Design, synthesis, and biological activity of prazosin-related antagonists. Role of the piperazine and furan units of prazosin on the selectivity for α1-adrenoreceptor subtypes

Article abstract of DOI:10.1021/jm9810654

Prazosin-related quinazolines 4-20 were synthesized, and their biological profiles at α₁-adrenoreceptor subtypes were assessed by functional experiments in isolated rat vas deferens (α(1A)), spleen (α(1B)), and aorta (α(1D)) and by binding assa

Full text of DOI:10.1021/jm9810654

4844
J . Med. Chem. 1998, 41, 4844-4853
Design , Syn th esis, a n d Biologica l Activity of P r a zosin -Rela ted An ta gon ists.
Role of th e P ip er a zin e a n d F u r a n Un its of P r a zosin on th e Selectivity for
r1-Ad r en or ecep tor Su btyp es
Maria L. Bolognesi,† Roberta Budriesi,† Alberto Chiarini,† Elena Poggesi,‡ Amedeo Leonardi,‡ and
Carlo Melchiorre*,†
Department of Pharmaceutical Sciences, University of Bologna, Via Belmeloro 6, 40126 Bologna, Italy, and
Research and Development Division, Recordati S.p.A., Via Civitali 1, 20148 Milano, Italy
Received J uly 30, 1998
Prazosin-related quinazolines 4-20 were synthesized, and their biological profiles at R1-
adrenoreceptor subtypes were assessed by functional experiments in isolated rat vas deferens
(R1A), spleen (R1B), and aorta (R1D) and by binding assays in CHO cells expressing human cloned
R1-adrenoreceptor subtypes. The replacement of piperazine and furan units of prazosin (1) by
1,6-hexanediamine and phenyl moieties, respectively, affording 3-20, markedly affected both
affinity and selectivity for R1-adrenoreceptor subtypes in functional experiments. Cystazosin
(3), bearing a cystamine moiety, was a selective R1D-adrenoreceptor antagonist being 1 order
of magnitude more potent at R1D-adrenoreceptors (pA2, 8.54 ( 0.02) than at the R1A- (pA2, 7.53
( 0.01) and R1B-subtypes (pA2, 7.49 ( 0.01). The insertion of substituents on the furan ring of
3, as in compounds 4 and 5, did not improve the selectivity profile. The simultaneous
replacement of both piperazine and furan rings of 1 gave 8 which resulted in a potent, selective
R1B-adrenoreceptor antagonist (85- and 15-fold more potent than at R1A- and R1D-subtypes,
respectively). The insertion of substituents on the benzene ring of 8 affected, according to the
type and the position of the substituent, affinity and selectivity for R1-adrenoreceptors.
Consequently, the insertion of appropriate substituents in the phenyl ring of 8 may represent
the basis of designing new selective ligands for R1-adrenoreceptor subtypes. Interestingly, the
finding that polyamines 11, 16, and 20, bearing a 1,6-hexanediamine moiety, retained high
affinity for R1-adrenoreceptor subtypes suggests that the substituent did not give rise to negative
interactions with the receptor. Finally, binding assays performed with selected quinazolines
(2, 3, and 14) produced affinity results, which were not in agreement with the selectivity profiles
obtained from functional experiments. This rather surprising and unexpected finding may be
explained by considering neutral and negative antagonism.
In tr od u ction
Current evidence indicates that rat submaxillary gland,5
human liver,6 and various tissues such as prostatic rat
vas deferens,7 rabbit prostate, and prostatic urethra8
contain predominantly the R1A-adrenoreceptor, whereas
rat liver and spleen9 are considered R1B-adrenoreceptor
preparations, and the R1D-adrenoreceptor mediates the
contraction in rat aorta.10,11 As a result, the effort to
design agents selective for each of the three R1-adreno-
receptor subtypes has been an active area of research.
Several relatively selective ligands for R1-adrenorecep-
tors are now available.12 For example, SNAP 5089,12
KMD-3213,13 Rec 15/2739,14 RS-17053,15 and (-)-me-
phendioxan16 are selective for R1A-adrenoreceptors, (+)-
It is clear now that R1-adrenoreceptors are comprised
of multiple subtypes that can be classified by both
pharmacological and binding studies into at least three
subtypes, that is R1A(R1a), R1B(R1b), and R1D(R1d), with
upper and lower case subscripts being used to designate
native or recombinant receptors, respectively.1-3 How-
ever, the situation appears to be more complex as, in
addition to R1A-, R1B-, and R1D-adrenoreceptor subtypes,
which share a high affinity for prazosin, the existence
of additional R1-adrenoreceptors has been proposed.
These are called R1L-adrenoreceptors and are character-
ized by a low activity for prazosin. However, these
receptors have not been cloned yet and their character-
ization is still difficult.4
The existence of multiple R1-adrenoreceptor subtypes
holds the promise of developing new molecules, which
target only one receptor while not affecting others.
Furthermore, the different localization of these receptor
subtypes brought about the possibility of designing
drugs that selectively interact with distinct subtypes,
thus avoiding the occurrence of possible side effects.
cyclazosin17 and L-765,31418 are selective for the R1B
-
subtype, and BMY-737819 and cystazosin20 are selective
for the R1D-subtype. Whereas it has been demonstrated
that R1A-adrenoreceptor antagonists can be useful in the
treatment of benign prostatic hyperplasia, a potential
therapeutic use for both R1B- and R1D-subtype antago-
nists has not been defined yet. Perhaps, the fact that
only recently so-called selective R1-adrenoreceptor an-
tagonists have become available has prevented the
physiological roles of R1B- and R1D-adrenoreceptor sub-
types in blood pressure control or other physiological
functions from being revealed. It should be emphasized
† University of Bologna.
‡ Recordati S.p.A.
10.1021/jm9810654 CCC: $15.00 © 1998 American Chemical Society
Published on Web 10/28/1998

Prazosin-Related Antagonists
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 24 4845
Ch a r t 1
as well that the ideal selective ligands, which recognize
only one among multiple receptor subtypes, are few and
it remains a formidable challenge to medicinal chemists
to realize useful drugs of this kind. For example, BMY-
7378 is by far the most selective R1d-adrenoreceptor
antagonist reported to date.19 It displayed a 75- and
80-fold selectivity for the R1d-adrenoreceptor relative to
R1b and R1a subtypes, respectively. However, BMY-7378
is more potent (14-fold) at 5-HT1A receptors and only
9-fold less potent at D2 receptors with respect to R1d-
adrenoceptors.12
Design Ra tion a le
The starting point of this investigation was the
observation that modifying the piperazine ring of pra-
zosin (1) may afford antagonists, which are able to
differentiate among R1-adrenoreceptor subtypes. It was
shown that the piperazine ring of prazosin is not
essential for activity and can be replaced with an R,ω-
alkanediamine chain.21 Among a series of quinazolines
bearing a polymethylene chain, compound 2 displayed
the highest affinity for rat vas deferens R1-adrenocep-
tors, being even more potent than prazosin. It was
suggested that the hexane chain of 2 might contribute
to the binding by interacting with a lipophilic site
located between the sites where quinazoline and furan
rings interact.21 More recently, it was demonstrated
that the hexane chain could be constrained into a
cyclohexyl moiety as in cyclazosin.22 The (+)-enanti-
omer of cyclazosin (Chart 1) can be considered the first
R1b-selective adrenoreceptor antagonist in binding as-
says, being about 100-fold selective versus the native
R1A and about 40-fold versus the recombinant R1a- and
R1d-subtypes.16 Following a design rationale similar to
that leading to cyclazosin, Patane et al.18 synthesized,
by incorporating new structural elements into the
prazosin piperazine moiety, L-765,314 (Chart 1) which
turned out to be a selective R1b-adrenoreceptor antago-
nist.
Thus, the finding that the affinity profile of prazosin-
related quinazolines can depend on the type of moiety
linking the two nitrogen atoms of the piperazine ring
of prazosin prompted us to further modify the structure
of analogue 2, in an attempt to improve the affinity and
selectivity for different R1-adrenoreceptor subtypes. In
particular, two types of structural modifications were
performed on the structure of 2, that is (a) replacement
of the hexane spacer with a cystamine moiety and (b)
insertion of substituents on the furan ring or its
replacement by an (un)substituted phenyl unit. Thus,
replacing the hexane chain of 2 by a cystamine moiety,
which is a structural feature of benextramine,23 an
irreversible R-adrenoreceptor antagonist, afforded cys-
tazosin (3) which displayed an interesting selectivity
profile in comparison with both (+)-cyclazosin and the
carbon analogue 2, owing to a significantly lower affinity
F igu r e 1. Design strategy for the synthesis of quinazolines
3-20 by replacing the hexane spacer and/or the furan ring of
2, an open analogue of prazosin (1), by (a) a cystamine moiety
and (b) an (un)substituted phenyl ring, respectively.
for R1A- and R1B-adrenoreceptor subtypes relative to the
R
1D-subtype.20 This observation formed the basis for
further structural modifications. We thought that
increasing the number of contacts between a ligand and
its receptor would hopefully also increase receptor
subtype selectivity. To this end we introduced a chlo-
romethyl substituent at position 5 of furan ring of 3 and
2, affording 4 and 6, respectively, because it can be
easily functionalized as in 5 and 7. According to its
properties, an amine function can be protonated at
physiological pH giving rise to a possible additional
interaction with a nucleophilic, complementary receptor
group, which would increase the possibility to achieve
receptor subtype selectivity. Subtle and unpredictable
differences in the binding pockets may account for
selectivity; thus incorporation of additional structural
elements in the structure of a nonselective ligand may
well lead to preferential recognition of a particular
receptor subtype. Next, the furan ring of 2 was replaced
by a phenyl ring affording 8, which offered us the
possibility to incorporate additional structural elements
at different positions, yielding 9-20. These 12 com-
pounds allow us to investigate the effect of the substit-
uents not only upon the affinity but also on the
selectivity for R1-adrenoreceptor subtypes. The design
strategy for our compounds is shown in Figure 1.
We describe here the synthesis and the pharmacologi-
cal profile of quinazolines 4-20 in functional and
binding experiments in comparison with prototypes
prazosin (1) and its open analogues 2 and 3.
A preliminary communication dealing with cystazosin
(3) has been published recently.20
Ch em istr y
The new compounds were synthesized by standard
methods as illustrated in Schemes 1 and 2 and charac-
1
terized by H NMR, IR, and elemental analysis.

4846 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 24
Bolognesi et al.
Sch em e 1
Sch em e 2
forded 11 whereas reduction of 25 with NaBH4 gave the
corresponding alcohol 26. The latter compound was
transformed into the chloride 9 which, in turn, was
alkylated to the amine 10.
Biology
F u n ction a l Stu d ies. The pharmacological profile of
prazosin-related quinazolines 2-20 was evaluated at R1-
adrenoreceptor subtypes and R2-adrenoreceptors on
different isolated tissues using prazosin (1) and BMY-
7378 as standard compounds. R1-Adrenoreceptor sub-
types blocking activity was assessed by antagonism of
(-)-noradrenaline-induced contraction of prostatic vas
27
28
deferens (R1A
)
or thoracic aorta (R1D
)
and by antago-
nism of (-)-phenylephrine-induced contraction of spleen
(R1B),28 while R2-adrenoreceptor blocking activity was
determined by antagonism of the clonidine-induced
depression of the twitch responses of the field-stimu-
lated prostatic portion of rat vas deferens. The poten-
cies of the drugs were expressed as pA2 values.29-31
Bin d in g Exp er im en ts. Receptor subtype selectivity
of few prazosin-related quinazolines was further deter-
mined by employing receptor binding assays. [3H]-
Prazosin was used to label cloned human R1-adrenore-
ceptors expressed in Chinese hamster ovary (CHO)
cells.32 Furthermore, [3H]rauwolscine and [3H]spiper-
one were used to label R2-adrenoreceptors in rat cortex
and D2 receptors in rat striatum, respectively, whereas
[3H]8-hydroxy-2-(di-n-propylamino)tetralin ([3H]8-OH-
DPAT) was the radioligand to label cloned human
5-HT1A receptors which were expressed in HeLa cells.33,34
4-Amino-2-chloro-6,7-dimethoxyquinazoline (21) was
the common starting material. Adapting the procedure
reported for 23,24 compound 22 was obtained by aro-
matic nucleophilic substitution of N,N′-dimethylcyst-
amine20 on 21. The reaction of 22 and 23 with 5-chlo-
romethyl-2-furoyl chloride (24)25 afforded 4 and 6,
respectively, which were transformed by reaction with
dimethylamine into the corresponding N,N-dimethyl-
amino analogues 5 and 7 (Scheme 1). Compound 8 was
synthesized by reaction of 23 and benzoyl chloride.
Similarly, 12 and 17 were obtained from 23 and 3- or
4-chloromethylbenzoyl chloride, respectively. Chlorides
12 and 17 were reacted with dimethylamine, thiazoli-
dine, or N,N′-dimethyl-1,6-hexanediamine26 to give the
corresponding amines 13-16 and 18-20.
Finally, 2-substituted derivatives 9-11 were synthe-
sized following the synthetic pathway shown in Scheme
2. Intermediate 25 was obtained through amidation of
23 with 2-formyl-benzoyl chloride, which was generated
in situ by treating 2-formylbenzoic acid with SOCl2.
Reductive amination of 25 by 1,6-hexanediamine af-
Resu lts a n d Discu ssion
The biological activity expressed as pA2 values, at R1-
adrenoreceptor subtypes and R2-adrenoreceptors of com-

Prazosin-Related Antagonists
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 24 4847
Ta ble 1. Antagonist Affinities, Expressed as pA2, of 2-20 at R1- and R2-Adrenoreceptors on Isolated Tissue from the Rat, Namely,
Prostatic Vas Deferens (R1A and R2), Spleen (R1B), and Thoracic Aorta (R1D), in Comparison to Reference Compounds Prazosin (1) and
BMY-7378
a
pA2
b
no
X
R
R1A
R1B
R1D
R2
1
2
3
4
5
6
7
8
(prazosin)
CH2
S
S
S
8.60 ( 0.07
9.04 ( 0.02
7.53 ( 0.01
7.78 ( 0.07
6.70 ( 0.08
8.17 ( 0.02
7.41 ( 0.04
7.42 ( 0.03
6.38 ( 0.05
7.04 ( 0.01
7.97 ( 0.01
7.10 ( 0.01
7.32 ( 0.01
7.26 ( 0.08
7.82 ( 0.09
8.99 ( 0.01
9.84 ( 0.01
7.49 ( 0.01
7.37 ( 0.05
7.27 ( 0.03
8.97 ( 0.06
8.62 ( 0.02
9.35 ( 0.02
8.73 ( 0.04
7.88 ( 0.04
9.17 ( 0.02
7.18 ( 0.01
8.61 ( 0.02
7.43 ( 0.02
8.03 ( 0.05
8.91 ( 0.04
9.19 ( 0.03
8.54 ( 0.02
8.03 ( 0.09
8.22 ( 0.06
9.39 ( 0.02
8.23 ( 0.01
8.16 ( 0.02
8.55 ( 0.02
8.26 ( 0.02
8.50 ( 0.04
7.29 ( 0.05
8.69 ( 0.01
8.26 ( 0.03
8.51 ( 0.06
5.43 ( 0.13
6.66 ( 0.03
H
H
<5
CH2Cl
CH2NMe2
CH2Cl
CH2NMe2
H
2-CH2Cl
2-CH2NMe2
2-CH2NH(CH2)6NH2
3-CH2Cl
<5
<5
<5
<5
CH2
CH2
6.52 ( 0.05
5.31 ( 0.01
<5
5.47 ( 0.01
<5
5.44 ( 0.01
<5
5.94 ( 0.03
9
10
11
12
13
14
15
3-CH2NH2
3-CH2NMe2
16
17
18
19
3-CH2N(Me)(CH2)6NHMe
4-CH2Cl
4-CH2NMe2
8.05 ( 0.07
7.11 ( 0.02
7.14 ( 0.01
7.23 ( 0.10
7.71 ( 0.05
9.15 ( 0.04
8.53 ( 0.01
8.01 ( 0.03
7.84 ( 0.04
7.71 ( 0.01
7.86 ( 0.08
8.17 ( 0.03
<5
5.54 ( 0.02
5.12 ( 0.06
<5
20
4-CH2N(Me)(CH2)6NHMe
6.72 ( 0.04
6.94 ( 0.08
9.21 ( 0.09
7.55 ( 0.07
8.46 ( 0.07
8.34 ( 0.05
5.60 ( 0.03
<5
BMY-7378
a
pA2 values ( SE were calculated from Schild plots,29 constrained to a slope of -1.0, unless otherwise specified.30 pA2 is the positive
value of the intercept of the line derived by plotting log(DR - 1) vs log [antagonist]. The log(DR - 1) was calculated at least at three
different antagonist concentrations, and each concentration was tested from four to six times. Dose-ratio (DR) values represent the ratio
of the potency of the agonist (EC50) in the presence of the antagonist and in its absence. Parallelism of dose-response curves was checked
b
by linear regression, and the slopes were tested for significance (p < 0.05). pA2 values were calculated at only one concentration (10 µM)
according to van Rossum.31
pounds used in the present study, is shown in Table 1.
To make relevant considerations on structure-activity
relationships, prototypes 1 and 2 and the reference
compound BMY-7378 were included for comparison. All
quinazolines behaved as competitive antagonists as
revealed by the slopes of their Schild plots, which were
not significantly different from unity (p > 0.05) (Table
1). By taking as a starting point the hexane analogue
2 of prazosin, it is possible to observe how affinity and
selectivity for R1-adrenoreceptor subtypes can be mark-
edly affected by replacing the hexane moiety and/or the
furan ring by a cystamine unit and an (un)substituted
phenyl unit, respectively. Interestingly, all prazosin-
related compounds of the present investigation dis-
played a weak, if any, affinity for R2-adrenoreceptors
in comparison with R1-adrenoreceptor subtypes.
An analysis of the results shown in Table 1 reveals
that the replacement of the hexane spacer or the furan
F igu r e 2. Affinity constants (pA2) in rat prostatic vas deferens
ring of 2 by a cystamine moiety or a phenyl group,
(R1A), spleen (R1B), and aorta (R1D) R1-adrenoreceptor subtypes
affording 3 and 8, respectively, caused a dramatic effect
of cystazosin (3) and 8 in comparison with prazosin (1) and 2.
in the affinity profile for R1-adrenoreceptor subtypes as
shown in Figure 2. Clearly, 2 is a very potent R1-
adrenoreceptor antagonist but, at the same time, is not
to selectivity. Thus, cystazosin (3) resulted in a selective
1D-adrenoreceptor antagonist, owing to a slight (4.5-
fold) decrease in affinity for the R1D-subtype and a large
drop in affinity (32- and 224-fold, respectively) for R1A
and R1B-subtypes in comparison with 2. On the other
selective, displaying only a slight preference for the R1B
subtype. Interestingly, the structural modifications
performed on 2 did not improve affinity for R1-adreno-
receptor subtypes but, what is more important, gave rise
-
R
-

4848 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 24
Bolognesi et al.
Ta ble 2. Affinity Constants (pKi) of 2, 3, and 14 for Cloned R1-Adrenoreceptor Subtypes and 5-HT1A Receptors, Native
R2-Adrenoreceptors, and D2 Receptors in Comparison to Reference Compoundsa
pKi, human cloned receptors
pKi, native receptors (rat)
R2 (cerebral cortex)
no.
R1a
R1b
R1d
5-HT1A
D2 (striatum)
1
2
3
9.23 ( 0.07
9.78 ( 0.04
9.38 ( 0.05
9.49 ( 0.06
6.36 ( 0.06
9.39 ( 0.10
9.96 ( 0.07
8.97 ( 0.09
9.78 ( 0.06
7.19 ( 0.04
9.65 ( 0.08
9.71 ( 0.01
9.14 ( 0.07
9.61 ( 0.11
8.89 ( 0.01
<6
<6
<6
6.80 ( 0.03
7.00 ( 0.16
6.23 ( 0.07
7.72 ( 0.25
5.98 ( 0.20
<5
5.63 ( 0.02
e5
14
6.13 ( 0.04
8.76 ( 0.28
5.71 ( 0.15
7.32 ( 0.04
BYb
a
Values are the mean ( SE of at least three separate experiments performed in triplicate. The pseudo-Hill coefficients (nH) were not
significantly different from unity (p > 0.05). Equilibrium inhibition constants (Ki) were derived using the Cheng-Prusoff equation.40
Scatchard plots were linear or almost linear in all preparations tested. The affinity estimates were derived from displacement of [3H]prazosin
from R1-adrenoreceptors, [3H]rauwolscine from R2-adrenoreceptors, [3H]spiperone from D2 receptors, and [3H]8-hydroxy-2-(di-n-
b
propylamino)tetraline from 5-HT1A receptors. BY, BMY-7378.
hand, the phenyl analogue 8 displayed a significantly
improved R1B-selectivity (85- and 15-fold relative to R1A
a marked effect on the selectivity profile prompted us
to further modify the amine function, as in 13, 15 and
19. Thiazolidine analogues 15 and 19 did not display
a better affinity profile than prototypes 14 and 18,
-
and R1D-subtypes, respectively), owing to a much larger
decrease in affinity for both R1A- and R1D-subtypes than
for the R1B-subtype in comparison with 2 (Figure 2).
The insertion of a 5-chloromethyl or a 5-N,N-di-
methylaminomethyl substituent on the furan ring of 3,
affording 4 and 5, respectively, did not improve the
selectivity profile. The same structural modification
performed on 2 to afford 6 and 7 caused a marked
decrease in affinity which was more pronounced for
5-N,N-dimethylaminomethyl group. However, com-
whereas 13 was as active as 14 at R1A- and R1D
-
adrenoreceptors, while showing a marked increase in
affinity for the R1B-subtype.
Finally, replacing the chlorine atom of 9, 12, and 17
with a substituted 1,6-diaminohexane moiety, affording
11, 16, and 20, respectively, resulted in a marked effect
on the affinity for R1-adrenoreceptors. Polyamine 11
was as active as, if not more active than, prototype 8,
whereas analogue 16, bearing the N,N′-dimethyl-1,6-
diaminohexane moiety in position 3, turned out to be
pound 6 was slightly more potent than 2 at R1D
-
adrenoreceptors while displaying a significantly lower
affinity at R1A- and R1B-subtypes as revealed by its pA2
values (R1A, 8.17 ( 0.02; R1B, 8.97 ( 0.06; R1D, 9.39 (
0.02). Clearly, this finding suggests that appropriate
substituents on the aromatic moiety may have a role in
achieving receptor subtype selectivity.
slightly or markedly less potent at R1D- and R1B
-
adrenoreceptors, respectively, while being more potent
at the R1A-subtype than 8, and 20 retained a comparable
affinity at R1B-adrenoreceptors while being markedly
less potent at the R1A-subtype and more potent at the
R
1D-subtype in comparison with 8.
Next, the insertion of a substituent on the benzene
ring of 8, affording 9-20, affected differently, according
to substituent type and position, the affinity and, as a
consequence, the selectivity for R1-adrenoreceptor sub-
types. The affinity profiles of 9, 12, and 17 reveal that
a chloromethyl substituent at any position did not
improve potency relative to 8, the exception being 9 with
a slightly higher affinity at R1D-adrenoreceptors. How-
ever, it should be noted that when the substituent is at
position 4, as in 17, both affinity and selectivity profiles
were not markedly influenced relative to 8, whereas at
position 2 and 3 the affinity for R1-adrenoreceptor
subtypes was affected in such a way that 9, bearing a
2-substituent, was highly selective (224-148-fold) for
A most intriguing finding of the present investigation
is the observation that polyamines 11, 16, and 20
retained high affinity for R1-adrenoreceptor subtypes,
which suggests clearly that a 1,6-diaminohexane chain
on the benzene ring did not give rise to negative
interactions with the receptor. This observation may
have relevance for the development of new quinazolines
bearing a polyamine backbone on which additional
substituents can be mounted to improve selectivity for
R1-adrenoreceptor subtypes. Clearly, the site where the
terminal aromatic ring of 8 interacts does not seem to
present steric hindrance and particularly stringent
requirements.
R
1B- and R1D-adrenoreceptors versus the R1A-subtype,
The binding affinities, expressed as pKi values, in
CHO cells expressing human cloned R1-adrenoreceptor
subtypes and HeLa cells expressing human 5-HT1A
receptors and in membranes of rat cerebral cortex (R2-
adrenoreceptors) and striatum (D2 receptors) of quinazo-
lines 2, 3, and 14 are shown in Table 2 in comparison
with those of prazosin (1) and BMY-7378. As antici-
pated for cystazosin (3),20 the results obtained in binding
experiments did not show the same selectivity profile
observed in functional assays. It can be seen that while
binding affinities of reference compounds prazosin (1),
2, and BMY-7378 are qualitatively and quantitatively
comparable with pA2 values derived from functional
experiments, those observed for 3 and 14 are not in
agreement at all from both a qualitative and a quanti-
tative point of view with functional affinities. Both
compounds were devoid of selectivity for R1-adrenore-
and 12, having a 3-substituent, resulted a weak and
nonselective R1-adrenoreceptor antagonist.
Replacing chlorine atom of 9, 12, and 17 with an N,N-
dimethylamino group afforded 10, 14, and 18, respec-
tively, which were almost as active as 8 at R1A- and R1D
adrenoreceptors while being significantly less potent at
1B-adrenoreceptors. Consequently, the selectivity pro-
-
R
file of these analogues was different from that of 8 and
parent chlorides. It turned out that amines 10 and 14
had a reversed selectivity profile relative to 8 and 9 or
12, respectively, being more potent at R1D-adrenorecep-
tors, whereas amine 18 retained a selectivity profile
similar to that of 8 and 17 owing to its higher affinity
for R1B-adrenoreceptors.
The finding that inserting an N,N-dimethylamino-
methyl moiety onto the phenyl group of 8 resulted into

Prazosin-Related Antagonists
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 24 4849
purified by chromatography. Eluting with methylene chloride-
methanol-aqueous 30% ammonia (9:1:0.05) afforded 0.4 g
(25% yield) of 22 as the free base: 1H NMR (CDCl3) δ 2.03-
2.39 (m, 2 + 1 exchangeable with D2O), 2.43 (s, 3), 2.79-3.08
(m, 6), 3.21 (s, 3), 3.93 (s, 3), 3.97 (s, 3), 5.59 (br s, 2,
exchangeable with D2O), 6.81 (s, 1), 6.94 (s, 1).
5-Ch lor om eth yl-2-fu r oyl Ch lor id e (24). A solution of
5-hydroxymethylfuran-2-carboxylic acid25 (0.3 g, 2.11 mmol)
and SOCl2 (1.5 mL, 10 mmol) in benzene (10 mL) was refluxed
for 1 h. Removal of the solvent under reduced pressure
afforded crude 24 in a quantitative yield.
5-Ch lor om eth ylfu r an -2-car boxylic Acid (2-{2-[(4-Am in o-
6,7-d im e t h oxyq u in a zolin -2-yl)m e t h yla m in o]e t h yld i-
su lfa n yl}eth yl)m eth yla m id e Hyd r och lor id e (4). A solu-
tion of 24 (0.19 g, 1.1 mmol) in THF (20 mL) was added
dropwise to a solution of 22 (0.4 g, 1.1 mmol) in THF (15 mL).
After the mixture was stirred at room temperature for 3 h,
the solvent was removed under reduced pressure to give a
residue that was purified by chromatography. Eluting with
methylene chloride-methanol-aqueous 30% ammonia (9.5:
0.5:0.05) afforded 0.34 g (65% yield) of 4 as the free base that
was transformed into the hydrochloride salt: mp 170 °C (from
EtOH/ether); 1H NMR (free base; CDCl3) δ 2.96-3.42 (m, 10),
3.82-4.05 (m, 4), 3.93 (s, 3), 3.97 (s, 3), 4.61 (s, 2) 5.32 (br s,
2, exchangeable with D2O), 6.48 (d, 1), 6.85 (m, 1), 6.92-7.07
(m, 2). Anal. (C22H29Cl2N5O4S) C, H, N.
ceptor subtypes in binding assays, owing to a marked
increase in affinity of about 2 orders of magnitude for
R1a- and R1b-adrenoreceptors and of about 1 order of
magnitude for the R1d-subtype. As a matter of fact, the
theory states that the affinity of an antagonist assessed
in functional assays should not differ from that deter-
mined in binding experiments using both native and
recombinant receptors. For this reason competitive
antagonists are considered better tools for receptor
characterization and classification than agonists be-
cause for the latter ones, in addition to affinity, other
pharmacological parameters must be taken into ac-
count.35,36 Consequently, there is no apparent explana-
tion for the discrepancy observed between our functional
and binding results. However, very recently we dis-
cussed the possibility that if an antagonist does not
adhere perfectly to the concept of neutral antagonism
in the interaction with the receptor but behaves as a
negative antagonist (inverse agonist), then its affinity
may not be, as assumed by theory, system-independent,
giving rise to affinity values which might be different
according to the system employed for the determina-
tion.35 In other words, as pointed out by Leff,36 the use
of inverse agonists as neutral antagonists may have, like
agonists, problems since their estimated affinities are
system-dependent. Thus, for inverse agonists the af-
finity values estimated in functional assays may not
necessarily be comparable with those obtained in bind-
ing experiments. Interestingly, a survey of literature
has revealed that some of the so-called competitive
antagonists behave as inverse agonists when tested in
the appropriate model. In the field of R1-adrenoreceptor
antagonists, prazosin (1), WB 4101, and benoxathian
were shown to be inverse agonists in a vascular model.37
Thus, the difference, which is often observed for func-
tional and binding affinities of antagonists, might be
explained by the fact that these compounds are inverse
agonists, and hence their affinity is system-dependent.
Work is in progress to determine the nature of antago-
nism displayed by 3 and 14 and the other quinazolines
of the present investigation.
5-Dim eth yla m in om eth ylfu r a n -2-ca r boxylic Acid (2-{2-
[(4-Am in o-6,7-d im eth oxyqu in a zolin -2-yl)m eth yla m in o]-
eth yld isu lfa n yl}eth yl)m eth yla m id e Dioxa la te (5). A 5.6
M ethanolic solution of dimethylamine (4 mL) was added to a
solution of 4 (0.08 g, 0.14 mmol) in absolute ethanol (10 mL).
After the mixture was stirred at room temperature for 2 h,
the solvent was removed under reduced pressure to give a
residue that was purified by chromatography. Eluting with
methylene chloride-methanol-aqueous 30% ammonia (9.5:
0.5:0.04) afforded 0.03 g (40% yield) of 5 as the free base that
was transformed into the dioxalate salt and crystallized: mp
1
106 °C (from EtOH/ether); H NMR (free base; CDCl3) δ 2.25
(s, 6), 2.93-3.01 (m, 4), 3.20 (s, 6), 3.50 (s, 2), 3.73-3.93 (m,
10), 5.49 (br s, 2, exchangeable with D2O), 6.28 (d, 1), 6.85-
7.01 (m, 3). Anal. (C26H38N6O12S2) C, H, N.
5-Ch lor om eth ylfu r a n -2-ca r boxylic Acid {6-[(4-Am in o-
6,7-d im e t h o x y q u in a zo lin -2-y l)m e t h y la m in o ]h e x y l}-
m eth yla m id e Hyd r och lor id e (6). A solution of 24 (0.3 g,
1.68 mmol) in THF (20 mL) was added dropwise to a solution
of 2324 (0.58 g, 1.7 mmol) in THF (15 mL). After the mixture
was stirred at room temperature for 2 h, the solid was collected
by filtration, washed with ether, and purified by chromatog-
raphy. Eluting with methylene chloride-methanol-aqueous
30% ammonia (9.5:0.5:0.03) gave 0.16 g (20% yield) of 6 as
the free base that was transformed into the hydrochloride salt
and crystallized: mp 130 °C (from EtOH/ether); 1H NMR (free
base; CDCl3) δ 1.18-1.69 (m, 8), 2.95-3.29 (m, 7), 3.42-3.71
(m, 5), 3.87 (s, 3), 3.92 (s, 3), 4.56 (s, 2), 5.49 (br s, 2,
exchangeable with D2O), 6.33-6.43 (m, 1), 6.81-7.03 (m, 3).
Anal. (C24H33Cl2N5O3) C, H, N.
5-Dim eth yla m in om eth ylfu r a n -2-ca r boxylic Acid {6-
[(4-Am in o-6,7-d im eth oxyqu in a zolin -2-yl)m eth yla m in o]-
h exyl}m eth yla m id e Dih yd r och lor id e (7). This was syn-
thesized from 6 and dimethylamine following the procedure
described for 5 and purified by chromatography. Eluting with
methylene chloride-methanol-aqueous 30% ammonia (9.5:
0.5:0.03) afforded 0.08 g (80% yield) of 7 as the free base that
was transformed into the dihydrochloride salt and crystal-
lized: mp 145 °C (from EtOH/ether); 1H NMR (free base;
CDCl3) δ 1.18-1.69 (m, 8), 2.25 (s, 6), 3.01-3.22 (m, 7), 3.42-
3.54 (m, 5), 3.66 (t, 2), 3.89 (s, 3), 3.94 (s, 3), 5.51 (br s, 2,
exchangeable with D2O), 6.27 (d, 1), 6.82-7.01 (m, 3). Anal.
(C26H40Cl2N6O4) C, H, N.
Exp er im en ta l Section
Ch em istr y. Melting points were taken in glass capillary
tubes on a Bu¨chi SMP-20 apparatus and are uncorrected. IR,
1
electron impact (EI) mass, and H NMR spectra were recorded
on Perkin-Elmer 297, VG 7070E, and Varian VXR 300 instru-
ments, respectively. Chemical shifts are reported in parts per
million (ppm) relative to tetramethylsilane (TMS), and spin
multiplicities are given as s (singlet), br s (broad singlet), d
(doublet), t (triplet), or m (multiplet). Although the IR spectra
data are not included (because of the lack of unusual features),
they were obtained for all compounds reported and were
consistent with the assigned structures. The elemental com-
positions of the compounds agreed to within (0.4% of the
calculated values. When the elemental analysis is not in-
cluded, crude compounds were used in the next step without
further purification. Chromatographic separations were per-
formed on silica gel columns (Kieselgel 40, 0.040-0.063 mm,
Merck) by flash chromatography. Compounds were named
following IUPAC rules as applied by AUTONOM, a PC
software for systematic naming in organic chemistry (Beil-
stein-Institut and Springer-Verlag).
6,7-Dim eth oxy-N2-m eth yl-N2-[2-(2-m eth yla m in oeth yl-
d isu lfa n yl)eth yl]qu in a zolin e-2,4-d ia m in e (22). A mixture
of 21 (1.0 g, 4.2 mmol) and N,N′-dimethylcystamine20 (3.5 g,
19.4 mmol) in i-AmOH (15 mL) was refluxed for 30 h. Removal
of the solvent under reduced pressure gave a residue that was
N-{6-[(4-Am in o-6,7-d im eth oxyqu in a zolin -2-yl)m eth yl-
a m in o]h exyl}-N-m eth ylben za m id e Hyd r och lor id e (8). A
solution of benzoyl chloride (0.06 mL, 0.5 mmol) in dioxane
(10 mL) was added dropwise to a solution of 2324 (0.17 g, 0.5
mmol) in dioxane (15 mL). After the mixture was stirred at

4850 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 24
Bolognesi et al.
room temperature for 4 h, the solid was filtered and washed
with ether affording 8: 90% yield; mp 133-135 °C (from EtOH/
ether); 1H NMR (DMSO-d6) δ 1.03-1.68 (m, 8), 2.86-2.94 (m,
3), 3.17-3.21 (m, 4), 3.32-3.44 (m, 1), 3.53-3.80 (m, 2), 3.84
(s, 3), 3.89 (s, 3), 7.31-7.43 (m, 5), 7.48 (s, 1), 7.72 (s, 1), 8.42
(br s, 1, exchangeable with D2O), 11.85 (br s, 1, exchangeable
with D2O). Anal. (C25H34ClN5O3) C, H, N.
N-{6-[(4-Am in o-6,7-d im eth oxyqu in a zolin -2-yl)m eth yl-
am in o]h exyl}-2-for m yl-N-m eth ylben zam ide (25). 2-Formyl-
benzoyl chloride was synthesized in a quantitative yield from
2-formylbenzoic acid following the procedure described for 24.
A solution of this chloride (0.12 g, 0.72 mmol) in dioxane (5
mL) was added dropwise to a solution of 23 (0.25 g, 0.72 mmol)
and triethylamine (0.1 mL, 0.72 mmol) in dioxane (10 mL).
After the mixture was stirred at room temperature for 24 h,
the solvent was removed under reduced pressure to give a
residue that was purified by chromatography. Eluting with
methylene chloride-methanol-aqueous 30% ammonia (9.5:
0.5:0.04) afforded 0.3 g (87% yield) of 25 as the free base: 1H
NMR (CDCl3) δ 1.20-1.82 (m, 8), 3.15-3.22 (m, 6), 3.86-3.93
(m, 4), 4.03 (s, 3), 4.10 (s, 3), 5.82 (br s, 2, exchangeable with
D2O), 7.19-7.15 (m, 2), 7.30-7.38 (m, 1), 7.89-7.95 (m, 2),
8.07 (d, 1), 10.11 (s, 1).
N-{6-[(4-Am in o-6,7-d im eth oxyqu in a zolin -2-yl)m eth yl-
a m in o]h exyl}-2-h yd r oxym eth yl-N-m eth ylben za m id e Hy-
d r och lor id e (26). Sodium borohydride (0.04 g, 1.0 mmol) was
added portionwise to a cooled (0 °C) and stirred solution of 25
(0.5 g, 1.0 mmol) in absolute ethanol (18 mL). After the
mixture was stirred and cooled for 3 h, water was added to
the solution, and the solvent was removed under reduced
pressure to give a residue that was purified by chromatogra-
phy. Eluting with methylene chloride-methanol-aqueous
30% ammonia (9.5:0.5:0.04) afforded 0.37 g (79% yield) of 26
as the free base that was transformed into the hydrochloride
salt: mp 172 °C (from EtOH/ether); 1H NMR (free base; CDCl3)
δ 1.20-1.79 (m, 8), 3.07-3.21 (m, 6), 3.52-3.74 (m, 4 + 1
exchangeable with D2O), 3.84 (s, 3), 3.92 (s, 3), 4.52 (s, 2), 5.38
(br s, 2, exchangeable with D2O), 6.77-6.92 (m, 2), 7.31-7.44
(m, 4).
raphy. Eluting with methylene chloride-ethanol-aqueous
30% ammonia (8:2:0.2) afforded 0.08 g (70% yield) of 11 as
the free base that was transformed into the trihydrochloride:
mp 148 °C (from EtOH/ether); 1H NMR (CD3OD) δ 1.10-1.89
(m, 16), 3.14-3.31 (m, 7), 3.38-3.47 (m, 3), 3.80-4.01 (m, 4),
4.10 (s, 3), 4.19 (s, 3), 4.38-4.42 (m, 2), 5.33 (br s, 2,
exchangeable with D2O), 7.50-7.57 (m, 1), 7.67-7.89 (m, 5).
Anal. (C32H52Cl3N7O3) C, H, N.
N-{6-[(4-Am in o-6,7-d im eth oxyqu in a zolin -2-yl)m eth yl-
a m in o]h exyl}-3-ch lor om eth yl-N-m eth ylben za m id e Hy-
d r och lor id e (12). This was synthesized from 23 (0.80 g, 2.3
mmol) and 3-chloromethylbenzoyl chloride (0.32 mL, 2.3 mmol)
following the procedure described for 8: 1.1 g (90% yield); mp
1
145 °C (from 2-PrOH/dioxane); H NMR (DMSO-d6) δ 1.03-
1.94 (m, 8), 2.83-2.95 (m, 3), 3.17-3.22 (m, 4), 3.33-3.45 (m,
1), 3.58-3.80 (m, 2), 3.84 (s, 3), 3.90 (s, 3), 4.80 (s, 2), 7.30-
7.45 (m, 5), 7.71 (s, 1), 8.56 (br s, 1, exchangeable with D2O),
8.81 (br s, 1, exchangeable with D2O), 11.57 (br s, 1, exchange-
able with D2O). Anal. (C26H35Cl2N5O3) C, H, N.
N-{6-[(4-Am in o-6,7-d im eth oxyqu in a zolin -2-yl)m eth yl-
a m in o]h exyl}-3-a m in om eth yl-N-m eth ylben za m id e Dih y-
d r och lor id e (13). A solution of 12 (0.05 g, 0.09 mmol) in
ethanol (10 mL) was saturated with gaseous NH3 at -10 °C.
After the mixture was stirred at room temperature for 48 h,
the solvent was removed under reduced pressure affording a
residue that was purified by chromatography. Eluting with
methylene chloride-methanol-aqueous 30% ammonia (9:1:
0.1) gave 13 as the free base that was transformed into the
dihydrochloride salt and crystallized: 0.04 g (80% yield); mp
150 °C (from EtOH/ether); 1H NMR (CD3OD) δ 1.10-1.83 (m,
8), 2.91-3.10 (m, 3), 3.17-3.36 (m, 4), 3.44-3.81 (m, 3), 3.92
(s, 3), 3.98 (s, 3), 4.18 (s, 2), 7.19-7.31 (m, 1), 7.39-7.63 (m,
5). Anal. (C26H38Cl2N6O3) C, H, N.
N-{6-[(4-Am in o-6,7-d im eth oxyqu in a zolin -2-yl)m eth yl-
a m in o]h exyl}-3-d im et h yla m in om et h yl-N-m et h ylb en z-
a m id e Dih yd r och lor id e (14). This was synthesized from
12 (0.30 g, 0.56 mmol) following the procedure described for 5
and purified by chromatography. Eluting with methylene
chloride-ethanol-aqueous 30% ammonia (9.2:0.8:0.04) af-
forded 0.15 g (87% yield) of 14 as the free base that was
transformed into the dihydrochloride salt and crystallized: mp
N-{6-[(4-Am in o-6,7-d im eth oxyqu in a zolin -2-yl)m eth yl-
a m in o]h exyl}-2-ch lor om eth yl-N-m eth ylben za m id e Hy-
d r och lor id e (9). A mixture of 26 (0.25 g, 0.52 mmol) and
SOCl2 (0.1 mL, 1.5 mmol) in anhydrous chloroform (20 mL)
was refluxed for 2 h. Removal of the solvent gave 0.26 g of 9:
1
165 °C (from EtOH/ether); H NMR (DMSO-d6) δ 1.03-1.73
(m, 8), 2.68 (s, 3), 2.70 (s, 3), 2.90-2.97 (m, 3), 3.21-3.25 (m,
4), 3.41-3.53 (m, 1), 3.62-3.80 (m, 2), 3.86 (s, 3), 3.90 (s, 3),
4.31-4.35 (m, 2), 7.38-7.63 (m, 5), 7.79 (s, 1), 8.56 (br s, 1,
exchangeable with D2O), 8.80 (br s, 1, exchangeable with D2O),
11.01 (br s, 1, exchangeable with D2O), 11.93 (br s, 1,
1
mp 155 °C (from EtOH/ether); H NMR (DMSO-d6) δ 1.10-
1.97 (m, 8), 2.86-2.97 (m, 3), 3.10-3.24 (m, 3), 3.35-3.48 (m,
2), 3.61-3.81 (m, 2), 3.91 (s, 3), 3.97 (s, 3), 4.85 (s, 2), 7.28-
7.45 (m, 5), 7.81 (s, 1), 8.58 (br s, 1, exchangeable with D2O),
8.85 (br s, 1, exchangeable with D2O), 11.85 (br s, 1, exchange-
able with D2O). Anal. (C26H35Cl2N5O3) C, H, N.
exchangeable with D2O); EI MS m/z 508 (M+). Anal. (C28H42
-
Cl2N6O3) C, H, N.
N-{6-[(4-Am in o-6,7-d im eth oxyqu in a zolin -2-yl)m eth yl-
a m in o]h exyl}-N-m et h yl-3-t h ia zolid in -3-ylm et h ylb en z-
a m id e Dioxa la te (15). A mixture of 12 (0.3 g, 0.56 mmol),
thiazolidine (0.05 mL, 0.62 mmol), triethylamine (0.09 mL,
0.62 mmol), and KI (few crystals) in absolute ethanol (15 mL)
was refluxed for 27 h. Removal of the solvent gave a residue
that was purified by chromatography. Eluting with methylene
chloride-methanol-aqueous 30% ammonia (9.5:0.5:0.04) af-
forded 0.16 g (52% yield) of 15 as the free base that was
transformed into the dioxalate salt and crystallized: mp 101-
103 °C (from (EtOH/ether); 1H NMR (free base; CDCl3) δ 1.19-
1.70 (m, 8), 2.95 (s, 3), 3.06 (s, 3), 3.16-3.25 (m, 4), 3.56-3.72
(m, 6), 3.89 (s, 3), 3.95 (s, 3), 4.03 (s, 2), 6.24 (br s, 1,
exchangeable with D2O), 6.60 (br s, 1, exchangeable with D2O),
7.10-7.21 (m, 2), 7.25-7.46 (m, 4). Anal. (C33H44N6SO4) C,
H, N.
N-{6-[(4-Am in o-6,7-d im eth oxyqu in a zolin -2-yl)m eth yl-
am in o]h exyl}-N-m eth yl-3-{[m eth yl-(6-m eth ylam in oh exyl)-
a m in o]m eth yl}ben za m id e Tr ioxa la te (16). A solution of
12 (0.13 g, 0.26 mmol) and N,N′-dimethyl-1,6-hexanediamine26
(0.38 g, 2.6 mmol) in ethanol (15 mL) was stirred at 60 °C for
1 h and at room temperature for 24 h. Removal of the solvent
under reduced pressure gave a residue that was purified by
chromatography. Eluting with methylene chloride-methanol-
aqueous 30% ammonia (9:1:0.1) afforded 0.11 g (70% yield) of
N-{6-[(4-Am in o-6,7-d im eth oxyqu in a zolin -2-yl)m eth yl-
a m in o]h exyl}-2-d im et h yla m in om et h yl-N-m et h ylb en z-
a m id e Dih yd r och lor id e (10). This was synthesized from 9
(0.20 g, 0.37 mmol) following the procedure described for 5 and
purified by chromatography. Eluting with methylene chloride-
ethanol-aqueous 30% ammonia (9.2:0.8:0.04) gave 0.14 g (75%
yield) of 10 as the free base that was transformed into the
dihydrochloride: mp 158 °C (from EtOH/ether); 1H NMR (CD3-
OD) δ 1.08-1.80 (m, 8), 2.86 (s, 6), 3.01-3.15 (m, 3), 3.22-
3.27 (m, 3), 3.60-3.81 (m, 4), 3.92 (s, 3), 3.98 (s, 3), 4.27 (s, 2),
7.29 (s, 1), 7.50-7.68 (m, 5), 8.58 (br s, 2, exchangeable with
D2O). Anal. (C28H42Cl2N6O3) C, H, N.
N-{6-[(4-Am in o-6,7-d im eth oxyqu in a zolin -2-yl)m eth yl-
a m in o]h exyl}-2-[(6-a m in oh exyla m in o)m eth yl]-N-m eth yl-
ben za m id e Tr ih yd r och lor id e (11). A solution of 24 (0.1 g,
0.2 mmol) and 1,6-hexanediamine (0.23 g, 2.0 mmol) in toluene
(50 mL) was refluxed, and the water formed was continuously
removed for 24 h. The cooled mixture was filtered and the
filtrate evaporated to give the corresponding Schiff base that
was dissolved in ethanol (10 mL) and treated with NaBH4 (0.07
g). The mixture was stirred at room temperature for 3 h,
acidified with 2 N HCl (6 mL), made basic with 2 N NaOH,
and finally extracted with chloroform. Removal of dried (Na2-
SO4) solvents gave a residue that was purified by chromatog-

Prazosin-Related Antagonists
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 24 4851
16 as the free base that was transformed into the trioxalate
salt: mp < 50 °C (from EtOH/ether); 1H NMR (free base;
CDCl3) δ 1.15-1.83 (m, 16), 2.16-2.30 (m, 5), 2.46 (s, 3), 2.54-
2.66 (m, 2), 2.85-3.04 (m, 3), 3.10-3.27 (m, 4), 3.38-3.77 (m,
5), 3.90 (s, 3), 3.94 (s, 3), 5.55 (br s, 2, exchangeable with D2O),
6.87-7.01 (m, 2), 7.19-7.38 (m, 5). Anal. (C40H59N7O15) C,
H, N.
had attained a maximal level and remained steady. Contrac-
tions were recorded by means of a force displacement trans-
ducer (FT.03 Grass and 7003 Basile) connected to a four-
channel pen recorder (Battaglia-Rangoni KV 380). In addition,
parallel experiments in which tissues did not receive any
antagonist were run in order to check any variation in
sensitivity.
N-{6-[(4-Am in o-6,7-d im eth oxyqu in a zolin -2-yl)m eth yl-
a m in o]h exyl}-4-ch lor om eth yl-N-m eth ylben za m id e Hy-
d r och lor id e (17). This was synthesized from 23 (0.8 g, 2.3
mmol) and 4-chloromethylbenzoyl chloride (0.43 g, 2.3 mmol)
following the procedure described for 8 and purified by
chromatography. Eluting with methylene chloride-ethanol-
aqueous 30% ammonia (9.5:0.5:0.03) afforded 0.45 g (40%
yield) of 17 as the free base that was transformed into the
hydrochloride salt and crystallized: mp 162-164 °C (from
Va s Defer en s P r osta tic P or tion . This tissue (from rats
of 200-230 g) was used to assess R1A-adrenergic antagonism.27
Prostatic portions of 2 cm length were mounted under 400-
450 g of tension at 37 °C in Tyrode solution of the following
composition (mM): NaCl, 130.0; KCl, 2.0; CaCl2‚2H2O, 1.8;
MgCl2, 0.89; NaHCO3, 25.0; NaH2PO4‚2H2O, 0.42; glucose, 5.6.
Desipramine hydrochloride (0.01 µM) was added to prevent
the neuronal uptake of (-)-noradrenaline. The preparations
were equilibrated for 45-60 min. During this time the bathing
solution was changed every 10 min. Concentration-response
curves for isotonic contractions in response to (-)-noradrena-
line were obtained at 30 min intervals: the first one being
discarded and the second one was taken as control. The
antagonist was allowed to equilibrate with the tissue for 30
min, and then a new concentration-response curve to the
agonist was obtained. (-)-Noradrenaline solutions contained
0.05% Na2S2O5 to prevent oxidation.
R2-Adrenoreceptor antagonist potency was determined also
on vas deferens prostatic portions of 1.5-2 cm length which
were set up in an organ bath containing Krebs solution of the
following composition (mM): NaCl, 118.4; KCl, 4.7; CaCl2,
2.52; MgSO4, 0.6; KH2PO4, 1.2; NaHCO3, 25.0; glucose, 11.1.
(()-Propranolol hydrochloride (1 µM) and desipramine (0.01
µM) were present in the Krebs solution throughout the
experiments to block â-adrenoreceptor and neuronal uptake
mechanisms, respectively. The medium was maintained at
37 °C. Field stimulation of the tissues was carried out by
means of two platinum electrodes placed near the top and
bottom of the vas deferens using 0.1 Hz square pulses of 3 ms
duration at voltage of 20-40 V. The tissues were allowed to
equilibrate for at least 1 h under a resting tension of 0.35 g
before addition of any drug. A first clonidine concentration-
response curve, taken as control, was obtained cumulatively,
avoiding the inhibition of more than 90% of twitch. Under
these conditions it was possible to obtain a second concentra-
tion-response curve not significantly different from the first
one. The antagonist was allowed to equilibrate with the tissue
for 30 min before a second cumulative concentration-response
curve with agonist was made. Parallel experiments without
any antagonist were run in order to determine the concentra-
tion of agonist causing 100% inhibition of twitch response.
Sp leen . This tissue (from rats of 250-300 g) was employed
to determine R1B-adrenoreceptor antagonist potency.28 The
spleen was removed and bisected longitudinally into two strips
which were suspended in tissue baths containing Krebs
solution of the following composition (mM): NaCl, 118.4; KCl,
4.7; CaCl2, 1.9; MgSO4, 1.2; NaHCO3, 25.0; NaH2PO4, 1.2;
glucose, 11.7. Desipramine hydrochloride (0.01 µM) and (()-
propranolol hydrochloride (1 µM) were added to prevent the
neuronal uptake of (-)-phenylephrine and to block â-adreno-
receptors, respectively. The spleen strips were placed under
1 g of resting tension and equilibrated for 1 h. The cumulative
concentration-response curves to phenylephrine were mea-
sured isometrically and obtained 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, and then a new concentration-response curve to the
agonist was constructed.
Aor ta . This tissue (from rats of 250-300 g) was used to
assess R1D-adrenoreceptor antagonist potency.28 Thoracic
aorta was cleaned from extraneous connective tissue and
placed in Krebs solution of the following composition (mM):
NaCl, 118.4; KCl, 4.7; CaCl2, 1.9; MgSO4 1.2; NaHCO3, 25.0;
NaH2PO4, 1.2; glucose, 11.7. Desipramine hydrochloride (0.01
µM) and (()-propranolol hydrochloride (1 µM) were added to
prevent the neuronal uptake of (-)-noradrenaline and to block
â-adrenoreceptors, respectively. Two helicoidal strips (15 mm
1
EtOH/ether); H NMR (DMSO-d6) δ 1.03-1.94 (m, 8), 2.85-
2.93 (m, 3), 3.17-3.22 (m, 4), 3.37-3.44 (m, 1), 3.58-3.78 (m,
2), 3.83 (s, 3), 3.87 (s, 3), 4.79 (s, 2), 7.31-7.42 (m, 2), 7.43-
7.50 (m, 2), 7.61-7.66 (m, 1) 7.75 (s, 1), 8.56 (br s, 1,
exchangeable with D2O), 8.50-8.62 (br s, 1, exchangeable with
D2O), 8.86 (br s, 1, exchangeable with D2O), 11.82 (br s, 1,
exchangeable with D2O). Anal. (C26H35Cl2N5O3) C, H, N.
N-{6-[(4-Am in o-6,7-d im eth oxyqu in a zolin -2-yl)m eth yl-
a m in o]h exyl}-4-d im et h yla m in om et h yl-N-m et h ylb en z-
a m id e Dih yd r och lor id e (18). This was synthesized from
17 (0.11 g, 0.2 mmol) following the procedure described for 5
and purified by chromatography. Eluting with methylene
chloride-ethanol-aqueous 30% ammonia (9.2:0.8:0.04) af-
forded 0.09 g (88% yield) of 18 as the free base that was
transformed into the dihydrochloride salt and crystallized: mp
162-164 °C (from EtOH/ether); 1H NMR (free base; CDCl3) δ
1.03-1.71 (m, 8), 2.21 (s, 6), 2.90-3.27 (m, 7), 3.40 (s, 2), 3.43-
3.60 (m, 3), 3.83 (s, 3), 3.92 (s, 3), 5.52 (br s, 2, exchangeable
with D2O), 6.88-6.93 (m, 2), 7.31 (s, 4). Anal. (C28H42Cl2N6O3)
C, H, N.
N-{6-[(4-Am in o-6,7-d im eth oxyqu in a zolin -2-yl)m eth yl-
a m in o]h exyl}-N-m et h yl-4-t h ia zolid in -4-ylm et h ylb en z-
a m id e Dih yd r och lor id e (19). This was synthesized from
17 (0.1 g, 0.19 mmol) following the procedure described for 15
and purified by chromatography. Eluting with methylene
chloride-methanol-aqueous 30% ammonia (9.5:0.5:0.04) af-
forded 0.06 g (50% yield) of 19 as the free base that was
transformed into the dihydrochloride salt and crystallized: mp
162 °C (from (EtOH/ether); 1H NMR (free base; CDCl3) δ 1.18-
1.73 (m, 8), 2.92-3.30 (m, 10), 3.49-3.70 (m, 6), 3.89 (s, 3),
3.95 (s, 3), 4.03 (s, 2), 5.50 (br s, 1, exchangeable with D2O),
6.86 (s, 1), 6.90-7.11 (m, 1), 7.38 (s, 4). Anal. (C29H42Cl2N6O3)
C, H, N.
N-{6-[(4-Am in o-6,7-d im eth oxyqu in a zolin -2-yl)m eth yl-
am in o]h exyl}-N-m eth yl-4-{[m eth yl-(6-m eth ylam in oh exyl)-
a m in o]m eth yl}ben za m id e Tr ioxa la te (20). This was syn-
thesized from 17 (0.14 g, 0.28 mmol) and N,N′-dimethyl-1,6-
hexanediamine26 following the procedure described for 16 and
purified by chromatography. Eluting with methylene chloride-
methanol-aqueous 30% ammonia (9:1:0.1) afforded 0.09 g
(57% yield) of 20 as the free base that was transformed into
the trioxalate salt: mp < 60 °C; 1H NMR (free base; CDCl3) δ
1.04-1.79 (m, 16), 2.10-2.33 (m, 5), 2.51 (s, 3), 2.58-2.68 (m,
2), 2.87-3.09 (m, 3), 3.13-3.32 (m, 4), 3.40-3.78 (m, 5), 3.93
(s, 3), 3.96 (s, 3), 5.60 (br s, 2, exchangeable with D2O), 6.91-
7.04 (m, 2), 7.30-7.41 (m, 5). Anal. (C40H59N7O15) C, H, N.
Biology. F u n ction a l An ta gon ism in Isola ted Ra t Tis-
su es. Male Sprague Dawley rats (Charles River, Italy) were
killed by cervical dislocation under ketamine anesthesia and
the organs required were isolated, freed from adhering con-
nective tissue, and set up rapidly under a suitable resting
tension in 15 mL organ baths containing physiological salt
solution kept at appropriate temperature (see below) and
aerated with 5% CO2:95% O2 at pH 7.4. Concentration-
response curves were constructed by cumulative addition of
agonist. The concentration of agonist in the organ bath was
increased approximately 5-fold at each step, with each addition
being made only after the response to the previous addition

4852 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 24
Bolognesi et al.
× 3 mm) were cut from each aorta beginning from the end
most proximal to the heart. The endothelium was removed
by rubbing with filter paper: the absence of acetylcholine-
induced relaxation was taken as an indicator that vessel was
denuded successfully. Vascular strips were then tied with
surgical thread and suspended in a jacketed tissue bath
containing Tyrode solution. Strips were secured at one end
to Plexiglas hooks and connected to a transducer for monitor-
ing changes in isometric contraction. After at least a 1 h
equilibration period under an optimal tension of 1 g, cumula-
tive (-)-noradrenaline concentration-response curves were
recorded at 30 min intervals, the first two being discarded the
third one taken as control. The antagonist was allowed to
equilibrate with the tissue for 30 min before the generation of
the fourth cumulative concentration-response curve to (-)-
noradrenaline.
membranes were resuspended in binding buffer of 50 mM Tris-
HCl (pH 7.4), 2.5 mM MgCl2, and 10 µM pargiline.34 Mem-
branes were incubated in a final volume of 1 mL for 30 min at
30 °C with 0.7-1.4 nM [3H]8-OH-DPAT, in the absence or
presence of competing drugs. Nonspecific binding was deter-
mined in the presence of 10 µM 5-HT. The incubation was
stopped by addition of ice-cold Tris-HCl buffer and rapid
filtration through 0.2% polyethyleneimine pretreated What-
man GF/B or Schleicher & Schuell GF52 filters.
Da ta An a lysis. The affinity constants (pA2 values, Table
1) were determined by Schild plots29 obtained from the dose
ratios at the EC50 values of the agonists calculated at three
antagonist concentrations. Each concentration was tested five
times, and Schild plots were constrained to a slope of -1.30
The compounds were tested at only one concentration when
determining R2-adrenoreceptor blocking activity because of
their low affinity for this receptor. In these cases, pA2 (-log
KB) values were calculated according to van Rossum.31 Func-
tional data were analyzed by pharmacological computer
programs38 and are presented as the mean ( SE of n experi-
ments. Differences between mean values were tested for
significance by Student’s t-test.
Ra d ioliga n d Bin d in g Assa ys. Na tive Recep tor s. Bind-
ing studies on native R2-adrenoreceptors and D2 receptors were
carried out in membranes of rat cerebral cortex and striatum,
respectively. Male Sprague Dawley rats (200-300 g, SD
Harlan/Nossan, Italy) were killed by cervical dislocation, and
different tissues were excised and immediately frozen and
stored at -70 °C until use. For R2 membrane preparations,
cerebral cortices were homogenized (2 × 20 s) in 50 volumes
of cold Tris-HCl buffer, pH 7.4, using a Politron homogenizer
(speed 7). Homogenates were centrifuged at 49000g for 10
min, resuspended in 50 volumes of the same buffer, incubated
at 37 °C for 15 min, and centrifuged and resuspended 2 more
times. The final pellets were suspended in 100 volumes of
Tris-HCl buffer, pH 7.4, containing 10 µM pargiline and 0.1%
ascorbic acid. Membranes were incubated in a final volume
of 1 mL for 30 min at 25 °C with 0.5-1.5 nM [3H]rauwolscine,
in absence or presence of competing drugs. For D2 membrane
preparations, rat striata were homogenized (2 × 20 s) in 30
volumes of cold Tris-HCl buffer, pH 7.4, using a Politron
homogenizer (speed 7) and centrifuged at 49000g for 10 min.
The final pellets were suspended in 200 volumes of Tris-HCl
incubation buffer containing 10 µM pargiline, 0.1% ascorbic
acid, and the following saline concentrations: NaCl, 120 mM;
KCl, 5 mM; CaCl2, 2 mM; MgCl2, 1 mM. The membranes were
then incubated for 15 min at 37 °C with 0.2-0.6 nM [3H]-
spiperone. Nonspecific binding was determined in the pres-
ence of 10 µM phentolamine (R2-adrenoreceptors) and 1 µM
(+)-butaclamol (D2 receptors). The incubation was stopped by
addition of ice-cold Tris-HCl buffer and rapid filtration through
0.2% polyethyleneimine pretreated Whatman GF/B or Schle-
icher & Schuell GF52 filters. The filters were then washed
with ice-cold buffer, and the radioactivity retained on the filters
was counted by liquid scintillation spectrometry.
Binding data were analyzed using the nonlinear curve-
fitting program Allfit.39 Scatchard plots were linear in all
preparations. All pseudo-Hill coefficients (nH) were not
significantly different from unity (p > 0.05). Equilibrium
inhibition constants (Ki) were derived from the Cheng-Prusoff
equation,40 Ki ) IC50/(1 + L/Kd), where L and Kd are the
concentration and the equilibrium dissociation constant of the
radioligand. pKi values (Table 2) are the mean ( SE of three
separate experiments performed in triplicate.
Ack n ow led gm en t . Supported by a grant from
MURST and the University of Bologna.
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