Searching for cyclazosin analogues as α1B-adrenoceptor antagonists

Article abstract of DOI:10.1016/S0014-827X(02)00025-3

A series of quinazoline derivatives, 2-20, structurally related to the racemic α₁-adrenoceptor antagonist cyclazosin (1), were synthesized and evaluated for their functional antagonism at α₁- and α₂-adrenoceptors and for t

Full text of DOI:10.1016/S0014-827X(02)00025-3

Il Farmaco 58 (2003) 477Á487
/
www.elsevier.com/locate/farmac
Searching for cyclazosin analogues as a1B-adrenoceptor antagonists
Dario Giardina` a,*, O. Polimanti a, G. Sagratini a, P. Angeli a, U. Gulini a, G. Marucci a,
C. Melchiorre b, E. Poggesi c, A. Leonardi c
a Department of Chemical Sciences, University of Camerino, Via S. Agostino, 1 62032 Camerino, Italy
b Department of Pharmaceutical Sciences, University of Bologna, Bologna, Italy
c Pharmaceutical R&D Division, Recordati SpA, Milan, Italy
Received 29 July 2002; accepted 14 September 2002
Abstract
A series of quinazoline derivatives, 2Á20, structurally related to the racemic a1-adrenoceptor antagonist cyclazosin (1), were
/
synthesized and evaluated for their functional antagonism at a1- and a2-adrenoceptors and for their binding affinity at human
cloned a1a-, a1b- and a1d-adrenoceptor subtypes. They displayed, like 1, preferential antagonism and selectivity for a1 versus a2-
adrenoceptors. Compounds 10, 13, and 18 showed high potency at a1-adrenoceptors similar to that of 1 (pKB values 8.47Á
/
8.89
versus 8.67), whereas 13 and 15 were endowed with the highest a1-adrenoceptor selectivity, only 3- to 4-fold lower than that of 1. In
binding experiments, all of the compounds displayed an affinity practically similar to that found for 1, with the exception of 19 and
20 that were definitely less potent. The s-triazine analogue 18 was the most potent of the series with pKi values of 10.15 (a1a), 10.22
(a1b) and 10.40 (a1d), resulting 77-fold more potent than 1 at a1a-adrenoceptors. In addition, the majority of compounds, like
prototype 1, showed the same trend of preferential affinity for a1d- and a1b-adrenoceptors that a1a-subtype. In conclusion, we
identified compounds 2Á
interesting subtype-selectivity profile, which makes them suitable candidates for their resolution as enantiomers structurally related
to (ꢀ)-cyclazosin.
/5, 10, 12 and 13, bearing either an aliphatic- or an arylalkyl- or aryloxyalkyl-acyl function, with an
/
´
# 2002 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
Keywords: a1-Adrenoceptor subtypes; a1-Adrenoceptor antagonists; Quinazoline compounds; Cyclazosin; Cyclazosin analogues
1. Introduction
[4]. However, the functional role of specific a1-adreno-
ceptor subtypes is not completely defined. Many phar-
macological evidences indicate that the a1A
a1-Adrenoceptors constitute a heterogeneous family
of receptors belonging to the superfamily of G-protein
coupled receptors. At present, three native and func-
tional subtypes, a1A, a1B and a1D, can be pharmacolo-
gically distinguished which exhibit equivalency to the
cloned a1a-, a1b- and a1d-adrenoceptors expressed in
various tissues and cell lines [1]. Also a fourth a1-
adrenoceptor, a1L, displaying low affinity for prazosin
[2] and conformationally related to the a1A-subtype [3],
has been reported.
-
adrenoceptor is the most important subtype in mediat-
ing the human prostate smooth muscle contraction
making it a potential important target for treatment of
benign prostatic hypertrophy (BPH) [5]. In addition,
functional studies seem to indicate that, in animal
species, the a1A- and a1D-adrenoceptors are those
mainly involved in the contraction of main arteries,
whereas the a1B-subtype controls the small resistance
vessels [6]. On the contrary, available information
regarding a1-adrenoceptor subtypes mediating vasocon-
striction in humans is still very scarce [6].
a1-Adrenoceptors own therapeutic interest because of
their important role in control of blood pressure and
contraction and growth of smooth and cardiac muscle
Thus, the quantitative evaluation of subtype dis-
tribution and the definition of the proper functional role
in different tissues need further studies with new potent
and subtype-selective a1-adrenoceptor ligands. Particu-
* Corresponding author.
E-mail address: dario.giardina@unicam.it (D. Giardina`).
´
0014-827X/02/$ - see front matter # 2002 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
doi:10.1016/S0014-827X(02)00025-3

478
D. Giardina` et al. / Il Farmaco 58 (2003) 477Á487
/
larly, selective a1B- and a1D-antagonists are needed
because, in comparison with a1A antagonists, only a
few of them are available.
2. Chemistry
As shown in Scheme 1, the 6,7-dimemoxy-2-cis-
perhydro-1-quinoxalinyl-4-quinazolinamine 19 was first
synthesized and used as common precursor for all other
compounds. It was obtained as already reported [14] by
reaction of 2-chloro-6,7-dimethoxy-4-quinazolinylamine
With respect to a1B-selective ligands, besides the
alkylating agent chloroethylclonidine (CEC) [7], the
compounds spiperone, risperidone and AH11110A
were first indicated as reversible selective ligands, but
their selectivity was lowered or not confirmed in
subsequent experiments [8]. In contrast, the chiral 2,4-
diaminoquinazoline compound L-765,314 was shown to
be a1B-selective both in binding and functional experi-
ments [9]. In addition, we recently discovered [10] an
with the cis-perhydroquinoxaline [15]. Compounds 2Á
/
11, 12Á15 [14], and 16 were synthesized by acylation of
/
19 with appropriate acyl chloride in dichloromethane
and in presence of triethylamine. Most of acyl chlorides
were commercial, other were synthesized as reported in
literature or by standard procedure. The alkylation of 19
with cyanuric acid and with 2-chloro-4,6-dimethoxy-s-
triazine [16], in basic medium, gave the respective
triazine analogues 17 and 18. Finally, the methyl
derivative 20 was synthesized, by reductive alkylation
of 19 with formaldehyde and sodium cyanoborohydride,
in acetonitrile. With the exception of 17 and 18 all
compounds were obtained as hydrochloride salt and
purified by crystallization with proper solvent.
other chiral quinazoline derivative, (ꢀ
dowed with high binding affinity (pKiꢁ
/
)-cyclazosin, en-
9.16Á9.87) and
/
/
selectivity for native and cloned a1B/b-adrenoceptor with
respect to the a1A/a- and a1d-subtypes (up to 90- and 40-
fold, respectively). However, a functional study in
isolated rat tissues reported that (ꢀ)-cyclazosin was
/
endowed with low antagonist activity at all three a1-
adrenoceptors, while being unexpectedly devoid of
selectivity for the a1B-subtype [11]. At present, a further
pharmacological investigation is in progress to clarify
the discrepancy observed between binding and func-
tional results obtained for (ꢀ
/
)-cyclazosin. In compar-
ison with the (ꢀ)-enantiomer, racemic cyclazosin, ([4-(4-
/
3. Biology
amino-6,7-dimethoxyquinazolin-2-yl)-cis-perhydroqui-
noxalin-1-yl]furan-2-ylmethanone), (1) (Fig. 1) dis-
played a moderate selectivity towards two specific
subtypes. In particular it showed a preferential binding
affinity for cloned a1d- and a1b- adrenoceptors relative
to the a1a-subtype both in animal and human species
(10- to 15-fold [12] and 17- to 32-fold [13], respectively).
3.1. Functional experiments
Compounds 2Á20 were studied in animal isolated
/
tissues in order to test their a-adrenoceptor antagonist
activity and selectivity, in comparison with cyclazosin
used as reference. The a1-adrenoceptor antagonism was
assessed evaluating the inhibition of the noradrenaline
(NA) induced contraction of guinea pig spleen, a tissue
expressing the a1B-subtype [17]. The a2-adrenoceptor
blocking activity was determined by antagonism of the
clonidine-induced inhibition of the twitch response on
field-stimulated prostatic portion of rat vas deferens
[18,19].
The a1-adrenoceptor antagonist potency was ex-
pressed as pA2 values according to Arunlakshana and
Schild [20,21]. However, when the slope of the Schild
plot was significantly different from unity, the potency
was expressed as pKB values, according to van Rossum
[22]. The pKB values, representing the negative loga-
rithm of the apparent dissociation constant KB, were
calculated at the lowest antagonist concentration giving
The aim of present work was to search for new (ꢀ)-
/
cyclazosin analogues possibly endowed with increased
affinity and selectivity for a1b-adrenoceptors. The ap-
proach to this project consisted in the synthesis and the
pharmacological evaluation at a1-adrenoceptor sub-
types of a number of racemic compounds, bearing
different substituents as an alternative to the 2-furoyl
moiety of cyclazosin. Among these racemic derivatives,
molecules displaying a pharmacological profile compar-
able or, hopefully, better than that of racemic cyclazosin
will be selected for a subsequent development as
optically pure enantiomers.
Here we report the synthesis and the pharmacological
data of racemic cyclazosin analogues (2Á
the 2-furoyl moiety of cyclazosin has been exchanged for
aliphatic and alicyclic acyl groups (2Á6), a benzoyl
/
20), in which
/
function (7), a series of substituted and non-substituted
arylalkyl-, benzyloxy- or phenoxyalkyl-carbonyl groups
a significant rightward shift of the NA concentrationÁ
response curve.
/
(8Á
/
13), 5-methyl-2-furyl-, tetrahydro-2-furyl-, chroman-
16), disubstituted s-triazines
At a2-adrenoceptor the antagonism (pKB) was eval-
uated at a single concentration for compound 2, whereas
only an estimated affinity was given for the majority of
compounds because of their low activity.
2-yl-carbonyl groups (14Á
(17 and 18), an hydrogen atom (19), or a methyl group
(20) (Fig. 1).
/

D. Giardina` et al. / Il Farmaco 58 (2003) 477Á
/487
479
Fig. 1.
3.2. Binding studies
The pharmacological results are reported in Tables 1
and 2.
The pharmacological profile of studied compounds
was also evaluated in binding assays on cloned a1-
adrenoceptors, in comparison with the standard cycla-
zosin. Competition assays were performed using
[3H]prazosin to label cloned human a1-adrenoceptors
on membranes prepared from chinese hamster ovary
(CHO) cells expressing human a1a-, a1b- and a1d-
subtypes [23]. Binding affinities were expressed as pKi
4. Results and discussion
Compounds 2Á20 inhibited a1-adrenoceptors in a
/
non-competitive manner with the exception of 7, 14
and 15, which in the guinea pig spleen behaved as
competitive antagonists, albeit in a narrow range of
concentrations (10-fold) (Table 1). They displayed, like
cyclazosin (1), a preferential antagonism and selectivity
for a1- versus a2-adrenoceptors. The majority of com-
pounds were less potent than 1 with pKB values ranging
values derived using the ChengÁ
Experimental data were subjected to statistical analy-
sis by means of Student’s t-test. A P value B0.05 was
taken to indicate a statistically significant difference.
/Prusoff equation [24].
/

480
D. Giardina` et al. / Il Farmaco 58 (2003) 477Á487
/
Scheme 1. Reagents: (a) i-AmOH, reflux; (b) RCOCl, Et3N, CH2Cl2; (c) cyanuric chloride, Na2CO3, CH2Cl2 for 17; 2-chloro-4,6-dimethoxy-s-
triazine, Et3N, CH2Cl2 for 18; (d) 37% formaldehyde, NaBH3CN, CH3CN. Specifications for R in 2Á16 are as reported in Fig. 1.
/
from 6.52 to 7.99. However, compounds 10, 13, and 18
showed high a1-adrenoceptor antagonism similar to that
selectivity, at all three subtypes. Their affinity at a1b-
and a1d-adrenoceptor was comparable with that of 1
and other acylated compounds but was 16- to 77-fold
higher at a1a-subtype. The 4,6-dimethoxy-substituted
triazine 18 was the most potent compound of the series
at a1a-, a1b- and a1d-adrenoceptors with pKi values of
10.15, 10.22 and 10.40, respectively. On the contrary, the
N-unsubstituted compound 19 and its N-methyl deri-
vative 20 showed very low affinity at all three subtypes.
This latter result clearly suggests that a carboxamide
displayed by 1 (pKBꢁ8.89, 8.71, 8.47 and 8.67, respec-
/
tively). Compounds 13 and 15 were the most selective
antagonists for a1-adrenoceptors.
Binding experiments on cloned human a1a, a1b and
a1d-adrenoceptor subtypes showed that all the acylated
and s-triazine non-acylated cyclazosin analogues dis-
played an affinity similar to that found for 1 (Table 2).
Among the acylated analogues, the affinity at the a1a-
subtype was slightly higher (2- to 9-fold) than that of 1,
with the exception of the short-chain aliphatic acyl
function (2Á16) or an electron rich moiety like an s-
/
triazine ring (17 and 18) may play an important role in
the interaction of these cyclazosin-related compounds
with the receptor.
analogues 2Á4 and the 2-naphtylmethyl-carbonyl com-
/
pound 10, which were almost equiactive to 1. At the a1b-
subtype, the majority of compounds were practically as
potent as 1, whereas compounds 2, 4, 8 and 10 were
slightly less active (4 to 8-fold). Similarly, most of the
compounds displayed at a1d-adrenoceptors an affinity
similar to that of cyclazosin with the exception of 4 and
16, which were about 5-fold less potent than 1, and
compound 13, which resulted 3-fold more potent.
Altogether, the binding results indicate that the
affinity for a1b- and a1d-adrenoceptors can be retained
by exchanging the hydrogen atom of the piperazine NH
function of compound 19 for a variety of substituents,
supporting the view of a receptor binding site with no
particular constraint for the substituent linked to the
piperazine ring. On the contrary, the a1a-adrenoceptor
would express a more specific structural demand in the
N-substituent for optimum interaction, as revealed by
Concerning the non-acylated analogues 17Á
/
20, s-
triazines 17 and 18 showed high affinity, but not

D. Giardina` et al. / Il Farmaco 58 (2003) 477Á
/487
481
Table 1
resolution in the chiral form structurally related to
(ꢀ)-cyclazosin.
Functional a1- and a2-adrenoceptor antagonist activity of compounds
2-20, and cyclazosin (1) as reference, at guinea pig spleen and prostatic
rat vas deferens, respectively
/
Comp. a Adrenoceptors
pKB a1 versus NA a pKB a2 versus clo- a1/a2
Selectivity ratio c
5. Experimental
nidine b
1
8.679
7.569
7.059
7.939
7.999
7.499
7.749
7.339
7.869
8.899
6.529
7.369
8.719
7.959
7.779
7.379
7.889
8.479
6.889
7.179
/
0.06 d
0.17
0.08
0.04
0.05
0.04
0.06 f
0.10
0.08
0.07
0.05
0.05
0.07
0.03 g
0.02 h
0.06
0.12 i
0.13
0.05
0.04
4.909
/
0.03 e
0.18 e
5890
371
2
/
4.999
/
5.1. Chemistry
3
/
B/5
ꢀ
/
112
850
295
93
4
/
B/5
ꢀ
/
Melting points were taken in glass capillary tubes on a
Buchi SMP-20 apparatus and are unconnected. IR and
5
/
B
/
5.52
5.52
5.52
5.52
6
ꢀ
/
6
/
B
/
ꢀ/
7
/
B
/
ꢀ
ꢀ/
/
166
64
NMR spectra were recorded on PerkinÁElmer 297 and
/
8
/
B
/
Varian VXR 300 instruments, respectively. The IR
spectra, not included, were consistent with all the
assigned structures. The elemental analyses of com-
pounds agreed with the calculated values within the
9
/
B/
ꢀ/
72
10
11
12
13
14
15
16
17
18
19
20
/
B/6
ꢀ
ꢀ/
/
776
10
/
B
/
5.52
5.52
5.52
5.52
4.52
5.52
5.52
5.52
5
/
B
/
ꢀ/
69
/
B
/
ꢀ
/
1549
269
1780
71
range 9
/
0.4%. Chromatographic separations were per-
/
B
/
ꢀ
/
formed on silica gel columns (Kieselgel 40, 0.040Á
/0.063
/
B
/
ꢀ
/
mm, Merck) by flash chromatography, whereas the
analytical control was made with silica gel TLC plates
(Kieselgel 60 F254, layer thickness 0.25 mm, Merck). The
composition and volumetric ratio of eluting mixtures
/
B
/
ꢀ/
/
B
/
ꢀ
/
229
891
76
/
B
/
ꢀ
/
/
B/
ꢀ/
/
B/5
ꢀ/
148
were: (A) petrol etherÁ
ammonia (8:6:2:0.2); (B) ethyl acetateÁ
chloroform (6:3:1); (C) ethyl acetateÁpetrol etherÁ
methanolÁ7% ammonia (7:7:1:0.05); (D) cyclohexaneÁ
ethyl acetateÁmethanolÁ7% ammonia (6:3:1:0.05); (E)
petrol etherÁethyl acetateÁmethanolÁ7% ammonia
(9:7:1:0.1); (F) ethyl acetateÁcyclohexaneÁmethanol
(6:3:1); (G) petrol etherÁethyl acetateÁmethanolÁ14%
ammonia (8:8:2:0.2); (H) petrol etherÁ
methanolÁ14% ammonia (8:5:0.6:0.025);
petrol etherÁethyl acetateÁmethanolÁ
(8:4:1:0.05); (L) ethyl acetateÁpetrol etherÁ
28% ammonia (8:8:2:0.1); (M) petrol etherÁ
ateÁmethanolÁ14% ammonia (8:6:2:0.1);
petrol etherÁethyl acetateÁmethanolÁ7% ammonia
(8:6:2:0.2); (O) ethyl acetateÁdichloromethaneÁacetone
(5:3:2); (P) ethyl acetateÁcyclohexaneÁmethanol
(6.5:3:0.5); (Q) chloroformÁmethanol (9:1); (R)
petrol etherÁchloroformÁmethanolÁ28% ammonia
(8:6:2:0.15). Petroleum ether refers to the fraction with
a boiling point of 40Á60 8C. The term ‘dried’ refers to
the use of anhydrous sodium sulfate. Compounds were
named following IUPAC rules as applied by ACD/
Name, a PC IUPAC name generator, version 1997,
Advanced Chemistry Development, Toronto, Canada.
Most of acyl chlorides were commercial, other were
prepared as indicated in the literature. The 2-(2,6-
dimethoxyphenyl)ethanoyl chloride and 2-(2-isopropyl-
6-methoxyphenoxy)ethanoyl chloride were synthesized
as usual with SOCl2 in dry benzene starting from the
corresponding carboxylic acid.
/
ethyl acetateÁ
/
methanolÁ
/
28%
a
pKB values 9
log[B] [22], at the lowest significant antagonist concentration: 1
mM (3Á5, 11, 19, 20), 0.1 mM (2, 6, 8, 12, 16, 17), 0.03 mM (9), 0.01 mM
/
SE were calculated by the equation pKBꢁ
/
log(DRꢂ
/
/
methanolÁ
/
1)ꢂ
/
/
/
/
/
/
(1, 3, 13, 18), 0.003 mM (10), tested three to six times.
b
/
/
Estimated pKB values because compounds were inactive at low
concentrations whereas at higher concentrations (from 3 to 100 mM)
they gave inhibition of the twitch responses of the electrically
stimulated tissue.
/
/
/
/
/
/
/
/
c
Calculated by the antilog of the difference between pKB values at
a1 and a2-adrenoceptors.
/
ethyl acetateÁ
/
d
/
(I)
Calculated at the concentration 0.01 mM.
e
Calculated at the concentration 30 mM.
/
/
/
7% ammonia
methanolÁ
ethyl acet-
(N)
f
Normalized pA2 value; nꢁ
/
ꢂ/
1.059
0.959
0.9490.10.
A concomitant 30% depression of NA curve was observed.
/0.21.
/
/
/
g
h
i
Normalized pA2 value; nꢁ
/
ꢂ
/
/
0.12.
/
Normalized pA2 value; nꢁ
/
ꢂ/
/
/
/
/
/
/
/
/
the high affinity observed for 17 and 18, which bear a
substituted s-triazine moiety.
/
/
/
Concerning the selectivity towards a1-adrenoceptor
subtypes, the majority of compounds showed a trend of
preferential affinity for a1d- and a1b-adrenoceptors, in
analogy with the precursor cyclazosin. In particular the
acetyl and the benzyloxycarbonyl analogues, 2 and 12,
displayed a slight to moderate selectivity for a1d-
adrenoceptors over a1a- and a1b-subtypes (96- to 13-
fold and 13- to 6-fold, respectively).
/
/
/
/
In conclusion, we identified compounds 2Á5, 10, 12
/
and 13, bearing either an aliphatic acyl function or an
arylalkyl- or aryloxyalkyl-carbonyl group, that, in
binding assays, showed an interesting subtype-selectivity
profile, which makes them suitable candidates for

482
D. Giardina` et al. / Il Farmaco 58 (2003) 477Á487
/
Table 2
a
Binding affinity constants, expressed as pKi of compounds 2Á
/
20, and cyclazosin (1) as reference, for human cloned a1-adrenoceptor subtypes
Comp
a1-Adrenoceptors
b
pKi
Selectivity ratio
a1a
a1b
a1d
a1d/a1a
a1b/a1a
a1d/a1b
1
8.269
7.749
8.299
8.079
8.779
9.179
9.139
8.939
9.219
8.069
8.899
8.839
9.119
8.909
8.549
8.859
9.469
10.159
/
0.05
0.08
0.03
0.10
0.04
0.01
0.04
0.01
0.06
0.12
0.03
0.01
0.06
0.02
0.02
0.05
0.03
0.01
9.499
8.619
9.159
8.929
9.749
9.689
9.579
8.959
9.499
8.979
9.389
9.199
9.799
9.629
9.389
9.429
8.869
10.229
7.009
7.139
/
0.01
0.06
0.04
0.06
0.07
0.03
.05
9.779
9.729
9.649
9.069
9.929
9.779
10.059
9.689
9.519
9.369
9.749
9.969
10.309
9.969
9.429
9.149
9.629
10.409
6.969
7.459
/
0.04
0.01
0.03
0.02
0.01
0.03
0.02
0.01
0.04
0.06
0.02
0.03
0.10
0.01
0.02
0.07
0.04
0.02
0.05
0.05
31
96
22
10
14
4
17
7
2
13
3
2
/
/
/
3
/
/
/
7
4
/
/
/
7
1.5
5
/
/
/
9
1.5
1
6
/
/
/
3
7
/
/
/
8
3
3
8
/
/
0.09
0.02
.02
/
5
1
5
9
/
/
/
2
2
1
10
11
12
13
14
15
16
17
18
19
20
/
/
/
20
7
8
2
/
/
0.05
0.04
0.04
0.07
0.01
0.01
0.11
0.02
0.01
0.01
/
3
2
/
/
/
13
16
11
8
2
6
/
/
/
5
3
/
/
/
5
2
/
/
/
7
1
/
/
/
2
4
0.5
6
/
/
/
1.5
2
0.2
1
/
/
/
1.5
1
B/6
/
/
ꢀ/9
ꢀ
/
10
1
7.059
/
0.08
/
/
2
2
a
Equilibrium dissociation constants (Ki) were derived from IC50 values using the ChengÁ
/
Prusoff equation [24]. The affinity estimates were derived
from displacement of [3H]prazosin binding from a1-adrenoceptors. pKi values are the mean 9
performed in triplicate.
/SE of two to three separate experiments each
b
The selectivity ratio is the antilog of the difference between pKi values at different a1-adrenoceptor subtypes.
5.1.1. 6,7-Dimethoxy-2-cis-perhydro-1-quinoxalinyl-4-
quinazolinamine dihydrochloride (19)
cooled (0 8C) solution of 19 free base (0.5 g, 1.4 mmol)
and Et3N (0.2 g, 2.1 mmol) in dry CH2C12 (10 ml). The
mixture was stirred 3 h at room temperature (r.t.) then
cooled at 0 8C for a night. The precipitate was collected
and purified by column chromatography eluting with
the appropriate solvent mixture. The eluted free base
was transformed into the hydrochloride salt and crystal-
lized from specific solvent. In the case of compounds 7
and 8 the initial precipitate obtained after cooling was
transformed into the hydrochloride salt and crystallized.
Concerning compound 13, after 3 h stirring the reaction
mixture was distilled and the residue purified by column
chromatography.
A mixture of 2-chloro-6,7-dimethoxy-4-quinazolina-
mine (7.85 g, 32.80 mmol), cis-perhydroquinoxaline
(11.5 g, 82 mmol), triethylamine (13.3 g, 131 mmol)
and dimethylamino pyridine (0.4 g, 3.2 mmol) was
refluxed for 72 h in i-AmOH (80 ml) then the solvent
removed in vacuo and the residue purified by column
chromatography eluting with mixture of solvent A. The
coarse solid was transformed into the hydrochloride salt
and crystallized from MeOHÁ
14.7 g (73%) of product as dihydrochloride, m.p.: 290Á
294 8C.1H NMR (DMSO-d6): d 1.35Á
2.40 (m, 8H, H5Á8
perhydroquinoxaline), 3.00Á3.70 (m, 4H, H2Á3 perhy-
droquinoxaline), 3.86 (s, 3H, OCH3), 3.90 (s, 3H,
OCH3), 4.63Á4.86 (m, 1H, H4a perhydroquinoxaline),
4.92Á5.12 (m, 1H, H8a perhydroquinoxaline), 7.68 (s,
/i-PrOH (1:1). Obtained
/
/
/
/
5.1.3. 1-[4-(4-Amino-6,7-dimethoxy-2-quinazolinyl)-cis-
perhydro-1-quinoxalinyl]-1-ethanone hydrochloride (2)
Acetyl chloride was used; eluting mixture B; 69%
/
1H, arom), 7.65 (s, 1H, arom), 8.77 (s, br, 1H, NH
exchangeable with D2O), 9.00 (s, br, 1H, NH exchange-
able with D2O), 9.56 (s, br, 1H, NH exchangeable with
D2O), 9.95 (s, br, 1H, NH exchangeable with D2O),
12.70 (s, br, 1H, NH exchangeable with D2O). Anal.
yield; m.p.: 212Á
d6): d 1.30Á2.30 (m, 11H, H5Á8 perhydroquinoxaline
and CH3CO), 3.60Á4.40 (m, 11H, H2Á3 and H4a
perhydroquinoxaline, OCH3), 4.60Á4.80 (m, 1H, H8a
/
215 8C (i-PrOH);1H NMR (DMSO-
/
/
/
(C18H25N5O2×
/
2HCl×/2.5H2O) (C, H, N).
perhydroquinoxaline), 7.65 (s, 1H, arom), 7.78 (s, 1H,
arom), 8.64 (s, br, 1H, NH exchangeable with D2O),
8.96 (s, br, 1H, NH exchangeable with D2O), 12.21 (s,
br, 1H, NH exchangeable with D2O). Anal.
5.1.2. General procedure for the synthesis of 2Á
/
16
The proper acyl chloride (1.47 mmol) was dissolved in
dry CH2Cl2 (5 ml) and added dropwise to a stirred and
(C20H27N5O3×
/
HCl×
/
0.25i-PrOH×
/
2.5H2O) (C, H, N).

D. Giardina` et al. / Il Farmaco 58 (2003) 477Á
/487
483
5.1.4. 1-[4-(4-Amino-6,7-dimethoxy-2-quinazolinyl)-cis-
perhydro-1-quinoxalinyl]-2-methyl-1-propanone
hydrochloride (3)
(s, 1H, arom), 8.68 (s, br, 1H, NH exchangeable with
D2O), 8.90 (s, br, 1H, NH exchangeable with D2O),
11,96 (s, br, 1H, NH exchangeable with D2O). Anal.
The 2-methylpropanoyl chloride was used; eluting
(C25H35N5O3×
/
HCl×
/
0.25H2O) (C, H, N).
mixture C; 22% yield; m.p.: 275Á
NMR (DMSO-d6): d 0.98Á1.12 (m, 6H, CH(CH3)2),
1.30Á
3.03 (m, 1H, CH(CH3)2), 3.65Á
/
278 8C (EtOH);1H
/
5.1.8. [4-(4-Amino-6,7-dimethoxy-2-quinazolinyl)-cis-
perhydro-1-quinoxalinyl](phenyl)methanone
hydrochloride (7)
/
2.25 (m, 8H, H5Á8 perhydroquinoxaline), 2.78Á
/
/4.15 (m, 10H, OCH3
and H2Á3 perhydroquinoxaline), 4.20Á/4.38 (m, 1H, H4a
Benzoyl chloride was used; 43% yield; m.p.: 280Á
282 8C (EtOH); 1H NMR (DMSO-d6): d 1.40Á
1.61
(m, 4H, H5Á8 perhydroquinoxaline), 1.70Á2.12 (m, 4H,
H5Á8 perhydroquinoxaline), 3.70Á4.19 (m, 10H, H2Á3
perhydroquinoxaline, OCH3), 4.32Á4.44 (m, 1H, H4a
perhydroquinoxaline), 4.61Á4.80 (m, 1H, H8a perhydro-
quinoxaline), 7.43Á7.62 (m, 6H, arom), 7.75 (s, 1H,
/
perhydroquinoxaline), 4.55Á4.72 (m, 1H, H8a perhydro-
/
/
quinoxaline), 7.50 (s, 1H, arom), 7.73 (s, 1H, arom), 8.68
(s, br, 1H, NH exchangeable with D2O), 8.88 (s, br, 1H,
NH exchangeable with D2O), 11.91 (s, br, 1H, NH
/
/
/
exchangeable with D2O). Anal. (C22H31N5O3×
/
HCl×
/
/
H2O) (C, H, N).
/
arom), 8.67 (s, br, 1H, NH exchangeable with D2O),
8.89 (s, br, 1H, NH exchangeable with D2O), 11.74 (s,
br, 1H, NH exchangeable with D2O). Anal.
5.1.5. 1-[4-(4-Amino-6,7-dimethoxy-2-quinazolinyl)-cis-
perhydro-1-quinoxalinyl]-2,2-dimethyl-1-propanone
hydrochloride (4)
(C25H29N5O3×
/
HCl×/0.5H2O) (C, H, N).
The 2,2-dimethylpropanoyl chloride was used; eluting
mixture C; 23% yield; m.p.: 308Á
PrOH);1H NMR (DMSO-d6): d 1.20Á
5Á8 perhydroquinoxaline and C(CH3)3), 2.10Á
1H, H5Á8 perhydroquinoxaline), 3.65Á4.00 (m, 9H,
OCH3 and H2Á3 perhydroquinoxaline), 4.08Á4.34 (m,
2H, H2Á3 and H4a perhydroquinoxaline), 4.53Á4.75 (m,
1H, H8a perhydroquinoxaline), 7.40 (s, 1H, arom), 7.70
(s, 1H, arom), 8.62 (s, br, 1H, NH exchangeable with
D2O), 8.81 (s, br, 1H, NH exchangeable with D2O),
11.72 (s, br, 1H, NH exchangeable with D2O). Anal.
/
310 8C (MeOHÁ
2.00 (m, 16H,
2.30 (m,
/
i-
/
5.1.9. 1-[4-(4-Amino-6,7-dimethoxy-2-quinazolinyl)-cis-
perhydro-1-quinoxalinyl]-2-(4-methoxyphenyl)-1-
ethanone hydrochloride (8)
H
/
/
/
The 2-(4-methoxyphenyl)ethanoyl chloride was used;
52% yield; m.p.: 204Á
(DMSO-d6): d 1.30Á2.30 (m, 8H, H5Á8 perhydroqui-
noxaline), 3.57Á4.13 (m, 15H, H2Á3 perhydroquinoxa-
line, OCH3 and CH2CO), 4.25Á4.45 (m, 1H, H4a
perhydroquinoxaline), 4.53Á4.72 (m, 1H, H8a perhydro-
quinoxaline), 6.88 (d, Jꢁ9.0 Hz, 2H, arom), 7.20 (d,
Jꢁ9.0 Hz, 2H, arom), 7.49 (s, br, 1H, arom), 7.77 (s, br,
/
207 8C (EtOH); 1H NMR
/
/
/
/
/
(C23H33N5O3×
/
HCl×
/
0.5H2O×/0.25i-PrOH) (C, H, N).
/
/
5.1.6. 1-[4-(4-Amino-6,7-dimethoxy-2-quinazolinyl)-cis-
perhydro-1-quinoxalinyl]-1-hexanone hydrochloride (5)
Hexanoyl chloride was used; eluting mixture D; 20%
1H, arom), 8.68 (s, br, 1H, NH exchangeable with D2O),
8.90 (s, br, 1H, NH exchangeable with D2O), 11.93 (s,
br, 1H, NH exchangeable with D2O). Anal.
yield; m.p.: 215Á
d6): d 0.90Á1.00 (m, 3H, CH3), 1.12Á
perhydroquinoxaline and CO(CH2)4), 3.60Á
10H, H2Á3 perhydroquinoxaline and OCH3), 4.20Á
(m, 1H, H4a perhydroquinoxaline), 4.55Á4.78 (m, 1H,
/
217 8C (i-PrOH);1H NMR (DMSO-
2.45 (m, 16H, H5Á8
4.18 (m,
4.41
(C27H33N5O4×
/
HCl×/2H2O) (C, H, N).
/
/
/
/
5.1.10. 1-[4-(4-Amino-6,7-dimethoxy-2-quinazolinyl)-
cis-perhydro-1-quinoxalinyl]-2-(2,6-dimethoxyphenyl)-
1-ethanone hydrochloride (9)
The 2-(2,6-dimethoxyphenyl)ethanoyl chloride was
synthesized from the 2-(2,6-dimethoxyphenyl)acetic
acid [25] and SOCl2 by standard method and used for
/
H8a perhydroquinoxaline), 7.52 (s br, 1H, arom), 7.76 (s
br, 1H, arom), 8.64 (s, br, 1H, NH exchangeable with
D2O), 8.89 (s, br, 1H, NH exchangeable with D2O),
11.98 (s, br, 1H, NH exchangeable with D2O). Anal.
(C24H35N5O3×
/
HCl×
/
2H2O) (C, H, N).
reaction. Eluting mixture F; 70% yield; m.p.: 208Á
210 8C (i-PrOH). 1H NMR (DMSO-d6): d 1.30Á
2.37
(m, 8H, H5Á8 perhydroquinoxaline), 3.65 (s, 2H,
CH2CO), 3.68Á4.18 (m, 16H, H2Á3 perhydroquinoxa-
line and OCH3), 4.22Á4.40 (m, 1H, H4a perhydroqui-
noxaline), 4.57Á4.73 (m, 1H, H8a perhydroquinoxaline),
6.62 (d, Jꢁ9.5 Hz, 2H, arom), 7.21 (t, Jꢁ9.5 Hz, 1H,
arom), 7.50 (s, 1H, arom), 7.78 (s, 1H, arom), 8.69 (s, br,
1H, NH exchangeable with D2O), 8.87 (s, br, 1H, NH
exchangeable with D2O), 11.90 (s, br, 1H, NH exchange-
/
/
5.1.7. [4-(4-Amino-6,7-dimethoxy-2-quinazolinyl)-cis-
perhydro-1-quinoxalinyl](cyclohexyl)methanone
hydrochloride (6)
/
/
Cyclohexyl chloride was used; eluting mixture E; 67%
/
yield; m.p.: 283Á
d 1.00Á2.22 (m, 18H, H5Á8 perhydroquinoxaline and
cyclohexyl), 2.52Á2.71 (m, 1H, cyclohexyl), 3.62Á4.15
(m, 10H, H2Á3 perhydroquinoxaline and OCH3), 4.24Á
4.38 (m, 1H, H4a perhydroquinoxaline), 4.60Á4.75 (m,
1H, H8a perhydroquinoxaline), 7.53 (s, 1H, arom), 7.77
/
286 8C (MeOH); 1H NMR (DMSO-d6):
/
/
/
/
/
/
/
able with D2O). Anal. (C28H35N5O5×
/
HCl×/2H2O) (C, H,
N).

484
D. Giardina` et al. / Il Farmaco 58 (2003) 477Á487
/
5.1.11. [4-(4-Amino-6,7-dimethoxy-2-quinazolinyl)-cis-
perhydro-1-quinoxalinyl](2-naphthyl)-1-ethanone
hydrochloride (10)
1.13 (d, Jꢁ
H5Á8 perhydroquinoxaline), 3.20Á
(CH3)2CH), 3.70Á3.95 (m, 11H, H2Á3 perhydroqui-
noxaline and OCH3), 4.00Á4.30 (m, 2H, H2Á3 perhy-
droquinoxaline), 4.31Á4.43 (m, 1H, H4a
perhydroquinoxaline), 4.60 (m, 2H, COCH2), 4.64Á
4.80 (m, 1H, H8a perhydroquinoxaline), 6.83Á6.95 (m,
2H, arom), 7.08 (t, Jꢁ 7.90 Hz, 1H, arom), 7.40 (s, 1H,
/
6.90 Hz, 6H, (CH3)2CH), 1.30Á
/
2.40 (m, 8H,
/
3.45 (m, 1H,
/
The 2-(2-naphthyl)ethanoyl chloride [26] was used;
/
eluting mixture G, 10% yield, m.p.: 256Á
/
258 8C
/
1
(MeOH); H NMR (DMSO-d6): d 1.28Á
H5Á8 perhydroquinoxaline), 3.68Á
perhydroquinoxaline, OCH3 and CH2CO), 4.32Á
(m, 1H, H4a perhydroquinoxaline), 4.60Á4.76 (m, 1H,
H8a perhydroquinoxaline), 7.36Á7.57 (m, 4H, arom),
7.70Á7.97 (m, 5H, arom), 8.62 (s, br, 1H, NH exchange-
/2.32 (m, 8H,
/
/4.18 (m, 12H, H2Á3
/
/
4.48
/
/
arom), 7.75 (s, 1H, arom), 8.70 (s, br, 1H, NH
exchangeable with D2O), 8.88 (s br, 1H, NH exchange-
able with D2O), 11.77 (s br, 1H, NH exchangeable with
/
/
able with D2O), 8.90 (s, br, 1H, NH exchangeable with
D2O), 11.97 (s, br, 1H, NH exchangeable with D2O).
D2O). Anal. (C30H39N5O5×
/
HCl×
/
2.5H2O) (C, H, N).
Anal. (C30H33N5O3×
/
HCl×
/
2H2O) (C, H, N).
5.1.15. [4-(4-Amino-6,7-dimethoxy-2-quinazolinyl)-cis-
perhydro-1-quinoxalinyl](5-methyl-2-furyl) methanone
hydrochloride (14)
5.1.12. (E)-1-[4-(4-amino-6,7-dimethoxy-2-
quinazolinyl)-cis-perhydro-1-quinoxalinyl]-3-phenyl-2-
propen-1-one hydrochloride (11)
The 5-methyl-2-furancarbonyl chloride [28] was used;
eluting mixture L; 27% yield; m.p.: 220Á
PrOH); 1H NMR (DMSO-d6): d 1.20Á
2.10 (m, 7H,
H5Á8 perhydroquinoxaline), 2.30Á2.47 (m, 4H, H5Á8
perhydroquinoxaline and CH3), 3.73Á4.32 (m, 10H,
H2Á3 perhydroquinoxaline, OCH3), 4.38Á4.50 (m, 1H,
H4a perhydroquinoxaline), 4.57Á4.75 (m, 1H, H8a
/
223 8C (i-
The (E)-3-phenyl-2-propenoyl chloride was used;
/
eluting mixture E; 41% yield; m.p.: 246Á
(EtOHÁ
2.37 (m, 8H, H5Á8 perhydroquinoxaline), 3.78Á
/
250 8C
/
/
AcOEt, 1:1); 1H NMR (DMSO-d6): d 1.35Á
/
/
/
4.12
/
(m, 9H, OCH3 and H2Á3 perhydroquinoxaline), 4.20Á
4.40 (m, 1H, H2.3 perhydroquinoxaline), 4.46Á4.60 (m,
1H, H4a perhydroquinoxaline), 4.63Á4.80 (m, 1H, H8a
perhydroquinoxaline), 7.20 (d, Jꢁ16.7 Hz, 1H,
C6H5CH ÄCH), 7.32Á7.48 (m, 4H, arom), 7.56 (d, Jꢁ
16.7 Hz, 1H, C6H5CHÄCH), 7.67Á7.80 (m, 3H, arom),
/
/
/
perhydroquinoxaline), 6.30 (m, 1H, H4 furan), 7.05 (m,
1H, H3 furan), 7.58 (s, 1H, arom), 7.76 (s, 1H, arom),
8.68 (s, br, 1H, NH exchangeable with D2O), 8.90 (s, br,
1H, NH exchangeable with D2O), 11.95 (s, br, 1H, NH
/
/
/
/
/
/
/
exchangeable with D2O). Anal. (C24H29N5O4×
/
HCl×
/
8.70 (s, br, 1H, NH exchangeable with D2O), 8.88 (s, br,
2.5H2O) (C, H, N).
1H, NH exchangeable with D2Q), 11.82 (s, br, 1H, NH
exchangeable with D2O). Anal. (C27H31N5O3×
/
HCl×
/
5.1.16. [4-(4-Amino-6,7-dimethoxy-2-quinazolinyl)-cis-
perhydro-1-quinoxalinyl](tetrahydro-2-furanyl)
methanone hydrochloride (15)
0.75H2O) (C, H, N).
5.1.13. Benzyl 4-(4-amino-6,7-dimethoxy-2-
quinazolinyl)-cis-perhydro-1-quinoxaline carboxylate
hydrochloride (12)
Tetrahydro-2-furancarbonyl chloride [29] was used;
eluting mixture M; 21% yield; m.p.: 220Á
/
223 8C
1
(EtOH); H NMR (DMSO-d6): d 1.24Á/2.35 (m, 12H,
Benzyl chloromethanoate was used; eluting mixture
H; 14% yield; m.p.: 243Á
245 8C (EtOH); 1H NMR
(DMSO-d6): d 1.20Á2.50 (m, 8H, H5Á8 perhydroqui-
noxaline), 3.60Á4.30 (m, 11H, H2Á3 and H4a perhydro-
quinoxaline, OCH3), 4.62Á4.82 (m, 1H, H8a
perhydroquinoxaline), 5.18 (s, 2H, OCH2), 7.25Á7.42
(m, 5H, arom), 7.70 (s, 1H, arom), 7.80 (s, 1H, arom),
8.60 (s, br, 1H, NH exchangeable with D2O), 8.95 (s, br,
1H, NH exchangeable with D2O), 12.18 (s, br, 1H, NH
H5Á8 perhydroquinoxaline and H3Á4 tetrahydrofuran),
3.62Á4.18 (m, 12H, H2Á3 perhydroquinoxaline, H5
tetrahydrofuran and OCH3), 4.22Á4.3 8 (m, 2H, H4a
perhydroquinoxaline and H2 tetrahydrofuran), 4.55Á
4,80 (m, 1H, H8a perhydroquinoxaline), 7.40 (s, 1H,
arom), 7.70 (s, 1H, arom), 8.66 (s, br, 1H, NH
exchangeable with D2O), 8.85 (s, br, 1H, NH exchange-
able with D2O), 11,78 (s, br, 1H, NH exchangeable with
/
/
/
/
/
/
/
/
D2O). Anal. (C23H31N5O4×
/
HCl×
/
2.5H2O) (C, H, N).
exchangeable with D2O). Anal. (C26H31N5O4×
/
HCl×
/
0.75H2O) (C, H, N).
5.1.17. [4-(4-Amino-6,7-dimethoxy-2-quinazolinyl)-cis-
perhydro-1-quinoxalinyl] (3,4-dihydro-2H-2-chromenyl)
methanone hydrochloride (16)
5.1.14. 1-[4-(4-Amino-6,7-dimethoxy-2-quinazolinyl)-
cis-perhydro-1-quinoxalinyl]-2-(2-isopropyl-6-
methoxyphenoxy)-1-ethanone hydrochloride (13)
The 2-(2-isopropyl-6-methoxyphenoxy)ethanoyl chlo-
ride was synthesized from 2-(2-isopropyl-6-methoxyphe-
noxy)acetic acid [27] and SOCl2 by standard method
The 2-chromanecarbonyl chloride [30] was used;
eluting mixture N; 44% yield; m.p.: 238Á
/
240 8C
1
(MeOH); H NMR (DMSO-d6): d 1.02Á
/
2.42 (m, 10H,
H5Á8 perhydroquinoxaline and CHCH2 chromenyl),
2.68Á2.95 (m, 2H, CH2Ar chromenyl), 3.71Á4.40 (m,
11H, H2Á3 and H4a perhydroquinoxaline, OCH3), 4.60Á
4.92 (m, 1H, H8a perhydroquinoxaline), 5.08Á5.27 (m,
/
/
and used for reaction. Eluting mixture I; 21% yield;
1
/
m.p.: 208Á
/
209 8C (i-PrOH). H NMR (DMSO-d6): d
/

D. Giardina` et al. / Il Farmaco 58 (2003) 477Á
/487
485
1H, CH chromenyl), 6.80Á
/
6.94 (m, 2H, arom), 7.02Á
/
NaBH3CN (0.073 g, 1.16 mmol) and acidification (pH
7.20 (m, 2H, arom), 7.61 (s, 1H, arom), 7.75 (s, 1H,
arom), 8.66 (s, br, 1H, NH exchangeable with D2O),
8.91 (s, br, 1H, NH exchangeable with D2O), 12.15 (s,
br, 1H, NH exchangeable with D2O). Anal.
6.5) with acetic acid. The mixture was stirred at r.t. for 1
h then the solvent removed and the residue purified by
column chromatography eluting with the mixture R.
The free base was transformed into the hydrochloride
(C28H33N5O4×
/
HCl×
/
0.5H2O) (C, H, N).
salt and crystallized from MeOHÁ
tained 0.08 g (31%); m.p.: 290Á
293 8C; 1H NMR
(DMSO-d6): d 1.40Á1.95 (m, 6H, H5Á8 perhydroqui-
noxaline), 2.20Á2.45 (m, 2H, H5Á8 perhydroquinoxa-
line), 2.80 (s, 3H, CH3), 3.10Á3.70 (m, 4H, H2Á3
perhydroquinoxaline), 3.87 (s, 3H, OCH3), 3.90 (s, 3H,
OCH3), 4.70Á4.95 (m, 1H, H4a perhydroquinoxaline),
5.10Á5.40 (m, 1H, H8a perhydroquinoxaline), 7.80 (s,
/
i-PrOH (1:1). Ob-
/
5.1.18. 2-[4-(4,6-Dichloro-1,3,5-triazin-2-yl)-cis-
perhydro-1-quinoxalinyl]-6,7 dimethoxy-4-
quinazolinamine (17)
Compound 19 (0.5 g, 1.4 mmol) and Na2CO3 (0.16 g,
1.5 mmol) were added at 0Á
/
/
/
/
5 8C to a solution of 2,4,6-
/
trichloro-1,3,5-triazine (0.36 g, 1.95 mmol) in waterÁ
/
/
acetone (14/10 v/v). The mixture was stirred at r.t. for
1 h then the organic solvent evaporated and the residue
extracted with chloroform. The removal of dried solvent
gave a crude product that was purified by column
chromatography eluting with mixture O. Obtained 0.9 g
1H, arom), 7.85 (s, 1H, arom), 8.78 (s, br, 1H, NH
exchangeable with D2O), 9.10 (s, br, 1H, NH exchange-
able with D2O), 11.20 (s, br, 1H, NH exchangeable with
D2O), 13.06 (s, br, 1H, NH exchangeable with D2O).
Anal. (C19H27N5O2×
/
2HCl×/3H2O) (C, H, N).
(63%) of product; m.p.: 137Á
(CDCl3-d6): d 1.36Á2.17 (m, 7H, H5Á8 perhydroqui-
noxaline), 2.32Á2.55 (m, 1H, H5Á8 perhydroquinoxa-
line), 3.70Á3.88 (m, 2H, H2Á3 perhydroquinoxaline),
3.94 (s, 3H, OCH3), 3.98 (s, 3H, OCH3), 4.0Á4.20 (m,
1H, H2Á3 perhydroquinoxaline), 4.56Á4.80 (m, 3H,
2Á3, H4a and H8a perhydroquinoxaline), 5.14 (s, br,
2H, NH exchangeable with D2O), 6.81 (s, 1H, arom),
6.93 (s, 1H, arom). Anal. (C21H24Cl2N8O2×0.5C4H8O2)
(C, H, N).
/
140 8C (dec); 1H NMR
/
5.2. Biology
/
/
5.2.1. Functional antagonism in isolated tissues
Male guinea-pigs (250Á300 g) and male albino rats
(CD, BR, 125Á150 g) were used for the experiments at
/
/
/
/
H
a1- and a2-adrenoceptor, respectively. Animals were
killed by cervical dislocation, the required organs
isolated, freed from adhering connective tissue and set
up rapidly, under a suitable tension, in 20 ml organ
baths containing physiological salt solution (pH 7.4)
kept at 37 8C and aerated with 5% CO2: 95% O2.
/
5.1.19. 2-[4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-cis-
perhydro-1-quinoxalinyl]-6,7-dimethoxy-4-
quinazolinamine (18)
ConcentrationÁresponse curves were constructed by
/
cumulative addition of agonist. The agonist concentra-
tion in the bath was increased ca. 3-fold at each step,
with each addition being made only after the response of
previous addition had attained a maximal level and
remained steady. Contractions were recorded by means
of a force displacement transducer connected to the
MacLab System PowerLab/800. In addition parallel
experiments in which tissues did not receive any
antagonist were run in order to check any variation in
sensitivity.
A solution of 2-chloro-4,6-dimethoxy-s-triazine [16]
(0.31 g, 1.74 mmol) in CH2Cl2 (5 ml) was added
dropwise to a stirred solution of 19 (0.5 g, 1.46 mmol)
and Et3N (0.26 g, 2.6 mmol) dissolved in CH2Cl2 (10
ml). The mixture was stirred at reflux for 3 h then cooled
and left at 0 8C for a night. The crude precipitate was
collected and purified by two consecutive column
chromatography eluting with mixture P in the first and
Q in the second. Obtained 0.14 g (18% yield) of product;
1
m.p.: 147Á
/
150 8C (dec); H NMR (CDCl3-d6): d 1.34Á
/
2.12 (m, 7H, H5Á8 perhydroquinoxaline), 2.30Á
1H, H5Á8 perhydroquinoxaline), 3.70Á3.88 (m, 2H,
H2Á3 perhydroquinoxaline), 3.91Á4.01 (m, 12H,
OCH3), 4.10Á4.34 (m, 1H, H2Á3 perhydroquinoxaline),
4.48Á4.73 (m, 3H, H2Á3, H4a and H8a perhydroquinoxa-
line), 5.08 (s, br, 2H, NH exchangeable with D2O), 6.80
/
2.51 (m,
5.2.2. Guinea-pig spleen
/
a1-Adrenoceptor antagonism was determined on the
spleen [17]. The tissue was removed and bisected long-
itudinally into two strips which were suspended in tissue
baths containing Krebs solution of the following
composition (mM): NaCl, 120; KCl, 5.5; CaCl2, 2.5;
MgCl2, 1.2; NaH2PO4, 1.2; NaHCO3, 20; glucose, 11.
This solution contained also 0.1 mM cocaine hydrochlo-
/
/
/
(s, 1H, arom), 6.92 (s, 1H, arom). Anal. (C23H30N8O4×
/
0.5C4H8O2) (C, H, N).
ride and 1 mM (9
/
) normetanephrine to prevent the
5.1.20. 2-[4-Methyl-cis-perhydro-1-quinoxalinyl]-6,7-
dimethoxy-4-quinazolinamine dihydrochloride (20)
A solution of 37% formaldehyde (0.11 g, 3.64 mmol)
was added dropwise into a solution of 19 (0.25 g, 0.73
mmol) in acetonitrile (15 ml), followed of addition of
neuronal uptake of (ꢂ
/
)-NA and 1 mM (9)-propranolol
/
hydrochloride to block the b-adrenoceptors. The spleen
strips were placed under 1 g resting tension and
equilibrated for 45 min. The cumulative con-
centrationÁ
/
response curves to (ꢂ)-NA were obtained
/

486
D. Giardina` et al. / Il Farmaco 58 (2003) 477Á487
/
at 30 min intervals, the first one being discarded and the
second one taken as control. The antagonist was
allowed to equilibrate with the tissue for 30 min then a
values were obtained at three antagonist concentrations
and each concentration was tested three to six times.
Schild plots were constrained to the slope of ꢂ1, as
/
new concentrationÁ
obtained. The results were expressed as dissociation
constants.
/
response curve to the agonist was
requested by theory [21], since the experimental data
generated a line with a slope not significantly different
from the unity (P ꢀ0.05). For all other tested com-
/
pounds, the slope appeared to be significantly different
from unity, therefore an affinity estimate, as pKB value,
was obtained using the above equation according to van
Rossum [22]. The lowest active concentration of an-
tagonist was used for this calculation.
Concerning the a2-adrenoceptor antagonism the ma-
jority of compounds were inactive at low concentra-
tions, however, higher concentrations could not be
assayed because of the twitch inhibition of the electri-
cally stimulated tissue. Thus, an experimental antagonist
activity was evaluated only for compound 2 at its lower
active concentration (30 mM). It was investigated four
times and the result given as pKB value according to van
Rossum. For all other compounds only an estimated
affinity (pKB) was reported from the analysis of their
behavior at tested concentrations. Data from functional
5.2.3. Rat vas deferens
The a2-adrenoceptor blocking activity was assessed
on prostatic portion by antagonism to clonidine, which
inhibits the twitch responses of the field-stimulated
tissue by acting on the a2-adrenoceptor [18,19]. A first
clonidine doseÁresponse curve, taken as control, was
/
obtained cumulatively avoiding the inhibition of more
than 90% of twitch responses, while the concentration of
clonidine causing 100% inhibition was deduced from the
second doseÁ
experiments. Under these conditions it was possible to
obtain a second doseÁresponse curve, which was not
significantly different from the first one. Thus, after
incubation with antagonist for 30 min, a doseÁresponse
/response curve obtained from parallel
/
/
curve was obtained. The results were expressed as
dissociation constants.
assays are presented as the mean 9
Differences between mean values were tested for sig-
nificance by Student’s t-test. A P value B0.05 was
/SE of n experiments.
5.2.4. Radioligand binding assays at cloned receptors
Competition binding assays to cloned human a1-
adrenoceptor subtypes were performed in membrane
preparations from CHO cell lines transfected by electro-
poration with DNA expressing the gene encoding each
a1-adrenoceptor subtypes. Cloning and stable expres-
sion of the human a1-adrenoceptor gene was performed
as previously described [23]. CHO cells membranes (30
/
taken to indicate a statistically significant difference.
Binding data were analyzed using a non-linear curve-
fitting program Allfit [31]. Scatchard plots were linear in
all preparations and the pseudo-Hill coefficients non
significantly different from the unity (P ꢀ0.05). The
/
inhibition of the radioligand specific binding by tested
compounds allowed the estimation of IC50 values that
were converted to an affinity constant (Ki) by the
mg proteins) were incubated in 50 mM TrisÁ
pH 7.4, with 0.1Á0.4 nM [H]prazosin, in a final volume
of 1.02 ml for 30 min at 25 8C, in the absence or presence
of competing drugs (1 pMÁ
/
HCl buffer,
/
ChengÁ
/
Prusoff equation [24]: Kiꢁ
/
IC50/(1ꢀL/Kd),
/
where L and Kd are the concentration and the equili-
brium dissociation constant of the radioligand. Data are
/
10 mM). Non-specific
binding was determined in the presence of 10 mM
phentolamine. The incubation was stopped by addition
expressed as mean of pKi values 9SE of two to three
/
separate experiments performed in triplicate.
of ice-cold TrisÁHCl buffer and rapid filtration through
/
0.2% poly(ethylenimine)-pretreated Whatman GF/B or
Schleicher & Schuell GF52 filters.
Acknowledgements
5.2.5. Data analysis
In functional studies responses were expressed as
percentage of the maximal contraction observed in the
The authors acknowledge the University of Camerino
(Italy) and Recordati S.p.A of Milan (Italy) for financial
support.
agonist concentrationÁ
Pharmacological computer programs analyzed the ago-
nist concentrationÁresponse curves.
/response curve taken as control.
/
In the a1-adrenoceptor antagonism, pA2 values for
compounds 7, 14, 15 were determined by Schild plots
obtained by computer program calculations according
to Arunlakshana and Schild [20]. The linear regressional
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