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P64 Antiaggregatory and antithrombotic activities of pyrazole-1,2-dioxides
Posters / European Journal of Pharmaceutical Sciences 2 (1994) 117-194
134
P61
OESTRADIOL-LOWERINGACTIvfr'Y OF AMINOGLUTETHIMIDE(AG) AND
P62 AMINOGLUTETHIMIDEAND SOMEANALOGUESAS INHIBITORSOF 1713-HYDROXYSTEROIDDEHYDROGENASE R. LeLain,R. Whomsley,P.J. Nicholls,H.J. Smith WeFshSchoolof Pharmacy,UWCC,CardiffCF1 3XF,U.K.
3-(4'-AI~NOPHENYL)PYRROUDINE-2,S-DIONE (WSP3) R.Sadri,P.J.Nicholls,M Ahmadi,R.LeLain,H.J.Smith WelshSchoolofPharmacy,UWCC,CardiffCF13XF,U.K. In vitro, WSP3 is a more selective aromatase inhibitor than AG and possesses a similar order of potency (Daly et al. 1986). The present study compares their oestradiol (E2)-lowering activity in female rats. Adult animals were injected with pregnant mares' serum gonadotrophin (PMSG 100iu, sc) 5 days before blood was collected by cardiac puncture under ether anaesthesia and plasma E 2 levels were determined by RIA. On these occasions the blood sampling was performed 3h after administration of the aromatase inhibitor. After a single dose, AG (25mg kg q, po) caused an 85% fall in E 2 which was not maintained, levels rising to control values by 6h. In control rats, the plasma t0.s of both agents was determined using an hplc assay as 4.4h (AG) and 0.7h (WSP3). On repeated daily dosing, the E 2 lowering activity of WSP3 (at 3h) was maintained for at least 45 days whereas tolerance had developed to the action of AG by this time (Table).
17[~-Hydroxysteroid dehydrogenase (1713-HSD) is the enzyme responsible for the conversion of androstenedione and oestrone to the highly potent steroid hormones testosterone and 17~-oestradiol (Ea) respectively. Testosterone (as dibydrotestosterone) has been implicated in the maintenance of prostate cancer and E 2 in the development and growth of breast cancer. Consequently, inhibitors of 17~-HSD could be useful in the management of these hormone-dependent neoplasms. Aminoglutethimide (AG), in clinical use for the treatment of breast cancer in postmenopausal women, is effective because it lowers E 2 levels by competitive inhibition of aromatase, the enzyme responsible for the final step in oestrogen biosynthesis. Since structure-activity relationships for 1713-HSD by nonsteroidal compounds are not defined at present, it was of interest to determine whether AG and some of its analogues had an inhibitory effect on this enzyme using rat testicular microsomes. Enzyme activity was determined by measuring the formation of 3H-testosterone from 3Handrostenedione using tlc (silica gel 60) for separation of the steroids. At a substrate concentration of 0.25~M and a protein concentration of 0.194mg/ml, AG (200gM) inhibited testosterone formation by 13% (mean of n=3) compared to control. The greater inhibition of the enzyme by 4nitroglutethimide and glutethimide (49 and 55% (n=3) respectively at 200~tM) demonstrates that a 4-amino substituent is not essential for this activity. A Lineweaver-Burke plot showed the inhibition caused by these two compounds to be competitive. While it is unlikely that inhibition of 17~-HSD by AG would contribute significantly to the reduction of E 2 levels in vivo, these structureactivity relationships provide a basis for the design of more potent inhibitors.
Table. Plasma E 2 (pM) in PMSG-primed rats receiving either AG (25mg kg-l) or WSP3 (50mg kg-l) orally each day Day of treatment Control
1
35
1013+ 199
45
743+202
641 +265
WSP3
*480+78 "144+63 "251+178 (53) (81) (61) AG "152+34 "251+33 368+61 (85) (66) (44) Values are means + sere (n=6); values in parenthesis are % reduction compared to controls; *P<0.05. Part of the explanation may be altered phannacokinetics of AG; its to.5 had declined by 30% by day 45 while that of WSP3 was unchanged. However, despite this advantage on repeat dosing, the data indicate that WSP3 is less potent than AG in reducing E2 levels probably because of its short t0.5 and it is therefore unlikely to be a candidate for further development. M.I. Daly et al. (1986) J. Med. Chem. 29: 520-523. This work was funded by the Cancer Research Campaign.
P63 NITHOSOHYDRAZINES l~lOSTRUCTURALLY RELATED COMPOUNDS WITH ANTIPLATELET AND ANTITHROMBOTIC ACTIVITY K. Rehse, P. K6nig InstitutforPharmazie,FreieUniversitStBerlin,14195Berlin,Germany
Five classes of compounds involving a N-N-N-O structure (1-5) were
prepared: N=O I H2N
ci-
/O
f/O .. "
l
4- N N --
I
N --
N I
R
RI---N --N
,o. .s~
"
" "~' 4
C o m p o u n d s ta (R=Ph) and activities in concentrations
.,
H
.N "N
3
2
I __R 2
R,~I - -
I~.~ R,
~.-.%, ~
"
"'o
P64 ANTIAGGREGATORYAND ANTITHROMBOTICACTIVITIESOF PYRAZOLE-1,2-DIOXlDES K. Rehse,U, Mfiller rnstitutfdr Ph,~rmazie,FreieUniversit~tBerlin,14195Berlin,Germany
A series of pyrazole-4-one-l,2-dioxides (1) and the corresponding oximes (2) were prepared. Pharmacological effects of these stable redcoloured compounds have-not been reported hitherto. The effects on platelet aggregation induced by collagen were studied in human plateletrich plasma by the Born-test (Nature, 194: 927; 1962). The results of selected examples are summarized below. All ketones 1 a-e inhibit platelet aggregation in a dose-dependent manner (p~nol/L). The antiplatclet activities of 2 a-e are somewhat weaker. The influence of le
5
2a (RI=Ph. R2=SO3K) showed antiplatelet down to IC50 = 50 nmol/L (Born-Test.
Collagen). The soluble guanylyl-cyclase (sGC) was stimulated by l a (KM = 7.Spmol/L) and 2a (KM = 10 nmol/L). The antithrombotic activity was investigated in an in vivo thrombosis model. Thrombi were induced by an argon laser. After i.v. administration of l a (10mg/kg) the incidence of thrombi was reduced by 44% in arterioles (A) and 26% in venoles (V), whereas with substance 2b (60mg/kg/i.v., R 1= Ph, R2=COPh, Na) the incidence o f thrombi was reduced by 70% (A) and 56% (V). After oral administration of 4a (60mg/kg, 1-OH-Benzotriazole) the incidence of thrombi was reduced by 70°,6 (A) and 30°/6 (V). Many compounds showed the release o f nitrous oxide, which was determined by a gaschromatographic method. There was a reciprocal correlation between the amount of nitrous oxide formed and the IC5o values. This result suggests that the nitroxylate ion might be involved in the antiplatelet mechanism.
Ph 4-NC-Ph Ph-(CH2) 2
Ph CH3
a 1.5 b 0.2
a 94 b 14
e
e
0.25
50
n'
i -o
COOC2H5 d 0.7 x 2.X-N-OII H(:X:)C-CH2-O-Ph [CH 3 • 0.5 on soluble guanylate cyclase (sGC-')was determined by a standard method ('Methods of Enzymatic Analysis, 3rd Edit., VoL IV, S. 379, Verlag Chemie, Weinheim 1984). A Kin-value of 30 ~rnol/L was oJTAained: Stimulation of sGC demonstrates that Pyrazole-l,2-dioxides are likely to exert their antiplatelet activities via the NO/cGMP pathway. Antithrombotic effects after oral administration were determined in an in vivo thrombosis model. In rat mesenteric vessels thrombi were induced by a number of shots with an argon laser (Arch. Pharm. (Weinheim), 324: 301; 1991). After a single dose of 60 mg/kg of la a 40 % inhibition of platelet aggregation was observed in venoles. In arterioles the effect was even more pronounced (70 %).
134
P61
OESTRADIOL-LOWERINGACTIvfr'Y OF AMINOGLUTETHIMIDE(AG) AND
P62 AMINOGLUTETHIMIDEAND SOMEANALOGUESAS INHIBITORSOF 1713-HYDROXYSTEROIDDEHYDROGENASE R. LeLain,R. Whomsley,P.J. Nicholls,H.J. Smith WeFshSchoolof Pharmacy,UWCC,CardiffCF1 3XF,U.K.
3-(4'-AI~NOPHENYL)PYRROUDINE-2,S-DIONE (WSP3) R.Sadri,P.J.Nicholls,M Ahmadi,R.LeLain,H.J.Smith WelshSchoolofPharmacy,UWCC,CardiffCF13XF,U.K. In vitro, WSP3 is a more selective aromatase inhibitor than AG and possesses a similar order of potency (Daly et al. 1986). The present study compares their oestradiol (E2)-lowering activity in female rats. Adult animals were injected with pregnant mares' serum gonadotrophin (PMSG 100iu, sc) 5 days before blood was collected by cardiac puncture under ether anaesthesia and plasma E 2 levels were determined by RIA. On these occasions the blood sampling was performed 3h after administration of the aromatase inhibitor. After a single dose, AG (25mg kg q, po) caused an 85% fall in E 2 which was not maintained, levels rising to control values by 6h. In control rats, the plasma t0.s of both agents was determined using an hplc assay as 4.4h (AG) and 0.7h (WSP3). On repeated daily dosing, the E 2 lowering activity of WSP3 (at 3h) was maintained for at least 45 days whereas tolerance had developed to the action of AG by this time (Table).
17[~-Hydroxysteroid dehydrogenase (1713-HSD) is the enzyme responsible for the conversion of androstenedione and oestrone to the highly potent steroid hormones testosterone and 17~-oestradiol (Ea) respectively. Testosterone (as dibydrotestosterone) has been implicated in the maintenance of prostate cancer and E 2 in the development and growth of breast cancer. Consequently, inhibitors of 17~-HSD could be useful in the management of these hormone-dependent neoplasms. Aminoglutethimide (AG), in clinical use for the treatment of breast cancer in postmenopausal women, is effective because it lowers E 2 levels by competitive inhibition of aromatase, the enzyme responsible for the final step in oestrogen biosynthesis. Since structure-activity relationships for 1713-HSD by nonsteroidal compounds are not defined at present, it was of interest to determine whether AG and some of its analogues had an inhibitory effect on this enzyme using rat testicular microsomes. Enzyme activity was determined by measuring the formation of 3H-testosterone from 3Handrostenedione using tlc (silica gel 60) for separation of the steroids. At a substrate concentration of 0.25~M and a protein concentration of 0.194mg/ml, AG (200gM) inhibited testosterone formation by 13% (mean of n=3) compared to control. The greater inhibition of the enzyme by 4nitroglutethimide and glutethimide (49 and 55% (n=3) respectively at 200~tM) demonstrates that a 4-amino substituent is not essential for this activity. A Lineweaver-Burke plot showed the inhibition caused by these two compounds to be competitive. While it is unlikely that inhibition of 17~-HSD by AG would contribute significantly to the reduction of E 2 levels in vivo, these structureactivity relationships provide a basis for the design of more potent inhibitors.
Table. Plasma E 2 (pM) in PMSG-primed rats receiving either AG (25mg kg-l) or WSP3 (50mg kg-l) orally each day Day of treatment Control
1
35
1013+ 199
45
743+202
641 +265
WSP3
*480+78 "144+63 "251+178 (53) (81) (61) AG "152+34 "251+33 368+61 (85) (66) (44) Values are means + sere (n=6); values in parenthesis are % reduction compared to controls; *P<0.05. Part of the explanation may be altered phannacokinetics of AG; its to.5 had declined by 30% by day 45 while that of WSP3 was unchanged. However, despite this advantage on repeat dosing, the data indicate that WSP3 is less potent than AG in reducing E2 levels probably because of its short t0.5 and it is therefore unlikely to be a candidate for further development. M.I. Daly et al. (1986) J. Med. Chem. 29: 520-523. This work was funded by the Cancer Research Campaign.
P63 NITHOSOHYDRAZINES l~lOSTRUCTURALLY RELATED COMPOUNDS WITH ANTIPLATELET AND ANTITHROMBOTIC ACTIVITY K. Rehse, P. K6nig InstitutforPharmazie,FreieUniversitStBerlin,14195Berlin,Germany
Five classes of compounds involving a N-N-N-O structure (1-5) were
prepared: N=O I H2N
ci-
/O
f/O .. "
l
4- N N --
I
N --
N I
R
RI---N --N
,o. .s~
"
" "~' 4
C o m p o u n d s ta (R=Ph) and activities in concentrations
.,
H
.N "N
3
2
I __R 2
R,~I - -
I~.~ R,
~.-.%, ~
"
"'o
P64 ANTIAGGREGATORYAND ANTITHROMBOTICACTIVITIESOF PYRAZOLE-1,2-DIOXlDES K. Rehse,U, Mfiller rnstitutfdr Ph,~rmazie,FreieUniversit~tBerlin,14195Berlin,Germany
A series of pyrazole-4-one-l,2-dioxides (1) and the corresponding oximes (2) were prepared. Pharmacological effects of these stable redcoloured compounds have-not been reported hitherto. The effects on platelet aggregation induced by collagen were studied in human plateletrich plasma by the Born-test (Nature, 194: 927; 1962). The results of selected examples are summarized below. All ketones 1 a-e inhibit platelet aggregation in a dose-dependent manner (p~nol/L). The antiplatclet activities of 2 a-e are somewhat weaker. The influence of le
5
2a (RI=Ph. R2=SO3K) showed antiplatelet down to IC50 = 50 nmol/L (Born-Test.
Collagen). The soluble guanylyl-cyclase (sGC) was stimulated by l a (KM = 7.Spmol/L) and 2a (KM = 10 nmol/L). The antithrombotic activity was investigated in an in vivo thrombosis model. Thrombi were induced by an argon laser. After i.v. administration of l a (10mg/kg) the incidence of thrombi was reduced by 44% in arterioles (A) and 26% in venoles (V), whereas with substance 2b (60mg/kg/i.v., R 1= Ph, R2=COPh, Na) the incidence o f thrombi was reduced by 70% (A) and 56% (V). After oral administration of 4a (60mg/kg, 1-OH-Benzotriazole) the incidence of thrombi was reduced by 70°,6 (A) and 30°/6 (V). Many compounds showed the release o f nitrous oxide, which was determined by a gaschromatographic method. There was a reciprocal correlation between the amount of nitrous oxide formed and the IC5o values. This result suggests that the nitroxylate ion might be involved in the antiplatelet mechanism.
Ph 4-NC-Ph Ph-(CH2) 2
Ph CH3
a 1.5 b 0.2
a 94 b 14
e
e
0.25
50
n'
i -o
COOC2H5 d 0.7 x 2.X-N-OII H(:X:)C-CH2-O-Ph [CH 3 • 0.5 on soluble guanylate cyclase (sGC-')was determined by a standard method ('Methods of Enzymatic Analysis, 3rd Edit., VoL IV, S. 379, Verlag Chemie, Weinheim 1984). A Kin-value of 30 ~rnol/L was oJTAained: Stimulation of sGC demonstrates that Pyrazole-l,2-dioxides are likely to exert their antiplatelet activities via the NO/cGMP pathway. Antithrombotic effects after oral administration were determined in an in vivo thrombosis model. In rat mesenteric vessels thrombi were induced by a number of shots with an argon laser (Arch. Pharm. (Weinheim), 324: 301; 1991). After a single dose of 60 mg/kg of la a 40 % inhibition of platelet aggregation was observed in venoles. In arterioles the effect was even more pronounced (70 %).