A new polyamine derivative, a structural analog of spermine, with in vivo activity as an inhibitor of ethanol appetite

Article abstract of DOI:10.1016/j.bmc.2005.04.067

This report describes the design and synthesis of the synthetic polyamine DCD (N,N′-bis-(3-aminopropyl)cyclohexane-1,4-diamine, tetramethanesulfonate), a structural analog of spermine, and its in vivo activity as an inhibitor of alcohol consumption in a f

Full text of DOI:10.1016/j.bmc.2005.04.067

Bioorganic & Medicinal Chemistry 13 (2005) 4375–4382
A new polyamine derivative, a structural analog of spermine, with
in vivo activity as an inhibitor of ethanol appetite
a,
Marıa Font, Carmen Sanmartın, Hernan Garcıa, Selfa Contreras,
b
c
c
*
´
´
´
´
Carlos Paeilec and Norberto Bilbenyc
a
´ ´
Modelizacion Molecular, Departamento de Quımica Organica y Farmaceutica, Universidad de Navarra,
´
Irunlarrea no. 1, E-31008 Pamplona, Spain
´
b
´ ´
Seccion de Sıntesis, Departamento de Quımica Organica y Farmaceutica, Universidad de Navarra,
´
´
´
Irunlarrea no. 1, E-31008 Pamplona, Spain
´
Garbil Pharma Investigacion Ltda., Santiago de Chile, Chile
c
Received 6 July 2004; accepted 22 April 2005
Available online 31 May 2005
Abstract—This report describes the design and synthesis of the synthetic polyamine DCD (N,N0-bis-(3-aminopropyl)cyclohexane-
1,4-diamine, tetramethanesulfonate), a structural analog of spermine, and its in vivo activity as an inhibitor of alcohol consumption
in a free-choice paradigm carried out on genetically high-ethanol-consuming UChB rats. After acute treatment with DCD (daily
single dose, 20 mg/kg, p.o., 3 days), a 19% decrease in ethanol intake was obtained, without affecting the levels of food and water
intake. After chronic treatment (daily single dose, 20 mg/kg, p.o., 60 days) a decrease of up to 60% in ethanol intake with respect to
the basal period was provoked; this effect was significantly maintained during the post-treatment period and, according to the data
obtained from the determination of acetaldehyde levels in blood, was not related to a possible disulfiram-like effect. The design of
this new compound was carried out using molecular modeling techniques, with the structures of natural polyamines (putrescine,
spermidine, and spermine) and biosynthetically related diamines (1,3-diaminopropane; DAP) as templates. These polyamines have
shown activity as inhibitors of ethanol appetite in the same experimental model.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
From the therapeutic point of view, recent advances
made in the neurobiological area permit identification
of the neurotransmitters involved in the establishment
and maintenance of alcoholism11,12 and show that alco-
hol impacts multiple neurochemical receptors in the cen-
tral nervous system. Several studies showed that ethanol
is a potent and selective inhibitor of the N-methyl-D-as-
partate receptors (NMDAr) and that prolonged expo-
sure to ethanol leads to a compensatory ꢀup-regulationꢁ
of these receptors, resulting in enhanced NMDA recep-
tor-mediated functions after the removal of ethanol. It is
assumed that these alterations contribute to the develop-
ment of a tolerance and dependence of ethanol as well as
the acute and delayed signs of ethanol withdrawal.13,14
Alcohol dependence affects the biological, psychologi-
cal, and social aspects of personal life. The therapies
developed for treating this problem generally consist
of using drugs affecting different mechanisms of action,
which attenuate the psychoactive effects of alcohol and
reduce cravings for this substance. These drug associa-
tions attempt to reduce the manifestations of abstinence
(using benzodiazepines and other tranquilizers), prolong
and maintain the period of abstinence (adversive com-
pounds, such as disulfiram1 and acamprosate2 or cyana-
mide,3,4 or other compounds, such as naltrexone5), and
contribute to the treatment of problems such as anxiety
or depression,6,7 which frequently accompany the prob-
lem of alcohol dependence.8–10
NMDAr are large hetero-oligomeric complexes includ-
ing at least two copies of an NR1 subunit and two cop-
ies of an NR2 subunit,15 with a group of modulating
sites, among which the polyamine site is exposed.16–18
Keywords: Spermine; Inhibitor of ethanol appetite; NMDA receptor;
Disulfiram-like effect.
*
The endogenous polyamines, spermidine (SPD) and
spermine (SP) and their diamine precursor, putrescine
Corresponding author. Tel.: +34 948 425 600; fax: +34 948 425
649; e-mail: mfont@unav.es
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.04.067

4376
M. Font et al. / Bioorg. Med. Chem. 13 (2005) 4375–4382
NH2
H2N
1,3-propanodiamine, DAP
H2N
putrescine, Pu
NH2
H2N
NH2
spermidine, SPD
N
H
H
H2N
N
spermine, SP
DCD
NH2
N
H
+
NH2
+
NH2
+
NH3
+
NH3
4. CH3SO3H
Figure 1. Structures for reference and DCD compounds.
(Pu), (Fig. 1) are intracellular cationic molecules that are
essential for the growth, division, and differentiation of
cells.19,20 SPD and SP modulate the NMDA-receptor
complex, with low micromolar levels potentiating and
high micromolar concentrations inhibiting its func-
tion.21,22 The affinity of the polyamines for many types
of receptors and ion channels at which ethanol is also
believed to act suggests many potential interactions
among these agents.23,24 In addition, interactions be-
tween ethanol and polyamines are of potential impor-
tance with regard to many of the sequels of ethanol
abuse, including different states of ethanol withdrawal.
NMDA-receptor-mediated mechanisms may be crucial
in alcoholism and they provide a target for novel anti-
alcoholism drugs.25,26
the NMDAr, initially, it appears that the activity is con-
trolled by a wide range of factors.28 Therefore, since the
endogenous amines contain at least two amino groups,
these factors can include (a) the total number of amino
groups, (b) the number of carbon atoms that separate
these groups, (c) the distance between the amino groups,
and (d) the steric surroundings of these groups. The ba-
sicity of the amino groups as well as other properties,
such as the total steric volume, lipophilia, and polarity,
are factors that should be taken into consideration.
With regard to the structural factors, it has been demon-
strated that diamine compounds with chain lengths be-
tween C2 and C3 are partial agonists, while those that
have a chain length between C4 and C7 act as selective
antagonists. Compounds with longer chains act as re-
verse agonists. For total agonism, the required interac-
tion takes place with three amine-type points, two of
which are situated within a distance of approximately
These facts raise the possibility of developing new effec-
tive compounds for the treatment of alcoholism, which
would act by interacting with this NMDA receptor
through polyamine-sensitive sites.
˚
5 A from each other, with the third point being at a dis-
˚
tance of 5–6 A from the other two points. While a coor-
In a previous phase of our work, we demonstrated the in
vivo activity of a series of natural polyamines, such as
SPD and SP, and diamines, such as PU, and related
compounds, such as 1,3-propanodiamine (DAP, Fig.
1), as inhibitors of ethanol appetite in a genetically alco-
holic rat model. The activity shown by these compounds
was significant, but with a limited time of action. This
conclusion was based on the fact that a fast recovery
was observed once the treatment period ended.27
dinated action involving all three sites might explain the
partial agonism found for diamines of shorter length, it
was suggested that the antagonism may be attributed to
an interaction with only two of the three sites. A fourth
amine point of interaction, situated at an approximate
˚
distance of 12 A, would be necessary in order to explain
the reverse agonism of the long-chained diamines.29
Considering those previous data and in order to design
new compounds, an indirect drug design approach was
used. Following this approach, and starting from the
analysis and characterization of the de novo tri-dimen-
sional models (InsigthII software on SiliconGraphics
workstations) of the diamines DAP and Pu as well as
the polyamines SPD and SP, selected as the templates,
a series of elements that make up the pharmacophore30
were determined. Keeping in mind the polycationic
character of the molecules under study and the data ob-
tained from the reference literature, the descriptors con-
sidered for the design were (a) distance between the
nitrogens present, (b) value of the charge and the dis-
tance between charged nitrogens, (c) values, distances,
and geometric relationships among the molecular elec-
Keeping in mind the aforementioned, new compounds
were designed which would hopefully maintain the tar-
get biological activity, the inhibition of alcohol appetite,
as well as possess an improved biological profile and a
more favorable level of toxicity.
2. Design considerations
The pharmacology of polyamines has been reviewed
quite extensively and this has contributed to the estab-
lishment of structure–activity relationships. Considering
the different effects that these compounds produce on

M. Font et al. / Bioorg. Med. Chem. 13 (2005) 4375–4382
Table 1. Decrease in ethanol intake (%)a for the reference compounds
and DCD
4377
Compound
T-Basal
0–6 h
0–24 h
DAP
PU
ꢀ32.7**
ꢀ27.6**
ꢀ29.1**
ꢀ42.3**
ꢀ19.3**
ꢀ21.0**
ꢀ23.7**
ꢀ16.5*
ꢀ27.9**
ꢀ6.09*
SPD
SP
DCD
Acute treatment (20 mg/kg, p.o. · 3 days).
aValues are means (n = 8) of % changes of ethanol intake during the
first 6 h (0–6 h) and the whole day (0–24 h) following drug adminis-
tration, **p <0.005, *p <0.05, with relation to ethanol intake during the
basal period (paired Studentꢁs t-test).
Figure 2. 3D-models for DCD and SP.
Table 2. Descriptors obtained for the reference diamines DAP, PU,
and DAC (AM1 over the more stable conformationsa)
REF
Distances
˚
N1–N2 (A)
Charge
MEPb
N1
N2
N1
N2
DAP
PU
DAC
3.37–5.03
4.93–6.27
5.70
ꢀ0.35
ꢀ0.35
ꢀ0.33
ꢀ0.34
ꢀ0.34
ꢀ0.33
ꢀ109.65
ꢀ113.56
ꢀ113.27
ꢀ109.85
ꢀ110.80
ꢀ113.27
DAP: 1,3-propanodiamine; PU: putrescine; DAC: cyclohexane-1,4-
diamine.
a See Experimental for details.
b Molecular electrostatic potential.
trostatic potential MEP, and (d) localization of HOMO
and LUMO orbitals.
With regard to the in vivo activity shown by DAP and
Pu (Table 1), these structures were taken to be active
minimum fragments. For the design of new compounds,
an additional approach has been made in which SP can
be considered a central Pu with two lateral propylamine
fragments. Following this approach, a preliminary
structural modification was carried out, replacing the
central Pu by another structural element, such as 1,4-
diaminocyclohexyl, DAC, which maintains the previ-
ously cited descriptors within the permitted limits (Table
2). A new SP analog, N,N0-bis-(3-aminopropyl)cyclo-
hexane-1,4-diamine (DCD, Figs. 1 and 2) was proposed
as a target of synthesis.
Scheme 1.
analysis and HPLC chromatography. The melting
points of the solids were determined as complementary
data.
4. Biological evaluation
4.1. Inhibition of ethanol appetite
In order to evaluate the target activity, we studied the ef-
fects of DCD and the reference endogenous polyamines
on alcohol consumption in a free-choice paradigm car-
ried out on genetically high-ethanol-consuming UChB
rats; this strain has innate tolerance to ethanol,31,32
due to genetic differences, including changes in acetalde-
hyde metabolism.33–35 In the first series of experiments,
the effect of acute treatment on ethanol, water, and food
consumption was established using the selected com-
pounds (DAP, Pu, SP, SPD, DCD). For this assay, three
periods of tests were considered: (a) the 3-day basal per-
iod, during which basal consumption was established;
(b) the 3-day treatment period, during which a single
daily dose of 20 mg/kg of the compound was adminis-
tered p.o. (vehicle in the control group) and ethanol in-
take was measured daily during the first 6 h (0–6 h) as
3. Chemistry
The synthesis of DCD was carried out following Scheme
1. Basically, the synthesis occurs upon addition of the
two nucleophilic centers of the starting product, 1,4-
cyclohexanodiamine, to the proximal carbon of acrylo-
nitrile, and after subsequent reduction of the intermedi-
ate by means of catalytic hydrogenation with Ni-Raney
as the catalyst. The product, DCD, obtained as the free
base, was subsequently treated in order to obtain the
corresponding methanosulfonic salt derivative. The
structural elucidation and characterization of the prod-
ucts have been carried out by infrared and H and 13C
nuclear magnetic resonance spectroscopy. The purity
of the products was determined by C.H.N. elemental
1

4378
M. Font et al. / Bioorg. Med. Chem. 13 (2005) 4375–4382
well as throughout the whole day (0–24 h), establishing
time zero as the hour at which the compound was
administered; and (c) the post-treatment period, which
covers the days immediately following the treatment
period.
vertigo, blurred vision, confusion) that deters consump-
tion of alcohol.36,37
In order to discard the possibility that the inhibitory ef-
fect on ethanol appetite shown by compound DCD and
reference polyamines might be related to possible inhib-
itory activity of ALDC, a study was carried out on addi-
tional groups of UChB rats, which were administered
(i.p.) a standard dose of ethanol (2.76 g/kg) preceded
by p.o. administration of 20 mg/kg of polyamine or a
standard 300 mg/kg dose of disulfiram 30 min before
the ethanol challenge. Blood was drawn from the tail
vein and the acetaldehyde levels were determined by
gas chromatography.
In a second experiment carried out on different groups
of UChB rats, the variation in ethanol, water, and food
intake throughout a long-term administration of DCD
was studied. For this assay, once a 3-day basal period
of intake was established, the rats were subjected to a
60-day treatment period, during which a single daily
dose of 20 mg/kg of DCD was administered p.o. Intake
of ethanol, water, and food was measured once daily
throughout the 0–24 h period in relation to time zero.
The post-treatment period for this assay was extended
long enough to assure recovery from DCD treatment.
5. Results and discussion
4.2. Disulfiram-like effect
5.1. Inhibition of ethanol appetite (acute and chronic
treatment)
The search for an ideal drug for the treatment of alco-
holism is principally centered on compounds that lack
adverse effects, meaning disulfiram-like effects or effects
linked to the blocking of the acetaldehyde dehydroge-
nase enzyme (ALDC), the enzyme following alcohol
dehydrogenase in the major pathway of alcohol metab-
olism. Disulfiram inhibits ALDC, blocking oxidation of
alcohol and allowing acetaldehyde to accumulate in the
blood; accumulation of acetaldehyde produces the
highly unpleasant disulfiram–alcohol reaction (flushing,
throbbing in head and neck, throbbing headaches, respi-
ratory difficulty, nausea, copious vomiting, sweating,
thirst, chest pain, palpitations, dyspnea, hyperventila-
tion, tachycardia, hypotension, syncope, weakness,
The results obtained in these assays are shown in Tables
1, 3, and 4. With respect to the activity shown with acute
treatment (Table 1), all of the compounds tested signifi-
cantly decreased ethanol intake in the UChB rats; the ef-
fects were more marked during the first 6 h after drug
administration (0–6 h interval) as compared to those ob-
served during the whole day (0–24 h interval). No signif-
icant variations were observed with regard to food and
water intake.
In this assay, upon comparing the activity shown by
DCD with that shown by SP, it was observed that recu-
peration from ethanol intake was faster with DCD.
Table 3. Effecta of DCD (chronic p.o. administration, 20 mg/kg · 60 days) on ethanol intake (measurement interval: 0–24 h)
Basal Cycle
1
2
3
4
5
6
7
8
9
10
Mean
9.2
0.62
7.9
0.89
8.1
0.86
8.0
0.63
6.9
0.83
7.3
0.77
6.5
0.78
6.2
0.61
6.5
0.40
5.6
0.65
5.3
0.81
SE
%
ꢀ14.13
11
ꢀ11.96
12
ꢀ13.04
13
ꢀ25.00
14
ꢀ20.65
15
ꢀ29.35
16
ꢀ32.61
17
ꢀ29.35
18
ꢀ39.13
19
ꢀ42.39
20
Mean
SE
9.2
0.62
3.6
0.70
4.6
0.77
4.3
0.58
4.0
0.73
3.3
0.52
3.2
0.59
3.6
0.41
4.1
0.33
3.0
0.49
3.7
0.16
%
ꢀ60.87
ꢀ50.00
ꢀ53.26
ꢀ56.52
ꢀ64.13
ꢀ65.22
ꢀ60.87
ꢀ55.44
ꢀ67.39
ꢀ59.78
a Values are means SEM (n = 8) of changes of daily ethanol intake, expressed as ml/100 g/day, in relation to the basal values.
Table 4. Effecta of DCD (chronic p.o. administration 20 mg/kg · 60 days) on water intake (measurement interval: 0–24 h)
Basal
Cycle
1
2
3
4
5
6
7
8
9
10
Mean
SE
2.9
0.76
2.9
0.64
0
3.0
2.5
0.64
2.5
0.76
2.8
0.72
3.1
3.3
0.53
3.4
0.47
4.1
0.41
4.1
0.80
41.38
0.72
3.45
0.67
6.89
%
ꢀ13.79
13
ꢀ13.79
14
ꢀ3.44
15
13.79
17.24
41.38
11
4.0
12
4.5
16
5.5
17
18
19
20
Mean
SE
2.9
0.76
4.9
0.67
68.96
4.5
0.36
55.17
4.7
0.37
62.07
4.6
0.54
58.62
4.6
0.55
58.62
4.9
0.63
68.92
5.8
0.59
100
0.67
37.93
0.70
55.17
0.60
89.66
%
a Values are means SEM (n = 8) of changes of daily water intake, expressed as ml/100 g/day, in relation to the basal values.

M. Font et al. / Bioorg. Med. Chem. 13 (2005) 4375–4382
4379
120
100
80
EtOH intake H2O intake
60
40
20
0
-20
-40
-60
-80
cycle number
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38
Figure 3. Percentage change of ethanol and H2O intake during and after chronic DCD treatment (20 mg/kg, p.o.). DCD was administered daily from
day 0 (cycle 1) to day 60 (cycle 20), as indicated by the arrow. Values are means of daily intake with relation to basal values.
Table 5. Acetaldehyde levels (lg/100 ml) in blooda
Therefore, a new study was performed. The study in-
volved single daily administration (p.o.) of 20 mg/kg of
DCD (chronic treatment) for 60 days. Intake volume
was measured in 3-day cycles (1–20, for the T period
and 21–38 for the PT period). The data obtained appear
in Tables 3 and 4 and in Figure 3. The decrease in eth-
anol intake (Table 3) was highly significant, reaching a
maximum value of approximately 67%, and a gradual
recovery of basal scores of ethanol intake occurred after
stopping the treatment. However, in spite of the pro-
longed PT period, the basal values of intake were not
recuperated.
Compound Dose
(mg/kg, p.o.)
Time (h)b
0.5
1
2
Vehicleb
PU
—
53
83
5
7
49
45
6
6
18
35
5
3
20
20
20
40
SPD
SP
138 21
204 4*
155 24*
50 14
42 22
44
29
8
4
DCD
32.
3
30
2
Disulfiram 300
725 82** 816 53** 594 65**
a See Experimental for details.
b After ethanol administration.
* p <0.05.
** p <0.005.
Table 4 shows that long-term treatment with DCD
greatly influenced water intake in the rats. According
to the free-choice paradigm used, more than 70% of
the total liquid intake was alcohol solution, and taking
into account that the decrease in the volume of ethanol
ingested was accompanied by an increase of water in-
take, it could be considered that there was no notable
modification in the total volume of liquids ingested.
No significant variations with regard to food intake
were observed.
As expected, after ethanol administration, a significant in-
crease in the acetaldehyde levels was observed in the
group treated with disulfiram, while there were only slight
enhancements of acetaldehyde concentration in the
groups treated with the different polyamines; said
enhancements were very similar to those observed in the
control group and were considered nonsignificant. In
the case of DCD, the acetaldehyde levels detected were
lower than those corresponding to the control group.
Daily observation of the animals during the chronic
treatment with DCD revealed no detectable changes in
exploratory behavior and mobility of the animals.
These results indicate that the inhibitory activities of
voluntary ethanol consumption in UChB rats of the
polyamine compounds studied are not related to ALDC
enzyme inhibition.
5.2. Disulfiram-like effect
Table 5 shows the results corresponding to the acetalde-
hyde levels found in the blood samples obtained from
animals treated with different reference polyamines
(PU, SPD, and SP, 20 mg/kg p.o.) and DCD (40 mg/
kg p.o.). As a reference for this test, a standard dose
(300 mg/kg) of disulfiram is administered, to which the
alcohol consumption being practically reduced to zero
in UChB rats.35
6. Conclusions
Based on the data regarding inhibitory activity of etha-
nol appetite obtained for a group of endogenous poly-
amines, a structural analog, DCD, was designed. In
this design, the butyl central fragment of SP was substi-
tuted by a cyclohexane ring.

4380
M. Font et al. / Bioorg. Med. Chem. 13 (2005) 4375–4382
The results showed that upon an acute 3-day treatment,
DCD was able to reduce the ethanol intake in UChB
rats (the decrease was higher during the 0–6 h interval)
subjected to a free-choice paradigm, without signifi-
cantly affecting water and food intake. The long-term
treatment (60 days) with DCD provoked a decrease in
ethanol consumption of approximately 67%. Concomi-
tantly, the long-term DCD treatment also provoked a
moderate but significant and reversible decrease of water
intake.
(e) Obtainment of MEP values and their geometric
relationship (MEPMIN module in software pack-
age, MEPSIM 9742).
7.2. Synthesis
Chemicals were purchased from Aldrich, BDH Chemi-
cals, Fluka, etc. All of the products of the intermediate
and final reactions were characterized by infrared (Per-
kin Elmer FT 681) and nuclear magnetic resonance
(Bruker AC-200e, 200 MHz, for 1H and 50 MHz for
C13) spectroscopic techniques. The melting points were
determined using a Mettler FP82 system equipped with
a FP800/FP80 processor, an Olympus 8091 microscope,
and a video system; they have not been corrected. Ele-
mental analyses were obtained using a CHN-900 ele-
mental analyzer (LECO). The interval of purity
accepted was 0.4%.
Based on the data obtained it can be concluded that the
inhibitory effect of DCD and the reference polyamines
on ethanol intake is unrelated to an ALDC enzyme inhi-
bition mechanism.
The reduction of ethanol intake by polyamines in UChB
rats is possibly the result of NMDAr modulation in re-
gions of the brain that are involved in the craving for
different abuse substances, including alcohol.38 It has
been demonstrated that the endogenous polyamines
can behave as NMDAr antagonists when administered
at concentrations higher than the physiological levels.39
The structural modification carried out on SP in order
to obtain DCD presumably causes a modification of
the logP of DCD with respect to the logP obtained for
SP, which could favor the access of DCD to the target
NMDA receptors.
7.2.1.
N,N0-Bis(2-cianoethyl)cyclohexano-1,4-diamine
(1). Cyclohexane-1,4-diamine (5.7 g, 50 mmol, mixture
of isomers, Aldrich 33,9980-9) was placed in a flask pro-
vided with both stirring and refrigeration systems and
heated to 80 ꢁC. Simultaneously, 7.23 ml (110 mmol)
of acrylonitrile (BDH Chemicals 270471) was added
dropwise, with energetic stirring. The mixture was main-
tained at 80 ꢁC for 1 h, and then it was heated to 100 ꢁC
for 2 h. The mixture was allowed to cool and the solid
was collected with ethyl ether, filtered, and purified.
The desired compound (1) was obtained as a crystalline
white solid (21.05 g; 95% yield). Mp = 102–105 ꢁC
(EtOH). IR (KBr) cmꢀ1 = 3299 (N–H), 2925, 2852,
2800 (C–H aliphat.), 2245 (CN). 1H NMR (CDCl3,
200 MHz) d (ppm): 0.94–1.49 (m, 6H, CH2 and NH);
1.85–1.89 (m, 4H, CH2); 2.40–2.46 (m, 6H, CH2–NH2
and CH); 2.84–290 (m, 4H, CH2–NH2). 13C NMR
(CDCl3, 50 MHz) d (ppm): 19.2 (CH2CN); 31.9 (CH2);
42.4 (CH–NH2); 56.0 (CH); 118.7 (CN). C.H.N.
(C12H20N4) Calcd 65.49, 9.09, 25.45; Found: 65.00,
9.07, 25.52.
7. Experimental
7.1. Molecular modeling
The de novo construction of the reference molecule
models was carried out by applying the software pack-
age, InsightII,40 on SiliconGraphics workstations. The
protocol can be summarized as follows:
(a) Initial construction of the model.
(b) Hierarchized systematic conformational analysis:
Determination of the rotation-sensitive bonds; elec-
tion of a 30ꢁ window to check each dihedron. First
filtration: elimination of the conformations that are
indistinguishable by symmetry. Second filtration:
elimination of conformations that present steric
impediments. Third filtration: calculation of the
energy of conformation and elimination of those
conformations whose relative energy is greater than
10 kcal/mol at global minimum. Fourth filtration:
optimization of the geometry of the conformation
and elimination of those whose relative energy is
greater than 10 kcal/mol at global minimum. All
of the calculations were made within the framework
of molecular mechanics, with the CVFF force field,
(Search and Compare module, InsightII).
7.2.2. N,N0-Bis(3-aminopropyl)cyclohexano-1,4-diamine
(2). A dissolution of N,N0-bis(2-cianoethyl)cyclohex-
ano-1,4-diamine (1) in 150 ml of methanol saturated
with NH3 was placed in a Parr hydrogenating reactor
provided with both stirring and refrigeration systems.
Niquel-Raney (Fluka, 83440) was added (50 mg) and
the mixture was maintained at 50 ꢁC under hydrogen
pressure (40–50 psi) for 6 days, with stirring periods of
8 h/day. The catalyst was eliminated by filtration and
the solvent was eliminated under reduced pressure.
The desired compound (2) was obtained as a greenish-
colored syrup (5.48 g, 80% yield). IR (KBr)
cmꢀ1 = 3290, 3354 (NH2 and NH), 2855, 2928 (C–H ali-
1
phat.). H NMR (CDCl3, 200 MHz) d (ppm): 1.11–1.20
(m, 4H, CH2); 1.48–1.66 (m, 10H, CH2, NH2 and NH);
1.92–2.00 (m, 4H, CH2); 2.41–2.59 (m, 4H, CH2); 2.65–
2.79 (m, 8H, CH2); 13C NMR (CDCl3, 50 MHz) d
(ppm): 31.9 (CH2); 33.9 (CH2); 40.27 (CH–NH2); 44.9
(CH–NH); 58.7 (CH).
(c) Mechanoquantics optimization of the conforma-
tions obtained in the previous step, with the molec-
ular orbital calculations package, Mopac (AM141
semi-empirical
InsightII module).
approach,
AMPAC/MOPAC
(d) Obtainment of geometric descriptors, charges, and
orbitals.
7.2.3. Tetramethanosulfonate of N,N0-bis(3-aminopro-
pyl)cyclohexano-1,4-diamine (3). N,N0-Bis(3-aminopro-

M. Font et al. / Bioorg. Med. Chem. 13 (2005) 4375–4382
4381
pyl)cyclohexano-1,4-diamine (2, 2.14 g, 9.4 mmol) dis-
solved in 50 ml of methanol was mixed, under stirring,
with 2.6 ml (40 mmol) of methanosulfonic acid (Aldrich,
M-860-6) dissolved in 10 ml of ethyl ether. The mixture
was stirred at room temperature for 2 h and then fil-
tered, dried, and purified. Compound (3) was obtained
were considered the basal values; (b) the treatment per-
iod, T, during which (3 days for the acute treatment and
60 for the chronic treatment) a single 20 mg/kg dose of
the compound under evaluation was administered p.o.
daily at time zero; the corresponding volume of the vehi-
cle was administered p.o. to the control group; and (c)
the post-treatment period, PT, consisted of the days
immediately following the treatment period, in which
the animals were maintained under basal conditions,
without receiving any treatment.
as
a white crystalline solid: (1.5 g, 25% yield).
Mp = 215–217 ꢁC (EtOH). IR (KBr) cmꢀ1 = 2920,
2860 (C–H aliphat.); 1196 (SO3); 1H NMR (CDCl3,
200 MHz) d (ppm): 1.80–1.90 (m, 4H, CH2); 1.92–1.96
(m, 4H, CH2); 2.14–2.20 (m, 4H, CH2, NH2); 2.24 (s,
12H, CH3); 2.90–3.02 (m, 10H, CH2 and CH); 7.84 (br
In each 3-day period, ethanol intake was measured daily
during the first 6 h as well for the whole day (0–24 h) in
relation to time zero. The daily intake of water and food
(0–24 h) was evaluated throughout the three periods.
Daily intake measurements of ethanol, water, and food
during the basal period normalized at ml/100 g/day for
water and ethanol solution, and at g/100 g/day for food.
Differences in ethanol, water, and food intake between
the T and B periods, as well as between the PT and B
periods, were calculated for each rat. The results were
expressed as means SEM of changes in alcohol and
water consumption, resulting from comparing the in-
takes during the T and PT period to those of the B per-
iod. The significance of the differences was established
by means of the paired Studentꢁs t-test.
s, 6H, NH+ and NH+2), 13C NMR (CDCl3, 50 MHz)
3
d (ppm): 23.6 (CH2); 25.9 (CH2); 36.0 (CH2); 39.4
(CH3); 40.0 (CH–NH2); 54.3 (CH). C.H.N.
(C12H28N4Æ4CH3SO3HÆH2O) Calcd: 30.48, 7.30, 8.88;
Found: 30.72, 6.99, 8.40.
7.3. Inhibition of ethanol intake
UChB Wistar male and female alcohol-na¨ıve rats,
approximately 90 days old at the beginning of the assay,
were selected for their high level of voluntary consump-
tion of aqueous ethanol solution (10% v/v). They were
obtained from the animal breeding center at the Univer-
sity of Chile (Santiago de Chile, Chile). The rats were
individually housed in standard steel cages with free ac-
cess to an ethanol solution (10% v/v), water, and food.
The temperature was maintained at 22 1 ꢁC, with peri-
odic cycles of air changes. A regimen of light/darkness
of 12 h each and a relative humidity of approximately
60% was also maintained.
7.4. Disulfiram-like effect
UChB Wistar male and female alcohol-na¨ıve adult rats
(250–300 g) were obtained from the animal breeding
center at the University of Chile (Santiago de Chile,
Chile). They were distributed in three homogeneous
groups (n = 6–8). Physiological serum (administration
vehicle) was administered p.o. to the control group
30 min before ethanol administration (2.76 g/kg, i.p.).
DCD (20 mg/kg) was administered p.o. to the second
group 30 min before receiving the ethanol dose. Disulfi-
ram (300 mg/kg) was administered p.o. to the third
group 30 min before administration of the ethanol.
Blood was drawn from the tail vein and the acetalde-
hyde levels were determined by gas chromatography
(ꢀHead-Spaceꢁ method44). The significance of the differ-
ences was established by means of a one-way ANOVA
followed by a Dunnettꢁs test for multiple comparisons.
All of the procedures regarding the care and use of ani-
mals and animal experimentation reported in this paper
complied with norms set by our institutions and with the
Guide for the Care and Use of Laboratory Animals
(National Research Council, 1996).
Throughout the duration of the experiment, alcohol was
offered in the homecage. A two-bottle free-choice regi-
men between a 10% (v/v) alcohol solution and water
was offered, with unlimited access for 24 h/day. The bot-
tles were refilled every day with fresh solutions and their
relative position was interchanged in order to avoid
development of position preference. Standard chow pel-
lets were available ad libitum throughout the study.
References and notes
Rats (n = 8–10) were assigned to each experimental
group in a manner that ensured that the groups did
not differ in basal alcohol intake prior to drug treat-
ment. Physiological serum was the administration vehi-
cle. The doses chosen for natural polyamines are far
below the acute oral toxic doses cited in the reference
literature.43
1. Hughes, J. C.; Cook, C. C. H. Addiction 1997, 92, 381–
395.
2. Littleton, J. Addiction 1995, 90, 1179–1188.
3. Deitrich, R. A.; Troxell, P. A.; Worth, W. S.; Erwin, V. G.
Biochem. Pharmacol. 1976, 25, 2733–2737.
4. De Master, E. G.; Shirota, F. N.; Nagasawa, H. T. Alcohol
1985, 2, 117–121.
5. Volpicelli, J. R.; Watson, N. T.; King, A. C.; Sherman, C.
E.; OꢁBrien, C. P. Am. J. Psychiatry 1995, 152, 613–615.
6. Naranjo, C. A.; Bremmer, K. E. EXS 1994, 71, 209–219.
7. Kranzler, H. R.; Burleson, J. A.; Korner, P.; Del Boca, F.
K.; Bohn, M. J.; Brown, J.; Liebowitz, N. Am. J.
Psychiatry 1995, 152, 391–397.
The data regarding alcohol, water, and food intake were
with reference to the time zero, the start of the dark
phase of day 1 (at 19.00 h). Three periods of study were
established: (a) the basal period, B, in which the animals
were maintained under standard housing conditions,
without receiving any treatment; daily measurements
of ethanol, food, and water intake during this period
8. West, S. L.; Garbutt, J. C.; Carey, T. S.; Lux, L. J.;
Jackman, A. N.; Tolleson-Rinehart, S.; Lohr, K. N.;

4382
M. Font et al. / Bioorg. Med. Chem. 13 (2005) 4375–4382
Crews, F. T. Pharmacotherapy for Alcohol Dependence.
25. Naassila, M.; Hammoumi, S.; Legrand, E.; Durbin, P.;
Daoust, M. Alcohol. Clin. Exp. Res. 1998, 22, 802–809.
26. Rammes, G.; Mahal, B.; Putzke, J.; Parsons, C.;
Spielmanns, P.; Pestel, E.; Spanagel, R.; Zieglga¨nsber-
ger, W.; Schadrack, J. Neuropharmacology 2001, 40,
749–760.
Evidence Report/Technology Assessment Number 3,
Pharmacotherapy for Alcohol Dependence (AHCPR
Publication No. 99-E004). Rockville, MD: Agency for
Health Care Policy and Research, 1999.
9. Garbutt, J. C.; West, S. L.; Carey, T. S.; Lohr, K. N.;
Crews, F. T. JAMA 1999, 281, 1318–1325.
10. Kiefer, F.; Jahn, H.; Tarnaske, T.; Helwig, H.; Briken, P.;
Holzbach, R.; Ka¨mpf, P.; Stracke, R.; Baehr, M.; Naber,
D.; Wiedemann, K. Arch. Gen. Psychiatry 2003, 60, 92–99.
11. Swift, R. M. NEJM 1999, 340, 1482–1490.
12. Grant, K. A.; Lovinger, D. M. Clin. Neurosci. 1995, 3,
155–164.
13. Kiefer, F.; Jahn, H.; Koester, A.; Montkowski, A.;
Reinscheid, R. K.; Wiedemann, K. Biol. Psychiatry
2003, 53, 345–351.
14. Nagy, J. Curr. Drug Targets—CNS Neurol. Disorders
2004, 3, 169–179.
´
27. Bilbeny, N.; Garcıa, H. WO Patent 03026634, 2003.
28. Marton, L. J.; Pegg, A. E. Annu. Rev. Pharmacol. Toxicol.
1995, 35, 55–91.
29. Romano, C.; Williams, K.; De Priest, S.; Seshadri, R.;
Marshall, G. R.; Israel, M.; Molinoff, P. B. Mol.
Pharmacol. 1992, 41, 785–792.
30. Ehrlich, P. Dtsch. Chem. Ges. 1909, 42, 17.
31. Mardones, J.; Segovia-Riquelme, N. Neurobehav. Toxicol.
Teratol. 1983, 5, 171–178.
32. Mouse Genome Informatics (MGI) http://www.informat-
ics.jax.org/.
33. Sapag, A.; Tampier, L.; Valle-Prieto, A.; Quintanilla, M. E.;
Moncada, C.; Israel, Y. Pharmacogenetics 2003, 13, 509–
515.
15. Mayer, M. L.; Vyklicky, L.; Sernagor, E. Drug Dev. Res.
1989, 17, 263–280.
16. Williams, K.; Dawson, V. L.; Romano, C.; Dichter, M.
A.; Molinoff, P. B. Neuron 1990, 5, 99–208.
17. Araneda, R. C.; Lan, J.; Zheng, X.; Zukin, R. S.; Bennet,
M. V. L. Biophys. J. 1999, 76, 2899–2911.
18. Gibson, A. D.; Harris, B. R.; Rogers, D. T.; Littleton, J.
M. Brain Res. 2002, 952, 71–77.
34. Quintanilla, M. E.; Sepulveda, S.; Tampier, L. Alcohol
1993, 10, 381–385.
35. Tampier, L.; Quintanilla, M. E.; Mardones, J. Alcohol
Alcohol. 1994, 29, 751–755.
36. Tampier, L.; Quintanilla, M. E. J. Stud. Alcohol 2002, 63,
257–262.
19. Pegg, A. E. Cancer Res. 1988, 48, 759–774.
20. Marton, L. J.; Pegg, A. E. Annu. Rev. Pharmacol. 1995, 35,
55–91.
21. Berger, M. L.; Scho¨dl, C.; Noe, C. Eur. J. Med. Chem.
1998, 33, 3–14.
22. Lovinger, D. Alcohol. Clin. Exp. Res. (Suppl) 2000, 24,
183A.
23. Littleton, J. M.; Lovinger, D.; Liljequist, S.; Ticku, R.;
Matsumoto, I.; Barron, S. Alcohol. Clin. Exp. Res. 2001,
25, 132S–136S.
24. Littleton, J. M.; Dunnage, J.; Pagonis, C. Alcohol
Dependence and Modulation of Calcium Channels. In
Biomedical and Social Aspects of Alcohol and Alcohol-
ism; Kuriyama, K., Ed.; Elsevier: Amsterdam, 1988, pp
229–234.
37. Dietrich, R. A.; Erwin, V. G. Mol. Pharmacol. 1971, 7,
301–307.
38. Pontieri, F. E.; Zocchi, A.; Orzi, F. Funct. Neurol. 1998,
13, 311–326.
39. Williams, K. Biochem. J. 1997, 325, 289–297.
40. InsightII (r); Accelrys Inc.
41. Dewar, M. J. S.; Zoebisch, E. G.; Healy, E. F.; Stewart, J.
J. P. J. Am. Chem. Soc. 1985, 107, 3902–3909.
42. Sanz, F.; Manaut, F.; Rodriguez, J.; Lozoya, E.;
Lopez-de-Brinas, E. J. Comput. Aided Mol. Des. 1993,
7, 337.
43. Til, H. P.; Falke, H. E.; Prinsen, M. K.; Willems, M. I.
Food Chem. Toxicol. 1997, 35, 337–348.
44. Eriksson, P. C. J.; Sippel, H. W.; Forsander, O. A. Anal.
Biochem. 1977, 80, 116–124.