HK1213265B - New iridium-based complexes for ecl - Google Patents

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 made over 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 X and Y are C-R18 and C-R19, respectively, or wherein X is N and Y is C-R19, or wherein Y is N and X is C-R18,

wherein R1-R19 are each 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, sulfinyl, sulfeno, sulfonate, sulfinate, sulfenate, sulfamoyl, sulfoxide, phosphonyl, hydroxyphosphinyl, hydroxy-alkyl-phosphinoyl, phosphonate, phosphinate or R20, wherein R20 is aryl, substituted aryl, alkyl, substituted alkyl, branched alkyl, substituted branched alkyl, aralkyl, phosphonato or R20, wherein R20 is aryl, substituted aryl, alkyl, substituted alkyl, branched alkyl, substituted branched alkyl, aralkyl, or substituted alkyl Substituted aralkyl, alkaryl, substituted alkaryl, 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-R19, or/and between R16 and R19, respectively, two adjacent R's may 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 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, sulfinyl, sulfenyl, sulfenate, sulfonate, sulfinate, sulfenate, sulfamoyl, sulfoxide, phosphonyl, hydroxyphosphinolene, hydroxy-alkyl-phosphinoyl, hydroxyl-alkyl-phosphinoyl, hydroxyl-substituted alkyl-nitro, a hydrophilic group such as amino, substituted amino, alkylamino, substituted alkylamino, alkylsulfinyl, A phosphonate, a phosphinate, or,

wherein in R1-R12, or/and in R13-R16, or/and in R17-R19, or/and between R16 and R19, respectively, two adjacent R's may form an aliphatic ring or a substituted aliphatic ring, wherein the substituents are 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, sulfinyl, sulfenyl, sulfenate, sulfonate, sulfinate, sulfenate, sulfamoyl, sulfoxide, phosphonyl, hydroxyphosphinolene, hydroxy-alkyl-phosphinoyl, hydroxyl-substituted alkyl, halogen, cyano or nitro, a hydrophilic group such as amino, substituted amino, alkylamino, substituted or unsubstituted aryl, A phosphonate, a phosphinate,

wherein, if present in any of R1-R19, the substituents in R1-R19 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, sulfeno, sulfonate, sulfinate, sulfenoate, sulfamoyl, sulfoxide, phosphonyl, hydroxyphosphinolylene, hydroxy-alkyl-phosphinoyl, phosphonate, phosphinate,

wherein alkyl as used herein is a straight or branched alkyl chain of 1 to 20 carbon atoms in length or a heteroalkyl chain of 1 to 20 atoms in length 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-R19 is-Q-Z, wherein Q is a linking group or a covalent bond, and wherein Z is a functional group.

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

The invention further relates to the use of the compounds or conjugates disclosed in the invention for performing luminescence measurements or electrochemiluminescence reactions in aqueous solutions, in particular in electrochemiluminescence devices or electrochemiluminescence detection systems.

Furthermore, the present invention discloses a method for measuring an analyte by an in vitro method, comprising the steps of: (a) providing a sample suspected or known to contain an analyte; (b) contacting the sample with a conjugate of the invention under conditions suitable for formation of an analyte conjugate complex; 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 X and Y are C-R18 and C-R19, respectively, or wherein X is N and Y is C-R19, or wherein Y is N and X is C-R18,

wherein R1-R19 are each 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, sulfenyl, sulfonate, sulfinate, sulfenate, sulfamoyl, sulfoxide, phosphonyl, hydroxyphosphinolene, hydroxy-alkyl-phosphinoylene, phosphonate, phosphinate or R20, wherein R20 is aryl, substituted aryl, alkyl, substituted alkyl, branched alkyl, substituted branched alkyl, aralkyl, substituted aralkyl, or R20, 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-R19, or/and between R16 and R19, respectively, two adjacent R's 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, sulfinyl, sulfenyl, sulfenate, sulfonate, sulfinate, sulfenate, sulfamoyl, sulfoxide, phosphonyl, hydroxyphosphinolene, hydroxy-alkyl-phosphinoyl, hydroxyl-substituted alkyl, halogen, cyano or nitro, a hydrophilic group such as amino, substituted or unsubstituted arylsulfonyl, sulfo, A phosphonate, a phosphinate, or,

wherein in R1-R12, or/and in R13-R16, or/and in R17-R19, or/and between R16 and R19, respectively, two adjacent R's 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, sulfinyl, sulfenyl, sulfenate, sulfonate, sulfinate, sulfenate, sulfamoyl, sulfoxide, phosphonyl, hydroxyphosphinolene, hydroxy-alkyl-phosphinoyl, hydroxyl-substituted alkyl, halogen, cyano or nitro, a hydrophilic group such as amino, substituted or unsubstituted arylsulfonyl, sulfo, A phosphonate, a phosphinate,

wherein, if present in any of R1-R19, the substituents in R1-R19 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, sulfeno, sulfonate, sulfinate, sulfenoate, sulfamoyl, sulfoxide, phosphonyl, hydroxyphosphinolylene, hydroxy-alkyl-phosphinoyl, phosphonate, phosphinate,

wherein alkyl as used herein is a straight or branched alkyl chain of 1 to 20 carbon atoms in length or a heteroalkyl chain of 1 to 20 atoms in length 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-R19 is-Q-Z, wherein Q is a linking group or a covalent bond, and wherein Z is a functional group.

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

In one embodiment, one of R13 to R19 of formula I is-Q-Z.

The substituents in R1-R20 may be further substituted, for example, the alkyl group in an aminoalkyl group may be further substituted with a hydroxy, amino, carboxy or sulfo group, as known to the skilled person.

As used herein, including the appended claims, a substituent has a meaning well known to the skilled artisan.

The alkyl group 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 of 1 to 20 atoms in length containing 1 to 4 heteroatoms selected from O, N, P and S, preferably having a length of 1 to 10 carbon atoms. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, the isomeric pentyl groups, the isomeric hexyl groups, the isomeric heptyl groups, the isomeric octyl groups, and dodecyl groups. 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 refer to moieties of the 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, a preferred substituent for a substituted alkoxy group is a vinyloxy chain containing 1 to 40 vinyloxy (ethyleneoxy) units, or containing 1 to 20 vinyloxy units or containing 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, in formula I, each R1-R19 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 acid, sulfamoyl, or sulfoxide.

In one embodiment, in formula I, R1-R19 are each 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, in formula I, R1-R19 are each 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 to R19 of the compound of formula I is substituted with at least one hydrophilic group.

In one embodiment, at least one of R1 to 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, alkyl meaning 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 with an additional hydrophilic group such as hydroxyl or sulfo, preferably such substituted alkylamino containing two hydroxypropyl or hydroxyethyl residues; arylamino, aryl refers to an aromatic residue, such as phenyl or naphthyl, preferably phenyl; substituted arylamino having an aryl group as defined above and an additional residue formed from a hydrophilic group; alkylammonium, the alkyl group being as defined above, 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 alkyloxy, alkyl and substituted alkyl are as defined above; or aryloxy or substituted aryloxy, aryl and substituted aryl being 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.

Preferably, such hydrophilic groups are selected from amino, alkylamino, substituted alkylamino, arylamino, substituted arylamino, alkylammonium, substituted alkylammonium, carboxyl, hydroxyl, sulfo, sulfeno, sulfamoyl, sulfoxide and phosphonate, each preferably as defined in the preceding paragraph when 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 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, arylsulfonyl-alkyl, or arylsulfonyl-aryl, or arenesulfonyl, Sulfonamido-alkyl, sulfonamido-aryl, sulfonamido-alkoxy, sulfonamido-aryloxy, sulfonamido, alkanesulfonylamino-alkyl, alkanesulfonylamino-aryl, alkanesulfonylamino-alkoxy, alkanesulfonylamino-aryloxy, alkanesulfonylamino-alkyl, arenesulfonylamino-aryl, arenesulfonylamino-alkoxy, arenesulfonylamino-aryloxy, arenesulfonylamino-alkyl, alkanesulfinylamino-aryl, alkanesulfinylamino-alkoxy, alkanesulfinylamino-aryloxy, alkanesulfonylamino-aryl, arenesulfonylamino-alkyl, alkanesulfinylamino-aryl, alkanesulfinylamino-alkoxy, alkanesulfinylamino-aryloxy, alkanesulfonylamino-, Alkanesulfinylamino, arenesulfinylamino-alkyl, arenesulfinylamino-aryl, arenesulfinylamino-alkoxy, arenesulfinylamino-aryloxy, arenesulfinylamino, phosphono-alkyl, phosphono-aryl, phosphono-alkoxy, phosphono-aryloxy, phosphono, hydroxyphosphinonyl-alkyl, hydroxyphosphinonyl-aryl, hydroxyphosphinonyl-alkoxy, hydroxyphosphinonyl-aryloxy, hydroxyphosphinonyl, hydroxy-alkyl-phosphono-alkyl, hydroxy-alkyl-phosphono-aryl, hydroxy-alkyl-phosphono-alkoxy, hydroxy-alkyl-phosphono-aryloxy, alkoxyphosphono, phosphono, alkoxyaryl, phosphono, hydroxy-alkyl-phosphono, phosphonoamino-alkyl, phosphonoamino-aryl, phosphonoamino-alkoxy, phosphonoamino-aryloxy, phosphonoamino, or a salt of the above substituents when chemically matched, wherein alkyl is a straight or branched alkyl chain of 1 to 20 carbon atoms in length or a heteroalkyl chain of 1 to 20 atoms in length containing 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 containing 1 to 3 heteroatoms selected from O, S and N.

In one embodiment, at least one of R1 through 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 when chemically matched, salts of the above substituents, wherein alkyl is a straight or branched alkyl chain of 1 to 20 carbon atoms in length or contains 1 to 4 substituents selected from O, n, P and S is a heteroalkyl chain of 1-20 atoms in length, 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 containing 1-3 heteroatoms selected from O, S and N.

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

In one embodiment, at least one of R1 to 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 to R12 is a sulfo-methyl group, a sulfo-alkoxy group having an alkyl chain of C2 to 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 to R12 of formula I is a sulfo group.

In one embodiment, one to three of R1 to 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 the group consisting of 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 those 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 of the invention, Q is a covalent bond or a linking group having a backbone length of 1 to 200 atoms. In other words, if the backbone length is from 1 to 200 atoms, the shortest connection between the ring system of the third ligand of formula I and the functional group Z consists of from 1 to 200 atoms.

In the case of a ring system, the shortest number of atoms in the ring system is taken in evaluating the linker length. For example, a phenylene ring occupies the length of four atoms 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 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, or having as a backbone a chain having the 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 having as a backbone a straight or branched saturated, unsaturated, unsubstituted or substituted C1-C100 alkyl chain, 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, or as previously described having a backbone comprising one or more cyclic or heterocyclic aromatic or non-aromatic ring systems.

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-C50 alkyl chain, or a1 to 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 as previously described having a backbone containing one or more cyclic or heterocyclic aromatic or non-aromatic ring systems.

In yet another embodiment, Q is 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 a1 to 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 as previously described having as a backbone a chain of an aromatic or non-aromatic ring system containing one or more rings or heterocycles.

In one embodiment, Q, e.g., the linking group Q, 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 four carbon atoms in length, or having two or more atoms selected from O, N, P and S, or a substituted N, P or S atom, or a1 to 20 atom chain comprising at least one aryl, heteroaryl, substituted aryl or substituted heteroaryl group (wherein, for example, the phenylene ring occupies a length of four 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.

In one embodiment, Q in the compounds of the invention, e.g., the linking group Q, is a saturated C1-C12 alkyl chain, or a C1-C12 arylalkyl chain, or a1 to 12 atom chain having a backbone 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 a1 to 12 atom chain 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 (where, for example, the phenylene ring occupies a length of four 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 to R20. In one embodiment, at least one of R13 to 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 of the present invention 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.

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

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 N-hydroxysuccinimide ester and maleimido group.

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

wherein one to three of R1 to R12 of the phenylphenanthridine residue are independently sulfo-alkyl, sulfo-aryl, sulfo-alkoxy, sulfo-aryloxy, sulfo, or a salt thereof (= sulfonate), wherein the counterion is preferably a cation from the alkali metal group, and the remaining groups R1 to R12 are hydrogen,

wherein one of R13-R19 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 through R19 in formula I is hydrogen or R20, wherein R20 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 given below

Wherein one of R13-R19 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 through R19 in formula I is hydrogen or R20, wherein R20 is alkyl, and

wherein X and Y are N.

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

wherein R1 to 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 groups R13 to R20 in formula I are 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 chemiluminescent compounds of formula I are suitable as labels for future high sensitivity ECL-based detection methods.

Process for preparing compounds of formula I

The present invention relates in one aspect to a process for the preparation of compounds of formula I.

The compounds of formula I can be synthesized, for example, as follows or as shown in example 3.

The compounds of formula I are synthesized by first generating carbenes from precursors of compounds of formula iii (a) or formula iii (b) by heating or by using, for example, silver oxide, wherein R13-R17, X, Y, Z and Q are as defined above for the compounds of formula I.

The precursors are based on the general structures iii (a) and iii (b):

wherein R13-R17, X, Y, Z and Q are as defined above for compounds of formula I.

The carbene derivative is further reacted with an iridium dimer complex (bis-iridium complex).

Thus, in one aspect, the present invention relates to a process for the preparation of a compound of formula I, comprising the steps of:

(a) a carbene for preparing a compound of formula III (a) or formula III (b), and

(b) reacting the thus obtained carbene with an iridium dimer complex of the formula IV (bis-iridium complex) to obtain a compound of the formula I as defined above,

wherein each X is independently chlorine, bromine, iodine, hydroxy, methoxy, ethoxy, phenoxy, cyanato, or diphenylphosphinoalkyl, and

wherein R1-R12 are as defined above via the definition of the compounds of formula I.

In process step (a), the carbene may be obtained by heating a compound of formula iii (a) or formula iii (b), wherein R13-R17 are as defined above via definition of the compound of formula I, or by reacting said compound of formula iii (a) or formula iii (b) with a base, preferably with silver oxide, if appropriate in the presence of a solvent.

In one embodiment, process step (a) is carried out in dioxane.

The iridium dimer complex of the formula IV (bis-iridium complex) used as starting material in process step (b) can be synthesized, for example, according to Nonoyama, M., J. organomet. chem. 86 (1975) 263-267 and as shown, for example, in example 2 and as described, for example, in EP 12179056.2.

In one embodiment, the two "bridging groups" X in formula IV are each independently selected from chlorine, bromine, iodine, hydroxyl, methoxy, and cyanato.

In one embodiment, the two "bridging groups" X in formula IV are each independently selected from chlorine, bromine, and iodine.

In one embodiment, the two "bridging groups" X in formula IV are each chlorine.

As the skilled artisan will recognize, in certain embodiments, the two "bridging groups" in formula IV may be the same and are defined above.

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

Scheme 1: synthesis of Compounds of formula I

Novel iridium-based chemiluminescent compounds of formula II

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

Wherein in formula I (a) and in formula I (b), respectively and independently, R1 to R19 are as defined for formula I except that Q of formula I is Q1 or Q2, respectively, of formula II, wherein Q1 is a linking group, preferably wherein at least one of R13-R19 in formula I (a) is-Q1-Z, and wherein Q1 is a linking group;

wherein at least one of R13-R19 in formula I (b) is Q2, and each Q2 is independently a linking group or a covalent bond,

wherein X and Y are as defined for formula I,

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

wherein Z is a functional group.

In one embodiment, one of R13 to R19 of formula i (a) in formula II is Q1-Z.

In one embodiment, one of R13 to R19 in each formula i (b) in formula II is Q2.

In one embodiment, one of R13 to R19 in formula i (a) in formula II is Q1-Z, and one of R13 to R19 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 phenylphenanthridine as defined via the definitions given above for formula I and one third ligand, respectively.

In other embodiments, R1 to R19 have the same meaning as described above for R1 to R19 of the compounds of formula I.

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 readily understands, the linking group Q1 of formula II comprises n branching sites at which Q2 is bonded.

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 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, 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 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, 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, 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 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, or 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 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 in the electrochemiluminescent complex 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 four carbon atoms in length, or having two or more atoms selected from O, N, P and S, or a substituted N, P or S atom, or a1 to 20 atom chain comprising at least one aryl, heteroaryl, substituted aryl or substituted heteroaryl group (wherein, for example, the phenylene ring occupies a length of four 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.

In one embodiment, the linking group Q1 in the compounds of the invention is a saturated C1-C12 alkyl chain, or a C1-C12 arylalkyl chain, or a1 to 12 atom chain having a backbone 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 a1 to 12 atom chain 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 (where, for example, the phenylene ring occupies a length of four atoms).

Formulae I (b) and Q2 are present (n) times in the compounds of formula II, (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 a1 to 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 an aromatic or non-aromatic ring system containing one or more rings or heterocycles as described above.

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 a1 to 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 described above 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 a1 to 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 described above 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 a1 to 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 above 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-C12 alkyl chain, or a1 to 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 as described above 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 chain of 1 to 12 atoms having a backbone 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.

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, Q2 is a linking group and comprises one or more amino acids.

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

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

In one embodiment, Q2 is a linking group and comprises one or more nucleotides.

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

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

In formula II, (n) is an integer from 1 to 50, indicating that formulas I (b) and Q2 are present (n) times in the compound of formula II. In certain 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 formulas I (b) and Q2 are present (n) times in the compound of formula II. In certain embodiments, (n) is an integer of 1 to 49, 1 to 48, 1 to 47, 1 to 46, 1 to 45, 1 to 44, 1 to 43, 1 to 42, 1 to 41, 1 to 40, 2 to 50, 2 to 49, 2 to 48, 2 to 47, 2 to 46, 2 to 45, 2 to 44, 2 to 43, 2 to 42, 2 to 41, 2 to 40, 3 to 39, 3 to 38, 3 to 37, 3 to 36, 3 to 35, 3 to 34, 3 to 33, 3 to 32, 3 to 31, 3 to 30, 4 to 29, 4 to 28, 4 to 27, 4 to 26, 4 to 25, 4 to 24, 4 to 23, 4 to 22, 4 to 21, 4 to 20, 5 to 19, 5 to 18, 5 to 17, 5 to 16, 5 to 15, 5 to 14, 5 to 13, 5 to 12, 5 to 11, or 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 of the present invention 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.

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

Any combination of any embodiment of 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 preparing compounds of formula II

In one aspect, the invention relates to a process for preparing a compound of formula II.

The compounds of formula II may be synthesized in one embodiment in the following manner: substituted phenylphenanthridine dimer iridium complexes (see e.g. example 2.2) are first further reacted with a derivative of a third ligand containing a functional group (-Q-) Z to obtain monomeric iridium complexes. Monomeric iridium complexes are given, for example, in formula I. The monomeric iridium complex is then further reacted with a precursor of Q containing from 1 to 50 groups that can react with the functional groups of the monomeric iridium complex to form covalent bonds; this way the compound of formula II is obtained after the covalent bond is formed again.

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

Scheme 2: synthesis of Compounds of formula II

Conjugates comprising novel compounds of formula I or 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 of the invention, i.e. the biological substance covalently bound to the compound of 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 various conditions, e.g. aqueous media under standard conditions. Molecules that may be involved 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 appreciate, in the conjugates of the invention, the functional group Z of the compound of 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 reagent 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 means of 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 carried out 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 one 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, the 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, respectively, and an affinity binding agent selected from the group consisting of proteins, antigens, antibodies, biotin analogs, avidin, streptavidin, sugars, lectins, enzymes, polypeptides, amino groups, nucleic acids, nucleic acid analogs, complementary nucleic acids, nucleotides, polynucleotides, Peptide Nucleic Acids (PNA), polysaccharides, metal ion chelators, receptor agonists, and receptor antagonists 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 10⁹Affinity of l/mol.

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 of the invention comprises a covalently linked compound of 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 refers to a short, usually 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.

The nucleic acid analog may contain, for example, a substituted nucleotide having a substituent at the standard bases deoxyadenosine (dA), deoxyguanosine (dG), deoxycytidine (dC), deoxythymidine (dT), deoxyuridine (dU). Examples of such substituted nucleobases 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; pseudo-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)), 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.

The oligonucleotide may, for example, 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).

Sugar analogs are, for example, 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).

Numerous modifications of the internucleoside phosphate moiety are also known not to interfere with hybridization properties, and such backbone modifications can also be combined with substituted nucleotides or nucleotide analogs. 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 covers 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 some embodiments, the antibody is purified (1) to greater than 95% by weight of the antibody, as determined by, for example, the Lowry method, in certain embodiments to greater than 99% by weight; (2) to a degree 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 the 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' 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 linker enables the scFv to form the required structure for antigen binding. For an overview of scFv, see, e.g., Plueckthun, In: 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, 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, respectively, disclosed in the present invention in an electrochemiluminescence based detection method.

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

The analyte of the present invention may be any inorganic or organic molecule, including any biological substance of interest. 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 to 2,000, still preferably 10 to 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 disclosed in the present invention, respectively, under conditions suitable for the formation of an analyte conjugate complex; (c) measuring the complex formed in step (b), thereby obtaining a measure of the analyte.

In one embodiment, measuring the analyte refers to detecting the amount of 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 is to be understood 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 ℃, 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 arylacyl chloride 2 (0.01 mol) and stirred at room temperature under inert conditions for 30 minutes. The resulting mixture was refluxed for a further 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)Alkane/ethyl acetate) to obtain pure product 3 in 66% yield.

Benzamido-2-biphenyl 3 (0.01 mol) and POCl in 20 ml of toluene was mixed in accordance with the procedure described in 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, with 25% NH₄OH is washed with distilled water. The organic layer was MgSO₄Dried and concentrated in vacuo, followed by flash chromatography (silica gel, 1:1 hexanes/ethyl acetate) to afford 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

The procedure described in Nicolai, E.in chem. Pharm. Bull. 42 (1994) 1617-1630 can be used by placing the arylaniline (0.01 mole) and 2-sulfobenzoic acid cyclic anhydride (0.01 mole) in CH₃CN is heated at moderate temperature for 6 hours, thereby synthesizing the 6- (2-sulfophenyl) phenanthridine.

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

Example 1.4

Procedure for the Synthesis of 6-phenyl-alkylsulfonylphenanthridines

6-phenyl-alkylsulfonylphenanthridines can be synthesized by mild heating of alkylsulfonyl-arylanilines (0.01 mol) with benzoyl chloride (0.01 mol) in chloroform using the procedure described in Lion, C. in Bull. Soc. Chim. Belg. 98 (1989) 557-566 (see example 1.2).

After purification, the product can be converted into the appropriate phenanthridine based on the method described in 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。

The 6- (4-methylsulfonylphenyl) phenanthridine can also be prepared according to the procedure described by 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 dry 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 80 ml of HCl (1M) and then with water. The extract was purified over anhydrous MgSO₄Dried, filtered and the filtrate evaporated. 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 129 (2007) 13364) Compound 2,5,8, 11-Tetraoxatridecan-13-yl 4-methylbenzenesulfonic acid Ethyl ester (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. Extract with H₂O 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 obtain 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 DMF in 30 ml dry 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 ℃ under an inert atmosphere, 20 ml of (phenyl-4- (2,5,8, 11-tetraoxatridec-13-yloxy) benzoyl chloride (3.1 g, 9.14 mmol) were 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 then quenched with HCl (1M, 2 × 100 ml), NaHCO₃(100 ml) and water (50 ml). The organic phase is MgSO₄Dried 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 480.2386 found 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 the mixture was washed with NH₄OH (20%) was neutralized. The organic phase is extracted and washed successively with distilled water and brine and over MgSO₄And (5) drying. The resulting solution was purified by flash chromatography (silica gel, in ethyl acetate/hexane 1:1, Rf = 0.14).

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 hours. 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 chromatography on a column (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: IrCl₃•3H₂O and 2.5 equivalents of 6-phenylphenanthridine were heated at 120 ℃ under nitrogen in a 2-ethoxyethanol/water mixture (3: 1 v/v) for 18 hours. After cooling to room temperature, the precipitate was filtered off and washed successively with methanol and Et2O and dried to obtain 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

Mixing 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 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 were added to the residue. The mixture was stirred for 1 hour to obtain a reddish 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 Et was added₂And O is precipitated. Without further treatmentUsed in the next step in the case of 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 cesium (500 mg, 0.92 mmol) and IrCl₃A mixture of (159.5 mg, 0.45 mmol) in a 2-etoeotoh: water (3: 1, 16 ml) mixture was refluxed under nitrogen for 36 hours. 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

The 3-ethoxycarbonylmethyl-4-methyl-1-phenyl-1H- [1,2,4] triazol-4-ium iodide salt, a precursor of the carbene ligand, can be synthesized according to the procedure described in Moderhack, d, et al, j. Heterocyclic chem. 44 (2007) 393.

Synthesis (6-phenylphenanthridine)₂Ir- (3-ethoxycarbonylmethyl-4-methyl-1-phenyl-1H- [1,2, 4)]Triazole complexes

50 mg of 3-ethoxycarbonylmethyl-4-methyl-1-phenyl-1H- [1,2,4]Triazol-4-ium iodide salt and 20 mg of Ag₂O was stirred in 5 ml dioxane at room temperature under an inert atmosphere for 40 hours. 25 mg of (6-phenylphenanthridine) are added₂IrCl]₂And the mixture was refluxed for 24 hours. After cooling to room temperature, the residue was isolated by filtration and further purified by preparative HPLC.

Claims (16)

1. Iridium-based chemiluminescent compounds of formula I

Wherein X and Y are C-R18 and C-R19, respectively, or wherein X is N and Y is C-R19, or wherein Y is N and X is C-R18,

wherein each R1-R19 is independently hydrogen, halogen, cyano or nitro, amino, substituted amino, carboxyl, carboxylate, carbamoyl, hydroxyl, substituted or unsubstituted alkoxy, substituted or unsubstituted aryloxy, sulfanyl, substituted or unsubstituted alkylsulfonyl, substituted or unsubstituted arylsulfonyl, sulfo, sulfino, sulfenyl, sulfonate, sulfinate, sulfenate, sulfamoyl, phosphonyl, hydroxyphosphinonyl, hydroxy-alkyl-phosphinoyl, phosphonate, phosphinate, or R20, wherein R20 is aryl, substituted aryl, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, amino-alkyl, substituted amino-alkyl, amino-alkoxy, substituted amino-alkoxy, amino-aryloxy, carboxy, sulfo, sulfinyl, sulfinate, sulfamoyl, phosphonyl, phosphono, hydroxy-alkyl-phosphinoyl, phosphonate, phosphinate, or R20, A substituted amino-aryloxy group having a substituent group,

wherein in R1-R12, or/and in R13-R16, or/and in R17-R19, or/and between R16 and R19, respectively, two adjacent R may form an aliphatic ring or a substituted aliphatic ring, wherein the substituent is selected from hydrogen, alkyl, substituted alkyl, halogen, cyano or nitro, a hydrophilic group selected from: amino, substituted amino, alkylamino, substituted alkylamino, 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, sulfeno, sulfamoyl, phosphono, hydroxyphosphinonyl, hydroxy-alkyl-phosphinoyl, phosphonate, phosphinate,

wherein, if substitution is present in any one of R1-R19, the substituents in R1-R19 are each independently selected from halogen, cyano, or nitro, a hydrophilic group selected from: amino, alkylamino, alkylammonium, carboxyl, carboxylate, carbamoyl, hydroxyl, alkoxy, arylalkoxy, aryloxy, alkylaryloxy, polyethyleneoxy, polypropyleneoxy, sulfanyl, alkylsulfonyl, arylsulfonyl, sulfo, sulfino, sulfoxo, sulfonate, sulfinate, sulfenamide, sulfamoyl, phosphonyl, hydroxyphosphinolylene, hydroxy-alkyl-phosphinidene, phosphonate, phosphinate,

wherein the alkyl group is a straight or branched alkyl chain of 1 to 20 carbon atoms in length or a heteroalkyl chain of 1 to 20 atoms in length containing 1 to 4 heteroatoms selected from O, N, P and S, wherein the aryl group 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-R19 is-Q-Z;

wherein Q is a covalent bond or a linear or branched saturated, unsubstituted or substituted C1-C200 alkyl chain, or a chain of 1 to 200 atoms consisting of carbon atoms, substituted carbon atoms and/or one or more atoms selected from the group consisting of O, N, P and S, or substituted N, P, S atoms as backbone, or a linear or branched saturated, unsubstituted or substituted C1-C200 alkyl chain, or a chain of 1 to 200 atoms consisting of carbon atoms, substituted carbon atoms and/or one or more atoms selected from the group consisting of O, N, P and S, or substituted N, P, S atoms as backbone and the backbone contains 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.

2. The compound according to claim 1, wherein the substituted amino group is an alkylamino group, a substituted alkylamino group, an arylamino group or a substituted arylamino group.

3. The compound according to claim 1, wherein the substituted alkyl is arylalkyl, substituted arylalkyl, amino-alkyl or substituted amino-alkyl.

4. The compound according to claim 1, wherein the substituted aryl is amino-aryl, substituted amino-aryl, alkylaryl, substituted alkylaryl, amino-aryl or substituted amino-aryl.

5. A compound according to any one of claims 1 to 4, wherein Q is a covalent bond or a straight or branched saturated, unsubstituted or substituted C1-C100 alkyl chain, 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, as a backbone, or a straight or branched saturated, unsubstituted or substituted C1-C100 alkyl chain, 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, as a backbone and the backbone contains one or more cyclic or heterocyclic aromatic or non-aromatic ring systems.

6. A compound as claimed in claim 5 wherein Q is a covalent bond or a straight or branched saturated, unsubstituted or substituted C1-C50 alkyl chain, 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, as the backbone, or a straight or branched saturated, unsubstituted or substituted C1-C50 alkyl chain, 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, as the backbone and the backbone contains one or more cyclic or heterocyclic aromatic or non-aromatic ring systems.

7. A compound as claimed in any one of claims 1 to 4 wherein Q is a covalent bond or a straight or branched saturated, 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, as the backbone, or a straight or branched saturated, 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, as the backbone and which contains one or more cyclic or heterocyclic aromatic or non-aromatic ring systems.

8. A conjugate comprising a compound of any one of claims 1 to 7 covalently bonded to an affinity binding agent.

9. The conjugate of claim 8, wherein the affinity binding agent is selected from the group consisting of antigens and antibodies, biotin or biotin analogues selected from the group consisting of aminobiotin, iminobiotin and desthiobiotin and avidin or streptavidin, sugars and lectins, nucleic acids or nucleic acid analogues and complementary nucleic acids comprising substituted nucleic acid bases selected from the group consisting of 5-substituted pyrimidines, N-substituted purines, 8-substituted purines and 2-substituted dA, and receptors and ligands.

10. The conjugate of claim 8 or 9, wherein the affinity binding agent is a nucleic acid or an antibody.

11. Use of a compound according to any one of claims 1 to 7 or a conjugate according to any one of claims 8 to 10 as a label for performing an electrochemiluminescence reaction in an aqueous solution.

12. Use of a compound according to any one of claims 1 to 7 or a conjugate according to any one of claims 8 to 10 as a label in an electrochemiluminescence-based detection method.

13. Use of a compound according to any one of claims 1 to 7 or a conjugate according to any one of claims 8 to 10 as a label in the detection of an analyte.

14. 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 of any one of claims 8 to 10 as a label under conditions suitable for formation of an analyte conjugate complex; and

c) measuring the complex formed in step (b) and thereby obtaining a measure of the analyte.

15. A compound of formula II

Wherein R1 to R20 are as defined for formula I in claim 1, with the exception that Q of formula I is Q1 or Q2, respectively, of formula II, wherein Q1 is a linking group, wherein at least one of R13 to 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, and wherein Z is a functional group,

wherein Q1 has as a skeleton a straight or branched saturated, unsubstituted or substituted C1-C200 alkyl chain, or a 1-200 atom chain of carbon atoms, substituted carbon atoms, and/or one or more atoms selected from the group consisting of O, N, P and S, or substituted N, P, S atoms, or has as a skeleton a straight or branched saturated, unsubstituted or substituted C1-C200 alkyl chain, or a 1-200 atom chain of carbon atoms, substituted carbon atoms, and/or one or more atoms selected from the group consisting of O, N, P and S, or substituted N, P, S atoms, and which contains one or more cyclic or heterocyclic aromatic or nonaromatic ring systems, and

q2 is a covalent bond or a linking group having as a backbone a straight or branched saturated, unsubstituted or substituted C1-C200 alkyl chain, or a1 to 200 atom chain of carbon atoms, substituted carbon atoms and/or one or more atoms selected from the group consisting of O, N, P and S, or substituted N, P, S atoms, or a straight or branched saturated, unsubstituted or substituted C1-C200 alkyl chain, or a1 to 200 atom chain of carbon atoms, substituted carbon atoms and/or one or more atoms selected from the group consisting of O, N, P and S, or substituted N, P, S atoms, and the backbone contains one or more cyclic or heterocyclic aromatic or non-aromatic ring systems.

16. The compound of claim 15, wherein 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.