Synthesis, biological evaluation and structure-activity relationships of new phthalazinedione derivatives with vasorelaxant activity

Article abstract of DOI:10.1016/j.ejmech.2014.05.052

Five series of 1,4-phthalazinedione derivatives were synthesized in good yields. Vasorelaxant activity of these new derivatives was measured on either intact or endothelium-denuded isolated rat thoracic aortic rings pre-contracted with phenylephrine. Most of studied compounds, substituted in both nitrogen atoms, attained practically the total relaxation of the organ at low micromolar concentrations. The presence of functional endothelium significantly reduced the EC₅₀ values for most of studied compounds. Some structure-activity relationships were established and compounds 2d and 5d can be considered as new leads for further modifications.

Full text of DOI:10.1016/j.ejmech.2014.05.052

European Journal of Medicinal Chemistry 82 (2014) 407e417
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, biological evaluation and structureeactivity relationships of
new phthalazinedione derivatives with vasorelaxant activity
Javier Munín a , Elías Quezada b , Andrea Cui n~ as , Manuel Campos-Toimil ,
,
b
,
*
a
a
b
b
a, *
Eugenio Uriarte , Lourdes Santana , Dolores Vi n~ a
Farmacologí
a de las Enfermedades Cr oꢀ nicas, Centro de Investigaci oꢀ n en Medicina Molecular y Enfermedades Cr oꢀ nicas, Universidad de Santiago de
a
Compostela, 15782 Santiago de Compostela, Spain
b
Departamento de Quí
mica Org aꢀ nica, Facultad de Farmacia, Universidad de Santiago de Compostela, 15782 Santiago de Compostela, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 20 October 2013
Received in revised form
Five series of 1,4-phthalazinedione derivatives were synthesized in good yields. Vasorelaxant activity of
these new derivatives was measured on either intact or endothelium-denuded isolated rat thoracic aortic
rings pre-contracted with phenylephrine. Most of studied compounds, substituted in both nitrogen
atoms, attained practically the total relaxation of the organ at low micromolar concentrations. The
presence of functional endothelium significantly reduced the EC50 values for most of studied compounds.
Some structureeactivity relationships were established and compounds 2d and 5d can be considered as
new leads for further modifications.
19 May 2014
Accepted 22 May 2014
Available online 24 May 2014
Keywords:
Phthalazinedione derivatives
Synthesis
©
2014 Elsevier Masson SAS. All rights reserved.
Vasorelaxant activity
Endothelium
Rat aortic rings
SAR
1
. Introduction
antihypertensive agents acting on different mechanisms to control
blood pressure: diuretics [7], angiotensin-converting enzyme in-
hibitors [8,9], angiotensin II receptor blockers [9,10], calcium
Cardiovascular disorders are one of the main reasons for
morbidity and death in recent years in developed countries. Among
cardiovascular disorders, hypertension is the most common risk
factor and it can cause coronary disease, myocardial infarction,
stroke and sudden death and is the major contributor to cardiac
failure and renal insufficiency [1].
Hypertension affects billions of people around the world. It is
characterized by a persistently elevated blood pressure (BP)
exceeding 140/90 mmHg or greater [2]. Hypertension induces an
endothelial dysfunction via the release of superoxide anion by the
vascular wall [3,4]. This free radical, together with its derivatives,
counteracts the relaxing activity of endothelium-derived nitric
channel blockers [11], centrally sympathetic
and drugs that prevent the action of peripheral sympathetic ac-
tivity as -adrenergic [13,14] and -adrenergic [15] blocking agents.
2
a -adrenoceptors [12]
b
a
Antihypertensive drugs such as prazosin (I), terazosin (II) and
doxazosin (III), which have in their structure a quinazoline ring, are
considered the most effective and clinically useful class of selective
a
1
-AR antagonists (Fig. 1) [16e18].
The phthalazine ring is the isostere and positional isomer of
quinazoline ring and it is the nucleus of other well known vasodi-
lator, hydralazine (IV) (Fig. 2). Hydralazine is an established anti-
hypertensive drug that exerts its action via arterial dilation,
although concentrations of hydralazine that present in vitro vaso-
dilator effects in rat aorta (~1 mM) [19] are significantly higher than
plasma concentrations which cause hypotension in awake rats
(~100 nM) [20].
2
oxide and prostacyclin (PGI ) [5,6].
Due to the associated morbidity and mortality and cost to so-
ciety, preventing and treating hypertension is an important public
health challenge. Great efforts have been made on obtaining novel
It is not used as a primary drug for treating hypertension
because it also elicits sympathetic stimulation and salt retention,
which may lead to the development of congestive heart failure and
increases plasma rennin activity [21]. However, concomitant use of
hydralazine together with a venodilatory nitrate has been shown to
*
Corresponding authors.
E-mail addresses: elias.quezada@usc.es (E. Quezada), mdolores.vina@usc.es
(
D. Vi n~ a).
http://dx.doi.org/10.1016/j.ejmech.2014.05.052
223-5234/© 2014 Elsevier Masson SAS. All rights reserved.
0

408
J. Munín et al. / European Journal of Medicinal Chemistry 82 (2014) 407e417
anhydride (A) or 4-chlorophthalic anhydride (B) (Scheme 1)
37e39].
[
Firstly, 2,3-dihydro-2-methyl-1,4-phthalazinedione (1a) was
synthesized by reaction of phthalic anhydride (A) and methyl-
hydrazine under MW irradiation (320 W) and using Montmo-
rillonite KSF as a catalyzer. The same procedure was used
starting from B and yielded a mixture of 8-chloro-2,3-dihydro-2-
methyl-1,4-phthalazinedione (1b) and 5-chloro-2,3-dihydro-2-
methyl-1,4-phthalazinedione (1c) isomers in 1.8:1 proportion
which were separated by HPLC using a Partisil Si 10 (Whatman)
column.
Identification and characterization of both isomers was per-
formed using 1D and 2D NMR experiments (HMQC and HMBC). For
compound 1b
155.49 ppm) and protons of CH
however for compound 1c this correlation is observed between C-1
a
long-distance correlation between C-1
(
3
(3.49 ppm) is observed by HMBC,
Fig. 1. Clinically used
1
a -ARs antagonists IeIII.
(
(
156.21 ppm) and protons of CH
Fig. 4).
tert-Butoxycarbonylaminealkyl-2,3-dihydro-1,4-
3
(3.52 ppm) and H-8 (8.19 ppm)
prevent the development of nitrate tolerance and to maintain the
favorable hemodynamic effect of nitrates in patients with conges-
tive heart failure [22]. Isosorbide dinitrate/hydralazine combina-
tion is advocated as a reasonable option in the treatment of patients
with advanced, but stable heart failure and who remain symp-
tomatic despite optimal standard therapy [23]. Furthermore, hy-
dralazine (IV) is commonly used for severe pregnancy hypertension
phthalazinedione derivatives 2aef were obtained starting from 1,4-
phthalanzinediones 1aec by reaction with N-Boc-n-bromoalkyl-
amine and K
achieved for propyl derivatives 2b, 2d and 2f (83%, 79% and 86%
respectively) resulted slightly higher than those for the corre-
sponding ethyl derivatives 2a, 2c and 2e (67%, 78% and 78%
respectively). Deprotection of the above mentioned derivatives was
2
performed by treating with Et O/HCl, obtaining the corresponding
aminoalkyl-2,3-dihydro-1,4-phthalazinedione derivatives 3aef in
high yield [41].
ꢀ
2
CO
3
/KI in DMF and heating at 125 C [40]. Yields
[24].
Structural modification of hydralazine led to the discovery of
other phthalazine derivatives (VeVII, Fig. 2) [25e27] with vaso-
relaxant activity and some pyridazinone derivatives (VIIIeX, Fig. 3)
which are known drugs with interesting effects in cardiovascular
system due to their platelet aggregation inhibition as well as their
antihypertensive activity and cardiotonic properties [28e34].
Most of described pyridazinone derivatives, showing activity on
cardiovascular system, include aryl residues at 6 position (Fig. 3).
However, in the last few years new pyridazinone derivatives in
which the phenyl group at C6 was removed or replaced (compound
Bromoalkyl-2,3-dihydro-1,4-phthalazinedione derivatives 4aef
were obtained by reaction of 1,4-phthalazinediones 1aec and the
corresponding alkyldibromide and K
2
CO
3
in DMF and heating at
ꢀ
1
25 C [42]. Afforded yields were, in all the cases, higher for bro-
mopropyl-2,3-dihydro-1,4-phthalazinediones 4b, 4d and 4f (46%,
6% and 80% respectively) than those afforded for the corre-
8
X in Fig. 3) were described such as
1
a -ARs antagonists or platelet
sponding bromoethyl-2,3-dihydro-1,4-phthalazinediones 4a, 4c
and 4e (32%, 60% and 17% respectively). Furthermore, reaction time
had to be shorter from 24 h to obtain bromopropyl-2,3-dihydro-
aggregation inhibitors [28,29,35,36].
Considering the pyridazinone residue as the pharmacophoric
group for the activity, we focused on the synthesis of a series of new
derivatives of its benzene condensed analogue, phthalazinone,
with the aim of testing their vasorelaxant activity during in vitro
assays and try to establish a preliminary SAR. Experiments were
performed on both endothelium-intact and endothelium-denuded
aortic rings in order to elucidate whether achieved relaxation is or
not endothelium dependent.
1
,4-phthalazinediones 4b, 4d and 4f to 2 h for the corresponding
bromoethyl-2,3-dihydro 1,4-phthalazinediones 4a, 4c and 4e
because longer reaction time led to decomposition of these
derivatives.
Reaction of bromoalkyl-2,3-dihydro-1,4-phthalazinedione de-
rivatives 4aef with sodium azide and sodium iodide in DMSO at
1
derivatives 5aef in good yields [43]. Due to the instability
showed for derivatives 4a, 4c and 4e, the reaction with sodium
azide was heating just for 3 h, while using derivatives 4b, 4d and 4f
time reaction was prolonged for 24 h.
ꢀ
00 C afforded the azidoalkyl-2,3-dihydro-1,4-phthalazinedione
Additionally, since overall these compounds showed interesting
vasorelaxant activity, cytotoxicity test on cells of smooth muscle
derived from rat aorta fibroblasts (line A7r5) were performed.
2
. Results and discussion
Reduction of azidoalkyl-2,3-dihydro-1,4-phthalazinedione de-
ꢀ
rivatives 5aef with Ph
3
P in methanol at 80 C for 1 h, let to obtain
2.1. Chemistry
the aminoalkyl-2,3-dihydro-1,4-phthalazinedione derivatives 3aef
with excellent yield (70e84%), following an alternative route to
that previously described (Scheme 1) [44].
A series of differently substituted 1,4-phthalazinedione de-
rivatives were synthesized in good yield starting from phthalic
Fig. 2. Chemical structure of hydralazine (IV) and phthalazine derivatives (VeVII) with vasorelaxant activity.

J. Mun
ín et al. / European Journal of Medicinal Chemistry 82 (2014) 407e417
409
Fig. 3. Representative pyridazinone derivatives with activity on cardiovascular system.
ꢀ
Scheme 1. Reagents and conditions: (a) CH
3
NHNH
2
, Montmorillonite KSF, MW, 15 min; (b) Br(CH
2
)
2
NHBoc or Br(CH
2
ꢀ
)
3
NHBoc, K
2
CO
3
, KI, DMF, 125 C, 24 h; (c) sat. HCl/Et
2
O, CH
2
Cl
2
,
ꢀ
ꢀ
ꢀ
0
C, 30 min, then rt, 1 h; (d) Br(CH
2
)
2
Br or Br(CH
2
)
3
Br, K
2
CO
3
, DMF, 125 C, 2 or 24 h; (e) NaN
3
, NaI, DMSO, 100 C, 3 or 24 h; (f) (Ph)
3
P, CH
3
OH, 80 C, 1 h.
2
2
.2. Pharmacology
Most of new 1,4-phthalazinedione derivatives bearing sub-
stituents in both nitrogen atoms significantly relaxed the aortic
rings pre-contracted with PE, in a concentration-dependent
manner, in the absence of endothelium but affording a major vas-
orelaxant effect in the presence of endothelium (Table 1). Our re-
sults suggest that the relaxation induced by the above mentioned
compounds is caused by two mechanisms: a direct effect on
vascular smooth muscle and a mechanism that is dependent on the
presence of a functional endothelium. Most of the studied com-
pounds resulted to be more potent than hydralazine and others
vasorelaxant agents such as amrinone or diazepam. However,
contrary to hydralazine or diazepam and as in the case of amrinone,
the endothelium-dependent component resulted more significant.
.2.1. Vasorelaxant activity
Vasorelaxant
properties
of
the
synthesized
1,4-
phthalazinedione derivatives (series 1e5) were investigated using
isolated rat thoracic aortic rings pre-contracted with phenylephrine
(PE) following a standard procedure previously reported by us
[45,46] and compared to the reference drugs hydralazine, amrinone
[47] and diazepam [48]. Experiments were performed on both
endothelium-intact and endothelium-denuded aortic rings. PE
M) produced a sustained contraction in the rat isolated aortic
rings with or without endothelium. The maximal tensions reached
mg) were 1491 ± 46 and 2048 ± 69, respectively (p < 0.01, n ¼ 50).
(1 m
(

410
J. Munín et al. / European Journal of Medicinal Chemistry 82 (2014) 407e417
Fig. 4. HMBC spectra of compounds 1b and 1c.
The relaxation was expressed as a percentage of the reverse
contraction produced by PE.
Analyzing structural features of the new molecules and their
vasorelaxant activity on intact isolated rat aorta rings, some
considerations of structureeactivity relationship can be estab-
lished. Compounds 1aec resulted inactive as vasorelaxant agents,
demonstrating that position of the N-methyl substitution and the
chloro atom on the phenyl ring of the 1,4-phthalazinedione moiety,

J. Mun
ín et al. / European Journal of Medicinal Chemistry 82 (2014) 407e417
411
Table 1
Vasorelaxant activity of 1,4-phthalazinedione derivatives (series 1e5) in rat aortic
rings pre-contracted with PE (1 M).
m
Compound
EC50
(
mM) with
EC50 (mM) without
endothelium
endothelium
1
1
1
2
2
2
2
2
2
3
3
3
3
3
3
4
4
4
4
4
4
5
5
5
5
5
5
a
b
c
a
b
c
d
e
f
a
b
c
d
e
f
a
b
c
d
e
f
a
b
c
d
e
f
*
*
*
*
*
*
a
2.44 ± 0.57
3.96 ± 1.02
2.53 ± 0.57
2.15 ± 0.30
3.45 ± 0.78
4.78 ± 1.13
**
14.56 ± 3.05
23.61 ± 4.90
5.57 ± 0.91
4.85 ± 0.24
13.30 ± 1.99
18.23 ± 2.78
*
*
b
b
a
a
a
**
a
48.72 ± 10.64
8.51 ± 2.52
**
*
a
30.18 ± 2.81
*
*
a
44.68 ± 4.13
1.70 ± 0.29
3.10 ± 0.53
7.35 ± 1.20
6.61 ± 1.91
7.95 ± 1.06
3.36 ± 0.83
10.00 ± 2.12
2.35 ± 0.70
4.12 ± 0.63
2.33 ± 0.37
6.35 ± 0.74
4.31 ± 0.87
(1.18 ± 0.09) ꢁ 10
17 ± 6
a
8.46 ± 1.59
a
a
a
a
15.45 ± 2.55
32.55 ± 5.90
27.16 ± 4.96
20.16 ± 2.51
Fig. 5. Concentrationerelaxation curves for compound 3d in endothelium rat aortic
rings precontracted with PE (1
mM). In brackets, % of the maximal contraction afforded
after addition of ACh (1 M) which is relative to the removed endothelium.
m
a
6.71 ± 1.15
15.94 ± 2.52
3.71 ± 0.73
13.10 ± 1.40
11.71 ± 0.97
13.19 ± 2.39
7.44 ± 1.00b
remarkable loss of activity and a high dependence on endothelium
to produce vasorelaxation (Fig. 5). For these derivatives, the pres-
ence of a chloro atom on the phenyl ring resulted determinant for
the activity, being compounds 3a and 3b practically inactive as
vasorelaxant agents.
For all studied derivatives, the vasorelaxant activity was found
lower on endothelium-denuded isolated rat aorta rings pre-
contracted with PE, remaining the structural considerations
above mentioned for all studied derivatives.
a
a
b
3
3
Hydralazine
Amrinone [47]
Diazepam [48]
(1.32 ± 0.12) ꢁ 10
a
28 ± 8
69.0 ± 6.5
77.2 ± 8.3
*
Inactive at 200
m
M (highest concentration tested). **200
m
M produced relaxation
a
b
about 50%. P < 0.01 or P < 0.05 vs rings with endothelium.
tert-Butoxycarbonylaminealkyl-2,3-dihydro-1,4-
phthalazinedione derivatives 2aef, aminoalkyl-2,3-dihydro-1,4-
phthalazinedione derivatives 3aef, bromoalkyl-2,3-dihydro-1,4-
phthalazinedione derivatives 4aef and azidoalkyl-2,3-dihydro-
by themselves, are irrelevant to the activity. Potential interest of the
presence of a chlorine atom on 1,4-phthalazinedione structure was
studied because different compounds showing vasorelaxant activ-
ity as azelastine or diazepam show this substituent in their struc-
ture [26,48].
1,4-phthalazinedione derivatives 5aef resulted more potent vaso-
relaxant agents than the reference compound hydralazine in both
the presence or absence of endothelium (Fig. 6).
Introduction of an additional N-substitution on the second ni-
trogen atom of the phthalazinedione ring is necessary to afford
active derivatives. With the incorporation of this second N-substi-
tution, presence of chloro atom and position of the N-substituents
allow to observe some differences on the activity of these
derivatives.
In all of N,N-disubstituted studied series, activity of compounds
is higher when alkyl linker is incorporated on N2. Only one
exception is observed in bromo derivatives series in which com-
pound 4f resulted more potent than 4d as vasorelaxant agent.
Length of the alkyl linker on N2 or N3 also modifies the activity
of these derivatives. In general, when a chloro atom is present in the
structure, a N-propyl linker resulted more effective for the substi-
tution than an ethyl linker, with the only exception of compounds
2
.2.2. Cytotoxicity
The cytotoxic effects of two concentrations (25 and 50 mM) of
compounds 1aec, 2aef, 3aef, 4aef and 5aef on A7r5, a well
established vascular smooth muscle cell line obtained from em-
bryonic rat aorta, were studied. As depicted in Fig. 7, compounds
2
cef and 5f (at 50
m
M concentration) significantly decreased the
viability of A7r5 cells. However for a lower concentration (25
mM),
these compounds were well tolerated by the cells (Fig. 8). Consid-
ering the EC50 values obtained for vasorelaxant activity, this result
justifies the interest of these derivatives.
3. Conclusion
2
e and 2f (EC50 values resulted 3.45 and 4.78
m
M for 2e and 2f
Five series of 2 or 3-methyl-1,4-phthalazinedione derivatives N-
monosubstituted (1aec) or N,N-disubstituted with a variety of tert-
butoxycarbonylaminealkyl (2aef), aminoalkyl (3aef), bromoalkyl
(4aef) or azidoalkyl (5aef) groups linked to one of the N-atoms
have been synthesized in good yields. Substituted N-monomethyl
phthalazinediones lack of activity and additional substitution on
the second nitrogen atom is necessary.
Most of the compounds (2aef, 3ced and 3f, 4aef, 5aef) have
been found to cause relaxation of the aortic rings pre-contracted
with PE in a concentration-dependent manner, both in the pres-
ence or in the absence of endothelium. The relaxation caused by
our compounds was reduced after removal of the endothelium
respectively). When a chloro atom is not present in the structure,
N-ethyl linker resulted more effective than the propyl one, with the
exception of compounds 5a and 5b (EC50 values resulted 10.00 and
2
.35
mM for 5a and 5b respectively).
Regarding to the nature of the alkyl linker, tert-butox-
ycarbonylamine, bromo and azide alkyl substituents on the one of
the nitrogen atoms of the phthalazinedione ring, led to derivatives
with interesting vasorelaxant activity on intact isolated rat aortic
rings pre-contracted with PE.
However, aminoalkyl substitution in the same positions affor-
ded new derivatives (compounds 3aef) which showed
a

412
J. Munín et al. / European Journal of Medicinal Chemistry 82 (2014) 407e417
Fig. 6. Concentrationerelaxation curves for some of most active compounds (2d, 3d, 4f and 5d) in the described series on endothelium-denuded (a) and intact (b) rat thoracic aortic
rings precontracted with PE (1 M). Each point represents the mean value ± SEM (indicated by vertical bars) from, at least, five experiments.
m
layer. Further experimentation is needed in order to understand the
precise mechanism of action involved in the vascular smooth
muscle effects of these derivatives.
All compounds are non-toxic on A7r5 cells in concentration
showing vasorelaxant effects.
Compounds 2d and 5d can be considered as new leads for
further modifications. Both derivatives have a chloro atom at 5
position, methyl on the N3 and NHBoc group or azide group on N2
separated by a propyl linker. Replacement by different functional
groups of the chlorine atom as well as NHBoc and azide in the
propyl linker, will be performed in the future in order to obtain new
and more potent vasorelaxant derivatives.
4. Experimental section
4.1. Chemistry
All reactions utilizing air- or moisture-sensitive reagents were
carried out in flame dried glassware under an argon atmosphere,
unless otherwise stated. CH Cl , (CH CO, MeOH and DMSO were
2
2
3 2
)
distilled prior to use according to the standard protocols. Other
reagents were purchased and used as received without further
purification unless otherwise stated. Reactions were magnetically
stirred and monitored by thin layer chromatography (TLC) with
Fig. 7. Cytotoxic effects of a 24 h incubation with the studied compounds (50 mM) on
A7r5 vascular myocytes. Results are expressed as mean ± S.E.M from at least 5 ex-
periments. *Compounds decreased the viability versus the corresponding control
group treated with DMSO (P < 0.05).
0.15e0.2 mm pre-coated silica gel (10e40 mm) plates. Compounds
were visualized with UV light and/or by staining with ethanolic
phosphomolybdic acid (PMA) followed by heating on a hot plate
whilst ninhydrin was used as developer for amines. Flash chro-
matography (FC) was performed with silica gel (60e200 mesh)
under pressure. NMR spectra were recorded on Bruker-250 or AMX
5
00 spectrometers in CDCl
3
or DMSO with TMS as the internal
) are given in ppm and coupling con-
standard. Chemical shifts (
d
stants (J) in Hz. Multiplicity is indicated as follows: s, singlet; d,
doublet; t, triplet; q, quartet; m, multiplet; br, broad.
EIMS and HR-EIMS were carried out on a VG AutoSpec (Fison,
Ipswich, United Kingdom) instrument; the data are reported as m/z
(percentage of relative intensity of the most important fragments).
4.1.1. 2,3-Dihydro-2-methyl-1,4-phthalazinedione (1a)
Phthalic anhydride (A, 1.50 g, 10.13 mmol), methylhydrazine
(
0.53 ml, 0.46 g, 10.13 mmol) and montmorillonite KSF (10.0 g,
ꢀ
previously activated at 120 C for 8 h), were mixed and ground in a
mortar and placed in a clean and dry beaker. The reaction mixture
was irradiated in a microwave oven for 15 min in 1-min period
(
320 W), the reaction was monitored by TLC. After completion of
the reaction, it was cooled to room temperature and the resulting
product was extracted into CH Cl
(4 ꢁ 50 ml). The KSF
Fig. 8. Cytotoxic effects of a 24 h incubation with compounds 2cef and 5f (25 mM) on
A7r5 vascular myocytes. Results are expressed as mean ± S.E.M from at least 5
experiments.
2
2

J. Mun
ín et al. / European Journal of Medicinal Chemistry 82 (2014) 407e417
413
Montmorillonite was filtered out and the solvent was evaporated
under vacuum. The residue was purified by FC (CH Cl /CH OH
5:05, 90:10) affording 1a [39] (1.46 g, 82% yield) as a white solid,
4.1.5. 2-[2-(tert-Butoxycarbonyl)amine]ethyl-5-chloro-2,3-
dihydro-3-methyl-1,4-phthalazinedione (2c)
2
2
3
9
The compound 2c was obtained from 1b and N-Boc-2-
bromoethylamine in a similar manner as for the preparation of 2a
ꢀ
1
m.p. 231e232 C. H NMR (250 MHz, DMSO-d
6
)
d
¼ 11.75 (br, 1H,
ꢀ
1
NH), 8.17e8.21 (m, 1H, AreH), 7.84e7.96 (m, 3H, AreH), 3.38 (s, 3H,
3
(78%) as a white solid, m.p. 114e115 C. H NMR (250 MHz, CDCl )
1
3
CH
1
3
). C NMR (62.9 MHz, DMSO-d
6
)
d
¼ 157.41, 150.28, 132.98,
d
¼ 7.91 (d, J ¼ 7.8, 1H, AreH), 7.74 (d, J ¼ 7.8, 1H, AreH), 7.62 (t,
32.31, 128.83, 126.36, 124.83, 124.25, 37.73 (CH ). EIMS: m/z 176
3
J ¼ 7.8, 1H, AreH), 5.11 (br,1H, NH), 4.34 (t, J ¼ 5.0, 2H, NeCH
2
), 3.68
þ
ꢂ
13
[M] .
3 2 3
(s, 3H, NeCH ), 3.62 (m, 2H, NHeCH ), 1.45 (s, 9H, 3CH ). C NMR
(
1
(
[
62.9 MHz, CDCl
26.91, 124.79, 122.49, 79.53 ((CH
NHeCH ), 39.14 (NeCH ), 28.29 ((CH
M] ; calcd for C16 20ClN 4, 353.1142.
3
)
d
¼ 156.82, 155.85, 148.33, 134.88, 134.81, 132.60,
C), 66.38 (NeCH ), 39.59
C). HR-EIMS: m/z 353.1130
3
)
)
3 3
3
2
4.1.2. 8-Chloro-2,3-dihydro-2-methyl-1,4-phthalazinedione (1b)
2
3
and 5-chloro-2,3-dihydro-2-methyl-1,4-phthalazinedione (1c)
Following the previously described procedure to obtain 1a, re-
action of 3-chlorophthalic anhydride (B, 1.50 g, 8.22 mmol),
methylhydrazine (0.43 ml, 0.38 g, 8.22 mmol) and montmorillonite
KSF (8.0 g), afforded a mixture of 1b and 1c (0.56 g, 48% yield).
Mixture was purified by HPLC using a Partisil Si 10 column
þ
ꢂ
H
3
O
4
.1.6. 2-[3-(tert-Butoxycarbonyl)amine]propyl-5-chloro-2,3-
dihydro-3-methyl-1,4-phthalazinedione (2d)
The compound 2d was obtained from 1b and N-Boc-3-
bromopropylamine (0.11 g, 0.46 mmol) in a similar manner as for
the preparation of 2a (79%) as a white solid, m.p. 161e162 C. H
NMR (250 MHz, CDCl
(
(
(
Whatman) and CHCl
0.20 g) as white solids, ratio1.8:1. (1b): m.p. 223e224 C. H NMR
500 MHz, DMSO-d
¼ 11.72 (br, 1H, NH), 7.89 (d, J ¼ 7.8, 1H, H-5),
.84 (d, J ¼ 7.8, 1H, H-7), 7.78 (t, J ¼ 7.8, 1H, H-6), 3.49 (s, 3H, CH ).
¼ 155.49, 148.98, 134.92, 133.46,
33.08, 127.25, 124.49, 123.60, 38.24 (CH ). HR-EIMS: m/z 210.0192
3
/iPrOH (99:1), isolating 1b (0.36 g) and 1c
ꢀ
1
ꢀ
1
3
)
d
¼ 7.83 (d, J ¼ 7.8,1H, AreH), 7.67 (d, J ¼ 7.8,
6
) d
1
H, AreH), 7.56 (t, J ¼ 7.8, 1H, AreH), 5.00 (br, 1H, NH), 4.29 (t,
7
3
1
3
J ¼ 6.1, 2H, NeCH
2
), 3.61 (s, 3H, NeCH ), 2.00
3
), 3.33 (m, 2H, NHeCH
2
6
C NMR (125.7 MHz, DMSO-d ) d
13
(m, 2H, CH
2
eCH
2
eCH ), 1.40 (s, 9H, 3CH ). C NMR (62.9 MHz,
2
3
1
3
þ
ꢂ
ꢀ
1
CDCl
24.80, 122.41, 79.05 ((CH
7.70 (NHeCH ), 29.11 (CH
m/z 367.1299 [M] ; calcd for C17
)
d
¼ 156.71, 155.90, 148.46, 134.77, 134.75, 132.53, 127.05,
C), 64.65 (NeCH ), 39.02 (NeCH ),
eCH eCH ), 28.31 ((CH C). HR-EIMS:
22ClN , 367.1299.
3
[M] ; calcd for C
9
H
7
ClN
2
O
2
, 210.0196. (1c): m.p. 222e223 C. H
¼ 11.72 (br, 1H, NH), 8.19 (d, J ¼ 7.8, 1H,
1
3
3
)
3
2
3
NMR (500 MHz, DMSO-d
6
) d
2
2
2
2
3 3
)
H-8), 7.92 (d, J ¼ 7.8, 1H, H-6), 7.78 (t, J ¼ 7.8, 1H, H-7), 3.52 (s, 3H,
þ
ꢂ
1
3
H
3 4
O
CH
1
3
). C NMR (125.7 MHz, DMSO-d
6
)
d
¼ 156.21, 148.84, 135.78,
). HR-EIMS: m/z
32.72, 131.59, 130.38, 126.00, 121.97, 37.47 (CH
3
þ
ꢂ
4.1.7. 3-[2-(tert-Butoxycarbonyl)amine]ethyl-5-chloro-2,3-
dihydro-2-methyl-1,4-phthalazinedione (2e)
210.0196 [M] ; calcd for C
9
7
H ClN
2 2
O , 210.0196.
The compound 2e was obtained from 1c and N-Boc-3-
bromoethylamine in a similar manner as for the preparation of 2a
4
.1.3. 2-[2-(tert-Butoxycarbonyl)amine]ethyl-2,3-dihydro-3-
methyl-1,4-phthalazinedione (2a)
ꢀ
1
(
78%) as a white solid, m.p. 107e108 C. H NMR (250 MHz, CDCl )
3
2 3
A mixture of 1a (0.10 g, 0.57 mmol) and K CO (0.08 g,
.57 mmol) in anhydrous DMF (6 ml) was stirred at room tem-
d
¼ 8.37 (d, J ¼ 7.9, 1H, AreH), 7.78 (d, J ¼ 7.9, 1H, AreH), 7.64 (t,
0
J ¼ 7.9, 1H, AreH), 5.16 (br, 1H, NH), 4.32 (t, J ¼ 5.0, 2H, NeCH
2
), 3.70
perature for 2 h and kept under argon. Likewise, a solution of N-
Boc-2-bromoethylamine (0.13 g, 0.57 mmol) and KI (0.09 g,
0
Then, this solution was added on the first one and the reaction
mixture was stirred at 125 C for 24 h and kept under argon. The
mixture was cooled to room temperature, filtered and the precip-
itate was washed several times with anhydrous acetone. The sol-
vent was removed under vacuum and the residue was purified by
13
(s, 3H, NeCH
3
), 3.63 (m, 2H, NHeCH
2
), 1.46 (s, 9H, 3CH
3
). C NMR
(
1
(
[
62.9 MHz, CDCl
30.45, 126.36, 121.90, 79.42 ((CH
NHeCH ), 38.67 (NeCH ), 28.31 ((CH
M] ; calcd for C16 20ClN , 353.1142.
3
)
d
¼ 157.52, 155.77, 147.95, 135.74, 131.82, 131.50,
C), 66.59 (NeCH ), 39.48
C). HR-EIMS: m/z 353.1134
.57 mmol) in DMF (8 ml) was stirred for 2 h and kept under argon.
3
)
)
3 3
3
2
2
3
ꢀ
þ
ꢂ
H
3 4
O
4.1.8. 3-[3-(tert-Butoxycarbonyl)amine]propyl-5-chloro-2,3-
dihydro-2-methyl-1,4-phthalazinedione (2f)
The compound 2f was obtained from 1c and N-Boc-3-
bromopropylamine in a similar manner as for the preparation of
FC (CH
2 2
Cl /MeOH 99:1, 98:2) to provide 2a (0.12 g, 67% yield) as a
ꢀ
1
white solid, m.p. 170e171 C. H NMR (250 MHz, CDCl
d
3
)
¼ 8.30e8.34 (m, 1H, AreH), 7.90e7.96 (m, 1H, AreH), 7.70e7.75
ꢀ
1
2
a (86%) as a white solid, m.p. 142e143 C. H NMR (250 MHz,
CDCl
¼ 8.39 (d, 1H, J ¼ 7.9, AreH), 7.80 (d, 1H, J ¼ 7.9, AreH), 7.65
t, J ¼ 7.9, 1H, AreH), 4.80 (br, 1H, NH), 4.35 (t, J ¼ 6.0, 2H, NeCH ),
.71 (s, 3H, NeCH ), 3.39 (m, 2H, NHeCH ), 2.05 (m, 2H,
CH eCH eCH ), 1.43 (s, 9H, 3CH ). C NMR (62.9 MHz, CDCl
¼ 157.48, 155.94, 148.25, 135.71, 131.70, 131.43, 130.52, 126.25,
22.00, 78.99 ((CH C), 65.14 (NeCH ), 38.62 (NeCH ), 37.99
NHeCH ), 28.83 (CH eCH eCH ), 28.31 ((CH C). HR-EIMS: m/z
367.1312 [M] ; calcd for C17 22ClN , 367.1299.
(
m, 2H, AreH), 5.1 (br, 1H, NH), 4.33 (t, J ¼ 4.9, 2H, NeCH
2
), 3.68 (s,
), 1.43 (s, 9H, 3CH ). C NMR
2 3
3
) d
1
3
3
H, NeCH
3
), 3.59 (m, 2H, NHeCH
¼ 158.58, 155.77, 149.27, 132.39, 131.74, 128.80,
26.81, 124.37, 123.21, 79.42 ((CH C), 66.12 (NeCH ), 39.65
NHeCH ), 38.63 (NeCH ), 28.25 ((CH C). HR-EIMS: m/z 319.1523
M] ; calcd for C16 , 319.1532.
(
3
2
(
3
62.9 MHz, CDCl ) d
3
2
1
(
[
3
)
3
2
13
2
2
2
3
3
)
2
3
3 3
)
d
þ
ꢂ
21 3 4
H N O
1
(
3
)
3
2
3
2
2
2
2
3 3
)
þ
ꢂ
4
.1.4. 2-[3-(tert-Butoxycarbonyl)amine]propyl-2,3-dihydro-3-
H
3 4
O
methyl-1,4-phthalazinedione (2b)
The compound 2b was obtained from 1a and N-Boc-3-
bromopropylamine in a similar manner as for the preparation of
4.1.9. 2-(2-Aminoethyl)-2,3-dihydro-3-methyl-1,4-
phthalazinedione (3a)
ꢀ
1
2
a (83%), as a white solid, m.p. 153e154 C. H NMR (250 MHz,
CDCl
¼ 8.35e8.41 (m, 1H, AreH), 7.91e8.00 (m, 1H, AreH),
.74e7.78 (m, 2H, AreH), 4.80 (br, 1H, NH), 4.36 (t, J ¼ 6.0, 2H,
NeCH ), 3.72 (s, 3H, CH ), 3.34 (m, 2H, NHeCH ), 2.04 (m, 2H,
CH eCH eCH )), 1.43 (s, 9H, 3CH ). C NMR (62.9 MHz, CDCl
¼ 158.55, 155.86, 149.47, 132.39, 131.67, 128.76, 126.76, 124.49,
23.17, 79.49 ((CH C), 64.46 (NeCH ), 38.60 (NeCH ), 37.74
NHeCH ), 29.03 (CH eCH eCH ), 28.26 (3CH ). HR-EIMS: m/z
33.1685 [M] ; calcd for C17 , 333.1689.
To a solution of 2a (0.10 g, 0.31 mmol) in CH
2
Cl
2
(25 ml) stirring
ꢀ
3
)
d
at 0 C, sat. HCl/Et
2
O (2 ml) was added. Resulted solution was kept
ꢀ
7
to 0 C for 30 min. Then, it was carried out to room temperature and
stirred for 1 h. After the reaction, ion exchange Ambersep 900 OH
resin (3 g) was added and kept under stirring for another 30 min.
Finally, it was filtered and solvent was evaporated under vacuum.
2
3
2
13
(
2
2
2
3
3
)
d
1
3
)
3
2
3
The residue was purified by FC (CH
affording 3a (0.06 g, 88% yield) as a white solid, m.p. 100e101 C. H
NMR (250 MHz, CDCl
¼ 8.40 (d, J ¼ 9.3, 1H, AreH), 7.98 (d,
2 2
Cl /MeOH 90:10, 85:15)
ꢀ
1
(
3
2
2
2
H
2
3
þ
ꢂ
23
N
3
O
4
3
) d

414
J. Munín et al. / European Journal of Medicinal Chemistry 82 (2014) 407e417
J ¼ 9.3, 1H, AreH), 7.77 (m, 2H, AreH), 4.34 (t, J ¼ 5.3, 2H, NeCH
2
),
), 1.25 (br, 2H,
¼ 158.71, 149.60, 132.48, 131.80,
), 41.09 (NH eCH ),
). HR-EIMS: m/z 219.1003 [M] ; calcd for C11
CDCl
121.83, 64.79 (NeCH
(CH eCH eCH ). HR-EIMS: m/z 267.0777 [M] ; calcd for
14ClN , 267.0775.
3
)
d
¼ 157.59, 148.43, 135.80, 131.79, 131.10, 130.47, 126.00,
3
.72 (s, 3H, NeCH
3
), 3.30 (t, J ¼ 5.3, 2H, NH
2
eCH
2
2
), 38.61 (NeCH ), 38.50 (NH eCH ), 30.92
3
2
2
13
þ
ꢂ
NH
2
). C NMR (62.9 MHz, CDCl
3
)
d
2
H
2
2
1
28.93, 126.93, 124.63, 123.21, 69.18 (NeCH
2
2
2
C
12
3 2
O
þ
ꢂ
3
8.74 (NeCH
3
13 3 2
H N O ,
219.1008.
4.1.15. 2-(2-Bromoethyl)-2,3-dihydro-3-methyl-1,4-
phthalazinedione (4a)
4.1.10. 2-(3-Aminopropyl)-2,3-dihydro-3-methyl-1,4-
2 3
A mixture of 1a (0.20 g, 1.14 mmol) and K CO (0.24 g,
.73 mmol) in anhydrous DMF (10 ml) was kept under argon and
phthalazinedione (3b)
1
The compound 3b was obtained from 2b, following the previ-
ously described procedure to obtain 3a, (87%) as a white solid, m.p.
stirring for 2 h at room temperature. Then, 1,2-dibromoethane
(0.15 ml, 0.32 g, 1.70 mmol) was added and resulted solution was
ꢀ
1
9
7e98 C. H NMR (250 MHz, CDCl
.91 (d, J ¼ 9.0, 1H, AreH), 7.72 (m, 2H, AreH), 4.35 (t, J ¼ 6.8, 2H,
NeCH ), 3.69 (s, 3H, NeCH eCH ), 1.97 (m,
), 2.91 (t, J ¼ 6.8, 2H, NH
eCH eCH ), 1.72 (br, 2H, NH ). C NMR (62.9 MHz, CDCl
¼ 158.60, 149.62, 132.40, 131.66, 128.76, 126.75, 124.49, 123.16,
4.55 (NeCH ), 39.01 (NH eCH ), 38.66 (NeCH ), 32.43
). HR-EIMS: m/z 233.1167 [M] ; calcd for
233.1164.
3
)
d
¼ 8.35 (d, J ¼ 9.0, 1H, AreH),
ꢀ
stirred for 2 h at 125 C. The reaction mixture was then cooled to
7
room temperature, filtered and washed with anhydrous acetone
several times. Solvent was removed under vacuum and residue was
purified by FC (hexane/AcOEt 85:15), affording 4a (0.10 g, 32% yield)
2
3
2
2
13
2
d
H, CH
2
2
2
2
3
)
ꢀ
1
3
as a white solid, m.p. 105e106 C. H NMR (250 MHz, CDCl )
6
2
2
2
3
þ
ꢂ
d
¼ 8.40 (d, J ¼ 9.7, 1H, AreH), 8.00 (d, J ¼ 9.7, 1H, AreH), 7.79 (m,
H, AreH), 4.63 (t, J ¼ 6.0, 2H, NeCH ), 3.75 (t, J ¼ 6.0, 2H, BreCH ),
). C NMR (62.9 MHz, CDCl
¼ 158.66, 148.82,
), 38.71
). HR-EIMS: m/z 281.9993 [M] ; calcd for
, 282.0004.
(
CH
2
2
eCH eCH
2
2
3
1
(
C
2
2
C
12
15
H N
3
O
2
13
.70 (s, 3H, NeCH
3
3
) d
32.57, 131.92, 128.88, 126.84, 124.27, 123.30, 66.09 (NeCH
2
4.1.11. 2-(2-Aminoethyl)-5-chloro-2,3-dihydro-3-methyl-1,4-
þ
ꢂ
NeCH
3
), 28.90 (BreCH
2
phtalazinedione (3c)
The compound 3c was obtained from 2c following the previ-
ously described procedure to obtain 3a, (85%) as a white solid, m.p.
11
2 2
H11BrN O
ꢀ
1
1
27e128 C. H NMR (250 MHz, CDCl
AreH), 7.80 (d, J ¼ 7.9, 1H, AreH), 7.70 (t, J ¼ 7.9, 1H, AreH), 4.37 (t,
J ¼ 5.2, 2H, NeCH ), 3.70 (s, 3H, NeCH ), 3.16 (t, J ¼ 5.2, 2H,
NH eCH ), 1.26 (br, 2H, NH C NMR (62.9 MHz, CDCl
¼ 156.93, 148.62, 134.87, 134.51, 132.77, 126.76, 124.42, 122.30,
8.25 (NeCH ), 40.05 (NH eCH ), 38.97 (NeCH ). HR-EIMS: m/z
53.0617 [M] ; calcd for C11 12ClN , 253.0618.
3
)
d
¼ 7.99 (d, J ¼ 7.9, 1H,
4
.1.16. 2-(3-Bromopropyl)-2,3-dihydro-3-methyl-1,4-
phthalazinedione (4b)
The compound 4b was obtained from 1a and 1,3-
dibromopropane in a similar manner as for the preparation of 4a
but it was purified by FC using hexane/AcOEt 8:2, (46%) as a white
2
3
13
2
2
2
).
3
)
d
6
2
2
2
2
3
ꢀ
1
þ
ꢂ
solid, m.p. 86e87 C. H NMR (250 MHz, CDCl
H, AreH), 7.93 (d, J ¼ 9.3, 1H, AreH), 7.76 (m, 2H, AreH), 4.45 (t,
J ¼ 5.9, 2H, NeCH ), 3.73 (s, 3H, NeCH ), 3.62 (t, J ¼ 6.5, 2H,
BreCH ), 2.42 (m, 2H, CH ). C NMR (62.9 MHz, CDCl
¼ 158.64, 149.28, 132.45, 131.77, 128.94, 126.92, 124.54, 123.12,
4.39 (NeCH ), 38.70 (NeCH ), 31.84 (CH eCH eCH ), 29.63
). HR-EIMS m/z 296.0167 [M] ; calcd for C12 13BrN
96.0160.
3
)
d
¼ 8.39 (d, J ¼ 9.3,
H
3 2
O
1
2
3
4.1.12. 2-(3-Aminopropyl)-5-chloro-2,3-dihydro-3-methyl-1,4-
1
3
2
2
eCH
2
eCH
2
3
)
phthalazinedione (3d)
d
The compound 3d was obtained from 2d following the previ-
ously described procedure to obtain 3a, (82%) as a white solid, m.p.
6
(
2
2
3
2
2
2
þ
ꢂ
BreCH
2
H
2 2
O ,
ꢀ
1
1
11e112 C. H NMR (250 MHz, CDCl
3
)
d
¼ 7.90 (d, J ¼ 7.8,1H, AreH),
7
2
.74 (d, J ¼ 7.8, 1H, AreH), 7.63 (t, J ¼ 7.8, 1H, AreH), 4.37 (t, J ¼ 6.2,
H, NeCH
2
), 3.69 (s, 3H, NeCH
eCH eCH ), 1.33 (br, 2H, eNH
3
), 2.94 (t, J ¼ 6.2, 2H, NH eCH ),
2
2
13
2
.00 (m, 2H, CH
2
2
2
2
). C NMR
¼ 156.80, 148.59, 134.78, 134.74, 132.53, 127.18,
24.85, 122.40, 64.81 (NeCH ), 39.09 (NeCH ), 39.06 (NH eCH ),
). HR-EIMS: m/z 267.0767 [M] ; calcd for
, 267.0775.
4
.1.17. 2-(2-Bromoethyl)-5-chloro-2,3-dihydro-3-methyl-1,4-
phthalazinedione (4c)
The compound 4c was obtained from 1b and 1,2-dibromoetane
in a similar manner as for the preparation of 4a but it was purified
by FC using hexane/AcOEt 9:1, (60%) as a white solid, m.p.
(
3
62.9 MHz, CDCl ) d
1
3
C
2
3
2
2
þ
ꢂ
2.50 (CH
2 2 2
eCH eCH
12
H14ClN O
3 2
ꢀ
1
1
10e111 C. H NMR (250 MHz, CDCl
AreH), 7.77 (d, J ¼ 7.9, 1H, AreH), 7.66 (t, J ¼ 7.9, 1H, AreH), 4.62 (t,
J ¼ 5.9, 2H, NeCH ), 3.74 (t, J ¼ 5.9, 2H, BreCH ), 3.69 (s, 3H,
). C NMR (62.9 MHz, CDCl
¼ 156.87, 147.84, 135.03,
34.93, 132.75, 126.85, 124.95, 122.52, 66.30 (NeCH ), 39.13
). HR-EIMS: m/z 315.9619 [M] ; calcd for
, 315.9614.
3
)
d
¼ 7.95 (d, J ¼ 7.9, 1H,
4.1.13. 3-(2-Aminoethyl)-5-chloro-2,3-dihydro-2-methyl-1,4-
phthalazinedione (3e)
2
2
The compound 3e was obtained from 2e following the previ-
ously described procedure to obtain 3a, (70%) as a white solid, m.p.
13
NeCH
3
3
) d
1
(
C
2
ꢀ
1
þ
ꢂ
5
4e55 C. H NMR (250 MHz, CDCl
.82 (d, J ¼ 7.9, 1H, AreH), 7.67 (t, J ¼ 7.9, 1H, AreH), 4.33 (t, J ¼ 5.1,
H, NeCH ), 3.71 (s, 3H, NeCH eCH ), 1.25
), 3.16 (t, J ¼ 5.1, 2H, NH
br, 2H, NH ). C NMR (62.9 MHz, CDCl
¼ 157.64, 148.31, 135.85,
31.92, 131.24, 130.36, 126.17, 121.82, 68.69 (NeCH ), 40.25
3
)
d
¼ 8.35 (d, J ¼ 7.9, 1H, AreH),
NeCH
H
3 2
), 28.83 (BreCH
2 2
10BrClN O
7
2
(
1
11
2
3
2
2
13
2
3
) d
2
4.1.18. 2-(3-Bromopropyl)-5-chloro-2,3-dihydro-3-methyl-1,4-
phthalazinedione (4d)
The compound 4d was obtained from 1b and 1,3-
dibromopropane in a similar manner as for the preparation of 4c
þ
ꢂ
(
C
NH
2
eCH
12ClN
2
), 38.64 (NeCH
, 253.0618.
3
). HR-EIMS: m/z 253.0617 [M] ; calcd for
11
H
3
O
2
ꢀ
1
4.1.14. 3-(3-Aminopropyl)-5-chloro-2,3-dihydro-2-methyl-1,4-
(86%) as a white solid, m.p. 115e116 C. H NMR (250 MHz, CDCl
¼ 7.89 (d, J ¼ 7.9, 1H, AreH), 7.76 (d, J ¼ 7.9, 1H, AreH), 7.64 (t,
J ¼ 7.9, 1H, AreH), 4.44 (t, J ¼ 5.9, 2H, NeCH ), 3.70 (s, 3H, NeCH ),
3.61 (t, J ¼ 6.5, 2H, BreCH ), 2.40 (m, 2H, CH ). C NMR
(62.9 MHz, CDCl
¼ 156.88, 148.29, 134.97, 134.91, 132.63, 127.06,
124.96, 122.33, 64.62 (NeCH ), 39.17 (NeCH ), 31.73
(CH eCH eCH ), 29.60 (BreCH ). HR-EIMS: m/z 329.9770 [M] ;
calcd for C12 12BrClN , 329.9771.
3
)
phthalazinedione (3f)
d
The compound 3f was obtained from 2f following the previously
described procedure to obtain 3a, (68%) as a white solid, m.p.
2
3
13
2
2 2 2
eCH eCH
ꢀ
1
7
9e80 C. H NMR (250 MHz, CDCl
3
)
d
¼ 8.32 (d, J ¼ 7.8, 1H, AreH),
3
) d
7
.79 (d, J ¼ 7.8, 1H, AreH), 7.51 (t, J ¼ 7.8, 1H, AreH), 4.36 (t, J ¼ 5.9,
2
3
þ
ꢂ
2
H, NeCH
2
), 3.70 (s, 3H, NeCH
eCH eCH ),1.25 (br, 2H, NH
3
), 3.03 (t, J ¼ 5.9, 2H, NH eCH ),
2
2
2
2
2
2
13
2
.05 (m, 2H, CH
2
2
2
2
). C NMR (62.9 MHz,
H
2 2
O

J. Mun
ín et al. / European Journal of Medicinal Chemistry 82 (2014) 407e417
415
4
.1.19. 3-(2-Bromoethyl)-5-chloro-2,3-dihydro-2-methyl-1,4-
122.59, 65.86 (NeCH
m/z 279.0519 [M] ; calcd for C11
2
), 49.88 (N
3
eCH
2
), 39.17 (NeCH
, 279.0523.
3
). HR-EIMS:
þ
ꢂ
phthalazinedione (4e)
H10ClN O
5 2
The compound 4e was obtained from 1c and 1,2-dibromoethane
in a similar manner as for the preparation of 4c (17%) as a white
4.1.24. 2-(3-Azidopropyl)-5-chloro-2,3-dihydro-3-methyl-1,4-
phthalazinedione (5d)
The compound 5d was obtained from 4d following the previ-
ously described procedure to obtain 5b and after purification by FC
ꢀ
1
solid, m.p. 89e90 C. H NMR (250 MHz, CDCl
1
3
)
d
¼ 8.37 (d, J ¼ 7.8,
H, AreH), 7.80 (d, J ¼ 7.8, 1H, AreH), 7.65 (t, J ¼ 7.8, 1H, AreH), 4.62
), 3.71 (s, 3H,
¼ 157.65, 147.69, 135.90,
), 38.70
(
t, J ¼ 6.0, 2H, NeCH
2
), 3.75 (t, J ¼ 6.0, 2H, BreCH
2
13
ꢀ
1
NeCH
3
). C NMR (62.9 MHz, CDCl
3
)
d
using hexane/AcOEt 9:1 (67%) as a white solid, m.p. 115e116 C. H
NMR (250 MHz, CDCl
¼ 7.89 (d, J ¼ 7.9,1H, AreH), 7.76 (d, J ¼ 7.9,
1H, AreH), 7.65 (t, J ¼ 7.9, 1H, AreH), 4.39 (t, J ¼ 6.1, 2H, NeCH ),
3.70 (s, 3H, NeCH eCH ), 2.14 (m, 2H,
), 3.54 (t, J ¼ 6.6, 2H, N
¼ 156.86, 148.29,
134.95, 134.90, 132.63, 127.05, 124.94, 122.31, 63.83 (NeCH ), 48.29
(N eCH ), 39.14 (NeCH ), 28.13 (CH eCH eCH ). HR-EIMS: m/z
293.0679 [M] ; calcd for C12 12ClN , 293.068.
131.96, 131.57, 130.91, 126.26, 121.91, 66.73 (NeCH
2
3
) d
þ
ꢂ
(
NeCH
3
), 28.55 (BreCH
2
). HR-EIMS: m/z 315.9609 [M] ; calcd for
2
C
11
H
10BrClN
2
O
2
, 315.9614.
3
3
2
13
2 2 2 3
CH eCH eCH ). C NMR (62.9 MHz, CDCl ) d
4.1.20. 3-(3-Bromopropyl)-5-chloro-2,3-dihydro-2-methyl-1,4-
2
phthalazinedione (4f)
The compound 4f was obtained from 1c and 1,3-
dibromopropane in a similar manner as for the preparation of 4c
3
2
3
2
2
2
þ
ꢂ
H
5 2
O
ꢀ
1
(
d
80%) as a white solid, m.p. 113e114 C. H NMR (250 MHz, CDCl
¼ 8.38 (d, J ¼ 7.9, 1H, AreH), 7.79 (d, J ¼ 7.9, 1H, AreH), 7.64 (t,
J ¼ 7.9, 1H, AreH), 4.42 (t, J ¼ 5.7, 2H, NeCH ), 3.72 (s, 3H, NeCH ),
.70 (t, J ¼ 6.4, 2H, BreCH ), 2.40 (m, 2H, CH ). C NMR
62.9 MHz, CDCl
¼ 157.61, 148.16, 135.80, 131.82, 131.59, 130.56,
26.37, 122.08, 64.75 (NeCH ), 38.73 (NeCH ), 31.72
CH eCH eCH ), 30.24 (BreCH ). HR-EIMS: m/z 329.9755 [M] ;
calcd for C12 12BrClN , 329.9771.
3
)
4.1.25. 3-(2-Azidoethyl)-5-chloro-2,3-dihydro-2-methyl-1,4-
phthalazinedione (5e)
The compound 5e was obtained from 4e following the previ-
ously described procedure to obtain 5c (56%) as a white solid, m.p.
2
3
13
3
(
1
(
2
2 2 2
eCH eCH
ꢀ
1
3
)
d
106e107 C. H NMR (250 MHz, CDCl
3
)
d
¼ 8.37 (d, J ¼ 7.9, 1H,
2
3
AreH), 7.80 (d, J ¼ 7.9, 1H, AreH), 7.65 (t, J ¼ 7.9, 1H, AreH), 4.44 (t,
þ
ꢂ
13
2
2
2
2
J ¼ 5.0, 2H, NeCH
(62.9 MHz, CDCl
26.26, 121.87, 65.33 (NeCH
2
), 3.72 (m, 5H, NeCH
¼ 157.59, 147.80, 135.86, 131.94, 131.51, 130.71,
), 49.84 (N eCH ), 38.65 (NeCH ). HR-
EIMS: m/z 279.0517 [M] ; calcd for C11 10ClN , 279.0523.
3
þ N
3 2
eCH ). C NMR
H
2
O
2
3
) d
1
2
3
2
3
þ
ꢂ
4
.1.21. 2-(2-Azidoethyl)-2,3-dihydro-3-methyl-1,4-
phthalazinedione (5a)
To a stirred solution of 4a (0.10 g, 0.35 mmol) in DMSO (2 ml)
H
5 2
O
4.1.26. 3-(3-Azidopropyl)-5-chloro-2,3-dihydro-2-methyl-1,4-
were added NaN
3
(0.03 g, 0.46 mmol) and NaI (5 mg, 0.03 mmol).
phthalazinedione (5f)
ꢀ
Then, the mixture was kept under stirring for 3 h at 100 C. After
cooling, water was added (20 ml) and the mixture was extracted
with ether (4 ꢁ 20 ml). The ether extracts were washed with brine
The compound 5f was obtained from 4f following the previously
described procedure to obtain 5c (67%) as a white solid, m.p.
ꢀ
1
61e62 C. H NMR (250 MHz, CDCl
7.79 (d, J ¼ 7.9, 1H, AreH), 7.64 (t, J ¼ 7.9, 1H, AreH), 4.37 (t, J ¼ 5.7,
2H, NeCH ), 3.72 (s, 3H, NeCH eCH ), 2.13
), 3.61 (t, J ¼ 6.7, 2H, N
(m, 2H, CH ). C NMR (62.9 MHz, CDCl
¼ 157.55,
148.10, 135.77,131.79,131.55,130.50,126.33, 122.03, 63.89 (NeCH ),
48.37 (N eCH ), 38.67 (NeCH ), 28.11 (CH eCH eCH ). HR-EIMS:
m/z 293.0690 [M] ; calcd for C12 12ClN , 293.068.
3
)
d
¼ 8.38 (d, J ¼ 7.9, 1H, AreH),
(
3 ꢁ 10 ml) and dried over Na
2 4
SO . The solvent was removed under
vacuum and the crude oil obtained was purified by FC (hexane/
AcOEt 90:10e85:15), affording 5a (0.08 g, 93% yield) as a white
2
3
3
2
13
2
eCH
2
eCH
2
3
) d
ꢀ
1
solid, m.p.115e116 C. H NMR (250 MHz, CDCl
H, AreH), 7.95 (d, J ¼ 9.2, 1H, AreH), 7.78 (m, 2H, AreH), 4.50 (t,
J ¼ 5.0, 2H, NeCH ), 3.73 (s, 3H, NeCH ), 3.67 (t, J ¼ 5.0, 2H,
). C NMR (62.9 MHz, CDCl
¼ 158.61, 148.93, 132.60,
31.87, 128.82, 126.79, 124.17, 123.30, 65.64 (NeCH ), 49.82
3
)
d
¼ 8.39 (d, J ¼ 9.2,
2
1
3
2
3
2
2
2
þ
ꢂ
2
3
H
5 2
O
13
N
3
eCH
2
3
) d
1
2
4.1.27. General procedure for reduction from azides (5aef) to
amines (3aef)
þ
ꢂ
(
C
N
3
eCH
2
), 38.62 (NeCH
, 245.0913.
3
). HR-EIMS: m/z 245.0910 [M] ; calcd for
11
H
11
N
5
O
2
Triphenylphosphine (0.75 mmol, 74 mg) was added to a solution
of the corresponding azide derivatives 5aef (0.5 mmol) in dry
methanol (7 ml). Reaction mixture was heated under refluxing at
4.1.22. 2-(3-Azidopropyl)-2,3-dihydro-3-methyl-1,4-
ꢀ
phthalazinedione (5b)
80 C for 1 h. After completion of the reaction, it was cooled to room
Following the previously described procedure to obtain 5a, but
keeping the stirring for 24 h, reaction of 4b afforded 5b (100%) as a
temperature and the solvent was removed under vacuum. The
residue was purified by FC (CH
2 2
Cl /MeOH 90:10; 85:15), to obtain
ꢀ
1
white solid, m.p. 62e63 C. H NMR (250 MHz, CDCl
J ¼ 9.2, 1H, AreH), 7.92 (d, J ¼ 9.2, 1H, AreH), 7.76 (m, 2H, AreH),
), 3.72 (s, 3H, NeCH
3
)
d
¼ 8.38 (d,
3a (82%), 3b (84%), 3c (73%), 3d (70%), 3e (76%), 3f (71%). Chemical
1
13
structures of these compounds were confirmed by H and C RMN
experiments, obtaining similar spectra for the compounds 3aef to
those obtained by deprotecting of compounds 2aef.
4
.39 (t, J ¼ 6.1, 2H, NeCH
2
3
), 3.54 (t, J ¼ 6.7, 2H,
13
N
3
eCH
2
), 2.13 (m, 2H, CH
2
eCH
2
eCH
2
). C NMR (62.9 MHz, CDCl
¼ 158.49, 149.17, 132.35, 131.64, 128.78, 126.75, 124.39, 123.01,
3.48 (NeCH ), 48.21 (N eCH ), 38.56 (NeCH ), 28.07
). HR-EIMS: m/z 259.1060 [M] ; calcd for
, 259.1069.
3
)
d
6
2
3
2
3
4.2. Animals
þ
ꢂ
(
CH
2
eCH
2
eCH
2
C
12
13
H N
5
O
2
The animals used throughout this study (Male WistareKyoto
(WKY) rats (Iffa-Credo)), purchased from Criffa (Barcelona, Spain)
4
.1.23. 2-(2-Azidoethyl)-5-chloro-2,3-dihydro-3-methyl-1,4-
were housed, cared for and acclimatized (before the experiments)
as indicated previously [49].
phthalazinedione (5c)
The compound 5c was obtained from 4c, following the previ-
ously described procedure to obtain 5a and after purification by FC
4.2.1. Ethical approval
ꢀ
1
using hexane/AcOEt 9:1, (75%) as a white solid, m.p. 112e113 C. H
NMR (250 MHz, CDCl
All experiments were carried out in accordance with European
regulations on the protection of animals (Directive 2010/63/UE),
the Spanish Real Decreto 53/2013 (1 February) and/or the Guide for
the Care and Use of Laboratory Animals as adopted and promul-
gated by the US.
3
)
d
¼ 7.93 (d, J ¼ 7.8,1H, AreH), 7.78 (d, J ¼ 7.8,
H, AreH), 7.67 (t, J ¼ 7.8, 1H, AreH), 4.49 (t, J ¼ 5.0, 2H, NeCH ),
). C NMR
¼ 156.99, 148.08, 135.11, 135.04, 132.86, 126.85,
1
3
2
13
.70 (s, 3H, NeCH
3
), 3.67 (t, J ¼ 5.0, 2H, N
3 2
eCH
(
3
62.9 MHz, CDCl ) d

416
J. Mun
í
n et al. / European Journal of Medicinal Chemistry 82 (2014) 407e417
4
.2.2. Vasorelaxant activity
Vasorelaxant activity of newly synthesized compounds was
studied using thoracic aortic rings of Wistar rats precontracted
with PE (1 M). Male Wistar rats weighing 300e400 g were killed
solution was removed and 100 ml of dimethyl sulfoxide was added
to dissolve the crystals formed. Then, absorbance at 540 nm was
read using a microplate reader. The percentage cell viability was
m
calculated
as
[Absorbance(treatment)/Absorbance(negative
by a blow to the head and exsanguinated. The thoracic aorta was
rapidly dissected and transferred to a Petri dish with Krebs bicar-
bonate solution (KBS composition: 119 mM NaCl, 4.7 mM KCl,
control)] ꢁ 100%.
Acknowledgments
1
.5 mM CaCl
2 2 4 2 3
$2H O, 1.2 mM MgSO $7H O, 25 mM NaHCO ,
0
.03 mM EDTA-Na
2
, 11 mM glucose; pH ¼ 7.4). Excess of fat and
We are grateful to the Xunta de Galicia (10PXIB203303PR) for
financial support. Elías Quezada thanks Xunta de Galicia for the
Angeles Alvari n~ o fellowship.
connective tissue was removed and for the endothelium free ex-
periments was made a scraping of the arterial lumen with thick
cotton thread. The aorta was cut into rings (4e5 mm in length,
0
.9e1.0 mm in thickness) and each ring was placed in a 10 ml
ꢀ
References
organ-bath in KBS, maintained at 37 C and bubbled with carbogen.
Each aortic ring was mounted between two stainless steel hooks
passed through its lumen. The lower hook was fixed between two
plates and the upper one was attached to a force displacement
transducer (Pioden Controls Ltd., Canterbury, UK) to record the
isometric tension with a MacLab/16s and Chart v.3.6.3 software or a
PowerLab/8sp and Chart v.4.1.2 software Data Acquisition system
[
1] A.E. Kümmerle, J.M. Raimundo, C.M. Leal, G.S. daSilva, T.L. Balliano,
M.A. Pereira, C.A. de Simone, R.T. Sudo, G. Zapata-Sudo, C.A.M. Fraga,
E.J. Barreiro, Studies towards the identification of putative bioactive confor-
mation of potent vasodilator arylidene N-acylhydrazone derivatives, Eur. J.
Med. Chem. 44 (2009) 4004e4009.
[
[
2] A.S. Go, D. Mozaffarian, V.L. Roger, E.J. Benjamín, J.D. Berry, W.B. Borden, Heart
disease and stroke statistics-2013 update. A report from the American Heart
Association, Circulation 127 (2013) e6ee245.
3] C. Berry, M.J. Brosnan, J. Fennell, C.A. Hamilton, A. Dominiczak, Oxidative
stress and vascular damage in hypertension, Curr. Opin. Neph. Hypertens. 10
(2001) 247e255.
(
AD Instruments Castle Hill, Australia).
Preparations were equilibrated at a resting tension of 2 g for at
least 1 h. Thereafter, isometric contractions were induced by the
addition of PE (1 M). Once the contraction stabilized, a single
concentration of ACh (1 M) was added to the bath in order to
assess the endothelial integrity of the preparations. Endothelium
was considered to be intact when this drug elicited a vasorelaxation
[
[
4] B. Lass eꢁ gue, K. Griendling, Reactive oxygen species in hypertension. An up-
m
date, Am. J. Hypertens. 17 (2004) 852e860.
m
5] S. Cuzzocrea, E. Mazzon, L. Dugo, R. Di Paola, A.P. Caputti, D. Salvemini, Su-
peroxide: a key player in hypertension, FASEB J. 18 (2004) 94e101.
[6] Z.S. Katusic, Superoxide anion and endothelial regulation of arterial tone, Free
Radic. Biol. Med. 20 (1996) 443e448.
>
50% of the maximal contraction obtained in vascular rings pre-
[7] M.E. Ernst, M. Moser, Use of diuretics in patients with hypertension, N. Engl. J.
contracted with PE. The absence of ACh relaxant action in the
vessels indicated the total removal of endothelial cells. After
assessing the integrity of the endothelium, vascular tissues were
allowed to recuperate for 1 h, during which the physiological so-
lution was replaced every 15 min, before any experiment protocol
was started.
Med. 361 (2009) 2153e2164.
[8] R. Donnelly, Angiotensin-converting enzyme inhibitors and coronary heart
disease prevention, J. Renin Angiotensin Aldosterone Syst. 8 (2007) 13e22.
[
9] H.T. Ong, Are angiotensin-converting enzyme inhibitors and angiotensin re-
ceptor blockers especially useful for cardiovascular protection? J. Am. Board
Fam. Med. 22 (2009) 686e697.
[
10] F.H. Messerli, S. Bangalore, F. Ruschitzka, Angiotensin receptor blockers:
baseline therapy in hypertension? Eur. Heart J. 30 (2009) 2427e2430.
11] M. Pahor, B.M. Psaty, W.B. Alderman, W.B. Applegate, J.D. Williamson,
C. Cavazzini, C.D. Furberg, Health outcomes associated with calcium antago-
nists compared with other first-line antihypertensive therapies: a meta-
analysis of randomized controlled trials, Lancet 356 (2000) 1949e1954.
12] B. Szabo, Imidazoline antihypertensive drugs: a critical review on their
mechanism of action, Pharmacol. Ther. 93 (2002) 1e35.
After equilibration aortic rings were contracted by single con-
[
centration of PE (1
pounds of series 1e5 were added in progressively increasing
cumulative concentrations (10 nM-200 M) at 10 min intervals.
mM). Once the contractions stabilized, com-
m
[
[
Only one compound was tested in each ring. All compounds were
initially dissolved in DMSO to prepare a 100 mM stock solution.
Further solutions were made in KBS. Control experiments were
performed in presence of DMSO alone, at the same concentration as
those used with the derivatives tested, which demonstrated that
the solvent did not affect the contractile response of isolated aorta.
All the results are expressed as means ± the standard error of
the mean (S.E.M). The response of the aortic rings to the all com-
pounds was expressed as a percentage of the initial contraction to
13] L.H. Lindholm, B. Carlberg, O. Samuelsson, Should beta blockers remain first
choice in the treatment of primary hypertension? A meta-analysis, Lancet 366
(
2005) 1545e1553.
[14] L. Kalinowski, L.W. Dobrucki, M. Szczepanska-Konkel, M. Jankowski,
L. Martyniec, S. Angielski, T. Malinski, Third-generation -blockers stimulate
nitric oxide release from endothelial cells through ATP efflux, Circulation 107
2003) 2747e2752.
b
(
[
15] A.T. Remaley, Old drug, new tricks: the unexpected effect of doxazosin on
high-density lipoprotein, Circ. Res. 101 (2007) 116e118.
16] J. Handzlik, D. Maciag, M. Kubacka, S. Mogliski, B. Filipek, K. Stadnicka, K. Kiec-
Kononowicz, Synthesis, alpha 1-adrenoceptor antagonist activity, and SAR
study of novel arylpiperazine derivatives of phenytoin, Bioorg. Med. Chem. 16
(2008) 5982e5998.
17] V. Alabaster, S. Campbell, J. Danilewicz, C. Greengrass, R.M. Plews, 2,4-
Diamino-6,7-dimethoxyquinazolines. 2. 2-(4-Carbamoylpiperidino) de-
rivatives as alpha 1-adrenoceptor antagonists and antihypertensive agents,
J. Med. Chem. 30 (1987) 999e1003.
[
1 mM PE. Dose-response curves were analyzed by a sigmoidal curve
fitting analysis to give EC50 values (50% of the Emax) obtained in the
presence of blockers and the Emax value (the maximal effect).
[
4.2.3. Cell proliferation assay
[
[
18] H. Lepor, Role of alpha-adrenergic blockers in the treatment of benign pros-
tatic hyperplasia, Prostate (Suppl. 3) (1990) 75e84.
19] M.J. Moina, B. Bard aꢀ n, M. Campos-Toimil, A.F. Alzueta, J. Gil-Longo, F. Orallo,
A7r5, a vascular smooth muscle cell line obtained from em-
bryonic rat aorta (American Type Culture Collection, CRL-1444,
Rockville, MD, USA) were grown and kept in culture as described
elsewhere [50].
Effects of hydralazine on contractile responses to alpha 1- and alpha 2-
adrenoceptor agonists in isolated rubbed rat aorta, Gen. Pharmacol. 25
(1994) 165e172.
3
2
For experiments A7r5 cells (20 ꢁ 10 /cm ) were seeded in each
[
[
20] T. Ogiso, M. Iwaki, N. Ohtsuki, Pharmacokinetics of formation and excretion of
some metabolites of hydralazine and their hypotensive effect in rats,
J. Pharmacol. Exp. Ther. 233 (1985) 485e490.
21] J.G. Gerber, A.S. Nies, Antihypertensive agents and the drug therapy of hy-
pertension, in: A.G. Gilman, T.W. Rall, A.S. Nies, P. Taylor (Eds.), The Phar-
macological Basis of Therapeutics, Pergamon Press, New York (, 1990, pp.
784e813.
22] H. Gogia, A. Mehra, S. Parikh, M. Raman, J. Ajit-Uppal, J.V. Johnson, U. Elkayam,
Prevention of tolerance to hemodynamic effects of nitrates with concomitant
use of hydralazine in patients with chronic heart failure, J. Am. Coll. Cardiol.
26 (1995) 1575e1580.
well of 96-well culture plate. After overnight incubation, DMSO
(
1%) was added to the cells in the presence or absence of various
concentrations of compounds 1aec, 2aef, 3aef, 4aef and 5aef.
Cells without the addition of compounds were used as negative
control. After further incubation for 24 h, 3-(4,5-dimethyl-thiazol-
[
2
-yl)-2,5-diphenyltetrazolium (MTT) assay was performed to
measure cell viability [51]. Briefly, 10
PBS) was added to each well. After incubation for 2 h at 37 C, MTT
m
l of MTT solution (5 mg/ml in
ꢀ

J. Mun
ín et al. / European Journal of Medicinal Chemistry 82 (2014) 407e417
417
[
23] R.T. Cole, A.P. Kalogeropoulos, V.V. Georgiopoulou, M. Gheorghiade,
A. Quyyumi, C. Yancy, J. Butler, Hydralazine and isosorbide dinitrate in heart
failure: historical perspective, mechanisms and future directions, Circulation
arylpiperazine moiety and cyclic substituents at the pyridazinone nucleus,
Bioorg. Med. Chem. Lett. 13 (2003) 171e173.
[37] D. Vi n~ a, E. del Olmo, J.L. L oꢀ pez-P eꢀ rez, A. San Feliciano, Pyrazolo[3,4,5-de]
1
23 (2011) 2414e2422.
24] A.R. Vest, L.S. Cho, Hypertension in pregnancy, Cardiol. Clin. 30 (2012)
07e423.
phthalazine. Syntheses of a practically unknown heterocyclic system, Tetra-
hedron 65 (2009) 1574e1580.
[
[
4
[38] L.M. Bedoya, E. del Olmo, R. Sancho, B. Barboza, M. Beltr aꢀ n, A.E. García-
25] S. Demirayak, A.C. Karaburun, R. Beis, Some pyrrole substituted aryl pyr-
idazinone and phthalazinone derivatives and their antihypertensive activities,
Eur. J. Med. Chem. 39 (2004) 1089e1095.
Cadenas, S. S ꢀa nchez-Palomino, J.L. L oꢀ pez-P eꢀ rez, E. Mu n~ oz, A. San Feliciano,
J. Alcamí, Anti-HIV activity of stilbene-related heterocyclic compounds, Bio-
org. Med. Chem. Lett. 16 (2006) 4075e4079.
[
[
[
26] E. del Olmo, B. Barboza, M.I. Ybarra, J.L. L oꢀ pez-P eꢀ rez, R. Carr oꢀ n, M.A. Sevilla,
[39] D. Habibi, O. Marvi, Montmorillonite KSF clay as an efficient catalyst for the
synthesis of 1,4-dioxo-3,4-dihydrophthalazine-2(1H)-carboxamides and car-
bothioamides under solvent-free conditions using microwave irradiation,
Catal. Commun. 8 (2007) 127e130.
[40] K. Ling, L.M. Sayre, Discovery of a sensitive, selective, and tightly binding
fluorogenic substrate of bovine plasma amine oxidase, J. Org. Chem. 74 (2009)
339e350.
[41] P. Wipf, H. Kim, Total synthesis of cyclotheonamide A, J. Org. Chem. 58 (1993)
5592e5594.
[42] Z. Tian, S. Xie, Y. Du, Y. Ma, J. Zhao, W. Gao, C. Wang, Synthesis, cytotoxicity
and apoptosis of naphthalimide polyamine conjugates as antitumor agents,
Eur. J. Med. Chem. 44 (2009) 393e399.
C. Boselli, A. San Feliciano, Vasorelaxant activity of phthalazinones and related
compounds, Bioorg. Med. Chem. Lett. 16 (2006) 2786e2790.
27] F.M. Awadallah, W.I. El-Eraky, D.O. Saleh, Synthesis, vasorelaxant activity and
molecular modeling study of new phthalazine derivatives, Eur. J. Med. Chem.
5
2 (2012) 14e21.
28] A. Coelho, E. Ravi n~ a, N. Fraiz, M. Ya n~ ez, R. Laguna, E. Cano, E. Sotelo, Design,
synthesis, and structureeactivity relationships of novel series of 5-
a
alkylidenepyridazin-3(2H)-ones with a non-cAMP-based antiplatelet activ-
ity, J. Med. Chem. 50 (2007) 6476e6484.
29] C. Meyers, M. Ya n~ ez, A. Elmaatougi, T. Verhelst, A. Coelho, N. Fraiz,
G.L.F. Lami eꢁ re, X. García-Mera, R. Laguna, E. Cano, B.U.W. Maes, E. Sotelo, 2-
[
Substituted 4-, 5-, and 6-[(1E)-3-oxo-3-phenylprop-1-en-1-yl]pyridazin-
[43] P.H. Lambert, M. Vaultier, R. Carrig, Application of the intramolecular aza-
Wittig reaction to the synthesis of vinylogous urethanes and amides, J. Org.
Chem. 50 (1985) 5352e5356.
[44] B. Pal, P. Jaisankar, V.S. Gir, Versatile reagent for reduction of azides to amines,
Synth. Commun. 34 (2004) 1317e1323.
[45] A. Cuí n~ as, J. Elíes, F. Orallo, M. Campos-Toimil, Cyclic AMP relaxation of rat
3(2H)-ones and 2-substituted 4,5-bis[(1E)-3-oxo-3-phenylprop-1-en-1-yl]
pyridazin-3(2H)-ones as potent platelet aggregation inhibitors: design, syn-
thesis, and SAR studies, Bioorg. Med. Chem. Lett. 18 (2008) 793e797.
[
30] A. Siddiqui, R. Mishra, M. Shaharyar, Synthesis, characterization and antihy-
pertensive activity of pyridazinone derivatives, Eur. J. Med. Chem. 45 (2010)
2
283e2290.
aortic smooth muscle is mediated in part by decrease of depletion of intra-
cellular Ca2 stores and inhibition of capacitative calcium entry, Vasc. Phar-
þ
[
31] C. Schudt, S. Winder, B. Mueller, D. Ukena, Zardaverine as a selective inhibitor
of phosphodiesterase isozymes, Biochem. Pharmacol. 42 (1991) 153e162.
32] W. von der Saal, J.P. Holck, W. Kampe, A. Mertens, B. Muller-Beckmannf,
Nonsteroidal cardiotonics. 2. The inotropic activity of linear, tricyclic 5-6-5
fused heterocycles, J. Med. Chem. 32 (1989) 1481e1491.
macol. 58 (2013) 98e104.
[46] E. Quezada, G. Delogu, C. Picciau, L. Santana, G. Podda, F. Borges, V. García-
Morales, D. Vi n~ a, F. Orallo, Synthesis and vasorelaxant and platelet anti-
[
aggregatory activities of a new series of 6-halo-3-phenylcoumarins, Molecules
15 (2010) 270e279.
[
33] P. Pollesello, M. Ovaska, J. Kaivola, C. Tilgmann, K. Lundstr o€ m, N. Kalkkinen,
2þ
I. Ulmanen, E. Nissinen, J. Taskinen, Binding of a new Ca sensitizer, levosi-
mendan, to recombinant human cardiac troponin C. A molecular modelling,
fluorescence probe, and proton nuclear magnetic resonance study, J. Biol.
Chem. 269 (1994) 28584e28590.
[47] K. Mori, S. Takeuchi, H. Moritoki, K. Tsuchiya, Y. Nakaya, Matsuoka, Y. Kuroda,
Endothelium-dependent relaxation of rat thoracic aorta by amrinone-induced
nitric oxide release, Eur. Heart J. 17 (1996) 308e316.
[48] A. Galindo, M.L. Vargas, J. García Estan, T. Fuentes, J. Hern ꢀa ndez, Synergistic
0
0
[
[
34] E.N. Amin, A.-A.M. Abdel-Alim, S.G. Abdel-Moty, A.-N.A. El-Shorbagi, M.
Sh Abdel-Rahman, Synthesis of new 4,5-3(2H)pyridazinone derivatives and
their cardiotonic, hypotensive, and platelet aggregation inhibition activities,
Arch. Pharmacol. Res. 33 (2010) 25e46.
35] L. Betti, F. Corelli, M. Floridi, G. Giannaccini, L. Maccari, F. Manetti,
G. Strappaghetti, M. Botta, Alpha 1-adrenoceptor antagonists. 6. Structural
optimization of pyridazinone-arylpiperazines. Study of the influence on af-
finity and selectivity of cyclic substituents at the pyridazinone ring and alkoxy
groups at the arylpiperazine moiety, J. Med. Chem. 46 (2003) 3555e3558.
36] L. Betti, M. Floridi, G. Giannaccini, F. Manetti, G. Strappaghetti, A. Tafi, M. Botta,
alpha 1-Adrenoceptor antagonists. 5. Pyridazinone-arylpiperazines. Probing
the influence on affinity and selectivity of both ortho-alkoxy groups at the
interaction of diazepam with 3 ,5 -cyclic adenosine monophosphate-elevating
agents on rat aortic rings, Eur. J. Pharmacol. 428 (2001) 269e275.
ꢀ
~
ꢀ
ꢀ
[49] F. Orallo, E. Alvarez, M. Camina, J.M. Leiro, E. Gomez, P. Fernandez, The
possible implication of trans-resveratrol in the cardioprotective effects of
long-term moderate wine consumption, Mol. Pharmacol. 61 (2002) 294e302.
[50] M. Campos-Toimil, J. Elíes, F. Orallo, Trans and cis-resveratrol increase cyto-
plasmic calcium levels in A7r5 vascular smooth muscle cells, Mol. Nutr. Food
Res. 49 (2005) 396e404.
[51] T. Mosmann, Rapid colorimetric assay for cellular growth and survival:
application to proliferation and cytotoxicity assays, J. Immunol. Methods 65
(1983) 55e63.
[