Bioorthogonal reactions suitable for click / non-click applications

Abstract

Disclosed are compounds suitable for cell labeling or treating cancer, as well as pharma- ceutically acceptable salts and stereoisomers thereof, as well as pharmaceutical compositions containing same, and methods of making and using said compounds.

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 63 / 170,705, filed April 5, 2021, and U.S. Provisional Patent Application No. 63 / 315,328, filed March 1, 2022, each of which is incorporated by reference herein in its entirety.

[0002] government support This invention was made with government support under Grant No. 1DP2 ES030448 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCHII 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 6 KB in size. [Background technology]

[0004] The copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction, which debuted two decades ago (Rostovtsev et al., Angew. Chem., Int. Ed. 41(14):2596-2599 (2002)), has proven indispensable in fields ranging 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)). Its rapid kinetics, ease of implementation, and near-universal compatibility of its reaction components make it a widely appealing reaction. Relatively inert azides and terminal alkynes are two of the smallest functional groups available, and either component can be readily incorporated into biological macromolecules, metabolites, and probes without significantly perturbing the system under evaluation.The ability to incorporate these motifs into biological systems via unnatural amino acids using orthogonal tRNA systems and methionine auxotrophs (Kiick et al., Proc. Natl. Acad. Sci. USA 99(1):19-24 (2002); Prescher et al., Nat. Chem. Biol. 1(1):13-21 (2005); Plass et al., Angew. Chem., Int. Ed. 50(17):3878-3881 (2011)), 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., Chem. Soc. Rev. 49:4602-4614 (2020)), or 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)) has been transformative.

[0005] Bertozzi and coworkers have since extended the application of the azide-alkyne cycloaddition to living cells and in vivo systems by using strained cyclooctynes ​​instead of copper catalysts (Baskin et al., Proc. Natl. Acad. Sci. USA 104(43):16793-16797 (2007)). Strain-promoted reactions are currently being used to synthesize trans-cyclooctenes (Blackman et al., J. Am. Chem. Soc. 130(41):13518-13519 (2008)), norbornenes (Devaraj et al., Bioconjugate Chem. 19(12):2297-2299 (2008)), quadricyclanes (Sletten et al., J. Am. Chem. Soc. 133(44):17570-17573 (2011)), and cyclopropanes (Patterson et al., J. Am. Chem. Soc. 134(45):18638-18643 (2012); Row et al., J. Am. Chem. Soc. 139(21):7370-7375 (2017)) has become a staple of bioorthogonal compendia, with notable examples featuring:Importantly, cyclooctynes ​​undergo significant geometrical changes (Dommerholt et al., 2004) to enhance their reaction kinetics with azides (Agard et al., J. Am. Chem. Soc. 126(46):15046-15047 (2004)), tetrazines (Blackman 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)) compounds. 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 electronically (Agard et al., ACS Chem. Biol. 1(10):644-648 (2006); Baskin et al., Proc. Natl. Acad. Sci. USA 104(43):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)) with significant modifications.

[0006] The growing catalog of bioorthogonal reactions allows for the visualization, isolation, and manipulation of biomolecules in complex biological environments, both 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 involve the synthesis of sugars (Baskin et al., Proc. Natl. Acad. Sci. USA 104(43):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 (2019)). (2020)) and biopolymers for which modification by genetic means is neither practical nor possible (George et al., Chem. Commun.56:12307-12318 (2020)). Consequently, there is a need for additional bioorthogonal tools, particularly 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)), rapid (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)), stable (Row et al., J. Am. Chem. Soc. 139(21):7370-7375 (2017); Tu et al., Angew. Chem., Int. Ed. 58(27):9043-9048 (2019)), regioselective (Grost et al., Org. Biomol. Chem. 13:3866-3870(2015)), and functionally diverse (Volker 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., Int. 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 both biology and themselves (Liang et al., J. Am. Chem. Soc. 134(43):17904-17907 (2012); Patterson et al., Curr. Opin. Chem. Biol. 28:141-149 (2015)).

[0007] The development of new reactions that proceed along not just one or two but several of these axes simultaneously has been a fascinating but elusive aspiration (Row et al., Acc. Chem. Res. 51(5):1073-1081 (2018); Devaraj, NK, ACS Cent. Sci. 4(8):952-959 (2018)). Summary of the Invention [Means for solving the problem]

[0008] A first aspect of the present invention is directed to a compound represented by the structure of formula (I): [ka] wherein R1, R1', R2, and A1 are as defined herein, or a pharmaceutically acceptable salt or stereoisomer thereof.

[0009] Another aspect of the present invention relates to compounds represented by formulas (II) and (III): [ka] (wherein R4, R5, R6, R7, R7', R8, X, Y, and n are as defined herein), or a pharmaceutically acceptable salt or stereoisomer thereof.

[0010] Yet another aspect of the present invention relates to enamine N-oxide compounds represented by formulas (IV) and (V): [ka] (wherein R1, R1', R2, A1, R4, R5, R6, R7, R7', R8, X, Y, and n are as defined herein), or a pharmaceutically acceptable salt or stereoisomer thereof. The compounds of formulas (IV) and (V) each contain at least two active moieties.

[0011] The compounds of the present invention are particularly suitable for clinical applications where it is advantageous to deliver two active agents or moieties. Thus, in some embodiments, the compounds of formula (IV) or (V) are antibody-drug conjugates, in which one of the two active moieties is a binding moiety and the other 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 target a given protein for selective degradation, in which both active moieties are binding moieties. One of the binding moieties binds to the target protein, and the other binding moiety binds to a cellular enzyme that catalyzes the degradation of the target protein. Although both active moieties are binding moieties, the compounds themselves are therapeutic. In other embodiments, the compounds of formula (IV) or (V) are theranostic agents, in which one of the two active moieties is a diagnostic agent and the other is a therapeutic agent.

[0012] A further aspect of the present invention relates to a method for preparing bifunctional enamine N-oxide compounds of formula (IV) and (V) having two different active moieties. The method for producing the compound of formula (IV) requires reacting a compound of formula (I) with a compound of formula (II). The method for producing the compound of formula (V) requires reacting a compound of formula (I) with a compound of formula (III). The process or synthesis method for producing the compounds of formula (IV) and (V) involves a bioorthogonal reaction between two reagents, namely, the compound of formula (I) and the compounds of formulas (II) and (III). More specifically, it is a catalyst-free conjugate reverse Cope elimination reaction that allows for bioorthogonal ligation of the two active moieties.

[0013] Another aspect of the present invention is directed to a pharmaceutical composition comprising a therapeutically effective amount of a compound of Formula (IV) or a pharmaceutically acceptable salt or stereoisomer thereof, and a pharmaceutically acceptable carrier.

[0014] A further aspect of the invention relates to methods for diagnosing and treating diseases and disorders. In some embodiments, the disease is cancer. Another aspect of the invention relates to methods for protein labeling. In some embodiments, the methods relate to labeling cancer-associated antigens.

[0015] The biorthogonal reaction is rapid and connects (ligates) these two active moieties via a cleavable linker. The biorthogonal reaction can occur in vivo before administration to a subject or after administration of the individual reagents. That is, compounds (IV) and (V) can be administered to a subject. Alternatively, these compounds can be formed in vivo after administration of a compound of formula (I) and a compound of formula (II) or (III). [Brief explanation of the drawings]

[0016] [Figure 1] Figure 1 is a schematic illustrating the bioorthogonal reverse Cope elimination reaction between cyclooctyne and N,N-dialkylhydroxylamine.

[0017] [Figures 2A-2D] Figures 2A-2D show a computational study of the reverse Cope elimination reaction between cyclooctyne (COT) and N,N-dimethylhydroxylamine. The geometry was optimized at the M06-2X / 6-31G(d,p) level of theory, and the single-point energies were calculated at the M06-2X / 6-311G(2d,p) level of theory. Figure 2A shows a computational reaction model for assessing the reactivity of cyclooctyne. Figure 2B shows the calculated transition state structure and activation energy for cyclooctyne hydroamination. Figure 2C shows that additional ring strain in bicyclo[6.1.0]nonyne resulted in a lower activation barrier. Figure 2D is a table of the calculated free energy of activation (ΔG‡) as well as the strain (ΔE‡dist.) and interaction energies (ΔE‡int.), highlighting the rapidity of the reverse Cope elimination reaction and the central role of the strain energy of the hydroxylamine and alkyne in lowering the activation barrier. R = p-NO2Ph.

[0018] [Figure 3] Figure 3 shows the second-order rate constants for the hydroamination of cyclooctynes ​​2–10 with N,N-diethylhydroxylamine (1). Second-order kinetic studies were carried out using equimolar concentrations of cyclooctyne and hydroxylamine in CHCN at room temperature. The rate constant for difluorocyclooctyne 10 was derived from a competition experiment with carbamate 9.

[0019] [Figures 4A-4E] Figure 4A-4E show protein labeling using the reverse Cope elimination reaction. Figure 4A shows the synthetic route for the fluorophore-hydroxylamine conjugate 13. Figure 4B shows the modification of lysozyme with N-hydroxysuccinimide (NHS)-ester 14 to obtain cyclooctyne-containing lysozyme 15. The modified protein, lysozyme-COT15, was labeled with fluorescent hydroxylamine 13. Figure 4C shows an in-gel fluorescence analysis of lysozyme COT15 (0.14 mg / mL) incubated with various concentrations of hydroxylamine 13 (10-200 μM) in phosphate-buffered saline (PBS) for 2 h at room temperature. Figure 4D shows an in-gel fluorescence analysis of lysozyme COT15 (0.14 mg / mL) incubated with hydroxylamine 13 (200 μM) for 1-120 min in PBS at room temperature. Figure 4E shows that complete conjugation was observed by intact mass spectrometry analysis of lysozyme fluorophore conjugate 16 obtained by incubating lysozyme COT 15 (0.58 mg / mL) and hydroxylamine 13 (200 μM) in PBS for 6 h at room temperature.

[0020] [Figures 5A-5D]Figures 5A-5D demonstrate bioorthogonality. Figure 5A shows the synthesis of enamine N-oxide 17. Figure 5B is a bar graph showing the stability of hydroxylamine 13 and enamine N-oxide 17 studied in PBS at pH 7.4 in the presence of glutathione (5 mM), cell lysate (1 mg / mL), microsomes (0.2 mg / mL), or without additives. The protective effect of sodium ascorbate (5 mM) was further evaluated for hydroxylamine 13. Figure 5C is an in-gel fluorescence analysis of the 2-hour reaction between hydroxylamine 13 (200 μM) and lysozyme-COT15 in the presence of cell lysate (2.5 mg / mL), demonstrating exclusive labeling of lysozyme. Figure 5D shows cross-reactivity between different sets 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.

[0021] [Figures 6A-6H] Figures 6A-6H are reaction plots used to calculate second-order rate constants between N,N-diethylhydroxylamine and cyclooctynes ​​2 (Figure 6A), 3 (Figure 6B), 4 (Figure 6C), 5 (Figure 6D), 6 (Figure 6E), 7 (Figure 6F), 8 (Figure 6G), and 9 (Figure 6H). Each panel shows three separate experiments.

[0022] [Figure 7] Figure 7 shows a competition experiment performed between cyclooctyne carbamate 9 and difluorocyclooctyne 10 in a 1:4 ratio to determine the second-order rate constant of the latter with N,N-diethylhydroxylamine.

[0023] [Figure 8] Figure 8 shows images of the complete Coomassie stained (left) and in-gel fluorescent (right) images for a concentration-dependent protein labeling experiment. Both images are from the same gel.

[0024] [Figure 9]Figure 9 shows images of the complete Coomassie stained (left) and in-gel fluorescent (right) images for a concentration-dependent protein labeling experiment. Both images are from the same gel.

[0025] [Figures 10A-10C] Figures 10A-10C show mass spectrometry confirming the bioorthogonal reaction between hydroxylamine 13 and lysozyme-COT 15. ESI mass spectra of unmodified lysozyme (Figure 10A), the lysozyme-cyclooctyne conjugate (Figure 10B), and the reaction mixture of the hydroamination between hydroxylamine 13 and lysozyme-cyclooctyne conjugate 15 (Figure 10C) (single adduct: expected 15073.3 Da, observed 15073.3 Da; double adduct: expected 15840.6 Da, observed 15841.7 Da).

[0026] [Figures 11A-11C] Figures 11A-11C show alkyne activation. Figure 11A shows metal-catalyzed azide-alkyne cycloaddition. Figure 11B shows Train-promoted alkyne hydroamination. Figure 11C shows push-pull activated linear alkyne hydroamination.

[0027] [Figures 12A-12B] Figures 12A-B show the effect of terminal and propargyl modifications. Figure 12A shows the reactivity screening using alkynes 8'-15'. NMR conversion using trifluorotoluene as an internal standard. Figure 12B shows the synthesis of alkynes 9'-15'. R = OPMB. PMB = p-methoxybenzyl.

[0028] [Figures 13A-13B]Figures 13A-13B show the reaction rates and stabilities of selected alkynes and enamine N-oxides. Figure 13A is a table of second-order rate constants for alkynes 11'-15' in CD3CN at room temperature. Figure 13B is a graph showing the stability of alkynes 13', 14', 15' and enamine N-oxide 20' in 50% CD3CN / PBS in the presence or absence of glutathione (GSH) or HEK293T cell lysate.

[0029] [Figures 14A-14E] Figures 14A-14E show in vitro and live-cell labeling by bioorthogonal hydroamination. Figure 14A shows HaloTag protein conjugated to chloroalkyne 21', modified with TAMRA-hydroxylamine 22', and then visualized by in-gel fluorescence or fluorescence microscopy. Figure 14B shows the structures of chloroalkyne 21' and TAMRA-hydroxylamine 22'. Figure 14C shows time-dependent in-gel fluorescence analysis of hydroamination between alkyne 21' and hydroxylamine 22' (200 μM) for 1-60 min at room temperature. Figure 14D shows concentration-dependent in-gel fluorescence analysis of hydroamination between alkyne 21' (30 μM) and hydroxylamine 22' (25-200 μM) upon incubation for 2 h at room temperature. Figure 14E is a series of images showing that cell-surface HaloTag-GFP expressed on HEK293T cells was labeled with TAMRA via bioorthogonal hydroamination between alkyne 21' and hydroxylamine 22'. The merge is a composite of the Hoechst33342, GFP, and TAMRA channels. Scale bar = 50 μm. TAMRA = tetramethylrhodamine.

[0030] [Figures 15A-15B] Figures 15A-15B show a computational study of the effect of alkyne halogenation. Figure 15A is a table of the s-character (s-char) of the analyzed alkyne sp carbon, and the activation free energy (ΔG‡) was calculated for the reaction of the alkyne with hydroxylamine 24'. Figure 15B is a graph showing the correlation between s-character and activation free energy.

[0031] [Figures 16A-16E] Figures 16A-16E are a series of reaction plots used to calculate second-order rate constants between alkynes (11'-15') and N,N-diethylhydroxylamine. Figure 16A is a graph for alkyne 11' (2 mM) and hydroxylamine 2' (20-40 mM). Figure 16B is a graph for alkyne 12' (2 mM) and hydroxylamine 2' (18-37 mM). Figure 16C is a graph for alkyne 13' (10 mM) and hydroxylamine 2' (10 mM). Figure 16D is a graph for alkyne 15' (10 mM) and hydroxylamine 2' (10 mM). Figure 16E is a graph for alkyne 14' (10 mM) and hydroxylamine 2' (10 mM). Each panel shows the results of an experiment performed in triplicate.

[0032] [Figure 17] FIG. 17 is a series of 19F NMR spectra showing that compound 14' (2 mM) is stable in 50% CD3CN / PBS (pH 7.0) for 2 weeks.

[0033] [Figure 18] Figure 18 is a series of 19F NMR spectra showing that compound 14' (500 μM) has a half-life of 14 hours in 50% CD3CN / PBS (pH 7.0) in the presence of glutathione (2 mM) and is sufficiently stable for bioorthogonal transformation over an 8 hour period.

[0034] [Figure 19] FIG. 19 is a series of 19F NMR spectra showing that compound 15' (2 mM) is stable in 50% CD3CN / PBS (pH 7.0) for 1 week.

[0035] [Figure 20]Figure 20 is a series of 19F NMR spectra showing that compound 15' (500 μM) has a half-life of 43 hours in 50% CD3CN / PBS (pH 7.0) in the presence of glutathione (2 mM) and is sufficiently stable for bioorthogonal transformation over an 8-hour period.

[0036] [Figures 21A-21B] Figures 21A-B show the complete in-gel fluorescence image (Figure 21A) and the Coomassie-stained image (Figure 21B) for a time-dependent protein labeling experiment. Both images are from the same gel. The molecular weight of the ladder in the in-gel fluorescence image was identified and labeled using contrast-enhanced imaging settings.

[0037] [Figures 22A-22B] Figures 22A-B show the complete in-gel fluorescence image (Figure 22A) and the Coomassie-stained image (Figure 22B) for a time-dependent protein labeling experiment. Both images are from the same gel. The molecular weight of the ladder in the in-gel fluorescence image was identified and labeled using contrast-enhanced imaging settings.

[0038] [Figures 23A-23C] Figures 23A-C show mass spectrometry confirmation of the bioorthogonal reaction between hydroxylamine 22' and alkyne S15'. ESI mass spectra of the unmodified HaloTag protein (Figure 23A), the HaloTag-alkyne conjugate (expected 34,981 Da, observed 34,981 Da) (Figure 23B), and the reaction mixture of the hydroamination between hydroxylamine 22' and the HaloTag-alkyne conjugate (expected 35,527 Da, observed 35,529 Da) (Figure 23C).

[0039] [Figure 24] FIG. 24 shows the s character of the enamine N-oxide sp2 carbon (C2).

[0040] [Figures 25A-25D]Figures 25A-D show bioorthogonal transformations. Figure 25A shows associative bioorthogonal transformation. Figure 25B shows dissociative bioorthogonal transformation. Figure 25C shows chemically reversible bioconjugation. Figure 25D shows rapid and complete sequential bioorthogonal hydroamination and traceless release of biomolecules via enamine N-oxides.

[0041] [Figures 26A-26B] Figures 26A-B show an evaluation of the effect of hydroxylamine substituents on the biorthogonal reverse Cope elimination reaction. Figure 26A shows the synthetic route to obtain TAMRA-hydroxylamine conjugates 6"-9". Figure 26B shows a series of in-gel fluorescence and Coomassie stained images of lysozyme-cyclooctyne conjugate 11" (10 μM) incubated with TAMRA-hydroxylamine conjugates 6"-10" (200 μM) in PBS at room temperature for 1-72 hours.

[0042] [Figures 27A-27D] Figures 27A-D show computational studies investigating the formation and decomposition of enamine N-oxide structures. Figure 27A shows a computational reaction model exploring the effect of steric hindrance on the hydroamination and Cope elimination reactions. Figure 27B shows the calculated Gibbs free energy and free energy of activation. The reaction coordinates for Pathways A and B are colored blue and red, respectively. Figure 27C shows the three-dimensional structures of 17" and 18" and the transition state structures 17"-TSa and 18"-TSb for Pathways A and B. Figure 27D shows the reaction between cyclooctyne 22- (2 mM) and hydroxylamine 4- (2 mM) monitored by LCMS A220 absorbance.

[0043] [Figures 28A-28E]Figures 28A-28E show diboron-mediated enamine N-oxide reduction and payload release. Figure 28A is a reaction scheme for lysozyme TAMRA conjugates 6"conj, 9"conj, and 10"conj bearing enamine N-oxides, treated with diboron reagent in PBS at room temperature to induce fluorophore release. Figure 28B is a series of in-gel fluorescence and silver stain images for concentration-dependent cleavage of lysozyme TAMRA conjugates 6"conj, 9"conj, and 10"conj (480 nM) at room temperature over 1 h with B2pin2 (5-50 μM), along with time-dependent cleavage over 5-60 min with 5 μM B2pin2, analyzed by in-gel fluorescence. Silver staining serves as a loading control. Figure 28C is a series of graphs showing quantification of fluorescence in bands from time-dependent diboron-induced cleavage experiments. Figure 28D shows the complete conjugation and removal of TAMRA from lysozyme by mass spectrometry. Lys-COT11″ (10 μM), featuring 0 to 3 modifications, was mixed with hydroxylamine 6″ (200 μM) in PBS for 6 hours at room temperature. Figure 28E shows in-gel fluorescence and silver stain images for structurally diverse diboron reagents 27″–31″ (5 or 50 μM) incubated with N-methyllysozyme-TAMRA conjugate 6″ (240 nM) for 60 minutes at room temperature.

[0044] [Figures 29A-29B] Figures 29A-B show the characterization of diboron-mediated reductive cleavage of enamine N-oxides. Figure 29A shows the progress of the reaction between 4 mM p-nitrophenol-derived enamine N-oxide 32" and 10 mM B2(OH)4 in 10% DMSO-d6 / 23% CD3OD / 67% d-PBS, pH 7.4, which was monitored by H NMR spectroscopy over a 24-hour period. Figure 29B shows the progress of the reaction between p-nitrophenyl thioether 38" and p-nitrophenyl carbamate 39".

[0045] [Figures 30A-30E]Figures 30A-30E show an investigation of the reaction scope and payload release kinetics for diboron-mediated cleavage of enamine N-oxides. Figure 30A shows the reaction scheme for the synthesis of lysozyme-fluorescein conjugate 41" by hydroamination of Lys-COT11" with fluorescein hydroxylamine 40" in PBS at room temperature. Figure 30B shows the kinetics of diboron-mediated enamine N-oxide cleavage, as determined by fluorescence polarization under pseudo-first-order conditions, when lysozyme-fluorescein conjugate 41" (500 nM) was treated with B2pin2 (25-200 μM) in PBS at room temperature. Figure 30C is a graph showing the effect of buffer pH on the cleavage rate. Lysozyme-fluorescein conjugate 41" (500 nM) was reduced with B2pin2 (100 μM) in PBS, pH 4-10, and conversion was measured by fluorescence polarization. Figure 30D is a graph showing the effect of buffer composition on cleavage rate. Lysozyme-fluorescein conjugate 41" (500 nM) was reduced with B2pin2 (50 μM) in several buffers, and conversion was measured by fluorescence polarization. Figure 30E is a series of graphs showing the effect of leaving group composition on cleavage rate.

[0046] [Figures 31A-31D] Figures 31A-31D show the synthesis and cellular evaluation of chemically cleavable enamine N-oxide-linked antibody-drug conjugates. Figure 31A shows the synthesis of ADC61" and 62". Figure 31B is a graph of a cell viability assay of trastuzumab-derived ADC61" in the presence or absence of 50 μM B2pin2 on SK-BR-3 HER2+ breast cancer cells. Figure 31C is a graph of a cell viability assay of trastuzumab-derived ADC61" in the presence or absence of 50 μM B2pin2 on MDA-MB-231 HER2- breast cancer cells. Figure 31D is a graph of a cell viability assay of IgG isotype control-derived ADC62" in the presence or absence of 50 μM B2pin2 on SK-BR-3 HER2+ breast cancer cells. ND = not determined. Error bars represent standard deviation (n = 3).

[0047] [Figure 32A-32B] Figures 32A-B show that protein modification using enamine N-oxide chemistry is traceless and reversible. Figure 32A is a schematic of the sequential conjugation of a small molecule onto lysozyme. Figure 32B shows that clean and complete click and release were observed by intact mass spectrometry.

[0048] [Figure 33] Figure 33 shows the reductive release of p-nitrothiophenol (S3") from enamine N-oxide 38" by B2(OH)4 at room temperature. 1H NMR spectra of the reaction in the presence of caffeine internal standard (A) before diboron addition, (B) 4 min after addition, and (C) 30 min after addition.

[0049] [Figure 34] Figure 34 shows the reductive release of p-nitroaniline (24") from enamine N-oxide 39" by B2(OH)4 at room temperature. 1H NMR spectra of the reaction in the presence of caffeine internal standard (A) before diboron addition, (B) 5 min after addition, and (C) 30 min after addition.

[0050] [Figure 35] Figure 35 shows the complete Coomassie stained (top) and in-gel fluorescence (bottom) images of the time-dependent protein labeling experiment for compounds 6" and 10". Both images are from the same gel.

[0051] [Figure 36] Figure 36 shows the complete Coomassie stained (top) and in-gel fluorescence (bottom) images of the time-dependent protein labeling experiment for compounds 7" and 8". Both images are from the same gel.

[0052] [Figure 37]FIG. 37 shows the complete Coomassie stained (left) and in-gel fluorescence (right) images of a time-dependent protein labeling experiment for compound 9″. Both images are from the same gel.

[0053] [Figure 38] Figure 38 shows complete in-gel fluorescence (left) and Oriole staining (right) images of the stability assay of enamine N-oxide protein conjugates 6"conj, 9"conj, and 10"conj in PBS (pH 7.4). Both images are from the same gel.

[0054] [Figure 39] Figure 39 shows complete in-gel fluorescence (left) and Oriole staining (right) images for the stability assay of enamine N-oxide protein conjugates 6"conj, 9"conj, and 10"conj in RPMI. Both images are from the same gel.

[0055] [Figure 40] Figure 40 shows complete in-gel fluorescence (left) and Oriole staining (right) images of the stability assay of enamine N-oxide protein conjugates 6"conj, 9"conj, and 10"conj in RPMI supplemented with 10% fetal bovine serum. Both images are from the same gel.

[0056] [Figure 41A-41B] Figures 41A-B show evaluation of diboron reagents for cleavage of enamine N-oxide linked lysozyme-fluorophore conjugate 6"conj. Figure 41A shows the structures of diboron substrates 27"-31". Figure 41B is an in-gel fluorescence and silver stain image of the diboron reagent shown in Figure 41A. Both images are from the same gel.

[0057] [Figures 42A-42C]Figures 42A-C are a series of complete in-gel fluorescence and quantification of each band for enamine N-oxide linked lysozyme-fluorophore conjugates 6" conj, 9" conj, and 10" conj. Figure 42A is the complete in-gel fluorescence and quantification of 6" conj. Figure 42B is the complete in-gel fluorescence and quantification of 10" conj. Figure 42C is the complete in-gel fluorescence and quantification of 9" conj.

[0058] [Figure 43] FIG. 43 shows monitoring of the reaction between cyclooctyne 22″ (2 mM) and hydroxylamine 3″ (2 mM) by LCMS A220 absorbance.

[0059] [Figure 44A-44B]Figures 44A-B show confirmation of the bioorthogonal click and release reactions of structurally diverse enamine N-oxides by mass spectrometry. Figure 44A is a series of ESI mass spectra of unmodified lysozyme, lysozyme-cyclooctyne conjugate 11", the hydroamination ligation reaction between hydroxylamine 6" and lysozyme-COT11" (single adduct: expected 15073.1 Da, observed 15074.0 Da; double adduct: expected 15840.5 Da, observed 15842.7 Da), and the diboron-induced cleavage reaction of enamine N-oxide-linked conjugate 6" (single adduct: expected 14376.8 Da, observed 14376.9 Da; double adduct: expected 14447.8 Da, observed 14447.3 Da). Figure 44B shows the hydroamination ligation reaction between hydroxylamine 9" and lysozyme-COT11" (single adduct: expected 15041.1 Da, observed 15042.1 Da; double adduct: expected 15776.4 Da, observed 15778.5 Da), the diboron-induced cleavage reaction of the enamine N-oxide linked conjugate 9" (single adduct: expected 14376.8 Da, observed 14377.7 Da; double adduct: expected 15992.5 Da, observed 15994.0 Da), and the hydroxylamine 9" (single adduct: expected 14376.8 Da, observed 14377.7 Da; double adduct: expected 15992.5 Da, observed 15994.0 Da). A series of ESI mass spectra of the hydroamination ligation reaction between 10′ and lysozyme-COT11′ (single adduct: expected 15149.1 Da, observed 15149.7 Da; double adduct: expected 15992.5 Da, observed 15994.0 Da), and the diboron-induced cleavage reaction of the enamine N-oxide-linked conjugate 10′ (single adduct: expected 14376.8 Da, observed 14376.1 Da; double adduct: expected 14447.8 Da, observed 14447.3 Da).

[0060] [Figure 45] FIG. 45 is a series of gels showing that N-oxide-bearing amine-fluorescent conjugates 41″ and 48″-52″ induced fluorophore release when treated with B2pin2 in PBS at room temperature.

[0061] [Figure 46]FIG. 46 is a graph showing the effect of diboron reagent structure on cleavage rate.

[0062] [Figure 47] FIG. 47 shows the structures of antibody-nitroaniline conjugates S22″-S24″.

[0063] [Figure 48A-48B] Figures 48A-B are a series of diboron reagent dose-response curves from a cell viability assay in SK-BR-3 cells. Cells were treated with B2pin2 (Figure 48A) or B2(OH)4 (Figure 48B) for 72 hours. Error bars represent the mean ± SEM of data from biological replicates (n=3).

[0064] [Figure 49A-49B] Figures 49A-B are a series of diboron reagent dose-response curves from a cell viability assay in MDA-MB-231 cells. Cells were treated with B2pin2 (Figure 49A) or B2(OH)4 (Figure 49B) for 96 hours. Error bars represent the mean ± SEM of data from biological replicates (n=3).

[0065] [Figure 50] Figure 50 is the in-gel fluorescence of enamine N-oxide with lysozyme fluorescein conjugate 65" treated with B2pin2 in PBS at room temperature to trigger release of the fluorophore.

[0066] [Figure 51] FIG. 51 shows a series of reaction coordinates for the bioorthogonal hydroamination reaction between cyclooctyne 12″ and hydroxylamines 14″ and 15″.

[0067] [Figure 52] FIG. 52 is a graph showing the kinetic assay for enamine N-oxide linked lysozyme-fluorescein conjugate 41″. DETAILED DESCRIPTION OF THE INVENTION

[0068] 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 the subject matter of this specification belongs. As used in this specification and the appended claims, unless specified to the contrary, the following terms have the meanings indicated to facilitate understanding of the invention.

[0069] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a composition" includes mixtures of two or more such compositions; reference to "an inhibitor" includes mixtures of two or more such inhibitors, etc.

[0070] Unless otherwise specified or clear from the context, the term "about" as used herein is understood to mean within the normal tolerance in the art, for example, within 2 standard deviations of the mean. "About" can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values ​​provided herein are modified by the term "about."

[0071] The transitional phrase "comprising," which is synonymous with "including," "containing," or "characterized by," is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. When used in the context of the number of heteroatoms in a heterocyclic structure, it means that the heterocyclic group has that minimum number of heteroatoms. In contrast, the transitional phrase "consisting of" excludes any element, step, or ingredient not specified in the claim. The transitional phrase "consisting essentially of" limits the claim to certain materials or steps "and those that do not materially affect the basic and novel characteristic(s)" of the claimed invention.

[0072] The term "bi-orthogonal reaction" refers to any chemical reaction that can occur within a biological system without interfering with natural biochemical processes.

[0073] With respect to the compounds of the present invention, to the extent the following terms are used herein to further describe them, the following definitions apply.

[0074] As used herein, the term "alkyl" refers to a saturated, straight- or branched-chain monovalent hydrocarbon radical. In one embodiment, an alkyl group is a C1-C 18 In other embodiments, the alkyl group is a C0-C6, C0-C5, C0-C3, C1-C 12, C1-C8, C1-C6, C1-C5, C1-C4 or C1-C3 group (where C0 alkyl means a bond). Examples of alkyl groups include methyl, ethyl, 1-propyl, 2-propyl, i-propyl, 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 to C3 alkyl group. In some embodiments, the alkyl group is a C1 to C2 alkyl group or a methyl group.

[0075] As used herein, the term "alkylene" refers to a straight or branched divalent hydrocarbon chain, consisting solely of carbon and hydrogen, containing no unsaturation, having 1 to 12 carbon atoms, linking the rest of the molecule to a radical group, e.g., methylene, ethylene, propylene, n-butylene, etc. The alkylene chain may be attached to the rest of the molecule through a single bond and to the radical group through a single bond. In some embodiments, an alkylene group contains 1 to 8 carbon atoms (C1-C8 alkylene). In other embodiments, an alkylene group contains 1 to 5 carbon atoms (C1-C5 alkylene). In other embodiments, an alkylene group contains 1 to 4 carbon atoms (C1-C4 alkylene). In other embodiments, an alkylene group contains 1 to 3 carbon atoms (C1-C3 alkylene). In other embodiments, an alkylene group contains 1 to 2 carbon atoms (C1-C2 alkylene). In other embodiments, the alkylene group contains one carbon atom (C1 alkylene).

[0076] As used herein, the term "alkenyl" refers to a straight- or branched-chain monovalent hydrocarbon radical having at least one carbon-carbon double bond. Alkenyl includes radicals having "cis" and "trans" orientations, alternatively "E" and "Z" orientations. In one example, an alkenyl radical is a C2-C6 alkyl group. 18 In another embodiment, the alkenyl group is a C-C 12 , C2~C 10 , C2-C8, C2-C6 or C2-C3 groups. Examples include ethenyl or vinyl, prop-1-enyl, prop-2-enyl, 2-methylprop-1-enyl, but-1-enyl, but-2-enyl, but-3-enyl, buta-1,3-dienyl, 2-methylbuta-1,3-dienyl, hex-1-enyl, hex-2-enyl, hex-3-enyl, hex-4-enyl and hexa-1,3-dienyl.

[0077] As used herein, the term "alkynyl" refers to a straight- or branched-chain monovalent hydrocarbon radical having at least one carbon-carbon triple bond. In one example, an alkynyl radical is a C2-C 18 In another example, the alkynyl radical is a C-C group. 12 , C2~C 10 , C2-C8, C2-C6 or C2-C3. Examples include ethynylprop-1-ynyl, prop-2-ynyl, but-1-ynyl, but-2-ynyl and but-3-ynyl.

[0078] The term "alkoxyl" or "alkoxy" as used herein refers to an alkyl group, as defined above, attached to an oxygen radical, which is the point of attachment. Representative alkoxyl groups include methoxy, ethoxy, propyloxy, tert-butoxy, and the like. An "ether" is two hydrocarbyl groups covalently linked by an oxygen. Thus, the substituent of an alkyl that makes it an ether is or resembles an alkoxyl, as can be represented by one of -O-alkyl, -O-alkenyl, and -O-alkynyl.

[0079] As used herein, the term "halogen" (or "halo" or "halide") refers to fluorine, chlorine, bromine, or iodine.

[0080] As used herein, the term "cyclic group," used alone or as part of a larger moiety, broadly refers to any group containing a saturated, partially saturated, or aromatic ring system, such as carbocyclic (cycloalkyl, cycloalkenyl), heterocyclic (heterocycloalkyl, heterocycloalkenyl), aryl, and heteroaryl groups. A cyclic group can have one or more (e.g., fused) ring systems. Thus, for example, a cyclic group can contain one or more carbocyclic, heterocyclic, aryl, or heteroaryl groups.

[0081] As used herein, the term "carbocyclic" (also referred to as "carbocyclyl"), used alone or as part of a larger moiety, refers to a group containing a saturated, partially unsaturated, or aromatic ring system having 3 to 20 carbon atoms, alone or as part of a larger moiety (e.g., an alkcarbocyclic group). The term carbocyclyl includes mono-, di-, tri-, fused, bridged, and spiro-ring systems, and combinations thereof. In one embodiment, a carbocyclyl is a ring system containing 3 to 15 carbon atoms (C3-C4). 15 In one embodiment, the carbocyclyl contains 3 to 12 carbon atoms (C 12 In another embodiment, carbocyclyl is C3 to C8, C3 to C 10 or C5~C 10 In another embodiment, the carbocyclyl as a monocycle includes C3-C8, C3-C6, or C5-C6. In some embodiments, the carbocyclyl as a bicycle includes C7-C 12 In another embodiment, the carbocyclyl as a spiro system is C5-C 12Includes. Representative examples of monocyclic carbocyclyls include cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl, perdeuteriocyclohexyl, 1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl, cyclohexadienyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl, phenyl, and cyclododecyl; bicyclic carbocyclyls having 7 to 12 ring atoms include [4,3], [4,4], [4,5], [5,5], [5,6], or [6,6] ring systems, such as, for example, bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane, naphthalene, and bicyclo[3.2.2]nonane. Representative examples of spirocarbocyclyls include spiro[2.2]pentane, spiro[2.3]hexane, spiro[2.4]heptane, spiro[2.5]octane, and spiro[4.5]decane. The term carbocyclyl includes aryl ring systems as defined herein. The term carbocyclyl also includes cycloalkyl rings (e.g., saturated or partially unsaturated mono-, bi-, or spiro-carbocyclic rings). The term carbocyclic group also includes carbocyclic rings fused to one or more (e.g., 1, 2, or 3) different cyclic groups (e.g., aryl or heterocyclic rings), where the groups or points of attachment are on the carbocyclic ring.

[0082] Thus, the term carbocyclic, as used herein, also refers to a group of the formula -R c -carbocyclyl (wherein R c is an alkylene chain). The term carbocyclic, as used herein, also includes carbocyclylalkyl groups, which refer to groups of the formula -OR c -carbocyclyl (wherein R c Also included are carbocyclylalkoxy groups, which refer to groups attached through an oxygen atom of a alkylene group (wherein is an alkylene chain).

[0083] The term "carbocyclic" also encompasses "aryl" groups. As used herein, the term "aryl" (e.g., "aralkyl" where the terminal carbon atom on the alkyl is the point of attachment, e.g., a benzyl group), "aralkoxy" where the oxygen is the point of attachment, or "aralkoxyalkyl" where the point of attachment is on the aryl) used alone or as part of a larger moiety refers to a group containing a monocyclic, bicyclic, or tricyclic carbocyclic ring system, including fused rings, where at least one ring in the system is aromatic. In some embodiments, an aralkoxy group is a benzoxy group. The term "aryl" may be used interchangeably with the term "aryl ring." In one embodiment, aryl includes groups having 6 to 18 carbon atoms. In another embodiment, aryl includes groups having 6 to 10 carbon atoms. Examples of aryl groups include phenyl, naphthyl, anthracyl, biphenyl, phenanthrenyl, naphthacenyl, 1,2,3,4-tetrahydronaphthalenyl, 1H-indenyl, 2,3-dihydro-1H-indenyl, naphthyridinyl, and the like, which may be substituted or independently substituted by one or more substituents described herein. A particular aryl is phenyl. In some embodiments, an aryl group comprises an aryl ring fused to one or more (e.g., one, two, or three) different cyclic groups (e.g., carbocyclic or heterocyclic rings), where the radical or point of attachment is on the aryl ring.

[0084] Thus, the term aryl refers to a group of the formula -R c -aryl (wherein R c is an alkylene chain such as methylene or ethylene), and encompasses aralkyl groups (e.g., benzyl). In some embodiments, the aralkyl group is an optionally substituted benzyl group. The term aryl, as used herein, refers to a group of the formula -OR c -aryl (wherein R c Also included are aralkoxy groups, which refer to groups attached through an oxygen atom of a alkylene chain such as methylene or ethylene.

[0085] As used herein, the term "heterocyclyl," used alone or as part of a larger moiety, refers to a "carbocyclyl" containing a saturated, partially unsaturated, or aromatic ring system in which one or more (e.g., 1, 2, 3, or 4) carbon atoms are replaced with a heteroatom (e.g., O, N, N(O), S, S(O), or S(O)). The term heterocyclyl includes monocyclic, bicyclic, tricyclic, fused, bridged, and spiro ring systems, and combinations thereof. In some embodiments, heterocyclyl refers to a 3- to 15-membered heterocyclyl ring system. In some embodiments, heterocyclyl refers to a 3- to 12-membered heterocyclyl ring system. In some embodiments, heterocyclyl refers to a saturated ring system, such as a 3- to 12-membered saturated heterocyclyl ring system. The term heterocyclyl also includes C3-C8 heterocycloalkyl, which are saturated or partially unsaturated monocyclic, bicyclic, or spirocyclic ring systems containing 3 to 8 carbons and one or more (1, 2, 3, or 4) heteroatoms.

[0086] In some embodiments, heterocyclyl groups contain 3 to 12 ring atoms, including monocyclic, bicyclic, tricyclic, and spirocyclic ring systems in which the ring atoms are carbon and 1 to 5 ring atoms are heteroatoms such as nitrogen, sulfur, or oxygen. In some embodiments, heterocyclyls contain 3 to 7-membered monocyclic rings having one or more heteroatoms selected from nitrogen, sulfur, or oxygen. In some embodiments, heterocyclyls contain 4 to 6-membered monocyclic rings having one or more heteroatoms selected from nitrogen, sulfur, or oxygen. In some embodiments, heterocyclyls contain 3-membered monocyclic rings. In some embodiments, heterocyclyls contain 4-membered monocyclic rings. In some embodiments, heterocyclyls contain 5 to 6-membered monocyclic rings. In some embodiments, heterocyclyl groups contain 0 to 3 double bonds. In any of the foregoing embodiments, heterocyclyls contain 1, 2, 3, or 4 heteroatoms. The nitrogen or sulfur heteroatom may optionally be oxidized (e.g., NO, SO, SO), and the nitrogen heteroatom may optionally be quaternized (e.g., [NR]). + Cl - , [NR4] +OH -Representative examples of heterocyclyl include oxiranyl, aziridinyl, thiiranyl, azetidinyl, oxetanyl, thietanyl, 1,2-dithietanyl, 1,3-dithietanyl, pyrrolidinyl, dihydro-1H-pyrrolyl, dihydrofuranyl, tetrahydropyranyl, dihydrothienyl, tetrahydrothienyl, imidazolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, 1,1-dioxo-thiomorpholinyl, dihydropyranyl, tetrahydropyranyl, hexahydrothiopyranyl, hexahydropyrimidinyl, oxazolidinyl, and the like. Nanyl, thiazinanyl, thioxanyl, homopiperazinyl, homopiperidinyl, azepanyl, oxepanyl, thiepanyl, oxazepinyl, oxazepanyl, diazepanyl, 1,4-diazepanyl, diazepinyl, thiazepinyl, thiazepanyl, tetrahydrothiopyranyl, oxazolidinyl, thiazolidinyl, isothiazolidinyl, 1,1-dioxoisothiazolidinonyl, oxazolidinonyl, imidazolidinonyl, 4,5,6,7-tetrahydro[2H]indazolyl, tetrahydrobenzimidazolyl, 4,5,6,7-tetrahydrobenzo [d]imidazolyl, 1,6-dihydroimidazole[4,5-d]pyrrolo[2,3-b]pyridinyl, thiazinyl, thiophenyl, oxazinyl, thiadiazinyl, oxadiazinyl, dithiazinyl, dioxazinyl, oxathiazinyl, thiatriazinyl, oxatriazinyl, dithiadiazinyl, imidazolinyl, dihydropyrimidyl, tetrahydropyrimidyl, 1-pyrrolinyl, 2-pyrrolinyl, 3-pyrrolinyl, indolinyl, thiapyranyl, 2H-pyranyl, 4H-pyranyl, dioxanyl, 1,3-dioxolanyl, pyrazolinyl, pyrimidyl Azalidinyl, dithianyl, dithiolanyl, pyrimidinonyl, pyrimidindionyl, pyrimidin-2,4-dionyl, piperazinonyl, piperazinedionyl, pyrazolidinylimidazolinyl, 3-azabicyclo[3.1.0]hexanyl, 3,6-diazabicyclo[3.1.1]heptanyl, 6-azabicyclo[3.1.1]heptanyl, 3-azabicyclo[3.1.1]heptanyl, 3-azabicyclo[4.1.0]heptanyl, azabicyclo[2.2.2]hexanyl, 2-azabicyclo[3.2.1]octanyl, 8-azabicyclo[3.2.1]octanyl, 2-azabicyclo[2.2.2]octanyl, 8-azabicyclo[2.2.2]octanyl, 7-oxabicyclo[2.2.1]heptane, azaspiro[3.5]nonanyl, azaspiro[2.5]octanyl, azaspiro[4.5]decanyl, 1-azaspiro[4.5]decane-2-only, azaspiro[5.5]undecanyl, tetrahydroindolyl, octahydroindolyl, tetrahydroisoindolyl, tetrahydroindazolyl, 1,1-dioxohexahydrothiopyranyl. Examples of 5-membered heterocyclyls containing a sulfur or oxygen atom and 1 to 3 nitrogen atoms include thiazolyl, including thiazol-2-yl and thiazol-2-yl N-oxide, thiadiazolyl, including 1,3,4-thiadiazol-5-yl and 1,2,4-thiadiazol-5-yl, oxazolyl, such as oxazol-2-yl, and oxadiazolyl, such as 1,3,4-oxadiazol-5-yl and 1,2,4-oxadiazol-5-yl. Examples of 5-membered heterocyclyls containing 2 to 4 nitrogen atoms include imidazolyls such as imidazol-2-yl, triazolyls such as 1,3,4-triazol-5-yl, and tetrazolyls such as 1,2,3-triazol-5-yl, 1,2,4-triazol-5-yl, and 1H-tetrazol-5-yl. Representative examples of benzo-fused 5-membered heterocyclyls are benzoxazol-2-yl, benzothiazol-2-yl, and benzimidazol-2-yl. Examples of 6-membered heterocyclyls contain 1 to 3 nitrogen atoms and optionally sulfur or oxygen atoms, such as pyridyls, such as pyrid-2-yl, pyrid-3-yl, and pyrid-4-yl; pyrimidyls, such as pyrimid-2-yl and pyrimid-4-yl; triazinyls, such as 1,3,4-triazin-2-yl and 1,3,5-triazin-4-yl; pyridazinyls, especially pyridazin-3-yl, and pyrazinyl. Pyridine N-oxide and pyridazine N-oxide and pyridyl, pyrimid-2-yl, pyrimid-4-yl, pyridazinyl, and 1,3,4-triazin-2-yl groups are further examples of heterocyclyl groups. In some embodiments, heterocyclic groups comprise a heterocyclic ring fused to one or more (e.g., 1, 2, or 3) different cyclic groups (e.g., carbocyclic or heterocyclic rings) where the group or point of attachment is on the heterocyclic ring, and in some embodiments, the point of attachment is a heteroatom contained in the heterocyclic ring.

[0087] Thus, the term heterocyclic, as used herein, refers to a heterocyclyl group containing at least one nitrogen atom, and includes N-heterocyclyl groups, in which the heterocyclyl group is attached to the rest of the molecule through a nitrogen atom of the heterocyclyl group. Representative examples of N-heterocyclyl groups include 1-morpholinyl, 1-piperidinyl, 1-piperazinyl, 1-pyrrolidinyl, pyrazolidinyl, imidazolinyl, and imidazolidinyl. The term heterocyclic, as used herein, also refers to a heterocyclyl group containing at least one heteroatom, and includes C-heterocyclyl groups, in which the heterocyclyl group is attached to the rest of the molecule through a carbon atom of the heterocyclyl group. Representative examples of C-heterocyclyl groups include 2-morpholinyl, 2-, 3-, or 4-piperidinyl, 2-piperazinyl, and 2- or 3-pyrrolidinyl. The term heterocyclic also refers to a group of the formula -R c -heterocyclyl (wherein R c is an alkylene chain). The term heterocyclic, as used herein, also includes heterocyclylalkyl groups of the formula -OR c -heterocyclyl (wherein R c Also included are heterocyclylalkoxy groups, which refer to groups attached through an oxygen atom of a heterocyclic group (wherein is an alkylene chain).

[0088] The term "heterocycle" also encompasses "heteroaryl" groups. In some embodiments, heterocyclyl refers to heteroaryl ring systems, such as 5- to 14-membered heteroaryl ring systems. As used herein, the term "heteroaryl," used alone or as part of a larger moiety (e.g., "heteroarylalkyl" (also known as "heteroaralkyl") or "heteroarylalkoxy" (also known as "heteroaralkoxy"), refers to a monocyclic, bicyclic, or tricyclic ring system having 5 to 14 ring atoms, in which at least one ring is aromatic and contains at least one heteroatom. In one embodiment, heteroaryl includes 5- to 6-membered monocyclic aromatic groups in which one or more ring atoms is nitrogen, sulfur, or oxygen. Representative examples of heteroaryl groups include thienyl, furyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, triazolyl, thiadiazolyl, oxadiazolyl, tetrazolyl, thiatriazolyl, oxatriazolyl, pyridyl, pyrimidinyl, imidazolyl, pyrazolyl, pyrimidin ... Examples of heteroaryl include dazopyridyl, pyrazinyl, pyridazinyl, triazinyl, tetrazinyl, tetrazolo[1,5-b]pyridazinyl, purinyl, deazapurinyl, benzoxazolyl, benzofuryl, benzothiazolyl, benzothiadiazolyl, benzotriazolyl, benzimidazolyl, indolyl, 1,3-thiazol-2-yl, 1,3,4-triazol-5-yl, 1,3-oxazol-2-yl, 1,3,4-oxadiazol-5-yl, 1,2,4-oxadiazol-5-yl, 1,3,4-thiadiazol-5-yl, 1H-tetrazol-5-yl, 1,2,3-triazol-5-yl, and pyrid-2-yl N-oxide. The term "heteroaryl" also includes groups in which the heteroaryl is fused to one or more cyclic (e.g., carbocyclyl or heterocyclyl) rings, where the radical or point of attachment is on the heteroaryl ring.Non-limiting examples include indolyl, indolizinyl, isoindolyl, benzothienyl, benzothiophenyl, methylenedioxyphenyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzodioxazolyl, benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and pyrido[2,3-b]-1,4-oxazin-3(4H)-one. Heteroaryl groups can be monocyclic, bicyclic, or tricyclic. In some embodiments, heteroaryl groups comprise a heteroaryl ring fused to one or more (e.g., 1, 2, or 3) different cyclic groups (e.g., carbocyclic or heterocyclic rings) where the group or point of attachment is on the heteroaryl ring, and in some embodiments, the point of attachment is a heteroatom contained in the heterocyclic ring.

[0089] Thus, the term heteroaryl, as used herein, refers to a heteroaryl group as defined above that contains at least one nitrogen and encompasses N-heteroaryl groups, in which the point of attachment of the heteroaryl group to the rest of the molecule is through a nitrogen atom of the heteroaryl group. The term heteroaryl, as used herein, also refers to a heteroaryl group as defined above and encompasses C-heteroaryl groups, in which the point of attachment of the heteroaryl group to the rest of the molecule is through a carbon atom of the heteroaryl group. The term heteroaryl also refers to heteroaryl groups of the formula -R c -heteroaryl (wherein R c is an alkylene chain as defined above. The term heteroaryl as used herein also includes groups of the formula -OR c -heteroaryl (wherein R c Also included are heteroaralkoxy (or heteroarylalkoxy) groups, which refer to groups attached through an oxygen atom of a heteroaryl group (wherein is an alkylene group as defined above).

[0090] Unless otherwise stated and further defined for any particular group, any of the groups described herein can be substituted or unsubstituted. As used herein, the term "substituted" refers broadly to all permissible substituents, provided that such substitution is in accordance with the permissible valences of the substituted atom and substituent, and with the implicit proviso that the substitution results in a stable compound, i.e., a compound that does not spontaneously undergo transformation by rearrangement, cyclization, elimination, and the like. Representative substituents include halogens, hydroxyl groups, and any other organic group containing any number of carbon atoms, e.g., 1 to 14 carbon atoms, and which may contain one or more (e.g., 1, 2, 3, or 4) heteroatoms, such as oxygen, sulfur, and nitrogen atoms, grouped in a linear, branched, or cyclic structural format.

[0091] Unless otherwise disclosed for any particular group, representative examples of substituents include alkyl, substituted alkyl (e.g., C1-C6, C1-C5, C1-C4, C1-C3, C1-C2, C1), alkoxy (e.g., C1-C6, C1-C5, C1-C4, C1-C3, C1-C2, C1), substituted alkoxy (e.g., C1-C6, C1-C5, C1-C4, C1-C3, C1), haloalkyl (e.g., CF3), alkenyl (e.g., C2-C6, C2-C5, C2-C4, C2-C3, C2), substituted alkynyl (e.g., C2-C6, C2-C5, C2-C4, C2-C3, C2), carbocycle (e.g., C3-C 12 , C5-C6), substituted carbocyclic rings (e.g., C3-C 12 , C5-C6), heterocyclic rings (e.g., C3-C 12 , C5-C6), substituted heterocycle (e.g., C3-C6, C5-C6), aryl (e.g., benzyl and phenyl), substituted aryl (e.g., substituted benzyl or phenyl), heteroaryl (e.g., pyridyl or pyrimidyl), substituted heteroaryl (e.g., substituted pyridyl or pyrimidyl), aralkyl (e.g., benzyl), substituted aralkyl (e.g., substituted benzyl), halo, hydroxyl, aryloxy (e.g., C6-C 12 , C6), substituted aryloxy (e.g., C6-C 12, C6), alkylthio (e.g., C1 to C6), substituted alkylthio (e.g., C1 to C6), arylthio (e.g., C6 to C 12 , C6), substituted arylthio (e.g., C6-C 12 , C6), cyano, carbonyl, substituted carbonyl, carboxyl, substituted carboxyl, amino, substituted amino, amido, substituted amido, thio, substituted thio, sulfinyl, substituted sulfinyl, sulfonyl, substituted sulfinamide, substituted sulfinamide, sulfonamide, substituted sulfonamide, urea, substituted urea, carbamate, substituted carbamate, amino acid, and peptide groups.

[0092] As used herein, the term "π-electron withdrawing group" refers to a functional group containing π-electrons with a formal +ve or δ+ve charge, such as a carbonyl or nitro group, that attracts electron density.

[0093] As used herein, the term "inductive electron-withdrawing group" refers to atoms or functional groups containing electronegative atoms that attract more electron density from the atom to which they are attached, e.g., fluoro or alkoxy groups.

[0094] As used herein, the term "small molecule" refers to a molecule having a relatively low molecular weight, whether naturally occurring or artificially created (e.g., via chemical synthesis). Typically, small molecules are organic compounds (i.e., contain carbon). Small molecules may contain multiple carbon-carbon bonds, stereocenters, and other functional groups (e.g., amines, hydroxyls, carbonyls, heterocycles, etc.).

[0095] As used herein, the term active moiety refers to a distinct, definable portion or unit of a compound of the invention that performs some function or activity or is reactive with another molecule. Representative types of active moieties include binding moieties, therapeutic moieties, diagnostic moieties, and immobilization moieties.

[0096] As used herein, the term "immobilizing moiety" refers to a part of a compound of the invention that is insoluble to which the remainder of the compound of the invention is attached (e.g., via covalent bonding or encapsulation in a polymer matrix).

[0097] As used herein, the term "binding moiety" refers to the part of a compound of the invention that targets it to the appropriate site of action, for example, a cancer-associated antigen on a solid tumor cell.

[0098] As used herein, "therapeutic moiety" refers to a portion of a compound of the present invention that provides a therapeutic effect with respect to a disease or disorder when it reaches its intended site of action.

[0099] As used herein, the terms "diagnostic moiety" and "detectable moiety" are used interchangeably and refer to portions of the compounds of the invention that provide a diagnostic effect in connection with a disease or disorder and allow visualization of cells or tissues in which the compounds of the invention accumulate.

[0100] In one embodiment, the compound of the present invention is a compound represented by formula (I): [ka] or a pharmaceutically acceptable salt or stereoisomer thereof; During the ceremony: R1' is a linking group; R1 is either not present or R1 and R2 together with the nitrogen atom to which they are attached form a heterocyclyl; R2 is an optionally substituted (C1-C8) alkyl, —C(O)R″, —C(O)OR″, —C(O)NR″R″, —S(O)R″, —S(O)R″, (C3-C 10 ) carbocyclyl, 4- or 7-membered heterocyclyl, or a substituted polyethylene glycol chain, and each R" is independently hydrogen, (C1-C6) alkyl, (C3-C 10) carbocyclyl, 4- or 7-membered heterocyclyl, wherein said alkyl, carbocyclyl, or heterocyclyl is optionally substituted; A1 is an active moiety further defined herein below.

[0101] In some embodiments, R is absent and R is an alkylene chain, which is -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 and / or terminated (at either or both termini) by at least one of carbocyclyl, 3- to 12-membered heterocyclyl, 5- to 12-membered heteroaryl, or any combination thereof, and each R' is independently H or an optionally substituted C1-C 24 alkyl, and the intermediate and one or both terminal groups may be the same or different.

[0102] In some embodiments, the alkylene chain is C1-C 24 In some embodiments, the alkylene chain is a C1 to C 18 In some embodiments, the alkylene chain is a C1 to C 12In some embodiments, the alkylene chain is a C1 to C 10 It is an 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 or both ends) by at least one 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 heterocyclyl, or a combination thereof. In some embodiments, the alkylene chain is interrupted and / or terminated (at either or both ends) by -N(R')-. In some embodiments, the alkylene chain is interrupted and / or terminated (at either or both ends) by -C(O)-. In some embodiments, the alkylene chain is interrupted and / or terminated (at either or both ends) by -C(O)O-. In some embodiments, the alkylene chain is interrupted and / or terminated (at either or both ends) by -C(O)N(R')-. In some embodiments, the alkylene chain is interrupted and / or terminated (at either or both ends) by -N(R')S(O)2-. In some embodiments, the alkylene chain is interrupted and / or terminated (at either or both ends) by a 4- to 6-membered heterocyclyl. In some embodiments, the alkylene chain is pyrrolidine-2,5-dione ( [ka] ) and ends with

[0103] In some embodiments, R is absent and R is an alkylene chain, which is -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 and / or terminated (at either or both termini) by at least one of carbocyclyl, 3- to 12-membered heterocyclyl, 5- to 12-membered heteroaryl, or any combination thereof, and each R' is independently H or an optionally substituted C1-C 24 alkyl, and the intermediate and one or both terminal groups may be the same or different.

[0104] 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, the polyethylene glycol is interrupted and / or terminated (at either or both ends) by at least one 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)-, -N(R')S(O)-, -S(O)N(R')-, 4- to 6-membered heterocyclyl, or a combination thereof. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either or both ends) by -N(R')-. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either or both ends) by -C(O)-. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either or both ends) by -C(O)O-. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either or both ends) by -C(O)N(R')-. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either or both ends) by -N(R')S(O)2-. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either or both ends) by a 4- to 6-membered heterocyclyl. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either or both ends) by a pyrrolidine-2,5-dione ( [ka] ) and ends with

[0105] In some embodiments, R1 and R2, together with the nitrogen atom to which they are attached, form a 3- to 16-membered heterocyclyl containing 1-8 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 heterocyclyl containing 1-4 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 heterocyclyl containing 1-3 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 heterocyclyl containing 1-2 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 piperazinyl group.

[0106] In some embodiments, R1 is absent and R1' is C1-C 24 is an alkylene chain. R2 is methyl, ethyl, isopropyl, or t-butyl. In some embodiments, R1 is absent and R1' is a C1-C 18 In some embodiments, R is an alkylene chain and R is methyl, ethyl, isopropyl, or t-butyl. In some embodiments, R is absent and R is a C-C 12 In some embodiments, R is an alkylene chain and R is methyl, ethyl, isopropyl, or t-butyl. In some embodiments, R is absent and R is a C-C 10In some embodiments, R1 is absent, R1' is a C1-C8 alkylene chain, and R2 is methyl, ethyl, isopropyl, or t-butyl. In some embodiments, R1 is absent, R1' is a C1-C6 alkylene chain, and R2 is methyl, ethyl, isopropyl, or t-butyl. In some embodiments, R1 is absent, R1' is a C1-C4 alkylene chain, and R2 is methyl, ethyl, isopropyl, or t-butyl. In some embodiments, R1 is absent, R1' is a C1-C2 alkylene chain, and R2 is methyl, ethyl, isopropyl, or t-butyl. In some embodiments, R1 is absent, R1' is 1 to 20 -(CH2CH2-O)- units, and R2 is methyl, ethyl, isopropyl, or t-butyl. In some embodiments, R1 is absent, R1' is 1 to 15 -(CH2CH2-O)- units, and R2 is methyl, ethyl, isopropyl, or t-butyl. In some embodiments, R1 is absent, R1' is 1 to 10 -(CH2CH2-O)- units, and R2 is methyl, ethyl, isopropyl, or t-butyl. In some embodiments, R1 is absent, R1' is 1 to 5 -(CH2CH2-O)- units, and R2 is methyl, ethyl, isopropyl, or t-butyl. In some embodiments, R1 is absent, R1' is 1 to 2 -(CH2CH2-O)- units, and R2 is methyl, ethyl, isopropyl, or t-butyl.

[0107] In some embodiments, the active moiety is a binding moiety. Representative examples of binding moieties include moieties that bind to ubiquitin ligase enzymes or other cellular enzymes that catalyze the degradation of cellular proteins. For example, the ubiquitin-proteasome pathway (UPP) is an important cellular pathway that regulates important regulatory proteins and degrades misfolded or abnormal proteins. The UPP is central to multiple cellular processes. The covalent attachment of ubiquitin to specific protein substrates is achieved through the action of E3 ubiquitin ligases. These ligases include over 500 different proteins and are classified into multiple classes defined by the structural elements of their E3 functional activity.

[0108] In some embodiments, the binding moiety is a small molecule that binds to the E3 ligase cereblon (CRBN). Representative examples of small molecules that bind to CRBN are represented by any one of structures (D1-a) through (D1-d): [ka] [ka] wherein X2 is CH2 or C(O), X3 is CR"1R"2, NR"1, O, or S, and 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 to which they are attached form a C3-C7 carbocyclic or C3-C7 heterocyclic ring (e.g., azetidine, piperidine, pyrrolidine, cyclobutane, cyclohexane).

[0109] Still other small molecules that bind to cereblon and may be suitable for use in the present invention are disclosed in U.S. Pat. No. 9,770,512, as well as U.S. Patent Application Publication Nos. 2018 / 0015087, 2018 / 0009779, 2016 / 0243247, 2016 / 0235731, 2016 / 0235730, and 2016 / 0176916, and International Patent Publication Nos. WO2017 / 197055, WO2017 / 197051, WO2017 / 197036, WO2017 / 197056, and WO2017 / 197046.

[0110] In some embodiments, the binding moiety is a small molecule that binds to the von Hippel-Lindau (VHL) tumor suppressor E3 ligase. Representative examples of small molecules that bind to VHL are represented by any one of structures (D2-a) through (D2-j): [ka] [ka] In the formula, Z1 is a C5-C6 carbocyclic group or a C5-C6 heterocyclic group, [ka] [ka] wherein Y' is a bond, CH2, NH, NMe, O, or S, or a stereoisomer thereof.

[0111] In some embodiments, Z is phenyl, pyrrolyl, furanyl, thiophenyl, pyrazolyl, imidazolyl, oxazolyl, thiazolyl, pyridinyl, pyridazinyl, or pyrimidinyl. [ka] is.

[0112] Still other small molecules that bind to VHL and may be suitable for use in the present invention are disclosed in U.S. Patent Application Publication Nos. 2017 / 0121321 and 2014 / 0356322.

[0113] In some embodiments, the binding moiety is a small molecule that binds to an E3 ligase that is an inhibitor of apoptosis protein (IAP). Representative examples of small molecules that bind to IAPs are represented by any one of structures (D3-a) through (D3-f): [ka] [ka]

[0114] Still other small molecules that bind to IAPs and may be suitable for use in the present invention are disclosed in International Patent Application Publication Nos. WO2008128171, WO2008 / 016893, WO2014 / 060768, and WO2014 / 060767.

[0115] In some embodiments, the binding moiety is a small molecule that binds to the E3 ligase mouse double minute 2 (MDM2). Representative examples of small molecules that bind to MDM2 are represented by structures (D4-a) and (D4-b): [ka]

[0116] Still other small molecules that bind to MDM2 and may be suitable for use in the present invention are disclosed in U.S. Patent No. 9,993,472 B2. MDM2 is known in the art to function as a ubiquitin-E3 ligase.

[0117] In some embodiments, the binding moiety is a small molecule that binds to the ubiquitin receptor RPN13. Representative examples of small molecules that bind to RPN13 are represented by the structures (D5-a), (D5-b), (D5-c), and (D5-d): [ka]

[0118] Still other small molecules that bind to RPN13 and may be suitable for use in the present invention are disclosed in International Publication No. PCT / US2020 / 012825. RPN13 is known in the art to function as a ubiquitin receptor.

[0119] In some embodiments, A1 is a binding moiety that binds to a cellular protein other than a cellular enzyme that catalyzes the degradation of a cellular protein (such as a ubiquitin ligase). Representative examples of cellular proteins that can be targeted by the compounds of the invention containing a binding moiety include kinases, BET bromodomain-containing proteins, cytoplasmic signaling proteins (e.g., FKBP12), nuclear proteins, histone deacetylases (HDACs), lysine methyltransferases, aryl hydrocarbon receptors (AHRs), estrogen receptors, androgen receptors, glucocorticoid receptors, and transcription factors (e.g., SMARCA4, SMARCA2, TRIM24).

[0120] In certain embodiments, the binding moiety is a tyrosine kinase (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, FGFR2, FGFR3, FGFR4, FGR, FLT1, FLT3, FLT4, FRK , FYN, GSG2, HCK, IGF1R, 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, Aurora C, CHK1, CHK2, CLK1, CLK2, CLK3, DAPK1, 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, NEK11, PAK1, PAK2, PA K3, PAK4, PAK5, PAK6, PIM1, PIM2, PLK1, RIP2, RIP5, RSK1, RSK2, SGK2, SGK3, SIK1, STK33, TAO1, TAO2, TGF-beta, TLK2, TSSK1, TSSK2, ULK1, or ULK2),Examples of such kinases include cyclin-dependent kinases (e.g., Cdk1 to Cdk11) and leucine-rich repeat kinases (e.g., LRRK2).

[0121] In some embodiments, the binding moiety binds to a bromodomain and extra terminal (BET) protein, representative examples of which include ATPase family AAA domain-containing protein 2 (ATAD2), bromodomain adjacent zinc finger domain protein 1A (BAZ1A), BAZ1B, BAZ2A, BAZ2B, bromodomain-containing protein 1 (BRD1), BRD2, BRD3, BRD4, BRD5, BRD6, BRD7, BRD8, BRD9, BRD10, bromodomain testis-specific protein (BRDT), bromodomain and PHD finger -containing protein 1 (BRPF1), BRPF3, bromodomain and PHD finger-containing protein 3 (BRWD3), cat eye syndrome critical region protein 2 (CECR2), CREB binding protein (CREBBP), E1A binding protein P300 (EP300), general control of amino acid synthesis 5-like 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 Related, Matrix Associated, Actin Dependent Regulator of Chromatin, Subfamily A, Member 2 (SMARCA2), SMARCA4, SP100 nuclear body protein (SP100), SP110, SP140, transcription initiation factor TFIID subunit 1 (TAF1), TAF1L, TIF1a, tripartite motif-containing 28 (TRIM28), TRIM33, TRIM66, WD repeat protein 9 (WDR9), zinc finger MYND domain-containing protein 11 (ZMYND11), and mixed lineage leukemia-like protein 4 (MLL4). In one embodiment, the BET bromodomain-containing protein is BRD4.

[0122] In certain embodiments, the binding moiety binds to BRD2, BRD3, BRD4, antennapedia homeodomain protein, BRCA1, BRCA2, CCAAT-binding enhancer protein, histone, polycomb group protein, high mobility group protein, telomere binding protein, 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.

[0123] In some embodiments, the binding moiety binds to a BRD. Representative examples of small molecules that bind to a BRD include: [ka] During the ceremony: R is the point at which the linking group is attached; R' is methyl or ethyl.

[0124] In some embodiments, the binding moiety binds to CREBBP. Representative examples of small molecules that bind to CREBBP include: [ka] [ka] During the ceremony: R is the point at which the linking group is attached; A is N or CH; m is 0, 1, 2, 3, 4, 5, 6, 7, or 8.

[0125] In some embodiments, the binding moiety binds to SMARCA4 / PB1 / SMARCA2. Representative examples of small molecules that bind to SMARCA4 / PB1 / SMARCA2 include: [ka] During the ceremony: R is the point at which the linking group is attached; A is N or CH; m is 0, 1, 2, 3, 4, 5, 6, 7, or 8.

[0126] In some embodiments, the binding moiety binds to TRIM24 / BRPF1. Representative examples of small molecules that bind to TRIM24 / BRPF1 include: [ka] [ka] During the ceremony: R is the point at which the linking group is attached; m is 0, 1, 2, 3, 4, 5, 6, 7, or 8.

[0127] In some embodiments, the binding moiety binds to the glucocorticoid receptor. Representative examples of small molecules that bind to the glucocorticoid receptor include: [ka] [ka] During the ceremony: R is the point of attachment of the linking group.

[0128] In some embodiments, the binding moiety binds to the estrogen / androgen receptor. Representative examples of small molecules that bind to the estrogen / androgen receptor include: [ka] [ka] During the ceremony: R is the point of attachment of the linking group.

[0129] In some embodiments, the binding moiety binds to DOT1L. Representative examples of small molecules that bind to DOT1L are as follows: [ka] [ka] During the ceremony: R is the point at which the linking group is attached; A is N or CH; m is 0, 1, 2, 3, 4, 5, 6, 7, or 8.

[0130] In some embodiments, the binding moiety binds to Ras. Representative examples of small molecules that bind to Ras are: [ka] [ka] During the ceremony: R is the point of attachment of the linking group.

[0131] In some embodiments, the binding moiety binds to RasG12C. Representative examples of small molecules that bind to RasG12C are as follows: [ka] [ka] During the ceremony: R is the point of attachment of the linking group.

[0132] In some embodiments, the binding moiety binds to Her3. Representative examples of small molecules that bind to Her3 are as follows: [ka] [ka] During the ceremony: R is the point at which the linking group is attached; R' is [ka] is.

[0133] In some embodiments, the binding moiety binds to Bcl-2 / Bcl-XL. Representative examples of small molecules that bind to Bcl-2 / Bcl-XL are as follows: [ka] During the ceremony: R is the point of attachment of the linking group.

[0134] In some embodiments, the binding moiety binds to HDAC. Representative examples of small molecules that bind to HDAC are: [ka] During the ceremony: R is the point of attachment of the linking group.

[0135] In some embodiments, the binding moiety binds to PPAR-gamma. Representative examples of small molecules that bind to PPAR-gamma are as follows: [ka] During the ceremony: R is the point of attachment of the linking group.

[0136] In some embodiments, the binding moiety binds to RXR. Representative examples of small molecules that bind to RXR are as follows: [ka] [ka] During the ceremony: R is the point of attachment of the linking group.

[0137] In some embodiments, the binding moiety binds to DHFR. Representative examples of small molecules that bind to DHFR are as follows: [ka] [ka] During the ceremony: R is the point of attachment of the linking group.

[0138] In some embodiments, the binding moiety binds to BCL2. Representative examples of small molecules that bind to BCL2 are as follows: [ka] [ka] During the ceremony: R is the point of attachment of the linking group.

[0139] Still other small molecules that bind to cellular proteins and may be suitable for use as binding moieties in the present invention are disclosed in U.S. Patent Application Publication Nos. 2017 / 0121321 and 2014 / 0356322.

[0140] In some embodiments, the binding moiety is biotin or a biotin derivative. Biotin derivatives are known in the art. See, e.g., 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 labels in the biotechnology industry for many years. Representative examples of biotin derivatives that may be suitable for use in the present invention include desthiobiotin, pyrimethamine biotin, rac selenobiotin, biocytin, 2-iminobiotin, biocytin-L-proline, biotin cystamine, and biotin tobramycin amide. Other biotin derivatives that may be suitable for use in the present invention are described in the art, for example, in U.S. Patent No. 8,318,696 and U.S. Patent Application Publication No. 2007 / 0020206, each of which is incorporated by reference.

[0141] In some embodiments, the binding moiety is a short peptide sequence (e.g., 2-50 amino acids in length, e.g., 4-20 amino acids in length, where the amino acid residues in the peptide can be the same or different). Representative examples include α-amanitin, antipain, ceruletide, glutathione, leupeptin, netropsin, pepstatin, peptide T, phalloidin, teprotide, 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, softag1, softag3, Spot tag, Strep tag, T7 tag, TC tag, Ty tag, V5 tag, VSV tag, and Xpress tag.

[0142] In some embodiments, the binding moiety is a protein. Representative examples of proteinaceous binding moieties include chitin-binding protein (CBP), maltose-binding protein (MBP), glutathione-S-transferase (GST), thioredoxin, poly(NANP), biotin carboxyl carrier protein (BCCP), green fluorescent protein (GFP), HaloTag, SNAP tag, CLIP tag, HUH tag, Nus tag, Fc tag, and carbohydrate recognition domain tag. In some embodiments, the binding moiety is HaloTag.

[0143] In yet other embodiments, the binding moiety is an antibody (e.g., a monoclonal antibody) or 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 a tumor-associated antigen on cancer cells, such as solid tumor cells. Representative examples of monoclonal antibodies include muromonab-CD3, abciximab, rituximab, palivizumab, infliximab, trastuzumab, alemtuzumab, adalimumab, ibritumomab, omalizumab, cetuximab, bevacizumab, natalizumab, panitumumab, ranibizumab, eculizumab, certolizumab, ustekinumab, and canakinumab. , golimumab, ofatumumab, tocilizumab, denosumab, belimumab, ipilimumab, brentuximab, pertuzumab, raxibacumab, obinutuzumab, siltuximab, ramucirumab, vedolizumab, blinatumomab, nivolumab, pembrolizumab, idarucizumab, necitumumab, dinutuximab, secukinumab, mepolizumab, alirocumab, Evolocumab, daratumumab, elotuzumab, ixekizumab, reslizumab, olaratumab, bezlotoxumab, atezolizumab, oviltoxiximab, inotuzumab, brodalumab, guselkumab, dupilumab, sarilumab, avelumab, ocrelizumab, emicizumab, benralizumab, gemtuzumab, durvalumab, burosumab, and ranaldemab In some embodiments, the monoclonal antibody or binding fragment thereof is gemtuzumab, brentuximab, trastuzumab, inotuzumab, moxetumomab, polatuzumab, enfortumab, or belantamab.

[0144] In some embodiments, the binding moiety is a fragment of an antibody, e.g., a monoclonal antibody. For example, the fragment may be a variable fragment, such as a single-chain variable fragment (scFv) of a monoclonal antibody. Representative examples of scFvs include pexelizumab, duvortuxizumab, efangumab, gancotamab, letolizumab, oportuzumab monatox, bovalilizumab, and brolucizumab.

[0145] In some embodiments, the active moiety is a binding moiety that is a solubility-enhancing group. Examples of solubilizing groups include groups that are easily ionized in water in the pH range of 0 to 14, ionizable groups that can form salts, and substituents containing highly polar substituents that have a high dipole moment and can form strong interactions with water molecules. In some embodiments, the solubility-enhancing group is α-chloroacetyl.

[0146] In some embodiments, the active moiety is a therapeutic moiety. The therapeutic moiety, in some embodiments, may be a small molecule. In certain embodiments, the molecular weight of the small molecule is about 1,000 g / mol or less, about 900 g / mol or less, about 800 g / mol or less, about 700 g / mol or less, about 600 g / mol or less, about 500 g / mol or less, about 400 g / mol or less, about 300 g / mol or less, about 200 g / mol or less, or about 100 g / mol or less. In certain 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 certain embodiments, the therapeutic moiety is a therapeutically active agent such as a drug (eg, a molecule approved by the US Food and Drug Administration as provided in the Code of Federal Regulations (CFR)).

[0147] In some embodiments, the therapeutic moiety is an anti-cancer agent. Representative types of anti-cancer agents include anti-angiogenic agents, alkylating agents, antimetabolites, microtubulin polymerization disruptors, platinum coordination complexes, anthracenediones, substituted ureas, methylhydrazine derivatives, adrenocortical suppressants, hormones and antagonists, anti-cancer polysaccharides and anthracyclines (e.g., aclarubicin, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, pirarubicin, valrubicin, and derivatives and analogs thereof), and kinase inhibitors (e.g., pan-Her inhibitors (e.g., HKI-272, BIBW-2992, PF299, SN29926, and PR-509E)).

[0148] In some embodiments, the therapeutic moiety is a non-targeted cancer drug, which, as known in the art, refers to an agent with a relatively broad mechanism of action. Representative examples of non-targeted anti-cancer drugs include alkylating agents (e.g., busulfan, chlorambucil, cyclophosphamide, ifosfamide, mechlorethamine, melphalen, carmustine, streptozocin, dacarbazine, temozolomide, altretamine, and thioTEPA), antimetabolites (e.g., capecitabine, cytarabine, 5'-fluorouracil, gemcitabine, cladribine, fludarabine, 6-mercaptopurine, and pentostatin), folate antagonists (e.g., methotrexate and pemetrexed), mitotic inhibitors (e.g., cetaxel, paclitaxel, and cetaxel). These include cyclosporine, vinblastine, vincristine, vindesine, and vinorelbine), DNA inhibitors (e.g., hydroxyurea, carboplatin, cisplatin, oxaliplatin, mitomycin C, and pyrrolobenzodiazepines), toposomerase inhibitors (e.g., topotecan, irinotecan, daunorubicin, doxorubicin, etoposide, teniposide, and mitoxantrone), inducers of DNA breaks (e.g., bleomycin), ozogamicin, vedotin, emtansine, pasudotox, deruxtecan, govitecan, and mafodotin, or derivatives thereof.

[0149] In some embodiments, the therapeutic moiety is a targeted anti-cancer agent, which, as known in the art, refers to an agent that has a specific mechanism of action. Representative examples of non-targeted anticancer drugs 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), idelalisib (PI3Kδ), imatinib (KIT, PDGFR, ABL), lapatinib (HER2, EGFR), lenvatinib (VEGFR2), nilotinib (ABL), olaparib (PARP), and palbociclib (C DK4, CDK6), panobinostat (HDAC), pazopanib (VEGFR, PDGFR, KIT), ponatinib (ABL, FGFR1-3, FLT3, VEGFR2), regorafenib (KIT, PDGFRβ, RAF, RET, VEGFR1 / 2 / 3), romidepsin (HDAC), ruxolitinib (JAK1 / 2), sorafenib (VEGFR, PDGFR, KIT, RAT), temsirolimus (mTOR), trametinib (MEK), vandetanib (EGFR, RET, VEGFR2), vemurafenib (BRAF), vismodegib (PTCH), and vorinostat (HDAC). In some embodiments, the targeted anticancer drug is a kinase inhibitor.Representative examples of kinase inhibitors include abemaciclib, acalabrutinib, afatinib, alectinib, avapritinib, axitinib, baricitinib, binimetinib, bosutinib, brigatinib, cabozantinib, ceritinib, capmatinib, cobimetinib, crizotinib, dabrafenib, dacomitinib, dasatinib, encorafenib, entrectinib, erdafitinib, erlotinib, everolimus, fedratinib, fostamatinib, gefitinib, giltritanib, ibrotinib, icotinib, imatinib, lapatinib, and larolimus. These include trectinib, lenvatinib, lorlatinib, midostaurin, neratinib, netarsudil, nilotinib, nintedanib, osimertinib, palbociclib, pazopanib, pemigatinib, pexidartinib, ponatinib, pralsetinib, regorafenib, ribociclib, ripretinib, ruxolitinib, selpercatinib, selumetinib, sirolimus, sorafenib, sunitinib, temsirolimus, tofacitinib, tremetinib, tucatinib, upadacitinib, vandetanib, vemurafenib, and zanubrutinib.

[0150] In some embodiments, the therapeutic moiety is an antibacterial agent. Representative examples of antibacterial agents include plazomycin, eravacycline, selacycline, omadacycline, rifamycin, imipenem, cilastatin, relebactam, pretomanid, lefamrin, cefiderocol, sulfaquinoxaline, oxytetracycline, hygromycin B, tylosin, chlortetracycline, virginiamycin, neomycin, luncomycin, pyrantel, melengestrol, lasalocid, fenbendazole, semduramycin, decoquinate, ractopamine, laidlomycin, diclazuril, halifuginone, robenidine, clopidol, zilpaterol, monensin, zoalen, rubabegron, and bacitracin.

[0151] In some embodiments, the therapeutic moiety is a nonsteroidal anti-inflammatory drug (NSAID). Representative examples of NSAID agents include celecoxib, diclofenac, diflunisal, etodolac, fenoprofen, flurbiprofen, ibuprofen, indomethacin, ketoprofen, ketorolac, mefenamic acid, meloxicam, nabumetone, naproxen, oxaprozin, piroxicam, sulindac, and tolmetin.

[0152] In some embodiments, the therapeutic moiety is a corticosteroid. Representative examples of corticosteroid agents include deflazacort, dexamethasone, betamethasone, triamcinolone, hydrocortisone, methylprednisolone, and prednisone.

[0153] In some embodiments, the therapeutic moiety 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.

[0154] In some embodiments, the active moiety is a diagnostic moiety. Diagnostic moieties typically contain a detectable moiety, such as a label. Representative examples of diagnostic moieties include dyes, chromogenic agents, positron emission tomography (PET) tracers, and magnetic resonance imaging (MRI) contrast agents. The term "label" includes any moiety that allows for the capture, detection, or visualization of the compound to which it is attached. A label may be directly detectable (i.e., does not require further reaction or manipulation to become detectable; e.g., a fluorophore or chromophore is directly detectable) or indirectly detectable (i.e., is made detectable via reaction with or binding to another detectable entity; e.g., a hapten is detectable by immunostaining after reaction with an appropriate antibody containing a reporter such as a fluorophore). Representative examples of types of labels include affinity tags, radiometric labels (e.g., radionuclides (e.g., 32 P, 35 S, 3 H, 14 C. 125 I, 131 I, etc.), fluorescent dyes, phosphorescent dyes, chemiluminescent agents (e.g., acridinium esters, stabilized dioxetanes, 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., those used in ELISA, i.e., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, etc.), colorimetric labels (e.g., dyes, colloidal gold, etc.), magnetic labels (e.g., Dynabeads™, etc.), and haptens.

[0155] In some embodiments, the label comprises a fluorescent dye. Representative examples of fluorescent dyes include fluorescein and fluorescein dyes (e.g., fluorescein isothiocyanate (FITC), naphthofluorescein, 4',5'-dichloro-2',7'-dimethoxy-fluorescein, 6-carboxyfluorescein or FAM), carbocyanines, merocyanines, styryl dyes, oxonol dyes, phycoerythrin, erythrosine, eosin, rhodamine dyes (e.g., 5-carboxytetramethylrhodamine (TAMRA), carboxyrhodamine 6G, carboxy-X-rhodamine (ROX), Lissamine rhodamine B, rhodamine 6G, rhodamine green, rhodamine red, or tetramethylrhodamine (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 510, Oregon Green 520, Oregon Green 530, Oregon Green 540, Oregon Green 550, Oregon Green 560, Oregon Green 570, Oregon Green 580, Oregon Green 590, Oregon Green 591, Oregon Green 592, Oregon Green 593, Oregon Green 594, Oregon Green 595, Oregon Green 596, Oregon Green 597, Oregon Green 598, Oregon Green 59 ... 500, Oregon Green 514), Texas Red, Texas Red-X, Spectrum Red™, Spectrumgreen™, cyanine dyes (e.g., Cy-3™, Cy-5™, Cy-3.5™, Cy-5.5™), Alexa 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), and the like.For further examples of suitable fluorescent dyes and methods for attaching fluorescent dyes to other chemicals, see, e.g., 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.

[0156] In some embodiments, the diagnostic moiety comprises a rhodamine dye. In some embodiments, the diagnostic moiety comprises tetramethylrhodamine (TAMRA) or a derivative thereof.

[0157] In some embodiments, the diagnostic moiety is a color former, which refers to a chemical compound that induces a color reaction, as known in the art. Representative examples of color formers include azo reagents such as methyl orange and methyl red, nitrophenols, phthales such as phenolphthalein or thymolphthalein, sulfonephthaleins such as bromophenol blue or bromocresol green, indophenols such as 2,6-dichlorophenolindophenol, azine reagents such as the thiazine dye methylene blue, indigo carmine, diphenylamine derivatives such as diphenylamine-4-sulfonic acid and variamine blue, arsenazo III, catechol violet, dithizone, 1-(2'-pyridylazo)-2-naphthol, 4-(2'-pyridylazo)resorcinol, chrome azurol S, eriochrome black T, eriochrome blue black B, pyrogallol red, alizarin complexone, methylthymol blue, and xylenol orange.

[0158] In some embodiments, the diagnostic moiety is a PET tracer, which refers to a radioactive ligand used for imaging purposes, as known in the art.Representative examples include acetate (C-11), choline (C-11), fludeoxyglucose (F-18), sodium fluoride (F-18), fluoro-ethyl-spirpersone (F-18), methionine (C-11), prostate-specific membrane antigen (PSMA) (Ga-68), DOTATOC / DOTANOC / DOTATATE (Ga-68), florbetaben / florbetapir (F-18), rubidium (Rb-82), and FDDNP (F-18).

[0159] In some embodiments, the diagnostic moiety is an MRI contrast agent, which, as known in the art, refers to an agent used to improve the visibility of internal body structures. Representative examples include gadoteric acid, gadodiamide, gadobenic acid, gadopentetic acid, gadoteridol, gadofosveset, gadoveresetamide, gadoxetate, and gadobutrol.

[0160] Labels suitable for use in the present invention may be detectable by any of a variety of means, including spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, and chemical means.

[0161] In some embodiments, the active moiety is an immobilization moiety. Representative examples of immobilization moieties include polystyrene beads, magnetic agarose beads, cross-linked agarose beads, and TENTAGEL® beads.

[0162] In some embodiments, R2 is methyl, ethyl, isopropyl, or t-butyl.

[0163] In some embodiments, the compound of formula (I) is [ka] [ka] or a pharmaceutically acceptable salt or stereoisomer thereof.

[0164] In some embodiments, the optional substituents for compounds of Formula (I) are alkyl, alkenyl, alkynyl, halo, haloalkyl, cycloalkyl, heterocycloalkyl, hydroxy, alkoxy, cycloalkoxy, heterocycloalkoxy, haloalkoxy, aryloxy, heteroaryloxy, aralkyloxy, alkenyloxy, alkynyloxy, amino, alkylamino, cycloalkylamino, heterocycloalkylamino, arylamino, heteroarylamino, aralkylamino, N-alkyl-N-arylamino, N-alkyl-N-heteroarylamino, N-alkyl-N-aralkylamino, hydroxyalkyl, aminoalkyl, alkylthio, haloalkylthio, alkylsulfonyl, haloalkylsulfonyl, cycloalkylsulfonyl, heterocycloalkylsulfonyl, arylsulfonyl, heteroarylsulfonyl, aminosulfonyl, alkylaminosulfonyl, cycloalkylaminosulfonyl, heterocycloalkylaminosulfonyl, arylaminosulfonyl, heteroarylaminosulfonyl, N-alkyl-N-aryl and arylsulfonylamino, N-alkyl-N-heteroarylaminosulfonyl, formyl, alkylcarbonyl, haloalkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, carboxy, alkoxycarbonyl, alkylcarbonyloxy, amino, alkylsulfonylamino, haloalkylsulfonylamino, cycloalkylsulfonylamino, heterocycloalkylsulfonylamino, arylsulfonylamino, heteroarylsulfonylamino, aralkylsulfonylamino, alkylcarbonylamino, haloalkylcarbonylamino, cycloalkylcarbonylamino, heterocycloalkylcarbonylamino, arylcarbonylamino, heteroarylcarbonylamino, aralkylsulfonylamino, aminocarbonyl, alkylaminocarbonyl, cycloalkylaminocarbonyl, heterocycloalkylaminocarbonyl, arylaminocarbonyl, heteroarylaminocarbonyl, N-alkyl-N-arylaminocarbonyl, N-alkyl-N-heteroarylaminocarbonyl, cyano, nitro, and azido.

[0165] In some embodiments, the compound of Formula (I) is a compound of Formula Ia′, Ib, or Ic′: [ka] or a pharmaceutically acceptable salt or stereoisomer thereof; During the ceremony: R1' is a linking group; R1 is either not present or R1 and R2 together with the nitrogen atom to which they are attached form a heterocyclyl; R2 is an optionally substituted (C1-C8) alkyl, —C(O)R′, —C(O)OR′, —C(O)NR′R′, —S(O)R′, —S(O)R′, (C3-C 10 )carbocyclyl, or 4- or 7-membered heterocyclyl, where each R' is independently hydrogen, (C1-C6)alkyl, (C3-C 10 ) carbocyclyl, 4- or 7-membered heterocyclyl, wherein said alkyl, carbocyclyl, or heterocyclyl may be substituted; A1' is an antibody or antibody fragment.

[0166] In some embodiments, R1 is absent.

[0167] In some embodiments, R is absent and R is an alkylene chain, which is -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 and / or terminated (at either or both termini) by at least one of carbocyclyl, 3- to 12-membered heterocyclyl, 5- to 12-membered heteroaryl, or any combination thereof, and each R' is independently H or an optionally substituted C1-C 24 alkyl, and the intermediate and one or both terminal groups may be the same or different.

[0168] In some embodiments, the alkylene chain is C1-C 24 In some embodiments, the alkylene chain is a C1 to C 18 In some embodiments, the alkylene chain is a C1 to C 12 In some embodiments, the alkylene chain is a C1 to C 10It is an 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 or both ends) by at least one 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')-, or a combination thereof. In some embodiments, an alkylene chain is interrupted and / or terminated (at either or both ends) by -N(R')-. In some embodiments, an alkylene chain is interrupted and / or terminated (at either or both ends) by -C(O)-. In some embodiments, an alkylene chain is interrupted and / or terminated (at either or both ends) by -C(O)O-. In some embodiments, an alkylene chain is interrupted and / or terminated (at either or both ends) by -C(O)N(R')-. In some embodiments, an alkylene chain is interrupted and / or terminated (at either or both ends) by -N(R')S(O)2-.

[0169] In some embodiments, R is absent and R is a polyethylene glycol chain, which is -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 and / or terminated (at either or both termini) by at least one of carbocyclyl, 3- to 12-membered heterocyclyl, 5- to 12-membered heteroaryl, or any combination thereof, and each R' is independently H or an optionally substituted C1-C 24 alkyl, and the intermediate and one or both terminal groups may be the same or different.

[0170] 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, the polyethylene glycol is interrupted and / or terminated (at either or both ends) by at least one 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)-, -N(R')S(O)-, -S(O)N(R')-, or a combination thereof. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either or both ends) by -N(R')-. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either or both ends) by -C(O)-. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either or both ends) by -C(O)O-. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either or both ends) by -C(O)N(R')-. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either or both ends) by -N(R')S(O)2-.

[0171] In some embodiments, R1 and R2, together with the nitrogen atom to which they are attached, form a 3- to 16-membered heterocyclyl containing 1-8 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 heterocyclyl containing 1-4 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 heterocyclyl containing 1-3 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 heterocyclyl containing 1-2 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 piperazinyl group.

[0172] In some embodiments, R1 is absent and R1' is C1-C 24 In some embodiments, R is an alkylene chain and R is methyl or benzyl. In some embodiments, R is absent and R is a C-C 18 is an alkylene chain, and R2 is methyl or benzyl. In some embodiments, R1 is absent and R1' is C1-C 12 In some embodiments, R is an alkylene chain and R is methyl or benzyl. In some embodiments, R is absent and R is a C-C 10In 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.

[0173] In some embodiments, A1' is selected from the group consisting of muromonab-CD3, abciximab, rituximab, palivizumab, infliximab, trastuzumab, alemtuzumab, adalimumab, ibritumomab, omalizumab, cetuximab, bevacizumab, natalizumab, panitumumab, ranibizumab, eculizumab, certolizumab, ustekinumab, canakinumab, gonokinumab, gonadotropin ... Limumab, ofatumumab, tocilizumab, denosumab, belimumab, ipilimumab, brentuximab, pertuzumab, raxibacumab, obinutuzumab, siltuximab, ramucirumab, vedolizumab, blinatumomab, nivolumab, pembrolizumab, idarucizumab, necitumumab, dinutuximab, secukinumab, mepolizumab, alirocumab, evo Rokumab, daratumumab, elotuzumab, ixekizumab, reslizumab, olaratumab, bezlotoxumab, atezolizumab, obiltoxaximab, inotuzumab, brodalumab, guselkumab, dupilumab, sarilumab, avelumab, ocrelizumab, emicizumab, benralizumab, gemtuzumab, durvalumab, burosumab, ranaldemab, mogamulizumab In some embodiments, A1' is murizumab, erenumab, galcanezumab, tildrakizumab, cemiplimab, emapalumab, fremanezumab, ibalizumab, moxetumomab, ravulizumab, caplacizumab, romosozumab, risankizumab, polatuzumab, brolucizumab, crizanlizumab, sacituzumab, belantamab, or enfortumab, or an antigen-binding fragment thereof. In some embodiments, A1' is trastuzumab.

[0174] In some embodiments, the compound of formula (Ia') [ka] [ka] or a pharmaceutically acceptable salt or stereoisomer thereof.

[0175] In some embodiments, the compound of formula (Ib') is [ka] or a pharmaceutically acceptable salt or stereoisomer thereof.

[0176] In some embodiments, the compound of formula (Ic') [ka] [ka] or a pharmaceutically acceptable salt or stereoisomer thereof.

[0177] Other inventive compounds of the present invention are represented by formulas (II) and (III): [ka] or a pharmaceutically acceptable salt or stereoisomer thereof; During the ceremony: 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 a substituent; Y is absent or [ka] and; A2 is the active moiety; R4 is hydrogen, a substituent, or [ka] is a linking group attached to a group, or R4 and R5 together with the carbon atom to which they are attached form a carbocyclyl or heterocyclyl, where R4 is also [ka] attached to the group; R5 is hydrogen or an electron-withdrawing group; R6 is hydrogen, a π-electron donating group, or [ka] is a linking group attached to the group; R7 and R7' are independently hydrogen or an electron-withdrawing group, or R7 and R7' together with the carbon atom to which they are attached form C(O); R8 is hydrogen, a substituent, or [ka] is a linking group attached to the group; and n is 1, 2, or 3; However, each of formulas II and III is [ka] It has a group.

[0178] In some embodiments, n is 2.

[0179] In some embodiments, X is CR9R9'. In some embodiments, R9 and R9' are each hydrogen.

[0180] In some embodiments, R and R are independently hydrogen, (C-C) alkyl, (C-C) alkoxy, (C-C) haloalkyl, (C-C) haloalkoxy, -C(O)R 10 , N.R. 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 ) carbocyclyl, 4- or 7-membered heterocyclyl, -O(CH2) 0-3 (C3~C10 ) carbocyclyl or —O(CH2) containing 1 to 3 heteroatoms selected from O, N, and S 0-3 - 4- or 7-membered heterocyclyl, where each R 10 is independently hydrogen or (C1-C6) alkyl; wherein said alkyl, carbocyclyl or heterocyclyl may be further substituted.

[0181] In some embodiments, R4 is [ka] In some embodiments, R is a linking group bonded to the group. In some embodiments, R is O. In some embodiments, R is S. In some embodiments, R is NR 11 where R 11 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 is hydrogen or (C1-C6) alkyl.

[0182] In some embodiments, R4 is an alkylene chain, which is -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 and / or terminated (at either or both termini) by at least one of carbocyclyl, 3- to 12-membered heterocyclyl, 5- to 12-membered heteroaryl, or any combination thereof, and each R' is independently H or an optionally substituted C1-C 24 alkyl, and the intermediate and one or both terminal groups may be the same or different.

[0183] In some embodiments, the alkylene chain is C1-C 24 In some embodiments, the alkylene chain is a C1 to C 18 In some embodiments, the alkylene chain is a C1 to C 12 In some embodiments, the alkylene chain is a C1 to C 10It is an 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 or both ends) by at least one 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')-, or a combination thereof. In some embodiments, an alkylene chain is interrupted and / or terminated (at either or both ends) by -N(R')-. In some embodiments, an alkylene chain is interrupted and / or terminated (at either or both ends) by -C(O)-. In some embodiments, an alkylene chain is interrupted and / or terminated (at either or both ends) by -C(O)O-. In some embodiments, an alkylene chain is interrupted and / or terminated (at either or both ends) by -C(O)N(R')-. In some embodiments, an alkylene chain is interrupted and / or terminated (at either or both ends) by -N(R')S(O)2-.

[0184] In some embodiments, R4 is a polyethylene glycol chain, which is -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 and / or terminated (at either or both termini) by at least one of carbocyclyl, 3- to 12-membered heterocyclyl, 5- to 12-membered heteroaryl, or any combination thereof, and each R' is independently H or an optionally substituted C1-C 24 alkyl, and the intermediate and one or both terminal groups may be the same or different.

[0185] 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, the polyethylene glycol is interrupted and / or terminated (at either or both ends) by at least one 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)-, -N(R')S(O)-, -S(O)N(R')-, or a combination thereof. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either or both ends) by -N(R')-. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either or both ends) by -C(O)-. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either or both ends) by -C(O)O-. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either or both ends) by -C(O)N(R')-. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either or both ends) by -N(R')S(O)2-.

[0186] In some embodiments, R4 and R5, together with the carbon atom to which they are attached, form a 3- to 16-membered carbocyclyl or a 3- to 16-membered heterocyclyl containing 1-8 heteroatoms selected from N, O, and S. In some embodiments, R4 and R5, together with the carbon atom to which they are attached, form a 4- to 12-membered carbocyclyl or a 4- to 12-membered heterocyclyl containing 1-4 heteroatoms selected from N, O, and S. In some embodiments, R4 and R5, together with the carbon atom to which they are attached, form a 5- to 10-membered carbocyclyl or a 5- to 10-membered heterocyclyl containing 1-3 heteroatoms selected from N, O, and S. In some embodiments, R4 and R5, together with the carbon atom to which they are attached, form a 5- to 6-membered carbocyclyl or a 5- to 6-membered heterocyclyl containing 1-2 heteroatoms selected from N, O, and S.

[0187] In some embodiments, R4 and R5 together with the carbon atom to which they are attached form a 5-membered heterocyclyl containing two oxygen atoms.

[0188] In some embodiments, R5 is hydrogen.

[0189] In some embodiments, R5 is an electron-withdrawing group.

[0190] In some embodiments, R5 is an inductive electron withdrawing group. In some embodiments, the inductive electron withdrawing group is a halogen, OR 5’ , S.R. 5’ , or NR 5’ R 5’ where each R 5’ are independently hydrogen, C1-C6 alkyl, C6-C 12 It is aryl, 5- to 10-membered heteroaryl, carbonyl, sulfonyl, sulfinyl, or phosphoryl.

[0191] 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)NR 5” R 5” , -OP(O)OR 5” OR 5” , -P(O)NR 5” R 5” NR 5” R 5” where each R 5” are independently hydrogen, C1-C6 alkyl, C6-C 12 aryl, and 5- to 10-membered heteroaryl.

[0192] In some embodiments, R6 is hydrogen.

[0193] In some embodiments, R6 is a π-electron donor group.

[0194] In some embodiments, R6 is OR 12 , S.R. 12 , N.R. 12 NR 12 or a cyclic or acyclic amide, and each R 12 are independently hydrogen, (C1-C6) alkyl, (C3-C 10 ) carbocyclyl, 4- or 7-membered heterocyclyl, wherein said alkyl, carbocyclyl, or heterocyclyl may be optionally substituted.

[0195] In some embodiments, R7 and R7' are independently hydrogen or an inductive electron-withdrawing group. In some embodiments, the inductive electron-withdrawing group is a halogen, OR5’ , S.R. 5’ , or NR 5’ R 5’ where each R 5’ are independently hydrogen, C1-C6 alkyl, C6-C 12 It is aryl, 5- to 10-membered heteroaryl, carbonyl, sulfonyl, sulfinyl, or phosphoryl.

[0196] In some embodiments, R7 and R7' are independently 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)NR 5” R 5” , -OP(O)OR 5” OR 5” , -P(O)NR 5” R 5” NR 5” R 5” where each R 5” are independently hydrogen, C1-C6 alkyl, C6-C 12 aryl, and 5- to 10-membered heteroaryl.

[0197] In some embodiments, R8 is [ka] In some embodiments, R is a linking group bonded to a C-C group. In some embodiments, R is CH. In some embodiments, R is a C-C 12 In some embodiments, R8 is O.

[0198] In some embodiments, R8 is an alkylene chain, which is -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 and / or terminated (at either or both termini) by at least one of carbocyclyl, 3- to 12-membered heterocyclyl, 5- to 12-membered heteroaryl, or any combination thereof, and each R' is independently H or an optionally substituted C1-C 24 alkyl, and the intermediate and one or both terminal groups may be the same or different.

[0199] In some embodiments, the alkylene chain is C1-C 24 In some embodiments, the alkylene chain is a C1 to C 18 In some embodiments, the alkylene chain is a C1 to C 12 In some embodiments, the alkylene chain is a C1 to C 10It is an 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 or both ends) by at least one 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')-, or a combination thereof. In some embodiments, an alkylene chain is interrupted and / or terminated (at either or both ends) by -N(R')-. In some embodiments, an alkylene chain is interrupted and / or terminated (at either or both ends) by -C(O)-. In some embodiments, an alkylene chain is interrupted and / or terminated (at either or both ends) by -C(O)O-. In some embodiments, an alkylene chain is interrupted and / or terminated (at either or both ends) by -C(O)N(R')-. In some embodiments, an alkylene chain is interrupted and / or terminated (at either or both ends) by -N(R')S(O)2-.

[0200] In some embodiments, R8 is a polyethylene glycol chain, which is -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 and / or terminated (at either or both termini) by at least one of carbocyclyl, 3- to 12-membered heterocyclyl, 5- to 12-membered heteroaryl, or any combination thereof, and each R' is independently H or an optionally substituted C1-C 24 alkyl, and the intermediate and one or both terminal groups may be the same or different.

[0201] 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, the polyethylene glycol is interrupted and / or terminated (at either or both ends) by at least one 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)-, -N(R')S(O)-, -S(O)N(R')-, or a combination thereof. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either or both ends) by -N(R')-. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either or both ends) by -C(O)-. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either or both ends) by -C(O)O-. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either or both ends) by -C(O)N(R')-. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either or both ends) by -N(R')S(O)2-.

[0202] The moiety A2 is an active moiety defined similarly to A1 above in relation to compounds of formula (I).

[0203] In some embodiments, the compound of Formula (II) is represented by a compound of Formula (IIa): [ka] 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 is hydrogen or (C1-C6) alkyl, an optionally substituted alkylene chain, or an optionally substituted polyethylene glycol chain, and / or R5 is hydrogen, fluoro, or OR 5’ where OR 5’ is hydrogen or (C1-C6) alkyl, and / or A2 is a binding moiety, a therapeutic moiety, or a diagnostic moiety.

[0204] In some embodiments, the compound of formula (II) [ka] [ka] or a pharmaceutically acceptable salt or stereoisomer thereof.

[0205] In some embodiments, the compound of formula (II') is represented by a compound of formula (II'): [ka] or a pharmaceutically acceptable salt or stereoisomer thereof; During the ceremony: Each X is independently CR9R 9’ , NR9, O, S, C(O), S(O), or SO2, wherein the ring system contains 0-3 heteroatoms; R9 and R 9’ are independently hydrogen or a substituent; R4 is a linking group; A 2’ is a therapeutic small molecule; n is 1, 2, or 3.

[0206] In some embodiments, X is CR9R 9’ In some embodiments, R and R 9’ In some embodiments, R and R are independently selected from hydrogen, (C-C) alkyl, (C-C) alkoxy, (C-C) haloalkyl, (C-C) haloalkoxy, -C(O)R 10 , N.R. 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 ) carbocyclyl, 4- or 7-membered heterocyclyl, -O(CH2) 0-3 (C3~C 10 ) carbocyclyl or —O(CH2) containing 1 to 3 heteroatoms selected from O, N, and S 0-3 - 4- or 7-membered heterocyclyl, where each R 10 is independently hydrogen or (C1-C6) alkyl; wherein said alkyl, carbocyclyl or heterocyclyl may be further substituted.

[0207] In some embodiments, R4 is O, S, NR 10 , OC(O), NR 10 C(O), or OC(O)NR5, where R 10 is hydrogen or C1-C6 alkyl.

[0208] In some embodiments, R4 is an alkylene chain, which is -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 and / or terminated (at either or both termini) by at least one of carbocyclyl, 3- to 12-membered heterocyclyl, 5- to 12-membered heteroaryl, or any combination thereof, and each R' is independently H or an optionally substituted C1-C 24 alkyl, and the intermediate and one or both terminal groups may be the same or different.

[0209] In some embodiments, the alkylene chain is C1-C 24 In some embodiments, the alkylene chain is a C1 to C 18 In some embodiments, the alkylene chain is a C1 to C 12 In some embodiments, the alkylene chain is a C1 to C 10It is an 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 or both ends) by at least one 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')-, or a combination thereof. In some embodiments, an alkylene chain is interrupted and / or terminated (at either or both ends) by -N(R')-. In some embodiments, an alkylene chain is interrupted and / or terminated (at either or both ends) by -C(O)-. In some embodiments, an alkylene chain is interrupted and / or terminated (at either or both ends) by -C(O)O-. In some embodiments, an alkylene chain is interrupted and / or terminated (at either or both ends) by -C(O)N(R')-. In some embodiments, an alkylene chain is interrupted and / or terminated (at either or both ends) by -N(R')S(O)2-.

[0210] In some embodiments, R4 is a polyethylene glycol chain, which is -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 and / or terminated (at either or both termini) by at least one of carbocyclyl, 3- to 12-membered heterocyclyl, 5- to 12-membered heteroaryl, or any combination thereof, and each R' is independently H or an optionally substituted C1-C 24 alkyl, and the intermediate and one or both terminal groups may be the same or different.

[0211] 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, the polyethylene glycol is interrupted and / or terminated (at either or both ends) by at least one 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)-, -N(R')S(O)-, -S(O)N(R')-, or a combination thereof. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either or both ends) by -N(R')-. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either or both ends) by -C(O)-. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either or both ends) by -C(O)O-. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either or both ends) by -C(O)N(R')-. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either or both ends) by -N(R')S(O)2-.

[0212] In some embodiments, n is 2. In some embodiments, n is 2, each X is CH2, and the structure has formula II'a: [ka] or a pharmaceutically acceptable salt or stereoisomer thereof.

[0213] In some embodiments, A2' is an anti-cancer agent. In some embodiments, A2' is an auristatin, a maytansinoid, a tubulysin, an anthracycline, paclitaxel or docetaxel or a derivative thereof, a calicheamicin or a derivative thereof, a pyrrolobenzodiazepine dimer (PBD) or a derivative thereof, a duocarmycin or a derivative thereof, an eribulin or a derivative thereof, a camptothecin or a derivative thereof, or an exatecan or a derivative thereof.

[0214] Representative examples of auristatins include: [ka] Includes:

[0215] Representative examples of maytansinoids include: [ka] Includes:

[0216] Representative examples of tubulysins include: [ka] [ka] wherein R1 is CH2CH(CH3)2, CH2CH2CH3, CH2CH3, CH=C(CH3)2, or CH3; and R2 is [ka] is.

[0217] Representative examples of anthracyclines include: [ka] Includes:

[0218] For example, suitable sites for conjugation on anticancer drugs such as those described above are readily identified by those skilled in the art and are otherwise described in the literature (see Kostova et al., Pharmaceuticals, 14:442 (2021)).

[0219] In some embodiments, the compound of formula (IIa') is [ka] [ka] or a pharmaceutically acceptable salt or stereoisomer thereof.

[0220] In some embodiments, the compound of formula (III) is represented by a compound of formula (IIIa): [ka] or a pharmaceutically acceptable salt or stereoisomer thereof. In some embodiments, R6 is hydrogen, chloro, bromo, iodo, OR 12 , or SR 12 where each R 12 are independently hydrogen, (C1-C6) alkyl, (C3-C 10 ) carbocyclyl, 4- or 7-membered heterocyclyl; and / or R7 is hydrogen, fluoro, or OR 5’ where R 5’ is hydrogen or (C1-C6) alkyl; and / or R 7’ is hydrogen, fluoro, or OR 5’ where R 5’ is hydrogen or (C1-C6) alkyl; and R8 is CH2, O, C6-C 12is an aryl, a 5- to 10-membered heteroaryl, 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.

[0221] In some embodiments, the compound of formula (III) [ka] [ka] or a pharmaceutically acceptable salt or stereoisomer thereof.

[0222] In some embodiments, the optional substituents for a compound of Formula (II) or (III) are alkyl, alkenyl, alkynyl, halo, haloalkyl, cycloalkyl, heterocycloalkyl, hydroxy, alkoxy, cycloalkoxy, heterocycloalkoxy, haloalkoxy, aryloxy, heteroaryloxy, aralkyloxy, alkenyloxy, alkynyloxy, amino, alkylamino, cycloalkylamino, heterocycloalkylamino, arylamino, heteroarylamino, aralkylamino, N-alkyl-N-arylamino, N-alkyl-N-heteroarylamino, N-alkyl-N-aralkylamino, hydroxyalkyl, aminoalkyl, alkylthio, haloalkylthio, alkylsulfonyl, haloalkylsulfonyl, cycloalkylsulfonyl, heterocycloalkylsulfonyl, arylsulfonyl, heteroarylsulfonyl, aminosulfonyl, alkylaminosulfonyl, cycloalkylaminosulfonyl, heterocycloalkylaminosulfonyl, arylaminosulfonyl, heteroarylaminosulfonyl, N-alkyl- and arylsulfonylamino, N-alkyl-N-heteroarylaminosulfonyl, formyl, alkylcarbonyl, haloalkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, carboxy, alkoxycarbonyl, alkylcarbonyloxy, amino, alkylsulfonylamino, haloalkylsulfonylamino, cycloalkylsulfonylamino, heterocycloalkylsulfonylamino, arylsulfonylamino, heteroarylsulfonylamino, aralkylsulfonylamino, alkylcarbonylamino, haloalkylcarbonylamino, cycloalkylcarbonylamino, heterocycloalkylcarbonylamino, arylcarbonylamino, heteroarylcarbonylamino, aralkylsulfonylamino, aminocarbonyl, alkylaminocarbonyl, cycloalkylaminocarbonyl, heterocycloalkylaminocarbonyl, arylaminocarbonyl, heteroarylaminocarbonyl, N-alkyl-N-arylaminocarbonyl, N-alkyl-N-heteroarylaminocarbonyl, cyano, nitro, and azido.

[0223] Still other compounds of the present invention are compounds represented by formulas (IV) and (V): [ka] or a pharmaceutically acceptable salt or stereoisomer thereof; During the ceremony: R1' is a linking group; R1 is either not present or R1 and R2 together with the nitrogen atom to which they are attached form a heterocyclyl; R2 is an optionally substituted (C1-C8) alkyl, —C(O)R″, —C(O)OR″, —C(O)NR″R″, —S(O)R″, —S(O)R″, (C3-C 10 ) carbocyclyl, 4- or 7-membered heterocyclyl, or a substituted polyethylene glycol chain, and each R" is independently hydrogen, (C1-C6) alkyl, (C3-C 10 ) carbocyclyl, 4- or 7-membered heterocyclyl, wherein said alkyl, carbocyclyl, or heterocyclyl is optionally 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 a substituent; A1 is an active moiety as defined above in relation to compounds of formula (I); Y is absent or [ka] and; A2 is an active moiety as defined above for A1; R4 is hydrogen, a substituent, or [ka] is a linking group attached to a group, or R4 and R5 together with the carbon atom to which they are attached form a carbocyclyl or heterocyclyl, where R4 is also [ka] attached to the group; R5 is hydrogen or an electron-withdrawing group; R6 is hydrogen, a π-electron donating group, or [ka] is a linking group attached to the group; R7 and R7' are independently hydrogen or an electron-withdrawing group, or R7 and R7' together with the carbon atom to which they are attached form C(O); R8 is hydrogen, a substituent, or [ka] is a linking group attached to the group; and n is 1, 2, or 3; Each of the compounds of formula (IV) and (V) comprises at least one [ka] It has a group.

[0224] In some embodiments, R is absent and R is an alkylene chain, which is -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 and / or terminated (at either or both termini) by at least one of carbocyclyl, 3- to 12-membered heterocyclyl, 5- to 12-membered heteroaryl, or any combination thereof, and each R' is independently H or an optionally substituted C1-C 24 alkyl, and the intermediate and one or both terminal groups may be the same or different.

[0225] In some embodiments, the alkylene chain is C1-C 24 In some embodiments, the alkylene chain is a C1 to C 18 In some embodiments, the alkylene chain is a C1 to C 12 In some embodiments, the alkylene chain is a C1 to C 10It is an 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 or both ends) by at least one 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 heterocyclyl, or a combination thereof. In some embodiments, the alkylene chain is interrupted and / or terminated (at either or both ends) by -N(R')-. In some embodiments, the alkylene chain is interrupted and / or terminated (at either or both ends) by -C(O)-. In some embodiments, the alkylene chain is interrupted and / or terminated (at either or both ends) by -C(O)O-. In some embodiments, the alkylene chain is interrupted and / or terminated (at either or both ends) by -C(O)N(R')-. In some embodiments, the alkylene chain is interrupted and / or terminated (at either or both ends) by -N(R')S(O)2-. In some embodiments, the alkylene chain is interrupted and / or terminated (at either or both ends) by a 4- to 6-membered heterocyclyl. In some embodiments, the alkylene chain is pyrrolidine-2,5-dione ( [ka] ) and ends with

[0226] In some embodiments, R is absent and R is a polyethylene glycol chain, which is -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 and / or terminated (at either or both termini) by at least one of carbocyclyl, 3- to 12-membered heterocyclyl, 5- to 12-membered heteroaryl, or any combination thereof, and each R' is independently H or an optionally substituted C1-C 24 alkyl, and the intermediate and one or both terminal groups may be the same or different.

[0227] 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, the polyethylene glycol is interrupted and / or terminated (at either or both ends) by at least one 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)-, -N(R')S(O)-, -S(O)N(R')-, 4- to 6-membered heterocyclyl, or a combination thereof. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either or both ends) by -N(R')-. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either or both ends) by -C(O)-. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either or both ends) by -C(O)O-. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either or both ends) by -C(O)N(R')-. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either or both ends) by -N(R')S(O)2-. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either or both ends) by a 4- to 6-membered heterocyclyl. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either or both ends) by a pyrrolidine-2,5-dione ( [ka] ) and ends with

[0228] In some embodiments, R1 and R2, together with the nitrogen atom to which they are attached, form a 3- to 16-membered heterocyclyl containing 1-8 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 heterocyclyl containing 1-4 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 heterocyclyl containing 1-3 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 heterocyclyl containing 1-2 heteroatoms selected from N, O, and S.

[0229] In some embodiments, R2 is methyl, ethyl, isopropyl, or t-butyl.

[0230] In some embodiments, R1 is C1-C 24 In some embodiments, R is a C-C alkylene chain, and R is methyl, ethyl, isopropyl, or t-butyl. 18 In some embodiments, R is a C-C alkylene chain, and R is ethyl, isopropyl, or t-butyl. 12 In some embodiments, R is a C-C alkylene chain, and R is methyl, ethyl, isopropyl, or t-butyl. 10In some embodiments, R1 is an alkylene chain and R2 is methyl, ethyl, isopropyl, or t-butyl. In some embodiments, R1 is a C1-C8 alkylene chain and R2 is methyl, ethyl, isopropyl, or t-butyl. In some embodiments, R1 is a C1-C6 alkylene chain and R2 is methyl, ethyl, isopropyl, or t-butyl. In some embodiments, R1 is a C1-C4 alkylene chain and R2 is methyl, ethyl, isopropyl, or t-butyl. In some embodiments, R1 is a C1-C2 alkylene chain and R2 is methyl, ethyl, isopropyl, or t-butyl. In some embodiments, R1 is 1 to 20 —(CH2CH2—O)— units and R2 is methyl, ethyl, isopropyl, or t-butyl. In some embodiments, R1 is 1 to 15 —(CH2CH2—O)— units and R2 is methyl, ethyl, isopropyl, or t-butyl. In some embodiments, R1 is 1 to 10 -(CH2CH2-O)- units and R2 is methyl, ethyl, isopropyl, or t-butyl. In some embodiments, R1 is 1 to 5 -(CH2CH2-O)- units and R2 is methyl, ethyl, isopropyl, or t-butyl. In some embodiments, R1 is 1 to 2 -(CH2CH2-O)- units and R2 is methyl, ethyl, isopropyl, or t-butyl.

[0231] In some embodiments, n is 2.

[0232] In some embodiments, X is CR9R9'. In some embodiments, R9 and R9' are each hydrogen.

[0233] In some embodiments, R and R are independently hydrogen, (C-C) alkyl, (C-C) alkoxy, (C-C) haloalkyl, (C-C) haloalkoxy, -C(O)R 10 , N.R. 10 R 10 , -C(O)NR 10 R 10 , -OC(O)NR10 R 10 , -NR 10 C(O)R 10 , -NR 10 C(O)OR 10 , halogen, OH, CN, amino, (C3-C 10 ) carbocyclyl, 4- or 7-membered heterocyclyl, -O(CH2) 0-3 (C3~C 10 ) carbocyclyl or —O(CH2) containing 1 to 3 heteroatoms selected from O, N, and S 0-3 - 4- or 7-membered heterocyclyl, where each R 10 is independently hydrogen or (C1-C6) alkyl; wherein said alkyl, carbocyclyl or heterocyclyl may be further substituted.

[0234] In some embodiments, R4 is [ka] In some embodiments, R is a linking group bonded to the group. In some embodiments, R is O. In some embodiments, R is S. In some embodiments, R is NR 11 where R 11 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 is hydrogen or (C1-C6) alkyl.

[0235] In some embodiments, R4 is an alkylene chain, which is -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 and / or terminated (at either or both termini) by at least one of carbocyclyl, 3- to 12-membered heterocyclyl, 5- to 12-membered heteroaryl, or any combination thereof, and each R' is independently H or an optionally substituted C1-C 24 alkyl, and the intermediate and one or both terminal groups may be the same or different.

[0236] In some embodiments, the alkylene chain is C1-C 24 In some embodiments, the alkylene chain is a C1 to C 18 In some embodiments, the alkylene chain is a C1 to C 12 In some embodiments, the alkylene chain is a C1 to C 10It is an 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 or both ends) by at least one 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')-, or a combination thereof. In some embodiments, an alkylene chain is interrupted and / or terminated (at either or both ends) by -N(R')-. In some embodiments, an alkylene chain is interrupted and / or terminated (at either or both ends) by -C(O)-. In some embodiments, an alkylene chain is interrupted and / or terminated (at either or both ends) by -C(O)O-. In some embodiments, an alkylene chain is interrupted and / or terminated (at either or both ends) by -C(O)N(R')-. In some embodiments, an alkylene chain is interrupted and / or terminated (at either or both ends) by -N(R')S(O)2-.

[0237] In some embodiments, R4 is a polyethylene glycol chain, which is -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 and / or terminated (at either or both termini) by at least one of carbocyclyl, 3- to 12-membered heterocyclyl, 5- to 12-membered heteroaryl, or any combination thereof, and each R' is independently H or an optionally substituted C1-C 24 alkyl, and the intermediate and one or both terminal groups may be the same or different.

[0238] 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, the polyethylene glycol is interrupted and / or terminated (at either or both ends) by at least one 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)-, -N(R')S(O)-, -S(O)N(R')-, or a combination thereof. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either or both ends) by -N(R')-. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either or both ends) by -C(O)-. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either or both ends) by -C(O)O-. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either or both ends) by -C(O)N(R')-. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either or both ends) by -N(R')S(O)2-.

[0239] In some embodiments, R4 and R5, together with the carbon atom to which they are attached, form a 3- to 16-membered carbocyclyl or a 3- to 16-membered heterocyclyl containing 1-8 heteroatoms selected from N, O, and S. In some embodiments, R4 and R5, together with the carbon atom to which they are attached, form a 4- to 12-membered carbocyclyl or a 4- to 12-membered heterocyclyl containing 1-4 heteroatoms selected from N, O, and S. In some embodiments, R4 and R5, together with the carbon atom to which they are attached, form a 5- to 10-membered carbocyclyl or a 5- to 10-membered heterocyclyl containing 1-3 heteroatoms selected from N, O, and S. In some embodiments, R4 and R5, together with the carbon atom to which they are attached, form a 5- to 6-membered carbocyclyl or a 5- to 6-membered heterocyclyl containing 1-2 heteroatoms selected from N, O, and S.

[0240] In some embodiments, R4 and R5 together with the carbon atom to which they are attached form a 5-membered heterocyclyl containing two oxygen atoms.

[0241] In some embodiments, R5 is hydrogen.

[0242] In some embodiments, R5 is an electron-withdrawing group.

[0243] In some embodiments, R5 is an inductive electron withdrawing group. In some embodiments, the inductive electron withdrawing group is a halogen, OR 5’ , S.R. 5’ , or NR 5’ R 5’ where each R 5’ are independently hydrogen, C1-C6 alkyl, C6-C 12 It is aryl, 5- to 10-membered heteroaryl, carbonyl, sulfonyl, sulfinyl, or phosphoryl.

[0244] 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)NR 5” R 5” , -OP(O)OR 5” OR 5” , -P(O)NR 5” R 5” NR 5” R 5” where each R 5” are independently hydrogen, C1-C6 alkyl, C6-C 12 aryl, and 5- to 10-membered heteroaryl.

[0245] In some embodiments, R6 is hydrogen.

[0246] In some embodiments, R6 is a π-electron donor group.

[0247] In some embodiments, OR 12 , S.R. 12 , N.R. 12 NR 12 or a cyclic or acyclic amide, and each R 12 are independently hydrogen, (C1-C6) alkyl, (C3-C 10 ) carbocyclyl, 4- or 7-membered heterocyclyl, wherein said alkyl, carbocyclyl, or heterocyclyl may be optionally substituted.

[0248] In some embodiments, R7 and R7' are independently hydrogen or an inductive electron-withdrawing group. In some embodiments, the inductive electron-withdrawing group is a halogen, OR5’ , S.R. 5’ , or NR 5’ R 5’ where each R 5’ are independently hydrogen, C1-C6 alkyl, C6-C 12 It is aryl, 5- to 10-membered heteroaryl, carbonyl, sulfonyl, sulfinyl, or phosphoryl.

[0249] In some embodiments, R and R 7’ are independently 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)NR 5” R 5” , -OP(O)OR 5” OR 5” , -P(O)NR 5” R 5” NR 5” R 5” where each R 5” are independently hydrogen, C1-C6 alkyl, C6-C 12 aryl, and 5- to 10-membered heteroaryl.

[0250] In some embodiments, R8 is [ka] In some embodiments, R is a linking group bonded to a group. In some embodiments, R is CH. In some embodiments, R is aryl. In some embodiments, R is O.

[0251] In some embodiments, R8 is an alkylene chain, which is -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 and / or terminated (at either or both termini) by at least one of carbocyclyl, 3- to 12-membered heterocyclyl, 5- to 12-membered heteroaryl, or any combination thereof, and each R' is independently H or an optionally substituted C1-C 24 alkyl, and the intermediate and one or both terminal groups may be the same or different.

[0252] In some embodiments, the alkylene chain is C1-C 24 In some embodiments, the alkylene chain is a C1 to C 18 In some embodiments, the alkylene chain is a C1 to C 12 In some embodiments, the alkylene chain is a C1 to C 10It is an 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 or both ends) by at least one 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')-, or a combination thereof. In some embodiments, an alkylene chain is interrupted and / or terminated (at either or both ends) by -N(R')-. In some embodiments, an alkylene chain is interrupted and / or terminated (at either or both ends) by -C(O)-. In some embodiments, an alkylene chain is interrupted and / or terminated (at either or both ends) by -C(O)O-. In some embodiments, an alkylene chain is interrupted and / or terminated (at either or both ends) by -C(O)N(R')-. In some embodiments, an alkylene chain is interrupted and / or terminated (at either or both ends) by -N(R')S(O)2-.

[0253] In some embodiments, R8 is a polyethylene glycol chain, which is -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 and / or terminated (at either or both termini) by at least one of carbocyclyl, 3- to 12-membered heterocyclyl, 5- to 12-membered heteroaryl, or any combination thereof, and each R' is independently H or an optionally substituted C1-C 24 alkyl, and the intermediate and one or both terminal groups may be the same or different.

[0254] 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, the polyethylene glycol is interrupted and / or terminated (at either or both ends) by at least one 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)-, -N(R')S(O)-, -S(O)N(R')-, or a combination thereof. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either or both ends) by -N(R')-. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either or both ends) by -C(O)-. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either or both ends) by -C(O)O-. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either or both ends) by -C(O)N(R')-. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either or both ends) by -N(R')S(O)2-.

[0255] In some embodiments, A1 is a therapeutic moiety and A2 is a diagnostic moiety.

[0256] In some embodiments, A1 is a diagnostic moiety and A2 is a therapeutic moiety.

[0257] In some embodiments, A1 is a therapeutic moiety and A2 is a binding moiety.

[0258] In some embodiments, A1 is a binding moiety and A2 is a therapeutic moiety.

[0259] In some embodiments, A1 is a binding moiety and A2 is a binding moiety.

[0260] In some embodiments, the compound of formula (IV) is a compound represented by formula (IVa): [ka] 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 t-butyl; or R1 and R2 together with the nitrogen atom to which they are attached form a 3-16 membered heterocyclyl containing 1-8 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 is hydrogen or (C1-C6) alkyl, an optionally substituted alkylene chain, or an optionally substituted polyethylene glycol chain; and / or R5 is hydrogen, fluoro, or OR 5’ where OR 5’ is hydrogen or (C1-C6) alkyl; and / or A1 is a binding moiety, a therapeutic moiety, or a diagnostic moiety; and / or A2 is a binding moiety, a therapeutic moiety, or a diagnostic moiety.

[0261] In some embodiments, the compound of formula (IV) is [ka] [ka] or a pharmaceutically acceptable salt or stereoisomer thereof.

[0262] In some embodiments, the compound of formula (IV) is [ka] or a pharmaceutically acceptable salt or stereoisomer thereof.

[0263] In some embodiments, the compound of formula (IV) is a compound represented by formula IVa′, IVb′, or IVc′: [ka] or a pharmaceutically acceptable salt or stereoisomer thereof During the ceremony: R1' is a linking group; R1 is either not present or R1 and R2 together with the nitrogen atom to which they are attached form a heterocyclyl; R2 is an optionally substituted (C1-C8) alkyl, —C(O)R′, —C(O)OR′, —C(O)NR′R′, —S(O)R′, —S(O)R′, (C3-C 10 )carbocyclyl, or 4- to 7-membered heterocyclyl, where each R' is independently hydrogen, (C1-C6)alkyl, (C3-C 10 ) carbocyclyl, 4- to 7-membered heterocyclyl, wherein the alkyl, carbocyclyl, or heterocyclyl may be substituted; A1' is an antibody or antibody fragment; 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 a substituent; R4 is a linking group; A2' is a therapeutic small molecule; n is 1, 2, or 3.

[0264] In some embodiments, R is absent and R is an alkylene chain, which is -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 and / or terminated (at either or both termini) by at least one of carbocyclyl, 3- to 12-membered heterocyclyl, 5- to 12-membered heteroaryl, or any combination thereof, and each R' is independently H or an optionally substituted C1-C 24 alkyl, and the intermediate and one or both terminal groups may be the same or different.

[0265] In some embodiments, the alkylene chain is C1-C 24 In some embodiments, the alkylene chain is a C1 to C 18 In some embodiments, the alkylene chain is a C1 to C 12 In some embodiments, the alkylene chain is a C1 to C 10It is an 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 or both ends) by at least one 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')-, or a combination thereof. In some embodiments, an alkylene chain is interrupted and / or terminated (at either or both ends) by -N(R')-. In some embodiments, an alkylene chain is interrupted and / or terminated (at either or both ends) by -C(O)-. In some embodiments, an alkylene chain is interrupted and / or terminated (at either or both ends) by -C(O)O-. In some embodiments, an alkylene chain is interrupted and / or terminated (at either or both ends) by -C(O)N(R')-. In some embodiments, an alkylene chain is interrupted and / or terminated (at either or both ends) by -N(R')S(O)2-.

[0266] In some embodiments, R is absent and R is a polyethylene glycol chain, which is -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 and / or terminated (at either or both termini) by at least one of carbocyclyl, 3- to 12-membered heterocyclyl, 5- to 12-membered heteroaryl, or any combination thereof, and each R' is independently H or an optionally substituted C1-C 24 alkyl, and the intermediate and one or both terminal groups may be the same or different.

[0267] 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, the polyethylene glycol is interrupted and / or terminated (at either or both ends) by at least one 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)-, -N(R')S(O)-, -S(O)N(R')-, or a combination thereof. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either or both ends) by -N(R')-. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either or both ends) by -C(O)-. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either or both ends) by -C(O)O-. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either or both ends) by -C(O)N(R')-. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either or both ends) by -N(R')S(O)2-.

[0268] In some embodiments, R and R together with the nitrogen atom to which they are attached form a 3-16 membered heterocyclyl containing 1-8 heteroatoms selected from N, O, and S; and R′ is C-C 24an alkylene chain or 1 to 20 -(CHCH-O)- units, where R' is optionally substituted. In some embodiments, R and R together with the nitrogen atom to which they are attached form a 4- to 12-membered heterocyclyl containing 1 to 4 heteroatoms selected from N, O, and S; and R' is a C1-C 18 an alkylene chain or 1 to 15 -(CHCH-O)- units, where R' is optionally substituted. In some embodiments, R and R together with the nitrogen atom to which they are attached form a 5-10 membered heterocyclyl containing 1 to 3 heteroatoms selected from N, O, and S; and R' is a C1-C 12 an alkylene chain or 1 to 10 -(CHCH-O)- units, where R' is optionally substituted. In some embodiments, R and R together with the nitrogen atom to which they are attached form a 5-6 membered heterocyclyl containing 1 to 2 heteroatoms selected from N, O, and S; and R' is a C1-C 10 an alkylene chain or 1 to 5 -(CHCH-O)- units, where R is optionally substituted. In some embodiments, R and R together with the nitrogen atom to which they are attached form a piperazinyl group; and R is a C-C 10 An alkylene chain or 1 to 5 -(CH2CH2-O)- units, wherein R1' is optionally substituted.

[0269] In some embodiments, R1 is absent and R1' is C1-C 24 In some embodiments, R is an alkylene chain and R is methyl or benzyl. In some embodiments, R is absent and R is a C-C 18 In some embodiments, R is an alkylene chain and R is methyl or benzyl. In some embodiments, R is absent and R is a C-C 12 In some embodiments, R is an alkylene chain and R is methyl or benzyl. In some embodiments, R is absent and R is a C-C 10In 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.

[0270] In some embodiments, A1' is selected from the group consisting of muromonab-CD3, abciximab, rituximab, palivizumab, infliximab, trastuzumab, alemtuzumab, adalimumab, ibritumomab, omalizumab, cetuximab, bevacizumab, natalizumab, panitumumab, ranibizumab, eculizumab, certolizumab, ustekinumab, canakinumab, gonokinumab, gonadotropin ... Limumab, ofatumumab, tocilizumab, denosumab, belimumab, ipilimumab, brentuximab, pertuzumab, raxibacumab, obinutuzumab, siltuximab, ramucirumab, vedolizumab, blinatumomab, nivolumab, pembrolizumab, idarucizumab, necitumumab, dinutuximab, secukinumab, mepolizumab, alirocumab, evo Rokumab, daratumumab, elotuzumab, ixekizumab, reslizumab, olaratumab, bezlotoxumab, atezolizumab, obiltoxaximab, inotuzumab, brodalumab, guselkumab, dupilumab, sarilumab, avelumab, ocrelizumab, emicizumab, benralizumab, gemtuzumab, durvalumab, burosumab, ranaldemab, mogamulizumab In some embodiments, A1' is murizumab, erenumab, galcanezumab, tildrakizumab, cemiplimab, emapalumab, fremanezumab, ibalizumab, moxetumomab, ravulizumab, caplacizumab, romosozumab, risankizumab, polatuzumab, brolucizumab, crizanlizumab, sacituzumab, belantamab, or enfortumab, or an antigen-binding fragment thereof. In some embodiments, A1' is trastuzumab.

[0271] 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 , N.R. 10 R 10 , -C(O)NR 10 R 10 , -OC(O)NR 10 R 10 , -NR 10 C(O)R10 , -NR 10 C(O)OR 10 , halogen, OH, CN, amino, (C3-C 10 ) carbocyclyl, 4- or 7-membered heterocyclyl, -O(CH2) 0-3 (C3~C 10 ) carbocyclyl or —O(CH2) containing 1 to 3 heteroatoms selected from O, N, and S 0-3 - 4- or 7-membered heterocyclyl, where each R 10 is independently hydrogen or (C1-C6) alkyl; wherein said alkyl, carbocyclyl or heterocyclyl may be further substituted.

[0272] In some embodiments, R4 is O, S, NR 10 , OC(O), NR 10 C(O), or OC(O)NR5, where R 10 is hydrogen or C1-C6 alkyl.

[0273] In some embodiments, R4 is an alkylene chain, which is -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 12and / or terminated (at either or both termini) by at least one of carbocyclyl, 3- to 12-membered heterocyclyl, 5- to 12-membered heteroaryl, or any combination thereof, and each R' is independently H or an optionally substituted C1-C 24 alkyl, and the intermediate and one or both terminal groups may be the same or different.

[0274] In some embodiments, the alkylene chain is C1-C 24 In some embodiments, the alkylene chain is a C1 to C 18 In some embodiments, the alkylene chain is a C1 to C 12 In some embodiments, the alkylene chain is a C1 to C 10It is an 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 or both ends) by at least one 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')-, or a combination thereof. In some embodiments, an alkylene chain is interrupted and / or terminated (at either or both ends) by -N(R')-. In some embodiments, an alkylene chain is interrupted and / or terminated (at either or both ends) by -C(O)-. In some embodiments, an alkylene chain is interrupted and / or terminated (at either or both ends) by -C(O)O-. In some embodiments, an alkylene chain is interrupted and / or terminated (at either or both ends) by -C(O)N(R')-. In some embodiments, an alkylene chain is interrupted and / or terminated (at either or both ends) by -N(R')S(O)2-.

[0275] In some embodiments, R4 is a polyethylene glycol chain, which is -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 and / or terminated (at either or both termini) by at least one of carbocyclyl, 3- to 12-membered heterocyclyl, 5- to 12-membered heteroaryl, or any combination thereof, and each R' is independently H or an optionally substituted C1-C 24 alkyl, and the intermediate and one or both terminal groups may be the same or different.

[0276] 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, the polyethylene glycol is interrupted and / or terminated (at either or both ends) by at least one 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)-, -N(R')S(O)-, -S(O)N(R')-, or a combination thereof. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either or both ends) by -N(R')-. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either or both ends) by -C(O)-. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either or both ends) by -C(O)O-. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either or both ends) by -C(O)N(R')-. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either or both ends) by -N(R')S(O)2-.

[0277] In some embodiments, n is 2. In some embodiments, n is 2 and each X is CH2.

[0278] In some embodiments, A2' is an anti-cancer agent. In some embodiments, A2' is an auristatin, a maytansinoid, a tubulysin, an anthracycline, paclitaxel or docetaxel or a derivative thereof, a calicheamicin or a derivative thereof, a pyrrolobenzodiazepine dimer (PBD) or a derivative thereof, a duocarmycin or a derivative thereof, an eribulin or a derivative thereof, a camptothecin or a derivative thereof, or an exatecan or a derivative thereof.

[0279] In some embodiments, the antibody is a monoclonal antibody, R1 and R2 together with the nitrogen atom to which they are attached form a piperazinyl, and the compound has a structure represented by formula IVa'1: [ka] or a pharmaceutically acceptable salt or stereoisomer thereof.

[0280] In some embodiments, the antibody is a monoclonal antibody, R1 is absent, R2 is methyl, and the compound has a structure represented by formula IVa'2: [ka] or a pharmaceutically acceptable salt or stereoisomer thereof.

[0281] In some embodiments, the compound of formula (V) is represented by a compound of formula (Va): [ka] 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 t-butyl; or R1' is CH2, and R1 and R2, together with the nitrogen atom to which they are attached, form a 3-16 membered heterocyclyl containing 1-8 heteroatoms selected from N, O, and S; and / or R6 is hydrogen, chloro, bromo, iodo, OR 12 , or SR 12 where each R 12 are independently hydrogen, (C1-C6) alkyl, (C3-C 10 ) carbocyclyl, 4- or 7-membered heterocyclyl; and / or R7 is hydrogen, fluoro, or OR 5’ where R 5’ is hydrogen or (C1-C6) alkyl, an optionally substituted alkylene chain, or an optionally substituted polyethylene glycol chain; and / or R8 is CH2, O, C6-C 12 aryl, 5- to 10-membered heteroaryl; and / or R8 is O, S, NR 11 , OPh, OC(O), OC(O)NR 11 where R 11 is hydrogen or (C1-C6) alkyl, an optionally substituted alkylene chain, or an optionally substituted polyethylene glycol chain; and / or A1 is a binding moiety, a therapeutic moiety, or a diagnostic moiety; and / or A2 is a binding moiety, a therapeutic moiety, or a diagnostic moiety.

[0282] In some embodiments, the compound of formula (V) [ka] [ka] [ka] [ka] or a pharmaceutically acceptable salt or stereoisomer thereof.

[0283] In some embodiments, the compound of formula (V) is [ka] or a pharmaceutically acceptable salt or stereoisomer thereof.

[0284] In some embodiments, the optional substituents for a compound of Formula (IV) or (V) are alkyl, alkenyl, alkynyl, halo, haloalkyl, cycloalkyl, heterocycloalkyl, hydroxy, alkoxy, cycloalkoxy, heterocycloalkoxy, haloalkoxy, aryloxy, heteroaryloxy, aralkyloxy, alkenyloxy, alkynyloxy, amino, alkylamino, cycloalkylamino, heterocycloalkylamino, arylamino, heteroarylamino, aralkylamino, N-alkyl-N-arylamino, N-alkyl-N-heteroarylamino, N-alkyl-N-aralkylamino, hydroxyalkyl, aminoalkyl, alkylthio, haloalkylthio, alkylsulfonyl, haloalkylsulfonyl, cycloalkylsulfonyl, heterocycloalkylsulfonyl, arylsulfonyl, heteroarylsulfonyl, aminosulfonyl, alkylaminosulfonyl, cycloalkylaminosulfonyl, heterocycloalkylaminosulfonyl, arylaminosulfonyl, heteroarylaminosulfonyl, N-alkyl-N -arylaminosulfonyl, N-alkyl-N-heteroarylaminosulfonyl, formyl, alkylcarbonyl, haloalkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, carboxy, alkoxycarbonyl, alkylcarbonyloxy, amino, alkylsulfonylamino, haloalkylsulfonylamino, cycloalkylsulfonylamino, heterocycloalkylsulfonylamino, arylsulfonylamino, heteroarylsulfonylamino, aralkylsulfonylamino, alkylcarbonylamino, haloalkylcarbonylamino, cycloalkylcarbonylamino, heterocycloalkylcarbonylamino, arylcarbonylamino, heteroarylcarbonylamino, aralkylsulfonylamino, aminocarbonyl, alkylaminocarbonyl, cycloalkylaminocarbonyl, heterocycloalkylaminocarbonyl, arylaminocarbonyl, heteroarylaminocarbonyl, N-alkyl-N-arylaminocarbonyl, N-alkyl-N-heteroarylaminocarbonyl, cyano, nitro, and azido.

[0285] In an embodiment, [ka] and [ka] where one is a therapeutic moiety and the other is a diagnostic moiety, the compounds of formula (IV) and (V) are sometimes referred to as "theranostic" agents.

[0286] In some embodiments, the diagnostic moiety is a fluorophore and the therapeutic moiety is an anti-cancer drug.

[0287] In some embodiments, the diagnostic moiety is a fluorophore and the therapeutic moiety is a non-targeted anti-cancer drug.

[0288] In some embodiments, the diagnostic moiety is a fluorophore and the therapeutic moiety is a targeted anti-cancer drug.

[0289] In some embodiments, the diagnostic moiety is a fluorophore and the therapeutic moiety is a kinase inhibitor.

[0290] In some embodiments, the diagnostic moiety is a fluorophore and the therapeutic moiety is an antimicrobial agent.

[0291] In some embodiments, the diagnostic moiety is a fluorophore and the therapeutic moiety is an NSAID.

[0292] In some embodiments, the diagnostic moiety is a fluorophore and the therapeutic moiety is a DMARD.

[0293] In some embodiments, the diagnostic moiety is a chromogenic agent and the therapeutic moiety is an anti-cancer agent.

[0294] In some embodiments, the diagnostic moiety is a chromogenic agent and the therapeutic moiety is a non-targeted anti-cancer agent.

[0295] In some embodiments, the diagnostic moiety is a chromogenic agent and the therapeutic moiety is a targeted anti-cancer agent.

[0296] In some embodiments, the diagnostic moiety is a chromogenic agent and the therapeutic moiety is a kinase inhibitor.

[0297] In some embodiments, the diagnostic moiety is a color-forming agent and the therapeutic moiety is an antimicrobial agent.

[0298] In some embodiments, the diagnostic moiety is a color forming agent and the therapeutic moiety is an NSAID.

[0299] In some embodiments, the diagnostic moiety is a color-forming agent and the therapeutic moiety is a DMARD.

[0300] In some embodiments, the diagnostic moiety is a PET tracer and the therapeutic moiety is an anti-cancer drug.

[0301] In some embodiments, the diagnostic moiety is a PET tracer and the therapeutic moiety is a non-targeted anti-cancer drug.

[0302] In some embodiments, the diagnostic moiety is a PET tracer and the therapeutic moiety is a targeted anti-cancer drug.

[0303] In some embodiments, the diagnostic moiety is a PET tracer and the therapeutic moiety is a kinase inhibitor.

[0304] In some embodiments, the diagnostic moiety is a PET tracer and the therapeutic moiety is an antimicrobial agent.

[0305] In some embodiments, the diagnostic moiety is a PET tracer and the therapeutic moiety is an NSAID.

[0306] In some embodiments, the diagnostic moiety is a PET tracer and the therapeutic moiety is a DMARD.

[0307] In some embodiments, the diagnostic moiety is an MRI contrast agent and the therapeutic moiety is an anti-cancer agent.

[0308] In some embodiments, the diagnostic moiety is an MRI contrast agent and the therapeutic moiety is a non-targeted anti-cancer agent.

[0309] In some embodiments, the diagnostic moiety is an MRI contrast agent and the therapeutic moiety is a targeted anti-cancer agent.

[0310] In some embodiments, the diagnostic moiety is an MRI contrast agent and the therapeutic moiety is a kinase inhibitor.

[0311] In some embodiments, the diagnostic moiety is an MRI contrast agent and the therapeutic moiety is an antimicrobial agent.

[0312] In some embodiments, the diagnostic moiety is an MRI contrast agent and the therapeutic moiety is an NSAID.

[0313] In some embodiments, the diagnostic moiety is an MRI contrast agent and the therapeutic moiety is a DMARD.

[0314] In some embodiments [ka] and [ka] Compounds of formula (IV) and (V), in which one of the moieties is a binding moiety and the other is a different binding moiety, may be referred to as proteolytic targeting chimeras (also known as PROTACs or degraders) that target a given protein for selective degradation.

[0315] In some embodiments, the first binding moiety binds to an 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.

[0316] In some embodiments, the first binding moiety binds to an E3 ubiquitin ligase and 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 an IAP. In some embodiments, the E3 ligase is MDM2.

[0317] In some embodiments, the first binding moiety binds to an E3 ubiquitin ligase and 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.

[0318] In some embodiments, the first binding moiety binds to an E3 ubiquitin ligase and 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.

[0319] In some embodiments, the first binding moiety binds to an E3 ubiquitin ligase and the second binding moiety binds to an 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 an IAP. In some embodiments, the E3 ligase is MDM2.

[0320] In some embodiments, the first binding moiety binds to an E3 ubiquitin ligase and 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.

[0321] In some embodiments, the first binding moiety binds to an E3 ubiquitin ligase and 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.

[0322] In some embodiments [ka] and [ka] wherein one of the moieties is a therapeutic moiety and the other is a binding moiety comprising an antibody or a (cell target) binding fragment thereof, the compounds of formula (IV) and (IV') may be referred to as antibody-drug conjugates, and in some embodiments, the therapeutic moiety of the antibody-drug conjugate is an anti-cancer drug.

[0323] In some embodiments, the binding moiety of the antibody-drug conjugate is a monoclonal antibody or fragment thereof and the therapeutic moiety is a non-targeted anti-cancer drug.

[0324] In some embodiments, the binding moiety of the antibody-drug conjugate is a monoclonal antibody or fragment thereof and the therapeutic moiety is a targeted anti-cancer drug.

[0325] In some embodiments, the binding moiety of the antibody-drug conjugate is a monoclonal antibody or binding fragment thereof and the therapeutic moiety is a kinase inhibitor.

[0326] In some embodiments, the binding moiety of the antibody-drug conjugate is a monoclonal antibody or binding fragment thereof and the therapeutic moiety is an antibacterial agent.

[0327] In some embodiments, the binding moiety of the antibody-drug conjugate is a monoclonal antibody or binding fragment thereof and the therapeutic moiety is an NSAID.

[0328] In some embodiments, the binding moiety of the antibody-drug conjugate is a monoclonal antibody or binding fragment thereof and the therapeutic moiety is a DMARD.

[0329] In some embodiments, the binding moiety is biotin or a derivative thereof and the therapeutic moiety is a targeted anti-cancer drug.

[0330] In some embodiments, the binding moiety is biotin or a derivative thereof and the therapeutic moiety is a kinase inhibitor.

[0331] In some embodiments, the binding moiety is biotin or a derivative thereof and the therapeutic moiety is an antibacterial agent.

[0332] In some embodiments, the binding moiety is biotin or a derivative thereof and the therapeutic moiety is an NSAID.

[0333] In some embodiments, the binding moiety is biotin or a derivative thereof and the therapeutic moiety is a DMARD.

[0334] The compounds of the present invention may be in the form of free acid or free base, or in the form of a pharmaceutically acceptable salt. As used herein, the term "pharmaceutically acceptable" in the context of a salt refers to a salt of a compound that does not abolish the biological activity or properties of the compound and is relatively non-toxic, i.e., the salt of a compound can be administered to a subject without causing undesirable biological effects (such as dizziness or stomach upset) or interacting in a harmful manner with any of the other components of the composition in which it is contained. The term "pharmaceutically acceptable salt" refers to the product obtained by reacting a compound 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, Na, K, Ca, Mg, Fe, Cu, Al, Zn, and Mn salts. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts of amino groups formed with inorganic acids such as hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, isonicotinate, acetate, lactate, salicylate, citrate, tartrate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisate, fumarate, gluconate, glucuronate, saccharinate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, 4-methylbenzenesulfonate, or p-toluenesulfonate. Certain compounds of the present 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.

[0335] The compounds of the present invention may have at least one chiral center and therefore, as used herein, may be in the form of stereoisomers, including all isomers of individual compounds that differ only in the orientation of their atoms in space. The term stereoisomer includes enantiomers (enantiomers containing the (R-) or (S-) configuration of a compound), mixtures of enantiomers of a compound (physical mixtures of enantiomers, and racemates or racemic mixtures), geometric (cis / trans or E / Z, R / S) isomers of a compound, and isomers of a compound with two or more chiral centers that are not mirror images of each other (diastereoisomers). Because the chiral centers of a compound may undergo epimerization in vivo, for these compounds, administration of the compound in its (R-) form is considered equivalent to administration of the compound in its (S-) form. Thus, the compounds of the present invention may be prepared and used in the form of individual isomers, substantially free of other isomers, or in the form of mixtures of various isomers, such as racemic mixtures of stereoisomers.

[0336] In some embodiments, the compounds of the present invention are isotopic derivatives in that they have at least one desired isotopic substitution of an atom at an amount greater than the natural abundance of the isotope, i.e., enriched. In one embodiment, the compound contains deuterium or multiple deuterium atoms. Deuterium, i.e., 2 Substitution with heavier isotopes, such as H, may confer certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements, and therefore may be advantageous in some circumstances.

[0337] The compounds of the present invention may be prepared by crystallization under different conditions and may exist as one or a combination of polymorphic forms. For example, different polymorphs may be identified and / or prepared by using different solvents or different solvent mixtures for recrystallization, by carrying out crystallization at different temperatures, or by using various cooling modes ranging from very fast to very slow cooling during crystallization. Polymorphs may also be obtained by heating or melting the compound followed by gradual or rapid cooling. The presence of polymorphs may be determined by solid probe NMR spectroscopy, IR spectroscopy, differential scanning calorimetry, powder X-ray diffraction, and / or other known techniques.

[0338] In some embodiments, the pharmaceutical composition comprises a co-crystal of a compound of the present invention. As used herein, the term "co-crystal" refers to a stoichiometric multi-component system comprising a compound of the present invention and a co-crystal former, wherein the compound of the present invention and the co-crystal former are bound by non-covalent interactions. As used herein, the term "co-crystal former" refers to a compound that can form an intermolecular interaction with a compound of the present invention and co-crystallize therewith. Representative examples of co-crystal formers include benzoic acid, succinic acid, fumaric acid, glutaric acid, trans-cinnamic acid, 2,5-dihydroxybenzoic acid, glycolic acid, trans-2-hexanoic acid, 2-hydroxycaproic acid, lactic acid, sorbic acid, tartaric acid, ferulic acid, suberic acid, picolinic acid, salicylic acid, maleic acid, saccharin, 4,4'-bipyridine p-aminosalicylic acid, nicotinamide, urea, isonicotinamide, methyl 4-hydroxybenzoate, adipic acid, terephthalic acid, resorcinol, pyrogallol, phloroglucinol, hydroxyquinol, isoniazid, theophylline, adenine, theobromine, phenacetin, phenazone, etophylline, and phenobarbital.

[0339] Synthesis method In another aspect, the present invention is directed to methods for making the compounds of the present invention, or their pharmaceutically acceptable salts or stereoisomers. Generally, the compounds of the present invention and their pharmaceutically acceptable salts and stereoisomers can be prepared by any process known to be applicable to the preparation of chemically related compounds. The compounds of the present invention will be better understood in connection with the synthetic schemes described in the various examples, which illustrate non-limiting methods by which the compounds, e.g., compounds of Formulas I-III, can be prepared.

[0340] In one of these aspects, the present invention relates to a method of preparing a compound of formula IV: [ka] Formula I: [ka] with a compound of formula II: [ka] In some embodiments, a compound of formula (I) may be administered together with a compound of formula (II) to form a compound of formula (IV) in vivo.

[0341] In one of these aspects, the present invention relates to a method of preparing a compound of formula V: [ka] Formula I: [ka] with a compound of formula III: [ka] The method includes reacting a compound of formula (I) with a compound of formula (III). In some embodiments, a compound of formula (I) can be administered together with a compound of formula (III) to form a compound of formula (V) in vivo. Synthetic schemes for linking active moieties to compounds are known in the art. See, for example, Agarwal et al., Bioconjugate Chem. 26(2):176-192 (2015).

[0342] In one of these aspects, the invention relates to a method for preparing a compound of formula IVa'. [ka] Formula Ia': [ka] with a compound of formula II': [ka] with a compound of formula (I).

[0343] In one of these aspects, the invention relates to a method for preparing a compound of formula IVb'. [ka] Formula Ib': [ka] with a compound of formula II': [ka] with a compound of formula (I).

[0344] In one of these aspects, the invention is directed to a method of preparing a compound of formula IVc'. [ka] Formula Ic': [ka] with a compound of formula II': [ka] with a compound of formula (I).

[0345] In some embodiments, the reacting is carried out in the presence of a solvent.

[0346] In some embodiments, the solvent is an aprotic solvent, hi some embodiments, the aprotic solvent is DCM, CHCl3, CCl4, DCE, toluene, MeCN, or THF.

[0347] In some embodiments, the solvent is a protic solvent, hi some embodiments, the protic solvent is MeOH, EtOH, iPrOH, nBuOH, TFE, or HFIP.

[0348] 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% protic to aprotic. In some embodiments, the solvent mixture is 0-100% TFE in CHCl3. In some embodiments, the solvent mixture is about 20% TFE in CHCl3.

[0349] In some embodiments, the reaction is carried out in the presence of an aqueous buffer. In some embodiments, the aqueous buffer is an acidic buffer. In some embodiments, the aqueous buffer is an alkaline buffer.

[0350] In some embodiments, the reaction is carried out in the presence of a biological fluid, hi some embodiments, the biological fluid is blood, synovial fluid, lymphatic fluid, or vitreous fluid.

[0351] In some embodiments, the reaction is carried out in the presence of an aqueous solution containing biological components such as cell lysates, proteins, nucleic acids, or lipids.

[0352] In some embodiments, the reaction is carried out by adding a buffering reagent.Representative examples of buffering reagents include ascorbic acid, glutathione, citric acid, acetic acid, monopotassium phosphate, N-cyclohexyl-2-aminoethanesulfonic acid (CHES), and borate.In some embodiments, the buffering reagent is ascorbic acid or glutathione.

[0353] In some embodiments, the reacting is carried out at a temperature of about -40°C to 80°C. In some embodiments, the reacting is carried out at a temperature of 0°C to 60°C. In some embodiments, the reacting is carried out at a temperature of about 60°C. In some embodiments, the reacting is carried out at a temperature of about 20°C to 25°C.

[0354] In some embodiments, the compound of Formula (I) is in excess of 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.

[0355] In some embodiments, the reaction is carried out over a period of 1 week. In some embodiments, the reaction is carried out over a period of 5 days. In some embodiments, the reaction is carried out over a period of 3 days. In some embodiments, the reaction is carried out over a period of 24 hours. In some embodiments, the reaction is carried out over a period of 18 hours. In some embodiments, the reaction is carried out over a period of 12 hours. In some embodiments, the reaction is carried out over a period of 6 hours. In some embodiments, the reaction is carried out over a period of 3 hours. In some embodiments, the reaction is carried out over a period of 2 hours. In some embodiments, the reaction is carried out over a period of 1 hour. In some embodiments, the reaction is carried out over a period of 45 minutes. In some embodiments, the reaction is carried out over a period of 30 minutes. In some embodiments, the reaction is carried out over a period of 15 minutes. In some embodiments, the reaction is carried out over a period of 5 minutes. In some embodiments, the reaction is carried out over a period of 1 minute.

[0356] Pharmaceutical Composition Another aspect of the present invention is directed to a pharmaceutical composition comprising a therapeutically effective amount of a compound of the present invention, 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 vehicle suitable for administering a compound of the present invention to a mammal. Suitable carriers 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 a compound from one organ or part of the body to another. A carrier is "acceptable" in the sense of being physiologically inert and compatible with the other ingredients of the formulation and not harmful to the subject or patient. Depending on the type of formulation, the composition may also include one or more pharmaceutically acceptable excipients.

[0357] Generally, the compounds of the present invention and their pharmaceutically acceptable salts, or stereoisomers, can be formulated into a given type of composition according to conventional pharmaceutical practices such as conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, and compression processes (see, e.g., Remington: The Science and Practice of Pharmacy (20th ed.), ed. A.R. Gennaro, Lippincott Williams & Wilkins, and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J.C. Boylan, 1988-1999, Marcel Dekker, New York). The type of formulation depends on the mode of administration, which may include enteral (e.g., oral, buccal, sublingual, and rectal), parenteral (e.g., subcutaneous (sc), intravenous (iv), intramuscular (im), and intrasternal injection or infusion techniques, intraocular, intraarterial, intramedullary, intrathecal, intraventricular, transdermal, intradermal, intravaginal, intraperitoneal, mucosal, intranasal, intratracheal instillation, bronchial instillation, and inhalation), and topical (e.g., transdermal). Generally, the most appropriate administration route depends on various factors, including, for example, the nature of the agent (e.g., its stability in the environment of the gastrointestinal tract) 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 in that the compound may be administered relatively quickly, such as in single-dose treatments and / or acute conditions.

[0358] In some embodiments, the compounds are formulated for oral or intravenous administration (eg, systemic intravenous injection).

[0359] Thus, the compounds of the present invention can be formulated into solid compositions (e.g., powders, tablets, dispersible granules, capsules, cachets, and suppositories), liquid compositions (e.g., solutions in which the compound is dissolved, suspensions in which solid particles of the compound are dispersed, 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 rapid, intermediate, or extended release.

[0360] Oral solid dosage forms include capsules, tablets, pills, powders, and granules. In these solid dosage forms, the active compound is mixed with a carrier such as sodium citrate or dicalcium phosphate, and a) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and silicic acid; b) binders, such as methylcellulose, microcrystalline cellulose, hydroxypropylmethylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidinone, sucrose, and acacia; c) humectants, such as glycerol; d) disintegrants, such as cross-linked polymers (e.g., cross-linked polyvinylpyrrolidone (crospovidone), cross-linked carboxymethylcellulose, and the like). They are mixed with additional carriers or excipients such as sodium (croscarmellose sodium), sodium starch glycolate, agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; e) solution retarders, such as paraffin; f) absorption accelerators, such as quaternary ammonium compounds; g) wetting agents, such as cetyl alcohol and glycerol monostearate; h) absorbents, such as kaolin and bentonite clay; and i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may also contain buffering agents. Similar types of solid compositions can also be used as fillers for soft and hard gelatin capsules, using excipients such as lactose or milk sugar and high molecular weight polyethylene glycols. Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells, such as enteric coatings and other coatings. They may further contain an opacifying agent.

[0361] 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 stearyl fumarate, anhydrous lactose, microcrystalline cellulose, and croscarmellose sodium.The gelatin shell can contain gelatin, titanium dioxide, iron oxide, and coloring agents.

[0362] The liquid dosage form for oral administration includes solution, suspension, emulsion, microemulsion, syrup and elixir.In addition to compound, liquid dosage form can contain water or other solvent, solubilizer and emulsifier, for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oil (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol and fatty acid ester of sorbitan, and their mixtures, and the like, and the like.Oral composition can also contain excipients such as wetting agent, suspending agent, coloring agent, sweetener, flavoring agent and fragrance.

[0363] Injectable preparations for parenteral administration may include sterile aqueous or oily suspensions. These may be formulated according to standard techniques using appropriate dispersing or wetting agents and suspending agents. Sterile injectable preparations may also be sterile injectable solutions, suspensions, or emulsions in non-toxic parenterally acceptable diluents or solvents, such as solutions in 1,3-butanediol. Acceptable vehicles and solvents that may be used include water, Ringer's solution, USP, and isotonic sodium chloride solution. Additionally, sterile, fixed oils are conventionally used as solvents or suspending media. For this purpose, any brand of fixed oil, including synthetic mono- or diglycerides, may be used. In addition, fatty acids such as oleic acid are used in the preparation of injectables. Injectable preparations can be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved or dispersed in sterile water or other sterile injectable media prior to use. The effect of a compound can be prolonged by slowing its absorption, which can be accomplished by using liquid suspensions or crystalline or amorphous material with poor water solubility. Prolonged absorption of a compound from parenterally administered formulations can also be achieved by suspending the compound in an oil vehicle.

[0364] In certain embodiments, the compounds of the present invention can be administered locally rather than systemically, often in depot or sustained-release formulations, for example, by directly injecting the conjugate into an organ. In specific embodiments, long-acting formulations are administered by implantation (e.g., subcutaneous or intramuscular) or intramuscular injection. Injectable depot forms are made by forming microencapsule matrices of the compound in biodegradable polymers, such as polylactide-polyglycolide, poly(orthoesters), and poly(anhydrides). The compound release rate can be controlled by varying the ratio of compound to polymer and the nature of the particular polymer used. Depot injectable formulations can also be prepared by encapsulating the compound in liposomes or microemulsions that are compatible with body tissues. In yet other embodiments, the compound is delivered in a targeted drug delivery system, for example, liposomes coated with organ-specific antibodies. In such embodiments, the liposomes target and are selectively taken up by the organ.

[0365] The compositions may be formulated for buccal or sublingual administration and include examples of such compositions include tablets, lozenges and gels.

[0366] The compounds of the present invention can be formulated for administration by inhalation.Various forms suitable for administration by inhalation include aerosol, mist or powder.The pharmaceutical composition can be delivered in the form of an aerosol spray presentation from a pressurized pack or nebulizer 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 metered amount.In some embodiments, capsules and cartridges containing, for example, gelatin for use in an inhaler or insufflator can be formulated, containing a powder mixture of the compound and a suitable powder base such as lactose or starch.

[0367] The compounds of the present invention can be formulated for topical administration, which as used herein refers to intradermal administration by the invention of formulations to the epidermis. These types of compositions are typically in the form of ointments, pastes, creams, lotions, gels, solutions and sprays.

[0368] Representative examples of carriers useful for formulating compounds for topical application include solvents (e.g., alcohol, polyalcohol, water), creams, lotions, ointments, oils, plasters, liposomes, powders, emulsions, microemulsions, and buffer solutions (e.g., hypotonic or buffered saline).Cream can be formulated using saturated or unsaturated fatty acids such as stearic acid, palmitic acid, oleic acid, palmitoleic acid, cetyl or oleyl alcohol.Cream can also contain nonionic surfactants such as polyoxy-40-stearate.

[0369] In some embodiments, topical formulations can also contain excipients, such as penetration enhancers.These agents can transport pharmacologically active compounds through the stratum corneum to the epidermis or dermis, preferably with little or no systemic absorption.A wide variety of compounds have been evaluated for their effectiveness in increasing the penetration rate of drugs through the skin.For example, see Percutaneous Penetration Enhancers, Maibach HI and Smith HE (eds.), CRC Press, Inc., Boca Raton, Fla. (1995), which surveys 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 SI (Eds.), Interpharm Press Inc., Buffalo Grove, Ill. (1997), which surveys the use and testing of various skin penetration enhancers. Representative examples of penetration enhancers include triglycerides (e.g., soybean oil), aloe compositions (e.g., aloe vera gel), ethyl alcohol, isopropyl alcohol, octriphenyl polyethylene glycol, oleic acid, polyethylene glycol 400, propylene glycol, N-decylmethyl sulfoxide, fatty acid esters (e.g., isopropyl myristate, methyl laurate, glyceryl monooleate, and propylene glycol monooleate), and N-methylpyrrolidone.

[0370] Representative examples of other excipients that can be included in topical and other types of formulations (to the extent they are compatible) include preservatives, antioxidants, moisturizers, emollients, buffers, solubilizers, skin protectants, and surfactants. Suitable preservatives include alcohols, quaternary amines, organic acids, parabens, and phenols. Suitable antioxidants include ascorbic acid and its esters, sodium bisulfite, butylhydroxytoluene, butylhydroxyanisole, tocopherol, and chelating agents such as EDTA and citric acid. Suitable moisturizers include glycerin, sorbitol, polyethylene glycol, urea, and propylene glycol. Suitable buffers include citric acid, hydrochloric acid, and lactic acid buffers. Suitable solubilizers include quaternary ammonium chloride, cyclodextrin, benzyl benzoate, lecithin, and polysorbates. Suitable skin protectants include vitamin E oil, allatoin, dimethicone, glycerin, petrolatum, and zinc oxide.

[0371] Transdermal formulations typically use transdermal delivery devices and transdermal delivery patches, in which compounds are formulated in lipophilic emulsions or buffered aqueous solutions and dissolved and / or dispersed in polymers or adhesives.Patches can be configured for continuous, pulsatile, or on-demand delivery of pharmaceuticals.Transdermal delivery of compounds can be achieved by iontophoretic patches.Transdermal patches can provide controlled delivery of compounds by using rate-controlling membranes or by trapping compounds in polymer matrices or gels, slowing the absorption rate.Absorption enhancers can be used to increase absorption, and examples include absorbable pharmaceutically acceptable solvents that aid in skin passage.

[0372] Ophthalmic preparations include eye drops.

[0373] 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, as well as 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 the compound 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 therefore will melt in the rectum or vaginal cavity and release the compound.

[0374] Dosage As used herein, the term "therapeutically effective amount" refers to an amount of a compound of the present invention (which contains a therapeutic moiety or is therapeutic), or a pharmaceutically acceptable salt or stereoisomer thereof, effective to produce a desired therapeutic response in a patient. The term "therapeutic response" includes an amount of a compound of the present invention, or a pharmaceutically acceptable salt or stereoisomer thereof, that, when administered, is sufficient to induce a positive alteration in the disease or disorder being treated, or to prevent the onset or progression of the disease or disorder, or to alleviate to some extent one or more of the symptoms of the disease or disorder being treated in a subject, or to inhibit the proliferation of diseased cells.

[0375] As used herein, the term "diagnostically effective amount" refers to an amount of a compound of the invention (containing an amount of a diagnostic moiety), or a pharmaceutically acceptable salt or stereoisomer thereof, effective to produce a desired detectable response in a patient.

[0376] The total daily dosage of compound and its use can be determined according to standard medical practice, for example, by attending physician, using sound medical judgment.The specific therapeutically effective dose for any specific subject depends on various factors, including: the disease or disorder to be treated and its severity (for example, its current state); the activity of the compound used; the specific composition used; the age, weight, general health, sex and diet of subject; the administration time, administration route and excretion rate of the compound used; treatment period; the drug used in combination with or simultaneously with the specific compound used; and similar factors well known in the medical field (for example, see Hardman et al., eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th Edition, McGraw-Hill Press, 155-173, 2001).

[0377] The compounds of the present invention may be effective over a wide dosage range. In some embodiments, the total daily dose (e.g., for an adult human) may range from about 0.001 to about 1600 mg, 0.01 to about 1000 mg, 0.01 to about 500 mg, about 0.01 to about 100 mg, about 0.5 to about 100 mg, 1 to about 100 to about 400 mg per day, about 1 to about 50 mg per day, about 5 to about 40 mg per day, and in still other embodiments, about 10 to about 30 mg per day. Individual dosages may be formulated to contain the desired dosage, depending on the number of times the compound is administered per day. For example, capsules may be formulated containing about 1 to about 200 mg of compound (e.g., 1, 2, 2.5, 3, 4, 5, 10, 15, 20, 25, 50, 100, 150, and 200 mg). In some embodiments, the compound may be administered at a dose ranging from about 0.01 mg to about 200 mg / kg body weight per day. In some embodiments, doses of 0.1 to 100, e.g., 1 to 30 mg / kg per day, administered in one or more doses per day may be effective. By way of example, suitable doses for oral administration may range from 1 to 30 mg / kg body weight per day, and suitable doses for intravenous administration may range from 1 to 10 mg / kg body weight per day.

[0378] How to use In some embodiments, the present invention provides [ka] and [ka] In some embodiments, the present invention is directed to a method of treating a disease or disorder involving administering a therapeutically effective amount of a compound of Formula (IV or V), wherein one of is a therapeutic agent, or a pharmaceutically acceptable salt or stereoisomer thereof, to a subject in need thereof. In some embodiments, the disease is cancer.

[0379] In some aspects, the present invention relates to a method of treating cancer, comprising administering to a subject in need thereof 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 symmetric diboron reagent. In some embodiments, the diboron reagent is an asymmetric diboron reagent. In some embodiments, the diboron reagent is B2(OH)4, B 2pin2 , [ka] Other representative examples of diboron reagents include bis(catecholato)diboron, bis(hexylene glycolato)diboron, bis[(-)pinanediolate]diboron, bis(diisopropyl-1-tartrate glycolato)diboron, bis(N,N,N',N'-tetramethyl-d-tartramidoglycolate)diboron, and 2,2'-bi-132-dioxaborinane. Still other diboron reagents suitable for use in the present invention are described in Ali et al., Studies in Inorganic Chemistry, "Chapter 1 - Chemistry of the diboron 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 diboron reagent is administered at a concentration of about 1 pM to about 1 M. In some embodiments, the diboron reagent is administered at a concentration of about 1 pM to about 100 mM. In some embodiments, the diboron reagent is administered at a concentration of about 1 pM to about 10 mM. In some embodiments, the diboron reagent is administered at a concentration of about 1 pM to about 1 mM. In some embodiments, the diboron reagent is administered at a concentration of about 1 pM to about 100 μM. In some embodiments, the diboron reagent is administered at a concentration of about 1 pM to about 10 μM. In some embodiments, the diboron reagent is administered at a concentration of about 1 pM to about 1 μM. In some embodiments, the diboron reagent is administered at a concentration of about 1 pM to about 100 nM. In some embodiments, the diboron reagent is administered at a concentration of about 1 pM to about 10 nM. In some embodiments, the diboron reagent is administered at a concentration of about 1 pM to about 1 nM. In some embodiments, the diboron reagent is administered at a concentration of about 1 pM to about 100 pM.

[0380] In some embodiments, the methods involve administering a compound of formula (I) and a compound of formula (II or III), or a pharmaceutically acceptable salt or stereoisomer thereof; [ka] and [ka] Either of the compounds is a therapeutic agent, or the compounds formed by the reaction between the compounds of Formula (I) and (II) and between the compounds of Formula (I) and (III) are therapeutic to a subject in need thereof. The compounds of Formula (I) and the compounds of Formula (II or III) and their pharmaceutically acceptable salts and stereoisomers can be used in combination or simultaneously in the treatment of a disease or disorder. The terms "in combination" and "simultaneously" in this context mean that the compounds are administered simultaneously, including substantially contemporaneous administration via the same or separate dosage forms and via the same or different modes of administration, or sequentially, e.g., as part of the same regimen. The order and time intervals can be determined so that they can react together. For example, the compounds can be administered simultaneously or sequentially in any order at different times, but if not administered simultaneously, they can be administered sufficiently close in time to provide the desired therapeutic effect. In some embodiments, the method is directed to treating cancer.

[0381] In some aspects, the present invention is directed to methods of both treating and diagnosing a disease or disorder involving administering to a subject in need thereof a compound of Formula (IV or V), or a pharmaceutically acceptable salt or stereoisomer thereof, wherein the compound is in the form of a theranostic agent. In some embodiments, the disease is cancer.

[0382] In some embodiments, the present invention is directed to theranostic agents used to treat and diagnose diseases or disorders, such as cancer, involving administration of compounds of Formula (I) and Formula (II or III), or pharmaceutically acceptable salts or stereoisomers thereof, to a subject in need thereof.

[0383] In some aspects, the present invention is directed to a method of protein labeling, which involves administering to a subject in need thereof 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 relates to labeling a cancer-associated antigen. Tumor-associated antigens that may be suitable for use in the present invention include those described in Ilyas et al., J. Immunol. 195(11):5117-5122 (2015) and Haen et al., Nat. Rev. Clin. Oncol. 17:595-610 (2020).

[0384] A "disease" is generally considered to be a state of health in a subject in which the subject is unable to maintain homeostasis and, if the disease is not improved, the subject's health will continue to deteriorate. In contrast, a "disorder" in a subject is a state in which the subject is able to maintain homeostasis, but the subject's health is less favorable than it would be in the absence of the disorder. If left untreated, the disorder does not necessarily result in a further decline in the subject's health. In some embodiments, the compounds of the present invention may be useful in treating cell proliferative diseases and disorders (e.g., cancer or benign neoplasms). As used herein, the term "cell proliferative disease or disorder" refers to a condition characterized by deregulated or abnormal cell proliferation, or both, including non-cancerous conditions such as neoplasms, precancerous conditions, benign tumors, and cancers.

[0385] The term "subject" (or "patient") as used herein includes all members of the animal kingdom susceptible to or suffering from the indicated disease or disorder. In some embodiments, the subject is a mammal, e.g., a human or non-human mammal. The method is also applicable to companion animals such as dogs and cats, as well as livestock such as cows, horses, sheep, goats, and pigs, and other domestic and wild animals. A subject "in need" of treatment according to the present invention may be "suffering from or suspected of suffering from" a particular disease or disorder, or may have been positively diagnosed with or exhibit a sufficient number of risk factors or a sufficient number or combination of signs or symptoms that allow a medical professional to diagnose or suspect the subject as suffering from the disease or disorder. Thus, subjects suffering from and suspected of suffering from a particular disease or disorder are not necessarily two distinct groups.

[0386] The compounds of the invention can be used to treat and / or diagnose a wide variety of diseases and disorders, including cancer and non-cancerous conditions, etc. Exemplary types of non-cancerous (e.g., cell proliferative) diseases or disorders that may be suitable for treatment with the compounds of the invention include inflammatory diseases and conditions, autoimmune diseases, cardiac diseases, viral diseases, chronic and acute kidney disease or injury, metabolic diseases, and allergic and genetic diseases.

[0387] Representative examples of specific non-cancerous diseases and disorders include rheumatoid arthritis, alopecia areata, lymphoproliferative conditions, autoimmune blood disorders (e.g., hemolytic anemia, aplastic anemia, anhidrotic ectodermal dysplasia, true erythrocytic anemia, and idiopathic thrombocytopenia), cholecystitis, acromegaly, rheumatoid spondylitis, osteoarthritis, gout, scleroderma, sepsis, septic shock, dacryoadenitis, cryopyrin-associated periodic syndromes (CAPS), endotoxic shock, endometritis, gram-negative sepsis, keratoconjunctivitis sicca, toxic shock syndrome, asthma, and rheumatoid arthritis. Adult respiratory distress syndrome, chronic obstructive pulmonary disease, chronic pulmonary inflammation, chronic transplant rejection, hidradenitis suppurativa, inflammatory bowel disease, Crohn's disease, Behçet's syndrome, systemic lupus erythematosus, glomerulonephritis, multiple sclerosis, juvenile-onset diabetes, autoimmune uveoretinitis, autoimmune vasculitis, thyroiditis, Addison's disease, lichen planus, appendicitis, bullous pemphigoid, pemphigus vulgaris, pemphigus foliaceus, paraneoplastic pemphigus, myasthenia gravis, immunoglobulin A nephropathy, Hashimoto's disease, Sjögren's syndrome, vitiligo, Wegener's granulomatosis, testicular sarcoidosis, autoimmune Epidemic oophoritis, sarcoidosis, rheumatic carditis, ankylosing spondylitis, Graves' disease, autoimmune thrombocytopenic purpura, psoriasis, psoriatic arthritis, eczema, dermatitis herpetiformis, ulcerative colitis, pancreatic fibrosis, hepatitis, liver fibrosis, CD14-mediated sepsis, non-CD14-mediated sepsis, acute and chronic kidney disease, irritable bowel syndrome, heartburn, restenosis, cervicitis, stroke and ischemic injury, neurotrauma, acute and chronic pain, allergic rhinitis, allergic conjunctivitis, chronic heart failure, congestive heart failure, acute coronary syndrome, cachexia, malaria, Han Sen's disease, leishmaniasis, Lyme disease, Reiter's syndrome, acute synovitis, muscle degeneration, bursitis, tendinitis, tenosynovitis, herniated, ruptured or prolapsed disc syndrome, osteopetrosis, rhinosinusitis, thrombosis, silicosis, pulmonary sarcosis, bone resorption diseases such as osteoporosis, fibromyalgia, AIDS and other viral diseases such as shingles, herpes simplex type I or II, influenza virus and cytomegalovirus, diabetes type I and type II, obesity, insulin resistance and diabetic retinopathy, 22q11.2 deletion syndrome, Angelman syndrome, Canavan disease, celiac disease, Charcot-Marie-Tooth disease, color blindness, cri-the-cat syndrome, Down syndrome, cystic fibrosis, Duchenne muscular dystrophy, hemophilia, Klinefelter syndrome, neurofibromatosis, phenylketonuria, Prader-Willi syndrome, sickle cell disease, Tay-Sachs disease, Turner syndrome, urea cycle disorders, thalassemia, otitis media, pancreatitis, parotitis, pericarditis, peritonitis, pharyngitis, pleuritis, phlebitis, pneumonia, uveitis, polymyositis, proctitis, interstitial pulmonary fibrosis, dermatomyositis, atherosclerosis, arteriosclerosis, amyotrophic lateral sclerosis, asociality, varicose veins, depression, and sudden infant death syndrome.

[0388] In some embodiments, the method relates to treating a subject with cancer. Generally, the compounds of the present invention may be effective in treating carcinomas (solid tumors, including both primary and metastatic tumors), sarcomas, melanomas, and hematological cancers (cancers that develop in the blood, including lymphocytes, bone marrow, and / or lymph nodes), such as leukemia, lymphoma, and multiple myeloma. Adult tumors / cancers and pediatric tumors / cancers are included. The cancer may be a vascularized tumor, a tumor that has not yet been substantially vascularized, or a non-vascularized tumor.

[0389] Representative examples of cancer include adrenocortical carcinoma, AIDS-related cancer (e.g., Kaposi's and AIDS-related lymphoma), appendix cancer, childhood cancer (e.g., childhood cerebellar astrocytoma, childhood cerebral astrocytoma), basal cell carcinoma, skin cancer (non-melanoma), biliary tract cancer, extrahepatic bile duct cancer, intrahepatic bile duct cancer, bladder cancer, and urinary bladder cancer. cancer), brain cancer (e.g., glioma and glioblastoma, e.g., brain stem glioma, gestational trophoblastic tumor glioma, cerebellar astrocytoma, cerebral astrocytoma / malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumor, optic pathway and hypothalamic glioma), breast cancer, bronchial adenoma / carcinoid, carcinoid tumor, nervous system cancer (e.g., central nervous system cancer, central nervous system lymphoma), cervical cancer, chronic myeloproliferative disorder, colorectal cancer (e.g., colon cancer, rectal cancer), polycythemia vera, lymphoma Neoplasms of the liver, mycosis fungoides, Sézary syndrome, endometrial cancer, esophageal cancer, extracranial germ cell tumors, extragonadal germ cell tumors, extrahepatic bile duct cancer, eye cancer, intraocular melanoma, retinoblastoma, gallbladder cancer, gastrointestinal cancer (e.g., gastric cancer, small intestine cancer, gastrointestinal carcinoid, gastrointestinal stromal tumor (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, eye cancer, islet cell tumors (endocrine pancreas), renal cancer (e.g., Wilms' tumor, ulcerative colitis) alveolar renal carcinoma), liver cancer, lung cancer (e.g., non-small cell lung cancer and small cell lung cancer), Waldenstrom's macroglobulinemia, melanoma, intraocular (eye) melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous cell carcinoma of the neck of unknown primary site, multiple endocrine neoplasia (MEN), myelodysplastic syndrome, essential thrombocythemia, myelodysplastic / myeloproliferative disorders, nasopharyngeal carcinoma, neuroblastoma, oral cancer (e.g., oral cancer, lip cancer, oral cavity cancer, tongue cancer, oropharynx cancer, pharynx cancer, laryngeal cancer), ovarian cancer (e.g., ovarian epithelial carcinoma, ovarian germ cell tumors, ovarian low malignant potential tumors), pancreatic cancer, islet cell pancreatic cancer, cancer of the paranasal sinuses and nasal cavity, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineoblastoma, pituitary tumors, plasma cell neoplasms, pleuropulmonary blastoma, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, uterine cancer (e.g., endometrial carcinoma, uterine sarcoma, endometrial carcinoma), squamous cell carcinoma, testicular cancer, thymoma, thymic carcinoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter and other urinary tract, urethral cancer, gestational trophoblastic tumor, vaginal cancer, and vulvar cancer.

[0390] Sarcomas that may be treatable with the compounds of the invention include both soft tissue and bone cancers alike, representative examples of which include osteosarcoma or osteogenic sarcoma (bone) (e.g., Ewing's sarcoma), chondrosarcoma (cartilage), leiomyosarcoma (smooth muscle), rhabdomyosarcoma (skeletal muscle), mesothelioma or mesothelioma (membrane lining of body cavities), fibrosarcoma (fibrous tissue), angiosarcoma or hemangioendothelioma (blood vessels), liposarcoma (fatty tissue), glioma or astrocytoma (neurogenic connective tissue found in the brain), myxosarcoma (primitive embryonic connective tissue), and mesenchymal or mixed mesodermal tumor (mixed connective tissue type).

[0391] In some embodiments, the methods of the present invention involve treating a subject with a cell proliferative disease or disorder of the blood system, liver, brain, lung, colon, pancreas, prostate, ovary, breast, skin, and endometrium.

[0392] As used herein, "cell proliferative diseases or disorders of the blood system" includes lymphoma, leukemia, myeloid neoplasms, mast cell neoplasms, myelodysplasia, benign monoclonal gammopathy, polycythemia vera, chronic myeloid leukemia, myeloid metaplasia of unknown etiology, and essential thrombocythemia. Thus, representative examples of hematological cancers include multiple myeloma, lymphomas (T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma (diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL) and ALK+ anaplastic large cell lymphoma (e.g., 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's lymphoma / leukemia, mantle cell lymphoma, mediastinal (thymic) large B-cell lymphoma, follicular lymphoma, marginal zone lymphoma, lymphoplasmacytic lymphoma / Waldenstrom's macroglobulinemia, metastatic pancreatic adenocarcinoma, B-cell non-Hodgkin's lymphoma selected from refractory B-cell non-Hodgkin's lymphoma and relapsed B-cell non-Hodgkin's lymphoma, childhood lymphoma, and lymphomas of lymphocytic and cutaneous origin, such as small lymphocytic lymphoma, leukemia (including childhood leukemia, hairy cell leukemia, acute lymphocytic leukemia, acute myeloid leukemia, acute myeloid leukemia (e.g., acute monocytic leukemia), chronic lymphocytic leukemia, small lymphocytic leukemia, chronic myelocytic leukemia, chronic myelogenous leukemia, and mast cell leukemia), myeloid neoplasms, and mast cell neoplasms.

[0393] As used herein, " liver cell proliferative disease or disorder " includes all forms of liver cell proliferative disorder.Hepatic cell proliferative disorder can include liver cancer (for example, hepatocellular carcinoma, intrahepatic cholangiocarcinoma and hepatoblastoma), liver precancer or precancerous condition, liver benign growth or lesion, liver malignant growth or lesion, and metastatic lesion in the tissues and organs of the body other than the liver.Hepatic cell proliferative disorder can include liver hyperplasia, metaplasia, and dysplasia.

[0394] As used herein, " brain cell proliferative disease or disorder " includes all forms of cell proliferative disorder that affect the brain.Brain cell proliferative disorder can include brain cancer (for example, glioma, glioblastoma, meningioma, pituitary adenoma, vestibular schwannoma, and primitive neuroectodermal tumor (medulloblastoma)), brain precancer or precancerous condition, brain benign growth or lesion, brain malignant growth or lesion, and metastatic lesion in the tissues and organs of the body other than the brain.Brain cell proliferative disorder can include brain hyperplasia, metaplasia, and dysplasia.

[0395] As used herein, "pulmonary cell proliferative diseases or disorders" include all forms of cell proliferative disorders affecting lung cells. Pulmonary cell proliferative disorders include lung cancer, precancerous and precancerous conditions of the lung, benign growths or lesions of the lung, hyperplasia, metaplasia, and dysplasia of the lung, and metastatic lesions in tissues and organs other than the lung. Lung cancer includes all forms of lung cancer, such as malignant lung neoplasms, 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 may include "scar carcinoma," bronchioloalveolar carcinoma, giant cell carcinoma, spindle cell carcinoma, and large cell neuroendocrine carcinoma. Lung cancer also includes lung neoplasms 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 squamous cell lung cancer).

[0396] As used herein, "colon cell proliferative disease or disorder" includes all forms of cell proliferative disorders affecting colon cells, including colon cancer, colon precancer or precancerous conditions, colon adenomatous polyps, and colon metachronous lesions.Colon cancer includes sporadic and hereditary colon cancer, malignant colon neoplasms, intraepithelial carcinoma, typical carcinoid tumors, atypical carcinoid tumors, adenocarcinoma, squamous cell carcinoma, and squamous cell carcinoma.Colon cancer may be associated with genetic syndromes such as hereditary nonpolyposis colorectal cancer, familial adenomatous polyposis, MYH-associated polyposis, Gardner syndrome, Peutz-Jeghers syndrome, Turcot syndrome, and juvenile polyposis.Colon cell proliferative disorders may also be characterized by colon hyperplasia, metaplasia, or dysplasia.

[0397] As used herein, "pancreatic cell proliferative disease or disorder" includes all forms of cell proliferative disorders that affect pancreatic cells.Pancreatic cell proliferative disorders may include pancreatic cancer, precancerous or precancerous conditions of the pancreas, pancreatic hyperplasia, pancreatic dysplasia, benign pancreatic growths or lesions, and malignant pancreatic growths or lesions, as well as metastatic lesions in tissues and organs other than the pancreas.Pancreatic cancer includes all forms of pancreatic cancer, including ductal adenocarcinoma, adenosquamous carcinoma, pleomorphic giant cell carcinoma, mucinous adenocarcinoma, osteoclast-like 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 histochemical and ultrastructural heterogeneity (e.g., mixed cell types).

[0398] As used herein, "prostate cell proliferative disease or disorder" includes all forms of cell proliferative disorders that affect the prostate.Prostate cell proliferative disorders can include prostate cancer, prostate precancer or precancerous conditions, benign prostate growths or lesions, and malignant prostate growths or lesions, as well as metastatic lesions in body tissues and organs other than the prostate.Prostate cell proliferative disorders can include prostate hyperplasia, metaplasia, and dysplasia.

[0399] As used herein, "cell proliferative diseases or disorders of the ovary" includes all forms of cell proliferative disorders affecting cells of the ovary. Cell proliferative disorders of the ovary can include ovarian precancer or precancerous conditions, benign ovarian growths or lesions, ovarian cancer, and metastatic lesions in tissues and organs of the body other than the ovaries. Cell proliferative disorders of the ovary can include ovarian hyperplasia, metaplasia, and dysplasia.

[0400] As used herein, "cell proliferative diseases or disorders of the breast" include all forms of cell proliferative disorders affecting breast cells. Cell proliferative disorders of the breast can include breast cancer, precancer or precancerous conditions of the breast, benign growths or lesions of the breast, and metastatic lesions in tissues and organs of the body other than the breast. Cell proliferative disorders of the breast can include hyperplasia, metaplasia, and dysplasia of the breast.

[0401] As used herein, "cell proliferative diseases or disorders of the skin" includes all forms of cell proliferative disorders affecting skin cells. Cell proliferative disorders of the skin can include precancerous or precancerous conditions of the skin, benign growths or lesions of the skin, melanoma, malignant melanoma, or other malignant growths or lesions of the skin, as well as metastatic lesions in tissues and organs of the body other than the skin. Cell proliferative disorders of the skin can include hyperplasia, metaplasia, and dysplasia of the skin.

[0402] As used herein, "endometrial cell proliferative diseases or disorders" include all forms of cell proliferative disorders affecting cells of the endometrium. Endometrial cell proliferative disorders may include precancer or precancerous conditions of the endometrium, benign endometrial proliferations or lesions, endometrial cancer, and metastatic lesions in tissues and organs in the body other than the endometrium. Endometrial cell proliferative disorders may include endometrial hyperplasia, metaplasia, and dysplasia.

[0403] The compounds of the present invention and their pharmaceutically acceptable salts and stereoisomers can be administered to patients, e.g., cancer patients, as monotherapy or in combination therapy. The treatment can be a "front / first-line" treatment, i.e., as an initial treatment in patients who have not received a previous anticancer treatment regimen, alone or in combination with other treatments; a "second-line" treatment, i.e., as a treatment in patients who have received a previous anticancer treatment regimen, alone or in combination with other treatments; or a "third-line," "fourth-line," etc. treatment, alone or in combination with other treatments. The treatment can also be given to patients who have previously undergone unsuccessful or partially successful treatments but have become non-responsive or intolerant to the particular treatment. The treatment can also be given as an adjuvant treatment, i.e., to prevent cancer recurrence in patients who currently have no detectable disease or after surgical removal of a tumor. Thus, in some embodiments, the compounds can be administered to patients who have undergone previous therapy, such as chemotherapy, radioimmunotherapy, surgical therapy, immunotherapy, radiation therapy, targeted therapy, or any combination thereof.

[0404] The methods of the present invention may require administering a compound of the present invention or a pharmaceutical composition thereof to a patient in a single dose or multiple doses (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 10, 15, 20 or more doses). For example, the dosing frequency may range from once daily to about once every 8 weeks. In some embodiments, the dosing frequency ranges from about once daily for 1, 2, 3, 4, 5, or 6 weeks, and in other embodiments, involves at least one 28-day cycle comprising daily administration for 3 weeks (21 days) followed by a 7-day rest period. In other embodiments, the compound may be administered twice daily (BID) for 2.5 days (a total of 5 doses) or once daily (QD) for 2 days (a total of 2 doses). In other embodiments, the compound may be administered once daily (QD) for 5 days.

[0405] Medicine Kit The composition of the present invention can be assembled into a kit or pharmaceutical system.The kit or pharmaceutical system according to this aspect of the present invention comprises a carrier or package such as a box, a carton, a tube, etc., which has one or more containers such as a vial, a tube, an ampoule or a bottle sealed therein, and which contain the compound of the present invention or the pharmaceutical composition containing the compound and a pharmaceutically acceptable carrier, and wherein the compound and the carrier can be placed in the same or separate containers.The kit or pharmaceutical system of the present invention can also comprise printed instructions for using the compound and the composition.

[0406] These and other aspects of the present invention will be further understood by consideration of the following examples, which are intended to illustrate certain embodiments of the invention but are not intended to limit the scope of the invention as defined by the claims. [Example]

[0407] Example 1: General information, materials, and equipment.

[0408] General information All reactions were carried out in flame-dried round-bottom flasks under a positive nitrogen pressure unless otherwise noted. Gas-tight syringes with stainless steel needles or cannulas were used for the transfer of air- or moisture-sensitive liquids. Flash column chromatography was performed using granular silica gel (60 Å pore size, 40-63 μm, Silicycle). Analytical thin-layer chromatography (TLC) was performed using glass plates precoated with 0.25 mm silica gel impregnated with a fluorescent indicator (254 nm, Silicycle). TLC plates were visualized by short-wave ultraviolet light (254 nm) and / or exposure to an aqueous solution of potassium permanganate (KMnO). Organic solutions were concentrated at 20 °C on a rotary evaporator capable of achieving a minimum pressure of approximately 2 Torr unless otherwise noted. Room temperature is defined as 22.5 ± 2.5 °C. Reaction heating was performed using a UCON™ fluid heating bath.

[0409] Common Chemicals All solvents were purchased from Fisher Scientific or Sigma-Aldrich. Unless otherwise noted, chemical reagents were purchased from Fisher Scientific, Sigma-Aldrich, Alfa Aesar, Oakwood Chemical, Acros Organics, Combi-Blocks, or TCI America. CMA refers to an 80:18:2 v / v / v solution of chloroform:methanol (MeOH):ammonium hydroxide (28–30% ammonia solution). Chloroform used in the CMA solution and as a co-eluent in silica gel column chromatography was stabilized with 0.75% v / v ethanol. Chloroform used in all hydroamination reactions was stabilized with pentane.

[0410] General chemical equipment Proton nuclear magnetic resonance (NMR) measurements recorded on a 500 MHz Avance III spectrometer equipped with a multinuclear smart probe. 1 H NMR) spectra are reported in parts per million on the NMR spectrum. δ scale and NMR solvents (CDCl3: δ 7.24, CD3OD: δ 3.31 (CHD2OD), CD3CN: δ 1.94). Data are reported as follows: chemical shift [multiplicity (s = singlet, d = doublet, t = triplet, dd = double doublet, dt = double triplet, dq = double quartet, ddd = double double doublet, tt = triple triplet, td = triple doublet, tq = triple quartet, m = multiplet), coupling constants in Hertz, integrals, assignments]. Carbon-13 Nuclear Magnetic Resonance ( 13 C NMR) spectra are referenced to the carbon resonance of the solvent (CDCl3: δ 77.23, CD3OD: δ 49.15, CD3CN: δ 1.37). Fluorine-19 nuclear magnetic resonance ( 19F NMR was calibrated from the fluorine resonances of benzotrifluoride (CDCl: δ -62.76, CD3OD: δ -64.24, CD3CN: δ -63.22). Data are reported as follows: chemical shifts (assignments). Infrared data (IR) were obtained on a Cary 630 Fourier transform infrared spectrometer equipped with a diamond ATR objective and are reported as follows: absorption frequencies (cm -1 ), and the intensity of absorption (s = strong, m = medium, w = weak, br = broad). High-resolution mass spectra (HRMS) were recorded on a Q Exactive™ Plus Hybrid Quadrupole-Orbitrap™ mass spectrometer using electrospray ionization (ESI), atmospheric pressure ionization (API), or electron ionization (EI) sources. Automated C purification was performed using the Isolera™ One (Biotage®) purification system. 18 Reverse-phase chromatography was performed. High-performance liquid chromatography (HPLC) purification was performed using an Agilent 1260 Infinity system. In-gel fluorescence imaging was performed on a GE Healthcare Life Sciences Typhoon™ FLA 9500. Images were processed with Fiji ImageJ software.

[0411] General biological materials and methods All solvents and reagents were purchased from commercial suppliers and used as received. Deionized water (>18.2 μΩ) was used for the preparation of all aqueous buffers and solutions. Short oligonucleotide primers (<80 bp) were synthesized by MilliporeSigma (St. Louis, MO), and gene blocks (>80 bp) were synthesized by Twist Bioscience (South San Francisco, CA). Oligonucleotides were used as received without further desalting. Chemically competent E. coli DH5α and BL21(DE3) cells were purchased from New England Biolabs®. All plasmid isolations were performed using miniprep or midiprep kits from Zymo Research. DNA cleanup and concentration and DNA gel purification kits were purchased from Zymo Research. All enzymes used for standard restriction enzyme cloning (Q5® Hot Start DNA polymerase, restriction enzymes, T4 DNA ligase, and Antarctic phosphatase), NEBuilder® HiFi DNA Assembly Master Mix for Gibson Assembly® cloning, and the Q5® Mutagenesis Kit used to perform all site-directed mutagenesis reactions were purchased from New England Biolabs®. DNA sequencing services were performed by Quintarabio (Cambridge, MA). TransIT®-293 transfection reagent was purchased from Mirus Bio™.

[0412] General biological instruments All polymerase chain reactions (PCRs) were performed on a Bio-Rad Laboratories C1000 thermal cycler. Cells were lysed using a Fisherbrand™ Sonicator Model 505. Proteins were purified using a Bio-Rad NGC chromatography system. UV / vis absorbance measurements for protein A280 were acquired on an Agilent Technologies Cary 60 UV-Vis. In-gel fluorescence imaging was performed on a GE Healthcare Life Sciences Typhoon™ FLA 9500. Coomassie-stained gels were analyzed on a Bio-Rad Molecular Imager Gel Doc XR+ Imaging System.

[0413] Example 2: Bioorthogonal reactions of cycloalkynes

[0414] The reverse Cope elimination reaction 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)). Described below is the bioorthogonal reaction of N,N-dialkylhydroxylamines and cyclooctynes ​​to form stable enamine N-oxidoligation products in a rapid and regioselective manner using reactants containing as few as three non-hydrogen atoms.

[0415] The reverse Cope elimination reaction was evaluated using density functional theory calculations (Zhao et al., Theor. Chem. Acc. 120:215-241 (2008)) to confirm the activation barriers for the reaction of N,N-dimethylhydroxylamine with various cyclooctynes ​​(Figure 2A). The calculated activation energy for unmodified cyclooctyne was 18.9 kcal / mol, which was low enough for the reaction to proceed at room temperature. Also noteworthy was the absence of steric factors affecting the initial O··H··C2 bond in the transition state structure, suggesting the importance of steric ambivalence toward the propargyl substituent on the compatibility, mutual orthogonality, and reactivity of cyclooctynes.

[0416] Calculations on bicyclo[6.1.0]nonyne showed that additional strain could be utilized to the same effect as in the cycloaddition reaction (Figure 2C) (Dommerholt et al., Angew. Chem. Int. Ed. 49(49):9422-9425 (2010)). Instead, electronic accommodation of cyclooctyne (Baskin et al., Proc. Natl. Acad. Sci. USA 104(43):16793-16797 (2007); Agard et al., ACS Chem. Biol. 1(10):644-648 (2006)) proved more pronounced (Figure 2D). Further strain / interaction energy analysis showed a significant reduction in strain energy (Ess et al., Org. Lett. 10(8):1633-1636 (2008); Liu et al., Acc. Chem. Res. 50(9):2297-2308 (2017)). However, this was demonstrated in studies of cyclooctyne and its linear counterparts, where, unlike cycloadditions, this reaction did not benefit from an enhancement in interaction energy due to the addition of electronegative substituents; the rate acceleration was driven by the reduction in strain energy of both components. The counterintuitive increase in interaction energy was likely due to a reactant-oriented shift of the transition state in accordance with Hammond's postulate. The corresponding increase in the C1··N and C2··H bond lengths further supported this interpretation (Example 30).

[0417] Kinetic experiments confirmed the reactivity trends predicted by the calculations. Using NMR spectroscopy to monitor the reaction progress, the second-order rate constant for the reaction of N,N-diethylhydroxylamine (1) with a panel of cyclooctynes ​​2–10 in d3-acetonitrile at room temperature was determined (Figure 3). Cyclooctyne (2) proved to be remarkably reactive, with a kinetic constant of 3.25 × 10. -2 M -1 s -1 , an order of magnitude faster than the reaction with benzyl azide (Agard et al., J. Am. Chem. Soc. 126(46):15046-15047 (2004)). Further strain enhancement provided the predicted 6.7-fold rate acceleration for bicyclo[6.1.0]nonyne 3, but far from the 100-fold increase observed for the analogous azide-alkyne cycloaddition (Dommerholt et al., Angew. Chem. Int. Ed. 49(49):9422-9425 (2010)). Nevertheless, the 2.17 × 10 -1 M -1 s -1 This second-order rate constant compares favorably with the fastest azide-based reaction involving BARAC (Jewett et al., J. Am. Chem. Soc. 132(11):3688-3690 (2010)).

[0418] The hydroamination reaction of cyclooctyne was particularly sensitive to the inductive effect of the propargyl substituent, and a progressive rate enhancement was observed with increasing electronegativity (Figure 3). The most reactive of the cyclooctynol-derivative substrates was 3.87 M. -1 s -1Carbamate 9 was characterized by a rate constant of 0.05, a 120-fold improvement over cyclooctyne (2). Importantly, the minimal cyclooctyne-1-ol substructure proved versatile and easy to synthesize and derivatize; it was amenable to conjugation via ester, carbamate, or ketal linkages without incurring significant costs in size or reactivity. Indeed, elaborate functionalization of the core cyclooctyne proved not only unnecessary but sometimes detrimental. The reaction of N,N-diethylhydroxylamine with dibenzoazacyclooctyne 8 (DIBAC) (Debets et al., Chem. Commun. 46:97-99 (2010)) was rapid, but the reaction rate was still inferior to that of carbamate 9 and not appreciably superior to its milder counterpart. In particular, the ligation product of dibenzoazacyclooctyne 8 with N,N-diethylhydroxylamine was prone to decomposition and uniquely unstable to purification by both standard and reversed-phase flash chromatography.

[0419] Difluorocyclooctyne 10 was previously reported to act at the limit of bioorthogonality (Baskin et al., Proc. Natl. Acad. Sci. USA 104(43):16793-16797 (2007); Kim et al., Carbohydr. Res. 377:18-27 (2013)), providing a reasonable upper limit for the hydroamination kinetics that could be achieved using electronically tuned cyclooctynes ​​in a biological setting. Remarkably, competition experiments with cyclooctyne 9 revealed that its rate constant was 83.6 M -1 s -1 It was revealed that...

[0420] The reverse Cope elimination reaction was highly directed by substrate electronics and produced only a single observable regioisomer for cyclooctynes ​​2-7, 9, and 10. Thus, when symmetric N,N-dialkylhydroxylamines were used, a single product was selectively formed.

[0421] To test the bioorthogonality of the hydroamination reaction, in vitro protein labeling experiments were performed. Fluorophore-conjugated hydroxylamine 13 was first assembled from 6-carboxytetramethylrhodamine and hydroxylamine 12, which was then synthesized by nucleophilic substitution of iodide 11 with N-methylhydroxylamine hydrochloride (Figure 4A). Separately, lysozyme was functionalized with cyclooctyne via N-hydroxysuccinimide ester 14 (Figure 4B). With both reaction components in hand, cyclooctyne-functionalized lysozyme 15 was treated with hydroxylamine 13 (0-200 μM) in PBS for 2 h and analyzed by in-gel fluorescence (Figure 4C). Labeling occurred in a concentration-dependent manner, with saturation at 100 μM hydroxylamine. The reaction proceeded in a time-dependent manner (Figure 4D). Modified lysozyme 15 was treated with hydroxylamine 13 (200 μM) and quenched for various time points with N,N-diethylhydroxylamine (20 mM). In-gel fluorescence analysis revealed signal saturation by 1 h. The formation of the desired adducts on the protein was confirmed by mass spectrometry. Lysozyme 15 was incubated with hydroxylamine 13 (100 μM) in PBS, and the complete conversion of mono- and dicyclooctyne-functionalized lysozyme 15 to mono- and dienamine N-oxides 16 was confirmed by ESI-MS (Figure 4E).

[0422] The stability of both the enamine N-oxide and hydroxylamine species under various biologically relevant conditions was examined at various time points (Figure 5A-B). Hydroxylamine 13 was first incubated in PBS at room temperature, and HPLC analysis of the solution showed that the compound remained >86% intact for up to 8 hours. However, approximately 40% of the hydroxylamine had decomposed by the 24-hour time point. The primary degradation product was consistent with hydrolysis of the regioisomeric nitrone, likely generated by autoxidation. Therefore, this degradation pathway could be abrogated by the addition of 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 decomposition was observed over 24 hours. Hydroxylamine 13 was stable in HEK293T cell lysate (1 mg / mL) and showed no decomposition above background over 24 h, even when unbuffered with exogenous reducing agents.

[0423] Similar to the hydroxylamine, the stability of enamine N-oxide 17 was assessed by HPLC under biologically relevant conditions. It showed no evidence of decomposition over the course of 24 h at room temperature, alone or in the presence of 5 mM glutathione in PBS. Furthermore, N-oxides undergo heme protein-dependent reduction under hypoxic conditions, a process that is fully inhibited by aerobic conditions (Raleigh et al., Int. J. Radiat. Oncol. Biol. Phys. 42(4):763-767 (1998)). Incubation of enamine N-oxide 17 with human liver microsomes (0.2 mg / mL) under ambient air resulted in negligible decomposition after 24 h.

[0424] To further demonstrate the bioorthogonality of the reaction, TAMRA-hydroxylamine 13 and lysozyme-COT 15 were combined for 2 h in the presence and absence of HEK293T cell lysate in PBS (Figure 5C). In-gel fluorescence showed that lysozyme was exclusively labeled, and the degree of labeling was not perturbed by the presence of lysate. Under these conditions, there appeared to be no cross-reactivity between dialkylhydroxylamines and other proteins.

[0425] Finally, we explored the cross-compatibility of this reaction with other bioorthogonal systems to identify mutually orthogonal substrate combinations that could be used in tandem (Figure 5D) (Patterson et al., Curr. Opin. Chem. Biol. 28:141-149 (2015)). Tetrazines were first evaluated to see whether they would be compatible with the sterically congested cyclooctynes, which feature tetra-substitution at the propargyl position. Indeed, when cyclooctyne ketal 5 and tetrazine 18 were mixed in d3-acetonitrile at a concentration of 5 mM for 1 hour, no product was detected. To determine whether steric hindrance imposed by the fully substituted carbon (Liu et al., J. Am. Chem. Soc. 136(32):11483-11493 (2014)) or the electronic nature of the ketal were primarily responsible for the inhibition of the inverse electron-demand cycloaddition, electron-deficient cyclooctynes ​​9 and 10 were evaluated for similar effects under the same conditions. Electronic factors, alone or in combination with steric factors, make the two reactions orthogonal. Hydroxylamine reagents were evaluated to determine their compatibility with strained alkenes. 5 mM N,N-diethylhydroxylamine (1) (Patterson et al., J. Am. Chem. Soc. 134(45):18638-18643 (2012)) or trans-cyclooctene 19 (Blackman et al., J. Am. Chem. Soc. 130(41):13518-13519 (2008)) in combination with 5 mM cyclopropene 21 was found to be unreactive in d3-acetonitrile. N,N-dialkylhydroxylamines do not react with aldehydes and do not participate in copper-catalyzed azide-alkyne cycloaddition (Example 29) (Hein et al., Chem. Soc. Rev. 39:1302-1315 (2010)).

[0426] We identified a novel bioorthogonal ligation reaction between N,N-dialkylhydroxylamines and cyclooctynes. -1 s -1The reaction was characterized by rapid reaction kinetics with high second-order rate constants, exquisite regioselectivity, and small reactant components. N,N-Dialkylhydroxylamine reagents could reduce the number of non-hydrogen atoms to as few as three, and cyclooctynes ​​were highly effective, even when unfunctionalized. Cyclooctynes ​​could be conveniently attached to their propargyl positions without compromising reactivity. The hydroxylamine reagent and enamine N-oxide product were sufficiently stable under aqueous conditions in the presence of thiols or components of the cellular environment found in cell lysates, particularly on timescales germane to the ligation of small molecules to biomolecules. However, both components have their sensitivities: hydroxylamines to air, and enamine N-oxides to oxygen-deprived microsomes. Factors that mitigate these processes were identified, ensuring the bioorthogonality of the reaction.

[0427] Example 3: Synthesis of (E)-(cyclooct-1-en-1-yloxy)trimethylsilane [ka]

[0428] A round-bottom flask was sequentially charged with tetrahydrofuran (THF, 200 mL) and a solution of lithium bis(trimethylsilyl)amide (1 M in THF, 52.3 mL, 52.3 mmol), then cooled to −78 °C. A solution of cyclooctanone S1 (6.00 g, 47.5 mmol) in THF (200 mL) was added to this solution via cannula over 20 min at −78 °C. After 1.5 h, chlorotrimethylsilane (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 h, the reaction was quenched with saturated aqueous ammonium chloride (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.

[0429] Example 4: Synthesis of 2-((trimethylsilyl)oxy)cyclooctan-1-one (S3) [ka]

[0430] A round-bottom flask was charged with crude S2 (47.5 mmol) from the previous step and dissolved in dichloromethane (DCM, 200 mL). A solution of dimethyldioxirane (DMDO, 0.11 M in acetone, 595 mL, 60.0 mmol) was added to the solution at room temperature. After 15 min, the reaction mixture was concentrated and azeotroped with MeOH (2 × 200 mL). The resulting oil was dissolved in DCM (500 mL). To this solution were added 4-dimethylaminopyridine (DMAP, 581 mg, 4.75 mmol), trimethylamine (9.94 mL, 71.3 mmol), and chlorotrimethylsilane (7.24 mL, 57.1 mmol) sequentially at room temperature. After 2.5 h, the reaction mixture was washed with aqueous HCl (1 N, 500 mL), the organic layer was separated, and the aqueous layer was extracted with DCM (75 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude mixture was purified by flash column chromatography on silica gel (eluent: 5% ethyl acetate in hexanes) to give ketone S3 (8.35 g, 82% over three steps). 1 H NMR (500 MHz, CDCl3, 25℃): δ 4.15 (dd, J = 6.9, 3.4 Hz, 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). 13 C 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): calc'd for C 11 H 23 O2Si [M+H] + : 215.1467, found: 215.1463. TLC (5% ethyl acetate in hexanes), Rf: 0.70 (I2).

[0431] Example 5: Synthesis of (E)-8-((trimethylsilyl)oxy)cyclooct-1-en-1-yl trifluoromethanesulfonate (S4) [ka]

[0432] A round-bottom flask was charged with cyclooctanone S3 (2.06 g, 9.61 mmol) and THF (100 mL) in that order and then cooled to -78 °C. A solution of lithium bis(trimethylsilyl)amide (1 M in THF, 11.5 mL, 11.5 mmol) was added to the mixture via cannula at -78 °C. After 1 h, N-(5-chloro-2-pyridyl)bis(trifluoromethanesulfonimide) (4.15 g, 10.6 mmol) was added, and the dry ice bath was removed. After 2 h, the reaction mixture was diluted with hexane (200 mL) and washed sequentially with aqueous sodium hydroxide (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 flash column chromatography on silica gel (eluent: 7.5% DCM in hexanes) to give vinyl triflate S4 (3.1 g, 95%) as a colorless oil. 1H NMR (500 MHz, CDCl3, 25℃): δ 5.68 (t, J = 9.0 Hz, 1H), 4.66 (dd, J = 10.3, 5.4 Hz, 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 (126 MHz, CDCl3, 25°C): δ 150.6, 118.8 (q, J = 319.6 Hz), 120.0, 67.3, 37.0, 29.9, 26.2, 24.8, 23.6, -0.1. 19 F NMR (471 MHz, CDCl3, 25℃): δ -75.2. FTIR (thin film) cm -1 : 2933 (w), 1416 (m), 1200 (s), 1144 (m), 932 (m), 839 (s). HRMS (ESI) (m / z): calc'd for C 12 H 21 F3NaO4SSi [M+Na] + : 369.0774, found: 369.0776. TLC (100% hexanes), Rf: 0.42 (I2).

[0433] Example 6: Synthesis of シクロオクト-2-イン-1-オール(4)

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[0434] A round-bottom flask was charged sequentially with vinyl triflate S4 (3.03 g, 8.75 mmol) and THF (88 mL), then cooled to -78 °C. A solution of lithium diisopropylamide (2.0 M in THF / heptane / ethylbenzene, 8.75 mL, 17.5 mmol) was then added to the solution via syringe. The dry ice bath was removed, and the solution was allowed to warm to room temperature. After 2.5 h, tetrabutylammonium fluoride (1 M in THF, 17.5 mL, 17.5 mmol) was added to the reaction mixture via syringe. After 1 h, the reaction mixture was diluted with hexane (100 mL) and washed with saturated aqueous ammonium chloride (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 flash column chromatography on silica gel (eluent: 100% DCM) to give cyclooctynol 4 (566 mg, 52%) as a clear, colorless oil. The physical properties and spectral data were identical to those reported in the literature (Hagendorn, T., Eur. J. Org. Chem. 2014(6):1280-1286 (2014)). TLC (100% DCM), Rf: 0.31 (KMnO4).

[0435] Example 7: Synthesis of (E)-8-oxocyclooct-1-en-1-yl trifluoromethanesulfonate (S5) [ka]

[0436] A round-bottom flask was charged sequentially with vinyl triflate S4 (94.3 mg, 272 μmol) and DCM (1.4 mL). Trifluoroacetic acid (600 μL) was added to the solution at room temperature. After 30 min, 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 Dess-Martin periodinane (DMP, 231 mg, 544 μmol) were added sequentially at room temperature. After 30 min, the reaction mixture was diluted with hexane (2 mL) and purified by flash column chromatography on silica gel (eluent: 15% ethyl acetate in hexane) to give cyclooctenone S5 (64.1 mg, 87%) as a clear film. 1 H NMR (500 MHz, CDCl3, 25℃): δ 6.58 (t, J = 9.2 Hz, 1H), 2.85 (t, J = 7.3 Hz, 2H), 2.70 (dt, J = 9.3, 7.0 Hz, 2H), 1.84-1.75 (m, 2H), 1.74-1.68 (m, 2H), 1.61-1.54 (m, 2H). 13 C NMR (126 MHz, CDCl3, 25°C): δ 192.8, 149.7, 133.6, 118.8 (q, J = 320.1 Hz), 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 : 2937 (w), 1685 (m), 1416 (s), 1200 (s), 1141 (s), 1062 (s), 969 (s). HRMS (ESI) (m / z): calc'd for C9H 12 F3O4S [M+H] + : 273.0403, found: 273.0402. TLC (15% ethyl acetate in hexanes), Rf: 0.30 (KMnO4).

[0437] Example 8: Synthesis of (E)-1,4-dioxaspiro[4.7]dodec-6-en-6-yl trifluoromethanesulfonate (S6) [ka]

[0438] A round-bottom flask was charged with cyclooctenone S5 (150 mg, 551 μmol), ethylene glycol (302 μL, 5.51 mmol), and benzene (10 mL) at room temperature. p-Toluenesulfonic acid monohydrate (10.5 mg, 55.1 μmol) was then added to the solution. The flask was equipped with a Dean-Stark trap and a reflux condenser, and the reaction mixture was heated to reflux. After 23 h, the reaction mixture was cooled to room temperature and diluted with hexane. The crude mixture was purified by flash column chromatography on silica gel (eluent: 5% ethyl acetate in hexane) to afford ketal S6 (125 mg, 71%) as a clear, colorless oil. 1 H NMR (500 MHz, CDCl3, 25℃): δ 5.75 (t, J = 9.5 Hz, 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 (126 MHz, CDCl3, 25°C): δ 150.1, 123.0, 118.7 (q, J = 319.5 Hz), 107.3, 65.7, 37.7, 27.2, 23.1, 22.7, 21.9. 19 F NMR (471 MHz, CDCl3, 25℃): δ -75.4. FTIR (thin film) cm -1 : 2930 (w), 1409 (s), 1245 (w), 1200 (s), 1141 (s), 977 (s). HRMS (ESI) (m / z): calc'd for C 11 H 16 F3O5S [M+H] + : 317.0665, found: 317.0664.

[0439] Example 9: Synthesis of 1,4-dioxaspiro[4.7]dodeca-6-yne (5) [ka]

[0440] A round-bottom flask was charged sequentially with ketal S6 (70.1 mg, 222 μmol) and THF (4 mL), then cooled to −78 °C. A solution of lithium diisopropylamide (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 allowed to warm to room temperature. After 2.5 h, the solution was cooled to −78 °C, and additional lithium diisopropylamide solution (2 M in THF / heptane / ethylbenzene, 111 μL, 222 μmol) was added via syringe. The dry ice bath was removed, and the solution was allowed to warm to room temperature. After 1.5 h, the reaction was quenched with MeOH (1.0 mL) and concentrated under reduced pressure. The crude mixture was purified by flash column chromatography on silica gel (eluent: 5% ethyl acetate in hexane) to afford cyclooctyne 5 (25.5 mg, 69%) as a clear film. 1 H NMR (500 MHz, CDCl3, 25℃): δ 3.98-3.84 (m, 4H), 2.22 (t, J = 6.4 Hz, 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 C NMR (126 MHz, 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): calc'd for C 10 H 15 O2[M+H]+ : 167.1067, found: 167.1067. TLC (5% ethyl acetate in hexanes) Rf: 0.38 (KMnO4).

[0441] Example 10: Synthesis of cyclooct-2-yn-1-yl acetate (6) [ka]

[0442] A round-bottom flask was charged sequentially with cyclooctynol 4 (80.5 mg, 648 μmol), 4-dimethylaminopyridine (6.3 mg, 51.9 μmol), and DCM (3.0 mL) at room temperature. The solution was then cooled to 0 °C in an ice-water bath, and pyridine (261 μL, 3.24 mmol) was added dropwise to the solution. Acetic anhydride (73.5 μL, 778 μmol) was then added dropwise to the solution. The ice bath was removed, and the solution was allowed to warm to room temperature. After 16 h, the reaction was quenched with saturated aqueous ammonium chloride (1 mL) and 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 flash column chromatography on silica gel (eluent: 50% DCM in hexane) to give cyclooctyne 6 (95.5 mg, 87%) as a clear, colorless oil. 1 H NMR (500 MHz, CDCl3, 25℃): δ 5.34-5.26 (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 C NMR (126 MHz, 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): calc'd for C 10 H 15 O2[M+H] + : 167.1067, found: 167.1068. TLC (100% CH2Cl2), Rf: 0.57 (I2).

[0443] Example 11: Synthesis of 3-fluorocyclooct-1-yne (7) [ka]

[0444] A round-bottom flask was charged sequentially with cyclooctynol 4 (40.8 mg, 329 μmol) and DCM (3.0 mL), then cooled to 0 °C. Diethylaminosulfur trifluoride (DAST, 45.6 μL, 345 μmol) was then added to the solution via syringe. After 1 h, the reaction mixture was concentrated under reduced pressure. The crude mixture was purified by flash column chromatography on silica gel (eluent: 100% pentane) to afford fluorocyclooctyne 7 (21.0 mg, 51%) as a clear, colorless oil. 1 H NMR (500 MHz, CDCl3, 25℃): δ 5.12 (dt, J = 50.5, 5.1 Hz, 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 C NMR (126 MHz, CDCl3, 25℃): δ 104.9 (d, J = 10.5 Hz), 90.5 (d, J = 30.0 Hz), 84.7 (d, J = 171.2 Hz), 43.0 (d, J = 22.9 Hz), 34.3 (d, J = 1.9 Hz), 29.6, 25.5 (d, J = 2.9 Hz), 20.9 (d, J = 2.9 Hz). 19F NMR (471 MHz, CDCl3, 25℃): 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): calc'd for C8H 12 F [M+H] + : 127.0918, found: 127.0916. TLC (100% pentane), Rf: 0.26 (KMnO4).

[0445] Example 12: Synthesis of cyclooct-2-yn-1-yl(4-nitrophenyl)carbamate (9) [ka]

[0446] A round-bottom flask was charged with cyclooctynol 4 (10.8 mg, 87.0 μmol) and DCM (1 mL) sequentially. To this solution, 1-isocyanato-4-nitrobenzene (14.3 mg, 87.0 μmol) and trimethylamine (1.2 μL, 8.70 μmol) were added at room temperature. After 100 min, the reaction mixture was diluted with hexane. The crude mixture was purified by flash column chromatography on silica gel (eluent: 30% diethyl ether in hexane) to give carbamate 9 (16.7 mg, 67%) as a white solid. 1 H NMR (500 MHz, CD3CN, 25℃): δ 8.26 (s, 1H), 8.16 (d, J = 9.3 Hz, 2H), 7.62 (d, J = 9.3 Hz, 2H), 5.32 (tq, J = 5.1, 2.2 Hz, 1H), 2.35-2.24 (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). 13C NMR (126 MHz, 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): calc'd for C 15 H 17 N2O4[M+H] + : 289.1183, found: 289.1188. TLC (50% DCM in hexanes), Rf: 0.23 (KMnO4).

[0447] Example 13: Synthesis of (E)-N,N-diethylcyclooct-1-ene-1-amine oxide (S7) [ka]

[0448] N,N-Diethylhydroxylamine (28.4 μL, 276 μmol) was added via syringe to a solution of cyclooctyne 2 (Fairbanks et al., Macromolecules 43(9):4113-4119 (2010)) (19.9 mg, 184 μmol) in acetonitrile (1.8 mL) at room temperature. After 10 min, the reaction mixture was concentrated under reduced pressure. The crude mixture was purified by flash column chromatography on silica gel (eluent: 15 → 30% CMA in chloroform) to give enamine N-oxide S7 (36.0 mg, 99%) as a clear film. 1H NMR (500 MHz, CDCl3, 25℃): δ 6.65 (t, J = 8.8 Hz, 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 = 7.1 Hz, 6H). 13 C NMR (126 MHz, CDCl3, 25°C): 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): calc'd for C 12 H 24 NO [M+H] + : 198.1852, found: 198.1853. TLC (50% CMA in chloroform), Rf: 0.38 (KMnO4).

[0449] Example 14: Synthesis of (1R,8S,9S,E)-N,N-diethyl-9-(hydroxymethyl)bicyclo[6.1.0]non-4-ene-4-amine oxide (S8) [ka]

[0450] N,N-Diethylhydroxylamine (30.8 μL, 300 μmol) was added via syringe to a solution of cyclooctyne 3 (30.0 mg, 200 μmol) in MeOH (500 μL) and acetonitrile (2.0 mL) at room temperature. After 10 min, the reaction mixture was concentrated under reduced pressure. The crude mixture was purified by flash column chromatography on silica gel (eluent: 20→40% CMA in chloroform) to afford the enamine N-oxide S8 (45.6 mg, 95%) as a clear, colorless oil. 1H NMR (500 MHz, CD3OD, 25℃): δ 6.65 (t, J = 8.4 Hz, 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, 5.9 Hz, 1H), 2.55-2.40 (m, 2H), 2.26-2.12 (m, 2H), 2.15-2.02 (m, 1H), 1.74-1.57 (m, 2H), 1.21 (td, J = 7.1, 2.5 Hz, 6H), 1.19-1.10 (m, 1H), 1.11-1.01 (m, 2H). 13 C NMR (126 MHz, CD3OD, 25°C): 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): calc'd for C 14 H 26 NO2[M+H] + : 240.1958, found: 240.1957. TLC (50% CMA in chloroform), Rf: 0.16 (KMnO4).

[0451] Example 15: Synthesis of (E)-N,N-diethyl-3-hydroxychlorooct-1-ene-1-amine oxide (S9) [ka]

[0452] N,N-Diethylhydroxylamine (30.8 μL, 300 μmol) was added via syringe to a solution of cyclooctyne 3 (24.8 mg, 200 μmol) in acetonitrile (2.0 mL) at room temperature. After 5 min, the reaction mixture was concentrated under reduced pressure. The crude mixture was purified by flash column chromatography on silica gel (eluent: 20→40% CMA in chloroform) to afford enamine N-oxide S9 (35.4 mg, 83%) as a clear, colorless oil. 1 H NMR (500 MHz, CD3OD, 25℃): δ 6.40 (d, J = 7.3 Hz, 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 = 7.1 Hz, 3H), 1.18 (t, J = 7.1 Hz, 3H). 13 C NMR (126 MHz, CD3OD, 25°C): 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): calc'd for C 12 H 24 NO2[M+H] + : 214.1802, found: 214.1801. TLC (50% CMA in CHCl3), Rf: 0.14 (KMnO4).

[0453] Example 16: Synthesis of (E)-N,N-diethyl-1,4-dioxaspiro[4.7]dodec-6-ene-7-amine oxide (S10) [ka]

[0454] N,N-Diethylhydroxylamine (17.0 μL, 165 μmol) was added via syringe to a solution of cyclooctyne 7 (18.3 mg, 110 μmol) in acetonitrile (1.0 mL) at room temperature. After 10 min, the reaction mixture was concentrated under reduced pressure. The crude mixture was purified by flash column chromatography on silica gel (eluent: 15→30% CMA in chloroform) to afford the enamine N-oxide S10 (25.6 mg, 91%) as a clear film. 1 H NMR (500 MHz, CD3OD, 25℃): δ 6.67 (s, 1H), 3.99-3.88 (m, 4H), 3.55-3.33 (m, 4H), 2.78 (t, J = 6.7 Hz, 2H), 2.01-1.87 (m, 2H), 1.80-1.61 (m, 6H), 1.21 (t, J = 7.1 Hz, 6H). 13 C NMR (126 MHz, CD3OD, 25°C): 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): calc'd for C 14 H 26 NO3[M+H] + : 256.1907, found: 256.1906. TLC (50% CMA in chloroform), Rf: 0.35 (KMnO4).

[0455] Example 17: Synthesis of (E)-3-acetoxy-N,N-diethylcyclooct-1-ene-1-amine oxide (S11) [ka]

[0456] 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) at room temperature. After 5 min, the reaction mixture was concentrated under reduced pressure. The crude mixture was purified by flash column chromatography on silica gel (eluent: 10→30% CMA in chloroform) to afford the enamine N-oxide S11 (46.1 mg, 90%) as a clear, colorless oil. 1 H NMR (500 MHz, CD3OD, 25℃): δ 6.40 (d, J = 7.8 Hz, 1H), 5.47 (ddd, J = 11.7, 7.7, 4.8 Hz, 1H), 3.62 (dq, J = 12.5, 7.1 Hz, 1H), 3.48 (dq, J = 12.4, 7.2 Hz, 1H), 3.42-3.28 (m, 2H), 2.58-2.45 (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 = 7.1 Hz, 3H), 1.12 (t, J = 7.1 Hz, 3H). 13 C NMR (126 MHz, 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): calc'd for C 14 H 26 NO3[M+H] +: 256.1907, found: 256.1905. TLC (50% CMA in chlorofrom), Rf: 0.16 (KMnO4).

[0457] Example 18: Synthesis of (E)-N,N-diethyl-3-fluorocyclooct-1-ene-1-amine oxide (S12) [ka]

[0458] 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) at room temperature. After 10 min, the reaction mixture was concentrated under reduced pressure. The crude mixture was purified by flash column chromatography on silica gel (eluent: 15→30% CMA in chloroform) to afford the enamine N-oxide S12 (8.3 mg, 88%) as a clear film. 1 H NMR (500 MHz, CD3OD, 25℃): δ 6.64 (dd, J = 19.8, 6.5 Hz, 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 = 7.1 Hz, 3H), 1.17 (t, J = 7.1 Hz, 3H). 13 C NMR (126 MHz, CD3OD, 25℃): δ 147.34 (d, J = 13.4 Hz), 128.83 (d, J = 33.4 Hz), 92.09 (d, J = 161.6 Hz), 63.69, 62.13, 36.82 (d, J = 21.9 Hz), 30.47, 26.87, 26.60, 23.98 (d, J = 12.9 Hz), 8.99, 8.85. 19F NMR (471 MHz, CD3OD, 25℃): 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): calc'd for C 12 H 23 FNO [M+H] + : 216.1758, found: 216.1758. TLC (30% CMA in chloroform), Rf: 0.13 (KMnO4).

[0459] Example 19: Synthesis of (E)-N,N-diethyl-3-(((4-nitrophenyl)carbamoyl)oxy)cyclooct-1-ene-1-amine oxide (S13) [ka]

[0460] 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) at room temperature. After 10 min, the reaction mixture was concentrated under reduced pressure. The crude mixture was purified by flash column chromatography on silica gel (eluent: 30% CMA in chloroform) to afford the enamine N-oxide S13 (16.3 mg, 94%) as a clear film. 1H NMR (500 MHz, CD3OD, 25℃): δ 8.16 (d, J = 9.3 Hz, 2H), 7.63 (d, J = 9.3 Hz, 2H), 6.53 (d, J = 7.6 Hz, 1H), 5.53 (ddd, J = 11.9, 7.7, 4.9 Hz, 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 = 7.1 Hz, 3H), 1.15 (t, J = 7.2 Hz, 3H). 13 C NMR (126 MHz, 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): calc'd for C 19 H 28 N3O5[M+H] + : 378.2023, found: 378.2021. TLC (30% CMA in chloroform), Rf: 0.26 (KMnO4).

[0461] Example 20: Synthesis of (E)-N,N-diethyl-3,3-difluorocyclooct-1-ene-1-amine oxide (S14) [ka]

[0462] 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) at room temperature. After 5 min, the reaction mixture was concentrated under reduced pressure. The crude mixture was purified by flash column chromatography on silica gel (eluent: 10 → 30% CMA in chloroform) to afford the enamine N-oxide S14 (43.8 mg, 94%) as a clear film. 1 H NMR (500 MHz, CD3OD, 25℃): δ 7.02 (t, J = 11.7 Hz, 1H), 3.61-3.50 (m, 2H), 3.48-3.36 (m, 2H), 2.73 (t, J = 6.9 Hz, 2H), 2.27 (tt, J = 15.6, 6.5 Hz, 2H), 1.85-1.61 (m, 6H), 1.21 (t, J = 7.1 Hz, 6H). 13 C NMR (126 MHz, CD3OD, 25℃): δ 151.9 (t, J = 11.7 Hz), 126.8 (t, J = 35.0 Hz), 123.7 (t, J = 233.7 Hz), 63.3, 38.2, 28.3, 24.9, 24.5, 22.2, 8.8. 19 F NMR (471 MHz, CD3OD, 25℃): δ -83.4. FTIR (thin film) cm -1 : 3232 (br), 2937 (m), 1692 (m), 1457 (m), 1316 (m), 988 (s). HRMS (ESI) (m / z): calc'd for C 12 H 22 F2NO [M+H] + : 234.1664, found: 234.1664. TLC (30% CMA in chloroform), Rf: 0.059 (KMnO4).

[0463] Example 21: Synthesis of tert-butyl (2-(2-(hydroxy(methyl)amino)ethoxy)ethyl)carbamate (12) [ka]

[0464] Triethylamine (1.34 mL, 9.59 mmol) was added to a solution of 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) in dimethyl sulfoxide (2.4 mL) at room temperature. The reaction mixture was heated to 70 °C. After 1.5 h, the solution was cooled to room temperature, diluted with water, and purified by automatic C. 18 Reverse phase column chromatography (30g C 18 Purification on silica gel, 25 μm spherical particles, eluent: H2O + 0.1% TFA (2 CV), gradient 0 → 100% CH3CN / H2O + 0.1% TFA (10-15 CV) gave hydroxylamine 12 (348 mg, 62%) as a white solid. 1 H NMR (500 MHz, CDCl3, 25℃) δ 3.82 (ddd, J = 11.1, 7.3, 3.9 Hz, 1H), 3.63 (dt, J = 11.0, 4.2 Hz, 1H), 3.52-3.35 (m, 4H), 3.32-3.18 (m, 2H), 3.07 (s, 3H), 1.38 (s, 9H). 13 C NMR (126 MHz, CDCl3, 25℃) δ 164.1 (q, J = 37.5 Hz), 156.7, 116.5 (q, J = 289.2 Hz), 79.5, 70.9, 63.6, 60.2, 46.5, 40.4, 28.5. 19 F NMR (471 MHz, 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): calc'd for C 10 H 23 N2O4[M+H] + : 235.1652, found: 235.1650. TLC (40% CMA in chloroform), Rf: 0.58 (I2).

[0465] Example 22: Synthesis of 3',6'-bis(dimethylamino)-N-(2-(2-(hydroxy(methyl)amino)ethoxy)ethyl)-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-6-carboxamide (13) [ka]

[0466] 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-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU, 29.5 mg, 77.7 μmol) in N,N-dimethylformamide (DMF, 700 μL) at room temperature. 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 h and then concentrated under reduced pressure. The resulting residue was dissolved in N,N-dimethylformamide (500 μL) and added to the reaction mixture containing 6-TAMRA using a pipette. Using additional N,N-dimethylformamide (200 μL), the hydroxylamine solution was quantitatively transferred to the reaction mixture. The reaction mixture was stirred at room temperature for 4 hours. Additional portions of HATU (29.5 mg, 77.7 μmol) and DIPEA (49.2 μL, 282 μmol) were added to the reaction mixture. The solution was then stirred for an additional 2.5 hours. The resulting mixture was diluted with water and purified using an automatic C 18 Reverse phase column chromatography (30g C 18 Purification by silica gel, 25 μm spherical particles, eluent: HO + 0.1% TFA (2 CV), gradient 0 → 100% CHCN / HO + 0.1% TFA (10–15 CV) and flash column chromatography on silica gel (eluent: 70% CMA in CHCl) gave TAMRA-hydroxylamine 13 (22.8 mg, 59%) as a purple solid. 1H NMR (500 MHz, D2O, 25℃) δ 7.99 (d, J = 8.3 Hz, 1H), 7.87 (d, J = 8.1 Hz, 1H), 7.67-7.59 (m, 1H), 7.07 (d, J = 9.5 Hz, 2H), 6.73 (dd, J = 9.5, 2.4 Hz, 2H), 6.41 (d, J = 2.3 Hz, 2H), 3.84-3.63 (m, 4H), 3.53 (t, J = 5.4 Hz, 2H), 3.11-2.89 (m, 14H), 2.71 (s, 3H). 13 C NMR (126 MHz, 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): calc'd for C 30 H 35 N4O6[M+H] + : 547.2551, found: 547.2544. TLC (100% CMA), Rf: 0.37 (visual).

[0467] Example 23: Synthesis of 3-(((cyclooct-2-yn-1-yloxy)carbonyl)amino)propanoic acid (S16) [ka]

[0468] 3-Aminopropanoic acid (18.5 mg, 207 μmol) was added to a solution of carbonate S15 (Plass, et al., Angew. Chem., Int. Ed. 50(17):3878-3881 (2011)) (50.0 mg, 173 μmol) in MeOH (2.0 mL) at room temperature. N,N-Diisopropylethylamine (90.3 μL, 519 μmol) was then added to the solution. After 1 h, the reaction mixture was concentrated under reduced pressure. The crude mixture was purified by flash column chromatography on silica gel (eluent: hexane / ethyl acetate / acetic acid, v / v / v = 65:30:5) to give carbamate S16 (40.4 mg, 98%) as a clear, colorless oil. 1 H NMR (500 MHz, CD3OD, 25℃): δ 5.23-5.10 (m, 1H), 3.37-3.31 (m, 2H), 2.49 (t, J = 6.9 Hz, 3H), 2.29-2.20 (m, 1H), 2.21-2.07 (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 C NMR (126 MHz, CD3OD, 25℃): δ175.5, 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): calc'd for C 12 H 18 NO4[M+H] + : 240.1230, found: 240.1229. TLC (5% MeOH in DCM + 0.1% acetic acid), Rf: 0.19 (I2).

[0469] Example 24: Synthesis of 2,5-dioxopyrrolidin-1-yl 3-(((cyclooct-2-yn-1-yloxy)carbonyl)amino)propanoate (14) [ka]

[0470] To a solution of carboxylic acid S16 (18.3 mg, 76.5 μmol) in DCM (1.0 mL) was added 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) sequentially at room temperature. After 12 h, additional NHS (44.0 mg, 382 μmol) and EDC·HCl (73.4 mg, 382 μmol) were added to the reaction mixture. After 4 h, the reaction mixture was concentrated under reduced pressure. The crude mixture was purified by flash column chromatography on silica gel (eluent: 30% acetone in hexane) to give NHS-ester 14 (7.1 mg, 28%) as a clear, colorless oil. 1 H NMR (500 MHz, CDCl3, 25℃): δ 5.45-5.19 (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 C NMR (126 MHz, CDCl3, 25f℃): 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): calc'd for C 16 H 21 N2O6[M+H] + : 337.1394, found: 337.1391. TLC (50% ethyl acetate in hexanes), Rf: 0.25 (I2).

[0471] Example 25: Kinetic studies

[0472] All kinetic experiments were carried out in CD3CN at room temperature. Reactions were monitored by NMR spectroscopy using an internal standard. Second-order kinetics was performed by combining cyclooctyne with N,N-diethylhydroxylamine in a 1:1 ratio. Table 1 shows the experimental conditions for each cyclooctyne. Reported errors for rate constants are based on the standard deviation of the mean for triplicate experiments. Table 1. Kinetic studies of cyclooctyne [Table 1]

[0473] The second-order rate constant of difluorocyclooctyne 10 was determined using a competition experiment with carbamate 9. N,N-diethylhydroxylamine (1 equivalent; final concentration 1.9 mM) was added to a solution containing a 1:4 ratio of difluorocyclooctyne 10 (5 equivalents; final concentration 9.5 mM) and cyclooctyne carbamate 9 (20 equivalents; final concentration 38 mM) in CHCN at room temperature (Figure 7). The solution was transferred to an NMR tube and analyzed using 1,3,5-trimethoxybenzene as an internal standard. 1 The product ratio (S14:S13) was determined by H NMR spectroscopy. The second-order rate constant (k2) for difluorocyclooctyne 10 was calculated by multiplying the observed product ratio by the second-order rate constant (k2) for carbamate 9, yielding a value of 83.6 ± 14.9 M. -1 s -1The reported errors for the rate constants are the standard deviation of the mean for experiments performed in triplicate.

[0474] Example 26: Protein labeling experiments

[0475] Synthesis of lysozyme-COT15 Lysozyme (CAS 12650-88-3, 50 mg / mL in deionized HO) was diluted in phosphate-buffered saline (PBS, pH 7.4) to a final concentration of 10 mg / mL. A solution of cyclooctyne NHS-ester 13 (65 μL, 8.5 mM in DMSO) and DMSO (10 μL) was added to the lysozyme solution (250 μL, 10 mg / mL). The reaction solution was incubated at room temperature for 1 h. Excess cyclooctyne NHS-ester 13 was removed by spin filtration (3 kDa MWCO, 5 × 1:5 dilution). The concentration of lysozyme was measured by UV-vis spectrophotometry using A in denaturing buffer (pH 7.0, 6 M guanidinium, 30 mM MOPS). 280 The concentration of ATP was determined by measurement. For labeling experiments, the solution was diluted with PBS (pH 7.4) to a final concentration of 0.15 mg / mL or 0.60 mg / mL. The protein solution was flash frozen under liquid nitrogen and stored at -20°C.

[0476] Concentration-dependent protein labeling experiments A solution of lysozyme-COT15 (5.0 μL, 0.15 mg / mL) was dispensed into six samples. An aqueous solution of hydroxylamine 13 (0.21 μL; 0.25, 0.625, 1.25, 2.5, and 5 mM in deionized water; final concentrations of 10, 25, 50, 100, and 200 μM) was added to each of five aliquot samples. As a vehicle control, deionized water (0.21 μL) was added instead of hydroxylamine to one sample. In control samples requiring conditions without lysozyme-COT15, unmodified lysozyme was treated with hydroxylamine 13 (0.21 μL, 5 mM in water; 200 μM final concentration) or deionized water (0.21 μL). The reaction mixture was incubated in the dark at room temperature for 2 h. The reaction mixture was quenched with 5x sodium dodecyl sulfate (SDS) sample loading buffer (1.30 μL). Each solution (5 μL) was loaded onto a 15-well 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) gel. The gel was run at room temperature and 175 V for 50 minutes. In-gel fluorescence was imaged with a Typhoon™ FLA 9500 (GE) at 532 nM using a photomultiplier tube (PMT) setting of 500 V. Experiments were performed in triplicate (Figure 8).

[0477] Time-dependent protein labeling experiments A solution of lysozyme-COT15 (5.0 μL, 0.15 mg / mL) was aliquoted into six samples each. An aqueous solution of hydroxylamine 13 (0.21 μL, 5 mM in deionized water; 200 μM final concentration) was added to each of the five aliquot samples. For the vehicle control, deionized water (0.21 μL) was added to one sample instead of hydroxylamine 13. For control samples requiring conditions without lysozyme-COT15, unmodified lysozyme was treated with hydroxylamine 13 (0.21 μL, 5 mM in water; 200 μM final concentration) or deionized water (0.21 μL). The reaction mixture was incubated in the dark at room temperature and quenched by adding N,N-diethylhydroxylamine (1.30 μL, 100 mM in deionized HO; final concentration 20 mM) followed by 5×SDS sample loading buffer (1.63 μL) at the indicated time points. Samples were flash-frozen under liquid nitrogen until all samples were ready to be loaded onto the gel. After 2 hours, all reactions were quenched. All samples were thawed, and 5 μL of each solution was loaded onto a 15-well 12% SDS-PAGE gel. The gel was run at room temperature and 175 V for 50 minutes. In-gel fluorescence was imaged on a Typhoon™ FLA 9500 (GE) at 532 nM using a photomultiplier tube (PMT) setting of 500 V. Experiments were performed in triplicate (Figure 9).

[0478] Intact mass analysis A solution of hydroxylamine 13 (0.83 μL, 5 mM in deionized water) was added to a solution of lysozyme-COT15 (20 μL, 0.60 mg / mL in deionized water) to generate a reaction sample. Deionized HO (0.83 μL) was added to lysozyme-COT15 (20 μL, 0.60 mg / mL in deionized water) to generate a vehicle control. Unmodified lysozyme (20 μL, 0.60 mg / mL in deionized water) was added to deionized water (0.83 μL) to generate a blank background sample. The reaction was incubated in the dark at room temperature for 6 h. Samples were flash-frozen using liquid nitrogen and stored at -80 °C until further analysis. Electrospray ionization mass spectrometry (ESI-MS) analysis was performed on an LTQ XL™ ion trap mass spectrometer (ThermoFisher Scientific™, San Jose, CA) (FIG. 10).

[0479] Example 27: Protein labeling experiments in the presence of cell lysates

[0480] Cell culture: HEK-293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Corning®) containing 10% fetal bovine serum (FBS, Sigma), 100 units / mL penicillin, and 0.1 mg / mL streptomycin (Sigma) at 37°C in a humidified chamber with 5% CO2. Cells were passaged and dissociated with 0.25% trypsin, 0.1% ethylenediaminetetraacetic acid (EDTA) in Hank's balanced salt solution (HBSS) (Corning®). Cells tested negative for mycobacteria using the MycoAlert™ PLUS Mycoplasma Detection Kit (Lonza) according to the manufacturer's protocol.

[0481] Cell lysate: Cell culture medium was aspirated before lysis. Cells (10 cm dish, approximately 80% confluent) were lysed by adding lysis buffer (1.0 mL, 4°C; 150 mM NaCl, 50 mM Tris (pH 8.0), 1% Triton X-100). After centrifugation (13,000 × g) at 4°C, the supernatant was transferred to a clean tube, and the protein concentration was determined by BCA (bicinchoninic acid) protein assay (Pierce™ BCA Protein Assay Kit). Aliquots of cell lysate (7.1 mg / mL) were flash-frozen under liquid nitrogen and stored at -20°C.

[0482] Protein labeling experiments: Lysozyme-COT15 (0.75 μg, 5.0 μL, 0.15 mg / mL in deionized water) was aliquoted to form four samples. Cell lysate (20 μg, 2.83 μL, 7.1 mg / mL) was added to two samples, and deionized water (2.83 μL) was added to the remaining two samples. A fifth control sample lacking lysozyme-COT15 was prepared by adding cell lysate (20 μg, 2.83 μL, 7.1 mg / mL) to deionized water (5.0 μL). Then, either hydroxylamine 13 (0.33 μL, 5 mM in deionized water) or deionized water (0.33 μL) was added to the sample according to the conditions shown in Figure 5B. The reaction mixture was incubated in the dark at room temperature for 2 h. The reaction mixture was quenched with 5x SDS sample loading buffer (1.30 μL). 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. In-gel fluorescence was imaged with a Typhoon™ FLA9500 (GE) at 532 nM using a photomultiplier tube (PMT) setting of 500 V. Experiments were performed in triplicate.

[0483] Example 28: Stability studies

[0484] All reactions were monitored by HPLC at 0, 1, 2, 4, 8, and 24 hours.

[0485] HPLC analysis: The reaction with enamine N-oxide 17 was analyzed by HPLC (Pursuit 200 Å C 18 , 4.6 × 150 mm, 10 μm particles, 1 mL / min flow rate, eluent: isocratic 0% MeCN / HO + 0.1% TFA (1 min), gradient 0 → 100% MeCN / HO + 0.1% TFA (14 min), isocratic 100% MeCN / HO + 0.1% TFA (1 min), quantified using its absorbance at 280 nm. Reaction with hydroxylamine 13 was monitored by HPLC (Pursuit 200 Å C). 18 , 4.6 × 150 mm, 10 μm particles, 1 mL / min flow rate, eluent: isocratic 0% MeCN / H2O + 0.1% TFA (1 min), gradient 0 → 20% MeCN / H2O + 0.1% TFA (1 min), gradient 20 → 50% MeCN / H2O + 0.1% TFA (16 min), gradient 50 → 100% MeCN / H2O + 0.1% TFA (2 min), isocratic 100% MeCN / H2O + 0.1% TFA (1 min), quantified using its absorbance at 254 nm.

[0486] 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; 500 μM final concentration). Separately, hydroxylamine 13 (3 μL, 20 mM in deionized HO) was added to PBS (117 μL, pH 7.4; 500 μM final concentration). Each solution was monitored by HPLC at each time point.

[0487] Stability in the presence of glutathione, sodium ascorbate, and cell lysate: Reactions were performed as described above for stability in PBS, except that the PBS solution was first supplemented with glutathione (5 mM final concentration), sodium ascorbate (5 mM final concentration), or HEK293T cell lysate (1 mg / mL final concentration), and the pH was adjusted to 7.4 before the addition of enamine N-oxide 17 (500 μM final concentration) or hydroxylamine 13 (500 μM final concentration).

[0488] Microsome assay I: A solution of human liver microsomes (8 μL, 20 mg / mL in phosphate buffer, pH 7.4, Corning®; final concentration 200 μg / mL) and a solution of NADPH (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. A solution of enamine N-oxide 17 (26.7 μL, 15 mM in 25% MeOH / PBS, pH 7.4; 500 μM final concentration) was added to solution A. The cap of the microcentrifuge tube was pierced with a 16G needle to maintain an aerobic system. The reaction was incubated at room temperature in the dark. At each time point, 100 μL of sample was transferred to a clean 2.0 mL microcentrifuge tube, and the reaction was quenched with acetonitrile (100 μL). The mixture was centrifuged (13,000 xg) for 5 minutes at 4°C, and the supernatant was then transferred to a clean HPLC vial for analysis.

[0489] Microsome assay II: Human liver microsome solution (8 μL, 20 mg / mL in phosphate buffer, pH 7.4, Corning®; final concentration 200 μg / mL) and NADPH solution (13.4 μL, 60 mM in 10 mM NaOH; final concentration 1 mM) were added sequentially to PBS (358.6 μL, pH 7.4) and incubated at room temperature for 1 h to form solution B. Sodium ascorbate solution (400 μL, 10 mM in PBS, pH 7.4) and a solution of hydroxylamine 13 (20 μL, 20 mM in deionized HO) were added to solution B. The caps of the microcentrifuge tubes were pierced with a 16G needle to maintain an aerobic system. The reactions were incubated at room temperature in the dark. At each time point, 100 μL of the sample was transferred to a clean 2.0 mL microcentrifuge tube, and the reaction was quenched with acetonitrile (100 μL). The mixture was centrifuged (13,000 × g) for 5 min at 4°C, and the supernatant was transferred to a clean HPLC vial for analysis.

[0490] Example 29: Cross-reactivity studies

[0491] Me-tetrazine and cyclooctyne: A solution of cyclooctyne 5, 9, or 10 (125 μL, 20 mM in CD3CN, 1 equivalent; 5 mM final concentration) was added to each of the NMR tubes. A solution containing the internal standard 1,3,5-trimethoxybenzene (TMB, 50 μL, 50 mM in CD3CN; 5 mM final concentration) was then added to each of the samples via syringe. CD3CN (275 μL) was added to each tube to bring the volume of each solution to 450 μL. The tubes were inverted three times to mix the solutions. 1 A reference spectrum was obtained by H NMR spectroscopy. A solution of tert-butyl-(4-(6-methyl-1,2,4,5-tetrazin-3-yl)benzyl)carbamate (18, 50 μL, 50 mM solution in CD3CN, 2.00 equivalents; 10 mM final concentration) was added to a final volume of 500 μL. The tube was immediately inverted three times to mix the reaction solution, which was then incubated at room temperature for 1 hour. 1 The product was analyzed by 1 H NMR spectroscopy, and no spectral changes were observed. [ka]

[0492] Solutions of various hydroxylamines containing the following components were added to separate NMR tubes: (E)-cyclooct-4-en-1-yl(3-aminopropyl)carbamate (19, 16 mM in 6.2% DO in CD3CN; 5 mM final concentration), 4-methylbenzaldehyde (20, 100 mM in CD3CN; 5 mM final concentration), (2-methylcycloprop-2-en-1-yl)methyl isopropylcarbamate (21, 50 mM in CD3CN; 5 mM final concentration), or hex-5-ynoic acid (22, 50 mM in CD3CN; 5 mM final concentration). Then, 50 μL of a solution containing the internal standard 1,3,5-trimethoxybenzene (TMB, 50 mM in CD3CN; 5 mM final concentration) was added to each of the samples via syringe. CD3CN was added to each tube to bring the volume of each solution to 475 μL. Mix the solution by inverting the tube three times. 1Reference spectra were obtained by H NMR spectroscopy. For each reaction, a solution of N,N-diethylhydroxylamine (25 μL, 100 mM in CD3CN, 2.00 equivalents; 10 mM final concentration) was added to a final volume of 500 μL. The tube was immediately inverted three times to mix the reaction solution, which was then incubated at room temperature for 1 hour. 1 The product was analyzed by 1 H NMR spectroscopy, and no spectral changes were observed. ...

Claims

1. A compound having a structure represented by formula IV or V: 【Chemical 22】 or a pharma- ceutically acceptable salt or stereoisomer thereof; During the ceremony: R １ ' is a linking group; R １ does not exist or R １ and R ２ together with the nitrogen atom to which they are attached form a heterocyclyl; R ２ may be substituted (C １ ~C ８ ) alkyl, -C(O)R", -C(O)OR", -C(O)NR"R", -S(O)R", -S(O) ２ R”, ​​(C ３ ~C １０ ) carbocyclyl, 4- or 7-membered heterocyclyl, or a substituted polyethylene glycol chain, each R″ is independently hydrogen, (C １ ~C ６ ) alkyl, (C ３ ~C １０ ) carbocyclyl, 4-membered or 7-membered heterocyclyl, wherein said alkyl, carbocyclyl or heterocyclyl may be substituted; Each X is independently CR ９ R ９ ', N.R. ９ , O, S, C(O), S(O), or SO ２ wherein the ring system contains 0-3 heteroatoms; R ９ and R ９ ' is independently hydrogen or a substituent; A １ is the active moiety; Y is absent, or 【Chemistry 23】 and A ２ is the active moiety; R ４ is hydrogen, a substituent, or 【Chemistry 24】 is a linking group bonded to or R ４ and R ５ together with the carbon atom to which they are attached form a carbocyclyl or heterocyclyl, where R ４ Also 【Chemistry 25】 binds to; R ５ is hydrogen or an electron withdrawing group; R ６ is hydrogen, a π-electron donating group, or 【Chemistry 26】 is a linking group bonded to R ７ and R ７ ' is independently hydrogen or an electron withdrawing group, or R ７ and R ７ ' together with the carbon atom to which they are attached form C(O); R ８ is hydrogen, a substituent, or 【Chemistry 27】 is a linking group attached to n is 1, 2, or 3; wherein the compound comprises at least one 【Chemistry 28】 having a group, compound.

2. a) R １ is absent and R 1 ' is an alkylene chain, which is -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) ２ O-, -N(R')S(O) ２ -, -S(O) ２ N(R')-, -N(R')S(O)-, -S(O)N(R')-, -N(R')S(O) ２ N(R')-, -N(R')S(O)N(R')-, -OP(O)O(R')O-, -N(R')P(O)N(R'R')N(R')-, C ３ ~C １２ and / or terminated (at either or both termini) by at least one of carbocyclyl, 3- to 12-membered heterocyclyl, 5- to 12-membered heteroaryl, or any combination thereof; each R' is independently H or an optionally substituted C １ ~C ２４ alkyl, wherein the intermediate and one or both terminal groups may be the same or different; Optionally, the alkylene chain is a C 1 -C 12 alkylene chain; b) R 1 is absent and R 1 ' is a polyethylene glycol chain, which is any of -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) 2 O-, -N(R')S(O) 2 -, -S(O) 2 N(R')-, -N(R')S(O)-, -S(O)N(R')-, -N(R')S(O) 2 N(R')-, -N(R')S(O)N(R')-, -OP(O)O(R')O-, -N(R')P(O)N(R'R')N(R')-, C 3 to C 12 and / or terminated (at either or both termini) by at least one of carbocyclyl, 3- to 12-membered heterocyclyl, 5- to 12-membered heteroaryl, or any combination thereof, each R' is independently H or an optionally substituted C 1 -C 24 alkyl, and the interrupted and one or both termini may be the same or different; Optionally, the polyethylene glycol chain has 1 to 20 -(CH 2 CH 2 -O)- units; c) R 1 and R 2 together with the nitrogen atom to which they are attached form a 5-10 membered heterocyclyl containing 1 to 3 heteroatoms selected from N, O, and S; or d) The compound of claim 1, wherein R 2 is methyl, ethyl, isopropyl, or t-butyl.

3. A １ but a) a binding moiety, Optionally, the binding moiety is a small molecule, a short amino acid sequence, a protein, or an antibody or fragment thereof that binds to a predetermined target; Optionally, the antibody is a monoclonal antibody or a binding fragment thereof; Optionally, the small molecule is biotin or a derivative thereof, a small molecule that binds to an E3 ligase, or a small molecule that binds to a cellular protein. b) a therapeutic portion, Optionally, the therapeutic moiety is a non-targeted cancer drug, a targeted anti-cancer drug, an antibacterial drug, a non-steroidal anti-inflammatory drug (NSAID), a corticosteroid, or a disease-modifying anti-rheumatic drug (DMARD); Optionally, the targeted anti-cancer agent is a kinase inhibitor; or c) a diagnostic portion, 3. The compound of claim 1 or 2, optionally wherein the diagnostic moiety is a fluorophore, a chromogenic agent, a positron emission tomography (PET) tracer, or a magnetic resonance imaging (MRI) contrast agent.

4. a) n is 2, b) X is CR 9 R 9 '; Optionally, R 9 and R 9 ' are each hydrogen; c) R 9 and R 9 ' are independently hydrogen, (C 1 -C 6 )alkyl, (C 1 -C 6 )alkoxy, (C 1 -C 6 )haloalkyl, (C 1 -C 6 )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, (C 3 -C 10 )carbocyclyl, 4- or 7-membered heterocyclyl, -O(CH 2 ) 0-3 (C 3 -C 10 )carbocyclyl, or -O(CH 2 ) 0-3 -4- or 7-membered heterocyclyl containing 1 to 3 heteroatoms selected from O, N, and S, where each R 10 is independently hydrogen or (C 1 -C 6 )alkyl; where said alkyl, carbocyclyl, or heterocyclyl may be further substituted; or d) R 4 and R 5 together with the carbon atom to which they are attached form a 5- to 10-membered carbocyclyl or a 5- to 10-membered heterocyclyl containing 1 to 3 heteroatoms selected from N, O, and S; 2. The compound of claim 1, optionally wherein R4 and R5, together with the carbon atom to which they are attached, form a 5-membered heterocyclyl containing two oxygen atoms.

5. R 4 is 【Chemical 29】 is a linking group bonded to Optionally, R 4 is a) is O; b) is S; c) NR 11 , where R 11 is hydrogen or (C 1 -C 6 )alkyl; d) OPh; e) OC(O), f) OC(O)NR 11 , where R 11 is hydrogen or (C 1 -C 6 )alkyl; g) an alkylene chain, which is -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) 2 O-, -N(R')S(O) 2 -, -S(O) 2 N(R')-, -N(R')S(O)-, -S(O)N(R')-, -N(R')S(O) 2 N(R')-, -N(R')S(O)N(R')-, -OP(O)O(R')O-, -N(R')P(O)N(R'R')N(R')-, C 3 to C 12 and / or terminated (at either or both termini) by at least one of carbocyclyl, 3- to 12-membered heterocyclyl, 5- to 12-membered heteroaryl, or any combination thereof, each R' is independently H or an optionally substituted C 1 -C 24 alkyl, and the interrupted and one or both termini may be the same or different; Optionally, said alkylene chain is a C 1 -C 12 alkylene chain; or h) a polyethylene glycol chain, which is -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) 2 O-, -N(R')S(O) 2 -, -S(O) 2 N(R')-, -N(R')S(O)-, -S(O)N(R')-, -N(R')S(O) 2 N(R')-, -N(R')S(O)N(R')-, -OP(O)O(R')O-, -N(R')P(O)N(R'R')N(R')-, C 3 to C 12 and / or terminated (at either or both termini) by at least one of carbocyclyl, 3- to 12-membered heterocyclyl, 5- to 12-membered heteroaryl, or any combination thereof, each R' is independently H or an optionally substituted C 1 -C 24 alkyl, and the interrupted and one or both termini may be the same or different; 5. The compound according to any one of claims 1 to 4, optionally wherein the polyethylene glycol chain has from 1 to 20 -(CH 2 CH 2 -O)- units.

6. R ５ but a) hydrogen, or b) an electron-withdrawing group, Optionally, the electron-withdrawing group is an inductive electron-withdrawing group or a π-electron-withdrawing group; Optionally, said inductive electron withdrawing group is halogen, OR 5' , SR 5' , or NR 5' R 5' , where each R 5' is independently hydrogen, C 1 -C 6 alkyl, C 6 -C 12 aryl, 5-10 membered heteroaryl, carbonyl, sulfonyl, sulfinyl, or phosphoryl; Optionally, 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", NO 2 , CN, N 3 , -S(O)R 5", -S(O) 2 R 5", -S(O)OR 5", -S(O) 2 OR 5", -S(O)NR 5" R 5", -S(O) 2 NR 5" R 5", -OP(O)OR 5" OR 5", -P(O)NR 5" R 5" NR 5" R 5", wherein each R 5" is independently hydrogen, C 1 -C 6 alkyl, C The compound according to any one of claims 1 to 5, which is 6-C 12 aryl, 5-10 membered heteroaryl.

7. a) R ６ is hydrogen, b) R 6 is a π-electron donor group; Optionally, said π-electron donor group is OR 12 , SR 12 , NR 12 NR 12 , or a cyclic or acyclic amide, each R 12 independently being hydrogen, (C 1 -C 6 ) alkyl, (C 3 -C 10 ) carbocyclyl, 4- or 7-membered heterocyclyl, said alkyl, carbocyclyl, or heterocyclyl being optionally substituted; c) R 7 and R 7 ' are independently hydrogen or an inductive electron withdrawing group; Optionally, said inductive electron withdrawing group is halogen, OR 5' , SR 5' , or NR 5' R 5' , where each R 5' is independently hydrogen, C 1 -C 6 alkyl, C 6 -C 12 aryl, 5-10 membered heteroaryl, carbonyl, sulfonyl, sulfinyl, or phosphoryl; or d) R 7 and R 7 ' are independently hydrogen or a π-electron withdrawing group; Optionally, 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", NO 2 , CN, N 3 , -S(O)R 5", -S(O) 2 R 5", -S(O)OR 5", -S(O) 2 OR 5", -S(O)NR 5" R 5", -S(O) 2 NR 5" R 5", -OP(O)OR 5" OR 5", -P(O)NR 5" R 5" NR 5" R 5", wherein each R 5" is independently hydrogen, C 1 -C 6 alkyl, C The compound according to any one of claims 1 to 6, which is 6-C 12 aryl, 5-10 membered heteroaryl.

8. R ８ but, 【Chemistry 30】 is a linking group bonded to Optionally, R 8 is a) is CH2; b) is aryl; c) is O; d) an alkylene chain, which is -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) 2 O-, -N(R')S(O) 2 -, -S(O) 2 N(R')-, -N(R')S(O)-, -S(O)N(R')-, -N(R')S(O) 2 N(R')-, -N(R')S(O)N(R')-, -OP(O)O(R')O-, -N(R')P(O)N(R'R')N(R')-, C 3 to C 12 and / or terminated (at either or both termini) by at least one of carbocyclyl, 3- to 12-membered heterocyclyl, 5- to 12-membered heteroaryl, or any combination thereof, each R' is independently H or an optionally substituted C 1 -C 24 alkyl, and the interrupted and one or both termini may be the same or different; Optionally, said alkylene chain is a C 1 -C 12 alkylene chain; or e) A polyethylene glycol chain, which is -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) 2 O-, -N(R')S(O) 2 -, -S(O) 2 N(R')-, -N(R')S(O)-, -S(O)N(R')-, -N(R')S(O) 2 N(R')-, -N(R')S(O)N(R')-, -OP(O)O(R')O-, -N(R')P(O)N(R'R')N(R')-, C 3 to C 12 and / or terminated (at either or both termini) by at least one of carbocyclyl, 3- to 12-membered heterocyclyl, 5- to 12-membered heteroaryl, or any combination thereof, each R' is independently H or an optionally substituted C 1 -C 24 alkyl, and the interrupted and one or both termini may be the same or different; The compound according to any one of claims 1 to 7, optionally wherein the polyethylene glycol chain has from 1 to 10 -(CH 2 CH 2 -O)- units.

9. A ２ but a) a binding moiety, Optionally, the binding moiety is a small molecule, a short amino acid sequence, a protein, or an antibody or fragment thereof that binds to a predetermined target; Optionally, the antibody is a monoclonal antibody or a binding fragment thereof; Optionally, the small molecule is biotin or a derivative thereof, a small molecule that binds to an E3 ligase, or a small molecule that binds to a cellular protein. b) a therapeutic portion, Optionally, the therapeutic moiety is a non-targeted cancer drug, a targeted anti-cancer drug, an antibacterial drug, a non-steroidal anti-inflammatory drug (NSAID), a corticosteroid, or a disease-modifying anti-rheumatic drug (DMARD); Optionally, the targeted anti-cancer agent is a kinase inhibitor; or c) a diagnostic portion, 9. The compound of any one of claims 1 to 8, optionally wherein the diagnostic moiety is a fluorophore, a chromogenic agent, a positron emission tomography (PET) tracer, or a magnetic resonance imaging (MRI) contrast agent.

10. a) 【Chemistry 31】 【Chemistry 32】 【Chemical 33】 or a pharma- ceutically acceptable salt or stereoisomer thereof; Optionally, 【Chemical 34】 or a pharma- ceutically acceptable salt or stereoisomer thereof; or b) 【Chemistry 35】 【Chemical 36】 【Chemical 37】 【Chemical 38】 or a pharma- ceutically acceptable salt or stereoisomer thereof; Optionally, 【Chemical Formula 39】 or a pharma- ceutically acceptable salt or stereoisomer thereof.

11. a) 【Chemistry 40】 and 【Chemistry 41】 one of which is a diagnostic agent and the other is a therapeutic agent, and the compound is in the form of a theranostic agent; b) 【Chemistry 42】 and 【Chemistry 43】 one of which is an antibody or binding fragment thereof and the other is a therapeutic agent, the compound being in the form of an antibody-drug conjugate; or c) 【Chemistry 44】 and 【Chemistry 45】 2. The compound of claim 1, wherein both of are binders and the compound is a degrader.

12. The compound of formula IV has the formula IVa′, IVb′, or IVc′, 【Chemistry 46】 or a pharma- ceutically acceptable salt or stereoisomer thereof; During the ceremony: R 1 ' is a linking group; R 1 is absent, or R 1 and R 2 together with the nitrogen atom to which they are attached form a heterocyclyl; R 2 is optionally substituted (C 1 -C 8 )alkyl, —C(O)R′, —C(O)OR′, —C(O)NR′R′, —S(O)R′, —S(O) 2 R′, (C 3 -C 10 )carbocyclyl, or 4- to 7-membered heterocyclyl, where each R′ is independently hydrogen, (C 1 -C 6 )alkyl, (C 3 -C 10 )carbocyclyl, 4- to 7-membered heterocyclyl, said alkyl, carbocyclyl, or heterocyclyl being optionally substituted; A 1 ' is an antibody or an antibody fragment; each X is independently CR 9 R 9 ', NR 9 , O, S, C(O), S(O), or SO 2 , where the ring system contains 0-3 heteroatoms; R 9 and R 9 ' are independently hydrogen or a substituent; R 4 is a linking group; A 2 ' is a therapeutic small molecule; n is 1, 2, or 3; Optionally, a) a compound represented by formula IVa'1; 【Chemistry 47】 or a pharma- ceutically acceptable salt or stereoisomer thereof; or b) a compound represented by formula IVa'2, 【Chemistry 48】 or a pharma- ceutically acceptable salt or stereoisomer thereof.

13. A compound having a structure represented by formula I: 【Chemistry 1】 or a pharma- ceutically acceptable salt or stereoisomer thereof; During the ceremony: R １ ' is a linking group; R １ does not exist or R １ and R ２ together with the nitrogen atom to which they are attached form a heterocyclyl; R ２ may be substituted (C １ ~C ８ ) alkyl, -C(O)R", -C(O)OR", -C(O)NR"R", -S(O)R", -S(O) ２ R”, ​​(C ３ ~C １０ ) carbocyclyl, 4- or 7-membered heterocyclyl, or a substituted polyethylene glycol chain, each R″ is independently hydrogen, (C １ ~C ６ ) alkyl, (C ３ ~C １０ ) carbocyclyl, 4-membered or 7-membered heterocyclyl, wherein said alkyl, carbocyclyl or heterocyclyl may be substituted; A １ is the active moiety, Compound, or b) Formula II or III: 【Chemistry 7】 or a pharma- ceutically acceptable salt or stereoisomer thereof, During the ceremony, each X is independently CR 9 R 9 ', NR 9 , O, S, C(O), S(O), or SO 2 , where the ring system contains 0-3 heteroatoms; R 9 and R 9 ' are independently hydrogen or a substituent; Y is absent, or 【Chemistry 8】 and A 2 is an active moiety; R 4 is hydrogen, a substituent, or 【Chemistry 9】 is a linking group bonded to or R 4 and R 5 together with the carbon atom to which they are attached form a carbocyclyl or heterocyclyl, where R 4 is also 【Chemistry 10】 attached to a group; R 5 is hydrogen or an electron withdrawing group; R 6 is hydrogen, a π-electron donating group, or 【Chemistry 11】 is a linking group bonded to R 7 and R 7 ' are independently hydrogen or an electron withdrawing group; or R 7 and R 7 ' together with the carbon atom to which they are attached form C(O); R 8 is hydrogen, a substituent, or 【Chemistry 12】 is a linking group bonded to n is 1, 2, or 3; wherein the compound is 【Chemistry 13】 having a group, compound.

14. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of claims 1 to 13 or a pharma- ceutically acceptable salt or stereoisomer and a pharma- ceutically acceptable carrier.

15. A method for treating a disease or disorder comprising: Optionally, the disease is cancer, or b) A method for labeling a protein, in which one active moiety binds to the protein and the other active moiety is a diagnostic moiety (label), Optionally, the protein is a cancer associated antigen.

20. A compound, or a pharma- ceutically acceptable salt or stereoisomer thereof, for use in

16. A method for preparing a compound of formula IV comprising the steps of: 【Chemistry 49】 Formula I: 【Chemistry 50】 is reacted with a compound of formula II: 【Chemistry 51】 or A method for preparing a compound of formula V, comprising: 【Chemistry 52】 Formula I: 【Chemistry 53】 with a compound of formula III: 【Chemical 54】 with a compound of formula (I). Where: Optionally, the reacting step is carried out in the presence of a solvent; Optionally, the solvent is an aprotic solvent, a protic solvent, or a mixture of solvents; Optionally, the aprotic solvent is DCM, CHCl 3 , CCl 4 , DCE, toluene, MeCN, or THF; Optionally, the protic solvent is MeOH, EtOH, iPrOH, nBuOH, TFE, or HFIP; Optionally, the solvent mixture is a mixture of an aprotic solvent and a protic solvent; Further optionally, the solvent mixture is 0-100% TFE in CHCl 3 ; Optionally, the solvent mixture is about 20% TFE in CHCl 3 ; Optionally, the reacting step is carried out in the presence of an aqueous buffer; Optionally, the aqueous buffer is an acidic or alkaline buffer; Optionally, the reacting step is carried out in the presence of a biological fluid or an aqueous solution comprising a biological component; Optionally, the biological fluid is blood, synovial fluid, lymphatic fluid, or vitreous humor; Optionally, the reacting step is carried out at a temperature between 0° C. and 60° C.; Further optionally, the temperature is about 20° C. to 25° C.; Optionally, the compound of formula (I) is in excess of the compound of formula (II) or (III); Further optionally, the excess is about 5 equivalents; Optionally, the reacting step is performed with the addition of a buffer reagent; or Optionally, the method, wherein the buffering agent is ascorbic acid or glutathione.

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