DK145608B - ANALOGY PROCEDURE FOR PREPARING THE NICKETIC ACID ESTER OF N- (2-HYDROXYETHYL) NICOTINAMIDE - Google Patents

Description

(19) DANMARK (®)(19) DENMARK (®)

|p (12) FREMLÆGGELSESSKRIFT <n> 1^5608 B| p (12) PUBLICATION <n> 1 ^ 5608 B

DIREKTORATET FORDIRECTORATE OF

PATENT- OG VAREMÆRKEVÆSENETTHE PATENT AND TRADEMARKET SYSTEM

(21) Ansøgning nr. 1282/77 (61) Int.CI* C 07 D 213/82 (22) Indleveringsdag 23· mar. 1977 (24) Løbedag 23· mar. 1977 (41) Aim. tilgængelig 3- okt. 1977 (44) Fremlagt 27 * dec. 1982 (86) International ansøgning nr.(21) Application No. 1282/77 (61) Int.CI * C 07 D 213/82 (22) Filing date 23 · Mar. 1977 (24) Race day 23 · Mar. 1977 (41) Aim. available Oct. 3; 1977 (44) Presented 27 * Dec. 1982 (86) International application no.

(86) International indleveringsdag (85) Videreførelsesdag - (62) Stamansøgning nr. -(86) International filing day (85) Continuation day - (62) Master application no. -

(30) Prioritet 2. apr. 1976, 36101/76, JP 13· Jul. 1976, 82458/76, JP(30) Priority 2 Apr 1976, 36101/76, JP 13 · Jul. 1976, 82458/76, JP

15. Jul. 1976, 83522/76, JP15. Jul. 1976, 83522/76, JP

(71) Ansøger CHUGAl SEIYAKU KABUSHIKI KAISHA, Tokyo, JP.(71) Applicant CHUGAl SEIYAKU KABUSHIKI KAISHA, Tokyo, JP.

(72) Opfinder Hiroyuki Nagano, JP: Takashi Mori, JP: Sakae Takaku, JP: Isao Matsunaga, JP: Tatsuo Kujirai, JP: m. fl.(72) Inventor Hiroyuki Nagano, JP: Takashi Mori, JP: Sakae Takaku, JP: Isao Matsunaga, JP: Tatsuo Kujirai, JP: et al.

(74) Fuldmægtig Internationalt Patent-Bur eau.(74) Clerk of the International Patent Office.

(54) Analogifremgangsmåde til frem= stilling af salpetersyreesteren af N-(2-hydroxyethyl)nicotinamid.(54) Analogous process for preparing the nitric ester of N- (2-hydroxyethyl) nicotinamide.

Opfindelsen angår en analogifremgangsmåde til fremstilling af den hidtil ukendte salpetersyreester af N-(2-hydroxyethyl)nico-tinamid med den i patentkravet angivne formel I eller et farmaceutisk acceptabelt salt deraf.The present invention relates to an analogous process for the preparation of the novel nitric acid ester of N- (2-hydroxyethyl) nicotinamide having the formula I or the pharmaceutically acceptable salt thereof as claimed.

Nogle nicotinsyrederivater eller nicotinamidderivater er blevet beskrevet i litteraturen,f.eks. japansk offentliggørelses- ® skrift nr. 31352/1977, USA-patentbeskrivelse nr. 3.036.074, USA-Some nicotinic acid derivatives or nicotinamide derivatives have been described in the literature, e.g. Japanese Patent Publication No. 31352/1977, U.S. Patent No. 3,036,074, U.S. Pat.

OISLAND

3. patentbeskrivelse nr. 3.092.634, USA-patentbeskrivelse nr. 3.168.438 ^ og "Mie Medical Journal" vol. 16(3), side 207-211, (1967).3. Patent Specification No. 3,092,634, U.S. Patent Specification No. 3,168,438 and "Mie Medical Journal" Vol. 16 (3), pages 207-211, (1967).

± Fra det japanske offentliggørelsesskrift er det kendt at fremstille nicotinesterderivater ved omsætning af 1-nicotinyl-glyce-rin eller l-nicotinyl-2,3-isopropylidenglycerin med rygende sal- 3 145608 2 petersyre, hvorved der opnås l-nicotinylglycerin-2,3-dinitrat, hvorefter forbindelsen omdannes til dens dioxanadditionsforbindel-se, der har en coronar vasodilaterende virkning.From the Japanese publication specification it is known to prepare nicotine ester derivatives by reacting 1-nicotinyl-glycerin or 1-nicotinyl-2,3-isopropylidene glycerine with fuming nitric acid to give l-nicotinylglycerine-2.3 -dinitrate, after which the compound is converted to its dioxane addition compound having a coronary vasodilating effect.

I USA patentskrift nr. 3.036.074 beskrives bl.a. fremstilling af dinitroxydiethyl-nicotinamid ved omsætning af dihydroxydiethyl-nicontinamid med rygende salpetersyre; forbindelsen angives at bevirke en forøgelse af den coronare blodgennemstrømning.U.S. Patent 3,036,074 discloses, inter alia, preparation of dinitroxydiethyl-nicotinamide by reaction of dihydroxydiethyl-nicontinamide with fuming nitric acid; the compound is said to cause an increase in the coronary blood flow.

I USA-patentbeskrivelse nr. 3.092.634 og USA-patentbeskri-velse nr. 3.168.438 beskrives fremstilling af bis-salpetersyre-esteren af N,N-bis-(p-hydroxyethyl)nicotinamid, der har en coronar vasodilaterende virkning, ved at omsætte salpetersyreesteren af diethanolamin med nicotinsyrechlorid.In U.S. Patent No. 3,092,634 and U.S. Patent No. 3,168,438, the preparation of the bis-nitric acid ester of N, N-bis (p-hydroxyethyl) nicotinamide having a coronary vasodilating effect is described by reacting the nitric ester of diethanolamine with nicotinic acid chloride.

Imidlertid har de omtalte forbindelser en kortvarig virkning eller de har en uhensigtsmæssig virkning på blodtrykket eller hjertefunktionen, og de er således ikke velegnede som lægemidler til stimulering af kredsløbssystemet, f.eks. når det drejer sig om iskæmisk hjertesygdom. I betragtning heraf har det været ønskeligt at udvikle et ideelt lægemiddel.However, the compounds mentioned have a short-term effect or they have an adverse effect on blood pressure or heart function, and thus are not suitable as drugs for stimulating the circulatory system, e.g. when it comes to ischemic heart disease. Given this, it has been desirable to develop an ideal drug.

I "Mie Medical Journal", vol. 16(3), side 207-211 (1967) nævnes 2-nicotinamidoethanol. Imidlertid er forbindelsen blot omtalt som en forsøgsforbindelse for antitumorvirkning, og litteraturstedet rapporterer ingen bemærkelsesværdig farmakologisk virkning.Mie Medical Journal, Vol. 16 (3), pages 207-211 (1967) mentions 2-nicotinamidoethanol. However, the compound is merely referred to as a test compound for antitumor action, and the literature site reports no remarkable pharmacological effect.

Opfinderne af foreliggende opfindelse har gennem længere tid udført research-arbejde og testning for at finde frem til en forbindelse, der er værdifuld til behandling af kredsløbssygdomme, og er til slut nået frem til ovennævnte forbindelse med formlen I.The inventors of the present invention have, over a long period of time, performed research and testing to find a compound that is valuable for the treatment of circulatory diseases and has finally reached the above-mentioned compound of formula I.

Den omhandlede forbindelse og dens farmaceutisk acceptable salte udviser forbedret virkning ved behandling af kredsløbssygdomme, såsom coronar vasodilaterende virkning, antihypertensiv virkning, antiarrhythmisk virkning, antikoagulativ virkning og perifer vasodilaterende virkning. Den er således værdifuld ved behandlingen af iskæmisk hjertesygdom, som et antihypertensivt lægemiddel, anti-koagulativt lægemiddel, antiarrhytmisk lægemiddel og som et perifert vasodilaterende, herunder cerebralt vasodilaterende og renalt vasodilaterende, middel.The subject compound and its pharmaceutically acceptable salts exhibit improved efficacy in the treatment of circulatory disorders such as coronary vasodilatory, antihypertensive, antiarrhythmic, anticoagulant and peripheral vasodilating. It is thus valuable in the treatment of ischemic heart disease, as an antihypertensive drug, anticoagulant drug, antiarrhythmic drug, and as a peripheral vasodilating, including cerebral vasodilating and renal vasodilating agent.

Fremgangsmåden ifølge Opfindelsen er ejendommelig ved, 145608 3 a) at man omsætter nicotinsyre med den i kravet angivne formel II eller dens reaktionsdygtige derivat ved carboxylgruppen med mono-ethanolaminnitrat med den i kravet angivne formel III eller dets reaktionsdygtige derivat ved aminogruppen, eller ved b) at man omsætter N-(2-hydroxyethylJnicotinamid med den i kravet angivne formel IV med salpetersyre eller nitrylchlorid#hvorefter man om ønsket overfører den dannede forbindelse i et farmaceutisk acceptabelt salt deraf .The process of the invention is characterized by (a) reacting nicotinic acid with the formula II or its reactive derivative as claimed in the mono-ethanolamine nitrate carboxyl group with the formula III or its amino-reactive derivative as claimed in the claim, or at b) reacting N- (2-hydroxyethyl-nicotinamide of the formula IV as claimed in the claim with nitric acid or nitric chloride) and then transferring the compound, if desired, into a pharmaceutically acceptable salt thereof.

De reaktionsdygtige derivater af nicotinsyre ved carboxylgruppen omfatter f.eks. syrehalogenider, et syreanhydrid, et aktivt amid, en aktiv ester og lignende. De derivater, der sædvanligvis anvendes, er syrechloridet, syreazidet, syreanhydrider, såsom anhy-drider afledt af 2 mol af forbindelsen II og anhydrider af forbindelsen med en anden syre, f.eks. dialkylphosphorsyre, phenylphos-phorsyre, diphenylphosphorsyre, benzylphosphorsyre, halogeneret phosphorsyre, dialkylphosphorsyrling, thiosvovlsyre, svovlsyre, alkylkulsyre, fede syrer, såsom pivalinsyre, pentansyre, isopentan-syre, 2-ethylsmørsyre eller trichloreddikesyre, eller aromatiske carboxylsyrer, som f.eks. benzoesyre, amider, f.eks. amider med imidazol, 4-substitueret imidazol, dimethylpyrazol, triazol eller tetrazol; og estere, f.eks. cyanomethylester, 4-nitrophenylester, 2,4-dinitrophenylester, trichlorphenylester, pentachlorphenylester, methansulfonylphenylester, phenylazophenylester, phenylthioester, 4-nitrophenylthioester, p-cresylthioester, carboxymethylthioester, pyranylester, pyridylester, 8-quinolylthioester og estere med N,N-dimethylhydroxylamin, l-hydroxyl-2-(IH)-pyridon, N-hydroxysuccin-imid eller N-hydroxyphthalimid.The reactive derivatives of nicotinic acid in the carboxyl group include, for example, acid halides, an acid anhydride, an active amide, an active ester and the like. The derivatives commonly used are the acid chloride, acid azide, acid anhydrides such as anhydrides derived from 2 moles of compound II and anhydrides of the compound with another acid, e.g. dialkylphosphoric acid, phenylphosphoric acid, diphenylphosphoric acid, benzylphosphoric acid, halogenated phosphoric acid, dialkylphosphoric acid, thiosulfuric acid, sulfuric acid, alkyl carbonic acid, fatty acids such as pivalic acid, pentanoic acid, isopentanoic acid, benzoic acid, amides, e.g. amides with imidazole, 4-substituted imidazole, dimethylpyrazole, triazole or tetrazole; and esters, e.g. cyanomethyl ester, 4-nitrophenyl ester, 2,4-dinitrophenyl ester, trichlorophenyl ester, pentachlorophenyl ester, methanesulfonyl phenyl ester, phenylazophenyl ester, phenylthioester, 4-nitrophenylthioester, p-cresylthioester, carboxymethylthioester, pyranyl hydroxyl-2- (1H) -pyridone, N-hydroxysuccin-imide or N-hydroxyphthalimide.

Som reaktionsdygtige derivater af udgangsforbindelsen III kan nævnes forbindelser, hvis aminogruppe er aktiveret ved anvendelse af f.eks. phosphortrichlorid, ethylchlorphosphit eller methyl-chlorphosphit.As reactive derivatives of the starting compound III may be mentioned compounds whose amino group is activated using e.g. phosphorus trichloride, ethyl chlorophosphite or methyl chlorophosphite.

Fremgangsmåden angivet under a) ovenfor kan f.eks. udføres ved, at man omsætter et reaktionsdygtigt derivat af forbindelsen II ved carboxylgruppen med forbindelsen III, således at man kondenserer dem ved en temperatur på fra -10 til 50°C, fortrinsvis 0 til 10°C i 0,5 til 4 timer. Opløsningsmidlerne, der kan anvendes til denne omsætning, omfatter bl.a. vand, benzen, toluen, tetrahydro-furan, diethylether, dioxan, dimethylformamid, chloroform, methy-lenchlorid, acetonitril, acetone, carbontetrachlorid og ethylace- 4 1-45608 tat. Der kan ved kondensationen anvendes en accelerator, som omfatter uorganiske basiske forbindelser, f.eks. hydroxidet, carbonatet eller acetatet af et alkalimetal eller jordalkalimetal, som f.eks. natriumacetat, natriumcarbonat, kaliumacetat, kaliumcarbonat, natriumhydroxid, calciumacetat, calciumcarbonat;eller aminoforbin-delser, såsom pyridin, triethylamin, dimethylanilin og picolin.The process set out in (a) above may e.g. is carried out by reacting a reactive derivative of compound II at the carboxyl group with compound III so as to condense them at a temperature of from -10 to 50 ° C, preferably 0 to 10 ° C for 0.5 to 4 hours. The solvents that can be used for this reaction include water, benzene, toluene, tetrahydrofuran, diethyl ether, dioxane, dimethylformamide, chloroform, methylene chloride, acetonitrile, acetone, carbon tetrachloride and ethyl acetate. In the condensation, an accelerator may be used which comprises inorganic basic compounds, e.g. the hydroxide, carbonate or acetate of an alkali metal or alkaline earth metal, such as e.g. sodium acetate, sodium carbonate, potassium acetate, potassium carbonate, sodium hydroxide, calcium acetate, calcium carbonate; or amino compounds such as pyridine, triethylamine, dimethylaniline and picoline.

Kondensationsreaktionen mellem forbindelsen II og forbindelsen III, hvis aminogruppe er blevet aktiveret med f.eks. phos-phortrichlorid, ethylchlorphosphit eller methylchlorphosph.it, kan bekvemt udføres ved temperaturer fra stuetemperatur til det anvendte opløsningsmiddels tilbagesvalingstemperatur i 0,5-3 timer. Det normalt anvendte opløsningsmiddel til denne fremgangsmåde omfatter et neutralt opløsningsmiddel, som f.eks. benzen, toluen, xylen, dioxan eller tetrahydrofuran; eller et basisk opløsningsmiddel, som f.eks. pyridin, triethylamin, dimethylamin, dimethylanilin eller picolin. Dersom der anvendes et neutralt opløsningsmiddel, foretrækkes det at tilsætte aminforbindelser, som f.eks. pyridin, triethylamin, dimethylanilin eller picolin.The condensation reaction between Compound II and Compound III, whose amino group has been activated with e.g. Phosphorus trichloride, ethyl chlorophosphite or methyl chlorophosphite, can conveniently be carried out at room temperature to the reflux temperature of the solvent used for 0.5-3 hours. The solvent normally used for this process comprises a neutral solvent such as e.g. benzene, toluene, xylene, dioxane or tetrahydrofuran; or a basic solvent such as pyridine, triethylamine, dimethylamine, dimethylaniline or picoline. If a neutral solvent is used, it is preferred to add amine compounds such as e.g. pyridine, triethylamine, dimethylaniline or picoline.

Ifølge en anden udførelsesform for opfindelsen kan forbindelsen II omsættes med forbindelsen III i et inert opløsningsmiddel i nærværelse af en amiddannelsesaccelerator, f.eks. en imidforbindel-se, såsom N,N'-dicyclohexylcarbodiimid, N-cyclohexyl-N'-morpholino-ethylcarbodiimid eller NjN’-diethylcarbodiimid, en iminforbindelse, som f.eks. diphenylketen-N-cyclohexylimin eller pentamethylenketen-N-cyclohexylimin, eller et phosphat eller phosphit, som f.eks. tri-ethylphosphit, ethylpolyphosphat eller isopropylphosphat, ved fra stuetemperatur til opløsningsmidlets tilbagesvalingstemperatur, idet man anvender 1-5 timer. Det inerte opløsningsmiddel, der kan anvendes ved denne reaktion, omfatter f.eks. benzen, toluen, tetrahydrofuran, . chloroform, dioxan, acetonitril og dimethylformamid.According to another embodiment of the invention, compound II can be reacted with compound III in an inert solvent in the presence of an amide formation accelerator, e.g. an imide compound such as N, N'-dicyclohexylcarbodiimide, N-cyclohexyl-N'-morpholinoethylcarbodiimide or NjN'-diethylcarbodiimide, an imine compound such as e.g. diphenylketene-N-cyclohexylimine or pentamethylene-ketene-N-cyclohexylimine, or a phosphate or phosphite, e.g. triethyl phosphite, ethyl polyphosphate or isopropyl phosphate, at from room temperature to the reflux temperature of the solvent, using 1-5 hours. The inert solvent which may be used in this reaction comprises, e.g. benzene, toluene, tetrahydrofuran ,. chloroform, dioxane, acetonitrile and dimethylformamide.

Den ovenfor omtalte fremgangsmåde b) kan udføres ved, at man omsætter forbindelsen IV med salpetersyre eller nitrylchlorid i et inert opløsningsmiddel, som f.eks. chloroform eller dichlormethan, ved en temperatur på fra -5°C til stuetemperatur i 1-3 timer. Det nitrerende middel, der sædvanligvis anvendes ved denne omsætning, er rygende salpetersyre.The above-mentioned process b) can be carried out by reacting the compound IV with nitric acid or nitryl chloride in an inert solvent, such as e.g. chloroform or dichloromethane, at a temperature of from -5 ° C to room temperature for 1-3 hours. The nitrating agent commonly used in this reaction is fuming nitric acid.

Forbindelsen fremstillet ved fremgangsmåde a) eller fremgangsmåde b) kan omdannes til et organisk eller uorganisk syre- 145608 5 additionssalt,såsom f.eks^hydrochloridet, nitratet, oxalatet, p-toluen-sulfonatet eller maleatet.The compound prepared by process a) or process b) can be converted to an organic or inorganic acid addition salt such as, for example, the hydrochloride, nitrate, oxalate, p-toluene sulfonate or maleate.

Det omhandlede pyridinderivat kan formuleres ved en hvilken som helst almindelig anvendt fremgangsmåde til et farmaceutisk præparat i form af tabletter, granuler, pulvere, kapsler, suspensioner, parenterale injektionspræparater, suppositorier eller lignende. Til fremstillingen af tabletter, pulvere, granuler eller kapsler fyldt med pulver eller granuler kan den omhandlede forbindelse blandes med ét eller flere farmaceutiske bærestoffer, som f.eks. lactose, stivelse, mannitol, kaolin, krystallinsk cellulose, talkum, calcium-carbonat, magnesiumstearat eller lignende. Til fremstilling af bløde kapsler, der er fyldt med flydende præparater, kan den omhandlede forbindelse opløses i en olie. Den omhandlede forbindelse kan også suspenderes i gummi arabicum eller en vandig sucroseopløsning, og pH-værdien justeres. Den omhandlede forbindelse kan også blandes med mannitol til fremstilling af et passende parenteralt injektionspræparat.The present pyridine derivative may be formulated by any commonly used method for a pharmaceutical composition in the form of tablets, granules, powders, capsules, suspensions, parenteral injection preparations, suppositories or the like. For the preparation of tablets, powders, granules or capsules filled with powder or granules, the subject compound may be mixed with one or more pharmaceutical carriers, such as e.g. lactose, starch, mannitol, kaolin, crystalline cellulose, talc, calcium carbonate, magnesium stearate or the like. For the preparation of soft capsules filled with liquid preparations, the subject compound may be dissolved in an oil. The subject compound may also be suspended in gum arabic or an aqueous sucrose solution and the pH adjusted. The subject compound may also be admixed with mannitol to produce a suitable parenteral injection preparation.

Detomhandlede pyridinderivat kan foreligge i en hvilken som helst form for farmaceutiske præparater i en mængde,der er tilstrækkelig til at udøve virkninger til behandling eller forhindring af kredsløbssygdomme, men som ikke medfører nogen uhensigtsmæssig virkning ved administrering af præparatet. En enhedsdosis, scan f.eks. en tablet eller en kapsel, kan sædvanligvis indeholde 5-20 mg af den aktive forbindelse, dersom præparatet administreres peroralt. Dersom præparatet indgives parenteralt, er enhedsdosen pr. ampul sædvanligvis på 1-10 mg af forbindelsen.The present pyridine derivative may be present in any form of pharmaceutical composition in an amount sufficient to exert effects in the treatment or prevention of circulatory diseases, but which does not cause any adverse effect in administering the composition. A unit dose, scan e.g. a tablet or capsule may usually contain 5-20 mg of the active compound if the preparation is administered orally. If the preparation is administered parenterally, the unit dose per ampoule usually of 1-10 mg of the compound.

Det er klart, at den aktuelle dosis afhænger af den pågældende patients tilstand, og at dosis derfor skal bestemmes for hvert enkelt tilfælde. Det vil imidlertid i almindelighed være bekvemt og sikkert at anvende en dosering svarende til 10-100 mg, fortrinsvis 10-60 mg, aktiv forbindelse pr dag til voksne personer, når der administreres peroralt, og 1-100 mg, fortrinsvis 1-50 mg, pr. dag til voksne personer, dersom der administreres parenteralt ved injektion.It is clear that the actual dose depends on the patient's condition and that the dose should therefore be determined for each case. However, it will generally be convenient and safe to use a dosage of 10-100 mg, preferably 10-60 mg, active compound per day for adults when administered orally, and 1-100 mg, preferably 1-50 mg. , pr. per day for adults if administered parenterally by injection.

Fig. 1 viser i grafisk afbildning den procentvise forøgelse af den coronare blodgennemstrømning, dersom forbindelsen fremstillet ifølge nedenstående eksempel 1 eller nitroglycerin administreres' intravenøst ifølge nedenstående forsøg 2 .FIG. 1 shows in graphical view the percentage increase in coronary blood flow if the compound prepared according to Example 1 or nitroglycerin is administered 'intravenously' according to Experiment 2 below.

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Fig. 2 er en grafisk afbildning af varigheden af forøgelsen af den coronare blodgennemstrømning, når forbindelsen fra eksempel 1 eller nitroglycerin administreres intravenøst ifølge forsøg 2.FIG. Figure 2 is a graphic representation of the duration of the increase in coronary blood flow when the compound of Example 1 or nitroglycerin is administered intravenously according to Experiment 2.

Fig. 3 er en grafisk afbildning af forandringen af tensionen i det venstre hjertekammer, når forbindelsen fra eksempel 1 eller nitroglycerin administreres intravenøst ifølge forsøg 2.FIG. Figure 3 is a graphic representation of the change in tension in the left ventricle when the compound of Example 1 or nitroglycerin is administered intravenously according to Experiment 2.

Fig. 4 er en grafisk afbildning af forandringen i hjerterytmen, når forbindelsen fra eksempel 1 eller nitroglycerin administreres intravenøst som i forsøg 2·FIG. 4 is a graphic representation of the change in heart rhythm when the compound of Example 1 or nitroglycerin is administered intravenously as in Experiment 2 ·

Fig. 5, 6 og 7 er grafiske afbildninger, der viser forandringen i elektrocardiogrammerne, når forbindelsen fra eksempel 1 administreres intravenøst.FIG. 5, 6 and 7 are graphs depicting the change in the electrocardiograms when the compound of Example 1 is administered intravenously.

Fig. 8 er en grafisk afbildning, der viser forandringen i systemisk blodtryk, når forbindelsen fra eksempel 1 eller nitroglycerin administreres intravenøst ifølge forsøg 2.FIG. Figure 8 is a graph showing the change in systemic blood pressure when the compound of Example 1 or nitroglycerin is administered intravenously according to Experiment 2.

Fig. 9 og 10 er grafiske afbildninger, der viser forandringen i det systemiske blodtryk, når forbindelsen fra eksempel 1 administreres intravenøst ifølge forsøg 2.FIG. Figures 9 and 10 are graphs showing the change in systemic blood pressure when the compound of Example 1 is administered intravenously according to Experiment 2.

Fig. 11 og 12 er grafiske afbildninger, der viser forandringen i blodstrømmen i aorta, når forbindelsen fra eksempel 1 administreres intravenøst.FIG. 11 and 12 are graphs showing the change in blood flow in the aorta when the compound of Example 1 is administered intravenously.

Fig. 13 er forsøgskurver, der viser virkningen af for bindelsen fra eksempel 1 på blodpladekoagulationen forårsaget af ade-nosindiphosphat ifølge forsøg 7.FIG. Figure 13 is experimental curves showing the effect of the compound of Example 1 on the platelet coagulation caused by adenosine diphosphate of Experiment 7.

Fig. 14 er forsøgskurver, der viser virkningen af for bindelsen på blodpladekoagulationen forårsaget af collagen ifølge forsøg 7.FIG. 14 are experimental curves showing the effect of the binding on the platelet coagulation caused by the collagen of Experiment 7.

Det bemærkes, at i angivelsen x - SD (N=5) i figurerne 1-12 betyder x gennemsnitsværdien, SD standardafvigelsen og N=5 antal forsøgsdyr.It should be noted that in the indication x - SD (N = 5) in Figures 1-12, x means the mean value, SD standard deviation and N = 5 number of test animals.

Forsøg 1Experiment 1

Den akutte toksicitet (LDj-g) af forbindelsen fra eksempel 2 bestemtes, idet man anvendte SD-stamme han- og hunrotter (4 uger gamle) , der peroralt eller intravenøst fik administreret forbindelsen. LDj-q over for både han- og hunrotter lå fra 1200 til 1300 mg/kg ved peroral administrering og fra 800 til 1000 mg/kg ved intravenøs administrering.The acute toxicity (LD 2 -g) of the compound of Example 2 was determined using SD strain male and female rats (4 weeks old) administered orally or intravenously. LDj-q for both male and female rats ranged from 1200 to 1300 mg / kg for oral administration and from 800 to 1000 mg / kg for intravenous administration.

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Forsøg 2Experiment 2

Voksne bastardhunde, der var blevet bedøvet ved Intravenøs administrering af 30-40 mg/kg natriumpentobarbital, underkastedes tho-racotomi under ventilering med oxygen, idet man anvendte Bird's respirator. Herefter måltes forskellige fysiske fænomener på følgende måde.Adult bastard dogs that had been anesthetized by Intravenous administration of 30-40 mg / kg sodium pentobarbital were subjected to tho-racotomy under ventilation with oxygen using Bird's respirator. Thereafter, various physical phenomena were measured as follows.

1) Coronar blodgennemstrømning (CBF).1) Coronary blood flow (CBF).

En elektromagnetisk flowmetersonde fastgjortes til en circumflex gren eller et udspring af en forreste nedadgående gren af den venstre coronare arterie.An electromagnetic flowmeter probe is attached to a circumflex branch or a projection of an anterior descending branch of the left coronary artery.

2) Coronar perfusionstryk (CBP).2) Coronary perfusion pressure (CBP).

Det fine kateter forbundet til en tryktransducer blev indstuk-ket i den ydre ende af sonden i den circumflexe gren af den venstre coronare arterie.The fine catheter connected to a pressure transducer was inserted into the outer end of the probe in the circumflex branch of the left coronary artery.

3) Aortisk blodgennemstrømning (AoBF).3) Aortic blood flow (AoBF).

En elektromagnetisk flowmeter sonde blev fastgjort til aortas udspring.An electromagnetic flowmeter probe was attached to the aortic origin.

4) Venstre ventrikulære tension (LVT).4) Left ventricular tension (LVT).

En strain gauge påsyedes den forreste væg af den venstre ventrikel.A strain gauge is applied to the anterior wall of the left ventricle.

5) Systemisk blodtryk (SBP).5) Systemic blood pressure (SBP).

Et kateter forbundet til en tryktransducer indførtes i den højre femorale arterie.A catheter connected to a pressure transducer was inserted into the right femoral artery.

6) Hjerterytme (HR).6) Heart rhythm (HR).

Hjerterytmen måltes med et cardiotachometer, idet man anvendte pulstrykket som triggerpuls.Heart rate was measured with a cardiotachometer, using pulse pressure as a trigger pulse.

7) Elektrocardiogram (ECG).7) Electrocardiogram (ECG).

Et elektrocardiogram afledtes med en unipolær afledning på hjerteoverfladen, idet man anvendte et andet elektrodesæt på en del af den forreste væg af venstre ventrikel.An electrocardiogram was derived with a unipolar conduction to the heart surface, using a different electrode set on a portion of the anterior wall of the left ventricle.

Ud over forsøgspunkterne 1-7 ovenfor underkastedes nogle af dyrene måling af den renale blodgennaitstrøming (KBF) og den femorale blodgennem-strømning (FBF) ved at tilknytte en elektromagnetisk flowmeterscnde til den venstre renale arterie og den venstre femorale arterie.In addition to test points 1-7 above, some of the animals were subjected to measurement of renal blood flow (KBF) and femoral blood flow (PDB) by connecting an electromagnetic flow meter scan to the left renal artery and left femoral artery.

Forsøgsforbindelsen fra eksempel 1 opløstes i fysiologisk saltvandsopløsning eller i destilleret vand, hvorefter man administrerede intravenøst, peroralt eller sublingualt.Nitroglycerin administreredes på samme måde som en aktiv kontrolforbindelse for at sammenlignes med forsøgsforbindelsen.The test compound of Example 1 was dissolved in physiological saline or distilled water and then administered intravenously, orally or sublingually. Nitroglycerin was administered in the same way as an active control compound to be compared with the test compound.

145608 8145608 8

Resultater.Results.

(I) Intravenøs administrering.(I) Intravenous administration.

a) Forandring i den coronare blodgennemstrømning.a) Change in coronary blood flow.

Den diastoliske coronare blodgennemstrømning begyndte at forøges 10-20 sekunder efter intravenøs administrering af forsøgsforbindelsen i en dosis på 10 yg/kg eller mere. Den systoliske coronare blodgennemstrømning forøgedes ved en dosis på 250 yg/kg eller mere efterfulgt af forbigående nedgang lige efter administreringen. Mid-delcoronargennemstrømningen viste en vedvarende forøgelse efter administrering af forsøgsforbindelsen.The diastolic coronary blood flow began to increase 10-20 seconds after intravenous administration of the test compound at a dose of 10 µg / kg or more. The systolic coronary blood flow increased at a dose of 250 µg / kg or more, followed by transient decline just after administration. The mid-coronary flow showed a sustained increase after administration of the test compound.

Forøgelsen af coronargennemstrømningen er afbildet i fig. 1 og 2 udtrykt som maksimum % forandring og varighed over for værdierne før administreringen.The increase in coronary flow is depicted in FIG. 1 and 2 expressed as maximum% change and duration against the values prior to administration.

Administrering af den omhandlede forbindelse selv på dosisniveauer på 10 yg/kg intravenøst frembragte en signifikant forøgelse i coronargennemstrømningen, og i et dosisniveau på 500 yg/kg intravenøst forårsagedes en så bemærkelsesværdig forøgelse, at den viste sig at være egentlig hyperæmi. I det sidstnævnte tilfælde var varigheden af forøgelsen af den coronare gennemstrømning næsten 3 timer. Virkningen af den omhandlede forbindelse var bedre end virkningen af nitroglycerin, især med hensyn til varigheden af virkningen.Administration of the subject compound even at dose levels of 10 µg / kg intravenously produced a significant increase in coronary flow, and at a dose level of 500 µg / kg intravenously caused such a remarkable increase that it proved to be actual hyperemia. In the latter case, the duration of the increase in coronary flow was nearly 3 hours. The effect of the subject compound was superior to that of nitroglycerin, especially in terms of duration of action.

b) Forandring i venstre ventrikulære tension.b) Left ventricular tension change.

Som vist i fig. 3 udviste den venstre ventrikulære tension en ringe nedgang efter intravenøs administrering af forbindelsen fra eksempel 1 i en dosismængde på 50 yg/kg eller mere. Nedgangen var næsten ækvivalent med den for nitroglycerin i det afDrrivede dosisområde. Imidlertid var maksimumprocentnedgangen 33% eller mindre, selv i en dosismængde på 500 yg/kg intravenøs.As shown in FIG. 3, the left ventricular tension showed a slight decrease after intravenous administration of the compound of Example 1 at a dosage rate of 50 µg / kg or more. The decrease was almost equivalent to that of nitroglycerin in the driven dose range. However, the maximum percentage decrease was 33% or less, even at a dose of 500 µg / kg intravenously.

c) Forandring i hjerterytmen.c) Change in heart rhythm.

Som vist i fig. 4 viste hjerterytmen næsten ingen forandring ved administrering af den omhandlede forbindelse i en dosismængde på indtil 20 yg/kg intravenøst, medens en dosismængde på 50 yg/kg intravenøst eller mere udviste en lille dosisafhængig nedgang, der nåede 17% ved en dosismængde på 500 yg/kg intravenøst. Derimod udviste administrering af nitroglycerin i en dosismængde på mere end 10 yg/kg intravenøst forøget hjerterytme.As shown in FIG. 4, the heart rate showed almost no change in administration of the subject compound at a dosage rate of up to 20 µg / kg intravenously, while a dose rate of 50 µg / kg intravenously or more exhibited a small dose-dependent decrease reaching 17% at a dose rate of 500 µg. / kg intravenously. In contrast, administration of nitroglycerin at doses greater than 10 µg / kg showed intravenously increased heartbeat.

d) Forandring i elektrocardiogrammet.d) Change in the electrocardiogram.

Som vist i figurerne 5 og 7 forlængedes PP-og QTc-intervaller-ne dosisafhængigt ved intravenøs administrering af forbindelsen fra 145608 9 eksempel 1 i en dosis på mere end 50 yg/kg, medens PQ-intervallet næsten ikke forandredes ved administrering af en dosis indtil 1000 yg/kg intravenøst.As shown in Figures 5 and 7, the PP and QTc intervals were dose dependent extended by intravenous administration of the compound of Example 1 at a dose greater than 50 µg / kg, while the PQ interval was virtually unchanged by administering a dose. up to 1000 µg / kg intravenously.

e) Forandring i det systemiske blodtryk.e) Change in systemic blood pressure.

Som vist i figurerne 8, 9 og 10 udviste det systemiske blodtryk et signifikant dosisafhængigt fald ved intravenøs administrering af forsøgsforbindelsen i en dosismængde på mere end 50 yg/kg. Virkningen af forbindelsen på blodtrykket var ikke stærkere, end virkningen udvist af nitroglycerin, men varigheden var længere, end den der fremkommer med nitroglycerin.As shown in Figures 8, 9 and 10, the systemic blood pressure showed a significant dose-dependent decrease in intravenous administration of the test compound at a dose rate greater than 50 µg / kg. The effect of the compound on blood pressure was not stronger than the effect of nitroglycerin, but the duration was longer than that produced by nitroglycerin.

f) Forandring i aortisk blodgennemstrømning.f) Change in aortic blood flow.

Som vist i figurerne 11 og 12 udviser den aortiske blodgennemstrømning en lille dosisafhængig forøgelse ved intravenøs administrering af forbindelsen fra eksempel 1 i en dosismængde på 10 yg/kg eller mere. Den procentvise forøgelse ved en dosismængde på 500 yg/kg intravenøst nåede maksimum 40%, men varigheden var ikke så lang sammenlignet med den forøgelse, der fandt sted af den coronare gennemstrømning.As shown in Figures 11 and 12, aortic blood flow exhibits a small dose-dependent increase in intravenous administration of the compound of Example 1 at a dose rate of 10 µg / kg or more. The percentage increase at a dose rate of 500 µg / kg intravenously reached a maximum of 40%, but the duration was not that long compared to the increase that occurred by the coronary flow.

g) Forandring i renal eller femoral gennemstrømning.g) Change in renal or femoral flow.

De renale og femorale gennemstrømninger forøgedes på trods af et fald i det systemiske blodtryk ved intravenøs administrering af forbindelsen fra eksempel 1 i en dosismængde på 100-250 yg/kg. Imidlertid var forøgelsen i de renale og femorale gennemstrømninger mindre end forøgelsen i den coronare gennemstrømning med hensyn til størrelse og varighed.The renal and femoral flows increased despite a decrease in systemic blood pressure by intravenous administration of the compound of Example 1 at a dosage rate of 100-250 µg / kg. However, the increase in the renal and femoral flows was smaller than the increase in the coronary flow in size and duration.

Af de ovenforstående forsøgsresultater vil det fremgå, at den omhandlede forbindelse har en forøgende virkning på den coronare gennemstrømning og betydeligt bedre end virkningen af nitroglycerin. Yderligere frembringer den omhandlede forbindelse et fald i blodtrykket, en forøgelse i hjerterytmen og en reduktion af hjertetensionen, som ikke var for stor selv ved høje dosismængder.From the above test results, it will be seen that the compound in question has an increasing effect on the coronary flow and significantly better than the effect of nitroglycerin. In addition, the compound of the invention produces a decrease in blood pressure, an increase in heart rate and a reduction in cardiac tension, which was not excessive even at high dose levels.

Yderligere viser den kendsgerning, at den omhandlede forbindelse ikke forstyrrer overførslen af excitation i hjertet, således som det vises ved, at der ikke findes forlængede PQ-interval-ler på elektrocardiogrammet, at den kan være meget værdifuld som et lægemiddel ved behandling af iskæmisk hjertesyge.Further, the fact that the subject compound does not interfere with the transmission of excitation in the heart, as evidenced by the absence of extended PQ intervals on the electrocardiogram, may be very valuable as a drug in the treatment of ischemic heart disease. .

Forbindelsen kan anvendes som et antihypertensivt lægemiddel eller et perifert vasodilaterende middel, fordi den har en længe virkende og mild antihypertensiv virkning og en dilaterende virkning på de femorale og renale kar, såvel som en ønskelig effekt på det 145608 ίο iskæmiske hjerte.The compound can be used as an antihypertensive drug or a peripheral vasodilating agent because it has a long acting and mild antihypertensive effect and a dilating effect on the femoral and renal vessels, as well as a desirable effect on the ischemic heart.

(II) Peroral og subli^igCtal administrering.(II) Oral and subli ^ igCtal administration.

Forbindelsen fremstillet ifølge eksempel 2 administreredes sub-lingoaLt til hunde som en tablet, der indeholdt 1-10 mg af forbindelsen. Ca. 2 minutter efter administreringen var den coronare gennemstrømning tydelig forøget. Forbindelsen udviste i en dosismængde på 10 mg næsten ingen forandring i det systemiske blodtryk, h j ertetensio-nen 0g den aortiske gennemstrømning. Derimod udviste sublingual administrering af en tablet, der indeholdt hydrochloridet af den aktive forbindelse, ikke nogen væsentlig forøgelse i den coronare gennemstrømning. Dersom forbindelsen fremstillet ifølge eksempel 1 eller 2 i enten den fri form eller som hydrochloridet administreredes intra-duodenalt i en dosismængde på 50 yg/kg eller mere, fandtes en tydelig langvarig forøgelse i den coronare gennemstrømning.The compound prepared according to Example 2 was administered sub-lingually to dogs as a tablet containing 1-10 mg of the compound. Ca. Two minutes after administration, the coronary flow was clearly increased. The compound showed, at a dose of 10 mg, almost no change in the systemic blood pressure, the higher the tension and the aortic flow. In contrast, sublingual administration of a tablet containing the hydrochloride of the active compound did not show any significant increase in coronary flow. If the compound prepared according to Examples 1 or 2 in either the free form or as the hydrochloride was administered intravenously at a dose rate of 50 µg / kg or more, a marked long-term increase in the coronary flow was found.

Som det vil fremgå af forsøgsresultaterne, kan de omhandlede forbindelser anvendes i forskellige præparater til parenteral injektion; eller i form af kapsler, tabletter, granuler eller pulvere til peroral administrering eller i sublingual administrerbar form.As will be seen from the test results, the compounds of this invention can be used in various parenteral injection preparations; or in the form of capsules, tablets, granules or powders for oral administration or in sublingually administrable form.

I modsætning hertil absorberes nitroglycerin ikke gennem tarmvæggen og er derfor kun tilgængelig i sublingual administreringsform.In contrast, nitroglycerin is not absorbed through the intestinal wall and is therefore only available in sublingual administration form.

Forsøg 3Experiment 3

Voksne bastardhunde, der var blevet bedøvet ved intravenøs administrering af 30-40 mg/kg natriumpentobarbital, underkastedes thoracotomi, idet man ventilerede med oxygen under anvendelse afAdult bastard dogs that had been anesthetized by intravenous administration of 30-40 mg / kg sodium pentobarbital were subjected to thoracotomy, ventilating with oxygen using

Bird's respirator. Et kateter indsattesproximalt i en gren af den venstre coronararteries udspring, og man indsprøjtede et kontrast-Bird's respirator. A catheter was inserted proximally into a branch of the left coronary artery and a contrast injection was injected.

R RR R

stof som f.eks. Conlaxin H eller Angiocorei gennem kateteret.substance such as Conlaxin H or Angiocorei through the catheter.

De morphologiske forandringer i den venstre coronararterie filmedes, idet man anvendte en 35 mm film før og efter injiceringen af forbindelsen. Ved analyse fandtes det, at den coronararterie var betydeligt dilateret ved intravenøs administrering af den omhandlede forbindelse i et dosisniveau på 100/tg/kg eller mere.The morphological changes in the left coronary artery were filmed using a 35 mm film before and after the injection of the compound. By analysis, it was found that the coronary artery was significantly dilated by intravenous administration of the subject compound at a dose level of 100 µg / kg or more.

Forsøg 4Experiment 4

Man anvendte samme forsøgsopstilling som i forsøg 2, idet man anvendte voksne bastardhunde, der var bedøvet ved intravenøs administrering af natriumpentobarbital. Den venstre forreste nedadgående gren eller den circumflexe gren af den coronare arterie forsnævredes eller aflukkedes mekanisk på den distale side 145608 11 af det pågældende sted, idet man indførte sonden fra et elektromagnetisk flowmetér, for at udvirke et hjerteiskæmisk tilfælde,'hvorefter man observerede virkningen af forbindelsen fra eksempel 1” på tilfældet.The same test set was used as in Experiment 2, using adult bastard dogs stunned by intravenous administration of sodium pentobarbital. The left anterior descending branch or the circumflex branch of the coronary artery narrowed or closed mechanically on the distal side of the site, introducing the probe from an electromagnetic flowmeter to effect a cardiac ischemic event, and then observing the effect of the compound of Example 1 ”on the case.

Onder ufuldstændig forsnævring forøgedes den coronare strøm lidt, når forbindelsen fra eksempel 1 administreredes intravenøst i en dosismængde på lOO^g/kg eller mindre, medens den..... ' faldt i ringe grad, når man administrerede en mængde på mere end 250,Ag/kg. Efter administrering af den omhandlede forbindelse blev ST-forhøjelse af elektrokardiogrammet i den hjerteiskamiské . Λ del øjensynligt forbedret og på samme tid observerede ^ man også bedring af den ventriculære tensioni en iskæmisk del.Under incomplete constriction, the coronary flow increased slightly when the compound of Example 1 was administered intravenously at a dosage rate of 100 µg / kg or less, while it fell slightly when administered more than 250 , Ag / kg. Following administration of the subject compound, ST elevation of the electrocardiogram was performed in the cardiac chamois. Λ part apparently improved and at the same time ^ improvement of the ventricular tension in an ischemic part was also observed.

Ved fuldstændig afsnøring forbedrede forbindelsen ikke ST-for- r øgeisen eller den ventriculære tension i midtpunktet af den iskafiiiSfcé del, men man fandt en forbedring af de omgivende punkter i den iskasniske del.By complete lubrication, the connection did not improve the ST-front elevation or ventricular tension at the midpoint of the icecap section, but an improvement of the surrounding points of the Iscasian portion was found.

På lignende måde fandt man,at der, når en tablet indeholdende 10 mg af den fri form af forbindelsen fremstillet i eksempel 1 · administreredes sublingualt, skete en forøgelse af den coronare gennemstrømning ,og at ST-hævning af elektrokardiogrammet og reduktion af den ventriculære tension forbedredes.Similarly, when a tablet containing 10 mg of the free form of the compound prepared in Example 1 was administered sublingually, an increase in coronary flow occurred and ST elevation of the electrocardiogram and reduction of ventricular tension improved.

Forsøg 5 (I) En papillærmuskel, der var isoleret fra et marsvin, anbragtes i et organbad fyldt med oxygeneret Tyrode-opløsning * (ca: 1,8 mM, Ki 2,7 inM) ved 30°C. Den ene ende af musklen fiksére-des i organbadet, og den anden ende forbandtes til et tensiometer med en tråd. Virkningen af forbindelsen fra eksempel 1 på muskel" kontraktionskraften undersøgtes ved at give elektriske stimuli (2 0V,5 msec., 1Hz) til en muskel med en Ag-AgCl-elektrodeVMan undersøgte også den antagonistiske virkning af forbindelsen over for calciumioner eller isoproterenol. --1Experiment 5 (I) A papillary muscle isolated from a guinea pig was placed in an organ bath filled with oxygenated Tyrode solution * (approx. 1.8 mM, Ki 2.7 inM) at 30 ° C. One end of the muscle is fixed in the organ bath and the other end is connected to a tensiometer with a thread. The effect of the compound of Example 1 on muscle "The contraction force was investigated by providing electrical stimuli (20V, 5 msec, 1Hz) to a muscle with an Ag-AgCl electrodeVMan also investigated the antagonistic effect of the compound on calcium ions or isoproterenol. -1

Af forsøgsresultaterne fremgik det, at kontraktionen af den papillære muskel påført ved elektriské stimuli hæmmedes ved tilførsel af den omhandlede forbindelse i én mængde på mere end l^g/ml, og at calciumionen havde antagonistisk virkning over for en sådan hæmmende virkning. På den anden side"udviste den papillære muskel, når man tilførte isoproterenol i ett koncentråtion på^v 0,08 2-g/ml, meget kraftig excitation, hvorved der fremkom to eller flere uregelmæssige kontraktioner ved hvér enkelt elektrisk' påi-r : 12 145608 virkning. Under disse forhold bevirkede en tilsætning af den omhandlede forbindelse til opløsningen i en koncentration på mere end 2/tg/ml en hæmning af den for stærke excitation.From the test results, it was found that the contraction of the papillary muscle applied by electrical stimuli was inhibited by supplying the subject compound in an amount greater than 1 µg / ml, and that the calcium ion had antagonistic action against such inhibitory effect. On the other hand, when administering isoproterenol in a concentration concentration of 0.08 2-g / ml, the papillary muscle exhibited very strong excitation, resulting in two or more irregular contractions in each single electrical operation: Under these conditions, addition of the subject compound to the solution at a concentration greater than 2 µg / ml caused an inhibition of the excessive excitation.

(II) En spiralformet strimmel af en coronararterie fra en hund anbragtes i et organbad fyldt med Ca**-fri Lock's-opløs-ning. Den ene ende af strimmelen fikseredes til organbadet, og den anden ende forbandtes til et tensiometer. Man undersøgte virkningen af forbindelsen fra eksempel 1 på kontraktionen af strimlen, induceret ved polarisering af opløsningen ved tilsætning af 43 mmol K+ og 1 mmol Ca+ .(II) A helical strip of a coronary artery from a dog is placed in an organ bath filled with Ca ** free Lock's solution. One end of the strip was fixed to the organ bath and the other end was bandaged to a tensiometer. The effect of the compound of Example 1 on the contraction of the strip induced by polarization of the solution was investigated by the addition of 43 mmol K + and 1 mmol Ca +.

Forsøgsresultaterne viste, at kontraktionerne, der var induceret ved K+ depolarisering, hæmmedes af den omhandlede forbindelse, og at en sådan hæmmende virkning formindskedes ved tilsæt- • _ ++ ning af Ca (III) Virkningen af Ca+ på forøgelse i coronargennemstrøm-ningen undersøgtes, idet man anvendte en hund, der var bedøvet med natriumpentobarbital ved samme fremgangsmåde som beskrevet i forsøg 2.The test results showed that the contractions induced by K + depolarization were inhibited by the subject compound and that such inhibitory effect was diminished by the addition of Ca (III). The effect of Ca + on increase in coronary flow was investigated. using a dog anesthetized with sodium pentobarbital by the same procedure as described in Experiment 2.

(I) Forøgelsen i den coronare blodgennemstrømning induceret ved administrering af den omhandlede forbindelse hæmmedes ved store doser Ca (IV) Colon taeniae fra et marsvin anbragtes i et organbad fyldt med Tyrode-opløsning, og virkningen af forbindelsen fra eksempel 1 på den spontane kontraktion og kontraktionen induceret ved K+ depolarisering undersøgtes.(I) The increase in coronary blood flow induced by administration of the subject compound was inhibited by large doses of Ca (IV) Colon taeniae from a guinea pig placed in an organ bath filled with Tyrode solution, and the effect of the compound of Example 1 on the spontaneous contraction and the contraction induced by K + depolarization was investigated.

Forsøgsresultaterne viste, at den spontane kontraktion og kontraktion induceret ved K* depolarisering hæmmedes ved tilførsel af forbindelsen i en koncentrationsmængde på 2/&g/ml, og at enThe test results showed that the spontaneous contraction and contraction induced by K * depolarization were inhibited by the application of the compound at a concentration of 2 µg / ml and that a

XXXX

sådan hæmmende virkning genvandtes ved tilsætning af Casuch inhibitory effect was restored by the addition of Ca

Analyse af forsøgsresultaterne angiver, at den omhandlede forbindelse har en antagonistisk virkning i forhold til Ca**, således at den hæmmende virkning over for hjertekontraktion, den dilaterende virkning over for glatte muskler fra en hunds coronare arterie og den hæmmende virkning over for kontraktion af colon taeniae fra marsvin fremkaldt ved anvendelse af forbindelsen modvirkedes ved tilsætning af Ca**. Yderligere fandt man, at den omhandlede forbindelse havde en antiarrhytmisk virkning, fordi forbindelsen hæmmede den for stærke excitation af den papillære muskel, der var induceret ved administrering af isoproterenol.Analysis of the test results indicates that the subject compound has an antagonistic effect over Ca **, such that the inhibitory effect on heart contraction, the dilating effect on smooth muscles of a dog's coronary artery and the inhibitory effect on colon contraction guinea pig taeniae induced using the compound were counteracted by the addition of Ca **. Furthermore, the compound of this invention was found to have an antiarrhythmic effect because the compound inhibited the excessive excitation of the papillary muscle induced by the administration of isoproterenol.

13 14560813 145608

Forsøg 6Experiment 6

Blodgennemstrømningen blev gjort utilstrækkelig ved eksperimentelt at belaste coronararterien hos en hund, f.eks. ved at sammensnøre coronararterien i en vis periode, og lade arterien forblive i denne tilstand. Ved denne behandling bestemtes det perifere blodtryk og den perifere blodgennemstrømning} og spontane fluctuationer inden for et tidsrum af størrelsesordenen minutter efterfulgtes af ST-forhøjelse i elektrokardiogrammet.Blood flow was rendered insufficient by experimentally loading the coronary artery of a dog, e.g. by constricting the coronary artery for a certain period of time, leaving the artery in this state. In this treatment, peripheral blood pressure and peripheral blood flow} and spontaneous fluctuations were determined within a period of the order of minutes, followed by ST elevation in the electrocardiogram.

Dette fænomen er meget lig anfaldet af en art angina pectoris i et klinisk tilfælde. Den periodiske fluctuation har vist sig at forårsage periodiske spasmer i en forsnævret coronararterie. Man undersøgte virkningen af forbindelsen på en sådan angina pectoris model.This phenomenon is very similar to the attack of a species of angina pectoris in a clinical case. The periodic fluctuation has been shown to cause periodic spasms in a narrowed coronary artery. The effect of the compound on such an angina pectoris model was investigated.

Forsøgsresultaterne viste, at periodiske fluctuationer i det coronare blodtryk og i den coronare blodgennemstrømning hæmmedes ved intravenøs administrering af forbindelsen fra eksempel 1 i en dosismængde på 250Ag/kg eller mere. Det vil sige, at tilsyneladende reduceredes den periodiske fluctuation i blodgennemstrømningen og der forblev kun små fluctuationer tilbage. Den effektive virkningstid af det hæmmende middel lå fra 25 til 40 minutter.The test results showed that periodic fluctuations in the coronary blood pressure and in the coronary blood flow were inhibited by intravenous administration of the compound of Example 1 at a dosage rate of 250 Ag / kg or more. That is, it appears that the periodic fluctuation in blood flow was reduced and only small fluctuations remained. The effective time of action of the inhibitor was from 25 to 40 minutes.

Som angivet ovenfor er den omhandlede forbindelse virksom ved behandlingen af lidelsen angina pectoris.As indicated above, the compound in question is effective in treating the disorder angina pectoris.

Forsøg 7Experiment 7

Ved fremgangsmåden omtalt ovenfor undersøgtes forbindelsen fra eksempel 1 til konstatering af om den udviste antikoagulerende virkning over for blodplader, nemlig antiembolisk virkning.In the method described above, the compound of Example 1 was examined to determine whether the anticoagulant effect was shown against platelets, namely antiembolic effect.

1. Fremstilling af forsøgsopløsningen.1. Preparation of the test solution.

1-1) Indstilling af blodpladerig plasma (PRP-opløsning). Kaninblod indeholdende 38% natriumcitrat (blod: vandig natrium-citratopløsning = 9:1) underkastedes centrifugering ved 1000 omdrejninger pr. minut i 10 minutter. Den supernatante væske anvendtes som PRP-opløsning.1-1) Platelet rich plasma setting (PRP solution). Rabbit blood containing 38% sodium citrate (blood: aqueous sodium citrate solution = 9: 1) was subjected to centrifugation at 1000 rpm. minute for 10 minutes. The supernatant liquid was used as PRP solution.

1-2) Indstilling af en adenosin diphosphatopløsning (ADP-opløsning).1-2) Setting of an adenosine diphosphate solution (ADP solution).

Adenosindiphosphat (Sigma Chemical Compahy, Ltd.) opløstes’ i fysiologisk saltvandsopløsning i en koncentration på 100^g/ml.Adenosine diphosphate (Sigma Chemical Compahy, Ltd.) was dissolved in physiological saline solution at a concentration of 100 µg / ml.

1-3) Indstilling af collagensuspension.1-3) Adjustment of collagen suspension.

50 mg collagen opnået fra kvægsene (Sigma Chemical Co., Ltd.) opslæmmedes i 5 ml fysiologisk saltvandsopløsning, og homogeniseredes 145608 14 1 en glashomogenizer i 5 minutter. Dernæst centrifugerede man med 500 omdrejninger pr. minut i 5 minutter, og den supernatante væske anvendte som collagen opslæmning.50 mg of collagen obtained from the cattle (Sigma Chemical Co., Ltd.) was slurried in 5 ml of physiological saline solution and homogenized in a glass homogenizer for 5 minutes. Then they were centrifuged at 500 rpm. and the supernatant liquid used as collagen slurry.

1-4) Forbindelsen fra eksempel 1 opløstes i fysiologisk saltvandsopløsning for at opnå en forsøgsopløsning.1-4) The compound of Example 1 was dissolved in physiological saline solution to obtain a test solution.

2. Målingsmetode.2. Measurement method.

Målingen udførtes, idet man anvendte aggregometer (Evans Electroselenium Ltd., model 169).The measurement was performed using an aggregometer (Evans Electroselenium Ltd., model 169).

PRP-opløsningen (0,5 ml) og forsøgsopløsningen (0,025 ml) eller en fysiologisk saltvandsopløsning som kontrol (0,025 ml) anbragtes hver i to cuvetter, og dernæst inkuberede man ved 37°C i 2 minutter. Herefter tilsattes 0,025 ml ADP-opløsning eller 0,025 ml collagenopløsning til hver cuvette. Virkningen af forbindelsen på blodpladeaggregation induceret af ADP eller collagen blev målt.The PRP solution (0.5 ml) and the test solution (0.025 ml) or a physiological saline solution as a control (0.025 ml) were each placed in two cuvettes and then incubated at 37 ° C for 2 minutes. Then 0.025 ml of ADP solution or 0.025 ml of collagen solution was added to each cuvette. The effect of the compound on platelet aggregation induced by ADP or collagen was measured.

Som vist i fig. 13 og 14 var den initiale hastighed for ADP-induceret aggregation i forsøgsforbindelsen den samme som i kontrollen, men forsøgsforbindelsen accelererede dissocieringen af de aggregerede blodplader. På den anden side forsinkede forsøgsforbindelsen påbegyndelsen af collageninduceret aggregation, og graden af maksimumaggregation var lavere end hos kontrollen.As shown in FIG. 13 and 14, the initial rate of ADP-induced aggregation in the test compound was the same as in the control, but the test compound accelerated the dissociation of the aggregated platelets. On the other hand, the test compound delayed the onset of collagen-induced aggregation and the degree of maximum aggregation was lower than in the control.

Ud fra disse resultater bekræftedes den hæmmende virkning af forbindelsen på blodpladeaggregation. Man fandt således, at den omhandlede forbindelse også var værdifuld som antikoagulans.From these results, the inhibitory effect of the compound on platelet aggregation was confirmed. Thus, it was found that the subject compound was also valuable as anticoagulant.

Forsøg 8Experiment 8

Ved samme fremgangsmåde som beskrevet i forsøg 2, punkt 5, undersøgtes forandringen i det systemiske blodtryk, når forsøgsforbindelserne administreredes intravenøst. De opnåede resultater er vist i nedenforstående tabel.By the same procedure as described in Experiment 2, Section 5, the change in systemic blood pressure was examined when the test compounds were administered intravenously. The results obtained are shown in the table below.

TabelTable

Forsøgsforbindelse Dosis Procentforan- Varighed _Q4g/kg)_dring af S.B.P. (min.)Test compound Dose Percentage Duration _Q4g / kg) change of S.B.P. (mine.)

Forbindelse fra eksempel 1 50 16 22Compound of Example 1 50 16 22

Nitroglycerin 50 45 8 145608 15Nitroglycerin 50 45 8 145608 15

Forbindelsen fremstillet ifølge opfindelsen er endvidere ned hensyn til virkning mod angina pectoris blevet sammenlignet med den kendte forbindelse, der må anses for at være kemisk nærmest beslægtet dermed, nemlig dinitroxydiethylnicotinamid omhandlet i eksempel 8 i USA-patentskrift nr. 3.036.074.Furthermore, the compound of the invention has been compared to its effect against angina pectoris compared to the known compound which must be considered to be chemically closely related thereto, namely dinitroxydiethylnicotinamide disclosed in Example 8 of U.S. Patent 3,036,074.

Sammenligningsforsøg I overensstemmelse med de metoder, der er beskrevet ovenfor under forsøg 2, punkt 1, 5 og 6 - bortset fra, at der som forsøgsdyr blev benyttet beagle-hunde, der blev bedøvet ved intravenøs administrering af 30-40 mg/kg natriumpentobarbital - måltes den coronare blodgennemstrømning (CBF), det systemiske blodtryk (SBP) og hjerterytmen (HR) efter administrering af forbindelsen fremstillet ifølge foreliggende opfindelse eller af den kendte forbindelse. Endvidere måltes, i overensstemmelse med de metoder, der er beskrevet ovenfor i forsøgene 4 og 6, de to forbindelsers virkning på ændringen af den coronare blodgennemstrømning fremkaldt ved forsnævring af coronararterien som beskrevet i forsøg 4 eller ved den i forsøg 6 beskrevne angina pectoris model.Comparative Experiments In accordance with the methods described above under Experiments 2, Points 1, 5 and 6 - except that as test animals, beagle dogs anesthetized by intravenous administration of 30-40 mg / kg sodium pentobarbital were used - the coronary blood flow (CBF), systemic blood pressure (SBP) and heartbeat (HR) were measured after administration of the compound of the present invention or of the known compound. Furthermore, in accordance with the methods described above in experiments 4 and 6, the effect of the two compounds on the change in coronary blood flow induced by narrowing the coronary artery as described in experiment 4 or by the angina pectoris model described in experiment 6 was measured.

Resultater.Results.

a) Forandring af den coronare blodgennemstrømning (CBF):a) Change in coronary blood flow (CBF):

Forøgelsen af CBF og varigheden af forøgelsen af CBF ved administrering af forbindelsen fremstillet ifølge foreliggende opfindelse og af den kendte forbindelse er vist i henholdsvis fig. 15 og fig. 16.The increase of CBF and the duration of the increase of CBF in administering the compound of the present invention and of the known compound are shown in Figs. 15 and FIG. 16th

Intravenøs administrering af forbindelsen fremstillet ifølge op-, findelsen i en dosis på blot 2o pgAg viste en signifikant forøgelse af den coronare blodgennemstrømning. Endvidere var virkningen af denne forbindelse på forøgelsen af den coronare blodgennemstrømning signifikant højere end virkningen af den kendte forbindelse ved alle de anvendte doser.Intravenous administration of the compound prepared according to the invention at a dose of only 20 µgAg showed a significant increase in coronary blood flow. Furthermore, the effect of this compound on the increase of the coronary blood flow was significantly higher than the effect of the known compound at all doses used.

Den intravenøse administrering af forbindelsen fremstillet ifølge opfindelsen i en dosis på 100 yg/kg viste endvidere en virkningsvarighed på over 20 minutter, medens virkningsvarigheden opnået ved intravenøs administrering af den kendte forbindelse var bemærkelsesværdig kort. Forskellene mellem de to forbindelsers virkninger er således betragtelige.Furthermore, the intravenous administration of the compound of the invention at a dose of 100 µg / kg showed a duration of action of more than 20 minutes, while the duration of action obtained by intravenous administration of the known compound was remarkably short. Thus, the differences between the effects of the two compounds are considerable.

b) Forandring i det systemiske blodtryk (SBP).b) Change in systemic blood pressure (SBP).

Som vist i fig. 17 var det fald i det systemiske blodtryk, der blev fremkaldt ved administrering af den her omhandlede for- 145608 16 bindelse/ lavere end det af den kendte forbindelse fremkaldte fald.As shown in FIG. 17 was the decrease in systemic blood pressure caused by the administration of the present compound / lower than the decrease caused by the known compound.

c) Forandring i hjerterytme (HR).c) Change in heart rate (HR).

Som vist i fig. 18 viste hjerterytmen næsten ingen forandring ved administrering af den omhandlede forbindelse, medens den blev væsentligt forøget ved administrering af den kendte forbindelse.As shown in FIG. 18, the heartbeat showed almost no change in the administration of the subject compound, while it was substantially increased by the administration of the known compound.

d) Ændring af den coronare blodgennemstrømning under forsnævring af coronararterien.d) Change of coronary blood flow during narrowing of the coronary artery.

Som vist i fig. 19 viste den coronare gennemstrømning en forøgelse ved intravenøs administrering af forbindelsen fremstillet ifølge opfindelsen, men, i modsætning hertil, et fald ved administrering af den kendte forbindelse.As shown in FIG. 19, the coronary flow showed an increase in the intravenous administration of the compound of the invention, but, in contrast, a decrease in the administration of the known compound.

e) Virkning af forbindelserne på angina pectoris-model.e) Effect of compounds on angina pectoris model.

Intravenøs administrering af forbindelsen fremstillet ifølge opfindelsen i en dosis på mere end 150 yg/kg havde en hæmmende virkning på unormal ændring af elektrokardiogrammet (ST-forhøjelse) og på den periodiske fluktuation af den coronare blodgennemstrømning. I modsætning hertil viste intravenøs administrering af den kendte forbindelse selv i en dosis på 300 yg/kg ikke en sådan hæmmende virkning.Intravenous administration of the compound of the invention at a dose greater than 150 µg / kg had an inhibitory effect on abnormal change of the electrocardiogram (ST elevation) and on the periodic fluctuation of coronary blood flow. In contrast, intravenous administration of the known compound even at a dose of 300 µg / kg did not show such an inhibitory effect.

Diskussion I almindelighed er de farmakologiske virkninger, der kræves af en forbindelse som anti-anginalt lægemiddel, hvilket er formålet med den her omhandlede forbindelse, en forøgelse af den coronare blodgennemstrømning over en længere tidsperiode uden væsentlig ændring af hjerterytmen og blodtrykket. I betragtning heraf er forbindelsen fremstillet ifølge opfindelsen, som det fremgår af de ovenfor beskrevne prøveresultater a), b) og c), den kendte forbindelse langt overlegen, hvad angår den virkning, der kræves af et anti-anginalt lægemiddel.Discussion In general, the pharmacological effects required by a compound as an anti-anginal drug, which is the object of the present invention, are an increase in coronary blood flow over a prolonged period of time without significant change in heart rate and blood pressure. In view of this, the compound prepared according to the invention, as can be seen from the test results described above a), b) and c), the known compound is far superior in terms of the effect required by an anti-anginal drug.

Endvidere viste den her omhandlede forbindelse udmærkede virkninger ved de prøver, hvorved der anvendtes patologiske modeller, især ved den prøve hvor der anvendtes en angina pectoris-model ved sammensnøring af coronararterien, medens der ikke kunne iagttages signifikante virkninger ved anvendelse af den kendte forbindelse.Furthermore, the present compound showed excellent effects in the samples using pathological models, especially in the sample using an angina pectoris model in constricting the coronary artery, while no significant effects could be observed using the known compound.

-v 17 145608-v 17 145608

Eksempel 1Example 1

Til en blanding af 5 g natriumhydrogencarbonat, 15 ml vand, 1,69 g salpetersyreester af monoethanolaminnitrat og 20 ml chloroform sattes langsomt 2,5 g nicotinylchloridhydrochlorid i løbet af 10 til 30 minutter under omrøring ved 0 til 5°C.To a mixture of 5 g of sodium bicarbonate, 15 ml of water, 1.69 g of nitric acid ester of monoethanolamine nitrate and 20 ml of chloroform was slowly added 2.5 g of nicotinyl chloride hydrochloride over 10 to 30 minutes with stirring at 0 to 5 ° C.

Omrøringen fortsattes i yderligere 30 minutter, hvorefter chloro-formlaget fraskiltes. De tilbageblevne vandige lag ekstraheredes med chloroform, og ekstrakten blev blandet med det fraskilte chloroformlag. Det organiske lag vaskedes med en vandig opløsning af kaliumcarbonat, tørredes over vandfrit natriumsulfat og inddampedes under reduceret tryk til tørhed. Resten opløstes i ether-isopropanol (1:1), og herefter bobledes hydrogenchlorid igennem opløsningen under afkøling, hvorved man opnåede 2,35 g af salpetersyreesteren af N-(2-hydroxyethyl)nicotinamidhydrochlorid. Omkrystallisation med ethanol gav farveløse nålelignende krystaller, der havde et smeltepunkt på 132°C.Stirring was continued for another 30 minutes, after which the chloroform layer was separated. The remaining aqueous layers were extracted with chloroform and the extract was mixed with the separated chloroform layer. The organic layer was washed with an aqueous solution of potassium carbonate, dried over anhydrous sodium sulfate and evaporated under reduced pressure to dryness. The residue was dissolved in ether-isopropanol (1: 1) and then hydrogen chloride was bubbled through the solution under cooling to give 2.35 g of the nitric ester of N- (2-hydroxyethyl) nicotinamide hydrochloride. Recrystallization with ethanol gave colorless needle-like crystals having a melting point of 132 ° C.

Analyse:Analysis:

Beregnet for C8HioN304C1: C 38,80, H 4,07, N 16,96 (%)Calcd for C 8 H 10 N 3 O 4 Cl: C, 38.80; H, 4.07; N, 16.96 (%)

Fundet : C 38,89, H 4,02, N 16,72 (%) IR(cm_1) : NH, 3255m C= o., 1669, 0N02, 1640Found: C 38.89, H 4.02, N 16.72 (%) IR (cm -1): NH, 3255m C = 0.1669, NO2, 1640

Eksempel 2Example 2

Til en opløsning af 1,69 g salpetersyreester af monoethanol-amin i 20 ml pyridin sattes langsomt 2,5 g nicotinylchloridhydrochlorid i løbet af 10-30 minutter under omrøring ved 5°C. Efter yderligere omrøring i 30 minutter, inddampedes reaktionsblandingen til tørhed. Resten opløstes i chloroform, og opløsningen vaskedes med en vandig opløsning af natriumhydrogencarbonat. Det organiske lag fraskiltes, tørredes over vandfrit natriumsulfat og inddampedes under reduceret tryk til tørhed. Resten chromatograferedes på silicagel (Wakogel C-200, Wako Pure Chemical Industries, Ltd.,To a solution of 1.69 g of nitric acid ester of monoethanol-amine in 20 ml of pyridine was slowly added 2.5 g of nicotinyl chloride hydrochloride over 10-30 minutes with stirring at 5 ° C. After further stirring for 30 minutes, the reaction mixture was evaporated to dryness. The residue was dissolved in chloroform and the solution was washed with an aqueous solution of sodium bicarbonate. The organic layer was separated, dried over anhydrous sodium sulfate and evaporated under reduced pressure to dryness. The residue was chromatographed on silica gel (Wakogel C-200, Wako Pure Chemical Industries, Ltd.,

Japan)»hvorefter der elueredes med benzen-ethanol (5:1). Eluerings-midlet inddampedes til en halvfast masse, der omkrystalliseredes med diethylether, hvorved man opnåede 1,97 g af salpetersyreesteren af N-(2-hydroxyethyl)nicotinamid.Japan) 'and then eluted with benzene-ethanol (5: 1). The eluent was evaporated to a semi-solid mass which was recrystallized from diethyl ether to give 1.97 g of the nitric ester of N- (2-hydroxyethyl) nicotinamide.

Omkrystallisation af krystallerne med diethylether-ethanol gav farveløse nålelignende krystaller, der havde et smeltepunkt på 92 til 93°C.Recrystallization of the crystals with diethyl ether ethanol gave colorless needle-like crystals having a melting point of 92 to 93 ° C.

145608 18145608 18

Eksempel 3Example 3

En opløsning sif 10 g af salpetersyreesteren af N-(2-hydroxyethyl)nicotinamidhydrochlorid i vand neutraliseredes med en vandig opløsning af natriumhydrogencarbonat. Opløsningen ekstra-heredes med chloroform, og ekstrakten tørredes over vandfrit natriumsulfat og inddampedes under reduceret tryk til tørhed. Resten om-krystalliseredes med diethylether, hvorved man opnåede 7 g af salpetersyreesteren af N-(2-hydroxyethvl)nicotinamid. Omkrystallisation med isopropanol-diethylether gave farveløse nålelignende krystaller, der havde et smeltepunkt på 93°C.A solution of 10 g of the nitric ester of N- (2-hydroxyethyl) nicotinamide hydrochloride in water was neutralized with an aqueous solution of sodium bicarbonate. The solution was extracted with chloroform and the extract was dried over anhydrous sodium sulfate and evaporated under reduced pressure to dryness. The residue was recrystallized with diethyl ether to give 7 g of the nitric ester of N- (2-hydroxyethyl) nicotinamide. Recrystallization with isopropanol-diethyl ether gave colorless needle-like crystals having a melting point of 93 ° C.

IR (cm"1): NH 3250, 0N02 1630IR (cm -1): NH 3250, NO2 1630

Eksempel 4 N-(2-hydroxyethyl)nicotinamidnitrat (1,145 g) sattes efterhånden til 3 ml rygende. salpetersyre, der var afkølet til en temperatur på -10 til -5°C under omrøring. Efter yderligere omrøring i 1 time ved 0-5°C, tilsætter man diethylether til opløsningen for at udfælde 1,15 g salpetersyreester af N (-2-hydroxy ethyl) nico tinamid-nitrat, i form af farveløse krystaller. Krystallerne opløses i en vandig natriumcarbonatopløsning, hvorefter man tilsætter ethylacetat til opløsningen. Ethylacetatlaget fraskilles, tørres over natriumsulfat og inddampes under reduceret tryk. Resten om-krystalleres med diethylether, hvorved man opnår salpetersyreesteren af N- (2-hydroxyethyl) nicotinamid. Omkrystallisation med diethylether giver farveløse krystaller, der har et smp. på 90-92°C.Example 4 N- (2-hydroxyethyl) nicotinamide nitrate (1.145 g) was gradually added to 3 ml of smoking. nitric acid which was cooled to a temperature of -10 to -5 ° C with stirring. After further stirring for 1 hour at 0-5 ° C, diethyl ether is added to the solution to precipitate 1.15 g of nitric acid ester of N (-2-hydroxy ethyl) nico-tinamide nitrate, in the form of colorless crystals. The crystals are dissolved in an aqueous sodium carbonate solution and then ethyl acetate is added to the solution. The ethyl acetate layer is separated, dried over sodium sulfate and evaporated under reduced pressure. The residue is recrystallized from diethyl ether to give the nitric ester of N- (2-hydroxyethyl) nicotinamide. Recrystallization with diethyl ether gives colorless crystals having a m.p. at 90-92 ° C.

Analyse:Analysis:

Beregnet for CgHgNgO^ C 45,50, H 4,29, N 19,89 (%)Calculated for C CHHNgO ^ C 45.50, H 4.29, N 19.89 (%)

Fundet : C 45,37, H 4,09, H 19,71 (%)Found: C 45.37, H 4.09, H 19.71 (%)