WO2007128873A1 - A method for the preparation of maleimido derivatives of biomolecule labeling reactants and conjugates derived thereof - Google Patents

A method for the preparation of maleimido derivatives of biomolecule labeling reactants and conjugates derived thereof Download PDF

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WO2007128873A1
WO2007128873A1 PCT/FI2007/050247 FI2007050247W WO2007128873A1 WO 2007128873 A1 WO2007128873 A1 WO 2007128873A1 FI 2007050247 W FI2007050247 W FI 2007050247W WO 2007128873 A1 WO2007128873 A1 WO 2007128873A1
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chelate
chelating agent
compound
formula
lanthanide
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French (fr)
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Jari Hovinen
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Wallac Oy
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D255/00—Heterocyclic compounds containing rings having three nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D249/00 - C07D253/00
    • C07D255/02—Heterocyclic compounds containing rings having three nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D249/00 - C07D253/00 not condensed with other rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D257/00—Heterocyclic compounds containing rings having four nitrogen atoms as the only ring hetero atoms
    • C07D257/02—Heterocyclic compounds containing rings having four nitrogen atoms as the only ring hetero atoms not condensed with other rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/13—Labelling of peptides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials

Definitions

  • This invention relates to chelating agents or metal chelates, such as lanthanide(III) chelates tethered to a maleimido function and biomolecule conjugates derived thereof.
  • Time-resolved fluorometry exploits the unique fluorescence properties of lanthanide ⁇ i) chelates.
  • the long fluorescence decay after excitation of these molecules allows time-delayed signal detection. This eliminates background signal originating e.g. from microplates or buffer components.
  • the large Stokes shift i.e. the difference in the chelate's excitation and emission lines, in turn, results in a high signal-to-background ratio.
  • Thiols are know to react with haloacetamides, maleimides, disulfides and their analogues, mercurials, vinyl sulfones, aryl halides, aziridines and oxiranes [Brocklehurst, K. 1979, Int. J. Biochem., 10, 258].
  • the reactions of haloacetamides, maleimides and disulfides are the most commonly employed [Aslam, M., Dent, A., Bioconjugation, Macmillan Reference Ltd, London, 1998]. From them, the haloacetyl derivatives display a wide range of other reactivities, while disulfides are practically completely specific to thiols.
  • maleimides The reactivity of maleimides is between them.
  • the problem of the use of disulfides is the lability of the bioconjugate in the presence of reducing agents which are often present in the conjugation reaction.
  • reducing agents which are often present in the conjugation reaction.
  • Various mercapto selective biomolecule labeling reactants are currently commercially available.
  • the known mercapto selective luminescent lanthanide(III) chelates are haloacetamido or maleimido [Takalo, H., Mukkala, V.-M., Mikola, H., Liitti, P., Hemmila, L, 1994, Bioconjugate Chem., 5, 278; Chen, J., Selvin, P.R., 1999, Bioconjugate Chem. 10, 311] derivatives.
  • the labeling reaction is often performed in the presence of a reducing agent, most commonly tris-carboxyethyl phosphine (TCEP).
  • TCEP tris-carboxyethyl phosphine
  • the reducing agent reacts with the haloacetamido chelate reducing markedly the yield of the desired biomolecule conjugate.
  • TCEP also reduces disulfide derivatized labeling reactants, such as pyridylthioates.
  • maleimido group is stable under the reaction conditions required in solid phase oligopeptide synthesis [Machan, V., 2006, Chemiedozententagung, A26]. Furthermore, maleim ⁇ de reacts faster at neutral pH with mercaptans than haloacetamides, which became exploitable at considerably higher pH values [Schelte, P., Boeckler, C, Frisch. B., Schuber, F., 2000, Bioconjugate Chem., 11, 118].
  • Lanthanide(i ⁇ ) chelates are most commonly prepared by treatment of the free ligands with a slight excess of lanthanide chloride at pH 6-7. When the chelation is completed, the excess of lanthanide is removed by increasing pH of the reaction mixture to ca 11 where the uncomplexed lanthanide precipitates as lanthanide(III) hydroxide.
  • this procedure cannot be used with activated chelates due to the lability of the activating groups under basic conditions. This problem can be solved by using an ion transfer loaded with the desired metal ion as the ion source [Stavrianpoulous, US 4,767,609].
  • the main object of the present invention is to provide a method for the preparation chelating agents and metal chelates thereof tethered to maleimido function, useful for labeling of mercapto functions of biomolecules for use as probes in time resolved fluorescence spectroscopy, magnetic resonance imaging (MRT) or positron emission tomography (PET) and single positron emission computed tomography (SPECT).
  • MRT magnetic resonance imaging
  • PET positron emission tomography
  • SPECT single positron emission computed tomography
  • the major advantages of the present invention are: (i) The maleimido function can be coupled to the protected Hgand giving rise to hydrophobic, stable conjugates which can be synthesized in large scale and purified on classical column chromatography.
  • the protection groups can be removed with acid, and the free ligand can be converted to the corresponding lanthanide(IH) chelate by treatment with ion exchange resin loaded with lanthanide ions.
  • the invention concerns a method for the preparation of a chelating agent or metal chelate of formula (I),
  • X is a chelating agent able to bind a metal ion or a metal chelate
  • L is a linker
  • L 1 and L are linkers or are missing, A 1 and A 2 are reactive groups, and G is a chelating agent comprising a chelating part having carboxylic acid or phosphonic acid groups which are protected, and optionally a chromophoric moiety, optionally in the presence of an activator or a catalyst, to form a compound of formula (IT)
  • the invention concerns a labeling reactant, which is a chelating agent or a lanthanide chelate of formula (I),
  • L is a linker and X is a luminescent or non-luminescent lanthanide chelate, or a chelating agent able to chelate a metal to create said lanthanide chelate, wherein said chelating agent comprises a chelating part and optionally a chromophoric moiety comprising of one or more noncondensed aromatic units, wherein said aromatic units optionally are substituted.
  • the invention concerns a compound of formula (II)
  • L is a linker and G is a chelating agent comprising a chelating part having carboxylic acid or phosphonic acid groups which are protected, and optionally a chromophoric moiety.
  • the invention concerns a biospecific binding reactant conjugated with the labeling reactant according to this invention.
  • the reactive groups A 1 and A 2 can be any groups able to react with each other and form a covalent bond in the presence or absence of an activator or a catalyst.
  • the reactive groups are preferably selected from the group consisting of carboxyl, carbonyl, active ester, acid halide, aromatic halide, aminooxy, thioester, amino, alkynyl, azido, hydroxyl, and a group with pKg ⁇ 14.
  • the nature of the activator and the catalyst is dependent on the coupling reaction.
  • a coupling reaction between the maleimido derivative tethered to a carboxylic acid group and the amino derivative of the protected chelating agent in the presence of an activator is those commonly used in the formation of peptide bonds, including DCC, HOBT and HATU.
  • the maleimido derivative has to be preactivated in situ prior to the addition of the addition of the protected chelating agent, and the reaction is performed in the presence of an organic base, most commonly DIPEA.
  • the most suitable solvents are DMF, acetonitrile, THF and chlorinated hydrocarbons.
  • the protected chelating agents tethered to the maleimido group of formula (II) are hydrophopic, stable organic molecules, they can be purified by means of chromatography.
  • chromatographic methods are column chromatography on silica gel and aluminum oxide.
  • the reactive groups required for the reactions disclosed in (i)-(xii) can be directly attached to maleimide or to the protected chelating agent, or via linkers L 1 and lA After completed reaction the linker L is formed.
  • the protected chelating agent G is able to chelate a metal ion after it is deprotected and treated to the said metal ion, and comprises an optional chromophoric moiety and at least three protected carboxylic acid or phosphonic acid groups.
  • the protected chelating agent G is selected from a group consisting of wherein Z is independently selected from ftiryl, thienyl, phenyl or phenylethynyl, where phenyl is substituted or unsubstitued, or Z is not present, wherein G is a radical of any of the aforementioned structures and tethered to L, and R" an alkyl ester of an allyl ester.
  • Z is substituted phenyl, said phenyl is preferably trialkoxysubstituted, more preferably trimethoxysubstituted.
  • this invention concerns the method for the conversion of the said protected chelating agent tethered to the maleimido function (II) to the corresponding metal chelate of formula (I), and comprises (i) removal of the said ester protecting groups; (ii) and treatment of the resulting deprotected ligand with the appropriate metal salt.
  • acid labile protecting groups are the most suitable protecting groups.
  • Particularly preferable protecting group is /-butyl group.
  • the preferable method for the conversion of the said deprotected chelating agent to the corresponding metal chelate is by treatment with ion exchange resin loaded with the corresponding metal ions.
  • the most preferable ion exchange resin is Dowex-50.
  • this invention concerns chelating agents or lanthanide( ⁇ i) chelates of formula (I)
  • Ln is lanthanide, or is not present .
  • the most preferable lanthanides are europium, terbium, samarium, dysprosium, gadolinium and holmium.
  • the chelating agent or the chelate is preferably one of the following structures
  • Z is independently selected from furyl, thienyl, phenyl or phenylethynyl, where phenyl is substituted or unsubstitued, or Z is not present, wherein X is a radical of any of the aforementioned structures and tethered to the linker L and Ln is lanthanide.
  • Ln 3+ is missing.
  • Z is substituted phenyl, said phenyl is preferably trialkoxysubstituted. more preferably trimethoxysubstituted.
  • Lanthanide is most preferably selected from the group consisting of europium, terbium, samarium, dysprosium, gadolinium, and holmium.
  • the target i.e the biospecific binding reactant
  • the target can be a oligopeptide, protein or any bioactive molecule containing one or more mercapto groups.
  • the invention is further elucidated by the following non-restricting examples.
  • the structures and synthetic routes employed in the experimental part are depicted in Schemes 1 and 2.
  • Experimental details are given in Examples 1-4.
  • Scheme 1 illustrates synthesis of luminescent lanthanide(i ⁇ ) chelates tethered to a maleimido group.
  • Experimental details are given in Examples 1-5.
  • Scheme 2 illustrates the reaction between the conjugate molecule and a model peptide containing a single cysteine residue in its structure. Experimental details are given in Example 5.
  • Adsorption column chromatography was performed on columns packed with silica gel 60 (Merck).
  • the cation exchange resin (Dowex 50 X 8, 20-50 mesh, H + form) was converted to the Ln 3+ form by treatment with the appropriate lanthanide(i ⁇ ) chloride followed by washing with water until pH of the eluent was neutral.
  • 6-(2,5- dioxo-2H-pyrrol-l(5H)-yl)hexanoic acid, and the model peptide (CVEIDK) were purchased from Fluka and Sigma-Genosys, respectively.
  • NMR spectra were recorded on a Bracker 600 spectrometer operating at 600.13 MHz for H. The signal of TMS was used as an internal ( 1 H) reference.
  • Example 1 The synthesis of tetra(tert-butyl) 2,2',2",2'" ⁇ 4-(4-(6-(2,5-dioxo-2H- pyrrol- 1 (5H)-yl)hexylcarboxamido)phenylethynyl]pyridine-2,6-diyl]bis- (methylenenitrilo) ⁇ tetrakis(acetate), 1.
  • 6-(2,5-dioxo-2H-pyrrol-l(5H)-yl)hexanoic acid 60 mg, 0.28 mmol
  • O-(7- azabenzotriazol-l-yl)-l,l,3,3-tetramethyluronium hexafluorophosphate 100 mg, 0.28 mmol
  • ⁇ N-diisopropylethylamine 73 ⁇ L, 0.56 mmol
  • Tetra(t ⁇ rt-butyl) 2,2 ',2 ",2 ' ' '-[[4-[(4-aminophenyl)ethynyl]pyridine-2,6- diyl]bis(methylenenitrilo)]tetrakisacetate (100 mg, 0.18 mmol), synthesized as disclosed in Takalo, H., Hanninen, E., Kankare, J., 1993, HeIv. Chim. Acta, 76, 877, was added and the reaction was allowed to proceed overnight at RT. The reaction mixture was diluted with dichloromethane (50 mL) and washed with 10% citric acid. The organic layer was dried over Na 2 SO 4 and concentrated.
  • Example 2 The synthesis of tetra(tert-butyl) 2,2',2",2'"- ⁇ 6,6'- ⁇ 4"-2-[4-(6-(2,5- dioxo-2H-pyrrol-l (5H)-yl)hexyl)carboxamido ⁇ henyl)ethyl]pyrazole-l ",3 "- diyl ⁇ bis(pyridine)-2,2 r -diyl ⁇ bis(methylenenitrilo) ⁇ tetrakis(acetate) 2.
  • Example 3 The synthesis of 2,2'.2" 5 2'" ⁇ 4-(4-(6-(2,5-dioxo-2-H-pyrrol-l(5H)- yl)hexylcarboxamido)phenylethynyl]pyridine-2,6-diyl]bis- (methylenenitrilo) ⁇ tetrakis(acetic acid) euro ⁇ ium(HI) 3.
  • Example 4 The synthesis of 2,2 ⁇ 2 ",2'"- ⁇ ⁇ 6,6 '- ⁇ 4 ' '-2-[4-(6-(2,5-dioxo-2H-pyrrol- 1 (5H)-yl)hexyl)carboxamidophenyl)ethyl]pyrazole-l ",3 ' '-diyl ⁇ bis(pyridine)-2,2 '- diyl ⁇ bis(methylenenitrilo) ⁇ tetrakis(acetic acid) terbium( ⁇ i) s 4.
  • Example 5 Labeling of the oligopeptide with the europium chelate 3.
  • the model peptide (AC-CVEIDK-CONH 2 ) (1 mg) was dissolved in Tris HCl buffer (1 mL; pH 7).
  • Compound 3 (1 equiv.) was added, and the reaction was allowed to proceed for 1 h at RT. Purification on HPLC yielded the desired oligopeptide conjugate. t R 18.14 min.

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Abstract

This invention relates to a new method for the preparation of chelating agents or metal chelates, particularly lanthanide(III) chelates, tethered to a maleimido function and to novel end products and intermediates produced in said method. The invention concerns also biomolecule conjugates derived thereof.

Description

A METHOD FOR THE PREPARATION OF MALEIMH)O DERIVATIVES OF BIOMOLECULE LABELING REACTANTS AND CONJUGATES DERIVED THEREOF
FIELD OF THE INVENTION
This invention relates to chelating agents or metal chelates, such as lanthanide(III) chelates tethered to a maleimido function and biomolecule conjugates derived thereof.
BACKGROUND OF THE INVENTION
Time-resolved fluorometry exploits the unique fluorescence properties of lanthanide{πi) chelates. The long fluorescence decay after excitation of these molecules allows time-delayed signal detection. This eliminates background signal originating e.g. from microplates or buffer components. The large Stokes shift (i.e. the difference in the chelate's excitation and emission lines), in turn, results in a high signal-to-background ratio. These unique properties of lanthanide(IH) chelates can be exploited particularily in homogenous assays, when the use of conventional chromophores causes very high background. The different photochemical properties of europium, terbium, dysprosium and samarium chelates enable even development of multiparametric homogenous assays [Hemmila, L; Mukkala, V.-M. 2001, Crit. Rev. Clin. Lab. Sci. 441]. Accordingly, a number of attempts have been made to develop highly luminescent chelate labels suitable for time-resolved fluorometric applications. These include e.g. stabile chelates composed of derivatives of pyridines [US 4,920,195, US 4,801,722, US 4,761,481, PCT/FI91/00373, US 4,459,186, EP A-0770610, Remuinan et al,J. Chem. Soc. Perkin Trans 2, 1993, 1099], bipyridines [US 5,216,134], terpyridines [US 4,859,777, US 5,202,423, US 5,324,825] or various phenolic compounds [US 4,670,572, US 4,794,191, Ital Pat. 42508 A789] as the energy mediating groups and polycarboxylic acids as chelating parts. In addition, various dicarboxylate derivatives [US 5,032,677, US 5,055,578, US 4,772,563] macrocyclic cryptates [US 4,927,923, WO 93/5049, EP-A-493745] and macrocyclic Schiff bases [EP-A-369-000] have been disclosed. Also a method for the labelling of biospecific binding reactant such as hapten, a peptide, a receptor ligand, a drug or PNA oligomer with luminescent labels by using solid-phase synthesis has been published [US 6,080,839]. Similar strategy has also been exploited in multilabeling of oligonucleotides on solid phase [US 6,949,639].
Thiols are know to react with haloacetamides, maleimides, disulfides and their analogues, mercurials, vinyl sulfones, aryl halides, aziridines and oxiranes [Brocklehurst, K. 1979, Int. J. Biochem., 10, 258]. For the coupling purposes, the reactions of haloacetamides, maleimides and disulfides are the most commonly employed [Aslam, M., Dent, A., Bioconjugation, Macmillan Reference Ltd, London, 1998]. From them, the haloacetyl derivatives display a wide range of other reactivities, while disulfides are practically completely specific to thiols. The reactivity of maleimides is between them. The problem of the use of disulfides is the lability of the bioconjugate in the presence of reducing agents which are often present in the conjugation reaction. Various mercapto selective biomolecule labeling reactants are currently commercially available.
The known mercapto selective luminescent lanthanide(III) chelates are haloacetamido or maleimido [Takalo, H., Mukkala, V.-M., Mikola, H., Liitti, P., Hemmila, L, 1994, Bioconjugate Chem., 5, 278; Chen, J., Selvin, P.R., 1999, Bioconjugate Chem. 10, 311] derivatives. These derivatives have been synthesized by allowing the corresponding amino chelates or ligands to react with the appropriate activating agents (such as haloacetic anhydride, haloacetic acid N- hydroxysuccinate or 3-maleimidopropionic acid iV-hydroxysuccinimide ester). However, the current methods have several drawbacks, since first, the mercapto seletive chelates are reactive, hydrophilic compounds and their purification procedures are time consuming and laborius often including several HPLC purifications as well as extractions and precipitations. Secondly, in the case of haloacetamides, pH of the conjugation reaction has to be carefully controlled. At too low pH, the reaction is slow, while at too high pH amino functions, often abundantly present in the bioactive molecule to be labeled, overcompete the reaction of the desired mercapto function. Third, to avoid oxidation of the mercapto function to be labeled, the labeling reaction is often performed in the presence of a reducing agent, most commonly tris-carboxyethyl phosphine (TCEP). The reducing agent in turn, reacts with the haloacetamido chelate reducing markedly the yield of the desired biomolecule conjugate. Naturally, TCEP also reduces disulfide derivatized labeling reactants, such as pyridylthioates.
It has been demonstrated that the maleimido group is stable under the reaction conditions required in solid phase oligopeptide synthesis [Machan, V., 2006, Chemiedozententagung, A26]. Furthermore, maleimϊde reacts faster at neutral pH with mercaptans than haloacetamides, which became exploitable at considerably higher pH values [Schelte, P., Boeckler, C, Frisch. B., Schuber, F., 2000, Bioconjugate Chem., 11, 118].
Lanthanide(iπ) chelates are most commonly prepared by treatment of the free ligands with a slight excess of lanthanide chloride at pH 6-7. When the chelation is completed, the excess of lanthanide is removed by increasing pH of the reaction mixture to ca 11 where the uncomplexed lanthanide precipitates as lanthanide(III) hydroxide. However, this procedure cannot be used with activated chelates due to the lability of the activating groups under basic conditions. This problem can be solved by using an ion transfer loaded with the desired metal ion as the ion source [Stavrianpoulous, US 4,767,609].
SUMMARY OF THE INVENTION
The main object of the present invention is to provide a method for the preparation chelating agents and metal chelates thereof tethered to maleimido function, useful for labeling of mercapto functions of biomolecules for use as probes in time resolved fluorescence spectroscopy, magnetic resonance imaging (MRT) or positron emission tomography (PET) and single positron emission computed tomography (SPECT).
The major advantages of the present invention are: (i) The maleimido function can be coupled to the protected Hgand giving rise to hydrophobic, stable conjugates which can be synthesized in large scale and purified on classical column chromatography.
(ii) When desired, the protection groups can be removed with acid, and the free ligand can be converted to the corresponding lanthanide(IH) chelate by treatment with ion exchange resin loaded with lanthanide ions.
(iii) These molecules do not react with phosphines.
Thus, in one aspect, the invention concerns a method for the preparation of a chelating agent or metal chelate of formula (I),
Figure imgf000005_0001
wherein X is a chelating agent able to bind a metal ion or a metal chelate, and L is a linker, said method comprising the steps of a) reacting a compound of formula (Ia)
Figure imgf000005_0002
with a compound of formula (Ib)
A2-L2-G (Ib)
wherein L1 and L are linkers or are missing, A1 and A2 are reactive groups, and G is a chelating agent comprising a chelating part having carboxylic acid or phosphonic acid groups which are protected, and optionally a chromophoric moiety, optionally in the presence of an activator or a catalyst, to form a compound of formula (IT)
Figure imgf000006_0001
b) purifying of the compound formula (S), c) removal of the protecting groups, and d) optionally converting of the deprotected chelating agent to the corresponding metal chelate to form a chelate of formula (I).
According to another aspect, the invention concerns a labeling reactant, which is a chelating agent or a lanthanide chelate of formula (I),
Figure imgf000006_0002
wherein L is a linker and X is a luminescent or non-luminescent lanthanide chelate, or a chelating agent able to chelate a metal to create said lanthanide chelate, wherein said chelating agent comprises a chelating part and optionally a chromophoric moiety comprising of one or more noncondensed aromatic units, wherein said aromatic units optionally are substituted.
According to a third aspect, the invention concerns a compound of formula (II)
Figure imgf000006_0003
wherein L is a linker and G is a chelating agent comprising a chelating part having carboxylic acid or phosphonic acid groups which are protected, and optionally a chromophoric moiety.
According to a fourth aspect, the invention concerns a biospecific binding reactant conjugated with the labeling reactant according to this invention. DETAILED DESCRIPTION OF THE INVENTION
According to a preferable embodiment, the linkers L1, L2 and L are formed from one to ten moieties, each moiety being selected from the group consisting of phenyl, alkyl containing 1-12 carbon atoms, ethynediyl (-C≡C-), ethylenediyl (-C=C-); ether (-O-), thioether (-S-), amide (-CO-NH- and -NH-CO- and -CO-NR and -NR-CO-), carbonyl (-CO-), ester (-C00- and -00C-), disulfide (-SS-), diaza (-N=N-) or a tertiary amine (-NR-), where R represents an alkyl containing less than 5 carbon atoms, and 1,2,3-triazole.
In case both of the linkers L1 and L2 in the reagents (Ia) and (Ib) are missing, the linker L in compounds (I) and QX) is created from the reactive groups A1 and A2.
The reactive groups A1 and A2 can be any groups able to react with each other and form a covalent bond in the presence or absence of an activator or a catalyst. The reactive groups are preferably selected from the group consisting of carboxyl, carbonyl, active ester, acid halide, aromatic halide, aminooxy, thioester, amino, alkynyl, azido, hydroxyl, and a group with pKg < 14. The nature of the activator and the catalyst is dependent on the coupling reaction.
The compounds of formula (E), which are protected chelating agents tethered to a maleimido group, are new.
Figure imgf000007_0001
They can be synthesized using the following nonrestricted reactions:
(i) A coupling reaction between the maleimido derivative tethered to a carboxylic acid group and the amino derivative of the protected chelating agent in the presence of an activator. Suitable activators are those commonly used in the formation of peptide bonds, including DCC, HOBT and HATU. In certain cases, the maleimido derivative has to be preactivated in situ prior to the addition of the addition of the protected chelating agent, and the reaction is performed in the presence of an organic base, most commonly DIPEA. The most suitable solvents are DMF, acetonitrile, THF and chlorinated hydrocarbons.
(ii) A coupling reaction between the maleimido derivative tethered to a amino group and the carboxylic acid derivative of the protected chelating agent in the presence of an activator. Reaction conditions, and requirements are the same as disclosed in (i).
(iii) A coupling reaction between an active ester derivative or an acid halide of the maleimido derivative and the amino derivative of the protected ligand. The most suitable activating groups are active esters including N- hydroxysuccinimide, JV-hydroxybenzotriazole and pentafluorophenyl; and acid chlorides and acid fluorides.
(iv) A coupling reaction between the amino derivative of maleimido derivative and an active ester derivative or an acid halide of the protected ligand. Reaction conditions, and requirements are the same as disclosed in (iii).
(v) A palladium catalyzed Sonogashira reaction between the maleimido derivative tethered to an alkynyl group and the protected chelating agent tethered to an aromatic halide, wherein the halide is either bromine or iodide.
(vi) A palladium catalyzed Sonogashira reaction between the maleimido derivative tethered to an aromatic halide wherein the halide is either bromine or iodide, and the protected chelating agent tethered to alkynyl group.
(vii) A palladium catalyzed Heck reaction between the maleimido derivative tethered to an alkenyl group and the protected chelating agent tethered to an aromatic chloride.
(viii) A palladium catalyzed Heck reaction between the maleimido derivative tethered to an aromatic chloride and the protected chelating agent tethered to an alkenyl group. (ix) A Mitsunobu reaction between maleimide or a maleimido derivative tethered to a group of pK* < 14 and a protected chelating agent tethered to a primary hydroxyl group, (x) A Mitsunobu reaction between the protected chelating agent tethered to a group of pKa < 14 and the maleimido derivative tethered to a primary hydroxyl group, (xi) A Cu+ or Ru(AcO)2(PPh.3)2 catalyzed Huisgen's cycloaddition reaction between the protected chelating agent tethered to an alkynyl group and the maleimide derivative tethered to an azido group, (xii) A Cu+ or Ru(AcO)2(PPli3)2 catalyzed Huisgen's cycloaddition reaction between the protected chelating agent tethered to an azido group and the maleimide derivative tethered to an alkynyl group.
Since the protected chelating agents tethered to the maleimido group of formula (II) are hydrophopic, stable organic molecules, they can be purified by means of chromatography.
Most preferable chromatographic methods are column chromatography on silica gel and aluminum oxide.
The reactive groups required for the reactions disclosed in (i)-(xii) can be directly attached to maleimide or to the protected chelating agent, or via linkers L1 and lA After completed reaction the linker L is formed.
The protected chelating agent G is able to chelate a metal ion after it is deprotected and treated to the said metal ion, and comprises an optional chromophoric moiety and at least three protected carboxylic acid or phosphonic acid groups.
According to a preferable embodiment, the protected chelating agent G is selected from a group consisting of
Figure imgf000010_0001
wherein Z is independently selected from ftiryl, thienyl, phenyl or phenylethynyl, where phenyl is substituted or unsubstitued, or Z is not present, wherein G is a radical of any of the aforementioned structures and tethered to L, and R" an alkyl ester of an allyl ester. In case Z is substituted phenyl, said phenyl is preferably trialkoxysubstituted, more preferably trimethoxysubstituted.
According to one embodiment, this invention concerns the method for the conversion of the said protected chelating agent tethered to the maleimido function (II) to the corresponding metal chelate of formula (I), and comprises (i) removal of the said ester protecting groups; (ii) and treatment of the resulting deprotected ligand with the appropriate metal salt.
Since maleimido function is stable under acidic conditions, but not under prolonged treatment at high pH, acid labile protecting groups are the most suitable protecting groups. Particularly preferable protecting group is /-butyl group.
The preferable method for the conversion of the said deprotected chelating agent to the corresponding metal chelate is by treatment with ion exchange resin loaded with the corresponding metal ions.
The most preferable ion exchange resin is Dowex-50.
According to one embodiment, this invention concerns chelating agents or lanthanide(πi) chelates of formula (I)
Figure imgf000011_0001
wherein, the linker L is formed from one to ten moieties, each moiety being selected from the group consisting of phenyl, alkyl containing 1-12 carbon atoms, ethynediyl (-C≡C-), ethylenediyl (-C=C-); ether (-O-), thioether (-S-), amide (-CONH- and -NH-CO- and -CO-NR and -NR-CO-), carbonyl (-CO-), ester (-C00- and -OOC-), disulfide (-SS-), diaza (-N=N-) or a tertiary amine (-NR-), where R represents an alkyl containing less than 5 carbon atoms, and 1 ,2,3-triazole; and X is (i) a luminescent lanthanide(πi) chelate or a chelating agent able to chelate a lanthanide ion to create the said lanthanide chelate, wherein said chelating agent comprises a a chelating part and a chromophoric moiety having one or more noncondensed aromatic units, which are either substituted or unsubstituted, or (ii) X is a structure selected from
Figure imgf000012_0001
wherein Ln is lanthanide, or is not present .
The most preferable lanthanides are europium, terbium, samarium, dysprosium, gadolinium and holmium.
The chelating agent or the chelate is preferably one of the following structures
Figure imgf000013_0001
wherein Z is independently selected from furyl, thienyl, phenyl or phenylethynyl, where phenyl is substituted or unsubstitued, or Z is not present, wherein X is a radical of any of the aforementioned structures and tethered to the linker L and Ln is lanthanide. In case the structure represents a chelating agent, Ln3+ is missing. In case Z is substituted phenyl, said phenyl is preferably trialkoxysubstituted. more preferably trimethoxysubstituted.
Lanthanide is most preferably selected from the group consisting of europium, terbium, samarium, dysprosium, gadolinium, and holmium.
In the conjugate according to this invention, the target (i.e the biospecific binding reactant) can be a oligopeptide, protein or any bioactive molecule containing one or more mercapto groups.
EXPERIMENTAL SECTION
The invention is further elucidated by the following non-restricting examples. The structures and synthetic routes employed in the experimental part are depicted in Schemes 1 and 2. Experimental details are given in Examples 1-4. Scheme 1 illustrates synthesis of luminescent lanthanide(iπ) chelates tethered to a maleimido group. Experimental details are given in Examples 1-5. Scheme 2 illustrates the reaction between the conjugate molecule and a model peptide containing a single cysteine residue in its structure. Experimental details are given in Example 5.
Procedures
Adsorption column chromatography was performed on columns packed with silica gel 60 (Merck). The cation exchange resin (Dowex 50 X 8, 20-50 mesh, H+ form) was converted to the Ln3+ form by treatment with the appropriate lanthanide(iπ) chloride followed by washing with water until pH of the eluent was neutral. 6-(2,5- dioxo-2H-pyrrol-l(5H)-yl)hexanoic acid, and the model peptide (CVEIDK) were purchased from Fluka and Sigma-Genosys, respectively. NMR spectra were recorded on a Bracker 600 spectrometer operating at 600.13 MHz for H. The signal of TMS was used as an internal (1H) reference. Coupling constants are given in Hertz. ESI-TOF mass spectra were recorded on an Applied Biosystems Mariner instrument. HPLC purifications were performed using a Shimadzu LC 10 AT instrument equipped with a diode array detector, a fraction collector and a reversed phase column (LiChrocart 125-3 Purospher RP- 18e 5 μm). Mobile phase: (Buffer A): 0.02 M triethylammonium acetate (pH 7.0); (Buffer B): A in 50 % (v/v) acetonitrile. Gradient: from 0 to 1 min 95% A, from 1 to 21 min from 95% A to 100% B. Flow rate was 0.6 mL min"1.
Example 1. The synthesis of tetra(tert-butyl) 2,2',2",2'"{4-(4-(6-(2,5-dioxo-2H- pyrrol- 1 (5H)-yl)hexylcarboxamido)phenylethynyl]pyridine-2,6-diyl]bis- (methylenenitrilo)}tetrakis(acetate), 1.
6-(2,5-dioxo-2H-pyrrol-l(5H)-yl)hexanoic acid (60 mg, 0.28 mmol), O-(7- azabenzotriazol-l-yl)-l,l,3,3-tetramethyluronium hexafluorophosphate (100 mg, 0.28 mmol) and ΛζN-diisopropylethylamine (73 μL, 0.56 mmol) were dissolved in dry DMF (3 mL), and the mixture was stirred for 30 min at RT. Tetra(tørt-butyl) 2,2 ',2 ",2 ' ' '-[[4-[(4-aminophenyl)ethynyl]pyridine-2,6- diyl]bis(methylenenitrilo)]tetrakisacetate (100 mg, 0.18 mmol), synthesized as disclosed in Takalo, H., Hanninen, E., Kankare, J., 1993, HeIv. Chim. Acta, 76, 877, was added and the reaction was allowed to proceed overnight at RT. The reaction mixture was diluted with dichloromethane (50 mL) and washed with 10% citric acid. The organic layer was dried over Na2SO4 and concentrated. Purification on silica gel (eluent: methanol: dichloromethane, 5:95, v/v) yielded the title compound 1H NMR (CDCl3): 7.62 (2H, d, J8.5); 7.49 (2H, d, J 8.5); 7.17 (2H, s); 6.70 (2H, d, J 15.5); 3.91 (4H, s); 3.54 (2H51, Jl.3); 3.43 (8H5 s); 2.40 (2H, t, J7.3); 1.78 (2H, m); 1.65 (2H, m); 1.47 (36H, s); 1.39 (4H, m). ESI TOF MS for C49H68N5On + (M+H)+, calcd. 902.49; found 902.53.
Example 2. The synthesis of tetra(tert-butyl) 2,2',2",2'"-{{6,6'-{4"-2-[4-(6-(2,5- dioxo-2H-pyrrol-l (5H)-yl)hexyl)carboxamidoρhenyl)ethyl]pyrazole-l ",3 "- diyl}bis(pyridine)-2,2 r-diyl}bis(methylenenitrilo)}tetrakis(acetate) 2.
The title compound was synthesized using the method described in Example 1 but using tetra(tørt-butyl) 2,2 ',2 " ,2 " '- { {6,6 '- {4 ' '-[2-(4-aminophenyl)ethyl]ρyrazole- 1 ",3 "-diyl}bis(ρyridine)-2,2'-diyl}bis(methylenenitrilo)}tetrakis(acetate) (0.20 g, 0.23 mmol) synthesized as disclosed in US 5,859,215. The reaction was completed in 2h at RT. Purification was performed on silica gel (eluent CH2Cl2 / MeOH 95:5, v/v). 1H NMR (CDCl3): 8.35 (IH, s); 7.94 (IH, d, J6.8); 7.91 (IH, d, J6.8); 7.78 (1HΛJ7.6); 7.74 (lH, t, J7.8); 7.62 (IH, d,J7.6); 7.47 (lH, d, J7.4); 7.41 (2H, d, J8.3); 7.17 (2H, d, J8.3); 6.69 (2H5 d, J8.3), 4.10 (2H, s); 4.05 (2H, s); 3.53 (4H, s); 3.51 (4H, s); 3.49 (2H, m); 3.25 (2H, m); 2.87 82H5 m); 2.34 (2H, t, J 7.5); 1.78 (2H, m); 1.76-1.35 (8H, m); 1.47 (18H, s); 1.44 (18H, s). ESI TOF MS for C57H77N8On+ (M+H)+, calcd. 1049.57; found 1049.55.
Example 3. The synthesis of 2,2'.2"52'"{4-(4-(6-(2,5-dioxo-2-H-pyrrol-l(5H)- yl)hexylcarboxamido)phenylethynyl]pyridine-2,6-diyl]bis- (methylenenitrilo)}tetrakis(acetic acid) euroρium(HI) 3.
Compound 1 (50 mg) was dissolved in TFA (1 mL) and stirred for 2 h at RT before being concentrated in vacuo. The residue was triturated with diethyl ether, collected by filtration and dried in vacuo. The residue was dissolved in water (pH was adjusted to 7 with solid NaHCO3). The solution was passed through a Dowex-50 resin (Eu3+ form) using water as the eluent. Fractions containing the desired chelate were combined and concentrated in vacuo. ESI TOF MS for C33H31 EuN5On" (M- H)", calcd, 826.12, found 826.12.
Example 4. The synthesis of 2,2 \2 ",2'"-{ {6,6 '- {4 ' '-2-[4-(6-(2,5-dioxo-2H-pyrrol- 1 (5H)-yl)hexyl)carboxamidophenyl)ethyl]pyrazole-l ",3 ' '-diyl}bis(pyridine)-2,2 '- diyl}bis(methylenenitrilo)}tetrakis(acetic acid) terbium(πi)s 4.
Compound 2 was converted to the title compound as described in Example 3 but using Dowex 50-resin loaded with Tb3+. ESI TOF MS for C41H40N8O11Tb" (M-H)": calcd, 979.21, found 979.22.
Example 5. Labeling of the oligopeptide with the europium chelate 3. The model peptide (AC-CVEIDK-CONH2) (1 mg) was dissolved in Tris HCl buffer (1 mL; pH 7). Compound 3 (1 equiv.) was added, and the reaction was allowed to proceed for 1 h at RT. Purification on HPLC yielded the desired oligopeptide conjugate. tR 18.14 min. ESI TOF MS for C64H85EuNi3O22S" (M-H)", calcd, 1572.49, found 1572.67.
1:
Figure imgf000018_0001
Figure imgf000018_0002
SCHEME 1
Figure imgf000019_0001
SCHEME 2

Claims

Claims
1. A method for the preparation of a chelating agent or metal chelate of formula (I),
Figure imgf000020_0001
wherein X is a chelating agent able chelate a metal or a metal chelate and L is a linker, said method comprising the steps of a) reacting a compound of formula (Ia)
Figure imgf000020_0002
with a compound of formula (Ib)
A2-L2-G (Ib)
wherein L1 and L2 are linkers or are missing, A1 and A2 are reactive groups, and G is a chelating agent comprising a chelating part having carboxylic acid or phosphonic acid groups which are protected, and optionally a chromophoric moiety, optionally in the presence of an activator or a catalyst, to form a compound of formula (H)
Figure imgf000020_0003
b) purifying of the compound formula (IT), c) removal of the protecting groups, and d) optionally converting of the deprotected chelating agent to the corresponding metal chelate to form a chelate of formula (I).
2. The method according to claim 1 wherein the protecting groups in compound (II) are tert-butyl.
3. The method according to claim 1 wherein the compound (D) is purified by chromatography.
4. The method according to claim 1 wherein the protecting groups are removed by acidolysis.
5. The method according to claim 1 wherein the deprotected ligand is converted to the corresponding metal chelate using an ion-exchange resin loaded with the said metal ion.
6. A compound of formula (H)
Figure imgf000021_0001
wherein L is a linker and G is a chelating agent comprising a chelating part having carboxylic acid or phosphonic acid groups which are protected, and optionally a chromophoric moiety.
7. The compound according to claim 6 wherein the protecting groups are tert-batyl.
8. The compound according to claim 6 wherein the linker L is formed from one to ten moieties, each moiety being selected from the group consisting of phenyl, alkyl containing 1-12 carbon atoms, ethynediyl (-C≡C-), ethylenediyl (-C=C-); ether (-O-), thioether (-S-), amide (-CO-NH- and -NH-CO- and -CO-NR and -NR-CO-), carbonyl (-CO-), ester (-COO- and -OOC-), disulfide (-SS-), diaza (-N=N-) or a tertiary amine (-NR-), where R represents an alkyl containing less than 5 carbon atoms, and 1,2,3-triazole.
9. The compound according to claim 6 wherein G is selected from a group consisting of
Figure imgf000022_0001
Figure imgf000022_0002
Figure imgf000022_0003
Figure imgf000022_0004
wherein Z is independently selected from furyl, thienyl, phenyl or phenylethynyl. where phenyl is substituted or unsubstitued, or Z is not present, wherein G is a radical of any of the aforementioned structures and tethered to L, and R" an alkyl ester of an ally! ester.
10. A labeling reactant, which is a chelating agent or a lanthanide chelate of formula (I)5
Figure imgf000023_0001
wherein L is a linker and X is a luminescent or non-luminescent lanthanide chelate, or a chelating agent able to chelate a metal to create said lanthanide chelate, wherein said chelating agent comprises a chelating part and optionally a chromophoric moiety comprising of one or more noncondensed aromatic units, wherein said aromatic units optionally are substituted.
11. The compound according to claim 10 wherein the linker L is as defined in claim 8 above.
12. The compound according to claim 10 wherein the chelating agent or the chelate X is one of the following structures
Figure imgf000024_0001
wherein Z is independently selected from furyl, thienyl, phenyl or phenylethynyl, where phenyl is substituted or unsubstitued, or Z is not present, wherein X is a radical of any of the aforementioned structures and tethered to the linker L and Ln is lanthanide, or in case X is a chelating agent, Ln3+ is missing.
13. The chelate according to claim 10 wherein the metal is a lanthanide selected from the group consisting of europium, terbium, samarium, dysprosium, gadolinium, and holmium.
14. The chelate according to claim 10, which is 2,2',2",2'"{4-(4-(6-(2,5-dioxo-2- H-pyrrol- 1 (5H)-yl)hexylcarboxamido)phenylethynyl]pyridine-2,6-diyl]bis- (methylenenitrilo)}tetrakis(acetic acid) europiumQU) or 2,2',2",2"'-{{6t6'-{4"-2- [4-(6-(2,5-dioxo-2H-pyrrol-l(5H)-yl)hexyl)carboxamidophenyl)ethyl]ρyrazole- r ',3 ' '-diyl}bis(ρyridine)-2.2 '-diyl}bis(methylenenitrilo)}tetrakis(acetic acid) terbium(ffl).
15. A conjugate comprising a labeling reactant according to any of the claims 6-14 conjugated to a biospecific binding reactant.
16. The conjugate according to claim 15, where the biospecific binding reactant is an oligopeptide, a protein or any bioactive molecule containing one or more mercapto groups.
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