Prazosin-Related Compounds. Effect of Transforming the Piperazinylquinazoline Moiety into an Aminomethyltetrahydroacridine System on the Affinity for α1-Adrenoreceptors

Article abstract of DOI:10.1021/jm030952q

In a search for structurally new α₁-adrenoreceptor (α₁-AR) antagonists, prazosin (1)-related compounds 2-11 were synthesized and their affinity profiles were assessed by functional experiments in isolated rat vas deferens (α_1A), spleen (β _1B), and aorta (α_1D) and by binding assays in CHO cells expressing human cloned α₁-AR subtypes. Transformation of the piperazinylquinazoline moiety of 1 into an aminomethyltetrahydroacridine system afforded compound 2, endowed with reduced affinity, in particular for the α_1A-AR subtype. Then, to investigate the optimal features of the tricyclic moiety, the aliphatic ring of 2 was modified by synthesizing the lower and higher homologues 3 and 4. An analysis of the pharmacological profile, together with a molecular modeling study, indicated the tetrahydroacridine moiety as the most promising skeleton for α ₁-antagonism. Compounds 5-8, where the replacement of the furoyl group of 2 with a benzoyl moiety afforded the possibility to evaluate the effect of the substituent trifluoromethyl on receptor binding, resulted, except for 7, in a rather surprising selectivity toward α_1B-AR, in particular vs the α_1A subtype. Also the insertion of the 2,6-dimethoxyphenoxyethyl function of WB 4101 on the tetrahydroacridine skeleton of 2, and/or the replacement of the aromatic amino function with a hydroxy group, affording derivatives 9-11, resulted in α_1B-AR selectivity also vs the α_1D subtype. On the basis of these results, the tetrahydroacridine moiety emerged as a promising tool for the characterization of the α₁-AR, owing to the receptor subtype selectivity achieved by an appropriate modification of the lateral substituents.

Full text of DOI:10.1021/jm030952q

J . Med. Chem. 2003, 46, 4895-4903
4895
P r a zosin -Rela ted Com p ou n d s. Effect of Tr a n sfor m in g th e
P ip er a zin ylqu in a zolin e Moiety in to a n Am in om eth yltetr a h yd r oa cr id in e System
on th e Affin ity for r1-Ad r en or ecep tor s
Michela Rosini,† Alessandra Antonello,† Andrea Cavalli,† Maria L. Bolognesi,† Anna Minarini,†
Gabriella Marucci,‡ Elena Poggesi,§ Amedeo Leonardi,§ and Carlo Melchiorre*,†
Department of Pharmaceutical Sciences, University of Bologna, Via Belmeloro 6, 40126 Bologna, Italy,
Department of Chemical Sciences, University of Camerino, Via S. Agostino 1, 62032 Camerino (MC), Italy,
and Research and Development Division, Recordati S.p.A., Via Civitali 1, 20148 Milano, Italy
Received J uly 8, 2003
In a search for structurally new R1-adrenoreceptor (R1-AR) antagonists, prazosin (1)-related
compounds 2-11 were synthesized and their affinity profiles 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-AR subtypes. Transformation of the
piperazinylquinazoline moiety of 1 into an aminomethyltetrahydroacridine system afforded
compound 2, endowed with reduced affinity, in particular for the R1A-AR subtype. Then, to
investigate the optimal features of the tricyclic moiety, the aliphatic ring of 2 was modified by
synthesizing the lower and higher homologues 3 and 4. An analysis of the pharmacological
profile, together with a molecular modeling study, indicated the tetrahydroacridine moiety as
the most promising skeleton for R1-antagonism. Compounds 5-8, where the replacement of
the furoyl group of 2 with a benzoyl moiety afforded the possibility to evaluate the effect of the
substituent trifluoromethyl on receptor binding, resulted, except for 7, in a rather surprising
selectivity toward R1B-AR, in particular vs the R1A subtype. Also the insertion of the
2,6-dimethoxyphenoxyethyl function of WB 4101 on the tetrahydroacridine skeleton of 2, and/
or the replacement of the aromatic amino function with a hydroxy group, affording derivatives
9-11, resulted in R1B-AR selectivity also vs the R1D subtype. On the basis of these results, the
tetrahydroacridine moiety emerged as a promising tool for the characterization of the R1-AR,
owing to the receptor subtype selectivity achieved by an appropriate modification of the lateral
substituents.
In tr od u ction
type of quinazoline-bearing compounds widely used as
a pharmacological tool for R1-AR subtypes characteriza-
tion and as an effective drug in the management of
hypertension. In previous studies both the piperazine
moiety and the furan ring of prototype 1 have been
modified, affording antagonists that are able to differ-
entiate among R1-AR subtypes.11 This observation may
form the basis to further modify the structure of these
analogues, in an attempt to improve the selectivity.
In 1987, Campbell and co-workers,14 in order to
rationalize the exceptional R1-AR affinity and R1-
competitive antagonism displayed by certain 2,4-di-
amino-6,7-dimethoxyquinazoline derivatives, visualized
these compounds as conformationally restricted ana-
logues of noradrenaline. Following X-ray analyses and
molecular mechanics calculations, they could advance
that a dominant role in the receptor recognition process
was played by the protonated N1 of the quinazoline
system, which should mime the amine function of the
neurotransmitter, protonated at physiological pH. In
particular, they pointed out a charged-reinforced H-bond
interaction involving the quinazoline-N1 function and
an anionic site of the receptor. The relevance of the N1-
protonation in receptor binding was also confirmed
through the synthesis of a series of quinoline-analogues
of 1, in which the removal of the N3 function resulted
in an increased R1-AR affinity, accordingly with a
In recent years, the heterogeneity of the R1-adreno-
receptors (R1-ARs) has been claimed both on a molecular
and pharmacological level. The latest picture of R1-ARs
shows at least three well-characterized subtypes, namely,
R
1A (R1a), R1B (R1b), and R1D (R1d), with upper and lower
case subscripts being used to designate native or
recombinant receptor, respectively,1,2 while evidence
with regard to a putative R1L-AR now suggests it is a
functional phenotype of the R1A-AR.3
The effort to design agents selective for each of the
three R1-AR subtypes has been an active area of
research. Whereas the efficacy of R1A-AR antagonists in
the treatment of benign prostatic hyperplasia has been
demonstrated4,5 and the role of the R1B-AR subtype in
the regulation of blood pressure has recently been
advanced,6 a potential therapeutic use for the R1D-AR
subtype has not been firmly established yet.
In continued efforts to identify high-affinity, selective
ligands for subtypes of the R1-AR, our research group
has long being involved in designing new R1-AR antago-
nists structurally related to prazosin (1),7-13 the proto-
* Corresponding author. Tel. +39-051-2099706. Fax: +39-051-
2099734. E-mail: camelch@kaiser.alma.unibo.it.
† University of Bologna.
‡ University of Camerino.
§ Recordati S.p.A.
10.1021/jm030952q CCC: $25.00 © 2003 American Chemical Society
Published on Web 10/10/2003

4896 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 23
Rosini et al.
To evaluate the effect of a different relative position
of N1 function and furan moiety on R1-AR affinity and
selectivity, analogues 3 and 4, where a cyclopentane and
a cycloheptane moiety replaced the cyclohexane ring,
respectively, were synthesized.
The presence of a phenyl in place of the furan ring of
1 in related analogues had previously afforded the
opportunity to examine the effect of substituents on both
potency and selectivity toward R1-AR subtypes, through
the insertion, in all positions of the phenyl ring, of
properly chosen groups.13 Since the unsubstitued ana-
logue, together with compounds with the trifluoromethyl
group in the ortho, meta, or para position, resulted in
the most intriguing molecules because of their R1D
selectivity, the same structural modifications were
performed on 2, providing tetrahydroacridine analogues
5-8.
Moreover, because the 2,6-dimethoxyphenoxyethyl
function of WB 4101 {N-[2-(2,6-dimethoxyphenoxy)-
ethyl]-2,3-dihydro-1,4-benzodioxin-2-methanamine}, the
prototype of benzodioxan-bearing compounds as R1-AR
selective antagonist, is a key pharmacophore at this
receptor type,18 we thought it of interest to combine, in
the same molecule, this moiety and the tetrahydroacri-
dine system of 2, affording the hybrid structure 9.
Finally, we synthesized compounds 10 and 11, where
the 4-amino group of 2 and 9, respectively, was replaced
with a hydroxyl moiety, with the aim of verifying if this
structural modification could differently affect affinity
for R1-AR subtypes.
F igu r e 1. Design strategy for the synthesis of 2-11 by
connecting the piperazine ring of 1 to a quinoline ring system.
significantly enhanced basicity of the N1 nitrogen, with
respect to the prototype.15 Moreover, the role of the N1
function in the receptor recognition process was also
assessed through a series of isoquinoline derivatives in
which the lack of this nitrogen atom resulted in no
significant affinity for R1-AR.16
Very recently, a further interaction model 1-R1-ARs
suggested the N1 function on the quinazoline ring to be
one of the key groups involved in receptor binding,
together with the two methoxy moieties, the 4-amino
group, and the carbonyl function. Docking studies
revealed that the N1 moiety should possibly interact,
by hydrogen-bond formation, with the hydroxyl group
of a serine residue of TM5, which is present in all of
the three subtypes. Moreover, an identical number of
binding sites for 1 within all the R1-AR subtypes was
advanced, 1 supposedly interacting with amino acids in
We describe here the synthesis and the pharmacologi-
cal profile of tetrahydroacridines 2-11 in functional
and binding experiments in comparison with prototypes
prazosin (1) and WB 4101.
Ch em istr y
the same positions and in identical helices of R1A, R1B
,
All the compounds were synthesized by standard
procedures (Schemes 1-3) and were characterized by
IR, 1H NMR, mass spectra, and elemental analysis. The
structure of key intermediates 12-14 and of isomers
18 and 19 was assigned by means of 1H NMR and COSY
experiments.
and R1D receptors, thus accounting for the nonselectivity
of this ligand within R1-ARs.17
With the aim of improving the selectivity profile at
R1-AR, in this study 1 was further modified, affording
a series of derivatives in which the N1 function is
preserved, even if included in a different cyclic system.
Actually, since the synthesis of constrained analogues
of a lead compound represents a useful approach when
searching for clues about binding site topography, we
modified the quinazoline system into a tetrahydroacri-
dine ring, affording 2 (Figure 1). This structural modi-
fication, in addition to preserving N1 basicity, could force
N1 and the furan moiety to assume a reciprocal ar-
rangement likely similar to that of prazosin in one of
its low-energy conformations. Moreover, since the N3
function is not essential for R1-AR affinity, we thought
that its removal should not negatively affect the activity
of these tetrahydroacridine derivatives at R1-ARs.
To test such a hypothesis, a molecular modeling study
was carried out. In particular, the three-dimensional
model of our target molecule, 2, was built and analyzed
by means of a Monte Carlo conformational search. Then,
this was superimposed onto the crystallographic 3D
structure of 1, as retrieved from the Cambridge Struc-
tural Databank. The quite good superimposition ob-
tained between 2 and 1 (Figure 2) prompted us to carry
out the synthesis of the present series of derivatives.
The synthesis of compounds 2-4 was achieved by
condensation of 2-amino-4,5-dimethoxybenzonitrile with
the appropriate cycloketone in the presence of ZnCl2,
followed by reduction of the nitro group of derivatives
12-14 and coupling of the obtained amines (15-17)
with 2-furoyl chloride. 3-(Nitromethyl)cyclopentanone,
3-(nitromethyl)cyclohexanone, and 3-nitromethylcyclo-
heptanone were synthesized using, as starting materi-
als, nitromethane and the appropriate R,â-unsaturated
cycloketone according to Rosini et al.19 Similarly, com-
pounds 5-8 were obtained through amidation of 15 with
benzoyl chloride or with the appropriate (trifluoro-
methyl)benzoyl chloride, which was generated in situ
by treating the corresponding (trifluoromethyl)benzoic
acid with SOCl2 (Scheme 1).
Condensation of 15 with 2-(2,6-dimethoxyphenoxy)-
acetaldehyde,20 followed by reduction of the intermedi-
ate Schiff base with NaBH4, afforded the hybrid struc-
ture 9, as depicted in Scheme 2.
Finally, the POCl3-mediated condensation between
4,5-dimethoxyanthranilic acid and 3-(nitromethyl)cy-
clohexanone afforded the two possible structural isomers

Prazosin-Related Compounds
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 23 4897
Sch em e 1
Sch em e 2
Bin d in g Exp er im en ts. The pharmacological profile
of compounds 2, 3, and 6-11 was further evaluated by
radioreceptor binding assays using 1 and WB 4101 as
reference standards. [3H]Prazosin was used to label
cloned human R1-ARs expressed in Chinese hamster
ovary (CHO) cells.24
Resu lts a n d Discu ssion
The affinity for the three R1-AR subtypes of com-
pounds used in the present study was expressed as pKb
and pKi values and values are shown in Table 1. To
make relevant considerations on structure-activity
relationships, prototype 1 and WB 4101 were included
for comparison. All the tetrahydroacridine derivatives
behaved, like 1, as competitive antagonists, as they did
not affect the maximal responses induced by the agonist
while causing a parallel shift to the right of the
concentration-response curves to the agonist. Although
compounds 2 and 9 bear in their structure the tetrahy-
droacridine moiety of tacrine, a well-known AChE
inhibitor, they were poor AChE inhibitors as revealed
by their pIC50 values of 5.30 ( 0.11 and 5.05 ( 0.07,
respectively.
Since the affinities of all of the novel analogues at
R1-adrenoreceptor subtypes were substantially lower
than the prototype 1, the enantiomers produced by the
introduction of the chiral center were not separated. In
molecular modeling studies to establish the putative
active enantiomer, either R or S of 3 was superimposed
onto the template, i.e., the energy-minimized crystal
18 and 19. Reduction of the nitro function of 19 with
Raney Ni afforded the corresponding amine 20, which,
in turn, was submitted to coupling reaction with 2-furoyl
chloride, providing 10, or condensed with 2-(2,6-di-
methoxyphenoxy)acetaldehyde,20 to give 11 (Scheme 3).
Biology
F u n ction a l Stu d ies. Receptor subtype selectivity of
compounds 2-11 was determined at R1-ARs on different
isolated rat tissues using 1 and WB 4101 as reference
standards. R1-AR subtypes blocking activity was as-
sessed by antagonism of (-)-noradrenaline-induced
21
contraction of prostatic vas deferens (R1A
)
or thoracic
22
aorta (R1D
)
and by antagonism of (-)-phenylephrine-
induced contraction of rat spleen (R1B).23 Furthermore,
compounds 2 and 9, owing to their structural analogy
with tacrine, were evaluated also for their inhibitory
activity of AChE from human serum.

4898 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 23
Rosini et al.
Sch em e 3
Ta ble 1. Affinity Constants, Expressed as pKB (Isolated
Tissues) or pKi (CHO Cells) Values, of 1-11 and WB4101 at
R1-ARs on Isolated Prostatic Vas Deferens (R1A), Spleen (R1B),
and Thoracic Aorta (R1D) and at Human Recombinant R1-AR
Subtypes (pKi)
tion for the tricyclic moiety was detected. It turned out
that the S-enantiomer fitted onto 1 better than the
R-enantiomer (RMSD equal to 0.945 and 1.706 Å,
respectively). Therefore, in the following, only S-enan-
tiomers of the present series of derivatives were taken
into account, since they were considered to be those
biologically active.
Once established that the likely active enantiomer of
this series of antagonists was the S one, we focused on
the lead compound of the present series, 2, synthesized
in an attempt to identify a new lead as R1-AR antago-
nist, based on a tetrahydroacridine skeleton. The tri-
cyclic moiety of the S-enantiomer of 2 provided two
different conformations, being the substituent in posi-
tion 3 of the tricyclic moiety either axial or equatorial.
The heats of formation of the two conformers showed
that the equatorial was more stable than the axial one
by almost 2 kcal/mol. More interestingly, the best fitting
equatorial conformer superimposed much better onto 1
than the best fitting axial one (RMSD ) 0.769 and 1.988
Å, respectively). This showed that the equatorial con-
former was most likely to be the active one.
a
pKB
pKib
compd
R1A
R1B
R1D
R1a R1b R1d
1
2
3
4
5
6
7
8
8.93 ( 0.09 9.06 ( 0.11 8.93 ( 0.10 9.23 9.39 9.65
6.52 ( 0.05 7.30 ( 0.20 7.47 ( 0.16 7.02 7.91 7.80
6.26 ( 0.08 6.82 ( 0.04 6.11 ( 0.12 6.80 7.10 6.70
6.34 ( 0.21 6.92 ( 0.11 6.94 ( 0.08
6.67 ( 0.22 7.19 ( 0.08 6.63 ( 0.18
6.47 ( 0.14 7.33 ( 0.25 6.85 ( 0.08 6.54 8.13 7.61
7.16 ( 0.08 7.36 ( 0.03 7.58 ( 0.23 7.50 7.90 7.60
6.08 ( 0.08 7.20 ( 0.14 6.67 ( 0.11 7.07 8.05 7.72
5.70 ( 0.11 7.20 ( 0.11 6.39 ( 0.09 6.01 7.40 6.70
5.76 ( 0.08 6.95 ( 0.09 6.41 ( 0.07 5.59 6.53 5.94
6.24 ( 0.13 7.05 ( 0.09 5.92 ( 0.05 5.77 6.66 6.41
9
10
11
WB4101 9.48 ( 0.09 8.20 ( 0.11 8.85 ( 0.12 9.37 8.0 9.29
a
pKB values ( SE were calculated at one concentration (in the
range of 0.1-10 µM) according to van Rossum.31 Each concentra-
tion was tested from four to five times. Equilibrium dissociation
b
constants (Ki) were derived from IC50 values using the Cheng-
Prusoff equation.33 Each experiment was performed in triplicate.
Ki values were from two or three experiments, which agreed within
(20%.
The intriguing similarity shown by 1 and 2, with
respect to the relative positions of the pharmacophoric
functions, suggested that 2 should possibly fit with the
same amino acids of the R1-AR involved in prazosin
structure of 1. Actually, 3 is the less flexible antagonist
of the present series, being the tricyclic system coplanar.
Therefore, for each enantiomer of 3 only one conforma-

Prazosin-Related Compounds
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 23 4899
for R1A- and R1D-ARs, thus inducing relative R1B-AR
subtype selectivity. For all the derivatives of this series,
the major negative effect of the tetrahydroacridine
skeleton is observed for the R1D-AR subtype, which looks
more sensitive to the steric hindrance enhancement
produced by this moiety relative to the quinazoline
system of 1.
The inclusion of the 2,6-dimethoxyphenoxyethyl func-
tion of the potent and selective R1A-AR antagonist WB
4101 onto the tetrahydroacridine skeleton of 2, giving
compound 9, led to a very different affinity profile at
the R1A-AR subtype with respect to prototypes: the R1A
pKB value for 9 was 4575-fold lower than that for WB
4101 and 7-fold lower than that for 2. An analysis of
the structural features of the tetrahydroacridine skel-
eton of 2 and of the benzodioxan moiety of WB 4101
clearly reveals key physicochemical differences. As a
matter of fact, in this contest, the presence, in 9, of a
second basic function, may assume a relevant role in
determining a different ligand-receptor recognition
process. Interestingly, however, 9 turned out to be as
potent as 2 at the R1B-AR, suggesting that the lateral
substituent inserted onto the tetrahydroacridine skel-
eton is not able to effectively tune affinity at this site.
The behavior at the R1D-AR subtype is intermediate
between R1A- and R1B-AR subtypes.
Replacement of the amino function of 9 with a
hydroxy group, affording compound 11, resulted in a 35-
fold higher affinity at R1A-AR in functional experiments.
The finding that the same modification, performed on
2 to give 10, resulted instead in a decreased potency at
the R1A-AR subtype, put in evidence the importance of
the lateral chain in determining affinity for this receptor
type.
In conclusion, the modification of the piperazi-
nylquinazoline moiety of 1 into an aminomethyltetrahy-
droacridine system afforded compounds 2-11. The
whole series of derivatives resulted to be less potent at
all R1-AR subtypes relative to prototype 1, most of them
displaying a tendency to selectivity for the R1B-AR
subtype both in functional and binding experiments.
The newly introduced tricyclic system represents an
interesting pharmacophore at the R1B-AR subtype, lead-
ing to a significant affinity, which does not seem to be
greatly influenced by the lateral substituent. On the
contrary, the pharmacological profile of these com-
pounds at R1A- and R1D-ARs is significantly influenced
by the nature of the lateral chain, thus providing the
opportunity to modulate selectivity for the R1B-AR by
means of an appropriate modification of their structure.
F igu r e 2. Superimposition of AM1-minimized low-energy
conformation of 2 (carbon atoms are green) onto the AM1-
minimized 1 crystal structure. The superimposition was ac-
complished by fitting the following atoms: the oxygen atoms
of the methoxy groups, the pyridine and the aniline nitrogen
atoms, the carbonyl oxygen, a carbon and the oxygen atoms
of the furan ring. The pharmacophoric functions of 2 fit well
those of 1. However, the steric hindrance of the aliphatic
portion of 2 could prevent optimal interactions with the
biological counterpart.
binding. Notwithstanding, the affinity profile of 2 at R1-
ARs resulted to be significantly different from that
displayed by the prototype. Actually, 2 was found to be
more than 2 orders of magnitude weaker at the R1A-AR
subtype and between one and 2 orders of magnitude less
potent at R1B- and R1D-AR subtypes in functional assays.
A similar trend was observed in binding experiments.
Although 2 appears to be capable of all of the key
antagonist-receptor interactions shown for 1 in the
models previously described, a 3D model of the super-
imposition showed that the loss of potency could be
ascribed to the steric hindrance induced by the newly
introduced aliphatic portion of the tetrahydroacridine
backbone of 2 (Figure 2). To verify such a hypothesis,
the aliphatic ring of 2 was modified by synthesizing its
lower and higher homologues, 3 and 4, respectively.
Both of these compounds were identified to be slightly
less potent than 2 at all the R1-AR subtypes, except for
the affinity of 3 at the R1D subtype, which was signifi-
cantly reduced. The worse capability of derivatives 3 and
4 to fit 1 (3, RMSD of 0.945 Å; 4, RMSD of 1.306 Å)
could be mainly responsible for this loss of affinity, thus
not allowing an appropriate evaluation of the effect of
steric hindrance in receptor binding. However, the
higher R1D affinity of 4 versus 3 cannot be easily
explained by the present biological data and molecular
modeling results.
The tetrahydroacridine moiety of 2 proved to be the
most promising skeleton for R1-AR antagonism and,
consequently, its structure was further modified in order
to explore affinity and selectivity toward R1-AR sub-
types.
The efficacy of the trifluoromethyl substituent on the
phenyl ring of prazosin analogues in inducing selectivity
toward the R1D-AR subtype has previously been re-
ported.13 The same structural modifications, performed
on 2, provided analogues 5-8. With the exception of 7,
rather surprisingly, these compounds displayed an
appreciable selectivity for the R1B-AR subtype. It is
evident that the replacement of the quinazoline system
with a tetrahydroacridine ring negatively affects affinity
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,
electron impact (EI) mass, and direct infusion ESI-MS spectra
were recorded on Perkin-Elmer 297, VG 7070E, and Waters
1
ZQ 4000 apparatus, respectively. H NMR and COSY experi-
ments were recorded on Mercury 400 and Varian VXR 300
instruments. Chemical shifts are reported in parts per million
(ppm) relative to tetramethylsilane (TMS), and spin multiplici-
ties 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 compositions of
the compounds agreed to within (0.4% of the calculated value.

4900 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 23
Rosini et al.
When the elemental analysis is not included, crude compounds
were used in the next step without further purification.
Chromatographic separations were performed on silica gel
columns by flash (Kieselgel 40, 0.040-0.063 mm; Merck) or
gravity column (Kieselgel 60, 0.063-0.200 mm; Merck) chro-
matography. Compounds were named following IUPAC rules
as applied by Beilstein-Institut AutoNom (version 2.1), a PC
integrated software package for systematic names in organic
chemistry.
6,7-Dim eth oxy-3-(n itr om eth yl)-1,2,3,4-tetr a h yd r oa cr i-
d in -9-yla m in e (12). A mixture of 2-amino-4,5-dimethoxyben-
zonitrile (1.0 g, 5.61 mmol), 3-(nitromethyl)cyclohexanone19
(0.88 g, 5.61 mmol), and dry ZnCl2 (1.7 g) was heated at 100
°C for 3 h. After cooling, the reaction mixture was treated with
1 N NaOH and the separated solid, collected by filtration, was
washed with ether (2 × 30 mL), CHCl3 (2 × 30 mL), and EtOAc
(2 × 30 mL), affording crude 12: 40% yield; 1H NMR (DMSO-
d6) δ 1.55-1.62 (m, 1), 1.92-2.02 (m, 1), 2.45-2.76 (m, 4),
2.82-2.91 (m, 1), 3.78 (s, 3), 3.81 (s, 3), 4.61 (d, 2), 6.20 (br s,
exchangeable with D2O, 2), 7.02 (s, 1), 7.40 (s, 1); EI MS m/z
317 (M+).
in dry EtOH (2 mL) was added dropwise to a solution of 15
(0.10 g, 0.35 mmol) and triethylamine (0.048 mL, 0.35 mmol)
in dry EtOH (10 mL). Stirring at room temperature for 24 h
afforded 2 as a white solid that was collected by filtration and
transformed into the hydrochloride salt: 80% yield; mp 220-
222 °C (from EtOH); 1H NMR (CD3OD) δ 1.60-1.78 (m, 1),
2.18-2.25 (m, 2), 2.51-2.82 (m, 3), 3.01-3.16 (m, 1), 3.46 (t,
2), 3.99 (s, 3), 4.01 (s, 3), 6.60-6.65 (m, 1), 7.03 (s, 1), 7.18 (d,
1), 7.61 (s, 1), 7.68-7.71 (m, 1); EI MS m/z 381 (M+). Anal.
(C21H24ClN3O4) C, H, N.
F u r a n -2-ca r boxylic a cid (9-a m in o-6,7-d im eth oxy-2,3-
d ih yd r o-1H-cyclop en ta [b]qu in olin -2-ylm eth yl)a m id e h y-
d r och lor id e (3) was synthesized from 16 (0.10 g, 0.37 mmol),
triethylamine (0.051 mL, 0.37 mmol), and 2-furoyl chloride
(0.036 mL, 0.37 mmol) by following the procedure described
for 2 and purified by flash chromatography. Elution with
CHCl3/MeOH/aqueous 30% ammonia (9:1:0.06) afforded crude
3 that was transformed into the hydrochloride salt: 65% yield;
mp 263-265 °C (from ether/EtOH); 1H NMR (CD3OD) δ 2.12-
2.19 (m, 1), 2.25-3.15 (m, 4), 3.48 (t, 2), 3.86 (s, 3), 3.91 (s, 3),
6.58-6.60 (m, 1), 7.02 (s, 1), 7.14 (d, 1), 7.28 (s, 1), 7.68 (s, 1);
MS (ESI+) m/z 368 (M+H)+. Anal. (C20H22ClN3O4) C, H, N.
F u r a n -2-ca r boxylic a cid (11-a m in o-2,3-d im eth oxy-
7,8,9,10-t e t r a h y d r o -6H -c y c lo h e p t a [b ]q u in o lin -7-y l-
m eth yl)a m id e h yd r och lor id e (4) was synthesized from 17
(0.10 g, 0.33 mmol), triethylamine (0.046 mL, 0.33 mmol), and
2-furoyl chloride (0.033 mL, 0.33 mmol) by following the
procedure described for 2 and purified by flash chromatogra-
phy. Elution with CH2Cl2/EtOH/aqueous 30% ammonia (8.5:
1.5:0.01) afforded crude 4 that was transformed into the
hydrochloride salt: 72% yield; mp 241-243 °C (from ether/
EtOH); 1H NMR (DMSO-d6) δ 1.10-1.61 (m, 2), 1.78-1.97 (m,
3), 2.60-2.77 (m, 1), 3.04-3.22 (m, 5), 4.95 (s, 3), 4.97 (s, 3),
6.58-6.65 (m, 1), 7.18 (d, 1), 7.25 (s, 1), 7.81 (d, 2), 8.20 (br s,
exchangeable with D2O, 2), 8.58 (t, exchangeable with D2O,
1); MS (ESI+) m/z 396 (M + H)+. Anal. (C22H26ClN3O4) C, H,
N.
N-(9-Am in o-6,7-d im et h oxy-1,2,3,4-t et r a h yd r oa cr id in -
3-ylm eth yl)ben za m id e h yd r oclor id e (5) was synthesized
from 15 (0.045 g, 0.16 mmol), triethylamine (0.022 mL, 0.16
mmol), and benzoyl chloride (0.018 mL, 0.16 mmol) by follow-
ing the procedure described for 2 and purified by flash
chromatography. Elution with CH2Cl2/MeOH/aqueous 30%
ammonia (9:1:0.1) afforded crude 5 that was transformed into
the hydrochloride salt: 78% yield; mp 272-278 °C (from ether/
EtOH);1H NMR (CD3OD) δ 1.40-1.58 (m, 1), 2.10-2.23 (m,
2), 2.42-2.78 (m, 3), 2.95-3.05 (m, 1), 3.43 (t, 2), 3.88 (s, 3),
3.92 (s, 3), 7.02 (s, 1), 7.30 (s, 1), 7.42-7.56 (m, 3), 7.86 (d, 2);
MS (ESI+) m/z 392 (M + H)+. Anal. (C23H26ClN3O3) C, H, N.
N-(9-Am in o-6,7-d im et h oxy-1,2,3,4-t et r a h yd r oa cr id in -
3-ylm et h yl)-2-(t r iflu or om et h yl)b en za m id e h yd r och lo-
r id e (6). A solution of 2-(trifluoromethyl)benzoic acid (0.033
g, 0.174 mmol) and SOCl2 (0.13 mL, 1.74 mmol) in CHCl3 was
refluxed for 1 h. Removal of the solvent under reduced pressure
afforded crude 2-(trifluoromethyl)benzoyl chloride in a quan-
titative yield. A solution of this chloride (0.036 g, 0.174 mmol)
in dry CHCl3 (2 mL) was added dropwise to a solution of 15
(0.050 g, 0.174 mmol) and triethylamine (0.036 mL, 0.260
mmol) in dry EtOH (8 mL). After the mixture was stirred at
room temperature for 12 h, the solvent was removed under
reduced pressure to give a residue that was purified by flash
chromatography. Elution with CHCl3/MeOH/aqueous 30%
ammonia (9:1:0.04) afforded crude 6 that was transformed into
the hydrochloride salt: 77% yield; mp 252-254 °C (from ether/
EtOH); 1H NMR (CD3OD) δ 1.42-1.78 (m, 1H), 2.15-2.28 (m,
2H), 2.42-2.81 (m, 3H), 2.98-3.16 (m, 1H), 3.43 (d, 2H), 3.94
(s, 3H), 3.97 (s, 3H), 7,01 (s, 1H), 7.38 (s, 1H), 7.58-7.81 (m,
4H); MS (ESI+) m/z 460 (M + H)+. Anal. (C24H25ClF3N3O3) C,
H, N.
6,7-Dim et h oxy-2-(n it r om et h yl)-2,3-d ih yd r o-1H-cyclo-
p en ta [b]qu in olin -9-yla m in e (13) was synthesized from
2-amino-4,5-dimethoxybenzonitrile (1.88 g, 10.55 mmol) and
3-(nitromethyl)cyclopentanone19 (1.51 g, 10.55 mmol) by fol-
lowing the procedure described for 12, affording crude 13: 35%
1
yield; H NMR (DMSO-d6) δ 2.58-2.79 (m, 2), 2.99-3.21 (m,
3), 3.90 (s, 3), 3.92 (s, 3), 4.60-4.82 (m, 2), 6.35 (br s,
exchangeable with D2O, 2); 7.10 (s, 1), 7.45 (s, 1).
2,3-Dim eth oxy-7-(n itr om eth yl)-7,8,9,10-tetr a h yd r o-6H-
cycloh ep ta [b]qu in olin -11-yla m in e (14) was synthesized
from 2-amino-4,5-dimethoxybenzonitrile (2.08 g, 11.67 mmol)
and 3-(nitromethyl)cycloheptanone19 (2 g, 11.67 mmol) by
following the procedure described for 12, affording crude 14:
47% yield; 1H NMR (DMSO-d6) δ 1.16-1.62 (m, 2), 1.78-1.98
(m, 2), 2.15-2.36 (m, 1), 2.42-2.61 (m, 1), 2.79-3.10 (m, 3),
3.82 (s, 3), 3.86 (s, 3), 4.45 (t, 2), 6.21 (br s, exchangeable with
D2O, 2), 7.05 (s, 1), 7.42 (s, 1).
3-(Am in om eth yl)-6,7-dim eth oxy-1,2,3,4-tetr ah ydr oacr i-
d in -9-yla m in e (15). A suspension of 12 (0.18 g, 0.57 mmol)
and Raney Ni (nickel sponge; suspension in water) (0.10 g) in
MeOH (20 mL) and CH3COOH (5 mL) was hydrogenated for
6 h at room temperature. Following catalyst removal, the
solvent was evaporated, yielding a residue that was dissolved
in water, treated with 40% NaOH, and continuously extracted
with CHCl3 (50 mL). Removal of the dried solvent gave a
residue that was purified by flash chromatography. Elution
with CHCl3/MeOH/aqueous 30% ammonia (6:4:0.4) afforded
15 as a yellow solid: 75% yield; mp 144-148 °C; 1H NMR
(DMSO-d6) δ 1.23-1.42 (m, 1), 1.61-1.75 (m, 1), 2.01-2.18
(m, 1), 2.33-2.78 (m, 5), 2.80-2.96 (m, 1), 3.83 (s, 3), 3.86 (s,
3), 6.15 (br s, exchangeable with D2O, 2), 7.02 (s, 1), 7.42 (s,
1).
2-(Am in om eth yl)-6,7-d im eth oxy-2,3-d ih yd r o-1H-cyclo-
p en ta [b]qu in olin -9-yla m in e (16) was synthesized from 13
(0.31 g, 1.02 mmol) by following the procedure described for
15 and purified by flash chromatography. Elution with a step
gradient system of CHCl3/MeOH/aqueous 30% ammonia (9:1:
0.1 to 8.5:1.5:0.15) afforded 16 as a white solid: 90% yield;
mp 148-150 °C; 1H NMR (CD3OD) δ 2.61-2.70 (m, 1), 2.72-
2.85 (m, 2), 2.97 (d, 2), 3.05-3.29 (m, 2), 3.98 (s, 3), 4.02 (s, 3),
7.18 (s, 1), 7.45 (s, 1); MS (ESI+) m/z 274 (M + H)+.
7-(Am in om et h yl)-2,3-d im et h oxy-7,8,9,10-t et r a h yd r o-
6H-cycloh ep ta [b]qu in olin -11-yla m in e (17) was synthe-
sized from 14 (0.35 g, 1.16 mmol) by following the procedure
described for 15 and purified by flash chromatography. Elution
with CHCl3/MeOH/aqueous 30% ammonia (8.5:1.5:0.15) af-
forded 17 as a white solid: 87% yield; mp 138-140 °C; 1H
NMR (CD3OD) δ 1.20-1.55 (m, 3), 1.75-2.03 (m, 2), 2.35-
2.58 (m, 3), 2.65-2.90 (m, 3), 3.90 (s, 3), 3.92 (s, 3), 7.05 (s, 1),
7.23 (s, 1).
N-(9-Am in o-6,7-d im et h oxy-1,2,3,4-t et r a h yd r oa cr id in -
3-ylm et h yl)-3-(t r iflu or om et h yl)b en za m id e h yd r och lo-
r id e (7) was synthesized from 3-(trifluoromethyl)benzoic acid
(0.040 g, 0.209 mmol) and 15 (0.06 g, 0.209 mmol) by following
the procedure described for 6 and purified by flash chroma-
F u r a n -2-ca r boxylic Acid (9-Am in o-6,7-d im eth oxy-
1,2,3,4-tetr a h yd r oa cr id in -3-ylm eth yl)a m id e h yd r och lo-
r id e (2). A solution of 2-furoyl chloride (0.034 mL, 0.35 mmol)

Prazosin-Related Compounds
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 23 4901
tography. Elution with CHCl3/MeOH/aqueous 30% ammonia
(9:1:0.04) afforded crude 7 that was transformed into the
hydrochloride salt: 65% yield; mp 250-252 °C (from ether/
1.84 (m, 1), 1.98-2.06 (m, 1), 2.30-2.46 (m, 2), 2.62-2.85 (m,
4), 3.82 (s, 3), 3.84 (s, 3), 6.63 (s, 1), 7.45 (s, 1).
F u r a n -2-ca r b oxylic a cid (9-h yd r oxy-6,7-d im et h oxy-
1,2,3,4-tetr a h yd r oa cr id in -3-ylm eth yl)-a m id e h yd r och lo-
r id e (10) was synthesized from 20 (0.40 g, 0.13 mmol),
triethylamine (0.017 mL, 0.13 mmol), and 2-furoyl chloride
(0.012 mL, 0.13 mmol) by following the procedure described
for 2 and purified by flash chromatography. Elution with a
step gradient system of CH2Cl2/EtOH (9.8:0.4 to 9:1) afforded
crude 10 that was transformed into the hydrochloride salt:
76% yield; mp 205-209 °C (from ether/EtOH); 1H NMR (CD3-
OD) δ 1.60-1.78 (m, 1), 2.18-2.40 (m, 2), 2.70-3.26 (m, 4),
3.42-3.55 (m, 2), 4.02 (s, 3), 4.05 (s, 3), 6.59-6.63 (m, 1), 7.12-
7.21 (m, 2), 7.67-7.72 (m, 2); MS (ESI+) m/z 383 (M + H)+.
Anal. (C21H23ClN2O5) C, H, N.
1
EtOH); H NMR (CD3OD) δ 1.46-1.70 (m, 1), 2.18-2.38 (m,
2), 2.57-2.84 (m, 3), 3.01-3.16 (m, 1), 3.52 (d, 2), 3.97 (s, 3),
4.01 (s, 3), 7.03 (s, 1), 7.40 (s, 1), 7.71 (t, 1), 7.84 (d, 1), 8.17-
8.22 (m, 2); MS (ESI+) m/z 460 (M+H)+. Anal. (C24H25
ClF3N3O3) C, H, N.
-
N-(9-Am in o-6,7-d im et h oxy-1,2,3,4-t et r a h yd r oa cr id in -
3-ylm et h yl)-4-(t r iflu or om et h yl)b en za m id e h yd r och lo-
r id e (8) was synthesized from 4-(trifluoromethyl)benzoic acid
(0.033 g, 0.174 mmol) and 15 (0.05 g, 0.174 mmol) by following
the procedure described for 6 and purified by flash chroma-
tography. Elution with CHCl3/MeOH/aqueous 30% ammonia
(9:1:0.04) afforded crude 8 that was transformed into the
hydrochloride salt: 70% yield; mp 253-255 °C (from ether/
EtOH);1H NMR (DMSO-d6) δ 1.46-1.67 (m, 1), 2.08-2.28 (m,
2), 2.73-2.81 (m, 3), 3.03-3.16 (m, 1), 3.44 (t, 2), 3.96 (s, 3),
3.99 (s, 3), 7.22 (s, 1), 7.83 (s, 1), 7.92 (d, 2), 8.16 (d, 2), 9.01 (t,
exchangeable with D2O, 1); MS (ESI+) m/z 460 (M + H)+. Anal.
(C24H25ClF3N3O3) C, H, N.
3-{[2-(2,6-Dim eth oxyp h en oxy)eth yla m in o]m eth yl}-6,7-
d im eth oxy-1,2,3,4-tetr a h yd r oa cr id in -9-ol d ih yd r och lo-
r id e (11) was synthesized from 20 (0.15 g, 0.52 mmol), 2-(2,6-
dimethoxyphenoxy)acetaldehyde20 (0.20 g, 0.98 mmol), and
NaBH4 (0.37 g, 0.98 mmol) by following the procedure de-
scribed for 9. Removal of the solvent gave crude 11 that was
transformed into the hydrochloride salt: 55% yield; mp 250-
3-{[2-(2,6-Dim eth oxyp h en oxy)eth yla m in o]m eth yl}-6,7-
d im eth oxy-1,2,3,4-tetr a h yd r oa cr id in -9-yla m in e d ih yd r o-
ch lor id e (9). A solution of 2-(2,6-dimethoxyphenoxy)acetal-
dehyde20 (0.14, 0.70 mmol) in dry EtOH (2 mL) was added
dropwise to a mixture of 15 (0.10 g, 0.35 mmol) and molecular
sieves (3 Å) in dry MeOH (20 mL) over a period of 30 min at
room temperature. NaBH4 (0.030 g, 0.70 mmol) was then
added and the stirring was continued for further 12 h.
Following removal of molecular sieves, the solution was made
acidic with 6 N HCl (1 mL). Removal of the solvent gave a
residue, which was dissolved in water (10 mL). The solution
was washed with ether (2 × 20 mL) to remove nonbasic
materials and then was made basic with 2 N NaOH and finally
extracted with CHCl3 (3 × 20 mL). Removal of the dried
solvent gave a residue that was purified by flash chromatog-
raphy. Elution with CHCl3/MeOH/aqueous 30% ammonia (9:
1:0.05) afforded crude 9, which was transformed into the
hydrochloride salt: 65% yield; mp 247-253 °C (from ether/
1
252 °C (from ether/EtOH); H NMR (CDCl3) δ 1.22-1.42 (m,
1), 1.92-2.18 (m, 2), 2.46-2.67 (m, 3), 2.80-3.10 (m, 5), 3.62-
3.86 (m, 12), 4.10-4.20 (m, 2), 6.53-6.61 (m, 2), 6.89-7.15
(m, 2), 7.63 (s, 1H), 11.82 (s, exchangeable with D2O, 1); MS
(ESI+) m/z 469 (M + H)+. Anal. (C26H34Cl2N2O6) C, H, N.
Biology. F u n ction a l An ta gon ism in Isola ted Tissu es.
Male Wistar rats (275-300 g) were killed by cervical disloca-
tion, and the organs required were isolated, freed from
adhering connective tissue, and set up rapidly under a suitable
resting tension in 20 mL organ baths containing physiological
salt solution kept at 37 °C 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 3-fold at each
step, with each addition being made only after the response
to the 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.
Ra t Va s Defer en s P r osta tic P or tion . This tissue was
used to assess R1A-adrenergic antagonism.21 Prostatic portions
of 2 cm length were mounted under 0.5 g tension in Tyrode
solution of the following composition (mM): NaCl, 130.0; KCl,
2.0; CaCl2, 1.8; MgCl2, 0.89; NaHCO3, 25.0; NaH2PO4, 0.42;
glucose, 5.6. Cocaine hydrochloride (0.1 µM) was added to the
Tyrode to prevent the neuronal uptake of (-)-noradrenaline.
The preparations were equilibrated for 60 min with washing
every 15 min. After the equilibration period, tissues were
primed two times by addition of 10 µM (-)-noradrenaline.
After another washing and equilibration period of 60 min, a
(-)-noradrenaline concentration-response curve was con-
structed (basal response). The antagonist was allowed to
equilibrate with the tissue for 30 min, then a new concentra-
tion-response curve to the agonist was obtained. (-)-Norad-
renaline solutions contained 0.05% Na2S2O5 to prevent oxida-
tion.
Ra t Sp leen . This tissue was used to assess R1B-adrenergic
antagonism.23 The spleen was removed and bisected longitu-
dinally into two strips that were suspended in tissue baths
containing Krebs solution of the following composition (mM):
NaCl, 120.0; KCl, 4.7; CaCl2, 2.5; MgSO4, 1.5; NaHCO3, 20.0;
KH2PO4, 1.2; glucose, 11.0; K2EDTA, 0.01. (()-Propranolol
hydrochloride (4.0 µM) was added to block â-adrenoreceptors.
The spleen strips were placed under 1 g resting tension and
equilibrated for 2 h. The cumulative concentration-response
curves to (-)-phenylephrine were measured isometrically and
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, then a new
concentration-response curve to the agonist was constructed.
1
EtOH); H NMR (CD3OD) δ 1.42-1.58 (m, 1), 2.02-2.26 (m,
2), 2.53-2.79 (m, 5), 2.83 (t, 2), 2.98-3.06 (m, 1), 3.81 (s, 6),
3.91 (s, 3), 3.95 (s, 3), 4.09-4.15 (m, 2), 6.63 (d, 2), 7.01 (t, 1),
7.06 (s, 1), 7.37 (s, 1); MS (ESI+) m/z 468 (M + H)+. Anal.
(C26H35Cl2N3O5) C, H, N.
6,7-Dim eth oxy-1-(n itr om eth yl)-1,2,3,4-tetr a h yd r oa cr i-
d in -9-ol (18) a n d 6,7-Dim eth oxy-3-(n itr om eth yl)-1,2,3,4-
tetr a h yd r oa cr id in -9-ol (19). A mixture of 4,5-dimethoxy-
anthranilic acid (2.5 g, 12.72 mmol) and 3-(nitromethyl)cyclohex-
anone (2.0 g, 12.72 mmol) in POCl3 (10 mL) was refluxed for
2 h. The reaction mixture was evaporated and treated with
ice and solid NaHCO3 until pH 7-8. The separated solid was
taken up in ether, washed with water, dried, and evaporated
to give a residue that was purified by flash chromatography.
Elution with a step gradient system of CH2Cl2/EtOH/aqueous
30% ammonia (9.8:0.2:0.02 to 9:1:0.06) afforded 18 and 19,
respectively.
18: 35% yield; Rf 0.55 [CHCl3/MeOH/aqueous 30% ammonia
1
(9:1:0.04)]; mp 218-220 °C (from MeOH); H NMR (DMSO-
d6) δ 1.82-1.93 (m, 4), 2.63 (t, 2), 3.58-3.75 (m, 1), 3.80 (s, 3),
3.82 (s, 3), 4.43 (t, 1), 4.83 (dd, 1), 6.84 (s, 1), 7.40 (s, 1), 11.40
(s, exchangeable with D2O, 1); MS (ESI+) m/z 319 (M + H)+.
19: 30% yield; Rf 0.47 [CHCl3/MeOH/aqueous 30% ammonia
1
(9:1:0.04)]; mp 258-260 °C (from CH2Cl2); H NMR (CD3OD)
δ 1.24-1.63 (m, 1), 2.02-2.18 (m, 1), 2.45-2.98 (complex m,
5), 3.88 (s, 3), 3.90 (s, 3), 4.58 (t, 2), 6.82 (s, 1), 7.61 (s, 1); MS
(ESI+) m/z 319 (M + H)+.
3-(Am in om eth yl)-6,7-dim eth oxy-1,2,3,4-tetr ah ydr oacr i-
d in -9-ol (20) was synthesized from 19 (0.22 g, 0.69 mmol) and
Raney Ni (nickel sponge; suspension in water) (0.10 g) by
following the procedure described for 15 and purified by flash
chromatography. Elution with CHCl3/MeOH/aqueous 30%
ammonia (9:1:0.1) afforded 20 as a yellow solid: 77% yield;
mp 245-250 °C; 1H NMR (CD3OD) δ 1.22-1.38 (m, 1), 1.72-

4902 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 23
Rosini et al.
Ra t Aor ta . This tissue was used to assess R1D-adrenergic
antagonism.22 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. Cocaine
hydrochloride (0.1 µM) and (()-propranolol hydrochloride (4.0
µM) were added to prevent the neuronal uptake of (-)-
noradrenaline and to block â-ARs, respectively. Two helicoidal
strips (15 mm × 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 acetyl-
choline (100 µM)-induced relaxation to preparations contracted
with (-)-noradrenaline (1 µM) was taken as an indicator that
the 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 transducer for
monitoring changes in isometric contraction. After at least a
2 h equilibration period under an optimal tension of 2 g,
cumulative (-)-noradrenaline concentration-response curves
were recorded at 1 h intervals, the first two being discarded
and 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. (-)-Noradrenaline solutions contained 0.05%
K2EDTA and 0.9% NaCl to prevent oxidation.
Con for m a tion a l Sea r ch . The conformational analyses
were carried out to sample the rotatable bonds of the furoyl
substituent bound to the tricyclic skeleton. This was ac-
complished by means of Monte Carlo analyses27 as imple-
mented in MacroModel software.28 In a Monte Carlo study,
the phase space of a molecule is sampled by randomly
changing dihedral angle rotations or atom positions. Then, the
trial conformation is accepted if its energy has decreased from
the previous one. If the energy is higher, various criteria can
be applied to accept or reject the Monte Carlo trial. In the
present simulations, the number of Monte Carlo steps was set
equal to 8000 and the trial conformation was accepted if the
energy was lower than that of the previous conformation or if
its energy was within an energy window of 100 kJ /mol. Then,
the conformations were classified by means of a cluster
analysis29 using geometrical parameters as filtering screens.
The combined usage of Monte Carlo and cluster analyses has
turned out to be a potent means for sampling the conforma-
tional space of highly flexible molecules.30
Da ta An a lysis. In functional studies responses were ex-
pressed as percentage of the maximal contraction observed in
the agonist concentration-response curve taken as a control.
Pharmacological computer programs analyzed the agonist
concentration-response curves. pKB values of compounds
2-11 were calculated according to van Rossum.31
Binding data were analyzed using the nonlinear curve-
fitting program Allfit.32 Scatchard plots were linear in all
preparations. All pseudo-Hill coefficients (nH) were not sig-
nificantly different from unity (p > 0.05). Equilibrium inhibi-
tion constants (Ki) were derived from the Cheng-Prusoff
equation,33 Ki ) IC50/(1 + L/Kd), where L and Kd are the
concentration and the equilibrium dissociation constant of the
radioligand. pKi values are the mean ( SE of two or three
separate experiments performed in triplicate.
In h ibition of ACh E. The method of Ellman et al. was
followed.25 Five different concentrations of each compound
were used in order to obtain inhibition of AChE activity
comprised between 20 and 80%. The assay solution consisted
of a 0.1 M phosphate buffer pH 8.0, with the addition of 340
µM 5,5′-dithiobis(2-nitrobenzoic acid), 0.035 unit/mL AChE
derived from human erythrocytes (0.39 and 5.9 UI/mg, respec-
tively; Sigma Chemical), and 550 µM acetylthiocholine iodide.
Test compounds were added to the assay solution and prein-
cubated at 37 °C with the enzyme for 20 min followed by the
addition of substrate. Assays were done with a blank contain-
ing all components except AChE in order to account for
nonenzymatic reaction. The reaction rates were compared and
the percent inhibition due to the presence of test compounds
was calculated. Each concentration was analyzed in triplicate,
and IC50 values were determined graphically from log concen-
tration-inhibition curves.
Ra d ioliga n d Bin d in g Assa ys. Binding to cloned human
R1-AR subtypes was performed in membranes from CHO
(chinese hamster ovary) cells transfected by electroporation
with DNA expressing the gene encoding each R1-AR subtype.
Cloning and stable expression of the human R1-AR gene was
performed as previously described.24 CHO cell membranes (30
µg of proteins) were incubated in 50 mM Tris-HCl, pH 7.4,
with 0.1-0.4 nM [3H]prazosin, in a final volume of 1.02 mL
for 30 min at 25 °C, in absence or presence of competing drugs
(1 pM to 10 µM). Nonspecific binding was determined in the
presence of 10 µM phentolamine. The incubation was stopped
by addition of ice-cold Tris-HCl buffer and rapid filtration
through 0.2% polyethyleneimine-pretreated Whatman GF/B
or Schleicher & Schuell GF52 filters.
Molecu la r Mod elin g. Con str u ction of th e Mod els.
Compound 1 was directly retrieved for the Cambridge Struc-
tural Database (CSD code YARTEQ) and its geometry was
optimized at semiempirical Hamiltonian level AM1,26 as
implemented in the SYBYL (Tripos Inc. St. Louis, MO) graphic
interface to MOPAC (keyword PRECISE). The molecular
models of 2 and 3 were built by properly modifying the
7-methoxy-9-amino-1,2,3,4-tetrahydroacridine and 2,3-trim-
ethylene-4-phenyl-6-chloropyridine skeletons retrieved from
the CSD (CSD codes WIDDAO and TIBLEV, respectively) and
by adding the proper fragments from the standard library of
SYBYL. The models were minimized first by using steepest
descent and then conjugate gradient until a convergence of
0.05 kcal mol-1 Å-1 on the gradient was reached. When needed,
conformational searches were carried out, and the minimum
energy conformers were further optimized by means of the
AM1 method.
Ack n ow led gm en t. Supported by grants from the
University of Bologna and MIUR.
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