Synthesis and vasorelaxant and antiplatelet activities of a new series of (4-Benzylphthalazin-1-ylamino)alcohol derivatives

Article abstract of DOI:10.1007/s00044-017-1879-9

A new series of phthalazine derivatives was synthesized by reaction of phthalic anhydride and different substituted phenylacetic acids to yield the benzyliden-3H-isobenzofuran-1-one intermediates 2a–d. Treatment of them with hydrazine afforded 4-benzyl-2H-phthalazin-1-one derivatives 3a–d, which were substituted with the corresponding aminoalkylalcohol to obtain the (4-benzylphthalazin-1-ylamino)alcohol derivatives 4a–h. In general, these phthalazine derivatives relaxed the contractions produced by phenylephrine both in intact or endothelium-denuded aortic rings. In addition, platelet aggregation induced by thrombin was also inhibited by compounds 4c and 4g.

Full text of DOI:10.1007/s00044-017-1879-9

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-017-1879-9
ORIGINAL RESEARCH
Synthesis and vasorelaxant and antiplatelet activities of a new
series of (4-Benzylphthalazin-1-ylamino)alcohol derivatives
1
,2 ●
,4 ●
2 ●
1 ●
3 ●
Javier Munín
Elías Quezada Manuel Campos-Toimil Ernesto Cano
Dolores Viña
2
1
Eugenio Uriarte
Received: 8 July 2016 / Accepted: 15 March 2017
Springer Science+Business Media New York 2017
©
Abstract A new series of phthalazine derivatives was
synthesized by reaction of phthalic anhydride and different
substituted phenylacetic acids to yield the benzyliden-3H-
isobenzofuran-1-one intermediates 2a–d. Treatment of them
with hydrazine afforded 4-benzyl-2H-phthalazin-1-one
derivatives 3a–d, which were substituted with the corre-
sponding aminoalkylalcohol to obtain the (4-benzylphtha-
lazin-1-ylamino)alcohol derivatives 4a–h. In general, these
phthalazine derivatives relaxed the contractions produced
by phenylephrine both in intact or endothelium-denuded
aortic rings. In addition, platelet aggregation induced by
thrombin was also inhibited by compounds 4c and 4g.
Introduction
Relaxation of vascular smooth muscle is one of the useful
strategies for the treatment of hypertension, which is the
most common cardiovascular disease. Hypertension repre-
sents the major risk factor for endothelial dysfunction,
congestive heart failure, coronary artery disease and stroke
(Stokes 2004; Kümmerle et al. 2009). Other factor that
contributes to the development of these diseases is the
occlusion of a coronary artery by a thrombus affecting the
normal blood flow (Mahmood et al. 2015). Platelets play a
pivotal role in hemostasis forming a plug and limiting the
blood loss at sites of vascular injury; nevertheless their
involvement in pathological conditions such as thrombosis
and atherosclerosis is also well established, making the
platelets a key target in therapies to treat cardiovascular
diseases (von Hundelshausen and Schmitt 2014). Therefore,
there is a continuous need to explore, search and develop
new agents with both vasorelaxant and antiplatelet activity
in order to reduce the risk of cardiovascular complications
associated to hypertension and thrombus formation.
●
●
●
Keywords Synthesis Phthalazine Vasorelaxant activity
●
●
Platelet aggregation Hypertension Thrombus
The phthalazine ring is the isostere and positional isomer
of quinazoline ring and it is also the nucleus of well known
vasodilators such as hydralazine (I, Fig. 1) and budralazine
(II, Fig. 1) (Zacest et al. 1972; Ellershaw and Gurney 2001;
Yoshioka et al. 1987). Therefore, nowadays the phthalazine
moiety continuous being used for the synthesis of new
derivatives with vasodilator effect (Awadallah et al. 2012;
del Olmo et al. 2006; Vila et al. 2015). Additionally, some
phthalazine derivatives have shown antiplatelet activity. 4-
Phenylphthalazines bearing an amine substituent at position
*
Javier Munín
jmunin12@gmail.com
1
Farmacología de las Enfermedades Crónicas, Centro de
investigación en Medicina Molecular y Enfermedades Crónicas,
Universidade de Santiago de Compostela, Santiago de Compostela
5782, Spain
1
2
3
4
Departamento de Química Orgánica, Facultad de Farmacia,
Universidade de Santiago de Compostela, Santiago de Compostela
1
5782, Spain
Departamento de Farmacología, Farmacia y Tecnología
Farmacéutica, Facultad de Farmacia, Universidade de Santiago de
Compostela, Santiago de Compostela 15782, Spain
1
of phthalazine ring (III, Fig. 1) have been described by
inhibiting platelet aggregation induced by both arachidonic
acid and adenosine diphosphate (Eguchi et al. 1991). Recent
modifications on phthalazine ring such as the fusion of a
Instituto de Ciencias Químicas Aplicadas, Facultad de Ingeniería,
Universidad Autónoma de Chile, Santiago 7500912, Chile

Med Chem Res
Fig. 1 Phthalazine derivatives
with vasorelaxant or antiplatelet
activities: hydralazine (I),
budralazine (II), ethyl 1-(2-
methoxyphenyl)-5,7-
dimethylphthalazine-4-
substituted-6-carboxylate (III),
6
-(4-chlorophenoxy)tetrazolo
[
5,1-a]phthalazine (IV), (4-
benzylphthalazin-1-ylamino)
alcohols (V)
tetrazole ring at positions 1 and 2 as well as the introduction
of a phenoxy group at position 4 (IV, Fig. 1) have lead to
new derivatives showing anticoagulant and antithrombotic
effects (Yu et al. 2014).
In a previous work, we have reported the vasorelaxant
effects of a series of phthalazin-1,4-dione derivatives
Chemical shifts (δ) are given in ppm and coupling constants
(J) in Hz. Multiplicity is indicated as follows: s, singlet; d,
doublet; t, triplet; q, quartet; m, multiplet; br, broad.
High resolution mass spectrometry (HR-MS) and elec-
trospray ionization mass spectrometry were carried out on a
VG AutoSpec (Fision, Ipswich, United Kingdom)
instrument.
(
Munín et al. 2014). Following our research and considering
the background previously described, the aim of this work
is the synthesis and study of the vasorelaxant and anti-
platelet activities of new phthalazine derivatives bearing a
benzyl ring at position 4 and a hydroxyalkylamino sub-
stituent at position 1 (V).
(
Z)-3-(4-tert-Butylbenzyliden)-3H-isobenzofuran-1-one (2d)
Phthalic anhydride (1.0 g, 6.75 mmol) and 2-(4-tert-butyl-
phenyl)acetic acid (1d) (1.56 g, 8.10 mmol) were melted
and subsequently KOAc (66 mg, 0.68 mmol) was added.
The mixture was heated under molecular weight (MW)
irradiation (350 W) for 15 min in 1 min periods, until
change of color, keeping the temperature between 210 and
Experimental
2
30 °C. Then it was extracted with ethyl acetate and washed
Chemistry and pharmacology
with Na CO 10% and H O until pH 7. Residue was pur-
2
3
2
ified by FC with silica gel (35–70 mesh) using hexane/
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,
EtOAc (97:3) as eluent to give 2d (79% yield), as a white
1
solid, m.p. 109–110 °C. H NMR (250 MHz, CDCl ) δ =
3
2
2
3 2
1
.34 (s, 9H, 3CH ), 6.41 (s, 1H, C=CH), 7.43 (d, J = 8.6,
3
MeOH and dimethyl sulfoxide (DMSO) were distilled prior
to use according to the standard protocols. Other reagents
were purchased and used as received without further pur-
ification unless otherwise stated. Reactions were magneti-
cally stirred and monitored by thin layer chromatography
with 0.15–0.2 mm pre-coated silica gel (10–40 µm) plates.
Compounds were visualized with UV light and/or by
staining with ethanolic phosphomolybdic acid followed by
heating on a hot plate. Flash chromatography (FC) was
performed with silica gel (60–200 mesh) under pressure.
Nuclear magnetic resonance (NMR) spectra were recorded
2
H, Ar–H), 7.53 (m, 1H, Ar–H), 7.75 (m, 4H, Ar–H), 7.92
1
3
(d, J = 7.8, 1H, Ar–H). C NMR (62.9 MHz, CDCl ) δ =
3
3
1
1
2
1.15, 34.72, 107.00, 119.69, 123.26, 125.47, 125.70,
29.50, 129.89, 130.24, 134.36, 140.63, 144.06, 151.72,
+
•
67.13. HR-MS: m/z 278.1304 [M] ; calcd for C H O ,
1
9 18 2
78.1307.
4-(4-Methylbenzyl)-2H-phthalazin-1-one (3b)
3-(4-Methylbenzyliden)-3H-isobenzofuran-1-one (2b) (1.0 g,
4.2 mmol) was dissolved in hydrazine 1M in THF (8.5 mL,
8.50 mmol). The mixture was stirred for 2 h at room
on Bruker-250 or AMX 500 spectrometers in CDCl or
3
DMSO-d with Trimethyl silane as the internal standard.
6

Med Chem Res
temperature following for 4 h at 60 °C, in a sealed tube. The
mixture was cooled to room temperature and evaporated to
dryness. The resulting solid was purified by crystallization
(m, 4H, CH –CH ), 4.37 (s, 2H, CH –Ar), 4.95 (bs, 1H,
2 2 2
OH), 7.02 (d, J = 8.0, 2H, Ar–H), 7.14 (d, J = 8.0, 2H,
Ar–H), 7.37 (bs, 1H, NH), 7.76 (m, 2H, Ar–H), 7.92 (d, J
1
3
in EtOAc, to give 3b (98% yield), as a white solid, m.p.
= 7.0, 1H, Ar–H), 8.28 (d, J = 7.0, 1H, Ar–H). C NMR
1
2
03–204 °C. H NMR (250 MHz, CDCl ) δ = 2.29 (s, 3H,
(62.9 MHz, DMSO-d ) δ = 20.56, 38.11, 43.98, 59.76,
3
6
CH ), 4.28 (s, 2H, CH –Ar), 7.10 (d, J = 8.0, 2H, Ar–H),
118.34, 122.45, 124.78, 125.68, 128.23 (2C), 128.97 (2C),
3
2
7
.18 (d, J = 8.0, 2H, Ar–H), 7.74 (m, 3H, Ar–H), 8.48 (d,
130.77, 131.38, 135.05, 136.61, 150.04, 153.46. HR-MS
(ESI): m/z 294.1590 (M H ); calcd for C H N O:
18 20 3
1
3
+ +
J = 7.6, 1H, Ar–H), 11.59 (bs, 1H, NH). C NMR (62.9
MHz, CDCl ) δ = 20.97, 38.45, 125.38, 126.91, 128.22,
294.1601.
3
1
1
28.31 (2C), 129.36 (2C), 129.79, 131.19, 133.36, 134.52,
36.28, 146.58, 161.00. HR-MS: m/z 250.1114 [M] ;
+
•
2-(4-(4-Isopropylbenzyl)phthalazin-1-ylamino)ethanol (4c)
calcd for C H N O: 250.1106.
1
6 14 2
The compound 4c was obtained from 3c and 2-
4
-(4-Isopropylbenzyl)-2H-phthalazin-1-one (3c)
aminoethanol following the previously described proce-
dure to obtain 4b (98%), as a white solid, m.p. 137–138 °C.
1
The compound 3c was obtained from 2c and hydrazine 1M
H NMR (250 MHz, DMSO-d ) δ = 1.11 (d, J = 7.0, 6H,
6
in THF, following the previously described procedure to
2CH , 2.76 (m, 1H, (CH ) CH), 3.67 (m, 4H, CH –CH ),
3
3 2
2
2
obtain 3b (0.52 g, 98%) as a white solid; m.p. 197–198 °C.
4.38 (s, 2H, CH –Ar), 5.0 (bs, 1H, OH), 7.08 (d, J = 8.1,
2
1
H NMR (250 MHz, CDCl ) δ = 1.21 (d, J = 7.5, 6H,
2H, Ar–H), 7.19 (d, J = 8.1, 2H, Ar–H), 7.38 (bs, 1H, NH),
7.79 (m, 2H, Ar–H), 7.99 (d, J = 7.6, 1H, Ar–H), 8.26 (d,
3
2
7
7
1
3
CH ), 2.86 (m, 1H, (CH ) CH), 4.28 (s, 2H, CH –Ar),
3 3 2 2
1
3
.18 (d, J = 8.2, 2H, Ar–H), 7.22 (d, J = 8.2, 2H, Ar–H),
.76 (m, 3H, Ar–H), 8.47 (d, J = 7.2, 1H, Ar–H), 10.93 (bs,
J = 7.9, 1H, Ar–H). C NMR (62.9 MHz, DMSO-d ) δ =
6
23.90 (2C), 33.01, 38.02, 44.00, 59.77, 118.36, 122.50,
124.81, 125.74, 126.31 (2C), 128.33 (2C), 130.83, 131.49,
136.99, 146.11, 150.11, 153.47. HR-MS (ESI): m/z
1
3
H, NH). C NMR (62.9 MHz, CDCl ) δ = 23.93 (2C),
3
3.64, 38.43, 125.51, 126.77 (2C), 126.95, 128.30, 128.38
+
+
(
1
2C), 129.88, 131.28, 133.44, 134.82, 146.51, 147.35,
60.65. HR-MS: m/z 278.1417 [M] ; calcd for
322.1904 (M H ); calcd for C H N O: 322.1913
20 24 3
+
•
C H N O: 278.1419.
2-(4-(4-tert-Butylbenzyl)phthalazin-1-ylamino)ethanol (4d)
1
8 18 2
4
-(4-tert-Butylbenzyl)-2H-phthalazin-1-one (3d)
The compound 4d was obtained from 3d and 2-
aminoethanol following the previously described proce-
dure to obtain 4b (83%), as a white solid, m.p. 202–203 °C.
The compound 3d was obtained from 2d and hydrazine 1M
in THF following the previously described procedure to
obtain 3b (99%), as a white solid, m.p. 218–219 °C. H
1
H NMR (250 MHz, DMSO-d ) δ = 1.19 (s, 9H, 3CH ),
6
3
1
3.64 (m, 4H, CH –CH ), 4.38 (s, 2H, CH –Ar), 4.96 (bs,
2 2 2
NMR (250 MHz, CDCl ) δ = 1.28 (s, 9H, 3CH ), 4.29 (s,
1H, OH), 7.19 (d, J = 8.4, 2H, Ar–H), 7.24 (d, J = 8.4, 2H,
Ar–H), 7.38 (bs, 1H, NH), 7.79 (m, 2H, Ar–H), 7.99 (d,
J = 9.3, 1H, Ar–H), 8.26 (d, J = 8.9, 1H, Ar–H). C NMR
3
3
2
H, CH –Ar), 7.23 (d, J = 8.5, 2H, Ar–H), 7.31 (d, J = 8.5,
2
1
3
2
H, Ar–H), 7.76 (m, 3H, Ar–H), 8.50 (d, J = 7.5, 1H,
1
3
Ar–H), 11.50 (bs, 1H, NH). C NMR (62.9 MHz, CDCl3)
(62.9 MHz, DMSO-d ) δ = 31.14 (3C), 34.06, 37.88, 43.99,
6
δ = 31.28 (3C), 34.36, 38.30, 125.49, 125.59 (2C), 126.92,
59.76, 118.34, 122.49, 124.80, 125.15 (2C), 125.74, 128.07
1
1
28.09 (2C), 128.24, 129.88, 131.21, 133.39, 134.53,
46.59, 149.55, 160.96. HR-MS: m/z 292.1572 [M] ;
(2C), 130.83, 131.49, 136.59, 148.35, 150.09, 153.45. HR-
MS (ESI): m/z 336.2000 (M H ); calcd for C H N O:
21 26 3
+
•
+ +
calcd for C H N O: 292.1576.
336.2070.
1
9 20 2
2
-(4-(4-Methylbenzyl)phthalazin-1-ylamino)ethanol (4b)
3-(4-(4-Methylbenzyl)phthalazin-1-ylamino)propan-1-ol
4f)
(
4
1
1
-(4-Methylbenzyl)-2H-phthalazin-1-one (3b) (0.30 g,
.20 mmol) was dissolved in 2-aminoethanol (0.70 mL,
2.00 mmol) and refluxed for 48 h. The mixture was
4-(4-Methylbenzyl)-2H-phthalazin-1-one (3b) (0.50 g, 2.00
mmol) was dissolved in 3-aminopropan-1-ol (1.50 mL,
20.00 mmol) and refluxed for 48 h. The mixture was cooled
to room temperature, CH Cl (100 mL) was added and
cooled to room temperature, CH Cl (100 mL) was added
and washed with H O and brine. Organic phase was dried
2
2
2
2
2
over Na SO , filtered and evaporated to dryness. The
washed with H O and brine. Organic phase was dried over
2
4
2
resulting solid was purified by crystallization using CH CN,
Na SO , filtered and evaporated to dryness. The resulting
3
2
4
to give 4b (0.25 g, 72%), as a white solid, m.p. 201–202 °C.
solid was purified by crystallization using CH CN, to give
4f (0.45 g, 74%), as a white solid, m.p. 167–168 °C. H
3
1
1
H NMR (250 MHz, DMSO-d ) δ = 2.18 (s, 3H, CH ), 3.66
6
3

Med Chem Res
NMR (250 MHz, DMSO-d6) δ = 1.85 (m, 2H,
CH –CH –CH ), 2.18 (s, 3H, CH ), 3.52 (t, J = 6.3, 2H,
(Barcelona, Spain) were housed, cared for and acclimatized
(before the experiments) as indicated previously (Orallo
et al. 2002).
2
2
2
3
CH –N), 3.61 (t, J = 6.4, 2H, CH –O), 4.37 (s, 2H,
2
2
CH –Ar), 4.72 (bs, 1H, OH), 7.02 (d, J = 8.0, 2H, Ar–H),
2
7
2
1
3
1
1
.14 (d, J = 8.0, 2H, Ar–H), 7.34 (bs, 1H, NH), 7.76 (m,
Ethical approval
H, Ar–H), 7.91 (d, J = 7.2, 1H, Ar–H), 8.24 (d, J = 7.1,
1
3
H, Ar–H). C NMR (62.9 MHz, DMSO-d ) δ = 20.57,
All experiments were carried out in accordance with Eur-
opean regulations on the protection of animals (Directive
2010/63/UE), the Spanish Real Decreto 53/2013 (1 Feb-
ruary). The experimental protocols were approved by the
Bioethics Committee of the University of Santiago de
Compostela (Spain) and the Bioethics Committee for
Research (CEIC) of the Xunta de Galicia (Spain).
6
2.06, 38.12, 38.27, 59.78, 118.33, 122.37, 124.79, 125.66,
28.24 (2C), 128.98 (2C), 130.76, 131.34, 135.06, 136.63,
+
+
49.89, 153.43. HR-MS (ESI): m/z 308.1749 (M H );
calcd for C H N O: 308.1757.
1
9 22 3
3
-(4-(4-Isopropylbenzyl)phthalazin-1-ylamino)propan-1-ol
(
4g)
Vasorelaxant activity
The compound 4g was obtained from 3c and 3-
aminopropan-1-ol following the previously described proce-
Vasorelaxant activity of newly synthesized compounds
4a–h was studied using thoracic aortic rings of WKY rats
pre-contracted with phenylephrine (PE) (1 µM). Male WKY
rats weighing 300–400 g were killed by a blow to the head
and exsanguinated. The thoracic aorta was rapidly dissected
and transferred to a Petri dish with Krebs bicarbonate
solution (KBS composition: 119 mM NaCl, 4.7 mM KCl,
1.5 mM CaCl • 2H O, 1.2 mM MgSO • 7H O, 25 mM
dure to obtain 4f (95%), as a white solid, m.p. 109–110 °C.
1
H NMR (250 MHz, DMSO-d ) δ = 1.10 (d, J = 6.9, 6H,
6
2
CH ), 1.83 (m, 2H, CH –CH –CH ), 2.76 (m, 1H,
3 2 2 2
(
CH ) CH), 3.52 (t, J = 6.2, 2H, CH –N), 3.60 (t, J = 6.1,
3 2 2
2
H, CH –O), 4.38 (s, 2H, CH –Ar), 4.8 (bs, 1H, OH), 7.08
2 2
(
(
d, J = 8.2, 2H, Ar–H), 7.19 (d, J = 8.2, 2H, Ar–H), 7.36
bs, 1H, NH), 7.77 (m, 2H, Ar–H), 7.98 (d, J = 9.4, 1H,
2
2
4
2
1
3
Ar–H), 8.23 (d, J = 9.4, 1H, Ar–H). C NMR (62.9 MHz,
NaHCO , 0.03 mM EDTA-Na , 11 mM glucose; pH 7.4).
3 2
DMSO-d ) δ = 23.90 (2C), 32.07, 33.00, 38.02, 38.28,
Excess of fat and connective tissue was removed. For the
endothelium free experiments a scraping of the arterial
lumen with a thick cotton thread was made. The aorta was
cut into rings (4–5 mm in length, 0.9–1.0 mm in thickness)
and each ring was placed in a 10 mL 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 displace-
ment transducer (Pioden Controls Ltd., Canterbury, UK) to
record the isometric tension with a MacLab/16s and Chart
6
5
8.79, 118.34, 122.42, 124.80, 125.71, 126.31 (2C), 128.33
(
2C), 130.82, 131.44, 137.00, 146.11, 149.93, 153.42. HR-
+
+
MS (ESI): m/z 336.2000 (M H ); calcd for C H N O:
2
1 26 3
3
36.2070.
3
-(4-(4-tert-Butylbenzyl)phthalazin-1-ylamino)propan-1-ol
(
4h)
The compound 4h was obtained from 3d and 3-
aminopropan-1-ol following the previously described proce-
dure to obtain 4f (82%), as a white solid, m.p. 130–131 °C.
v.3.6.3 software or
a
PowerLab/8sp and Chart
1
H NMR (250 MHz, DMSO-d ) δ = 1.18 (s, 9H, 3CH ),
v.4.1.2 software Data Acquisition system (AD Instruments
Castle Hill, Australia).
6
3
1
.84 (m, 2H, CH –CH –CH ), 3.52 (t, J = 6.2, 2H,
2 2 2
CH –N), 3.60 (t, J = 6.2, 2H, CH –O), 4.38 (s, 2H,
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 µM). Once the contraction
stabilized, a single concentration of acethylcholine (ACh)
(1 µM) was added to the bath in order to assess the endo-
thelial integrity of the preparations. Endothelium was con-
sidered to be intact when this drug elicited a vasorelaxation
˃50% of the maximal contraction obtained in vascular rings
pre-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 solution was replaced every 15
min, before any experiment protocol was started.
2
2
CH –Ar), 4.78 (bs, 1H, OH), 7.19 (d, J = 8.5, 2H, Ar–H),
2
7
2
1
.24 (d, J = 8.5, 2H, Ar–H), 7.36 (sa, 1H, NH), 7.78 (m,
H, Ar–H), 7.99 (d, J = 9.3, 1H, Ar–H), 8.25 (d, J = 9.3,
1
3
H, Ar–H). C NMR (62.9 MHz, DMSO-d ) δ = 31.15
6
(
3C), 32.07, 34.07, 37.90, 38.29, 58.79, 118.35, 122.43,
1
1
3
24.82, 125.16 (2C), 125.72, 128.09 (2C), 130.84, 131.48,
36.60, 148.36, 149.94, 153.41. HR-MS (ESI): m/z
+
+
50.2215 (M H ); calcd for C H N O: 350.2226.
2
2 28 3
Animals
The animals used throughout this study (Male Wistar-Kyoto
WKY) rats (Iffa-Credo)), purchased from Criffa
(

Med Chem Res
8
After equilibration, aortic rings were contracted by single
concentration of PE (1 µM). Once the contractions stabi-
lized, compounds of series 4a–h were added in progres-
sively increasing cumulative concentrations (1–200 µM) at
cell density of 2.5–3.5 × 10 platelet/ml. The calcium
concentration in the extracellular medium was adjusted to 2
mM by the addition of the appropriate amount of CaCl2.
1
0 min intervals. Only one compound was tested in each
Platelet aggregation studies Platelet aggregation was
measured using a dual channel aggregometer (Chrono-log,
Havertown, PA, USA). Each tested compound, dissolved in
DMSO, was incubated with washed platelets at 37 °C for
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 solvent did not affect the contractile response of iso-
lated aorta.
All the results are expressed as means ± the standard
error of the mean. The response of the aortic rings to the all
compounds was expressed as a percentage of the initial
contraction to 1 µM PE. Dose–response curves were ana-
lyzed by a sigmoidal curve fitting analysis to give EC50
values (50% of the Emax) obtained in the presence of the
tested compounds and the Emax value (the maximal effect).
5
min. A platelet activator (thrombin) was then added to
induce platelet aggregation and the light transmission was
monitored over 5 min period. Platelet aggregation is mea-
sured as the maximum change in light transmission during
this period. The 100% aggregation value was obtained
when vehicle (DMSO) was added instead of the com-
pounds. The final DMSO concentration was below 1% (v/v)
in all cases.
Results and discussion
Chemistry
Antiplatelet activity
Preparation of washed platelets Washed human platelets
were prepared from blood anticoagulated with citrate-
phosphate-dextrose, which was obtained from Centro de
Transfusion de Galicia (Santiago de Compostela, Spain).
Bags containing buffy coat from individuals donors were
diluted with the same volume of washing buffer (NaCl, 120
mM; KCl, 5 Mm; trisodium citrate, 12 mM; glucose, 10
mM; sucrose, 12.5 mM; pH = 6) and centrifuged at 400×g
for 8 min. The upper layer containing platelet (platelet-rich
plasma) was removed and centrifuged at 1100×g for 18
min. The resulting platelet pellet was recovered, resus-
pended with washing buffer, and centrifuged again at
A series of differently substituted (4-benzylphthalazin-1-
ylamino)alcohols were synthesized in good yields. Reaction
under microwave irradiation of phthalic anhydride and
different substituted phenylacetic acids 1a–d yielded the
corresponding benzyliden-3H-isobenzofuran-1-ones 2a–d
(Viña et al. 2009; Munín et al. 2015; Kumar et al. 2013;
Hjelmencrant et al. 2000). These benzyliden-3H-iso-
benzofuran-1-one derivatives were further transformed into
4-benzyl-2H-phthalazin-1-one derivatives 3a–d (Munín
et al. 2015) by treatment with hydrazine in THF (del Olmo
et al. 2006; Viña et al. 2009). Finally, reaction under reflux
of 3a–d derivatives with an excess of either 2-aminoethan-
1-ol or 3-aminopropan-1-ol lead to the desired (4-ben-
1
100×g for 15 min. Finally, the platelet pellet from this step
was resuspended in a modified Tyrode-HEPES buffer
HEPES, 10 mM; NaCl, 140 mM; KCl, 3 mM; MgCl , 0.5
(
zylphthalazin-1-ylamino)alcohol
(Scheme 1) (Munín et al. 2015).
derivatives
4a–h
2
mM; NaHCO , 5 mM; glucose, 10 mM; pH 7.4) to afford a
3
Scheme 1 General synthetic
route to obtain compounds
4
a–h. Reagents and conditions:
a AcOK, MW (350W),
2
2 2
10–230 °C; b H NNH
1
M THF, rt to 60 °C; c
H
H
2
NCH
NCH
2
CH
CH
2
OH or
CH OH, reflux
2
2
2
2

Med Chem Res
Table 1 Vasorelaxant activity of (4-benzylphthalazin-1-ylamino)
alcohol derivatives in rat aortic rings pre-contracted with PE (1 μM)
Table 2 Antiplatelet activity of (4-benzylphthalazin-1-ylamino)
alcohol derivatives using thrombin (0.25 U/ml) as stimulating agent
for aggregation
Compound EC50 (μM) with
EC50 (μM) without
endothelium
endothelium
Compound
IC50 (µM)
4
4
4
4
4
4
4
4
a
b
c
26.41 ± 4.29
31.35 ± 2.65
16.41 ± 3.35
43.34 ± 4.62
15.02 ± 3.05
23.09 ± 4.34
21.47 ± 2.71
b
59.54 ± 4.82**
48.79 ± 5.42**
21.89 ± 4.75
a
4a
*
4b
*
4c
110.6 ± 5.4
d
e
4d
**
63.23 ± 1.10*
44.34 ± 6.59**
23.92 ± 2.27
a
4e
*
f
4f
*
g
h
4g
31.59 ± 1.88
**
4h
3
3
Hydralazine (1.18 ± 0.09) × 10
(1.32 ± 0.12) × 10
Milrinone
4.70 ± 0.50
c
Amrinone
17 ± 6
28 ± 8*
*
Inactive at 200 µM (highest concentration tested). All reported IC50
values are the mean of at least five experiments employing human
blood from different individuals
*
*
P < 0.01
*P < 0.05 vs. ring with endothelium as determined by ANOVA/
*
*At 200 µM, platelet aggregation was produced by the compounds
Dunnet’s test. All reported EC50 values are the mean of at least five
experiments
a
Inactive at 200 μM (highest concentration tested)
the compounds 4c and 4g inhibited platelet aggregation
induced by thrombin (0.25 U/ml) (Table 2). This suggests
that both the substitution at para position of the 4-benzyl
ring as its nature is responsible for the antiplatelet activity.
Also the alkyl chain length at 1 position is important to
inhibitory activity, because the compound 4g bearing a
propyl chain is more than three times more potent than the
compound 4c which links an ethyl chain.
b
1
00 μM produced relaxation about 50%
c
Mori et al. (1996)
Pharmacology
The vasorelaxant effects of compounds 4a–h were studied
using isolated rat thoracic aortic rings pre-contracted with
PE following a standard procedure previously reported by
us (Cuíñas et al. 2013; Quezada et al. 2010) and compared
to the reference drugs hydralazine and amrinone (Mori et al.
Conclusion
1
996). The cumulative addition of the new compounds
A new series of (4-benzylphthalazin-1-ylamino)alcohol deri-
vatives has been synthesized in good yields. All the com-
pounds have been found to cause a concentration-dependent
relaxation of aortic rings pre-contracted with PE in the μM
range in the presence of an intact endothelium. However, this
relaxant effect was significantly reduced after removal of the
endothelium layer, except for compounds 4c and 4g. Both
compounds bearing an isopropyl group at the para position of
benzyl ring. Moreover, only these two compounds also
showed antiplatelet activity against thrombin, being com-
pound 4g the most potent derivative in this series.
(
1–200 µM) caused a sustained concentration-dependent
relaxation of the contractions induced by PE (1 µM) in
intact rat aortic rings. In the absence of endothelium, this
vasorelaxant effect decreases significantly, except for the
compounds 4c and 4g which showed nearly the same
relaxing activity in both cases. Both derivatives contain in
their structure an isopropyl group at the para position of 4-
benzyl ring, which seems determinant to show independent
endothelium activity. However, when this isopropyl group
was replaced by a tert-butyl group in the same position,
these derivatives 4d and 4h, lack vasorelaxant activity in the
absence of endothelium at the highest concentration tested
Acknowledgements We are grateful to the Xunta de Galicia
(
200 μM). Most of these derivatives present a vasorelaxant
(10PXIB203303PR) for the financial support.
effect in intact aortic rings similar to the amrinone and they
are much more potent vasorelaxant agents than hydralazine
in the in vitro studies. The described effects seem to be
reversible because a 60 min washout period allows a pro-
gressive recovery of the functionality of muscle and endo-
thelial cells of the rings. The corresponding EC50 values are
shown in Table 1.
Conflict of interest The authors declare that they have no competing
interests.
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