Search for the pharmacophore in prazosin for Transport-P

Article abstract of DOI:10.1016/j.bmc.2005.03.030

Partial structures of prazosin have been synthesised and tested for inhibition of Transport-P in order to identify the structural features of prazosin, which appear to be involved in binding to the putative transporter. It is shown that the pyrimidinyl 4-amino group is critically important for binding but that the 6,7-dimethoxy and 2-furoyl groups are not essential.

Full text of DOI:10.1016/j.bmc.2005.03.030

Bioorganic & Medicinal Chemistry 13 (2005) 3681–3689
Search for the pharmacophore in prazosin for Transport-P
Patricia A. Zunszain,a Cesare Federico,a Mario Sechi,a
Saad Al-Damlujib and C. Robin Ganellina,*
aDepartment of Chemistry, Christopher Ingold Laboratories, University College London, 20 Gordon Street, London WC1H 0AJ, UK
bDivisions of Endocrinology and Biochemistry, Royal Free and University College Medical School, Royal Free Campus,
Rowland Hill Street, London NW3 2PF, UK
Received 19 October 2004; accepted 15 March 2005
Available online 11 April 2005
Abstract—Partial structures of prazosin have been synthesised and tested for inhibition of Transport-P in order to identify the struc-
tural features of prazosin, which appear to be involved in binding to the putative transporter. It is shown that the pyrimidinyl 4-
amino group is critically important for binding but that the 6,7-dimethoxy and 2-furoyl groups are not essential.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
amine, which is neither completely neutral nor perma-
nently positively charged. Some examples of the com-
pounds are 4-benzylpiperidine, 4-phenylbutylamine
and imipramine, which had IC50 values of 4.4 0.5,
2.8 0.6 and 0.23 0.07 lM, respectively2 (Fig. 1).
Transport-P is an antidepressant-sensitive, proton-
dependent, V-ATPase-linked uptake process for amines
in peptidergic neurones of the hypothalamus. It is unu-
sual in its anatomical location in post-synaptic neurones
and in being activated by its ligand prazosin.1–8
Although prazosin is a potent antagonist at adrenergic
a1 receptors,9 this uptake effect of prazosin does not ap-
pear to involve adrenergic receptors.7 There are two
types of compounds that have so far been identified to
act as ligands for Transport-P: Group A compounds,
exemplified by prazosin, are accumulated in a coopera-
tive manner and Group B compounds, which include
phenylethylamines and some well established antide-
pressants, are accumulated noncooperatively by the
same uptake process. The structural properties of group
B have been studied2 by examining a large series of com-
pounds for their ability to inhibit competitively the
uptake of prazosin (1 lM) in immortalised gonado-
trophin-releasing hormone neurones (GT1-1 GnRH
cells) as previously described in detail.1
Presumably these compounds bind to a putative trans-
porter protein involved in the uptake process. The spe-
cial interest in characterising this transporter is that it
may provide clues for the development of a novel type
of antidepressant drug.10 Since these compounds differ
markedly from prazosin in chemical structure it is of
interest to examine in detail the extent to which the
structure of prazosin is required for the uptake process.
Initially, we have concentrated on seeking the structural
features, which appear to be involved in binding.
In seeking the pharmacophore for uptake of prazosin (1)
it is helpful to distinguish four main structural elements
of prazosin based on the four different rings that comprise
its structure (Fig. 2). These are labelled A: dime-
thoxy-benzenoid fusion, B: 4-aminopyrimidine, C: piper-
azine and D: 2-furoyl. Various notional fragmentations
Those group B compounds, which have the greatest
affinity for Transport-P appear to consist of a condensed
cyclic structure of 18–20 carbon atoms and a basic
NH
N
(CH2)4 NH2
Keywords: Transport-P; Prazosin; Peptidergic neurones; V-ATPase-
linked uptake.
(CH2)3
NMe2
*
Corresponding author. Tel.: +44 20 7679 4624; fax: +44 20 7679
7463; e-mail: c.r.ganellin@ucl.ac.uk
Figure 1. Structures of some inhibitors of Transport-P.
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.03.030

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P. A. Zunszain et al. / Bioorg. Med. Chem. 13 (2005) 3681–3689
(Fig. 3) have been made to the molecular structure of
O
O
D
prazosin to remove functional groups or particular
atoms in a stepwise manner. These led to a set of
compounds (Table 1), which have been synthesised
and tested for their influence on the uptake of prazosin
as follows.
N
C
H3CO
H3CO
N
B
N
A
N
NH2
1
The 2-furoyl group (D) has been replaced by benzoyl,
acetyl or phenyl to give compounds 2, 3 and 4, respec-
tively. In another example the 2-furoyl group (D) has
been removed and the amidic N of the piperazine ring
has been replaced by CHMe to give the 4-methylpiper-
idine 5. The 3-methylpiperidine isomer 6 has also been
made. The two methoxy groups on ring A have been re-
moved to afford 7 and then the 2-furoyl group (D) re-
placed by benzoyl to give 8; finally the 2-furoyl group
(D) has been removed and the amidic N of the piper-
azine ring C has been replaced by CH2 to provide a
piperidine 9.
Figure 2. The four rings, labelled A–D, in prazosin (1).
O
O
N
H3CO
H3CO
N
N
N
NH2
The 4-amino group on ring B has been replaced by a
hydrogen atom to give 10, and also replaced by 4-hydr-
Figure 3. Notional fragmentations of the prazosin structure.
Table 1. Structure and activities of prazosin analogues
X
X
C
C
R1
R2
N
B
N
R1
R2
N
B
N
R1
R2
N
A
A
N
X
B
C
N
N
N
H
R3
R3
III
I
II
R1
A
R2
R1
R2
Y
N
X
C
C
Y
N
X
A
V
IV
Compd
Struct
R1
R2
R3 or Y
X
Inhibitiona
%
c
1
I
MeO
MeO
MeO
MeO
MeO
MeO
H
MeO
MeO
MeO
MeO
MeO
MeO
H
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
H
NCOFurb
NCOPh
NCOMe
NPh
2
I
63
37
93 2d
5
5
3
I
4
I
5
I
CHMe
85
85
64
73
83
5
3
6
I
CHMee
NCOFur
NCOPh
CH2
3
7
I
5
8
I
H
H
7
9
I
H
H
3
10
10
11
12
13
14
15
16
17
18
19
I
MeO
MeO
H
MeO
MeO
H
NCOFur
NCOFur
NCOFur
NCOFur
NCOFur
NCOPh
CH2
I
OH
NH2
0
II
III
IV
IV
IV
IV
IV
V
44
36
4
6
H
H
MeO
MeO
MeO
MeO
Cl
MeO
MeO
MeO
MeO
Cl
CH2CH2–
CH2CH2–
CH2CH2–
SO2
10 13
16
91 2f
7
NCOFur
NCOFur
NCOFur
0
CH2
CH
47
5
7
7
H
H
a % inhibition of prazosin uptake by 10À4 M concentration of the test compound. Values are shown with SEM.
O
b Fur =
.
c Prazosin.
d IC50 = 5.2 2 lM.
e Isomer of 5 (3-methylpiperidine).
f IC50 = 23 12 lM.

P. A. Zunszain et al. / Bioorg. Med. Chem. 13 (2005) 3681–3689
3683
oxy, which tautomerises to the amide to give 11. The
dimethoxy-benzenoid ring A has been removed to afford
the disubstituted pyrimidine 12. Replacement of dime-
thoxy-aminoquinazoline (A and B) by benzimidazole
gives 13.
Compound 11 was prepared from 2-chloro-4,6,7-tri-
methoxyquinazoline (24) and 1-(2-furoyl)piperazine
(Scheme 2). Compound 12 was obtained as shown in
Scheme 3 using 4-amino-2-chloropyrimidine as interme-
diate. Reaction of 2,4-dichloropyrimidine with NH4OH,
followed by chromatographic separation, permitted the
isolation of the desired 4-amino-2-chloro isomer, which
was subsequently reacted with 1-(2-furoyl) piperazine to
give 12. Benzimidazole 13 was prepared by direct reaction
of 2-chloro benzimidazole with 1-(2-furoyl)piperazine
(Scheme 3). The 3,4-dimethoxyphenylethyl-piperazines
14, 15 and 3,4-dimethoxyphenylethyl-piperidine 16
(X = CH2) were synthesised as depicted in Scheme 4. Bro-
mination of 2-(3,4-dimethoxyphenyl)ethanol (25) with
N-bromosuccinimide in the presence of PPh3 afforded
the corresponding bromo derivative 26, which was then
substituted with the appropriate piperazines or piperidine
in the presence of K2CO3.
The amidino fragment (N3@C–NH2) of the quinazoline
in ring B has been removed and the N1@C replaced by
CH2–CH2 to provide the arylethylpiperazine 14 which,
upon further replacement of furoyl (D) by benzoyl, gives
15; then replacement of the furoylpiperazine (C and D)
by piperidine affords 16. The linkage has been further
shortened using a polar group SO2 to make a neutral
(i.e., nonbasic) molecule 17. In another approach to re-
duce the piperazine pKa but still leave it basic and to ex-
plore the chemical space potentially available to
contribute hydrophobically to binding, dichlorobenzyl
and fluorenyl groups (the latter approximating to a phe-
nyl benzyl group) have been attached directly to the
piperazine moiety giving 18 and 19, respectively.
Compounds 17–19 were prepared by direct replacement
of a chloro or bromo substituent in 3,4-dimethoxyben-
zene sulfonyl chloride, 3,4-dichlorobenzyl bromide or
9-bromofluorene, respectively, with 1-(2-furoyl)piper-
azine (Scheme 3).
2. Chemistry
The synthesis of 2-(1-piperazinyl)-4-aminoquinazolines
2–9 is summarised in Scheme 1. Treatment of 6,7-di-
methoxy-quinazoline-2,4(1H,3H)-dione (20a) with a mix-
ture of POCl3 and PCl5 afforded the corresponding
2,4-dichloro compound 21a, which upon reaction with
NH4OH led regioselectively to the 4-amino-2-chloro
derivative 22a. The same sequence starting from quinaz-
oline-2,4(1H,3H)-dione (20b) led to the corresponding
quinazoline 22b. Nucleophilic addition of the appropri-
ate piperazines, piperidines or furfurylamine was ef-
fected in refluxing 1-pentanol or isoamyl alcohol to
produce the desired final compounds.
3. Biological studies
The compounds were examined for their ability to inhi-
bit competitively the uptake of prazosin (1 lM) in
immortalised gonadotrophin-releasing hormone neuro-
nes (GT1-1 GnRH cells) as previously described in
detail.1 Each compound was tested for its ability to com-
pete with prazosin (at 10À6 M; inhibition of Transport-
P). Efficacy was defined as % inhibition of the uptake
of prazosin (at 10À6 M) when the test compound was
used in a concentration of 10À4 M. Efficacy was
expressed as % of the effect of a maximal inhibitory con-
centration of desipramine (10À4 M). IC50 values were
calculated for compounds, which achieved a maximal
inhibitory response, defined as 90% of the inhibitory
effect of desipramine 10À4 M. Each experimental point
Reduction of 2,4-dichloro-6,7-dimethoxyquinazoline
(21a) with NaBH4 led to 2-chloro-6,7-dimethoxyquinaz-
oline (23), which was then subjected to nucleophilic addi-
tion by 1-(2-furoyl)piperazine, obtained by treating
piperazine with 2-furoyl chloride, to give 10 (Scheme 2).
H
R1
R2
N
Cl
R1
R2
N
O
R1
R2
N
Cl
ii
i
N
NH
N
NH2
O
Cl
22a R1 = R2 = OCH3
22b R1 = R2 = H
20a R1 = R2 = OCH3
20b R1 = R2 = H
21a R1 = R2 = OCH3
21b R1 = R2 = H
X
R1
R2
N
N
iii
HN
X
N
NH2
2 - 9
Scheme 1. Experimental conditions: (i) POCl3, PCl5, PhN(CH3)2, reflux, 20 h; (ii) 35% NH4OH, THF, rt, 16 h; (iii) appropriate amine, 1-pentanol or
isoamyl alcohol, reflux, 3–20 h.

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P. A. Zunszain et al. / Bioorg. Med. Chem. 13 (2005) 3681–3689
O
O
N
H3CO
H3CO
N
Cl
H3CO
H3CO
N
Cl
H3CO
H3CO
N
N
ii
i
N
N
N
Cl
23
21a
10
iii
O
O
O
O
N
N
H3CO
H3CO
N
Cl
H3CO
H3CO
N
N
H3CO
H3CO
N
N
ii
N
OH
N
OCH3
NH
O
24
11
Scheme 2. Experimental conditions: (i) NaBH4, isopropanol, 70–80 ꢁC, 5 days; (ii) 1-(2-furoyl)piperazine, isoamyl alcohol, 150 ꢁC, 18 h; (iii)
NaOCH3, CH3OH, reflux, 12 h.
O
O
N
O
N
N
O
N
H
N
N
12
N
NH2
N
13
ii
iii
O
O
H3CO
H3CO
SO2
O
O
O
N
O
N
i
iv
HN
N
+
Cl
HN
NH
O
17
v
vi
Cl
Cl
N
N
O
N
N
O
18
O
19
Scheme 3. Experimental conditions: (i) 48% HBr, EtOH–H2O, 80 ꢁC, 1.5 h; (ii) 4-amino-2-chloropyrimidine, 1-pentanol, reflux, 3 h; (iii)
2-chlorobenzimidazole, 1-pentanol, 100 ꢁC, 6 h; (iv) 3,4-dimethoxybenzene sulfonyl chloride, Et3N, CH2Cl2, rt, 12 h; (v) 3,4-dichlorobenzyl bromide,
1-pentanol, reflux, 16 h; (vi) 9-bromofluorene, 1-pentanol, reflux, 12 h.
X
H3CO
H3CO
N
H3CO
H3CO
Br
HN
H3CO
H3CO
OH
ii
i
X
14 - 16
26
25
Scheme 4. Experimental conditions: (i) PPh3, N-bromosuccinimide, toluene, rt; 12 h; (ii) appropriate amine, K2CO3, EtOH, reflux; 24 ꢁC.
was carried out in triplicate and each experiment was
performed twice; the data are mean SEM of six
observations for each experimental point. Unlabelled
prazosin (at 10À6 M) increased the uptake of [3H]prazo-

P. A. Zunszain et al. / Bioorg. Med. Chem. 13 (2005) 3681–3689
3685
sin by 126.8 6.3%. Biological data and general struc-
tures of title compounds are reported in Table 1.
2. The presence of the 4-amino group in the pyrimidine
ring (B) is critically important presumably because it
raises the pKa >7 so that sufficient numbers of mole-
cules are protonated and positively charged at
pH 7.4.
4. Results and discussion
3. The piperazine ring (C) can be replaced by piperidine
and therefore the 2-furoyl group (D) is not required
for binding.
4. If the piperazine ring is retained, then the 2-furoyl
group (D) can be replaced by phenyl to achieve lM
affinity.
Previous work has demonstrated that compounds with a
basic amino group, which would be protonated (cati-
onic) at the physiological neutral pH of 7.4 are able to
inhibit the uptake of prazosin. In the present series, all
the compounds except 10, 11, 14, 17 and 19 bind to
some extent to the uptake system. Compound 10 (i.e.,
prazosin minus the quinazoline 4-amino group) was
not able to inhibit uptake at 10À4 M. Comparison with
2-amino-6,7-dimethoxy-4-methylquinazoline as a model
reference structure for the pKa (reported11 5.2) suggests
that 10 would probably have <1% of its molecules in the
protonated form at pH 7.4. Prazosin, on the other hand
(pKa 6.84)12 would be approximately 40% cationic (note
2,4-diaminoquinazoline has pKa 7.96 at 20 ꢁC).13 Com-
pound 11 would be even less basic than 10.
6. Experimental
6.1. Chemistry (general)
Melting points were carried out on an Electrothermalꢂ
melting point apparatus and are uncorrected. 1H
NMR spectra were recorded at 300 or 500 MHz on a
Bruker AMX-300 and a Bruker AVANCE-500, respec-
tively. The NMR experiments were carried out in
CDCl3, CD3OD or DMSO-d6 with tetramethylsilane
as an internal reference. Chemical shifts are quoted in
parts per million (ppm) and the coupling constants are
reported in Hertz (Hz). Analytical HPLC was carried
out on a Shimadzu HPLC apparatus with a Kromasil
C18 lm reversed column (250 · 4.6 mm) at a flow rate
of 1 mL/min and detected at 254 nm. The mobile phases
were mixtures of A = methanol + 0.1% trifluoroacetic
acid, and B = water + 0.1% trifluoroacetic acid, and
are indicated as the ratio A:B. Mass spectra were re-
corded on a VG 7070H Double Focusing Mass Spec-
trometer or a VG ZAB-SE Double Focusing Mass
Spectrometer, using atmospheric pressure chemical ioni-
sation (APCI), electrospray (ES) or fast atom bombard-
ment (FAB). Elemental analyses were determined on a
Perkin Elmer 2400 CHN elemental analyser and were
carried out at UCL Departmental Microanalysis Ser-
vice. Column chromatography was done using Merck
silica gel 60 (70–230 mesh). TLC was carried out using
Merck Kieselgel 60 F254 aluminium sheets, visualised
at 254 nm and stained with potassium iodoplatinate or
5% H2SO4 in ethanol prior to heating.
Compound 14, which was not active, and 15, which gave
low inhibition (10–16% at 10À4 M), are 1-dimethoxyphen-
ylethyl-4-acyl-piperazine derivatives and the result is sur-
prising in view of the inhibitory activity of the piperidine
analogue 16 (91% inhibition at 10À4 M). However, this
may be accounted for by the differences in pKa values
since acyl piperazines are reported to have substantially
lower pKaꢀs than piperidines (compare 1-methyl-4-ben-
zoyl-piperazine pKa 6.78 with 1-methylpiperidine pKa
10.19).14 Another acylpiperazine that gave low inhi-
bition is the benzimidazole 13 (36% inhibition at
10À4 M), which would certainly have a pKa value below
that of 2-aminobenzimidazole (whose pKa is 7.5 at
20 ꢁC).15
For the furoyl analogue, comparing prazosin with 7, it
appears that the dimethoxy groups contribute to affin-
ity. For the benzoylpiperazines, however, (compare 2
and 8) the dimethoxy groups do not appear to contri-
bute to binding affinity. The benzenoid fusion appears
to make a small contribution to affinity when comparing
the des-methoxy compounds 7 and 12. Compound 12,
which, in comparison with prazosin, no longer has the
benzenoid fusion or the dimethoxy groups still retains
some affinity but the two effects appear to be additive
since it has less than 50% affinity. Two compounds (4
and 16) were able to achieve a maximal inhibition of
prazosin binding and had their IC50 values determined
as 5.2 2 and 23 12 lM, respectively.
6.2. 4-Amino-2-(4-benzoyl-piperazin-1-yl)-6,7-dimeth-
oxyquinazoline hydrochloride hydrate (2)
A mixture of 4-amino-2-chloro-6,7-dimethoxyquinazo-
line (22a) (0.77 g, 3.2 mmol), prepared as previously de-
scribed16 and 1-benzoylpiperazine17 (0.61 g, 3.2 mmol)
in 1-pentanol (20 mL) was heated at reflux and stirred
for 3 h. A white solid was formed, and after cooling it
was filtered and washed with diethyl ether. The solid
was recrystallised from methanol to give 2 as white crys-
tals (0.45 g, 32%); mp 288–290 ꢁC; HPLC (A:B = 50:50),
Rt 9.33 min (99.7%); 1H NMR (DMSO-d6, 500 MHz): d
8.90 (br s, 1H, NH), 8.65 (br s, 1H, NH), 7.74 (s, 1H,
quin-8H), 7.53 (s, 1H, quin-5H), 7.45–7.49 (m, 5H,
Ph), 3.94–4.01 (br s, 4H, NCH2 · 2), 3.87 (s, 3H,
OCH3), 3.83 (s, 3H, OCH3), 3.42–3.76 (br s, 4H,
NCH2 · 2). MS (ESP) m/z 394 (M+1+, 100%). Anal.
Calcd for C21H23N5O3ÆHClÆ0.25H2O: C, 58.06; H,
Further studies are in progress to determine the struc-
tural features needed for a compound to activate the
transport system.
5. Conclusions
1. It is not necessary to retain the dimethoxy groups or
even the dimethoxy-benzenoid fusion (A) to achieve
10À4 M binding affinity to the putative uptake
system.

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P. A. Zunszain et al. / Bioorg. Med. Chem. 13 (2005) 3681–3689
5.68; N, 16.12; Cl, 8.16. Found: C, 58.16; H, 5.43; N,
16.13; Cl, 7.86.
4.69 (d, 2H, NCH2), 3.83 (s, 3H, OCH3), 3.79 (s,
3H, OCH3), 2.78 (t, 2H, NCH2), 1.59–1.66 (m, 1H),
1.01–1.11 (m, 4H, NCH2 · 2), 0.92 (d, 3H, CH3). MS
(FAB+), m/z 303 [M+H]+. Anal. Calcd for
C16H22N4O2: C, 63.56; H, 7.33; N, 18.53. Calcd for
5% inorganic: C, 60.3; H, 6.96; N, 17.60. Found: C,
60.16; H, 6.95; N, 17.42.
6.3. 2-(4-Acetylpiperazin-1-yl)-4-amino-6,7-dimethoxy-
quinazoline hydrochloride sesquihydrate (3)
4-Amino-2-chloro-6,7-dimethoxyquinazoline
(22a)
(0.15 g, 0.63 mmol) and 1-acetylpiperazine (0.08 g,
0.63 mmol) were dissolved in isoamyl alcohol (9 mL)
and the mixture was stirred overnight at 140 ꢁC under
nitrogen. A precipitate was formed and the mixture
was cooled and Et2O was added. The precipitate was fil-
tered off, washed with Et2O and recrystallised from
EtOH/Et2O to afford 3 as hygroscopic white crystals
(0.13 g, 54%); mp 240–244 ꢁC (lit.18 mp 244–245 ꢁC);
HPLC (A:B = 40:60), Rt 9.23 min (100%); 1H NMR
(DMSO-d6, 300 MHz): d 8.78 (br s, 2H, NH2), 7.74 (s,
1H, quin-8H), 7.44 (s, 1H, quin-5H), 3.89 (s, 3H,
OCH3), 3.84 (s, 3H, OCH3), 3.80 (br s, 4H,
NCH2 · 2), 3.61 (br s, 4H, NCH2 · 2), 2.06 (s, 3H,
COCH3). MS (FAB+) m/z 332 (M+, 100%). Anal.
Calcd. for C16H21N5O3ÆHClÆ1.5H2O: C, 48.67; H, 6.38;
N, 17.74; Cl, 8.98. Found: C, 49.07; H, 6.47; N, 17.38;
Cl, 8.58.
6.6. 4-Amino-6,7-dimethoxy-2-(3-methylpiperidin-1-
yl)quinazoline hydrochloride hydrate (6)
A mixture of 4-amino-2-chloro-6,7-dimethoxyquinazo-
line (22a) (0.11 g, 0.45 mmol) and 3-methylpiperidine
(0.045 g, 0.45 mmol) in isoamyl alcohol (10 mL) was
heated at 140 ꢁC under N2 and stirred for 12 h. After
being cooled, the mixture was diluted with diethyl ether
and the resulting white precipitate was collected and
washed with diethyl ether before being recrystallised
from EtOH/Et2O to afford a white solid (0.065 g,
42%); mp 246–249 ꢁC; HPLC (A:B = 30:70), Rt =
3.80 min (100%); 1H NMR (DMSO-d6, 300 MHz): d
8.64 (d, 2H, NH2), 7.72 (s, 1H, quin-8H), 7.54 (s, 1H,
quin-5H), 4.51 (d, 2H, NCH2), 3.87 (s, 3H, OCH3),
3.83 (s, 3H, OCH3), 3.06 (t, 2H, NCH2), 2.78 (m, 1H),
1.42–1.82 (m, 4H), 1.13 (d, 3H). MS (ES) m/z 303
[M+H]+. Anal. Calcd for C16H22N4O2ÆHClÆ0.25ÆH2O:
C, 55.97; H, 6.90; N, 16.32. Found: C, 55.93; H, 6.89;
N, 16.12.
6.4. 4-Amino-6,7-dimethoxy-2-(4-phenyl-piperazin-1-
yl)quinazoline hydrochloride (4)
A mixture of 4-amino-2-chloro-6,7-dimethoxyquinazo-
line (22a) (0.35 g, 1.46 mmol) and 1-phenylpiperazine
(0.24 g, 1.46 mmol) in 1-pentanol (20 mL) was heated
under reflux and stirred for 20 h. A solid appeared and
after cooling it was filtered and recrystallised from 1-
pentanol to give 4 as pale yellow crystals (0.18 g,
6.7. 4-Amino-2-[4-(2-furoyl)piperazin-1-yl]-quinazoline
hydrochloride (7)
Compound 7 was prepared as described for 2, from 4-
amino-2-chloroquinazoline19 (22b) (0.20 g, 1.1 mmol)
and 1-(2-furoyl)piperazine (0.22 g, 1.25 mmol), to give
30%);
mp
283–286 ꢁC;
HPLC
(A:B = 50:50),
Rt = 11.54 min (99.2%); 1H NMR (DMSO-d6,
500 MHz): d 8.96 (br s, 1H, NH2), 8.65 (br s, 1H,
NH2), 7.77 (s, 1H, quin-8H), 7.62 (s, 1H, quin-5H),
7.24 (t, 2H, Ph-2,6H), 7.00 (d, 2H, Ph-3,5H), 6.81 (t,
1H, Ph-4H), 4.01–4.03 (br s, 4H, NCH2 · 2), 3.87 (s,
3H, OCH3), 3.84 (s, 3H, OCH3), 3.27–3.29 (br s, 4H,
NCH2 · 2). MS (ES) m/z 366 [M+H]+. Anal. Calcd for
C20H23N5O2ÆHCl: C, 59.77; H, 6.02; N, 17.43; Cl, 8.82.
Found: C, 59.55; H, 5.93; N, 17.37; Cl, 8.55.
7
(0.25 g, 64%); mp 306–308 ꢁC/decomp.; HPLC
1
(A:B = 50:50), Rt 4.56 min (99.4%); H NMR (DMSO-
d6, 300 MHz): d 9.12 (br s, 1H, NH2), 8.91 (br s, 1H,
NH2), 8.20 (d, 1H, quin-8H), 7.94 (d, 1H, quin-5H),
7.80 (d, 1H, fur-5H), 7.73 (t, 1H, quin-8H), 7.34 (t,
1H, quin-6H), 7.00 (d, 1H, fur-3H), 6.58 (br s, 1H,
fur-4H), 3.96 (br s, 4H, NCH2 · 2), 3.78 (br s, 4H,
NCH2 · 2). MS (APCI) m/z 324.1 (M+, 100%). Anal.
Calcd for C17H17N5O2Æ1.1HCl: C, 56.18; H, 5.02; N,
19.27; Cl, 10.73. Found: C, 56.00; H, 5.07; N, 19.20;
Cl, 10.36.
6.5. 4-Amino-6,7-dimethoxy-2-(4-methylpiperidin-1-
yl)quinazoline (5)
6.8. 4-Amino-2-(4-benzoylpiperazin-1-yl)-quinazoline
hydrochloride (8)
A mixture of 4-amino-2-chloro-6,7-dimethoxyquinazo-
line (22a) (0.50 g, 2.09 mmol) and 4-methyl-piperazine
(0.41 g, 4.17 mmol) in 1-pentanol (20 mL) was heated
at reflux and stirred for 20 h. The solution was evapo-
rated and the amorphous solid was triturated with
diethyl ether and filtered. The solid was suspended in
water and a saturated solution of NaHCO3 was added
until pH 8 with stirring. The resulting solid was fil-
tered, washed with water and recrystallised from
EtOH/H2O to give 5 as pale brown crystals (0.075 g,
12%). The analysis of this material corresponds to
the presence of 5% inorganic material; mp 176–
178 ꢁC; HPLC (A:B = 70:30), Rt = 47.01 min (98.9%);
1H NMR (DMSO-d6, 500 MHz): d 7.43 (s, 1H, quin-
8H), 7.21 (br s, 2H, NH2), 6.78 (s, 1H, quin-5H),
Compound 8 was prepared as described for 2, from 4-
amino-2-chloro-quinazoline (22b) (0.24 g, 1.3 mmol)
and 1-(2-benzoyl)piperazine (0.25 g, 1.3 mmol), to give
8
(0.31 g, 65%); mp 310–312 ꢁC/decomp.; HPLC
1
(A:B = 50:50), Rt 27.91 min (99.4%); H NMR (DMSO-
d6, 300 MHz): d 9.19 (br s, 1H, NH2), 9.04 (br s, 1H,
NH2), 8.26 (d, 1H, quin-8H), 7.86 (m, 2H, quin-5,7H),
7.51 (m, 6H, quin-6H + Ph), 4.00 (br s, 4H,
NCH2 · 2), 3.81 (br s, 2H, NCH2), 3.59 (br s, 2H,
NCH2). MS (APCI) m/z 334.2 (M+1+, 100%). Anal.
Calcd for C19H19N5OÆ1.1HCl: C, 61.10; H, 5.42; N,
18.75; Cl, 10.44. Found: C, 61.13; H, 5.46; N, 18.41;
Cl, 10.15.

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3687
1
6.9. 4-Amino-2-(piperidin-1-yl)-quinazoline hydrochloride
(9)
Rt = 3.28 min (100%); H NMR (CDCl3, 300 MHz): d
7.49 (d, 1H, fur-5H), 7.40 (s, 1H, quin-5H), 7.06 (d,
1H, fur-3H), 6.55 (s, 1H, quin-8H), 6.50 (dd, 1H, fur-
4H), 3.95 (br s, 7H, OCH3 + NCH2 · 2), 3.91 (s, 3H,
OCH3), 3.78 (br s, 4H, NCH2 · 2). MS (FAB+), m/z
385 [M+H]+. Anal. Calcd for C16H22N4O2Æ0.25H2O:
C, 58.68; H, 5.31; N, 14.41. Found: C, 58.88; H, 5.24;
N, 14.41.
Compound 9 was prepared as described for 2, from 4-
amino-2-chloro-quinazoline (22b) (0.44 g, 2.44 mmol)
and piperidine (0.21 g, 2.44 mmol), to give 9 (0.25 g,
37%); mp 280–282 ꢁC/decomp.; HPLC (A:B = 50:50),
1
Rt 7.17 min (100%); H NMR (DMSO-d6, 300 MHz):
d 9.05 (br s, 1H, NH2), 8.90 (br s, 1H, NH2), 8.22 (d,
1H, quin-8H), 7.81 (t, 1H, quin-7H), 7.65 (d, 1H,
quin-5H), 7.43 (t, 1H, quin-6H), 3.86 (br s, 4H,
NCH2 · 2), 1.66 (br s, 6H, CH2 · 3). MS (APCI) m/z
229 (M+). Anal. Calcd for C13H16N4ÆHCl: C, 58.98; H,
6.47; N, 21.16; Cl, 13.39. Found: C, 58.82; H, 6.55; N,
20.96; Cl, 13.68.
6.12. 4-Amino-2-[4-(2-furoyl)piperazin-1-yl]-pyrimidine
hydrochloride hydrate (12)
4-Amino-2-chloropyrimidine20,21 (0.09 g, 0.72 mmol)
was heated with 1-(2-furoyl)piperazine (0.13 g, 0.72
mmol) in 1-pentanol (3 mL) under reflux for 3 h. The
mixture was then cooled and the filtrate was recrystal-
lised from EtOH–ether, to give 12 (0.14 g, 62%); mp
243–244 ꢁC/decomp.; HPLC (A:B = 30:70), Rt 8.11 min
(99.9%); 1H NMR (DMSO-d6, 300 MHz): d 7.89 (d,
1H, pyr-6H), 7.87 (d, 1H, fur-5H), 7.05 (d, 1H, fur-
3H), 6.65 (dd, 1H, fur-4H), 6.50 (d, 1H, pyr-5H), 3.81
(br s, 8H, NCH2 · 4). MS (FAB +) m/z 274 (M+1+).
Anal. Calcd for C13H15N5O2ÆHClÆH2O: C, 47.64; H,
5.54; N, 21.37; Cl, 10.82. Found: C, 47.65; H, 5.40; N,
21.27; Cl, 10.49.
6.10. 6,7-Dimethoxy-2-[4-(2-furoyl)-piperazin-1-yl]quin-
azoline hydrogen oxalate (10)
To a solution of 2,4-dichloro-6,7-dimethoxyquinazoline
(21a) (0.35 g, 1.37 mmol) in isopropanol (15 mL),
NaBH4 (0.05 g, 1.37 mmol) was added. The mixture
was heated at 70–80 ꢁC for 5 days, until HPLC examina-
tion showed the complete transformation of the starting
material. The solvent was evaporated and the residue
was purified by preparative HPLC using MeOH–H2O
70:30 as eluant. Evaporation of the appropriate fractions
gave an off-yellow residue, which was recrystallised from
EtOH to afford 0.043 g (14%) of pure 2-chloro-6,7-
dimethoxyquinazoline (23); HPLC (A:B = 70:30), Rt
6.13. 2-[4-(2-Furoyl)piperazin-1-yl]-benzimidazole hydro-
chloride hydrate (13)
A suspension of 2-chlorobenzimidazole (0.17 g, 1.15
mmol) and 1-(2-furoyl)piperazine (0.20 g, 1.15 mmol)
in 1-pentanol (5 mL) was heated at 100 ꢁC for 6 h. After
cooling, the solid was filtered off, washed thoroughly
with CH2Cl2, then recrystallised twice from EtOH, to
give 13 as a white solid (0.22 g, 59%); mp 274–276 ꢁC;
HPLC (A:B = 50:50), Rt 4.53 min (99.9%); 1H NMR
(DMSO-d6, 300 MHz): d 7.90 (d, 1H, fur-5H), 7.44 (br
s, 2H, benzim-6,7H), 7.29 (br s, 2H, benzim-5,6H),
7.12 (d, 1H, fur-3H), 6.68 (dd, 1H, fur-4H), 3.94 (br s,
4H, NCH2 · 2), 3.80 (m, 4H, NCH2 · 2). MS (FAB+)
m/z 297 (M+1+, 100%). Anal. Calcd for C16H15N4O2Æ
HClÆ0.25H2O: C, 57.15; H, 4.95; N, 16.66; Cl, 10.54.
Found: C, 57.17; H, 4.81; N, 16.56; Cl, 10.12.
1
4.13 min; H NMR (CDCl3, 300 MHz): d 9.02 (s, 1H,
quin-4H), 7.25 (s, 1H, quin-8H), 7.09 (s, 1H, quin-5H),
4.02 (s, 3H, OCH3), 4.00 (s, 3H, OCH3). MS (FAB+)
m/z 225 (M+). To a solution of 23 (0.04 g, 0.19 mmol)
in isoamyl alcohol (8 mL), 1-(2-furoyl)piperazine
(0.035 g, 0.19 mmol) was added. The mixture was heated
at 150 ꢁC for 18 h. The solvent was evaporated under
vacuum and the residue was dissolved in saturated NaH-
CO3 and extracted with CH2Cl2. The organic layer was
dried (Na2SO4) and evaporated to give a residue that
was purified by column chromatography using EtOAc–
petrol spirit 1:10. The product was then transformed into
its oxalate to give 10 as yellow crystals (0.03 g, 34%); mp
204–206 ꢁC; HPLC (A:B = 60:40), Rt 3.02 min (100%);
1H NMR (DMSO-d6, 300 MHz): d 9.00 (s, 1H, quin-
4H), 7.88 (d, 1H, fur-5H), 7.26 (s, 1H, quin-5H), 7.05
(d, 1H, fur-3H), 6.96 (s, 1H, quin-8H), 6.78 (br s, 1h,
fur-3H), 6.66 (dd, 1H, fur-4H), 3.91 (s, 3H, OCH3),
3.88 (d, 4H), 3.84 (s, 3H, OCH3). MS (ES+) m/z 369.3.
Anal. Calcd for C19H20N4O4ÆC2H2O4: C, 55.02; H,
4.84; N, 12.22. Found: C, 54.66; H, 4.79; N, 11.94.
6.14. 1-[2-(3,4-Dimethoxyphenyl)ethyl]-4-(2-furoyl)-
piperazine hydrogen oxalate (14)
An externally cooled solution of 2-(3,4-dimethoxyphen-
yl)ethanol (25) (2.80 g, 15.3 mmol) dissolved in toluene
(30 mL) was treated with PPh3 (4.48 g, 17.1 mmol) and
N-bromosuccinimide (2.85 g, 16 mmol) was added por-
tionwise. After 12 h of stirring at room temperature the
mixture was quenched with Na2S2O3 (10% w/v, 17 mL).
The organic layer was washed twice with NaOH (1 M)
and then with water, dried (MgSO4) and evaporated.
The residue was treated with diethyl ether, and precipi-
tated Ph3PO was removed by filtration. Column chroma-
tography purification afforded 4-(2-bromoethyl)-1,2-
dimethoxybenzene (26) as an oil, which crystallised on
standing and was recrystallised from EtOH. A solution
of 26 (0.13 g, 0.53 mmol) and 1-(2-furoyl) piperazine
(0.11 g, 0.64 mmol) in ethanol (3 mL) was stirred under
reflux in the presence of K2CO3 (0.04 g, 0.28 mmol)
6.11. 6,7-Dimethoxy-2-[4-(2-furoyl)-piperazin-1-yl]quin-
azoline-4-one hydrate (11)
A mixture of 2-chloro-4,6,7-trimethoxyquinazoline (24)
(0.069 g,
0.27 mmol)
and
1-(2-furoyl)piperazine
(0.049 g, 0.27 mmol) in isoamyl alcohol (10 mL) was
heated at 150 ꢁC under N2 and stirred for 12 h. The
solution was cooled but no precipitate formed; the sol-
vent was then evaporated to afford an off-white solid
that was recrystallised twice from EtOH/Et2O to give
11 (0.05 g, 54%); mp 266–268 ꢁC; HPLC (A:B = 50:50),

3688
P. A. Zunszain et al. / Bioorg. Med. Chem. 13 (2005) 3681–3689
during 24 h. The reaction mixture was filtered and the
filtrate was evaporated in vacuo. The resulting residue
was purified by column chromatography using EtOAc–
MeOH 50:1 to give an oil, which was converted into the
oxalate salt. Recrystallised from EtOH–diethyl ether gave
14 (0.23 g, 99%); mp 135–136 ꢁC; HPLC (A:B = 55:45),
Rt 11.2 min; 1H NMR (DMSO-d6, 300 MHz): d 7.87 (br
s, 1H, fur-5H), 7.06 (d, 1H, fur-3H), 6.88 (d, 1H, Ph-
5H), 6.86 (s, 1H, Ph-2H), 6.75 (dd, 1H, Ph-6H), 6.66
(dd, 1H, fur-4H), 3.81 (br s, 4H, NCH2 · 2), 3.75 (s, 3H,
OCH3), 3.72 (s, 3H, OCH3), 2.82 (br s, 6H, NCH2 · 3),
2.79 (m, 2H, CH2). MS (APCI) m/z 345.2 (M+1+, 100%).
Anal. Calcd for C19H24N2O4ÆC2H2O4: C, 58.06; H, 6.03;
N, 6.45. Found: C, 57.80; H, 6.00; N, 6.33.
Calcd for C17H19N2O6S: C, 53.82; H, 5.55; N, 7.38.
Found: C, 53.70; H, 5.55; N, 7.38.
6.18. 1-(3,4-Dichlorobenzyl)-4-(2-furoyl)-piperazine
hydrogen oxalate (18)
A solution of 3,4-dichlorobenzyl bromide (0.402 g,
1.67 mmol)
and
1-(2-furoyl)piperazine
(0.302 g,
1.67 mmol) in 1-pentanol (7 mL) was heated at reflux
for 16 h. Then, the solvent was evaporated and the solid
dissolved and extracted with NaOH and CH2Cl2. The
organic phase was dried and evaporated. Oxalic acid
was added to an ethanolic solution and the oxalate pre-
cipitated upon addition of diethyl ether. The solid was
filtered and recrystallised twice from EtOH/diethyl ether
to give 18 as white crystals (34%); mp 164–166 ꢁC;
HPLC (A:B = 60:40), Rt 4.75 min (100%); 1H NMR
(DMSO-d6, 300 MHz): d 7.82 (d, 1H, fur-H5), 7.63 (d,
1H, Ph-2H), 7.61 (s, 1H, Ph-5H), 7.35 (dd, 1H, Ph-
6H), 6.98 (d, 1H, fur-H3), 6.62 (dd, 1H, fur-H4), 3.69
(s, 4H, NCH2 · 2), 2.58 (t, 4H, NCH2 · 2). MS
(FAB+) m/z 339 (M+1+, 100%). Anal. Calcd for
C16H26Cl2N2O2Æ1.1C2H2O4: C, 49.88; H, 4.19; N, 6.39;
Cl, 16.18. Found: C, 50.15; H, 4.08; N, 6.41; Cl, 16.0.
6.15. 1-[2-(3,4-Dimethoxyphenyl)ethyl]-4-benzoyl-piper-
azine hydrogen oxalate (15)
Compound 15 was prepared as described for 14, from 26
(0.25 g, 1 mmol) and benzoyl piperazine (0.19 g,
1 mmol). Yield 86%; mp 145–146 ꢁC; HPLC (A:B =
45:55), Rt 13.7 min (100%); 1H NMR (DMSO-d6,
300 MHz): d 7.58 (m, 5H, Ph), 7.00 (d, 1H, Ph-4H),
6.99 (s, 1H, Ph-2H), 6.88 (d, 1H, Ph-5H), 3.87 (s, 3H,
OCH3), 3.84 (s, 3H, OCH3), 3.58 (m, 4H, NCH2 · 2),
3.03 (m, 6H, NCH2 · 3), 2.82 (m, 2H, CH2). MS (APCI)
m/z 355.2 (M+1+, 100%). Anal. Calcd for C21H26N2O3Æ
C2H2O4: C, 62.15; H,6.35; N, 6.30. Found: C, 61.73; H,
6.38; N, 6.23.
6.19. 4-(9-Fluorenyl)-1-(2-furoyl)piperazine (19)
A mixture of 9-bromofluorene (0.32 g, 1.30 mmol) and
1-(2-furoyl)piperazine (0.23 g, 1.30 mmol) in 1-pentanol
(5 mL) was heated under reflux and stirred for 12 h.
Evaporation of the solvent afforded a yellow sticky oil,
which was triturated with petrol spirit (bp 60–80 ꢁC).
The resulting precipitate was collected and purified by
extraction. It was dissolved in a solution of NaHCO3
and extracted with CH2Cl2. Evaporation of the organic
solvent gave a white product, which was recrystallised
from MeOH to give 0.16 g (13%) of final product; mp
161–163 ꢁC; HPLC (A:B = 70:30), Rt = 4.09 min
6.16. 1-[2-(3,4-Dimethoxyphenyl)ethyl]piperidine hydro-
gen oxalate (16)
Compound 16 was prepared as described for 14 except
that no K2CO3 was used, reacting 26 (0.25 g, 1 mmol)
and piperidine (0.85 g, 10 mmol). Yield 57%; mp 194–
1
196 ꢁC; HPLC (A:B = 65:35), Rt 7.93 min (100%); H
NMR (DMSO-d6, 300 MHz): d 6.90 (d, 1H, Ph-5H),
6.88 (s, 1H, Ph-2H), 6.77 (d, 1H, Ph-4H), 3.72 (s, 3H,
OCH3), 3.59 (s, 3H, OCH3), 3.18 (m, 6H, NCH2 · 3),
2.90 (m, 2H, CH2), 1.74 (m, 4H, NCH2 · 2), 1.59 (m,
2H, CH2). MS (ES) m/z 250 (M+, 100%). Anal. Calcd
for C15H23NO2ÆC2H2O4: C, 60.12; H, 7.42; N, 4.13.
Found: C, 59.67; H, 7.49; N, 4.06.
1
(100%); H NMR (CDCl3, 300 MHz): d 7.52–7.62 (d,
4H, Ar-H · 4), 7.20–7.29 (m, 5H, Ar-H · 4 + fur-5H),
6.85 (d, 1H, fur-3H), 6.34 (t, 1H, fur-H4), 4.80 (s, 1H,
NCH), 3.68 (br s, 4H, NCH2 · 2), 2.60 (br s, 4H,
NCH2 · 2). MS (FAB+), m/z 385 [M+H]+. Anal. Calcd
for C22H20N2O2: C, 76.41; H, 5.82; N, 8.18. Found: C,
76.72; H, 5.85; N, 8.13.
6.17. 1-(3,4-Dimethoxybenzenesulfonyl)-4-(2-furoyl)-
piperazine (17)
6.20. Pharmacology
A mixture of 3,4-dimethoxybenzenesulfonyl chloride
(0.37 g, 1.56 mmol), 1-(2-furoyl)piperazine (0.28 g,
1.56 mmol) and triethylamine (2 mL) in anhydrous
CH2Cl2 (8 mL) was stirred at room temperature for
12 h. After cooling a white precipitate was formed and
it was filtered off and the resulting solution evaporated.
The oil was crystallised upon addition of EtOH and the
precipitate was recrystallised twice to give 17. Yield
72%; mp 115–116 ꢁC; HPLC (A:B = 50:50), Rt
The compounds were tested for their ability to inhibit the
uptake of prazosin (at 10À6 M) in GT1-1 immortalised
gonadotrophin-releasing hormone (GnRH) peptidergic
neurones, as has been described in detail.1 Briefly, the cells
were grown at 37 ꢁC in Corning 175 cm2 flasks in culture
medium consisting of Dulbeccoꢀs modified Eagleꢀs
medium (DMEM) and Hamꢀs F-12 (ratio 1:1) containing
10% foetal bovine serum and sodium bicarbonate 3.7 g/L,
in a humidified atmosphere containing 5% CO2 in air.
Culture media were changed at 48-h intervals. After
7 days, the cells were dispersed in the presence of trypsin,
deoxyribonuclease I and ethylenediaminetetraacetic acid
and incubated in Corning or Nunc 12-well plates
(ꢀ2 · 106 cells/well). The wells had been coated with
poly-D-lysine (2.5 lg/cm2; Sigma P-6407; MW 70,000–
1
9.19 min (100%); H NMR (CDCl3, 300 MHz): d 7.38
(d, 1H, fur-H5), 7.29 (dd, 1H, Ph-2H), 7.12 (d, 1H,
fur-3H), 6.93 (d, 1H, Ph-5H), 6.88 (d, 1H, Ph-6H),
6.39 (dd, 1H, fur-H4), 3.87 (s, 3H, OCH3), 3.85 (s, 3H,
OCH3), 3.81 (t, 4H, NCH2 · 2), 3.01 (t, 4H,
NCH2 · 2). MS (APCI) m/z 381 (M+1+, 100%). Anal.

P. A. Zunszain et al. / Bioorg. Med. Chem. 13 (2005) 3681–3689
3689
150,000) and laminin (0.25 lg/cm2; Sigma L-2020). Drugs
were dissolved in buffer consisting of DMEM with
25 mM HEPES and 0.5 mM sodium ascorbate, pH 7.4.
Uptake studies were performed on the intact cells. After
2–4 days in culture, the cells were washed twice with buf-
fer at 25 ꢁC then incubated at 37 ꢁC for 60 min in the pres-
ence of [3H]prazosin 2 · 10À10 M and unlabelled prazosin
10À6 M. The test compounds were present in the indi-
cated concentrations. At the end of the incubation period,
the culture plates were placed on ice to inhibit the release
of amines.8 After 30 s, the buffer was removed and the
cells were washed twice with buffer at 0 ꢁC. The cold
buffer was then removed and the cells were solubilised
with 2 mL of a warm solution of 0.1% sodium dodecyl sul-
phate and 0.1 M sodium hydroxide. Fifty microlitres
aliquots were removed for protein assay and 10 mL of
scintillation liquid was then added to the cell extract,
mixed and radioactivity was measured in a scintillation
spectrometer with an efficiency of 50%. Protein content
was measured by the bicinchoninic acid modification of
the biuret reaction using albumin standards (Perbio,
Chester, Cheshire, England). Nonspecific uptake was
defined as uptake in the presence of the antidepressant
desipramine (at 10À4 M) and specific (desipramine-sensi-
tive) uptake was obtained by subtracting nonspecific from
total uptake. Efficacy was defined as % inhibition of the
uptake of prazosin (at 10À6 M) when the test compound
was used in a concentration of 10À4 M. Efficacy was
expressed as % of the effect of a maximal inhibitory con-
centration of desipramine (10À4 M). Half-maximal inhib-
itory concentrations (IC50 values) were calculated from
the concentration–response curves. IC50 values were cal-
culated only for compounds, which achieved a maximal
inhibitory response, defined as 90% of the inhibitory effect
of desipramine (10À4 M). When a compound did not
achieve the maximal inhibitory response, IC50 values were
not calculated and the data were expressed only as efficacy
(% inhibition relative to desipramine 10À4 M). The experi-
ments were carried out in triplicate and each experiment
was performed twice; the data are therefore presented as
the mean SEM of six observations.
come Trust (Sir Henry Wellcome Commemorative
Award for Innovative Research). We also gratefully
acknowledge Mr. Wasyl Tertiuk for his generous help
in this work.
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Acknowledgements
Financial support was gratefully received from Funda-
cion Antorchas, and CONICET (Argentina), CNR
(Council of National Research—Italy) and the Well-
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