New potential uroselective NO-donor α1-antagonists

Article abstract of DOI:10.1021/jm030825u

A recent uroselective α₁-adrenoceptor antagonist, REC15/2739, has been joined with nitrooxy and furoxan NO-donor moieties to give new NO-donor α₁-antagonists. All the compounds studied proved to be potent and selective ligands of hum

Full text of DOI:10.1021/jm030825u

3762
J . Med. Chem. 2003, 46, 3762-3765
New P oten tia l Ur oselective NO-Don or r1-An ta gon ists
Donatella Boschi,† Gian Cesare Tron,‡ Antonella Di Stilo,† Roberta Fruttero,† Alberto Gasco,*,† Elena Poggesi,§
Gianni Motta,§ and Amedeo Leonardi§
Dipartimento di Scienza e Tecnologia del Farmaco, Universita` degli Studi di Torino, via Pietro Giuria 9, 10125 Torino, Italy,
Dipartimento di Scienze Chimiche, Alimentari, Farmaceutiche e Farmacologiche, Universita` degli Studi del Piemonte
Orientale, V. le Ferrucci 33, I-28100, Novara, Italy, and Pharmaceutical Research and Development Division, Recordati S.p.A.,
via M. Civitali 1, I-20148 Milano, Italy
Received February 25, 2003
A recent uroselective R1-adrenoceptor antagonist, REC15/2739, has been joined with nitrooxy
and furoxan NO-donor moieties to give new NO-donor R1-antagonists. All the compounds studied
proved to be potent and selective ligands of human cloned R1a-receptor subtype. Derivatives 6
and 7 were able to relax the prostatic portion of rat vas deferens contracted by (-)-noradrenaline
because of both their R1A-antagonist and their NO-donor properties.
In tr od u ction
and 10 tested for their R1-, R2-, and 5-HT1A-receptor
affinities and for the R1A-adrenoceptor antagonism on
rat vas deferens.
Benign prostatic hyperplasia (BPH) is a widely dif-
fused pathology in the aging male population. It consists
of a progressive enlargement of the prostate that results
in an obstruction of the proximal urethra.1 The hyper-
plastic prostate tissue contracts under sympathic stimu-
lation, principally mediated by R1-receptors. At present,
native R1-adrenoceptors appear to exist as three sub-
types encoded by three genes, R1A/R1a, R1B/R1b, R1D/R1d,
where upper and lower case letters indicate subtypes
from animal or human tissue and cloned subtypes,
respectively.2 There is functional evidence for an ad-
ditional R1-subtype (R1L) with a low affinity for pra-
Resu lts a n d Discu ssion
Scheme 1 briefly describes the standard procedure
used for the preparation of derivatives of 1. The thiol-
induced NO generation by the final products was
indirectly evaluated by determining, through Griess
reaction, the amount of nitrite ion formed by NO
-
oxidation.8 The results expressed as % NO2 (mol/mol)
are reported in Table 1. Nitrite production is strongly
dependent on the medium, the concentration, and the
nature of the thiol employed, and thus, it is only
indicative of the NO production that might occur in a
cellular environment. In addition, it does not give
information about the NO-redox form(s) involved in the
release. The ability to produce nitrite ion follows the
series 7 > 6 > 5 = 10. In tissues and cells, thiol-
dependent NO production and/or enzymatic activation
has been proposed for nitrate. Enzymatic activation
cannot be excluded for furoxans either.9
The affinities of the furoxan and nitrooxy derivatives,
as well as those of the reference compound 1 for cloned
R1-adrenoceptors, were evaluated in binding assays on
membranes prepared from CHO cells (Chinese hamster
ovary cells) expressing human R1a-, R1b-, R1d-subtypes.
Competition assays were performed using [3H]prazosin
to label the cloned receptors.10 Similarly, the affinities
of the products for the human cloned 5HT1A-serotonin-
ergic receptor were evaluated in membranes prepared
from human HeLa cells expressing this receptor. [3H]-
8-hydroxy-2-(di-n-propylamino)tetralin ([3H]-8-OH-DPAT)
was used as radioligand in the competition binding
experiments.10 The affinities of the products for native
R2-adrenergic receptors were carried out in membranes
of rat cerebral cortex with [3H]rauwolscine as radioli-
gand.11 The results expressed as Ki are reported in Table
1.
zosin.3 It may represent a different affinity state of R1A
-
adrenoceptors. In the human prostate, R1A-adrenoceptors
are mainly present but the R1B-subtype also seems to
play a role. Consequently a pharmacological approach
used in the symptomatic treatment of BPH involves the
employment of R1A-antagonists.3
Nitric oxide NO• is an important biological messenger
that elicits a surprisingly wide range of physiological
effects on the cardiovascular system, the central and
peripheral nervous systems, and the immune system.4
In the peripheral nervous system, it is the neurotrans-
mitter at some nonadrenergic noncholinergic (NANC)
neuroeffector junctions, and consequently, it is impli-
cated in many genitourinary tract activities. In particu-
lar in the prostate, NO• as a neurotransmitter and as a
paracrine factor can modulate smooth muscular tone
and secretory functions.5
On these bases, to develop our previous work on NO-
donor R1-antagonists,6 we designed a series of “hybrid”
drugs in which we joined the structure of REC15/2739
(1), a recent uroselective R1-adrenoceptor antagonist,
discovered in Recordati S.p.A. laboratories,7 with ni-
trooxy and furoxan NO-donor moieties. In this paper,
we report the synthesis, the structural characterization,
and the pharmacological profile of novel compounds 5-7
Analysis of the data shows that the nitrooxy deriva-
tive 10 displays a very high affinity for the R1a-
adrenoceptor subtype. Its Ki value is 3.6-fold lower than
that of the reference 1. This product also shows a
* To whom correspondence should be addressed. Telephone: 0039
011 6707670. Fax: 0039 011 6707286. E-mail: alberto.gasco@unito.it.
† Universita` degli Studi di Torino.
‡ Universita` degli Studi del Piemonte Orientale.
§ Recordati S.p.A.
10.1021/jm030825u CCC: $25.00 © 2003 American Chemical Society
Published on Web 07/23/2003

Brief Articles
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 17 3763
Sch em e 1
Ta ble 1. Pharmacological Profile and Nitrite Formation of 5-7
and 10 and Reference 1a
ner. In the case of derivative 5, which is a feeble NO
donor, there was a parallel shift without any reduction
of the maximal effect. The other two furoxan derivatives
6 and 7, which are more potent NO donors, behaved
differently. In fact, the shift to the right of the concen-
tration-response curve of NA was accompanied by a
decrease in the maximum effect (see Figure 1, panels
A and B). In the case of cyano derivative 7, the most
potent NO donor, the decrease had already occurred at
the first concentration tested. The maximal response
was restored when the experiments were repeated in
the presence of 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-
1-one (ODQ), a well-known inhibitor of the soluble
guanilate cyclase (sGC). Simple NO-donor furoxans
showed similar behavior when tested under the same
experimental conditions (data not shown). Thus, this
decrease is most likely due to the dilating properties of
NO on the tissue mediated by sGC. For each compound,
we calculated pA2 values at the concentrations reported
in the footnote of Table 1, using the equation pA2 ) log-
(CR - 1) - log[B].12 The average values are entered in
Table 1. The nitrooxy derivative 10 behaved in quite a
different manner. This product also induced a shift to
the right of the concentration-response curve of NA but
was accompanied at the high concentrations tested by
a decrease of the maximal response that was not
abolished by the presence of ODQ (Figure 1C). Conse-
quently, this effect cannot be NO-dependent. This same
decrease of the maximum effect with the increase of the
concentration of the product is shown by reference 1 and
its analogues in human prostate.12 For these two
products, we determined apparent pA2 values at the
lowest surmountable concentration giving a significant
rightward shift of the concentration-response curves
of the NA, using the above-reported equation. pA2 values
rank in the order 10 > 1 > 5 > 6 > 7. Analysis of the
results reveals that R1a binding affinities (pKi) of the
compounds poorly correlate (r2 ) 0.49) with their
antagonist potencies observed in NA-induced contrac-
tions of the prostatic portion of rat vas deference (pA2).
The discrepancy often observed between functional and
binding affinities may be due to the different conditions
of the receptors utilized in the assays. In binding
procedures, homogenates of cell membranes bearing a
homogeneous population of human cloned receptors are
used, whereas in functional assays, rat native receptors
inhibition of
% NO2- ( SEd
receptor binding, Ki c (nM)
pA2 ( SEb
(mol/mol)
compd
R1A
R1a
R1b R1d
R2 5-HT1A
Cys 5×
1
5
6
7
10
8.42 ( 0.10 0.34
8.21 ( 0.06 0.16
7.97 ( 0.06 1.8
7.46 ( 0.07 0.73
3.9 1.5 33.3
4.7 4.2 53.5
15.9 20.4 359.0
6.5 3.9 81.1
5.9
1.6
1.2
2.2
7.2
1.11 ( 0.32
5.28 ( 0.24
32.3 ( 0.7
<1
8.64 ( 0.06 0.094 1.2 3.5 81.8
a
Functional R1-adrenoceptor antagonism on the prostatic por-
tion of rat vas deferens (R1A) and receptor binding affinity for
human cloned R1-adrenoceptor subtypes, 5HT1A-serotoninergic
b
receptors, and rat cortex R2-adrenoceptors. pA2 values are the
mean of 6-14 determinations. They were estimated at two
concentrations for 5-7 (3 × 10-9 and 1 × 10-8 M for 5; 3 × 10-8
and 1 × 10-7 M for 6 and 7; in the case of derivatives 6 and 7,
when the NO relaxing effect was observed, we determined pA2
values in the presence of 1 µM ODQ, which was added to the bath
at least 10 min before the addition of the antagonist). Since pA2
values obtained at both concentrations were similar, the antago-
nism was assumed to be competitive. For 1 and 10, we determined
an apparent pA2 value at one antagonist concentration (3 × 10-9
and 1 × 10-8 M, respectively). c Ki values were obtained from two
to three experiments (each performed in triplicate), which cor-
d
respond to within 20%. A solution of the appropriate compound
(20 µL) in dimethyl sulfoxide was added to 1980 µL of a 1:1 v/v
mixture of 50 mM phosphate buffer (pH 7.4) and MeOH, contain-
ing 5 × 10-4 M L-cysteine. The final concentration of the compound
was 10-4 M. After 1 h at 37 °C, 1 mL of the reaction mixture was
treated with 250 µL of Griess reagent.8 No production of nitrite
was observed in the absence of L-cysteine.
significantly improved R1a-selectivity (37-, 77-, and 870-
fold relative to the R1d- subtype, cloned 5HT1A receptor,
and native R2-adrenergic receptors, respectively) in
comparison with the reference (4-, 17-, and 98-fold,
respectively). By contrast, R1a-selectivities relative to the
R1b-subtype are similar in the two products (13- and 12-
fold, respectively). As far as the furoxan derivatives are
concerned, their affinity for R1a-subtype is lower than
that of 10 and, compared to reference, it follows the
order 5 > 1 > 7 > 6. The most active methyl derivative
5 also shows the best selectivity profile for R1a-subtype.
The functional R1A antagonistic activity of the prod-
ucts was evaluated by antagonism of (-)-noradrenaline
(NA) induced contractions of the prostatic portion of rat
vas deferens in comparison with the antagonism of the
reference 1. All the substances were able to shift the
cumulative concentration-response curve of NA to the
right in a concentration-dependent and reversible man-

3764 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 17
Brief Articles
they display potent antagonist properties at the R1A
-
adrenoceptor subtype present in the rat vas deferens.
Derivatives 6 and 7 are able to relax this tissue
contracted by NA because of both their R1A-antagonist
properties and their abilities to release NO under the
experimental conditions adopted.
Since KT-1,14 a hybrid obtained by combination of
prazosin and nitrooxy moieties, also retains (in vivo as
well) cardiovascular effects similar to those of both
nitrates and R1-adrenoceptor blocking agents, all the
products described in the present work might be of
interest for further in vivo studies on their potentialities
in the treatment of BPH. This symbiotic approach could
have advantages on the simultaneous administration
of two single active drugs because the resulting hybrid
should display a more balanced pharmacokinetic profile
during the entire course of its action and, possibly, an
increased activity.
Exp er im en ta l Section
The results of elemental analyses of the new compounds are
within (0.4% of the theoretical values.
N-(3-(4-(2-H yd r oxy)p h en yl)p ip er a zin -1-yl)p r op yl)-3-
m eth yl-4-oxo-2-p h en yl-4H-ch r om en e-8-ca r boxa m id e (2).
A 1 M Br3B solution (55 mL) was added dropwise to a stirred
solution of 1 (3.13 g, 5.00 mmol) in dry CH2Cl2 (60 mL), and
the mixture was refluxed under N2 for 4 h. The mixture was
cooled to 5 °C, and a KHCO3 saturated solution (120 mL) was
added dropwise. The two phases were stirred for 1 h until the
solid formed was dissolved. Then they were separated. The
aqueous phase was extracted with CH2Cl2, and the organic
layers were collected, washed with brine, dried, and evapo-
rated. The yellow solid obtained (2.43 g, 98%) was character-
ized by comparison with an authentic sample supplied by
Recordati S. p. A. and used for the next synthesis without
further purification.
N-(3-(4-(2-(3-Meth ylfu r oxa n -4-ylm eth oxy)p h en yl)p ip -
er azin -1-yl)pr opyl)-3-m eth yl-4-oxo-2-ph en yl-4H-ch r om en e-
8-ca r boxa m id e Dih yd r och lor id e (5). Compound 3 (0.77 g,
4.00 mmol) was added to a stirred suspension of 2 (1.00 g, 2.00
mmol) and K2CO3 (1.10 g, 8.00 mmol) in dry DMF (13 mL).
After being stirred at room temperature for 24 h, the solution
was poured into ice-cooled water and the solid formed was
filtered, washed with ice-cooled water, dried, and purified by
flash chromatography (eluent, CH2Cl2/MeOH, 9.5:0.5). The
resulting product (0.70 g, 50%) was dissolved in dry MeOH,
and HCl-saturated MeOH was added to the solution. The
addition of dry ethyl ether gave a white precipitate that was
filtered and dried under vacuum at 40 °C for 3 days, affording
the title derivative: mp 145-148 °C (dec). Anal. (C34H35N5O6‚
2HCl‚H2O) C, H, N, Cl.
N-(3-(4-(2-(3-Ca r ba m oylfu r oxa n -4-ylm eth oxy)p h en yl)-
p ip er a zin -1-yl)p r op yl)-3-m eth yl-4-oxo-2-p h en yl-4H-ch r o-
m en e-8-ca r boxa m id e (6). The title compound was prepared
as described for 5 starting from 4 (0.88 g, 4.00 mmol). The
obtained precipitate was purified by MPLC (eluent, CH2Cl2/7
N NH3 in MeOH, 9.75:0.25). The resulting beige solid (0.80 g,
60%) was characterized as a free base: mp 189-195 °C (dec).
Anal. (C34H34N6O7‚1.5H2O) C, H, N.
N-(3-(4-(2-(3-Cya n ofu r oxa n -4-ylm eth oxy)p h en yl)p ip er -
a zin -1-yl)p r op yl)-3-m eth yl-4-oxo-2-p h en yl-4H-ch r om en e-
8-ca r boxa m id e (7). A solution of 6 (1.33 g, 2.00 mmol) in dry
THF (75 mL) and dry pyridine (4.5 mL) was stirred under N2
and cooled at 0 °C. To this solution trifluoroacetic anhydride
(1.5 mL, 6.00 mmol) was added. After 15 min, the reaction
was quenched by adding saturated NaHCO3 solution (15 mL)
and the THF was evaporated. The residue was extracted with
EtOAc, and the combined organic layers were washed with
water and brine and then dried and evaporated. The residue
purified by MPLC (eluent, CH2Cl2/MeOH, 97.5:2.5) gave 0.71
F igu r e 1. (A) Effect of 6 on contraction to NA in rat vas
deferens: control cumulative concentration-response curve
(square) and 6 at 3 × 10-8 M (solid triangle), 1 × 10-7 M (solid
diamond), and 1 × 10-7 M after ODQ incubation (open
diamond). (B) Effect of 7 on contraction to NA in rat vas
deferens: control cumulative concentration-response curve
(square) and 7 at 3 ×10-8 M (solid triangle), 3 × 10-8 M after
ODQ incubation (open triangle), 1 × 10-7 M (solid circle), and
1 × 10-7 M after ODQ incubation (open circle). (C) Effect of
10 on contraction to NA in rat vas deferens: control cumulative
concentration-response curve (square) and 10 at 1 × 10-8
M
(solid triangle), 3 × 10-8 M (solid circle), and 3 × 10-8 M after
ODQ incubation (open circle).
expressed in their intact original tissue are utilized. It
would seem that receptors may be organized differently
under the two respective conditions, and consequently,
their biological behavior may be different. Or more
simply, a different bioavailability of the compounds at
the receptor level may exist.13 In addition, radioreceptor
binding measures simple displacement of a ligand from
the binding site whereas the functional response in-
volves multiple coordinated steps of a more complex
system.
Con clu sion s
The reported compounds are potent and selective
ligands of a human cloned R1a-receptor subtype, and

Brief Articles
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 17 3765
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1283.
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g (57%) of the product as a pale-yellow solid: mp 77 °C (dec).
Anal. (C34H32N6O6‚0.5H2O) C, H, N.
N-(3-(4-(2-(3-Br om op r op oxy)p h en yl)p ip er a zin -1-yl)-
p r op yl)-3-m eth yl-4-oxo-2-p h en yl-4H-ch r om en e-8-ca r box-
a m id e (9). 1,3-Dibromopropane (8, 0.49 mL, 4,80 mmol) and
6 N NaOH (0.80 mL, 4,80 mmol) were added to a stirred
solution of 2 (1.20 g, 2.40 mmol) in THF (15 mL). The solution
was heated at 45 °C until the disappearance of starting
material by TLC (24 h). The solution was then diluted with
EtOAc, washed with water and brine, and then dried and
evaporated. The crude material, purified by column chroma-
tography (eluent, EtOAc/PE/7 N NH3 in MeOH, 6:3.5:0.5), gave
0.80 g (54%) of the product as white powder that was
crystallized from ethanol and dried at 40 °C under vacuum
for 3 days: mp (EtOH) 131-133 °C. Anal. (C33H36BrN3O4‚
0.25H2O) C, H, N.
N-(3-(4-(2-(3-Nitr ooxyp r op oxy)p h en yl)p ip er a zin -1-yl)-
p r op yl)-3-m eth yl-4-oxo-2-p h en yl-4H-ch r om en e-8-ca r box-
a m id e (10). To a suspension of 9 (0.62 g, 1.00 mmol) in
acetonitrile (10 mL), silver nitrate (0.34 g, 2.00 mmol) was
added, and the reaction mixture was refluxed for 4 h. The
silver bromine formed was filtered off, and the solvent was
carefully evaporated. The residue was taken up in EtOAc,
washed with water and brine, and then dried and evaporated.
The crude residue was purified by column chromatography
(eluent, EtOAc/PE/7 N NH3 in MeOH, 6:3.5:0.5) and afforded
0.25 g (41%) of a pale-yellow solid that was further purified
by two crystallizations with 95% EtOH: mp (EtOH) 139-142
°C (dec). Anal. (C33H36N4O7) C, H, N.
Ack n ow led gm en t. Financial support from the Ital-
ian MIUR is gratefully acknowledged.
Su p p or tin g In for m a tion Ava ila ble: Detailed experi-
mental procedures on the radioligand binding assays and
functional studies of compounds 1, 5-7, and 10, including a
description of the standard techniques and instrument used
for the synthesis of the compounds, and their NMR data. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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