Enamine N-oxides: synthesis and applications as hypoxia-responsive prodrugs and imaging agents

Abstract

Disclosed are compounds and pharma- ceutically acceptable salts and stereoisomers thereof suitable for the diagnosis and treatment of diseases and disorders characterized by, associated with, or exhibiting tissue hypoxia, such as solid tumors, etc. Also disclosed are pharmaceutical compositions containing same, and methods of making and using the 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 / 165,992, filed March 25, 2021, which is incorporated herein by reference in its entirety.

[0002] government support This invention was made with government support under Grant No. DP2 OD025643 awarded by the National Institutes of Health. The government has certain rights in this invention. [Background technology]

[0003] Tumor hypoxia refers to a state of oxygen deficiency in tumor tissue resulting from inappropriate and irregular angiogenesis of rapidly proliferating cancer cells (Hockel et al., J. Natl. Cancer Inst. 93(4):266-276 (2001); Harris, AL, Rev. Cancer 2(1):38-47 (2002); Eales et al., Oncogenesis 5(1):e190 (2016); Carreau et al., J. Cell. Mol. Med. 15(6):1239-1253 (2011)). Diffusion and perfusion limitations in these regions can result in both sustained and transient levels of hypoxia characterized by oxygen tensions below 2%, and in the most severe cases of radiobiological hypoxia, below 0.1% (Mistry et al., Int. J. Radiat. Oncol., Biol., Phys. 98(5):1183-1196 (2017); McKeown, SR, Br. J. Radiol. 87(1035):20130676 (2014)). Malnutrition and inadequate oxygenation of hypoxic tissues leads to extensive adaptive reprogramming of cancer cells, transforming them into highly invasive and metastatic species with profoundly altered metabolism and enhanced potential for proliferation and survival (Schito et al., Trends Cancer 2(12):758-770 (2016); Terry, et al., Int. J. Mol. Sci. 19(10):3044 (2018); Shah et al., Cancer Lett. 492:63-70 (2020)).

[0004] The development of hypoxia in tumors is highly correlated with poor survival and prognosis in cancer patients with advanced solid tumors, and current treatments have drawbacks. For example, given the essential role that oxygen plays as a radiosensitizer, radiation therapy is ineffective against hypoxic tissues; chemotherapy, which targets actively proliferating cells, is ineffective against cells in a hypoxia-induced quiescent state; and surgical options are often limited by the enhanced metastatic potential of cancers that exhibit hypoxia (Terry, et al., Int. J. Mol. Sci. 19(10):3044 (2018); Teicher, BA, Cancer Metastasis Rev. 13(2):139-168 (1994); Rohwer et al., Drug Resist. Updates 14(3):191-201 (2011); Eckert et al., Front. Immunol. 10:407 (2019); Rankin et al., Science 352(6282):175-180 (2016)).

[0005] Additionally, over the past 60 years, dozens of hypoxia-activated prodrugs (HAPs) have been developed, with 11 entering the clinic (Brown et al., Nat. Rev. Cancer 4(6):437-447 (2004); Wilson et al., Nat. Rev. Cancer 11(6):393-410 (2011); Hunter et al., Br. J. Cancer 114(10):1071-1077 (2016); Phillips, RM, Cancer Chemother. Pharmacol. 77(3):441-457 (2016); Sharma et al., Chem. Soc. Rev. 48(3):771-813 (2019)). However, clinical success has been slow (Spiegelberg et al., Clin. Transl. Radiat. Oncol. 15:62-69 (2019)). Recently, the HAP tirapazamine (Brown, JM, Br. J. Cancer 67(6):1163-1170 (1993)) and TH-302 (Duan et al., J. Med. Chem. 51(8):2412-2420 (2008)), respectively, failed to meet their primary endpoints in phase 3 clinical trials (Rischin et al., J. Clin. Oncol. 28(18):2989-2995 (2010); Tap et al., Lancet Oncol. 18(8):1089-1103 (2017)), stimulating a reevaluation of approaches to prodrug development (Hunter et al., Br. J. Cancer 114(10):1071-1077 (2016); Spiegelberg et al., Clin. Transl. Radiat. Oncol. 15:62-69 (2019)). In particular, these studies emphasize the importance of patient stratification (Wilson et al., Nat. Rev. Cancer 11(6):393-410 (2011); Spiegelberg et al., Clin. Transl. Radiat. Oncol. 15:62-69 (2019)).

[0006] Given the above, therapeutic agents targeting tumor hypoxia are urgently needed. The presence, extent, and severity of hypoxia vary widely among patients, but no clinical factors, such as size or stage, or genomic markers that adequately predict hypoxia have been identified (Hunter et al., Br. J. Cancer 114(10):1071-1077 (2016); Spiegelberg et al., Clin. Transl. Radiat. Oncol. 15:62-69 (2019)). This heterogeneity in hypoxia, which can occur among tumors of the same type as well as across patient subpopulations, complicates the development of effective treatments as well as diagnostic agents. Retrospective studies from tirapazamine trials have not established efficacy in the general population, but [ 18 revealed that among a subgroup of patients in whom tumor hypoxia was detected by [F]misonidazole (MISO)-based positron emission tomography (PET) imaging, a significant reduction in locoregional failure was observed in the treated cohort versus the control cohort (Rischin et al., J. Clin. Oncol. 24(13):2098-2104 (2006)). The results of this study highlight the need to utilize hypoxia for the simultaneous development of therapeutic and companion noninvasive diagnostic agents. Summary of the Invention [Means for solving the problem]

[0007] The present invention provides novel branched enamine N-oxide compounds, methods for their preparation, and their use in the diagnosis and treatment of diseases and disorders characterized by or manifesting tissue hypoxia, particularly solid tumors. Their dual functionality confers clinical versatility; therefore, they are referred to herein as theranostic agents. The compounds of the present invention undergo hypoxia-selective and heme protein-dependent reduction to induce simultaneous activation of active agents, such as drugs and / or diagnostic imaging agents, once the compounds reach the site of hypoxic tissue. Thus, compounds of the present invention containing therapeutic agents, such as anticancer agents, can be considered hypoxia-responsive prodrugs. As shown in various examples, the enamine N-oxide caged cytotoxin staurosporine exhibited a hypoxic-to-normoxic cytotoxicity ratio that is advantageous and complementary to that of AQ4N, a well-studied aliphatic amine N-oxide hypoxia-activated prodrug. Other examples confirm the dual functionality of the enamine N-oxides of the invention and demonstrate that compounds of the invention containing near-infrared probes selectively labeled hypoxic tumor tissue in both cell and in vivo tumor xenograft mouse models.

[0008] The enamine N-oxide group acts as a cage for the active moiety. The sensitivity of the compounds of the present invention to hypoxic environments facilitates cleavage of the therapeutic moiety and its release from the enamine N-oxide to hypoxic tissue. In doing so, the compounds of the present invention provide a mode of localized delivery to sites of tissue hypoxia, such as the tumor microenvironment, which may otherwise be difficult to target.

[0009] Accordingly, an aspect of the present invention is compounds represented by formulas I, II, III, and IV: [ka] and pharmaceutically acceptable salts, stereoisomers thereof. wherein R1, R2, R3, R4, R7, R8, and A are as defined herein.

[0010] Another aspect of the present invention is directed to a process or method for preparing compounds of formula I, II, III, and IV. The compounds of formula (I) and (II) are positional isomers. The method for producing compounds of formula (I and II) includes preparing compounds of formula (V) [ka] with a compound of formula (VII) [ka] The compound of formula (III) and the compound of formula (IV) are positional isomers. A method for preparing the compounds of formula (III) and (IV) comprises reacting the compound of formula (VI) [ka] with a compound of formula (VII).

[0011] Yet another aspect of the present invention is directed to pharmaceutical compositions comprising a therapeutically and / or diagnostically effective amount of a compound of Formulas (I-IV) and pharmaceutically acceptable salts and stereoisomers thereof, and a pharmaceutically acceptable carrier.

[0012] A further aspect of the present invention is directed to a method of treating a disease or disorder characterized by, associated with, or manifested by tissue hypoxia, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of any one of Formulas (I-IV) containing a therapeutic moiety, e.g., an anticancer agent. In some embodiments, the method is directed to a method of treating a solid tumor characterized by a hypoxic tumor microenvironment.

[0013] A further aspect of the present invention relates to a method of diagnosing a disease or disorder characterized by, associated with, or indicative of tissue hypoxia, comprising administering to a subject in need thereof a diagnostically effective amount of a compound of any one of Formulas (I-IV) comprising at least one diagnostic moiety, e.g., a diagnostic imaging agent. [Brief explanation of the drawings]

[0014] [Figures 1A-1D] A series of schematic diagrams of the mechanism and design of hypoxia-activated prodrugs are shown. Figure 1A shows that hypoxia-activated prodrugs utilize a generally futile redox cycle to achieve selectivity. Oxygen sequentially reverses the reduction of the 1e-reductase prodrug. Figure 1B shows that two sequential 2e-reductions by a heme protein convert the aliphatic N-oxide prodrug AQ4N to the cytotoxic agent AQ4. Figure 1C shows the design of a novel hypoxia-activated prodrug called an enamine N-oxide. The enamine N-oxide can release a small molecule upon selective 2e-bioreducation under hypoxic conditions. The resulting unsaturated iminium ion can readily react with biological nucleophiles. Figure 1D shows that the reverse Cope elimination between an alkyne and a dialkylhydroxylamine provides access to novel enamine N-oxides under mild reaction conditions. LG = leaving group. [Figure 2A-2B] Figure 2 shows the hydroamination reaction between alkynes and N,N-dialkylhydroxylamines. Figure 2A shows the alkyne substrate range for the reaction. Figure 2B shows the hydroxylamine substrate range. The major regioisomer of the product is shown, and yields are reported as the average isolated yield from two experiments. The regioisomer ratio (rr) represents the ratio of the major to minor product as determined by H NMR analysis. If rr is not shown, only the indicated regioisomer is observed. The reaction was monitored by thin-layer chromatography for the disappearance of the limiting reagent, and the reaction time is provided. a) Alkyne (1 equiv.), N,N-diethylhydroxylamine (5.0 equiv.); b) N,N-dialkylhydroxylamine (1 equiv.), alkyne (2.0 equiv.). [Figures 3A-3F] We demonstrate that enamine N-oxides are bioreduced in an oxygen-dependent manner in vitro and in cells in tissue culture. Figure 3A shows a panel of chromogenic enamine N-oxide probes that release 2-nitroaniline upon reduction. These probes were incubated with human liver microsomes under hypoxic conditions (0.1% pO2), and their initial reduction rates were measured and reported as relative reduction rates (krel) normalized to probe 32a. Figure 3B shows the time-dependent reduction of enamine N-oxide probe 32c by human liver microsomes under hypoxic and normoxic conditions. The data represent the concentration of released 2-nitroaniline based on absorbance measurements at λ = 412 nm, demonstrating a 21-fold enhancement of the initial rate of reduction under hypoxic conditions. Figure 3C shows a bar graph of a panel of CYP450 inhibitors evaluated for their ability to inhibit the reduction of enamine N-oxide probe 32c in the A412 microsomal assay using human liver microsomes, as well as oxygen, NADPH depletion, and microsomal heat inactivation. Figure 3D shows the chemical synthesis of enamine N-oxide caged staurosporine 37 and the nonreducible control compound 36. Figure 3E shows dose-response curves for prodrug 37, staurosporine, and the nonreducible alkyne derivative 36 under both normoxic and hypoxic conditions in A431 cells (epidermoid carcinoma cells). The pO2 for each condition is shown in parentheses. Figure 3F shows a comparison table of HCR values ​​for compound 37 and AQ4N in H460 and A431 cells. TFA = trifluoroacetic acid; HCR = hypoxic-to-normoxic cytotoxicity ratio; NADPH = nicotinamide adenine dinucleotide phosphate; HI = heat inactivated; DPI = diphenyleneiodonium chloride; TAO = troleandomycin; DDC = diethyldithiocarbamate. [Figures 4A-4F]We demonstrate that hypoxia-specific in vivo reduction of enamine N-oxides results in intracellular and in vivo intracellular protein labeling. Figure 4A shows a workflow diagram for visualizing hypoxia-dependent cell or tumor tissue section labeling with alkyne-containing enamine N-oxide probes. Under hypoxic conditions, these probes are reduced and covalently modify proteins with the alkyne handle. Cell lysates or tumor tissue sections from probe-treated samples are labeled with a TAMRA-azide fluorophore via copper-catalyzed azide-alkyne cycloaddition (CuAAC) click chemistry. Figure 4B shows the structures of the alkyne-containing enamine N-oxide imaging probe and pimonidazole. Figure 4C shows A431 cells treated with probes 38-40 for 48 hours and visualized by in-gel fluorescence after CuAAC using a TAMRA-azide fluorophore. Figure 4D shows the activation of enamine N-oxide probe 39 over 48 hours at approximately 1% pO2 and its oxygen-dependent labeling in cell culture with the BxPC-3 pancreatic cancer cell line. Figure 4E shows the labeling profile of probe 39, demonstrating its hypoxia selectivity across a range of cancer cell lines. The scale bar represents 100 μm. H / N = hypoxic-to-normoxia ratio; Nec = necrotic area; TAMRA = tetramethylrhodamine; DAPI = 4',6-diamidino-2-phenylindole. Figure 4F shows immunofluorescence images of tumor tissue sections obtained from BxPC-3 xenografts in mice intraperitoneally inoculated with enamine N-oxide 39 and pimonidazole. The localization of compound 39 strongly correlates with the staining patterns from immunofluorescent labeling of known hypoxia markers, GLUT1, HIF1α, CAIX, and pimonidazole. [Figure 5A-5B] We demonstrate that hypoxia-responsive bioreduction of enamine N-oxides enables near-infrared (NIR) fluorescence imaging of tumors in vivo. Figure 5A shows the structure of alkyne-containing enamine N-oxide NIR probe 42. Figure 5B shows a series of NIR images of a BxPC-3 xenograft mouse model demonstrating preferential accumulation of enamine N-oxide probe 42 in tumors. hpi = hours post-injection. [Figure 6]We demonstrate that enamine N-oxides undergo thermal decomposition via Cope elimination. When the model substrate S1 was heated in CDCl3 at 60 °C for 1 h, two new products were isolated in addition to the starting material. The presence of the α,β-unsaturated nitrone S2 and alkene S3 suggested that Cope elimination was the decomposition pathway for these enamine N-oxides. [Figures 7A-7B] Figures 7A-7B show that enamine N-oxide reduction results in the release of a leaving group. Figure 7A shows that incubation of enamine N-oxide 6 with reducing agent B2(OH)4 or Fe2+ in 100 mM HEPES (pH 7.4) resulted in the release of p-cresol, as analyzed by HPLC. For aerobic oxidation of Fe2+, sodium dithionite (Na2SO4) was used to reduce Fe3+ to Fe2+. Figure 7B shows that incubation of alkyl N-oxide S4 did not result in the release of p-cresol. Reduction resulted in the production of tertiary amine S5, confirming the essential role of an internal olefin in converting the N-oxide reduction event to the release of a leaving group. Values ​​represent the mean ± SD (n = 2). [Figure 8A-8B] Nucleophilic capture experiments of in situ generated α,β-unsaturated iminium ions are shown. Figure 8A shows that enamine N-oxide S6 is not reduced in the absence of diboron. Figure 8B shows that enamine N-oxide S6 was reduced with B2(OH)4 in the presence of benzyl mercaptan (10 equivalents) at room temperature. The Michael adduct S7 was obtained in 94% yield. The aldehyde-containing product S7 is consistent with the nucleophilic addition of benzyl mercaptan to the α,β-unsaturated iminium ion intermediate generated upon extrusion of the leaving group. Subsequent hydrolysis of the iminium ion yielded the isolated product. [Figures 9A-9C]Figure 9A shows a series of graphs depicting the enamine N-oxide 32c in the presence of different reducing agents. Figure 9A shows probe 32c (200 μM) incubated with tetrahydroxydiboron (B2(OH)4, 1 equivalent, 200 μM) at room temperature. Figure 9B shows probe 32c (200 μM) incubated with 5 mM cysteine ​​in phosphate buffer, pH 7.4, at 37 °C. Figure 9C shows probe 32c (200 μM) incubated with 5 mM glutathione (GSH) in phosphate buffer, pH 7.4, at 37 °C. The probe is completely reduced by diboron within 10 minutes but is unresponsive to cysteine ​​or glutathione over a 2-hour period. [Figure 10] The structure-dependent effect of enamine N-oxides on microsomal reduction rates is shown. Enamine N-oxide probes (32a-f) were incubated with human liver microsomes under both anaerobic (line graph) and aerobic conditions and measured for the release of 2-nitroaniline by UV spectroscopy. Relative reduction rates (krel) are normalized to the reduction rate of the dimethyl-containing probe 32a. Microsomal reduction rates under both anaerobic and aerobic conditions are quantified in the table. Values ​​represent the mean ± SD (n = 3). [Figure 11] This is a bar graph showing the stability of enamine N-oxide 6 in the presence of different metal cations. Enamine N-oxides (200 μM) containing a p-cresol leaving group were incubated with 2 mM of the following cations for 1 h at room temperature in 100 mM HEPES, pH 7.4. Only Fe2+ was able to reduce the enamine N-oxide. Fe2+ incubations included 10 mM sodium dithionite (see Figure 7 for the effect of sodium dithionite). Values ​​represent the mean ± SD (n = 2). [Figure 12] Figure 1 shows the dose-response cell viability curves of prodrug 37 in A431 and H460 cell lines. Cells were treated with the derivative for 48 hours under normoxic (20% pO2) or hypoxic (0.1% pO2) conditions and then tested for cell viability by MTT assay. Values ​​represent the mean ± SEM of data from biological replicates (n = 3). [Figure 13] Dose-response cell viability curves for AQ4N in H460 and A431 cell lines are shown. Cells were treated with AQ4N under normoxic (20% pO2) or hypoxic (0.1% pO2) conditions for 24 hours and then grown under normoxic conditions for an additional 72 hours. Cell viability was determined by MTT assay. Values ​​represent the mean ± SEM of data from biological replicates (n=3). [Figure 14] 1 is a graph showing the metabolism of compound 37 in the presence of A431 cells. 7.5×10 cells were incubated with 100 μM compound 37 in serum-free RPMI medium, and the presence of metabolites was analyzed by HPLC at the indicated time points. A431 cells specifically converted enamine N-oxide 37 to staurosporine at an approximately 4-fold higher rate under hypoxic conditions. Values ​​represent the mean ± SEM (n=3). [Figures 15A-15B] Figure 15A shows a dose-response study of proapoptotic caspase activation in the A431 cell line by enamine N-oxides. Figure 15A is a graph of the Caspase-Glo® assay used to measure the activity of caspases 3 and 7. Cells were treated with various concentrations of each compound under normoxic (20% pO2) or hypoxic (0.1% pO2) conditions for 24 hours, and the extent of apoptosis was then measured using the Caspase-Glo® assay. Values ​​represent the mean ± SEM of data from four biological replicates (n = 4). Figure 15B is a Western blot of procaspase 3, cleaved caspase 3, cleaved PARP, and actin. Cells were treated with the indicated concentrations of each compound under normoxic (20% pO2) or hypoxic (0.1% pO2) conditions for 24 hours, and then analyzed by Western blot using the apoptosis Western blot cocktail. [Figures 16A-16B]Figure 16A shows images of gels demonstrating that increased oxygen tensions used in probe activation studies provided finer resolution of oxygen dependence. Figure 16A shows Bx-PC3 cells treated with enamine N-oxide probe 39 at the indicated oxygen tensions, lysed, labeled with TAMRA-azide fluorophore using CuAAC, and then visualized by in-gel fluorescence. Hypoxic to normoxia (H / N) ratios are displayed. Figure 16B shows Coosmassie staining of the gel, which serves as a loading control. [Figure 17] Western blot showing pimonidazole activation in BxPC-3 cells at different oxygen tensions. Cells were treated with 10 μM pimonidazole for 48 h, lysed, and analyzed by Western blot. Pimonidazole reaches its apparent half-maximal labeling capacity (0.1% pO2) only after 1% pO2. H / N = hypoxic to normoxic labeling ratio. [Figure 18] This is a series of images showing the comparison of probe 39, pimonidazole, and Hoechst 33342 localization. Tumor tissue sections were obtained from BxPC-3 xenografts in nude mice intraperitoneally inoculated with enamine N-oxide 39, pimonidazole, and Hoechst 33342. Probe 39 was visualized by copper-catalyzed azide-alkyne cycloaddition using the TAMRA-azide fluorophore. Pimonidazole and GLUT1 were visualized by immunofluorescence. Enamine N-oxide 39 colocalized with GLUT1 and pimonidazole hypoxia markers. These hypoxia markers localize away from the perfusion marker Hoechst 33342. This pattern is consistent with hypoxia-specific labeling of probe 39 in regions distal to well-perfused regions. TAMRA = tetramethylrhodamine; GLUT1 = glucose transporter 1. Scale bar = 100 μm. [Figure 19]This is a series of images showing a comparison of probe 39, CD31, and DAPI localization. Tumor tissue sections were obtained from BxPC-3 xenografts in nude mice intraperitoneally inoculated with enamine N-oxide 39. Probe 39 was visualized by copper-catalyzed azide-alkyne cycloaddition using the TAMRA-azide fluorophore. Immunofluorescence imaging of the CD31 endothelial cell marker shows that vasculature is primarily localized in regions distal to areas well labeled by enamine N-oxide 39 staining, and even further away from areas of necrosis. This pattern supports hypoxia-specific activation of enamine N-oxide in poorly vascularized areas of tumors. TAMRA = tetramethylrhodamine; DAPI = 4',6-diamidino-2-phenylindole. Scale bar = 100 μm. [Figures 20A-20C] Figure 20A shows a series of hematoxylin and eosin (H&E) stained and negative control images of BxPC-3 tumor xenograft tissue. Figure 20A shows a series of H&E and immunofluorescence images generated from slices adjacent to the image presented in Figure 4D. Figure 20B shows a series of H&E and immunofluorescence images generated from slices adjacent to the image presented in Figure 16. Figure 20C shows a series of H&E and immunofluorescence images generated from slices adjacent to the image presented in Figure 17. Control images for the visualized images were generated via a copper-mediated azide-alkyne click reaction in the absence of copper sulfate. Secondary control-only images for the immunofluorescence images are also presented. H&E = hematoxylin and eosin; TAMRA = tetramethylrhodamine; DAPI = 4',6-diamidino-2-phenylindole. Scale bar = 100 μm. [Figure 21]This is a series of H&E staining and immunofluorescence images of BxPC-3 tumor xenograft tissue from a mouse injected with a saline control. Images for each hypoxia marker were generated from adjacent tissue sections. Secondary-only controls for CAIX and GLUT1 are from the same images but processed with the same image settings as those used for the primary-containing samples in the same row. H&E = hematoxylin and eosin; DAPI = 4',6-diamidino-2-phenylindole. Scale bar = 100 μm. *Antigen retrieval was performed by incubating slides in 10 mM citrate, pH 6.0, at 90°C for 15 minutes. [Figure 22] Figure 22A is a gel image showing that probe 42 labeled Bx-PC3 cells in a hypoxia-dependent manner in cell culture. Bx-PC3 cells were treated with probe 42 (10 μM) for 12 hours under normoxia and hypoxia, then lysed and visualized by in-gel fluorescence after CuAAC using the TAMRA-azide fluorophore. Figure 22B is a series of images confirming the presence of hypoxia in the mouse tumor imaged with near-infrared (NIR) probe 42 in Figure 5. Copper-catalyzed azide-alkyne cycloaddition-mediated fluorescent labeling of enamine N-oxide NIR probe 42 and immunofluorescent labeling of pimonidazole in tumor tissue sections show strong colocalization, confirming the presence of hypoxia. TAMRA = tetramethylrhodamine; DAPI = 4',6-diamidino-2-phenylindole. Scale bar = 100 μm. [Figure 23] 1 is a bar graph showing the relative mean radiative efficiency of tumor regions on the right flank compared to tumor-free regions on the left flank over time (n=3). The 0-hour post-injection (hpi) time point represents measurements taken before probe injection. DETAILED DESCRIPTION OF THE INVENTION

[0015] 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.

[0016] 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.

[0017] Unless otherwise specified, the term "about" means within 10% (eg, within 5%, 2%, or 1%) of the particular value that is modified by the term "about."

[0018] 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 methods or 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 of the claimed invention, "and those that do not materially affect the basic and novel characteristics."

[0019] 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.

[0020] As used herein, the term "alkyl" refers to a saturated straight-chain 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.

[0021] 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).

[0022] 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.

[0023] 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.

[0024] The term "alkoxyl" or "alkoxy" as used herein refers to an alkyl group, as defined above, having an oxygen radical attached thereto, 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 alkyl substituent that makes the alkyl an ether is an alkoxyl, as can be represented by one of -O-alkyl, -O-alkenyl, and -O-alkynyl, or an analog thereof.

[0025] As used herein, the term "halogen" (or "halo" or "halide") refers to fluorine, chlorine, bromine, or iodine.

[0026] 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, e.g., 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.

[0027] As used herein, the term "carbocycle" (also "carbocyclyl"), used alone or as part of a larger moiety, refers to a group containing a saturated, partially unsaturated, or aromatic ring system of 3 to 20 carbon atoms, either alone or as part of a larger moiety (e.g., an alkyl carbocyclic group). The term carbocyclyl includes monocyclic, bicyclic, tricyclic, 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 to C6). 15 In one embodiment, carbocyclyl is a group having 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 to C8, C3 to C6, or C5 to C6. In some embodiments, the carbocyclyl, as a bicycle, includes C7 to C 12 In another embodiment, the carbocyclyl, as a spiro system, includes C5-C 12Representative examples of monocyclic carbocyclyls include cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl, perdeuterated cyclohexyl, 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 bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane, naphthalene, and bicyclo[3.2.2]nonane. Representative examples of spirocarbocyclyl 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 monocyclic, bicyclic, or spirocyclic carbocycles). 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 a radical or point of attachment is on the carbocyclic ring.

[0028] Thus, the term carbocycle as used herein includes rings of the formula -R c -carbocyclyl (wherein R c The term "carbocycle" as used herein also includes carbocyclylalkyl groups, which refer to groups of the formula -OR c -carbocyclyl (wherein R c is an alkylene chain).

[0029] As used herein, the term "aryl" used alone or as part of a larger moiety (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) 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.

[0030] Thus, the term aryl includes aralkyl groups (e.g., benzyl), which, as disclosed above, have the formula -R c -aryl (wherein R c means a group of an alkylene chain, e.g., methylene or ethylene. 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 includes aralkoxy groups, which refers to groups attached through an oxygen atom of a alkylene chain such as methylene or ethylene.

[0031] 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. In some embodiments, heterocyclyl refers to a heteroaryl ring system, such as a 5- to 14-membered heteroaryl 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.

[0032] In some embodiments, heterocyclyl groups contain 3 to 12 ring atoms, including monocyclic, bicyclic, tricyclic, and spirocyclic ring systems, where the ring atoms are carbon and 1 to 5 ring atoms are heteroatoms (e.g., 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. Any nitrogen or sulfur heteroatom may be optionally oxidized (e.g., NO, SO, SO), and any nitrogen heteroatom may be optionally 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, oxazinanyl, Thiazinanyl, thioxanyl, homopiperazinyl, homopiperidinyl, azepanyl, oxepanyl, thiepanyl, oxazepinyl, oxazepanyl, 1,4-diazepanyl, 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-dihydroisothiazolidinonyl midazole[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, pyrazolidinyl, dithianyl, dithiolanyl, Pyrimidinonyl, pyrimidoindionyl, pyrimidine-2,4-dionyl, piperazinonyl, piperazedionyl, 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]decan-2-onyl, azaspiro[5.5]undecanyl, tetrahydroindolyl, octahydroindolyl, tetrahydroisoindolyl, tetrahydroindazolyl, and 1,1-dioxohexahydrothiopyranyl. Examples of 5-membered heterocyclyls containing sulfur or oxygen atoms and 1 to 3 nitrogen atoms include thiazolyls, including thiazol-2-yl and thiazol-2-yl N-oxide, thiadiazolyls, including 1,3,4-thiadiazol-5-yl and 1,2,4-thiadiazol-5-yl, oxazolyls, such as oxazol-2-yl, and oxadiazolyls, 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; 1,2,3-triazol-5-yl, 1,2,4-triazol-5-yl, and tetrazolyls, such as 1H-tetrazol-5-yl. Representative examples of benzofused 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 pyridyl, e.g., pyrid-2-yl, pyrid-3-yl, and pyrid-4-yl; pyrimidyl, e.g., pyrimid-2-yl and pyrimid-4-yl; triazinyl, e.g., 1,3,4-triazin-2-yl and 1,3,5-triazin-4-yl; pyridazinyl, especially pyridazin-3-yl and pyrazinyl. Pyridine N-oxide and pyridazine N-oxide, as well as 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, a heterocyclic group comprises a heterocycle fused to one or more (e.g., 1, 2, or 3) different cyclic groups (e.g., carbocycles or heterocycles), where the radical or point of attachment is on the heterocycle, and in some embodiments, the point of attachment is a heteroatom contained in the heterocycle.

[0033] Thus, the term "heterocyclic" encompasses N-heterocyclyl groups, which as used herein refer to heterocyclyl groups containing at least one nitrogen, where the point of attachment of the heterocyclyl group to the remainder of the molecule is through a nitrogen atom in 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" also encompasses C-heterocyclyl groups, which as used herein refer to heterocyclyl groups containing at least one heteroatom, where the point of attachment of the heterocyclyl group to the remainder of the molecule is through a carbon atom in the heterocyclyl group. Representative examples of C-heterocyclyl groups include 2-morpholinyl, 2- or 3- or 4-piperidinyl, 2-piperazinyl, and 2- or 3-pyrrolidinyl. The term "heterocyclic" also refers to groups of the formula -R, as disclosed above. c -heterocyclyl (wherein R c is an alkylene chain). The term "heterocyclic" as used herein also includes heterocyclylalkyl groups, which means groups of the formula -OR c -heterocyclyl (wherein R c is an alkylene chain).

[0034] As used herein, the term "heteroaryl," used alone or as part of a larger moiety (e.g., "heteroarylalkyl" (also "heteroaralkyl"), or "heteroarylalkoxy" (also "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, pyrimidyl, imidazolyl, and the like. Included are pyridyl, 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, benzothiazolyl, 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, a heteroaryl group comprises a heteroaryl ring fused to one or more (e.g., 1, 2, or 3) different cyclic groups (e.g., carbocyclic or heterocyclic rings), where the radical 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.

[0035] Thus, the term heteroaryl encompasses N-heteroaryl groups, as used herein, which refer to heteroaryl groups as defined above containing at least one nitrogen, where the point of attachment of the heteroaryl group to the remainder of the molecule is through a nitrogen atom in the heteroaryl group. The term heteroaryl, as used herein, also encompasses C-heteroaryl groups, which refer to heteroaryl groups as defined above, where the point of attachment of the heteroaryl group to the remainder of the molecule is through a carbon atom in the heteroaryl group. The term heteroaryl also encompasses heteroarylalkyl groups, which, as disclosed above, can be represented by the formula -R c -heteroaryl (wherein R c is an alkylene chain as defined above. The term heteroaryl also includes heteroaralkoxy (or heteroarylalkoxy) groups, which as used herein have the formula -OR c heteroaryl (wherein R c refers to a group attached through an oxygen atom of a group (which is an alkylene group as defined above).

[0036] Unless otherwise specified, and unless 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, with the implicit proviso that such substitution, subject to the allowed valences of the substituted atom and substituent, 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, which may contain one or more (e.g., 1, 2, 3, or 4) heteroatoms such as oxygen, sulfur, and nitrogen, organized in a linear, branched, or cyclic structural format.

[0037] 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-C2, 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-C12 , 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.

[0038] As used herein, the term "π-electron withdrawing group" refers to a π-electron-containing functional group with a formal +ve or δ+ve charge, such as a carbonyl or nitro group, that attracts electron density.

[0039] 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.

[0040] 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.).

[0041] 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. Exemplary types of active moieties include therapeutic moieties and diagnostic moieties.

[0042] As used herein, the term "therapeutic moiety" refers to the 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, which in this case is hypoxic tissue.

[0043] As used herein, the terms "diagnostic moiety" and "detectable moiety" are used interchangeably and refer to a portion of a compound of the invention that provides a diagnostic effect in connection with a disease or disorder and allows visualization of cells or tissues in which the compound of the invention accumulates (in this case, hypoxic tissues).

[0044] In one embodiment, the compounds of the present invention are compounds represented by Formulas I and II or pharmaceutically acceptable salts or stereoisomers thereof: [ka] During the ceremony, R1 is hydrogen, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, OH, CN, NO2, NH2, (C1-C6 alkyl)NH, (C1-C6 alkyl)2N, C3-C6 carbocyclyl, a 5- to 6-membered heterocyclyl, an inductive electron-withdrawing group, a leaving group, or a -[L]-diagnostic moiety, wherein R1 is optionally substituted; [L] is absent or an optionally substituted linking group that can carry multiple diagnostic moieties, which may be the same or different; R2 is hydrogen, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, OH, CN, NO2, NH2, (C1-C6 alkyl)NH, (C1-C6 alkyl)2N, C3-C6 carbocyclyl, 5- to 6-membered heterocyclyl, an inductive electron-withdrawing group, a π-electron-withdrawing group, or a -[L]-diagnostic moiety, wherein R2 is optionally substituted; [L] is a linking group that can carry zero or multiple diagnostic moieties, which can be the same or different; R3 is hydrogen, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, OH, CN, NO2, NH2, (C1-C6 alkyl)NH, (C1-C6 alkyl)2N, C3-C6 carbocyclyl, 5- to 6-membered heterocyclyl, an inductive electron-withdrawing group, a π-electron-withdrawing group, or a -[L]-diagnostic moiety, wherein R3 is optionally substituted; [L] is a linking group that can carry zero or multiple diagnostic moieties, which can be the same or different; R4 is hydrogen, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, OH, CN, NO2, NH2, (C1-C6 alkyl)NH, (C1-C6 alkyl)2N, C3-C6 carbocyclyl, 5- to 6-membered heterocyclyl, an inductive electron-withdrawing group, a leaving group, a cleavable linking group, or a -[L]-diagnostic moiety, wherein R4 is optionally substituted; [L] is a linking group that can carry zero or multiple diagnostic moieties, which can be the same or different; R7 is (C1-C8) alkyl, (C3-C 10 ) carbocyclyl, or a 4- or 10-membered heterocyclyl containing 1 to 3 heteroatoms selected from O, N, and S, wherein said alkyl, carbocyclyl, or heterocyclyl is further optionally substituted; or R7 and R8, together with the nitrogen atom to which they are attached, form a 4-7 membered heterocyclyl containing 1-3 heteroatoms selected from O, N and S; R8 is (C1-C8) alkyl, (C3-C 10 ) carbocyclyl, or a 4- or 10-membered heterocyclyl containing 1 to 3 heteroatoms selected from O, N, and S, wherein said alkyl, carbocyclyl, or heterocyclyl may be further substituted; A is absent or is a therapeutic moiety; provided that the compound of formula (I or II) contains at least one -[L]-diagnostic or therapeutic moiety. When A is a therapeutic moiety, R4 is a cleavable linking group; When the compound contains at least one -[L]-diagnostic moiety and A is absent, R1 and / or R4 are leaving groups.

[0045] In some embodiments, R1 is hydrogen.

[0046] In some embodiments, R1 is C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, OH, CN, NO2, NH2, (C1-C6 alkyl)NH, (C1-C6 alkyl)2N, C3-C6 carbocyclyl, or 5-6 membered heterocyclyl.

[0047] In some embodiments, R1 is an inductive electron-withdrawing group. In some embodiments, the inductive electron-withdrawing group is halogen, OR5, SR5, NR5R5, or a cyclic or acyclic amide, where each R5 is independently hydrogen, (C1-C6) alkyl, (C3-C 10 ) carbocyclyl, or 4- to 7-membered heterocyclyl, wherein said alkyl, carbocyclyl, or heterocyclyl is optionally substituted.

[0048] In some embodiments, R is a leaving group, which, as known in the art, refers to an atom or group of atoms that leaves the remainder of a molecule, taking with it the electron pair that was the bond between the leaving group and the remainder of the molecule. In the context of the present disclosure, a leaving group leaves the compound upon contact with hypoxic tissue. Representative examples of leaving groups include iodo, bromo, chloro, OR, SR, -OC(O)R, OC(O)OR, OC(O)NR, -OC(S)R, OC(S)OR, OC(S)NR, -OS(O)R, -OS(O)OR, -OP(O)OR, -OP(O)R, -SC(O)R, -SC(O)SR, or -SC(S)SR, where each R is independently hydrogen, (C-C)alkyl, (C-C 10 ) carbocyclyl, or 4- to 7-membered heterocyclyl, wherein the alkyl, carbocyclyl, or heterocyclyl may be substituted.

[0049] In some embodiments, R1 is -[L]-diagnostic moiety. Diagnostic moieties typically contain a detectable moiety, such as a label. Representative examples of diagnostic moieties include dyes, chromogens, 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 labels include affinity tags, radiometric labels (e.g., radionuclides (e.g., 32 P, 35 S, 3 H, 14 C. 125 I, 131I, 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., enzymes 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.

[0050] 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 TM , Spectrum Green TM , cyanine dyes (e.g., Cy-3 TM , Cy-5 TM , Cy-3.5 TM , Cy-5.5 TM), 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.

[0051] In some embodiments, the diagnostic moiety comprises a rhodamine dye. In some embodiments, the diagnostic moiety comprises tetramethylrhodamine (TAMRA) or a derivative thereof.

[0052] In some embodiments, the diagnostic moiety is an affinity tag, which refers to an agent that participates in an interaction (e.g., antigen and antibody, enzyme and substrate, receptor and ligand) that facilitates the capture and / or purification of a molecule, as known in the art. Representative examples include small chemical compounds (e.g., biotin and its derivatives), short amino acid sequences (e.g., 2-20 amino acids in length, 4-12 amino acids in length, e.g., (His)6 tag, (His)4 tag, (His)3 tag, (His)2 tag, (Leu)4 tag, (Leu)3 tag, (Leu)2 tag, HA tag, FLAG tag, VSV-G tag, HSV tag, and V5 tag), chitin-binding protein (CBP), maltose-binding protein (MBP), Strep-tag, and glutathione-S-transferase (GST).

[0053] In some embodiments, the diagnostic moiety is a coloring agent, which refers to a chemical compound that induces a color reaction, as known in the art. Representative examples 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, derivatives of diphenylamine 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.

[0054] 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), PSMA (Ga-68), DOTATOC / DOTANOC / DOTATATE (Ga-68), florbetaben / florbetapir (F-18), rubidium (Rb-82), and FDDNP (F-18).

[0055] 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, gadoversetamide, gadoxetate, and gadobutrol.

[0056] In some embodiments, the diagnostic moiety is an intercalating agent, which, as known in the art, refers to an agent that inserts between stacked base pairs of DNA. Intercalating agents are hydrophobic heterocyclic ring molecules that resemble the ring structure of base pairs. Representative examples include ethidium bromide, acridine orange, and actinomycin D.

[0057] In some embodiments, R1 is a -[L]-diagnostic moiety, where [L] is a linking group that may be replaced by the same or a different -[L]-diagnostic moiety. In some embodiments, [L] is -O-, -S-, -N(R')-, -C≡C-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -C(NOR')-, -C(O)N(R')-, -N(R')C(O)N(R')-, -N(R')C(O)O-, -OC(O)N(R')-, -C(NR')-, -N(R')C(R')-, -N(R')C(O)N(R')-, -N(R')S(O)N(R')-, -OP(O)O(R')-, -N(R')P(O)N(R'R')N(R')-, C3-C 12 an alkylene chain optionally interrupted and / or terminated (at either or both ends) by at least one of carbocyclene, 3- to 12-membered heterocyclene, 5- to 12-membered heteroarylene, or any combination thereof; and each R' is independently H or an optionally substituted C1-C 24 In some embodiments, the alkylene chain is C1 to C6. 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 by and / or terminated (at either or both ends) with at least one of -O-, -S-, -N(R')-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(R')-, -N(R')C(O)-, or combinations thereof. In some embodiments, the alkylene chain is interrupted by and / or terminated (at either or both ends) with -N(R')-. In some embodiments, the alkylene chain is interrupted and / or terminated (at either or both ends) by -O-, hi some embodiments, the alkylene chain is interrupted and / or terminated (at either or both ends) by -S-.

[0058] In some embodiments, [L] is -O-, -S-, -N(R')-, -C≡C-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -C(NOR')-, -C(O)N(R')-, -N(R')C(O)N(R')-, -N(R')C(O)N(R')-, -N(R')C(O)N(R')-, -N(R')S(O)N(R')-, -OP(O)O(R')-, -N(R')P(O)N(R'R')N(R')-, C3-C 12 a polyethylene glycol chain optionally interrupted and / or terminated (at either or both ends) by at least one of carbocyclene, 3- to 12-membered heterocyclene, 5- to 12-membered heteroarylene, or any combination thereof, wherein each R' is independently H or an optionally substituted C1-C 24The alkyl groups are the interrupting and one or both terminal groups may be the same or different. 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 by and / or terminates (at either or both ends) in at least one of -S-, -N(R')-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(R')-, -N(R')C(O)-, or a combination thereof. In some embodiments, the polyethylene glycol chain is interrupted by -C(O)- and / or terminates (at either or both ends) with -C(O)-. In some embodiments, the polyethylene glycol chain is interrupted by -N(R')- and / or terminates (at either or both ends) with -N(R')-. In some embodiments, the polyethylene glycol chain is interrupted by -S- and / or terminates (at either or both ends) with -S-.

[0059] 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.

[0060] In some embodiments, R2 is hydrogen.

[0061] In some embodiments, R2 is C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, OH, CN, NO2, NH2, (C1-C6 alkyl)NH, (C1-C6 alkyl)2N, C3-C6 carbocyclyl, or 5-6 membered heterocyclyl.

[0062] In some embodiments, R2 is an inductive electron-withdrawing group. In some embodiments, the inductive electron-withdrawing group is halogen, OR6, SR6, or NR6R6, where each R6 is independently hydrogen, C1-C6 alkyl, C6-C6 alkyl, or C6-C6 alkyl. 12 In some embodiments, the inductive electron-withdrawing group is aryl, 5- to 10-membered heteroaryl, carbonyl, sulfonyl, sulfinyl, or phosphoryl. In some embodiments, the inductive electron-withdrawing group is halogen. In some embodiments, the halogen is fluoro or chloro.

[0063] In some embodiments, R2 is a π-electron withdrawing group. In some embodiments, the π-electron withdrawing group is -C(O)R6, -C(O)NR6R6, -C(O)NR6R6, -C(O)OR6, -S(O)R6, -S(O)2R6, -S(O)OR6, -S(O)NR6R6, -S(O)2NR6R6, -O-P(O)OR6OR6, or -P(O)NR6R6NR6R6, where each R6 is independently hydrogen, C1-C6 alkyl, C6-C 12 aryl, or 5- to 10-membered heteroaryl.

[0064] In some embodiments, R2 is a -[L]-diagnostic moiety. In some embodiments, R2 is a -[L]-diagnostic moiety as described above for R1. In some embodiments, R2 is a -[L]-diagnostic moiety, where [L] is a linking group optionally replaced by the same or a different -[L]-diagnostic moiety.

[0065] In some embodiments, R3 is hydrogen.

[0066] In some embodiments, R3 is C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, OH, CN, NO2, NH2, (C1-C6 alkyl)NH, (C1-C6 alkyl)2N, C3-C6 carbocyclyl, or 5-6 membered heterocyclyl.

[0067] In some embodiments, R3 is an inductive electron-withdrawing group. In some embodiments, the inductive electron-withdrawing group is halogen, OR6, SR6, or NR6R6, where each R6 is independently hydrogen, C1-C6 alkyl, C6-C6 alkyl, or C6-C6 alkyl. 12 In some embodiments, the inductive electron-withdrawing group is aryl, 5- to 10-membered heteroaryl, carbonyl, sulfonyl, sulfinyl, or phosphoryl. In some embodiments, the inductive electron-withdrawing group is halogen. In some embodiments, the halogen is fluoro or chloro.

[0068] In some embodiments, R3 is a π-electron withdrawing group. In some embodiments, the π-electron withdrawing group is -C(O)R6, -C(O)NR6R6, -C(O)NR6R6, -C(O)OR6, -S(O)R6, -S(O)2R6, -S(O)OR6, -S(O)NR6R6, -S(O)2NR6R6, -O-P(O)OR6OR6, or -P(O)NR6R6NR6R6, where each R6 is independently hydrogen, C1-C6 alkyl, C6-C 12 aryl, or 5- to 10-membered heteroaryl.

[0069] In some embodiments, R3 is a -[L]-diagnostic moiety. In some embodiments, R3 is a diagnostic moiety as described above for R1. In some embodiments, R3 is a -[L]-diagnostic moiety, where [L] is a linking group optionally replaced by the same or a different -[L]-diagnostic moiety.

[0070] In some embodiments, R1 and R3 are each -[L]-diagnostic moieties, which may be the same or different. In some embodiments, R1, R2, and R3 are each -[L]-diagnostic moieties, which may be the same or different from each other.

[0071] In some embodiments, R4 is hydrogen, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, OH, CN, NO2, NH2, (C1-C6 alkyl)NH, (C1-C6 alkyl)2N, C3-C6 carbocyclyl, or 5-6 membered heterocyclyl.

[0072] In some embodiments, R4 is a leaving group.

[0073] In some embodiments, R4 is a -[L]-diagnostic moiety. In some embodiments, R4 is a diagnostic moiety as described above for R1. In some embodiments, R4 is a -[L]-diagnostic moiety, where [L] is a linking group optionally replaced by the same or a different -[L]-diagnostic moiety.

[0074] In some embodiments, R4 is a cleavable linking group.

[0075] In some embodiments, R4 is -O-, -S-, -N(R')-, -C≡C-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -C(NOR')-, -C(O)N(R')-, -N(R')C(O)N(R')-, -N(R')C(O)O-, -OC(O)N(R')-, -C(NR')-, -N(R')C(R')-, -N(R')C(O)N(R')-, -N(R')S(O)N(R')-, -OP(O)O(R')-, -N(R')P(O)N(R'R')N(R')-, C3-C 12an alkylene chain interrupted and / or terminated (at either or both ends) by at least one of carbocyclene, 3- to 12-membered heterocyclene, 5- to 12-membered heteroarylene, or any combination thereof; and each R' is independently H or an optionally substituted C1-C 24 In some embodiments, the alkylene chain is C1 to C6. 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 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: -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')-, -OP(O)O(R')O-, -N(R')P(O)N(R'R')N(R')-, or a combination thereof. In some embodiments, the alkylene chain is interrupted and / or terminated (at either or both ends) by -OC(O)-. In some embodiments, the alkylene chain is interrupted by -OC(O)O- and / or terminated (at either or both ends) with -OC(O)O-. In some embodiments, the alkylene chain is interrupted by -OC(O)N(R')- and / or terminated (at either or both ends) with -OC(O)N(R')-.

[0076] In some embodiments, R4 is -O-, -S-, -N(R')-, -C≡C-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -C(NOR')-, -C(O)N(R')-, -N(R')C(O)N(R')-, -N(R')C(O)N(R')-, -N(R')C(O)N(R')-, -N(R')S(O)N(R')-, -OP(O)O(R')-, -N(R')P(O)N(R'R')N(R')-, C3-C 12 a polyethylene glycol chain interrupted and / or terminated (at either or both ends) by at least one of carbocyclene, 3- to 12-membered heterocyclene, 5- to 12-membered heteroarylene, or any combination thereof, wherein each R' is independently H or an optionally substituted C1-C 24The intermediate and one or both terminal groups are alkyl, and may be the same or different. 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: -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')-, -OP(O)O(R')O-, -N(R')P(O)N(R'R')N(R')-, or a combination thereof. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either or both ends) by -OC(O)-. In some embodiments, the polyethylene glycol chain is interrupted by -OC(O)O- and / or terminates (at either or both ends) with -OC(O)O-. In some embodiments, the polyethylene glycol chain is interrupted by -OC(O)N(R')- and / or terminates (at either or both ends) with -OC(O)N(R')-.

[0077] In some embodiments, R7 is Me, Et, n Bu, iPr, Cy, [ka] is.

[0078] In some embodiments, R7 and R8, together with the nitrogen atom to which they are attached, form a 6-membered heterocyclyl containing two heteroatoms selected from O and N. In some embodiments, R7 and R8, together with the nitrogen atom to which they are attached, form a piperidinyl, piperazinyl, or morpholinyl group.

[0079] In some embodiments, R8 is Me, Et, nBu, iPr, Cy, [ka] is.

[0080] In some embodiments, the optional substituents for R, R, R, R, R, and R are alkyl, alkenyl, alkynyl, halo, haloalkyl, cycloalkyl, heterocycloalkyl, hydroxy, alkoxy, cycloalkoxy, heterocycloalkoxy, haloalkoxy, aryloxy, heteroaryloxy, aralkyloxy, alkylenyloxy, 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 aryl, N-alkyl-N-arylaminosulfonyl, N-alkyl-N-heteroarylaminosulfonyl, formyl, alkylcarbonyl, haloalkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, carboxy, alkoxycarbonyl, alkylcarbonyloxy, amino, alkylaminosulfonyl, cycloalkylaminosulfonyl, heterocycloalkylaminosulfonyl, 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, cyan, nitro, azideand -[L]-diagnostic moieties. In some embodiments, any substituent may be one or more additional -[L]-diagnostic moieties, which may be the same or different.

[0081] In some embodiments, A is a therapeutic moiety. Generally, the therapeutic moiety can be any agent effective in treating a disease or disorder characterized by, associated with, or exhibiting tissue hypoxia. In some embodiments, the therapeutic moiety is 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 small molecule is a therapeutically active agent such as a drug (e.g., a molecule approved by the U.S. Food and Drug Administration as provided in the Code of Federal Regulations (CFR)).

[0082] 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)).

[0083] In some embodiments, the anti-cancer agent is a non-targeting agent, which, as known in the art, refers to an agent with a relatively broad mode of action that does not involve one or more specific molecular targets. Representative examples of non-targeting anti-cancer agents include alkylating agents (e.g., busulfan, chlorambucil, cyclophosphamide, ifosfamide, mechlorethamine, melphalen, carmustine, streptozocin, dacarbazine, temozolomide, altretamine, and thiotepa), antimetabolites (e.g., capecitabine, cytarabine, 5'-fluorouracillin, gemcitabine, cladribine, fludarabine, 6-mercaptopurine, and pentostatin), folate antagonists (e.g., methotrexate and pemetrexed), mitotic inhibitors (e.g., cetaxel, paclitaxel, vincristine ... blastine, 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.

[0084] Representative examples of 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 (CD K4, 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 agent is a kinase inhibitor.Representative examples of kinase inhibitors include abemaciclib, acalabrutinib, afatinib, alectinib, avapritinib, axitinib, baricitinib, benimetinib, bosutinib, brigatinib, cabozantinib, ceritinib, capmatinib, cobimetinib, crizotinib, dabrafenib, dacomitinib, dasatinib, encorafenib, entrectinib, erdafitinib, erlotinib, everolimus, fedratinib, fostamatinib, gefitinib, giltritanib, ibrotinib, icotinib, imatinib, and lapatinib. , larotrectinib, 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.

[0085] In some embodiments, the therapeutic moiety is a hypoxia-inducible factor inhibitor (HIF). Representative types of HIF include daprodustat, desidustat, molidustat, roxadustat, vadadustat, wortmannin, LY94002, GDC-0941, PI-103, rapamycin, PP242, aminoflavones, glyceollins (e.g., glyceollin I, glyceollin II, glyceollin III, and glyceollin IV), topotecan, EZN-2968, ENMD-1198, geldanamycin, vorinostat, and the like. Examples of HIF inhibitors that may be suitable for use in the present invention include thiazol-1, YC-1, PX-478, pleurotin, cardiac glycosides (e.g., convallotoxin, antalin, oabain, digoxin, digitoxin, oleandrin, adonitoxin, diglemmontianin, scillarenin, and proscillaridin A), FM19G11, acriflavine, PT2385, echinomycin, chetomin, bortezomib, amphotericin B, triptolide, and AJM290. Still other HIF inhibitors that may be suitable for use in the present invention are disclosed in U.S. Patent Application Publication Nos. 2017 / 0157112 and 2017 / 0157111.

[0086] In some embodiments, the therapeutic moiety is an apoptotic agent, which, as known in the art, refers to an agent that acts to cause apoptosis activation by stopping cells from cycling and blocking proliferation and survival, also known as programmed cell death. Representative examples include staurosporine, raptinal, anthracyclines (e.g., doxorubicin, daunorubicin, epirubicin, and idarubicin), prodigiosins (e.g., prodigiosin, nonylprodigiosin, undecylprodigiosin, metacycloprodigiosin, streptorubin B, and obatoclax), bortezomib, HGS-ETR1, HGS-ETR2, HGS-TR2J, PRO1762, TRA-8, CD95-Fc, adalimumab, and the like. These include rifabutin, etanercept, Remicade, CDP571, IDN-6556, IDN-6734, VX-799, MX1013, VX-740, VX-756, M-920, M-826, immunocaps-3, immunocaps-6, Ad-G / iCasp3, TWX024, embelin, LY2181308, chelerythrine, antimycin A, HA14-1, genasense, chalcone, ONYX-015, CDB3, amifostine, and pifithrin-α.

[0087] Representative examples of NSAIDs include celecoxib, diclofenac, diflunisal, etodolac, fenoprofen, flurbiprofen, ibuprofen, indomethacin, ketoprofen, ketorolac, mefenamic acid, meloxicam, nabumetone, naproxen, oxaprozin, piroxicam, sulindac, and tolmetin.

[0088] 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.

[0089] In some embodiments, the compound of Formula (I) is a compound represented by any one of the following structures or a pharmaceutically acceptable salt or stereoisomer thereof: [ka] [ka] In some embodiments, R1 is hydrogen, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 haloalkoxy, CN, NO2, NH2, (C1-C6 alkyl)NH, halogen, OR5, SR5, NR5R5, or a -[L]-diagnostic moiety, where each R5 is independently hydrogen, (C1-C6)alkyl, (C3-C 10 ) carbocyclyl, 4- to 7-membered heterocyclyl; and / or R2 is halogen, OR6, SR6, NR6R6, —C(O)R6, —C(O)NR6R6, —C(O)NR6R6, —C(O)OR6, —S(O)R6, —S(O)2R6, —S(O)OR6, —S(O)NR6R6, —S(O)2NR6R6, —O-P(O)OR6OR6, —P(O)NR6R6NR6R6, or a —[L]-diagnostic moiety, where each R6 is independently hydrogen, C1-C6 alkyl, C6-C 12 aryl, or 5- to 10-membered heteroaryl; and / or R3 is halogen, OR6, SR6, NR6R6, -C(O)R6, -C(O)NR6R6, -C(O)NR6R6, -C(O)OR6, -S(O)R6, -S(O)2R6, -S(O)OR6, -S(O)NR6R6, -S(O)2NR6R6, -O-P(O)OR6OR6, -P(O)NR6R6NR6R6, or -[L]-diagnostic moiety, where each R6 is independently hydrogen, C1-C6 alkyl, C6-C 12 aryl, or 5-10 membered heteroaryl; and / or R4 is a leaving group, a cleavable linking group, or a -[L]-diagnostic moiety; and / or A is absent or a therapeutic moiety. In some embodiments, halogen is fluoro or chloro. In some embodiments, C6-C 12Aryl is phenyl. In some embodiments, the 5-10 membered heteroaryl is pyrrole, furan, thiophene, pyridine, or pyrimidine. In some embodiments, C1-C6 alkyl is methyl, ethyl, or propyl or isopropyl. In some embodiments, A is an anti-cancer agent. In some embodiments, the diagnostic moiety is a fluorescent dye.

[0090] In some embodiments of a compound of Formula (I), R1 is hydrogen, C1-C6 alkyl, CN, NH2, (C1-C6 alkyl)NH, halogen, OR5, SR5, or NR5R5, where each R5 is independently hydrogen, (C1-C6) alkyl, (C3-C 10 ) carbocyclyl, or 4- to 7-membered heterocyclyl; and / or R2 is halogen, OR6, SR6, NR6R6, or a -[L]- diagnostic moiety, where each R6 is independently hydrogen, C1-C6 alkyl, C6-C 12 aryl, or 5- to 10-membered heteroaryl; and / or R3 is halogen, OR6, SR6, or NR6R6, where each R6 is independently hydrogen, C1-C6 alkyl, C6-C 12 aryl, or 5-10 membered heteroaryl; and / or R4 is a cleavable linking group; and / or A is a therapeutic moiety. In some embodiments, halogen is fluoro or chloro. In some embodiments, C6-C 12 Aryl is phenyl. In some embodiments, the 5-10 membered heteroaryl is pyrrole, furan, thiophene, pyridine, or pyrimidine. In some embodiments, C1-C6 alkyl is methyl, ethyl, or propyl or isopropyl. In some embodiments, A is an anticancer agent.

[0091] In some embodiments, the compound of Formula (I) is provided as a compound represented by any one of Formulas Ia-If, or a pharmaceutically acceptable salt or stereoisomer thereof. [ka]

[0092] In some embodiments, the compound of Formula (Ia) is a compound represented by any one of the following structures or a pharmaceutically acceptable salt or stereoisomer thereof: [ka] [ka] .

[0093] In some embodiments, the compound of Formula (Ib) is a compound represented by any one of the following structures or a pharmaceutically acceptable salt or stereoisomer thereof: [ka] [ka]

[0094] In some embodiments, the compound of Formula (Ic) is a compound represented by any one of the following structures or a pharmaceutically acceptable salt or stereoisomer thereof: [ka] [ka]

[0095] In some embodiments, the compound of Formula (Id) is a compound represented by any one of the following structures or a pharmaceutically acceptable salt or stereoisomer thereof: [ka] [ka]

[0096] In some embodiments, the compound of Formula (Ie) is a compound represented by any one of the following structures or a pharmaceutically acceptable salt or stereoisomer thereof: [ka] [ka]

[0097] In some embodiments, the compound of formula (If) is a compound represented by any one of the following structures or a pharmaceutically acceptable salt or stereoisomer thereof: [ka] [ka]

[0098] In some embodiments, the compound of formula (II) is a compound represented by any one of the following structures or a pharmaceutically acceptable salt or stereoisomer thereof: [ka] In some embodiments, R1 is hydrogen, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 haloalkoxy, CN, NO2, NH2, (C1-C6 alkyl)NH, halogen, OR5, SR5, NR5R5, or a -[L]-diagnostic moiety, where each R5 is independently hydrogen, (C1-C6)alkyl, (C3-C 10) carbocyclyl, or 4- to 7-membered heterocyclyl; and / or R2 is halogen, OR6, SR6, NR6R6, —C(O)R6, —C(O)NR6R6, —C(O)NR6R6, —C(O)OR6, —S(O)R6, —S(O)2R6, —S(O)OR6, —S(O)NR6R6, —S(O)2NR6R6, —O-P(O)OR6OR6, —P(O)NR6R6NR6R6, or a —[L]-diagnostic moiety, where each R6 is independently hydrogen, C1-C6 alkyl, C6-C 12 aryl, or 5- to 10-membered heteroaryl; and / or R3 is halogen, OR6, SR6, NR6R6, -C(O)R6, -C(O)NR6R6, -C(O)NR6R6, -C(O)OR6, -S(O)R6, -S(O)2R6, -S(O)OR6, -S(O)NR6R6, -S(O)2NR6R6, -O-P(O)OR6OR6, -P(O)NR6R6NR6R6, or -[L]-diagnostic moiety, where each R6 is independently hydrogen, C1-C6 alkyl, C6-C 12 aryl, or 5-10 membered heteroaryl; and / or A is absent or a therapeutic moiety. In some embodiments, halogen is fluorine or chloro. In some embodiments, C6-C 12 Aryl is phenyl. In some embodiments, the 5-10 membered heteroaryl is pyrrole, furan, thiophene, pyridine, or pyrimidine. In some embodiments, C1-C6 alkyl is methyl, ethyl, or propyl or isopropyl. In some embodiments, A is an anti-cancer agent. In some embodiments, the diagnostic moiety is a fluorescent dye.

[0099] In some embodiments of compounds of Formula (II), R1 is hydrogen, C1-C6 alkyl, CN, NH2, (C1-C6 alkyl)NH, halogen, OR5, SR5, or NR5R5, where each R5 is independently hydrogen, (C1-C6) alkyl, (C3-C 10) carbocyclyl, or 4- to 7-membered heterocyclyl; and / or R2 is halogen, OR6, SR6, NR6R6, or a -[L]- diagnostic moiety, where each R6 is independently hydrogen, C1-C6 alkyl, C6-C 12 aryl, or 5- to 10-membered heteroaryl; and / or R3 is halogen, OR6, SR6, or NR6R6, where each R6 is independently hydrogen, C1-C6 alkyl, C6-C 12 aryl, or 5- to 10-membered heteroaryl; and / or R4 is a cleavable linking group, and / or A is a therapeutic moiety. In some embodiments, halogen is fluorine or chloro. In some embodiments, C6-C 12 Aryl is phenyl. In some embodiments, the 5-10 membered heteroaryl is pyrrole, furan, thiophene, pyridine, or pyrimidine. In some embodiments, C1-C6 alkyl is methyl, ethyl, or propyl or isopropyl. In some embodiments, A is an anticancer agent.

[0100] In some embodiments, the compound of Formula (II) is a compound represented by Formula IIa-IIf or a pharmaceutically acceptable salt or stereoisomer thereof. [ka]

[0101] In some embodiments, the compound of Formula (IIa) is a compound represented by any one of the following structures or a pharmaceutically acceptable salt or stereoisomer thereof: [ka] [ka]

[0102] In some embodiments, the compound of Formula (IIb) is a compound represented by any one of the following structures or a pharmaceutically acceptable salt or stereoisomer thereof: [ka] [ka]

[0103] In some embodiments, the compound of Formula (IIc) is a compound represented by any one of the following structures or a pharmaceutically acceptable salt or stereoisomer thereof: [ka] [ka]

[0104] In some embodiments, the compound of Formula (IId) is a compound represented by any one of the following structures or a pharmaceutically acceptable salt or stereoisomer thereof: [ka] [ka]

[0105] In some embodiments, the compound of Formula (IIe) is a compound represented by any one of the following structures or a pharmaceutically acceptable salt or stereoisomer thereof: [ka] [ka]

[0106] In some embodiments, the compound of Formula (IIf) is a compound represented by any one of the following structures or a pharmaceutically acceptable salt or stereoisomer thereof: [ka] [ka]

[0107] In another embodiment, the compounds of the present invention are represented by Formulas III and IV, or pharmaceutically acceptable salts or stereoisomers thereof: [ka] where: R1 is hydrogen, CH2, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, OH, CN, NO2, NH2, (C1-C6 alkyl)NH, (C1-C6 alkyl)2N, C3-C6 carbocyclyl, 5- to 6-membered heterocyclyl, an inductive electron-withdrawing group, a cleavable linking group, or a -[L]-diagnostic moiety, wherein R1 is optionally substituted; [L] is a linking group that can carry zero or multiple diagnostic moieties, which can be the same or different; R2 is hydrogen, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, OH, CN, NO2, NH2, (C1-C6 alkyl)NH, (C1-C6 alkyl)2N, C3-C6 carbocyclyl, 5- to 6-membered heterocyclyl, an inductive electron-withdrawing group, a π-electron-withdrawing group, a leaving group, or a -[L]-diagnostic moiety, wherein R2 is optionally substituted; [L] is a linking group that can carry zero or multiple diagnostic moieties, which can be the same or different; R3 is hydrogen, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, OH, CN, NO2, NH2, (C1-C6 alkyl)NH, (C1-C6 alkyl)2N, C3-C6 carbocyclyl, 5- to 6-membered heterocyclyl, an inductive electron-withdrawing group, a π-electron-withdrawing group, a leaving group, or a -[L]-diagnostic moiety, wherein R3 is optionally substituted; [L] is a linking group that can carry zero or multiple diagnostic moieties, which can be the same or different; R4 is hydrogen, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, OH, CN, NO2, NH2, (C1-C6 alkyl)NH, (C1-C6 alkyl)2N, C3-C6 carbocyclyl, 5- to 6-membered heterocyclyl, an inductive electron-withdrawing group, a π-electron-withdrawing group, a leaving group, or a -[L]-diagnostic moiety, wherein R4 is optionally substituted; [L] is a linking group that can carry zero or multiple diagnostic moieties, which can be the same or different; R7 is (C1-C8) alkyl, (C3-C 10 ) carbocyclyl, or a 4- or 10-membered heterocyclyl containing 1 to 3 heteroatoms selected from O, N, and S, wherein said alkyl, carbocyclyl, or heterocyclyl may be further substituted; or R7 and R8, together with the nitrogen atom to which they are attached, form a 4-7 membered heterocyclyl containing 1-3 heteroatoms selected from O, N and S; R8 is (C1-C8) alkyl, (C3-C 10 ) carbocyclyl, or a 4- or 10-membered heterocyclyl containing 1 to 3 heteroatoms selected from O, N, and S, wherein said alkyl, carbocyclyl, or heterocyclyl may be further substituted; A is absent, a leaving group, or a therapeutic moiety provided that the compound of formula (III or IV) contains at least one -[L]-diagnostic or therapeutic moiety. When A is a therapeutic moiety, R1 is a cleavable linking group; When at least one of R2, R3, and R4 is a -[L]-diagnostic moiety of a compound of formula (III), R1 is CH2 and A is a leaving group; When at least one of R1, R2, and R3 is a -[L]-diagnostic moiety and A is absent for compounds of formula (IV), R4 is a leaving group.

[0108] In some embodiments, R1 is hydrogen, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, OH, CN, NO2, NH2, (C1-C6 alkyl)NH, (C1-C6 alkyl)2N, C3-C6 carbocyclyl, or 5-6 membered heterocyclyl.

[0109] In some embodiments, R1 is CH2.

[0110] In some embodiments, R1 is -[L]-diagnostic moiety. Diagnostic moieties typically contain a detectable moiety, such as a label. In certain embodiments, the label comprises a fluorescent dye. In some embodiments, the diagnostic moiety comprises a rhodamine dye. In some embodiments, the diagnostic moiety comprises tetramethylrhodamine (TAMRA) or a derivative thereof.

[0111] In some embodiments, the diagnostic moiety is an affinity tag.

[0112] In some embodiments, the diagnostic moiety is chromogenic.

[0113] In some embodiments, the diagnostic moiety is a PET tracer.

[0114] In some embodiments, the diagnostic moiety is an MRI contrast agent.

[0115] In some embodiments, the diagnostic moiety is an intercalating agent.

[0116] In some embodiments, R1 is a -[L]-diagnostic moiety, where [L] is a linking group that may be replaced by the same or a different -[L]-diagnostic moiety. In some embodiments, [L] is -O-, -S-, -N(R')-, -C≡C-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -C(NOR')-, -C(O)N(R')-, -N(R')C(O)N(R')-, -N(R')C(O)O-, -OC(O)N(R')-, -C(NR')-, -N(R')C(R')-, -N(R')C(O)N(R')-, -N(R')S(O)N(R')-, -OP(O)O(R')-, -N(R')P(O)N(R'R')N(R')-, C3-C 12 an alkylene chain optionally interrupted and / or terminated (at either or both ends) by at least one of carbocyclene, 3- to 12-membered heterocyclene, 5- to 12-membered heteroarylene, or any combination thereof, wherein each R' is independently H or an optionally substituted C-C 24 In some embodiments, the alkylene chain is C1 to C6. 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 by and / or terminated (at either or both ends) with at least one of -O-, -S-, -N(R')-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(R')-, -N(R')C(O)-, or combinations thereof. In some embodiments, the alkylene chain is interrupted by and / or terminated (at either or both ends) with -N(R')-. In some embodiments, the alkylene chain is interrupted and / or terminated (at either or both ends) by -O-, hi some embodiments, the alkylene chain is interrupted and / or terminated (at either or both ends) by -S-.

[0117] In some embodiments, [L] is -O-, -S-, -N(R')-, -C≡C-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -C(NOR')-, -C(O)N(R')-, -N(R')C(O)N(R')-, -N(R')C(O)N(R')-, -N(R')C(O)N(R')-, -N(R')S(O)N(R')-, -OP(O)O(R')-, -N(R')P(O)N(R'R')N(R')-, C3-C 12 a polyethylene glycol chain optionally interrupted and / or terminated (at either or both ends) by at least one of carbocyclene, 3- to 12-membered heterocyclene, 5- to 12-membered heteroarylene, or any combination thereof, wherein each R' is independently H or an optionally substituted C-C 24The alkyl groups are the interrupting and one or both terminal groups may be the same or different. 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 by and / or terminates (at either or both ends) in at least one of -S-, -N(R')-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)N(R')-, -N(R')C(O)-, or a combination thereof. In some embodiments, the polyethylene glycol chain is interrupted by -C(O)- and / or terminates (at either or both ends) with -C(O)-. In some embodiments, the polyethylene glycol chain is interrupted by -N(R')- and / or terminates (at either or both ends) with -N(R')-. In some embodiments, the polyethylene glycol chain is interrupted by -S- and / or terminates (at either or both ends) with -S-.

[0118] In some embodiments, R1 is a cleavable linking group.

[0119] In some embodiments, R1 is -O-, -S-, -N(R')-, -C≡C-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -C(NOR')-, -C(O)N(R')-, -N(R')C(O)N(R')-, -N(R')C(O)O-, -OC(O)N(R')-, -C(NR')-, -N(R')C(R')-, -N(R')C(O)N(R')-, -N(R')S(O)N(R')-, -OP(O)O(R')-, -N(R')P(O)N(R'R')N(R')-, C3-C 12 an alkylene chain interrupted and / or terminated (at either or both ends) by at least one of carbocyclene, 3- to 12-membered heterocyclene, 5- to 12-membered heteroarylene, or any combination thereof; and each R' is independently H or an optionally substituted C1-C 24 In some embodiments, the alkylene chain is C1 to C6. 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: -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')-, -OP(O)O(R')O-, -N(R')P(O)N(R'R')N(R')-, or a combination thereof. In some embodiments, the alkylene chain is interrupted and / or terminated (at either or both ends) by -OC(O)-. In some embodiments, the alkylene chain is interrupted by -OC(O)O- and / or terminated (at either or both ends) with -OC(O)O-. In some embodiments, the alkylene chain is interrupted by -OC(O)N(R')- and / or terminated (at either or both ends) with -OC(O)N(R')-.

[0120] In some embodiments, R1 is -O-, -S-, -N(R')-, -C≡C-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -C(NOR')-, -C(O)N(R')-, -N(R')C(O)N(R')-, -N(R')C(O)N(R')-, -N(R')C(O)N(R')-, -N(R')S(O)N(R')-, -OP(O)O(R')-, -N(R')P(O)N(R'R')N(R')-, C3-C 12 a polyethylene glycol chain interrupted and / or terminated (at either or both ends) by at least one of carbocyclene, 3- to 12-membered heterocyclene, 5- to 12-membered heteroarylene, or any combination thereof, wherein each R' is independently H or an optionally substituted C-C24 The intermediate and one or both terminal groups are alkyl, and may be the same or different. 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: -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')-, -OP(O)O(R')O-, -N(R')P(O)N(R'R')N(R')-, or a combination thereof. In some embodiments, the polyethylene glycol chain is interrupted and / or terminated (at either or both ends) by -OC(O)-. In some embodiments, the polyethylene glycol chain is interrupted by -OC(O)O- and / or terminates (at either or both ends) with -OC(O)O-. In some embodiments, the polyethylene glycol chain is interrupted by -OC(O)N(R')- and / or terminates (at either or both ends) with -OC(O)N(R')-.

[0121] In some embodiments, R2 is hydrogen.

[0122] In some embodiments, R2 is C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, OH, CN, NO2, NH2, (C1-C6 alkyl)NH, (C1-C6 alkyl)2N, C3-C6 carbocyclyl, or 5-6 membered heterocyclyl.

[0123] In some embodiments, R2 is a leaving group.

[0124] In some embodiments, R2 is an inductive electron-withdrawing group. In some embodiments, the inductive electron-withdrawing group is halogen, OR6, SR6, or NR6R6, where each R6 is independently hydrogen, C1-C6 alkyl, C6-C6 alkyl, or C6-C6 alkyl. 12 In some embodiments, the inductive electron-withdrawing group is aryl, 5- to 10-membered heteroaryl, carbonyl, sulfonyl, sulfinyl, or phosphoryl. In some embodiments, the inductive electron-withdrawing group is halogen. In some embodiments, the halogen is fluoro or chloro.

[0125] In some embodiments, R2 is a π-electron withdrawing group. In some embodiments, the π-electron withdrawing group is -C(O)R6, -C(O)NR6R6, -C(O)NR6R6, -C(O)OR6, -S(O)R6, -S(O)2R6, -S(O)OR6, -S(O)NR6R6, -S(O)2NR6R6, -O-P(O)OR6OR6, or -P(O)NR6R6NR6R6, where each R6 is independently hydrogen, C1-C6 alkyl, C6-C 12 aryl, or 5- to 10-membered heteroaryl.

[0126] In some embodiments, R2 is a -[L]-diagnostic moiety. In some embodiments, R2 is a diagnostic moiety as described above for R1. In some embodiments, R2 is a -[L]-diagnostic moiety, where [L] is a linking group optionally replaced by the same or a different -[L]-diagnostic moiety.

[0127] In some embodiments, R3 is hydrogen.

[0128] In some embodiments, R3 is C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, OH, CN, NO2, NH2, (C1-C6 alkyl)NH, (C1-C6 alkyl)2N, C3-C6 carbocyclyl, or 5-6 membered heterocyclyl.

[0129] In some embodiments, R3 is a leaving group.

[0130] In some embodiments, R3 is an inductive electron-withdrawing group. In some embodiments, the inductive electron-withdrawing group is halogen, OR6, SR6, or NR6R6, where each R6 is independently hydrogen, C1-C6 alkyl, C6-C6 alkyl, or C6-C6 alkyl. 12 In some embodiments, the inductive electron-withdrawing group is aryl, 5- to 10-membered heteroaryl, carbonyl, sulfonyl, sulfinyl, or phosphoryl. In some embodiments, the inductive electron-withdrawing group is halogen. In some embodiments, the halogen is fluoro or chloro.

[0131] In some embodiments, R3 is a π-electron withdrawing group. In some embodiments, the π-electron withdrawing group is -C(O)R6, -C(O)NR6R6, -C(O)NR6R6, -C(O)OR6, -S(O)R6, -S(O)2R6, -S(O)OR6, -S(O)NR6R6, -S(O)2NR6R6, -O-P(O)OR6OR6, or -P(O)NR6R6NR6R6, where each R6 is independently hydrogen, C1-C6 alkyl, C6-C 12 aryl, or 5- to 10-membered heteroaryl.

[0132] In some embodiments, R3 is a -[L]-diagnostic moiety. In some embodiments, R3 is a diagnostic moiety as described above for R1. In some embodiments, R3 is a -[L]-diagnostic moiety, where [L] is a linking group optionally replaced by the same or a different -[L]-diagnostic moiety.

[0133] In some embodiments, R4 is hydrogen.

[0134] In some embodiments, R4 is C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, OH, CN, NO2, NH2, (C1-C6 alkyl)NH, (C1-C6 alkyl)2N, C3-C6 carbocyclyl, or 5-6 membered heterocyclyl.

[0135] In some embodiments, R4 is a leaving group.

[0136] In some embodiments, R4 is an inductive electron-withdrawing group. In some embodiments, the inductive electron-withdrawing group is halogen, OR6, SR6, or NR6R6, where each R6 is independently hydrogen, C1-C6 alkyl, C6-C 12 In some embodiments, the inductive electron-withdrawing group is aryl, 5- to 10-membered heteroaryl, carbonyl, sulfonyl, sulfinyl, or phosphoryl. In some embodiments, the inductive electron-withdrawing group is halogen. In some embodiments, the halogen is fluoro or chloro.

[0137] In some embodiments, R4 is a -[L]-diagnostic moiety. In some embodiments, R4 is a diagnostic moiety as described above for R1. In some embodiments, R4 is a -[L]-diagnostic moiety, where [L] is a linking group optionally replaced by the same or a different -[L]-diagnostic moiety.

[0138] In some embodiments, R4 is hydrogen.

[0139] In some embodiments, R7 is Me, Et, n Bu, iPr, Cy, [ka] is.

[0140] In some embodiments, R7 and R8, together with the nitrogen atom to which they are attached, form a 6-membered heterocyclyl containing two heteroatoms selected from O and N. In some embodiments, R7 and R8, together with the nitrogen atom to which they are attached, form a piperidinyl, piperazinyl, or morpholinyl group.

[0141] In some embodiments, R8 is Me, Et, nBu, iPr, Cy, [ka] is.

[0142] In some embodiments, the optional substituents for R, R, R, R, R, and R are alkyl, alkenyl, alkynyl, halo, haloalkyl, cycloalkyl, heterocycloalkyl, hydroxy, alkoxy, cycloalkoxy, heterocycloalkoxy, haloalkoxy, aryloxy, heteroaryloxy, aralkyloxy, alkylenyloxy, 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 aryl, N-alkyl-N-arylaminosulfonyl, N-alkyl-N-heteroarylaminosulfonyl, formyl, alkylcarbonyl, haloalkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, carboxy, alkoxycarbonyl, alkylcarbonyloxy, amino, alkylaminosulfonyl, cycloalkylaminosulfonyl, heterocycloalkylaminosulfonyl, 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, cyan, nitro, azideand -[L]-diagnostic moieties. In some embodiments, any substituent may be one or more additional -[L]-diagnostic moieties, which may be the same or different.

[0143] In some embodiments, A is a therapeutic moiety. In some embodiments, the therapeutic moiety is 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 small molecule 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)).

[0144] In some embodiments, the therapeutic moiety is an anti-cancer agent.

[0145] In some embodiments, the anti-cancer agent is a non-targeting agent.

[0146] In some embodiments, the therapeutic moiety is a targeted anti-cancer agent. In some embodiments, the targeted anti-cancer agent is a kinase inhibitor.

[0147] In some embodiments, the therapeutic moiety is a hypoxia inducible factor inhibitor (HIF).

[0148] In some embodiments, the therapeutic moiety is an apoptotic agent.

[0149] In some embodiments, the therapeutic moiety is a nonsteroidal anti-inflammatory drug (NSAID).

[0150] In some embodiments, the therapeutic moiety is a disease-modifying antirheumatic drug (DMARD).

[0151] In some embodiments, A is absent.

[0152] In some embodiments, A is a leaving group.

[0153] In some embodiments, the compound of Formula (III) is represented by any one of the following structures: [ka] or a pharmaceutically acceptable salt or stereoisomer thereof. In some embodiments, R1 is CH2, a cleavable linking group, or -[L]-diagnostic moiety; and / or R2 is halogen, OR6, SR6, NR6R6, -C(O)R6, -C(O)NR6R6, -C(O)NR6R6, -C(O)OR6, -S(O)R6, -S(O)2R6, -S(O)OR6, -S(O)NR6R6, -S(O)2NR6R6, -OP(O)OR6OR6, -P(O)NR6R6NR6R6, or -[L]-diagnostic moiety, wherein each R6 is independently hydrogen, C1-C6 alkyl, C6-C 12 aryl, or 5- to 10-membered heteroaryl; and / or R3 is halogen, OR6, SR6, NR6R6, -C(O)R6, -C(O)NR6R6, -C(O)NR6R6, -C(O)OR6, -S(O)R6, -S(O)2R6, -S(O)OR6, -S(O)NR6R6, -S(O)2NR6R6, -O-P(O)OR6OR6, -P(O)NR6R6NR6R6, or -[L]-diagnostic moiety, where each R6 is independently hydrogen, C1-C6 alkyl, C6-C 12aryl, or 5-10 membered heteroaryl; and / or R4 is hydrogen, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 haloalkoxy, CN, NO2, NH2, (C1-C6 alkyl)NH, halogen, OR5, SR5, NR5R5, or a -[L]-diagnostic moiety, where each R5 is independently hydrogen, (C1-C6) alkyl, (C3-C 10 ) carbocyclyl, 4- to 7-membered heterocyclyl; and / or A is absent, a leaving group, or a therapeutic moiety. In some embodiments, halogen is fluorine or chloro. In some embodiments, C6-C 12 Aryl is phenyl. In some embodiments, the 5-10 membered heteroaryl is pyrrole, furan, thiophene, pyridine, or pyrimidine. In some embodiments, C1-C6 alkyl is methyl, ethyl, or propyl or isopropyl. In some embodiments, A is an anti-cancer agent. In some embodiments, the diagnostic moiety is a fluorescent dye.

[0154] In some embodiments of compounds of Formula (III), R1 is a cleavable linking group; and / or R2 is halogen, OR6, SR6, NR6R6, or a -[L]-diagnostic moiety, where each R6 is independently hydrogen, C1-C6 alkyl, C6-C 12 aryl, or a 5- to 10-membered heteroaryl; and / or R3 is halogen, OR6, SR6, NR6R6, or -[L]-diagnostic moiety, where each R6 is independently hydrogen, C1-C6 alkyl, CN, NH2, (C1-C6 alkyl)NH, halogen, OR5, SR5, or NR5R5; and / or A is a therapeutic moiety. In some embodiments, halogen is fluorine or chloro. In some embodiments, C6-C 12Aryl is phenyl. In some embodiments, the 5-10 membered heteroaryl is pyrrole, furan, thiophene, pyridine, or pyrimidine. In some embodiments, C1-C6 alkyl is methyl, ethyl, or propyl or isopropyl. In some embodiments, A is an anticancer agent.

[0155] In some embodiments, the compound of Formula (III) is a compound represented by any one of Formulas IIIa-IIId, or a pharmaceutically acceptable salt or stereoisomer thereof: [ka] [ka] where LG=leaving group.

[0156] In some embodiments, the compound of Formula (IIIa) is a compound represented by any one of the following structures or a pharmaceutically acceptable salt or stereoisomer thereof: [ka] [ka] [ka]

[0157] In some embodiments, the compound of Formula (IIIb) is a compound represented by any one of the following structures or a pharmaceutically acceptable salt or stereoisomer thereof: [ka] [ka] or a pharmaceutically acceptable salt or stereoisomer thereof.

[0158] In some embodiments, the compound of Formula (IIIc) is a compound represented by any one of the following structures or a pharmaceutically acceptable salt or stereoisomer thereof: [ka] [ka]

[0159] In some embodiments, the compound of Formula (IIId) is a compound represented by any one of the following structures or a pharmaceutically acceptable salt or stereoisomer thereof: [ka] [ka]

[0160] In some embodiments, the compound of formula (IV) is a compound represented by any one of the following structures or a pharmaceutically acceptable salt or stereoisomer thereof: [ka] In some embodiments, R1 is CH2, a cleavable linking group, or a -[L]-diagnostic moiety; and / or R2 is halogen, OR6, SR6, NR6R6, -C(O)R6, -C(O)NR6R6, -C(O)NR6R6, -C(O)OR6, -S(O)R6, -S(O)2R6, -S(O)OR6, -S(O)NR6R6, -S(O)2NR6R6, -O-P(O)OR6OR6, -P(O)NR6R6NR6R6, or a -[L]-diagnostic moiety, where each R6 is independently hydrogen, C1-C6 alkyl, C6-C8 alkyl, C8-C9 alkyl, C10-C9 alkyl, C12-C9 alkyl, C14-C9 alkyl, C16-C9 alkyl, C18 ... 12aryl, or 5- to 10-membered heteroaryl; and / or R3 is halogen, OR6, SR6, NR6R6, -C(O)R6, -C(O)NR6R6, -C(O)NR6R6, -C(O)OR6, -S(O)R6, -S(O)2R6, -S(O)OR6, -S(O)NR6R6, -S(O)2NR6R6, -O-P(O)OR6OR6, -P(O)NR6R6NR6R6, or -[L]-diagnostic moiety, where each R6 is independently hydrogen, C1-C6 alkyl, C6-C 12 aryl, or 5-10 membered heteroaryl; and / or R4 is hydrogen, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 haloalkoxy, CN, NO2, NH2, (C1-C6 alkyl)NH, halogen, OR5, SR5, NR5R5, or a -[L]-diagnostic moiety, where each R5 is independently hydrogen, (C1-C6) alkyl, (C3-C 10 ) carbocyclyl, 4- to 7-membered heterocyclyl; and / or A is absent, a leaving group, or a therapeutic moiety. In some embodiments, halogen is fluorine or chloro. In some embodiments, C6-C 12 Aryl is phenyl. In some embodiments, the 5-10 membered heteroaryl is pyrrole, furan, thiophene, pyridine, or pyrimidine. In some embodiments, C1-C6 alkyl is methyl, ethyl, or propyl or isopropyl. In some embodiments, A is an anti-cancer agent. In some embodiments, the diagnostic moiety is a fluorescent dye.

[0161] In some embodiments of compounds of Formula (IV), R1 is a cleavable linking group; and / or R2 is halogen, OR6, SR6, NR6R6, or a -[L]-diagnostic moiety, where each R6 is independently hydrogen, C1-C6 alkyl, C6-C 12 aryl, or 5- to 10-membered heteroaryl; and / or R3 is halogen, OR6, SR6, NR6R6, or a -[L]-diagnostic moiety, where each R6 is independently hydrogen, C1-C6 alkyl, C6-C 12aryl, or 5- to 10-membered heteroaryl; and / or R4 is hydrogen, C1-C6 alkyl, CN, NH2, (C1-C6 alkyl)NH, halogen, OR5, SR 5、 or NR5R5; and / or A is a therapeutic moiety. In some embodiments, halogen is fluorine or chloro. In some embodiments, C6-C 12 Aryl is phenyl. In some embodiments, the 5-10 membered heteroaryl is pyrrole, furan, thiophene, pyridine, or pyrimidine. In some embodiments, C1-C6 alkyl is methyl, ethyl, or propyl or isopropyl. In some embodiments, A is an anticancer agent.

[0162] In some embodiments, the compound of formula (IV) is a compound represented by formula IVa-IVd or a pharmaceutically acceptable salt or stereoisomer thereof. [ka]

[0163] In some embodiments, the compound of formula (IVa) is a compound represented by any one of the following structures or a pharmaceutically acceptable salt or stereoisomer thereof: [ka] [ka]

[0164] In some embodiments, the compound of Formula (IVb) is a compound represented by any one of the following structures or a pharmaceutically acceptable salt or stereoisomer thereof: [ka] [ka]

[0165] In some embodiments, the compound of Formula (IVc) is a compound represented by any one of the following structures or a pharmaceutically acceptable salt or stereoisomer thereof: [ka] [ka]

[0166] In some embodiments, the compound of Formula (IVd) is a compound represented by any one of the following structures or a pharmaceutically acceptable salt or stereoisomer thereof: [ka] [ka]

[0167] In some embodiments, the compounds of the present invention are [ka] or a pharmaceutically acceptable salt or stereoisomer thereof.

[0168] In some embodiments, the compounds of the present invention are [ka] or a pharmaceutically acceptable salt or stereoisomer thereof.

[0169] As used herein, the term "pharmaceutically acceptable" in the context of a salt refers to a salt of a compound that does not abrogate the biological activity or properties of the compound and is relatively non-toxic; i.e., the salt form of the compound can be administered to a subject without causing undesired biological effects (such as dizziness or stomach upset) or interacting in a deleterious 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 an appropriate 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 include 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, saccharate, 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.

[0170] The compounds of the present invention may have at least one chiral center and therefore may be in the form of stereoisomers, which as used herein encompasses 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 compounds with two or more chiral centers that are not mirror images of one another (diastereoisomers). The chiral centers of a compound may undergo epimerization in vivo; therefore, for these compounds, administration of a compound in its (R-) form is considered equivalent to administration of a 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.

[0171] In some embodiments, the compounds of the present invention may be in the form of 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. In one embodiment, the compound is 11 C or multiple 11 In one embodiment, the compound comprises 13 N or multiple 13 In one embodiment, the compound comprises: 15 O or multiple 15 In one embodiment, the compound contains 18 F or multiple 18 Contains F 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.

[0172] 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.

[0173] 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 polycrystal. The compound of the present invention comprises a system of components including a compound of the present invention and a co-crystal former, wherein the compound of the present invention and the co-crystal former are connected 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. Synthesis method

[0174] In another aspect, the present invention is directed to methods for making the compounds of the present invention, or pharmaceutically acceptable salts or stereoisomers thereof. Generally, the compounds of the present invention, or pharmaceutically acceptable salts or stereoisomers thereof, can be prepared by any method 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 of the present invention can be prepared.

[0175] In another aspect, the present invention provides compounds of formula I and II [ka] 1. A method for preparing a compound of formula V: [ka] with a compound of formula VII: [ka] This includes reacting with

[0176] In another aspect, the present invention provides a compound of formula III or IV [ka] 1. A method for preparing a compound of formula VI: [ka] with a compound of formula VII: [ka] The method includes reacting an alkynyl group with a hydroxyl group. Synthetic schemes for attaching an active moiety to a compound (e.g., an alkynyl group-containing compound) are known in the art. For example, see Wang et al., J. Am. Chem. Soc. 141(43):17133-17141 (2019) and Guerrant et al., Bioorg. Med. Chem. Lett. 23(11):3283-3287 (2013).

[0177] In some embodiments, the reacting is carried out in the presence of a solvent.

[0178] In some embodiments, the solvent is an aprotic solvent, hi some embodiments, the aprotic solvent is DCM, CHCl3, CCl4, DCE, toluene, MeCN, or THF.

[0179] In some embodiments, the solvent is a protic solvent. In some embodiments, the protic solvent is MeOH, EtOH, iPrOH, nBuOH, TFE, or HFIP.

[0180] In some embodiments, the solvent is a solvent mixture. In some embodiments, the solvent mixture is a mixture of an aprotic solvent and a protic solvent. 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.

[0181] 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.

[0182] 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.

[0183] 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.

[0184] 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.

[0185] In some embodiments, the reaction is carried out at a temperature of about -40°C to 80°C. In some embodiments, the reaction is carried out at a temperature of about 0°C to 60°C. In some embodiments, the reaction is carried out at a temperature of about 20°C to 60°C. In some embodiments, the reaction is carried out at a temperature of about 60°C. In some embodiments, the reaction is carried out at a temperature of about 20°C to 25°C.

[0186] In some embodiments, the compound of Formula (VII) is in excess of the compound of Formula (V) or (VI). In some embodiments, the excess is about 10 equivalents. In some embodiments, the excess is about 5 equivalents.

[0187] 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.

[0188] In some embodiments, the reaction is carried out at a temperature of about 60° C., and / or the solvent mixture is about 20% TFE in CHCl 3 , and / or the reaction is carried out for 18 hours. Pharmaceutical Composition

[0189] 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.

[0190] 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, 2000 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.

[0191] In some embodiments, the compounds are formulated for oral or intravenous administration (eg, systemic intravenous injection).

[0192] 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); semisolid compositions (e.g., gels, suspensions, and creams); and gases (e.g., propellants for aerosol compositions). The compounds may also be formulated for immediate, intermediate, or sustained release.

[0193] 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) a filler or extender such as starch, lactose, sucrose, glucose, mannitol, and silicic acid, b) a binder such as methylcellulose, microcrystalline cellulose, hydroxypropylmethylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) a humectant such as glycerol, d) a cross-linked polymer (e.g., cross-linked polyvinylpyrrolidone (crospovidone), cross-linked sodium carboxymethylcellulose, e.g., hydroxypropylmethylcellulose ... They are mixed with additional carriers or excipients such as disintegrating agents such as (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, dosage forms may also contain buffering agents. Solid compositions of a similar type may also be used as fillers in soft- and hard-filled 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.

[0194] In some embodiments, the compounds of the present invention can be formulated in hard or soft gelatin capsules.Representative excipients that can be used include pregelatinized starch, magnesium stearate, mannitol, sodium stearyl fumarate, lactose anhydrous, microcrystalline cellulose, and croscarmellose sodium.The gelatin shell can contain gelatin, titanium dioxide, iron oxide, and coloring agents.

[0195] 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.

[0196] Injectable preparations for parenteral administration may include sterile aqueous or oily suspensions. They 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 bland, fixed oil, including synthetic mono- or diglycerides, may be used. In addition, fatty acids such as oleic acid are used in injectable preparations. Injectable preparations may 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 before use. The effect of a compound can be prolonged by slowing its absorption, which can be accomplished by using a liquid suspension with poor water solubility or crystalline or amorphous material. Prolonged absorption of a compound from a parenterally administered formulation can also be achieved by suspending the compound in an oil vehicle.

[0197] In certain embodiments, the compounds of the present invention can be administered locally rather than systemically, for example, via injection of the conjugate directly into an organ, often in a depot or sustained-release formulation. In certain embodiments, long-acting formulations are administered by implantation (e.g., subcutaneously or intramuscularly) or by 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 release rate of the compound can be controlled by varying the compound-to-polymer ratio and the properties of the particular polymer used. Depot injectable formulations can also be prepared by entrapping the compound in liposomes or microemulsions that are compatible with body tissues. Furthermore, in other embodiments, the compound is delivered in a targeted drug delivery system, for example, liposomes coated with organ-specific antibodies. In such embodiments, the liposomes are targeted to and selectively taken up by the organ.

[0198] The compositions may be formulated for buccal or sublingual administration and include, for example, tablets, lozenges and gels.

[0199] The compound of the present invention can be formulated for administration by inhalation.Various forms suitable for administration by inhalation include aerosol, mist or powder.Pharmaceutical compositions can be delivered in the form of aerosol spray presentation from pressurized pack or nebulizer using suitable propellant (for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas).In some embodiments, the dosage unit of pressurized aerosol can be determined by providing a valve to deliver a metered amount.In some embodiments, for example, capsules and cartridges containing gelatin for use in inhaler or insufflator can be formulated to contain a powder mixture of compound and suitable powder base such as lactose or starch.

[0200] The compounds of the present invention can be formulated for topical administration, which as used herein refers to intradermal administration of a formulation according to the present invention to the epidermis. These types of compositions are typically in the form of ointments, pastes, creams, lotions, gels, solutions and sprays.

[0201] Typical 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 with saturated or unsaturated fatty acids such as stearic acid, palmitic acid, oleic acid, palmito-oleic acid, cetyl, or oleyl alcohol.Cream can also contain nonionic surfactants such as polyoxy-40-stearate.

[0202] In some embodiments, topical formulations may also contain excipients, such as penetration enhancers. These agents can preferably transport pharmacologically active compounds through the stratum corneum to the epidermis or dermis 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 reviews 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). Representative examples of penetration enhancers include triglycerides (e.g., soybean oil), aloe compositions (e.g., aloe vera gel), ethyl alcohol, isopropyl alcohol, octylphenyl polyethylene glycol, oleic acid, polyethylene glycol 400, propylene glycol, N-decylmethyl sulfoxide, fatty acid esters (e.g., isopropyl myristate, methyl laurate, glycerol monooleate, and propylene glycol monooleate), and N-methylpyrrolidone.

[0203] Representative examples of other excipients that may be included in topical and other types of formulations (to the extent 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, butylated hydroxytoluene, butylated hydroxyanisole, 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 chlorides, cyclodextrins, benzyl benzoate, lecithin, and polysorbates. Suitable skin protectants include vitamin E oil, allatoin, dimethicone, glycerin, petrolatum, and zinc oxide.

[0204] Transdermal formulations typically use transdermal delivery devices and transdermal delivery patches, in which compounds are formulated in lipophilic emulsions or buffered aqueous solutions dissolved and / or dispersed in polymers or adhesives. Patches can be constructed 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, where the absorption rate is slowed by using rate-controlling membranes or by trapping the compound in a polymer matrix or gel. Absorption enhancers can be used to increase absorption, and examples include absorbable pharmaceutically acceptable solvents that aid in passage through the skin.

[0205] Ophthalmic preparations include eye drops.

[0206] Preparations for rectal administration include enemas, rectal gels, rectal foams, rectal aerosols, and retention enemas, which may contain conventional suppository bases such as cocoa butter or other glycerides, and synthetic polymers such as polyvinylpyrrolidone, PEG, etc. Compositions for rectal or vaginal administration can also be formulated as suppositories, which can be prepared by mixing the compound with suitable non-irritating carriers and excipients, such as cocoa butter, a mixture of fatty acid glycerides, polyethylene glycol, suppository wax, and combinations thereof, all of which are solid at ambient temperature but liquid at body temperature, and therefore melt in the rectum or vaginal cavity and release the compound. Dosage

[0207] As used herein, the term "therapeutically effective amount" refers to an amount of a compound of the present invention (containing a therapeutic moiety), or a pharmaceutically acceptable salt or stereoisomer thereof, effective to produce a desired therapeutic response in a patient suffering from a disease or disorder characterized by, associated with, or exhibiting tissue hypoxia. Thus, the term "therapeutically effective amount" 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 change 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 symptoms of the disease or disorder being treated in a subject, or to inhibit the proliferation of diseased cells.

[0208] As used herein, the term "diagnostically effective amount" refers to an amount of a compound of the present invention (including a diagnostic moiety), or a pharmaceutically acceptable salt or stereoisomer thereof, effective to produce a desired detectable response in a patient suffering from a disease or disorder characterized by, associated with, or exhibiting tissue hypoxia. Thus, the term "diagnostically effective amount" includes an amount of a compound of the present invention, or a pharmaceutically acceptable salt or stereoisomer thereof, that, when administered, induces a signal that can be detected or visualized by any of a variety of means, including spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, or chemical means.

[0209] The total daily dosage of compound and its use can be determined according to standard medical practice, for example, by attending physician with 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).

[0210] 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. How to use

[0211] In some embodiments, the present invention is directed to a method for labeling hypoxic tissue, e.g., a solid tumor, comprising administering to a subject in need thereof a compound of Formula (I, II, III, or IV), or a pharmaceutically acceptable salt or stereoisomer thereof. The active moiety contained in the compound of the present invention is selected accordingly.

[0212] In some embodiments, the present invention is directed to a method for treating a disease or disorder characterized by, associated with, or exhibiting tissue hypoxia, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of Formula (I, II, III, or IV), or a pharmaceutically acceptable salt or stereoisomer thereof. The active moiety contained in the compound of the present invention is selected accordingly.

[0213] The term "subject" (or "patient") as used herein includes all members of the animal kingdom susceptible to or afflicted with solid tumors. The methods are also applicable to companion animals such as dogs and cats, as well as livestock such as cattle, horses, sheep, goats, pigs, and other domesticated and wild animals. A subject "in need" of treatment according to the present invention may be one who has tested positive for or is "suffering from or suspected of having" a disease or disorder that exhibits a sufficient number of risk factors or a sufficient number or combination of signs or symptoms such that a medical professional can diagnose or suspect the subject of having the disease or disorder.

[0214] Diseases and disorders characterized by, associated with, or exhibiting tissue hypoxia are known in the art. See, for example, Wigerup et al., Pharm. Thera. 164:152-169 (2016); Sharma et al., Chem. Soc. Rev. 48(3):771-813 (2019); Bernauer et al., Brit. J. Cancer 124:539-551 (2021); Jing et al., Mol. Cancer 18:157 (2019). Tissue hypoxia can be an inherent characteristic of a disease or disorder. In some embodiments, localized tissue hypoxia can be induced by administration of a hypoxia-activating agent.

[0215] Exemplary classes of non-cancerous (e.g., cell proliferative) diseases or disorders that are characterized by, associated with, or exhibit tissue hypoxia and may be suitable for treatment with the compounds of the invention include inflammatory diseases and conditions, anemia, renal failure, cardiovascular disease, reperfusion injury, and metabolic diseases.

[0216] Representative examples of specific non-cancerous diseases and disorders include coronary heart disease, stroke, peripheral arterial heart disease, aortic disease, cerebrovascular disease, rheumatic heart disease, congenital heart disease, deep vein thrombosis, pulmonary embolism, arrhythmias, hyperlactemia, metabolic brain diseases, DNA repair deficiencies, porphyrias, metabolic skin diseases, and proteostasis deficiencies.

[0217] In some embodiments, the method relates to treating a subject with cancer. Generally, the compounds of the present invention can be effective in treating carcinomas (solid tumors, including both primary and metastatic tumors), sarcomas, melanomas, and hematological cancers (cancers affecting the blood, including lymphocytes, bone marrow, and / or lymph nodes), such as leukemia, lymphoma, and multiple myeloma. This includes adult tumors / cancers and pediatric tumors / cancers. The cancer can be a tumor that is vascularized, or not yet substantially vascularized, or not vascularized.

[0218] Representative examples of cancer include adrenocortical carcinoma, AIDS-related cancer (e.g., Kaposi's sarcoma and AIDS-related lymphoma), appendix cancer, childhood cancer (e.g., childhood cerebellar astrocytoma, childhood cerebral astrocytoma), basal cell carcinoma, skin (non-melanoma) cancer, biliary tract cancer, extrahepatic bile duct cancer, intrahepatic bile duct cancer, bladder cancer, brain tumors (e.g., brain stem glioma, gestational trophoblastic tumor glioma, cerebellar astrocytoma, cerebral astrocytoma / malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumor, visual pathway and hypothalamic glioma), breast cancer, and bronchial adenoma / carcinoid. , carcinoid tumors, nervous system cancers (e.g., central nervous system cancer, central nervous system lymphoma), cervical cancer, chronic myeloproliferative disorders, colorectal cancers (e.g., colon cancer, rectal cancer), polycythemia vera, lymphoid neoplasms, 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 cancers (e.g., gastric cancer, small intestine cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors (GIST)), germ cell tumors, ovarian germ cell tumors, head and neck cancer, Hodgkin's lymphoma, leukemia , lymphoma, multiple myeloma, hepatocellular carcinoma, hypopharyngeal carcinoma, intraocular melanoma, eye cancer, pancreatic islet cell tumor (pancreatic endocrine), kidney cancer (e.g., Wilms' tumor, renal clear cell carcinoma), liver cancer, lung cancer (e.g., non-small cell lung cancer, small cell lung carcinoma), Waldenstrom's macroglobulinoma, melanoma, intraocular (ocular) melanoma, Merkel cell carcinoma, mesothelioma, primary occult metastatic squamous cell carcinoma, multiple endocrine neoplasia (MEN), myelodysplastic syndrome, essential thrombocythemia, myelodysplastic / myeloproliferative disorders, nasopharyngeal carcinoma, neuroblastoma, oral cancer (e.g., mouth cancer, lip cancer, oral cancer, tongue cancer) , oropharyngeal cancer, pharyngeal cancer, laryngeal cancer), ovarian cancer (e.g., ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor), pancreatic cancer, pancreatic islet cell carcinoma, paranasal sinus and nasal cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pinealoma, pituitary tumor, plasma cell neoplasm, pleuropulmonary blastoma, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, uterine cancer (e.g., endometrial cancer, 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 organs, urethral cancer, gestational trophoblastic tumor, vaginal cancer, and vulvar cancer.

[0219] Sarcomas that may be treatable with the compounds of the invention include both soft tissue and bone cancers, 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 (membranous 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).

[0220] In some embodiments, the methods of the 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.

[0221] As used herein, "cell proliferative diseases or disorders of the blood system" includes lymphoma, leukemia, myeloid neoplasm, mast cell neoplasm, myelodysplasia, benign monoclonal gammopathy, polycythemia vera, chronic myeloid leukemia, myeloid metaplasia of unknown etiology, and essential thrombocythemia. Representative examples of hematological cancers are therefore multiple myeloma, lymphomas (including 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., B-cell non-Hodgkin's lymphoma selected from diffuse large B-cell lymphoma (e.g., germinal center B-cell-like diffuse large B-cell lymphoma or activated B-cell-like diffuse large B-cell lymphoma)), Burkitt's lymphoma / leukemia, mantle cell lymphoma, mediastinal (thymic) large B-cell lymphoma, follicular lymphoma, ... ALK+ anaplastic large cell lymphoma (e.g., B-cell non-Hodgkin's lymphoma), ALK+ anaplastic large cell lymphoma (e.g., B-cell non-Hodgkin's lymphoma), ALK+ anaplastic large cell lymphoma (e.g., B-cell non-Hodgkin's lymphoma), ALK+ anaplastic large cell lymphoma (e.g., B-cell non-Hodgkin's lymphoma), ALK lymphoma, marginal zone lymphoma, lymphoplasmacytic lymphoma / Waldenstrom's macroglobulinemia, metastatic pancreatic adenocarcinoma, refractory B-cell non-Hodgkin's lymphoma, and relapsed B-cell non-Hodgkin's lymphoma, childhood lymphoma, and lymphomas of lymphocytic and cutaneous origin (e.g., 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 myelogenous leukemia, chronic myeloid leukemia, and mast cell leukemia), myeloid neoplasms and mast cell neoplasms.

[0222] 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.

[0223] 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 tumor (for example, glioma, glioblastoma, meningioma, pituitary adenoma, vestibular schwannoma, and primitive neuroectodermal tumor (medulloblastoma)), brain precancerous 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.

[0224] 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 lung growths or lesions, pulmonary hyperplasia, metaplasia, and dysplasia, as well as 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," bronchoalveolar 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).

[0225] 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.

[0226] 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).

[0227] 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.

[0228] 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.

[0229] 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.

[0230] 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.

[0231] 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.

[0232] In some embodiments, the labeling method is directed to labeling solid tumors in a hypoxic tumor microenvironment, involving administration of a compound of Formula (I, II, III, or IV), or a pharmaceutically acceptable salt or stereoisomer thereof, to a subject in need thereof. In some embodiments, the solid tumor is in the brain, breast, cervix, kidney, liver, lung, pancreas, or rectum. In some embodiments, the solid tumor is epidermoid carcinoma, lung carcinoma, glioblastoma, or pancreatic adenocarcinoma.

[0233] In some embodiments, the method of treatment is directed to a method of treating a solid tumor in a hypoxic tumor microenvironment, comprising administering a therapeutically effective amount of a compound of Formula (I, II, III, or IV), or a pharmaceutically acceptable salt or stereoisomer thereof, to a subject in need thereof. In some embodiments, the solid tumor is in the brain, breast, cervix, kidney, liver, lung, pancreas, or rectum. In some embodiments, the solid tumor is epidermoid carcinoma, lung carcinoma, glioblastoma, or pancreatic adenocarcinoma. Hypoxic tumor microenvironments are known in the art. See, e.g., Petrova et al., Oncogenesis 7:10 (2018); Muz et al., Hypoxia (Auckl). 3:83-92 (2015); Hockel et al., J. Natl. Cancer Inst. 93(4):266-276 (2001).

[0234] 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 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.

[0235] 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. Combination therapy

[0236] The compounds of the present invention and their pharmaceutically acceptable salts or stereoisomers can be used in combination or simultaneously with at least one other active agent, such as an anti-cancer agent or regimen, in the treatment of diseases and disorders. In this context, the terms "in combination" and "simultaneously" mean that the agents are administered simultaneously, including substantially simultaneous administration in the same or separate dosage forms and by the same or different administration modes, or sequentially, for example, as part of the same treatment regimen or by a sequential treatment regimen. Thus, when administered sequentially, at the start of administration of the second agent, the first of the two agents may still be detectable at an effective concentration at the treatment site in some cases. The order and time interval can be determined so that they can act together (e.g., synergistically to provide increased benefit than if they were administered separately). For example, the agents 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, which may be synergistic.

[0237] In some embodiments, a treatment regimen may include administering a compound of the present invention, or a pharmaceutically acceptable salt or stereoisomer thereof, in combination with one or more additional therapeutic agents known to be used in the treatment of a disease or disorder (e.g., cancer). See Hardman et al., eds., Goodman & Gilman's The Pharmacological Basis of Basis of Therapeutics, 10th ed., McGraw-Hill, New York, 2001; Physician's Desk Reference, 60th ed., 2006. For example, anticancer agents that can be used in combination with the compounds of the present invention are known in the art. See, for example, U.S. Pat. No. 9,101,622 (section 5.2 thereof) and U.S. Pat. No. 9,345,705 B2 (columns 12-18 thereof). Representative examples of additional anti-cancer agents and treatment regimens include radiation therapy, chemotherapeutic agents (e.g., antimitotic agents, antiangiogenic agents, antihormones, autophagy inhibitors, alkylating agents, intercalating antibiotics, growth factor inhibitors, antiandrogens, signal transduction pathway inhibitors, anti-microtubule agents, platinum coordination complexes, HDAC inhibitors, proteasome inhibitors, and topoisomerase inhibitors), immunomodulatory agents, therapeutic antibodies (e.g., monospecific and bispecific antibodies), and CAR-T therapy.

[0238] In some embodiments, the compounds of the present invention and the additional anticancer therapeutic agent may be administered less than 5 minutes apart, less than 30 minutes apart, less than 1 hour apart, about 1 hour apart, about 1 to about 2 hours apart, about 2 to about 3 hours apart, about 3 to about 4 hours apart, about 4 to about 5 hours apart, about 5 to about 6 hours apart, about 6 to about 8 hours apart, about 8 to about 9 hours apart, about 9 to about 10 hours apart, about 10 to about 11 hours apart, about 11 to about 12 hours apart, about 12 to 18 hours apart, 18 to 24 hours apart, 24 to 36 hours apart, 36 to 48 hours apart, 48 to 52 hours apart, 52 to 60 hours apart, 60 to 72 hours apart, 72 to 84 hours apart, 84 to 96 hours apart, or 96 to 120 hours apart. Two or more anticancer therapeutic agents may be administered within the same patient visit.

[0239] In some embodiments, the compounds of the present invention and the additional therapeutic agents (e.g., anticancer therapeutic agents) are administered cyclically. As an example, in the context of cancer treatment, cycling therapy involves administering a first anticancer therapeutic agent for a period of time, followed by administering a second anticancer therapeutic agent for a period of time, optionally administering a third anticancer therapeutic agent for a period of time, etc., with this sequential administration, i.e., cycle, being repeated to reduce the development of resistance to one of the anticancer therapeutic agents, avoid or reduce side effects of one of the anticancer therapeutic agents, and / or improve the efficacy of the treatment. As an example, in the context of cancer treatment, cycling therapy involves administering a first anticancer therapeutic agent for a period of time, followed by administering a second anticancer therapeutic agent for a period of time, optionally administering a third anticancer therapeutic agent for a period of time, etc., with this sequential administration, i.e., cycle, being repeated to reduce the development of resistance to one of the anticancer therapeutic agents, avoid or reduce side effects of one of the anticancer therapeutic agents, and / or improve the efficacy of the treatment.

[0240] In some embodiments, the compounds of the present invention can be administered together with an agent that locally creates a hypoxic region (for example, 10% or less oxygen) in a tumor or a defined region containing one or more tumors.Representative types of such agents include antiangiogenic agents and vascular disrupting agents.See, for example, US Patent Application Publication No. 2017 / 0224693A1. Medicine Kit

[0241] 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.

[0242] These and other aspects of the present invention will be further understood by consideration of the following examples, which are intended to illustrate certain particular embodiments of the invention, but are not intended to limit the scope of the invention as defined by the claims. [Example]

[0243] Example 1: General information, materials, and equipment. General information

[0244] 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. material

[0245] 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: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. General chemical equipment

[0246] Proton nuclear magnetic resonance (NMR) measurements recorded on a 500 MHz Avance III spectrometer equipped with a multinuclear smart probe. 1H NMR) spectra are reported in parts per million on the NMR spectrum. δ scale and NMR solvents (CDCl3: δ 7.24 (chloroform), CD3OD: δ 3.31 (CHD2OD)). Data are reported as follows: chemical shift [multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, p = quintet, sx = sextet, sp = septet, dd = double doublet, dt = double triplet, ddd = double double doublet, dtd = double triple doublet, td = triple doublet, tdd = triple double doublet, qd = double quartet, m = multiplet), coupling constants in Hertz, integrals, assignments]. Carbon-13 Nuclear Magnetic Resonance ( 13 C NMR) spectra are based on the carbon resonance of the solvent (CDCl3: δ 77.23, CD3OD: δ 49.15). Fluorine-19 nuclear magnetic resonance ( 19 F NMR was calibrated from the fluorine resonance of benzotrifluoride (CDCl: δ -62.76, CD3OD: δ -64.24). Data are reported as follows: chemical shift (assignment). 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 frequency (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. An automated C purification system was used to purify the Isolera One (Biotage). 18 Reverse phase chromatography was performed. High performance liquid chromatography (HPLC) purification was performed using an Agilent 1260 Infinity system. General biological instruments

[0247] UV / vis absorbance measurements were acquired on an Agilent Technologies Cary 60 UV-Vis spectrophotometer. In-gel fluorescence imaging was performed on a GE Healthcare Life Sciences Typhoon™ FLA 9500. Slides were imaged at the Dana-Farber Cancer Institute's Confocal and Light Microscopy Core using a Nikon Ti Eclipse. Images were acquired with a Hamamatsu camera using a 10x objective at 0.64 microns / pixel. Hoechst 33342 and DAPI were imaged with a 405 / 20 filter (excitation) and a 460 / 50 filter (emission) and blue pseudocolor; FITC and Alexa Fluor 488 were imaged with a 482 / 35 filter (excitation) and a 536 / 75 filter (emission) and green pseudocolor; and TAMRA was imaged with a 560 / 40 filter (excitation) and a 630 / 75 filter (emission) and red pseudocolor. In vivo imaging was obtained using Living Image (version 4.2) on a Xenogen IVIS 100. Images were processed with Fiji ImageJ software.

[0248] Example 2: Design of hypoxia-responsive chemical motifs with drug-releasing and labeling properties

[0249] Introduction of α,β-unsaturation onto the amine N-oxide provided the enamine N-oxide structure (Figure 1C). By embedding a leaving group at the allylic position, a signal output mechanism was engineered into the structure. Reduction of the enamine N-oxide generates an enamine, from which β-elimination generates two functionally related species: 1) the leaving group and 2) the electrophilic α,β-unsaturated iminium ion. The allylic leaving group (LG) can be a caged drug if a prodrug is desired, a fluorophore if a probe, or an inert halogen or chalcogen if none is present. The function of the electrophilic component is similarly defined by the payload attached to the allylic position (R3). It can be an affinity tag such as biotin or an alkyne, a probe such as a fluorophore, or a [18 A PET tracer, such as a [F] fluorine atom, is introduced as appropriate for the application. In designing the enamine N-oxide, both labeling and release possibilities are captured.

[0250] Example 3: Synthesis of Enamine N-Oxides

[0251] Intermolecular reverse Cope elimination of unactivated alkynes is complicated by the tendency of enamine N-oxides to undergo Cope elimination (Bourgeois et al., J. Am. Chem. Soc. 131(3):874-875 (2009); Beauchemin, AM, Org. Biomol. Chem. 11(41):7039-7050 (2013)) and [1,2]-Meissenheimer rearrangement (Bernier et al., J. Org. Chem. 73(11):4229-4232 (2008); Castagnoli et al., Tetrahedron 26(18):4319-4327 (1970); Bottle et al., J. Chem. Soc., Perkin Trans. 2(7):1001-1007 (1991)). Activation by induction effects was investigated (Figure 1D). Using p-fluorophenyl propargyl ether (3) as a model substrate, an initial temperature screen ranging from room temperature to 80 °C in chloroform (CHCl3) demonstrated that the desired enamine N-oxide could be obtained, but there was a significant trade-off between conversion and decomposition, with a sharp drop in yield within a narrow ±10 °C window. Even so, the maximum yield was 50% (Table 1). [Table 1]

[0252] Solvent screening showed that although the hydroamination rate was fastest, the product was most susceptible to decomposition in less polar aprotic solvents (CHCl, CHCl, CCl, DCE, PhMe), whereas reaction conversion was lower in polar protic solvents (MeOH, EtOH, iPrOH, nBuOH), and fewer decomposition products were observed (Tables 2-4). Given the importance of the N-oxide oxygen in both the Cope (Figure 6) and Meissenheimer decomposition processes, we investigated the function of solvent pKa and found that 2,2,2-trifluoroethanol (TFE) mitigated decomposition better than the less acidic alcohols isopropyl alcohol and n-butanol, likely through increased enamine N-oxide stabilization. However, lower pKa solvents adversely affected the reaction rate, likely due to inhibition of the hydroxylamine reagent via protonation, as observed for 1,1,1,3,3,3-hexafluoroisopropanol (HFIP). [Table 2] [Table 3] [Table 4]

[0253] Finally, the optimal balance between reaction rate and product stability was achieved by using a low-polarity solvent supplemented with a minimal amount of a strong hydrogen-bond-donating solvent additive (entries 10 and 11, Table 5). Hydroamination of alkyne 3 with N,N-diethylhydroxylamine in 20% TFE / CHCl3 (v / v) at 60 °C for 18 h afforded the corresponding enamine N-oxide in 96% yield (entry 14, Tables 5 and 6). [Table 5] [Table 6]

[0254] Example 4: Reaction optimization

[0255] Hydroamination reactions performed under the indicated conditions were carried out in a 1-dram vial, flushed with nitrogen, sealed with parafilm, and heated using a UCON™ oil bath. The vial was charged with alkyne 3 (30.0 mg, 0.200 mmol) as a 0.4 M solution in the reaction solvent at room temperature. N,N-diethylhydroxylamine was then added via syringe as a 2 M solution in the reaction solvent at room temperature. The vial was sealed and heated to the indicated temperature for the indicated time. After heating, the reaction mixture was concentrated under reduced pressure and dissolved in CDCl3. The amounts of enamine N-oxide (5 and 5') and p-fluorophenol (4) were determined using benzotrifluoride as an internal standard. 1 H and 19 The positional isomer ratios in different solvents were determined by F-NMR integration. 1 Determined by H-NMR integration.

[0256] Decomposition studies were performed using the isolated enamine N-oxides (5:5', 15:1). An LC-MS vial was charged with a 0.1 M solution of the enamine N-oxide (23.9 mg, 100 μmol) in the desired reaction solvent and heated to 60 °C for 6 h. After heating, the reaction mixture was concentrated under reduced pressure and dissolved in CDCl3. The amounts of enamine N-oxides (5 and 5') and p-fluorophenol (4) were determined using benzotrifluoride as an internal standard. 1 H and 19 The positional isomer ratios in different solvents were determined by F-NMR integration. 1 Determined by H-NMR integration.

[0257] Example 5: General Procedure A: Hydroxylamine-Alkyne Hydroamination (Alkyne Range)

[0258] A 1-dram glass vial was charged with alkyne (50.0 mg, 1) at room temperature. A solution of N,N-diethylhydroxylamine (1.00 M, 5.00 equiv.) in 20% v / v trifluoroethanol in chloroform was then added via syringe. The vial was then flushed with nitrogen, capped, and sealed with parafilm. The reaction mixture was heated to 60°C in a UCON™ fluid heating bath until completion, as determined by TLC. Upon completion, the oil bath was removed, and the reaction was cooled to room temperature. The reaction mixture was directly purified by flash chromatography on silica gel (eluent: CMA in chloroform). Fractions containing the target compound were combined, and the solvent was removed under reduced pressure using a rotary evaporator at 0°C.

[0259] Example 6: General Procedure B: Hydroxylamine-Alkyne Hydroamination (Hydroxylamine Range)

[0260] To a 1-dram glass vial was added benzyl prop-2-yn-1-yl carbonate (Achard et al., Angew. Chem., Int. Ed. 50:3552-3556 (2011)) (2.00 equiv.) at room temperature. A solution of hydroxylamine (1.00 M, 20.0 mg, 1 equiv.) in 20% v / v trifluoroethanol in chloroform was then added via syringe. The vial was flushed with nitrogen, capped, and sealed with parafilm. The reaction mixture was heated to 60° C. in a UCON™ fluid heating bath until completion, as determined by thin-layer chromatography. Upon completion, the oil bath was removed and the reaction was cooled to room temperature. The reaction mixture was then concentrated under reduced pressure. The resulting residue was purified by automated C chromatography. 18 Reverse phase column chromatography (30g C 18 Purification was performed using silica gel (25 μm spherical particles) with eluent: HO + 0.1% TFA (2 CV), gradient 0 → 100% MeCN / HO + 0.1% TFA (10-15 CV). Fractions containing the target compound were combined and the solvent was removed under reduced pressure using a rotary evaporator at 0 °C.

[0261] Example 7: Synthesis of (E)-N,N-diethyl-3-(4-fluorophenoxy)prop-1-ene-1-amine oxide (5) and N,N-diethyl-3-(4-fluorophenoxy)prop-1-ene-2-amine oxide (5'). [ka]

[0262] Enamine N-oxide 5 was synthesized according to general procedure A using 1-fluoro-4-(prop-2-yn-1-yloxy)benzene (3) (Tsuzuki et al., Bioorg. Med. Chem. Lett. 20:7269-7273, (2010)). The reaction mixture was stirred at 60 °C for 18 h and purified by flash column chromatography on silica gel (eluent: 30% CMA in chloroform) to give regioisomeric enamine N-oxides 5 and 5' (Test 1: 77.2 mg, 97%; Test 2: 78.4 mg, 98%) as white solids. The regioisomeric ratios (5:5', Test 1: 4.4:1, Test 2: 4.6:1) were determined by the C1 vinyl proton of the major isomer and the C1 vinyl proton of the minor isomer. 1 It was determined by taking the ratio of the 1 H-NMR integrals. 1 H NMR (500 MHz, CD3OD, 25 ℃): [5] δ 7.06-6.92 (m, 4H), 6.60 (dt, J = 13.3, 4.9 Hz, 1H), 6.35 (dt, J = 13.1, 1.8 Hz, 1H), 4.70 (dd, J = 5.0, [5'] δ 7.08-6.92 (m, 4H), 5.98 (d, J = 1.7 Hz, 1H), 5.76 (d, J = 1.5 Hz, 1H), 4.78 (d, J = 1.1 Hz, 2H), 3.64 (dq, J = 12.7, 7.2 Hz, 2H), 3.49-3.43 (m, 2H), 1.28 (t, J = 7.1 Hz, 6H). 13C NMR (126 MHz, CD3OD, 25℃): [5] δ 159.1 (d, J = 237.5 Hz), 155.9 (d, J = 2.4 Hz), 140.1, 126.5, 117.4 (d, J = 8.1 Hz), 116.9 (d, J = 23.4 Hz), 66.4, 65.3, 8.7. [5'] δ 159.3 (d, J = 238.0 Hz), 155.8 (d, J = 2.9 Hz), 151.1, 117.4 (d, J = 8.1 Hz), 117.1 (d, J = 23.5 Hz), 115.8, 67.1, 64.7, 8.9. 19 F NMR (471 MHz, CD3OD, 25℃) [5] δ -125.3. [5'] δ -124.8. FTIR (thin film) cm -1 : 3232 (w), 1506 (s), 1461 (w), 1200 (s), 962 (w), 828 (m) HRMS (ESI) (m / z): calc'd for C 13 H 19 FNO2[M+H] + : 240.1400, found: 240.1395. TLC (30% CMA in chloroform), Rf: 0.057 (UV, KMnO4).

[0263] Example 8: Synthesis of (E)-3-(((benzyloxy)carbonyl)oxy)-N,N-dimethylprop-1-ene-1-amine oxide (6) and N,N-diethyl-3-(p-tolyloxy)prop-1-ene-2-amine oxide (6′). [ka]

[0264] Enamine N-oxide 6 was synthesized according to general procedure A using 1-methyl-4-(prop-2-yn-1-yloxy)benzene (Efe et al., Chem. Commun. 47:803-805 (2011)). The reaction mixture was stirred at 60 °C for 24 h and purified by flash column chromatography on silica gel (eluent: 30% CMA in chloroform) to give regioisomeric enamine N-oxides 6 and 6' (Test 1: 77.0 mg, 96%; Test 2: 76.4 mg, 95%) as white solids. The regioisomeric ratio (6:6', Test 1: 3.8:1, Test 2: 4.6:1) was determined by the C1 vinyl proton of the major isomer and the C1 vinyl proton of the minor isomer. 1 It was determined by taking the ratio of the 1 H-NMR integrals. 1 H NMR (500 MHz, CD3OD, 25℃): [6] δ 7.04 (d, J = 8.0 Hz, 2H), 6.80 (d, J = 8.6 Hz, 2H), 6.55 (dt, J = 13.2, 4.8 Hz, 1H), 6.27 (dt, J = 13.2, [6'] δ 7.06 (d, J = 8.3 Hz, 2H), 6.84 (d, J = 8.6 Hz, 2H), 5.93 (d, J = 1.6 Hz, 1H), 5.70 (d, J = 1.4 Hz, 1H), 4.72 (d, J = 1.0 Hz, 2H), 3.63-3.54 (m, 2H), 3.45-3.37 (m, 2H), 2.23 (s, 3H), 1.23 (t, J = 7.1 Hz, 6H). 13C NMR (126 MHz, CD3OD, 25℃): [6] δ 157.5, 151.3, 139.8, 131.1, 126.8, 115.9, 65.8, 65.3, 20.7, 8.7. [6'] δ 157.4, 132.2, 131.8, 131.2, 115.9, 115.7, 66.6, 64.7, 20.7, 8.9. FTIR (thin film) cm -1 : 2982 (w), 2941 (w), 1513 (s), 1238 (m), 1014 (w), 961 (w), 816 (w). HRMS (ESI) (m / z): calc'd for C 14 H 22 NO2[M+H] + : 236.1645, found: 236.1641. TLC (30% CMA in chloroform), Rf: 0.20 (UV, KMnO4).

[0265] Example 9: Synthesis of (E)-N,N-diethyl-3-(4-nitrophenoxy)prop-1-ene-1-amine oxide (7) and N,N-diethyl-3-(4-nitrophenoxy)prop-1-ene-2-amine oxide (7'). [ka]

[0266] Enamine N-oxide 7 was synthesized according to general procedure A using 1-nitro-4-(prop-2-yn-1-yloxy)benzene (Tsuzuki et al., Bioorg. Med. Chem. Lett. 20:7269-7273, (2010)). The reaction mixture was stirred at 60 °C for 16 h and purified by flash column chromatography on silica gel (eluent: 30% CMA in chloroform) to give regioisomeric enamine N-oxides 7 and 7' (Test 1: 74.5 mg, 99%; Test 2: 73.4 mg, 97%) as yellow oils. The regioisomeric ratio (7:7', Test 1: 15:1, Test 2: 15:1) was determined by the C1 vinyl proton of the major isomer and the C1 vinyl proton of the minor isomer. 1 It was determined by taking the ratio of the 1 H-NMR integrals. 1 H NMR (500 MHz, CD3OD, 25℃): [7] δ 8.21 (d, J = 9.3 Hz, 2H), 7.13 (d, J = 9.3 Hz, 2H), 6.65 (dt, J = 13.3, 5.0 Hz, 1H), 6.43 (dt, J = 13.1, [7'] δ 8.21 (d, J = 9.3 Hz, 2H), 7.17 (d, J = 9.3 Hz, 2H), 6.01 (d, J = 2.1 Hz, 1H), 5.86-5.79 (m, 1H), 4.96 (d, J = 0.9 Hz, 2H), 3.70-3.46 (m, 4H), 1.31 (t, J = 7.1 Hz, 6H). 13 C NMR (126 MHz, CD3OD, 25℃): [7] δ 163.2, 141.8, 139.2, 125.5, 124.1, 114.7, 65.0, 63.8, 7.1. [7'] δ 162.9, 149.1, 142.0, 125.8, 115.6, 114.7, 65.5, 63.3, 7.4. FTIR (thin film) cm -1: 3213 (br), 2944 (w), 1591 (s), 1509 (s), 1334 (s), 1260 (s), 1110 (m). HRMS (ESI) (m / z): calc'd for C 13 H 19 N2O4[M+H] + : 267.1345, found: 267.1337. TLC (30% CMA in chloroform), Rf: 0.097 (UV, KMnO4).

[0267] Example 10: Synthesis of (E)-N,N-diethyl-3-(4-methoxyphenoxy)prop-1-ene-1-amine oxide (8) and N,N-diethyl-3-(4-methoxyphenoxy)prop-1-ene-2-amine oxide (8'). [ka]

[0268] Enamine N-oxide 8 was synthesized according to general procedure A using 1-methoxy-4-(prop-2-yn-1-yloxy)benzene (Achard et al., Angew. Chem., Int. Ed. 50:3552-3556 (2011)). The reaction mixture was stirred at 60 °C for 41 h and purified by flash column chromatography on silica gel (eluent: 30% CMA in chloroform) to give regioisomeric enamine N-oxides 8 and 8' (Test 1: 70.4 mg, 91%; Test 2: 71.7 mg, 93%) as white solids. The regioisomeric ratio (8:8', Test 1: 9.3:1, Test 2: 4.5:1) was determined by the C1 vinyl proton of the major isomer and the C1 vinyl proton of the minor isomer. 1 It was determined by taking the ratio of the 1 H-NMR integrals. 1H NMR (500 MHz, CD3OD, 25 ℃): [8] δ 6.97-6.82 (m, 4H), 6.60 (dt, J = 13.1, 4.9 Hz, 1H), 6.34 (dt, J = 13.1, 1.9 Hz, 1H), 4.69 (dd, J = 4.9, [8'] δ 6.97-6.86 (m, 4H), 5.97 (d, J = 1.7 Hz, 1H), 5.75 (d, J = 1.4 Hz, 1H), 4.74 (s, 2H), 3.75 (s, 3H), 3.69-3.57 (m, 2H), 3.50-3.40 (m, 2H), 1.27 (t, J = 7.1 Hz, 6H). 13 C NMR (126 MHz, CD3OD, 25℃): [8] δ 155.9, 153.7, 139.9, 126.8, 117.1, 115.9, 66.4, 65.3, 56.3 8.7. [8'] δ 156.1, 153.6, 151.3, 117.1, 115.9, 115.7, 67.2, 64.6, 56.3, 8.9. FTIR (thin film) cm -1 : 3232 (br), 2945 (w), 1506 (d), 1461 (m), 1223 (s), 1033 (m), 828 (m). HRMS (ESI) (m / z): calc'd for C 14 H 22 NO3[M+H] + : 252.1600, found: 252.1593. TLC (30% CMA in chloroform), Rf: 0.13 (UV, KMnO4).

[0269] Example 11: Synthesis of (E)-N,N-diethyl-3-(octyloxy)prop-1-ene-1-amine oxide (9) and N,N-diethyl-3-(octyloxy)prop-1-ene-2-amine oxide (9'). [ka]

[0270] Enamine N-oxide 9 was synthesized according to general procedure A using 1-(prop-2-yn-1-yloxy)octane (Sahoo et al., Org. Biomol. Chem. 12:2615-2625, (2014)). The reaction mixture was heated for 64 h and purified by flash column chromatography on silica gel (eluent: 30% CMA in chloroform) to give regioisomeric enamine N-oxides 9 and 9' (Run 1: 65.1 mg, 85%; Run 2: 63.0 mg, 82%) as clear, colorless oils. The regioisomeric ratio (9:9', Run 1: 3.5:1, Run 2: 2.3:1) was determined by the C1 vinyl proton of the major isomer and the C1 vinyl proton of the minor isomer. 1 It was determined by taking the ratio of the 1 H-NMR integrals. 1 H NMR (500 MHz, CD3OD, 25℃): [9] δ 6.45 (dt, J = 13.1, 5.1 Hz, 1H), 6.21 (dt, J = 13.1, 1.8 Hz, 1H), 4.12 (dd, J = 5.0, 1.8 Hz, 2H), 3.48 (t, J = 6.6 Hz, 2H), 3.45-3.31 (m, 4H), 1.39-1.28 (m, 12H), 1.26 (t, J = 7.2 Hz, 6H), 0.90 (m, 3H). [9'] δ 5.95 (d, J = 1.0 Hz, 1H), 5.59 (d, J = 1.1 Hz, 1H), 4.18 (d, J = 0.9 Hz, 2H), 3.64-3.55 (m, 2H), 3.50 (t, J = 6.5 Hz, 2H), 3.45-3.31 (m, 2H), 1.63-1.55 (m, 6H), 1.39-1.28 (m, 6H), 1.23 (t, J = 7.0 Hz, 6H), 0.90 (m, 3H). 13C NMR (126 MHz, CD3OD, 25℃): [9] δ 139.2, 127.8, 72.1, 68.3, 65.2, 33.2, 30.9, 30.7, 30.6, 27.4, 23.9, 14.6, 8.7. [9'] δ 151.8, 115.5, 72.4, 69.3, 64.4, 33.1, 30.8, 30.6, 30.6, 27.5, 23.9, 14.6, 8.8.FTIR cm -1 : 2929 (s), 2855 (m), 1464 (w), 1367 (w), 1107 (m), 961 (w). HRMS (ESI) (m / z): calc'd for C 15 H 32 NO2[M+H] + : 258.2428, found: 258.2422. TLC (30% CMA in chloroform), Rf: 0.10 (KMnO4).

[0271] Example 12: Synthesis of (E)-N,N-diethyl-3-hydroxyprop-1-ene-1-amine oxide (10) and N,N-diethyl-3-hydroxyprop-1-ene-2-amine oxide (10'). [ka]

[0272] Enamine N-oxide 10 was synthesized according to general procedure A using prop-2-yn-1-ol (Sigma Aldrich). The reaction mixture was stirred at 60 °C for 43 h and purified by flash column chromatography on silica gel (eluent: 40% CMA in chloroform) to give enamine N-oxide 10 (Test 1: 82.9 mg, 64%; Test 2: 77.2 mg, 60%) as a brown oil and 10' (Test 1: 39.5 mg, 30%; Test 2: 40.9 mg, 32%) as a brown oil. The regioisomeric ratios (10:10', Test 1: 2.1:1, Test 2: 1.9:1) were determined by taking the ratio of the amounts of the isolated regioisomers. 1H NMR (500 MHz, CD3OD, 25℃):

[10] δ 6.48 (dt, J = 13.1, 4.7 Hz, 1H), 6.18 (dt, J = 13.3, 2.1 Hz, 1H), 4.23 (dd, J = 4.7, 2.0 Hz, 2H), 3.50-3.33 (m, 4H), 1.27 (t, J = 7.2 Hz, 6H). [10'] δ 5.78 (d, J = 1.5 Hz, 1H), 5.58 (d, J = 1.2 Hz, 1H), 3.60 (dq, J = 12.4, 7.0 Hz, 2H), 3.45 - 3.34 (m, 2H), 1.25 (t, J = 7.1 Hz, 4H). 13 C NMR (126 MHz, CD3OD, 25℃):

[10] δ 138.1, 130.6, 65.2, 60.0, 8.7. [10'] δ 155.1, 112.7, 64.9, 61.1, 8.8. FTIR (thin film) cm -1 :

[10] 3213 (br), 2989 (m), 1659 (m), 1450 (m), 1103 (m), 954 (s). [10'] 3198 (br), 2989 (m), 1662 (m), 1450 (m), 1379 (m), 954 (s). HRMS (ESI) (m / z):

[10] calc'd for C7H 16 NO2[M+H] + : 146.1181, found: 146.1176. [10'] calc'd for C7H 16 NO2[M+H] + : 146.1181, found: 146.1176. TLC (60% CMA in chloroform), Rf:

[10] 0.14 (KMnO4). [10'] 0.11 (KMnO4).

[0273] Example 13: Synthesis of (E)-N,N-diethyl-3-(pivaloyloxy)prop-1-ene-1-amine oxide (11) and N,N-diethyl-3-(pivaloyloxy)prop-1-ene-2-amine oxide (11′) [ka]

[0274] Enamine N-oxide 11 was synthesized using prop-2-yn-1-yl pivalate according to general procedure A (Achard et al., Angew. Chem., Int. Ed. 50:3552-3556 (2011)). The reaction mixture was heated for 32 h and purified by flash column chromatography on silica gel (eluent: 30% CMA in chloroform) to give regioisomeric enamine N-oxides 11 and 11' (Run 1: 60.5 mg, 74%; Run 2: 63.6 mg, 78%) as clear yellow oils. The regioisomeric ratios (11:11', Run 1: 9.1:1, Run 2: 11.1:1) were determined by the C1 vinyl proton of the major isomer and the C1 vinyl proton of the minor isomer. 1 It was determined by taking the ratio of the 1 H-NMR integrals. 1 H NMR (500 MHz, CD3OD, 25℃):

[11] δ 6.50 (dt, J = 13.2, 6.1 Hz, 1H), 6.29 (dt, J = 13.2, 1.5 Hz, 1H), 4.72 (dd, J = 6.1, 1.5 Hz, 2H), 3.43-3.32 (m, 4H), 1.29-1.22 (m, 6H), 1.22 (s, 9H). [11'] δ 5.92 (d, J = 2.0 Hz, 1H), 5.61-5.56 (m, 1H), 4.85 (d, J = 2.0 Hz, 2H), 3.64-3.52 (m, 2H), 3.49-3.41 (m, 2H), 1.29-1.22 (m, 6H), 1.22 (s, 9H). 13 C NMR (126 MHz, CD3OD, 25℃):

[11] δ 179.4, 141.3, 125.5, 65.3, 62.0, 27.6, 8.6. [11'] δ 179.0, 150.8, 115.3, 64.5, 40.0, 28.2, 8.8. FTIR (thin film) cm -1: 2974 (m), 1729 (s), 1461 (w), 1367 (w), 1282 (m), 1155 (s), 957 (w). HRMS (ESI) (m / z): calc'd for C 12 H 24 NO3[M+H] + : 230.1751, found: 230.1746. TLC (30% CMA in chloroform), Rf: 0.10 (KMnO4).

[0275] Example 14: Synthesis of (E)-N,N-diethyl-3-((phenylcarbamoyl)oxy)prop-1-ene-1-amine oxide (12) and N,N-diethyl-3-((phenylcarbamoyl)oxy)prop-1-ene-2-amine oxide (12′). [ka]

[0276] Enamine N-oxide 12 was synthesized using prop-2-yn-1-ylphenylcarbamate according to general procedure A (Newton et al., Aust. J. Chem. 61:432-437 (2008)). The reaction mixture was heated for 22 hours and purified by flash column chromatography on silica gel (eluent: 30% CMA in chloroform) to give regioisomeric enamine N-oxides 12 and 12' (Run 1: 74.4 mg, 99%; Run 2: 71.2 mg, 94%) as clear, colorless oils. The regioisomeric ratio (12:12', Run 1: 8.5:1, Run 2: 8.3:1) was determined by the C1 vinyl proton of the major isomer and the C1 vinyl proton of the minor isomer. 1 It was determined by taking the ratio of the 1 H-NMR integrals. 1H NMR (500 MHz, CD3OD, 25℃):

[12] δ 7.43 (d, J = 8.0 Hz, 2H), 7.27 (t, J = 8.0 Hz, 2H), 7.02 (t, J = 7.4 Hz, 1H), 6.55 (dt, J = 13.2, 5.7 Hz, [12'] δ 7.45 (d, 2H), 7.29 (t, 2H), 7.03 (t, 1H), 5.91 (d, J = 1.9, 1H), 5.67 (s, 1H), 4.92 (s, 2H), 3.66-3.43 (m, 4H), 1.26 (t, J = 7.2, 6H). 13 C NMR (126 MHz, CD3OD, 25℃):

[12] δ 153.2, 140.7, 140.2, 130.0, 125.9, 124.3, 120.0, 65.3, 62.2, 8.6. [12'] δ 153.2, 151.3, 140.2, 130.0, 124.4, 120.0, 114.8, 64.6, 62.0, 8.8. FTIR (thin film) cm -1 : 2944 (w), 1714 (m), 1599 (m), 1546 (m), 1446 (m), 1315 (m), 1218 (s), 1054 (m), 756 (s). HRMS (ESI) (m / z): calc'd for C 14 H 21 N2O3[M+H] + : 265.1547, found: 265.1541. TLC (30% CMA in chloroform), Rf: 0.20 (UV, KMnO4).

[0277] Example 15: Synthesis of (E)-3-(((benzyloxy)carbonyl)oxy)-N,N-diethylprop-1-en-1-amine oxide (13). [ka]

[0278] Enamine N-oxide 13 was synthesized using benzylprop-2-yn-1-yl carbonate according to general procedure A (Achard et al., Angew. Chem., Int. Ed. 50:3552-3556 (2011)). The reaction mixture was heated for 9 h and purified by flash column chromatography on silica gel (eluent: 30% CMA in chloroform) to give enamine N-oxide 13 (Run 1: 61.0 mg, 83%; Run 2: 58.2 mg, 79%) as a clear, colorless oil. The regioisomeric ratio (>20:1) was determined between the C1 vinyl proton of the major isomer and the C1 vinyl proton of the minor isomer. 1 It was determined by taking the ratio of the 1 H-NMR integrals. 1 H NMR (500 MHz, CD3OD, 25℃): δ 7.41-7.32 (m, 5H), 6.51 (dtd, J = 13.2, 5.1, 1.3 Hz, 1H), 6.30 (dt, J = 13.5, 2.1 Hz, 1H), 5.17 (s, 2H), 4.80 (d, J = 5.7 Hz, 2H), 3.41-3.31 (m, 4H), 1.22 (t, J = 7.2 Hz, 6H). 13 C NMR (126 MHz, CD3OD, 25℃): δ 156.3, 141.1, 137.1, 129.8, 129.7, 129.5, 125.1, 71.0, 65.4, 65.3, 8.6. FTIR (thin film) cm -1 : 1748 (m), 1666 (w), 1394 (w), 1244 (m), 1177 (s), 1129 (s), 946 (m). HRMS (ESI) (m / z): calc'd for C 15 H 22 NO4[M+H] + : 280.1543, found: 280.1538. TLC (30% CMA in chloroform), Rf: 0.30 (UV, KMnO4).

[0279] Example 16: Synthesis of (E)-3-((diethoxyphosphoryl)oxy)-N,N-diethylprop-1-en-1-amine oxide (14) [ka]

[0280] Enamine N-oxide 14 was synthesized using diethylprop-2-yn-1-yl phosphate according to general procedure A (Jones et al., Org. Lett. 7:3271-3274, (2005)). The reaction mixture was stirred at 60° C. for 16 hours and purified by flash column chromatography on silica gel (eluent: 30% CMA in chloroform) to give enamine N-oxide 14 (Test 1: 58.1 mg, 77%; Test 2: 59.4 mg, 78%) as a yellow oil. 1 H NMR (500 MHz, CD3OD, 25℃): δ 6.55 (dtd, J = 13.1, 5.4, 1.1 Hz, 1H), 6.37 (dt, J = 13.1, 1.7 Hz, 1H), 4.71 (ddd, J = 8.4, 5.4, 1.7 Hz, 2H), 4.15 (dq, J = 8.2, 7.0 Hz, 4H), 3.49-3.32 (m, 4H), 1.35 (td, J = 7.1, 1.1 Hz, 6H), 1.27 (t, J = 7.2 Hz, 6H). 13 C NMR (126 MHz, CD3OD, 25℃): δ 141.0, 125.8 (d, J = 6.7 Hz), 65.8 (d, J = 6.2 Hz), 65.4, 65.3, 16.6 (d, J = 6.7 Hz), 8.6. 31 P NMR (202 MHz, CD3OD, 25℃) δ -1.5. FTIR (thin film) cm -1: 3399 (br), 2989 (m), 1599 (m), 1260 (m), 1029 (s), 805 (w). HRMS (ESI) (m / z): calc'd for C 11 H 25 NO5P [M+H] + : 282.1470, found: 282.1463. TLC (30% CMA in chloroform), Rf: 0.079 (KMnO4).

[0281] Example 17: Synthesis of (E)-N,N-diethyl-3-hydroxydec-1-en-1-amine oxide (15) [ka]

[0282] Enamine N-oxide 15 was synthesized using deca-1-yn-3-ol according to general procedure A (Ye et al., J. Am. Chem. Soc. 132:8550-8551 (2010)). The reaction mixture was stirred at 60° C. for 98 hours and purified by flash column chromatography on silica gel (eluent: 40% CMA in chloroform) to give enamine N-oxide 15 (Test 1: 49.0 mg, 62%, Test 2: 48.0 mg, 61%) as a yellow oil. 1 H NMR (500 MHz, CD3OD, 25℃): δ 6.39 (dd, J = 13.1, 5.6 Hz, 1H), 6.13 (dd, J = 13.1, 1.7 Hz, 1H), 4.25 (qd, J = 6.4, 1.6 Hz, 1H), 3.42-3.31 (m, 4H), 1.61-1.53 ​​(m, 2H), 1.47-1.29 (m, 10H), 1.27 (td, J = 7.2, 5.0 Hz, 6H), 0.95-0.85 (m, 3H). 13C NMR (126 MHz, CD3OD, 25℃): δ 138.1, 134.0, 70.1, 65.3, 38.6, 33.1, 30.7, 30.5, 26.6, 23.9, 14.6, 8.7. FTIR (thin film) cm -1 : 3220 (br), 2926 (s), 2855 (s), 1595 (w), 1461 (m), 1379 (m), 965 (m). HRMS (ESI) (m / z): calc'd for C8H 17 NO2[M+H] + : 244.2271, found: 244.2271. TLC (50% CMA in chloroform), Rf: 0.11 (KMnO4).

[0283] Example 18: (Synthesis of 3-fluorodec-1-yne (S9)) [ka]

[0284] A round-bottom flask was charged with dec-1-yn-3-ol (Ye et al., J. Am. Chem. Soc. 132:8550-8551 (2010)) (300 mg, 1.94 mmol, 1 equiv.) dissolved in dichloromethane (19 mL) and cooled to −10 °C using a calcium chloride-ice bath. Diethylaminosulfur trifluoride (308 μL, 2.33 mmol, 1.20 equiv.) was added dropwise via syringe. After 3 h, water (10 mL) was added to the reaction mixture. The organic layer was then washed with water (2 × 10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude residue was purified by flash column chromatography on silica gel (eluent: 100% hexane) to afford fluoroalkyne S9 (240 mg, 79%) as a clear, colorless oil. 1H NMR (500 MHz, CDCl3, 25℃): δ 5.06 (dtd, J = 48.2, 6.4, 2.0 Hz, 1H), 2.64 (dd, J = 5.6, 2.1 Hz, 1H), 1.92-1.70 (m, 2H), 1.52-1.39 (m, 2H), 1.34-1.22 (m, 8H), 0.86 (t, J = 7.0 Hz, 3H). 13 C NMR (126 MHz, CDCl3, 25℃): δ 82.65 (d, J = 167.2 Hz), 80.73 (d, J = 25.9 Hz), 76.28 (d, J = 10.5 Hz), 36.01 (d, J = 22.2 Hz), 31.9, 29.3, 29.3, 24.57 (d, J = 4.2 Hz), 22.8, 14.3. 19 F NMR (471 MHz, CDCl3, 25℃): δ 175.0. FTIR (thin film) cm -1 : 3306 (m), 2926 (s), 2858 (s), 1461 (m). HRMS (GC-MS) (m / z): calc'd for C 10 H 17 F [M] + : 156.1309, found: 156.1309. TLC (100 % hexanes), Rf: 0.70 (KMnO4).

[0285] Example 19: Synthesis of (E)-N,N-diethyl-3-fluorodec-1-en-1-amine oxide (16) [ka]

[0286] N-oxide 16 was synthesized using 3-fluorodec-1-yne (S9) according to general procedure A. The reaction mixture was heated for 16 h and purified by flash column chromatography on silica gel (eluent: 30% CMA in chloroform) to give enamine N-oxide 16 (Run 1: 43.3 mg, 55%; Run 2: 35.1 mg, 45%) as a clear, colorless oil. 1 H NMR (500 MHz, CD3OD, 25℃): δ 6.49 (ddd, J = 18.3, 13.1, 5.3 Hz, 1H), 6.30 (dt, J = 13.1, 1.8 Hz, 1H), 5.24-5.07 (m, 1H), 3.46-3.33 (m, 4H), 1.80-1.67 (m, 2H), 1.49-1.41 (m, 2H), 1.39-1.29 (m, 8H), 0.95-0.87 (m, 3H). 13 C NMR (126 MHz, CD3OD, 25℃): δ 139.61 (d, J = 12.4 Hz), 129.89 (d, J = 18.2 Hz), 91.52 (d, J = 170.7 Hz), 65.36 (d, J = 9.0 Hz), 36.58 (d, J = 21.2 Hz), 33.06, 30.53, 30.45, 25.80 (d, J = 4.2 Hz), 23.84, 14.56, 8.67. 19 F NMR (471 MHz, CDCl3, 25℃): δ 181.2. FTIR (thin film) cm -1 : 2929 (s), 2858 (s), 1684 (w), 1464 (m), 1375 (m), 1129 (w), 965 (s). HRMS (ESI) (m / z): calc'd for C 14 H 29 FNO [M+H] + : 246.2228, found: 246.2222. TLC (30% CMA in chloroform), Rf: 0.30 (KMnO4).

[0287] Example 20: Synthesis of 3-chlorodec-1-yne (S10) [ka]

[0288] A round-bottom flask was charged with dec-1-yn-3-ol (Ye et al., J. Am. Chem. Soc. 132:8550-8551 (2010)) (250 mg, 1.62 mmol, 1 equiv.) and dissolved in tetrahydrofuran (16 mL) at room temperature. Solid N-chlorosuccinimide (303 mg, 2.27 mmol, 1.40 equiv.) and triphenylphosphine (638 mg, 2.43 mmol, 1.50 equiv.) were then added to the reaction mixture. After 14 h, the reaction was concentrated under reduced pressure. The crude product was purified by flash column chromatography on silica gel (eluent: 10% dichloromethane in hexanes) to afford chloroalkyne S10 (134 mg, 48%) as a clear, colorless oil. 1 H NMR (500 MHz, CDCl3, 25℃): δ 4.48 (td, J = 6.7, 2.3 Hz, 1H), 2.57 (d, J = 2.3 Hz, 1H), 1.97-1.89 (m, 2H), 1.54-1.47 (m, 2H), 1.35-1.23 (m, 8H), 0.87 (t, J = 7.1 Hz, 3H). 13 C NMR (126 MHz, CDCl3, 25℃): δ 82.3, 74.3, 48.1, 39.2, 31.9, 29.3, 29.0, 26.3, 22.8, 14.3. FTIR (thin film) cm -1 : 3302 (m), 2926 (s), 2855 (s), 1461 (m). HRMS (GC-MS) (m / z): calc'd for C 10 H 17 Cl [M]: 172.1013, found: 172.1014. TLC (10% dichloromethane in hexanes), Rf: 0.80 (KMnO4).

[0289] Example 21: Synthesis of (E)-3-chloro-N,N-diethyldec-1-en-1-amine oxide (17) [ka]

[0290] Enamine N-oxide 17 was synthesized using 3-chlorodec-1-yne (S10) according to general procedure A. The reaction mixture was heated for 16 h and purified by flash column chromatography on silica gel (eluent: 30% CMA in chloroform) to give enamine N-oxide 17 (Run 1: 27.2 mg, 36%; Run 2: 36.2 mg, 48%) as a clear, colorless oil. 1 H NMR (500 MHz, CD3OD, 25℃): δ 6.49 (dd, J = 12.9, 8.7 Hz, 1H), 6.36 (d, J = 12.9 Hz, 1H), 4.63 (dt, J = 8.9, 6.9 Hz, 1H), 3.44-3.31 (m, 4H), 1.92-1.83 (m, 2H), 1.52-1.40 (m, 2H), 1.37-1.29 (m, 8H), 1.26 (q, J = 7.2 Hz, 6H), 0.90 (m, 3H). 13 C NMR (126 MHz, CD3OD, 25℃): δ 140.3, 131.8, 65.5, 65.5, 59.2, 39.8, 33.0, 30.4, 30.2, 27.6, 23.8, 14.6, 8.7, 8.6. FTIR (thin film) cm -1 : 2929 (s), 2855 (s), 1681 (w), 1461 (w), 1375 (m), 969 (s). HRMS (ESI) (m / z): calc'd for C 14 H 29 ClNO [M+H] + : 262.1932, found: 262.1927. TLC (30% CMA in chloroform), Rf: 0.20 (KMnO4).

[0291] Example 22: Synthesis of (E)-3,3-diethoxy-N,N-diethylprop-1-en-1-amine oxide (18) [ka]

[0292] Enamine N-oxide 18 was synthesized according to general procedure A using 3,3-diethoxyprop-1-yne. The reaction mixture was stirred at 60 °C for 11 h and purified by flash column chromatography on silica gel (eluent: 20% CMA in chloroform) to give enamine N-oxide 18 (Test 1: 76.5 mg, 90%; Test 2: 77.2 mg, 91%) as a white solid. The regioisomeric ratio (>20:1 rr) was determined between the C1 vinyl proton of the major isomer and the C1 vinyl proton of the minor isomer. 1 It was determined by taking the ratio of the 1 H-NMR integrals. 1 H NMR (500 MHz, CD3OD, 25℃): δ 6.43-6.32 (m, 2H), 5.16 (d, J = 3.7 Hz, 1H), 3.67 (dq, J = 9.5, 7.0 Hz, 2H), 3.55 (dq, J = 9.5, 7.0 Hz, 2H), 3.44-3.35 (m, 4H), 1.26 (t, J = 7.2 Hz, 6H), 1.21 (t, J = 7.0 Hz, 6H). 13 C NMR (126 MHz, CD3OD, 25℃): δ 141.8, 128.6, 99.6, 65.2, 62.5, 15.7, 8.7. FTIR (thin film) cm -1 : 3362 (br), 2978 (m), 1684 (w), 1375 (w), 1126 (m), 1051 (s), 969 (m). HRMS (ESI) (m / z): calc'd for C 11 H 24 NO3[M+H] + : 218.1756, found: 218.1750. TLC (30% CMA in chloroform), Rf: 0.13 (KMnO4).

[0293] Example 23: Synthesis of N,N-diethyl-7-hydroxyhept-1-ene-2-amine oxide (19) [ka]

[0294] Enamine N-oxide 19 was synthesized according to general procedure A using hept-6-yn-1-ol. The reaction mixture was stirred at 60 °C for 240 h and purified by flash column chromatography on silica gel (eluent: 30% CMA in chloroform) to give enamine N-oxide 19 (Test 1: 37.8 mg, 42%; Test 2: 35.7 mg, 40%) as a white solid. The regioisomeric ratio (>20:1) was determined between the C1 vinyl proton of the major isomer and the C1 vinyl proton of the minor isomer. 1 It was determined by taking the ratio of the 1 H-NMR integrals. 1 H NMR (500 MHz, CD3OD, 25℃): δ 5.93 (d, J = 1.2 Hz, 1H), 5.30 (d, J = 1.5 Hz, 1H), 3.56 (dt, J = 12.7, 6.7 Hz, 4H), 3.37-3.30 (m, 2H), 2.24 (t, J = 7.8 Hz, 2H), 1.69-1.56 (m, 4H), 1.53-1.44 (m, 2H), 1.19 (t, J = 7.1 Hz, 6H). 13 C NMR (126 MHz, CD3OD, 25℃): δ 155.3, 111.3, 63.4, 62.8, 33.5, 30.3, 29.0, 26.8, 8.7. FTIR (thin film) cm -1 : 3258 (br), 2937 (s), 2863 (s), 1670 (m), 1461 (m), 1375 (m), 958 (s). HRMS (ESI) (m / z): calc'd for C 11 H 24 NO2[M+H] +: 202.1807, found: 202.1802. TLC (30% CMA in chloroform), Rf: 0.060 (KMnO4).

[0295] Example 24: Synthesis of 3-(dibutylamino)-N,N-diethylprop-1-ene-2-amine oxide (20) and (E)-3-(dibutylamino)-N,N-diethylprop-1-ene-1-amine oxide (20′) [ka]

[0296] Enamine N-oxide 20 was synthesized according to general procedure A using N-butyl-N-(prop-2-yn-1-yl)butan-1-amine (Acquaah-Harrison et al., J. Comb. Chem. 12:491-496 (2010)). The reaction mixture was heated for 145 h and purified by flash column chromatography on silica gel (eluent: 50% CMA in chloroform) to give regioisomeric enamine N-oxides 20 and 20' (Run 1: 43.6 mg, 57%, Run 2: 49.7 mg, 65%) as clear, colorless oils. The regioisomeric ratios (20:20', Run 1: 1: 1.2, Run 2: 1: 1.2) were determined by the cleavage of the C1 vinyl proton of the major isomer and the C1 vinyl proton of the minor isomer. 1 Determined by taking the ratio of H-NMR integrals. Further purification by flash chromatography on silica gel (eluent: 30% CMA in chloroform) afforded the regioisomers separately for analytical characterization. 1H NMR (500 MHz, CD3OD, 25℃):

[20] δ 5.92 (s, 1H), 5.67 (s, 1H), 3.59 (dq, J = 12.5, 7.0 Hz, 2H), 3.39 (dq, J = 12.5, 7.1 Hz, 2H), 3.22 (s, 2H), 2.51-2.44 (m, 2H), 1.51-1.41 (m, 4H), 1.34 (dq, J = 14.2, 7.3 Hz, 4H), 1.22 (t, J = 7.0 Hz, 6H), 0.93 (t, J = 7.3 Hz, 6H). [20'] δ 6.45 (dt, J = 13.6, 6.9 Hz, 1H), 6.19 (dt, J = 13.1, 1.4 Hz, 1H), 3.43-3.31 (m, 4H), 3.28 (dd, J = 6.9, 1.4 Hz, 2H), 2.52-2.45 (m, 4H), 1.53-1.43 (m, 4H), 1.34 (dq, J = 14.6, 7.3 Hz, 4H), 1.26 (t, J = 7.1 Hz, 6H), 0.94 (t, J = 7.4 Hz, 6H). 13 C NMR (126 MHz, CD3OD, 25℃):

[20] δ 153.2, 114.4, 63.8, 55.7, 55.0, 30.5, 21.8, 14.6, 8.8. [20'] δ 140.38, 126.83, 65.12, 54.73, 52.36, 30.07, 21.75, 14.37, 8.64. FTIR (thin film) cm -1 :

[20] 2959 (s), 2870 (m), 2803 (w), 1669 (w), 1461 (m), 1375 (m), 954 (w). [20'] 2955 (s), 2862 (m), 2803 (w), 1461 (m), 1375 (m), 976 (w). HRMS (ESI) (m / z):

[20] calc’d for C 15 H 33 N2O [M+H] + : 257.2587, found: 257.2582. [20'] calc’d for C15 H 33 NO [M+H] + : 257.2587, found: 257.2581. TLC (50% CMA in chloroform), Rf: 0.40 (20, KMnO4), 0.35 (20', KMnO4).

[0297] Example 25: Synthesis of 3-((tert-butoxycarbonyl)amino)-N,N-diethylprop-1-ene-2-amine oxide (21) and (E)-3-((tert-butoxycarbonyl)amino)-N,N-diethylprop-1-ene-1-amine oxide (21′). [ka]

[0298] Enamine N-oxide 21 was synthesized using tert-butyl prop-2-yn-1-ylcarbamate according to general procedure A (Wipf et al., Org. Lett. 6:3593-3595 (2004)). The reaction mixture was stirred at 60 °C for 155 h and purified by flash column chromatography on silica gel (eluent: 30% CMA in chloroform) to give regioisomeric enamine N-oxides 21 and 21' (Test 1: 48.8 mg, 62%; Test 2: 47.6 mg, 61%) as yellow oils. The regioisomeric ratio (21:21', Test 1: 1.5:1, Test 2: 1.5:1) was determined by taking the ratio of the H-NMR integrals between the C1 vinyl proton of the major isomer and the C1 vinyl proton of the minor isomer. 1H NMR (500 MHz, CD3OD, 25℃):

[21] δ 5.78 (s, 1H), 5.43 (s, 1H), 3.88 (s, 2H), 3.44-3.31 (m, 4H), 1.45 (s, 9H), 1.25 (t, J = 7.1 Hz, 6H). [21'] δ 6.33 (dt, J = 12.4, 6.0 Hz, 1H), 6.09 (d, J = 13.3 Hz, 1H), 3.77 (d, J = 6.0 Hz, 2H), 3.58 (dq, J = 14.0, 7.0 Hz, 4H), 1.44 (s, 9H), 1.26 (t, J = 7.2 Hz, 6H). 13 C NMR (126 MHz, CD3OD, 25℃):

[21] δ 158.1, 153.4, 111.9, 80.7, 65.3, 41.0, 28.9, 8.7. [21'] δ 158.3, 139.2, 128.0, 80.4, 64.6, 39.6, 28.9, 8.6. FTIR (thin film) cm -1 : 3343 (br), 2978 (m), 1696 (s), 1521 (m), 1167 (s). HRMS (ESI) (m / z): calc'd for C 12 H 25 N2O3[M+H] + : 245.1865, found: 245.1859. TLC (30% CMA in chloroform), Rf: 0.054 (KMnO4).

[0299] Example 26: Synthesis of N,N-diethyl-3-(phenylthio)prop-1-ene-2-amine oxide (22) and (E)-N,N-diethyl-3-(phenylthio)prop-1-ene-1-amine oxide (22′) [ka]

[0300] Enamine N-oxide 22 was synthesized according to general procedure A using phenyl(prop-2-yn-1-yl)sulfane. The reaction mixture was stirred at 60 °C for 105 h and purified by flash column chromatography on silica gel (eluent: 30% CMA in chloroform) to give regioisomeric enamine N-oxides 22 and 22' (Test 1: 46.4 mg, 57%; Test 2: 44.9 mg, 55%) as yellow oils. The regioisomeric ratio (22:22', Test 1: 2.5:1, Test 2: 1.3:1) was determined by taking the ratio of the H-NMR integrals between the C1 vinyl proton of the major isomer and the C1 vinyl proton of the minor isomer. 1 H NMR (500 MHz, CD3OD, 25℃):

[22] δ 7.53-7.13 (m, 5H), 5.84 (d, J = 1.8 Hz, 1H), 5.60-5.53 (m, 1H), 3.91 (d, J = 1.2 Hz, 2H), 3.58 (dq, J = [22'] δ 7.57-7.13 (m, 5H), 6.39 (dt, J = 13.0, 7.6 Hz, 1H), 6.07-5.97 (m, 1H), 3.68 (dd, J = 7.6, 1.3 Hz, 2H), 3.25-3.13 (m, 4H), 1.05 (t, J = 7.2 Hz, 6H). 13 C NMR (126 MHz, CD3OD, 25℃):

[22] δ 151.9, 136.4, 132.0, 130.4, 128.5, 114.8, 64.0, 34.4, 8.8. [22'] δ 140.1, 136.0, 132.0, 130.3, 128.1, 126.7, 65.3, 32.9, 8.5. FTIR (thin film) cm -1 : 3243 (br), 1655 (w), 1439 (m), 738 (s), 693 (s). HRMS (ESI) (m / z): calc'd for C 13 H 20 NOS [M+H]+ : 238.1266, found: 238.1260. TLC (30% CMA in chloroform), Rf: 0.071 (UV, KMnO4).

[0301] Example 27: Synthesis of (E)-3-(((benzyloxy)carbonyl)oxy)-N,N-dimethylprop-1-en-1-amine oxide (23). [ka]

[0302] Enamine N-oxide 23 was synthesized using N,N-dimethylhydroxylamine according to general procedure A (Liu et al., Chinese Patent No. 2009 / 101503374 (2009)). The reaction mixture was heated for 10 h, concentrated under reduced pressure, and purified by automated C 18 Reverse phase column chromatography (30g C 18 Purification on silica gel, 25 μm spherical particles, eluent: HO + 0.1% TFA (2 CV), gradient 0 → 100% MeCN / HO + 0.1% TFA (15 CV), t = 6.1 CV) afforded enamine N-oxide 23 (Run 1: 82.2 mg, 100%; Run 2: 80.5 mg, 98%) as a clear, colorless oil. The regioisomeric ratio (>20:1) was determined by taking the ratio of the H-NMR integrals between the C1 vinyl proton of the major isomer and the C1 vinyl proton of the minor isomer. 1 H NMR (500 MHz, CD3OD, 25℃): δ 7.43-7.31 (m, 5H), 6.65 (dt, J = 13.2, 1.6, 1H), 6.54 (dt, J = 13.3, 5.5, 1H), 5.17 (s, 2H), 4.77 (dd, J = 5.6, 1.5, 2H), 3.26 (s, 6H). 13C NMR (126 MHz, CD3OD, 25℃): δ 156.2, 144.7, 137.1, 129.8, 129.7, 129.5, 121.3, 71.0, 65.1, 60.5. FTIR (thin film) cm -1 : 1744 (s), 1453 (w), 1394 (w), 1267 (s), 957 (w). HRMS (ESI) (m / z): calc'd for C 13 H 18 NO4[M+H] + : 252.1230, found: 252.1227. TLC (30% CMA in chloroform), Rf: 0.30 (UV, KMnO4).

[0303] Example 28: Synthesis of N,N-dibutylhydroxylamine (S11) [ka]

[0304] Acrylonitrile (180 μL, 2.74 mmol, 1.00 equiv) was added via syringe to a solution of dibutylamine (354 mg, 2.74 mmol, 1 equiv) in methanol (135 mL) at room temperature. After 25 h, the reaction mixture was concentrated and used without further purification. The crude product was dissolved in dichloromethane (27 mL), and solid sodium carbonate (581 mg, 5.48 mmol, 2.00 equiv) was added in one portion. The resulting suspension was cooled to 0 °C in an ice-water bath, after which 39% peracetic acid / acetic acid (465 μL, 2.74 mmol, 1.00 equiv) was added via syringe, and the reaction mixture was allowed to warm to room temperature. After 3.5 h, methanol (0.5 mL) was added, and the reaction mixture was loaded directly onto a silica gel column. The reaction mixture was purified by flash column chromatography (eluent: 10% CMA in chloroform) to give the hydroxylamine S11 (316 mg, 79%) as a white solid. 1H NMR (500 MHz, CD3OD, 25℃): δ 2.62 (t, J = 7.4, 4H), 1.60-1.49 (m, 4H) 1.34 (sxt, J = 7.8, 7.3, 4H), 0.90 (t, J = 7.3, 6H). 13 C NMR (126 MHz, CD3OD, 25℃): δ 60.8, 29.7, 20.8, 14.3. FTIR (thin film) cm -1 :3165 (br), 2956 (s), 2870 (s), 1465 (s), 1372 (s), 1070 (s), 742 (s). HRMS (ESI) (m / z): calc'd for C8H 20 NO [M+H] + : 146.1539, found: 146.1537. TLC (10% CMA in chloroform), Rf: 0.33 (KMnO4).

[0305] Example 29: Synthesis of (E)-N-(3-(((benzyloxy)carbonyl)oxy)prop-1-en-1-yl)-N-butylbutan-1-amine oxide (24) [ka]

[0306] Enamine N-oxide 24 was synthesized using N,N-dibutylhydroxylamine (S11) according to general procedure B. The reaction mixture was heated for 24 h, concentrated under reduced pressure, and purified by automated C 18 Reverse phase column chromatography (30g C 18 Purification on silica gel, 25 μm spherical particles, eluent: HO + 0.1% TFA (2 CV), gradient 0 → 100% MeCN / HO + 0.1% TFA (15 CV), t = 8.3 CV) afforded enamine N-oxide 24 (Test 1: 42.1 mg, 91%; 46.2 mg, 99%) as a clear, colorless oil. The regioisomeric ratio (>20:1) was determined by taking the ratio of the H-NMR integrals between the C1 vinyl proton of the major isomer and the C1 vinyl proton of the minor isomer.1 H NMR (500 MHz, CD3OD, 25℃): δ 7.42-7.31 (m, 5H), 6.51 (dt, J = 13.2, 5.6, 1H), 6.36 (dt, J = 13.0, 1.6, 1H), 5.17 (s, 2H), 4.79 (dd, J = 5.7, 1.5, 2H), 3.35-3.21 (m, 4H), 1.86-1.54 (m, 4H), δ 1.40-1.29 (m, 4H), 0.95 (t, J = 7.4, 6H). 13 C NMR (126 MHz, CD3OD, 25℃): δ 156.3, 142.1, 137.1, 129.8, 129.7, 129.4, 124.3, 70.9, 70.7, 65.3, 26.0, 21.1, 14.3. FTIR (thin film) cm -1 : 2959 (m), 2873 (w), 1748 (s), 1457 (w), 1394 (w), 1267 (s), 950 (w). HRMS (ESI) (m / z): calc'd for C 19 H 30 NO4[M+H] + : 336.2169, found: 336.2167. TLC (30% CMA in chloroform), Rf: 30.40 (UV, KMnO4).

[0307] Example 30: Synthesis of (E)-N-(3-(((benzyloxy)carbonyl)oxy)prop-1-en-1-yl)-N-cyclohexylcyclohexaneamine oxide (25) [ka]

[0308] Enamine N-oxide 25 was synthesized using N,N-dicyclohexylhydroxylamine according to general procedure B (Murray et al., Synth. Commun. 19:3509-3522 (1989)). The reaction mixture was heated for 25 h, concentrated under reduced pressure, and purified using automated C18 Reverse phase column chromatography (30g C 18 Purification on silica gel, 25 μm spherical particles, eluent: HO+0.1% TFA (2 CV), gradient 0→100% MeCN / HO+0.1% TFA (15 CV), tR=7.0 CV) afforded enamine N-oxide 25 (Run 1: 10.8 mg, 27%; Run 2: 15.7 mg, 40%) as a clear, colorless oil. 1 H NMR (500 MHz, CD3OD, 25℃): δ 7.42-7.30 (m, 5H), 6.43 (dt, J = 12.1, 5.9, 1H), 6.21 (d, J = 13.2, 1H), 5.17 (s, 2H), 4.79 (d, J = 5.8, 2H), 3.37 (tt, J = 11.4, 2.9, 2H), 2.19 (d, J = 11.9, 2H), 1.96-1.80 (m, 6H), 1.65 (d, J = 13.0, 2H), 1.51-1.38 (m, 4H), 1.39-1.25 (m, 4H), 1.18-1.04 (m, 2H). 13 C NMR (126 MHz, CD3OD, 25℃): δ 156.4, 139.4, 137.2, 129.8, 129.7, 129.4, 125.8, 74.5, 70.9, 65.5, 28.4, 27.0, 26.7, 26.6, 26.4. FTIR (thin film) cm -1 : 2929 (m), 2855 (m), 1744 (s), 1453 (m), 1394 (w), 1263 (s), 950 (m). HRMS (ESI) (m / z): calc'd for C 23 H 34 NO4[M+H] + : 388.2482, found: 388.2476. TLC (30% CMA in chloroform), Rf: 0.50 (UV, KMnO4).

[0309] Example 31: Synthesis of N-ethyl-N-isopropylhydroxylamine (S12) [ka]

[0310] Acrylonitrile (1.07 mL, 16.4 mmol, 1.00 equiv.) was added via syringe to a solution of N-methylpropan-2-amine (1.00 g, 13.7 mmol, 1 equiv.) in methanol (135 mL) at room temperature. After 13 h, the reaction mixture was concentrated and used without further purification. The crude product was dissolved in dichloromethane (135 mL), and solid sodium carbonate (2.88 g, 27.3 mmol, 2.00 equiv.) was added in one portion. The resulting suspension was cooled to 0 °C in an ice-water bath, and then 39% peracetic acid / acetic acid (2.32 mL, 13.7 mmol, 1.00 equiv.) was added via syringe, and the reaction mixture was allowed to warm to room temperature. After 3 h, methanol (2.5 mL) was added, and the reaction mixture was loaded directly onto a silica gel column. The reaction mixture was purified by flash column chromatography (eluent: 15% CMA in chloroform). Fractions containing the desired product were collected and concentrated under reduced pressure. The resulting liquid was then purified again on a short silica plug (1 inch) to remove excess acetic acid (eluent: 50% CMA in chloroform) to give hydroxylamine S12 (953 mg, 78%) as a yellow liquid. 1 H NMR (500 MHz, CD3OD, 25℃): δ 5.72 (bs, 1H), 2.91 (sx, J = 6.5, 1H), 2.71 (q, J = 7.1, 2H), 1.13 (t, J = 7.1, 3H), 1.08 (d, J =6.5, 6H). 13 C NMR (126 MHz, CD3OD, 25℃): δ 56.7, 49.2, 18.3, 12.4. FTIR (thin film) cm -1 : 3191 (br), 2971 (s), 2851 (s), 1457 (s), 1379 (s), 1141 (s), 742 (s). HRMS (ESI) (m / z): calc'd for C5H 14 NO [M+H] +: 104.1070, found: 104.1068. TLC (20% CMA in chloroform), Rf: 0.20 (KMnO4).

[0311] Example 32: Synthesis of (E)-3-(((benzyloxy)carbonyl)oxy)-N-ethyl-N-isopropylprop-1-en-1-amine oxide (26) [ka]

[0312] Enamine N-oxide 26 was synthesized using N-ethyl-N-isopropylhydroxylamine (S12) according to general procedure B. The reaction mixture was heated for 19 h, concentrated under reduced pressure, and purified by automated C 18 Reverse phase column chromatography (30g C 18 Purification on silica gel, 25 μm spherical particles, eluent: HO+0.1% TFA (2 CV), gradient 0→100% MeCN / HO+0.1% TFA (15 CV), tR=7.5 CV) afforded enamine N-oxide 26 (Run 1: 23.4 mg, 41%; Run 2: 23.6 mg, 41%) as a clear, colorless oil. 1 H NMR (500 MHz, CD3OD, 25℃): δ 7.43-7.31 (m, 5H), 6.53 (dt, J = 13.4, 5.2, 1H), 6.44 (dt, J = 13.5, 1.5, 1H), 5.19 (s, 2H), 4.85 (d, 2H), 4.04 (sp, J = 6.5, 1H), 3.81 (qd, J = 7.1, 1.2, 2H), 1.40 (d, J = 6.4, 3H), 1.37 (d, J = 6.6, 3H), 1.32 (t, J = 7.1, 3H). 13 FTIR (thin film)cm-1 :1751 (m), 1684 (s), 1457 (w), 1397 (w), 1267 (s), 1200 (s), 1133 (s). HRMS (ESI) (m / z): calc'd for C 16 H 24 NO4[M+H] + : 294.1700, found: 294.1696. TLC (30% CMA in chloroform), Rf: 0.30 (UV, KMnO4).

[0313] Example 33: Synthesis of 4-(hydroxy(methyl)amino)butan-1-ol (S13) [ka]

[0314] A round-bottom flask was sequentially charged with 4-bromobutan-1-ol (860 mg, 5.36 mmol, 1 equiv.), N-methylhydroxylamine hydrochloride (540 mg, 6.45 mmol, 1.20 equiv.), and sodium carbonate (1.40 g, 16.1 mmol, 3.00 equiv.). N,N'-Dimethylpropyleneurea (0.5 mL) was added to the flask via syringe, and the reaction mixture was stirred at 40 °C. After 24 h, the reaction mixture was directly purified by flash column chromatography on silica gel (eluent: 5% methanol in dichloromethane) to give product S13 (174 mg, 28%) as a white solid. 1 H NMR (500 MHz, CDCl3, 25℃): δ 3.65-3.57 (m, 2H), 2.81-2.50 (m, 5H), 1.80-1.61 (m, 4H). 13 C NMR (126 MHz, CDCl3, 25℃): δ 63.0, 62.0, 48.6, 31.5, 25.3. FTIR (thin film) cm -1: 3295 (br, m), 2944 (m), 2870 (m), 1476 (m), 1397 (m), 1036 (s), 805 (m). HRMS (ESI) (m / z): calc'd for C5H 14 NO2[M+H] + : 120.1019, found: 120.1017. TLC (5% methanol in dichloromethane), Rf: 0.25 (KMnO4).

[0315] Example 34: Synthesis of (E)-N-(3-(((benzyloxy)carbonyl)oxy)prop-1-en-1-yl)-4-hydroxy-N-methylbutan-1-amine oxide (27). [ka]

[0316] Enamine N-oxide 27 was synthesized using 4-(hydroxy(methyl)amino)butan-1-ol (S13) according to general procedure B. The reaction mixture was heated for 7 h, concentrated under reduced pressure, and purified by automated C 18 Reverse phase column chromatography (30g C 18 Purification on silica gel, 25 μm spherical particles, eluent: HO + 0.1% TFA (2 CV), gradient 0 → 100% MeCN / HO + 0.1% TFA (15 CV), t = 6.5 CV) afforded enamine N-oxide 27 (Test 1: 34.6 mg, 67%; Test 2: 33.4 mg, 64%) as a clear, colorless oil. The regioisomeric ratio (>20:1) was determined by taking the ratio of the H-NMR integrals between the C1 vinyl proton of the major isomer and the C1 vinyl proton of the minor isomer. 1H NMR (500 MHz, CD3OD, 20℃): δ 7.44-7.28 (m, 5H), 6.59-6.47 (m, 2H), 5.17 (s, 2H), 4.78 (d, J = 4.2, 2H), 3.57 (t, J = 6.3, 2H), 3.43-3.31 (m, 2H), 3.21 (s, 3H), 1.92-1.65 (m, 2H), 1.54 (p, J = 7.1, 6.9, 2H). 13 FTIR (thin film)cm -1 : 2952 (w), 1748 (s), 1453 (w), 1267 (s), 943 (w). HRMS (ESI) (m / z): calc'd for C 16 H 24 NO5[M+H] + : 310.1649, found: 310.1644. TLC (30% CMA in chloroform), Rf: 0.10 (UV, KMnO4).

[0317] Example 35: Synthesis of 5-(hydroxy(methyl)amino)pentanenitrile (S14) [ka]

[0318] A round-bottom flask was charged sequentially with 5-bromovaleronitrile (720 μL, 6.17 mmol, 1 equiv.), triethylamine (1.71 mL, 12.3 mmol, 2.00 equiv.), N-methylhydroxylamine hydrochloride (1.03 g, 12.3 mmol, 2.00 equiv.), and tetrabutylammonium iodide (342 mg, 926 μmol, 0.150 equiv.). N,N'-Dimethylpropyleneurea (5 mL) was then added to the flask via syringe, and the reaction mixture was stirred at 40 °C. After 14 h, diethyl ether (30 mL) and water (30 mL) were added to the reaction mixture. The aqueous phase was then washed with diethyl ether (10 × 30 mL). The combined organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by flash column chromatography on silica gel (eluent: 10% CMA in chloroform) to afford hydroxylamine S14 (336 mg, 42%) as a white solid. 1 H NMR (500 MHz, CDCl3, 25℃): δ 2.67-2.59 (m, 5H), 2.38 (t, J = 6.6 Hz, 2H), 1.75-1.70 (m, 4H). 13 C NMR (126 MHz, CDCl3, 25℃): δ 119.8, 61.1, 49.0, 26.4, 23.4, 17.4. FTIR (thin film) cm -1 : 3201 (br, m), 2952 (s), 2870 (s), 2247 (m), 1438 (s), 1382 (m), 1028 (m), 805 (m). HRMS (ESI) (m / z): calc'd for C6H 13 NO [M+H] + : 129.1022, found: 129.1020. TLC (30% CMA in chloroform), Rf: 0.30 (KMnO4).

[0319] Example 36: Synthesis of (E)-N-(3-(((benzyloxy)carbonyl)oxy)prop-1-en-1-yl)-4-cyano-N-methylbutan-1-amine oxide (28). [ka]

[0320] Enamine N-oxide 28 was synthesized using 5-(hydroxy(methyl)amino)pentanenitrile (S14) according to general procedure B. The reaction mixture was heated for 9 h, concentrated under reduced pressure, and purified by automated C 18 Reverse phase column chromatography (30g C 18 Purification on silica gel, 25 μm spherical particles, eluent: HO + 0.1% TFA (2 CV), gradient 0 → 100% MeCN / HO + 0.1% TFA (10 CV), t = 5.6 CV) afforded enamine N-oxide 28 (Test 1: 39.2 mg, 79%; Test 2: 43.2 mg, 87%) as a clear, colorless oil. The regioisomeric ratio (>20:1) was determined by taking the ratio of the H-NMR integrals between the C1 vinyl proton of the major isomer and the C1 vinyl proton of the minor isomer. 1 H NMR (500 MHz, CD3OD, 25℃): δ 7.44-7.32 (m, 5H), 6.70-6.60 (m, 2H), 5.19 (s, 2H), 4.85 (d, J = 3.4, 2H), 3.87-3.72 (m, 2H), 3.61 (s, 3H), 2.52 (t, J = 7.1, 2H), 2.03-1.81 (m, 2H), 1.77-1.66 (m, 2H). 13 C NMR (126 MHz, CD3OD, 25℃): δ 156.1, 137.8, 136.9, 129.8, 129.8, 129.6, 126.9, 120.6, 71.2, 70.2, 64.6, 57.4, 23.3, 23.2, 17.1. FTIR (thin film) cm -1 : 2952 (s), 2855 (m), 1748 (s), 1572 (m), 1457 (m), 1394 (w), 1267 (s), 946 (w). HRMS (ESI) (m / z): calc'd for C 17 H 23 N2O4[M+H] +: 319.1652, found: 319.1650. TLC (30% CMA in chloroform), Rf: 0.30 (UV, KMnO4).

[0321] Example 37: Synthesis of N-(4-azidobutyl)-N-methylhydroxylamine (S15) [ka]

[0322] To a round-bottom flask was added 1-azido-4-bromobutane (Satyanarayana et al., Chem. Commun. 48:1461-1463 (2012)) (220 mg, 1.23 mmol, 1 equiv.), N-methylhydroxylamine hydrochloride (113 mg, 1.35 mmol, 1.10 equiv.), and sodium iodide (27.8 mg, 0.185 mmol, 0.150 equiv.). N,N'-dimethylpropylene urea (500 μL) and triethylamine (190 μL, 1.35 mmol, 1.10 equiv.) were then added sequentially via syringe, and the reaction mixture was stirred at 40 °C. After 16 h, the residue was directly purified by flash column chromatography on silica gel (eluent: 5% CMA in chloroform) to afford hydroxylamine S15 (100 mg, 57%) as a clear, colorless oil. 1 H NMR (500 MHz, CDCl3, 25℃): δ 3.25 (t, J = 6.3, 2H), 2.67-2.57 (m, 5H), 1.67-1.61 (m, 4H). 13 C NMR (126 MHz, CDCl3, 25℃): δ 61.5, 51.5, 48.8, 26.8, 24.6. FTIR (thin film) cm -1 : 3220 (br, w), 2929 (m), 2870 (m), 2094 (s), 1457 (w), 1256 (m). HRMS (ESI) (m / z): calc'd for C5H 13 NO [M+H] +: 145.1084, found: 145.1083. TLC (30% CMA in chloroform), Rf: 0.55 (KMnO4).

[0323] Example 38: Synthesis of (E)-4-azido-N-(3-(((benzyloxy)carbonyl)oxy)prop-1-en-1-yl)-N-methylbutan-1-amine oxide (29). [ka]

[0324] Enamine N-oxide 29 was synthesized using N-(4-azidobutyl)-N-methylhydroxylamine (S15) according to general procedure B. The reaction mixture was heated for 9 h, concentrated under reduced pressure, and purified by automated C 18 Reverse phase column chromatography (30g C 18 Purification on silica gel, 25 μm spherical particles, eluent: HO + 0.1% TFA (2 CV), gradient 0 → 100% MeCN / HO + 0.1% TFA (10 CV), t = 6.0 CV) afforded enamine N-oxide 29 (Test 1: 32.0 mg, 69%; Test 2: 26.8 mg, 58%) as a clear, colorless oil. The regioisomeric ratio (>20:1) was determined by taking the ratio of the H-NMR integrals between the C1 vinyl proton of the major isomer and the C1 vinyl proton of the minor isomer. 1 H NMR (500 MHz, CD3OD, 25℃): δ 7.42-7.32 (m, 5H), 6.71-6.58 (m, 2H), 5.19 (s, 2H), 4.85 (d, J = 3.5 Hz, 2H), 3.85-3.71 (m, 2H), 3.60 (s, 3H), 3.37 (t, J = 6.6 Hz, 2H), 1.96-1.75 (m, 2H), 1.70-1.58 (m, 2H). 13FTIR (thin film)cm -1 : 2952 (w), 2098 (s), 1748 (s), 1453 (w), 1263 (s), 943 (w). HRMS (ESI) (m / z): calc'd for C 16 H 23 N4O4[M+H] + : 335.1714, found: 335.1710. TLC (30% CMA in chloroform), Rf: 0.30 (UV, KMnO4).

[0325] Example 39: Synthesis of N-benzyl-5-bromopentanamide (S16) [ka]

[0326] To a solution of trimethylamine (3.66 mL, 26.3 mmol, 2.10 equiv.) in diethyl ether (125 mL) at room temperature, benzylamine hydrochloride (1.80 g, 12.5 mmol, 1.00 equiv.) was added as a solid. The solution was cooled to 0 °C in an ice-water bath. After 15 min, 5-bromovaleryl chloride (2.50 g, 12.5 mmol, 1 equiv.) was then added dropwise via syringe. The ice-water bath was removed, and the reaction mixture was allowed to warm to room temperature. After 3 h, the reaction mixture was filtered through Celite® and concentrated under reduced pressure. The crude residue was purified by flash column chromatography on silica gel (eluent: 50% ethyl acetate in hexanes) to afford pentanamide S16 (1.05 g, 31%) as an off-white solid. 1H NMR (500 MHz, CDCl3, 25℃): δ 7.38-7.20 (m, 5H), 5.88 (s, 1H), 4.41 (d, J = 5.7, 2H), 3.39 (t, J = 6.6, 2H), 2.22 (t, J = 7.3, 2H), 1.94-1.74 (m, 4H). 13 C NMR (126 MHz, CDCl3, 25℃): δ 172.3, 138.4, 128.9, 128.0, 127.7, 43.8, 35.7, 33.4, 32.3, 24.4. FTIR (thin film) cm -1 : 3265 (m), 3063 (w), 2929 (m), 1640 (s), 1543 (s), 1453 (s), 1230 (m), 1029 (m), 731 (m), 697 (s). HRMS (ESI) (m / z): calc'd for C 12 H 17 BrNO [M+H] + : 270.0488, found: 270.0485. TLC (30% ethyl acetate in hexanes), Rf: 0.25 (UV, KMnO4).

[0327] Example 40: Synthesis of N-benzyl-5-(hydroxy(methyl)amino)pentanamide (S17) [ka]

[0328] A round-bottom flask was charged with N-benzyl-5-bromopentanamide (S17, 1.05 g, 3.88 mmol, 1 equiv.) and N-methylhydroxylamine hydrochloride (648 mg, 7.76 mmol, 2.00 equiv.). N,N'-dimethylpropyleneurea (5 mL) and triethylamine (1.08 mL, 7.76 mmol, 2.00 equiv.) were then added sequentially via syringe, and the reaction mixture was stirred at 40 °C. After 14 h, the reaction mixture was directly purified by flash column chromatography on silica gel (eluent: 10% methanol in dichloromethane) to afford hydroxylamine S17 (889 mg, 97%) as a white solid. 1 H NMR (500 MHz, CDCl3, 25℃): δ 7.38-7.21 (m, 5H), 6.78 (t, J = 5.9, 1H), 4.40 (d, J = 5.6 Hz, 2H), 3.25 (t, J = 7.3, 1H), 2.30 (td, J = 7.2, 7.2, 2.3, 2H), 2.60 (s, 3H), 2.22 (t, J = 7.1, 2H), 1.77 (dq, J = 40.8, 7.4, 7.0, 4H). 13 C NMR (126 MHz, CDCl3, 25℃): δ 173.1, 138.6, 128.9, 128.0, 127.6, 61.9, 48.8, 43.7, 36.5, 26.9, 23.5. FTIR (thin film) cm -1 : 3295 (m), 2926 (m), 1632 (s), 1539 (s), 1453 (m), 745 (m), 697 (s). HRMS (ESI) (m / z): calc'd for C 13 H 21 N2O2[M+H] + : 237.1598, found: 237.1594. TLC (10% methanol in dichloromethane), Rf: 0.45 (UV, KMnO4).

[0329] Example 41: Synthesis of (E)-5-(benzylamino)-N-(3-(((benzyloxy)carbonyl)oxy)prop-1-en-1-yl)-N-methyl-5-oxopentan-1-amine oxide (30). [ka]

[0330] Enamine N-oxide 30 was synthesized using N-benzyl-5-(hydroxy(methyl)amino)pentanamide (S17) according to general procedure B. The reaction mixture was heated for 9 h, concentrated under reduced pressure, and purified by automated C 18 Reverse phase column chromatography (30g C 18 Purification on silica gel, 25 μm spherical particles, eluent: HO + 0.1% TFA (2 CV), gradient 0 → 100% MeCN / HO + 0.1% TFA (10 CV), t = 6.4 CV) afforded enamine N-oxide 30 (Test 1: 27.3 mg, 76%; Test 2: 23.9 mg, 66%) as a clear, colorless oil. The regioisomeric ratio (>20:1) was determined by taking the ratio of the H-NMR integrals between the C1 vinyl proton of the major isomer and the C1 vinyl proton of the minor isomer. 1 H NMR (500 MHz, CD3OD, 25℃): δ 7.48-7.18 (m, 10H), 6.64 (dt, J = 13.4, 1.3, 1H), 6.59 (dt, J = 13.5, 4.5, 1H), 5.19 (s, 2H), 4.83 (dd, J = 4.5, 1.3, 2H), 4.36 (s, 2H), 3.84-3.68 (m, 2H), 3.56 (s, 3H), 2.30 (t, J = 7.1, 2H), 1.87-1.72 (m, 2H), 1.73-1.64 (m, 2H). 13C NMR (126 MHz, CD3OD, 25℃): δ 175.0, 156.1, 140.2, 137.9, 136.9, 129.9, 129.8, 129.7, 129.6, 128.8, 128.4, 126.8, 71.3, 70.9, 64.6, 57.2, 44.3, 36.1, 23.6, 23.4. FTIR (thin film) cm -1 : 3067 (w), 1751 (s), 1654 (s), 1546 (m), 1263 (s), 1200 (s), 1133 (s), 719 (m). HRMS (ESI) (m / z): calc'd for C 24 H 31 N2O5[M+H] + : 427.2227, found: 427.2221. TLC (40% CMA in chloroform), Rf: 0.25 (UV, KMnO4).

[0331] Example 42: Synthesis of (E)-4-(3-(((benzyloxy)carbonyl)oxy)prop-1-en-1-yl)morpholine 4-oxide (31). [ka]

[0332] Enamine N-oxide 31 was synthesized using morpholin-4-ol according to general procedure B (O'Neil et al., Tetrahedron Lett. 42:8247-8249 (2001)). The reaction mixture was heated for 24 h, concentrated under reduced pressure, and purified by automated C 18 Reverse phase column chromatography (30g C 18 Purification on silica gel, 25 μm spherical particles, eluent: HO+0.1% TFA (2 CV), gradient 0→100% MeCN / HO+0.1% TFA (15 CV), tR=6.1 CV) afforded enamine N-oxide 31 (Run 1: 48.6 mg, 85%; Run 2: 45.4 mg, 80%) as a clear, colorless oil. 1H NMR (500 MHz, CD3OD, 25℃): δ 7.41-7.30 (m, 5H), 6.62 (t, J = 1.3 Hz, 2H), 5.17 (s, 2H), 4.80 (d, J = 2.9, 2H), 4.26 (ddd, J = 7.1, 2H), 3.81 (d, J = 12.4, 2H), 3.64 (td, J = 11.7, 3.7, 2H), 2.98 (d, J = 11.5, 2H). 13 C NMR (126 MHz, CD3OD, 25℃): δ 156.2, 144.7, 137.1, 129.8, 129.7, 129.5, 122.6, 71.0, 66.4, 65.2, 62.5. FTIR (thin film) cm -1 : 2944 (w), 1744 (s), 1457 (w), 1394 (w), 1263 (s), 1118 (m), 950 (m). HRMS (ESI) (m / z): calc'd for C 15 H 20 NO5[M+H] + : 294.1336, found: 294.1331. TLC (30% CMA in chloroform), Rf: 0.20 (UV, KMnO4).

[0333] Example 43: Synthesis of 1-(ナフタレン-1-イル)ブタ-3-イン-2-オール (S18)

change

[0334] A round-bottom flask was charged with 2-(1-naphthyl)ethanol (300 mg, 1.74 mmol, 1 equiv.) and dissolved in dichloromethane (17 mL) at room temperature. Pyridinium chlorochromate (413 mg, 1.92 mmol, 1.10 equiv.) was added in one portion, and the flask was stirred at room temperature. After 22 h, silica gel (1.00 g) was added, and the reaction mixture was stirred for 10 min. The slurry was then loaded directly onto a short silica gel plug, and the crude product was eluted with diethyl ether (100 mL). The eluent was concentrated under reduced pressure. The crude residue was then dissolved in tetrahydrofuran (17 mL) and cooled to 0 °C using an ice-water bath. A solution of ethynylmagnesium bromide (500 mM, 3.48 mL, 1.74 mmol, 1.00 equiv.) in tetrahydrofuran was then added dropwise via syringe. After 1 h, saturated aqueous ammonium chloride solution (10 mL) and ethyl acetate (50 mL) were added sequentially. The organic layer was washed with water (2×50 mL) and brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by flash column chromatography on silica gel (eluent: 15% ethyl acetate / hexane) to give the product S18 (82.0 mg, 24%) as a clear, colorless oil. 1 H NMR (500 MHz, CDCl3, 25℃): δ 8.07 (dd, J = 8.4, 1.2, 1H), 7.86 (dd, J = 8.0, 1.5, 1H), 7.78 (dd, J = 7.0, 2.4, 1H), 7.56-7.39 (m, 4H), 4.75 (ddd, J = 7.8, 6.0, 2.1, 1H), 3.58-3.41 (m, 2H), 2.48 (d, J = 2.1, 1H), 2.00 (s, 1H). 13 C NMR (126 MHz, CDCl3, 25℃): δ 134.1, 132.6, 132.3, 129.1, 128.4, 128.1, 126.3, 125.9, 125.6, 123.8, 84.5, 74.1, 62.7, 41.2. FTIR (thin film)cm -1: 3369 (br-w), 3287 (m), 3045 (w), 1025 (s), 775 (s). HRMS (ESI) (m / z): calc'd for C 14 H 12 NaO [M+Na] + : 219.0780, found: 219.0778. TLC (15% ethyl acetate in hexanes), Rf: 0.30 (UV, KMnO4).

[0335] Example 44: Synthesis of 1-(naphthalen-1-yl)but-3-yn-2-one (S19) [ka]

[0336] A round-bottom flask was charged with 1-(naphthalen-1-yl)but-3-yn-2-ol (S18, 15.0 mg, 76.4 μmol, 1 equiv.) and dissolved in dichloromethane (1 mL) at room temperature. Dess-Martin periodinane (32.4 mg, 76.4 μmol, 1.00 equiv.) was added to the flask in one portion. After 15 h, a 1:1 aqueous solution of saturated sodium thiosulfate and sodium bicarbonate (1 mL) was added. The mixture was diluted with ethyl acetate (20 mL), and the organic layer was washed with water (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by flash column chromatography on silica gel (eluent: 5% ethyl acetate / hexane) to give product S19 (11.0 mg, 74%) as a clear, colorless oil. 1 H NMR (500 MHz, CDCl3, 25℃): δ 7.91-7.79 (m, 3H), 7.56-7.37 (m, 4H), 4.28 (s, 2H), 3.14 (s, 1H). 13C NMR (126 MHz, CDCl3, 25℃): δ 184.6, 134.1, 132.4, 129.1, 129.0, 128.8, 128.8, 126.8, 126.2, 125.7, 123.9, 81.3, 80.4, 50.0. FTIR (thin film)cm -1 : 3272 (m), 3049 (w), 2094 (s), 1677 (s), 1088 (s), 786 (s). HRMS (ESI) (m / z): calc'd for C 14 H 10 NaO [M+Na] + : 217.0624, found: 217.0621. TLC (20% ethyl acetate in hexanes), Rf: 0.70 (UV).

[0337] Example 45: Synthesis of 1-(2,2-difluorobut-3-yn-1-yl)naphthalene (S20) [ka]

[0338] A round-bottom flask was charged with 1-(naphthalen-1-yl)but-3-yn-2-one (S19, 522 mg, 2.69 mmol, 1 equiv.) and cooled to 0 °C in an ice-water bath. With stirring, neat diethylaminosulfur trifluoride (728 μL, 5.51 mmol, 2.05 equiv.) was added dropwise via syringe. The ice-water bath was removed, and the reaction mixture was stirred at room temperature. After 16 h, the reaction was directly loaded onto silica gel and purified by flash column chromatography (eluent: 100% hexanes) to give the product S20 (206 mg, 35%) as a white solid. 1 H NMR (500 MHz, CDCl3, 25℃): δ 8.13 (d, J = 8.5, 1H), 7.95-7.85 (m, 2H), 7.64-7.45 (m, 4H), 3.88 (t, J =14.7, 2H), 2.70 (t, J = 5.0, 1H). 13C NMR (126 MHz, CDCl3, 25℃): δ 134.1, 132.9, 130.0, 128.9, 128.9, 128.0 (t, J = 3.5), 126.4, 125.9, 125.4, 124.4, 113.5 (t, J = 235.5), 76.7 (t, J = 6.7), 76.5 (t, J = 40.5), 41.8 (t, J = 26.8). 19 F NMR (471 MHz, CDCl3, 25℃): δ 81.8. FTIR (thin film) cm -1 : 3295 (w), 2135 (w), 1271 (w), 1159 (m), 1054 (m), 1025 (m), 779 (s). HRMS (ESI) (m / z): calc'd for C 14 H 11 F2 [M+H] + : 217.0823, found: 217.0822. TLC (100% hexanes), Rf: 0.15 (UV, KMnO4).

[0339] Example 46: Synthesis of 5-bromo-N-(naphthalen-2-ylmethyl)pentanamide (S21) [ka]

[0340] 5-Bromovaleryl chloride (1.01 mL, 7.52 mmol, 1 equiv.) was added via syringe to a round-bottom flask containing diethyl ether (75 mL) cooled to 0 °C using an ice-water bath. Triethylamine (2.20 mL, 15.8 mmol, 2.10 equiv.) and 1-naphthalenemethylamine (1.10 mL, 7.52 mmol, 1.00 equiv.) were then added dropwise to the reaction mixture via syringe. The ice-water bath was removed, and stirring was continued at room temperature. After 7 h, the reaction mixture was concentrated under reduced pressure. The resulting residue was purified by flash column chromatography on silica gel (eluent: 50% ethyl acetate in hexanes) to afford pentanamide S21 (2.07 g, 86%) as a white solid. 1 H NMR (500 MHz, CDCl3, 25℃): δ 7.98 (d, J = 8.2, 1H), 7.86 (dd, J = 7.8, 1.6, 1H), 7.80 (dd, J = 5.5, 4.0, 1H), 7.52 (dddd, J = 17.5, 8.1, 6.8, 1.4, 2H), 7.57-7.46 (m, 2H), 5.68 (s, 1H), 4.87 (d, J = 5.3, 2H), 3.36 (t, J = 6.6, 2H), 2.19 (t, J = 7.2, 2H), 1.94-1.73 (m, 4H). 13 C NMR (126 MHz, CDCl3, 25℃): δ 172.0, 134.1, 133.7, 131.6, 129.0, 128.9, 127.1, 126.9, 126.3, 125.6, 123.7, 42.1, 35.7, 33.3, 32.3, 24.4. FTIR (thin film) cm -1 : 3280 (m), 3056 (w), 2933 (w), 1640 (s), 1543 (s), 1267 (m), 775 (s). HRMS (ESI) (m / z): calc'd for C 16 H 19 BrNO [M+H] +: 320.0645, found: 320.0641. TLC (40% ethyl acetate in hexanes), Rf: 0.30 (UV, KMnO4).

[0341] Example 47: Synthesis of 5-(hydroxy(methyl)amino)-N-(naphthalen-2-ylmethyl)pentanamide (S22) [ka]

[0342] A round-bottom flask was charged with 5-bromo-N-(naphthalen-2-ylmethyl)pentanamide (S15, 1.00 g, 3.12 mmol, 1 equiv.) and N-methylhydroxylamine hydrochloride (522 mg, 6.25 mmol, 2.00 equiv.). N,N'-Dimethylpropyleneurea (3 mL) and triethylamine (871 μL, 6.25 mmol, 2.00 equiv.) were then added sequentially via syringe, and the reaction mixture was stirred at 40 °C. After 19 h, the residue was directly purified by flash column chromatography on silica gel (eluent: 5% methanol in dichloromethane) to afford hydroxylamine S22 (638 mg, 71%) as a white solid. 1 H NMR (500 MHz, CD3OD, 25℃): δ 8.04 (d, J = 8.4, 1H), 7.91-7.85 (m, 1H), 7.81 (d, J = 7.8, 1H), 7.59-7.39 (m, 4H), 4.82 (s, 2H), 2.61-2.55 (m, 2H), 2.53 (s, 3H), 2.25 (t, J = 7.4, 2H), δ 1.73-1.63 (m, 2H), 1.62-1.52 (m, 2H). 13 C NMR (126 MHz, CD3OD, 25℃): δ 175.8, 135.5, 135.2, 132.9, 129.9, 129.4, 127.5, 127.4, 127.0, 126.5, 124.7, 63.0, 49.1, 42.4, 37.0, 27.9, 24.9. FTIR (thin film) cm-1 : 3283 (br, m) 3062 (w), 2952 (m), 1640 (s), 1543 (m), 779 (m). HRMS (ESI) (m / z): calc'd for C 17 H 23 N2O2[M+H] + : 287.1754, found: 287.1751. TLC (5% methanol in dichloromethane), Rf: 0.30 (UV, KMnO4).

[0343] Example 48: Synthesis of (E)-N-(3,3-difluoro-4-(naphthalen-1-yl)but-1-en-1-yl)-N-methyl-5-((naphthalen-2-ylmethyl)amino)-5-oxopentan-1-amine oxide (S1). [ka]

[0344] A glass 1-dram vial was charged with 1-(2,2-difluorobut-3-yn-1-yl)naphthalene (S20, 50.0 mg, 231 μmol, 1 equiv.) and dissolved in 20% v / v trifluoroethanol in chloroform (116 μL) at room temperature. 5-(hydroxy(methyl)amino)-N-(naphthalen-2-ylmethyl)pentanamide (S22, 99.0 mg, 347 μmol, 1.50 equiv.) was then added in one portion. The vial was flushed with nitrogen, capped, sealed with parafilm, and heated to 60 °C. After 12 h, the reaction mixture was removed from the oil bath and allowed to cool to room temperature. The reaction mixture was directly purified by flash column chromatography on silica gel (eluent: 50% CMA in chloroform). Fractions containing the desired compound were combined and the solvent was removed under reduced pressure at 0° C. using a rotary evaporator to give enamine N-oxide S1 (97.9 mg, 84%) as an off-white solid. 1H NMR (500 MHz, CD3OD, 25℃): δ 8.10 (d, J = 8.5, 1H), 8.04 (dd, J = 8.4, 1.2, 1H), 7.91-7.77 (m, 4H), 7.57-7.38 (m, 8H), 6.63 (q, J = 12.2, 1H), 6.41 (dt, J = 13.1, 2.0, 1H), 4.85-4.77 (m, 2H), 3.84 (t, J = 15.3, 2H), 3.05-2.97 (m, 2H), 2.88 (s, 3H), 2.15 (t, J = 7.3, 2H), 1.53-1.36 (m, 3H), 1.28-1.16 (m, 1H). 13 C NMR (126 MHz, CD3OD, 25℃): δ 175.0, 145.5 (t, J = 9.4 Hz), 135.5, 135.5, 135.3, 134.1, 132.9, 131.2, 130.1 (t, J = 4.6 Hz), 129.9, 129.9, 129.7, 129.5, 127.6, 127.5, 127.5, 127.0, 127.0, 127.0, 126.6, 126.5, 125.6, 124.7, 123.9 (t, J = 26.8 Hz), 122.1 (t, J = 241.9 Hz), 71.4, 58.5, 42.4, 40.9 (t, J = 27.1 Hz), 36.4, 23.8, 23.6. 19 F NMR (470.5 MHz, CDCl3, 25℃): δ -94.3 (q, J = 244.2). FTIR (thin film) cm -1 : 3049 (m), 2937 (m), 1647 (m), 1550 (m), 1028 (m), 779 (s). HRMS (ESI) (m / z): calc’d for C 31 H 33 F2N2O2[M+H] + : 503.2505, found: 503.2503. TLC (50% CMA in chloroform), Rf: 0.30 (UV, KMnO4).

[0345] Example 49: Synthesis of (1E,2Z)-3-fluoro-N-methyl-4-(naphthalen-1-yl)but-2-en-1-imine oxide (S2). [ka]

[0346] A 1-dram vial was charged with enamine N-oxide S1 (90.0 mg, 179 μmol, 1 equiv.) and dissolved in chloroform-d (1.79 mL). The reaction vial was flushed with nitrogen, sealed with a cap and parafilm, and heated to 60°C. After 1 h, the oil bath was removed and the reaction was cooled to room temperature. The reaction mixture was purified by flash column chromatography on silica gel (eluent: 5% CMA in chloroform). Fractions containing the desired compound were combined, and the solvent was removed under reduced pressure at 0°C using a rotary evaporator to give product S2 (14.9 mg, 34%) as an off-white solid. 1 H NMR (500 MHz, CDCl3, 25℃): δ 7.94 (d, J = 8.3, 1H), 7.86 (d, J = 7.3, 1H), 7.79 (d, J = 7.9, 1H), 7.55-7.35 (m, 4H), 7.31 (d, J = 9.6, 1H), 6.16 (dd, J = 36.1, 9.7, 1H), 4.08 (d, J = 14.0, 2H), 3.65 (s, 3H). 13 C NMR (126 MHz, CDCl3, 25℃): δ 165.7 (d, J = 277.1), 134.2, 132.0, 130.7 (d, J = 3.0), 130.4 (d, J = 3.2), 129.1, 128.6, 128.1, 126.8, 126.1, 125.7, 123.6, 100.2 (d, J = 8.1), 52.6, 36.5 (d, J = 26.2). 19 F NMR (471 MHz, CDCl3, 25℃): δ -89.5. FTIR (thin film) cm -1: 2922 (m), 2855 (w), 1662 (m), 1569 (w), 1408 (m), 1140 (s), 779 (s). HRMS (ESI) (m / z): calc'd for C 15 H 15 FNO [M+H] + : 244.1132, found: 244.1129. TLC (5% CMA / chloroform), Rf: 0.30 (UV, KMnO4).

[0347] Example 50: Synthesis of N-(naphthalen-2-ylmethyl)pent-4-enamide (S3).

[0348] A 1-dram vial was charged with enamine N-oxide S1 (90.0 mg, 179 μmol, 1 equiv.) and dissolved in chloroform-d (1.79 mL). The reaction vial was flushed with nitrogen, capped, sealed with parafilm, and heated to 60°C. After 1 h, the oil bath was removed and the reaction was cooled to room temperature. The reaction mixture was purified by flash column chromatography on silica gel (eluent: 100% chloroform). Fractions containing the desired compound were combined, and the solvent was removed under reduced pressure at 0°C using a rotary evaporator to give olefin S3 (8.10 mg, 19%) as a white solid. 1 H NMR (500 MHz, CDCl3, 25℃): δ 7.98 (d, J = 8.1, 1H), 7.86 (d, J = 9.1, 1H), 7.80 (dd, J = 6.4, 3.0, 1H), 7.56-7.46 (m, 2H), 7.41 (q, J = 3.7, 2H), 5.79 (ddt, J = 16.8, 10.2, 6.5, 1H), 5.65 (s, 1H), 5.02 (dd, J = 17.2, 1.6, 1H), 4.95 (dd, J = 10.2, 1.5, 1H), 4.88 (d, J = 5.3, 2H), 2.40 (q, J = 7.3, 2H), 2.27 (dd, J = 8.2, 6.6, 2H). 13C NMR (126 MHz, CDCl3, 25℃): δ 172.0, 137.1, 134.1, 133.8, 131.6, 129.0, 128.9, 127.1, 126.9, 126.2, 125.6, 123.8, 115.9, 42.0, 36.1, 29.8. FTIR (thin film) cm -1 : 3291 (s), 2926 (w), 1636 (s), 1535 (s), 775 (s). HRMS (ESI) (m / z): calc'd for C 16 H 18 NO [M+H] + : 240.1383, found: 240.1380. TLC (20% ethyl acetate in hexanes), Rf: 0.15 (UV).

[0349] Example 51: Synthesis of prop-2-yn-1-yl(2-nitrophenyl)carbamate (S23) [ka]

[0350] A round-bottom flask was charged with 2-nitroaniline (300 mg, 2.17 mmol, 1 equiv.) and dissolved in dichloromethane (20 mL) at room temperature. Triethylamine (1.51 mL, 10.9 mmol, 5.00 equiv.) was then added via syringe, and the flask was cooled to 0 °C in an ice-water bath. Triphosgene (258 mg, 869 μmol, 0.400 equiv.) was added as a solid in one portion, and the ice-water bath was removed. After 1.5 h at room temperature, the reaction was cooled to 0 °C, and propargyl alcohol (192 μL, 3.26 mmol, 1.50 equiv.) was added dropwise via syringe. The ice-water bath was removed, and the reaction mixture was allowed to warm to room temperature. After 1.5 h, the reaction mixture was diluted with dichloromethane (20 mL), and aqueous hydrogen chloride solution (1 N, 10 mL) was added. The organic layer was washed sequentially with water (2 × 20 mL) and brine (20 mL). The organic layer was then dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting crude product was purified by flash column chromatography on silica gel (eluent: 5% ethyl acetate in hexane) to afford alkyne S23 (421 mg, 88%) as a yellow solid. 1 H NMR (500 MHz, CDCl3, 25℃): δ 9.94 (s, 1H), 8.54 (dd, J = 8.6, 1.3, 1H), 8.20 (dd, J = 8.5, 1.6, 1H), 7.63 (ddd, J = 8.7, 7.2, 1.6, 1H), 7.14 (ddd, J = 8.5, 7.2, 1.3, 1H), 4.80 (d, J = 2.4, 2H), 2.53 (t, J = 2.4, 7.2 1H). 13 C NMR (126 MHz, CD3OD, 25℃): δ 152.1, 139.6, 136.4, 135.2, 126.9, 124.7, 123.2, 78.8, 76.8, 54.2. FTIR (thin film) cm -1: 3339 (m), 3272 (m), 1744 (s), 1610 (m), 1498 (s), 1435 (m), 1334 (s), 1237 (s), 1054 (s), 976 (m), 738 (m). HRMS (ESI) (m / z): calc'd for C 10 H9N2O4[M+H] + : 221.0557, found: 221.0555. TLC (10% ethyl acetate in hexanes), Rf: 0.30 (UV, KMnO4).

[0351] Example 52: Synthesis of (E)-N,N-dimethyl-3-(((2-nitrophenyl)carbamoyl)oxy)prop-1-en-1-amine oxide (32a). [ka]

[0352] A 2 mL glass LC-MS vial was charged with prop-2-yn-1-yl (2-nitrophenyl)carbamate (S23, 20.0 mg, 90.8 μmol, 1 equiv.) at room temperature. A solution of N,N-dimethylhydroxylamine (Liu et al., Chinese Patent No. 2009 / 101503374 A (2009)) (2.27 M, 200 μL, 454 μmol, 5.00 equiv.) in 20% v / v trifluoroethanol in chloroform was then added via syringe. The vial was flushed with nitrogen, sealed with a septum cap and parafilm, and heated to 60 °C. After 19 h, the reaction mixture was removed from the heat, cooled to room temperature, and loaded directly onto a silica gel column. The reaction mixture was purified by flash column chromatography (eluent: 30% CMA in chloroform). Fractions containing the desired compound were combined and the solvent was removed under reduced pressure on a rotary evaporator at 0° C. to give enamine N-oxide 32a (23.0 mg, 90%) as a yellow solid. 1H NMR (500 MHz, CD3OD, 25℃): δ = 8.18 (dd, J = 8.4, 1.3, 1H), 8.14 (dd, J = 8.4, 1.6, 1H), 7.70 (ddd, J = 8.6, 7.3, 1.6, 1H), 7.29 (ddd, J = 8.5, 7.3, 1.3, 1H), 6.88 (dt, J = 13.5, 1.8, 1H), 6.72 (dt, J = 13.5, 5.0, 1H), 4.90 (dd, J = 5.0, 1.9, 2H), 3.66 (s, 6H). 13 C NMR (126 MHz, CD3OD, 25℃): δ 154.3, 140.4, 139.7, 136.3, 134.8, 126.9, 126.2, 125.2, 123.8, 62.4, 58.8. FTIR (thin film) cm -1 : 1744 (m), 1610 (m), 1505 (s), 1341 (m), 1192 (s), 1133 (m). HRMS (ESI) (m / z): calc'd for C 12 H 16 N3O5[M+H] + : 282.1084, found: 282.1081. TLC (30% CMA in chloroform), Rf: 0.20 (UV, KMnO4).

[0353] Example 53: Synthesis of (E)-4-(3-(((2-nitrophenyl)carbamoyl)oxy)prop-1-en-1-yl)morpholine 4-oxide (32b). [ka]

[0354] A 2 mL glass LC-MS vial was charged with prop-2-yn-1-yl (2-nitrophenyl)carbamate (S23, 20.0 mg, 90.8 μmol, 1 equiv.) at room temperature. A solution of morpholin-4-ol (O'Neil et al., Tetrahedron Lett. 42:8247-8249 (2001)) (1.00 M, 454 μL, 454 μmol, 5.00 equiv.) was added via syringe. The vial was flushed with nitrogen, sealed with a septum cap and parafilm, and heated to 60 °C. After 23 h, the reaction was removed from the heat, cooled to room temperature, and loaded directly onto a silica gel column. The reaction mixture was purified by flash column chromatography (eluent: 30% CMA in chloroform). Fractions containing the desired compound were combined and the solvent was removed under reduced pressure on a rotary evaporator at 0° C. to give enamine N-oxide 32b (16.6 mg, 57%) as a clear yellow oil. 1 H NMR (500 MHz, CD3OD, 25℃): δ 8.16 (ddd, J =16.9, 8.4, 1.4, 2H), 7.70 (ddd, J = 8.6, 7.3, 1.6, 1H), 7.30 (ddd, J = 8.5, 7.3, 1.3, 1H), 6.89 (dt, J= 13.6, 1.8, 1H), 6.78 (dt, J = 13.6, 4.9, 1H), 4.93 (dd, J = 4.9, 1.8, 2H), 4.20 (ddd, J = 13.6, 10.7, 2.2, 2H), 4.07-3.96 (m, 4H), 3.73 (dt, J = 10.9, 2.3, 2H). 13 C NMR (126 MHz, CD3OD, 25℃): δ 154.3, 140.5, 139.1, 136.3, 134.8, 127.9, 126.9, 125.2, 123.8, 65.6, 62.6, 62.3. FTIR (thin film) cm -1 : 1744 (m) 1684 (m), 1610 (m), 1505 (s), 1341 (m), 1200 (s), 1129 (m). HRMS (ESI) (m / z): calc'd for C14 H 18 N3O6[M+H] + : 324.1190, found: 324.1187. TLC (30% CMA in chloroform), Rf: 0.10 (UV, KMnO4).

[0355] Example 54: Synthesis of (E)-N,N-diethyl-3-(((2-nitrophenyl)carbamoyl)oxy)prop-1-en-1-amine oxide (32c). [ka]

[0356] A 2 mL glass LC-MS vial was charged with prop-2-yn-1-yl (2-nitrophenyl)carbamate (S23, 300 mg, 1.36 mmol, 1 equiv.) and dissolved in 20% v / v trifluoroethanol in chloroform (6.81 mL) at room temperature. N,N-Diethylhydroxylamine (700 μL, 6.81 mmol, 5.00 equiv.) was then added via syringe. The vial was flushed with nitrogen, sealed with a septum cap and parafilm, and heated to 60 °C. After 8.5 h, the reaction was removed from the heat, cooled to room temperature, and loaded directly onto a silica gel column. The reaction mixture was purified by flash column chromatography (eluent: 30% CMA in chloroform). Fractions containing the desired compound were combined and the solvent was removed under reduced pressure on a rotary evaporator at 0° C. to give enamine N-oxide 32c (357 mg, 85%) as a yellow solid. 1H NMR (500 MHz, CD3OD, 25℃): δ 8.15 (ddd, J = 12.7, 8.4, 1.3, 2H), 7.70 (ddd, J = 8.7, 7.3, 1.6, 1H), 7.29 (ddd, J = 8.5, 7.3, 1.3, 1H), 6.62 (dt, J = 13.5, 5.0, 1H), 6.47 (dt, J = 13.4, 1.8, 1H), 4.92 (dd, J = 5.0, 1.8, 2H), 3.83 (p, J = 7.1, 4H), 1.39 (t, J = 7.1, 6H). 13 C NMR (126 MHz, CD3OD, 25℃): δ 154.5, 140.6, 136.2, 134.8, 128.9, 126.8, 125.2, 123.9, 120.0, 65.4, 62.8, 8.3. FTIR (thin film) cm -1 : 1725 (s), 1610 (m), 1528 (s), 1356 (m), 1237 (s). HRMS (ESI) (m / z): calc'd for C 14 H 20 N3O5[M+H] + : 310.1397, found: 310.1393. TLC (30% CMA in chloroform), Rf: 0.20 (UV, KMnO4).

[0357] Example 55: Synthesis of (E)-N-butyl-N-(3-(((2-nitrophenyl)carbamoyl)oxy)prop-1-en-1-yl)butan-1-amine oxide (32d). [ka]

[0358] A 2 mL glass LC-MS vial was charged with prop-2-yn-1-yl (2-nitrophenyl)carbamate (S23, 40.0 mg, 182 μmol, 2.00 equiv.) at room temperature. A solution of N,N-dibutylhydroxylamine (S11, 454 mM, 200 μL, 90.8 μmol, 1 equiv.) in 20% v / v trifluoroethanol in chloroform was then added via syringe. The vial was flushed with nitrogen, sealed with a septum cap and parafilm, and heated to 60 °C. After 28 h, the reaction was removed from the heat, cooled to room temperature, and loaded directly onto a silica gel column. The reaction mixture was purified by flash column chromatography (eluent: 30% CMA in chloroform). Fractions containing the desired compound were combined and the solvent was removed under reduced pressure on a rotary evaporator at 0° C. to give enamine N-oxide 32d (19.7 mg, 63%) as a clear yellow oil. 1 H NMR (500 MHz, CD3OD, 25℃): δ 8.14 (ddd, J = 8.2, 6.6, 1.4, 2H), 7.70 (ddd, J = 8.6, 7.3, 1.5, 1H), 7.29 (ddd, J = 8.5, 7.3, 1.3, 1H), 6.60 (dt, J = 13.5, 4.9, 1H), 6.50 (dt, J = 13.4, 1.7, 1H), 4.90 (dd, J = 5.0, 1.7, 2H), 3.41-3.31 (m, 2H), 3.31-3.23 (m, 2H), 1.88-1.59 (m, 4H), 1.42 (sx, J = 7.4, 4H), 0.98 (t, J = 7.4, 6H). 13 C NMR (126 MHz, CD3OD, 25℃): δ 154.5, 140.3, 136.2, 135.7, 134.8, 128.2, 126.9, 125.3, 123.9, 70.1, 62.7, 25.6, 20.5, 14.1. FTIR (thin film)cm -1: 2907 (w), 1744 (m), 1610 (m), 1505 (s), 1341 (m), 1196 (s). HRMS (ESI) (m / z): calc'd for C 18 H 28 N3O5[M+H] + : 366.2023, found: 366.2016. TLC (30% CMA in chloroform), Rf: 0.50 (UV, KMnO4).

[0359] Example 56: Synthesis of (E)-N-ethyl-N-isopropyl-3-(((2-nitrophenyl)carbamoyl)oxy)prop-1-en-1-amine oxide (32e). [ka]

[0360] A 2 mL glass LC-MS vial was charged with prop-2-yn-1-yl (2-nitrophenyl)carbamate (S23, 20.0 mg, 90.8 μmol, 1 equiv.) at room temperature. Then, a 20% v / v solution of N-ethyl-N-isopropylhydroxylamine (S12, 2.27 M, 200 μL, 454 μmol, 5.00 equiv.) in chloroform (trifluoroethanol) was added via syringe. The vial was flushed with nitrogen, sealed with a septum cap and parafilm, and heated to 60 °C. After 23 h, the reaction mixture was removed from the heat, cooled to room temperature, and loaded directly onto a silica gel column. The reaction mixture was purified by flash column chromatography (eluent: 30% CMA in chloroform). Fractions containing the desired compound were combined and the solvent was removed under reduced pressure on a rotary evaporator at 0° C. to give enamine N-oxide 32e (12.0 mg, 41%) as a clear yellow oil. 1H NMR (500 MHz, CD3OD, 25℃): δ 8.14 (ddd, J = 8.4, 3.3, 1.4, 2H), 7.70 (ddd, J = 8.7, 7.3, 1.6, 1H), 7.30 (ddd, J = 8.5, 7.3, 1.3, 1H), 6.60 (dt, J = 13.5, 5.0, 1H), 6.50 (dt, J = 13.5, 1.6, 1H), 4.92 (dd, J = 5.0, 1.6, 2H), 4.08 (sp, J = 6.5, 1H), 3.85 (q, J = 7.1, 2H), 1.45 (dd, J = 9.8, 6.5, 6H), 1.38 (t, J = 7.1, 3H). 13 FTIR (thin film)cm -1 : 1744 (m), 1684 (m) 1613 (m), 1505 (s), 1341 (m), 1196 (s), 1133 (m). HRMS (ESI) (m / z): calc'd for C 15 H 22 N3O5[M+H] + : 324.1554, found: 324.1550. TLC (30% CMA in chloroform), Rf: 0.20 (UV, KMnO4).

[0361] Example 57: Synthesis of (E)-N-cyclohexyl-N-(3-(((2-nitrophenyl)carbamoyl)oxy)prop-1-en-1-yl)cyclohexaneamine oxide (32f). [ka]

[0362] A 2 mL glass LC-MS vial was charged with prop-2-yn-1-yl (2-nitrophenyl)carbamate (S23, 40.0 mg, 0.182 mmol, 1 equiv.) at room temperature. A solution containing N,N-dicyclohexylhydroxylamine (Murray et al., Synth. Commun. 19:3509-3522 (1989)) (2.73 M, 200 μL, 546 μmol, 3.00 equiv.) was added via syringe. The vial was flushed with nitrogen, sealed with a septum cap and parafilm, and heated to 60 °C. After 24 h, the reaction was removed from the heat, cooled to room temperature, and loaded directly onto a silica gel column. The reaction mixture was purified by flash column chromatography (eluent: 10% CMA in chloroform). Fractions containing the desired compound were combined and the solvent was removed under reduced pressure on a rotary evaporator at 0° C. to give enamine N-oxide 32f (20.0 mg, 26%) as a clear yellow oil. 1 H NMR (500 MHz, CD3OD, 25℃): δ 8.12 (ddd, J = 8.3, 6.8, 1.4, 2H), 7.70 (ddd, J = 8.7, 7.3, 1.6, 1H), 7.30 (ddd, J = 8.4, 7.3, 1.3, 1H), 6.51 (dt, J = 13.4, 5.1, 1H), 6.41 (dt, J = 13.4, 1.7, 1H), 4.90 (dd, J = 5.2, 1.6, 2H), 3.93 (tt, J = 11.7, 3.4, 2H), 2.22 (dt, J = 11.9, 3.3, 2H), 2.12 (dt, J = 11.8, 3.4, 2H), 1.96 (tdd, J = 16.4, 6.3, 2.9, 4H), 1.70 (dt, J = 13.5, 3.2, 2H), 1.56 (dddd, J = 15.1, 11.9, 9.3, 3.3, 4H), 1.42 (qq, J = 13.2, 3.5, 4H), 1.19 (qt, J = 13.1, 3.8, 2H). 13C NMR (126 MHz, CD3OD, 25℃): δ 154.7, 141.0, 136.2, 134.7, 132.4, 129.4, 126.8, 125.4, 124.1, 75.9, 63.0, 28.1, 26.8, 26.3, 26.1, 26.0. FTIR (thin film) cm -1 HRMS (ESI) (m / z): calc'd for C 22 H 32 N3O5[M+H] + : 418.2336, found: 418.2330. TLC (30% CMA in chloroform), Rf: 0.50 (UV, KMnO4).

[0363] Example 58: Synthesis of N,N-diethyl-3-(p-tolyloxy)propan-1-amine (S5) [ka]

[0364] A round-bottom flask was charged sequentially with 3-diethylamino-1-propanol (0.300 g, 2.29 mmol, 1.40 equiv.), triphenylphosphine (428 mg, 1.63 mmol, 1.00 equiv.), and p-cresol (177 mg, 1.63 mmol, 1 equiv.). Tetrahydrofuran (16 mL) was then added to the flask, and the solution was cooled to 0 °C in an ice-water bath. After 5 min, diisopropyl azodicarboxylate (330 mg, 1.63 mmol, 1.00 equiv.) was added dropwise via syringe. The ice-water bath was removed, and the reaction mixture was allowed to warm to room temperature. After 31 h, the reaction mixture was concentrated under reduced pressure. The resulting crude product was purified by flash column chromatography on silica gel (eluent: 20% CMA in chloroform) to afford amine S5 (181 mg, 50%) as an off-white solid. 1H NMR (500 MHz, CDCl3, 25℃): δ 7.05 (d, J = 8.5, 2H), 6.78 (d, J = 8.5, 2H), 3.96 (t, J = 6.3, 2H), 2.60 (t, J = 7.4, 2H), 2.54 (q, J = 7.2, 4H), 2.26 (s, 3H), 1.96-1.83 (m, 2H), 1.02 (t, J = 7.2 6H). 13 C NMR (126 MHz, CDCl3, 25℃): δ 156.9, 130.0, 129.9, 114.5, 66.3, 49.5, 47.1, 26.8, 20.6, 11.5. FTIR (thin film) cm -1 : 2967 (m), 1513 (s), 1472 (m), 1241 (s), 1058 (m), 816 (m). HRMS (ESI) (m / z): calc'd for C 14 H 24 NO [M+H] + : 222.1852, found: 222.1850. TLC (30% CMA in chloroform), Rf: 0.40 (UV, KMnO4).

[0365] Example 59: Synthesis of N,N-diethyl-3-(p-tolyloxy)propan-1-amine oxide (S4) [ka]

[0366] A round-bottom flask was charged with N,N-diethyl-3-(p-tolyloxy)propan-1-amine (S5, 100 mg, 824 μL, μmmol, 1 equiv.) at room temperature. Dichloromethane (8.24 mL) was then introduced into the flask, and the solution was cooled to 0 °C in an ice-water bath. m-Chloroperoxybenzoic acid (142 mg, 824 μmmol, 1.00 equiv.) was then added as a solid to the flask in one portion at 0 °C. After 30 min, the reaction was concentrated under reduced pressure. The resulting crude product was purified by flash column chromatography on silica gel (eluent: 30% CMA in chloroform) to afford N-oxide S4 (105 mg, 97%) as a clear, colorless oil. 1 H NMR (500 MHz, CDCl3, 25℃): δ 6.99 (d, J = 8.4, 2H), 6.70 (d, J = 8.5, 2H), 3.97 (t, J = 5.7, 2H), 3.28-3.20 (m, 2H), 3.16 (q, J = 7.3, 4H), 2.24 (dq, J = 11.5, 5.6 Hz, 2H), 2.20 (s, 3H), 1.25 (t, J = 7.3, 6H). 13 C NMR (126 MHz, CDCl3, 25℃): δ 156.3, 130.2, 129.9, 114.2, 65.8, 62.7, 60.2, 23.2, 20.4, 8.7. FTIR (thin film) cm -1 : 2948 (m) 1513 (s), 1461 (m), 1237 (s), 961 (m), 816 (m). HRMS (ESI) (m / z): calc'd for C 14 H 24 NO2[M+H] + : 238.1802, found: 238.1798. TLC (30% CMA in chloroform), Rf: 0.10 (UV, KMnO4).

[0367] Example 60: Synthesis of 1-phenyl-4-(triisopropylsilyl)but-3-yn-2-ol (S24). [ka]

[0368] (Triisopropylsilyl)acetylene (2.24 mL, 9.99 mmol, 1.20 equiv.) was added via syringe to a round-bottom flask containing dichloromethane (83 mL) and cooled to −78° C. in an acetone-dry ice bath. A solution of 2.5 M n-butyllithium (3.66 mL, 9.15 mmol, 1.10 equiv.) in hexanes was added dropwise to the reaction mixture via syringe. The solution was then warmed to 0° C. in an ice-water bath. After 30 min, the solution was cooled to −78° C., and phenylacetaldehyde (973 μL, 8.32 mmol, 1 equiv.) was added dropwise via syringe. After 1 h, aqueous hydrogen chloride (1N, 50 mL) and ethyl acetate (100 mL) were added sequentially. The aqueous layer was extracted with ethyl acetate (2 × 30 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by flash column chromatography on silica gel (eluent: 2% ethyl acetate in hexanes) to afford alkyne S24 (1.76 g, 70%) as a clear, colorless oil. 1 H NMR (500 MHz, CDCl3, 25℃): δ 7.37-7.23 (m, 5H), 4.64 (q, J = 6.0, 1H), 3.04 (d, J = 6.4, 2H), 2.01 (d, J = 5.4, 1H), 1.10-1.06 (m, 21H). 13 C NMR (126 MHz, CDCl3, 25℃): δ 136.7, 130.1, 128.5, 127.0, 108.1, 86.8, 63.8, 44.3, 18.7, 11.3. FTIR (thin film) cm -1 : 3339 (br-w), 2944 (s), 2862 (s), 2173 (w), 1461 (m), 1032 (s), 883 (s). HRMS (ESI) (m / z): calc'd for C 19 H 31 OSi [M+H] +: 303.2139, found: 303.2137. TLC (5% ethyl acetate in hexanes), Rf: 0.27 (UV, KMnO4).

[0369] Example 61: Synthesis of 1-methyl-4-((1-phenylbut-3-yn-2-yl)oxy)benzene (S25). [ka]

[0370] A round-bottom flask was charged sequentially with 1-phenyl-4-(triisopropylsilyl)but-3-yn-2-ol (S24, 1.00 g, 3.31 mmol, 1.40 equiv.), triphenylphosphine (868 mg, 3.31 mmol, 1.40 equiv.), and p-cresol (255 mg, 2.36 mmol, 1 equiv.). Tetrahydrofuran (23 mL) was then added to the flask, and the solution was cooled to 0 °C in an ice-water bath. After 5 min, diisopropyl azodicarboxylate (652 μL, 3.31 mmol, 1.40 equiv.) was added dropwise via syringe. The ice-water bath was removed, and the reaction mixture was allowed to warm to room temperature. After 17 h, the reaction was diluted with ethyl acetate (200 mL), and the organic layer was washed successively with water (50 mL) and brine (50 mL). The organic layer was concentrated under reduced pressure. The crude product was then dissolved in tetrahydrofuran (23 mL) and cooled to 0 °C in an ice-water bath. Tetrabutylammonium fluoride (1.40 mL, 1.40 mmol, 0.600 equiv) was added dropwise via syringe, and the reaction mixture was allowed to warm to room temperature after removing the ice-water bath. After 30 min, the reaction was concentrated under reduced pressure. The resulting crude product was purified by flash column chromatography on silica gel (eluent: 100% hexanes) to afford alkyne S25 (213 mg, 38%) as a clear, colorless oil. 1H NMR (500 MHz, CDCl3, 25℃): δ 7.39-7.23 (m, 5H), 7.06 (d, J = 8.1, 2H), 6.87 (d, J = 8.5, 2H), 4.84 (td, J = 6.8, 2.0, 1H), 3.22 (ddd, J = 49.0, 13.8, 6.7, 2H), 2.47 (d, J =1.9, 1H), 2.26 (s, 3H). 13 C NMR (126 MHz, CDCl3, 25℃): δ 155.4, 136.7, 131.1, 130.0, 129.9, 128.5, 127.1, 116.0, 82.0, 75.4, 69.2, 42.3, 20.7. FTIR (thin film) cm -1 : 3287 (m), 3030 (w), 1509 (s), 1226 (s), 1021 (m), 700 (m). HRMS (ESI) (m / z): calc'd for C 17 H 17 O [M+H] + : 237.1274, found: 237.1271. TLC (100% hexanes), Rf: 0.20 (UV, KMnO4).

[0371] Example 62: Synthesis of (E)-N,N-diethyl-4-phenyl-3-(p-tolyloxy)but-1-ene-1-amine oxide (S6) [ka]

[0372] A glass 1-dram vial was charged with 1-methyl-4-((1-phenylbut-3-yn-2-yl)oxy)benzene (S25, 100 mg, 423 μmol, 1 equiv.) and dissolved in 20% v / v trifluoroethanol in chloroform (2.12 mL) at room temperature. N,N-Diethylhydroxylamine (218 μL, 2.12 mmol, 5.00 equiv.) was then added via syringe. The vial was flushed with nitrogen, sealed with a septum cap and parafilm, and heated to 60 °C. After 30 h, the reaction was removed from the heat, cooled to room temperature, and loaded directly onto a silica gel column. The reaction mixture was purified by flash column chromatography (eluent: 30% CMA in chloroform). Fractions containing the desired compound were combined, and the solvent was removed under reduced pressure on a rotary evaporator at 0 °C to give enamine N-oxide S6 (116 mg, 84%) as an off-white solid. 1 H NMR (500 MHz, CD3OD, 25℃): δ 7.32-7.18 (m, 5H), 7.08 (d, J = 8.2, 2H), 6.85 (d, J = 8.6, 2H), 6.44 (dd, J = 13.4, 6.0, 1H), 6.13 (dd, J = 13.4, 1.3, 1H), 5.20 (qd, J = 6.3, 1.3, 1H), 3.70-3.53 (m, 4H), 3.23-3.08 (m, 2H), 2.25 (s, 3H), 1.05 (td, J = 7.2, 5.1, 6H). 13 C NMR (126 MHz, CD3OD, 25℃): δ 156.5, 138.0, 134.7, 133.0, 132.6, 131.2, 131.0, 129.7, 128.0, 117.6, 77.3, 65.3, 65.4, 42.0, 20.7, 8.2, 8.1. FTIR (thin film) cm -1 : 3030 (w), 1666 (m), 1509 (s), 117 (s), 1136 (s). HRMS (ESI) (m / z): calc'd for C 21 H 28 NO2[M+H] +: 326.2115, found: 326.2111. TLC (30% CMA in chloroform), Rf: 0.15 (UV, KMnO4).

[0373] Example 63: Synthesis of 1-methyl-4-((1-phenylbut-3-yn-2-yl)oxy)benzene (S7) [ka]

[0374] A round-bottom flask was charged with (E)-N,N-diethyl-4-phenyl-3-(p-tolyloxy)but-1-ene-1-amine oxide (S6, 26.0 mg, 79.8 μmol, 1 equiv.) and dissolved in chloroform-d (773 μL) at room temperature. Benzyl mercaptan (93.7 μL, 798 μmol, 10.0 equiv.) was then added via syringe. After 5 min, analysis by TLC showed no reduction by benzyl mercaptan. Solid-state bis(pinacolato)diboron (20.3 mg, 79.8 μmol, 1.00 equiv.) was then added in one portion at room temperature. After 15 min, the reaction mixture was loaded directly onto a silica gel column. The reaction mixture was purified by flash column chromatography (eluent: 100% chloroform) to give aldehyde S7 (20.2 mg, 94%) as an off-white solid. 1 H NMR (500 MHz, CDCl3, 25℃): δ 9.51 (dd, J = 2.5, 1.4 Hz, 1H), 7.33-7.18 (m, 8H), 7.14-7.08 (m, 2H), 3.68 (q, J = 13.4, 10.4 Hz, 2H), 3.20 (tdd, J = 8.1, 6.5, 5.6 Hz, 1H), 2.97 (dd, J = 13.7, 6.4 Hz, 1H), 2.78 (dd, J =13.8, 8.2 Hz, 1H), 2.60-2.40 (m, 2H). 13FTIR (thin film) cm -1 : 3026 (w), 2922 (w), 1722 (s), 1494 (m), 1453 (m), 700 (s). HRMS (ESI) (m / z): calc'd for C 17 H 19 OS [M+H] + : 271.1151, found: 271.1149. TLC (100% chloroform), Rf: 0.45 (UV, KMnO4).

[0375] Example 64: Synthesis of Boc-propargyl-staurosporine (35) [ka]

[0376] To a round-bottom flask, Boc-staurosporine (Zimmermann et al., International Patent No. WO 1999 / 9902532) (85.0 mg, 150 μmol, 1 equiv.) and 4-nitrophenylprop-2-yn-1-yl carbonate (Egami et al., Org. Biomol. Chem. 9:7667-7670 (2011)) (66.3 mg, 300 μmol, 2.00 equiv.) were sequentially added. Tetrahydrofuran (10 mL) was then introduced via syringe, and the reaction mixture was cooled to 0 °C in an ice-water bath. After 5 min, solid sodium hydride (18.0 mg, 0.750 mmol, 5.00 equiv.) was added in one portion at 0 °C. After 2 h, the reaction mixture was added dropwise to aqueous hydrogen chloride solution (1 N, 50 mL) at 0 °C. The mixture was then diluted with dichloromethane (100 mL). The aqueous layer was washed with dichloromethane (2 × 50 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude solid was purified by flash chromatography on silica gel (eluent: 40% ethyl acetate in hexane) to give alkyne 35 (91.9 mg, 94%) as a white solid. 1 H NMR (500 MHz, CDCl3, 25℃): δ 9.37 (d, J = 8.0 Hz, 1H), 7.94 (d, J = 7.6 Hz, 1H), 7.74 (d, J = 8.4 Hz, 1H), 7.51-7.42 (m, 2H), 7.37 (t, J = 7.4 Hz, 1H), 7.32 (t, J = 7.3 Hz, 1H), 7.18 (t, J = 10.0 Hz, 1H), 6.58 (dd, J = 8.5, 4.5 Hz, 1H), 5.33-5.23 (m, 1H), 5.21-5.07 (m, 1H), 5.09-4.96 (m, 2H), 4.84-4.56 (m, 1H), 3.91 (d, J = 59.1 Hz, 1H), 2.66 (s, 3H), 2.62-2.53 (m, 2H), 2.51-2.39 (m, 7H), 1.59 (s, 3H), 1.48 (s, 6H). 13C NMR (126 MHz, CDCl3, 25℃): δ 167.3, 156.1, 151.8, 139.0, 136.8, 131.9, 130.3, 127.0, 126.1, 125.6, 124.7, 123.3, 121.7, 121.2, 120.4, 117.6, 116.6, 114.0, 112.9, 112.6, 108.0, 95.1, 85.2, 82.7, 80.5, 77.8, 75.8, 60.7, 54.0, 50.0, 49.5, 30.5, 29.9, 29.5, 28.9, 28.7, 28.4. FTIR (thin film) cm -1 : 2974 (w), 2933 (w), 1774 (m), 1722 (m), 1688 (m), 1632 (w), 1584 (w), 1341 (s), 1308 (s), 1282 (s), 1140 (m), 741 (m). HRMS (ESI) (m / z): calc'd for C 37 H 37 N4O7[M+H] + : 649.2657, found: 649.2647. TLC (50% ethyl acetate in hexanes), Rf: 0.50 (UV).

[0377] Example 65: Synthesis of propargyl-staurosporine (36) [ka]

[0378] A 20 mL glass flash vial was charged with Boc-propargyl-staurosporine (35, 8.0 mg, 12.3 μmol, 1 equiv.) at room temperature. A solution of 20% trifluoroacetic acid in dichloromethane (2.40 mL) was introduced via syringe. After 1 h, the reaction mixture was concentrated under a stream of nitrogen. The crude residue was then purified by automated C 18 Reverse phase column chromatography (30g C 18Purification by silica gel, 25 μm spherical particles, eluent: HO+0.1% TFA (2 CV), gradient 0→100% MeCN / HO+0.1% TFA (15 CV, tR=8.5 CV) gave staurosporine-alkyne 36 (6.6 mg, 98%) as a white solid. 1 H NMR (500 MHz, CD3OD, 25℃): δ 9.13 (d, J = 7.9 Hz, 1H), 7.99 (d, J = 8.5 Hz, 1H), 7.82 (d, J = 7.6 Hz, 1H), 7.59 (ddd, J = 8.5, 7.2, 1.3 Hz, 1H), 7.47 (t, J = 7.4 Hz, 1H), 7.40 (ddd, J = 8.2, 7.0, 1.2 Hz, 1H), 7.25 (t, J = 7.5 Hz, 1H), 7.04 (d, J = 8.1 Hz, 1H), 6.19 (dd, J = 9.5, 2.7Hz, 1H), 5.03-4.95 (m, 3H), 4.41 (d, J = 16.5 Hz, 1H), 4.14 (s, 1H), 3.98-3.89 (m, 1H), 3.22-3.08 (m, 2H), 2.76 (s, 3H), 2.57 (s, 3H), 2.09-1.94 (m, 4H). 13 C NMR (126 MHz, CD3OD, 25℃): δ 169.3, 152.2, 139.5, 137.6, 132.8, 131.3, 127.7, 127.3, 127.0, 126.9, 125.6, 123.9, 122.8, 122.7, 121.0, 117.7, 116.5, 114.8, 113.8, 109.7, 94.4, 81.7, 81.5, 78.9, 77.5, 60.5, 55.8, 54.7, 49.9, 31.5, 28.8, 28.8. FTIR (thin film) cm -1 : 2929 (m), 1774 (s), 1718 (s), 1636 (w), 1580 (m), 1345 (s), 1312 (s), 1278 (s), 1095 (s), 741 (s). HRMS (ESI) (m / z): calc'd for C32 H 29 N4O5[M+H] + : 549.2132, found: 549.2127. TLC (5% methanol in dichloromethane), Rf: 0.40 (UV).

[0379] Example 66: Synthesis of N,N-diethylenamine N-oxide staurosporine (37) [ka]

[0380] A 2 mL glass LC-MS vial was charged with N,N-diethylhydroxylamine (15.8 μL, 154 μmol, 10.0 equiv.) at room temperature. A solution of Boc-propargylstaurosporine (36, 10.0 mg, 15.4 μmol, 1 equiv.) in 20% v / v trifluoroethanol in chloroform (200 μL) was introduced via syringe. The mixture was transferred to a 4" NMR tube, flushed with nitrogen, sealed with a cap and parafilm, and heated to 50 °C. After 20 h, the reaction was removed from the oil bath and cooled to room temperature. The reaction mixture was then concentrated under reduced pressure. A solution of 20% trifluoroacetic acid in dichloromethane (200 μL) was then added via syringe at room temperature. After 1 h, the reaction was concentrated under reduced pressure. The resulting residue was purified by C 18 Purification was performed by preparative high-performance liquid chromatography (HPLC) using a reverse-phase column (250 × 21.2 mm, 5 μm particle size, 20 mL / min flow rate, eluent: HO + 0.1% TFA (2 min), gradient 0 → 100% MeCN / HO + 0.1% TFA (18 min), t = 14.0 min). Fractions containing the desired compound were combined, and the solvent was removed under reduced pressure on a rotary evaporator at 0 °C to give enamine N-oxide 37 (9.40 mg, 96%) as a white solid. 1H NMR (500 MHz, CD3OD, 25℃): δ 9.14 (d, J = 7.9 Hz, 1H), 7.90 (d, J = 8.6 Hz, 1H), 7.46 (td, J = 7.5, 2.1 Hz, 2H), 7.29 (t, J = 7.5 Hz, 1H), 7.18-7.00 (m, 4H), 6.80 (dt, J = 13.5, 4.2 Hz, 1H), 5.98 (dd, J = 9.5, 2.5 Hz, 1H), 5.27 (d, J = 16.3 Hz, 1H), 5.10-4.90 (m, 3H), 4.20-4.10 (m, 2H), 4.09 (s, 1H), 4.04-3.91 (m, 2H), 3.94-3.85 (m, 1H), 3.14 (dt, J = 13.1, 9.0 Hz, 1H), 2.73 (s, 3H), 2.61 (s, 3H), 1.88 (s, 4H), 1.62 (t, J = 7.1 Hz, 3H), 1.52 (t, J = 7.0 Hz, 3H). 13 C NMR (125.8 MHz, CD3OD, 25℃): δ 169.8, 152.2, 139.4, 137.8, 134.5, 132.7, 131.8, 128.9, 128.0, 127.1, 126.8, 126.4, 125.1, 123.9, 122.5, 122.4, 121.2, 117.6, 116.1, 115.0, 113.5, 110.4, 94.5, 82.0, 81.6, 65.7, 63.3, 60.4, 55.7, 50.4, 31.4, 28.9, 28.8, 8.7, 8.6. FTIR (thin film) cm -1 : 1785 (m), 1681 (s), 1349 (m), 1282 (m), 1200 (s), 1133 (s), 801 (w). HRMS (ESI) (m / z): calc’d for C 36 H 40 N5O6[M+H] + : 638.2973, found: 638.2964. TLC (50% CMA in chloroform), Rf: 0.20 (UV).

[0381] Example 67: Synthesis of (E)-N,N-diethyl-3-hydroxynon-1-en-8-yn-1-amine oxide (38) [ka]

[0382] A glass 1-dram vial was charged with nona-1,8-diyn-3-ol (Feldman et al., Heterocycles 81:117-143 (2010)) (50.0 mg, 367 μmol, 1 equiv.) and dissolved in 20% v / v trifluoroethanol in chloroform (1.84 mL) at room temperature. N,N-Diethylhydroxylamine (189 μL, 1.84 mmol, 5.00 equiv.) was then added via syringe. The vial was flushed with nitrogen, sealed with a septum cap and parafilm, and heated to 60 °C. After 43 h, the reaction was removed from the heat, cooled to room temperature, and loaded directly onto a silica gel column. The reaction mixture was purified by flash column chromatography (eluent: 50% CMA in chloroform). Fractions containing the desired compound were combined and the solvent was removed under reduced pressure on a rotary evaporator at 0° C. to give enamine N-oxide 38 (25.5 mg, 31%) as a white solid. 1 H NMR (500 MHz, CD3OD, 25℃): δ 6.39 (dd, J = 13.1, 5.7, 1H), 6.14 (dd, J = 13.1, 1.5, 1H), 4.27 (q, J = 5.8, 1H), 3.42-3.31 (m, 4H), 2.22-2.14 (m, 3H), 1.63-1.47 (m, 6H), 1.27 (td, J = 7.1, 4.6, 6H). 13 C NMR (126 MHz, CD3OD, 25℃): δ 138.3, 133.8, 70.0, 69.8, 65.3, 65.3, 38.0, 29.7, 25.7, 19.1, 8.8. FTIR (thin film) cm -1: 3227 (br-s), 2940 (s), 2862 (s), 1591 (w), 1461 (m), 965 (m). HRMS (ESI) (m / z): calc'd for C 13 H 24 NO2[M+H] + : 226.1802, found: 226.1799. TLC (50% CMA in chloroform), Rf: 0.10 (KMnO4).

[0383] Example 67: Synthesis of 3-fluoronona-1,8-diyne (S26) [ka]

[0384] A round-bottom flask was charged with nona-1,8-diyn-3-ol (Feldman et al., Heterocycles 81: 117-143 (2010)) (100 mg, 734 μmol, 1 equiv.) at room temperature. Dichloromethane (8 mL) was added to the flask, and the solution was cooled to 0 °C in an ice-water bath. After 5 min, diethylaminosulfur trifluoride (107 μmol, 808 μmol, 1.10 equiv.) was slowly added dropwise via syringe. After 5 h, the reaction mixture was directly loaded onto a silica gel column. The crude mixture was purified by flash chromatography (eluent: 1% DCM / pentane) to afford fluoroalkyne S26 (81.2 mg, 80%) as a clear, colorless oil. 1 H NMR (500 MHz, CDCl3, 25℃): δ 5.06 (dtd, J = 48.2, 6.3, 2.1, 1H), 2.65 (dd, J = 5.6, 2.1, 1H), 2.19 (td, J = 6.6, 2.6, 2H), 1.92 (t, J = 2.7, 1H), 1.91-1.70 (m, 2H), 1.64-1.49 (m, 4H). 13C NMR (126 MHz, CDCl3, 25℃): δ 84.1, 82.4 (d, J = 167.8 Hz), 80.4 (d, J = 25.9 Hz), 76.5 (d, J = 10.5 Hz), 68.8, 35.4 (d, J = 22.2 Hz), 28.1, 23.71 (d, J = 3.9 Hz), 18.4. 19 F NMR (471 MHz, CDCl3, 25℃): δ 175.2. FTIR (thin film) cm -1 : 3298 (s), 2926 (s), 2855 (s), 1729 (m), 984 (m). HRMS (ACPI) (m / z): calc'd for C9H 12 F [M+H] + : 139.0918, found: 139.0918. TLC (100% hexanes), Rf: 0.10 (KMnO4).

[0385] Example 68: Synthesis of (E)-N,N-diethyl-3-fluoronon-1-en-8-yn-1-amine oxide (39) [ka]

[0386] 3-Fluoronona-1,8-diyne (S26, 46.7 mg, 338 μmol, 1 equiv.) was placed in a glass 1-dram vial and dissolved in 20% v / v trifluoroethanol in chloroform (1.69 mL) at room temperature. N,N-Diethylhydroxylamine (174 μL, 1.69 mmol, 5.00 equiv.) was then added via syringe. The vial was flushed with nitrogen, sealed with a septum cap and parafilm, and heated to 60 °C. After 11 h, the reaction was removed from the heat, cooled to room temperature, and loaded directly onto a silica gel column. The reaction mixture was purified by flash column chromatography (eluent: 40% CMA in chloroform). Fractions containing the desired compound were combined, and the solvent was removed under reduced pressure on a rotary evaporator at 0 °C to give enamine N-oxide 39 (38.2 mg, 50%) as a white solid. 1 H NMR (500 MHz, CD3OD, 25℃): δ 6.50 (ddd, J = 18.3, 13.1, 5.3, 1H), 6.31 (dt, J = 13.1, 1.8, 1H), 5.26-5.10 (m, 1H), 3.45-3.33 (m, 4H), 2.26-2.16 (m, 3H), 1.84-1.70 (m, 2H), 1.63-1.51 (m, 4H), 1.27 (td, J = 7.1, 5.7, 6H). 13 C NMR (126 MHz, CD3OD, 25℃): δ 139.7 (d, J = 12.5 Hz), 129.7 (d, J = 19.0 Hz), 91.4 (d, J = 170.7 Hz), 84.8, 69.9, 65.4 (d, J = 9.3 Hz), 36.0 (d, J = 21.8 Hz), 29.4, 24.9 (d, J = 4.7 Hz), 19.1, 8.7. 19 F NMR (471 MHz, CDCl3, 25℃): δ 181.3. FTIR (thin film) cm -1: 3298 (s), 2944 (s), 2866 (m), 1684 (w), 1461 (m), 1375 (m), 965 (s). HRMS (ESI) (m / z): calc'd for C 13 H 23 FNO [M+H] + : 228.1758, found: 228.1754. TLC (50% CMA in chloroform), Rf: 0.20 (KMnO4).

[0387] Example 69: Synthesis of 3-chloronona-1,8-diyne (S27) [ka]

[0388] A round-bottom flask was charged with nona-1,8-diyn-3-ol (Feldman et al., Heterocycles 81: 117-143 (2010)) (40.0 mg, 294 μmol, 1 equiv.) and dissolved in tetrahydrofuran (3 mL) at room temperature. Solid N-chlorosuccinimide (59.0 mg, 441 μmol, 1.50 equiv.) and triphenylphosphine (93.0 mg, 353 μmol, 1.20 equiv.) were then added together in one portion. After 4 h, the reaction mixture was heated to 50° C. for an additional 2 h. The reaction was then removed from the oil bath and allowed to cool to room temperature. Ethyl acetate (10 mL) was added, and the organic layer was washed with a 1:1 aqueous solution of saturated sodium thiosulfate and saturated sodium bicarbonate (2 × 5 mL). The organic layer was then dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude mixture was purified by flash column chromatography on silica gel (eluent: 30% dichloromethane in hexanes) to afford alkyne S27 (41.8 mg, 92%) as a clear, colorless oil. 1H NMR (500 MHz, CDCl3, 25℃): δ 4.49 (td, J = 6.7, 2.3, 1H), 2.58 (d, J = 2.3, 1H), 2.19 (td, J = 6.9, 2.7, 2H), 1.99-1.90 (m, 3H), 1.70-1.48 (m, 4H). 13 C NMR (126 MHz, CDCl3, 25℃): δ 84.1, 82.0, 74.6, 68.9, 47.9, 38.6, 27.9, 25.4, 18.5. FTIR (thin film) cm -1 : 3298 (s), 2922 (s), 2847 (m) 1438 (m) 1118 (m). HRMS (APCI) (m / z): calc'd for C9H 12 Cl [M+H] + : 155.0622, found: 155.0621. TLC (60% dichloromethane in hexanes), Rf: 0.85 (KMnO4).

[0389] Example 70: Synthesis of (E)-N,N-diethyl-3-fluoronon-1-en-8-yn-1-amine oxide (40) [ka]

[0390] 3-Chloronona-1,8-diyne (S27, 50.0 mg, 323 μmol, 1 equiv.) was placed in a glass 1-dram vial and dissolved in 20% v / v trifluoroethanol in chloroform (3.23 mL) at room temperature. N,N-Diethylhydroxylamine (166 μL, 1.62 mmol, 5.00 equiv.) was then added via syringe. The vial was flushed with nitrogen, sealed with a septum cap and parafilm, and heated to 60 °C. After 8.5 h, the reaction was removed from the heat, cooled to room temperature, and loaded directly onto a silica gel column. The reaction mixture was purified by flash column chromatography (eluent: 30% CMA in chloroform). Fractions containing the desired compound were combined, and the solvent was removed under reduced pressure on a rotary evaporator at 0 °C to afford enamine N-oxide 40 (70.0 mg, 89%) as a white solid. 1 H NMR (500 MHz, CD3OD, 25℃): δ 6.52 (dd, J = 13.0, 8.7, 1H), 6.37 (dd, J = 13.0, 0.8, 1H), 4.65 (dt, J = 8.4, 6.7, 1H), 3.46-3.32 (m, 4H), 2.23-2.17 (m, 3H), 1.94-1.86 (m, 2H), 1.62-1.51 (m, 4H), 1.27 (td, J = 7.1, 5.9, 6H). 13 C NMR (126 MHz, CD3OD, 25℃): δ 140.4, 131.7, 84.8, 70.0, 65.5, 59.0, 39.2, 29.1, 26.7, 19.0, 8.8, 8.7. FTIR (thin film) cm -1 : 3298 (s), 2944 (s), 2866 (m), 1681 (w), 1461 (m), 1375 (m), 969 (s). HRMS (ESI) (m / z): calc'd for C 13 H 23 ClNO [M+H] + : 244.1463, found: 244.1459. TLC (30% CMA in chloroform), Rf: 0.25 (KMnO4).

[0391] Example 71: Synthesis of tert-butyl (6-hydroxyhexyl) (prop-2-yn-1-yl)carbamate (S28) [ka]

[0392] A round-bottom flask was charged with 6-amino-1-hexanol (2.00 g, 17.1 mmol, 1 equiv.) and dissolved in dichloromethane (100 mL) at room temperature. Di-tert-butyl dicarbonate (4.31 mL, 18.8 mmol, 1.10 equiv.) was then added via syringe. After 14 h, the reaction mixture was concentrated under reduced pressure. The crude residue was used without further purification and dissolved in dichloromethane (100 mL) at room temperature. Pyridine (4.15 mL, 51.3 mmol, 3.00 equiv.), tert-butyldimethylsilyl chloride (2.84 g, 18.8 mmol, 1.10 equiv.), and 4-(dimethylamino)pyridine (0.209 g, 1.71 mmol, 0.100 equiv.) were then added sequentially to the flask. After 8 h, saturated aqueous ammonium chloride solution (30 mL) was added. The organic phase was further washed with water (30 mL) and brine (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude residue was used without further purification and was dissolved in dimethylformamide (100 mL) at room temperature. The reaction mixture was cooled to 0 °C in an ice-water bath, and sodium hydride (1.23 g, 51.3 mmol, 3.00 equiv.) was added in one portion. After 30 min, propargyl bromide (2.86 mL, 25.7 mmol, 1.50 equiv.) was added dropwise via syringe. The ice-water bath was removed, and the reaction was allowed to warm to room temperature. After 6 h, saturated aqueous ammonium chloride (10 mL), ethyl acetate (200 mL), and water (200 mL) were added sequentially. The aqueous phase was washed with ethyl acetate (2 × 100 mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and dried in vacuo. The crude residue was used without further purification and was dissolved in tetrahydrofuran (100 mL) at room temperature. The reaction mixture was cooled to 0°C in an ice-water bath, and tetrabutylammonium fluoride in tetrahydrofuran (1.00 M, 17.1 mL, 17.1 mmol, 1.00 equiv) was added dropwise. After 1 h, the reaction mixture was concentrated under reduced pressure, and the crude mixture was purified by flash column chromatography on silica gel (20% ethyl acetate in hexanes) to afford alkyne S28 (1.93 g, 44%) as a clear, colorless oil. 1H NMR (500 MHz, DMSO-d6, 75℃): δ 3.98 (t, J = 3.3 Hz, 3H), 3.40 (d, J = 6.7 Hz, 2H), 3.23 (t, J = 7.3 Hz, 2H), 3.07 (s, 1H), 1.53 (q, J = 6.9 Hz, 2H), 1.47-1.35 (m, 11H), 1.36-1.25 (m, 4H). 13 FTIR (thin film) cm -1 : 3298 (s), 2944 (s), 2866 (m), 1681 (w), 1461 (m), 1375 (m), 969 (s). HRMS (ESI) (m / z): calc'd for C 14 H 25 NNaO3[M+Na] + : 278.1727, found: 278.1724. TLC (20% ethyl acetate in hexanes), Rf: 0.20 (KMnO4).

[0393] Example 73: Synthesis of tert-butyl (6-hydroxyoct-7-yn-1-yl)(prop-2-yn-1-yl)carbamate (S29). [ka]

[0394] A round-bottom flask was charged with alcohol S28 (187 mg, 730 μmol, 1 equiv.) and dissolved in dichloromethane (7 mL) at room temperature. Pyridinium chlorochromate (0.173 g, 803 μmol, 1.10 equiv.) was added in one portion, and the flask was stirred at room temperature. After 14 h, silica gel (1.00 g) was added, and the reaction mixture was stirred for 10 min. The slurry was then loaded directly onto a short silica gel plug, and the crude product was eluted with diethyl ether (100 mL). The eluent was concentrated under reduced pressure. The crude residue was then dissolved in tetrahydrofuran (7 mL) and cooled to 0 °C using an ice-water bath. A solution of ethynylmagnesium bromide in tetrahydrofuran (500 mM, 1.46 mL, 730 μmol, 1.00 equiv.) was then added dropwise via syringe. After 1 h, saturated aqueous ammonium chloride (10 mL) and ethyl acetate (50 mL) were added sequentially. The organic layer was washed with water (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by flash column chromatography on silica gel (eluent: 20% ethyl acetate in hexane) to give product S29 (88.0 mg, 43%) as a clear, colorless oil. 1 H NMR (500 MHz, DMSO-d6, 75℃): δ 5.30-5.24 (m, 1H), 4.16 (qd, J = 6.6, 2.1 Hz, 1H), 3.97 (s, 2H), 3.23-2.99 (m, 3H), 1.63-1.44 (m, 4H), 1.42-1.33 (m, 11H), 1.28-1.16 (m, 2H). 13 C NMR (126 MHz, MeOD, 45℃): δ 155.5, 85.0, 80.2, 79.3, 72.0, 71.2, 61.2, 46.5, 37.5, 35.8, 27.6, 27.4, 26.1, 24.6. FTIR (thin film) cm -1 : 3298 (s), 2944 (s), 2866 (m), 1681 (w), 1461 (m), 1375 (m), 969 (s). HRMS (ESI) (m / z): calc'd for C 16 H 25NNaO3[M+H] + : 302.1727, found: 302.1723. TLC (20% ethyl acetate in hexanes), Rf: 0.20 (KMnO4).

[0395] Example 74: Synthesis of tert-butyl (6-fluorooct-7-yn-1-yl)(prop-2-yn-1-yl)carbamate (S30) [ka]

[0396] A round-bottom flask was charged with fluoroalkyne S30 (510 mg, 1.83 mmol, 1 equiv.), dissolved in dichloromethane (18 mL), and cooled to 0 °C using an ice-water bath. Diethylaminosulfur trifluoride (253 μL, 1.92 mmol, 1.20 equiv.) was added dropwise via syringe. After 3 h, the reaction mixture was concentrated under reduced pressure. The resulting residue was purified by flash column chromatography on silica gel (eluent: 5% ethyl acetate in hexanes) to afford fluoroalkyne S30 (412 mg, 60%) as a clear, colorless oil. 1 H NMR (500 MHz, DMSO-d6, 75℃): δ 5.40-5.04 (m, 1H), 3.99 (d, J = 2.5 Hz, 2H), 3.74-3.45 (m, 1H), 3.30-3.13 (m, 2H), 2.99-2.78 (m, 1H), 1.86-1.72 (m, 2H), 1.59-1.30 (m, 15H). 13 C NMR (126 MHz, DMSO-d6, 75℃): δ 153.9, 81.8 (d, J = 165.0 Hz), 80.2 (d, J = 25.1 Hz), 80.0, 78.7, 78.4, 72.7, 45.7, 35.5, 34.8 (d, J = 22.0 Hz), 27.6, 26.9, 25.2, 23.1 (d, J = 4.1 Hz). 19F NMR (471 MHz, DMSO-d6, 75℃): δ -173.6. FTIR (thin film) cm -1 : 3298 (s), 2944 (s), 2866 (m), 1681 (w), 1461 (m), 1375 (m), 969 (s). HRMS (ESI) (m / z): calc'd for C 16 H 24 FNNaO2[M+H] + : 304.1683, found: 304.1681. TLC (10% ethyl acetate in hexanes), Rf: 0.50 (KMnO4).

[0397] Example 75: Synthesis of Si700-fluoroalkyne (S31) [ka]

[0398] A round-bottom flask was charged with tert-butyl (6-fluorooct-7-yn-1-yl)(prop-2-yn-1-yl)carbamate (S30, 14.5 mg, 51.5 μmol, 1 equiv.) and dissolved in a mixture of 20% trifluoroacetic acid in dichloromethane (2 mL) at room temperature. After 30 min, the mixture was concentrated under reduced pressure. The crude mixture was then dissolved in dimethylformamide (2 mL) at room temperature, and N,N-diisopropylethylamine (35.2 μL, 206 μmol, 4.00 equiv.) was added via syringe. After 5 min, Si700-acid (24.1 mg, 51.5 μmol, 1.00 equiv.) and HATU (23.5 mg, 61.8 μmol, 1.20 equiv.) were added sequentially and stirred in the dark at room temperature. After 2 h, the reaction mixture was diluted with dichloromethane (100 mL). The organic layer was washed with saturated aqueous sodium bicarbonate (2×20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. 18Purification by preparative high-performance liquid chromatography (HPLC) using a reverse-phase column (250 × 21.2 mm, 5 μm particle size, 20 mL / min flow rate, eluent: 40% MeCN / HO+0.1% TFA (2 min), gradient 40 → 100% MeCN / HO+0.1% TFA (18 min), t = 15.0 min) afforded fluoroalkyne S31 (17.4 mg, 54%) as a blue solid. 1 H NMR (500 MHz, CD3CN, 65℃): δ 7.49-7.38 (m, 2H), 7.16 (d, J = 7.7 Hz, 1H), 7.07 (s, 2H), 6.70 (s, 2H), 5.14 (dd, J = 47.6, 6.8 Hz, 1H), 4.27 (s, 2H), 3.81 (t, J = 8.0 Hz, 4H), 3.54 (s, 2H), 3.19 (s, 6H), 3.04-2.84 (m, 5H), 2.62-2.53 (m, 1H), 2.08 (s, 3H), 1.89-1.63 (m, 4H), 1.58-1.27 (m, 4H), 0.57 (d, J = 5.8 Hz, 6H). 13 C NMR (126 MHz, CD3CN, 65℃): δ 171.9, 165.8, 158.5, 151.8, 142.4, 138.1, 137.7, 134.8, 133.6, 130.3, 129.7, 129.5, 125.3, 116.1, 83.8 (d, J = 165.2 Hz), 81.7 (d, J = 25.6 Hz), 80.8, 78.0 (d, J = 10.5 Hz), 73.5, 55.8, 36.7 (d, J = 22.1 Hz), 34.3, 30.5, 28.6, 27.4, 27.2, 25.0 (d, J = 4.2 Hz), 19.7, 12.9, -0.9, -1.0. 19 F NMR (471 MHz, CD3CN, 55℃): δ -76.6, -175.3. FTIR (thin film) cm -1HRMS (ESI) (m / z): calc'd for C 40 H 45 FN3OSi [M] + : 630.3310, found: 630.3299.

[0399] Example 76: Synthesis of Si700-enamine N-oxide (42) [ka]

[0400] A 4-inch NMR tube was syringed with a solution of Si700-fluoroalkyne (S31, 2.00 mg, 3.10 μmol, 1 equiv) in 20% v / v trifluoroethanol in chloroform (250 μL) and N,N-diethylhydroxylamine (2 M, 250 μL, 500 μmol, 161 equiv) in 20% v / v trifluoroethanol in chloroform. The tube was sealed, flushed with nitrogen, and heated to 40 °C in the dark. After 22 h, the reaction mixture was concentrated under reduced pressure. The crude residue was purified by C 18 Purification was performed by preparative high-performance liquid chromatography (HPLC) using a reverse-phase column (250 × 21.2 mm, 5 μm particle size, 20 mL / min flow rate, eluent: 40% MeCN / HO + 0.1% TFA (2 min), gradient 40 → 100% MeCN / HO + 0.1% TFA (18 min), t = 11.0 min). Fractions containing the desired compound were combined, and the solvent was removed under reduced pressure on a rotary evaporator at 0 °C to give enamine N-oxide 42 (1.48 mg, 64%) as a blue solid. Structural assignment was performed using additional information from COSY, HSQC, and HMBC experiments. 1H NMR (500 MHz, CD3OD, 45℃): δ 7.49 (s, 2H), 7.21 (d, J = 7.7 Hz, 1H), 7.14 (s, 2H), 6.71-6.52 (m, 3H), 6.43 (d, J = 13.4 Hz, 1H), 5.26 (d, J = 47.7 Hz, 1H), 4.24 (s, 2H), 3.83 (t, J = 7.9 Hz, 8H), 3.67 (s, 2H), 3.23 (s, 6H), 2.97-2.90 (m, 4H), 2.84 (s, 1H), 2.09 (s, 3H), 1.81 (s, 4H), 1.63-1.32 (m, 10H), 0.57 (d, J = 11.8 Hz, 6H). 13 C NMR (126 MHz, CD3OD, 45℃): δ 173.3, 165.7, 158.7, 152.2, 143.2, 138.1, 137.7, 135.0, 134.1 (d, J = 14.6 Hz), 133.5, 132.9 (d, J = 18.9 Hz), 130.7, 129.8, 129.6, 125.4, 116.1, 91.1 (d, J = 173.4 Hz), 80.0, 74.8, 65.4 (d, J = 6.8 Hz), 55.8, 47.2, 40.9, 36.0 (d, J = 21.1 Hz), 33.9, 30.8, 27.7, 27.4, 25.5 (d, J = 4.0 Hz), 19.5, 8.4, 8.3, -1.0, -1.4 19 F NMR (470.5 MHz, CD3OD, 25 ℃): δ -76.9, -184.5. FTIR (thin film) cm -1 : 2922 (w), 1673 (s), 1446 (w), 1192 (s), 1133 (s), 846 (m), 801 (m), 723 (m). HRMS (ESI) (m / z): calc’d for C 44 H 56 FN4O2Si [M+H] + : 719.4151, found: 719.4144.

[0401] Example 77: Biological Procedures cell culture

[0402] Cells were cultured in RPMI (A431, Bx-PC3, H460, HeLa, MDA-MB-23) or DMEM (U251) containing 10% FBS (Sigma), 100 units / mL penicillin, and 0.1 mg / mL streptomycin (Sigma) in a humidified chamber at 37 °C under ambient atmosphere with 5% CO2, unless otherwise noted. Cells were passaged and dissociated with 0.25% trypsin, 0.1% EDTA in HBSS (Corning). The following cell lines were obtained from other laboratories: U251, HeLa, and A431 (Charles D. Stiles, Dana-Farber Cancer Institute); BxPC3 (Nathanael Gray, Dana-Farber Cancer Institute); and MDA-MB-231 (Jun Qi, Dana-Farber Cancer Institute). All cells tested negative for mycobacteria using the MycoAlert PLUS Mycoplasma Detection Kit (Lonza) according to the manufacturer's protocol. Staurosporine Enamine N-Oxide Cell Viability Studies

[0403] A431 or H460 cells were seeded at a density of 12,000–13,000 cells per well in clear 96-well plates in medium [100 μL of RPMI supplemented with 5% heat-inactivated human serum (Sigma), penicillin (100 units / mL), and streptomycin (0.1 mg / mL)]. PBS (100 μL) was added to the edge wells. Cells were incubated at 37°C in an ambient atmosphere containing 5% CO2. After 24 h, the medium was aspirated and replaced with medium containing the desired staurosporine derivative [50 μL of RPMI supplemented with 5% HS, penicillin (100 units / mL), and streptomycin (0.1 mg / mL)]. For A431 cells, staurosporine or staurosporine derivative treatment concentrations started at 50 μM and were serially diluted fourfold across nine wells. For H460 cells, staurosporine or staurosporine derivative treatment concentrations started at 100 μM and were serially diluted fourfold across nine wells. Treatment compounds were prepared from ethanol-containing stock solutions (10 mM in ethanol; 0.5-1.0% ethanol final concentration), so vehicle control wells contained 1% ethanol. Plates were then incubated at 37°C under ambient (20% pO2) or hypoxic (0.1% pO2) atmosphere with 5% CO2 for 48 hours. Hypoxic conditions were maintained using a ProOx C21 chamber (Biosferix) in a cell culture incubator. After 48 hours, the medium was aspirated, the wells were washed once with PBS (130 μL), and cell viability was determined using an MTT assay. Thiazolyl blue tetrazolium bromide in FluoroBrite DMEM (100 μL, 0.5 mg / mL, Gibco) was added to each well, and the plate was incubated for 4 hours at 37°C under ambient atmosphere containing 5% CO2. After 4 hours, the medium was partially removed (75 μL) and replaced with DMSO (50 μL). The plate was incubated for 10 minutes on a shaker at room temperature. The absorbance at 570 nm was measured using a microplate reader (Clariostar Plus, BMG Labtech). AQ4N cell viability assay

[0404] These studies were performed as previously reported (Manley et al., J. Pharmacol. Exp. Ther. 344:368-377 (2013)). H460 or A431 cells were seeded at a density of 5,000 cells per well in clear 96-well plates in medium [100 μL of RPMI supplemented with 5% FBS, penicillin (100 units / mL), and streptomycin (0.1 mg / mL)]. PBS (100 μL) was added to the edge wells. Cells were incubated at 37°C under ambient atmosphere with 5% CO2. After 24 hours, the medium was aspirated and replaced with medium containing AQ4N (Sigma) [50 μL of RPMI supplemented with 5% FBS, penicillin (100 units / mL), and streptomycin (0.1 mg / mL)]. For both cell lines, the AQ4N treatment concentration started at 200 μM and was serially diluted four-fold across nine wells. AQ4N solutions were prepared from a DMSO-containing stock solution (20 mM in DMSO; 1% DMSO final concentration); therefore, our vehicle control wells contained 1% DMSO. Plates were then incubated at 37°C for 24 hours in an ambient (20% pO2) or hypoxic (0.1% pO2) atmosphere containing 5% CO2. Hypoxic conditions were maintained using a hypoxic incubator chamber (StemCell Technologies) in a cell culture incubator. After 24 hours, the medium was aspirated and replaced with fresh medium [100 μL of RPMI supplemented with 5% FBS, penicillin (100 units / mL), and streptomycin (0.1 mg / mL)]. Plates were returned to 37°C for 72 hours in an ambient atmosphere containing 5% CO2. After 72 hours, cell viability was measured using the MTT assay. Thiazolyl blue tetrazolium bromide in FluoroBrite DMEM (100 μL, 0.5 mg / mL, Gibco) was added to each well, and the plate was incubated for 4 hours at 37°C under ambient atmosphere with 5% CO2. After 4 hours, the medium was partially removed (75 μL) and replaced with DMSO (50 μL). The plate was incubated for 10 minutes on a shaker at room temperature.The absorbance at 570 nm was then measured using a microplate reader (Clariostar Plus, BMG Labtech). Staurosporine release by A431 cells

[0405] In a sterile 2 mL HPLC vial, add 7.5 x 10 6 Each vial was filled with a cell suspension and sealed with a septum-containing cap. One vial contained RPMI (1 mL) alone as a cell-free control. Staurosporine enamine N-oxide 37 stock solution (10 μL, 10 mM in ethanol; 100 μM final concentration) was then added to each vial. The septum was pierced with an open 22-gauge needle for normoxic conditions. Hypoxic conditions were maintained using a nitrogen line connected to a vacuum gas manifold. The vials were maintained at 37°C in a water bath and shaken every 15 min. At different time points (0, 1, 2, 4, and 8 h), aliquots (100 μL) of the cell suspension were removed from each vial and immediately added to acetonitrile (100 μL) containing p-nitrophenol (10 μM) as an internal standard. The quenched samples were then cooled to -20°C for 1 h and centrifuged at 10,000 × g for 5 min at 4°C. The supernatant was collected and analyzed by HPLC (C 18 The column was 4.6 × 250 mm, 5 μm particle size, with a flow rate of 1 mL / min, and the eluent was a gradient of 0 → 20% MeCN / HO + 0.1% TFA (1 min), gradient of 20 → 40% MeCN / HO + 0.1% TFA (7 min), gradient of 40 → 100% MeCN / HO + 0.1% TFA (3 min). Staurosporine was quantified using its absorbance at 280 nm. In vitro microsome depletion assay

[0406] All reactions were prepared and performed in semi-microspectrophotometer quartz cells equipped with septum screw caps (Starna Cells). For anaerobic conditions, all solutions were degassed for 30 minutes by sparging with nitrogen. Cells were maintained under anaerobic conditions using a nitrogen line connected to a nitrogen gas manifold or using a nitrogen-filled balloon. For experiments requiring normoxic conditions, the quartz cells were left open to ambient atmosphere. Unless otherwise noted, reactions were prepared as follows: A solution of human liver microsomes (10 μL, 20 mg / mL in phosphate buffer, pH 7.4, Corning; final concentration of 200 μg / mL) was added to phosphate buffer (928.3 μL, 100 mM phosphate buffer, pH 7.4; final volume of 1 mL) at room temperature, followed by a solution of NADPH (16.7 μL, 60 mM in 10 mM NaOH; final concentration of 1 mM). After 1 h, the reaction was initiated by adding a stock solution of 2-nitroaniline enamine N-oxide probes 32a–f (50 μL, 4 mM in phosphate buffer, pH 7.4; 200 μM final concentration). The absorbance of the solution at 430 nm was immediately blanked and then recorded at 0 and 2 h using a UV-vis spectrophotometer (Cary 60, Agilent).

[0407] Heat-inactivated microsomes were prepared for control experiments by heating a solution of human liver microsomes (20 mg / mL in phosphate buffer, pH 7.4, Corning) to 45°C for 30 min.

[0408] For studies involving CYP450 inhibitors, a solution of the CYP450 inhibitor (10 μL, 20 mM in DMSO; 200 μM final concentration) was added to a solution of NADPH and human liver microsomes under anaerobic conditions 30 min before the addition of the 2-nitroaniline enamine N-oxide probe.

[0409] To determine the initial rate of microsomal depletion, absorbance at 430 nm was measured every 0.1 or 0.5 seconds over the first 5 minutes of reaction at room temperature. Enamine N-oxide with reducing agent

[0410] A stock solution of 2-nitroaniline enamine N-oxide probe 32c (50 μL, 4 mM in 100 mM phosphate buffer, pH 7.4; 200 μM final concentration) was added to phosphate buffer (850 μL, 100 mM phosphate, pH 7.4; 1 mL final volume) in a semi-micro quartz cell open to the ambient atmosphere. A solution of glutathione (100 μL, 50 mM in phosphate buffer, pH 7.4; 5 mM final concentration), cysteine ​​(100 μL, 50 mM in phosphate buffer, pH 7.4; 5 mM final concentration), or tetrahydroxydiboron (100 μL, 20 mM in methanol; 200 μM final concentration) was added to the solution. Reactions with glutathione or cysteine ​​were incubated at 37 °C. Reactions with tetrahydroxydiboron were carried out at room temperature. Wavelength scans (400-550 nm) were recorded at several time points (0, 1, and 2 h). Assays to assess the importance of enamine N-oxide unsaturation

[0411] Enamine N-oxide 6 or alkyl N-oxide S4 (50 μL, 4 mM in water; 200 μM final concentration) was added to a HEPES buffer solution (750 μL, 100 mM HEPES, pH 7.4; 1 mL final volume) in an HPLC vial. Reduction of the N-oxide was initiated by adding a solution of tetrahydroxydiboron (200 μL, 200 nmol, 1 equiv., 1 mM in ethanol), and the resulting solution was incubated at room temperature. After 1 h, the solution was analyzed by HPLC (C). 18 The column was 4.6 × 50 mm, 2.4 μm particle size, with a flow rate of 1 mL / min, and the eluent was a gradient of HO + 0.1% TFA (1 min), followed by a gradient of 0 → 100% MeCN / HO + 0.1% TFA (4 min), followed by MeCN + 0.1% TFA (1 min). p-Cresol was quantified by its absorbance at 280 nm. Assay to evaluate the stability of enamine N-oxides to metal cations

[0412] Enamine N-oxide 3a (50 μL, 4 mM in water; 200 μM final concentration) was added to a HEPES buffer solution (946 μL, 100 mM HEPES, pH 7.4; 1 mL final volume) in an HPLC vial. Then, a solution of metal ions (4 μL, 500 mM in water, 2 mM final concentration) was added, and the solution was incubated at room temperature. After 1 h, the solution was analyzed by HPLC (C 18 The column was 4.6 × 50 mm, 2.4 μm particle size, and flow rate was 1 mL / min. The eluent was a gradient of HO + 0.1% TFA (1 min), followed by a gradient of 0 → 100% MeCN / HO + 0.1% TFA (4 min), followed by MeCN + 0.1% TFA (1 min). p-Cresol was quantified by absorbance at 280 nm.

[0413] The following cations were assayed using this procedure: sodium chloride, potassium chloride, calcium chloride, magnesium chloride, manganese(II) chloride, cobalt(II) sulfate, zinc(II) chloride, nickel(II) acetate, and iron(III) chloride.

[0414] For iron(II) sulfate, a solution of iron(II) sulfate (2 μL, 1 M in 1 N HCl; 2 mM final concentration) was added, followed by a solution of sodium dithionite (10 μL, 1 M in 1 N NaOH; 10 mM final concentration). Western blot of pimonidazole-labeled cells

[0415] Cells were seeded at a density of 200,000 cells per well in 6-well plates in RPMI medium supplemented with 2 mL of 5% heat-inactivated human serum (Sigma), penicillin (100 units / mL), and streptomycin (0.1 mg / mL). The cells were incubated at 37°C under ambient atmosphere with 5% CO2. After 24 hours, the medium was aspirated and replaced with 1 mL of RPMI medium supplemented with 5% heat-inactivated HS, penicillin (100 units / mL), and streptomycin (0.1 mg / mL). The plates were then incubated at 37°C under ambient conditions (20% pO2) or oxygenated conditions (5%, 1%, or 0.1% pO2) with 5% CO2 for 48 hours. Oxygenated conditions were maintained using a hypoxic chamber (ProOx C21, Biosferix) in a cell culture incubator. After 48 h, the medium was aspira...

Claims

1. A compound having the structure represented by Formula I or II below, or a pharma- ceutically acceptable salt or stereoisomer thereof: 【Chemistry 1】 R １ is hydrogen, C １ ~C ６ Alkyl, C １ ~C ６ Haloalkyl, C １ ~C ６ Alkoxy, C １ -C ６ Haloalkoxy, OH, CN, NO ２ , N.H. ２ , (C １ ~C ６ alkyl)NH, (C １ ~C ６ Alkyl) ２ N.C. ３ ~C ６ carbocyclyl, 5- to 6-membered heterocyclyl, an inductive electron withdrawing group, a leaving group, or a -[L]-diagnostic moiety, where R １ is optionally substituted; [L] is an optionally substituted linking group that can be absent or carry multiple diagnostic moieties, which can be the same or different; R ２ is hydrogen, C １ ~C ６ Alkyl, C １ ~C ６ Haloalkyl, C １ ~C ６ Alkoxy, C １ ~C ６ Haloalkoxy, OH, CN, NO ２ , N.H. ２ , (C １ ~C ６ alkyl)NH, (C １ ~C ６ Alkyl) ２ N.C. ３ ~C ６ carbocyclyl, 5- to 6-membered heterocyclyl, an inductive electron withdrawing group, a π-electron withdrawing group, or -[L]-diagnostic moiety, where R ２ is optionally substituted; [L] is a linking group which may be absent or may carry multiple diagnostic moieties, which may be the same or different; R ３ is hydrogen, C １ ~C ６ Alkyl, C １ ~C ６ Haloalkyl, C １ ~C ６ Alkoxy, C １ ~C ６ Haloalkoxy, OH, CN, NO ２ , N.H. ２ , (C １ ~C ６ alkyl)NH, (C １ ~C ６ Alkyl) ２ N.C. ３ ~C ６ carbocyclyl, 5- to 6-membered heterocyclyl, an inductive electron withdrawing group, a π-electron withdrawing group, or -[L]-diagnostic moiety, where R ３ is optionally substituted; [L] is a linking group which may be absent or may carry multiple diagnostic moieties, which may be the same or different; R ４ is hydrogen, C １ ~C ６ Alkyl, C １ ~C ６ Haloalkyl, C １ ~C ６ Alkoxy, C １ ~C ６ Haloalkoxy, OH, CN, NO ２ , N.H. ２ , (C １ ~C ６ alkyl)NH, (C １ ~C ６ Alkyl) ２ N.C. ３ ~C ６ carbocyclyl, 5- to 6-membered heterocyclyl, an inductive electron withdrawing group, a leaving group, a cleavable linking group, or -[L]-diagnostic moiety, where R ４ is optionally substituted; [L] is a linking group which may be absent or may carry multiple diagnostic moieties, which may be the same or different; R ７ (C １ ~C ８ ) alkyl, (C ３ ~C １０ ) carbocyclyl, or a 4- or 10-membered heterocyclyl containing 1 to 3 heteroatoms selected from O, N, and S, said alkyl, carbocyclyl, or heterocyclyl being further optionally substituted; or R ７ and R ８ together with the nitrogen atom to which they are attached form a 4- to 7-membered heterocyclyl containing 1 to 3 heteroatoms selected from O, N and S; R ８ (C １ ~C ８ ) alkyl, (C ３ ~C １０ ) carbocyclyl, or a 4- or 10-membered heterocyclyl containing 1 to 3 heteroatoms selected from O, N, and S, said alkyl, carbocyclyl, or heterocyclyl being optionally further substituted; A is absent or is a therapeutic moiety; provided that the compound of formula (I or II) contains at least one -[L]-diagnostic or therapeutic moiety. When A is a therapeutic moiety, R ４ is a cleavable linking group; If the compound contains at least one -[L]-diagnostic moiety and A is absent, then R １ and / or R ４ is a leaving group.

2. R １ but a) hydrogen; b) is an inductive electrophilic group; Optionally, said derivative group is OR 5 , SR 5 , NR 5 R 5 , or a cyclic or acyclic amide, where each R 5 is independently hydrogen, (C 1 -C 6 ) alkyl, (C 3 -C 10 ) carbocyclyl, or 4- to 7-membered heterocyclyl, where said alkyl, carbocyclyl, or heterocyclyl is optionally substituted; c) a leaving group, Optionally, the leaving group is iodo, bromo, chloro, OR 9 , SR 9 , -OC(O)R 9 , OC(O)OR 9 , OC(O)NR 9 R 9 , -OC(S)R 9 , OC(S)OR 9 , OC(S)NR 9 R 9 , -OS(O) 2 R 9 , -OS(O) 2 OR 9 , -OP(O)OR 9 OR 9 , -OP(O)R 9 R 9 , -SC(O)R 9 , -SC(O)SR 9 , or -SC(S)SR 9 , where each R 9 is independently hydrogen, (C 1 -C 6 )alkyl, (C 3 -C 10 ) carbocyclyl, or 4- to 7-membered heterocyclyl, wherein said alkyl, carbocyclyl, or heterocyclyl is optionally substituted; or d) an optionally substituted -[L]-diagnostic moiety; Optionally, the diagnostic moiety is a fluorescent dye, a chromogenic agent, a positron emission tomography (PET) tracer, or a magnetic resonance imaging (MRI) contrast agent; Optionally, [L] is -O-, -S-, -N(R')-, -C≡C-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -C(NOR')-, -C(O)N(R')-, -N(R')C(O)N(R')-, -N(R')C(O)O-, -OC(O)N(R')-, -C(NR')-, -N(R')C(R')-, -N(R')C(O)N(R')-, -N(R')S(O)N(R')-, -OP(O)O(R')-, -N(R')P(O)N(R'R')N(R')-, C 3 to C 12 an alkylene chain optionally interrupted and / or terminated (at either or both termini) by at least one of a carbocyclene, a 3- to 12-membered heterocyclene, a 5- to 12-membered heteroarylene, or any combination thereof, where each R' is independently H or an optionally substituted C 1 -C 24 alkyl, and the interruptions and one or both termini can be the same or different; wherein, further optionally, the alkylene chain is a C 1 -C 12 alkylene chain; or Optionally, [L] is -O-, -S-, -N(R')-, -C≡C-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -C(NOR')-, -C(O)N(R')-, -N(R')C(O)N(R')-, -N(R')C(O)N(R')-, -N(R')C(O)N(R')-, -N(R')S(O)N(R')-, -OP(O)O(R')-, -N(R')P(O)N(R'R')N(R')-, C 3 to C 12 a polyethylene glycol chain optionally interrupted and / or terminated (at either or both termini) by at least one of a carbocyclene, a 3- to 12-membered heterocyclene, a 5- to 12-membered heteroarylene, or any combination thereof, where 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; Further optionally, the polyethylene glycol chain has 1 to 10 -(CH 2 CH 2 -O)- units; The compound of claim 1.

3. R ２ but a) hydrogen; b) an inductive electron-withdrawing group, Optionally, said inductive electron withdrawing group is halogen, OR 6 , SR 6 , or NR 6 R 6 , where each R 6 is independently hydrogen, C 1 -C 6 alkyl, C 6 -C 12 aryl, 5- to 10-membered heteroaryl, carbonyl, sulfonyl, sulfinyl, or phosphoryl; Further optionally, the halogen is fluoro or chloro; c) a π-electron withdrawing group, Optionally, the π-electron withdrawing group is -C(O)R 6 , -C(O)NR 6 R 6 , -C(O)NR 6 R 6 , -C(O)OR 6 , -S(O)R 6 , -S(O) 2 R 6 , -S(O)OR 6 , -S(O)NR 6 R 6 , -S(O) 2 NR 6 R 6 , -OP(O)OR 6 OR 6 , or -P(O)NR 6 R 6 NR 6 R 6 , where each R 6 is independently hydrogen, C 1 -C 6 alkyl, C 6 -C 12 aryl, or 5-10 membered heteroaryl; d) an optionally substituted -[L]-diagnostic moiety; Optionally, the diagnostic moiety is a fluorescent dye, a chromogenic agent, a positron emission tomography (PET) tracer, or a magnetic resonance imaging (MRI) contrast agent.

3. A compound according to claim 1 or 2.

4. R 3 is a) hydrogen; b) an inductive electron-withdrawing group, Optionally, said inductive electron withdrawing group is halogen, OR 6 , SR 6 , or NR 6 R 6 , where each R 6 is independently hydrogen, C 1 -C 6 alkyl, C 6 -C 12 aryl, 5- to 10-membered heteroaryl, carbonyl, sulfonyl, sulfinyl, or phosphoryl; Further optionally, the halogen is fluoro or chloro; c) a π-electron withdrawing group, Optionally, the π-electron withdrawing group is -C(O)R 6 , -C(O)NR 6 R 6 , -C(O)NR 6 R 6 , -C(O)OR 6 , -S(O)R 6 , -S(O) 2 R 6 , -S(O)OR 6 , -S(O)NR 6 R 6 , -S(O) 2 NR 6 R 6 , -OP(O)OR 6 OR 6 , or -P(O)NR 6 R 6 NR 6 R 6 , where each R 6 is independently hydrogen, C 1 -C 6 alkyl, C 6 -C 12 aryl, or 5-10 membered heteroaryl; d) an optionally substituted -[L]-diagnostic moiety; Optionally, the diagnostic moiety is a fluorescent dye, a chromogenic agent, a positron emission tomography (PET) tracer, or a magnetic resonance imaging (MRI) contrast agent. The compound according to any one of claims 1 to 3.

5. R １ and R ３ is each -[L]-diagnostic moiety, which may be the same or different.

6. R １ , R ２ and R ３ is each -[L]-diagnostic moiety, which may be the same or different.

7. R ４ but a) a leaving group, Optionally, the leaving group is iodo, bromo, chloro, OR 9 , SR 9 , -OC(O)R 9 , OC(O)OR 9 , OC(O)NR 9 R 9 , -OC(S)R 9 , OC(S)OR 9 , OC(S)NR 9 R 9 , -OS(O) 2 R 9 , -OS(O) 2 OR 9 , -OP(O)OR 9 OR 9 , -OP(O)R 9 R 9 , -SC(O)R 9 , -SC(O)SR 9 , or -SC(S)SR 9 , where each R 9 is independently hydrogen, (C 1 -C 6 )alkyl, (C 3 -C 10 ) carbocyclyl, or 4- to 7-membered heterocyclyl, wherein said alkyl, carbocyclyl, or heterocyclyl is optionally substituted; or b) a cleavable linking group, Optionally, the cleavable linking group is selected from the group consisting of -O-, -S-, -N(R')-, -C≡C-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -C(NOR')-, -C(O)N(R')-, -N(R')C(O)N(R')-, -N(R')C(O)O-, -OC(O)N(R')-, -C(NR')-, -N(R')C(R')-, -N(R')C(O)N(R')-, -N(R')S(O)N(R')-, -OP(O)O(R')-, -N(R')P(O)N(R'R')N(R')-, C 3 to C 12 an alkylene chain interrupted and / or terminated (at either or both termini) by at least one of a carbocyclene, a 3- to 12-membered heterocyclene, a 5- to 12-membered heteroarylene, or any combination thereof, where each R' is independently H or an optionally substituted C 1 -C 24 alkyl, and the interruptions and one or both termini may be the same or different; Further optionally, said alkylene chain is a C 1 -C 12 alkylene chain; or Optionally, the cleavable linking group is selected from the group consisting of -O-, -S-, -N(R')-, -C≡C-, -C(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -C(NOR')-, -C(O)N(R')-, -N(R')C(O)N(R')-, -N(R')C(O)N(R')-, -N(R')C(O)N(R')-, -N(R')S(O)N(R')-, -OP(O)O(R')-, -N(R')P(O)N(R'R')N(R')-, C3 to C12 a polyethylene glycol chain interrupted and / or terminated (at either or both termini) by at least one of a carbocyclene, a 3- to 12-membered heterocyclene, a 5- to 12-membered heteroarylene, or any combination thereof, where 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; Further optionally, the polyethylene glycol chain has 1 to 10 -(CH 2 CH 2 -O)- units; The compound according to any one of claims 1 to 6.

8. a) R ７ But, Me, Et, ｎ Bu, iPr, Cy, 【Chemistry 2】 That is, b) R 8 is Me, Et, nBu, iPr, Cy, 【Chemistry 3】 is, or c) R 7 and R 8 together with the nitrogen atom to which they are attached form a 6-membered heterocyclyl containing two heteroatoms selected from O and N; A compound according to any one of claims 1 to 7.

9. A a) does not exist, or b) a therapeutic portion, wherein optionally, said therapeutic moiety is an anti-cancer agent, a hypoxia inducible factor inhibitor, or an apoptotic agent; Optionally, the anti-cancer drug is a targeted anti-cancer drug; Optionally, the targeted anti-cancer agent is a kinase inhibitor. The compound according to any one of claims 1 to 6.

10. 【Chemical 4】 【Chemistry 5】 2. The compound of claim 1, wherein:

11. 【Chemical 6】 2. The compound of claim 1, wherein:

12. A compound having a structure represented by formula III or IV, or a pharma- ceutically acceptable salt or stereoisomer thereof: 【Chemistry 7】 where R １ is hydrogen, CH ２ , C １ ~C ６ Alkyl, C １ ~C ６ Haloalkyl, C １ ~C ６ Alkoxy, C １ ~C ６ Haloalkoxy, OH, CN, NO ２ , N.H. ２ , (C １ ~C ６ alkyl)NH, (C １ ~C ６ Alkyl) ２ N.C. ３ ~C ６ carbocyclyl, 5- to 6-membered heterocyclyl, an inductive electron withdrawing group, a cleavable linking group, or -[L]-diagnostic moiety, where R １ is optionally substituted; [L] is a linking group which may be absent or may carry multiple diagnostic moieties, which may be the same or different; R ２ is hydrogen, C １ ~C ６ Alkyl, C １ ~C ６ Haloalkyl, C １ ~C ６ Alkoxy, C １ ~C ６ Haloalkoxy, OH, CN, NO ２ , N.H. ２ , (C １ ~C ６ alkyl)NH, (C １ ~C ６ Alkyl) ２ N.C. ３ ~C ６ carbocyclyl, 5- to 6-membered heterocyclyl, an inductive electron withdrawing group, a π-electron withdrawing group, a leaving group, or a -[L]-diagnostic moiety, where R ２ is optionally substituted; [L] is a linking group which may be absent or may carry multiple diagnostic moieties, which may be the same or different; R ３ is hydrogen, C １ ~C ６ Alkyl, C １ ~C ６ Haloalkyl, C １ ~C ６ Alkoxy, C １ ~C ６ Haloalkoxy, OH, CN, NO ２ , N.H. ２ , (C １ ~C ６ alkyl)NH, (C １ ~C ６ Alkyl) ２ N.C. ３ ~C ６ carbocyclyl, 5- to 6-membered heterocyclyl, an inductive electron withdrawing group, a π-electron withdrawing group, a leaving group, or a -[L]-diagnostic moiety, where R ３ is optionally substituted; [L] is a linking group which may be absent or may carry multiple diagnostic moieties, which may be the same or different; R ４ is hydrogen, C １ ~C ６ Alkyl, C １ ~C ６ Haloalkyl, C １ ~C ６ Alkoxy, C １ ~C ６ Haloalkoxy, OH, CN, NO ２ , N.H. ２ , (C １ ~C ６ alkyl)NH, (C １ ~C ６ Alkyl) ２ N.C. ３ ~C ６ carbocyclyl, 5- to 6-membered heterocyclyl, an inductive electron withdrawing group, a π-electron withdrawing group, a leaving group, or a -[L]-diagnostic moiety, where R ４ is optionally substituted; [L] is a linking group which may be absent or may carry multiple diagnostic moieties, which may be the same or different; R ７ (C １ ~C ８ ) alkyl, (C ３ ~C １０ ) carbocyclyl, or a 4- or 10-membered heterocyclyl containing 1 to 3 heteroatoms selected from O, N, and S, said alkyl, carbocyclyl, or heterocyclyl being optionally further substituted; or R ７ and R ８ together with the nitrogen atom to which they are attached form a 4- to 7-membered heterocyclyl containing 1 to 3 heteroatoms selected from O, N and S; R ８ (C １ ~C ８ ) alkyl, (C ３ ~C １０ ) carbocyclyl, or a 4- or 10-membered heterocyclyl containing 1 to 3 heteroatoms selected from O, N, and S, said alkyl, carbocyclyl, or heterocyclyl being optionally further substituted; A is absent, a leaving group, or a therapeutic moiety; provided that the compound of formula (III or IV) contains at least one -[L]-diagnostic or therapeutic moiety. When A is a therapeutic moiety, R １ is a cleavable linking group; R ２ , R ３ , and R ４ is a -[L]-diagnostic moiety of the compound of formula (III), then R １ is CH ２ and A is a leaving group; R １ , R ２ , and R ３ is a -[L]-diagnostic moiety and A is absent for compounds of formula (IV), then R ４ is a leaving group.

13. A pharmaceutical composition comprising a therapeutically effective amount of a compound or a pharma- ceutically acceptable salt or stereoisomer of any one of claims 1 to 12 and a pharma- ceutically acceptable carrier.

14. A method of treating a disease or disorder characterized by, associated with, or exhibiting tissue hypoxia, comprising: Optionally, the disease or disorder is a solid tumor; Optionally, the solid tumor is in the brain, breast, cervix, kidney, liver, lung, pancreas, or rectum; Further optionally, the solid tumor is epidermoid carcinoma, lung carcinoma, glioblastoma, or pancreatic adenocarcinoma. b) a method for treating and diagnosing a disease or disorder characterized by, associated with, or indicative of tissue hypoxia, wherein said compound comprises at least one [L]-diagnostic and therapeutic moiety, or c) A method for diagnosing a disease or disorder characterized by, associated with, or indicative of tissue hypoxia, wherein said compound comprises at least one [L]-diagnostic moiety. wherein optionally said disease or disorder is a solid tumor characterized by a hypoxic tumor microenvironment; Optionally, the solid tumor is in the brain, breast, cervix, kidney, liver, lung, pancreas, or rectum; Further optionally, the solid tumor is epidermoid carcinoma, lung carcinoma, glioblastoma, or pancreatic adenocarcinoma.

13. A compound or a pharma- ceutically acceptable salt or stereoisomer according to any one of claims 1 to 12 for use in Claim 15: a) a compound of formula I or II: 【Chemistry 8】 A method for preparing a compound of formula V: 【Chemistry 9】 with a compound of formula VII: 【Chemistry 10】 or b) a compound of formula III or IV: 【Chemistry 11】 A method for preparing a compound of formula VI: 【Chemistry 12】 with a compound of formula VII: 【Chemistry 13】 reacting with Where: Optionally, the reaction of a) or b) is carried out in the presence of a solvent. Optionally, the solvent is an aprotic solvent, a protic solvent, or a solvent mixture; 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 reaction of a) or b) is carried out at a temperature between 0° C. and 80° C.; Further optionally, the temperature is about 60° C. Optionally, the compound of formula (VII) is in excess of the compound of formula (V) or (VI); Further optionally, the excess is about 5 equivalents; or Optionally, the reaction of a) or b) is carried out for a period of 18 hours. method.

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