10e)
1
a pale yellow oil. H NMR (CDCl3): 4.76 (s, 2H); 7.25 (d,
1H, J ¼ 5.04 Hz); 7.9 (s, 1H); 8.25 (d, 2H, J ¼ 8.77 Hz); 8.3
The residue was chromatographed on silica gel using light
petroleum/EtOAc (7:3) as an eluent yielding 10e (70%) as
1
a pale yellow oil. H NMR (CDCl3): 4.61 (s, 2H); 6.91 (dd,
(d, 2H, J ¼ 8.77 Hz); 8.75 (d, 1H, J ¼ 5.04 Hz).
1H, J ¼ 2.63e7.89 Hz); 7.3 (d, 1H, J ¼ 4.82 Hz); 7.34 (d,
1H, J ¼ 7.89 Hz); 7.43 (d, 1H, J ¼ 7.89 Hz); 7.55 (t, 1H,
J ¼ 1.97 Hz); 7.69 (s, 1H); 8.67 (d, 1H, J ¼ 4.82 Hz).
6.3.10. (2-(3-Nitrophenyl)pyridin-4-yl)methanol (9l)
The residue was diluted with water and extracted with
EtOAc. The organic layer was dried over anhydrous
Na2SO4, filtered and the solvent evaporated to give a residue
which was then subjected to flash chromatography. Elution
with light petroleum/EtOAc (7:3e2:8) afforded 9l (69%), as
6.4.4. 4-(Chloromethyl)-2-p-tolylpyridine (10f)
The residue was chromatographed on silica gel using light
petroleum/EtOAc (8:2) as an eluent yielding 10f (80%) as
a pale oil. 1H NMR (CDCl3): 2.41 (s, 3H); 4.61 (s, 2H);
7.28e7.32 (m, 2H); 7.75 (s, 1H); 7.91 (d, 2H, J ¼ 8.77);
8.68 (d, 1H, J ¼ 4.82 Hz).
1
a pale yellow oil. H NMR (CDCl3): 4.88 (s, 2H); 7.34 (d,
1H, J ¼ 5.04 Hz); 7.68 (t, 1H, J ¼ 7.89); 7.87 (s, 1H); 8.3 (d,
1H, J ¼ 7.89 Hz); 8.42 (d, 1H, J ¼ 7.89 Hz); 8.72 (d, 1H,
J ¼ 5.04 Hz); 8.91 (s, 1H).
6.4.5. 4-(Chloromethyl)-2-m-tolylpyridine (10g)
The residue was chromatographed on silica gel using light
petroleum/EtOAc (75:25) as an eluent yielding 10g (81%) as
6.3.11. 2-(2-Phenylpyiridin-4-yl)methanol (9m)
The residue was diluted with water and extracted with
CH2Cl2. The organic layer was dried over anhydrous
Na2SO4, filtered and the solvent evaporated affording 9m
1
a pale yellow oil. H NMR (CDCl3): 2.49 (s, 3H); 4.56 (s,
2H); 7.41e7.44 (m, 2H); 7.79e7.82 (m, 2H); 7.88 (s, 1H);
8.43 (d, 2H, J ¼ 8.77); 8.73 (d, 1H, J ¼ 4.82 Hz).
1
(96%), as a pale oil. H NMR (CDCl3): 4.85 (s, 2H); 7.27
(d, 1H,J ¼ 6.36 Hz); 7.44e7.55 (m, 3H); 7.78 (s, 1H); 8.04
6.4.6. N-(4-(4-(Chloromethyl)pyridin-2-yl)phenyl)
acetamide (10h)
(d, 2H, J ¼ 7.02 Hz); 8.70 (d, 1H, J ¼ 5.04 Hz).
The residue was chromatographed on silica gel using light
petroleum/EtOAc (6:4) as an eluent yielding 10h (73%) as
6.3.12. 3-Biphenylmethanol (9n)
1
a pale yellow oil. H NMR (CDCl3): 2.44 (s, 3H); 4.48 (s,
The residue was diluted with water and extracted with
CH2Cl2. The organic layer was dried over anhydrous
Na2SO4, filtered and the solvent evaporated affording 9n
2H); 7.25 (d, 1H, J ¼ 5.26 Hz); 7.36 (s, 1H); 7.66 (d, 2H,
J ¼ 8.77 Hz); 7.73 (s, 1H); 8.02 (d, 2H, J ¼ 8.77 Hz); 8.67
(d, 1H, J ¼ 5.26 Hz).
1
(98%). H NMR (CDCl3): 4.81 (s, 2H); 7.36e7.74 (m, 9H).

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R. Filosa et al. / European Journal of Medicinal Chemistry 42 (2007) 293e306
6.4.7. N-(3-(4-(Chloromethyl)pyridin-2-yl)phenyl)
acetamide (10i)
The residue was chromatographed on silica gel using light
petroleum/EtOAc (6:4) as an eluent yielding 10i (75%) as
1
a pale yellow oil. H NMR (CDCl3): 2.44 (s, 3H); 4.44 (s,
2H); 7.43e7.48 (m, 2H); 7.79e7.82 (m, 2H); 7.88 (s, 1H);
8.43 (d, 2H, J ¼ 8.77); 8.77 (d, 1H, J ¼ 4.82 Hz).
dried over anhydrous Na2SO4, filtered and the solvent evaporated
to give a residue which was then subjected to flash chromatog-
raphy. Elution with CH2Cl2/Et2O (95:5) afforded 12 (77%) as
a pale yellow oil. 1H NMR (CDCl3): 2.40 (s, 3H); 3.87 (s, 3H);
6.94e7.04 (m, 3H); 7.49 (br s, 1H); 7.94 (d, 2H, J ¼ 8 Hz);
8.51 (d, 1H, J ¼ 5.2 Hz).
6.4.14. 2-(4-Methoxyphenyl)pyridine-4-carboxylic acid (13)
KMnO4 (370 mg, 1.1 eq) was added to a rapidly mechani-
cally stirred solution of 12 (0.425 g, 2.13 mmol) in a mixture
of tert-butyl alcohol (2.55 mL) and water (1 mL). The reaction
mixture was refluxed for 2.5 h. An additional 202.4 mg
(0.6 eq) of KMnO4 was added together with 1 mL of a 1:1
mixture of tert-butyl alcohol/water. The reaction mixture
was refluxed until the permanganate color disappeared (about
2.5 h). An additional 202.4 mg (0.6 eq) of KMnO4 was added
together with 2 mL of a 1:1 mixture of tert-butyl alcohol/wa-
ter. After 3.5 h 370 mg of KMnO4 (1.1 eq) was added together
with 2 mL of a 1:1 mixture of tert-butyl alcohol/water. The re-
action mixture was refluxed overnight. Finally, an additional
720 mg of KMnO4 (2 eq) was added and the reaction mixture
was refluxed until the permanganate color disappeared. The
solution was cooled at room temperature, filtered on Celite
and the filtrate was washed more number of times with hot wa-
ter. The aqueous phase was washed with diethyl ether and then
acidified with acetic acid to pH 3.6. The precipitate was col-
lected affording 13 (76%) as a white solid. 1H NMR
(CD3OD): 3.87 (s, 3H); 7.01 (d, 2H, J ¼ 9 Hz); 7.79 (d, 1H,
J ¼ 6.0 Hz); 8.02 (d, 2H, J ¼ 9 Hz); 8.23 (s, 1H); 8.73 (d,
1H, J ¼ 6.0 Hz).
6.4.8. 4-(Chloromethyl)-2-(4-chlorophenyl)pyridine (10j)
The residue was chromatographed on silica gel using light
petroleum/EtOAc (7:3) as an eluent yielding 10j (94%) as
1
a pale yellow oil. H NMR (CDCl3): 4.64 (s, 2H); 7.30 (d,
1H, J ¼ 5.1 Hz); 7.47 (d, 2H, J ¼ 8.6 Hz); 7.76 (s, 1H); 7.98
(d, 2H, J ¼ 8.6 Hz); 8.71 (d, 1H, J ¼ 5.1 Hz).
6.4.9. 4-(Chloromethyl)-2-(4-nitrophenyl)pyridine (10k)
The residue was chromatographed on silica gel using light
petroleum/EtOAc (6:4) as an eluent yielding 10k (79%) as
1
a pale yellow oil. H NMR (CDCl3): 4.47 (s, 2H); 7.25 (d,
1H, J ¼ 5.26 Hz); 7.36 (s, 1H); 7.66 (d, 2H, J ¼ 8.77 Hz);
7.73 (s, 1H); 8.01 (d, 2H, J ¼ 8.77 Hz); 8.67 (d, 1H,
J ¼ 5.26 Hz).
6.4.10. 4-(Chloromethyl)-2-(3-nitrophenyl)pyridine (10l)
The residue was chromatographed on silica gel using light
petroleum/Et2O (9:1e8:2) as an eluent yielding 10l (79%) as
1
a pale yellow oil. H NMR (CDCl3): 4.55 (s, 2H); 7.37 (d,
1H, J ¼ 5.04 Hz); 7.69 (t, 1H, J ¼ 7.89 Hz); 7.85 (s, 1H);
8.32 (d, 1H, J ¼ 7.89 Hz); 8.41 (d, 1H, J ¼ 7.89 Hz); 8.74
(d, 1H, J ¼ 5.04 Hz).
6.4.11. 4-(Chloromethyl)-2-phenylpyridine (10m)
6.4.15. (2-(4-Methoxyphenyl)pyridin-4-yl)methanol (15)
Thionyl chloride (1.5 mL, 21 mmol) was added to 15
(0.16 g, 0.7 mmol). The reaction mixture was stirred at
60 ꢀC for 4 h. Thionyl chloride in excess was removed. The
residue was cooled at 0 ꢀC and methanol was added dropwise.
The reaction mixture was stirred at room temperature for 2 h.
The solvent was removed and the residue was diluted with 5%
NaHCO3 and extracted with CHCl3. The organic layer was
dried over anhydrous Na2SO4, filtered and the solvent evapo-
rated to give methyl 2-(4-methoxyphenyl)pyridine-4-carboxyl-
ate 14 (82%) as a yellow oil. 1H NMR (CDCl3): 9.88 (s, 3H);
3.99 (s, 3H); 7.01 (d, 2H, J ¼ 8.99 Hz); 7.71 (d, 1H,
J ¼ 5.04 Hz); 8.02 (d, 2H, J ¼ 8.8 Hz); 8.25 (s, 1H); 8.79 (d,
1H, J ¼ 5.04 Hz).
The residue was chromatographed on silica gel using light
petroleum/EtOAc (8:2) as an eluent yielding 10m (71%) as
1
a pale oil. H NMR (CDCl3): 4.50 (s, 2H); 7.4e7.6 (m, 4H);
7.78 (s, 1H); 8.04 (d, 2H, J ¼ 7.23 Hz); 8.72 (d, 1H,
J ¼ 5.04 Hz).
6.4.12. 3-(Chloromethyl)biphenyl (10n)
The combined organic phases were dried over anhydrous
Na2SO4, filtered and the solvent evaporated in vacuo to give
a residue which was then subjected to flash chromatography.
Elution with n-hexane/EtOAc (9:1e8:2) afforded 10n (97%).
1H NMR (CDCl3): 4.69 (s, 2H); 7.35e7.90 (m, 9H).
6.4.13. 1-(4-Methoxyphenyl)-4-methylpyridine (12)
A solution of 1.5 M DIBALH in toluene (0.89 mL,
1.32 mmol) was added dropwise to a magnetically stirred so-
lution of 14 (0.106 g, 0.44 mmol) in toluene (8 mL) and
cooled at 0 ꢀC. The reaction mixture was stirred at room tem-
perature for 1 h. The reaction was quenched with a saturated
solution of potassium tartrate (0.297 g). The mixture was fil-
tered and diluted with diethyl ether. The organic layer was
dried over anhydrous Na2SO4, filtered and the solvent evapo-
rated to give a residue which was then subjected to flash chro-
matography. Elution with light petroleum/EtOAc (1:1)
2-Bromo-4-methyl pyridine (1g, 5.8 mmol) was added to
a magnetically stirred solution of NiCl2(dppe) (0.266 g,
0.505 mmol) in dry THF (21 mL).
The reaction mixture was magnetically stirred at room tem-
perature under argon atmosphere for 5 min and then a solution
of 4-methoxyphenyl magnesium bromide (1.48 g, 7.0 mmol)
in dry THF (10 mL) was added dropwise. The reaction mix-
ture was stirred at room temperature for 18 h. The solvent
was removed and the residue was diluted with a concentrated
solution of NH3 (27 mL) and the aqueous phase was extracted
with CH2Cl2 (3 ꢂ 30 mL). The combined organic phases were
1
afforded 15 (79%) as a pale oil. H NMR (CDCl3): 3.89 (s,
3H); 4.81 (s, 2H); 7.02 (d, 2H, J ¼ 8.99 Hz); 7.19 (d, 1H,

R. Filosa et al. / European Journal of Medicinal Chemistry 42 (2007) 293e306
303
J ¼ 5.04 Hz); 7.70 (s, 1H); 7.98 (d, 2H, J ¼ 8.99 Hz); 8.63
(1H, J ¼ 5.04 Hz).
layer was dried over anhydrous Na2SO4, filtered and the
solvent evaporated to give a residue which was then subjected
to flash chromatography. Elution with light petroleum/EtOAc
(8:2e1:1) afforded (2-(3-methoxyphenyl)pyridin-4-yl)metha-
6.4.16. 4-(Chloromethyl)-2-(4-methoxyphenyl)pyridine (10b)
Thionyl chloride (0.473 mL, 6.48 mmol) was added to a so-
lution of 15 (0.08 g, 0.37 mmol) in CH2Cl2 (3.5 mL). The re-
action mixture was stirred at room temperature for 4 h. The
solvent was removed and the residue was diluted with Et2O
and washed with saturated NaHCO3. The organic layer was
dried over anhydrous Na2SO4, filtered and the solvent evapo-
rated affording 10b (85%) as a pale yellow oil. 1H NMR
(CDCl3): 3.91 (s, CH3O); 4.80 (s, CH2); 7.01 (d, 2H,
J ¼ 8.99 Hz); 7.20 (d, 1H, J ¼ 5.04 Hz); 7.74 (s, 1H); 8.00
(d, 2H, J ¼ 8.99 Hz); 8.7 (1H, J ¼ 5.04 Hz).
1
nol 19 (86%) as a pale yellow oil. H NMR (CDCl3): 3.93
(s, 3H); 4.85 (s, 2H); 7.01 (d, 1H, J ¼ 8.11 Hz); 7.28 (d, 1H,
J ¼ 4.82 Hz) 7.41 (t, 1H, J ¼ 8.11 Hz); 7.59 (d, 1H,
J ¼ 8.11 Hz); 7.62 (s, 1H); 7.76 (s, 1H); 8.67 (d, 1H,
J ¼ 4.82 Hz). Thionyl chloride (1.26 mL, 17.2 mmol) was
added to a solution of 19 (0.212 g, 0.98 mmol) in CH2Cl2
(3.5 mL). The reaction mixture was stirred at room tempera-
ture for 4 h.
The solvent was removed and the residue was diluted with
CH2Cl2 and washed with saturated NaHCO3. The organic
layer was dried over anhydrous Na2SO4, filtered and the sol-
vent evaporated to give a residue which was then subjected
to flash chromatography. Elution with light petroleum/EtOAc
(7:3) afforded 10c (96%) as a pale yellow oil. 1H NMR
(CDCl3): 3.94 (s, 3H); 4.64 (s, 2H); 7.02 (d, 1H,
J ¼ 7.89 Hz); 7.30 (br, 1H); 7.43 (t, 1H, J ¼ 7.89 Hz); 7.58
(d, 1H, J ¼ 7.89 Hz); 7.63 (d, 1H, J ¼ 4.82 Hz); 7.86 (s,
1H); 8.67 (1H, J ¼ 4.82 Hz).
6.4.17. (2-(3-Methoxyphenyl)pyridin-4-yl)methoxy-
tert-butyl-dimethylsilane (18)
Imidazole (0.197 g, 2.9 mmol) and TBDSiCl (0.219 g,
1.93 mmol) were added to a cold (0 ꢀC) solution of (2-bromo-
pyridin-4-yl)methanol (16) in dry DMF. The resulting mixture
was magnetically stirred at 25 ꢀC for 13 h. The solvent was re-
moved and the residue was diluted with water (13 mL) and the
aqueous phase was extracted with Et2O (3 ꢂ 20 mL). The
combined organic phases were dried over anhydrous
Na2SO4, filtered and the solvent evaporated to give a residue
which was then subjected to flash chromatography. Elution
with light petroleum/EtOAc (9:1e1:1) afforded 17 (89%) as
6.5. General procedures for 2aer
DBO (8) (1 eq), K2CO3 (4 eq), NaI (4 eq), alkyl chlorides
(2 eq), DMF (5 mL) were taken in a 6-mL glass tube with
a magnetic stir bar.
For derivatives 2o, 2p and 2q the corresponding alkyl ha-
lides such as benzyl chloride, 4-phenyl benzyl chloride and
1-(chloromethyl)naphthalene were purchased from Sigmae
Aldrich.
For derivative 2r 1-bromomethylphenanthrene was synthe-
sized as described in the literature [11].
The vessel was sealed with a septum and placed into the
microwave cavity. The reaction mixture was then subjected
to microwave irradiation under conditions of time, power
and temperature as indicated in Table 2.
At the end of irradiation the reaction mixture was cooled to
room temperature.
The solvent was removed and the residuewas diluted with wa-
ter and extracted with CH2Cl2. The organic layer was dried over
anhydrous Na2SO4, filtered and the solvent evaporated to give
a residue which was then subjected to flash chromatography.
1
colorless oil. H NMR (CDCl3): 0.18 (s, 6H); 1.01 (s, 9H);
4.77 (s, 2H); 7.23 (d, 1H; J ¼ 5.04 Hz); 7.39 (s, 1H); 8.3
(1H, J ¼ 5.04 Hz).
To a solution of NiCl2(dppe) (0.132 g, 0.26 mmol) in dry
THF (28 mL), kept under argon atmosphere at room tempera-
ture and magnetically stirred, was added 17 (0.88 g,
2.9 mmol). The reaction mixture was stirred for 5 min and
then a solution of 3-methoxyphenyl magnesium bromide
(0.6 g, 3.49 mmol) in THF (7 mL) was added dropwise. The
solution was stirred at room temperature for 18 h. The solvent
was removed and the residue was diluted with NH3 (14 mL)
and water (10 mL). The aqueous phase was extracted with
CH2Cl2 (3 ꢂ 20 mL). The combined organic phases were dried
over anhydrous Na2SO4, filtered and the solvent evaporated to
give a residue which was then subjected to flash chromatogra-
phy. Elution with hexane/EtOAc (9:1) afforded 18 (65%) as
1
a white solid. H NMR (CDCl3): 0.2 (s, 6H); 1.01 (s, 9H);
3.93 (s, 3H); 4.84 (s, 2H); 7.00 (dd, 1H, J ¼ 8.11, 2.64 Hz);
7.23 (d, 1H, J ¼ 4.88 Hz) 7.41 (t, 1H, J ¼ 7.89 Hz); 7.58 (d,
1H, J ¼ 7.89 Hz); 7.62 (t, 1H, J ¼ 2.19); 7.73 (s, 1H); 8.66
(d, 1H, J ¼ 4.82 Hz).
6.5.1. 3,8-Bis((2-(3,4,5-trimethoxyphenyl)pyridin-4-yl)
methyl)-3,8-diazabicyclo[3.2.1]octane (2a)
The residue was chromatographed on silica gel using light
petroleum/EtOAc (2:8) to provide 2a (70%) as an orange
solid. Compound 2a was converted into its hydrochloric salt.
6.4.18. 4-(Chloromethyl)-2-(3-methoxyphenyl)pyridine (10c)
A solution of TBAF (4.58 mL, 4.58 mmol) in THF was
added to a cold (0 ꢀC) solution of 18 (0.58 g, 1.76 mmol) in
dry THF (20 mL). The reaction mixture was stirred for
45 min at 0 ꢀC and then for 3 h at 25 ꢀC.
The reaction was diluted with water (40 mL) and stirred for
10 min. The solvent was removed and the residue was diluted
with water (10 mL) and extracted with CH2Cl2. The organic
1
Mp: 195.4 ꢀC; H NMR (D2O): 2.2e2.4 (m, 4H); 2.92e2.98
(m, 4H); 3.83 (s, 3H); 3.84 (s, 3H); 3.91 (s, 6H); 3.93 (s,
2H); 4.00 (s, 2H); 4.17 (s, 2H); 4.6 (s, 2H); 7.16 (s, 2H);
7.23 (s, 2H); 803 (dd, 1H, J ¼ 5.92, 1.53 Hz); 8.09 (dd, 1H,
J ¼ 5.92, 1.53 Hz); 8.23 (s, 1H); 8.49 (s, 1H); 8.64 (d, 1H,
J ¼ 5.92 Hz); 8.81 (d, 1H, J ¼ 5.92 Hz). 13C NMR (D2O):
22.1; 50.2; 54.3; 55.2; 57.5; 59.1; 60.1; 103.1; 103.2; 123.1;

304
R. Filosa et al. / European Journal of Medicinal Chemistry 42 (2007) 293e306
123.7; 124.1; 125.1; 125.7; 129.1; 141.3; 147.1; 149.8; 151.4;
158.1. MS (ESI) m/z: 627.3; 382.0.
138.7; 138.7; 147.7; 147.8; 147.9; 148.7; 154.5; 154.6;
156.3. MS (ESI) m/z: 479.2.
6.5.2. 3,8-Bis((2-(4-methoxyphenyl)pyridin-4-yl)methyl)-
3,8-diazabicyclo[3.2.1]octane (2b)
6.5.6. 3,8-Bis((2-p-tolylpyridin-4-yl)methyl)-3,8-diaza bicyclo
[3.2.1]octane (2f)
Elution with hexane/EtOAc (7:3) afforded 2f (48%) as
The residue was chromatographed on silica gel using
CH2Cl2/MeOH (95:5) as an eluent to provide 2b (82%) as
a yellow solid. Compound 2b was converted into its hydro-
1
a pale oil. Mp: 264 ꢀC; H NMR (CDCl3): 1.93e2.04 (m,
4H); 2.44 (s, 6H); 2.49 (d, 2H, J ¼ 10.8 Hz); 2.66 (dd, 2H,
J ¼ 10.8e2.85 Hz); 3.18 (br, 2H); 3.58 (s, 2H); 3.61 (s, 2H);
7.22 (d, 1H, J ¼ 5.26 Hz); 7.27e7.35 (m, 5H); 7.73 (s, 1H);
7.80 (s, 1H); 7.93 (d, 4H, J ¼ 7.23); 8.63 (d, 2H,
J ¼ 5.26 Hz). 13C NMR (CDCl3): 21.1; 24.1; 55.7; 56.9;
58.7; 63.1; 124.1; 124.9; 125.1; 125.7; 128.9; 132.1; 140.9;
148.3; 154.2. MS (ESI) m/z: 475.2.
1
chloric salt. Mp: 198.5 ꢀC; H NMR (CDCl3): 2.4 (s, 4H);
2.86 (d, 2H, J ¼ 12.72 Hz); 2.97 (d, 2H, J ¼ 12.72 Hz); 3.87
(s, 3H); 3.88 (s, 3H); 3.93 (s, 2H); 4.04 (s, 2H); 4.55 (s,
2H); 7.16 (d, 2H, J ¼ 6.58 Hz); 7.19 (d, 2H, J ¼ 7.02 Hz);
7.80 (d, 2H, J ¼ 8.99 Hz); 7.88 (d, 2H, J ¼ 8.77 Hz); 7.90
(d, 1H, 6.14 Hz); 8.00 (d, 1H, J ¼ 6.14 Hz); 8.16 (s, 1H);
8.36 (s, 1H); 8.54 (d, 1H, J ¼ 6.14 Hz); 8.72 (d, 1H,
J ¼ 6.14 Hz). 13C NMR (CDCl3): 26.3; 55.3; 56.2; 59.2;
59.9; 60.9; 114.1; 119.6; 121.5; 128.1; 123.1; 132.1; 149.4;
149.5; 157.1; 160.4. MS (ESI) m/z: 507.5; 1035.0.
6.5.7. 3,8-Bis((2-m-tolylpyridin-4-yl)methyl)-3,8-diaza bicyclo
[3.2.1]octane (2g)
Elution with petroleum ether/EtOAc (9:1e8:2) afforded 2g
1
(75%) as a white solid. Mp (2g$HCl): 266.3 ꢀC; H NMR
6.5.3. 3,8-Bis((2-(3-methoxyphenyl)pyridin-4-yl)methyl)-
3,8-diazabicyclo[3.2.1]octane (2c)
(CDCl3): 2.03 (br, 4H); 2.45 (s, 6H); 2.5 (d, 2H,
J ¼ 10.8 Hz); 2.6 (d, 2H, J ¼ 10.8 Hz); 3.22 (s, 2H); 3.61 (s,
2H); 3.66 (s, 2H); 7.26e7.28 (m, 4H); 7.41 (t, 2H,
J ¼ 7.87 Hz); 7.55 (s, 2H); 7.79 (d, 2H, J ¼ 7.87); 7.86 (s,
2H); 8.69 (d, 2H, J ¼ 5.04 Hz). 13C NMR (CDCl3): 21.1;
24.1; 55.7; 56.9; 58.7; 63.1; 124.1; 124.7; 125.3; 125.7;
128.7; 132.4; 140.8; 148.2; 154.1. MS (ESI) m/z : 475.4.
The residue was chromatographed on silica gel using light
petroleum/EtOAc (2:8) as an eluent to provide 2c (62%) as
a yellow solid. Compound 2c was converted into its hydro-
chloric salt. Mp: 198.7 ꢀC; 1H NMR (D2O): 2.4 (br, 4H);
2.95 (d, 2H, J ¼ 12.72 Hz); 2.97 (d, 2H, J ¼ 12.72 Hz); 3.89
(s, 3H); 3.90 (s, 3H); 3.99 (s, 2H); 4.07 (s, 2H); 4.6 (s, 2H);
7.25e7.33 (m, 2H); 7.37e7.5 (m, 4H); 7.55e7.64 (m, 2H);
8.02 (dd, 1H, J ¼ 1.53e6.36 Hz); 8.11 (dd, 1H, J ¼ 1.53e
6.36 Hz); 8.25 (s, 1H); 8.42 (s, 1H); 8.65 (d, 1H,
J ¼ 6.14 Hz); 8.33 (d, 1H). 13C NMR (D2O): 23.4; 54.1;
55.0; 56.1; 59.3; 64.1; 114.1; 114.7; 117.3; 118.1; 121.7;
122.1; 125.0; 126.1; 126.8; 127.9; 132.1; 133.2; 142.3;
144.1; 149.1; 153.4; 160.1. MS (ESI) m/z: 507.2.
6.5.8. 3,8-Bis((2-(4-acetamidophenyl)pyridin-4-yl)methyl)-
3,8-diazabicyclo[3.2.1]octane (2h)
Elution with CHCl3/MeOH (95:5e9:1) afforded 2j (77%)
1
as a white solid. Mp (2h$HCl): 254.3 ꢀC; H NMR (CDCl3):
1.95e2.10 (m, 4H); 2.24 (s, 6H); 2.5 (d, 2H, J ¼ 10.8 Hz);
2.66 (dd, 2H, J ¼ 10.8e2.63 Hz); 3.18 (s, 2H); 3.60 (s, 2H);
3.64 (s, 2H); 4.0 (br, 2NH2); 4.48 (s, 4H); 7.25 (d, 2H,
J ¼ 5.26 Hz); 7.36 (s, 2H); 7.66 (d, 4H, J ¼ 8.77 Hz); 7.73
(s, 2H); 8.01 (d, 4H, J ¼ 8.77 Hz); 8.67 (d, 2H, J ¼ 5.26 Hz).
13C NMR (CDCl3): 22.7; 23.5; 55.7; 56.9; 58.7; 63.1; 118.7;
120.3; 124.1; 124.7; 130.1; 134.3; 134.4; 141.9; 148.4;
150.1; 155.6. MS (ESI) m/z: 561.3.
6.5.4. 3,8-Bis((2-(4-hydroxyphenyl)pyridin-4-yl)methyl)-
3,8-diazabicyclo[3.2.1]octane (2d)
Elution with hexane/EtOAc (1:1e2:8) afforded 2d (65%) as
a pale oil. Mp: 263.2 ꢀC; 1H NMR (DMSO): 1.99 (m, 4H); 2.38
(d, 2H, J ¼ 9.21 Hz); 2.60 (d, 2H, J ¼ 9.21 Hz); 3.12 (s, 2H);
3.58 (s, 2H); 3.60 (s, 2H); 6.87 (d, 4H, J ¼ 8.11 Hz); 7.21 (d,
1H, J ¼ 5.04 Hz); 7.27 (d, 1H, J ¼ 5.04 Hz); 7.74 (s, 1H); 7.8
(s, 1H); 7.91 (d, 4H, J ¼ 8.11 Hz); 8.51 (t, 2H, J ¼ 4.6 Hz);
9.72 (d, 2H, J ¼ 3.51 Hz). 13C NMR (DMSO): 24.6; 53.8;
57.2; 57.8; 58.5; 108.9; 114.0; 117.1; 119.9; 126.3; 128.2;
147.4; 147.6, 148.4; 154.5; 157. MS (ESI) m/z: 478.5.
6.5.9. 3,8-Bis((2-(3-acetamidophenyl)pyridin-4-yl)methyl)-
3,8-diazabicyclo[3.2.1]octane (2i)
Elution with CHCl3/MeOH (95:5e9:1) afforded 2i (63%)
1
as a pale oil. Mp: 176 ꢀC; H NMR (CDCl3): 1.95e2.04 (m,
4H); 2.2 (s, 6H); 2.48 (d, 2H, J ¼ 10.8); 2.63 (d, 2H,
J ¼ 10.8); 3.15 (s, 2H); 3.58 (s, 2H); 3.62 (s, 2H); 7.01 (d,
2H, J ¼ 5.26 Hz); 7.57 (s, 2H); 7.73 (d, 4H, J ¼ 8.77 Hz);
7.97 (d, 4H, J ¼ 8.77 Hz); 8.60 (d, 2H, J ¼ 5.26 Hz). 13C
NMR (CDCl3): 22.7; 23.5; 55.7; 56.9; 58.7; 63.1; 118.7;
120.3; 124.1; 124.7; 130.1; 134.3; 134.4; 141.9; 148.4;
150.1; 155.6. MS (ESI) m/z: 561.3.
6.5.5. 3,8-Bis((2-(3-hydroxyphenyl)pyridin-4-yl)methyl)-
3,8-diazabicyclo[3.2.1]octane (2e)
Elution with petroleum ether/EtOAc (1:1e8:2) afforded 2e
1
(63%) as a white solid. Mp: 93.3 ꢀC; H NMR (CDCl3): 1.99
(br, 4H); 2.38 (d, 2H, J ¼ 9.21 Hz); 2.6 (d, 2H, J ¼ 9.21 Hz);
3.12 (s, 2H); 3.58 (s, 2H); 3.60 (s, 2H); 6.83 (d, 2H,
J ¼ 7.89 Hz); 7.31 (m, 4H); 7.46 (d, 2H, J ¼ 7.89 Hz) 7.51
(s, 1H); 7.80 (s, 1H); 7.86 (s, 2H); 8.57 (t, 2H, J ¼ 4.37 Hz);
9.56 (d, 2H, J ¼ 4.6 Hz). 13C NMR (CDCl3): 24.6; 53.7;
57.2; 57.9; 58.4; 11.9; 114.5; 115.7; 118.1; 120.8; 128.3;
6.5.10. 3,8-Bis((2-(4-chlorophenyl)pyridin-4-yl)methyl)-3,8-
diazabicyclo[3.2.1]octane (2j)
Elution with light petroleum/EtOAc (9:1e7:3) afforded 2j
(82%) as a white solid. Mp (2j$HCl): 245.3 ꢀC; rt: 17.84 min;
1H NMR (CDCl3): 2.07 (m, 4H); 2.50 (d, 2H, J ¼ 10.8 Hz);

R. Filosa et al. / European Journal of Medicinal Chemistry 42 (2007) 293e306
305
2.65 (d, 2H, J ¼ 10.8 Hz); 3.16 (s, 2H); 3.61 (s, 2H); 3.63 (s,
6.5.15. 3,8-Bis((biphenyl-1-yl)methyl)-3,8-diaza bicyclo
[3.2.1]octane (2o)
2H); 7.22 (d, 2H, J ¼ 4.92 Hz); 7.5 (d, 4H, J ¼ 8.11 Hz); 7.65
(s, 1H); 7.80 (s, 1H); 7.96 (d, 4H, J ¼ 8.11 Hz); 8.67 (d, 2H,
J ¼ 4.92 Hz). 13C NMR (CDCl3): 26.3; 30.5; 54.7; 59.1; 60.2;
62.1; 121.1; 122.8; 124.1; 124.7; 130.1; 134.3; 134.4; 141.9;
148.4; 150.1; 155.6. MS (ESI) m/z: 515.2.
The residue was chromatographed on silica gel using light
petroleum/EtOAc (9:1e1:1) as an eluent to provide 2o (60%)
as a white solid. Mp (2o$HCl) 254.3 ꢀC dec; 1H NMR
(CDCl3): 1.88e2.02 (m, 4H); 2.48 (d, 2H, J ¼ 10.2 Hz);
2.63 (d, 2H, J ¼ 10.2 Hz); 3.24 (s, 2H); 3.51 (s, 2H); 3.54
(s, 2H); 7.3e7.61 (m, 18H). 13C NMR (CDCl3): 28; 56; 59;
63; 127.5; 127.7; 127.8; 128.3; 129.3; 134.5; 134.7; 140.5;
143.3; 143.9. MS (ESI) m/z: 445.1.
6.5.11. 3,8-Bis((2-(4-nitrophenyl)pyridin-4-yl)methyl)-3,8-
diazabicyclo[3.2.1]octane (2k)
Elution with light petroleum/EtOAc (1:1e2:8) afforded 2k
1
(77%) as a pale yellow solid. Mp (2k$HCl): 198.4 ꢀC; H
NMR (CDCl3): 2.03 (br, 4H); 2.45 (s, 6H); 2.5 (d, 2H,
J ¼ 10.8 Hz); 2.6 (d, 2H, J ¼ 10.8 Hz); 3.22 (s, 2H); 3.61 (s,
2H); 3.66 (s, 2H); 7.39 (d, 2H, J ¼ 5.26 Hz); 7.65 (s, 1H);
7.67 (s, 1H); 8.22 (d, 4H, J ¼ 8.77 Hz); 8.36 (d, 4H,
J ¼ 8.77 Hz); 8.77 (d, 2H, J ¼ 5.26 Hz). 13C NMR (CDCl3):
26.3; 30.5; 54.7; 59.1; 60.2; 62.1; 121.1; 122.8; 124.1;
124.7; 130.1; 134.3; 134.4; 141.9; 148.4; 150.1; 155.6. MS
(ESI) m/z: 537.1.
6.5.16. 3,8-Bis((phenyl-1-yl)methyl)-3,8-diaza bicyclo
[3.2.1]octane (2p)
Elution with CHCl3/MeOH/NH3 (95:5:0.05) afforded 2p
1
(74%) as a white solid. H NMR (CDCl3): 1.65e1.97 (m,
4H); 2.2 (d, 2H, J ¼ 12 Hz); 6.45 (d, 2H, J ¼ 9.2 Hz); 3.42
(m, 2H); 3.46 (s, 4H); 7.18e7.38 (m, 10H).
6.5.17. 3,8-Bis(2-(naphthalen-1-yl)methyl)-3,8-diaza
bicyclo[3.2.1]octane (2q)
6.5.12. 3,8-Bis((2-(3-nitrophenyl)pyridin-4-yl)methyl)-3,8-
diazabicyclo[3.2.1]octane (2l)
Elution with petroleum ether/EtOAc (1:1e2:8) afforded 2l
The residue was chromatographed on silica gel using light
petroleum/EtOAc (9:1e1:1) as an eluent to provide 3 (62%) as
1
a white solid. Mp (2q$HCl) 278 ꢀC dec; H NMR (CDCl3):
1
(72%) as a yellow solid. Mp (2l$HCl): 194.7 ꢀC; H NMR
1.84e1.94 (m, 4H); 2.58 (d, 2H, J ¼ 10.08 Hz); 2.70e2.85
(m, 4H); 3.22e3.42 (m, 4H); 7.37e7.60 (m, 8H); 7.76 (d,
2H, J ¼ 7.02 Hz); 7.89 (d, 2H, J ¼ 4.85 Hz); 8.12 (t, 2H,
J ¼ 7.67 Hz). 13C NMR (CDCl3): 30; 33; 45; 56; 59; 124.1;
124.7; 125.2; 125.6; 126.3; 126.8; 127.3; 132.3; 133.4. MS
(ESI) m/z: 421.2.
(CDCl3): 1.94e2.10 (m, 4H); 2.53 (d, 2H, J ¼ 10.08 Hz);
2.68 (d, 2H, J ¼ 10.52 Hz); 3.2 (s, 2H); 3.65 (s, 2H); 3.88
(s, 2H); 7.35 (d, 1H, J ¼ 5.04 Hz); 7.42 (d, 1H,
J ¼ 5.04 Hz); 7.69 (t, 2H, J ¼ 7.89 Hz); 7.84 (s, 1H); 7.92 (s,
1H); 8.3 (d, 2H, J ¼ 7.89); 8.38e8.46 (m, 2H); 8.69 (d, 2H,
J ¼ 5.04 Hz); 8.90 (s, 2H). 13C NMR (CDCl3): 26.1; 30.1;
55.7; 59.1; 60.2; 62.1; 121.1; 122.3; 124.1; 124.7; 130.1;
134.1; 134.3; 141.9; 148.7; 150.1; 155.3. MS (ESI) m/z: 537.1.
6.5.18. 3,8-Bis(phenanthren-3-yl)methyl-3,8-diazabicyclo
[3.2.1]octane (2r)
Elution with petroleum ether/EtOAc (7:3) afforded 2r
6.5.13. 3,8-Bis((2-phenylpyridin-4-yl)methyl)-3,8-diaza
bicyclo[3.2.1]octane (2m)
1
(69%) as a pale oil. H NMR (CDCl3): 1.99e2.04 (m, 4H);
2.54 (d, 2H, J ¼ 10.8); 2.69 (d, 2H, J ¼ 10.8); 3.25 (s, 2H);
3.78 (s, 2H); 3.82 (s, 2H); 7.5e8.0 (m, 14H); 8.6e8.74 (m,
4H). 13C NMR (CDCl3): 21.1; 41.3; 52.3; 56.4; 60.3; 122.1;
122.4, 126.6; 127.2; 128.3; 130; 131.5; 133.1; 133.4; 134.1.
MS (ESI) m/z: 493.1.
The residue was chromatographed on silica gel using light
petroleum/EtOAc (7:3) as an eluent to provide 2m (62%) as
a pale yellow solid. Compound 2m was converted into its hy-
1
drochloric salt. Mp: 198.1 ꢀC; H NMR (D2O): 2.3e2.4 (m,
4H); 2.8 (d, 2H, J ¼ 10.8 Hz); 3.0 (d, 2H, J ¼ 10.8 Hz); 3.9
(s, 2H); 4.2 (s, 2H); 4.5 (s, 2H); 7.51e7.58 (m, 4H); 7.76e
7.85 (m, 6H); 7.9 (d, 1H, J ¼ 2.8 Hz); 8.1 (d, 1H,
J ¼ 2.8 Hz); 8.27 (d, 2H, J ¼ 8.11 Hz); 8.64 (d, 1H,
J ¼ 5.04 Hz); 8.77 (d, 1H, J ¼ 5.04 Hz). 13C NMR (D2O):
21.1; 41.3; 52.3; 56.4; 60.3; 123.3; 124.1; 124.7; 125.6;
126.2; 128.1; 130.0; 131.0; 132.1; 140.0; 142.3; 150.1;
154.2; 155.3. MS (ESI) m/z: 447.1.
7. Materials and methods
Test compounds were solubilised in DMSO at 100 mM and
then diluted into culture medium.
7.1. Cells
6.5.14. 3,8-Bis(3-phenylbenzyl)-3,8-diaza bicyclo[3.2.1]
octane (2n)
Elution with petroleum ether/EtOAc (7:3) afforded 2c
Cell lines were purchased from American Type Culture
Collection (ATCC). Haematological tumor-derived cells
were grown in RPMI-1640 medium supplemented with 10%
FCS, 100 Units/mL penicillin G and 100 mg/mL streptomycin.
Solid tumor-derived cells were grown in their specific media
supplemented with 10% FCS and antibiotics. Cell cultures
were incubated at 37 ꢀC in a humidified 5% CO2 atmosphere.
The absence of mycoplasma contamination was checked
periodically by the Hoechst staining method.
1
(78%) as a pale oil. H NMR (CDCl3): 1.96e2.04 (m, 4H);
2.48 (d, 2H, J ¼ 10.8); 2.63 (d, 2H, J ¼ 10.8); 3.15 (s, 2H);
3.58 (s, 2H); 3.62 (s, 2H); 7.25e7.5 (m, 12H); 7.6e7.74 (m,
6H). 13C NMR (CDCl3): 21.1; 41.3; 52.3; 56.4; 60.3; 123.3;
124.1; 124.7; 125.6; 126.2; 128.1; 130.0; 131.0; 132.1;
140.0; 142.3; 150.1; 154.2; 155.3. MS (ESI) m/z: 445.1.

306
R. Filosa et al. / European Journal of Medicinal Chemistry 42 (2007) 293e306
7.2. Antiproliferative assays
analysis by raising the forward scatter threshold, and the DNA
content of the nuclei was registered on a logarithmic scale.
The percentage of cellsin thehypodiploidregionwas calculated.
Exponentially growing was re-suspended in growth medium
containing serial dilutions of the drugs. Cell viability was deter-
mined after 96 h at 37 ꢀC by the 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyl-tetrazolium bromide (MTT) method [12,13].
7.6. Cell-cycle analysis
For analysis of cell cycle distribution, the DNA content of
individual cells was measured by flow cytometry. Cells were
seeded at a density of 5 ꢂ 105 cells/well in six-well plates. Af-
ter 24 h, cells were stimulated with 2a (10 mM). After 24 h,
cells and supernatants were collected together and incubated
with Nicoletti reagent. Core DNA content was measured using
linear amplification in the FL2-channel of a FACScan flow cy-
tometer using CellQuest software. Cell cycle was analysed by
ModFit LT software.
7.3. Cells’ culture
MCF-7 cells were cultured in DMEM supplemented with
2 mM L-glutamine, 10% heat-inactivated foetal bovine serum
(FBS), 10,000 Uints/mL penicillin and 10 mg/mL streptomycin
(all from CambrexBioscience, Verviers, Belgium) at 37 ꢀC in an
atmosphere of 95% O2 and 5% CO2. The cells were plated at
a density of 5 ꢂ 105 cells in 6-cm cell culture plates the day be-
fore treatment. At the end of the incubation time cells were pro-
cessed for Western blotting and FACS analysis.
7.7. Statistical analysis
All results are shown as mean ꢁ SEM of three experiments
performed in triplicate. The optical density of the bands of
ANXA1 protein expression detected by Western blot was nor-
malized with a-tubulin. Statistical comparison between groups
was made using parametric Bonferroni test. P-values < 0.05
were considered significant.
7.4. Western blot analysis
Expression of p21CIP1/Waf-1 was detected by SDS-PAGE.
Total intracellular protein was extracted from the cells by
freezing and thawing in lysis buffer containing protease and
phosphatase inhibitors (1 mM phenylmethylsulfonyl fluoride,
1 mg/mL leupeptin, 1 mg/mL pepstatin, 1 mg/mL aprotinin,
1 mM NaF in 50 mM TrizmaeHCl) as described [14].
References
Protein content was estimated by Biorad protein assay (BIO-
RAD, Milan, Italy). Samples (30 mg protein) were loaded onto
10% acrylamide gel and separated by SDS-PAGE in denaturating
conditions at 100 V. The separated proteins were then transferred
electrophoretically (100 mA/blot; Trans Blot Semi-Dry, BIO-
RAD) to nitrocellulose paper (Immobilon-NC, Millipore, USA)
soaked in transfer buffer [25 mM TRIS, 192 mM glycine and
20% methanol vol/vol (SigmaeAldrich, Gallarate, Italy)]. Non-
specific binding was blocked by incubation of the blot in 5%
non-fat dry-milk (BIO-RAD, Milan, Italy) in TBSeTween
[25 mM Tris, 150 mM NaCl, 0.1% Tween vol/vol (all from Sig-
maeAldrich, Gallarate, Italy)] for 60 min. The blots were incu-
bated overnight at 4 ꢀC with the primary monoclonal antibody
against p21CIP/Waf1 (diluted 1:200) (Santa Cruz Biotechnology,
Inc., Germany) and then at room temperature (1 h) with an appro-
priate secondary mouse antibody (1:10,000) (SigmaeAldrich,
Gallarate, Italy). Immunoreactive protein bands were detected
by chemiluminescence reagents (ECL) and exposed to hyperfilm
(both from Amersham Biosciences, Milan, Italy). The blots were
then scanned and analysed (Gel-Doc 2000 BIO-RAD).
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