Conformationally restrained analogues of sympathomimetic catecholamines: Synthesis and adrenergic activity of 5,6- and 6,7-dihydroxy-3,4-dihydrospiro[naphthalen-1(2H)-2′,5′- morpholines]

Article abstract of DOI:10.1016/S0223-5234(01)01322-8

The 5,6- (10a) and 6,7-dihydroxy-3,4-dihydrospiro[naphthalen-1(2H)-2′,5′-morpholine] (11a) and their N-isopropyl derivatives (10b and 11b) (DDSNMs), which can be viewed as the result of the combination of the structure of the 2-(3,4-dihydroxyphenyl)morpholine 5a or 5b (DPMs) with the structure of the corresponding 1-(aminomethyl)-5,6-dihydroxy- (8a or 8b) or 1-(aminomethyl)-6,7-dihydroxy-1,2,3,4-tetrahydro-1-naphthalen-ol (9a or 9b) (1-AMDTNs) were synthesised. The new compounds DDSNMs 10a,b and 11a,b were assayed for their α- and β-adrenergic properties by means of binding experiments and functional tests and the results were compared with those obtained for catecholamines 1a, b and the previously described morpholine (5) and tetrahydronaphthalene (8, 9) derivatives. The affinity and activity indices thus obtained indicate in general a low ability of the new compounds 10 and 11 to interact with the α- and β-adrenoceptors, which, in all cases, was lower than that of the corresponding morpholine (5) and tetrahydronaphthalene (8, 9) analogues.

Full text of DOI:10.1016/S0223-5234(01)01322-8

Eur. J. Med. Chem. 37 (2002) 11–22
Original article
Conformationally restrained analogues of sympathomimetic
catecholamines
Synthesis and adrenergic activity of 5,6- and 6,7-dihydroxy-3,4-
dihydrospiro[naphthalen-1(2H)-2%,5%-morpholines]ꢀ
Aldo Balsamo a,*, Annalina Lapucci a, Clementina Manera a, Adriano Martinelli a,
Susanna Nencetti a, Elisabetta Orlandini a, Vincenzo Calderone b, Gino Giannaccini b,
Paola Nieri b
a Dipartimento di Scienze Farmaceutiche, Facolta´ di Farmacia, Uni6ersita´ di Pisa, Via Bonanno 6, 56100 Pisa, Italy
b Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, Facolta´ di Farmacia, Uni6ersita´ di Pisa, Via Bonanno 6,
56100 Pisa, Italy
Received 27 July 2001; received in revised form 10 October 2001; accepted 7 November 2001
Abstract
The 5,6- (10a) and 6,7-dihydroxy-3,4-dihydrospiro[naphthalen-1(2H)-2%,5%-morpholine] (11a) and their N-isopropyl derivatives
(10b and 11b) (DDSNMs), which can be viewed as the result of the combination of the structure of the 2-(3,4-dihydroxy-
phenyl)morpholine 5a or 5b (DPMs) with the structure of the corresponding 1-(aminomethyl)-5,6-dihydroxy- (8a or 8b) or
1-(aminomethyl)-6,7-dihydroxy-1,2,3,4-tetrahydro-1-naphthalen-ol (9a or 9b) (1-AMDTNs) were synthesised. The new com-
pounds DDSNMs 10a,b and 11a,b were assayed for their a- and b-adrenergic properties by means of binding experiments and
functional tests and the results were compared with those obtained for catecholamines 1a, b and the previously described
morpholine (5) and tetrahydronaphthalene (8, 9) derivatives. The affinity and activity indices thus obtained indicate in general a
low ability of the new compounds 10 and 11 to interact with the a- and b-adrenoceptors, which, in all cases, was lower than that
´
of the corresponding morpholine (5) and tetrahydronaphthalene (8, 9) analogues. © 2002 Editions scientifiques et me´dicales
Elsevier SAS. All rights reserved.
Keywords: catecholamines; a- and b-adrenoceptors; morpholine; N-isopropyl derivatives
1. Introduction
Abbre6iations: NE, norepinephrine; ISO, isoprenaline; 1-AMDICs,
1-(aminomethyl)-5,6- and 1-(aminomethyl)-6,7-dihydroxyisochro-
mane; 3-DPPs, 3-(3,4-dihydroxyphenyl)-3-piperidinols; 2-DPMs,
One of the methods most frequently used in order to
obtain information about the pharmacophoric confor-
2-(3,4-dihydroxyphenyl)morpholines;
2-ADTNs,
2-amino-5,6-
mation of conformationally mobile drugs consists of
the synthesis and the study of the pharmacological
properties of their analogues in which the presumed
pharmacophoric groups are introduced into rigid or
semirigid structures [2–14].
Natural catecholamines and the structurally related
adrenergic drugs are flexible molecules which, at the
moment of interacting with the receptor, may assume
and 2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalen-1-ol; 1-
AMDTNs, 1-(aminomethyl)-5,6- and 1-(aminomethyl)-6,7-dihydroxy-
1,2,3,4-tetrahydro-1-naphthalen-ol; DDSNMs, 5,6- and 6,7-di-
hydroxy-3,4-dihydrospiro[naphthalen-1(2H)-2%,5%-morpholine]; DHA,
dihydroalprenolol; NOE, nuclear Overhauser effect.
ꢀ 21st paper in the series Conformational effects in the acti6ity of
drugs. For preceding paper see Ref. [1].
* Correspondence and reprints.
E-mail address: balsamo@far.unipi.it (A. Balsamo).
´
0223-5234/02/$ - see front matter © 2002 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
PII: S0223-5234(01)01322-8

12
A. Balsamo et al. / European Journal of Medicinal Chemistry 37 (2002) 11–22
different spatial arrangements of the presumed active
groups (aryl moiety, amino nitrogen and alcoholic hy-
droxyl) by rotation around the single CaꢀC1 and/or
C1ꢀC2 bond [15–19]. The exploitation of the method of
the conformationally restrained analogues on this class
of drugs has led to the synthesis of numerous semirigid
and rigid compounds, some of which have been found
to maintain, towards one or more subtypes of adrener-
gic receptors, the adrenergic properties of the corre-
sponding conformationally free drugs [3,4,7,8,11–14].
Our contribution in this context has been the synthesis
and the study of some types of semirigid analogues of
norepinephrine (NE, 1a) and isoproterenol (ISO, 1b), in
which the conformational freedom of catecholamines
1a and 1b was limited around the single carbon–carbon
CaꢀC1 bond, as in 1-AMDIC derivatives 2 and 3 by
means of an isochromanic structure [12], around the
C1ꢀC2 bond, as in 3-DPP derivatives 4 by means of a
piperidinic structure [11], and in 2-DPM derivatives 5
with a morpholinic structure [11], or around both the
CaꢀC1 and C1ꢀC2 bonds, as in 2-ADTN derivatives 6
and 7, by means of a tetrahydronaphthalenic structure
[11] (Fig. 1).
tive in their low-energy conformations allowing the
spatial coincidence of the pharmacophoric groups, it
was possible to construct steric models for interaction
with the a1-, a2- and b-adrenergic receptors [11,12] (Fig.
2).
Following this study, another semirigid analogue of
the tetrahydronaphthalenic type, the 1-(aminomethyl)-
5,6-dihydroxy-1,2,3,4-tetrahydro-1-naphthalen-1-ol (1-
AMDTN, 8a) was found to be active at the level of the
a2 receptor (pD2=5.78 and Ki=46 nM) [20–23]. For
this compound, the arrangement of the pharma-
cophoric groups, with the exception of the phenyl ring,
which appears to be clearly in a different rotameric
position, corresponds to the one indicated by the a2
model [23].
5,6-Dihydroxy-3,4-dihydrospiro[naphthalen-1(2H)-2%,
5%-morpholine] (DDSNM, 10a) (Fig. 3) may be viewed
as the result of the combination of the structure of the
2-DPM 5a, used for the construction of the a2 model in
Fig. 2, with the structure of the 1-AMDTN 8a, in
which the aryl is blocked in a conformation which, even
if pharmacophoric, is different from the one indicated
by the model in Fig. 2.
This paper describes the synthesis and the adrenergic
properties of the DDSNM 10a, together with those of
its N-isopropyl derivative 10b and their respective re-
gioisomers characterised by the different position of
one of the two catecholic hydroxyls (11a and 11b).
DDSNMs 11a and 11b may be considered as conforma-
tionally restrained analogues of the previously de-
scribed 1-AMDTNs 9a and 9b, respectively.
By superimposing the stereostructures of the
molecules that proved to be biopharmacologically ac-
2. Chemistry
The DDSNMs 10a and 11a and their N-isopropyl
derivatives 10b and 11b were synthesised as outlined in
Fig. 4. The reaction of aminoalcohols 12 [20] and 13
[21] with chloroacetyl chloride yielded the chloroac-
etamides 14 and 15. Base-catalysed (KOt-Bu) cyclisa-
tion of 14 and 15 gave the spiromorpholones 16 and 17,
which were reduced with LiAlH4 to the amines 18 and
19. While catalytic hydrogenolysis of 18 and 19 with
palladium on charcoal yielded the catecholic com-
pounds 10a and 11a, respectively, alkylation of the
same compounds with isopropyl bromide in the pres-
ence of potassium hydroxide afforded the N-isopropyl
derivatives 20 and 21, which by catalytic hydrogenolysis
gave the N-isopropyl DDSNMs 10b and 11b.
3. Pharmacology
The affinity of DDSNMs 10 and 11 for a-adrenocep-
tors was determined by binding tests carried out on rat
brain membrane preparations. [3H]Prazosin and
Fig. 1. Structures of catecholamines NE and ISO and of some their
semirigids analogues.

A. Balsamo et al. / European Journal of Medicinal Chemistry 37 (2002) 11–22
13
Fig. 2. Steric models for the interaction with the a1- (A) a2- (B) and b- (C) adrenergic receptors. The models arise from the superimposition of
the pharmacophoric groups (aryl moiety, aminic nitrogen, alcoholic or ethereal oxygen) of adrenergic agonist catecholamine derivatives in the
conformations in which they should interact with the receptors. The common arylethanolaminic portion is colored in green; the other portions
are colored depending on which drug they arise from (see [12]).
[3H]rauwolscine were used as specific tritiated ligands
for a1 and a2 receptors, respectively. The affinity for
b1- and b2-adrenergic receptors of 10 and 11 was de-
termined by binding tests on rat brain for b1- and on
bovine lung membranes for b2-receptors, respectively.
[3H]CGP 26505 was used as the specific tritiated lig-
and for rat brain b1 receptors, while [3H]DHA in the
presence of 50 nM CGP 26505 which displaced
[3H]DHA binding from the b1-adrenoceptor subpopu-
lation, which represented 17% in the bovine lung, was
used to label bovine lung b2 receptors. The binding
affinity indices of DDSNMs 10 and 11 are reported
in Table 1, together with those obtained in the same
tests with NE and ISO (1) and 2-DPM (5) and 1-
AMDTN (8 and 9).
4. Results
As for a1-receptors (Table 1), the DDSNM analogue
of 8a (10a) was found to be completely devoid of any
affinity, while 8a and 2-DPM 5a showed affinity in-
dices, respectively 55- and 16-fold higher than that of
NE. Also the regioisomer of 10a (11a) proved to be
completely devoid of affinity for this a1-receptor, while
1-AMDTN 9a was practically devoid of affinity, with a
Ki value more than 150 times higher than that of NE.
Both the N-isopropyl-substituted DDSNMs 10b and
11b and 1-AMDTN 9b were completely lacking in
affinity for a1-receptors, while 1-AMDTN 8b and 2-
DPM 5b showed affinity indices about 1.5 times higher
or fivefold lower than that of ISO.
The activity of DDSNMs 10 and 11 was tested on
isolated vas deferens for a1-adrenoceptors, and on
isolated, electrically-stimulated guinea-pig ileum for
a2-adrenoceptors. The activity of DDSNMs 10 and 11
was tested on isolated guinea-pig atria and on iso-
lated guinea-pig tracheal strips for b1- and b2-adreno-
ceptors, respectively. The activity indices towards a-
and b-adrenoceptors are shown in Table 2, together
with those obtained in the same functional tests for
catecholamines 1, 2-DPM (5) and 1-AMDTN (8 and
9).
As for a2-adrenoceptors (Table 1), DDSNM 10a
showed a certain affinity, with an index about 60 or 110
times higher than that of 1-AMDTN 8a or 2-DPM 5a,
respectively. These last compounds in turn showed a
lower affinity for this receptor by about half or one
order of magnitude than NE. The regioisomer of the
DDSNM derivative 10a (11a) showed a Ki value 20- or
13-fold higher than that of 10a and of the correspond-
ing 1-AMDTN 9a, respectively.
Both the N-isopropyl-substituted DDSNM 10b and
ISO (1b) exhibited a similar affinity index for this

14
A. Balsamo et al. / European Journal of Medicinal Chemistry 37 (2002) 11–22
receptor that is about half an order of magnitude
higher than 1-AMDTN 8b and 2-DPM 5b. The re-
gioisomer of DDSNM 10b (11b) was completely devoid
of any affinity, while the corresponding 1-AMDTN 9b
showed an affinity similar to that of ISO.
At the level of b1-adrenoceptors (Table 1), both
DDSNMs 10a and 10b and their regioisomers 11a and
11b were practically lacking in affinity. On the contrary,
on the same b1-adrenoceptors, both 2-DPM 5 and
1-AMDTNs 8 and 9 proved to possess a certain
affinity, even if considerably lower than that of the
corresponding catecholamines 1a and 1b.
As regards b2-adrenoceptors (Table 1), the DDSNM
10a appeared to possess an affinity index is about six
times higher than those of the 2-DPM 5a and the
corresponding 1-AMDTN 8a. 2-DPM 5a and 1-
AMDTN 8a exhibited affinity indices similar to that of
the natural catecholamine NE (1a). The regioisomer of
DDSNM 10a (11a) was found to be lacking in affinity;
the same result was obtained with the corresponding
1-AMDTN 9a.
Neither of the N-isopropyl-substituted DDSNMs 11a
and 11b showed an appreciable affinity for b2-adreno-
ceptors; also 1-AMDTN 9b appeared to be devoid of
affinity on these adrenoceptors, whereas the 2-DPM 5b
and the 1-AMDTN 8b exhibited a certain affinity,
albeit considerably lower than that of ISO (1b).
As regards the results of the functional tests (Table
2), on a1-adrenoceptors the DDSNM 10a appeared to
be practically inactive with a pD2 value 53.5; the
2-DPM 5a and 1-AMDTN 8a, on the contrary, proved
to possess an appreciable activity, with pD2 values of
4.88 and 4.12, respectively, and an intrinsic activity
(i.a.) close to 1.00, while the natural catecholamine NE
1a exhibited a pD2 value of 5.12. Also the regioisomer
of 10a (11a) showed a pD2 value 53.5, while the
corresponding 1-AMDTN 9a was completely inactive.
On the same a1-adrenoceptors, both the N-isopropyl-
substituted DDSNMs 10b and 11b were completely
inactive, like 2-DPM 5b and 1-AMDTNs 8b and 9b,
which had proved to be practically inactive.
As for the activity on a2-adrenoceptors (Table 2),
DDSNM 10a showed a pD2 value of 5.01 and an
intrinsic activity of 1.00, while the 2-DPM (5a) and the
corresponding 1-AMDTN 8a exhibited a pD2 value of
7.34 and 5.78, and an i.a. of 1.08 and 0.73, respectively.
On the same adrenoceptor, NE 1a showed a pD2 of
6.56. The regioisomer of 10a (11a) showed a pD2 value
of 3.50, with a low i.a. value (0.60), while the corre-
sponding 1-AMDTN 9a was slightly more active, with
a pD2 value and an i.a. index of 4.31 and 1.00,
respectively.
Fig. 3. Structure of title compounds.

A. Balsamo et al. / European Journal of Medicinal Chemistry 37 (2002) 11–22
15
Fig. 4. Synthesis of title compounds.
Table 1
Radioligand binding affinities of compounds 1, 5, 8–11.
Compound
Ki (nM) a
a-adrenergic binding affinity
Rat brain (a1)
b-adrenergic binding affinity
Rat brain (b1)
Rat brain (a2)
Bovine lung (b2)
1a (NE)
5a (2-DPM)
450 (390–520) b
7400 (7000–7800) b
25 000 (23 000–27 000) c
75 000 (68 500–81 000)
\100 000
4.8 (4.5–7.1) b
126 (108–144) b
6000 (5200–6600) b
9200 (8500–9800) b
11 000 (10 000–13 000) c
\100 000
26 (17–28) b
15 000 (12 000–18 000) b
27 000 (24 000–29 000) c
54 000 (47 500–60 500)
\100 000
8a (1-AMDTN)
9a (1-AMDTN)
10a (DDSNM)
11a (DDSNM)
1b (ISO)
46 (43–48) c
4300 (3500–5000)
2900 (2200–3500)
58 000 (50 000–65 000)
21 000 (16 000–24 000) b
5100 (4300–5600) b
3000 (2700–3300) c
29 000 (21 000–36 000)
22 000 (17 000–27 000)
\100 000
53 000 (46 000–61 000)
\100 000
\100 000
\100 000
35 500 (32 000–39 500) b
6700 (6000–7500) b
52 000 (47 000–56 000) c
\100 000
80 (53–100) b
110 (100–130) b
36 000 (32 000–40 000) b
9000 (8400–9700) c
\100 000
5b (2-DPM)
48 000 (42 000–53 500) b
24 000 (22 000–26 000) c
47 000 (39 000–54 000)
\100 000
8b (1-AMDTN)
9b (1-AMDTN)
10b (DDSNM)
11b (DDSNM)
\100 000
\100 000
\100 000
\100 000
\100 000
a Geometric means of five separate determinations with confidence limits shown in parentheses.
b Ref. [11].
c Ref. [20].
The N-isopropyl-substituted DDSNM 10b exhibited
a pD2 value and an i.a. index similar to those of 2-DPM
5b (5.27 and 0.97 vs. 5.00 and 1.02), while ISO showed
a pD2 value of 4.95 and an i.a. of 0.74. The DDSNM
regioisomer of 10b (11b) was slightly less active than
DDSNM 10b (pD2 and i.a. 4.53 and 0.94, respectively).

16
A. Balsamo et al. / European Journal of Medicinal Chemistry 37 (2002) 11–22
Table 2
Adrenergic activities a of compounds 1, 5, 8–11 on isolated preparations.
Compound
a-adrenergic activity a
b-adrenergic activity a
Isolated rat vas deferens
Isolated guinea-pig ileum
Isolated guinea-pig atria
Isolated guinea-pig tracheal
(a1)
(a2)
(b1)
strip (b2)
pD2
i.a. b
pD2
i.a. b
pD2
i.a. b
pD2
i.a. b
1a (NE)
5a (2-DPM)
5.12 (90.10) c
4.88 (90.09) c
4.12 (90.05) d
–
1.00 c
0.91 c
6.56 (90.11) c
7.34 (90.12) c
1.00 c
1.08 c
0.73
1.00
1.00
6.32 (90.07) c
5.31 (90.30) c
4.04 (90.18) d
4.08 (90.08)
53.50
1.00 c
0.91 c
6.03 (90.06) c
4.76 (90.20) c
1.00 c
0.70 c
0.60 d
0.61
8a (1-AMDTN)
9a (1-AMDTN)
10a (DDSNM)
11a (DDSNM)
1b (ISO)
1.01 d 5.78 (90.10) e
0.98 d B3.50 d
–
–
–
4.31 (90.06)
5.01 (90.17)
3.50 (90.26)
4.95 (90.14) c
5.00 (90.21) c
4.16 (90.01)
–
0.73
4.08 (90.11)
53.50
4.10 (90.11)
53.50
0.48
53.50
0.60
–
3.50 (90.14) c
0.83 c
0.74 c
1.02 c
0.86
8.45 (90.12) c
1.00 c
8.33 (90.18) c
1.00 c
0.87 c
c
5b (2-DPM)
–
–
–
–
–
–
–
4.62 (90.08) c
0.77 c
5.91 (90.91) c
d
d
d
8b (1-AMDTN)
9b (1–AMDTN)
10b (DDSNM)
11b (DDSNM)
–
–
–
–
–
–
53.50
–
–
–
–
–
4.45 (90.35)
53.50
53.50
0.36
5.27 (90.11)
4.53 (90.22)
0.97
0.94
a The values represent the mean of 4–6 experiments for each drug9standard error in parentheses.
b Intrinsic activity, i.e. the ratio between the maximal response elicited by the compound under test and that elicited by the full agonist, namely
NE and ISO for a and b adrenoceptors, respectively.
c Ref. [11].
d Ref. [20].
e Ref. [23].
5. Conformational studies
On the same adrenoceptors, 1-AMDTN 8b exhibited an
activity slightly lower than that of 11b, while 9b ap-
peared to be inactive.
With the aim of determining the real conformational
situation of the DDSNM 10a and thus understanding
the spatial relationship between its active groups and
those of its conformationally more free analogues 2-
DPM 5a and 1-AMDTN 8a, conformational studies
were carried out by means of both experimental and
theoretical methods.
Regarding b1-adrenoceptors (Table 2) both
DDSNMs 10a and 11a proved to be practically devoid
of activity, while 1-AMDTNs 8a and 9a showed a
certain activity (a pD2 of about 4 and an i.a. of 0.98
and 0.73, respectively). The 2-DPM 5a exhibited a pD2
value about one order of magnitude lower than that of
NE (5.31 vs. 6.32 and an i.a. slightly lower (0.91 vs
1.00).
1
As a first approach, a H-NMR study was carried
out on the dibenzyloxy derivative of 10a (18), chosen in
view of the relatively limited stability of its free cate-
cholic nuclei in solution. As regards the conformation
of the tetrahydronaphthalene portion, the results ob-
tained for compound 18 are in agreement with those
found for type 8 and 9 analogues, for which a half-
chair preferred conformation was found ([23] and refer-
ence therein citated). As regards the conformational
situation of the spiromorpholine ring, studies on 18
were unsuccessful, due to the superimposition of the
signals of the diagnostic H6%a and H6%b (Fig. 5) protons
on the signals of the other ring protons. However, the
signals of these two protons were well separated in the
spectrum of the N-isopropyl derivative of 18 (20). In
this compound, the saturation of the proton H6%a (2.28
ppm), which lies in the pseudoaxial position, produced
not only the expected NOE (21%) on the signal of its
geminal proton H6%b (2.68 ppm), but also an appreciable
NOE (9%) on the signal at 7.45 ppm attributable to the
aromatic proton H8, thus indicating that the proton
On the same b1-adrenoceptors, both N-isopropyl-
substituted DDSNMs 10b and 11b were completely
inactive, as were the corresponding 1-AMDTNs 8b and
9b; N-isopropyl- substituted 2-DPM 5b appeared to
possess an activity much lower than that of ISO (1b),
with a pD2 and an i.a. value of 4.62 and 0.77 vs 8.45
and 1.00 of 1b. On b2-adrenoceptors (Table 2), the
DDSNM 10a, like the 2-DPM 5a, proved to possess a
modest activity (a pD2 of 4.10 vs 4.76 and an i.a. of
0.48 vs 0.70, respectively); the regioisomer of 10a (11a)
and the corresponding 1-AMDTN (9a) exhibited still
lower values both of pD2 and of i.a. Also the N-isopro-
pyl-substituted DDSNMs 10b and 11b, like the 1-
AMDTN 8b, were practically inactive on this
adrenoceptor; only the 1-AMDTN 9b showed a certain
activity, albeit characterised by a very low i.a. value.
The 2-DPM 5b exhibited pD2 and i.a. values of 5.81
and 0.87, respectively, considerably lower than those of
ISO 1b, i.e. 8.33 and 1.00, respectively.

A. Balsamo et al. / European Journal of Medicinal Chemistry 37 (2002) 11–22
17
at a temperature of 1000 K with a step of 1 fs for 50 ps
after an equilibration of 1 ps; for every ps of the
simulation, a conformation was sampled and minimised
with 10−4 kcal mol−1 A on RMS derivative as conver-
,
gence criteria. The dielectric constant was fixed to 4,
and was distance-dependent, in order to simulate an aq.
environment. All the 50 minimised conformations were
then compared and it was found that they can be
reduced to four, corresponding to the two possible
chair arrangements that both the cyclohexenic and
morpholinic ring can assume. The conformation shown
in Fig. 6, possessing the morpholinic oxygen in a pseu-
doequatorial position, is the preferred one, with energy
differences of almost 3.4 kcal mol−1, with respect to all
others. These results are thus in agreement with those
1
obtained by H-NMR because the distance between the
Fig. 5. Compound 20 in its preferred conformation.
,
H4 and H8 in this conformation is only 2.7 A.
H6%a is in the proximity of H8. On the basis of these
data, it is thus possible to assign to compound 20 the
conformation shown in Fig. 5 and consequently to
attribute the same type of conformation also to com-
pound 10b. This type of conformation is presumably
extensible also to 10a, 11a, 11b, which differ from 10b
only in the position of the phenolic hydroxyl (11b) or in
the presence of an hydrogen in the place of the N-iso-
propyl (10a and 11a).
As regards conformational analysis of 10a carried
out by theoretical methods, this was performed by
molecular mechanics and dynamics calculations using
the DISCOVER program [24] with the CVFF force field,
in which partial atomic changes are also defined. The
starting geometries of DTNSM 10a, considered in their
protonated form, were built by using the Insight pro-
gramme [24] A molecular dynamics simulation was run
6. Discussion
The DDSNM 10a presents a certain affinity for the
a2-adrenoceptor, which, however, is lower than that of
both 2-DPM 5a and 1-AMDTN 8a; the N-isopropyl
derivative of 10a (10b) showed a level of affinity of
about one order of magnitude lower than that of both
10a and of 2-DPM and the 1-AMDTN analogues 5b
and 8b. The regioisomeric DDSNMs of 10a and 10b
(11a and 11b) are practically devoid of any affinity for
the a2-adrenoceptor; the same result was also found for
the 1-AMDTN analogue 9b while the 1-AMDTN ana-
logue 9a exhibited a modest affinity.
Although, the low potency values recorded by func-
tional tests for several compounds can reflect a poor
selectivity towards receptor subtypes and suggest a
Fig. 6. Compound 10a in its preferred conformation.

18
A. Balsamo et al. / European Journal of Medicinal Chemistry 37 (2002) 11–22
1-AMDTN 8a. Fig. 7 shows a superimposition of the
structure of 10a, arranged in the low-energy conforma-
1
tion resulting from the combination of both H-NMR
studies and theoretical calculations, on those of 5a and
8a, arranged in the low-energy conformations found in
previous studies.
The good coincidence of the common molecular por-
tions of these compounds allows us to exclude the
possibility that the differences in the a2-adrenergic
properties between 10a and 5a, and 8a may be due to
differences in the spatial arrangement of the active
groups. On the contrary, more probable explanations
may be sought in the greater rigidity of the DDSNM
system of 10a with respect to the 2-DPM or 1-AMDTN
of 5a and 8a, respectively, and/or in the steric hin-
drance due to the additional atoms that are necessary
to reduce the conformational freedom of the same
2-DPM 5a and 1-AMDTN 8a, which may partially
hinder a good fit with the a2-adrenoceptor.
Fig. 7. Superimposition of the compound 10a (grey) in its preferred
conformation with compound 5a (cyan) and compound 8a (magenta)
in low energy conformations; nitrogen and oxygen atoms are blue
and red, respectively, the hydrogen atoms are not reported.
cautious interpretation of these experimental data,
however it should be observed that the trend of the
activity indices corresponded to that of the affinity
indices. In particular, the DDSNM 10a proved to pos-
sess a moderate agonistic activity towards the a2-
adrenoceptor, which was in all cases clearly lower than
that of the 2-DPM 5a and the 1-AMDTN analogue 8a;
the N-isopropyl-substituted DDSNM 10b exhibited a
similar activity to that of 10a, whereas the DDSNM
regioisomers of 10a and 10b (11a and 11b) appeared to
be almost completely inactive.
At the level of the other adrenoceptors studied (a1, b1
and b2), the DDSNM 10a, like its N-isopropyl deriva-
tive 10b and their regioisomers 11a and 11b, proved to
be almost completely devoid of any affinity or activity,
in line with the fact that on these receptors, the 1-
AMDTNs 8a and 8b, and to an even greater extent
their regioisomers 9a and 9b, had not revealed any
adrenergic properties.
8. Experimental
8.1. Chemistry
Melting points were determined on a Kofler hot stage
apparatus and are uncorrected. IR spectra for compari-
son between compounds were recorded with a Perkin–
Elmer mod. 1310, as nujol mulls in the case of solid
substances, or as liquid film in the case of liquids.
1H-NMR spectra were routinely recorded with a CFT
20 spectrometer operating at 80 MHz in ca. 5% solu-
tion of CDCl3 (for neutral compounds or the free
bases) or D2O (for the salts), using Me4Si or
Me3Si(CH2)3SO3Na as the internal standard, respec-
1
tively. The H-NMR spectra for compounds 10a,b and
11a,b (as salts) were detected in ca. 2% D2O with a
Bruker AC-200 instrument. The 1H-NMR for com-
pounds 18 and 20 were recorded using a Varian VXR-
300 spectrometer in ca. 0.08 M CDCl3 solution, and the
temperature was controlled to 90.1 °C. All the solu-
tions were accurately degassed by freeze-pump-thaw
cycles. Evaporations were made in vacuo (rotating
evaporator). Magnesium sulphate was always used as
the drying agent. Elemental analysis were performed by
our analytical laboratory and agreed with theoretical
values to within 90.4%.
7. Conclusions
This study, and in particular the synthesis of
DDSNM 10a, was undertaken with the aim of verifying
the effects on the a2-adrenergic properties, of the com-
bination in a single molecule of the structure of two
semirigid analogues of NE, both possessing appreciable
properties on this receptor, like 2-DPM 5a, and 1-
AMDTN 8a. The results shown in Tables 1 and 2
indicate that, while partly maintaining the affinity and
the activity with respect to 2-DPM 5a, and the corre-
sponding 1-AMDTN 8a, the new DDSNM analogue of
NE (10a) appears to possess a lower ability to interact
with this receptor at the molecular level.
A possible explanation for these results might have
lain in differences in the spatial arrangement of the
pharmacophoric groups of DDSNM 10a, compared
with the positions that the same groups occupy in the
conformationally less rigid analogues 2-DPM 5a and
8.1.1. 1-Hydroxy-1%-(chloroacetaminomethyl)-5,6-
dibenzyloxy-1,2,3,4-tetrahydronaphthalene (14)
A solution of NaOH (0.74 g, 18.5 mmol) in H2O (4
mL) was added to a solution of aminoalcohol 12 [20]
(1.20 g, 3.08 mmol) in CH2Cl2 (33 mL) Chloro-
acetylchloride (0.695 g, 6.16 mmol) was then added
dropwise and the mixture was stirred for 2 h at room
temperature (r.t.). The mixture was then poured into

A. Balsamo et al. / European Journal of Medicinal Chemistry 37 (2002) 11–22
19
water (18 mL). The separated aq. layer was extracted
with CH2Cl2 and the combined organic extracts were
washed with water, dried and evapored to yield a solid
that was crystallised from EtOAc to afford 14 (0.95 g,
66%): m.p. 118–119 °C; IR w 1660 cm−1(CONH);
1H-NMR l 1.50–2.06 (m, 4H), 2.36–2.51 (br, 1H),
2.56–2.90 (m, 2H), 3.40–3.76 (m, 2H), 4.05 (s, 2H),
5.01 (s, 2H), 5.13 (s, 2H), 6.83–7.53 (m, 12H).
C27H28ClNO4 (C, H, N).
filtered and the solid washed with ether. The organic
solvents were dried and evaporated to give an oil
1
consisting of 18 (0.56 g, 96%): H-NMR l 1.56–1.88
(m, 4H), 2.13–3.12 (m, 6H), 3.48–4.12 (m, 2H), 5.00 (s,
2H), 5.16 (s, 2H), 6.94 (d, 1H, J=8.80 Hz), 7.12–7.60
(m, 11H).
The resulting oil 18 was dissolved in a mixture of
Et2O–MeOH (8:2) and added of a solution of H2C2O4
in the same mixture. The resulting oxalate salt was
filtered and recrystallised from MeOH–Et2O to give
18·H2C2O4; m.p. 153–155 °C. C29H31NO7 (C, H, N).
8.1.2. 1-Hydroxy-1-(chloroacetaminomethyl)-6,7-
dibenzyloxy-1,2,3,4-tetrahydronaphthalene (15)
This compound was prepared from 13 [21] (1.10 g,
2.82 mmol) following the same procedure described
above for the preparation of compound 14. The crude
product was an oil consisting essentially of 15 (1.10 g,
84%) which was used for the following reaction without
further purification; IR w 1660 cm−1 (CONH); 1H-
NMR l 1.56–2.46 (m, 5H), 2.53–2.85 (m, 2H), 3.33–
3.70 (m, 2H), 4.05 (s, 2H), 5.15 (s, 4H), 6.70 (s, 1H),
7.16 (s, 1H), 7.43 (br, 10H). C27H28ClNO4 (C, H, N).
8.1.6. (6,7-Dibenzyloxy)-3,4-dihydrospiro[naphthalene-
1(2H)-2%,5%-morpholine] oxalate (19·H2C2O4)
Reduction of the morpholone 17 (0.66 g, 1.53 mmol)
with LiAlH4 as described above for 18 gave the desired
morpholine 19 as an oil (0.58 g, 90%): 1H-NMR l
1.50–2.16 (m, 4H), 2.56–3.16 (m, 6H), 3.50–4.23 (m,
2H), 5.25 (s, 2H), 5.31 (s, 2H), 6.80 (s, 1H), 7.43 (s,
1H), 7.50–7.83 (m, 10H).
Compound 19 was converted to the oxalate salt and
after recrystallisation from MeOH–Et2O yielded
19·H2C2O4; m.p. 186–188 °C. C29H31NO7 (C, H, N).
8.1.3. (5,6-Dibenzyloxy)-3,4-dihydrospiro-
[naphthalene-1(2H)-2%,5%-morpholin-4%-one] (16)
KOt-Bu (1.20 g, 10.70 mmol) was added portionwise
over 1 h to a stirred solution of the chloroacetamide 14
(0.95 g, 2.04 mmol) in anhydrous C6H6 (46 mL). The
reaction mixture was stirred at r.t. for 1.5 h, and then
diluted with water. The separated organic layer was
washed with H2O, dried and evaporated to give a solid
that was crystallised from EtOAc to yield 16 (0.50 g,
57%): m.p. 192–194 °C; IR w 1670 cm−1 (CONH);
1H-NMR l 1.63–2.96 (m, 6H), 3.33–3.73 (m, 2H), 4.26
(s, 2H), 5.02 (s, 2H), 5.13 (s, 2H), 6.76–7.56 (m, 12H).
C27H27NO4 (C, H, N).
8.1.7. (5,6-Dihydroxy)-3,4-dihydrospiro[naphthalene-
1(2H)-2%,5%-morpholine] oxalate (10a·H2C2O4)
A solution of 18·H2C2O4 (0.20 g, 0.40 mmol) in
MeOH (20 mL) was hydrogenated at r.t. and atmo-
spheric pressure in the presence of 10% Pd on charcoal
(0.07 g). When the absorption stopped the catalyst was
removed by filtration and the resulting solution was
concentrated and anhydrous Et2O was added. The re-
sulting precipitate was filtered and then crystallised
from MeOH–Et2O to give 10a·H2C2O4 (0.08 g, 62%):
m.p. 180–183 °C (dec.); 1H-NMR l 1.58–2.08 (m,
4H), 2.25–3.40 (m, 6H), 3.90–4.37 (m, 2H), 6.86 (d,
1H, J=8.77 Hz), 7.10 (d, 1H, J=8.77 Hz). C15H19NO7
(C, H, N).
8.1.4. (6,7-Dibenzyloxy)-3,4-dihydrospiro-
[naphthalene-1(2H)-2%,5%-morpholin-4%-one] (17)
The chloroacetamide 15 (0.90 g, 1.93 mmol) was
converted to the spiromorpholone 17 as described
above for preparation of compound 16. The crystallisa-
tion of the crude solid from AcOEt gave 17 (0.66 g,
80%): m.p. 171–173 °C; IR w 1670 cm−1 (CONH);
1H-NMR l 1.50–2.83 (m, 6H), 3.20–3.60 (m, 2H), 4.31
(s, 2H), 5.18 (s, 4H), 6.63 (s, 1H), 7.16 (s, 1H), 7.36 (br,
10H). C27H27NO4 (C, H, N).
8.1.8. (6,7-Dihydroxy)-3,4-dihydrospiro[naphthalene-
1(2H)-2%,5%-morpholine] oxalate (11a·H2C2O4)
This compound was prepared from 19·H2C2O4 (0.25
g, 0.60 mmol) dissolved in a mixture of MeOH (10 mL)
and CH2Cl2 (3 mL) following the same procedure de-
scribed for compound 10a·H2C2O4. The crude product
was purified by crystallisation from MeOH–Et2O to
yield 11a·H2C2O4 (0.20 g, 66%): m.p. 164–167 °C.
1H-NMR l 1.54–2.09 (m, 4H), 2.30–3.51 (m, 6H),
3.85–4.27 (m, 2H), 6.69 (s, 1H), 7.10 (s, 1H).
C15H19NO7 (C, H, N).
8.1.5. (5,6-Dibenzyloxy)-3,4-dihydrospiro[naphthalene-
1(2H)-2%,5%-morpholine] oxalate (18·H2C2O4)
A solution of the morpholone 16 (0.60 g, 1.40 mmol)
in anhydrous THF (15 mL) was added dropwise to a
stirred suspension of LiAlH4 (0.21 g, 5.54 mmol) in
anhydrous THF (9 mL). The reaction mixture was
heated at reflux for 3 h and then cooled to 10 °C. The
excess of LiAlH4 was destroyed by careful addition of
H2O, 10% NaOH solution, H2O. The mixture was
8.1.9. (5,6-Dibenzyloxy)-3,4-dihydro-5%-N-isopropyl-
spiro[naphthalene-1(2H)-2%,5%-morpholine] oxalate
(20·H2C2O4)
A suspension of 18 (0.35 g, 0.84 mmol), 2-bromo-
propane (0.35 mL, 3.7 mol) and KOH (0.09 g, 6.2

20
A. Balsamo et al. / European Journal of Medicinal Chemistry 37 (2002) 11–22
mmol) in absolute EtOH (2 mL) was stirred at 60 °C
for 4 days. Then the reaction mixture was supple-
mented with Et2O, filtered and evaporated to dryness to
rat cerebral cortex membranes as elsewhere reported
[25].
1
give 20 as an oil (0.32 g, 83%): H-NMR l 1.00 and
8.2.2. Rat brain i1 receptors
1.03 (2d, 6H, J=6.40 Hz), 1.13–1.76 (m, 4H), 2.16–
2.93 (m, 7H), 3.68–4.12 (m, 2H), 4.99 (s, 2H), 5.13 (s,
2H), 6.91 (d, 1H, J=8.80 Hz), 7.03–7.60 (m, 11H).
Compound 20 was converted to the corresponding
oxalate salt of the amine which was recrystallised from
MeOH–Et2O to give 20·H2C2O4: m.p. 173–175 °C.
C32H37NO7 (C, H, N).
b1 receptors were assayed in rat cortical membranes
following the procedure previously described [11].
8.2.3. Bo6ine lung i2 receptors
b2 receptor binding was studied in bovine lung using
[3H]DHA (dihydroalprenolol) as the ligand (DuPont de
Nemours, New England Nuclear Division, specific ac-
tivity=48.1 Ci mmol−1).
8.1.10. (6,7-Dibenzyloxy)-3,4-dihydro-5%-N-isopropyl-
spiro[naphthalene-1(2H)-2%,5%-morpholine] oxalate
(21·H2C2O4)
Membranes were obtained by lung homogenisation
in 1:20 volumes of 0.32 M sucrose, followed by cen-
trifugation at 800×g for 10 min at 5 °C. The resulting
pellet was suspended in 50 mM phosphate buffer at pH
7.4 containing 0.02% ascorbic acid and then cen-
trifuged. This step was repeated twice. Crude lung
membranes were suspended in ($4 mg mL−1 proteins)
and incubated with 1 nM [3H]DHA in the presence of
50 nM CGP 26505. After incubation at 25 °C for 30
min, the samples were filtered on Whatman GF/B
glass-fiber filters and washed with 3×5 mL of phos-
phate buffer, dried and added to 8 mL of Ready
Protein Beckman scintillation cocktail. Non-specific
binding was measured in the presence of 35 mM l-
isoprenaline.
The affinity of drugs for specific binding sites was
expressed as the molar concentration inhibiting the
specific binding by 50% (IC50). These values were calcu-
lated from the displacement curves by log probit analy-
sis. The dissociation constant (Ki) was derived from the
equation of Cheng Prusoff [26]. The ligand affinity (Kd)
of [3H]DHA was 1 nM.
The reaction was carried out on compound 19 (0.65
g, 1.56 mmol) as described for compound 20 to obtain
the desired N-isopropyl derivative 21 as an oil (0.68 g,
1
95%) H-NMR l 0.96 and 0.99 (2d, 6H, J=6.45 Hz),
1.55–1.90 (m, 4H), 2.33–3.01 (m, 7H), 3.40–4.02 (m,
2H), 5.09 (s, 2H), 6.15 (s, 2H), 6.61 (s, 1H), 7.13 (s,
1H), 7.18–7.48 (m, 10H).
Conversion to the oxalate salt and recrystallisation
from MeOH–Et2O gave 21·H2C2O4; m.p. 164–168 °C.
C32H37NO7 (C, H, N).
8.1.11. (5,6-Dihydroxy)-3,4-dihydro-5%-N-isopropyl-
spiro[naphthalene-1(2H)-2%,5%-morpholine] oxalate
(10b·H2C2O4)
A solution of 20·H2C2O4 (0.30 g, 0.55 mmol) in
MeOH (28 mL) was hydrogenated at r.t. and atmo-
spheric pressure in the presence of 10% Pd on charcoal
(0.10 g). When the absorption stopped the catalyst was
removed by filtration and the resulting solution was
evaporated to dryness. The resulting solid was then
crystallised from MeOH–Et2O to give 10b·H2C2O4
1
(0.15 g, 60%): m.p. 185–188 °C. H-NMR l 1.35 and
8.3. Functional test methods
1.38 (2d, 6H, J=6.40 Hz), 1.52–2.10 (m, 4H), 2.38–
3.58 (m, 7H), 3.95–4.28 (m, 2H), 6.87 (d, 1H, J=8.40
Hz), 7.06 (d, 1H, J=8.40 Hz). C18H25NO7 (C, H, N).
The assays were conducted on isolated mammalian
preparations, in accordance with the legislation of the
Italian Authorities (D.L. 27/01/92, no. 116) concerning
animal experimentation. The animals, under light Et2O
anesthesia, were killed by cervical dislocation and bled.
The thoracic and subsequently the abdominal cavities
were opened by midline incision. The organs were
immediately explanted and placed in Tyrode solution
[composition (mM): NaCl (136.8), KCl (2.95), CaCl2
(1.80), MgSO4·7H2O (1.05), NaH2PO4 (0.41), NaHCO3
(11.9), Glucose (5.5)] at r.t. and gassed with carbogen
(95% O2–5% CO2), or with O2.
Adrenoceptor activity was assayed for a1-receptors
on rat 6as deferens, for a2-receptors on guinea-pig
ileum, for b1-receptors on guinea-pig atria and for b2-re-
ceptors on guinea-pig trachea. When not otherwise
described, agonist and antagonist activity was tested on
resting tone of the organ.
8.1.12. (6,7-Dihydroxy)-3,4-dihydro-5%-N-isopropyl-
spiro[naphthalene-1(2H)-2%,5%-morpholine] oxalate
(11b·H2C2O4)
The reaction was performed on 21·H2C2O4 (0.30 g,
0.55 mmol) as described for 10b·H2C2O4. The amine
salt was then recrystallised from MeOH–Et2O to give
1
11b·H2C2O4 (0.16 g, 80%): m.p. 203–206 °C. H-NMR
l 1.34 and 1.38 (2d, 6H, J=6.45 Hz), 1.58–2.06 (m,
4H), 2.50–3.60 (m, 7H), 3.95–4.38 (m, 2H), 7.08 (s,
1H), 6.69 (s, 1H). C18H25NO7 (C, H, N).
8.2. Radioligand binding methods
8.2.1. Rat brain h1 and h2 receptors
a1 and a2 receptor binding was determined in

A. Balsamo et al. / European Journal of Medicinal Chemistry 37 (2002) 11–22
21
8.3.1. Rat 6as deferens
8.3.4. Guinea-pig trachea
Vas deferens was taken from Sprague–Dawley male
albino rats (200–250 g body weight). Both vasa defer-
entia were removed without stretching from the epi-
didymis to the prostatic urethra, after moving the
intestine to one side. The intact duct was carefully
separated from extraneous surrounding tissues and
placed in a 10 mL organ bath containing Tyrode solu-
tion (pH 7.4) at 37 °C, bubbled with carbogen. The
preparation was suspended longitudinally between the
organ holder and a force displacement transducer
(Basile Model 7006), loaded with 0.5 g, connected to a
unirecord microdynamometer (Basile Model 7050). The
organ was left to stabilise for 30 min before beginning
the experiment.
Tracheae were obtained from the same animals de-
scribed above. The trachea was removed, freed from
extraneous tissue, and after locating the smooth muscle
tissue, the cartilage was cut on at the opposite side,
obtaining a rectangular piece of tissue with the muscu-
lar layer in the middle. The trachea was then cut
transversally at equally spaced intervals up to 1 mm
from the left and the right border, resulting in zig–zag
strips. The strip, tied by threads at the opposite ends,
was placed in a 10 mL organ bath as described for
previous preparations, filled with Krebs solution (com-
position (mM): NaCl (118), KCl (4.75), CaCl2 (2.50),
MgSO4·7H2O (1.19), KH2PO4 (1.19), NaHCO3 (25),
Glucose (11.5)) at 37 °C, and gassed with carbogen.
The organs, held at a tension of 0.5 g, were tied to an
isotonic transducer (Basile Model 7006) and the last in
turn to a unirecord microdynamometer (Basile Model
7050) to record the response of the smooth muscula-
ture. After a 1 h stabilizing time interval, the organ was
contracted with carbacol (5.5×10−6 M) to obtain a
marked muscular tone. The agonistic action of the
compounds under test was assessed as the ability to
inhibit the constant level of tracheal smooth muscle
tone induced by carbachol.
The concentration–response curves were obtained
using the method of cumulative concentrations for all
organs except the atria. Agonist activity was expressed
in terms of pD2 values (−log ED50 i.e. the negative
logarithm of a drug molar concentration producing
50% of the maximal response) and intrinsic activity (the
ratio between the maximal response of a test compound
and that of the reference agonist, which was NE for a
receptors and ISO for b receptors). Antagonist activity
was evaluated as the ability of the compounds under
test to reduce the response to a submaximal concentra-
tion of ISO after an incubation period of 30 min and
expressed as −log IC50, i.e. the negative logarithm of
the concentration that reduced the agonist response by
50%.
8.3.2. Guinea-pig ileum
Dunkin–Hartley male guinea pigs weighing 250–300
g, were deprived of food intake for 24 h before the
experiments. Portions of ileum 2–3 cm in length, about
10 cm distal to the ileocecal valve, were carefully dis-
sected, freed from the surrounding mesenteric tissue,
attached with thread to the organ holder and to the
recording system by opposite sides of their open ends,
and suspended in a 10 mL organ bath containing
Tyrode solution at 37 °C gassed with carbogen. The
ileum preparations were placed between two platinum
electrodes (4×45 mm) set at a distance of 7 mm in the
bath. The tissues were preloaded with a tension of 0.5 g
and left to stabilise for 45–60 min before beginning
electrical stimulation, which was carried out with a digit
stimulator (Biomedica Mangoni Model BM-ST3) using
the following parameters: single rectangular pulses, 0.1
Hz frequency, 0.3 ms pulse width, 12 V supramaximal
voltage. The activity of the tested drugs on a2-adreno-
ceptors was evaluated as their ability to inhibit acetyl-
choline release evoked by electrical stimulation of nerve
fibers. The effects of the released mediator on intestinal
smooth muscle were recorded as longitudinal contrac-
tions by an isotonic transducer (Basile Model 7006)
connected to a unirecord microdynamometer (Basile
Model 7050).
The following drugs were used as salts: 1a (l-NE) as
a bitartrate, 1b (l-ISO) as a hydrochloride, compounds
10a,b and 11a,b as oxalates.
8.3.3. Guinea-pig atria
The atria, like the tracheae, were obtained from the
same animals employed in the previous preparation.
After sacrifice, the heart was removed, both the atria
were separated from the ventriculi and a strip was
obtained. The ends of the specimen were tied and
suspended in an organ bath containing Tyrode solu-
tion, at 32 °C aerated with pure O2. The upper part of
the organ was attached to an isometric transducer
(Basile Model 7003) connected to a unirecord micrody-
namometer (Basile Model 7050). The organ was left to
stabilise for 30 min before beginning the experiment.
Acknowledgements
This work was supported in part by a grant from the
Consiglio Nazionale delle Richerche and the Ministero
dell’Universita` e della Ricerca Scientifica e Tecnologica.
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