CA1215707A - Aromatic prostacyclin analogues - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

Stable biologically active aromatic prostacyclin analogues of the formula wherein X represents

Description

~ 7~

BACKGROUND OF TIIE INVENTION
..

Field of The Invention:
_, This invention relates to stable aromatic prostacyclin . analogues which are active as inhibitors of blood platelet aggregation and which show arterial dilation activities.

Description of the Prior Art:

The prostaglandins were first discovered in the 1920's and have proven since then to be among the most ubiquitous pharma-ceutically active compounds ever tested. Their use and the use of analogues and derivatives thereof, has been suggested in as wide a range of applications as fertility control, induction of labor, regulation of blood pressure, regulation of blood clott-ing, control of asthma, anticonvulsion, antidepressing action and many others. A new compound has recently been discovered (Nature 263, 663 tl976~; Prosta~landins, vol. 12, 685 and 715 : (1976); Chem. and Engineering News, December 20, 1976) which belongs to the general family of prostaglàndins. The compound ha~ been named prostacyclin and its structure has been Droven by synthesis (Johnson, et al, Prostaglandins, 12, 915 (1976);
Corey, et al, J. Amer. Chem. Soc., 99, 2006 (1977) to be that , _ . ~ _ ., . . , ?

of formula I. (The numbering system for prostacyclins is given for reference):

' <~

1 ~ 20 HO o~
, `

Its generic name is 6, 9 ~ -oxido~ , 15 ~-dihydroxyprosta ~Z) 5, E(13)-dienoic acid~ Prostacyclin is the most votent inhibitor of blood platelet aggregation of all the prostaglandins dis-covered to date. It has also been shown that prostacyclin - destroys platelet aggregates after they have formed and that it has, in addition, a powerful action as a dilator of blood vessels.
Prostacyclin thus appears to act in exactly opposite ways to thromboxane A2~ another recently discovered member of the : prostaglandin family. Thromboxane A2 causes platelet aggrega-tion and simultaneously acts as powerful constrictor of arteries.
Both prostacyclin and thromboxane A2 are derived biosynthetically . from a common intermediate called endoperoxide, and they aredecomposed by water to prostaglandins (Scheme I). The balance between the levels of prostacyclin and thromboxane ~2~ appears to maintain a finely tuned equilibrium between blood platelet aggregation versus dissolution and~arterial constriction versus _ _ . . _ . , . . . , _ . .. . _ _ _ . .. . _ .. _ _ ~ _ ~57~7 dllation .
Scheme I: -.
prostacyclins From fatty ~ \
acid ~ Endoperoxides ~prostaglandins precursors , ~ 7 thromboxanes Thromboxane A2 generatecl by pla-telets promotes aggregation while prostacyclin produced by vascular endothelium inhibits aggregation. It has also been proposed (S. Moncada et al, Lancet I, 18 (1977)) that 1) the basal formation of prostacyclin by S vascular endothelium may be important in the maintenance of the normal integrity of vessel walls by inhibiting the adherence of . platelets, 2) prostacyclin may normally limit thrombus formation, and 3) when a vessel wall is damaged, the formation of a normal hemostatic plug may be assisted by diminished prostacyclin pro-duction.

I In addition to its effects on platelets, prostacyclin may play a crucial role in preventing gastric ulceration by inhibit-ing secretion, in blood pressure regulation by control of vascula tone, and in inflammation by inhibiting protease secretion of 15 polymorphonuclear leucocytes. These and several other important ¦
physiological processes may be regulated by the opponent actions ¦
of t~ mb~ne ~2 (~'A2) and prostacycl n (PCIz).

. I

~ 5707 The use of prostacyclins has therefore been suggested in the treatment of blood clotting in diseased vessels of patients with eardiovaseular problems. Since prostac~clin has retroactive aetion and'notonly inhibits blood elotting but also dissolves already ~ormed clots, i,t can be used in heart attaek eases and . artherosclerosis. Increased suseeptibility of platelets to aggregation accompanies vaseular eomplications in diabetes, in eerebxal strokes assoeiated with essential hypertension and in post heart attack eases. These are other areas where prosta- !
eyelin or its analogues ean be highly benefieial. The main draw-baek of the use of prostacyelin for these a~plications is its very short biologi-cal half-life of 2 minutes. This prev2nts the externally provided drug from reaching its target tissues intact. The need to maintain the ~drug in a totally anhydrous eondition also prevents its ready shipment, storage and testing for pharmaeologieal applications. If an analogue or derivative of prostaeyelin ean be found whieh is stable and shows similar effects on blood platelets and arteries, it would have wide applieations in pharmaeology and the treatment o~ eardiovaseular and related diseases.

~LZ~7~7 Another application for a stable analogue of prostacyclin would be i-ts use as an inhibitor of blood platelet aggregation in externally circulated blood by kidney dialysis machines or heart~lung machines.
..
Prostacyclin analogues may also be useful in the -treatment of several types of shock. For example, in hemorrhagic shock, vasoconstriction may severely limit the flow of blood to the gut and kidneys. Replacement of the blood volume lost will I easily redilate the gut vessels but f-low through the kidney maybe impeded for a long period of time. During this interval the . kidneys may infarct. In addition, prolonged vasoconstriction in the gut region may release proteases from liver and pancreatic lysozomes. Prostacyclin or its analogues may attenuate these deleterious effects by maintaining a small amount of blood flow through these areas.
~ .
A number of stable analogues of prostacyclin of varying bio-: logical activity has been ~repared. See for example Nicolaou et al, Angew. Chem., Int. Ed. Engl. 17,293 (1978) fox a general review. Among these are thia-prostacyclins (II), discussed by Nicolaou and cowor~ers in J. Amer. Che~D Soc. 99, 7736 (1977).

~2~

~o~{
s~
`- ! ~

HO
O~ OH

(thia pxostacyclins II) (~ - PGI2, III) Another analogue of natural prostacyclin is ~ 6 prostacyclin (~6 _ PGI2, (III)) prepared by Shimoji and co-workers (J. Amer. Chem. Soc., 100, 2547 (1978). Compound III, ~6 _ PGI2, appears to have recently been isolated from rat stomach homogenates and its presumed structure was iden-tified by Sih et al (J. Amer. Chem. Soc., 100, 643 (1978)).

Upjohn scientists have recently reported the nitrogen-containing prostacyclin analogues IV and V; (Bundy, G., Tetrahedron Letters, 1371 (1978)):

l -7-~2~7~

,~OOli N 5C-- ~OOH
N

bH HO
OH

~IV) (V) Analogues IV and V showed high stability and po-tency in inhibiting platelet aggregation.

All of the aforementioned analogues ~ V) showed biological activity mimicking prostacyclin action. Other, somewhat less t active analogues of prostacyclin have also been prepared: dihydroprostacyclin (VI) (Corey et al, J.
Amer. Chem. Soc, 99, 2006 (1977)) and (4E) - isoprostacyclin (VII).

COOH
< ~ OOH
: ' /
~ ~

llO Oll HO

~VI~ IVII~

?
. .

~ 7 The present inventors have compared the biological activity for all stable prostacyclin analogues rep~r-ted to'~ate (II-VII, plus others listed in the aEoremen-tioned Nicolaou et al article in Angewandte Chemie). They have sought to elucidate the struc~ures which yield the highest biological activity and have discovered that the most potent ones are those retaining Sp - hybridized C-6. Natural prostacyclin, of course, contains ¦
an Sp hybidized C-6;-compounds II-V do so as well., Compounds VI and VII on the other hand do not have a C-6 Sp -carbon and show less biological activity. Based on this discovery,' the present inventors have discovered that both high stability and biological activity can be obtained generally ~ when the labile cyclic ether rings of natural prostacyclin is ; replaced by a fully aromatic ring,a fact which among others , assures the presence of an Sp2 hybidized C-6. The Pre~sence of an aromatic ring instead of a labile cyclic enol ether~ring I also ensures stability in aqueous, physiological media and renders the,analogues useful for a large number of applications.
:' SUMMARY OF THE INVENTION

Accordingly it is an object of the invention to provide pharmacologically active compounds useful in the treatment of blood clotting and other related cardiovascular diseases. A
further object of the invention is to provi,de pharmacolo~ically ' ~ ii7~7 active compounds which are stable analogs and/or derivatives of prostacyclin.

Another object of the invention is -to provide stable bio-.. logically active prosta~yclin analogues which contain an aromatic nucleus in replacement of the cyclic ether ring of natural prostacyclin. Still another object of the invention is to provide stable, biologically active prostacyclin analogues which contain as part of the aforementioned aromatic nucleus a pyridazine system. A Eurther object of the invention is to provide stable r active prostacyclin analogues which contain as part of the aforementioned aromatic nucleus a furan system, or a pyrrole system,or a thiophene system. These and other objects of the inventlon which will become obvious hereinafter have been attained by providing stable biologically active pro~
1.5 stacyclin derivatives of formula VIII:

~ ~COO~ , ( Vl I I ) ; OH

wherein X = -N=N-;
-N=N-; r -o-; -S-; or -NH-; and wherein R = represents -10- ' .

~ 7 hydrogen, a pharmaceutically acceptable cation or a pharmaceutically acceptable lower alkyl group.

DETAILED DESCRIPTION OF I~ IR~ RrD EULDDIMENTS

.. The compounds of the present invention comprise prostacyclin analogues of the formula \J~

OH

- wherein X = -N=N-; -N=N-, ~O~ ; ~S~ or NH and wherein R =
hydrogen or a pharmaceutically acceptable cation or a pharmaceu-; tically acceptable lower alkyl group. -Pharmaceutically acceptable cations useful for the purposes 10 ~ of this invention are those with pharmaceutically acceptable metal cations, ammonium, amine cations or quaternary ammonium cations.
Especially preferred metal cations are those derived from the alkali metals, e.g., lithium~ sodiwn and potassium, and from the alkaline earth metals, e.g., magnesium and calcium, although cationic forms of other metals, e.g., aluminum, zinc, and iron, are within the scope of this invention.
.' __ .. ., . . .. , . . _ .... .. .. . _ ...?

~ lZ15~07 Pharmacolo~ically acceptable amine cations are those derived from primary, secondary, or tertiary amines. Examples of suitable amines are methylamine, dimethylamine, trimethylamine ethylamine, dibutylamine, triisopropylamine, N-me~hylhexylamine, decylamine, dodecylamine, allylamine, crotylamine, cyclopentyla-. mine, dicyclohexylamine, benzylamine, dibenzvlamine, ~ -phenyl-ethylamine, ~ -phenylethylamine, ethylenediamine, diethylene-triamine, and like aliphatic, cycloaliphatic, and araliphatic amines containing up to and includinq about 18 carbon atoms, as well as heterocyclic amines, e.g., piperidine, morpholine, pyrrolidine, piperazine, and lower-alkyl derivatives thereof, e.g., l-methylpiperidine, 4-ethylmorpholine, l-isopropylpyrroli-dine, 2-methylpyrrolidine, 1, 4-dimethylpiperazine, 2-methyl-piperidine, and the like, as well as amines containing water solubilizing or hydrophilic groups, e.g~, mono-, di-, and tri~
ethanolamine, ethyldietha~olamine, N-butylethanolamine, 2-amino-l-butanol, 2-am~no-2-ethyl-1, 3-propanediol, 2-amino-2~methyl~
propanol, tris(hydroxymethyl) aminomethane, N-phenylethanolamine, ; N-(p-tert-amylphenyl~diethanolamine, galactamine, N-methyl-2b glucamine, N-methylglucosamine, ephedrine, phenylephrine, epinephrine, procaine, and the like.

Examples of suitable pharmacologically acceptable quaternary ammonium cations are tetramethylammonium, tetraethylammonium, , . ... .. _ . . .. .. ,, ,.. ,.. . ........ , - -- _. . . ,. ___ ,_ I
.

benzyltrimethylammonium, phenyltriethylammonium, and the like.

Pharmaceutically acceptable lower alkyl groups are those derived from Cl - ClO hydrocarbyl residues, especi~ly Cl - C4.
Most preferred are methyl and ethyl groups.
. .

When X = -N=N- or N=N(0)-, the aromaticity of the ring is given by a full sextet of ~ electrons.
..
When X = O:;\S/or -~ the resultin~ aromaticity o~ the rinc - is given }~y ~ ~ electrons, -~ of which are contributed by two double bonds and two are provided by the lone pairs on ox~gen, sulfur or nitrogen.
; Representative compoundswithin the scope of this invention are:
6, 9 - pyridaza prostacyclin ~X = -~=N-), metl-yl ester and Sodium Salt 0 6, 9-pyridaza prostacyclin - N- oxide (X= -N=N-; 2 isomers), methyl ester and sodium salts ; 6, 9 -pyrrole prostacyclin (X = NHj, methyl ester and Sodium Salt 6, 9- furan prostacyclin (X = 0), methyl ester and Sodium Salt 6, 9-thiophene prostacyclin (X=S); methyl ester and .
~, 1 ~2~L~7~

Sodium Salt.

The compounds of the present invention may all be pre-pared from a common intermediate prostanoid diketone IX. This diketone is reacted using one of the appropriate reagents a - e to obtain the cyclized Droducts of the invention or their immediate protected precursors:

O\I~ ~/c Reagents > Products of the R~( o~2 invention (IX) Rl may be H or lower alkyl, preferably Cl - C4 alkyl; R may be H or a hydroxy group - protecting agent, such as an acyl grouP
or a silyl group. Preferably R is -CH3 and R is tert-butyl-dimethylsilyl or tetrahydropyran. Reagents a - e are given in Table 1: - ~
.

T A B L ~

Reagent Product , .............. ..... _ . .. ... _ .__ I
a) NH2NH2 followed by oxidation Pyridaza prostacyclin 1 15 b) Reagent aJ followed by further oxidation N-oxides of pyridaza prostacyclin c) H or dehydrating agent, heat furan prostacyclin d) P255 or P2S3, heat thiopheneDrostacyclin e) NH3, at low temperatures pyrrole prostacyclln _ ... .... ., . . . . . . . . . .. _ _ ~Z~7~' Cyclizations of diketones to yield pyridazines, furans, pyrroles and thiopenes are well known in the art.
For example, the general methodology of R.M. Acheson, "An introduction to the Chemistry of Heterocyclic Compounds", 3rd ed., Wiley, N.Y., 1976 and references cited herein can be applied for the construction of the furan prostacyclin~
for the construction of the pyrrole prostacyclin; for the preparation of thiophene prostacyclin and for the preparation of pyridazine-prostacyclin. Protecting groups on the acid functionality as well as the hydroxy functionality are pref-erably used to avoid side reactions which complicate the synthesis and yield mixtures of products. General methods for the protection and deprotection of hydroxy groups with acyl or silyl groups can be followed. These methods, are found for example in McOmie, Protective Groups in Organic Chemistry, (1976).
Reagent a) For the preparation of the pyridaza prosta-cyclin, for example, diketone IX
(Rl = lower alkyl, R2 = H) is treated with 0.95 - 102 equiv-alents, preferably 1.0 equivalents, of hydrazine at room temperature for small periods of time to obtain the corresponding 7,8-dihydro-6,9-pyridaza prostacyclin X:

COORl ~N~ (X)

2 ~' ~ ~ ~ /

Ot~

This reaction is equally successful when Rl is hydrogen. It is preferable to use 95% ~ hydrazine for the condensation reaction. The reaction proceeds well in an organic solvent or an ~queous/organic solvent. Examples of such solvents are ethers, including cyclic ethers, alcohols, hydrocarbons, halogenated hydrocarbons, and mixtures thereof with or with-out water. The preferred solvents are ethers and aqueous ethers. Most preferred is tetrahydrofuran - H2O (9:1). Pre-ferred temperatures for the reaction are -10 to ~40C, most preferably +10 to +30C.

In order to obtain the fully aromatic pyridaza prostacyclin derivative, the 7,8-dihydro-6,9-pyridaza prostacyclin (X) (Rl = lower alkyl) may be oxidized, for ex-ample over PtO2, and isolated by common methods~ The free lS acid form of 6,9-pyridaza prostacyclin VIII,(X=-N=N-, R=H) cannot be prepared from the corresponding methyl ester VIII
(X=-N=N-, R= lower alkyl) by aqueous base hydrolysis in high yields, due to the base sensitivity of the compound~ It is therefore prepared directly from the ~ree acid form of the diketone prostanoid precursor IX (Rl = H~ ~y similar conden-sation with hydrazine and oxidation. However, it is within the present invention to selectively hydrolyze the methyl ester VIII (X=-N=N-, R= lower alkyl) to the free acid or salt (R = H or cation) by a selective agent which will not destroy the remainder of the molecule.

. , . I

2LZ157~7 Reagent b) Further oxida~ion oE 6, 9-pyridaza prostacyclin (VIII, X=-N=N-) yields the corresponding N-oxides (VIII, X= -N=N(0)-) which are purified chromato~raphically as a mixture. Oxidation may be carried out with any mild oxidation agent such as m-chloroperbenzoic acid (rlCPBA) or other common oxidants; (0.8 - 1.5 eauivalents oxidant :1 equivalent of pyri-pyridaza compound) this reaction may be carried ou-t with equal success on the esterified compounds or on the corresponding free acid forms. The oxidation yields about 60~ yield of a 10 1:1 mixture of the N-oxide next to position 6, and the ~l-oxide next to position 9. Both the N-oxide esters (2 compounds) and the - N-oxide acids (2 compounds) are stable in solution or neat under !
neutral conditions, although basic media leads to destruction over extended periods of time. The non-oxidized pyridaza 15 prostacyclins are slightly more unstable. It is Possible that the obserYed relative instability of 6, 9-pyridaza Prostacyclin and its oxides arises from the self-promoting tendency of the non-oxidized compound to eliminate water due to its basic nature.l Removal of the basic character by oxidation to the corresponding !
2~ N-oxides leads to enhanced stability.
Reagent c) For the preparation of the furan ~rostacyclin VIII (X = -0-), starting diketone IX may he treated with an acid ¦
such as Hc~ or the like,or a dehydrating aqent such as P2O5, or .I

-- -- _ ~. , ........ . .. __ . .. __ . _ ... --.. _ _ . . _ _ ____ ~S7~7 Pc~, under anhydrous conditions. Preferred solvents are ben-zene, toluene or other similar ones. This reaction first causes cyclization and then further loss of a mole of water to yeld the aromatic furan. The reaction is preferably carried out on the protected diketone IX ~Rl=C~3, R2=tert-butyldimethylsilyl) and under anhydrous conditions. Other possible dehydrating agents are for example: molecular sieves, Ca CO3, P205. Another scheme for $he preparation of furan prostacyclin is treatment of the~,B -unsaturated lac- -tone XI (~2=H) (ob~ained ~y treatment of the corresp~ndingsaturated lactone with phenylselenenyl halide in base~
followe~ by oxidation with H202~, with a lithium alkyl or Grignar~ reagent, followed by dehydration:

C)~ 1) LiCH2~ ~;!C~}2CH2 5VIII,X- -0-~
2) Dehydration -~~
R O
oR2 The saturated lactone used as starting material has been dis-closed by E.J~ Corey and coworkers in J. Amer. Chem. SocO~
93, 1491 tl971) and J.S. Bindra and R. Bindra? "Creativity in Organie Synthesis, Volume 1", pg 210y Academic Press, New York~ (197~)o ~8-I
~ ;7~

Rea~ent d) Thiophene prostacyclin (VIII, X=S) may be obtained by treating the starting diketone IX with a rea~ent such as : P2S5 or P2S3 in a solvent such as benzene, toluene, and at a temperature in the range of 25- 200C . An excess of P2S5 (or P2S3) is used ovex the amount of diketone, preferably 1-2 equivalents,most preferably 10 eguivalents. Heat treat-ment at 100 to 200C then yields the thiophene prostacyclin ¦ product~ It is preferred to use a protected starting diketone methyl ester (Rl = CH3-, R2 = tertbutyldimethyl silyl) and 1~ ¦ carry out the reaction under an inert gas, such as N2; to pre-vent oxidation of -the final product. I

Reaqent e) Preparation of pyrrole prostacyclin (VIII; X=NH) may ¦
be~carried out by treating the starting protected diketone IX
with ammonia, in a solvent such as ether, or THF at low temperatures, preferably at 0 to 250C, most preferably at . 20 to 25 C. Such low temperatures are necessary in~order to prevent further reaction of the ester at carbon 1 with the . amine functionality of the pyrrole ring. Another method to : prevent the formation of an amide to C-l carboxy group, is -to use as the starting material the partially protected diketone VIII (R = H, R = tertbutyl dimethylsilyl).

. The starting prostanoid diketones VIII may be prepared by a : sequence of hydrolysis and oxidation reactions on a protected prostacyclin derivative XII:

~ 15~

R ~ ; R dlket XII XIII

I this sequellce of reaCtlonS R is preferably a lower alkyl group and R may be any - OH protecting group, such as trialkylsilyl, tetrahydropyranyl, acyl or the like. Preferably R is -CH3 and R is the tetrahydropyranyl (THP) group.
Prostacyclin XII may be prepared from the corresponding pro-tected iodide XV by dehydroiodination in base.

,~COORl (Y ~ ) The hydroxyl -deprotected Iodide XV (R = -CH3, R = H) has been . prepared by various investigators and is readily available (Nicolaou et al, J. Chem. Soc., Chem Comm., 630 (1977);
Johnson, ~, et al, J. ~mer. Chem. Soc., 99, 4182 (197t);
Wittaker, N., Tetr. Lett., 2805 (1977)). Standard methods of ... . .. .... .. . ...... . ..

~ ~S7~;~

protecting hydroxy groups may be used for the protection reaction of the -OH groups of deprotected XV. The hydrolysis of prostacyclin derivative XII may be carried out in mild acid or mild base. Preferably it is performed in acid, most preferably it is carried out in acetic acid (traces) /THF (room tempera-. ture, few minutes). T~e oxidation of XIII to the diketone VIII
may be carried out with any standard o~idation reagent, such as Jones reagent or a variation thereof. Preferably the oxidant is pyridinium chloroch-romate in an organic solvent,such as for example methylene chloride at room temperature; or CrO3 in ace-tone at -20C. ~ields of diketone VIII are better than 70~.

. The compounds of this invention can be administered by any means that effects palliating conditions of cardiovascular complications in warm-blooded animals. For example, administra-tion can be parenterally, subcutaneously, intravenously, intra-muscularly or intraperitoneally. Alternatively or concurr~ntly, administration can be by the oral route. The dosage administered¦
will be dependent upon the age~ health and weight of the reci-: pient, kind of concurrent -treatment if any, frequency of treat- ¦
ment, ancl the nature of the effect desired. Cenerally, daily do-sage of active ingredient compounds ~ill be from about 0.5 mcJ to 50 mg per kg of body weight. Normally, f~om 1 to 30 mg per kg per day, in one or more applications per day is effective to :, .. . ., ! !

I ~S7~ 1 obtain the desired result. The compounds can be employed in I dosage forms such as tablets, capsules, powder packets, or liquid solutions, suspensions, or elixirs, for oral administra-tion, or sterile liquid for formulations such as solutions or 1 5 suspensions for parenteral use. In such compositions, the . active ingredient will ordinarily always be present in an amount of at least 0.5~ by weight based on the to-tal weight of the composition and not more than 90~ by weight. Compounds of ~
; the present invention are useful in the treatment of blood clotting in heart attack cases, artherosclerosis, diabetes and cerebral strokes. They are useful in the treatment of various types of shock, such as hemorrhagic shock. in preventing . gastric ulceration, by inhibiting secretion; in blood pressure regulation, and in inflamation.
.
Having generally described the invention, a more complete understanding can be obtained by reference to certain examples, which are included for purposes of illus-tration only and are not intended to be limiting unless otherwise specified.
:
Experimental Methods o _Bioloc~ical Testin~

Effects of prostacyclin analogs on platelet aggregation . wer~ evaluated using human and rabbit citrated platelet-rich ,~
.

.,, ......... . . .. -- . .. . , .... - _ _ _ ~ _ _ l ~Z~S~17 I
plasma in a chronolog aggregometer at 37. Each of the analogs was tested at two concentrations (20 mM and 2 uM) for agonistic activity (inhibi~.ion of aggregation induced by 2 url ADP) and antagonistic activity (preven-tion of the inhibition of ADP-induced aggregation by 5 mM prostacyclin).
.. ~ ' Effects of prostacyclin analogs on isolated perfused cat , coronary arteries were measured as follows.

Cats of either sex (%.5-3.5 kg) were anesthetized with ~, sodium pentobarbital (30 mg/kg) given intravenously. Hearts ~ were rapidly excised and placed in oxygenated ~95% 2 ~ 5~ C2) . ice-cold Krebs-Henseleit (K-H) solution of the following millimolar composition: NaC1~ 118; KCl, 4.75; CaC12~ H2O, 2-54 KH2PO4, 1-19; MgSO4 7H2O, 1.19; NaHCO3, 12O5; glucose, 10.00. A 20-gauge stainless steel cannula was inserted into the right coronary artery via the coronar,y ostium~ Distal t~
the cannula, approximately 1 cm of coronary artery was dissected free'of surrounding tissue. The section of right : coronar,y artery with the cannula in place was excised from the heart and immediately transferred to a constant flow perfusion apparatus.

-23~

~ 7 The perfusion apparatus consists of a reservoir containing 20 ml of warm (37~C) oxygenated (95% 2 ~ 5~ CO2) K-H solution which bathes the coronary artery and serves as recirculating perfusate. An increase in perfusion pressure indicates vaso-constriction, whereas a decrease in perfusion pressure signifies ¦ vasodilation. Following an initial 1 hr. equilibrium period,vascular responsiveness was established by adding 25 mM KCl.
After washing with fresh K-H solution Eor 20-30 mins., the preparation achieved a relatively constant low basal tone.
lO` Basal perfusion pressure averaged 50~ 2.5 mm Hg. Fresh K-H
dilutions of stock prostacyclin analog concentrations were added to the perfusate reservoir in 0.1-0.2 ml volumes. Changes in-perfusion pressure in response to prostacyclin analogue addition generally plat-eaus within 5 min. of administration.

Example 1 Preparation of 6-keto PG~ methyl ester (IX, R- =
2 ---- _ CH3, R _- H) 1.1 Preparation of protected 11, 15 bis (THP) ~rostacyclin, methyl ester ~ OOC~3 COOCH3 HO TN
OTHP
XVI XVII

-2~- ~

_ _ , ,, .. _ . ... . _ I
.
~Z~7 COOCll3 . ,~o~ . ' .. > ~;

; TIIPO
~ . , OT~P
':
XVIII

Iodide XVI was prepared as described by Nicolaou et al, in J. Chem. Soc., Chem. Comm., 630 (1977). The iodlde was treated for minutes witll dihydropyran ( 3 equivalents) in Cil2C12 at 25C in tl~e presence oE 0.02 equiv~l~nts OL p-toluene-sulfonic acid. The protected iodide XVII was then treated witll !
excess DBU in toluene solution at 115C for 60 minutes and prostacyclin XVIII was obtained in L00% yield, by TLC. ¦
. . I
1.2 Preparation of 6 keto PG~, methyl ester COOCIl3 ~ OOC~13 : XVIII ~ ~ ~ 5:1~L 3 ~

TIIPO OTHP IIO

XI,~ XX

Prostacyclin XVIlI was treated witll traces of acetic acid in wet lOTI~F at 25C ~or 15 millutes ~o o~tain 78~ oL 11, 15 ~is ~ P)-6-keto PGFl (XIX); oxidatio~ of X~X with 2 ~quival~nts of pyridinium chlorochromate in CH2Clz at 25C led to an bis 11, 15 (TIIP) diketone in 76~ yield wllich was further deprotected witll ¦

-25- 1l ' 1, ~ ~5~

AcOH - THF-H20 (3:2:2) at 45C to yield 90~ of the diketo methyl ester XX.
.
This intermediate and its protected deriva-tives are useful : for the synthesis of va,rious of the compounds of the present ~ 5. invention.
.
. Example 2 . Preparatlon of 6, 9~yridaza prostacyclin, free acid 2.1 Preparation of 6-keto PGE~ free acid _ .. _ ._ .. _ ... .. . ._.. _ I

IX~ ~ O

TlIPO OTHP HO -. OH

XXI XXII

Methyl ester XIX (supra) was hydrolyzed with equivalents of LiOH in CH30H : H20 ( : ) to yield a lithium salt which was then acidified to free acid XXI. - This acid was oxidized with Jones reagent in acetone at -20~C and finally the THP groups~
were removed with acetic acid -TIIF - H20 (3:2:2) at 45~C (9o~

yield o glve 6-keto PG- free acid ~XXII).

¦ -26-~, ~
I

. .

2.2 ~r~parati~-1 o~ 6, 9 ,COOII

_iZ 112~ ~ ~9~ 0 ¦ 0}1 ~b ¦ XXIII XXIV

Treatment of the diketone free acid XXII with hvdrazi1le ~ ¦1 (l.0 equivalent, 95% ~) in l'Hl~ ~ ~12 (9:1) at 25C for 5 mi~utes I led to a new compound XXIII (Rf = 0.22 silica - 10% CH3011 in CH2Cl2) - This compound was directly oxidized by stirring over PtO2 at 25~C for 60 minutes to yielci 60~ of the aromatic free acid XXIV . T11is acid was metllylated with diazo- !
m2thane and isolated by careEul ureL~arative layer chromatograp11y to avoid decompoaltion (Rf=0.29 sllica - 10% CEi301-1 in CH2Cl2).
lo I l~lNMR
¦¦ ~ree acid XXIV was stable in aqueous solutions at 25C for at least 30 days. I-ts biological activity as well as that of intermediate XXIII were tested by the assay described under "Experimental Methods...".
, . ' I
- !
`-1~ -27- 1 I
I
,The results are as follows:

t~
¦ Saline Biological ~ctivity (25) _ . ~
¦ Compound Platelet Aggregation Cat Coronary Artery . ~ _ _ __ ~
1! prostacyclin 2 min- Inhibitor (Potent)(1) Dilator (Potent)~
, I Compound XXIV ~ 24h Inhibitor (Potent)(0.5) Dilator(Potent)~0.5) l Compound XXIII > 24h Inhibitor (Potent.)(O.l) Dilator(Potent)(O.l) I ! _ . __ . _ ¦¦ Example 3 Il Preparation of 6, 9-pyridaza Prostacyclin, N-oxides ¦ Oxidation of pyrldaza prostacyclin XXIV with 1.2 equivalents . 11 of m-CPBA in CH2C12 at 25C led to 60~ of a mixture of 2 oxides ¦! xxv G~and ~ . These oxides were not separated but were purified ¦¦ chxomatographically, Rf = 0.34, silica -20% CH30H in CH2Cl2, ca l! 1 1 by H NMR.
~ ~ - .
¦ The mixture of N-oxides was tested for bioloaical activity I and the results were as. follows:
I
¦ Biological Activity - t~ ~
saline (25C) Platelet Aggre~ation Cat Coronary Artery ~ .... ~ ~
N-oxides XXV>24h Small effect Small effect 1, _ . . .

20 ' !I Having now fully describcd this invention, it will be Il apparent to one of ordinary skill in the art that many modi~ica-¦¦ tions and variations can be made thereto without changing the scope thereof.

Claims (30)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:-

1. A process for preparing a stable biologically active aromatic prostacyclin analogue having the formula (VIII) wherein X represents or ; and R represents hydrogen, a pharmaceutically acceptable cation or a pharmaceutically acceptable lower alkyl group, which comprises a) reacting a prostanoid diketone of formula (IX) wherein R1 is hydrogen or a lower alkyl, R2 is hydrogen or a hydroxy group-protecting agent, with NH2 NH2 followed by oxidation; or b) reacting a prostanoid diketone of formula (IX) wherein R1 and R2 are the same as defined in (a) with NH2NH2 followed by oxidation, followed by further oxidation;
c) reacting a prostanoid diketone of formula (IX) wherein R1 and R2 are the same as defined in (a) with acid or a dehydrating agent; or d) reacting a prostanoid diketone of formula (IX) wherein R1 amd R2 are the same as defined in (a) with P2S5 or P2S3;
and optionally converting compound (VIII) into the free acid or pharmaceutically acceptable salt thereof.

2. A process for preparing a stable biologically active aromatic prostacyclin analogue having the formula (VIII) wherein X represents -N=N- and R represents -CH3 which comprises reacting a pros-tanoid diketone of formula (IX) wherein R1 is -CH3, R2 is hydrogen or a hydroxy group-protecting agent, with NH2NH2 followed by oxidation.

3. Process according to claim 2, which comprises convert-ing the biologically active aromatic prostacyclin analogue of formula (VIII) in which X=-N=N- and R=-CH3, into the sodium salt thereof.

4. Process according to claim 2, which comprises further oxidizing the biologically active aromatic prostacyclin anal-ogue of formula (VIII) in which X=-N-N and R=-CH3 to give compounds of formula (VIII) in which and R=-CH3.

5. Process according to claim 4 which comprises convert-ing the biologically active aromatic prostacyclin analogue of formula (VIII) in which and R=-CH3 into the sodium salt thereof.

6. A process for preparing a stable biologically active aromatic prostacyclin analogue having the formula having the formula (VIII) wherein IMAGE and R=-CH3, which comprises reacting a prostanoid diketone of formula (IX) wherein R1 is -CH3, R2 is hydrogen or a hydroxy group protect-ing agent, with an acid or a dehydrating agent.

7. Process according to claim 6, which comprises convert-ing the biologically active aromatic prostacyclin analogue of formula (VIII) in which and R=-CH3, into the sodium salt thereof.

8. A process for preparing a stable biologically active aromatic prostacyclin analogue having the formula (VIII) wherein and R=-CH3, which comprises reacting a prostanoid diketone of formula (IX) wherein R1 is -CH3, R2 is hydrogen or a hydroxy group protecting agent, with P2S5 or P2S3.

9. Process according to claim 8, which comprises convert-ing the biologically active aromatic prostacyclin analogue of formula (VIII) in which and R=-CH3, into the sodium salt thereof.

10. Process for preparing a stable biologically active aromatic prostacyclin analogue having the formula (VIII) wherein X= -NH and R= CH3, which comprises reacting a prostanoid diketone of formula (IX) as defined in claim 1 wherein R1 is -CH3, R2 is hydrogen or a hydroxy group-protecting agent, with ammonia at low temperatures.

11. Process according to claim 10, which comprises converting the biologically active aromatic prostacyclin anal-ogue of formula (VIII) in which X= and R=-CH3, into the sodium salt thereof.

12. A process for preparing a prostacyclin analogue of the formula (X) wherein R represents H, a pharmaceutically acceptable cation or a pharmaceutically acceptable lower alkyl, which comprises treating a diketone of formula (IX) wherein R1 = lower alkyl, R2=H with hydrazine at room temperatures for small periods of time, and selectively hydrolyzing the lower alkyl ester (IX ) to the free acid or salt (R=H or pharmaceutically acceptable cation).

13. Process for preparing a prostacyclin analogue of the formula (X) wherein R= -CH3 which comprises treating a diketone of formula (IX) wherein R1=-CH3, R2=H with hydrazine at room temperatures for small periods of time.

14. Process according to claim 13, which comprises converting the prostacyclin analogue of formula (X), wherein R=-CH3, into the sodium salt thereof.

15. Stable biologically active aromatic prostacyclin analogues having the formula (VIII) wherein X represents ; and R represents hydrogen, a pharmaceutically acceptable cation or a pharmaceutically acceptable lower alkyl group, whenever prepared by the process of claim 1, or its obvious chemical equivalents.

16. The prostacyclin analogue of claim 15 wherein X=-N=N-and R=-CH3,whenever prepared by the process of claim 2, or its obvious chemical equivalents.

17. The prostacyclin analogue of claim 15 wherein X=-N=N-and R= Na, whenever prepared by the process of claim 3, or its obvious chemical equivalents.

18. The prostacyclin analogue of claim 15 wherein and R= -CH3, whenever prepared by the process of claim 4, or its obvious chemical equivalents.

19. The prostacyclin,analogue of claim 15 wherein and R=Na, whenever prepared by the process of claim 5, or its obvious chemical equivalents.

20. The prostacyclin analogue of claim 15 wherein and R= -CH3, whenever prepared by the process of claim 6, or its obvious chemical equivalents.

21. The prostacyclin analogue of claim 15 wherein and R=Na, whenever prepared by the process of claim 7, or its obvious chemical equivalents.

22. The prostacyclin analogue of claim 15 wherein and R= -CH3, whenever prepared by the process of claim 8, or its obvious chemical equivalents.

23. The prostacyclin analogue of claim 15 wherein and R= -Na, whenever prepared by the process of claim 9, or its obvious chemical equivalents.

24. The prostacyclin analogue of formula (VIII) as defined in claim 15 wherein X= and R= -CH3, whenever prepared by the process of claim 10, or its obvious chemical equivalents.

25. The prostacyclin analogue of formula (VIII) as defined in claim 15 wherein X= and R=Na, whenever prepared by the process of claim 11, or its obvious chemical equivalents.

26. A prostacyclin analogue of the formula (X) wherein R represents H, a pharmaceutically acceptable cation or a pharmaceutically acceptable lower alkyl group, whenever prepared by the process of claim 12, or its obvious chemical equivalents.

27. The prostacyclin analogue of claim 26 wherein R= -CH3, whenever prepared by the process of claim 13, or its obvious chemical equivalents.

28. The prostacyclin analogue of claim 26 wherein R= Na, whenever prepared by the process of claim 14 or its obvious chemical equivalents.

29. A process for preparing a stable biologically active aromatic prostacyclin analogue having the formula (VIII) wherein X represents ; or ; and R represents hydrogen, a pharmaceutically acceptable cation or a pharmaceutically acceptable lower alkyl group, which comprises a) when X represents or i) reacting a prostanoid diketone of formula (IX) wherein R1 is hydrogen or a lower alkyl, R2 is hydrogen or a hydroxy group-protecting agent, with NH2 NH2 followed by oxidation; or ii) reacting a prostanoid diketone of formula (IX) wherein R1 and R2 are the same as defined in (a) with NH2NH2 followed by oxidation, followed by further oxidation;
iii) reacting a prostanoid diketone of formula (IX) wherein R1 and R2 are the same as defined in (a) with an acid or a dehydrating agent; or iv) reacting a prostanoid diketone of formula (IX) wherein R1 and R2 are the same as defined in (a) with P2S5 or P2S3;
and optionally converting compound (VIII) into the free acid or pharmaceutically acceptable salt thereof;
b) when X = -NH and R = CH3, which comprises re-acting prostanoid diketone of formula (IX) wherein R1 is -CH3, R2 is hydrogen or a hydroxy group-protecting agent, with ammonia at low temperatures.

39 .

30. A stable biologically active aromatic prostacyclin analogue of formula (VIII) as defined in claim 29, wherein a) R represents hydrogen, a pharmaceutically acceptable cation or a pharmaceutically acceptable lower alkyl group, and X represents whenever prepared by the process of claim 29 (a) or its obvious chemical equivalents, b) X represents -NH and R is -CH3 , whenever prepared by the process of claim 29(b) or its obvious chemical equivalents.