HK1212369B

HK1212369B - New iridium-based complexes for ecl

Info

Publication number: HK1212369B
Application number: HK16100055.4A
Authority: HK
Inventors: Frank Bergmann; Robert CYSEWSKI; Luisa De Cola; Sebastian Dziadek; Jesus Miguel Fernandez Hernandez; Hans-Peter Josel; Christoph Seidel
Original assignee: F. Hoffmann-La Roche Ag
Priority date: 2012-08-02
Filing date: 2013-08-02
Publication date: 2018-07-13

Description

Novel iridium-based complexes for ECL

Background

The present invention relates to novel iridium-based ir (iii) luminescent complexes, conjugates comprising these complexes as labels and their use, for example, in the electrochemiluminescence-based detection of analytes.

Electrochemiluminescence (also known as electrochemiluminescence and abbreviated as ECL) is a process whereby species generated at an electrode undergo a high-energy electron transfer reaction to form a lasing state that emits light. The first detailed ECL study was described by Hercules and Bard et al in the mid 1960 s. After about 50 years of research, ECL has now become a very powerful analytical technique and is widely used in fields such as immunoassays, food and water testing, and biological warfare agent testing.

There are a large number of compounds that appear to be useful in Organic Light Emitting Devices (OLEDs). These compounds are suitable for use as solid materials or may be dissolved in organic fluids. However, there is no conclusion about its utility in aqueous media, as is required for the detection of analytes from biological samples.

Generally, ECL-based detection methods are based on the use of water-soluble ruthenium complexes containing Ru (II +) as metal ion.

Despite significant advances in the past decades, there remains a great need for more sensitive in vitro diagnostic assays based on electrochemiluminescence.

It has now surprisingly been found that certain iridium-based Ir (III +) luminescent complexes represent extremely promising labels for future high-sensitivity ECL-based detection methods.

Summary of The Invention

The invention discloses an iridium-based chemiluminescent compound of formula I

Wherein each R1-R20 is independently hydrogen, halogen, cyano or nitro, amino, substituted amino, alkylamino, substituted alkylamino, arylamino, substituted arylamino, alkylammonium, substituted alkylammonium, carboxyl, carboxylate, carbamoyl, hydroxyl, substituted or unsubstituted alkoxy, substituted or unsubstituted aryloxy, sulfanyl, substituted or unsubstituted alkylsulfonyl, substituted or unsubstituted arylsulfonyl, sulfo, sulfino, sulfeno, sulfonate, sulfinate, sulfenoate, sulfamoyl, sulfoxide, phosphonyl, hydroxyphosphinoyl, hydroxy-alkyl-phosphinoyl (phosphinoyl), phosphonate, phosphinate or R21, wherein R21 is aryl, substituted aryl, alkyl, substituted alkyl, branched alkyl, substituted branched alkyl, or R21, Arylalkyl, substituted arylalkyl, alkylaryl, substituted alkylaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, amino-alkyl, substituted amino-alkyl, amino-alkoxy, substituted amino-alkoxy, amino-aryl, substituted amino-aryl, amino-aryloxy, substituted amino-aryloxy,

wherein in R1-R12, or/and in R13-R16 or/and in R17-R20, respectively, two adjacent R may form an aromatic ring or a substituted aromatic ring, wherein the substituent is selected from the group consisting of hydrogen, alkyl, substituted alkyl, halogen, cyano or nitro, a hydrophilic group, such as amino, substituted amino, alkylamino, substituted alkylamino, alkylammonium, substituted alkylammonium, carboxyl, carboxylate, carbamoyl, hydroxyl, substituted or unsubstituted alkoxy, substituted or unsubstituted aryloxy, sulfanyl, substituted or unsubstituted alkylsulfonyl, substituted or unsubstituted arylsulfonyl, sulfo, sulfino, sulfoxo, sulfonate, sulfinate, sulfenate, sulfamoyl, sulfoxide, phosphonyl, hydroxyphosphinolylenoyl, hydroxy-alkyl-phosphinoylene, sulfenyl, sulfenate, sulfamoyl, sulfoxide, phosphonyl, hydroxyphosphinoyl, hydroxyl-alkyl-phosphinoylene, sulfenyl, sulf, A phosphonate, a phosphinate or,

wherein in R1-R12, or/and in R13-R16, or/and in R17-R20, respectively, two adjacent R may form an aliphatic ring or a substituted aliphatic ring, wherein the substituent is selected from the group consisting of hydrogen, alkyl, substituted alkyl, halogen, cyano or nitro, a hydrophilic group, such as amino, substituted amino, alkylamino, substituted alkylamino, alkylammonium, substituted alkylammonium, carboxyl, carboxylate, carbamoyl, hydroxyl, substituted or unsubstituted alkoxy, substituted or unsubstituted aryloxy, sulfanyl, substituted or unsubstituted alkylsulfonyl, substituted or unsubstituted arylsulfonyl, sulfo, sulfino, sulfenic, sulfonate, sulfinate, sulfenic acid, sulfamoyl, sulfoxide, phosphonyl, hydroxyphosphinolene, hydroxy-alkyl-phosphinoylene, hydroxyl-alkyl-phosphinoylene, sulfenic acid, sulfamoyl, sulfoxide, phosphonyl, phosphono, hydroxyl-alkyl-phosphinoylene, hydroxyl-sulfonylene, A phosphonate, a phosphinate,

wherein, if a substitution is present in any of R1-R21, the substituents in R1-R21 are each independently selected from the group consisting of halogen, cyano or nitro, a hydrophilic group such as amino, alkylamino, alkylammonium, carboxyl, carboxylate, carbamoyl, hydroxyl, alkoxy, arylalkoxy, aryloxy, alkylaryloxy, polyethyleneoxy, polypropylenoxy, sulfanyl, alkylsulfonyl, arylsulfonyl, sulfo, sulfino, sulfoxyl, sulfonate, sulfinate, sulfamoyl, sulfoxide, phosphonyl, hydroxyphosphinoyl, hydroxy-alkyl-phosphinoyl, phosphonate, phosphinate,

wherein alkyl as used herein is a straight or branched alkyl chain having a length of 1 to 20 carbon atoms or a heteroalkyl chain having a length of 1 to 20 atoms comprising 1 to 4 heteroatoms selected from O, N, P and S, wherein aryl is a 5,6 or 7 membered aryl ring system, or a 5,6 or 7 membered heteroaryl ring system comprising 1 to 3 heteroatoms selected from O, S and N,

wherein at least one of R13-R20 is-Q-Z, wherein Q is a linking group or a covalent bond, and wherein Z is a functional group, and,

wherein at least one of X and Y is N and the other of X or Y is independently N or C.

Also disclosed are conjugates comprising the above compounds and an affinity binding agent covalently bound thereto.

The invention further relates to the use of a compound or conjugate as disclosed in the present invention, in particular in an electrochemiluminescence device or an electrochemiluminescence detection system, for performing luminescence measurements or electrochemiluminescence reactions in aqueous solutions.

Furthermore, the present invention discloses a method for measuring an analyte by an in vitro method, the method comprising the steps of: (a) providing a sample suspected or known to contain the analyte; (b) contacting the sample with a conjugate according to the invention under conditions suitable for the formation of a conjugate complex of the analyte, and (c) measuring the complex formed in step (b) and thereby obtaining a measure of the analyte.

Detailed Description

As noted above, there is a need for novel metal-based chemiluminescent compounds that are suitable for use in vitro diagnostic assays.

Novel iridium-based chemiluminescent compounds of formula I

The present invention relates to iridium-based chemiluminescent compounds of formula I

Wherein each R1-R20 is independently hydrogen, halogen, cyano or nitro, amino, substituted amino, alkylamino, substituted alkylamino, arylamino, substituted arylamino, alkylammonium, substituted alkylammonium, carboxyl, carboxylate, carbamoyl, hydroxyl, substituted or unsubstituted alkoxy, substituted or unsubstituted aryloxy, sulfanyl, substituted or unsubstituted alkylsulfonyl, substituted or unsubstituted arylsulfonyl, sulfo, sulfino, sulfeno, sulfonate, sulfinate, sulfenoate, sulfamoyl, sulfoxide, phosphonyl, hydroxyphosphinolene, hydroxy-alkyl-phosphinoyl, phosphonate, phosphinate or R21, wherein R21 is aryl, substituted aryl, alkyl, substituted alkyl, branched alkyl, substituted branched alkyl, arylalkyl, substituted arylalkyl, or R21, Alkylaryl, substituted alkylaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, amino-alkyl, substituted amino-alkyl, amino-alkoxy, substituted amino-alkoxy, amino-aryl, substituted amino-aryl, amino-aryloxy, substituted amino-aryloxy,

wherein, if a substitution is present in any of R1-R21, the substituents in R1-R21 are each independently selected from the group consisting of halogen, cyano or nitro, a hydrophilic group such as amino, alkylamino, alkylammonium, carboxyl, carboxylate, carbamoyl, hydroxyl, alkoxy, arylalkoxy, aryloxy, alkylaryloxy, polyethyleneoxy, polypropylenyloxy, sulfanyl, alkylsulfonyl, arylsulfonyl, sulfo, sulfino, sulfenyl, sulfonate, sulfinate, sulfenate, sulfamoyl, sulfoxide, phosphonyl, hydroxyphosphinolylene, hydroxy-alkyl-phosphinoyl, phosphonate, phosphinate,

The compounds of formula I comprise two ligands derived from phenylphenanthridines as defined by the definitions given via formula I and one third ligand.

In one embodiment, one of R13-R20 of formula I is-Q-Z.

As known to those skilled in the art, the substituents in R1-R21 may be further substituted, for example, the alkyl-group in an aminoalkyl-group may be further substituted with a hydroxyl, amino, carboxyl, or sulfo group.

As used herein, including the appended claims, substituents have meanings that are well known to the skilled artisan.

Alkyl is preferably a straight or branched alkyl chain having a length of 1 to 20 carbon atoms, preferably having a length of 1 to 10 carbon atoms, particularly preferably having a length of 1 to 6 carbon atoms; or a heteroalkyl chain having a length of 1 to 20 atoms, preferably 1 to 10 carbon atoms, comprising 1 to 4 heteroatoms selected from O, N, P and S. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, the isomeric pentyls, the isomeric hexyls, the isomeric heptyls, the isomeric octyls, and dodecyls. In a particularly preferred embodiment, alkyl is methyl or ethyl.

The terms alkoxy and alkyloxy, and substituted alkyl and substituted alkoxy, respectively, may be used interchangeably. Alkoxy and alkyloxy represent moieties of formula-OR, wherein R is preferably an alkyl moiety as defined above. Examples of alkoxy moieties include, but are not limited to, methoxy, ethoxy, and isopropoxy.

In one embodiment, preferred substituents for the substituted alkoxy group are vinyloxy chains containing from 1 to 40 vinyloxy (ethyleneoxy) units, or containing from 1 to 20 vinyloxy units or containing from 1 to 10 vinyloxy units.

Aryl is preferably a 5-, 6-or 7-membered aryl ring system, preferably a 6-membered aryl ring system, or a 5-, 6-or 7-membered heteroaryl ring system comprising 1-3 heteroatoms selected from O, S and N, preferably a 6-membered heteroaryl ring system. In a particularly preferred embodiment, aryl is phenyl.

In one embodiment, each R1-R20 in formula I is independently hydrogen, hydroxy, substituted or unsubstituted alkoxy, substituted or unsubstituted aryloxy, sulfanyl, substituted or unsubstituted alkylsulfonyl, substituted or unsubstituted arylsulfonyl, sulfo, sulfino, sulfenic, sulfonate, sulfinate, sulfenic, sulfamoyl, or sulfoxide.

In one embodiment, each R1-R20 in formula I is independently hydrogen, substituted or unsubstituted alkoxy, substituted or unsubstituted aryloxy, substituted or unsubstituted alkylsulfonyl, substituted or unsubstituted arylsulfonyl, sulfonate, sulfinate, sulfenate, sulfamoyl, or sulfoxide.

In one embodiment, each R1-R20 in formula I is independently hydrogen, substituted or unsubstituted alkoxy, substituted or unsubstituted alkylsulfonyl, substituted or unsubstituted arylsulfonyl, sulfonate, or sulfoxide.

In one embodiment, at least one of R1-R20 of the compound according to formula I is substituted with at least one hydrophilic group.

In one embodiment, at least one of R1-R12 of the phenylphenanthridine residues comprised in formula I, formula I (a) of formula II and/or formula I (b) as defined herein, respectively, is substituted by at least one hydrophilic group, in particular by at least one hydrophilic group as defined below.

Preferred hydrophilic groups are amino groups; alkylamino, wherein alkyl represents a linear chain, such as methyl, ethyl, propyl, butyl, pentyl or a branched alkyl chain, such as isopropyl, isobutyl, tert-butyl, preferably a linear alkyl chain, such as methyl or ethyl; substituted alkylamino, containing, for example, one or two branched or straight chains bonded to the N-atom, substituted by another hydrophilic group, such as hydroxyl or sulfo, preferably containing two hydroxypropyl or hydroxyethyl residues; arylamino, wherein aryl represents an aromatic residue, such as phenyl or naphthyl, preferably phenyl; substituted arylamino having aryl groups as defined above and additional residues consisting of hydrophilic groups; alkylammonium, wherein alkyl is as defined above, and preferably a trimethylammonium residue or a triethylammonium residue; substituted alkylammonium; a carboxyl group; carboxylic acid esters, preferably alkyl esters, such as methyl or ethyl esters; a carbamoyl group; a hydroxyl group; substituted or unsubstituted alkoxy, wherein alkyl and substituted alkyl are as defined above; or an aryloxy group or a substituted aryloxy group, wherein aryl and substituted aryl are as defined above; a sulfanyl group; substituted or unsubstituted alkylsulfonyl; substituted or unsubstituted arylsulfonyl; a sulfo group; a sulfino group; a sulfenyl group; a sulfamoyl group; a sulfoxide; phosphono; a hydroxyphosphinolene group; hydroxy-alkyl-phosphonoidene; a phosphonate; a phosphonite salt.

Such hydrophilic groups are preferably selected from amino, alkylamino, substituted alkylamino, arylamino, substituted arylamino, alkylammonium, substituted alkylammonium, carboxyl, hydroxyl, sulfo, sulfoxyl, sulfamoyl, sulfoxide and phosphonate, each preferably as defined in the preceding paragraph, where applicable.

In a preferred embodiment, the hydrophilic group is selected from the group consisting of alkylamino, alkylammonium, substituted alkylammonium, carboxyl, hydroxyl, sulfo, sulfoxyl, sulfamoyl, sulfoxide, and phosphonate.

In a more particularly preferred embodiment, the hydrophilic group is selected from sulfo and sulfamoyl.

In one embodiment, at least one of R1-R12 is a substituted or unsubstituted group selected from sulfo-alkyl, sulfo-aryl, sulfo-alkoxy, sulfo-aryloxy, sulfo, sulfino-alkyl, sulfino-aryl, sulfino-alkoxy, sulfino-aryloxy, sulfino, sulfenyl-alkyl, sulfenyl-aryl, sulfenyl-alkoxy, sulfenyl-aryloxy, sulfenyl, sulfamoyl-alkyl, sulfamoyl-aryl, sulfamoyl-alkoxy, sulfamoyl-aryloxy, sulfamoyl, alkylsulfonyl-alkyl, alkylsulfonyl-aryl, alkylsulfonyl, arenesulfonyl-alkyl, or arenesulfonyl-aryl, or arenesulfonyl, or, Sulfoamino-alkyl, sulfoamino-aryl, sulfoamino-alkoxy, sulfoamino-aryloxy, sulfoamino, sulfinylamino-alkyl, sulfinylamino-aryl, sulfinylamino-alkoxy, sulfinylamino-aryloxy, sulfinamino, alkanesulfonylamino-alkyl, alkanesulfonylamino-aryl, alkanesulfonylamino-alkoxy, alkanesulfonylamino-aryloxy, alkanesulfonylamino-alkyl, arenesulfonylamino-aryl, arenesulfonylamino-alkoxy, arenesulfonylamino-aryloxy, arenesulfonylamino, alkanesulfinylamino-alkyl, alkanesulfinylamino-aryl, alkanesulfinylamino-alkoxy, alkanesulfonylamino-alkoxy, sulpho-aryl, alkanesulfonylamino-aryloxy, arenesulfonylamino-alkyl, alkanesulfinylamino-aryl, alkanesulfinylamino-alkoxy, alkanesulfonylamino-alkoxy, alkanesulfonyl, Alkanesulfinylamino-aryloxy, alkanesulfinylamino, arenesulfinylamino-alkyl, arenesulfinylamino-aryl, arenesulfinylamino-alkoxy, arenesulfinylamino-aryloxy, arenesulfinylamino, phosphono-alkyl, phosphono-aryl, phosphono-alkoxy, phosphono-aryloxy, phosphono, hydroxyphosphinoyl-alkyl, hydroxyphosphinoyl-aryl, hydroxyphosphinoyl-alkoxy, hydroxyphosphinoyl-aryloxy, hydroxyphosphinoyl, hydroxy-alkyl-phosphono-alkyl, hydroxy-alkyl-phosphono-aryl, hydroxy-alkyl-phosphono-alkoxy, hydroxy-alkyl-phosphono-aryloxy, alkoxyphosphono, Hydroxy-alkyl-phosphono, phosphonoamino-alkyl, phosphonoamino-aryl, phosphonoamino-alkoxy, phosphonoamino-aryloxy, phosphonoamino, or, where chemically matched, a salt of the above substituents, wherein alkyl is a straight or branched alkyl chain having a length of 1 to 20 carbon atoms or a heteroalkyl chain having a length of 1 to 20 atoms, comprising 1 to 4 heteroatoms selected from O, N, P and S, and wherein aryl as used herein is a 5,6 or 7 membered aryl ring system, or a 5,6 or 7 membered heteroaryl ring system comprising 1 to 3 heteroatoms selected from O, S and N.

In one embodiment, at least one of R1-R12 is a substituted or unsubstituted group selected from sulfo-alkyl, sulfo-aryl, sulfo-alkoxy, sulfo-aryloxy, sulfo, sulfamoyl-alkyl, sulfamoyl-aryl, sulfamoyl-alkoxy, sulfamoyl-aryloxy, sulfamoyl, alkanesulfonyl-alkyl, alkanesulfonyl-aryl, alkanesulfonyl, arenesulfonyl-alkyl, arenesulfonyl-aryl, arenesulfonyl, alkanesulfonylamino-alkyl, alkanesulfonylamino-aryl, alkanesulfonylamino-alkoxy, alkanesulfonylamino-aryloxy, alkanesulfonylamino, arenesulfonylamino-alkyl, arenesulfonylamino-aryl, arenesulfonyl-aryl, arenesulfonylamino-aryloxy, arenesulfonylamino, Arenesulfonylamino-alkoxy, arenesulfonylamino-aryloxy, arenesulfonylamino, phosphono-alkyl, phosphono-aryl, phosphono-alkoxy, phosphono-aryloxy, phosphono, hydroxyphosphinoyl-alkyl, hydroxyphosphinoyl-aryl, hydroxyphosphinoyl-alkoxy, hydroxyphosphinoyl-aryloxy, hydroxyphosphinoyl, hydroxy-alkyl-phosphono-alkyl, hydroxy-alkyl-phosphono-aryl, hydroxy-alkyl-phosphono-alkoxy, hydroxy-alkyl-phosphono-aryloxy, hydroxy-alkyl-phosphono, or, in the case of chemical matching, salts of the abovementioned substituents, where alkyl is a straight-chain or branched alkyl chain having a length of from 1 to 20 carbon atoms or having 1- A 20 atom length, a heteroalkyl chain comprising 1-4 heteroatoms selected from O, N, P and S, and wherein aryl as used herein is a 5,6 or 7 membered aryl ring system, or a 5,6 or 7 membered heteroaryl ring system comprising 1-3 heteroatoms selected from O, S and N.

In one embodiment, at least one of R1-R12 is sulfo-alkyl, sulfo-aryl, sulfo-alkoxy, sulfo-aryloxy, sulfo, or a salt thereof (═ sulfonate), wherein the counter ion is preferably a cation from the alkali metal group.

In one embodiment, at least one of R1-R12 is sulfo-alkyl, sulfo-alkoxy, sulfo, or a salt thereof (═ sulfonate), wherein the counterion is a cation from the alkali metal group.

In one embodiment, at least one of R1-R12 is sulfo-methyl, sulfo-alkoxy having an alkyl chain of C2-C4, or a salt thereof (═ sulfonate), wherein the counterion is a cation from the alkali metal group.

In one embodiment, at least one of the groups R1-R12 of formula I is a sulfo group.

In one embodiment, 1 to 3 of R1-R12 are not hydrogen.

In one embodiment, the counter ion is an alkali metal cation selected from the group consisting of lithium, sodium, potassium and cesium cations.

In one embodiment, the counter ion is an alkali metal cation selected from sodium cation and cesium cation.

In one embodiment, the counter ion is a cesium cation.

In one embodiment, the phenylphenanthridine residues comprised in formula I are selected from the substituted phenylphenanthridines given below.

The term "linker" as used herein has the meaning known to the person skilled in the art and relates to a molecule or group of molecules for linking molecular fragments. The linking group is characterized by having two or more chemically orthogonal functionalities on a flexible or rigid backbone. Covalent bonds are not linking groups in the sense of the present invention.

In the compounds according to the invention, Q is either a covalent bond or a linking group having a backbone length of between 1 and 200 atoms. In other words, if the backbone length is between 1 and 200 atoms, the shortest connection between the aromatic ring of the third ligand of formula I and the functional group Z consists of 1 to 200 atoms.

In the case of the presence of a ring system, the shortest number of atoms in the ring system is used when evaluating the linker length. For example, a phenylene ring can be 4 atoms long in the linking group.

In one embodiment, Q is a covalent bond or a linking group having as a backbone a straight or branched saturated, unsaturated, unsubstituted or substituted C1-C200 alkyl chain, or a 1-200 atom chain consisting of carbon atoms, substituted carbon atoms, and/or one or more atoms selected from O, N, P and S, or substituted N, P, S atoms, or a chain having as a backbone of an aromatic or non-aromatic ring system containing one or more rings or heterocycles as previously described.

In one embodiment, Q is a covalent bond or a linking group and has as a backbone a straight or branched saturated, unsaturated, unsubstituted or substituted C1-C100 alkyl chain, or a 1-100 atom chain consisting of carbon atoms, substituted carbon atoms and/or one or more atoms selected from O, N, P and S, or substituted N, P or S atoms, or a chain as previously described having a backbone of an aromatic or non-aromatic ring system containing one or more rings or heterocycles.

In one embodiment, Q is a covalent bond or a linking group and has as a backbone a straight or branched saturated, unsaturated, unsubstituted or substituted C1-C50 alkyl chain, or a 1-50 atom chain consisting of carbon atoms, substituted carbon atoms and/or one or more atoms selected from O, N, P and S, or substituted N, P or S atoms, or a chain as previously described having a backbone containing one or more cyclic or heterocyclic aromatic or non-aromatic ring systems.

In a further embodiment Q is a covalent bond or a linking group and has as backbone a straight or branched saturated, unsaturated, unsubstituted or substituted C1-C20 alkyl chain, or a 1-20 atom chain consisting of carbon atoms, substituted carbon atoms and/or one or more atoms selected from O, N, P and S, or substituted N, P or S atoms, or a chain as described before having a backbone comprising one or more cyclic or heterocyclic aromatic or non-aromatic ring systems.

In one embodiment, Q in the electrochemiluminescent complex of the invention, e.g., the linking group Q, is a straight or branched saturated, unsaturated, unsubstituted or substituted C1-C20 alkyl chain, or a C1-C20 arylalkyl chain in which, for example, the phenylene ring occupies 4 carbon atoms in length, or having an alkyl group consisting of carbon atoms, substituted carbon atoms, and/or one or more atoms selected from O, N, P and S, or a chain of 1 to 20 atoms of the skeleton consisting of substituted N, P or S atoms, or having a skeleton consisting of carbon atoms, substituted carbon atoms and one or more atoms selected from O, N, P and S, or substituted N, P or S atoms, which comprises a chain of 1 to 20 atoms of at least one aryl, heteroaryl, substituted aryl or substituted heteroaryl group (wherein, for example, the phenylene ring occupies a length of 4 atoms).

In one embodiment, Q in the compounds according to the invention, e.g. the linking group Q, is a saturated C1-C12 alkyl chain, or a C1-C12 arylalkyl chain, or a chain of 1-12 atoms having a backbone consisting of carbon atoms, substituted carbon atoms and one or more atoms selected from O, N, P and S, or substituted N, P or S atoms, or a chain of 1-12 atoms having a backbone consisting of carbon atoms, substituted carbon atoms and one or more atoms selected from O, N, P and S, or substituted N, P or S atoms, comprising at least one aryl, heteroaryl, substituted aryl or substituted heteroaryl group (wherein, e.g. the phenylene ring occupies a length of 4 atoms).

In one embodiment, Q is a covalent bond. Where Q is a covalent bond, the functional group Z is at least one of R13-R20. In one embodiment, one of R13-R20 is Z.

In one embodiment, Q-Z is maleimide.

In one embodiment, the linking group Q comprises one or more amino acids.

In one embodiment, the linking group Q comprises one or more nucleotides.

In one embodiment, both X and Y in formula I are N.

In one embodiment, the functional group Z comprised in the iridium-based complex of formula I according to the invention is selected from the group consisting of aldehydes, carboxylic acids, carboxylic esters, epoxides, N-hydroxysuccinimide esters, amino groups, halogens, hydrazines, hydroxyl, thiol, maleimido, alkynyl, azide, isocyanate, isothiocyanate and phosphoramidite.

In one embodiment, the functional group Z comprised in the iridium-based complex of formula I according to the invention is selected from carboxylic acids, N-hydroxysuccinimide esters, amino groups, halogens, thiol groups, maleimido groups, alkynyl groups, azides, isocyanates, isothiocyanates and phosphoramidites.

In a particularly preferred embodiment, the functional group Z comprised in the iridium-based complex of formula I according to the invention is selected from carboxylic acids, N-hydroxysuccinimide esters and maleimido groups.

In a particularly preferred embodiment, the functional group Z comprised in the iridium-based complex of formula I according to the invention is selected from the group consisting of N-hydroxysuccinimide ester and maleimido group.

In one embodiment, the invention relates to compounds of formula I,

wherein 1 to 3 of R1-R12 of the phenylphenanthridine residues are independently sulfo-alkyl, sulfo-aryl, sulfo-alkoxy, sulfo-aryloxy, sulfo, or salts thereof (═ sulfonates), wherein the counter ion is preferably a cation from the alkali metal group, and the other groups of R1-R12 are hydrogen,

wherein one of R13-R20 is-Q-Z, wherein Q is a linking group or a covalent bond, and wherein Z is a functional group, and the other of R13-R20 in formula 1 is hydrogen or R21, wherein R21 is alkyl, and

wherein X and Y are N.

In one embodiment, the present invention relates to compounds of formula I wherein the phenylphenanthridine residues comprised in formula I are selected from the group of substituted phenylphenanthridines as given below

And

wherein X and Y are N.

In one embodiment, the invention relates to compounds of formula I,

wherein R1-R12 are hydrogen,

wherein one of R13-R20 is-Q-Z, wherein Q is a linking group or a covalent bond, and wherein Z is a functional group, preferably a carboxylic acid, and the other of R13-R20 in formula 1 is hydrogen or R21, wherein R21 is alkyl, and

wherein X and Y are N.

Any combination of any embodiment of compounds of formula I as defined above is considered to be within the scope of the present invention.

It has now been surprisingly and unexpectedly found that iridium-based electrochemiluminescent compounds of formula I are suitable as labels for future high sensitivity ECL-based detection methods.

Novel iridium-based chemiluminescent compounds of formula II

In one embodiment, the invention relates to a compound according to formula II

Wherein in formula I (a) and in formula I (b), respectively and independently, R1-R20 are as defined for formula I,

except for the following: q of formula I is Q1 or Q2 of formula II, respectively, wherein Q1 is a linking group, preferably wherein at least one of R13-R20 of formula I (a) is-Q1-Z and wherein Q1 is a linking group;

wherein at least one of R13-R20 in formula I (b) is Q2, and each Q2 is independently a linking group or a covalent bond,

wherein (n) is an integer of 1 to 50,

wherein X and Y are as defined for formula I,

and wherein Z is a functional group.

In one embodiment, one of R13-R20 of formula I (a) in formula II is Q1-Z.

In one embodiment, one of R13-R20 in each of formulas i (b) in formula II is Q2.

In one embodiment, one of R13-R20 of formula i (a) in formula II is Q1-Z, and one of R13-R20 in each of formula i (b) in formula II is Q2.

The compounds of formula I (a) and formula I (b) comprise two ligands derived from phenylphenanthridines as defined by the definition given hereinbefore via formula I and one third ligand, respectively.

In one embodiment, R1-R20 have the same meaning as described above for R1-R20 of the compound of formula II.

In one embodiment, formula i (a) and formula i (b) are the same except for Q1-Z in formula i (a) and Q2 in formula i (b), respectively.

As the skilled person will readily recognize, the linking group Q1 of formula II comprises n branching sites where Q2 is attached.

In one embodiment, Q1 of formula II has as a backbone a straight or branched saturated, unsaturated, unsubstituted or substituted C1-C200 alkyl chain, or a 1-200 atom chain consisting of carbon atoms, substituted carbon atoms and/or one or more atoms selected from O, N, P and S, or substituted N, P, S atoms, or a chain as previously described having a backbone containing one or more cyclic or heterocyclic aromatic or non-aromatic ring systems.

In one embodiment, the linking group Q1 of formula II has as a backbone a straight or branched saturated, unsaturated, unsubstituted or substituted C1-C100 alkyl chain, or a 1-100 atom chain consisting of carbon atoms, substituted carbon atoms and/or one or more atoms selected from O, N, P and S, or substituted N, P or S atoms, or a chain as previously described having a backbone containing one or more cyclic or heterocyclic aromatic or non-aromatic ring systems.

In one embodiment, the linking group Q1 of formula II has as a backbone a straight or branched saturated, unsaturated, unsubstituted or substituted C1-C50 alkyl chain, or a 1-50 atom chain consisting of carbon atoms, substituted carbon atoms and/or one or more atoms selected from O, N, P and S, or substituted N, P or S atoms, or a chain as previously described having a backbone containing one or more cyclic or heterocyclic aromatic or non-aromatic ring systems.

In a further embodiment, the linking group Q1 of formula II has as a backbone a straight or branched, saturated, unsaturated, unsubstituted or substituted C1-C20 alkyl chain, or a chain of 1 to 20 atoms consisting of carbon atoms, substituted carbon atoms and/or one or more atoms selected from O, N, P and S, or substituted N, P or S atoms, or a chain as described before having a backbone comprising one or more cyclic or heterocyclic aromatic or non-aromatic ring systems.

In one embodiment, the linking group Q1 of formula II in the electrochemiluminescent complexes of the invention is a straight or branched, saturated, unsaturated, unsubstituted, substituted C1-C20 alkyl chain, or a C1-C20 arylalkyl chain in which, for example, the phenylene ring occupies 4 carbon atoms in length, or having an alkyl group consisting of carbon atoms, substituted carbon atoms, and/or one or more atoms selected from O, N, P and S, or a substituted N, P or S atom, or a 1-20 atom chain of the backbone, or a 1-20 atom chain, or having at least one of a carbon atom, a substituted carbon atom and one or more atoms selected from the group consisting of O, N, P and S, or a substituted N, P or S atom, comprising at least one aryl, heteroaryl, substituted aryl or substituted heteroaryl group (wherein, for example, the phenylene ring is 4 atoms in length).

In one embodiment, Q1, e.g. the linking group Q1, in the compounds according to the invention is a saturated C1-C12 alkyl chain, or a C1-C12 arylalkyl chain, or a chain of 1-12 atoms with a backbone consisting of carbon atoms, substituted carbon atoms and one or more atoms selected from O, N, P and S, or substituted N, P or S atoms, or a chain of 1-12 atoms with a backbone consisting of carbon atoms, substituted carbon atoms and one or more atoms selected from O, N, P and S, or substituted N, P or S atoms, comprising at least one aryl, heteroaryl, substituted aryl or substituted heteroaryl group (wherein, e.g. the phenylene ring occupies a length of 4 atoms).

In the compounds according to formula II, formulae i (b) and Q2 are present (n) times, wherein (n) is an integer from 1 to 50. These (n) Q2 are each independently a covalent bond or a linking group having as a backbone a straight or branched saturated, unsaturated, unsubstituted or substituted C1-C200 alkyl chain, or a 1-200 atom chain consisting of carbon atoms, substituted carbon atoms, and/or one or more atoms selected from O, N, P and S, or substituted N, P, S atoms, or a chain having as a backbone containing one or more cyclic or heterocyclic aromatic or non-aromatic ring systems as previously described.

In one embodiment, each Q2 of formula II is independently a covalent bond or a linking group having as a backbone a straight or branched saturated, unsaturated, unsubstituted or substituted C1-C100 alkyl chain, or a 1-100 atom chain consisting of carbon atoms, substituted carbon atoms, and/or one or more atoms selected from O, N, P and S, or substituted N, P or S atoms, or a chain as previously described having a backbone comprising one or more cyclic or heterocyclic aromatic or non-aromatic ring systems.

In one embodiment, each Q2 of formula II is independently a covalent bond or a linking group having as a backbone a straight or branched saturated, unsaturated, unsubstituted or substituted C1-C50 alkyl chain, or a 1-50 atom chain consisting of carbon atoms, substituted carbon atoms, and/or one or more atoms selected from O, N, P and S, or substituted N, P or S atoms, or a chain as previously described having a backbone comprising one or more cyclic or heterocyclic aromatic or non-aromatic ring systems.

In one embodiment, each Q2 of formula II is independently a covalent bond or a linking group having as a backbone a straight or branched saturated, unsaturated, unsubstituted or substituted C1-C20 alkyl chain, or a 1-20 atom chain consisting of carbon atoms, substituted carbon atoms, and/or one or more atoms selected from O, N, P and S, or substituted N, P or S atoms, or a chain as previously described having a backbone comprising one or more cyclic or heterocyclic aromatic or non-aromatic ring systems.

In one embodiment, each Q2 of formula II is independently a covalent bond or a linking group having as a backbone a saturated C1-C12 alkyl chain, or a 1-12 atom chain consisting of carbon atoms, substituted carbon atoms, and/or one or more atoms selected from O, N, P and S, or substituted N, P or S atoms, or a chain having as a backbone containing one or more cyclic or heterocyclic aromatic or non-aromatic ring systems as previously described.

In one embodiment, the linking group Q1 comprises one or more amino acids.

In one embodiment, the linking group Q1 comprises a peptide chain.

In one embodiment, the linking group Q2 comprises one or more amino acids.

In one embodiment, both the linking groups Q1 and Q2 comprise one or more amino acids.

In one embodiment, the linking group Q1 comprises one or more nucleotides.

In one embodiment, the linking group Q2 comprises one or more nucleotides.

In one embodiment, both the linking groups Q1 and Q2 comprise one or more nucleotides.

In one embodiment, the linking group Q2 is selected from-C₆H₄-(CH₂)₂-and-C₆H₄-(CH₂)₂-CO-。

In formula II, (n) is an integer from 1 to 50, indicating that formulae I (b) and Q2 are present (n) times in the compound according to formula II. In particular embodiments, (n) is an integer from 2 to 50, or from 1 to 40, or from 2 to 40, or from 3 to 31.

In formula II, (n) is an integer from 1 to 50, indicating that formulae I (b) and Q2 are present (n) times in the compound according to formula II. In particular embodiments, (n) is from 1 to 49, from 1 to 48, from 1 to 47, from 1 to 46, from 1 to 45, from 1 to 44, from 1 to 43, from 1 to 42, from 1 to 41, from 1 to 40, from 2 to 50, from 2 to 49, from 2 to 48, from 2 to 47, from 2 to 46, from 2 to 45, from 2 to 44, from 2 to 43, from 2 to 42, from 2 to 41, from 2 to 40, from 3 to 39, from 3 to 38, from 3 to 37, from 3 to 36, from 3 to 35, from 3 to 34, from 3 to 33, from 3 to 32, from 3 to 31, from 3 to 30, from 4 to 29, from 4 to 28, from 4 to 27, from 4 to 26, from 4 to 25, from 4 to 24, from 4 to 23, from 4 to 22, from 4 to 21, from 4 to 20, from 5 to 19, from 4 to 27, from 4 to 26, from 4 to 25, from 4 to 24, from 4 to 23, from 4 to 22, from 4 to 21, from 4 to 20, from 5 to 19, from 5 to 5, from 5 to 15, from 5 to 12, from 5 to 13, an integer from 5 to 11, or from 5 to 10.

In one embodiment, in formula II, (n) is 1.

In one embodiment, in formula II, (n) is 2.

In one embodiment, in formula II, (n) is 3.

In one embodiment, the functional group Z comprised in the iridium-based complex of formula II according to the present invention is selected from the group consisting of aldehydes, carboxylic acids, carboxylic esters, epoxides, N-hydroxysuccinimide esters, amino groups, halogens, hydrazines, hydroxyl, thiol, maleimido, alkynyl, azide, isocyanate, isothiocyanate and phosphoramidite.

In one embodiment, the functional group Z comprised in the iridium-based complex of formula II according to the invention is selected from the group consisting of carboxylic acid, N-hydroxysuccinimide ester, amino group, halogen, thiol, maleimido, alkynyl, azide, isocyanate, isothiocyanate and phosphoramidite.

In a particularly preferred embodiment, the functional group Z comprised in the iridium-based complex of formula II according to the invention is selected from the group consisting of N-hydroxysuccinimide ester and maleimido group.

Any combination of any embodiment of the compounds of formula II as defined above is considered to be within the scope of the present invention.

It has now been surprisingly and unexpectedly found that iridium-based chemiluminescent compounds of formula II are suitable as labels for future high sensitivity ECL-based detection methods.

Process for the preparation of compounds of formulae I and II

The present invention relates in one aspect to novel processes for the preparation of compounds of formula I and compounds of formula II, respectively.

The compounds according to formula I can be synthesized, for example, in accordance with the following method (based on Lamansky, S., Inorg. chem. 40 (2001) 1704-1711): synthesizing a substituted phenyl-phenanthridine dimer iridium complex; reacting the dimer with a precursor of Q-Z to produce a product according to formula I.

According to this process, the compounds of formula I can be obtained, for example, as shown in scheme 1 below.

Schematic diagram 1: synthesis of compounds of formula I. Reagents and conditions: (i) the method comprises the following steps Na (Na)₂CO₃2-ethoxyethanol; m is an integer from 1 to 17.

The substituted phenyl-phenanthridine dimer iridium complexes used as starting materials can be obtained, for example, by the methods shown in the examples (see example 2) and as described, for example, in EP 12179056.2.

The compounds according to formula II can be synthesized, for example, in accordance with the following method (based on Lamansky, S., Inorg. chem. 40 (2001) 1704-1711): synthesizing a substituted phenyl-phenanthridine dimer iridium complex; reacting the dimer with a precursor of a linking group Q containing 1-50 third ligand moieties to produce a product according to formula II.

According to this process, the compound of formula II can be obtained, for example, as shown in scheme 2 below.

Schematic diagram 2: synthesis of compounds of formula II. Reagents and conditions: cs₂CO₃，DMF。

The compounds according to formula II can also be synthesized in another way: the substituted phenyl-phenanthridine dimer iridium complex (see, e.g., example 2.2) is first further reacted with a derivative of a third ligand containing a functional group (-Q-) Z to give a monomeric iridium complex. Monomeric iridium complexes are given, for example, in formula I. Then reacting the monomeric iridium complex further with a precursor of Q containing 1 to 50 groups reactive with the functional groups of the monomeric iridium complex to form a covalent bond; in this way, after formation of the covalent bond, a compound according to formula II is likewise obtained.

According to this process, the compound of formula II can be obtained, for example, as shown in scheme 3 below.

Schematic diagram 3: synthesis of compounds of formula II.

Conjugates comprising novel compounds of formula I or formula II and other aspects of the invention

In one aspect, the present invention relates to a conjugate comprising an iridium-based electrochemiluminescent compound of formula I or formula II as disclosed and defined above, respectively, and a biological substance covalently bonded thereto. Examples of suitable biological substances are cells, viruses, subcellular particles, proteins, lipoproteins, glycoproteins, peptides, polypeptides, nucleic acids, Peptide Nucleic Acids (PNA), oligosaccharides, polysaccharides, lipopolysaccharides, cell metabolites, haptens, hormones, pharmacological substances, alkaloids, steroids, vitamins, amino acids and sugars.

In one embodiment, the biological substance of the conjugate according to the invention, i.e. the biological substance covalently bound to the compound according to formula I or formula II, respectively, is an affinity binding agent. Affinity binders are molecules that are capable of binding a molecule to another molecule via attraction between the molecules and thereby obtaining a stable association in which the molecules are in close proximity to each other. The result of the molecular association is the formation of a molecular complex (complex). Attractive bonding between the components of the complex is generally weaker than in covalent bonding. In this case, the binding agent is an affinity binding agent, which means that it is capable of binding an affinity complex (complex), i.e. a complex that is stable under the respective conditions (e.g. aqueous medium under standard conditions). Molecules that may participate in molecular binding include, but are not limited to, proteins, nucleic acids, carbohydrates, lipids, and small organic molecules, such as drugs. Thus, the types of complexes formed by molecular association include: protein-protein, protein-DNA, protein-hormone, protein-drug, antigen-antibody, receptor-ligand, biotin-avidin or streptavidin (streptavidin), nucleic acid-complementary nucleic acid, or receptor-receptor (antagonist) agonist.

As the skilled person will understand, in the conjugate according to the invention the functional group Z of the compound according to formula I or formula II, respectively, is used to form a covalent bond with a group on the affinity binding agent and is no longer present in this form. In case the affinity binding agent itself does not contain a group suitable for binding to or reacting with group Z, such a group can be easily introduced into the affinity binding agent by relying on established procedures.

In one aspect, the invention relates to the preparation of conjugates by reacting a functional group Z of a compound of formula I or formula II with an appropriate group of an affinity binding agent as defined herein, which is reactive with the functional group Z.

This process can be performed by the skilled person using standard methods known to the skilled person.

In one aspect, the invention relates to a conjugate obtainable by the above-described method of preparing a conjugate.

While not wishing to be further limited, for clarity, the affinity binding agent may comprise any of the following: antigens, proteins, antibodies, biotin or biotin analogues and avidin or streptavidin, sugars and lectins, enzymes, polypeptides, amino groups, nucleic acids or nucleic acid analogues and complementary nucleic acids, nucleotides, polynucleotides, Peptide Nucleic Acids (PNA), polysaccharides, metal-ion chelators, receptor agonists or receptor antagonists. For example, an affinity binding agent can be one partner of a specific binding pair (partner), wherein the other partner of the binding pair is associated with a cell surface or intracellular structure, or is a target on a cell surface or intracellular structure.

In one embodiment, the conjugate comprises a compound of formula I or formula II, and an affinity binding agent selected from the group consisting of a protein, antigen, antibody, biotin analog, avidin, streptavidin, sugar, lectin, enzyme, polypeptide, amino group, nucleic acid analog, complementary nucleic acid, nucleotide, polynucleotide, Peptide Nucleic Acid (PNA), polysaccharide, metal ion chelator, receptor agonist, or receptor antagonist bound thereto.

Preferably, the affinity binding agent is a partner or member of an affinity binding pair, or a partner or member of a specific binding pair also known to those skilled in the art.

An affinity binder has at least 10 for its target, e.g., one member of a specific binding pair (e.g., an antibody) for the other member of the specific binding pair (e.g., an antigen thereof)⁷Affinity of l/mol. The affinity binder preferably has 10 for its target⁸l/mol or even more preferably 10⁹l/mol ofAffinity force.

In one embodiment, the invention relates to a conjugate wherein the affinity binding agent is selected from the group consisting of an antigen, an antibody, biotin or a biotin analogue, avidin or streptavidin, a sugar, a lectin, a nucleic acid or nucleic acid analogue and a complementary nucleic acid, receptor and ligand.

In one embodiment, the invention relates to a conjugate wherein the affinity binding agent is selected from the group consisting of an antibody, biotin or a biotin analogue, avidin or streptavidin, and a nucleic acid.

In one embodiment, the conjugate comprises a compound of formula I or formula II, and a protein, antigen, antibody, biotin analog, avidin, streptavidin, sugar, lectin, enzyme, polypeptide, amino group, nucleic acid analog, complementary nucleic acid, nucleotide, polynucleotide, Peptide Nucleic Acid (PNA), polysaccharide, metal-ion chelator, receptor agonist, or receptor antagonist.

In one embodiment, the conjugate according to the invention comprises a covalently linked compound according to formula I or formula II, respectively as disclosed and defined above, and an affinity binding agent which is an oligonucleotide or an antibody.

The biotin analogue is aminobiotin, iminobiotin or desthiobiotin.

The term "oligonucleotide" or "nucleic acid" as used herein generally denotes a short, generally single-stranded, polynucleotide comprising at least 8 nucleotides and up to about 1000 nucleotides. In a preferred embodiment, the oligonucleotide will have a length of at least 9, 10, 11, 12, 15, 18, 21, 24, 27 or 30 nucleotides. In a preferred embodiment, the oligonucleotide will have a length of no more than 200, 150, 100, 90, 80, 70, 60, 50, 45, 40, 35 or 30 nucleotides.

The term oligonucleotide is to be understood broadly and includes DNA and RNA, as well as analogs and modifications thereof.

For example, a nucleic acid analog may contain substituted nucleotides that carry substituents at the standard bases deoxyadenosine (dA), deoxyguanosine (dG), deoxycytidine (dC), deoxythymidine (dT), deoxyuridine (dU). Examples of such substituted nucleotides are: 5-substituted pyrimidines, such as 5-methyldc, aminoallyl dU or dC, 5- (aminoethyl-3-acryloylimido) -dU, 5-propynyl-dU or-dC, 5-halo-dU or-dC; n-substituted pyrimidines, such as N4-ethyl-dC; n-substituted purines, such as N6-ethyl-dA, N2-ethyl-dG; 8-substituted purines, such as 8- [ (6-amino) -hex-1-yl ] -8-amino-dG or-dA, 8-halo-dA or-dG, 8-alkyl-dG or-dA; and 2-substituted dAs, such as 2-amino-dA.

The nucleic acid analog may contain a nucleotide or a nucleoside analog. That is, naturally occurring nucleobases can be exchanged by using nucleobase analogs such as 5-nitroindole-d-nucleosides; 3-nitro-pyrrole-d-nucleoside, deoxyinosine (dI), deoxyadenosine (dX); 7-deaza-dG, -dA, -dI or-dX; 7-deaza-8-aza-dG, -dA, -dI or-dX; 8-aza-dA, -dG, -dI, or-dX; d-m-type mycin; false dU; a pseudo-differential dC; 4-thio-dT; 6-thio-dG; 2-thio-dT; iso-dG; 5-methyl-iso-dC; N8-linked-8-aza-7-deaza-dA; 5, 6-dihydro-5-aza-dC; and vinylidene-dA or pyrrolo-dC. As will be apparent to those skilled in the art, the nucleobases in the complementary strand must be selected in such a way that duplex formation is specific. For example, if 5-methyl-iso-dC is used in one strand (e.g., (a)), then iso-dG must be used in the complementary strand (e.g., (a')).

In nucleic acid analogs, the oligonucleotide backbone must be modified to contain a substituted sugar residue in the internucleoside phosphate moiety, a sugar analog, a modification, and/or be PNA.

For example, the oligonucleotide may contain a nucleotide having a substituted deoxyribose sugar such as 2' -methoxy, 2' -fluoro, 2' -methylseleno, 2' -allyloxy, 4' -methyl dN (where N is a nucleobase, e.g., A, G, C, T or U).

For example, the carbohydrate analog is xylose; 2',4' -bridged ribose such as (2'-O, 4' -C methylene) - (oligomers known as LNA) or (2'-O, 4' -C ethylene) - (oligomers known as ENA); l-ribose, L-d-ribose, hexitol (oligomer known as HNA); cyclohexenyl (oligomer known as CeNA); altritol (oligomer known as ANA); tricyclic ribo-saccharide analogs fused to a cyclopropane ring, in which the C3 'and C5' atoms are connected by an ethylene bridge (referred to as oligomers of tricyclic DNA); glycerol (oligomer known as GNA); glucopyranose (oligomer called Homo DNA); carbaibose (replacement of tetrahydrofuran subunits with cyclopentane); hydroxymethyl-morpholine (an oligomer known as morpholino DNA).

A large number of modifications of the internucleoside phosphate moiety are also known not to interfere with the hybridisation properties, and such backbone modifications can also be combined with substituted nucleotides or nucleotide analogues. Examples are phosphorothioate oligonucleotides, phosphorodithioate oligonucleotides, phosphoramidate oligonucleotides and methylphosphonate oligonucleotides.

PNAs (with a backbone free of phosphate and d-ribose) can also be used as DNA analogs.

The above-described modified nucleotides, nucleotide analogs, and oligonucleotide backbone modifications may be incorporated into the oligonucleotides as desired within the meaning of the present invention.

The term "antibody" is used herein in the broadest sense and specifically encompasses monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity.

An "isolated" antibody is one that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of their natural environment are materials that interfere with the research, diagnostic, or therapeutic use of the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In certain embodiments, the antibody is purified (1) to greater than 95 wt% of the antibody, and in certain embodiments to greater than 99 wt%, as determined by, for example, the Lowry method; (2) to the extent sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence by employing, for example, a rotating cup sequencer, or (3) to homogeneity obtained by SDS-PAGE under reducing or non-reducing conditions using, for example, coomassie blue or silver stain. Isolated antibodies include antibodies that are in situ in recombinant cells, as at least one component of the antibody's natural environment will not be present. Typically, however, the isolated antibody will be prepared by at least one purification step.

"native antibodies" are typically heterotetrameric glycoproteins of about 150,000 daltons, consisting of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, and the number of disulfide bonds varies among heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable domain (VH) at one end, followed by multiple constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain and the light chain variable domain is aligned with the variable domain of the heavy chain. Specific amino acid residues are thought to constitute the interface between the light and heavy chain variable domains.

The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of that antibody. The variable domain of the heavy chain may be referred to as "VH". The variable domain of the light chain may be referred to as "VL". These domains are usually the most variable parts of an antibody and contain an antigen binding site.

The term "variable" refers to the fact that: certain portions of the variable domains differ widely in sequence among antibodies and are used for the binding and specificity of each particular antibody for its particular antigen. However, this variability is not evenly distributed throughout the variable domains of the antibodies. It is concentrated in three segments, which are called hypervariable regions (HVRs) in both the light and heavy chain variable domains. The more highly conserved portions of the variable domains are called Framework Regions (FR). The variable regions of native heavy and light chains each comprise four FR regions, mostly in a β -sheet configuration, connected by three HVRs, which form loops connecting, or in some cases forming part of, the β -sheet structure. The HVRs in each chain are held in close proximity to each other by the FR region and, together with HVRs from the other chain, contribute to the formation of the antigen-binding site of the antibody (see Kabat et al, Sequences of proteins of Immunological Interest, fifth edition, National Institute of Health, Bethesda, Md. (1991)). The constant domains are not directly involved in binding the antibody to the antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity.

The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two distinct types, called kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains.

Antibodies (immunoglobulins) can be assigned to different classes according to the amino acid sequence of the constant domains of their heavy chains. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA 2. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known and are generally described, for example, in Abbas et al, Cellular and mol. The antibody may be part of a larger fusion molecule consisting of covalent or non-covalent binding of the antibody to one or more other proteins or peptides.

The terms "full-length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody in its substantially intact form, rather than an antibody fragment as defined below. The term particularly refers to antibodies having heavy chains with Fc regions.

An "antibody fragment" comprises a portion of an intact antibody, which portion preferably comprises the antigen binding region thereof. Examples of antibody fragments include Fab, Fab ', F (ab')2, and Fv fragments; a diabody; a linear antibody; a single chain antibody molecule; and multispecific antibodies composed of antibody fragments.

Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each of which has a single antigen-binding site, and a residual "Fc" fragment, the name of which reflects its ability to crystallize readily. Pepsin treatment produced F (ab')2 fragments that had two antigen binding sites and were still capable of cross-linking antigens.

"Fv" is the smallest antibody fragment that contains the entire antigen-binding site. In one embodiment, a two-chain Fv species consists of a dimer of one heavy and one light chain variable domain in tight, non-covalent association. In single chain Fv (scfv) species, one heavy and one light chain variable domain may be covalently linked by a flexible peptide linker such that the light and heavy chains may bind in a "dimeric" structure similar to that in a two chain Fv species. In this configuration, the three HVRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. Overall, these six HVRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three HVRs specific for an antigen) is able to recognize and bind antigen, although with lower affinity than the intact binding site.

The Fab fragment contains both the heavy and light chain variable domains and also contains the constant domain of the light chain and the first constant domain of the heavy chain (CH 1). Fab's fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab '-SH is a marker herein for Fab' in which the cysteine residues of the constant domains carry a free thiol group. F (ab ')2 antibody fragments were initially prepared as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

"Single chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Typically, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the required structure for antigen binding. For an overview of scFv, see, e.g., Plueckthun, n: the Pharmacology of Monoclonal Antibodies, Vol 113, Rosenburg and Moore (eds.), Springer-Verlag, New York (1994), p.269-315.

The term "diabodies" refers to antibody fragments having two antigen-binding sites, which fragments comprise a heavy chain variable domain (VH) linked to a light chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between two domains on the same strand, the domain is forced to pair with the complementary domain of the other strand and two antigen binding sites are created. Diabodies may be bivalent or bispecific. Diabodies are more fully described in e.g. EP 0404097; WO 1993/01161; hudson, P.J. et al, nat. Med. 9 (2003) 129-; and Holliger, P. et al, PNAS USA 90 (1993) 6444-. Tri-and tetrabodies are also described in Hudson, P.J. et al, nat. Med. 9 (2003) 129-134.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprised in the population are identical except for possible mutations (e.g., naturally occurring mutations that may be present in minor amounts). Thus, the modifier "monoclonal" indicates the character of the antibody as not being a discrete mixture of antibodies. In certain embodiments, such monoclonal antibodies generally include antibodies comprising a polypeptide sequence that binds a target, wherein the target-binding polypeptide sequence is obtained by a method comprising selecting a single target-binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection process may be to select a unique clone from a plurality of clones (e.g., a collection of hybridoma clones, phage clones, or recombinant DNA clones). It will be appreciated that the selected target-binding sequence may be further altered, for example, to improve avidity for the target, humanise the target-binding sequence, improve its production in cell culture, reduce its immunogenicity in vivo, produce multispecific antibodies, etc., and that antibodies comprising the altered target-binding sequence are also monoclonal antibodies of the invention. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibody preparations are advantageous in that they are generally free from contamination by other immunoglobulins.

As noted above, the compounds and conjugates disclosed herein have quite advantageous properties. For example, the disclosed compounds or conjugates, respectively, exhibit high ECL efficiency. This high efficiency is also exhibited if the corresponding measurements are performed in an aqueous system, compared to many ECL markers which show high ECL efficiency when analyzed only in organic solvents. For example, many OLED dyes are typically analyzed in acetonitrile and do not dissolve in aqueous solutions or, if soluble, do not exhibit efficient electrochemiluminescence in aqueous solutions.

In a preferred embodiment, the invention relates to the use of the compounds or conjugates disclosed in the invention, respectively, for performing an electrochemiluminescence reaction in an aqueous solution. An aqueous solution is any solution comprising at least 90% water (weight to weight). Obviously, such aqueous solutions may additionally contain ingredients such as buffer compounds, detergents and tertiary amines, e.g. tripropylamine, as electron donors in ECL reactions.

In one aspect, the invention relates to the use of a compound or conjugate as disclosed in the present invention, respectively, in an electrochemiluminescence based detection method.

In one aspect, the invention relates to the use of a compound or conjugate as disclosed in the invention, respectively, in the detection of an analyte.

The analyte according to the present invention may be any inorganic or organic molecule, including any relevant biological substance. Examples of suitable biological substances which represent analytes in the sense of the present invention are cells, viruses, subcellular particles, proteins, lipoproteins, glycoproteins, peptides, polypeptides, nucleic acids, oligosaccharides, polysaccharides, lipopolysaccharides, cell metabolites, haptens, hormones, pharmacological substances, alkaloids, steroids, vitamins, amino acids and sugars.

The analyte may be selected from polypeptides, carbohydrates, and inorganic or organic drug molecules.

A polypeptide or protein is a molecule consisting essentially of amino acids and having at least two amino acids joined by peptide bonds. Where the analyte of interest is to be studied in the methods disclosed herein, the polypeptide will preferably consist of at least 5,6, 7, 8, 9, 10, 12, 15, 20, 25 and 30 up to about 10,000 amino acids. Preferably, the polypeptide will contain 5-2,000 amino acids, still preferably 10-1,000 amino acids.

Where the analyte is a nucleic acid, these are preferably naturally occurring DNA or RNA oligonucleotides.

In one aspect, the invention relates to a method of measuring an analyte by an in vitro method, the method comprising the steps of: (a) providing a sample suspected or known to contain the analyte; (b) contacting the sample with a conjugate between an affinity binding agent and a compound according to formula I or formula II, respectively, as disclosed in the present invention under conditions suitable for the formation of an analyte conjugate complex; and (c) measuring the complex formed in step (b) and thereby obtaining a measure of the analyte.

In one embodiment, measuring the analyte means measuring the amount of the analyte in the sample.

In one embodiment, the measurement for detecting the analyte in the above method is performed by using an electrochemiluminescence based detection procedure. It is also preferred to carry out the process in aqueous solution.

The following examples are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It will be appreciated that modifications may be made in the procedures set forth without departing from the spirit of the invention.

All patents and publications identified herein are hereby incorporated by reference in their entirety.

Examples

Example 1

Synthesis of substituted phenyl-phenanthridines

Example 1.1

General procedure for the synthesis of substituted 2-aminobiphenyls:

the appropriate 2-aminobiphenyls, which are required for further reactions to phenanthridines, can be synthesized using a Suzuki-Miyaura coupling reaction between commercially available 2-bromoaniline derivatives and the corresponding arylboronic acids, as described by Youn, S.W. in Tetrahedron Lett.50 (2009) 4598-4601.

Typical procedure:

a 10 mol% PdCl₂(PPh₃)₂，K₂CO₃，DMF/H₂O (5/1), 80 ℃ for 24 hours.

Other examples are as follows:

。

example 1.2

General procedure for the synthesis of substituted phenanthridines:

to an ice-cooled solution of 2-arylaniline 1 (0.01 mol) in chloroform (20 ml) was added arylacid chloride 2 (0.01 mol) and stirred at room temperature for 30 minutes under inert conditions. The resulting mixture was refluxed for an additional 2 hours with stirring. The reaction mixture was treated by dropwise addition of pyridine (0.02 mol in 10 ml chloroform) over a period of 60 minutes. The mixture was allowed to cool to room temperature and stirred overnight. The mixture was washed thoroughly with 0.5M HCl, over MgSO₄Dried and concentrated in vacuo. The crude product was purified by flash chromatography on silica gel (3: 2 hexane/ethyl acetate) to give pure product 3 in 66% yield.

Benzamido-2-biphenyl 3 (0.01 mol) and POCl in 20 ml of toluene was reacted according to the procedure described by Lion, C, in Bull. Soc. Chim. Belg. 98 (1989) 557-566₃(5 ml) was refluxed and stirred under nitrogen for 18 hours. By CH₂Cl₂The cooled reaction mixture was diluted (30 ml) and poured into ice, over 25% NH₄OH and distilled water. The organic layer was MgSO₄Dried and concentrated in vacuo, then flash chromatographed (silica gel, 1: 1 hexane/ethyl acetate) to give the product 4, 6-phenylphenanthridine.

Yield: 52 percent. A white solid.¹H NMR (CDCl₃, 400 MHz) 7.54-7.85 (m, 9H), 8.10(d, J = 8.0 Hz, 1H), 8.28 (d, J = 7.9 Hz, 1H), 8.62 (d, J = 8.4 Hz, 1H), 8.67(d, J = 8.4 Hz, 1H)。

Obtained using 2-naphthalen-2-yl-phenylamine instead of 2-arylaniline:

¹H-NMR (400 MHz, CDCl₃) 8.64 (d, J = 9.1 Hz, 2H), 8.29 (d, J = 8.1Hz, 1H), 8.16 (d, J = 8.92 Hz, 1H), 7.92 (d, J = 7.48 Hz, 1H), 7.79-7.75 (m,2H), 7.69 (t, J = 14.0, 8.2 Hz, 1H), 7.63-7.61 (m, 2H), 7.53-7.46 (m, 4H),7.19 (t, J = 14.3, 7.2 Hz, 1H).

MS: [M+H]⁺306.3。

using naphthalene-carbonyl chloride instead of phenyl acid chloride:

¹H-NMR (400 MHz, CDCl₃) 8.74(d, J = 8.3 Hz, 1H), 8.65 (d, J = 8.1Hz, 1H), 8.27 (d, J = 8.1 Hz, 1H), 8.23 (s, 1H), 8.15 (d, J = 8.3 Hz, 1H),8.03 (d, J = 8.4 Hz, 1H), 7.97-7.94 (m, 2H), 7.90-7.85 (m, 2H), 7.80-7.69 (m,2H), 7.62 (t, J= 14.2, 7.1 Hz, 1H), 7.59-7.55 (m, 2H).

MS: [M+H]⁺306.3。

example 1.3

Procedure for the Synthesis of 6- (2-sulfophenyl) phenanthridine

By reacting an arylaniline (0.01 mol) with 2-sulfobenzoic acid cyclic anhydride (0.01 mol) in CH, using the procedure described, for example, in Nicolai, E, chem, Pharm, Bull, 42 (1994) 1617-₃CN is heated at moderate temperature for 6 hours to synthesize the 6- (2-sulfophenyl) phenanthridine.

After purification, the product can be converted into the appropriate phenanthridine based on the procedure described in example 1.2.

Example 1.4

Procedure for the Synthesis of 6-phenyl-alkylsulfonylphenanthridines

Using the procedure as described in Lion, C. in Bull. Soc. Chim. Belg. 98 (1989) 557-566, 6-phenyl-alkylsulfonylphenanthridines can be synthesized by reacting alkylsulfonyl-arylanilines (0.01 mol) with benzoyl chloride (0.01 mol) in chloroform at moderate temperatures and with heating, see example 1.2.

¹H-NMR (400 MHz, CDCl₃) 8.92 (d, J = 8.7 Hz, 1H), 8.75 (d, J = 1.9Hz, 1H), 8.68 (d, J = 7.0 Hz, 1H), 8.35 (dd, J = 8.7, 2.0 Hz, 1H), 8.30 (d, J= 7.2 Hz, 1H), 7.89 (t, J = 15.3, 7.1 Hz, 1H), 7.81-7.73 (m, 3H), 7.64-7.56(m, 3H) 3.12 (s, 3H).

MS: [M+H]+ 334,3。

6- (4-Methylsulfophenyl) phenanthridine was also prepared by following the procedure described in Cymerman, J. in J. chem. Soc. (1949) 703-707.

Example 1.5

Synthesis of 6- [4- (2- {2- [2- (2-methoxy-ethoxy) -ethoxy ] -ethoxy } -ethoxy) -phenyl ] -phenanthridine

Synthesis of 2,5,8, 11-Tetraoxatridecan-13-ol tosylate

The procedure is as follows: (JACS, 2007, 129, 13364) 2,5,8, 11-tetraoxatridecan-13-ol (7 g, 33.6 mmol) and triethylamine (4.9 ml, 35.3 mmol) in anhydrous CH₂Cl₂To a solution in (100 ml) was added 4-toluenesulfonyl chloride (6.7 g, 35.3 mmol) and DMAP (120 mg). The mixture was stirred at room temperature for 20 hours. The reaction mixture was washed with 80mL of HCl (1M) followed by water. The extract was purified over anhydrous MgSO₄Dry, filter and evaporate the filtrate. The residue was used in the next step without further purification.

Yield: 11.0 g (90%)

NMR:

¹H NMR (400 MHz, CDCl₃) 7.75 – 7.64 (m, 2H), 7.31 – 7.26 (m, 2H),4.16 – 4.06 (m, 2H), 3.62 (m 2H), 3.59 – 3.40 (m, 10H), 3.30 (s, 3H), 2.38(s, 3H).

¹³C{¹H} NMR (101 MHz, CDCl₃) 144.75 (s), 132.90 (s), 129.77 (s),127.8 (s), 71.82 (s), 70.60 (s), 70.48 (s), 70.47 (s), 70.41 (s), 70.39 (s),69.23 (s), 68.55 (s), 58.90 (s), 21.53 (s)。

Synthesis of 4-PEG 4-Ethyl benzoate:

the procedure is as follows: (JACS, 2007, 129, 13364) Synthesis of Ethyl 2,5,8, 11-tetraoxatridecan-13-yl 4-methylbenzenesulfonate (8.1 g, 22.3 mmol), Ethyl 4-hydroxybenzoate (3.7 g, 22.3 mmol), K₂CO₃A mixture of (15.4 g, 111.5 mmol) and 18-crown-6 (0.59 g, 2.2 mmol) was refluxed in acetone (120 ml) for 22 hours. The reaction mixture was concentrated and extracted with ethyl acetate. Extracting with hydrogen₂O washing, over anhydrous MgSO₄Dried and filtered. The filtrate was evaporated to dryness and the residue was purified by column chromatography on silica gel (dichloromethane/methanol ═ 100: 1) to give the compound（1.93 g，88％）。

Yield: 7 g (88%)

NMR:

¹H NMR (400 MHz, CDCl₃) 8.01 – 7.84 (m, 2H), 6.96 – 6.85 (m, 2H),4.29 (q,J= 7.1 Hz, 2H), 4.12 (dd,J= 5.4, 4.3 Hz, 2H), 3.82 (dd,J= 5.4,4.2 Hz, 2H), 3.71 – 3.56 (m, 10H), 3.51 – 3.45 (m, 2H), 3.32 (s, 3H), 1.32(t,J= 7.1 Hz, 3H).

¹³C{¹H} NMR (101 MHz, CDCl₃) 166.29 (s), 162.47 (s), 131.45 (s),123.01 (s), 114.11 (s), 71.90 (s), 70.84 (s), 70.60 (s), 70.59 (s), 70.58(s), 70.48 (s), 69.51 (s), 67.54 (s), 60.57 (s), 58.98 (s), 14.35 (s).

MS(+):

[M+Na⁺]⁺= calculated value 379.1727, found value 379.1743.

Synthesis of 4-PEG 4-benzoic acid:

the procedure is as follows: (JACS, 2007, 129, 13364) Compound 4- (2,5,8, 11-Tetraoxatridecan-13-yloxy) benzoic acid Ethyl ester (7 g, 19.6 mmol) and KOH (2.3 g, 41.24 mmol) in 200 mL EtOH/H₂The mixture in O (1: 1, v/v) was refluxed overnight. After cooling, the mixture was neutralized with HCl (2N). The resulting mixture was extracted with EtOAc and evaporated to dryness. The resulting white solid was recrystallized from EtOAc/hexanes.

Yield: 5.3 g (85%)

NMR:

¹H NMR (300 MHz, CDCl₃) 11.17 (s, 1H), 8.14 – 7.89 (m, 2H), 7.03 –6.75 (m, 2H), 4.29 – 4.02 (m, 2H), 3.92 – 3.81 (m, 2H), 3.78 – 3.57 (m, 10H),3.57 – 3.46 (m, 2H), 3.35 (s, 3H).

¹³C{¹H} NMR (75 MHz, CDCl₃) 171.46 (s), 163.24 (s), 132.30 (s),121.98 (s), 114.33 (s), 71.96 (s), 70.91 (s), 70.67 (s), 70.66 (s), 70.64(s), 70.54 (s), 69.55 (s), 67.66 (s), 59.08 (s).

MS(-):

[M-H]^-= calculated value 327.1438, found value 327.1456.

Synthesis of N-biphenyl-2-yl-4- (2- {2- [2- (2-methoxy-ethoxy) -ethoxy ] -ethoxy } -ethoxy) -benzamide:

the procedure is as follows: to a solution of 4- (2,5,8, 11-tetraoxatridec-13-yloxy) benzoic acid (3 g, 9.14 mmol), 0.2 mL of DMF in 30 mL of anhydrous DCM at 0 deg.C was added oxalyl chloride (1.05 mL, 12.34 mmol). The reaction mixture was stirred at 0 ℃ for 1 hour. The solution was concentrated to dryness. The oily residue was used in the next step without further purification.

A solution of 2-phenylaniline (1.6 g), pyridine (2.4 mL) in chloroform (80 mL) was cooled to 0 deg.C under an inert atmosphere, 20 mL of (phenyl-4- (2,5,8, 11-tetraoxatridec-13-yloxy) benzoyl chloride (3.1 g, 9.14 mmol) was slowly added to the solution and the final mixture was brought to room temperature, the solution was refluxed for 2 hours and stirred at room temperature overnight the reaction mixture was washed with HCl (1M, 2 × 100 mL), NaHCO₃(100 mL) and water (50 mL). With MgSO₄The organic phase was dried and purified by chromatography on silica gel (EtOAc/hexanes).

Yield: 4.1 (90%)

NMR:

¹H NMR (400 MHz, CDCl₃) 8.49 (dd,J= 8.3, 0.9 Hz, 1H), 7.94 (s,1H), 7.61 – 7.35 (m, 9H), 7.33 – 7.25 (m, 1H), 7.19 (m, 1H), 6.91 – 6.84 (m,2H), 4.16 – 4.10 (m, 2H), 3.85 (m, 2H), 3.77 – 3.58 (m, 10H), 3.56 – 3.49 (m,2H), 3.36 (s, 3H).

¹³C{¹H} NMR (101 MHz, CDCl₃) 164.56 (s), 161.65 (s), 138.18 (s),135.12 (s), 132.32 (s), 129.97 (s), 129.39 (s), 129.22 (s), 128.66 (s),128.57 (s), 128.16 (s), 127.13 (s), 124.18 (s), 121.23 (s), 114.57 (s), 71.95(s), 70.89 (s), 70.64 (s), 70.63 (s), 70.54 (s), 69.54 (s), 67.63 (s), 59.04(s), 53.51 (s).

MS(+)

[M+H]⁺= calculated value 480.2386, found value 480.2383; [ M + Na;)]⁺= calculated value 502.2200, found value 502.2204.

Synthesis of 6- [4- (2- {2- [2- (2-methoxy-ethoxy) -ethoxy ] -ethoxy } -ethoxy) -phenyl ] -phenanthridine:

the procedure is as follows: reacting N-biphenyl-2-yl-4- (2- {2- [2- (2-methoxy-ethoxy) -ethoxy ] -ethyl]-ethoxy } -ethoxy) -benzamide (4 g, 8.34 mmol), POCl₃(10 ml) was refluxed in 10 ml of toluene for 20 hours. The mixture was cooled to room temperature and 100 ml of dichloromethane were added. The solution was poured into ice and combined with NH₄The mixture was neutralized with OH (20%). The organic phase is extracted and washed successively with distilled water and brine and with MgSO₄And (5) drying. The resulting solution was purified by flash chromatography (silica gel, in 1: 1 ethyl acetate/hexane, R_f0.14).

Yield: 1 g (25%)

NMR:

¹H NMR (300 MHz, CDCl₃) 8.68 (d,J= 8.3 Hz, 1H), 8.59 (dd,J= 8.1,1.4 Hz, 1H), 8.23 (dd,J= 8.1, 1.1 Hz, 1H), 8.15 (dd,J= 8.3, 0.7 Hz, 1H),7.84 (ddd,J= 8.3, 7.1, 1.3 Hz, 1H), 7.79 – 7.57 (m, 5H), 7.15 – 7.03 (m,2H), 4.29 – 4.19 (m, 2H), 3.93–3.90 (m, 2H), 3.80 – 3.60 (m, 12H), 3.59 –3.49 (m, 2H), 3.37 (s, 3H).

¹³C{¹H} NMR (75 MHz, CDCl₃) 160.92 (s), 159.45 (s), 143.84 (s),133.59 (s), 131.26 (s), 130.61 (s), 130.26 (s), 129.05 (s), 128.90 (s),127.19 (s), 126.85 (s), 125.39 (s), 123.70 (s), 122.29 (s), 122.01 (s),114.68 (s), 72.02 (s), 70.97 (s), 70.74 (s), 70.72 (s), 70.69, 70.62 (s),69.80 (s), 67.68 (s), 59.15 (s).

MS (+) JM358-F5, [M+H]⁺Calculated = 462,2280, found 462.2275.

Synthesis of 3- (4-phenanthridin-6-yl-phenoxy) -propane-1-sulfonic acid cesium salt

6- (4-methoxyphenyl) phenanthridine was prepared by cyclization of N- (biphenyl-2-yl) -4-methoxybenzamide (2 g, 6.59 mmol) according to the procedure described above. The compound was purified by chromatography in dichloromethane/hexane (gradient 1: 5 to 1: 1). Yield: 87 percent.

NMR:¹H NMR (300 MHz, DMSO) 8.94 (d,J= 8.2 Hz, 1H), 8.84 (dd,J=8.2, 1.2 Hz, 1H), 8.18 – 8.05 (m, 2H), 7.97 (ddd,J= 8.3, 7.1, 1.3 Hz, 1H),7.86 – 7.62 (m, 5H), 7.23 – 7.07 (m, 2H), 3.88 (s, 3H).

¹H NMR (300 MHz, CDCl₃) 8.70 (d,J= 8.3 Hz, 1H), 8.61 (dd,J= 8.1,1.3 Hz, 1H), 8.28 (d,J= 8.0 Hz, 1H), 8.18 (dd,J= 8.3, 0.7 Hz, 1H), 7.86(ddd,J= 8.3, 7.1, 1.3 Hz, 1H), 7.81 – 7.56 (m, 5H), 7.18 – 7.02 (m, 2H),3.92 (s, 3H).

¹³C NMR (75 MHz, CDCl₃) 160.95 (s), 160.33 (s), 143.72 (s), 133.67(s), 132.12 (s), 131.36 (s), 130.71 (s), 130.20 (s), 129.13 (s), 128.97 (s),127.23 (s), 126.92 (s), 125.40 (s), 123.73 (s), 122.33 (s), 122.03 (s),114.03 (s), 55.57 (s).

MS [ESI-MS (+)]: [M+H⁺]^-Found 286.1231, calculated 286.1226.

4-phenanthridin-6-yl-phenol: deprotection of 6- (4-methoxyphenyl) phenanthridine is achieved by using HBr. A suspension of 6- (4-methoxyphenyl) phenanthridine (1 g, 3.5 mmol) in 15 mL (HBr, 47%) is refluxed at 100 ℃ for 12 h. The mixture was allowed to cool to room temperature, poured into ice water, and taken over Na₂CO₃And (4) neutralizing. The resulting precipitate was filtered off and washed with water and Et₂And O washing. The solid was purified by column chromatography using dichloromethane/MeOH. Yield: 90 percent.

NMR:¹H NMR (300 MHz, DMSO) 9.84 (s, 1H), 8.92 (d,J= 8.2 Hz, 1H),8.82 (dd,J= 8.2, 1.2 Hz, 1H), 8.20 – 8.11 (m, 1H), 8.08 (dd,J= 8.1, 1.2Hz, 1H), 8.02 – 7.88 (m, 1H), 7.84 – 7.64 (m, 3H), 7.64 – 7.49 (m, 2H), 7.06– 6.89 (m, 2H).

MS [ESI-MS (-)]: [M-H⁺]^-Found 270.0922, calculated 270.0924.

To a solution of 4- (phenanthridin-6-yl) phenol (320 mg, 1.18 mmol) in DMF (4 ml) was added Cs₂CO₃(482.2 mg, 1.48 mmol) and 1, 3-propylsultone (159 mg, 1.30 mmol). The reaction mixture was stirred at room temperature overnight. The reaction mixture was concentrated to dryness and the residue was purified by column chromatography (silica) using dichloromethane/MeOH (gradient 10: 1 to 5: 1). Yield: 72 percent

NMR: 1H NMR (300 MHz, DMSO-d₆) 8.98 – 8.87 (m, 1H), 8.83 (dd, J =7.9, 1.6 Hz, 1H), 8.12 (m, 2H), 7.97 (ddd, J = 8.3, 7.0, 1.3 Hz, 1H), 7.85 –7.69 (m, 3H), 7.67 (d, J = 8.6 Hz, 2H), 7.14 (d, J = 8.7 Hz, 2H), 4.19 (t, J= 6.5 Hz, 2H), 2.64 – 2.57 (m, 2H), 2.15 – 1.97 (m, 2H).

MS [EI-MS (-)]: [M-Cs⁺]Calculated 392.0956, found 392.0962.

Example 2

General procedure for the synthesis of chloro-crosslinked dimer complexes:

the general procedure is disclosed in Nonoyama, M.J. organomet. chem. 86 (1975) 263-267.

The iridium dimer was synthesized as follows: IrCl under nitrogen₃•3H₂O and 2.5 equivalents of 6-phenylphenanthridine were heated at 120 ℃ for 18 hours in a 2-ethoxyethanol/water mixture (3: 1, v/v). After cooling to room temperature, the precipitate was filtered off and successively treated with methanol and Et₂O washed and dried to provide the desired dimer.

Example 2.1

Complexes with unsubstituted phenylphenanthridines

[ (6-Phenylphenanthridine)₂IrCl]₂。

Yield: 71 percent. Brown solid.¹H NMR (DMSO-d₆, 400 MHz) 6.45 (d, J = 6.8, 4H),6.58 (t, J = 7.1, 13.9 Hz, 4H), 6.95 (t, J = 7.1, 14.2 Hz, 4H), 7.56 (t, J =7.4, 16.0 Hz, 4H), 7.68 (t, J = 8.1, 16.2 Hz, 4H), 7.93 (t, J = 8.0, 14.6 Hz,4H), 8.07-8.13 (m, 8H), 8.80 (d, J = 7.3 Hz, 4H), 8.93-9.01 (m, 12H)。

Example 2.2

Complexes with substituted phenylphenanthridines

Reacting 6- [4- (2- {2- [2- (2-methoxy-ethoxy) -ethoxy ] -ethyl]-ethoxy } -ethoxy) -phenyl]Phenanthridine (1 g, 2.16 mmol), IrCl₃·3H₂O (346 mg, 0.98 mmol) in 16 ml of 2-EtOEtOH: H₂The mixture in O (12: 4) was refluxed overnight under a nitrogen atmosphere. The reaction mixture was cooled to room temperature, and 60 ml of water was added to obtain an oily precipitate. The supernatant was discarded, and 50 ml of water was added to the residue. The mixture was stirred for 1 hour to obtain a red-brown precipitate. The solid was filtered and washed with water (50 ml) and Et₂O (30 ml) wash. The brown solid was dissolved in a smaller amount of dichloromethane and washed with water by adding Et₂And (4) precipitating O. It was used in the subsequent steps without further purification.

Yield: 550 mg (50%)

NMR:

¹H NMR (300 MHz, CDCl₃) 8.74 (d, J = 8.1 Hz, 4H), 8.36 (dd, J = 8.0,5.2 Hz, 8H), 7.90 (dd, J = 14.7, 7.7 Hz, 8H), 7.81 (d, J = 9.0 Hz, 4H), 7.79-7.67 (m, 4H), 6.78-6.65 (m, 4H), 6.32 (dd, J = 8.8, 2.5 Hz, 4H), 5.89-5.83 (m, 4H), 5.28 (d, J = 2.5 Hz, 4H), 3.67-3.10 (m, 100H, PEG chain, containing certain impurities)

MS(ESI-MS(+)):

[M+2Na⁺]²⁺Calculated value 1171.3463, found value 1171.3473; [ (C ^ N)₂Ir]⁺= calculated value 1113.3877, found value 1113.3892.

Synthesis of bis-iridium complexes with 3- (4-phenanthridin-6-yl-phenoxy) -propane-1-sulfonic acid cesium salt

Ligand 3- (4- (phenanthridin-6-yl) -phenoxy) propane-1-sulfonic acid caesium (500 mg, 0.92 mmol) and IrCl₃(159.5 mg, 0.45 mmol) in 2-EtOEtOH: H₂O (3: 1, 16 ml) mixtureThe mixture in the composition was refluxed for 36 hours under a nitrogen atmosphere. The reaction mixture was filtered and the filtrate was concentrated to dryness. The residue was used in the next step without further purification.

MS [ESI-MS(-)]：[Ir(C^N)₂-2Cs⁺]^-Calculated 975.13858, found 975.13882.

Example 3

Synthesis of Ir (6-Phenylphenanthridine) as chloride salt₂Carboxypropylphenyl-bipyridine complexes

Under nitrogen atmosphere, dimer [ (6-phenylphenanthridine)₂IrCl]₂A mixture of (163 mg, 0.110 mmol) and 4- (4-methyl-2, 2-bipyridin-4' -yl) -butyric acid (60 mg, 0.232 mmol) was heated in 2-ethoxyethanol (15 ml) at reflux overnight. The product was precipitated with water and filtered off. With Et₂O washing the residue. The complex has R in TLC (dichloromethane/MeOH 10: 1)_f= 0.57. The product was purified by column chromatography (silica) using dichloromethane/MeOH (gradient 10: 1 to 5: 1). Yield: 25 percent of

NMR:¹H NMR (300 MHz, DMSO-d₆) 9.22 – 89.17 (t, J = 6.0 Hz, 2H),8.86-8.80 (t, J = 9.0 Hz, 2H), 8.66-8.60 (t, J = 6.0 Hz, 2H), 8.53-8.48 (m,2H), 8.40 – 8.38 (m, 2H), 8.11-7.99 (m, 6H), 7.62-7.59 (d, J = 9.0 Hz, 2 H),7.52-7.46 (t, J = 9.0 Hz, 2H), 7.38-7.34(t, J = 6.0 Hz, 2H), 7.29-7.24(t, J =9.0 Hz, 2H),7.13-7.09 (m, 2H), 6.90 – 6.85 (m, 2H), 6.90 – 6.85 (m, 2H), 6.88– 6.75 (t, J = 9.0 Hz, 2H), 2.21 – 2.17 (m, 2H), 1.83 – 1.77 (m, 2H), 1.55 –1.42 (m, 2H).

MS [ESI-MS(+)]: [M]⁺Calculated value 957.27936, found value957.27754。

Claims

1. Iridium-based chemiluminescent compounds of formula I

Wherein each R1-R20 is independently hydrogen, halogen, cyano or nitro, amino, substituted amino, alkylamino, substituted alkylamino, arylamino, substituted arylamino, alkylammonium, substituted alkylammonium, carboxyl, carboxylate, carbamoyl, hydroxyl, substituted or unsubstituted alkoxy, substituted or unsubstituted aryloxy, sulfanyl, substituted or unsubstituted alkylsulfonyl, substituted or unsubstituted arylsulfonyl, sulfo, sulfino, sulfeno, sulfonate, sulfinate, sulfenoate, sulfamoyl, sulfoxide, phosphonyl, hydroxyphosphinolene, hydroxy-alkyl-phosphinoyl, phosphonate, phosphinate or R21, wherein R21 is aryl, substituted aryl, alkyl, substituted alkyl, branched alkyl, substituted branched alkyl, arylalkyl, substituted arylalkylalkyl, Alkylaryl, substituted alkylaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, amino-alkyl, substituted amino-alkyl, amino-alkoxy, substituted amino-alkoxy, amino-aryl, substituted amino-aryl, amino-aryloxy, substituted amino-aryloxy,

wherein in R1-R12, or/and in R13-R16 or/and in R17-R20, respectively, two adjacent R optionally form an aromatic ring or a substituted aromatic ring, wherein the substituents are selected from hydrogen, alkyl, substituted alkyl, halogen, cyano or nitro, a hydrophilic group, or,

wherein in R1-R12, or/and in R13-R16, or/and in R17-R20, respectively, two adjacent R optionally form an aliphatic ring or a substituted aliphatic ring, wherein the substituents are selected from hydrogen, alkyl, substituted alkyl, halogen, cyano or nitro, hydrophilic groups,

wherein, if there is a substitution in any of R1-R21, the substituents in R1-R21 are each independently selected from halogen, cyano or nitro, hydrophilic groups,

wherein the alkyl used is a straight or branched alkyl chain having a length of 1 to 20 carbon atoms or a heteroalkyl chain having a length of 1 to 20 atoms and containing 1 to 4 heteroatoms selected from O, N, P and S, wherein aryl is a 5,6 or 7 membered aryl ring system, or a 5,6 or 7 membered heteroaryl ring system containing 1 to 3 heteroatoms selected from O, S and N,

wherein at least one of R13-R20 is-Q-Z, wherein Q is a linking group, and wherein Z is a functional group, and,

wherein at least one of X and Y is N and the other of X or Y is independently N or C,

wherein Q has as a backbone a straight or branched saturated, unsaturated, unsubstituted or substituted C1-C200 alkyl chain; a chain of 1 to 200 atoms consisting of carbon atoms, substituted carbon atoms and/or one or more atoms selected from O, N, P and S, or substituted N, P, S atoms; a linear or branched, saturated, unsaturated, unsubstituted or substituted C1-C200 alkyl chain having a backbone comprising one or more cyclic or heterocyclic aromatic or non-aromatic ring systems; or a chain of 1 to 200 atoms consisting of carbon atoms, substituted carbon atoms and/or one or more atoms selected from O, N, P and S, or substituted N, P, S atoms, having a skeleton comprising one or more cyclic or heterocyclic aromatic or non-aromatic ring systems,

wherein the functional group Z is selected from the group consisting of aldehyde, carboxylic acid ester, epoxide, N-hydroxysuccinimide ester, amino, halogen, hydrazine, hydroxyl, thiol, maleimido, alkynyl, azide, isocyanate, isothiocyanate and phosphoramidite,

wherein at least one of R1-R12 is sulfo-alkyl, sulfo-aryl, sulfo-alkoxy, sulfo-aryloxy, sulfo, or a sulfonate thereof, wherein the counter ion is preferably a cation from the alkali metal group.

2. The compound of claim 1, wherein Q has as a backbone a linear or branched saturated, unsaturated, unsubstituted or substituted C1-C100 alkyl chain; a chain of 1 to 100 atoms consisting of carbon atoms, substituted carbon atoms and/or one or more atoms selected from O, N, P and S, or substituted N, P or S atoms; a linear or branched, saturated, unsaturated, unsubstituted or substituted C1-C100 alkyl chain having a backbone comprising one or more cyclic or heterocyclic aromatic or non-aromatic ring systems; or a chain of 1 to 100 atoms consisting of carbon atoms, substituted carbon atoms and/or one or more atoms selected from O, N, P and S, or substituted N, P or S atoms, having a skeleton comprising one or more cyclic or heterocyclic aromatic or non-aromatic ring systems.

3. The compound of claim 2, wherein Q has as a backbone a straight or branched saturated, unsaturated, unsubstituted or substituted C1-C50 alkyl chain; a chain of 1 to 50 atoms consisting of carbon atoms, substituted carbon atoms and/or one or more atoms selected from O, N, P and S, or substituted N, P or S atoms; a linear or branched, saturated, unsaturated, unsubstituted or substituted C1-C50 alkyl chain having a backbone comprising one or more cyclic or heterocyclic aromatic or non-aromatic ring systems; or a chain of 1 to 50 atoms consisting of carbon atoms, substituted carbon atoms and/or one or more atoms selected from O, N, P and S, or substituted N, P or S atoms, having a skeleton comprising one or more cyclic or heterocyclic aromatic or non-aromatic ring systems.

4. The compound of claim 1, wherein Q has as a backbone a straight or branched saturated, unsaturated, unsubstituted or substituted C1-C20 alkyl chain; a chain of 1 to 20 atoms consisting of carbon atoms, substituted carbon atoms and/or one or more atoms selected from O, N and S; a linear or branched, saturated, unsaturated, unsubstituted or substituted C1-C20 alkyl chain having a backbone comprising one or more cyclic or heterocyclic aromatic or non-aromatic ring systems; or a chain of 1 to 20 atoms consisting of carbon atoms, substituted carbon atoms and/or one or more atoms selected from O, N and S, having a skeleton comprising one or more cyclic or heterocyclic aromatic or non-aromatic ring systems.

5. A conjugate comprising a compound according to any one of claims 1-4 covalently bonded to an affinity binding agent.

6. A conjugate according to claim 5, wherein the affinity binding agent is selected from the group consisting of antigens and antibodies, biotin or aminobiotin, iminobiotin or desthiobiotin, and avidin or streptavidin, sugars and lectins, nucleic acids or nucleotides and complementary nucleic acids containing substitutions carrying substituents at the standard bases deoxyadenosine (dA), deoxyguanosine (dG), deoxycytidine (dC), deoxythymidine (dT), deoxyuridine (dU), and receptors and ligands.

7. The conjugate of claim 5 or 6, wherein the affinity binding agent is a nucleic acid or an antibody.

8. Use of a compound according to any one of claims 1 to 4 or a conjugate according to any one of claims 5 to 7 for performing an electrochemiluminescence reaction in an aqueous solution.

9. Use of a compound according to any one of claims 1 to 4 or a conjugate according to any one of claims 5 to 7 in an electrochemiluminescence-based detection method.

10. Use of a compound according to any one of claims 1 to 4 or a conjugate according to any one of claims 5 to 7 in the detection of an analyte.

11. A method of measuring an analyte by an in vitro method, the method comprising the steps of:

a) providing a sample suspected or known to contain an analyte;

b) contacting the sample with a conjugate according to any one of claims 5 to 7 under conditions suitable for formation of an analyte conjugate complex; and

c) measuring the complex formed in step b) by using an electrochemiluminescence based detection procedure and thereby obtaining the amount of the analyte in the sample.

12. A compound according to formula II

Wherein R1-R20 are as defined for formula I in claim 1, with the exception that Q of formula I is Q1 or Q2 in formula II, respectively, wherein Q1 is a linking group, wherein at least one of R13-R20 in formula I (b) is Q2, and each Q2 is independently a linking group or a covalent bond, wherein n is an integer from 1 to 50, wherein X and Y are as defined for formula I, and wherein Z is a functional group.