DK171437B1 - Chemiluminescent acridine derivatives and process for their preparation - Google Patents
Chemiluminescent acridine derivatives and process for their preparation Download PDFInfo
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- DK171437B1 DK171437B1 DK437187A DK437187A DK171437B1 DK 171437 B1 DK171437 B1 DK 171437B1 DK 437187 A DK437187 A DK 437187A DK 437187 A DK437187 A DK 437187A DK 171437 B1 DK171437 B1 DK 171437B1
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Abstract
Description
DK 171437 B1DK 171437 B1
Opfindelsen angår hidtil ukendte, kemiluminescerende acridinderivater og en fremgangsmåde til deres fremstilling.The invention relates to novel chemiluminescent acridine derivatives and to a process for their preparation.
Luminescerende forbindelser finder allerede mangesidig anvendelse. De anvendes som indikatorer ved bioanalyser, 5 enzymimmunanalyser og luminescensimmunanalyser (jfr. W.P. Collins "Alternative Immunoassays", forlag John Wiley & Sons Ltd., Chichester, 1985), men de anvendes også ved nuclein-syre-hybridiseringsanalyser (jfr. J.A. Matthews et al. "Analytical Biochemistry", 151, 205-209, 1985). Desuden 10 anvendes kemiluminescerende forbindelser ved "flow injection analysis", til detektorer, der er indskudt efter kolonnen ved væskechromatografi, i strømningsforskningen og i kunstige lyskilder.Luminescent compounds are already widely used. They are used as indicators in bioassays, 5 enzyme immunoassays and luminescence immunoassays (cf. WP Collins "Alternative Immunoassays", publisher John Wiley & Sons Ltd., Chichester, 1985), but they are also used in nucleic acid hybridization assays (cf. JA Matthews et al. "Analytical Biochemistry", 151, 205-209, 1985). In addition, chemiluminescent compounds are used in "flow injection analysis", for detectors inserted after the column by liquid chromatography, in the flow research and in artificial light sources.
Ved kemiluminescensimmunanalyserne har især to struk-15 turtyper af kemiluminescerende mærkningssubstanser opnået større betydning. Det drejer sig her på den ene side om luminol- og isoluminolderivaterne, der er beskrevet af H.R. Schroeder et al., "Methods in Enzymology", Academic Press Inc., New York, vol. LVII, 1978, side 424 ff, samt i GB-20 -patentskrifter nr. 2.008.247 og 2.041.920 og i DE-patent-skrifter 2.618.419 og 2.618.511, samt i EP-offentiiggørelses-skrift nr. 135.071. Man får et overblik over den praktiske anvendelse af isoluminolforbindelserne som luminescensindikatorer hos W.G. Wood, J. Clin. Chem. Clin. Biochem. 22, 25 1984, side 905-918.In chemiluminescence immunoassays, in particular, two structural types of chemiluminescent labeling substances have gained greater importance. Here, on the one hand, these are the luminol and isoluminol derivatives described by H.R. Schroeder et al., "Methods in Enzymology", Academic Press Inc., New York, Vol. LVII, 1978, pages 424 et seq., And in GB-20 Patent Nos. 2,008,247 and 2,041,920 and in DE Patent Nos. 2,618,419 and 2,618,511, as well as in EP Publication No. 135,071. An overview of the practical use of the isoluminol compounds as luminescence indicators by W.G. Wood, J. Clin. Chem. Clin. Biochem. 22, 25 1984, pages 905-918.
På den anden side har også acridiniumesterforbindelser fundet anvendelse som kemiluminescerende mærkningssubstanser. Sådanne acridiniumestere kendes fra US-patentskrift nr. 3.352.791, GB-patentskrifter nr. 1.316.363 og 1.461.877 30 samt fra EP-offentiiggørelsesskrift nr. 82.636. Anvendelsen af acridiniumestere som mærkningssubstanser i immunanalyser er beskrevet af Weeks et al., Clin. Chem. 29/8 (1983), 1474--1479. Også anvendelsen af phenanthridiniumestere som mærkningssubstans i luminescensimmunanalyser er kendt fra EP-35 -offentliggørelsesskrift nr. 170.415.On the other hand, acridinium ester compounds have also been used as chemiluminescent labeling substances. Such acridinium esters are known from U.S. Patent Nos. 3,352,791, GB Patent Nos. 1,316,363 and 1,461,877, and from EP Publication No. 82,636. The use of acridinium esters as labeling substances in immunoassays has been described by Weeks et al., Clin. Chem. 29/8 (1983), 1474--1479. Also, the use of phenanthridinium esters as labeling substance in luminescence immunoassays is known from EP-35 Publication No. 170,415.
Kemiluminescensen fra acridiniumestere kan startes 2 DK 171437 B1 ved tilsætning af en basisk H202-opløsning. For kemilumi-nescensens mekanisme har F. McCapra, Acc. Chem. Res. 9, 201, 1976, givet en overbevisende forklaring. Derved er den fraspaltelige gruppes art tydeligvis afgørende for såvel lys-5 kvanteudbyttet som for den hydrolytiske stabilitet.The chemiluminescence from acridinium esters can be started by adding a basic H 2 O 2 solution. For the mechanism of chemiluminescence, F. McCapra, Acc. Chem. Res. 9, 201, 1976, given a convincing explanation. Thus, the nature of the leaving group is obviously crucial for both light quantum yield and hydrolytic stability.
De hidtil kendte acridiniumestere har fordelen af et højere lyskvanteudbytte i forhold til luminol- og isoluminol-forbindelserne, og lyskvanteudbyttet bliver heller ikke påvirket uheldigt af proteiner, der er bundet til indikatoren 10 (jfr. Weeks et al., Clin. Chem. 29/8 (1983), 1474-1479).The known acridinium esters have the advantage of a higher light quantum yield over the luminol and isoluminol compounds, and the light quantum yield is also not adversely affected by proteins bound to the indicator 10 (cf. Weeks et al., Clin. Chem. 29 / 8 (1983), 1474-1479).
Skønt de fra EP-offentliggørelsesskrift nr. 82.636 kendte acridiniumphenylestere ved stimulering af kemiluminescensen ved hjælp af milde oxidationsmidler udmærker sig ved en stor påvisningsfølsomhed, udviser de forstyrrende 15 ulemper ved den praktiske anvendelse. Frem for alt er phenyl-esterbindingen i vandige systemer også allerede meget labil ved stuetemperatur. Hertil kommer, at acridiniumphenylesteren under de der angivne oxidationsbetingelser har en lysemission, der først forsvinder efter ca. 10 sekunder i stor ud-20 strækning, dvs. op til over 95%. 1 sammenligning hermed har andre ikke-isotopiske analysefremgangsmåder meget kortere måletider og herved gives mulighed for en større prøvekapacitet.Although the acridinium phenyl esters known from EP Publication No. 82,636 known to stimulate chemiluminescence by means of mild oxidants are characterized by a high detection sensitivity, they exhibit disruptive drawbacks in the practical application. Above all, the phenyl ester bond in aqueous systems is also already very labile at room temperature. In addition, under the oxidation conditions stated, the acridinium phenyl ester has a light emission that disappears only after approx. 10 seconds in large extent, ie. up to over 95%. In comparison, other non-isotopic assay methods have much shorter measurement times and thus allow for a larger sample capacity.
Det er derfor formålet med den foreliggende opfindelse 25 at tilvejebringe hidtil ukendte, kemiluminescerende acridi-niumderivater, der på grund af et større lyskvanteudbytte har en hurtigere reaktionskinetik, og dermed muliggøres korte måletider ved en luminescensimmunanalyse.It is therefore the object of the present invention to provide novel chemiluminescent acridium derivatives which, due to greater light quantum yield, have a faster reaction kinetics, thus enabling short measurement times by luminescence immunoassay.
Dette formål er opnået med de her omhandlede, kemi-30 luminescerende acridiniumderivater, som er ejendommelige ved, at de har den almene formel IThis object has been achieved with the above-described chemiluminescent acridinium derivatives, which are characterized in that they have the general formula I
R1 A® 35 *2 -QCU- 4R1 A® 35 * 2 -QCU- 4
O = C - RO = C - R
3 DK 171437 B13 DK 171437 B1
hvor R1 betyder hydrogen, en alkyl-, alkenyl- eller alkynyl-gruppe med 1-10 C-atomer, en benzyl-, phenyl- eller napth-ylgruppe, R2 og R3 betyder hydrogen, en alkylgruppe med 1-4 C-atomer, en usubstitueret aminogruppe, en eventuelt i alkyl-5 grupperne med hydroxy monosubstitueret C1_4-alkyl- eller Ci-A-dialkylaminogruppe, en cyclisk aminogiruppe, f.eks. mor-pholin, en carboxy-, C1_4-alkoxy-, cyano- eller nitrogruppe eller halogen, R4 betyder en substitueret sulfonamidgruppe med den almene formel Vwherein R 1 is hydrogen, an alkyl, alkenyl or alkynyl group having 1-10 C atoms, a benzyl, phenyl or napthyl group, R 2 and R 3 are hydrogen, an alkyl group having 1-4 C atoms, an unsubstituted amino group, one optionally in the alkyl groups having hydroxy monosubstituted C 1-4 alkyl or C 1-6 dialkylamino group, a cyclic amino group, e.g. morpholine, a carboxy, C1-4 alkoxy, cyano or nitro group or halogen, R4 means a substituted sulfonamide group of the general formula V
10 X - R5 / -N (V) 15 S02 - R610 X - R5 / -N (V) SO 2 - R6
eller med den almene formel VIor of general formula VI
20 R6 / -N (VI) \ 5 25 S02 - X - R520 R6 / -N (VI) \ 5 25 SO2 - X - R5
eller en thioalkyl- eller thioarylgruppe med den almene formel IIor a thioalkyl or thioaryl group of the general formula II
30 - S - X - R5 (II) hvor X betyder en forgrenet eller ikke-forgrenet Ci-5~alky-lengruppe eller en ortho-, meta- eller para-phenylengruppe, der også kan indeholde heteroatomer, R5 er en reaktiv gruppe, 35 der under skånende betingelser selektivt kan indgå en binding med amino-, carboxy-, thiol- eller andre funktionelle grupper i substanser af biologisk interesse, R6 i den almene formel V betyder en alkyl- eller alkenylgruppe med 1-10 C-atomer, en med C1_4-alkyl- mono- eller disubstitueret aminogruppe, 40 en benzyl- eller en arylgruppe, der også kan være substitueret med hydroxy, amino, alkoxy med 1-4 C-atomer eller aryl- DK 171437 B1 4- S - X - R5 (II) wherein X is a branched or unbranched C1-5 alkylene group or an ortho, meta or para-phenylene group which may also contain heteroatoms, R5 is a reactive group, Which under selective conditions can selectively form a bond with amino, carboxy, thiol or other functional groups in substances of biological interest, R 6 in the general formula V means an alkyl or alkenyl group having 1-10 C atoms, a with C 1-4 alkyl mono- or disubstituted amino group, a benzyl or an aryl group which may also be substituted by hydroxy, amino, alkoxy of 1-4 C atoms or aryl-
oxy, og R6 i den almene formel VI ud over de for formel Voxy, and R6 of general formula VI in addition to those of formula V
Θ nævnte substituenter desuden kan betyde hydrogen, og A er en anion, der ikke påvirker kemiluminescensen.Subst said substituents may also mean hydrogen and A is an anion that does not affect chemiluminescence.
Blandt substanserne af biologisk interesse forstås 5 frem for alt antigener. Under dette begreb hører også hormoner, steroider, lægemidler, lægemiddelmetabolitter, toxi-ner, alkaloider og også antistoffer.Among the substances of biological interest are 5 above all antigens. This term also includes hormones, steroids, drugs, drug metabolites, toxins, alkaloids and also antibodies.
Den anion, der ikke påvirker kemiluminescensen uheldigt, kan være en tetrafluorborat-, perchlorat-, halo-10 gen-, alkylsulfat-, halogensulfonat-, alkylsulfonat- eller arylsulfonat-anion. Også enhver anden anion kan anvendes, såfremt den ikke hindrer eller svækker kemiluminescensen.The anion which does not adversely affect the chemiluminescence may be a tetrafluoroborate, perchlorate, halogen, alkyl sulfate, halogen sulfonate, alkyl sulfonate or arylsulfonate anion. Also, any other anion can be used if it does not hinder or impair chemiluminescence.
Ved aryl forstås aromatiske carbonhydrider, især phenyl og naphthyl. Når sådanne grupper indeholder hetero-15 atomer, er der tale om et nitrogen- eller oxygenatom, især quinolin, indol, pyrrol og pyridin. Ved halogener forstås fluor, chlor, brom og iod.By aryl is meant aromatic hydrocarbons, especially phenyl and naphthyl. When such groups contain heteroatoms, it is a nitrogen or oxygen atom, especially quinoline, indole, pyrrole and pyridine. Halogens are fluorine, chlorine, bromine and iodine.
De substituerede aminogrupper er C^-C^-alkyl- eller C^-C^dialkylaminer, hvor alkylgrupperne kan være monosubs-20 titueret med hydroxy. Ligeledes kan der være tale om en morpholingruppe. De ved R2 og R3 angivne alkoxygrupper har 1-4 C-atomer i alkyldelen.The substituted amino groups are C ^-C ^ alkyl or C ^-C ^ dialkylamines wherein the alkyl groups may be monosubstituted with hydroxy. Likewise, there may be a morpholine group. The alkoxy groups represented by R 2 and R 3 have 1-4 C atoms in the alkyl moiety.
I almindelighed vælges acridiniumderivater, i hvilke X betyder en methylen-, ethylen-, propylen- eller en or-25 tho-, metha- eller para-phenylengruppe. Til forbedring af acridiniumderivaternes vandopløselighed kan disse grupper også have hydrofile substituenter, der indeholder hetero-atomer.Generally, acridinium derivatives are selected in which X represents a methylene, ethylene, propylene or an ortho, metha or para-phenylene group. To improve the water solubility of the acridinium derivatives, these groups may also have hydrophilic substituents containing heteroatoms.
Af særlig betydning for anvendelsesmulighederne af 30 acridiniumderivaterne ifølge opfindelsen er substituenten R5. Gennem et egnet valg af denne gruppe får acridinderi-vatet en så stor reaktivitet, at det under skånende betingelser selektivt kan indgå en binding med en funktionel gruppe i den biologiske substans, der skal påvises. Egnede 35 reaktive grupper ses af den følgende opstilling: 5 DK 171437 B1Of particular importance to the uses of the 30 acridinium derivatives of the invention is the substituent R5. Through a suitable choice of this group, the acridine derivative gains such a high reactivity that under sparing conditions it can selectively enter into a bond with a functional group in the biological substance to be detected. Suitable 35 reactive groups are seen in the following arrangement: 5 DK 171437 B1
OISLAND
0 °Vi0 ° We
a) -C - 0 - Na) -C - 0 - N
<r<r
. o _^SO,M. o _ ^ SO, M
5 il f i *5 il f i *
b) -C - O - Nb) -C - O - N
)— Y( + )= H^ + \ alkali(+)) - Y (+) = H + + alkali (+)
OISLAND
oisland
c) -C - O - N Ic) -C - O - N I
10 \ I I10 \ I I
O' d) -S02 - CH = CH2 (+/-Λ 15 e) -SOp-CHp-CH?-N/ M . .O 'd) -SO2 - CH = CH2 (+/- Λ e) -SOp-CHp-CH2 -N / M. .
2 2 2 Whalogenid(")2 2 2 Whalogenide (")
f) -N = C = Sf) -N = C = S
β) 20 \=/β) 20 \ = /
MM
^NH2 halogenid ^ ^ h) -C^^ NH2 halide ^ ^ h) -C ^
N0-RN0-R
25 0y-i)25y-i)
i) -Ni) -N
Cl Cl O \_f k) -c - o -/ V τιCl Cl O \ _f k) -c - o - / V τι
30 W30 W
Cl Cl o l) -c - o HØ-N02Cl Cl o l) -c - o HE-NO2
? /^N? / ^ N
35 m) -C- N^J35 m) -C- N ^ J
o n5 n) -C - C - CFj 6 DK 171437 B1o n5 n) -C - C - CFj 6 DK 171437 B1
I mange tilfælde har acridiniumderivater ifølge opfindelsen, hvor R5 betyder en gruppe med formlen IIIIn many cases, acridinium derivatives according to the invention have where R5 represents a group of formula III
0 5 0 } (III)0 5 0} (III)
- C - o - N- C - o - N
0 vist sig egnede.0 proved to be suitable.
10 Desuden foretrækkes også acridiniumforbindelser,In addition, acridinium compounds are also preferred.
hvor R1 betyder en methylgruppe. Især anvendes derfor ofte acridiniumforbindelser ifølge opfindelsen, der svarer til den almene formel IVwhere R1 represents a methyl group. In particular, acridinium compounds according to the invention, which correspond to the general formula IV, are therefore often used
15 f3 ri T ί Λ <iv>15 f3 ri T ί Λ <iv>
20 A - x -1 - o - *Q20 A - x -1 - o - * Q
o o hvor A og X har de ovenfor nævnte betydninger.o o where A and X have the above meanings.
25 Når gruppen R4 betyder en sulfonamid-gruppe i acri-When the group R 4 means a sulfonamide group in acrylic
diniumderivaterne ifølge opfindelsen, har gruppen den almene formel Vof the dinium derivatives of the invention, the group has the general formula V
X - R5 30 / -N (V) S02 - R6X - R5 30 / -N (V) SO2 - R6
eller den almene formel VIor the general formula VI
35 7 DK 171437 B1 R6 / -N (VI) \ 5 5 S02 - X - R5 hvor X, R5 og R6 har de ovenfor nævnte betydninger.35 7 DK 171437 B1 R6 / -N (VI) \ 5 5 SO2 - X - R5 where X, R5 and R6 have the above meanings.
Typiske repræsentanter for denne klasse af acridinium-derivater ifølge opfindelsen har den almene formel VIITypical representatives of this class of acridinium derivatives of the invention have the general formula VII
10 CH, • 2 o <vii>10 CH, • 2 o <vii>
Wo rWo r
20 hvor A og X har de ovenfor nævnte betydninger.20 where A and X have the above meanings.
Ved foreliggende opfindelse tilvejebringes to hidtil ukendte klasser af acridiniumforbindelser, hvor den ene klasse indeholder en thiolestergruppering og den anden en sulfonamidstruktur. I forhold til de kendte acridiniumphenyl-25 ester-forbindelser har acridiniumacylsulfonamid-derivaterne en større stabilitet, og både disse derivater og thiolesteme har en hurtigere kinetik. Begge forbindelsesklasser udmærker sig desuden ved et større lysudbytte.The present invention provides two novel classes of acridinium compounds, one class containing a thiol ester group and the other a sulfonamide structure. Compared to the known acridinium phenyl ester compounds, the acridinium acylsulfonamide derivatives have a greater stability, and both these derivatives and the thiol esters have a faster kinetics. Both connection classes are also distinguished by a greater light output.
En betydelig fordel ved de acridiniumforbindelser 30 ifølge opfindelsen, der indeholder thiolesterholdige, fraspaltelige grupper, i forhold til de fra den EP-offentiiggø-relsesskrift nr. 82.636 kendte acridiniumphenylestere ligger i lysemissionens væsentligt hurtigere reaktionskinetik. Således giver den efter eksempel 1 fremstillede acridinium-35 thioester ifølge opfindelsen (forbindelse 6) ved en måletid på 1 sekund, et lysudbytte, der er en faktor 10 større under de samme oxidationsbetingelser. Disse store lysudbytter samtidig med korte måletider muliggør en væsentlig større 8 DK 171437 B1 prøvekapacitet i luminometeret.A significant advantage of the acridinium compounds 30 of the invention, which contain thiol ester-containing leaving groups, over the acridinium phenyl esters known from EP Publication No. 82,636 are in the substantially faster reaction kinetics of the light emission. Thus, the acridinium thioester of the invention (Compound 6) prepared according to Example 1, at a measurement time of 1 second, gives a light yield which is a factor 10 greater under the same oxidation conditions. These large light yields, together with short measurement times, allow for a significantly larger sample capacity in the luminometer.
Således viser fig. 1 kinetikken af lysemissionen af et antistofkonjugat af en ifølge eksempel l fremstillet acridiniumthioester (forbindelse 6), og fig. 2 viser kine-5 tikken af lysemissionen af et antistofkonjugat af 4-(2-suc-cinimidyl-oxycarbony lethyl) phenyl-10-methylacridinium-9-carboxylat-methosulfat (EP-of fenti iggørelsesskrift nr. 82.636, side 10). 100 μΐ af hver af traceropløsningerne stimuleres ved tilsætning af 350 μΐ 0,05 M KCl/NaOH-puffer 10 pH 13 + 0,1% H2O2 til kemiluminescens, og der registreres over et tidsrum på 10 sekunder.Thus, FIG. 1 shows the kinetics of the light emission of an antibody conjugate of an acridinium thioester prepared according to Example 1 (compound 6); and FIG. Figure 2 shows the kinetics of the light emission of an antibody conjugate of 4- (2-succinimidyl-oxycarbonylethyl) phenyl-10-methylacridinium-9-carboxylate methosulfate (EP-A-Publication No. 82,636, page 10). 100 μΐ of each of the tracer solutions is stimulated by adding 350 μΐ 0.05 M KCl / NaOH buffer 10 pH 13 + 0.1% H2O2 to chemiluminescence and recorded over a period of 10 seconds.
I fig. 1 nås den maksimale lysemission allerede efter 0,66 sekunder, og den er allerede efter 0,88 sekunder igen faldet til halvdelen. I forhold hertil nås den maksimale 15 lysemission først efter 1,77 sekunder i fig. 2, og den falder først efter 2,88 sekunder igen til halvdelen.In FIG. 1, the maximum light emission is reached already after 0.66 seconds and it has already fallen to half again after 0.88 seconds. In this respect, the maximum light emission is only reached after 1.77 seconds in FIG. 2, and it only drops to half again after 2.88 seconds.
Det var fuldstændig uventet, at på amidnitrogenet sulfonylsubstituerede acridinium-9-carboxylsyreamider har en udmærket kemiluminescens, da det vides, at acridinium-20 9-carboxylsyreamid i modsætning til acridinium-9-carboxyl-syreestere ikke viser nogen kemiluminescens (jfr. F. McCapra i W. Carruthers og J.K. Sutherland; Progress in Organic Chem., vol. 8, 231-277, 1973, Butterworth, London).It was completely unexpected that the amide nitrogen sulfonyl-substituted acridinium-9-carboxylic acid amides have an excellent chemiluminescence, since it is known that, unlike acridinium-9-carboxylic acid esters, acridinium-9-carboxylic acid esters show no chemiluminescence F. in W. Carruthers and JK Sutherland; Progress in Organic Chem., Vol. 8, 231-277, 1973, Butterworth, London).
Acridinium-9-carboxylsyrethioestere ifølge opfindelsen 25 fremstilles ved den her omhandlede fremgangsmåde, som er ejendommelig ved det i de kendetegnende dele af kravene 7 og 8 angivne.Acridinium-9-carboxylic acid ethioesters of the invention 25 are prepared by the process of this invention, which is characterized by the features of claims 7 and 8.
Fremstillingen sker på følgende måde:The preparation is done as follows:
Acridin eller derivater heraf med R2 og/eller R3 i 30 de påkondenserede phenylringe omsættes ifølge den af Lehm-stedt og Hundertmark i Ber. 63, 1229 (1930), angivne fremgangsmåde i ethanol/iseddikesyre og kaliumcyanid til 9-cya-noacridin. Herfra udvindes efter omkrystallisering acridin--9-carboxylsyre henholdsvis R2/R3-substitueret acridin-9-35 -carboxylsyre ved omsætning med svovlsyre og natriumnitrit svarende til den af Lehmstedt og Wirth i Ber. 61, 2044 (1928) 9 DK 171437 B1 beskrevne fremgangsmåde. Ved omsætning af acridin-9-carboxyl-syre henholdsvis R2/R3-substitueret acridin-9-carboxylsyre med thionylchlorid udvindes forbindelsen med den almene formel VIII 5Acridine or derivatives thereof with R2 and / or R3 in the condensed phenyl rings are reacted according to that of Lehm-stedt and Hundertmark in Ber. 63, 1229 (1930), disclose the process in ethanol / glacial acetic acid and potassium cyanide for 9-cyanoacridine. From this, after recrystallization, acridine-9-carboxylic acid and R2 / R3-substituted acridine-9-35-carboxylic acid, respectively, are recovered by reaction with sulfuric acid and sodium nitrite similar to that of Lehmstedt and Wirth in Ber. 61, 2044 (1928) disclosed in accordance with the method described. By reacting acridine-9-carboxylic acid or R2 / R3-substituted acridine-9-carboxylic acid with thionyl chloride, the compound of general formula VIII is recovered.
R2 4 11^ RR2 4 11 ^ R
(VIII) Λ(VIII) Λ
10 0 Y10 0 Y
hvor Y betyder chlor. I stedet for et halogen kan der også i forbindelsen VIII indføres en oxycarbonyl-C1-C5-alkyl-, oxycarbonylaryl- eller imidazolidgruppe i stedet for Y.where Y means chlorine. Instead of a halogen, an oxycarbonyl-C1-C5 alkyl, oxycarbonylaryl or imidazolidine group may also be substituted in compound VIII for Y.
15 De R2/R3-substituerede acridinderivater kan synteti seres enkelt efter i litteraturen kendte fremgangsmåder. Sådanne synteser er f.eks. beskrevet i: Comprehensive Heterocyclic Chemistry, Editors A. Katritzky, C.W. Rees, Vol. 2, side 395 ff, Pergamon Press, 1984 eller Heterocyclic Com-20 pounds, Vol. 9, Acridines and Cond. 2. udgave, R.H. Acheson,The R2 / R3-substituted acridine derivatives can be synthesized simply according to methods known in the literature. Such syntheses are e.g. described in: Comprehensive Heterocyclic Chemistry, Editors A. Katritzky, C.W. Rees, Vol. 2, page 395 et seq., Pergamon Press, 1984 or Heterocyclic Com-20 pounds, Vol. 9, Acridines and Cond. 2nd Edition, R.H. Acheson,
John Wiley and Sons, 1973.John Wiley and Sons, 1973.
Syrechloridet (VIII) omsættes derpå med en thiocarb-oxylsyre med den almene formel IX, 25 HS - X - COOH (IX) f.eks. med 2-mercaptobenzoesyre, under basiske betingelser til dannelse af en thiolestercarboxylsyre, der herefter forestres med en til dannelse af gruppen R5 egnet forbin-30 delse, f.eks. med N-hydroxysuccinimid. Herefter alkyleres acridinforbindelsen i 10-stillingen efter i litteraturen kendte fremgangsmåder. Egnet til en methylering er frem for alt trimethyloxoniumtetrafluorborat, men der kan dog også med dimethyl sul fat, methylfluorsulfonat, toluensul fonsy remethyl-35 ester eller trifluormethansulfonsyremethylester opnås gode udbytter af en kemiluminiserende acridiniumforbindelse.The acid chloride (VIII) is then reacted with a thiocarboxylic acid of the general formula IX, HS - X - COOH (IX) e.g. with 2-mercaptobenzoic acid, under basic conditions to form a thiol ester carboxylic acid, which is then esterified with a compound suitable for the group R5, e.g. with N-hydroxysuccinimide. Thereafter, the acridine compound is alkylated at the 10-position according to methods known in the literature. Suitable for methylation is primarily trimethyloxonium tetrafluoroborate, however, good yields of a chemiluminescent acridinium compound can also be obtained with dimethyl sulphate, methyl fluorosulphonate, toluene sulphonic remethyl ester or trifluoromethanesulphonic acid methyl ester.
10 DK 171437 B110 DK 171437 B1
Til fremstilling af acridiniumsulfonamidderivater ifølge opfindelsen gås ligeledes ud fra acridin-9-carboxyl-syrechloridet (VIII). Denne forbindelse omsættes med et primært eller sekundært sulfonamid, fortrinsvis med en be-5 skyttet sulfonamidcarboxylsyre med den almene formel XFor the preparation of acridinium sulfonamide derivatives according to the invention, the acridine-9-carboxylic acid chloride (VIII) is also used. This compound is reacted with a primary or secondary sulfonamide, preferably with a protected sulfonamide carboxylic acid of the general formula X
H OH O
6 1 11 R® - S02 - N - X - C - OZ (X) 10 eller med den almene formel XI 156 1 11 R® - SO2 - N - X - C - OZ (X) 10 or of general formula XI 15
HH
R6 - N - S02 - X - COOZ (XI) 20 hvor X og R*> har de ovenfor nævnte betydninger, og Z betyder en gruppe, der beskytter carboxygruppen, hvilken beskyttelsesgruppe til sidst fraspaltes. F.eks. kan N-benzensulfonyl-glycinbenzylester anvendes ved denne reaktion som beskyttelsesgruppe. Den efter fraspaltning af beskyttelsesgruppen 25 fremkomne syre omdannes med en egnet forbindelse, f.eks. N-hydroxysuccinimid, til gruppen R5. Herfra udvindes den kemiluminescerende acridiniumforbindelse efter i litteraturen kendte fremgangsmåder ved alkylering på nitrogenet i 10--stillingen.R6 - N - SO2 - X - COOZ (XI) 20 where X and R *> have the above meanings and Z means a group protecting the carboxy group, which protecting group is eventually split off. For example. For example, N-benzenesulfonyl-glycine benzyl ester can be used as a protecting group. The acid obtained after cleavage of the protecting group 25 is converted with a suitable compound, e.g. N-hydroxysuccinimide, to the group R5. From this, the chemiluminescent acridinium compound is recovered according to methods known in the literature by alkylation on the nitrogen at the 10-position.
30 De fremkomne acridiniumforbindelser lader sig herefter omsætte med en substans af biologisk interesse, f.eks. et antigen, et antistof, et hormon, et lægemiddel, en læge-middelmetabolit, et toxin eller et alkaloid til en lumin-escerende forbindelse. Derved er acridiniumderivatet enten 35 direkte eller over et bromolekyle, såsom polylysin, poly-glutaminsyre eller polyvinylamin, bundet til den biologisk interessante substans under dannelse af et stabilt immunologisk aktivt konjugat. Dette konjugat betegnes også som en tracer, og det anvendes i de i det efterfølgende beskrevne 40 luminescensimmunanalyser. Til luminescens immunanalysen ifølge 11 DK 171437 B1 opfindelsen til bestemmelse af en antigen-substans i en væskeprøve efter en kompetitiv-eller en sandwich-fremgangsmåde er det nødvendigt med mindst én immunologisk aktiv komponent, der er immobiliseret på en fast fase og desuden 5 den luminescerende tracer.The resulting acridinium compounds can then be reacted with a substance of biological interest, e.g. an antigen, an antibody, a hormone, a drug, a drug metabolite, a toxin or an alkaloid for a luminescent compound. Thereby, the acridinium derivative, either directly or over a bromolecule such as polylysine, polyglutamic acid or polyvinylamine, is bonded to the biologically interesting substance to form a stable immunologically active conjugate. This conjugate is also referred to as a tracer and it is used in the 40 luminescence immunoassays described below. For the luminescence immunoassay of the invention to determine an antigenic substance in a liquid sample following a competitive or sandwich process, at least one immunologically active component immobilized on a solid phase is required and in addition the luminescent tracer.
Både den luminescerende forbindelse og luminescens-immunanalysen er genstand for patentkrav i DK-A nr. 0684/92.Both the luminescent compound and the luminescence immunoassay are subject to claims in DK-A No. 0684/92.
Luminescensimmunanalysen kan gennemføres på forskellige måder.The luminescence immunoassay can be performed in various ways.
10 En mulighed består i, at det med antigenet specifikt reagerende immobiliserede antistof inkuberes med en prøve af den væske, der skal undersøges, og et konjugat af antigenet og et kemiluminescerende acridiniumderivat (antigen-tracer) , hvorefter prøven og den ikke-bundne tracer skilles 15 fra, og den bundne tracer bringes sammen med et oxidationsmiddel for at fremkalde en lysemission, hvorefter man ud fra den målte styrke af lysemissionen bestemmer mængden af tilstedeværende antigen.One possibility is that the antigen specifically reacting immobilized antibody is incubated with a sample of the liquid to be tested and a conjugate of the antigen and a chemiluminescent acridinium derivative (antigen tracer), after which the sample and the unbound tracer are separated. 15, and the bound tracer is brought together with an oxidizing agent to induce a light emission, after which the amount of antigen present is determined from the measured strength of the light emission.
En anden mulighed for gennemførelse af luminescensim-20 munanalysen består i, at et med antigenet specifikt reagerende immobiliseret antistof inkuberes med en prøve af den væske, der skal undersøges, med et konjugat med et andet specifikt reagerende antistof og et kemiluminescerende acridiniumderivat, hvorefter prøven og det ikke-bundne mærkede 25 konjugat skilles fra, og det bundne mærkede konjugat bringes sammen med et oxidationsmiddel for at fremkalde en lysemission, hvorefter man ud fra den målte styrke af lysemissionen bestemmer mængden af tilstedeværende antigen.Another possibility for carrying out the luminescence immunoassay consists in incubating an antigen-specific reacting immobilized antibody with a sample of the liquid to be tested, with a conjugate with another specific reacting antibody, and a chemiluminescent acridinium derivative, and the unbound labeled 25 conjugate is separated, and the bound labeled conjugate is brought together with an oxidizing agent to induce a light emission, after which the amount of antigen present is determined from the measured strength of the light emission.
De ovenfor nævnte luminescensimmunanalyser kan også 30 gennemføres på den måde, at man skiller den væske, der skal undersøges, fra det immobiliserede antistof før tilsætningen af det mærkede konjugat.The above-mentioned luminescence immunoassays may also be performed by separating the liquid to be assayed from the immobilized antibody prior to the addition of the labeled conjugate.
I andre gennemførlige luminescensimmunanalyser ifølge opfindelsen er det ikke antistoffet men antigenet, der er 35 immobiliseret. Således kan et med antistoffet specifikt reagerende immobiliseret antigen inkuberes med en prøve af 12 DK 171437 B1 den væske, der skal undersøges, og en opløsning af et kon-jugat af antistoffet og et kemiluminescerende acridinium-derivat, hvorefter prøven og det ikke-bundne mærkede konjugat skilles fra, hvorefter det bundne mærkede konjugat bringes 5 sammen med et oxidationsmiddel. Der forekommer nu en lysemission, hvorefter man ud fra den målte styrke af lysemissionen bestemmer mængden af tilstedeværende antigen.In other feasible luminescence immunoassays of the invention, it is not the antibody but the antigen that is immobilized. Thus, an antibody specifically reacting immobilized antigen can be incubated with a sample of the liquid to be tested and a solution of a conjugate of the antibody and a chemiluminescent acridinium derivative, after which the sample and the unbound labeled the conjugate is separated and the bound labeled conjugate is brought together with an oxidizing agent. A light emission now occurs, after which the amount of antigen present is determined from the measured strength of the light emission.
En yderligere variant består i, at et med antistoffet specifikt reagerende immobiliseret antigen inkuberes med en 10 opløsning af et konjugat af antistoffet og et kemilumin-iscerende acridiniumderivat, hvorefter det ikke-omsatte mærkede konjugat skilles fra, og der tilsættes en prøve af den væske, der skal undersøges, hvorefter prøven igen skilles fra, og det bundne mærkede konjugat bringes sammen med et 15 oxidationsmiddel for at fremkalde en lysemission, og ud fra styrken af lysemissionen bestemmes mængden af tilstedeværende antigen.A further variant consists in incubating an antibody specifically reacting immobilized antigen with a solution of a conjugate of the antibody and a chemiluminescent acridinium derivative, then separating the unreacted labeled conjugate and adding a sample of the liquid. it is to be investigated, after which the sample is again separated and the bound labeled conjugate is brought together with an oxidizing agent to induce a light emission, and from the strength of the light emission the amount of antigen present is determined.
Endelig er luminescensimmunanalysen også gennemførlig på den måde, at et med antistoffet specifikt reagerende 20 immobiliseret antigen inkuberes med en opløsning af et konjugat af antistoffet og et kemiluminescerende acridiniumderivat, hvortil der sættes en prøve af den væske, der skal undersøges, og prøven og det ikke-bundne konjugat skilles fra, og det bundne mærkede konjugat bringes sammen med et 25 oxidationsmiddel for at fremkalde en lysemission, hvorefter man ud fra den målte styrke af lysemissionen bestemmer mængden af tilstedeværende antigen.Finally, the luminescence immunoassay is also feasible in that an antibody specifically reacting with the antibody is incubated with a solution of a conjugate of the antibody and a chemiluminescent acridinium derivative to which a sample of the liquid to be tested is added and the sample and the -bonded conjugate is separated and the bound labeled conjugate is brought together with an oxidizing agent to induce a light emission, after which the amount of antigen present is determined from the measured strength of the light emission.
Fremstillingen af acridiniuraforbindelser ifølge opfindelsen er illustreret i eksemplerne 1-3.The preparation of acridiniura compounds of the invention is illustrated in Examples 1-3.
3030
Eksemel 1 9-Cvanoacridin (liExample 1 9-Cvanoacridine (li
Til acridin (10 g) i 45 ml ethanol sættes 3,3 ml iseddikesyre og dråbevis en opløsning af 5,25 g kaliumcyanid 35 i 8 ml vand, hvorefter reaktionsblandingen opvarmes i 2 timer under tilbagesvaling, hvorefter der afkøles, og de flygtige DK 171437 Bl 13 bestanddele fjernes under vakuum. Remanensen røres sammen med 30 ml 2 N NaOH, hvorefter der frafiltreres ved sugning, og efter vask to gange med 2 N NaOH og vand tillades henstand i nogen tid i fugtig tilstand. Råproduktet røres sammen i 5 methylenchlorid, og uopløst substans frafiltreres ved sugning, og der vaskes med methylenchlorid, de samlede organiske faser koncentreres, og det rå 9-cyanoacridin omkrystalliseres ud fra n-butylacetat.To acridine (10 g) in 45 ml of ethanol is added 3.3 ml of glacial acetic acid and dropwise a solution of 5.25 g of potassium cyanide 35 in 8 ml of water, then the reaction mixture is heated at reflux, then cooled and the volatiles DK 171437 Bl 13 ingredients are removed under vacuum. The residue is stirred with 30 ml of 2N NaOH, filtered off by suction and after washing twice with 2N NaOH and water allowed to stand for some time in a moist state. The crude product is stirred in methylene chloride and the undissolved substance is filtered off by suction and washed with methylene chloride, the combined organic phases are concentrated and the crude 9-cyanoacridine is recrystallized from n-butyl acetate.
Udbytte: 50%, smp.: 183-185°C.Yield: 50%, mp: 183-185 ° C.
10 IR: 2230 cm+1.IR: 2230 cm + 1.
Acridin-9-carboxvlsvre (2) 9-Cyanoacridin (5 g) sættes langsomt portionsvis til 40 ml koncentreret H2SO4, og blandingen opvarmes i 2 timer 15 til 90-95°C, hvorefter der tilsættes 8,5 g NaN02, hvorefter der omrøres i yderligere 2 timer ved denne temperatur. Den varme opløsning sættes under hurtig omrøring til 620 ml isvand, hvorefter udfældningen frafiltreres ved sugning, og den opløses i mindst mulig 2 N NaOH. Opløsningen filtreres, 20 og den gøres sur med 50%'s H2S04, hvorefter den udfældende acridin-9-carboxylsyre frafiltreres ved sugning, og der tørres under vakuum.Acridine-9-carboxylic acid (2) 9-Cyanoacridine (5 g) is slowly added portionwise to 40 ml of concentrated H2SO4 and the mixture is heated for 2 hours 15 to 90-95 ° C, then 8.5 g of NaNO2 is added and then stirred. for another 2 hours at this temperature. The hot solution is added with rapid stirring to 620 ml of ice water, after which the precipitate is filtered off by suction and dissolved in as little as 2 N NaOH. The solution is filtered, acidified with 50% H 2 SO 4, then the precipitated acridine-9-carboxylic acid is filtered off with suction and dried under vacuum.
Udbytte: 95%, smp.: 288-289°C.Yield: 95%, mp: 288-289 ° C.
IR: 3440(br), 3200(br), 2600-2500(br), 1980; 1650? 25 1605? 1420 cm+1.IR: 3440 (br), 3200 (br), 2600-2500 (br), 1980; 1650? 25 1605? 1420 cm + 1.
Acridin-9-carboxvlsvrechlorid-hydrochlorid (3)Acridine-9-carboxylic acid chloride hydrochloride (3)
Acridin-9-carboxylsyre (5 g) sættes portionsvis til 50 ml frisk destilleret SOCl2, og der opvarmes 5 timer til 30 tilbagesvaling; den nu klare opløsning koncentreres ved destillation til begyndende udfældning, udfældningen fuldstændiggøres ved tilsætning af cyclohexan og afkøling. Udfældningen frafiltreres ved sugning, og den tørres under vakuum, hvorved der fås acridin-9-carboxylsyrechlorid-hydro-35 chlorid.Acridine-9-carboxylic acid (5 g) is added portionwise to 50 ml of freshly distilled SOCl2 and heated to reflux for 5 hours; the now clear solution is concentrated by distillation to the initial precipitate, the precipitate is completed by the addition of cyclohexane and cooling. The precipitate is filtered off by suction and dried under vacuum to give acridine-9-carboxylic acid chloride hydrochloride.
Udbytte: 90%, smp.: 223°C.Yield: 90%, mp: 223 ° C.
14 DK 171437 B114 DK 171437 B1
Elementaranalyse (beregnet som C14H9CINO x HC1) beregnet: C 60,5 H 2,8 N 5,0 Cl 25,5 fundet: C 59,4 H 3,3 N 5,0 Cl 25,2.Elemental analysis (calculated as C 14 H 9 ClNO x HCl) calculated: C 60.5 H 2.8 N 5.0 Cl 25.5 found: C 59.4 H 3.3 N 5.0 Cl 25.2.
5 fPhenvl-21-carboxylsyre!acridin-9-thiocarboxvlat M)5 Phenyl-21-carboxylic acid acridine-9-thiocarboxylate M)
Acridin-9-carboxylsyrechlorid-hydrochlorid (30 g) suspenderes i 720 ml methylenchlorid, og hertil sættes thio-salicylsyre (17,7 g) og 50 ml triethylamin, og den fremkomne opløsning, som bliver klar, efteromrøres i 10 minutter ved 10 stuetemperatur. Efter fjernelse af opløsningsmidlet sættes til remanensen 35 g natriumcarbonat og 1400 ml vand, og den resulterende opløsning koncentreres indtil der fremkommer en udfældning, som frafiltreres ved sugning. Filtratet mættes med NaCl, og det derved udfældende bundfald frafiltreres 15 ligeledes ved sugning. Den vandige opløsning af de samlede udfældninger gøres sur ved 80°C med iseddikesyre, hvorefter det udfældende produkt frafiltreres ved sugning, og det tørres under vakuum.Acridine-9-carboxylic acid chloride hydrochloride (30 g) is suspended in 720 ml of methylene chloride, to which is added thio-salicylic acid (17.7 g) and 50 ml of triethylamine, and the resulting solution which becomes clear is stirred for 10 minutes at room temperature. . After removal of the solvent, 35 g of sodium carbonate and 1400 ml of water are added to the residue and the resulting solution is concentrated until a precipitate is obtained which is filtered off by suction. The filtrate is saturated with NaCl and the precipitate thus precipitated is also filtered off by suction. The aqueous solution of the total precipitates is acidified at 80 ° C with glacial acetic acid, after which the precipitated product is filtered off by suction and dried under vacuum.
Udbytte: 80%, smp.: 261-265°C.Yield: 80%, mp: 261-265 ° C.
20 NMR (DMSO, 100 MHz): δ = 7,6-8,4 ppm, kompleks multiplet IR: 1680 cm"1 (S), 1720 (m) 1260 (s).NMR (DMSO, 100 MHz): δ = 7.6-8.4 ppm, complex multiple IR: 1680 cm -1 (S), 1720 (m) 1260 (s).
21 -(Succinimidovloxvcarbonvl)phenylacridin-9-thiocarboxvlat (5) .21 - (Succinimidovloxycarbonyl) phenylacridine-9-thiocarboxylate (5).
25 Til en suspension af 10 g af thiolestercarboxylsyren i 190 ml tørt tetrahydrofuran sættes ved 0°C 3,2 g N-hyd-roxysuccinimid, og herefter tilsættes ved -20°C 6,9 g di-cyclohexylcarbodiimid (DCC), og der efteromrøres i 2 timer ved -20°C, hvorefter der omrøres natten over ved stuetempera-30 tur. Efter tilsætning af 0,28 ml iseddikesyre omrøres der i 1 time, hvorefter der tilsættes ethylacetat (25 ml), og udfældningen frafiltreres. Filtratet koncentreres, og efter omkrystallisation ud fra chlorbenzen fås bleggult 2'-(suc-cinimidoyloxycarbonyl)phenylacridin-9-thiocarboxylat.To a suspension of 10 g of the thiol ester carboxylic acid in 190 ml of dry tetrahydrofuran is added 3.2 g of N-hydroxysuccinimide and then added at -20 ° C 6.9 g of di-cyclohexylcarbodiimide (DCC), and after stirring for 2 hours at -20 ° C, then stirring overnight at room temperature. After the addition of 0.28 ml glacial acetic acid, the mixture is stirred for 1 hour, then ethyl acetate (25 ml) is added and the precipitate is filtered off. The filtrate is concentrated and, after recrystallization from chlorobenzene, pale yellow 2 '- (sucimimidoyloxycarbonyl) phenylacridine-9-thiocarboxylate is obtained.
35 15 DK 171437 B135 15 DK 171437 B1
Udbytte: 80%, smp.: 198-200*C.Yield: 80%, mp: 198-200 ° C.
IR: 1810 cm-1, 1780, 1745, 1225, 1205 NMR (DMSO), 100 MHz): S = 2,95 ppm (s, 4H), 7,7-8,4 ppm (m, 12H).IR: 1810 cm -1, 1780, 1745, 1225, 1205 NMR (DMSO), 100 MHz): δ = 2.95 ppm (s, 4H), 7.7-8.4 ppm (m, 12H).
5 2 1 - (Succinimidovloxvcarbonvl)phenvl-10-methvlacridinium-9-thiocarboxvlat-tetrafluorborat (6) 3 g N-hydroxysuccinimidester (5) opvarmes med 7,8 g trimethyloxoniumtetrafluorborat i 40 ml 1,2-dichlorethan i 10 8 timer ved 80’C, og der efteromrøres natten over ved stue temperatur. Udfældningen filtreres fra, og der udkoges med 1,2-dichlorethan. De samlede organiske faser koncentreres, og der omkrystalliseres ud fra acetone-diisopropylether. Udbytte: 40%, smp.: 245*C.2 2 - (Succinimidovloxycarbonyl) phenyl-10-methylacridinium-9-thiocarboxylate 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 10 hours. C and stirring overnight at room temperature. The precipitate is filtered off and boiled with 1,2-dichloroethane. The combined organic phases are concentrated and recrystallized from acetone diisopropyl ether. Yield: 40%, mp: 245 ° C.
15 IR: 3440 cm"1 (br), 1800, 1780, 1740(s), 1670, 1065(s) NMR (DMSO, 100 MHz); δ * 3,0 ppm (s, 4H), 4,95 ppm (s, let udbredt, 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); δ * 3.0 ppm (s, 4H), 4.95 ppm (s, slightly widespread, 3H), 7.9-8.6 (m, 10H), 9.9 ppm (d, 2H).
Eksempel 2 20 Fremstillingen af succinimidoyloxycarbonylmethyl-10- -methylacridinium-9-thiocarboxylat-tetrafluorborat, hvor der gås ud fra syrechlorid (3) og thioglycolsyre sker analogt med syntesen af (6). Udbyttet af de enkelte syntesetrin samt den spektroskopiske karakterisering af produkterne (7) 25 til (9) er anført i det efterfølgende:Example 2 The preparation of succinimidoyloxycarbonylmethyl-10-methylacridinium-9-thiocarboxylate tetrafluoroborate, starting from acid chloride (3) and thioglycolic acid, is analogous to the synthesis of (6). The yield of the individual synthesis steps as well as the spectroscopic characterization of the products (7) 25 to (9) are given below:
Carboxvmethvlacridin-9-thiocarboxvlat (7).Carboxymethylacridine-9-thiocarboxylate (7).
Udbytte: 60%, Smp.: 218*C (under sønderdeling) IR: 3440 cm"1 (br), 2400(br), 1950(br), 1710(m), 1660(s), 30 1070(m) NMR (DMSO, 100 MHz): S = 4,25 ppm (s, 2H); 7,6-8,4 (m, 8H).Yield: 60%, Mp: 218 ° C (decomp.) IR: 3440 cm -1 (br), 2400 (br), 1950 (br), 1710 (m), 1660 (s), 30,1070 (m) NMR (DMSO, 100 MHz): S = 4.25 ppm (s, 2H); 7.6-8.4 (m, 8H).
Succiniroidovloxvcarbonvlmethvlacridin-9-thiocarboxYlat (8). Udbytte: 80%.Succiniroidovloxycarbonylmethylacridine-9-thiocarboxylate (8). Yield: 80%.
35 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, 16 DK 171437 B1 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, 16, 7.6-8.3 ppm (m, 8H).
Succinimidovloxvcarbonvlmethvl-10-methvlacridinium-9-thio-carboxvlat-tetrafluorborat (9).Succinimidovloxycarbonylmethyl-10-methylacridinium-9-thio-carboxylate tetrafluoroborate (9).
5 Udbytte: 40%, Smp.: 250’C.Yield: 40%, mp: 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).
1010
Eksempel 3 N-Benzensulfonvl-N-(benzvloxycarbonvlmethvl)acridin-9-carb-oxvlsvreamid (10).Example 3 N-Benzenesulfonyl-N- (benzyloxycarbonylmethyl) acridine-9-carboxylic acid amide (10).
Til 3,3 g N-benzensulfonylglycinbenzylester i 110 ml 15 tetrahydrofuran sættes 130 mg 4-(dimethylamino)-pyridin og 6 ml triethylamin, og efter 10 minutter sættes hertil 3 g acridin-9-carboxylsyrechlorid-hydrochlorid, og den fremkomne suspension opvarmes i 6 timer til tilbagesvaling. Udfældningen frafiltreres ved sugning, og opløsningsmidlet fjernes, 20 hvorefter remanensen optages i methylenchlorid, og der sammenrøres i kort tid med 2 N NaOH. Den organiske fase koncentreres efter tørring over MgS04, og den fremkomne remanens omkrystalliseres ud fra toluen/heptan.To 3.3 g of N-benzenesulfonylglycine benzyl ester in 110 ml of tetrahydrofuran are added 130 mg of 4- (dimethylamino) -pyridine and 6 ml of triethylamine, and after 10 minutes 3 g of acridine-9-carboxylic acid chloride hydrochloride are added and the resulting suspension is heated in 6 hours for reflux. The precipitate is filtered off by suction and the solvent is removed, the residue is taken up in methylene chloride and stirred briefly with 2N NaOH. The organic phase is concentrated after drying over MgSO 4 and the resulting residue is recrystallized from toluene / heptane.
Udbytte: 70%, Smp.: 58“C 25 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).Yield: 70%, Mp: 58 ° C IR: 3440 cm -1 (br), 1735, 1680, 1357, 1165 NMR (DMSO, 100 MHz): δ 5.2 ppm (s, 2H), δ, 3 ppm (s, 2H), 7.0-8.4 ppm (m, 18H).
N-Benzensulfonvl-N- (carboxvmethvl) acridin-9-carboxvlsvreamid 30 (11).N-Benzenesulfonyl-N- (carboxymethyl) acridine-9-carboxylic acid amide (11).
1 g N-benzensulfonyl-N-(benzyloxycarbonylmethyl)acri-din-9-carboxylsyreamid i 60 ml iseddikesyre hydrogeneres under tilsætning af 2 ml koncentreret HC1 og Pd/C (10%) ved stuetemperatur og under normalt tryk; efter endt reaktion 35 frafiltreres katalysatoren ved sugning, og ud fra filtratet fås ved koncentrering carboxylsyren som et gult fast stof.1 g of N-benzenesulfonyl-N- (benzyloxycarbonylmethyl) acridine-9-carboxylic acid amide in 60 ml of glacial acetic acid is hydrogenated with the addition of 2 ml of concentrated HCl and Pd / C (10%) at room temperature and under normal pressure; upon completion of reaction 35, the catalyst is filtered off by suction and from the filtrate is obtained by concentrating the carboxylic acid as a yellow solid.
17 DK 171437 B1 NMR (DMSO, 100 MHz): δ 5,0 ppm (s, 2H) , 7,1-8,5 ppm (ro, 13H) .17 NM 171437 B1 NMR (DMSO, 100 MHz): δ 5.0 ppm (s, 2H), 7.1-8.5 ppm (ro, 13H).
Forbindelsen (11) omsættes med N-hydroxysuccinimid analogt med fremstillingen af (5) til N-benzensulfonyl-N-5 (succinimidoyloxycarbonylmethyl) acridin-9-carboxylsyreamid (12) . (12) levaterniseres med trimethyloxoniumtetrafluorborat, som det er beskrevet ved (6), til N-benzensulfonyl-N-(succinimidoyloxycarbonylmethyl) -10-methylacridinium-9-carboxyl-syreamidtetrafluorborat (13).Compound (11) is reacted with N-hydroxysuccinimide analogously to the preparation of (5) to N-benzenesulfonyl-N-5 (succinimidoyloxycarbonylmethyl) acridine-9-carboxylic acid amide (12). (12) levaternized with trimethyloxonium tetrafluoroborate, as described by (6), for N-benzenesulfonyl-N- (succinimidoyloxycarbonylmethyl) -10-methylacridinium-9-carboxylic acid amide tetrafluoroborate (13).
10 EKsempel 4 N-Phenvl-N- (4-benzvloxvcarbonylbenzensulfonvl^ acridin-9-carb-pxylgyrewd (14).Example 4 N-Phenyl-N- (4-benzyloxycarbonylbenzenesulfonyl) -acridine-9-carb-pyxylglycide (14).
Til 11 g 4-(N-phenylsulfamido)benzoesyrebenzylester 15 i 300 ml methylenchlorid sættes 360 mg 4-(dimethylamino)-pyridin og 16,6 ml triethylamin, efter 10 minutter sættes hertil 8,34 g acridin-9-carboxylsyrechlorid-hydrochlorid (3), og der opvarmes i 16 timer til tilbagesvaling. Den afkølede opløsning sammenrøres kortvarigt med 2N NaOH, hvor-20 efter den fraskilte organiske fase vaskes med vand, og der tørres over Na2SC>4, og der koncentreres. Remanensen omkrystalliseres fra toluen/heptan.To 11 g of 4- (N-phenylsulfamido) benzoic acid benzyl ester 15 in 300 ml of methylene chloride are added 360 mg of 4- (dimethylamino) pyridine and 16.6 ml of triethylamine, after 10 minutes 8.34 g of acridine-9-carboxylic acid chloride hydrochloride ( 3) and heated for 16 hours to reflux. The cooled solution is briefly stirred with 2N NaOH, after which the separated organic phase is washed with water and dried over Na 2 SO 4> and concentrated. The residue is recrystallized from toluene / heptane.
Udbytte: 70%, smp.: 161-163*C.Yield: 70%, mp: 161-163 ° C.
NMR (DMSO, 100 MHz): δ - 5,5 ppm (s,2H), δ = 6,8-8,6 ppm 25 (m,22H).NMR (DMSO, 100 MHz): δ - 5.5 ppm (s, 2H), δ = 6.8-8.6 ppm (m, 22H).
N-Phenvl-N-(4-carboxvbenzensulfonyHacridin-9-carboxvlsvre-amid-hvdrobromid (15).N-Phenyl-N- (4-carboxybenzenesulfonylacridine-9-carboxylic acid amide hydrobromide (15)).
8,58 g N-phenyl-N- (4-benzyloxycarbonylbenzensulfonyl) -30 acridin-9-carboxylsyreamid (14) opvarmes i 30 ml 33%*s HBr i iseddikesyre til 60*C i 2 timer, efter afkøling sættes hertil 60 ml diisopropylether, hvorefter udfældningen fra-filtreres ved sugning, og den tørres under vakuum.8.58 g of N-phenyl-N- (4-benzyloxycarbonylbenzenesulfonyl)-acridine-9-carboxylic acid amide (14) are heated in 30 ml of 33% HBr in glacial acetic acid to 60 ° C for 2 hours, after cooling to 60 ml. diisopropyl ether, after which the precipitate is filtered off by suction and dried under vacuum.
Udbytte: 95%, smp.: 255‘C.Yield: 95%, mp: 255 ° C.
35 NMR(DMSO, 100 MHz): 5 - 6,8-9 ppm (m).NMR (DMSO, 100 MHz): δ - 6.8-9 ppm (m).
18 DK 171437 B1 N-Phenvl-N-14-succinimidovloxycarbonvlbenzensulfonvll acridin- 9-carboxvlsvreamid (16).18 DK 171437 B1 N-Phenyl-N-14-succinimidovloxycarbonylbenzenesulfonyl acridine-9-carboxylic acid amide (16).
Til 5,63 g N-phenyl-N-(4-carboxybenzensulfonyl)acri-din-9-carboxylsyreamid-hydrobromid (15) i 250 ml tetrahydro-5 furan sættes 2,8 ml triethylamin, hvorefter der afkøles til -15*C, og der sættes hertil 0,96 ml chlormyresyreethylester.To 5.63 g of N-phenyl-N- (4-carboxybenzenesulfonyl) acridine-9-carboxylic acid amide hydrobromide (15) in 250 ml of tetrahydrofuran is added 2.8 ml of triethylamine and then cooled to -15 ° C. and to this is added 0.96 ml of chloroformic acid ethyl ester.
Der efteromrøres i 20 minutter, hvorefter der tilsættes 1,15 g N-hydroxysuccinimid, og der omrøres i 3 timer ved-15"C, hvorefter man tør op til stuetemperatur, og der efter-10 omrøres natten over. Udfældningen frafiltreres ved sugning, hvorefter filtratet koncentreres, og remanensen optages med methylenchlorid, hvorefter den resulterende opløsning vaskes med vand, en NaHC03-opløsning og vand, hvorefter der tørres over Na2S04. Den organiske fase koncentreres, og remanensen 15 omkrystalliseres fra toluen.Stir for 20 minutes, then add 1.15 g of N-hydroxysuccinimide and stir for 3 hours at -15 ° C, then dry to room temperature and stir overnight 10. The precipitate is filtered off by suction, then the filtrate is concentrated and the residue is taken up with methylene chloride, then the resulting solution is washed with water, a NaHCO 3 solution and water, then dried over Na 2 SO 4. The organic phase is concentrated and the residue is recrystallized from toluene.
Udbytte: 50%, smp.: 226eC (sønderdeling).Yield: 50%, mp: 226 ° C (dec.).
NMR(DMS0, 100 MHz): δ = 2,95 ppm (s,4H), δ 6,8-8,7 ppm (m, 17H) .NMR (DMSO, 100 MHz): δ = 2.95 ppm (s, 4H), δ 6.8-8.7 ppm (m, 17H).
20 N-Phenvl-N- (4-succinimidovloxvcarbonvlbenzensulfonvll -10-methvlacridinium-9-carboxylsvreamid-fluorsulfonat (17).N-Phenyl-N- (4-succinimidovloxycarbonylbenzenesulfonyl-10-methylacridinium-9-carboxylic acid amide fluorosulfonate (17)).
1,16 g N-phenyl-N-(4-succinimidoyloxycarbonylbenzen-sulfonyl)acridin-9-carboxylsyreamid (16) omrøres i 60 ml 1,2-dichlorethan med 0,3 ml methyl fluorsul fonat i 24 timer 25 ved stuetemperatur, hvorefter den udfældende udfældning frafiltreres ved sugning, og den tørres under vakuum.1.16 g of N-phenyl-N- (4-succinimidoyloxycarbonylbenzenesulfonyl) acridine-9-carboxylic acid amide (16) are stirred in 60 ml of 1,2-dichloroethane with 0.3 ml of methyl fluorosulfonate for 24 hours at room temperature, after which the precipitated precipitate is filtered off by suction and dried under vacuum.
Udbytte: 65%.Yield: 65%.
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), 30 NMR(DMSO, 100 MHz): δ - 2,95 ppm (s,4H), 6 = 4,75 ppm (s,br, 3H), 6 = 7,0-9,0 (m,17H).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), 30 NMR (DMSO, 100 MHz): δ - 2.95 ppm (s, 4H), δ = 4.75 ppm (s, br, 3H), δ = 7.0 -9.0 (m, 17H).
Massespektrum: m/e - 594 : M+ (kation).Mass spectrum: m / e - 594: M + (cation).
Eksempel 5 35 Fremstillingen af N-(4-methoxyphenyl)-N-(4-succinimi- doyloxycarbonylbenzensulfonyl) -10-methylacridinium-9-carb- 19 DK 171437 B1 oxylsyreamid-fluorsulfonat (21) ud fra 4-[N-(4'-methoxyphe-nyl)sulfamido]-benzoesyrebenzylester og acridin-9-carboxyl-syrechlorid-hydrochlorid (3) sker analogt med syntesen af (17) (se eksempel 4). Udbytterne af de enkelte syntesetrin 5 samt den spektroskopiske karakterisering er anført i det efterfølgende N-f 4-Methoxvphenvl)-N-(4-benzvloxvcarbonvl-benzensulfonvl)-acridin-9-carboxvlsvreamid (18).Example 5 The preparation of N- (4-methoxyphenyl) -N- (4-succinimidoyloxycarbonylbenzenesulfonyl) -10-methylacridinium-9-carb-oxylic acid amide fluorosulfonate (21) from 4- [N- (4) (methoxyphenyl) sulfamido] benzoic acid benzyl ester and acridine-9-carboxylic acid chloride hydrochloride (3) occur analogously to the synthesis of (17) (see Example 4). The yields of the individual synthesis steps 5 as well as the spectroscopic characterization are given in the following N-f 4-Methoxyphenyl) -N- (4-benzyloxycarbonyl-benzenesulfonyl) -acridine-9-carboxylic acid amide (18).
10 Udbytte: 70%, Smp.: 182-183*C.Yield: 70%, mp: 182-183 ° C.
NMR(DMSO, 100 MHz): δ - 3,5 ppm (s,3H), δ = 5,5 ppm (s,2H), δ 6,35-6,63 ppm (d,br,2H), δ = 7,05-7,2 ppm (d,br,2H), δ - 7,35-8,5 ppm (m,17H).NMR (DMSO, 100 MHz): δ - 3.5 ppm (s, 3H), δ = 5.5 ppm (s, 2H), δ 6.35-6.63 ppm (d, br, 2H), δ = 7.05-7.2 ppm (d, br, 2H), δ - 7.35-8.5 ppm (m, 17H).
15 N- (4-Methoxvphenvli -N- (4-carboxvbenzensulfonvH acridin-9-carboxvlsvrearoid-hvdrobromid (19).N- (4-Methoxyphenyl) -N- (4-carboxybenzenesulfonyl) acridine-9-carboxylic acid thyroid hydrobromide (19).
Udbytte: 95%, Smp.: 273’C (sønderdeling).Yield: 95%, Mp: 273 ° C (dec.).
NMR(DMSO, 100 MHz): 5 = 3,5 ppm (s,3H), δ = 6,4-6,6 ppm (d,br,2H), δ = 7,05-7,2 ppm (d,br,2H), δ = 7,7-8,5 ppm 20 (m,12H).NMR (DMSO, 100 MHz): δ = 3.5 ppm (s, 3H), δ = 6.4-6.6 ppm (d, br, 2H), δ = 7.05-7.2 ppm (d , br, 2H), δ = 7.7-8.5 ppm (m, 12H).
N-(4-Methoxvphenvl)-N-(4-succinimidovloxvcarbonvlbenzensul-fonvl)acridin-9-carboxvlsvreamid (20).N- (4-Methoxyphenyl) -N- (4-succinimidovloxycarbonylbenzenesulfonyl) acridine-9-carboxylic acid amide (20).
Udbytte: 50%, Smp.: 232-234*C.Yield: 50%, mp: 232-234 ° C.
25 NMR(DMSO,100 MHz): δ = 2,95 ppm (s,4H), 6 = 3,5 ppm (s,3H), δ = 6,4-6,6 ppm (d,br,2H), δ - 7,05-7,25 ppm (d,br,2H), δ - 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.NMR (DMSO, 100 MHz): δ = 2.95 ppm (s, 4H), 6 = 3.5 ppm (s, 3H), δ = 6.4-6.6 ppm (d, br, 2H) , δ - 7.05-7.25 ppm (d, br, 2H), δ - 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.
30 N-(4-Methoxvphenvl)-N-(4-succinimidoYloxvcarbonvlbenzensul-fonvl)-10-methvlacridinium-9-carboxvlsvreamid-fluorsulfonat (21).N- (4-Methoxyphenyl) -N- (4-succinimidoyloxycarbonylbenzenesulfonyl) -10-methylacridinium-9-carboxylic acid amide fluorosulfonate (21).
Substansen udfælder ikke ved reaktionen, men den udvindes ved koncentrering af opløsningen og sammenrøring 35 af remanensen med diisopropylether.The substance does not precipitate by the reaction, but it is recovered by concentrating the solution and stirring the residue with diisopropyl ether.
20 DK 171437 B120 DK 171437 B1
Udbytte: 80%.Yield: 80%.
NMR(DMSO, 100 MHz): δ = 2,95 ppm (s,4H), δ - 3,5 ppm (s,3H), δ - 4,8 ppm (s,br,3H), δ - 6,45-6,7 ppm (d,br,2H), δ - 7,2--7,4 ppm (d,br,2H), δ = 7,7-9 ppm (m,12H).NMR (DMSO, 100 MHz): δ = 2.95 ppm (s, 4H), δ - 3.5 ppm (s, 3H), δ - 4.8 ppm (s, br, 3H), δ - 6, 45-6.7 ppm (d, br, 2H), δ - 7.2--7.4 ppm (d, br, 2H), δ = 7.7-9 ppm (m, 12H).
5 Massespektrum: m/e = 624 M+ (kation).Mass spectrum: m / e = 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).
10 Eksempel 6Example 6
Fremstillingen af N-(4-methoxyphenyl)-N-(3-succin-imidoy 1 oxycarbonyl-benzensulfonyl) -10-methylacridinium-9-carboxylsyreamid-fluorsulfonat(25) ud fra 3-[N-(4'-methoxy-phenyl)sulfamido]-benzoesyrebenzylester og acridin-9-carb-15 oxylsyrechlorid-hydrochlorid (3) udføres analogt med syntesen af (17) (se eksempel 4). Udbytterne af de enkelte syntesetrin samt den spektroskopiske karakterisering er anført i det efterfølgende.Preparation of N- (4-methoxyphenyl) -N- (3-succinimidoxycarbonylbenzenesulfonyl) -10-methylacridinium-9-carboxylic acid amide fluorosulfonate (25) from 3- [N- (4'-methoxy-phenyl) ) sulfamido] benzoic acid benzyl ester and acridine-9-carboxylic acid chloride hydrochloride (3) are carried out analogously to the synthesis of (17) (see Example 4). The yields of the individual synthesis steps as well as the spectroscopic characterization are given below.
20 N- (4 -Methoxvphenvl) -N (3 -benzvloxvcarbonvlbenzensulfonvl) -acridin-9-carboxvlsvreamid (22).N- (4-Methoxyphenyl) -N (3-benzyloxycarbonylbenzenesulfonyl) -acridine-9-carboxylic acid amide (22).
Udbytte: 70%, Smp.: 168-170°C.Yield: 70%, mp: 168-170 ° C.
NMR(DMS0, 100 MHz): δ - 3,5 ppm (s,3H), δ = 5,45 ppm (s,2H), 6=6,5 ppm (s,br,2H), δ = 7,1 ppm (br,2H), δ = 7,3-8,8 ppm 25 (m,17H).NMR (DMSO, 100 MHz): δ - 3.5 ppm (s, 3H), δ = 5.45 ppm (s, 2H), δ = 6.5 ppm (s, br, 2H), δ = 7, 1 ppm (br, 2H), δ = 7.3-8.8 ppm (m, 17H).
N- (4-Methoxvphenvl) -N-(3-carboxvbenzensulfonyllacridin-9-carboxvlsvreamid-hvdrobromid (23).N- (4-Methoxyphenyl) -N- (3-carboxybenzenesulfonylacridine-9-carboxylic acid amide hydrobromide (23).
Udbytte: 90%, Smp.: 264eC.Yield: 90%, mp: 264 ° C.
30 NMR(DMSO, 100 MHz): δ = 3,5 ppm (s,3H), δ = 6,4-6,6 ppm (d,br,2H), δ = 7,0-7,2 ppm (d,br,2H), δ = 7,6-8,8 ppm (m,12H).NMR (DMSO, 100 MHz): δ = 3.5 ppm (s, 3H), δ = 6.4-6.6 ppm (d, br, 2H), δ = 7.0-7.2 ppm ( d, br, 2H), δ = 7.6-8.8 ppm (m, 12H).
21 DK 171437 B1 N-(4-Methoxvphenvl)-N-f3-succinimidovloxvcarbonvlbenzensul-fonvl)acridin-9-carboxvlsvreamid f24).B1 N- (4-Methoxyphenyl) -N-f3-succinimidovloxycarbonylbenzenesulfonyl) acridine-9-carboxylic acid amide (24).
Udbytte: 50%, Smp.: 223-225‘C.Yield: 50%, mp: 223-225 ° C.
NMR (DMSO, 100 MHz), δ = 2,95 ppm (s,4H), δ - 3,5 ppm (s,3H), 5 5 = 6,4-6,6 ppm (d,br,2H), δ = 7,0-7,2 ppm (d,br,2H), δ = 7,4-8,9 ppm (m,12H).NMR (DMSO, 100 MHz), δ = 2.95 ppm (s, 4H), δ - 3.5 ppm (s, 3H), δ = 6.4-6.6 ppm (d, br, 2H) , δ = 7.0-7.2 ppm (d, br, 2H), δ = 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).
10 N-(4-Methoxvphenvll-N-f3-succinimidovloxvcarbonvlbenzensul-fonvl)-10-methvlacridinium-9-carboxylsvreamidfluorsulfonat (25).10 N- (4-Methoxyphenyl) N- (3-succinimidovloxycarbonylbenzenesulfonyl) -10-methylacridinium-9-carboxylic acid amide fluorosulfonate (25).
Udbytte: 90%.Yield: 90%.
15 NMR (DMSO, 100 MHz): δ = 2,95 ppm (s,4H), δ = 3,55 ppm (s,3H), δ = 4,8 ppm (s,br,3H), 5 - 6,45-6,7 (d,br,2H), 5 = 7,05-7,3 ppm (d,br,2H), δ = 7,5-8,9 ppm (m,12H).NMR (DMSO, 100 MHz): δ = 2.95 ppm (s, 4H), δ = 3.55 ppm (s, 3H), δ = 4.8 ppm (s, br, 3H), 5-6 , 45-6.7 (d, br, 2H), δ = 7.05-7.3 ppm (d, br, 2H), δ = 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) 20 Masse: m/e = 624 M+ (kation).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) Mass: m / e = 624 M + (cation).
Eksempel 7Example 7
Tracer-fremstillina til a-fetoprotein-kemiluminescensim-munanalvse.Tracer preparation for α-fetoprotein chemiluminescence immunoassay.
25 100 μΐ antistof (1 mg/ml), 11,5 μΐ af den ifølge eksempel 1 fremstillede acridiniumthioester (forbindelse (6)) (1 mg/ml i DMSO og 600 μΐ konjugationspuffer (0,01 M phosp-hat, pH 8,0) inkuberes i 15 minutter. Herefter sættes hertil 200 μΐ lysin (10 mg/ml), og der inkuberes i yderligere 15 30 minutter. Denne blanding overføres til en PD 10-søjle ("Sephadex® G 25 Medium"), 0,1 M phosphat, pH 6,3 som løbe-middel). 10 dråber pr. fraktion samles. De enkelte fraktioner undersøges efter en egnet fortynding for deres kemilumine-scensaktivitet (350 μΐ oxidationsmiddel: 0,1% H202 i 0,1 N 35 NaOH). Tracerfraktionen (1. aktivitetstop) samles og opbevares ved 4°C.25 100 μΐ antibody (1 mg / ml), 11.5 μΐ of the acridinium thioester prepared according to Example 1 (compound (6)) (1 mg / ml in DMSO and 600 μΐ conjugation buffer (0.01 M phosp hat, pH 8 , Incubate for 15 minutes, then add 200 μΐ of lysine (10 mg / ml) and incubate for a further 15 30 minutes. This mixture is transferred to a PD 10 column ("Sephadex® G 25 Medium"), 0 , 1 M phosphate, pH 6.3 as a running agent). 10 drops per fraction is collected. The individual fractions are assayed for a suitable dilution for their chemiluminescence activity (350 μΐ oxidant: 0.1% H 2 O 2 in 0.1 N 35 NaOH). The tracer fraction (1st activity peak) is collected and stored at 4 ° C.
22 DK 171437 B122 DK 171437 B1
Eksempel 8Example 8
Gennemførelse af a-fetoprotein-kemiluminescensimmunanalvsen.Implementation of the α-fetoprotein chemiluminescence immunoassay.
50 μΐ standard/prøve og 150 μΐ puffer (phosphat; 0,1 M pH 6,3, 1% "Tween® 20", 0,1% kvægserumalbumin, 0,1 M NaCl, 5 0,01% NaN3) rystes i 15 minutter i med monoklonalt anti-AFP- -antistof belagte analyserør. Herefter vaskes der to gange med 1 ml puffer. 200 μΐ tracer tilsættes i en egnet fortynding, og der omrystes i 15 minutter. Der vaskes igen to gange med 1 ml puffer. Kemiluminescensmålingen startes med 10 350 μΐ oxidationsmiddel (0,1% H2O2 i 0,1 N NaOH, måletid = 2 sekunder).Shake 50 μΐ standard / sample and 150 μΐ buffer (phosphate; 0.1 M pH 6.3, 1% "Tween® 20", 0.1% bovine serum albumin, 0.1 M NaCl, 0.01% NaN 3) 15 minutes in assay tube with monoclonal anti-AFP antibody. Then wash twice with 1 ml of buffer. Add 200 μΐ tracer in a suitable dilution and shake for 15 minutes. Wash again twice with 1 ml of buffer. The chemiluminescence measurement is started with 10 350 μΐ of oxidant (0.1% H2O2 in 0.1 N NaOH, measurement time = 2 seconds).
Fig. 3 viser det typiske forløb af en standardkurve af en immuno-kemiluminemetrisk analyse (IKMA) for e-fetopro-teinet (AFP).FIG. Figure 3 shows the typical course of a standard curve of an immunochemilumuminometric assay (IKMA) for the e-feto protein (AFP).
Claims (7)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DK199200684A DK173875B1 (en) | 1986-08-22 | 1992-05-25 | New chemiluminescent 9-carboxy-acridinium cpds. - and their conjugates with biologically interesting substances, used in luminescence immunoassay |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE3628573 | 1986-08-22 | ||
| DE3628573A DE3628573C2 (en) | 1986-08-22 | 1986-08-22 | Chemiluminescent acridine derivatives, processes for their preparation and their use in luminescence immunoassays |
Publications (3)
| Publication Number | Publication Date |
|---|---|
| DK437187D0 DK437187D0 (en) | 1987-08-21 |
| DK437187A DK437187A (en) | 1988-02-23 |
| DK171437B1 true DK171437B1 (en) | 1996-10-28 |
Family
ID=6307971
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| DK437187A DK171437B1 (en) | 1986-08-22 | 1987-08-21 | Chemiluminescent acridine derivatives and process for their preparation |
Country Status (12)
| Country | Link |
|---|---|
| EP (2) | EP0647628B1 (en) |
| JP (2) | JPH0699401B2 (en) |
| AT (2) | ATE132490T1 (en) |
| CA (1) | CA1341402C (en) |
| DE (4) | DE3645292C2 (en) |
| DK (1) | DK171437B1 (en) |
| ES (2) | ES2083949T3 (en) |
| FI (1) | FI90542C (en) |
| GR (1) | GR3019470T3 (en) |
| IE (1) | IE74678B1 (en) |
| NO (1) | NO171725C (en) |
| PT (1) | PT85563B (en) |
Families Citing this family (28)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0082636B2 (en) * | 1981-12-11 | 2006-10-18 | The Welsh National School of Medicine | Luminescent labelling materials and procedures |
| US4745181A (en) * | 1986-10-06 | 1988-05-17 | Ciba Corning Diagnostics Corp. | Polysubstituted aryl acridinium esters |
| ATE111083T1 (en) * | 1986-10-22 | 1994-09-15 | Abbott Lab | CHEMILUMINESCENT ACRIDINIUM AND PHENANTRIDINIUM SALTS. |
| NZ227506A (en) * | 1987-12-31 | 1992-06-25 | London Diagnostics Inc | Specific binding assays using chemiluminescent compounds |
| FR2625565A1 (en) * | 1987-12-31 | 1989-07-07 | London Diagnostics Inc | Improved chemiluminescent esters, thioesters and amides, and analyses employing them |
| DE3844954C2 (en) * | 1988-02-20 | 1998-07-16 | Hoechst Ag | Special chemiluminescent acridine derivatives and their use in luminescent immunoassays |
| US6002007A (en) * | 1988-02-20 | 1999-12-14 | Dade Behring Marburg Gmbh | Special chemiluminescent acridine derivatives and the use thereof in luminescence immunoassays |
| US4950613A (en) * | 1988-02-26 | 1990-08-21 | Gen-Probe Incorporated | Proteted chemiluminescent labels |
| US5227489A (en) * | 1988-08-01 | 1993-07-13 | Ciba Corning Diagnostics Corp. | Stable hydrophilic acridinium esters suitable for liposome encapsulation |
| AU634716B2 (en) * | 1988-08-01 | 1993-03-04 | Ciba Corning Diagnostics Corp. | Method for detection of an analyte using acridinium esters and liposomes |
| US5241070A (en) * | 1988-09-26 | 1993-08-31 | Ciba Corning Diagnostics Corp. | Nucleophilic polysubstituted aryl acridinium esters and uses thereof |
| US5663074A (en) * | 1988-09-26 | 1997-09-02 | Chiron Diagnostics Corporation | Nucleophilic polysubstituted aryl acridinium ester conjugates and syntheses thereof |
| WO1992009580A1 (en) * | 1990-11-21 | 1992-06-11 | Beckman Instruments, Inc. | Chemiluminescent compounds |
| DE4041080A1 (en) | 1990-12-21 | 1992-06-25 | Behringwerke Ag | METHOD FOR DETERMINING AN ANALYT |
| DE4228839A1 (en) | 1992-08-29 | 1994-03-03 | Behringwerke Ag | Methods for the detection and determination of mediators |
| US5491072A (en) * | 1993-05-17 | 1996-02-13 | Lumigen, Inc. | N-alkylacridan carboxyl derivatives useful for chemiluminescent detection |
| BE1008216A4 (en) * | 1994-01-25 | 1996-02-20 | Biocode Sa | HETEROCYCLIC CHEMOLUMINESCENT DERIVATIVES. |
| AU689920B2 (en) * | 1994-06-24 | 1998-04-09 | Dade Behring Marburg Gmbh | Method of stabilizing molecules, or parts of molecules, which are sensitive to hydrolysis |
| AU2775395A (en) * | 1994-07-15 | 1996-02-16 | Abbott Laboratories | Chemiluminescent signal enhancement |
| WO1996027785A1 (en) * | 1995-03-03 | 1996-09-12 | Abbott Laboratories | Article and method for conducting chemiluminescent reaction |
| US5731148A (en) * | 1995-06-07 | 1998-03-24 | Gen-Probe Incorporated | Adduct protection assay |
| EP0761652A1 (en) * | 1995-08-01 | 1997-03-12 | Mochida Pharmaceutical Co., Ltd. | Acridinium compound having a plurality of luminescent groups and binding groups, and conjugate thereof |
| WO1997033884A1 (en) * | 1996-03-15 | 1997-09-18 | Ss Pharmaceutical Co., Ltd. | Reagents for labeling sh groups, process for the preparation of them, and method for labeling with them |
| DK0915851T3 (en) * | 1996-07-16 | 2002-04-29 | Nederlanden Staat | Dibenzodihydropyridine carboxyl esters and their use in chemiluminescence assay methods |
| BE1011206A4 (en) * | 1997-06-11 | 1999-06-01 | Biocode Sa | HETEROCYCLIC DERIVATIVES chemiluminescent. |
| EP1496050A1 (en) | 2003-07-08 | 2005-01-12 | Roche Diagnostics GmbH | Novel chemiluminescent compounds and their use |
| EP1658495A1 (en) | 2003-07-30 | 2006-05-24 | Roche Diagnostics GmbH | Novel chemiluminescent compounds and their use |
| WO2005015215A1 (en) | 2003-07-30 | 2005-02-17 | Roche Diagnostics Gmbh | Novel chemiluminescent compounds and their use |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB1461877A (en) * | 1974-02-12 | 1977-01-19 | Wellcome Found | Assay method utilizing chemiluminescence |
| GB2008247B (en) * | 1977-11-17 | 1982-12-15 | Welsh Nat School Med | Detecting or quantifying substances using labelling techniques |
| EP0082636B2 (en) * | 1981-12-11 | 2006-10-18 | The Welsh National School of Medicine | Luminescent labelling materials and procedures |
-
1986
- 1986-08-22 DE DE3645292A patent/DE3645292C2/en not_active Expired - Lifetime
- 1986-08-22 DE DE3628573A patent/DE3628573C2/en not_active Expired - Lifetime
-
1987
- 1987-08-19 ES ES87112022T patent/ES2083949T3/en not_active Expired - Lifetime
- 1987-08-19 AT AT87112022T patent/ATE132490T1/en not_active IP Right Cessation
- 1987-08-19 EP EP94119395A patent/EP0647628B1/en not_active Expired - Lifetime
- 1987-08-19 DE DE3751659T patent/DE3751659D1/en not_active Expired - Lifetime
- 1987-08-19 ES ES94119395T patent/ES2182835T3/en not_active Expired - Lifetime
- 1987-08-19 AT AT94119395T patent/ATE223903T1/en not_active IP Right Cessation
- 1987-08-19 EP EP87112022A patent/EP0257541B1/en not_active Expired - Lifetime
- 1987-08-19 DE DE3752357T patent/DE3752357D1/en not_active Expired - Lifetime
- 1987-08-20 PT PT85563A patent/PT85563B/en unknown
- 1987-08-20 FI FI873609A patent/FI90542C/en not_active IP Right Cessation
- 1987-08-20 NO NO873520A patent/NO171725C/en not_active IP Right Cessation
- 1987-08-21 CA CA000545036A patent/CA1341402C/en not_active Expired - Lifetime
- 1987-08-21 IE IE224587A patent/IE74678B1/en not_active IP Right Cessation
- 1987-08-21 DK DK437187A patent/DK171437B1/en not_active IP Right Cessation
- 1987-08-21 JP JP62206622A patent/JPH0699401B2/en not_active Expired - Lifetime
-
1994
- 1994-06-14 JP JP6131617A patent/JP2766780B2/en not_active Expired - Lifetime
-
1996
- 1996-03-29 GR GR960400849T patent/GR3019470T3/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| NO873520L (en) | 1988-02-23 |
| IE960911L (en) | 1988-02-22 |
| DE3752357D1 (en) | 2002-10-17 |
| DK437187D0 (en) | 1987-08-21 |
| FI90542C (en) | 1994-02-25 |
| DE3628573C2 (en) | 1994-10-13 |
| PT85563B (en) | 1990-05-31 |
| FI873609A (en) | 1988-02-23 |
| JPH0699401B2 (en) | 1994-12-07 |
| PT85563A (en) | 1987-09-01 |
| EP0647628A1 (en) | 1995-04-12 |
| NO171725C (en) | 1993-04-28 |
| DK437187A (en) | 1988-02-23 |
| EP0257541A2 (en) | 1988-03-02 |
| DE3751659D1 (en) | 1996-02-15 |
| NO873520D0 (en) | 1987-08-20 |
| FI90542B (en) | 1993-11-15 |
| GR3019470T3 (en) | 1996-06-30 |
| ATE132490T1 (en) | 1996-01-15 |
| DE3628573A1 (en) | 1988-02-25 |
| ATE223903T1 (en) | 2002-09-15 |
| ES2182835T3 (en) | 2003-03-16 |
| FI873609A0 (en) | 1987-08-20 |
| JPH07179428A (en) | 1995-07-18 |
| JPS6357572A (en) | 1988-03-12 |
| ES2083949T3 (en) | 1996-05-01 |
| NO171725B (en) | 1993-01-18 |
| EP0647628B1 (en) | 2002-09-11 |
| EP0257541A3 (en) | 1988-11-30 |
| DE3645292C2 (en) | 1997-12-11 |
| CA1341402C (en) | 2002-11-26 |
| EP0257541B1 (en) | 1996-01-03 |
| IE872245L (en) | 1988-02-22 |
| JP2766780B2 (en) | 1998-06-18 |
| IE74678B1 (en) | 1997-07-30 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| B1 | Patent granted (law 1993) | ||
| PUP | Patent expired |