FI90537B - Chemiluminescent acridine derivatives, their preparation and their use in luminescence immunoassay - Google Patents

Abstract

Chemiluminescent acridinium derivatives of the formula I <IMAGE> in which R<4> is a radical of the formula II or III <IMAGE> and A<(-)>, X-, R<1>-R<3>, R<5> and R<6> have the specified meanings, and processes for the preparation of the compounds of the formula (I) and the use thereof in chemiluminescence immunoassays.

Description

1 90537

This invention relates to chemiluminescent acridine derivatives, to a process for their preparation and to their use in luminescence-based immunoassays.

Luminescent compounds already have a wide range of uses. They are used as indicators in biological assays, enzymatic immunoassays, and luminescence immunoassays (see W.P. Collins, Alternative Immunoassays, John Wiley & Sons Ltd., Chichester, 1985) but also in nucleic acid hybridization assays [see AND. Matthews et al., Analytical Biochemistry 151 15 (1985) 205-209]. In addition, chemiluminescent compounds are used in "flow injection analysis", post-column detectors in liquid chromatography, flow studies and artificial light sources.

In particular, two structural types of chemiluminescent markers 20 have become more important in chemiluminescence immunoassays. These are, firstly, the luminol and isoluminol derivatives described in H.R. Schroeder et al., Methods in Enzymology 57 (1978) 424 (Academic Press Inc., New York) and 25 GB Patents 2,008,247 and 2,041,920, DE Patents 2,618,419 and 2,618,511, and EP application publication 135 071. An overview of the use of isoluminol compounds as luminescence indicators in practice can be found in the article list WG Wood, J. Clin, Chem. Clin. Biochem. 22 (1984) • 30 905-918.

On the other hand, acridinium ester compounds have also been used as chemiluminescent markers. Such acridinium esters are known from U.S. Pat. No. 3,352,791, GB Patents 1,316,363 and 1,461,877, and EP-A-35 from Muscle Publication 82,636. The use of acridinium esters 2,90537 as markers in immunoassays is described by Weeks et al., Chem. 29/8 (1983) 1474-1479. The use of phenanthridinium esters as markers in luminescence immunoassays is also already known from EP-A-5 170 415.

The chemiluminescence of acridinium esters can be initiated by the addition of a basic H 2 O 2 solution. The mechanism of chemiluminescence has been convincingly explained by F. McCapra, Acc. Chem. Res. 9 (1976) 201. The quality of the leaving groups is apparently decisive 10 in terms of both photantum yield and hydrolytic stability.

The advantage of the acridinium esters already known over luminol and isoluminol compounds is that they have a higher amount of photonquants, which is not reduced by the proteins bound to the indicator [see Weeks et al., Clin. Chem. 29/8 (, 983) 1474-1479].

Although acridinium phenyl esters known from EP-A-82 636 have a high detection sensitivity in the excitation of chemiluminescence by weak oxidants, they have drawbacks which in practice interfere with their use. Above all, the phenyl ester compound is very labile in aqueous systems already at room temperature. In addition, under the oxidation conditions indicated therein, acridinylphenyl esters exhibit light emission which ceases for the most part, i. more than 95%, only after about 10 seconds. In addition, in other non-isotope determination methods, the measurement times are relatively shorter and thus allow a larger sample throughput.

It has already been proposed to use chemiluminescent acridinium derivatives which have faster reaction kinetics with high photonquant yield and thus allow short measurement times in the luminescence ____ trans assay (see DE application P 3 628 573.0).

35 These are acridinium derivatives of the formula 3 90537 rA αθ rb -CT rc

5 D

O = C R

wherein Ra is hydrogen, an alkyl, alkenyl or alkynyl group containing 1 to 10 carbon atoms, or a benzyl or aryl group; R 1 * and R c are each independently hydrogen, an alkyl group having 1 to 4 carbon atoms, a substituted or unsubstituted amino group, a carboxyl, alkoxyl, cyano or nitro group or halogen; RD is a group in which the sulfonamide group is directly attached to the carbonyl group via nitrogen, or a thioalkyl or thioaryl group of the formula -SY-re, in which Y is a branched or unbranched aliphatic or aromatic group which may also contain heteroatoms, and Re is a reactive group which, under mild conditions, is capable of selectively forming a bond with amino, carboxyl or thiol groups or other functional groups present in substances of biological interest; and fp is an anion that does not adversely affect chemil-25 minescence.

It has now been found that certain acridinium derivatives are particularly well suited for use as chemiluminescent compounds due to their excellent stability and unexpectedly high detection sensitivity. Accordingly, the invention relates to chemiluminescent acridinium derivatives of formula (I), 4 90537 R A® 5 1 (I) 1 4

o = C - R

wherein R 1 is hydrogen or an alkyl group containing 1 to 10 carbon atoms, R 4 is a group of formula (II) or (III)

R

/

5 (ID

SO -X-R 2 15 5 -t

X-R

'N \ 6 i111 »SO -R 2 20 where R5 is a group of formula V: 0 ° * _ 25«

- C - 0 - N

J-J <V>

O

R6 is a phenyl group which may be substituted by alkyl or alkoxyl groups having 1 to 4 carbon atoms or a morpholino (C1-C4) alkoxy group, X is a phenyl group bonded directly to a nitrogen or sulfur atom and via a C1-4 alkyl group to R5, and / P is an anion that does not adversely affect chemiluminescence.

1, 5 9G537

Substances of biological interest are primarily antigens. This term includes, for example, hormones, steroids, drugs, drug metabolites, toxins, alkaloids, and also antibodies.

Examples of suitable aminocarboxylic acids are glycine, alanine, serine, phenylalanine, histidine, α-aminobutyric acid, methionine, valine, norvaline, leucine, isoleucine, norleucine, aspartic acid, glutamic-10-nitric acid, 4-aminobenzoic acid, 4-aminobenzoic acid, 4-aminobenzoic acid, aminophenylacetic acid, 4-aminophenoxyacetic acid and 3- (4-amino) phenylpropionic acid.

The anion which does not adversely affect the chemiluminescence may be, for example, a tetrafluoroborate, perchlorate, halide, alkyl sulfate, halosulfonate, alkylsulfonate or arylsulfonate anion. Any other anion may also be used as long as it does not suppress or attenuate chemiluminescence.

Heteroalkyl groups or heterocyclic groups preferably contain heteroatoms which contribute to improving the water solubility of the compounds of the invention, such as nitrogen, oxygen, sulfur, phosphorus or combinations thereof. Particularly suitable heterocyclic rings include, for example, morpholine, piperazine, piperidine, tetrahydrofuran, dioxane, and the like.

Of particular interest are the acridinium derivatives according to claim 1, characterized in that X is a group of formula IV

(R) wherein R7 is a substituent of the formula - (CH2) n, wherein n is 0-4.

6 90537

The substituent R5 is particularly important for the use of the acridinium derivatives according to the invention. By appropriately selecting this group, the activity of the acridinium derivative becomes so high that it is already able, under mild conditions, to selectively form a bond with one of the functional groups of the biological substance which determines it. In the compounds of the invention R5 is:

O

0 V-,

Il /

10 -C - O - N

(v) o

In addition, acridinium compounds of formula VI have proved to be particularly suitable. In the formula,

15 VI

CH _ '.3 A® R ^ ^ 2 20 a ASf1 is N — f o (vi)

2 yJ

O

X is a group of formula VII, - <S> - <CVn- <VII) wherein n is 2 or 4, R 12 and R 13 are each independently hydrogen, an alkyl or alkoxyl group containing 1 to 4 C atom, or a morpholino- (C 1 -C 4) alkoxy group, and / ft has the meaning mentioned in claim 1. These compounds are readily water-soluble products.

tl 7 90537

Of the latter compounds, which correspond to formula VI, very particularly preferred are those in which X is a p-ethylenephenyl group, R12 is H and R13 is a p-methoxyl group or R12 is an o-methoxyl group and R13 is a p-methoxyl group, such as, for example, having the formula CH Αθ

«rL

10 λ / sy

IS . O

\ nr \ s 0 SO -CH -C ^ _ 2 2 2 \ r-

IS

15 0 ^

In addition, acridinium derivatives of the formula VIII are particularly suitable

, n CH Αθ 20 I 3 oco I o // \ / X ~ ‘c \ λ ~ | (VIII)

C 25 0 \ 6 ° - VJ

SO - R ry 2 wherein R6 is a phenyl group which may be substituted by up to three alkyl or alkoxy groups or morpho. A (C 1 -C 4) alkoxy group, and X has the meaning given in claim 1 or 2, or R 6 is a phenyl group which may be substituted by up to three C 1-4 alkyl groups, and X is an o, m or p-phenylene group . These compounds are also readily water-soluble products.

8 90537

Surprisingly, acridinium-9-carboxylic acid amide containing a sulfonyl-substituted amide nitrogen exhibits excellent chemiluminescence, as it is known that acridinium-9-carboxylic acid amides, in contrast to 5. Sutherland, Progress in Organic Chemistry, Vol. 8, Butterworth, London, 1973, pp. 231-277).

One significant advantage of the acridinium compounds according to the invention over the acridinium phenyl esters known from EP-A-82 636 is the considerably faster reaction kinetics associated with light emission (see DE-application P 3 628 573.0).

Another advantage is the stability of the tracer prepared by the compounds 15 of the invention. Figure 1 shows the results of a stability test in which the intensity of the respective chemiluminescent signal after storage at elevated temperature (50 ° C) was measured. Curve a relates to N- (4-methoxyphenyl) -N- [4- (2-succinimidoyloxycarbonylethyl) benzenesulfonyl] -10-methylcridinium-9-carboxylic acid amide fluorosulfonate (6), curve b relates to N- (4-methoxyphenyl) - N- [4- (4-Succinimidoyloxycarbonylbutyl) benzenesulfonyl] -10-methylacridinium-9-carboxylic acid amide fluorosulfonate (11) and curve c of 4- (2-Succinimidoyloxycarbonyl ethyl) phenyl-10-methylacridine -carboxylate-thimetosulfate (EP-A-82 636, p. 10) to a prepared tracer.

It can be clearly seen that the tracers according to the invention obtained from compounds a and b are more stable than the corresponding compound obtained from c.

Similar results are obtained in a similar experiment at 4 ° C. Figure 2 shows the signal intensity at 4 ° after storage. Again about the compound. The tracer prepared from 35a appears to be crucially more stable than that prepared from compound c.

li 9 90 537

In the preparation of the acridinium sulfonamide derivatives according to the invention, acridine-9-carboxylic acid chloride (IX) can be used as a starting material. To prepare this compound, for example, acridine is reacted according to the method of Leh-5 mstedt and Hundertmark [Ber. 63 (1930) 1229] with potassium cyanide in ethanol-acetic acid to give 9-cyanoacridine. It is preferably obtained after recrystallization according to the method proposed by Lehmtedt and Wirth [Ber. 61 (1928) 10 2044] by reaction with sulfuric acid and sodium nitrite acridine-9-carboxylic acid. Reaction of acridine-9-carboxylic acid with, for example, thionyl chloride gives a compound of formula IX, 15oo (IX) # \

O Y

20 wherein Y is chlorine. Y in compound IX may also have an oxycarbonylalkyl, oxycarbonylaryl or imidazole group instead of halogen.

The acid chloride (IX) can then be reacted with a protected sulfonamide carboxylic acid corresponding to ... Formula X

HO

. 1 R-SO 2 -N-X-C-OZ (X) 30 or formula XI,

B

R 6 N-S02-X-C00Z

... · 35 wherein X and R6 have the above-mentioned roots and Z is a carboxyl protecting group which is subsequently removed. For example, N- (4-benzoxycarbonylphenyl) -N-4-toluenesulfonic acid amide can be used in this reaction. Preferably, a t-butyl ester is used, the protecting group of which can be introduced into the molecule and again removed under particularly mild conditions.

The acid group formed after deprotection is then converted to R5 with a suitable compound, for example N-hydroxysuccinimide. From this compound, a chemiluminescent acridine compound is obtained by alkylation of the nitrogen in the 10-position.

The resulting acridinium compounds can then be reacted with an agent of biological interest, for example, an antigen, antibody, hormone, drug, drug metabolite, toxin, or alkaloid, to provide a luminescent compound. In this case, the acridinium derivative binds either directly or via a bridging molecule, such as an amino acid, oligo- or poly-amino acid, peptide or synthetic polymer, to the biologically interesting substance to form a stable, immunologically active conjugate. This conjugate is also called a tracer and is used in the luminescence immunoassays described below. At least one immunologically active component immobilized on a solid phase, as well as a luminescent label, are required to determine the antigenic agent in a fluid-sample sample by a competitive or layered method for the luminescence immunoassay of the invention.

After the completion of the immune reaction and any washing steps required, the light emission is triggered by the sequential or simultaneous addition of one or more reagents, with at least one reagent containing an oxidant in bound or unbound form. The Lumine-35 sensory immunoassay can then be performed in a variety of ways.

l! 11 90537

One possibility is that the immobilized antibody that specifically reacts with the antigen is incubated with a sample of the test fluid and a conjugate of the antigen fractionating luminescent acridinium derivative (antigenic marker), the sample and unbound marker are separated, contacted with the label, then the amount of antigen present is determined from the measured intensity of the light-10 sion.

Another possibility for performing luminescence immunoassays is to incubate an immunized antibody that specifically reacts with the antigen with a sample of the test fluid and a conjugate of another specific reactive antibody and a chemiluminescent acridine derivative conjugate, a sample, and a sample. contacting the necessary reagents to effect light emission and determining the amount of antigen present from the measured light emission intensity.

The above luminescence immunoassays can also be performed by separating the test fluid from the immobilized antibody prior to the addition of the labeled conjugate.

Other luminescence immunoassays according to the invention do not immobilize the antibody but the antigen. The immunized antigen that specifically reacts with the antibody is then incubated with a sample of the liquid taken from the test and a solution of the antibody and chemiluminescent acridinium derivative, then the sample and unbound labeled conjugate are separated and the bound labeled conjugate is contacted. . A light emission then occurs, the intensity of which can determine the amount of antigen present.

Yet another alternative is to incubate the immobilized antigen 5 that specifically reacts with the antibody with a solution of the conjugate consisting of the antibody and the chemiluminescent acridinium derivative, separating the unreacted labeled conjugate, adding the bound sample to the test fluid, then separating the labeled liquid again. with the reagents required for contacting to effect light emission, and the amount of antigen present is then determined from the intensity of the emission.

The luminescence immunoassay can also be performed by incubating the immobilized antigen that specifically reacts with the antibody with a solution of the conjugate consisting of the antibody and the chemiluminescent acridinium derivative. reagents and then determine the amount of antigen present from the measured light emission.

The preparation of the acridinium compounds of the invention is further illustrated by Examples 1-7.

Example 1 . '25 N- (4-Methoxyphenyl) -4- [4- (2-benzoxycarbonylmethyl) benzenesulfonyl] lacridine-9-carboxylic acid amide (3)

To a solution of 17 g of 4 '- [N- (4-methoxyphenyl) sulfonamido] -3-phenylpropionic acid benzyl ester (1) in 400 ml of dichloromethane is stirred 460 mg of 4-30 (Ν, Ν-dimethylamino) pyridine and 22 g of , 1 ml of triethylamine, 10 minutes later 11.12 g of acridine-9-carboxylic acid hydrochloride (2) are added and the mixture is refluxed for 6 hours. The cooled solution is stirred briefly with 2 N NaOH, and the separated organic phase is washed with water, dried over magnesium sulfate and concentrated. The residue is purified by column chromatography. Yield: 60%

Melting point: 130-132 ° C

NMR (DMSO, 100 MHz); S = 2.7-3.0 (d, broad, 2H); 3.0-3.3 δ (d, broad, 2H); 3.5 (s, broad, 3H); 5.1 (s. 2H); 6.5 (d, broad, 2H); 7.1 (d, broad, 2H); 7.35 (s, 5H); 7.5-8.3 (m, 12 H) ppm N- (4-methoxyphenyl) -N-Γ4- (2-carboxyethyl) benzenesulfonylacridine-9-carboxylic acid amide hydrobromide (4) 10 6.3 g of a compound 3 is heated in 30 ml of a mixture of 33% HBr in glacial acetic acid at 60 ° C for 2 hours, and after cooling, 60 ml of diisopropyl ether are added and the precipitate is filtered off with suction and dried under reduced pressure.

15 Yield: 90%

Melting point: 237 ° C (decomposition) NMR (DMSO, 100 MHz); S- 2.7 (d, broad, 2H); 3.1 (d, broad, 2H); 3.5 (s, broad, 3H); 6.5 (d, broad, 2H); 7.0-8.4 (m, 15 H) ppm 20 N- (4-Methoxyphenyl) -N- [4- [2-succinimidoxycarbonyl] ethyl) benzenesulfonyl] Acridine-9-carboxylic acid amide (5)

To a solution of 3.1 g of compound 4 in 50 ml of tetrahydrofuran is stirred 1.41 ml of triethylamine, the mixture is cooled to -20 ° C and 0.474 ml of chloroformic acid ethyl ester is added. The mixture is stirred for 20 minutes, 575 mg of N-hydroxysuccinimide are added, this mixture is stirred at -20 ° C for 3 hours and allowed to warm to room temperature overnight while stirring. The precipitate is filtered off with suction, the filtrate is concentrated, the residue is dissolved in dichloromethane or ethyl acetate and the resulting solution is washed with water, NaHCO3 solution and water and dried over MgSO4. The organic phase is concentrated and the residue is recrystallized from toluene.

14 90537

Yield: 50% NMR (DMSO, 100 MHz): δ = 2.8 (s, 4 H); 3.2 (s, broad, 4H); 3.5 (s, broad, 3H); 6.5 (d, broad, 2H); 7.2 (d, broad, 2H); 7.6-8.4 (m, 12H) ppm δ IR: 3400 (broad), 3060, 2930, 1815 (w), 1780 (w), 1740 (s), 1690 (m), 1600 (w), 1510 (n), 1370 (m), 1250 (m), 1203 (m), 1175 (m) cm-1 N- (4-Methoxyphenyl) -N- [4- [2-succinimidoxycarbonyl] ethyl] benzenesulfonyl-10-methylcrididinium-9-carboxylic acid sulfuric acid amide fluorosulfonate (6)

To a solution of 1.27 g of compound 5 in 60 ml of dichloromethane is stirred at -20 ° C 0.4 ml of methyl fluorosulfonate. The mixture is stirred at -20 ° C for 2 hours and allowed to warm to room temperature overnight. After the addition of toluene, a yellow solid precipitates which is separated by suction filtration and dried under reduced pressure.

Yield: 80% NMR (DMSO, 100 MHz): δ = 2.9 (s, 4 H); 3.2 (s, broad, 4H); 3.5 (s, broad, 3H); 4.8 (s, broad, 3H); 6.5 (broad, 2H); 7.2 (broad, 2H); 7.6-9.0 (m, 12 H) ppm IR: 3400 (broad), 3160, 2970, 1810 (w), 1785 (w), 1740 (S), 1695 (m), 1600 (W), 1555 (w), 1510 (m), 1370 (m), 1290 (m), 1250 (m), 1219 (m), 1170 (m) cm-1 Mass spectrum (m / z): 652 (M *, cation)

Example 2 Preparation of N (4-methoxyphenyl) -N- [4- (4-succinimidoxycarbonylbutyl] benzenesulfonyl] -10-methylcrididinium-9-carboxylic acid amide fluorosulfonate (11) 4 '- [N- (4-methoxy) sulfoxy] benzyl ester (7) and acridine-9-carboxylic acid hydrochloride (2) are carried out in a similar manner to the synthesis of compound 6. The yields of the various synthetic steps and the characteristic spectral data are given below.

I; is 90537 N- (4-methoxyphenyl) -N- [4- (4-benzyloxycarbonylbutyl) benzenesulfonyl] acridine-9-carboxylic acid amide (8)

Yield: 40% (viscous oil, partially solidified) δ NMR (CDCl 3 100 MHz): δ = 2.85 (m, 4 H); 2.45 (t, broad, 2H); 2.8 (6, broad, 2H); 3.5 (s, 3H); 5.15 (s, 2H); 6.3 (d, 2H): 6.9 (d, 2H); 7.3-8.3 (m, 17 H) ppm N- (4-methoxyphenyl-N- [4-carboxybutyl) benzenesulfonyl] acridine-9-carboxylic acid dihydrobromide 10 (9)

Yield: 95%

Melting point: 153-155 ° C (decomposition) NMR (DMSO, 100 MHz): δ = 1.7 (s, broad, 4 H; 2.3 (t, broad, 2 H); 2.8 (s, broad , 2H), 3.5 (s, broad, 3H), 6.5 (broad, 2H), 7.05 (broad, 2H), 7.5-8.5 (m, 12 H) ppm N- (4-Flethoxyphenyl) -β- (4- (4-succinimidoxycarbonylbutyl) benzenesulfonyl] acridine-9-carboxylic acid amide (10)

Yield: 25% Melting point: 75-80 ° C (decomposition) NMR (DMSO, 100 MHz): δ = 1.8 (broad, 4 H), 2.3 (s, 2 H); 2.85 (s, broad, 6H); 3.5 (s, broad, 3H); 6.5 (d, broad, 2H); 7.05 (d, broad, 2H); 7.5 -8.3 (m, 12 H) ppm N- (4-methoxyphenyl) -N- [4- (4-succinimidoxycarbonyl] butyl] benzenesulfonyl] -10-methylcrididinium-9-carboxylic acid amide difluorosulfonate (11)

Yield: 90% NMR (DMSO, 100 MHz); δ = 1.8 broad, 4 H); 2.3 (s, broad, 2H); 2.8 (s, broad, 6H); 3.5 (s, broad, 3H); 4.8 (broad, .30 3 H); 6.5 (broad, 2H); 7.05 (broad, 2H); 7.5-9.0 (m, 12 H) ppm IR: 3340 (broad), 3060 (w), 2930 (m), 2870 (w), 1810 (w), 1785 (w), 1740 (s) , 1695 (m), 1600 (w), 1550 (w), 1510 (m), 1460 (w), 1370 (m), 1290 (s), 1250 (s), 1205 (s), 1170 35 (s) ) cm Mass spectrum (m / z): 680 (M +, cation) 16 9 0 537

Example 3 Preparation of N- (2,4-dimethoxyphenyl) -β- (4- (2-succinimidoyloxycarbonyl) benzenesulfonyl-10-methylcrididinium-9-carboxylic acid pyramide difluorosulfonate (16a) 4 '- [N- (2,4- dimethoxyphenyl) sulfamido] -3-phenylpropionic acid benzyl ester (12a) and acridine-9-carboxylic acid hydrochloride (2) proceed in a similar manner to the synthesis of compound 6. The yields and characteristic spectral data for the various synthetic steps are shown below.

N- (2,4-Dimethoxyphenyl) -N- [4- (2-benzyloxycarbonyl) benzenesulfonyl] acridine-9-carboxylic acid amide (13a)

Yield: 50%

Melting point: 74 ° C

NMR (DMSO, 100 MHz): δ = 2.9 (d, broad, 2 H); 3.1 (broad d, 2H); 3.3 (s. 3H); 3.4 (s. 3H); 5.1 (s. 2H); 5.9-6.2 δ (m, 2H); 7.0 (d, 1H); 7.35 (s, 5H); 7.5-8.2 (m, 12 H) ppm N- (2,4-dimethoxyphenyl) -N- [4- [2-carboxyethyl} benzenesulfonyl] lacridine-9-carboxylic acid amide hydrobromide (14a) Yield: 95% NMR (DMSO, 100 MHz): δ = 2.75 (d, broad, 2H), 3.05 (d, broad, 2H), 3.3 (s, 3H), 3.5 (s, 3H), 5, 95-6.3 (m, 2H), 7.05 (d, 1H), 7.6-8.6 (m, 12H), 9.2 (s, broad, 2H) ppm N- (2,4-Dimethoxyphenyl) -4- [4- (2-succinimidoxycarbo-30-methyl] benzenesulfonyl] 1-acridine-9-carboxylic acid pyramide (15a)

Yield: 45%

Melting point: ca. 105 ° C (decomposition) NMR (DMSO, 100 MHz): δ = 2.9 (s, 4 H); 3 (broad, 2H); 3.2.35 (s, 3H); 3.4 (s, 3H); 5.9-6.3 (m, 2H); 7.0 (d, 1H); 17 9 G 5 3 7 7.5-8.4 (m, 12 H) ppm IR (KBr tablet): 3440 (broad), 3060 (w), 2930 (w), 2850 (w), 1815 (w ), 1785 (w), 1740 (s), 1695 (m), 1600 (w), 1510 (m), 1460 (w), 1440 (w), 1365 (m), 1320 (w), 1290 (w) ), Δ 1240 (m), 1210 (s), 1165 (m), 1085 (m) cm-1 N- [2,4-dimethoxyphenyl] -N- [4- (2-succinimidovloxycarbonyl] benzenesulfonyl] -110-methylcrididinium-9 -Carboxylic acid amide fluorosulfonate (16a)

Yield: 80% Melting point: ca. 135 ° C (decomposition) NMR (DMSO, 100 MHz); δ = 2.9 (s, 4 H); 2.95-4.2 (m, 10H); 4.8-5.0 (s, s, 3H); 6.05-6.25 (m, 1H); 7.6-9.0 (m, 14 H) ppm IR (KBr tablet: 3430 (m), 2950 (w), 2870 (w), 2825 (w), 1810 (w), 1780 (m)) , 1750 (s), 1695 (m), 1610 (m), 1555 (w), 1510 (m 1465 (m), 1380 (m), 1285 (m), 1250 (m), 1210 (s), 1170 (m) cm-1

Example 4 (not a compound of the invention) N- (3,4-Ethylenedioxyphenyl) -β- [4- (2-succinimido-20-hydroxycarbonyl) benzenesulfonyl] -10-methylacridine-9-carboxylic acid amide acid 1 f The preparation of onate (16b) from 4 '- [N- (3,4-ethylenedioxyphenyl) sulfamido] -3-phenylpropionic acid benzyl ester (12b) and acridine-9-carboxylic acid chloride hydrochloride (2) is carried out according to Example 1. The yields and characteristic spectral data of the different phases are shown below.

N- (3,4-ethylenedioxyphenyl) -N- [4- (2-benzyloxycarbonyl) benzenesulfonyl] acridine-9-carboxylic acid amide (13b) 30 Yield: 50%

Melting point: 91.5 ° C

NMR (DMSO, 100 MHz); S = 2.9 (d, broad, 2H); 3.2 d, broad, 2H); 4.0 (s, broad, 4H); 5.1 (s, 2H); 6.3-6.8 (m, 3H); (s, 5H); 7.6-8.3 (m, 12 H) ppm is 90537 N- (3,4-ethylenedioxyphenyl) -Ν-Γ4- (2-carbonyloxyethyl) -benzenesulfonylacridine-9-carboxylic acid amide hydrochloride (14b)

Yield: 95%

5 Melting point:> 200 ° C

NMR (DMSO, 100 MHz): δ = 2.7 (m, 2 H), 3.05 (m, 2 H); 4.0 (s, broad, 4H); 6.3-6.8 (m, 3H); 7.5-8.6 (m, 12H); 9.6 (s, broad, 2H) ppm N- (3,4-ethylenedioxyphenyl) -N-Γ 4-fuccinimidovloxycarb-10-methylethyl-benzenesulfonyl-1-acridine-9-carboxylic acid amide (15b)

Yield: 45%

Melting point: 140 ° C (decomposition) NMR (DMSO, 100 MHz) δ = 2.7-2.9 (d, s, overlapping, δ 15 H); 3.0 (d, 2H); 4.0 (s, broad, 4H); 6.3-6.8 (m, 3H); 7.5-8.4 (m, 12 H) ppm IR (KBr tablet): 3420 (broad), 3060 (m) 3060 (m), 2980 (m), 293 0 (m), 1810 ( w), 1790 (w), 1740 (m). 1695 (s), 1590 (m), 1460 (w), 1430 (w), 1410 (w), 1370 (m), 1300 (m), 1225 (s), 20 1175 (S).

N- (3,4-Ethylenedioxyphenyl) -N- [4- (succinimidoxycarbonyl) benzenesulfonyl] -10-methylchridinium-9-carboxylic acid amide fluorosulfonate (16b)

Yield: 80% Melting point: ca. 110 ° C (decomposition) NMR (DMSO, 100 MHz): δ = 2.85 (s, 4 H); 3.0-3.3 (s, s broad, 4H); 3.8-4.5 (m, broad, 4H); 4.75-5.1 (s, broad with shoulders, 3 H); 6.3-9.0 (m, 15 H) ppm Example 5 ·. N- (4-Carboxyphenyl) -4-toluenesulfonic acid amide

To a mixture of 252 g (3 mol) of sodium bicarbonate and 139.1 g (1 mol) of 4-aminobenzoic acid in 2.5 liters of water is added dropwise at 20-30 ° C 190.5 g. · (1 mol) ) toluene sulfochloride in 300 ml of i-propyl ether. The mixture is stirred vigorously for 2-4 hours until the l 19 90 537 sulfochloride is consumed. The pH of the separated aqueous solution is adjusted to 1 with concentrated hydrochloric acid, and the precipitate is dissolved in propyl acetate. The extract is washed twice with 2N hydrochloric acid and once with water, dried over sodium sulfate and evaporated. 240 g (82.5% of theory) of N- (4-carboxyphenyl) -4-toluenesulfonic acid amide are obtained.

1 H-NMR (DMSO-d 6): δ = 2.3 (s, CH 3); 7.1 (d, 2 aromatic Hs); 7.3 (d, 2 arom. H); 7.6-7.9 (m, 4 arom. H); 10.75 (broad); 12.7 (broad) 10 N- (4-Benzyloxycarbonylphenyl) -4-toluenesulfonic acid pyramide

A solution of 11.64 g (40 mmol) of N- (4-carboxyphenyl) -4-toluenesulfonic acid amide, 5.06 g (40 mmol) of benzyl chloride and 5.20 g (44 mmol) of diisopropylethylamine In 100 m of dimethylformamide, heated for 4 hours at 140 ° C. After completion of the reaction, the mixture is evaporated under reduced pressure, the residue is dissolved in propyl acetate, and the solution is washed twice with 2N hydrochloric acid and twice with saturated NaHCO3 solution, dried over sodium sulfate and evaporated. 11.8 g (78% of theory) of N- (4-benzyloxycarbonylphenyl) -4-toluenesulphonic acid amide are obtained, which is recrystallized from methanol. 1 H-NMR (DMSO-d 6): δ = 2.3 (s, CH 3); 5.3 (s, CH 2); 7.1-7.3 (dd, 4 arom. H); 7.4 (s, Cl 2); 7.7-7.9 (dd, 4 arom.

25 H); 10.8 (broad, NH) N- (4-Benzyloxycarbonylphenyl) -N- (4-toluenesulfonyl) acridine-9-carboxylic acid amide

To a solution of 1.5 g (4 mmol) of N- (4-benzyloxycarbonylphenyl) -4-toluenesulfonic acid amide, 1.23 g (4.4 mmol) of acridinic acid hydrochloride and 0.02 g of dimethylaminopyridine in 20 ml of anhydrous tetra -hydrofuran, 2.1 ml (15 mmol) of triethylamine in 10 ml of tetrahydrofuran are added dropwise at 25 ° C. The temperature is raised to 60 ° C. After completion of the reaction, the crystallized product is stirred with methanol, separated by suction filtration and recrystallized from ethyl acetate.

Yield: 1.57 g (67.0% of theory) 1 H-NMR (CDCl 3): δ = 2.5 (s, CH 3); 5.2 (s, CH 2); (s, C 6 H 5); 7.0-8.2 (m, 16 aromatic Hs) N- (4-carboxyphenyl-N- (4-toluenesulfonyl) acridine-9-carboxylic acid amide hydrobromide A batch of 1.17 g (2 mmol) of N- ( 4-Benzyloxycarbonyl-phenyl) -N- (4-toluenesulfonyl) -acridine-9-carboxylic acid acid amide is heated in 10 ml of 33% hydrogen bromide glacial acetic acid solution with stirring for 4 hours at 60 [deg.] C. After cooling, the precipitate is filtered off with suction. dried under reduced pressure.

Yield: 1.00 g (87% of theory) 15 H-NMR (TFA): δ = 2.6 (s, CH 3); 7.3-8.6 (m, 16 arom. H); 11.65 (s, NH) MS: 496 (M +) N- (4-Succinimidoxycarbonylphenyl] -N- (4-toluenesulfonylacridine-9-carboxylic acid amide) To a solution containing 0.57 g (1 mmol) of N- ( 4-Carboxyphenyl) -N- (4-toluenesulfonyl) acridine-9-carboxylic acid amide hydrobromide and 0.21 g (2 mmol) of triethylamine in 25 ml of anhydrous tetrahydrofuran are added at -15 ° C with stirring. 0.11 g (1 mmol) of ethyl ester of chloroformate The mixture is stirred at the same temperature for 1 hour, after which 0.12 g (1 mmol) of N-hydroxysuccinimide are added, and after a further hour the mixture is allowed to stand at room temperature for 15 hours. evaporate under reduced pressure, dissolve the residue in ethyl acetate, wash the solution with sodium hydrogen carbonate solution and water and dry over sodium sulfate, after evaporation to give 0.42 g (70.8% of theory) of the desired product.

1 H-NMR (TFA): δ = 2.6 (s, CH 3)? 3.1 (s, CH 2 -CH 2); 7.0-35 8.6 (m, 16 aromatic Hs) 21,90537 N- (4-Succinimidovloxycarbonylphenyl) -N-C4-toluenesulfonyl-10-methylcrididinium-9-carboxylic acid amide fluorosulfonate

To a solution of 0.59 g (1 mmol) of N- (4-succin-5-quinimidoyloxycarbonylphenyl) -N- (4-toluenesulfonyl) acridine-9-carboxylic acid amide in 30 ml of 1,2-dichloroethane is added At 25 ° C with stirring 0.85 g (7.5 mmol) of fluorosulfonic acid methyl ester. The reaction product precipitates within 4 hours. After suction filtration separation 10 and drying, 0.43 g (60.8% of theory) of the desired product are obtained.

1 H-NMR (TFA): δ = 2.6 (s, Ar-CH 3); 3.1 (s, CH 2 -CH 2); 4.9 (s, N-CH 2); 7.3-8.8 (m, 16 arom. H)

Example 6 In the same manner as in Example 5, N- (4-succinimidoyloxycarbonylmethylphenyl) -N- (4-toluenesulfonyl) -1O-methylacridinium-9-carboxylic acid fluoride is obtained from N- (4-carboxymethylphenyl) -4-toluenesulfonic acid amide. N- (4-carboxymethylphenyl) -4-toluenesulfonic acid amide 1 H-NMR (DMSO): δ = 2.3 (s, CH 3); 3.5 (s, CH 2); 7.0 AB, C6H4); 7.3-7.6 (AB, C6H4); 10.2 (s, NH) N- (4-Benzyloxycarbonylmethyl-phenyl) -4-toluenesulfonic acid amide Yield: 35% of theory 1 H-NMR (DMSO): δ = 2.3 (s, CH 3); 3.6 (s, COCH 2); (s, OCH 2); 6.9-7.7 (m, 13 arom. H); 10.2 (s, NH)

Yield: 35% of theory 1 H-NMR (CDCl 3): δ = 2.55 (s, CH 3); 3.3 (s, COCH 2); 5.0 (s, OCH 2); 6.7-8.2 (m, 21 aromatic Hs) N- (4-carboxymethylphenyl) -N- (4-toluenesulfonyl) acridine-9-carboxylic acid amide hydrobromide

Yield: 95% of theory ... 1 H-NMR (TFA): δ = 2.65 (s, CH 3); 3.55 (s, CH 2); 6.8-8.6 (m, 35 16 arom. H) 22 90537 N- (4-Succinimidoyloxycarbonylmethylphenyl) -N- (4-toluenesulfonyl) acridine-9-carboxylic acid amide Yield: 71% of theory 1 H-NMR (TFA): δ = 2.65 (s, CH3 of toluene); 3.0 (s, CH2-5 CH2); 3.6 (light, COCH 2); 6.9-8.5 (m, aroma).

N- (4-succinimidoyloxycarbonylmethylphenyl) -N- (4-toluenesulfonyl) -10-methylacridinium-9-carboxylic acid amide fluorosulfonate Yield: 80% of theory 10 1 H-NMR (TFA): δ = 2.6 (s, toluene CH3); 2.9 (s, CH 2 -CH 2); 3.6 (broad, COCH2); 4.9 (s, N-CH 3); 6.8-8.9 (m, aroma). Example 7

In the same manner as in Example 5, N- [4- (2-carboxyethyl) phenyl] -4-toluenesulfonic acid amide was obtained from N-15 [4- (2-succinimidoyloxycarbonyl) phenyl] -N-4-toluene]. enesulfonyl) -10-methylacridinium-9-carboxylic acid amide fluorosulfonate.

N- [4- (2-carboxyethyl) phenyl] -4-toluenesulfonic acid amide Yield: 44% of theory 1 H-NMR (DMSO): δ = 2.3 (s, CH 3); 2.4-2.8 (m, CH 2 -CH 2); 7.0 (AB, 4H); 7.2-7.8 (AB, 4H); 10.1 (s, NH) N- [4- (2-Benzyloxycarbonylethyl) phenyl] -4-toluenesulfonic acid amide Yield: 78% of theory 1 H-NMR (CDCl 3): δ = 2.35 (s, CH3); 2.4-3.0 (m, CH 2 -CH 2); 5.1 (s, OCH 2); 7.0-7.8 (13 arom. H, NH) N- [4- (2-Benzyloxycarbonylethyl) phenyl] -N- (4-toluenesulfonyl) acridine-9-carboxylic acid amide Yield: 77% theoretical 1 H-NMR (CDCl 3): δ = 2.2-2.8 (m, 7 H); 5.0 (s, CH 2); 6.65-7.0 (AB, 4 arom. H); 7.3-8.3 (m, 17 aromatic Hs) N- (4- (2-carboxyethyl) phenyl] -N- (4-toluenesulfonyl) -acridine-9-carboxylic acid amide hydrobromide 35 Yield: 65% of theory 23 90537 1 H-NMR (CD 3 OD): δ = 2.1-2.8 (m, CH 2 -CH 2), 2.5 (s, CH 3), 6.7-8.5 (16 arom. H) ) N- [4- (2-Succinimidoyloxycarbonylethyl) phenyl] -N- (4-toluenesulfonyl) acridine-9-carboxylic acid amide. Yield: 90% of theory 1 H-NMR (CDCl 3): δ = 2.6 (s, CH 3) 2.7 (broad, CH 2 -CH 2), 2.8 (s, CH 2 -CH 2), 6.6-8.3 (16 aro H) 4- [4- (2-succinimidoyloxycarbonyl) Phenyl] -N- (4-toluenesulfonyl) -10-methylacridinium-9-carboxylic acid 10-pyramidofluorosulfonate

Yield: 84% of theory 1 H-NMR (TFA): δ = 2.6 (s, CH 3); 2.3-3.3 (broad background, 11 H; s at 3.1); 4.9 (s, N-CH 3); 6.7-8.8 (16 arom. H).

15 Example 7a

In a manner similar to the previous examples, N-Γ4- (N-methylmorpholino-N-2-ethoxy) phenyl-N- Γ4- (2-succinimidoylcarboxyethyl) phenylsulfonyl-1-10-methylglutridine-9-carboxylic acid amidium sulfonate difluoruloxide is obtained. Deviating procedures are shown in the following reaction steps.

(4-Chlorosulfonyl) -2-phenylpropionic acid t-butyl ester 25 g (0.1 mol) of (4-chlorosulfonyl) -2-phenylpropionic acid, 12 ml of t-butanol, 60 ml of isobutene and 3 ml of concentrated sulfuric acid are combined At -15 ° C and stir vigorously in an autoclave at room temperature for 24 hours. The reaction mixture, again cooled to -15 ° C, is stirred with an excess of sodium hydrogen carbonate solution and the mixture is extracted with methylene chloride and finally evaporated under reduced pressure.

Yield: 20.4 g (67% of theory). The product is recrystallized from hexane.

1 H-NMR (CDCl 3): δ = 1.4 (s); 2.6 (m) 3.0 (t); (M); Δ 7.95 (m) MS (m / z); 305 (M * H) 24 9 0 537 (4-Chlorosulfonyl) -2-phenylpropionic acid was prepared in a known manner from 2-phenylpropionic acid and chlorosulfonic acid.

4- (Morpholino-N-2-ethoxy) aniline 5 25 g (0.1 mol) of 4- (morpholino-N-2-ethoxy) nitrobenzene are allowed to boil with 75 g of tin granules in 400 ml. half-concentrated hydrochloric acid under reflux for 4 hours. After cooling, the mixture is poured into 400 ml of 33% hydrochloric acid and extracted with isopropyl ether and the organic phase is evaporated after drying. 20 g (90% of theory) of the desired product are obtained. 1 H-NMR (CDCl 3): δ = 2.5 (t); 2.7 (7); 3.5 (wide); 3.7 (t); 4.0 (t); 6.6 (m) MS (m / z): 222 (M +) The same product is obtained by hydrogenation of a nitro compound in methanol in the presence of a palladium on carbon catalyst. The nitrogen compound was prepared from Bull. Soc. Chim. France 1955, 1353-62.

After standing at room temperature for 10 hours, N-Γ4- (morpholino-N-2-ethoxyphenyl) -N--4- (2-t-butoxycarbonyl) phenylsulfonamide was treated with 9.3 g (30 mmol) of 4-chlorosulfonylphenylpropyl. p-butyl ester of pionic acid, 6.9 g of 4- (morpholino-N-2-25 ethoxy) aniline, 0.3 g of dimethylaminopyridine and 150 ml of methylene chloride are washed with saturated sodium hydrogen carbonate solution, evaporated to one third of its volume and chromatographed on a diatomaceous earth column using a mixture of 90% methylene chloride and 10% methanol as eluent, and the main fraction of the eluate is evaporated.

Yield: 9 g (61.2% of theory) 1 H-NMR (CDCl 3): δ = 1.35 (s); 2.5 (m); 2.7-3.0 (m); 3.7 (m); 4.0 (t); 6.7-7.0 (m); 7.2-7.7 (m) MS (m / z); 491 (M + H) 35 N- [4- (Morpholino-N-2-ethoxy) phenyl] -N- [2-t-butoxycarboxylic acid ethylsulfonyl] -9-acridinecarboxylic acid oxide

To a solution of 2.0 g (4.1 mmol) of N- [4- (morpholino-N-2-ethoxy) phenyl] -N- (4- (2-t-butoxycarbonyl-5-ethyl) phenylsulfonamide) in 50 ml methylene chloride, 50 ml of 33% sodium hydroxide, 30 mg of dimethylaminopyridine, 1.2 g of tetrabutylammonium chloride and 1.55 g (5.6 mmol) of 9-acridinecarboxylic acid hydrochloride are stirred successively with vigorous stirring. After 10 hours, the orane phase is separated off, washed with water, dried over sodium sulphate and evaporated.

Yield: 2.8 g (98% of theory) 1 H-NMR (CDCl 3): δ = 1.4 (s); 2.3-2.5 (m); 2.5-2.8 (m);

3.0-3.3 (t); 3.6-3.9 (m); 6.3 (d); 6.9 (d); 7.4-8.3 (m) MS

15 (m / z): 695) M +) N- (4- (morpholino-N-2-ethoxy) phenyl) -Nf 4- [2-carboxyethyl] -phenylsulfonyl] -9-acridinecarboxylic acid pyramide 0.7 g (1 mmol) N - [4- (morpholino-N-2-ethoxy) phenyl] -N- [4- (2-t-butoxycarbonylethyl) phenylsulfonyl] -9-ac-. The trifluorocarboxylic acid amide is dissolved in any trifluoroacetic acid and the solution is allowed to stand at room temperature overnight. The solution is evaporated off with a water jet, the residue is dissolved in water and the pH of the filtered solution is adjusted to 4 with sodium acetate. The precipitated product is extracted into methylene chloride, the solution is dried over sodium sulphate and evaporated.

Yield: 0.6 g (94% of theory) 1 H-NMR (DMSO): δ = 2.6-3.0 (m); 3.0.3.3 (m); 3.6.4.0 (m); 4.0-4.4 (m); 5.8-6.6 (m); 6.8-7.0 (m); 7.3-8.4 (m).

MS: m / 2 = 639 (M +) N- [4-morpholino-N-2-ethoxy) phenyl] -N- [4- (2-succinimidoylcarboxyethyl) phenylsulfonyl] -9-acridinecarboxylamide

Yield: 95% of theory 26 90 537 1 H-NMR (CDCl 3): δ = 2,3.2.7 (m); 2.8 (s); 2.9-3.4 (m); 3.5-3.9 (m); 3.9-4.3 (m); 6.2-7.0 (m); 7.3-8.3 (m).

MS: 736 (M *) N- [4- (N-methylmorpholino-N-2-ethoxy) phenyl] -N- [4- (2-succin-5-quinimidoylcarboxyethyl) phenylsulfonyl] -10-methylacridine-9- carboxylic acid amidium difluorosulfonate Yield: 85% of theory

The compound is clearly water soluble in color. 1 H-NMR (DMSO): δ = 2.85 (s); 3.1 (s); 3.2-4.4 (m); 4.75 (s); 6.4-8.3 (m)

Example 8

Tracer preparation for TSH chemiluminescence im transformation assay

The antibody (91 μΐ, 100 μg), the acridinium derivative prepared according to Example 1 (Compound 6; 20 μΐ, 1 mg / ml in acetonitrile and conjugation buffer (0.01 M phosphate buffer, pH 8; 600 μΐ) is incubated for 15 minutes. 200 μ 200 lysine (10 mg / ml in conjugation buffer) is then added and the incubation is continued for 15 minutes 20 This mixture is applied to a PD 20 column [Selphadex® G 25 pack (spherical cross-linked dextran gel, manufactured by Pharmacia, Sweden)] and eluted using 0 as eluent. , 1 M phosphate, pH 6,3 The chemiluminescent activity of the individual fractions is tested after an appropriate dilution (350 μΐ oxidant: 0.1% H 2 O 2 in 0.1 N NaOH).

Tracer fractions (activity peak 1) are pooled and stored at 4 ° C. The tracer ready for use in the h-TSH chemiluminescence assay is suitably prepared with phosphate buffer [0.1 M phosphate, pH 6.3, 1% Tween * 20 (polyethylene sorbitan monolaurate, manufactured e.g. by ICI American Inc., USA), 0, 1% bovine serum albumin, 0.1 M NaCl, 0.01% NaN3 / dilution.

27 90537

Example 9 Implementation of h-TSH chemiluminescence immunoassays

The standard / sample (50 μΐ) and tracer (200 μΐ) are shaken for 2 hours at room temperature in monoclonal-5 anti-TSH antibody-coated tubes.

Then wash three times with 1 ml of buffer or distilled water. Light emission is achieved by adding 300 μ put of activating reagent (buffer, pH 1, 0.5% H 2 O 2) and 300 μ ja of starting reagent (0.2 N 10 NaOH) to each of the tubes through the two dispensers in the luminescence meter. The measurement time is 1 s.

Figure 3 shows a typical course of a standard curve of human thyroid stimulating hormone (h-TSH) immunological chemiluminometric assays (ICMA).

Claims (15)

1. Chemiluminescent acridinium derivative of formula (I) 1%> Αθ (I) O = C - R 4 where R 1 is hydrogen or an alkyl group containing 1-10 carbon atoms, R 4 is a group of formula (II) or (III) ) 6 R -N 2 (II) 2 SO -X-R 2 X-R 20 / -N \ 6 (HI) SO -R 2 where R 5 is a group of formula ·. 25 _ 0? R 6 is a phenyl group which may be substituted by alkyl or alkoxy groups containing 1-4 C atoms or with a morpholino (C 1 -C 3 alkoxy group, X is a phenyl group which is directly bonded to the nitrogen or sulfur atom and by a CM alkyl group to the group RA, and A 'is an anion which does not have a detrimental effect on chemiluminescence. 2. A compound according to claim 1, characterized in that X is a group of formula IV -R7 - <(0) (iv) where R7 is a substituent of formula - (CH 2) 0, where n is 0 -4. 3. Rs - SO2 - N - X - C - OZ (X) or formula XI. Ί R6 - N - SO2 - X - COOZ (XI) wherein 37 Y is halogen or an oxycarbonylalkyl, oxycarbonylaryl or imidazolidine group, X and R6 are the same as in claim 1 and Z is a carboxy protecting group which is subsequently removed , and the acid so obtained is converted to a desired acridinium derivative containing the group R5, in which the nitrogen in the acridine body is then still quaternized. Compound according to any one of the preceding claims, characterized in that it has the formula VI 10 CH 0 N - R 0 * \ *> - SO - X— CON V-1 o 2. where X is a group of formula VII -0 · “Λ · 25 where n is 2 or 4, R n and R 13 are each independently is an alkyl or alkoxy group containing 1-4 C atoms, or a morpholine (C1-C4) alkoxy group, and A 'represents the same as in claim 1. 4. A compound according to claim 3, characterized in that X is a p-ethylene phenyl group, R 12 is H and R 13 is a p-methoxyl group or R 12 is an o-methoxyl group and R 13 is a p-methoxyl group. 36 90537 5. A compound according to claim 1 or 2, characterized in that it has the formula Vili CH Αθ 5 .c X- - ° x \ (Vili) ° N 60 0 N \ J Process for the preparation of compounds according to claim 1, characterized in that a compound having the formula IX cco c (ix) // s o Y is reacted with a protected sulfonamide carboxylic acid having the formula X H 0. I II Process for the preparation of a luminescent compound, characterized in that an acridinium derivative of the formula I according to claim 1 binds directly or by means of a bridge molecule to a protein, polypeptide or some other biological substance, thereby forming a stable, immunologically active conjugate. 8. Luminescence immunoassay to determine an antigenic substance in a liquid sample, characterized in that in performing it with a competitive or sandwich method, at least one immunologically active component is immobilized on a solid phase and at least another component, the label, is a luminescent compound which can be prepared according to claim 7. 9. Luminescence immunoassay according to claim 8, characterized in that a) an immobilized antibody reacting specifically with an antigen is incubated with a sample taken from the liquid to be examined and with a conjugate of the antigen a chemiluminescent acridinium derivative according to claim 1; b) separating the sample and the unbound labeled conjugate; C) the bound labeled conjugate is successively or simultaneously contacted with one or more reagents to cause light emission; and d) the amount of antigen present is determined starting from the measured light emission power. 38 90537 10. Luminescence immunoassay according to claim 9, characterized in that, before the labeled immunoconjugate is added, the liquid to be examined is separated from the immobilized antibody. Wherein X represents the same as in claim 1 or 2 and R 6 is a phenyl group which may be substituted by a maximum of three alkyl or alkoxyl groups each containing from 1 to 4 C atoms, or with a morpholino (C 1 -C 4) alkoxy group, and X is an o-, m- or p-phenylene group. Luminescence immunoassay according to claim 8, characterized in that a) an immobilized antibody reacting specifically with an antigen is incubated with a sample taken from the liquid to be examined and with a conjugate of another, specifically reacting antibody and a chemiluminescent acridinium derivative according to claim 1; b) separating the sample and the unbound labeled conjugate; c) the bound, labeled conjugate is successively or simultaneously contacted with one or more reagents to cause light emission; and d) the amount of antigen present is determined starting from the measured light emission power. 12. Luminescence immunoassay according to claim 11, characterized in that before the labeled conjugate is added, the liquid to be examined is separated from the immobilized antibody. A luminescence immunoassay according to claim 8, characterized in that - a) an immobilized antigen that reacts specifically with an antibody is incubated with a sample taken from the liquid to be examined and with a solution of a conjugate of the antibody and a chemiluminescent acridinium derivative of claim 1; B) separating the sample and the unbound labeled conjugate; c) the bound labeled conjugate is successively or simultaneously contacted with one or more reagents to cause light emission; and (d) the amount of antigen present is determined starting from the measured light emission strength. A luminescence immunoassay according to claim 8, characterized in that a) an immobilized antigen that reacts specifically with an antibody is incubated with a solution of a conjugate of the antibody and a chemiluminescent acridinium derivative according to claim 1, b) the unreacted labeled conjugate is separated; C) the sample taken from the liquid to be examined is added; d) then the sample is separated again; e) the bound labeled conjugate is contacted successively or simultaneously with one or more reagents to cause light emission; and f) the amount of antigen present is determined starting from the measured light emission power. A luminescence immunoassay according to claim 8, characterized in that a) an immobilized antigen that reacts specifically with an antibody is incubated with a solution of a conjugate of the antibody and a chemiluminescent acridinium derivative according to claim 1; b) the sample taken from the liquid to be examined is added: (c) the sample and the unbound conjugate are separated; d) the bound, labeled conjugate is brought successively ... or simultaneously in contact with one or more reagents to cause light emission; and e) the amount of antigen present is determined starting from the measured light emission power.