Giuliana Leoncini

Coumarin, chromone, and 4(3H)-pyrimidinone novel bicyclic and tricyclic derivatives as antiplatelet agents: synthesis, biological evaluation, and comparative molecular field analysis.

As a further part of our chemical and biological studies in this field, we describe the multistep preparations of the properly substituted 2-(1-piperazinyl)chromone 1b, 4-(1-piperazinyl)coumarins 5c-h, their linear benzo-fused analogues 4a,b and 8a,b, bicyclic (15e-g) and tricyclic (15h,i) fused derivatives of 6-(1-piperazinyl)pyrimidin-4(3H)-one, and of the 4H-pyrido[1,2-a]pyrimidine derivatives 9b,c. The in vitro evaluation of their inhibitory properties towards human platelet aggregation induced in platelet-rich plasma by ADP, collagen, or the Ca (2+)ionophore A23187 showed the high activity of compounds 5d-g and 15f,g,i, among which the coumarins 5g and 5d proved to be, in that order, the most effective in vitro antiplatelet agents until now synthesized by us. Thus, in order to consider also the 4-aminocoumarin structural class, we developed a new statistically significant 3-D QSAR model, more general than the one previously obtained, through a further CoMFA study based on the antiplatelet activity data and molecular steric and electrostatic potentials of both the previously studied and herein described compounds.

THE EFFECT OF METHYLGLYOXAL ON THE GLYCOLYTIC ENZYMES

with free arginine and have found that the reaction rate, much faster with methylglyoxal than with phenylglyoxal, depends on the buffer type and increases by increasing the pH. Data on the inactivation mechanism of aldolase and glyceraldehyde-3-phosphate dehydrogenase by methyl- glyoxal have shown that one arginine residue is involved in the interaction of the enzyme with the ketoalde- hyde [S ,6].Since all glycolytic enzymes,except triose- phosphate isomerase, contain essential arginine resi- dues [7-l 11, it seemed worthwhile to compare the effect of methylglyoxal on the enzymes of the glyco- lytic pathway. 2. Materials and methods Substrates, enzymes and coenzymes were pur- chased from either Sigma or Boehringer Mannheim . Methylglyoxal (Fluka) freshly distilled and phenyl- glyoxal (Sigma), prepared every time before use were tested according to [ 121 and [ 131, respectively. Lactatt dehydrogenase (EC 1 .l .1.27) from rabbit muscle was assayed in the presence of 50 mM phosphate buffer (pH 7 .O), 3 .O mM pyruvate and 0.2 mM NADH; alco- hol dehydrogenase (EC 1 .l .l .l) and hexokinase (EC 2.7.1.1) from yeast were tested according to [14] and [

Activation of p38 MAPKinase/cPLA2 pathway in homocysteine-treated platelets.

Summary.  Hyperhomocysteinemia is considered a risk factor in arterial and venous thrombosis. The mechanism by which homocysteine (HCy) supports athereothrombosis is still unknown and may be multifactorial. Earlier in vitro studies demonstrated that HCy induced arachidonic acid release and increased thromboxane B2 (TXB2) formation. In this work, we found that HCy stimulated the rapid and sustained phosphorylation of platelet p38 mitogen‐activated protein kinase (p38 MAPK). The effect was time‐ and dose‐dependent. The HCy effect on p38 MAPK phosphorylation was prevented by N‐acetyl‐l‐cysteine and iloprost and was partially inhibited by nordihydroguaiaretic acid. Moreover, the incubation of platelets with HCy led to the phosphorylation of cytosolic phospholipase A2 (cPLA2). In addition HCy promoted cPLA2 activation, assessed as arachidonic acid release. The cPLA2 phosphorylation and activation were both impaired by the inhibition of  p38 MAPK through SB203580. This effect was not complete, reaching at the most the 50% of the total. In FURA 2‐loaded platelets, HCy induced a dose‐dependent intracellular calcium rise suggesting that the calcium elevation promoted by HCy could participate in the cPLA2 activation, leading to arachidonic acid release and TXB2 formation. In conclusion, our data provide insight into the mechanisms of platelet activation induced by HCy, suggesting that the p38 MAPK/cPLA2 pathway could play a relevant role in platelet hyperactivity described in hyperhomocysteinemia.

Synthesis, antiplatelet activity and comparative molecular field analysis of substituted 2-amino-4H-pyrido[1,2-a]pyrimidin-4-ones, their congeners and isosteric analogues

2-(1-Piperazinyl)-4H-pyrido[1,2-a]pyrimidin-4-one (5a) is a recently described in vitro inhibitor of human platelet aggregation which specifically inhibits the activity of high affinity cAMP phosphodiesterase. A number of substitution derivatives, isosteres, and analogues of 5a were now synthesized and tested in vitro for their inhibitory activity on human platelet aggregation induced in platelet-rich plasma by ADP, collagen, or the Ca2+ ionophore A23187. Among the most effective compounds, the 6-methyl, 8-methyl and 6,8-dimethyl derivatives of 5a resulted nearly as active as the lead when platelet aggregation was induced by ADP or A23187, but less active when collagen was the inducer. On the basis of present results and those previously obtained by us in this and 2-aminochromone structural fields, we have developed a statistically significant 3-D QSAR model, using comparative molecular field analysis (CoMFA), describing the variation of the antiplatelet activity in terms of molecular steric and electrostatic potential changes.

Synthesis and anti-platelet activity of some 2-(dialkylamino)chromones

Substituted 2-(diethylamino) or 2-(ethylamino)chromones 5a—g were obtained from the reaction of suitable phenols with the reagent ethyl N,N-diethyl- or N-ethylmalonamate/POCl3 (2a or 2b). The above compounds, together with other N-substituted 2-aminochromones, either prepared from related chromones by simple reactions (5h—k) or previously obtained by us (5l—w), were tested in vitro for their inhibitory activities against human platelet aggregation induced by collagen, ADP and arachidonic acid. Many compounds showed activity and some were more active than acetylsalicylic acid in the tests with ADP and arachidonic acid. When the 2-amino substituent of tested chromones was a diethylamino group, the highest activity was found. The presence in position 7 of electron releasing substituents (OH, OCH3, CH3) led to an increase of activity, whereas a decrease occurred when an electron withdrawing substituent was present in position 3 (NO2) or 6 (NO2, Cl).

Alkaline extraction and reverse-phase high-performance liquid chromatography of adenine and pyridine nucleotides in human platelets

The levels of adenine (ATP, ADP, AMP) and pyridine (NAD, NADH) nucleotides in human platelets have been measured by a simple and reproducible method. A rapid alkaline extraction allows a complete recovery of the compounds concerned. The metabolic ATP and ADP in the cytosolic fraction, the amount released upon thrombin stimulation, and the ADP bound to F-actin have also been evaluated. Analysis was performed by reverse-phase, isocratic high-performance liquid chromatography on a 5-microns Lichrosorb RP-18 column with uv detection at 254 nm.

Homocysteine decreases platelet NO level via protein kinase C activation

Hyperhomocysteinaemia has been associated with increased risk of thrombosis and atherosclerosis. Homocysteine produces endothelial injury and stimulates platelet aggregation. Several molecular mechanisms related to these effects have been elucidated. The study aimed to deeply investigate the homocysteine effect on nitric oxide formation in human platelets. The homocysteine-induced changes on nitric oxide, cGMP, superoxide anion levels and nitrotyrosine formation were evaluated. The enzymatic activity and the phosphorylation status of endothelial nitric oxide synthase (eNOS) at thr495 and ser1177 residues were measured. The protein kinase C (PKC), assayed by immunofluorescence confocal microscopy technique and by phosphorylation of p47pleckstrin, and NADPH oxidase activation, tested by the translocation to membrane of the two cytosolic subunits p47(phox) and p67(phox), were assayed. Results show that homocysteine reduces platelet nitric oxide and cGMP levels. The inhibition of eNOS activity and the stimulation of NADPH oxidase primed by PKC appear to be involved. PKC stimulates the eNOS phosphorylation of the negative regulatory residue thr495 and the dephosphorylation of the positive regulatory site ser1177. GF109203X and U73122, PKC and phospholipase Cgamma2 pathway inhibitors, respectively, reverse this effect. Moreover, homocysteine stimulates superoxide anion elevation and NADPH oxidase activation. These effects are significantly decreased by GF109203X and U73122, suggesting the involvement of PKC in NADPH oxidase activation. Homocysteine induces formation of the peroxynitrite biomarker nitrotyrosine. Taken together these results suggest that the homocysteine-mediated responses leading to nitric oxide impairment are mainly coupled to PKC activation. Thus homocysteine stimulates platelet aggregation and decreases nitric oxide bioavailability.

Synthesis and antiplatelet activity of 2-(diethylamino)-7-ethoxychromone and related compounds

Abstract 2-(Diethylamino)-7-ethoxychromone 3a and its 2-(1-piperidinyl)analogue 3b were synthesized by reaction of 3-ethoxyphenol 1 with 3-(dialkylamino)-3-oxo-propanoic acid ethyl ester 2 in the presence of phosphorus oxychloride. With a view to improve their biological activity the above 7-ethoxychromones 3 were submitted to some structural modifications firstly involving the 4-CO group. The 4H-chromenes 4 and the 4-thiochromones 5 were obtained by action of suitable reagents. The compounds 5 were then easily transformed to 4-(methylthio)chromenylium iodides 6 . Then from the 2-(diethylamino)-7-ethoxychromone 3a were obtained with suitable reactions the 3,6-diamino derivative 8 , the 3- and 6-formyl derivatives 9a,b and the Mannich base 10 . By action of acetic anhydride this latter compound yielded the methylenebis derivative 11 . Most of the above compounds were tested in vitro for their inhibitory activities against human platelet aggregation induced by collagen, ADP and arachidonic acid. Among the tested compounds the 2-(diethylamino)-7-ethoxychromone 3a showed the highest activity.

Effect of 2-(1-piperazinyl)-4H-pyrido[1,2-a]pyrimidin-4-one (AP155) on human platelets in vitro.

The effect on human platelets of 2-(1-piperazinyl)-4H-pyrido[1,2-a]pyrimi din-4-one (AP155) was tested in vitro by measuring cyclic adenosine monophosphate (cAMP) level, cytosolic Ca++, [(125I)]fibrinogen binding as well as aggregation induced by several agonists. AP155 dose-dependently inhibited aggregation both in platelet rich plasma (PRP) and in washed platelets (WP), exerting its maximal power in the presence of collagen, ADP and platelet activating factor (PAF). It specifically inhibited the activity of cAMP high affinity phosphodiesterase (PDE), resulting in a sufficient increase in cAMP levels to activate cAMP-dependent protein kinase. AP155 was able to inhibit aggregation, the increase in cytosolic Ca++ induced by thrombin, and fibrinogen binding to ADP or thrombin-stimulated platelets. Thus, this new pyridopyrimidine derivative exerts its antiplatelet activity by increasing cAMP intracellular concentration.

1,2-Fused pyrimidines VII. 3-(Dialkylamino)-1H-pyrimido[1,2-a]quinolin-1-ones and 2-(dialkylamino)-4H-pyrimido[2,1-a]isoquinolin-4-ones as antiplatelet compounds

Summary A number of 3-(dialkylamino)-1 H -pyrimido[1,2- a ]quinolin-1-ones 3 and 2-(dialkylamino)-4 H -pyrimido[2,1- a ] iso-quinolin-4-ones 4 were prepared by treating the corresponding chloro derivatives with an excess of dialkylamines. The highest in vitro antiplatelet activity was obtained when the dialkylamino substituent was 1-piperazinyl (compounds 3g and 4e ). The novel 2-(1-piperazinyl)-4 H -pyrido[1,2- a ]pyrimidin-4-one 2a was also prepared by an analogous procedure, which resulted in the most active compound towards all the platelet aggregation inducers used (ADP, collagen, A 23187). Moreover, some examples of 1-(dialkylamino)-3 H -pyrimido [1,2- a ]quinolin-3-ones 5 and 4-(dialkylamino)-2 H -pyrimido[2,1- a ]isoquinolin-2-ones 6 were also obtained (together with negligible or lower amounts of the corresponding isomers 3 and 4 , respectively) from the cyclocondensation of the appropriate ethyl N,N -dialkylmalonamate/phosphorus oxychloride reagents 13 with 2-aminoquinoline or 1-aminoisoquinoline. These latter compounds showed a rather low antiplatelet activity.