Pharmacological Research Communications, Vol. 9, No. 10, 1977
INHIBITION OF HUMANPLATELETAGGREGATIONBY STABLE ANALOGSOF PROSTACYCLIN G. Togna(1), C. Gandolfi(2), A. Andreoni(2), A. Fumagalli(2), C. Passarotti (2), F. Faustini (2) and C. Patrono(1) (1)Department of Pharmacology and Centro di Studio per la Fisiopatologia dello Shock C.N.R., Catholic University of Rome, and (2)Carlo Erba Research Institute, Milan, Italy. Received in fina/ form 10 November 1977
SUMMARY A novel chemical synthesis of nat- and rac-Prostacyclin (PGI2) and 5,6-dihydro-PGI2 is described. Like PGI2, both stable analogs inhibit human platelet aggregation induced by ADP, Arachidonic Acid (AA), Collagen and Adrenaline. In all forms of aggregation, 5,6~-dihydro-PGI2 resulted lO times more potent than 5,6~-dihydro-PGI2. The potency ratio of 5,6a-dihydro-PGI2 to PGI2 was variably estimated between l:lO0, in the case of ADP-induced aggregation, and l:lO in the case of AA-induced aggregation. The availability of stable mimics of PGI2 should be of considerable value in future studies of this substance and may have therapeutic implications. INTRODUCTION Prostacyclin or Prostaglandin 12 (PGI2: 9-deoxy-6,9a-epoxy-Z~PGFIa) is a newly discovered product of AA transformations via the "PG-synthe tase" complex (Johnson et al., 1976), originally described as PGX by Vane's group (Moncada et al., 1976). Although this compound had been previously detected as a minor component in rat stomach fundus extracts (Pace-Asciak and Wolfe, 1971), i t was not until recently that the biological significance of the 6(9)oxy pathwaywas established in the vessel wall microsomes through a series of elegant studies by Vane and his colleagues. These Authors have shown that, besides causing relaxation of some vascular smooth muscle, PAI2 is the most powerful naturally occurring inhibitor of platelet aggregation
909
910
Pharmacological Research Communications, VoL 9, IVo. 10, 1977
(Gryglewski et a l . , 1976). Although this discovery is of major pathophysiological importance, the chemical i n s t a b i l i t y of PGI2 (which is rapidly hydrated, at physiological pH, to the stable product 6-keto-PGFla) should theoretically narrow the therapeutic perspective for its potent plateletantiaggregating properties. Therefore, several attempts were made to modify it s chemical structure in order to increase its s t a b i l i t y . This report describes a novel chemical synthesis of 5,6m-dihydro-PGI2 and 5,6#-dihydro-PGI2 and compares their inhibitory action on human platelet aggregation with those of similarly synthetized PGI2 and 6-keto-PGFl~. MATERIALS AND METHODS The relatively inexpensive (rac- and nat-) dimethoxycyclopentyl-eptenoic acid methyl ester (1) has been used to synthetize PGI2 (rac- and nat-) and their 5,6-dihydro analogs. The novel chemical route is outlined in Figure l , and can be summarized as follows: a) I I . l , I I I . l (bX=Br; cX=I): halocyclization of I with N-Bromo or N-Iodo succinimide (I.2 equiv) in CCl4 and selective depyranylization with p-TsOH acid in dry methanol. b) I I ' . l , I I I . l (aX=H): dehalogenation of the halo compounds I I . l , I I I . l (b,c) with t r i - n - b u t y l t i n hydride (I.2 equiv) in benzene at 55° C for 12 hrs. c) 11.2 (a,b,c) and I I I . 2 (a,,b,c): deacetilization of I I , I I I (a,b,c) mixture of 5,6a and 5,6#-isomers in aqueous acetone with 0.2 N oxalic acid for 6 hrs. at 45° C, alkylation of the aldehydes with the sodium salt of the 2oxo-heptyl-dimethyl-phosphonate (1.2 equiv) in benzene, and then chromatographic separation of the (5,6~)a,#-unsaturated ketones I I . 2 (a,b,c) from 5,6#-compounds I I I . 2 (a,b,c). d) I I . 3 - I I . 4 ; I I I , 3 - I I I . 4 (a,b~c): reduction of the ketones II.2 and I I I . 2 (a,b,c) with ethereal zinc-borohydride, chromatographic separation of 15epimeric alcohols (M=CH3) and saponification with LiOH in aqueous methanol. e) IVa, IVb: dehydrohalogenation of the haloacids I I . 3 , II.4 (b,c) with potassium t-butoxide (2.1 equiv) in t-BuOH for 2 hrs. at room temperature. Potassium salts of PGI2 (rac-, nat-) were obtained as an amorphous powder and kept at -20° C. Rac-5,6m-dihydro-PGI2 ( I I . 3 ) was an o i l ; nat-5,6m-dihydro-PGI2 ( I I . 3 , m.p. 78-80° C; /a/D= +32.5°, /m/365= + I l l . 6 °, in EtOH) and 5,6#-dihydroPGI2 ( I I I . 3 : rac- m.p. I05-I06 ° C; nat- m.p. lOl-102 °, /a/D= +6,4°, /a/365 = +33.2° in CHCI3) were crystalline compounds. The optically active compounds obtained by this route are identical with those prepared from nat-PGF2mas starting material,according to the procedures
Pharmacological Research Communications, VoL 9, No. 10, 1977
911
X
HO -~.~j~~OOCH3 °i
•
o °°
.~CH(OCH3)
2
THP(~
HO II (1,2,3,4) a,b,c
~
0
COOM
~
..
HO
Rl
R HO
R2
IV a,b
X
I I l (1,2,3,4) a,b,c
II, I l l n
a b c
COOM
•
~
e
X
H Br I
1 2 3 4
I
IV CH(OCH3)2 tCH=CH-CO-C5HII tCH=CH-CHOH-C5HIl (S-OH) tCH=CH-CHOH'CsHIl (R -OH)
II : S,6e (6~-H) I l l : S,6B (6~-H)
a
b
Rl
R2
OH H
H OH
M: CH3, H, K T.p
Fig.
1.
Pharrnacolog/cal Research Commun/cat/ons, VoL 9, No. 10, 1977
912
disclosed in the meantime by Corey et al. (1977), Nicolaou et al. (1977), T~moskUsi et al. (1977) and Whittaker (1977). Blood from healthy donors, who had not taken any drug for at least one week, was collected from the antecubital vein into I/lO volume of 3.8% trisodium citrate. After centrifugation at 150 x g for 20 min at room temperature, platelet rich plasma (PRP) was removed by aspiration. For each experiment, 3 to 5 samples of PRP from different subjects were pooled and divided into l ml aliquots. Platelet aggregation was monitored by continuous recording of light transmission in a Born aggregometer (Born, 1962). For both isomers of 5,6-dihydro-PGI 2 and PGI2, the optically active compounds as well as the racemic mixtures were tested; 6-keto-PGFle was tested as a racemic mixture. These substances were dissolved either in 95% ethanol or in
Collagen 38pM 0
T_
5.6 #- dihydro-PGI2 lpg/ml 5.6 a-dihydro. PGI= 0.t pg/ml
.o V~ ,m
E
= 50
(I=
. w
control
100
.I
0
L
2
_
.I
.
4
I.
6
I
_
8-
time (min) Fig. 2. Comparison of inhibition of Collagen-induced human platelet aggregation by 5,6a- and 5,6~-dihydro-PGI~. The superimposed tracings were obtained in PRP fro~ the same donor.
Pharmacological Research Communications, Vo/. 9, No, lO, 1977
913
0.02 M PO4 buffer pH 7.4 and added to PRP in a volume of 5-I0 ~l. For each experiment, a control was run with the same volume of the appropriate solvent alone. The compounds to be investigated were incubated for 2-3 min at 37° C in PRP prior to the addition of the aggregating agent. AA (0.4 mM), ADP (lO ~/~), Collagen (38 ~M) and Adrenaline (15 ~M) were used to induce aggregation.
A,,A.
0.4ram
Or
2ng
I ng 0.5 no .o .w
E
PGI2
f~
¢tZ z,.,. ,e.,..=
~e
control
1001
"!
0
. . . . . .
J .........
1
i
I
2
3
..............
time (min)
Fig. 3. Dose-dependent inhibition of AA-induced human platelet aggregation by PGI9. The superimposed tracings were obtained in PRP from the same donor. RESULTS Like PGI2, both stable analogs i n h i b i t platelet aggregation induced by ADP, AA, Collagen and Adrenaline. In all forms of aggregation, 5,6~-dihydroPGI2 resulted lO times more potent than 5,6#-dihydro-PGI 2 (Fig. 2). The 5,6aisomer was active in the range of 0.01 - l pg/ml PRP, the lowest concentration referring to AA-induced aggregation. PGI2 was active at considerably lower concentrations: I00% irlhibition was obtained at lO ng/ml in the case of Adrenaline-induced aggregation, at 5 ng/ml in the case of both Collagen- and ADP-induced aggregation, at 2 ng/ml in the
Pharmacological Research Communications, VoL 9, No. 10, 1977
914
case of AA-induced aggregation. In this form of aggregation, concentrations as low as 0.5 ng/ml vlhich do not reduce the maximum % change of light trans~ mission do appreciably retard the aggregation process (Fig. 3). The activity of the racemic mixtures resulted 30 to 50% lower than that of the optically active compounds. A racemic mixture of 6-keto-PGFla displayed qualitatively similar antiaggregating properti'es to PGI2, although its potency was roughly estimated as one hundredth that of the parent compound. 100 ¢=
..
('0
i
¢ZJ ww.
=,,
80-
¢0
m
=
60-
E
._c
40
i
¢t
=
20 GI=,
° ~
. . . . .
0.1
I ............
1
I,
10
_
I
100
........
I
1000
concentration (ng/ml)
Fig. 4. Dose (log scale)-response of the inhibitory effect of PGI2 and 5,6~-dihydro-PGI2 on ADP-induced humanplatelet aggregation. When directly compared in the same experiments, the potency ratio of 5,Radihydro-PGI2 to PGI2 was variably estimated between l:lO0, in the case of ADP-induced aggregation (Fig. 4), and l:lO in the case of AAinduced aggregation (Fig. 5). The latter is a rough estimate, since the response to increasing concentrations of the inhibitor was not as graded as in the case of the former and prevented the drawing of a dose-response curve.
Pharmacological Research Communications, Vol. 9, No. 1(7, 1977 A+A.0,4 rnM Y •
915
PGI2 2ng 5,6 a-dihydro-PGI2 20ng
e.-
E e.-
5(] ° ~
5,6 e-dihydro-PGI2 lOng control
PGI2 lng
10C
+J
0
....
|,
1
_
!
2
,,,1 . . . . . . .
3
1
.
4
time (rain)
Fig. 5. Comparison of inhibition of AA-induced human platelet aggregation by PGI~ and 5,6a-dihydro-PGIg. The superimposed tracings w~re obtained in PRP from the same donor. DISCUSSION The results presented indicate that two newly synthetized stable analogs of PGI2 are mimic of the naturally occurring compound with reference to its platelet-antiaggregating properties, though lO to lO0 times less potent. Similar results have been reported by Crane et al. (1977) with respect to ADP-induced aggregation. That 6-keto-PGFla is also active in inhibiting human platelet aggregation - though less potent than PGI2 - is an interesting observation, which confirms a similar finding by Pace-Asciak (1977), and raises the possibility that i t may have physiological
significance, in view of the presumably longer half-
l i f e of 6-keto-PGFl: in vivo. A proper consideration of the potential clinical use of stable analogs of PGI2 , such as those described in this paper, must await a more general survey of their biological activities, particularly on the cardiovascular and gastrointestinal systems. Studies of this nature are now in progress in our laboratories. ACKNOWLEDGEMENTS We are indebted to Professor L. Caprino for helpful discussions and criticism.
916
Pharmacological Research Communications, VoL 9, No. 10, 1977
REFERENCES Born G.V.R., Nature (London) 194, 927, 1962. Corey E.J., Keck G.E. and Sz~kely I . , J. Am. Chem. Soc. 9_99,2006, 1977. Crane B.H., Maish T.L., Maddox Y.T., Corey E.J. and Ramwell P., Clin. Res. 25, 337A, 1977. Gryglewski R.J., Bunting S., Moncada S., Flower R.J. and Vane J.R., Prostaglandins 12, 685, 1976. Johnson R.A., Morton D.R., Kinner J.H., Gorman R.R., McGuire J.C., Sun F.F., Whittaker N., Bunting S., Salmon J., Moncada S. and Vane J.R., Prostaglandins 12, 915, 1976. Moncada S., Gryglewski R.J., Bunting S. and Vane J.R., Nature 263, 663, 1976. Nicolaou K.C., Barnette W.E., Gasic G.P., Magolda R.L., Sipio W.J., Silver M. J., Smith J.B. and Ingerman C.M., Lancet I, I058, 1977. Pace-Asciak C.R., Fed. Proc., in press. Pace-Asciak C.R. and Wolfe L.S., Biochemistry lO, 3657, 1971. T~m~sk~zi I . , Galambos G., Simonidesz V. and Kovacs G., Tetrahedron Letters 2627, 1977. Whittaker N., Tetrahedron Letters 2805, 1977.
INHIBITION OF HUMANPLATELETAGGREGATIONBY STABLE ANALOGSOF PROSTACYCLIN G. Togna(1), C. Gandolfi(2), A. Andreoni(2), A. Fumagalli(2), C. Passarotti (2), F. Faustini (2) and C. Patrono(1) (1)Department of Pharmacology and Centro di Studio per la Fisiopatologia dello Shock C.N.R., Catholic University of Rome, and (2)Carlo Erba Research Institute, Milan, Italy. Received in fina/ form 10 November 1977
SUMMARY A novel chemical synthesis of nat- and rac-Prostacyclin (PGI2) and 5,6-dihydro-PGI2 is described. Like PGI2, both stable analogs inhibit human platelet aggregation induced by ADP, Arachidonic Acid (AA), Collagen and Adrenaline. In all forms of aggregation, 5,6~-dihydro-PGI2 resulted lO times more potent than 5,6~-dihydro-PGI2. The potency ratio of 5,6a-dihydro-PGI2 to PGI2 was variably estimated between l:lO0, in the case of ADP-induced aggregation, and l:lO in the case of AA-induced aggregation. The availability of stable mimics of PGI2 should be of considerable value in future studies of this substance and may have therapeutic implications. INTRODUCTION Prostacyclin or Prostaglandin 12 (PGI2: 9-deoxy-6,9a-epoxy-Z~PGFIa) is a newly discovered product of AA transformations via the "PG-synthe tase" complex (Johnson et al., 1976), originally described as PGX by Vane's group (Moncada et al., 1976). Although this compound had been previously detected as a minor component in rat stomach fundus extracts (Pace-Asciak and Wolfe, 1971), i t was not until recently that the biological significance of the 6(9)oxy pathwaywas established in the vessel wall microsomes through a series of elegant studies by Vane and his colleagues. These Authors have shown that, besides causing relaxation of some vascular smooth muscle, PAI2 is the most powerful naturally occurring inhibitor of platelet aggregation
909
910
Pharmacological Research Communications, VoL 9, IVo. 10, 1977
(Gryglewski et a l . , 1976). Although this discovery is of major pathophysiological importance, the chemical i n s t a b i l i t y of PGI2 (which is rapidly hydrated, at physiological pH, to the stable product 6-keto-PGFla) should theoretically narrow the therapeutic perspective for its potent plateletantiaggregating properties. Therefore, several attempts were made to modify it s chemical structure in order to increase its s t a b i l i t y . This report describes a novel chemical synthesis of 5,6m-dihydro-PGI2 and 5,6#-dihydro-PGI2 and compares their inhibitory action on human platelet aggregation with those of similarly synthetized PGI2 and 6-keto-PGFl~. MATERIALS AND METHODS The relatively inexpensive (rac- and nat-) dimethoxycyclopentyl-eptenoic acid methyl ester (1) has been used to synthetize PGI2 (rac- and nat-) and their 5,6-dihydro analogs. The novel chemical route is outlined in Figure l , and can be summarized as follows: a) I I . l , I I I . l (bX=Br; cX=I): halocyclization of I with N-Bromo or N-Iodo succinimide (I.2 equiv) in CCl4 and selective depyranylization with p-TsOH acid in dry methanol. b) I I ' . l , I I I . l (aX=H): dehalogenation of the halo compounds I I . l , I I I . l (b,c) with t r i - n - b u t y l t i n hydride (I.2 equiv) in benzene at 55° C for 12 hrs. c) 11.2 (a,b,c) and I I I . 2 (a,,b,c): deacetilization of I I , I I I (a,b,c) mixture of 5,6a and 5,6#-isomers in aqueous acetone with 0.2 N oxalic acid for 6 hrs. at 45° C, alkylation of the aldehydes with the sodium salt of the 2oxo-heptyl-dimethyl-phosphonate (1.2 equiv) in benzene, and then chromatographic separation of the (5,6~)a,#-unsaturated ketones I I . 2 (a,b,c) from 5,6#-compounds I I I . 2 (a,b,c). d) I I . 3 - I I . 4 ; I I I , 3 - I I I . 4 (a,b~c): reduction of the ketones II.2 and I I I . 2 (a,b,c) with ethereal zinc-borohydride, chromatographic separation of 15epimeric alcohols (M=CH3) and saponification with LiOH in aqueous methanol. e) IVa, IVb: dehydrohalogenation of the haloacids I I . 3 , II.4 (b,c) with potassium t-butoxide (2.1 equiv) in t-BuOH for 2 hrs. at room temperature. Potassium salts of PGI2 (rac-, nat-) were obtained as an amorphous powder and kept at -20° C. Rac-5,6m-dihydro-PGI2 ( I I . 3 ) was an o i l ; nat-5,6m-dihydro-PGI2 ( I I . 3 , m.p. 78-80° C; /a/D= +32.5°, /m/365= + I l l . 6 °, in EtOH) and 5,6#-dihydroPGI2 ( I I I . 3 : rac- m.p. I05-I06 ° C; nat- m.p. lOl-102 °, /a/D= +6,4°, /a/365 = +33.2° in CHCI3) were crystalline compounds. The optically active compounds obtained by this route are identical with those prepared from nat-PGF2mas starting material,according to the procedures
Pharmacological Research Communications, VoL 9, No. 10, 1977
911
X
HO -~.~j~~OOCH3 °i
•
o °°
.~CH(OCH3)
2
THP(~
HO II (1,2,3,4) a,b,c
~
0
COOM
~
..
HO
Rl
R HO
R2
IV a,b
X
I I l (1,2,3,4) a,b,c
II, I l l n
a b c
COOM
•
~
e
X
H Br I
1 2 3 4
I
IV CH(OCH3)2 tCH=CH-CO-C5HII tCH=CH-CHOH-C5HIl (S-OH) tCH=CH-CHOH'CsHIl (R -OH)
II : S,6e (6~-H) I l l : S,6B (6~-H)
a
b
Rl
R2
OH H
H OH
M: CH3, H, K T.p
Fig.
1.
Pharrnacolog/cal Research Commun/cat/ons, VoL 9, No. 10, 1977
912
disclosed in the meantime by Corey et al. (1977), Nicolaou et al. (1977), T~moskUsi et al. (1977) and Whittaker (1977). Blood from healthy donors, who had not taken any drug for at least one week, was collected from the antecubital vein into I/lO volume of 3.8% trisodium citrate. After centrifugation at 150 x g for 20 min at room temperature, platelet rich plasma (PRP) was removed by aspiration. For each experiment, 3 to 5 samples of PRP from different subjects were pooled and divided into l ml aliquots. Platelet aggregation was monitored by continuous recording of light transmission in a Born aggregometer (Born, 1962). For both isomers of 5,6-dihydro-PGI 2 and PGI2, the optically active compounds as well as the racemic mixtures were tested; 6-keto-PGFle was tested as a racemic mixture. These substances were dissolved either in 95% ethanol or in
Collagen 38pM 0
T_
5.6 #- dihydro-PGI2 lpg/ml 5.6 a-dihydro. PGI= 0.t pg/ml
.o V~ ,m
E
= 50
(I=
. w
control
100
.I
0
L
2
_
.I
.
4
I.
6
I
_
8-
time (min) Fig. 2. Comparison of inhibition of Collagen-induced human platelet aggregation by 5,6a- and 5,6~-dihydro-PGI~. The superimposed tracings were obtained in PRP fro~ the same donor.
Pharmacological Research Communications, Vo/. 9, No, lO, 1977
913
0.02 M PO4 buffer pH 7.4 and added to PRP in a volume of 5-I0 ~l. For each experiment, a control was run with the same volume of the appropriate solvent alone. The compounds to be investigated were incubated for 2-3 min at 37° C in PRP prior to the addition of the aggregating agent. AA (0.4 mM), ADP (lO ~/~), Collagen (38 ~M) and Adrenaline (15 ~M) were used to induce aggregation.
A,,A.
0.4ram
Or
2ng
I ng 0.5 no .o .w
E
PGI2
f~
¢tZ z,.,. ,e.,..=
~e
control
1001
"!
0
. . . . . .
J .........
1
i
I
2
3
..............
time (min)
Fig. 3. Dose-dependent inhibition of AA-induced human platelet aggregation by PGI9. The superimposed tracings were obtained in PRP from the same donor. RESULTS Like PGI2, both stable analogs i n h i b i t platelet aggregation induced by ADP, AA, Collagen and Adrenaline. In all forms of aggregation, 5,6~-dihydroPGI2 resulted lO times more potent than 5,6#-dihydro-PGI 2 (Fig. 2). The 5,6aisomer was active in the range of 0.01 - l pg/ml PRP, the lowest concentration referring to AA-induced aggregation. PGI2 was active at considerably lower concentrations: I00% irlhibition was obtained at lO ng/ml in the case of Adrenaline-induced aggregation, at 5 ng/ml in the case of both Collagen- and ADP-induced aggregation, at 2 ng/ml in the
Pharmacological Research Communications, VoL 9, No. 10, 1977
914
case of AA-induced aggregation. In this form of aggregation, concentrations as low as 0.5 ng/ml vlhich do not reduce the maximum % change of light trans~ mission do appreciably retard the aggregation process (Fig. 3). The activity of the racemic mixtures resulted 30 to 50% lower than that of the optically active compounds. A racemic mixture of 6-keto-PGFla displayed qualitatively similar antiaggregating properti'es to PGI2, although its potency was roughly estimated as one hundredth that of the parent compound. 100 ¢=
..
('0
i
¢ZJ ww.
=,,
80-
¢0
m
=
60-
E
._c
40
i
¢t
=
20 GI=,
° ~
. . . . .
0.1
I ............
1
I,
10
_
I
100
........
I
1000
concentration (ng/ml)
Fig. 4. Dose (log scale)-response of the inhibitory effect of PGI2 and 5,6~-dihydro-PGI2 on ADP-induced humanplatelet aggregation. When directly compared in the same experiments, the potency ratio of 5,Radihydro-PGI2 to PGI2 was variably estimated between l:lO0, in the case of ADP-induced aggregation (Fig. 4), and l:lO in the case of AAinduced aggregation (Fig. 5). The latter is a rough estimate, since the response to increasing concentrations of the inhibitor was not as graded as in the case of the former and prevented the drawing of a dose-response curve.
Pharmacological Research Communications, Vol. 9, No. 1(7, 1977 A+A.0,4 rnM Y •
915
PGI2 2ng 5,6 a-dihydro-PGI2 20ng
e.-
E e.-
5(] ° ~
5,6 e-dihydro-PGI2 lOng control
PGI2 lng
10C
+J
0
....
|,
1
_
!
2
,,,1 . . . . . . .
3
1
.
4
time (rain)
Fig. 5. Comparison of inhibition of AA-induced human platelet aggregation by PGI~ and 5,6a-dihydro-PGIg. The superimposed tracings w~re obtained in PRP from the same donor. DISCUSSION The results presented indicate that two newly synthetized stable analogs of PGI2 are mimic of the naturally occurring compound with reference to its platelet-antiaggregating properties, though lO to lO0 times less potent. Similar results have been reported by Crane et al. (1977) with respect to ADP-induced aggregation. That 6-keto-PGFla is also active in inhibiting human platelet aggregation - though less potent than PGI2 - is an interesting observation, which confirms a similar finding by Pace-Asciak (1977), and raises the possibility that i t may have physiological
significance, in view of the presumably longer half-
l i f e of 6-keto-PGFl: in vivo. A proper consideration of the potential clinical use of stable analogs of PGI2 , such as those described in this paper, must await a more general survey of their biological activities, particularly on the cardiovascular and gastrointestinal systems. Studies of this nature are now in progress in our laboratories. ACKNOWLEDGEMENTS We are indebted to Professor L. Caprino for helpful discussions and criticism.
916
Pharmacological Research Communications, VoL 9, No. 10, 1977
REFERENCES Born G.V.R., Nature (London) 194, 927, 1962. Corey E.J., Keck G.E. and Sz~kely I . , J. Am. Chem. Soc. 9_99,2006, 1977. Crane B.H., Maish T.L., Maddox Y.T., Corey E.J. and Ramwell P., Clin. Res. 25, 337A, 1977. Gryglewski R.J., Bunting S., Moncada S., Flower R.J. and Vane J.R., Prostaglandins 12, 685, 1976. Johnson R.A., Morton D.R., Kinner J.H., Gorman R.R., McGuire J.C., Sun F.F., Whittaker N., Bunting S., Salmon J., Moncada S. and Vane J.R., Prostaglandins 12, 915, 1976. Moncada S., Gryglewski R.J., Bunting S. and Vane J.R., Nature 263, 663, 1976. Nicolaou K.C., Barnette W.E., Gasic G.P., Magolda R.L., Sipio W.J., Silver M. J., Smith J.B. and Ingerman C.M., Lancet I, I058, 1977. Pace-Asciak C.R., Fed. Proc., in press. Pace-Asciak C.R. and Wolfe L.S., Biochemistry lO, 3657, 1971. T~m~sk~zi I . , Galambos G., Simonidesz V. and Kovacs G., Tetrahedron Letters 2627, 1977. Whittaker N., Tetrahedron Letters 2805, 1977.
