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    • 专利摘要:
    CHEMIOLUMINESCENT COMPOUND BASED ON IRIDIOUS, CONJUGATED, USES OF A COMPOUND AND METHOD FOR MEASURING ANALYTICALThe present invention relates to new luminescent complexes based on iridium Ir (III), conjugates that comprise these complexes as a marker and their applications, for example, in the detection of an electrolyte-based analyte.
    公开号:BR112013019503A2
    申请号:R112013019503-7
    申请日:2012-02-07
    公开日:2020-11-10
    发明作者:Hans-Peter Josel；Eloisa Lopez-Calle；Jesus Miguel Fernandez Hernandez；Luisa De Cola；Robert Cysewski；Toralf Zarnt
    申请人:F. Hoffmann-La Roche Ag；
    IPC主号:
    专利说明:

    [001] [001] The present invention relates to new luminescent complexes based on iridium Ir (III), conjugates that comprise these complexes as a marker and their applications, for example, in the detection of an analyte based on electrochemiluminescence. BACKGROUND OF THE INVENTION
    [002] [002] Electrogenerated chemiluminescence (also called electrochemiluminescence and abbreviated as ECL) is the process by which species generated on electrodes undergo high-energy electron transfer reactions to form excited states that emit light. The first detailed studies of ECL were described by Hercules and Bard et al. in the mid-1960s. After about 40 years of study, ECL has now become a very powerful analytical technique and is widely used in the areas of, for example, immunoassay, food and water testing, and biological warfare.
    [003] [003] There are a huge number of compounds that appear to be of interest for use in organic light-emitting devices (OLEDs).
    [004] [004] In general, ECL-based detection methods are based on the use of water-soluble ruthenium complexes, which comprise Ru (II +) as a metal ion.
    [005] [005] Despite significant improvements made over the past decades, there is still a huge need for in vitro diagnostic tests based on more sensitive electrochemiluminescence.
    [006] [006] Nowadays it has surprisingly been discovered that certain luminescent complexes based on iridium ir (III +), represent very promising markers for future detection methods based on high sensitivity ECL. BRIEF DESCRIPTION OF THE INVENTION
    [007] [007] The present invention discloses a chemiluminescent compound based on iridium of Formula I R6 R7 R5 R4 R8 R3 R9 R10 N R2 R1 R11
    [008] [008] The present invention also discloses a conjugate comprising the above compound and covalently attached to it an affinity binding agent.
    [009] [009] The present invention also relates to the use of a compound or conjugate as disclosed in the present invention to perform a luminescence measurement or an electrochemiluminescence reaction in an aqueous solution, especially in an electrochemiluminescent device or an electrochemical chemiluminescent detection system.
    [010] [010] In addition, the present invention discloses a method for measuring an analyte by an in vitro method, the method of which comprises the steps of (a) supplying a suspected or known sample comprising the analyte, (b) placing said sample contacting a conjugate according to the present invention under conditions suitable for the formation of a conjugate-analyte complex, and (c) measuring the complex formed in step (b), and thereby obtaining a measure of the analyte. DETAILED DESCRIPTION OF THE INVENTION
    [011] [011] The present invention relates to an iridium-based chemiluminescent compound of Formula I R6 R7 R5 R4 R8 R3 R9 R10 N R2 R1 R11
    [012] [012] In one embodiment, at least one of the R1 to R16 of the compound according to Formula I is replaced by at least one hydrophilic group.
    [013] [013] In one embodiment, the preferred substituents for substituted alkyloxy are ethylenoxy chains that comprise from 1 to 40 ethylenoxy units or that comprise from 1 to 20 ethylenoxy units or that comprise from 1 to 10 ethylenoxy units.
    [014] [014] Preferred hydrophilic groups are amino, alkylamino, with alkyl meaning a straight chain such as methyl, ethyl, propyl, pentyl, butyl or a branched alkyl chain, such as isopropyl, isobutyl, tertiary butyl, preferably a linear alkyl chain such as methyl or ethyl, substituted alkylamino, it contains one or two, for example, a branched or straight chain attached to the N atom, which are replaced by an additional hydrophilic group, such as hydroxyl or sulfo, preferably that substituted alkylamino contains two hydroxypropyl residues or hydroxyethyl, arylamino, with aryl, which refers to an aromatic residue, such as phenyl or naphthyl, preferably phenyl, substituted arylamino, with aryl as defined above and an additional residue formed by a hydrophilic group, alkylammonium, with alkyl as defined above and preferably being a trimethylammonium or triethylammonium, substituted alkylammonium, carb residue oxy, carboxylic acid ester, preferably an alkyl ester, such as methyl or ethyl ester, carbamoyl, hydroxy, alkyl unsubstituted or substituted with alkyl and substituted alkyl being as defined above or aryloxy or aryloxy substituted with aryl and substituted aryl being as defined above, sulfanyl, alkylsulfonyl, arylsulfonyl, sulfo, sulfine, sulfene, sulfonamide, sulfoxide, sulfodioxide, phosphonate, phosphinate.
    [015] [015] Preferably, this hydrophilic group is selected from amino, alkylamino, substituted alkylamino, substituted arylamino, alkylammonium, substituted alkylammonium, carboxy, hydroxy, sulfo, sulfene, sulfonamide, sulfoxide, sulfodioxide and phosphonate, when applicable, each preferably , as defined in the paragraph above.
    [016] [016] In an additional embodiment, the hydrophilic group is selected from sulfo, sulfonamide, sulfodioxide.
    [017] [017] In one embodiment, at least one of the groups R1 to R12 of Formula I is a sulfo group.
    [018] [018] In one embodiment, at least one of the R1 to R12 of the phenylphenanthridine residues comprised in Formula I is replaced by at least one hydrophilic group.
    [019] [019] In one embodiment, the phenylphenanthridine residues comprised in Formula I are selected from the substituted phenylphenanthridines given below.
    [020] [020] In the compound according to the present invention, the linker Q preferably has a length of the main chain between 1 and 20 atoms. In other words, the shortest connection between the Formula I pyridyl ring and the functional group Y consists of 1 to 20 atoms. In one embodiment, the Q linker in the electrochemical chemiluminescent complex of that invention is a saturated, unsaturated, unsubstituted, substituted, substituted, straight or branched C1-C20 alkyl chain or a C1-C20 arylalkyl chain (for example, where a phenylene ring counts for one length of four carbon atoms) or a chain of 1 to 20 atoms, with a main chain consisting of carbon atoms and one or more hetero atoms selected from O, N and S, or a chain of 1 to 20 atoms, with a backbone consisting of carbon atoms and one or more heteroatoms selected from O, N and S comprising at least one aryl, heteroaryl, substituted aryl or substituted heteroaryl group (for example, where a phenylene ring counts for a length of four atoms). In one embodiment, the linker Q in a compound according to the present invention is a saturated C1-C12 alkyl chain, or a C1-C12 arylalkyl chain or a chain of 1 to 12 atoms, with a main chain consisting of atoms of carbon and one or more hetero atoms selected from O, N and S, or a chain of 1 to 12 atoms, with a main chain consisting of carbon atoms and one or more hetero atoms selected from O, N and S that comprise at least one aryl, heteroaryl, substituted aryl or substituted heteroaryl group (for example, where a phenylene ring counts for a length of four atoms).
    [021] [021] In one embodiment, the functional group Y comprised in the iridium-based complex according to the present invention is selected from the group consisting of carboxylic acid, N-
    [022] [022] A conjugate comprising an electrochemiluminescent compound based on iridium of Formula I, as disclosed and defined in the present application above and a biological substance covalently bound therein. 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.
    [023] [023] In one embodiment, the biological substance of a conjugate according to the present invention, that is, covalently linked to a compound according to Formula I, is an affinity binding agent.
    [024] [024] Not wishing to be further limited, but, in the interest of objectivity, the affinity binding agent may comprise any of the following: an antigen, a protein, an antibody, biotin or analogous to biotin and avidin or streptavidin, sugar and lectin, an enzyme, a polypeptide, an amino group, a nucleic acid or nucleic acid analog and complementary nucleic acid, a nucleotide, a polynucleotide, a peptide nucleic acid (PNA), a polysaccharide, a metal ion sequestering agent , a receptor agonist, receptor antagonist, or any combination thereof. For example, the affinity binding agent may be a partner of a specific binding pair, in which the other partner of said binding pair is associated with or is the target on a cell surface or an intracellular structure.
    [025] [025] Preferably, an affinity liaison officer is, a partner or member of an affinity liaison pair, or as it is also called by the person skilled in the art, a partner or member of a specific liaison pair.
    [026] [026] An affinity linker has at least an 107 L / mol affinity for its target, for example, a member of a specific binding pair, such as an antibody, to the other member of the specific binding pair, as its antigen. An affinity binding agent preferably has an affinity of 108 L / mol or even more preferably 109 L / mol for its target.
    [027] [027] In one embodiment, the present invention relates to a conjugate in which the affinity binding agent is selected from the group consisting of antigen, antibody, biotin or biotin analog, avidin or streptavidin, sugar, lectin , nucleic acid or nucleic acid analogue and complementary nucleic acid, receptor and ligand.
    [028] [028] In one embodiment, the present invention relates to a conjugate in which the affinity binding agent is selected from the group consisting of antibody, biotin or biotin analog, avidin or streptavidin and nucleic acid.
    [029] [029] In one embodiment, the conjugate according to the present invention comprises a compound according to Formula I covalently linked, as disclosed and defined in the present application above, and an affinity binding agent that is both an oligonucleotide and an antibody .
    [030] [030] Biotin analogs are aminobiotin, iminobiotin or destiobiotin.
    [031] [031] The term "oligonucleotide" or "nucleic acid", as used in the present application, generally refers to single-stranded polynucleotides comprising at least 8 nucleotides and a maximum of about 1000 nucleotides. In a preferred embodiment, an oligonucleotide will have a length of at least 9, 10, 11, 12, 15, 18, 21, 24, 27 or 30 nucleotides. In a preferred embodiment, an oligonucleotide will have a length of not more than 200, 150, 100, 90, 80, 70, 60, 50, 45, 40, 35 or 30 nucleotides.
    [032] [032] The term oligonucleotide is to be understood widely and includes DNA and RNA, as well as analogues and modifications thereof.
    [033] [033] A nucleic acid analogue can contain, for example, a substituted nucleotide carrying a substituent on the standard bases of deoxyadenosine (dA), deoxyguanosine (dG), deoxycytosine (dC), deoxythymidine (dT), deoxyuracil (dU). Examples of such substituted nucleobases are: 5-substituted pyrimidines such as 5 methyl dC, aminoalyl dU or dC, 5- (aminoethyl-3-acrylamide) -dU, 5-propynyl-dU or -dC, 5 halogenated -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 halogenated dA or dG, 8 – dG or dA alkyl; and 2 substituted dA as 2 amino dA.
    [034] [034] A nucleic acid analog can contain a nucleotide or a nucleoside analog. That is, naturally occurring nucleobases can be exchanged using nucleobase analogs such as 5-Nitroindole d riboside; 3 nitro pyrrole d riboside, deoxyinosine (dI), deoxyxanthinesine (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 Formicin; pseudo dU; pseudo iso dC; 4 uncle dT; 6 uncle dG; 2 uncle dT; iso dG; 5-methyl-iso-dC; N8-linked 8-aza-7 – deaza-dA; 5,6-dihydro-5-aza-dC; and ethylene-dA or pyrol-dC. Obvious to a person skilled in the art, the nucleobase in the complementary tape must be selected in such a way that the duplex formation is specific. If, for example, 5-methyl-iso-dC is used on a tape (for example, (a)) iso dG must be on the complementary tape (for example, (a ')).
    [035] [035] In a nucleic acid analogue the oligonucleotide backbone can be modified to contain substituted sugar residues, sugar analogs, changes in the internucleoside phosphate component and / or be a PNA.
    [036] [036] An oligonucleotide may, for example, contain a nucleotide with a substituted deoxy ribose such as 2'-methoxy, 2'-fluoro, 2'-methylethelene, 2'-allyoxy, 4'-methyl dN (where N is a nucleobase, for example, A, G, C, T or U).
    [037] [037] Sugar analogues are, for example, xylose; 2 ', 4' with Ribose bridge as (2'-O, 4'-C methylene) - (oligomer known as LNA) or (2'-O, 4'-C ethylene) - (oligomer known as ENA); L-ribose, L-d-ribose, hexitol (oligomer known as HNA); cyclohexenyl (oligomer known as CeNA); altritol (an oligomer known as ANA), a tricyclic ribose analog where the atoms C3 'and C5' are connected by an ethylene bridge that is fused to a cyclopropane ring (oligomer known as tricycle-DNA), glycerin (oligomer known as GNA); Glycopyranose (oligomer known as Homo-DNA); carbaribose (with a cyclopentane instead of a tetrahydrofuran subunit); hydroxymethyl-morpholine (oligomers known as morpholine DNA).
    [038] [038] A large number of modifications of the internucleoside phosphate component are also known to not interfere with hybridization properties and these modifications of the main chain can also be combined with substituted nucleotides or nucleotide analogues. Examples are phosphorioate, phosphorditioate, phosphoramidate and methylphosphonate oligonucleotides.
    [039] [039] PNA (which has a phosphate and d-ribose backbone) can also be used as a DNA analog.
    [040] [040] The aforementioned modified nucleotides, nucleotide analogs as well as modifications of the oligonucleotide backbone can be combined as desired into an oligonucleotide in the sense of the present invention.
    [041] [041] The term "antibody" in this application is used in the broadest sense and specifically encompasses monoclonal antibodies, polyclonal antibodies, multispecific antibodies (for example, bispecific antibodies) formed by at least two intact antibodies and antibody fragments, as long as they exhibit the desired biological activity.
    [042] [042] An "isolated" antibody is one that has been identified and separated and / or recovered from a component of its natural environment.
    [043] [043] "Native antibodies" are generally heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by a covalent disulfide bridge, although the number of disulfide bonds varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has disulfide bridges between regularly spaced chains. Each heavy chain has, at one end, a variable domain (VH) followed by several constant domains. Each light chain has a variable domain at one end (VL) 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 variable domain of the light chain is aligned with the variable domain of the heavy chain. Specific amino acid residues are believed to form an interface between the light and heavy chain variable domains.
    [044] [044] The "variable region" or "variable domain" of an antibody refers to the amino terminal domains of the antibody's heavy chain or light chain. The variable domain of the heavy chain can be referred to as "VH". The variable domain of the light chain can be referred to as “VL”. These domains are usually the most variable parts of an antibody and contain the antigen binding sites.
    [045] [045] The term "variable" refers to the fact that certain portions of the variable domain differ extensively in sequence between antibodies and are used in the binding and specificity of each specific antibody to its specific antigen. However, variability is not evenly distributed across all variable domains of antibodies. It is concentrated in three segments called hypervariable regions (HVRs), both in the light chain and in the variable domains of the heavy chain.
    [046] [046] The "light chains" of antibodies (immunoglobulins) of any species of vertebrate can be attributed to one of the two clearly distinct types, called kappa () and lambda (), based on the amino acid sequence of their constant domains.
    [047] [047] Depending on the amino acid sequences of the domains contained in their heavy chains, antibodies (immunoglobulins) can be assigned to different classes. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these can also be divided into subclasses (isotypes), for example, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known and generally described, for example, in Abbas et al., Cellular and Mol. Immunology, 4th ed. W. B. Saunders, Co. (2000). An antibody can be part of a larger fusion molecule, formed by covalent or non-covalent association of the antibody with one or more proteins or peptides.
    [048] [048] The terms "whole antibody", "whole antibody" and "whole antibody" are used interchangeably in this application to refer to an antibody in its substantially intact form, and not to antibody fragments as defined below. The terms refer specifically to an antibody with heavy chains that contain an Fc region.
    [049] [049] "Antibody fragments" comprise a portion of an intact antibody, preferably comprising the antigen binding region thereof. Examples of antibody fragments include Fab, Fab ', F (ab') 2 fragments, and Fv fragments; diabodies; linear antibodies; single chain antibody molecules and multispecific antibodies formed from antibody fragments.
    [050] [050] Antibody digestion with papain 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 readily crystallize . Pepsin treatment produces an F (ab ') 2 fragment that has two antigen combining sites and is also capable of cross-linking with the antigen.
    [051] [051] "Fv" is the minimum antibody fragment that contains a complete antigen binding site. In one embodiment, a double-stranded Fv species consists of a variable domain dimer of a heavy chain and a light chain in close non-covalent association. In a single-chain Fv species (scFv), a variable domain of the light chain and one of the heavy chain can be covalently linked by a flexible peptide linker, so that the light and heavy chains can associate in a "dimeric" structure analogous to a double-stranded Fv species. It is in this configuration that the three HVRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer.
    [052] [052] The Fab fragment contains the light and heavy chain variable domains and also contains the light chain constant domain and the first heavy chain constant (CH1) domain. Fab 'fragments differ from Fab fragments by adding some residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is the designation in the present application for Fab 'where the cysteine residue (s) of the constant domains carry a free thiol group. The F (ab ') 2 antibody fragments were originally produced as pairs of Fab' fragments that have cysteine hinges between them. Other chemical couplings of antibody fragments are also known.
    [053] [053] Fragments of "Fv single chain" or "scFv" antibodies comprise the VH and VL domains of an antibody, where these domains are present in a single polypeptide chain. Generally, the scFv polypeptide also comprises a polypeptide linker between the VH and VL domains that allows scFv to form the desired structure for antigen binding.
    [054] [054] The term "diabody" refers to antibody fragments with two antigen binding sites, whose fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) on the same polypeptide chain (VH-VL). Using a linker that is extremely short to allow pairing between the two domains on the same chain, the domains are forced to pair with complementary domains on another chain and create two antigen binding sites. Diabodies can be bivalent or bispecific. Diabodies are more fully described, for example, in EP 0404 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. Tribodies and tetrabodies are also described in Hudson P. J. et al., Nat. Med. 9 (2003) 129-134.
    [055] [055] The term "monoclonal antibody", as used in the present application, refers to an antibody obtained from a substantially homogeneous population of antibodies, that is, the individual antibodies that comprise the population are identical, except for possible mutations, for example, naturally occurring mutations that may be present in smaller quantities. Thus, the “monoclonal” modifier indicates the character of the antibody as not being a mixture of distinct antibodies. In certain embodiments, that monoclonal antibody typically includes an antibody that comprises a sequence of polypeptides that binds to a target, wherein the sequence of target binding polypeptides has been obtained by a process that includes the selection of a single sequence of binding polypeptides target, from a plurality of polypeptide sequences. For example, the selection process may be the selection of a single clone, from a plurality of clones, such as a cluster of hybridoma clones, phage clones or recombinant DNA clones. It should be understood that the selected target binding sequence can also be changed, for example, to improve affinity for that target, humanize the target binding sequence, improve its production in cell culture, reduce its immunogenicity in vivo,
    [056] [056] As mentioned, the compounds and conjugates as disclosed in the present application have very favorable properties. For example, the compounds or conjugates disclosed, respectively, show high ECL efficiency. This high efficiency is also present if the corresponding measurements are carried out in an aqueous system compared to many ECL markers that have only shown high ECL efficiency when analyzed in an organic solvent. For example, many OLED dyes are usually analyzed in acetonitrile and are either not soluble in an aqueous solution or, if soluble, because they do not show efficient electrochemiluminescence in an aqueous solution.
    [057] [057] In a preferred embodiment, the present invention relates to the use of a compound or a conjugate, respectively, as disclosed in the present invention for carrying out an electrochemiluminescence reaction in an aqueous solution. An aqueous solution is a solution that comprises at least 90% water (by weight).
    [058] [058] In one embodiment, the present invention relates to the use of a compound or a conjugate, respectively, as disclosed in the present invention in a detection method based on electrochemiluminescence.
    [059] [059] In one embodiment, the present invention relates to the use of a compound or a conjugate, respectively, as disclosed in the present invention in the detection of an analyte.
    [060] [060] An analyte according to the present invention can be any organic or inorganic molecule, which includes any biological substance of interest. Examples of suitable biological substances that represent an analyte 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, substances pharmacological, alkaloids, steroids, vitamins, amino acids and sugars.
    [061] [061] The analyte can be selected from the group consisting of a polypeptide, a carbohydrate and an inorganic or organic drug molecule.
    [062] [062] A polypeptide or protein is a molecule that is essentially composed of amino acids and that has at least two amino acids linked by peptide bond. In the event that the analyte of interest is investigated in a method disclosed in the present application, the polypeptide preferably will consist of at least 5, 6, 7, 8, 9, 10, 12, 15, 20, 25 and 30 to up to about 10,000 amino acids . Preferably, the polypeptide will contain from 5 to 2,000, also preferably from 10 to 1,000 amino acids.
    [063] [063] If the analyte is a nucleic acid, these nucleic acids are preferably naturally occurring DNA or RNA oligonucleotides.
    [064] [064] In one embodiment, the present invention relates to a method for measuring an analyte by an in vitro method, the method of which comprises the steps of (a) providing a suspected or known sample comprising the analyte, (b) placing said sample in contact with a conjugate according to, between an affinity binding agent and a compound according to Formula I, as disclosed in the present invention under conditions suitable for formation of a conjugate-analyte complex, and (c) measuring the complex formed in step (b), and thereby obtain a measurement of the analyte.
    [065] [065] In one embodiment, the measurement in the above method for detecting an analyte is performed using a detection procedure based on electrochemiluminescence. Also preferred, the method is practiced in an aqueous solution.
    [066] [066] The following examples are provided to aid the understanding of the present invention, the true scope of which is presented in the attached claims. It is understood that modifications can be made to the procedures presented without departing from the spirit of the invention.
    [067] [067] With the Suzuki-Miyaura coupling reaction described by Youn, S.W., in Tetrahedron Lett. 50 (2009) 4598-4601, between the commercially available 2-bromoaniline derivatives and the corresponding arylboronic acid, 2-aminodiphenyls can be synthesized, which are necessary for the additional reactions to phenyltridines.
    [068] [068] Typical procedure:
    [069] [069] For the cooled solution of 2-arylaniline 1 (0.01 mol) in chloroform (20 mL), aryl acid chloride 2 (0.01 mol) was added and stirred under inert condition for 30 min at room temperature. The resulting mixture was refluxed with stirring for the next 2 h. The reaction mixture was treated by adding dropwise pyridine (0.02 mol in 10 ml of chloroform) over a period of 60 min. The mixture was cooled to room temperature and stirred overnight. The mixture was washed well with 0.05M HCl, dried over MgSO 4 and concentrated in vacuo. The crude product was purified by flash chromatography on silica gel, 3: 2 hexane / ethyl acetate to provide crude product 3 at 66% of production.
    [070] [070] Benzamido-2-diphenyl 3 (0.01 mol) and POCl3 (5 ml) in 20 ml of toluene were refluxed and stirred under nitrogen for 18 h, following the procedure described by Lion, C., in Bull. Soc. Chim. Belg.
    [071] [071] Using 2-naphthalen-2-yl-phenylamine instead of 2-arylaniline: 1 H-NMR (400 MHz, CDCl3) 8.64 (d, J = 9.1 Hz, 2H) , 8.29 (d, J = 8.1 Hz, 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).
    [072] [072] Using naphthalene-carbonyl chloride instead of phenyl acid chloride: 1 H-NMR (400 MHz, CDCl3) 8.74 (d, J = 8.3 Hz, 1H), 8.65 (d, J = 8.1 Hz, 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).
    [073] [073] 6- (2-sulfophenyl) phenanthridine can be synthesized by lightly heating arylaniline (0.01 mol) with cyclic 2-sulfobenzoic acid (0.01 mol) in CH3CN for 6 h using the procedure described by Nicolai, E., in Chem. Pharm. Bull. 42 (1994) 1617-1630.
    [074] [074] After purification, the product can be converted to the appropriate phenanthridine based on the method described in Example 1.2.
    [075] [075] 6-Phenyl-alkylsulfonyl-phenanthridine can be synthesized by lightly heating alkylsulfonyl-arylaniline (0.01 mol) with benzoic acid 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.
    [076] [076] After purification, the product can be converted to the appropriate phenanthridine based on the method described in Example 1.2.
    [077] [077] 6- (4-methylsulfophenyl) phenanthridine can also be prepared by the following procedure, described by Cymerman, J., in J. Chem.
    [078] [078] Procedure: (JACS, 2007, 129, 13364) For a solution of 2,5,8,11-tetraoxatridecan-13-ol (7 g, 33.6 mmol) and triethylamine (4.9 mL, 35, 3 mmol) in dry CH2Cl2 (100 mL), 4-toluenesulfonyl chloride (6.7 g,
    [079] [079] Procedure: (JACS, 2007, 129, 13364) A mixture of ethyl 4-methylbenzenesulfonate 2,5,8,11-tetraoxatridecan-13-yl compound (8.1 g, 22.3 mmol), ethyl ester of 4-hydroxybenzoic acid (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 h. 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 obtain the compound (1.93 g, 88%).
    [080] [080] Procedure: (JACS, 2007, 129, 13364) A mixture of the compound 4- (2,5,8,11-tetraoxatridecan-13-yl-oxy) ethyl 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 / hexanes.
    [081] [081] Procedure: For a solution of 4- (2,5,8,11- tetraoxatridecan-13-yl-oxy) benzoic acid (3 g, 9.14 mmol), 0.2 mL of DMF in 30 mL of DCM dried at 0 ° C, oxalyl chloride (1.05 mL, 12.34 mmol) was added. The reaction mixture was stirred at 0 ° C for 1 h. The solution was concentrated to dryness. The oily residue was used without further purification in the next step.
    [082] [082] A solution of 2-phenylaniline (1.6 g) and pyridine (2.4 ml) in chloroform (80 ml) under an inert atmosphere was cooled to 0ºC. (Phenyl-4- chloride
    [083] [083] Procedure: N-diphenyl-2-yl-4- (2- {2- [2- (2-methoxy-ethoxy) - ethoxy] -ethoxy} -ethoxy) -benzamide (4 g, 8.34 mmol ), POCl3 (10 mL) in 10 mL of toluene were refluxed for 20 h. The reaction mixture was cooled to room temperature and 100 ml of dichloromethane was added. The solution was poured onto ice and the mixture neutralized with NH4OH (20%). 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).
    [084] [084] The general procedure was published by Nonoyama, M., J.
    [085] [085] Iridium dimers were synthesized as follows: IrCl3 • 3H2O and 2.5 equiv of 6-phenylphenanthridine were heated at 120 ° C for 18 h under nitrogen in a mixture of 2-ethoxyethanol / water (3: 1, v / v). After being cooled to room temperature, the precipitate was filtered and successively washed with methanol and Et2O, and dried to provide the desired dimer.
    [086] [086] 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 obtain an oily precipitate. The supernatant was discarded and 50 ml of water was added to the residue. The mixture was stirred for 1 h to obtain 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 by adding Et2O, and was used in the next step without further purification.
    [087] [087] A mixture of 3-hydroxy-2-pyridinecarboxylic acid (0.01 mol), ethyl 4-bromobutanoate or ethyl 6-bromohexanoate (0.021 mol), and a mixture of potassium carbonate (5 eq.) In DMF (20 mL) was heated at 90ºC for 20 h under nitrogen. After cooling, the reaction mixture was poured into an ice-water mixture and extracted three times with dichloromethane (30 ml), dried over anhydrous MgSO4, filtered, and the solvent was evaporated to dryness. Purification took place by flash chromatography (silica, hexane / ethyl acetate 3: 1) to provide the product (based on US Patent 5,219,847).
    [088] [088] The ester formed was hydrolyzed by NaOH in MeOH (pH = 10).
    [089] [089] Under an argon atmosphere, 4-ml of 1,2-dimethoxyethane 5-bromo-pyridine-2-carboxylic acid (93 mg, 0.46 mmol), 4- (2-carboxyethyl) benzene boronic acid ( 106 mg, 0.55 mmol), 0.51 mL of a 2M aqueous sodium carbonate solution and dichlorobis- (triphenylphosfin) palladium (II) (20 mg, 0.03 mmol). The mixture is stirred at 90ºC overnight, cooled and processed with water.
    [090] [090] A dimeric complex of cross-linked 0.5 mmol chlorine, 1.25 mmol picolinate and 3 mmol Na2CO3 are mixed in 2-ethoxyethanol (12 mL) and heated at 120ºC for 15 h. Distilled water (25 ml) was added to the cooled mixture, and then the crude product was filtered and washed with water, followed by portions of n-hexane and Et2O. The product was purified by flash chromatography (silica, n-hexane / dichloromethane) to provide a red powder.
    [091] [091] A suspension of Ir dimer (150 mg, 0.065 mmol), picolinic acid (17 mg, 0.137 mmol) and Na2CO3 (70 mg, 0.65 mmol) in 20 mL of dichloromethane / ethanol (4: 1) refluxed overnight. After cooling, the mixture was concentrated to dryness. The residue was purified by flash chromatography in dichloromethane / MeOH (gradient 100: 0 to 10: 1). The compound was recrystallized from dichloromethane / Et2O.
    [092] [092] The electrochemiluminescence signal of several metal complexes was evaluated on an ELECSYS analyzer (Roche Diagnostics
    [093] [093] ECL Results: Reference Ru (bpy) 3 = 10,000 counts in 10 nmolar concentration - JM 360 = 31258 counts in 10 nmolar concentration - RC 72 = 45512 counts in 10 nmolar concentration N
    [094] [094] Ir (6-phenylphenanthridine) 2 2- (carboxyethyl-phenyl) pyridine-2-carboxylic acid (15 mg) was dissolved in a mixture of 5 ml of dry acetonitrile and 0.01 ml of dry pyridine. Disuccinimidyl carbonate (DSC) (1.5 eq) was added and the mixture was stirred under nitrogen at room temperature overnight. The solution was added to chloroform (10 ml), washed with 0.05M HCl (1 x 2 ml), saturated aqueous NaHCO 3 (1 x 2 ml) and water (2 x 5 ml), dried over MgSO 4 and concentrated to vacuum to provide a red powder.
    [095] [095] Ir (6-phenylphenanthridine) 2 2- (carboxyethyl-phenyl) pyridine-2-carboxylic acid NHS ester (12 mg) and 4 mg of N-biotinyl-3,6-dioxaoctane-1,8-diamine trifluoroacetate were dissolved in 5 ml of dry DMF. Pyridine (0.016 ml in 2 ml of DMF) was added and the mixture was stirred under nitrogen at room temperature overnight. The solution was added to chloroform (10 ml), washed with 0.5M HCl (1 x 2 ml), saturated aqueous NaHCO3 (1 x 2 ml) and water (2 x 5 ml), dried over MgSO4 and concentrated in vacuo to provide a red powder. The product was purified by column chromatography (silica, n-hexane / ethyl acetate) to provide a red powder.
    MS: [M + H] + 1328.6
    权利要求:
    Claims (11)
    [1]
    1. CHEMIOLUMINESCENT COMPOUND BASED ON IRIDIOUS, characterized by being Formula I R6 R7 R5 R4 R8 R3 R9 R10 N R2 R1 R11
    OO R12 R2 N R16 Ir R3 R1 R13 R15 R4 N R14 R12 R5 R9 R11 R6 R8 R7 R10 where R1-R16 are hydrogen, halide, cyano or nitro group, amino, alkylamino, substituted alkylamino, arylamino, substituted arylamino, alkylammonium, substituted alkylammonium, carboxy, carboxylic acid ester, carbamoyl, hydroxy, substituted or unsubstituted alkyloxy, substituted or unsubstituted aryloxy, sulfanyl, alkylsulfonyl, arylsulfonyl, sulfo, sulfine, sulfene, sulfonamide, sulfoxide, sulfodioxide, phosphonate, phosphonate or R17, where R17 is aryl, substituted aryl, alkyl, substituted alkyl, branched alkyl, substituted branched alkyl, arylalkyl, substituted arylalkyl, alkylaryl, substituted alkylaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, where the substituent is selected from hydrogen, halide, cyano or nitro group, a hydrophilic group, such as amino, alkylamino, substituted alkylamino, alkylammonium, substituted alkylammonium, carboxy, carboxylic acid ester, carbamoyl, hydroxy, substituted or unsubstituted alkyloxy, substituted or unsubstituted aryloxy, sulfanyl, alkylsulfonyl, arylsulfonyl, sulfo, sulfine, sulfene, sulfonamide, sulfoxide, sulfodioxide, phosphonate, phosphinate or,
    where within R1-R12 and / or within R13-R16, respectively, two adjacent Rs can form an aromatic ring or a substituted aromatic ring, where the substituent is selected from hydrogen, halide, cyano or nitro group, a hydrophilic group, such as amino, alkylamino, substituted alkylamino, alkylammonium, substituted alkylammonium, carboxy, carboxylic acid ester, carbamoyl, hydroxy, substituted or unsubstituted alkyloxy, substituted or unsubstituted aryloxy, sulfanyl, alkylsulfonyl, arylsulfonyl, sulphine, sulfine, sulfene, sulfonamide, sulfoxide, sulfodioxide, phosphonate, phosphinate or, where within R1-R12 and / or within R13-R16, respectively, two adjacent Rs can form an aliphatic ring or a substituted aliphatic ring, where the substituent is selected from hydrogen, halide, cyano or nitro group, a hydrophilic group, such as amino, alkylamino, substituted alkylamino, alkylammonium, substituted alkylammonium, carboxy, car acid ester boxyl, carbamoyl, hydroxy, substituted or unsubstituted alkyloxy, substituted or unsubstituted aryloxy, sulfanyl, alkylsulfonyl, arylsulfonyl, sulfo, sulfine, sulfene, sulfonamide, sulfoxide, sulfodioxide, phosphonate, phosphinate and where at least one of the R13-R16 is –QY, where Q represents a ligand and Y is a functional group.
    [2]
    2. COMPOUND according to claim 1, characterized in that the linker Q is a saturated, unsaturated, unsubstituted, unsubstituted or substituted C1-C20 alkyl chain or a chain of 1 to 20 atoms with a main chain consisting of atoms carbon and one or more heteroatoms selected from O, N and S.
    [3]
    COMPOSITE according to claim 1, characterized in that the linker Q is a saturated C1-C12 alkyl chain or a chain with 1 to 12 atoms with a main chain consisting of carbon atoms and one or more hetero atoms selected from O, N and S.
    [4]
    COMPOSITE according to one of claims 1 to 2, characterized in that the functional group Y is selected from the group consisting of carboxylic acid, N-hydroxysuccinimide ester, amino group, halogen, sulfhydryl, maleimido, alkynyl, azide and phosphoramidite.
    [5]
    CONJUGATE, characterized in that it comprises a compound, as defined in one of claims 1 to 4, and covalently attached to it an affinity binding agent.
    [6]
    CONJUGATE according to claim 5, characterized in that the affinity binding agent is selected from the group consisting of antigen and antibody, biotin or biotin and avidin or streptavidin, sugar and lectin, nucleic acid or nucleic acid and complementary nucleic acid and receptor and ligand.
    [7]
    CONJUGATE according to one of claims 5 to 6, characterized in that the affinity binding agent is a nucleic acid or an antibody.
    [8]
    8. USE OF A COMPOUND, as defined in one of claims 1 to 4, or of a conjugate, as defined in one of claims 5 to 7, characterized in that it is for carrying out an electrochemiluminescence reaction in an aqueous solution.
    [9]
    9. USE OF A COMPOUND, as defined in one of claims 1 to 4, or of a conjugate, as defined in one of claims 5 to 7, characterized in that it is a detection method based on electrochemiluminescence.
    [10]
    10. USE OF A COMPOUND, as defined in one of claims 1 to 4, or of a conjugate, as defined in one of claims 5 to 7, characterized in that it is in the detection of an analyte.
    [11]
    11. METHOD FOR MEASURING ANALYTICS by an in vitro method, the method characterized by comprising the steps of:
    (a) providing a sample suspected or known to comprise the analyte;
    (b) placing said sample in contact with a conjugate,
    as defined in one of claims 5 to 7, under conditions suitable for the formation of a conjugate-analyte complex;
    (c) measure the complex formed in step (b) and thereby obtain a measurement of the analyte.
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    同族专利:
    公开号 | 公开日
    CN103347888A|2013-10-09|
    AU2012215497A1|2013-07-04|
    JP5786040B2|2015-09-30|
    EP2673284A1|2013-12-18|
    CA2822899A1|2012-08-16|
    US8835637B2|2014-09-16|
    ES2645765T3|2017-12-07|
    MX2013008418A|2013-09-13|
    SG192675A1|2013-09-30|
    US20160145281A1|2016-05-26|
    BR112013019503B1|2021-05-25|
    KR20140053834A|2014-05-08|
    MX342921B|2016-10-19|
    CN103347888B|2016-12-21|
    JP2014506571A|2014-03-17|
    WO2012107419A1|2012-08-16|
    AU2012215497B2|2016-07-28|
    EP2673284B1|2017-08-30|
    US20130323719A1|2013-12-05|
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    法律状态:
    2020-11-24| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|
    2020-12-22| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|
    2021-04-06| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|
    2021-05-25| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 07/02/2012, OBSERVADAS AS CONDICOES LEGAIS. |
    优先权:
    申请号 | 申请日 | 专利标题
    EP11153913|2011-02-09|
    EP11153913.6|2011-02-09|
    PCT/EP2012/051996|WO2012107419A1|2011-02-09|2012-02-07|New iridium-based complexes for ecl|
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