Europium(III) complexes as pH sensors

Abstract

The present invention relates to a compound of formula (I): [C1] TIFF2023547195000217.tif69156 [wherein R1, R2, R3 and R4 are as defined in the specification.] The present invention relates to the compound The present invention also relates to europium(III) complexes obtainable from compounds of formula (I) or from complexing agents comprising said compounds, and to the use of said complexes for labelling organic or biological molecules.

Description

[Technical Field]

[0001] The present invention is based on a series of Eu(III) complexes incorporating novel chromophores based on a 4-O-alkyl-3-N,N-dialkyl-arylethynylpyridine structure whose luminescence emission switches on by over two orders of magnitude during acidification after excitation in the 330-370 nm range. Such complexes have utility in monitoring acidification in living cells or in any chemical / biological event that results in pH alteration. [Background technology]

[0002] Intracellular pH (pHi) is an important parameter for studying biological phenomena. In fact, intracellular pH plays a number of essential roles in cellular, enzymatic, and tissue activity, including proliferation and apoptosis, multidrug resistance, ion transport, endocytosis, and muscle contraction. Studying pH changes within living cells is also crucial for studying cell internalization pathways, such as phagocytosis, endocytosis, and ligand internalization of a given receptor. Changes in pH also affect the nervous system, affecting synaptic transmission, neuronal excitability, intercellular connections via gap junctions, and signaling cascades. Abnormal pH values ​​are associated with improper cellular function, growth, and division, and are observed in several common diseases, such as cancer and Alzheimer's disease. In cell biology, a low intracellular pH can be used to denature proteins or activate enzyme and protein functions that would be too slow at pH levels around 7. For example, the acidic environment of lysosomes (pH 4.5–5.5) can promote protein degradation. For this reason, cellular dysfunction is often associated with abnormal pH values ​​in cellular compartments.

[0003] Therefore, by labeling the internalized species (receptor or substrate) with a pH-sensitive luminescent dye whose luminescence intensity or lifetime varies with pH, ​​the internalization and endosomal uptake processes can be followed over time. Ideally, the observed change should be as large as possible, and in the limit, this is similar to the concept of a "light switch," although this term is frequently misused in the literature, with too many smaller luminescence intensity fluctuations being unfairly praised as "switch-on" sensors (Chem. Rev. 1997, 97, 1515 and Chem. Rev. 2010, 110, 2709).

[0004] For decades, academics and industrialists have developed pH sensors to measure pH. In 1982, Roger Tsien introduced fluorescein derivatives to assess cytoplasmic pH (Cell Biol. 1982, 95, 189). Several improvements have been made by chemically modifying fluorescein to target cellular compartments or improve solubility in biological media. However, these fluorescein pH sensor derivatives are subject to photobleaching due to singlet oxygen generation, which can also damage cells (Am. J. Physiol. 1989, 256, F957).

[0005] In fact, the majority of pH sensors reported in the literature are pK a This involves adapting the molecular structures of common fluorophores (organic dyes) such as rhodamine, BODIPY™, and various cyanine dyes to adjust the values ​​and enable conjugation.

[0006] In fact, pHrodo™ Green and Red are commercially available from Thermofisher. These dyes belong to the rhodamine family, and some researchers have made some modifications to the backbone to target some biological applications. For this reason, N,N'-trifluoroethylrhodamine derivatives (RH-PEF) have been used to monitor the internalization of antibodies, and their core structure has a pK of around 5.1.a It has been reported that the intensity increase is 58-fold between pH 7.4 and 5.0 (Angew. Chem. Int. Ed. 2014, 53, 6085).

[0007] The pentafluorinated reagent provides AcidiFluor™ ORANGE with a rhodamine skeleton. AcidiFluor™ ORANGE is a fluorescence imaging probe that significantly enhances fluorescence in acidic environments such as lysosomes, late endosomes, and granules.

[0008] CypHer5 is currently commercially available from GE (Amersham) and belongs to the class of cyanine dyes. In related research, cyanine fluorescent dye labels have been used and an assay for tracking the internalization of the HER-2 receptor has been developed (ACS Chem. Biol. 2014, 9, 2237). However, when Grover investigated GPCR labeling with cyanine dyes, an intensity enhancement that was 5-fold smaller was obtained between pH 5 and 8 (Angew. Chem. Int. Ed. 2012, 51, 4838).

[0009] Presumably, the most promising approach reported so far uses a modified BODIPY™ core, and Nagano has reported switching multiples of up to 300-fold using these systems. Here, pK a is easily adjusted by variation of the aniline substituent. As the lone pair conjugation becomes increasingly difficult and the stabilization strength of the conjugate acid weakens due to solvation, pK a increases from 3.8 to 6.0 in the order of H < Me < Et. Such systems have been used to investigate breast cancer tissue samples, and by labeling the immunotherapeutic agent Herceptin™ (trastuzumab) with a BODIPY™ dye, it has become possible to microscopically track the internalization of the HER-2 antibody receptor in mice (Nat. Med. 2009, 15, 104).

[0010] These fluorescent organic dyes (fluorescein, rhodamine, rosamine, cyanine, and BODIPY™) do not offer significant lifetime tuning or ratiometric response and have inherent drawbacks associated with autofluorescence and photobleaching. Therefore, to circumvent the photobleaching, autofluorescence, and small Stokes shift of organic dyes (fluorescein, rhodamine, rosamine, BODIPY™, cyanine, etc.), other researchers have sought to use luminescent lanthanide labeling agents to benefit from the well-established advantages (e.g., large Stokes shift, long emission lifetime) sometimes associated with time-resolved spectroscopy or microscopy.

[0011] Yuan has reported the synthesis of red / green Eu in modified acyclic chelates based on terpyridine ligands. 3+ vs. Tb 3+ By monitoring the change in the emission intensity ratio, we report a system with a seven-fold ratiometric variation (Anal. Chim. Acta 2013, 761, 149). Because this system is based on two complexes (europium and terbium), the use of chemical tools is limited, making the experimental procedure complicated. Furthermore, these complexes are not functionalized, making any bioconjugation reactions impossible.

[0012] Papkovsky et al. described a rather weakly luminescent Eu(III)DTPA complex based on a carbostyril sensitizer, in which the lifetime and emission intensity at 614 nm changed 7-fold between pH 7.5 and 6.5 (pK a 6.5, λ exc 370 nm), a series of high-throughput assays have been developed to monitor extracellular acidification (Anal. Biochem. 2009, 390, 21 and US 2002 / 0058793). This europium complex is designed to monitor only extracellular pH changes induced by glycolytic events. Therefore, this system cannot be used to measure pH.

[0013] Smith observed that the Eu / Tb emission intensity ratio changed from 1.3 at pH 4.6 to 2.9 at pH 6.5 (λ exc describe a method that allows real-time monitoring of lysosomal pH using confocal microscopy, based on the analysis of fluorescence intensity at wavelengths of 355 nm (Chem. Commun. 2012, 48, 8520). The system operates with two different (europium- and terbium-based) complexes of a common ligand, which makes it somewhat complicated from an industrial point of view. Compared to existing organic dye pH sensors, the system behaves in an inverse manner, with fluorescence emission being higher at neutral pH and decreasing as the pH decreases. From a microscopy perspective, this is a significant drawback.

[0014] Using a kinetically stable macrocyclic ligand system based on triazacyclononane, McMahon (Chem. Commun. 2013, 49, 5363) devised a Eu(III) complex that allows for monitoring the pH of the endoplasmic reticulum in living cells based on luminescence lifetime fluctuations (a 75% change in the europium lifetime was observed over the pH range 6.5–7.5). The authors improved the brightness of the complex compared to that described by Smith et al. However, because one of the pendant arms (sulfonamide moieties) is essential for pH sensing, this system does not allow for any change in the chelating moiety around the europium center. Furthermore, this complex is not designed for bioconjugation. Finally, because the luminescence of this europium complex occurs when the pH is increased from pH 4 to 8, this system is not well suited for microscopy purposes.

[0015] Recently, Patra et al. developed a pH-sensitive lysosome-targeting luminescent europium probe (New J. Chem. 2020, 44, 3570). The absorption wavelength is 445 nm, which is incompatible with most commercially available excitation sources (flash lamp excitation between 300 and 360 nm, or laser excitation at 337 nm). Furthermore, the complex is luminescent even at pH 7, an undesirable property for biological and microscopy applications. Again, this lanthanide system does not allow for conjugation of the luminescent probe to target vectors or proteins.

[0016] Takalo has studied the photophysical properties of arylethynylpyridine complexes (Helv. Chim. Acta 1993, 76, 877), and Latva et al. have summarized their results in a paper (J. Lumin. 1997-75-149) discussing the properties of europium chelates. Neither paper mentions that the fluorescence of such systems is pH sensitive. Summary of the Invention [Problem to be solved by the invention]

[0017] Therefore, there is a strong need for lanthanide-based pH-sensitive fluorescent probes that are compatible with biological media and allow monitoring of biological events accompanied by pH fluctuations. The excitation of the probes must be possible between 320 nm and 365 nm, wavelengths widely used in plate readers (flash lamps and lasers). [Means for solving the problem]

[0018] Therefore, we set out to create a new family of compounds that exhibit a 100% change in excited state lifetime and a two-order of magnitude change in long-lived emission intensity over the pH range 8-4. These desirable properties have been shown to be previously unobtainable. To obtain these desirable properties over the pH range 8-4, the pK of the emitting species to be protonated must be increased. aIt was found that the α-to-β ratio (α,β-to-β) must be between 4.5 and 6.5. This result was achieved in accordance with the present invention by designing a chromophore consisting of a diaryl-substituted arylethynylpyridinyl moiety, where one of the substituents is a 4-O-alkyl group and the second is a 3-N,N-dialkylamine moiety (e.g., 3-N,N-dialkylaniline). Such chromophores, when incorporated into macrocycles such as triazacyclononane (TACN), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), or related chelating systems, can form kinetically stable complexes with europium(III).

[0019] The present invention relates to a compound of formula (I): [ka] wherein R1, R2, R3, and R4 are as defined in the detailed description below. The present invention relates to the compound

[0020] The present invention also provides a compound represented by formula (II) or (III): [ka] [In the formula, R a , R b , R c , R d , R e , Chrom1, Chrom2 and Chrom3 are as defined in the detailed description below.] The present invention relates to a complexing agent.

[0021] The present invention also relates to a europium complex comprising a compound of formula (I) or a complexing agent of formula (II) or (III) and a europium(III) ion.

[0022] Furthermore, the present invention relates to a conjugate obtained by reacting a europium complex with a molecule of interest. [Brief explanation of the drawings]

[0023] [Figure 1] FIG. 1 shows the absorption spectrum of complex 14 at pH 4 to 10. [Figure 2] FIG. 1 shows the emission spectrum of complex 14 at pH 4 to 10. [Figure 3] FIG. 1 shows the pH versus emission lifetime variation allowing the determination of the pKa value for complex 14. [Figure 4] FIG. 1 shows the absorption spectrum of complex 18 at pH 4 to 10. [Figure 5] FIG. 1 shows the emission spectrum of complex 18 at pH 4 to 10. [Figure 6] FIG. 1 shows the pH vs. emission lifetime variation allowing the determination of the pKa value for complex 18. [Figure 7] FIG. 1 shows the absorption spectrum of complex 21 at pH 4 to 10. [Figure 8] FIG. 1 shows the emission spectrum of complex 21 at pH 4 to 10. [Figure 9] FIG. 10 shows the pH vs. emission lifetime variation allowing the determination of the pKa value for complex 21. [Figure 10] FIG. 10 shows the absorption spectrum of complex 78a at pH 4 to 10. [Figure 11] FIG. 1 shows the emission spectrum of complex 78a at pH 4 to 10. [Figure 12] FIG. 10 shows the pH vs. emission lifetime variation allowing the determination of the pKa value for complex 78a. [Figure 13] FIG. 10 is a diagram showing the absorption spectrum of complex 78b at pH 4 to 10. [Figure 14] FIG. 10 shows the emission spectrum of complex 78b at pH 4 to 10. [Figure 15] FIG. 10 shows the pH vs. emission lifetime variation allowing the determination of the pKa value for complex 78b. [Figure 16] FIG. 10 is a diagram showing the absorption spectrum of complex 88a at pH 4 to 10. [Figure 17]FIG. 10 shows the emission spectrum of complex 88a at pH 4 to 10. [Figure 18] FIG. 10 shows the pH vs. emission lifetime variation allowing the determination of the pKa value for complex 88a. [Figure 19] FIG. 10 is a diagram showing the absorption spectrum of complex 88b at pH 4 to 10. [Figure 20] FIG. 10 shows the emission spectrum of complex 88b at pH 4 to 10. [Figure 21] FIG. 10 shows the pH vs. emission lifetime variation allowing the determination of the pKa value for complex 88b. [Figure 22] FIG. 1 shows the absorption spectrum of complex 93 at pH 4 to 10. [Figure 23] FIG. 1 shows the emission spectrum of complex 93 at pH 4 to 10. [Figure 24] FIG. 10 shows the pH vs. emission lifetime variation allowing the determination of the pKa value for complex 93. [Figure 25] FIG. 1 shows the absorption spectrum of complex 104 at pH 4 to 10. [Figure 26] FIG. 1 shows the emission spectrum of complex 104 at pH 4 to 10. [Figure 27] FIG. 1 shows the pH vs. emission lifetime variation allowing the determination of the pKa value for complex 104. [Figure 28] (Top) Luminescence intensity of complex 18 measured at different pH values ​​(295 K) with different time delays (red = 60 μs, orange = 460 μs, purple = 1000 μs). Data are normalized to a 60 μs delay at pH 4. (Bottom) Luminescence intensity of complex 18 measured at various pH values ​​with different time windows (red = 60–460 μs, orange = 1000–2000 μs, purple = 1500–2500 μs). Data are normalized to a 60–460 μs time window at pH 4. Measurements were performed in NH4CH3CO2 (pH 4 and 5), MES (pH 5.5, 6, and 6.5), HEPES (pH 7), and NH4HCO3 (pH 8) buffers (all 0.1 M containing 0.1 M NaCl). [Figure 29]FIG. 1 is a graph showing the change in fluorescence intensity at 620 nm as a function of pH for the complexes of the present invention and comparative complexes. DETAILED DESCRIPTION OF THE INVENTION

[0024] In one aspect, the present invention provides a compound of formula (I): [ka] [In the formula, R1 is —CO2H, —PO(OH)R5 or —CH2N(CH2CO2H)2; R2 is -CH2OH, -CH2OSO2CH3, Br, Cl, or -CH2N(CH2CO2H)2; R3 is (C1-C6) alkyl optionally substituted with an -L1-E group or a G group; R4 is -(CH2) m -NR6R7; R5 is (C1-C4) alkyl, preferably methyl; -SO3 - phenyl optionally substituted with a group, preferably at the meta or para position; or benzyl; R6 is H or (C1-C4) alkyl; R7 is (C1-C6) alkyl optionally substituted with an -L1-E group; Alternatively, R6 and R7 together with the nitrogen atom to which they are attached form a piperidine, morpholine, or piperazine, which may be N-protected with a tert-butoxycarbonyl group or N-substituted with R8; R8 is (C1-C6) alkyl optionally substituted with an -L1-E group; L1 is a direct bond, -CONH-(CH2) n - or -NHCO-(CH2) n - and; E is -SO3H, -SO2(OCH2CF3), -N + Alk1Alk2Alk3, a carbohydrate residue, or a sulfobetaine; G is a carboxyl group which may be protected in the form of an ester (e.g., -CO2Me or -CO2t Bu group), an amino group optionally protected by a tert-butoxycarbonyl group, a succinimidyl ester, a haloacetamide group, a hydrazine group, an isothiocyanate group, or a maleimide group; Alk1, Alk2, and Alk3 each independently represent (C1-C6) alkyl, preferably (C1-C4) alkyl; m is 0, 1, 2 or 3; n is 0, 1, 2 or 3. The present invention relates to the compound

[0025] In the context of this disclosure, a carbohydrate residue is a glucose residue in cyclic or linear form, or a carbohydrate residue of the formula -(CHOH) k is understood to mean the group -CH2OH, where k is an integer from 3 to 12, preferably k is 3 or 4.

[0026] In the context of the present disclosure, sulfobetaines have the following formula: [ka] wherein R represents (C1-C6) alkyl, preferably methyl or ethyl, and t is an integer from 1 to 6, preferably t is 1 or 2. In one embodiment, sulfobetaines have the formula -(CH2)2N + (CH3)2-(CH2)3-SO3 - It is based on.

[0027] In the context of the present disclosure, the groups -SO3H, -CO2H and -PO(OH)2 are in deprotonated or non-deprotonated form depending on the pH. Therefore, these groups are referred to below and in the appended claims as -SO3 - , -CO2 - and -PO(OH)O - It also refers to the base and vice versa.

[0028] In the context of this disclosure, the embodiments described herein can be combined.

[0029] In one embodiment, R1 is -CO2H or -PO(OH)R5, where R5 is (C1-C4)alkyl, preferably methyl, and R2 is -CH2OH or -CH2OSO2CH3. In another embodiment, R1 and R2 are each -CH2N(CH2COOH)2.

[0030] In one embodiment, R3 is (C1-C4) alkyl optionally substituted with an -L1-E or G group.

[0031] In one embodiment, R4 is -NR6R7, where R6 and R7 are each independently (C1-C4) alkyl, or R6 and R7 together with the nitrogen atom to which they are attached form a piperidine that may be N-protected with a tert-butoxycarbonyl group or N-substituted with an R8 group.

[0032] In one embodiment, R8 is (C1-C4) alkyl optionally substituted with an -L1-E group.

[0033] In one embodiment, L1 is a direct bond or -CONH-(CH2) n -It is.

[0034] In one embodiment, E is —SO 3 H or —SO 2 (OCH 2 CF 3 ).

[0035] In one embodiment, G is a carboxyl group optionally protected in the form of an ester (e.g., -COMe or -CO t and an amino group which may be protected by a tert-butoxycarbonyl group.

[0036] In one embodiment, m is 0. In another embodiment, m is 1. In yet another embodiment, m is 2. In yet another embodiment, m is 3.

[0037] In one embodiment, n is 0. In another embodiment, n is 1. In yet another embodiment, n is 2. In yet another embodiment, n is 3.

[0038] In another aspect, the present invention provides a compound represented by formula (II) or (III): [ka] [In the formula, R a is absent or is -CH2NH2; R b、 R c , R d and R e one (and only one) of the groups is a group of formula (IV), and the others are each independently —CHCOOR f and -CH2PO(OH)R g Selected from; R f is H, (C1-C4) alkyl, -NHCH(R h )-(C1-C4) alkyl or -NHCH(R h )-C(O)OR j and; R g is (C1-C4) alkyl; R h is (C1-C4) alkyl or phenyl; R j is H or (C1-C4) alkyl; Chrom1, Chrom2, and Chrom3 each independently represent a group of formula (IV) and a group of formula (V): [ka] (In the formula, each R1 is -CO2H or -PO(OH)R5; R3 is (C1-C6) alkyl optionally substituted with an -L1-E group or a G group; R4 is -(CH2) m -NR6R7; R5 is (C1-C4) alkyl, preferably methyl; -SO3- phenyl optionally substituted with a group, preferably at the meta or para position; or benzyl; R6 is H or (C1-C4) alkyl; R7 is (C1-C6) alkyl optionally substituted with an -L1-E group; Alternatively, R6 and R7 together with the nitrogen atom to which they are attached form a piperidine, morpholine, or piperazine, which may be N-protected with a tert-butoxycarbonyl group or N-substituted with an R8 group; R8 is (C1-C6) alkyl optionally substituted with an -L1-E group; R9 is (C1-C6) alkyl optionally substituted with a -L1-E group or a G group; L1 is a direct bond, -CONH-(CH2) n - or -NHCO-(CH2) n and; E is -SO3H, -SO2(OCH2CF3), -N + Alk1Alk2Alk3, a carbohydrate residue, or a sulfobetaine; G is a carboxyl group which may be protected in the form of an ester (e.g., -CO2Me or -CO2 t Bu group), an amino group optionally protected by a tert-butoxycarbonyl group, a succinimidyl ester, a haloacetamide group, a hydrazine group, an isothiocyanate group, or a maleimide group; Alk1, Alk2, and Alk3 each independently represent (C1-C6) alkyl, preferably (C1-C4) alkyl; m is 0, 1, 2 or 3; n is 0, 1, 2, or 3. Selected from.] wherein the compound of formula (II) contains at least one group of formula (IV).

[0039] A preferred family of complexing agents comprises compounds of formula (II): Another preferred family of compounds comprises compounds of formula (III):

[0040] In one embodiment, the complexing agent of formula (II) contains only one group of formula (IV), i.e., one of Chrom1, Chrom2 and Chrom3 is a group of formula (IV) and the other two are groups of formula (V), which may be the same or different, preferably the same.

[0041] In one embodiment, the complexing agent of formula (II) comprises two groups of formula (IV), i.e., two of Chrom1, Chrom2 and Chrom3 are groups of formula (IV), which may be the same or different, preferably the same, and the other is a group of formula (V).

[0042] In one embodiment, the complexing agent of formula (II) comprises three groups of formula (IV), i.e., Chrom1, Chrom2 and Chrom3 are each groups of formula (IV) and may be the same or different, preferably the same.

[0043] In one embodiment, each R1 is a -PO(OH)R5 group, where R5 is (C1-C4) alkyl, preferably methyl.

[0044] In one embodiment, R3 is (C1-C4) alkyl optionally substituted with an -L1-E or G group.

[0045] In one embodiment, R4 is -NR6R7, where R6 and R7 are each independently (C1-C4) alkyl, or R6 and R7 together with the nitrogen atom to which they are attached form a piperidine that may be N-protected with a tert-butoxycarbonyl group or N-substituted with an R8 group.

[0046] In one embodiment, R8 is (C1-C4) alkyl optionally substituted with an -L1-E group.

[0047] In one embodiment, L1 is a direct bond or -CONH-(CH2) n -It is.

[0048] In one embodiment, E is —SO 3 H or —SO 2 (OCH 2 CF 3 ).

[0049] In one embodiment, G is a carboxyl group optionally protected in the form of an ester (e.g., -COMe or -CO t and an amino group which may be protected by a tert-butoxycarbonyl group.

[0050] In one embodiment, m is 0. In another embodiment, m is 1, and in yet another embodiment, m is 2. In yet another embodiment, m is 3.

[0051] In one embodiment, n is 0. In another embodiment, n is 1, and in yet another embodiment, n is 2. In yet another embodiment, n is 3.

[0052] In another aspect, the present invention provides a method for producing a europium(III) ion (Eu 3+ and a compound of formula (I) wherein R1 and R2 are each —CH2N(CH2COOH)2.

[0053] In another aspect, the present invention relates to a europium complex comprising a europium(III) ion and a compound of formula (II) or (III) disclosed herein.

[0054] The europium complexes of the present invention can be prepared by contacting a compound of Formula (I), (II), or (III) disclosed herein with a europium salt. Typically, 1 equivalent of the compound is reacted with 1 to 5 equivalents of a europium salt (e.g., chloride, acetate, or triflate) in a solvent (acetonitrile, methanol, or other solvent compatible with these salts) or an aqueous buffer at room temperature for several minutes to yield the corresponding complex.

[0055] Representative europium complexes of the present invention have the following structure: [ka]

[0056] Another representative europium complex of the present invention has the following structure: [ka] [Wherein Z is [ka] , or [ka] ].

[0057] The compounds of formulas (I), (II) and (III) disclosed herein (and their corresponding europium complexes) containing a G group are particularly suitable for labeling organic or biological molecules that contain functional groups capable of reacting with the reactive group to form a covalent bond.

[0058] Thus, in one aspect, the present invention relates to a conjugate obtained by reacting (i) a europium complex disclosed herein having a G group with (ii) a molecule of interest. Any organic or biological molecule can be conjugated to the europium complex disclosed herein, so long as it has a functional group capable of reacting with the G group of the complex.

[0059] In one embodiment, the molecule of interest is selected from amino acids, peptides, proteins, antibodies, sugars, carbohydrate chains, nucleosides, nucleotides (DNA, RNA), oligonucleotides, and enzyme substrates, in particular enzyme suicide substrates, such as benzylguanine or benzylcytosine (enzyme substrates commercially available under the names Snaptag and Cliptag), chloroalkanes (enzyme substrates commercially available under the name Halotag), or coenzyme A (enzyme substrates commercially available under the name ACPtag or MCPtag).

[0060] The europium complexes of the present invention, when protonated, have a pK in the range of 4.5 to 6.5. a Usually, if a molecule has several protonation sites, each protonation site has a pK a Surprisingly, the complexes of the present invention exhibit only one observable pK, independent of the number of chromophores (i.e., compounds of formula (I), (IV), or (V)) immobilized on the complex. a It was found to exhibit value.

[0061] The europium complexes of the present invention can also be bioconjugated to molecules of interest at sites that do not perturb the fluorescence intensity upon pH fluctuations in the medium. The europium complexes of the present invention have excellent luminescence brightness, which is highly desirable for detecting biological events at low concentrations.

[0062] As mentioned above, the europium complexes of the present invention can contain one to three chromophores and can be functionalized to allow the introduction of water-soluble moieties and functional groups for bioconjugation. Depending on the desired complex, a corresponding synthetic route can be selected (Schemes 1 to 3). For complexes with one chromophore, two chromophores, or three non-identical chromophores, two options are possible, as illustrated in Schemes 1 and 2 and exemplified in more detail in the experimental section. For complexes with three identical chromophores, the synthesis is illustrated in Scheme 3.

[0063] For example, triazacyclononane is alkylated with either a chromophore or a pyridine moiety, depending on the desired number of chromophores required in the complex. After deprotection of the Boc group, the final chromophore or pyridine moiety is introduced into the di-chromophore or di-pyridinyl alkylated macrocycle. The phosphinate ethyl ester functionality is hydrolyzed, and europium complexes are formed by the addition of europium(III) chloride. Optionally, the complexes can be functionalized with sulfonate moieties to allow for better water solubility and functional groups for conjugation with proteins, antibodies, peptides, and enzyme substrates.

[0064] General synthetic scheme for complexes containing one, two, and three chromophores (Option 1) [ka] Scheme 1

[0065] General synthetic scheme for complexes containing one and two chromophores (option 2) [ka] Scheme 2

[0066] A general synthetic scheme for complexes containing three identical chromophores. [ka] Scheme 3

[0067] General synthesis of acyclic complexes The backbone synthesis has been described previously (Helvetica Chimica Acta, 1993, 76, 877). This synthesis is supplemented by the introduction of an N,N-dialkyl moiety at the meta position of the triple bond and an O-alkyl group at the para position. An example is shown in Scheme 4. [ka] Scheme 4

[0068] General synthesis of DOTA macrocycle-based complexes To highlight the general features of the present invention, a chromophore was introduced into the DOTA system using a classical synthetic strategy described elsewhere (WO 2006 / 120444, WO 2009 / 010580, WO 2010 / 084090, Acc. Chem. Res 2009, 42, 925). The strategy used is illustrated in Scheme 5, which leads to the corresponding europium complexes decorated with one chromophore. [ka] Scheme 5

[0069] Other routes for preparing the complexing agents disclosed herein and the corresponding europium complexes are generally described below and further illustrated in the Examples.

[0070] Synthesis of non-functionalized chromophores [ka] Scheme 6

[0071] Commercially available compound 1 was reduced to the corresponding aniline and alkylated in the presence of iodoethane for 48 h. The chromophore scaffold was obtained by two Sonogashira reactions, yielding compound 7. The resulting alcohol was converted to the corresponding mesylate 8, which was used for subsequent macrocycle alkylation.

[0072] Synthesis of unfunctionalized complexes containing one chromophore. [ka] Scheme 7

[0073] The synthesis of a europium complex containing one chromophore is illustrated in Scheme 7. Triazacyclonane 9 was alkylated with mesylated pyridine 10. The third nitrogen atom was deprotected with TFA and alkylated with chromophore 8. The phosphinate ethyl ester was hydrolyzed and the macrocycle was complexed with europium to give europium(III) complex 14.

[0074] Synthesis of non-functionalized complexes containing two chromophores. [ka] Scheme 8

[0075] To obtain complexes containing two chromophores, such as the europium complex 18, the strategy applied was identical to the previous one, except that the order of introducing the pyridine derivatives (compounds 8 and 10) was reversed, as presented in Scheme 8.

[0076] Synthesis of non-functionalized complexes containing three chromophores. [ka] Scheme 9

[0077] A complex with three chromophores was synthesized using unprotected triazacyclononane 19 and chromophore 8 to give the corresponding europium complex 21.

[0078] Synthesis of ethyl ester functionalized chromophores [ka] Scheme 10

[0079] To functionalize chromophore 8, the methyl group of the methoxy group was replaced with an aliphatic chain containing an ester moiety for later introduction of sulfonate or other hydrophilic functional groups. For this purpose, bromonitrophenol 22 was alkylated, followed by reduction of the nitro group and subsequent alkylation with iodoethane. Two Sonogashira reactions gave the activated chromophore as the mesylate derivative 30.

[0080] Synthesis of NHBoc-functionalized chromophores [ka] Scheme 11

[0081] Instead of functionalizing with a protected carboxylic acid in the form of an ethyl ester, the chromophore was functionalized with a protected amine in the form of a classical carbamate (Boc group). The synthetic route was the same as the previous one using N-Boc-protected bromopropylamine (Scheme 11).

[0082] Synthesis of ethyl ester functionalized chromophores [ka] Scheme 12

[0083] The alkylation of aniline was controlled by selecting the molar ratio of the alkylating reagent to the aniline derivative. Compound 36 was then mono-alkylated with iodoethane. The resulting secondary aniline was methylated using reductive amination. The final step of the synthesis was the same as described above.

[0084] Synthesis of cyclic amine systems on chromophores [ka] Scheme 13

[0085] Aniline derivatives were converted to cyclic amines (piperidine, piperazine, morpholine). In the case of compounds 46a-d, the secondary amines are useful for attaching soluble moieties or can serve as bioconjugation points.

[0086] Synthesis of N,N-Alkylpropylsulfonate Precursor Chromophores [ka] Scheme 14

[0087] Aniline was substituted on one side with an alkyl chain bearing a terminally capped sulfonate and on the other side with an alkyl chain. To this end, the corresponding chromophore was prepared using the same methodology as in the previous scheme, except that the corresponding bromopropyl sulfonate substituted with a trifluoroethyl moiety was employed as the protecting group. The synthetic scheme is illustrated in Scheme 14.

[0088] Synthesis of NHBoc-functionalized pyridines [ka] Scheme 15

[0089] Synthesis of methyl ester-functionalized pyridines [ka] Scheme 16

[0090] Compounds 60 and 63 were synthesized from pyridine 6. The first C-C bond formation reaction was carried out using the Heck reaction. The resulting double bond was hydrogenated in a classical manner. Activation of the alcohol function gave the corresponding mesylate derivatives 60 and 63 (Schemes 15 and 16).

[0091] Synthesis of functionalized complexes containing one chromophore. [ka] Scheme 17

[0092] The synthesis of complex 65 incorporating one chromophore is described in Scheme 17.

[0093] Synthesis of functionalized complexes containing one chromophore. [ka] Scheme 18

[0094] The synthesis of complex 71 is illustrated in Scheme 18. At the end of the synthesis, a sulfonate moiety was introduced onto the pyridine heterocycle.

[0095] Synthesis of functionalized complexes containing one chromophore. [ka] Scheme 19

[0096] Scheme 19 illustrates the synthesis of a complex that can be functionalized via the piperazinyl moiety on the secondary amine (compound 73b).

[0097] Synthesis of functionalized complexes containing two chromophores. [ka] Scheme 20

[0098] The synthesis of two complexes with two chromophores is described in Scheme 20. The pK of each complex is a was adjusted by the nature of the alkyl group substituting the nitrogen atom. The synthesis began with alkylation of triazacyclononane with the mesylate chromophore. Deprotection of the Boc group followed by a third alkylation afforded compounds 76a-b. Hydrolysis of the phosphinate and europium complexation afforded complexes 77a-b, which were treated with homotaurine to afford the desired water-soluble europium(III) complexes.

[0099] Synthesis of functionalized complexes containing two chromophores. [ka] Scheme 21

[0100] The synthesis of complex 84 bearing a piperazinyl moiety is described in Scheme 21. The same strategy as previously described was followed. Commercially available deprotected triazacyclononane 79 was monoalkylated. Deprotection of both Boc groups was carried out in the presence of trifluoroacetic acid. The chromophore was introduced at this stage, and the phosphinate ester functionality was then hydrolyzed, forming the europium complex after the addition of europium chloride. Reaction of a secondary amine with propane sultone afforded the desired complex 84.

[0101] Synthesis of functionalized complexes containing two chromophores. [ka] Scheme 22

[0102] As previously described, a sulfonate group was introduced at the terminal moiety of one of the alkyl groups of the aniline functionality. The synthesis followed the same strategy with chromophores 57a-b, i.e., alkylation of the macrocycle, deprotection of the Boc group, introduction of the final pyridine derivative, hydrolysis of the phosphinate ester, and complexation with europium. Furthermore, final basic hydrolysis of the phosphinate ester allowed deprotection of the trifluoroethyl protecting group to give the free sulfonate functionality (Scheme 22).

[0103] Synthesis of functionalized complexes containing three chromophores. [ka] Scheme 23

[0104] A complex containing three chromophores was prepared as described in Scheme 23. The synthetic route was based on the same strategy as previously described, leading to complex 91.

[0105] [ka] Scheme 24

[0106] Scheme 24 shows the preparation of asymmetric complex 93 using the same strategy as above, which has two identical chromophores (N,N-ethylpropylsulfonate) and the third chromophore is a moiety bearing an N,N-diethylaniline group. This complex can be used for subsequent bioconjugation.

[0107] Synthesis of functionalized complexes containing three chromophores. [ka] Scheme 25

[0108] Another method to obtain a complex containing three chromophores was to use a triazacyclononane with a pendant methylene NHBoc arm, as previously disclosed in US 9,981,967. This synthesis was simple and afforded the desired complex 98 containing three identical chromophores.

[0109] Synthesis of bioconjugated complexes containing two chromophores [ka] Scheme 26

[0110] Scheme 26 illustrates the conjugation of europium complexes 78a-b with the well-known enzyme substrate benzylguanine methylbenzamide NHS ester (BG-MB-NHS) (WO 2010 / 034931) to give benzylguanine complexes 100a-b, or with maleimides to give complexes 101a-b, which can be used for antibody or protein labeling.

[0111] Synthesis of acyclic complexes [ka] Scheme 27

[0112] This scheme illustrates the synthesis of acyclic compounds. The synthesis of pyridine ethynylaryl derivatives has been previously described by Takalo (ibid.). Appropriate building blocks were used to obtain europium chelates functionalized to enable bioconjugation. The corresponding maleimide and benzylguanine derivatives 105 and 106 were prepared in a similar manner.

[0113] Experimental section The following abbreviations are used in the experimental section: 9-N3 1,4,7-triazacyclononane Appearance BG Benzylguanine Boc tert-butoxycarbonyl br Broad (NMR) BSA Bovine serum albumin CPL circularly polarized light d days (reaction time) d Double line (NMR) DCM dichloromethane dd doublet doublet (NMR) ddq doublet doublet quartet (NMR) DIPEA Diisopropylethylamine dm doublet multiplet (NMR) DMF Dimethylformamide DMSO dimethyl sulfoxide dppf 1,1'-bis(diphenylphosphino)ferrocene EDTA Ethylenediaminetetraacetic acid ESI electrospray ionization EtOAc ethyl acetate EtOH ethanol equiv. equivalent amount FA formic acid GPCR G protein-coupled receptor h Time (reaction time) HATU (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HRMS high resolution mass spectrometry ICP-MS inductively coupled plasma mass spectrometry ICT Intramolecular charge transfer LRMS low resolution mass spectrometry Ln Lanthanides LTG LysoTracker Green m multiplet (NMR) MeOH Methanol MES 2-(N-morpholino)ethanesulfonic acid Mp melting point MS mass spectrometry NMR nuclear magnetic resonance PBS Phosphate Buffered Saline PDA Photodiode Array Pd / C Palladium Carbon Pd(dppf)Cl2 [1,1'-bis(diphenylphosphino)ferrocene]palladium(II) dichloride q quartet (NMR) quant. RT room temperature s Singlet (NMR) S n nth singlet state (energy level) t Triple line (NMR) TEEAc Triethylamine TFA trifluoroacetic acid THF tetrahydrofuran TLC thin layer chromatography TMS trimethylsilyl UV ultraviolet light Vis visible

[0114] compound 2 [ka] 4-Iodo-1-methoxy-2-nitrobenzene 1 (1.00 g, 3.58 mmol), glacial acetic acid (5 mL, 87 mmol), and MeOH (5 mL) were mixed under argon. Iron powder (1.02 g, 18.3 mmol) was added, and the mixture was heated to 50 °C for 1 h. The mixture was filtered, and the solvent was removed under reduced pressure. The resulting residue was dissolved in CHCl (100 mL), and aqueous NaOH (25 mL, 2 M) was added, and the subsequent precipitate was removed by filtration. The aqueous layer was separated and extracted with CHCl (5 × 15 mL). The combined organic layers were dried over MgSO, and the solvent was removed under reduced pressure to give 2 (674 mg, 76%) as a light brown solid.

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[0115] compound 3 [ka] Compound 2 (2.83 g, 11.4 mmol), iodoethane (7 mL, 87 mmol), and KCO (6.3 g, 45.6 mmol) were mixed in anhydrous CHCN (15 mL) under argon. The reaction mixture was heated to 60 °C for 48 h. After this time, the solvent was removed under reduced pressure, and the residue was dissolved in CHCl (30 mL), washed with water (5 × 20 mL), and dried over KCO. The solvent was removed under reduced pressure to give a pale orange oil (2.16 g, 62%).

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[0116] compound 4 [ka] Compound 3 (548 mg, 1.80 mmol), trimethylsilylacetylene (0.5 mL, 3.6 mmol), Pd(dppf)Cl, DCM (150 mg, 0.184 mmol), and pyrrolidine (0.45 mL, 5.4 mmol) were mixed in anhydrous THF (3 mL) under argon. The reaction mixture was heated to 50 °C for 19 h, and then the solvent was removed under reduced pressure. The resulting residue was dissolved in CHCl (30 mL), washed with HO (4 × 30 mL), and dried over KCO. Removal of the solvent under reduced pressure gave the crude product, which was purified by column chromatography (SiO, 100% hexane to 6% EtOAc in hexane) to give a pale orange oil (323 mg, 65%).

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[0117] compound 5 [ka] Triethylamine trihydrofluoride (0.75 mL, 3.28 mmol) was added to a solution of compound 4 (115 mg, 0.306 mmol) in anhydrous THF (3 mL) under argon. The solution was heated to 30 °C for 37 h, and then the solvent was removed under reduced pressure. The resulting oil was dissolved in CHCl (30 mL) and washed with water (3 × 40 mL). The combined aqueous layers were extracted with CHCl (5 × 40 mL), and the combined organic layers were dried over KCO. The solvent was removed under reduced pressure to give a pale orange oil (81 mg, 87%).

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[0118] compound 6 [ka] Compound 6 was synthesized in nine steps from 2-bromo-6-methylpyridine according to the procedure described in US2015 / 361116.

[0119] compound 7 [ka] A solution of compound 5 (120 mg, 0.59 mmol) and compound 6 (175 mg, 0.60 mmol) in anhydrous THF (2.5 mL) was treated under argon with pyrrolidine (0.1 mL, 1.22 mmol) and Pd(dppf)Cl 2· DCM (60 mg, 0.073 mmol) was added. The reaction mixture was heated to 50 °C for 18 h, then the solvent was removed under reduced pressure and the resulting residue was dissolved in CHCl (40 mL) and washed with water (3 × 40 mL). The organic layer was dried over KCO and the solvent was removed under reduced pressure to give a brown residue. This crude residue was purified by reverse-phase HPLC (10-100% CHCN in H0, 10 min, t r =10.7 min) to give a pale yellow oil (118 mg, 48%).

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[0120] compound 8 [ka] Compound 7 (67 mg, 0.161 mmol), methanesulfonic anhydride (56 mg, 0.322 mmol), and DIPEA (0.07 mL, 0.402 mmol) were mixed in anhydrous THF (1.5 mL) under argon and stirred at room temperature for 90 minutes. After this time, the solvent was removed under reduced pressure. To the resulting residue were added CHCl (30 mL) and HO (30 mL). The organic layer was separated and washed with HO (2 × 30 mL), and the combined aqueous layers were extracted with CHCl (1 × 30 mL). The combined organic layers were dried over KCO, and the solvent was removed under reduced pressure to give a pale orange oil (69 mg, 87%), which was used directly in the next step without further purification.

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[0121] Compound 9 was synthesized in three steps according to the procedure described in WO2014 / 111661.

[0122] compound 10 [ka] Compound 10 was synthesized in five steps from 2-bromo-6-methylpyridine according to the procedure described in Inorg. Chem., 2012, 51, 8042.

[0123] compound 11 [ka] The dihydrochloride salt of 1-(tert-butoxycarbonyl)-1,4,7-triazacyclononane 9 (28 mg, 0.0926 mmol), compound 10 (64 mg, 0.218 mmol), and KCO (40 mg, 0.289 mmol) were mixed in anhydrous CHCN (2 mL) under argon and heated at 65 °C for 18 h. After this time, the solution was separated from the inorganic salts and purified by reverse-phase HPLC (10–100% CHCN in HO for 10 min, t r =11.8 min) to give a pale yellow oil (33 mg, 57%).

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[0124] compound 12 [ka] A solution of compound 11 (33 mg, 0.053 mmol) in trifluoroacetic acid and CHCl (20% v / v, 4 mL total) was prepared. The solution was stirred at room temperature for 25 minutes, and then the solvent was removed under reduced pressure to give an orange residue. CHCl (30 mL) was added to the residue, and the solvent was again removed under reduced pressure. This procedure was repeated five times to give a pale orange oil (28 mg, quant.).

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[0125] compound 13 [ka] Compound 12 (8.5 mg, 16.2 μmol), compound 8 (16 mg, 32.4 μmol), and KCO (5 mg, 35.4 μmol) were mixed in anhydrous CHCN (1 mL) under argon and heated to 60 °C for 18 h. After this time, the crude mixture was separated from inorganic salts by filtration. The resulting solution was directly analyzed by reverse-phase HPLC (10-100% CHCN in HO for 10 min, t r =11.0 min) to give compound 13 as a pale yellow oil (7.4 mg, 50%).

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[0126] Complex 14 [ka] Ligand 13 (2 mg, 2 μmol) was dissolved in a mixture of CHOH / HO (1:1, 2 mL total), and the pH was adjusted to 12 with aqueous NaOH. The solution was heated to 60 °C for 14 h. After cooling and adjusting the pH to 7 with dilute hydrochloric acid (0.1 M), EuCl3.6HO (3 mg, 8 μmol) was added, and the reaction mixture was heated to 60 °C for 15 h. The reaction mixture was analyzed by reverse-phase HPLC (10–100% CH3CN in HO, 10 min, t r =8.5 min) to give a yellow solid (2 mg, 93%).

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[0127] compound 15 [ka] The dihydrochloride salt 9 (31 mg, 0.10 mmol) of 1-(t-butoxycarbonyl)-1,4,7-triazacyclononane, the mesylate 8 (122 mg, 0.26 mmol), and KCO (60 mg, 0.43 mmol) were mixed in anhydrous CHCN (2 mL) under argon. The resulting mixture was heated to 60 °C for 14 h, after which the crude solution was separated from the inorganic salts and the solvent removed under reduced pressure to give an orange oil, which was purified by reverse-phase HPLC (10–100% CHCN in HO for 10 min, t r =15.6 min) to give a pale yellow oil (66 mg, 62%).

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[0128] compound 16 [ka] A solution of compound 15 (66 mg, 0.0643 mmol) in trifluoroacetic acid and CHCl (10% v / v, 3 mL total) was prepared. The solution was stirred at room temperature for 1 hour, and then the solvent was removed under reduced pressure to give an orange residue. CHCl (30 mL) was added to the residue, and the solvent was again removed under reduced pressure. This procedure was repeated five times to give a pale orange oil (67 mg, quant.).

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[0129] compound 17 [ka] Compound 16 (39 mg, 0.0375 mmol), mesylate 10 (36 mg, 0.123 mmol), and KCO (30 mg, 0.217 mmol) were mixed in anhydrous CHCN (2 mL) under argon and heated to 60 °C for 18 h. After this time, the crude mixture was separated from the inorganic salts by centrifugation, and the resulting solution was directly analyzed by reverse-phase HPLC (10–100% CHCN in 25 mM ammonium bicarbonate buffer, 10 min, t r =11.8 min) to give a pale yellow oil (23 mg, 55%).

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[0130] Complex 18 [ka] Ligand 17 (11.5 mg, 0.01024 mmol) was dissolved in a mixture of CHOH / HO (1:1, 4 mL total), and the pH was adjusted to 12 with 1 M aqueous NaOH. The solution was heated to 60 °C for 15 h. After cooling and adjusting the pH to 6 with dilute hydrochloric acid (0.1 M), EuCl3.6HO (6 mg, 0.0164 mmol) was added, and the reaction mixture was heated to 60 °C for 17 h. After this time, the solution was separated from the inorganic salts by centrifugation and analyzed by reverse-phase HPLC (10–100% CH3OH in HO, 10 min, t r =13.6 min) to give a yellow solid (7 mg, 58%).

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[0131] Compound 19 is commercially available.

[0132] compound 20 [ka] The trihydrochloride salt of 1,4,7-triazacyclononane 19 (4.5 mg, 0.0189 mmol), compound 8 (33 mg, 0.067 mmol), and KCO (20 mg, 0.145 mmol) were mixed in anhydrous CHCN (1.5 mL) under argon and heated at 60 °C for 17 h. After this time, the crude mixture was separated from the inorganic salts by filtration. The resulting solution was directly analyzed by reverse-phase HPLC (10–100% CHCN in HO for 10 min, t r =16.5 min) to give a pale yellow oil (8 mg, 32%).

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[0133] Complex 21 [ka] Ligand 20 (8 mg, 6 μmol) was dissolved in a mixture of CHOH / HO (1:1, 2 mL total), and the pH was adjusted to 12 with aqueous NaOH (1.0 M). The solution was heated to 60 °C for 4 h. After cooling and adjusting the pH to 7 with hydrochloric acid (1.0 M), EuCl3.6HO (3 mg, 8 μmol) was added, and the reaction mixture was heated to 60 °C for 19 h. The reaction mixture was analyzed by reverse-phase HPLC (10–100% CH3CN in HO, 10 min, t r =12.4 min) to give a yellow solid (4 mg, 48%).

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[0134] compound 24 [ka] To a mixture of 4-bromo-2-nitrophenol 22 (2.49 g, 11.4 mmol) and K2CO3 (2.31 g, 16.7 mmol) in anhydrous CH3CN (40 mL) was added ethyl 4-bromobutyrate 23 (2.3 mL, 17.2 mmol) under argon. The mixture was heated to 70 °C for 64 h, and then the solvent was removed under reduced pressure. CHCl2 (50 mL) was added to the residue, and the resulting suspension was washed with H2O (5 × 50 mL). The organic layer was dried over K2CO3, and the solvent was removed under reduced pressure to give a crude residue that was purified by column chromatography (SiO2, 1:1 hexane / CHCl2 to 100% CHCl2) to give a pale yellow oil (3.38 g, 98%).

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[0135] compound 25 [ka] Compound 24 (4.30 g, 12.95 mmol), iron powder (3.60 g, 64.5 mmol), and glacial acetic acid (3.7 mL, 64.6 mmol) were mixed in EtOH (20 mL) under argon and heated to 50 °C for 6 h. The mixture was allowed to cool, filtered, and the solvent removed under reduced pressure. DCM (50 mL) was added to the resulting mixture, and the resulting solution was washed with saturated aqueous NaEDTA (3 × 40 mL) and HO (2 × 40 mL). The combined aqueous layers were extracted with CHCl (6 × 40 mL). The combined organic layers were dried over KCO, and the solvent was removed under reduced pressure to give a pale golden oil (3.5 g, 90%).

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[0136] compound 26 [ka] To a solution of compound 25 (3.19 g, 10.6 mmol) and K2CO3 (3.73 g, 27.0 mmol) in anhydrous CH3CN (10 mL) was added iodoethane (4 mL, 50 mmol) under argon. The mixture was heated to 70 °C for 65 h, and then the solvent was removed under reduced pressure. The resulting residue was dissolved in C2Cl2 (50 mL), washed with HO (4 × 50 mL), and then dried over K2CO3. The solvent was removed under reduced pressure to give a crude residue that was purified by column chromatography (SiO2, neat C2Cl2 to 1% CH3OH in C2Cl2) to give a pale red oil (2.51 g, 66%).

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[0137] compound 27 [ka] Compound 26 (3.03 g, 8.46 mmol) and PdCl(allyl) (310 mg, 0.85 mmol) were mixed in a vessel, and the vessel was evacuated and refilled with argon three times. Anhydrous CHCN (15 mL) was then added. To this mixture were added P(t-Bu) (0.31 mL, 1.28 mmol), trimethylsilylacetylene (2.4 mL, 17.3 mmol), and piperidine (2.1 mL, 21.3 mmol), in that order. The reaction mixture was stirred at 34 °C for 30 h. The solvent was then removed under reduced pressure, and the residue was dissolved in CHCl (50 mL). The solution was washed with H0 (4 × 50 mL) and dried over KCO. The solvent was removed under reduced pressure to give a brown oil, which was purified by column chromatography (SiO 2 , 100% hexanes to 6% EtOAc in hexanes) to give a yellow oil (2.29 mg, 75%).

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[0138] compound 28 [ka] Triethylamine trihydrofluoride (6.25 mL, 38 mmol) was added to a solution of compound 27 (960 mg, 2.56 mmol) in anhydrous THF (8 mL) under argon. The solution was stirred at 30 °C for 24 h, and then the solvent was removed under reduced pressure. The residue was then dissolved in CHCl (30 mL) and washed with water (6 × 30 mL). The combined aqueous layers were extracted with CHCl (2 × 30 mL) and then dried over KCO to give a yellow oil (749 mg, 97%). This product was used in the next step without further purification.

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[0139] compound 29 [ka] Compound 28 (528 mg, 1.74 mmol) and ethyl (6-(hydroxymethyl)-4-(bromopyridin-2-yl)(methyl)phosphinate 19 (486 mg, 1.65 mmol) were mixed in anhydrous CHCN (10 mL) under argon. To this solution was added PdCl(allyl) (70 mg, 0.191 mmol), P( t Bu)3 (0.06 mL, 0.25 mmol) and piperidine (0.43 mL, 4.70 mmol) were added in that order. The resulting mixture was stirred under argon at 40 °C for 36 h, then the solvent was removed under reduced pressure. The residue was dissolved in CHCl2 (40 mL), washed with H2O (3 × 40 mL), dried over KCO3, and the solvent was removed under reduced pressure. The crude product was purified by reverse-phase HPLC (10–100% CH3CN in H2O, 10 min, t r =12.2 min) to give a pale orange oil (359 mg, 40%).

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[0140] compound 30 [ka] Compound 29 (250 mg, 0.484 mmol), methanesulfonic anhydride (126 mg, 0.72 mmol), and DIPEA (0.25 mL, 1.44 mmol) were mixed in anhydrous THF (2 mL) under argon. The reaction mixture was stirred at room temperature for 1 h while monitoring the progress of the reaction by TLC. After complete conversion, the solvent was removed under reduced pressure, and the crude residue obtained was dissolved in CHCl (40 mL), washed with water (3 × 40 mL), and the organic layer was dried over KCO. The solvent was removed under reduced pressure to give an orange oil (288 mg, quant.).

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[0141] compound 32 [ka] To a solution of 4-bromo-2-nitrophenol 22 (10 g, 45 mmol) in anhydrous CH3CN (240 mL) was added 3-(Boc-amino)propyl bromide 31 (14.1 g, 58.4 mmol), Cs2CO3 (22.2 g, 67.4 mmol), and NaI (1.7 g, 11.2 mmol) under an inert atmosphere. The reaction mixture was stirred at 70 °C for 16 h, filtered, and washed with CH3CN. The mother liquor was concentrated under reduced pressure. Purification by chromatography on silica gel (cyclohexane / EtOAc 8 / 2, then 7 / 3, then 6 / 4) gave compound 32 as a yellow oil (15.9 g, 94%), which solidified upon standing.

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[0142] compound 33 [ka] To a solution of compound 32 (15.9 g, 42.4 mmol) in dioxane (133 mL) and HO (26 mL) was added zinc (14.1 g, 211 mmol) and NHCI (11.4 g, 211 mmol) at 0 °C. After 15 min at 0 °C, the reaction mixture was stirred at 50 °C for 20 h. It was then filtered through Celite® and washed with EtOAc. The organic layer was washed successively with HO and brine, dried over NaSO, filtered, and concentrated under reduced pressure to give crude compound 33 (14.4 g, 99%) as a brown oil.

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[0143] compound 34 [ka] To a solution of compound 33 (7.3 g, 21.1 mmol) in CHCN (23 mL) in a sealed flask was added KCO (7.38 g, 52.9 mmol) and EtI (8.11 mL, 99.4 mmol). The reaction mixture was stirred at 70 °C for 16 h, filtered through a Buchner funnel, and washed with EtOAc. The mother liquor was washed with water, dried over NaSO, filtered, and concentrated under reduced pressure. Purification by chromatography on silica gel (cyclohexane / EtOAc 8:2) gave compound 34 (7.04 g, 83%) as a slightly yellow oil.

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[0144] compound 35 [ka] A solution of compound 34 (5.4 g, 13.5 mmol) in anhydrous THF (18.9 mL) was degassed with argon in a sealed flask. Triethylamine (18.9 mL, 135 mmol), trimethylsilylacetylene (12.2 mL, 85.4 mmol), CuI (1.09 g, 5.73 mmol), and Pd(PPh3)2Cl2 (2 g, 2.85 mmol) were then added, and the mixture was degassed with argon and then stirred at 65 °C for 16 h. The reaction mixture was filtered through Celite®, washed with EtOAc, and concentrated under reduced pressure. The residue was solubilized in EtOAc, and the organic layer was washed successively with HO and brine, dried over Na2SO4, filtered, and concentrated under reduced pressure. Purification by flash chromatography on silica gel (cyclohexane / EtOAc 10:0 to 8:2) afforded compound 35 (6.4 g) in 80% purity. A second purification by flash chromatography on silica gel (CH2Cl2 / EtOAc 100:0 to 95:5) gave compound 35 as an orange oil (3.5 g, 61%).

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[0145] compound 36 [ka] Triethylamine trihydrofluoride (1.1 mL, 6.75 mmol) was added to a solution of compound 35 (185 mg, 0.442 mmol) in anhydrous THF (5 mL) under argon. The solution was stirred at 30 °C for 38 h, and then the solvent was removed under reduced pressure. The residue was then dissolved in CHCl (30 mL) and washed with water (3 × 30 mL). The combined aqueous layers were extracted with CHCl (2 × 30 mL), and the combined organic layers were dried over KCO to give a pale yellow oil (74 mg, 48%). This product was used in the next step without further purification.

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[0146] compound 37 [ka] Compound 36 (172 mg, 0.496 mmol) and ethyl (6-(hydroxymethyl)-4-(bromopyridin-2-yl)(methyl)phosphinate 6 (139 mg, 0.473 mmol) were mixed in anhydrous CHCN (3 mL) under argon. To this solution were added PdCl(allyl) (20 mg, 0.055 mmol), P(tBu) (0.02 mL, 0.082 mmol), and piperidine (0.12 mL, 1.22 mmol), in that order. The resulting mixture was stirred under argon at 35 °C for 24 h, and then the solvent was removed under reduced pressure. The residue was dissolved in CHCl (40 mL), washed with H0 (3 × 40 mL), and dried over KCO. The solvent was removed under reduced pressure to give a brown oil, which was purified by reverse-phase HPLC (10–100% CHCN, 10 min, t r =11.7 min) to give a pale yellow oil (64 mg, 24%).

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[0147] compound 38 [ka] Compound 37 (40 mg, 0.072 mmol), methanesulfonic anhydride (25 mg, 0.143 mmol), and DIPEA (0.025 mL, 0.025 mmol) were mixed in anhydrous THF (2 mL) under argon. The reaction mixture was stirred at room temperature for 90 minutes, and then the solvent was removed under reduced pressure. The resulting residue was dissolved in CHCl (40 mL) and washed with water (3 × 40 mL), and the combined aqueous layers were extracted with CHCl (2 × 40 mL). The combined organic layers were dried over KCO, and the solvent was removed under reduced pressure to give a yellow oil (45 mg, 100%).

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[0148] compound 39 [ka] To a solution of compound 24 (3.50 g, 11.6 mmol) and KCO (4.8 g, 34.5 mmol) in anhydrous CHCN (3 mL) under argon, iodoethane (1.21 mL, 15.0 mmol) was added. The mixture was heated to 55 °C for 70 h, filtered to remove inorganic salts, and the solvent was then removed under reduced pressure. The resulting oil was purified by column chromatography (SiO, 100% CHCl) to give a pale yellow oil (1.65 g, 43%).

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[0149] compound 40 [ka] Compound 39 (939 mg, 2.84 mmol), paraformaldehyde (415 mg, 4.61 mmol), and a few drops of acetic acid were mixed in anhydrous EtOH (25 mL). The mixture was stirred at room temperature under argon for 20 minutes, at which point NaBHCN (640 mg, 10 mmol) was added, and the reaction mixture was stirred for an additional 48 hours. After the solvent was removed under reduced pressure, the resulting residue was dissolved in CHCl (30 mL) and washed with NaHCO solution (1 × 30 mL) and water (3 × 30 mL). The organic layer was dried over KCO, and the solvent was removed under reduced pressure to give a pale yellow oil (833 mg, 85%).

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[0150] compound 41 [ka] Compound 40 (1.47 g, 4.27 mmol) and PdCl(allyl) (160 mg, 0.44 mmol) were mixed in a vessel, and the vessel was evacuated and refilled with argon three times. Anhydrous CHCN (8 mL) was then added. To this mixture were added P(t-Bu) (0.16 mL, 0.66 mmol), trimethylsilylacetylene (1.2 mL, 8.66 mmol), and piperidine (1.0 mL, 10.1 mmol), in that order. The reaction mixture was stirred at 35 °C for 19 h. The solvent was then removed under reduced pressure, and the residue was dissolved in CHCl (50 mL). The solution was washed with HO (2 × 50 mL) and dried over KCO. The solvent was removed under reduced pressure to give a brown oil, which was purified by column chromatography (SiO2, 100% hexane to 7% EtOAc in hexane) to give the expected compound as a pale orange oil (1.19 g, 77%).

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[0151] compound 42 [ka] Triethylamine trihydrofluoride (3.6 mL, 22.0 mmol) was added to a solution of compound 41 (530 mg, 1.47 mmol) in anhydrous THF (5 mL) under argon. The solution was stirred at 30 °C for 17 h, and then the solvent was removed under reduced pressure. The residue was then dissolved in CHCl (30 mL) and washed with water (3 × 30 mL). The combined aqueous layers were extracted with CHCl (2 × 30 mL), and the combined organic layers were dried over KCO to give a yellow oil (410 mg, 97%). This product was used in the next step without further purification.

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[0152] compound 43 [ka] Compound 42 (396 mg, 1.37 mmol) and ethyl (6-(hydroxymethyl)-4-(bromopyridin-2-yl)(methyl)phosphinate 19 (362 mg, 1.23 mmol) were mixed in anhydrous CHCN (10 mL) under argon. To this solution was added PdCl(allyl) (50 mg, 0.137 mmol), P( t Bu)3 (0.05 mL, 0.21 mmol) and piperidine (0.4 mL, 4.37 mmol) were added in that order. The resulting mixture was stirred under argon at 40 °C for 36 h, and then the solvent was removed under reduced pressure. The residue was dissolved in CHCl2 (40 mL), washed with H2O (3 × 40 mL), dried over KCO3, and the solvent was removed under reduced pressure to give an orange oil, which was purified by reverse-phase HPLC (10–100% CH3CN, 10 min, t r =13.2 min) to give a pale orange oil (185 mg, 30%).

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[0153] compound 44 [ka] Compound 43 (160 mg, 0.318 mmol), methanesulfonic anhydride (111 mg, 0.637 mmol), and DIPEA (0.11 mL, 0.632 mmol) were mixed in anhydrous THF (4 mL) under argon. The reaction mixture was stirred at room temperature for 1 h, at which point the solvent was removed under reduced pressure. The resulting crude residue was dissolved in CHCl (40 mL) and washed with water (3 × 40 mL), and the combined aqueous layers were extracted with CHCl (3 × 40 mL). The combined organic layers were dried over KCO, and the solvent was removed under reduced pressure to give an orange oil (167 mg, 90%).

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[0154] Compounds 45a-d are commercially available products.

[0155] Compound 46a was prepared according to the procedure described in Angew Chem Int Ed 2018,57 5110 (see supplementary material) for the formation of N-Boc piperazine derivatives.

[0156] Compound 46b was prepared according to the procedure described in WO2005 / 075410 for the formation of N-methylpiperazine derivatives (see Example 5).

[0157] Compound 46c was prepared according to the procedure described in Journal Heterocyclic chemistry 2013, 50, 995.

[0158] Compound 46d was prepared as per the procedure described in Organic Biomolecular & Chemistry 2010, 8, 4077 (see supplementary material).

[0159] Compounds 47a-d were prepared as described for compound 4.

[0160] Compounds 48a-d were prepared as described for compound 5.

[0161] Compounds 49a-d were prepared as described for compound 7.

[0162] Compounds 50a-d were prepared as described for compound 8.

[0163] compound 51 [ka] Oxalyl chloride (0.75 mL, 8.75 mmol) was added dropwise to a mixture of 3-bromopropanesulfonic acid sodium salt (1 g, 4.44 mmol), anhydrous CHCN (4 mL), and anhydrous DMF (0.1 mL) cooled to 0 °C under argon. The mixture was stirred at 0 °C for 2 h, then the solvent was removed under reduced pressure (bath temperature <20 °C). The resulting residue was dried under high vacuum. To this residue was added anhydrous CHCN (4 mL) and 2,2,2-trifluoroethanol (9 mL, 125 mmol) under argon. Triethylamine (3 mL, 21.5 mmol) was added dropwise while cooling to 0 °C. The mixture was stirred at 0 °C for 5 h, then the solvent was removed under reduced pressure. Ethyl acetate (40 mL) and HO (40 mL) were added to the crude residue, and the organic layer was washed with HO (3 × 40 mL). The organic layer was dried over Na2SO4 and the solvent was removed under reduced pressure to give a pale orange oil (1.1 g, 87%).

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[0164] compound 52 [ka] 5-Iodo-o-anisidine (245 mg, 0.984 mmol), compound 51 (1.1 g, 3.86 mmol), KCO (425 mg, 3.08 mmol), and NaI (40 mg, 0.27 mmol) were mixed in anhydrous CHCN (5 mL) under argon. The resulting mixture was heated to 78 °C for 72 h, and then the solvent was removed under reduced pressure. DCM (40 mL) and HO (40 mL) were added to the crude residue, and the organic layer was washed with HO (2 × 40 mL). The organic layer was dried over KCO, and the solvent was removed under reduced pressure to give the crude product as a light brown residue, which was purified by column chromatography (SiO, 100% hexanes to 20% EtOAc in hexanes) to give a pale yellow oil (283 mg, 63%).

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[0165] Compound 53a [ka] Compound 52 (310 mg, 0.684 mmol) and acetaldehyde (0.46 mL, 8.16 mmol) were mixed with two drops of glacial acetic acid in anhydrous EtOH (3 mL) under argon. The resulting mixture was stirred at room temperature for 15 min, and then NaBHCN (170 mg, 2.71 mmol) was added. The mixture was stirred again at room temperature under argon for 16 h, after which the solvent was removed under reduced pressure to give a yellow residue. DCM (40 mL) and HO (40 mL) were added to the residue, and the organic layer was washed with HO (3 × 40 mL). The combined aqueous layers were extracted with DCM (1 × 40 mL), and the combined organic layers were dried over KCO. The solvent was removed under reduced pressure to give a pale yellow oil (247 mg, 75%).

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[0166] Compound 53b [ka] Compound 52 (269 mg, 0.594 mmol) and paraformaldehyde (800 mg, 8.89 mmol) were mixed with two drops of glacial acetic acid in anhydrous EtOH (8 mL) under argon. The resulting mixture was stirred at room temperature for 15 min, and then NaBHCN (300 mg, 4.77 mmol) was added. The mixture was stirred again at room temperature under argon for 18 h, after which the solvent was removed under reduced pressure to give a yellow residue. DCM (40 mL) and HO (40 mL) were added to the residue, and the organic layer was washed with HO (3 × 40 mL). The combined aqueous layers were extracted with DCM (2 × 40 mL), and the combined organic layers were dried over KCO. The solvent was removed under reduced pressure to give a pale yellow oil (249 mg, 90%).

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[0167] Compound 54a [ka] Compound 53a (176 mg, 0.366 mmol) and trimethylsilylacetylene (0.1 mL, 0.72 mmol) were mixed in anhydrous CHCN (3 mL) under argon. To this solution was added PdCl(allyl) (14 mg, 0.038 mmol), P( t Bu)3 (0.015 mL, 0.062 mmol) and piperidine (0.09 mL, 0.91 mmol) were added in that order. The resulting mixture was stirred under argon at 35 °C for 20 h, then the solvent was removed under reduced pressure to give the crude product as a brown residue, which was purified by column chromatography (SiO2, 100% hexanes to 20% EtOAc in hexanes) to give a pale orange oil (151 mg, 91%).

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[0168] Compound 54b [ka] Compound 53b (240 mg, 0.514 mmol) and trimethylsilylacetylene (0.14 mL, 1.01 mmol) were mixed in anhydrous CHCN (2.5 mL) under argon. To this solution was added PdCl(allyl) (20 mg, 0.055 mmol), P( t Bu)3 (0.013 mL, 0.054 mmol) and piperidine (0.13 mL, 1.32 mmol) were added in that order. The resulting mixture was stirred under argon at 35 °C for 16 h, then the solvent was removed under reduced pressure to give the crude product as a brown residue, which was purified by column chromatography (SiO2, 100% hexanes to 20% EtOAc in hexanes) to give a pale orange oil (157 mg, 70%).

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[0169] Compound 55a [ka] Triethylamine trihydrofluoride (3 mL, 6.75 mmol) was added to a solution of compound 54a (341 mg, 0.755 mmol) in anhydrous THF (5 mL) under argon. The solution was stirred at 30 °C for 72 h, and then the solvent was removed under reduced pressure. The residue was dissolved in CHCl (30 mL) and washed with water (4 × 30 mL). The combined aqueous layers were extracted with CHCl (1 × 30 mL), and the combined organic layers were dried over KCO to give a light brown oil (207 mg, 72%) after solvent evaporation. This product was used in the next step without further purification.

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[0170] Compound 55b [ka] Triethylamine trihydrofluoride (1.1 mL, 6.75 mmol) was added to a solution of compound 54b (195 mg, 0.446 mmol) in anhydrous THF (3 mL) under argon. The solution was stirred at 30 °C for 48 h, and then the solvent was removed under reduced pressure. The residue was dissolved in CHCl (30 mL) and washed with water (3 × 30 mL). The combined aqueous layers were extracted with CHCl (2 × 30 mL), and the combined organic layers were dried over KCO to give a light brown oil (160 mg, 98%). This product was used in the next step without further purification.

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[0171] Compound 56a [ka] Compound 55a (207 mg, 0.546 mmol) and ethyl (6-(hydroxymethyl)-4-(bromopyridin-2-yl)(methyl)phosphinate 6 (152 mg, 0.517 mmol) were mixed in anhydrous CHCN (5 mL) under argon. To this solution was added PdCl(allyl) (20 mg, 0.055 mmol), P( t Bu)3 (0.02 mL, 0.082 mmol) and piperidine (0.11 mL, 1.11 mmol) were added in that order. The resulting mixture was stirred under argon at 35 °C for 18 h, then the solvent was removed under reduced pressure. The residue was dissolved in CHCl2 (40 mL) and washed with H2O (3 × 40 mL), and the combined aqueous layers were extracted with CHCl2 (2 × 40 mL). The combined organic layers were dried over KCO3, and the solvent was removed under reduced pressure to give an orange oil, which was purified by reverse-phase HPLC (10–100% CH3CN, 10 min, t r =11.3 min) to give a pale yellow oil (111 mg, 36%).

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[0172] Compound 56b [ka] Compound 55b (160 mg, 0.438 mmol) and ethyl (6-(hydroxymethyl)-4-(bromopyridin-2-yl)(methyl)phosphinate 6 (116 mg, 0.395 mmol) were mixed in anhydrous CHCN (3 mL) under argon. To this solution was added PdCl(allyl) (16 mg, 0.044 mmol), P( t Bu)3 (0.016 mL, 0.066 mmol) and piperidine (0.09 mL, 0.92 mmol) were added in that order. The resulting mixture was stirred under argon at 35 °C for 16 h, and then the solvent was removed under reduced pressure. The residue was dissolved in CHCl2 (40 mL) and washed with H2O (3 × 40 mL), and the combined aqueous layers were extracted with CHCl2 (2 × 40 mL). The combined organic layers were dried over KCO3, and the solvent was removed under reduced pressure to give a brown oil, which was purified by reverse-phase HPLC (10–100% CH3CN, 10 min, t r =10.8 min) to give a pale yellow oil (119 mg, 52%).

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[0173] Compound 57a [ka] Compound 56a (110 mg, 0.186 mmol), methanesulfonic anhydride (65 mg, 0.373 mmol), and DIPEA (0.065 mL, 0.373 mmol) were mixed in anhydrous THF (3 mL) under argon. The reaction mixture was stirred at room temperature for 2 h, and then the solvent was removed under reduced pressure. The resulting residue was dissolved in CHCl (40 mL) and washed with water (3 × 40 mL), and the organic layer was dried over KCO. The solvent was removed under reduced pressure to give a yellow oil (125 mg, 100%).

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[0174] Compound 57b [ka] Compound 56b (119 mg, 0.206 mmol), methanesulfonic anhydride (72 mg, 0.413 mmol), and DIPEA (0.072 mL, 0.413 mmol) were mixed in anhydrous THF (3 mL) under argon. The reaction mixture was stirred at room temperature for 2 hours, and then the solvent was removed under reduced pressure. The resulting residue was dissolved in CHCl (40 mL) and washed with water (3 × 40 mL), and the organic layer was dried over KCO. The solvent was removed under reduced pressure to give a clear, colorless oil (88 mg, 65%).

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[0175] Compound 58 (E,Z mixture) [ka] Compound 6 (74 mg, 0.25 mmol), tert-butyl N-allylcarbamate (155 mg, 0.99 mmol), palladium(II) acetate (11 mg, 0.05 mmol), and triphenylphosphine (20 mg, 0.08 mmol) were mixed in toluene (2 mL) under an argon atmosphere. The reaction mixture was degassed by bubbling argon through the solution for 15 min. Triethylamine (0.3 mL, 2.2 mmol) was added, and the mixture was heated to 80 °C under argon for 18 h. After cooling, the solvent was removed under reduced pressure, and the resulting residue was dissolved in CHCl (30 mL), washed with H0 (4 × 30 mL), and dried over MgSO. Removal of the solvent under reduced pressure gave the crude product, which was purified by reverse-phase HPLC (10–100% CHCN in H0, 10 min, t r =9.1 min) to give a yellow oil as a mixture of E / Z isomers (93 mg, 63%).

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[0176] compound 59 [ka] In a vessel, the E / Z isomer mixture of compound 58 (210 mg, 0.57 mmol) was dissolved in EtOH (60 mL) with Pd / C (10% Pd content, 10 mg). The vessel was then loaded into a Parr hydrogenation apparatus (40 bar H2 pressure) and the reaction mixture was stirred for 8 h. After this time, the catalyst was removed by filtration and the solvent was removed under reduced pressure to give a pale yellow oil (211 mg, quant.).

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[0177] compound 60 [ka] Compound 59 (25 mg, 0.067 mmol), methanesulfonic anhydride (20 mg, 0.115 mmol), and DIPEA (0.03 mL, 0.172 mmol) were mixed in anhydrous THF (0.5 mL) under argon. The reaction mixture was stirred at room temperature for 1 h while being monitored by TLC. After complete conversion, the solvent was removed under reduced pressure, and the crude residue obtained was dissolved in CHCl (40 mL), washed with water (3 × 40 mL), and the organic layer was dried over KCO. The solvent was removed under reduced pressure to give a clear oil (30 mg, quant.).

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[0178] Compounds 61, 62 and 63 were prepared according to the procedure described in WO2018 / 229408 for carboxylate derivatives.

[0179] Compound 64 was synthesized as described for compound 13.

[0180] Complex 65 was synthesized as described for complex 14.

[0181] Compound 66 was synthesized as described for compound 11.

[0182] Compound 67 was synthesized as described for compound 12.

[0183] Compound 68 was synthesized as described for compound 13.

[0184] Complex 69 was synthesized as described for complex 14.

[0185] Complex 70 was synthesized as described for complexes 78a-b.

[0186] Complex 71 was synthesized as described for complexes 99a-b.

[0187] Compound 72 was synthesized as described for compound 13.

[0188] Complex 73a was synthesized as described for complex 14.

[0189] Complex 73b was synthesized as described for complexes 78a-b.

[0190] Compound 74a [ka] 1-tert-Butoxycarbonyl-1,4,7-triazacyclononane dihydrochloride (66 mg, 0.218 mmol), mesylate 30 (288 mg, 0.484 mmol), and K2CO3 (122 mg, 0.833 mmol) were mixed in anhydrous CH3CN (2.5 mL) under argon. The resulting mixture was heated to 70 °C for 16 h and then cooled to room temperature. The solution was separated from the inorganic salts by filtration, and the solvent was removed under reduced pressure to give the crude product as a viscous residue. This was purified by reverse-phase HPLC (10–100% CH3CN in HO for 10 min, t r =17.5 min) to give an orange oil (160 mg, 27%).

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[0191] Compound 74b [ka] 1-tert-Butoxycarbonyl-1,4,7-triazacyclononane dihydrochloride (38 mg, 0.126 mmol), mesylate 44 (167 mg, 0.288 mmol), and KCO (104 mg, 0.753 mmol) were mixed in anhydrous CHCN (4 mL) under argon. The resulting mixture was heated to 60 °C for 48 h and then cooled to room temperature. The solution was separated from the inorganic salts by filtration, and the solvent was removed under reduced pressure to give the crude product. This was purified by reverse-phase HPLC (10–100% CHCN in HO for 10 min, t r =12.2 min) to give a pale orange oil (110 mg, 73%).

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[0192] compound 75a [ka] A solution of compound 74a (19 mg, 0.016 mmol) in trifluoroacetic acid and CHCl was prepared (10% v / v, 3 mL total) and stirred under argon for 40 minutes. Immediately after this, the solvent was removed under reduced pressure. Additional CHCl (approximately 50 mL) was added and the solvent was removed under reduced pressure. This procedure was repeated three times. The residue was dissolved in CHCl (30 mL), washed with aqueous NaCO (4%, pH = 11, 3 × 30 mL), and dried over KCO. The solvent was removed under reduced pressure to give an orange oil (17 mg, quant.).

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[0193] Compound 75b [ka] A solution of compound 74b (110 mg, 0.092 mmol) in trifluoroacetic acid and CHCl was prepared (10% v / v, 3 mL total) and stirred under argon for 60 minutes. The solvent was removed under reduced pressure, and additional CHCl (approximately 50 mL) was added, and the solvent was removed under reduced pressure. This process was repeated four times. The residue was dissolved in CHCl (30 mL), washed with aqueous NaCO (4%, pH = 11, 3 × 30 mL), and dried over KCO. The solvent was removed under reduced pressure to give an orange oil (111 mg, quant.).

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[0194] Compound 76a [ka] Compound 75a (43 mg, 0.038 mmol), compound 60 (45 mg, 0.10 mmol), and KCO (30 mg, 0.22 mmol) were mixed in anhydrous CHCN (2 mL) under argon and heated to 70 °C for 18 h, after which LCMS analysis confirmed complete conversion of the macrocycle. The reaction mixture was allowed to cool and then separated from the inorganic salts by filtration. The solvent was removed under reduced pressure to give an orange solid (88 mg), which was used directly in the next step without further purification.

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[0195] Compound 76b [ka] Compound 75b (111 mg, 0.0919 mmol), compound 60 (59 mg, 0.130 mmol), and KCO (45 mg, 0.33 mmol) were mixed in anhydrous CHCN (3 mL) under argon and heated to 65 °C for 16 h. After this time, LCMS analysis confirmed complete conversion of the macrocycle. The reaction mixture was allowed to cool, and the solution was separated from the inorganic salts by filtration. The solvent was removed under reduced pressure to give an orange oil (165 mg), which was used directly in the next step without further purification.

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[0196] Complex 77a [ka] The crude ligand 76a (88 mg) from the previous step was dissolved in a mixture of CHOH / HO (4:1, 2.5 mL total) and the pH was adjusted to 12 with aqueous NaOH. The solution was heated to 60 °C for 1.5 h, after which LCMS analysis confirmed complete hydrolysis of the phosphinate and chromophore head ester groups. After cooling and adjusting the pH to 7 with hydrochloric acid (0.1 M), EuCl3.6HO (42 mg, 0.115 mmol) was added and the reaction mixture was heated to 60 °C for 18 h. The reaction mixture was analyzed by reverse-phase HPLC (10–100% CH3CN in HO for 10 min, t r =8.5 min) to give a yellow solid (22 mg, 39% over three steps).

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[0197] Complex 77b [ka] The crude ligand 76b (165 mg) from the previous step was dissolved in a mixture of CHOH / HO (1:1, 2 mL total) and the pH was adjusted to 12 with aqueous NaOH. The solution was heated to 60 °C for 1.5 h, after which LCMS analysis confirmed complete hydrolysis of the phosphinate and chromophore head ester groups. After cooling and adjusting the pH to 7 with hydrochloric acid (0.1 M), EuCl.6HO (34 mg, 0.093 mmol) was added and the reaction mixture was heated to 60 °C for 17 h. The reaction mixture was analyzed by reverse-phase HPLC (10–100% CHCN in HO for 10 min, t r =8.7 min) to give a yellow solid (50 mg, 37% over three steps).

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[0198] Complex 78a [ka] Complex 77a (3.9 mg, 2.6 μmol) and DIPEA (6 μL, 34 μmol) were mixed in anhydrous DMSO (0.4 mL) under argon. To this solution were added HATU (5 mg, 13 μmol), homotaurine (2 mg, 14 μmol), and water (40 μL). The mixture was stirred at room temperature for 19 h. After dilution with water, the reaction mixture was analyzed by reverse-phase HPLC (10–100% CHOH in HO for 10 min, t r =11.6 min) to give a yellow solid (4 mg, 89%).

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[0199] Complex 78b [ka] Complex 77b (20 mg, 13.7 μmol) and DIPEA (15 μL, 85 μmol) were mixed in anhydrous DMSO (0.5 mL) under argon. To this solution, HATU (12 mg, 31 μmol), homotaurine (4.5 mg, 31 μmol), and water (50 μL) were added, and the mixture was stirred at room temperature for 16 h. After dilution with water, the reaction mixture was analyzed by reverse-phase HPLC (10–100% CHOH in HO for 10 min, t r =10.6 min) to give a yellow solid (9 mg, 38%).

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[0200] Compound 80 was synthesized as described for compound 11.

[0201] Compound 81 was synthesized as described for compound 12.

[0202] Compound 82 was synthesized as described for compound 17.

[0203] Complex 83 was synthesized as described for complex 18.

[0204] Complex 84 was functionalized using the method described in WO2004 / 113275.

[0205] Compound 85a [ka] The dihydrochloride salt 9 (24 mg, 0.0794 mmol) of 1-(tert-butoxycarbonyl)-1,4,7-triazacyclononane, mesylate 57a (125 mg, 0.186 mmol), and KCO (44 mg, 0.32 mmol) were mixed in anhydrous CHCN (3 mL) under argon. The reaction mixture was heated to 65 °C for 16 h, after which the crude solution was separated from the inorganic salts and the solvent removed under reduced pressure to give an orange oil, which was purified by reverse-phase HPLC (10–100% CHCN in HO containing 0.1% formic acid, 10 min, t r=13.5 min) to give a pale yellow oil (88 mg, 80%).

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[0206] Compound 85b [ka] The dihydrochloride salt 9 (18 mg, 0.06 mmol) of 1-(tert-butoxycarbonyl)-1,4,7-triazacyclononane, mesylate 57b (88 mg, 0.134 mmol), and KCO (35 mg, 0.25 mmol) were mixed in anhydrous CHCN (3 mL) under argon. The resulting mixture was heated to 65 °C for 18 h, after which the crude solution was separated from the inorganic salts and the solvent removed under reduced pressure to give an orange oil, which was purified by reverse-phase HPLC (10–100% CHCN in HO for 10 min, t r =14.2 min) to give a pale yellow oil (58 mg, 72%).

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[0207] Compound 86a [ka] A solution of compound 85a (88 mg, 0.064 mmol) in trifluoroacetic acid and CHCl was prepared (10% v / v, 3 mL total) and stirred under argon for 60 minutes. Immediately after this, the solvent was removed under reduced pressure. Additional CHCl (approximately 50 mL) was added and the solvent was removed under reduced pressure. This procedure was repeated three times. Drying under high vacuum for several hours gave an orange oil (88 mg, quant.).

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[0208] Compound 86b [ka] A solution of compound 85b (29 mg, 0.021 mmol) in trifluoroacetic acid and CHCl was prepared (10% v / v, 3 mL total) and stirred under argon for 60 minutes. Immediately after this, the solvent was removed under reduced pressure. Additional CHCl (approximately 50 mL) was added and the solvent was removed under reduced pressure. This procedure was repeated three times. Drying under high vacuum for several hours yielded an orange oil (29 mg, quant.).

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[0209] Compound 87a [ka] Compound 86a (44 mg, 0.032 mmol), compound 60 (30 mg, 0.067 mmol), and KCO (30 mg, 0.22 mmol) were mixed in anhydrous CHCN (2 mL) under argon and heated to 60 °C for 15 h. After this time, LC-MS analysis confirmed complete conversion of the macrocycle. The reaction mixture was allowed to cool, and the solution was separated from the inorganic salts by filtration. The solvent was removed under reduced pressure to give a yellow oil (72 mg), which was used directly in the next step without further purification.

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[0210] Compound 87b [ka] Compound 86b (29 mg, 0.021 mmol), compound 60 (19 mg, 0.042 mmol), and KCO (20 mg, 0.15 mmol) were mixed in anhydrous CHCN (2 mL) under argon and heated to 60 °C for 15 h. After this time, LCMS analysis confirmed complete conversion of the macrocycle. The reaction mixture was allowed to cool, and the solution was separated from the inorganic salts by filtration. The solvent was removed under reduced pressure to give a yellow oil (47 mg), which was used directly in the next step without further purification.

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[0211] Complex 88a [ka] Crude ligand 87a (57 mg) was dissolved in a mixture of CH3OH / aqueous NaOH (0.1 M, 3:1, 2 mL total). The solution was heated to 70 °C for 24 h, after which LCMS analysis confirmed complete hydrolysis of the phosphinate and sulfonate ester groups. After cooling and adjusting the pH to 6 with dilute hydrochloric acid (0.1 M), EuCl3.6HO (14 mg, 0.038 mmol) was added, and the reaction mixture was heated to 75 °C for 24 h. The reaction mixture was analyzed by reverse-phase HPLC (10–100% CH3CN in HO for 10 min, t r =7.9 min) to give a pale yellow solid (13 mg, 27% over two steps).

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[0212] Complex 88b [ka] Crude ligand 87b (23 mg) was dissolved in a mixture of CH3OH / aqueous NaOH (0.1 M, 3:1, 2 mL total). The solution was heated to 70 °C for 48 h, after which LCMS analysis confirmed complete hydrolysis of the phosphinate and sulfonate ester groups. After cooling and adjusting the pH to 5.5 with hydrochloric acid (0.1 M), EuCl3.6HO (8 mg, 0.022 mmol) was added, and the reaction mixture was heated to 75 °C for 24 h. The reaction mixture was analyzed by reverse-phase HPLC (10–100% CH3CN in HO for 10 min, t r =7.9 min) to give a dark yellow solid (1.8 mg, 11% over two steps).

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[0213] Compound 89 was synthesized as described for compound 92.

[0214] Compound 90 was synthesized as described for compound 93.

[0215] Compound 91 was synthesized as described for compound 78a.

[0216] compound 92 [ka] Compound 85a (44 mg, 0.032 mmol), compound 38 (31 mg, 0.049 mmol), and KCO (30 mg, 0.22 mmol) were mixed in anhydrous CHCN (2 mL) under argon and heated to 60 °C for 14 h. After this time, LCMS analysis confirmed complete conversion of macrocycle 85a. The reaction mixture was allowed to cool, and the solution was separated from inorganic salts by filtration. The solvent was removed under reduced pressure to give a yellow oil (47 mg), which was used directly in the next step without further purification.

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[0217] Complex 93 [ka] Crude ligand 92 (38 mg) was dissolved in a mixture of CH3OH / aqueous NaOH (0.1 M, 3:1, 2 mL total). The solution was heated to 70 °C for 24 h, after which LCMS analysis confirmed complete hydrolysis of the phosphinate and sulfonate ester groups. After cooling and adjusting the pH to 6.5 with hydrochloric acid (0.1 M), EuCl3.6HO (14 mg, 0.038 mmol) was added, and the reaction mixture was heated to 75 °C for 24 h. The reaction mixture was analyzed by reverse-phase HPLC (10–100% CH3CN in HO for 10 min, t r =8.8 min) to give a pale yellow solid (11 mg, 25% over two steps).

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[0218] Compound 94 was obtained according to the procedure described in US Pat. No. 9,981,967.

[0219] Compound 95 was prepared as described for compound 20.

[0220] Complex 96 was prepared as described for complex 21.

[0221] Complex 97 was prepared as described for complex 78a.

[0222] Complex 98 was prepared as described for complex 99a.

[0223] Complex 99a [ka] To complex 78a (6.93 mg, 4 μmol) was added trifluoroacetic acid (200 μL). The mixture was stirred at room temperature for 1 h and then purified by preparative HPLC (Waters Xbridge C column). 18, 5μm, 20×100mm-A / H2O 25mM TEAAc pH7 B / CH3CN t=0min 2%Bt=18min 40%B-20mL.min -1 ), yielding a yellow solid (3.5 μmol, 87%).

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[0224] Complex 99b [ka] To complex 78b (9 mg, 5283 nmol) was added trifluoroacetic acid (1 mL). The mixture was stirred at room temperature for 1 h and then purified by preparative HPLC (Waters Xbridge C column). 18 , 5μm, 20×100mm-A / H2O 25mM TEAAc pH7 B / CH3CN t=0min 2%Bt=18min 40%B-20mL.min -1 ), yielding a yellow solid (4256 nmol, 81%).

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[0225] BG-MB-NHS (Structure Scheme 26)

[0226] The compound was obtained as described in Inorganic Chemistry-2014-53-1854.

[0227] Complex 100a [ka] A solution of BG-MB-NHS (0.615 mg, 1 μmol) in dry DMSO (100 μL) was added to complex 99a (1.403 mg, 860 nmol). DIPEA (0.5 μL, 2862 nmol) was added to the resulting solution, and the mixture was stirred at room temperature for 1 h and purified by preparative HPLC (Waters Xbridge C column). 18, 5μm, 20×100mm-A / H2O 25mM TEAAc pH7 B / CH3CN t=0min 2%Bt=18min 40%B-20mL.min -1 ), yielding a white powder (370 nmol, 43%).

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[0228] Complex 100b A solution of BG-MB-NHS (0.738 mg, 1.20 μmol) in dry DMSO (430 μL) was added to complex 99b (1.924 mg, 1.2 μmol). DIPEA (0.5 μL, 2862 nmol) was added to the resulting solution, and the mixture was stirred at room temperature for 1 h and purified by preparative HPLC (Waters Xbridge C column). 18 , 5μm, 20×100mm-A / H2O 25mM TEAAc pH7 B / CH3CN t=0min 2%Bt=18min 40%B-20mL.min -1 ), a white powder was obtained (0.728 μmol, yield 60%).

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[0229] Complex 101a [ka] To a solution of complex 99a (1.403 mg, 860 nmol) in dry DMSO (100 μL) was added a solution of DIPEA (0.150 μL, 860 nmol) and 6-maleimidohexanoic acid N-hydroxysuccinimide ester (0.271 mg, 860 nmol) in anhydrous DMSO (5 μL). The mixture was stirred at room temperature for 1 h and purified by preparative HPLC (Waters Xbridge C column). 18 , 5μm, 20×100mm-A / H2O 25mM TEAAc pH5 B / CH3CN t=0min 2%Bt=18min 40%B-20mL.min -1), a yellow powder was obtained (761 nmol, 89% yield).

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[0230] Complex 101b [ka] To a solution of complex 99b (1.283 mg, 800 nmol) in dry DMSO (100 μL) was added a solution of DIPEA (0.140 μL, 800 nmol) and 6-maleimidohexanoic acid N-hydroxysuccinimide ester (0.252 mg, 800 nmol) in anhydrous DMSO (5 μL). The mixture was stirred at room temperature for 1 h and purified by preparative HPLC (Waters Xbridge C column). 18 , 5μm, 20×100mm-A / H2O 25mM TEAAc pH5 B / CH3CN t=0min 2%Bt=18min 40%B-20mL.min -1 ), a yellow powder was obtained (726 nmol, 91% yield).

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[0231] Compound 102 was synthesized according to the procedure described in Helvetica Chimica Acta, 1993, 76, 877.

[0232] compound 103 [ka] Compound 36 (74 mg, 0.214 mmol) and compound 102 (140 mg, 0.195 mmol) were mixed in anhydrous CHCN (2.5 mL) under argon. To this solution was added PdCl(allyl) (10 mg, 0.027 mmol), P( tBu) (0.01 mL, 0.041 mmol) and piperidine (0.06 mL, 0.66 mmol) were added in that order. The resulting mixture was stirred under argon at 40 °C for 17 h, and then the solvent was removed under reduced pressure. The crude product was purified by reverse-phase HPLC (10-100% CHCN in HO, t r =19.8 min) to give a pale orange oil (73 mg, 40%).

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[0233] Complex 104 [ka] A solution of complex 102 (36 mg, 0.038 mmol) in trifluoroacetic acid and CHCl (20% v / v, 4 mL total) was prepared. The solution was stirred at room temperature for 6 h, and then the solvent was removed under reduced pressure to give an orange residue. This residue was purified by reverse-phase HPLC (10–100% CHCN in H0, 10 min, t r The solution was purified by reverse-phase HPLC (10-100% CHCN in HO for 10 min, t = 1.2 min) to give the free amine, which was dissolved in HO (3 mL), adjusted to pH 6.5, and then EuCl3.6HO (14 mg, 0.038 mmol) was added. The mixture was heated to 60 °C for 24 h, and then the solution was separated from the inorganic salts by filtration. The solution was purified by reverse-phase HPLC (10-100% CH3CN in HO for 10 min, t r =1.7 min) to give a yellow solid (27 mg, 93% over two steps).

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[0234] Complex 105 [ka] To a solution of complex 104 (0.655 mg, 860 nmol) in dry DMSO (100 μL) was added a solution of DIPEA (0.150 μL, 860 nmol) and 6-maleimidohexanoic acid N-hydroxysuccinimide ester (0.271 mg, 860 nmol) in anhydrous DMSO (5 μL). The mixture was stirred at room temperature for 1 h and purified by preparative HPLC (Waters Xbridge C column). 18 , 5μm, 20×100mm-A / H2O 25mM TEAAc pH6 B / CH3CN t=0min 5%Bt=19min 40%B-20mL.min -1 ), yielding a yellow powder.

number

[0235] Complex 106 [ka] A solution of BG-MB-NHS (783 μg, 1274 nmol) in dry DMSO (430 μL) was added to complex 104 (970 μg, 1274 nmol). DIPEA (0.5 μL, 2862 nmol) was added to the resulting solution, and the mixture was stirred at room temperature for 1 h and purified by preparative HPLC (Waters Xbridge C column). 18 , 5μm, 20×100mm-A / H2O 25mM TEAAc pH6 B / CH3CN t=0min 5%Bt=19min 40%B-20mL.min -1 ), a white powder was obtained (0.728 μmol, 60% yield).

number

[0236] Representative complexes of the present invention (complexes 14, 18, 21, 78a, 78b, 88a, 88b, 93, and 104) were characterized. The presence of the Boc group was not expected to alter these properties. For this reason, experiments were performed using Boc groups in functionalized complexes. Absorption and emission spectra were recorded over a pH gradient (from pH 4 to pH 10).

[0237] 1) Photophysical properties The photophysical properties are summarized in Tables 1 and 2. [Table 1]

[0238] Traditionally, the molar extinction coefficient ε is calculated using the Beer-Lambert equation, which involves measuring the absorbance of a series of solutions of the complex at different known concentrations. A plot of absorbance at a specific wavelength against concentration allows the determination of ε from the slope of the linear correlation. The quantum yield (QY) of a complex increases as the pH decreases. For complexes 78b, 88a, 88b, and 93, the quantum yield at pH 8 approaches 0, thus indicating that the complexes are barely luminescent at physiological pH; conversely, these complexes are very bright at pH 4.

[0239] Rather unexpectedly, an isosbestic point was observed at 332 nm. This may be useful because excitation at or near this wavelength allows equivalent excitation of both the unprotonated and protonated complexes, which exist in a pH-dependent equilibrium in solution. No isosbestic point was observed for complex 14.

[0240] [Table 2]

[0241] 2) Luminescence of the complex For all emission spectra, a striking "switch-on" of emission intensity is observed upon decreasing pH across the physiological range (Nat. Rev. Mol. Cell. Biol. 2010, 11, 50). The ΔJ = 2 band (ΔJ assignment shown in Figure 2) is clearly the most intense in the emission spectra, especially for excitation at higher wavelengths, and is therefore the most appropriate reference band. The change in emission intensity with pH occurs in all complexes of the present invention. The replacement of an extended chromophore with a single pyridine and the installation of a long head group around the chromophore had no effect on the shape of the europium emission spectrum. In fact, extensive modifications made to the complexes do not affect the immediate coordination environment around the europium(III) center, which remains virtually unperturbed.

[0242] As the most luminescent band, the maximum wavelength of the ΔJ=2 band (ΔJ assignment shown in Figure 2) was selected as the wavelength of emission to be monitored.

[0243] 3) pK of the complex a value The pKa values ​​determined for the complexes of the present invention are shown in Table 3. [Table 3]

[0244] The experimentally measured pH dependence of the excited state lifetime has a very similar profile, independent of the number of chromophores incorporated into the europium complex.

[0245] The variability of luminescence lifetime with pH is shown in Figures 3, 6, 9, 12, 15, 18, 21, 24, and 27. The luminescence lifetime values ​​at each pH were obtained by plotting the luminescence lifetime decay over time and fitting the curves to a simple exponential decay model. The pKa values ​​of the complexes were determined by reversible protonation. Protonation of one chromophore in the complexes (complexes 18 and 21) does not affect the pKa of the other chromophore.

[0246] Chemical modifications on the chromophore result in some change in the pKa value (4.32-6.75). This pKa value can be fine-tuned to specifically target a desired value. This result is highly desirable, as it can be envisioned for monitoring pH fluctuations within cells, e.g., in lysosomes, endosomes, and the trans-Golgi network.

[0247] 4) Time Gate Experiment One advantage of the long emission lifetimes for lanthanides is the possibility of performing time-gated measurements. In addition, as a result of the "switch-on" nature of complex 18, the unprotonated and protonated forms have different emission lifetimes. These properties offer a unique opportunity to control to some extent the relative degree of "switch-on" of the probe by varying the time window, as well as its apparent pKa value. This aspect is demonstrated in Figure 28 using appropriate buffers of different pHs (in the presence of 0.1 M NaCl).

[0248] By measuring the emission intensity of the highly sensitive ΔJ=2 band at different pH values ​​at longer delay times after excitation (FIG. 28, top), a shallower curve was obtained (lower apparent pKa).

[0249] This concept was demonstrated in three different time windows (Fig. 28, bottom panel): 60-460 μs, 1000-2000 μs, and 1500-2500 μs.

[0250] In addition to slight tuning of apparent pKa, these time-gated results also demonstrate the possibility of altering the magnitude of the probe's on / off ratio by varying the time window.

[0251] For complex 18, the luminescence intensity (λ) at pH 4 (sensor on) and pH 8 (sensor off) was measured during different time windows. em A summary of the ratios (at 613.5 nm) is shown in Table 4. [Table 4]

[0252] For comparative purposes, the degree of switch-on response of the complex can be quantified as the ratio of the emission intensity at the maximum of the ΔJ=2 band at pH 4, when the sensor is fully switched "on," to the emission intensity at the maximum of the ΔJ=2 band at pH 8, when the sensor is effectively "off." These values ​​are summarized in Table 4.

[0253] The relative switch-on response increases dramatically as longer time windows are used.

[0254] It was noted that the water solubility of complexes 78a-b, 88a-b and 93 was significantly higher than that of complexes 14, 18 and 21 as a result of the presence of hydrophilic sulfonate groups at the periphery of the complexes.

[0255] 5) Fluorescence intensity The fluorescence intensities of representative complexes of the present invention (78a, 78b, 104) were measured using a Pherastar FS plate reader equipped with a flash lamp. Buffer solutions (pH 4 to pH 7.8) were dispensed into a white 96-well plate, and then lanthanide complexes (10 μL at 110 nM) were dispensed into an aqueous solution containing 0.1% BSA. Immediately after, the fluorescence intensities were recorded between pH 4 and pH 7.8, and the results are reported in Figure 29. For comparison, the fluorescence intensities of the following complexes were also measured:

[0256] [ka] [ka] [ka] [ka] Complexes 107-110 were synthesized as described in WO2014 / 111661. Complex 111 is commercially available under the trade name Lumi4-Tb.

[0257] As can be seen from Figure 29, the complexes of the present invention (78a, 78b, and 104) are pH-sensitive, and the fluorescence intensity of these complexes increases with decreasing pH. In contrast, the fluorescence intensity of the comparative complexes (107, 108, 109, 110, and 111) does not change significantly with pH fluctuations, indicating that these complexes are not pH-sensitive.

Claims

1. Formula (I): 【Chemistry 1】 [In the formula, R １ Ha-CO ２ H, -PO(OH)R ５ or -CH ２ N (CH ２ CO ２ H) ２ and R ２ Ha-CH ２ OH, -CH ２ OSO ２ CH ３ , Cl, Br or —CH ２ N (CH ２ CO ２ H) ２ and R ３ Ha-L １ -E group or G group (C １ -C ６ ) alkyl; R ４ Ha-(CH ２ ) ｍ -NR ６ R ７ and R ５ (C １ -C ４ ) alkyl; —SO ３ － phenyl optionally substituted by a group; or benzyl; R ６ is H or (C １ -C ４ ) alkyl; R ７ Ha-L １ -E group (C １ -C ６ ) alkyl; Or, R ６ and R ７ may be N-protected with a tert-butoxycarbonyl group together with the nitrogen atom to which they are attached, or R ８ to form an optionally N-substituted piperidine, morpholine, or piperazine; R ８ Ha-L １ -E group (C １ -C ６ ) alkyl; L １ is a direct bond, -CONH-(CH ２ ) ｎ - or -NHCO-(CH ２ ) ｎ - and; E is -SO ３ H, -SO ２ (OCH ２ CF ３ ), -N ＋ Alk １ Alk ２ Alk ３ , a carbohydrate residue, or a sulfobetaine; G is a carboxyl group which may be protected in the form of an ester, an amino group which may be protected with a tert-butoxycarbonyl group, a succinimidyl ester, a haloacetamide group, a hydrazine group, an isothiocyanate group, or a maleimide group; Alk １ , Alk ２ , and Alk ３ are each independently (C １ -C ６ ) represents alkyl; m is 0, 1, 2 or 3; n is 0, 1, 2 or 3. Compound.

2. R １ Ga-CO ２ H or -PO(OH)R ５ [In the formula, R ５ (C １ -C ４ ) alkyl. ２ Ga-CH ２ OH or -CH ２ OSO ２ CH ３ 2. The compound of claim 1, wherein:

3. R １ and R ２ are -CH ２ N (CH ２ COOH) ２ 2. The compound of claim 1, wherein:

4. Formula (II) or (III): 【Chemistry 2】 [In the formula, R ａ is absent or -CH ２ NH ２ and R ｂ、 R ｃ , R ｄ and R ｅ is a group of formula (IV), and the others are each independently —CH ２ COOR ｆ and -CH ２ PO(OH)R ｇ Selected from: R ｆ is H, (C １ -C ４ ) alkyl, —NHCH(R ｈ )-(C １ -C ４ ) alkyl or —NHCH(R ｈ )-C(O)OR ｊ and R ｇ (C １ -C ４ ) alkyl; R ｈ (C １ -C ４ ) alkyl or phenyl; R ｊ is H or (C １ -C ４ ) alkyl; Chrome １ , Chrom ２ , and Chrom ３ are each independently a group of formula (IV) and a group of formula (V): 【Transformation 3】 (In the formula, Each R １ Ha-CO ２ H or -PO(OH)R ５ and R ３ Ha-L １ -E group or G group (C １ -C ６ ) alkyl; R ４ Ha-(CH ２ ) ｍ -NR ６ R ７ and R ５ (C １ -C ４ ) alkyl; —SO ３ － phenyl optionally substituted by a group; or benzyl; R ６ is H or (C １ -C ４ ) alkyl; R ７ Ha-L １ -E group (C １ -C ６ ) alkyl; Or, R ６ and R ７ may be N-protected with a tert-butoxycarbonyl group together with the nitrogen atom to which they are attached, or R ８ forming piperidine, morpholine, or piperazine optionally N-substituted with a group; R ８ Ha-L １ -E group (C １ -C ６ ) alkyl; R ９ Ha-L １ -E group or G group (C １ -C ６ ) alkyl; L １ is a direct bond, -CONH-(CH ２ ) ｎ - or -NHCO-(CH ２ ) ｎ and E is -SO ３ H, -SO ２ (OCH ２ CF ３ ), -N ＋ Alk １ Alk ２ Alk ３ , a carbohydrate residue, or a sulfobetaine; G is a carboxyl group which may be protected in the form of an ester, an amino group which may be protected with a tert-butoxycarbonyl group, a succinimidyl ester, a haloacetamide group, a hydrazine group, an isothiocyanate group, or a maleimide group; Alk １ , Alk ２ , and Alk ３ are each independently (C １ -C ６ ) represents alkyl; m is 0, 1, 2 or 3; n is 0, 1, 2 or 3. is selected from. provided that the compound of formula (II) contains at least one group of formula (IV).

5. The compound of claim 4 which is a compound of formula (II).

6. 6. The compound of claim 5, containing only one group of formula (IV).

7. 6. The compound of claim 5, comprising two groups of formula (IV).

8. 6. The compound of claim 5, comprising three groups of formula (IV).

9. Each R １ Ga-PO(OH)R ５ The group [wherein, R ５ (C １ -C ４ ) alkyl.

10. R ４ Ga-NR ６ R ７ [In the formula, R ６ and R ７ are each independently (C １ -C ４ ) alkyl, or R ６ and R ７ may be N-protected with a tert-butoxycarbonyl group together with the nitrogen atom to which they are attached, or R ８ The compound according to claim 5, wherein the compound is a piperidine optionally N-substituted with a group.

11. L １ is a direct bond or -CONH-(CH ２ ) ｎ The compound according to claim 5, wherein

12. E is -SO ３ H or -SO ２ (OCH ２ CF ３ 6. The compound of claim 5, wherein

13. 6. The compound according to claim 5, wherein G is a carboxyl group which may be protected in the form of an ester or an amino group which may be protected with a tert-butoxycarbonyl group.

14. The compound according to claim 4, wherein R 5 is phenyl substituted at the meta or para position with a methyl or an —SO 3 − group.

15. The compound of claim 9, wherein each R 1 is a —PO(OH)R 5 group, where R 5 is methyl.

16. A europium complex comprising a europium(III) ion and a compound of formula (I), (II) or (III) as defined in any one of claims 3 to 15.

17. The following structure: 【Chemistry 4】 17. The complex of claim 16, having the formula:

18. The following structure: 【Transformation 5】 wherein Z is 【Transformation 6】 , or 【Transformation 7】 It is.] 17. The complex of claim 16, having the formula:

19. A conjugate obtained by reacting (i) a europium complex as defined in claim 16 having a G group with (ii) a molecule of interest.

20. The complex of claim 1, wherein the complex has the structure: 【Transformation 8】 20. The conjugate of claim 19, having the formula:

21. The complex of claim 20, wherein the complex has the structure: 【Chemistry 9】 wherein Z is 【Chemistry 10】 , or 【Chemistry 11】 It is.] 20. The conjugate of claim 19, having the formula:

22. 20. The conjugate of claim 19, wherein the molecule of interest is an amino acid, a peptide, a protein, an antibody, a sugar, a carbohydrate chain, a nucleoside, a nucleotide, an oligonucleotide, or an enzyme substrate.

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