Phenylpiperazinylalkylamino substituted pyridazinones as potent α1 adrenoceptor antagonists

Article abstract of DOI:10.1021/jm0009336

QSAR models have been used for designing a series of compounds characterized by a N-phenylpiperazinylalkylamino moiety linked to substituted pyridazinones, which have been synthesized. Measurements of the binding affinities of the new compounds toward the

Full text of DOI:10.1021/jm0009336

J . Med. Chem. 2001, 44, 2403-2410
2403
P h en ylp ip er a zin yla lk yla m in o Su bstitu ted P yr id a zin on es a s P oten t r1
Ad r en ocep tor An ta gon ists
Daniela Barlocco,*,§ Giorgio Cignarella,§ Vittorio Dal Piaz,† M. Paola Giovannoni,† Pier G. De Benedetti,*,‡
Francesca Fanelli,‡ Federica Montesano,# Elena Poggesi,# and Amedeo Leonardi#
Istituto Chimico Farmaceutico e Tossicologico, Viale Abruzzi 42, 20131 Milano, Italy, Dipartimento di Scienze Farmaceutiche,
Via G. Capponi 9, 50121 Firenze, Italy, Dipartimento di Chimica, Via Campi 183, 41100 Modena, Italy, and Pharmaceutical
R & D Division, Recordati s.p.a., Via Civitali 1, 20148 Milano, Italy
Received February 28, 2000
QSAR models have been used for designing a series of compounds characterized by a
N-phenylpiperazinylalkylamino moiety linked to substituted pyridazinones, which have been
synthesized. Measurements of the binding affinities of the new compounds toward the R1a-,
R1b-, and R1d-AR cloned subtypes as well as the 5-HT1A receptor have been done validating, at
least in part, the estimations of the theoretical models. This study provides insight into the
structure activity relationships of the R1-ARs ligands and their R1-AR/5-HT1A selectivity.
In tr od u ction
ported11 2-methyl-4-nitro-5-acetyl-6-substituted-3(2H)-
pyridazinones Ia ,b, by nucleophilic substitution at
position 4. Several different substituents at position 5
(compounds 1e-h ,j,l,n ) have been also considered. The
binding affinity assays of the new compounds toward
the human cloned R1a-, R1b-, and R1d-AR subtypes as well
as the human cloned 5-HT1A receptor have been also
carried out.
R1-Adrenergic receptors (R1-AR) are members of the
superfamily of G protein coupled receptors (GPCR) that
transduce signals across the cell membrane. The R1-ARs
mediate the functional effects of catecholamines such
as epinephrine and norepinephrine by coupling to the
GRq that induces the activation of phospholipase C,
culminating into the phosphoinositide (PI) hydrolysis.
Molecular biology techniques allowed the identifica-
tion of cDNAs encoding three R1-ARs (R1a, R1b, and
R1d).1-5 The three recombinant R1-ARs correlate closely
with the three R1-AR subtypes that were identified in
native tissues and which mediate their functional
responses (R1A, R1B, and R1D).6-9 The presence of these
different R1-AR subtypes in blood vessels and other
smooth muscles points to the importance of developing
selective drugs for receptor classification and charac-
terization as well as for therapeutic effectiveness.
A relevant aspect of chemical research is to predict
the behavior of new molecules from their structure, prior
to synthesis. It is generally assumed that noncovalent
forces control ligand-receptor interactions and that
these forces can be described in terms of electrostatic
and steric effects, which depend on molecular size and
shape. Recently, quantitative structure activity rela-
tionship (QSAR) analysis based on ad hoc defined
supermolecules and the corresponding size-shape de-
scriptors has been employed for deciphering the molec-
ular features responsible for the affinity and selectivity
in a wide ranging series of noncongeneric antagonists
of the cloned bovine R1a-, hamster R1b-, and rat R1d-
ARs.10 The QSAR models previously obtained have been
herein used to design and estimate the binding affinities
of a new molecular series of potential R1-AR ligands.
This work describes the design and the syn-
thesis of a series of phenylpiperazine derivatives
(1a -d ,k ,i,m ,o-r ) obtained from the previously re-
The results of this study provide insight into the
structure activity relationships (SAR) of R1-ARs ligands
and their R1-AR/5-HT1A selectivity.
Ch em istr y
Compounds 1a -d ,k ,i,m ,o-q were obtained in good
yields by nucleophilic displacement of the 4-nitro group
of the known pyridazinones Ia ,b,11 by stirring for 30 min
at room temperature an ethanolic solution of the ap-
propriate I with an excess of the required N,ω-ami-
noalkyl-N1-phenylpiperazine. The latter were in turn
prepared by reduction of the corresponding cyano-
derivatives or by deprotection of the appropriate ph-
thalimide, following standard procedures. (See Scheme
1). Compound 1r (see Table 1) was obtained in the same
way by directly condensing Ia and N-(2-methoxyphe-
nyl)piperazine. The homologues 1e,f were prepared
according to the same scheme starting from the previ-
ously reported 4-nitro-5-propanonyl- (Ic)11 and 4-nitro-
5-butanonyl (Id )12 analogues. The 5-unsubstituted
1g,h ,j,l,n (see Table 1) were obtained from the ap-
propriate 4-chloropyridazinones,13,14 by heating at 140
°C with an excess of the required N,ω-aminoalkyl-N1-
phenylpiperazine.
Resu lts a n d Discu ssion
In this work, a theoretical QSAR approach based on
size and shape descriptors has been employed to design
novel R1-AR ligands, taking also advantage of the
information inferred from a previous study on monocy-
clic and bicyclic substituted pyridazinones.15
A typical R1-blocker contains an unsaturated or
aromatic ring on each side of a protonated nitrogen
atom, one of these two moieties being closer to the
* To whom correspondence should be addressed. Tel: 39.02.29502223.
Fax: 39.02.29514197. E-mail: daniela.barlocco@unimi.it.
§ Istituto Chimico Farmaceutico e Tossicologico.
† Dipartimento di Scienze Farmaceutiche.
‡ Dipartimento di Scienze Chimiche.
#
Pharmaceutical R & D Division, Recordati s.p.a.
10.1021/jm0009336 CCC: $20.00 © 2001 American Chemical Society
Published on Web 06/16/2001

2404 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 15
Ta ble 1. Physicochemical Data of Compounds 1a -r
Barlocco et al.
compd
n
R
R1
R2
R3
% yield
mp (°C)
oil
147-148
162-163
oil
oil
oil
133-134
oil
formulaa
1a
1b
1c
1d
1e
1f
1g
1h
1k
1i
2
2
2
2
2
2
2
2
3
3
3
3
2
2
3
3
4
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
CH3
COCH3
COCH3
COCH3
COCH3
COC2H5
COCH(CH3)2
H
OCH3
F
Cl
H
H
H
Cl
H
H
H
Cl
H
Cl
H
Cl
H
H
H
Cl
Cl
84
91
98
83
76
92
53
48
95
72
23
41
79
22
94
79
79
95
C26H30N4O3
C25H28FN5O2
C25H28ClN5O2
C26H30ClN5O3
C27H33N5O3
C28H35N5O3
C24H29N5O2
C24H28ClN5O2
C27H33N4O3
C27H32ClN5O3
C25H31N5O2
C25H30ClN5O2
C21H29N5O3
C19H27N5O2
C22H31N5O3
C22H30ClN5O2
C22H30ClN5O3
C24H26N4O3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
H
COCH3
COCH3
H
oil
128-129
130-131
oil
133-134
177-178
83-84
101-102
101-102
173-175
1j
1l
H
1m
1n
1o
1p
1q
1r
COCH3
H
COCH3
COCH3
COCH3
CH3
CH3
CH3
CH3
a
Elemental analyses for C,H,N were within ( 0.4% of the calculated data.
Sch em e 1
cophoric element for the long-range electrostatic recog-
nition and productive interaction with the aspartate of
the R1-AR binding site, its contribution to the binding
energy is almost constant.16 On the contrary, short-
range forces (repulsive, dispersive, inductive, etc.) modu-
late both ligand affinity and selectivity. Thus, the
modulation of the binding affinity by a wide nonconge-
neric series of R1-AR ligands can be described and
explained by the variation of the ligand size-shape
features that are related to the short range acting forces.
These constitute the foundations of the supermolecule-
based QSAR approach previously proposed.10,17,18
According to the ligand based three-dimensional (3D)
pharmacophore, the supermolecule approach assumes
that the volume obtained by superimposing the most
structurally different ligands that show the highest
affinities for the same receptor (supermolecule) might
reflect the overall shape and conformational flexibility
of the high-affinity receptor binding site. Therefore, size
and shape descriptors can be defined ad hoc (that is, on
a specific molecular series and in connection with a
specific bioactivity) with respect to the supermolecule.
The supermolecule for the R1a-AR subtype (Figure 1a)
is based on six structurally different compounds (1-6,
Chart 1). Fewer compounds yielded the supermolecules
for the R1b- and the R1d-AR subtypes (7-10 for the R1b-
AR and 7, 8, 10-12 for the R1d-AR; Chart 1, Figure 1b,c);
these two supermolecules share three ligands: com-
pounds 7, 8, and 10 (Chart 1).
protonated nitrogen atom than the other. The proto-
nated nitrogen atom of the ligands is thought to interact
with the conserved aspartate on helix 3 of the receptor
according to a precise geometry dictated by a directional
charge reinforced hydrogen bonding interaction.15,16 The
protonated nitrogen atom of the R1-AR ligands may be
found as part of aliphatic chains, of conjugated systems
(i.e. quinazoline, isoquinoline, imidazoline) as well as
of aliphatic rings (i.e. piperazine and piperidine). While
the protonated nitrogen atom is an essential pharma-
The selected size and shape descriptors of the com-
pounds considered in this study are listed in Table 2.
In particular, the table reports the inner (Vin1a,Vin1b, and
Vin1d), the outer (Vout1a, Vout1b, and Vout1d), and the
difference (Vdif1a, Vdif1b, and Vdif1d) van der Waals (vdw)
volumes relative to the reference volume of ad hoc
defined supermolecules, one for each subtype (Figure
1). According to the definition of these molecular

New R1 Adrenoceptor Antagonists
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 15 2405
F igu r e 1. (a) Frontal (top) and side (bottom) views of the supermolecule obtained by superimposing the most active and structurally
different antagonists for the cloned bovine R1a-AR. The same side views have been employed for representing the volume of the
a1a supermolecule embedding all the ligands considered in this study superimposed on the a1a supermolecule. The same description
of point (a) is valid for points (b) and (c) with the difference that they refer, respectively, to the a1b and a1d supermolecules. The
van der Waals volumes of the a1a, a1b, and a1d supermolecules are 1016.00 Å3, 680.75 Å3, and 754.13 Å3, respectively.
descriptors, higher affinities are realized by maximizing
Vin and minimizing Vout. For its formulation Vdif [Vdif
Vdif by reducing Vin more than Vout, as compared to
compound 1a .
)
(Vin-Vout)/Vsup] is a size and shape descriptor that takes
into account the information content codified by both
Vin and Vout normalized by the volume of the supermol-
ecule (Vsup). By definition, the ligands constituting the
supermolecules show the following singularities: Vmol
) Vin, Vout ) 0, and Vin/Vmol ) 1, where Vmol is the vdw
molecular volume. The van der Waals volumes of the
R1a, R1b, and R1d supermolecules are 1016.00 Å3, 680.75
Å3, and 754.13 Å3, respectively. In general, a Vdif value
close to that of one of the supermolecule components
corresponds to high binding affinity value.
As for the R1a-AR subtype, theoretical size-shape
descriptors predict that a two carbon atom linker is
compatible with the structural variability of the sub-
stituent in position 5 of the pyridazinone ring. In other
words, changing the size-shape of the substituent in
position 5 of the pyridazinone ring invariantly produces
high Vdif. In fact, 1a , 1e, and 1f mainly realize relatively
The fluoro or chloro substitutions for the methoxy
group in position 2′ of the phenyl ring (1b and 1c) as
well as the introduction of a chlorine in position 5′ of
the phenyl ring (i.e. 1d ) do not induce relevant changes
in Vdif, as compared to compound 1a .
Prolonging the two carbon atom spacer of 1a to three
carbon atoms (1k , 1i), while leaving the phenyl sub-
stituent in position 6, increases the outer volume,
lowering Vdif. Thus, the computed indices predict that
size-shape variability in position 5 of the pyridazinone
is well tolerated, whereas increasing the length of the
spacer from 2 to 3 methylenic units or substituting the
phenyl ring in position 6 of the pyridazinone with a
methyl group slightly lowers the R1a-AR binding affinity.
As for the R1b-AR, the different substitutions in
position 5 of the pyridazinone ring, combined with an
ethylenic spacer (1a , 1e, and 1f), produce relatively high
Vdif. Halogen substitutions in positions 2′ or 5′ of the
phenyl ring (1b-d ) produce Vdif comparable with those
of the 2′-methoxy derivative (1a ). Replacing the phenyl
ring in position 6 by a methyl group (1m ) or reducing
the size in both positions 5 and 6 (1n ) lowers the inner
volume more than the outer volume, lowering Vdif.
high Vdif, by means of high Vin and intermediate Vout
whereas compound 1g realizes high Vdif through low Vin
and Vout
,
.
Reducing the size-shape of the substituent in position
6 (1m ) or in both positions 5 and 6 (1n ) slightly lowers

2406 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 15
Barlocco et al.
Ch a r t 1

New R1 Adrenoceptor Antagonists
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 15 2407
Ta ble 2. Ad Hoc Defined Size and Shape Descriptors (Vin, Vout, Vin/Vmol, and Vdif) of the Set of Molecules Considered in This Study
compd
Vin1a (Å3)
Vout1a (Å3)
Vdif1a
Vin1b (Å3)
Vout1b (Å3)
Vdif1b
Vin1d (Å3)
Vout1d (Å3)
Vdif1d
1a
1b
1c
1d
1e
1f
1g
1h
1k
1i
351.75
335.00
341.38
365.88
355.50
358.75
332.25
348.63
336.50
351.38
307.38
320.00
317.25
295.88
333.63
347.13
358.00
302.88
45.00
43.75
45.37
46.87
56.75
66.38
31.63
33.37
73.88
74.00
69.50
73.38
26.88
14.00
25.50
27.87
29.88
52.25
0.3158
0.2998
0.3047
0.3284
0.3076
0.3010
0.3095
0.3245
0.2704
0.2856
0.2449
0.2539
0.2989
0.2902
0.3172
0.3287
0.3378
0.2580
350.13
334.13
343.75
358.75
349.38
356.75
327.00
336.00
307.00
314.00
296.63
297.75
309.25
283.88
306.63
324.13
317.63
296.75
47.62
48.50
43.88
53.13
59.87
68.25
37.13
45.88
105.00
111.75
82.87
95.13
35.63
27.75
55.87
51.62
70.87
60.88
0.4641
0.4382
0.4600
0.4688
0.4441
0.4426
0.4447
0.4451
0.3099
0.3103
0.3279
0.3108
0.4197
0.3929
0.3847
0.4180
0.3785
0.3618
347.75
329.00
340.63
358.00
342.38
365.50
318.00
326.38
325.38
332.38
313.38
316.00
318.88
287.25
322.13
333.75
293.13
304.75
50.25
53.50
47.12
53.88
66.87
59.63
46.38
55.50
86.50
93.25
66.12
77.00
26.00
24.38
40.25
41.75
95.00
52.63
0.4124
0.3819
0.4069
0.4216
0.3819
0.4240
0.3765
0.3755
0.3311
0.3315
0.3428
0.3313
0.4060
0.3644
0.3908
0.4048
0.2747
0.3495
1j
1l
1m
1n
1o
1p
1q
1r
Ta ble 3. Calculated by Vdif Based QSAR Models (pK1a calc, pK1b calc, pK1d calc) and Experimental (pK1a exp, pK1b exp, pK1d exp) Binding
Affinities
compd
pK1a calc
pK1a exp
∆
pK1b calc
pK1b exp
∆
pK1d calc
pK1d exp
∆
1a
1b
1c
1d
1e
1f
1g
1h
1k
1i
8.82
8.74
8.77
8.89
8.78
8.75
8.79
8.87
8.59
8.67
8.46
8.50
8.74
8.69
8.83
8.89
8.94
8.53
9.46
9.49
9.52
9.55
9.72
9.74
9.66
9.00
8.82
8.64
8.59
8.70
8.64
7.73
9.09
9.29
8.52
6.79
-0.64
-0.75
-0.75
-0.66
-0.94
-0.99
-0.87
-0.13
-0.23
0.03
8.98
8.83
8.96
9.01
8.87
8.86
8.87
8.87
8.08
8.08
8.18
8.04
8.72
8.57
8.52
8.71
8.48
8.38
8.48
8.42
9.12
8.40
8.84
9.19
8.70
8.75
7.69
8.85
8.26
8.70
7.30
7.48
7.27
8.10
7.39
6.59
0.50
0.41
-0.16
0.61
0.03
-0.33
0.17
0.12
0.39
-0.77
-0.08
-0.66
1.42
1.09
1.25
0.61
1.09
9.19
8.89
9.13
9.28
8.89
9.30
8.84
8.83
8.39
8.40
8.51
8.40
9.12
8.72
8.98
9.11
7.84
8.57
9.89
9.31
9.60
9.39
9.60
9.37
7.93
8.97
8.37
8.41
7.93
8.53
8.50
7.75
7.46
8.27
8.30
5.00
-0.70
-0.42
-0.47
-0.11
-0.71
-0.07
0.91
-0.14
0.02
-0.01
0.58
-0.13
0.62
0.97
1.52
0.84
-0.46
3.57
1j
1l
-0.13
-0.20
0.10
1m
1n
1o
1p
1q
1r
0.96
-0.26
-0.40
0.42
1.74
1.79
a
pK1a values have been calculated according to the following regression equation: pK1a ) 5.18 ((0.83) Vdif + 7.19 ((0.27), n ) 34, r
) 0.74, s ) 0.53, and F ) 39.01, where n is the number of compounds, r is the correlation coefficient, s is the saturated deviation, and F
b
is the Fisher ratio. pK1b values have been calculated according to the following regression equation: pK1b ) 5.87 ((0.72) Vdif + 6.26
((0.22), n ) 30, r ) 0.83, s ) 0.51, and F ) 66.08. c pK1d values have been calculated according to the following regression equation: pK1d
) 0.74((1.14) Vdif + 5.17((0.33), n ) 31, r ) 0.84, s ) 0.57, and F ) 72.86.
Moreover, the elongation of the ethylenic spacer of 1a
to three carbon atoms (1k ) reduces Vin and increases
The predicted and experimental binding affinities for
the three R1-AR subtypes, together with their differences
(∆), are reported in Table 3. Recalling that the simple
approach used is mainly devoted to ligand design
purposes and is based on QSAR models generated by a
highly noncongeneric molecular series,10 the agreement
between the predicted and measured binding affinity
data values can be considered satisfactory. In fact, we
consider these QSAR models as useful ligand design and
decision making tools. In this case, these tools showed
low predictive resolution (about 0.5, 1, and 1.5 respec-
tively, in a logarithmic scale). Nevertheless, they al-
lowed the selection among the many hypothetical ligands
designed by estimating their affinities for the R1-ARs
according to three levels: (a) low affinity, pKi 7-8; (b)
high affinity, pKi 8-9; and (c) very high affinity, pKi >
9. On these bases, a few wrong estimations (∆ >1.1)
have been done (pK1a: compound 1r ; pK1b: compounds
1m , 1o, 1r ; pK1d: compounds 1o and 1r ). The molecular
descriptor Vdif completely failed in estimating the af-
finities of 1r for all the three R1-AR subtypes, suggesting
that the low binding affinity of 1r does not depend on
Vout, lowering Vdif. In general, compounds with a three
or four carbon atom spacer show lower Vdif than the
corresponding two carbon atom spacer compounds
(Tables 1-4).
Thus, for the R1b-AR, size-shape indices predict that,
similarly to the R1a-AR, structural variability in position
5 of the pyridazinone is better tolerated than that in
position 6. Moreover, the elongation of the alkyl spacer
is predicted to lower the R1b-AR binding affinity.
As for the R1d-AR, size and shape descriptors show a
behavior almost similar to that of the R1a-AR.
The linear regression equations previously obtained
(Table 3)10 have been used for estimating the binding
affinities of the new compounds. On the basis of both
the predicted binding affinities data values (nanomolar
range) and the smaller variance of the estimated
R1a-AR binding affinities (about 0.5 in a logarithmic
scale) with respect to the other two subtypes (about 1.0
and 1.5, respectively), we decided to synthesize the
designed compounds.

2408 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 15
Barlocco et al.
Ta ble 4. Affinity Constants (Ki, nM) of Compounds 1a -r ; Prazosin, BMY7378, and 8-OH-DP AT toward Human Cloned R1
Adrenoceptor Subtypes and 5-HT1A Receptora
Ki (nM)a
compd
R-1a
0.63
0.32
0.30
R-1b
3.29
3.77
0.75
R-1d
0.13
0.49
0.25
5-HT1A
bindingb [35S] GTPγS
1a
1b
1c
1d
1e
1f
2.47
24.12
11.60
n.a
0.28
0.19
0.18
3.95
1.45
0.64
0.41
0.25
0.43
76.23
4.06
10.20
pEC50 ) 8.6; 19%
n.a.
1g
1h
1k
1i
1j
1l
1m
1n
1o
1p
1q
1r
0.22
1.0
1.51
0.44
2.59
2.0
2.29
18.56
0.89
0.53
2.99
162.80
0.61
381.05
1757.20
2.01
1.76
20.28
1.44
5.51
11.78
1.07
4.32
3.92
11.79
2.92
13.98
322.1
134.50
1000.00
126.72
2.01
>1000
49.84
33.13
53.39
8.04
49.08
258.92
0.42
3.20
13.00
32.10
1.48
11.24
1.39
17.69
34.45
5.34
pEC50 ) 8.8; 17%
5.96
>10.000
>1000
P r a zosin
BMY7378
8-OH-DP AT
0.23
1.43
>10.000
68.97
5975.00
0.93
3.44
pEC50 ) 9.27; 26%
pEC50 ) 76; 100%
>1000
a
Equilibrium dissociation constants (Ki) were derived from IC50 using the Cheng-Prusoff equation.23 The affinities estimated were
derived from displacement of [3H]prazosin binding for R1-adrenoceptors and [3H]-8-hydroxy-2-(di-n-propylamino)tetraline for 5-HT1A
b
receptor. Each experiment was performed in triplicate. Ki values were from 2 to 3 experiments which agreed within 20%. n.a. ) not
active, indicative of neutral antagonism. For partial agonists the pEC50 and percent of maximum effect are given. Values are from 2 to
3 experiments which agreed within 20%.
the ligand size-shape features. In fact, the very low
affinity of this compound for all the three R1-ARs is
probably due to the fact that no nitrogen atom suitable
for protonation, at physiological pH, is present in this
compound, thus preventing the ligand to perform the
proper salt-bridge interaction with the aspartate of the
receptor binding site. The overestimation of the R1b-AR
binding affinity of 1m is due to the fact that reducing
the size of the pyridazinone moiety, with respect to 1a ,
lowers both the inner and outer volumes of this com-
pound with respect to the reference R1b-AR supermol-
ecule but not enough for producing a good estimation
of the binding affinity of this compound. The overesti-
mation of the R1b- and the R1d-AR binding affinity of 1o
is due to the fact that reducing the size of the pyridazi-
none moiety increases Vin and reduces Vout with respect
to 1l, thus producing a Vdif too high for predicting
correctly the binding affinity of this compound. As a
consequence, QSAR models fail in estimating the R1a-
AR selectivity of 1o.
In general, R1a-AR binding affinities are slightly
underestimated (Table 3) whereas, the binding affinities
for the other two R1-AR subtypes, in particular the R1b-
AR, are rather overestimated (Table 3) by the theoreti-
cal model. For these reasons, the theoretical model does
not estimate properly the R1a/R1b selectivity of com-
pounds 1m , 1o, 1p , and 1q and the R1a/R1d selectivity
of compounds 1g, 1o, and 1p .
SAR analysis shows that three compounds, i.e., 1g,
1o, and 1p , are selective for the R1a-AR as compared to
the other two R1-AR subtypes. Selectivity of 1g is mainly
due to the lack of substitution in position 5 of the
pyridazinone that primarily lowers the affinity for the
R1d-AR and, by a lower extent, that for the R1b-AR. The
R1a-AR selectivity of compound 1o is mainly due to the
replacement of the phenyl ring in position 6 of the
pyridazinone with a methyl group, on one hand, that
lowers the affinity for the R1b-AR, and to the elongation
of the ethylenic spacer to three carbon atoms, on the
other one, that lowers the affinity for the R1d-AR.
However, the presence of a phenyl ring in position 6
increases the affinity of this compound for the 5-HT1A
receptor. This drawback is overcome by introducing a
chlorine in position 5′ of the phenyl ring (i.e. 1p ), that
lowers the affinity for the serotoninergic receptor while
also retaining lower affinities for the R1b- and R1d-AR
subtypes, than for the R1a-AR.
Interestingly, the majority of the compounds proposed
in this work show low affinities for the 5-HT1A receptor.
This is mainly due to the presence of a chlorine
substituent in position 5′ of the phenyl ring and/or to
the elongation of the alkyl spacer. However, the latter
needs the presence of a phenyl ring in position 6 of the
pyridazinone to produce a low affinity for the 5-HT1A
receptor.
As for functional studies, compound 1i was tested for
its functional activity on rat aorta contractions induced
by (-)-noradrenaline and, as expected, proved to be an
antagonist with pKb of 8.21.
Furthemore, compounds showing a relevant affinity
for the 5-HT1A receptor were tested for their ability to
influence the binding of [35S]GTPγS to HeLa cell mem-
branes expressing the human 5-HT1A receptor. Com-
pounds 1a , 1f, and 1q behaved as neutral antagonists,
whereas compounds 1e and 1o proved to be partial
agonists with a pEC50 of 8.6 and 8.8 and a percent
maximum effect of 19 and 17%, respectively. (See
Table 4.)
In conclusion, the QSAR models previously obtained
on a very heterogeneous series of protonated R1-AR
antagonists have been challenged in their capability to
design and estimate the R1-AR binding affinities of a
set of N-arylpiperazines with satisfactory results.
This study indicates that substitutions in positions 5
and/or 6 of the pyridazinone ring as well as in positions
2′ and 5′ of the phenyl ring together with the length of

New R1 Adrenoceptor Antagonists
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 15 2409
were resuspended in a small volume of ice-cold Tris 5 mM and
EDTA 5 mM buffer (pH 7.4) and immediately frozen and stored
at -70 °C until use.
the linker are responsible for modulating the ligand R1-
AR binding affinities and R1-AR/5-HT1A selectivities.
On the experimental day, cell membranes were resuspended
in binding buffer 50 mM Tris (pH 7.4), 2.5 mM MgCl2, 10 µM
pargiline.21 Membranes were incubated in a final volume of 1
mL for 30 min at 30 °C with 1.2 nM [3H]8-OH-DPAT, in the
absence or presence of competing drugs; nonspecific binding
was determined in the presence of 10 µM 5-HT. The incubation
was stopped by addition of ice-cold Tris buffer and rapid
filtration through 0.2% polyethyleneimine pretreated Schle-
icher and Schuell GF52 filters.
Exp er im en ta l Section
Ch em istr y. Melting points were determined on a Bu¨chi 510
capillary melting points apparatus and are uncorrected.
Analyses indicated by the symbols were within ( 0.4 of the
theoretical values. 1H NMR spectra were recorded on a Bruker
AC200 spectrometer; chemical shifts are reported as δ (ppm)
relative to tetramethylsilane. TLC on silica gel plates was used
to check product purity. Silica gel 60 (Merck; 230-400 mesh)
was used for flash chromatography.
The inhibition of specific binding of the radioligands by the
tested drugs was analyzed to estimate the IC50 value by using
the nonlinear curve-fitting program Allfit.22 The IC50 value is
converted to an affinity constant (Ki) by the equation of Cheng
and Prusoff.23
2-Meth yl-4-[(4-a r ylp ip er a zin -1-yl)a m in oa lk yl]-5-a cetyl-
6-p h en yl(m eth yl)-3-(2H)-p yr id a zin on es 1a -d ,k ,i,m ,o-q.
A solution of the required Ia ,b11 (1 mmol) and the appropriate
N,ω-aminoalkyl-N1-phenylpiperazine15 (2.5 mmol) in EtOH (6
mL) was stirred at room temperature for 30 min. Water (5
mL) was added, the solvent was evaporated under vacuum,
and the residue was extracted with CH2Cl2. The organic layer
was dried over Na2SO4, the solid was filtered off, and the
solvent was evaporated under vacuum. The residue was
purified by flash chromatography eluting with cyclohexane/
ethyl acetate 4/6 to give as the first run the desired compounds
1. (See Table 1 for data.)
F u n ction a l Stu d ies. Evaluation of compounds for R1-
antagonism was performed as previously published.24 Briefly,
Sprague Dawley rats (350-450 g body weight) were sacrificed
by cervical dislocation. The aorta was isolated, freed of
adhering connective tissue, and placed in Krebs solution
containing NaCl (112 mM), glucose (11.1 mM), NaHCO3 (25
mM), KCl (4.7 mM), CaCl2 (2.5 mM), KH2PO4 (1.2 mM), and
MgSO4 (1.2 mM). Desmethylimipramine (0.1 µM) and corti-
costerone (1 µM) to block neuronal and extraneuronal uptake
of noradrenaline, (()-propranolol (1µM) to block â-receptors,
and yohimbine (0.1 µM) to block R2-receptors were added to
the Krebs solution. Aortic strips (2 × 30 mm long) were
mounted for isotonic tension recording in 20 mL-organ bath
containing Krebs buffer aerated constantly with 95% O2-5%
CO2 and maintained at 37 °C and loaded with a resting tension
of 1.5 g. The strips were allowed to equilibrate for 60 min with
washing every 20 min. After the equilibration period, tissues
were primed twice (every 60 min) by addition of 10 µM
noradrenaline. After another washing and equilibration period
of 60 min, a noradrenaline concentration-response curve was
constructed (basal curve). Following washout of noradrenaline,
single concentrations of the compounds were incubated for 30
min before repeating the noradrenaline concentration-
response curve. Responses were expressed as percentage of
the maximal contraction observed in the basal noradrenaline
concentration-response curve and analyzed by nonlinear
curve fitting according to the method reported by De Lean et
al.22 Schild-plot parameters were evaluated by linear-regres-
sion analysis according to Tallarida and Murray.25
Stim u la tion of [35S]GTP γS Bin d in g a t Clon ed 5-HT1A
Recep tor . The effects of the compounds tested with [35S]-
GTPγS binding were evaluated according to the method of
Stanton and Beer26 with minor modifications. On the experi-
mental day, cell membranes from HeLa cells transfected with
human cloned 5-HT1A receptors, prepared as above-described,
were resuspended in buffer containing 20 mM HEPES, 3 mM
MgSO4, and 120 mM NaCl (pH 7.4). The membranes were
incubated with 30 µM GDP and decreasing concentrations of
test drugs (from 100 µM to 0.1 nM) or decreasing concentra-
tions of 5-HT, from 100 µM to 0.1 nM (reference curve) for 20
min at 30 °C in a final volume of about 0.5 mL. Samples were
then transferred to ice, added with [35S]GTPγS (150-250 pM),
and then incubated for a further 30 min at 30 °C. Nonspecific
binding was determined in the presence of 10 µM GTPγS. The
incubation was stopped by addition of ice-cold HEPES and
rapid filtration on Schleicher and Schuell GF52 filters, using
a Brandel cell harvester. The filters were washed three times
with a total of 5 mL of the same buffer. Radioactivity was
counted by liquid scintillation spectrometry with efficiency >
90.
F or 1a : 1H NMR (CDCl3) δ: 1.9.(s, 3H); 2.7-2.8 (m, 6H);
3.1-3.2 (m, 4H); 3.3-3.4 (m, 2H); 3.8 (s, 3H); 3.9 (s, 3H); 6.8-
7.0 (m, 4H); 7.4 (s, 5H); 8.0 (br. S, 1H, exch. with D2O).
The higher homologues 1e,f were prepared according to the
same method starting from 2-methyl-4-nitro-5-propanonyl-
(Ic)11 or isobutanonyl- (Id ),12 respectively.
F or 1e: 1H NMR (CDCl3) δ: 0.8 (t, 3H, J ) 8 Hz); 2.1 (q,
2H; J ) 8 Hz); 2.6-2.8 (m, 6H); 3.0-3.2 (m, 6H); 3.8 (s, 3H);
3.9 (s, 3H); 6.8-7.0 (m, 4H); 7.3-7.5 (m, 6H)
F or 1f: 1H NMR (CDCl3) δ: 0.8 (2s, 6H); 2.2-2.3 (m 1H);
2.6-2.7 (m, 6H); 3.0-3.1 (m, 4H); 3.2-3.6 (m, 2H); 3.8 (s, 3H);
3.9 (s, 3H); 6.8-7.1 (m, 4H); 7.3-7.5 (m, 6H).
Compound 1r was obtained by condensation in the same
conditions of Ia and N-(2-methoxyphenyl)piperazine. 1H NMR
(CDCl3) δ: 2.1 (s, 3H); 3.1-3.2 (m, 4H); 3.5-3.6 (m, 4H); 3.9
(2s, 6H); 6.9-7.0 (m, 4H); 7.4 (app.s, 5H). (See Table 1 for
data.)
2-Meth yl-4-[(4-ar ylpiper azin -1-yl)am in oalkyl]-6-ph en yl-
(m eth yl)-3-(2H)-p yr id a zin on es 1g,h ,j,l,n . A mixture of the
required 4-chloro-3(2H)pyridazinone13,14 and an excess of the
appropriate phenylpiperazinalkylamine was heated at 140 °C
for 12 h. The residue was first purified by flash chromatog-
raphy eluting with CH2Cl2/MeOH 95/5 and then by gravimetric
chromatography, eluting with toluene/methanol 9/1. (See Table
1 for data.)
Biology. Ra d ioliga n d Bin d in g Assa y a t Clon ed r1-
Ad r en ocep tor s. Binding to cloned human R1-adrenoceptor
subtypes was performed in membranes from CHO cells (chi-
nese hamster ovary cells) transfected by electroporation with
DNA expressing the gene encoding each R1-adrenoceptor
subtype. Cloning and stable expression of the human R1-
adrenoceptor gene was performed as previously described.19
CHO cell membranes (30 µg 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 mL for 30 min at 25 °C, in the absence or presence
of competing drugs (1 pM-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 pre-
treated Whatman GF/B or Schleicher and Schuell GF52 filters.
Ra d ioliga n d Bin d in g Assa y a t Hu m a n Clon ed 5HT1A
-
Ser oton in er gic Recep tor s. Genomic clone G-21 coding for
the human 5HT1A-serotoninergic receptor was stably trans-
fected in a human cell line (HeLa).20 HeLa cells were grown
as monolayers in Dulbecco’s modified Eagle’s medium (DMEM),
supplemented with 10% fetal calf serum and gentamicin (100
µg/mL), 5% CO2 at 37 °C. Cells were detached from the growth
flask at 95% confluence by a cell scraper and were lised in
ice-cold Tris 5 mM and EDTA 5 mM buffer (pH 7.4). Homo-
genates were centrifuged at 40 000 × g × 20 min, and pellets
Stimulation of [35S]GTPγS binding induced by the com-
pounds tested was expressed as percent increase in binding
above basal value, being the maximal stimulation observed
with 5-HT taken as 100%. The concentration-response curve
of the agonistic activity was analyzed by nonlinear fitting
program Allfit.22 The maximal stimulation of [35S]GTPγS

2410 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 15
Barlocco et al.
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Weinshank, R. L.; Gluchowski, C.; Branchek, T. A. Faseb J .
1994, 8, A353.
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Langer, S. Z.; Lefkowitz, R. J .; Minneman, K. P.; Ruffolo, R. R.
International Union of Pharmacology X. Recommendation for
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Structure-Affinity/Selectivity Relationships. Methods Companion
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(11) Dal Piaz, V.; Ciciani, G.; Turco, G. Oxidative cleavage of 7-oxo-
6,7-dihydroisoxazolo[3,4-d]pyridazines by ceric(IV) ammonium
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6-phenyl-2,4-substituted-3(2H)-pyridazinones and their rigid
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binding (Emax) achieved for each drug and the concentration
required to obtain 50% of Emax (pEC50 value) were evaluated.
Molecu la r Mod elin g. The protonated structures of the
ligands considered in this study were fully optimized by means
of semiempirical molecular orbital calculations (AM1),27 using
the MOPAC 6.0 (QCPE 455) program.
The ad hoc modeling consisted of comparing the vdw volume
of the minimized structure of each ligand (in its extended
conformation) with the vdw volume of a supermolecule chosen
as a template. Three different supermolecules were modeled
for the three R1-AR subtypes. In the series of structurally
heterogeneous ligands considered in a previous study,10 subsets
of analogues can be identified. For each subset, the ligand
showing the highest affinity for the specific R1-AR subtype was
chosen as a component of the respective reference supermol-
ecule. Thus, the ligands used for the R1a supermolecule are
compounds 1-6, the ligands for the R1b supermolecule are
compounds 7-10, and the ligands for the R1d supermolecule
are 7, 8, 10-12 (Chart 1). The ligands chosen as components
of each supermolecule were superimposed by a topologic rigid
body fit procedure based on the following pharmacophoric
criteria: (a) the hydrogen of the protonated nitrogen atom and
(b) the aromatic rings closest to and farthest from the proto-
nated nitrogen. All the other compounds were rigidly super-
imposed on the appropriate supermolecule with each ligand
being superimposed on the analogue compound present in the
supermolecule or on its structurally closest compound. Match-
ing involved the moieties carrying the protonated nitrogen,
i.e., the piperazinic ring.
QUANTA molecular modeling package (release 96; Molec-
ular Simulation Inc., 200 Fifth Avenue, Waltham, MA 02154)
was used for molecular comparison and computation of the
vdw volumes. We considered the following size and shape
descriptors: Vin and Vout, which are, respectively, the intersec-
tion and the outer van der Waals volume of the ligand
considered with respect to the vdw volume of the reference
supermolecule; and Vdif, which is computed according to the
formula Vdif ) (Vin-Vout)/Vsup, where Vsup is the molecular
volume of the reference supermolecule.
(14) Schonbeck, R.; Kloimstein, E. Uber Pyridazine mit kerngebun-
denem. Chlor. Monatsh. Chem. 1968, 99, 15-48.
(15) Montesano, F.; Barlocco, D.; Dal Piaz, V.; Leonardi, A.; Poggesi,
E.; Fanelli, F.; De Benedetti, P. G. Isoxazolo-[3,4-d]-pyridazin-
7-(6H)-ones and their corresponding 4,5-disubstituted-3-(2H)
pyridazinone analogues as new substrates for R1-adrenoceptor
selective antagonists: synthesis, modeling and binding studies.
Bioorg Med. Chem. 1998, 6, 925-935.
(16) Cavalli, A.; Fanelli, F.; Taddei, C.; De Benedetti, P. G.; Cotecchia,
S. Amino acids of the R1B-adrenergic receptor involved in agonist
binding: further differences in docking catecholamines to recep-
tor subtypes. FEBS Lett. 1996, 399, 9-13.
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R.; Leonardi, A. R1-Adrenoceptor Subtype Selectivity: Molecular
Modelling and Theoretical Quantitative Structure-Affinity Re-
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Ack n ow led gm en t. Financial support from CNR
and technical support from CICAIA (University of
Modena) are acknowledged.
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