NO171725B - CHEMILUMINESCING ACRIDIN DERIVATIVES - Google Patents

CHEMILUMINESCING ACRIDIN DERIVATIVES Download PDF

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NO171725B
NO171725B NO873520A NO873520A NO171725B NO 171725 B NO171725 B NO 171725B NO 873520 A NO873520 A NO 873520A NO 873520 A NO873520 A NO 873520A NO 171725 B NO171725 B NO 171725B
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carboxylic acid
acridine
acridinium
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Peter Molz
Hans-Juergen Skrzipczyk
Henning Luebbers
Helmut Strecker
Gerd Schnorr
Tonio Kinkel
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Hoechst Ag
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D219/00—Heterocyclic compounds containing acridine or hydrogenated acridine ring systems
    • C07D219/04—Heterocyclic compounds containing acridine or hydrogenated acridine ring systems with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to carbon atoms of the ring system
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label

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Abstract

The invention relates to novel acridinium derivatives of the formula I: <IMAGE> in which R<1> is hydrogen, an alkyl, alkenyl or alkynyl radical having 1 to 10 carbon atoms, a benzyl or aryl group, R<2> and R<3> are hydrogen, an alkyl group having 1 to 4 carbon atoms, a substituted or unsubstituted amino group, a carboxyl, C1-C4-alkoxy, cyano or nitro group or halogen, R<4> is a radical in which a sulphonamide group is bonded directly to the carbonyl group via the nitrogen and A<(-)> is an anion which does not interfere with the chemiluminescence; and their use in luminescence immunoassays.

Description

Oppfinnelsens gjenstand er kjemiluminescerende acridinderivater. The object of the invention is chemiluminescent acridine derivatives.

Luminescerende forbindelser har allerede en utbredt anvendelse. De anvendes som indikatorer i bioanalyser, enzymimmu-noanalyser og luminescensimmunoanalyser (sammenlign W.P. Collins "Alternative Immunoassays", Verlag John Wiley & Sons Ltd., Chichester, 1985), de anvendes imidlertid også i nukleinsyre-hybridiseringsanalyser (sammenlign J.A. Matthews et al. "Analytical Biochemistry", 151, 205-209, 1985). Dessuten anvendes kjemiluminescerende forbindelser ved "flow injection analysis", i "post-column"-detektorer i vaeske-kromatografi, i strømningsforskning og i kunstige lyskilder. Luminescent compounds are already widely used. They are used as indicators in bioassays, enzyme immunoassays and luminescence immunoassays (compare W.P. Collins "Alternative Immunoassays", Verlag John Wiley & Sons Ltd., Chichester, 1985), however, they are also used in nucleic acid hybridization assays (compare J.A. Matthews et al. " Analytical Biochemistry", 151, 205-209, 1985). Chemiluminescent compounds are also used in "flow injection analysis", in "post-column" detectors in liquid chromatography, in flow research and in artificial light sources.

Ved kjemiluminescensimmunoanalyser har spesielt to struktur-typer av kjemiluminescerende markeringsstoffer fått større betydning. Det dreier seg herved på den ene side om luminol-henholdsvis isoluminolderivater, som er beskrevet ved H.R. Schroeder et al., "Methods in Enzymology", Academic Press Inc., New York, Vol. LVII, 1978, 424 og følgende, samt i GB-PS 2 008 247 og 2 041 920, i DE-PS 26 18 419 og 26 18 511, samt i EP-publ. 135 071. Et oversyn over den praktiske anvendelse av isoluminolforbindelser som luminescens-indikatorer finnes hos W.G. Wood, "J. Cl in. Chem. Cl in. Biochem." 22, 1984, 905-918. In chemiluminescence immunoassays, two structural types of chemiluminescent markers in particular have gained greater importance. This concerns, on the one hand, luminol or isoluminol derivatives, which are described by H.R. Schroeder et al., "Methods in Enzymology", Academic Press Inc., New York, Vol. LVII, 1978, 424 et seq., as well as in GB-PS 2 008 247 and 2 041 920, in DE-PS 26 18 419 and 26 18 511, as well as in EP publ. 135 071. A review of the practical use of isoluminol compounds as luminescence indicators can be found in W.G. Wood, "J. Cl in. Chem. Cl in. Biochem." 22, 1984, 905-918.

På den annen side har også acridiniumesterforbindelser funnet anvendelse som kjemiluminescerende markeringsstoffer. Slike acridiniumestere er kjent fra US-PS 3 352 791, GB-PS 1 316 363 og 1 461 877, samt f ra EP-publ. 82 636. Anvendelsen av acridiniumestere som markeringsstoffer i immunoanalyser er omtalt i weeks et al., "Clin. Chem." 29/8 (1983), 1474-1479. Også anvendelsen av fenantridiniumestere som markeringsstoff i luminisensimmunoanalyser er allerede kjent fra EP-publ. 170 415. On the other hand, acridinium ester compounds have also found use as chemiluminescent markers. Such acridinium esters are known from US-PS 3 352 791, GB-PS 1 316 363 and 1 461 877, as well as from EP publ. 82 636. The use of acridinium esters as markers in immunoassays is discussed in Weeks et al., "Clin. Chem." 29/8 (1983), 1474-1479. The use of phenanthridine esters as markers in luminescence immunoassays is also already known from EP-publ. 170,415.

Kjemiluminescensen av acridiniumestere kan startes ved tilsetning av alkalisk EtøC^-oppløsning. For mekanismen av kjemiluminescensen har F. McCapra, "Acc.Chem.Res." 9, 201, 1976, gitt en overbevisende forklaring. Derved er tydelig typen av den avspaltbare gruppe avgjørende så vel for lyskvant-utbyttet som også for den hydrolytiske stabilitet. The chemiluminescence of acridinium esters can be started by the addition of alkaline Et 2 C 2 solution. For the mechanism of chemiluminescence, F. McCapra, "Acc.Chem.Res." 9, 201, 1976, provided a convincing explanation. Thereby, the type of the cleavable group is clearly decisive both for the light quantum yield and also for the hydrolytic stability.

De hittil allerede kjente acridiniumestere har i forhold til luminol- og isoluminolforbindelsene den fordel en høyere lyskvant-utbytte, som også ikke påvirkes av til indikatoren bundne proteiner (sammenlign Weeks et al., "Clin. Chem." 29/8 Compared to the luminol and isoluminol compounds, the previously known acridinium esters have the advantage of a higher light quantum yield, which is also not affected by proteins bound to the indicator (compare Weeks et al., "Clin. Chem." 29/8

(1983), 1474-1479). (1983), 1474-1479).

Skjønt de fra den EP-søknad 82 636 kjente acridiniumfenyl-estere ved energisering av kjemiluminescensen ved milde oksydasjonsmidler, utmerker seg ved en høy påvisningsfølsom-het, har de for den praktiske anvendelse forstyrrende ulemper. Fremfor alt er fenylesterbindingene i vandige systemer også allerede ved romtemperatur meget labil. Dertil kommer at under de der angitte oksydasjonsbetingelsene viser acridiniumfenylesterene en lysemisjon som er først sterkt avsluttet efter ca. 10 sek. , det vil si til over 95%. Sammenlignet med dette har andre ikke-isotopiske analysefrem-gangsmåter meget kortere måletider, og muliggjør derved en høyere prøveproduksjon. Although the acridinium phenyl esters known from EP application 82 636, when energizing the chemiluminescence by mild oxidizing agents, are distinguished by a high detection sensitivity, they have disturbing disadvantages for practical use. Above all, the phenyl ester bonds in aqueous systems are also very labile even at room temperature. In addition, under the oxidation conditions specified there, the acridinium phenyl esters show a light emission which is only strongly terminated after approx. 10 sec. , i.e. to over 95%. Compared to this, other non-isotopic analysis methods have much shorter measurement times, thereby enabling a higher sample production.

Oppfinnelsens oppgave var derfor å stille til disposisjon nye acridiniumderivater som ved høy lyskvant-utbytte har en hurtigere reaksjonskinetik og dermed muliggjør kortere måletider for en luminescensimmunoanalyse. The task of the invention was therefore to make available new acridinium derivatives which, with a high light quantum yield, have faster reaction kinetics and thus enable shorter measurement times for a luminescence immunoassay.

Det ble nå funnet at disse krav oppfylles av kjemiluminescerende acridiniumderivater med formel I It was now found that these requirements are met by chemiluminescent acridinium derivatives of formula I

der R<1>er hydrogen, Ci-C4-alkyl, R<2>ogR<3>er hydrogen, R<4>er en substituert sulfonamidgruppe med formel V eller med formel VI eller en tioalkyl- eller tioarylrest med formel II where R<1>is hydrogen, C1-C4 alkyl, R<2>and R<3>are hydrogen, R<4>is a substituted sulfonamide group of formula V or of formula VI or a thioalkyl or thioaryl residue of formula II

hvorved whereby

X er en metylen-, etylen-, propylen- eller en orto-, meta-eller parafenylengruppe og R^ er en reaktiv gruppe, X is a methylene, ethylene, propylene or an ortho, meta or paraphenylene group and R^ is a reactive group,

A~ er et anion som ikke påvirker kjemiluminescensen, og R^ i formel V og VI er fenyl eller naftyl som også kan være substituert med C^^alkoksy. A~ is an anion which does not affect the chemiluminescence, and R^ in formulas V and VI is phenyl or naphthyl which may also be substituted with C 1-2 alkoxy.

Anionene som ikke påvirker kjemiluminescensen kan være et tetrafluoroborat-, perklorat-, halogenid-, alkylsulfat-, halosulfonat-, alkylsulfonat- eller arylsulfonatantion. Også et hvert annet anion kan anvendes så vidt det ikke slukker eller svekker kjemiluminescensen. The anions that do not affect the chemiluminescence can be a tetrafluoroborate, perchlorate, halide, alkyl sulfate, halosulfonate, alkylsulfonate or arylsulfonate anion. Any other anion can also be used as long as it does not extinguish or weaken the chemiluminescence.

Dessuten foretrekkes også acridiniumforbindelser hvori R<*>er en metylgruppe. Spesielt ofte anvendes acridiniumforbindelser ifølge oppfinnelsen som tilsvarer formel IV In addition, acridinium compounds in which R<*> is a methyl group are also preferred. Particularly often used are acridinium compounds according to the invention which correspond to formula IV

hvori A og X har ovennevnte betydning. wherein A and X have the above meanings.

Typiske representanter av disse klasser av acridiniumderivater ifølge oppfinnelsen tilsvarer formel VII Typical representatives of these classes of acridinium derivatives according to the invention correspond to formula VII

hvori A og X har ovennevnte betydninger. wherein A and X have the above meanings.

Ved foreliggende oppfinnelse stilles det altså til disposisjon to ny klasser av acridiniumforbindelser, i det den ene klasse utmerker seg ved en tiolestergruppering og den andre ved en sulfonamidstruktur. Acridiniumacylsulfonamid-derivatene har en høyere stabilitet, tiolesteren en hurtigere kinetik enn de tidligere kjente acridiniumfenylesterforbind-elsene. Begge forbindelsesklasser utmerker seg dessuten ved et høyere lysutbytte. The present invention thus makes available two new classes of acridinium compounds, in that one class is characterized by a thiol ester grouping and the other by a sulfonamide structure. The acridinium acylsulfonamide derivatives have a higher stability, the thiol ester a faster kinetic than the previously known acridinium phenyl ester compounds. Both connection classes are also distinguished by a higher light output.

En betydelig fordel ved acridiniumforbindelsen ifølge oppfinnelsen med tiolesterholdige avspaltbare grupper, i forhold til de i fra EP-søknad 82 636 kjente acridinium- fenylestere, ligger i den vesentlig hurtigere reaksjonskinetik av lysemisjonen. Således gir den ifølge eksempel 1 fremstilte acridiniumtioester ifølge oppfinnelsen (forbindelse 6) ved 1 sekunds måletid, et rundt faktor 10 høyere lysutbytte under de samme oksydasjonsbetingelsene. Dette høye lysutbytte ved samtidig korte måletider, muliggjør et vesentlig høyere prøveproduksjon til luminometeret. A significant advantage of the acridinium compound according to the invention with thiol ester-containing cleavable groups, compared to the acridinium phenyl esters known from EP application 82,636, lies in the substantially faster reaction kinetics of the light emission. Thus, the acridinium thioester according to the invention prepared according to example 1 (compound 6) gives, at a measurement time of 1 second, an approximately factor 10 higher light yield under the same oxidation conditions. This high light yield at the same time short measurement times enables a significantly higher sample production for the luminometer.

Således viser fig. 1 kinetiken av lysemisjonen av antistoff-konjugat fra den ifølge eksempel 1 fremstilte acridiniumtioester (forbindelse 6) og fig. 2 denne av antistoffkonjugatet av 4-(2-succinimidyl-oksykarbonyletyl)-fenyl-10-metylacridinium-9-karboksylat-metosulfat (EP-søknad 82 636, side 10). 100 jjI av traceroppløsningene energiseres ved tilsetning av 350 pl (0,05 m KC1/NaOH-buf f er, pH 13 + 0, 1% H202til kjemiluminescens og opptegnes over et tidsrom på 10 sekunder. Thus, fig. 1 the kinetics of the light emission of antibody conjugate from the acridinium thioester prepared according to example 1 (compound 6) and fig. 2 this of the antibody conjugate of 4-(2-succinimidyl-oxycarbonylethyl)-phenyl-10-methylacridinium-9-carboxylate methosulfate (EP application 82 636, page 10). 100 µl of the tracer solutions are energized by the addition of 350 µl (0.05 m KC1/NaOH buffer, pH 13 + 0.1% H 2 O 2 ) to chemiluminescence and recorded over a period of 10 seconds.

I fig. 1 oppnås den maksimale lysemisjon allerede efter 0,66 sek. og er efter 0,88 sek. allerede falt igjen til halvparten. Derimot oppnås i fig. 2 den maksimale lysemisjon først efter 1,77 sek. og er først efter 2,88 sek. igjen falt til halvparten. In fig. 1, the maximum light emission is achieved already after 0.66 sec. and is after 0.88 sec. already fell again to half. In contrast, in fig. 2 the maximum light emission only after 1.77 sec. and is only after 2.88 sec. again fell to half.

Helt uventet er at på amidnitrogenet sulfonylsubstituerte acridinium-9-karboksylsyreamider har en utmerket kjemiluminescens, for det er kjent at acridinium-9-karboksylsyreamid i motsetning til acridinium-9-karboksylsyreesteren ikke viser kjemiluminescens (se F. McCapra i W. Carruthers og J.K. Sutherland: "Progress in Organic Chem.", vol. 8, 231-277, 1973, Butterworth, London). Quite unexpectedly, sulfonyl-substituted acridinium-9-carboxylic acid amides on the amide nitrogen have an excellent chemiluminescence, because it is known that acridinium-9-carboxylic acid amide, in contrast to the acridinium-9-carboxylic acid ester, does not show chemiluminescence (see F. McCapra in W. Carruthers and J.K. Sutherland : "Progress in Organic Chem.", vol. 8, 231-277, 1973, Butterworth, London).

Acridinium-9-karboksylsyretioesterene ifølge oppfinnelsen lar seg fremstille på følgende måte: Acridin eller dets derivater med R<2>og/eller R<3>i de påkon-denserte fenylringer omsettes efter den av Lehmstedt og Hundertmark i "Ber." 63, 1229 (1930) angitte fremgangsmåte i etanol/iseddik med kaliumcyanid til 9-cyanacridin. Herav fremstilles efter omkrystallisering ved omsetning med svovelsyre og natriumnitrit tilsvarende den av Lehmstedt og Wirth i "Ber." 61, 2044, (1928), omtalte fremgangsmåte acridin-9-karboksylsyre henholdsvis R<2>/R<3->substituerte acridin-9-karboksylsyre. Ved omsetning av acridin-9-karbok-sylsyren respektiv R<2>/R<3->ubstituerte acrIdin-9-karboksylsy-ren med tionylklorid fremstilles forbindelsen med formel VIII The acridinium-9-carboxylic acid thioesters according to the invention can be prepared in the following way: Acridine or its derivatives with R<2> and/or R<3> in the condensed phenyl rings are reacted according to Lehmstedt and Hundertmark in "Ber." 63, 1229 (1930) stated method in ethanol/glacial vinegar with potassium cyanide to 9-cyanacridine. This is produced after recrystallization by reaction with sulfuric acid and sodium nitrite similar to that by Lehmstedt and Wirth in "Ber." 61, 2044, (1928), mentioned method acridine-9-carboxylic acid respectively R<2>/R<3->substituted acridine-9-carboxylic acid. By reacting the acridine-9-carboxylic acid or R<2>/R<3->substituted acridine-9-carboxylic acid with thionyl chloride, the compound of formula VIII is prepared

hvori Y betyr klor. I stedet for et halogen kan det i forbindelsen VIII for Y også innføres en oksykarbonyl-C^-Cs-alkyl-, oksokarbonylaryl- eller imidazolidgruppe. where Y means chlorine. Instead of a halogen, an oxycarbonyl-C 1 -C 8 -alkyl, oxocarbonylaryl or imidazolide group can also be introduced in the compound VIII for Y.

De R<2>/R<3->substituerte acridinderivater kan enkelt fremstilles efter litteraturkjente fremgangsmåter. Slike synteser er eksempel omtalt i: "Comprehensive Heterocyclic Chemistry"; Editors Å. Katritzky, C.W. Rees, vol. 2, s. 395ff; "Pergamon Press", 1984; eller "Heterocyclic Compounds", vol. 9, "Acridines and Cond." 2<nd>Edition, R.M. Acheson, John Wiley and Sons, 1973. The R<2>/R<3->substituted acridine derivatives can be easily prepared according to methods known in the literature. Examples of such syntheses are discussed in: "Comprehensive Heterocyclic Chemistry"; Editors Å. Katritzky, C.W. Rees, vol. 2, pp. 395ff; "Pergamon Press", 1984; or "Heterocyclic Compounds", vol. 9, "Acridines and Cond." 2<nd>Edition, R.M. Acheson, John Wiley and Sons, 1973.

Syrekloridet (VIII) omsettes derefter med en tiolkarboksyl-syre med formel IX, The acid chloride (VIII) is then reacted with a thiolcarboxylic acid of formula IX,

for eksempel med 2-merkaptobenzosyre under alkaliske betingelser til tiolesterkarboksylsyre, som derefter forestres med en for fremstilling av resten R^ egnet for example with 2-mercaptobenzoic acid under alkaline conditions to thiol ester carboxylic acid, which is then esterified with a suitable for the preparation of the residue R

forbindelse, for eksempel med N-hydroksysuccinimid. Derefter alkyleres acridinforbindelsene efter litteraturkjente fremgangsmåter i 10-stilling. For en metylering egner seg fremfor alt trimetyloksoniumtetrafluoroborat, Imidlertid får man også med dimetylsulfonat, metylfluorsulfonat, toluensul-fonsyremetylester eller trifluorometansulfonsyrmetylester gode utbytter av den kjemiluminescerende acridiniumfor-bindelse. compound, for example with N-hydroxysuccinimide. The acridine compounds are then alkylated in the 10-position according to methods known in the literature. Trimethyloxonium tetrafluoroborate is above all suitable for methylation. However, good yields of the chemiluminescent acridinium compound are also obtained with dimethylsulfonate, methylfluorosulfonate, toluenesulfonic acid methyl ester or trifluoromethanesulfonic acid methyl ester.

Til fremstilling av acridiniumsulfonamid-derivatene ifølge oppfinnelsen går man likeledes ut fra acridin-9-karboksyl-syreklorid (VIII). Denne forbindelse omsettes derefter med et primært eller sekundært sulfonamid, fortrinnsvis med en beskyttet sulfonamidkarboksylsyre med formel X For the production of the acridinium sulfonamide derivatives according to the invention, one also starts from acridine-9-carboxylic acid chloride (VIII). This compound is then reacted with a primary or secondary sulfonamide, preferably with a protected sulfonamide carboxylic acid of formula X

eller formel XI or formula XI

hvori X og R<*>>har ovennevnte betydning og at Z betyr en rest som beskytter karboksygruppen og som derefter avspaltes. For eksempel kan det for denne reaksjon som beskyttelsesgruppe anvendes en benzensulfonylglycinbenzylester. Den efter avspaltning av beskyttelsesgruppen dannede syre, overføres derefter med en egnet, eksempelvis med N-hydroksysuccinimid til resten R^. Herav fremstilles de kjemiluminescerende acridiniumforbindelser ifølge litteraturkjente fremgangsmåter ved alkylering ved nitrogenet I 10-stilling. in which X and R<*>> have the above meaning and that Z means a residue which protects the carboxy group and which is then cleaved off. For example, a benzenesulfonyl glycine benzyl ester can be used as a protecting group for this reaction. The acid formed after cleavage of the protective group is then transferred with a suitable, for example with N-hydroxysuccinimide to the residue R . From this, the chemiluminescent acridinium compounds are prepared according to methods known in the literature by alkylation at the nitrogen in the 10 position.

De dannede acridiniumforbindelser lar seg derefter omsette med et stoff av biologisk interesse, for eksempel et antigen, et antistoff, et hormon, et legemiddel, en legemiddel-metabolitt, et toksin, eller et alkaloid til en lumin escerende forbindelse. Derved bindes acridiniumderivatet enten direkte eller over et bromolekyl, som polylysin, polyglutaminsyre eller polyvinylamid, til det biologiske interessante stoff under dannelse av et stabilt immunologisk aktivt konjugat. Dette konjugat betegnes også som tracer og anvendes i den nedenfor omtalte luminescensimmunoanalyse. The formed acridinium compounds can then be reacted with a substance of biological interest, for example an antigen, an antibody, a hormone, a drug, a drug metabolite, a toxin, or an alkaloid to a luminescent compound. Thereby, the acridinium derivative is bound either directly or via a bridge molecule, such as polylysine, polyglutamic acid or polyvinylamide, to the biologically interesting substance, forming a stable immunologically active conjugate. This conjugate is also referred to as a tracer and is used in the luminescence immunoassay described below.

Fremstillingen av acridiniumforbindelsen ifølge oppfinnelsen vises ved eksemplene 1 til 6. The production of the acridinium compound according to the invention is shown in examples 1 to 6.

EKSEMPEL 1 EXAMPLE 1

9- cvanacridin ( 1) 9- cvanacridine ( 1)

Til acridin (10 g) i 45 ml etanol setter man 3,3 ml iseddik og dråpevis en oppløsning av 5,25 g kaliumcyanid i 8 ml vann, oppvarmer reaksjonsblandingen i 2 timer under tilbakeløp, avkjøler og fjerner de flyktige bestanddeler i vakuum. Resten røres ut med 30 ml 2 N NaOH, suges fra og, efter to gangers vasking med 2 N NaOH og vann, hensettes noen tid fuktig i luften. Råproduktet røres ut i metylenklorid, uoppløst stoff suges fra og vaskes med metylenklorid, de forenede organiske faser sammenføres og det urene 9-cyanacridin omkrystalliseres fra n-butylacetat. To acridine (10 g) in 45 ml of ethanol, add 3.3 ml of glacial acetic acid and dropwise a solution of 5.25 g of potassium cyanide in 8 ml of water, heat the reaction mixture for 2 hours under reflux, cool and remove the volatile components in vacuo. The residue is stirred out with 30 ml of 2 N NaOH, sucked off and, after washing twice with 2 N NaOH and water, left for some time moist in the air. The crude product is stirred in methylene chloride, undissolved matter is sucked off and washed with methylene chloride, the combined organic phases are combined and the impure 9-cyanacridine is recrystallized from n-butyl acetate.

Utbytte: 50% Smeltepunkt: 183-5°C. Yield: 50% Melting point: 183-5°C.

IR: 2 230cm_<1>. IR: 2230cm_<1>.

Acridin- 9- karboksylsvre ( 2 ) Acridine-9-carboxylic acid ( 2 )

9-cyanacridin (5 g) has langsomt porsjonsvis til 40 ml konsentrert H2SO4og blandingen oppvarmes i 2 timer ved 90-95°C, efter tilsetning av 8,5 g NaN02»og omrøres ytterligere i 2 timer ved denne temperatur. Den varme oppløsning helles under hurtig omrøring i 620 ml isvann, utfellingen suges fra og oppløses i minst mulig 2 N NaOH. Oppløsningen filtreres og surgjøres med 50#-ig H2SO4, den utfelte acridin-9-karbok-sylsyre suges fra og tørkes under vakuum. 9-cyanacridine (5 g) is added slowly in portions to 40 ml of concentrated H2SO4 and the mixture is heated for 2 hours at 90-95°C, after the addition of 8.5 g of NaN02" and stirred for a further 2 hours at this temperature. The hot solution is poured with rapid stirring into 620 ml of ice water, the precipitate is sucked off and dissolved in as little as possible 2 N NaOH. The solution is filtered and acidified with 50% H2SO4, the precipitated acridine-9-carboxylic acid is sucked off and dried under vacuum.

Ubytte: 95% Smeltepunkt: 288-9°C. Yield: 95% Melting point: 288-9°C.

IR: 3440(br), 3200(br), 2600-2500(br), 1980; 1650; 1605; 1420"<1>. IR: 3440(br), 3200(br), 2600-2500(br), 1980; 1650; 1605; 1420"<1>.

Acridln- 9- karboksvlsvreklorid- hvdroklorld ( 3) Acridln-9-karboksylsyre (5 g) has porsjonsvis til 50 ml nydestlllert SOCI2og oppvarmes 1 5 timer under tilbakeløp, den derefter klare oppløsning konsentreres ved destillering inntil begynnende utfelling, utfellingen fullstendiggjøres ved cykloheksantilsetning og avkjøling. Frasugning av utfellingen og tørking under vakuum gir acridin-9-karboksyl-syrekloridhydroklorid. Acridln-9-carboxylic acid hydrochloride (3) Acridln-9-carboxylic acid (5 g) is added in portions to 50 ml of freshly distilled SOCl2 and heated for 15 hours under reflux, the then clear solution is concentrated by distillation until precipitation begins, the precipitation is completed by addition of cyclohexane and cooling . Suctioning off the precipitate and drying under vacuum gives acridine-9-carboxylic acid chloride hydrochloride.

Utbytte: 90% Smeltepunkt: 233°C Yield: 90% Melting point: 233°C

Elementanalyse (beregnet som C14H9CINO x HC1) Elemental analysis (calculated as C14H9CINO x HC1)

( Fenvl- 2'- karboksvlsvre) acridin- 9- tiokarboksvlat ( 4 ) Acridin-9-karboksylsyreklorid-hydroklorid (30 g) suspenderes i 720 ml metylenklorid, tilsettes tiosalicylsyre (17,7 g) og 50 ml trietylamin, oppløsningen som er blitt klar omrører man i 10 minutter ved værelsestemperatur. Efter fjerning av oppløsningsmidlet blandes resten med 35 g soda og 1 400 ml vann, den resulterende oppløsning dampes inn inntil opptreden av en utfelling, denne suges fra. Filtratet mettes med NaCl og den derved dannede utfelling suges likeledes fra. Den vandige oppløsning av de forenede utfellinger surgjøres ved 80°C med iseddik og det dannede produkt suges fra og tørkes under vakuum. ( Phenyl-2'-carboxylic acid) acridine-9-thiocarboxylic acid ( 4 ) Acridine-9-carboxylic acid chloride hydrochloride (30 g) is suspended in 720 ml of methylene chloride, thiosalicylic acid (17.7 g) and 50 ml of triethylamine are added, the solution which has been ready, stir for 10 minutes at room temperature. After removal of the solvent, the residue is mixed with 35 g of soda and 1,400 ml of water, the resulting solution is evaporated until a precipitate appears, which is suctioned off. The filtrate is saturated with NaCl and the resulting precipitate is likewise sucked off. The aqueous solution of the combined precipitates is acidified at 80°C with glacial acetic acid and the product formed is sucked off and dried under vacuum.

Utbytte: 80% Smeltepunkt: 261-5°C Yield: 80% Melting point: 261-5°C

NMR (DMS0, 100 MHz): S = 7,6-8,4 ppm, kompleks multiplett NMR (DMS0, 100 MHz): S = 7.6-8.4 ppm, complex multiplet

IR: 1680 cm-<1>(s), 1720 (m) 1260 (s). IR: 1680 cm-<1>(s), 1720 (m) 1260 (s).

2 ' - ( succinimidoyl oksykarbonyl ) fenylacridin- 9- tlokarboksylat 151 2'-(succinimidoyl oxycarbonyl)phenylacridine-9-tlocarboxylate 151

Til en suspensjon av 10 g av tiolesterkarboksylsyren i 190 ml tørr tetrahydrofuran setter man ved 0°C 3,2 g N-hydroksysuccinimid, derefter ved -20"C 6,9 g dicykloheksyl-karbodiimid (DCC) og lar det efteromrøre ved -20"C i 2 timer, og derefter over natt ved romtemperatur. Efter tilsetning av 0,28 ml iseddik omrøres det i 1 time, derefter tilsettes eddikester (25 ml) og utfellingen filtreres fra. Filtratet dampes inn og gir efter omkrystallisering fra klorbenzen et blekgult 2'(succinimidoyloksykarbonyl)fenylacridin-9-tiokarboksylat. To a suspension of 10 g of the thiol ester carboxylic acid in 190 ml of dry tetrahydrofuran, 3.2 g of N-hydroxysuccinimide are added at 0°C, then at -20°C 6.9 g of dicyclohexylcarbodiimide (DCC) and allowed to stir at -20 "C for 2 hours, and then overnight at room temperature. After adding 0.28 ml of glacial acetic acid, it is stirred for 1 hour, then acetic acid (25 ml) is added and the precipitate is filtered off. The filtrate is evaporated and after recrystallization from chlorobenzene gives a pale yellow 2'(succinimidoyloxycarbonyl)phenylacridine-9-thiocarboxylate.

Utbytte: 80% Smeltepunkt: 198-200°C. Yield: 80% Melting point: 198-200°C.

IR: 1810 cm-<1>, 1780, 1745, 1225, 1205 IR: 1810 cm-<1>, 1780, 1745, 1225, 1205

NMR (DMSO), 100 MHz): S = 2,95 ppm (s, 4H), 7,7-8,4 pm (m, 12H). 2 ' ( succinimidoyloksykarbonyl) fenyl- 10- metylacrldinlum- 9-tlokarboksylat- tetrafluoroborat ( 6) 3 g N-hydroksysuccinimidester (5) oppvarmes med 7,8 g trimetyloksoniumtetrafluoroborat i 40 ml 1,2-dikloretan I 8 timer ved 80° C og efteromrøres over natten ved romtemperatur. Utfellingen filtreres fra og kokes ut med 1,2-dikloretan. De forenede organiske faser dampes inn og omkrystalliseres fra aceton:diisopropyleter. NMR (DMSO), 100 MHz): S = 2.95 ppm (s, 4H), 7.7-8.4 pm (m, 12H). 2' (succinimidoyloxycarbonyl)phenyl-10-methylacrdinlum-9-tlocarboxylate-tetrafluoroborate (6) 3 g of N-hydroxysuccinimide ester (5) are heated with 7.8 g of trimethyloxonium tetrafluoroborate in 40 ml of 1,2-dichloroethane for 8 hours at 80° C and stirred overnight at room temperature. The precipitate is filtered off and boiled off with 1,2-dichloroethane. The combined organic phases are evaporated and recrystallized from acetone:diisopropyl ether.

Utbytte: 4 0% Smeltepunkt: 245°C. Yield: 40% Melting point: 245°C.

IR: 3440 cm-<1>(br), 1800, 1780, 1740 (s), 1670, 1065 (s), NMR (DMSO, 100 MHz); S = 3,0 ppm (s, 4H), 4,95 ppm (s, svakt utbredt, 3H), 7,9-8,6 (m, 10H), 9,9 ppm (d, 2H). IR: 3440 cm-<1>(br), 1800, 1780, 1740 (s), 1670, 1065 (s), NMR (DMSO, 100 MHz); S = 3.0 ppm (s, 4H), 4.95 ppm (s, weakly prevalent, 3H), 7.9-8.6 (m, 10H), 9.9 ppm (d, 2H).

EKSEMPEL 2 EXAMPLE 2

Fremstillingen av succinimidoyloksykarbonylmetyl-10-metylacridinium-9-tiokarboksylat-tetrafluoroborat, fra syreklorid (3) og tioglykolsyre foregår analogt fremstillingen av (6). Utbyttene av de enkelte syntesetrinn, samt den spektro skopiske karakterisering av produkt (7) til (9) er angitt nedenfor: The preparation of succinimidoyloxycarbonylmethyl-10-methylacridinium-9-thiocarboxylate-tetrafluoroborate, from acid chloride (3) and thioglycolic acid proceeds analogously to the preparation of (6). The yields of the individual synthesis steps, as well as the spectroscopic characterization of products (7) to (9) are stated below:

Karboksvmetvlacridin- 9- tiokarboksylat ( 7) Carboxvmetvlacridine-9-thiocarboxylate (7)

Utbytte: 6056 Smeltepunkt: 218° C (under spaltning) IR: 3440 cm-<1>(br), 2400(br), 1950(br), 1710(m), 1660(s), 1070(m) Yield: 6056 Melting point: 218° C (under decomposition) IR: 3440 cm-<1>(br), 2400(br), 1950(br), 1710(m), 1660(s), 1070(m)

NMR (DMSO, 100 MHz): S = 4,25 ppm (s, 2H); 7,6-8,4 (m, 8H). NMR (DMSO, 100 MHz): S = 4.25 ppm (s, 2H); 7.6-8.4 (m, 8H).

Succinimldo. vloksykarbon. ylmetylacridin- 9- tlokarboksylat ( 8) Utbytte: 80% Succinimldo. oxycarbon. ylmethylacridin-9-tlocarboxylate (8) Yield: 80%

IR: 3440 cm-<1>(br), 2930, 1820, 1785, 1740(s), 1205, 1165 NMR (DMSO, 100 MHz); S = 2,95 ppm (s, 4H), 4,77 ppm (s, 2H), 7,6-8,3 ppm (m, 8H). IR: 3440 cm-<1>(br), 2930, 1820, 1785, 1740(s), 1205, 1165 NMR (DMSO, 100 MHz); S = 2.95 ppm (s, 4H), 4.77 ppm (s, 2H), 7.6-8.3 ppm (m, 8H).

Succinimldoyloksykarbonylmetyl- 10- metylacridinium- 9- tiokarboksylat- tetrafluoroborat ( 9) Succinimldoyloxycarbonylmethyl- 10- methylacridinium- 9- thiocarboxylate- tetrafluoroborate ( 9)

Utbytte: 40% Smeltepunkt: 250°C Yield: 40% Melting point: 250°C

IR: 3440 cm-<1>(br), 1810, 1780, 1735(s), 1538, 1350, 1060 NMR (DMSO, 100 MHz): S =2,9 ppm (s, 4H), 4,8 (s, 2H), 4,9 ppm, (s, 3H), 7,7-9,0 (m, 8H). IR: 3440 cm-<1>(br), 1810, 1780, 1735(s), 1538, 1350, 1060 NMR (DMSO, 100 MHz): S =2.9 ppm (s, 4H), 4.8 ( s, 2H), 4.9 ppm, (s, 3H), 7.7-9.0 (m, 8H).

EKSEMPEL 3 EXAMPLE 3

N- benzensulfonyl- N-( benzyloksykarbonylmetyl ) acrldln- 9-karboksylsyreamid ( 10) N-benzenesulfonyl-N-(benzyloxycarbonylmethyl)acrldln-9-carboxylic acid amide (10)

3,3 g N-benzensulfonylglycinbenzylester blandes i 110 ml tetrahydrofuran med 130 mg 4(dimetylamino)-pyridin og 6 ml trietylamid, efter 10 minutter tilsetter man 3 g acridin-9-karboksylsyreklorid-hydroklorid og oppvarmer den dannede suspensjon i 6 timer under tilbakeløp. Utfellingen suges fra, oppløsningsmidlet fjernes, resten tas opp i metylenklorid og røres kort ut med 2 N NaOH. Den organiske fase dampes inn efter tørking over MgS04og den resulterende rest omkrystalliseres fra toluen:heptan. 3.3 g of N-benzenesulfonylglycine benzyl ester are mixed in 110 ml of tetrahydrofuran with 130 mg of 4(dimethylamino)-pyridine and 6 ml of triethylamide, after 10 minutes 3 g of acridine-9-carboxylic acid chloride hydrochloride are added and the resulting suspension is heated for 6 hours under reflux . The precipitate is suctioned off, the solvent is removed, the residue is taken up in methylene chloride and stirred briefly with 2 N NaOH. The organic phase is evaporated after drying over MgSO 4 and the resulting residue is recrystallized from toluene:heptane.

Utbytte: 70% Smeltepunkt: 58°C Yield: 70% Melting point: 58°C

IR: 3440 cm-<1>(br), 1735, 1680, 1357, 1165 IR: 3440 cm-<1>(br), 1735, 1680, 1357, 1165

NMR (DMSO, 100 MHz): S = 5,2 ppm (s, 2H), 5,3 ppm (s, 2H), 7,0-8,4 ppm (m, 18H). NMR (DMSO, 100 MHz): S = 5.2 ppm (s, 2H), 5.3 ppm (s, 2H), 7.0-8.4 ppm (m, 18H).

N- benzensulfonvl- N-( karboksvmetvl) acr idin- 9- karboksvlsvreamid Lill 1 g N-benzensulfonyl-N(benzyloksykarbonylmetyl)acridin-9-karboksylsyreamld 1 60 ml iseddlk hydrogeneres under tilsetning av 2 ml konsentrert HC1 og Pd/C (1056) ved romtemperatur og normaltrykk; efter reaksjonens avslutning frasuges katalysatoren, fra filtratet får man med inndampning karbok-sylsyren som gult faststoff. N-benzenesulfonyl-N-(carboxymethyl)acridine-9-carboxylamide Little 1 g of N-benzenesulfonyl-N(benzyloxycarbonylmethyl)acridine-9-carboxylic acid amld 1 60 ml of ice-cold water are hydrogenated with the addition of 2 ml of concentrated HCl and Pd/C (1056) at room temperature and normal pressure; after the end of the reaction, the catalyst is sucked off, from the filtrate the carboxylic acid is obtained by evaporation as a yellow solid.

NMR (DMSO, 100 MHz): S = 5,0 ppm (s, 2H), 7,1-8,5 ppm (m, 13H). NMR (DMSO, 100 MHz): S = 5.0 ppm (s, 2H), 7.1-8.5 ppm (m, 13H).

N- benzensulfonvl- N-( succinimidovioksvkarbonylmetvi) acridin- 9-karboksvlsvreamid (12) N-benzenesulfonyl-N-(succinimidovioxcarbonylmethyl)acridine-9-carboxylsulfreamide (12)

501 mg av forbindelse 11 i 25 ml tørr tetrahydrofuran er behandlet med 277 pl trietylamin, avkjøles til -15°C og behandles med 96 pl klormaursyreetylester. Man omrører i 1 time ved denne temperatur, tilsetter så 115 mg N-hydroksysuccinimid, lar det hele langsomt oppvarmes til romtemperatur og omrører reaksjonsblandingen ved denne temperatur over natten. Bunnfallet suges av, filtratet dampes inn under vakuum og resten tas opp i eddiksyreetylester. Den resulterende oppløsning ekstraheres med vann, NaHC03-oppløsning og vann, tørkes med natriumsulfat og oppløsningsmidlet destil-leres av under vakuum. Efter behandling av den oljeaktige rest med diisopropyleter oppnår man et svakt gult pulver. 501 mg of compound 11 in 25 ml of dry tetrahydrofuran is treated with 277 µl of triethylamine, cooled to -15°C and treated with 96 µl of ethyl chloroformate. The mixture is stirred for 1 hour at this temperature, then 115 mg of N-hydroxysuccinimide is added, the whole is allowed to slowly warm to room temperature and the reaction mixture is stirred at this temperature overnight. The precipitate is sucked off, the filtrate is evaporated under vacuum and the residue is taken up in ethyl acetate. The resulting solution is extracted with water, NaHCO 3 solution and water, dried with sodium sulfate and the solvent is distilled off under vacuum. After treatment of the oily residue with diisopropyl ether, a faint yellow powder is obtained.

Utbytte: 40% ; Smeltepunkt: 196°C (dekomponering). Dividend: 40% ; Melting point: 196°C (decomposition).

NMR (DMSO, 100 MHz): S (ppm) = 2,94 (s, 4H); 5,65 (s, 2H); 7,0-8,4 (m, 13H). NMR (DMSO, 100 MHz): S (ppm) = 2.94 (s, 4H); 5.65 (s, 2H); 7.0-8.4 (m, 13H).

N- benzensulfon. vl- N-( succinimidovioksykarbonylmetyl )- 10-metvlacridinium- 9- karboksylsvre- amldfluorsulfonat (13 ) N-benzene sulfone. vl- N-( succinimidovioxycarbonylmethyl )- 10-metvlacridinium- 9- carboxylic acid- amldfluorosulfonate (13 )

497 mg av forbindelse 12 omrøres i 25 ml 1,2-dikloretan med 503 pl metylfluorsulfonat i 18 timer ved romtemperatur hvorefter det dannede gule bunnfall suges av og tørkes under vakuum. 497 mg of compound 12 is stirred in 25 ml of 1,2-dichloroethane with 503 µl of methyl fluorosulfonate for 18 hours at room temperature, after which the yellow precipitate formed is sucked off and dried under vacuum.

Utbytte: 45%. Yield: 45%.

NMR (DMSO, 100 MHz): S (ppm) = 2,90 (s, br, 2H); 4,95 (s, br, 3H); 5,75 (s, br, 2H); 6,73-9,1 (m, 13H). NMR (DMSO, 100 MHz): S (ppm) = 2.90 (s, br, 2H); 4.95 (s, br, 3H); 5.75 (s, br, 2H); 6.73-9.1 (m, 13H).

EKSEMPEL 4 EXAMPLE 4

N- fenyl- N( 4- benzyloksykarbonylbenzensulfonyl ) acridin- 9-karboksylsyreamid ( 14) 11 g 4(N-fenylsulfamido)benzosyrebenzylester blandes i 300 ml metylenklorid med 360 mg 4(dimetylamino)pyridin og 16,6 ml trietylamin, efter 10 minutter tilsetter man 8,34 g acridin-9-karboksylsyreklorid-hydroklorid (3) og oppvarmer i 16 timer under tilbakeløp. Den avkjølte oppløsning utrøres kort med 2 N NaøH, den adskilte organiske fase vaskes med vann, tørkes over Na2S04og inndampes. Resten omkrystalliseres fra toluen:heptan. N-phenyl-N(4-benzyloxycarbonylbenzenesulfonyl)acridine-9-carboxylic acid amide (14) 11 g of 4(N-phenylsulfamido)benzoic acid benzyl ester are mixed in 300 ml of methylene chloride with 360 mg of 4(dimethylamino)pyridine and 16.6 ml of triethylamine, after 10 minutes add 8.34 g of acridine-9-carboxylic acid chloride hydrochloride (3) and heat for 16 hours under reflux. The cooled solution is stirred briefly with 2 N NaOH, the separated organic phase is washed with water, dried over Na2SO4 and evaporated. The residue is recrystallized from toluene:heptane.

Utbytte: 70% Smeltepunkt: 161-163°C Yield: 70% Melting point: 161-163°C

NMR (DMSO, 100 MHz): S = 5,5 ppm (s, 2H), S = 6,8-8,6 ppm (m, 22H). NMR (DMSO, 100 MHz): S = 5.5 ppm (s, 2H), S = 6.8-8.6 ppm (m, 22H).

jj- f enyl - N-( 4- karboksybenzensulfonyl ) acridin- 9- karboksylsyreamid- hydrobromid ( 15) jj-phenyl-N-(4-carboxybenzenesulfonyl)acridine-9-carboxylic acid amide hydrobromide (15)

8,58 g N-fenyl-N-(4-benzyloksykarbonylbenzensulfonyl )acridin-9-karboksylsyreamid (14) oppvarmes i 30 ml 33% HBr i iseddik i 2 timer ved 60°C, efter avkjøling tilsetter man 60 ml diisopropyleter, suger fra utfellingen og tørker i vakuum. 8.58 g of N-phenyl-N-(4-benzyloxycarbonylbenzenesulfonyl)acridine-9-carboxylic acid amide (14) is heated in 30 ml of 33% HBr in glacial acetic acid for 2 hours at 60°C, after cooling one adds 60 ml of diisopropyl ether, suction from the precipitate and dried in vacuum.

Utbytte: 9556 Smeltepunkt 255° C Yield: 9556 Melting point 255° C

NMR (DMSO, 100 MHz): S = 6,8-9 ppm (m) NMR (DMSO, 100 MHz): S = 6.8-9 ppm (m)

N- fenyl- N-( 4 - suks in Imi dovi ok syk arb on yl benzen sul f on vi )-acrldin- 9- karboksvlsvreamid ( 16) N- phenyl- N-( 4 - succin imido oxy carbonyl benzene sulfon vi )-acrldin- 9- carboxyl amide ( 16)

5,63 g N-fenyl-N-(4-karboksybenzensulfonyl)acridin-9-karboksylsyreamid-hydrobromid (15) i 250 ml tetrahydrofuran blandes med 2,8 ml trietylamin og avkjøles til -15°C og blandes så med 0,96 ml klormaursyreetylester. Man efteromrører i 20 minutter, tilsetter 1,15 g N-hydroksysuksinimid, omrører i 3 timer ved -15°C, lar det tine til romtemperatur og efter-omrører natten over. Utfellingen suges fra, filtratet dampes inn, resten opptas med metylenklorid, den resulterende oppløsning vaskes med vann, NaHC03-oppløsning og vann og tørkes over Na2S04. Den organiske fase dampes inn og resten omkrystalliseres fra toluen. 5.63 g of N-phenyl-N-(4-carboxybenzenesulfonyl)acridine-9-carboxylic acid amide hydrobromide (15) in 250 ml of tetrahydrofuran is mixed with 2.8 ml of triethylamine and cooled to -15°C and then mixed with 0.96 ml chloroformate ethyl ester. The mixture is stirred for 20 minutes, 1.15 g of N-hydroxysuccinimide is added, stirred for 3 hours at -15°C, allowed to thaw to room temperature and stirred overnight. The precipitate is suctioned off, the filtrate is evaporated, the residue is taken up with methylene chloride, the resulting solution is washed with water, NaHCO 3 solution and water and dried over Na 2 SO 4 . The organic phase is evaporated and the residue is recrystallized from toluene.

Utbytte: 5056 Smeltepunkt: 226°C (under spaltning). NMR (DMSO, 100 MHz): S = 2,95 ppm (s, 4H), S = 6,8-8,7 ppm (m, 17H). Yield: 5056 Melting point: 226°C (under decomposition). NMR (DMSO, 100 MHz): S = 2.95 ppm (s, 4H), S = 6.8-8.7 ppm (m, 17H).

N- fenyl- N-( 4- succinimidoyloksykarbonylbenzensulfonyl )- 10-metvlacridinium- 9- karboks. vlsyreamid- f luorosulfonat ( 17 ) N-phenyl-N-(4-succinimidoyloxycarbonylbenzenesulfonyl)-10-metvlacridinium-9-carbox. hydrochloric acid amide fluorosulfonate ( 17 )

1,16 g N-fenyl-N-(4-succinimidoyloksykarbonylbenzensulfonyl)-acridin-9-karboksylsyreamid (16) omrøres i 60 ml 1,2-dikloretan med 0,3 ml metylf luorosulfonat i 24 timer ved romtemperatur, den dannede utfelling suges fra og tørkes i vakuum. 1.16 g of N-phenyl-N-(4-succinimidoyloxycarbonylbenzenesulfonyl)-acridine-9-carboxylic acid amide (16) is stirred in 60 ml of 1,2-dichloroethane with 0.3 ml of methyl fluorosulfonate for 24 hours at room temperature, the precipitate formed is suction from and dried in vacuum.

Utbytte: 6556 Dividend: 6556

IR: 3420 cm"<1>(br), 3100(br), 1805(w), 1770(m), 1745(s), 1700(m), 1385(m), 1280(m), 1255(s), 1230(s), 1205(s) IR: 3420 cm"<1>(br), 3100(br), 1805(w), 1770(m), 1745(s), 1700(m), 1385(m), 1280(m), 1255(s ), 1230(s), 1205(s)

NMR (DMSO, 100 MHz): S = 2,95 ppm (s, 4H), S = 4,75 ppm (s, br, 3H), S = 7,0-9,0 ppm (m, 17H) NMR (DMSO, 100 MHz): S = 2.95 ppm (s, 4H), S = 4.75 ppm (s, br, 3H), S = 7.0-9.0 ppm (m, 17H)

Massespektrum: m/z = 594 : M<+>(kation). Mass spectrum: m/z = 594 : M<+>(cation).

EKSEMPEL 5 EXAMPLE 5

Fremstillingen av N-(4-metoksyfenyl)-N-(4-succinimidoyloksy-karbonylbenzensul f onyl) -10-metylacridinium-9-karboksylsyreamid-fluorosulfonat (21), fra 4-[N-(4'-metoksyfenyl)sulf-amido]-benzosyrebenzylester og acridin-9-karboksylsyreklorid-hydroklorid (3), foregår analogt fremstillingen av (17) (se eksempel 4). Utbyttene av de enkelte syntesetrinn samt den spektroskop!ske karakterisering er angitt i det følgende. The preparation of N-(4-methoxyphenyl)-N-(4-succinimidoyloxy-carbonylbenzenesulfonyl)-10-methylacridinium-9-carboxylic acid amide fluorosulfonate (21), from 4-[N-(4'-methoxyphenyl)sulfamido ]-benzoic acid benzyl ester and acridine-9-carboxylic acid chloride-hydrochloride (3), the preparation of (17) takes place analogously (see example 4). The yields of the individual synthesis steps as well as the spectroscopic characterization are stated below.

N - ( 4- metoksyf enyl ) - N- ( 4 - benzvloksvkarbonvl- benzensulf onyl )-acridin- 9- karboksylsyreamid ( 18) N - ( 4-Methoxyphenyl ) - N - ( 4 - benzenesulfonyl ) - acridine - 9 - carboxylic acid amide ( 18 )

Utbytte: 70% Smeltepunkt: 182-183°C Yield: 70% Melting point: 182-183°C

NMR (DMSO, 100 MHz): S = 3,5 ppm (s, 3H), S = 5,5 ppm (s, 2H), S = 6,35-6,63 ppm (d, br, 2H), S = 7,05-7,2 ppm (d, br, 2H), S - 7,35-8,5 ppm (m, 17H). NMR (DMSO, 100 MHz): S = 3.5 ppm (s, 3H), S = 5.5 ppm (s, 2H), S = 6.35-6.63 ppm (d, br, 2H), S = 7.05-7.2 ppm (d, br, 2H), S - 7.35-8.5 ppm (m, 17H).

N-( 4- metoksyfenyl)- N-( 4- karboksybenzensulfonyl ) acridln- 9-karboksylsyreamid- hydrobromid ( 19) N-(4-Methoxyphenyl)-N-(4-carboxybenzenesulfonyl) acridln-9-carboxylic acid amide hydrobromide (19)

Utbytte: 95% Smeltepunkt 273°C (under spaltning) NMR (DMSO, 100 MHz): S = 3,5 ppm (s, 3H), S = 6,4-6,6 ppm (d, br, 2H), S = 7,05-7,2 ppm (d, br, 2H), S = 7,7-8,5 ppm (m, 12H). Yield: 95% Melting point 273°C (under decomposition) NMR (DMSO, 100 MHz): S = 3.5 ppm (s, 3H), S = 6.4-6.6 ppm (d, br, 2H), S = 7.05-7.2 ppm (d, br, 2H), S = 7.7-8.5 ppm (m, 12H).

N - ( 4- metoksyf enyl ) - N- ( 4 - succlnimidoyloksykarbonylbenzensul-fonyl) acridln- 9- karboksylsyreamid ( 20) N - ( 4- methoxy enyl ) - N - ( 4 - succinimidoyloxycarbonylbenzenesulfonyl) acridln-9- carboxylic acid amide ( 20 )

Utbytte: 50* Smeltepunkt: 232-234°C Yield: 50* Melting point: 232-234°C

NMR (DMSO, 100 MHz): S = 2,95 ppm (s, 4H), S = 3,5 ppm (s, 3H), S 6,4-6,6 ppm (d, br, 2H), S = 7,05-7,25 ppm (d, br, 2H), S 7,8-8,6 ppm (m, 12H). NMR (DMSO, 100 MHz): S = 2.95 ppm (s, 4H), S = 3.5 ppm (s, 3H), S 6.4-6.6 ppm (d, br, 2H), S = 7.05-7.25 ppm (d, br, 2H), S 7.8-8.6 ppm (m, 12H).

IR: 3050 cm-<1>, 1805(w), 1780(m), 1740(s), 1700(m), 1505(m), 1370(m), 1250(m), 1200(s), 1185. IR: 3050 cm-<1>, 1805(w), 1780(m), 1740(s), 1700(m), 1505(m), 1370(m), 1250(m), 1200(s), 1185 .

N- ( 4- metoksyf enyl ) - N- ( 4 - succlnimldoyloksykarbonylbenzensul-fonyl ) - 10- metylacridinlum- 9- karboksylsyreamid- fluorosulfonat 1211 N-( 4- methoxy enyl ) - N-( 4 - succinylmoyloxycarbonylbenzenesulfonyl ) - 10- methylacridinum-9- carboxylic acid amide fluorosulfonate 1211

Stoffet faller ikke ut ved reaksjonen, det utvinnes ved inndampning av oppløsning og utrøring av resten med diisopropyleter. The substance does not precipitate during the reaction, it is recovered by evaporating the solution and stirring the residue with diisopropyl ether.

Utbytte: 80* Yield: 80*

NMR (DMSO, 100 MHz): S = 2,95 ppm (s, 4H), S - 3,5 ppm (s, 3H), S = 4,8 ppm (s, br, 3H), S = 6,45-6,7 ppm (d, br, 2H), S = 7,2-7,4 ppm (d, br, 2H); S = 7,7-9 ppm (m, 12H). NMR (DMSO, 100 MHz): S = 2.95 ppm (s, 4H), S - 3.5 ppm (s, 3H), S = 4.8 ppm (s, br, 3H), S = 6, 45-6.7 ppm (d, br, 2H), S = 7.2-7.4 ppm (d, br, 2H); S = 7.7-9 ppm (m, 12H).

Massespektrum: m/z = 624 M<+>(kation) Mass spectrum: m/z = 624 M<+>(cation)

IR: 3440 cm-<1>(br), 3100, 2950, 1805(w), 1775(m), 1740(s), 1695(m), 1610(m), 1505(m), 1375(m), 1280(m), 1250(s), 1205(s ). IR: 3440 cm-<1>(br), 3100, 2950, 1805(w), 1775(m), 1740(s), 1695(m), 1610(m), 1505(m), 1375(m) , 1280(m), 1250(s), 1205(s ).

EKSEMPEL 6 EXAMPLE 6

Fremstillingen av N-(4-metoksyfenyl)-N-(3-succinimidoyloksykarbonyl -benzensulfonyl)-10-metylacridinium-9-karboksylsyreamid-fluorsulfonat (25) fra 3-[N-(4'-metoksyfenyl)sulf-amido]-benzosyrebenzylester og acridin-9-karboksylsyreklorid-hydroklorid (3) foregår analogt til fremstillingen av (17) The preparation of N-(4-methoxyphenyl)-N-(3-succinimidoyloxycarbonyl-benzenesulfonyl)-10-methylacridinium-9-carboxylic acid amide fluorosulfonate (25) from 3-[N-(4'-methoxyphenyl)sulfamido]-benzoic acid benzyl ester and acridine-9-carboxylic acid chloride-hydrochloride (3) proceeds analogously to the preparation of (17)

(se eksempel 4). Utbyttet av de enkelte syntesetrinn, samt den spektroskopiske karakterisering er angitt i det følgende. (see example 4). The yield of the individual synthesis steps, as well as the spectroscopic characterization is indicated in the following.

N-( 4- metoksyf enyl )- N- ( 3- benzy loksykarbonylbenzensulf onyl )-acridin- 9- karboksylsyreamid ( 22) N-(4-methoxyphenyl)-N-(3-benzyloxycarbonylbenzenesulfonyl)-acridine-9-carboxylic acid amide (22)

Utbytte: 70* Smeltepunkt: 168-170°C Yield: 70* Melting point: 168-170°C

NMR (DMSO, 100 MHz): S = 3,5 ppm (s, 3H), S = 5,45 ppm (s, 2H), S = 6,5 ppm (s, br, 2H), S = 7,1 ppm (br, 2H), S = 7,3-8,8 ppm (m, 17H). NMR (DMSO, 100 MHz): S = 3.5 ppm (s, 3H), S = 5.45 ppm (s, 2H), S = 6.5 ppm (s, br, 2H), S = 7, 1 ppm (br, 2H), S = 7.3-8.8 ppm (m, 17H).

N-( 4- metoksyfenyl )- N-( 3- karboksybenzensulfonyl) acridln- 9-karboksylsyreamid- hydrobromid ( 23) N-(4-Methoxyphenyl)-N-(3-carboxybenzenesulfonyl) acridln-9-carboxylic acid amide hydrobromide (23)

Utbytte: 90* Smeltepunkt: 264°C Yield: 90* Melting point: 264°C

NMR (DMSO, 100 MHz): S = 3,5 ppm (s, 3H), S = 6,4-6,6 ppm (d, br, 2H), S = 7,0-7,2 ppm (d, br, 2H), å = 7,6-8,8 ppm (m, 12H). NMR (DMSO, 100 MHz): S = 3.5 ppm (s, 3H), S = 6.4-6.6 ppm (d, br, 2H), S = 7.0-7.2 ppm (d , br, 2H), δ = 7.6-8.8 ppm (m, 12H).

N- ( 4- metoksvf enyl ) - N- ( 3- succinimidoyloksykarbonylbenzensulfonyl) acridln- 9- karboksylsyreamld ( 24) N-(4-methoxyphenyl)-N-(3-succinimidoyloxycarbonylbenzenesulfonyl)acridln-9-carboxylic acid amld (24)

Utbytte: 50* Smeltepunkt: 223-225°C Yield: 50* Melting point: 223-225°C

NMR (DMSO, 100 MHz): S = 2,95 ppm (s, 4H), S = 3,5 ppm (s, 3H), S = 6,4-6,6 ppm (d, br, 2H), S = 7,0-7,2 ppm (d, br, 2H), S = 7,4-8,9 ppm (m, 12H). NMR (DMSO, 100 MHz): S = 2.95 ppm (s, 4H), S = 3.5 ppm (s, 3H), S = 6.4-6.6 ppm (d, br, 2H), S = 7.0-7.2 ppm (d, br, 2H), S = 7.4-8.9 ppm (m, 12H).

IR: 3500 cm-<1>(br), 3060, 2950, 2840, 1805(w), 1785(m), 1740(s), 1700(m), 1510(m), 1380(m), 1250(m), 1205(m), 1165(m). IR: 3500 cm-<1>(br), 3060, 2950, 2840, 1805(w), 1785(m), 1740(s), 1700(m), 1510(m), 1380(m), 1250( m), 1205(m), 1165(m).

N- ( 4- metoksyf enyl ) - N- ( 3- succinimidoyloksykarbonylbenzensulfonyl )- 10- metylacrldlnlum- 9- karboksylsyreamid- fluorosulfonat 1251 N-( 4- methoxy enyl ) - N-( 3- succinimidoyloxycarbonylbenzenesulfonyl )- 10- methyl acrylamide- 9- carboxylic acid amide fluorosulfonate 1251

Utbytte: 90* Yield: 90*

NMR (DMSO, 100 MHz): S = 2,95 ppm (s, 4H), S = 3,55 ppm (s, 3H), S = 4,8 ppm (s, br, 3H), S = 6,45-6,7 (d, br, 2H), 5 = 7,05-7,3 ppm (d, br, 2H), S = 7,5-8,9 ppm (m, 12H). NMR (DMSO, 100 MHz): S = 2.95 ppm (s, 4H), S = 3.55 ppm (s, 3H), S = 4.8 ppm (s, br, 3H), S = 6, 45-6.7 (d, br, 2H), δ = 7.05-7.3 ppm (d, br, 2H), S = 7.5-8.9 ppm (m, 12H).

IR: 3500 cm-<1>(br), 3080, 2950, 1805(w), 1780(m), 1740(s), 35 1700(m), 1610(m), 1510(m), 1380(m), 1250(s), 1205(s), 1170(s). IR: 3500 cm-<1>(br), 3080, 2950, 1805(w), 1780(m), 1740(s), 35 1700(m), 1610(m), 1510(m), 1380(m ), 1250(s), 1205(s), 1170(s).

Masse: m/z = 624 M<+>(kation) Mass: m/z = 624 M<+>(cation)

Claims (2)

1. Kjemiluminescerende acridiniumderivat med formel I, 1. Chemiluminescent acridinium derivative of formula I, derR<1>er hydrogen, Ci-C4-alkyl,R<2>ogR3 er hydrogen, R<4>er en substituert sulfonamidgruppe med formel V where R<1>is hydrogen, C1-C4 alkyl, R<2>and R3 are hydrogen, R<4>is a substituted sulfonamide group of formula V eller med formel VI or with formula VI eller en tioalkyl- eller tioarylrest med formel II or a thioalkyl or thioaryl radical of formula II hvorved X er en metylen-, etylen-, propylen- eller en orto-, meta-eller parafenylengruppe og R<5>er en reaktiv gruppe, A~ er et anion som ikke påvirker kjemiluminescensen, og R*> i formel V og VI er f enyl eller naftyl som også kan være substituert med Ci_4alkoksy.whereby X is a methylene, ethylene, propylene or an ortho, meta or paraphenylene group and R<5> is a reactive group, A~ is an anion which does not affect the chemiluminescence, and R*> in formulas V and VI are f phenyl or naphthyl which can also be substituted with C 1-4 alkoxy. 2. Acridiniumderivat ifølge krav 1,karakterisertved formel IV 2. Acridinium derivative according to claim 1, characterized by formula IV der A og X har den i krav 1 angitte betydning.where A and X have the meaning specified in claim 1.
NO873520A 1986-08-22 1987-08-20 CHEMILUMINESCING ACRIDIN DERIVATIVES NO171725C (en)

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