Synthesis, characterization and antiproliferative studies of the enantiomers of cis-[(1,2-camphordiamine)dichloro]platinum(II) complexes

Article abstract of DOI:10.1016/j.bmc.2007.11.021

The platinum(II) complex cis-[(1S,2R,3S)-1,7,7-trimethylbicyclo[2.2.1]heptane-2,3-diamine]dichloroplatinum(II) (1) and its enantiomer (2) have been synthesized and physically and spectroscopically characterized. To obtain the enantiopure complexes the chiral pool approach was applied. The synthetic pathway has four steps, starting from (±)-diphenylethylenediamine (DPEDA) (3) and the natural products (1S)-camphorquinone or (1R)-camphorquinone to obtain enantiomers 1 and 2, respectively. The interaction of the Pt(II) complexes with DNA was studied by several techniques: circular dichroism, electrophoresis on agarose gel and atomic force microscopy (AFM). These studies showed differences in the degree of interaction between both enantiomers and DNA (calf thymus DNA and plasmid pBR322 DNA). The cytotoxicity of enantiomers 1 and 2 against the HL-60 cell line was studied by in vitro tests of antiproliferative activity, incubating during both 24 h and 72 h. An important difference of activity was found between both enantiomers regarding the IC₅₀ data at 24 h of incubation. Thus, complex 1 showed to be much more active than its enantiomer 2.

Full text of DOI:10.1016/j.bmc.2007.11.021

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 1721–1737
Synthesis, characterization and antiproliferative studies
of the enantiomers of cis-[(1,2-camphordiamine)
dichloro]platinum(II) complexes
a,
a
d
a
´
*
´
´
Angel M. Montana, Francisco J. Bernal, Julia Lorenzo, Carlos Farnos,
˜
a
b
c
d
Consuelo Batalla, Maria J. Prieto, Virtudes Moreno, Francesc X. Aviles,
e
e
´
Juan M. Mesas and Marıa-Teresa Alegre
a
`
Departamento de Quı´mica Orga´nica, Universidad de Barcelona, Martı´ i Franques 1-11, 08028 Barcelona, Spain
bDepartamento de Microbiolog´ıa, Universidad de Barcelona, Campus de Pedralbes, 08028 Barcelona, Spain
c
`
Departamento de Quı´mica Inorga´nica, Universidad de Barcelona, Martı´ i Franques 1-11, 08028 Barcelona, Spain
dInstituto de Biotecnologı´a y Biomedicina Vicent Villar Palası´, Universidad Auto´noma de Barcelona,
Bellaterra, 08193 Barcelona, Spain
eEscuela Polite´cnica Superior, Universidad de Santiago de Compostela, Campus de Lugo, 27002 Lugo, Spain
Received 10 July 2007; revised 2 November 2007; accepted 7 November 2007
Available online 13 November 2007
Abstract—The platinum(II) complex cis-[(1S,2R,3S)-1,7,7-trimethylbicyclo[2.2.1]heptane-2,3-diamine]dichloroplatinum(II) (1) and
its enantiomer (2) have been synthesized and physically and spectroscopically characterized. To obtain the enantiopure complexes
the chiral pool approach was applied. The synthetic pathway has four steps, starting from ( )-diphenylethylenediamine (DPEDA) (3)
and the natural products (1S)-camphorquinone or (1R)-camphorquinone to obtain enantiomers 1 and 2, respectively. The interac-
tion of the Pt(II) complexes with DNA was studied by several techniques: circular dichroism, electrophoresis on agarose gel and
atomic force microscopy (AFM). These studies showed differences in the degree of interaction between both enantiomers and
DNA (calf thymus DNA and plasmid pBR322 DNA). The cytotoxicity of enantiomers 1 and 2 against the HL-60 cell line was stud-
ied by in vitro tests of antiproliferative activity, incubating during both 24 h and 72 h. An important difference of activity was found
between both enantiomers regarding the IC50 data at 24 h of incubation. Thus, complex 1 showed to be much more active than its
enantiomer 2.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
dilution which conditions its efficiency.3–6 Moreover
many cancer types have been able to develop resistance
against cisplatin7,8 which considerably decreases its activ-
ity against cancer cells by several mechanisms, among
them: (a) reduction of the drug concentration inside the
cell by irreversible reaction with biomolecules containing
nucleophilic sulfur groups, (b) reparation of the structural
modifications undergone by DNA or (c) inactivation of
the apoptotic mechanism of programmed cancer cell
death.
The development of new platinum alkylating agents, dif-
ferent from the classical model of cisplatin1,2 (Fig. 1), is
actually an important research line in the search of less
toxic, more potent and more selective chemotherapeutic
anticancer agents. The high nephrotoxicity of cisplatin
(partially avoided by using highly diluted saline solutions)
and its high neurotoxicity (specially if administered to-
gether with paclitaxel) condition the therapeutic use of
this active principle. It is used at low doses and in high
In the last 10 years new alternative analogues of cisplatin
have appeared such as carboplatin,9–12 oxaliplatin13–15
and other different types of platinum compounds (water
soluble platinum compounds, multinuclear platinum
complexes, compound with trans geometry, complexes
having bioactive molecules (themselves) as ligands,
Keywords: 1,2-Camphordiamines; Enantiomers; Platinum(II) com-
plexes; DNA; Atomic force microscopy; Circular dichroism; Anticancer
agents; Cytotoxicity; Apoptosis.
*
Corresponding author. Tel.: +34 93 402 16 81; fax: +34 93 339 78
78; e-mail: angel.montana@ub.edu
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.11.021

´
A. M. Montan˜a et al. / Bioorg. Med. Chem. 16 (2008) 1721–1737
1722
1IV
1IV
1'
1V
1V
7
7
6a
5a
H
1'', 2''
NH2
H
1''', 2'''
NH2
H3N
H3N
Cl
Cl
6b
5b
1
H
H
4
2
3
5a
Cl
Cl
6a
6b
Pt
Cl
Cl
1
3
H
H
2
H
H
Pt
4
Pt
5b
N
N
H2
1''', 2'''
H2
1'', 2''
1'
Cisplatin
Figure 1. Structure of cisplatin and of complexes 1 and 2.
1
2
etc.),16–19 in an effort to overcome the limitations of the
actual platinum based therapeutic agents.20
the cellular machinery as a consequence of these confor-
mational and/or configurational differences.25
There are in the literature references to structure–activ-
ity relationship studies, SAR, about the influence of ste-
reochemistry in the activity of platinum(II) complexes.
Thus, different antitumour activities have been found
between diastereoisomers of ethylenediamino com-
plexes, both in vitro and in vivo.21 Also, a wide study
on the influence of cis versus trans configuration on
the cytotoxic activity of 1,2-diamino-cyclohexane plati-
num(II) complexes (DACH)22 has been carried out,
and very interesting differences were appreciated. More-
over, studies on the change of erythro into threo config-
uration in dihydroxylated DACH complexes showed a
clear influence of stereochemistry in the molecular rec-
ognition of drugs by the pockets or receptors of target
biomolecules, in this case, the bases of DNA.
In the present work, we have synthesized, isolated and
tested both enantiomers of camphor-1,2-diamine plati-
num(II) complexes (see Fig. 1) in order to evaluate the
different cytotoxic activity of both enantiomers and to
initiate the structure–activity relationship in this kind
of compounds.
The synthesis of both enantiomeric camphane ligands
was carried out in parallel and the corresponding plati-
num(II) complexes were prepared by reaction with
potassium tetrachloroplatinate. Studies of interaction
of these complexes with DNA were performed by three
techniques: (a) Circular dichroism, that allowed us to
study the changes produced on the secondary structure
of DNA, by the bonding of platinum complexes to the
DNA bases. (b) Electrophoresis on agarose gel, tech-
nique that allowed us to obtain information on the mod-
ification of the tertiary structure of DNA after
interaction with platinum(II) complexes. (c) Atomic
force microscopy (AFM), which showed, in an intuitive
form, the topology and morphology of DNA when it
interacts with platinum complexes. The evaluation of
the cytotoxic activity of both enantiomers was studied
in vitro against a HL-60 cancer cell line (leukaemia).
Interesting differences were observed in the antiprolifer-
ative activity of both enantiomers and in the apoptotic
death mechanism, as it is discussed below.
In pharmacology, chirality is an important factor in
drug efficacy. About 56% of the drugs currently in use
are chiral compounds, and about 88% of these chiral
synthetic drugs are used therapeutically as racemates.
Unfortunately, there are many racemic drugs where
the stereospecificity of the metabolism and/or the phar-
macodynamic effects of the enantiomers are not
known.23
Prior to Ariens’ critical review of ‘Sophisticated nonsense
in pharmacokinetics and clinical pharmacology’,24 pub-
lished in 1984, neglect of stereochemistry in drug devel-
opment was widespread and only in the last decade or so
has it achieved a prominent place in druce the publica-
tion of this seminal work it is well established that there
is a molecular recognition of enantiomers of chiral drugs
by the chiral receptors in cells, so one of the enantio-
mers, the eutomer, is the active or more active one,
but the other enantiomer, the distomer, is the inactive
or less active one.
2. Results and discussion
2.1. Synthesis and characterization of 1,2-diamine ligands
and platinum complexes
The synthesis of ligands 3 and 4 was performed by the
same synthetic pathway26 (see Scheme 2). The starting
material for 3 was (1S)-camphorquinone and ( )-1,2-
diphenylethane-1,2-diamine (5) [( )-DPEDA], mean-
while, 4 was prepared from (1R)-camphorquinone and
( )-DPEDA. A great advantage in the preparation of
enantiomers 3 and 4 is the commercial availability of
both enantiomers of the precursory camphorquinone.
All intermediates and final products were physically
In the field of chiral platinum complexes, the interaction
with DNA and formation of cross-links with adjacent
purine bases are considered to be the crucial steps in
the differentiation of antitumour activity between enan-
tiomeric complexes. Because double-helical DNA has a
chiral nature, interaction of DNA molecule with enan-
tiomeric platinum complexes should lead to diastereo-
isomeric adducts. It has been demonstrated that DNA
cross-links of platinum complexes with enantiomeric
amine ligands not only can exhibit different conforma-
tional features but also can be processed differently by
1
and spectroscopically characterized by FT-IR, H and
13C NMR, as well as 2-D correlation experiments, and
mass spectrometry and elemental analysis. Also, the
optical activity was measured on enantiopure samples
of products. The preparation of ( )-DPEDA (5) was

´
A. M. Montan˜a et al. / Bioorg. Med. Chem. 16 (2008) 1721–1737
1723
carried out in two steps (see Scheme 1) 27: (1) Condensa-
tion reaction of dibenzoyl, cyclohexanone and ammo-
nium acetate. (2) Reduction of the bisimine obtained
from the previous reaction by the use of Li, NH3/
THF, EtOH at ꢀ78 ꢁC, resulting in a racemic mixture
of 1,2-diphenylethane-1,2-diamine (5).
The second step in the synthesis of ligands was the
reduction of imino groups in 6, 7, 13 and 14 with
NaBH4. We found out that it was very important to
work under mild reaction conditions and to establish a
short reaction time to avoid the acid–base abstraction
of benzylic hydrogens of 6, 7, 13 and 14 by hydride ions.
Otherwise, the bisimine system of these substrates
should afford pyrazine systems instead of undergoing a
reduction process to generate piperazines 9, 10 or 15,
16. For this reason, in order to get higher chemoselectiv-
ity, it was preferred to run the reactions for short reac-
tion times, even though, the conversion was not
completed and to recover the unconverted starting
material which could be submitted again to the reduc-
tion conditions in a subsequent different process. In
the piperazines the secondary amine groups adopt a
cis relationship with respect to the camphane framework
because in the reduction process the hydride ions can
The first step in the synthesis of camphordiamines is the
condensation between camphorquinone and ( )-DPED-
A (5) facilitated by the azeotropic distillation of water/tol-
uene system in a Dean–Stark apparatus. The formation of
diastereoisomers 6, 7, 13 and 14 (see Fig. 2) was due to the
use of 5 as a racemic mixture. The byproduct 8, isolated
from the condensation reaction of (1S)-camphorquinone,
is a reaction intermediate precursor of diimine 6. This
intermediate is a hemiaminal that was transformed into
6, in quantitative yield, by a dehydration process under
the same condensation reaction conditions.
O
a) Li, NH3/THF, EtOH
Ar, -78ºC, 1h
CH3COONH4
N
N
O
H2N
+
CH3COOH
N2, reflux, 2h
b) HCl
c) NaOH
H2N
O
62%
rac-5
91%
Scheme 1. Synthetic methodology for the preparation of diamine 5.
benzene
N2, reflux, 12h
Dean-Stark
14
2
14
14
11
8
13
11
11
8
13
13
12
12
12
1.-NaBH4, MeOH, 0ºC
N2, r.t., 1h
10
2''
6''
3''
3''
2''
9
10
2''
3''
5''
1
NH
3
H2N
H2N
N
N
8
1
O
+
benzene
1
1''
6'
2
6
2
6
N
3
7
3
7
4
4''
4''
4''
4
5
9
10
+
9
1''
6'
7
6
5
4
5
1''
N2, reflux, 12h
Dean-Stark
2.-HCl
3.-NaOH
HN
HO
HN
5''
6''
6'' 5''
5'
6'
O
1'
1'
1'
2'
5'
(1S)-Camphorquinone
2'
5'
5
2'
9, 10
6, 7
8
4'
4'
3'
4'
3'
3'
HCl (2M)
14
13
9
11
8
1''
1'
1'''
12
10
2''
6''
3''
+
1
NH2
7
2
3
4
6a
Pd/C (10 %)
MeOH (anh.), N2, 1h
7
H
H
4''
·2 Cl-
NH3+Cl-
NH3+Cl-
1
HCOONH4
+
1''
6'
6
6b
5
2
+H2N
5''
5a
H
H
4
3
1'
5b
5'
2'
3
11, 12
3'
4'
14
14
2
11
11
13
13
10
8
8
7
1.-NaBH4, MeOH, 0ºC
N2, r.t., 1h
7
9
9
2'
3'
2'
3'
N
O
NH
H2N
benzene
1
1
6
2
6
5
4'
5
4'
10
+
3
3
4
4
1''
6''
1''
6''
N2, reflux, 12h
Dean-Stark
N
12
HN
12
2.-HCl
3.-NaOH
6' 5'
5''
6' 5'
5''
H2N
1''
2''
1''
2''
O
(1R)-Camphorquinone
5
13, 14
15, 16
4''
3''
4''
3''
HCl 2M
14
1''
1'''
11
13
10
8
7
7
9
+
2'
3'
5a
4
NH2
H
1
6
4
2
H
NH3+Cl-
NH3+Cl-
5b
Pd/C (10 %)
MeOH (anh.), N2, 1h
5
4'
3
6a
6b
1
3
HCOONH4
+
1''
6''
2
H
H
+H2N
12
6' 5'
5''
·2 Cl-
1''
2''
1'
4
17, 18
4''
3''
Scheme 2. Synthetic path for the preparation of ligands 3 and 4.

´
A. M. Montan˜a et al. / Bioorg. Med. Chem. 16 (2008) 1721–1737
1724
14
14
14
2
13
13
11
13
9
11
8
11
12
12
12
2
3''
2''
9
10
2''
3''
5''
2''
3''
5''
N
10
N
3
4
N
3
4
8
1
1
1
1''
6
7
3
2
10
5
4''
4''
4''
8
9
7
7
6
N
6
N
1''
6'
4
5
5
1''
HO
HN
6''
6'
6''
5'
6''
5''
6'
1'
1'
1'
5'
2'
5'
2'
2'
6
7
8
4'
3'
4'
3'
3'
4'
14
14
13
13
11
11
8
8
7
7
2'
3'
2'
3'
9
9
N
N
1
1
6
6
2
2
10
10
5
4'
5
4'
3
3
4
1'
4
1'
N
N
6'
5'
6'
5'
12
12
1''
1''
6''
4''
6''
2''
2''
5''
5''
13
14
3''
3''
4''
Figure 2. Structure of diastereoisomeric pairs (6, 7) and (13, 14) and of byproduct 8.
R1
R1
NH3+Cl-
NH2
K2PtCl4
Cl
Pt
NH3+Cl-
H2O r.t., 1 h
N
R2
R2
Cl
H2
3, R1 = Me, R2 = H
4, R1 = H, R2 = Me
1, R1 = Me, R2 = H
2, R1 = H, R2 = Me
Scheme 3. Preparation of Pt(II) complexes of 1,2-diamines 3 and 4.
only attack the imino groups of 6, 7, 13 and 14 by the
endo face of the bicyclic system.
2.2. Studies of interaction of enantiomeric platinum
complexes with DNA
The next step in the obtention of ligands 3 and 4 was the
transformation of piperazines 9, 10, 15 and 16 into the
hydrochlorides 11, 12, 17 and 18 by using 2 M HCl
and ulterior concentration to dryness. Finally, the deb-
enzylation process of these hydrochlorides was carried
out by hydrogenolysis using 10% Pd/C as a catalyst
and ammonium formate (HCOONH4) as a source of
hydrogen. In this reaction it is quite important the com-
plete absence of O2 (not only from the system atmo-
sphere but also the possible oxygen dissolved in the
absolute methanol used as solvent) in order to avoid
the passivation of catalyst. The resulting products were
the 1,2-diamines 3 or 4 as ammonium hydrochlorides,
whose purification was performed by crystallization in
MeOH.
With the purpose of performing a preliminary evalua-
tion of the ability of complexes 1 and 2 to interact with
DNA molecules as the main biological targets, several
biochemical assays were recorded by using three tech-
niques: circular dichroism, electrophoresis on agarose
gel and atomic force microscopy. These techniques al-
lowed us to observe the interaction of platinum com-
plexes and DNA molecules at different levels.
2.3. Circular dichroism (CD) studies
This technique is quite sensitive to the changes in the
secondary structure of nucleic acids, which allows ana-
lyzing any conformational modification of DNA pro-
voked by its interaction with platinum complexes.
When the complexes produce variations in the molar
ellipticity Dh this means that some modifications are also
produced on the bases stacking and on the magnitude of
the winding angle between adjacent base pairs, that is,
on the bending and winding of DNA helix.28,29
The synthesis of platinum(II) complexes 1 and/or 2 was
carried out by reaction of 3 and/or 4 with potassium tet-
rachloroplatinate in aqueous media, under nitrogen
atmosphere and in the absence of light (see Scheme 3).
Complexes 1 and/or 2 precipitated once they were
formed due to their low solubility in water, and they
were isolated by filtration.
In these CD studies, the spectra of compounds 1, 2, 3, 4
and cisplatin were registered, after incubation with Calf
Thymus DNA at three molar ratios ri (molar ratio of the

´
A. M. Montan˜a et al. / Bioorg. Med. Chem. 16 (2008) 1721–1737
1725
compound per nucleotide): 0.1, 0.3 and 0.5. The wave-
lengths used were in between 220 and 320 nm. The val-
ues obtained of molar ellipticity for cisplatin and for the
ligands 3, 4 and complexes 1 and 2 are illustrated in Fig-
ure 3 and data are quoted in Table 1.
The ligands 3 and 4 did not provoke significant changes
on the ellipticity (see Fig. 4A), however, the complexes 1
and 2 modified the bases stacking in DNA molecules,
but to a lesser extent than cisplatin did. In the graphical
representation of the net variation of maximum elliptic-
6000
4000
2000
0
8000
6000
4000
2000
0
220
240
260
280
300
320
-2000
-4000
-6000
-8000
220
240
260
280
300
320
-2000
-4000
-6000
-8000
Wavelength (nm)
Wavelength (nm)
DNA
ri=0.1
ri=0.3
ri=0.5
DNA
ri=0.1
ri=0.3
ri=0.5
3000
2000
1000
0
3500.00
2500.00
1500.00
500.00
230
250
270
290
310
330
-1000
-2000
-3000
-4000
-500.00
-1500.00
-2500.00
-3500.00
230
250
270
290
310
330
Wavelength (nm)
Wavelength (nm)
DNA
DNA-2 . ri = 0.3
DNA-2 . ri = 0.1
DNA-2 . ri = 0.5
DNA
DNA-4. ri = 0.3
DNA-4. ri = 0.1
DNA-4. ri = 0.5
Figure 3. (A) CD curves of native DNA and DNA incubated with diamine 3 at ri = 0.1, 0.3 and 0.5. (B) CD curves of native DNA and DNA
incubated with platinum complex 1 at ri = 0.1, 0.3 and 0.5. (C) CD curves of native DNA and DNA incubated with diamine 4 at ri = 0.1, 0.3 and 0.5.
(D) CD curves of native DNA and DNA incubated with platinum complex 2 at ri = 0.1, 0.3 and 0.5.
Table 1. Ellipticity variations DH and maximum and minimum wavelengths k observed for 1, 2, 3, 4 and cisplatin
Compound
ri
DHmax
kmax
DHmin
kmin
Dhmax (compound-DNA) ꢀ Dhmax
kmax (compound-DNA) ꢀ kmax
(free DNA)
(free DNA)
DNA
4985
5006
5097
4964
7060
274.4
275.6
275.2
277.0
278.6
ꢀ6556
ꢀ6203
ꢀ6277
ꢀ5920
ꢀ5746
246.6
245.6
246.0
245.2
247.8
0.1
0.3
0.5
0.1
21
112
1.2
0.8
2.6
4.2
Diamine 3
ꢀ21
2075
Complex 1
Diamine 4
Complex 2
Cisplatin
0.3
0.5
0.1
6284
5587
5405
280.0
281.6
274.4
ꢀ4926
ꢀ4042
ꢀ6546
ꢀ6368
ꢀ6332
ꢀ5633
ꢀ4882
ꢀ5633
ꢀ5352
247.6
247.6
246.6
1299
602
5.6
7.2
0
420
0.3
0.5
0.1
5461
5253
7057
274.4
275.6
276.6
246.6
246.6
246
476
268
0
1.2
2.2
2072
0.3
0.5
0.1
6435
5665
7373
276.2
276.2
276.6
247
246
1450
680
1.8
1.8
2.2
247.0
2388
0.3
0.5
8355
7942
279.6
278.0
ꢀ5158
248.4
248.2
3370
2957
5.2
3.6
ꢀ4473

´
A. M. Montan˜a et al. / Bioorg. Med. Chem. 16 (2008) 1721–1737
1726
8
7
6
5
4
3
2
1
0
4000
3500
3000
2500
2000
1500
1000
500
0
0.1
0.3
0.5
-500
0.1
0.3
0.5
Molar ratio ri
Molar ratio ri
1
2
3
4
Cisplatin
1
2
3
4
Cisplatin
Figure 4. (A) Variations of maximum ellipticity Dh of diamines 3 and 4, complexes 1 and 2 and cisplatin versus native DNA. (B) Variations of
maximum wavelength Dkmax of diamines 3 and 4, complexes 1 and 2 and cisplatin versus native DNA.
ity versus ri (Fig. 4A and B) it is possible to appreciate
the different mode of interaction of enantiomers 1 and
2 with DNA.
complexes 1 and 2 could have a slight different behav-
iour comparing maximum wavelength differences (at
high molar ratios), which could be consistent with a pos-
sible different interaction mode for both enantiomeric
platinum complexes with the chiral DNA target.
When observing the ellipticity variation Dh it is possible
to appreciate that complexes 1 and 2 have behaviour
quite similar to each other and quite different from that
of cisplatin. When increasing the molar ratio ri from 0.1
to 0.3 a progressive decrease of the ellipticity values is
observed for 1 and 2 and an increase of ellipticity can
be observed for cisplatin. This different behaviour could
be interpreted by the easiness of cisplatin to form intra-
strand cis-bifunctional bonds between the platinum
atom and DNA due to steric reasons. When increasing
the molar ratio from 0.3 to 0.5 the trend of the three
complexes is similar (even maintaining the differences)
and a decrease of molar ellipticity is observed in all
cases. This fact could be related to the unwinding and
bending of the DNA helix due to both, the formation
of inter-strand bonds between DNA threads and to a
possible change of the DNA B-form into a Z-form.
2.4. Agarose gel electrophoresis
The studies of electrophoresis on the samples of DNA
incubated with the enantiomeric platinum complexes 1
and 2 allowed us to observe modifications on the tertiary
structure of DNA molecules induced by the platinum
complexes. In this study, plasmid pBR322 DNA was
used, which presents a circular shape with to main
forms: a relaxed open circular form (OC) and a super-
coiled covalently closed form (CCC) that is much more
compacted than the former one. Cis-platinum com-
plexes interact with this type of DNA and produce a
relaxation of the CCC form (causing a decrease of com-
paction) and, at the same time, they produce a certain
folding of the OC form.31–33 This phenomenon produces
that the migration difference between the bands corre-
sponding to both DNA forms should decrease when
increasing the concentration of complexes. At suffi-
ciently high drug concentrations, the coalescence point
is reached in which both bands get superposed34 at a
medium position of the electrophoretic lane (see Fig. 5).
When analyzing the variations of wavelength at the max-
imum absorption, the trends are different for complexes 1
and 2 and the standard of cisplatin. In this case, complex 1
and cisplatin produce an increase of the wavelength when
increasing the molar ratio from 0.1 to 0.3, meanwhile
complex 2 induces a slight decrease of k. The bathochro-
mic effect observed for 1 could be interpreted as a higher
possibility of 1 to form intra-strand cis-bifunctional
bonds between platinum atom and DNA than complex
2.30 However, when going from 0.3 to 0.5 molar ratio
the behaviour of both complexes and that of cisplatin
are completely different. This phenomenon could be inter-
preted, as mentioned before, as due to the different degree
of unwinding and bending of the DNA helix by the three
compounds due to both, the formation of inter-strand
bonds between DNA threads and to a possible change
of the DNA B-form into a Z-form.
The 1,2-camphordiamine ligands 3 and 4 did not pro-
duce any change in the electrophoretic mobility of the
plasmid DNA, which confirms the results obtained from
the circular dichroism: the ligands do not interact with
DNA. However, the enantiomeric complexes 1 and 2
clearly produced a change in the mobility of OC and
CCC bands, reaching the coalescence point at both eval-
uated ri. The only difference in the electrophoretic
behaviour of both complexes 1 and 2 between ri = 0.1
and ri = 0.5 is that at the lower concentration both com-
plexes seem to induce a less intense folding in the OC
form, which would justify that their migration should
be lower and, in consequence, the coalescence point
should be slightly in an upper position with respect to
the coalescence point induced at ri = 0.5.
It is possible to conclude that the behaviour of the three
complexes is highly dependent on the molar ratio ri.
From this study we can withdraw the conclusion that

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A. M. Montan˜a et al. / Bioorg. Med. Chem. 16 (2008) 1721–1737
1727
Figure 5. Agarose gel electrophoresis. Lane 1: pBR322 incubated with 1 at ri = 0.1. Lane 2: pBR322 incubated with 1 at ri = 0.5. Lane 3: pBR322 + 3
at ri = 0.5. Lane 4: pBR322 + 4 at ri = 0.5. Lane 5: pBR322 + 2 at ri = 0.1. Lane 6: pBR322 + 2 at ri = 0.5. Lane 7: free native pBR322. Lane 8:
pBR322 incubated with cisplatin at ri = 0.5.
2.5. Tapping-mode atomic force microscopy (TMAFM)
cisplatin. However, complex 2 produces a higher degree
of fragmentation of DNA chains and a lower number of
knots than complex 1. There are also some differences in
the other morphological effects observed. This behav-
iour seems to indicate that the type and degree of inter-
action with DNA of both enantiomeric platinum
complexes is slightly different.
The atomic force microscopy resulted to be an excellent
tool to study the surface relief, morphology and topol-
ogy of the DNA molecules, before and after incubation
with the compounds whose interaction with DNA was
evaluated. With this technique it is possible to obtain
three-dimensional pictures of DNA, which makes the
observations very informative and intuitive. In our case
the Tapping Mode AFM was used in order to minimize
the contact between the tip of the probe and the surface
to be analyzed. With this technique, the spoiling of the
sample by contact and the risk of dragging and sweeping
sample particles during the analyses were avoided.35
2.6. Studies of the cytotoxicity in vitro of platinum
complexes against HL-60 cells
Antiproliferative studies of enantiomers 1 and 2 were
carried out with HL-60 cell line (acute promyelocytic
leukaemia).36,37 The cell viability was measured after
incubation of cells in the presence of compounds to be
tested at 37 ꢁC for 24 and 72 h. In Figure 7 the viability
(in %) versus concentration (lM) of complex 1 or 2 is
represented. The IC50 values obtained for both enantio-
mers and cisplatin (as a standard or reference) after 24
and 72 h of incubation are quoted in Table 3.
The evaluation of the degree of interaction of complexes
1 and 2 with DNA by TMAFM was carried out at a mo-
lar ratio ri = 0.005. Also, diamine 3, cisplatin (as stan-
dard or reference) and free plasmid DNA were studied
by this technique. In the case of the free DNA test,
the sample was heated up to 60 ꢁC for 5 min to induce
the adoption of the OC form by the DNA molecules.
In all cases the same ri concentration was used to make
the test results comparable. The 2D photographic
pictures of the AFM observations are illustrated in
Figure 6.
Complex 1 is about twice as active as enantiomer 2, at
short incubation time. As a matter of fact, for a 100
lM concentration of 1, after 24 h of incubation, only
10% of cells remain alive and functional. Meanwhile,
for the same concentration of complex 2 and for the
same incubation time 40% of cells are still alive. How-
ever, at higher incubation times both complexes im-
prove their cytotoxic activities and reach similar IC50
values.
The ligand 3 produced a higher aggregation degree than
the free plasmid DNA but without inducing the forma-
tion of as many nodes and cross-links as the correspond-
ing platinum complex 1. The morphological singularities
generated by every tested compound on the plasmid
DNA are quoted in Table 2. The data from Table 2 rep-
resent the number of morphological changes undergone
by DNA per 100 molecules evaluated. This number of
molecules was taken into account for statistical reasons.
The explanation of this difference of activity between
both enantiomers only could be based on their difference
of chirality because all studies and tests have been car-
ried out under strictly the same experimental conditions.
The level at which this difference is produced is not
known yet. It could be at the level of absorption of drug
by the cell due to a higher ability of enantiomer 1 to pass
through the cell membrane. Also, a possibility is that in-
side the cells enantiomer 2 should have more rapid deac-
tivation kinetics in front of metallothioneins,
glutathione and other biomolecules having nucleophilic
thio groups. A simpler and alternative explanation is
that enantiomer 1 could act as an eutomer inducing a
better molecular recognition at the interaction sites of
DNA bases inside the cell nucleus.
According to the previous studies of DC and electro-
phoresis, the ligand 3 does not interact with DNA.
Thus, the morphological changes observed for this com-
pound in AFM could be due to weak interactions like
hydrogen bonds between the amines and the DNA
bases. No covalent bonds are expected due to the low
number of knots or cross-links observed. On the other
hand, complexes 1 and 2 induced morphology changes
on DNA molecules quite similar to that generated by

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A. M. Montan˜a et al. / Bioorg. Med. Chem. 16 (2008) 1721–1737
1728
Figure 6. TMAFM images of plasmid DNA, free and incubated with 1, 2, 3 and cisplatin.
Table 2. Effects produced on pBR322 DNA, observed by AFM, after incubation with complexes 1, 2 and cisplatin
Morphological effects
Free DNA
Ligand 3
Complex 1
Complex 2
Cisplatin
Number of molecules of DNA evaluated
Number of fragments
Number of knots
100
0
100
8
100
10
100
51
100
18
3
19
27
22
89
186
72
123
53
212
35
Number of crossings
Number of supercoilings
Number of foldings
4
0
30
55
256
35
281
47
312
Ligand 3 was incubated with DNA and analyzed by AFM as a reference or standard for comparison. The data represent percentages.

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A. M. Montan˜a et al. / Bioorg. Med. Chem. 16 (2008) 1721–1737
1729
120
100
80
60
40
20
0
72 h
24 h
100
80
24 h
72 h
60
40
20
0
0
20
40
60
80
100
0
50
100
150
200
250
Concentration (µM)
Complex 1
Concentration (µM)
Complex 2
Figure 7. Viability of HL-60 cells in the presence of enantiomeric platinum complexes 1 and 2 after 24 and 72 h of incubation.
Table 3. IC50 data after 24 h and 72 h of incubation
These assays showed that the studied compounds, in
general, induced apoptosis and produced the death of
the larger amount of cells by this pathway. In general
low percentages of cell death by necrosis were observed.
It is worth noting, as can be appreciated in Table 4 and
Figure 8, the different behaviour of enantiomers 1 and 2
in the flow cytometry studies.
Complex
IC50 (lM) a 24 h
IC50 (lM) a 72 h
1
35.25 2.68
71.48 9.21
15.61 1.15
14.57 2.38
12.54 1.34
2.15 1.01
2
Cisplatin
Complex 1 has a very different activity respect to its enantiomer 2 after
24 h of incubation.
3. Conclusions
2.7. Studies of apoptosis by flow cytometry
The work presented deals with the synthesis, evaluation
of the interaction with DNA and the cytotoxic activity
of two enantiomers of a new class of platinum(II) com-
plexes with antiproliferative activity. These enantiomers
showed interesting differences in their biological behav-
iour which demonstrate the molecular recognition of
chirality by DNA as the target biomolecule.
In order to study in which way the platinum compounds
produced the cellular death (necrosis or apoptosis) studies
of flow cytometry38–44 were performed on enantiomers 1,
2 and cisplatin as a reference. These compounds were
incubated for 24 h at a concentration close to the IC50
and results are shown in Table 4. In Figure 8 the diagrams
obtained by this technique, corresponding to the com-
pounds 1, 2 and cisplatin, as well as the control test, are
illustrated. In these graphics, it is possible to appreciate
the populations of cells in the four possible status: the
population of alive cells (i.e., cells that present negative
controls for annexin and propidium iodide PI (left square
at the bottom), the population of cells that undergo apop-
tosis (negative for PI and positive for annexin, right
square at the bottom), cells involved in a process of late
apoptosis or necrosis (positive for both dyes, right square
on the top) and necrotic cells (positive for PI, left square
on the top).
We have carried out the enantioselective synthesis of li-
gands 3 and 4 with structure of 1,7,7-trimethylbicy-
clo[2.2.1]heptane-2,3-diamine. The enantiomeric excess
obtained in both syntheses was of 90% for an overall yield
of 30%. The preparation of enantiomeric platinum(II)
complexes 1 and 2 was performed by reaction with
K2PtCl4, affording pure samples of enantiomeric com-
plexes which were purified and physically and spectro-
scopically characterized.
The interaction of both enantiomers with DNA was stud-
ied by three techniques: circular dichroism (to evaluate
modifications on the secondary structure), electrophore-
sis on agarose gel (to estimate the modifications of the ter-
tiary structure of DNA) and atomic force microscopy to
observe the changes in morphology and topology of
DNA. In the first assay we used Calf Thymus DNA and
in the other two techniques pBR322 plasmid DNA.
From Figure 8 it is possible to observe the different
behaviour of compounds 1 and 2. Thus, 1 produces a
higher population of apoptotic cells than 2, even higher
than cisplatin. This is the desirable death mechanism for
a cytotoxic drug.
Table 4. Percentages of the cell death pathways observed by the flow cytometry assay
Treatment (IC50 24 h, lM)
% Vital ells
% Apoptotic ells
% Late apoptotic/necrotic cells
% Necrotic cells
Control
85.39
53.89
64.59
44.75
8.43
40.68
29.21
47.23
5.95
4.89
5.86
7.13
0.24
0.55
0.34
0.90
Cisplatin (15.61)
2 (78.41)
1 (35.25)

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A. M. Montan˜a et al. / Bioorg. Med. Chem. 16 (2008) 1721–1737
1730
100
101
102
103
104
100
101
102
103
104
Annexin
Complex 1
Annexin
Complex 2
100
101
102
103
104
100
101
102
103
104
Annexin
Control
Annexin
Cisplatin
Figure 8. Images of flow cytometry diagrams for compounds 1, 2, cisplatin and control blank.
In vitro studies of antiproliferative activity were per-
formed against HL-60 cancer cell line (leukaemia). More-
over, flow cytometry studies allowed us to evaluate the
death mechanism induced by the platinum complexes.
In all biochemical assays we have observed changes in
the structure of DNA similar to that induced by cisplatin,
drug used as a reference or standard in our studies. This
behaviour was confirmed by the in vitro cytotoxicity tests.
It is worth noting that the IC50 value of compound 1 is half
of the IC50 value for 2 at 24 h of incubation. In other
words, 1 is twice as active as 2 for such incubation period.
Finally, by flow cytometry it was observed that 1 induced
apoptosis at a level considerably higher than 2 and even
than cisplatin and a low level of necrosis, which is desir-
able in an antiproliferative drug. Summarizing, there is
a clear difference of behaviour between enantiomers 1
and 2 observed by all techniques and in all assays carried
out. The reasons, at the molecular level, for this phenom-
enon are under study in our laboratory.
distilled from sodium/benzophenone prior to use. CH2Cl2
was dried by refluxing it over CaH2 under nitrogen. Ele-
mental analyses (C, H, N, S) were carried out on a Carlo
Erba EA1108 apparatus. Infrared spectra were recorded
on a FT-IR NICOLET 510 spectrophotometer in a
4000–400 cmꢀ1 range. The oily samples were recorded
as films on NaCl plates and in the case of solids as KBr
plates. NMR spectra were obtained on a Varian Gem-
ini-200, a Varian Unity-300 plus or a Varian VXR-500,
1
using CDCl3, [D7]DMF or [D6]DMSO as solvents. H
NMR spectra were obtained at 200 MHz, 300 MHz or
400 MHz frequencies and chemical shifts are given in
ppm, relative to tetramethylsilane (TMS). 13C NMR
and DEPT experiments were recorded at 50 MHz,
75 MHz or 100 MHz and were referenced to the
77.0 ppm resonance of CDCl3. Mass spectra were ob-
tained by a Fisons VG Quattro triple quadrupole analyzer
in the 1800–200 m/z range, fitted with a canon working at
10 kV. The mass spectra were carried out using MeCN–
H2O as solvent under electrospray (ESP-MS) conditions
or by the FAB(+) technique using NBA as a matrix. In
this last technique, the samples were dissolved in DMSO
and bombarded with accelerated caesium atoms. In the
case of working with a chemical ionization technique, a
Hewlett–Packard 5890 mass spectrometer was used (the
operating conditions are specified for each case). Melting
points were measured on a Galenkamp and on a Stuart
Scientific SMP3 apparatus. Circular dichroism (CD) spec-
4. Experimental
4.1. Materials and methods
Reactions that required an inert atmosphere were con-
ducted under dry nitrogen or argon and the glassware
was oven-dried (120 ꢁC). THF, Et2O and benzene were

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A. M. Montan˜a et al. / Bioorg. Med. Chem. 16 (2008) 1721–1737
1731
1
tra were obtained at room temperature on a JASCO J720
spectropolarimeter with a 450 W xenon lamp using a
computer for spectral subtraction and noise reduction.
Each sample was scanned three times in a range of wave-
length between 220 and 360 nm at a 50 nm minꢀ1 rate.
The CD spectra obtained are the mean of the three inde-
pendent scans. The data are expressed as mean residue
molar ellipticity H in deg cm2 molꢀ1. Atomic Force
Microscopy (AFM). The samples were imaged in a Nano-
scope III Multimode atomic force microscope (Digital
Instrumentals Inc., Santa Barbara, CA) operating in tap-
ping mode in air, at a scan rate of 1–3 Hz. The microscope
probes were 125-lm long monocrystalline silicon cantile-
vers with integrated conical-shaped Si tips (Nanosensors
GmbH, Germany) with an average resonance frequency
f0 ꢁ 330 kHz and spring constant K ꢁ 50 N mꢀ1. The
cantilever is rectangular and the tip radius given by sup-
plier is ꢁ5 nm, with a 35ꢁ cone angle and a high aspect ra-
tio. In general the images were obtained at room
temperature (23 2 ꢁC) and the relative humidity was
typically 55%.
a light brown solid. Yield: 0.042 g, 57%. The H NMR,
13C NMR and IR spectra were, as expected, identical to
those of enantiomer 1. MS [FAB(+), NBA,]: m/z (%)
397.8 (100) [M–Cl–H+], 434.3 (43) [M], 513.4 (64)
[M+DMSO+H+]. Anal. Calcd for C10H20Cl2N2Pt: C,
27.7; H, 4.6; N, 6.4. Found: C, 28.0; H, 4.7; N, 6.3.
4.4. Synthesis of (1S,2R,3S)-1,7,7-trimethylbicyclo[2.2.1]-
heptane-2,3-diammonium dichloride (3)
In a 25-mL round-bottomed flask, hydrochlorides 11 and/
or 12 (0.279 g, 0.66 mmol) and ammonium formate
(0.601 g, 9.53 mmol) were dissolved in anhydrous MeOH
(14 mL) under nitrogen atmosphere and the resulting
solution was purged five times by N2/vacuum cycles to
eliminate the traces of oxygen dissolved in methanol.
On the other hand, in a three-necked 50-mL flask, fitted
with septa, 10% Pd/C (0.126 g, 45% w/w) was weighed un-
der argon. Next a Dimroth condenser was adapted and
the system pumped out and back filled with nitrogen five
times. The solution of 11 (or 12) and ammonium formate
in methanol was transferred via cannula to the second
flask and the mixture was purged, as mentioned before,
10 times. The reaction mixture was heated to reflux of sol-
vent for 1.25 h (control by TLC). The crude mixture was
let to cool down to room temperature and it was filtered
through a two-layers short pad of Celiteꢂ and activated
neutral alumina. The resulting clear solution was concen-
trated to dryness in vacuo, obtaining a white solid. The
resulting solid was dissolved in 2 M HCl (25 mL) to gen-
erate the corresponding hydrochloride. The aqueous
solution was extracted with ethyl acetate (2 · 15 mL) to
remove dibenzoyl. The resulting aqueous solution was
4.2. Synthesis of cis-[(1S,2R,3S)-1,7,7-trimethylbicyclo-
[2.2.1]heptane-2,3-diamine]dichloroplatinum(II) (1)
In a 10 mL cone-shaped flask, diamine 3 hydrochloride
(0.066 g, 0.27 mmol) was dissolved in H2O (2.5 mL) un-
der nitrogen atmosphere. In other flask, a solution of
K2PtCl4 (0.113 g, 0.27 mmol) in H2O (1 mL) was pre-
pared under nitrogen. The aqueous diamine solution
was transferred via cannula to the second solution.
The mixture was stirred for 1 h at room temperature
and it was kept to stand in the dark at room temperature
for 24 h. The formed brown solid was filtered out by
using a sintered-glass plate (10–16 lm of pore size)
and it was dried in vacuo and afterwards into a desicca-
tor in the presence of P2O5. Yield: 0.061 g, 51%.
concentrated to dryness in vacuo obtaining 3 as a pure
23
D
white solid. Yield: 0.106 g, 85%. ½aꢂ þ 27:1 (c = 0.98,
MeOH). IR (KBr): mmax 3434 (mNH2), 2957 ðmC3 ꢀ HÞ,
sp
3000–2000 ðmNHþ3 Þ, 1514 ðdNHþ3 Þ, 1461 ðdCs3p ꢀ HÞ
cmꢀ1. H NMR (400 MHz, D2O, 25 ꢁC): d 0.80 (s, 3 H;
1
IR (KBr): mmax 3506, 3215 (mNH2), 2958, 2879 ðmC3 ꢀ HÞ,
sp
H-100 or H-1000), 0.93 (s, 3H; H-100 or H-1000), 0.95 (s, 3H;
H-10), 0.99–1.24 (m, 2H; H-5b, H-6b), 1.56–1.63 (m,1H;
1593 (dNH2), 1460 ðdC3sp ꢀ HÞ cmꢀ1
.
1H NMR
H-6a), 1.75–1.90 (m, 1H; H-5a), 2.07–2.08 (d, J4–5a
=
(400 MHz, [D6]DMSO, 25 ꢁC, TMS): d 0.67 (s, 3 H;
H-1IV or H-1V), 0.85–0.89 (m, 1H; H-6 b), 0.89 (s, 3 H;
H-1IV or H-1V), 0.92–0.97 (m, 1H; H-5 b), 1.18 (s, 3 H;
H-10), 1.25–1.33 (m, 1H; H-6a), 1.40–1.51 (m, 1H;
H-5a), 1.73–1.74 (d, J4–5a = 4.4 Hz, 1H; H-4), 2.81–2.87
(m, 1H; H-2), 2.93–2.99 (m, 1H; H-3), 4.22–4.29 (m, 1H;
H100 or H-200), 4.66–4.73 (m, 1H; H-1000 or H-2000), 5.75–
5.80 (m, 1H; H-100 or H-200), 5.83–5.89 (m, 1H; H-1000 or
H-2000) ppm. 13C NMR (100 MHz, [D7]DMF, 25 ꢁC): d
10.8 (C-1IV or C-1V), 20.8 (C-1IV or C-1V), 21.3 (C-10),
26.3 (C-5), 35.2 (C-6), 47.5 (C-7), 50.2 (C-4), 51.3 (C-1),
70.8 (C-3), 75.1 (C-2) ppm. MS [FAB(+), NBA]:
m/z (%) 476.8 (83) [M–Clꢀ+DMSO], 397.8 (100) [M–
Clꢀ–H+], 362.9 (56) [M–2Clꢀ–H+]. Anal. Calcd for
C10H20Cl2N2Pt: C, 27.7; H, 4.6; N, 6.4. Found: C, 27.9;
H, 4.5; N, 6.0.
4.8 Hz, 1H; H-4), 3.54–3.56 (d, J2–3 = 9.2 Hz, 1H; H-2),
3.62–3.65 (d, J3–2 = 9.2 Hz, 1H; H-3) ppm. 13C NMR
(100 MHz, D2O, 25 ꢁC): d 10.6 (C-100 or C-1000), 20.1 (C-
100 or C-1000), 20.5 (C-10), 25.7 (C-5), 34.6 (C-6), 47.6 (C-
7), 49.0 (C-4), 49.2 (C-1), 56.6 (C-3), 60.4 (C-2) ppm.
MS (EI, 70 eV): m/z (%) 168.9 (100) [M–2HCl]. Anal.
Calcd for C10H22Cl2N2: C, 49.80; H, 9.19; N, 11.61.
Found: C, 49.93; H, 9.21; N, 11.58.
4.5. Synthesis of (1R,2S,3R)-1,7,7-trimethylbicyclo[2.2.1]-
heptane-2,3-diammonium dichloride (4)
Following an experimental procedure similar to the pre-
viously described, substrates 17 and/ or 18 (0.210 g,
0.50 mmol) were reacted with ammonium formate
(0.451 g, 7.22 mmol) and 10% Pd/C (0.095 g, 45% w/w)
4.3. Synthesis of cis-[(1R,2S,3R)-1,7,7-trimethylbicy-
clo[2.2.1]heptane-2,3-diamine]dichloroplatinum(II) (2)
in MeOH (12 mL), obtaining product 4 as a pure white
23
solid. Yield: 0.109 g, 90%. ½aꢂD ꢀ 26:8 (c = 0.21,
MeOH). Anal. Calcd for C10H22Cl2N2: C, 49.80; H,
9,19; N, 11.61. Found: C, 49.73; H, 9.25; N, 11.68.
Substrate 4 (0.041 g, 0.17 mmol) and K2PtCl4 (0.079 g,
0.19 mmol) were submitted to the same experimental
conditions as that in the preceding procedure, obtaining
1
The H NMR, 13C NMR, IR and MS spectra of 4 were
identical to those of enantiomer 3.

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A. M. Montan˜a et al. / Bioorg. Med. Chem. 16 (2008) 1721–1737
1732
4.6. Preparation of ( )-1,2-diphenylethane-1,2-diamine,
( )-DPEDA (5)
C-60 or C-200, C-600), 127.8 (C-20, C-60 or C-200, C-600),
127.9 (C-30, C-50 or C-300, C-500), 128.1 (C-30, C-50 or C-
300, C-500), 137.2 (C-10 or C-100), 138.1 (C-10 or C-100),
168.3 (C-2 or C-7), 169.5 (C-2 or C-7) ppm. MS (EI,
70 eV): m/z (%) = 343 (27) [M+H+], 342 (100) [M], 341
(24) [M–H+], 340 (59) [M–2H+]. Anal. Calcd for
C24H26N2: C, 84.17; H, 7.65; N, 8.18. Found: C, 84.21;
H, 7.62; N, 8.13.
This compound was prepared according to the synthetic
methodology described in Ref. 27.
4.7. Synthesis of 6, 7 and of intermediate 8
In a 250-mL round-bottomed flask, fitted with a Dean–
Stark apparatus, (1S)-camphorquinone (1 g, 6.02 mmol)
and ( )-DPEDA (5) (1.277 g, 6.02 mmol) dissolved in
anhydrous benzene (140 mL) were placed, under nitrogen
atmosphere. The solution was heated to reflux of solvent,
under inert atmosphere for 17 h (control by TLC). The
reaction mixture was let to reach room temperature and
it was concentrated to dryness. The resulting crude prod-
uct was submitted to flash column chromatography on sil-
ica gel, eluting with mixtures of hexane/EtOAc/MeOH of
increasing polarity, isolating the following fractions:
0.497 g of 8 (hexane/AcOEt 85:15), 1.098 g of 6 (hexane/
AcOEt 85:15 y 8:2) and 0.096 g of 7 (hexane/AcOEt
75:25 and 7:3) as yellowish oils.
4.7.3. (2S,8S)-4,5-diphenyl-8,11,11-trimethyl-3,6-diazatri-
cyclo[6.2.1.02,7]undec-6-en-2-ol (8). IR (film): mmax 3344
(mO-H), 3030 ðmC2sp ꢀ HÞ, 2960 ðmCs3p ꢀ HÞ, 1746
ðmC2sp ꢀ NÞ, 1453 ðdCs3p ꢀ HÞ cmꢀ1. 1H NMR (400 MHz,
CDCl3, 25 ꢁC): d 0.99 (s, 3H; H-12), 1.05 (s, 3H; H-13 or
H-14), 1.17 (s, 3H; H-13 or H-14), 1.41–1.48 (m, 1H;
H-9), 1.65–1.72 (m, 1H; H-9), 1.78–1.87 (m, 1H; H-10),
2.06–2.07 (d, J1–10a = 4 Hz, 1H; H-1), 2.17–2.24 (m, 1H;
H-10), 2.47 (s, 1H; H-1000), 3.94–3.96 (d, J5–4 = 9.2 Hz,
1H; H-5), 4.48–4.50 (d, J4–5 = 9.2 Hz, 1H; H-4), 7.21–
7.26 (m, 4H; H-30, H-50, H-300, H-500), 7.28–7.32 (m, 2H;
H-40, H-400), 7.36–7.38 (m, 4H; H-20, H-60, H-200, H-600)
ppm. 13C NMR (100 MHz, CDCl3, 25 ꢁC): d 9.7 (C-12),
20.4 (C-13 or C-14), 22.9 (C-13 or C-14), 23.0 (C-10),
31.3 (C-9), 45.1 (C-11), 55.7 (C-1), 59.0 (C-8), 70.1 (C-5),
71.2 (C-4), 84.6 (C-2), 127.1 (C-40 or C-400), 127.2 (C-40 or
C-400), 127.7 (C-20, C-60 or C-200, C-600), 127.8 (C-20, C-60
or C-200, C-600), 128.7 (C-30, C-50 or C-300, C-500), 128.9 (C-
30, C-50 or C-300, C-500), 139.7 (C-10 or C-100), 140.5 (C-10
or C-100), 166.3 (C-7) ppm. MS (EI, 70 eV): m/z
(%) = 361.1 (100) [M+H+], 343.1 (28) [M–OH]+]. Anal.
Calcd for C25H32N2O: C, 79.75; H, 8.57; N, 7.44. Found:
C,79.80; H, 8.55; N, 7.39.
4.7.1. (1S,4R,5R)-4,5-diphenyl-1,11,11-trimethyl-3,6-diaz-
atricyclo[6.2.1.02,7]undeca-2,6-diene (6). IR (film): mmax
3062, 3033 ðmCs2p ꢀ HÞ, 2961, 2879 ðmCs3p ꢀ HÞ, 1654
ðmC2sp ꢀ NÞ, 1452 ðdCs3p ꢀ HÞ cmꢀ1. 1H NMR (500 MHz,
CDCl3, 25 ꢁC): d 0.92 (s, 3 H; H-12), 1.05 (s, 3H; H-13 or
H-14), 1.14 (s, 3H; H-13 or H-14), 1.62–1.65 (m, 2H; H-
10), 1.86–1.92 (m, 1H; H-9b), 2.17–2.24 (m, 1H; H-9a),
2.59 (d, J8–9a = 4.5 Hz, 1H; H-8), 4.38 (d, J5–4 = 11 Hz,
1H; H-4 or H-5), 4.41 (d, J5-4 = 11 Hz, 1H; H-4 or H-5),
6.92–6.96 (m, 4H; H-30, H-50, H-300, H-500), 7.18–7.22 (m,
6H; H-20, H-40, H-60, H-200, H-400, H-600) ppm. 13C NMR
(50 MHz, CDCl3, 25 ꢁC): d9.9 (C-12), 17.7 (C-13 or C-
14), 20.6 (C-13 or C-14), 25.4 (C-9), 31.6 (C-10), 46.4 (C-
11), 52.7 (C-8), 53.0 (C-1), 65.0 (C-5), 67.2 (C-4), 126.9
(C-40 or C-400), 127.0 (C-40 or C-400), 127.6 (C-20, C-60 or
C-200, C-600), 127.7 (C-20, C-60 or C-200, C-600), 128.1 (C-30,
C-50 or C-300, C-500), 142.3 (C-10 or C-100), 142.4 (C-10 or
C-100), 165.8 (C-2 or C-7), 167.8 (C-2 or C-7) ppm. MS
(EI, 70 eV): m/z (%) 343 (27) [M+H+], 342 (100) [M], 341
(24) [M–H+], 340 (59) [M–2H+]. Anal. Calcd for
C24H26N2: C, 84.17; H, 7.65; N, 8.18. Found: C, 84.15;
H, 7.67; N, 8.20.
4.8. Synthesis of 9 and 10
In a 25-mL flask, fitted with septa and a magnetic stirring
bar, substrate 6 (or 7) (0.459 g, 1.34 mmol) was dissolved
in anhydrous methanol (16 mL) under argon atmosphere.
The solution was cooled down to 0 ꢁC in an ice bath and
sodium borohydride (0.259 g, 6.83 mmol) was added at
once. After addition the system was purged with argon,
the cooling bath was removed and the reaction mixture
was stirred for 1 h at room temperature (controlled by
TLC). To generate the hydrochloride, 2 M HCl was
added (15 mL), and methanol was removed by the rotary
evaporator. The resulting aqueous solution was extracted
with EtOAc (4 · 10 mL). The organic phases were com-
bined together, dried on anhydrous magnesium sulfate,
filtered and concentrated to dryness, resulting in 0.276 g
of dark oil mainly formed by non-reacted substrate 6
(or 7). The aqueous phase was treated with 2.5 M NaOH
(20 mL) to regenerate the free amines which formed a
white precipitate. EtOAc (10 mL) was added to dissolve
the solid and the aqueous phase was extracted with more
EtOAc (4 · 10 mL). All organic phases were combined to-
gether, dried on anhydrous magnesium sulfate, filtered
and concentrated to dryness, resulting in 0.207 g of col-
ourless oil corresponding to product 9 (or 10). The frac-
tion of dark oil corresponding to non-converted
substrate 6 (or 7) was reacted a second time with NaBH4
(0.155 g, 4.10 mmol), under the same previous reaction
conditions, affording additional 0.072 g of 9 (or 10). Over-
all yield: 0.279 g, 99.9%.
4.7.2. (1S,4S,5S)-4,5-diphenyl-1,11,11-trimethyl-3,6-diaz-
atricyclo[6.2.1.02,7]undeca-2,6-diene (7). IR (film): mmax
3062, 3033 ðmC2sp ꢀ HÞ, 2961, 2879 cmꢀ1 ðmCs3p ꢀ HÞ,
1
1654 cmꢀ1 ðmCs2p ꢀ NÞ, 1452 cmꢀ1 ðdCs3p ꢀ HÞ cmꢀ1. H
NMR (500 MHz, CDCl3, 25 ꢁC): d 0.91 (s, 3 H; H-12),
1.08 (s, 3H; H-13 or H-14), 1.17 (s, 3H; H-13 or H-14),
1.81 (qd, J10–9a = 10 Hz, J10–12 = 2.5 Hz, 2H; H-10),
1.97–2.03 (m, 1H; H-9b), 2.19–2.24 (m, 1H; H-9a), 2.62
(d, J8–9a = 4.5 Hz, 1H; H-8), 4.94 (d, J4–5 = 8 Hz, 1H;
H-4 or H-5), 4.98 (d, J4–5 = 8.5 Hz, 1H; H-4 or H-5),
6.74–6.76 (m, 2H; H-30, H-50 or H-300, H-500), 6.82–6.84
(m, 2H; H-30, H-50 or H-300, H-500), 7.05–7.07 (m, 6H; H-
20, H-40, H-60, H-200, H-400, H-600) ppm. 13C NMR
(50 MHz, CDCl3, 25 ꢁC): d 9.9 (C-12), 17.5 (C-13 or C-
14), 20.7 (C-13 or C-14), 24.5 (C-9), 32.5 (C-10), 47.0
(C-11), 53.0 (C-8), 53.2 (C-1), 63.4 (C-5), 63.7 (C-4),
126.7 (C-40 or C-400), 126.9 (C-40 or C-400), 127.6 (C-20,

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1733
1
4.8.1. (1S,2R,4R,5R,7S)-4,5-diphenyl-1,11,11-trimethyl-
3,6-diazatricyclo[6.2.1.02,7]undecane (9). IR (film): mmax
ðdC3sp ꢀ HÞ cmꢀ1. H NMR (400 MHz, D2O, 25 ꢁC): d
0.84 (s, 3 H; H-13 or H-14), 0.95 (s, 3H; H-13 or H-14),
1.15–1.30 (m, 2H; H-10), 1.23 (s, 3H; H-12), 1.58–1.72
(m, 1H; H-9a or H-9b), 1.78–1.90 (m, 1H; H-9a or H-9b),
2.02 (d, J8-9a = 4.0 Hz, 1H; H-8), 3.82 (d, J2–7 = 8.4 Hz,
1H; H-2 or H-7), 3.92 (d, J2–7 = 8.4 Hz, 1H; H-2 or H-7),
3062, 3031 ðmC2sp ꢀ H, 2927 ðmCs3p ꢀ HÞ, 1491 ðmCs3p ꢀ NÞ,
1452 ðdC3sp ꢀ HÞ cmꢀ1
.
1H NMR (400 MHz, CDCl3,
25 ꢁC): d 0.88 (s, 3 H; H-13 or H-14), 0.90 (s, 3H; H-13 or
H-14), 1.03–1.10 (m, 2H; H-10), 1.48–1.59 (m, 2H; H-9),
1.69 (s, 3H; H-12), 1.78–1.90 (m, 3H; H-3, H-6, H-8),
5.00 (d, J4–5 = 11.6 Hz, 1H; H-4 or H-5), 5.04 (d, J4–5
=
11.2 Hz, 1H; H-4 or H-5), 7.20–7.22 (m, 2H; H-40, H-400),
7.24–7.26 (m, 4H; H-20, H-60, H-200, H-600), 7.29–7.34 (m,
4H; H-30, H-50, H-300, H-500) ppm. 13C NMR (100 MHz,
D2O, 25 ꢁC): d 10.6 (C-12), 20.5 (C-13 or C-14), 21.2 (C-
13 or C-14), 26.3 (C-9), 34.5 (C-10), 48.1 (C-11), 48.8 (C-
1), 49.0 (C-8), 56.0 (C-7), 59.5 (C-4 or C-5), 60.2 (C-4 or
C-5), 66.7 (C-2), 128.5 (C-40, C-400), 128.8 (C-20, C-200 or
C-60, C-600), 128.9 (C-20, C-200 or C-60, C-600), 129.6 (C-30,
C-300 or C-50, C-500), 129.7 (C-30, C-300 or C-50, C-500),
142.3 (C-10 or C-100), 142.5 (C-10 or C-100) ppm. MS-FAB
[FAB(+), NBA,]: m/z (%) 349 (47) [(M–2HCl)+2H+], 348
(71) [(M–2HCl)+H+], 347 (100) [M–2HCl]. Anal. Calcd
for C24H32Cl2N2: C, 68.73; H, 7.69; N, 6.68. Found: C,
68.80; H, 7.65; N, 6.71.
2.88 (d, J7–2 = 7.3 Hz, 1H; H-2 or H-7), 3.16 (d, J2–7
=
7.3 Hz, 1H; H-2 or H-7), 3.60 (d, J4–5 = 8.4 Hz, 1H; H-4
or H-5), 4.06 (d, J4–5 = 8.4 Hz, 1H; H-4 or H-5), 7.21–
7.23 (m, 2H; H-40, H-400), 7.26 (m, 4H; H-20, H-60, H-200,
H-600), 7.25–7.27 (m, 4H; H-30, H-50, H-300, H-500) ppm.
13C NMR (100 MHz, CDCl3, 25 ꢁC): d 11.4 (C-13 or C-
14), 21.8 (C-12), 22.8 (C-13 or C-14), 27.5 (C-10), 35.7
(C-9), 47.4 (C-1 or C-11), 48.5 (C-1 or C-11), 50.8 (C-8),
58.3 (C-2 or C-7), 63.9 (C-4 or C-5), 65.9 (C-2 or C-7),
70.7 (C-4 or C-5), 126.8 (C-40 or C-400), 127.5 (C-40 or C-
400), 127.6 (C-20, C-200 or C-60, C-600), 127.8 (C-20, C-200 or
C-60, C-600), 128.3 (C-30, C-300 or C-50, C-500), 128.6 (C-30,
C-300 or C-50, C-500), 144.2 (C-10 or C-100), 144.9 (C-10 or
C-100) ppm. MS (EI, 70 eV): m/z (%) = 347 (27) [M+H+],
346 (100) [M], 342 (32) [M–4H+]. Anal. Calcd for
C24H30N2: C, 83.19; H, 8.73; N, 8.08. Found: C, 83.23;
H, 8.77; N, 8.12.
4.9.2.
(1S,2R,4S,5S,7S)-4,5-diphenyl-1,11,11-trimethyl-
3,6-diazatricyclo[6.2.1.02,7]undecane dihydrochloride (12).
IR (KBr): mmax 3348 (mN–H), 3041 ðmC2sp ꢀ HÞ, 2968
ðmC3sp ꢀ HÞ, 2958–2387 ðmNHþ2 Þ, 1570 (dN–H), 1451
4.8.2. (1S,2R,4S,5S,7S)-4,5-diphenyl-1,11,11-trimethyl-
3,6-diazatricyclo[6.2.1.02,7]undecane (10). IR (film): mmax
ðdC3sp ꢀ HÞ cmꢀ1. H NMR (400 MHz, D2O, 25 ꢁC): d
1
3062, 3031 ðmCs2p ꢀ HÞ, 2927 ðmCs3p ꢀ HÞ, 1491 ðmCs3p ꢀ NÞ,
0.86 (s, 3H; H-13 or H-14), 0.95 (s, 3H; H-13 or H-14),
1.15–1.30 (m, 2H; H-10), 1.26 (s, 3H; H-12), 1.58–1.72
(m, 1H; H-9a or H-9b), 1.78–1.90 (m, 1H; H-9a or H-9b),
1.98 (d, J8–9a = 4.0 Hz, 1H; H-8), 3.82 (d, J2–7 = 8.4 Hz,
1H; H-2 or H-7), 3.92 (d, J2–7 = 8.4 Hz, 1H; H-2 or H-7),
1452 ðdC3sp ꢀ HÞ cmꢀ1
.
1H NMR (400 MHz, CDCl3,
25 ꢁC): d 0.86 (s, 3H; H-13 or H-14), 0.96 (s, 3H; H-13
or H-14), 1.05–1.21 (m, 2H; H-10), 1.52–1.74 (m, 2H;
H-9), 1.65 (s, 3H; H-12), 1.75–1.80 (m, 1H; H-8), 1.95–
2.10 (m, 1H; H-3 or H-6), 2.26–2.39 (m, 1H; H-3 or H-
6), 2.97 (d, J7–2 = 7.2 Hz, 1H; H-2 or H-7), 3.02 (d,
J2–7 = 7.3 Hz, 1H; H-2 or H-7), 3.65 (d, J4–5 = 7.9 Hz,
1H; H-4 or H-5), 4.18 (d, J5–4 = 7.9 Hz, 1H; H-4 or H-
5), 7.16–7.24 (m, 2H; H-40, H-400), 7.25 (m, 4H; H-20,
H-60, H-200, H-600), 7.26–7.27 (m, 4H; H-30, H-50, H-300,
H-500) ppm. 13C NMR (100 MHz, CDCl3, 25 ꢁC): d 12.1
(C-13 or C-14), 21.6 (C-12), 23.1 (C-13 or C-14), 27.0
(C-10), 36.4 (C-9), 47.5 (C-1 or C-11), 48.4 (C-1 or C-
11), 50.6 (C-8), 61.5 (C-2 or C-7), 63.0 (C-4 or C-5), 65.9
(C-2 or C-7), 66.3 (C-4 or C-5), 126.8 (C-40 or C-400),
127.4 (C-40 or C-400), 127.5 (C-20, C-200 or C-60, C-600),
127.7 (C-20, C-200 or C-60,00C-600), 128.4 (C-30, C-300 or C-
50, C-500), 128.7 (C-30, C-3 or C-50, C-500), 144.5 (C-10 or
C-100), 145.0 (C-10 or C-100) ppm. MS (EI, 70 eV): m/z
(%) = 347 (33) [M+H+], 346 (100) [M], 342 (32)
[M–4H+]. Anal. Calcd for C24H30N2: C, 83.19; H, 8.73;
N, 8.08. Found: C, 83.17; H, 8.79; N, 8.03.
4.83 (d, J4–5 = 6.4 Hz, 1H; H-4 or H-5), 4.87 (d, J4–5
=
6.0 Hz, 1H; H-4 or H-5), 7.20–7.22 (m, 2H; H-40, H-400),
7.24–7.26 (m, 4H; H-20, H-60, H-200, H-600), 7.29–7.34 (m,
4H; H-30, H-50, H-300, H-500) ppm. 13C NMR (100 MHz,
D2O, 25 ꢁC): d 10.1 (C-12), 20.5 (C-13 or C-14), 21.3 (C-
13 or C-14), 25.4 (C-9), 35.0 (C-10), 47.8 (C-11), 47.9 (C-
1), 49.7 (C-8), 56.9 (C-7), 59.2 (C-4 or C-5), 59.8 (C-4 or
C-5), 66.7 (C-2), 128.3 (C-40, C-400), 128.9 (C-20, C-200 or
C-60, C-600), 129.4 (C-20, C-200 or C-60, C-600), 129.5 (C-30,
C-300 or C-50, C-500), 129.8 (C-30, C-300 or C-50, C-500),
140.1 (C-10 or C-100), 140.8 (C-10 or C-100) ppm. MS-FAB
[FAB(+), NBA,]: m/z (%) 349 (47) [(M–2HCl)+2H+], 348
(71) [(M–2HCl)+H+], 347 (100) [M–2HCl]. Anal. Calcd
for C24H32Cl2N2: C, 68.73; H, 7.69; N, 6.68. Found: C,
68.70; H, 7.71; N, 6.66.
4.10. Synthesis of 13 and 14
Following the same experimental procedure previously
described for the synthesis of 6 and 7, (1R)-camphorqui-
none (0.501 g, 2.98 mmol) was reacted with ( )-DPEDA
(0.633 g, 2.98 mmol) in anhydrous benzene (70 mL). The
crude product was submitted to flash column chroma-
tography, isolating 0.764 of 13 (with hexane/AcOEt
85:15) and 0.110 g of diastereoisomer 14 (with hexane/
AcOEt 85:15 and 8:2). Yield: 85%.
4.9. Synthesis of 11 and 12
Compounds 9 and/or 10 were treated with 2 M HCl and
the resulting hydrochlorides 11 and/or 12 were formed.
The aqueous solution was concentrated to dryness in a
dry ice rotary evaporator, affording products 11 and/
or 12 as yellowish solids in quantitative yield.
4.9.1. (1S,2R,4R,5R,7S)-4,5-diphenyl-1,11,11-trimethyl-
4.10.1. (1R,4S,5S)-4,5-diphenyl-1,11,11-trimethyl-3,6-diaz-
atricyclo[6.2.1.02,7]undeca-2,6-diene (13). IR (film): mmax
3062, 3033 ðmCs2p ꢀ HÞ, 2961, 2879 ðmCs3p ꢀ HÞ, 1654
ðmC2sp ꢀ NÞ, 1452 ðdCs3p ꢀ HÞ cmꢀ1. 1H NMR (500 MHz,
3,6-diazatricyclo[6.2.1.02,7]undecane dihydrochloride (11).
IR (KBr): mmax3348 (mN–H), 3041 ðmC2 ꢀ HÞ, 2968
sp
ðmC3sp ꢀ HÞ, 2958–2387 ðmNHþ2 Þ, 1570 (dN–H), 1451

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A. M. Montan˜a et al. / Bioorg. Med. Chem. 16 (2008) 1721–1737
1734
CDCl3, 25 ꢁC): d 0.92 (s, 3H; H12), 1.05 (s, 3H; H-13 or H-
14), 1.14 (s, 3H; H-13 or H-14), 1.62–1.65 (m, 2H; H-10),
1.86–1.92 (m, 1H; H-9b), 2.17–2.24 (m, 1H; H-9a), 2.59
(d, J8–9a = 4.5 Hz, 1H; H-8), 4.38 (d, J5–4 = 11 Hz, 1H; H-
4 or H-5), 4.41 (d, J5–4 = 11 Hz, 1H; H-4 or H-5), 6.92–
6.96 (m, 4H; H-30, H-50, H-300, H-500), 7.18–7.22 (m, 6H;
H-20, H-40, H-60, H-200, H-400, H-600) ppm. 13C NMR
(50 MHz, CDCl3, 25 ꢁC): d 9.9 (C-12), 17.7 (C-13 or C-
14), 20.6 (C-13 o C-14), 25.4 (C-9), 31.6 (C-10), 46.4 (C-
11), 52.7 (C-8), 53.0 (C-1), 65.0 (C-5), 67.2 (C-4), 126.9
(C-40 or C-400), 127.0 (C-40 or C-400), 127.6 (C-20, C-60 or
C-2000, C-600), 127.7 (C-20, C-60 or C-200, C-600), 128.1 (C-30,
C-5 or C-300, C-500), 142.3 (C-10 or C-100), 142.4 (C-10 or
C-100), 165.8 (C-2 or C-7), 167.8 (C-2 or C-7) ppm. MS
(EI, 70 eV): m/z (%) = 343 (27) [M+H+], 342 (100) [M],
341 (24) [M–H+], 340 (59) [M–2H+]. Anal. Calcd for
C24H26N2: C, 84.17; H, 7.65; N, 8.18. Found: C, 84.23;
H, 7.61; N, 8.15.
5), 7.21–7.23 (m, 2H; H-40, H-400), 7.26 (m, 4H; H-20, H-
60, H-200, H-600), 7.25–7.27 (m, 4H; H-30, H-50, H-300, H-
500) ppm. 13C NMR (100 MHz, CDCl3, 25 ꢁC): d 11.4
(C-13 or C-14), 21.8 (C-12), 22.8 (C-13 or C-14), 27.5
(C-10), 35.7 (C-9), 47.4 (C-1 or C-11), 48.5 (C-1 or C-
11), 50.8 (C-8), 58.3 (C-2 or C-7), 63.9 (C-4 or C-5), 65.9
(C-2 or C-7), 70.7 (C-4 or C-5), 126.8 (C-40 or C-400),
127.5 (C-40 or C-400), 127.6 (C-20, C-200 or C-60, C-600),
127.8 (C-20, C-200 or C-60,00C-600), 128.3 (C-30, C-300 or C-
50, C-500), 128.6 (C-30, C-3 or C-50, C-500), 144.2 (C-10 or
C-100), 144.9 (C-10 or C-100) ppm. MS (EI, 70 eV): m/z
(%) = 347 (27) [M+H+], 346 (100) [M], 342 (32) [M–
4H+]. Anal. Calcd for C24H30N2 (346.5): C, 83.19; H,
8.73; N, 8.08. Found: C, 83.27; H, 8.80; N, 8.10.
4.11.2. (1R,2S,4R,5R,7R)-4,5-diphenyl-1,11,11-trimethyl-
3,6-diazatricyclo[6.2.1.02,7]undecane (16). IR (film): mmax
3062, 3031 ðmCs2p ꢀ HÞ, 2927 ðmCs3p ꢀ HÞ, 1491 ðmCs3p ꢀ NÞ,
1452 ðdC3sp ꢀ HÞ cmꢀ1
.
1H NMR (400 MHz, CDCl3,
25 ꢁC): d 0.86 (s, 3H; H-13 or H-14), 0.96 (s, 3H; H-13
or H-14), 1.05–1.21 (m, 2H; H-10), 1.52–1.74 (m, 2H;
H-9), 1.65 (s, 3H; H-12), 1.75–1.80 (m, 1H; H-8), 1.95–
2.10 (m, 1H; H-3 or H-6), 2.26–2.39 (m, 1H; H-3 or H-
6), 2.97 (d, J7–2 = 7.2 Hz, 1H; H-2 o H-7), 3.02 (d,
J2–7 = 7.3 Hz, 1H; H-2 or H-7), 3.65 (d, J4–5 = 7.9 Hz,
1H; H-4 or H-5), 4.18 (d, J5–4 = 7.9 Hz, 1H; H-4 or H-
5), 7.16–7.24 (m, 2H; H-40, H-400), 7.25 (m, 4H; H-20,
H-60, H-200, H-600), 7.26–7.27 (m, 4H; H-30, H-50, H-300,
H-500) ppm. 13C NMR (100 MHz, CDCl3, 25 ꢁC): d 12.1
(C-13 or C-14), 21.6 (C-12), 23.1 (C-13 or C-14), 27.0
(C-10), 36.4 (C-9), 47.5 (C-1 or C-11), 48.4 (C-1 or C-
11), 50.6 (C-8), 61.5 (C-2 or C-7), 63.0 (C-4 or C-5), 65.9
(C-2 or C-7), 66.3 (C-4 or C-5), 126.8 (C-40 or C-400),
127.4 (C-40 or C-400), 127.5 (C-20, C-200 or C-60, C-600),
127.7 (C-20, C-200 or C-60, C-600), 128.4 (C-30, C-300 or C-
50, C-500), 128.7 (C-30, C-300 or C-50, C-500), 144.5 (C-10 or
C-100), 145.0 (C-10 or C- cmꢀ1 100) ppm. MS (EI, 70 eV):
m/z (%) = 347 (33) [M+H+], 346 (100) [M], 342 (32) [M–
4H+]. Anal. Calcd for C24H30N2: C, 83.19; H, 8.73; N,
8.08. Found: C, 83.14; H, 8.68; N, 8.12.
4.10.2. (1R,4R,5R)-4,5-diphenyl-1,11,11-trimethyl-3,6-
diazatricyclo[6.2.1.02,7]undeca-2,6-diene (14). IR (film):
m
max 3062, 3033 ðmC2 ꢀ HÞ, 2961, 2879 ðmCs3p ꢀ HÞ, 1654
sp
ðmC2sp ꢀ NÞ, 1452 ðdCs3p ꢀ HÞ cmꢀ1. 1H NMR (500 MHz,
CDCl3, 25 ꢁC): d 0.91 (s, 3H; H-12), 1.08 (s, 3H; H-13 or
H-14), 1.17 (s, 3H; H-13 or H-14), 1.81 (qd,
J10–9a = 10 Hz, J10–12 = 2.5 Hz, 2H; H-10), 1.97–2.03
(m, 1H; H-9b), 2.19–2.24 (m, 1H; H-9a), 2.62 (d,
J8–9a = 4.5 Hz, 1H; H-8), 4.94 (d, J4–5 = 8 Hz, 1H; H-4
or H-5), 4.98 (d, J4–5 = 8.5 Hz, 1H; H-4 or H-5), 6.74–
6.76 (m, 2H; H-30, H-50 or H-300, H-500), 6.82–6.84 (m,
2H; H-30, H-50 or H-300, H-500), 7.05–7.07 (m, 6H; H-20,
H-40, H-60, H-200, H-400, H-600) ppm. 13C NMR (50 MHz,
CDCl3, 25 ꢁC): d 9.9 (C-12), 17.5 (C-13 or C-14), 20.7
(C-13 or C-14), 24.5 (C-9), 32.5 (C-10), 47.0 (C-11), 53.0
(C-8), 53.2 (C-1), 63.4 (C-5), 63.7 (C-4), 126.7 (C-40 or
C-400), 126.9 (C-40 or C-400), 127.6 (C-20, C-60 or C-200, C-
600), 127.8 (C-20, C-60 or C-200, C-600), 127.9 (C-30, C-50 or
C-300, C-500), 128.1 (C-30, C-50 or C-300, C-500), 137.2 (C-10
or C-100), 138.1 (C-10 or C-100), 168.3 (C-2 or C-7), 169.5
(C-2 or C-7) ppm. MS (EI, 70 eV): m/z (%) = 343 (27)
[M+H+], 342 (100) [M], 341 (24) [M–H+], 340 (59) [M–
2H+]. Anal. Calcd for C24H26N2: C, 84.17; H, 7.65; N,
8.18. Found: C, 84.13; H, 7.60; N, 8.19.
4.12. Synthesis of 17 and 18
Compounds 15 and/or 16 were treated with 2 M HCl
and the resulting hydrochlorides 17 and/or 18 were
formed. The aqueous solution was concentrated to dry-
ness in a dry-ice rotary evaporator, affording products
17 and/or 18 as yellowish solids in quantitative yield.
4.11. Synthesis of 15 and 16
The preparation of compounds 15 and 16 was carried out
by the same procedure previously described for products 9
and 10. Thus, substrates 13 and/or 14 (0.783 g,
2.29 mmol) were reacted with NaBH4 (0.452 g,
11.73 mmol) in anhydrous methanol (27 mL), obtaining
products 15 or 16 as a colourless oil. Yield: 0.325 g, 41%.
4.12.1. (1R,2S,4S,5S,7R)-4,5-diphenyl-1,11,11-trimethyl-
3,6-diazatricyclo[6.2.1.02,7]undecane dihydrochloride (17).
IR (KBr): mmax 3348 (mN–H), 3041 ðmC2sp ꢀ HÞ, 2968
ðmC3sp ꢀ HÞ, 2958–2387 ðmNHþ2 Þ, 1570 (dN–H), 1451
1
ðdC3sp ꢀ HÞ cmꢀ1. H NMR (400 MHz, D2O, 25 ꢁC): d
4.11.1. (1R,2S,4S,5S,7R)-4,5-diphenyl-1,11,11-trimethyl-
3,6-diazatricyclo[6.2.1.02,7]undecane (15). IR (film):
0.84 (s, 3H; H-13 or H-14), 0.95 (s, 3H; H-13 or H-
14), 1.15–1.30 (m, 2H; H-10), 1.23 (s, 3H; H-12), 1.58–
1.72 (m, 1H; H-9a or H-9b), 1.78–1.90 (m, 1H; H-9a
or H-9b), 2.02 (d, J8–9a = 4.0 Hz, 1H; H-8), 3.82 (d,
J2–7 = 8.4 Hz, 1H; H-2 or H-7), 3.92 (d, J2–7 = 8.4 Hz,
1H; H-2 or H-7), 5.00 (d, J4–5 = 11.6 Hz, 1H; H-4 or
H-5), 5.04 (d, J4–5 = 11.2 Hz, 1H; H-4 or H-5), 7.20–
7.22 (m, 2H; H-40, H-400), 7.24–7.26 (m, 4H; H-20, H-
60, H-200, H-600), 7.29–7.34 (m, 4H; H-30, H-50, H-300,
m
max3062, 3031ðmCs2p-H), 2927 ðmC3sp-H), 1491 ðmCs3p-N),
1452 (dC3sp-H) cmꢀ1 1H NMR (400 MHz, CDCl3,
.
25 ꢁC): d 0.88 (s, 3H; H-13 or H-14), 0.90 (s, 3H; H-13
or H-14), 1.03–1.10 (m, 2H; H-10), 1.48–1.59 (m, 2H;
H-9), 1.69 (s, 3H; H-12), 1.78–1.90 (m, 3H; H-3, H-6,
H-8), 2.88 (d, J7–2 = 7.3 Hz, 1H; H-2 or H-7), 3.16 (d,
J2–7 = 7.3 Hz, 1H; H-2 or H-7), 3.60 (d, J4–5 = 8.4 Hz,
1H; H-4 or H-5), 4.06 (d, J4–5 = 8.4 Hz, 1H; H-4 or H-

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A. M. Montan˜a et al. / Bioorg. Med. Chem. 16 (2008) 1721–1737
1735
H-500) ppm. 13C NMR (100 MHz, D2O, 25 ꢁC): d 10.6
(C-12), 20.5 (C-13 or C-14), 21.2 (C-13 or C-14), 26.3
(C-9), 34.5 (C-10), 48.1 (C-11), 48.8 (C-1), 49.0 (C-8),
56.0 (C-7), 59.5 (C-4 or C-5), 60.2 (C-4 or C-5), 66.7
(C-2), 128.5 (C-40, C-400), 128.8 (C-20, C-200 or C-60, C-
600), 128.9 (C-20, C-200 or C-60, C-600), 129.6 (C-30, C-300
or C-50, C-500), 129.7 (C-30, C-300 or C-50, C-500), 142.3
(C-10 or C-100), 142.5 (C-10 or C-100) ppm. MS-FAB
[FAB(+), NBA,]: m/z (%) = 349 (47) [(M–2HCl)+2H+],
348 (71) [(M–2HCl)+H+], 347 (100) [M–2HCl]. Anal.
Calcd for C24H32Cl2N2: C, 68.73; H, 7.69; N, 6.68.
Found: C, 68.65; H, 7.73; N, 6.61.
tris-(hydroxymethyl)aminomethane
hydrochloride
(Tris–HCl), 0.1 mM EDTA]:DMSO (98:2) mixture was
prepared. The use of DMSO is to facilitate the dissolu-
tion of compounds to be evaluated. The pH of the solu-
tion was adjusted to 7.4 with 0.1 M NaOH (prepared
with milli-Q water). A stock solution of CT DNA (Calf
Thymus DNA) in TE was prepared (20 lg mLꢀ1) and
kept at 4 ꢁC before use. The final concentration of
DNA was determined by measuring the absorbance at
260 nm in an UV–vis spectrophotometer. Drug-DNA
complex formation was accomplished by addition of ali-
quots of the compound at different concentrations in TE
buffer to the appropriate volume of the CT DNA solu-
tion (5 mL). The samples were prepared with an input
molar ratio of the complex to nucleotide, ri = 0.1, 0.3,
0.5. As a blank, a solution in TE of free native DNA
was used. The reactions were run at 37 ꢁC for 24 h in
the dark.
4.12.2. (1R,2S,4R,5R,7R)-4,5-diphenyl-1,11,11-trimethyl-
3,6-diazatricyclo[6.2.1.02,7]undecane dihydrochloride (18).
IR (KBr): mmax 3348 (mN–H), 3041 ðmC2sp ꢀ HÞ, 2968
ðmC3sp ꢀ HÞ, 2958–2387 ðmNHþ2 Þ, 1570 (dN–H), 1451
ðdC3sp ꢀ HÞ cmꢀ1. H NMR (400 MHz, D2O, 25 ꢁC): d
1
0.86 (s, 3H; H-13 or H-14), 0.95 (s, 3H; H-13 or H-14),
1.15–1.30 (m, 2H; H-10), 1.26 (s, 3H; H-12), 1.58–1.72
(m, 1H; H-9a or H-9b), 1.78–1.90 (m, 1H; H-9a or H-
4.13.3. Sample preparation for agarose gel electrophore-
sis. Stock solutions of the platinum complexes
(1 mg mLꢀ1) in TE (50 mM NaCl, 10 mM Tris–HCl,
0.1 mM EDTA, pH 7.4): DMSO (98:2) mixture were pre-
pared. Drug-DNA complex formation was accomplished
by addition of the appropriate volume of the DNA
pBR322 (0.25 mg/ mL) stock solution to aliquots of the
compound at different concentrations in TE buffer. The
samples were prepared with an input molar ratio of the
complex to nucleotide, ri = 0.1, 0.3, 0.5. The reactions
were run at 37 ꢁC for 24 h in the dark. Twenty microliters
aliquots of complex-DNA compounds were submitted to
0.5% agarose gel electrophoresis for 5 h at 1.5 V/cm in
0.5XTBE (45 mM Tris–borate, 1 mM EDTA, pH = 8.0)
buffer. The Gel was subsequently stained in the same buf-
fer containing ethidium bromide (1 mg/mL) and photo-
graphed with a Fujifilm FTI-500 system.
9b), 1.98 (d, J8–9a = 4.0 Hz, 1H; H-8), 3.82 (d, J2–7
=
8.4 Hz, 1H; H-2 or H-7), 3.92 (d, J2–7 = 8.4 Hz, 1H; H-
2 or H-7), 4.83 (d, J4–5 = 6.4 Hz, 1H; H-4 or H-5), 4.87
(d, J4–5 = 6.0 Hz, 1H; H-4 or H-5), 7.20–7.22 (m, 2H;
H-40, H-400), 7.24–7.26 (m, 4H; H-20, H-60, H-200, H-600),
7.29–7.34 (m, 4H; H-30, H-50, H-300, H-500) ppm. 13C
NMR (100 MHz, D2O, 25 ꢁC): d 10.1 (C-12), 20.5 (C-
13 or C-14), 21.3 (C-13 or C-14), 25.4 (C-9), 35.0 (C-
10), 47.8 (C-11), 47.9 (C-1), 49.7 (C-8), 56.9 (C-7), 59.2
(C-4 or C-5), 59.8 (C-4 or C-5), 66.7 (C-2), 128.3 (C-40,
C-400), 128.9 (C-20, C-200 or C-60, C-600), 129.4 (C-20, C-
200 or C-60, C-600), 129.5 (C-30, C-300 or C-50, C-500), 129.8
(C-30, C-300 or C-50, C-500), 140.1 (C-10 or C-100), 140.8
(C-10 or C-100); MS-FAB [FAB(+), NBA,]: m/z
(%) = 349 (47) [(M–2HCl)+2H+], 348 (71) [(M–
2HCl)+H+], 347 (100) [M–2HCl]. Anal. Calcd for
C24H32Cl2N2: C, 68.73; H, 7.69; N, 6.68. Found: C,
68.81; H, 7.71; N, 6.72.
4.13.4. Sample preparation for atomic force microscopy
(AFM). An HEPES solution was prepared [40 mM of 4-
(2-hydroxyethyl)-1-piperazinemethanesulfonic
acid
(HEPES), 10 mM MgCl2, pH 7.4]. The DNA standard
was prepared as follows: 1 lL of pBR322 DNA (Boeh-
ringer Mannheim GmbH) was dissolved in 39 lL of
HEPES, the solution was heated at 60 ꢁC for 30 min,
to obtain a high number of OC forms, and incubated
at 37 ꢁC for 24 h in the dark. Stock solutions
(1 mg mLꢀ1) of the complex in HEPES/DMSO (40:60)
mixture were prepared. One microliter of DNA
pBR322 (0.25 lg lLꢀ1) and an appropriate aliquot of
the stock solution (ri = 0.5) were diluted to 40 lL in
HEPES. The samples were centrifuged for 2 min before
incubation. The stock solutions of HEPES and of the
complexes were filtered through a sterile 0.2 lm filter
FP 030/3 to remove possible particles or aggregates in
suspension. Finally, the samples were incubated at
37 ꢁC for 24 h in the dark. The observation slides were
prepared by placing a drop (6 lL) of DNA solution or
DNA-complex solution onto a freshly cleaved mica disc
(Ashville–Schoonmaker Mica Co., Newport New, V.A).
After absorption of the sample on the mica surface for
5 min at room temperature, the discs were rinsed for
10 s in a jet of deionized water of 18 MX cmꢀ1 from a
Milli-Q water purification system (Millipore, Molshem,
4.13. Biological studies
4.13.1. Determination of the concentration of the com-
pounds under evaluation. In order to express the concen-
tration of the compounds with respect to the DNA base
pairs, the molar ratio of compound to nucleotide (ri)
was used. This parameter reflects the proportion be-
tween the platinum complex and the base pairs of
DNA (mol of compound/mol of nucleotide). The value
of ri was determined as follows:
m ꢃ Mnucl ꢃ Am
ri ¼
C ꢃ Mr ꢃ V
where; m, mass of compound (lg); Mnucl, average for-
mula weight per nucleotide (330 g/mol), Am, number
of metallic atoms per compound (for the complexes,
we considered Am = 1), C, DNA concentration (lg/
mL), Mr, formula weight of each compound (g/mol)
and V, volume of sample (mL).
4.13.2. Sample preparation for DNA interaction studies
by circular dichroism. A stock solution of the platinum
complex (1 mg mLꢀ1) in a TE [50 mM NaCl, 10 mM

´
A. M. Montan˜a et al. / Bioorg. Med. Chem. 16 (2008) 1721–1737
1736
France) directed onto the surface with a squeeze bottle.
The samples were blow-dried with a flow of dry argon
for 10 min before imaging by AFM.
CTQ2005-01195/BQU, UB-ACIU-2007, CTQ2007-
64843/BQU and BIO2004-05-879). The authors thank
´
Dr. Francisca Garcıa (Cell Culture Facility) and Manu-
ela Costa (Flow Cytometry Facility) for technical
assistance.
4.13.5. Cytotoxicity against HL-60 cells. The growth
inhibitory effect of platinum complexes on the leukae-
mia HL-60 cell line was measured by the MTT, [3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide] assay. The cell line HL60 (acute promyelocytic
leukaemia, ATTCC No. CLL-240) was cultivated in
RPMI-1640 supplemented with 2 mM L-glutamine
and 10% FBS in sterile cultivation flasks into an incu-
bator at 37 ꢁC and under a controlled atmosphere (5%
of CO2 and 95% of humidity). To carry out the cell
proliferation assays, the cells growing in the logarith-
mic phase were seeded in 96-well plates in a 100 lL
volume of culture medium with a density of 104 cells/
well. The cells were let to stand into the incubator
for 2–3 h before performing any further treatment
and then they were treated with varying doses of plat-
inum complexes and the reference drug cisplatin at
37 ꢁC for 24 h and 72 h. The treatment with the differ-
ent complexes was carried out by adding 100 lL of
medium to an aliquot of the standard solution of the
complex in such a way that the final concentration in
the well should be in between 0 and 200 lM. For each
of the variants tested, four wells were used. Aliquots of
20 lL of MTT solution (per 200 lL of culture medium)
were then added to each well. The plates were incu-
bated a 37 ꢁC and under 5% CO2 for 2–3 h, depending
on the metabolic capability of cells. After this time, the
colour formed was quantified by a spectrophotometric
plate reader (ELISA) at 495 nm wavelength, with a ref-
erence filter of 620 nm that allows the correction of
background absorption produced by cell residues.
These values of absorbance were also corrected by
the average of the absorbance of blanks (having only
culture medium). All experiments of cytotoxicity were
independently performed in triplicate with quadrupli-
cates of samples (4 wells per sample). The cell viability
percentage was calculated by dividing the average
absorbance of the cells treated with a platinum com-
plex by that of the control. The IC50 values (drug con-
centration at which 50% of the cells are viable relative
to the control) were obtained by GraphPad Prism soft-
ware, version 4.0.
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