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Comparative effects of some 3-amino 4,6-diarylpyridazines on the biosynthesis in vitro of TXA2 and PGI2- and on the TXA2- and PGI2-synthesizing activities of cardiac tissue
Pmstaghndii Lcukottienegand Essential Fatty Acids (1990) 39,19-2.5 @I!J Longman Group UK Ltd 1990
Comparative Effects of some 3-Amino 4,6=Diarylpyridazines on the Biosynthesis in vitro of TX& and PG12- and on the TXA2- and PGI,-Synthesizing Activities of Cardiac Tissue Pham Huu Chanh*, V. Dossou-Gbete*, Coudert+ and C. Rubat’
R. Kaiser*, B. Lasserre*,
R. Chahine*, J. Couquelet+, P.
de la R&dation”, CNRS Institute de Physiologic-2F, Magendie, F-31400, Toulouse, France and ‘Laboratoire de Chimie ThLrapeutique, Groupe de Recherche en Pharmacochimie, Universitt? de Clermont-Ferrand, F-63001, Clermont-Fd Cedex, France (Reprint requests to PHC)
*"Pharmacologic
ABSTRACT.
Some 3-amino 4,Cdiarylpyridazine derivatives were tested for their effects on TXAz and PG12 biosyntheses in vitro and on the TXA2- and PGITsynthesizing activities of cardiac tissue. Horse platelet and aorta microsomes were used as sources of thromboxane and prostacyclin synthetases respectively. The TXA2and PGIrsynthesizhtg activities of cardiac tissue were studied on isolated perfused rabbit hearts (the heart microsomes being used both as TXAz synthetase and PGIz synthetase sources). TXBz and 6-keto PGFt, were determined by RIA. Among. the compounds under study, 3-morpholino 4,6-diphenylpyridaxine (ZZZ)was shown to inhibit specitlcally the TXA2 synthetase. Substitution of the morpholino group by a diiethylamino one (Z) reinforced the inhibiting effects on TXAz synthetase but it also revealed a slight anti-prostacyclin synthetase action of the molecule. Replacement of 3-morpholmo moieties by either a 3-hydra&to (IV), or a 2-dimethylaminoethylamino (V), or a 2-morpholinoethylamio group (VZ) abolished completely the effects of the molecule on TXA2 and PGIz synthetases. Likewise the addition of chlorine on the para-position on the phenyl ring of Z neutralized all its inhibitory effects both on TXAz and PGIz synthetases in vitro. None of the S-amino 4,6_diarylpyridazine derivatives was active on either the TXAr or PGIrsynthesizing activities of cardiac tissue.
INTRODUCTION
Chemistry
Thromboxane A2 (TXA*) and prostacyclin (PGI*) are broadly involved in the physiology and physiopathology of the cardiovascular system: they play a prominent role in homeostasis; a lack of balance in their production may give rise to cardiovascular disorders such as hypertension, thromboses, myocardial ischaemia and diabetes (l4). Modulators of eicosanoid biosynthesis may be valuable as potential therapeutic agents for cardiovascular diseases. Earlier data obtained in this area with pyridazinic compounds (5-7) prompted us to extend our investigations to other derivatives of the series. This study aimed to show the effects of 3-amino, 4,6-diarylpyridazines on both TXAz and PGI, biosyntheses in vitro, and TXA2- and PG12 synthesizing activities of isolated rabbit heart tissue.
Six 3-amino, 4,6_diarylpyridazines were selected for our study (Fig. 1). They were prepared from chalcones according to the scheme represented in Figure 2. In a first step, additional of hydrogen cyanide to chalcones was performed via an exchange with acetone cyanhydrin. Hydrolysis of the intermediate ar-keto nitriles led to 2,4-diary1 4-oxobutanoic acids which in turn, by condensation with hydrazine hydrate, were converted into 4,6-diary1 4,5-dihydro 3(2H)-pyridazinones (8). The latter were dehydrogenated by bromination and dehydrobromination, then treated with phosphorus oxychloride. The resulting 3-chloro pyridazines reacted with either amines or hydrazine hydrate, or diamines to give the expected 3-amino pyridazine derivatives (I to VI). Preparation and analysis of these compounds have been previously reported (9).
19
-
,.(z-dl.ethylan,naethylamlno) 4,6_dLphe”yl
pyridazlne
)_(Z_norpholinoethyIsmino)
4,6-diphe”yl
pyr.dsz*“e
VI
V
Fig. 1 Chemical structure of six 3-amino 4,6_diarylpyridazines
under study
or one fixed concentration of enzyme and various concentration ranges of arachidonic acid (from 32.8 x 10e6 M to 26.2 x lo- M). The incubation ivas carried out for 2 minutes at 4°C. The reaction was stopped by addition of 0.2 M citric acid so that the pH was 3.5. Compounds under study were preincubated with enzyme sources for 10 min at 37°C: the enzyme reaction was started by addition of arachidonic acid. At the end of the incubation (2 min), TXBz or 6keto PGFr, were extracted twice by 850 ~1 diethylether. The ether phase of both extractions ‘(850 ~1 X 2) was centrifuged at 4°C for 10 minutes (600 x g, J 21B Beckman refrigerated Centrifuge). The supernatant (organic phase) was evaporated’ under nitrogen to dryness then dissolved in 200 ~1 of RIA buffer for RIA determination of TXBz or 6-keto PGFi,. Arachidonic acid was dissolved in hexane then evaporated to dryness before being diluted in RIA buffer for experimentation. As 3-amino 4,6_diarylpyridazine derivatives were not hydrosoluble, they were dissolved with just one drop of hydrochloric acid; the obtained acidified solvent did not alter either the chemical structure of the molecules under study or the biosyntheses of TXAz or PGIz generated by incubation of AA with either HPM or HAoM. Moreover when perfused into the coronary circulation of rabbit isolated hearts this solvent did not change either the heart mechanism (contractile force, rhythm or output), or the TXA2-synthesizing and PG12-synthesizing activities of cardiac tissue.
TXA2- and PG12-synthesizing activities of cardiac tissue
$: NH
Fig. 2
Scheme representing the various stages in the synthesis of 3-amino 4,6-diarylpyridazines
MATERIALS AND METHODS Biosyntheses of TXAz and PGIz in vitro TXA:, and PGI2 biosyntheses were carried out by incubation of arachidonic acid (substrate) with horse platelet microsomes (HPM) or horse aorta microsomes (HoAM), sources of TXA;! and PGIZ synthetases respectively (6, 10). For each compound, 2 series of experiments were carried out with: - either one fixed concentration of arachidonic acid (32.8 x 10m6M) and increasing concentrations of enzymes (HPM, HAoM).
Isolated rabbit hearts used as an experimental model were perfused according to Langendorff’s method as previously described (11). Perfusion was performed in a retrograde way, via the aorta and the coronary circulation. Thus the heart can be considered as a cardiac tissue model kept alive by a Tyrode solution perfusion. Hearts having fulfilled the selection standards (steady rhythm, contractile force, and output) after a 20 min resting period, were allotted at random into various groups: - group I: control hearts perfused with a standard Tyrode solution - other groups: hearts perfused for 10 minutes with a single concentration of compounds under study. Then they were washed by a 2 min perfusion of standard Tyrode solution. The hearts were then removed from the perfusion device and immediately frozen in liquid nitrogen. Each heart was used once either as a control heart or as a single-dose-treated heart. Microsomes from both control and treated hearts were used as
Comparative Effects of some 3-Amino 4,6_Diarylpyridazines
enzyme sources to initiate the biosyntheses of TXA2 and PG12. The ability of heart microsomes to generate the formation of either TXA;? or PG12 when they were incubated with arachidonic acid represents the TXA2-synthesizing or PG12-synthesizing activities of cardiac tissue. The differences between the control and drug-perfused hearts in their eicosanoid-synthesizing ability enabled us to assess the effects of these compounds on the TXA?and PGIT synthesizing activities of cardiac tissue. RIA determination of TXBz and CKeto PGFI, The method of RIA determination of TXB2 and 6keto PGFi, has been described elsewhere (5, 7). Reagents were dissolved in standard RIA buffer containing 0.1% gelatine (pH 7.4). The volume of incubation mixture was 250 ~1: 100 ~1 RIA buffer, either 50 ~1 standard solution (containing either l1000 pg TXB*, or lo-600 pg 6-keto PGF,,) or 50 ~1 extract, 50 ~1 labelled TXB,! or 6-keto PGFi, (1.11 x lo4 dpm), 50 ~1 immun-serum (which bound 50% of 3H TXB2 or 3H 6-keto PGFt, in the absence of competition induced by unlabelled TXB2 or 6-keto PGF,,). Vials were vortexed, incubated at room temperature for 1 hr, then overnight (over 16 h) at 4°C. Antibody-bound and -free TXB2 or 6-keto PGF,, were separated using dextran-coated charcoal suspension (5 mg Norit A/O.5 mg dextran T70/1 ml phosphate buffer) at 4°C for 13 minutes. After a 2,000 x g centrifugation (J 21B Beckman refrigerated centrifuge) at 4°C for 10 minutes, an aliquot of the antibody-bound TXB2 or 6-keto PGF,, from supernatants was sampled into scintillating fluid and the radioactivity was counted in a Packard Tri-carb spectrometer 2450tR). The binding of various samples to the maximal binding ratio was calculated for each sample, the concentration of which was determined from a standard curve after logit transformation. The detection limits of RIA procedures were 10 and 15 pg for TXB2 and 6-keto PGF,, respectively.
21
blood. The pellet was thereafter suspended in Tris buffer (100 mM, pH 7.5). The platelet suspension was frozen and thawed at least three times, then centrifuged at 5,000 x g for 15 min. The supernatant was then centrifuged at 105,000 x g for 60 min (Beckman L5-65 ultracentrifuge) (12). The pellet was suspended again in Tris buffer (pH 7.5) and sampled into vials and lyophilized. Horse aorta microsomes (HAoM) Horse aorta was dissected free of surrounding tissues, chopped into pieces, ground and homogenized in ice cold phosphate buffer at pH 7.5. The homogenate was centrifuged at 12,000 x g for 10 min. The supernatant was filtered through gauzes then centrifuged at 105,000 x g for 60 min. The resulting pellet was homogenized in phosphate buffer and lyophilized (5-7, 10). Rabbit heart microsomes Isolated rabbit hearts were thawed and dissected to separate adjacent vessels and tissues. Then they were chopped, ground and homogenized in the Potter homogenizer in ice cold phosphate buffer at pH 7.5. Heart microsomes were obtained by a 60 min centrifugation at 105,000 xg as in the preparation of horse aorta and platelet microsomes. Protein was determined according to the method described by Lowry et al (13) and modified by Hartree (14) by using bovine serum albumin as a standard, Data analysis Significance of the results was statistically assessed by using two-way analysis of variance with Student’s t test for unpaired and paired data. Statistical methods of regression analysis were used in enzyme kinetics estimation (15). For the calculation of the regression equations and effective doses, probit transformation of the inhibition percentages was used. All values in Tables and Figures were reported as means *SEM.
Preparation of microsomes Horse blood and aorta were taken from a slaughter house. Horse platelet microsomes (HP&f) Blood was withdrawn directly from the carotid artery immediately after the death of the animal, and poured into plastic jars containing 0.11 M 5,5 HZ0 sodium citrate (1 vol citrate for 9 vol blood). Platelet rich plasma (PRP) was obtained from a first 10 min centrifugation at 200 x g then another 15 min centrifugation at 2,000 x g of the titrated
RESULTS TXAz biosynthesis in vitro Incubation of a fixed concentration of AA (32.8 x 10m6M) with HPM concentrations (expressed in protein equivalence) ranging from 250 to 1,000 pg protein, yielded amounts of TXBz which rose with increasing HPM concentrations: 122.9 + 4.2, 172.0 + 4.5, 170.8 f 2.7 and 189.9 f 8.3 pg TXBz vs. 250, 500, 750 and 1,00 pg protein respectively. When preincubated with HPM for 10 min prior to addition of arachidonic acid, compounds II, IV,
22
Prostaglandins Leukotrienes and Essential Fatty Acids Table 1 Comparative effects of compounds I and III on TXA, biosynthesis in vitro when using a fixed concentration of arachidonic acid (AA: 32.8 x lo-” M) against increasing concentrations of horse platelet microsomes (HPM): determination of the amounts (in picograms, pg) of TXB2. n = 7 p <: ** 0.01. 2 x 10-j M HPM (pg Pr)
Control
250 500 750 1000
122.9 172.0 170.8 189.9
I x lo_.3M
I
+ + + +
4.2 4.5 2.7 8.3
113.3 117.6 141.5 156.6
III + f f +
4.4 8.2** 4.6** 12.1**
I
114.0 131.2 138.7 136.3
f + f f
4.8 5.1** 8.0** 5.9**
111
41.9 64.5 55.3 66.8
f 5.4** I?I 8.0** + 7.8** f 9.8**
60.8 66.7 79.0 52.4
+ + + +
4.1** 8.9** 8.7** 4.1**
Table 2 Comparative effects of compounds I and Ill on the biosynthesis of TXA2 induced by the incubation of one fixed concentration of HPM (125 fig Pr) with different concentrations of arachidonic acid: the amounts of TXB, formed were expressed in pg (m f ESm; n = 5; p< * 0.01; ** 0.001) Arachidonic Acid (AA)
32.8 65.6 13.1 19.6 26.2
x x x x x
10-hM lo-” M lo-‘M lo-’ M lo-” M
I Control
52.5 103.2 178.6 187.3 200.2
III
I
1 x W4M
+ + ? + +
3.0 9.9 13.0 15.2 16.3
35.0 70.3 113.4 131.4 148.6
+ + + + +
3.0* 7.4* 8.0* 8.0’ 8.0*
37.5 72.0 112.1 124.3 136.4
f 2.0* k 7.6% I!z 8.0* z.t 8.1* f 9.7*
24.8 48.0 82.8 81.6 97.7
111 5 xMI IO-’ M
f f f zt +
2.4** 5.9** 8.4** 8.8** 10.6**
I
III 1 x IO” M
32.0 64.2 102.0 111.5 120.9
?z + + + +
2.0** 6.7** 5.4** 9.5** 10.4**
21.2 41..0 72.6 66.3 85.1
+ + f + +
2.2** 5.2** 7.8** 7.9** 8.0**
22.8 45.9 72.6 72.4 80.3
f f + + +
1.8** 3.9** 5.1** 7.8** 7.9**
Table 3 Inhibitory effects of compounds I and III on TXA, biosynthesis in vitro: IDS0 (dose capable of inhibiting by 50% the thromboxane synthetase activity at various concentrations of arachidonic acid, AA). AA
6.56 1.31 1.97 2.62
I
x lo-’ M x 10-j M x 1O-5M x 10-j M
4.16 3.77 2.53 4.75
Table 4 Kinetics of the TXAz-synthetase
Control
III
x 1O-4M x lO-‘M x 10-j M x 10-j M
+ + f +
2.78 3.59 3.67 2.39
x x x x
lo-’ h? lo-‘M lo-‘M lo-‘M
8.95 5.54 5.34 6.25
x 1O-4M x 1O-4M x 1O-4M x 1O-4M
f + f f
4.81 4.92 5.49 5.44
x x x x
lo-’ M lo-‘M lo-’ M lo-‘M
inhibiting action of compounds of I and Ill
Km
V??laX
1.215 x 1O-4M + 3.290 x lo-’ M
3.460 x lOmyM.min-’ f 4.460 x lo-“’ M.min-’
1.649 x 1O-4M f 2.499 x lo-’ M 1.288 x 1O-4M + 3.925 x lo-‘M 1.274 x 1O-4M + 5.109 x lo-’ M
2.760 X lOmyM.min? f 2.13 x lo-“‘M.min-’ 1.64 x lO_‘M.min-’ rt 2.39 x lO_“‘M.min-’ 1.398 x lo-’ M.min-’ + 2.669 x lo-“’ M.min~’
1.209 x 1O-4M + 3.289 x lo-’ M 1.212 x 1O-4M f 2.545 x lo-‘M 9.78 x lo-‘M + 2.655 x lo-’ M
2.29 x lO_‘M.min- f 1.77 x lO_“‘M.min-’ 2.05 x lOmyM.min-’ + 1.98 x lo-“‘M.min-’ 1.27 x lo-‘M.minI! 1.45 x lO_“‘M.min-’
I
1 x 1O-4M 5 x 1O-4M 1 x 10-j M Ill
1 x lo-’ M 5 x lo-’ M 1 x 10” M
V and VZ did not significantly change the amounts of TXB2 formed. They neither stimulated nor inhibited TXA2 and PG12 biosyntheses. Under the influence of compounds Z and III, when incubated with arachidonic acid, HPM synthesized smaller amounts of TXB2 compared to those synthesized to control experiments without preincubation with the compounds under study. Compounds Z and ZZZinhibited TXA2 biosynthesis, this inhibition increased with increasing doses. Table I reports the most important effects observed at various doses of Z and
111. Similar data were obtained when using a fixed concentration of HPM with increasing concentrations of arachidonic acid: in fact, a concentration of HPM equivalent to 125 pg protein incubated with various dose ranges of arachidonic acid (32.8 x lo6 M, 65.6 x 1O-6M, 13.1 x 1O-5M, 19.6 x l(r5 M, 26.2 x lop5 M) generated the formation of 52.5 + 3.0, 103.2 k9.9, 178.6 f 13.0, 187.3 f 15.2 and 200.2 + 16.3 pg TXB2 respectively. Under these experimental conditions, Z and ZZZonce more inhibited the formation of TXA;! (Table 2). Table 3 reports
Comparative Effects of some 3-Amino 4,6-Diarylpyridazines
23
PGI2 biosynthesis in vitro
Fig. 3 Comparative effects of I and Ill on the biosynthesis of TXAz in vitro: Lineweaver-Burk plot showed the relationship between the reciprocals of TXBZformation velocity (l/V) and the reciprocals of substrate concentrations (l/S) in the absence (control) and in the presence of various concentrations of I and III for a concentration of HPM equivalent to 125 pgprotein. Straight lines were fitted by linear regression analysis using a computer program. Each point represents the mean of 5 samples with SEm (not shown) being less than 10% of this value. The correlation coefficients for the lines were between 0.989 and 0.995.
the inhibiting effects (IDsa) of Z and 111 on TXA2 synthetase: compound I was found to be more active than III. Graphical Lineweaver-Burk representation of l/v (reciprocals of TXB2 formation rates) vs. l/S (reciprocals of substrate concentrations) showed that the slopes of regression lines obtained with drug-pretreated HPM were higher than the slope of the control regression line, thus demonstrating that both compounds Z and 111 inhibited the biosynthesis of TXA;! (Fig. 3). Table 4 r&presents the kinetic parameters of the inhibitory effects of compounds Z and ZZZon TXA2 biosynthesis. These data showed that both compounds inhibited the TXAZ synthetase, their action being of a non-competitive type,
PG12 biosynthesis generated by incubation of a fixed concentration of arachidonic acid (32.8 x 10-b M) with horse aorta microsomes (HAoM) was not significantly modified by III, but it was inhibited by I: this inhibition of PGIZ synthetase occurred in both cases; when incubating either a fixed concentration of arachidonic acid with increasing concentrations of HAoM, or a fixed concentration of HAoM with increasing concentrations of AA (Table 5). Graphical Lineweaver-Burk representation showed that the inhibitory effects of Z on PG12 synthetase in vitro was of competitive type (Fig. 4). Using Dixon’s method, representing the relationship between the reciprocals of 6-keto PGF1, formation vs. various concentration ranges of Z fed us to the same conciusion, namely that inhibition of the PG12
1 I
0
3.8
I
5.07
,
7.61
Inhibitor effects of I on the biosynthesis of PGIz in vitro. Lineweaver-Burk graphical representation obtained by plotting l/V (V = 6-keto PGFla formation rate) vs. l/S (S = concentrations of the substrate i.e. arachidonic acid, AA) in the absence (control C) and in ‘4 presence of varifus conceflrations of I (1 = I x lo- M, 2 = 5 x It)- M, 3 = 1 x 10 M) Fig. 4
Table 5 Inhibitory effects of I on the PGll synthetase in vitro yielded by the incubation or arachidonic acid with HAoM amounting to 100 pg protein. Each value (in pg of 6-keto PGF,(u) represented the mean (m + ESm) of 5 experiments. p <: * 0.05; ** 0.01. Arachidonic Acid
Control
129.7 + 4.9
165.6 f 5.5
178.1 f 5.4
189.7 f 5.7
113.0 + 8.9 90.0 + 3.1* 87.0 t 6.3
150.2 f 8.4 128.0 f 3.3** 123.0 + 6.0**
161.7 + 6.1 145.6 f 7.2** 142.2 f 7.3**
176.4 + 4.5 161.2 f 9.X* 147.9 + 8.3**
( HAoM)
I 1 x lO_‘M 5 x 1OY’M 1 x 10-j M
Table 6 Kinetics of the PGI, synthetase-inhibiting
Control ( HAom) 1 x 1O-4M 5 x 1W4M 1 x 10-l M
action of compound I
Km
Vmax
4.47 x 1O-5M ? 2.72 x 10-h M
2.51
X
IO?
M.min-’ f 3.60
X
lo-” M.min-’
5.64 x IO-’ M + 5.75 x 10-h M 9.06 x lo-’ M + 5.34 x lo-+ M 7.79 x lo-’ M f 8.12 x lO+ M
2.38
x x
lWy M.min-’ + 6.70 10“ M.min-’ f 5.12 10” M.min-’ * 7.56
x
2.42 2.18
10.” M.min-’ lo-” M.min-’ lo-” M.min-’
x
x x
24
Prostaglandins Leukotrienes and Essential Fatty Acids Table 7 Data representing
the inhibitory effects of I on the biosynthesis of PG12 in vitro generated by the incubation of a fixed concentration of arachidonic acid (32.8 x lo-” M) with increasing concentrations of HAoM (expressed in pg protein). HAo (Dg Pr)
MID,,,
25 50 lot) 200
3.69 2.15 6.45 9.27
x x x x
IO-‘M lo-’ M 1O-JM 10m4M
+ + + ?
2.59 7.64 8.77 5.71
x x x x
10.’ M lo-‘M IO-’ M lo-‘M
Regression equation
Correlation
y y y y
‘r r r r
= = = =
0.925x 0.625x 0.417x 0.753x
-
1.60 0.35 1.63 0.77
= = = =
0.999** 0.999** 0.999** 0.999**
** : p < 0.01
synthetase induced by Z was of competitive type: The kinetic data of the PG12 synthetase in the presence of Z are represented in Table 6. This action was however weak: at the concentration ranges used, Z did not inhibit the PG12 synthetase by more than 30% (Table 7).
hearts: the PG12-synthesizing activity hearts was not significantly different found in control hearts.
TXA+ynthesizing
Our investigations aimed to study the effects of some 3-amino 4,6_diarylpyridazine derivatives, lirstly on the TXAz and PGIz-synthetases in vitro, and secondly on the TXA2- and PGI,?-synthesizing activities of the cardiac tissue of isolated and non-working rabbit hearts kept alive by means of a standard Tyrode perfusion into the coronary circulation. Under the experimental conditions adopted, our study showed that among the six 3-amino 4,6-diarylpyridazine derivatives tested, only 3-dimethylamino ,4,6_diphenylpyridazine (Z) and 3-morpholino 4,6diphenylpyridazine (ZZZ) were able to inhibit the TXA2 synthetase in vitro: Z was the more active with an IDsc = 2.5 x 10e4 M f 3.7 x 10m8,whereas the IDso of ZZZwas 5.3 x 1O_4M f 5.5 x lo_, M. Unlike ZZZwhich was completely inactive on the PG12 synthetase, Z inhibited it. ZZ,ZV, V and VI were not active either on the TXA2 synthetase or on the PGIz synthetase in vitro. Moreover none of these 3-amino 4,6_diarylpyridazine derivatives modified either the TXAZ-synthesizing activity or the PGITsynthesizing activity of cardiac tissue. From the data thus obtained, the following conclusions could be drawn: 1) As Z inhibited both that TXAz and PG12 synthetases in vitro it would suggest that Z may act on the cyclooxygenase. But owing to the fact that Z had quite different effects on the TXA2 and PGI2 synthetases, the direct effects of Z on both enzymes were obvious but it was not excluded that Z may also have a partial inhibitory effect on PG endoperoxide synthetase. 2) The morpholino group in the 3-position of pyridazine appeared to be necessary to give specific anti-TXA2 synthetase activity to the molecule since its replacement by a dimethylamine group stimulated the inhibitory effects on TXA, synthetase, but simultaneously it conferred inhibitory effects on the PG12 synthetase in vitro to the molecule.
activity of cardiac tissue
Microsomes from control isolated non-working rabbit hearts (perfused with a standard Tyrode solution) induced the formation of TXB2 from arachidonic acid: the RIA-determined amounts of TXB2 increased with the increasing concentrations of microsomal fractions from hearts under study. 21.7 It: 1.9, 37.6 f 3.5, 65.7 k 5.1, 78.2 & 7.4, 99.7 f 8.9 and 118.8 f 8.3 pg TXB2 were found to result from 25, 50, 100, 150, 200 and 300 pg protein respectively. Thus microsomes from rabbit hearts were endowed with TXATsynthesizing activity. Moreover we demonstrated that this TXAZ-synthesizing activity of cardiac tissue could be modified (either stimulated or inhibited) if the heart was previously perfused with chemical compounds (16). But when perfused at concentrations ranging from 1 X 10e5 M to 1 X 10e3 M into the coronary circulation, the compounds under study did not significantly alter the TXATsynthesizing activity of drug-perfused-heart microsomes: they neither inhibited nor stimulated the TXAZ-synthesizing activity of cardiac tissue. PG12-synthesizing activity of cardiac tissue Heart microsomes were also found to have a PGI. synthesizing activity. Among the 6 compounds under investigation none of them was active on the PGI*-synthesizing activity of cardiac tissue. In fact, when incubated with arachidonic acid (32.8 X locontrol from fractions microsomal (jM) (Tyrode-perfused) hearts amounting to 25, 50, 100, 150, 200 and 300 pg protein, induced the formation of 65.1 + 6.4, 82.9 k 7.8, 120.0 f 9.5, 140.7 rt 13.7, 170.2 + 15.7 and 177.7 + 15.5 pg 6-keto PGFlti respectively. Microsomes from the hearts ‘perfused with compounds under study at concentrations ranging from 1 x 10m5M to 1 x 10e3 M, yielded the same data as those obtained in control
of treated from that
DISCUSSION
Comparative Effects of some 3-Amino 4,6_Diarylpyridazines
3) The replacement of the dimethylamino group by either a hydrazino group, or a 2-dimethylaminoethylamino or 2-morpholinoethylamino chain suppressed any activity of the molecule on both TXA2 and PGIz synthetases. 4) Likewise, the introduction of a chlorine in the .para-position on the phenyl ring of I abolished any effect of the molecule on the TXAz synthetase and PG12 synthetase. 5) None of the 3-amino 4,6_diarylpyridazine derivatives was active on either the TXAz-synthesizing or PG12-synthesizing activities of cardiac tissue. 6) This study confirmed once more the existence of the TXAz-synthesizing and PGIr synthesizing activities of the cardiac tissue. Such activities have been revealed in various experiments and could be modified (either stimulated or inhibited) by synthetic organic compounds (16). Among the six 3-amino 4,6_diarylpyridazine derivatives under study, I and III were not capable of modifying either the TXAz-synthesizing or the PGIz-synthesizing activity of cardiac tissue, but there were active on the TXA2 and PG12 synthetase in vitro. II, ZV, V and Vf being inactive on both enzymes in vitro were also devoided of any activity on the TXAT and PGIz-synthesizing activities of cardiac tissue. Besides, many other compounds (16) which inhibited the TXAZ-synthesizing activity of cardiac tissue did not have any activity of TXAz and PG12 synthetases in vitro. These data contributed to demonstrate the entity of the TXA? and PGITsynthesizing activities of cardiac tissue i.e. these activities could evolve quite independently from those affecting the TXA? and PGI:, synthetases. Acknowledgements We thank Prof. P. Tronche for his encouragement and continued support of this project, together with the Rotary International Club Toulouse-Sud (France) for its aid and support.
References I. Moncada S.. Prostacyclin from discovery to clinical applications. J Pharmacol (Paris) 16 Suppl 5: 71-88, 198.5.
2. Pham Huu Chanh., Prostaglandines et hypertension. Paroi ArtCrielle/Arterial Wall 6: 239-248,198O. 3. Pham Huu Chanh, Lasserre B, Dossou-Gbete V. Kaiser R, Courtault A, Glock Y, JPh Nacher., Angor spastique. Arch Mal Coeur Vaisseaux 79 (4): 605. 1986. 4. Pham Huu Chanh, Lasserre B, Pham Huu Chanh A.. Icosanoids and diabetes. VIth Int Conf on PGs and Related Compounds. Florence (Italy) June 3-6, 1986. 5. Pham Huu Chanh, Lasserre B, Pham Huu Chanh A. Dossou-Gbete V. Palhares de Miranda A L. Kaiser R. Tronche P. Couquelet J, Inhibition of thromboxane biosynthesis by 3-dimethylamino 5-(3’-trifluoromethyl benzylidene) 6-methyl (4H) pyridazine (PCSS). IRCS Med Sci 13: 690-691, 1985. 6 Pham Huu Chanh. Lasserre B, Tronche P, Couquelet J, Dossou-Gbete V, Palhares de Miranda A L, Enhanced prostacyclin biosynthesis and decreased thromboxane formation by 5-(2’.6’-dichloro benzylidene) 6-methyl (4H) pyridazine (PC89). PG LT Med 19: 37-49, 1985. 7. Pham Huu Chanh. Lasserre B. Dossou-Gbete V. Couquelet J. Tronche P.. An inhibitor of prostacyclin biosynthesis derived from aminopyridazine. PG LT Med 30: l-7, 1987. 8. Coudert P. Couquelet J. Tronche P.. A new synthetic route to 4,6-diarylpyridazinones. J Heterocyclic Chem 25: 799-802. 1988. 9. Coudert P, Rubat C, Couquelet J M. Bastide J., Synthuse et etude de I’effet antidepresseur de diaryl-4.6 amino-3 pyridazines. analogues de la minaprine. II Farmaco Ed Sci 43: 793-800, 1988. 10. Tai H H, Yuan B. Wu A T.. Transformation of arachidonate into 6-keto PGFl,. thromboxane Bz and prostaglandin E2 by sheep lung microsomal fraction. Biochem J 170: 441-444. 1978. Il. Pham Huu Chanh. Junstad M, Wennmalm A.. Augmented noradrenaline release following nerve stimulation after inhibition of prostaglandin synthesis with indomethacin. Acta Physiol Stand 86: 563-567. 1972. 12. Needleman P. Moncada S, Bunting S, Vane J R, Hamberg M. Samuelsson B., Identification of an enzyme in platelet microsomes which generates thromboxane A? from prostaglandin eidoperoxides. Nature 261: 5588560. 1976. 13. Lowry 0 H. Rosenbrough N J. Farr A L. Randall R J.. Protein measurement with the Folin phenol reagent. J Biol them 193: 265-275. 1951. 14. Hartree E F. A modification of the Lowry method that gives a linear photometric response. Annal Biochem 48: 422-427. 1972. IS. Wilkinson G N.. Statistical estimations in enzyme kinetics. Biochem J 80: 324-332. 1961. 16 Pham Huu Chanh. Lasserre B, Pham Huu Chanh A. Palhares de Miranda A L. Tronche P, Couquelet J. Rubat C. A selective inhibitor ol ‘thromboxane synthetase activity of rabbit heart tissue: a pyridazinic derivative. PG LT & Ess Fiitty Acids 33: 143-147, 19X8.
25
Comparative Effects of some 3-Amino 4,6=Diarylpyridazines on the Biosynthesis in vitro of TX& and PG12- and on the TXA2- and PGI,-Synthesizing Activities of Cardiac Tissue Pham Huu Chanh*, V. Dossou-Gbete*, Coudert+ and C. Rubat’
R. Kaiser*, B. Lasserre*,
R. Chahine*, J. Couquelet+, P.
de la R&dation”, CNRS Institute de Physiologic-2F, Magendie, F-31400, Toulouse, France and ‘Laboratoire de Chimie ThLrapeutique, Groupe de Recherche en Pharmacochimie, Universitt? de Clermont-Ferrand, F-63001, Clermont-Fd Cedex, France (Reprint requests to PHC)
*"Pharmacologic
ABSTRACT.
Some 3-amino 4,Cdiarylpyridazine derivatives were tested for their effects on TXAz and PG12 biosyntheses in vitro and on the TXA2- and PGITsynthesizing activities of cardiac tissue. Horse platelet and aorta microsomes were used as sources of thromboxane and prostacyclin synthetases respectively. The TXA2and PGIrsynthesizhtg activities of cardiac tissue were studied on isolated perfused rabbit hearts (the heart microsomes being used both as TXAz synthetase and PGIz synthetase sources). TXBz and 6-keto PGFt, were determined by RIA. Among. the compounds under study, 3-morpholino 4,6-diphenylpyridaxine (ZZZ)was shown to inhibit specitlcally the TXA2 synthetase. Substitution of the morpholino group by a diiethylamino one (Z) reinforced the inhibiting effects on TXAz synthetase but it also revealed a slight anti-prostacyclin synthetase action of the molecule. Replacement of 3-morpholmo moieties by either a 3-hydra&to (IV), or a 2-dimethylaminoethylamino (V), or a 2-morpholinoethylamio group (VZ) abolished completely the effects of the molecule on TXA2 and PGIz synthetases. Likewise the addition of chlorine on the para-position on the phenyl ring of Z neutralized all its inhibitory effects both on TXAz and PGIz synthetases in vitro. None of the S-amino 4,6_diarylpyridazine derivatives was active on either the TXAr or PGIrsynthesizing activities of cardiac tissue.
INTRODUCTION
Chemistry
Thromboxane A2 (TXA*) and prostacyclin (PGI*) are broadly involved in the physiology and physiopathology of the cardiovascular system: they play a prominent role in homeostasis; a lack of balance in their production may give rise to cardiovascular disorders such as hypertension, thromboses, myocardial ischaemia and diabetes (l4). Modulators of eicosanoid biosynthesis may be valuable as potential therapeutic agents for cardiovascular diseases. Earlier data obtained in this area with pyridazinic compounds (5-7) prompted us to extend our investigations to other derivatives of the series. This study aimed to show the effects of 3-amino, 4,6-diarylpyridazines on both TXAz and PGI, biosyntheses in vitro, and TXA2- and PG12 synthesizing activities of isolated rabbit heart tissue.
Six 3-amino, 4,6_diarylpyridazines were selected for our study (Fig. 1). They were prepared from chalcones according to the scheme represented in Figure 2. In a first step, additional of hydrogen cyanide to chalcones was performed via an exchange with acetone cyanhydrin. Hydrolysis of the intermediate ar-keto nitriles led to 2,4-diary1 4-oxobutanoic acids which in turn, by condensation with hydrazine hydrate, were converted into 4,6-diary1 4,5-dihydro 3(2H)-pyridazinones (8). The latter were dehydrogenated by bromination and dehydrobromination, then treated with phosphorus oxychloride. The resulting 3-chloro pyridazines reacted with either amines or hydrazine hydrate, or diamines to give the expected 3-amino pyridazine derivatives (I to VI). Preparation and analysis of these compounds have been previously reported (9).
19
-
,.(z-dl.ethylan,naethylamlno) 4,6_dLphe”yl
pyridazlne
)_(Z_norpholinoethyIsmino)
4,6-diphe”yl
pyr.dsz*“e
VI
V
Fig. 1 Chemical structure of six 3-amino 4,6_diarylpyridazines
under study
or one fixed concentration of enzyme and various concentration ranges of arachidonic acid (from 32.8 x 10e6 M to 26.2 x lo- M). The incubation ivas carried out for 2 minutes at 4°C. The reaction was stopped by addition of 0.2 M citric acid so that the pH was 3.5. Compounds under study were preincubated with enzyme sources for 10 min at 37°C: the enzyme reaction was started by addition of arachidonic acid. At the end of the incubation (2 min), TXBz or 6keto PGFr, were extracted twice by 850 ~1 diethylether. The ether phase of both extractions ‘(850 ~1 X 2) was centrifuged at 4°C for 10 minutes (600 x g, J 21B Beckman refrigerated Centrifuge). The supernatant (organic phase) was evaporated’ under nitrogen to dryness then dissolved in 200 ~1 of RIA buffer for RIA determination of TXBz or 6-keto PGFi,. Arachidonic acid was dissolved in hexane then evaporated to dryness before being diluted in RIA buffer for experimentation. As 3-amino 4,6_diarylpyridazine derivatives were not hydrosoluble, they were dissolved with just one drop of hydrochloric acid; the obtained acidified solvent did not alter either the chemical structure of the molecules under study or the biosyntheses of TXAz or PGIz generated by incubation of AA with either HPM or HAoM. Moreover when perfused into the coronary circulation of rabbit isolated hearts this solvent did not change either the heart mechanism (contractile force, rhythm or output), or the TXA2-synthesizing and PG12-synthesizing activities of cardiac tissue.
TXA2- and PG12-synthesizing activities of cardiac tissue
$: NH
Fig. 2
Scheme representing the various stages in the synthesis of 3-amino 4,6-diarylpyridazines
MATERIALS AND METHODS Biosyntheses of TXAz and PGIz in vitro TXA:, and PGI2 biosyntheses were carried out by incubation of arachidonic acid (substrate) with horse platelet microsomes (HPM) or horse aorta microsomes (HoAM), sources of TXA;! and PGIZ synthetases respectively (6, 10). For each compound, 2 series of experiments were carried out with: - either one fixed concentration of arachidonic acid (32.8 x 10m6M) and increasing concentrations of enzymes (HPM, HAoM).
Isolated rabbit hearts used as an experimental model were perfused according to Langendorff’s method as previously described (11). Perfusion was performed in a retrograde way, via the aorta and the coronary circulation. Thus the heart can be considered as a cardiac tissue model kept alive by a Tyrode solution perfusion. Hearts having fulfilled the selection standards (steady rhythm, contractile force, and output) after a 20 min resting period, were allotted at random into various groups: - group I: control hearts perfused with a standard Tyrode solution - other groups: hearts perfused for 10 minutes with a single concentration of compounds under study. Then they were washed by a 2 min perfusion of standard Tyrode solution. The hearts were then removed from the perfusion device and immediately frozen in liquid nitrogen. Each heart was used once either as a control heart or as a single-dose-treated heart. Microsomes from both control and treated hearts were used as
Comparative Effects of some 3-Amino 4,6_Diarylpyridazines
enzyme sources to initiate the biosyntheses of TXA2 and PG12. The ability of heart microsomes to generate the formation of either TXA;? or PG12 when they were incubated with arachidonic acid represents the TXA2-synthesizing or PG12-synthesizing activities of cardiac tissue. The differences between the control and drug-perfused hearts in their eicosanoid-synthesizing ability enabled us to assess the effects of these compounds on the TXA?and PGIT synthesizing activities of cardiac tissue. RIA determination of TXBz and CKeto PGFI, The method of RIA determination of TXB2 and 6keto PGFi, has been described elsewhere (5, 7). Reagents were dissolved in standard RIA buffer containing 0.1% gelatine (pH 7.4). The volume of incubation mixture was 250 ~1: 100 ~1 RIA buffer, either 50 ~1 standard solution (containing either l1000 pg TXB*, or lo-600 pg 6-keto PGF,,) or 50 ~1 extract, 50 ~1 labelled TXB,! or 6-keto PGFi, (1.11 x lo4 dpm), 50 ~1 immun-serum (which bound 50% of 3H TXB2 or 3H 6-keto PGFt, in the absence of competition induced by unlabelled TXB2 or 6-keto PGF,,). Vials were vortexed, incubated at room temperature for 1 hr, then overnight (over 16 h) at 4°C. Antibody-bound and -free TXB2 or 6-keto PGF,, were separated using dextran-coated charcoal suspension (5 mg Norit A/O.5 mg dextran T70/1 ml phosphate buffer) at 4°C for 13 minutes. After a 2,000 x g centrifugation (J 21B Beckman refrigerated centrifuge) at 4°C for 10 minutes, an aliquot of the antibody-bound TXB2 or 6-keto PGF,, from supernatants was sampled into scintillating fluid and the radioactivity was counted in a Packard Tri-carb spectrometer 2450tR). The binding of various samples to the maximal binding ratio was calculated for each sample, the concentration of which was determined from a standard curve after logit transformation. The detection limits of RIA procedures were 10 and 15 pg for TXB2 and 6-keto PGF,, respectively.
21
blood. The pellet was thereafter suspended in Tris buffer (100 mM, pH 7.5). The platelet suspension was frozen and thawed at least three times, then centrifuged at 5,000 x g for 15 min. The supernatant was then centrifuged at 105,000 x g for 60 min (Beckman L5-65 ultracentrifuge) (12). The pellet was suspended again in Tris buffer (pH 7.5) and sampled into vials and lyophilized. Horse aorta microsomes (HAoM) Horse aorta was dissected free of surrounding tissues, chopped into pieces, ground and homogenized in ice cold phosphate buffer at pH 7.5. The homogenate was centrifuged at 12,000 x g for 10 min. The supernatant was filtered through gauzes then centrifuged at 105,000 x g for 60 min. The resulting pellet was homogenized in phosphate buffer and lyophilized (5-7, 10). Rabbit heart microsomes Isolated rabbit hearts were thawed and dissected to separate adjacent vessels and tissues. Then they were chopped, ground and homogenized in the Potter homogenizer in ice cold phosphate buffer at pH 7.5. Heart microsomes were obtained by a 60 min centrifugation at 105,000 xg as in the preparation of horse aorta and platelet microsomes. Protein was determined according to the method described by Lowry et al (13) and modified by Hartree (14) by using bovine serum albumin as a standard, Data analysis Significance of the results was statistically assessed by using two-way analysis of variance with Student’s t test for unpaired and paired data. Statistical methods of regression analysis were used in enzyme kinetics estimation (15). For the calculation of the regression equations and effective doses, probit transformation of the inhibition percentages was used. All values in Tables and Figures were reported as means *SEM.
Preparation of microsomes Horse blood and aorta were taken from a slaughter house. Horse platelet microsomes (HP&f) Blood was withdrawn directly from the carotid artery immediately after the death of the animal, and poured into plastic jars containing 0.11 M 5,5 HZ0 sodium citrate (1 vol citrate for 9 vol blood). Platelet rich plasma (PRP) was obtained from a first 10 min centrifugation at 200 x g then another 15 min centrifugation at 2,000 x g of the titrated
RESULTS TXAz biosynthesis in vitro Incubation of a fixed concentration of AA (32.8 x 10m6M) with HPM concentrations (expressed in protein equivalence) ranging from 250 to 1,000 pg protein, yielded amounts of TXBz which rose with increasing HPM concentrations: 122.9 + 4.2, 172.0 + 4.5, 170.8 f 2.7 and 189.9 f 8.3 pg TXBz vs. 250, 500, 750 and 1,00 pg protein respectively. When preincubated with HPM for 10 min prior to addition of arachidonic acid, compounds II, IV,
22
Prostaglandins Leukotrienes and Essential Fatty Acids Table 1 Comparative effects of compounds I and III on TXA, biosynthesis in vitro when using a fixed concentration of arachidonic acid (AA: 32.8 x lo-” M) against increasing concentrations of horse platelet microsomes (HPM): determination of the amounts (in picograms, pg) of TXB2. n = 7 p <: ** 0.01. 2 x 10-j M HPM (pg Pr)
Control
250 500 750 1000
122.9 172.0 170.8 189.9
I x lo_.3M
I
+ + + +
4.2 4.5 2.7 8.3
113.3 117.6 141.5 156.6
III + f f +
4.4 8.2** 4.6** 12.1**
I
114.0 131.2 138.7 136.3
f + f f
4.8 5.1** 8.0** 5.9**
111
41.9 64.5 55.3 66.8
f 5.4** I?I 8.0** + 7.8** f 9.8**
60.8 66.7 79.0 52.4
+ + + +
4.1** 8.9** 8.7** 4.1**
Table 2 Comparative effects of compounds I and Ill on the biosynthesis of TXA2 induced by the incubation of one fixed concentration of HPM (125 fig Pr) with different concentrations of arachidonic acid: the amounts of TXB, formed were expressed in pg (m f ESm; n = 5; p< * 0.01; ** 0.001) Arachidonic Acid (AA)
32.8 65.6 13.1 19.6 26.2
x x x x x
10-hM lo-” M lo-‘M lo-’ M lo-” M
I Control
52.5 103.2 178.6 187.3 200.2
III
I
1 x W4M
+ + ? + +
3.0 9.9 13.0 15.2 16.3
35.0 70.3 113.4 131.4 148.6
+ + + + +
3.0* 7.4* 8.0* 8.0’ 8.0*
37.5 72.0 112.1 124.3 136.4
f 2.0* k 7.6% I!z 8.0* z.t 8.1* f 9.7*
24.8 48.0 82.8 81.6 97.7
111 5 xMI IO-’ M
f f f zt +
2.4** 5.9** 8.4** 8.8** 10.6**
I
III 1 x IO” M
32.0 64.2 102.0 111.5 120.9
?z + + + +
2.0** 6.7** 5.4** 9.5** 10.4**
21.2 41..0 72.6 66.3 85.1
+ + f + +
2.2** 5.2** 7.8** 7.9** 8.0**
22.8 45.9 72.6 72.4 80.3
f f + + +
1.8** 3.9** 5.1** 7.8** 7.9**
Table 3 Inhibitory effects of compounds I and III on TXA, biosynthesis in vitro: IDS0 (dose capable of inhibiting by 50% the thromboxane synthetase activity at various concentrations of arachidonic acid, AA). AA
6.56 1.31 1.97 2.62
I
x lo-’ M x 10-j M x 1O-5M x 10-j M
4.16 3.77 2.53 4.75
Table 4 Kinetics of the TXAz-synthetase
Control
III
x 1O-4M x lO-‘M x 10-j M x 10-j M
+ + f +
2.78 3.59 3.67 2.39
x x x x
lo-’ h? lo-‘M lo-‘M lo-‘M
8.95 5.54 5.34 6.25
x 1O-4M x 1O-4M x 1O-4M x 1O-4M
f + f f
4.81 4.92 5.49 5.44
x x x x
lo-’ M lo-‘M lo-’ M lo-‘M
inhibiting action of compounds of I and Ill
Km
V??laX
1.215 x 1O-4M + 3.290 x lo-’ M
3.460 x lOmyM.min-’ f 4.460 x lo-“’ M.min-’
1.649 x 1O-4M f 2.499 x lo-’ M 1.288 x 1O-4M + 3.925 x lo-‘M 1.274 x 1O-4M + 5.109 x lo-’ M
2.760 X lOmyM.min? f 2.13 x lo-“‘M.min-’ 1.64 x lO_‘M.min-’ rt 2.39 x lO_“‘M.min-’ 1.398 x lo-’ M.min-’ + 2.669 x lo-“’ M.min~’
1.209 x 1O-4M + 3.289 x lo-’ M 1.212 x 1O-4M f 2.545 x lo-‘M 9.78 x lo-‘M + 2.655 x lo-’ M
2.29 x lO_‘M.min- f 1.77 x lO_“‘M.min-’ 2.05 x lOmyM.min-’ + 1.98 x lo-“‘M.min-’ 1.27 x lo-‘M.minI! 1.45 x lO_“‘M.min-’
I
1 x 1O-4M 5 x 1O-4M 1 x 10-j M Ill
1 x lo-’ M 5 x lo-’ M 1 x 10” M
V and VZ did not significantly change the amounts of TXB2 formed. They neither stimulated nor inhibited TXA2 and PG12 biosyntheses. Under the influence of compounds Z and III, when incubated with arachidonic acid, HPM synthesized smaller amounts of TXB2 compared to those synthesized to control experiments without preincubation with the compounds under study. Compounds Z and ZZZinhibited TXA2 biosynthesis, this inhibition increased with increasing doses. Table I reports the most important effects observed at various doses of Z and
111. Similar data were obtained when using a fixed concentration of HPM with increasing concentrations of arachidonic acid: in fact, a concentration of HPM equivalent to 125 pg protein incubated with various dose ranges of arachidonic acid (32.8 x lo6 M, 65.6 x 1O-6M, 13.1 x 1O-5M, 19.6 x l(r5 M, 26.2 x lop5 M) generated the formation of 52.5 + 3.0, 103.2 k9.9, 178.6 f 13.0, 187.3 f 15.2 and 200.2 + 16.3 pg TXB2 respectively. Under these experimental conditions, Z and ZZZonce more inhibited the formation of TXA;! (Table 2). Table 3 reports
Comparative Effects of some 3-Amino 4,6-Diarylpyridazines
23
PGI2 biosynthesis in vitro
Fig. 3 Comparative effects of I and Ill on the biosynthesis of TXAz in vitro: Lineweaver-Burk plot showed the relationship between the reciprocals of TXBZformation velocity (l/V) and the reciprocals of substrate concentrations (l/S) in the absence (control) and in the presence of various concentrations of I and III for a concentration of HPM equivalent to 125 pgprotein. Straight lines were fitted by linear regression analysis using a computer program. Each point represents the mean of 5 samples with SEm (not shown) being less than 10% of this value. The correlation coefficients for the lines were between 0.989 and 0.995.
the inhibiting effects (IDsa) of Z and 111 on TXA2 synthetase: compound I was found to be more active than III. Graphical Lineweaver-Burk representation of l/v (reciprocals of TXB2 formation rates) vs. l/S (reciprocals of substrate concentrations) showed that the slopes of regression lines obtained with drug-pretreated HPM were higher than the slope of the control regression line, thus demonstrating that both compounds Z and 111 inhibited the biosynthesis of TXA;! (Fig. 3). Table 4 r&presents the kinetic parameters of the inhibitory effects of compounds Z and ZZZon TXA2 biosynthesis. These data showed that both compounds inhibited the TXAZ synthetase, their action being of a non-competitive type,
PG12 biosynthesis generated by incubation of a fixed concentration of arachidonic acid (32.8 x 10-b M) with horse aorta microsomes (HAoM) was not significantly modified by III, but it was inhibited by I: this inhibition of PGIZ synthetase occurred in both cases; when incubating either a fixed concentration of arachidonic acid with increasing concentrations of HAoM, or a fixed concentration of HAoM with increasing concentrations of AA (Table 5). Graphical Lineweaver-Burk representation showed that the inhibitory effects of Z on PG12 synthetase in vitro was of competitive type (Fig. 4). Using Dixon’s method, representing the relationship between the reciprocals of 6-keto PGF1, formation vs. various concentration ranges of Z fed us to the same conciusion, namely that inhibition of the PG12
1 I
0
3.8
I
5.07
,
7.61
Inhibitor effects of I on the biosynthesis of PGIz in vitro. Lineweaver-Burk graphical representation obtained by plotting l/V (V = 6-keto PGFla formation rate) vs. l/S (S = concentrations of the substrate i.e. arachidonic acid, AA) in the absence (control C) and in ‘4 presence of varifus conceflrations of I (1 = I x lo- M, 2 = 5 x It)- M, 3 = 1 x 10 M) Fig. 4
Table 5 Inhibitory effects of I on the PGll synthetase in vitro yielded by the incubation or arachidonic acid with HAoM amounting to 100 pg protein. Each value (in pg of 6-keto PGF,(u) represented the mean (m + ESm) of 5 experiments. p <: * 0.05; ** 0.01. Arachidonic Acid
Control
129.7 + 4.9
165.6 f 5.5
178.1 f 5.4
189.7 f 5.7
113.0 + 8.9 90.0 + 3.1* 87.0 t 6.3
150.2 f 8.4 128.0 f 3.3** 123.0 + 6.0**
161.7 + 6.1 145.6 f 7.2** 142.2 f 7.3**
176.4 + 4.5 161.2 f 9.X* 147.9 + 8.3**
( HAoM)
I 1 x lO_‘M 5 x 1OY’M 1 x 10-j M
Table 6 Kinetics of the PGI, synthetase-inhibiting
Control ( HAom) 1 x 1O-4M 5 x 1W4M 1 x 10-l M
action of compound I
Km
Vmax
4.47 x 1O-5M ? 2.72 x 10-h M
2.51
X
IO?
M.min-’ f 3.60
X
lo-” M.min-’
5.64 x IO-’ M + 5.75 x 10-h M 9.06 x lo-’ M + 5.34 x lo-+ M 7.79 x lo-’ M f 8.12 x lO+ M
2.38
x x
lWy M.min-’ + 6.70 10“ M.min-’ f 5.12 10” M.min-’ * 7.56
x
2.42 2.18
10.” M.min-’ lo-” M.min-’ lo-” M.min-’
x
x x
24
Prostaglandins Leukotrienes and Essential Fatty Acids Table 7 Data representing
the inhibitory effects of I on the biosynthesis of PG12 in vitro generated by the incubation of a fixed concentration of arachidonic acid (32.8 x lo-” M) with increasing concentrations of HAoM (expressed in pg protein). HAo (Dg Pr)
MID,,,
25 50 lot) 200
3.69 2.15 6.45 9.27
x x x x
IO-‘M lo-’ M 1O-JM 10m4M
+ + + ?
2.59 7.64 8.77 5.71
x x x x
10.’ M lo-‘M IO-’ M lo-‘M
Regression equation
Correlation
y y y y
‘r r r r
= = = =
0.925x 0.625x 0.417x 0.753x
-
1.60 0.35 1.63 0.77
= = = =
0.999** 0.999** 0.999** 0.999**
** : p < 0.01
synthetase induced by Z was of competitive type: The kinetic data of the PG12 synthetase in the presence of Z are represented in Table 6. This action was however weak: at the concentration ranges used, Z did not inhibit the PG12 synthetase by more than 30% (Table 7).
hearts: the PG12-synthesizing activity hearts was not significantly different found in control hearts.
TXA+ynthesizing
Our investigations aimed to study the effects of some 3-amino 4,6_diarylpyridazine derivatives, lirstly on the TXAz and PGIz-synthetases in vitro, and secondly on the TXA2- and PGI,?-synthesizing activities of the cardiac tissue of isolated and non-working rabbit hearts kept alive by means of a standard Tyrode perfusion into the coronary circulation. Under the experimental conditions adopted, our study showed that among the six 3-amino 4,6-diarylpyridazine derivatives tested, only 3-dimethylamino ,4,6_diphenylpyridazine (Z) and 3-morpholino 4,6diphenylpyridazine (ZZZ) were able to inhibit the TXA2 synthetase in vitro: Z was the more active with an IDsc = 2.5 x 10e4 M f 3.7 x 10m8,whereas the IDso of ZZZwas 5.3 x 1O_4M f 5.5 x lo_, M. Unlike ZZZwhich was completely inactive on the PG12 synthetase, Z inhibited it. ZZ,ZV, V and VI were not active either on the TXA2 synthetase or on the PGIz synthetase in vitro. Moreover none of these 3-amino 4,6_diarylpyridazine derivatives modified either the TXAZ-synthesizing activity or the PGITsynthesizing activity of cardiac tissue. From the data thus obtained, the following conclusions could be drawn: 1) As Z inhibited both that TXAz and PG12 synthetases in vitro it would suggest that Z may act on the cyclooxygenase. But owing to the fact that Z had quite different effects on the TXA2 and PGI2 synthetases, the direct effects of Z on both enzymes were obvious but it was not excluded that Z may also have a partial inhibitory effect on PG endoperoxide synthetase. 2) The morpholino group in the 3-position of pyridazine appeared to be necessary to give specific anti-TXA2 synthetase activity to the molecule since its replacement by a dimethylamine group stimulated the inhibitory effects on TXA, synthetase, but simultaneously it conferred inhibitory effects on the PG12 synthetase in vitro to the molecule.
activity of cardiac tissue
Microsomes from control isolated non-working rabbit hearts (perfused with a standard Tyrode solution) induced the formation of TXB2 from arachidonic acid: the RIA-determined amounts of TXB2 increased with the increasing concentrations of microsomal fractions from hearts under study. 21.7 It: 1.9, 37.6 f 3.5, 65.7 k 5.1, 78.2 & 7.4, 99.7 f 8.9 and 118.8 f 8.3 pg TXB2 were found to result from 25, 50, 100, 150, 200 and 300 pg protein respectively. Thus microsomes from rabbit hearts were endowed with TXATsynthesizing activity. Moreover we demonstrated that this TXAZ-synthesizing activity of cardiac tissue could be modified (either stimulated or inhibited) if the heart was previously perfused with chemical compounds (16). But when perfused at concentrations ranging from 1 X 10e5 M to 1 X 10e3 M into the coronary circulation, the compounds under study did not significantly alter the TXATsynthesizing activity of drug-perfused-heart microsomes: they neither inhibited nor stimulated the TXAZ-synthesizing activity of cardiac tissue. PG12-synthesizing activity of cardiac tissue Heart microsomes were also found to have a PGI. synthesizing activity. Among the 6 compounds under investigation none of them was active on the PGI*-synthesizing activity of cardiac tissue. In fact, when incubated with arachidonic acid (32.8 X locontrol from fractions microsomal (jM) (Tyrode-perfused) hearts amounting to 25, 50, 100, 150, 200 and 300 pg protein, induced the formation of 65.1 + 6.4, 82.9 k 7.8, 120.0 f 9.5, 140.7 rt 13.7, 170.2 + 15.7 and 177.7 + 15.5 pg 6-keto PGFlti respectively. Microsomes from the hearts ‘perfused with compounds under study at concentrations ranging from 1 x 10m5M to 1 x 10e3 M, yielded the same data as those obtained in control
of treated from that
DISCUSSION
Comparative Effects of some 3-Amino 4,6_Diarylpyridazines
3) The replacement of the dimethylamino group by either a hydrazino group, or a 2-dimethylaminoethylamino or 2-morpholinoethylamino chain suppressed any activity of the molecule on both TXA2 and PGIz synthetases. 4) Likewise, the introduction of a chlorine in the .para-position on the phenyl ring of I abolished any effect of the molecule on the TXAz synthetase and PG12 synthetase. 5) None of the 3-amino 4,6_diarylpyridazine derivatives was active on either the TXAz-synthesizing or PG12-synthesizing activities of cardiac tissue. 6) This study confirmed once more the existence of the TXAz-synthesizing and PGIr synthesizing activities of the cardiac tissue. Such activities have been revealed in various experiments and could be modified (either stimulated or inhibited) by synthetic organic compounds (16). Among the six 3-amino 4,6_diarylpyridazine derivatives under study, I and III were not capable of modifying either the TXAz-synthesizing or the PGIz-synthesizing activity of cardiac tissue, but there were active on the TXA2 and PG12 synthetase in vitro. II, ZV, V and Vf being inactive on both enzymes in vitro were also devoided of any activity on the TXAT and PGIz-synthesizing activities of cardiac tissue. Besides, many other compounds (16) which inhibited the TXAZ-synthesizing activity of cardiac tissue did not have any activity of TXAz and PG12 synthetases in vitro. These data contributed to demonstrate the entity of the TXA? and PGITsynthesizing activities of cardiac tissue i.e. these activities could evolve quite independently from those affecting the TXA? and PGI:, synthetases. Acknowledgements We thank Prof. P. Tronche for his encouragement and continued support of this project, together with the Rotary International Club Toulouse-Sud (France) for its aid and support.
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