Pyridazines. Part 31: synthesis and antiplatelet activity of 4,5-disubstituted-6-phenyl-3(2H)-pyridazinones.

Article abstract of DOI:10.1248/cpb.50.1574

This paper describes the synthesis and the antiplatelet activity of a series of 4,5-disubstituted-6-phenyl-3(2H)-pyridazinones. Some of these compounds show a dose-dependent activity and were found to be more active than their 5-substituted analogues.

Full text of DOI:10.1248/cpb.50.1574

1574
Chem. Pharm. Bull. 50(12) 1574—1577 (2002)
Vol. 50, No. 12
Pyridazines. Part 311): Synthesis and Antiplatelet Activity of
4,5-Disubstituted-6-phenyl-3(2H)-pyridazinones
a
b
b
b
b
,a
*
Eddy SOTELO, Nuria FRAIZ, Matilde YÁÑEZ, Reyes LAGUNA, Ernesto CANO, and Enrique RAVIÑA
a Departamento de Química Orgánica, Laboratorio de Química Farmacéutica: and b Departamento de Farmacología,
Facultad de Farmacia, Universidad de Santiago de Compostela; 15782-Santiago de Compostela, España.
Received July 18, 2002; accepted September 24, 2002
This paper describes the synthesis and the antiplatelet activity of a series of 4,5-disubstituted-6-phenyl-
3(2H)-pyridazinones. Some of these compounds show a dose-dependent activity and were found to be more active
than their 5-substituted analogues.
Key words pyridazinone; platelet; platelet aggregation; platelet aggregation inhibitor
111) and 212) and the strategy is based on the modification of
The homeostatic system is designed to maintain fluid
blood flow under physiological conditions and to react
rapidly to form a clot at sites of vascular damage. Neverthe-
less, the intravascular formation of platelet aggregates is an
important pathogenic factor in widespread cardiovascular
diseases, such as myocardial and cerebral circulatory disor-
ders, venous thromboses, cardiac and cerebral infarcts and
arteriosclerosis, as well as contributing to the risk of em-
bolism in surgery on the heart and blood vessels. In these
fields, the discovery of aggregation-inhibiting drugs has led
to a new approach to therapy and prophylaxis.2,3) The active
compounds hitherto available for this function, such as
acetylsalicylic acid, sulfinpyrazone and dipyridamole, have a
relatively low activity and, consequently, extended therapy is
unsatisfactory and the search for promising new drugs in this
therapeutic area is of great interest. One of the most studied
targets in this field is represented by the PDE III inhibitors,
which have received a great deal of attention in recent years,
not only because of their antiplatelet properties but also on
the basis that they could be used as cardiotonic agents due to
the absence of safe and orally effective ionotropic agents for
the treatment of congestive heart failure.
the reactivity at the 4-position caused by the electronic effect
of the neighbouring group. Some preliminary results related
to the compounds studied here as antiplatelet agents have
been published in communication form.13,14)
As part of our detailed study into the structure/reactivity
relationships in these series of compounds we recently de-
scribed a new synthetic route to 4,5-disubstituted-3(2H)-
pyridazinones based on the high reactivity of aldehyde 1 to-
ward the cyanide anion.13) This route affords compounds
bearing a cyano group at position 4 of the heterocyclic ring.
The synthetic exploitation of this transformation (involving
the cyanohydrin intermediate 1a) has allowed the efficient
preparation of several new derivatives (Chart 1).
Bearing this result in mind, we have then studied several
transformations that involve the use of cyanohydrin 1a as an
intermediate. One of the best known of these procedures is
the preparation of carboxylic acids by oxidation of cyanohy-
drins. The selective action of silver oxide (obtained by addi-
tion of sodium hydroxide to silver nitrate) on the hydroxyl
group at position 5 of cyanohydrin 1a provided an excellent
preparative method to obtain 5-carboxy-4-cyano-6-phenyl-
3(2H)-pyridazinone 3a as a crystalline solid in 88% yield.
This step occurs through nucleophilic attack of the hydroxyl
ion on the acylcyanide intermediate. Another important pro-
cedure involving cyanohydrin formation is the one-pot three-
step transformation of a,b-unsaturated aldehydes into the
In this regard a considerable number of 3(2H)-pyridazi-
nones and their 4,5-dihydroderivatives with anti-aggregating
properties have been described.4,5) Zardaverine and Pimoben-
dan are probably the most representative examples of this
class of compounds.
As part of our ongoing medicinal chemistry project aimed
at exploring the platelet inhibitory activity of a series of 5-
substituted-6-aryl-3(2H)-pyridazinones,6—10) we recently de-
scribed the potent antiplatelet activity of compounds in this
series along with some preliminary pharmacological studies.
These studies reveal that the substituent in the 5-position not
only determines the potency of the antiplatelet activity
(quantitative aspect) but also the mechanism of action (quali-
tative aspect).9,10) These results have prompted us to synthe-
sise and evaluate the antiplatelet activity of several 4,5-disub-
stituted-6-phenyl-3(2H)-pyridazinones in order to determine
the pharmacological profile induced by the introduction of a
substituent at position 4 of this system.
Fig. 1. Examples of Pharmacologically Useful 3(2H)-Pyridazinones
Chemistry
The general synthetic strategy used to prepare the 4,5-dis-
ubstituted pyridazinones 3 is shown in Charts 1 and 2. The
precursors in these routes are the 5-substituted pyridazinones Fig. 2. 4,5-Disubstituted-6-phenyl-3(2H)-pyridazinones 3
* To whom correspondence should be addressed. e-mail: qofara@usc.es
© 2002 Pharmaceutical Society of Japan

December 2002
1575
Reagents: (a) Br2/AcOH, (b): I2/dioxane, (c) NaNO2/AcOH, (d) H2/Pt2O.
Chart 2
Table 1. Antiplatelet Activity of the 4,5-Disubstituted-6-phenyl-3(2H)-
pyridazinones 3
Reagents: (a) NaCN/EtOH, (b) AgNO3/NaOH, (c) MnO2/ROH, (d) (i) NaCN/EtOH
(ii) AgNO3/NaOH, (e) (i) NaCN/ROH, (ii) MnO2.
Chart 1
Compound
X
Y
IC50 (mM)
3a
3b
3c
3d
3e
3f
3g
3h
2
COOH
COOMe
COOEt
COOiPr
NH2
NH2
NH2
NH2
NH2
CN
CN
CN
CN
Br
0.71
—
1.10
corresponding esters by treatment with manganese dioxide
and sodium cyanide.15,16) The formation of the ester group
takes place without isolation of the cyanohydrin, which is ox-
idised to an activated acylcyanide that, in turn, undergoes al-
coholysis by the solvent. On the basis of these references, we
performed the oxidation of cyanohydrin 1a with manganese
dioxide in different alcohols, to obtain the corresponding 5-
alkoxycarbonyl-4-cyano-6-phenyl-3(2H)-pyridazinones
3b—d in high yields as yellow crystalline solids. Is interest-
ing to note that compounds 3a—d can be prepared in one-
pot procedures (Chart 1).
0.53
a)
I
—
NO
NH2
H
H
—
0.17
b)
—
—
0.0047
4
COOH
—
Milrinone
a) Precipitate at the studied doses. b) Induces platelet aggregation. —ϭInactive.
During the spectroscopic characterisation (1H-, 13C-NMR)
of different 5-amino-3(2H)-pyridazinones12) we have ob- versatile intermediates to prepare other 4,5-disubstituted- and
served the nucleophilic character generated at C4 produced 4,5-heterofused-pyridazinones.
by the push–pull effect operating in these compounds. In ac-
cordance with these observations, enaminone 2 readily re- Results and Discussions
acted with different electrophiles to afford 5-amino-4-substi-
The platelet aggregation inhibitory activities of the 4,5-
tuted-3(2H)pyridazinones 3e—h. Thus, compound 2 was disubstituted-3(2H)-pyridazinone derivatives described here
easily halogenated in the 4-position by treatment with were examined on washed human platelets using the turbidi-
bromine in acetic acid or iodine in dioxane to afford com- metric method of Born17) and thrombin as inducer of platelet
pounds 3e and 3f, respectively. In turn, nitrosation of 2 gives aggregation. The results of these experiments are sum-
5-amino-4-nitroso-6-phenyl-3(2H)-pyridazinone (3g) as a marised in Table 1.
deep-red solid in excellent yield. Catalytic hydrogenation of
the nitroso group in 3g gave high yields of the diamine 3h.
Several of the studied pyridazinones showed antiplatelet
activity, although in each case it was lower than the reference
The 3(2H)-pyridazinones 3a—h were fully characterised compound milrinone. A few of the compounds studied in-
by their analytical and spectroscopic data. The IR spectra of hibit platelet aggregation in a dose-dependent manner. Com-
compounds 3 showed the presence of NH and carbonyl parison of these results with the antiplatelet activity of the 5-
groups (3380—3280 cmϪ1, 1680 cmϪ1) respectively. The substituted-6-phenyl-3(2H)-pyridazinones (4, 2) (Table 1)
structures of these compounds was confirmed by the absence shows that the introduction of a substituent at position 4 of
of the H4 signal in the 1H-NMR spectra. Further experiments these compounds produces an increase in the platelet in-
are currently in progress because these compounds constitute hibitory activity; this effect is particularly significant in com-

1576
Vol. 50, No. 12
slowly sodium cyanide (0.14 g, 3.0 mmol) and the stirring was maintained
for 15 min at room temperature. To this suspension was added silver nitrate
(0.50 g, 3.0 mmol) and the pH of the mixture was increased to 12 by the ad-
dition of 10% sodium hydroxide. The reaction mixture was stirred for 24 h at
room temperature, diluted with 30 ml of dichloromethane and filtered
pounds 3a, 3c, 3d and 3g. For the cyanoesters 3b—d, a slight
increase in activity was observed within the series due to the
modification of the alkoxy group in the ester function; the
isopropyl derivative 3d is the most active. Curiously, the di-
amine 3h has the opposite activity and actually induces through silica. The filtrate was treated with 10% hydrochloric acid and then
extracted with dichloromethane. The extracts were dried (Na2SO4) and the
solvent was removed under reduced pressure to afford a solid that was puri-
fied by recrystallization from isopropanol (0.20 g, 80%). mp 253—255 °C.
platelet aggregation.
As can be observed from the results in Table 1, the most
potent compound within this series is the nitroso derivative
3g. Bearing in mind the fact that nitroso derivatives are fatal
in medical applications,18) we are now preparing new com-
pounds bearing substituents that can mimic the nitroso group
(e.g. CHO, COR).
Although the antiplatelet activity of these derivatives is
only modest, the real value of the data in Table 1 is to pro-
vide useful information to complete our overview of the SAR
and mechanistic pharmacological studies of these series’.9,10)
These studies are still in progress and will be published in
due course.
IR (KBr): nmax cmϪ1 3000, 2235, 1688, 1648, 1589. 1H-NMR: dH (300
MHz, DMSO-d6): 7.17 (1H, br s, deuterium oxide exchangeable OH), 7.43
(5H, m, Aromatics), 13.47 (1H, br s, deuterium oxide exchangeable, NH).
C12H7N3O3 requires C, 59.75; H, 2.92; N, 17.43, Found: C, 59.86; H, 3.14;
N, 17.76.
5-Alkoxycarbonyl-4-cyano-6-phenyl-3(2H)-pyridazinones 3b-d, Gen-
eral Procedure To a stirred solution of aldehyde 1 (0.20 g, 1.0 mmol) in
the corresponding alcohol (30 ml) was added slowly sodium cyanide (0.14 g,
3.0 mmol) and the stirring was maintained for 15 min at room temperature.
To this suspension was added activated manganese dioxide (1.2 g, 12.9
mmol) and the stirring was maintained for 15 min at room temperature. The
reaction mixture was diluted with dichloromethane and filtered through sil-
ica gel. The solvents were removed under reduced pressure and the resulting
solid purified by recrystallization from isopropanol.
4-Cyano-5-methoxycarbonyl-6-phenyl-3(2H)-pyridazinone (3b)
(0.24 g, 95%). mp 190—191 °C. IR (KBr): nmax cmϪ1 2230, 1735, 1668,
1588. 1H-NMR: dH (300 MHz, DMSO-d6): 3.76 (1H, s, OCH3), 7.40 (2H,
m, Aromatics), 7.50 (3H, m, Aromatics), 14.63 (1H, br s, NH, deuterium
oxide exchangeable). C13H9N3O3 requires C, 61.17; H, 3.55; N, 16.46,
Found: C, 61.24; H, 3.55; N, 16.56.
Experimental
Melting points were measured on a Gallenkamp melting point apparatus
and are uncorrected. Infrared spectra (IR) were recorded on a Perkin-Elmer
1640 FTIR spectrophotometer. 1H-NMR spectra were obtained on Bruker
WM250 and AM300 Hz spectrometers using tetramethylsilane as the inter-
nal standard (chemical shifts are in d values, J in Hz). Mass spectra were de-
termined on a Varian MAT-711 instrument. Elemental analyses were per-
formed on a Perkin-Elmer 240B apparatus at the Microanalysis Service of
the University of Santiago de Compostela. The progress of the reactions was
monitored by thin layer chromatography with 2.5 mm Merck silica gel GF
254 strips, and the purified compounds each showed a single spot; unless
otherwise stated iodine vapour and/or UV light were used for detection.
Chromatographic separations were performed on silica gel columns by flash
chromatography (Kieselgel 40, 0.040—0.063 mm).
Preparation of Washed Platelets Human platelet concentrates from
blood anticoagulated with citrate-phosphate-dextrose were obtained from the
Centro de Transfusión de Galicia (Santiago de Compostela, Spain). Platelets
were purified by sedimentation through a discontinuous metrizamide gradi-
ent. For this purpose 8 ml of platelet concentrate was layered onto a
10%/25% (1 ml/1 ml) metrizamide gradient and centrifuged at 1000ϫg for
20 min. The resulting platelet band was recovered, diluted to 8 ml with wash-
ing buffer (NaCl, 140 mM; KCl, 5 mM; trisodium citrate, 12 mM; glucose,
10 mM; sucrose, 12.5 mM; pH 6) and centrifuged again at 1000ϫg for
20 min. Finally, the platelet band recovered from this step was resuspended
in a modified Tyrode-HEPES buffer (HEPES, 10 mM; NaCl, 140 mM; KCl,
3 mM; MgCl, 0.5 mM; NaHCO3, 5 mM; glucose, 10 mM; pH 7.4) to afford a
concentration of 3—3.5 10Ϫ8 platelets/ml. The calcium concentration in the
extracellular medium was 2 mM.
4-Cyano-5-ethoxycarbonyl-6-phenyl-3(2H)-pyridazinone (3c) (0.25 g,
1
95%). mp 181—183 °C. IR (KBr): nmax cmϪ1 2230, 1720, 1666, 1569. H-
NMR: dH (300 MHz, DMSO-d6): 1.02 (t, Jϭ6.9 Hz, 3H, CH3), 4.24 (q,
Jϭ6.9 Hz, 2H, OCH2), 7.45 (2H, m, Aromatics), 7.48 (3H, m, Aromatics),
14.35 (1H, br s, NH, deuterium oxide exchangeable). C14H11N3O3 requires
C, 62.45; H, 4.12; N, 15.61, Found: C, 62.81; H, 4.03; N, 15.78.
4-Cyano-5-isopropyloxycarbonyl-6-phenyl-3(2H)-pyridazinone (3d)
(0.26 g, 93%). mp 184—186 °C. IR (KBr): nmax cmϪ1 2233, 1731, 1669,
1587. 1H-NMR: dH (300 MHz, DMSO-d6): 1.11 (d, Jϭ6.0 Hz, 6H, 2ϫCH3),
5.04 (m, Jϭ6.0 Hz, 1H, OCH), 7.49 (5H, m, Aromatics), 14.34 (1H, br s,
NH, deuterium oxide exchangeable). C15H13N3O3 requires C, 63.59; H, 4.62;
N, 14.83, Found: C, 63.71; H, 4.57; N, 14.96.
5-Amino-4-bromo-6-phenyl-3(2H)-pyridazinone (3e) To a mixture of
5-amino-6-phenyl-3(2H)-pyridazinone 2 (0.70 g, 3.7 mmol) and sodium ac-
etate trihydrate (0.66 g, 4.8 mmol) in acetic acid (15 ml) at 0 °C was slowly
added a solution of bromine (0.65 ml, 4.4 mmol) in acetic acid (3 ml). The
resulting mixture was stirred at room temperature for 45 min and then added
to ice/water. The precipitated solid was collected by filtration. Further purifi-
cation was carried out by recrystallization from isopropanol to give 3e as a
white solid (0.91 g, 92%). mp 323—325 °C (dec.). IR: nmax cmϪ1 3422—
3185 (NH2), 1643 (CO), 1578 (Aromatics). 1H-NMR: dH (300 MHz,
DMSO-d6): 6.04 (2H, br s, NH2, deuterium oxide exchangeable), 7.48 (5H,
m, Aromatics), 12.64 (1H, br s, NH, deuterium oxide exchangeable).
C10H8BrN3O requires C, 45.13; H, 3.03; N, 15.79, Found: C, 45.21; H, 3.17;
N, 15.72.
Platelet Aggregation Studies Platelet aggregation was measured using
a dual channel aggregometer (Chrono-log, Havertown, PA, U.S.A). Each test
compound was incubated with washed platelets at 37 °C for 5 min. Stimulus
was then added to induce platelet aggregation, and the light transmission
was monitored over a 5 min period. Platelet aggregation is expressed as the
maximum change in light transmission during this period, with a 100%
value being obtained when only stimulus, and not compound, was added.
4-Cyano-5-(1؅-cyanohydroxymethyl)-6-phenyl-3(2H)-pyridazinone
(1a) To a stirred solution of aldehyde 1 (0.10 g, 0.5 mmol) in ethanol
(10 ml) was added, in several portions, sodium cyanide (0.07 g, 0.15 mmol)
and the stirring was maintained for 1 h. The reaction mixture was poured
into ice and then extracted with ethyl acetate. The organic extracts were
dried (Na2SO4) and concentrated under reduced pressure to give an oily
residue that was purified by column chromatography (hexane/ethyl acetate,
1 : 1). Subsequent recrystallization from isopropanol afforded the cyanohy-
drin 1a as yellow prisms (0.12 g, 90%). mp 145.5—147.0 °C (dec.). IR
5-Amino-4-iodo-6-phenyl-3(2H)-pyridazinone (3f) To a solution of 5-
amino-6-phenyl-3(2H)-pyridazinone
2 (0.20 g, 1.06 mmol) in dioxane
(15 ml) was added iodine (0.27 g, 2.13 mmol) and 0.75 M nitric acid (1.5 ml).
The mixture was heated under reflux for 12 h, the solvent was removed
under reduced pressure and the resulting residue was poured into ice to af-
ford a solid, which was purified by recrystallization from ethanol (0.27 g,
81%). mp 254—256 °C (dec.). IR: nmax cmϪ1 3500—3200 (NH2), 1639
(CO), 1581 (Aromatics). 1H-NMR: dH (300 MHz, DMSO-d6): 5.93 (2H,
br s, NH2, deuterium oxide exchangeable), 7.55 (5H, m, Aromatics), 12.57
(1H, br s, NH, deuterium oxide exchangeable). C10H8IN3O requires C,
38.36; H, 2.58; N, 13.42, Found: C, 38.72; H, 2.47; N, 13.48.
5-Amino-4-nitroso-6-phenyl-3(2H)-pyridazinone (3g) To a solution
of 5-amino-6-phenyl-3(2H)-pyridazinone 2 (0.50 g, 2.7 mmol) in a 1 : 1 mix-
ture of acetic acid/water (25 ml) at 65 °C was added slowly a solution of
sodium nitrite (0.36 g, 5.3 mmol) in water (4 ml). The reaction mixture was
stirred at 65 °C during 2 h and the solid that precipitated was collected by fil-
tration. Further purification was carried out by recrystallization from a 1 : 1
mixture of ethyl acetate/methanol to give 3g as a red solid (0.45 g, 80%). mp
238—240 °C (dec.). IR: nmax cmϪ1 3422—3185 (NH2), 1633 (CO), 1588
(KBr): nmax cmϪ1 3808—3065, 2230, 1663, 1590. H-NMR: dH (300 MHz,
1
DMSO-d6): 3.21 (br s, 1H, OH), 4.10 (br s, 1H, CH), 7.55—7.48 (5H, m,
Aromatics), 13.94 (1H, br s, NH, deuterium oxide exchangeable). HR-MS of
sodium salt of 1a m/z Calcd for C13H7N4 NaO (Mϩ): 274.0467, Found:
274.0491.
5-Carboxy-4-cyano-6-phenyl-3(2H)-pyridazinone (3a) To a stirred
solution of aldehyde 1 (0.20 g, 1.0 mmol) in ethanol (30 ml) was added
1
(Aromatics). H-NMR: dH (300 MHz, DMSO-d6): 3.38 (2H, br s, NH2, deu-

December 2002
1577
terium oxide exchangeable), 7.40 (3H, m, Aromatics), 7.90 (2H, m, Aromat-
ics), 12.39 (1H, br s, NH, deuterium oxide exchangeable). HR-MS m/z Calcd
for C10H8N4O2 (Mϩ): 216.1963, Found: 216.1952.
5) Frank H., Heinisch G., “Progress in Medicinal Chemistry,” ed. by Ellis
G. P., Luscombe D. K., Elsevier, Amsterdam, 29, 141 (1992).
6) Laguna R., Montero A., Cano E., Raviña E., Sotelo E., Estévez I., Acta
Pharmaceutica Hungarica, 66, S43—S45 (1996) [Chem. Abstr., 126,
165993 (1996)].
7) Laguna R., Rodriguez-Liñares B., Cano E., Estévez I., Raviña E.,
Sotelo E., Chem. Pharm. Bull., 45, 1151—1155 (1997).
8) Montero-Lastres A., Fraiz N., Laguna R., Cano E., Estévez I., Raviña
E., Biol. Pharm. Bull., 22, 1376—1379 (1999).
4,5-Diamino-6-phenyl-3(2H)-pyridazinone (3h) To a solution of 5-
amino-4-nitroso-6-phenyl-3(2H)-pyridazinone (3g) (0.25 g, 1.1 mmol) in a
1 : 1 mixture of methanol/ethyl acetate was added a catalytic amount of plat-
inum (IV) oxide. The reaction mixture was stirred at room temperature
under a hydrogen atmosphere for 24 h. The mixture was filtered through sil-
ica, the solvents were removed under reduced pressure and the resulting
residue was purified by recrystallization from isopropanol (0.14 g, 60%). mp
189—191 °C (dec.). IR: nmax cmϪ1 3457 (NH2), 1636 (CO), 1585 (Aromat-
9) Sotelo E., Fraiz N., Yañez M., Laguna R., Cano E., Brea J. M., Raviña
E., Bioorg. & Med. Chem. Lett., 12, 1575—1577 (2002).
ics). 1H-NMR: dH (300 MHz, DMSO-d6): 5.63 (2H, br s, NH2, deuterium 10) Sotelo E., Fraiz N., Yáñez M., Terrades V., Laguna R., Cano E.,
oxide exchangeable), 7.60 (3H, m, Aromatics), 7.65 (2H, m, Aromatics),
Raviña E., Bioorg. & Med. Chem., 10, 2873—2882 (2002).
12.63 (1H, br s, NH, deuterium oxide exchangeable). C10H10N4O requires C, 11) Sotelo E., Raviña E., Estévez I., J. Heterocyclic Chem., 36, 985—990,
59.39; H, 4.98; N, 27.70, Found: C, 59.29; H, 4.77; N, 28.13.
(1999).
12) Estévez I., Raviña E., Sotelo E., J. Heterocyclic Chem., 35, 1421—
Acknowledgements We thank the Spanish Instituto de Cooperacion
Iberoamericana (ICI) for Eddy Sotelo’s Doctoral Fellowship.
1428 (1998).
13) Sotelo E., Pita B., Raviña E., Tetrahedron Lett., 41, 2863—2866
(2000).
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