HK1249760B - New iridium-based complexes for ecl - Google Patents

Description

New iridium-based complexes for ECL

This application is a divisional application of the following application: Application date: August 2, 2013; Application number: 201380051227.2 (PCT/EP2013/002322); Invention name: same as above.

Background of the Invention

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 detection of analytes based on electrochemiluminescence.

Electrochemiluminescence (also known as electrochemiluminescence and abbreviated as ECL) is a process in which species generated at an electrode undergo a high-energy electron transfer reaction to form an excited state that emits light. The first detailed studies of ECL were described by Hercules and Bard et al. in the mid-1960s. After approximately 50 years of research, ECL has become a very powerful analytical technique with widespread use in fields such as immunoassays, food and water testing, and detection of biological warfare agents.

A large number of compounds appear to be useful in organic light-emitting devices (OLEDs). These compounds are available in solid materials or soluble in organic fluids. However, their utility in aqueous media, such as those required for detecting analytes in biological samples, remains uncertain.

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

Despite significant improvements over the past decades, there remains a significant need for more sensitive electrochemiluminescence-based in vitro diagnostic assays.

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

SUMMARY OF THE INVENTION

The present invention discloses an iridium-based chemiluminescent compound of formula II

wherein in Formula I(a) and Formula I(b), respectively and independently, each R1-R18 is independently hydrogen, halogen, cyano or nitro, amino, substituted amino, alkylamino, substituted alkylamino, arylamino, substituted arylamino, alkylammonium, substituted alkylammonium, carboxyl, carboxylate, 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, phosphono ( phosphono), hydroxyphosphinoyl, hydroxy-alkyl-phosphinoyl, phosphonate, phosphinate, or R19, wherein R19 is aryl, substituted aryl, alkyl, substituted alkyl, branched alkyl, substituted branched alkyl, arylalkyl, substituted arylalkyl, alkylaryl, substituted alkylaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, amino-alkyl, substituted amino-alkyl, amino-alkoxy, substituted amino-alkoxy, amino-aryl, substituted amino-aryl, amino-aryloxy, substituted amino-aryloxy,

wherein in R1-R12, or/and in R13-R16, and/or in R17 and R18, respectively, two adjacent R 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, carboxylate, carboxylate, carbamoyl, hydroxy, substituted or unsubstituted alkoxy, substituted or unsubstituted aryloxy, sulfanyl, substituted or unsubstituted alkylsulfonyl, substituted or unsubstituted arylsulfonyl, sulfo, sulfinyl, sulfenyl, sulfonate, sulfinate, sulfenate, sulfamoyl, sulfoxide, phosphono, hydroxyphosphinyl, hydroxy-alkyl-phosphinyl, phosphonate, phosphinate, or

wherein, in R1-R12, or/and in R13-R16, and/or in R17 and R18, respectively, two adjacent R groups may form an aliphatic ring or a substituted aliphatic 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, carboxylate, carboxylate, carbamoyl, hydroxy, substituted or unsubstituted alkoxy, substituted or unsubstituted aryloxy, sulfanyl, substituted or unsubstituted alkylsulfonyl, substituted or unsubstituted arylsulfonyl, sulfo, sulfinyl, sulfenyl, sulfonate, sulfinate, sulfenate, sulfamoyl, sulfoxide, phosphono, hydroxyphosphinyl, hydroxy-alkyl-phosphinyl, phosphonate, phosphinate,

wherein, if substitution is present in any of R1-R19, the substituents in R1-R19 are each independently selected from halogen, cyano or nitro, a hydrophilic group such as amino, alkylamino, alkylammonium, carboxyl, carboxylate, carboxylate, carboxylate, carbamoyl, hydroxy, alkoxy, arylalkoxy, aryloxy, alkylaryloxy, polyethyleneoxy, polypropyleneoxy, sulfanyl, alkylsulfonyl, arylsulfonyl, sulfo, sulfinyl, sulfenyl, sulfonate, sulfinate, sulfenate, sulfamoyl, sulfoxide, phosphono, hydroxyphosphinyl, hydroxy-alkyl-phosphinyl, phosphonate, phosphinate,

wherein alkyl as used herein is a linear 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,

Where X represents C or N,

Where Y represents C or N,

wherein at least one of R13-R18 in formula I(a) is -Q1-Z and wherein Q1 is a linking group,

wherein at least one of R13-R18 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.

The present invention also discloses a conjugate comprising the above compound and an affinity binding agent covalently bonded thereto.

The present invention also relates to the use of the compounds or conjugates disclosed in the present invention for performing luminescence measurements or electrochemiluminescence reactions in aqueous solution, especially in an electrochemiluminescent device or an electrochemiluminescent detection system.

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

Detailed Description of the Invention

As indicated above, there is a need for new metal-based chemiluminescent compounds suitable for use in in vitro diagnostic assays.

Novel iridium-based chemiluminescent compound of formula II

The present invention relates to an iridium-based chemiluminescent compound of formula II

wherein in Formula I(a) and Formula I(b), each R1-R18 is independently hydrogen, halogen, cyano or nitro, amino, substituted amino, alkylamino, substituted alkylamino, arylamino, substituted arylamino, alkylammonium, substituted alkylammonium, carboxyl, carboxylate, carboxylate, carboxylate, carbamoyl, hydroxyl, substituted or unsubstituted alkoxy, substituted or unsubstituted aryloxy, sulfanyl, substituted or unsubstituted alkylsulfonyl, substituted or unsubstituted arylsulfonyl, sulfo, sulfinyl, sulfenyl, sulfonate, sulfinate, sulfenate, sulfamoyl, sulfoxide, phosphonyl, hydroxyphosphinyl, hydroxy-alkyl-phosphinyl, phosphonate, phosphinate or R19, wherein R19 is aryl, substituted aryl, alkyl, substituted alkyl, branched alkyl, substituted branched alkyl, arylalkyl, substituted arylalkyl, alkylaryl, substituted alkylaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, amino-alkyl, substituted amino-alkyl, amino-alkoxy, substituted amino-alkoxy, amino-aryl, substituted amino-aryl, amino-aryloxy, substituted amino-aryloxy,

wherein in R1-R12, or/and in R13-R16, and/or in R17 and R18, respectively, two adjacent R 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, carboxylate, carboxylate, carbamoyl, hydroxy, substituted or unsubstituted alkoxy, substituted or unsubstituted aryloxy, sulfanyl, substituted or unsubstituted alkylsulfonyl, substituted or unsubstituted arylsulfonyl, sulfo, sulfinyl, sulfenyl, sulfonate, sulfinate, sulfenate, sulfamoyl, sulfoxide, phosphono, hydroxyphosphinyl, hydroxy-alkyl-phosphinyl, phosphonate, phosphinate, or

wherein, in R1-R12, or/and in R13-R16, and/or in R17 and R18, respectively, two adjacent R groups may form an aliphatic ring or a substituted aliphatic 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, carboxylate, carboxylate, carbamoyl, hydroxy, substituted or unsubstituted alkoxy, substituted or unsubstituted aryloxy, sulfanyl, substituted or unsubstituted alkylsulfonyl, substituted or unsubstituted arylsulfonyl, sulfo, sulfinyl, sulfenyl, sulfonate, sulfinate, sulfenate, sulfamoyl, sulfoxide, phosphono, hydroxyphosphinyl, hydroxy-alkyl-phosphinyl, phosphonate, phosphinate,

wherein, if substitution is present in any of R1-R19, the substituents in R1-R19 are each independently selected from halogen, cyano or nitro, a hydrophilic group such as amino, alkylamino, alkylammonium, carboxyl, carboxylate, carboxylate, carboxylate, carbamoyl, hydroxy, alkoxy, arylalkoxy, aryloxy, alkylaryloxy, polyethyleneoxy, polypropyleneoxy, sulfanyl, alkylsulfonyl, arylsulfonyl, sulfo, sulfinyl, sulfenyl, sulfonate, sulfinate, sulfenate, sulfamoyl, sulfoxide, phosphono, hydroxyphosphinyl, hydroxy-alkyl-phosphinyl, phosphonate, phosphinate,

wherein alkyl as used herein is a linear 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,

Where X represents C or N,

Where Y represents C or N,

wherein at least one of R13-R18 in formula I(a) is -Q1-Z and wherein Q1 is a linking group,

wherein at least one of R13-R18 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.

In one embodiment, one of R13 to R18 of Formula I(a) is Q1-Z.

In one embodiment, one of R13 to R18 in each of Formula I(b) is Q2.

In one embodiment, one of R17 or R18 of Formula I(a) is -Q1-Z.

In one embodiment, one of R17 or R18 in each Formula I(b) is Q2.

In one embodiment, one of R13 to R18 of Formula I(a) is Q1-Z and one of R13 to R18 in each Formula I(b) is Q2.

In one embodiment, one of R17 or R18 of Formula I(a) is Q1-Z and one of R17 or R18 in each Formula I(b) is Q2.

In one embodiment, Formula I(a) and Formula I(b) are identical except for Q1-Z in Formula I(a) and Q2 in Formula I(b), respectively.

As known to those skilled in the art, the substituents in R1-R19 may be further substituted. For example, the alkyl group in the aminoalkyl group may be further substituted with a hydroxyl group, an amino group, a carboxyl group or a sulfo group.

As used herein, including in the appended claims, substituents have the meanings commonly 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 1 to 10 carbon atoms, and particularly preferably 1 to 6 carbon atoms; or a heteroalkyl chain having a length of 1 to 20 carbon atoms, preferably 1 to 10 carbon atoms, containing 1 to 4 heteroatoms selected from O, N, P, and S. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, isomeric pentyls, isomeric hexyls, isomeric heptyls, isomeric octyls, and dodecyl. In a particularly preferred embodiment, the alkyl group is methyl or ethyl.

The terms alkoxy and alkyloxy and substituted alkyl and substituted alkoxy, respectively, are 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, preferred substituents for substituted alkyloxy groups are ethyleneoxy chains comprising 1 to 40 ethyleneoxy units, or comprising 1 to 20 ethyleneoxy units, or comprising 1 to 10 ethyleneoxy 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, preferably a 6-membered heteroaryl ring system comprising 1 to 3 heteroatoms selected from O, S and N. In a particularly preferred embodiment, aryl is phenyl.

In one embodiment, in Formula I(a) and Formula I(b), each R1-R18, separately and independently, is independently hydrogen, hydroxy, substituted or unsubstituted alkoxy, substituted or unsubstituted aryloxy, sulfanyl, substituted or unsubstituted alkylsulfonyl, substituted or unsubstituted arylsulfonyl, sulfo, sulfinyl, sulfenyl, sulfonate, sulfinate, sulfenate, sulfamoyl, or sulfoxide.

In one embodiment, in Formula I(a) and Formula I(b), each R1-R18, respectively and independently, is independently hydrogen, substituted or unsubstituted alkoxy, substituted or unsubstituted aryloxy, substituted or unsubstituted alkylsulfonyl, substituted or unsubstituted arylsulfonyl, sulfonate, sulfinate, sulfenate, sulfamoyl, or sulfoxide.

In one embodiment, in Formula I(a) and Formula I(b), each R1-R18, respectively and independently, is independently hydrogen, substituted or unsubstituted alkoxy, substituted or unsubstituted alkylsulfonyl, substituted or unsubstituted arylsulfonyl, sulfonate, or sulfoxide.

In one embodiment, at least one of R1 to R18 of the compound according to Formula I(a) and/or Formula I(b) is substituted with at least one hydrophilic group.

In one embodiment, at least one of R1 to R12 of the phenylphenanthridine residue contained in formula I(a) and/or formula I(b) is substituted by at least one hydrophilic group, in particular by at least one hydrophilic group as defined below.

Preferred hydrophilic groups are amino, alkylamino (wherein alkyl refers to a straight chain, such as methyl, ethyl, propyl, butyl, pentyl chain, or a branched alkyl chain, such as isopropyl, isobutyl, tert-butyl, preferably a straight alkyl chain, such as methyl or ethyl), substituted alkylamino (this contains, for example, one or two branches or straight chains bonded to the N-atom, which are substituted by additional hydrophilic groups, such as hydroxyl or sulfo groups, such substituted alkylamino preferably contains two hydroxypropyl or hydroxyethyl residues), arylamino (wherein aryl refers to an aromatic residue, such as phenyl or naphthyl, preferably phenyl), substituted arylamino (having an aryl group as defined above and formed by a hydrophilic group ), alkylammonium (having an alkyl group as defined above and preferably a trimethylammonium residue or a triethylammonium residue), substituted alkylammonium, carboxyl, carboxylate (preferably an alkyl ester such as a methyl or ethyl ester), carbamoyl, hydroxy, substituted or unsubstituted alkoxy (wherein alkyl and substituted alkyl are as defined above) or aryloxy or substituted aryloxy (wherein aryl and substituted aryl are as defined above), sulfanyl, substituted or unsubstituted alkylsulfonyl, substituted or unsubstituted arylsulfonyl, sulfo, sulfinyl, sulfenyl, sulfamoyl, sulfoxide, phosphono, hydroxyphosphinyl, hydroxy-alkyl-phosphinyl, phosphonate or phosphinate.

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

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

In another particularly preferred embodiment, the hydrophilic group is selected from sulfo and sulfamoyl groups.

In one embodiment, at least one of R1-R12 is selected from sulfo-alkyl, sulfo-aryl, sulfo-alkoxy, sulfo-aryloxy, sulfo, sulfinyl-alkyl, sulfinyl-aryl, sulfinyl-alkoxy, sulfinyl-aryloxy, sulfinyl, sulfenyl-alkyl, sulfenyl-aryl, sulfenyl-alkoxy, sulfenyl-aryloxy, sulfenyl, sulfamoyl-alkyl, sulfamoyl-aryl, sulfamoyl-alkoxy, sulfamoyl-aryloxy, sulfamoyl, alkanesulfonyl-alkyl, alkanesulfonyl-aryl, alkanesulfonyl, arenesulfonyl-alkyl or arenesulfonyl-aryl or arenesulfonyl, sulfoamino-alkane alkyl, sulfoamino-aryl, sulfoamino-alkoxy, sulfoamino-aryloxy, sulfoamino, sulfoamino-alkyl, sulfoamino-aryl, sulfoamino-alkoxy, sulfoamino-aryloxy, sulfoamino, alkanesulfonylamino-alkyl, alkanesulfonylamino-aryl, alkanesulfonylamino-alkoxy, alkanesulfonylamino-aryloxy, alkanesulfonylamino, arenesulfonylamino-alkyl, arenesulfonylamino-aryl, arenesulfonylamino-alkoxy, arenesulfonylamino-aryloxy, arenesulfonylamino, alkanesulfinylamino-alkyl, alkanesulfinylamino-aryl, alkanesulfinylamino-alkoxy, Alkylsulfinylamino-aryloxy, alkanesulfinylamino, arenesulfinylamino-alkyl, arenesulfinylamino-aryl, arenesulfinylamino-alkoxy, arenesulfinylamino-aryloxy, arenesulfinylamino, phosphono-alkyl, phosphono-aryl, phosphono-alkoxy, phosphono-aryloxy, phosphono, hydroxyphosphinyl-alkyl, hydroxyphosphinyl-aryl, hydroxyphosphinyl-alkoxy, hydroxyphosphinyl-aryloxy, hydroxyphosphinyl, hydroxy-alkyl-phosphinyl-alkyl, hydroxy-alkyl-phosphinyl-aryl, hydroxy-alkyl-phosphinyl-alkoxy, hydroxy-alkyl- a substituted or unsubstituted group of phosphono-aryloxy, hydroxy-alkyl-phosphinyl, phosphonoamino-alkyl, phosphonoamino-aryl, phosphonoamino-alkoxy, phosphonoamino-aryloxy, phosphonoamino, or, if 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 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 to R12 is selected from the group consisting of 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, arenesulfonylamino-alkoxy, arenesulfonylamino-aryloxy, arenesulfonylamino, phosphonyl-alkyl, phosphonyl-aryl, phosphonyl-alkoxy , phosphonyl-aryloxy, phosphonyl, hydroxyphosphinyl-alkyl, hydroxyphosphinyl-aryl, hydroxyphosphinyl-alkoxy, hydroxyphosphinyl-aryloxy, hydroxyphosphinyl, hydroxy-alkyl-phosphinyl-alkyl, hydroxy-alkyl-phosphinyl-aryl, hydroxy-alkyl-phosphinyl-alkoxy, hydroxy-alkyl-phosphinyl-aryloxy, hydroxy-alkyl-phosphinyl or, if 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 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 to R12 is sulfo-alkyl, sulfo-aryl, sulfo-alkoxy, sulfo-aryloxy, sulfo or a salt thereof (= sulfonate), wherein the counterion is preferably a cation selected from alkali metals.

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

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

In one embodiment, at least one of the groups R1 to R12 of formula I(a) and/or formula I(b) is a sulfo group.

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

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

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

In one embodiment, the counterion is a cesium cation.

In one embodiment, the phenylphenanthridine residue contained in Formula I(a) and/or Formula I(b) is selected from the substituted phenylphenanthridines given below.

In a preferred embodiment of formula I(a) and formula I(b), respectively, X and Y do not simultaneously represent N.

In one embodiment of formula I(a) and formula I(b), respectively, X represents C and Y represents N.

In one embodiment of formula I(a) and formula I(b), respectively, Y represents C and X represents N.

The term "linker" as used herein has a meaning known to those skilled in the art and refers to a molecule or group of molecules used to connect molecular fragments. A linker is characterized by having two or more chemically orthogonal functional groups on a flexible or rigid backbone. A covalent bond is not a linker within the meaning of the present invention.

In the compounds of the present invention, the linker Q1 preferably has a backbone of 1 to 200 atoms. A skilled person will readily recognize that the linker Q1 of Formula II comprises n branch points at which Q2 is bonded. In other words, the shortest connection between the ring system of the third ligand in Formula I(a) and the functional group Z consists of 1 to 200 atoms.

In one embodiment, Q1 has as its backbone a linear 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 described above having a backbone containing one or more cyclic or heterocyclic aromatic or non-aromatic ring systems.

If a ring system is present, the number of atoms in the shortest ring system is used in estimating the length of the linker. As an example, a phenylene ring would be 4 atoms long in the linker.

In one embodiment, the linker Q1 has as its backbone a linear 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 described above having a backbone containing one or more cyclic or heterocyclic aromatic or non-aromatic ring systems.

In one embodiment, the linker Q1 has as its backbone a linear 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 a chain as described above having a backbone containing one or more cyclic or heterocyclic aromatic or non-aromatic ring systems.

In another embodiment, the linker Q1 has as its backbone a linear or branched saturated, unsaturated, unsubstituted or substituted C1-C20 alkyl chain or a 1 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 containing one or more cyclic or heterocyclic aromatic or non-aromatic ring systems.

In one embodiment, the linker Q1 in the electrochemiluminescent complex of the present invention is a linear or branched saturated, unsaturated, unsubstituted or substituted C1-C20 alkyl chain or C1-C20 arylalkyl chain (wherein, for example, the phenylene ring is 4 carbon atoms in length), or a 1 to 20 atom chain having a skeleton 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 1 to 20 atom chain having a skeleton consisting of carbon atoms, substituted carbon atoms and one or more atoms selected from O, N, P and S or substituted N, P or S atoms containing at least one aryl, heteroaryl, substituted aryl or substituted heteroaryl group (wherein, for example, the phenylene ring is 4 atoms in length).

In one embodiment, the linker Q1 comprises a peptide chain.

In one embodiment, Q2 is selected from —C ₆ H ₄ —(CH ₂ ) ₂ — and —C ₆ H ₄ —(CH ₂ ) ₂ —CO—.

In one embodiment, the linker Q1 in the compounds of the present invention is a saturated C1-C12 alkyl chain or a C1-C12 arylalkyl chain, or a 1 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 a 1 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 containing at least one aryl, heteroaryl, substituted aryl or substituted heteroaryl group (wherein, for example, the phenylene ring is 4 atoms in length).

Formula I (b) and Q2 are present (n) times in the compound according to formula II, wherein (n) is an integer from 1 to 50. These (n) Q2 are each independently a covalent bond or a linker having as a backbone a linear or branched saturated, unsaturated, unsubstituted or substituted C1-C200 alkyl chain or a 1 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 as described above having a backbone containing one or more cyclic or heterocyclic aromatic or non-aromatic ring systems.

In one embodiment, each Q2 is independently a covalent bond or a linker having as its backbone a linear 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 described above having a backbone containing one or more cyclic or heterocyclic aromatic or non-aromatic ring systems.

In one embodiment, each Q2 is independently a covalent bond or a linker having as its backbone a linear 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 a chain as described above having a backbone containing one or more cyclic or heterocyclic aromatic or non-aromatic ring systems.

In one embodiment, each Q2 is independently a covalent bond or a linker having as its backbone a linear or branched saturated, unsaturated, unsubstituted or substituted C1-C20 alkyl chain or a chain of 1 to 20 atoms consisting of carbon atoms, substituted carbon atoms and/or one or more atoms selected from O, N, P and S or substituted N, P or S atoms, or a chain as described above having a backbone containing one or more cyclic or heterocyclic aromatic or non-aromatic ring systems.

In one embodiment, each Q2 is independently a covalent bond or a linker having as its backbone a saturated C1-C12 alkyl chain or a 1 to 12 atom chain having as its backbone 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 linker Q1 comprises one or more amino acids.

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

In one embodiment, linkers Q1 and Q2 both comprise one or more amino acids.

In one embodiment, the linker Q1 comprises one or more nucleotides.

In one embodiment, linker Q2 comprises one or more nucleotides.

In one embodiment, linkers Q1 and Q2 both comprise one or more nucleotides.

In Formula II, (n) is an integer from 1 to 50, indicating that Formula I (b) and Q2 are present (n) times in the compound according to 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 Formula I (b) and Q2 are present (n) times in the compound according to Formula II. In certain embodiments, (n) is 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 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 contained in the iridium-based complex of formula II of the present invention is selected from aldehyde, carboxylic acid, carboxylic acid ester, epoxide, N-hydroxysuccinimide ester, amino, halogen, hydrazine, hydroxyl, sulfhydryl, maleimido, alkynyl, azide, isocyanate, isothiocyanate and phosphoramidite.

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

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

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

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

wherein Formula I(a) and Formula I(b) are identical except for Q1-Y in Formula I(a) and Q2 in Formula I(b), respectively,

wherein in formulae I(a) and I(b), respectively, 1 to 3 of R1 to R12 are independently sulfo-alkyl, sulfo-aryl, sulfo-alkoxy, sulfo-aryloxy, sulfo or salts thereof (= sulfonates), wherein the counterion is preferably a cation selected from alkali metals, and the other groups R1 to R12 are hydrogen,

Where X represents C or N,

Where Y represents C or N,

wherein one of R13 to R18 in formula I(a) is -Q1-Z and the other groups R13 to R18 in formula I(a) are hydrogen,

wherein one of R13 to R18 in formula I(b) is Q2, the other groups R13 to R18 in formula I(a) are hydrogen, wherein Q1 is a linking group and Q2 is a linking group or a covalent bond,

(n) is an integer from 1 to 50, and

Z is a functional group.

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

Novel iridium-based chemiluminescent compound of formula I

In another aspect, the present invention relates to compounds according to formula I

wherein each R1-R18 is independently hydrogen, halogen, cyano or nitro, amino, substituted amino, alkylamino, substituted alkylamino, arylamino, substituted arylamino, alkylammonium, substituted alkylammonium, carboxyl, carboxylate, carboxylate, carboxylate, carbamoyl, hydroxy, substituted or unsubstituted alkoxy, substituted or unsubstituted aryloxy, sulfanyl, substituted or unsubstituted alkylsulfonyl, substituted or unsubstituted arylsulfonyl, sulfo, sulfinyl, sulfenyl, sulfonate, sulfinate, sulfenate, sulfamoyl, sulfonyl, sulfinyl, sulfenyl, sulfonate, sulfenyl, sulfonate, sulfonate, sulfonate, sulfonamide, sulfonyl, sulfonyl, sulfinyl, sulfenyl, sulfonate, sulfonate, sulfonate, sulfonate, sulfonamide, sulfonyl, sulfinyl, sulfenyl, sulfonate ... R19, wherein R19 is aryl, substituted aryl, alkyl, substituted alkyl, branched alkyl, substituted branched alkyl, arylalkyl, substituted arylalkyl, alkylaryl, substituted alkylaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, amino-alkoxy, substituted amino-alkoxy, amino-aryl, substituted amino-aryl, amino-aryloxy, substituted amino-aryloxy,

wherein in R1-R12, or/and in R13-R16, and/or in R17 and R18, respectively, two adjacent R 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, carboxylate, carboxylate, carbamoyl, hydroxy, substituted or unsubstituted alkoxy, substituted or unsubstituted aryloxy, sulfanyl, substituted or unsubstituted alkylsulfonyl, substituted or unsubstituted arylsulfonyl, sulfo, sulfinyl, sulfenyl, sulfonate, sulfinate, sulfenate, sulfamoyl, sulfoxide, phosphono, hydroxyphosphinyl, hydroxy-alkyl-phosphinyl, phosphonate, phosphinate, or

wherein, in R1-R12, or/and in R13-R16, and/or in R17 and R18, respectively, two adjacent R groups may form an aliphatic ring or a substituted aliphatic 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, carboxylate, carboxylate, carbamoyl, hydroxy, substituted or unsubstituted alkoxy, substituted or unsubstituted aryloxy, sulfanyl, substituted or unsubstituted alkylsulfonyl, substituted or unsubstituted arylsulfonyl, sulfo, sulfinyl, sulfenyl, sulfonate, sulfinate, sulfenate, sulfamoyl, sulfoxide, phosphono, hydroxyphosphinyl, hydroxy-alkyl-phosphinyl, phosphonate, phosphinate,

wherein, if substitution is present in any of R1-R19, the substituents in R1-R19 are each independently selected from halogen, cyano or nitro, a hydrophilic group such as amino, alkylamino, alkylammonium, carboxyl, carboxylate, carboxylate, carboxylate, carbamoyl, hydroxy, alkoxy, arylalkoxy, aryloxy, alkylaryloxy, polyethyleneoxy, polypropyleneoxy, sulfanyl, alkylsulfonyl, arylsulfonyl, sulfo, sulfinyl, sulfenyl, sulfonate, sulfinate, sulfenate, sulfamoyl, sulfoxide, phosphono, hydroxyphosphinyl, hydroxy-alkyl-phosphinyl, phosphonate, phosphinate,

wherein alkyl as used herein is a linear 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,

Where X represents C or N,

Where Y represents C or N,

wherein at least one of R13-R18 in formula I is -Q-Z and wherein Q-Z is maleimide or wherein Q is a covalent bond or a linear or branched saturated, unsaturated, unsubstituted or substituted C21-C200 alkyl chain or a 21 to 200 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 a chain as described above having a backbone containing one or more cyclic or heterocyclic aromatic or non-aromatic ring systems, and wherein Z is a functional group.

The compounds of the formula I, formula I(a) and formula I(b) each comprise two ligands derived from phenylphenanthridine as defined by the definition given for formula I and a third ligand.

In other embodiments, R1 to R19 have the same meanings as described above for R1 to R19 of the compound of Formula II.

In one embodiment, Q-Z is maleimide.

In one embodiment, Q is a covalent bond.

In one embodiment, Q is a linear or branched saturated, unsaturated, unsubstituted or substituted C21-C200 alkyl chain or a 21 to 200 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 a chain as described above having a backbone containing one or more cyclic or heterocyclic aromatic or non-aromatic ring systems.

In one embodiment, Q is a linear or branched saturated, unsaturated, unsubstituted or substituted C21-C100 alkyl chain or a 21 to 100 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 a chain as described above having a backbone containing one or more cyclic or heterocyclic aromatic or non-aromatic ring systems.

In one embodiment, Q is a linear or branched saturated, unsaturated, unsubstituted or substituted C21-C50 alkyl chain or a 21 to 50 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 a chain as described above having a backbone containing one or more cyclic or heterocyclic aromatic or non-aromatic ring systems.

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

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

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

Any combination of any of the embodiments of the 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 the formula I are suitable as labels for future highly sensitive ECL-based detection methods.

Preparation methods of compounds of formula II and I

One aspect of the present invention relates to novel processes for preparing compounds of formula I and compounds of formula II, respectively.

Compounds according to formula I can be synthesized, for example, as follows (based on Lamansky, S., Inorg. Chem. 40 (2001) 1704-1711): synthesize a substituted phenyl-phenanthridine dimer iridium complex; react this dimer with a precursor of Q-Z to produce a product according to formula I.

According to this method, compounds of formula I can be obtained, for example, as shown in Scheme 1 below.

Scheme 1: Synthesis of compounds of formula I. Reagents and conditions: (i): Na ₂ CO ₃ , 2-ethoxyethanol; m is an integer from 1 to 20.

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

The compounds used as starting materials for the preparation of the phenyl-phenanthridine dimer iridium complex are commercially available or can be obtained by methods known to the skilled person, as shown, for example, in the examples (see Example 1).

Compounds according to formula II can be synthesized, for example, as follows (based on Lamansky, S., Inorg. Chem. 40 (2001) 1704-1711): a substituted phenyl-phenanthridine dimer iridium complex is synthesized; this dimer is reacted with a precursor of a linker Q containing 2-50 pyridyl-oxazolyl groups to produce a product according to formula II.

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

Scheme 2: Synthesis of the compound of formula II. Reagents and conditions: Cs ₂ CO ₃ , DMF.

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

The compounds used as starting materials for the preparation of the phenyl-phenanthridine dimer iridium complex are commercially available or can be obtained by methods known to the skilled person, as shown, for example, in the examples (see Example 1).

Compounds according to Formula II can also be synthesized in another manner: a substituted phenyl-phenanthridine dimer iridium complex (see, for example, Example 2.2) is first reacted with an oxazolyl-pyridine derivative containing the functional group (-Q-)Z to produce a monomeric iridium complex. For example, a monomeric iridium complex is given in Formula I. However, for use in synthesizing complexes according to Formula II, the compound given in Formula I additionally contains those wherein the linker Q is Q2 as defined for Formula II. This monomeric iridium complex is then reacted with a precursor of Q2 containing 2-50 groups that can react with the functional groups of the monomeric iridium complex to form a covalent bond; thus, after covalent bond formation, the compound according to Formula II is obtained.

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

Scheme 3: Synthesis of compounds of Formula II.

Conjugates comprising novel compounds of formula II or formula I and further 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, respectively, as disclosed and defined above, 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, cellular metabolites, haptens, hormones, pharmacological substances, alkaloids, steroids, vitamins, amino acids, and sugars.

In one embodiment, the conjugate of the present invention, i.e., the biological substance covalently bonded to the compound according to Formula II or Formula I, respectively, is an affinity binder. Affinity binders are molecules capable of molecularly binding to one another due to attractive interactions between the molecules, resulting in a stable association in which the molecules are in close proximity. Molecular binding results in the formation of a molecular complex. The attractive bond between the components of a complex is generally weaker than that of a covalent bond. In this case, the binder is an affinity binder, meaning that it is capable of binding to affinity complexes, i.e., complexes that are stable under the respective conditions (e.g., in aqueous medium under standard conditions). Molecules that can participate in molecular binding include, but are not limited to, proteins, nucleic acids, carbohydrates, lipids, and small organic molecules, such as drugs. Thus, types of complexes formed due to molecular binding include: protein-protein, protein-DNA, protein-hormone, protein-drug, antigen-antibody, receptor-ligand, biotin-avidin or streptavidin, nucleic acid-complementary nucleic acid, or receptor-receptor agonist (antagonist).

The skilled person will recognize that in the conjugates of the present invention, the functional group Z of the compound according to formula II or formula I has been used to form a covalent bond with a group on the affinity binding agent and is no longer present as such. If the affinity binding agent itself does not contain a group suitable for binding or reacting with group Z, such a group can be readily introduced into the affinity binding agent by means of well-established procedures.

In one aspect, the present invention relates to the preparation of a conjugate by reacting a functional group Z of a compound of formula II or formula I with a group suitable for reacting with the functional group Z of an affinity binding agent as defined herein.

The skilled person can carry out this process using standard methods known to the skilled person.

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

Without wishing to be further limited, for clarity, affinity binding agents may comprise any of the following: an antigen, a protein, an antibody, biotin or a biotin analog and avidin or streptavidin, a sugar and a lectin, an enzyme, a polypeptide, an amino group, a nucleic acid or a nucleic acid analog and a complementary nucleic acid, a nucleotide, a polynucleotide, a peptide nucleic acid (PNA), a polysaccharide, a metal ion chelator, a receptor agonist, or a receptor antagonist. For example, the affinity binding agent may be one partner of a specific binding pair, wherein the other partner of the binding pair is associated with or is a target on the cell surface or an intracellular structure.

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

The affinity binding agent is preferably a partner or member of an affinity binding pair, or what the skilled person also calls a specific binding pair.

An affinity binding agent has an affinity for its target, e.g., one member of a specific binding pair, such as an antibody, and the other member of the specific binding pair, such as its antigen, of at least 10 ⁷ l/mol. An affinity binding agent preferably has an affinity for its target of 10 ⁸ l/mol or more preferably 10 ⁹ l/mol.

In one embodiment, the present 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 analog, avidin or streptavidin, a sugar, a lectin, a nucleic acid or a nucleic acid analog and a complementary nucleic acid, a receptor and a ligand.

In one embodiment, the present 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 II or Formula I and a protein, antigen, antibody, biotin, a biotin analog, avidin, streptavidin, a sugar, a lectin, an enzyme, a polypeptide, an amino group, a nucleic acid, a nucleic acid analog, a complementary nucleic acid, a nucleotide, a polynucleotide, a peptide nucleic acid (PNA), a polysaccharide, a metal ion chelator, a receptor agonist, a receptor antagonist, or any combination thereof.

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

Biotin analogs are aminobiotin, iminobiotin or desthiobiotin.

As used herein, the term "oligonucleotide" or "nucleic acid" generally refers to a short, typically single-stranded polynucleotide comprising at least 8 nucleotides and up to about 1000 nucleotides. In a preferred embodiment, the oligonucleotide has a length of at least 9, 10, 11, 12, 15, 18, 21, 24, 27, or 30 nucleotides. In a preferred embodiment, the oligonucleotide has 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 construed broadly and includes DNA and RNA and analogs and modifications thereof.

Nucleic acid analogs can, for example, contain substituted nucleotides with substituents at the standard bases deoxyadenosine (dA), deoxyguanosine (dG), deoxycytidine (dC), deoxythymidine (dT), and deoxyuridine (dU). Examples of such substituted nucleobases are: 5-substituted pyrimidines, such as 5-methyl dC, 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 dA, such as 2-amino dA.

Nucleic acid analogs may contain nucleotides or nucleotide analogs. That is, naturally occurring nucleobases may be replaced with nucleobase analogs, such as 5-nitroindole-d-nucleoside; 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-metamorphine; pseudo-dU; pseudo-iso-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 the skilled artisan, the nucleobases in the complementary strands must be chosen so 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')).

Among nucleic acid analogs, the oligonucleotide backbone can be modified to contain substituted sugar residues, sugar analogs, modifications in the internucleoside phosphate moiety, and/or be a PNA.

Oligonucleotides may, for example, contain nucleotides with substituted deoxyribose sugars, such as 2'-methoxy, 2'-fluoro, 2'-methylseleno, 2'-allyloxy, 4'-methyldN (wherein N is a nucleobase, such as A, G, C, T or U).

Examples of sugar analogs are xylose; 2',4'-bridged riboses such as (2'-O, 4'-Cmethylene)- (oligomer known as LNA) or (2'-O, 4'-Cethylidene)- (oligomer known as ENA); L-ribose, L-d-ribose, hexitol (oligomer known as HNA); cyclohexenyl (oligomer known as CeNA); altritol (oligomer known as ANA); tricyclic ribose analogs in which the C3' and C5' atoms are linked by an ethylene bridge fused to a cyclopropane ring (oligomer known as tricyclic DNA); glycerol (oligomer known as GNA); glucopyranose (oligomer known as Homo DNA); carbaribose (having cyclopentane instead of tetrahydrofuran subunits); and hydroxymethyl-morpholine (oligomer known as morpholino DNA).

A number of modifications of the internucleoside phosphate moiety are also known that do not interfere with hybridization properties, and such backbone modifications can also be combined with substituted nucleotides or nucleotide analogs. Examples are phosphorothioate oligonucleotides, phosphorodithioate oligonucleotides, phosphoramidite oligonucleotides, and methylphosphonate oligonucleotides.

PNA (which has a backbone without phosphate and d-ribose) can also be used as a DNA analog.

The modified nucleotides, nucleotide analogs and oligonucleotide backbone modifications mentioned above can be combined as desired in the oligonucleotides in the sense of the present invention.

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

An "isolated" antibody is one that has been determined to be separated and/or recovered from components of its natural environment. Contaminant components of its natural environment are materials that interfere with research, diagnostic, or therapeutic applications of the antibody and may include enzymes, hormones, and other proteinaceous or non-proteinaceous 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 some embodiments to greater than 99% by weight; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence using, for example, a spinning cup sequenator, or (3) to homogeneity as determined by SDS-PAGE under reducing or non-reducing conditions using, for example, Coomassie blue or silver stain. An isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. However, generally, an isolated antibody is prepared by at least one purification step.

"Native antibodies" are usually heterotetrameric glycoproteins of approximately 150,000 daltons composed 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, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. 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 believed to form 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 generally the most variable parts of an antibody and contain the antigen-binding site.

The term "variable" refers to the fact that certain portions of the variable domain differ widely in sequence among antibodies and contribute to the binding and specificity of each particular antibody for its particular antigen. However, this variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments—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 (FRs). The variable domains of native heavy and light chains each contain four FR regions, which generally adopt a β-pleated sheet configuration, connected by three HVRs. These FRs form loops that connect or, in some cases, form part of the β-pleated sheet structure. The HVRs in each chain are held together by the FR regions and, together with the HVRs from the other chain, contribute to the formation of the antibody's antigen-binding site (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., 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 the antibody's participation in antibody-dependent cellular cytotoxicity.

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

Antibodies (immunoglobulins) can be classified into different classes based on the amino acid sequence of the constant domain of their heavy chains. There are five major 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 IgA2. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known and generally described in, for example, Abbas et al., Cellular and Mol. Immunology, 4th ed., W.B. Saunders, Co. (2000). Antibodies can be part of a larger fusion molecule formed by covalent or non-covalent association of an antibody with 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 antibody fragments as defined below. The term specifically refers to antibodies having heavy chains that contain an Fc region.

"Antibody fragments" comprise a portion of an intact antibody, which preferably comprises its antigen-binding region. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

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

"Fv" is the smallest antibody fragment that contains a complete 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 are covalently linked by a flexible peptide linker, allowing the light and heavy chains to associate in a "dimeric" structure similar to that in 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. Collectively, these six HVRs provide the antibody with antigen-binding specificity. However, even a single variable domain (or half of an Fv comprising only three antigen-specific HVRs) has the ability to recognize and bind antigen, albeit with lower affinity than the entire binding site.

The Fab fragment contains the heavy and light chain variable domains, as well as the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' fragments with free thiol groups on the cysteine residue(s) in their constant domains. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

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

The term "diabody" refers to an antibody fragment with two antigen-binding sites, which comprises a heavy chain variable domain (VH) connected 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 the two domains on the same chain, these domains are forced to pair with the complementary domains of another chain and produce two antigen-binding sites. Diabodies can be bivalent or bispecific. Diabodies are more fully described in, for example, EP 0 404 097; WO 1993/01161; Hudson, P.J. et al., Nat. Med. 9 (2003) 129-134; and Holliger, P. et al., PNAS USA 90 (1993) 6444-6448. Triabodies and tetrabodies are also described in Hudson, P.J. et al., Nat. Med. 9 (2003) 129-134.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible mutations (e.g., naturally occurring mutations that may be present in small amounts). Thus, the modifier "monoclonal" indicates the characteristic of the antibody as not being a mixture of different antibodies. In certain embodiments, such monoclonal antibodies generally include antibodies comprising a polypeptide sequence that binds to a target, wherein the target-binding polypeptide sequence is obtained by a process comprising selecting a single target-binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection process can involve selecting a unique clone from a plurality of clones (e.g., a collection of hybridoma clones, phage clones, or recombinant DNA clones). It should be understood that the selected target-binding sequence can be further altered to, for example, improve affinity for the target, humanize the target-binding sequence, improve its production in cell culture, reduce its in vivo immunogenicity, generate multispecific antibodies, etc., and that antibodies comprising such altered target-binding sequences are also monoclonal antibodies of the present 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, an advantage of monoclonal antibody preparations is that they are generally free of contamination by other immunoglobulins.

As mentioned, the compounds and conjugates disclosed herein possess considerable advantages. For example, the disclosed compounds or conjugates, respectively, exhibit high ECL efficiencies. In contrast to many ECL labels, which exhibit high ECL efficiencies only when analyzed in organic solvents, these high efficiencies are also present when the corresponding measurements are performed in aqueous systems. For example, many OLED dyes are typically analyzed in acetonitrile and are either insoluble in aqueous solutions or, even if soluble, do not exhibit efficient electrochemiluminescence in aqueous solutions.

In a preferred embodiment, the present invention relates to the use of the compounds or conjugates, respectively, as disclosed herein, for performing an electrochemiluminescent reaction in an aqueous solution. An aqueous solution is any solution comprising at least 90% water (weight/weight). Such aqueous solutions may obviously also contain other ingredients, such as buffer compounds, detergents, and, for example, tertiary amines, such as tripropylamine, which serve as electron donors in the ECL reaction.

In one aspect, the present invention relates to the use of the compounds or conjugates, respectively, as disclosed in the present invention in a detection method based on electrochemiluminescence.

In one aspect, the present invention relates to the use of the compounds or conjugates, respectively, as 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 representing analytes in the sense of the present invention are cells, viruses, subcellular particles, proteins, lipoproteins, glycoproteins, peptides, polypeptides, nucleic acids, oligosaccharides, polysaccharides, lipopolysaccharides, cellular 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 composed essentially of amino acids and having at least two amino acids linked by peptide bonds. If the relevant analyte is to be studied in the methods disclosed herein, the polypeptide preferably consists of at least 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, and 30 up to about 10,000 amino acids. The polypeptide preferably contains 5 to 2,000, and more preferably 10 to 1,000 amino acids.

If the analytes are nucleic acids, these are preferably naturally occurring DNA or RNA oligonucleotides.

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

In one embodiment, measuring an analyte refers to detecting the amount of the analyte in a sample.

In one embodiment, the measurement in the above-described analyte detection method is performed using a detection procedure based on electrochemiluminescence.The method is also preferably performed 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 understood that modifications can be made in the procedures described without departing from the spirit of the invention.

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

Example

Example 1

Synthesis of Substituted Phenyl-Phenanthridines

Example 1.1

General procedure for the synthesis of substituted 2-aminobiphenyls:

The appropriate 2-aminobiphenyls required for further reaction to the phenanthridines were synthesized via Suzuki-Miyaura coupling reactions 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 procedures:

a: 10 mol % PdCl ₂ (PPh ₃ ) ₂ , K ₂ CO ₃ , DMF/H ₂ O (5/1), 80℃, 24 h

Other embodiments:

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 aryl acid chloride 2 (0.01 mol) and stirred at room temperature under inert conditions for 30 minutes. The resulting mixture was then refluxed with stirring for 2 hours. The reaction mixture was treated by dropwise addition of pyridine (0.02 mol in 10 ml of chloroform) over 60 minutes. The mixture was allowed to cool to room temperature and stirred overnight. The mixture was washed thoroughly with 0.5 M HCl, dried over _MgSO₄ , and concentrated in vacuo. The crude product was purified by flash chromatography on silica gel using 3:2 hexane/ethyl acetate to yield pure product 3 in 66% yield.

Benzamido-2-biphenyl 3 (0.01 mol) and _POCl₃ (5 ml) were refluxed in 20 ml of toluene under nitrogen and stirred for 18 hours according to the procedure described by Lion, C. in Bull. Soc. Chim. Belg. 98 (1989) 557-566. The cooled reaction mixture was _diluted with _CH₂Cl₂ (30 ml) and poured onto ice, washed with 25% _NH₄OH and distilled water. The organic layer was dried over _MgSO₄ and concentrated in vacuo, followed by flash chromatography (silica gel, 1:1 hexane/ethyl acetate) to yield the product, 4,6-phenylphenanthridine.

Using 2-naphth-2-yl-phenylamine instead of 2-aryl-aniline yields:

MS: [M+H] ⁺ 306.3.

Using naphthalene-carbonyl chloride instead of phenylacyl chloride yields:

MS: [M+H] ⁺ 306.3.

Example 1.3

Synthesis procedure of 6-(2-sulfophenyl)phenanthridine

6-(2-Sulfophenyl)phenanthridine can be synthesized by gently heating arylaniline (0.01 mol) with 2-sulfobenzoic acid cyclic anhydride (0.01 mol) in _CH3CN for 6 hours using the procedure described by Nicolai, E. in Chem. Pharm. Bull. 42 (1994) 1617-1630.

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

Example 1.4

Synthesis procedure of 6-phenyl-alkylsulfonylphenanthridine

6-Phenyl-alkylsulfonylphenanthridine can be synthesized by gently heating alkylsulfonyl-arylaniline (0.01 mol) with benzoyl chloride (0.01 mol) in chloroform using the procedure described by 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.

6-(4-Methylsulfonphenyl)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:

Procedure: (JACS, 2007, 129, 13364) To a solution of 2,5,8,11-tetraoxatridecan-13-ol (7 g, 33.6 mmol) and triethylamine (4.9 mL, 35.3 mmol) in anhydrous _{CH₂Cl₂ (} 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 1M HCl and then with water. The extract was dried over anhydrous _MgSO₄ , filtered, and the filtrate evaporated. The residue was used in the next step without further purification.

Synthesis of 4-PEG4-ethyl benzoate:

Procedure: (JACS, 2007, 129, 13364) A mixture of ethyl 2,5,8,11-tetraoxatridecan-13-yl 4-methylbenzenesulfonate (8.1 g, 22.3 mmol), ethyl 4-hydroxybenzoate (3.7 g, 22.3 mmol), _{K₂CO₃ (} 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. The extract was washed with _H₂O , dried over anhydrous _MgSO₄ , and filtered. The filtrate was evaporated to dryness, and the residue was purified by column chromatography on silica gel (dichloromethane/methanol = 100:1) to yield the compound (1.93 g, 88%).

Synthesis of 4-PEG4-benzoic acid:

Procedure: (JACS, 2007, 129, 13364) A mixture of ethyl 4-(2,5,8,11-tetraoxatridecan-13-yloxy)benzoate (7 g, 19.6 mmol) and KOH (2.3 g, 41.24 mmol) in 200 mL of EtOH/ _H₂O (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/hexane.

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

Procedure: To a solution of 4-(2,5,8,11-tetraoxatridecan-13-yloxy)benzoic acid (3 g, 9.14 mmol) and 0.2 mL of DMF in 30 mL of anhydrous DCM was added oxalyl chloride (1.05 mL, 12.34 mmol) at 0°C. The reaction mixture was stirred at 0°C 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) and pyridine (2.4 ml) in chloroform (80 ml) was cooled to 0°C under an inert atmosphere. (Phenyl-4-(2,5,8,11-tetraoxatridecan-13-yloxy)benzoyl chloride (3.1 g, 9.14 mmol) in 20 ml was slowly added to the solution, and the resulting mixture was allowed to reach room temperature. The solution was refluxed for 2 hours and stirred at room temperature overnight. The reaction mixture was extracted with HCl (1 M, 2 x 100 ml), _NaHCO₃ (100 ml), and water (50 ml). The organic phase was dried over _MgSO₄ and purified by chromatography on silica gel (EtOAc/hexane).

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

Procedure: N-Biphenyl-2-yl-4-(2-{2-[2-(2-methoxy-ethoxy)-ethoxy]-ethoxy}-ethoxy)-benzamide (4 g, 8.34 mmol) and _POCl₃ (10 ml) were refluxed in 10 ml of toluene for 20 h. The mixture was cooled to room temperature and 100 ml of dichloromethane were added. The solution was poured onto ice and neutralized with 20% _NH₄OH . The organic phase was extracted and washed successively with distilled water and brine, and dried over _MgSO₄ . The resulting solution was purified by flash chromatography (silica gel, in ethyl acetate/hexane 1:1, _Rf = 0.14).

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

6-(4-Methoxyphenyl)phenanthridine was prepared by cyclizing 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%.

4-Phenanthridin-6-yl-phenol: Deprotection of 6-(4-methoxyphenyl)phenanthridine was achieved using HBr. A suspension of 6-(4-methoxyphenyl)phenanthridine (1 g, 3.5 mmol) in 15 mL of HBr (47%) was refluxed at 100°C for 12 hours. The mixture was cooled to room temperature, poured into ice water, and _neutralized with _Na₂CO₃ . The resulting precipitate was filtered and washed with water and _Et₂O . The solid was purified by column chromatography using dichloromethane/MeOH. Yield: 90%.

To a solution of 4-(phenanthridin-6-yl)phenol (320 mg, 1.18 mmol) in DMF (4 mL ₎ were added _Cs₂CO₃ (482.2 mg, 1.48 mmol) and 1,3-propylsultone (159 mg, 1.30 mmol). The reaction mixture was stirred at room temperature overnight. The reaction mixture was concentrated to dryness, and the residue was purified by column chromatography (silica gel) using a dichloromethane/MeOH gradient (10:1 to 5:1). Yield: 72%.

Example 2

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

The general procedure is disclosed in Nonoyama, M., J. Organomet. Chem. 86 (1975) 263-267.

Iridium dimer was synthesized as follows: IrCl ₃ •3H ₂ O and 2.5 equivalents of 6-phenylphenanthridine were heated in a 2-ethoxyethanol/water mixture (3:1, v/v) at 120° C. for 18 hours under nitrogen. After cooling to room temperature, the precipitate was filtered off and washed sequentially with methanol and Et ₂ O, and dried to provide the desired dimer.

Example 2.1

Complex with unsubstituted phenylphenanthridine

[(6-phenylphenanthridine) ₂ IrCl] ₂ .

Example 2.2

Complexes with substituted phenylphenanthridines

A mixture of 6-[4-(2-{2-[2-(2-methoxy-ethoxy)-ethoxy]-ethoxy}-ethoxy)-phenyl]-phenanthridine (1 g, 2.16 mmol), _IrCl₃ · _3H₂O (346 mg, 0.98 mmol) in 16 mL of 2-EtOEtOH: _H₂O (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 afford an oily precipitate. The supernatant was discarded, and 50 mL of water was added to the residue. The mixture was stirred for 1 hour to afford a reddish-brown precipitate. The solid was filtered and washed with water (50 mL) and _Et₂O (30 mL). The brown solid was dissolved in a smaller amount of dichloromethane and precipitated upon addition of _Et₂O . It was used in the next step without further purification.

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

A mixture of cesium 3-(4-(phenanthridin-6-yl)phenoxy)propane-1-sulfonate (500 mg, 0.92 mmol) and _IrCl₃ (159.5 mg, 0.45 mmol) in a 3:1 mixture of 2-EtOEtOH and water (16 mL) was refluxed under a nitrogen atmosphere 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.

Example 3 :

Synthesis of iridium complexes

a) (6-phenylphenanthridine) ₂ Ir (2-(4-pyridin-2-yl-[1,2,3]triazol-1-yl)-ethanol) complex:

50 mg of [(6-phenylphenanthridine) ₂ IrCl] ₂ , 16 mg of 2-(4-pyridin-2-yl-[1,2,3]triazol-1-yl)-ethanol (synthesized as described in WO 2011/067401 A1), and 18 mg of Na ₂ CO ₃ were mixed in 2-ethoxyethanol (0.2 ml) and heated to 135°C under an inert atmosphere for 18 hours. Distilled water (20 ml) was added to the cooled mixture, and the crude product was then extracted with DCM. The organic phase was dried and the solvent evaporated to a 5 ml solution. Diethyl ether was added, the residue was filtered off, and washed with a 2% ether:methanol solution. Dissolution in DCM and precipitation with hexane yielded 32 mg of a red powder.

(Based on Lamansky, S., Inorg. Chem. 40 (2001) 1704-1711)

b) (6-phenylphenanthridine) ₂ Ir (3-(5-pyridin-2-yl-1H-pyrazol-3-yl)-propan-1-ol):

This compound was synthesized under the conditions described in Example 3 a).

Example 4 :

Synthesis of iridium-polylabel

ß-Alaninyl-ß-alaninyl-azidohomoalaninyl-ß-alaninyl-glutaminyl-ß-alaninyl-azidohomoalaninyl-ß-alaninyl-glutaminyl-ß-alaninyl-azidohomoalaninyl-ß-alaninyl-glutaminyl-ß-alaninyl-azidohomoalaninyl-ß-alaninyl-ß-alanine or NH2-UUZUEUZUEUZUU-OH (F)

Compound (F) was prepared by Fmoc-(fluorenylmethoxycarbonyl)-solid phase peptide synthesis on a Multisynthec multi-peptide synthesizer SYRO II using several reactors of 15 mg of 4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxy-resin from Novabiochem/Merck with a loading of 0.5 mmol/g. For each position containing Y in the amino acid sequence, N-Fmoc-azidohomoalanine (Azido-Abu was Fmoc-protected according to prior art methods) (Bachem), for each position containing U, Fmoc-ß-alanine, and for each position containing E, Fmoc-glutamic acid (tert-butyl ester) were coupled to the growing peptide immobilized on the synthesis resin. 90 micromoles of each N-Fmoc amino acid were coupled twice by dissolving it with 100 micromoles of 1-hydroxybenzotriazole in 270 microliters of dimethylformamide, then adding 100 micromoles of N,N-diisopropylcarbodiimide as a coupling agent, and then dispersing the resin into this solution in the reactor of the peptide synthesizer. Each coupling step lasted for 1 hour. After each coupling step, the temporary Fmoc group was cleaved using a 50% solution of piperidine in dimethylformamide over 20 minutes. After each reaction step, a dimethylformamide wash step was performed. After synthesis, the final peptide (F) was cleaved from the resin using a mixture of 95% trifluoroacetic acid and 5% ethanedithiol over 2 hours to remove the permanent tert-butyl ester protecting group. After filtering off the resin beads, the product was precipitated by adding cold diisopropyl ether, isolated by filtration, redissolved in acetic acid, and lyophilized. The crude material was purified by reverse phase HPLC to at least 95% pure material. Characterization was performed by analytical reverse phase HPLC and ESI-MS.

The 2-(1H-[1,2,3]triazol-4-yl)-pyridine derivative of the linker Q can be synthesized by "click-chemistry," as described in WO 2011/067401 A1, a copper-catalyzed Huisgen cycloaddition for the synthesis of 2-(4-pyridin-2-yl-[1,2,3]triazol-1-yl)-ethanol. In the next step, the iridium complex dimer described above is further reacted in molar excess with a linker containing 2-(1H-[1,2,3]triazol-4-yl)-pyridine in DMF at 80° C. under a nitrogen atmosphere overnight. The iridium-linker derivative can be purified by reverse phase chromatography and the product characterized by HPLC-ESI MS.

Claims (15)

1. Iridium-based chemiluminescent compounds of formula I Formula I Each of R1-R18 is independently hydrogen, halogen, cyano or nitro, amino, substituted amino, alkylamino, substituted alkylamino, arylamino, substituted arylamino, alkylammonium, substituted alkylammonium, carboxyl, carboxylate, carboxylate ester, carbamoyl, hydroxyl, substituted or unsubstituted alkoxy, substituted or unsubstituted aryloxy, thioalkyl, substituted or unsubstituted alkylsulfonyl, substituted or unsubstituted arylsulfonyl, sulfonyl, sulfinyl, sulfonate, sulfinate The following groups are included: alkyl, sulfenyl, aminosulfonyl, phosphonyl, hydroxyphosphonoyl, hydroxy-alkyl-phosphonoyl, phosphonate, phosphonate, or R19, wherein R19 is aryl, substituted aryl, alkyl, substituted alkyl, aralkyl, substituted aralkyl, alkylaryl, substituted alkylaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, amino-alkoxy, substituted amino-alkoxy, amino-aryl, substituted amino-aryl, amino-aryloxy, substituted amino-aryloxy. In R1-R12, or/and in R13-R16, and/or R17 and R18, two adjacent Rs may form an aromatic ring or a substituted aromatic ring, wherein the substituent is selected from hydrogen, alkyl, substituted alkyl, halogen, cyano or nitro, hydrophilic group, wherein the hydrophilic group is selected from amino, substituted amino, alkylamino, substituted alkylamino, alkylammonium, substituted alkylammonium, carboxyl, carboxylate, carboxylate, carbamoyl, hydroxy, substituted or unsubstituted alkoxy, substituted or unsubstituted aryloxy, thioalkyl, substituted or unsubstituted alkylsulfonyl, substituted or unsubstituted arylsulfonyl, sulfonyl, sulfinyl, sulfonate, sulfinate, sulfonate, aminosulfonyl, phosphono, hydroxyphosphono, hydroxy-alkyl-phosphono, phosphonate and phosphonate, or In R1-R12, or/and in R13-R16, and/or R17 and R18, two adjacent Rs may form an aliphatic ring or a substituted aliphatic ring, wherein the substituent is selected from hydrogen, alkyl, substituted alkyl, halogen, cyano or nitro, hydrophilic group, wherein the hydrophilic group is selected from amino, substituted amino, alkylamino, substituted alkylamino, alkylammonium, substituted alkylammonium, carboxyl, carboxylate, carboxylate, carbamoyl, hydroxy, substituted or unsubstituted alkoxy, substituted or unsubstituted aryloxy, thioalkyl, substituted or unsubstituted alkylsulfonyl, substituted or unsubstituted arylsulfonyl, sulfonyl, sulfinyl, sulfonate, sulfinate, sulfonate, aminosulfonyl, phosphono, hydroxyphosphono, hydroxy-alkyl-phosphono, phosphonate and phosphonate. Wherein, if substitution is present in any of R1-R19, the substituents in R1-R19 are each independently selected from halogen, cyano or nitro, and hydrophilic groups, wherein the hydrophilic groups are selected from amino, alkylamino, alkylammonium, carboxyl, carboxylate, carboxylate ester, carbamoyl, hydroxy, alkoxy, arylalkoxy, aryloxy, alkylaryloxy, polyvinyloxy, polyacryloxy, thioalkyl, alkylsulfonyl, arylsulfonyl, sulfonyl, sulfinyl, sulfenyl, sulfonate, sulfinate, sulfenate, aminosulfonyl, phosphono, hydroxyphosphono, hydroxy-alkyl-phosphono, phosphonate, phosphonate. The alkyl group is a straight-chain or branched alkyl chain with a length of 1-20 carbon atoms or a heteroalkyl chain with a length of 1-20 atoms containing 1-4 heteroatoms selected from O, N, P, and S; the aryl group is a 5, 6, or 7-membered aryl ring system or a 5, 6, or 7-membered heteroaryl ring system containing 1-3 heteroatoms selected from O, S, and N. Where X represents C or N. Where Y represents C or N. In Formula I, at least one of R13-R18 is –Q–Z, where Q–Z is maleimide or where Q is a covalent bond, or a straight or branched saturated, unsubstituted or substituted C21-C200 alkyl chain, or a 21- to 200-atom chain with a skeleton 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 the chain whose skeleton comprises one or more cyclic or heterocyclic aromatic or non-aromatic ring systems, where Z is a functional group. The functional group Z is selected from aldehyde, carboxylic acid, carboxylic acid ester, epoxide, N-hydroxysuccinimide ester, amino, halogen, hydrazine, hydroxy, mercapto, maleimide, alkynyl, azide, isocyanate, isothiocyanate, and phosphoramidite. 2. The compound according to claim 1, wherein R19 is a branched alkyl group or a substituted branched alkyl group. 3. The compound of claim 1, wherein Q is a straight or branched saturated, unsubstituted or substituted C21-C200 alkyl chain, or a 21 to 200-atom chain with a skeleton 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 the skeleton comprises one or more cyclic or heterocyclic aromatic or non-aromatic ring systems. 4. The compound of claim 3, wherein Q is a straight or branched saturated, unsubstituted or substituted C21-C50 alkyl chain, or a 21 to 50-atom chain with a skeleton 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 the skeleton contains one or more cyclic or heterocyclic aromatic or non-aromatic ring systems. 5. The compound according to any one of claims 1 to 4, wherein the functional group Z is selected from carboxylic acid group, N-hydroxysuccinimide group, amino group, halogen group, mercapto group, maleimide group, alkynyl group, azide group, isocyanate group, isothiocyanate group and phosphoramidide group. 6. The compound according to any one of claims 1 to 4, wherein the functional group Z is selected from N-hydroxysuccinimide ester group and maleimide group. 7. The compound according to any one of claims 1 to 4, wherein Q-Z are maleimides. 8. The compound according to any one of claims 1 to 4, wherein at least one of R1-R12 is a substituted or unsubstituted group selected from the following: sulfonyl-alkyl, sulfonyl-aryl, sulfonyl-alkoxy, sulfonyl-aryloxy, sulfonyl, sulfinyl-alkyl, sulfinyl-aryl, sulfinyl-alkoxy, sulfinyl-aryloxy, sulfinyl, sulfenyl-alkyl, sulfenyl-aryl, sulfenyl-alkoxy, sulfenyl-aryloxy, sulfenyl, aminosulfonyl-alkyl, aminosulfonyl-aryl, aminosulfonyl-alkoxy, aminosulfonyl-aryloxy, aminosulfonyl, alkylsulfonyl-alkyl, alkylsulfonyl-aryl, alkylsulfonyl, aromatic sulfonyl-alkyl alkyl, or aryl sulfonyl-aryl, or aryl sulfonyl, sulfonamino-alkyl, sulfonamino-aryl, sulfonamino-alkoxy, sulfonamino-aryl, sulfonamino-aryl, sulfonamino-aryl, sulfonamino-aryl, sulfonamino-aryl, sulfonamino-aryl, sulfonamino-aryl, alkyl sulfonylamino-alkyl, alkyl sulfonylamino-aryl, alkyl sulfonylamino-alkoxy, alkyl sulfonylamino-aryl, aryl sulfonylamino-aryl, aryl sulfonylamino-aryl, aryl sulfonylamino-aryl, aryl sulfonylamino-aryl, aryl sulfonylamino-aryl, aryl sulfonylamino-aryl, aryl sulfonylamino-aryl, aryl sulfonylamino-aryl, aryl sulfonylamino-aryl, aryl sulfonylamino-aryl, aryl sulfonylamino, alkyl sulfonylamino alkyl-alkyl, alkyl sulfinylamino-aryl, alkyl sulfinylamino-alkoxy, alkyl sulfinylamino-aryl, alkyl sulfinylamino, aromatic sulfinylamino-alkyl, aromatic sulfinylamino-aryl, aromatic sulfinylamino-alkoxy, aromatic sulfinylamino-aryl, aromatic sulfinylamino, phosphonyl-alkyl, phosphonyl-aryl, phosphonyl-alkoxy, phosphonyl-aryl, phosphonyl, hydroxyphosphonyl-alkyl, hydroxyphosphonyl-aryl, hydroxyphosphonyl-alkoxy, hydroxyphosphonyl-aryl, hydroxyphosphonyl-alkyl, hydroxy-alkyl-phosphonyl-alkyl, hydroxy-alkyl-phosphonyl The alkyl group is a salt of the above-mentioned substituents, including alkyl-aryl, hydroxy-alkyl-phosphinico-alkoxy, hydroxy-alkyl-phosphinico-aryl, hydroxy-alkyl-phosphinico-, phosphinicoamino-alkyl, phosphinicoamino-aryl, phosphinicoamino-alkoxy, phosphinicoamino-aryl, phosphinicoamino, or phosphinicoamino, wherein "alkyl" is a straight-chain or branched alkyl chain of 1-20 carbon atoms or a heteroalkyl chain of 1-20 atoms containing 1-4 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 1-3 heteroatoms selected from O, S and N. 9. The compound according to any one of claims 1 to 4, wherein at least one of R1-R12 is a substituted or unsubstituted group selected from the following: sulfonyl-alkyl, sulfonyl-aryl, sulfonyl-alkoxy, sulfonyl-aryloxy, sulfonyl, aminosulfonyl-alkyl, aminosulfonyl-aryl, aminosulfonyl-alkoxy, aminosulfonyl-aryloxy, aminosulfonyl, alkylsulfonyl-alkyl, alkylsulfonyl-aryl, alkylsulfonyl, aromatic sulfonyl-alkyl, aromatic sulfonyl-aryl, aromatic sulfonyl, alkylsulfonylamino-alkyl, alkylsulfonylamino-aryl, alkylsulfonylamino-alkoxy, alkylsulfonylamino-aryloxy, alkylsulfonylamino, aromatic sulfonylamino-alkyl, aromatic sulfonylamino-aryl, aromatic sulfonylamino-alkoxy, aromatic sulfonylamino-aryloxy, aromatic sulfonylamino-aryloxy, aromatic sulfonylamino-aryloxy, phosphonyl The following are salts of the above substituents under chemical matching: alkyl-phosphono-aryl, phosphono-alkoxy, phosphono-aryl, phosphono-hydroxyphosphono-alkyl, hydroxyphosphono-aryl, hydroxyphosphono-alkoxy, hydroxyphosphono-aryl, hydroxy-alkyl-phosphono-aryl, hydroxy-alkyl-phosphono-alkoxy, hydroxy-alkyl-phosphono-aryl, hydroxy-alkyl-phosphono-alkoxy, hydroxy-alkyl-phosphono-aryl, hydroxy-alkyl-phosphono-aryl, or salts of the above substituents under chemical matching, wherein "alkyl" is a straight-chain or branched alkyl chain of 1-20 carbon atoms or a heteroalkyl chain of 1-20 atoms containing 1-4 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 1-3 heteroatoms selected from O, S and N. 10. A conjugate comprising the compound according to any one of claims 1 to 9 and an affinity binder covalently bonded thereto. 11. The conjugate of claim 10, wherein the affinity binding agent is selected from antigens and antibodies, biotin or biotin analogs and avidin or streptavidin, sugars and lectins, nucleic acids or nucleic acid analogs and complementary nucleic acids, and receptors and ligands, wherein the biotin analog is aminobiotin, iminobiotin or desulfobiotin, and the nucleic acid analog is a substituted nucleotide with a substituent at a standard base of deoxyadenosine (dA), deoxyguanosine (dG), deoxycytidine (dC), deoxythymidine (dT), or deoxyuridine (dU). 12. The conjugate of claim 10 or 11, wherein the affinity binding agent is a nucleic acid or an antibody. 13. Use of the compound according to any one of claims 1 to 9 or the conjugate according to any one of claims 10 to 12 for carrying out an electrochemiluminescence reaction in aqueous solution. 14. Use of the compound according to any one of claims 1 to 9 or the conjugate according to any one of claims 10 to 12 in an electrochemiluminescence-based detection method. 15. A method for measuring an analyte by an in vitro method, the method comprising the steps of a) Provide a sample that is suspected or known to contain the analyte. b) Contact the sample with the conjugate according to any one of claims 10 to 12 under conditions suitable for forming an analyte-conjugate complex, and c) Measure the complex formed in step (b) and thereby obtain the quantity of the analyte.