EP0988288A1

EP0988288A1 - Heterocyclic chemoluminescent derivatives

Info

Publication number: EP0988288A1
Application number: EP98925343A
Authority: EP
Inventors: Gianangelo Ghitti; Michel Kohl; Robert Lejeune; Roger Renotte; Guy Sarlet
Original assignee: Biocode SA
Current assignee: Immunodiagnostic Systems SA
Priority date: 1997-06-11
Filing date: 1998-06-10
Publication date: 2000-03-29
Also published as: BE1011206A4; WO1998056765A1

Abstract

The invention concerns chemoluminescent acridinium derivatives of formula (I) in which R1 to R13 are substituents not hindering the expression of chemoluminescence, provided that at least one of the substituents R12 and R13 comprises, as single element or element binding with an acridium derivative, an atom other than carbon.

Description

HETEROCYCLIOUS CHEMOLUMINESCENT DERIVATIVES. Object of the invention.

The present invention relates to chemiluminescent derivatives of acridinium as well as a conjugate comprising one of these derivatives, linked to a specific biological component.

Another aspect of the invention relates to the diagnostic and / or assay kit comprising this conjugate or one of these derivatives, and the use of the conjugate or of one of these chemiluminescent derivatives for the assay and / or detection of a specific biological compound. Technological background underlying the invention.

Chemiluminescence is a formation of light obtained by a chemical reaction. The general mechanism of this reaction has been described in particular by Schuster et al (Advances in Physical organic Chemistry,

187-238 (1984)) by the following reaction:

A → B * → B + hv

Compound A, by a chemical, physical or biochemical reaction (most often by oxidation with a peroxide) gives a product in an excited state ("B *") which returns to the ground state by the emission of light

(H v).

This chemiluminescence process is widely used in analytical chemistry and clinical biology, especially for immunoassays (chemoluminescence imrauno assay or "CLIA").

Patent applications EP-A-273115, EP-A-257541 and EP-A-263657 describe heterocyclic chemiluminescent compounds derived from acridinium, quinolenium phenantridiniumn and isoquinolenium and their isomers, possibly conjugated to antigens, antibodies or nucleic acids for stretch used in chemiluminescence tests.

The chemiluminescent power of these derivatives is calculated by the emission of light generated in contact with hydrogen peroxide in an alkaline medium. Studies by F. McCapra (Accounts of Chemical Research (1976), 9, 6, p. 201-209) have established a mechanism for chemiluminescence of acridinium compounds. He postulates the formation of an excited reaction intermediate (dioxetane) which breaks down into C0 ₂ and methylacridone. It also involves the displacement by H ₂ 0 ₂ of a leaving group Y. This displacement would, according to generally accepted theory, be possible for derivatives having a group Y having a pKa lower than that of hydrogen peroxide (pKa <12 ).

Patent application WO95 / 19976 describes heterocyclic derivatives of acridinium, phenanthridinium, quinolinium and isoquinolenium as well as their isomers and any derivatives obtained by substitution, improved, in particular stable derivatives and / or having a high chemiluminescence, in particular when the pKa value of the leaving group is high, preferably when the pKa is greater than 12. Objects of the invention.

The object of the present invention is to obtain acridinium derivatives chemically related to those described in the abovementioned patent application 095/19976, which are easy to synthesize, which also offer numerous possibilities for adaptation, notably including a series of chemical modifications. such as grafting an arm (spacer), the aim being inter alia to orient the coupling towards functions that are usually difficult to access when labeling proteins such as phenol and thiol groups.

The present invention also aims to obtain chemiluminescent derivatives characterized by a dose-response curve allowing their use in the assay and / or diagnosis over a wide concentration range.

A final object of the present invention is to obtain a diagnostic kit comprising said active and / or stable chemiluminescent derivative. Characteristic elements of the invention.

The present invention relates to chemiluminescent acridinium derivatives of formula:

in which R _x to R ₁₃ are substituents which do not prevent the expression of chemiluminescence, it being understood that at least one of the substituents R ₁₂ and R ₁₃ comprises, as a single element or as a binding element to the derivative of acridinium, an atom other than carbon.

Preferably, in the chemiluminescent derivatives according to the invention, A is a complementary ion chosen from the halogen, nitrate, sulfate, iodomercurate, trifluoromethanesulfonate, tartrate and phthalate ions, R _x to R ₁₃ . are hydrogen or radicals of type R ₁₄ or BR ₁₄ where B represents a group or a connecting atom and R _X4 represents a hydrocarbon radical which may contain one or more heteroatoms, R ₁₂ and R ₁₃ are identical or different substituents whose function relates to chemiluminescence and to the coupling, one of the substituents R ₁₂ and R ₁₃ comprising as a single element a halogen atom or comprising as connecting element to the acridinium derivative an atom chosen from azotides and chalcogens, while the other of the substituents R ₁₂ and R ₁₃ is similar to the substituents R _x to R _X1 , with the exception of hydrogen.

Preferably, in the chemiluminescent derivatives according to the invention, B represents in the formula BR ₁₄ , a group - (CH ₂ ) n - where n represents an integer from 1 to 5, B also represents a group containing an azotide or represents a chalcogen, preferably a nitrogen group or twice-linked oxygen, R _X4 being as defined above. And more particularly, R ₁₄ is a radical chosen from the group consisting of al yl, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl and heteroarylkyl optionally substituted.

More particularly still, in these chemiluminescent derivatives, R _x and R _lx are hydrogen, one of the substituents R ₁₂ and R ₁₃ is a substituent chosen from halogens and BR ₁₄ groups, the other of the substituents R ₁₂ and R ₁₃ being similar to BR ₁₄ or R ₁₄ , B and R ₁₄ being as defined above.

According to a preferred embodiment of the invention, the chemiluminescent derivatives are the compounds;

[1-ethoxy-l- (4-carboxyphenyl) -1-iminomethane] -10- methylacridinium-9-carboxylate or {l-ethoxy-l- [4- (2-bromoethoxy) -phenyl] -l- iminomethane} - 10-methylacridinium-9-carboxylate or (l-chloro-l-phenyl-ι-iminomethane) -lθ- methylacridinium-9-carboxylate or

[1-benzyloxy-1- (4-carboxyphenyl) -1-iminomethane] - 10-methylacridinium-9-carboxylate or

[1-chloro-l- (3-cyanophenyl) -l-iminomethane] -10- methylacridinium-9-carboxylate or [l-chloro-l- (2,4,5, -trifluorophenyl) -l- iminomethane] -10 -methylacridinium-9-carboxylate or

(1-chloro-1-perfluorophenyl-1-iminomethane) -10- methylacridinium-9-carboxylate. These derivatives are designated by the references 0X_, to 0X ₇ respectively and their structural formulas are given in Table 1 below.

Another aspect of the invention relates to the conjugate comprising a chemiluminescent derivative according to the invention linked to a specific biological component.

The term "specific biological compound" means any biological molecule (lipid, saccharide, protein, peptide, nucleic acid, ...) or set of biological molecules, specific to a species (viral, bacterial, plant, animal or other) , an individual, a pathology (such as cancer or caused by a viral, bacterial or other agent), an activity or a biochemical system (such as an enzyme reaction, ...) .

Said biological component is capable of being detected and / or measured, or capable of being used for assaying and / or detecting a specific biological component. Preferably, this specific biological compound is chosen from the group consisting of antibodies, haptens, antigens, nucleic acids, agonists, cell transporters, fatty acids, lipids and / or a mixture of them. The present invention also relates to the diagnostic and / or assay kit comprising the conjugate or one of the chemiluminescent derivatives according to the invention and the use of the conjugate and / or of a chemiluminescent derivative according to the invention for the assay and / or the detection of a specific biological compound.

Brief description of the Figures.

FIG. 1 represents a general diagram for the preparation of the OX derivatives.

FIG. 2 represents the diagram of synthesis of the derivative 0X7.

FIG. 3 illustrates the kinetics of the Ab (antibodies) labeled with 0X3, 0X5, 0X6 and 0X7.

Figures 4 to 6 illustrate the stability of the Ac marked with 0X6, 0X7 and 0X5 respectively. In Figures 3 to 6, the signal is expressed by the ratio between the intensity (in RLU) measured at time t and the intensity (in RLU) measured at time t = 0. FIG. 7 represents the evolution of the concentration of product 0x7 as a function of time in a buffer at pH 8.

FIG. 8 represents an example of TSH assay with Ac labeled with 0x7.

Figure 9 shows the infrared spectrum of pentafluorobenzaldehyde.

Figure 10 shows the infrared spectrum of pentafluorobenzaldoxime. Figure 11 shows the infrared spectrum of chlorinated pentafluorobenzaldoxime.

Figure 12 shows the infrared spectrum of the coupling product of chlorinated pentafluorobenzaldoxime with 9-acridine carboxylic acid chloride, being 1-chloro-1-perfluorophenyl-1-iminomethane 9-acridine carboxylate.

FIGS. 13 to 15 represent the infrared spectra of the markers 0X7, 0X5 and 0X6.

Figures 16 to 18 show the mass spectra of the markers 0X5, 0X6 and 0X7.

The OX compounds, the formulas of which are given in Table 1, were all prepared by following the general preparation scheme described in FIG. 1. Their syntheses required the synthesis of the starting aldehyde (II) when the latter does not was not directly available. The first step is to form the oxime (III) by reaction between the aldehyde (II) and hydroxylamine. The reaction is carried out in a buffer at pH 4.5-5.

Chloroxime (IV) is prepared by bubbling chlorine through an oxime solution.

The chloro-derivatives were obtained directly by coupling of chloroxime to the acridine nucleus. The other 3 derivatives were previously treated with an equivalent of sodium alcoholate before the step of coupling to the acridine nucleus. The alcoholate is prepared from sodium and the corresponding alcohol. Compounds IV or IVb are coupled to the carboxylic acid chloride in an organic solvent supplemented with NET ₃ .

The products V or Vb are methylated by the pair of reagents CH ₃ I / HgCl ₂ or methyl triflate.

EXAMPLES EXAMPLE 1: OPERATING MODES COMMON TO CERTAIN DERIVATIVES (FIG. 1). 1. Synthesis of aldehyde II (for OX6 and OX7 derivatives).

The aldehydes used in the synthesis of the 0X6 and 0X7 derivatives were produced by reduction of the corresponding acid chlorides (2,4,5-trifluorobenzoic acid chloride or 2,3,4,5,6-pentafluorobenzoic acid). The protocol used is as follows: one equivalent of acid chloride is placed in the presence of 1.1 equivalents of Bu ₃ SnH in THF. After reaction, the THF is evaporated in vacuo and the residue treated with water. The latter is then extracted with petroleum ether (Eb 40 ° C) and then passed over charcoal. The aldehyde is obtained by evaporation under vacuum.

2. Formation of oxime III (for all derivatives).

One equivalent of aldehyde dissolved in ethanol is added with water and five equivalents of hydroxylamine hydrochloride. The pH is adjusted to a value of 5 by adding 5% NaOH. The organic solvent is evaporated and the oxime formed is extracted with toluene. The organic solution is dried over Na ₂ S0 ₄ and passed over charcoal. It is then concentrated under vacuum and added with 40 ° C petrolein. Cooling the solution to -20 ^° C causes the oxime to crystallize.

3. Transformation into chloroxime IV (for all derivatives).

The chloroxime is prepared by bubbling chlorine into an oxime solution in a CHCl ₃ / dioxane mixture previously cooled to 0 ° C. The reaction is complete and the chloroxime (III) is directly obtained by evaporation of the solvents followed, when the chloroxime is solid at room temperature, recrystallization by adding 40 ° C petrolein (the chloroxime of derivatives 0X6 and 0X7 is liquid).

4. Reaction step with an alcoholate (for OX1, OX2 and 0X4 derivatives).

Obtaining the product IVb.

The alcoholate is prepared by reaction between an equivalent of sodium and an equivalent of alcohol (ethanol or phenol). The reaction is carried out in alcohol when possible (ethanol) or in acetone

(phenol).

One equivalent of chloroxime dissolved in acetone is directly reacted with one equivalent of alcoholate. The pH of the medium is adjusted to 4.5 by adding concentrated HCl and the precipitate formed (NaCl) is removed by filtration or by centrifugation. Water is added to the solution. Evaporation of the organic solvents causes the product to crystallize.

5. Coupling to acridine (for all derivatives). Obtaining the VTb product.

The acridine carboxylic acid is transformed into acid chloride in thionyl chloride brought to reflux. After complete transformation of the acid, the thionyl chloride is evaporated under vacuum. An equivalent of the compounds prepared in steps 3

(0X3, 0X5, 0X6 and 0X7) or 4 (0X1, 0X2 and 0X4) are coupled with an equivalent of acridine carboxylic acid chloride in THF supplemented with an excess of pyridine. The THF is evaporated in vacuo and the residue taken up in toluene. The toluene phase is washed with water and then dried over anhydrous sodium sulfate. The product formed is either crystallized by adding petroleum ether (Eb 40 ° C) to the solution (0X1, 0X2, 0X3, 0X4 and 0X5) or chromatographed on a silica column using a toluene / ethyl acetate phase / acetic acid in propotions 2/1 / 0.01 (0X6 and 0X7). The fractions corresponding to the first product leaving the column are recovered and concentrated in vacuo. The addition of petroleum ether (Eb 40 ° C) causes the product to crystallize.

6. Methylation of the compounds (for all derivatives).

The products obtained in step 5 are methylated with the pair of reactants CH ₃ I / HgCl ₂ . The reaction is carried out on an intimate mixture of 50 mg of product with 50 mg of HgCl ₂ added with 1 ml of CH ₃ I. It takes place in a bomb brought to 110 ^° C for 2 h 30 min. After reaction, the bomb is refrigerated at -20 ° C. After cooling and decanting of the contents of the bomb, the supernatant is removed and the crystals of methylated product are washed successively with CH ₃ I and then with diethyl ether. The methylated product can optionally be recrystallized from an acetone / diethyl ether mixture. EXAMPLE 2: SYNTHESIS OF THE 0X7 PROBE (FIG. 2) OR

(1-C2ÏLORCH1-PERFLUOROPHENΥL-1-IMINOMETHANE) -10- METHYLACRIDINIUM-9-CARBOXYLATE.

1. Preparation of pentafluorobenzaldoxime (I).

20 g of pentafluorobenzaldehyde are dissolved in 400 ml of ethanol. On the other hand, 25 g of hydroxylamine hydrochloride are dissolved in 200 ml of water. The aqueous solution is added to the alcoholic solution and the pH of the resulting solution is brought to 5 by addition of 2N NaOH. After complete transformation (TLC), the oxime is crystallized by evaporation of the ethanol from the solution. The product is isolated by filtration and washed thoroughly with water. 17.8 g of pentafluorobenzaldoxime are recovered after drying of the precipitate (yield 82%).

2. Preparation of chloropentafluorobenzaldoxime (II).

1 g of pentafluorobenzaldoxime is dissolved in 15 ml of a dioxane-chloroform mixture (1: 1). This solution is cooled to 0 ° C and saturated with chlorine. After 90 minutes of reaction, the transformation is complete (TLC). Solvents and excess chlorine are removed under reduced pressure and the residue is used as is in the next step. 3. Coupling of chloropentafluorobenzaldoxime (II) to 9-acridine carboxylic acid chloride

(III) -

1.05 g of 9-acridine carboxylic acid is transformed into acid chloride by treatment with thionyl chloride (10 ml) containing a trace of N, N-dimethylformamide for 90 minutes at 85 ° C. The excess chloride of thionyl is removed under vacuum. The acid chloride (III) obtained in the form of the hydrochloride is suspended in 20 ml of tetrahydrofuran and neutralized with 5 equivalents (2400 μl) of triethylamine. The product obtained in the previous step is diluted in 10 ml of tetrahydrofuran and added to the acid chloride solution. After 30 minutes of reaction, the solvent is removed under vacuum and the residue is dissolved in a minimum of chloroform. The organic phase is washed three times with 40 ml of water, decanted and dried over anydre sodium sulfate. The solvent is removed on a rotary evaporator and the residue is diluted with a minimum of toluene and left to crystallize at 0 ° C overnight. The product is filtered, washed with cold toluene and then with petroleum ether. It is then dried under vacuum in the presence of a desiccant. 1.8 g of product (IV) are recovered after drying (yield 85%). 4. N-methylation of the product (IV) and obtaining of the chemiluminescent tracer OX7 (V).

50 mg of the product (IV) are dissolved in 2 ml of anhydrous dichloromethane and treated with 800 μl of a 10% solution of methyl trifluoromethanesulfonate in dichloromethane. After 4 hours of reaction, the desired product is precipitated by moderate addition of ether to the reaction medium. The tracer is isolated by filtration and washed successively with diethyl ether and then with petroleum ether. 50 mg of product (V) are recovered (yield 73%). Melting points and chromatographic data.

Compound N ° Melting point (° C) R _f CCM R _f CCM SIL SIL G UVjgj RP18W / Uy _2S ι

I 13 1 0.87 0.70 IV 206-207 0.77 0.31 V 210-213 0.00 0.27

Mobile phases CCM SIL G / UV _2S4 : toluene / ethyl acetate / acetic acid (2: 1: 0.04).

CCM DIL RP18W / UV ₂₅ _,: acetonitrile / 0.1 M phosphate buffer pH = 3 (60: 40 sodium heptanesulfonate: 0.1%). EXAMPLE 3: EXPERIMENTAL TESTS ON OX PROBES. 1. Chemiluminescence yield

The chemiluminescence yield of the OX derivatives cannot be determined when the molecules are in the free state. It is measured after coupling of these products to a protein. For example, for the derivatives 0X5, 0X6 and 0X7, the rate of incorporation on the protein is measured by a fluorescent method.

For 0x7, we can also quantify the fluorescence emitted by a degradation derivative: acridone. The quantum yield of the molecule of 0x7 conjugated to an anti-hCG antibody was evaluated at 1.10 ¹⁹ RLU / mole. 2. Emission kinetics

All the emission kinetics recorded with OX derivatives are very fast. More than 95% of the signal is emitted in the second following the injection of the reagent triggering the chemiluminescent reaction. Even when coupled, chloroxime derivatives retain this particularly rapid kinetics. For example, Figure 3 shows the kinetics observed on antibodies (Ac) labeled with chloro-derivatives. 3. Stability study

Of all the oximes synthesized, chloroximes appear by far the most stable in the presence of proteins. Measurements carried out by mass spectrometry on 0x7 show no significant variation in the spectrum of the molecule after an incubation of 45 minutes in an acetonitrile / water mixture (proportions 50/50). This chemical stability makes it possible to envisage coupling the probe to the proteins via the halogenated function of the molecule. All the stability measurements carried out show that the oximes 0X5, 0X6 and 0X7 greatly increase their stability in an aqueous medium after coupling to the proteins. The first results of a stability study on antibodies labeled with these probes are given in Figures 4, 5 and 6. This study is carried out at 25 ° C in buffers (0.1 M phosphate, 0.15 M NaCl , 0.1% NaN ₃ , 0.1% BSA) at pH 5, 6 and 7. Under the experimental conditions described below, the first results show that the stability of the tracers decreases with the basicity of the medium, which is linked to the nature of the probes. They also show that the 0X5 tracer is significantly less stable than the 0X6 and 0X7 tracers. The breaks observed between t = 470 and t = 560 hours on the curves of FIGS. 4 and 5 at pH 5 and 6 make it difficult to conclude on the exact stability of the tracers 0X6 and 0X7 at pH 5 and 6. However, the tracer 0X7 appears as being the most stable of the three, and this more particularly at pH 5. This important stability at pH 5 makes it possible to envisage a long conservation of the tracer at 4 ^° C. On the other hand, the stability of the latter at pH 7 allows to consider all possible immunoassays at this pH. The stability of the 0X7 probe is very sensitive to pH. In an acid medium (pH 5) the stability of the compound is much higher than in a basic medium (pH 8). When the probe is attached to a protein, its stability greatly increases regardless of the pH tested. 3.1 STABILITY OF THE UNCoupled OX7 PROBE AT PH 8.

The stability of the free probe was tested at 25 ° C in a medium identical to that used in the labeling reactions (pH of the medium: 8). The degradation of the product was followed by HPLC analysis using as a chromatographic support a column (4x125 mm) of RP-18 Lichrospheres (5 μm) and as mobile phase an acetonitrile / water mixture added with decane sulfonic acid, hydroxide tetramethyl ammonium and trifluoroacetic acid (final pH: 2.5).

FIG. 7 shows the evolution of the concentration of OX7 product as a function of time in a buffer at pH 8.

Parametric adjustment of the data presented by a function of type: Y = A * exp (-B * X) + C has led to the following results:

- = 0.992, - yx =,

The half-life of the free probe obtained using the parametric adjustment is 17 minutes. 3.2 STABILITY OF THE OX7 PROBE COUPLED TO ANTIBODY.

The stability of the OX7 probe coupled to an antibody was tested at pH 5, 6 and 7. It was evaluated by measuring the chemiluminescent activity of samples of labeled protein aged in phosphate / NaCl buffers supplemented with proteins. Parametric adjustment of the data collected at pH 6 and 7 leads to half-life times of the order of 600 and 375 hours. On the other hand, at pH 5, the half-life time is much longer (the small signal variations recorded over a period of approximately 2 months do not allow a valid parametric adjustment).

4.1 LABELING OF ANTI-TSH ANTIBODIES BY THE OX7 PROBE 25 μg of antibody in solution in 100 μl of labeling buffer (0.1 M sodium phosphate solution, 0.15 M NaCl adjusted to pH 8.0) are added 10 μl of a solution of 0x7-methyltriflate in acetonitrile (solution 0.5 mg / ml). After homogenization, the reaction mixture is incubated for 15 minutes at room temperature. The excess of 0X7 probe is neutralized by adding 100 μl of a lysine solution to the labeling buffer (solution 1.5 mg / ml). After a further 5 minute incubation at room temperature, the medium is chromatographed on a column (50 × 1 cm) of Sephadex G25. The chromatographic column is balanced and eluted with a 0.1 M phosphate buffer, 0.15 M NaCl. 0.1% NaN ₃ containing BSA (lg / 1) and adjusted to p 5.0. The chromatographic fractions are read by measurement of chemiluminescence. The fractions corresponding to the antibody peak are pooled and stored in the purification buffer at a temperature of 4 ° C.

4.2 DETERMINATION OF TSH WITH ANTIBODIES MARKED BY OX7.

A non-optimized TSH assay was carried out following a protocol identical to a commercial IRMA assay with anti-TSH antibodies labeled with the 0X7 probe. The protocol used includes the following steps:

• preparation of the tracer by diluting the peak of labeled antibodies in a tracer buffer at pH 7.0 (50x dilution);

• filling of the various coated tubes with 200 μl of standards (0, 0.15, 0.5, 1.5, 4, 15, 50 and 85 μl / ml); • stirring for 2 hours at room temperature;

• suction of the contents of each tube;

• rinsing of the tubes with a washing buffer (operation carried out twice) and elimination of the liquid phase by suction; • reading of the chemiluminescence of the different tubes.

The calibration curve obtained (Fig. 8) shows that antibodies labeled with the 0X7 probe can be perfectly suitable as a chemiluminescent tracer in the context of an assay requiring high sensitivity.

Claims

Chemiluminescent acridinium derivatives of formula:

in which R _x to R ₁₃ are substituents which do not prevent the expression of chemiluminescence, it being understood that at least one of the substituents R _X2 and R ₁₃ comprises, as a single element or as a binding element to the derivative of acridinium, an atom other than carbon.

2. Chemiluminescent derivatives according to claim 1, characterized in that:

A is a complementary ion chosen from the halogen, nitrate, sulfate, sulfonate, iodomercurate, trifluoromethane sulfonate, tartrate and phthalate ions,

R _x to R _xx are hydrogen or radicals of type R ₁₄ or BR ₁₄ where B represents a group or a bonding atom and R _X4 represents a hydrocarbon radical which may contain one or more heteroatoms,

R ₁₂ and R _i3 are identical or different substituents the function of which relates to chemiluminescence and to coupling, one of the substituents R ₁₂ and R ₁₃ comprising a halogen atom as a single element or comprising as a derivative binding element acridinium an atom chosen from azotides and chalcogens, while the other of the substituents R ₁₂ and R ₁₃ is similar to the substituents R _x at Ru, with the exception of hydrogen.

3. Chemiluminescent derivatives according to claim 2, characterized in that, in formula B-

B represents a group - (CH ₂ ) n-, where n represents an integer from 1 to 5, B also represents a group containing an azotide or represents a chalcogen preferably a nitrogen group or oxygen twice bonded, R ₁₄ being as defined above.

4. Chemiluminescent derivatives according to claim 3, characterized in that:

R ₁ is a radical chosen from the group consisting of optionally substituted alkyl, alkenyl, aryl, arylalkyl, heteroaryl and heteroarylkyl.

5. Chemiluminescent derivatives according to claims 3 and 4, characterized in that: R _x to R _xx are hydrogen, one of the substituents R _X2 and R _x3 is a substituent chosen from halogens and BR groups ₄ , l the other of the substituents R _X2 and R _X3 being similar to BR _X4 or to R _X4 , B and R _X4 being as defined above.

6. Chemiluminescent derivatives according to claim 5, of formula: [1-ethoxy-1- (4-carboxyphenyl) -1-iminomethane] -10- methylacridinium-9-carboxylate or

{l-ethoxy-1- [4- (2-bromoethoxy) -phenyl] -1-iminomethane} -10-methylacridinium-9-carboxylate or

(l-chloro-l-phenyl-l-iminomethane) -lθ- methylacridinium-9-carboxylate or

[1-benzyloxy-1- (4-carboxyphenyl) -1-iminomethane] - 10-methylacridinium-9-carboxylate or [1-chloro-1- (3-cyanophenyl) -1-iminomethane] -10- methylacridinium-9-carboxylate or

[l-chloro-l- (2,4,5-trifluorophenyl) -l- iminomethane] -10-methylacridinium-9-carboxylate or

(1-chloro-1-perfluorophenyl-1-iminomethane) -10- methylacridinium-9-carboxylate.

7. Conjugate comprising a chemiluminescent derivative according to any one of the claims

1 to 6 optionally linked via an arm to a specific biological component chosen from the group consisting of antibodies, haptens, antigens, nucleic acids, agonists, cell transporters, fatty acids, lipids and / or a mixture of them.

8. A diagnostic and / or assay kit comprising the conjugate according to claim 7 or a derivative according to any one of claims 1 to 6.

9. Use of the conjugate according to claim

7 or a derivative according to any one of claims 1 to 6, for the labeling of proteins, the assay and / or the detection of a specific biological component chosen from the group consisting of antibodies, haptens, antigens , fatty acids, lipids and / or a mixture of them.