europium(III) complex as a pH sensor

Abstract

This invention relates to a compound of formula (I), wherein R1, R2, R3, and R4 are as defined in the specification. The invention also relates to europium (III) complexes obtained from compounds of formula (I) or from complexing agents comprising said compounds, and to the use of such complexes for labeling organic or biomolecules.

Description

Technical Field

[0001] This invention is based on a series of Eu(III) complexes comprising a novel chromophore based on the structure 4-O-alkyl-3-N,N-dialkyl-arylethynylpyridine, which, upon excitation in the 330 to 370 nm range during acidification, exhibits luminescence emission onset by more than two orders of magnitude. Such complexes can be used to monitor acidification in living cells, or any chemobiological event that produces pH modification. Background Technology

[0002] Intracellular pH (pHi) is a key parameter for studying biological phenomena. In fact, intracellular pH plays many important roles in cellular, enzymatic, and tissue activities, including proliferation and apoptosis, multidrug resistance, ion transport, endocytosis, and muscle contraction. Studying pH changes within living cells is also crucial for investigating cellular internalization pathways, such as phagocytosis, endocytosis, and ligand internalization at given receptors. Changes in PHi also affect the nervous system, influencing synaptic transmission, neuronal excitability, and cell-cell coupling via gap junctions and signal cascades. Abnormal PHi values ​​are associated with inappropriate cell function, growth, and division, and are seen in some common diseases such as cancer and Alzheimer's disease. In cell biology, low pH compartments can be used to denature proteins or activate enzymes and protein functions, which would be too slow at around pH 7. For example, the acidic environment of lysosomes (pH 4.5 to 5.5) can promote protein degradation. Therefore, cellular dysfunction is often associated with abnormal pH values ​​in cellular compartments.

[0003] Therefore, if the species to be internalized (receptor or substrate) is labeled with a luminescent pH-sensitive dye, the emission intensity or lifetime of which varies with pH, ​​the process of internalization and endosome uptake can be tracked in real time. Ideally, the observed change should be as large as possible; in extreme cases, this is similar to the concept of a "light switch," although this term is often misused in the literature, and too many small changes in emission intensity are inappropriately referred to as "switch" sensors (Chem. Rev. 1997, 97, 1515 and Chem. Rev. 2010, 110, 2709).

[0004] For decades, academics and practitioners have developed pH sensors to measure pHi. Roger Tsien introduced a luciferin derivative for assessing cytoplasmic pH in 1982 (Cell Biol. 1982, 95, 189). Some improvements have been made to luciferin by chemically modifying it to target cellular compartments or increase its solubility in biological media. However, these luciferin pH sensor derivatives are susceptible to photobleaching due to the generation of singlet oxygen, which can potentially damage cells (Am. J. Physiol. 1989, 256, F957).

[0005] In fact, most pH sensors reported in the literature involve detection of common fluorophores (organic dyes, such as rhodamine and bodily dyes). TM Adjusting the molecular structure of various anthocyanin dyes to modify pK a Values ​​and allow combination.

[0006] In fact, pHrodo TM Green and pHrodo TM Red dyes are available from Thermofisher. These dyes belong to the rhodamine family, and some researchers have modified the backbone to target specific biological applications. Therefore, N,N'-trifluoroethyl rhodamine derivatives (RH-PEF) have been used to monitor antibody internalization; their core structure's pK... a It is approximately 5.1, and its strength has been reported to increase 58-fold between pH 7.4 and 5.0 (Angew. Chem. Int. Ed. 2014, 53, 6085).

[0007] Goryo Chemicals provides AcidiFluor TM ORANGE, which has a Rosamine skeleton. AcidiFluor TM ORANGE is a fluorescent imaging probe that can significantly enhance fluorescence in acidic environments such as lysosomes, late nucleosomes, and particles.

[0008] CypHer5, now available from GE (Amersham), is an anthocyanin dye. Related research has used fluorescent anthocyanin dye labeling and developed a detection method to track HER-2 receptor internalization (ACS Chem. Biol. 2014, 9, 2237). Grover examined GPCRs labeled with anthocyanin dyes and showed a small 5-fold increase in intensity between pH 5 and 8 (Angew. Chem. Int. Ed. 2012, 51, 4838).

[0009] It can be stated that the most promising approach reported to date is the use of modified BODIPY TM cores, and Nagano reported that these systems achieve a conversion factor of up to 300-fold. Herein, the pK a can be readily tuned by modifying the aniline substituent, and the pK a increases from 3.8 to 6.0 in the order H<Me<Et, since the conjugation of the lone pair becomes increasingly unfavorable and the conjugate acid is less stabilized by solvation. Such systems have been used to examine breast cancer tissue samples, by labeling the immunotherapeutic agent Herceptin TM with BODIPY TM (trastuzumab), enabling tracking of the internalization of the HER-2 antibody receptor in mice via microscopy (Nat. Med. 2009, 15, 104).

[0010] These fluorescent organic dyes (fluorescein, rhodamine, rosamine, cyanine and BODIPY TM ) provide neither significant lifetime modulation nor ratiometric response, and have inherent drawbacks associated with autofluorescence and photobleaching. Therefore, to avoid photobleaching, autofluorescence and small Stokes shifts of organic dyes (fluorescein, rhodamine, rosamine, BODIPY TM , cyanine, etc.), others have sought the use of luminescent lanthanide labels, and benefited from their recognized advantages, e.g., large Stokes shifts, long emission lifetimes, sometimes in conjunction with time-resolved spectroscopy or microscopy methods.

[0011] Yuan reported monitoring the change in the ratio of red / green Eu 3+ to Tb 3+ emission intensities in modified acyclic chelates based on terpyridine ligands, obtaining a system with a 7-fold change in the ratiometric ratio (Anal. Chim. Acta 2013, 761, 149). Since the system is based on two complexes (europium and terbium), it is difficult to employ chemical tools and complicates the experimental procedure. Furthermore, these complexes are not functionalized, thereby precluding any bioconjugation reactions.

[0012] Papkovsky et al. described a rather weakly emissive Eu(III) DTPA complex based on a quinolone sensitizer, wherein the lifetime and emission intensity at 614 nm changed by 7-fold between pH 7.5 and 6.5 (pK a 6.5, λ excThe system (370 nm) is capable of monitoring extracellular acidification using a series of high-throughput assays (Anal. Biochem. 2009, 390, 21 and US2002 / 0058793). The europium complex is designed to monitor only extracellular pH changes induced by glycolytic events. Therefore, this system cannot be used for pHi measurements.

[0013] Smith described a method for real-time monitoring of lysosomal pH using confocal microscopy, based on analysis of the Eu / Tb emission intensity ratio, from 1.3 at pH 4.6 to 2.9 at pH 6.5 (λ). exc (355nm)(Chem. Commun. 2012, 48, 8520). From an industrial perspective, this system works with two different complexes (based on europium and terbium) sharing a common ligand, which makes the system somewhat complex. Compared to existing organic dye pHi sensors, this system behaves in the opposite way, as fluorescence emission is higher at neutral pH and decreases as the pH decreases. From a microscopic perspective, this is a significant drawback.

[0014] Using a kinetically stable macrocyclic ligand system based on triazacyclononane, McMahon (Chem. Commun. 2013, 49, 5363) designed an Eu(III) complex capable of monitoring the pH of the endoplasmic reticulum in living cells based on changes in emission lifetime; a 75% change in europium lifetime was observed in the pH range of 6.5 to 7.5. The authors improved the brightness of the complex compared to that described by Smith et al. However, the system does not allow for any changes in the chelate moiety around the europium center because one of the cantilevers (the sulfonamide moiety) is mandatory in the pH sensor. Furthermore, the complex was not designed for biological conjugation. Finally, europium complex emission occurs when the pH increases from pH 4 to pH 8, making the system less suitable for microscopic purposes.

[0015] Recently, Patra et al. developed a luminescent, pH-sensitive lysosomal-targeting 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 remains emissive at pH 7, an undesirable property for biological and microscopic applications. Similarly, this lanthanide system does not allow the emission probe to conjugate with targeting vectors or proteins.

[0016] Takalo studied the photophysical properties of arylethynylpyridine complexes (Helv. Chim. Acta 1993, 76, 877), and Latva et al. compiled their results in an article discussing the properties of europium chelates (J. Lumin. 1997-75-149). Neither article mentioned that the fluorescence of such systems is pH sensitive.

[0017] Therefore, there is a strong need for lanthanide-based fluorescent pH-sensitive probes that are compatible with biological media and capable of monitoring biological events involving pH changes. The probe excitation should ideally be between 320 and 365 nm, wavelengths widely used in plate readers (flash lamps and lasers).

[0018] Therefore, the inventors set out to create a new family of compounds that exhibit 100% variation in excited-state lifetime and two orders of magnitude longer lifetime emission intensity variation within a pH range of 8 to 4. These desired properties have so far proven impossible to obtain. It has been found that, in order to obtain these desired properties within a pH range of 8 to 4, the pK of the protonated emitting species... a The required concentration is between 4.5 and 6.5. According to the invention, this result can be achieved by designing a chromophore consisting of an arylethynylpyridinyl moiety, wherein the aryl group is disubstituted, one substituent being a 4-O-alkyl group and the second substituent being a 3-N,N-dialkylamine moiety (e.g., 3-N,N-dialkylaniline). When incorporated into macrocyclic compounds such as triazacyclononane (TACN), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), or related chelate systems, this chromophore can form kinetically stable complexes with europium (III). Summary of the Invention

[0019] This invention relates to a compound of formula (I):

[0020]

[0021] R1, R2, R3 and R4 are defined in the following detailed description.

[0022] The present invention also relates to a complexing agent of formula (II) or (III):

[0023]

[0024] Where R a R b R c R d R e Chrom1, Chrom2 and Chrom3 are defined in the following detailed description.

[0025] 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.

[0026] The present invention further relates to a conjugate obtained by a reaction between a europium complex and a target molecule. Attached Figure Description

[0027] Figure 1 , Figure 4 , Figure 7 , Figure 10 , Figure 13 , Figure 16 , Figure 19 , Figure 22 and Figure 25 The absorption spectra of complexes 14, 18, 21, 78a, 78b, 88a, 88b, 93, and 104 from pH 4 to 10 are described.

[0028] Figure 2 , Figure 5 , Figure 8 , Figure 11 , Figure 14 , Figure 17 , Figure 20 , Figure 23 and Figure 26 The emission spectra of complexes 14, 18, 21, 78a, 78b, 88a, 88b, 93, and 104 from pH 4 to 10 are described.

[0029] Figure 3 , Figure 6 , Figure 9 , Figure 12 , Figure 15 , Figure 18 , Figure 21 , Figure 24 and Figure 27 The emission lifetime as a function of pH was described, thus enabling the determination of the pK values ​​of complexes 14, 18, 21, 78a, 78b, 88a, 88b, 93, and 104. a value.

[0030] Figure 28 (Top) The emission intensity of complex 18 is shown, measured at different pH values ​​(295 K) with different time delays (red = 60 μs, orange = 460 μs, purple = 1000 μs). Data were normalized to a 60 μs delay at pH 4.

[0031] Figure 28(Bottom) shows the emission intensity of complex 18 measured at different pH values ​​using different time windows (red = 60–460 μs, orange = 1000–2000 μs, purple = 1500–2500 μs). Data were 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 in 0.1 M NaCl).

[0032] Figure 29 The changes in 620 nm fluorescence intensity of the complex of the present invention and the comparative complex are shown as a function of pH. Detailed Implementation

[0033] In one aspect, the present invention relates to a compound of formula (I):

[0034]

[0035] in:

[0036] R1 is –CO2H, -PO(OH)R5 or –CH2N(CH2CO2H)2;

[0037] R2 is –CH2OH, -CH2OSO2CH3, Br, Cl or –CH2N(CH2CO2H)2;

[0038] R3 is an (C1-C6) alkyl group optionally substituted with a group –L1-E or a group G;

[0039] R4 is –(CH2) m -NR6R7;

[0040] R5 is an (C1-C4) alkyl group, preferably methyl; optionally, it is bound by a –SO3 group. - Substituted phenyl group, the group –SO3 - Preferably at the meta or para position; or benzylic;

[0041] R6 is H or (C1-C4) alkyl;

[0042] R7 is an alkyl group that is optionally substituted with a –L1-E group (C1-C6);

[0043] Alternatively, R6 and R7 together with the nitrogen atom to which they are attached form piperidine, morpholine, or piperazine, wherein the piperazine is optionally protected by a tert-butoxycarbonyl N- or optionally substituted by R8 N-.

[0044] R8 is an (C1-C6) alkyl group optionally substituted with the group –L1-E;

[0045] L1 is a bond, –CONH-(CH2) n -or –NHCO-(CH2) n -;

[0046] E is -SO3H, -SO2(OCH2CF3), -N + Alk1Alk2Alk3, carbohydrate residues or sulfobetaine;

[0047] G is a carboxyl group optionally protected in the form of an ester (e.g., the group –CO2Me or -CO2). t Bu), an amino group, succinimide ester, haloacetamido group, hydrazine group, isothiocyanate group or maleimide group optionally protected by tert-butoxycarbonyl group;

[0048] Alk1, Alk2 and Alk3 each independently represent (C1-C6) alkyl groups, preferably (C1-C4) alkyl groups;

[0049] m can be 0, 1, 2, or 3;

[0050] n can be 0, 1, 2, or 3.

[0051] In this disclosure, carbohydrate residues are understood to refer to glucose residues in cyclic or linear form, or of the formula –(CHOH). k -CH2OH group, wherein k is an integer from 3 to 12, preferably k is 3 or 4.

[0052] In the context of this disclosure, sulfobetaine is understood to refer to a group having one of the following formulas:

[0053]

[0054] Wherein R represents (C1-C6) alkyl, preferably methyl or ethyl, and t is an integer from 1 to 6, preferably 1 or 2. In one embodiment, sulfo-betaine is of the formula –(CH2)2N + (CH3)2-(CH2)3-SO3 - Group.

[0055] In the context of this disclosure, the -SO3H, -CO2H, and -PO(OH)2 groups are in or not in a deprotonated form, depending on the pH. Therefore, these groups also refer to the -SO3 group in the following description and the appended claims. - -CO2 - and -PO(OH)O - ,vice versa.

[0056] Under the circumstances of this disclosure, the embodiments described herein can be combined.

[0057] In one embodiment, R1 is –CO2H or -PO(OH)R5, wherein R5 is (C1-C4)alkyl, preferably methyl, and R2 is –CH2OH or -CH2OSO2CH3. In another embodiment, R1 and R2 are each –CH2N(CH2COOH)2.

[0058] In one embodiment, R3 is an (C1-C4) alkyl group optionally substituted with a group –L1-E or a group G.

[0059] In one embodiment, R4 is –NR6R7, wherein 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 piperazine, wherein the piperazine is optionally protected by a tert-butoxycarbonyl N- or optionally substituted by a group R8 N-.

[0060] In one embodiment, R8 is an (C1-C4) alkyl group optionally substituted with the group –L1-E.

[0061] In one implementation, L1 is a bond or –CONH-(CH2). n -

[0062] In one implementation, E is -SO3H or -SO2(OCH2CF3).

[0063] In one embodiment, G is a carboxyl group optionally protected in the form of an ester (e.g., the group –CO2Me or -CO2). t Bu) or an amino group optionally protected by a tert-butoxycarbonyl group.

[0064] In one implementation, m is 0. In another implementation, m is 1. In another implementation, m is 2. In another implementation, m is 3.

[0065] In one implementation, n is 0. In another implementation, n is 1. In another implementation, n is 2. In another implementation, n is 3.

[0066] In another aspect, the present invention relates to a complexing agent of formula (II) or (III):

[0067]

[0068] in:

[0069] R a It either does not exist or is –CH2NH2;

[0070] R b R c R d and Re One (and only one) of them is a group of formula (IV), and the others are each independently selected from –CH2COOR f and -CH2PO(OH)R g ;

[0071] R f H, (C1-C4)alkyl, -NHCH(R) h -(C1-C4)alkyl or -NHCH(R) h )-C(O)OR j ;

[0072] R g It is an (C1-C4) alkyl group;

[0073] R h It is (C1-C4) alkyl or phenyl;

[0074] R j It is H or (C1-C4) alkyl;

[0075] Chrom1, Chrom2, and Chrom3 are each independently selected from groups of formula (IV) and formula (V):

[0076]

[0077] in:

[0078] Each R1 is –CO2H or -PO(OH)R5;

[0079] R3 is an (C1-C6) alkyl group optionally substituted with a group –L1-E or a group G;

[0080] R4 is –(CH2) m -NR6R7;

[0081] R5 is an (C1-C4) alkyl group, preferably methyl; optionally, it is bound by a –SO3 group. - Substituted phenyl group, the group –SO3 - Preferably at the meta or para position; or benzylic;

[0082] R6 is H or (C1-C4) alkyl;

[0083] R7 is an alkyl group that is optionally substituted with a –L1-E group (C1-C6);

[0084] Alternatively, R6 and R7 together with the nitrogen atom to which they are attached form piperidine, morpholine, or piperazine, wherein the piperazine is optionally protected by a tert-butoxycarbonyl N- or optionally substituted by a group R8 N-;

[0085] R8 is an (C1-C6) alkyl group optionally substituted with the group –L1-E;

[0086] R9 is an (C1-C6) alkyl group optionally substituted with a group –L1-E or a group G;

[0087] L1 is a bond, –CONH-(CH2) n -or –NHCO-(CH2) n ;

[0088] E is -SO3H, -SO2(OCH2CF3), -N + Alk1Alk2Alk3, carbohydrate residues or sulfobetaine;

[0089] G is a carboxyl group optionally protected in the form of an ester (e.g., the group –CO2Me or -CO2). t Bu), an amino group, succinimide ester, haloacetamido group, hydrazine group, isothiocyanate group, or maleimide group optionally protected by a tert-butoxycarbonyl group;

[0090] Alk1, Alk2 and Alk3 each independently represent (C1-C6) alkyl groups, preferably (C1-C4) alkyl groups;

[0091] m can be 0, 1, 2, or 3;

[0092] n is 0, 1, 2, or 3;

[0093] The condition is that the compound of formula (II) contains at least one group of formula (IV).

[0094] Preferred complexing agents include compounds of formula (II). Another preferred family of compounds includes compounds of formula (III).

[0095] In one embodiment, the complexing agent of formula (II) contains only one group of formula (IV), that is, 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.

[0096] In one embodiment, the complexing agent of formula (II) comprises two formula (IV) groups, namely, two of Chrom1, Chrom2 and Chrom3 are formula (IV) groups, which may be the same or different, preferably the same, and the other is a formula (V) group.

[0097] In one embodiment, the complexing agent of formula (II) comprises three formula (IV) groups, namely, Chrom1, Chrom2 and Chrom3 are each of formula (IV) groups, which may be the same or different, preferably the same.

[0098] In one embodiment, each R1 is -PO(OH)R5, wherein R5 is (C1-C4) alkyl, preferably methyl.

[0099] In one embodiment, R3 is an (C1-C4) alkyl group optionally substituted with a group –L1-E or a group G.

[0100] In one embodiment, R4 is –NR6R7, wherein 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 piperazine, wherein the piperazine is optionally protected by a tert-butoxycarbonyl N- or optionally substituted by a group R8 N-.

[0101] In one embodiment, R8 is an (C1-C4) alkyl group optionally substituted with the group –L1-E.

[0102] In one implementation, L1 is a bond or –CONH-(CH2). n -

[0103] In one implementation, E is -SO3H or -SO2(OCH2CF3).

[0104] In one embodiment, G is a carboxyl group optionally protected in the form of an ester (e.g., the group –CO2Me or -CO2). t Bu) or an amino group optionally protected by a tert-butoxycarbonyl group.

[0105] In one implementation, m is 0. In another implementation, m is 1; in yet another, m is 2; and in still another, m is 3.

[0106] In one implementation, n is 0. In another implementation, n is 1; in yet another, n is 2; and in still another, n is 3.

[0107] In another aspect, the present invention relates to a europium complex comprising europium(III) ions (Eu). 3+ ) and compounds of formula (I), wherein R1 and R2 are each –CH2N(CH2COOH)2.

[0108] In another aspect, the present invention relates to a europium complex comprising a europium(III) ion and a compound of formula (II) or (III) as disclosed herein.

[0109] The europium complexes of the present invention can be prepared by contacting compounds of formula (I), (II), or (III) of the invention disclosed herein with europium salts. Typically, 1 equivalent of the compound is reacted with 1 to 5 equivalents of a europium salt (e.g., a chloride, acetate, or trifluoromethanesulfonate) in an aqueous solution of a solvent (acetonitrile, methanol, or other solvent compatible with these salts) or a buffer solution at room temperature for several minutes to generate the corresponding complex.

[0110] The representative europium complex of this invention has the following structure:

[0111]

[0112] Another representative europium complex of the present invention has the following structure:

[0113]

[0114] Where Z is:

[0115]

[0116]

[0117] Compounds of formulas (I), (II), and (III) disclosed herein (and their corresponding europium complexes) containing the group G are particularly suitable for labeling organic or biomolecules containing functional groups capable of reacting with reactive groups to form covalent bonds.

[0118] Therefore, in one aspect, the present invention relates to conjugates obtained by (i) a europium complex disclosed herein having a group G, and (ii) a reaction of a target molecule. Any target organic or biological molecule can be conjugated with the europium complex disclosed herein, provided that it has a functional group capable of reacting with the group G of the complex.

[0119] In one embodiment, the target molecule is selected from: amino acids, peptides, proteins, antibodies, sugars, carbohydrate chains, nucleosides, nucleotides (DNA, RNA), oligonucleotides, and enzyme substrates, especially suicide enzyme substrates, such as benzylguanine or benzylcytosine (enzyme substrates sold under the names Snaptag and Cliptag), chloroalkyl (enzyme substrates sold under the name Halotag), or coenzyme A (enzyme substrates sold under the names ACPtag or MCPtag).

[0120] When protonated, the europium complex of the present invention exhibits a pK value of 4.5 to 6.5. a Value. Typically, when a molecule has multiple protonation sites, each protonation site exhibits a pK value. a Value. Surprisingly, the complexes of this invention exhibit only one observable pK.a The value is independent of the number of chromophores (i.e., compounds of formula (I), (IV) or (V)) fixed on the complex.

[0121] The europium complexes of this invention can also bioconjugate to target molecules at sites that do not disrupt fluorescence intensity in response to changes in the pH of the medium. The europium complexes of this invention exhibit excellent emission luminosity, making them ideal for detecting low concentrations of biological events.

[0122] As described above, the europium complexes of the present invention can contain one to three chromophores and can be functionalized to allow the introduction of a water-soluble moiety and functional groups for bioconjugation. Depending on the desired complex, appropriate synthetic routes (Schemes 1 to 3) can be selected. For complexes having one, two, or three different chromophores, two possible options are described in Schemes 1 to 2 and illustrated in more detail in the Experimental Section. For complexes having three identical chromophores, the synthesis is described in Scheme 3.

[0123] For example, triazacyclononane is partially alkylated with a chromophore or pyridine depending on the desired number of chromophores in the complex. After deprotection of the Boc group, the last chromophore or pyridine moiety is introduced onto the dichromophore or dipyridyl-alkylated macrocyclic compound. The ethyl phosphonate functional group is hydrolyzed to form a europium complex by adding europium(III) chloride. Optionally and if appropriate, the complex can be partially functionalized with a sulfonate group to allow for better water solubility, and functionalized with functional groups conjugated to proteins, antibodies, peptides, and enzyme substrates.

[0124] General synthetic schemes for complexes with 1, 2, and 3 chromophores (Option 1)

[0125]

[0126] Option 1

[0127] General synthetic schemes for complexes with 1 or 2 chromophores (Option 2)

[0128]

[0129] Option 2

[0130] General synthetic scheme for complexes with 3 identical chromophores

[0131]

[0132] Option 3

[0133] General Synthesis of Acyclic Complexes

[0134] The skeletal synthesis has been previously described (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 moiety at the para position. Scheme 4 provides an example.

[0135]

[0136] Option 4

[0137] General Synthesis of Complexes Based on DOTA Macrocyclic Compounds

[0138] To emphasize the general features of the invention, the chromophore has been introduced into the DOTA system using classical synthetic strategies described elsewhere (WO 2006 / 120444, WO 2009 / 010580, WO 2010 / 084090, Acc.Chem.Res2009, 42, 925). Embodiment 5 describes the strategy used to obtain the corresponding europium complex with one chromophore.

[0139]

[0140] Option 5

[0141] Other methods for preparing the complexing agents and corresponding europium complexes disclosed herein are illustrated below and further explained in the examples.

[0142] Nonfunctionalized chromophore synthesis

[0143]

[0144] Option 6

[0145] Commercially available compound 1 was reduced to the corresponding aniline, which was then alkylated in ethane iodide for 48 hours. The chromophore skeleton was obtained by using two stalk reactions to yield compound 7. The resulting alcohol was converted to the corresponding methanesulfonate 8 and used for subsequent macrocyclic alkylation.

[0146] Synthesis of nonfunctionalized complexes with one chromophore

[0147]

[0148] Option 7

[0149] Scheme 7 describes the synthesis of a europium complex with one chromophore. Triazacyclononane 9 is alkylated with pyridine methanesulfonate 10. The third nitrogen atom is deprotected with TFA and alkylated with chromophore 8. Ethyl phosphonate is hydrolyzed, and the macrocyclic compound is complexed with europium to obtain europium(III) complex 14.

[0150] Synthesis of nonfunctionalized complexes with two chromophores

[0151]

[0152] Option 8

[0153] To obtain a complex with two chromophores, such as europium complex 18, the same strategy was used as before, but the order of introducing pyridine derivatives (compounds 8 and 10) was reversed, as shown in scheme 8.

[0154] Synthesis of nonfunctionalized complexes with 3 chromophores

[0155]

[0156] Option 9

[0157] A complex with three chromophores was synthesized using unprotected triazacyclononane 19 and chromophore 8, yielding the corresponding europium complex 21.

[0158] Synthesis of chromophores functionalized with ethyl ester

[0159]

[0160] Option 10

[0161] To functionalize chromophore 8, the methyl group of the methoxy group is replaced with an aliphatic chain having an ester moiety, thereby subsequently introducing a sulfonate group or other hydrophilic functional group. Thus, bromonitrophenol 22 is alkylated, followed by reduction of the nitro group, and then alkylated with iodoethane. Two shavings reactions are performed to obtain the chromophore, which is then activated to methanesulfonate derivative 30.

[0162] NHBoc functionalized chromophore synthesis

[0163]

[0164] Option 11

[0165] The chromophore is functionalized with a protected amine in the form of a classic carbamate (Boc group), instead of a protected carboxylic acid in the form of an ethyl ester. The synthetic route is the same as that used previously with N-Boc-protected bromopropylamine (Scheme 11).

[0166] Synthesis of chromophores functionalized with ethyl ester

[0167]

[0168] Option 12

[0169] The alkylation of aniline is controlled by selecting the molar ratio of the alkylating agent and the aniline derivative. Therefore, compound 36 was monoalkylated with iodoethane. The resulting secondary aniline was then methylated using reductive amination. The final steps of the synthesis were the same as described above.

[0170] Synthesis of cyclic amine systems on chromophores

[0171]

[0172] Option 13

[0173] Aniline derivatives can be converted into cyclic amines (piperidine, piperazine, morpholine). In the case of compounds 46a-d, secondary amines can be used to connect soluble moieties or as biological conjugation sites.

[0174] Synthesis of N,N-alkylpropylsulfonate precursor chromophores

[0175]

[0176] Option 14

[0177] One side of the aniline is replaced with an alkyl chain of a sulfonate with terminal protection, and the other side is replaced with an alkyl chain. Thus, the corresponding chromophore is prepared using the same method as in the previous scheme, but with the corresponding bromopropyl sulfonate partially substituted with a trifluoroethyl group as a protecting group. The synthetic scheme is described in Scheme 14.

[0178] Synthesis of NHBoc-functionalized pyridine

[0179]

[0180] Option 15

[0181] Synthesis of pyridine with methyl ester functionalization

[0182]

[0183] Option 16

[0184] 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. The alcohol functional group was activated to give the corresponding methanesulfonate derivatives 60 and 63 (Schemes 15 and 16).

[0185] Synthesis of functionalized complexes with a chromophore

[0186]

[0187] Option 17

[0188] Scheme 17 describes the synthesis of complex 65 incorporating a chromophore.

[0189] Synthesis of functionalized complexes with a chromophore

[0190]

[0191] Option 18

[0192] Scheme 18 describes the synthesis of complex 71. The sulfonate moiety is introduced onto the pyridine heterocycle at the end of the synthesis.

[0193] Synthesis of functionalized complexes with a chromophore

[0194]

[0195] Option 19

[0196] Scheme 19 elucidates the synthesis of a complex that can be functionalized via a piperazine moiety on a secondary amine (compound 73b).

[0197] Synthesis of functionalized complexes with two chromophores

[0198]

[0199] Option 20

[0200] Scheme 20 describes the synthesis of two complexes with two chromophores. The pK of each complex is... a The properties of the alkyl group replacing the nitrogen atom are adjusted. The synthesis begins with the alkylation of triazacyclononane with the methanesulfonate chromophore. A third alkylation is performed after deprotection of the Boc group to obtain compounds 76a-b. Hydrolysis and europium complexation of the phosphonate yield complexes 77a-b, which are treated with high-taurine to obtain the desired water-soluble europium(III) complexes.

[0201] Synthesis of functionalized complexes with two chromophores

[0202]

[0203] Option 21

[0204] Scheme 21 describes the synthesis of complex 84 having a piperazine moiety. It follows the same strategy as previously described. A commercially available diprotected triazacyclononane 79 is monoalkylated. Deprotection of the two Boc groups is carried out in the presence of trifluoroacetic acid. A chromophore is introduced at this stage, followed by hydrolysis of the hypophosphite functional group, forming a europium complex upon addition of europium chloride. The secondary amine is reacted with propanesulfonate to obtain the desired complex 84.

[0205] Synthesis of functionalized complexes with two chromophores

[0206]

[0207] Option 22

[0208] As previously described, a sulfonate group is introduced into the terminal portion of one of the alkyl groups of the aniline functional group. The synthesis follows the same strategy used with chromophores 57a-b: macrocyclic alkylation, deprotection of the Boc group, introduction of the final pyridine derivative, hydrolysis of the hypophosphite, and complexation with europium. Furthermore, the final basic hydrolysis of the hypophosphite allows for the deprotection of the trifluoroethyl protecting group, yielding the free sulfonate functional group moiety (Scheme 22).

[0209] Synthesis of functionalized complexes with three chromophores

[0210]

[0211] Option 23

[0212] A complex with three chromophores was prepared as described in Scheme 23. The synthetic route was based on the same strategy as previously described, yielding complex 91.

[0213]

[0214] Option 24

[0215] Scheme 24 illustrates the preparation of asymmetric complex 93 using the same strategy as described above. Complex 93 has two identical chromophores (N,N-ethylpropylsulfonate) and a third chromophore consisting of an N,N-diethylaniline group. This complex can be used for subsequent bioconjugation.

[0216] Synthesis of functionalized complexes with three chromophores

[0217]

[0218] Option 25

[0219] Another method to obtain a complex with three chromophores is to use a triazacyclononane with a methylene side arm NHBoc, previously disclosed in US 9,981,967. This synthesis is simple and can yield the desired complex with three identical chromophores.

[0220] Synthesis of bioconjugated complexes with two chromophores

[0221]

[0222] Option 26

[0223] Scheme 26 elucidates the conjugation of europium complex 78a-b with benzylguanine methylbenzamide NHS ester (BG-MB-NHS, a well-known enzyme substrate (WO 2010 / 034931)) to obtain benzylguanine complex 100a-b, or the conjugation of europium complex 78a-b with maleimide to obtain complex 101a-b, which can be used for antibody or protein labeling.

[0224] Synthesis of acyclic complexes

[0225]

[0226] Option 27

[0227] This scheme demonstrates the synthesis of acyclic compounds. Takalo (ibid.) has previously described the synthesis of pyridine ethynyl aryl derivatives. Europium chelates are obtained using suitable structural units, which are then functionalized to allow for bioconjugation. The corresponding maleimide and benzylguanine derivatives 105 and 106 are prepared in a similar manner.

[0228] Experimental Section

[0229] The following abbreviations are used in the experimental section.

[0230] 9-N3 1,4,7-Triazacyclononane

[0231] app. (Obviously)

[0232] BG Benzylguanine

[0233] Boc tert-butoxycarbonyl

[0234] br broad peak (NMR)

[0235] BSA (Bovine Serum Albumin)

[0236] CPL circularly polarized light

[0237] d days (reaction time)

[0238] d doublet (NMR)

[0239] DCM dichloromethane

[0240] dd double doublet (NMR)

[0241] ddq quartet doublet (NMR)

[0242] DIPEA (diisopropylethylamine)

[0243] dm doublet of multiplets (NMR)

[0244] DMF (dimethylformamide)

[0245] DMSO (dimethyl sulfoxide)

[0246] dppf 1,1'-bis(diphenylphosphine)ferrocene

[0247] EDTA (ethylenediaminetetraacetic acid)

[0248] ESI Electrospray Ionization

[0249] EtOAc (ethyl acetate)

[0250] EtOH (ethanol)

[0251] equiv. equivalent

[0252] FA Formic acid

[0253] GPCR G protein-coupled receptor

[0254] h hours (reaction time)

[0255] HATU (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate)

[0256] HEPES 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid

[0257] HRMS high-resolution mass spectrometry

[0258] ICP-MS (Inductively Coupled Plasma Mass Spectrometry)

[0259] Intramolecular charge transfer in ICT

[0260] LRMS low-resolution mass spectrometry

[0261] Ln Lanthanides

[0262] LTG LysoTracker Green

[0263] m multiplet (NMR)

[0264] MeOH (methanol)

[0265] MES 2-(N-morpholino)ethanesulfonic acid

[0266] Mp melting point

[0267] MS mass spectrometry

[0268] NMR (Nuclear Magnetic Resonance)

[0269] PBS phosphate buffer

[0270] PDA photodiode array

[0271] Pd / C Palladium on Carbon

[0272] Pd(dppf)Cl2[1,1'-bis(diphenylphosphino)ferrocene]palladium(II) dichloride

[0273] q quartet (NMR)

[0274] quantification

[0275] RT room temperature

[0276] s-s singlet (NMR)

[0277] S n The nth singlet state (energy level)

[0278] t triplet (NMR)

[0279] TEEAc triethylamine

[0280] TFA (trifluoroacetic acid)

[0281] THF Tetrahydrofuran

[0282] TLC (Thin Layer Chromatography)

[0283] TMS (trimethylsilyl)

[0284] UV ultraviolet rays

[0285] Vis visible light

[0286] Compound 2

[0287]

[0288] 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 combined under argon atmosphere. Iron powder (1.02 g, 18.3 mmol) was added, and the mixture was heated to 50 °C for 1 hour. The mixture was filtered, and the solvent was removed under reduced pressure. The resulting residue was dissolved in CH2Cl2 (100 mL), and NaOH aqueous solution (25 mL, 2 M) was added; the precipitate was removed by filtration. The aqueous layer was separated and extracted with CH2Cl2 (5 × 15 mL). The organic layers were combined, dried over MgSO4, and the solvent was removed under reduced pressure to give a light brown solid 2 (674 mg, 76%). 1H-NMR (700MHz, CDCl3) δ 7.00 (dd, 3 J H-H =8.3, 4 J H-H =2.2,1H),6.98(d, 4 J H-H =2.2,1H),6.51(d, 3 J H-H =8.3,1H),3.81(s,3H); 13 C-NMR(176MHz, CDCl3)δ147.2,138.0,126.9,123.0,112.3,83.4,55.6; ESI-LRMS(+)m / z 250[M+H] + ; ESI-HRMS(+)[C7H9NO 127 I] + Calculated value: 249.9729, Actual measured value: 249.9750; mp: 90℃.

[0289] Compound 3

[0290]

[0291] Compound 2 (2.83 g, 11.4 mmol), iodoethane (7 mL, 87 mmol), and K₂CO₃ (6.3 g, 45.6 mmol) were combined in anhydrous CH₃CN (15 mL) under argon atmosphere. The reaction mixture was heated to 60 °C for 48 hours. Afterward, the solvent was removed under reduced pressure, and the residue was dissolved in CH₂Cl₂ (30 mL), washed with water (5 × 20 mL), and dried over K₂CO₃. Removal of the solvent under reduced pressure yielded a pale orange oil (2.16 g, 62%). 1 H-NMR (600MHz, CDCl3) δ 7.24 (dd, 3 J H-H =8.5, 4 J H-H =1.9,1H),7.15(d, 4 J H-H =1.9,1H),6.58(d, 3 J H-H =8.5,1H),3.81(s,3H),3.12(q, 3 J H-H =7.1,4H),1.02(t, 3 J H-H =7.1,6H); 13C-NMR(151MHz, CDCl3)δ153.5,141.0,131.0,130.0,113.4,83.0,55.4,45.9,11.9; ESI-LRMS(+)m / z 306[M+H] + ; ESI-HRMS(+)[C 11 H 17 NO 127 I] + Calculated value: 306.0355; Actual value: 306.0363.

[0292] Compound 4

[0293]

[0294] Compound 3 (548 mg, 1.80 mmol), trimethylsilylacetylene (0.5 mL, 3.6 mmol), Pd(dppf)Cl2, DCM (150 mg, 0.184 mmol), and pyrrolidine (0.45 mL, 5.4 mmol) were combined in anhydrous THF (3 mL) under argon atmosphere. The reaction mixture was heated to 50 °C for 19 hours, and then the solvent was removed under reduced pressure. The resulting residue was dissolved in CH2Cl2 (30 mL), washed with H2O (4 × 30 mL), and dried over K2CO3. The solvent was removed under reduced pressure to obtain a crude product, which was purified by column chromatography (SiO2, 100% hexane to 6% EtOAc in hexane solution) to give a pale orange oil (323 mg, 65%). 1 H-NMR (700MHz, CDCl3) δ 7.12 (d, 3 J H-H =8.5,1H),7.03(s,1H),6.75(d, 3 J H-H =8.5,1H),3.85(s,3H),3.14(q, 3 J H-H =7.1,4H),1.01(t, 3 J H-H =7.1,6H),0.24(s,9H); 13 C-NMR (176MHz, CDCl3) δ154.5,139.1,127.2,125.3,115.1,111.2,105.9,91.9,55.6,46.1,12.1,0.3; 29 Si-NMR(139MHz, CDCl3)δ-18.3; ESI-LRMS(+)m / z 276[M+H] + ; ESI-HRMS(+)[C 16 H26 NOSi] + Calculated value: 276.1784; Actual measurement: 276.1790; R f =0.40 (SiO2, 10% EtOAc in hexane solution).

[0295] Compound 5

[0296]

[0297] Under argon atmosphere, triethylamine trihydrofluoric acid (0.75 mL, 3.28 mmol) was added to an anhydrous THF (3 mL) solution of compound 4 (115 mg, 0.306 mmol). The solution was heated to 30 °C for 37 hours, and then the solvent was removed under reduced pressure. The resulting oil was dissolved in CH₂Cl₂ (30 mL) and washed with water (3 × 40 mL). The combined aqueous layers were extracted with CH₂Cl₂ (5 × 40 mL), and the organic layers were combined and dried over K₂CO₃. The solvent was removed under reduced pressure to obtain a pale orange oil (81 mg, 87%). 1 H-NMR (700MHz, CDCl3) δ 7.14 (dd, 3 J H-H =8.4, 4 J H-H =2.0,1H),7.06(d, 4 J H-H =2.0,1H),6.78(d, 3 J H-H =8.4,1H),3.86(s,3H),3.15(q, 3 J H-H =7.1,4H),2.98(s,1H),1.02(t, 3 J H-H =7.1,6H); 13 C-NMR (176MHz, CDCl3) δ154.6,139.2,127.1,125.3,113.9,111.3,84.4,75.3,55.7,46.1,12.1; ESI-LRMS(+)m / z 204[M+H] + ; ESI-HRMS(+)[C 13 H 18 NO] + Calculated value: 204.1388; Actual value: 204.1401.

[0298] Compound 6

[0299]

[0300] Compound 6 was synthesized from 2-bromo-6-methylpyridine in nine steps according to the procedure described in US 2015 / 361116.

[0301] Compound 7

[0302]

[0303] Under argon atmosphere, pyrrolidine (0.1 mL, 1.22 mmol) and Pd(dppf)Cl2·DCM (60 mg, 0.073 mmol) were added to anhydrous THF (2.5 mL) solution of compound 5 (120 mg, 0.59 mmol) and compound 6 (175 mg, 0.60 mmol). The reaction mixture was heated to 50 °C for 18 hours, and then the solvent was removed under reduced pressure. The resulting residue was dissolved in CH2Cl2 (40 mL) and washed with water (3 × 40 mL). The organic layer was dried over K2CO3 and the solvent was removed under reduced pressure to obtain a brown residue. The crude residue was subjected to reversed-phase HPLC (10 to 100% CH3CN in H2O solution, 10 min, t r Purification was carried out over 10.7 min to obtain a pale yellow oily substance (118 mg, 48%). 1 H-NMR (600MHz, CDCl3) δ 8.03 (d, 3 J H-P =6.0, 1H), 7.49 (s, 1H), 7.18 (d, 3 J H-H =8.5,1H),7.09(s,1H),6.82(d, 3 J H-H =8.5,1H),4.80(s,2H),4.14–3.82(m,2H),3.87(s,3H),3.16(q, 3 J H-H =6.9,4H),1.77(d, 2 J H-P =15,3H),1.26(t, 3 J H-H =7.0,3H),1.03(t, 3 J H-H =6.9,6H); 13 C-NMR (151MHz, CDCl3) δ 160.8 (d, 3 J C-P =19),155.2,153.2(d, 1 J C-P =155),139.5,133.3(d, 3 J C-P =11),128.3(d,2 J C-P =22),127.2,125.0,124.1(d, 4 J C-P =3),113.5,111.5,97.0,84.9,64.2,61.3(d, 2 J C-P =6), 55.7, 46.0, 16.5(d, 3 J C-P =6),13.6(d, 1 J C-P =105), 12.0; 31 P{ 1 H}-NMR(243MHz, CDCl3)δ+39.4; ESI-LRMS(+)m / z 417[M+H] + ; ESI-HRMS(+)[C 22 H 30 N2O4P] + Calculated value: 417.1943; Actual value: 417.1954.

[0304] Compound 8

[0305]

[0306] Compound 7 (67 mg, 0.161 mmol), methanesulfonic anhydride (56 mg, 0.322 mmol), and DIPEA (0.07 mL, 0.402 mmol) were combined in anhydrous THF (1.5 mL) under argon atmosphere and stirred at room temperature for 90 minutes. The solvent was then removed under reduced pressure. CH₂Cl₂ (30 mL) and H₂O (30 mL) were added to the resulting residue. The organic layers were separated and washed with H₂O (2 × 30 mL). The combined aqueous layers were extracted with CH₂Cl₂ (1 × 30 mL). The combined organic layers were dried over K₂CO₃ and the solvent was removed under reduced pressure to obtain a pale orange oil, which was used directly for the next step without further purification (69 mg, 87%). 1 H-NMR (400MHz, CDCl3) δ 8.09 (dd, 3 J H-P =6.0, 4 J H-H =1.5,1H),7.64–7.62(m,1H),7.19(dd, 3 J H-H =8.5, 4 J H-H =2.1,1H),7.10(d, 4 J H-H=2.1,1H),6.84(d, 3 J H-H =8.5,1H),5.36(s,2H),4.19–3.81(m,5H),3.17(q, 3 J H-H =7.1,4H),3.13(s,3H),1.77(d, 2 J H-P =15,3H),1.27(t, 3 J H-H =7.0,3H),1.04(t, 3 J H-H =7.1,6H); ESI-LRMS(+)m / z 495[M+H] + .

[0307] Compound 9 was synthesized in three steps according to the process described in WO 2014 / 111661.

[0308] Compound 10

[0309]

[0310] Compound 10 was synthesized from 2-bromo-6-methylpyridine in five steps, following the procedure described in Inorg.Chem., 2012, 51, 8042.

[0311] Compound 11

[0312]

[0313] Under argon atmosphere, 1-(tert-butoxycarbonyl)-1,4,7-triazacyclononane dihydrochloride 9 (28 mg, 0.0926 mmol), compound 10 (64 mg, 0.218 mmol), and K₂CO₃ (40 mg, 0.289 mmol) were combined in anhydrous CH₃CN (2 mL) and heated to 65 °C for 18 hours. Subsequently, the solution was separated from the inorganic salts by reversed-phase HPLC (10 to 100% CH₃CN in H₂O solution, 10 min, t). r Purification was carried out over 11.8 min to obtain a pale yellow oily substance (33 mg, 57%). 1 H-NMR(700MHz, CDCl3)δ7.92–7.88(m,2H),7.78–7.72(m,2H),7.62(d, 3 J H-H =7.9,1H),7.55(d, 3 J H-H=7.9,1H),4.11–4.03&3.87–3.79(m,4H),3.95–3.90m,4H),3.40–3.29(m,4H),3.11–2.96(m,4H),2.72–2.59(m,4H),1.75(d, 2 J H-P =15,3H),1.74(d, 2 J H-P =15,3H),1.44(s,9H),1.25–1.21(2×t, 3 J H-H =7.1,6H); 13 C-NMR(176MHz, CDCl3)δ161.3(br s),155.7,153.9(d, 1 J C-P =160),153.7(d, 1 J C-P =160),136.5(d, 3 J C-P =10),136.4(d, 3 J C-P =10),125.9(2×d, 2 J C-P =20),125.5,125.3,79.5,63.4,63.1,61.0(2×d, 2 J C-P =6),56.4),55.3,54.8,54.2,50.1,49.7,28.7,16.6,16.5,13.5(d, 1 J C-P =104),13.4(d, 1 J C-P =104); 31 P{ 1 H}-NMR(162MHz, CDCl3)δ+40.3,+40.2; ESI-LRMS(+)m / z 624[M+H] + ; ESI-HRMS(+)[C 29 H 48 [N5O6P2] + Calculated value: 624.3080; Actual value: 624.3105.

[0314] Compound 12

[0315]

[0316] A solution of compound 11 (33 mg, 0.053 mmol) was prepared in trifluoroacetic acid and CH2Cl2 (20% v / v, 4 mL total). The solution was stirred at room temperature for 25 minutes, and then the solvent was removed under reduced pressure to obtain an orange residue. CH2Cl2 (30 mL) was added to the residue, and the solvent was removed under reduced pressure again. This process was repeated five times to obtain a pale orange oil (28 mg, quantified). 1 H-NMR (700MHz, CDCl3) δ7.84–7.79(m,4H),7.48–7.43(m,2H),4.30(s,4H),4.14–3.87(m,4H),3.57–3.38(m,8H),3.30–3.17(m,4H),1.73(d, 2 J H-P =15,6H),1.28(t, 3 J H-H =7.0,6H); 13 C-NMR (176MHz, CDCl3) δ 156.8 (d, 3 J C-P =20),153.2(d, 1 J C-P =160),137.6(d, 3 J C-P =10),126.2(d, 2 J C-P =21),62.0(d, 2 J C-P =6),59.9,51.6,49.4,44.5(2×s),16.3(d, 3 J C-P =6),13.6(d, 1 J C-P =102); 31 P{ 1 H}-NMR(162MHz, CDCl3)δ+40.8,+40.7; ESI-LRMS(+)m / z 524[M+H] + ; ESI-HRMS(+)[C 24 H 40 [N5O4P2] + Calculated value: 524.2556; Actual value: 524.2563.

[0317] Compound 13

[0318]

[0319] Compound 12 (8.5 mg, 16.2 μmol), compound 8 (16 mg, 32.4 μmol), and K₂CO₃ (5 mg, 35.4 μmol) were combined in anhydrous CH₃CN (1 mL) under argon atmosphere and heated to 60 °C for 18 hours. Subsequently, the crude mixture was separated from the inorganic salts by filtration. The resulting solution was directly subjected to reversed-phase HPLC (10 to 100% CH₃CN in H₂O solution, 10 min, t). r =11.0 min), to obtain a pale yellow oily compound 13 (7.4 mg, 50%); 1 H-NMR (400MHz, CDCl3) δ 8.01 (d, 3 J H-P =6.0,1H),7.94–7.87(m,2H),7.82–7.74(m,2H),7.67–7.61(m,3H),7.18(d, 3 J H-H =8.4, 1H), 7.09 (s, 1H), 6.84 (d, 3 J H-H =8.4,1H),4.18–3.76(m,15H),3.17(q, 3 J H-H =6.9,4H),2.97–2.80(m,12H),1.76(d, 2 J H-P =15,3H),1.75(d, 2 J H-P =15,3H),1.29–1.20(m,9H),1.04(t, 3 J H-H =6.9,6H); 13 C-NMR(101MHz,CDCl3)δ161.5(d+d, 3 J C-P =12),155.0,153.6(d+d, 1 J C-P =159),139.4,136.4(d, 3 J C-P =9),133.3(d, 3 J C-P =16),127.8(d, 2 J C-P =22),127.0,126.4(d, 4 J C-P =4),125.9,125.6(d, 2 J C-P=20),124.8,113.4,111.3,96.4,85.1,64.4(2×s),60.9(d+d, 2 J C-P =6), 55.6, 45.9, 16.5(d+d, 3 J C-P =6.0), 13.4(d+d, 1 J C-P =104), 11.9; 31 P{ 1 H}-NMR(162MHz, CDCl3)δ+40.2(2P),+40.0(1P); ESI-LRMS(+)m / z 922[M+H] + ; ESI-HRMS(+)[C 46 H 67 N7O7P3] + Calculated value: 922.4315; Actual value: 922.4355.

[0320] Complex 14

[0321]

[0322] Ligand 13 (2 mg, 2 μmol) was dissolved in a CH3OH / H2O mixture (1:1, 2 mL total), and the pH was adjusted to 12 using an aqueous NaOH solution. The solution was heated to 60 °C for 14 hours. After cooling and adjusting the pH to 7 using dilute hydrochloric acid (0.1 M), EuCl3·6H2O (3 mg, 8 μmol) was added, and the reaction mixture was heated to 60 °C for 15 hours. The reaction mixture was then subjected to reversed-phase HPLC (10 to 100% CH3CN in H2O solution, 10 min, t). r Purification was carried out over 8.5 min to obtain a yellow solid (2 mg, 93%); ESI-LRMS (+) m / z 986 [M+H] + 495[M+2H] 2+ ; ESI-HRMS(+)[C 40 H 52 N7O7P3 151 Eu] + Calculated value: 986.2340; Actual measurement: 986.2327; τ H2O (ms)=0.50 (pH 9), 0.53 (pH 8), 0.74 (pH 7), 1.06 (pH 6), 1.15 (pH 5), 1.16 (pH 4); ε 331nm =11450M -1 cm -1 ;Ф pH8 = 0.5%, Ф pH 4 = 16.8% (λ) exc =331nm).

[0323] Compound 15

[0324]

[0325] Under argon atmosphere, 1-(tert-butoxycarbonyl)-1,4,7-triazacyclononane dihydrochloride 9 (31 mg, 0.10 mmol), methanesulfonate 8 (122 mg, 0.26 mmol), and K₂CO₃ (60 mg, 0.43 mmol) were combined in anhydrous CH₃CN (2 mL). The resulting mixture was heated to 60 °C for 14 hours, and then the crude solution was separated from the inorganic salts. The solvent was removed under reduced pressure to obtain an orange oily substance, which was then passed by reversed-phase HPLC (10 to 100% CH₃CN in H₂O solution, 10 min, t). r Purification was carried out over 15.6 min to obtain a pale yellow oily substance (66 mg, 62%). 1 H-NMR (400MHz, CDCl3) δ8.03–7.96(m,2H),7.66(s,1H),7.58(s,1H),7.17(d,2H),7.08(s,2H),6.82(d,2H),4.15–4.02&3.91–3.78(m,1 0H),3.94(2×s,4H),3.44–3.29(m,4H),3.20–3.04(m,12H),2.76–2.61(m,4H),1.76(2×d,6H),1.48(s,9H),1.24(2×t,6H),1.02(t,12H); 13 C-NMR (101MHz, CDCl3) δ 161.7 (d, 3 J C-P =21),161.5(d, 3 J C-P =21),155.7,155.1,155.0,153.8(d, 1 J C-P =157),153.6(d, 1 J C-P =157),139.9(2×s),132.8(d, 3 J C-P =12),132.7(d, 3 J C-P =12),128.0(d, 2 J C-P =22),127.9(d, 2 JC-P =22),127.1,126.4(d, 4 J C-P =3),126.1(d, 4 J C-P =3),125.0(2×s),113.7,113.6,111.4(2×s),96.5,96.2,85.3(d, 4 J C-P =2),85.1(d, 4 J C-P =2),79.5,62.9,62.6,61.1(2×d, 2 J C-P =6),56.1,55.7,55.0,54.6,54.0,50.0,49.7,46.0,28.8,16.6(d),13.5(d, 1 J C-P =1.5), 13.4(d, 1 J C-P =1.5), 12.0; 31 P{ 1 H}-NMR(162MHz, CDCl3)δ+40.2(1P),+40.1(1P); ESI-LRMS(+)m / z 1027[M+H] + ; ESI-HRMS(+)[C 55 H 78 N7O8P2] + Calculated value: 1026.539; Actual value: 1026.543.

[0326] Compound 16

[0327]

[0328] A solution of compound 15 (66 mg, 0.0643 mmol) was prepared in trifluoroacetic acid and CH2Cl2 (10% v / v, 3 mL total). The solution was stirred at room temperature for 1 hour, and then the solvent was removed under reduced pressure to obtain an orange residue. CH2Cl2 (30 mL) was added to the residue, and the solvent was removed again under reduced pressure. This process was repeated five times to obtain a pale orange oil (67 mg, quantified). 1 H-NMR(600MHz,CD3OD)δ7.97(s,2H),7.95(dd, 3 J H-P =6, 4 J H-H =1.0,2H),7.79(dd, 3 J H-H =8.7,4 J H-H =1.9,2H),7.72–7.70(m,2H),7.41(d, 3 J H-H =8.7,2H),4.24(s,4H),4.17–4.10&4.01–3.95(m,4H),4.07(s,6H),3.70(q, 3 J H-H =7.2,8H),3.42–3.35(m,4H),3.29–3.23(m,4H),3.06–2.99(m,4H),1.82(d, 2 J H-P =15,6H),1.30(t, 3 J H-H =7.0,6H),1.12(t, 3 J H-H =7.2,12H); 13 C-NMR(151MHz,CD3OD)δ160.8(d, 3 J C-P =20),155.6,155.2(d, 1 J C-P =159),136.8,133.8(d, 3 J C-P =12),128.6,128.5(d, 2 J C-P =24),127.5,126.2,116.9,114.9,94.6,87.3(d, 4 J C-P =2),63.1(d, 2 J C-P =7),60.6,57.7,55.0,52.3,49.9,45.5,16.8(d, 3 J C-P =6.2),13.6(d, 1 J C-P =103),10.4, 31 P{ 1 H}-NMR(162MHz,CD3OD)δ+40.8;ESI-LRMS(+)m / z 926[M+H] + ,464[M+2H] 2+ ,309[M+3H] 3+ ;ESI-HRMS(+)[C 50 H 70 N7O6P2] +Calculated value: 926.4863; Actual value: 926.4865.

[0329] Compound 17

[0330]

[0331] Compound 16 (39 mg, 0.0375 mmol), mesylate 10 (36 mg, 0.123 mmol), and K₂CO₃ (30 mg, 0.217 mmol) were combined in anhydrous CH₃CN (2 mL) under argon atmosphere and heated to 60 °C for 18 hours. Subsequently, the crude mixture was separated from the inorganic salts by centrifugation, and the resulting solution was directly subjected to reversed-phase HPLC (10 to 100% CH₃CN in 25 mM ammonium bicarbonate buffer, 10 min, t). r =11.8 min), a pale yellow oily substance (23 mg, 55%) was obtained; 1 H-NMR (600MHz, CDCl3) δ 8.00 (dd, 3 J H-P =6.0, 4 J H-H =1.3,2H),7.93–7.88(m,1H),7.83–7.76(m,1H),7.73–7.67(m,1H),7.65(s,2H),7.18(dd, 3 J H-H =8.5, 4 J H-H =1.8,2H),7.09(d, 4 J H-H =1.8,2H),6.83(d, 3 J H-H =8.5,2H),4.14–4.04&3.90–3.82(m,12H),4.01–3.91(m,4H),3.16(q, 3 J H-H =7.1,8H),3.07–2.83(m,12H),1.76(d, 2 J H-P =15,6H),1.75(d, 2 J H-P =15,3H),1.25(t, 3 J H-H =7.0,6H),1.23(t, 3 J H-H =7.0,3H),1.03(t, 3 J H-H=7.1,12H); ESI-LRMS(+)m / z 1124[M+H] + 563[M+2H] 2+ 375 [M+3H] 3+ ; ESI-HRMS(+)[C 59 H 82 N8O8P3] + Calculated value: 1123.547; Actual measurement: 1123.548.

[0332] Complex 18

[0333]

[0334] Ligand 17 (11.5 mg, 0.01024 mmol) was dissolved in a CH3OH / H2O mixture (1:1, 4 mL total), and the pH was adjusted to 12 using 1 M NaOH aqueous solution. The solution was heated to 60 °C for 15 hours. After cooling and adjusting the pH to 6 using dilute hydrochloric acid (0.1 M), EuCl3·6H2O (6 mg, 0.0164 mmol) was added, and the reaction mixture was heated to 60 °C for 17 hours. Subsequently, the solution was separated from the inorganic salts by centrifugation and analyzed by reversed-phase HPLC (10 to 100% CH3OH in H2O solution, 10 min, t). r Purification was carried out over 13.6 min to obtain a yellow solid (7 mg, 58%); ESI-LRMS (+) m / z 1189 [M+H] + 595[M+2H] 2+ ,397[M+3H] 3+ ; ESI-HRMS(+)[C 53 H 68 N8O8P3 151 Eu] 2+ Calculated value: 595.1796; Actual measurement: 595.1741; τ H2O (ms)=0.34(pH 9), 0.34(pH 8), 0.47(pH 7), 0.78(pH 6), 0.96(pH 5), 1.00(pH 4).

[0335] Compound 19 is commercially available.

[0336] Compound 20

[0337]

[0338] Under argon atmosphere, 1,4,7-triazacyclononane trihydrochloride 19 (4.5 mg, 0.0189 mmol), compound 8 (33 mg, 0.067 mmol), and K₂CO₃ (20 mg, 0.145 mmol) were combined in anhydrous CH₃CN (1.5 mL) and heated to 60 °C for 17 hours. Subsequently, the crude mixture was separated from the inorganic salts by filtration. The resulting solution was directly subjected to reversed-phase HPLC (10 to 100% CH₃CN in H₂O solution, 10 min, t). r =16.5min), a pale yellow oily substance (8mg, 32%) was obtained; 1 H-NMR (600MHz, CDCl3) δ 8.01 (d, 3 J H-P =5.0,3H),7.68(s,3H),7.18(dd, 3 J H-H =8.5, 4 J H-H =1.9,3H),7.09(d, 4 J H-H =1.9,3H),6.82(d, 3 J H-H =8.5,3H),4.13–4.05&3.87–3.81(m,6H),3.92(s,6H),3.88(s,9H),3.16(q, 3 J H-H =7.2,12H),2.94(br s,12H),1.76(d, 2 J H-P =15,9H),1.24(t, 3 J H-H =7.0,9H),1.03(t, 3 J H-H =7.2,18H); 13 C-NMR (151MHz, CDCl3) δ 161.9 (d, 3 J C-P =20),155.4,154.0(d, 1 J C-P =157),139.8,133.0(d, 3 J C-P =12),128.0(d, 2 J C-P =23),127.3,126.8,125.2,113.9,111.7,96.6,85.5,64.2,61.3(d, 2 J C-P=7),56.1,55.9,46.3,16.8(d, 3 J C-P =6),13.7(d, 1 J C-P =104), 12.3; 31 P{ 1 H}-NMR(162MHz, CDCl3)δ+40.1; ESI-LRMS(+)m / z1325[M+H] + ,663[M+2H] 2+ ,443{M+3H] 3+ ; ESI-HRMS(+)[C 72 H 98 N9O9P3] 2+ The calculated value is 662.8350, and the actual measured value is 662.8334.

[0339] Complex 21

[0340]

[0341] Ligand 20 (8 mg, 6 μmol) was dissolved in a CH3OH / H2O mixture (1:1, 2 mL total), and the pH was adjusted to 12 using a 1.0 M NaOH aqueous solution. The solution was heated to 60 °C for 4 hours. After cooling and adjusting the pH to 7 using 1.0 M hydrochloric acid, EuCl3·6H2O (3 mg, 8 μmol) was added, and the reaction mixture was heated to 60 °C for 19 hours. The reaction mixture was then subjected to reversed-phase HPLC (10 to 100% CH3CN in H2O solution, 10 min, t). r Purification was carried out over 12.4 min to obtain a yellow solid (4 mg, 48%); ESI-LRMS (+) m / z 1390 [M+H] + 696[M+2H] 2+ ,464[M+3H] 3+ ; ESI-HRMS(+)[C 66 H 82 N9O9P3 151 Eu] + Calculated value: 1390.467; Actual measurement: 1390.470; τ H2O (ms)=0.24 (pH 9), 0.25 (pH 8), 0.32 (pH 7), 0.59 (pH 6), 0.83 (pH 5), 0.84 (pH 4); ε 331nm =45000L M -1 cm -1 .

[0342]

[0343] Under argon atmosphere, ethyl 4-bromobutyrate 23 (2.3 mL, 17.2 mmol) was added to a mixture of 22 (2.49 g, 11.4 mmol) of 4-bromo-2-nitrophenol and 2.31 g (16.7 mmol) of K₂CO₃ in anhydrous CH₃CN (40 mL). The mixture was heated to 70 °C for 64 hours, and then the solvent was removed under reduced pressure. CH₂Cl₂ (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 then the solvent was removed under reduced pressure to obtain a crude residue, which was purified by column chromatography (SiO₂, 1:1 hexane / CH₂Cl₂ to 100% CH₂Cl₂) to give a pale yellow oil (3.38 g, 98%). 1 H-NMR (700MHz, CDCl3) δ 7.95 (d, 4 J H-H =2.5,1H),7.60(dd, 3 J H-H =8.9, 4 J H-H =2.5,1H),6.97(d, 3 J H-H =8.9,1H),4.17–4.12(m,4H),2.55(t, 3 J H-H =7.0,2H),2.18–2.12(m,2H),1.25(t, 3 J H-H =7.1); 13 C-NMR (176MHz, CDCl3) δ173.1,151.6,140.4,137.0,128.5,116.3,112.1,68.9,60.8,30.3,24.3,14.4; ESI-LRMS(+)m / z 332[M+H] + ; ESI-HRMS(+)[C 12 H 15 NO5Br] + Calculated value: 332.0134; Actual measured value: 332.0146; R f =0.47 (SiO2, 100% CH2Cl2).

[0344] Compound 25

[0345]

[0346] 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 combined in EtOH (20 mL) under argon atmosphere and heated to 50 °C for 6 hours. The mixture was cooled, filtered, and the solvent was removed under reduced pressure. DCM (50 mL) was added to the resulting mixture, and the resulting solution was washed with saturated Na₄EDTA aqueous solution (3 × 40 mL) and H₂O (2 × 40 mL). The combined aqueous layers were extracted with CH₂Cl₂ (6 × 40 mL). The combined organic layers were dried over K₂CO₃ and the solvent was removed under reduced pressure to obtain a pale golden oil (3.5 g, 90%). 1 H-NMR (700MHz, CDCl3) δ 6.75 (d, 4 J H-H =2.4,1H),6.70(dd, 3 J H-H =8.4, 4 J H-H =2.4,1H),6.55(d, 3 J H-H =8.4,1H),4.11(q, 3 J H-H =7.2,2H),3.93(t, 3 J H-H =6.1,2H),3.89(br s,2H),2.46(t, 3 J H-H =7.1,1H),2.11–2.06(m,2H),1.22(t, 3 J H-H =7.2,3H); 13 C-NMR (176MHz, CDCl3) δ173.1,145.3,138.0,120.3,117.2,113.3,112.6,67.4,60.5,31.0,24.6,14.2; ESI-LRMS(+)m / z 302[M+H] + ; ESI-HRMS(+)[C 12 H 17 NO3Br] + Calculated value: 302.0392; Actual value: 302.0402.

[0347]

[0348] Under argon atmosphere, iodoethane (4 mL, 50 mmol) was added to an anhydrous CH3CN (10 mL) solution of compound 25 (3.19 g, 10.6 mmol) and K2CO3 (3.73 g, 27.0 mmol). The mixture was heated to 70 °C for 65 hours, and then the solvent was removed under reduced pressure. The resulting residue was dissolved in CH2Cl2 (50 mL), washed with H2O (4 × 50 mL), and then dried over K2CO3. The solvent was removed under reduced pressure to obtain a crude residue, which was purified by column chromatography (SiO2, pure CH2Cl2 to 1% CH3OH in CH2Cl2 solution) to give a light red oil (2.51 g, 66%). 1 H-NMR(700MHz,CDCl3)δ6.99–6.96(m,2H),6.69(d, 3 J H-H =8.4,1H),4.13(q, 3 J H-H =7.2,2H),3.99(t, 3 J H-H =6.3,2H),3.13(q, 3 J H-H =7.0,4H),2.51(t, 3 J H-H =7.4,2H),2.15–2.10(m,2H),1.25(t, 3 J H-H =7.0,3H),1.04(t, 3 J H-H =7.0,6H); 13 C-NMR (176MHz, CDCl3) δ173.2,151.8,141.8,124.4,124.1,114.8,113.4,67.8,60.6,45.7,31.0,24.9,14.4,12.5; ESI-LRMS(+)m / z 358[M+H] + ; ESI-HRMS(+)[C 16 H 25 NO3Br] + Calculated value: 358.1018; Actual measurement: 358.1020; R f =0.04 (SiO2, pure CH2Cl2), 0.3 (1% CH3OH in CH2Cl2 solution).

[0349] Compound 27

[0350]

[0351] Compound 26 (3.03 g, 8.46 mmol) and Pd₂Cl₂(allyl)₂ (310 mg, 0.85 mmol) were combined in a container, which was degassed and then refilled with argon for three cycles. Anhydrous CH₃CN (15 mL) was then added. P(t-Bu)₃ (0.31 mL, 1.28 mmol), trimethylsilylacetylene (2.4 mL, 17.3 mmol), and piperidine (2.1 mL, 21.3 mmol) were added sequentially to the mixture. The reaction mixture was stirred at 34 °C for 30 hours. The solvent was then removed under reduced pressure, and the residue was dissolved in CH₂Cl₂ (50 mL). The solution was washed with H₂O (4 × 50 mL) and then dried over K₂CO₃. The solvent was removed under reduced pressure to obtain a brown oily substance, which was then purified by column chromatography (SiO2, 100% hexane to 6% EtOAc hexane solution) to obtain a yellow oily substance (2.29 mg, 75%). 1 H-NMR (600MHz, CDCl3) δ 7.06 (dd, 3 J H-H =8.4, 4 J H-H =2.0,1H),7.01(d, 4 J H-H =2.0,1H),6.74(d, 3 J H-H =8.4,1H),4.14(q, 3 J H-H =7.1,2H),4.03(t, 3 J H-H =6.1,2H),3.13(q, 3 J H-H =7.2,4H),2.52(t, 3 J H-H =7.3,2H),2.17–2.11(m,2H),1.27(t, 3 J H-H =7.1,3H),1.03(t, 3 J H-H =7.2,6H),0.24(s,9H); 13 C-NMR (151MHz, CDCl3) δ173.1,153.3,139.7,126.6,125.0,115.2,112.6,105.8,91.7,67.3,60.4,45.6,30.9,24.6,14.2,12.3,0.10; 29 Si NMR(139MHz, CDCl3)δ-16.0; ESI-LRMS(+)m / z 376[M+H]+ ; ESI-HRMS(+)[C 21 H 34 NO3Si] + Calculated value: 376.2308; Actual measured value: 376.2299; R f =0.3 (SiO2, 10% EtOAc hexane solution).

[0352] Compound 28

[0353]

[0354] Under argon atmosphere, triethylamine trihydrofluoric acid (6.25 mL, 38 mmol) was added to an anhydrous THF (8 mL) solution of compound 27 (960 mg, 2.56 mmol). The solution was stirred at 30 °C for 24 hours, and then the solvent was removed under reduced pressure. The residue was then dissolved in CH₂Cl₂ (30 mL) and washed with water (6 × 30 mL). The combined aqueous layers were extracted with CH₂Cl₂ (2 × 30 mL) and dried over K₂CO₃ to give a yellow oil (749 mg, 97%). The product was used for the next step without further purification. 1 H-NMR (700MHz, CDCl3) δ 7.07 (dd, 3 J H-H =8.3, 4 J H-H =2.0,1H),7.03(d, 4 J H-H =2.0,1H),6.75(d, 3 J H-H =8.3,1H),4.13(q, 3 J H-H =7.2,2H),4.03(t, 3 J H-H =6.3,2H),3.13(q, 3 J H-H =7.2,4H),2.97(s,1H),2.52(t, 3 J H-H =7.4,2H),2.17–2.12(m,2H),1.25(t, 3 J H-H =7.2,3H),1.03(t, 3 J H-H =7.1,6H); 13C-NMR (176MHz, CDCl3) δ173.1,153.4,139.8,126.4,125.0,114.0,112.7,84.3,75.2,67.3,60.4,45.6,30.9,24.6,14.2,12.3; ESI-LRMS(+)m / z 304[M+H] + ; ESI-HRMS(+)[C 18 H 26 NO3] + Calculated value: 304.1913; Actual value: 304.1924.

[0355] Compound 29

[0356]

[0357] Compound 28 (528 mg, 1.74 mmol) and (6-(hydroxymethyl)-4-(bromopyridin-2-yl)(methyl)phosphinic acid ethyl ester 19 (486 mg, 1.65 mmol) were combined in anhydrous CH3CN (10 mL) under argon atmosphere. Pd2Cl2(allyl)2 (70 mg, 0.191 mmol) and P( t Bu)3 (0.06 mL, 0.25 mmol) and piperidine (0.43 mL, 4.70 mmol). The resulting mixture was stirred at 40 °C for 36 hours under argon atmosphere, followed by solvent removal under reduced pressure. The residue was dissolved in CH2Cl2 (40 mL), washed with H2O (3 × 40 mL), dried over K2CO3, and then solvent removed under reduced pressure. The crude product was subjected to reversed-phase HPLC (10 to 100% CH3CN in H2O solution, 10 min, t). r Purification was carried out over 12.2 min to obtain a light orange oil (359 mg, 40%). 1 H-NMR (400MHz, CDCl3) δ 8.04 (d, 3 J H-P =6.0, 1H), 7.47 (s, 1H), 7.13 (d, 3 J H-H =8.4, 1H), 7.07 (s, 1H), 6.82 (d, 3 J H-H =8.4,1H),4.81(s,2H),4.22–3.78(m,6H),3.17(q, 3 J H-H =6.9,4H),2.53(t, 3 J H-H =7.2,2H),2.22–2.11(m,2H),1.78(d,2 J H-P =15,3H),1.31–1.22(m,6H),1.06(t, 3 J H-H =6.9,6H); 13 C-NMR(101MHz,CDCl3)δ173.0,160.4(d, 3 J C-P =19),153.9,153.1(d, 1 J C-P =155),140.0,133.2(d, 3 J C-P =11),128.3(d, 2 J C-P =22),126.5,124.8,124.0(d, 4 J C-P =3),113.6,112.8,97.1,84.7,67.4,64.0,61.2(d, 2 J C-P =6.2),60.5,45.6,30.8,24.6,16.5(d, 3 J C-P =6.0), 14.2, 13.5(d, 1 J C-P =105), 12.3; 31 P{ 1 H}-NMR(162MHz, CDCl3)δ+39.4; ESI-LRMS(+)m / z 517[M+H] + ; ESI-HRMS(+)[C 27 H 38 N2O6P] + Calculated value: 517.2468; Actual value: 517.2451.

[0358] Compound 30

[0359]

[0360] Compound 29 (250 mg, 0.484 mmol), methanesulfonic anhydride (126 mg, 0.72 mmol), and DIPEA (0.25 mL, 1.44 mmol) were combined in anhydrous THF (2 mL) under argon atmosphere. The reaction mixture was stirred at room temperature for 1 hour, and the reaction progress was monitored by TLC. After complete conversion, the solvent was removed under reduced pressure, and the resulting crude residue was dissolved in CH2Cl2 (40 mL), washed with water (3 × 40 mL), and the organic layer was dried over K2CO3. The solvent was removed under reduced pressure to obtain an orange oil (288 mg, quantitative). 1 H-NMR (400MHz, CDCl3) δ 8.10 (d, 3 J H-P =6.0, 1H), 7.64 (s, 1H), 7.15 (d, 3 J H-H =8.4, 1H), 7.09 (s, 1H), 6.83 (d, 3 J H-H =8.4,1H),5.37(s,2H),4.19–3.82(m,6H),3.18(q, 3 J H-H =7.0,4H),3.14(s,3H),2.54(t, 3 J H-H =7.2,2H),2.22–2.13(m,2H),1.78(d, 2 J H-P =15,3H),1.32–1.24(m,6H),1.07(t, 3 J H-H =6.9,6H); ESI-LRMS(+)m / z 595[M+H] + ; ESI-HRMS(+)[C 28 H 40 N2O8SP] + Calculated value: 595.2242; Actual measurement: 595.2236; R f =0.4 (SiO2, 5% CH3OH in CH2Cl2 solution).

[0361] Compound 32

[0362]

[0363] Under an inert atmosphere, 3-(Boc-amino)propylbromide 31 (14.1 g, 58.4 mmol), Cs₂CO₃ (22.2 g, 67.4 mmol), and Nal (1.7 g, 11.2 mmol) were added to an anhydrous CH₃CN (240 mL) solution of 4-bromo-2-nitrophenol 22 (10 g, 45 mmol). 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 was performed by silica gel chromatography (cyclohexane / EtOAc 8 / 2, then 7 / 3, then 6 / 4) to give compound 32 (15.9 g, 94%) as a yellow oil that solidified upon standing. 1 H NMR (CDCl3, 400MHz) δ (ppm) 7.98 (d, J = 2.5Hz, 1H), 7.62 (dd, J = 8.9Hz, J = 2.5Hz, 1H), 6.98 (d, J = 8.9Hz, 1H), 4.95 (br s,1H),4.15(t,J=5.9Hz,2H),3.36-3.32(m,2H),2.06-2.01(m,2H),1.43(s,9H); 13 C NMR (CDCl3, 100MHz) δ (ppm) 156.3, 151.6, 140.2, 137.1, 128.6, 116.2, 112.2, 79.4, 68.3, 38.0, 29.3, 28.5.

[0364] Compound 33

[0365]

[0366] Zinc (14.1 g, 211 mmol) was added to a solution of compound 32 (15.9 g, 42.4 mmol) in dioxane (133 mL) and H₂O (26 mL), followed by NH₄Cl (11.4 g, 211 mmol) at 0 °C. After 15 minutes at 0 °C, the reaction mixture was stirred at 50 °C for 20 hours. Then it was subjected to… The mixture was filtered and washed with EtOAc. The organic layer was washed successively with H2O and brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain crude compound 33 (14.4 g, 99%), which was a brown oily substance. 1H NMR (CDCl3, 400MHz) δ (ppm) 6.84-6.81 (d, J = 2.3Hz, 1H), 6.79-6.76 (dd, J = 2.3Hz, J = 8.5Hz, 1H), 6.62 (d, J = 8.5Hz, 1H), 4.69 (br s,1H),4.02(t,J=6.0Hz,2H),3.70(br s,2H),3.34-3.31(m,2H),2.01-1.96(m,2H),1.44(s,9H); 13 C NMR (CDCl3, 100MHz) δ (ppm) 156.1, 145.2, 138.1, 120.7, 117.6, 113.6, 113.0, 79.5, 66.3, 37.9, 29.8, 28.5; ESI-LRMS (+) m / z (M) + 344.49.

[0367] Compound 34

[0368]

[0369] In a sealed flask, K₂CO₃ (7.38 g, 52.9 mmol) and Etl (8.11 mL, 99.4 mmol) were added to a CH₃CN (23 mL) solution of compound 33 (7.3 g, 21.1 mmol). The reaction mixture was stirred at 70 °C for 16 hours, filtered through a Büchner filter, and washed with EtOAc. The mother liquor was washed with water, dried over Na₂SO₄, filtered, and concentrated under reduced pressure. Purification was performed by silica gel chromatography (cyclohexane / EtOAc 8:2) to give compound 34 (7.04 g, 83%) as a pale yellow oil. 1 H NMR (CDCl3, 400MHz) δ (ppm) 7.07-7.03 (m, 2H), 6.70 (d, J = 8.9Hz, 1H), 5.7 (br s,1H),4.03(t,J=5.8Hz,2H),3.37-3.33(m,2H),3.13(q,J=7.1Hz,4H),2.00-1.95(m,2H),1.45(s,9H),1.01(t,J=7.1Hz,6H); 13 C NMR (CDCl3, 100MHz) δ (ppm) 156.3, 152.7, 141.4, 125.4, 125.0, 114.6, 113.4, 79.0, 68.5, 46.2, 39.2, 29.6, 28.6, 12.0; ESI-LRMS (+) m / z (M+H) + 403.0.

[0370] Compound 35

[0371]

[0372] In a sealed flask, an anhydrous THF solution (18.9 mL) of compound 34 (5.4 g, 13.5 mmol) was degassed with argon. Then, triethylamine (18.9 mL, 135 mmol), trimethylsilylacetylene (12.2 mL, 85.4 mmol), Cul (1.09 g, 5.73 mmol), and Pd(PPh3)2Cl2 (2 g, 2.85 mmol) were added. The mixture was then degassed with argon and stirred at 65 °C for 16 hours. The reaction mixture was then subjected to… The mixture was filtered, washed with EtOAc, and concentrated under reduced pressure. The residue was dissolved in EtOAc, and the organic layer was washed successively with H2O and brine, dried over Na2SO4, filtered, and concentrated under reduced pressure. Purification was performed by silica gel chromatography (cyclohexane / EtOAc 10:0 to 8:2) to obtain compound 35 (6.4 g) with a purity of 80%. A second purification was performed by silica gel chromatography (CH2Cl2 / EtOAc 100:0 to 95:5) to obtain compound 35 (3.5 g, 61%) as an orange oil. 1 H NMR (CDCl3, 400MHz) δ (ppm) 7.12 (d, J = 8.3Hz, 1H), 7.08 (s, 1H), 6.74 (d, J = 8.3Hz, 1H), 5.96 (br s,1H),4.08-4.03(t,J=5.7Hz,2H),3.37-3.32(m,2H),3.15-3.08(q,J=7.0 Hz, 4H), 2.01-1.96 (m, 2H), 1.45 (s, 9H), 0.99 (t, J = 7.1Hz, 6H), 0.24 (s, 9H); 13 C NMR(CDCl3,100MHz)δ(ppm)156.4,154.4,139.6,127.6,125.9,115.6,112.7,10 5.8,92.1,79.0,68.4,46.4,39.3,29.7,28.7,12.1,0.2;ESI-LRMS(+)m / z(M+H) + 419.15.

[0373] Compound 36

[0374]

[0375] Under argon atmosphere, triethylamine trihydrofluoric acid (1.1 mL, 6.75 mmol) was added to an anhydrous THF (5 mL) solution of compound 35 (185 mg, 0.442 mmol). The solution was stirred at 30 °C for 38 hours, and then the solvent was removed under reduced pressure. The residue was then dissolved in CH₂Cl₂ (30 mL) and washed with water (3 × 30 mL). The combined aqueous layers were extracted with CH₂Cl₂ (2 × 30 mL), and the combined organic layers were dried over K₂CO₃ to give a pale yellow oil (74 mg, 48%). The product was used for the next step without further purification. 1 H-NMR (600MHz, CDCl3) δ 7.13 (d, 3 J H-H =8.2,1H),7.10(s,1H),6.76(d, 3 J H-H =8.2,1H),4.06(t, 3 J H-H =5.7,2H),3.40–3.31(m,2H),3.12(q, 3 J H-H =7.0,4H),2.98(s,1H),2.02–1.95(m,2H),1.45(s,9H),0.99(t, 3 J H-H =7.0,6H); 13 C-NMR (151MHz, CDCl3) δ156.4,154.5,139.6,127.5,125.9,114.4,112.7,84.3,79.0,75.5,68.4,46.3,39.3,29.7,28.6,12.0; ESI-LRMS(+)m / z 347[M+H] + ; ESI-HRMS(+)[C 20 H 31 N2O3] + Calculated value: 347.2353; Actual value: 347.2350.

[0376] Compound 37

[0377]

[0378] Compound 36 (172 mg, 0.496 mmol) and ethyl 6-(6-(hydroxymethyl)-4-(bromopyridin-2-yl)(methyl)phosphinic acid ester (139 mg, 0.473 mmol) were combined in anhydrous CH3CN (3 mL) under argon atmosphere. Pd2Cl2(allyl)2 (20 mg, 0.055 mmol), P(tBu)3 (0.02 mL, 0.082 mmol), and piperidine (0.12 mL, 1.22 mmol) were added sequentially to this solution. The resulting mixture was stirred at 35 °C for 24 hours under argon atmosphere, followed by solvent removal under reduced pressure. The residue was dissolved in CH2Cl2 (40 mL), washed with H2O (3 × 40 mL), and dried over K2CO3. Solvent removal under reduced pressure yielded a brown oily substance, which was subjected to reversed-phase HPLC (10 to 100% CH3CN, 10 min, t r Purification was carried out over 11.7 min to obtain a light yellow oily substance (64 mg, 24%). 1 H-NMR (700MHz, CDCl3) δ 8.03 (d, 3 J H-P =6.0, 1H), 7.50 (s, 1H), 7.17 (d, 3 J H-H =8.2,1H),7.12(s,1H),6.81(d, 3 J H-H =8.2,1H),5.83(br s,1H),4.80(s,2H),4.14–4.06&3.89–3.82(m,2H),4.08(t, 3 J H-H =5.8,2H),3.38–3.32(m,2H),3.15(q, 3 J H-H =7.0,4H),2.03–1.97(m,2H),1.77(d, 2 J H-P =15,3H),1.44(s,9H),1.26(t, 3 J H-H =7.1,3H),1.01(t, 3 J H-H =7.0,6H); 13 C-NMR (176MHz, CDCl3) δ 160.8 (d, 3 J C-P =19),156.4,155.0,153.3(d, 1 J C-P =155),139.8,133.2(d, 3 J C-P=11),128.3(d, 2 J C-P =22),127.5,125.7,124.1(d, 4 J C-P =3),113.9,112.8,96.9,85.0(d, 4 J C-P =2),79.1,68.4,64.2,61.3(d, 2 J C-P =7),46.2,39.2,29.6,28.6,16.5(d, 3 J C-P =6), 13.5(d, 1 J C-P =105),12.1; 31 P{ 1 H}-NMR(162MHz, CDCl3)δ+39.5; ESI-LRMS(+)m / z 560[M+H] + ; ESI-HRMS(+)[C 29 H 43 N3O6P] + Calculated value: 560.2889; Actual value: 560.2879.

[0379] Compound 38

[0380]

[0381] Compound 37 (40 mg, 0.072 mmol), methanesulfonic anhydride (25 mg, 0.143 mmol), and DIPEA (0.025 mL, 0.025 mmol) were combined in anhydrous THF (2 mL) under argon atmosphere. 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 CH2Cl2 (40 mL), washed with water (3 × 40 mL), and the combined aqueous layers were extracted with CH2Cl2 (2 × 40 mL). The combined organic layers were dried over K2CO3 and the solvent was removed under reduced pressure to give a yellow oil (45 mg, 100%). 1 H-NMR (400MHz, CDCl3) δ 8.09 (dd, 3 J H-P =6.0, 4 J H-H =1.4,1H),7.63(s,1H),7.19(dd, 3 J H-H =8.2, 4 J H-H =1.6,1H),7.14(d,4 J H-H =1.6,1H),6.82(d, 3 J H-P =8.2,1H),5.86(br s,1H),5.36(s,2H),4.18–4.02&3.94–3.80(m,4H),3.40–3.31(m,2H),3.19–3.12(m,7H),2.03–1.99(m,2H),1.77(d, 2 J H-P =15,3H),1.44(s,9H),1.27(t, 3 J H-H =7.1,3H),1.04–1.00(m,6H); ESI-LRMS(+)m / z 638[M+H] + ; ESI-HRMS(+)[C 30 H 45 N3O8SP] + Calculated value: 638.2665; Actual measurement: 638.2676.

[0382] Compound 39

[0383]

[0384] Under argon atmosphere, iodoethane (1.21 mL, 15.0 mmol) was added to an anhydrous CH3CN (3 mL) solution of compound 24 (3.50 g, 11.6 mmol) and K2CO3 (4.8 g, 34.5 mmol). The mixture was heated to 55 °C for 70 hours, and the inorganic salts were removed by filtration, followed by solvent removal under reduced pressure. The resulting oil was purified by column chromatography (SiO2, 100% CH2Cl2) to give a pale yellow oil (1.65 g, 43%). 1 H-NMR (700MHz, CDCl3) δ 6.70 (dd, 3 J H-H =8.3, 1H), 6.67 (s, 1H), 6.57 (d, 3 J H-H =8.3,1H),4.15(q, 3 J H-H =7.1,2H),4.00(t, 3 J H-H =6.1,2H),3.13(q, 3 J H-H =7.2,2H),2.49(t, 3 J H-H=7.1,2H),2.18–2.11(m,2H),1.29(t, 3 J H-H =7.2,3H),1.25(t, 3 J H-H =7.1,3H); 13 C-NMR (176MHz, CDCl3) δ173.3,145.0,139.8,118.4,114.3,112.7,111.6,67.6,60.7,38.2,31.3,24.7,14.7,14.4; ESI-LRMS(+)m / z 331[M+H] + ; ESI-HRMS(+)[C 14 H 21 NO3Br] + Calculated value: 330.0705; Actual measured value: 330.0714; R f =0.4 (SiO2, pure CH2Cl2).

[0385] Compound 40

[0386]

[0387] Compound 39 (939 mg, 2.84 mmol), paraformaldehyde (415 mg, 4.61 mmol), and a few drops of acetic acid were combined in anhydrous EtOH (25 mL). The mixture was stirred at room temperature for 20 minutes under argon atmosphere, at which point NaBH3CN (640 mg, 10 mmol) was added, and the reaction was stirred for another 48 hours. The solvent was removed under reduced pressure, and the resulting residue was dissolved in CH2Cl2 (30 mL) and washed with NaHCO3 solution (1 × 30 mL) and water (3 × 30 mL). The organic layer was dried over K2CO3, and the solvent was removed under reduced pressure to obtain a pale yellow oil (833 mg, 85%). 1 H-NMR (400MHz, CDCl3) δ 7.00 (dd, 3 J H-H =8.3, 4 J H-H =2.3,1H),6.98(d, 4 J H-H =2.3,1H),6.70(d, 3 J H-H =8.3,1H),4.16(q, 3 J H-H =7.1,2H),4.02(t, 3 J H-H =6.3,2H),3.13(q, 3 JH-H =7.2,2H),2.76(s,2H),2.54(t, 3 J H-H =7.3,2H),2.17–2.10(m,2H),1.27(t, 3 J H-H =7.1,3H),1.12(t, 3 J H-H =7.2,3H); 13 C-NMR (176MHz, CDCl3) δ172.9,150.6,143.3,123.9,118.0,113.8,113.3,67.4,60.5,49.2,39.0,30.8,24.7,14.2,12.2; ESI-LRMS(+)m / z 344[M+H] + ESI-HRMS(+)[C 15 H 23 NO3Br] + Calculated value: 344.0861; Actual value: 344.0872.

[0388] Compound 41

[0389]

[0390] Compound 40 (1.47 g, 4.27 mmol) and Pd₂Cl₂(allyl)₂ (160 mg, 0.44 mmol) were combined in a container, which was degassed and then refilled with argon for three cycles. Anhydrous CH₃CN (8 mL) was then added. P(t-Bu)₃ (0.16 mL, 0.66 mmol), trimethylsilylacetylene (1.2 mL, 8.66 mmol), and piperidine (1.0 mL, 10.1 mmol) were added sequentially to the mixture. The reaction mixture was stirred at 35 °C for 19 hours. The solvent was then removed under reduced pressure, and the residue was dissolved in CH₂Cl₂ (50 mL). The solution was washed with H₂O (2 × 50 mL) and dried over K₂CO₃. The solvent was removed under reduced pressure to obtain a brown oily substance, which was purified by column chromatography (SiO2, 100% hexane to 7% EtOAc hexane solution) to obtain the desired compound as a pale orange oily substance (1.19 g, 77%). 1 H-NMR (600MHz, CDCl3) δ 7.05 (d, 3 J H-H =8.2,1H),7.00(s,1H),6.73(d, 3 J H-H =8.2,1H),4.14(q, 3 J H-H=7.1,2H),4.04(t, 3 J H-H =6.2,2H),3.09(d, 3 J H-H =7.0,2H),2.75(s,3H),2.53(t, 3 J H-H =7.3,2H),2.20–2.13(m,2H),1.25(t, 3 J H-H =7.1,3H),1.09(t, 3 J H-H =7.0,3H),0.24(s,9H); 13 C-NMR (151MHz, CDCl3) δ173.2,152.3,141.7,126.4,122.7,115.4,112.1,105.9,92.0,67.3,60.6,49.5,39.3,31.0,24.8,14.4,12.3,0.2; 29 Si-NMR(139MHz, CDCl3)δ-18.3; ESI-LRMS(+)m / z 362[M+H] + ; ESI-HRMS(+)[C 20 H 32 NO3Si] + Calculated value: 362.2151; Actual measured value: 362.2162; R f =0.2 (SiO2, 10% EtOAc hexane solution).

[0391] Compound 42

[0392]

[0393] Under argon atmosphere, triethylamine trihydrofluoric acid (3.6 mL, 22.0 mmol) was added to an anhydrous THF (5 mL) solution of compound 41 (530 mg, 1.47 mmol). The solution was stirred at 30 °C for 17 hours, and then the solvent was removed under reduced pressure. The residue was then dissolved in CH₂Cl₂ (30 mL) and washed with water (3 × 30 mL). The combined aqueous layers were extracted with CH₂Cl₂ (2 × 30 mL), and the combined organic layers were dried over K₂CO₃ to give a yellow oil (410 mg, 97%). The product was used for the next step without further purification. 1 H-NMR (600MHz, CDCl3) δ 7.07 (dd, 3 J H-H =8.3, 4 J H-H=2.0,1H),7.02(d, 4 J H-H =2.0,1H),6.75(d, 3 J H-H =8.3,1H),4.14(q, 3 J H-H =7.1,2H),4.04(t, 3 J H-H =6.3,2H),3.10(q, 3 J H-H =7.2,2H),2.97(s,1H),2.75(s,3H),2.53(t, 3 J H-H =7.3,2H),2.21–2.14(m,2H),1.25(t, 3 J H-H =7.1,3H),1.10(t, 3 J H-H =7.2,3H); 13 C-NMR (151MHz, CDCl3) δ173.2,152.5,141.8,126.3,122.8,114.3,112.2,84.4,75.4,67.3,60.6,49.4,39.2,31.0,24.8,14.4,12.4; ESI-LRMS(+)m / z 290[M+H] + ; ESI-HRMS(+)[C 17 H 24 NO3] + Calculated value: 290.1756; Actual measurement: 290.1752.

[0394] Compound 43

[0395]

[0396] Compound 42 (396 mg, 1.37 mmol) and (6-(hydroxymethyl)-4-(bromopyridin-2-yl)(methyl)phosphinic acid ethyl ester 19 (362 mg, 1.23 mmol) were combined in anhydrous CH3CN (10 mL) under argon atmosphere. Pd2Cl2(allyl)2 (50 mg, 0.137 mmol) and P( tBu)3 (0.05 mL, 0.21 mmol) and piperidine (0.4 mL, 4.37 mmol). The resulting mixture was stirred at 40 °C for 36 hours under argon atmosphere, followed by solvent removal under reduced pressure. The residue was dissolved in CH2Cl2 (40 mL), washed with H2O (3 × 40 mL), dried over K2CO3, and the solvent removed under reduced pressure to obtain an orange oil. This oil was then subjected to reversed-phase HPLC (10 to 100% CH3CN, 10 min, t). r Purification was carried out over 13.2 min to obtain a light orange oil (185 mg, 30%). 1 H-NMR (600MHz, CDCl3) δ 8.02 (d, 3 J H-P =6.0,1H),7.47(s,1H),7.12(dd, 3 J H-H =8.3, 4 J H-H =2.0,1H),7.05(d, 4 J H-H =2.0,1H),6.80(d, 3 J H-H =8.3,1H),4.80(s,2H),4.14(q, 3 J H-H =7.2,2H),4.12–4.08&3.90–3.83(m,2H),4.07(t, 3 J H-H =6.4,2H),3.12(q, 3 J H-H =7.0,2H),2.78(s,3H),2.53(t, 3 J H-H =7.3,2H),2.21–2.15(m,2H),1.77(d, 2 J H-P =15,3H),1.27(t, 3 J H-H =7.0,3H),1.25(t, 3 J H-H =7.2,3H),1.12(t, 3 J H-H =7.0,3H); 13 C-NMR(151MHz,CDCl3)δ173.1,160.7(d, 3 J C-P =19),153.3(d, 1 J C-P=155),153.1,142.1,133.3(d, 3 J C-P =11),128.3(d, 2 J C-P =22),126.4,124.1(d, 4 J C-P =3),122.6,113.9,112.3,97.1,84.9,67.4,64.2,61.3(d, 2 J C-P =6),60.6,49.4,39.2,31.0,24.7,16.6(d, 3 J C-P =6),14.3,13.6(d, 1 J C-P =104), 12.4; 31 P{ 1 H}-NMR(162MHz, CDCl3)δ+39.5; ESI-LRMS(+)m / z 503[M+H] + ; ESI-HRMS(+)[C 26 H 36 N2O6P] + Calculated value: 503.2311; Actual value: 503.2297.

[0397] Compound 44

[0398]

[0399] Compound 43 (160 mg, 0.318 mmol), methanesulfonic anhydride (111 mg, 0.637 mmol), and DIPEA (0.11 mL, 0.632 mmol) were combined in anhydrous THF (4 mL) under argon atmosphere. The reaction mixture was stirred at room temperature for 1 hour, during which time the solvent was removed under reduced pressure. The resulting crude residue was dissolved in CH2Cl2 (40 mL), washed with water (3 × 40 mL), and the combined aqueous layers were extracted with CH2Cl2 (3 × 40 mL). The combined organic layers were dried over K2CO3 and the solvent was removed under reduced pressure to give an orange oil (167 mg, 90%). 1 H-NMR (400MHz, CDCl3) δ 8.10 (d, 3 J H-P =6.0, 1H), 7.64 (s, 1H), 7.15 (d, 3 J H-H =8.4, 1H), 7.09 (s, 1H), 6.83 (d, 3 J H-H=8.4,1H),5.37(s,2H),4.19–3.82(m,6H),3.18(q, 3 J H-H =7.0,4H),3.14(s,3H),2.54(t, 3 J H-H =7.2,2H),2.22–2.13(m,2H),1.78(d, 2 J H-P =15,3H),1.32–1.24(m,6H),1.07(t, 3 J H-H =6.9,6H); ESI-LRMS(+)m / z 595[M+H] + ; ESI-HRMS(+)[C 27 H 38 N2O8SP] + Calculated value: 581.2087; Actual measured value: 581.2099; R f =0.4 (SiO2, 5% CH3OH in CH2Cl2 solution).

[0400] Compound 45a-d is commercially available.

[0401] Compound 46a was prepared according to the process for forming N-Boc piperazine derivatives described in Angew Chem Int Ed 2018, 57 5110 (see Supplementary Material).

[0402] Compound 46b was prepared according to the process for forming N-methylpiperazine derivatives described in WO 2005 / 075410 (see Example 5).

[0403] Compound 46c was prepared according to the process described in Journal Heterocyclic Chemistry 2013, 50, 995.

[0404] Compound 46d was prepared according to the process described in Organic Biomolecular & Chemistry 2010, 8, 4077 (see Supplementary Material).

[0405] Compound 47a-d was prepared as described in Compound 4.

[0406] Compound 48a-d was prepared as described in Compound 5.

[0407] Compound 49a-d was prepared as described in Compound 7.

[0408] Compound 50a-d was prepared as described in Compound 8.

[0409] Compound 51

[0410]

[0411] Under argon atmosphere, oxalyl chloride (0.75 mL, 8.75 mmol) was added dropwise to a mixture of sodium 3-bromopropanesulfonate (1 g, 4.44 mmol), anhydrous CH3CN (4 mL), and anhydrous DMF (0.1 mL) cooled to 0 °C. The mixture was stirred at 0 °C for 2 hours, and then the solvent was removed under reduced pressure (bath temperature <20 °C). The resulting residue was dried under high vacuum. Under argon atmosphere, anhydrous CH3CN (4 mL) and 2,2,2-trifluoroethanol (9 mL, 125 mmol) were added to the residue. Triethylamine (3 mL, 21.5 mmol) was added dropwise while cooling to 0 °C. The mixture was stirred at 0 °C for 5 hours, and then the solvent was removed under reduced pressure. Ethyl acetate (40 mL) and H2O (40 mL) were added to the crude residue, and the organic layer was washed with H2O (3 × 40 mL). The organic layer was dried with Na2SO4 and the solvent was removed under reduced pressure to obtain a pale orange oil (1.1 g, 87%). 1 H-NMR(600MHz,CDCl3)δ4.53(q,2H, 3 J H-F =8.0), 3.54(t,2H, 3 J H-H =6.2),3.45–3.41(m,2H),2.47–2.42(m,2H); 13 C-NMR (151MHz, CDCl3) δ 122.0 (q, 1 J C-F =280),64.0(q, 2 J C-F =40), 49.9, 30.1, 26.7; 19 F-NMR (151MHz, CDCl3) δ-74.0.

[0412] Compound 52

[0413]

[0414] Under argon atmosphere, 5-iodo-o-anisidine (245 mg, 0.984 mmol), compound 51 (1.1 g, 3.86 mmol), K₂CO₃ (425 mg, 3.08 mmol), and NaI (40 mg, 0.27 mmol) were combined in anhydrous CH₃CN (5 mL). The resulting mixture was heated to 78 °C for 72 hours, and then the solvent was removed under reduced pressure. DCM (40 mL) and H₂O (40 mL) were added to the crude residue, and the organic layer was washed with H₂O (2 × 40 mL). The organic layer was dried over K₂CO₃, and the solvent was removed under reduced pressure to obtain a crude product, a light brown residue, which was purified by column chromatography (SiO₂, 100% hexane to 20% EtOAc in hexane solution) to obtain a pale yellow oil (283 mg, 63%). 1 H-NMR (400MHz, CDCl3) δ 6.99 (dd, 3 J H-H =8.3, 4 J H-H =2.0,1H),6.82(d, 4 J H-H =2.0,1H),6.50(d, 3 J H-H =8.3,1H),4.53(q, 3 J H-F =8.0,2H),3.86(s,3H),3.39–3.29(m,4H),2.26–2.17(m,2H); 13 C-NMR (101MHz, CDCl3) δ146.9,138.9,126.0,122.2(q, 1 J C-F =280),118.3,111.5,84.1,63.9(q, 2 J C-F =40), 55.7, 49.1, 41.4, 23.5; 19 F-NMR(376MHz, CDCl3)δ-73.9; ESI-LRMS(+)m / z 454[M+H] + ; ESI-HRMS(+)[C 12 H 16 NO4SF3I] + Calculated value: 453.9797; Actual value: 453.9792; R f (SiO2, 20% EtOAc hexane solution) = 0.3.

[0415] Compound 53a

[0416]

[0417] Compound 52 (310 mg, 0.684 mmol), acetaldehyde (0.46 mL, 8.16 mmol), and two drops of glacial acetic acid were combined in anhydrous EtOH (3 mL) under argon atmosphere. The resulting mixture was stirred at room temperature for 15 minutes, and then NaBH3CN (170 mg, 2.71 mmol) was added. The mixture was stirred at room temperature for another 16 hours under argon atmosphere, and then the solvent was removed under reduced pressure to obtain a yellow residue. DCM (40 mL) and H2O (40 mL) were added to the residue, and the organic layer was washed with H2O (3 × 40 mL). The combined aqueous layer was extracted with DCM (1 × 40 mL), and the combined organic layer was dried over K2CO3. The solvent was removed under reduced pressure to obtain a pale yellow oil (247 mg, 75%). 1 H-NMR (400MHz, CDCl3) δ 7.29 (dd, 3 J H-H =8.4, 4 J H-H =2.1,1H),7.18(d, 4 J H-H =2.1,1H),6.60(d, 3 J H-H =8.4,1H),4.46(q, 3 J H-F =8.0,2H),3.79(s,3H),3.37–3.30(m,2H),3.15(t, 3 J H-H =6.6,2H),3.07(q, 3 J H-H =7.1,2H),2.06–1.97(m,2H),1.00(t, 3 J H-H =7.1,3H); 13 C-NMR (101MHz, CDCl3) δ154.1,140.2,132.3,130.9,122.0(q, 1 J C-F =280),113.7,82.9,63.7(q, 2 J C-F =40), 55.4, 49.8, 49.0, 46.6, 21.5, 12.1; 19 F-NMR(376MHz, CDCl3)δ-74.0; ESI-LRMS(+)m / z 482[M+H] + ; ESI-HRMS(+)[C 14 H 20 NO4SF3I]+ Calculated value: 482.0110; Actual value: 482.0106.

[0418]

[0419] Compound 52 (269 mg, 0.594 mmol), paraformaldehyde (800 mg, 8.89 mmol), and two drops of glacial acetic acid were combined in anhydrous EtOH (8 mL) under argon atmosphere. The resulting mixture was stirred at room temperature for 15 minutes, and then NaBH3CN (300 mg, 4.77 mmol) was added. The mixture was stirred at room temperature for another 18 hours under argon atmosphere, and then the solvent was removed under reduced pressure to obtain a yellow residue. DCM (40 mL) and H2O (40 mL) were added to the residue, and the organic layer was washed with H2O (3 × 40 mL). The combined aqueous layer was extracted with DCM (2 × 40 mL), and the combined organic layer was dried over K2CO3. The solvent was removed under reduced pressure to obtain a pale yellow oil (249 mg, 90%). 1 H-NMR (400MHz, CDCl3) δ 7.27 (dd, 3 J H-H =8.3, 4 J H-H =2.0,1H),7.17(d, 4 J H-H =2.0,1H),6.59(d, 3 J H-H =8.3,1H),4.50(q, 3 J H-F =8.0,2H),3.81(s,3H),3.44–3.37(m,2H),3.11(t, 3 J H-H =6.7,2H),2.72(s,3H),2.18–2.07(m,2H); 13 C-NMR (176MHz, CDCl3) δ152.6,143.0,131.8,128.4,122.2(q, 1 J C-F =280),113.5,83.4,63.8(q, 2 J C-F =40), 55.5, 53.1, 49.1, 39.5, 21.6; 19 F-NMR(376MHz, CDCl3)δ-73.9; ESI-LRMS(+)m / z 468[M+H] + ; ESI-HRMS(+)[C 13 H 18 NO4SF3I]+ Calculated value: 467.9953; Actual value: 467.9952.

[0420] Compound 54a

[0421]

[0422] Compound 53a (176 mg, 0.366 mmol) and trimethylsilylacetylene (0.1 mL, 0.72 mmol) were combined in anhydrous CH3CN (3 mL) under argon atmosphere. Pd2Cl2(allyl)2 (14 mg, 0.038 mmol) and P( t Bu)3 (0.015 mL, 0.062 mmol) and piperidine (0.09 mL, 0.91 mmol). The resulting mixture was stirred at 35 °C for 20 hours under argon atmosphere, and then the solvent was removed under reduced pressure to obtain a crude product, a brown residue, which was purified by column chromatography (SiO2, 100% hexane to 20% EtOAc in hexane solution) to give a pale orange oil (151 mg, 91%). 1 H-NMR (400MHz, CDCl3) δ 7.16 (dd, 3 J H-H =8.4, 4 J H-H =2.0,1H),7.06(d, 4 J H-H =2.0,1H),6.77(d, 3 J H-H =8.4,1H),4.45(q, 3 J H-F =8.0,2H),3.83(s,3H),3.38–3.30(m,2H),3.16(t, 3 J H-H =6.5,2H),3.08(q, 3 J H-H =7.2,2H),2.06–1.94(m,2H),0.99(t, 3 J H-H =7.2,3H),0.24(s,9H); 13 C-NMR (101MHz, CDCl3) δ154.9,138.2,128.3,126.1,122.0(q, 1 J C-F =280),115.3,111.5,105.3,92.4,63.8(q, 2 J C-F=40), 55.5, 49.9, 49.1, 46.9, 21.6, 12.2, 0.2; 19 F-NMR(376MHz, CDCl3)δ-74.0; ESI-LRMS(+)m / z 452[M+H] + ; ESI-HRMS(+)[C 19 H 29 NO4F3SSi] + Calculated value: 452.1539; Actual measured value: 452.1545; R f (SiO2, 20% EtOAc in hexane solution) = 0.3.

[0423] Compound 54b

[0424]

[0425] Compound 53b (240 mg, 0.514 mmol) and trimethylsilylacetylene (0.14 mL, 1.01 mmol) were combined in anhydrous CH3CN (2.5 mL) under argon atmosphere. Pd2Cl2(allyl)2 (20 mg, 0.055 mmol) and P( t Bu)3 (0.013 mL, 0.054 mmol) and piperidine (0.13 mL, 1.32 mmol). The resulting mixture was stirred at 35 °C for 16 hours under argon atmosphere, and then the solvent was removed under reduced pressure to obtain a crude product, a brown residue, which was purified by column chromatography (SiO2, 100% hexane to 20% EtOAc in hexane solution) to give a pale orange oil (157 mg, 70%). 1 H-NMR (700MHz, CDCl3) δ 7.13 (dd, 3 J H-H =8.3, 4 J H-H =1.8,1H),7.05(d, 4 J H-H =1.8,1H),6.76(d, 3 J H-H =8.3,1H),4.49(q, 3 J H-F =8.0,2H),3.84(s,3H),3.42–3.38(m,2H),3.10(t, 3 J H-H =6.7,2H),2.73(s,3H),2.14–2.08(m,2H),0.24(s,9H); 13C-NMR (151MHz, CDCl3) δ153.2,141.0,127.5,123.3,122.2(q, 1 J C-F =280),115.6,111.2,105.4,92.4,63.8(q, 2 J C-F =40), 55.5, 53.1, 49.1, 39.6, 21.5, 0.2; 19 F-NMR(376MHz, CDCl3)δ-74.0; ESI-LRMS(+)m / z 438[M+H] + ; ESI-HRMS(+)[C 18 H 27 NO4F3SSi] + Calculated value: 438.1382; Actual measurement: 438.1377; R f (SiO2, 20% EtOAc in hexane solution) = 0.3.

[0426] Compound 55a

[0427]

[0428] Under argon atmosphere, triethylamine trihydrofluoric acid (3 mL, 6.75 mmol) was added to an anhydrous THF (5 mL) solution of compound 54a (341 mg, 0.755 mmol). The solution was stirred at 30 °C for 72 hours, and then the solvent was removed under reduced pressure. The residue was then dissolved in CH₂Cl₂ (30 mL) and washed with water (4 × 30 mL). The combined aqueous layers were extracted with CH₂Cl₂ (1 × 30 mL), and the combined organic layers were dried over K₂CO₃. The solvent was evaporated, and a light brown oil (207 mg, 72%) was obtained. This product was used for the next step without further purification. 1 H-NMR (400MHz, CDCl3) δ 7.18 (dd, 3 J H-H =8.4, 4 J H-H =2.0,1H),7.08(d, 4 J H-H =2.0,1H),6.80(d, 3 J H-H =8.4,1H),4.46(q, 3 J H-F =8,2H),3.84(s,3H),3.38–3.31(m,2H),3.17(t, 3 J H-H =6.6,2H),3.09(q,3 J H-H =7.1,2H),2.99(s,1H),2.05–1.95(m,2H),0.99(t, 3 J H-H =7.1,3H), 13 C-NMR (101MHz, CDCl3) δ155.0,138.3,128.2,126.1,122.2(q, 1 J C-F =280),114.2,111.6,83.9,75.8,63.8(q, 2 J C-F =40), 55.5, 49.9, 49.1, 46.7, 21.6, 12.2; 19 F-NMR(376MHz, CDCl3)δ-74.0; ESI-LRMS(+)m / z 380[M+H] + ; ESI-HRMS(+)[C 16 H 21 NO4SF3] + Calculated value: 380.1143; Actual value: 380.1155.

[0429] Compound 55b

[0430]

[0431] Under argon atmosphere, triethylamine trihydrofluoric acid (1.1 mL, 6.75 mmol) was added to an anhydrous THF (3 mL) solution of compound 54b (195 mg, 0.446 mmol). The solution was stirred at 30 °C for 48 hours, and then the solvent was removed under reduced pressure. The residue was then dissolved in CH₂Cl₂ (30 mL) and washed with water (3 × 30 mL). The combined aqueous layers were extracted with CH₂Cl₂ (2 × 30 mL), and the combined organic layers were dried over K₂CO₃ to give a light brown oil (160 mg, 98%). This product was used for the next step without further purification. 1 H-NMR (400MHz, CDCl3) δ 7.15 (dd, 3 J H-H =8.3, 4 J H-H =1.9,1H),7.07(d, 4 J H-H =1.9,1H),6.78(d, 3 J H-H =8.3,1H),4.50(q, 3 J H-F=8.0,2H),3.85(s,3H),3.43–3.37(m,2H),3.10(t, 3 J H-H =6.6,2H)2.99(s,1H),2.73(s,3H),2.17–2.07(m,2H); 13 C-NMR (101MHz, CDCl3) δ153.3,141.1,127.4,123.2,122.2(q, 1 J C-F =280),114.4,111.2,83.9,75.8,63.8(q, 2 J C-F =40), 55.5, 53.0, 49.0, 39.4, 21.5; 19 F-NMR(376MHz, CDCl3)δ-74.0; ESI-LRMS(+)m / z 366[M+H] + ; ESI-HRMS(+)[C 15 H 19 NO4SF3] + Calculated value: 366.0987; Actual value: 366.0986.

[0432] Compound 56a

[0433]

[0434] Compound 55a (207 mg, 0.546 mmol) and (6-(hydroxymethyl)-4-(bromopyridin-2-yl)(methyl)phosphinic acid ethyl ester 6 (152 mg, 0.517 mmol) were combined in anhydrous CH3CN (5 mL) under argon atmosphere. Pd2Cl2(allyl)2 (20 mg, 0.055 mmol) and P( t Bu)3 (0.02 mL, 0.082 mmol) and piperidine (0.11 mL, 1.11 mmol). The resulting mixture was stirred at 35 °C for 18 hours under argon atmosphere, followed by solvent removal under reduced pressure. The residue was dissolved in CH2Cl2 (40 mL), washed with H2O (3 × 40 mL), and the combined aqueous layers were extracted with CH2Cl2 (2 × 40 mL). The combined organic layers were dried over K2CO3, and the solvent was removed under reduced pressure to obtain an orange oil, which was then subjected to reversed-phase HPLC (10 to 100% CH3CN, 10 min, t). r Purification was carried out over 11.3 min to obtain a light yellow oily substance (111 mg, 36%). 1 H-NMR (400MHz, CDCl3) δ 8.02 (d, 3 JH-P =6.0,1H),7.51(s,1H),7.21(dd, 3 J H-H =8.4, 4 J H-H =2.0,1H),7.11(d, 4 J H-H =2.0,1H),6.84(d, 3 J H-H =8.4,1H),4.81(s,2H),4.46(q, 3 J H-F =8,2H),4.16–4.05&3.91–3.79(m,2H),3.86(s,3H),3.38–3.31(m,2H),3.19(t, 3 J H-H =6.6,2H),3.11(q, 3 J H-H =7.1,2H),2.08–1.99(m,2H),1.77(d, 2 J H-P =15,3H),1.31–1.14(t, 3 J H-H =7.0,3H),1.01(t, 3 J H-H =7.1,3H); 13 C-NMR(101MHz,CDCl3)δ161.0(d, 3 J C-P =19),155.6,153.2(d, 1 J C-P =155),138.6,133.1(d, 3 J C-P =11),128.2(d, 2 J C-P =22),128.1,125.8,124.1(d, 4 J C-P =3),122.1(q, 1 J C-F =280),113.8,111.8,96.4,85.2(d, 4 J C-P =2),64.2,63.7(q, 2 J C-F =40),61.3(d, 2 J C-P =6),55.6,49.8,49.1,46.7,21.6,16.5(d,3 J C-P =6), 13.5(d, 1 J C-P =104)12.2; 19 F-NMR (376MHz, CDCl3) δ-74.0; 31 P-NMR(162MHz, CDCl3)δ+39.5; ESI-LRMS(+)m / z 593[M+H] + ; ESI-HRMS(+)[C 25 H 33 N2O7SF3P] + Calculated value: 593.1698; Actual value: 593.1709.

[0435] Compound 56b

[0436]

[0437] Compound 55b (160 mg, 0.438 mmol) and (6-(hydroxymethyl)-4-(bromopyridin-2-yl)(methyl)phosphinic acid ethyl ester 6 (116 mg, 0.395 mmol) were combined in anhydrous CH3CN (3 mL) under argon atmosphere. Pd2Cl2(allyl)2 (16 mg, 0.044 mmol) and P( t Bu)3 (0.016 mL, 0.066 mmol) and piperidine (0.09 mL, 0.92 mmol). The resulting mixture was stirred at 35 °C for 16 hours under argon atmosphere, followed by solvent removal under reduced pressure. The residue was dissolved in CH2Cl2 (40 mL), washed with H2O (3 × 40 mL), and the combined aqueous layers were extracted with CH2Cl2 (2 × 40 mL). The combined organic layers were dried over K2CO3, and the solvent was removed under reduced pressure to obtain a brown oily substance, which was then subjected to reversed-phase HPLC (10 to 100% CH3CN, 10 min, t). r Purification was carried out over 10.8 min to obtain a light yellow oily substance (119 mg, 52%). 1 H-NMR (700MHz, CDCl3) δ 8.00 (d, 3 J H-P =6.0,1H),7.50(s,1H),7.18(dd, 3 J H-H =8.4, 4 J H-H =2.0,1H),7.09(d, 4 J H-H =2.0,1H),6.82(d, 3 J H-H=8.4,1H),4.80(s,2H),4.49(q, 3 J H-F =8.0,2H),4.13–4.06&3.91–3.82(m,2H),3.86(s,3H),3.42–3.38(m,2H),3.12(t, 3 J H-H =6.3,2H),2.75(s,3H),2.16–2.09(m,2H),1.76(d, 2 J H-P =15,3H),1.26(t, 3 J H-H =7.1,3H); 13 C-NMR(101MHz,CDCl3)δ161.0(d, 3 J C-P =19),153.9,153.3(d, 1 J C-P =155),141.4,133.1(d, 3 J C-P =11),128.2(d, 2 J C-P =22),127.5,124.1(d, 4 J C-P =3),123.0,122.2(q, 1 J C-F =280),114.0,111.5,96.4,85.2(d, 4 J C-P =2),64.2,63.8(q, 2 J C-F =40),61.3(d, 2 J C-P =7),55.5,53.0,49.0,39.4,21.5,16.5(d, 3 J C-P =6),13.5(d, 1 J C-P =104); 19 F-NMR(376MHz,CDCl3)δ-73.9; 31 P{ 1 H}-NMR(162MHz,CDCl3)δ+39.6;ESI-LRMS(+)m / z 579[M+H] + ;ESI-HRMS(+)[C 24 H 31 N2O7SF3P] +Calculated value: 579.1542; Actual value: 579.1543.

[0438] Compound 57a

[0439]

[0440] Compound 56a (110 mg, 0.186 mmol), methanesulfonic anhydride (65 mg, 0.373 mmol), and DIPEA (0.065 mL, 0.373 mmol) were combined in anhydrous THF (3 mL) under argon atmosphere. 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 CH2Cl2 (40 mL), washed with water (3 × 40 mL), and the organic layer was dried over K2CO3. The solvent was removed under reduced pressure to give a yellow oil (125 mg, 100%). 1 H-NMR (400MHz, CDCl3) δ 8.09 (dd, 3 J H-P =6.0, 4 J H-H =1.3,1H),7.63(s,1H),7.24(dd, 3 J H-H =8.4, 4 J H-H =1.9,1H),7.13(d, 4 J H-H =1.9,1H),6.86(d, 3 J H-H =8.4,1H),5.36(s,2H),4.46(q, 3 J H-F =8.0,2H),4.18–4.06&3.93–3.83(m,2H),3.87(s,3H),3.38–3.32(m,2H),3.20(t, 3 J H-H =6.4,2H),3.15–3.09(m,5H),2.09–1.99(m,2H),1.77(d, 2 J H-P =15),1.27(t, 3 J H-H =7.1,3H),1.02(t, 3 J H-H =7.0,3H); ESI-LRMS(+)m / z 671[M+H] + ; ESI-HRMS(+)[C 26 H 35 N2O9S2F3P] +Calculated value: 671.1474; Actual value: 671.1483.

[0441] Compound 57b

[0442]

[0443] Compound 56b (119 mg, 0.206 mmol), methanesulfonic anhydride (72 mg, 0.413 mmol), and DIPEA (0.072 mL, 0.413 mmol) were combined in anhydrous THF (3 mL) under argon atmosphere. 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 CH2Cl2 (40 mL), washed with water (3 × 40 mL), and the organic layer was dried over K2CO3. The solvent was removed under reduced pressure to obtain a clear, colorless oil (88 mg, 65%). 1 H-NMR (400MHz, CDCl3) δ 8.08 (d, 3 J H-P =6.0,1H),7.63(s,1H),7.21(dd, 3 J H-H =8.3, 4 J H-H =2.0,1H),7.11(d, 4 J H-H =2.0,1H),6.84(d, 3 J H-H =8.3,1H),5.36(s,2H),4.50(q, 3 J H-F =8.0,2H),4.17–4.08&3.93–3.83(m,2H),3.87(s,3H),3.44–3.38(m,2H),3.17–3.11(m,5H),2.77(s,3H),2.18–2.09(m,2H),1.76(d, 2 J H-P =15,3H),1.27(t, 3 J H-H =7.1,3H).

[0444] Compound 58 (mixture E, Z)

[0445]

[0446] Compound 6 (74 mg, 0.25 mmol), N-allylcarbamate tert-butyl ester (155 mg, 0.99 mmol), palladium(II) acetate (11 mg, 0.05 mmol), and triphenylphosphine (20 mg, 0.08 mmol) were combined in toluene (2 mL) under an argon atmosphere. The reaction mixture was degassed by bubbling argon in the solution for 15 minutes. Triethylamine (0.3 mL, 2.2 mmol) was added, and the mixture was heated to 80 °C under argon for 18 hours. After cooling, the solvent was removed under reduced pressure, and the resulting residue was dissolved in CH2Cl2 (30 mL), washed with H2O (4 × 30 mL), and dried over MgSO4. The solvent was removed under reduced pressure to obtain the crude product, which was then subjected to reversed-phase HPLC (10 to 100% CH3CN in H2O solution, 10 min, t). r Purification was carried out over 9.1 min to obtain a yellow oily substance, which was an E / Z isomer mixture (93 mg, 63%). 1 H-NMR(400MHz, CDCl3)δ8.03–7.91(2×d,1H),7.45–7.28(2×s,1H),6.61–6.44(m,2H),4.83–4.77(2× s,2H),4.14–4.03&3.89–3.77(m,2H),3.97–3.93(m,2H),1.80–1.74(2×d,3H),1.48–1.40(2×t,3H); 31 P{ 1 H}-NMR(298K,162MHz,CDCl3)δ+40.5,+40.4; ESI-LRMS(+)m / z 371[M+H] + ; ESI-HRMS(+)[C 17 H 28 N2O5P] + Calculated value: 371.1736; Actual value: 371.1746.

[0447] Compound 59

[0448]

[0449] In a container, an E / Z isomer mixture of compound 58 (210 mg, 0.57 mmol) was dissolved in EtOH (60 mL), and Pd / C (10% Pd, 10 mg) was added. The container was then loaded onto a Parr hydrogenator (40 bar H2 pressure), and the reaction mixture was stirred for 8 hours. Subsequently, the catalyst was removed by filtration, and the solvent was removed under reduced pressure to obtain a pale yellow oil (211 mg, measured amount). 1 H-NMR (600MHz, CDCl3) δ 7.82 (d,3 J H-P =6.0,1H),7.24(s,1H),4.78(s,2H),4.66(br s,1H),4.12–3.79(m,2H),3.18–3.11(m,2H),2.73–2.68(m,2H),1.87–1.81(m,2H),1.76(d, 2 J H-P =15,3H),1.43(s,9H),1.26(t, 3 J H-H =7.0,3H); 13 C-NMR (151MHz, CDCl3) δ 160.5 (d, 3 J C-P =19),156.1,153.0(d, 1 J C-P =156),152.2(d, 3 J C-P =10),127.0(d, 2 J C-P =21),123.0,79.5,64.1,61.2(d, 2 J C-P =6),40.0,32.6,30.8,28.5,16.6(d, 3 J C-P =6),13.7(d, 1 J C-P =104); 31 P{ 1 H}-NMR(243MHz, CDCl3)δ+39.9; ESI-LRMS(+)m / z 373[M+H] + ; ESI-HRMS(+)[C 17 H 30 N2O5P] + Calculated value: 373.1892; Actual measurement: 373.1880; R f =0.4 (SiO2, 5% CH3OH in CH2Cl2 solution).

[0450] Compound 60

[0451]

[0452] Compound 59 (25 mg, 0.067 mmol), methanesulfonic anhydride (20 mg, 0.115 mmol), and DIPEA (0.03 mL, 0.172 mmol) were combined in anhydrous THF (0.5 mL) under argon atmosphere. The reaction mixture was stirred at room temperature for 1 hour and monitored by TLC. After complete conversion, the solvent was removed under reduced pressure, and the resulting crude residue was dissolved in CH2Cl2 (40 mL), washed with water (3 × 40 mL), and the organic layer was dried over K2CO3. The solvent was removed under reduced pressure to obtain a clear oil (30 mg, quantitative). 1 H-NMR (400MHz, CDCl3) δ 7.86 (d, 3 J H-P =6.0,1H),7.41(s,1H),5.33(s,2H),4.66(br s,1H),4.15–3.77(m,2H),3.19–3.09(m,5H),2.76–2.67(m,2H),1.89–1.79(m,2H),1.74(d, 2 J H-P =15,3H),1.42(s,9H),1.25(t, 3 J H-H =7.1,3H); ESI-LRMS(+)m / z 373[M+H] + ; ESI-HRMS(+)[C 18 H 32 N2O7PS] + Calculated value: 451.1668; Actual measurement: 451.1666; R f =0.5 (SiO2, 5% CH3OH in CH2Cl2 solution).

[0453] Compounds 61, 62 and 63 were prepared according to the process for carboxylic acid ester derivatives described in WO2018 / 229408.

[0454] Compound 64 was synthesized as described in Compound 13.

[0455] As described in complex 14, synthesize complex 65.

[0456] Compound 66 was synthesized as described in Compound 11.

[0457] Compound 67 was synthesized as described in Compound 12.

[0458] Compound 68 was synthesized as described in Compound 13.

[0459] As described in complex 14, synthesize complex 69.

[0460] As described in complexes 78a-b, synthesize complex 70.

[0461] As described in complexes 99a-b, synthesize complex 71.

[0462] Compound 72 was synthesized as described in Compound 13.

[0463] As described in complex 14, synthesize complex 73a.

[0464] As described in complexes 78a-b, synthesize complex 73b.

[0465] Compound 74a

[0466]

[0467] Under argon atmosphere, 1-tert-butoxycarbonyl-1,4,7-triazacyclononane dihydrochloride (66 mg, 0.218 mmol), methanesulfonate 30 (288 mg, 0.484 mmol), and K₂CO₃ (122 mg, 0.833 mmol) were combined in anhydrous CH₃CN (2.5 mL). The resulting mixture was heated to 70 °C for 16 hours and then cooled to room temperature. The solution was separated from the inorganic salt by filtration, and the solvent was removed under reduced pressure to obtain a crude product, which was a viscous residue. This residue was then subjected to reversed-phase HPLC (10 to 100% CH₃CN in H₂O solution, 10 min, t). r Purification was carried out over 17.5 min to obtain an orange oil (160 mg, 27%). 1 H-NMR(700MHz, CDCl3)δ8.01–7.97(m,2H),7.65(s,1H),7.56(s,1H),7.11( d,2H),7.05(s,2H),6.80(d,2H),4.13(q,4H),4.11–4.06&3.88–3.80(m,4H) ,4.05(t,4H),3.94(s,2H),3.93(s,2H),3.41–3.31(m,4H),3.15(q,8H),3. 12–3.04(m,4H),2.74–2.62(m,4H),2.51(t,4H),2.18–2.11(m,4H),1.76(d, 2 J H-P =15),1.75(d, 2 J H-P =15),1.47(s,9H),1.26–1.22(m,12H),1.04(t,12H); 13 C-NMR(176MHz,CDCl3)δ173.1,161.7(d, 3 J C-P =20),161.4(d,3 J C-P =20),155.7,154.1,153.9(d, 1 J C-P =157),153.6(d, 1 J C-P =157),140.1,132.8(app.t, 3 J C-P =11),127.9(app.t, 2 J C-P =21),126.5,126.4(d, 4 J C-P =3),126.2(d, 4 J C-P =3),124.8,114.0,113.8,112.8,96.5,96.3,85.2,85.1,79.5,67.5,62.9,62.6,61.1(d, 2 J C-P =6.4),61.0(d, 2 J C-P =6.4),60.6,56.1,55.0,54.7,54.0,50.0,49.7,45.7,30.9,28.8,24.7,16.6(d),14.3,13.4(d, 1 J C-P =104),13.3(d, 1 J C-P =104), 12.4; 31 P{ 1 H}-NMR(283MHz, CDCl3)δ+40.1,+40.0; ESI-LRMS(+)m / z1226[M+H] + ; ESI-HRMS(+)[C 65 H 94 N7O 12 P2] + Calculated value: 1226.644; Actual measurement: 1226.646.

[0468] Compound 74b

[0469]

[0470] Under argon atmosphere, 1-tert-butoxycarbonyl-1,4,7-triazacyclononane dihydrochloride (38 mg, 0.126 mmol), methanesulfonate 44 (167 mg, 0.288 mmol), and K₂CO₃ (104 mg, 0.753 mmol) were combined in anhydrous CH₃CN (4 mL). The resulting mixture was heated to 60 °C for 48 hours and then cooled to room temperature. The solution was separated from the inorganic salt by filtration, and the solvent was removed under reduced pressure to obtain the crude product. This crude product was then subjected to reversed-phase HPLC (10 to 100% CH₃CN in H₂O solution, 10 min, t). r Purification was carried out over 12.2 min to obtain a pale orange oil (110 mg, 73%). 1 H-NMR(400MHz, CDCl3)δ8.00(d,2H),7.65(s,1H),7.59(s,1H),7.12(dd,2H),7.06(s,2H),6.80(d,2H),4.18–3.78(m,16H),3.47–3.32(m ,4H),3.21–3.06(m,8H),2.83–2.72(m,10H),2.53(t,4H),2.23–2.14(m,4H),1.76(d,6H),1.48(s,9H),1.28–1.22(m,12H),1.11(t,6H); 31 P{ 1 H}-NMR(162MHz, CDCl3)δ+40.2,+40.1; ESI-LRMS(+)m / z 400[M+3H] 3+ 600 [M+2H] 2+ 1198[M+H] + ; ESI-HRMS(+)[C 63 H 90 N7O 12 P2] + Calculated value: 1198.612; Actual measurement: 1198.607.

[0471] Compound 75a

[0472]

[0473] A solution of compound 74a (19 mg, 0.016 mmol) in trifluoroacetic acid and CH₂Cl₂ (10% v / v, 3 mL total) was prepared and stirred under argon for 40 min. Immediately afterwards, the solvent was removed under reduced pressure. Another 50 mL of CH₂Cl₂ was added, and the solvent was removed under reduced pressure. This process was repeated three times. The residue was dissolved in CH₂Cl₂ (30 mL), washed with an aqueous solution of Na₂CO₃ (4%, pH = 11, 3 × 30 mL), and dried over K₂CO₃. The solvent was removed under reduced pressure to obtain an orange oil (17 mg, quantified). 1 H-NMR (400MHz, CDCl3) δ 7.95 (dd, 3 J H-P =6.0, 4 J H-H =1.3,2H),7.38(app.s,2H),7.12(dd, 3 J H-H =8.4, 4 J H-H =2.0,2H),7.06(d, 4 J H-H =2.0,2H),6.81(d, 3 J H-H =8.4,2H),4.23–4.10&3.96–3.86(m,8H),4.05(t, 3 J H-H =6.4,4H),3.99(s,4H),3.31–3.24(m,4H),3.16(q, 3 J H-H =7.1,8H),3.13–3.07(m,4H),2.73(br s,4H),2.52(t, 3 J H-H =7.2,4H),2.20–2.11(m,4H),1.77(d, 2 J H-P =15,6H),1.31–1.22(m,12H),1.05(t, 3 J H-H =7.1,12H); 13 C-NMR(101MHz,CDCl3)δ173.2,159.4(d, 3 J C-P =20),154.6(d, 1 J C-P =157),154.2,140.2,133.4(d, 3 J C-P =12),127.9(d,2 J C-P =22),126.7,126.3,124.8,113.5,112.8,97.5,84.7,67.5,61.3(d, 2 J C-P =6),60.7,60.0,53.9,51.6,50.1,49.4,49.2,45.7,44.8,31.0,24.7,16.7(d, 3 J C-P =6),14.4,13.8(d, 1 J C-P =103), 12.5; 31 P{ 1 H}-NMR(162MHz, CDCl3)δ+39.2; ESI-LRMS(+)m / z 1127[M+H] + ; ESI-HRMS(+)[C 60 H 86 N7O 10 P2] + Calculated value: 1126.592; Actual value: 1126.592.

[0474] Compound 75b

[0475]

[0476] A solution of compound 74b (110 mg, 0.092 mmol) in trifluoroacetic acid and CH₂Cl₂ (10% v / v, 3 mL total) was prepared and stirred under argon for 60 min. The solvent was removed under reduced pressure, and another 50 mL of CH₂Cl₂ was added, followed by further solvent removal under reduced pressure. This process was repeated four times. The residue was dissolved in CH₂Cl₂ (30 mL), washed with an aqueous solution of Na₂CO₃ (4%, pH = 11, 3 × 30 mL), and dried over K₂CO₃. The solvent was removed under reduced pressure to obtain an orange oil (111 mg, quantified). 1 H-NMR (700MHz, CDCl3) δ 7.89 (d, 3 J H-P =6.0,2H),7.77(s,2H),7.64(dd, 3 J H-H =8.5, 4 J H-H =1.3,2H),7.57(s,2H),7.13(d, 3 J H-H =8.5,2H),4.39(s,4H),4.22(t, 3 J H-H=6.5,4H),4.15–4.07&3.97–3.90(m,8H),3.68(q, 3 J H-H =7.2,4H),3.64–3.51(m,8H),3.43–3.35(m,4H),3.29(s,6H),2.51(t, 3 J H-H =6.8,4H),2.21–2.16(m,4H),1.75(d, 2 J H-P =15,6H),1.27(td,, 3 J H-H =7.1, 4 J H-P =3.0,6H),1.23(t, 3 J H-P =7.1,6H),1.18(t, 3 J H-H =7.2,6H); 13 C-NMR(176MHz,CDCl3)δ173.1,156.2(d, 3 J C-P =21),153.3(d, 1 J C-P =160),152.5,135.6,128.2,128.1(d, 2 J C-P =20),127.7,127.1(d, 4 J C-P =4),115.4,114.2,94.6,86.2(d, 4 J C-P =2.6),69.1,62.6(d, 2 J C-P =7.0),61.0,59.7,54.1,53.6,51.5,50.0,44.2,30.5,24.0,16.3(d, 3 J C-P =6.2),14.2,13.4(d, 1 J C-P =102),10.3; 31 P-NMR(162MHz,CDCl3)δ+40.8;ESI-LRMS(+)m / z1098[M+H] + ;ESI-HRMS(+)[C 58 H 82 N7O 10 P2] +Calculated value: 1098.5598; Actual value: 1098.5573.

[0477] Compound 76a

[0478]

[0479] Compounds 75a (43 mg, 0.038 mmol), 60 (45 mg, 0.10 mmol), and K₂CO₃ (30 mg, 0.22 mmol) were combined in anhydrous CH₃CN (2 mL) under argon atmosphere and heated to 70 °C for 18 hours. After this period, LCMS analysis confirmed the complete conversion of the macrocyclic compounds. The reaction mixture was cooled 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 for the next step without further purification; ESI-LRMS (+) m / z 1481 [M+H] + ; ESI-HRMS(+)[C 77 H 113 N9O 14 P3] + Calculated value: 1480.762; Actual measurement: 1480.760.

[0480] Compound 76b

[0481]

[0482] Compounds 75b (111 mg, 0.0919 mmol), 60 (59 mg, 0.130 mmol), and K₂CO₃ (45 mg, 0.33 mmol) were combined in anhydrous CH₃CN (3 mL) under argon atmosphere and heated to 65 °C for 16 hours. After this period, LCMS analysis confirmed the complete conversion of the macrocyclic compounds. The reaction mixture was cooled, and the solution was separated from the inorganic salts by filtration. The solvent was removed under reduced pressure to obtain an orange oil (165 mg), which was used directly for the next step without further purification; ESI-LRMS(+) m / z 1453 [M+H] + ; ESI-HRMS(+)[C 75 H 109 N9O 14 P3] + Calculated value: 1452.731; Actual value: 1452.729.

[0483] Complex 77a

[0484]

[0485] The crude ligand 76a (88 mg) from the previous step was dissolved in a CH3OH / H2O mixture (4:1, 2.5 mL total), and the pH was adjusted to 12 using an aqueous NaOH solution. The solution was heated to 60 °C for 1.5 h. After this period, LCMS analysis confirmed the complete hydrolysis of the phosphonate and chromophore ester groups. After cooling and adjusting the pH to 7 using hydrochloric acid (0.1 M), EuCl3·6H2O (42 mg, 0.115 mmol) was added, and the reaction mixture was heated to 60 °C for 18 h. The reaction mixture was then subjected to reversed-phase HPLC (10 to 100% CH3CN in H2O solution, 10 min, t... r Purification was carried out over 8.5 min to obtain a yellow solid (22 mg, 39% in three steps); ESI-LRMS (+) m / z 1490 [M+H] + 746[M+2H] 2+ ; ESI-HRMS(+)[C 67 H 91 151 EuN9O 14 P3] 2+ Calculated value: 745.7561; Actual measurement: 745.7562; τ H2O (ms)=0.27 (pH=8), 0.39 (pH=7), 0.53 (pH=6), 0.95 (pH=5), 0.97 (pH=4).

[0486] Complex 77b

[0487]

[0488] The crude ligand 76b (165 mg) from the previous step was dissolved in a CH3OH / H2O mixture (1:1, 2 mL total), and the pH was adjusted to 12 using an aqueous NaOH solution. The solution was heated to 60 °C for 1.5 h. After this period, LCMS analysis confirmed the complete hydrolysis of the phosphonate and chromophore ester groups. After cooling and adjusting the pH to 7 using hydrochloric acid (0.1 M), EuCl3·6H2O (34 mg, 0.093 mmol) was added, and the reaction mixture was heated to 60 °C for 17 h. The reaction mixture was then subjected to reversed-phase HPLC (10 to 100% CH3CN in H2O solution, 10 min, t... r Purification was carried out over 8.7 min to obtain a yellow solid (50 mg, 37% in three steps); ESI-LRMS (+) m / z 1462 [M+H] + 732[M+2H] 2+ ; ESI-HRMS(+)[C 65 H 86 151EuN9O 14 P3] + Calculated value: 1462.473; Actual measurement: 1462.468.

[0489] Complex 78a

[0490]

[0491] Under argon atmosphere, complex 77a (3.9 mg, 2.6 μmol) and DIPEA (6 μL, 34 μmol) were combined in anhydrous DMSO (0.4 mL). HATU (5 mg, 13 μmol), high taurine (2 mg, 14 μmol), and water (40 μL) were added to this solution. The mixture was stirred at room temperature for 19 hours. After dilution with water, the reaction mixture was passed through reversed-phase HPLC (10 to 100% CH3OH in H2O solution, 10 min, t). r Purification was carried out over 11.6 min to obtain a yellow solid (4 mg, 89%); ESI-LRMS (-) m / z 1730 [MH] - 865 [M-2H] 2- ; ESI-HRMS(+)[C 73 H 101 151 EuN 11 O 18 P3S2] 2- Calculated value: 864.7601; Actual measurement: 864.7610; τ H2O (ms)=0.25 (pH 8), 0.34 (pH 7), 0.70 (pH 6), 0.98 (pH 5), 1.04 (pH 4); ε 330nm =39000LM -1 cm -1 ;Ф pH 8 = 0.3%, Ф pH 4 = 16% (λ) exc =330nm).

[0492] Complex 78b

[0493]

[0494] Under argon atmosphere, complex 77b (20 mg, 13.7 μmol) and DIPEA (15 μL, 85 μmol) were combined in anhydrous DMSO (0.5 mL). HATU (12 mg, 31 μmol), high taurine (4.5 mg, 31 μmol), and water (50 μL) were added to this solution, and the mixture was stirred at room temperature for 16 hours. After dilution with water, the reaction mixture was passed through reversed-phase HPLC (10 to 100% CH3OH in H2O solution, 10 min, t). r Purification was carried out over 10.6 min to obtain a yellow solid (9 mg, 38%); ESI-LRMS (-) m / z 170 1 [MH] - 850 [M-2H] 2- ; ESI-HRMS(+)[C 71 H 99 151 EuN 11 O 18 P3S2] 2+ Calculated value: 852.7530; Measured value: 852.7601; τ H2O (ms)=0.30 (pH 8), 0.29 (pH 7), 0.42 (pH 6), 0.77 (pH 5), 1.05 (pH 4); ε 330nm =39000L M -1 cm -1 ;Ф pH 8 = 0.01%, Ф pH 4 = 14.5% (λ) exc =330nm).

[0495] Compound 80 was synthesized as described in Compound 11.

[0496] Compound 81 was synthesized as described in Compound 12.

[0497] Compound 82 was synthesized as described in Compound 17.

[0498] As described in complex 18, synthesize complex 83.

[0499] The complex 84 was functionalized using the method described in WO 2004 / 113275.

[0500] Compound 85a

[0501]

[0502] Under argon atmosphere, 1-(tert-butoxycarbonyl)-1,4,7-triazacyclononane dihydrochloride 9 (24 mg, 0.0794 mmol), methanesulfonate 57a (125 mg, 0.186 mmol), and K₂CO₃ (44 mg, 0.32 mmol) were combined in anhydrous CH₃CN (3 mL). The resulting mixture was heated to 65 °C for 16 hours, and then the crude solution was separated from the inorganic salts. The solvent was removed under reduced pressure to obtain an orange oil, which was then passed by reversed-phase HPLC (10 to 100% CH₃CN in H₂O containing 0.1% formic acid, t� ... r Purification was carried out over 13.5 min to obtain a pale yellow oily substance (88 mg, 80%). 1 H-NMR(700MHz, CDCl3)δ8.01(s,2H),7.66(s,1H),7.61(s,1H),7.22(d,2H),7.12(s,2H),6.84(d,2H),4.46(q,4H),4.17–3.77(m,14 H),3.46–3.31(m,8H),3.25–3.04(m,12H),2.87–2.65(m,4H),2.11–1.98(m,4H),1.77(d,6H),1.48(s,9H),1.25(t,6H),1.01(t,6H); 13 C-NMR (101MHz, CDCl3) δ161.5(d),155.6(d),155.5,153.1,138.6,132.7(d),128.1,127.9(d),126.5(d),125.8,122.1(q),113.9(d),111.8 ,96.1,85.4,79.7,63.8(q)62.5,61.1(d),55.7,55.6,54.8,54.4,53. 7,49.8,49.4,49.1,46.7,46.0,28.7,21.6,16.6(d),13.4(2×d),12.2; 19 F-NMR (376MHz, CDCl3) δ-74.0; 31 P-NMR(162MHz, CDCl3)δ+40.1(s); ESI-LRMS(+)m / z 1379[M+H] + ; ESI-HRMS(+)[C 61 H 84 F6N7O 14 P2S2] + Calculated value: 1378.490; Actual value: 1378.489.

[0503] Compound 85b

[0504]

[0505] Under argon atmosphere, 1-(tert-butoxycarbonyl)-1,4,7-triazacyclononane dihydrochloride 9 (18 mg, 0.06 mmol), methanesulfonate 57b (88 mg, 0.134 mmol), and K₂CO₃ (35 mg, 0.25 mmol) were combined in anhydrous CH₃CN (3 mL). The resulting mixture was heated to 65 °C for 18 hours, and then the crude solution was separated from the inorganic salts. The solvent was removed under reduced pressure to obtain an orange oily substance, which was then passed by reversed-phase HPLC (10 to 100% CH₃CN in H₂O solution, 10 min, t). r Purification was carried out over 14.2 min to obtain a pale yellow oily substance (58 mg, 72%). 1 H-NMR(700MHz, CDCl3)δ8.02–7.98(m,2H),7.65(s,1H),7.58(s,1H),7.20(d ,2H),7.11(s,2H),6.83(d,2H),4.50(q,4H),4.15–4.06&3.89–3.81(m,10H) ,3.95(2×s,4H),3.44–3.32(m,8H),3.16–3.05(m,8H),2.76(s,6H),2.73–2. 65(m,4H),2.17–2.10(m,4H),1.76(2×d,6H),1.48(s,9H),1.28–1.22(m,6H); 19 F-NMR (376MHz, CDCl3) δ-73.9; 31 P-NMR(162MHz, CDCl3)δ+40.1(s); ESI-LRMS(+)m / z1350[M+H] + ; ESI-HRMS(+)[C 59 H 80 F6N7O 14 P2S2] + Calculated value: 1350.458; Actual value: 1350.455.

[0506] Compound 86a

[0507]

[0508] A solution of compound 85a (88 mg, 0.064 mmol) in trifluoroacetic acid and CH₂Cl₂ (10% v / v, 3 mL total) was prepared and stirred under argon for 60 min. Immediately afterwards, the solvent was removed under reduced pressure. Another 50 mL of CH₂Cl₂ was added, and the solvent was removed under reduced pressure. This process was repeated three times. The solution was dried under high vacuum for several hours to obtain an orange oil (88 mg, quantified). 1 H-NMR (400MHz, CD3OD) δ 8.00 (d, 4 J H-H =1.9,2H),7.98(dd, 3 J H-P =6.0, 4 J H-H =1.1,2H),7.82(dd, 3 J H-H =8.8, 4 J H-H =1.9,2H),7.75–7.74(m,2H),7.42(d, 3 J H-H =8.8,2H),5.20–5.08(m,12H),4.70(q, 3 J H-F =8.3,4H),4.43(s,4H),4.16–3.90(m,10H),3.88–3.80(m,4H),3.75(q, 3 J H-H =7.1,4H),3.49(t, 3 J H-H =7.2,4H),2.04–1.94(m,4H),1.80(d, 2 J H-P =15,6H),1.30(t, 3 J H-H =7.0,6H),1.13(t, 3 J H-H =7.1,6H); 13 C-NMR (101MHz, CD3OD) δ 161.3 (d, 3 J C-P =20),155.5,155.1(d, 1 J C-P =157),137.0,134.2(d, 3 J C-P =12),128.9(d, 4 J C-P =2.5),128.8(d, 2 J C-P=22),127.8,126.1,123.8(q, 1 J C-F =280),116.8,115.0,94.9,87.2(d, 4 J C-P =2.2),65.7(q, 2 J C-F =38),63.1(d, 2 J C-P =6.5),60.6,57.7,56.6,55.3,52.4,51.0,48.0,45.5,32.7,26.7,23.7,20.8,16.8(d, 3 J C-P =6.2),13.7(d, 1 J C-P =103), 10.4; 19 F-NMR(376MHz,CD3OD)δ-73.5(-SO2CH2C F 3), -74.9 (TFA salt); 31 P-NMR(162MHz,CD3OD)δ+40.8; ESI-LRMS(+)m / z 1279[M+H] + ; ESI-HRMS(+)[C 56 H 76 F6N7O 12 P2S2] + Calculated value: 1278.437; Actual measurement: 1278.440.

[0509] Compound 86b

[0510]

[0511] A solution of compound 85b (29 mg, 0.021 mmol) in trifluoroacetic acid and CH₂Cl₂ (10% v / v, 3 mL total) was prepared and stirred under argon for 60 min. Immediately afterwards, the solvent was removed under reduced pressure. Another 50 mL of CH₂Cl₂ was added, and the solvent was removed under reduced pressure. This process was repeated three times. The solution was dried under high vacuum for several hours to obtain an orange oil (29 mg, quantified). 1 H-NMR (400MHz, CDCl3) δ 7.87 (d, 3 J H-P =6.0,2H),7.57–7.43(m,6H),7.00(d, 3 J H-H =8.5,2H),4.51(q, 3 J H-F=7.9,4H),4.27–4.09&4.03–3.87(m,14H),3.67–3.53(m,6H),3.47–3.26(m,12H),3.13–3.03(m,8H),2.14–2.05(m,4H),1.77(d, 2 J H-P =15,6H),1.31(t, 3 J H-H =7.4,6H); 19 F-NMR(376MHz,CDCl3)δ-74.6(-SO2CH2C F 3), -76.3 (TFA salt); 31 P-NMR(162MHz, CDCl3)δ+40.3; ESI-LRMS(+)m / z 1250[M+H] + ; ESI-HRMS(+)[C 54 H 72 N7F6O 12 P2S2] + Calculated value: 1250.406; Actual measurement: 1250.408.

[0512] Compound 87a

[0513]

[0514] Compound 86a (44 mg, 0.032 mmol), compound 60 (30 mg, 0.067 mmol), and K₂CO₃ (30 mg, 0.22 mmol) were combined in anhydrous CH₃CN (2 mL) under argon atmosphere and heated to 60 °C for 15 hours. After this period, LC-MS analysis confirmed the complete conversion of the macrocyclic compounds. The reaction mixture was cooled, and the solution was separated from the inorganic salts by filtration. The solvent was removed under reduced pressure to obtain a yellow oil (72 mg), which was used directly in the next step without further purification. 1 H-NMR (400MHz, CDCl3) δ 7.96 (d, 3 J H-P =6.2,1H),7.78(d, 3 J H-P =6.2,2H),7.42(s,1H),7.24(s,2H),7.19(dd, 3 J H-H =8.4, 4 J H-H =2.0,2H),7.09(d, 4 J H-H =2.0,2H),6.82(d,3 J H-H =8.4,2H),4.76(s,6H)4.68(br s,1H),4.45(q, 3 J H-F =8.0,4H),4.12–3.76(m,16H),3.37–3.30(m,4H),3.20–3.05(m,10H),2.94–2.82(m,8H),2.72–2.64(m,2H),1.83–1.79(m,4H),1.73(d, 2 J H-P =15,9H),1.41(s,9H),1.26–1.20(m,9H),0.99(t, 3 J H-H =7.1,6H); 19 F-NMR (376MHz, CDCl3) δ-74.0; 31 P-NMR(162MHz, CDCl3)δ+40.0; ESI-LRMS(+)m / z 1633[M+H] + ,817[M+2H] 2+ ; ESI-HRMS(+)[C 73 H 104 F6N9O 16 P3S2] 2+ Calculated value: 816.8080; Actual value: 816.8085.

[0515] Compound 87b

[0516]

[0517] Compound 86b (29 mg, 0.021 mmol), compound 60 (19 mg, 0.042 mmol), and K₂CO₃ (20 mg, 0.15 mmol) were combined in anhydrous CH₃CN (2 mL) under argon atmosphere and heated to 60 °C for 15 hours. After this period, LCMS analysis confirmed the complete conversion of the macrocyclic compounds. The reaction mixture was cooled, and the solution was separated from the inorganic salts by filtration. The solvent was removed under reduced pressure to obtain a yellow oil (47 mg), which was used directly in the next step without further purification. 1 H-NMR (400MHz, CDCl3) δ 7.99 (d, 3 J H-P =6.0,1H),7.80(dd, 3 J H-P =6.0, 4 J H-H=1.4,2H),7.44–7.42(m,1H),7.25–7.22(m,2H),7.22–7.16(m,2H),7.09(d, 4 J H-H =1.8,2H),6.83(d, 3 J H-H =8.4,2H),4.77(s,6H),4.50(q, 3 J H-F =8.0,4H),4.13–3.76(m,16H),3.43–3.38(m,2H),3.17–3.09(m,14H),2.75(s,6H),2.71–2.66(m,2H),1.85–1.80(m,4H),1.74(d, 2 J H-P =15,9H),1.42(s,9H),1.25(t, 3 J H-H =7.1,9H); 19 F-NMR (376MHz, CDCl3) δ-73.9; 31 P-NMR(162MHz, CDCl3)δ+40.0; ESI-LRMS(+)m / z 1605[M+H] + ; ESI-HRMS(+)[C 71 H 99 F6N9O 16 P3S2] + Calculated value: 1604.577; Actual value: 1604.577.

[0518] Complex 88a

[0519]

[0520] Crude ligand 87a (57 mg) was dissolved in a mixture of CH3OH / NaOH aqueous solution (0.1 M, 3:1, total 2 mL). The solution was heated to 70 °C for 24 hours. After this period, LCMS analysis confirmed the complete hydrolysis of the phosphonate and sulfonate groups. After cooling and adjusting the pH to 6 with dilute hydrochloric acid (0.1 M), EuCl3·6H2O (14 mg, 0.038 mmol) was added, and the reaction mixture was heated to 75 °C for 24 hours. The reaction mixture was then subjected to reversed-phase HPLC (10 to 100% CH3CN in H2O solution, 10 min, t). r =7.9 min) purification, yielding a pale yellow solid (13 mg, 27% in two steps); τ H2O(ms)=0.31 (pH9), 0.31 (pH8), 0.29 (pH 7), 0.41 (pH 6), 0.76 (pH 5), 1.07 (pH 4); ε 332nm =35000M -1 cm -1 ;Ф pH 8 = 0.06%, Ф pH 4 = 13.4% (λ) exc =332nm).

[0521] Complex 88b

[0522]

[0523] Crude ligand 87b (23 mg) was dissolved in a mixture of CH3OH / NaOH aqueous solution (0.1 M, 3:1, total 2 mL). The solution was heated to 70 °C for 48 hours. After this period, LCMS analysis confirmed the complete hydrolysis of the phosphonates and sulfonates. After cooling and adjusting the pH to 5.5 with hydrochloric acid (0.1 M), EuCl3·6H2O (8 mg, 0.022 mmol) was added, and the reaction mixture was heated to 75 °C for 24 hours. The reaction mixture was then subjected to reversed-phase HPLC (10 to 100% CH3CN in H2O solution, 10 min, t... r =7.9 min) purification, yielding a deep yellow solid (1.8 mg, 11% in two steps); τ H2O (ms)=0.30(pH6.5),0.31(pH6),0.40(pH 5),0.83(pH 4),1.01(pH 3).ε 336nm =35000M -1 cm -1 ;Ф pH 6 = 0.06%, Ф pH 3 = 13% (λ) exc =336nm).

[0524] Compound 89 was synthesized as described in Compound 92.

[0525] Compound 90 was synthesized as described in Compound 93.

[0526] Compound 91 was synthesized as described in compound 78a.

[0527] Compound 92

[0528]

[0529] Compound 85a (44 mg, 0.032 mmol), compound 38 (31 mg, 0.049 mmol), and K₂CO₃ (30 mg, 0.22 mmol) were combined in anhydrous CH₃CN (2 mL) under argon atmosphere and heated to 60 °C for 14 hours. After this period, LCMS analysis confirmed the complete conversion of macrocyclic compound 85a. The reaction mixture was cooled, and the solution was separated from the inorganic salt by filtration. The solvent was removed under reduced pressure to obtain a yellow oil (47 mg), which was used directly in the next step without further purification. 1 H-NMR (400MHz, CD3OD) δ7.91–7.82(m,5H),7.30–7.16(m,7H),6.99–6.93(m,3H),4.84(s,6H),4.64(q, 3 J H-F =8.1,4H),4.14–3.84(m,22H),3.43–3.37(m,2H),3.30–3.24(m,2H),3.19–3.07(m,8H),2.91–2.83(m,8H),2.02–1.89(m,6H),1.77(d, 2 J H-P =15,9H),1.44(s,9H),1.26–1.21(m,9H),1.03–0.98(m,12H); 19 F-NMR (376MHz, CD3OD) δ -76.8; 31 P-NMR(162MHz,CD3OD)δ+41.8; ESI-LRMS(+)m / z 1821[M+H] + ,911[M+2H] 2+ ; ESI-HRMS(+)[C 85 H 117 F6N 10 O 17 P3S2] 2+ Calculated value: 910.8594; Actual value: 910.8554.

[0530] Complex 93

[0531]

[0532] Crude ligand 92 (38 mg) was dissolved in a mixture of CH3OH / NaOH aqueous solution (0.1 M, 3:1, total 2 mL). The solution was heated to 70 °C for 24 hours. After this period, LCMS analysis confirmed the complete hydrolysis of the phosphonates and sulfonates. After cooling and adjusting the pH to 6.5 with hydrochloric acid (0.1 M), EuCl3·6H2O (14 mg, 0.038 mmol) was added, and the reaction mixture was heated to 75 °C for 24 hours. The reaction mixture was then subjected to reversed-phase HPLC (10 to 100% CH3CN in H2O solution, 10 min, t). r Purification was carried out over 8.8 min to obtain a pale yellow solid (11 mg, 25% in two steps); τ H2O (ms)=0.20 (pH 9), 0.21 (pH 8), 0.22 (pH 7), 0.35 (pH 6), 0.69 (pH 5), 0.98 (pH 4), 0.98 (pH 4), 1.01 (pH 3.5).ε 332nm =75000M -1 cm -1 ;Ф pH 8 = 0.01%, Ф pH 4 = 15.9% (λ) exc =332nm).

[0533] Compound 94 was obtained according to the process described in US9,981,967.

[0534] Compound 95 was prepared as described in Compound 20.

[0535] Complex 96 was prepared as described in complex 21.

[0536] Complex 97 was prepared as described in complex 78a.

[0537] Complex 98 was prepared as described in complex 99a.

[0538] Complex 99a

[0539]

[0540] Trifluoroacetic acid (200 μL) was added to complex 78a (6.93 mg, 4 μmol). The mixture was stirred at room temperature for 1 hour, and then analyzed by preparative HPLC (Waters Xbridge C10 column). 18 ,5μm,20×100mm–A / H2O 25mM TEAAc pH7B / CH3CN t=0min 2%B–t=18min 40%B–20mL.min -1Purification yielded a yellow solid (3.5 μmol, 87%). ESI-LRMS(-) m / z 814.70 [M-2H] 2- .

[0541] Complex 99b

[0542]

[0543] Trifluoroacetic acid (1 mL) was added to complex 78b (9 mg, 5283 nmol). The mixture was stirred at room temperature for 1 hour, and then analyzed by preparative HPLC (Waters Xbridge C10 column). 18 , 5μm, 20×100mm–A / H2O 25mM TEAAc pH7B / CH3CN t=0min 2%B–t=18min 40%B–20mL.min -1 Purification yielded a yellow solid (4256 nmol, 81%). ESI-LRMS(-) m / z 800.83 [M-2H] 2- .

[0544] BG-MB-NHS (Structural Scheme 26)

[0545] The compound was obtained as described in Inorganic Chemistry-2014-53-1854.

[0546] Complex 100a

[0547]

[0548] 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 hour. The solution was then analyzed by preparative HPLC (Waters Xbridge C10 column). 18 , 5μm, 20×100mm–A / H2O 25mM TEAAcpH 7B / CH3CN t=0min 2%B–t=18min 40%B–20mL.min -1 Purification yielded a white powder (370 nmol, 43%). ESI-LRMS (-) m / z 1064.89 [M-2H] 2- .

[0549] Complex 100b

[0550] 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 hour. The solution was then analyzed by preparative HPLC (Waters Xbridge C10 column). 18 ,5μm,20×100mm–A / H2O 25mM TEAAcpH 7B / CH3CN t=0min 2%B–t=18min 40%B–20mL.min -1 Purification yielded a white powder (0.728 μmol, 60% yield). ESI-LRMS(-) m / z 1051.09 [M+H] 2- .

[0551] Complex 101a

[0552]

[0553] To a solution of complex 99a (1.403 mg, 860 nmol) in dry DMSO (100 μL), add a solution of DIPEA (0.150 μL, 860 nmol) and N-hydroxysuccinimide 6-maleimide hexanoate (0.271 mg, 860 nmol) in anhydrous DMSO (5 μL). Stir the mixture at room temperature for 1 hour and then analyze by preparative HPLC (Waters Xbridge C10 column). 18 ,5μm,20×100mm–A / H2O 25mM TEAAc pH 5B / CH3CN t=0min 2%B–t=18min 40%B–20mL.min -1 Purification yielded a yellow powder (761 nmol, yield 89%). ESI-LRMS(-) m / z 911.75 [M+H] 2- .

[0554] Complex 101b

[0555]

[0556] To a solution of complex 99b (1.283 mg, 800 nmol) in dry DMSO (100 μL), add DIPEA (0.140 μL, 800 nmol) and anhydrous DMSO (5 μL) solution of 6-maleimide hexanoic acid N-hydroxysuccinimide ester (0.252 mg, 800 nmol). Stir the mixture at room temperature for 1 hour and then analyze by preparative HPLC (Waters Xbridge C10 column).18 ,5μm,20×100mm–A / H2O 25mM TEAAc pH 5B / CH3CN t=0min 2%B–t=18min 40%B–20mL.min -1 Purification yielded a yellow powder (726 nmol, yield 91%). ESI-LRMS(-) m / z 898.2 [M+H] 2- .

[0557] Compound 102 was synthesized according to the process described in Helvetica Chimica Acta, 1993, 76, 877.

[0558] Compound 103

[0559]

[0560] Compound 36 (74 mg, 0.214 mmol) and compound 102 (140 mg, 0.195 mmol) were combined in anhydrous CH3CN (2.5 mL) under argon atmosphere. Pd2Cl2(allyl)2 (10 mg, 0.027 mmol) and P( t Bu)3 (0.01 mL, 0.041 mmol) and piperidine (0.06 mL, 0.66 mmol). The resulting mixture was stirred at 40 °C for 17 hours under argon atmosphere, followed by solvent removal under reduced pressure. The crude product was subjected to reversed-phase HPLC (10 to 100% CH3CN in H2O solution, t... r Purification was carried out over 19.8 min to obtain a pale orange oil (73 mg, 40%). 1 H-NMR(700MHz, CDCl3)δ7.59(s,2H),7.18–7.11(m,2H),6.81(d, 3 J H-H =8.2,1H),4.09(t, 3 J H-H =5.4,2H),4.01(s,4H),3.49(s,8H),3.40–3.34(m,2H),3.15(q, 3 J H-H =6.7,4H),2.03–1.98(m,2H),1.46(s,36H),1.45(s,9H),1.02(t, 3 J H-H =6.7,6H); 13C-NMR (176MHz, CDCl3) δ170.7,159.1,156.4,154.6,139.6,133.1,127.3,125.8,122.8,114.9,11 2.8,94.0,86.4,81.2,79.0,68.5,59.9,56.0,46.4,39.3,29.7,28.7,28.3,12.0; ESI-LRMS(+)m / z 939[M+H] + ; ESI-HRMS(+)[C 51 H 80 N5O 11 ] + Calculated value: 938.5854; Actual value: 938.5851.

[0561] Complex 104

[0562]

[0563] A solution of complex 102 (36 mg, 0.038 mmol) was prepared in trifluoroacetic acid and CH2Cl2 (20% v / v, 4 mL total). The solution was stirred at room temperature for 6 hours, and then the solvent was removed under reduced pressure to obtain an orange residue. The residue was subjected to reversed-phase HPLC (10 to 100% CH3CN in H2O solution, 10 min, t... r Purification was performed (1.2 min) to obtain free amine. The free amine was dissolved in H2O (3 mL), the pH was adjusted to 6.5, and then EuCl3·6H2O (14 mg, 0.038 mmol) was added. The mixture was heated to 60 °C for 24 hours, and then the solution was separated from the inorganic salts by filtration. The solution was then subjected to reversed-phase HPLC (10 to 100% CH3CN in H2O solution, 10 min, t). r Purification was carried out over 1.7 min to obtain a yellow solid (27 mg, 93% in two steps); ESI-LRMS (+) m / z 763 [M+H] + ; ESI-HRMS(+)[C 30 H 36 151 EuN5O9] + Calculated value: 763.1729; Actual measurement: 763.1731; τ H2O (ms)=0.30 (pH 8), 0.32 (pH 7), 0.35 (pH 6), 0.37 (pH 5), 0.38 (pH 4); ε 330nm =12000M -1 cm -1 ;Ф pH 8 = 0.17%, Ф pH4 = 3.2% (λ) exc =330nm).

[0564] Complex 105

[0565]

[0566] To a solution of complex 104 (0.655 mg, 860 nmol) in dry DMSO (100 μL), add a solution of DIPEA (0.150 μL, 860 nmol) and N-hydroxysuccinimide 6-maleimide hexanoate (0.271 mg, 860 nmol) in anhydrous DMSO (5 μL). Stir the mixture at room temperature for 1 hour and then analyze by preparative HPLC (Waters Xbridge C100 column). 18 ,5μm,20×100mm–A / H2O 25mM TEAAc pH 6B / CH3CN t=0min 5%B–t=19min 40%B–20mL.min -1 Purification yielded a yellow powder. ESI-LRMS(-) m / z 956.85 [M+H] - .

[0567] Complex 106

[0568]

[0569] 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 hour. The mixture was then analyzed by preparative HPLC (Waters Xbridge C10 column). 18 ,5μm,20×100mm–A / H2O 25mM TEAAc pH6B / CH3CN t=0min 5%B–t=19min 40%B–20mL.min -1 Purification yielded a white powder (0.728 μmol, yield 60%). ESI-LRMS(-) m / z 1262.06 [M+H] - .

[0570] The properties of representative complexes of this invention (complexes 14, 18, 21, 78a, 78b, 88a, 88b, 93, and 104) were investigated. The presence of the Boc group was not expected to alter these properties. Therefore, functionalized complexes were tested with the Boc group. Absorption and emission spectra were recorded across a pH gradient (pH 4 to pH 10).

[0571] 1) Photophysical properties

[0572] Tables 1 and 2 summarize the photophysical properties.

[0573] Table 1: Photophysical properties of some europium complexes

[0574] Complex 14 18 21 78a 78b 88a 88b 93 104 <![CDATA[λ exc (nm)]]> 331 328 331 330 330 332 336 332 330 <![CDATA[ε(M -1 .cm -1 )]]> 11450 35000 45000 39000 39000 35000 35000 75000 12000 QYpH 8 0.5 0.2 0.1 0.3 0.01 0.06 0.06 0.01 0.17 QYpH 4 16.8 17.6 17 16 14.5 13.4 13 15.9 3.2

[0575] Traditionally, the molar extinction coefficient ε is calculated using the Beer-Lambert equation, which involves measuring the absorbance of a series of complex solutions at different known concentrations. A graph showing absorbance versus concentration at a specific wavelength allows ε to be determined based on a linearly correlated gradient. The quantum yield (QY) of the complex increases with decreasing pH. For complexes 78b, 88a, 88b, and 93, the quantum yield is close to 0 at pH 8, meaning these complexes are almost non-luminescent at physiological pH; conversely, they are very bright at pH 4.

[0576] Unexpectedly, an isoabsorbance point was observed at 332 nm. This could be useful because excitation at or near this wavelength would allow for isoexcitation of both unprotonated and protonated complexes existing in a pH-dependent equilibrium in solution. No isoabsorbance point was observed for complex 14.

[0577] Table 2: Excited-state lifetimes (ms) of complexes at different pH values

[0578] Complex 14 18 21 78a 78b 88a 88b 93 104 pH 9 0.5 0.34 0.24 - - 0.31 - 0.2 pH 8 0.53 0.34 0.25 0.25 0.3 0.31 - 0.21 0.3 pH 7 0.74 0.47 0.32 0.34 0.29 0.29 0.3 0.22 0.32 pH 6 1.06 0.78 0.59 0.7 0.42 0.41 0.31 0.35 0.35 pH 5 1.15 0.96 0.83 0.98 0.77 0.76 0.4 0.69 0.37 pH 4 1.16 1 0.84 1.04 1.05 1.07 0.83 0.98 0.38 pH 3 - - - - - - 1.01 1.01 -

[0579] 2) Emission of complexes

[0580] For all emission spectra, an impressive emission intensity “switch” was observed as pH decreased beyond the physiological range (Nat. Rev. Mol. Cell. Biol. 2010, 11, 50). ΔJ = 2 bands (( Figure 2 The ΔJ distribution shown is clearly the strongest in the emission spectrum and therefore the most suitable reference band, especially under higher wavelength excitation. For all complexes of this invention, the emission intensity varies with pH. Replacing an extended chromophore with a single pyridine and attaching a long-headed group around the chromophore have no effect on the form of the europium emission spectrum. In fact, significant modifications to the complexes do not affect the direct harmonic environment around the europium(III) center, which remains virtually undisturbed.

[0581] As the most emissive band, the maximum wavelength of the ΔJ = 2 band is selected ( Figure 2The ΔJ allocation shown in the figure is used as the emission wavelength to be monitored.

[0582] 3) pK of the complex a value

[0583] Table 3 shows the pK values ​​of the complexes of the present invention. a value.

[0584] Table 3: pK of the complex a Value (295K, I = 0.1M NaCl)

[0585] Complex 14 18 21 78a 78b 88a 88b 93 104 <![CDATA[pK a ]]> 6.75 6.3 6.21 6.20 5.34 5.2 4.32 5.19 6.35

[0586] The experimentally measured excited-state lifetimes exhibited very similar pH-dependent curves, independent of the number of chromophores incorporated into the europium complex.

[0587] Emission lifetime as a function of pH Figure 3 , Figure 6 , Figure 9 , Figure 12 , Figure 15 , Figure 18 , Figure 21 , Figure 24 and Figure 27 As shown. The emission lifetime value for each pH was obtained by plotting the decay of emission lifetime over time and fitting the curve to a simple exponential decay model. For reversible protonation, the pK of the complex was determined. a Value. The pK of the protonation of one chromophore to the other chromophore in the complexes (complexes 18 and 21). a No impact.

[0588] Chemical modification of chromophores involves pK a Some changes in the value (from 4.32 to 6.75). This pK can be fine-tuned. a The value was specifically obtained to obtain the desired value. This result is highly desirable because it is conceivable to monitor changes in pH organelles within cells, such as in lysosomes, endosomes, and the trans-Golgi network.

[0589] 4) Time-gated experiment

[0590] One advantage of the long emission lifetimes of lanthanides is the ability to perform time-gated measurements. Furthermore, due to the “switching” properties of complex 18, the emission lifetimes of the unprotonated and protonated forms differ. These properties offer unique opportunities to, to some extent, control the apparent pK by altering the time window. a The value and the relative degree of probe "on / off". In practice, this is achieved using appropriate buffer solutions of different pH values ​​(in the presence of 0.1M NaCl). Figure 28 This was explained in the text.

[0591] The emission intensity of the ultrasensitive ΔJ=2 band was measured at different pH values ​​with longer excitation delay times. Figure 28 (top), resulting in a shallower curve (lower apparent pK) a ).

[0592] Using three different time windows ( Figure 28 The bottom section illustrates this concept: 60–460, 1000–2000, and 1500–2500 μs.

[0593] Besides apparent pK a Beyond the fine-tuning, these time-gating results also demonstrate the possibility of altering the magnitude of the probe's on / off ratio by changing the time window.

[0594] Table 4 summarizes the emission intensity ratio (λ) of complex 18 at pH 4 (sensor on) and pH 8 (sensor off). em =613.5nm), where the transmitted signal was measured between different time windows.

[0595] Table 4

[0596]

[0597] For comparative purposes, the on / off response of the complex can be quantified as the ratio of the emission intensity of the maximum value in the ΔJ=2 band at pH 4 (sensor fully "on") and pH 8 (sensor effectively "off"). These values ​​are summarized in Table 4.

[0598] With the use of a longer time window, the relative switching response increases significantly.

[0599] It is worth noting that, due to the presence of hydrophilic sulfonate groups on the periphery of the complexes, the water solubility of complexes 78a-b, 88a-b, and 93 is significantly higher than that of complexes 14, 18, and 21.

[0600] 5) Fluorescence intensity

[0601] The fluorescence intensity of representative complexes (78a, 78b, 104) of the present invention was measured using a Pherastar FS plate reader equipped with a flash lamp. Buffer solutions (pH 4 to pH 7.8) were distributed in white 96-well plates, and then lanthanide complexes (10 μL, 110 nM) were distributed in an aqueous solution containing 0.1% BSA. Fluorescence intensities were then recorded between pH 4 and pH 7.8, and the results are reported in [the table / document / etc.]. Figure 29 In addition, the fluorescence intensity of the following complexes was also measured by comparison:

[0602]

[0603]

[0604] Complexes 107 to 110 are synthesized as described in WO 2014 / 111661. Complex 111 is commercially available under the trade name Lumi4-Tb.

[0605] from Figure 29 It can be seen that the complexes (78a, 78b, and 104) of the present invention are pH sensitive: the fluorescence intensity of these complexes increases when the pH decreases. In contrast, the comparative complexes (107, 108, 109, 110, and 111) are not pH sensitive because their fluorescence intensity does not change significantly with pH.

Claims

1. A compound of formula (I): in: R1 is –CO2H, -PO(OH)R5 or –CH2N(CH2CO2H)2; R2 is –CH2OH, –CH2OSO2CH3 or –CH2N(CH2CO2H)2; R3 is an alkyl group that is optionally substituted with a –L1-E group (C1-C6); R4 is -NR6R7; R5 is an (C1-C4) alkyl group; optionally, it is enclosed by a SO3 group. - Substituted phenyl; R6 is a (C1-C4) alkyl group; R7 is an alkyl group that is optionally substituted with a –L1-E group (C1-C6); L1 is a bond, –CONH-(CH2) n -or –NHCO-(CH2) n -; E is -SO3H or -SO2(OCH2CF3); n can be 0, 1, 2, or 3.

2. The compound according to claim 1, wherein R1 is –CO2H or -PO(OH)R5, wherein R5 is (C1-C4) alkyl, and R2 is –CH2OH or -CH2OSO2CH3.

3. The compound according to claim 1, wherein R1 and R2 are each –CH2N(CH2COOH)2.

4. The compound according to claim 1, wherein R4 is –NR6R7, and wherein R6 and R7 are each independently (C1-C4) alkyl.

5. The compound according to claim 1, wherein L1 is a bond or –CONH-(CH2). n - 6. A compound of formula (II): (II) in: R a It either does not exist or is –CH2NH2; Chrom1, Chrom2, and Chrom3 are each independently selected from groups of formula (IV) and formula (V): (IV) (V) in: Each R1 is –CO2H or -PO(OH)R5; R3 is an alkyl group that is optionally substituted with a –L1-E group (C1-C6); R4 is -NR6R7; R5 is an (C1-C4) alkyl group; optionally, it is enclosed by a SO3 group. - Substituted phenyl; R6 is a (C1-C4) alkyl group; R7 is an alkyl group that is optionally substituted with a –L1-E group (C1-C6); R9 is an (C1-C6) alkyl group optionally substituted with a group –L1-E or a group G; L1 is a bond, –CONH-(CH2) n -or –NHCO-(CH2) n ; E is -SO3H or -SO2(OCH2CF3); G is a carboxyl group optionally protected in the form of an ester, an amino group optionally protected by a tert-butoxycarbonyl group, a succinimide ester group, a haloacetamido group, a hydrazine group, an isothiocyanate group, or a maleimide group. n is 0, 1, 2, or 3; The condition is that the compound of formula (II) contains at least one group of formula (IV).

7. The compound according to claim 6, wherein it contains only one group of formula (IV).

8. The compound according to claim 6, comprising two groups of formula (IV).

9. The compound according to claim 6, comprising three formula (IV) groups.

10. The compound according to claim 6, wherein each R1 is -PO(OH)R5, wherein R5 is (C1-C4) alkyl.

11. The compound according to claim 6, wherein R4 is –NR6R7, and wherein R6 and R7 are each independently (C1-C4) alkyl.

12. The compound according to claim 6, wherein L1 is a bond or –CONH-(CH2). n - 13. The compound according to claim 6, wherein G is an amino group optionally protected by a tert-butoxycarbonyl group.

14. The compound of claim 6, wherein G is a carboxyl group optionally protected in the form of an ester or an amino group optionally protected by a tert-butoxycarbonyl group.

15. A compound of formula (III): (III) in: R b R c R d and R e One of them is a group of formula (IV), and the others are each independently selected from –CH2COOR. f and -CH2PO(OH)R g ; R f H, (C1-C4)alkyl, -NHCH(R) h -(C1-C4)alkyl or -NHCH(R) h )-C(O)OR j ; R g It is an (C1-C4) alkyl group; R h It is (C1-C4) alkyl or phenyl; R j It is H or (C1-C4) alkyl; (IV) in: Each R1 is –CO2H or -PO(OH)R5; R3 is an (C1-C6) alkyl group optionally substituted with a group –L1-E or a group G; R4 is -NR6R7; R5 is an (C1-C4) alkyl group; optionally, it is enclosed by a SO3 group. - Substituted phenyl; R6 is a (C1-C4) alkyl group; R7 is an alkyl group that is optionally substituted with a –L1-E group (C1-C6); L1 is a bond, –CONH-(CH2) n -or –NHCO-(CH2) n ; E is -SO3H or -SO2(OCH2CF3); G is a carboxyl group optionally protected in the form of an ester, an amino group optionally protected by a tert-butoxycarbonyl group, a succinimide ester group, a haloacetamido group, a hydrazine group, an isothiocyanate group, or a maleimide group. n can be 0, 1, 2, or 3.

16. A europium complex comprising europium(III) ions and a compound of formula (I) or (II) according to any one of claims 1 to 14.

17. A europium complex comprising europium(III) ions and a compound of formula (III) according to claim 15.

18. A compound containing europium(III) ions having any of the following structures: 、 、 、 、 、 、 、 、 ， Where Z is: 。 19. A conjugate obtained by (i) a europium complex and (ii) a target molecule, wherein the europium complex comprises a europium(III) ion and a compound according to claim 7 or 8, wherein R9 is a (C1-C6) alkyl group substituted with a group G.

20. The conjugate according to claim 19, wherein the target molecule is an amino acid, protein, carbohydrate chain, nucleoside, nucleotide or enzyme substrate.

21. The conjugate according to claim 20, wherein the target molecule is an enzyme substrate.

22. A conjugate comprising (i) a europium complex with the following structure ， (ii) The target molecule is obtained through reaction.

23. The conjugate according to claim 22, wherein the target molecule is an amino acid, protein, carbohydrate chain, nucleoside, nucleotide or enzyme substrate.

24. The conjugate according to claim 23, wherein the target molecule is an enzyme substrate.

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