Bioorthogonal reactions suitable for click / unclick applications

Abstract

Disclosed are compounds useful for cell labeling or cancer treatment, and pharmaceutically acceptable salts and stereoisomers thereof. Also disclosed are pharmaceutical compositions containing the compounds, as well as methods of making and using the compounds.

Description

[0001] Related applications

[0002] This application claims priority claims under 35 U.S. SC §119(e) to U.S. Provisional Application No. 63 / 170,705, filed April 5, 2021, and U.S. Provisional Application No. 63 / 315,328, filed March 1, 2021, each of which is incorporated herein by reference in its entirety.

[0003] Government support

[0004] This invention was completed with government funding granted by the National Institutes of Health (NIH) under grant number 1DP2 ES030448. The government holds certain rights to this invention.

[0005] sequence list

[0006] This application contains a sequence list that has been electronically submitted in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on April 4, 2022, is named 52095-726001WO_ST25.txt and is 6KB in size. Background of the Invention

[0008] Two decades ago, the advent of copper-catalyzed cycloaddition of azides and alkynes (CuAAC) reactions (Rostovtsev et al., Angew. Chem., Int. Ed. 41(14): 2596-2599(2002)) proved indispensable in various fields from materials science to chemical biology (Hein et al., Chem. Soc. Rev. 39: 1302-1315(2010); Neumann et al., Macromol. Rapid Commun. 41: 1900359(2020)). It is a popular reaction due to its rapid kinetics, ease of execution, and tunability with virtually universal reaction components. The relatively inert azides and terminal alkynes are two of the smallest functional groups available, and either component can be readily incorporated into biomolecules, metabolites, and probes without significantly interfering with the system being evaluated. Using orthogonal tRNA systems and methionine auxotrophs via non-natural amino acids (Kiick et al., Proc. Natl. Acad. Sci. USA 99(1): 19-24 (2002); Prescheret et al., Nat. Chem. Biol. 1(1): 13-21 (2005); Plass et al., Angew. Chem., Int. Ed. 50(17): 3878-3881 (2011)), via lipid and nucleotide modifications (Parker et al., Cell 180(4): 605-632 (2020); Hang et al., Acc. Chem. Res. 44(9): 699-708 (2011); Laguerre et al., Curr. Opin. Cell Biol. 53: 97-104 (2018); Flores et al. The ability to incorporate these motifs into biological systems has been altered through metabolic engineering (Parker et al., Cell 180(4): 605-632(2020); Agard et al., Acc. Chem. Res. 42(6): 788-797(2009); Laughlin et al., Proc. Natl. Acad. Sci. USA 106(1): 12-17(2009)).

[0009] Subsequently, Bertozzi and colleagues have extended the application of azide-alkyne cycloaddition reactions to living cells and in vivo systems by using strained cyclooctyne instead of copper catalysts (Baskin et al., Proc. Natl. Acad. Sci. USA 104(43): 16793-16797(2007)). Currently, strain-promoted reactions are a major component of the bioorthogonal program, with notable examples including trans-cyclooctene (Blackman et al., J.Am.Chem.Soc.130(41):13518-13519(2008)), norbornene (Devaraj et al., Bioconjugate Chem.19(12):2297-2299(2008)), tetracycloane (Sletten et al., J.Am.Chem.Soc.133(44):17570-17573(2011)) and cyclopropane (Patterson et al., J.Am.Chem.Soc.134(45):18638-18643(2012); Row et al.). The reverse electron demand of Diels-Alder cycloaddition, dipole cycloaddition and phosphine linkages (al., J.Am.Chem.Soc.139(21):7370-7375(2017)).Importantly, cyclooctyne has undergone extensive geometric (Dommerholt et al., Angew. Chem., Int. Ed. 49(49): 9422-9425 (2010); Ning et al., Angew. Chem., Int. Ed. 47(12): 2253-2255 (2008); Mbua et al., ChemBioChem 12(12): 1912-1921 (2011); Jewett et al., J. Am. Chem. Soc. 132(11): 3688-3690 (2010); de Almeida et al., Angew. Chem., Int. Ed. 51(10): 2443-2447 (2012)) and electronic (Agard et al., ACS) studies. Chem.Biol.1(10):644-648(2006); Baskin et al., Proc.Natl.Acad.Sci.USA104(4):16793-16797(2007); Ni et al., Angew.Chem., Int.Ed.54(4):1190-1194(2015); Hu et al., J.Am.Chem.Soc.142(44):18826-18835(2020)) to enhance with azides (Agard et al., J.Am.Chem.Soc.126(46):15046-15047(2004)), tetrazine (Blackman et al. The reaction kinetics of sydnone (Tao et al., Chem. Commun. 54(40): 5082-5085(2018)) and diazo (Andersen et al., J. Am. Chem. Soc. 130(41): 13518-13519(2008); Yang et al., Angew. Chem., Int. Ed. 51(30): 7476-7479(2012)), sydnone (Tao et al., Chem. Commun. 54(40): 5082-5085(2018)) and diazo (Andersen et al., J. Am. Chem. Soc. 137(7): 2412-2415(2015)).

[0010] The increasing number of bioorthogonal reaction programs makes it possible to visualize, separate and manipulate biomolecules in complex biological environments in vitro and in vivo (Sletten et al., Angew. Chem., Int. Ed. 48(38): 6974-6998 (2009); Parker et al., Cell 180(4): 605-632 (2020); Takayama et al., Molecules 24(1): 172 (2019)). These reactions are helpful for studying primary and secondary metabolites, such as sugars (Baskin et al., Proc. Natl. Acad. Sci. USA 104(4): 16793-16797 (2007); Agard et al., Acc. Chem. Res. 42(6): 788-797 (2009); Cioce et al., Curr. Opin. Chem. Biol. 60: 66-78 (2021)) and lipids (Hang et al., Acc. Chem. Res. 44(9): 699-708 (2011); Laguerre et al., Curr. Opin. Cell Biol. 53: 97-104 (2018); Flores et al., Chem. Soc. Rev. 49: 4602-4612 (2020)) as well as biomacromolecules (George According to et al., Chem. Commun. 56: 12307-12318 (2020), modifying it through genetic means is neither practical nor possible. Therefore, the need for other biological orthogonal tools remains, especially those that are more compact (Shih et al., J.Am.Chem.Soc.137(32):10036-10039(2015); Andersen et al., J.Am.Chem.Soc.137(7):2412-2415(2015)), faster (Jewett et al., J.Am.Chem.Soc.132(11):3688-3690(2010); Darko et al., Chem.Sci.5:3770-3776(2014); Hu et al., J.Am.Chem.Soc.142(44):18826-18835(2020)), and more stable (Row et al.). al., J.Am.Chem.Soc.139(21):7370-7375(2017); Tu et al., Angew.Chem., Int.Ed.58(27):9043-9048(2019)), regional selectivity ( et al., Org. Biomol. Chem. 13: 3866-3870 (2015)), functional diversification ( et al., Angew. Chem., Int. Ed. 53(39): 10536-10540 (2014); Li et al., Nat. Chem. Biol. 12(3): 129-137 (2016); Versteegen et al., Angew. Chem., Iht. Ed. 57(33): 10494-10499 (2018); Carlson et al., J. Am. Chem. Soc. 140(10): 3603-3612 (2018); Ji et al., Chem. Soc. Rev. 48: 1077-1094 (2019) and orthogonal to biology and itself (Liang et al.) The tools of J. Am. Chem. Soc. 134(43): 17904-17907 (2012); Patterson et al. Curr. Opin. Chem. Biol. 28: 141-149 (2015).

[0011] The development of new responses, not just along one or two axes, but along several of these axes simultaneously, has always been an attractive but difficult aspiration (Row et al., Acc. Chem. Res. 51(5): 1073-1081 (2018); Devaraj, NK, ACS Cent. Sci. 4(8): 952-959 (2018)). Invention Overview

[0013] The first aspect of the present invention relates to compounds represented by the structure of formula (I):

[0014]

[0015] R1, R1', R2 and A1 are as defined herein, or pharmaceutically acceptable salts or stereoisomers thereof.

[0016] Other aspects of the invention relate to compounds represented by formulas (II) and (III):

[0017]

[0018] R4, R5, R6, R7, R7', R8, X, Y, and n are as defined herein, or pharmaceutically acceptable salts or stereoisomers thereof.

[0019] Another aspect of the present invention relates to enamine N-oxides represented by formulas (IV) and (V):

[0020]

[0021] R1, R1', R2, A1, R4, R5, R6, R7, R7', R8, X, Y, and n are as defined herein, or pharmaceutically acceptable salts or stereoisomers thereof. The compounds of formulas (IV) and (V) each contain at least two active moieties.

[0022] The compounds of the present invention are particularly suitable for clinical applications in which the delivery of two active agents or portions is advantageous. Thus, in some embodiments, the compounds of formula (IV) or (V) are antibody-drug conjugates, wherein one of the two active portions is a binding moiety, and the other active portion is a therapeutic agent. In other embodiments, the compounds of formula (IV) or (V) are proteolytic targeting chimeras (also known as PROTACs or degraders) that selectively degrade a given protein, wherein both active portions are binding moiety. One binding moiety binds to the target protein, while the other binding moiety binds to a cellular enzyme that catalyzes the degradation of the target protein. Although both active portions are binding moiety, the compound itself is therapeutic. In other embodiments, the compounds of formula (IV) or (V) are therapeutic diagnostic agents, wherein one of the two active portions is a diagnostic agent, and the other active portion is a therapeutic agent.

[0023] Another aspect of this invention relates to a process for preparing bifunctional enamine N-oxide compounds of formulas (IV) and (V) carrying two different active moieties. The process for producing compound (IV) requires reacting compound (I) with compound (II). The process for producing compound (V) requires reacting compound (I) with compound (III). The processes or synthetic methods for producing compounds (IV) and (V) involve two reagents, namely, a bioorthogonal reaction between compound (I) and compounds (II) and (III). More specifically, it is a non-catalytic conjugate retro-Cope elimination reaction that enables the biorthogonal connection of the two active moieties.

[0024] Another aspect of the invention relates to pharmaceutical compositions comprising a therapeutically effective amount of a compound of formula (IV) or a pharmaceutically acceptable salt or stereoisomer thereof, and a pharmaceutically acceptable carrier.

[0025] Further aspects of the invention relate to methods for diagnosing and treating diseases and conditions. In some embodiments, the disease is cancer. Other aspects of the invention relate to methods for protein labeling. In some embodiments, these methods involve labeling cancer-associated antigens.

[0026] The biorthogonal reaction is rapid and binds (links) the two active moieties together via a cleavable linker. The biorthogonal reaction may occur before administration to the subject or in vivo after administration of a single reagent. That is, compounds (IV) and (V) can be administered to the subject. Alternatively, these compounds can be formed in vivo after administration of compound (I) and compound (II) or (III).

[0027] Brief description of the attached figures

[0028] Figure 1 This is a schematic diagram depicting the bioorthogonal retro-Cope elimination reaction between cyclooctyne and N,N-dialkylhydroxylamine.

[0029] Figures 2A-2D The computational study of the retro-Cope elimination reaction between cyclooctyne (COT) and N,N-dimethylhydroxylamine is illustrated. Geometric optimizations were performed at the M06-2X / 6-31G(d,p) theoretical level, and single-point energy calculations were performed at the M06-2X / 6-311G(2d,p) theoretical level. Figure 2A It is a computational reaction model for evaluating the reactivity of cyclooctyne. Figure 2B The calculated transition state structure and activation energy of cyclooctyne hydroamination are shown. Figure 2C The additional ring strain of bicyclic [6.1.0]nonyne was shown to result in a lower activation barrier. Figure 2D It is the calculated activation free energy. and distortion free energy and interaction energy The table highlights the rapidity of the retro-Cope elimination reaction and the central role of hydroxylamine and alkyne distortion in lowering the activation barrier. R = p-NO2Ph.

[0030] Figure 3 The second-order rate constants for the hydroamination of cyclooctyne 2-10 by N,N-diethylhydroxylamine (1) are shown. Second-order kinetics were performed at room temperature in CD3CN using equimolar concentrations of cyclooctyne and hydroxylamine. The rate constant for difluorocyclooctyne 10 was derived from a competition experiment with carbamate 9.

[0031] Figures 4A-4E The retro-Cope elimination reaction was demonstrated for protein labeling. Figure 4A This is the synthetic route for fluorophore-hydroxylamine conjugate 13. Figure 4B The modification of lysozyme with N-hydroxysuccinimide (NHS)-ester 14 to provide cyclooctyne-containing lysozyme 15 is shown. The modified protein lysozyme-COT 15 is labeled with fluorescent hydroxylamine 13. Figure 4CThe fluorescence analysis of lysozyme-COT 15 (0.14 mg / mL) and different concentrations of hydroxylamine 13 (10 μM-200 μM) in phosphate-buffered saline (PBS) at room temperature for 2 hours was performed. Figure 4D This is an in-gel fluorescence analysis of lysozyme-COT 15 (0.14 mg / mL) and hydroxylamine 13 (200 μM) in PBS incubated at room temperature for 1 min–120 min. Figure 4E The results showed that complete coupling was observed by full mass spectrometry of lysozyme-fluorophore conjugate 16, which was obtained by incubating lysozyme-COT 15 (0.58 mg / mL) with hydroxylamine 13 (200 μM) in PBS at room temperature for 6 hours.

[0032] Figures 5A-5D The diagram illustrates biological orthogonality. Figure 5A The synthesis of enamine N-oxide 17 is shown. Figure 5B The bar graph shows the stability of hydroxylamine 13 and enamine N-oxide 17. This study was conducted in PBS at pH 7.4 with glutathione (5 mM), cell lysis buffer (1 mg / mL), and microsomes (0.2 mg / mL), or in PBS at pH 7.4 without additives. The protective effect of sodium ascorbate (5 mM) on hydroxylamine 13 was also evaluated. Figure 5C The in-gel fluorescence analysis of hydroxylamine 13 (200 μM) and lysozyme-COT 15 after reacting for 2 hours in the presence of cell lysate (2.5 mg / mL) showed the proprietary labeling of lysozyme. Figure 5D Showing the cross-reactivity between different groups of bioorthogonal components evaluated in CD3CN at room temperature. R1=CH2NHBoc, R2=C(O)NH(CH2)3NH2, Ar=p-methylphenyl, R3=C(O)NHCH(CH3)2, R4=(CH2)2COOH.

[0033] Figures 6A-6H It is used to calculate N,N-diethylhydroxylamine and cyclooctyne 2 ( Figure 6A ), cyclooctylene 3 ( Figure 6B ), cyclooctylene 4 ( Figure 6C ), cyclooctylene 5 ( Figure 6D ), cyclooctylene 6 ( Figure 6E ), Cyclooctyne 7 ( Figure 6F ), cyclooctylene 8 ( Figure 6G ) and cyclooctylene 9 ( Figure 6H The reaction plots show the second-order rate constants between n=3 independent experiments.

[0034] Figure 7A competition experiment was conducted between cyclooctyne carbamate 9 and difluorocyclooctyne 10 in a 1:4 ratio to determine the second-order rate constant of the latter relative to N,N-diethylhydroxylamine.

[0035] Figure 8 These are images of full Coomassie brilliant blue staining (left) and intragel fluorescence (right) from a concentration-dependent protein labeling experiment. Both images are from the same gel.

[0036] Figure 9 These are images of full Coomassie brilliant blue staining (left) and intragel fluorescence (right) from a concentration-dependent protein labeling experiment. Both images are from the same gel.

[0037] Figures 10A-10C The results showed that mass spectrometry confirmed the bioorthogonal reaction between hydroxylamine 13 and lysozyme-COT 15. Unmodified lysozyme ( Figure 10A ), lysozyme-cyclooctyne conjugate ( Figure 10B The reaction mixture for the hydroamination reaction between hydroxylamine 13 and lysozyme-cyclooctylene conjugate 15 ( Figure 10C ESI mass spectra of (monadduct: expected value 15073.3 Da, observed value 15073.3 Da; diadduct: expected value 15840.6 Da, observed value 15841.7 Da).

[0038] Figures 11A-11C This demonstrates alkyne activation. Figure 11A This demonstrates a metal-catalyzed azide-alkyne cycloaddition reaction. Figure 11B The strain-promoted hydroamylation of alkynes was demonstrated. Figure 11C The hydroamination reaction of push-pull activated linear alkynes is shown.

[0039] Figures 12A-12B The effects of terminal and propargyl modifications are shown. Figure 12A Reactivity screening using alkynes 8′–15′ was shown. NMR conversion was performed with trifluorotoluene as an internal standard. Figure 12B The synthesis of alkynes 9′–15′ is shown. R = OPMB. PMB = p-methoxybenzyl.

[0040] Figures 13A-13B The reaction kinetics and stability of selected alkynes and enamine N-oxides are shown. Figure 13A This is a table of second-order rate constants for alkynes 11′-15′ in CD3CN at room temperature. Figure 13B The graph shows the stability of alkynes 13′, 14′, 15′ and enamine N-oxide 20′ in 50% CD3CN / PBS with or without glutathione (GSH) or HEK293T cell lysate.

[0041] Figures 14A-14E In vitro and live-cell labeling via bioorthogonal hydroammoniation reaction is shown. Figure 14A The HaloTag protein was shown to be coupled with chloroacetylene 21′ and modified with TAMRA-hydroxylamine 22′, and then visualized by in-gel fluorescence or fluorescence microscopy. Figure 14B The structures of chloroacetylenes 21′ and TAMRA-hydroxylamine 22′ are described. Figure 14C This is a time-dependent in-gel fluorescence analysis of the hydroamination reaction between alkyne 21′ and hydroxylamine 22′ (200 μM) at room temperature, from 1 min to 60 min. Figure 14D It is a concentration-dependent in-gel fluorescence analysis of the hydroamination reaction between alkyne 21′ (30 μM) and hydroxylamine 22′ (25 μM-200 μM) after incubation at room temperature for 2 hours. Figure 14E This is a series of images showing HaloTag-GFP expressed on the cell surface of HEK293T cells labeled with TAMRA via a bioorthogonal hydroamination reaction between alkyne 21′ and hydroxylamine 22′. The merged image is a mixture of Hoechst33342, GFP, and TAMRA channels. Scale bar = 50 μm. TAMRA = tetramethylrhodamine.

[0042] Figures 15A-15B A computational study describing the effects of alkyne halogenation was conducted. Figure 15A This is an s-char table of the sp carbons of the analyzed alkynes, and the activation free energy of the reaction between alkynes and hydroxylamine 24′ was calculated. Figure 15B The graph shows the correlation between the sigmoid curve and the activation free energy.

[0043] Figures 16A-16E It is a series of reaction diagrams used to calculate the second-order rate constant between alkynes (11′-15′) and N,N-diethylhydroxylamine. Figure 16A This is a chart of alkynes 11′ (2mM) and hydroxylamines 2′ (20mM-40mM). Figure 16B This is a chart of alkynes 12′ (2mM) and hydroxylamines 2′ (18mM-37mM). Figure 16C This is a graph of alkyne 13′ (10 mM) and hydroxylamine 2′ (10 mM). Figure 16D This is a graph of alkyne 15′ (10 mM) and hydroxylamine 2′ (10 mM). Figure 16E These are graphs of alkynes 14′ (10 mM) and hydroxylamine 2′ (10 mM). Each graph shows the results of three replicate experiments.

[0044] Figure 17 It is a series 19 F NMR spectrum, showing that compound 14′ (2 mM) is stable for 2 weeks in 50% CD3CN / PBS (pH 7.0).

[0045] Figure 18 It is a series 19 F NMR spectra showed that compound 14′ (500 μM) had a half-life of 14 hours in 50% CD3CN / PBS (pH 7.0) in the presence of glutathione (2 mM) and was sufficiently stable for 8 hours of bioorthogonal transformation.

[0046] Figure 19 It is a series 19 F NMR spectrum, showing that compound 15′ (2 mM) is stable for 1 week in 50% CD3CN / PBS (pH 7.0).

[0047] Figure 20 It is a series 19 F NMR spectra showed that compound 15′ (500 μM) had a half-life of 43 hours in 50% CD3CN / PBS (pH 7.0) in the presence of glutathione (2 mM) and was sufficiently stable for 8 hours of bioorthogonal transformation.

[0048] Figures 21A-21B These are whole-gel fluorescence images used in time-dependent protein labeling experiments. Figure 21A ) and Coomassie brilliant blue staining images ( Figure 21B Both images are from the same gel. Images with increased contrast settings were used to identify and label the molecular weight gradients in the intragel fluorescence images.

[0049] Figures 22A-22B These are whole-gel fluorescence images used in time-dependent protein labeling experiments. Figure 22A ) and Coomassie brilliant blue staining images ( Figure 22B Both images are from the same gel. Images with increased contrast settings were used to identify and label the molecular weight gradients in the intragel fluorescence images.

[0050] Figures 23A-23C The results showed that mass spectrometry confirmed the bioorthogonal reaction between hydroxylamine 22′ and alkyne S15′. Unmodified HaloTag protein ( Figure 23A ), HaloTag-alkyne coupling (expected value 34981 Da, observed value 34981 Da) Figure 23B The reaction mixture of hydroxylamine 22′ and the HaloTag-alkyne coupling for hydroamination (expected value 35527 Da, observed value 35529 Da) Figure 23C ESI mass spectrum of ).

[0051] Figure 24 Showing enamine N-oxide sp 2 - The s-type of carbon (C2).

[0052] Figures 25A-25D It describes biological orthogonal transformations. Figure 25A This demonstrates the associative biological orthogonal transformation. Figure 25B This demonstrates the biological orthogonal transformation of dissociation. Figure 25C This demonstrates chemically reversible bioconjugation. Figure 25D It demonstrates a rapid and complete continuous biorthogonal hydroamination reaction and the traceless release of biomolecules through enamine N-oxides.

[0053] Figures 26A-26B This section evaluates the effect of hydroxylamine substituents on the biorthogonal retro-Cope elimination reaction. Figure 26A This demonstrates the synthetic route for obtaining TAMRA-hydroxylamine conjugate 6”-9”. Figure 26B Showing the effect of TAMRA-hydroxylamine conjugate 6” conj -10” conj A series of in-gel fluorescence images and Coomassie brilliant blue staining images of lysozyme-cyclooctyne conjugate 11 (10 μM) incubated in PBS at room temperature for 1 h–72 h.

[0054] Figures 27A-27D The illustration shows a computational study investigating the formation and degradation of enamine N-oxide structures. Figure 27A A computational reaction model is shown to explore the effect of steric hindrance on hydroamination and Cope elimination reactions. Figure 27B The calculated Gibbs free energy and activation free energy are displayed. The reaction coordinates for paths A and B are blue and red, respectively. Figure 27C The 3D structures of 17” and 18” are shown, as well as the transitional structures 17”-TSa and 18”-TSb for paths A and B. Figure 27D The reaction between cyclooctyne 22” (2 mM) and hydroxylamine 4” (2 mM) was monitored by A220 absorption on LCMS.

[0055] Figures 28A-28E The diagram illustrates the diboron-mediated reduction of enamine N-oxides and load release. Figure 28A It is a lysozyme-TAMRA conjugate containing enamine N-oxide 6” conj 9” conj and 10” conj A reaction protocol for inducing fluorophore release by treatment with diboron reagent in PBS at room temperature. Figure 28B It is a lysozyme TAMRA conjugate 6” conj 9” conj and 10” conjA series of in-gel fluorescence and silver staining images of concentration-dependent lysis of (480 nM) B2pin2 co-occurred at room temperature for 1 h, as well as time-dependent lysis of 5 μM B2pin2 co-occurred for 5 min–60 min, analyzed by in-gel fluorescence. Silver staining was provided as a loading control. Figure 28C It is a series of graphs that show the quantification of fluorescence in bands from time-dependent diboron-induced lysis experiments. Figure 28D The complete binding and removal of TAMRA from lysozyme by mass spectrometry was demonstrated. Lys-COT11 (10 μM) with 0–3 modifications was bound to hydroxylamine 6 (200 μM) in PBS at room temperature for 6 h. Figure 28E Describes structurally diverse diboron reagents 27”-31” (5 μM or 50 μM) coupled with N-methyllysozyme-TAMRA conjugates 6” conj Fluorescence and silver staining images of the gel after incubation at room temperature for 60 min at (240 nM).

[0056] Figures 29A-29B Characterization of the diboron-mediated reductive cleavage of enamine N-oxides is shown. Figure 29A The reaction process of 4 mM p-nitrophenol-derived enamine N-oxide 32” with 10 mM B2(OH)4 is shown under the conditions of 10% DMSO-d6 / 23% CD3OD / 67% d-PBS, pH 7.4. 1 It was monitored by H NMR spectroscopy for 24 hours. Figure 29B The reaction process between p-nitrophenyl sulfide 38” and p-nitrophenyl carbamate 39” is shown.

[0057] Figures 30A-30E The study demonstrates the reaction range and load release kinetics of diboron-mediated enamine N-oxide cleavage. Figure 30A The reaction scheme for synthesizing lysozyme-luciferin conjugate 41” by hydroamylation of Lys-COT 11” with luciferin hydroxylamine 40” in PBS at room temperature is shown. Figure 30B The kinetics of diboron-mediated cleavage of enamine N-oxides under pseudo-first-order conditions, as determined by fluorescence polarization, were shown when lysozyme-luciferin conjugate 41” (500 nM) was treated with B2pin2 (25 μM–200 μM) in PBS at room temperature. Figure 30C The graph illustrates the effect of buffer pH on lysis rate. Lysozyme-luciferin conjugate 41” (500 nM) was reduced in PBS at pH 4–10 with B2pin2 (100 μM), and the conversion rate was measured by fluorescence polarization. Figure 30DThe graph depicts the effect of buffer composition on lysis rate. Lysozyme-luciferin conjugate 41” (500 nM) was reduced with B2pin2 (50 μM) in several buffers, and the conversion rate was measured by fluorescence polarization. Figure 30E It is a series of charts showing the effect of leaving group composition on cleavage rate.

[0058] Figures 31A-31D The synthesis and cellular evaluation of chemically cleavable enamine N-oxide-linked antibody-drug conjugates are shown. Figure 31A The synthesis of ADC 61” and 62” is shown. Figure 31B Trastuzumab-derived ADC 61” acts on SK-BR-3HER2 in the presence or absence of 50 μM B2pin2. + A graph showing the cell viability of breast cancer cells. Figure 31C This is a cell viability assay of MDA-MB-231HER2 breast cancer cells with or without 50 μM B2pin2, showing the effect of trastuzumab-derived ADC 61. Figure 31D With or without 50 μM B2pin2, the IgG isotype-derived ADC 62” acts on SK-BR-3HER2. + Graph showing cell viability of breast cancer cells. ND = Not measured. Error bars represent standard deviation (n=3).

[0059] Figures 32A-32B The results show that protein modification using enamine N-oxide chemistry is traceless and reversible. Figure 32A This is a schematic diagram illustrating the sequential coupling of small molecules on lysozyme. Figure 32B The clean and complete click and release observed by whole mass spectrometry are depicted.

[0060] Figure 33 The reaction was shown to reduce enamine N-oxides with B₂(OH)₄ at room temperature, releasing p-nitroaniline (S₃”). The reaction was carried out in the presence of caffeine as an internal standard. 1 1H NMR spectra, (A) before diboron addition, (B) 4 min after addition, and (C) 30 min after addition.

[0061] Figure 34 The reaction was shown to reduce p-nitroaniline from enamine N-oxides with B2(OH)4 at room temperature (24”). The reaction was carried out in the presence of caffeine as an internal standard. 1 1H NMR spectra (A) before diboron addition, (B) 5 min after addition, and (C) 30 min after addition.

[0062] Figure 35These are images of full Coomassie Brilliant Blue staining (top) and intragel fluorescence (bottom) in a time-dependent protein labeling experiment of compounds 6” and 10”. Both images are from the same gel.

[0063] Figure 36 These are images of full Coomassie Brilliant Blue staining (top) and intragel fluorescence (bottom) in a time-dependent protein labeling experiment of compounds 7” and 8”. Both images are from the same gel.

[0064] Figure 37 These are images of full Coomassie Brilliant Blue staining (left) and intragel fluorescence (right) in a time-dependent protein labeling experiment of compound 9. Both images are from the same gel.

[0065] Figure 38 It is an enamine N-oxide protein conjugate 6” conj 9” conj and 10” conj Images of fluorescence (left) and Oriole staining (right) within the whole gel for stability assay in PBS (pH 7.4). Both images are from the same gel.

[0066] Figure 39 It is an enamine N-oxide protein conjugate 6” conj 9” conj and 10” conj Images of intragel fluorescence (left) and Oriole staining (right) for stability assay in RPMI. Both images are from the same gel.

[0067] Figure 40 It is an enamine N-oxide protein conjugate 6” conj 9” conj and 10” conj Images of intragel fluorescence (left) and Oriole staining (right) for stability assay in RPMI supplemented with 10% fetal bovine serum. Both images are from the same gel.

[0068] Figures 41A-41B The 6” lysozyme-fluorophore conjugate for cleaving enamine N-oxide linkages is shown. conj Evaluation of diboron reagents. Figure 41A The structure of diboron substrates 27”–31” was depicted. Figure 41B yes Figure 41A The images shown are of intragel fluorescence and silver staining with diboron reagent. Both images are from the same gel.

[0069] Figures 42A-42C It is an enamine N-oxide-linked lysozyme i fluorescent conjugate 6” conj 9” conj and 10” conjSerial whole-gel fluorescence and quantification for each band. Figure 42A It is 6” conj Intragel fluorescence and quantification. Figure 42B It is 10” conj Intragel fluorescence and quantification. Figure 42C It is 9” conj Intragel fluorescence and quantification.

[0070] Figure 43 The reaction between cyclooctyne 22” (2 mM) and hydroxylamine 3” (2 mM) was monitored by A220 absorption on LCMS.

[0071] Figures 44A-44B This demonstrates the confirmation of bioorthogonal click and release reactions of structurally diverse enamine N-oxides by mass spectrometry. Figure 44A It is the hydroamination linkage reaction between unmodified lysozyme, lysozyme-cyclooctyne coupling 11”, hydroxylamine 6” and lysozyme-COT11” (monadduct: expected value 15073.1 Da, observed value 15074.0 Da; diadduct: expected value 15840.5 Da, observed value 15842.7 Da) and diboron-induced enamine N-oxide-linked coupling 6”. conj A series of ESI mass spectra of the cleavage reaction (monadduct: expected value 14376.8 Da, observed value 14376.9 Da; diadduct: expected value 14447.8 Da, observed value 14447.3 Da). Figure 44B It is the hydroamination linkage reaction between hydroxylamine 9” and lysozyme-COT 11” (monadduct: expected value 15041.1 Da, observed value 15042.1 Da; diadduct: expected value 15776.4 Da, observed value 15778.5 Da), and the diboron-induced enamine N-oxide-linked conjugate 9”. conj The cleavage reaction of hydroxylamine 10” and lysozyme-COT 11” (monadduct: expected value 14376.8 Da, observed value 14377.7 Da; diadduct: expected value 14447.8 Da, observed value 14447.3 Da), the hydroamylation linkage between hydroxylamine 10” and lysozyme-COT 11” (monadduct: expected value 15149.1 Da, observed value 15149.7 Da; diadduct: expected value 15992.5 Da, observed value 15994.0 Da), and the diboron-induced enamine N-oxide-linked coupling compound 10” conj A series of ESI mass spectra of the cleavage reaction (monadduct: expected value 14376.8 Da, observed value 14376.1 Da; diadduct: expected value 14447.8 Da, observed value 14447.3 Da).

[0072] Figure 45It is a series of gels that show that when enamine N-oxides carrying lysozyme-luciferin conjugates 41” and 48”-52” are treated with B2pin2 in PBS at room temperature, it induces the release of fluorophores.

[0073] Figure 46 The chart shows the effect of the structure of the diboron reagent on the pyrolysis rate.

[0074] Figure 47 The structure of the antibody-nitroaniline conjugate S22”-S24” is shown.

[0075] Figures 48A-48B These are a series of diboron reagent dose-response curves for SK-BR-3 cell viability assays. Using B2pin2 ( Figure 48A ) or B2(OH)4( Figure 48B Cells were treated for 72 hours. The error bar represents the mean ± SEM of data from biological replicates (n=3).

[0076] Figures 49A-49B These are a series of diboron reagent dose-response curves for the cell viability assay of MDA-MB-231 cells. Using B2pin2 ( Figure 49A ) or B2(OH)4( Figure 49B Cells were treated for 96 hours. The error bar represents the mean ± SEM of data from biological replicates (n=3).

[0077] Figure 50 It is the intragel fluorescence of enamine N-oxide carrying lysozyme-luciferin conjugate 65”, which was induced to release the fluorophore by treatment with B2pin2 in PBS at room temperature.

[0078] Figure 51 It is a series of reaction coordinates for the bioorthogonal hydroamination reaction between cyclooctyne 12” and hydroxylamine 14” and 15”.

[0079] Figure 52 The graph shows the kinetics of enamine N-oxide-linked lysozyme-luciferin conjugate 41”. Invention Details

[0081] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this subject matter pertains. As used in the specification and appended claims, unless otherwise stated, the following terms have the indicated meanings for the purpose of understanding the invention.

[0082] As used in this description and the appended claims, the singular forms “a” and “the” include plural references unless the context clearly specifies otherwise. Thus, for example, reference to “a composition” includes a mixture of two or more such compositions, reference to “an inhibitor” includes a mixture of two or more such inhibitors, and so on.

[0083] Unless otherwise specified or obvious from the context, as used herein, the term “about” shall be understood to mean within the normal tolerance range in the field, such as within 2 standard deviations of the mean. “About” may be understood to mean within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the value indicated. Unless the context clearly specifies otherwise, all numerical values ​​provided herein are modified by the term “about”.

[0084] The transitional term "comprising," synonymous with "including," "containing," or "characterized by," is inclusive or open-ended and does not exclude other unreferenced elements or method steps. When used in the context of the number of heteroatoms in a heterocyclic structure, it signifies the minimum number of heteroatoms in the heterocyclic group. In contrast, the transitional phrase "composed of" excludes any element, step, or component not specified in the claims. The transitional phrase "substantially composed of" limits the scope of the claims to the specified materials or steps of the invention "and materials or steps that do not substantially affect the essential and novel features of the invention."

[0085] The term "biorthogonal reaction" refers to any chemical reaction that can occur within a living system without interfering with natural biochemical processes.

[0086] Regarding the compounds of the present invention, and to the extent that they are further described herein using the following terms, the following definitions apply.

[0087] As used herein, the term "alkyl" refers to a saturated straight-chain or branched monovalent hydrocarbon group. In one embodiment, the alkyl group is C1-C2. 18 Alkyl groups. In other embodiments, the alkyl group is C0-C6, C0-C5, C0-C3, C1-C6. 12C1-C8, C1-C6, C1-C5, C1-C4 or C1-C3 groups (where C0 alkyl refers to a bond). Examples of alkyl groups include methyl, ethyl, 1-propyl, 2-propyl, isopropyl, 1-butyl, 2-methyl-1-propyl, 2-butyl, 2-methyl-2-propyl, 1-pentyl, n-pentyl, 2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl, 1-hexyl, 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl, 2,3-dimethyl-2-butyl, 3,3-dimethyl-2-butyl, heptyl, octyl, nonyl, decyl, undecyl, and dodecyl. In some embodiments, the alkyl group is a C1-C3 alkyl group. In some embodiments, the alkyl group is a C1-C2 alkyl group or methyl.

[0088] As used herein, the term "alkylene" refers to a straight-chain or branched divalent hydrocarbon chain that links the remainder of a molecule to a free radical, consisting only of carbon and hydrogen, containing no unsaturated bonds, and having 1 to 12 carbon atoms, such as methylene, ethylene, propylene, n-butylene, etc. The alkylene chain can be linked to the remainder of the molecule by a single bond and to a free radical by a single bond. In some embodiments, the alkylene contains 1 to 8 carbon atoms (C1-C8 alkylene). In other embodiments, the alkylene contains 1 to 5 carbon atoms (C1-C5 alkylene). In other embodiments, the alkylene contains 1 to 4 carbon atoms (C1-C4 alkylene). In other embodiments, the alkylene contains 1 to 3 carbon atoms (C1-C3 alkylene). In other embodiments, the alkylene contains 1 to 2 carbon atoms (C1-C2 alkylene). In other embodiments, the alkylene contains 1 carbon atom (C1 alkylene).

[0089] As used herein, the term "alkenyl" refers to a straight-chain or branched monovalent hydrocarbon group having at least one carbon-carbon double bond. Alkenyl groups include radicals having "cis" and "trans" orientations, or "E" and "Z" orientations. In one instance, the alkenyl group is C2-C. 18 Alkenyl groups. In other embodiments, the alkenyl group is C2-C. 12 C2-C 10 C2-C8, C2-C6, or C2-C3 groups. Examples include ethenyl (or vinyl), propenyl, propenyl, 2-methylpropenyl, butenyl, butenyl, butenyl, 3-butenyl, butenyl, 2-methylbutenyl, hexenyl, hexenyl, 3-butenyl, hexenyl, and 1,3-dienyl.

[0090] As used herein, the term "alkynyl" refers to a straight-chain or branched monovalent hydrocarbon group having at least one carbon-carbon triple bond. In one example, the alkynyl group is C2-C. 18 Group. In other examples, the alkynyl group is C2-C. 12 C2-C 10 C2-C8, C2-C6, or C2-C3 groups. Examples include ethynylprop-1-ynyl, prop-2-ynyl, but-1-ynyl, but-2-ynyl, and but-3-ynyl.

[0091] As used herein, the term "alkoxyl" or "alkoxy" refers to an alkyl group to which an oxygen radical is attached, as described above, and the oxygen radical is the linker. Representative alkoxy groups include methoxy, ethoxy, propoxy, tert-butoxy, etc. An "ether" is two hydrocarbon groups covalently linked by oxygen. Therefore, the substituents of the alkyl group that make it an ether are or similar to alkoxy groups, and can be represented, for example, by one of -O-alkyl, -O-alkenyl, and -O-ynyl.

[0092] As used in this article, the term "halogen" (or "halogenated" or "halogenated") refers to fluorine, chlorine, bromine, or iodine.

[0093] As used herein, the term "cyclogroup" broadly refers to any group containing a saturated, partially saturated, or aromatic ring system, whether used alone or as part of a larger part, such as carbocyclic (cycloalkyl, cycloalkenyl), heterocyclic (heterocyclic alkyl, heterocyclic alkenyl), aryl, and heteroaryl. A cyclogroup may have one or more (e.g., fused) ring systems. Thus, for example, a cyclogroup may contain one or more carbocyclic, heterocyclic, aryl, or heteroaryl groups.

[0094] As used herein, the term "carbocyclic" (also referred to as "carbocyclic group") refers to a group having 3 to 20 carbon atoms (e.g., alkcarbocyclic) that, alone or as a significant part of a saturated, partially unsaturated, or aromatic system, either alone or as a significant part of it. The term carbocyclic group includes monocyclic, bicyclic, tricyclic, fused, bridging, and spirocyclic systems and combinations thereof. In one embodiment, the carbocyclic group comprises 3 to 15 carbon atoms (C3-C4). 15 In one embodiment, the carbocyclic group comprises 3 to 12 carbon atoms (C3-C4). 12 In another embodiment, the carbocyclic group includes C3-C8, C3-C... 10 Or C5-C 10 In another embodiment, the carbocyclic group as a monocyclic ring includes C3-C8, C3-C6, or C5-C6. In some embodiments, the carbocyclic group as a bicyclic ring includes C7-C8. 12In another embodiment, the carbocyclic group as the spirocyclic system includes C5-C... 12 Representative examples of monocyclic carbocyclic groups include cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl, per-deuteriumcyclohexyl, 1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl, cyclohexadienyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl, phenyl, and cyclododecyl; bicyclic carbocyclic groups having 7 to 12 ring atoms include [4,3], [4,4], [4,5], [5,5], [5,6], or [6,6] ring systems, such as bicyclic [2.2.1]heptane, bicyclic [2.2.2]octane, naphthalene, and bicyclic [3.2.2]nonane. Representative examples of spirocyclocyclic groups include spiro[2.2]pentane, spiro[2.3]hexane, spiro[2.4]heptane, spiro[2.5]octane, and spiro[4.5]decane. The term carbocyclic includes aryl ring systems as defined herein. The term carbocyclic also includes cycloalkyl rings (e.g., saturated or partially unsaturated single-carbon, double-carbon, or spirocyclocyclic rings). The term carbocyclic group also includes a carbocyclic ring fused with one or more (e.g., 1, 2, or 3) different cyclic groups (e.g., aromatic or heterocyclic rings), wherein a radical or linker is located on the carbocyclic ring.

[0095] Therefore, the term carbocyclic also includes carbocyclic alkyl groups, as used herein, which refer to the formula -R c - A carbocyclic group, wherein R c It is an alkylene chain. The term carbocyclic also includes carbocyclic alkoxy groups, as used herein, which refer to those derived through the formula --O--R c - A group bonded to the oxygen atom of a carbocyclic group, wherein R c It is an alkylene chain.

[0096] The term "carbocyclic" also includes "aryl" groups. As used herein, the term "aryl" (e.g., "aranediol," where the terminal carbon atom on the alkyl group is the linking point, such as benzyl, "araneoxy," where the oxygen atom is the linking point, or "araneoxyalkyl," where the linking point is on the aryl group), used alone or as part of a larger part, refers to a group comprising a monocyclic, bicyclic, or tricyclic carbocyclic system, including fused rings, wherein at least one ring in the system is aromatic. In some embodiments, the araneoxy group is a benzoyloxy group. The term "aryl" may be used interchangeably with the term "aromatic ring." In one embodiment, an aryl group comprises a group having 6 to 18 carbon atoms. In another embodiment, an aryl group comprises a group having 6 to 10 carbon atoms. Examples of aryl groups include phenyl, naphthyl, anthracene, biphenyl, phenanthrene, naphthacenyl, 1,2,3,4-tetrahydronaphthyl, 1H-indenyl, 2,3-dihydro-1H-indenyl, and naphthyridinyl, which may be substituted by one or more substituents described herein or substituted independently. A particular aryl group is phenyl. In some embodiments, the aryl group comprises an aromatic ring fused to one or more (e.g., 1, 2, or 3) different cyclic groups (e.g., carbocyclic or heterocyclic), wherein a free radical or linker is located on the aromatic ring.

[0097] Therefore, the term aryl includes aralkyl (e.g., benzyl), as mentioned above, aralkyl refers to formula -R c -aryl groups, where R c It is an alkylene chain, such as methylene or ethylene. In some embodiments, the aryl group is optionally substituted with a benzyl group. The term aryl also includes arylalkoxy, as used herein, which refers to a chain of the formula -OR c -A group bonded to the oxygen atom of an aryl group, wherein Rc is an alkylene chain, such as methylene or ethylene.

[0098] As used herein, the term "heterocyclic group" refers to a "carbocyclic group" used alone or as part of a larger portion, containing a saturated, partially unsaturated, or aromatic ring system, wherein one or more (e.g., 1, 2, 3, or 4) carbon atoms are substituted with heteroatoms (e.g., O, N, N(O), S, S(O) or S(O)₂). The term heterocyclic group includes monocyclic, bicyclic, tricyclic, fused, bridging, and spirocyclic systems and combinations thereof. In some embodiments, a heterocyclic group refers to a 3- to 15-membered heterocyclic system. In some embodiments, a heterocyclic group refers to a 3- to 12-membered heterocyclic system. In some embodiments, a heterocyclic group refers to a saturated ring system, such as a 3- to 12-membered saturated heterocyclic system. The term heterocyclic group also includes C3-C8 heterocyclic alkyl groups, which are saturated or partially unsaturated monocyclic, bicyclic, or spirocyclic systems containing 3 to 8 carbons and one or more (1, 2, 3, or 4) heteroatoms.

[0099] In some embodiments, the heterocyclic group comprises 3 to 12 ring atoms and includes monocyclic, bicyclic, tricyclic, and spirocyclic systems, wherein the ring atoms are carbon and 1 to 5 ring atoms are heteroatoms, such as nitrogen, sulfur, or oxygen. In some embodiments, the heterocyclic group comprises a 3- to 7-membered monocyclic ring having one or more heteroatoms selected from nitrogen, sulfur, or oxygen. In some embodiments, the heterocyclic group comprises a 4- to 6-membered monocyclic ring having one or more heteroatoms selected from nitrogen, sulfur, or oxygen. In some embodiments, the heterocyclic group comprises a 3-membered monocyclic ring. In some embodiments, the heterocyclic group comprises a 4-membered monocyclic ring. In some embodiments, the heterocyclic group comprises a 5- to 6-membered monocyclic ring. In some embodiments, the heterocyclic group comprises 0 to 3 double bonds. In any of the foregoing embodiments, the heterocyclic group comprises 1, 2, 3, or 4 heteroatoms. Any nitrogen or sulfur heteroatom may optionally be oxidized (e.g., NO, SO, SO2), and any nitrogen heteroatom may optionally be quaternized (e.g., [NR4]). + Cl - [NR4] + OH -Representative examples of heterocyclic groups include ethylene oxide, acridine, thiiranyl, aziridine, oxadiazine, thioheterobutyl, 1,2-dithioheterobutyl, 1,3-dithioheterobutyl, pyrrolidine, dihydro-1H-pyrrolyl, dihydrofuranyl, tetrahydropyranyl, dihydrothiophene, tetrahydrothiophene, imidazoalkyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, 1,1-dioxothiomorpholinyl, dihydropyranyl, tetrahydropyranyl, hexahydrothiopyranyl, hexahydropyrimidinyl, oxazinyl, thiazinyl, thiaoxazinyl, homopiperazinyl, homopiperidine, aziridine-heptyl, oxadiazine, thioheptanyl, oxazepinyl yl), oxazepanyl, diazacycloheptanyl, 1,4-di-azacycloheptanyl, diazacycloheptanyl, thiazozacycloheptanyl, thiazozacycloheptanyl, tetrahydrothiopyranyl, oxazolyl, thiazoyl, isothiazolyl, 1,1-dioxoisothiazoline dinonyl, oxazolyl dinonyl, imidazoline dinonyl, 4,5,6,7-tetrahydro[2H]inzolyl, tetrahydrobenzimidazolyl, 4,5,6,7-tetrahydrobenzimidazolyl, 1,6-dihydroimidazo[4,5-D]pyrrolo[2,3-b]pyridyl, thiazinyl, phenylthio, oxazinyl, thiadiazinyl, oxadiazinyl, dithiazinyl, dioxazinyl, oxazinyl, oxazinyl, thiazinyl, thiazolyl, thiazolyl thiatriazinyl, oxatriazinyl, dithiadiazinyl, imidazolinyl, dihydropyrimidinyl, tetrahydropyrimidinyl, 1-pyrrolinyl, 2-pyrrolinyl, 3-pyrrolinyl, dihydroindolyl, thiapyranyl, 2H-pyranyl, 4H-pyranyl, dioxane, 1,3-dioxolane, pyrazolinyl, pyrazolylalkyl, dithiazopentyl, pyrimidinyl, pyrimidinyl-2,4-diketone, piperazine-nonyl, piperazine-dinonyl, pyrazolylalkylimidazolinyl, 3-azabicyclo[3.1.0]hexane, 3,6-diazabicyclo[3.1.1]heptane, 6-azabicyclo[3.1.1]heptane, 3-azabicyclo[3.1.1]heptane Cyclo[3.1.1]heptyl, 3-azabicyclo[4.1.0]heptyl, azabicyclo[2.2.2]hexyl, 2-azabicyclo[3.2.1]octyl, 8-azabicyclo[3.2.1]octyl, 2-azabicyclo[2.2.2]octyl, 8-azabicyclo[2.2.2]octyl, 7-oxabicyclo[2.2.1]heptane, azaspiro[3.5]nonyl, azaspiro[2.5]octyl, azaspiro[4.5]decyl, 1-azaspiro[4.5]dec-2-one, azaspiro[5.5]undecyl, tetrahydroindolyl, octahydroindolyl, tetrahydroisoindolyl, tetrahydroindazoleyl, 1,1-dioxohexahydrothiopyranyl.Examples of five-membered heterocyclic groups containing sulfur or oxygen atoms and one to three nitrogen atoms include thiazolyl (including thiazolyl-2-yl and thiazolyl-2-yl N-oxide), thiadiazolyl (including 1,3,4-thiadiazol-5-yl and 1,2,4-thiadiazol-5-yl), oxazolyl (e.g., oxazol-2-yl), and oxadiazolyl (e.g., 1,3,4-oxadiazol-5-yl and 1,2,4-oxadiazol-5-yl). Examples of five-membered heterocyclic groups containing two to four nitrogen atoms include imidazolel, such as imidazole-2-yl; triazolyl, such as 1,3,4-triazol-5-yl; 1,2,3-triazol-5-yl, 1,2,4-triazol-5-yl, and tetrazolyl, such as 1H-tetrazole-5-yl. Representative examples of benzo-fused 5-membered heterocyclic groups are benzoxazol-2-yl, benzothiazol-2-yl, and benzimidazol-2-yl. Example 6-membered heterocyclic groups contain one to three nitrogen atoms and optionally sulfur or oxygen atoms, such as pyridyl groups (e.g., pyridin-2-yl, pyridin-3-yl, and pyridin-4-yl); pyrimidinyl groups (e.g., pyrimidin-2-yl and pyrimidin-4-yl); triazinyl groups (e.g., 1,3,4-triazin-2-yl and 1,3,5-triazin-4-yl); and pyridazinyl groups, particularly pyridazin-3-yl and pyrazinyl groups. Pyridine N-oxides and pyridazine N-oxides, as well as pyridyl, pyrimidin-2-yl, pyrimidin-4-yl, pyridazinyl, and 1,3,4-triazin-2-yl, are other examples of heterocyclic groups. In some embodiments, the heterocyclic group comprises a heterocycle fused to one or more (e.g., 1, 2 or 3) different cyclic groups (e.g., carbocyclic or heterocyclic), wherein the radical or linker is located on the heterocycle, and in some embodiments, the linker is a heteroatom contained in the heterocycle.

[0100] Therefore, the term heterocyclic includes N-heterocyclic groups, as used herein, which refer to a heterocyclic group containing at least one nitrogen atom, and wherein the connection point between the heterocyclic group and the remainder of the molecule is through a nitrogen atom in the heterocyclic group. Representative examples of N-heterocyclic groups include 1-morpholinyl, 1-piperidinyl, 1-piperazinyl, 1-pyrrolidinyl, pyrazolyl, imidazolinyl, and imidazoalkyl. The term heterocyclic also includes C-heterocyclic groups, as used herein, which refer to a heterocyclic group containing at least one heteroatom, and wherein the connection point between the heterocyclic group and the remainder of the molecule is through a carbon atom in the heterocyclic group. Representative examples of C-heterocyclic groups include 2-morpholinyl, 2-or 3-or 4-piperidinyl, 2-piperazinyl, and 2-or 3-pyrrolidinyl. The term heterocyclic also includes heterocyclic alkyl groups, as disclosed above, where a heterocyclic alkyl group refers to a group of formula -R c - A heterocyclic group, wherein Rc is an alkylene chain. The term heterocyclic also includes heterocyclic alkoxy groups, as used herein, which refer to those derived via the formula -OR c - A free radical bonded to the oxygen atom of a heterocyclic group, wherein R c It is an alkylene chain.

[0101] The term "heterocyclic" also includes "heteroaryl" groups. In some embodiments, a heterocyclic group refers to a heteroaryl ring system, such as a 5- to 14-membered heteroaryl ring system. As used herein, the term "heteroaryl," used alone or as part of a larger part, such as "heteroarylalkyl" (also called "heteroaralkyl") or "heteroarylalkoxy" (also called "heteroaralkoxy"), refers to a monocyclic, bicyclic, or tricyclic ring system having 5 to 14 ring atoms, wherein at least one ring is aromatic and contains at least one heteroatom. In one embodiment, a heteroaryl comprises a 5- to 6-membered monocyclic aromatic group, wherein one or more ring atoms are nitrogen, sulfur, or oxygen. Representative examples of heteroaryl groups include thienyl, furanyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, triazolyl, thiadiazolyl, oxadiazolyl, tetrazolyl, thiatriazolyl, oxtriazolyl, pyridyl, pyrimidinyl, imidazolylpyridyl, pyrazinyl, pyridazinyl, triazinyl, tetraazinyl, tetrazo[1,5-b]pyridazinyl, purinyl, denitropurinyl, benzoxazolyl, and benzyl. The terms "heteroaryl" also include groups in which the heteroaryl group is fused to one or more cyclic (e.g., carbocyclic or heterocyclic) rings, with a radical or linker on the heteroaryl ring. Non-limiting examples include indolyl, indolazinyl, isoindolyl, benzothiophene, benzobenzyl, methylenedioxyphenyl, benzofuranyl, dibenzofuranyl, indazole, benzimidazolyl, benzodioxazolyl, benzothiazolyl, quinolinyl, isoquinolinyl, terazinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolinyl, carbazole, acridineyl, phenazinyl, phenthiazinyl, phenoxazinyl, tetrahydroisoquinolinyl, and pyrido[2,3-b]-1,4-oxazin-3(4H)-one. Heteroaryl groups can be monocyclic, bicyclic, or tricyclic. In some embodiments, the heteroaryl group comprises a heteroaryl ring fused to one or more (e.g., 1, 2 or 3) different cyclic groups (e.g., carbocyclic or heterocyclic), wherein the radical or linker is located on the heteroaryl ring, and in some embodiments, the linker is a heteroatom contained in the heterocyclic ring.

[0102] Therefore, the term heteroaryl includes N-heteroaryl, which, as used herein, refers to a heteroaryl as described above containing at least one nitrogen atom, and wherein the connection point between the heteroaryl and the remainder of the molecule is through a nitrogen atom in the heteroaryl. The term heteroaryl also includes C-heteroaryl, which, as used herein, refers to a heteroaryl as described above, and wherein the connection point between the heteroaryl and the remainder of the molecule is through a carbon atom in the heteroaryl. The term heteroaryl also includes heteroarylalkyl, which, as described above, refers to the formula -R c - A heteroaryl group, wherein R c It is an alkylene chain as described above. The term heteroaryl also includes a heteroaralkoxy group, which, as used herein, refers to a group with the formula --O--R c - A heteroaryl group bonded to an oxygen atom, wherein R c It is an alkylene chain as defined above.

[0103] Unless otherwise stated, and to the extent that no particular group is further defined, any group described herein may be substituted or not substituted. As used herein, the term “substituted” broadly refers to all permissible substituents, implicitly provided that such substituent conforms to the permissible valence of the substituted atom and the substituent, and that such substituent produces a stable compound, i.e., a compound that does not spontaneously undergo rearrangement, cyclization, elimination, or other transformations. Representative substituents include halogens, hydroxyl groups, and any other organic group grouped in linear, branched, or cyclic structures containing any number of carbon atoms (e.g., 1 to 14 carbon atoms), and may include one or more (e.g., 1, 2, 3, or 4) heteroatoms, such as oxygen, sulfur, and nitrogen.

[0104] Representative examples of substituents may include alkyl groups, substituted alkyl groups (e.g., C1-C6, C1-C5, C1-C4, C1-C3, C1-C2, C1), alkoxy groups (e.g., C1-C6, C1-C5, C1-C4, C1-C3, C1-C2, C1), and substituted alkoxy groups (e.g., C1-C6, C1-C5, C1-C4, C1-C3, C1-C2, C1). Haloalkyl (e.g., CF3), alkenyl (e.g., C2-C6, C2-C5, C2-C4, C2-C3, C2), substituted alkenyl (e.g., C2-C6, C2-C5, C2-C4, C2-C3, C2), alkynyl (e.g., C2-C6, C2-C5, C2-C4, C2-C3, C2), substituted alkynyl (e.g., C2-C6, C2-C5, C2-C4, C2-C3, C2), cyclic (e.g., C3-C4). 12 (C5-C6), substituted rings (e.g., C3-C6), and substituted rings (e.g., C3-C6). 12C5-C6), carbon rings (e.g., C3-C6), and carbon rings (e.g., C5-C6). 12 C5-C6), substituted carbon rings (e.g., C3-C6), and substituted carbon rings (e.g., C5-C6). 12 (C5-C6), heterocyclic rings (e.g., C3-C6), and heterocyclic rings. 12 (C5-C6), substituted heterocycles (e.g., C3-C6), and substituted heterocycles. 12 C5-C6), aryl (e.g., benzyl and phenyl), substituted aryl (e.g., substituted benzyl or phenyl), heteroaryl (e.g., pyridyl or pyrimidinyl), substituted heteroaryl (e.g., substituted pyridyl or pyrimidinyl), aralkyl (e.g., benzyl), substituted aralkyl (e.g., substituted benzyl), halogenated, hydroxylated, aryloxy (e.g., C6-C6), arylyl (e.g., C5-C6), aryl (e.g., C5-C6), aryl (e.g., C5-C6), substituted ...substituted aryl (e.g., C5-C6 12 C6), substituted aryl groups (e.g., C6-C4), and substituted aryl groups. 12 C6), thioalkyl (e.g., C1-C6), substituted thioalkyl (e.g., C1-C6), arylthio (e.g., C6-C6), ...substituted thioalkyl (e.g., C1-C6), arylthioalkyl 12 C6), substituted aryl thiols (e.g., C6-C4), and substituted aryl thiols (e.g., C6-C4). 12 (C6), cyano, carbonyl, substituted carbonyl, carboxyl, substituted carboxyl, amino, substituted amino, amide, substituted amide, thio, substituted thio, sulfinyl, substituted sulfinyl, sulfonyl, substituted sulfonyl, sulfinamide, substituted sulfinamide, sulfonamide, substituted sulfonamide, urea, substituted urea, carbamate, substituted carbamate, amino acid and peptide.

[0105] As used herein, the term "π-electron-withdrawing group" refers to a functional group containing π electrons that has an electron-attracting charge form of +ve or δ+ve, such as a carbonyl or nitro group.

[0106] As used herein, the term “induced electron-withdrawing group” refers to an atom or functional group containing an electronegative atom that attracts a greater electron density from the atom it is attached to (e.g., a fluorine group or an alkoxy group).

[0107] As used herein, the term "small molecule" refers to a molecule with a relatively low molecular weight, whether naturally occurring or artificially created (e.g., through chemical synthesis). Typically, a small molecule is an organic compound (i.e., it contains carbon). The small molecule may contain multiple carbon-carbon bonds, stereocenters, and other functional groups (e.g., amines, hydroxyl groups, carbonyl groups, and heterocycles).

[0108] As used herein, the term active moiety refers to a unique, definable portion or unit of a compound of the present invention that performs certain functions or activities or reacts with other molecules. Representative types of active moieties include binding moieties, therapeutic moieties, diagnostic moieties, and fixation moieties.

[0109] As used herein, the term "fixed portion" refers to a portion of the compound of the present invention that is insoluble and to which the remainder of the compound of the present invention is bound (e.g., by covalent bonding or encapsulation in a polymer matrix).

[0110] As used herein, the term "binding portion" refers to the portion of the compound of the present invention that targets it to an appropriate site of action (e.g., cancer-associated antigens on solid tumor cells).

[0111] As used herein, "therapeutic portion" refers to the portion of the compound of the present invention that provides a therapeutic effect on a disease or symptom when it reaches its intended site of action.

[0112] As used herein, the terms “diagnostic portion” and “detectable portion” are used interchangeably and refer to the portion of the compound of the invention that provides a diagnostic effect in relation to a disease or condition and allows visualization of the cells or tissue in which the compound of the invention is accumulated.

[0113] In one aspect, the compounds of the present invention are represented by formula (I):

[0114]

[0115] Or its pharmaceutically acceptable salt or stereoisomer, wherein:

[0116] R1′ is a linking group;

[0117] R1 does not exist, or

[0118] R1 and R2, together with the nitrogen atoms they are attached to, form a heterocyclic group;

[0119] R2 is an optionally substituted (C1-C8) alkyl group, -C(O)R”, -C(O)OR”, -C(O)NR”R”, -S(O)R”, -S(O)2R”, (C3-C 10 A carbocyclic, 4- or 7-membered heterocyclic or substituted polyethylene glycol chain, wherein each R” is independently hydrogen, (C1-C6)alkyl, (C3-C6)alkyl, or substituted. 10 The alkyl group, carbocyclic group, or heterocyclic group may be substituted with a carbocyclic, 4-membered, or 7-membered heterocyclic group; and the alkyl, carbocyclic, or heterocyclic group may be substituted with a carbocyclic, 4-membered, or 7-membered heterocyclic group.

[0120] A1 is the active portion, as further defined below.

[0121] In some embodiments, R1 is absent, and R1′ is an alkylene chain that can be -O-, -S-, -N(R′)-, -C≡C-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -C(NOR′)-, -C(O)N(R′)-, -C(O)N(R′)C(O)-, -R′C(O)N(R′)R′, -C(O)N(R′)C(O)N(R′)-, -N(R′)C(O)N(R′)-, -N(R′)C(O)O-, -OC(O)N(R′)-, -C(NR′)-, -N(R′) C(NR′)-, -C(NR′)N(R′)-, -N(R′)C(NR′)N(R′)-, -OB(Me)O-, -S(O)2-, -OS(O)-, -S(O)O-, -S(O)-, -OS(O)2-, -S(O)2O-, -N(R′)S(O)2-, - S(O)2N(R′)-, --N(R′)S(O)-, -S(O)N(R′)-, -N(R′)S(O)2N(R′)-, -N(R′)S(O)N(R′)-, -OP(O)O(R′)O-, -N(R′)P(O)N(R′R′)N(R′)-, C3-C 12 At least one of carbocyclic, 3- to 12-membered heterocyclic, 5- to 12-membered heteroaryl groups, or any combination thereof, is interrupted and / or terminated (at either end or both ends), wherein each R′ is independently H or optionally substituted C1-C. 24 Alkyl groups, wherein the interrupting group and one or two terminating groups may be the same or different.

[0122] In some implementations, the alkylene chain is C1-C. 24 Alkylene chain. In some embodiments, the alkylene chain is C1-C2. 18 Alkylene chain. In some embodiments, the alkylene chain is C1-C2. 12 Alkylene chain. In some embodiments, the alkylene chain is C1-C2. 10Alkylene chain. In some embodiments, the alkylene chain is a C1-C8 alkylene chain. In some embodiments, the alkylene chain is a C1-C6 alkylene chain. In some embodiments, the alkylene chain is a C1-C4 alkylene chain. In some embodiments, the alkylene chain is a C1-C2 alkylene chain. In some embodiments, the alkylene chain is interrupted and / or terminated (at either end or both ends) by at least one or a combination of -N(R′)-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(R′)-, -N(R′)C(O)-, --N(R′)C(O)O-, -OC(O)N(R′)-, -S(O)2-, -N(R′)S(O)2-, -S(O)2N(R′)-, 4- to 6-membered heterocyclic groups. In some embodiments, the alkylene chain is interrupted and / or terminated (at either end or both ends) by -N(R′)-. In some embodiments, the alkylene chain is interrupted and / or terminated by -C(O)- (at either end or both ends). In some embodiments, the alkylene chain is interrupted and / or terminated by -C(O)O- (at either end or both ends). In some embodiments, the alkylene chain is interrupted and / or terminated by -C(O)N(R′)- (at either end or both ends). In some embodiments, the alkylene chain is interrupted and / or terminated by -N(R′)S(O)2- (at either end or both ends). In some embodiments, the alkylene chain is interrupted and / or terminated by a 4- to 6-membered heterocyclic group (at either end or both ends). In some embodiments, the alkylene chain is pyrrolidine-2,5-dione. termination.

[0123] In some implementations, R1 is absent, and R1′ is a polyethylene glycol chain that can be represented by -O-, -S-, -N(R′)-, -C≡C-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -C(NOR′)-, -C(O)N(R′)-, -C(O)N(R′)C(O)-, -R′C(O)N(R′)R′-, -C(O)N(R′)C(O)N(R′)-, -N(R′)C(O)N(R′)-, -N(R′)C(O)O-, -OC(O)N(R′)-, -C(NR′)-, -N(R ′)C(NR′)-, -C(NR′)N(R′)-, -N(R′)C(NR′)N(R′)-, -OB(Me)O-, -S(O)2-, -OS(O)-, -S(O)O-, -S(O)-, -OS(O)2-, -S(O)2O-, -N(R′)S(O)2- , -S(O)2N(R′)-, -N(R′)S(O)-, -S(O)N(R′)-, -N(R′)S(O)2N(R′)-, -N(R′)S(O)N(R′)-, -OP(O)O(R′)O-, -N(R′)P(O)N(R′R′)N(R′)-, C3-C 12 At least one of carbocyclic, 3- to 12-membered heterocyclic, 5- to 12-membered heteroaryl groups, or any combination thereof, is interrupted and / or terminated (at either end or both ends), wherein each R′ is independently H or optionally substituted C1-C. 24 Alkyl groups, wherein the interrupting group and one or two terminating groups may be the same or different.

[0124] In some embodiments, the polyethylene glycol chain has 1 to 20 -(CH2CH2-O)- units. In some embodiments, the polyethylene glycol chain has 1 to 15 -(CH2CH2-O)- units. In some embodiments, the polyethylene glycol chain has 1 to 10 -(CH2CH2-O)- units. In some embodiments, the polyethylene glycol chain has 1 to 5 -(CH2CH2-O)- units. In some embodiments, the polyethylene glycol chain has 1 to 2 -(CH2CH2-O)- units. In some embodiments, polyethylene glycol is interrupted and / or terminated (at either end or both ends) by at least one or a combination of -N(R′)-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(R′)-, -N(R′)C(O)-, -N(R′)C(O)O-, -OC(O)N(R′)-, -S(O)2-, --N(R′)S(O)2-, -S(O)2N(R′)-, 4- to 6-membered heterocyclic groups. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either end or both ends) by -N(R′)-. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either end or both ends) by -C(O)-. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either end or both ends) by -C(O)O-. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated by -C(O)N(R′)- (at either end or both ends). In some embodiments, the polyethylene glycol chain is interrupted and / or terminated by -N(R′)S(O)2- (at either end or both ends). In some embodiments, the polyethylene glycol chain is interrupted and / or terminated by a 4- to 6-membered heterocyclic group (at either end or both ends). In some embodiments, the polyethylene glycol chain is pyrrolidine-2,5-dione. termination.

[0125] In some embodiments, R1 and R2, together with the nitrogen atom to which they are attached, form a 3- to 16-membered heterocyclic group containing one to eight heteroatoms selected from N, O, and S. In some embodiments, R1 and R2, together with the nitrogen atom to which they are attached, form a 4- to 12-membered heterocyclic group containing one to four heteroatoms selected from N, O, and S. In some embodiments, R1 and R2, together with the nitrogen atom to which they are attached, form a 5- to 10-membered heterocyclic group containing one to three heteroatoms selected from N, O, and S. In some embodiments, R1 and R2, together with the nitrogen atom to which they are attached, form a 5- to 6-membered heterocyclic group containing one to two heteroatoms selected from N, O, and S. In some embodiments, R1 and R2, together with the nitrogen atom to which they are attached, form a piperazine group.

[0126] In some implementations, R1 does not exist, and R1′ is C1-C 24The chain is alkylene, and R2 is methyl, ethyl, isopropyl, or tert-butyl. In some embodiments, R1 is absent, and R1′ is C1-C. 18 The chain is alkylene, and R2 is methyl, ethyl, isopropyl, or tert-butyl. In some embodiments, R1 is absent, and R1′ is C1-C. 12 The alkylene chain, and R2, are methyl, ethyl, isopropyl, or tert-butyl. In some embodiments, R1 is absent, and R1′ is C1-C. 10 In some embodiments, R1 is absent, R1′ is a C1-C8 alkylene chain, and R2 is methyl, ethyl, isopropyl, or tert-butyl. In some embodiments, R1 is absent, R1′ is a C1-C6 alkylene chain, and R2 is methyl, ethyl, isopropyl, or tert-butyl. In some embodiments, R1 is absent, R1′ is a C1-C4 alkylene chain, and R2 is methyl, ethyl, isopropyl, or tert-butyl. In some embodiments, R1 is absent, R1′ is a C1-C2 alkylene chain, and R2 is methyl, ethyl, isopropyl, or tert-butyl. In some embodiments, R1 is absent, R1′ is one to 20 -(CH2CH2-O)- units, and R2 is methyl, ethyl, isopropyl, or tert-butyl. In some embodiments, R1 is absent, R1′ is 1 to 15 -(CH2CH2-O)- units, and R2 is methyl, ethyl, isopropyl, or tert-butyl. In some embodiments, R1 is absent, R1′ is 1 to 10 -(CH2CH2-O)- units, and R2 is methyl, ethyl, isopropyl, or tert-butyl. In some embodiments, R1 is absent, R1′ is 1 to 5 -(CH2CH2-O)- units, and R2 is methyl, ethyl, isopropyl, or tert-butyl. In some embodiments, R1 is absent, R1′ is 1 to 2 -(CH2CH2-O)- units, and R2 is methyl, ethyl, isopropyl, or tert-butyl.

[0127] In some implementations, the active part is the binding part. Representative examples of binding parts include those that bind ubiquitin ligases or other cellular enzymes that catalyze the degradation of cellular proteins. For example, the ubiquitin-proteasome pathway (UPP) is a major cellular pathway that regulates key regulatory proteins and degrades misfolded or malformed proteins. UPP is central to many cellular processes. The covalent linking of ubiquitin to a specific protein substrate is achieved through the action of E3 ubiquitin ligases. These ligases comprise more than 500 different proteins and are categorized into several classes based on the structural elements that enable their E3 functional activity.

[0128] In some implementations, the binding moiety is a small molecule that binds to an E3 ligase (which is cereblon (CRBN)). Representative examples of small molecules that bind CRBN are represented by any of the structures (D1-a) to (D1-d):

[0129]

[0130] Where X2 is CH2 or C(O), and X3 is CR”1R”2, NR”1, O or S, where R” i R”1 and R”2 are independently hydrogen, halogen, OH, NH2, C1-C3 alkyl, C1-C3 alkoxy or C1-C3 alkylamine, or R”1 and R”2 together with the atoms they are bonded to form a C3-C7 carbocyclic or C3-C7 heterocyclic (e.g., aziridine, piperidine, pyrrolidine, cyclobutane, cyclohexane).

[0131] Other small molecules that can be incorporated into cereblon and are applicable to this invention are also disclosed in U.S. Patent 9,770,512 and U.S. Patent Publications 2018 / 0015087, 2018 / 0009779, 2016 / 0243247, 2016 / 0235731, 2016 / 0237730 and 2016 / 0176916, and International Patent Publications WO 2017 / 197055, WO 2017 / 199051, WO 2017 / 17036, WO 2017197056 and WO 2017 / 197046.

[0132] In some implementations, the binding moiety is a small molecule that binds to an E3 ligase (a von Hippel-Lindau (VHL) tumor inhibitor). Representative examples of small molecules that bind to VHL are represented by any of the structures (D2-a) to (D2-j):

[0133] Where Z1 is a C5-C6 carbon ring or a C5-C6 heterocyclic group. Where Y′ is a bond, CH2, NH, NMe, O or S, or a stereoisomer thereof.

[0134] In some embodiments, Z1 is phenyl, pyrrolyl, furanyl, phenylthio, pyrazolyl, imidazolyl, oxazolyl, thiazolyl, pyridinyl, pyridazinyl, or pyrimidinyl. In some embodiments, Z is...

[0135] Other small molecules that combine with VHL and are applicable to the present invention are also disclosed in U.S. Patent Publications 2017 / 0121321 and 2014 / 0356322.

[0136] In some implementations, the binding moiety is a small molecule that binds to an E3 ligase (an inhibitor of apoptosis protein (IAP)). Representative examples of small molecules that bind IAP are represented by any of the structures (D3-a) to (D3-f):

[0137]

[0138] International patent publications WO 2008128171, WO 2008 / 016893, WO 2014 / 060768 and WO 2014 / 060767 also disclose other small molecules that combine with IAP and are applicable to the present invention.

[0139] In some implementations, the binding moiety is a small molecule that binds to an E3 ligase (which is mouse bimicrosome 2 (MDM2)). Representative examples of small molecules that bind to MDM2 are represented by structures (D4-a) and (D4-b):

[0140]

[0141] U.S. Patent No. 9,993,472B2 also discloses other small molecules that can be used in conjunction with MDM2 and are applicable to the present invention. MDM2 is known in the art to have the function of a ubiquitin-E3 ligase.

[0142] In some implementations, the binding moiety is a small molecule that binds to the ubiquitin receptor RPN13. Representative examples of small molecules that bind RPN13 are represented by structures (D5-a), (D5-b), (D5-c), and (D5-d):

[0143]

[0144] International Publication No. PCT / US2020 / 012825 also discloses other small molecules that bind to RPN13 and are applicable to this invention. RPN13 is known in the art to have the function of a ubiquitin receptor.

[0145] In some embodiments, A1 is a binding moiety that binds to cellular proteins other than cellular enzymes that catalyze the degradation of cellular proteins (such as ubiquitin ligases). Representative examples of cellular proteins that can be targeted by the compounds of the present invention containing the binding moiety include kinases, proteins with a bromine-containing BET domain, cytoplasmic signaling proteins (e.g., FKBP12), nucleoproteins, histone deacetylases (HDACs), lysine methyltransferases, aryl hydrocarbon receptors (AHRs), estrogen receptors, androgen receptors, glucocorticoid receptors, and transcription factors (e.g., SMARCA4, SMARCA2, TRIM24).

[0146] In some embodiments, it binds partially bound tyrosine kinases (e.g., AATK, ABL, ABL2, ALK, AXL, BLK, BMX, BTK, CSF1R, CSK, DDR1, DDR2, EGFR, EPHA1, EPHA2, EPHA3, EPHA4, EPHA5, EPHA6, EPHA7, EPHA8, EPHA10, EPHB1, EPHB2, EPHB3, EPHB4, EPHB6, ERBB2, ERBB3, ERBB4, FER, FES, FGFR1, FGFR7, FGFR3, FGFR4, FGR, FLT1, FLT3, FLT4, FRK, FYN, GSG2, HCK, IGF). 1R, ILK, INSR, INSRR, IRAK4, ITK, JAK1, JAK2, JAK3, KDR, KIT, KSR1, LCK, LMTK2, LMTK3, LTK, LYN, MATK, MERTK, MET , MLTK, MST1R, MUSK, NPR1, NTRK1, NTRK2, NTRK3, PDGFRA, PDGFRB, PLK4, PTK2, PTK2B, PTK6, PTK7, RET, ROR1, ROR2, ROS1, RYK, SGK493, SRC, SRMS, STYK1, SYK, TEC, TEK, TEX14, TIE1, TNK1, TNK2, TNNI3K, TXK, TYK2, TYRO3, YES1 or ZAP70), serine / threonine kinases (e.g., casein kinase 2, protein kinase A, protein kinase B, protein kinase C, Raf kinase, CaM kinase, AKT1, AKT2, AKT3, ALK1, ALK2, ALK3, ALK4, Aurora A, Aurora B),

[0147] Aurora C, CHK1, CHK2, CLK1, CLK2, CLK3, DAPK 1, DAPK2, DAPK3, DMPK, ERK1, ERK2, ERK5, GCK, GSK3, HIPK, KHS1, LKB1, LOK, MAPKAPK2, MAPKAPK, MNK1, MSSK1, MST1, MST2, MST4, NDR, NEK2, NEK3, NEK6, NEK7, NEK9, NEK1 1, PAK1, PAK2, PAK3, PAK4, PAK5, PAK6, PIM 1, PIM2, PLK 1, RIP2, RIP5, RSK1, RSK2, SGK2, SGK3, SIK1, STK33, TAO1, TAO2, TGF-beta, TLK2, TSSK1, TSSK2, ULK1, or ULK2), cyclin-dependent kinases (e.g., Cdk1-Cdk11) or leucine-rich repeat kinases (e.g., LRRK2).

[0148] In some implementations, proteins containing partially bound bromo-binding domains and superterminal domains (BET) are included. Representative examples include protein 2 (ATAD2) containing the AAA domain of the ATPase family, protein 1A (BAZ1A), BAZ1B, BAZ2A, BAZ2B with a bromo-binding domain adjacent to the zinc finger domain, protein 1 (BRD1), BRD2, BRD3, BRD4, BRD5, BRD6, BRD7, BRD8, BRD9, BRD10, testis-specific protein with a bromo-binding domain (BRDT), PHD finger protein 1 (BRPF1), BRPF3, protein with a bromo-binding domain and WD3 repeat (BRWD3), cat's eye syndrome borderline protein 2 (CECR2), CREB-binding protein (CREBBP), E1A-binding protein P300 (EP300), and amino acid synthesis 5. The protein-like proteins include universal regulatory protein 2 (GCN5L2), histone-lysine N-methyltransferase 2A (KMT2A), P300 / CBP-associated factor (PCAF), pH-interacting protein (PHIP), protein kinase C-binding protein 1 (PRKCBP1), SWI / SNF-associated matrix-associated actin-dependent regulatory factor subfamily A member 2 (SMARCA2), SMARCA4, Sp100 nucleosomal protein (SP100), SP110, SP140, transcription initiation factor TFIID subunit 1 (TAF1), TAF1L, TIF1a, tridomain protein 28 (TRIM28), TRIM33, TRIM66, WD repeat protein 9 (WDR9), zinc finger MYND domain protein 11 (ZMYND11), and mixed lineage leukemia-like protein 4 (MLL4). In some embodiments, the BET bromine-containing domain protein is BRD4.

[0149] In some implementations, the following proteins are bound: BRD2, BRD3, BRD4, antennal homologous domain proteins, BRCA1, BRCA2, CCAAT-enhancing binding proteins, histones, polycomb-group proteins, high-mobility group proteins, telomere-binding proteins, FANCA, FANCD2, FANCE, FANCF, HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, HDAC11, hepatocyte nuclear factor, Mad2, NF-κB, nuclear receptor coactivator, CREB-binding protein, p55, p107, p130, p53, c-fos, c-jun, c-mdm2, c-myc, or c-rel.

[0150] In some implementations, the binding moiety binds to the BRD. Representative examples of small molecules that bind to BRDs include:

[0151]

[0152] in:

[0153] R is the point to which the linking group is attached; and

[0154] R′ is methyl or ethyl.

[0155] In some implementations, the binding moiety binds to CREBBP. Representative examples of small molecules that bind to CREBBP include:

[0156]

[0157]

[0158] in:

[0159] R is the point to which the linking group is attached;

[0160] A is N or CH; and

[0161] m can be 0, 1, 2, 3, 4, 5, 6, 7 or 8.

[0162] In some implementations, the binding moiety binds to SMARCA4 / PB1 / SMARCA2. Representative examples of small molecules binding to SMARCA4 / PB1 / SMARCA2 include:

[0163]

[0164] in:

[0165] R is the point to which the linking group is attached;

[0166] A is N or CH; and

[0167] m can be 0, 1, 2, 3, 4, 5, 6, 7 or 8.

[0168] In some implementations, the binding moiety binds to TRIM24 / BRPF1. Representative examples of small molecules that bind to TRIM24 / BRPF1 include:

[0169]

[0170] in:

[0171] R is the point where the linking group is attached: and

[0172] m can be 0, 1, 2, 3, 4, 5, 6, 7 or 8.

[0173] In some implementations, the binding moiety binds to the glucocorticoid receptor. Representative examples of small molecules that bind to the glucocorticoid receptor include:

[0174]

[0175]

[0176] in:

[0177] R is the point to which the linking group is attached.

[0178] In some implementations, the binding moiety binds to estrogen / androgen receptors. Representative examples of small molecules that bind to estrogen / androgen receptors include:

[0179]

[0180]

[0181] in:

[0182] R is the point to which the linking group is attached.

[0183] In some implementations, the binding moiety binds to DOT1L. Representative examples of small molecules that bind to DOT1L include:

[0184]

[0185]

[0186] in:

[0187] R is the point to which the linking group is attached;

[0188] A is N or CH; and

[0189] m can be 0, 1, 2, 3, 4, 5, 6, 7 or 8.

[0190] In some implementations, the binding moiety binds to Ras. Representative examples of small molecules that bind to Ras include:

[0191]

[0192]

[0193] in:

[0194] R is the point to which the linking group is attached.

[0195] In some implementations, the binding moiety binds to RasG12C. Representative examples of small molecules that bind to RasG12C include:

[0196]

[0197] in:

[0198] R is the point to which the linking group is attached.

[0199] In some implementations, the binding moiety binds to Her3. Representative examples of small molecules that bind to Her3 include:

[0200]

[0201] in:

[0202] R is the point to which the linking group is attached; and

[0203] R′ is

[0204] In some implementations, the binding moiety binds to Bcl-2 / Bcl-XL. Representative examples of small molecules that bind to Bcl-2 / Bcl-XL include:

[0205]

[0206] in:

[0207] R is the point to which the linking group is attached.

[0208] In some implementations, the binding moiety binds to HDAC. Representative examples of small molecules that bind to HDAC include:

[0209]

[0210] in:

[0211] R is the point to which the linking group is attached.

[0212] In some implementations, the binding moiety binds to PPAR-γ. Representative examples of small molecules that bind to PPAR-γ include:

[0213]

[0214] in:

[0215] R is the point to which the linking group is attached.

[0216] In some implementations, the binding moiety binds to RXR. Representative examples of small molecules that bind to RXR include:

[0217]

[0218]

[0219] in:

[0220] R is the point to which the linking group is attached.

[0221] In some implementations, the binding moiety binds to DHFR. Representative examples of small molecules that bind to DHFR include:

[0222]

[0223]

[0224] in:

[0225] R is the point to which the linking group is attached.

[0226] In some implementations, the binding moiety binds to BCL2. Representative examples of small molecules that bind to BCL2 include:

[0227]

[0228]

[0229]

[0230] in:

[0231] R is the point to which the linking group is attached.

[0232] Other small molecules that bind to cellular proteins and are suitable for use as binding motifs in this invention are also disclosed in U.S. Patent Publications 2017 / 0121321 and 2014 / 0356322.

[0233] In some embodiments, the binding portion is biotin or a biotin derivative. Biotin derivatives are known in the art. See, for example, Molecular Probes Handbook, A Guide to Fluorescent Probes and Labeling Technologies, 11 thEd., Life Technologies Corporation, 2010. Biotin and its derivatives have been widely used as molecular markers in the biotechnology industry for many years. Representative examples of biotin derivatives suitable for use in this invention include desulfobiotin, pyrimethamine biotin, rac-selenobiotin, biocytidine, 2-iminobiotin, biocytidine-L-proline, biotincystamine, and biotin-tobramycinamide. Other biotin derivatives suitable for use in this invention are described in the art, for example, in U.S. Patent No. 8,318,696 and U.S. Patent Publication No. 2007 / 0020206, each of which is incorporated herein by reference.

[0234] In some implementations, the binding portion is a short peptide sequence (e.g., 2 to 50 amino acids in length, such as 4 to 20 amino acids in length, wherein the amino acid residues in the peptide may be the same or different). Representative examples include α-amaminine, analgesic, tadpoles, glutathione, leucosterol, pepsin, peptidase, peptide T, phalloidin, tepropeptide, tuftsin, ALFA tag, Avi tag, C-tag, calmodulin tag, polyglutamic acid tag, polyarginine tag, E-tag, FLAG-tag, HA tag, His tag, Myc tag, NE tag, Rho1D4 tag, S-tag, SBP tag, softag 1, softag 3, Spot tag, Strep tag, T7 tag, TC tag, Ty tag, V5 tag, VSV tag, and Xpress tag.

[0235] In some embodiments, the binding moiety is a protein. Representative examples of protein-binding moieties include chitin-binding protein (CBP), maltose-binding protein (MBP), glutathione S-transferase (GST), thioredoxin, poly(NANP), biotinylate carboxyl carrier protein (BCCP), green fluorescent protein (GFP), Halo tags, SNAP tags, CLIP tags, HUH tags, Nus tags, Fc tags, and carbohydrate recognition domain tags. In some embodiments, the binding moieties are HaloTags.

[0236] In other embodiments, the binding portion is an antibody (e.g., a monoclonal antibody) or a fragment thereof that binds to the intended target. In some embodiments, the monoclonal antibody binds to a cell surface receptor present on diseased cells. In some embodiments, the monoclonal antibody binds to tumor-associated antigens on cancer cells, such as solid tumor cells. Representative examples of monoclonal antibodies include moromuzumab-CD3, abciximab, rituximab, palizumab, infliximab, trastuzumab, alemtuzumab, adalimumab, teimozumab, omalizumab, cetuximab, bevacizumab, natezumab, panitumumab, ranibizumab, eculizumab, cetrus, ustekinumab, canatumab, and golimumab. Ophamumab, Tocilizumab, Denosumab, Belimumab, Ipilimumab, Bentuximab, Pertuximab, Raxicurumab, Ophamumab, Sestoxicurumab, Ramucirumab, Vedolizumab, Bonatumab, Nivolumab, Pembrolizumab, Idacilumab, Nexicurumab, Denocicurumab, Sestoxicurumab, Mepleribumab, Alirobumab, Illoxicurumab Eutumumab, Daremumab, Erotozumab, Isebemab, Relizumab, Olanzab, Belottosuzumab, Atezolizumab, Otosaxizine, Oga-Itozumab, Brodatumab, Gusekumab, Duprexumab, Thaliruzumab, Averuzumab, Omepizumab, Emecilizumab, Benalizumab, Gelotuzumab, Dvorumab, Ibuprofen Lanaruzumab, Moglicililumab, Erenelumab, Ganecililumab, Tectogizumab, Cimiprilumab, Ipatavuzumab, Remannetumab, Ibacililumab, Mosetumab, Revuricililumab, Capsulesumab, Lomosocililumab, Rissacililumab, Polotocililumab, Broxetinecililumab, Lizanililumab, Cetirizumab, Belantacililumab, or Enroximatelumab.

[0237] In some implementations, the binding portion is a fragment of an antibody (e.g., a monoclonal antibody). For example, the fragment can be a variable fragment, such as a single-chain variable fragment (scFv) of a monoclonal antibody. Representative examples of scFvs include peroxyzumab, dutuximab, efungumab, gantuzumab, lelizumab, oportuzumab monatox, vobalizumab, and broxol.

[0238] In some embodiments, the active portion is the binding portion, which is a solubility-enhancing group. Examples of solubility-enhancing groups include substituents containing groups that are ionizable in water at pH 0-14, ionizable groups capable of forming salts, and highly polar substituents with high dipole moments capable of forming strong interactions with water molecules. In some embodiments, the solubility-enhancing group is an α-chloroacetyl group.

[0239] In some embodiments, the active portion is a therapeutic portion. In some embodiments, the therapeutic portion may be a small molecule. In some embodiments, the molecular weight of the small molecule is not more than about 1,000 g / mol, not more than about 900 g / mol, not more than about 800 g / mol, not more than about 700 g / mol, not more than about 600 g / mol, not more than about 500 g / mol, not more than about 400 g / mol, not more than about 300 g / mol, not more than about 200 g / mol, or not more than about 100 g / mol. In some embodiments, the molecular weight of the small molecule is at least about 100 g / mol, at least about 200 g / mol, at least about 300 g / mol, at least about 400 g / mol, at least about 500 g / mol, at least about 600 g / mol, at least about 700 g / mol, at least about 800 g / mol, or at least about 900 g / mol, or at least about 1,000 g / mol. In some implementations, the therapeutic component is a therapeutically active agent, such as a drug (e.g., a molecule approved by the U.S. Food and Drug Administration as defined in the Federal Regulatory Regulations (CFR)).

[0240] In some implementations, the treatment component is an anticancer agent. Representative types of anticancer agents include antiangiogenic agents, alkylating agents, antimetabolites, tubulin polymerization disruptors, platinum coordination complexes, anthraquinones, substituted ureas, methylhydrazine derivatives, adrenocortical inhibitors, hormones and antagonists, anticancer polysaccharides, and anthracycline antibiotics (e.g., arubicin, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, pirarubicin, pentoxyverine and their derivatives and analogues) and kinase inhibitors (e.g., pan-Her inhibitors (e.g., HKI-272, BIBW-2992, PF299, SN29926 and PR-509E)).

[0241] In some implementations, the treatment component is a non-targeted cancer agent, as is known in the art, which refers to an agent with a relatively broad mode of action. Representative examples of non-targeted anticancer drugs include alkylating agents (e.g., busulfan, chlorambucil, cyclophosphamide, ifosfamide, nitrogen mustard, melphalan, carmustine, streptozotocin, dacarbazine, temozolomide, hexamethylmelamine, and thiotepa), antimetabolites (e.g., capecitabine, cytarabine, 5′-fluorouracil, gemcitabine, cladribine, fludarabine, 6-mercaptopurine, and pentostatin), folic acid antagonists (e.g., methotrexate and pemetrexed), and mitotic inhibitors (e.g., docetaxel, paclitaxel, vinblastine, vincristine, vinblastine, etc.). Dixin and vinorelbine), DNA inhibitors (e.g., hydroxyurea, carboplatin, cisplatin, oxaliplatin, mitomycin C and pyrrole-benzoheptatriene), topoisomerase inhibitors (e.g., topotecan, irinotecan, daunorubicin, doxorubicin, etoposide, teniposide and mitoxantrone), DNA breakage inducers (e.g., bleomycin), ozomicin, vedotin, emtansine, pasudotox, delutec, govitecan and mafodotin or derivatives thereof).

[0242] In some implementations, the treatment component is a targeted anticancer agent, as is known in the art, referring to an agent with a specific mode of action. Representative examples of non-targeted anticancer agents include afatinib (EGFR, HER2), axitinib (KIT, PDGFRβ, VEGFR1 / 2 / 3), bosutinib (ABL), cabozantinib (FLT3, KIT, MET, RET, VEGFR2), ceritinib (ALK), crizotinib (ALK, MET), dabrafenib (ABL), erlotinib (EGFR), ibrutinib (BTK), ederaris (PI3Kδ), imatinib (KIT, PDGFR, ABL), lapatinib (HER2, EGFR), lenvatinib (VEGFR2), nilotinib (ABL), olaparib (PARP), and pebucixirib (CDK). 4. Drugs targeted at cancer include CDK6), pabisostat (HDAC), pazopanib (VEGFR, PDGFR, KIT), panatinib (ABL, FGFR1-3, FLT3, VEGFR2), regorafenib (KIT, PDGFRβ, RAF, RET, VEGFR1 / 2 / 3), romidesin (HDAC), ruxolitinib (JAK1 / 2), sorafenib (VEGFR, PDGFR, KIT, RAT), tamsilimus (mTOR), trametinib (MEK), vandetanib (EGFR, RET, VEGFR2), vemurafenib (BRAF), vemodega (PTCH), and vorinostat (HDAC). In some implementations, the targeted anticancer agent is a kinase inhibitor. Representative examples of kinase inhibitors include abexicillin, acalatinib, afatinib, alectinib, avatinib, axitinib, baricitinib, bimetinib, bosutinib, brigatinib, cabozantinib, ceritinib, carmatinib, cobitinib, crizotinib, dabrafenib, dacomitinib, dasatinib, cannefenib, entrectinib, erdatinib, erlotinib, everolimus, fizzotinib, fotatinib, gefitinib, gilteritinib, ibrutinib, icotinib, imatinib, and others. Patinib, larotrectinib, lenvatinib, lorlatinib, midotolin, neratinib, netarsudil, nilotinib, nintedanib, osimertinib, pabuxirib, pazopanib, permitinib, percidatinib, ponatinib, pralitinib, regorafenib, ribocicib, riprepitinib, ruxotetinib, sertinib, sumetinib, sirolimus, sorafenib, sunitinib, tesimolimus, tolvatinib, trametinib, taucapinib, utpatinib, vandetanib, vemurafenib, and zanubrutinib.

[0243] In some implementations, the treatment component is an antibacterial agent. Representative examples of antibacterial agents include prazomicin, ilacycline, sarecycline free base, ometracycline, rifamycin, imipenem, cilastatin, relebactam, premani, lefamoline, cefiderocol, sulfaquinoxaline, oxytetracycline, hygromycin B, tylosin, chlortetracycline, virginiamycin, neomycin, lincomycin, pyrantel pamoate, melemone, lasalomycin, fenbendazole, serodomicin, decaoxyquin ester, ractopamine, leprosycin, diclazuril, halifuginone, clopidogrel, clopidogrel, zipateroside, monensin, zoalene, lubabegron, and bacitracin.

[0244] In some implementations, the treatment component is a nonsteroidal anti-inflammatory drug (NSAID). Representative examples of NSAIDs include celecoxib, diclofenac, diflunisal, etodoxacin, phenoprofen, flurbiprofen, ibuprofen, indomethacin, ketoprofen, ketorolac, mefenamic acid, meloxicam, nabumetone, naproxen, oxapzin, pyrimethamine, sulindac, and tomatine.

[0245] In some implementations, the treatment component is a corticosteroid. Representative examples of corticosteroids include defcodone, dexamethasone, betamethasone, triamcinolone, hydrocortisone, methylprednisolone, and prednisone.

[0246] In some implementations, the treatment component is a disease-modifying antirheumatic drug (DMARD). Representative examples of DMARDs include hydroxychloroquine, leflunomide, methotrexate, sulfasalazine, minocycline, penicillamine, cyclophosphamide, azathioprine, cyclosporine, apremilast, and mycophenolate mofetil.

[0247] In some implementations, the active portion is a diagnostic portion. Diagnostic portions typically contain detectable components, such as markers. Representative examples of diagnostic portions include dyes, chromogenic agents, positron emission tomography (PET) tracers, and magnetic resonance imaging (MRI) contrast agents. The term "marker" includes any portion that allows the compound to which it is attached to to be captured, detected, or visualized. Markers can be directly detectable (i.e., they are detectable without any further reaction or manipulation, e.g., fluorophores or chromophores are directly detectable) or they can be indirectly detectable (i.e., they are made detectable by reacting with or binding to another detectable entity, e.g., haptens can be detected by immunostaining following a reaction with a suitable antibody containing a reporter gene (e.g., a fluorophore)). Representative examples of marker types include affinity tags, radiation markers (e.g., radionuclides, e.g., for example, 32 P, 35 S, 3 H,14 C 125 I, 131 (e.g., I), fluorescent dyes, phosphorescent dyes, chemiluminescent agents (e.g., acridinium esters, stabilized dioxanes, etc.), spectrally resolvable inorganic fluorescent semiconductor nanocrystals (i.e., quantum dots), metal nanoparticles (e.g., gold, silver, copper, and platinum) or nanoclusters, enzymes (e.g., enzymes used in ELISA, such as horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), colorimetric labels (e.g., dyes, colloidal gold, etc.), magnetic labels (e.g., Dynabeads). TM ) and haptens.

[0248] In some implementations, the marker contains a fluorescent dye. Representative examples of fluorescent dyes include fluorescein and fluorescein dyes (e.g., fluorescein isothiocyanate (FITC), naphthalene fluorescein, 4′,5′-dichloro-2′,7′-dimethoxyfluorescein, 6-carboxyfluorescein, or FAM), carbonyl cyanide, anthocyanins, styryl dyes, oxonol dyes, phycoerythrin, erythrosine, eosin, rhodamine dyes (e.g., 5-carboxytetramethylrhodamine (TAMRA), carboxyrhodamine 6G, carboxy-X-rhodamine hydrochloride (ROX), rhodamine B, rhodamine 6G, rhodamine green, rhodamine red, or tetramethylrosine (TMR)), coumarin and coumarin dyes (e.g., methoxycoumarin, dialkylaminocoumarin, hydroxycoumarin, and aminomethylcoumarin, or AMCA), Oregon green dyes (e.g., Oregon Green 488, Oregon Green 500, Oregon Green 514), Texas Red, Texas Red-X, Spectrum Red TM Spectrum Green TM cyanine dyes (e.g., Cy-3) TM Cy-5 TM Cy-3.5 TM Cy-5.5 TMAlexa Fluor dyes (e.g., Alexa Fluor 350, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 660 and Alexa Fluor 680), BODIPY dyes (e.g., BODIPY FL, BODIPY R6G, BODIPY TMR, BODIPY TR, BODIPY 530 / 550, BODIPY 558 / 568, BODIPY 564 / 570, BODIPY 576 / 589, BODIPY 581 / 591, BODIPY 630 / 650, BODIPY 650 / 665), IRDyes (e.g., IRD40, IRD 700, IRD 800), etc. For more examples of suitable fluorescent dyes and methods for coupling fluorescent dyes with other chemical entities, see, for example, The Handbook of Fluorescent Probes and Research Products, 9th Ed., Molecular Probes, Inc., Eugene, Oregon and Molecular Probes Handbook, A Guide to Fluorescent Probes and Labeling Technologies, 11. th Ed., Life Technologies.

[0249] In some embodiments, the diagnostic portion includes a rhodamine dye. In some embodiments, the diagnostic portion includes tetramethylrhodamine (TAMRA) or a derivative thereof.

[0250] In some embodiments, the diagnostic component is a chromogenic agent, as is known in the art, referring to a compound that induces a colorimetric reaction. Representative examples of chromogenic agents include azo reagents such as methyl orange and methyl red, nitrophenol, phthalides such as phenolphthalein or thymolphthalein, sulfonphthales such as bromophenol blue or bromocresol green, indophenols such as 2,6-dichloroindophenol, azazine reagents such as thiazide dyes methylene blue, indigo carmine, diphenylamine derivatives such as diphenylamine-4-sulfonic acid and valamine blue, azoarsine III, catechol violet, dithizone, 1-(2′-pyridineazo)-2-naphthol, 4-(2′-pyridineazo)resorcinol, chrome azure S, chrome black T, chrome blue black B, pyrogallol red, alizarin complexes, methyl thymol blue, and xylenol orange.

[0251] In some implementations, the diagnostic component is a PET tracer, which, as is known in the art, refers to a radioligand used for imaging purposes. Representative examples include acetate (C-11), chline (C-11), fluorodeoxyglucose (F-18), sodium fluoride (F-18), fluoro-ethyl-spiperone (F-18), methionine (C-11), prostate-specific membrane antigen (PSMA) (Ga-68), DOTATOC / DOTANOC / DOTATATE (Ga-68), flubitaban / flubetapyr (F-18), rubidium (Rb-82), and FDDNP (F-18).

[0252] In some implementations, the diagnostic component is an MRI contrast agent, as known in the art, referring to an agent used to improve the visibility of internal body structures. Representative examples include gadoterate, gadodiamide, gadobenate, gadopentetate, gadoterol, gadophosphine, gadofosamide, gadoxetate, and gadobutrol.

[0253] The markers applicable to this invention can be detected by any of a variety of means, including spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, and chemical methods.

[0254] In some implementations, the active portion is a stationary portion. Representative examples of stationary portions include polystyrene beads, agarose magnetic beads, cross-linked agarose beads, and... Pearl.

[0255] In some implementations, R2 is methyl, ethyl, isopropyl, or tert-butyl.

[0256] In some implementations, the compound of formula (I) is

[0257] Or its pharmaceutically acceptable salts or stereoisomers.

[0258] In some embodiments, the optional substituents of the compound of formula (I) are selected from the group consisting of: alkyl, alkenyl, alkynyl, halogen, haloalkyl, cycloalkyl, heterocycloalkyl, hydroxyl, alkoxy, cycloalkoxy, heterocycloalkoxy, haloalkoxy, aryloxy, heteroaryloxy, aralkyloxy, alkenyloxy, alkynyloxy, amino, alkylamino, cycloalkylamino, heterocycloalkylamino, arylamino, heteroarylamino, arylalkylamino, N-alkyl-N-arylamino, N-alkyl-N-heteroarylamino, N-alkyl-N-arylalkylamino, hydroxyalkyl, aminoalkyl, alkylthio, haloalkylthio, alkylsulfonyl, haloalkylsulfonyl, cycloalkylsulfonyl, heterocycloalkylsulfonyl, arylsulfonyl, heteroarylsulfonyl, aminosulfonyl, alkylaminosulfonyl, cycloalkylaminosulfonyl, heterocycloalkylaminosulfonyl, arylaminosulfonyl Acyl, heteroarylaminosulfonyl, N-alkyl-N-arylaminosulfonyl, N-alkyl-N-heteroarylaminosulfonyl, formyl, alkyl carbonyl, haloalkyl carbonyl, alkenyl carbonyl, alkynyl carbonyl, carboxyl, alkoxy carbonyl, alkyl carbonyloxy, amino, alkylsulfonylamino, haloalkylsulfonylamino, cycloalkylsulfonylamino, heterocyclic alkylsulfonylamino, arylsulfonylamino, heteroarylsulfonylamino, aralkylsulfonylamino, alkylcarbonylamino, haloalkylcarbonylamino, cycloalkylcarbonylamino, heterocyclic alkylcarbonylamino, arylcarbonylamino, heteroarylcarbonylamino, aralkylsulfonylamino, aminocarbonyl, alkylaminocarbonyl, cycloalkylaminocarbonyl, heterocyclic alkylaminocarbonyl, arylaminocarbonyl, heteroarylaminocarbonyl, N-alkyl-N-arylaminocarbonyl, N-alkyl-N-heteroarylaminocarbonyl, cyano, nitro, and azide.

[0259] In some embodiments, the compound of formula (I) is of formula Ia′, Ib, or Ic′:

[0260]

[0261] Or its pharmaceutically acceptable salts or stereoisomers.

[0262] in:

[0263] R1′ is a linking group;

[0264] R1 does not exist, or

[0265] R1 and R2, together with the nitrogen atoms they are attached to, form a heterocyclic group;

[0266] R2 is an optionally substituted (C1-C8) alkyl group, -C(O)R′, -C(O)OR′, -C(O)NR′R′, -S(O)R′, -S(O)2R′, (C3-C 10 A carbocyclic or 4- or 7-membered heterocyclic group, wherein each R” is independently hydrogen, (C1-C6)alkyl, (C3-C6)alkyl, or (C7-C8)alkyl.10 The alkyl group, carbocyclic group, or heterocyclic group may be substituted with a carbocyclic, 4-membered, or 7-membered heterocyclic group; and the alkyl, carbocyclic, or heterocyclic group may be substituted with a carbocyclic, 4-membered, or 7-membered heterocyclic group.

[0267] A1′ is an antibody or antibody fragment.

[0268] In some implementations, R1 is absent.

[0269] In some embodiments, R1 is absent, and R1′ is an alkylene chain that can be substituted with -O-, -S-, -N(R′)-, -C-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -C(NOR′)-, -C(O)N(R′)-, -C(O)N(R′)C(O)-, -R′C(O)N(R′)R′-, -C(O)N(R′)C(O)N(R′)-, -N(R′)C(O)-, -N(R′)C(O)N(R′)-, -N(R′)C(O)O-, -OC(O)N(R′)-, -C(NR′)-, -N(R ′)C(NR′)-, -C(NR′)N(R′)-, -N(R′)C(NR′)N(R′)-, -OB(Me)O-, -S(O)2-, -OS(O)-, -S(O)O-, -S(O)-, -OS(O)2-, -S(O)2O-, -N(R′)S(O)2-, -S(O)2N(R′)-, --N(R′)S(O)-, -S(O)N(R′)-, -N(R′)S(O)2N(R′)-, -N(R′)S(O)N(R′)-, -OP(O)O(R′)O-, -N(R′)P(O)N(R′R′)N(R′)-, C3-C 12 At least one of carbocyclic, 3- to 12-membered heterocyclic, 5- to 12-membered heteroaryl groups, or any combination thereof, is interrupted and / or terminated (at either end or both ends), wherein each R′ is independently H or optionally substituted C1-C. 24 Alkyl groups, wherein the interrupting group and one or two terminating groups may be the same or different.

[0270] In some implementations, the alkylene chain is C1-C. 24 Alkylene chain. In some embodiments, the alkylene chain is C1-C2. 18 Alkylene chain. In some embodiments, the alkylene chain is C1-C2. 12 Alkylene chain. In some embodiments, the alkylene chain is C1-C2. 10Alkylene chain. In some embodiments, the alkylene chain is a C1-C8 alkylene chain. In some embodiments, the alkylene chain is a C1-C6 alkylene chain. In some embodiments, the alkylene chain is a C1-C4 alkylene chain. In some embodiments, the alkylene chain is a C1-C2 alkylene chain. In some embodiments, the alkylene chain is interrupted and / or terminated (at either end or both ends) by at least one or a combination of -N(R′)-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(R′)-, -N(R′)C(O)-, -OC(O)N(R′)-, -S(O)2-, -N(R′)S(O)2-, and -S(O)2N(R′)-. In some embodiments, the alkylene chain is interrupted and / or terminated (at either end or both ends) by -N(R′)-. In some embodiments, the alkylene chain is interrupted and / or terminated by -C(O)- (at either end or both ends). In some embodiments, the alkylene chain is interrupted and / or terminated by -C(O)O- (at either end or both ends). In some embodiments, the alkylene chain is interrupted and / or terminated by -C(O)N(R′)- (at either end or both ends). In some embodiments, the alkylene chain is interrupted and / or terminated by -N(R′)S(O)2- (at either end or both ends).

[0271] In some implementations, R1 is absent, and R1′ is a polyethylene glycol chain that can be substituted with -O-, -S-, --N(R′)-, -C≡C-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -C(NOR′)-, -C(O)N(R′)-, -C(O)N(R′)C(O)-, -R′C(O)N(R ′)R′-, -C(O)N(R′)C(O)N(R′)-, -N(R′)C(O)-, -N(R′)C(O)N(R′)-, -N(R′)C(O)O -, -OC(O)N(R′)-, -C(NR′)-, -N(R′)C(NR′)-, -C(NR′)N(R′)-, -N(R′)C(NR′)N(R ′)-, -OB(Me)O-, -S(O)2-, -OS(O)-, -S(O)O-, -S(O)-, -OS(O)2-, -S(O)2O-, -N( R′)S(O)2-, -S(O)2N(R′)-, --N(R′)S(O)-, -S(O)N(R′)-, -N(R′)S(O)2N(R′)-, – At least one or any combination of N(R′)S(O)N(R′)-, -OP(O)O(R′)O-, -N(R′)P(O)N(R′R′)N(R′)-, C3-C12 carbocyclic group, 3- to 12-membered heterocyclic group, 5- to 12-membered heteroaryl group, or any combination thereof, is interrupted and / or terminated (at either end or both ends), wherein each R′ is independently H or optionally substituted C1-C 24 Alkyl groups, wherein the interrupting group and one or two terminating groups may be the same or different.

[0272] In some embodiments, the polyethylene glycol chain has 1 to 20 -(CH2CH2-O)- units. In some embodiments, the polyethylene glycol chain has 1 to 15 -(CH2CH2-O)- units. In some embodiments, the polyethylene glycol chain has 1 to 10 -(CH2CH2-O)- units. In some embodiments, the polyethylene glycol chain has 1 to 5 -(CH2CH2-O)- units. In some embodiments, the polyethylene glycol chain has 1 to 2 -(CH2CH2-O)- units. In some embodiments, polyethylene glycol is interrupted and / or terminated (at either end or both ends) by at least one or a combination of -N(R′)-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(R′)-, -N(R′)C(O)-, -N(R′)C(O)O-, -OC(O)N(R′)-, -S(O)2-, -N(R′)S(O)2-, and -S(O)2N(R′)-. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either end or both ends) by -N(R′)-. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either end or both ends) by -C(O)-. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either end or both ends) by -C(O)O-. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated by -C(O)N(R′)- (at either end or both ends). In some embodiments, the polyethylene glycol chain is interrupted and / or terminated by -N(R′)S(O)2- (at either end or both ends).

[0273] In some embodiments, R1 and R2, together with the nitrogen atom to which they are attached, form a 3- to 16-membered heterocyclic group containing one to eight heteroatoms selected from N, O, and S. In some embodiments, R1 and R2, together with the nitrogen atom to which they are attached, form a 4- to 12-membered heterocyclic group containing one to four heteroatoms selected from N, O, and S. In some embodiments, R1 and R2, together with the nitrogen atom to which they are attached, form a 5- to 10-membered heterocyclic group containing one to three heteroatoms selected from N, O, and S. In some embodiments, R1 and R2, together with the nitrogen atom to which they are attached, form a 5- to 6-membered heterocyclic group containing one to two heteroatoms selected from N, O, and S. In some embodiments, R1 and R2, together with the nitrogen atom to which they are attached, form a piperazine group.

[0274] In some implementations, R1 does not exist, and R1′ is C1-C 24 The chain is alkylene, and R2 is methyl or benzyl. In some embodiments, R1 is absent, and R1′ is C1-C. 18 The chain is alkylene, and R2 is methyl or benzyl. In some embodiments, R1 is absent, and R1′ is C1-C. 12The chain is alkylene, and R2 is methyl or benzyl. In some embodiments, R1 is absent, and R1′ is C1-C. 10 In some embodiments, R1 is absent, R1′ is a C1-C8 alkylene chain, and R2 is methyl or benzyl. In some embodiments, R1 is absent, R1′ is a C1-C6 alkylene chain, and R2 is methyl or benzyl. In some embodiments, R1 is absent, R1′ is a C1-C4 alkylene chain, and R2 is methyl or benzyl. In some embodiments, R1 is absent, R1′ is a C1-C2 alkylene chain, and R2 is methyl or benzyl. In some embodiments, R1 is absent, R1′ is 1 to 20 -(CH2CH2-O)- units, and R2 is methyl or benzyl. In some embodiments, R1 is absent, R1′ is 1 to 15 -(CH2CH2-O)- units, and R2 is methyl or benzyl. In some embodiments, R1 is absent, R1′ is 1 to 10 -(CH2CH2-O)- units, and R2 is methyl or benzyl. In some embodiments, R1 is absent, R1′ is 1 to 5 -(CH2CH2-O)- units, and R2 is methyl or benzyl. In some embodiments, R1 is absent, R1′ is 1 to 2 -(CH2CH2-O)- units, and R2 is methyl or benzyl.

[0275] In some implementation schemes, A1' is moromarab-CD3, abciximab, rituximab, pallizumab, infliximab, trastuzumab, alenzab, adalimumab, tiimozumab, omalizumab, cetuximab, bevacizumab, natezumab, panitumumab, ranibizumab, ikulizumab, cetozumab, ustekinumab, canatumab, golimumab, Ophamumab, Tocilizumab, Denosumab, Belimumab, Ipilimumab, Bentuximab, Pertuximab, Raxicurumab, Ophamumab, Sestoxicurumab, Ramucirumab, Vedolizumab, Bonatumab, Nivolumab, Pembrolizumab, Idacilumab, Nexicurumab, Denocicurumab, Sestoxicurumab, Mepleribumab, Alilobumarab, Evodocin Anti-, Daremumab, Erotozumab, Isebemab, Relizumab, Olanzab, Belottosuzumab, Atezolizumab, Otosaximab, Ogaituzumab, Brodatumab, Gusekumab, Duprexumab, Thaliru, Averu, Omega, Emezabumab, Benalizumab, Getozumab, Dvalumab, Ibuprofen, Ranaru Monoclonal antibodies, including mogliflozin, elenocumab, ganezumab, tetrazolizumab, cimiprizumab, epavazumab, remannetumab, ibazine, mosetumab, revulizumab, cappserizumab, lomoxoluzumab, resalizumab, polotozumab, broxelizumab, lizanilizumab, cetozumab, belantazumab, or enrofloxacin, or fragments thereof. In some embodiments, A1′ is trastuzumab.

[0276] In some embodiments, the compound of formula (Ia′) is:

[0277]

[0278] Or its pharmaceutically acceptable salts or stereoisomers.

[0279] In some embodiments, the compound of formula (Ib′) is:

[0280] Or its pharmaceutically acceptable salts or stereoisomers.

[0281] In some embodiments, the compound of formula (Ic′) is:

[0282] Or its pharmaceutically acceptable salts or stereoisomers.

[0283] Other inventive compounds of this invention are represented by formulas (II) and (III):

[0284]

[0285] Or its pharmaceutically acceptable salts or stereoisomers.

[0286] in:

[0287] Each X is independently CR9R9′, NR9, O, S, C(O), S(O) or SO2, wherein the ring system contains 0 to 3 heteroatoms;

[0288] R9 and R9′ are independently hydrogen or substituents;

[0289] Y does not exist or

[0290] A2 is the active component;

[0291] R4 is hydrogen, a substituent, or a component bound to... Linking groups on the group, or

[0292] R4 and R5, together with the carbon atoms they are attached to, form a carbocyclic or heterocyclic group, wherein R4 is also bonded to... On the group;

[0293] R5 is hydrogen or an electron-withdrawing group;

[0294] R6 is hydrogen, a π-electron-donating group, or a group bound to hydrogen. Linking groups on the group;

[0295] R7 and R7′ are independently hydrogen or electron-withdrawing groups, or

[0296] R7 and R7', together with the nitrogen atoms they are attached to, form C(O);

[0297] R8 is hydrogen, a substituent, or bound to... Linking groups on the group; and

[0298] n is 1, 2, or 3.

[0299] The prerequisite is that equations II and III each contain one Group.

[0300] In some implementations, n is 2.

[0301] In some embodiments, X is CR9R9′. In some embodiments, R9 and R9′ are each hydrogen.

[0302] In some embodiments, R9 and R9' are independently hydrogen, (C1-C6)alkyl, (C1-C6)alkoxy, (C1-C6)haloalkyl, (C1-C6)haloalkoxy, -C(O)R 10 -NR 10 R 10 -C(O)NR 10 R 10 -OC(O)NR 10 R 10 -NR 10 C(O)R 10 -NR 10 C(O)OR 10 Halogen, OH, CN, amino, (C3-C) 10 ) carbocyclic group, 4- or 7-membered heterocyclic group, -O(CH2)O-3(C3-C 10 A carbocyclic group containing one to three heteroatoms selected from O, N, and S -O(CH2). 0-3 -4- or 7-membered heterocyclic groups, where each R 10 Independently, it is hydrogen or (C1-C6) alkyl; wherein the alkyl, carbocyclic or heterocyclic group is further optionally substituted.

[0303] In some implementations, R4 is incorporated into A linking group on the group. In some embodiments, R4 is O. In some embodiments, R4 is S. In some embodiments, R4 is NR. 11 , where R 11 It is hydrogen or (C1-C6) alkyl. In some embodiments, R4 is OPh. In some embodiments, R4 is OC(O). In some embodiments, R4 is OC(O)NR. 11 R11 is hydrogen or (C1-C6) alkyl.

[0304] In some embodiments, R4 is an alkylene chain that can be -O-, -S-, -N(R′)-, -C≡C-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -C(NOR′)-, -C(O)N(R′)C(O)-, -R′C(O)N(R′)R′-, -C(O)N(R′)C(O)N(R′)-, -N(R′)C(O)N(R′)-, -N(R′)C(O)N(R′)-, -N(R′)C(O)O-, -OC(O)N(R′)-, -C(NR′)-, -N(R′)C(N R′)-, -C(NR′)N(R′)-, -N(R′)C(NR′)N(R′)-, -OB(Me)O-, -S(O)2-, -OS(O)-, -S(O)O-, -S(O)-, -OS(O)2-, -S(O)2O-, -N(R′)S(O)2-, -S (O)2N(R′)-, -N(R′)S(O)-, -S(O)N(R′)-, -N(R′)S(O)2N(R′)-, -N(R′)S(O)N(R′)-, -OP(O)O(R′)O-, -N(R′)P(O)N(RμR′)N(R′)-, C3-C 12 At least one of carbocyclic, 3- to 12-membered heterocyclic, 5- to 12-membered heteroaryl groups, or any combination thereof, is interrupted and / or terminated (at either end or both ends), wherein each R′ is independently H or optionally substituted C1-C. 24 Alkyl groups, wherein the interrupting group and one or two terminating groups may be the same or different.

[0305] In some implementations, the alkylene chain is C1-C. 24 Alkylene chain. In some embodiments, the alkylene chain is C1-C2. 18 Alkylene chain. In some embodiments, the alkylene chain is C1-C2. 12 Alkylene chain. In some embodiments, the alkylene chain is C1-C2. 10Alkylene chain. In some embodiments, the alkylene chain is a C1-C8 alkylene chain. In some embodiments, the alkylene chain is a C1-C6 alkylene chain. In some embodiments, the alkylene chain is a C1-C4 alkylene chain. In some embodiments, the alkylene chain is a C1-C2 alkylene chain. In some embodiments, the alkylene chain is interrupted and / or terminated (at either end or both ends) by at least one or a combination of -N(R′)-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(R′)-, -N(R′)C(O)-, -OC(O)N(R′)-, -S(O)2-, -N(R′)S(O)2-, and -S(O)2N(R′)-. In some embodiments, the alkylene chain is interrupted and / or terminated (at either end or both ends) by -N(R′)-. In some embodiments, the alkylene chain is interrupted and / or terminated by -C(O)- (at either end or both ends). In some embodiments, the alkylene chain is interrupted and / or terminated by -C(O)O- (at either end or both ends). In some embodiments, the alkylene chain is interrupted and / or terminated by -C(O)N(R′)- (at either end or both ends). In some embodiments, the alkylene chain is interrupted and / or terminated by -N(R′)S(O)2- (at either end or both ends).

[0306] In some implementations, R4 is a polyethylene glycol chain that can be -O-, -S-, -N(R′)-, -C≡C-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -C(NOR′)-, -C(O)N(R′)-, -C(O)N(R′)C(O)-, -R′C(O)N(R′)R′-, -C(O)N(R′)C(O)N(R′)-, -N(R′)C(O)N(R′)-, -N(R′)C(O)O-, -OC(O)N(R′)-, -C(NR′)-, -N(R′)C( NR′)-, -C(NR′)N(R′)-, -N(R′)C(NR′)N(R′)-, -OB(Me)O-, -S(O)2-, -OS(O)-, -S(O)O-, -S(O)-, -OS(O)2-, -S(O)2O-, -N(R′)S(O)2-, - S(O)2N(R′)-, -N(R′)S(O)-, -S(O)N(R′)-, -N(R′)S(O)2N(R′)-, -N(R′)S(O)N(R′)-, -OP(O)O(R′)O-, -N(R′)P(O)N(R′R′)N(R′)-, C3-C 12At least one of carbocyclic, 3- to 12-membered heterocyclic, 5- to 12-membered heteroaryl groups, or any combination thereof, is interrupted and / or terminated (at either end or both ends), wherein each R′ is independently H or optionally substituted C1-C. 24 Alkyl groups, wherein the interrupting group and one or two terminating groups may be the same or different.

[0307] In some embodiments, the polyethylene glycol chain has 1 to 20 -(CH2CH2-O)- units. In some embodiments, the polyethylene glycol chain has 1 to 15 -(CH2CH2-O)- units. In some embodiments, the polyethylene glycol chain has 1 to 10 -(CH2CH2-O)- units. In some embodiments, the polyethylene glycol chain has 1 to 5 -(CH2CH2-O)- units. In some embodiments, the polyethylene glycol chain has 1 to 2 -(CH2CH2-O)- units. In some embodiments, polyethylene glycol is interrupted and / or terminated (at either end or both ends) by at least one or a combination of -N(R′)-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(R′)-, -N(R′)C(O)-, -N(R′)C(O)O-, -OC(O)N(R′)-, -S(O)2-, -N(R′)S(O)2-, and -S(O)2N(R′)-. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either end or both ends) by -N(R′)-. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either end or both ends) by -C(O)-. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either end or both ends) by -C(O)O-. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated by -C(O)N(R′)- (at either end or both ends). In some embodiments, the polyethylene glycol chain is interrupted and / or terminated by -N(R′)S(O)2- (at either end or both ends).

[0308] In some embodiments, R4 and R5, together with the carbon atoms they are attached to, form a 3- to 16-membered carbocyclic group or a 3- to 16-membered heterocyclic group containing one to eight heteroatoms selected from N, O, and S. In some embodiments, R4 and R5, together with the carbon atoms they are attached to, form a 4- to 12-membered carbocyclic group or a 4- to 12-membered heterocyclic group containing one to four heteroatoms selected from N, O, and S. In some embodiments, R4 and R5, together with the carbon atoms they are attached to, form a 5- to 10-membered carbocyclic group or a 5- to 10-membered heterocyclic group containing one to three heteroatoms selected from N, O, and S. In some embodiments, R4 and R5, together with the carbon atoms they are attached to, form a 5- to 6-membered carbocyclic group or a 5- to 6-membered heterocyclic group containing one to two heteroatoms selected from N, O, and S.

[0309] In some implementations, R4 and R5, together with the carbon atoms to which they are attached, form a 5-membered heterocyclic group containing a 2-oxygen atom.

[0310] In some implementations, R5 is hydrogen.

[0311] In some implementations, R5 is an electron-withdrawing group.

[0312] In some embodiments, R5 is an electron-withdrawing group. In some embodiments, the electron-withdrawing group is a halogen, OR 5′ SR 5′ or NR 5′ R 5′ , where each R 5′ Independently hydrogen, C1-C6 alkyl, C6-C 12 Aryl, 5- to 10-membered heteroaryl, carbonyl, sulfonyl, sulfinyl or phosphoryl.

[0313] In some embodiments, R5 is a π-electron-withdrawing group. In some embodiments, the π-electron-withdrawing group is -C(O)R. 5” -C(O)NR 5” R 5” -C(O)NR 5” R 5” -C(O)OR 5” NO2, CN, N3, -S(O)R 5” -S(O)2R 5” -S(O)OR 5” -S(O)2OR 5” -S(O)NR 5” R 5” ,-S(O)2NR5”R5”,-OP(O)OR 5” OR 5” -P(O)NR 5” R 5” NR 5” R 5” Each R5″ is independently hydrogen, C1-C6 alkyl, C6-C 12 Aryl, 5- to 10-membered heteroaryl.

[0314] In some implementations, R6 is hydrogen.

[0315] In some implementations, R6 is a π-electron-donating group.

[0316] In some implementations, R6 is OR 12 SR 12 NR 12 NR 12 Either cyclic amides or acyclic amides, wherein each R12 Independently hydrogen, (C1-C6)alkyl, (C3-C 10 ) carbocyclic, 4-membered or 7-membered heterocyclic group, wherein the alkyl, carbocyclic or heterocyclic group is optionally substituted.

[0317] In some embodiments, R7 and R7′ are independently hydrogen or electron-withdrawing groups. In some embodiments, the electron-withdrawing groups are halogens, OR... 5′ SR 5′ or NR 5′ R 5′ , where each R 5′ Independently hydrogen, C1-C6 alkyl, C6-C 12 Aryl, 5- to 10-membered heteroaryl, carbonyl, sulfonyl, sulfinyl or phosphoryl.

[0318] In some implementations, R7 and R 7′ It can be either hydrogen or a π-electron-withdrawing group. In some embodiments, the π-electron-withdrawing group is -C(O)R. 5” -C(O)NR 5” R 5” -C(O)NR 5” R 5” -C(O)OR 5” NO2, CN, N3, -S(O)R 5” -S(O)2R 5” -S(O)OR 5” -S(O)2OR 5” -S(O)NR 5” R 5” -S(O)2NR 5” R 5” -OP(O)OR 5” OR 5” -P(O)NR 5” R 5” NR 5” R 5” , where each R 5″ It is an independent hydrogen, C1-C6 alkyl, C6-C 12 Aryl, 5- to 10-membered heteroaryl.

[0319] In some implementations, R8 is incorporated into A linking group on the group. In some embodiments, R8 is CH2. In some embodiments, R8 is C6-C. 12 Aryl or 5- to 10-membered heteroaryl. In some embodiments, R8 is O.

[0320] In some embodiments, R8 is an alkylene chain that can be -O-, -S-, -N(R′)-, -C≡C-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -C(NOR′)-, -C(O)N(R′)C(O)-, -R′C(O)N(R′)R′-, -C(O)N(R′)C(O)N(R′)-, -N(R′)C(O)N(R′)-, -N(R′)C(O)N(R′)-, -N(R′)C(O)O-, -OC(O)N(R′)-, -C(NR′)-, -N(R′)C(NR) ′)-, -C(NR′)N(R′)-, -N(R′)C(NR′)N(R′)-, -OB(Me)O-, -S(O)2-, -OS(O)-, -S(O)O-, -S(O)-, -OS(O)2-, -S(O)2O-, -N(R′)S(O)2-, -S( O)2N(R′)-, -N(R′)S(O)-, -S(O)N(R′)-, -N(R′)S(O)2N(R′)-, --N(R′)S(O)N(R′)-, -OP(O)O(R′)O-, -N(R′)P(O)N(R′R′)N(R′)-, C3-C 12 At least one of carbocyclic, 3- to 12-membered heterocyclic, 5- to 12-membered heteroaryl groups, or any combination thereof, is interrupted and / or terminated (at either end or both ends), wherein each R′ is independently H or optionally substituted C1-C. 24 Alkyl groups, wherein the interrupting group and one or two terminating groups may be the same or different.

[0321] In some implementations, the alkylene chain is C1-C. 24 Alkylene chain. In some embodiments, the alkylene chain is C1-C2. 18 Alkylene chain. In some embodiments, the alkylene chain is C1-C2. 12 Alkylene chain. In some embodiments, the alkylene chain is C1-C2. 10Alkylene chain. In some embodiments, the alkylene chain is a C1-C8 alkylene chain. In some embodiments, the alkylene chain is a C1-C6 alkylene chain. In some embodiments, the alkylene chain is a C1-C4 alkylene chain. In some embodiments, the alkylene chain is a C1-C2 alkylene chain. In some embodiments, the alkylene chain is interrupted and / or terminated (at either end or both ends) by at least one or a combination of -N(R′)-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(R′)-, -N(R′)C(O)-, -OC(O)N(R′)-, -S(O)2-, -N(R′)S(O)2-, and -S(O)2N(R′)-. In some embodiments, the alkylene chain is interrupted and / or terminated (at either end or both ends) by -N(R′)-. In some embodiments, the alkylene chain is interrupted and / or terminated by -C(O)- (at either end or both ends). In some embodiments, the alkylene chain is interrupted and / or terminated by -C(O)O- (at either end or both ends). In some embodiments, the alkylene chain is interrupted and / or terminated by -C(O)N(R′)- (at either end or both ends). In some embodiments, the alkylene chain is interrupted and / or terminated by -N(R′)S(O)2- (at either end or both ends).

[0322] In some implementations, R8 is a polyethylene glycol chain that can be -O-, -S-, -N(R′)-, -C≡C-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -C(NOR′)-, -C(O)N(R′)-, -C(O)N(R′)C(O)-, -R′C(O)N(R′)R′-, -C(O)N(R′)C(O)N(R′)-, -N(R′)C(O)N(R′)-, -N(R′)C(O)O-, -OC(O)N(R′)-, -C(NR′)-, -N(R′)C(N R′)-, -C(NR′)N(R′)-, -N(R′)C(NR′)N(R′)-, -OB(Me)O-, -S(O)2-, -OS(O)-, -S(O)O-, -S(O)-, -OS(O)2-, -S(O)2O-, -N(R′)S(O)2-, -S (O)2N(R′)-, -N(R′)S(O)-, -S(O)N(R′)-, -N(R′)S(O)2N(R′)-, --N(R′)S(O)N(R′)-, -OP(O)O(R′)O-, -N(R′)P(O)N(R′R′)N(R′)-, C3-C 12At least one of carbocyclic, 3- to 12-membered heterocyclic, 5- to 12-membered heteroaryl groups, or any combination thereof, is interrupted and / or terminated (at either end or both ends), wherein each R′ is independently H or optionally substituted C1-C. 24 Alkyl groups, wherein the interrupting group and one or two terminating groups may be the same or different.

[0323] In some embodiments, the polyethylene glycol chain has 1 to 20 -(CH2CH2-O)- units. In some embodiments, the polyethylene glycol chain has 1 to 15 -(CH2CH2-O)- units. In some embodiments, the polyethylene glycol chain has 1 to 10 -(CH2CH2-O)- units. In some embodiments, the polyethylene glycol chain has 1 to 5 -(CH2CH2-O)- units. In some embodiments, the polyethylene glycol chain has 1 to 2 -(CH2CH2-O)- units. In some embodiments, polyethylene glycol is interrupted and / or terminated (at either end or both ends) by at least one or a combination of -N(R′)-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(R′)-, -N(R′)C(O)-, -N(R′)C(O)O-, -OC(O)N(R′)-, -S(O)2-, --N(R′)S(O)2-, and -S(O)2N(R′)-. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either end or both ends) by -N(R′)-. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either end or both ends) by -C(O)-. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either end or both ends) by -C(O)O-. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated by -C(O)N(R′)- (at either end or both ends). In some embodiments, the polyethylene glycol chain is interrupted and / or terminated by -N(R′)S(O)2- (at either end or both ends).

[0324] Part A2 is the active part, and its definition is the same as that of A1 for the compound of formula (I) above.

[0325] In some embodiments, compounds of formula (II) are represented by compounds of formula (IIa): (IIa), or a pharmaceutically acceptable salt or stereoisomer thereof. In some embodiments, R4 is O, S, NR. 11 OPh, OC(O), OC(O)NR 11 , where R 11 It is hydrogen or (C1-C6) alkyl, optionally substituted alkylene chains, or optionally substituted polyethylene glycol chains; and / or R5 is hydrogen, fluorine, or OR 5′ OR 5′It is hydrogen or (C1-C6) alkyl; and / or A2 is a binding part, a therapeutic part, or a diagnostic part.

[0326] In some implementations, the compound of formula (II) is

[0327] Or its pharmaceutically acceptable salts or stereoisomers.

[0328] In some embodiments, the compound of formula (II′) is represented by the compound of formula (II′):

[0329]

[0330] Or its pharmaceutically acceptable salts or stereoisomers.

[0331] in:

[0332] Each X is independently a CR9R 9′ NR9, O, S, C(O), S(O) or SO2, wherein the ring system contains 0 to 3 heteroatoms;

[0333] R9 and R 9′ It can be hydrogen or a substituent independently;

[0334] R4 is a linking group;

[0335] A2′ is a therapeutic small molecule; and

[0336] n is 1, 2, or 3.

[0337] In some implementations, X is CR9R 9′ In some embodiments, R9 and R9′ are each hydrogen. In some embodiments, R9 and R9′ are independently hydrogen, (C1-C6)alkyl, (C1-C6)alkoxy, (C1-C6)haloalkyl, (C1-C6)haloalkoxy, -C(O)R 10 -NR 10 R 10 -C(O)NR 10 R 10 -OC(O)NR 10 R 10 -NR 10 C(O)R 10 -NR 10 C(O)OR 10 Halogen, OH, CN, amino, (C3-C) 10 Carbocyclic groups, 4- or 7-membered heterocyclic groups, -O(CH2) 0-3 (C3-C 10A carbocyclic group containing one to three heteroatoms selected from O, N, and S -O(CH2). 0-3 -4- or 7-membered heterocyclic groups, where each R 10 Independently, it is hydrogen or (C1-C6) alkyl; wherein the alkyl, carbocyclic or heterocyclic group is further optionally substituted.

[0338] In some implementations, R4 is O, S, NR 10 OC(O), NR 10 C(O) or OC(O)NR5, where R 10 It is hydrogen or C1-C6 alkyl.

[0339] In some embodiments, R4 is an alkylene chain that can be -O-, -S-, -N(R′)-, -C-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -C(NOR′)-, -C(O)N(R′)C(O)-, -R′C(O)N(R′)R′-, -C(O)N(R′)C(O)N(R′)-, -N(R′)C(O)N(R′)-, -N(R′)C(O)N(R′)-, -N(R′)C(O)O-, -OC(O)N(R′)-, -C(NR′)-, -N(R′)C(N R′)-, -C(NR′)N(R′)-, -N(R′)C(NR′)N(R′)-, -OB(Me)O-, -S(O)2-, -OS(O)-, -S(O)O-, -S(O)-, -OS(O)2-, -S(O)2O-, -N(R′)S(O)2-, -S (O)2N(R′)-, -N(R′)S(O)-, -S(O)N(R′)-, -N(R′)S(O)2N(R′)-, --N(R′)S(O)N(R′)-, -OP(O)O(R′)O-, -N(R′)P(O)N(R′R′)N(R′)-, C3-C 12 At least one of carbocyclic, 3- to 12-membered heterocyclic, 5- to 12-membered heteroaryl groups, or any combination thereof, is interrupted and / or terminated (at either end or both ends), wherein each R′ is independently H or optionally substituted C1-C. 24 Alkyl groups, wherein the interrupting group and one or two terminating groups may be the same or different.

[0340] In some implementations, the alkylene chain is C1-C. 24 Alkylene chain. In some embodiments, the alkylene chain is C1-C2. 18 Alkylene chain. In some embodiments, the alkylene chain is C1-C2. 12 Alkylene chain. In some embodiments, the alkylene chain is C1-C2. 10Alkylene chain. In some embodiments, the alkylene chain is a C1-C8 alkylene chain. In some embodiments, the alkylene chain is a C1-C6 alkylene chain. In some embodiments, the alkylene chain is a C1-C4 alkylene chain. In some embodiments, the alkylene chain is a C1-C2 alkylene chain. In some embodiments, the alkylene chain is interrupted and / or terminated (at either end or both ends) by at least one or a combination of -N(R′)-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(R′)-, -N(R′)C(O)-, -OC(O)N(R′)-, -S(O)2-, -N(R′)S(O)2-, and -S(O)2N(R′)-. In some embodiments, the alkylene chain is interrupted and / or terminated (at either end or both ends) by -N(R′)-. In some embodiments, the alkylene chain is interrupted and / or terminated by -C(O)- (at either end or both ends). In some embodiments, the alkylene chain is interrupted and / or terminated by -C(O)O- (at either end or both ends). In some embodiments, the alkylene chain is interrupted and / or terminated by -C(O)N(R′)- (at either end or both ends). In some embodiments, the alkylene chain is interrupted and / or terminated by -N(R′)S(O)2- (at either end or both ends).

[0341] In some implementations, R4 is a polyethylene glycol chain that can be substituted with -O-, -S-, -N(R′)-, -C-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -C(NOR′)-, -C(O)N(R′)-, -C(O)N(R′)C(O)-, -R′C(O)N(R′)R′-, -C(O)N(R′)C(O)N(R′)-, -N(R′)C(O)N(R′)-, -N(R′)C(O)N(R′)-, -N(R′)C(O)O-, -OC(O)N(R′)-, -C(NR′)-, -N(R′)C(N R′)-, -C(NR′)N(R′)-, -N(R′)C(NR′)N(R′)-, -OB(Me)O-, -S(O)2-, -OS(O)-, -S(O)O-, -S(O)-, -OS(O)2-, -S(O)2O-, -N(R′)S(O)2-, -S (O)2N(R′)-, -N(R′)S(O)-, -S(O)N(R′)-, -N(R′)S(O)2N(R′)-, --N(R′)S(O)N(R′)-, -OP(O)O(R′)O-, -N(R′)P(O)N(R′R′)N(R′)-, C3-C 12At least one of carbocyclic, 3- to 12-membered heterocyclic, 5- to 12-membered heteroaryl groups, or any combination thereof, is interrupted and / or terminated (at either end or both ends), wherein each R′ is independently H or optionally substituted C1-C. 24 Alkyl groups, wherein the interrupting group and one or two terminating groups may be the same or different.

[0342] In some embodiments, the polyethylene glycol chain has 1 to 20 -(CH2CH2-O)- units. In some embodiments, the polyethylene glycol chain has 1 to 15 -(CH2CH2-O)- units. In some embodiments, the polyethylene glycol chain has 1 to 10 -(CH2CH2-O)- units. In some embodiments, the polyethylene glycol chain has 1 to 5 -(CH2CH2-O)- units. In some embodiments, the polyethylene glycol chain has 1 to 2 -(CH2CH2-O)- units. In some embodiments, polyethylene glycol is interrupted and / or terminated (at either end or both ends) by at least one or a combination of -N(R′)-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(R′)-, -N(R′)C(O)-, -N(R′)C(O)O-, -OC(O)N(R′)-, -S(O)2-, -N(R′)S(O)2-, and -S(O)2N(R′)-. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either end or both ends) by -N(R′)-. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either end or both ends) by -C(O)-. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either end or both ends) by -C(O)O-. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated by -C(O)N(R′)- (at either end or both ends). In some embodiments, the polyethylene glycol chain is interrupted and / or terminated by -N(R′)S(O)2- (at either end or both ends).

[0343] In some implementations, n is 2. In some implementations, n is 2 and each X is CH2, and the structure represented by equation II′a is:

[0344] Or its pharmaceutically acceptable salts or stereoisomers.

[0345] In some embodiments, A2' is an anticancer agent. In some embodiments, A2' is aurestatin, maytansine, scutellarin, anthracyclines, paclitaxel or docetaxel or derivatives thereof, calcitrazine or derivatives thereof, pyrrolobenzodiazepine dimer (PBD) or derivatives thereof, pyromycin or derivatives thereof, eribulin or derivatives thereof, camptothecin or derivatives thereof, or eczema or derivatives thereof.

[0346] Representative examples of aurateurines include dolastatin 10- Monomethylaurestatin E (MMAE) Monomethylaurestatin F (MMAF)- PF-06380101- and atorvastatin-OMe-

[0347] Representative examples of maytansine include maytansine- DM1- DM3- and DM4-

[0348] Representative examples of the tubulosin class include tubulosin A- Microtubulesin B- Microtubulesin C- Microtubulesin G- Microtubulesin I- Where R1 is CH2CH(CH3)2, CH2CH2CH3, CH2CH3, CH=C(CH3)2, or CH3; and

[0349] R2 is

[0350] Representative examples of anthracyclines include doxorubicin. PNU-159682- And Ladirubi Star -

[0351] For example, as mentioned above, suitable sites for conjugation to anticancer agents are readily identified by those skilled in the art and are described elsewhere in the literature. See Kostova et al., Pharmaceuticals, 14:442 (2021).

[0352] In some embodiments, the compound of formula (IIa′) is:

[0353] Or its pharmaceutically acceptable salts or stereoisomers.

[0354] In some embodiments, compounds of formula (III) are represented by compounds of formula (IIIa):

[0355]

[0356] Or a pharmaceutically acceptable salt or stereoisomer thereof. In some embodiments, R6 is hydrogen, chlorine, bromine, iodine, OR 12 or SR 12 , where each R 12 Independently hydrogen, (C1-C6)alkyl, (C3-C 10 R7 is a carbocyclic group, a 4- or 7-membered heterocyclic group; and / or R7 is hydrogen, fluorine, or OR 5′ , where R 5′ It is hydrogen or (C1-C6) alkyl; and / or R 7′ Is it hydrogen, fluorine, or OR? 5′ , where R 5′ It is hydrogen or (C1-C6) alkyl; and R8 is CH2, O, or C6-C. 12 A1 is an aryl group, a 5- to 10-membered heteroaryl group, an optionally substituted alkylene chain, or an optionally substituted polyethylene glycol chain; and / or A2 is a binding moiety, a therapeutic moiety, or a diagnostic moiety.

[0357] In some implementations, the compound of formula (III) is Or its pharmaceutically acceptable salts or stereoisomers.

[0358] In some embodiments, the optional substituents of the compound of formula (II) or (III) are selected from the group consisting of: alkyl, alkenyl, alkynyl, halogen, haloalkyl, cycloalkyl, heterocycloalkyl, hydroxyl, alkoxy, cycloalkoxy, heterocycloalkoxy, haloalkoxy, aryloxy, heteroaryloxy, arylalkyloxy, alkynoxy, alkynoxy, amino, alkylamino, cycloalkylamino, heterocycloalkylamino, arylamino, heteroarylamino, arylalkylamino, N-alkyl-N-arylamino, N-alkyl-N-heteroarylamino, N-alkyl-N-arylalkylamino, hydroxyalkyl, aminoalkyl, alkylthio, haloalkylthio, alkylsulfonyl, haloalkylsulfonyl, cycloalkylsulfonyl, heterocycloalkylsulfonyl, arylsulfonyl, heteroarylsulfonyl, aminosulfonyl, alkylaminosulfonyl, cycloalkylaminosulfonyl, heterocycloalkylaminosulfonyl, aryl Aminosulfonyl, heteroarylaminosulfonyl, N-alkyl-N-arylaminosulfonyl, N-alkyl-N-heteroarylaminosulfonyl, formyl, alkyl carbonyl, haloalkyl carbonyl, alkenyl carbonyl, alkynyl carbonyl, carboxyl, alkoxy carbonyl, alkyl carbonyloxy, amino, alkylsulfonylamino, haloalkylsulfonylamino, cycloalkylsulfonylamino, heterocyclic alkylsulfonylamino, arylsulfonylamino, heteroarylsulfonylamino, aralkylsulfonylamino, alkylcarbonylamino, haloalkylcarbonylamino, cycloalkylcarbonylamino, heterocyclic alkylcarbonylamino, arylcarbonylamino, heteroarylcarbonylamino, aralkylsulfonylamino, aminocarbonyl, alkylaminocarbonyl, cycloalkylaminocarbonyl, heterocyclic alkylaminocarbonyl, arylaminocarbonyl, heteroarylaminocarbonyl, N-alkyl-N-arylaminocarbonyl, N-alkyl-N-heteroarylaminocarbonyl, cyano, nitro, and azide.

[0359] However, other inventive compounds are represented by formulas (IV) and (V):

[0360]

[0361] Or its pharmaceutically acceptable salts or stereoisomers.

[0362] in:

[0363] R1′ is a linking group;

[0364] R1 does not exist, or

[0365] R1 and R2, together with the nitrogen atoms they are attached to, form a heterocyclic group;

[0366] R2 is an optionally substituted (C1-C8) alkyl group, -

[0367] C(O)R”, -C(O)OR”, -C(O)NR”R”, -S(O)R”, -S(O)2R”, (C3-C 10A carbocyclic, 4- or 7-membered heterocyclic or substituted polyethylene glycol chain, wherein each R” is independently hydrogen, (C1-C6)alkyl, (C3-C6)alkyl, or substituted. 10 ) carbocyclic, 4-membered or 7-membered heterocyclic group, and wherein said alkyl, carbocyclic or heterocyclic group is optionally substituted; and

[0368] Each X is independently CR9R9′, NR9, O, S, C(O), S(O) or SO2, wherein the ring system contains 0 to 3 heteroatoms;

[0369] R9 and R9′ are independently hydrogen or substituents;

[0370] A1 is the active part of the compound of formula (I) as described above;

[0371] Y does not exist or

[0372] A2 is the active portion relative to A1, as described above;

[0373] R4 is hydrogen, a substituent, or a component bound to... Linking groups on the group, or

[0374] R4 and R5, along with the carbon atoms they are attached to, form carbocyclic or heterocyclic groups, where R4 also binds to... On the group;

[0375] R5 is hydrogen or an electron-withdrawing group;

[0376] R6 is hydrogen, a π-electron-donating group, or a group bound to hydrogen. Linking groups on the group;

[0377] R7 and R7′ are independently hydrogen or electron-withdrawing groups, or

[0378] R7 and R7', together with the carbon atoms they are attached to, form C(O);

[0379] R8 is hydrogen, a substituent, or bound to... Linking groups on groups; and

[0380] n is 1, 2, or 3;

[0381] The prerequisite is that each of the compounds of formulas (IV) and (V) contains at least one Group.

[0382] In some embodiments, R1 is absent, and R1′ is an alkylene chain that can be -O-, -S-, -N(R′)-, -C≡C-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -C(NOR′)-, -C(O)N(R′)-, -C(O)N(R′)C(O)-, -R′C(O)N(R′)R′-, -C(O)N(R′)C(O)N(R′)-, -N(R′)C(O)N(R′)-, -N(R′)C(O)O-, -OC(O)N(R′)-, -C(NR′)-, -N(R′)- )C(NR′)-, -C(NR′)N(R′)-, -N(R′)C(NR′)N(R′)-, -OB(Me)O-, -S(O)2-, -OS(O)-, -S(O)O-, -S(O)-, -OS(O)2-, -S(O)2O-, -N(R′)S(O)2-, -S(O)2N(R′)-, --N(R′)S(O)-, -S(O)N(R′)-, -N(R′)S(O)2N(R′)-, -N(R′)S(O)N(R′)-, -OP(O)O(R′)O-, -N(R′)P(O)N(R′R′)N(R′)-, C3-C 12 At least one of carbocyclic, 3- to 12-membered heterocyclic, 5- to 12-membered heteroaryl groups, or any combination thereof, is interrupted and / or terminated (at either end or both ends), wherein each R′ is independently H or optionally substituted C1-C. 24 Alkyl groups, wherein the interrupting group and one or two terminating groups may be the same or different.

[0383] In some implementations, the alkylene chain is C1-C. 24 Alkylene chain. In some embodiments, the alkylene chain is C1-C2. 18 Alkylene chain. In some embodiments, the alkylene chain is C1-C2. 12 Alkylene chain. In some embodiments, the alkylene chain is C1-C2. 10Alkylene chain. In some embodiments, the alkylene chain is a C1-C8 alkylene chain. In some embodiments, the alkylene chain is a C1-C6 alkylene chain. In some embodiments, the alkylene chain is a C1-C4 alkylene chain. In some embodiments, the alkylene chain is a C1-C2 alkylene chain. In some embodiments, the alkylene chain is interrupted and / or terminated (at either end or both ends) by at least one or a combination of -N(R′)-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(R′)-, -N(R′)C(O)-, --N(R′)C(O)O-, -OC(O)N(R′)-, -S(O)2-, -N(R′)S(O)2-, -S(O)2N(R′)-, 4- to 6-membered heterocyclic groups. In some embodiments, the alkylene chain is interrupted and / or terminated (at either end or both ends) by -N(R′)-. In some embodiments, the alkylene chain is interrupted and / or terminated by -C(O)- (at either end or both ends). In some embodiments, the alkylene chain is interrupted and / or terminated by -C(O)O- (at either end or both ends). In some embodiments, the alkylene chain is interrupted and / or terminated by -C(O)N(R′)- (at either end or both ends). In some embodiments, the alkylene chain is interrupted and / or terminated by -N(R′)S(O)2- (at either end or both ends). In some embodiments, the alkylene chain is interrupted and / or terminated by a 4- to 6-membered heterocyclic group (at either end or both ends). In some embodiments, the alkylene chain is pyrrolidine-2,5-dione. termination.

[0384] In some implementations, R1 is absent, and R1′ is a polyethylene glycol chain that can be -O-, -S-, --N(R′)-, -C≡C-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -C(NOR′)-, -C(O)N(R′)-, -C(O)N(R′)C(O)-, -R′C(O)N(R′)R′, -C(O)N(R′)C(O)N(R′)-, -N(R′)C(O)N(R′)-, -N(R′)C(O)O-, -OC(O)N(R′)-, -C(NR′)-, -- ...N(R′)-, -C(NR′)-, --N(R′)C(O)N(R′)-, -N(R′)C(O)N(R′)-, -N(R′)C(O)N(R′)-, -C(NR′)-, -N(R′)C(O)C(R′)C(O)N(R′)-, -C(NR′)C(R′)C(O)C(R′)C(O)N(R′)-, -C(NR′)C(R′)C(O)C(R′)C(O)C(R′)C(O)C(R′)C(O)C(R′)C(O)C(R′)C(O)C(R′)C(O)C(R′)C(O)C(R′)C(O)C(R′)C(O)C(R′)C(O ′)C(NR′)-, -C(NR′)N(R′)-, -N(R′)C(NR′)N(R′)-, -OB(Me)O-, -S(O)2-, -OS(O)-, -S(O)O-, -S(O)-, -OS(O)2-, -S(O)2O-, -N(R′)S(O)2- , -S(O)2N(R′)-, -N(R′)S(O)-, -S(O)N(R′)-, -N(R′)S(O)2N(R′)-, -N(R′)S(O)N(R′)-, -OP(O)O(R′)O-, -N(R′)P(O)N(R′R′)N(R′)-, C3-C 12 At least one of carbocyclic, 3- to 12-membered heterocyclic, 5- to 12-membered heteroaryl groups, or any combination thereof, is interrupted and / or terminated (at either end or both ends), wherein each R′ is independently H or optionally substituted C1-C. 24 Alkyl groups, wherein the interrupting group and one or two terminating groups may be the same or different.

[0385] In some embodiments, the polyethylene glycol chain has 1 to 20 -(CH2CH2-O) units. In some embodiments, the polyethylene glycol chain has 1 to 15 -(CH2CH2-O) units. In some embodiments, the polyethylene glycol chain has 1 to 10 -(CH2CH2-O) units. In some embodiments, the polyethylene glycol chain has 1 to 5 -(CH2CH2-O) units. In some embodiments, the polyethylene glycol chain has 1 to 2 -(CH2CH2-O) units. In some embodiments, polyethylene glycol is interrupted and / or terminated (at either end or both ends) by at least one or a combination of -N(R′)-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(R′)-, -N(R′)C(O)-, -N(R′)C(O)O-, -OC(O)N(R′)-, -S(O)2-, --N(R′)S(O)2-, -S(O)2N(R′)-, 4- to 6-membered heterocyclic groups. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either end or both ends) by -N(R′)-. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either end or both ends) by -C(O)-. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either end or both ends) by -C(O)O-. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated by -C(O)N(R′)- (at either end or both ends). In some embodiments, the polyethylene glycol chain is interrupted and / or terminated by -N(R′)S(O)2- (at either end or both ends). In some embodiments, the polyethylene glycol chain is interrupted and / or terminated by a 4- to 6-membered heterocyclic group (at either end or both ends). In some embodiments, the polyethylene glycol chain is pyrrolidine-2,5-dione. termination.

[0386] In some embodiments, R1 and R2, together with the nitrogen atom to which they are attached, form a 3- to 16-membered heterocyclic group containing 1 to 8 heteroatoms selected from N, O, and S. In some embodiments, R1 and R2, together with the nitrogen atom to which they are attached, form a 4- to 12-membered heterocyclic group containing 1 to 4 heteroatoms selected from N, O, and S. In some embodiments, R1 and R2, together with the nitrogen atom to which they are attached, form a 5- to 10-membered heterocyclic group containing 1 to 3 heteroatoms selected from N, O, and S. In some embodiments, R1 and R2, together with the nitrogen atom to which they are attached, form a 5- to 6-membered heterocyclic group containing 1 to 2 heteroatoms selected from N, O, and S.

[0387] In some implementations, R2 is methyl, ethyl, isopropyl, or tert-butyl.

[0388] In some implementations, R1 is C1-C 24The alkylene chain, R2 is methyl, ethyl, isopropyl, or tert-butyl. In some embodiments, R1 is C1-C. 18 The alkylene chain, and R2 are methyl, ethyl, isopropyl, or tert-butyl. In some embodiments, R1 is C1-C. 12 The alkylene chain, and R2 are methyl, ethyl, isopropyl, or tert-butyl. In some embodiments, R1 is C1-C. 10 The alkylene chain, and R2, are methyl, ethyl, isopropyl, or tert-butyl. In some embodiments, R1 is a C1-C8 alkylene chain, and R2 is methyl, ethyl, isopropyl, or tert-butyl. In some embodiments, R1 is a C1-C6 alkylene chain, and R2 is methyl, ethyl, isopropyl, or tert-butyl. In some embodiments, R1 is a C1-C4 alkylene chain, and R2 is methyl, ethyl, isopropyl, or tert-butyl. In some embodiments, R1 is a C1-C2 alkylene chain, and R2 is methyl, ethyl, isopropyl, or tert-butyl. In some embodiments, R1 has 1 to 20 -(CH2CH2-O)- units, and R2 is methyl, ethyl, isopropyl, or tert-butyl. In some embodiments, R1 has 1 to 15 -(CH2CH2-O)- units, and R2 is methyl, ethyl, isopropyl, or tert-butyl. In some embodiments, R1 is 1 to 10 -(CH2CH2-O)- units, and R2 is methyl, ethyl, isopropyl, or tert-butyl. In some embodiments, R1 is 1 to 5 -(CH2CH2-O)- units, and R2 is methyl, ethyl, isopropyl, or tert-butyl. In some embodiments, R1 is 1 to 2 -(CH2CH2-O)- units, and R2 is methyl, ethyl, isopropyl, or tert-butyl.

[0389] In some implementations, n is 2.

[0390] In some embodiments, X is CR9R9′. In some embodiments, R9 and R9′ are each hydrogen.

[0391] In some embodiments, R9 and R9' are independently hydrogen, (C1-C6)alkyl, (C1-C6)alkoxy, (C1-C6)haloalkyl, (C1-C6)haloalkoxy, -C(O)R 10 -NR 10 R 10 -C(O)NR 10 R 10 -OC(O)NR 10 R 10 -NR 10 C(O)R 10 -NR 10 C(O)OR 10 Halogen, OH, CN, amino, (C3-C)10 Carbocyclic groups, 4- or 7-membered heterocyclic groups, -O(CH2) 0-3 (C3-C 10 A carbocyclic group containing one to three heteroatoms selected from O, N, and S -O(CH2). 0-3 -4- or 7-membered heterocyclic groups, where each R 10 It is independently hydrogen or (C1-C6) alkyl; wherein the alkyl, carbocyclic or heterocyclic group is further optionally substituted.

[0392] In some implementations, R4 is incorporated into A linking group on the group. In some embodiments, R4 is O. In some embodiments, R4 is S. In some embodiments, R4 is NR. 11 , where R 11 It is hydrogen or (C1-C6) alkyl. In some embodiments, R4 is OPh. In some embodiments, R4 is OC(O). In some embodiments, R4 is OC(O)NR. 11 , where R 11 It is hydrogen or (C1-C6) alkyl.

[0393] In some embodiments, R4 is an alkylene chain that can be -O-, -S-, -N(R′)-, -C-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -C(NOR′)-, -C(O)N(R′)C(O)-, -C(O)N(R′)C(O)-, -R′C(O)N(R′)R′-, -C(O)N(R′)C(O)N(R′)-, -N(R′)C(O)N(R′)-, -N(R′)C(O)O-, -OC(O)N(R′)-, -C(NR′)-, -N(R′)C( NR′)-, -C(NR′)N(R′)-, -N(R′)C(NR′)N(R′)-, -OB(Me)O-, -S(O)2-, -OS(O)-, -S(O)O-, -S(O)-, -OS(O)2-, -S(O)2O-, -N(R′)S(O)2-, - S(O)2N(R′)-, -N(R′)S(O)-, -S(O)N(R′)-, -N(R′)S(O)2N(R′)-, -N(R′)S(O)N(R′)-, -OP(O)O(R′)O-, -N(R′)P(O)N(R′R′)N(R′)-, C3-C 12 At least one of carbocyclic, 3- to 12-membered heterocyclic, 5- to 12-membered heteroaryl groups, or any combination thereof, is interrupted and / or terminated (at either end or both ends), wherein each R′ is independently H or optionally substituted C1-C. 24Alkyl groups, wherein the interrupting group and one or two terminating groups may be the same or different.

[0394] In some implementations, the alkylene chain is C1-C. 24 Alkylene chain. In some embodiments, the alkylene chain is C1-C2. 18 Alkylene chain. In some embodiments, the alkylene chain is C1-C2. 12 Alkylene chain. In some embodiments, the alkylene chain is C1-C2. 10 Alkylene chain. In some embodiments, the alkylene chain is a C1-C8 alkylene chain. In some embodiments, the alkylene chain is a C1-C6 alkylene chain. In some embodiments, the alkylene chain is a C1-C4 alkylene chain. In some embodiments, the alkylene chain is a C1-C2 alkylene chain. In some embodiments, the alkylene chain is interrupted and / or terminated (at either end or both ends) by at least one or a combination of -N(R′)-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(R′)-, -N(R′)C(O)-, -OC(O)N(R′)-, -S(O)2-, -N(R′)S(O)2-, and -S(O)2N(R′)-. In some embodiments, the alkylene chain is interrupted and / or terminated (at either end or both ends) by -N(R′)-. In some embodiments, the alkylene chain is interrupted and / or terminated by -C(O)- (at either end or both ends). In some embodiments, the alkylene chain is interrupted and / or terminated by -C(O)O- (at either end or both ends). In some embodiments, the alkylene chain is interrupted and / or terminated by -C(O)N(R′)- (at either end or both ends). In some embodiments, the alkylene chain is interrupted by -N(R′)S(O). 2- Interruption and / or termination (at either end or both ends).

[0395] In some implementations, R4 is a polyethylene glycol chain that can be substituted with -O-, -S-, -N(R′)-, -C-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -C(NOR′)-, -C(O)N(R′)C(O)-, -R′C(O)N(R′)R′-, -C(O)N(R′)C(O)N(R′)-, -N(R′)C(O)N(R′)-, -N(R′)C(O)N(R′)-, -N(R′)C(O)O-, -OC(O)N(R′)-, -C(NR′)-, -N(R′)C( NR′)-, -C(NR′)N(R′)-, -N(R′)C(NR′)N(R′)-, -OB(Me)O-, -S(O)2-, -OS(O)-, -S(O)O-, -S(O)-, -OS(O)2-, -S(O)2O-, -N(R′)S(O)2-, - S(O)2N(R′)-, -N(R′)S(O)-, -S(O)N(R′)-, -N(R′)S(O)2N(R′)-, -N(R′)S(O)N(R′)-, -OP(O)O(R′)O-, -N(R′)P(O)N(R′R′)N(R′)-, C3-C 12 At least one of carbocyclic, 3- to 12-membered heterocyclic, 5- to 12-membered heteroaryl groups, or any combination thereof, is interrupted and / or terminated (at either end or both ends), wherein each R′ is independently H or optionally substituted C1-C. 24 Alkyl groups, wherein the interrupting group and one or two terminating groups may be the same or different.

[0396] In some embodiments, the polyethylene glycol chain has 1 to 20 -(CH2CH2-O)- units. In some embodiments, the polyethylene glycol chain has 1 to 15 -(CH2CH2-O)- units. In some embodiments, the polyethylene glycol chain has 1 to 10 -(CH2CH2-O′)- units. In some embodiments, the polyethylene glycol chain has 1 to 5 -(CH2CH2-O)- units. In some embodiments, the polyethylene glycol chain has 1 to 2 -(CH2CH2-O)- units. In some embodiments, polyethylene glycol is interrupted and / or terminated (at either end or both ends) by at least one or a combination of -N(R′)-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(R′)-, -N(R′)C(O)-, -N(R′)C(O)O-, -OC(O)N(R′)-, -S(O)2-, -N(R′)S(O)2-, and -S(O)2N(R′)-. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either end or both ends) by -N(R′)-. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either end or both ends) by -C(O)-. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either end or both ends) by -C(O)O-. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated by -C(O)N(R′)- (at either end or both ends). In some embodiments, the polyethylene glycol chain is interrupted and / or terminated by -N(R′)S(O)2- (at either end or both ends).

[0397] In some embodiments, R4 and R5, together with the carbon atoms they are attached to, form a 3- to 16-membered carbocyclic group or a 3- to 16-membered heterocyclic group containing one to eight heteroatoms selected from N, O, and S. In some embodiments, R4 and R5, together with the carbon atoms they are attached to, form a 4- to 12-membered carbocyclic group or a 4- to 12-membered heterocyclic group containing one to four heteroatoms selected from N, O, and S. In some embodiments, R4 and R5, together with the carbon atoms they are attached to, form a 5- to 10-membered carbocyclic group or a 5- to 10-membered heterocyclic group containing one to three heteroatoms selected from N, O, and S. In some embodiments, R4 and R5, together with the carbon atoms they are attached to, form a 5- to 6-membered carbocyclic group or a 5- to 6-membered heterocyclic group containing one to two heteroatoms selected from N, O, and S.

[0398] In some implementations, R4 and R5, together with the carbon atoms to which they are attached, form a 5-membered heterocyclic group containing a 2-oxygen atom.

[0399] In some implementations, R5 is hydrogen.

[0400] In some implementations, R5 is an electron-withdrawing group.

[0401] In some embodiments, R5 is an electron-withdrawing group. In some embodiments, the electron-withdrawing group is a halogen, OR 5′ SR 5′ or NR 5′ R 5′ , where each R 5′ Independently hydrogen, C1-C6 alkyl, C6-C 12 Aryl, 5- to 10-membered heteroaryl, carbonyl, sulfonyl, sulfinyl or phosphoryl.

[0402] In some embodiments, R5 is a π-electron-withdrawing group. In some embodiments, the π-electron-withdrawing group is -C(O)R. 5” -C(O)NR 5” R 5” -C(O)NR 5” R 5” -C(O)OR 5” NO2, CN, N3, -S(O)R 5” -S(O)2R 5” -S(O)OR 5” -S(O)2OR 5” -S(O)NR 5” R 5” -S(O)2NR 5” R 5” -OP(O)OR 5” OR 5” -P(O)NR 5” R 5” NR 5” R 5” , where each R 5″ It is an independent hydrogen, C1-C6 alkyl, C6-C 12 Aryl, 5- to 10-membered heteroaryl.

[0403] In some implementations, R6 is hydrogen.

[0404] In some embodiments, R6 is a π-electron-donating group.

[0405] In some implementations, R6 is OR 12 SR 12 NR 12 NR 12 Either cyclic amides or acyclic amides, wherein each R 12 Independently hydrogen, (C1-C6)alkyl, (C3-C 10 ) carbocyclic, 4-membered or 7-membered heterocyclic group, wherein the alkyl, carbocyclic or heterocyclic group is optionally substituted.

[0406] In some embodiments, R7 and R7′ are independently hydrogen or electron-withdrawing groups. In some embodiments, the electron-withdrawing groups are halogens, OR... 5′ SR 5′ or NR 5′ R 5′ , where each R 5′ Independently hydrogen, C1-C6 alkyl, C6-C 12 Aryl, 5- to 10-membered heteroaryl, carbonyl, sulfonyl, sulfinyl or phosphoryl.

[0407] In some embodiments, R7 and R7′ are independently hydrogen or electron-withdrawing groups. In some embodiments, the electron-withdrawing group is -C(O)R. 5” -C(O)NR 5” R 5” -C(O)NR 5” R 5” ,-C(O)OR5”,NO2,CN,N3,-S(O)R 5” -S(O)2R 5” -S(O)OR 5” -S(O)2OR 5” -S(O)NR 5” R 5” -S(O)2NR 5” R 5” -OP(O)OR 5” OR 5” -P(O)NR 5” R 5” NR 5” R 5” , where each R 5″ It is an independent hydrogen, C1-C6 alkyl, C6-C 12 Aryl, 5- to 10-membered heteroaryl.

[0408] In some implementations, R8 is incorporated into A linking group on the group. In some embodiments, R8 is CH2. In some embodiments, R8 is aryl. In some embodiments, R8 is O.

[0409] In some embodiments, R8 is an alkylene chain that can be -O-, -S-, -N(R′)-, -C≡C-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -C(NOR′)-, -C(O)N(R′)-, -C(O)N(R′)C(O)-, -R′C(O)N(R′)R′-, -C(O)N(R′)C(O)N(R′)-, -N(R′)C(O)N(R′)-, -N(R′)C(O)O-, -OC(O)N(R′)-, -C(NR′)-, -N(R′)C(N R′)-, -C(NR′)N(R′)-, -N(R′)C(NR′)N(R′)-, -OB(Me)O-, -S(O)2-, -OS(O)-, -S(O)O-, -S(O)-, -OS(O)2-, -S(O)2O-, -N(R′)S(O)2-, -S (O)2N(R′)-, -N(R′)S(O)-, -S(O)N(R′)-, -N(R′)S(O)2N(R′)-, --N(R′)S(O)N(R′)-, -OP(O)O(R′)O-, -N(R′)P(O)N(R′R′)N(R′)-, C3-C 12 At least one of carbocyclic, 3- to 12-membered heterocyclic, 5- to 12-membered heteroaryl groups, or any combination thereof, is interrupted and / or terminated (at either end or both ends), wherein each R′ is independently H or optionally substituted C1-C. 24 Alkyl groups, wherein the interrupting group and one or two terminating groups may be the same or different.

[0410] In some implementations, the alkylene chain is C1-C. 24 Alkylene chain. In some embodiments, the alkylene chain is C1-C2. 18 Alkylene chain. In some embodiments, the alkylene chain is C1-C2. 12 Alkylene chain. In some embodiments, the alkylene chain is C1-C2. 10Alkylene chain. In some embodiments, the alkylene chain is a C1-C8 alkylene chain. In some embodiments, the alkylene chain is a C1-C6 alkylene chain. In some embodiments, the alkylene chain is a C1-C4 alkylene chain. In some embodiments, the alkylene chain is a C1-C2 alkylene chain. In some embodiments, the alkylene chain is interrupted and / or terminated (at either end or both ends) by at least one or a combination of -N(R′)-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(R′)-, -N(R′)C(O)-, -OC(O)N(R′)-, -S(O)2-, -N(R′)S(O)2-, and -S(O)2N(R′)-. In some embodiments, the alkylene chain is interrupted and / or terminated (at either end or both ends) by -N(R′)-. In some embodiments, the alkylene chain is interrupted and / or terminated by -C(O)- (at either end or both ends). In some embodiments, the alkylene chain is interrupted and / or terminated by -C(O)O- (at either end or both ends). In some embodiments, the alkylene chain is interrupted and / or terminated by -C(O)N(R′)- (at either end or both ends). In some embodiments, the alkylene chain is interrupted and / or terminated by -N(R′)S(O)2- (at either end or both ends).

[0411] In some implementations, R8 is a polyethylene glycol chain that can be -O-, -S-, -N(R′)-, -C≡C-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -C(NOR′)-, -C(O)N(R′)-, -C(O)N(R′)C(O)-, -R′C(O)N(R′)R′-, -C(O)N(R′)C(O)N(R′)-, -N(R′)C(O)N(R′)-, -N(R′)C(O)O-, -OC(O)N(R′)-, -C(NR′)-, -N(R′)C(N R′)-, -C(NR′)N(R′)-, -N(R′)C(NR′)N(R′)-, -OB(Me)O-, -S(O)2-, -OS(O)-, -S(O)O-, -S(O)-, -OS(O)2-, -S(O)2O-, -N(R′)S(O)2-, -S (O)2N(R′)-, -N(R′)S(O)-, -S(O)N(R′)-, -N(R′)S(O)2N(R′)-, --N(R′)S(O)N(R′)-, -OP(O)O(R′)O-, -N(R′)P(O)N(R′R′)N(R′)-, C3-C 12At least one of carbocyclic, 3- to 12-membered heterocyclic, 5- to 12-membered heteroaryl groups, or any combination thereof, is interrupted and / or terminated (at either end or both ends), wherein each R′ is independently H or optionally substituted C1-C. 24 Alkyl groups, wherein the interrupting group and one or two terminating groups may be the same or different.

[0412] In some embodiments, the polyethylene glycol chain has 1 to 20 -(CH2CH2-O)- units. In some embodiments, the polyethylene glycol chain has 1 to 15 -(CH2CH2-O)- units. In some embodiments, the polyethylene glycol chain has 1 to 10 -(CH2CH2-O)- units. In some embodiments, the polyethylene glycol chain has 1 to 5 -(CH2CH2-O)- units. In some embodiments, the polyethylene glycol chain has 1 to 2 -(CH2CH2-O)- units. In some embodiments, polyethylene glycol is interrupted and / or terminated (at either end or both ends) by at least one or a combination of -N(R′)-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(R′)-, -N(R′)C(O)-, -N(R′)C(O)O-, -OC(O)N(R′)-, -S(O)2-, -N(R′)S(O)2-, and -S(O)2N(R′)-. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either end or both ends) by -N(R′)-. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either end or both ends) by -C(O)-. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either end or both ends) by -C(O)O-. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated by -C(O)N(R′)- (at either end or both ends). In some embodiments, the polyethylene glycol chain is interrupted and / or terminated by -N(R′)S(O)2- (at either end or both ends).

[0413] In some implementations, A1 is the treatment component and A2 is the diagnostic component.

[0414] In some implementations, A1 is the diagnostic part and A2 is the treatment part.

[0415] In some implementations, A1 is the treatment portion and A2 is the binding portion.

[0416] In some implementations, A1 is the binding portion and A2 is the treatment portion.

[0417] In some implementations, A1 is the bonding portion and A2 is the bonding portion.

[0418] In some embodiments, compounds of formula (IV) are represented by compounds of formula (IVa): Or a pharmaceutically acceptable salt or stereoisomer thereof. In some embodiments, R1 is absent and R1′ is an optionally substituted alkylene chain or an optionally substituted polyethylene glycol chain; and / or R2 is methyl, ethyl, isopropyl, or tert-butyl; or R1 and R2, together with the nitrogen atom to which they are attached, form a 3- to 16-membered heterocyclic group containing 1 to 8 heteroatoms selected from N, O, and S; R1′ is CH2; and / or R4 is O, S, NR. 11 OPh, OC(O), OC(O)NR 11 , where R 11 It is hydrogen or (C1-C6) alkyl, optionally substituted alkylene chains, or optionally substituted polyethylene glycol chains; and / or R5 is hydrogen, fluorine, or OR. 5′ OR 5′ It is hydrogen or (C1-C6) alkyl; and / or A1 is a binding portion, therapeutic portion or diagnostic portion; and / or A2 is a binding portion, therapeutic portion or diagnostic portion.

[0419] In some implementations, the compound of formula (IV) is

[0420] Or its pharmaceutically acceptable salts or stereoisomers.

[0421] In some implementations, the compound of formula (IV) is

[0422] Or its pharmaceutically acceptable salts or stereoisomers.

[0423] In some embodiments, compounds of formula (IV) are represented by compounds of formula IVa′, IVb′, or IVc′:

[0424] Or its pharmaceutically acceptable salts or stereoisomers.

[0425] in:

[0426] R1′ is a linking group;

[0427] R1 does not exist, or

[0428] R1 and R2, together with the nitrogen atoms they are attached to, form a heterocyclic group;

[0429] R2 is an optionally substituted (C1-C8) alkyl group, -C(O)R′, -C(O)OR′, -C(O)NR′R′, -S(O)R′, -S(O)2R′, (C3-C 10 A carbocyclic or 4- or 7-membered heterocyclic group, wherein each R” is independently hydrogen, (C1-C6)alkyl, (C3-C6)alkyl, or (C7-C8)alkyl. 10 ) carbocyclic, 4-membered or 7-membered heterocyclic group, wherein the alkyl, carbocyclic or heterocyclic group is optionally substituted;

[0430] A1′ is an antibody or antibody fragment;

[0431] Each X is independently CR9R9′, NR9, O, S, C(O), S(O) or SO2, wherein the ring system contains 0 to 3 heteroatoms;

[0432] R9 and R9′ are independently hydrogen or substituents;

[0433] R4 is a linking group;

[0434] A2′ is a therapeutic small molecule; and

[0435] n is 1, 2, or 3.

[0436] In some embodiments, R1 is absent, and R1′ is an alkylene chain that can be -O-, -S-, --N(R′)-, -C≡C-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -C(NOR′)-, -C(O)N(R′)-, -C(O)N(R′)C(O)-, -R′C(O)N(R′)R′, -C(O)N(R′)C(O)N(R′)-, -N(R′)C(O)N(R′)-, -N(R′)C(O)O-, -OC(O)N(R′)-, -C(NR′)-, -N(R′)- )C(NR′)-, -C(NR′)N(R′)-, -N(R′)C(NR′)N(R′)-, -OB(Me)O-, -S(O)2-, -OS(O)-, -S(O)O-, -S(O)-, -OS(O)2-, -S(O)2O-, -N(R′)S(O)2-, -S(O)2N(R′)-, --N(R′)S(O)-, -S(O)N(R′)-, -N(R′)S(O)2N(R′)-, -N(R′)S(O)N(R′)-, -OP(O)O(R′)O-, -N(R′)P(O)N(R′R′)N(R′)-, C3-C 12At least one of carbocyclic, 3- to 12-membered heterocyclic, 5- to 12-membered heteroaryl groups, or any combination thereof, is interrupted and / or terminated (at either end or both ends), wherein each R′ is independently H or optionally substituted C1-C. 24 Alkyl groups, wherein the interrupting group and one or two terminating groups may be the same or different.

[0437] In some implementations, the alkylene chain is C1-C. 24 Alkylene chain. In some embodiments, the alkylene chain is C1-C2. 18 Alkylene chain. In some embodiments, the alkylene chain is C1-C2. 12 Alkylene chain. In some embodiments, the alkylene chain is C1-C2. 10 Alkylene chain. In some embodiments, the alkylene chain is a C1-C8 alkylene chain. In some embodiments, the alkylene chain is a C1-C6 alkylene chain. In some embodiments, the alkylene chain is a C1-C4 alkylene chain. In some embodiments, the alkylene chain is a C1-C2 alkylene chain. In some embodiments, the alkylene chain is interrupted and / or terminated (at either end or both ends) by at least one or a combination of -N(R′)-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(R′)-, -N(R′)C(O)-, -N(R′)C(O)O-, -OC(O)N(R′)-, -S(O)2-, -N(R′)S(O)2-, and -S(O)2N(R′)-. In some embodiments, the alkylene chain is interrupted and / or terminated (at either end or both ends) by -N(R′)-. In some embodiments, the alkylene chain is interrupted and / or terminated by -C(O)- (at either end or both ends). In some embodiments, the alkylene chain is interrupted and / or terminated by -C(O)O- (at either end or both ends). In some embodiments, the alkylene chain is interrupted and / or terminated by -C(O)N(R′)- (at either end or both ends). In some embodiments, the alkylene chain is interrupted and / or terminated by -N(R′)S(O)2- (at either end or both ends).

[0438] In some implementations, R1 is absent, and R1′ is a polyethylene glycol chain that can be substituted with -O-, -S-, -N(R′)-, -C-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -C(NOR′)-, -C(O)N(R′)-, -C(O)N(R′)C(O)-, -R′C(O)N(R′)R′, -C(O)N(R′)C(O)N(R′)-, -N(R′)C(O)-, -N(R′)C(O)N(R′)-, -N(R′)C(O)O-, -OC(O)N(R′)-, -C(NR′)-, -N(R′)-, -N(R′)- )C(NR′)-, -C(NR′)N(R′)-, -N(R′)C(NR′)N(R′)-, -OB(Me)O-, -S(O)2-, -OS(O)-, -S(O)O-, -S(O)-, -OS(O)2-, -S(O)2O-, -N(R′)S(O)2-, -S(O)2N(R′)-, --N(R′)S(O)-, -S(O)N(R′)-, -N(R′)S(O)2N(R′)-, -N(R′)S(O)N(R′)-, -OP(O)O(R′)O-, -N(R′)P(O)N(R′R′)N(R′)-, C3-C 12 At least one of carbocyclic, 3- to 12-membered heterocyclic, 5- to 12-membered heteroaryl groups, or any combination thereof, is interrupted and / or terminated (at either end or both ends), wherein each R′ is independently H or optionally substituted C1-C. 24 Alkyl groups, wherein the interrupting group and one or two terminating groups may be the same or different.

[0439] In some embodiments, the polyethylene glycol chain has 1 to 20 -(CH2CH2-O)- units. In some embodiments, the polyethylene glycol chain has 1 to 15 -(CH2CH2-O)- units. In some embodiments, the polyethylene glycol chain has 1 to 10 -(CH2CH2-O)- units. In some embodiments, the polyethylene glycol chain has 1 to 5 -(CH2CH2-O)- units. In some embodiments, the polyethylene glycol chain has 1 to 2 -(CH2CH2-O)- units. In some embodiments, polyethylene glycol is interrupted and / or terminated (at either end or both ends) by at least one or a combination of -N(R′)-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(R′)-, -N(R′)C(O)-, -N(R′)C(O)O-, -OC(O)N(R′)-, -S(O)2-, -N(R′)S(O)2-, and -S(O)2N(R′)-. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either end or both ends) by -N(R′)-. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either end or both ends) by -C(O)-. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either end or both ends) by -C(O)O-. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either end or both ends) by -C(O)N(R′)-. In some embodiments, the polyethylene glycol chain is interrupted by -N(R′)S(O). 2- Interruption and / or termination (at either end or both ends).

[0440] In some embodiments, R1 and R2, together with the nitrogen atom to which they are attached, form a 3- to 16-membered heterocyclic group containing 1 to 8 heteroatoms selected from N, O, and S; while R1′ is C1-C 24 An alkylene chain or one to 20 -(CH2CH2-O)- units, wherein R1′ is optionally substituted. In some embodiments, R1 and R2, together with the nitrogen atom to which they are attached, form a 4- to 12-membered heterocyclic group containing one to four heteroatoms selected from N, O, and S; while R1′ is C1-C 18 An alkylene chain or one to 15 -(CH2CH2-O)- units, wherein R1′ is optionally substituted. In some embodiments, R1 and R2, together with the nitrogen atom to which they are attached, form a 5- to 10-membered heterocyclic group containing one to three heteroatoms selected from N, O, and S; while R1′ is C1-C 12 An alkylene chain or one to ten -(CH2CH2-O)- units, wherein R1′ is optionally substituted. In some embodiments, R1 and R2, together with the nitrogen atom to which they are attached, form a 5- to 6-membered heterocyclic group containing one to two heteroatoms selected from N, O, and S; while R1′ is C1-C10 An alkylene chain or one to five -(CH2CH2-O)- units, wherein R1′ is optionally substituted. In some embodiments, R1 and R2, together with the nitrogen atom to which they are attached, form a piperazine group; while R1′ is C1-C. 10 An alkylene chain or one to five -(CH2CH2-O)- units, wherein R1′ is optionally substituted.

[0441] In some implementations, R1 does not exist, and R1′ is C1-C 24 The alkylene chain, and R2 is methyl or benzyl. In some embodiments, R1 is absent, and R1′ is C1-C. 18 The alkylene chain, and R2 is methyl or benzyl. In some embodiments, R1 is absent, and R1′ is C1-C. 12 The alkylene chain, and R2 is methyl or benzyl. In some embodiments, R1 is absent, and R1′ is C1-C. 10 In some embodiments, R1 is absent, R1′ is a C1-C8 alkylene chain, and R2 is methyl or benzyl. In some embodiments, R1 is absent, R1′ is a C1-C6 alkylene chain, and R2 is methyl or benzyl. In some embodiments, R1 is absent, R1′ is a C1-C4 alkylene chain, and R2 is methyl or benzyl. In some embodiments, R1 is absent, R1′ is a C1-C2 alkylene chain, and R2 is methyl or benzyl. In some embodiments, R1 is absent, R1′ is 1 to 20 -(CH2CH2-O)- units, and R2 is methyl or benzyl. In some embodiments, R1 is absent, R1′ is 1 to 15 -(CH2CH2-O)- units, and R2 is methyl or benzyl. In some embodiments, R1 is absent, R1′ is 1 to 10 -(CH2CH2-O)- units, and R2 is methyl or benzyl. In some embodiments, R1 is absent, R1′ is 1 to 5 -(CH2CH2-O)- units, and R2 is methyl or benzyl. In some embodiments, R1 is absent, R1′ is 1 to 2 -(CH2CH2-O)- units, and R2 is methyl or benzyl.

[0442] In some implementation schemes, A1' is moromarab-CD3, abciximab, rituximab, pallizumab, infliximab, trastuzumab, alenzab, adalimumab, tiimozumab, omalizumab, cetuximab, bevacizumab, natezumab, panitumumab, ranibizumab, ikulizumab, cetozumab, ustekinumab, canatumab, golimumab, Ophamumab, Tocilizumab, Denosumab, Belimumab, Ipilimumab, Bentuximab, Pertuximab, Raxicurumab, Ophamumab, Sestoxicurumab, Ramucirumab, Vedolizumab, Bonatumab, Nivolumab, Pembrolizumab, Idacilumab, Nexicurumab, Denocicurumab, Sestoxicurumab, Mepleribumab, Alilobumarab, Evodocin Anti-, Daremumab, Erotozumab, Isebemab, Relizumab, Olanzab, Belottosuzumab, Atezolizumab, Otosaximab, Ogaituzumab, Brodatumab, Gusekumab, Duprexumab, Thaliru, Averu, Omega, Emezabumab, Benalizumab, Getozumab, Dvalumab, Ibuprofen, Ranaru Monoclonal antibodies, including mogliflozin, elenocumab, ganezumab, tetrazolizumab, cimiprizumab, epavazumab, remannetumab, ibazine, mosetumab, revulizumab, cappserizumab, lomoxoluzumab, resalizumab, polotozumab, broxelizumab, lizanilizumab, cetozumab, belantazumab, or enrofloxacin, or fragments thereof. In some embodiments, A1′ is trastuzumab.

[0443] In some embodiments, X is CR9R9′. In some embodiments, R9 and R9′ are each hydrogen. In some embodiments, R9 and R9′ are independently hydrogen, (C1-C6)alkyl, (C1-C6)alkoxy, (C1-C6)haloalkyl, (C1-C6)haloalkoxy, -C(O)R 10 -NR 10 R 10 -C(O)NR 10 R 10 -OC(O)NR 10 R 10 -NR 10 C(O)R 10 -NR 10 C(O)OR 10 Halogen, OH, CN, amino, (C3-C) 10 Carbocyclic groups, 4- or 7-membered heterocyclic groups, -O(CH2) 0-3 (C3-C 10 A carbocyclic group containing one to three heteroatoms selected from O, N, and S -O(CH2). 0-3-4- or 7-membered heterocyclic groups, where each R 10 It is independently hydrogen or (C1-C6) alkyl; wherein the alkyl, carbocyclic or heterocyclic group is further optionally substituted.

[0444] In some implementations, R4 is O, S, NR 10 OC(O), NR 10 C(O) or OC(O)NR5, where R 10 It is hydrogen or C1-C6 alkyl.

[0445] In some embodiments, R4 is an alkylene chain that can be -O-, -S-, -N(R′)-, -C≡C-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -C(NOR′)-, -C(O)N(R′)-, -C(O)N(R′)C(O)-, -R′C(O)N(R′)R′-, -C(O)N(R′)C(O)N(R′)-, -N(R′)C(O)N(R′)-, -N(R′)C(O)O-, -OC(O)N(R′)-, -C(NR′)-, -N(R′)C( NR′)-, -C(NR′)N(R′)-, -N(R′)C(NR′)N(R′)-, -OB(Me)O-, -S(O)2-, -OS(O)-, -S(O)O-, -S(O)-, -OS(O)2-, -S(O)2O-, -N(R′)S(O)2-, - S(O)2N(R′)-, -N(R′)S(O)-, -S(O)N(R′)-, -N(R′)S(O)2N(R′)-, -N(R′)S(O)N(R′)-, -OP(O)O(R′)O-, -N(R′)P(O)N(R′R′)N(R′)-, C3-C 12 At least one of carbocyclic, 3- to 12-membered heterocyclic, 5- to 12-membered heteroaryl groups, or any combination thereof, is interrupted and / or terminated (at either end or both ends), wherein each R′ is independently H or optionally substituted C1-C. 24 Alkyl groups, wherein the interrupting group and one or two terminating groups may be the same or different.

[0446] In some implementations, the alkylene chain is C1-C. 24 Alkylene chain. In some embodiments, the alkylene chain is C1-C2. 18 Alkylene chain. In some embodiments, the alkylene chain is C1-C2. 12 Alkylene chain. In some embodiments, the alkylene chain is C1-C2. 10Alkylene chain. In some embodiments, the alkylene chain is a C1-C8 alkylene chain. In some embodiments, the alkylene chain is a C1-C6 alkylene chain. In some embodiments, the alkylene chain is a C1-C4 alkylene chain. In some embodiments, the alkylene chain is a C1-C2 alkylene chain. In some embodiments, the alkylene chain is interrupted and / or terminated (at either end or both ends) by at least one or a combination of -N(R′)-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(R′)-, -N(R′)C(O)-, -OC(O)N(R′)-, -S(O)2-, -N(R′)S(O)2-, and -S(O)2N(R′)-. In some embodiments, the alkylene chain is interrupted and / or terminated (at either end or both ends) by -N(R′)-. In some embodiments, the alkylene chain is interrupted and / or terminated by -C(O)- (at either end or both ends). In some embodiments, the alkylene chain is interrupted and / or terminated by -C(O)O- (at either end or both ends). In some embodiments, the alkylene chain is interrupted and / or terminated by -C(O)N(R′)- (at either end or both ends). In some embodiments, the alkylene chain is interrupted and / or terminated by -N(R′)S(O)2- (at either end or both ends).

[0447] In some embodiments, R4 is a polyethylene glycol chain that can be -O-, -S-, -N(R′)-, -C≡C-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -C(NOR′)-, -C(O)N(R′)-, -C(O)N(R′)C(O)-, -R′C(O)N(R′)R′-, -C(O)N(R′)C(O)N(R′)-, -N(R′)C(O)N(R′)-, -N(R′)C(O)O-, -OC(O)N(R′)-, -C(NR′)-, -N(R′)C(N R′)-, -C(NR′)N(R′)-, -N(R′)C(NR′)N(R′)-, -OB(Me)O-, -S(O)2-, -OS(O)-, -S(O)O-, -S(O)-, -OS(O)2-, -S(O)2O-, -N(R′)S(O)2-, -S (O)2N(R′)-, -N(R′)S(O)-, -S(O)N(R′)-, -N(R′)S(O)2N(R′)-, --N(R′)S(O)N(R′)-, -OP(O)O(R′)O-, -N(R′)P(O)N(R′R′)N(R′)-, C3-C 12At least one of carbocyclic, 3- to 12-membered heterocyclic, 5- to 12-membered heteroaryl groups, or any combination thereof, is interrupted and / or terminated (at either end or both ends), wherein each R′ is independently H or optionally substituted C1-C. 24 Alkyl groups, wherein the interrupting group and one or two terminating groups may be the same or different.

[0448] In some embodiments, the polyethylene glycol chain has 1 to 20 -(CH2CH2-O)- units. In some embodiments, the polyethylene glycol chain has 1 to 15 -(CH2CH2-O)- units. In some embodiments, the polyethylene glycol chain has 1 to 10 -(CH2CH2-O)- units. In some embodiments, the polyethylene glycol chain has 1 to 5 -(CH2CH2-O)- units. In some embodiments, the polyethylene glycol chain has 1 to 2 -(CH2CH2-O)- units. In some embodiments, polyethylene glycol is interrupted and / or terminated (at either end or both ends) by at least one or a combination of -N(R′)-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(R′)-, -N(R′)C(O)-, -N(R′)C(O)O-, -OC(O)N(R′)-, -S(O)2-, --N(R′)S(O)2-, and -S(O)2N(R′)-. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either end or both ends) by -N(R′)-. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either end or both ends) by -C(O)-. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either end or both ends) by -C(O)O-. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated by -C(O)N(R′)- (at either end or both ends). In some embodiments, the polyethylene glycol chain is interrupted and / or terminated by -N(R′)S(O)2- (at either end or both ends).

[0449] In some implementations, n is 2. In some implementations, n is 2, and each X is CH2.

[0450] In some embodiments, A2' is an anticancer agent. In some embodiments, A2' is aurestatin, maytansine, scutellarin, anthracycline, paclitaxel or docetaxel or a derivative thereof, calichimycin or a derivative thereof, pyrrolobenzodiazepine dimer (PBD) or a derivative thereof, pyromycin or a derivative thereof, eribulin or a derivative thereof, camptothecin or a derivative thereof, or eczema or a derivative thereof.

[0451] In some embodiments, the antibody is a monoclonal antibody, R1 and R2, together with the nitrogen atom to which they are attached, form a piperazine group, and the compound has a structure represented by formula IVa′1:

[0452] Or its pharmaceutically acceptable salts or stereoisomers.

[0453] In some embodiments, the antibody is a monoclonal antibody, R1 is absent, and R2 is methyl, and the compound has a structure represented by formula IVa′2:

[0454] Or its pharmaceutically acceptable salts or stereoisomers.

[0455] In some embodiments, the compound of formula (V) is represented by the compound of formula (Va):

[0456] Or a pharmaceutically acceptable salt or stereoisomer thereof. In some embodiments, R1 is absent: R1′ is an optionally substituted alkylene chain or an optionally substituted polyethylene glycol chain; and / or R2 is methyl, ethyl, isopropyl, or tert-butyl; or R1′ is CH2 or R1 and R2, together with the nitrogen atom to which they are attached, forming a 3- to 16-membered heterocyclic group containing 1 to 8 heteroatoms selected from N, O, and S; and / or R6 is hydrogen, chlorine, bromine, iodine, or OR. 12 or SR 12 , where each R 12 Independently hydrogen, (C1-C6)alkyl, (C3-C 10 R7 is a carbocyclic group, a 4- or 7-membered heterocyclic group; and / or R7 is hydrogen, fluorine, or OR 5′ , where R 5′ It is hydrogen or (C1-C6) alkyl; and / or R7 is hydrogen, fluorine or OR 5′ , where R 5′ It is hydrogen or (C1-C6) alkyl; and R8 is CH2, O, C6-C. 12 aryl or 5 to 10-membered heteroaryl; and / or R8 is O, S, NR 11 OPh, OC(O), OC(O)NR 11 , where R 11 It is a hydrogen or (C1-C6) alkyl, optionally substituted alkylene chain, or optionally substituted polyethylene glycol chain and / or A1 is a binding part, therapeutic part or diagnostic part; and / or A2 is a binding part, therapeutic part or diagnostic part.

[0457] In some implementations, the compound of formula (V) is

[0458]

[0459]

[0460] Or its pharmaceutically acceptable salts or stereoisomers.

[0461] In some implementations, the compound of formula (V) is

[0462]

[0463] Or its pharmaceutically acceptable salts or stereoisomers.

[0464] In some embodiments, the optional substituents of the compounds of formula (IV) or (V) are selected from the group consisting of: alkyl, alkenyl, alkynyl, halogen, haloalkyl, cycloalkyl, heterocycloalkyl, hydroxyl, alkoxy, cycloalkoxy, heterocycloalkoxy, haloalkoxy, aryloxy, heteroaryloxy, aralkyloxy, alkenyloxy, alkynyloxy, amino, alkylamino, cycloalkylamino, heterocycloalkylamino, arylamino, heteroarylamino, arylalkylamino, N-alkyl-N-arylamino, N-alkyl-N-heteroarylamino, N-alkyl-N-arylalkylamino, hydroxyalkyl, aminoalkyl, alkylthio, haloalkylthio, alkylsulfonyl, haloalkylsulfonyl, cycloalkylsulfonyl, heterocycloalkylsulfonyl, arylsulfonyl, heteroarylsulfonyl, aminosulfonyl, alkylaminosulfonyl, cycloalkylaminosulfonyl, heterocycloalkylaminosulfonyl, arylamino alkylsulfonyl, heteroarylaminosulfonyl, N-alkyl-N-arylaminosulfonyl, N-alkyl-N-heteroarylaminosulfonyl, formyl, alkyl carbonyl, haloalkyl carbonyl, alkenyl carbonyl, alkynyl carbonyl, carboxyl, alkoxy carbonyl, alkyl carbonyloxy, amino, alkylsulfonylamino, haloalkylsulfonylamino, cycloalkylsulfonylamino, heterocyclic alkylsulfonylamino, arylsulfonylamino, heteroarylsulfonylamino, aralkylsulfonylamino, alkylcarbonylamino, haloalkylcarbonylamino, cycloalkylcarbonylamino, heterocyclic alkylcarbonylamino, arylcarbonylamino, heteroarylcarbonylamino, aralkylsulfonylamino, aminocarbonyl, alkylaminocarbonyl, cycloalkylaminocarbonyl, heterocyclic alkylaminocarbonyl, arylaminocarbonyl, heteroarylaminocarbonyl, N-alkyl-N-arylaminocarbonyl, N-alkyl-N-heteroarylaminocarbonyl, cyano, nitro, and azide.

[0465] In the implementation plan, where and One is the therapeutic component, and the other is the diagnostic component; compounds of formulas (IV) and (V) can be referred to as "therapeutic diagnostic" agents.

[0466] In some implementations, the diagnostic component is a fluorophore, and the therapeutic component is an anticancer agent.

[0467] In some implementations, the diagnostic component is a fluorophore, and the therapeutic component is a non-targeted anticancer agent.

[0468] In some implementations, the diagnostic component is a fluorophore, and the therapeutic component is a targeted anticancer agent.

[0469] In some implementations, the diagnostic component is a fluorophore, and the therapeutic component is a kinase inhibitor.

[0470] In some implementations, the diagnostic component is a fluorophore, and the therapeutic component is an antibacterial agent.

[0471] In some implementations, the diagnostic component is a fluorophore, and the therapeutic component is an NSAID.

[0472] In some implementations, the diagnostic component is a fluorophore, and the therapeutic component is DMARD.

[0473] In some implementations, the diagnostic component is a chromogenic agent, and the therapeutic component is an anticancer agent.

[0474] In some implementations, the diagnostic component is a chromogenic agent, and the therapeutic component is a non-targeted anticancer agent.

[0475] In some implementations, the diagnostic component is a chromogenic agent, and the therapeutic component is a targeted anticancer agent.

[0476] In some implementations, the diagnostic component is a chromogenic agent, and the therapeutic component is a kinase inhibitor.

[0477] In some implementations, the diagnostic component is a chromogenic agent, and the therapeutic component is an antibacterial agent.

[0478] In some implementations, the diagnostic component is a chromogenic agent, and the therapeutic component is an NSAID.

[0479] In some implementations, the diagnostic component is a chromogenic agent, and the therapeutic component is a DMARD.

[0480] In some implementations, the diagnostic component is a PET tracer, and the therapeutic component is an anticancer agent.

[0481] In some implementations, the diagnostic component is a PET tracer, and the therapeutic component is a non-targeted anticancer agent.

[0482] In some implementations, the diagnostic component is a PET tracer, and the therapeutic component is a targeted anticancer agent.

[0483] In some implementations, the diagnostic component is a PET tracer, and the therapeutic component is a kinase inhibitor.

[0484] In some implementations, the diagnostic component is a PET tracer, and the therapeutic component is an antibacterial agent.

[0485] In some implementations, the diagnostic component is a PET tracer, and the therapeutic component is an NSAID.

[0486] In some implementations, the diagnostic component is a PET tracer, and the therapeutic component is a DMARD.

[0487] In some implementations, the diagnostic component is an MRI contrast agent, and the therapeutic component is an anticancer agent.

[0488] In some implementations, the diagnostic component is an MRI contrast agent, and the therapeutic component is a non-targeted anticancer agent.

[0489] In some implementations, the diagnostic component is an MRI contrast agent, and the therapeutic component is a targeted anticancer agent.

[0490] In some implementations, the diagnostic component is an MRI contrast agent, and the therapeutic component is a kinase inhibitor.

[0491] In some implementations, the diagnostic component is an MRI contrast agent, and the therapeutic component is an antibacterial agent.

[0492] In some implementations, the diagnostic component is an MRI contrast agent, and the therapeutic component is an NSAID.

[0493] In some implementations, the diagnostic component is an MRI contrast agent, and the therapeutic component is a DMARD.

[0494] In some implementation schemes, where and One is a binding part, and the other is a different binding part. Compounds of formulas (IV) and (V) can be called chimeras that target protein hydrolysis (also known as PROTACs or degraders), which target a given protein for selective degradation.

[0495] In some embodiments, the first binding moiety binds to E3 ubiquitin ligase, and the second binding moiety binds to ALK. In some embodiments, the E3 ubiquitin ligase is cereblon. In some embodiments, the E3 ubiquitin ligase is VHL. In some embodiments, the E3 ligase is IAP. In some embodiments, the E3 ligase is MDM2.

[0496] In some embodiments, the first binding moiety binds to E3 ubiquitin ligase, while the second binding moiety binds to BTK. In some embodiments, the E3 ubiquitin ligase is cereblon. In some embodiments, the E3 ubiquitin ligase is VHL. In some embodiments, the E3 ligase is IAP. In some embodiments, the E3 ligase is MDM2.

[0497] In some embodiments, the first binding moiety binds to E3 ubiquitin ligase, while the second binding moiety binds to BET. In some embodiments, the E3 ubiquitin ligase is cereblon. In some embodiments, the E3 ubiquitin ligase is VHL. In some embodiments, the E3 ligase is IAP. In some embodiments, the E3 ligase is MDM2.

[0498] In some embodiments, the first binding moiety binds to E3 ubiquitin ligase, while the second binding moiety binds to BRD4. In some embodiments, the E3 ubiquitin ligase is cereblon. In some embodiments, the E3 ubiquitin ligase is VHL. In some embodiments, the E3 ligase is IAP. In some embodiments, the E3 ligase is MDM2.

[0499] In some embodiments, the first binding moiety binds to E3 ubiquitin ligase, while the second binding moiety binds to HDAC. In some embodiments, the E3 ubiquitin ligase is cereblon. In some embodiments, the E3 ubiquitin ligase is VHL. In some embodiments, the E3 ligase is IAP. In some embodiments, the E3 ligase is MDM2.

[0500] In some embodiments, the first binding moiety binds to an E3 ubiquitin ligase, while the second binding moiety binds to an estrogen receptor. In some embodiments, the E3 ubiquitin ligase is cereblon. In some embodiments, the E3 ubiquitin ligase is VHL. In some embodiments, the E3 ligase is IAP. In some embodiments, the E3 ligase is MDM2.

[0501] In some embodiments, the first binding moiety binds to an E3 ubiquitin ligase, while the second binding moiety binds to an androgen receptor. In some embodiments, the E3 ubiquitin ligase is cereblon. In some embodiments, the E3 ubiquitin ligase is VHL. In some embodiments, the E3 ligase is IAP. In some embodiments, the E3 ligase is MDM2.

[0502] In some implementation schemes, where and One is the therapeutic portion, and the other is the binding portion comprising an antibody or a (cell target) binding fragment thereof; compounds of formulas (IV and IV′) and (V) may be called antibody-drug conjugates. In some embodiments, the therapeutic portion of the antibody-drug conjugate is an anticancer agent.

[0503] In some implementations, the binding portion of the antibody-drug conjugate is a monoclonal antibody or a fragment thereof, while the therapeutic portion is a non-targeted anticancer agent.

[0504] In some implementations, the binding portion of the antibody-drug conjugate is a monoclonal antibody or a fragment thereof, while the therapeutic portion is a targeted anticancer agent.

[0505] In some implementations, the binding portion of the antibody-drug conjugate is a monoclonal antibody or its binding fragment, while the therapeutic portion is a kinase inhibitor.

[0506] In some implementations, the binding portion of the antibody-drug conjugate is a monoclonal antibody or its binding fragment, while the therapeutic portion is an antibacterial agent.

[0507] In some implementations, the binding portion of the antibody-drug conjugate is a monoclonal antibody or a fragment thereof, while the therapeutic portion is an NSAID.

[0508] In some implementations, the binding portion of the antibody-drug conjugate is a monoclonal antibody or a fragment thereof, while the therapeutic portion is a DMARD.

[0509] In some implementations, the binding portion is biotin or a derivative thereof, while the therapeutic portion is a targeted anticancer agent.

[0510] In some implementations, the binding portion is biotin or a derivative thereof, while the therapeutic portion is a kinase inhibitor.

[0511] In some implementations, the binding portion is biotin or a derivative thereof, while the therapeutic portion is an antibacterial agent.

[0512] In some implementations, the binding portion is biotin or a derivative thereof, while the therapeutic portion is NSAID.

[0513] In some implementations, the binding portion is biotin or a derivative thereof, while the therapeutic portion is DMARD.

[0514] The compounds of the present invention may be in the form of free acids or free bases or pharmaceutically acceptable salts. As used herein, in the context of salts, the term "pharmaceutically acceptable" means a salt of a compound that does not eliminate the biological activity or properties of the compound and is relatively non-toxic; that is, the salt form of the compound can be administered to a subject without causing adverse biological effects (such as dizziness or stomach upset) or interacting harmfully with any other component of the composition containing it. The term "pharmaceutically acceptable salt" refers to a product obtained by reacting the compounds of the present invention with a suitable acid or base. Examples of pharmaceutically acceptable salts of the compounds of the present invention include those derived from suitable inorganic bases such as Li salts, Na salts, K salts, Ca salts, Mg salts, Fe salts, Cu salts, Al salts, Zn salts, and Mn salts. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts formed by amino groups and inorganic acids, such as hydrochlorides, hydrobroms, hydroiodates, nitrates, sulfates, hydrogen sulfates, phosphates, isonicotinates, acetates, lactates, salicylates, citrates, tartrates, pantothenates, hydrogen tartrates, ascorbic acid salts, succinates, maleates, gentianates, fumarates, gluconates, glucurons, glycosides, formates, benzoates, glutamates, methanesulfonates, ethanesulfonates, benzenesulfonates, 4-methylbenzenesulfonates, or p-toluenesulfonates. Certain compounds of this invention can form pharmaceutically acceptable salts with various organic bases such as lysine, arginine, guanidine, diethanolamine, or metformin. Suitable base salts include aluminum, calcium, lithium, magnesium, potassium, sodium, or zinc salts.

[0515] The compounds of the present invention may have at least one chiral center and therefore may be in the form of stereoisomers, which, as used herein, include all isomers of the respective compounds, differing only in the spatial orientation of their atoms. The term stereoisomer includes mirror-image isomers (enantiomers, including (R-) or (S-) configurations of the compound), mixtures of mirror-image isomers of the compound (physical mixtures of enantiomers and racemic mixtures or racemic mixtures), geometric (cis / trans or E / Z, R / S) isomers of the compound, and isomers of the compound having more than one chiral center and not being mirror images of each other (diastereomers). The chiral center of the compound may undergo epimerization in vivo; therefore, for these compounds, the application of the (R-) form is considered equivalent to the application of the (S-) form. Thus, the compounds of the present invention may be manufactured and used in the form of individual isomers and substantially free of other isomers, or in the form of mixtures of various isomers, such as racemic mixtures of stereoisomers.

[0516] In some embodiments, the compound is an isotopic derivative because it has at least one desired atomic isotope substituted, the amount of which is substituted above the natural abundance of the isotope, i.e., it is enriched. In one embodiment, the compound comprises deuterium or multiple deuterium atoms. Heavier isotopes such as deuterium (i.e., 2 H) substitution may have certain therapeutic advantages due to its higher metabolic stability, such as increased half-life in vivo or reduced dose requirements, and therefore may be advantageous in some cases.

[0517] The compounds of the present invention can be prepared by crystallization under different conditions and can exist as one or a combination of polymorphs of the compound. For example, different polymorphs can be identified and / or prepared by crystallization at different temperatures, or by recrystallization using different solvents or different solvent mixtures during crystallization using various cooling modes (from very rapid to very slow cooling) during crystallization. Polymorphs can also be obtained by heating or melting the compound and then cooling it gradually or rapidly. The presence of polymorphs can be determined by solid-state probe NMR spectroscopy, IR spectroscopy, differential scanning calorimetry, powder X-ray diffraction, and / or other known techniques.

[0518] In some embodiments, the pharmaceutical composition comprises a cocrystal of the compounds of the present invention. As used herein, the term "cocrystal" refers to a stoichiometric multicomponent system comprising the compounds of the present invention and a cocrystal formation, wherein the compounds of the present invention and the cocrystal formation are linked by non-covalent interactions. As used herein, the term "cocrystal formation" refers to a compound capable of forming intermolecular interactions with and co-crystallizing with the compounds of the present invention. Representative examples of eutectic formations include benzoic acid, succinic acid, fumaric acid, glutaric acid, trans-cinnamic acid, 2,5-dihydroxybenzoic acid, glycolic acid, trans-2-hexanoic acid, 2-hydroxyhexanoic acid, lactic acid, sorbic acid, tartaric acid, ferulic acid, succinic acid, pyridinecarboxylic acid, salicylic acid, maleic acid, saccharin, 4,4′-bipyridine-p-aminosalicylic acid, nicotinamide, urea, isonicotinamide, methyl 4-hydroxybenzoate, adipic acid, terephthalic acid, resorcinol, pyrogallol, phlorogallol, isoniazid, theophylline, adenine, theobromine, phenacetin, antipyrine, ethoxyphylline, and phenobarbital.

[0519] Synthesis method

[0520] In another aspect, the present invention relates to methods for manufacturing the compounds of the present invention or their pharmaceutically acceptable salts or stereoisomers. In a broader sense, the compounds of the present invention and their pharmaceutically acceptable salts and stereoisomers can be prepared by any process known to be suitable for the preparation of chemically related compounds. The compounds of the present invention will be better understood in conjunction with the synthetic schemes described in various working examples, which illustrate non-limiting methods for preparing compounds, such as those of formulas I-III.

[0521] In one of these aspects, the present invention relates to a method for preparing a compound of formula IV:

[0522]

[0523] Including compounds of formula I:

[0524] Reaction with compound of formula II:

[0525] In some embodiments, the compound of formula (I) may be administered together with the compound of formula (II) to form the compound of formula (IV) in vivo.

[0526] In one of these aspects, the present invention relates to a method for preparing compound V:

[0527]

[0528] Including compounds of formula I:

[0529] Reaction with compound of formula III:

[0530] In some embodiments, the compound of formula (I) may be administered together with the compound of formula (III) to form the compound of formula (V) in vivo. Synthetic schemes for attaching the active moiety to the compound are known in the art. See, for example, Agarwal et al., Bioconjugate Chem. 26(2): 176-192 (2015).

[0531] In one of these aspects, the present invention relates to a method for preparing compounds of formula IVa':

[0532]

[0533] Including compounds of formula Ia':

[0534] Reaction with compound of formula II':

[0535]

[0536] In one of these aspects, the present invention relates to a method for preparing compounds of formula IVb':

[0537]

[0538] Including compounds of formula Ib':

[0539] Reaction with compound of formula II':

[0540]

[0541] In one of these aspects, the present invention relates to a method for preparing compounds of formula IVc':

[0542]

[0543] Including compounds of formula Ic':

[0544] Reaction with compound of formula II':

[0545]

[0546] In some implementations, the reaction is carried out in the presence of a solvent.

[0547] In some embodiments, the solvent is an aprotic solvent. In some embodiments, the aprotic solvent is DCM, CHCl3, CCl4, DCE, toluene, MeCN, or THF.

[0548] In some embodiments, the solvent is a protic solvent. In some embodiments, the protic solvent is MeOH, EtOH, iPrOH, nBuOH, TFE, or HFIP.

[0549] In some embodiments, the solvent is a solvent mixture. In some embodiments, the solvent mixture is a mixture of aprotic and protic solvents. In some embodiments, the solvent mixture is 0-100% proton-to-aprotic solvent. In some embodiments, the solvent mixture is 0-100% TFE in CHCl3. In some embodiments, the solvent mixture is about 20% TFE in CHCl3.

[0550] In some embodiments, the reaction is carried out in the presence of a water-soluble buffer. In some embodiments, the water-soluble buffer is an acidic buffer. In some embodiments, the water-soluble buffer is an alkaline buffer.

[0551] In some embodiments, the reaction takes place in the presence of biological fluids. In some embodiments, the biological fluids are blood, synovial fluid, lymph, or vitreous fluid.

[0552] In some implementations, the reaction is carried out in the presence of an aqueous solution containing biological components such as cell lysates, proteins, nucleic acids, or lipids.

[0553] In some embodiments, the reaction is carried out with the addition of a buffer. Representative examples of buffers include ascorbic acid, glutathione, citric acid, acetic acid, potassium dihydrogen phosphate, N-cyclohexyl-2-aminoethanesulfonic acid (CHES), and borates. In some embodiments, the buffer is either ascorbic acid or glutathione.

[0554] In some embodiments, the reaction is carried out at a temperature of about -40°C to -80°C. In some embodiments, the reaction is carried out at a temperature between 0°C and 60°C. In some embodiments, the reaction is carried out at a temperature of about 60°C. In some embodiments, the reaction is carried out at a temperature of about 20°C to 25°C.

[0555] In some embodiments, the compound of formula (I) is in excess relative to the compound of formula (II) or (III). In some embodiments, the excess is about 10 equivalents. In some embodiments, the excess is about 5 equivalents.

[0556] In some implementations, the reaction occurs within one week. In some implementations, the reaction occurs within five days. In some implementations, the reaction occurs within three days. In some implementations, the reaction occurs within 24 hours. In some implementations, the reaction occurs within 18 hours. In some implementations, the reaction occurs within 12 hours. In some implementations, the reaction occurs within 6 hours. In some implementations, the reaction occurs within 3 hours. In some implementations, the reaction occurs within 2 hours. In some implementations, the reaction occurs within 1 hour. In some implementations, the reaction occurs within 45 minutes. In some implementations, the reaction occurs within 30 minutes. In some implementations, the reaction occurs within 15 minutes. In some implementations, the reaction occurs within 5 minutes. In some implementations, the reaction occurs within 1 minute.

[0557] Pharmaceutical Composition

[0558] Another aspect of the invention relates to pharmaceutical compositions comprising a therapeutically effective amount of the inventive compound or a pharmaceutically acceptable salt or stereoisomer thereof, and a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier," as known in the art, refers to a pharmaceutically acceptable material, composition, or carrier suitable for administration of the compound of the invention to mammals. Suitable carriers may include, for example, liquids (both aqueous and non-aqueous, and combinations thereof), solids, encapsulating materials, gases and combinations thereof (e.g., semi-solids), and gases, which function to carry or transport the compound from one organ or part of the body to another organ or part of the body. The carrier is "acceptable," meaning it is physiologically inert and compatible with other components of the formulation, and harmless to the subject or patient. Depending on the type of formulation, the composition may also include one or more pharmaceutically acceptable excipients.

[0559] In a broad sense, the compounds of the present invention and their pharmaceutically acceptable salts or stereoisomers can be formulated into compositions of a given type according to conventional pharmaceutical practices, such as conventional mixing, dissolving, granulation, sugar-coated pill making, pulverizing, emulsifying, encapsulating, and tableting processes (see, for example, Remington: The Science and Practice of Pharmacy (20th ed.), ed. ARGennaro, Lippincott Williams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and JCBoylan, 1988–1999, Marcel Dekker, New York). The type of formulation depends on the route of administration, which may include enteral (e.g., oral, sublingual, sublingual, and rectal), parenteral (e.g., subcutaneous (sc), intravenous (iv), intramuscular (im), and intrasternal injection, or infusion techniques, intraocular, intra-arterial, intramedullary, intrathecal, intracardiac, percutaneous, intradermal, intravaginal, intraperitoneal, mucosal, nasal, tracheal instillation, bronchial instillation, and inhalation), and topical (e.g., percutaneous). Generally, the most suitable route of administration will depend on a number of factors, including, for example, the nature of the formulation (e.g., its stability in the gastrointestinal environment) and / or the condition of the subject (e.g., whether the subject can tolerate oral administration). For example, parenteral (e.g., intravenous) administration may also be advantageous because, for example, in cases of single-dose treatment and / or acute discomfort, the compound can be administered relatively rapidly.

[0560] In some embodiments, the compound is formulated for oral or intravenous administration (e.g., systemic intravenous injection).

[0561] Therefore, the compounds of the present invention can be formulated into solid compositions (e.g., powders, tablets, dispersible granules, capsules, pouches, and suppositories), liquid compositions (e.g., solutions dissolving the compound, suspensions dispersing solid particles of the compound, emulsions and solutions containing liposomes, micelles or nanoparticles, syrups, and elixirs); semi-solid compositions (e.g., gels, suspensions, and creams); and gases (e.g., propellants for aerosol compositions). The compounds can also be formulated for immediate, intermediate, or sustained release.

[0562] Oral solid dosage forms include capsules, tablets, pills, powders, and granules. In these solid dosage forms, the active compound is coupled with a carrier such as sodium citrate or dicalcium phosphate, as well as other carriers or excipients such as a) fillers or extenders such as starch, lactose, sucrose, glucose, mannitol, and silica; b) binders such as methylcellulose, microcrystalline cellulose, hydroxypropyl methylcellulose, carboxymethyl cellulose, sodium carboxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and gum arabic; c) humectants such as glycerin; and d) disintegrants such as cross-linked polymers (e.g., cross-linked polyethylene glycol). The mixture contains: e) pyrrolidone (crosspovidone), croscarmellose sodium (crosspovidone carboxymethyl cellulose), sodium glycolate starch, agar, calcium carbonate, potato or cassava starch, alginate, certain silicates and sodium carbonate; f) solution retardants such as paraffin; f) absorption enhancers such as quaternary ammonium compounds; g) wetting agents such as cetyl alcohol and glyceryl monostearate; h) absorbents such as kaolin and bentonite; and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium dodecyl sulfate and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also include a buffer. Similar types of solid compositions can also be used as fillers for soft-filled and hard-filled gelatin capsules using excipients such as lactose (lactose or milk sugar) and high molecular weight polyethylene glycol. Solid dosage forms of tablets, sugar-coated pills, capsules, pills and granules can be prepared using coatings and shells such as enteric coatings and other coatings. They may also contain light-blocking agents.

[0563] In some embodiments, the compounds of the present invention can be formulated into hard or soft gelatin capsules. Representative excipients that can be used include pregelatinized starch, magnesium stearate, mannitol, sodium stearoyl fumarate, anhydrous lactose, microcrystalline cellulose, and croscarmellose sodium. The gelatin shell may include gelatin, titanium dioxide, iron oxide, and colorants.

[0564] Liquid dosage forms for oral administration include solutions, suspensions, emulsions, microemulsions, syrups, and elixirs. In addition to the compound, liquid dosage forms may contain aqueous or non-aqueous carriers commonly used in the art (depending on the solubility of the compound), such as water or other solvents, solubilizers, and emulsifiers such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butanediol, dimethylformamide, oils (particularly cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerin, tetrahydrofurfuryl alcohol, polyethylene glycol, and sorbitan fatty acid esters and mixtures thereof. Oral compositions may also include excipients such as wetting agents, suspending agents, coloring agents, sweeteners, flavoring agents, and aromatizers.

[0565] Injectable formulations intended for parenteral administration may include sterile aqueous solutions or oil-containing suspensions. They can be formulated using suitable dispersants or wetting agents and suspending agents according to standard techniques. Sterile injectable formulations are also sterile injectable solutions, suspensions, or emulsions in non-toxic, parenteral-acceptable diluents or solvents, such as solutions dissolved in 1,3-butanediol. Acceptable carriers and solvents that can be used are water, Ringer's solution, USP, and isotonic sodium chloride solution. In addition, sterile fixed oils are typically used as solvents or suspension media. For this purpose, any mild fixed oil can be used, including synthetic monoglycerides or diglycerides. Furthermore, fatty acids (such as oleic acid) are used in the preparation of injections. Injectable formulations are sterilized, for example, by filtration through a bacterial trap filter, or by incorporating a sterilizing agent into a sterile solid composition, which can then be dissolved or dispersed in sterile water or other sterile injection media prior to use. The duration of action can be prolonged by slowing down the absorption of the compound, which can be achieved by using liquid suspensions or crystalline or amorphous materials with poor water solubility. Prolonged absorption of compounds from parenteral administration can also be achieved by suspending the compounds in an oily solvent.

[0566] In some embodiments, the compounds of the present invention can be administered locally rather than systemically, for example, by directly injecting the conjugate into an organ, typically in the form of a reservoir formulation or a sustained-release formulation. In specific embodiments, long-acting formulations are administered via implantation (e.g., subcutaneous or intramuscular) or intramuscular injection. Injectable long-acting formulations are manufactured by forming a microcapsule matrix of the compound in a biodegradable polymer (e.g., polylactic-polyglycolic acid, polyorthoester, and polyanhydride). The release rate of the compound can be controlled by varying the ratio of the compound to the polymer and the properties of the specific polymer used. Injectable long-acting formulations are also prepared by encapsulating the compound in liposomes or microemulsions that are compatible with body tissues. Furthermore, in other embodiments, the compound is delivered in a targeted drug delivery system, for example, in liposomes coated with organ-specific antibodies. In such embodiments, the liposomes target the organ and are selectively taken up by the organ.

[0567] The composition can be formulated for sublingual or oral administration, examples of which include tablets, lozenges, and gels.

[0568] The compounds of this invention can be formulated for inhalation. Various forms suitable for inhalation include aerosols, sprays, or powders. The pharmaceutical composition can be delivered from a pressurized package or nebulizer in the form of an aerosol spray using a suitable propellant (e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas). In some embodiments, the dosage unit of the pressurized aerosol can be determined by providing a valve to deliver a measured amount. In some embodiments, capsules and cartridges comprising gelatin (e.g., for inhalers or blowpipes) can be formulated into a powder mixture containing the compound and a suitable powder base such as lactose or starch.

[0569] The compounds of this invention can be formulated for surface application, which, as used herein, means intradermal application of the formulations of this invention to the epidermis. These types of compositions are typically available in the form of ointments, pastes, creams, lotions, gels, solutions, and sprays.

[0570] Representative examples of carriers used to formulate surface-applied compounds include solvents (e.g., alcohols, polyols, water), creams, lotions, ointments, oils, plasters, liposomes, powders, emulsions, microemulsions, and buffers (e.g., hypotonic or buffered saline). For example, creams can be formulated using saturated or unsaturated fatty acids such as stearic acid, palmitic acid, oleic acid, palmitoleic acid, cetyl alcohol, or oleyl alcohol. Creams may also contain nonionic surfactants such as poly(40) stearate.

[0571] In some embodiments, the surface formulation may also include excipients, examples of which are penetration enhancers. These formulations are capable of transporting pharmacologically active compounds across the stratum corneum and into the epidermis or dermis, preferably with little or no systemic absorption. The effectiveness of various compounds in enhancing the rate of skin penetration of drugs has been evaluated. See, for example, Percutaneous Penetration Enhancers, Maibach HI and Smith HE (eds.), CRC Press, Inc., Boca Raton, Fla. (1995) (which investigated the use and testing of various skin penetration enhancers) and Buyuktimkin et al., Chemical Means of Transdermal Drug Permeation Enhancement in Transdermal and Topical Drug Delivery Systems, Gosh TK, Pfister WR, Yum S.I. (eds.), Interpharm Press Inc., Buffalo Grove, Ill. (1997). Representative examples of penetration enhancers include triglycerides (e.g., soybean oil), aloe vera compositions (e.g., aloe vera gel), ethanol, isopropanol, octylphenyl polyethylene glycol, oleic acid, polyethylene glycol 400, propylene glycol, N-decyl methyl sulfoxide, fatty acid esters (e.g., isopropyl myristate, methyl laurate, glyceryl monooleate, and propylene glycol monooleate), and N-methylpyrrolidone.

[0572] Representative examples of other excipients that may be included in surface formulations and other types of formulations (to the extent they are compatible) include preservatives, antioxidants, humectants, emollients, buffers, solubilizers, skin protectants, and surfactants. Suitable preservatives include alcohols, quaternary ammonium compounds, organic acids, benzoates, and phenols. Suitable antioxidants include ascorbic acid and its esters, sodium bisulfite, butylated hydroxytoluene, butylated hydroxyanisole, tocopherol, and chelating agents such as EDTA and citric acid. Suitable humectants include glycerin, sorbitol, polyethylene glycol, urea, and propylene glycol. Suitable buffers include citric acid, hydrochloric acid, and lactate buffers. Suitable solubilizers include quaternary ammonium hydrochloride, cyclodextrins, benzyl benzoate, lecithin, and polysorbate. Suitable skin protectants include vitamin E oil, allantoin, dimethyl silicone oil, glycerin, petrolatum, and zinc oxide.

[0573] Transdermal formulations typically employ transdermal delivery devices and transdermal delivery patches, where compounds are formulated into lipophilic emulsions or buffered aqueous solutions dissolved in and / or dispersed in a polymer or binder. Patches can be constructed for continuous, pulsatile, or on-demand delivery of agents. Transdermal delivery of compounds can be achieved via iontophoresis patches. Transdermal patches can provide controlled delivery of compounds, where absorption rates are slowed by using rate-controlled membranes or by trapping the compound within a polymer matrix or gel. Absorption enhancers can be used to increase absorption; examples include absorbable, pharmaceutically acceptable solvents that facilitate transdermal absorption.

[0574] Ophthalmic preparations include eye drops.

[0575] Formulations for rectal administration include enemas, rectal gels, rectal foams, rectal aerosols, and retention enemas, which may contain conventional suppository bases such as cocoa butter or other glycerides, and synthetic polymers such as polyvinylpyrrolidone, PEG, etc. Compositions for rectal or vaginal administration can also be formulated as suppositories, which can be prepared by mixing compounds with suitable non-irritating carriers and excipients such as cocoa butter, mixtures of fatty acid glycerides, polyethylene glycol, suppository waxes, and combinations thereof, all of which are solid at ambient temperature but liquid at body temperature, and thus melt and release the compounds in the rectal or vaginal cavity.

[0576] dose

[0577] As used herein, the term "therapeuticly effective amount" refers to the amount of the compound of the present invention (containing a therapeutic portion or being therapeutic) or a pharmaceutically acceptable salt or stereoisomer thereof that effectively produces the desired therapeutic response in a patient. The term "therapeutic response" includes an amount of the compound of the present invention or a pharmaceutically acceptable salt or stereoisomer thereof that, when administered, induces a positive change in the disease or discomfort to be treated, or is sufficient to prevent the development or progression of the disease or discomfort, or to some extent alleviates one or more symptoms of the disease or discomfort being treated in a subject, or inhibits the growth of diseased cells.

[0578] As used herein, the term "diagnostic effective amount" refers to the amount of the compound of the present invention (which contains a diagnostic portion) or a pharmaceutically acceptable salt or stereoisomer thereof that effectively produces the desired detectable response in a patient.

[0579] The total daily dose of the compound and its administration can be determined according to standard medical practice, for example, by the attending physician using reasonable medical judgment. The specific therapeutically effective dose for any particular subject will depend on a number of factors, including: the disease or discomfort to be treated and its severity (e.g., its current state); the activity of the compound used; the specific composition used; the subject's age, weight, general health condition, sex, and diet; the timing, route of administration, and excretion rate of the compound used; the duration of treatment; drugs used in combination with or concurrently with the specific compound used; and similar factors known in the medical community (see, for example, Hardman et al., eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th Edition, McGraw-Hill Press, 155-173, 2001).

[0580] The compounds of this invention are effective over a wide dosage range. In some embodiments, the total daily dose (e.g., for adults) can range from about 0.001 mg to about 1600 mg, 0.01 mg to about 1000 mg, 0.01 mg to about 500 mg, about 0.01 mg to about 100 mg, about 0.5 mg to about 100 mg, about 1 mg to about 100-400 mg daily, about 1 mg to about 50 mg daily, about 5 mg to about 40 mg daily, and in other embodiments about 10 mg to about 30 mg daily. Depending on the frequency of daily administration of the compound, a single dose containing the desired amount can be formulated. As an example, capsules can be formulated from about 1 mg to about 200 mg of the compound (e.g., 1 mg, 2 mg, 2.5 mg, 3 mg, 4 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 50 mg, 100 mg, 150 mg, and 200 mg). In some embodiments, the compound may be administered at doses ranging from about 0.01 mg to about 200 mg / kg body weight daily. In some embodiments, doses of 0.1 to 100 of one or more doses daily, such as 1 mg / kg to 30 mg / kg daily, may be effective. As an example, suitable doses for oral administration may be in the range of 1 mg / kg to 30 mg / kg body weight daily, while suitable doses for intravenous administration may be in the range of 1 mg / kg to 10 mg / kg body weight daily.

[0581] How to use

[0582] In some aspects, the present invention relates to a method for treating a disease or discomfort, which involves administering a therapeutically effective amount of a compound of formula (IV or V) to a subject in need, wherein... and One is a therapeutic agent, or one in which the compound or its pharmaceutically acceptable salt or stereoisomer is therapeutic. In some embodiments, the disease is cancer.

[0583] In some aspects, the present invention relates to a method of treating cancer, which involves administering to a subject in need a therapeutically effective amount of a compound of formula IV′ or a pharmaceutically acceptable salt or stereoisomer thereof and a diboron reagent. In some embodiments, the diboron reagent is a symmetrical diboron reagent. In some embodiments, the diboron reagent is an asymmetrical diboron reagent. In some embodiments, the diboron reagent is B2(OH)4, B2pin2, etc. Other representative examples of diboron reagents include bis(catechol)boron, bis(vinyl glycol)diboron, bis[(-)pinenediol]diboron, bis(diisopropyl-L-diethyl tartrate)diboron, bis(N,N,N′,N′-tetramethyl-D-tartrateamine glycol)diboron, and 2,2′-bi-1,3,2-oxoborane. However, other diboron reagents suitable for use in this invention are disclosed in Aliet al., Studies in Inorganic Chemistry, “Chapter 1-Chemistry of thediboron compounds” 22:1-57 (2005); Neeve et al., Chem. Rev. 116(16):9091-9161 (2016); Ding et al., Molecules 24(7):1325 (2019). In some embodiments, the boron reagent is applied at a concentration of about 1 pM to about 1 mM. In some embodiments, the boron reagent is applied at a concentration of about 1 pM to about 100 mM. In some embodiments, the boron reagent is applied at a concentration of about 1 pM to about 10 mM. In some embodiments, the boron reagent is applied at a concentration of about 1 pM to about 1 mM. In some embodiments, the boron reagent is applied at a concentration of about 1 pM to about 100 μM. In some embodiments, the boron reagent is applied at a concentration of about 1 pM to about 10 μM. In some embodiments, the boron reagent is applied at a concentration of about 1 pM to about 1 μM. In some embodiments, the boron reagent is applied at a concentration of about 1 pM to about 100 nM. In some embodiments, the boron reagent is applied at a concentration of about 1 pM to about 10 nM. In some embodiments, the boron reagent is applied at a concentration of about 1 pM to about 1 nM. In some embodiments, the boron reagent is applied at a concentration of about 1 pM to about 100 pM.

[0584] In some embodiments, the method of the present invention requires administering a compound of formula (I) and a compound of formula (II or III) or a pharmaceutically acceptable salt or stereoisomer thereof to a subject in need, wherein and One is a therapeutic agent, or a compound formed by a reaction between compounds of formula (I) and (II) and between compounds of formula (I) and (III) is therapeutic. Compounds of formula (I) and (II or III), and their pharmaceutically acceptable salts and stereoisomers, can be used in combination or simultaneously to treat a disease or discomfort. In this context, the terms "combination" and "simultaneously" mean that the compounds are administered together by the same or separate dosage forms, and by the same or different methods of administration, or sequentially, for example, as part of the same regimen, which includes substantially simultaneous administration. The order and time intervals can be determined such that they can react together. For example, the compounds can be administered sequentially in any order, either simultaneously or at different time points; however, if not simultaneously, they can be administered at sufficiently close time intervals to provide the desired therapeutic effect. Therefore, these terms are not limited to administering the active agent at exactly the same time. In some embodiments, the method relates to treating cancer.

[0585] In some aspects, the present invention relates to methods for treating and diagnosing diseases or discomforts, which require administering to a subject in need a compound of formula (IV or V) or a pharmaceutically acceptable salt or stereoisomer thereof, wherein the compound is present in the form of a therapeutic diagnostic agent. In some embodiments, the disease is cancer.

[0586] In some aspects, the present invention relates to therapeutic diagnostic agents for the treatment and diagnosis of diseases or ailments such as cancer, which require administration to a subject in need of a compound of formula (I) and a compound of formula (II or III) or a pharmaceutically acceptable salt or stereoisomer thereof.

[0587] In some aspects, the present invention relates to a method for protein labeling that involves administering to a subject in need a compound of formula (IV or V) or a pharmaceutically acceptable salt or stereoisomer thereof, wherein the compound of formula (IV or V) contains a diagnostic moiety and a binding moiety. In some embodiments, the method involves labeling cancer-associated antigens. Tumor-associated antigens suitable for use in the present invention are disclosed in Ilyas et al., J. Immunol. 195(11): 5117-5122 (2015) and Haen et al., Nat. Rev. Clin. Oncol. 17: 595-610 (2020).

[0588] "Disease" is generally considered to be a subject's state of health in which the subject is unable to maintain homeostasis, and in which the subject's health continues to deteriorate if the disease is not improved. In contrast, a subject's "discomfort" is a state of health in which the subject is able to maintain homeostasis, but the subject's health is not as good as it would be without discomfort. Discomfort does not necessarily lead to a further decline in the subject's health without treatment. In some embodiments, the compounds of the present invention can be used to treat proliferative disorders and discomfort (e.g., cancer or benign tumors). As used herein, the term "proliferative disorder or discomfort" refers to a condition characterized by disordered or abnormal cell growth, or both, including non-cancerous conditions such as growths, precancerous conditions, benign tumors, and cancer.

[0589] As used herein, the term "subject" (or "patient") includes all members of the animal kingdom who are susceptible to or suffer from the said disease or discomfort. In some embodiments, the subject is a mammal, such as a human or non-human mammal. The method is also applicable to companion animals, such as dogs and cats, and livestock, such as cattle, horses, sheep, goats, pigs, and other domestic and wild animals. According to the invention, a subject "in need" of treatment may "have or be suspected of having" a specific disease or discomfort, and may have been diagnosed or otherwise exhibit a sufficient number of risk factors or a sufficient number of signs or symptoms, or a combination thereof, to allow a medical professional to diagnose or suspect that the subject has the disease or discomfort. Therefore, subjects who have and are suspected of having a specific disease or discomfort are not necessarily two distinct groups.

[0590] The compounds of this invention can be used to treat and / or diagnose a variety of diseases and discomforts, including cancerous and non-cancerous conditions. Exemplary types of non-cancerous (e.g., proliferative) diseases or discomforts that can be treated with the compounds of this invention include inflammatory diseases and conditions, autoimmune diseases, heart disease, viral diseases, chronic and acute kidney disease or injury, metabolic diseases, and allergic and genetic diseases.

[0591] Specific examples of non-cancerous conditions and discomforts include rheumatoid arthritis, alopecia areata, lymphoproliferative disorders, autoimmune blood disorders (e.g., hemolytic anemia, aplastic anemia, anhidrotic ectodermal dysplasia, simple erythrocytic anemia, and idiopathic thrombocytopenic purpura), cholecystitis, acromegaly, rheumatoid spondylitis, osteoarthritis, gout, scleroderma, sepsis, septic shock, dacryoadenitis, cold pyridine-associated periodic syndrome (CAPS), endotoxic shock, endometritis, Gram-negative sepsis, keratoconjunctivitis sicca, and toxic shock. Syndrome, asthma, adult respiratory distress syndrome, chronic obstructive pulmonary disease, chronic lung inflammation, chronic graft rejection, hidradenitis suppurativa, inflammatory bowel disease, Crohn's disease, Behçet's syndrome, systemic lupus erythematosus, glomerulonephritis, multiple sclerosis, juvenile diabetes, autoimmune uveitis, autoimmune vasculitis, thyroiditis, Addison's disease, lichen planus, appendicitis, bullous pemphigus, pemphigus vulgaris, pemphigus foliaceus, paraneoplastic pemphigus, myasthenia gravis, immunoglobulin A nephropathy, Hashimoto's disease, Sjögren's syndrome, vitiligo, Wegener's granulation tissue Swelling, granulomatous orchitis, autoimmune oophoritis, sarcoidosis, rheumatic heart disease, ankylosing spondylitis, Graves' disease, autoimmune thrombocytopenic purpura, psoriasis, psoriatic arthritis, eczema, herpetic dermatitis, ulcerative colitis, pancreatic fibrosis, hepatitis, liver fibrosis, CD14-mediated sepsis, non-CD14-mediated sepsis, acute and chronic kidney disease, irritable bowel syndrome, fever (pyresis), restenosis, cervicitis, stroke and ischemic injury, neurotrauma, acute and chronic pain, allergic rhinitis, allergic conjunctivitis, chronic heart failure Heart failure, congestive heart failure, acute coronary syndrome, cachexia, malaria, leprosy, leishmaniasis, Lyme disease, Reiter's syndrome, acute synovitis, muscle degeneration, bursitis, tendinitis, tenosynovitis, intervertebral disc herniation, rupture or extrusion syndrome, osteosclerosis, sinusitis, thrombosis, silicosis, pulmonary sarcoma, bone resorption diseases such as osteoporosis, fibromyalgia, AIDS and other viral diseases such as herpes zoster, herpes simplex type I or II, influenza virus and cytomegalovirus, type I and type II diabetes, obesity, insulin resistance and diabetic retinopathy, 22q11.2. Deficiency syndromes, Angelman syndrome, Canavan disease, celiac disease, Charcot-Marie-Tooth disease, color blindness, Cri-Cry syndrome, Down syndrome, cystic fibrosis, Duchenne muscular dystrophy, hemophilia, Klinefelter syndrome, neurofibromatosis, phenylketonuria, Prad-Willi syndrome, sickle cell disease, Ty Sachs disease, Turner syndrome, urea cycle disorder, thalassemia, otitis media, pancreatitis, mumps, pericarditis, peritonitis, pharyngitis, pleurisy, phlebitis, pneumonia, uveitis, polymyositis, proctitis, interstitial pulmonary fibrosis, dermatomyositis, atherosclerosis, arteriosclerosis, amyotrophic lateral sclerosis, social inability, varicose veins, vaginitis, depression, and sudden infant death syndrome.

[0592] In some embodiments, the method involves treating a subject with cancer. Generally, the compounds of this invention are effective in treating cancers (including solid tumors, both primary and metastatic), sarcomas, melanomas, and blood cancers (cancers affecting the blood, including lymphocytes, bone marrow, and / or lymph nodes), such as leukemia, lymphoma, and multiple myeloma. This includes adult tumors / cancers and childhood tumors / cancers. Cancers can be vascularized, or substantially non-vascularized or non-vascularized tumors.

[0593] Representative examples of cancers include adrenocortical carcinoma, AIDS-related cancers (e.g., Kaposi's sarcoma and AIDS-related lymphoma), appendix cancer, childhood cancers (e.g., pediatric cerebellar astrocytoma, pediatric brain astrocytoma), basal cell carcinoma, skin cancer (non-melanoma), bile duct cancer, extrahepatic bile duct cancer, intrahepatic bile duct cancer, bladder cancer, urinary bladder cancer, brain cancer (e.g., gliomas and glioblastomas such as brainstem glioma, gestational trophoblastic tumor glioma, cerebellar astrocytoma, brain astrocytoma / malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumor). Tumors, visual pathway and hypothalamic gliomas, breast cancer, bronchial adenoma / carcinoid, carcinoid tumors, nervous system cancers (e.g., central nervous system cancers, central nervous system lymphomas), cervical cancer, chronic myeloproliferative disorders, colorectal cancers (e.g., colon cancer, rectal cancer), polycythemia vera, lymphoid tumors, mycosis fungoides, Sezary syndrome, endometrial cancer, esophageal cancer, extracranial ectodermal tumors, gonadal ectodermal tumors, extrahepatic bile duct cancer, ocular cancer, intraocular melanoma, retinoblastoma, gallbladder cancer, gastrointestinal cancers (e.g., gastric cancer, small bowel cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors (GIST)), germ cell tumors, ovarian germ cell tumors, head and neck cancer, Hodgkin's lymphoma, leukemia, lymphoma, multiple myeloma, hepatocellular carcinoma, hypopharyngeal cancer, intraocular melanoma, ocular cancer, islet cell carcinoma Tumors (endocrine pancreas), renal cell carcinoma (e.g., nephroblastoma, clear cell renal cell carcinoma), liver cancer, lung cancer (e.g., non-small cell lung cancer and small cell lung cancer), Waldenström macroglobulinemia, melanoma, intraocular (ocular) melanoma, Merkel cell carcinoma, mesothelioma, occult metastatic squamous cell carcinoma of the primary neck lesion, multiple endocrine adenomas (MEN), myelodysplastic syndrome, essential thrombocytosis, myelodysplastic / myeloproliferative disorders, nasopharyngeal carcinoma, neuroblastoma, oral cancer (oral Cancer (e.g., oral cancer, lip cancer, oral cavity cancer, tongue cancer, oropharyngeal cancer, pharyngeal cancer, laryngeal cancer), ovarian cancer (e.g., ovarian epithelial cancer, ovarian germ cell tumor, low-grade malignant potential ovarian tumor), pancreatic cancer, islet cell pancreatic cancer, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal blastoma, pituitary adenoma, plasma cell tumor, pleural pulmonary blastoma, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, uterine cancer (e.g.,Endometrial cancer, uterine sarcoma, uterine corpus cancer), squamous cell carcinoma, testicular cancer, thymoma, thymic carcinoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter and other urinary organs, urethral cancer, gestational trophoblastic tumor, vaginal cancer, and vulvar cancer.

[0594] Sarcomas that can be treated with the compounds of this invention include both soft tissue cancers and bone cancers, and representative examples include osteosarcoma or osteoblastoma (bone) (e.g., Ewing's sarcoma), chondrosarcoma (cartilage), leiomyosarcoma (smooth muscle), rhabdomyosarcoma (skeletal muscle), mesothelial sarcoma or mesothelioma (membranous lining of body cavities), fibrosarcoma (fibrous tissue), angiosarcoma or angioendothelioma (blood vessels), liposarcoma (adipose tissue), glioma or astrocytoma (neurogenic connective tissue found in the brain), myxosarcoma (primordial embryonic connective tissue), and mesenchymal tumor or mixed mesodermal tumor (mixed connective tissue type).

[0595] In some embodiments, the method of the present invention relates to treating a subject suffering from or in distress a proliferative disease of the blood system, liver, brain, lungs, colon, pancreas, prostate, ovaries, breasts, skin, and endometrium.

[0596] As used in this article, “proliferative disorders or conditions of the hematopoietic system” include lymphoma, leukemia, bone marrow tumors, mast cell tumors, myelodyplasia, benign monoclonal globulinosis, polycythemia vera, chronic myeloid leukemia, idiopathic extramedullary metaplasia, and essential thrombocythemia. Therefore, representative examples of hematologic malignancies may include multiple myeloma, lymphomas (including T-cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma (diffuse large B-cell lymphoma (DLBCL)), follicular lymphoma (FL), mantle cell lymphoma (MCL), and ALK+ anaplastic large cell lymphoma (e.g., B-cell non-Hodgkin lymphoma selected from diffuse large B-cell lymphoma (e.g., germinal center B-cell-like diffuse large B-cell lymphoma or activated B-cell-like diffuse large B-cell lymphoma)), Burkitt lymphoma / leukemia, mantle cell lymphoma, mediastinal (thymic) large B-cell lymphoma, follicular lymphoma, etc. Tumors, marginal zone lymphomas, lymphoplasmacytic lymphomas / Walden's macroglobulinemia, metastatic pancreatic cancer, refractory B-cell non-Hodgkin lymphomas and relapsed B-cell non-Hodgkin lymphomas, childhood lymphomas, and lymphomas of lymphocytic and cutaneous origin, such as small lymphocytic lymphomas, leukemias including childhood leukemia, hairy cell leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, acute myeloid leukemia (e.g., acute monocytic leukemia), chronic lymphocytic leukemia, small lymphocytic leukemia, chronic myeloid leukemia, chronic myeloid leukemia and mast cell leukemia, bone marrow tumors and mast cell tumors.

[0597] As used herein, “cell proliferative disorders or conditions of the liver” includes all forms of cell proliferative disorders affecting the liver. Cell proliferative disorders of the liver can include liver cancer (e.g., hepatocellular carcinoma, intrahepatic cholangiocarcinoma, and hepatoblastoma), precancerous lesions or precancerous states of the liver, benign growths or lesions of the liver, and malignant growths or lesions of the liver, as well as metastatic lesions in other body tissues and organs. Cell proliferative disorders of the liver can include hyperplasia, metaplasia, and developmental abnormalities of the liver.

[0598] As used herein, “brain cell proliferative disorders or conditions” encompasses all forms of proliferative disorders affecting the brain. Brain cell proliferative disorders can include brain cancers (e.g., gliomas, glioblastomas, meningiomas, pituitary adenomas, vestibular schwannomas, and primitive neuroectodermal tumors (medulloblastomas)), precancerous lesions or precancerous states of the brain, benign growths or lesions of the brain, malignant growths or lesions of the brain, and metastatic lesions of body tissues and organs other than the brain. Brain cell proliferative disorders can include brain hyperplasia, metaplasia, and developmental abnormalities.

[0599] As used herein, “pulmonary cell proliferative disorders or conditions” encompasses all forms of cell proliferative disorders affecting lung cells. Pulmonary cell proliferative disorders include lung cancer, precancerous lesions or precancerous conditions, benign growths or lesions of the lung, pulmonary hyperplasia, metaplasia, and developmental abnormalities, as well as metastatic lesions in body tissues and organs other than the lungs. Lung cancer includes all forms of lung cancer, such as malignant lung tumors, carcinoma in situ, typical carcinoid tumors, and atypical carcinoid tumors. Lung cancer includes small cell lung cancer (“SLCL”), non-small cell lung cancer (“NSCLC”), squamous cell carcinoma, adenocarcinoma, small cell carcinoma, large cell carcinoma, squamous cell carcinoma, and mesothelioma. Lung cancer can include “scar carcinoma”, bronchoalveolar carcinoma, giant cell carcinoma, spindle cell carcinoma, and large cell neuroendocrine carcinoma. Lung cancer also includes lung tumors with histological and ultrastructural heterogeneity (e.g., mixed cell types). In some embodiments, the compounds of the present invention can be used to treat non-metastatic or metastatic lung cancer (e.g., NSCLC, ALK-positive NSCLC, NSCLC with ROS1 rearrangement, lung adenocarcinoma, and lung squamous cell carcinoma).

[0600] As used in this article, “colonic proliferative disorders or conditions” encompasses all forms of proliferative disorders affecting colon cells, including colon cancer, precancerous lesions or precancerous states of the colon, colonic adenomatous polyps, and metachronic colonic lesions. Colon cancer includes sporadic and hereditary colon cancer, malignant colonic tumors, carcinoma in situ, typical and atypical carcinoid tumors, adenocarcinoma, squamous cell carcinoma, and squamous cell carcinoma. Colon cancer may be associated with hereditary syndromes such as hereditary nonpolyposis colorectal cancer, familial adenomatous polyposis, MYH-associated polyposis, Gardner syndrome, Peutz-Jeghers syndrome, Tecot syndrome, and juvenile polyposis. Colonic proliferative disorders may also be characterized by colonic hyperplasia, metaplasia, and developmental abnormalities.

[0601] As used herein, “pancreatic cell proliferative disorders or lesions” encompasses all forms of cell proliferative disorders affecting pancreatic cells. Pancreatic cell proliferative disorders include pancreatic cancer, pancreatic precancerous lesions or precancerous conditions, pancreatic hyperplasia, pancreatic dysplasia, benign growths or lesions of the pancreas, and malignant growths or lesions of the pancreas, as well as metastatic lesions in other body tissues and organs outside the pancreas. Pancreatic cancer includes all forms of cancer of the pancreas, including ductal adenocarcinoma, adenosquamous carcinoma, pleomorphic giant cell carcinoma, mucinous adenocarcinoma, osteoclastoid giant cell carcinoma, mucinous cystadenocarcinoma, acinar carcinoma, unclassified large cell carcinoma, small cell carcinoma, pancreatoblastoma, papillary tumor, mucinous cystadenoma, papillary cystic tumor, and serous cystadenoma, as well as pancreatic tumors with histological and ultrastructural heterogeneity (e.g., mixed cell types).

[0602] As used herein, “prostate cell proliferative disorders or conditions” includes all forms of cell proliferative disorders affecting the prostate. Prostate cell proliferative disorders can include prostate cancer, precancerous lesions or precancerous conditions of the prostate, benign growths or lesions of the prostate, and malignant growths or lesions of the prostate, as well as metastatic lesions in other body tissues and organs outside the prostate. Prostate cell proliferative disorders include benign prostatic hyperplasia, metaplasia, and developmental abnormalities.

[0603] As used in this article, "ovarian cell proliferative disorders or conditions" includes all forms of cell proliferative disorders affecting ovarian cells. Ovarian cell proliferative disorders include precancerous lesions or precancerous conditions of the ovary, benign growths or lesions of the ovary, ovarian cancer, and metastatic lesions in body tissues and organs outside the ovary. Ovarian cell proliferative disorders may include ovarian hyperplasia, metaplasia, and developmental abnormalities.

[0604] As used in this article, "breast cell proliferative disorders or dysplasia" includes all forms of cell proliferative dysplasia affecting breast cells. Breast cell proliferative dysplasia includes breast cancer, precancerous lesions or conditions, benign growths or lesions of the breast, and metastatic lesions in other tissues and organs outside the breast. Breast cell proliferative dysplasia can include breast hyperplasia, metaplasia, and developmental abnormalities.

[0605] As used herein, “dermatoproliferative disorders or conditions” include all forms of cell proliferation disorders affecting skin cells. Dermatoproliferative disorders include precancerous lesions or precancerous conditions of the skin, benign growths or lesions of the skin, malignant melanoma or other malignant growths or lesions of the skin, and metastatic lesions in body tissues and organs outside the skin. Dermatoproliferative disorders can include skin hyperplasia, metaplasia, and developmental abnormalities.

[0606] As used herein, "endometrial cell proliferative disorders or conditions" encompasses all forms of cell proliferative disorders affecting endometrial cells. Endometrial cell proliferative disorders include precancerous lesions or precancerous conditions of the endometrium, benign growths or lesions of the endometrium, endometrial cancer, and metastatic lesions in body tissues and organs outside the endometrium. Endometrial cell proliferative disorders can include endometrial hyperplasia, metaplasia, and developmental abnormalities.

[0607] The compounds of this invention, and their pharmaceutically acceptable salts and stereoisomers, can be administered to patients, such as cancer patients, as monotherapy or in combination therapy. Treatment can be "front-line," i.e., as initial treatment for patients who have not previously received anticancer therapy, whether used alone or in combination with other treatments; or "second-line," as treatment for patients who have previously received anticancer therapy, whether used alone or in combination with other treatments; or as "third-line," "fourth-line," etc., treatment, whether used alone or in combination with other treatments. Treatment can also be given to patients whose previous treatments were unsuccessful or partially successful but who are unresponsive to or intolerant of a particular treatment. Treatment can also be given as adjuvant therapy, i.e., to prevent cancer recurrence in patients with currently undetectable disease or after surgical removal of a tumor. Therefore, in some embodiments, the compounds can be administered to patients who have received prior treatments such as chemotherapy, radioimmunotherapy, surgery, immunotherapy, radiation therapy, targeted therapy, or any combination thereof.

[0608] The methods of the present invention may require administering the compounds of the present invention or pharmaceutical compositions thereof to a patient in single or multiple doses (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 10, 15, 20 or more doses). For example, the frequency of administration can range from once daily to approximately once every eight weeks. In some embodiments, the frequency of administration ranges from approximately once daily for 1, 2, 3, 4, 5 or 6 weeks, while in other embodiments, at least one 28-day cycle is required, which includes daily administration for 3 weeks (21 days), followed by a 7-day withdrawal period. In other embodiments, the compound may be administered twice daily (BID) for a two-and-a-half-day treatment course (5 doses in total) or once daily (QD) for a two-day treatment course (2 doses in total). In other embodiments, the compound may be administered once daily (QD) for a five-day treatment course.

[0609] Drug kit

[0610] The compositions of the present invention can be assembled into a kit or pharmaceutical system. According to this aspect of the invention, the kit or pharmaceutical system includes a carrier or packaging, such as a box, carton, tube, or the like, wherein one or more containers, such as vials, tubes, ampoules, or bottles, are tightly sealed and contain the compound of the present invention or a pharmaceutical composition containing the compound and a pharmaceutically acceptable carrier, wherein the compound and the carrier may be placed in the same or separate containers. The kit or pharmaceutical system of the present invention may also include printed instructions for using the compound and composition.

[0611] These and other aspects of the invention will be further understood when considering the following embodiments, which are intended to illustrate certain specific implementations of the invention but are not intended to limit its scope as defined by the claims. Example

[0612] Example 1: General information, materials and instruments.

[0613] General information

[0614] Unless otherwise specified, all reactions were carried out in flame-dried round-bottom flasks under positive pressure nitrogen. Air- and moisture-sensitive liquids were transferred using a gas-tight syringe with a stainless steel needle or cannula. Granular silica gel (60- Rapid column chromatography was performed using pore sizes of 40–63 μm (Silycle). Thin-layer chromatography (TLC) was performed using 0.25 mm silica gel pre-coated glass plates impregnated with a fluorescent indicator (254 nm, Silycle). The TLC plates were visualized by exposure to short-wave ultraviolet light (254 nm) and / or an aqueous potassium permanganate (KMnO4) solution. Unless otherwise specified, organic solutions were concentrated at 20 °C on a rotary evaporator capable of reaching a minimum pressure of approximately 2 Torr. Room temperature was defined as 22.5 ± 2.5 °C. UCON was used. TM The reaction is heated by a fluid heating bath.

[0615] General Chemical Materials

[0616] All solvents were purchased from Fisher Scientific or Sigma-Aldrich. Unless otherwise specified, chemical reagents were purchased from Fisher Scientific, Sigma-Aldrich, Alfa Aesar, Oakwood Chemical, AcrosOrganics, Combi Blocks, or TCI America. CMA refers to a solution of 80:18:2 v / v / v chloroform:methanol (MeOH):ammonium hydroxide (28-30% ammonia solution). The chloroform used in the CMA solution and the chloroform used as a co-elution in silica gel column chromatography were stabilized with 0.75% v / v ethanol. All chloroform used in the hydroamination reactions was stabilized with pentene.

[0617] General chemical instruments

[0618] Proton nuclear magnetic resonance (NMR) 1 ¹H NMR spectra were recorded using a 500 MHz Avance III spectrometer with a multinucleus smart probe, reported in parts per million (ppm) on the δ scale, with reference to residual protium in the NMR solvent (CDCl₃: δ 7.24, CD₃OD: δ 3.31 (CHD₂OD), CD₃CN: δ 1.94). Data are reported as follows: chemical shifts [multiplicity (s = singlet, d = doublet, t = triplet, dd = double doublet, dt = double triplet, dq = double quartet, ddd = double doublet, tt tripletet, td = triple doublet, tq = triple quartet, m = multiply], coupling constants in Hertz, integral, assigned values]. Carbon-13 NMR (… 13 The C NMR spectrum was referenced to the carbon resonance of the solvent (CDCl3: δ77.23, CD3OD: δ49.15, CD3CN: δ1.37). Fluorine-19 NMR (… 19F NMR was calibrated according to the fluorine resonance of benzene trifluoride (CDCl3: δ-62.76, CD3OD: δ-64.24, CD3CN: δ-63.22). Data are reported as follows: Chemical shifts (assigned values). Infrared data (IR) were obtained using a Cary 630 Fourier transform infrared spectrometer equipped with diamond ATR objectives and are reported as follows: Absorption frequencies (cm²). -1 Absorption intensity (s = strong, m = medium, w = weak, br = broad). Using electrospray ionization (ESI), atmospheric pressure ionization (API), or electron ionization (EI) sources, in QExactive... TM Plus Hybrid Quadrupole-OrbitTap TM High-resolution mass spectrometry (HRMS) was recorded on a mass spectrometer. Isolera was used. TM One The purification system performs automatic C 18 Reversed-phase chromatography. High-performance liquid chromatography (HPLC) purification was performed using an Agilent 1260 Infinity system. (At GE Healthcare Life Sciences Typhoon) TM Intragel fluorescence imaging was performed on an FLA 9500. Images were processed using Fiji ImageJ software.

[0619] General biomaterials and methods

[0620] All solvents and reagents were purchased from commercial suppliers and used immediately upon receipt. Deionized water (>18.2 μΩ) was used to prepare all aqueous buffers and solutions. Short oligonucleotide primers (<80 bp) were synthesized by MilliporeSigma (St. Louis, MO), while gene segments (>80 bp) were synthesized by Twist Bioscience (South San Francisco, CA). Oligonucleotides were ready for use immediately upon receipt without further desalting. Chemocompetent *E. coli* DH5α and BL21(DE3) cells were purchased from New England Biosciences. All plasmids were isolated using small-scale or medium-scale preparation kits from Zymo Research. DNA cleaners and concentrators, as well as DNA gel purification kits, were purchased from Zymo Research. All enzymes used for standard restriction enzyme cloning (…) Hot-start DNA polymerase, restriction endonuclease, T4 DNA ligase, and thermosensitive phosphatase (for Gibson) cloned HiFi DNA Assembly MasterMix and other technologies for performing all site-directed mutagenesis reactions. All mutagenesis kits were purchased from New England. DNA sequencing services were provided by Quintarabio (Cambridge, MA). Transfection reagent purchased from MirusBio TM .

[0621] General biological instruments

[0622] All polymerase chain reactions (PCR) were performed on a Bio-Rad Laboratories C 1000 thermal cycler. Fisherbrand was used. TM Cells were lysed using a Model 505 sonicator. Proteins were purified using a Bio-Rad NGC chromatography system. UV / vis absorbance measurements for protein A280 determination were obtained on an Agilent Cary 60 UV / vis spectrophotometer. (GE HealthcareLife Sciences Typhoon) TM Intragel fluorescence imaging was performed on an FLA 9500. Coomassie-stained gels were analyzed on a Bio-Rad MolecularImager Gel Doc XR+ imaging system.

[0623] Example 2: Bioorthogonal reaction of cycloalkynes

[0624] Retro-Cope elimination has proven useful in biorthogonal reactions. Figure 1 (Bourgeois et al., J.Am.Chem.Soc.131(3):874-875(2009); Beauchemin, AM, Org.Biomol.Chem.11:7039-7050(2013); O′Neil et al., Chem.Commun.50:7336-7339(2014)). The following describes the bioorthogonal reaction of N,N-dialkylhydroxylamine and cyclooctyne to form stable enamine N-oxide linked products in a rapid and regioselective manner, with the reaction components containing only three non-hydrogen atoms.

[0625] The retro-Cope elimination reaction was evaluated by density functional theory (Zhao et al., Theor. Chem. Acc. 120: 215-241 (2008)), and the activation barriers for the reaction of N,N-dimethylhydroxylamine with various cyclooctynes ​​were determined. Figure 2AThe calculated activation energy of the unmodified cyclooctyne is 18.9 kcal / mol, which is low enough to allow the reaction to proceed at room temperature. It is also noteworthy that there is no steric factor affecting the initial O··H··C2 bond in the transition state structure, suggesting that the steric contradiction of the propargyl substituent will be important for the adaptability, reciprocal orthogonality, and reactivity of the cyclooctyne.

[0626] Calculations for bicyclic [6.1.0]nonyne show that additional strain can be controlled to produce the same effect as a cycloaddition reaction. Figure 2C (Dommerholt et al., Angew. Chem. Int. Ed. 49(49): 9422-9425(2010)). Conversely, the electrotactic modulation of cyclooctyne has been shown to be more significant (Baskin et al., Proc. Natl. Acad. Sci. USA 104(43): 16793-16797(2007); Agard et al., ACS Chem. Biol. 1(10): 644-648(2006)). Figure 2D Further distortion / interaction energy analysis revealed that the distortion energy of cyclooctyne was significantly lower than that of its linear counterpart (Ess et al., Org. Lett. 10(8): 1633-1636 (2008); Liu et al., Acc. Chem. Res. 50(9): 2297-2308 (2017)), but this was not the case for cycloaddition, where the reaction did not benefit from the increased interaction energy upon the addition of an electronegative substituent; the rate acceleration was driven by the decrease in distortion energy of both components. According to the Hammond hypothesis, the paradoxical increase in interaction energy could be due to the reactant-ward shift of the transition state towards the reactant. The corresponding increase in the lengths of the C1··N and C2··H bonds further supports this explanation (Example 30).

[0627] Kinetic experiments confirmed the reactivity trend predicted by calculation. The reaction progress was monitored using NMR spectroscopy, and the second-order rate constants of the reaction between N,N-diethylhydroxylamine (1) and a set of cyclooctylenes in d3-acetonitrile were determined at room temperature. Figure 3 Cyclooctyne (2) has been shown to be highly reactive, exhibiting a reactivity of 3.25 × 10⁻⁶. -2 M -1 s -1The second-order rate constant is an order of magnitude faster than its reaction with benzyl azides (Agard et al., J. Am. Chem. Soc. 126(46): 15046-15047(2004)). As predicted for bicyclic [6.1.0]nonyne 3, further strain enhancement provides a 6.7-fold rate acceleration, but this is far less than the 100-fold increase observed in similar azide-alkyne cycloaddition reactions (Dommerholt et al., Angew. Chem. Int. Ed. 49(49): 9422-9425(2010)). Nevertheless, the second-order reaction rate constant is 2.17 × 10⁻⁶. - 1 M -1 s -1 It is superior to the fastest azide-based reaction involving BARAC (Jewett et al., J.Am.Chem.Soc.132(11):3688-3690(2010)).

[0628] The hydroamination of cyclooctyne is particularly sensitive to the inductive effect of the propargyl substituent, and a gradual increase in rate is observed with increasing electronegativity. Figure 3 Among the substrates derived from cyclooctanol, carbamate 9 is the most reactive, with a rate constant of 3.87 M. -1 s -1 The rate of reaction is 120-fold higher than that of cyclooctyne (2). Importantly, the minimized cyclooctyne-1-ol substructure has been shown to be versatile, both readily synthesized and readily derivatized; it is suitable for coupling via ester, carbamate, or ketal bonds without incurring significant costs in terms of size or reactivity. In fact, fine functionalization of the core cyclooctyne is not only unnecessary but has sometimes proven detrimental. The reaction of N,N-diethylhydroxylamine with dibenzozacyclooctyne 8 (DIBAC) (Debets et al., Chem. Commun. 46: 97-99 (2010)) is rapid, but still slower than carbamate 9 and not significantly superior to its more stringent counterpart. Notably, the linkage product of dibenzozacyclooctyne 8 with N,N-diethylhydroxylamine is readily degraded and is particularly unstable for purification by standard and reversed-phase rapid chromatography.

[0629] Previous reports have shown that difluorocyclooctyn 10 operates at the bioorthogonality limit (Baskin et al., Proc. Natl. Acad. Sci. USA 104(4): 16793-16797 (2007); Kim et al., Carbohydr. Res. 377: 18-27 (2013)), providing a reasonable upper limit for the hydroammoniation kinetics achievable with electronically tuned cyclooctyn in a biological environment. Surprisingly, competing experiments with cyclooctyn 9 showed a rate constant of 83.6 M. -1 s -1 .

[0630] The retro-Cope elimination reaction is largely guided by substrate electronics and produces only one observable regioisomer for cyclooctyne 2–7, 9, and 10. Therefore, when symmetrical N,N-dialkylhydroxylamines are used, a single product is selectively formed.

[0631] To test the bioorthogonality of the hydroamylation reaction, in vitro protein labeling experiments were performed. The fluorophore-conjugated hydroxylamine 13 was initially assembled from 6-carboxytetramethylrhodamine and hydroxylamine 12, which was then synthesized by nucleophilically substituting iodide 11 with N-methylhydroxylamine hydrochloride. Figure 4A Additionally, lysozyme functionalized with N-hydroxysuccinimide ester 14 ( ) Figure 4B With these two reaction components, cyclooctyne-functionalized lysozyme 15 was treated in PBS with hydroxylamine 13 (0 μM–200 μM) for 2 hours, and the results were analyzed by in-gel fluorescence analysis. Figure 4C The labeling was concentration-dependent, and saturation was achieved at 100 μM hydroxylamine. The reaction was time-dependent. Figure 4D Modified lysozyme 15 was treated with hydroxylamine 13 (200 μM) and quenched with N,N-diethylhydroxylamine (20 mM) at different time points. In-gel fluorescence analysis showed signal saturation after 1 hour. The desired adducts formed on the protein were verified by mass spectrometry. Lysozyme 15 was incubated with hydroxylamine 13 (100 μM) in PBS, and the complete conversion of monocyclic and bicyclic octyne-functionalized lysozyme 15 to monoamines and diene amine N-oxides 16 was verified by ESI-MS. Figure 4E ).

[0632] The stability of enamine N-oxides and hydroxylamines under various biologically relevant conditions was verified at different time points. Figures 5A-5BHydroxylamine 13 was first incubated in PBS at room temperature, and HPLC analysis of the solution showed that >86% of the compound remained intact for up to 8 hours. However, at the 24-hour timepoint, approximately 40% of the hydroxylamine had degraded. The primary degradation products were consistent with the hydrolysis of regioisomeric nitrones, which may be generated by auto-oxidation. Therefore, this degradation pathway could be eliminated by adding cellular reducing agents such as ascorbic acid (5 mM) or glutathione (5 mM) (Bobko et al., Free Radical Biol. Med. 42(3): 404-412 (2007)). Negligible degradation was observed within 24 hours. Hydroxylamine 13 was stable in HEK293T cell lysate (1 mg / mL) and no degradation above background occurred within 24 hours, even without buffering with exogenous reducing agents.

[0633] Similar to hydroxylamine, the stability of enamine N-oxide 17 was assessed by HPLC under biorelevant conditions. No signs of degradation were observed over a 24-hour timeframe, either alone in PBS or in the presence of 5 mM glutathione. Furthermore, while N-oxide did undergo heme protein-dependent reduction under hypoxic conditions, this process was adequately inhibited under aerobic conditions (Raleigh et al., Int. J. Radiat. Oncol. Biol. Phys. 42(4): 763-767 (1998)). Degradation of enamine N-oxide 17 with human liver microsomes (0.2 mg / mL) after 24 hours of incubation in ambient air was negligible.

[0634] To further demonstrate the bioorthogonality of the reaction, TAMRA-hydroxylamine 13 and lysozyme-COT 15 were bound in PBS for 2 hours in both the presence and absence of HEK293T cell lysates. Figure 5C In-gel fluorescence showed that only lysozyme was labeled, and the degree of labeling was not affected by the presence of lysates. Under these conditions, there appeared to be no cross-reactivity between dialkylhydroxylamine and other proteins.

[0635] Finally, the cross-compatibility of this reaction with other bioorthogonal systems was explored to identify mutually orthogonal substrate combinations that can be used in tandem. Figure 5D(Patterson et al., Curr. Opin. Chem. Biol. 28: 141-149 (2015)). First, tetrazines were evaluated to see if they were compatible with sterically crowded cyclooctynes ​​with a tetrasubstituted nucleotide at the propargyl position. In fact, no product was detected when cyclooctyne ketal 5 and tetrazine 18 were combined in d3-acetonitrile at a concentration of 5 mM for 1 hour. To determine whether the steric confinement imposed by the electrons of the fully substituted carbon (Liu et al., J. Am. Chem. Soc. 136 (32): 11483-11493 (2014)) or ketal was primarily responsible for suppressing the reverse electron-demanding cycloaddition, electron-deficient cyclooctynes ​​9 and 10 were evaluated under the same conditions to obtain a similar effect. The electrons (alone or in combination with stereochemistry) made the two reactions orthogonal. Hydroxylamine reagents were evaluated to see if they were compatible with strained alkenes. It has been shown that 5 mM N,N-diethylhydroxylamine (1) in combination with 5 mM cyclopropene 21 (Patterson et al., J.Am.Chem.Soc.134(45):18638-18643(2012)) or trans-cyclooctene 19 (Bilackman et al., J.Am.Chem.Soc.130(41):13518-13519(2008)) is unreactive in d3-acetonitrile. N,N-dialkylhydroxylamine does not react with aldehydes, nor does it participate in copper-catalyzed azide-alkyne cycloaddition reactions (Example 29) (Hein et al., Chem.Soc.Rev.39:1302-1315(2010)).

[0636] A novel bioorthogonal linkage reaction between N,N-dialkylhydroxyamine and cyclooctyne was identified. This reaction exhibits rapid kinetics, with a second-order rate constant as high as 84 M. -1 s -1 The reaction exhibits excellent regioselectivity and small reactant components. The N,N-dialkylhydroxylamine reagent can be reduced to only three non-hydrogen atoms, and cyclooctyne is highly effective even in its unfunctionalized state. Cyclooctyne can be readily attached to its propargyl position without sacrificing reactivity. Under aqueous conditions, the hydroxylamine reagent and the enamine N-oxide product are sufficiently stable in the presence of thiols or cellular environmental components found in cell lysates, particularly on the timescale of linking small molecules and biomolecules. However, both components have their sensitivities: hydroxylamine is sensitive to air, while enamine N-oxide is sensitive to anaerobic microsomes. Factors mitigating these processes were identified, and the bioorthogonality of the reaction was ensured.

[0637] Example 3: Synthesis of (E)-(cyclooctatetraen-1-en-1-yloxy)trimethylsilane

[0638]

[0639] Tetrahydrofuran (THF, 200 mL) and bis(trimethylsilyl)aminolithium solution (1 M in THF, 52.3 mL, 52.3 mmol) were added sequentially to a round-bottom flask, and the mixture was then cooled to -78 °C. Over 20 minutes, a solution of cyclooctanone S1 in THF (200 mL) (6.00 g, 47.5 mmol) was added to the -78 °C solution through a sleeve. After 1.5 hours, trimethylchlorosilane (TMSCl, 5.94 g, 54.7 mmol) was added, and the dry ice bath was removed. The solution was allowed to warm to room temperature. After 1 hour, the reaction was quenched with a saturated ammonium chloride aqueous solution (500 mL) and diluted with hexane (500 mL). The organic layer was washed with brine (200 mL), dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. Crude S2 was used in the next step without further purification.

[0640] Example 4: Synthesis of 2-(trimethylsiloxy)cyclooctanone (S3)

[0641]

[0642] Add crude S2 (47.5 mmol) from the previous step to a round-bottom flask and dissolve it in dichloromethane (DCM, 200 mL). Add dimethyldiethylene oxide (DMDO, 0.11 M in acetone, 595 mL, 60.0 mmol) to the solution at room temperature. After 15 minutes, concentrate the reaction mixture and azeotropically react with MeOH (2 × 200 mL). Dissolve the resulting oil in DCM (500 mL). At room temperature, add 4-dimethylaminopyridine (DMAP, 581 mg, 4.75 mmol), triethylamine (9.94 mL, 71.3 mmol), and trimethylchlorosilane (7.24 mL, 57.1 mmol) sequentially to the solution. After 2.5 hours, wash the reaction mixture with aqueous hydrochloric acid (1 N, 500 mL), separate the organic layer, and extract the aqueous layer with DCM (75 mL). Dry the combined organic layers with anhydrous sodium sulfate, filter, and concentrate under reduced pressure. The crude mixture was purified by silica gel rapid column chromatography (elution: 5% ethyl acetate in hexane) to provide ketone S3 (8.35 g, 82%, via 3 steps). 1 H NMR (500MHz, CDCl3, 25°C): δ4.15 (dd, J=6.9, 3.4Hz, 1H), 2.65-2.54 (m, 1H), 2.32-2.20 (m, 1H), 2.13-2.02 (m, 1H) ), 2.03-1.93(m, 1H), 1.86-1.79(m, 1H), 1.78-1.63(m, 2H), 1.57-1.37(m, 4H), 1.30-1.16(m, 1H), 0.08(s, 9H). 13C10 NMR (126 MHz, CDCl3, 25 °C): δ 217.6, 77.7, 39.1, 34.7, 27.2, 26.1, 25.3, 21.4, 0.2. FTIR (thin film) cm -1 :2930(b), 1707(w), 1252(m), 1111(m), 1051(m), 835(s).HRMS(ESI)(m / z): C 11 H 23 O2Si[M+H] + Calculated value: 215.1467, Actual value: 215.1463. TLC (5% ethyl acetate in hexane), Rf: 0.70 (I2).

[0643] Example 5: (E Synthesis of 8-((trimethylsilyl)oxy)cyclooct-1-en-1-yltrifluoromethanesulfonate (S4) become

[0644]

[0645] Cyclooctanone S3 (2.06 g, 9.61 mmol) and THF (100 mL) were added sequentially to a round-bottom flask, and the mixture was then cooled to -78 °C. At -78 °C, a solution of bis(trimethylsilylamine)-lithium (1 M in THF, 11.5 mL, 11.5 mmol) was added to the mixture through a sleeve. After 1 hour, N-(5-chloro-2-pyridyl)bis(trifluoromethanesulfonylimide) (4.15 g, 10.6 mmol) was added, and the dry ice bath was removed. After 2 hours, the reaction mixture was diluted with hexane (200 mL) and washed sequentially with aqueous sodium hydroxide solution (1 M, 2 × 150 mL) and brine (100 mL). The resulting organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude mixture was purified by rapid silica gel column chromatography (eluent: 7.5% DCM in hexane) to give a colorless oily vinyltrifluoromethanesulfonate S4 (3.1 g, 95%). 1 H NMR (500MHz, CDCl3, 25°C): δ5.68 (t, J=9.0Hz, 1H), 4.66 (dd, J=10.3, 5.4Hz, 1H), 2.37-2.21 (m, 1H), 2.11-1.97(m, 1H), 1.83-1.67(m, 4H), 1.65-1.53(m, 1H), 1.52-1.29(m, 3H), 0.14(s, 9H). 13 C NMR (126MHz, CDCl3, 25℃): δ150.6, 118.8 (q, J=319.6Hz), 120.0, 67.3, 37.0, 29.9, 26.2, 24.8, 23.6, -0.1. 19FTIR NMR (471 MHz, CDCl3, 25 °C): δ -75.2. FTIR (thin film) cm⁻¹ 1 :2933(w), 1416(m), 1200(s), 1144(m), 932(m), 839(s).HRMS(ESI)(m / z): C 12 H 21 F3NaO4SSi[M+Na] + Calculated value: 369.0774, Actual value: 369.0776. TLC (100% hexane), Rf: 0.42 (I2).

[0646] Example 6: Synthesis of 2-cyclooctyne-1-ol (4)

[0647]

[0648] Vinyltrifluoromethanesulfonate S4 (3.03 g, 8.75 mmol) and THF (88 mL) were added sequentially to a round-bottom flask, which was then cooled to -78 °C. A solution of lithium diisopropylamino (2 M in THF / heptane / ethylbenzene, 8.75 mL, 17.5 mmol) was added to the solution via syringe. The dry ice bath was immediately removed, and the solution was allowed to warm to room temperature. After 2.5 hours, tetrabutylammonium fluoride (1 M in THF, 17.5 mL, 17.5 mmol) was added to the reaction mixture via syringe. After 1 hour, the reaction mixture was diluted with hexane (100 mL) and washed with saturated ammonium chloride aqueous solution (100 mL) and brine (100 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude mixture was purified by rapid silica gel column chromatography (elution: 100% DCM) to give a colorless, transparent oily cyclooctanol 4 (566 mg, 52%). The physical properties and spectroscopic data are the same as those reported in the literature (Hagendorn, T., Eur. J. Org. Chem. 2014(6): 1280-1286(2014)). TLC (100% DCM), Rf: 0.31 (KMnO4).

[0649] Example 7: Synthesis of (E)-8-oxocyclooct-1-en-1-yltrifluoromethanesulfonate (S5)

[0650]

[0651] Vinyltrifluoromethanesulfonate S4 (94.3 mg, 272 μmol) and DCM (1.4 mL) were added sequentially to a round-bottom flask. Trifluoroacetic acid (600 μL) was added to the solution at room temperature. After 30 minutes, the reaction mixture was concentrated under reduced pressure. The crude mixture was dissolved in DCM (2.7 mL). Sodium bicarbonate (68.6 mg, 817 μmol) and Dysmart's high-iodine reagent (DMP, 231 mg, 544 μmol) were added sequentially at room temperature. After 30 minutes, the reaction mixture was diluted with hexane (2 mL) and purified by rapid silica gel column chromatography (elution: 15% ethyl acetate in hexane) to give a clear, thin-film cyclooctanone S5 (64.1 mg, 87%). 1 H NMR (500MHz, CDCl3, 25°C): δ6.58 (t, J=9.2Hz, 1H), 2.85 (t, J=7.3Hz, 2H), 2.70 ( dt, J=9.3, 7.0Hz, 2H), 1.84-1.75(m, 2H), 1.74-1.68(m, 2H), 1.61-1.54(m, 2H). 13 C NMR (126MHz, CDCl3, 25℃): δ192.8, 149.7, 133.6, 118.8 (q, J=320.1Hz), 40.7, 25.3, 23.5, 23.1, 22.0. 19 F NMR (471 MHz, CDCl3, 25 °C): δ -74.2. FTIR (thin film) cm⁻¹ -1 :2937(w), 1685(m), 1416(s), 1200(s), 1141(s), 1062(s), 969(s).HRMS(ESI)(m / z): C9H 12 F3O4S[M+H] + Calculated value: 273.0403, Actual value: 273.0402. TLC (15% ethyl acetate in hexane), Rf: 0.30 (KMnO4).

[0652] Example 8: ( E) Synthesis of -1,4-dioxospirocyclic [4.7]dodec-6-en-6-yltrifluoromethanesulfonate (S6)

[0653]

[0654] At room temperature, cyclooctanone S5 (150 mg, 551 μmol), ethylene glycol (302 μL, 5.51 mmol), and benzene (10 mL) were added sequentially to a round-bottom flask. Then, p-toluenesulfonic acid monohydrate (10.5 mg, 55.1 μmol) was added to the solution. The flask was equipped with a Dean-Stark separator and a reflux condenser, and the reaction mixture was heated to reflux. After 23 hours, the reaction mixture was cooled to room temperature and diluted with hexane. The crude mixture was purified by rapid silica gel column chromatography (eluent: 5% ethyl acetate in hexane) to give a colorless, transparent, oily ketal S6 (125 mg, 71%). 1 H NMR (500MHz, CDCl3, 25°C): δ5.75 (t, J=9.5Hz, 1H), 4.14-3.91 (m, 4H), 2.48-2.36 (m, 2H), 2.04-1.98 (m, 2H), 1.65-1.52 (m, 6H). 13 C NMR (126MHz, CDCl3, 25℃): δ150.1, 123.0, 118.7 (q, J=319.5Hz), 107.3, 65.7, 37.7, 27.2, 23.1, 22.7, 21.9. 19 F NMR (471 MHz, CDCl3, 25 °C): δ -75.4. FTIR (thin film) cm -1 :2930(w), 1409(s), 1245(w), 1200(s), 1141(s), 977(s).HRMS(ESI)(m / z): C 11 H 16 F3O5S[M+H] + Calculated value: 317.0665, Actual value: 317.0664.

[0655] Example 9: Synthesis of 1,4-dioxospirocyclic '4,716-dodecyne (5)

[0656]

[0657] Ketal S6 (70.1 mg, 222 μmol) and THF (4 mL) were added sequentially to a round-bottom flask, and the mixture was then cooled to -78 °C. A solution of lithium diisopropylaminoacetate (LDA, 2 M in THF / heptane / ethylbenzene, 222 μL, 443 μmol) was added to the solution via syringe. The dry ice bath was immediately removed, and the solution was heated to room temperature. After 2.5 hours, the solution was cooled to -78 °C, and an additional solution of lithium diisopropylaminoacetate (2 M in THF / heptane / ethylbenzene, 111 μL, 222 μmol) was added via syringe. The ice bath was immediately removed, and the solution was warmed to room temperature. After 1.5 hours, the reaction was quenched with MeOH (1.0 mL), and the mixture was concentrated under reduced pressure. The crude mixture was purified by rapid silica gel column chromatography (eluent: 5% ethyl acetate in hexane) to give a clear, thin-film cyclooctyne 5 (25.5 mg, 69%). 1 H NMR (500MHz, CDCl3, 25℃): δ3.98-3.84(m, 4H), 2.22(t, J=6.4Hz, 2H), 2.18-2.11(m, 2H), 1.95-1.87(m, 2H), 1.77-1.71(m, 2H), 1.68-1.61(m, 2H). 13 C10 NMR (126MHz, CDCl3, 25℃): δ 107.4, 105.3, 89.8, 64.7, 47.4, 34.2, 29.8, 27.0, 20.6. FTIR (thin film) cm -1 :2926(m), 2214(w), 1446(w)1275(w), 1170(m), 1129(s), 1029(s).HRMS(ESI)(m / z): C 10 H 15 O2[M+H] + Calculated value: 167.1067, Actual value: 167.1067. TLC (5% ethyl acetate in hexane) Rf: 0.38 (KMnO4).

[0658] Example 10: Synthesis of cyclooctyl-2-yn-1-yl acetate (6)

[0659]

[0660] At room temperature, cyclooctanol 4 (80.5 mg, 648 μmol), 4-dimethylaminopyridine (6.3 mg, 51.9 μmol), and DCM (3.0 mL) were sequentially added to a round-bottom flask. The solution was then cooled to 0 °C in an ice-water bath, and pyridine (261 μL, 3.24 mmol) was added dropwise. Acetic anhydride (73.5 μL, 778 μmol) was then added dropwise. The ice bath was immediately removed, and the solution was heated to room temperature. After 16 hours, the reaction was quenched with a saturated aqueous solution of ammonium chloride (1 L), and the solution was diluted with DCM (50 mL). The organic layer was washed with water (50 mL), dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The crude mixture was purified by rapid silica gel column chromatography (eluent: 50% DCM in hexane) to give a colorless, transparent, oily cyclooctyne 6 (95.5 mg, 87%). 1 H NMR (500MHz, CDCl3, 25℃): δ5.34-5.26(m, 1H), 2.31-2.21(m, 1H), 2.21-2.08(m, 2H), 2.02(s, 3H) , 2.02-1.93(m, 1H), 1.94-1.83(m, 2H), 1.83-1.72(m, 1H), 1.72-1.57(m, 2H), 1.57-1.46(m, 1H). 13 CNMR (126MHz, CDCl3, 25℃): δ 170.4, 102.0, 90.8, 66.7, 41.7, 34.4, 29.8, 26.4, 21.3, 20.9. FTIR (thin film) cm -1 :2930(m), 1737(s), 1450(w), 1230(s), 1025(m), 969(m).HRMS(ESI)(m / z): C 10 H 15 O2[M+H] + Calculated value: 167.1067, Actual value: 167.1068. TLC (100% CH2Cl2), Rf: 0.57 (I2).

[0661] Example 11: Synthesis of 3-fluorocyclooctyl-1-yne (7)

[0662]

[0663] Cyclooctanol 4 (40.8 mg, 329 μmol) and DCM (3.0 mL) were added sequentially to a round-bottom flask, and the mixture was then cooled to 0 °C. Diethylaminosulfur trifluoride (DAST, 45.6 μL, 345 μmol) was then added to the solution via syringe. After 1 hour, the reaction mixture was concentrated under reduced pressure. The crude mixture was purified by rapid silica gel column chromatography (elution: 100% pentane) to give a colorless, transparent, oily fluorocyclooctylene 7 (21.0 mg, 51%). 1 H NMR (500MHz, CDCl3, 25°C): δ5.12 (dt, J=50.5, 5.1Hz, 1H), 2.34-2.01 (m, 4H), 1.94-1.86 (m, 2H), 1.82-1.68 (m, 2H), 1.64-1.44 (m, 2H). 13 CNMR (126MHz, CDCl3, 25℃): δ104.9 (d, J=10.5Hz), 90.5 (d, J=30.0Hz), 84.7 (d, J=171.2 Hz), 43.0 (d, J=22.9Hz), 34.3 (d, J=1.9Hz), 29.6, 25.5 (d, J=2.9Hz), 20.9 (d, J=2.9Hz). 19 F NMR (471 MHz, CDCl3, 25 °C): δ -172.2. FTIR (thin film) cm -1 :2930(s), 2855(m), 2214(w), 1450(m), 1353(m), 1029(m), 988(s).HRMS(ESI)(m / z): C8H 12 F[M+H] + Calculated value: 127.0918, Actual value: 127.0916. TLC (100% pentane), Rf: 0.26 (KMnO4).

[0664] Example 12: Synthesis of cyclooctyl-2-yn-1-yl(4-nitrophenyl)carbamate (9)

[0665]

[0666] Cyclooctanol 4 (10.8 mg, 87.0 μmol) and DCM (1 mL) were added sequentially to a round-bottom flask. At room temperature, 4-p-nitrobenzene isocyanate (14.3 mg, 87.0 μmol) and triethylamine (1.2 μL, 8.70 μmol) were added to the solution. After 100 minutes, the reaction mixture was diluted with hexane. The crude mixture was purified by rapid silica gel column chromatography (eluent: 30% diethyl ether in hexane) to give a white solid carbamate 9 (16.7 mg, 67%). 1H NMR (500MHz, CD3CN, 25°C): δ8.26 (s, 1H), 8.16 (d, J=9.3Hz, 2H), 7.62 (d, J=9.3Hz, 2H), 5.32 (tq, J=5.1, 2.2Hz, 1H), 2.35-2.2 4(m, 1H), 2.24-2.15(m, 2H), 2.08-1.99(m, 1H), 1.94-1.86(m, 2H), 1.86-1.75(m, 1H), 1.73-1.63(m, 2H), 1.64-1.52(m, 1H). 13 C10 NMR (126MHz, CD3CN, 25℃): δ 153.5, 146.1, 143.8, 126.0, 118.8, 103.2, 91.6, 68.7, 42.5, 35.0, 30.4, 26.9, 21.1. FTIR (thin film) cm -1 :3321(m), 2930(m), 1722(s), 1566(s), 1510(s), 1327(s), 1226(s), 1055(s).HRMS(ESI)(m / z): C 15 H 17 N₂O₄[M+H] + Calculated value: 289.1183, Actual value: 289.1188. TLC (50% DCM in hexane), Rf: 0.23 (KMnO4).

[0667] Example 13: Synthesis of (E)-N,N-diethylcyclooct-1-ene-1-oxamine (S7)

[0668]

[0669] At room temperature, N,N-diethylhydroxylamine (28.4 μL, 276 μmol) was added via syringe to an acetonitrile solution of cyclooctyne 2 (1.8 mL) (Fairbanks et al., Macromolecules 43(9): 4113-4119(2010)) (19.9 mg, 184 μmol). After 10 minutes, the reaction mixture was concentrated under reduced pressure. The crude mixture was purified by rapid silica gel column chromatography (elution: 15% → 30% CMA in chloroform) to give a transparent thin film enamine N-oxide S7 (36.0 mg, 99%). 1 HNMR (500MHz, CDCl3, 25℃): δ6.65 (t, J=8.8Hz, 1H), 3.40-3.09 (m, 4H), 2.41 -2.27(m, 2H), 2.19-2.09(m, 2H), 1.67-1.39(m, 8H), 1.16(t, J=7.1Hz, 6H). 13C10 NMR (126MHz, CDCl3, 25℃): δ 146.8, 125.6, 61.8, 29.7, 28.3, 26.1, 26.0, 25.6, 25.3, 8.8. FTIR (thin film) cm -1 : 3340(br), 2926(s), 2855(m), 1655(w), 1466(m), 956(s).HRMS(ESI)(m / z): C 12 H 24 NO[M+H] + Calculated value: 198.1852, Actual value: 198.1853. TLC (chloroform with 50% CMA), Rf: 0.38 (KMnO4).

[0670] Example 14: (1R, 8S, 9S, E) -N， N- Diethyl-9-(hydroxymethyl)bicyclo[6.1.0]non-4-ene-4-oxide Synthesis of amine (S8)

[0671]

[0672] At room temperature, N,N-diethylhydroxylamine (30.8 μL, 300 μmol) was added via syringe to a solution of cyclooctyne 3 in methanol (500 μL) and acetonitrile (2.0 mL) (30.0 mg, 200 μmol). After 10 minutes, the reaction mixture was concentrated under reduced pressure. The crude mixture was purified by rapid silica gel column chromatography (elution buffer: 20% → 40% CMA in chloroform) to give a colorless, transparent, oily enamine N-oxide S8 (45.6 mg, 95%). 1 H NMR (500MHz, CD3OD, 25°C): δ6.65 (t, J=8.4Hz, 1H), 3.72-3.60 (m, 2H), 3.61-3.42 (m, 2H), 3.40-3.29 (m, 2H), 2.63 (dt, J=16.5, 5.9Hz, 1H), 2. 55-2.40(m, 2H), 2.26-2.12(m, 2H), 2.15-2.02(m, 1H), 1.74-1.57(m, 2 H), 1.21 (td, J=7.1, 2.5Hz, 6H), 1.19-1.10 (m, 1H), 1.11-1.01 (m, 2H). 13 CNMR (126MHz, CD3OD, 25℃): δ 148.4, 127.5, 62.8, 62.7, 59.7, 27.4, 25.9, 24.9, 24.7, 22.6, 20.9, 19.5, 8.9, 8.8. FTIR (thin film) cm -1: 3235(br), 2986(m), 2937(m), 2866(m), 1461(m), 1375(m), 1033(s).HRMS(ESI)(m / z): C 14 H 26 NO2[M+H] + Calculated value: 240.1958, Actual value: 240.1957. TLC (chloroform in 50% CMA), Rf: 0.16 (KMnO4).

[0673] Example 15: Synthesis of (E)-N,N-diethyl-3-hydroxycyclooct-1-ene-1-oxamine (S9)

[0674]

[0675] At room temperature, N,N-diethylhydroxylamine (30.8 μL, 300 μmol) was added via syringe to a solution of cyclooctyne 3 in methanol (500 μL) and acetonitrile (2.0 mL) (24.8 mg, 200 μmol). After 5 minutes, the reaction mixture was concentrated under reduced pressure. The crude mixture was purified by rapid silica gel column chromatography (elution buffer: 20% → 40% CMA in chloroform) to give a colorless, transparent, oily enamine N-oxide S9 (35.4 mg, 83%). 1 H NMR (500MHz, CD3OD, 25℃): δ6.40 (d, J=7-3Hz, 1H), 4.55-4.44 (m, 1H), 3.66-3.55 (m, 1H), 3.53-3.42 (m, 1H), 3.41-3.31 (m, 2H), 2.58-2 .48 (m, 1H), 2.48-2.36 (m, 1H), 2.00-1.92 (m, 1H), 1.88-1.76 (m, 1H), 1.76-1.43 (m, 6H), 1.31 (t, J=7.1Hz, 3H), 1.18 (t, J=7.1Hz, 3H). 13 C10 NMR (126MHz, CD3OD, 25℃): δ 146.1, 132.0, 69.9, 63.5, 61.8, 39.0, 30.9, 27.4, 27.0, 25.1, 9.1, 9.0. FTIR (thin film) cm -1 : 3310(br), 2930(s), 2490(br), 2065(w), 1454(s), 1062(s), 984(s).HRMS(ESI)(m / z): C 12 H 24 NO2[M+H] + Calculated value: 214.1802, Actual value: 214.1801. TLC (50% CMA in CHCl3), Rf: 0.14 (KMnO4).

[0676] Example 16: ( E) - N Synthesis of N-diethyl-1,4-dioxospirocyclic[4,7]6-dodecene-7-oxoamine (S10)

[0677]

[0678] At room temperature, N,N-diethylhydroxylamine (17.0 μL, 300 μmol) was added via syringe to a 1.0 mL solution of cyclooctyne 7 (18.3 mg, 110 μmol) in acetonitrile. After 10 minutes, the reaction mixture was concentrated under reduced pressure. The crude mixture was purified by rapid silica gel column chromatography (elution: 15% → 30% CMA in chloroform) to give a clear, thin-film enamine N-oxide S10 (25.6 mg, 91%). 1 H NMR (500MHz, CD3OD, 25℃): δ6.67 (s, 1H), 3.99-3.88 (m, 4H), 3.55-3.33 (m, 4H), 2. 78 (t, J=6.7Hz, 2H), 2.01-1.87 (m, 2H), 1.80-1.61 (m, 6H), 1.21 (t, J=7.1Hz, 6H). 13 C10 NMR (126MHz, CD3OD, 25℃): δ 148.9, 131.7, 110.0, 65.4, 63.1, 40.6, 29.9, 26.0, 24.1, 23.6, 9.0. FTIR (thin film) cm -1 :3355(br), 2933(m), 1677(w), 1454(m), 1081(s), 1029(s), 954(s).HRMS(ESI)(m / z): C 14 H 26 NO3[M+H] + Calculated value: 256.1907, Actual value: 256.1906. TLC (chloroform with 50% CMA), Rf: 0.35 (KMnO4).

[0679] Example 17: Synthesis of (E)-N,N-3-acetoxy-diethylcyclooct-1-ene-1-oxamine (S11)

[0680]

[0681] At room temperature, N,N-diethylhydroxylamine (30.8 μL, 300 μmol) was added via syringe to a solution of cyclooctyne 8 (33.2 mg, 200 μmol) in acetonitrile (2.0 mL). After 5 minutes, the reaction mixture was concentrated under reduced pressure. The crude mixture was purified by rapid silica gel column chromatography (elution: 10% → 30% CMA in chloroform) to give a colorless, transparent, oily enamine N-oxide S11 (46.1 mg, 90%). 1H NMR (500MHz, CD3OD, 25℃): δ6.40 (d, J=7.8Hz, 1H), 5.47 (ddd, J=11.7, 7.7, 4.8Hz, 1H ), 3.62 (dq, J=12.5, 7.1Hz, 1H), 3.48 (dq, J=12.4, 7.2Hz, 1H), 3.42-3.28 (m, 2H), 2.5 8-2.45(m, 2H), 2.05(s, 3H), 2.02-1.93(m, 1H), 1.93-1.84(m, 1H), 1.82-1.68(m, 3H) , 1.68-1.60 (m, 1H), 1.60-1.46 (m, 2H), 1.31 (t, J=7.1Hz, 3H), 1.12 (t, J=7.1Hz, 3H). 13 C10 NMR (126MHz, CD3OD, 25℃): δ 172.2, 147.8, 128.4, 73.2, 63.6, 62.1, 35.3, 30.6, 27.3, 27.3, 24.6, 21.1, 9.1, 8.8. FTIR (thin film) cm -1 : 3235(br), 2933(w), 1730(m), 1454(w), 1368(w), 1238(s), 1029(m).HRMS(ESI)(m / z): C 14 H 26 NO3[M+H] + Calculated value: 256.1907, Actual value: 256.1905. TLC (50% CMA in chloroform), Rf: 0.16 (MnO4).

[0682] Example 18: Synthesis of (E)-N,N-diethyl-3-fluorocyclooct-1-ene-1-oxamine (S12)

[0683]

[0684] At room temperature, N,N-diethylhydroxylamine (6.72 μL, 65.4 μmol) was added via syringe to a solution of cyclooctyne 7 (5.5 mg, 43.6 μmol) in acetonitrile (500 μL). After 10 minutes, the reaction mixture was concentrated under reduced pressure. The crude mixture was purified by rapid silica gel column chromatography (elution: 15% → 30% CMA in chloroform) to give a clear, thin-film enamine N-oxide S12 (8.3 mg, 88%). 1H NMR (500MHz, CD3OD, 25℃): δ6.64(dd, J=19.8, 6.5Hz, 1H), 5.47-5.29(m, 1H), 3.69-3.44(m, 2H), 3.43-3.32(m, 2H), 2.60-2.49(m , 1H), 2.48-2.36 (m, 1H), 2.20-2.06 (m, 1H), 1.85-1.75 (m, 2H), 1.73-1.55 (m, 5H), 1.30 (t, J=7.1Hz, 3H), 1.17 (t, J=7.1Hz, 3H). 13 C NMR (126MHz, CD3OD, 25°C): δ147.34 (d, J=13.4Hz), 128.83 (d, J=33.4Hz), 92.09 (d, J=161.6Hz ), 63.69, 62.13, 36.82 (d, J=21.9Hz), 30.47, 26.87, 26.60, 23.98 (d, J=12.9Hz), 8.99, 8.85. 19 F NMR (471 MHz, CD3OD, 25 °C): δ -172.0. FTIR (thin film) cm -1 :3373(br), 2937(s), 2863(m), 1595(m), 1454(m), 1379(m), 958(s).HRMS(ESI)(m / z): C 12 H 23 FNO[M+H] + Calculated value: 216.1758, Actual value: 216.1758. TLC (chloroform with 30% CMA), Rf: 0.13 (KMnO4).

[0685] Example 19: ( E) - N, N- Diethyl-3-(((4-nitrophenyl)carbamoyl)oxy)cyclooct-1-ene-1- Synthesis of amine oxide (S13)

[0686]

[0687] At room temperature, N,N-diethylhydroxylamine (7.1 μL, 69.2 μmol) was added via syringe to a solution of cyclooctyne 9 (13.3 mg, 46.1 μmol) in acetonitrile (1.0 mL). After 10 minutes, the reaction mixture was concentrated under reduced pressure. The crude mixture was purified by rapid silica gel column chromatography (elution: 30% CMA in chloroform) to give a clear thin film of enamine N-oxide S13 (16.3 mg, 94%). 1H NMR (500MHz, CD3OD, 25°C): δ8.16 (d, J=9.3Hz, 2H), 7.63 (d, J=9.3Hz, 2H), 6.53 (d, J=7.6Hz, 1H), 5.53 (ddd, J=11.9, 7.7, 4.9Hz, 1H), 3.69-3.57 (m, 1H), 3.55 -3.30(m, 3H), 2.61-2.49(m, 2H), 2.14-2.03(m, 1H), 1.94-1.86(m, 1H), 1.86- 1.71 (m, 3H), 1.69-1.49 (m, 3H), 1.33 (t, J = 7.1Hz, 3H), 1.15 (t, J = 7.2Hz, 3H). 13 C10 NMR (126MHz, CD3OD, 25℃): δ 154.6, 148.0, 146.9, 143.9, 128.4, 126.0, 119.0, 74.1, 63.6, 62.2, 35.5, 30.6, 27.3, 27.3, 24.6, 9.1, 8.9. FTIR (thin film) cm -1 : 3198(br), 2933(w), 1726(w), 1516(m), 1327(m), 1223(s), 1044(m). HRMS(ESI) (m / z): Calculated C 19 H 28 N3O5[M+H] + : 378.2023, Actual: 378.2021. TLC (chloroform with 30% CMA), Rf: 0.26 (KMnO4).

[0688] Example 20: Synthesis of (E)-N,N-diethyl-3,3-difluorocyclooct-1-ene-1-oxamine (S14)

[0689]

[0690] N,N-diethylhydroxylamine (30.8 μL, 300 μmol) was added via syringe to a solution of cyclooctyne 10 (Madea et al., Chem. Commun. 52: 12901-12904 (2016)) (28.8 mg, 200 μmol) in acetonitrile (1.84 mL). After 5 minutes, the reaction mixture was concentrated under reduced pressure. The crude mixture was purified by rapid silica gel column chromatography (elution: 10% → 30% CMA in chloroform) to give a clear thin film of enamine N-oxide S14 (43.8 mg, 94%). 1H NMR (500MHz, CD3OD, 25°C): δ7.02 (t, J=11.7Hz, 1H), 3.61-3.50 (m, 2H), 3.48-3.36 (m, 2H), 2. 73 (t, J=6.9Hz, 2H), 2.27 (tt, J=15.6, 6.5Hz, 2H), 1.85-1.61 (m, 6H), 1.21 (t, J=7.1Hz, 6H). 13 C NMR (126MHz, CD3OD, 25°C): δ151.9 (t, J=11.7Hz), 126.8 (t, J=35.0Hz), 123.7 (t, J=233.7Hz), 63.3, 38.2, 28.3, 24.9, 24.5, 22.2, 8.8. 19 F NMR (471 MHz, CD3OD, 25 °C): δ -83.4. FTIR (thin film) cm -1 : 3232(br), 2937(m), 1692(m), 1457(m), 1316(m), 988(s).HRMS(ESI)(m / z): C12H 22 F2NO[M+H] + Calculated value: 234.1664, Actual value: 234.1664. TLC (30% CMA in chloroform), Rf: 0.059 (KMnO4).

[0691] Example 21: (2-(2-(hydroxy(methyl)amino)ethoxy)ethyl)carbamate tert-butyl ester (12 )) Synthesis

[0692]

[0693] At room temperature, triethylamine (1.34 mL, 9.59 mmol) was added to a solution of dimethyl sulfoxide (2.4 mL) containing iodoalkane 11 (Heller et al., Angew. Chem., Int. Ed. 54(35): 10327-10330(2015)) (756 mg, 2.40 mmol) and N-methylhydroxylamine hydrochloride (401 mg, 4.80 mmol). The reaction mixture was then heated to 70 °C. After 1.5 hours, the solution was cooled to room temperature, diluted with water, and passed through an automated C... 18 Reversed-phase column chromatography (30g C) 18 Silica gel, 25 μm spherical particles, eluent: H2O + 0.1% TFA (2 CV), gradient 0% → 100% CH3CN / H2O + 0.1% TFA (10 CV to 15 CV)) purification to provide white solid hydroxylamine 12 (348 mg, 62%). 1H NMR (500MHz, CDCl3, 25°C) δ3.82 (ddd, J=11.1, 7.3, 3.9Hz, 1H), 3.63 (dt, J=11. 0, 4.2Hz, 1H), 3.52-3.35(m, 4H), 3.32-3.18(m, 2H), 3.07(s, 3H), 1.38(s, 9H). 13 C NMR (126MHz, CDCl3, 25℃) δ164.1 (q, J=37.5Hz), 156.7, 116.5 (q, J=289.2Hz), 79.5, 70.9, 63.6, 60.2, 46.5, 40.4, 28.5. 19 F NMR (471MHz, CDCl3, 25℃) δ -75.47. FTIR (thin film) cm -1 : 3351(br), 2945(w), 2900(w), 2236(s), 1361(m), 1290(m), 1185(s), 1129(s), 1085(s).HRMS(ESI)(m / z): C 10 H 23 N₂O₄[M+H] + Calculated value: 235.1652, Actual value: 235.1650. TLC (40% CMA in chloroform), Rf: 0.58 (I2).

[0694] Example 22: 3′,6′-bis(dimethylamino)-N-(2-(2-(hydroxy(methyl)amino)ethoxy)ethyl)-3- Synthesis of oxo-3H-spiro[isobenzofuran-1,9′-oxanthracene]-6-carboxamide (13)

[0695]

[0696] At room temperature, N,N-diisopropylethylamine (DIPEA, 49.2 μL, 282 μmol) was added to a solution of 6-carboxytetramethylrhodamine (6-TAMRA, 30.4 mg, 70.6 μmol) and 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolyl[4,5-b]pyridine 3-oxide hexafluorophosphate (HATU, 29.5 mg, 77.7 μmol) in N,N-dimethylformamide (DMF, 700 μL). In a separate vial, trifluoroacetic acid (100 μL) was added to a solution of hydroxylamine 12 (61.5 mg, 177 μmol) in DCM (400 μL). The resulting solution was stirred at room temperature for 1 hour, then concentrated under reduced pressure. The resulting residue was dissolved in N,N-dimethylformamide (500 μL) and then pipetteted into the reaction mixture containing 6-TAMRA. A separate volume of N,N-dimethylformamide (200 μL) was quantitatively transferred to the reaction mixture. The reaction mixture was stirred at room temperature for 4 hours. Another volume of HATU (29.5 mg, 77.7 μmol) and DIPEA (49.2 μL, 282 μmol) was added to the reaction mixture. The solution was then stirred for another 2.5 hours. The resulting mixture was diluted with water and analyzed by automated C... 18 Reversed-phase column chromatography (30gC) 18 Purification was performed using silica gel (25 μm spherical particles), eluent: H2O + 0.1% TFA (2 CV), gradient 0% → 100% CH3CN / H2O + 0.1% TFA (10 CV to 15 CV) and rapid silica gel column chromatography (eluent: 70% CMA in CHCl3) to obtain purple solid TAMRA-hydroxylamine 13 (22.8 mg, 59%). 1 H NMR (500MHz, D2O, 25℃) δ7.99 (d, J=8.3Hz, 1H), 7.87 (d, J=8.1Hz, 1H), 7.67-7.59 (m, 1H), 7.07 (d, J=9.5Hz, 2H), 6.73 (d d, J=9.5, 2.4Hz, 2H), 6.41 (d, J=2.3Hz, 2H), 3.84-3.63 (m, 4H), 3.53 (t, J=5.4Hz, 2H), 3.11-2.89 (m, 14H), 2.71 (s, 3H). 13 CNMR (126MHz, D₂O, 25℃) δ 173.2, 168.0, 157.6, 156.7, 156.6, 143.0, 133.5, 130.9, 130.6, 129.1, 128.9, 128.1, 113.6, 112.7, 96.1, 68.8, 66.6, 60.2, 47.5, 34.0, 39.7. FTIR (thin film) cm -1: 3280(br), 2926(w), 1648(w)1595(s), 1491(m), 1409(m), 1349(m), 1189(m).HRMS(ESI)(m / z): C 30 H 35 N4O6[M+H] + Calculated value: 547.2551, Actual value: 547.2544.TLC (100% CMA), Rf: 0.37 (visual).

[0697] Example 23: Synthesis of 3-(((cyclooctyl-2-yn-1-yloxy)carbonyl)amino)propionic acid (S16)

[0698]

[0699] At room temperature, 3-aminopropionic acid (18.5 mg, 207 μmol) was added to a solution of carbonate S15 (50.0 mg, 173 μmol) in methanol (2.0 mL) (Plass, et al., Angew. Chem., Int. Ed. 50(17): 3878-3881(2011)). Then, N,N-diisopropylethylamine (90.3 μL, 519 μmol) was added to the solution. After 1 hour, the reaction mixture was concentrated under reduced pressure. The crude mixture was purified by rapid silica gel column chromatography (elution: hexane / ethyl acetate / acetic acid, v / v / v = 65:30:5) to give a colorless, transparent, oily carbamate S16' (40.4 mg, 98%). 1 H NMR (500MHz, CD3OD, 25°C): δ5.23-5.10 (m, 1H), 3.37-3.31 (m, 2H), 2.49 (t, J=6.9Hz, 3H), 2.29-2.20 (m, 1H), 2.2 1-2.07(m, 2H), 2.03-1.94(m, 1H), 1.95-1.85(m, 2H), 1.86-1.75(m, 1H), 1.73-1.60(m, 2H), 1.60-1.50(m, 1H). 13 C10 NMR (126MHz, CD3OD, 25℃): δ 175.5, 158.2, 102.0, 92.4, 68.2, 43.1, 37.9, 35.5, 35.3, 31.0, 27.4, 21.3. FTIR (thin film) cm -1 :2930(m), 1700(s), 1528(m), 1252(m), 1137(w).HRMS(ESI)(m / z): C 12 H 18 NO4[M+H] +Calculated value: 240.1230, Actual value: 240.1229. TLC (5% methanol + 0.1% acetic acid in DCM), Rf: 0.19 (I2).

[0700] Example 24: 2,5-Dioxopyrrolidone-1-yl 3-(((cyclooct-2-yn-1-oxy)carbonyl)amino)propionate (14) Synthesis

[0701]

[0702] At room temperature, N-hydroxysuccinimide (NHS, 22.0 mg, 191 μmol), ethylcarbodiimide hydrochloride (EDC·HCl, 36.7 mg, 191 μmol), and N,N-diisopropylethylamine (53.3 μL, 306 μmol) were sequentially added to a solution of carboxylic acid S16 (18.3 mg, 76.5 μmol) in DCM (1.0 mL). After 12 hours, additional NHS (44.0 mg, 382 μmol) and EDC·HCl (73.4 mg, 382 μmol) were added to the reaction mixture. After 4 hours, the reaction mixture was concentrated under reduced pressure. The crude mixture was purified by rapid silica gel column chromatography (elution: 30% acetone in hexane) to give a colorless, transparent, oily NHS ester 14 (7.1 mg, 28%). 1 H NMR (500MHz, CDCl3, 25℃): δ5.45-5.19(m, 2H), 3.63-3.47(m, 2H), 2.94-2.67(m, 6H), 2.33-2.20(m, 1H), 2.21 -2.08(m, 2H), 2.04-1.94(m, 1H), 1.95-1.81(m, 2H), 1.81-1.71(m, 1H), 1.70-1.58(m, 2H), 1.58-1.45(m, 2H). 13 C10 NMR (126 MHz, CDCl3, 25 f ° C): δ 169.2, 167.7, 155.7, 101.9, 91.1, 67.6, 42.0, 36.6, 34.4, 32.3, 29.8, 26.4, 25.8, 20.9. FTIR (thin film) cm -1 :3358(w), 2930(w), 1782(w), 1733(s), 1517(m), 1245(m), 1200(s).HRMS(ESI)(m / z): C 16 H 21 N₂O₆[M+H] + Calculated value: 337.1394, Actual value: 337.1391. TLC (50% ethyl acetate in hexane), Rf: 0.25 (I2).

[0703] Example 25: Dynamics Study

[0704] All kinetic experiments were performed at room temperature in a CD3CN. Reactions were monitored by NMR spectroscopy using an internal standard. Second-order kinetics were performed by combining cyclooctyne and N,N-diethylhydroxylamine in a 1:1 ratio. Table 1 shows the experimental conditions for each cyclooctyne. The reported rate constant error is based on the standard deviation of the mean of three replicates.

[0705] Table 1. Kinetic studies of cyclooctyne

[0706]

[0707]

[0708] The second-order rate constant of difluorocyclooctyne 10 was determined using a competition experiment with carbamate 9. At room temperature, N,N-diethylhydroxylamine (1 equivalent; final concentration 1.9 mM) was added to a CD3CN solution containing difluorocyclooctyne 10 (5 equivalents; final concentration 9.5 mM) and cyclooctyne carbamate 9 (20 equivalents; final concentration 38 mM) in a 1:4 ratio. Figure 7 The solution was transferred to an NMR tube, and 1,3,5-trimethoxybenzene was used as an internal standard. 1 The product ratio (S14:S13) was determined by ¹H NMR spectroscopy. The second-order rate constant (k2) of difluorocyclooctyne 10 was calculated by multiplying the observed product ratio by the second-order rate constant (k2) of carbamate 9, yielding 83.6 × 14.9 M. -1 s -1 The reported rate constant error is the standard deviation of the mean of three repeated experiments.

[0709] Example 26: Protein Labeling Experiment

[0710] Synthesis of lysozyme-COT 15

[0711] Lysozyme (CAS12650-88-3, 50 mg / mL in deionized H2O) was diluted in phosphate-buffered saline (PBS, pH 7.4) to a final concentration of 10 mg / mL. Cyclooctyne NHS ester 13 solution (65 μL, 8.5 mM in DMSO) and DMSO (10 μL) were added to the lysozyme solution (250 μL, 10 mg / mL). The reaction solution was incubated at room temperature for 1 hour. Excess cyclooctyne NHS ester 13 was removed by spin filtration (3 kDa MWCO, 5 × 1:5 dilution). The concentration of lysozyme was measured on a UV-Vis spectrophotometer in denaturing buffer (pH 7.0, 6 M guanidine salt, 30 mM MOPS). 280To determine the final concentration, dilute the solution with PBS (pH 7.4) to a final concentration of 0.15 mg / mL or 0.60 mg / mL for the labeling experiment. Rapidly freeze the protein solution in liquid nitrogen and store at -20°C.

[0712] Concentration-dependent protein labeling experiments

[0713] Lysozyme-COT 15 solution (5.0 μL, 0.15 mg / mL) was divided into 6 aliquots. Hydroxylamine 13 aqueous solution (0.21 μL; 0.25 mM, 0.625 mM, 1.25 mM, 2.5 mM, and 5 mM in deionized water; final concentrations of 10 μM, 25 μM, 50 μM, 100 μM, and 200 μM) was added to each of the 5 aliquots. Deionized water (0.21 μL) was added to one sample instead of hydroxylamine as a solvent control. In control samples requiring lysozyme-COT 15-free conditions, unmodified lysozyme was treated with hydroxylamine 13 (0.21 μL, 5 mM in water; final concentration 200 μM) or deionized water (0.21 μL). The reaction mixture was incubated at room temperature in the dark for 2 hours. Quench the reaction mixture with 1.30 μL of 5× sodium dodecyl sulfate (SDS) loading buffer. Load each aliquot (5 μL) onto a 15-well 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) gel. Run the gel at room temperature and 175 V for 50 minutes. Use Typhoon. TM Fluorescence imaging within the gel was performed using an FLA 9500 (GE) at 532 nm with a photomultiplier tube (PMT) set to 500 V. The experiment was repeated three times. Figure 8 ).

[0714] Time-dependent protein labeling experiments

[0715] Lysozyme-COT 15 solution (5.0 μL, 0.15 mg / mL) was aliquoted into 6 aliquots. Hydroxylamine 13 aqueous solution (0.21 μL, 5 mM in deionized water; final concentration 200 μM) was added to each of the 5 aliquots. Deionized water (0.21 μL) was added to one sample as a solvent control instead of hydroxylamine 13. In control samples requiring lysozyme-COT 15-free conditions, unmodified lysozyme was treated with either hydroxylamine 13 (0.21 μL, 5 mM in water; final concentration 200 μM) or deionized water (0.21 μL). The reaction mixture was incubated at room temperature in the dark and quenched by adding N,N-diethylhydroxylamine (1.30 μL, 100 mM in deionized H₂O; final concentration 20 mM) at each specified time point, followed by the addition of 5×SDS loading buffer (1.63 μL). Samples were rapidly frozen in liquid nitrogen until all samples were ready for loading onto the gel. After 2 hours, all reactions were quenched. All samples were thawed and each solution (5 μL) was loaded onto a 15-well 12% SDS-PAGE gel. The gel was run at room temperature and 175 V for 50 minutes. Typhoon was used. TM Fluorescence imaging within the gel was performed using an FLA 9500 (GE) at 532 nm with a photomultiplier tube (PMT) set to 500 V. The experiment was repeated three times. Figure 9 ).

[0716] Complete mass spectrometry analysis

[0717] Hydroxylamine 13 solution (0.83 μL, 5 mM in deionized water) was added to lysozyme-COT 15 solution (20 μL, 0.60 mg / mL in deionized water) to generate the reaction sample. Deionized H2O (0.83 μL) was added to lysozyme-COT 15 (20 μL, 0.60 mg / mL in deionized water) to generate the solvent control. Unmodified lysozyme (20 μL, 0.60 mg / mL in deionized water) was added to deionized water (0.83 μL) to generate the blank background sample. The reaction was incubated at room temperature in the dark for 6 hours. The samples were then rapidly frozen with liquid nitrogen and stored at -80°C for further analysis. The reaction was performed using an LTQ XL... TM Ion trap mass spectrometer (ThermoFisher Scientific) TM Electrospray ionization mass spectrometry (ESI-MS) analysis was performed at the hospital (San Jose, CA) (Figure 10).

[0718] Example 27: Protein labeling experiment in the presence of cell lysates

[0719] Cell culture: HEK-293T cells were cultured in Duchenne Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS, Sigma), 100 units / mL penicillin, and 0.1 mg / mL streptomycin (Sigma) in a humidified incubator at 37°C and 5% CO2. Cultured in Hanks' balanced salt solution (HBSS). Cells were passaged and dissociated using 0.25% trypsin and 0.1% EDTA. Following the manufacturer's instructions, the cells were analyzed using MycoAlert. TM The PLUS Mycoplasma Detection Kit (Lonza) showed negative results for mycobacteria in the cells.

[0720] Cell lysates: Before lysis, aspirate the cell culture medium. Lyse cells (10cm culture dish, approximately 80% confluence) by adding lysis buffer (1.0mL, 4℃; 150mM NaCl, 50mM Tris (pH 8.0), 1% Triton X-100). After centrifugation at 4℃ (13,000×g), transfer the supernatant to a clean tube and perform BCA (diquinoline carboxylic acid) protein assay (Pierce method). TM Protein concentration was determined using the BCA Protein Assay Kit. Aliquots of cell lysate (7.1 mg / mL) were rapidly frozen in liquid nitrogen and stored at -20°C.

[0721] Protein labeling assay: Lysozyme-COT 15 (0.75 μg, 5.0 μL, 0.15 mg / mL in deionized water) was divided into four aliquots. Cell lysate (20 μg, 2.83 μL, 7.1 mg / mL) was added to two aliquots, and deionized water (2.83 μL) was added to the remaining two aliquots. A fifth control sample without lysozyme-COT 15 was prepared by adding cell lysate (20 μg, 2.83 μL, 7.1 mg / mL) to deionized water (5.0 μL). Then, according to... Figure 5B Under the conditions shown, add hydroxylamine 13 (0.33 μL, 5 mM in deionized water) or deionized water (0.33 μL) to the sample. Incubate the reaction mixture at room temperature in the dark for 2 hours. Quench the reaction mixture with 5×SDS loading buffer (1.30 μL). Load each solution (15 μL) onto a 15-well 12% SDS-PAGE gel. Run the gel at room temperature and 175 V for 50 min. Use Typhoon TM Fluorescence imaging within the gel was performed using an FLA 9500 (GE) at 532 nm with a photomultiplier tube (PMT) set to 500 V. The experiment was repeated three times.

[0722] Example 28: Stability Study

[0723] All reactions were monitored by HPLC at time points of 0, 1, 2, 4, 8 and 24 hours.

[0724] HPLC analysis: using HPLC (Pursuit) C 18 The reaction with enamine N-oxide 17 was analyzed using HPLC (Pursuite HPLC) at a flow rate of 1 mL / min and a particle size of 4.6 × 150 mm and 10 μm. The eluent was 0% MeCN / H₂O + 0.1% TFA (1 min) followed by a gradient of 0% → 100% MeCN / H₂O + 0.1% TFA (14 min) and then 100% MeCN / H₂O + 0.1% TFA (1 min). Quantification was performed using the absorbance at 280 nm. C 18 4.6 × 150 mm, 10 μm particles, flow rate 1 mL / min, eluent: isocratic elution 0% MeCN / H2O + 0.1% TFA (1 min), gradient elution 0% → 20% MeCN / H2O + 0.1% TFA (1 min), gradient elution 20% → 50% MeCN / H2O + 0.1% TFA (16 min), gradient elution 50% → 100% MeCN / H2O + 0.1% TFA (2 min)) The reaction with hydroxylamine 13 was analyzed and quantified using its absorbance at 254 nm.

[0725] Stability in PBS: Enamine N-oxide 17 (4 μL, 15 mM in 25% v / v MeOH / PBS, pH 7.4) was added to PBS (116 μL, pH 7.4; final concentration 500 μM). Hydroxylamine 13 (3 μL, 20 mM in deionized H₂O) was added separately to PBS (117 μL, pH 7.4; final concentration 500 μM). Each solution was monitored by HPLC at each time point.

[0726] Stability in the presence of glutathione, sodium ascorbate, and cell lysate: The reaction was carried out as described in the stability section in PBS above, except that before adding enamine N-oxide 17 (final concentration 500 μM) or hydroxylamine 13 (final concentration 500 μM), glutathione (final concentration 5 mM), sodium ascorbate (final concentration 5 mM), or HEK293T cell lysate (final concentration 1 mg / mL) was added to the PBS solution, and the pH was adjusted to 7.4.

[0727] Microsomal assay I: Human liver microsomal solution (8 μL, phosphate buffer, 20 mg / mL, pH 7.4) was prepared. A final concentration of 200 μg / mL and NADPH solution (13.4 μL, 60 mM in 10 mM NaOH solution) were sequentially added to PBS (751.9 μL, pH 7.4) in a 2.0 mL microcentrifuge tube. The solution was incubated at room temperature for 1 hour to obtain solution A. Enamine N-oxide 17 solution (26.7 μL, 15 mM in 25% MeOH / PBS, pH 7.4; final concentration 500 μM) was added to solution A. The cap of the microcentrifuge tube was punctured with a 16G needle to maintain an aerobic system. The reacti...

Claims

1. A compound having a structure represented by formula IV or V: (IV) or (V), Or its pharmaceutically acceptable salts or stereoisomers. in: R1' is an alkylene chain, wherein the alkylene chain is represented by –O–, –S–, –N(R')–, –C≡C–, –C(O)–, –C(O)O–, –OC(O)–, –OC(O)O–, –C(NOR')–, –C(O)N(R')–, –C(O)N(R')C(O)–, –R'C(O)N(R')R'–, –C(O)N(R')C(O)N(R')–, –N(R')C(O)N(R')–, –N(R')C(O)O–, –OC(O)N(R')–, –C(NR')–, –N(R')C(NR') –, –C(NR’)N(R’)–, –N(R’)C(NR’)N(R’)–, –OB(Me)O–, –S(O)2–, –OS(O)–, –S(O)O–, –S(O)–, –OS(O)2–, –S(O)2O–, –N(R’)S(O)2–, –S( O)2N(R')–, -N(R')S(O)–, -S(O)N(R')–, -N(R')S(O)2N(R')–, -N(R')S(O)N(R')–, -OP(O)O(R')O–, -N(R')P(O)N(R'R')N(R')–, C3-C 12 At least one of carbocyclic, 3- to 12-membered heterocyclic, 5- to 12-membered heteroaryl groups, or any combination thereof, is interrupted or terminated at one end and / or at the other end, wherein each R' is independently H or a substituted C1-C 24 Alkyl groups, wherein the interrupting group and the terminating group are the same or different; or R1' is a polyethylene glycol chain, wherein the polyethylene glycol chain is represented by –O–, –S–, –N(R')–, –C≡C–, –C(O)–, –C(O)O–, –OC(O)–, –OC(O)O–, –C(NOR')–, –C(O)N(R')–, –C(O)N(R')C(O)–, –R'C(O)N(R')R'–, –C(O)N(R')C(O)N(R')–, –N(R')C(O)–, –N(R')C(O)N(R')–, –N(R')C(O)O–, –OC(O)N(R')–, –C(NR')–, –N(R')C(NR') )–, –C(NR’)N(R’)–, –N(R’)C(NR’)N(R’)–, –OB(Me)O–, –S(O)2–, –OS(O)–, –S(O)O–, –S(O)–, –OS(O)2–, –S(O)2O–, –N(R’)S(O)2–, –S( O)2N(R')–, -N(R')S(O)–, -S(O)N(R')–, -N(R')S(O)2N(R')–, -N(R')S(O)N(R')–, -OP(O)O(R')O–, -N(R')P(O)N(R'R')N(R')–, C3-C 12 At least one of carbocyclic, 3- to 12-membered heterocyclic, 5- to 12-membered heteroaryl groups, or any combination thereof, is interrupted or terminated at one end and / or at the other end, wherein each R' is independently H or a substituted C1-C 24 Alkyl groups, wherein the interrupting group and the terminating group are the same or different; R1 does not exist, or R1 and R2, together with the nitrogen atoms they are attached to, form a heterocyclic group; R2 is a substituted C1-C8 alkyl group, -C(O)R”, -C(O)OR”, -C(O)NR”R”, -S(O)R”, -S(O)2R”, C3-C 10 A carbocyclic, 4- or 7-membered heterocyclic, or substituted polyethylene glycol chain, wherein each R” is independently hydrogen, C1-C6 alkyl, C3-C6 alkyl, C4-C6 alkyl, C5-C6 alkyl, C6-C6 alkyl, C7 ... 10 Carbocyclic, 4-membered or 7-membered heterocyclic groups, wherein the alkyl, carbocyclic or heterocyclic group is substituted; Each X is independently CR9R9', NR9, O, S, C(O), S(O) or SO2, wherein the ring system contains 0 to 3 heteroatoms; R9 and R9' are independently hydrogen or substituents; A1 is the combination part, the treatment part, or the diagnostic part; Y does not exist or ; A2 is a combination part, a treatment part, or a diagnostic part; R4 is hydrogen, a substituent, or a component bound to... Linking groups on the group, wherein the linking groups are: a) O, b) S, c) NR 11 , where R 11 It is hydrogen or C1-C6 alkyl. d) OPh, e)OC(O), f) OC(O)NR 11 , where R 11 It is hydrogen or C1-C6 alkyl. g) Alkylene chains, wherein the alkylene chains are represented by –O–, –S–, –N(R')–, –C≡C–, –C(O)–, –C(O)O–, –OC(O)–, –OC(O)O–, –C(NOR')–, –C(O)N(R')–, –C(O)N(R')C(O)–, –R'C(O)N(R')R'–, –C(O)N(R')C(O)N(R')–, –N(R')C(O)–, –N(R')C(O)N(R')–, –N(R')C(O)O–, –OC(O)N(R')–, –C(NR')–, –N(R')C(NR')– , –C(NR')N(R')–, –N(R’)C(NR’)N(R’)–, –OB(Me)O–, –S(O)2–, –OS(O)–, –S(O)O–, –S(O)–, –OS(O)2–, –S(O)2O–, –N(R’)S(O)2–, –S(O )2N(R')–, -N(R')S(O)–, -S(O)N(R')–, -N(R')S(O)2N(R')–, -N(R')S(O)N(R')–, -OP(O)O(R')O–, -N(R')P(O)N(R'R')N(R')–,, C3-C 12 At least one of carbocyclic, 3- to 12-membered heterocyclic, 5- to 12-membered heteroaryl groups, or any combination thereof, is interrupted or terminated at one end and / or at the other end, wherein each R' is independently H or a substituted C1-C 24 Alkyl groups, wherein the interrupting group and the terminating group are the same or different, or h) Polyethylene glycol chains, wherein the polyethylene glycol chains are represented by –O–, –S–, –N(R')–, –C≡C–, –C(O)–, –C(O)O–, –OC(O)–, –OC(O)O–, –C(NOR')–, –C(O)N(R')–, –C(O)N(R')C(O)–, –R'C(O)N(R')R'–, –C(O)N(R')C(O)N(R')–, –N(R')C(O)N(R')–, –N(R')C(O)O–, –OC(O)N(R')–, –C(NR')–, –N(R')C(NR') –, –C(NR’)N(R’)–, –N(R’)C(NR’)N(R’)–, –OB(Me)O–, –S(O)2–, –OS(O)–, –S(O)O–, –S(O)–, –OS(O)2–, –S(O)2O–, –N(R’)S(O)2–, –S( O)2N(R')–, -N(R')S(O)–, -S(O)N(R')–, -N(R')S(O)2N(R')–, -N(R')S(O)N(R')–, -OP(O)O(R')O–, -N(R')P(O)N(R'R')N(R')–, C3-C 12 At least one of carbocyclic, 3- to 12-membered heterocyclic, 5- to 12-membered heteroaryl groups, or any combination thereof, is interrupted or terminated at one end and / or at the other end, wherein each R' is independently H or a substituted C1-C 24 Alkyl groups, wherein the interrupting group and the terminating group are the same or different, or R4 and R5, together with the carbon atoms they are attached to, form a carbocyclic or heterocyclic group, wherein R4 is also bonded to... superior; R5 is hydrogen or an electron-withdrawing group; R6 is hydrogen, a π-electron-donating group, or a group bound to hydrogen. Linking groups on; R7 and R7' are independently hydrogen or electron-withdrawing groups, or R7 and R7' together with the carbon atoms they are attached to form C(O); R8 is hydrogen, a substituent, or bound to... The linking group on; and n is 1, 2, or 3; in: a) and One is a diagnostic agent, and the other is a therapeutic agent; b) of which and One is an antibody or its binding fragment, and the other is a therapeutic agent, or c) and All are binders. The prerequisite is that the compound contains at least one Group.

2. The compound according to claim 1, wherein R1 is absent and R1' is an alkylene chain, said alkylene chain being –O–, –S–, –N(R')–, –C≡C–, –C(O)–, –C(O)O–, –OC(O)–, –OC(O)O–, –C(NOR')–, –C(O)N(R')–, –C(O)N(R')C(O)–, –R'C(O)N(R')R'–, –C(O)N(R')C(O)N(R')–, –N(R')C(O)–, –N(R')C(O)N(R')–, –N(R')C(O)O–, –OC(O)N(R')–, –C(NR')– , –N(R')C(NR')–, –C(NR’)N(R’)–, –N(R’)C(NR’)N(R’)–, –OB(Me)O–, –S(O)2–, –OS(O)–, –S(O)O–, –S(O)–, –OS(O)2–, –S(O)2O–, –N(R’)S(O) 2–, –S(O)2N(R’)–, –N(R’)S(O)–, –S(O)N(R’)–, –N(R’)S(O)2N(R’)–, -N(R')S(O)N(R')–, -OP(O)O(R')O–, -N(R')P(O)N(R'R')N(R')–, C3-C 12 At least one of carbocyclic, 3- to 12-membered heterocyclic, 5- to 12-membered heteroaryl groups, or any combination thereof, is interrupted or terminated at one end and / or at the other end, wherein each R' is independently H or a substituted C1-C 24 Alkyl groups, wherein the interrupting group and the terminating group are the same or different.

3. The compound according to claim 2, wherein the alkylene chain is C1-C2. 12 Alkylene chain.

4. The compound according to claim 1, wherein R1 is absent and R1' is a polyethylene glycol chain, said polyethylene glycol chain being replaced by –O–, –S–, –N(R')–, –C≡C–, –C(O)–, –C(O)O–, –OC(O)–, –OC(O)O–, –C(NOR')–, –C(O)N(R')–, –C(O)N(R')C(O)–, –R'C(O)N(R')R'–, –C(O)N(R')C(O)N(R')–, –N(R')C(O)–, –N(R')C(O)N(R')–, –N(R')C(O)O–, –OC(O)N(R')–, –C(NR')– –, –N(R’)C(NR’)–, –C(NR’)N(R’)–, –N(R’)C(NR’)N(R’)–, –OB(Me)O–, –S(O)2–, –OS(O)–, –S(O)O–, –S(O)–, –OS(O)2–, –S(O)2O–, –N(R’)S(O )2–, –S(O)2N(R’)–, –N(R’)S(O)–, –S(O)N(R’)–, –N(R’)S(O)2N(R’)–, -N(R')S(O)N(R')–, -OP(O)O(R')O–, -N(R')P(O)N(R'R')N(R')–, C3-C 12 At least one of carbocyclic, 3- to 12-membered heterocyclic, 5- to 12-membered heteroaryl groups, or any combination thereof, is interrupted or terminated at one end and / or at the other end, wherein each R' is independently H or a substituted C1-C 24 Alkyl groups, wherein the interrupting group and the terminating group are the same or different.

5. The compound according to claim 4, wherein the polyethylene glycol chain has 2 to 20 -(CH2CH2-O)- units.

6. The compound according to claim 1, wherein R1 and R2, together with the nitrogen atom to which they are attached, form a 5- to 10-membered heterocyclic group containing 1 to 3 heteroatoms selected from N, O and S.

7. The compound according to claim 1, wherein R2 is methyl, ethyl, isopropyl or tert-butyl.

8. The compound according to any one of claims 1-7, wherein A1 is the binding moiety.

9. The compound of claim 8, wherein the binding portion is a small molecule, short amino acid sequence, protein, antibody, or antibody-binding fragment that binds to a predetermined target.

10. The compound of claim 9, wherein the antibody is a monoclonal antibody or a binding fragment thereof.

11. The compound according to claim 9, wherein the small molecule is biotin or a derivative thereof, a small molecule that binds to E3 ligase, or a small molecule that binds to cellular proteins.

12. The compound according to any one of claims 1-7, wherein A1 is the therapeutic component.

13. The compound of claim 12, wherein the therapeutic portion A1 is a non-targeted anticancer agent, a targeted anticancer agent, an antibacterial agent, a nonsteroidal anti-inflammatory drug (NSAID), a corticosteroid, or a disease-modifying antirheumatic drug (DMARD).

14. The compound of claim 12, wherein the therapeutic portion A1 is a targeted anticancer agent or a non-targeted anticancer agent.

15. The compound of claim 14, wherein the targeted anticancer agent is a kinase inhibitor.

16. The compound according to any one of claims 1-7, wherein A1 is a diagnostic portion.

17. The compound of claim 16, wherein the diagnostic fraction A1 is a fluorophore, a chromogenic agent, a positron emission tomography (PET) tracer, or a magnetic resonance imaging (MRI) contrast agent.

18. The compound of claim 17, wherein the diagnostic portion A1 is a fluorophore.

19. The compound of claim 17, wherein the diagnostic portion A1 is a positron emission tomography (PET) tracer.

20. The compound according to claim 1, wherein n is 2.

21. The compound according to claim 1, wherein X is CR9R9'.

22. The compound according to claim 21, wherein R9 and R9' are each hydrogen.

23. The compound according to claim 1, wherein R9 and R9' are independently hydrogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, C1-C6 haloalkoxy, -C(O)R 10 -NR 10 R 10 -C(O)NR 10 R 10 -OC(O)NR 10 R 10 -NR 10 C(O)R 10 -NR 10 C(O)OR 10 Halogen, OH, CN, amino, C3-C 10 Carbocyclic groups, 4- or 7-membered heterocyclic groups, -O(CH2) 0-3 C3-C 10 A carbocyclic group, containing one to three heteroatoms selected from O, N, and S, of the radical -O(CH2). 0-3 -4- or 7-membered heterocyclic groups, where each R 10 It is independently hydrogen or C1-C6 alkyl; wherein the alkyl, carbocyclic or heterocyclic group is further substituted.

24. The compound according to any one of claims 1-7, wherein R4 is bound to... Linking groups on.

25. The compound according to claim 24, wherein R4 is O.

26. The compound according to claim 24, wherein R4 is S.

27. The compound according to claim 24, wherein R4 is NR. 11 , where R 11 It is hydrogen or C1-C6 alkyl.

28. The compound according to claim 24, wherein R4 is OPh.

29. The compound according to claim 24, wherein R4 is OC(O).

30. The compound according to claim 24, wherein R4 is OC(O)NR. 11 , where R 11 It is hydrogen or C1-C6 alkyl.

31. The compound according to claim 24, wherein R4 is an alkylene chain, said alkylene chain being –O–, –S–, –N(R')–, –C≡C–, –C(O)–, –C(O)O–, –OC(O)–, –OC(O)O–, –C(NOR')–, –C(O)N(R')–, –C(O)N(R')C(O)–, –R'C(O)N(R')R'–, –C(O)N(R')C(O)N(R')–, –N(R')C(O)–, –N(R')C(O)N(R')–, –N(R')C(O)O–, –OC(O)N(R')–, –C(NR')–, –N (R')C(NR')–, –C(NR')N(R')–, –N(R’)C(NR’)N(R’)–, –OB(Me)O–, –S(O)2–, –OS(O)–, –S(O)O–, –S(O)–, –OS(O)2–, –S(O)2O–, –N(R’)S(O)2 –, –S(O)2N(R’)–, –N(R’)S(O)–, –S(O)N(R’)–, –N(R’)S(O)2N(R’)–, –N(R’)S(O)N(R’)–, –OP(O)O(R’)O–, –N(R’)P(O)N(R’R’)N(R’)–, C3-C 12 At least one of carbocyclic, 3- to 12-membered heterocyclic, 5- to 12-membered heteroaryl groups, or any combination thereof, is interrupted or terminated at one end and / or at the other end, wherein each R' is independently H or a substituted C1-C 24 Alkyl groups, wherein the interrupting group and the terminating group are the same or different.

32. The compound according to claim 31, wherein the alkylene chain is C1-C2. 12 Alkylene chain.

33. The compound according to claim 24, wherein R4 is a polyethylene glycol chain, said polyethylene glycol chain being coupled with –O–, –S–, –N(R')–, –C≡C–, –C(O)–, –C(O)O–, –OC(O)–, –OC(O)O–, –C(NOR')–, –C(O)N(R')–, –C(O)N(R')C(O)–, –R'C(O)N(R')R'–, –C(O)N(R')C(O)N(R')–, –N(R')C(O)–, –N(R')C(O)N(R')–, –N(R')C(O)O–, –OC(O)N(R')–, –C(NR')–, – N(R')C(NR')–, –C(NR')N(R')–, –N(R’)C(NR’)N(R’)–, –OB(Me)O–, –S(O)2–, –OS(O)–, –S(O)O–, –S(O)–, –OS(O)2–, –S(O)2O–, –N(R’)S(O)2 –, –S(O)2N(R’)–, –N(R’)S(O)–, –S(O)N(R’)–, –N(R’)S(O)2N(R’)–, –N(R’)S(O)N(R’)–, –OP(O)O(R’)O–, –N(R’)P(O)N(R’R’)N(R’)–, C3-C 12 At least one of carbocyclic, 3- to 12-membered heterocyclic, 5- to 12-membered heteroaryl groups, or any combination thereof, is interrupted or terminated at one end and / or at the other end, wherein each R' is independently H or a substituted C1-C 24 Alkyl groups, wherein the interrupting group and the terminating group are the same or different.

34. The compound of claim 33, wherein the polyethylene glycol chain has 2 to 20 -(CH2CH2-O)- units.

35. The compound according to claim 1, wherein R4 and R5 together with the carbon atoms to which they are attached form a 5- to 10-membered carbocyclic group or a 5- to 10-membered heterocyclic group containing one to three heteroatoms selected from N, O and S.

36. The compound according to claim 35, wherein R4 and R5, together with the carbon atoms to which they are attached, form a 5-membered heterocyclic group containing 2 oxygen atoms.

37. The compound according to any one of claims 1-7, wherein R5 is hydrogen.

38. The compound according to any one of claims 1-7, wherein R5 is an electron-withdrawing group.

39. The compound according to claim 38, wherein R5 is an electron-withdrawing group.

40. The compound according to claim 39, wherein the electron-withdrawing inducing group is a halogen, OR 5' SR 5' or NR 5' R 5' , where each R 5' Independently hydrogen, C1-C6 alkyl, C6-C 12 Aryl, 5- to 10-membered heteroaryl, carbonyl, sulfonyl, sulfinyl or phosphoryl.

41. The compound according to claim 38, wherein R5 is a π-electron-withdrawing group.

42. The compound according to claim 41, wherein the π-electron-withdrawing group is -C(O)R 5” -C(O)NR 5” R 5” -C(O)NR 5” R 5” -C(O)OR 5” NO2, CN, N3, -S(O)R 5” -S(O)2R 5” -S(O)OR 5” -S(O)2OR 5” -S(O)NR 5” R 5” -S(O)2NR 5” R 5” -OP(O)OR 5” OR 5” -P(O)NR 5” R 5” NR 5” R 5” , where each R 5'' Independently hydrogen, C1-C6 alkyl, C6-C 12 Aryl, 5- to 10-membered heteroaryl.

43. The compound according to any one of claims 1-7, wherein R6 is hydrogen.

44. The compound according to any one of claims 1-7, wherein R6 is a π-electron-donating group.

45. The compound according to claim 44, wherein R6 is OR 12 SR 12 NR 12 NR 12 Either cyclic amides or acyclic amides, wherein each R 12 Independently, it is hydrogen, C1-C6 alkyl, C3-C 10 A carbocyclic, 4-membered or 7-membered heterocyclic group, wherein the alkyl, carbocyclic or heterocyclic group is substituted.

46. ​​The compound according to any one of claims 1-7, wherein R7 and R7' are independently hydrogen or electron-withdrawing groups.

47. The compound according to claim 46, wherein the electron-withdrawing inducing group is a halogen, OR 5' SR 5' or NR 5' R 5' , where each R 5' Independently hydrogen, C1-C6 alkyl, C6-C 12 Aryl, 5- to 10-membered heteroaryl, carbonyl, sulfonyl, sulfinyl or phosphoryl.

48. The compound according to any one of claims 1-7, wherein R7 and R7' are independently hydrogen or electron-withdrawing groups.

49. The compound according to claim 48, wherein the π-electron-withdrawing group is -C(O)R 5” -C(O)NR 5” R 5” -C(O)NR 5” R 5” -C(O)OR 5” NO2, CN, N3, -S(O)R 5” -S(O)2R 5” -S(O)OR 5” -S(O)2OR 5” -S(O)NR 5” R 5” -S(O)2NR 5” R 5” -OP(O)OR 5” OR 5” -P(O)NR 5” R 5” NR 5” R 5” , where each R 5'' Independently hydrogen, C1-C6 alkyl, C6-C 12 Aryl, 5- to 10-membered heteroaryl.

50. The compound according to any one of claims 1-7, wherein R8 is bound to... Linking groups on.

51. The compound according to claim 50, wherein R8 is CH2.

52. The compound according to claim 50, wherein R8 is an aryl group.

53. The compound according to claim 50, wherein R8 is O.

54. The compound according to claim 50, wherein R8 is an alkylene chain, wherein the chain is modified by –O–, –S–, –N(R')–, –C≡C–, –C(O)–, –C(O)O–, –OC(O)–, –OC(O)O–, –C(NOR')–, –C(O)N(R')–, –C(O)N(R')C(O)–, –R'C(O)N(R')R'–, –C(O)N(R')C(O)N(R')–, –N(R')C(O)N(R')–, –N(R')C(O)N(R')–, –N(R')C(O)O–, –OC(O)N(R')–, –C(NR')–, –N(R') )C(NR')–, –C(NR’)N(R’)–, –N(R’)C(NR’)N(R’)–, –OB(Me)O–, –S(O)2–, –OS(O)–, –S(O)O–, –S(O)–, –OS(O)2–, –S(O)2O–, –N(R’)S(O)2–, -S(O)2N(R')–, -N(R')S(O)–, -S(O)N(R')–, -N(R')S(O)2N(R')–, -N(R')S(O)N(R')–, -OP(O)O(R')O–, -N(R')P(O)N(R'R')N(R')–, C3-C 12 At least one of carbocyclic, 3- to 12-membered heterocyclic, 5- to 12-membered heteroaryl groups, or any combination thereof, is interrupted or terminated at one end and / or at the other end, wherein each R' is independently H or a substituted C1-C 24 Alkyl group, wherein the interrupting group and the terminating group are the same or different.

55. The compound according to claim 54, wherein the alkylene chain is C1-C2. 12 Alkylene chain.

56. The compound according to claim 50, wherein R8 is a polyethylene glycol chain, said polyethylene glycol chain being coupled with –O–, –S–, –N(R')–, –C≡C–, –C(O)–, –C(O)O–, –OC(O)–, –OC(O)O–, –C(NOR')–, –C(O)N(R')–, –C(O)N(R')C(O)–, –R'C(O)N(R')R'–, –C(O)N(R')C(O)N(R')–, –N(R')C(O)–, –N(R')C(O)N(R')–, –N(R')C(O)O–, –OC(O)N(R')–, –C(NR')–, – N(R')C(NR')–, –C(NR')N(R')–, –N(R’)C(NR’)N(R’)–, –OB(Me)O–, –S(O)2–, –OS(O)–, –S(O)O–, –S(O)–, –OS(O)2–, –S(O)2O–, –N(R’)S(O)2 –, –S(O)2N(R’)–, –N(R’)S(O)–, –S(O)N(R’)–, –N(R’)S(O)2N(R’)–, –N(R’)S(O)N(R’)–, –OP(O)O(R’)O–, –N(R’)P(O)N(R’R’)N(R’)–, C3-C 12 At least one of carbocyclic, 3- to 12-membered heterocyclic, 5- to 12-membered heteroaryl groups, or any combination thereof, is interrupted or terminated at one end and / or at the other end, wherein each R' is independently H or a substituted C1-C 24 Alkyl groups, wherein the interrupting group and the terminating group are the same or different.

57. The compound of claim 56, wherein the polyethylene glycol chain has 1 to 10 -(CH2CH2-O)- units.

58. The compound according to any one of claims 1-7, wherein A2 is the binding moiety.

59. The compound of claim 58, wherein the binding portion A2 is a small molecule, short amino acid sequence, protein, antibody, or antibody-binding fragment that binds to a predetermined target.

60. The compound of claim 58, wherein the antibody is a monoclonal antibody or a binding fragment thereof.

61. The compound of claim 59, wherein the small molecule is biotin or a derivative thereof, a small molecule that binds to E3 ligase, or a small molecule that binds to cellular proteins.

62. The compound according to any one of claims 1-7, wherein A2 is the therapeutic component.

63. The compound of claim 62, wherein the therapeutic portion A2 is a non-targeted anticancer agent, a targeted anticancer agent, an antibacterial agent, a nonsteroidal anti-inflammatory drug (NSAID), a corticosteroid, or a disease-modifying antirheumatic drug (DMARD).

64. The compound of claim 63, wherein the therapeutic portion A2 is a targeted anticancer agent or a non-targeted anticancer agent.

65. The compound of claim 64, wherein the targeted anticancer agent is a kinase inhibitor.

66. The compound according to any one of claims 1-7, wherein A2 is the diagnostic portion.

67. The compound of claim 66, wherein the diagnostic portion A2 is a fluorophore, a chromogenic agent, a positron emission tomography (PET) tracer, or a magnetic resonance imaging (MRI) contrast agent.

68. The compound of claim 67, wherein the diagnostic portion A2 is a fluorophore.

69. The compound of claim 67, wherein the diagnostic portion A2 is a positron emission tomography (PET) tracer.

70. The compound according to claim 1, wherein the compound is 、 、 , , , , , , , , , , , Or, or its pharmaceutically acceptable salt or stereoisomer.

71. The compound according to claim 70, wherein the compound is Or, or its pharmaceutically acceptable salt or stereoisomer.

72. The compound according to claim 1, which is 、 、 、 、 、 、 、 、 、 、 、 、 、 , , , , , , , , , , , Or, or its pharmaceutically acceptable salt or stereoisomer.

73. The compound according to claim 72, which is Or, or its pharmaceutically acceptable salt or stereoisomer.

74. The compound according to any one of claims 1-7, wherein and One is a diagnostic agent, and the other is a therapeutic agent, and the compound is in the form of a therapeutic agent.

75. The compound according to any one of claims 1-7, wherein and One is an antibody or a binding fragment thereof, and the other is a therapeutic agent, wherein the compound is in the form of an antibody-drug conjugate.

76. The compound according to any one of claims 1-7, wherein and All of these are binders, and the compounds are degrading agents.

77. The compound according to claim 1, wherein the compound of formula IV has formula IVa', IVb' or IVc': (IVa')、 (IVb')、 (IVc'), or its pharmaceutically acceptable salt or stereoisomer.

78. The compound according to claim 77, wherein the antibody is a monoclonal antibody, and R1 and R2, together with the nitrogen atom to which they are attached, form a piperazine group and have a structure represented by formula IVa'1: (IVa'1) or its pharmaceutically acceptable salt or stereoisomer.

79. The compound according to claim 77, wherein the antibody is a monoclonal antibody, R1 is absent, and R2 is methyl, and has a structure represented by formula IVa'2: (IVa'2), or its pharmaceutically acceptable salt or stereoisomer.

80. A pharmaceutical composition comprising a therapeutically effective amount of the compound or pharmaceutically acceptable salt or stereoisomer according to any one of claims 1-73 and 77-79, and a pharmaceutically acceptable carrier.

81. Use of the compound or pharmaceutically acceptable salt or stereoisomer of any one of claims 77-79 in the preparation of a medicament for treating cancer, said medicament comprising the compound or pharmaceutically acceptable salt or stereoisomer of any one of claims 77-79 and a diborone reagent.

82. The use according to claim 81, wherein the diboron reagent is a symmetrical diboron reagent.

83. The use according to claim 81, wherein the diboron reagent is an asymmetric diboron reagent.

84. Use of the compound or pharmaceutically acceptable salt or stereoisomer of any one of claims 1-7, 12-57 and 62-73 in the preparation of a medicament for treating cancer.

85. Use of the compound or pharmaceutically acceptable salt or stereoisomer of any one of claims 1-11, 16-61, and 66-73 in the preparation of a reagent for protein labeling, said reagent comprising the compound or pharmaceutically acceptable salt or stereoisomer of any one of claims 1-11, 16-61, and 66-73, wherein one active portion binds to a protein and the other active portion is a diagnostic marker, and wherein said protein is a cancer-associated antigen.

86. A process for preparing the compound of formula IV according to claim 1: (IV), Including compounds of formula I (I) with compounds of formula II: (II) Reaction.

87. A process for preparing the compound of formula V according to claim 1: (V)， Including compounds of formula I (I) and Compound III (III) Reaction.

88. The process according to claim 86 or 87, wherein the reaction is carried out in the presence of a solvent.

89. The process according to claim 88, wherein the solvent is an aprotic solvent.

90. The process according to claim 89, wherein the aprotic solvent is DCM, CHCl3, CCl4, DCE, toluene, MeCN, or THF.

91. The process according to claim 88, wherein the solvent is a proton solvent.

92. The process according to claim 91, wherein the proton solvent is MeOH, EtOH, iPrOH, nBuOH, TFE or HFIP.

93. The process according to claim 88, wherein the solvent is a solvent mixture.

94. The process according to claim 93, wherein the solvent mixture is a mixture of aprotic solvent and protic solvent.

95. The process according to claim 87 or 88, wherein the reaction is carried out in the presence of an aqueous buffer solution.

96. The process according to claim 95, wherein the aqueous buffer solution is an acidic buffer solution.

97. The process according to claim 95, wherein the aqueous buffer solution is an alkaline buffer solution.

98. The process according to claim 87 or 88, wherein the reaction is carried out in the presence of a biological fluid.

99. The process according to claim 98, wherein the biofluid is blood, synovial fluid, lymph, or vitreous fluid.

100. The process according to claim 87 or 88, wherein the reaction is carried out in the presence of an aqueous solution containing biological components.

101. The process according to claim 87 or 88, wherein the reaction is carried out at a temperature between 0°C and 60°C.

102. The process according to claim 101, wherein the temperature is 20°C-25°C.

103. The process according to claim 87 or 88, wherein the compound of formula (I) is in excess relative to the compound of formula (II) or (III).

104. The process according to claim 103, wherein the excess is 5 equivalents.

105. The process according to claim 87 or 88, wherein the reaction is carried out with the addition of a buffer reagent.

106. The process according to claim 105, wherein the buffer reagent is ascorbic acid or glutathione.

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