IE83772B1 - Acridinium derivatives and their use in luminescence immunoassays - Google Patents

Abstract

ABSTRACT The invention relates to novel acridinium derivatives of the formula I: 1 l fi / in which R] is hydrogen, an alkyl, alkenyl or alkynyl radical with (1) to (10) carbon atoms or a benzyl or aryl group, R2 and R3 are hydrogen, an alkyl group with (1) to (4) carbon atoms, a substituted or unsubstituted amino group, a carboxcyl, C1-C4 alkoxy, cyano or nitro group or halogen, R4 is a radical in which a sulfonamide group is bonded via the nitrogen‘ directly to the carbonyl group and A8 is an anion which does no impair the chem_iluminescence; and their use in luminescence immunoassays. These compounds can advantageously be employed in the form of biologically active conjugates, for example in immunoluminescence assays as so—ca1led "labels".

Description

ACRIDINIUM DERIVATIVES AND THEIR USE IN LUMINESCENCE IMMUNOASSAYS DADE BEHRING MARBURG GMBH The invention relates to chemiluminescent acridine derivatives, to processes for preparing them and to their use in luminescence immunoassays.

Luminescent compounds already have widely varying uses.

They are employed as indicators in bioassays, enzyme immunoassa s and luminescence immunoassays (cf. W.P.

Y Collins "Alternative Immunoassays", publishers John ), but are also used in nucleic acid hybridization assays (cf. J.A. Matthews et al. 151, 205-209, l985).

Chemiluminescent moreover employed Wiley & Sons Ltd , Chichester, "Analytical Biochemistry", compounds are in in post column detectors in flow flow injection analysis, liquid chromatography, in research and in artificial light sources.

In chemiluminescence immunoassays, two structural types of chemiluminescent labeling substances in particular have achieved relatively great importance. These are on the one hand the in H.R. isoluminol derivatives Schroeder et al., "Methods Enzymology", Academic Press Inc., New York, Vol. LVII, 1978, 424 et seq. and in British Patents 2,008,247 and 2,041,920, German Patents 2,618,419 and 2,618,511 and European Patent Application 135,071. A review of the luminol and described in practical use of isoluminol compounds as luminescence indicators is to be found in W.G. Wood, J. Clin.Chem.

Clin. Biochem. 22, 1984 905-918.

On the other hand, acridinium ester compounds have also Such acridinium esters are known from US Patent 3,352,791, British Patents 1,316,363 Patent Application 82,636. as labeling substances in immunoassays is described in Weeks et al., Clin. Cheat 29/8 (1983), l474—1479. The use of phenanthridinium esters as a labeling substance found use as chemiluminescent labeling substances. and 1,461,877 and European The use of acridinium esters in luminescence immunoassays is also already known from European Patent Application 170,415. acridinium esters can be H202 mechanism of The chemiluminescence of alkaline solution. A started by addition of convincing explanation of chemiluminescence has been given by F.

Acc.Chem. Res. 9, 201, 1976. Thus, leaving group is evidently decisive both for the light Mccapra, the nature of the quantum yield and for the hydrolytic stability.

Compared with the luminol and isoluminol compounds, the acridinium esters already known to date have the advan— tage of a higher light quantum yield, which is also not impaired by proteins bound to the indicator (cf. Weeks et al., Clin. Chem. 29/8 (1983), 1474-1479).

Although the acridinium phenyl esters known from European Patent Application 82,636 are distinguished by a high detection sensitivity when the chemiluminescence is stimulated by mild which oxidizing with agents, they have disadvantages interfere their use in practice. In particular, the phenyl ester bond is very unstable in aqueous systems, even already at room temperature. Moreover, under the oxidation conditions stated therein, the acridinium phenyl esters exhibit that is to say to after about 10 photoemission which largely subsides, the than 95%, In comparison with this, extent of more only seconds. other non—isotopic assay methods have far shorter measurement times and thereby allow a higher sample throughput.

The object of the present invention. was therefore to provide new acridinium derivatives which exhibit faster reaction kinetics with a high light quantum yield and thereby allow short measurement times for a luminescence immunoassay.

It has now been found that these requirements are met by which acridinium derivative of the formula I luminescent compounds comprise an in which R; is hydrogen, an alkyl, alkenyl or alkynyl radical with 1 to 10 carbon atoms or a benzyl, phenyl or and R3 with 1 to 4 carbon atoms, naphthyl group, R2 are hydrogen, an alkyl group an amino group or a mono— or di-C1-C4 alkylamino group, where the alkyl radicals can be monosubstituted by hydroxyl, and morpholine, a carboxyl, C1-C4 alkoxy, cyano or nitro group or halogen, R4 is a radical in which a substituted sulfonamide group is bonded via the nitrogen directly to the group, and A‘ carbonyl is an anion which does not impair the chemiluminescence, excluding acridinium compounds of the formula I in which R1, R2 and R3 are each as defined above and R4 is a substituted sulfonamide group of the formula V or VI X“R5 Re -—N: (V) —-Ni (V0 023 :R5 025 --X'—R5 where X is a branched or unbranched C1—C5—alkylene group or is a phenylene or naphthylene group or is quinole, indole, pyrrole or pyridine, R5 is a reactive group which under mild conditions is capable of bonding selectively with amino, carboxyl, thiol or other functional groups in substances of biological interest, and R5 in the formula V is an alkyl or alkenyl radical having 1 to 10 carbon atoms, a C1—C4—alkyl—mono- substituted or —disubstituted amino group or is a morpholino, benzyl, phenyl or naphthyl group which is optionally substituted by hydroxyl, amino, alkoxy of 1 to 4 carbon atoms or phenoxy or naphthoxy groups, the hydrogen, and R5 substituents where the bonded to a protein, a formula VI, as well as additionally be in the mentioned, may acridinium derivative of formula I is directly or via a bridge molecule polypeptide or another substance of biological interest to form a stable immunologically active conjugate.

Substances of biological interest are to be understood as being, above all, antigens. This term also includes hormones, steroids, drugs, drug metabolites toxins, alkaloids and also antibodies.

The anion which does not impair the chemiluminescence halide, alkylsulfonate or can be a alkylsulfate, tetrafluoroborate, perchlorate, halosulfonate, arylsulfonate anion. Any other anion can also be used, as long as it does not extinguish or weaken the chemiluminescence.

By aryl there are to be understood aromatic hydrocarbons, in particular phenyl and naphthyl. By aromatic groups containing heteroatoms there are to be understood aromatic hydrocarbons which contain a nitrogen or oxygen atom, in particular quinoline, indole, pyrrole and pyridine.

Halogens are to be understood. as fluorine, chlorine, bromine and iodine.

The substituted amino groups are preferably C1—C4—alkyl— or C14;-dialkylamines, it being possible for the alkyl radicals in turn to be monosubstituted, for example by hydroxyl. The substituted amines can also be a nmrpho— line radical. The alkoxy radicals mentioned for R2 and R3 are preferably those with 1-4 carbon atoms in the alkyl part.

The branched. or unbranched aliphatic groups mentioned for X are preferably C1-C5—alkylene groups.

Acridinium derivatives in which X is a methylene, ethylene, propylene or an ortho—, meta- or para- phenylene group are in general chosen. These groups can also carry hydrophilic substituents containing heteroatoms in order to improve the water—solubility of the acridinium derivatives.

The substituent R5 is of particular importance for the possible uses of the acridinium derivatives according to the acridine derivative is given such a high reactivity that the invention. By suitable choice of this group, under mild conditions it is already capable of bonding selectively’ with. a functional group of the biological substance to be detected. Suitable reactive groups can be seen from the following compilation: c) 6) r) s) h) 1) lg) 1) '0 - O ' N)__ Y(+) HP) Alkali W O °>— / ..c — 0 - N . I 0% \ (+)/ ' _ \ 'S°2‘CH2"CH2 N:__> Halide (-) ..N : C = S —©-Na (+) ' NH Halide (-) // 2 _ .

.C\» 0-1? 0 —N (:1 cl _3 _ Q - - Cl c c1 I 020 I O I '2 O M ‘R /‘N -c— N /’ ‘$2 -C-C"CF3 In many cases, acridinium derivatives according to the invention in which R5 is a group of the formula III ..CN (HI 0 have proved to be suitable.

In addition, acridinium compounds in which R1 is a methyl group are also preferred. Acridinium compounds according to the invention which correspond to the formula IV .l3 N® \.

/' Ag _ O C 0 (IV) in which A and X have the abovementioned meanings, are therefore particularly frequently employed.

If the radical R4 in the acridinium derivatives according to the invention is a sulfonamide radical, it can then preferably correspond to the formula V x - R5 ’ (V) \ S02 or to the formula VI ‘ f _N/ NI) \ 5 S02-X-R where X and R5 have the abovementioned meanings and R5 in formula V is an alkyl or alkenyl radical having from 1 to 10 carbon atoms, an amino group which is preferably monosubstituted or disubstituted by C1~C4—alkyl, or a morpholino, benzyl or aryl group which can also be substituted by hydroxyl, amino, alkoxy having from 1 to carbon atoms or aryloxy, and R5 in formula VI can also additionally by hydrogen.

Typical representatives of this class of acridinium derivatives according to the invention correspond to the formula VII (EH3. . _ (VII) in which A and X have the abovementioned meanings.

The present invention thus provides two new classes of acridinium compounds, one class being distinguished by a thiol ester grouping and the other by a sulfonamide The have a higher stability and the thiol esters faster structure. acridinium acylsulfonamide derivatives kinetics than the previously known acridinium phenyl ester compounds. Both classes of compound are moreover distinguished by a higher light yield. acridinium with advantage of the according to the thiol A significant compounds leaving with invention groups the Patent containing esters in comparison esters known from European acridinium phenyl 82,636 kinetics of faster the lies in the considerably Thus, acridinium thioester according to the invention prepared Application reaction photoemission. according to Example 1 (compound 6) has a 10 times higher light yield at a neasurement time of 1. second This high light yield with measurement times which are at the same time under the same oxidation conditions. short allows a considerably higher sample throughput in the luminometer. thus shows the kinetics of the photoemission of the Fig. an antibody conjugate of acridinium thioester (compound 6), and Fig. 2 —(2— prepared according to Example 1 shows those of the antibody conjugate of succinimidyl-oxycarbonylethyl)-phenyl—lO—methylacri— Patent 100 pl of the particular dinium 9—carboxylate methosulfate (European Application 82,636, page 10). tracer solutions are stimulated to chemiluminescence by addition of 350 ul of 0.05 M KCl/NaOH buffer, pH 13 + .1% of Hxb and recorded over a 10 second period.

In Fig. 1, the maximum photoemission is already reached after 0.66 second, and has already fallen again to half after 0.88 second. In contrast, in Fig. 2 the maximum photoemission is reached only after 1.77 seconds and has fallen again to half only after 2.88 seconds.

It is completely unexpected that acridinium 9- carboxamides substituted by sulfonyl on the amide nitrogen have an excellent chemiluminescence, since it is known that acridinium 9-carboxamide, in contrast to the 9—carboxylic acid shows no all (cf. F. acridinium esters, chemiluminescence at Mccapra in W. _ 10 _ Sutherland: 1973, Butterworth, London).

Carruthers and J.K. Progress in Organic Chem., Vol. 8, 231-277, The acridinium 9—carboxylic acid thioesters according to the invention can be prepared in the following way: Acridine or its derivatives with R2 and/or R3 in the fused—on phenyl rings are converted into the 9-cyano- acridine in ethanol/glacial acetic acid and potassium cyanide by the process described by Lehmstedt and Hun- l229 (1930). After acridine—9-carboxylic dertmark in Ber. 63, recrystallization, the acid or R%G9—substituted acridine—9—carboxylic acid is obtained fronx this product by reaction with sulfuric acid and sodium nitrite in accordance with the process described by Lehmstedt and Wirth in Ber. 61, 2044 (I928). Reaction of the acridine—9—carboxylic acid or R2/R3—substituted acridinecarboxylic acid with thionyl chloride gives the compound of the formula VIII .n\ 3 C / \ 0/‘! (VIII) in which Y denotes chlorine. Instead of a halogen, an oxycarbonyl—C1—C5—alkyl, oxycarbonylaryl or imidazolide group can also be introduced for Y into compound VIII.

The R2/R3—substituted acridine derivatives can be syn- thesized in a simple manner by processes which are known from the literature. Such syntheses are described, for example, in: Comprehensive Heterocyclic Chemistry; Editors A. Katritzky, C.W. Rees, Vol. 2, p. 395 et seq , Pergamon Press, 1984 or Hetercyclic Compounds, Vol. 9, Acridines and Cond. 2nd Edition, R.M. Acheson, John Wiley and Sons, 1973.

The acid chloride (VIII) is then reacted with a thiol— carboxylic acid of the formula IX HS - X - COOH (IX) for example with 2-mercaptobenzoic acid, under alkaline conditions to give the thiol ester carboxylic acid, which is then esterified with a compound suitable for radial R5, with N- The then in the lO—position by processes which are the tetrafluoroborate is above all suitable for methylation, but the compound are also obtained with dimethyl sulfate, methyl trifluoromethanesulfonate. preparation of the for example hydroxysuccinimide. acridine alkylated known compound is from literature. Trimethyloxonium acridinium methyl methyl good yields of chemiluminescent fluorosulfonate, toluenesulfonate or sulfonamide derivatives the acridinium according to the (VIII) is This compound is then reacted with a primary To prepare invention, acridinecarbonyl chloride likewise used as the starting substance. or secondary sulfonamide, preferably with. a protected sulfonamide—carboxylic acid of the formula X Jr-x—3-oz (X) R — S02 or of the formula XI Rs _ 5, _ so (xx) - X — COOZ V in which X and R5 have the abovementioned meanings and Z is a radical which protects the carboxyl group and is subsequently split off. N—Benzenesulfonylglycine benzyl - 12 _ ester, for example, can be used as the protective group for this reaction. The acid formed after the protective group has been split off is then converted into the radical R5 with a suitable compound, for example with N- The compound is obtained from this product by alkylation on hydroxysuccinimide. chemiluminescent acridinium the nitrogen in the 10-position by processes which are known from the literature.

The acridiniunl compounds obtained 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 give a luminescent compound. The acridinium derivative is thereby bound such as to the stable either directly or via a bridge molecule, polylysine, polyglutamic acid or polyvinylamine, substance of biological interest to form a immunologically active conjugate. This conjugate is also called a the immunoassays below. At tracer and is used in luminescence described least one immunologically active component which is immobilized on a solid phase and also the luminescent tracer are required for the luminescence immunoassay according to the substance in a liquid sample by a competitive process or invention for the determination of an antigenic a sandwich process. The luminescence immunoassay can now be carried out in various ways.

One possibility is to incubate the immobilized antibody which reacts specifically with the antigen with a sample of the liquid under investigation and a conjugate of the acridinium derivative antigen and a chemiluminescent (antigen. tracer), to separate off the sample and the unbound tracer, to bring the bound tracer together with an oxidizing agent in order to cause photoemission and then to determine the amount of antigen present from the photoemission intensity measured. - 13 _ Another possibility for carrying out the luminescence immunoassay comprises incubating an immobilized antibody which reacts specifically with the antigen with a sample of the liquid under investigation, and a conjugate of a second, specifically reacting antibody and a chemiluminescent acridiniun1 derivative, separating off the sample and the unbound labeled conjugate, bringing the bound labeled conjugate together with an oxidizing agent in order‘ to cause photoemission and determining the amount of antigen present from the photoemission intensity measured.

The luminescence immunoassays mentioned above can also be carried out by separating off the the addition of the labeled conjugate. liquid under investigation from immobilized antibody before carried not the In other luminescence immunoassays which can be out according to the invention, Thus, the antigen and antibody‘ is immobilized. an immobilized antigen which reacts specifically with the can be antibody under of the incubated with a sample of liquid investigation and a solution of a conjugate antibody and. a chemiluminescent acridiniun1 derivative, the sample and the unbound labeled conjugate can then be separated off and the bound labeled conjugate can then be brought Photoemission then occurs, together with an oxidizing agent. and the amount of antigen present can be determined from its intensity.

Another variant comprises incubating an immobilized antigen which reacts specifically with the antibody with the chemiluminescent acridinium derivative, a solution of a conjugate of antibody and a separating off the unreacted labeled conjugate, adding a sample of the liquid under investigation, subsequently separating off the sample again, bringing the bound labeled conjugate together with an oxidizing agent in order to cause _ 14 _ photoemission and then determining the amount of antigen present from this photoemission.

Finally, the luminescence immunoassay can also be carried out by a procedure in which an immobilized antigen which reacts specifically with the antibody is incubated with a solution of a conjugate of the antibody and a chemiluminescent acridinium derivative, a sample of the liquid under investigation is added, the sample and the unbound conjugate are separated off, the bound labeled conjugate is brought together with an oxidizing agent and the amount of antigen present is then determined from the photoemission measured.

The following examples, which are already part of the subject matter of the main application, the serve as an introduction to preparation and use of the luminescent compounds according to the invention.

Example 1 —Cyanoacridine (1) 3.3 ml of glacial acetic acid are added to acridine (10 g) potassium cyanide in 8 ml of water is added dropwise, in 45 ml of ethanol and a solution of 5.25 g of the reaction mixture is heated under reflux for 2 hours and cooled. and. the ‘volatile constituents are stripped off in vacuo. The residue is stirred with 30 ml of 2 N NaOH, NaOH and water and left to stand in the moist state in filtered off with suction, washed twice with 2 N air for some time. The crude product is stirred into filtered the methylene chloride, undissolved substance is off with suction and washed with methylene chloride, combined organic phases are concentrated and the crude 9~cyanoacridine is recrystallized from n—butyl acetate.

Yield: 50% l83—5°C IR: 2230 cm'l melting point: _ 15 - Acridinecarboxylic acid (2) (5 g) 40 ml of concentrated H2804 90-95EC for‘ 2 hours and, —Cyanoacridine is slowly’ added in portions to and the mixture is heated at after addition. of 8.5 g of NaNOb is stirred at this temperature for a further 2 hours. The hot solution is poured onto 620 ml of ice water, with rapid stirring, and the precipitate is filtered off with suction and dissolved in the smallest possible amount of 2 N NaOH. The solution is filtered, the filtrate is acidified with 50% strength H2804 and the acridine—9—carboxylic acid which precipitates out is filtered off with suction and dried in vacuo.

Yield: 95% melting point: 288-9°C IR: 3440(br), 3200(br), 2600-2500(br), 1980; 1650; ; 1420 cm‘l Acridine—9—carbonyl chloride hydrochloride (3) Acridine—9—carboxylic acid (5 g) 50 ml of freshly distilled SOCl2 heated under reflux for 5 hours; is added in portions to and the mixture is the solution, which is then clear, is concentrated by distillation until a precipitate starts to form, and precipitation is brought to completion by addition of cyclohexane and cooling.

The precipitate is filtered off with suction and dried in vacuo to give acridinecarbonyl chloride hydrochloride.

Yield: 90% melting point: 223°C Elemental analysis (calculated as CMH9ClNO x HCl) Cl 25.5 C1 25.2 H 2.8 H 3.3 N 5.0 N 5.0 C 60.5 C 59.4 calculated found (Phenyl—2'—carboxylic acid) acridine—9—thiocarboxylate (4) Acridine-9—carbonyl chloride hydrochloride (30 g) is suspended in 720 ml of methylene chloride, thiosalicylic acid (17.7 g) and 50 ml of triethylamine are added and the solution, which becomes clear, is subsequently stirred at room temperature for 10 minutes. After the solvent has been stripped off, 35 g of sodium carbonate and 1400 ml of water are added to the residue, the resulting solution is concentrated until a precipitate filtered off with The appears and this is suction. filtrate is saturated with NaCl and the precipitate which thereby separates out is likewise filtered off with suction, The aqueous combined precipitates is acidified with glacial acetic acid at solution of the BOEC and the product which precipitates out is filtered off with suction and dried in vacuo.

Yield: 80% melting point: 26l—5°C NMR (DMSO, 100 MHZ): 5= 7.6-8.4 ppm, complex multiplet IR: 1680 cm‘1 (s), 1720 (m) 1260 (s) '-(Succinimidoyloxycarbonyl)phenylacridine—9-thiocar- boxylate (5) 3.2 g of N—hydroxysuccinimide are added to a suspension of 10 g of the thiol ester carboxylic acid in 190 ml of dry tetrahydrofuran at 0°C, 6.9 (DCC) are then added at mixture is subsequently stirred at —20°C for 2 hours and After addition of g of dicyclohexyl— carbodiimide —20°C and the then at roon1 temperature overnight. 0.28 ml of glacial acetic acid, the mixture is stirred (25 ml) is then added and the The filtrate ethyl acetate is filtered off. for 1 hour, precipitate is concen- trated and after recrystallization from chlorobenzene yellow 2'-(succinimidoyloxycarbonyl)- gives pale phenylacridinethiocarboxylate.

Yield: 80% melting point: 198-200°C IR: 1810 Cmfl, 1780, 1745, 1225, 1205 NMR (DMSO), 100 MHz: 6= 2.95 ppm (s, 4H), 7.7-8.4 ppm (m, 12H) '-(Succinimidoyloxycarbonyl)phenyl—l0—methylacridinium 9-thiocarboxylate tetrafluoroborate (6) 3 g of N—hydroxysuccinimide ester (5) are heated at 80°C _ 17 _ .8 40 ml of 1,2-dichloroethane for 8 hours and the mixture with g of trimethyloxonium tetrafluoroborate in is subsequently stirred overnight at room temperature.

The precipitate is filtered off and extracted by boiling with 1,2-dichloroethane. The combined organic phases are concentrated and the residue is recrystallized from acetonediisopropyl ether.

Yield: 40% melting point: 245°C IR: 3440 Cmd (br), 1800, 1780, 1740(s), 1670, l065(s) NMR (DMSO, 100 MHz): 5= 3.0 ppm (s, 4H), 4.95 ppm (s, slightly spread, 3H), 7.9-8.6 (m, 10H), 9.9 ppm (d, 2H) Example 2 The preparation of succinimidoyloxycarbonylmethyl-10— methylacridinium 9-thiocarboxylate tetrafluoroborate, starting from the acid chloride (3) and thioglycolic acid is carried out analogously to the synthesis of (6).

The the characterization of individual and yields of synthesis products (2) to (2) by steps spectroscopy are given below: Carboxymethylacridine—9-thiocarboxylate (7) Yield: 60% melting point: 218°C (with decomposition) IR: 3440 cm'1 (br), 2400(br), 1950(br), l7lO(m), (8), lO70(m) NMR (DMSO, 100 MHZ): 6: 4.25 ppm (s, 2H); 7.6-8.4 (m, 8H) Succinimidoyloxycarbonylmethylacridinethiocarboxylate (8) Yield: 80% IR: 3440 cmJ' (br), 2930, 1820, 1785, 1740(5), 1205, 1165 NMR (DMSO, 100 MHZ): 5: 2.95 ppm (5, 4H), 4.77 ppm (s, 2H), 7.6-8.3 ppm (m, 8H) Succinimidoyloxycarbonylmethyl-10—methylacridinium 9- thiocarboxylate tetrafluoroborate (9) Yield: 40% melting point: 250°C IR: 3440 cm‘1 (br), 1810, 1780, 1735(8), 1538, 1350, 1060 NMR (DMSO, 100 MHz): 5= 2.9 ppm (s, 4H), 4.8 (s, 2H), 4.9 ppm, (s, 3H), 7.7-9.0 (m, 8H) Example 3 N—Benzenesulfonyl-N-(benzyloxycarbonylmethyl)acridine—9— carboxamide (10) 130 mg of 4-(dimethylamino)—pyridine and 6 ml of added to 3.3 g fonylglycine benzyl ester in 110 ml of tetrahydrofuran, triethylamine are of N—benzenesul- 3 g of acridinecarbonyl chloride hydrochloride are added after 10 minutes and the suspension formed is heated. under reflux for 6 hours. The precipitate is filtered off with suction, the solvent is stripped off and the residue is taken up in methylene chloride and stirred briefly with 2 N NaOH. After drying over Mgsoh the organic phase is concentrated and the resulting residue is recrystallized from toluene/heptane.

Yield: 70% IR: 3440 cmJ'(br), 1735, 1680, 1357, 1165 NMR (DMSO, 100 MHZ): 5= 5.2 ppm (s, 2H), 5.3 ppm (s, 2H), 7.0—8.4 ppm (m, 18H) melting point: 58°C N—Benzenesulfonyl-N-(carboxymethyl)acridine carboxamide (11) 1 g of N—benzenesulfonyl—N(benzyloxycarbonylmethyl)— acridine—9—carboxamide in 60 ml of glacial acetic acid is hydrogenated at room temperature and normal pressure with the addition of 2 ml of concentrated HCl and Pd/C (10%); filtered off with filtrate gives the carboxylic acid as a yellow solid. when the reaction has ended, the catalyst is suction and concentration of the _ 19 _ NMR 2H), H) (DMSO, 100 MHZ): 5: 5.0 ppm (s, 7.1-8.5 ppm (m, sulfonyl-N-(succinimidoyloxycarbonylmethylacridine carboxamide (lg). (13) is quaternized with trimethyloxonium tetrafluoroborate, as described for (6), to give N—benzenesulfonyl—N—(succinimidoyloxy— carbonylmethyl)—lO—methylacridinium 9—carboxamide tetrafluoroborate (13).

Example 4: N—Phenyl—N(4—benzyloxycarbonylbenzenesulfonyl)acridine— (14) 4-(dimethylamino)pyridine —carboxamide 360 mg of triethylamine are added to 11 g of benzyl 4(N—phenyl- in 300 ml and 16.6 ml of sulfamido)benzoate of methylene chloride, 8.34 g of acridine—9-carbonyl chloride hydrochloride (3) are added after 10 xninutes and. the mixture is heated The stirred briefly with 2N NaOH and the organic phase is under reflux for 16 hours. cooled solution is separated off, washed with water, dried over NagSO4 and concentrated. The residue is recrystallized from toluene/heptane.

Yield: 70% melting point l6l—163°C NMR (DMSO, 100 MHZ): 6= 5.5 ppm (s, 2H), 5= 6.8—8.6 ppm (m, 22H) N—Phenyl—N-(4—carboxybenzenesulfonyl)acridine-9— (15) N-phenyl—N—(4—benzyloxycarbonylbenzene- (14) are heated at 60°C in 30 ml of 33% HBr in glacial acetic acid for 2 hours carboxamide hydrobromide 8.58 g of sulfonyl)acridine—9—carboxamide and, after cooling, 60 ml of diisopropyl ether are added and the precipitate is filtered off with. suction. and dried in vacuo. _ 20 _ Yield: 95% NMR (DMSO, melting point: 255°C 100 MHZ): 5= 6.8-9 ppm (m) N—Phenyl-N-(4—succinimidoyloxycarbonylbenzenesulfonyl)- (16) 2.8 ml of triethylamine are added to 5.63 g of N-phenyl- acridine—9-carboxamide N-(4—carboxybenzenesulfonyl)acridine—9—carboXamide (15) in 250 ml the mixture is cooled to —15°C and 0.96 ml of ethyl chloro- hydrobromide of tetrahydrofuran, formate is added. The ndxture is subsequently" stirred for 20 minutes, 1.15 g of N-hydroxysuccinimide are added -15°C for 3 hours, to thaw to room temperature and is subsequently stirred and the mixture is stirred at allowed overnight. The precipitate is filtered off with suction, the filtrate is concentrated, the residue is taken up in methylene chloride and the resulting solution is washed NaHCO3 Na2SO4. The organic phase is concentrated and the residue with water, solution and water and dried over is recrystallized from toluene.

Yield: 50% melting point: 226° (decomposition) NMR (DMSO, 100 MHZ): 6=2.95 ppm (s, 4H), 6: 6.8-8.7 ppm (m, 17H) N—Phenyl—N—(4—succinimidoyloxycarbonylbenzenesulfonyl)— —methylacridinium 9—carboxamide fluorosulfonate (17) .16 g of N—phenyl—N—(4—succinimidoyloxycarbonylbenzene— (16) with stirred in methyl sulfonyl)acridinecarboxamide 60 ml of 1,2—dichloroethane are 0.3 ml of fluorosulfonate at room temperature for 24 hours and the precipitate which separates out is filtered. off with suction and dried in vacuo.

Yield: 65% IR: 3420 cm'1 (br), 3100(br), 1805(w), 1770(m), 1745(s), 1700(m), 1385(m), 1280(m), 1255(s), 1230(5), 1205(s) NMR (DMSO, 100 MHZ): 5: 2.95 ppm (s, 4H), 5: 4.75 ppm (3, br, 3H), 5= 7.0-9.0 ppm (m, 17H) Mass spectrum: m/z = 594 : M+ (cation) _ 21 _ Example 5 The preparation of N—(4-methoxyphenyl)—N-(4—succin- imidoyloxycarbonylbenzenesulfonyl)-10—methylacridinium (21) 4-[N—(4'-methoxyphenyl)sulfamido]—benzoate and acridine- (3) is (17) The yields of the individual synthesis steps and the —carboxamide fluorosulfonate starting from benzyl —carbonyl chloride hydrochloride carried out analogously to the synthesis of (see Example 4). characterization by spectroscopy are given below.

N-(4—Methoxyphenyl)—N—(4—benzyloxycarbonyl—benzene— sulfonyl)acridine—9-carboxamide (18) Yield: 70% melting point: l82—l83°C NMR (DMSO, 100 MHz): 6: 3.5 ppm (s, 3H), 6: 5.5 ppm (5, 2H), 6: 6.35-6.63 ppm (d, br, 2H), 5: 7.05—7.2 ppm (d, br, 2H), 6: 7.35-8.5 ppm (m, 17H) N-(4—Methoxypheny1)—N—(4—carboxybenzenesulfonyl)- acridine-9—carboxamide hydrobromide (19) Yield: 95% melting point: 273°C (decomposition) NMR (DMSO, 100 MHZ): 6: 3.5 ppm (s, 3H), 6: 6.4-6.6 ppm (d, br, 2H), : 7.05—7.2 ppm (d, br, 2H), 6: 7.7-8.5 ppm (m, 12H) N-(4-Methoxyphenyl)—N~(4—succinimidoyloxycarbonyl— benzenesulfonyl)acridine—9-carboxamide (20) Yield: 50% melting point: 232—234°C NMR (DMSO, 100 MHZ): 6: 2.95 ppm (s, 4H), 6: 3.5 ppm (s, 3H), 6: 6.4-6.6 ppm (d, br, 2H), 6: 7.05-7.25 ppm (d, br, 2H), 6: 7.8-8.6 ppm (m, 12H) IR: 3050 cm'1 l805(W), l780(m), 1740(8), l700(m), l505(m), l370(m), 1250(m), l200(S), ll85 N—(4—Methoxyphenyl)—N—(4—succinimidoyloxycarbonyl— benzenesulfonyl)—l0—methylacridinium-9—carboxamide (21) The substance does not precipitate out on reaction, it fluorosulfonate is obtained by concentrating the solution and stirring _ 22 _ the residue with diisopropyl ether.

Yield: 80% NMR (DMSO, 100 MHz): 5= 2.95 ppm (s, 4H), 5: 3.5 ppm (s, 3H), 5= 4.8 ppm (s, br, 3H), 6= 6.45-6.7 ppm (d, br, 2H), = 7.2-7.4 ppm (d, br, 2H), 6: 7.7-9 ppm (m, 12H) Mass spectrum: m/z = 624 M* (cation) IR: 3440 Cmfi (br), 3100, 2950, l80S(w), l775(m), l740(s), l695(m), l6lO(m), l505(m), l375(m), l280(m), 125o(s), 12o5(s) Example 6 The preparation of N—(4—methoxyphenyl)—N—(3-succin- imidoyloxycarbonylbenzenesulfonyl)—lO—methylacridinium (25) -[N-(4'-methoxyphenyl)sulfamido]-benzoate and acridine— -carboxamide fluorosulfonate starting from benzyl —carbonyl chloride hydrochloride (3) is carried out (17) synthesis analogously to the synthesis of The yield of the (see Example 4). individual steps and the characterization by spectroscopy are given below.

N-(4—Methoxyphenyl)—N(3—benzyloxycarbonylbenzene— sulfonyl)acridine—9—carboxamide (22) Yield: 70% melting point: 168-170°C NMR (DMSO, 100 MHZ): 5= 3.5 ppm (s, 3H), 5: 5.45 ppm (s, 2H), 5= 6.5 ppm (s, br, 2H), 5= 7.1 ppm (br, 2H), : 7.3-8.8 ppm (m, 17H) N-(4—Methoxyphenyl)-N-(3—carboxybenzene- sulfonyl)acridine—9-carboxamide hydrobromide (23) Yield: 90% melting point: 264°C NMR (DMSO, lOO MHZ): 6= 3.5 ppm (s, 3H), 5= 6.4-6.6 ppm (d, br, 2H), 6= 7.0-7.2 ppm (d, br, 2H), 6= 7.6-8.8 ppm (m, 12H) N-(4—Methoxyphenyl)—N—(3—succinimidoyloxycarbonyl— benzenesulfonyl)acridinecarboxamide (24) Yield: 50% melting point: 223—225°C NMR (DMSO, 100 MHZ): 5: .95 ppm (5, 4H), 6= 3.5 ppm _ 23 _ (s, 3H), 6= 6.4-6.6 ppm (d, br, 2H), 5= 7.0-7.2 ppm (d, br, 2H), 5= 7.4—8.9 ppm (m, 12H) IR: 3500 cmfl (br), 3060, 2950, 2840, 1805(w), l785(m), 1740(s), 1700(m), 1510(m), l380(m), l250(m), 1205(m), 1165(m) N—(4—Methoxyphenyl)-N-(3—succinimidoyloxycarbonyl— benzenesulfonyl)methylacridinium 9—carboxamide fluorosulfonate (25) Yield: 90% NMR (DMSO, 100 MHZ): 6= 2.95 ppm (s, 4H), 6: 3.55 ppm (s, 3H), 5= 4.8 ppm (s, br, 3H), 6= 6.45-6.7 (d, br, 2H), 6: 7.05-7.3 ppm (d, br, 2H), 6= 7.5-8.9 ppm (m, 12H) IR: 3500 Cm4' (br), 3080, 2950, 1805(w), l780(m), 1740(8), 35 l700(m), l6l0(m), l5l0(m), l380(m), 1250(8). 1205(8), 1170(8) Mass: m/z = 624 M+ (cation) Example 7 Tracer preparation for d-fetoprotein chemiluminescence immunoassay 100 pl of 11.5 pl of acridinium thioester prepared according to Example 1 antibodies (1 mg/ml), the (compound (6)) (1 mg/ml in DMSO and 600 pl of conjugation buffer (0.01 M phosphate, pH 8.0)) are incubated for 15 ndnutes. 200 pl of lysine (10 mg/ml) are then added and the mixture is incubated for a This batch is transferred to a PD 10 .1 M phosphate, pH 6.3 as further 15 minutes. column (Sephadex G 25 medium, the mobile phase). 10 drops/fraction are collected. The individual fractions are tested for their chemiluminescence activity after appropriate dilution (350 pl of oxidizing agent: 0.1% of H202 in 0.1 N NaOH).

The tracer fractions stored at 4°C. (1st activity peak) are pooled and Example 8 d—Fetoprotein chemiluminescence immunoassay procedure pl of 150 pl of buffer (phosphate; 0.1 M pH 6.3, 1% of Tween 20, 0.1% of bovine 0.1 M NaCl, 0.01% of NaNfl coated with monoclonal standard/sample and are shaken for anti—AFP serum albumin, minutes in tubes antibody. Washing with 2 X 1 ml portions of buffer is then carried out. pl of tracer are added in an appropriate dilution Washing with The chemiluminescence measurement is started with 350 pl of and the mixture is shaken for 15 minutes. 2 x 1 ml portions of buffer is again carried out. oxidizing agent (0.1% of Hg» in 0.1 N NaOH, measurement time of 2 seconds).

Figure 3 shows the typical course of a standard curve of an immuno-chemiluminometric assay (ICMA) for the alpha- fetoprotein (AFP).

Claims (1)

1. Patent claims A luminescent compound which comprises an acridinium derivative of the formula I in which R1 is hydrogen, an alkyl, alkenyl or alkynyl radical with l to 10 carbon atoms or a benzyl, phenyl or naphthyl group, R2 and R3 are hydrogen, an alkyl group with 1 to 4 carbon atoms, an amino group or a mono— or di—C1-C4 alkylamino group, where the alkyl radicals can be monosubstituted by hydroxyl, and morpholine, a carboxyl, C1—C4—alkoxy, cyano or‘ nitro group or halogen, R4 is a radical in which a substituted sulfonamide group is bonded via the nitrogen directly to the carbonyl group, and A is an anion which does not impair the chemiluminescence, excluding acridinium compounds of the formula I in which R1, R2 and R3 are each as defined above and R4 is a substituted sulfonamide group of the formula V or VI X-——R5 R5 ~——N< (V) __~< (vi) O,S——-R6 O2S——X—-—-R5 where X is a branched or unbranched C1—C5—alkylene group or is a phenylene or naphthylene group or is quinole, indole, pyrrole or pyridine, R5 is a reactive group which under mild conditions is capable of bonding selectively with. amino, carboxyl, thiol or other functional groups in substances of biological interest, and R6 in the formula V is an alkyl or alkenyl radical having 1 to 10 carbon atoms, a C1-C4-alkyl—mono— substituted or —disubstituted amino group or is a morpholino, benzyl, phenyl or naphthyl group which is optionally substituted by hydroxyl, amino, alkoxy of l to 4 carbon atoms or phenoxy or naphthoxy groups, and R5 in the formula VI, as well as the substituents mentioned, may additionally be hydrogen, where the acridinium derivative of the formula I is bonded directly or via a bridge molecule to a protein, a polypeptide or another substance of biological interest to form a stable immunologically active conjugate. A compound as claimed in claim 1, wherein the substance of biological interest is selected from the group consisting of antigens, hormones, steroids, drugs, drug metabolites, toxins, alkaloids and antibodies. A luminescence immunoassay for the determination of an antigenic substance in a liquid sample, in which, in a competitive process or a sandwich process, at least one immunologically active component is immobilized on a solid phase and at least one other component, the tracer, is a luminescent compound as claimed in claim 1. A luminescence immunoassay as claimed in claim 3, which comprises a) incubating an immobilized antibody, which reacts specifically with an antigen, with a sample of the liquid under investigation and a conjugate of the antigen and a chemiluminescent acridinium derivative of the formula I as claimed in claim 1; b) separating off the sample and the unbound labeled conjugate; c) bringing the bound labeled. conjugate together with an oxidizing agent in order to cause photo- emission and d) determining the amount of antigen present from the photoemission intensity measured. A luminescence immunoassay as claimed in claim 4, wherein the liquid under investigation is separated off from the immobilized antibody before addition of the labeled conjugate. A luminescence immunoassay as claimed in claim 3, which comprises a) incubating an immobilized antibody, which reacts specifically with an antigen, with a sample of the liquid under investigation and ea conjugate of a second, specifically reacting antibody and a chemiluminescent acridinium derivative of the formula I as claimed in claim 1; b) separating off the sample and the unbound labeled conjugate; c) bringing the bound labeled conjugate together with an oxidizing agent in order to cause photo— emission and determining the amount of antigen present from the photoemission intensity measured. A luminescence immunoassay as claimed in claim 6, wherein the liquid under investigation is separated off from the immobilized antibody before addition of the labeled conjugate. A luminescence immunoassay as claimed in claim 3, which comprises a) incubating an immobilized antigen, which reacts specifically with an antibody, with a sample of the liquid under investigation and a solution of the conjugate of the antibody and a chemilumi- nescent acridinium derivative of the formula I as claimed in claim 1; separating off the sample and the unbound labeled conjugate; bringing the bound labeled conjugate together with an oxidizing agent in order to cause photo- emission and determining the amount of antigen present from the photoemission intensity measured. A luminescence immunoassay as claimed in claim 3, which comprises a) incubating an inmpbilized antigen which reacts specifically with an antibody, with a solution of the chemiluminescent acridinium derivative of the conjugate of the antibody and a formula I as claimed in claim 1; separating off the unreacted labeled conjugate; adding a sample of the liquid under investi- gation; subsequently separating off the sample again; bringing the bound labeled conjugate together with an oxidizing agent in order to cause photo- emission and determining the amount of antigen present from the photoemission intensity measured. A luminescence immunoassay as claimed in claim 3, which comprises incubating an immobilized antigen, which reacts specifically with the antibody, with a solution of the the chemiluminescent and a of the conjugate of antibody acridinium derivative formula I as claimed in claim 1; adding a sample of the liquid under investi- gation; bringing the bound labeled conjugate together with an oxidizing agent in order to cause photo- emission and determining the amount of antigen present from the photoemission intensity measured. F. R. KELLY & CO., AGENTS FOR THE APPLICANTS