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),

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)

with a compound of formula (Ib)

A ² -L ² -G (Ib)

wherein 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)

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),

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)

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 L ¹ , L ² 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 L ¹ and L ² in the reagents (Ia) and (Ib) are missing, the linker L in compounds (I) and QX) is created from the reactive groups A ¹ and A ² .

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.

The compounds of formula (E), which are protected chelating agents tethered to a maleimido group, are new.

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(PPli ₃ )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 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.

According to a preferable embodiment, 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.

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)

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

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

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, Ln ³⁺ 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 Ln ³⁺ 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 ( ¹ H) 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 Na ₂ SO ₄ and concentrated. Purification on silica gel (eluent: methanol: dichloromethane, 5:95, v/v) yielded the title compound ¹ H NMR (CDCl ₃ ): 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 (2H ₅ 1, Jl.3); 3.43 (8H ₅ 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 C ₄₉ H ₆₈ N ₅ O _n ⁺ (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)}tetraki s(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 CH ₂ Cl ₂ / MeOH 95:5, v/v). ¹ H NMR (CDCl ₃ ): 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 (2H ₅ 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 82H ₅ 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 C ₅₇ H ₇₇ N ₈ On ⁺ (M+H) ⁺ , calcd. 1049.57; found 1049.55.

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) 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 NaHCO ₃ ). The solution was passed through a Dowex-50 resin (Eu ³⁺ form) using water as the eluent. Fractions containing the desired chelate were combined and concentrated in vacuo. ESI TOF MS for C ₃₃ H ₃₁ EuN ₅ On ^" (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 Tb ³⁺ . ESI TOF MS for C ₄₁ H ₄₀ N ₈ O ₁₁ Tb ^" (M-H) ^" : calcd, 979.21, found 979.22.

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. ESI TOF MS for C ₆₄ H ₈₅ EuNi ₃ O ₂₂ S ^" (M-H) ^" , calcd, 1572.49, found 1572.67.

1:

SCHEME 1

SCHEME 2