6-thienyl and 6-phenylimidazo[2,1-b]thiazoles as inhibitors of mitochondrial NADH dehydrogenase

Article abstract of DOI:10.1016/S0223-5234(99)00203-2

Starting from the potent inhibitory effect of the previously described 2-methyl-6-(2-thienyl)imidazo[2,1-b]thiazole on mitochondrial complex I, we prepared a series of derivatives in order to study the effect of a different substitution at the positions 2, 5 and 6. The replacement of the thienyl group at position 6 with a phenyl group does not modify the biological behaviour of the lead compound, whereas the shift of the methyl group from position 2 to position 5 yields a compound devoid of inhibitory effects. In both the 6-thienyl and 6-phenyl series, the lengthening of the chain at position 2 has provided useful information to outline the structural determinants of the ubiquinone antagonist action in imidazothiazole derivatives.

Full text of DOI:10.1016/S0223-5234(99)00203-2

Eur. J. Med. Chem. 34 (1999) 883−889
© 1999 Éditions scientifiques et médicales Elsevier SAS. All rights reserved
883
Laboratory note
6-Thienyl and 6-phenylimidazo[2,1-b]thiazoles as inhibitors of mitochondrial
NADH dehydrogenase§
Aldo Andreania*, Mirella Rambaldia, Alberto Leonia, Rita Morigia, Alessandra Locatellia,
Gianluca Giorgib, Giorgio Lenazc, Anna Ghellid, Mauro Degli Espostie
aDipartimento di Scienze Farmaceutiche, Via Belmeloro 6, 40126 Bologna, Italy
bCentro Interdipartimentale di Analisi e Determinazioni Strutturali, Via A. Moro, 53100 Siena, Italy
cDipartimento di Biochimica “G. Moruzzi”, Via Irnerio 48, 40126 Bologna, Italy
dDipartimento di Biologia E.S., Via Irnerio 42, 40126 Bologna, Italy
eDepartment of Biochemistry and Molecular Biology, Monash University, Clayton, VIC, Australia
(Received 16 November 1998; accepted 10 February 1999)
Abstract – Starting from the potent inhibitory effect of the previously described 2-methyl-6-(2-thienyl)imidazo[2,1-b]thiazole on mitochon-
drial complex I, we prepared a series of derivatives in order to study the effect of a different substitution at the positions 2, 5 and 6. The
replacement of the thienyl group at position 6 with a phenyl group does not modify the biological behaviour of the lead compound, whereas
the shift of the methyl group from position 2 to position 5 yields a compound devoid of inhibitory effects. In both the 6-thienyl and 6-phenyl
series, the lengthening of the chain at position 2 has provided useful information to outline the structural determinants of the ubiquinone
antagonist action in imidazothiazole derivatives. © 1999 Éditions scientifiques et médicales Elsevier SAS
imidazo[2,1-b]thiazole / mitochondrial complex I / ubiquinone / rotenone
1. Introduction
different antagonistic actions are unclear [1, 8, 9], the
study of a series of derivatives in which the chemical
structures have been systematically modified is useful to
elucidate how complex I inhibitors interact with the
enzyme [10].
NADH-ubiquinone reductase, commonly known as
complex I, is the most complicated and least understood
of the respiratory complexes of mitochondria and bacte-
ria [1–3].
Recently, we have undertaken a systematic work to
study the structure-activity relationships in complex I
inhibitors bearing an indole [11, 12] and imidazothiazole
moiety [13]. 2-Methyl-6-(2-thienyl)imidazo[2,1-b]thia-
zole 1 (figure 1) was the most potent compound and was
found to have a mode of action overlapping that of the
classical inhibitor rotenone as well as that of the product-
like inhibitor myxothiazol [8, 13]. Using 1 as the lead
compound, we have evaluated first the effect of the
substitution of the thienyl group in position 6 with a
phenyl group (2) and the shift of the methyl group from
position 2 to 5 (3). The results indicated that the thienyl
group may be replaced by a phenyl group without loss of
potency, whereas the position of the methyl group is
critical (compound 3 is inactive). We next planned the
synthesis of 6-thienyl and 6-phenyl derivatives with
The interest in this intricate enzyme complex is in-
creasing due to its possible involvement in the pathogen-
esis of human neurodegenerative diseases such as Alzhe-
imer’s [4], Parkinson’s [5], diabetes [6] and the fact that
complex I is becoming a preferred target of commercial
pesticides, especially acaricides [7]. There is a plethora of
complex I inhibitors that act as antagonists of the hydro-
phobic substrate ubiquinone, and generally differ in their
action depending upon which quinone intermediate they
preferentially antagonize (quinone, semiquinone or
quinol) [8]. Because the chemical determinants of the
§Presented in part at the First Italian-Swiss Meeting on Medi-
cinal Chemistry (Torino, Italy, September 23–26, 1997).
*Correspondence and reprints

884
Figure 1. Synthesis of 6-thienyl and 6-phenylimidazo[2,1-b]thiazoles.
various substituents at position 2, to identify whether this
position in the imidazothiazole ring corresponded to the
attachment point for mimics of the polyisoprenoid side
chain of ubiquinone, whose length is known to be critical
for Complex I activity [14, 15].
2. Chemistry
The 5-alkyl-2-aminothiazoles 6 (figure 1) were pre-
pared from the appropriate aldehydes 4 which were
brominated at the α-position (5) and treated with thio-
urea. When compounds 6 (R = ethyl, propyl) were treated
with the bromoacetylthiophens 7–8, white solids were
isolated, corresponding to the intermediate salts 10–11
which were characterized only on the basis of their IR
spectrum (νC=O ≈ 1 670 cm–1). These compounds were
refluxed with dilute hydrochloric acid in order to obtain
the imidazothiazoles 13–17, whose spectroscopic data
(table I) are in agreement with the assigned structures.
Under the same experimental conditions, starting from 6
(R = butyl) and 3-(bromoacetyl)thiophene 8, the resulting
precipitate had a structure different from 10–11 since the
If this were the case, 2-substituents with increasing
hydrophobicity (13–17 and 19–24) were expected to have
an enhanced inhibitory potency, proportional to their
hydrophobicity as previously reported for 2-substituted
acridones [16]. However, the structure-activity results
indicated that, in imidazothiazoles, position 2 is sterically
critical for complex I inhibition, but is unlikely to be the
junction for mimics of the ubiquinone side chain. During
the synthesis, some unexpected compounds were isolated
(18 and 25–27) and two of them were tested (26 and 27).

885
Table I. Imidazothiazoles 13–27.
νmax, cm-1
δ (ppm); J (Hz) in DMSO-d6
a
Compound Formula(mw) M.p., °C
b
13
14
C11H10N2S2
(234.3)
C12H12N2S2
111–113
105–106
1 265, 850, 725, 685
1.25 (3H, t, CH3, J = 7.5) 2.77 (2H, q, CH2, J = 7.5) 7.06 (1H, m, T)
7.35 (1H, m, T) 7.41 (1H, m, T) 7.70 (1H, s, th) 8.01 (1H, s, im)
0.95 (3H, t, CH3, J = 7) 1.64 (2H, sex, CH2, J = 7) 2.73 (2H, t, CH2,
J = 7) 7.07 (1H, m, T) 7.36 (1H, m, T) 7.41 (1H, m, T) 7.71 (1H, s, th)
8.02 (1H, s, im)
c
1 270, 850, 720, 680
(248.4)
15
16
17
C12H12N2S2
(248.4)
C11H10N2S2
(234.3)
C12H12N2S2
(248.4)
108–112
103–105
126–130
1 660, 1 620, 1 560, 1 060 1.29 (6H, d, CH3, J = 7) 3.12 (1H, sep, CH, J = 7) 7.07 (1H, m, T) 7.36
(1H, m, T) 7.41 (1H, m, T) 7.71 (1H, s, th) 8.00 (1H, s, im)
1 270, 1 070, 780, 715
1 270, 1 260, 780, 725
1.25 (3H, t, CH3, J = 7.5) 2.76 (2H, q, CH2, J = 7.5) 7.46 (1H, m, T)
7.55 (1H, m, T) 7.69 (1H, m, T) 7.71 (1H, s, th) 7.98 (1H, s, im)
0.95 (3H, t, CH3, J = 7.5) 1.63 (2H, sex, CH2, J = 7.5) 2.72 (2H, t,
CH2, J = 7.5) 7.46 (1H, m, T) 7.56 (1H, m, T) 7.69 (1H, m, T) 7.71
(1H, s, th) 7.98 (1H, s, im)
18
19
20
21
22
23
24
25
26
27
C13H16N2O2S2 176–180
(296.4)
3 500–2 200, 1 620, 1 590, 0.85 (3H, t, CH3, J = 7) 1.32 (4H, m, CH2) 1.76 (2H, m, CH2) 3.94
1 070, 780
(1H, t, CH, J = 7) 7.44 (1H, m, T) 7.56 (1H, s, im) 7.58 (1H, m, T) 7.67
(1H, m, T)
d
C13H12N2S
(228.3)
136–140
142–145
136–140
128–130
130–133
109–111
1 600, 1 195, 1 065, 720 1.26 (3H, t, CH3, J = 7.5) 2.78 (2H, q, CH2, J = 7.5) 7.24 (1H, t, ar,
J = 8) 7.38 (2H, t, ar, J = 8) 7.72 (1H, s, th) 7.81 (2H, d, ar, J = 8) 8.13
(1H, s, im)
1 600, 1 260, 1 065, 720 0.94 (3H, t, CH3, J = 7.5) 1.63 (2H, sex, CH2, J = 7.5) 2.72 (2H, t,
CH2, J = 7.5) 7.23 (1H, t, ar, J = 8) 7.37 (2H, t, ar, J = 8) 7.71 (1H,
s, th) 7.80 (2H, d, ar, J = 8) 8.12 (1H, s, im)
C14H14N2S
(242.3)
C14H14N2S
(242.3)
1 595, 1 180, 770, 710
1.29 (6H, d, CH3, J = 7) 3.12 (1H, sep, CH, J = 7) 7.24 (1H, t, ar, J
= 8) 7.38 (2H, t, ar, J = 8) 7.71 (1H, s, th) 7.81 (2H, d, ar, J = 8) 8.11
(1H, s, im)
C15H16N2S
(256.4)
1 595, 1 535, 1 260, 720 0.89 (3H, t, CH3, J = 7.5) 1.34 (2H, sex, CH2, J = 7.5) 1.58 (2H, qui,
CH2, J = 7.5) 2.73 (2H, t, CH2, J = 7.5) 7.22 (1H, t, ar, J = 8) 7.36 (2H,
t, ar, J = 8) 7.71 (1H, s, th) 7.79 (2H, d, ar, J = 8) 8.12 (1H, s, im)
1 590, 1 250, 1 055, 710 0.88 (3H, t, CH3, J = 7) 1.32 (4H, m, CH2) 1.62 (2H, qui, CH2, J = 7)
2.75 (2H, t, CH2, J = 7) 7.24 (1H, t, ar, J = 8) 7.38 (2H, t, ar, J = 8)
7.73 (1H, s, th) 7.81 (2H, d, ar, J = 8) 8.13 (1H, s, im)
C16H18N2S
(270.4)
C17H20N2S
(284.4)
1 590, 1 255, 760, 715
0.86 (3H, t, CH3, J = 7) 1.29 (6H, m, CH2) 1.60 (2H, qui, CH2, J = 7)
2.74 (2H, t, CH2, J = 7) 7.23 (1H, t, ar, J = 8) 7.38 (2H, t, ar, J = 8)
7.71 (1H, s, th) 7.80 (2H, d, ar, J = 8) 8.13 (1H, s, im)
C15H18N2O2S 176–179
(290.4)
3 500–2 200, 1 615, 1 595, 0.85 (3H, t, CH3, J = 7) 1.33 (4H, m, CH2) 1.77 (2H, m, CH2) 3.93
1 060, 750
(1H, t, CH, J = 7) 7.20 (1H, t, ar, J = 8) 7.35 (2H, t, ar, J = 8) 7.65 (1H,
s, im) 7.72 (2H, d, ar, J = 8)
C16H20N2O2S 185–187
(304.4)
3 500–2 200, 1 610, 1 590, 0.84 (3H, t, CH3, J = 7) 1.26 (4H, m, CH2) 1.38 (2H, m, CH2) 1.77
1 055, 750
(2H, m, CH2) 3.94 (1H, t, CH, J = 7) 7.20 (1H, t, ar, J = 8) 7.36 (2H,
t, ar, J = 8) 7.64 (1H, s, im) 7.75 (2H, d, ar, J = 8)
3 500–2 200, 1 610, 1 595, 0.83 (3H, t, CH3, J = 7) 1.23 (6H, s, CH2) 1.38 (2H, t, CH2, J = 7) 1.79
C17H22N2O2S 185–188
(318.4)
1 060, 750
(2H, m, CH2) 3.94 (1H, t, CH, J = 7) 7.20 (1H, t, ar, J = 8) 7.36 (2H,
t, ar, J = 8) 7.64 (1H, s, im) 7.73 (2H, d, ar, J = 8)
aAbbreviations: T = thiophene, th = thiazole, im = imidazole, ar = aromatic. bRef. [24], m.p. 116–118. cRef. [23], m.p. not reported. dRef. [24,
25], m.p. 125–127.
IR showed an additional C=O stretching absorption. This
compound was considered as the iminothiazolone 12
which, after refluxing with dilute hydrochloric acid, gave
the imidazole 18. The structure of this new derivative was
The same reaction was repeated with 2-bromo-
acetophenone 9 and similar behaviour was observed:
when R was ethyl or propyl, the intermediate salt was
analogous to 11, leading to compounds 19–21, whereas,
when R was a longer chain, the precipitate was analogous
to 12 (it led to compounds 25–27) and the filtrate
1
confirmed by IR, H-NMR (table I) and 13C-NMR, MS
(see Experimental section).

886
contained the intermediate analogous to 11 which, after
the usual treatment with dilute hydrochloric acid, gave
the imidazothiazoles 22–24.
Contrary to 6-thienyl derivatives, 6-phenyl derivatives
with 2-substituents of increasing hydrophobicity either
maintained or reduced the inhibitory capacity of the lead
compound. There was no direct correlation between the
increase in length and hydrophobicity of the substituent
and inhibition, since the 2-ethyl (19) and the 2-butyl (22)
derivatives had a markedly reduced potency with respect
to the 2-propyl derivative (20). Nevertheless, 2-pentyl
(23) and mostly 2-esyl (24) derivatives showed an in-
creased inhibitory effect, thus suggesting that the length
of the chain is important for the interaction with the
complex I binding site.
For a deeper insight into the structure of the carboxylic
acids 18 and 25–27, one of these, 2-(4-phenyl-2-
imidazolylsulfanyl)heptanoic acid 26, was subjected to
DEPT, HETCOR, MS (see Experimental section) and to
crystallographic studies. Its crystal structure shows that
the imidazole and the phenyl ring are almost planar as
observed in analogous compounds [17, 18], whereas the
orientation of the carboxylic group is almost perpendicu-
lar to them. Further crystallographic details will be given
elsewhere.
The different behaviour of the thienyl or phenyl deriv-
atives indicated a slightly different interaction as inhibi-
tors of complex I. In previous work we studied in detail
the inhibitory properties of compound 1, which appeared
to act as a quinol antagonist [13]. In order to clarify the
relationship between the new phenyl derivatives and
quinone interaction in complex I, compounds 2 and 20
were tested with two different substrates, namely the
hydrophilic ubiquinone-1 (Q1) and the hydrophobic
undecyl-benzoquinone (UBQ). The potency of 2 was
higher with Q1 (I50 = 75 µM) than with UBQ (I50 = 130
µM), similarly to compound 1 [13]. Conversely, com-
pound 20 showed the same I50 for both Q1 and UBQ (120
µM). Thus, the inhibitory effect of the more hydrophobic
20 could depend on some internal reaction steps that are
not extensively influenced by the nature of the quinone
substrate for complex I activity. Indeed, compound 20
acted as a classical non-competitive inhibitor with respect
to both Q1 and UBQ, similarly to rotenone (figure 2) [8],
while 2 showed mixed competition with UBQ, as previ-
ously reported for 1 [13]. Taken together, the biological
data suggest that the substitution of a thienyl group with
a bulkier phenyl group at position 6 could shift the
inhibitory action of imidazothiazole compounds from
antagonists of the quinol product to antagonists of the
semiquinone intermediate. The latter would correspond to
the action of rotenone in complex I [8].
3. Biological results
Table II reports the biological activity of the reference
compounds (1–3) and of the newly synthesized imidazo-
thiazole derivatives (13–17 and 19–24). Among the
unexpected imidazole by-products, compounds 26–27
were quite inactive. Consequently, the analogues 18 and
25 were not tested.
The increase of hydrophobicity at position 2 in both
6-(2-thienyl) (13–15) and 6-(3-thienyl) derivatives (16
and 17) decreased the inhibitory potency on complex I
activity in comparison with the 2-methyl derivative 1.
Therefore, position 2 seems to be very critical for the
potency of complex I inhibition.
Table II. Biological activity of compounds 1–3, 13–17, 19–24 and
26–27.
Compound
Residual activity of NADH:UBQ
a
reductase (%)
1
2
3
12
24
80
63
91
63
63
100
82
17
100
73
46
0
114
107
13
14
15
16
17
19
20
21
22
23
24
26
27
4. Discussion
Structurally, most complex I inhibitors have a head-tail
module that is critical for the antagonist action versus the
ubiquinone substrate [8, 10]. The results of the present
study clarify that complex I inhibition is strictly depen-
dent on the substitutions at the extreme positions 2 and 6
of the imidazothiazole ring. The different potency of the
substituents at the 2 position, when position 6 is occupied
by either a thienyl or a phenyl group, indicates that steric
constraints in the binding site limit the length and
bulkiness of imidazothiazoles for their optimal interac-
aThe numbers are the average of at least three separate experi-
ments. The assay conditions are described in the experimental
section. UBQ (30 µM) was used as electron acceptor. The final
concentration of the compounds was 0.25 mM.

887
A
5. Experimental
5.1. Chemistry
Compounds 2–3 have been previously described [19].
The aldehydes 4 (butyraldehyde, valeraldehyde, hexanal,
heptanal, octanal) and 2-bromoacetophenone 9 are com-
mercially available. 2-(Bromoacetyl)thiophene 7 [20] and
3-(bromoacetyl)thiophene 8 [21] were prepared accord-
ing to the literature. The bromoaldehydes 5 were prepared
under experimental conditions analogous to those re-
ported for the synthesis of α-bromopropionaldehyde [22].
The imidazo[2,1-b]thiazoles 13 [23, 24], 14 [23] and
19 [25] were already reported in the literature.
The melting points are uncorrected. Analyses (C, H, N)
were within ± 0.4% of the theoretical values. TLC was
performed on Bakerflex plates (Silica gel IB2-F): the
eluent was a mixture of petroleum ether/acetone in
various proportions. The IR spectra (table I) were re-
B
1
corded in nujol on a Perkin-Elmer 683. The H-NMR
(table I) and the 13C-NMR spectra were recorded on a
Varian Gemini (300 MHz), and were referenced to
solvent signals (DMSO). The EI mass spectra were
recorded on a VG 7070E. X-ray crystallography: the data
were collected on a Siemens P4 four-circle diffractome-
ter.
5.1.1. 5-Alkyl-2-aminothiazoles 6
The appropriate bromoaldehyde 5 (100 mmol) was
treated with 80 mmol of thiourea and stirred at 90–100 °C
for 2 h. After cooling, the reaction mixture was treated
with 2 N NaOH until basic and extracted with chloro-
form. Compounds 6 thus obtained were used, without
further purification, in the following step.
5.1.2. Reaction of the 5-alkyl-2-aminothiazoles 6 with the
bromoacetylthiophenes 7–8 (synthesis of 13–18)
The appropriate compound 6 (45 mmol) was dissolved
in acetone (100 mL) and treated with 2-(bromo-
Figure 2. Effect of compound 2 and 20 on the Q1 (A) and UBQ
(B) titration of the NADH:Q reductase activity. (●) Control.
( ) In the presence of 0.18 mM compound 2. ([) In the
presence of 0.18 mM compound 20.
acetyl)thiophene
7
or 3-(bromoacetyl)thiophene
8
(45 mmol). The reaction mixture was refluxed for 3–6 h
(according to a TLC test) and the resulting salt 10–12 was
treated, without further purification, with 200 mL of 2 N
HCl. After 1 h reflux, the solution was cautiously basified
by adding dropwise 15% NH4OH. The resulting base
(13–18) was collected by filtration and crystallized from
ethanol with a yield of 35–40% (table I).
tion with complex I. In phenyl derivatives, the aliphatic
chain substituents in position 2 might mimic the hydro-
phobic tail of ubiquinone. A further lengthening of the
chain which resembles the polyprenil tail of ubiquinone
should better clarify if position 2 corresponds to the
attachment point for hydrophobic substituents.
Future studies with systematic substitutions at other
positions such as nitrogen 7 will define the determinants
to optimize the ubiquinone antagonist action of imida-
zothiazoles and produce extremely potent inhibitors of
complex I.
The following spectra were also recorded:
1) as an example of compound 10, R = isopropyl was
1
chosen, H-NMR: 1.21 (6H, d, CH3, J = 7); 3.01 (1H,
sep, CH, J = 7); 5.69 (2H, s, CH2); 7.20 (1H, s, th); 7.38
(1H, m, T); 8.11 (1H, m, T); 8.18 (1H, m, T); 9.59 (1H,
s, NH).

888
2) compound 12, IR: 3 500–2 200, 1 760, 1 685, 1 640,
The redox activity of NADH:ubiquinone oxidoreductase
was measured by using Q1 or UBQ as electron acceptor.
The test compound, dissolved in DMSO, was added to
0.4 mg/mL of mitochondrial protein in 2 mL of 50 mM
K2HPO4 buffer containing 1 mM EDTA, 2 mM KCN (pH
7.4) and 0.1 mM NADH at room temperature. The
reaction was started by quinone and followed by decrease
in absorbance of NADH in the dual wavelength mode at
350 minus 410 nm with an extinction of 5.5 mM–1 cm–1
as previously described [27].
1 560 cm–1. 1H-NMR: 0.87 (3H, t, CH3, J = 7); 1.40 (4H,
m, CH2); 2.05 (2H, m, CH2); 4.90 (1H, t, CH, J = 7); 5.37
(2H, s, CH2); 7.58 (1H, m, T); 7.74 (1H, m, T); 8.78 (1H,
m, T). MS: 296 (M+ , 11), 278 (M-H2O, 4), 268 (M-CO,
9), 124 (C6H4SO, 30), 111 (C5H3SO, 100).
3) Compound 18, MS: 296 (M+ , 13), 278 (M-H2O,
100), 235 (278-C3H7, 85), 207 (235-CO, 40), 182 (49),
123 (26), 109 (27), 55 (34).
5.1.3. Reaction of the 5-alkyl-2-aminothiazoles 6 with
2-bromoacetophenone 9 (synthesis of 19–27)
The appropriate compound 6 (10 mmol) was dissolved
in acetone (50 mL) and treated with 10 mmol of
2-bromoacetophenone 9. The mixture was refluxed for
2–5 h (according to a TLC test) and worked up with two
procedures. For compounds 19–21, acetone was evapor-
ated under reduced pressure and the resulting residue was
refluxed for 1 h with 100 mL of 2 N HCl. The solution
thus obtained was then basified with 15% NH4OH and
the resulting base 19–21 was crystallized from ethanol
with a yield of 30–35% (table I). For Compounds 22–27
the precipitate was collected by filtration and worked up
as above. Compounds 25–27 were crystallized from
ethanol with a yield of 15%. The filtrate, evaporated and
worked up in the same manner, gave compounds 22–24
which were crystallized from ethanol with a yield of 3%.
The following data were also recorded for compound 26:
13C-NMR (with DEPT and HETCOR): 13.80 (CH3),
21.90 (CH2), 26.13 (CH2), 30.81 (CH2), 31.83 (CH2),
50.29 (S-CH), 118.04 (im-5), 124.34 (ar-2 + 6), 126.56
(ar-4), 128.59 (ar-3 + 5), 133.13 (im-4), 138.09 (ar-1),
139.67 (im-2), 172.43 (COOH). MS: 304 (M+ , 45), 286
(M-H2O, 8), 260 (M-CO2, 16), 229 (286-C4H9, 15), 203
(48), 190 (70), 176 (100), 117 (56). Crystal data:
C16H20N2O2S, M = 304.4, Monoclinic, a = 17.305 (2), b
= 8.5690 (10), c = 10.8620 (10) Å, â= 99.67 (1)°, V =
1587.8 (3) Å3 (by least-squares refinement on diffracto-
meter angles for 43 randomly selected and automatically
centred reflections), space group P21/c (n. 14), Z = 4, F
(000) = 648, Dc = 1.27 g cm–3, µ (Mo-Kα) = 0.21 mm–1.
Acknowledgements
This work was supported by grants from the Italian
National Research Council (CNR, Rome) and from the
University of Bologna (Funds for Selected Research
Topics).
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