Inhibitors of cyclin-dependent kinases

RELATED APPLICATIONS

0001This application is a national stage filing under 35 U.S.C. § 371 of international PCT application, PCT/US2016/024345, filed Mar. 25, 2016, which claims priority under 35 U.S.C. § 119(e) to U.S. provisional application, U.S. Ser. No. 62/139,352, filed Mar. 27, 2015, each of which is incorporated herein by reference.

GOVERNMENT SUPPORT

0002This invention was made with government support under grant number 1 R01 CA179483-01A1 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

0003The members of the cyclin-dependent kinase (CDK) family play critical regulatory roles in cell proliferation. There are currently twenty known mammalian CDKs. While CDK7-CDK13 have been linked to transcription, only CDK1, 2, 4, and 6 show demonstrable association with the cell cycle.

0004Unique among the mammalian CDKs, CDK7 has consolidated kinase activities, regulating both the cell cycle and transcription. In the cytosol, CDK7 exists as a heterotrimeric complex and is believed to function as a CDK1/2-activating kinase (CAK), whereby phosphorylation of conserved residues in CDK1/2 by CDK7 is required for full catalytic CDK activity and cell cycle progression (Desai et al., “Effects of phosphorylation by CAK on cyclin binding by CDC2 and CDK2.” Mol. Cell Biol. 15, 345-350 (1995); Kaldis et al., “Analysis of CAK activities from human cells.” Eur. J. Biochem. 267, 4213-4221 (2000); Larochelle et al., “Requirements for CDK7 in the assembly of CDK1/cyclin B and activation of CDK2 revealed by chemical genetics in human cells.” Mol. Cell 25, 839-850 (2007)). In the nucleus, CDK7 forms the kinase core of the RNA polymerase (RNAP) II general transcription factor complex and is charged with phosphorylating the C-terminal domain (CTD) of RNAP II, a requisite step in gene transcriptional initiation (Serizawa. et al., “Association of CDK-activating kinase subunits with transcription factor TFIIH.” Nature 374, 280-282 (1995); Shiekhattar et al., “CDK-activating kinase complex is a component of human transcription factor TFIIH.” Nature 374, 283-287 (1995); Drapkin et al., “Human cyclin-dependent kinase-activating kinase exists in three distinct complexes.” Proc. Natl. Acad. Sci. U.S.A. 93, 6488-6493 (1996); Liu. et al., “Two cyclin-dependent kinases promote RNA polymerase II transcription and formation of the scaffold complex.” Mol. Cell Biol. 24, 1721-1735 (2004); Akhtar et al., “TFIIH kinase places bivalent marks on the carboxy-terminal domain of RNA polymerase II.” Mol. Cell 34, 387-393 (2009); Glover-Cutter et al., “TFIIH-associated CDK7 kinase functions in phosphorylation of C-terminal domain Ser7 residues, promoter-proximal pausing, and termination by RNA polymerase II.” Mol. Cell Biol. 29, 5455-5464 (2009)). Together, the two functions of CDK7, i.e., CAK and CTD phosphorylation, support critical facets of cellular proliferation, cell cycling, and transcription.

0005CDK12 and CDK13 were identified in cDNA screens for cell cycle regulators. Because their cyclin partners were not yet known, they were initially named CRKRS and CDC2L5 (Ko et al., J. Cell Sci., 2001, 114, 2591-2603; Marques et al., Biochem Biophys Res Commun., 2000, 279(3):832-837), respectively. They were found to be 1490- and 1512-amino acid proteins, respectively, with a conserved central CTD kinase domain and degenerate RS domains identified in their N- and C-terminal regions (Even et al., J Cell Biochem., 2006, 99(3), 890-904).

0006Evidence has shown CDK12 and CDK13 play an important role in cancer development. A comprehensive genomic approach identified CDK12 to be one of the most frequently somatically mutated genes in high-grade serous ovarian cancer, the most fatal form of the disease (Erratum, Nature, 2011, 474(7353), 609-615). Several identified point mutations in the kinase domain point to the critical importance of the kinase activity of CDK12 for the development/progression of this disease. CDK12 has also been found to contribute to the development of breast cancer. Notably, CDK12 is located on chromosome 17, within the 17q21 locus that contains several candidate genes for breast cancer susceptibility (Kauraniemi et al., Cancer Res., 2001, 61(22), 8235-8240), and it is co-amplified with the tyrosine kinase receptor ERBB2, a protein amplified and overexpressed in about 20% of breast tumors. Gene fusion between CDK12 and ERBB2 was also detected in gastric cancer (Zang et al., Cancer Res., 2011, 71(1), 29-39). CDK12 is also implicated in the modification of tamoxifen sensitivity in estrogen-positive breast cancer via the modulation of the mitogen-activated protein kinase pathway (Iorns et al., Carcinogenesis, 2009, 30(10):1696-1701).

0007Due to the important regulatory functions of kinases, such as CDK7, CDK12, and CDK13, in cell cycle control, cell proliferation, differentiation, and apoptosis, it is important to develop modulators of the activities of these kinases, including selective modulators, for use as research tools as well as therapeutic agents in the treatment of diseases.

SUMMARY OF THE INVENTION

0008Cyclin dependent kinases (CDKs), e.g., CDK7, CDK12, and CDK13 are key regulators of the cell cycle. Their successive activation and inactivation drives the cycle forward. The activity of CDKs is regulated by multiple mechanisms such as positive and negative phosphorylation, binding of regulatory proteins like cyclins, and CDK inhibitors. Most CDKs require the phosphorylation of a threonine residue located in the T-loop to achieve full kinase activity. This threonine residue is conserved in all CDKs that function in cell cycle regulation. The enzyme responsible for this phosphorylation is therefore termed CDK-activating-kinase or CAK. CAK complexes have been found to be composed of CDK7, CDK12, CDK13, cyclin H, and MAT1. Besides its CAK function, CDK7, CDK12, and CDK13 also play a role in transcription and possibly in DNA repair. This suggests that the CDK7, CDK12, and CDK13 enzyme complexes are involved in multiple functions in the cell, e.g., cell cycle control, apoptosis, transcription regulation, and DNA repair.

0009The present invention provides compounds of Formulae (I)-(III), and pharmaceutically acceptable salts, solvates, hydrates, polymorphs, co-crystals, tautomers, stereoisomers, isotopically labeled derivatives, prodrugs, and compositions thereof. The compounds of Formulae (I)-(III), and pharmaceutically acceptable salts, solvates, hydrates, polymorphs, co-crystals, tautomers, stereoisomers, isotopically labeled derivatives, prodrugs, and compositions thereof, may inhibit the activity of a kinase. The compounds described herein can selectively inhibit specific CDK subtypes, for example, CDK7, CDK12, or CDK13. In certain embodiments, the compounds of Formulae (I)-(III) are selective for CDK7 compared to other kinases. In certain embodiments, the compounds of Formulae (I)-(III) are selective for CDK12 and/or CDK13 compared to other kinases. The present invention also provides methods of using the inventive compounds, and pharmaceutically acceptable salts, solvates, hydrates, polymorphs, co-crystals, tautomers, stereoisomers, isotopically labeled derivatives, prodrugs, and compositions thereof, to study the inhibition of a kinase (e.g., CDK7, CDK12, and/or CDK13) and as therapeutics for the prevention and/or treatment of diseases associated with the overexpression and/or aberrant activity of a kinase (e.g., CDK7, CDK12, and/or CDK13). In certain embodiments, the inventive compounds are used for the prevention and/or treatment of proliferative diseases (e.g., cancers (e.g., leukemia, acute lymphoblastic leukemia, lymphoma, Burkitt's lymphoma, melanoma, multiple myeloma, breast cancer, Ewing's sarcoma, osteosarcoma, brain cancer, neuroblastoma, lung cancer, colorectal cancer), benign neoplasms, diseases associated with angiogenesis, inflammatory diseases, autoinflammatory diseases, and autoimmune diseases) in a subject.

0010Since the discovery of selective inhibitors of CDK7, CDK12, and CDK13 has been hampered by the high sequence and structural similarities of the kinase domain of CDK family members, the development of selective inhibitors of the transcriptional cyclin-dependent kinases (tCDKs) will allow dissection of their individual contributions to the regulation of transcription and evaluation of their therapeutic potential. Without wishing to be bound by any particular theory, the inventive compounds' selectivity for CDK7, CDK12, and/or CDK13 may be due to the compounds' ability to covalently modify a specific cysteine residue of these kinases (e.g., Cys312 of CDK7, Cys1039 of CDK12, Cys1017 of CDK13).

0011In one aspect, the present invention provides compounds of Formula (I):

0012<chemistry id="CHEM-US-00001" num="00001"><img file="USRE50776E_D0001.tif" /></chemistry><br /> and pharmaceutically acceptable salts, solvates, hydrates, polymorphs, co-crystals, tautomers, stereoisomers, isotopically labeled derivatives, and prodrugs thereof, wherein R¹, R², R³, R⁵, Ring A, Ring B, L¹, and L²are as defined herein.

0013In one aspect, the present invention provides compounds of Formula (II):

0014<chemistry id="CHEM-US-00002" num="00002"><img file="USRE50776E_D0002.tif" /></chemistry><br /> and pharmaceutically acceptable salts, solvates, hydrates, polymorphs, co-crystals, tautomers, stereoisomers, isotopically labeled derivatives, and prodrugs thereof, wherein R¹, R², R³, R⁴, Ring A, Ring B, L¹, and L²are as defined herein.

0015In one aspect, the present invention provides compounds of Formula (III):

0016<chemistry id="CHEM-US-00003" num="00003"><img file="USRE50776E_D0003.tif" /></chemistry><br /> and pharmaceutically acceptable salts, solvates, hydrates, polymorphs, co-crystals, tautomers, stereoisomers, isotopically labeled derivatives, and prodrugs thereof, wherein R¹, R², R³, R⁴, Ring A, Ring B, L¹, and L²are as defined herein.

0017In another aspect, the present disclosure provides pharmaceutical compositions including a compound described herein, and optionally a pharmaceutically acceptable excipient. In certain embodiments, the pharmaceutical compositions described herein include a therapeutically or prophylactically effective amount of a compound described herein. The pharmaceutical composition may be useful for treating a proliferative disease in a subject in need thereof, preventing a proliferative disease in a subject in need thereof, inhibiting the activity of a protein kinase in a subject, biological sample, tissue, or cell, and/or inducing apoptosis in a cell. In certain embodiments, the proliferative disease is an acute inflammatory disease. In certain embodiments, the acute inflammatory disease is rheumatoid arthritis, Crohn's disease, or fibrosis.

0018In another aspect, the present invention provides methods for treating and/or preventing a proliferative disease. Exemplary proliferative diseases which may be treated include cancer, benign neoplasm, diseases associated with angiogenesis, inflammatory diseases, autoinflammatory diseases, and autoimmune diseases. In certain embodiments, the cancer is one or more selected from the group consisting of pancreatic cancer, lung cancer (e.g. small cell lung cancer (SCLC), and non-small cell lung cancer), prostate cancer, breast cancer, ovarian cancer, kidney cancer, liver cancer, Ewing's sarcoma, osteosarcoma, brain cancer, neuroblastoma, and colorectal cancer.

0019Another aspect of the invention relates to methods of inhibiting the activity of a kinase (e.g., CDK (e.g., CDK7, CDK12, CDK13)) in a biological sample or subject. In certain embodiments, the method involves the selective inhibition of CDK7. In certain embodiments, the method involves the selective inhibition of CDK12. In certain embodiments, the method involves the selective inhibition of CDK13.

0020Also provided by the present invention are methods of inhibiting transcription of one or more genes in the cell of a biological sample or subject. The transcription of genes affected by the activity of CDK7 may be inhibited by a compound of the invention. In certain embodiments, these genes are one or more selected from the group consisting of MYC, RUNX1, MYB, TAL1, GATA3, KLF2, HNRPDL, p21, ASCL1, MYCN, INSM1, NEUROD1, NEUROG1, FOXG1, FOXA1, SOX2, SOX4, BCL11A, OTX2, GAT2, PHOX2B, PLK2, TAF1, CTGF, WEE1, SDIM, JUN, PIM1, IL8, FOS1. The transcription of genes affected by the activity of CDK12 may be inhibited by a compound of the invention. In certain embodiments, these genes are one or more selected from the group consisting of BRCA1, FANCI, ATR, FANCD2, APEX1, NEK9, CHEK1, CHEK2, ATM, RAD51C, RAD51D, ORC3L, MDC1, TERF2, ERCC4, FANCF, PARP9, RUNX1, MYB, TAL1, MCL1, MYC, BCL2, ETS1, EWS-FLI. The transcription of genes affected by the activity of CDK13 may be inhibited by a compound of the invention. In certain embodiments, the gene is SNORA38.

0021The present invention also provides methods of inhibiting cell growth in a biological sample or subject. In still another aspect, the present invention provides methods of inducing apoptosis of a cell in a biological sample or subject.

0022In yet another aspect, the present invention provides compounds of Formulae (I)-(III), and pharmaceutically acceptable salts, solvates, hydrates, polymorphs, co-crystals, tautomers, stereoisomers, isotopically labeled derivatives, prodrugs, and compositions thereof, for use in the treatment of a disease (e.g., a proliferative disease such as cancer) in a subject.

0023Another aspect of the present disclosure relates to kits comprising a container with a compound, or pharmaceutical composition thereof, as described herein. The kits described herein may include a single dose or multiple doses of the compound or pharmaceutical composition. The kits may be useful in a method of the disclosure. In certain embodiments, the kit further includes instructions for using the compound or pharmaceutical composition. A kit described herein may also include information (e.g. prescribing information) as required by a regulatory agency such as the U.S. Food and Drug Administration (FDA).

0024The present invention provides methods for administering to a subject in need thereof an effective amount of a compound, or pharmaceutical composition thereof, as described herein. Also described are methods for contacting a cell with an effective amount of a compound, or pharmaceutical composition thereof, as described herein. In certain embodiments, a method described herein further includes administering to the subject an additional pharmaceutical agent. In certain embodiments, a method described herein further includes contacting the cell with an additional pharmaceutical agent. The methods described herein may further include performing radiotherapy, immunotherapy, and/or transplantation on the subject.

0025The details of one or more embodiments of the invention are set forth herein. Other features, objects, and advantages of the invention will be apparent from the Detailed Description, the Examples, and the Claims.

BRIEF DESCRIPTION OF THE DRAWINGS

0026The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments of the invention and together with the description, serve to explain the principles of the invention.

0027<figref idref="DRAWINGS">FIG. <b>1</b></figref> shows inhibition of Jurkat cell viability by compounds E9, E17, and dinaciclib. Specifically, compounds E9 and E11 irreversibly inhibit Jurkat cell viability. The antiproliferative effects of compounds E9 and E17 are present with and without washout, while dinaciclib's effects are abolished with washout. Cells were treated with each compound for 4 hours and either no washout (no washout) or had compound removed by washing cells with media (washout). Sixty-eight (68) hours after washout, the cells were assayed for antiproliferative effects. Error bars indicate +/−SD.

0028<figref idref="DRAWINGS">FIG. <b>2</b></figref> shows binding of compounds E9 and E17 with CDK12 at two concentrations. Compounds E9 and E17 were able to block pulldown of cyclin K at 1 μM and 200 nM. Specifically, Jurkat cells were treated with DMSO, compounds E9, E17, and dinaciclib at two concentrations for 4 hours. Decreased pulldown of cyclin K with biotin-THZ1 indicated a loss of CDK12 binding. Cyclin H pulldown was not affected, indicating that CDK7 binding is not affected.

0029<figref idref="DRAWINGS">FIG. <b>3</b></figref> shows the effect of compounds E9, E17, and Dinaciclib on the downstream protein targets. Specifically, compounds E9 and E17 show an irreversible effect on Polymerase II Serine 2 phosphorylation and MCL1 levels with and without washout. Dinaciclib is only effective when it is present in cells, and its effect is abolished by washout.

0030<figref idref="DRAWINGS">FIG. <b>4</b></figref> shows evaluation of the exemplified compounds in neuroblastoma (NB) cells. The IC50 values are shown in nM.

0031<figref idref="DRAWINGS">FIG. <b>5</b></figref> shows that Dinaciclib and Compounds E9, E17, and E18 inhibit Jurkat T-cell acute lymphoblastic leukemia (T-ALL) cell proliferation. Compounds E9, E17, and E18 irreversibly inhibit T-ALL cell proliferation while Dinaciclib acts as a reversible inhibitor. Jurkat cells were seeded at a density of 25,000 cells/well in 96-well plates. Cells were then treated with the indicated compounds in a 10-pt dose escalation format from 1 nM to 10 μM or DMSO control for 72 hours. After 72 hours, the cells were assayed using CellTiter-Glo Luminescent Cell Viability Assay (Promega) to determine cell viability by measuring the amount of ATP present in each sample cell population, which is an indicator of cell metabolic activity. Results were graphed as fraction of the DMSO control at 72 hours. All data points were performed in biological triplicate. Error bars are +/−SD.

0032<figref idref="DRAWINGS">FIG. <b>6</b></figref> shows the effect of Dinaciclib and compounds E9, E17, and E18 on the downstream protein targets. Specifically, reversible inhibitor Dinaciclib and covalent inhibitors Compounds E9, E17, and E18 are likely to downregulate mRNA transcripts of TAL1, RUNX1, and MYB in Jurkat T-cell acute lymphoblastic leukemia (T-ALL) cells. Jurkat cells were seeded at a density of 5 million cells/10 mL. Cells were then treated with 200 nM or 1 μM of the indicated compounds or with DMSO control for 6 hours. Total RNA was extracted from 5 million cells using the RNeasy Plus Mini Kit (Qiagen) with a gDNA eliminator mini column to remove genomic DNA. mRNA was reverse transcribed into cDNA using the SuperScript III First-Strand Synthesis Kit (Life Technologies) using an oligo-dT primer to capture polyadenylated mRNAs. Quantitative PCR (qPCR) using transcript—specific Taqman probes (Applied Biosystems) was used to assess the effect of compound treatment on the expression of the indicated mRNA transcripts. All experiments shown were performed in biological triplicate. Each individual biological sample was qPCR—amplified in technical duplicate. Error bars are +/−SD. Expression data from drug treatments were normalized to GAPDH probe.

0033<figref idref="DRAWINGS">FIG. <b>7</b></figref> shows electrospray ionization mass spectra of CDK12/CCNK complex after treatment with DMSO for 1 hr at room temperature. The zero-charge mass spectra in the upper panel and inset is the result of deconvolution of the raw mass spectrum in the lower panel. A, CCNK. C, phosphorylated CDK12. D, CDK12.

0034<figref idref="DRAWINGS">FIG. <b>8</b></figref> shows electrospray ionization mass spectra of CDK12/CCNK complex after treatment with E-9 for 1 hr at room temperature. The zero-charge mass spectra in the upper panel and inset is the result of deconvolution of the raw mass spectrum in the lower panel. A, CCNK. B, phosphorylated and E-9 labeled CDK12. C, E-9 labeled CDK12. Only the masses of CDK12 proteins shift after treatment.

0035<figref idref="DRAWINGS">FIG. <b>9</b></figref> shows pull down assay with exemplified compounds. The first and second wells are at the concentrations of 1 μM and 200 nM respectively.

0036<figref idref="DRAWINGS">FIG. <b>10</b></figref> shows KiNativ™ kinome profiling of compound E9.

0037<figref idref="DRAWINGS">FIG. <b>11</b></figref> shows exemplified downstream genes. Transcription of these downstream genes are affected by the activities of CDK7, CDK12, and CDK13.

0038<figref idref="DRAWINGS">FIG. <b>12</b></figref> shows an overview of the CRISPR-mediated strategy to introduce cysteine-to-serine mutations into the genomic loci of CDK12 and CDK13. Shown are gene tracks of RNA polymerase II ChIP-seq signal at CDK12 (left) and CDK13 (left) gene loci in Jurkat cells. Cartoons depict (1) the Cas9/guide RNA construct used to target Cas9-mediated DNA cutting to particular either CDK12 or CDK/13 gene loci and (2) a repair construct that contains DNA sequence encoding for a serine mutation in CDK12 (C1039S) and CDK13 (C1017S), which together introduce the desired cysteine-to-serine coding mutations into these loci.

0039<figref idref="DRAWINGS">FIG. <b>13</b></figref> shows genotype for wild type (top) CDK12 C1039S/CDK13 C1017S double mutant (bottom) cells. The sequencing results for CDK12 (left) and CDK13 (right) loci are depicted. Expression of these putatively inhibitor-refractory mutants is expected to rescue compound-induced proliferation defects that result from CDK12 and CDK13 covalent inhibition. For CDK12 WT loci, TGC codes for cysteine; while for CDK12 C1039S loci, TCC codes for serine. For CDK13 WT loci, TGT codes for cysteine; while for CDK13 C1017S loci, TCT codes for serine.

0040<figref idref="DRAWINGS">FIG. <b>14</b></figref> shows the 72 hour proliferation results from wild type and CDK12 C1039S/CDK13 C1017S HAP1 cells. Double mutant cells are 4-fold less sensitive to E17 compared to wild type cells indicating rescue of CDK12/13 inibition—induced proliferation defects. HAP1 cells were seeded at a density of 12,000 cells/well in 96-well plates. Twenty-four hours later, cells were treated with the indicated compounds in a 10-pt dose escalation format from 1 nM to 10 μM or DMSO control for 72 hours. After 72 hours, the cells were assayed using CellTiter-Glo Luminescent Cell Viability Assay (Promega) to determine cell viability by measuring the amount of ATP present in each sample cell population, which is an indicator of cell metabolic activity. Results were graphed as fraction of the DMSO control at 72 hours. All data points were performed in biological triplicate. Error bars are +/−SD.

0041<figref idref="DRAWINGS">FIG. <b>15</b></figref> shows the profiling of E-9 and E11 in the HAP1 cell proliferation assay. HAP1 cells expressing inhibitor-refractory mutations in CDK12 (C1039S) and CDK13 (C1017S) were 4-fold less sensitive to E17 compared to control wild type HAP1 cells. This result indicates that a significant portion of intracellular E17 activity comes from covalent inhibition of CDK12 and/or CDK13. Mutation of these targeted cysteines to a less nucleophilic serine is sufficient to rescue a significant portion of anti-prolierative activity at concentrations less than 350 nM.

DEFINITIONS

0042Definitions of specific functional groups and chemical terms are described in more detail below. The chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75^thEd., inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in Thomas Sorrell, Organic Chemistry, University Science Books, Sausalito, 1999; Smith and March, March's Advanced Organic Chemistry, 5^thEdition, John Wiley &amp; Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; and Carruthers, Some Modern Methods of Organic Synthesis, 3^rdEdition, Cambridge University Press, Cambridge, 1987. The disclosure is not intended to be limited in any manner by the exemplary listing of substituents described herein.

0043Compounds described herein can comprise one or more asymmetric centers, and thus can exist in various isomeric forms, e.g., enantiomers and/or diastereomers. For example, the compounds described herein can be in the form of an individual enantiomer, diastereomer or geometric isomer, or can be in the form of a mixture of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomer. Isomers can be isolated from mixtures by methods known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred isomers can be prepared by asymmetric syntheses. See, for example, Jacques et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen et al., Tetrahedron 33:2725 (1977); Eliel, Stereochemistry of Carbon Compounds (McGraw-Hill, N Y, 1962); and Wilen, Tables of Resolving Agents and Optical Resolutions, p. 268 (E. L. Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, Ind. 1972). The disclosure additionally encompasses compounds described herein as individual isomers substantially free of other isomers, and alternatively, as mixtures of various isomers.

0044In a formula, <img file="USRE50776E_D0004.tif" /> is a single bond where the stereochemistry of the moieties immediately attached thereto is not specified, - - - is absent or a single bond, and <img file="USRE50776E_D0005.tif" /> or <img file="USRE50776E_D0006.tif" /> is a single or double bond.

0045Unless otherwise stated, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures except for the replacement of hydrogen by deuterium or tritium, replacement of ¹⁹F with ¹⁸F, or the replacement of ¹²C with ¹³C or ¹⁴C are within the scope of the disclosure. Such compounds are useful, for example, as analytical tools or probes in biological assays.

0046When a range of values is listed, it is intended to encompass each value and sub-range within the range. For example “C_1-6” is intended to encompass, C₁, C₂, C₃, C₄, C₅, C₆, C_1-6, C_1-5, C_1-4, C_1-3, C_1-2, C_2-6, C_2-5, C_2-4, C_2-3, C_3-6, C_3-5, C_3-4, C_4-6, C_4-5, and C_5-6.

0047The term “aliphatic” includes both saturated and unsaturated, straight chain (i.e., unbranched), branched, acyclic, cyclic, or polycyclic aliphatic hydrocarbons, which are optionally substituted with one or more functional groups. As will be appreciated by one of ordinary skill in the art, “aliphatic” is intended herein to include, but is not limited to, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, and cycloalkynyl moieties. Thus, the term “alkyl” includes straight, branched and cyclic alkyl groups. An analogous convention applies to other generic terms such as “alkenyl”, “alkynyl”, and the like. Furthermore, the terms “alkyl”, “alkenyl”, “alkynyl”, and the like encompass both substituted and unsubstituted groups. In certain embodiments, “lower alkyl” is used to indicate those alkyl groups (cyclic, acyclic, substituted, unsubstituted, branched or unbranched) having 1-6 carbon atoms.

0048In certain embodiments, the alkyl, alkenyl, and alkynyl groups employed in the disclosure contain 1-20 aliphatic carbon atoms. In certain other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the disclosure contain 1-10 aliphatic carbon atoms. In yet other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the disclosure contain 1-8 aliphatic carbon atoms. In still other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the disclosure contain 1-6 aliphatic carbon atoms. In yet other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the disclosure contain 1-4 carbon atoms. Illustrative aliphatic groups thus include, but are not limited to, for example, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, —CH₂-cyclopropyl, vinyl, allyl, n-butyl, sec-butyl, isobutyl, tert-butyl, cyclobutyl, —CH₂-cyclobutyl, n-pentyl, sec-pentyl, isopentyl, tert-pentyl, cyclopentyl, —CH₂-cyclopentyl, n-hexyl, sec-hexyl, cyclohexyl, —CH₂-cyclohexyl moieties and the like, which again, may bear one or more substituents. Alkenyl groups include, but are not limited to, for example, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, and the like. Representative alkynyl groups include, but are not limited to, ethynyl, 2-propynyl (propargyl), 1-propynyl, and the like.

0049The term “alkyl” refers to a radical of a straight-chain or branched saturated hydrocarbon group having from 1 to 10 carbon atoms (“C_1-10alkyl”). In some embodiments, an alkyl group has 1 to 9 carbon atoms (“C_1-9alkyl”). In some embodiments, an alkyl group has 1 to 8 carbon atoms (“C_1-8alkyl”). In some embodiments, an alkyl group has 1 to 7 carbon atoms (“C_1-7alkyl”). In some embodiments, an alkyl group has 1 to 6 carbon atoms (“C_1-6alkyl”). In some embodiments, an alkyl group has 1 to 5 carbon atoms (“C_1-5alkyl”). In some embodiments, an alkyl group has 1 to 4 carbon atoms (“C_1-4alkyl”). In some embodiments, an alkyl group has 1 to 3 carbon atoms (“C_1-3alkyl”). In some embodiments, an alkyl group has 1 to 2 carbon atoms (“C_1-2alkyl”). In some embodiments, an alkyl group has 1 carbon atom (“C₁alkyl”). In some embodiments, an alkyl group has 2 to 6 carbon atoms (“C_2-6alkyl”). Examples of C_1-6alkyl groups include methyl (C₁), ethyl (C₂), propyl (C₃) (e.g., n-propyl, isopropyl), butyl (C₄) (e.g., n-butyl, tert-butyl, sec-butyl, iso-butyl), pentyl (C₅) (e.g., n-pentyl, 3-pentanyl, amyl, neopentyl, 3-methyl-2-butanyl, tertiary amyl), and hexyl (C₆) (e.g., n-hexyl). Additional examples of alkyl groups include n-heptyl (C₇), n-octyl (C₈), and the like. Unless otherwise specified, each instance of an alkyl group is independently unsubstituted (an “unsubstituted alkyl”) or substituted (a “substituted alkyl”) with one or more substituents (e.g., halogen, such as F). In certain embodiments, the alkyl group is an unsubstituted C_1-10alkyl (such as unsubstituted C_1-6alkyl, e.g., —CH₃). In certain embodiments, the alkyl group is a substituted C_1-10alkyl (such as substituted C_1-6alkyl, e.g., —CF₃). In certain embodiments, the alkyl group is unsubstituted C_1-10alkyl (such as unsubstituted C_1-6alkyl, e.g., —CH₃(Me), unsubstituted ethyl (Et), unsubstituted propyl (Pr, e.g., unsubstituted n-propyl (n-Pr), unsubstituted isopropyl (i-Pr)), unsubstituted butyl (Bu, e.g., unsubstituted n-butyl (n-Bu), unsubstituted tert-butyl (tert-Bu or t-Bu), unsubstituted sec-butyl (sec-Bu), unsubstituted isobutyl (i-Bu)). In certain embodiments, the alkyl group is substituted C_1-10alkyl (such as substituted C_1-6alkyl, e.g., —CF₃, Bn).

0050The term “haloalkyl” is a substituted alkyl group, wherein one or more of the hydrogen atoms are independently replaced by a halogen, e.g., fluoro, bromo, chloro, or iodo. In some embodiments, the haloalkyl moiety has 1 to 8 carbon atoms (“C_1-8haloalkyl”). In some embodiments, the haloalkyl moiety has 1 to 6 carbon atoms (“C_1-6haloalkyl”). In some embodiments, the haloalkyl moiety has 1 to 4 carbon atoms (“C_1-4haloalkyl”). In some embodiments, the haloalkyl moiety has 1 to 3 carbon atoms (“C_1-3haloalkyl”). In some embodiments, the haloalkyl moiety has 1 to 2 carbon atoms (“C_1-2haloalkyl”). Examples of haloalkyl groups include —CHF₂, —CH₂F, —CF₃, —CH₂CF₃, —CF₂CF₃, —CF₂CF₂CF₃, —CCl₃, —CFCl₂, —CF₂Cl, and the like.

0051The term “heteroalkyl” refers to an alkyl group, which further includes at least one heteroatom (e.g., 1, 2, 3, or 4 heteroatoms) selected from oxygen, nitrogen, or sulfur within (i.e., inserted between adjacent carbon atoms of) and/or placed at one or more terminal position(s) of the parent chain. In certain embodiments, a heteroalkyl group refers to a saturated group having from 1 to 10 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroC_1-10alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 to 9 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroC_1-9alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 to 8 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroC_1-8alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 to 7 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroC_1-7alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 to 6 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroC_1-6alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 to 5 carbon atoms and 1 or 2 heteroatoms within the parent chain (“heteroC_1-5alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 to 4 carbon atoms and for 2 heteroatoms within the parent chain (“heteroC_1-4alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 to 3 carbon atoms and 1 heteroatom within the parent chain (“heteroC_1-3alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 to 2 carbon atoms and 1 heteroatom within the parent chain (“heteroC_1-2alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 carbon atom and 1 heteroatom (“heteroC₁alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 2 to 6 carbon atoms and 1 or 2 heteroatoms within the parent chain (“heteroC_2-6alkyl”). Unless otherwise specified, each instance of a heteroalkyl group is independently unsubstituted (an “unsubstituted heteroalkyl”) or substituted (a “substituted heteroalkyl”) with one or more substituents. In certain embodiments, the heteroalkyl group is unsubstituted heteroC_1-10alkyl. In certain embodiments, the heteroalkyl group is substituted heteroC_1-10alkyl.

0052“Alkenyl” refers to a radical of a straight-chain or branched hydrocarbon group having from 2 to 20 carbon atoms, one or more carbon-carbon double bonds, and no triple bonds (“C_2-20alkenyl”). In some embodiments, an alkenyl group has 2 to 10 carbon atoms (“C_2-10alkenyl”). In some embodiments, an alkenyl group has 2 to 9 carbon atoms (“C_2-9alkenyl”). In some embodiments, an alkenyl group has 2 to 8 carbon atoms (“C_2-8alkenyl”). In some embodiments, an alkenyl group has 2 to 7 carbon atoms (“C_2-7alkenyl”). In some embodiments, an alkenyl group has 2 to 6 carbon atoms (“C_2-6alkenyl”). In some embodiments, an alkenyl group has 2 to 5 carbon atoms (“C_2-5alkenyl”). In some embodiments, an alkenyl group has 2 to 4 carbon atoms (“C_2-4alkenyl”). In some embodiments, an alkenyl group has 2 to 3 carbon atoms (“C_2-3alkenyl”). In some embodiments, an alkenyl group has 2 carbon atoms (“C₂alkenyl”). The one or more carbon-carbon double bonds can be internal (such as in 2-butenyl) or terminal (such as in 1-butenyl). Examples of C_2-4alkenyl groups include ethenyl (C₂), 1-propenyl (C₃), 2-propenyl (C₃), 1-butenyl (C₄), 2-butenyl (C₄), butadienyl (C₄), and the like. Examples of C_2-6alkenyl groups include the aforementioned C_2-4alkenyl groups as well as pentenyl (C₅), pentadienyl (C₅), hexenyl (C₆), and the like. Additional examples of alkenyl include heptenyl (C₇), octenyl (C₈), octatrienyl (C₈), and the like. Unless otherwise specified, each instance of an alkenyl group is independently optionally substituted, i.e., unsubstituted (an “unsubstituted alkenyl”) or substituted (a “substituted alkenyl”) with one or more substituents. In certain embodiments, the alkenyl group is unsubstituted C_2-10alkenyl. In certain embodiments, the alkenyl group is substituted C_2-10alkenyl. In an alkenyl group, a C═C double bond for which the stereochemistry is not specified (e.g., —CH═CHCH₃or

0053<chemistry id="CHEM-US-00004" num="00004"><img file="USRE50776E_D0007.tif" /></chemistry><br /> may be an (E)- or (Z)-double bond.

0054The term “heteroalkenyl” refers to an alkenyl group, which further includes at least one heteroatom (e.g., 1, 2, 3, or 4 heteroatoms) selected from oxygen, nitrogen, and sulfur within (i.e., inserted between adjacent carbon atoms of) and/or placed at one or more terminal position(s) of the parent chain. In certain embodiments, a heteroalkenyl group refers to a group having from 2 to 10 carbon atoms, at least one double bond, and 1 or more heteroatoms within the parent chain (“heteroC_2-10alkenyl”). In some embodiments, a heteroalkenyl group has 2 to 9 carbon atoms at least one double bond, and 1 or more heteroatoms within the parent chain (“heteroC_2-9alkenyl”). In some embodiments, a heteroalkenyl group has 2 to 8 carbon atoms, at least one double bond, and 1 or more heteroatoms within the parent chain (“heteroC_2-8alkenyl”). In some embodiments, a heteroalkenyl group has 2 to 7 carbon atoms, at least one double bond, and 1 or more heteroatoms within the parent chain (“heteroC_2-7alkenyl”). In some embodiments, a heteroalkenyl group has 2 to 6 carbon atoms, at least one double bond, and 1 or more heteroatoms within the parent chain (“heteroC_2-6alkenyl”). In some embodiments, a heteroalkenyl group has 2 to 5 carbon atoms, at least one double bond, and 1 or 2 heteroatoms within the parent chain (“heteroC_2-5alkenyl”). In some embodiments, a heteroalkenyl group has 2 to 4 carbon atoms, at least one double bond, and for 2 heteroatoms within the parent chain (“heteroC_2-4alkenyl”). In some embodiments, a heteroalkenyl group has 2 to 3 carbon atoms, at least one double bond, and 1 heteroatom within the parent chain (“heteroC_2-3alkenyl”). In some embodiments, a heteroalkenyl group has 2 to 6 carbon atoms, at least one double bond, and 1 or 2 heteroatoms within the parent chain (“heteroC_2-6alkenyl”). Unless otherwise specified, each instance of a heteroalkenyl group is independently unsubstituted (“unsubstituted heteroalkenyl”) or substituted (“substituted heteroalkenyl”) with one or more substituents. In certain embodiments, the heteroalkenyl group is unsubstituted heteroC_2-10alkenyl. In certain embodiments, the heteroalkenyl group is substituted heteroC_2-10alkenyl.

0055“Alkynyl” refers to a radical of a straight-chain or branched hydrocarbon group having from 2 to 20 carbon atoms, one or more carbon-carbon triple bonds, and optionally one or more double bonds (“C_2-20alkynyl”). In some embodiments, an alkynyl group has 2 to 10 carbon atoms (“C_2-10alkynyl”). In some embodiments, an alkynyl group has 2 to 9 carbon atoms (“C_2-9alkynyl”). In some embodiments, an alkynyl group has 2 to 8 carbon atoms (“C_2-8alkynyl”). In some embodiments, an alkynyl group has 2 to 7 carbon atoms (“C_2-7alkynyl”). In some embodiments, an alkynyl group has 2 to 6 carbon atoms (“C_2-6alkynyl”). In some embodiments, an alkynyl group has 2 to 5 carbon atoms (“C_2-5alkynyl”). In some embodiments, an alkynyl group has 2 to 4 carbon atoms (“C_2-4alkynyl”). In some embodiments, an alkynyl group has 2 to 3 carbon atoms (“C_2-3alkynyl”). In some embodiments, an alkynyl group has 2 carbon atoms (“C₂alkynyl”). The one or more carbon-carbon triple bonds can be internal (such as in 2-butynyl) or terminal (such as in 1-butynyl). Examples of C_2-4alkynyl groups include, without limitation, ethynyl (C₂), 1-propynyl (C₃), 2-propynyl (C₃), 1-butynyl (C₄), 2-butynyl (C₄), and the like. Examples of C_2-6alkenyl groups include the aforementioned C_2-4alkynyl groups as well as pentynyl (C₅), hexynyl (C₆), and the like. Additional examples of alkynyl include heptynyl (C₇), octynyl (C₈), and the like. Unless otherwise specified, each instance of an alkynyl group is independently optionally substituted, i.e., unsubstituted (an “unsubstituted alkynyl”) or substituted (a “substituted alkynyl”) with one or more substituents. In certain embodiments, the alkynyl group is unsubstituted C_2-10alkynyl. In certain embodiments, the alkynyl group is substituted C_2-10alkynyl.

0056The term “heteroalkynyl” refers to an alkynyl group that includes at least one heteroatom (e.g., 1, 2, 3, or 4 heteroatoms) selected from oxygen, nitrogen, and sulfur within (i.e., inserted between adjacent carbon atoms of) and/or placed at one or more terminal position(s) of the parent chain. In certain embodiments, a heteroalkynyl group refers to a group having from 2 to 10 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain (“heteroC_2-10alkynyl”). In some embodiments, a heteroalkynyl group has 2 to 9 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain (“heteroC_2-9alkynyl”). In some embodiments, a heteroalkynyl group has 2 to 8 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain (“heteroC_2-8alkynyl”). In some embodiments, a heteroalkynyl group has 2 to 7 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain (“heteroC_2-7alkynyl”). In some embodiments, a heteroalkynyl group has 2 to 6 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain (“heteroC_2-6alkynyl”). In some embodiments, a heteroalkynyl group has 2 to 5 carbon atoms, at least one triple bond, and 1 or 2 heteroatoms within the parent chain (“heteroC_2-5alkynyl”). In some embodiments, a heteroalkynyl group has 2 to 4 carbon atoms, at least one triple bond, and 1 or 2 heteroatoms within the parent chain (“heteroC_2-4alkynyl”). In some embodiments, a heteroalkynyl group has 2 to 3 carbon atoms, at least one triple bond, and 1 heteroatom within the parent chain (“heteroC_2-3alkynyl”). In some embodiments, a heteroalkynyl group has 2 to 6 carbon atoms, at least one triple bond, and 1 or 2 heteroatoms within the parent chain (“heteroC_2-6alkynyl”). Unless otherwise specified, each instance of a heteroalkynyl group is independently unsubstituted (“unsubstituted heteroalkynyl”) or substituted (“substituted heteroalkynyl”) with one or more substituents. In certain embodiments, the heteroalkynyl group is unsubstituted heteroC_2-10alkynyl. In certain embodiments, the heteroalkynyl group is substituted heteroC_2-10alkynyl.

0057“Carbocyclyl” or “carbocyclic” refers to a radical of a non-aromatic cyclic hydrocarbon group having from 3 to 10 ring carbon atoms (“C_3-10carbocyclyl”) and zero heteroatoms in the non-aromatic ring system. In some embodiments, a carbocyclyl group has 3 to 8 ring carbon atoms (“C_3-8carbocyclyl”). In some embodiments, a carbocyclyl group has 3 to 6 ring carbon atoms (“C_3-6carbocyclyl”). In some embodiments, a carbocyclyl group has 3 to 6 ring carbon atoms (“C_3-6carbocyclyl”). In some embodiments, a carbocyclyl group has 5 to 10 ring carbon atoms (“C_5-10carbocyclyl”). Exemplary C_3-6carbocyclyl groups include, without limitation, cyclopropyl (C₃), cyclopropenyl (C₃), cyclobutyl (C₄), cyclobutenyl (C₄), cyclopentyl (C₅), cyclopentenyl (C₅), cyclohexyl (C₆), cyclohexenyl (C₆), cyclohexadienyl (C₆), and the like. Exemplary C_3-8carbocyclyl groups include, without limitation, the aforementioned C_3-6carbocyclyl groups as well as cycloheptyl (C₇), cycloheptenyl (C₇), cycloheptadienyl (C₇), cycloheptatrienyl (C₇), cyclooctyl (C₈), cyclooctenyl (C₈), bicyclo[2.2.1]heptanyl (C₇), bicyclo[2.2.2]octanyl (C₈), and the like. Exemplary C_3-10carbocyclyl groups include, without limitation, the aforementioned C_3-8carbocyclyl groups as well as cyclononyl (C₉), cyclononenyl (C₉), cyclodecyl (C₁₀), cyclodecenyl (C₁₀), octahydro-1H-indenyl (C₉), decahydronaphthalenyl (C₁₀), spiro[4.5]decanyl (C₁₀), and the like. As the foregoing examples illustrate, in certain embodiments, the carbocyclyl group is either monocyclic (“monocyclic carbocyclyl”) or contain a fused, bridged or spiro ring system such as a bicyclic system (“bicyclic carbocyclyl”) and can be saturated or can be partially unsaturated. “Carbocyclyl” also includes ring systems wherein the carbocyclic ring, as defined above, is fused with one or more aryl or heteroaryl groups wherein the point of attachment is on the carbocyclic ring, and in such instances, the number of carbons continue to designate the number of carbons in the carbocyclic ring system. Unless otherwise specified, each instance of a carbocyclyl group is independently optionally substituted, i.e., unsubstituted (an “unsubstituted carbocyclyl”) or substituted (a “substituted carbocyclyl”) with one or more substituents. In certain embodiments, the carbocyclyl group is unsubstituted C_3-10carbocyclyl. In certain embodiments, the carbocyclyl group is substituted C_3-10carbocyclyl.

0058In some embodiments, “carbocyclyl” is a monocyclic, saturated carbocyclyl group having from 3 to 10 ring carbon atoms (“C_3-10cycloalkyl”). In some embodiments, a cycloalkyl group has 3 to 8 ring carbon atoms (“C_3-8cycloalkyl”). In some embodiments, a cycloalkyl group has 3 to 6 ring carbon atoms (“C_3-6cycloalkyl”). In some embodiments, a cycloalkyl group has 5 to 6 ring carbon atoms (“C_5-6cycloalkyl”). In some embodiments, a cycloalkyl group has 5 to 10 ring carbon atoms (“C_5-10cycloalkyl”). Examples of C_5-6cycloalkyl groups include cyclopentyl (C₅) and cyclohexyl (C₅). Examples of C_3-6cycloalkyl groups include the aforementioned C_5-6cycloalkyl groups as well as cyclopropyl (C₃) and cyclobutyl (C₄). Examples of C_3-8cycloalkyl groups include the aforementioned C_3-6cycloalkyl groups as well as cycloheptyl (C₇) and cyclooctyl (C₈). Unless otherwise specified, each instance of a cycloalkyl group is independently unsubstituted (an “unsubstituted cycloalkyl”) or substituted (a “substituted cycloalkyl”) with one or more substituents. In certain embodiments, the cycloalkyl group is unsubstituted C_3-10cycloalkyl. In certain embodiments, the cycloalkyl group is substituted C_3-10cycloalkyl.

0059“Heterocyclyl” or “heterocyclic” refers to a radical of a 3- to 10-membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon (“3-10 membered heterocyclyl”). In heterocyclyl groups that contain one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valency permits. A heterocyclyl group can either be monocyclic (“monocyclic heterocyclyl”) or a fused, bridged, or Spiro ring system, such as a bicyclic system (“bicyclic heterocyclyl”), and can be saturated or can be partially unsaturated. Heterocyclyl bicyclic ring systems can include one or more heteroatoms in one or both rings. “Heterocyclyl” also includes ring systems wherein the heterocyclic ring, as defined above, is fused with one or more carbocyclyl groups wherein the point of attachment is either on the carbocyclyl or heterocyclic ring, or ring systems wherein the heterocyclic ring, as defined above, is fused with one or more aryl or heteroaryl groups, wherein the point of attachment is on the heterocyclic ring, and in such instances, the number of ring members continue to designate the number of ring members in the heterocyclic ring system. Unless otherwise specified, each instance of heterocyclyl is independently optionally substituted, i.e., unsubstituted (an “unsubstituted heterocyclyl”) or substituted (a “substituted heterocyclyl”) with one or more substituents. In certain embodiments, the heterocyclyl group is unsubstituted 3-10 membered heterocyclyl. In certain embodiments, the heterocyclyl group is substituted 3-10 membered heterocyclyl.

0060In some embodiments, a heterocyclyl group is a 5-10 membered, non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon (“5-10 membered heterocyclyl”). In some embodiments, a heterocyclyl group is a 5-8 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-8 membered heterocyclyl”). In some embodiments, a heterocyclyl group is a 5-6 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-6 membered heterocyclyl”). In some embodiments, the 5-6 membered heterocyclyl has 1-3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heterocyclyl has 1-2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heterocyclyl has one ring heteroatom selected from nitrogen, oxygen, and sulfur.

0061Exemplary 3-membered heterocyclyl groups containing one heteroatom include, without limitation, azirdinyl, oxiranyl, thiiranyl. Exemplary 4-membered heterocyclyl groups containing one heteroatom include, without limitation, azetidinyl, oxetanyl and thietanyl. Exemplary 5-membered heterocyclyl groups containing one heteroatom include, without limitation, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl, and pyrrolyl-2,5-dione. Exemplary 5-membered heterocyclyl groups containing two heteroatoms include, without limitation, dioxolanyl, oxasulfuranyl, disulfuranyl, and oxazolidin-2-one. Exemplary 5-membered heterocyclyl groups containing three heteroatoms include, without limitation, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclyl groups containing one heteroatom include, without limitation, piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary 6-membered heterocyclyl groups containing two heteroatoms include, without limitation, piperazinyl, morpholinyl, dithianyl, and dioxanyl. Exemplary 6-membered heterocyclyl groups containing three heteroatoms include, without limitation, triazinanyl. Exemplary 7-membered heterocyclyl groups containing one heteroatom include, without limitation, azepanyl, oxepanyl and thiepanyl. Exemplary 8-membered heterocyclyl groups containing one heteroatom include, without limitation, azocanyl, oxecanyl and thiocanyl. Exemplary 5-membered heterocyclyl groups fused to a C₆aryl ring (also referred to herein as a 5,6-bicyclic heterocyclic ring) include, without limitation, indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl, benzoxazolinonyl, and the like. Exemplary 6-membered heterocyclyl groups fused to an aryl ring (also referred to herein as a 6,6-bicyclic heterocyclic ring) include, without limitation, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and the like. Exemplary bicyclic heterocyclyl groups include, without limitation, indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl, tetrahydrobenzothienyl, tetrahydrobenzofuranyl, tetrahydroindolyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, decahydroisoquinolinyl, octahydrochromenyl, octahydroisochromenyl, decahydronaphthyridinyl, decahydro-1,8-naphthyridinyl, octahydropyrrolo[3,2-b]pyrrole, indolinyl, phthalimidyl, naphthalimidyl, chromanyl, chromenyl, 1H-benzo[e][1,4]diazepinyl, 1,4,5,7-tetrahydropyrano[3,4-b]pyrrolyl, 5,6-dihydro-4H-furo[3,2-b]pyrrolyl, 6,7-dihydro-5H-furo[3,2-b]pyranyl, 5,7-dihydro-4H-thieno[2,3-c]pyranyl, 2,3-dihydro-1H-pyrrolo[2,3-b]pyridinyl, 2,3-dihydrofuro[2,3-b]pyridinyl, 4,5,6,7-tetrahydro-1H-pyrrolo[2,3-b]pyridinyl, 4,5,6,7-tetrahydrofuro[3,2-c]pyridinyl, 4,5,6,7-tetrahydrothieno[3,2-b]pyridinyl, 1,2,3,4-tetrahydro-1,6-naphthyridinyl, and the like.

0062“Aryl” refers to a radical of a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 pi electrons shared in a cyclic array) having 6-14 ring carbon atoms and zero heteroatoms provided in the aromatic ring system (“C_6-14aryl”). In some embodiments, an aryl group has six ring carbon atoms (“C₆aryl”; e.g., phenyl). In some embodiments, an aryl group has ten ring carbon atoms (“C₁₀aryl”; e.g., naphthyl such as 1-naphthyl and 2-naphthyl). In some embodiments, an aryl group has fourteen ring carbon atoms (“C₁₄aryl”; e.g., anthracyl). “Aryl” also includes ring systems wherein the aryl ring, as defined above, is fused with one or more carbocyclyl or heterocyclyl groups, wherein the radical or point of attachment is on the aryl ring, and in such instances, the number of carbon atoms continue to designate the number of carbon atoms in the aryl ring system. Unless otherwise specified, each instance of an aryl group is independently optionally substituted, i.e., unsubstituted (an “unsubstituted aryl”) or substituted (a “substituted aryl”) with one or more substituents. In certain embodiments, the aryl group is unsubstituted C_6-14aryl. In certain embodiments, the aryl group is substituted C_6-14aryl.

0063“Aralkyl” refers to an optionally substituted alkyl group substituted by an optionally substituted aryl group. “Aralkyl” is a subset of “alkyl” and refers to an alkyl group substituted with an aryl group, wherein the point of attachment is on the alkyl group. In certain embodiments, the aralkyl is optionally substituted benzyl. In certain embodiments, the aralkyl is benzyl. In certain embodiments, the aralkyl is optionally substituted phenethyl. In certain embodiments, the aralkyl is phenethyl.

0064“Heteroaryl” refers to a radical of a 5-10 membered, monocyclic or bicyclic 4n+2 aromatic ring system (e.g., having 6 or 10 pi electrons shared in a cyclic array) having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen and sulfur (“5-10 membered heteroaryl”). In heteroaryl groups that contain one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valency permits. Heteroaryl bicyclic ring systems can include one or more heteroatoms in one or both rings. “Heteroaryl” includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or more carbocyclyl or heterocyclyl groups wherein the point of attachment is on the heteroaryl ring, and in such instances, the number of ring members continue to designate the number of ring members in the heteroaryl ring system. “Heteroaryl” also includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or more aryl groups wherein the point of attachment is either on the aryl or heteroaryl ring, and in such instances, the number of ring members designates the number of ring members in the fused (aryl/heteroaryl) ring system. Bicyclic heteroaryl groups wherein one ring does not contain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl, and the like) the point of attachment can be on either ring, i.e., either the ring bearing a heteroatom (e.g., 2-indolyl) or the ring that does not contain a heteroatom (e.g., 5-indolyl).

0065In some embodiments, a heteroaryl group is a 5-10 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-10 membered heteroaryl”). In some embodiments, a heteroaryl group is a 5-8 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-8 membered heteroaryl”). In some embodiments, a heteroaryl group is a 5-6 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-6 membered heteroaryl”). In some embodiments, the 5-6 membered heteroaryl has 1-3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heteroaryl has 1-2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heteroaryl has 1 ring heteroatom selected from nitrogen, oxygen, and sulfur. Unless otherwise specified, each instance of a heteroaryl group is independently optionally substituted, i.e., unsubstituted (an “unsubstituted heteroaryl”) or substituted (a “substituted heteroaryl”) with one or more substituents. In certain embodiments, the heteroaryl group is unsubstituted 5-14 membered heteroaryl. In certain embodiments, the heteroaryl group is substituted 5-14 membered heteroaryl.

0066Exemplary 5-membered heteroaryl groups containing one heteroatom include, without limitation, pyrrolyl, furanyl, and thiophenyl. Exemplary 5-membered heteroaryl groups containing two heteroatoms include, without limitation, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary 5-membered heteroaryl groups containing three heteroatoms include, without limitation, triazolyl, oxadiazolyl, and thiadiazolyl. Exemplary 5-membered heteroaryl groups containing four heteroatoms include, without limitation, tetrazolyl. Exemplary 6-membered heteroaryl groups containing one heteroatom include, without limitation, pyridinyl. Exemplary 6-membered heteroaryl groups containing two heteroatoms include, without limitation, pyridazinyl, pyrimidinyl, and pyrazinyl. Exemplary 6-membered heteroaryl groups containing three or four heteroatoms include, without limitation, triazinyl and tetrazinyl, respectively. Exemplary 7-membered heteroaryl groups containing one heteroatom include, without limitation, azepinyl, oxepinyl, and thiepinyl. Exemplary 5,6-bicyclic heteroaryl groups include, without limitation, indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzoisofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, benzthiazolyl, benzisothiazolyl, benzthiadiazolyl, indolizinyl, and purinyl. Exemplary 6,6-bicyclic heteroaryl groups include, without limitation, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl. Exemplary tricyclic heteroaryl groups include, without limitation, phenanthridinyl, dibenzofuranyl, carbazolyl, acridinyl, phenothiazinyl, phenoxazinyl, and phenazinyl.

0067“Heteroaralkyl” is a subset of alkyl and heteroaryl and refers to an optionally substituted alkyl group substituted by an optionally substituted heteroaryl group.

0068“Unsaturated” or “partially unsaturated” refers to a group that includes at least one double or triple bond. A “partially unsaturated” ring system is further intended to encompass rings having multiple sites of unsaturation, but is not intended to include aromatic groups (e.g., aryl or heteroaryl groups). Likewise, “saturated” refers to a group that does not contain a double or triple bond, i.e., contains all single bonds.

0069The term “unsaturated bond” refers to a double or triple bond.

0070The term “unsaturated” or “partially unsaturated” refers to a moiety that includes at least one double or triple bond.

0071The term “saturated” refers to a moiety that does not contain a double or triple bond, i.e., the moiety only contains single bonds.

0072Alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl groups, which are divalent linking groups, are further referred to using the suffix -ene, e.g., alkylene, alkenylene, alkynylene, carbocyclylene, heterocyclylene, arylene, and heteroarylene. Thus, alkylene is the divalent moiety of alkyl, alkenylene is the divalent moiety of alkenyl, alkynylene is the divalent moiety of alkynyl, heteroalkylene is the divalent moiety of heteroalkyl, heteroalkenylene is the divalent moiety of heteroalkenyl, heteroalkynylene is the divalent moiety of heteroalkynyl, carbocyclylene is the divalent moiety of carbocyclyl, heterocyclylene is the divalent moiety of heterocyclyl, arylene is the divalent moiety of aryl, and heteroarylene is the divalent moiety of heteroaryl.

0073An atom, moiety, or group described herein may be unsubstituted or substituted, as valency permits, unless otherwise provided expressly. The term “optionally substituted” refers to substituted or unsubstituted.

0074A group is optionally substituted unless expressly provided otherwise. The term “optionally substituted” refers to being substituted or unsubstituted. In certain embodiments, alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl groups are optionally substituted (e.g., “substituted” or “unsubstituted” alkyl, “substituted” or “unsubstituted” alkenyl, “substituted” or “unsubstituted” alkynyl, “substituted” or “unsubstituted” carbocyclyl, “substituted” or “unsubstituted” heterocyclyl, “substituted” or “unsubstituted” aryl or “substituted” or “unsubstituted” heteroaryl group). In general, the term “substituted”, whether preceded by the term “optionally” or not, means that at least one hydrogen present on a group (e.g., a carbon or nitrogen atom) is replaced with a permissible substituent, e.g., a substituent which upon substitution results in a stable compound, e.g., a compound which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, or other reaction. Unless otherwise indicated, a “substituted” group has a substituent at one or more substitutable positions of the group, and when more than one position in any given structure is substituted, the substituent is either the same or different at each position. The term “substituted” is contemplated to include substitution with all permissible substituents of organic compounds, any of the substituents described herein that results in the formation of a stable compound. The present disclosure contemplates any and all such combinations in order to arrive at a stable compound. For purposes of this disclosure, heteroatoms such as nitrogen may have hydrogen substituents and/or any suitable substituent as described herein which satisfy the valencies of the heteroatoms and results in the formation of a stable moiety. In certain embodiments, the substituent is a carbon atom substituent. In certain embodiments, the substituent is a nitrogen atom substituent. In certain embodiments, the substituent is an oxygen atom substituent. In certain embodiments, the substituent is a sulfur atom substituent.

0075Exemplary carbon atom substituents include, but are not limited to, halogen, —CN, —NO₂, —N₃, —SO₂H, —SO₃H, —OH, —OR^aa, —ON(R^bb)₂, —N(R^bb)₂, —N(R^bb)₃⁺X⁻, —N(OR^cc)R^bb, —SH, —SR^aa, —SSR^cc, —C(═O)R^aa, —CO₂H, —CHO, —C(OR^cc)₂, —CO₂R^aa, —OC(═O)R^aa, —OCO₂R^aa, —C(═O)N(R^bb)₂, —OC(═O)N(R^bb)₂, —NR^bbC(═O)R^aa, —NR^bbCO₂R^aa, —NR^bbC(═O)N(R^bb)₂, —C(═NR^bb)R^aa, —C(═NR^bb)OR^aa, —OC(═NR^bb)R^aa, —OC(═NR^bb)OR^aa, —C(═NR^bb)N(R^bb)₂, —OC(═NR^bb)N(R^bb)₂, —NR^bbC(═NR^bb)N(R^bb)₂, —C(═O)NR^bbSO₂R^aa, —NR^bbSO₂R^aa, —SO₂N(R^bb)₂, —SO₂R^aa, —SO₂OR^aa, —OSO₂R^aa, —S(═O)R^aa, —OS(═O)R^aa, —Si(R^aa)₃, —OSi(R^aa)₃, —C(═S)N(R^bb)₂, —C(═O)SR^aa, —C(═S)SR^aa, —SC(═S)SR^aa, —SC(═O)SR^aa, —OC(═O)SR^aa, —SC(═O)OR^aa, —SC(═O)R^aa, —P(═O)(R^aa)₂, —P(═O)(OR^cc)₂, —OP(═O)(R^aa)₂, —OP(═O)(OR^cc)₂, —P(═O)(N(R^bb)₂)₂, —OP(═O)(N(R^bb)₂)₂, NR^bbP(═O)(R^aa)₂, —NR^bbP(O)(OR^cc)₂, —NR^bbP(═O)(N(R^bb)₂)₂, —P(R^cc)₂, —P(OR^cc)₂, —P(R^cc)₃⁺X⁻, —P(OR^cc)₃⁺X⁻, —P(R^cc)₄, —P(OR^cc)₄, —OP(R^cc)₂, —OP(R^cc)₃⁺X⁻, —OP(OR^cc)₂, —OP(OR^cc)₃⁺X⁻, —OP(R^cc)₄, —OP(OR^cc)₄, —B(R^aa)₂, —B(OR^cc)₂, —BR^aa(OR^cc), C_1-10alkyl, C_1-10perhaloalkyl, C_2-10alkenyl, C_2-10alkynyl, heteroC_1-10alkyl, heteroC_2-10alkenyl, heteroC_2-10alkynyl, C_3-10carbocyclyl, 3-14 membered heterocyclyl, C_6-14aryl, and 5-14 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R^ddgroups; or two geminal hydrogens on a carbon atom are replaced with the group ═O, ═S, ═NN(R^bb)₂, ═NNR^bbC(═O)R^aa, ═NNR^bbC(═O)OR^aa, ═NNR^bbS(═O)₂R^aa, ═NR^bb, or ═NOR^cc; wherein X⁻ is a counterion;

0076each instance of R^aais, independently, selected from C_1-10alkyl, C_1-10perhaloalkyl, C_2-10alkenyl, C_2-10alkynyl, C_3-10carbocyclyl, 3-14 membered heterocyclyl, C_6-14aryl, and 5-14 membered heteroaryl, or two R^aagroups are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R^ddgroups;

0077each instance of R^bbis, independently, selected from hydrogen, —OH, —OR^aa, —N(R^cc)₂, —CN, —C(═O)R^aa, —C(═O)N(R^cc)₂, —CO₂R^aa, —SO₂R^aa, —C(═NR^cc)OR^aa, —C(═NR^cc)N(R^cc)₂, —SO₂N(R^cc)₂, —SO₂R^cc, —SO₂OR^cc, —SOR^aa, —C(═S)N(R^cc)₂, —C(═O)SR^cc, —C(═S)SR^cc, —P(═O)(R^aa)₂, —P(═O)(OR^cc)₂, —P(═O)N(R^cc)₂)₂, C_1-10alkyl, C_1-10perhaloalkyl, C_2-10alkenyl, C_2-10alkynyl, heteroC_1-10alkyl, heteroC_2-10alkenyl, heteroC_2-10alkynyl, C_3-10carbocyclyl, 3-14 membered heterocyclyl, C_6-14aryl, and 5-14 membered heteroaryl, or two R^bbgroups are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R^ddgroups; wherein X is a counterion;

0078each instance of R^ccis, independently, selected from hydrogen, C_1-10alkyl, C_1-10perhaloalkyl, C_2-10alkenyl, C_2-10alkynyl, C_3-10carbocyclyl, 3-14 membered heterocyclyl, C_6-14aryl, and 5-14 membered heteroaryl, or two R^ccgroups are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R^ddgroups;

0079each instance of R^ddis, independently, selected from halogen, —CN, —NO₂, —N₃, —SO₂H, —SO₃H, —OH, —OR^ee, —ON(R^ff)₂, —N(R^ff)₂, —N(R^ff)₃⁺X⁻, —N(OR^ee)R^ff, —SH, —SR^ee, —SSR^ee, —C(═O)R^ee, —CO₂H, —CO₂R^ee, —OC(═O)R^ee, —OCO₂R^ee, —C(═O)N(R^ff)₂, —OC(═O)N(R^ff)₂, —NR^ffC(═O)R^ee, —NR^ffCO₂R^ee, —NR^ffC(═O)N(R^ff)₂, —C(═NR^ff)OR^ee, —OC(═NR^ff)R^ee, —OC(═NR^ff)OR^ee, —C(═NR^ff)N(R^ff)₂, —OC(═NR^ff)N(R^ff)₂, —NR^ffC(═NR^ff)N(R^ff)₂, —NR^ffSO₂R^ee, —SO₂N(R^ff)₂, —SO₂R^ee, —SO₂OR^ee, —OSO₂R^ee, —S(═O)R^ee, —Si(R^ee)₃, —OSi(R^ee)₃, —C(═S)N(R^ff)₂, —C(═O)SR^ee, —C(═S)SR^ee, —SC(═S)SR^ee, —P(═O)(OR^ee)₂, —P(═O)(R^ee)₂, —OP(═O)(R^ee)₂, —OP(═O)(OR^ee)₂, C_1-6alkyl, C_1-6perhaloalkyl, C_2-6alkenyl, C_2-6alkynyl, C_3-10carbocyclyl, 3-10 membered heterocyclyl, C_6-10aryl, 5-10 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R^gggroups, or two geminal R^ddsubstituents can be joined to form ═O or ═S; wherein X⁻ is a counterion;

0080each instance of R^eeis, independently, selected from C_1-6alkyl, C_1-6perhaloalkyl, C_2-6alkenyl, C_2-6alkynyl, C_3-10carbocyclyl, C_6-10aryl, 3-10 membered heterocyclyl, and 3-10 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R^gggroups;

0081each instance of 0 is, independently, selected from hydrogen, C_1-6alkyl, C_1-6perhaloalkyl, C_2-6alkenyl, C_2-6alkynyl, C_3-10carbocyclyl, 3-10 membered heterocyclyl, C_6-10aryl and 5-10 membered heteroaryl, or two 0 groups are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R^gggroups; and

0082each instance of R^ggis, independently, halogen, —CN, —NO₂, —N₃, —SO₂H, —SO₃H, —OH, —OC_1-6alkyl, —ON(C_1-6alkyl)₂, —N(C_1-6alkyl)₂, —N(C_1-6alkyl)₃⁺X⁻, —NH(C_1-6alkyl)₂⁺X⁻, —NH₂(C_1-6alkyl)⁺X⁻, —NH₃⁺X⁻, —N(OC_1-6alkyl)(C_1-6alkyl), —N(OH)(C_1-6alkyl), —NH(OH), —SH, —SC_1-6alkyl, —SS(C_1-6alkyl), —C(═O)(C_1-6alkyl), —CO₂H, —CO₂(C_1-6alkyl), —OC(═O)(C_1-6alkyl), —OCO₂(C_1-6alkyl), —C(═O)NH₂, —C(═O)N(C_1-6alkyl)₂, —OC(═O)NH(C_1-6alkyl), —NHC(═O)(C_1-6alkyl), —N(C_1-6alkyl)C(═O)(C_1-6alkyl), —NHCO₂(C_1-6alkyl), —NHC(═O)N(C_1-6alkyl)₂, —NHC(═O)NH(C_1-6alkyl), —NHC(═O)NH₂, —C(═NH)O(C_1-6alkyl), —OC(═NH)(C_1-6alkyl), —OC(═NH)OC_1-6alkyl, —C(═NH)N(C_1-6alkyl)₂, —C(═NH)NH(C_1-6alkyl), —C(═NH)NH₂, —OC(═NH)N(C_1-6alkyl)₂, —OC(NH)NH(C_1-6alkyl), —OC(NH)NH₂, —NHC(NH(C_1-6alkyl)₂, —NHC(═NH)NH₂, —NHSO₂(C_1-6alkyl), —SO₂N(C_1-6alkyl)₂, —SO₂NH(C_1-6alkyl), —SO₂NH₂, —SO₂C_1-6alkyl, —SO₂OC_1-6alkyl, —OSO₂C_1-6alkyl, —SOC_1-6alkyl, —Si(C_1-6alkyl)₃, —OSi(C_1-6alkyl)₃-C(═S)N(C_1-6alkyl)₂, C(═S)NH(C_1-6alkyl), C(═S)NH₂, —C(═O)S(C_1-6alkyl), —C(═S)SC_1-6alkyl, —SC(═S)SC_1-6alkyl, —P(═O)(OC_1-6alkyl)₂, —P(═O)(C_1-6alkyl)₂, —OP(═O)(C_1-6alkyl)₂, —OP(═O)(OC_1-6alkyl)₂, C_1-6alkyl, C_1-6perhaloalkyl, C_2-6alkenyl, C_2-6alkynyl, C_3-10carbocyclyl, C_6-10aryl, 3-10 membered heterocyclyl, 5-10 membered heteroaryl; or two geminal R^ggsubstituents can be joined to form ═O or ═S; wherein X⁻is a counterion.

0083A “counterion” or “anionic counterion” is a negatively charged group associated with a cationic quaternary amino group in order to maintain electronic neutrality. Exemplary counterions include halide ions (e.g., F⁻, Cl⁻, Br⁻, I⁻), NO₃⁻, ClO₄⁻, OH⁻, H₂PO₄⁻, HSO₄⁻, sulfonate ions (e.g., methansulfonate, trifluoromethanesulfonate, p-toluenesulfonate, benzenesulfonate, 10-camphor sulfonate, naphthalene-2-sulfonate, naphthalene-1-sulfonic acid-5-sulfonate, ethan-1-sulfonic acid-2-sulfonate, and the like), carboxylate ions (e.g., acetate, ethanoate, propanoate, benzoate, glycerate, lactate, tartrate, glycolate, and the like). Exemplary counterions further include BF₄⁻, PF₄⁻, PF₆⁻, AsF₆⁻, SbF₆⁻, B[3,5-(CF₃)₂C₆H₃]₄]⁻, B(C₆F₅)₄⁻, BPh₄⁻, Al(OC(CF₃)₃)₄⁻, and carborane anions (e.g., CB₁₁H₁₂⁻ or (HCB₁₁Me₅Br₆)⁻). Exemplary counterions which may be multivalent include CO₃²⁻, HPO₄²⁻, PO₄³, B₄O₇²⁻, SO₄²⁻, S₂O₃²⁻, carboxylate anions (e.g., tartrate, citrate, fumarate, maleate, malate, malonate, gluconate, succinate, glutarate, adipate, pimelate, suberate, azelate, sebacate, salicylate, phthalates, aspartate, glutamate, and the like), and carboranes.

0084“Halo” or “halogen” refers to fluorine (fluoro, —F), chlorine (chloro, —Cl), bromine (bromo, —Br), or iodine (iodo, —I).

0085The term “hydroxyl” or “hydroxy” refers to the group —OH. The term “substituted hydroxyl” or “substituted hydroxyl,” by extension, refers to a hydroxyl group wherein the oxygen atom directly attached to the parent molecule is substituted with a group other than hydrogen, and includes groups selected from —OR^aa, —ON(R^bb)₂, —OC(═O)SR^aa, —OC(═O)R^aa, —OCO₂R^aa, —OC(═O)N(R^bb)₂, —OC(═NR^bb)R^aa, —OC(═NR^bb)OR^aa, —OC(═NR^bb)N(R^bb)₂, —OS(═O)R^aa, —OSO₂R^aa, —OSi(R^aa)₃, —OP(R^cc)₂, —OP(R^cc)₃⁺X⁻, —OP(OR^cc)₂, —OP(OR^cc)₃⁺X⁻, —OP(═O)(R^aa)₂, —OP(═O)(OR^cc)₂, and —OP(═O)(N(R^bb))₂, wherein X⁻, R^aa, R^bb, and R^ccare as defined herein.

0086The term “amino” refers to the group —NH₂. The term “substituted amino,” by extension, refers to a monosubstituted amino, disubstituted amino, or trisubstituted amino. In certain embodiments, the “substituted amino” is a monosubstituted amino or disubstituted amino group.

0087The term “sulfonyl” refers to a group selected from —SO₂N(R^bb)₂, —SO₂R^aa, and —SO₂OR^aa, wherein R^aaand R^bbare as defined herein.

0088The term “sulfinyl” refers to the group —S(═O)R^aa, wherein R^aais as defined herein.

0089“Acyl” refers to a moiety selected from the group consisting of —C(═O)R^aa, —CHO, —CO₂R^aa, —C(═O)N(R^bb)₂, —C(═NR^bb)R^aa, —C(═NR^bb)OR^aa, —C(═NR^bb)N(R^bb)₂, —C(═O)NR^bbSO₂R^aa, —C(═S)N(R^bb)₂, —C(═O)SR^aa, or —C(═S)SR^aa, wherein R^aaand R^bbare as defined herein. In other instances, “acyl” refers to a group having the general formula: —C(═O)R^X1, C(═O)OR^X1, —C(═O)—O—C(═O)R^X1, —C(═O)SR^X1, —C(═O)N(R^X1)₂, —C(═S)R^X1), —C(═S)N(R^X1)₂, —C(═S)S(R^X1), —C(═NR^X1)R^X1, C(═NR^X1)OR^X1, —C(═NR^X1)SR^X1, or —C(═NR^X1)N(R^X1)₂, wherein R^X1is hydrogen; halogen; substituted or unsubstituted hydroxyl; substituted or unsubstituted thiol; substituted or unsubstituted amino; substituted or unsubstituted acyl, cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched alkyl; cyclic or acyclic, substituted or unsubstituted, branched or unbranched alkenyl; substituted or unsubstituted alkynyl; substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, aliphaticoxy, heteroaliphaticoxy, alkyloxy, heteroalkyloxy, aryloxy, heteroaryloxy, aliphaticthioxy, heteroaliphaticthioxy, alkylthioxy, heteroalkylthioxy, arylthioxy, heteroarylthioxy, mono- or di-aliphaticamino, mono- or di-heteroaliphaticamino, mono- or di-alkylamino, mono- or di-heteroalkylamino, mono- or di-arylamino, or mono- or di-heteroarylamino; or two R^X1groups taken together form a 5- to 6-membered heterocyclic ring. Exemplary acyl groups include aldehydes (—CHO), carboxylic acids (—CO₂H), ketones, acyl halides, esters, amides, imines, carbonates, carbamates, and ureas. Acyl substituents include, but are not limited to, any of the substituents described herein, that result in the formation of a stable moiety (e.g., aliphatic, alkyl, alkenyl, alkynyl, heteroaliphatic, heterocyclic, aryl, heteroaryl, acyl, oxo, imino, thiooxo, cyano, isocyano, amino, azido, nitro, hydroxyl, thiol, halo, aliphaticamino, heteroaliphaticamino, alkylamino, heteroalkylamino, arylamino, heteroarylamino, alkylaryl, arylalkyl, aliphaticoxy, heteroaliphaticoxy, alkyloxy, heteroalkyloxy, aryloxy, heteroaryloxy, aliphaticthioxy, heteroaliphaticthioxy, alkylthioxy, heteroalkylthioxy, arylthioxy, heteroarylthioxy, acyloxy, and the like, each of which may or may not be further substituted).

0090The term “carbonyl” refers a group wherein the carbon directly attached to the parent molecule is sp²hybridized, and is substituted with an oxygen, nitrogen, or sulfur atom, e.g., a group selected from ketones (—C(═O)R^aa), carboxylic acids (—CO₂H), aldehydes (—CHO), esters (—CO₂R^aa, —C(═O)SR^aa, —C(═S)SR^aa), amides (—C(═O)N(R^bb)₂, —C(═O)NR^bbSO₂R^aa, —C(═S)N(R^bb)₂), and imines (—C(═NR^bb)R^aa, —C(═NR^bb)OR^aa), —C(═NR^bb)N(R^bb)₂), wherein R^aaand R^bbare as defined herein.

0091The term “silyl” refers to the group —Si(R^aa)₃, wherein R^aais as defined herein.

0092The term “oxo” refers to the group ═O, and the term “thiooxo” refers to the group ═S.

0093Nitrogen atoms can be substituted or unsubstituted as valency permits, and include primary, secondary, tertiary, and quaternary nitrogen atoms. Exemplary nitrogen atom substituents include, but are not limited to, hydrogen, —OH, —OR^aa, —N(R^cc)₂, —CN, —C(═O)R^aa, —C(═O)N(R^cc)₂, —CO₂R^aa, —SO₂R^aa, —C(═NR^bb)R^aa, —C(═NR^cc)OR^aa, —C(═NR^cc)N(R^cc)₂, —SO₂N(R^cc)₂, —SO₂R^cc, —SO₂OR^cc, —SOR^aa, —C(═S)N(R^cc)₂, —C(═O)SR^cc, —C(═S)SR^cc, —P(═O)(OR^cc)₂, —P(═O)(R^aa)₂, —P(═O)(N(R^cc)₂)₂, C_1-10alkyl, C_1-10perhaloalkyl, C_2-10alkenyl, C_2-10alkynyl, C_3-10carbocyclyl, 3-14 membered heterocyclyl, C_6-14aryl, and 5-14 membered heteroaryl, or two R^ccgroups attached to a nitrogen atom are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R^ddgroups, and wherein R^aa, R^bb, R^cc, and R^ddare as defined above.

0094In certain embodiments, the substituent present on a nitrogen atom is a nitrogen protecting group (also referred to as an amino protecting group). Nitrogen protecting groups include, but are not limited to, —OH, —OR^aa, —N(R^cc)₂, —C(═O)R^aa, —C(═O)N(R^cc)₂, —CO₂R^aa, —SO₂R^aa, —C(═NR^cc)R^aa, —C(═NR^cc)OR^aa, —C(═NR^cc)N(R^cc)₂, —SO₂N(R^cc)₂, —SO₂R^cc, —SO₂OR^cc, —SOR^aa, —C(═S)N(R^cc)₂, —C(═O)SR″, —C(═S)SR^cc, C_1-10alkyl (e.g., aralkyl, heteroaralkyl), C_2-10alkenyl, C_2-10alkynyl, C_3-10carbocyclyl, 3-14 membered heterocyclyl, C_6-14aryl, and 5-14 membered heteroaryl groups, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aralkyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R^ddgroups, and wherein R^aa, R^bb, R^ccand are as defined herein. Nitrogen protecting groups are well R^ddknown in the art and include those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3^rdedition, John Wiley &amp; Sons, 1999, incorporated herein by reference.

0095For example, nitrogen protecting groups such as amide groups (e.g., —C(═O)R^aa) include, but are not limited to, formamide, acetamide, chloroacetamide, trichloroacetamide, trifluoroacetamide, phenylacetamide, 3-phenylpropanamide, picolinamide, 3-pyridylcarboxamide, N-benzoylphenylalanyl derivative, benzamide, p-phenylbenzamide, o-nitrophenylacetamide, o-nitrophenoxyacetamide, acetoacetamide, (N′-dithiobenzyloxyacylamino)acetamide, 3-(p-hydroxyphenyl)propanamide, 3-(o-nitrophenyl)propanamide, 2-methyl-2-(o-nitrophenoxy)propanamide, 2-methyl-2-(o-phenylazophenoxy)propanamide, 4-chlorobutanamide, 3-methyl-3-nitrobutanamide, o-nitrocinnamide, N-acetylmethionine derivative, o-nitrobenzamide, and o-(benzoyloxymethyl)benzamide.

0096Nitrogen protecting groups such as carbamate groups (e.g., —C(═O)OR^aa) include, but are not limited to, methyl carbamate, ethyl carbamate, 9-fluorenylmethyl carbamate (Fmoc), 9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluorenylmethyl carbamate, 2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methyl carbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc), 2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate (Teoc), 2-phenylethyl carbamate (hZ), 1-(1-adamantyl)-1-methylethyl carbamate (Adpoc), 1,1-dimethyl-2-haloethyl carbamate, 1,1-dimethyl-2,2-dibromoethyl carbamate (DB-t-BOC), 1,1-dimethyl-2,2,2-trichloroethyl carbamate (TCBOC), 1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc), 1-(3,5-di-t-butylphenyl)-1-methylethyl carbamate (t-Bumeoc), 2-(2′- and 4′-pyridyl)ethyl carbamate (Pyoc), 2-(N,N-dicyclohexylcarboxamido)ethyl carbamate, t-butyl carbamate (BOC or Boc), 1-adamantyl carbamate (Adoc), vinyl carbamate (Voc), allyl carbamate (Alloc), 1-isopropylallyl carbamate (Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc), 8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, alkyldithio carbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz), p-nitobenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzyl carbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzyl carbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate, 2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate, 2-(p-toluenesulfonyl)ethyl carbamate, [2-(1,3-dithianyl)]methyl carbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc), 2,4-dimethylthiophenyl carbamate (Bmpc), 2-phosphonioethyl carbamate (Peoc), 2-triphenylphosphonioisopropyl carbamate (Ppoc), 1,1-dimethyl-2-cyanoethyl carbamate, m-chloro-p-acyloxybenzyl carbamate, p-(dihydroxyboryl)benzyl carbamate, 5-benzisoxazolylmethyl carbamate, 2-(trifluoromethyl)-6-chromonylmethyl carbamate (Tcroc), m-nitrophenyl carbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate, 3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl(o-nitrophenyl)methyl carbamate, t-amyl carbamate, S-benzyl thiocarbamate, p-cyanobenzyl carbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentyl carbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate, 2,2-dimethoxyacylvinyl carbamate, o-(N,N-dimethylcarboxamido)benzyl carbamate, 1,1-dimethyl-3-(N,N-dimethylcarboxamido)propyl carbamate, 1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate, 2-furanylmethyl carbamate, 2-iodoethyl carbamate, isoborynl carbamate, isobutyl carbamate, isonicotinyl carbamate, p-(p′-methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl carbamate, 1-methylcyclohexyl carbamate, 1-methyl-1-cyclopropylmethyl carbamate, 1-methyl-1-(3,5-dimethoxyphenyl)ethyl carbamate, 1-methyl-1-(p-phenylazophenyl)ethyl carbamate, 1-methyl-1-phenylethyl carbamate, 1-methyl-1-(4-pyridyl)ethyl carbamate, phenyl carbamate, p-(phenylazo)benzyl carbamate, 2,4,6-tri-t-butylphenyl carbamate, 4-(trimethylammonium)benzyl carbamate, and 2,4,6-trimethylbenzyl carbamate.

0097Nitrogen protecting groups such as sulfonamide groups (e.g., —S(═O)₂R^aa) include, but are not limited to, p-toluenesulfonamide (Ts), benzenesulfonamide, 2,3,6,-trimethyl-4-methoxybenzenesulfonamide (Mtr), 2,4,6-trimethoxybenzenesulfonamide (Mtb), 2,6-dimethyl-4-methoxybenzenesulfonamide (Pme), 2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte), 4-methoxybenzenesulfonamide (Mbs), 2,4,6-trimethylbenzenesulfonamide (Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds), 2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide (Ms), β-trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide, 4-(4′,8′-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS), benzylsulfonamide, trifluoromethylsulfonamide, and phenacylsulfonamide.

0098Other nitrogen protecting groups include, but are not limited to, phenothiazinyl-(10)-acyl derivative, N-p-toluenesulfonylaminoacyl derivative, N′-phenylaminothioacyl derivative, N-benzoylphenylalanyl derivative, N-acetylmethionine derivative, 4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiasuccinimide (Dts), N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole, N-1,1,4,4-tetramethyldisilylazacyclopentane adduct (STABASE), 5-substituted 1,3-dimethyl-1,3,5-triazacyclohexan-2-one, 5-substituted 1,3-dibenzyl-1,3,5-triazacyclohexan-2-one, 1-substituted 3,5-dinitro-4-pyridone, N-methylamine, N-allylamine, N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine, N-(1-isopropyl-4-nitro-2-oxo-3-pyroolin-3-yl)amine, quaternary ammonium salts, N-benzylamine, N-di(4-methoxyphenyl)methylamine, N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr), N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr), N-9-phenylfluorenylamine (PhF), N-2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fcm), N-2-picolylamino N′-oxide, N-1,1-dimethylthiomethyleneamine, N-benzylideneamine, N-p-methoxybenzylideneamine, N-diphenylmethyleneamine, N-[(2-pyridyl)mesityl]methyleneamine, N—(N′,N-dimethylaminomethylene)amine, N,N-isopropylidenediamine, N-p-nitrobenzylideneamine, N-salicylideneamine, N-5-chlorosalicylideneamine, N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine, N-cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-1-cyclohexenyl)amine, N-borane derivative, N-diphenylborinic acid derivative, N-[phenyl(pentaacylchromium- or tungsten)acyl]amine, N-copper chelate, N-zinc chelate, N-nitroamine, N-nitrosoamine, amine N-oxide, diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt), diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates, dibenzyl phosphoramidate, diphenyl phosphoramidate, benzenesulfenamide, o-nitrobenzenesulfenamide (Nps), 2,4-dinitrobenzenesulfenamide, pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide, triphenylmethylsulfenamide, and 3-nitropyridinesulfenamide (Npys).

0099Exemplary oxygen atom substituents include, but are not limited to, —R^aa, —C(═O)SR^aa, —C(═O)R^aa, —CO₂R^aa, —C(═O)N(R^bb)₂, —C(—NR^bb)R^aa, —C(═NR^bb)OR^aa, —C(═NR^bb)N(R^bb)₂, —S(═O)R^aa, —SO₂R^aa, —Si(R^aa)₃, —P(R^cc)₂, —P(R^cc)₃⁺X⁻, —P(OR^cc)₂, —P(OR^cc)₃⁺X⁻, —P(═O)(R^aa)₂, —P(═O)(OR^cc)₂, and —P(═O)(N(R^bb)₂)₂, wherein X⁻, R^aa, R^bb, and R^ccare as defined herein. In certain embodiments, the oxygen atom substituent present on an oxygen atom is an oxygen protecting group (also referred to as a hydroxyl protecting group). Oxygen protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3^rdedition, John Wiley &amp; Sons, 1999, incorporated herein by reference. Exemplary oxygen protecting groups include, but are not limited to, methyl, t-butyloxycarbonyl (BOC or Boc), methoxylmethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl, (phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM), p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM), guaiacolmethyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM), siloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR), tetrahydropyranyl (THP), 3-bromotetrahydropyranyl, tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl (MTHP), 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranyl S,S-dioxide, 1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl (CTMP), 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl, 2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl, 1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl, 2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2-(phenylselenyl)ethyl, t-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl, benzyl (Bn), p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolyl N-oxido, diphenylmethyl, p,p′-dinitrobenzhydryl, 5-dibenzosuberyl, triphenylmethyl, a-naphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)phenylmethyl, tri(p-methoxyphenyl)methyl, 4-(4′-bromophenacyloxyphenyl)diphenylmethyl, 4,4′,4″-tris(4,5-dichlorophthalimidophenyl)methyl, 4,4′,4″-tris(levulinoyloxyphenyl)methyl, 4,4′,4″-tris(benzoyloxyphenyl)methyl, 3-(imidazol-1-yl)bis(4′,4″-dimethoxyphenyl)methyl, 1,1-bis(4-methoxyphenyl)-1′-pyrenylmethyl, 9-anthryl, 9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl, 1,3-benzodisulfuran-2-yl, benzisothiazolyl S,S-dioxido, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS), dimethylthexylsilyl, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formate, benzoylformate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate, 3-phenylpropionate, 4-oxopentanoate (levulinate), 4,4-(ethylenedithio)pentanoate (levulinoyldithioacetal), pivaloate, adamantoate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate, 2,4,6-trimethylbenzoate (mesitoate), alkyl methyl carbonate, 9-fluorenylmethyl carbonate (Fmoc), alkyl ethyl carbonate, alkyl 2,2,2-tfichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl carbonate (TMSEC), 2-(phenylsulfonyl) ethyl carbonate (Psec), 2-(triphenylphosphonio) ethyl carbonate (Peoc), alkyl isobutyl carbonate, alkyl vinyl carbonate alkyl allyl carbonate, alkyl p-nitrophenyl carbonate, alkyl benzyl carbonate, alkyl p-methoxybenzyl carbonate, alkyl 3,4-dimethoxybenzyl carbonate, alkyl o-nitrobenzyl carbonate, alkyl p-nitrobenzyl carbonate, alkyl S-benzyl thiocarbonate, 4-ethoxy-1-napththyl carbonate, methyl dithiocarbonate, 2-iodobenzoate, 4-azidobutyrate, 4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate, 2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl, 4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate, 2,6-dichloro-4-methylphenoxyacetate, 2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate, 2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate, isobutyrate, monosuccinoate, (E)-2-methyl-2-butenoate, o-(methoxyacyl)benzoate, a-naphthoate, nitrate, alkyl N,N,N′,N′-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate, borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate, sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate (Ts).

0100Exemplary sulfur atom substituents include, but are not limited to, —R^aa, —C(═O)SR^aa, —C(═O)R^aa, —CO₂R^aa, —C(═O)N(R^bb)₂, —C(═NR^bb)R^aa, —C(═NR^bb)OR^aa, —C(═NR^bb)N(R^bb)₂, —S(═O)R^aa, —SO₂R^aa, —Si(R^aa)₃, —P(R^cc)₂, —P(R^cc)₃, —P(R^cc)₃⁺X⁻, —P(OR^cc)₂, —(OR^cc)₃⁺X⁻, —P(═O)(R^aa)₂, —P(═O)(OR^cc)₂, and —P(═O)(N(R^bb)₂)₂, wherein R^aa, R^bb, and R^ccare as defined herein. In certain embodiments, the sulfur atom substituent present on a sulfur atom is a sulfur protecting group (also referred to as a thiol protecting group). Sulfur protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3^rdedition, John Wiley &amp; Sons, 1999, incorporated herein by reference.

0101A “hydrocarbon chain” refers to a substituted or unsubstituted divalent alkyl, alkenyl, or alkynyl group. A hydrocarbon chain includes (1) one or more chains of carbon atoms immediately between the two radicals of the hydrocarbon chain; (2) optionally one or more hydrogen atoms on the chain(s) of carbon atoms; and (3) optionally one or more substituents (“non-chain substituents,” which are not hydrogen) on the chain(s) of carbon atoms. A chain of carbon atoms consists of consecutively connected carbon atoms (“chain atoms” or “carbon units”) and does not include hydrogen atoms or heteroatoms. However, a non-chain substituent of a hydrocarbon chain may include any atoms, including hydrogen atoms, carbon atoms, and heteroatoms. For example, hydrocarbon chain —C^AH(C^BH₂C^CH₃)— includes one chain atom C^A, one hydrogen atom on C^A, and non-chain substituent —(C^BH₂C^CH₃). The term “C_xhydrocarbon chain,” wherein x is a positive integer, refers to a hydrocarbon chain that includes x number of chain atom(s) between the two radicals of the hydrocarbon chain. If there is more than one possible value of x, the smallest possible value of x is used for the definition of the hydrocarbon chain. For example, —CH(C₂H₅)— is a C₁hydrocarbon chain, and

0102<chemistry id="CHEM-US-00005" num="00005"><img file="USRE50776E_D0008.tif" /></chemistry><br /> is a C₃hydrocarbon chain. When a range of values is used, the meaning of the range is as described herein. For example, a C_3-10hydrocarbon chain refers to a hydrocarbon chain where the number of chain atoms of the shortest chain of carbon atoms immediately between the two radicals of the hydrocarbon chain is 3, 4, 5, 6, 7, 8, 9, or 10. A hydrocarbon chain may be saturated (e.g., —(CH₂)₄—). A hydrocarbon chain may also be unsaturated and include one or more C═C and/or C═C bonds anywhere in the hydrocarbon chain. For instance, —CH═CH—(CH₂)₂—, —CH₂—C═C—CH₂—, and —C═C—CH═CH— are all examples of a unsubstituted and unsaturated hydrocarbon chain. In certain embodiments, the hydrocarbon chain is unsubstituted (e.g., —C═C— or —(CH₂)₄—). In certain embodiments, the hydrocarbon chain is substituted (e.g., —CH(C₂H₅)— and —CF₂—). Any two substituents on the hydrocarbon chain may be joined to form an optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl ring. For instance,

0103<chemistry id="CHEM-US-00006" num="00006"><img file="USRE50776E_D0009.tif" /></chemistry><br /> are all examples of a hydrocarbon chain. In contrast, in certain embodiments,

0104<chemistry id="CHEM-US-00007" num="00007"><img file="USRE50776E_D0010.tif" /></chemistry><br /> are not within the scope of the hydrocarbon chains described herein. When a chain atom of a C_xhydrocarbon chain is replaced with a heteroatom, the resulting group is referred to as a C_xhydrocarbon chain wherein a chain atom is replaced with a heteroatom, as opposed to a C_x-1hydrocarbon chain. For example,

0105<chemistry id="CHEM-US-00008" num="00008"><img file="USRE50776E_D0011.tif" /></chemistry><br /> is a C₃hydrocarbon chain wherein one chain atom is replaced with an oxygen atom.

0106The term “leaving group” is given its ordinary meaning in the art of synthetic organic chemistry and refers to an atom or a group capable of being displaced by a nucleophile. Examples of suitable leaving groups include, but are not limited to, halogen (such as F, Cl, Br, or I (iodine)), alkoxycarbonyloxy, aryloxycarbonyloxy, alkanesulfonyloxy, arenesulfonyloxy, alkyl-carbonyloxy (e.g., acetoxy), arylcarbonyloxy, aryloxy, methoxy, N,O-dimethylhydroxylamino, pixyl, and haloformates. In some cases, the leaving group is a sulfonic acid ester, such as toluenesulfonate (tosylate, —OTs), methanesulfonate (mesylate, —OMs), p-bromobenzenesulfonyloxy (brosylate, —OBs), —OS(═O)₂(CF₂)₃CF₃(nonaflate, —ONf), or trifluoromethanesulfonate (triflate, —OTf). In some cases, the leaving group is a brosylate, such as p-bromobenzenesulfonyloxy. In some cases, the leaving group is a nosylate, such as 2-nitrobenzenesulfonyloxy. The leaving group may also be a phosphineoxide (e.g., formed during a Mitsunobu reaction) or an internal leaving group such as an epoxide or cyclic sulfate. Other non-limiting examples of leaving groups are water, ammonia, alcohols, ether moieties, thioether moieties, zinc halides, magnesium moieties, diazonium salts, and copper moieties. Other exemplary leaving groups include, but are not limited to, activated substituted hydroxyl groups (e.g., —OC(═O)SR^aa, —OC(═O)R^aa, —OCO₂R^aa, —OC(═O)N(R^bb)₂, —OC(═NR^bb)R^aa, —OC(═NR^bb)OR^aa, —OC(═NR^bb)N(R^bb)₂, —OS(═O)R^aa, —OSO₂R^aa, —OP(R^cc)₂, —OP(R^cc)₃, —OP(═O)₂R^aa, —OP(═O)(R^aa)₂, —OP(═O)(OR^cc)₂, —OP(═O)₂N(R^bb)₂, and —OP(═O)(NR^bb)₂, wherein R^aa, R^bb, and R^ccare as defined herein).

0107The term “heteroatom” refers to an atom that is not hydrogen or carbon. In certain embodiments, the heteroatom is nitrogen. In certain embodiments, the heteroatom is oxygen. In certain embodiments, the heteroatom is sulfur.

0108As used herein, the term “salt” refers to any and all salts, and encompasses pharmaceutically acceptable salts. The term “salt” refers to ionic compounds that result from the neutralization reaction of an acid and a base. A salt is composed of one or more cations (positively charged ions) and one or more anions (negative ions) so that the salt is electrically neutral (without a net charge). Salts of the compounds of this invention include those derived from inorganic and organic acids and bases. Examples of acid addition salts are salts of an amino group formed with inorganic acids, such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or with organic acids, such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid or by using other methods known in the art such as ion exchange. Other salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, hippurate, and the like. Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N⁺(C_1-4alkyl)₄salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further salts include ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate, and aryl sulfonate.

0109The term “pharmaceutically acceptable salt” refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, incorporated herein by reference. Pharmaceutically acceptable salts of the compounds described herein include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid or by using other methods known in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N⁺(C_1-4alkyl)₄⁻ salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate, and aryl sulfonate.

0110The term “solvate” refers to forms of the compound that are associated with a solvent, usually by a solvolysis reaction. This physical association may include hydrogen bonding. Conventional solvents include water, methanol, ethanol, acetic acid, DMSO, THF, diethyl ether, and the like. The compounds described herein may be prepared, e.g., in crystalline form, and may be solvated. Suitable solvates include pharmaceutically acceptable solvates and further include both stoichiometric solvates and non-stoichiometric solvates. In certain instances, the solvate will be capable of isolation, for example, when one or more solvent molecules are incorporated in the crystal lattice of a crystalline solid. “Solvate” encompasses both solution-phase and isolatable solvates. Representative solvates include hydrates, ethanolates, and methanolates.

0111The term “hydrate” refers to a compound that is associated with water. Typically, the number of the water molecules contained in a hydrate of a compound is in a definite ratio to the number of the compound molecules in the hydrate. Therefore, a hydrate of a compound may be represented, for example, by the general formula R·x H₂O, wherein R is the compound, and x is a number greater than 0. A given compound may form more than one type of hydrate, including, e.g., monohydrates (x is 1), lower hydrates (x is a number greater than 0 and smaller than 1, e.g., hemihydrates (R·0.5H₂O)), and polyhydrates (x is a number greater than 1, e.g., dihydrates (R·2H₂O) and hexahydrates (R·6H₂O)).

0112The term “tautomers” or “tautomeric” refers to two or more interconvertible compounds resulting from at least one formal migration of a hydrogen atom and at least one change in valency (e.g., a single bond to a double bond, a triple bond to a single bond, or vice versa). The exact ratio of the tautomers depends on several factors, including temperature, solvent, and pH. Tautomerizations (i.e., the reaction providing a tautomeric pair) may catalyzed by acid or base. Exemplary tautomerizations include keto-to-enol, amide-to-imide, lactam-to-lactim, enamine-to-imine, and enamine-to-(a different enamine) tautomerizations.

0113It is also to be understood that compounds that have the same molecular formula but differ in the nature or sequence of bonding of their atoms or the arrangement of their atoms in space are termed “isomers”. Isomers that differ in the arrangement of their atoms in space are termed “stereoisomers”.

0114Stereoisomers that are not mirror images of one another are termed “diastereomers” and those that are non-superimposable mirror images of each other are termed “enantiomers”. When a compound has an asymmetric center, for example, it is bonded to four different groups, a pair of enantiomers is possible. An enantiomer can be characterized by the absolute configuration of its asymmetric center and is described by the R- and S-sequencing rules of Calm and Prelog, or by the manner in which the molecule rotates the plane of polarized light and designated as dextrorotatory or levorotatory (i.e., as (+) or (−)-isomers respectively). A chiral compound can exist as either individual enantiomer or as a mixture thereof. A mixture containing equal proportions of the enantiomers is called a “racemic mixture”.

0115The term “polymorphs” refers to a crystalline form of a compound (or a salt, hydrate, or solvate thereof) in a particular crystal packing arrangement. All polymorphs have the same elemental composition. Different crystalline forms usually have different X-ray diffraction patterns, infrared spectra, melting points, density, hardness, crystal shape, optical and electrical properties, stability, and solubility. Recrystallization solvent, rate of crystallization, storage temperature, and other factors may cause one crystal form to dominate. Various polymorphs of a compound can be prepared by crystallization under different conditions.

0116The term “co-crystal” refers to a crystalline structure composed of at least two components. In certain embodiments, a co-crystal may contain a compound of the present invention and one or more other component, including but not limited to, atoms, ions, molecules, or solvent molecules. In certain embodiments, a co-crystal may contain a compound of the present invention and one or more components related to said compound, including not limited to, an isomer, tautomer, salt, solvate, hydrate, synthetic precursor, synthetic derivative, fragment or impurity of said compound. Co-crystals may be useful to improve the properties (e.g., solubility, stability, and ease of formulation) of a compound of the present invention.

0117The term “isotopes” refers to variants of a particular chemical element such that, while all isotopes of a given element share the same number of protons in each atom of the element, those isotopes differ in the number of neutrons. The term “radioactivity” or “radioactive decay” refers to the process by which a nucleus of an unstable isotope (e.g., ¹⁸F) loses energy by emitting particles or rays (e.g., alpha particles, beta particles, and gamma rays) of electromagnetic radiation. Such an unstable isotope or a material including the unstable isotope is referred to as “radioactive.” The Curie (Ci) is a non-SI (non-International System of Units) unit of radioactivity and is defined as 1 Ci=3.7×10¹⁰decays per second. The term “specific activity” refers to the unit radioactivity of a material (e.g., any compound disclosed herein, or a salt, tautomer, stereoisomer, or isotopically labeled derivative (e.g., ¹⁸F-labeled derivative) thereof). In certain embodiments, the term “specific activity” refers to the radioactivity of a material per micromole (μmol) of the material.

0118The term “isotopically labeled derivative” or “isotopically labeled” refers to a compound wherein one or more atoms in the compound (or in an associated ion or molecule of a salt, hydrate, or solvate) has been replaced with an isotope of the same element. For the given element or position in the molecule the isotope will be enriched, or present in a higher percentage of all atoms of the element or of all atoms at the position in the molecule in a sample, relative to an unlabeled variant. In certain embodiments, the enriched isotope will be a stable isotope. In certain embodiments, the enriched isotope will be an unstable or radioactive isotope (e.g., a radionuclide). In certain embodiments, the enriched isotope may be detected by a measurement technique, including but not limited to nuclear magnetic resonance, mass spectrometry, infrared spectroscopy, or a technique that measures radioactive decay.

0119The term “prodrugs” refers to compounds that have cleavable groups and become by solvolysis or under physiological conditions the compounds described herein, which are pharmaceutically active in vivo. Such examples include, but are not limited to, choline ester derivatives and the like, N-alkylmorpholine esters and the like. Other derivatives of the compounds described herein have activity in both their acid and acid derivative forms, but in the acid sensitive form often offer advantages of solubility, tissue compatibility, or delayed release in the mammalian organism (see, Bundgard, H., Design of Prodrugs, pp. 7-9, 21-24, Elsevier, Amsterdam 1985). Prodrugs include acid derivatives well known to practitioners of the art, such as, for example, esters prepared by reaction of the parent acid with a suitable alcohol, or amides prepared by reaction of the parent acid compound with a substituted or unsubstituted amine, or acid anhydrides, or mixed anhydrides. Simple aliphatic or aromatic esters, amides, and anhydrides derived from acidic groups pendant on the compounds described herein are particular prodrugs. In some cases it is desirable to prepare double ester type prodrugs such as (acyloxy)alkyl esters or ((alkoxycarbonyl)oxy)alkylesters. C₁-C₈alkyl, C₂-C₈alkenyl, C₂-C₈alkynyl, aryl, C₇-C₁₂substituted aryl, and C₇-C₁₂arylalkyl esters of the compounds described herein may be preferred.

0120The term “inhibition”, “inhibiting”, “inhibit,” or “inhibitor” refer to the ability of a compound to reduce, slow, halt or prevent activity of a particular biological process (e.g., activity of a bromodomain and/or a bromodomain-containing protein) in a cell relative to vehicle.

0121As used herein the term “inhibit” or “inhibition” in the context of enzymes, for example, in the context of CDK (e.g., CDK7, CDK12, CDK13), refers to a reduction in the activity of the enzyme. In some embodiments, the term refers to a reduction in the level of enzyme activity, e.g., CDK (e.g., CDK7, CDK12, CDK13) activity, to a level that is statistically significantly lower than an initial level, which may, for example, be a baseline level of enzyme activity. In some embodiments, the term refers to a reduction in the level of enzyme activity, e.g., CDK (e.g., CDK7, CDK12, CDK13) activity, to a level that is less than 75%, less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.1%, less than 0.01%, less than 0.001%, or less than 0.0001% of an initial level, which may, for example, be a baseline level of enzyme activity.

0122When a compound, pharmaceutical composition, method, use, or kit is referred to as “selectively,” “specifically,” or “competitively” binding a first protein or a first chromatin, the compound, pharmaceutical composition, method, use, or kit binds the first protein or the first chromatin with a higher binding affinity (e.g., not less than about 2-fold, not less than about 5-fold, not less than about 10-fold, not less than about 30-fold, not less than about 100-fold, not less than about 1,000-fold, or not less than about 10,000-fold) than binding a second protein or second chromatin that is different from the first protein and the first chromatin. When a compound, pharmaceutical composition, method, use, or kit is referred to as “selectively,” “specifically,” or “competitively” modulating (e.g., increasing or inhibiting) the activity of a bromodomain-containing protein, the compound, pharmaceutical composition, method, use, or kit modulates the activity of the bromodomain-containing protein to a greater extent (e.g., not less than about 2-fold, not less than about 5-fold, not less than about 10-fold, not less than about 30-fold, not less than about 100-fold, not less than about 1,000-fold, or not less than about 10,000-fold) than the activity of at least one protein that is different from the bromodomain-containing protein.

0123The term “aberrant activity” refers to activity deviating from normal activity, that is, abnormal activity. The term “increased activity” refers to activity higher than normal activity.

0124The terms “composition” and “formulation” are used interchangeably.

0125A “subject” to which administration is contemplated refers to a human (i.e., male or female of any age group, e.g., pediatric subject (e.g., infant, child, or adolescent) or adult subject (e.g., young adult, middle-aged adult, or senior adult)) or non-human animal. In certain embodiments, the non-human animal is a mammal (e.g., primate (e.g., cynomolgus monkey or rhesus monkey), commercially relevant mammal (e.g., cattle, pig, horse, sheep, goat, cat, or dog), or bird (e.g., commercially relevant bird, such as chicken, duck, goose, or turkey)). In certain embodiments, the non-human animal is a fish, reptile, or amphibian. The non-human animal may be a male or female at any stage of development. The non-human animal may be a transgenic animal or genetically engineered animal. A “patient” refers to a human subject in need of treatment of a disease.

0126The term “biological sample” refers to any sample including tissue samples (such as tissue sections and needle biopsies of a tissue); cell samples (e.g., cytological smears (such as Pap or blood smears) or samples of cells obtained by microdissection); samples of whole organisms (such as samples of yeasts or bacteria); or cell fractions, fragments or organelles (such as obtained by lysing cells and separating the components thereof by centrifugation or otherwise). Other examples of biological samples include blood, serum, urine, semen, fecal matter, cerebrospinal fluid, interstitial fluid, mucous, tears, sweat, pus, biopsied tissue (e.g., obtained by a surgical biopsy or needle biopsy), nipple aspirates, milk, vaginal fluid, saliva, swabs (such as buccal swabs), or any material containing biomolecules that is derived from another biological sample.

0127The terms “administer,” “administering,” or “administration” refers to implanting, absorbing, ingesting, injecting, inhaling, or otherwise introducing a compound described herein, or a composition thereof, into, in, or on a subject.

0128The terms “treatment,” “treat,” and “treating” refer to reversing, alleviating, delaying the onset of, or inhibiting the progress of a disease described herein. In some embodiments, treatment may be administered after one or more signs or symptoms of the disease have developed or have been observed. In other embodiments, treatment may be administered in the absence of signs or symptoms of the disease. For example, treatment may be administered to a susceptible subject prior to the onset of symptoms (e.g., in light of a history of symptoms and/or in light of exposure to a pathogen). Treatment may also be continued after symptoms have resolved, for example, to delay or prevent recurrence.

0129The term “prevent,” “preventing,” or “prevention” refers to a prophylactic treatment of a subject who has not and had not been diagnosed with a disease but is at risk of developing the disease. The term “prevent,” “preventing,” or “prevention” also refers to a prophylactic treatment of a subject who was suffering from a disease or has not been diagnosed with the disease, but is at risk of regression or recurrence of the disease. In certain embodiments, the subject is at a higher risk of developing the disease or at a higher risk of regression of the disease than an average healthy member of a population.

0130The terms “condition,” “disease,” and “disorder” are used interchangeably.

0131An “effective amount” of a compound described herein refers to an amount sufficient to elicit the desired biological response, i.e., treating the condition. As will be appreciated by those of ordinary skill in this art, the effective amount of a compound described herein may vary depending on such factors as the desired biological endpoint, the pharmacokinetics of the compound, the condition being treated, the mode of administration, and the age and health of the subject. In certain embodiments, an effective amount is a therapeutically effective amount. In certain embodiments, an effective amount is a prophylactic treatment. In certain embodiments, an effective amount is the amount of a compound described herein in a single dose. In certain embodiments, an effective amount is the combined amounts of a compound described herein in multiple doses.

0132A “therapeutically effective amount” of a compound described herein is an amount sufficient to provide a therapeutic benefit in the treatment of a condition or to delay or minimize one or more symptoms associated with the condition. A therapeutically effective amount of a compound means an amount of therapeutic agent, alone or in combination with other therapies, which provides a therapeutic benefit in the treatment of the condition. The term “therapeutically effective amount” can encompass an amount that improves overall therapy, reduces or avoids symptoms, signs, or causes of the condition, and/or enhances the therapeutic efficacy of another therapeutic agent. In certain embodiments, a therapeutically effective amount is an amount sufficient for inhibiting CDK (e.g., CDK7, CDK12, CDK13). In certain embodiments, a therapeutically effective amount is an amount sufficient for treating an acute inflammatory disease (e.g., rheumatoid arthritis, Crohn's disease, or fibrosis) and/or a proliferative disease (e.g., cancer, benign neoplasm, diseases associated with angiogenesis, inflammatory diseases, autoinflammatory diseases, autoimmune diseases, pancreatic cancer, lung cancer (e.g. small cell lung cancer (SCLC), non-small cell lung cancer), prostate cancer, breast cancer, ovarian cancer, kidney cancer, liver cancer, Ewing's sarcoma, osteosarcoma, brain cancer, neuroblastoma, and colorectal cancer). In certain embodiments, a therapeutically effective amount is an amount sufficient for inhibiting CDK (e.g., CDK7, CDK12, CDK13) and for treating an acute inflammatory disease (e.g., rheumatoid arthritis, Crohn's disease, or fibrosis) and/or a proliferative disease (e.g., cancer, benign neoplasm, diseases associated with angiogenesis, inflammatory diseases, autoinflammatory diseases, autoimmune diseases, pancreatic cancer, lung cancer (e.g. small cell lung cancer (SCLC), and non-small cell lung cancer), prostate cancer, breast cancer, ovarian cancer, kidney cancer, liver cancer, Ewing's sarcoma, osteosarcoma, brain cancer, neuroblastoma, and colorectal cancer).

0133A “prophylactically effective amount” of a compound described herein is an amount sufficient to prevent a condition, or one or more symptoms associated with the condition or prevent its recurrence. A prophylactically effective amount of a compound means an amount of a therapeutic agent, alone or in combination with other agents, which provides a prophylactic benefit in the prevention of the condition. The term “prophylactically effective amount” can encompass an amount that improves overall prophylaxis or enhances the prophylactic efficacy of another prophylactic agent. In certain embodiments, a prophylactically effective amount is an amount sufficient for inhibiting CDK (e.g., CDK7, CDK12, CDK13). In certain embodiments, a prophylactically effective amount is an amount sufficient for treating an acute inflammatory disease (e.g., rheumatoid arthritis, Crohn's disease, or fibrosis) and/or a proliferative disease (e.g., cancer, benign neoplasm, diseases associated with angiogenesis, inflammatory diseases, autoinflammatory diseases, autoimmune diseases, pancreatic cancer, lung cancer (e.g. small cell lung cancer (SCLC), and non-small cell lung cancer), prostate cancer, breast cancer, ovarian cancer, kidney cancer, liver cancer, Ewing's sarcoma, osteosarcoma, brain cancer, neuroblastoma, and colorectal cancer). In certain embodiments, a prophylactically effective amount is an amount sufficient for inhibiting CDK (e.g., CDK7, CDK12, CDK13) and for treating acute inflammatory disease (e.g., rheumatoid arthritis, Crohn's disease, or fibrosis) and/or a proliferative disease (e.g., cancer, benign neoplasm, diseases associated with angiogenesis, inflammatory diseases, autoinflammatory diseases, autoimmune diseases, pancreatic cancer, lung cancer (e.g. small cell lung cancer (SCLC), and non-small cell lung cancer), prostate cancer, breast cancer, ovarian cancer, kidney cancer, liver cancer, Ewing's sarcoma, osteosarcoma, brain cancer, neuroblastoma, and colorectal cancer).

0134A “proliferative disease” refers to a disease that occurs due to abnormal growth or extension by the multiplication of cells (Walker, Cambridge Dictionary of Biology; Cambridge University Press: Cambridge, UK, 1990). A proliferative disease may be associated with: 1) the pathological proliferation of normally quiescent cells; 2) the pathological migration of cells from their normal location (e.g., metastasis of neoplastic cells); 3) the pathological expression of proteolytic enzymes such as the matrix metalloproteinases (e.g., collagenases, gelatinases, and elastases); or 4) the pathological angiogenesis as in proliferative retinopathy and tumor metastasis. Exemplary proliferative diseases include cancers (i.e., “malignant neoplasms”), benign neoplasms, diseases associated with angiogenesis, inflammatory diseases, and autoimmune diseases.

0135The term “angiogenesis” refers to the physiological process through which new blood vessels form from pre-existing vessels. Angiogenesis is distinct from vasculogenesis, which is the de novo formation of endothelial cells from mesoderm cell precursors. The first vessels in a developing embryo form through vasculogenesis, after which angiogenesis is responsible for most blood vessel growth during normal or abnormal development. Angiogenesis is a vital process in growth and development, as well as in wound healing and in the formation of granulation tissue. However, angiogenesis is also a fundamental step in the transition of tumors from a benign state to a malignant one, leading to the use of angiogenesis inhibitors in the treatment of cancer. Angiogenesis may be chemically stimulated by angiogenic proteins, such as growth factors (e.g., VEGF). “Pathological angiogenesis” refers to abnormal (e.g., excessive or insufficient) angiogenesis that amounts to and/or is associated with a disease.

0136The terms “neoplasm” and “tumor” are used herein interchangeably and refer to an abnormal mass of tissue wherein the growth of the mass surpasses and is not coordinated as in the growth of normal tissue. A neoplasm or tumor may be “benign” or “malignant,” depending on the following characteristics: degree of cellular differentiation (including morphology and functionality), rate of growth, local invasion, and metastasis. A “benign neoplasm” is generally well differentiated, has characteristically slower growth than a malignant neoplasm, and remains localized to the site of origin. In addition, a benign neoplasm does not have the capacity to infiltrate, invade, or metastasize to distant sites. Exemplary benign neoplasms include, but are not limited to, lipoma, chondroma, adenomas, acrochordon, senile angiomas, seborrheic keratoses, lentigos, and sebaceous hyperplasias. In some cases, certain “benign” tumors may later give rise to malignant neoplasms, which may result from additional genetic changes in a subpopulation of the tumor's neoplastic cells, and these tumors are referred to as “pre-malignant neoplasms.” An exemplary pre-malignant neoplasm is a teratoma. In contrast, a “malignant neoplasm” is generally poorly differentiated (anaplasia) and has characteristically rapid growth accompanied by progressive infiltration, invasion, and destruction of the surrounding tissue. Furthermore, a malignant neoplasm generally has the capacity to metastasize to distant sites. The term “metastasis,” “metastatic,” or “metastasize” refers to the spread or migration of cancerous cells from a primary or original tumor to another organ or tissue and is typically identifiable by the presence of a “secondary tumor” or “secondary cell mass” of the tissue type of the primary or original tumor and not of that of the organ or tissue in which the secondary (metastatic) tumor is located. For example, a prostate cancer that has migrated to bone is said to be metastasized prostate cancer and includes cancerous prostate cancer cells growing in bone tissue.

0137The term “cancer” refers to a class of diseases characterized by the development of abnormal cells that proliferate uncontrollably and have the ability to infiltrate and destroy normal body tissues. See, e.g., Stedman's Medical Dictionary, 25th ed.; Hensyl ed.; Williams &amp; Wilkins: Philadelphia, 1990. Exemplary cancers include, but are not limited to, hematological malignancies. Additional exemplary cancers include, but are not limited to, acoustic neuroma; adenocarcinoma; adrenal gland cancer; anal cancer; angiosarcoma (e.g., lymphangiosarcoma, lymphangioendotheliosarcoma, hemangiosarcoma); appendix cancer; benign monoclonal gammopathy; biliary cancer (e.g., cholangiocarcinoma); bladder cancer; breast cancer (e.g., adenocarcinoma of the breast, papillary carcinoma of the breast, mammary cancer, medullary carcinoma of the breast, triple negative breast cancer (TNBC)); brain cancer (e.g., meningioma, glioblastomas, glioma (e.g., astrocytoma, oligodendroglioma), medulloblastoma); bronchus cancer; carcinoid tumor; cervical cancer (e.g., cervical adenocarcinoma); choriocarcinoma; chordoma; craniopharyngioma; colorectal cancer (e.g., colon cancer, rectal cancer, colorectal adenocarcinoma); connective tissue cancer; epithelial carcinoma; ependymoma; endotheliosarcoma (e.g., Kaposi's sarcoma, multiple idiopathic hemorrhagic sarcoma); endometrial cancer (e.g., uterine cancer, uterine sarcoma); esophageal cancer (e.g., adenocarcinoma of the esophagus, Barrett's adenocarcinoma); Ewing's sarcoma; ocular cancer (e.g., intraocular melanoma, retinoblastoma); familiar hypereosinophilia; gall bladder cancer; gastric cancer (e.g., stomach adenocarcinoma); gastrointestinal stromal tumor (GIST); germ cell cancer; head and neck cancer (e.g., head and neck squamous cell carcinoma, oral cancer (e.g., oral squamous cell carcinoma), throat cancer (e.g., laryngeal cancer, pharyngeal cancer, nasopharyngeal cancer, oropharyngeal cancer)); heavy chain disease (e.g., alpha chain disease, gamma chain disease, mu chain disease; hemangioblastoma; hypopharynx cancer; inflammatory myofibroblastic tumors; immunocytic amyloidosis; kidney cancer (e.g., nephroblastoma a.k.a. Wilms' tumor, renal cell carcinoma); liver cancer (e.g., hepatocellular cancer (HCC), malignant hepatoma); lung cancer (e.g., bronchogenic carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung); leiomyosarcoma (LMS); mastocytosis (e.g., systemic mastocytosis); muscle cancer; myelodysplastic syndrome (MDS); mesothelioma; myeloproliferative disorder (MPD) (e.g., polycythemia vera (PV), essential thrombocytosis (ET), agnogenic myeloid metaplasia (AMM) a.k.a. myelofibrosis (MF), chronic idiopathic myelofibrosis, chronic myelocytic leukemia (CML), chronic neutrophilic leukemia (CNL), hypereosinophilic syndrome (HES)); neuroblastoma; neurofibroma (e.g., neurofibromatosis (NF) type 1 or type 2, schwannomatosis); neuroendocrine cancer (e.g., gastroenteropancreatic neuroendocrine tumor (GEP-NET), carcinoid tumor); osteosarcoma (e.g., bone cancer); ovarian cancer (e.g., cystadenocarcinoma, ovarian embryonal carcinoma, ovarian adenocarcinoma); papillary adenocarcinoma; pancreatic cancer (e.g., pancreatic andenocarcinoma, intraductal papillary mucinous neoplasm (IPMN), Islet cell tumors); penile cancer (e.g., Paget's disease of the penis and scrotum); pinealoma; primitive neuroectodermal tumor (PNT); plasma cell neoplasia; paraneoplastic syndromes; intraepithelial neoplasms; prostate cancer (e.g., prostate adenocarcinoma); rectal cancer; rhabdomyosarcoma; salivary gland cancer; skin cancer (e.g., squamous cell carcinoma (SCC), keratoacanthoma (KA), melanoma, basal cell carcinoma (BCC)); small bowel cancer (e.g., appendix cancer); soft tissue sarcoma (e.g., malignant fibrous histiocytoma (MFH), liposarcoma, malignant peripheral nerve sheath tumor (MPNST), chondrosarcoma, fibrosarcoma, myxosarcoma); sebaceous gland carcinoma; small intestine cancer; sweat gland carcinoma; synovioma; testicular cancer (e.g., seminoma, testicular embryonal carcinoma); thyroid cancer (e.g., papillary carcinoma of the thyroid, papillary thyroid carcinoma (PTC), medullary thyroid cancer); urethral cancer; vaginal cancer; and vulvar cancer (e.g., Paget's disease of the vulva).

0138The term “hematological malignancy” refers to tumors that affect blood, bone marrow, and/or lymph nodes. Exemplary hematological malignancies include, but are not limited to, leukemia, such as acute lymphoblastic leukemia (ALL) (e.g., B-cell ALL, T-cell ALL), acute myelocytic leukemia (AML) (e.g., B-cell AML, T-cell AML), chronic myelocytic leukemia (CML) (e.g., B-cell CML, T-cell CML), and chronic lymphocytic leukemia (CLL) (e.g., B-cell CLL, T-cell CLL)); lymphoma, such as Hodgkin lymphoma (HL) (e.g., B-cell HL, T-cell HL) and non-Hodgkin lymphoma (NHL) (e.g., B-cell NHL, such as diffuse large cell lymphoma (DLCL) (e.g., diffuse large B-cell lymphoma (DLBCL, e.g., activated B-cell (ABC) DLBCL (ABC-DLBCL))), follicular lymphoma, chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), mantle cell lymphoma (MCL), marginal zone B-cell lymphoma (e.g., mucosa-associated lymphoid tissue (MALT) lymphoma, nodal marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma), primary mediastinal B-cell lymphoma, Burkitt's lymphoma, Waldenstrom's macroglobulinemia (WM, lymphoplasmacytic lymphoma), hairy cell leukemia (HCL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, central nervous system (CNS) lymphoma (e.g., primary CNS lymphoma and secondary CNS lymphoma); and T-cell NHL, such as precursor T-lymphoblastic lymphoma/leukemia, peripheral T-cell lymphoma (PTCL) (e.g., cutaneous T-cell lymphoma (CTCL) (e.g., mycosis fungoides, Sezary syndrome), angioimmunoblastic T-cell lymphoma, extranodal natural killer T-cell lymphoma, enteropathy type T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, and anaplastic large cell lymphoma); lymphoma of an immune privileged site (e.g., cerebral lymphoma, ocular lymphoma, lymphoma of the placenta, lymphoma of the fetus, testicular lymphoma); a mixture of one or more leukemia/lymphoma as described above; myelodysplasia; and multiple myeloma (MM).

0139The term “inflammatory disease” refers to a disease caused by, resulting from, or resulting in inflammation. The term “inflammatory disease” may also refer to a dysregulated inflammatory reaction that causes an exaggerated response by macrophages, granulocytes, and/or T-lymphocytes leading to abnormal tissue damage and/or cell death. An inflammatory disease can be either an acute or chronic inflammatory condition and can result from infections or non-infectious causes. Inflammatory diseases include, without limitation, atherosclerosis, arteriosclerosis, autoimmune disorders, multiple sclerosis, systemic lupus erythematosus, polymyalgia rheumatica (PMR), gouty arthritis, degenerative arthritis, tendonitis, bursitis, psoriasis, cystic fibrosis, arthrosteitis, rheumatoid arthritis, inflammatory arthritis, Sjogren's syndrome, giant cell arteritis, progressive systemic sclerosis (scleroderma), ankylosing spondylitis, polymyositis, dermatomyositis, pemphigus, pemphigoid, diabetes (e.g., Type I), myasthenia gravis, Hashimoto's thyroiditis, Graves' disease, Goodpasture's disease, mixed connective tissue disease, sclerosing cholangitis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, pernicious anemia, inflammatory dermatoses, usual interstitial pneumonitis (UIP), asbestosis, silicosis, bronchiectasis, berylliosis, talcosis, pneumoconiosis, sarcoidosis, desquamative interstitial pneumonia, lymphoid interstitial pneumonia, giant cell interstitial pneumonia, cellular interstitial pneumonia, extrinsic allergic alveolitis, Wegener's granulomatosis and related forms of angiitis (temporal arteritis and polyarteritis nodosa), inflammatory dermatoses, hepatitis, delayed-type hypersensitivity reactions (e.g., poison ivy dermatitis), pneumonia, respiratory tract inflammation, Adult Respiratory Distress Syndrome (ARDS), encephalitis, immediate hypersensitivity reactions, asthma, hay fever, allergies, acute anaphylaxis, rheumatic fever, glomerulonephritis, pyelonephritis, cellulitis, cystitis, chronic cholecystitis, ischemia (ischemic injury), reperfusion injury, allograft rejection, host-versus-graft rejection, appendicitis, arteritis, blepharitis, bronchiolitis, bronchitis, cervicitis, cholangitis, chorioamnionitis, conjunctivitis, dacryoadenitis, dermatomyositis, endocarditis, endometritis, enteritis, enterocolitis, epicondylitis, epididymitis, fasciitis, fibrositis, gastritis, gastroenteritis, gingivitis, ileitis, iritis, laryngitis, myelitis, myocarditis, nephritis, omphalitis, oophoritis, orchitis, osteitis, otitis, pancreatitis, parotitis, pericarditis, pharyngitis, pleuritis, phlebitis, pneumonitis, proctitis, prostatitis, rhinitis, salpingitis, sinusitis, stomatitis, synovitis, testitis, tonsillitis, urethritis, urocystitis, uveitis, vaginitis, vasculitis, vulvitis, vulvovaginitis, angitis, chronic bronchitis, osteomyelitis, optic neuritis, temporal arteritis, transverse myelitis, necrotizing fasciitis, and necrotizing enterocolitis.

0140An “autoimmune disease” refers to a disease arising from an inappropriate immune response of the body of a subject against substances and tissues normally present in the body. In other words, the immune system mistakes some part of the body as a pathogen and attacks its own cells. This may be restricted to certain organs (e.g., in autoimmune thyroiditis) or involve a particular tissue in different places (e.g., Goodpasture's disease which may affect the basement membrane in both the lung and kidney). The treatment of autoimmune diseases is typically with immunosuppression, e.g., medications which decrease the immune response. Exemplary autoimmune diseases include, but are not limited to, glomerulonephritis, Goodpasture's syndrome, necrotizing vasculitis, lymphadenitis, peri-arteritis nodosa, systemic lupus erythematosis, rheumatoid arthritis, psoriatic arthritis, systemic lupus erythematosis, psoriasis, ulcerative colitis, systemic sclerosis, dermatomyositis/polymyositis, anti-phospholipid antibody syndrome, scleroderma, pemphigus vulgaris, ANCA-associated vasculitis (e.g., Wegener's granulomatosis, microscopic polyangiitis), uveitis, Sjogren's syndrome, Crohn's disease, Reiter's syndrome, ankylosing spondylitis, Lyme disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, and cardiomyopathy.

0141The term “kinase” is a type of enzyme that transfers phosphate groups from high energy donor molecules, such as ATP, to specific substrates, referred to as phosphorylation. Kinases are part of the larger family of phosphotransferases. One of the largest groups of kinases are protein kinases, which act on and modify the activity of specific proteins. Kinases are used extensively to transmit signals and control complex processes in cells. Various other kinases act on small molecules such as lipids, carbohydrates, amino acids, and nucleotides, either for signaling or to prime them for metabolic pathways. Kinases are often named after their substrates. More than 500 different protein kinases have been identified in humans. Exemplary human protein kinases include, but are not limited to, AAK1, ABL, ACK, ACTR2, ACTR2B, AKT1, AKT2, AKT3, ALK, ALK1, ALK2, ALK4, ALK7, AMPKa1, AMPKa2, ANKRD3, ANPa, ANPb, ARAF, ARAFps, ARG, AurA, AurAps1, AurAps2, AurB, AurBps1, AurC, AXL, BARK1, BARK2, BIKE, BLK, BMPR1A, BMPR1Aps1, BMPR1Aps2, BMPR1B, BMPR2, BMX, BRAF, BRAFps, BRK, BRSK1, BRSK2, BTK, BUB1, BUBR1, CaMK1a, CaMK1b, CaMK1d, CaMK1g, CaMK2a, CaMK2b, CaMK2d, CaMK2g, CaMK4, CaMKK1, CaMKK2, caMLCK, CASK, CCK4, CCRK, CDC2, CDC7, CDK10, CDK11, CDK2, CDK3, CDK4, CDK4ps, CDK5, CDK5ps, CDK6, CDK7, CDK7ps, CDK8, CDK8ps, CDK9, CDKL1, CDKL2, CDKL3, CDKL4, CDKL5, CGDps, CHED, CHK1, CHK2, CHK2ps1, CHK2ps2, CK1a, CK1a2, CK1aps1, CK1aps2, CK1aps3, CK1d, CK1e, CK1g1, CK1g2, CK1g2ps, CK1g3, CK2a1, CK2a1-rs, CK2a2, CLIK1, CLIK1L, CLK1, CLK2, CLK2ps, CLK3, CLK3ps, CLK4, COT, CRIK, CRK7, CSK, CTK, CYGD, CYGF, DAPK1, DAPK2, DAPK3, DCAMKL1, DCAMKL2, DCAMKL3, DDR1, DDR2, DLK, DMPK1, DMPK2, DRAK1, DRAK2, DYRK1A, DYRK1B, DYRK2, DYRK3, DYRK4, EGFR, EphA1, EphA10, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphB1, EphB2, EphB3, EphB4, EphB6, Erk1, Erk2, Erk3, Erk3ps1, Erk3ps2, Erk3ps3, Erk3ps4, Erk4, Erk5, Erk7, FAK, FER, FERps, FES, FGFR1, FGFR2, FGFR3, FGFR4, FGR, FLT1, FLT1ps, FLT3, FLT4, FMS, FRK, Fused, FYN, GAK, GCK, GCN2, GCN22, GPRK4, GPRK5, GPRK6, GPRK6ps, GPRK7, GSK3A, GSK3B, Haspin, HCK, HER2/ErbB2, HER3/ErbB3, HER4/ErbB4, HH498, HIPK1, HIPK2, HIPK3, HIPK4, HPK1, HRI, HRIps, HSER, HUNK, ICK, IGF1R, IKKa, IKKb, IKKe, ILK, INSR, IRAK1, IRAK2, IRAK3, IRAK4, IRE1, IRE2, IRR, ITK, JAK1, JAK2, JAK3, JNK1, JNK2, JNK3, KDR, KHS1, KHS2, KIS, KIT, KSGCps, KSR1, KSR2, LATS1, LATS2, LCK, LIMK1, LIMK2, LIMK2ps, LKB1, LMR1, LMR2, LMR3, LOK, LRRK1, LRRK2, LTK, LYN, LZK, MAK, MAP2K1, MAP2K1ps, MAP2K2, MAP2K2ps, MAP2K3, MAP2K4, MAP2K5, MAP2K6, MAP2K7, MAP3K1, MAP3K2, MAP3K3, MAP3K4, MAP3K5, MAP3K6, MAP3K7, MAP3K8, MAPKAPK2, MAPKAPK3, MAPKAPK5, MAPKAPKps1, MARK1, MARK2, MARK3, MARK4, MARKps01, MARKps02, MARKps03, MARKps04, MARKps05, MARKps07, MARKps08, MARKps09, MARKps10, MARKps11, MARKps12, MARKps13, MARKps15, MARKps16, MARKps17, MARKps18, MARKps19, MARKps20, MARKps21, MARKps22, MARKps23, MARKps24, MARKps25, MARKps26, MARKps27, MARKps28, MARKps29, MARKps30, MAST1, MAST2, MAST3, MAST4, MASTL, MELK, MER, MET, MISR2, MLK1, MLK2, MLK3, MLK4, MLKL, MNK1, MNK1ps, MNK2, MOK, MOS, MPSK1, MPSK1ps, MRCKa, MRCKb, MRCKps, MSK1, MSK12, MSK2, MSK22, MSSK1, MST1, MST2, MST3, MST3ps, MST4, MUSK, MYO3A, MYO3B, MYT1, NDR1, NDR2, NEK1, NEK10, NEK11, NEK2, NEK2ps1, NEK2ps2, NEK2ps3, NEK3, NEK4, NEK4ps, NEK5, NEK6, NEK7, NEK8, NEK9, NIK, NIM1, NLK, NRBP1, NRBP2, NuaK1, NuaK2, Obscn, Obscn2, OSR1, p38a, p38b, p38d, p38g, p70S6K, p70S6Kb, p70S6Kps1, p70S6Kps2, PAK1, PAK2, PAK2ps, PAK3, PAK4, PAK5, PAK6, PASK, PBK, PCTAIRE1, PCTAIRE2, PCTAIRE3, PDGFRa, PDGFRb, PDK1, PEK, PFTAIRE1, PFTAIRE2, PHKg1, PHKg1ps1, PHKg1ps2, PHKg1ps3, PHKg2, PIK3R4, PIM1, PIM2, PIM3, PINK1, PITSLRE, PKACa, PKACb, PKACg, PKCa, PKCb, PKCd, PKCe, PKCg, PKCh, PKCi, PKCips, PKCt, PKCz, PKD1, PKD2, PKD3, PKG1, PKG2, PKN1, PKN2, PKN3, PKR, PLK1, PLK1ps1, PLK1ps2, PLK2, PLK3, PLK4, PRKX, PRKXps, PRKY, PRP4, PRP4ps, PRPK, PSKH1, PSKH1ps, PSKH2, PYK2, QIK, QSK, RAF1, RAF1ps, RET, RHOK, RIPK1, RIPK2, RIPK3, RNAseL, ROCK1, ROCK2, RON, ROR1, ROR2, ROS, RSK1, RSK12, RSK2, RSK22, RSK3, RSK32, RSK4, RSK42, RSKL1, RSKL2, RYK, RYKps, SAKps, SBK, SCYL1, SCYL2, SCYL2ps, SCYL3, SGK, SgK050ps, SgK069, SgK071, SgK085, SgK110, SgK196, SGK2, SgK223, SgK269, SgK288, SGK3, SgK307, SgK384ps, SgK396, SgK424, SgK493, SgK494, SgK495, SgK496, SIK (e.g., SIK1, SIK2), skMLCK, SLK, Slob, smMLCK, SNRK, SPEG, SPEG2, SRC, SRM, SRPK1, SRPK2, SRPK2ps, SSTK, STK33, STK33ps, STLK3, STLK5, STLK6, STLK6ps1, STLK6-rs, SuRTK106, SYK, TAK1, TAO1, TAO2, TAO3, TBCK, TBK1, TEC, TESK1, TESK2, TGFbR1, TGFbR2, TIE1, TIE2, TLK1, TLK1ps, TLK2, TLK2ps1, TLK2ps2, TNK1, Trad, Trb1, Trb2, Trb3, Trio, TRKA, TRKB, TRKC, TSSK1, TSSK2, TSSK3, TSSK4, TSSKps1, TSSKps2, TTBK1, TTBK2, TTK, TTN, TXK, TYK2, TYK22, TYRO3, TYRO3ps, ULK1, ULK2, ULK3, ULK4, VACAMKL, VRK1, VRK2, VRK3, VRK3ps, Wee1, Wee1B, Wee1Bps, Wee1ps1, Wee1ps2, Wnk1, Wnk2, Wnk3, Wnk4, YANK1, YANK2, YANK3, YES, YESps, YSK1, ZAK, ZAP70, ZC1/HGK, ZC2/TNIK, ZC3/MINK, and ZC4/NRK.

0142The term “SRC family kinase” refers to a family of non-receptor tyrosine protein kinases that includes nine members: SRCA subfamily that includes c-SRC (proto-oncogene tyrosine-protein kinase SRC), YES (proto-oncogene tyrosine-protein kinase Yes), FYN (proto-oncogene tyrosine-protein kinase FYN), and FGR (Gardner-Rasheed feline sarcoma viral (v-FGR) oncogene homolog); SRCB subfamily that includes LCK (lymphocyte-specific protein tyrosine kinase), HCK (tyrosine-protein kinase HCK, hemopoietic cell kinase), BLK (tyrosine-protein kinase BLK), and LYN (tyrosine-protein kinase LYN); and FRK (Fyn-related kinase).

0143The term “CDK” refers to a cyclin-dependent kinase. A CDK binds a cyclin (e.g., Cyclin H), which is a regulatory protein. CDKs phosphorylate their substrates at serines and threonines. The consensus sequence for the phosphorylation site in the amino acid sequence of a CDK substrate is [S/T*]PX[K/R], where S/T* is the phosphorylated serine or threonine, P is proline, X is any amino acid, K is lysine, and R is arginine CDKs include CDK1, CDK2, CDK3, CDK4, CDK5, CDK6, CDK7, CDK8, CDK9, CDK10, CDK11, CDK12, CDK13, CDK14, CDK15, CDK16, CDK17, CDK18, CDK19 and CDK20.

0144CDK7, cyclin-dependent kinase 7, is a CDK, wherein the substrate is Cyclin H, MAT1 (e.g., MNAT1), or Cyclin H and MAT1. CDK7 is alternatively referred to as CAK1, HCAK, MO15, STK1, CDKN7, and p39MO15. Non-limiting examples of the nucleotide and protein sequences for human CDK7 are described in GenBank Accession Number NP_001790, incorporated herein by reference. The amino acid sequence of this CDK7 is as follows:

0145<tables id="TABLE-US-00001" num="00001"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>MALDVKSRAKRYEKLDFLGEGQFATVYKARDKNTNQIVAIKKIKLGHRSEA</entry></row><row><entry></entry></row><row><entry>KDGINRTALREIKLLQELSHPNIIGLLDAFGHKSNISLVFDFMETDLEVII</entry></row><row><entry></entry></row><row><entry>KDNSLVLTPSHIKAYMLMTLQGLEYLHQHWILHRDLKPNNLLLDENGVLKL</entry></row><row><entry></entry></row><row><entry>ADFGLAKSFGSPNRAYTHQVVTRWYRAPELLFGARMYGVGVDMWAVGCILA</entry></row><row><entry></entry></row><row><entry>ELLLRVPFLPGDSDLDQLTRIFETLGTPTEEQWPDMCSLPDYVTFKSFPGI</entry></row><row><entry></entry></row><row><entry>PLHHIFSAAGDDLLDLIQGLFLFNPCARITATQALKMKYFSNRPGPTPGCQ</entry></row><row><entry></entry></row><row><entry>LPRPNCPVETLKEQSNPALAIKRKRTEALEQGGLPKKLIF</entry></row></tbody></tgroup></table></tables>

0146CDK12, cyclin-dependent kinase 12, is a CDK, wherein the substrate is Cyclin K or Flavopiridol. CDK12 is alternatively referred to as Cdc2-related kinase, CDC2-related protein kinase 7, Cell division cycle 2-related protein kinase 7, Cell division protein kinase 12, CRK7, CRKR, CRKRS, cyclin-dependent kinase 12, or KIAA0904. Non-limiting examples of the nucleotide and protein sequences for human CDK12 are described in Uniprot Number Q9NYV4, which is incorporated herein by reference. The amino acid sequence of this CDK12 is as follows: MPNSERHGGKKDGSGGASGTLQPSSGGGSSNSRERHRLVSKHKRHKSKHSKDMGLVTPEA

0147<tables id="TABLE-US-00002" num="00002"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>MPNSERHGGKKDGSGGASGTLQPSSGGGSSNSRERHRLVSKHKRHKSKDM</entry></row><row><entry></entry></row><row><entry>GLVTPEAASLGTVIKPLVEYDDISSDSDTFSDDMAFKLDRRENDERRGSD</entry></row><row><entry></entry></row><row><entry>RSDRLHKHRHHQHRRSRDLLKAKQTEKEKSQEVSSKSGSMKDRISGSSKR</entry></row><row><entry></entry></row><row><entry>SNEETDDYGKAQVAKSSSKESRSSKLHKEKTRKERELKSGHKDRSKSHRK</entry></row><row><entry></entry></row><row><entry>RETPKSYKTVDSPKRRSRSPHRKWSDSSKQDDSPSGASYGQDYDLSPSRS</entry></row><row><entry></entry></row><row><entry>HTSSNYDSYKKSPGSTSRRQSVSPPYKEPSAYQSSTRSPSPYSRRQRSVS</entry></row><row><entry></entry></row><row><entry>PYSRRRSSSYERSGSYSGRSPSPYGRRRSSSPFLSKRSLSRSPLPSRKSMK</entry></row><row><entry></entry></row><row><entry>RSSRSPAYSRHSSSHSKKKRSSSRSRHSSISPVRLPLNSSLGAELSRKKK</entry></row><row><entry></entry></row><row><entry>ERAAAAAAAKMDGKESKGSPVFLPRKENSSVEAKDSGLESKKLPRSVKLEK</entry></row><row><entry></entry></row><row><entry>SAPDTELVNVTHLNTEVKNSSDTGKVKLDENSEKHLVKDLKAQGTRDSKP</entry></row><row><entry></entry></row><row><entry>IALKEEIVTPKETETSEKETPPPLPTIASPPPPLPTTTPPPQTPPLPPLP</entry></row><row><entry></entry></row><row><entry>PIPALPQQPPLPPSQPAFSQVPASSTSTLPPSTHSKTSAVSSQANSQPPV</entry></row><row><entry></entry></row><row><entry>QVSVKTQVSVTAAIPHLKTSTLPPLPLPPLLPGDDDMDSPKETLPSKPVKK</entry></row><row><entry></entry></row><row><entry>EKEQRTRHLLTDLPLPPELPGGDLSPPDSPEPKAITPPQQPYKKRPKICCP</entry></row><row><entry></entry></row><row><entry>RYGERRQTESDWGKRCVDKFDIIGIIGEGTYGQVYKAKDKDTGELVALKKV</entry></row><row><entry></entry></row><row><entry>RLDNEKEGFPITAIREIKILRQLIHRSVVNMKEIVTDKQDALDFKKDKGAF</entry></row><row><entry></entry></row><row><entry>YLVFEYMDHDLMGLLESGLVHFSEDHIKSFMKQLMEGLEYCHKKNELHRDI</entry></row><row><entry></entry></row><row><entry>KCSNILLNNSGQIKLADEGLARLYNSEESRPYTNKVITLWYRPPELLLGEE</entry></row><row><entry></entry></row><row><entry>RYTPAIDVWSCGCILGELFTKKPIFQANLELAQLELISRLCGSPCPAVWPD</entry></row><row><entry></entry></row><row><entry>VIKLPYENTMKPKKQYRRRLREEFSFIPSAALDLLDHMLTLDPSKRCTAEQ</entry></row><row><entry></entry></row><row><entry>TLQSDFLKDVELSKMAPPDLPHWQDCHELWSKKRRRQRQSGVVVEEPPPSK</entry></row><row><entry></entry></row><row><entry>TSRKETTSGTSTEPVKNSSPAPPQPAPGKVESGAGDAIGLADITQQLNQSE</entry></row><row><entry></entry></row><row><entry>LAVLLNLLQSQTDLSIPQMAQLLNIHSNPEMQQQLEALNQSISALTEATSQ</entry></row><row><entry></entry></row><row><entry>QQDSETMAPEESLKEAPSAPVILPSAEQTTLEASSTPADMQNILAVLLSQL</entry></row><row><entry></entry></row><row><entry>MKTQEPAGSLEENNSDKNSGPQGPRRTPTMPQEEAAACPPHILPPEKRPP</entry></row><row><entry></entry></row><row><entry>EPPGPPPPPPPPPLVEGDLSSAPQELNPAVTAALLQLLSQPEAEPPGHLPH</entry></row><row><entry></entry></row><row><entry>EHQALRPMEYSTRPRPNRTYGNTDGPETGFSAIDTDERNSGPALTESLVQ</entry></row><row><entry></entry></row><row><entry>TLVKNRTFSGSLSHLGESSSYQGTGSVQFPGDQDLRFARVPLALHPVVGQ</entry></row><row><entry></entry></row><row><entry>PFLKAEGSSNSVVHAETKLQNYGELGPGTTGASSSGAGLHWGGPTQSSA</entry></row><row><entry></entry></row><row><entry>YGKLYRGPTRVPPRGGRGRGVPY</entry></row></tbody></tgroup></table></tables>

0148CDK13, cyclin-dependent kinase 13, is a CDK, wherein the relevant cyclin is cyclin K and a reference inhibitor is the pan-CDK inhibitor Flavopiridol and the c-terminal domain (CTD) of RNA-polymerase II is a physiological substrate. CDK13 is alternatively referred to as CHED; CDC2L; CDC2L5; or hCDK13. Non-limiting examples of the nucleotide and protein sequences for human CDK12 are described in GenBank Accession Number M80629, which is incorporated herein by reference. The amino acid sequence of this CDK13 is as follows:

0149<tables id="TABLE-US-00003" num="00003"><table frame="none" colsep="0" rowsep="0" tabstyle="monospace"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="217pt" align="left" /><tbody valign="top"><row><entry>MPSSSDTALGGGGGLSWAEKKLEERRKRRRFLSPQQPPLLLPLLQPQLLQP</entry></row><row><entry></entry></row><row><entry>PPPPPPLLFLAAPGTAAAAAAAAAASSSCFSPGPPLEVKRLARGKRRAGGR</entry></row><row><entry></entry></row><row><entry>QKRRRGPRAGQEAEKRRVFSLPQPQQDGGGGASSGGGVTPLVEYEDVSSQS</entry></row><row><entry></entry></row><row><entry>EQGLLLGGASAATAATAAGGTGGSGGSPASSSGTQRRGEGSERRPRRDRRS</entry></row><row><entry></entry></row><row><entry>SSGRSKERHREHRRRDGQRGGSEASKSRSRHSHSGEERAEVAKSGSSSSSG</entry></row><row><entry></entry></row><row><entry>GRRKSASATSSSSSSRKDRDSKAHRSRTKSSKEPPSAYKEPPKAYREDKTE</entry></row><row><entry></entry></row><row><entry>PKAYRRRRSLSPLGGRDDSPVSHRASQSLRSRKSPSPAGGGSSPYSRRLPR</entry></row><row><entry></entry></row><row><entry>SPSPYSRRRSPSYSRHSSYERGGDVSPSPYSSSSWRRSRSPYSPVLRRSGK</entry></row><row><entry></entry></row><row><entry>SRSRSPYSSRHSRSRSRHRLSRSRSRHSSISPSTLTLKSSLAAELNKNKKA</entry></row><row><entry></entry></row><row><entry>RAAEAARAAEAAKAAEATKAAEAAAKAAKASNTSTPTKGNTETSASASQTN</entry></row><row><entry></entry></row><row><entry>HVKDVKKIKIEHAPSPSSGGTLKNDKAKTKPPLQVTKVENNLIVDKATKKA</entry></row><row><entry></entry></row><row><entry>VIVGKESKSAATKEESVSLKEKTKPLTPSIGAKEKEQHVALVTSTLPPLPL</entry></row><row><entry></entry></row><row><entry>PPMLPEDKEADSLRGNISVKAVKKEVEKKLRCLLADLPLPPELPGGDDLSK</entry></row><row><entry></entry></row><row><entry>SPEEKKTATQLHSKRRPKICGPRYGETKEKDIDWGKRCVDKFDIIGIIGEG</entry></row><row><entry></entry></row><row><entry>TYGQVYKARDKDTGEMVALKKVRLDNEKEGFPITAIREIKILRQLTHQSII</entry></row><row><entry></entry></row><row><entry>NMKEIVTDKEDALDFKKDKGAFYLVFEYMDHDLMGLLESGLVHFNENHIKS</entry></row><row><entry></entry></row><row><entry>FMRQLMEGLDYCHKKNFLHRDIKCSNILLNNRGQIKLADFGLARLYSSEES</entry></row><row><entry></entry></row><row><entry>RPYTNKVITLWYRPPELLLGEERYTPAIDVWSCGCILGELFTKKPIFQANQ</entry></row><row><entry></entry></row><row><entry>ELAQLELISRICGSPCPAVWPDVIKLPYENTMKPKKQYRRKLREEFVFIPA</entry></row><row><entry></entry></row><row><entry>AALDLFDYMLALDPSKRCTAEQALQCEFLRDVEPSKMPPPDLPLWQDCHEL</entry></row><row><entry></entry></row><row><entry>WSKKRRRQKQMGMTDDVSTIKAPRKDLSLGLDDSRTNTPQGVLPSSQLKSQ</entry></row><row><entry></entry></row><row><entry>GSSNVAPVKTGPGQHLNHSELAILLNLLQSKTSVNMADFVQVLNIKVNSET</entry></row><row><entry></entry></row><row><entry>QQQLNKINLPAGILATGEKQTDPSTPQQESSKPLGGIQPSSQTIQPKVETD</entry></row><row><entry></entry></row><row><entry>AAQAAVQSAFAVLLTQLIKAQQSKQKDVLLEERENGSGHEASLQLRPPPEP</entry></row><row><entry></entry></row><row><entry>STPVSGQDDLIQHQDMRILELTPEPDRPRILPPDQRPPEPPEPPPVTEEDL</entry></row><row><entry></entry></row><row><entry>DYRTENQHVPTTSSSLTDPHAGVKAALLQLLAQHQPQDDPKREGGIDYQAG</entry></row><row><entry></entry></row><row><entry>DTYVSTSDYKDNFGSSSFSSAPYVSNDGLGSSSAPPLERRSFIGNSDIQSL</entry></row><row><entry></entry></row><row><entry>DNYSTASSHSGGPPQPSAFSESFPSSVAGYGDIYLNAGPMLFSGDKDHRFE</entry></row><row><entry></entry></row><row><entry>YSHGPIAVLANSSDPSTGPESTHPLPAKMHNYNYGGNLQENPSGPSLMHGQ</entry></row><row><entry></entry></row><row><entry>TWTSPAQGPGYSQGYRGHISTSTGRGRGRGLPY</entry></row></tbody></tgroup></table></tables>

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION

0150Cyclin dependent kinases (CDKs) are key regulators of the cell cycle. Their successive activation and inactivation drives the cycle forward. The activity of CDKs is regulated by multiple mechanisms such as positive and negative phosphorylation, binding of regulatory proteins like cyclins, and CDK inhibitors. CDK7 plays a critical role in the regulation of RNA polymerase II-mediated transcription of protein-encoding genes. Disruption of CDK7, CDK12, and/or CDK13 signaling cause defects in transcription. However, a complete understanding of how these disruptions affect global transcription is lacking. Furthermore, the absence of selective inhibitors of CDK7, CDK12, and CDK13 has hindered investigation of the transcriptional and functional consequences of acute and long-term inhibition of these kinases under normal and pathological conditions. The present invention provides selective CDK7, CDK12, and/or CDK13 inhibitors and analogs, which have the ability to covalently modify a cysteine residue located outside of the canonical kinase domain (i.e., Cys312 of CDK7, Cys1039 of CDK12, and Cys1017 of CDK13). Selective covalent inhibitors of these kinases may be useful in the treatment of various proliferative diseases including cancer.

0151The present invention provides compounds, which inhibit the activity of a kinase, for the prevention and/or treatment of a subject with a proliferative disease. In certain embodiments, the inventive compounds inhibit the activity of cyclin-dependent kinase (CDK). In certain embodiments, the inventive compounds inhibit the activity of a cyclin-dependent kinase 7 (CDK7). In certain embodiments, the inventive compounds inhibit the activity of a cyclin-dependent kinase 12 (CDK12). In certain embodiments, the inventive compounds inhibit the activity of a cyclin-dependent kinase 13 (CDK13). The present invention also provides methods of using the compounds described herein, e.g., as biological probes to study the inhibition of the activity of a kinase (e.g., CDK (e.g. CDK7, CDK12, and/or CDK13)), and as therapeutics, e.g., in the prevention and/or treatment of diseases associated with the overexpression and/or aberrant activity of a kinase (e.g., CDK (e.g. CDK7, CDK12, and/or CDK13)). In certain embodiments, the diseases are proliferative diseases. The proliferative diseases that may be treated and/or prevented include, but are not limited to, cancers (e.g., breast cancer, leukemia, melanoma, multiple myeloma), benign neoplasms, diseases associated with angiogenesis, inflammatory diseases, autoinflammatory diseases, and autoimmune diseases. Also provided by the present disclosure are pharmaceutical compositions, kits, methods, and uses including a compound of Formula (I) as described herein.

0000Compounds

0152Aspects of the present disclosure relate to the compounds described herein. The compounds described herein are purine, pyrazolo-triazine, and pyrazolo-pyrimidine containing compounds that are useful in treating and/or preventing proliferative diseases in a subject, inhibiting the activity of a protein kinase (e.g., CDK) in a subject or biological sample, and inducing apoptosis of a cell. In certain embodiments, a compound described herein is a compound of any one of Formulae (I)-(III), or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof. In certain embodiments, a compound described herein is a compound of Formula (I), or a pharmaceutically acceptable salt thereof. In certain embodiments, a compound described herein is a compound of Formula (II), or a pharmaceutically acceptable salt thereof. In certain embodiments, a compound described herein is a compound of Formula (III), or a pharmaceutically acceptable salt thereof.

0153In certain embodiments, a compound described herein is of Formula (I):

0154<chemistry id="CHEM-US-00009" num="00009"><img file="USRE50776E_D0012.tif" /></chemistry><br /> or a pharmaceutically acceptable salt thereof, wherein:

0155R¹is optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted heterocyclyl, optionally substituted heteroaryl, —NR^aR^b, —OR^b, —SR^b, —C(═O)R^b, —C(═O)OR^b, or —C(═O)NR^aR^b, wherein each instance of R^aand R^bis independently hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or a nitrogen protecting group when attached to nitrogen, or an oxygen protecting group when attached to oxygen, or a sulfur protecting group when attached to sulfur; or R^aand R^bare joined to form an optionally substituted heterocyclic or optionally substituted heteroaryl ring;

0156R³is hydrogen, halogen, or optionally substituted C₁-C₆alkyl;

0157R⁵is hydrogen, optionally substituted C₁-C₆alkyl, or a nitrogen protecting group;

0158L¹is a bond, —NR^L1—(CH₂)_t—, —O—, or —S—;

0159R^L1is hydrogen, optionally substituted C₁-C₆alkyl, or a nitrogen protecting group;

0160t is 0 or an integer between 1 and 5, inclusive;

0161Ring A is optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl;

0162L²is a bond, optionally substituted C_1-4alkylene, —C(═O)—, —NR^L2—, —C(═O)NR^L2—, —NR^L2C(═O)—, —O—, or —S—, wherein R^L2is hydrogen, optionally substituted C₁-C₆alkyl, or a nitrogen protection group;

0163Ring B is absent, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl; and

0164R²is any of Formulae (i-1)-(i-41):

0165<chemistry id="CHEM-US-00010" num="00010"><img file="USRE50776E_D0013.tif" /></chemistry><chemistry id="CHEM-US-00011" num="00011"><img file="USRE50776E_D0014.tif" /></chemistry><chemistry id="CHEM-US-00012" num="00012"><img file="USRE50776E_D0015.tif" /></chemistry><chemistry id="CHEM-US-00013" num="00013"><img file="USRE50776E_D0016.tif" /></chemistry><chemistry id="CHEM-US-00014" num="00014"><img file="USRE50776E_D0017.tif" /></chemistry><br /> wherein:

0166L³is a bond or an optionally substituted C_1-4hydrocarbon chain, optionally wherein one or more carbon units of the hydrocarbon chain are independently replaced with —C═O—, —O—, —S—, —NR^L3a—, —NR^L3aC(═O)—, —C(═O)NR^L3a—, —SC(═O)—, —C(═O)S—, —OC(═O)—, —C(═O)O—, —NR^L3aC(═S)—, —C(═S)NR^L3a—, trans-CR^L3b═CR^L3b—, cis-CR^L3b═CR^L3b—, —C═C—, —S(═O)—, —S(═O)O—, —OS(═O)—, —S(═O)NR^L3a—, —NR^L3aS(═O)—, —S(═O)₂—, —S(═O)₂O—, —OS(═O)₂—, —S(═O)₂NR^L3a—, or —NR^L3aS(═O)₂—, wherein R^L3ais hydrogen, substituted or unsubstituted C_1-6alkyl, or a nitrogen protecting group, and wherein each occurrence of R^L3bis independently hydrogen, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl, or two R^L3bgroups are joined to form an optionally substituted carbocyclic or optionally substituted heterocyclic ring;

0167L⁴is a bond or an optionally substituted, branched or unbranched C_1-6hydrocarbon chain;

0168each of R^E1, R^E2, and R^E3is independently hydrogen, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, —CN, —CH₂OR^EE, —CH₂N(R^EE)₂, —CH₂SR^EE, —OR^EE, —N(R^EE)₂, —Si(R^EE)₃, and —SR^EE, wherein each occurrence of R^EEis independently hydrogen, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl, or two R^EEgroups are joined to form an optionally substituted heterocyclic ring; or R^E1and R^E3, or R^E2and R^E3, or R^E1and R^E2are joined to form an optionally substituted carbocyclic or optionally substituted heterocyclic ring;

0169R^E4is a leaving group;

0170R^E5is halogen;

0171R^E6is hydrogen, substituted or unsubstituted C_1-6alkyl, or a nitrogen protecting group;

0172each instance of Y is independently O, S, or NR^E7, wherein R^E7is hydrogen, substituted or unsubstituted C_1-6alkyl, or a nitrogen protecting group;

0173a is 1 or 2; and

0174each instance of z is independently 0, 1, 2, 3, 4, 5, or 6, as valency permits; and

0175provided that the compound is not

0176<chemistry id="CHEM-US-00015" num="00015"><img file="USRE50776E_D0018.tif" /></chemistry>

0177In certain embodiments, a compound described herein is of Formula (II):

0178<chemistry id="CHEM-US-00016" num="00016"><img file="USRE50776E_D0019.tif" /></chemistry><br /> or a pharmaceutically acceptable salt thereof, wherein:

0179R¹is optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted heterocyclyl, optionally substituted heteroaryl, —NR^aR^b, —OR^b, —SR^b, —C(═O)R^b, —C(═O)OR^b, or —C(═O)NR^aR^b, wherein each instance of R^aand R^bis independently hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or a nitrogen protecting group when attached to nitrogen, or an oxygen protecting group when attached to oxygen, or a sulfur protecting group when attached to sulfur; or R^aand R^bare joined to form an optionally substituted heterocyclic or optionally substituted heteroaryl ring;

0180each of R³and R⁴is independently hydrogen, halogen, or optionally substituted C₁-C₆alkyl;

0181L¹is a bond, —NR^L1—(CH₂)_t—, —O—, or —S—;

0182R^L1is hydrogen, optionally substituted C₁-C₆alkyl, or a nitrogen protecting group;

0183t is 0 or an integer between 1 and 5, inclusive;

0184Ring A is optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl;

0185L²is a bond, optionally substituted C_1-4alkylene, —C(═O)—, —NR^L2—, —C(═O)NR^L2—, —NR^L2C(═O)—, —O—, or —S—, wherein R^L2is hydrogen, optionally substituted C₁-C₆alkyl, or a nitrogen protection group;

0186Ring B is absent, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl; and

0187R²is any of Formulae (i-1)-(i-41):

0188<chemistry id="CHEM-US-00017" num="00017"><img file="USRE50776E_D0020.tif" /></chemistry><chemistry id="CHEM-US-00018" num="00018"><img file="USRE50776E_D0021.tif" /></chemistry><chemistry id="CHEM-US-00019" num="00019"><img file="USRE50776E_D0022.tif" /></chemistry><chemistry id="CHEM-US-00020" num="00020"><img file="USRE50776E_D0023.tif" /></chemistry><chemistry id="CHEM-US-00021" num="00021"><img file="USRE50776E_D0024.tif" /></chemistry><br /> wherein:

0189L³is a bond or an optionally substituted C_1-4hydrocarbon chain, optionally wherein one or more carbon units of the hydrocarbon chain are independently replaced with —C═O—, —O—, —S—, —NR^L3a—, —NR^L3aC(═O)—, —C(═O)NR^L3a—, —SC(═O)—, —C(═O)S—, —OC(═O)—, —C(═O)O—, —NR^L3aC(═S)—, —C(═S)NR^L3a—, trans-CR^L3b═CR^L3b—, cis-CR^L3b═CR^L3b—, —C═C—, —S(═O)—, —S(═O)O—, —OS(═O)—, —S(═O)NR^L3a—, —NR^L3aS(═O)—, —S(═O)₂—, —S(═O)₂O—, —OS(═O)₂—, —S(═O)₂NR^L3a—, or —NR^L3aS(═O)₂—, wherein R^L3a is hydrogen, substituted or unsubstituted C_1-6alkyl, or a nitrogen protecting group, and wherein each occurrence of R^L3bis independently hydrogen, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl, or two R^L3bgroups are joined to form an optionally substituted carbocyclic or optionally substituted heterocyclic ring;

0190L⁴is a bond or an optionally substituted, branched or unbranched C_1-6hydrocarbon chain;

0191each of R^E1, R^E2, and R^E3is independently hydrogen, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, —CN, —CH₂OR^EE, —CH₂N(R^EE)₂, —CH₂SR^EE, —OR^EE, —N(R^EE)₂, —Si(R^EE)₃, and —SR^EE, wherein each occurrence of R^EEis independently hydrogen, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl, or two R^EEgroups are joined to form an optionally substituted heterocyclic ring;

0192or R^E1and R^E3, or R^E2and R^E3, or R^E1and R^E2are joined to form an optionally substituted carbocyclic or optionally substituted heterocyclic ring;

0193R^E4is a leaving group;

0194R^E5is halogen;

0195R^E6is hydrogen, substituted or unsubstituted C_1-6alkyl, or a nitrogen protecting group;

0196each instance of Y is independently O, S, or NR^E7, wherein R^E7is hydrogen, substituted or unsubstituted C_1-6alkyl, or a nitrogen protecting group;

0197a is 1 or 2; and

0198each instance of z is independently 0, 1, 2, 3, 4, 5, or 6, as valency permits.

0199In certain embodiments, a compound described herein is of Formula (III):

0200<chemistry id="CHEM-US-00022" num="00022"><img file="USRE50776E_D0025.tif" /></chemistry><br /> or a pharmaceutically acceptable salt thereof, wherein:

0201R¹is optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted heterocyclyl, optionally substituted heteroaryl, —NR^aR^b, —OR^b, —SR^b, —C(═O)R^b, —C(═O)OR^b, or —C(═O)NR^aR^b, wherein each instance of R^aand R^bis independently hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or a nitrogen protecting group when attached to nitrogen, or an oxygen protecting group when attached to oxygen, or a sulfur protecting group when attached to sulfur; or R^aand R^bare joined to form an optionally substituted heterocyclic or optionally substituted heteroaryl ring;

0202each of R³and R⁴is independently hydrogen, halogen, or optionally substituted C₁-C₆alkyl;

0203L¹is a bond, —NR^L1—(CH₂)_t—, —O—, or —S—;

0204R^L1is hydrogen, optionally substituted C₁-C₆alkyl, or a nitrogen protecting group;

0205t is 0 or an integer between 1 and 5, inclusive;

0206Ring A is optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl;

0207L²is a bond, optionally substituted C_1-4alkylene, —C(═O)—, —NR^L2—, —C(═O)NR^L2—, —NR^L2C(═O)—, —O—, or —S—, wherein R^L2is hydrogen, optionally substituted C₁-C₆alkyl, or a nitrogen protection group;

0208Ring B is absent, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl; and

0209R²is any of Formulae (i-1)-(i-41):

0210<chemistry id="CHEM-US-00023" num="00023"><img file="USRE50776E_D0026.tif" /></chemistry><chemistry id="CHEM-US-00024" num="00024"><img file="USRE50776E_D0027.tif" /></chemistry><chemistry id="CHEM-US-00025" num="00025"><img file="USRE50776E_D0028.tif" /></chemistry><chemistry id="CHEM-US-00026" num="00026"><img file="USRE50776E_D0029.tif" /></chemistry><chemistry id="CHEM-US-00027" num="00027"><img file="USRE50776E_D0030.tif" /></chemistry><br /> wherein:

0211L³is a bond or an optionally substituted C_1-4hydrocarbon chain, optionally wherein one or more carbon units of the hydrocarbon chain are independently replaced with —C═O—, —O—, —S—, —NR^L3a—, —NR^L3aC(═O)—, —C(═O)NR^L3a—, —SC(═O)—, —C(═O)S—, —OC(═O)—, —C(═O)O—, —NR^L3aC(═S)—, —C(═S)NR^L3a—, trans-CR^L3b═CR^L3b—, cis-CR^L3b═CR^L3b—, —C═C—, —S(═O)—, —S(═O)O—, —OS(═O)—, —S(═O)NR^L3a—, —NR^L3aS(═O)—, —S(═O)₂—, —S(═O)₂O—, —OS(═O)₂—, —S(═O)₂NR^L3a—, or —NR^L3aS(═O)₂—, wherein R^L3ais hydrogen, substituted or unsubstituted C_1-6alkyl, or a nitrogen protecting group, and wherein each occurrence of R^L3bis independently hydrogen, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl, or two R^L3bgroups are joined to form an optionally substituted carbocyclic or optionally substituted heterocyclic ring;

0212L⁴is a bond or an optionally substituted, branched or unbranched C_1-6hydrocarbon chain;

0213each of R^E1, R^E2, and R^E3is independently hydrogen, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, —CN, —CH₂OR^EE, —CH₂N(R^EE)₂, —CH₂SR^EE, —OR^EE, —N(R^EE)₂, —Si(R^EE)₃, and —SR^EE, wherein each occurrence of R^EEis independently hydrogen, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl, or two R^EEgroups are joined to form an optionally substituted heterocyclic ring;

0214or R^E1and R^E3, or R^E2and R^E3, or R^E1and R^E2are joined to form an optionally substituted carbocyclic or optionally substituted heterocyclic ring;

0215R^E4is a leaving group;

0216R^E5is halogen;

0217R^E6is hydrogen, substituted or unsubstituted C_1-6alkyl, or a nitrogen protecting group;

0218each instance of Y is independently 0, S, or NR^E7, wherein R^E7is hydrogen, substituted or unsubstituted C_1-6alkyl, or a nitrogen protecting group;

0219a is 1 or 2; and

0220each instance of z is independently 0, 1, 2, 3, 4, 5, or 6, as valency permits.

0221As generally defined herein in Formulae (I)-(III), R¹is optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted heterocyclyl, optionally substituted heteroaryl, —NR^aR^b, —OR^b, —SR^b, —C(═O)R^b, —C(═O)OR^b, or —C(═O)NR^aR^b, wherein each instance of R^aand R^bis independently hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or a nitrogen protecting group when attached to nitrogen, or an oxygen protecting group when attached to oxygen, or a sulfur protecting group when attached to sulfur; or R^aand R^bare joined to form an optionally substituted heterocyclic or optionally substituted heteroaryl ring.

0222In certain embodiments, R¹is optionally substituted alkyl. In certain embodiments, R¹is unsubstituted alkyl. In certain embodiments, R¹is substituted alkyl. In certain embodiments, R¹is substituted alkyl. In certain embodiments, R¹is optionally substituted carbocyclylalkyl, optionally substituted arylalkyl, optionally substituted heterocyclylalkyl, or optionally substituted heteroarylalkyl. In certain embodiments, R¹is optionally substituted heteroaryl. In certain embodiments, R¹is optionally substituted monocyclic heteroaryl. In certain embodiments, R¹is optionally substituted 5-membered heteroaryl. In certain embodiments, R¹is optionally substituted 6-membered heteroaryl. In certain embodiments, R¹is optionally substituted heterocyclyl. In certain embodiments, R¹is optionally substituted monocyclic heterocyclyl. In certain embodiments, R¹is optionally substituted 5-membered heterocyclyl. In certain embodiments, R¹is optionally substituted 6-membered heterocyclyl. In certain embodiments, R¹is —OR^b, wherein R^bis as defined herein. In certain embodiments, R¹is —OR^b, wherein R^bis optionally substituted alkyl, optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or an oxygen protecting group. In certain embodiments, R¹is —SR^b, wherein R^bis as defined herein. In certain embodiments, R¹is —SR^b, wherein R^bis optionally substituted alkyl, optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or a sulfur protecting group. In certain embodiments, R¹is —C(═O)R^b, wherein R^bis as defined herein. In certain embodiments, R¹is —C(═O)R^b, wherein R^bis optionally substituted alkyl, optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl. In certain embodiments, R¹is —C(═O)OR^b, wherein R^bis as defined herein. In certain embodiments, R¹is —C(═O)OR^b, wherein R^bis optionally substituted alkyl, optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or an oxygen protecting group. In certain embodiments, R¹is C(═O)NR^aR^b, wherein R^aand R^bare as defined herein. In certain embodiments, R¹is C(═O)NR^aR^b, wherein each instance of R^aand R^bis independently optionally substituted alkyl, optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or a nitrogen protecting group, or R^aand R^bare taken together with the nitrogen to form an optionally substituted heterocyclyl ring. In certain embodiments, R¹is C(═O)NR^aR^b, wherein R^ais hydrogen or optionally substituted alkyl, and R^bis independently optionally substituted alkyl, optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or a nitrogen protecting group, or R^aand R^bare taken together with the nitrogen to form an optionally substituted heterocyclyl ring.

0223In certain embodiments, R¹is —NR^aR^b, wherein R^aand R^bare as defined herein. In certain embodiments, R¹is —NR^aR^b, wherein each of R^aand R^bis independently hydrogen, optionally substituted alkyl, optionally substituted carbocyclyl, or a nitrogen protecting group. In certain embodiments, R¹is —NR^aR^b, wherein R^ais hydrogen, optionally substituted alkyl, or optionally substituted carbocyclyl; and R^bis independently hydrogen, optionally substituted alkyl, or a nitrogen protecting group.

0224In certain embodiments of Formulae (I)-(III), R¹is of Formula (n-1):

0225<chemistry id="CHEM-US-00028" num="00028"><img file="USRE50776E_D0031.tif" /></chemistry><br /> wherein:

0226each instance of R^1ais independently hydrogen, halogen, optionally substituted C₁-C₆alkyl, —N(R^N1)₂, or —OR^O1;

0227each instance of R^N1is independently hydrogen, optionally substituted C₁-C₆alkyl, or a nitrogen protecting group;

0228R^O1is independently hydrogen, optionally substituted C₁-C₆alkyl, or an oxygen protecting group; and

0229a1 is 0 or an integer between 1 and 6, inclusive.

0230In certain embodiments, a1 is 1. In certain embodiments, a1 is 2. In certain embodiments, a1 is 3.

0231In certain embodiments, R^1ais optionally substituted C₁-C₆alkyl. In certain embodiments, R^1ais unsubstituted C₁-C₆alkyl. In certain embodiments, R^1ais methyl or ethyl. In certain embodiments, R^1ais substituted C₁-C₆alkyl. In certain embodiments, R^1ais hydroxy C₁-C₆alkyl. In certain embodiments, R^1ais —CH₂OH. In certain embodiments, R^1ais —CH₂CH₂OH. In certain embodiments, R^1ais —N(R^N1)₂, wherein R^N1is as defined herein. In certain embodiments, R^1ais —NHR^N1, wherein R^N1is as defined herein. In certain embodiments, R^1ais —NHR^N1, wherein R^N1is hydrogen or optionally substituted C₁-C₆alkyl. In certain embodiments, R^1ais —NH₂. In certain embodiments, R^1ais —NHR^N1, wherein R^N1is optionally substituted C₁-C₆alkyl. In certain embodiments, R^1ais —NHR^N1, wherein R^N1is unsubstituted C₁-C₆alkyl. In certain embodiments, R^1ais —NHR^N1, wherein R^N1is methyl or ethyl. In certain embodiments, R^1ais —NHCH₃. In certain embodiments, R^1ais —NHR^N1, wherein R^N1is a nitrogen protecting group. In certain embodiments, R^1ais —N(CH₃)R^N1, wherein R^N1is optionally substituted C₁-C₆alkyl. In certain embodiments, R^1ais —N(CH₃)R^N1, wherein R^N1is unsubstituted C₁-C₆alkyl. In certain embodiments, R^1ais —N(CH₃)R^N1, wherein R^N1is methyl or ethyl. In certain embodiments, R^1ais —N(CH₃)₂. In certain embodiments, R^1ais —N(CH₃)R^N1, wherein R^N1is a nitrogen protecting group.

0232In certain embodiments of Formulae (I)-(III), R¹is —NR^aR^b, wherein R^aand R^bare joined to form an optionally substituted heterocyclic or optionally substituted heteroaryl ring. In certain embodiments, R¹is of one of the following formulae:

0233<chemistry id="CHEM-US-00029" num="00029"><img file="USRE50776E_D0032.tif" /></chemistry><br /> wherein:

0234each instance of R^NXis independently hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted heterocyclyl, optionally substituted heteroaryl, or a nitrogen protecting group;

0235each instance of R^1bis independently hydrogen, halogen, optionally substituted C₁-C₆alkyl, —N(R^N1)₂, or —OR^O1;

0236each instance of R^N1is independently hydrogen, optionally substituted C₁-C₆alkyl, or a nitrogen protecting group;

0237R^O1is independently hydrogen, optionally substituted C₁-C₆alkyl, or an oxygen protecting group; and

0238b1 is 0 or an integer between 1 and 6, inclusive, as valency permits.

0239In certain embodiments, R¹is of Formula (n-2):

0240<chemistry id="CHEM-US-00030" num="00030"><img file="USRE50776E_D0033.tif" /></chemistry><br /> wherein:

0241each instance of R^1bis independently hydrogen, halogen, optionally substituted C₁-C₆alkyl, —N(R^N1)₂, or —OR^O1;

0242each instance of R^N1is independently hydrogen, optionally substituted C₁-C₆alkyl, or a nitrogen protecting group;

0243R^O1is independently hydrogen, optionally substituted C₁-C₆alkyl, or an oxygen protecting group; and

0244b1 is 0 or an integer between 1 and 6, inclusive.

0245In certain embodiments, b1 is 1. In certain embodiments, b1 is 2. In certain embodiments, b1 is 3.

0246In certain embodiments, R^1bis optionally substituted C₁-C₆alkyl. In certain embodiments, R^1bis unsubstituted C₁-C₆alkyl. In certain embodiments, R^1bis methyl or ethyl. In certain embodiments, R^1bis substituted C₁-C₆alkyl. In certain embodiments, R^1bis hydroxy C₁-C₆alkyl. In certain embodiments, R^1bis —CH₂OH. In certain embodiments, R^1bis —CH₂CH₂OH. In certain embodiments, R^1bis —N(R^N1)₂, wherein R^N1is as defined herein. In certain embodiments, R^1bis —NHR^N1, wherein R^N1is as defined herein. In certain embodiments, R^1bis —NHR^N1, wherein R^N1is hydrogen or optionally substituted C₁-C₆alkyl. In certain embodiments, R^1bis —NH₂. In certain embodiments, R^1bis —NHR^N1, wherein R^N1is optionally substituted C₁-C₆alkyl. In certain embodiments, R^1bis —NHR^N1, wherein R^N1is unsubstituted C₁-C₆alkyl. In certain embodiments, R^1bis —NHR^N1, wherein R^N1is methyl or ethyl. In certain embodiments, R^1bis —NHCH₃. In certain embodiments, R^1bis —NHR^N1, wherein R^N1is a nitrogen protecting group. In certain embodiments, R^1bis —N(CH₃)R^N1, wherein R^N1is optionally substituted C₁-C₆alkyl. In certain embodiments, R^1bis —N(CH₃)R^N1, wherein R^N1is unsubstituted C₁-C₆alkyl. In certain embodiments, R^1bis —N(CH₃)R^N1, wherein R^N1is methyl or ethyl. In certain embodiments, R^1bis —N(CH₃)₂. In certain embodiments, R^1bis —N(CH₃)R^N1, wherein R^N1is a nitrogen protecting group.

0247In certain embodiments, R^NXis hydrogen. In certain embodiments, R^NXis optionally substituted alkyl. In certain embodiments, R^NXis unsubstituted C₁-C₆alkyl (e.g. methyl or ethyl). In certain embodiments, R^NXis substituted C₁-C₆alkyl. In certain embodiments, R^NXis a nitrogen protecting group.

0248In certain embodiments of Formulae (I)-(III), R¹is optionally substituted carbocyclyl. In certain embodiments, R¹is optionally substituted monocyclic carbocyclyl. In certain embodiments, R¹is optionally substituted cyclopentyl. In certain embodiments, R¹is optionally substituted cyclohexyl of Formula (n-3)

0249<chemistry id="CHEM-US-00031" num="00031"><img file="USRE50776E_D0034.tif" /></chemistry><br /> wherein:

0250each instance of R^1cis independently hydrogen, halogen, optionally substituted C₁-C₆alkyl, —N(R^N1)₂, or —OR^O1;

0251each instance of R^N1is independently hydrogen, optionally substituted C₁-C₆alkyl, or a nitrogen protecting group;

0252R^O1is independently hydrogen, optionally substituted C₁-C₆alkyl, or an oxygen protecting group; and

0253c1 is 0 or an integer between 1 and 6, inclusive.

0254In certain embodiments, c1 is 1. In certain embodiments, c1 is 2. In certain embodiments, c1 is 3.

0255In certain embodiments, R^1cis optionally substituted C₁-C₆alkyl. In certain embodiments, R^1cis unsubstituted C₁-C₆alkyl. In certain embodiments, R^1cis methyl or ethyl. In certain embodiments, R^1cis substituted C₁-C₆alkyl. In certain embodiments, R^1cis hydroxy C₁-C₆alkyl. In certain embodiments, R^1cis —CH₂OH. In certain embodiments, R^1cis —CH₂CH₂OH. In certain embodiments, R^1cis —N(R^N1)₂, wherein R^N1is as defined herein. In certain embodiments, R^1cis —NHR^N1, wherein R^N1is as defined herein. In certain embodiments, R^1cis —NHR^N1, wherein R^N1is hydrogen or optionally substituted C₁-C₆alkyl. In certain embodiments, R^1cis —NH₂. In certain embodiments, R^1cis —NHR^N1, wherein R^N1is optionally substituted C₁-C₆alkyl. In certain embodiments, R^1cis —NHR^N1, wherein R^N1is unsubstituted C₁-C₆alkyl. In certain embodiments, R^1cis —NHR^N1, wherein R^N1is methyl or ethyl. In certain embodiments, R^1cis —NHCH₃. In certain embodiments, R^1cis —NHR^N1, wherein R^N1is a nitrogen protecting group. In certain embodiments, R^1cis —N(CH₃)R^N1, wherein R^N1is optionally substituted C₁-C₆alkyl. In certain embodiments, R^1cis —N(CH₃)R^N1, wherein R^N1is unsubstituted C₁-C₆alkyl. In certain embodiments, R^1cis —N(CH₃)R^N1, wherein R^N1is methyl or ethyl. In certain embodiments, R^1cis —N(CH₃)₂. In certain embodiments, R^1cis —N(CH₃)R^N1, wherein R^N1is a nitrogen protecting group.

0256In certain embodiments of Formulae (I)-(III), R¹is optionally substituted aryl. In certain embodiments, R¹is optionally substituted monocyclic aryl. In certain embodiments, R¹is optionally substituted phenyl of Formula (n-4)

0257<chemistry id="CHEM-US-00032" num="00032"><img file="USRE50776E_D0035.tif" /></chemistry><br /> wherein:

0258each instance of R^1dis independently hydrogen, halogen, —CN, —NO₂, —N₃, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted heterocyclyl, optionally substituted heteroaryl, —OR^A, —N(R^B)₂, —SR^A, —C(═O)R^C, —C(═O)OR^A, —OC(═O)R^C, —C(═O)N(R^B)₂, —NR^BC(═O)R^C, —OC(═O)N(R^B)₂, —NR^BC(═O)OR^A, —NR^BC(═O)N(R^B)₂, S(═O)R^C, —SO₂R^C, —NR^BSO₂R^C, or —SO₂N(R^B)₂;

0259each instance of R^Ais hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, an oxygen protecting group when attached to oxygen, or a sulfur protecting group when attached to sulfur;

0260each instance of R^Bis independently hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted acyl, or a nitrogen protecting group, or two R^Bgroups are taken together with their intervening atoms to form an optionally substituted heterocyclic ring; and

0261d1 is 0 or an integer between 1 and 5, inclusive.

0262In certain embodiments, d1 is 0. In certain embodiments, d1 is 1. In certain embodiments, d1 is 2. In certain embodiments, d1 is 3.

0263In certain embodiments, R^1dis hydrogen. In certain embodiments, R^1dis halogen (e.g. F, Cl, Br, or I). In certain embodiments, R^1dis optionally substituted alkyl. In certain embodiments, R^1dis unsubstituted alkyl. In certain embodiments, R^1dis methyl or ethyl.

0264As generally defined herein in Formulae (I)-(III), R³is hydrogen, halogen, or optionally substituted C₁-C₆alkyl. In certain embodiments, R³is hydrogen. In certain embodiments, R³is halogen (e.g. F, Cl, Br, or I). In certain embodiments, R³is optionally substituted C₁-C₆alkyl. In certain embodiments, R³is unsubstituted C₁-C₆alkyl. In certain embodiments, R³is methyl, ethyl, n-propyl, or isopropyl. In certain embodiments, R³is substituted C₁-C₆alkyl.

0265As generally defined herein in Formulae (II)-(III), R⁴is hydrogen, halogen, or optionally substituted C₁-C₆alkyl. In certain embodiments, R⁴is hydrogen. In certain embodiments, R⁴is halogen (e.g. F, Cl, Br, or I). In certain embodiments, R⁴is optionally substituted C₁-C₆alkyl. In certain embodiments, R⁴is unsubstituted C₁-C₆alkyl. In certain embodiments, R⁴is methyl, ethyl, n-propyl, or isopropyl. In certain embodiments, R⁴is substituted C₁-C₆alkyl.

0266As generally defined herein in Formula (I), R⁵is hydrogen, optionally substituted C₁-C₆alkyl, or a nitrogen protecting group. In certain embodiments, R⁵is hydrogen. In certain embodiments, R⁵is optionally substituted C₁-C₆alkyl. In certain embodiments, R⁵is unsubstituted C₁-C₆alkyl. In certain embodiments, R⁵is methyl, ethyl, n-propyl, or isopropyl. In certain embodiments, R⁵is substituted C₁-C₆alkyl. In certain embodiments, R⁵is nitrogen protecting group. In certain embodiments, R⁵is nitrogen protecting group.

0267As generally defined herein in Formulae (I)-(III), L¹is a bond, —NR^L1—(CH₂)_t—, —O—, or —S—, wherein R^L1is hydrogen, optionally substituted C₁-C₆alkyl, or a nitrogen protecting group, wherein t is 0 or an integer between 1 and 5, inclusive. In certain embodiments, L¹is a bond. In certain embodiments, L¹is —O—. In certain embodiments, L¹is —S—. In certain embodiments, L¹is —NR^L1—(CH₂)_t—, wherein R^L1is hydrogen, optionally substituted C₁-C₆alkyl, or a nitrogen protecting group. In certain embodiments, t is 0. In certain embodiments, t is 1. In certain embodiments, t is 2. In certain embodiments, t is 3. In certain embodiments, t is 4. In certain embodiments, t is 5. In certain embodiments, L¹is —NR^L1—, wherein R^L1is hydrogen, optionally substituted C₁-C₆alkyl, or a nitrogen protecting group. In certain embodiments, L¹is —NH—. In certain embodiments, L¹is —NH—. In certain embodiments, L¹is —NR^L1—, wherein R^L1is optionally substituted C₁-C₆alkyl. In certain embodiments, L¹is —NR^L1—, wherein R^L1is unsubstituted C₁-C₆alkyl. In certain embodiments, L¹is —NR^L1—, wherein R^L1is methyl or ethyl. In certain embodiments, L¹is —NR^L1—, wherein R^L1is substituted C₁-C₆alkyl. In certain embodiments, L¹is —NR^L1—, wherein R^L1is a nitrogen protecting group (e.g. Boc). In certain embodiments, L¹is —NR^L1—CH₂—, wherein R^L1is hydrogen, optionally substituted C₁-C₆alkyl, or a nitrogen protecting group. In certain embodiments, L¹is —NHCH₂—. In certain embodiments, L¹is —NR^L1—CH₂—, wherein R^L1is optionally substituted C₁-C₆alkyl. In certain embodiments, L¹is —NR^L1—CH₂—, wherein R^L1is unsubstituted C₁-C₆alkyl. In certain embodiments, L¹is —NR^L1—CH₂—, wherein R^L1is methyl or ethyl. In certain embodiments, L¹is —NR^L1—CH₂—, wherein R^L1is substituted C₁-C₆alkyl. In certain embodiments, L¹is —NR^L1—CH₂—, wherein R^L1is a nitrogen protecting group (e.g. Boc).

0268As generally defined herein in Formulae (I)-(III), L²is a bond, optionally substituted C_1-4alkylene, —C(═O)—, —NR^L2—, —C(═O)NR^L2—, —NR^L2C(═O)—, —O—, or —S—, wherein R^L2is hydrogen, optionally substituted C₁-C₆alkyl, or a nitrogen protection group. In certain embodiments, L²is a bond. In certain embodiments, L²is —O—. In certain embodiments, L²is —S—. In certain embodiments, L²is optionally substituted C_1-4alkylene. In certain embodiments, L²is unsubstituted C_1-4alkylene. In certain embodiments, L²is —CH₂—. In certain embodiments, L²is —(CH₂)₂—. In certain embodiments, L²is —(CH₂)₃—. In certain embodiments, L²is —(CH₂)₄—. In certain embodiments, L²is —C(═O)—. In certain embodiments, L²is —NR^L2—, wherein R^L2is hydrogen, optionally substituted C₁-C₆alkyl, or a nitrogen protecting group. In certain embodiments, L²is —NH—. In certain embodiments, L²is —NH—. In certain embodiments, L²is —NR^L2—, wherein R^L2is optionally substituted C₁-C₆alkyl. In certain embodiments, L²is —NR^L2—, wherein R^L2is unsubstituted C₁-C₆alkyl. In certain embodiments, L²is —NR^L2—, wherein R^L2is methyl or ethyl. In certain embodiments, L²is —NR^L2—, wherein R^L2is substituted C₁-C₆alkyl. In certain embodiments, L²is —NR^L2—, wherein R^L2is a nitrogen protecting group (e.g. Boc). In certain embodiments, L²is —C(═O)NR^L2—, wherein R^L2is hydrogen, optionally substituted C₁-C₆alkyl, or a nitrogen protecting group. In certain embodiments, L²is —C(═O)NH—. In certain embodiments, L²is —C(═O) wherein R^L2is optionally substituted C₁-C₆alkyl. In certain embodiments, L²is —C(═O)NR^L2—, wherein R^L2is unsubstituted C₁-C₆alkyl. In certain embodiments, L²is —C(═O)NR^L2—, wherein R^L2is methyl or ethyl. In certain embodiments, L²is —C(═O)NR^L2—, wherein R^L2is substituted C₁-C₆alkyl. In certain embodiments, L²is —C(═O)NR^L2—, wherein R^L2is a nitrogen protecting group (e.g. Boc). In certain embodiments, L²is —NR^L2C(═O)—, wherein R^L2is hydrogen, optionally substituted C₁-C₆alkyl, or a nitrogen protecting group. In certain embodiments, L²is —NHC(═O)—. In certain embodiments, L²is —NR^L2C(═O)—, wherein R^L2is optionally substituted C₁-C₆alkyl. In certain embodiments, L²is —NR^L2C(O)—, wherein R^L2is unsubstituted C₁-C₆alkyl. In certain embodiments, L²is —NR^L2C(═O)—, wherein R^L2is methyl or ethyl. In certain embodiments, L²is —NR^L2C(═O)—, wherein R^L2is substituted C₁-C₆alkyl. In certain embodiments, L²is —NR^L2C(═O)—, wherein R^L2is a nitrogen protecting group (e.g. Boc).

0269In certain embodiments of Formula (I), R³is hydrogen and R⁵is optionally substituted C₁-C₆alkyl or a nitrogen protecting group. In certain embodiments of Formula (I), R³is hydrogen and R⁵is optionally substituted C₁-C₆alkyl. In certain embodiments of Formula (I), R³is hydrogen and R⁵is unsubstituted C₁-C₆alkyl. In certain embodiments of Formula (I), R³is hydrogen and R⁵is methyl, ethyl, n-propyl, or isopropyl.

0270In certain embodiments of Formula (II) or (III), R³is hydrogen and R⁴is optionally substituted C₁-C₆alkyl or a nitrogen protecting group. In certain embodiments of Formula (II) or (III), R³is hydrogen and R⁴is optionally substituted C₁-C₆alkyl. In certain embodiments of Formula (II) or (III), R³is hydrogen and R⁴is unsubstituted C₁-C₆alkyl. In certain embodiments of Formula (II) or (III), R³is hydrogen and R⁴is methyl, ethyl, n-propyl, or isopropyl.

0271In certain embodiments of Formula (I), R¹is optionally substituted carbocyclyl; R³is hydrogen; and R⁵is optionally substituted C₁-C₆alkyl or a nitrogen protecting group. In certain embodiments of Formula (I), R¹is optionally substituted cyclohexyl; R³is hydrogen; and R⁵is optionally substituted C₁-C₆alkyl. In certain embodiments of Formula (I), R¹is mono-substituted cyclohexyl; R³is hydrogen; and R⁵is unsubstituted C₁-C₆alkyl. In certain embodiments of Formula (I), R¹is optionally substituted aryl; R³is hydrogen; and R⁵is optionally substituted C₁-C₆alkyl or a nitrogen protecting group. In certain embodiments of Formula (I), R¹is optionally substituted phenyl; R³is hydrogen; and R⁵is optionally substituted C₁-C₆alkyl. In certain embodiments of Formula (I), R¹is mono-substituted phenyl; R³is hydrogen; and R⁵is unsubstituted C₁-C₆alkyl. In certain embodiments of Formula (I), R¹is —NR^aR^b, wherein R^aand R^bare as defined herein; R³is hydrogen; and R⁵is optionally substituted C₁-C₆alkyl or a nitrogen protecting group. In certain embodiments of Formula (I), R¹is —NR^aR^b, wherein R^ais optionally substituted carbocyclyl and R^bis hydrogen or optionally substituted alkyl; R³is hydrogen; and R⁵is optionally substituted C₁-C₆alkyl. In certain embodiments of Formula (I), R¹is —NR^aR^b, wherein R^ais optionally substituted cyclohexyl and R^bis hydrogen; R³is hydrogen; and R⁵is unsubstituted C₁-C₆alkyl. In certain embodiments of Formula (I), R¹is —NR^aR^b, wherein R^aand R^bare joined to form an optionally substituted heterocylic ring; R³is hydrogen; and R⁵is optionally substituted C₁-C₆alkyl. In certain embodiments of Formula (I), R¹is —NR^aR^b, wherein R^aand R^bare joined to form an optionally substituted piperidine ring; R³is hydrogen; and R⁵is unsubstituted C₁-C₆alkyl. In certain embodiments of Formula (I), R¹is —NR^aR^b, wherein R^aand R^bare joined to form a mono-substituted piperidine ring; R³is hydrogen; and R⁵is unsubstituted C₁-C₆alkyl.

0272In certain embodiments of Formula (II) or (III), R³is hydrogen and R⁴is optionally substituted C₁-C₆alkyl or a nitrogen protecting group. In certain embodiments of Formula (II) or (III), R³is hydrogen and R⁴is optionally substituted C₁-C₆alkyl. In certain embodiments of Formula (II) or (III), R³is hydrogen and R⁴is unsubstituted C₁-C₆alkyl. In certain embodiments of Formula (II) or (III), R³is hydrogen and R⁴is methyl, ethyl, n-propyl, or isopropyl.

0273In certain embodiments of Formula (II) or (III), R¹is optionally substituted carbocyclyl; R³is hydrogen; and R⁴is optionally substituted C₁-C₆alkyl or a nitrogen protecting group. In certain embodiments of Formula (II) or (III), R¹is optionally substituted cyclohexyl; R³is hydrogen; and R⁴is optionally substituted C₁-C₆alkyl. In certain embodiments of Formula (II) or (III), R¹is mono-substituted cyclohexyl; R³is hydrogen and R⁴is unsubstituted C₁-C₆alkyl. In certain embodiments of Formula (II) or (III), R¹is optionally substituted aryl; R³is hydrogen and R⁴is optionally substituted C₁-C₆alkyl or a nitrogen protecting group. In certain embodiments of Formula (II) or (III), R¹is optionally substituted phenyl; R³is hydrogen; and R⁴is optionally substituted C₁-C₆alkyl. In certain embodiments of Formula (II) or (III), R¹is mono-substituted phenyl; R³is hydrogen; and R⁴is unsubstituted C₁-C₆alkyl. In certain embodiments of Formula (II) or (III), R¹is —NR^aR^b, wherein R^aand R^bare as defined herein; R³is hydrogen; and R⁴is optionally substituted C₁-C₆alkyl or a nitrogen protecting group. In certain embodiments of Formula (II) or (III), R¹is —NR^aR^b, wherein R^ais optionally substituted carbocyclyl and R^bis hydrogen or optionally substituted alkyl; R³is hydrogen and R⁴is optionally substituted C₁-C₆alkyl. In certain embodiments of Formula (II) or (III), R¹is —NR^aR^b, wherein R^ais optionally substituted cyclohexyl and R^bis hydrogen; R³is hydrogen; and R⁴is unsubstituted C₁-C₆alkyl. In certain embodiments of Formula (II) or (III), R¹is —NR^aR^b, wherein R^aand R^bare joined to form an optionally substituted heterocylic ring; R³is hydrogen; and R⁴is optionally substituted C₁-C₆alkyl. In certain embodiments of Formula (II) or (III), R¹is —NR^aR^b, wherein R^aand R^bare joined to form an optionally substituted piperidine ring; R³is hydrogen; and R⁴is unsubstituted C₁-C₆alkyl. In certain embodiments of Formula (II) or (III), R¹is —NR^aR^b, wherein R^aand R^bare joined to form a mono-substituted piperidine ring; R³is hydrogen; and R⁴is methyl, ethyl, n-propyl, or isopropyl.

0274Compounds of any one of Formulae (I)-(III) include Ring A between linker L¹and linker L². Ring A may be optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl. In certain embodiments, Ring A is optionally substituted carbocyclyl. In certain embodiments, Ring A is optionally substituted heterocyclyl. In certain embodiments, Ring A is optionally substituted aryl. In certain embodiments, Ring A is optionally substituted heteroaryl. In certain embodiments, Ring A is optionally substituted phenyl. In certain embodiments, Ring A is phenyl substituted with only L¹and L². In certain embodiments, Ring A is optionally substituted cyclohexyl. In certain embodiments, Ring A is optionally substituted piperidinyl. In certain embodiments, Ring A is optionally substituted piperizinyl. In certain embodiments, Ring A is optionally substituted pyridinyl. In certain embodiments, Ring A is optionally substituted pyrimidinyl.

0275In certain embodiments of Formulae (I)-(III), linkers L¹and L²are attached “ortho” or 1.2 to Ring A. In certain embodiments, linkers L¹and L²are attached “meta” or 1.3 to Ring A. In certain embodiments, linkers L¹and L²are attached “para” or 1.4 to ring A.

0276In certain embodiments of Formulae (I)-(III), Ring A is

0277<chemistry id="CHEM-US-00033" num="00033"><img file="USRE50776E_D0036.tif" /></chemistry><br /> wherein each ring atom is optionally substituted. In certain embodiments, Ring A is

0278<chemistry id="CHEM-US-00034" num="00034"><img file="USRE50776E_D0037.tif" /></chemistry><br /> wherein each ring atom is optionally substituted. In certain embodiments, Ring A is

0279<chemistry id="CHEM-US-00035" num="00035"><img file="USRE50776E_D0038.tif" /></chemistry><br /> wherein each ring atom is optionally substituted. In certain embodiments, Ring A is

0280<chemistry id="CHEM-US-00036" num="00036"><img file="USRE50776E_D0039.tif" /></chemistry><br /> wherein each ring atom is optionally substituted, and L¹and L²may attach to ring A at either indicated position. In certain embodiments, Ring A is

0281<chemistry id="CHEM-US-00037" num="00037"><img file="USRE50776E_D0040.tif" /></chemistry><br /> wherein each ring atom is optionally substituted, and L¹and L²may attach to ring A at either indicated position. In certain embodiments, Ring A is

0282<chemistry id="CHEM-US-00038" num="00038"><img file="USRE50776E_D0041.tif" /></chemistry><br /> wherein each ring atom is optionally substituted, and L¹and L²may attach to ring A at either indicated position. In certain embodiments, Ring A is

0283<chemistry id="CHEM-US-00039" num="00039"><img file="USRE50776E_D0042.tif" /></chemistry><br /> wherein each ring atom is optionally substituted, and L¹and L²may attach to ring A at either indicated position.

0284In certain embodiments of Formulae (I)-(III), Ring A is

0285<chemistry id="CHEM-US-00040" num="00040"><img file="USRE50776E_D0043.tif" /></chemistry><br /> In certain embodiments, Ring A is

0286<chemistry id="CHEM-US-00041" num="00041"><img file="USRE50776E_D0044.tif" /></chemistry><br /> In certain embodiments, Ring A is

0287<chemistry id="CHEM-US-00042" num="00042"><img file="USRE50776E_D0045.tif" /></chemistry><br /> In certain embodiments, Ring A is

0288<chemistry id="CHEM-US-00043" num="00043"><img file="USRE50776E_D0046.tif" /></chemistry><br /> L¹and L²may attach to ring A at either indicated position. In certain embodiments, Ring A is

0289<chemistry id="CHEM-US-00044" num="00044"><img file="USRE50776E_D0047.tif" /></chemistry><br /> wherein L¹is attached to position a and L²is attached to position b. In certain embodiments, Ring A is

0290<chemistry id="CHEM-US-00045" num="00045"><img file="USRE50776E_D0048.tif" /></chemistry><br /> wherein L²is attached to position a and L¹is attached to position b. In certain embodiments, Ring A is

0291<chemistry id="CHEM-US-00046" num="00046"><img file="USRE50776E_D0049.tif" /></chemistry><br /> wherein L¹and L²may attach to ring A at either indicated position. In certain embodiments, Ring A is

0292<chemistry id="CHEM-US-00047" num="00047"><img file="USRE50776E_D0050.tif" /></chemistry><br /> wherein L¹and L²may attach to ring A at either indicated position.

0293Compounds of Formulae (I)-(III) include Ring B between linker L²and group R². In certain embodiments, linker L²is a bond, such that Ring B is directly attached to Ring A. Ring B may absent, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl. In certain embodiments, Ring B is absent, such that L²is directly attached to R². In certain embodiments, Ring B is absent and linker L²is a bond, such that Ring A is directly attached to R². In certain embodiments, Ring B is optionally substituted carbocyclyl. In certain embodiments, Ring B is optionally substituted heterocyclyl. In certain embodiments, Ring B is optionally substituted aryl. In certain embodiments, Ring B is optionally substituted heteroaryl. In certain embodiments, Ring B is optionally substituted phenyl. In certain embodiments, Ring B is optionally substituted cyclohexyl. In certain embodiments, Ring B is optionally substituted piperidinyl. In certain embodiments, Ring B is optionally substituted piperizinyl. In certain embodiments, Ring B is optionally substituted pyridinyl. In certain embodiments, Ring B is optionally substituted pyrimidinyl.

0294In certain embodiments of Formulae (I)-(III), linker L²and group R²are attached “ortho” or 1.2 to each other on Ring B. In certain embodiments, linkers L²and group R²are attached “meta” or 1.2 to each other on Ring B. In certain embodiments, linkers L²and R²are attached “para” or 1.4 to each other on Ring B.

0295In certain embodiments of Formulae (I)-(III), Ring B is

0296<chemistry id="CHEM-US-00048" num="00048"><img file="USRE50776E_D0051.tif" /></chemistry><br /> wherein each ring atom is optionally substituted. In certain embodiments, Ring B is

0297<chemistry id="CHEM-US-00049" num="00049"><img file="USRE50776E_D0052.tif" /></chemistry><br /> wherein each ring atom is optionally substituted. In certain embodiments, Ring B is

0298<chemistry id="CHEM-US-00050" num="00050"><img file="USRE50776E_D0053.tif" /></chemistry><br /> wherein each ring atom is optionally substituted. In certain embodiments, Ring B is

0299<chemistry id="CHEM-US-00051" num="00051"><img file="USRE50776E_D0054.tif" /></chemistry><br /> wherein each ring atom is optionally substituted, and L²and R²may attach to Ring B at either indicated position. In certain embodiments, Ring B is

0300<chemistry id="CHEM-US-00052" num="00052"><img file="USRE50776E_D0055.tif" /></chemistry><br /> wherein each ring atom is optionally substituted, and L²and R²may attach to Ring B at either indicated position. In certain embodiments, Ring B is

0301<chemistry id="CHEM-US-00053" num="00053"><img file="USRE50776E_D0056.tif" /></chemistry><br /> wherein each ring atom is optionally substituted, and L²and R²may attach to Ring B at either indicated position. In certain embodiments, Ring B is

0302<chemistry id="CHEM-US-00054" num="00054"><img file="USRE50776E_D0057.tif" /></chemistry><br /> wherein each ring atom is optionally substituted, and L²and R²may attach to Ring B at either indicated position.

0303In certain embodiments of Formulae (I)-(III), Ring B is

0304<chemistry id="CHEM-US-00055" num="00055"><img file="USRE50776E_D0058.tif" /></chemistry><br /> In certain embodiments, Ring B is

0305<chemistry id="CHEM-US-00056" num="00056"><img file="USRE50776E_D0059.tif" /></chemistry><br /> In certain embodiments, Ring B is

0306<chemistry id="CHEM-US-00057" num="00057"><img file="USRE50776E_D0060.tif" /></chemistry><br /> In certain embodiments, Ring B is

0307<chemistry id="CHEM-US-00058" num="00058"><img file="USRE50776E_D0061.tif" /></chemistry><br /> L²and R²may attach to Ring B at either indicated position. In certain embodiments, Ring B is

0308<chemistry id="CHEM-US-00059" num="00059"><img file="USRE50776E_D0062.tif" /></chemistry><br /> wherein L²and R²may attach to Ring B at either indicated position. In certain embodiments, Ring B is

0309<chemistry id="CHEM-US-00060" num="00060"><img file="USRE50776E_D0063.tif" /></chemistry><br /> wherein L²and R²may attach to Ring B at either indicated position.

0310Compounds of Formulae (I)-(III) include R²attached to Ring B. In certain embodiments, Ring B is absent, such that R²is directly attached to linker L². In certain embodiments, Ring B is absent and L²is a bond, such that R²is directly attached to Ring A. In certain embodiments, R²comprises an electrophilic moiety. In certain embodiments, R²comprises a Michael acceptor moiety. The electrophilic moiety (e.g., Michael acceptor moiety) may react with a cysteine residue of a kinase (e.g., CDK (e.g., CDK7)) to allow for covalent attachment of the compound to the kinase. In certain embodiments, the electrophilic moiety (e.g., Michael acceptor moiety) may react with a cysteine residue of a kinase (e.g., CDK (e.g., CDK7)). In certain embodiments, the electrophilic moiety (e.g., Michael acceptor moiety) may react with the Cys312 residue of CDK7. In certain embodiments, the covalent attachment is irreversible. In certain embodiments, the covalent attachment is reversible.

0311As generally defined herein in Formulae (I)-(III), R²may be any one of Formulae (i-1)-(i-41). In certain embodiments, R²is of Formula (i-1):

0312<chemistry id="CHEM-US-00061" num="00061"><img file="USRE50776E_D0064.tif" /></chemistry><br /> In certain embodiments, R²is of Formula (i-2):

0313<chemistry id="CHEM-US-00062" num="00062"><img file="USRE50776E_D0065.tif" /></chemistry><br /> In certain embodiments, R²is of Formula (i-3):

0314<chemistry id="CHEM-US-00063" num="00063"><img file="USRE50776E_D0066.tif" /></chemistry><br /> In certain embodiments, R²is of Formula (i-4):

0315<chemistry id="CHEM-US-00064" num="00064"><img file="USRE50776E_D0067.tif" /></chemistry><br /> In certain embodiments, R²is of Formula (i-5):

0316<chemistry id="CHEM-US-00065" num="00065"><img file="USRE50776E_D0068.tif" /></chemistry><br /> In certain embodiments, R²is of Formula (i-6):

0317<chemistry id="CHEM-US-00066" num="00066"><img file="USRE50776E_D0069.tif" /></chemistry><br /> In certain embodiments, R²is of Formula (i-7):

0318<chemistry id="CHEM-US-00067" num="00067"><img file="USRE50776E_D0070.tif" /></chemistry><br /> In certain embodiments, R²is of Formula (i-8):

0319<chemistry id="CHEM-US-00068" num="00068"><img file="USRE50776E_D0071.tif" /></chemistry><br /> In certain embodiments, R²is of Formula (i-9):

0320<chemistry id="CHEM-US-00069" num="00069"><img file="USRE50776E_D0072.tif" /></chemistry><br /> In certain embodiments, R²is of Formula (i-10):

0321<chemistry id="CHEM-US-00070" num="00070"><img file="USRE50776E_D0073.tif" /></chemistry><br /> In certain embodiments, R²is of Formula (i-11):

0322<chemistry id="CHEM-US-00071" num="00071"><img file="USRE50776E_D0074.tif" /></chemistry><br /> In certain embodiments, R²is of Formula (i-12):

0323<chemistry id="CHEM-US-00072" num="00072"><img file="USRE50776E_D0075.tif" /></chemistry><br /> In certain embodiments, R²is of Formula (i-13):

0324<chemistry id="CHEM-US-00073" num="00073"><img file="USRE50776E_D0076.tif" /></chemistry><br /> In certain embodiments, R²is of Formula (i-14):

0325<chemistry id="CHEM-US-00074" num="00074"><img file="USRE50776E_D0077.tif" /></chemistry><br /> In certain embodiments, R²is of Formula (i-15):

0326<chemistry id="CHEM-US-00075" num="00075"><img file="USRE50776E_D0078.tif" /></chemistry><br /> In certain embodiments, R²is of Formula (i-16):

0327<chemistry id="CHEM-US-00076" num="00076"><img file="USRE50776E_D0079.tif" /></chemistry><br /> In certain embodiments, R²is of Formula (i-17):

0328<chemistry id="CHEM-US-00077" num="00077"><img file="USRE50776E_D0080.tif" /></chemistry><br /> In certain embodiments, R²is of Formula (i-18):

0329<chemistry id="CHEM-US-00078" num="00078"><img file="USRE50776E_D0081.tif" /></chemistry><br /> In certain embodiments, R²is of Formula (i-19):

0330<chemistry id="CHEM-US-00079" num="00079"><img file="USRE50776E_D0082.tif" /></chemistry><br /> In certain embodiments, R²is of Formula (i-20):

0331<chemistry id="CHEM-US-00080" num="00080"><img file="USRE50776E_D0083.tif" /></chemistry><br /> In certain embodiments, R²is of Formula (i-21):

0332<chemistry id="CHEM-US-00081" num="00081"><img file="USRE50776E_D0084.tif" /></chemistry><br /> In certain embodiments, R²is of Formula (i-22):

0333<chemistry id="CHEM-US-00082" num="00082"><img file="USRE50776E_D0085.tif" /></chemistry><br /> In certain embodiments, R²is of Formula (i-23):

0334<chemistry id="CHEM-US-00083" num="00083"><img file="USRE50776E_D0086.tif" /></chemistry><br /> In certain embodiments, R²is of Formula (i-24):

0335<chemistry id="CHEM-US-00084" num="00084"><img file="USRE50776E_D0087.tif" /></chemistry><br /> In certain embodiments, R²is of Formula (i-25):

0336<chemistry id="CHEM-US-00085" num="00085"><img file="USRE50776E_D0088.tif" /></chemistry><br /> In certain embodiments, R²is of Formula (i-26):

0337<chemistry id="CHEM-US-00086" num="00086"><img file="USRE50776E_D0089.tif" /></chemistry><br /> In certain embodiments, R²is of Formula (i-27):

0338<chemistry id="CHEM-US-00087" num="00087"><img file="USRE50776E_D0090.tif" /></chemistry><br /> In certain embodiments, R²is of Formula (i-28):

0339<chemistry id="CHEM-US-00088" num="00088"><img file="USRE50776E_D0091.tif" /></chemistry><br /> In certain embodiments, R²is of Formula (i-29):

0340<chemistry id="CHEM-US-00089" num="00089"><img file="USRE50776E_D0092.tif" /></chemistry><br /> In certain embodiments, R²is of Formula (i-30):

0341<chemistry id="CHEM-US-00090" num="00090"><img file="USRE50776E_D0093.tif" /></chemistry><br /> In certain embodiments, R²is of Formula (i-31):

0342<chemistry id="CHEM-US-00091" num="00091"><img file="USRE50776E_D0094.tif" /></chemistry><br /> In certain embodiments, R²is of Formula (i-32):

0343<chemistry id="CHEM-US-00092" num="00092"><img file="USRE50776E_D0095.tif" /></chemistry><br /> In certain embodiments, R²is of Formula (i-33):

0344<chemistry id="CHEM-US-00093" num="00093"><img file="USRE50776E_D0096.tif" /></chemistry><br /> In certain embodiments, R²is of Formula (i-34):

0345<chemistry id="CHEM-US-00094" num="00094"><img file="USRE50776E_D0097.tif" /></chemistry><br /> In certain embodiments, R²is of Formula (i-35):

0346<chemistry id="CHEM-US-00095" num="00095"><img file="USRE50776E_D0098.tif" /></chemistry><br /> In certain embodiments, R²is of Formula (i-36):

0347<chemistry id="CHEM-US-00096" num="00096"><img file="USRE50776E_D0099.tif" /></chemistry><br /> In certain embodiments, R²is of Formula (i-37):

0348<chemistry id="CHEM-US-00097" num="00097"><img file="USRE50776E_D0100.tif" /></chemistry><br /> In certain embodiments, R²is of Formula (i-38):

0349<chemistry id="CHEM-US-00098" num="00098"><img file="USRE50776E_D0101.tif" /></chemistry><br /> In certain embodiments, R²is of Formula (i-39):

0350<chemistry id="CHEM-US-00099" num="00099"><img file="USRE50776E_D0102.tif" /></chemistry><br /> In certain embodiments, R²is of Formula (i-40):

0351<chemistry id="CHEM-US-00100" num="00100"><img file="USRE50776E_D0103.tif" /></chemistry><br /> In certain embodiments, R²is of Formula (i-41):

0352<chemistry id="CHEM-US-00101" num="00101"><img file="USRE50776E_D0104.tif" /></chemistry><br /> In certain embodiments, R²is of Formula (i-1a):

0353<chemistry id="CHEM-US-00102" num="00102"><img file="USRE50776E_D0105.tif" /></chemistry><br /> In certain embodiments, R²is of Formula (i-1b):

0354<chemistry id="CHEM-US-00103" num="00103"><img file="USRE50776E_D0106.tif" /></chemistry><br /> In certain embodiments, R²is of Formula (i-1c):

0355<chemistry id="CHEM-US-00104" num="00104"><img file="USRE50776E_D0107.tif" /></chemistry><br /> In certain embodiments, R²is of Formula (i-1d):

0356<chemistry id="CHEM-US-00105" num="00105"><img file="USRE50776E_D0108.tif" /></chemistry><br /> In certain embodiments, R²is of Formula (i-1e):

0357<chemistry id="CHEM-US-00106" num="00106"><img file="USRE50776E_D0109.tif" /></chemistry><br /> In certain embodiments, R²is of Formula (i-1f):

0358<chemistry id="CHEM-US-00107" num="00107"><img file="USRE50776E_D0110.tif" /></chemistry><br /> In certain embodiments, R²is of Formula (i-1g):

0359<chemistry id="CHEM-US-00108" num="00108"><img file="USRE50776E_D0111.tif" /></chemistry><br /> In certain embodiments, R²is

0360<chemistry id="CHEM-US-00109" num="00109"><img file="USRE50776E_D0112.tif" /></chemistry><br /> In certain embodiments, R²is of Formula (i-1h):

0361<chemistry id="CHEM-US-00110" num="00110"><img file="USRE50776E_D0113.tif" /></chemistry><br /> In certain embodiments,

0362<chemistry id="CHEM-US-00111" num="00111"><img file="USRE50776E_D0114.tif" /></chemistry>

0363In certain embodiments, R²is of Formula (i-1a):

0364<chemistry id="CHEM-US-00112" num="00112"><img file="USRE50776E_D0115.tif" /></chemistry><br /> In certain embodiments, R²is of Formula (i-1b):

0365<chemistry id="CHEM-US-00113" num="00113"><img file="USRE50776E_D0116.tif" /></chemistry><br /> In certain embodiments, R²is of Formula (i-1c):

0366<chemistry id="CHEM-US-00114" num="00114"><img file="USRE50776E_D0117.tif" /></chemistry>

0367In certain embodiments, R²is of Formula (i-18a):

0368<chemistry id="CHEM-US-00115" num="00115"><img file="USRE50776E_D0118.tif" /></chemistry><br /> In certain embodiments, R²is of Formula (i-18b):

0369<chemistry id="CHEM-US-00116" num="00116"><img file="USRE50776E_D0119.tif" /></chemistry><br /> In certain embodiments, R²is of Formula (i-18c):

0370<chemistry id="CHEM-US-00117" num="00117"><img file="USRE50776E_D0120.tif" /></chemistry>

0371In certain embodiments, R²is of Formula (i-15a):

0372<chemistry id="CHEM-US-00118" num="00118"><img file="USRE50776E_D0121.tif" /></chemistry><br /> In certain embodiments, R²is of Formula (i-15b):

0373<chemistry id="CHEM-US-00119" num="00119"><img file="USRE50776E_D0122.tif" /></chemistry><br /> In certain embodiments, R²is of Formula (i-15c):

0374<chemistry id="CHEM-US-00120" num="00120"><img file="USRE50776E_D0123.tif" /></chemistry>

0375R²may contain linker L³or L⁴. In certain embodiments, L³is a bond. L³is an optionally substituted C_1-4hydrocarbon chain. In certain embodiments, L³is an optionally substituted C_1-4hydrocarbon chain, wherein one or more carbon units of the hydrocarbon chain are independently replaced with —C(═O)—, —O—, —S—, —NR^L3a—, —NR^L3aC(═O)—, —C(═O)NR^L3a—, —SC(═O)—, —C(═O)S—, —OC(═O)—, —C(═O)O—, —NR^L3aC(═S)—, —C(═S)NR^L3a—, trans-CR^L3b═CR^L3b—, cis-CR^L3b═CR^L3b—, —C═C—, —S(═O)—, —S(═O)O—, —OS(═O)—, —S(═O)NR^L3a—, —NR^L3aS(═O)—, —S(═O)₂—, —S(═O)₂O—, —OS(═O)₂—, —S(═O)₂NR^L3a—, or —NR^L3aS(═O)₂—. In certain embodiments, L³is an optionally substituted C_1-4hydrocarbon chain, wherein one carbon unit of the hydrocarbon chain is replaced with —NR^L3a— (e.g., —NH—). In certain embodiments, L³is of the formula: —(CH₂)_1-4—NR^L3a— (e.g., —(CH₂)_1-4—NH—) or —NR^L3a—CH₂)_1-4— (e.g., —NH—CH₂)_1-4—). In certain embodiments, L³is —NR^L3a—. In certain embodiments, L³is —NR^L3a(C═O)—. In certain embodiments, L³is —(C═O)NR^L3a—. In certain embodiments, L³is —NH—. In certain embodiments, L³is —(C═O)—. In certain embodiments, L³is —NH(C═O)—. In certain embodiments, L³is —(C═O)NH—. In certain embodiments, L³is —O—. In certain embodiments, L³is —S—. In certain embodiments, L⁴is a bond. In certain embodiments, L⁴is an optionally substituted C_1-4hydrocarbon chain.

0376Linker L³may contain groups R^L3aor R^L3b. In certain embodiments, R^L3ais hydrogen. In certain embodiments, at least one instance of R^L3bis hydrogen. In certain embodiments, each instance of R^L3bis hydrogen. In certain embodiments, at least one instance of R^L3bis —Cl, —Br, or —I. In certain embodiments, each instance of R^L3bis —Cl, —Br, or —I. In certain embodiments, at least one instance of R^L3bis —F. In certain embodiments, each instance of R^L3bis —F. In certain embodiments, at least one instance of R^L3bis optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl. In certain embodiments, two R^EMgroups are joined to form an optionally substituted carbocyclic or optionally substituted heterocyclic ring.

0377R²may contain groups R^E1, R^E2, and/or R^E3. In certain embodiments, R^E1is hydrogen. In certain embodiments, R^E2is hydrogen. In certain embodiments, R^E3is hydrogen. In certain embodiments, R^E1is —Cl, —Br, or —I. In certain embodiments, R^E2is —Cl, —Br, or —I. In certain embodiments, R^E3is —Cl, —Br, or —I. In certain embodiments, R^E1is —F. In certain embodiments, R^E2is —F. In certain embodiments, R^E3is —F. In certain embodiments, R^E1is optionally substituted alkyl (e.g., substituted or unsubstituted C_1-6alkyl). In certain embodiments, R^E2is optionally substituted alkyl (e.g., substituted or unsubstituted C_1-6alkyl). In certain embodiments, R^E3is optionally substituted alkyl (e.g., substituted or unsubstituted C_1-6alkyl). In certain embodiments, R^E1is optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, —CN, —CH₂OR^EE, —CH₂N(R^EE)₂, —CH₂SR^EE, —OR^EE, —N(R^EE)₂, —Si(R^EE)₃, or —SR^EE. In certain embodiments, R^E2is optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted (heteroaryl, —CN, —CH₂OR^EE, —CH₂N(R^EE)₂—, —CH₂SR^EE, —OR^EE, —N(R^EE)₂, —Si(R^EE)₃, or —SR^EE. In certain embodiments, R^E3is optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, —CN, —CH₂OR^EE, —CH₂N(R^EE)₂, —CH₂SR^EE, —OR^EE, —N(R^EE)₂, —Si(R^EE)₃, or —SR^EE. In certain embodiments, R^E1is —N(R^EE)₂. In certain embodiments, R^E2is —N(R^EE)₂. In certain embodiments, R^E3is —N(R^EE)₂. In certain embodiments, R^E1is —N(CH₃)₂. In certain embodiments, R^E2is —N(CH₃)₂. In certain embodiments, R^E3is —N(CH₃)₂. In certain embodiments, R^E1is —CH₂N(R^EE)₂. In certain embodiments, R^E2is —CH₂N(R^EE) In certain embodiments, R^E3is —CH₂N(R^EE)₂. In certain embodiments, R^E1is —CH₂N(CH₃)₂. In certain embodiments, R^E2is —CH₂N(CH₃)₂. In certain embodiments, R^E3is —CH₂N(CH₃)₂. In certain embodiments, R^E1is —CN. In certain embodiments, R^E2is —CN. In certain embodiments, R^E3is —CN.

0378In certain embodiments, R^E1and R^E3are joined to form an optionally substituted carbocyclic ring. In certain embodiments, R^E1and R^E3are joined to form an optionally substituted heterocyclic ring. In certain embodiments, R^E2and R^E3are joined to form an optionally substituted carbocyclic ring. In certain embodiments, R^E2and R^E3are joined to form an optionally substituted heterocyclic ring. In certain embodiments, R^E1and R^E2are joined to form an optionally substituted carbocyclic ring. In certain embodiments, R^E1and R^E2are joined to form an optionally substituted heterocyclic ring.

0379R²may contain group R^E4, where R^E4is a leaving group. In certain embodiments, R^E4is —Cl, —Br, or —I. In certain embodiments, R^E4is —F. In certain embodiments, R^E4is —OS(═O)R^E4aor —OS(═O)₂R^E4a, wherein R^E4ais substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl. In certain embodiments, R^E4is —OR^E4a. In certain embodiments, R^E4is —OMs, —OTf, —OTs, —OBs, or 2-nitrobenzenesulfonyloxy. In certain embodiments, R^E4is —OR^E4a. In certain embodiments, R^E4is —OMe, —OCF₃, or —OPh. In certain embodiments, R^E4is —OC(═O)R^E4a. In certain embodiments, R^E4is —OC(═O)Me, —OC(═O)CF₃, —OC(═O)Ph, or —OC(═O)Cl. In certain embodiments, R^E4is —OC(═O)OR^E4a. In certain embodiments, R^E4is —OC(═O)OMe or —OC(═O)O(t-Bu).

0380R²may contain group R^E5, where R^E5is a halogen. In certain embodiments, R^E5is —Cl, —Br, or —I. In certain embodiments, R^E5is —F.

0381R²may contain group R^E6. In certain embodiments, R^E6is hydrogen. In certain embodiments, R^E6is substituted or unsubstituted C₁-C₆alkyl. In certain embodiments, R^E6is a nitrogen protecting group.

0382In certain embodiments, a is 1. In certain embodiments, a is 2.

0383In certain embodiments, z is 0. In certain embodiments, z is 1. In certain embodiments, z is 2. In certain embodiments, z is 3, 4, 5, or 6.

0384R²may contain group Y. In certain embodiments, Y is 0. In certain embodiments, Y is S. In certain embodiments, Y is NR^E7. In certain embodiments, Y is NH.

0385Compounds of Formula (I) may exist as tautomers or mixtures thereof of Formulae (I-a) and (I-b):

0386<chemistry id="CHEM-US-00121" num="00121"><img file="USRE50776E_D0124.tif" /></chemistry>

0387In each tautomer, R⁵is attached to different imidazole nitrogens in compounds of each formula. In certain embodiments, R⁵is attached to the nitrogen at the position labeled 9, as in Formula (I-a). In certain embodiments, R⁵is attached to the nitrogen at the position labeled 7, as in Formula (I-b). In certain embodiments, compounds of Formula (I) may exist as a mixture of compounds of Formulae (I-a) and (I-b), in which case R⁵is attached to the nitrogen at the position labeled 9 for components of the mixture corresponding to Formula (I-a), and R⁵is attached to the nitrogen at the position labeled 7 for components of the mixture corresponding to Formula (I-b).

0388In certain embodiments, a compound of Formula (I) is of Formula (I-1):

0389<chemistry id="CHEM-US-00122" num="00122"><img file="USRE50776E_D0125.tif" /></chemistry><br /> or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof, wherein L¹, L², Ring A, Ring B, R², R⁵, R^b, R^1a, and a1 are as defined herein.

0390In certain embodiments, a compound of Formula (I) is of Formula (I-1-a):

0391<chemistry id="CHEM-US-00123" num="00123"><img file="USRE50776E_D0126.tif" /></chemistry><br /> or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof, wherein R², R⁵, R^L1, R^b, R^1a, and a1 are as defined herein.

0392In certain embodiments, a compound of Formula (I) is of Formula (I-1-a-i):

0393<chemistry id="CHEM-US-00124" num="00124"><img file="USRE50776E_D0127.tif" /></chemistry><br /> or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof, wherein R², R⁵, R^L1, R^b, R^1a, and a1 are as defined herein.

0394In certain embodiments, a compound of Formula (I) is of Formula (I-1-b):

0395<chemistry id="CHEM-US-00125" num="00125"><img file="USRE50776E_D0128.tif" /></chemistry><br /> or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof, wherein R², L², R⁵, R^L1, R^b, R^1a, and a1 are as defined herein.

0396In certain embodiments, a compound of Formula (I) is of Formula (I-1-b-i):

0397<chemistry id="CHEM-US-00126" num="00126"><img file="USRE50776E_D0129.tif" /></chemistry><br /> or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof, wherein R², L², R⁵, R^L1, R^b, R^1a, and a1 are as defined herein.

0398In certain embodiments, a compound of Formula (I) is of Formula (I-1-b-ii):

0399<chemistry id="CHEM-US-00127" num="00127"><img file="USRE50776E_D0130.tif" /></chemistry><br /> or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof, wherein R², L², R⁵, R^L1, R^b, R^1a, and a1 are as defined herein.

0400In certain embodiments, a compound of Formula (I) is of Formula (I-1-c):

0401<chemistry id="CHEM-US-00128" num="00128"><img file="USRE50776E_D0131.tif" /></chemistry><br /> or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof, wherein R², R⁵, R^L1, R^b, R^1a, and a1 are as defined herein.

0402In certain embodiments, a compound of Formula (I) is of Formula (I-1-c-i):

0403<chemistry id="CHEM-US-00129" num="00129"><img file="USRE50776E_D0132.tif" /></chemistry><br /> or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof, wherein R², R⁵, R^L1, R^b, R^1a, and a1 are as defined herein.

0404In certain embodiments, a compound of Formula (I) is of Formula (I-2):

0405<chemistry id="CHEM-US-00130" num="00130"><img file="USRE50776E_D0133.tif" /></chemistry><br /> or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof, wherein R², L², L¹, Ring A, Ring B, R⁵, R^1b, and b1 are as defined herein

0406In certain embodiments, a compound of Formula (I) is of Formula (I-2-a):

0407<chemistry id="CHEM-US-00131" num="00131"><img file="USRE50776E_D0134.tif" /></chemistry><br /> or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof, wherein R², R^L1, R⁵, R^1b, and b1 are as defined herein.

0408In certain embodiments, a compound of Formula (I) is of Formula (I-2-a-i):

0409<chemistry id="CHEM-US-00132" num="00132"><img file="USRE50776E_D0135.tif" /></chemistry><br /> or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof, wherein R², R^L1, R⁵, R^1b, and b1 are as defined herein.

0410In certain embodiments, a compound of Formula (I) is of Formula (I-2-b):

0411<chemistry id="CHEM-US-00133" num="00133"><img file="USRE50776E_D0136.tif" /></chemistry><br /> or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof, wherein R², L², R^L1, R⁵, R^1b, and b1 are as defined herein.

0412In certain embodiments, a compound of Formula (I) is of Formula (I-2-b-i):

0413<chemistry id="CHEM-US-00134" num="00134"><img file="USRE50776E_D0137.tif" /></chemistry><br /> or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof, wherein R², L², R^L1, R⁵, R^1b, and b1 are as defined herein.

0414In certain embodiments, a compound of Formula (I) is of Formula (I-2-b-ii):

0415<chemistry id="CHEM-US-00135" num="00135"><img file="USRE50776E_D0138.tif" /></chemistry><br /> or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof, wherein R², L², R^L1, R⁵, R^1b, and b1 are as defined herein.

0416In certain embodiments, a compound of Formula (I) is of Formula (I-3):

0417<chemistry id="CHEM-US-00136" num="00136"><img file="USRE50776E_D0139.tif" /></chemistry><br /> or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof, wherein R², L², L¹, Ring A, Ring B, R⁵, R^1c, and c1 are as defined herein.

0418In certain embodiments, a compound of Formula (I) is of Formula (I-3-a):

0419<chemistry id="CHEM-US-00137" num="00137"><img file="USRE50776E_D0140.tif" /></chemistry><br /> or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof, wherein R², L¹, R⁵, R^1c, and c1 are as defined herein.

0420In certain embodiments, a compound of Formula (I) is of Formula (I-3-a-1):

0421<chemistry id="CHEM-US-00138" num="00138"><img file="USRE50776E_D0141.tif" /></chemistry><br /> or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof, wherein R², L¹, R⁵, R^1c, and c1 are as defined herein.

0422In certain embodiments, a compound of Formula (I) is of Formula (I-3-b):

0423<chemistry id="CHEM-US-00139" num="00139"><img file="USRE50776E_D0142.tif" /></chemistry><br /> or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof, wherein R², L¹, R⁵, R^1c, and c1 are as defined herein.

0424In certain embodiments, a compound of Formula (II) is of Formula (I-3-b-i):

0425<chemistry id="CHEM-US-00140" num="00140"><img file="USRE50776E_D0143.tif" /></chemistry><br /> or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof, wherein R², L¹, R⁵, R^1c, and c1 are as defined herein.

0426In certain embodiments, a compound of Formula (I) is one of the following formulae:

0427<chemistry id="CHEM-US-00141" num="00141"><img file="USRE50776E_D0144.tif" /></chemistry>

0428In certain embodiments, a compound of Formula (II) is of Formula (II-1):

0429<chemistry id="CHEM-US-00142" num="00142"><img file="USRE50776E_D0145.tif" /></chemistry><br /> or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof, wherein R², L¹, L², Ring A, Ring B, R⁴, R^b, R^1a, and a1 are as defined herein.

0430In certain embodiments, a compound of Formula (II) is of Formula (II-1-a):

0431<chemistry id="CHEM-US-00143" num="00143"><img file="USRE50776E_D0146.tif" /></chemistry><br /> or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof, wherein R², R^L1, R⁴, R^b, R^1a, and a1 are as defined herein.

0432In certain embodiments, a compound of Formula (II) is of Formula (II-1-a-i):

0433<chemistry id="CHEM-US-00144" num="00144"><img file="USRE50776E_D0147.tif" /></chemistry><br /> or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof, wherein R², R^L1, R⁴, R^b, R^1a, and a1 are as defined herein.

0434In certain embodiments, a compound of Formula (II) is of Formula (II-1-b):

0435<chemistry id="CHEM-US-00145" num="00145"><img file="USRE50776E_D0148.tif" /></chemistry><br /> or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof, wherein R², R^L1, R⁴, R^b, R^1a, and a1 are as defined herein.

0436In certain embodiments, a compound of Formula (II) is of Formula (II-1-b-i):

0437<chemistry id="CHEM-US-00146" num="00146"><img file="USRE50776E_D0149.tif" /></chemistry><br /> or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof, wherein R², R^L1, R⁴, R^b, R^1a, and a1 are as defined herein.

0438In certain embodiments, a compound of Formula (II) is of Formula (II-1-c):

0439<chemistry id="CHEM-US-00147" num="00147"><img file="USRE50776E_D0150.tif" /></chemistry><br /> or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof, wherein R², L², R^L1, R⁴, R^b, R^1a, and a1 are as defined herein.

0440In certain embodiments, a compound of Formula (II) is of Formula (II-2):

0441<chemistry id="CHEM-US-00148" num="00148"><img file="USRE50776E_D0151.tif" /></chemistry><br /> or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof, wherein R², L¹, L², Ring A, Ring B, R⁴, R^1b, and b1 are as defined herein.

0442In certain embodiments, a compound of Formula (II) is of Formula (II-2-a):

0443<chemistry id="CHEM-US-00149" num="00149"><img file="USRE50776E_D0152.tif" /></chemistry><br /> or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof, wherein R², R^L1, R⁴, R^1b, and b1 are as defined herein.

0444In certain embodiments, a compound of Formula (II) is of Formula (II-2-b):

0445<chemistry id="CHEM-US-00150" num="00150"><img file="USRE50776E_D0153.tif" /></chemistry><br /> or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof, wherein R², R^L1, R⁴, R^1b, and b1 are as defined herein.

0446In certain embodiments, a compound of Formula (II) is of Formula (II-2-c):

0447<chemistry id="CHEM-US-00151" num="00151"><img file="USRE50776E_D0154.tif" /></chemistry><br /> or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof, wherein R², R^L1, R⁴, R^1b, and b1 are as defined herein.

0448In certain embodiments, a compound of Formula (II) is of Formula (II-2-d):

0449<chemistry id="CHEM-US-00152" num="00152"><img file="USRE50776E_D0155.tif" /></chemistry><br /> or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof, wherein R², L², R^L1, R⁴, R^1b, and b1 are as defined herein.

0450In certain embodiments, a compound of Formula (II) is of Formula (II-2-d-i):

0451<chemistry id="CHEM-US-00153" num="00153"><img file="USRE50776E_D0156.tif" /></chemistry><br /> or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof, wherein R², L², R^L1, R⁴, R^1b, and b1 are as defined herein.

0452In certain embodiments, a compound of Formula (II) is of Formula (II-2-d-ii):

0453<chemistry id="CHEM-US-00154" num="00154"><img file="USRE50776E_D0157.tif" /></chemistry><br /> or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof, wherein R², L², R^L1, R⁴, R^1b, and b1 are as defined herein.

0454In certain embodiments, a compound of Formula (II) is of one of the following formulae:

0455<chemistry id="CHEM-US-00155" num="00155"><img file="USRE50776E_D0158.tif" /></chemistry>

0456In certain embodiments, a compound of Formula (II) is of one of the following formulae:

0457<chemistry id="CHEM-US-00156" num="00156"><img file="USRE50776E_D0159.tif" /></chemistry>

0458In certain embodiments, a compound of Formula (II) is of the formula:

0459<chemistry id="CHEM-US-00157" num="00157"><img file="USRE50776E_D0160.tif" /></chemistry><br /> or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof.

0460In certain embodiments, a compound of Formula (II) is of the formula:

0461<chemistry id="CHEM-US-00158" num="00158"><img file="USRE50776E_D0161.tif" /></chemistry><chemistry id="CHEM-US-00159" num="00159"><img file="USRE50776E_D0162.tif" /></chemistry><chemistry id="CHEM-US-00160" num="00160"><img file="USRE50776E_D0163.tif" /></chemistry><br /> or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof.

0462In certain embodiments, a compound of Formula (III) is of Formula (III-1):

0463<chemistry id="CHEM-US-00161" num="00161"><img file="USRE50776E_D0164.tif" /></chemistry><br /> or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof, wherein R², L², L¹, Ring A, Ring B, R⁴, R^b, R^1a, and a1 are as defined herein.

0464In certain embodiments, a compound of Formula (III) is of Formula (III-1-a):

0465<chemistry id="CHEM-US-00162" num="00162"><img file="USRE50776E_D0165.tif" /></chemistry><br /> or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof, wherein R², R^L1, R⁴, R^b, R^1a, and a1 are as defined herein.

0466In certain embodiments, a compound of Formula (III) is of Formula (III-1-a-ii):

0467<chemistry id="CHEM-US-00163" num="00163"><img file="USRE50776E_D0166.tif" /></chemistry><br /> or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof, wherein R², R^L1, R⁴, R^b, R^1a, and a1 are as defined herein.

0468In certain embodiments, a compound of Formula (III) is of Formula (III-1-b):

0469<chemistry id="CHEM-US-00164" num="00164"><img file="USRE50776E_D0167.tif" /></chemistry><br /> or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof, wherein R², R^L1, R⁴, R^b, R^1a, and a1 are as defined herein.

0470In certain embodiments, a compound of Formula (III) is of Formula (III-1-b-i):

0471<chemistry id="CHEM-US-00165" num="00165"><img file="USRE50776E_D0168.tif" /></chemistry><br /> or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof, wherein R², R^L1, R⁴, R^b, R^1a, and a1 are as defined herein.

0472In certain embodiments, a compound of Formula (III) is of Formula (III-1-c):

0473<chemistry id="CHEM-US-00166" num="00166"><img file="USRE50776E_D0169.tif" /></chemistry><br /> or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof, wherein R², L², R^L1, R⁴, R^b, R^1a, and a1 are as defined herein.

0474In certain embodiments, a compound of Formula (III) is of Formula (III-1-c-i):

0475<chemistry id="CHEM-US-00167" num="00167"><img file="USRE50776E_D0170.tif" /></chemistry><br /> or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof, wherein R², L², R^L1, R⁴, R^b, R^1a, and a1 are as defined herein.

0476In certain embodiments, a compound of Formula (III) is of Formula (III-1-c-ii):

0477<chemistry id="CHEM-US-00168" num="00168"><img file="USRE50776E_D0171.tif" /></chemistry><br /> or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof, wherein R², L², R^L1, R⁴, R^b, R^1a, and a1 are as defined herein.

0478In certain embodiments, a compound of Formula (III) is of Formula (III-2):

0479<chemistry id="CHEM-US-00169" num="00169"><img file="USRE50776E_D0172.tif" /></chemistry><br /> or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof, wherein R², L¹, L², Ring A, Ring B, R⁴, R^1b, and b1 are as defined herein.

0480In certain embodiments, a compound of Formula (III) is of Formula (III-2-a):

0481<chemistry id="CHEM-US-00170" num="00170"><img file="USRE50776E_D0173.tif" /></chemistry><br /> or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof, wherein R², R^L1, R⁴, R^1b, and b1 are as defined herein.

0482In certain embodiments, a compound of Formula (III) is of Formula (III-2-a-i):

0483<chemistry id="CHEM-US-00171" num="00171"><img file="USRE50776E_D0174.tif" /></chemistry><br /> or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof, wherein R², R^L1, R⁴, R^1b, and b1 are as defined herein.

0484In certain embodiments, a compound of Formula (III) is of Formula (III-2-a-ii):

0485<chemistry id="CHEM-US-00172" num="00172"><img file="USRE50776E_D0175.tif" /></chemistry><br /> or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof.

0486In certain embodiments, a compound of Formula (III) is of Formula (III-2-b):

0487<chemistry id="CHEM-US-00173" num="00173"><img file="USRE50776E_D0176.tif" /></chemistry><br /> or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof, wherein R², R^L1, R⁴, R^1b, and b1 are as defined herein.

0488In certain embodiments, a compound of Formula (III) is of Formula (III-2-b-i):

0489<chemistry id="CHEM-US-00174" num="00174"><img file="USRE50776E_D0177.tif" /></chemistry><br /> or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof, wherein R², R^L1, R⁴, R^1b, and b1 are as defined herein.

0490In certain embodiments, a compound of Formula (III) is of Formula (III-2-c):

0491<chemistry id="CHEM-US-00175" num="00175"><img file="USRE50776E_D0178.tif" /></chemistry><br /> or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof, wherein R², R^L1, R⁴, R^1b, and b1 are as defined herein.

0492In certain embodiments, a compound of Formula (III) is of Formula (III-2-d):

0493<chemistry id="CHEM-US-00176" num="00176"><img file="USRE50776E_D0179.tif" /></chemistry><br /> or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof, wherein R², L², R^L1, R⁴, R^1b, and b1 are as defined herein.

0494In certain embodiments, a compound of Formula (III) is of Formula (III-2-d-i):

0495<chemistry id="CHEM-US-00177" num="00177"><img file="USRE50776E_D0180.tif" /></chemistry><br /> or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof, wherein R², L², R^L1, R⁴, R^1b, and b1 are as defined herein.

0496In certain embodiments, a compound of Formula (III) is of Formula (III-2-d-ii):

0497<chemistry id="CHEM-US-00178" num="00178"><img file="USRE50776E_D0181.tif" /></chemistry><br /> or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof, wherein R², L², R^L1, R⁴, R^1b, and b1 are as defined herein.

0498In certain embodiments, a compound of Formula (III) is of one of the following formulae:

0499<chemistry id="CHEM-US-00179" num="00179"><img file="USRE50776E_D0182.tif" /></chemistry>

0500In certain embodiments, a compound of Formula (III) is of one of the following formulae:

0501<chemistry id="CHEM-US-00180" num="00180"><img file="USRE50776E_D0183.tif" /></chemistry><chemistry id="CHEM-US-00181" num="00181"><img file="USRE50776E_D0184.tif" /></chemistry>

0502In certain embodiments, a compound of Formula (III) is of the formula:

0503<chemistry id="CHEM-US-00182" num="00182"><img file="USRE50776E_D0185.tif" /></chemistry><chemistry id="CHEM-US-00183" num="00183"><img file="USRE50776E_D0186.tif" /></chemistry>

0504In certain embodiments, a compound of Formula (III) is of the formula:

0505<chemistry id="CHEM-US-00184" num="00184"><img file="USRE50776E_D0187.tif" /></chemistry><chemistry id="CHEM-US-00185" num="00185"><img file="USRE50776E_D0188.tif" /></chemistry><br /> or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof.

0506In some embodiments, a compound of Formula (I) is of the formula:

0507<chemistry id="CHEM-US-00186" num="00186"><img file="USRE50776E_D0189.tif" /></chemistry><chemistry id="CHEM-US-00187" num="00187"><img file="USRE50776E_D0190.tif" /></chemistry><br /> or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof.

0508In some embodiments, a compound of Formula (I) is of the formula:

0509<chemistry id="CHEM-US-00188" num="00188"><img file="USRE50776E_D0191.tif" /></chemistry><br /> or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof.

0510In some embodiments, a compound of Formula (II) is of the formula:

0511<chemistry id="CHEM-US-00189" num="00189"><img file="USRE50776E_D0192.tif" /></chemistry><br /> or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof.

0512In some embodiments, a compound of Formula (II) is of the formula:

0513<chemistry id="CHEM-US-00190" num="00190"><img file="USRE50776E_D0193.tif" /></chemistry><br /> or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof.

0514In some embodiments, a compound of Formula (III) is of the formula:

0515<chemistry id="CHEM-US-00191" num="00191"><img file="USRE50776E_D0194.tif" /></chemistry><br /> or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof.

0516In some embodiments, a compound of Formula (III) is of the formula:

0517<chemistry id="CHEM-US-00192" num="00192"><img file="USRE50776E_D0195.tif" /></chemistry><chemistry id="CHEM-US-00193" num="00193"><img file="USRE50776E_D0196.tif" /></chemistry><br /> or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof. <br /> Pharmaceutical Compositions, Kits, and Administration

0518The pharmaceutical compositions described herein are useful in treating and/or preventing proliferative diseases (e.g., cancers (e.g., leukemia, acute lymphoblastic leukemia, lymphoma, Burkitt's lymphoma, melanoma, multiple myeloma, breast cancer, Ewing's sarcoma, osteosarcoma, brain cancer, neuroblastoma, lung cancer, colorectal cancer), benign neoplasms, diseases associated with angiogenesis, inflammatory diseases, autoinflammatory diseases, and autoimmune diseases) in a subject. The compositions described herein are also useful for inhibiting the activity of a protein kinase (e.g., CDK (e.g., CDK7, CDK12, and/or CDK13)) in a subject, biological sample, tissue, or cell. The compositions described herein are also useful for inducing apoptosis in a cell.

0519The present disclosure provides pharmaceutical compositions comprising a compound described herein (e.g., a compound of any one of Formulae (I)-(III)), or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof, and optionally a pharmaceutically acceptable excipient. In certain embodiments, the pharmaceutical composition of the invention comprises a compound described herein, or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable excipient. In certain embodiments, a pharmaceutical composition described herein comprises a compound described herein, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient. In certain embodiments, the compound described herein, or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof, is provided in an effective amount in the pharmaceutical composition.

0520In certain embodiments, the effective amount is a therapeutically effective amount (e.g., amount effective for treating a proliferative disease in a subject in need thereof). In certain embodiments, the effective amount is an amount effective for inhibiting the activity of a protein kinase (e.g., CDK (e.g., CDK7, CDK12, and/or CDK13)) in a subject in need thereof. In certain embodiments, the effective amount is an amount effective for inhibiting the activity of a protein kinase (e.g., CDK (e.g., CDK7, CDK12, and/or CDK13)) in a cell. In certain embodiments, the effective amount is an amount effective for inducing apoptosis in a cell. In certain embodiments, the effective amount is a prophylactically effective amount (e.g., amount effective for preventing a proliferative disease in a subject in need thereof and/or for keeping a subject in need thereof in remission of a proliferative disease).

0521In certain embodiments, a protein kinase described herein is a CDK. In certain embodiments, a protein kinase described herein is CDK1, CDK2, CDK3, CDK4, CDK5, CDK6, CDK7, CDK8, CDK9, CDK10, CDK11, CDK12, CDK13, CDK14, CDK15, CDK16, CDK17, CDK18, CDK19, or CDK20. In certain embodiments, a protein kinase described herein is CDK7. In certain embodiments, a protein kinase described herein is CDK12. In certain embodiments, a protein kinase described herein is CDK13. In certain embodiments, a protein kinase described herein is a Src family kinase. In certain embodiments, a protein kinase described herein is SRC. In certain embodiments, a protein kinase described herein is FGR. In certain embodiments, a protein kinase described herein is BUB1B. In certain embodiments, a protein kinase described herein is CHEK2. In certain embodiments, a protein kinase described herein is HIPK4. In certain embodiments, a protein kinase described herein is PRKCQ. In certain embodiments, a protein kinase described herein is RET. In certain embodiments, a protein kinase described herein is MELK. In certain embodiments, a protein kinase described herein is IRAK1, IRAK4, BMX, or PI3K. In certain embodiments, a protein kinase described herein is ABL, ARG, BLK, CSK, EphB1, EphB2, FGR, FRK, FYN, SRC, YES, LCK, LYN, MAP2K5, NLK, p38a, SNRK, or TEC. In certain embodiments, a protein kinase described herein is ABL1(H396P)-phosphorylated, ABL1-phosphorylated, BLK, EPHA4, EPHB2, EPHB3, EPHB4, FGR, JAK3(JH1domain-catalytic), KIT, KIT(L576P), KIT(V559D), PDGFRB, SRC, YES, ABL1(H396P)-nonphosphorylated, ABL1(Y253F)-phosphorylated, ABL1-nonphosphorylated, FRK, LYN, ABL1(Q252H)-nonphosphorylated, DDR1, EPHB1, ERBB4, p38-alpha, ABL2, ABL1(Q252H)-phosphorylated, SIK, EPHA8, MEK5, ABL1(E255K)-phosphorylated, ABL1(F317L)-nonphosphorylated, FYN, LCK, EPHA2, ABL1(M351T)-phosphorylated, TXK, EGFR(L858R), EGFR(L861Q), ERBB2, ERBB3, EPHA5, ABL1(F317I)-nonphosphorylated, EGFR(L747-E749del, A750P), CSK, EPHA1, ABL1(F317L)-phosphorylated, BRAF(V600E), EGFR, KIT-autoinhibited, or EGFR(E746-A750del). In certain embodiments, a protein kinase described herein is ABL1(F317L)-nonphosphorylated, ABL1(H396P)-nonphosphorylated, ABL1(H396P)-phosphorylated, ABL1-phosphorylated, BLK, EPHA4, EPHB2, EPHB3, EPHB4, JAK3(JH1domain-catalytic), KIT, KIT(L576P), KIT(V559D), LYN, PDGFRB, SRC, YES, ABL1-nonphosphorylated, ABL1(Y253F)-phosphorylated, ERBB3, FGR, FRK, p38-alpha, ABL1(F317I)-nonphosphorylated, DDR1, EPHA2, ABL1(Q252H)-phosphorylated, MEK5, ABL1(Q252H)-nonphosphorylated, ABL2, FYN, EPHB1, ABL1(E255K)-phosphorylated, ABL1(F317L)-phosphorylated, EPHA1, ABL1(M351T)-phosphorylated, ERBB4, TXK, LCK, EPHA8, SIK, EPHA5, EGFR(L861Q), CSF1R-autoinhibited, BRAF(V600E), BRK, CSK, KIT(D816V), KIT-autoinhibited, EGFR(L747-T751del,Sins), EGFR(L858R), EGFR(L747-E749del, A750P), or CSF1R.

0522In certain embodiments, the effective amount is an amount effective for inhibiting the activity of a protein kinase (e.g., CDK (e.g., CDK7, CDK12, and/or CDK13)) by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 98%. In certain embodiments, the effective amount is an amount effective for inhibiting the activity of a protein kinase (e.g., CDK (e.g., CDK7, CDK12, and/or CDK13)) by not more than 10%, not more than 20%, not more than 30%, not more than 40%, not more than 50%, not more than 60%, not more than 70%, not more than 80%, not more than 90%, not more than 95%, or not more than 98%. In certain embodiments, the effective amount is an amount effective for inhibiting the activity of a protein kinase (e.g., CDK (e.g., CDK7, CDK12, and/or CDK13)) by a range between a percentage described in this paragraph and another percentage described in this paragraph, inclusive.

0523Pharmaceutical compositions described herein can be prepared by any method known in the art of pharmacology. In general, such preparatory methods include bringing the compound described herein (i.e., the “active ingredient”) into association with a carrier or excipient, and/or one or more other accessory ingredients, and then, if necessary and/or desirable, shaping, and/or packaging the product into a desired single- or multi-dose unit.

0524Pharmaceutical compositions can be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses. A “unit dose” is a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage, such as one-half or one-third of such a dosage.

0525Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition described herein will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered. The composition may comprise between 0.1% and 100% (w/w) active ingredient.

0526Pharmaceutically acceptable excipients used in the manufacture of provided pharmaceutical compositions include inert diluents, dispersing and/or granulating agents, surface active agents and/or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and/or oils. Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and perfuming agents may also be present in the composition.

0527Exemplary diluents include calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, cornstarch, powdered sugar, and mixtures thereof.

0528Exemplary granulating and/or dispersing agents include potato starch, corn starch, tapioca starch, sodium starch glycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose, and wood products, natural sponge, cation-exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (Veegum), sodium lauryl sulfate, quaternary ammonium compounds, and mixtures thereof.

0529Exemplary surface active agents and/or emulsifiers include natural emulsifiers (e.g., acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g., bentonite (aluminum silicate) and Veegum (magnesium aluminum silicate)), long chain amino acid derivatives, high molecular weight alcohols (e.g., stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g., carboxy polymethylene, polyacrylic acid, acrylic acid polymer, and carboxyvinyl polymer), carrageenan, cellulosic derivatives (e.g., carboxymethylcellulose sodium, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose), sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monolaurate (Tween® 20), polyoxyethylene sorbitan (Tween® 60), polyoxyethylene sorbitan monooleate (Tween® 80), sorbitan monopalmitate (Span® 40), sorbitan monostearate (Span® 60), sorbitan tristearate (Span® 65), glyceryl monooleate, sorbitan monooleate (Span® 80), polyoxyethylene esters (e.g., polyoxyethylene monostearate (Myrj® 45), polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and Solutol®), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g., Cremophor®), polyoxyethylene ethers, (e.g., polyoxyethylene lauryl ether (Brij® 30)), poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, Pluronic® F-68, poloxamer P-188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, and/or mixtures thereof.

0530Exemplary binding agents include starch (e.g., cornstarch and starch paste), gelatin, sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol, etc.), natural and synthetic gums (e.g., acacia, sodium alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage of isapol husks, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, microcrystalline cellulose, cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate (Veegum®), and larch arabogalactan), alginates, polyethylene oxide, polyethylene glycol, inorganic calcium salts, silicic acid, polymethacrylates, waxes, water, alcohol, and/or mixtures thereof.

0531Exemplary preservatives include antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, antiprotozoan preservatives, alcohol preservatives, acidic preservatives, and other preservatives. In certain embodiments, the preservative is an antioxidant. In other embodiments, the preservative is a chelating agent.

0532Exemplary antioxidants include alpha tocopherol, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and sodium sulfite.

0533Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA) and salts and hydrates thereof (e.g., sodium edetate, disodium edetate, trisodium edetate, calcium disodium edetate, dipotassium edetate, and the like), citric acid and salts and hydrates thereof (e.g., citric acid monohydrate), fumaric acid and salts and hydrates thereof, malic acid and salts and hydrates thereof, phosphoric acid and salts and hydrates thereof, and tartaric acid and salts and hydrates thereof. Exemplary antimicrobial preservatives include benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and thimerosal.

0534Exemplary antifungal preservatives include butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and sorbic acid.

0535Exemplary alcohol preservatives include ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and phenylethyl alcohol.

0536Exemplary acidic preservatives include vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and phytic acid.

0537Other preservatives include tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisol (BHA), butylated hydroxytoluened (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, Glydant® Plus, Phenonip®, methylparaben, German® 115, Germaben® II, Neolone®, Kathon®, and Euxyl®.

0538Exemplary buffering agents include citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, and mixtures thereof.

0539Exemplary lubricating agents include magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behanate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, and mixtures thereof.

0540Exemplary natural oils include almond, apricot kernel, avocado, babassu, bergamot, black current seed, borage, cade, camomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, corn, cotton seed, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon, litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, sasquana, savoury, sea buckthorn, sesame, shea butter, silicone, soybean, sunflower, tea tree, thistle, tsubaki, vetiver, walnut, and wheat germ oils. Exemplary synthetic oils include, but are not limited to, butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone oil, and mixtures thereof.

0541Liquid dosage forms for oral and parenteral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredients, the liquid dosage forms may comprise inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (e.g., cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents. In certain embodiments for parenteral administration, the conjugates described herein are mixed with solubilizing agents such as Cremophor®, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and mixtures thereof.

0542Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions can be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation can be a sterile injectable solution, suspension, or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution, U.S.P., and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or di-glycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables.

0543The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

0544In order to prolong the effect of a drug, it is often desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This can be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution, which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form may be accomplished by dissolving or suspending the drug in an oil vehicle.

0545Compositions for rectal or vaginal administration are typically suppositories which can be prepared by mixing the conjugates described herein with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol, or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active ingredient.

0546Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active ingredient is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or (a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, (b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, (c) humectants such as glycerol, (d) disintegrating agents such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, (e) solution retarding agents such as paraffin, (f) absorption accelerators such as quaternary ammonium compounds, (g) wetting agents such as, for example, 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 glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may include a buffering agent.

0547Solid compositions of a similar type can be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the art of pharmacology. They may optionally comprise opacifying agents and can be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of encapsulating compositions which can be used include polymeric substances and waxes. Solid compositions of a similar type can be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.

0548The active ingredient can be in a micro-encapsulated form with one or more excipients as noted above. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings, and other coatings well known in the pharmaceutical formulating art. In such solid dosage forms the active ingredient can be admixed with at least one inert diluent such as sucrose, lactose, or starch. Such dosage forms may comprise, as is normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such a magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms may comprise buffering agents. They may optionally comprise opacifying agents and can be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of encapsulating agents which can be used include polymeric substances and waxes.

0549Dosage forms for topical and/or transdermal administration of a compound described herein may include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, and/or patches. Generally, the active ingredient is admixed under sterile conditions with a pharmaceutically acceptable carrier or excipient and/or any needed preservatives and/or buffers as can be required. Additionally, the present disclosure contemplates the use of transdermal patches, which often have the added advantage of providing controlled delivery of an active ingredient to the body. Such dosage forms can be prepared, for example, by dissolving and/or dispensing the active ingredient in the proper medium. Alternatively or additionally, the rate can be controlled by either providing a rate controlling membrane and/or by dispersing the active ingredient in a polymer matrix and/or gel.

0550Suitable devices for use in delivering intradermal pharmaceutical compositions described herein include short needle devices. Intradermal compositions can be administered by devices which limit the effective penetration length of a needle into the skin. Alternatively or additionally, conventional syringes can be used in the classical mantoux method of intradermal administration. Jet injection devices which deliver liquid formulations to the dermis via a liquid jet injector and/or via a needle which pierces the stratum corneum and produces a jet which reaches the dermis are suitable. Ballistic powder/particle delivery devices which use compressed gas to accelerate the compound in powder form through the outer layers of the skin to the dermis are suitable.

0551Formulations suitable for topical administration include, but are not limited to, liquid and/or semi-liquid preparations such as liniments, lotions, oil-in-water and/or water-in-oil emulsions such as creams, ointments, and/or pastes, and/or solutions and/or suspensions. Topically administrable formulations may, for example, comprise from about 1% to about 10% (w/w) active ingredient, although the concentration of the active ingredient can be as high as the solubility limit of the active ingredient in the solvent. Formulations for topical administration may further comprise one or more of the additional ingredients described herein.

0552A pharmaceutical composition described herein can be prepared, packaged, and/or sold in a formulation suitable for pulmonary administration via the buccal cavity. Such a formulation may comprise dry particles which comprise the active ingredient and which have a diameter in the range from about 0.5 to about 7 nanometers, or from about 1 to about 6 nanometers. Such compositions are conveniently in the form of dry powders for administration using a device comprising a dry powder reservoir to which a stream of propellant can be directed to disperse the powder and/or using a self-propelling solvent/powder dispensing container such as a device comprising the active ingredient dissolved and/or suspended in a low-boiling propellant in a sealed container. Such powders comprise particles wherein at least 98% of the particles by weight have a diameter greater than 0.5 nanometers and at least 95% of the particles by number have a diameter less than 7 nanometers. Alternatively, at least 95% of the particles by weight have a diameter greater than 1 nanometer and at least 90% of the particles by number have a diameter less than 6 nanometers. Dry powder compositions may include a solid fine powder diluent such as sugar and are conveniently provided in a unit dose form.

0553Low boiling propellants generally include liquid propellants having a boiling point of below 65° F. at atmospheric pressure. Generally the propellant may constitute 50 to 99.9% (w/w) of the composition, and the active ingredient may constitute 0.1 to 20% (w/w) of the composition. The propellant may further comprise additional ingredients such as a liquid non-ionic and/or solid anionic surfactant and/or a solid diluent (which may have a particle size of the same order as particles comprising the active ingredient).

0554Pharmaceutical compositions described herein formulated for pulmonary delivery may provide the active ingredient in the form of droplets of a solution and/or suspension. Such formulations can be prepared, packaged, and/or sold as aqueous and/or dilute alcoholic solutions and/or suspensions, optionally sterile, comprising the active ingredient, and may conveniently be administered using any nebulization and/or atomization device. Such formulations may further comprise one or more additional ingredients including, but not limited to, a flavoring agent such as saccharin sodium, a volatile oil, a buffering agent, a surface active agent, and/or a preservative such as methylhydroxybenzoate. The droplets provided by this route of administration may have an average diameter in the range from about 0.1 to about 200 nanometers.

0555Formulations described herein as being useful for pulmonary delivery are useful for intranasal delivery of a pharmaceutical composition described herein. Another formulation suitable for intranasal administration is a coarse powder comprising the active ingredient and having an average particle from about 0.2 to 500 micrometers. Such a formulation is administered by rapid inhalation through the nasal passage from a container of the powder held close to the nares.

0556Formulations for nasal administration may, for example, comprise from about as little as 0.1% (w/w) to as much as 100% (w/w) of the active ingredient, and may comprise one or more of the additional ingredients described herein. A pharmaceutical composition described herein can be prepared, packaged, and/or sold in a formulation for buccal administration. Such formulations may, for example, be in the form of tablets and/or lozenges made using conventional methods, and may contain, for example, 0.1 to 20% (w/w) active ingredient, the balance comprising an orally dissolvable and/or degradable composition and, optionally, one or more of the additional ingredients described herein. Alternately, formulations for buccal administration may comprise a powder and/or an aerosolized and/or atomized solution and/or suspension comprising the active ingredient. Such powdered, aerosolized, and/or aerosolized formulations, when dispersed, may have an average particle and/or droplet size in the range from about 0.1 to about 200 nanometers, and may further comprise one or more of the additional ingredients described herein.

0557A pharmaceutical composition described herein can be prepared, packaged, and/or sold in a formulation for ophthalmic administration. Such formulations may, for example, be in the form of eye drops including, for example, a 0.1-1.0% (w/w) solution and/or suspension of the active ingredient in an aqueous or oily liquid carrier or excipient. Such drops may further comprise buffering agents, salts, and/or one or more other of the additional ingredients described herein. Other opthalmically-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form and/or in a liposomal preparation. Ear drops and/or eye drops are also contemplated as being within the scope of this disclosure.

0558Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for administration to humans, such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and/or perform such modification with ordinary experimentation.

0559The compounds provided herein are typically formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the compositions described herein will be decided by a physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject or organism will depend upon a variety of factors including the disease being treated and the severity of the disorder; the activity of the specific active ingredient employed; the specific composition employed; the age, body weight, general health, sex, and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific active ingredient employed; the duration of the treatment; drugs used in combination or coincidental with the specific active ingredient employed; and like factors well known in the medical arts.

0560The compounds and compositions provided herein can be administered by any route, including enteral (e.g., oral), parenteral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, interdermal, rectal, intravaginal, intraperitoneal, topical (as by powders, ointments, creams, and/or drops), mucosal, nasal, bucal, sublingual; by intratracheal instillation, bronchial instillation, and/or inhalation; and/or as an oral spray, nasal spray, and/or aerosol. Specifically contemplated routes are oral administration, intravenous administration (e.g., systemic intravenous injection), regional administration via blood and/or lymph supply, and/or direct administration to an affected site. In general, the most appropriate route of administration will depend upon a variety of factors including 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 is able to tolerate oral administration). In certain embodiments, the compound or pharmaceutical composition described herein is suitable for topical administration to the eye of a subject.

0561The exact amount of a compound required to achieve an effective amount will vary from subject to subject, depending, for example, on species, age, and general condition of a subject, severity of the side effects or disorder, identity of the particular compound, mode of administration, and the like. An effective amount may be included in a single dose (e.g., single oral dose) or multiple doses (e.g., multiple oral doses). In certain embodiments, when multiple doses are administered to a subject or applied to a biological sample, tissue, or cell, any two doses of the multiple doses include different or substantially the same amounts of a compound described herein. In certain embodiments, when multiple doses are administered to a subject or applied to a biological sample, tissue, or cell, the frequency of administering the multiple doses to the subject or applying the multiple doses to the tissue or cell is three doses a day, two doses a day, one dose a day, one dose every other day, one dose every third day, one dose every week, one dose every two weeks, one dose every three weeks, or one dose every four weeks. In certain embodiments, the frequency of administering the multiple doses to the subject or applying the multiple doses to the tissue or cell is one dose per day. In certain embodiments, the frequency of administering the multiple doses to the subject or applying the multiple doses to the tissue or cell is two doses per day. In certain embodiments, the frequency of administering the multiple doses to the subject or applying the multiple doses to the tissue or cell is three doses per day. In certain embodiments, when multiple doses are administered to a subject or applied to a biological sample, tissue, or cell, the duration between the first dose and last dose of the multiple doses is one day, two days, four days, one week, two weeks, three weeks, one month, two months, three months, four months, six months, nine months, one year, two years, three years, four years, five years, seven years, ten years, fifteen years, twenty years, or the lifetime of the subject, biological sample, tissue, or cell. In certain embodiments, the duration between the first dose and last dose of the multiple doses is three months, six months, or one year. In certain embodiments, the duration between the first dose and last dose of the multiple doses is the lifetime of the subject, biological sample, tissue, or cell. In certain embodiments, a dose (e.g., a single dose, or any dose of multiple doses) described herein includes independently between 0.1 μg and 1 μg, between 0.001 mg and 0.01 mg, between 0.01 mg and 0.1 mg, between 0.1 mg and 1 mg, between 1 mg and 3 mg, between 3 mg and 10 mg, between 10 mg and 30 mg, between 30 mg and 100 mg, between 100 mg and 300 mg, between 300 mg and 1,000 mg, or between 1 g and 10 g, inclusive, of a compound described herein. In certain embodiments, a dose described herein includes independently between 1 mg and 3 mg, inclusive, of a compound described herein. In certain embodiments, a dose described herein includes independently between 3 mg and 10 mg, inclusive, of a compound described herein. In certain embodiments, a dose described herein includes independently between 10 mg and 30 mg, inclusive, of a compound described herein. In certain embodiments, a dose described herein includes independently between 30 mg and 100 mg, inclusive, of a compound described herein.

0562In certain embodiments, an effective amount of a compound for administration one or more times a day to a 70 kg adult human comprises about 0.0001 mg to about 3000 mg, about 0.0001 mg to about 2000 mg, about 0.0001 mg to about 1000 mg, about 0.001 mg to about 1000 mg, about 0.01 mg to about 1000 mg, about 0.1 mg to about 1000 mg, about 1 mg to about 1000 mg, about 1 mg to about 100 mg, about 10 mg to about 1000 mg, or about 100 mg to about 1000 mg, of a compound per unit dosage form.

0563In certain embodiments, the compound of the invention is administered orally or parenterally at dosage levels sufficient to deliver from about 0.001 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 50 mg/kg, preferably from about 0.1 mg/kg to about 40 mg/kg, preferably from about 0.5 mg/kg to about 30 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, and more preferably from about 1 mg/kg to about 25 mg/kg, of subject body weight per day, one or more times a day, to obtain the desired therapeutic effect.

0564Dose ranges as described herein provide guidance for the administration of provided pharmaceutical compositions to an adult. The amount to be administered to, for example, a child or an adolescent can be determined by a medical practitioner or person skilled in the art and can be lower or the same as that administered to an adult.

0565A compound or composition, as described herein, can be administered in combination with one or more additional pharmaceutical agents (e.g., therapeutically and/or prophylactically active agents) useful in treating and/or preventing a proliferative disease. The compounds or compositions can be administered in combination with additional pharmaceutical agents that improve their activity (e.g., activity (e.g., potency and/or efficacy) in treating a proliferative disease in a subject in need thereof, in preventing a proliferative disease in a subject in need thereof, and/or in inhibiting the activity of a protein kinase (e.g., CDK (e.g., CDK7, CDK12, and/or CDK13)) in a subject, biological sample, tissue, or cell), improve bioavailability, improve safety, reduce drug resistance, reduce and/or modify metabolism, inhibit excretion, and/or modify distribution in a subject, biological sample, tissue, or cell. It will also be appreciated that the therapy employed may achieve a desired effect for the same disorder, and/or it may achieve different effects. In certain embodiments, a pharmaceutical composition described herein including a compound described herein and an additional pharmaceutical agent shows a synergistic effect that is absent in a pharmaceutical composition including one of the compound and the additional pharmaceutical agent, but not both.

0566The compound or composition can be administered concurrently with, prior to, or subsequent to one or more additional pharmaceutical agents, which may be useful as, e.g., combination therapies in treating and/or preventing a proliferative disease. Pharmaceutical agents include therapeutically active agents. Pharmaceutical agents also include prophylactically active agents. Pharmaceutical agents include small organic molecules such as drug compounds (e.g., compounds approved for human or veterinary use by the U.S. Food and Drug Administration as provided in the Code of Federal Regulations (CFR)), peptides, proteins, carbohydrates, monosaccharides, oligosaccharides, polysaccharides, nucleoproteins, mucoproteins, lipoproteins, synthetic polypeptides or proteins, small molecules linked to proteins, glycoproteins, steroids, nucleic acids, DNAs, RNAs, nucleotides, nucleosides, oligonucleotides, antisense oligonucleotides, lipids, hormones, vitamins, and cells. In certain embodiments, the additional pharmaceutical agent is a pharmaceutical agent useful in treating a proliferative disease. In certain embodiments, the additional pharmaceutical agent is a pharmaceutical agent useful in preventing a proliferative disease. In certain embodiments, the additional pharmaceutical agent is a pharmaceutical agent useful in inhibiting the activity of a protein kinase (e.g., CDK (e.g., CDK7, CDK12, and/or CDK13)) in a subject, biological sample, tissue, or cell. In certain embodiments, the additional pharmaceutical agent is a pharmaceutical agent useful in inducing apoptosis in a cell. In certain embodiments, the additional pharmaceutical agent is a pharmaceutical agent approved by a regulatory agency (e.g., the US FDA) for treating and/or preventing a proliferative disease. Each additional pharmaceutical agent may be administered at a dose and/or on a time schedule determined for that pharmaceutical agent. The additional pharmaceutical agent(s) may also be administered together with each other and/or with the compound or composition described herein in a single dose or administered separately in different doses. The particular combination to employ in a regimen will take into account compatibility of the compound described herein with the additional pharmaceutical agent(s) and/or the desired therapeutic and/or prophylactic effect to be achieved. In general, it is expected that the additional pharmaceutical agent(s) in combination be utilized at levels that do not exceed the levels at which they are utilized individually. In some embodiments, the levels utilized in combination will be lower than those utilized individually.

0567In certain embodiments, the additional pharmaceutical agent is an anti-proliferative agent (e.g., anti-cancer agent). In certain embodiments, the additional pharmaceutical agent is an anti-leukemia agent. In certain embodiments, the additional pharmaceutical agent is ABITREXATE (methotrexate), ADE, Adriamycin RDF (doxorubicin hydrochloride), Ambochlorin (chlorambucil), ARRANON (nelarabine), ARZERRA (ofatumumab), BOSULIF (bosutinib), BUSULFEX (busulfan), CAMPATH (alemtuzumab), CERUBIDINE (daunorubicin hydrochloride), CLAFEN (cyclophosphamide), CLOFAREX (clofarabine), CLOLAR (clofarabine), CVP, CYTOSAR-U (cytarabine), CYTOXAN (cyclophosphamide), ERWINAZE (Asparaginase Erwinia Chrysanthemi), FLUDARA (fludarabine phosphate), FOLEX (methotrexate), FOLEX PFS (methotrexate), GAZYVA (obinutuzumab), GLEEVEC (imatinib mesylate), Hyper-CVAD, ICLUSIG (ponatinib hydrochloride), IMBRUVICA (ibrutinib), LEUKERAN (chlorambucil), LINFOLIZIN (chlorambucil), MARQIBO (vincristine sulfate liposome), METHOTREXATE LPF (methorexate), MEXATE (methotrexate), MEXATE-AQ (methotrexate), mitoxantrone hydrochloride, MUSTARGEN (mechlorethamine hydrochloride), MYLERAN (busulfan), NEOSAR (cyclophosphamide), ONCASPAR (Pegaspargase), PURINETHOL (mercaptopurine), PURIXAN (mercaptopurine), Rubidomycin (daunorubicin hydrochloride), SPRYCEL (dasatinib), SYNRIBO (omacetaxine mepesuccinate), TARABINE PFS (cytarabine), TASIGNA (nilotinib), TREANDA (bendamustine hydrochloride), TRISENOX (arsenic trioxide), VINCASAR PFS (vincristine sulfate), ZYDELIG (idelalisib), or a combination thereof. In certain embodiments, the additional pharmaceutical agent is an anti-lymphoma agent. In certain embodiments, the additional pharmaceutical agent is ABITREXATE (methotrexate), ABVD, ABVE, ABVE-PC, ADCETRIS (brentuximab vedotin), ADRIAMYCIN PFS (doxorubicin hydrochloride), ADRIAMYCIN RDF (doxorubicin hydrochloride), AMBOCHLORIN (chlorambucil), AMBOCLORIN (chlorambucil), ARRANON (nelarabine), BEACOPP, BECENUM (carmustine), BELEODAQ (belinostat), BEXXAR (tositumomab and iodine I 131 tositumomab), BICNU (carmustine), BLENOXANE (bleomycin), CARMUBRIS (carmustine), CHOP, CLAFEN (cyclophosphamide), COPP, COPP-ABV, CVP, CYTOXAN (cyclophosphamide), DEPOCYT (liposomal cytarabine), DTIC-DOME (dacarbazine), EPOCH, FOLEX (methotrexate), FOLEX PFS (methotrexate), FOLOTYN (pralatrexate), HYPER-CVAD, ICE, IMBRUVICA (ibrutinib), INTRON A (recombinant interferon alfa-2b), ISTODAX (romidepsin), LEUKERAN (chlorambucil), LINFOLIZIN (chlorambucil), Lomustine, MATULANE (procarbazine hydrochloride), METHOTREXATE LPF (methotrexate), MEXATE (methotrexate), MEXATE-AQ (methotrexate), MOPP, MOZOBIL (plerixafor), MUSTARGEN (mechlorethamine hydrochloride), NEOSAR (cyclophosphamide), OEPA, ONTAK (denileukin diftitox), OPPA, R-CHOP, REVLIMID (lenalidomide), RITUXAN (rituximab), STANFORD V, TREANDA (bendamustine hydrochloride), VAMP, VELBAN (vinblastine sulfate), VELCADE (bortezomib), VELSAR (vinblastine sulfate), VINCASAR PFS (vincristine sulfate), ZEVALIN (ibritumomab tiuxetan), ZOLINZA (vorinostat), ZYDELIG (idelalisib), or a combination thereof. In certain embodiments, the additional pharmaceutical agent is an anti-myelodysplasia agent. In certain embodiments, the additional pharmaceutical agent is REVLIMID (lenalidomide), DACOGEN (decitabine), VIDAZA (azacitidine), CYTOSAR-U (cytarabine), IDAMYCIN (idarubicin), CERUBIDINE (daunorubicin), or a combination thereof.

0568In certain embodiments, the additional pharmaceutical agent is an anti-macroglobulinemia agent. In certain embodiments, the additional pharmaceutical agent is LEUKERAN (chlorambucil), NEOSAR (cyclophosphamide), FLUDARA (fludarabine), LEUSTATIN (cladribine), or a combination thereof. In certain embodiments, the additional pharmaceutical agent is ABITREXATE (methotrexate), ABRAXANE (paclitaxel albumin-stabilized nanoparticle formulation), AC, AC-T, ADE, ADRIAMYCIN PFS (doxorubicin hydrochloride), ADRUCIL (fluorouracil), AFINITOR (everolimus), AFINITOR DISPERZ (everolimus), ALDARA (imiquimod), ALIMTA (pemetrexed disodium), AREDIA (pamidronate disodium), ARIMIDEX (anastrozole), AROMASIN (exemestane), AVASTIN (bevacizumab), BECENUM (carmustine), BEP, BICNU (carmustine), BLENOXANE (bleomycin), CAF, CAMPTOSAR (irinotecan hydrochloride), CAPDX, CAPRELSA (vandetanib), CARBOPLATIN-TAXOL, CARMUBRIS (carmustine), CASODEX (bicalutamide), CEENU (lomustine), CERUBIDINE (daunorubicin hydrochloride), CERVARIX (recombinant HPV bivalent vaccine), CLAFEN (cyclophosphamide), CMF, COMETRIQ (cabozantinib-s-malate), COSMEGEN (dactinomycin), CYFOS (ifosfamide), CYRAMZA (ramucirumab), CYTOSAR-U (cytarabine), CYTOXAN (cyclophosphamide), DACOGEN (decitabine), DEGARELIX, DOXIL (doxorubicin hydrochloride liposome), DOXORUBICIN HYDROCHLORIDE, DOX-SL (doxorubicin hydrochloride liposome), DTIC-DOME (dacarbazine), EFUDEX (fluorouracil), ELLENCE (epirubicin hydrochloride), ELOXATIN (oxaliplatin), ERBITUX (cetuximab), ERIVEDGE (vismodegib), ETOPOPHOS (etoposide phosphate), EVACET (doxorubicin hydrochloride liposome), FARESTON (toremifene), FASLODEX (fulvestrant), FEC, FEMARA (letrozole), FLUOROPLEX (fluorouracil), FOLEX (methotrexate), FOLEX PFS (methotrexate), FOLFIRI, FOLFIRI-BEVACIZUMAB, FOLFIRI-CETUXIMAB, FOLFIRINOX, FOLFOX, FU-LV, GARDASIL (recombinant human papillomavirus (HPV) quadrivalent vaccine), GEMCITABINE-CISPLATIN, GEMCITABINE-OXALIPLATIN, GEMZAR (gemcitabine hydrochloride), GILOTRIF (afatinib dimaleate), GLEEVEC (imatinib mesylate), GLIADEL (carmustine implant), GLIADEL WAFER (carmustine implant), HERCEPTIN (trastuzumab), HYCAMTIN (topotecan hydrochloride), IFEX (ifosfamide), IFOSFAMIDUM (ifosfamide), INLYTA (axitinib), INTRON A (recombinant interferon alfa-2b), IRESSA (gefitinib), IXEMPRA (ixabepilone), JAKAFI (ruxolitinib phosphate), JEVTANA (cabazitaxel), KADCYLA (ado-trastuzumab emtansine), KEYTRUDA (pembrolizumab), KYPROLIS (carfilzomib), LIPODOX (doxorubicin hydrochloride liposome), LUPRON (leuprolide acetate), LUPRON DEPOT (leuprolide acetate), LUPRON DEPOT-3 MONTH (leuprolide acetate), LUPRON DEPOT-4 MONTH (leuprolide acetate), LUPRON DEPOT-PED (leuprolide acetate), MEGACE (megestrol acetate), MEKINIST (trametinib), METHAZOLASTONE (temozolomide), METHOTREXATE LPF (methotrexate), MEXATE (methotrexate), MEXATE-AQ (methotrexate), MITOXANTRONE HYDROCHLORIDE, MITOZYTREX (mitomycin c), MOZOBIL (plerixafor), MUSTARGEN (mechlorethamine hydrochloride), MUTAMYCIN (mitomycin c), MYLOSAR (azacitidine), NAVELBINE (vinorelbine tartrate), NEOSAR (cyclophosphamide), NEXAVAR (sorafenib tosylate), NOLVADEX (tamoxifen citrate), NOVALDEX (tamoxifen citrate), OFF, PAD, PARAPLAT (carboplatin), PARAPLATIN (carboplatin), PEG-INTRON (peginterferon alfa-2b), PEMETREXED DISODIUM, PERJETA (pertuzumab), PLATINOL (cisplatin), PLATINOL-AQ (cisplatin), POMALYST (pomalidomide), prednisone, PROLEUKIN (aldesleukin), PROLIA (denosumab), PROVENGE (sipuleucel-t), REVLIMID (lenalidomide), RUBIDOMYCIN (daunorubicin hydrochloride), SPRYCEL (dasatinib), STIVARGA (regorafenib), SUTENT (sunitinib malate), SYLATRON (peginterferon alfa-2b), SYLVANT (siltuximab), SYNOVIR (thalidomide), TAC, TAFINLAR (dabrafenib), TARABINE PFS (cytarabine), TARCEVA (erlotinib hydrochloride), TASIGNA (nilotinib), TAXOL (paclitaxel), TAXOTERE (docetaxel), TEMODAR (temozolomide), THALOMID (thalidomide), TOPOSAR (etoposide), TORISEL (temsirolimus), TPF, TRISENOX (arsenic trioxide), TYKERB (lapatinib ditosylate), VECTIBIX (panitumumab), VEIP, VELBAN (vinblastine sulfate), VELCADE (bortezomib), VELSAR (vinblastine sulfate), VEPESID (etoposide), VIADUR (leuprolide acetate), VIDAZA (azacitidine), VINCASAR PFS (vincristine sulfate), VOTRIENT (pazopanib hydrochloride), WELLCOVORIN (leucovorin calcium), XALKORI (crizotinib), XELODA (capecitabine), XELOX, XGEVA (denosumab), XOFIGO (radium 223 dichloride), XTANDI (enzalutamide), YERVOY (ipilimumab), ZALTRAP (ziv-aflibercept), ZELBORAF (vemurafenib), ZOLADEX (goserelin acetate), ZOMETA (zoledronic acid), ZYKADIA (ceritinib), ZYTIGA (abiraterone acetate), or a combination thereof. In certain embodiments, the additional pharmaceutical agent is a protein kinase inhibitor (e.g., tyrosine protein kinase inhibitor). In certain embodiments, the additional pharmaceutical agent is an inhibitor of a Src family kinase. In certain embodiments, the additional pharmaceutical agent is a CDK inhibitor. In certain embodiments, the additional pharmaceutical agent is a CDK7 inhibitor. In certain embodiments, the additional pharmaceutical agent is a CDK12 inhibitor. In certain embodiments, the additional pharmaceutical agent is a CDK13 inhibitor. In certain embodiments, the additional pharmaceutical agent is an inhibitor of one or more protein kinases selected from the group consisting of IRAK1, IRAK4, BMX, and PI3K. In certain embodiments, the additional pharmaceutical agent is an inhibitor of one or more protein kinases selected from the group consisting of BUB1B, CDK2, CDK9, CHEK2, FGR, HIPK4, PRKCQ, RET, SRC, or MELK. In certain embodiments, the additional pharmaceutical agent is an inhibitor of one or more protein kinases selected from the group consisting of ABL, ARG, BLK, CSK, EphB1, EphB2, FGR, FRK, FYN, SRC, YES, LCK, LYN, MAP2K5, NLK, p38a, SNRK, and TEC. In certain embodiments, the additional pharmaceutical agent is an inhibitor of one or more protein kinases selected from the group consisting of ABL1(H396P)-phosphorylated, ABL1-phosphorylated, BLK, EPHA4, EPHB2, EPHB3, EPHB4, FGR, JAK3(JH1domain-catalytic), KIT, KIT(L576P), KIT(V559D), PDGFRB, SRC, YES, ABL1(H396P)-nonphosphorylated, ABL1(Y253F)-phosphorylated, ABL1-nonphosphorylated, FRK, LYN, ABL1(Q252H)-nonphosphorylated, DDR1, EPHB1, ERBB4, p38-alpha, ABL2, ABL1(Q252H)-phosphorylated, SIK, EPHA8, MEK5, ABL1(E255K)-phosphorylated, ABL1(F317L)-nonphosphorylated, FYN, LCK, EPHA2, ABL1(M351T)-phosphorylated, TXK, EGFR(L858R), EGFR(L861Q), ERBB2, ERBB3, EPHA5, ABL1(F317I)-nonphosphorylated, EGFR(L747-E749del, A750P), CSK, EPHA1, ABL1(F317L)-phosphorylated, BRAF(V600E), EGFR, KIT-autoinhibited, and EGFR(E746-A750del). In certain embodiments, the additional pharmaceutical agent is an inhibitor of one or more protein kinases selected from the group consisting of ABL1(F317L)-nonphosphorylated, ABL1(H396P)-nonphosphorylated, ABL1(H396P)-phosphorylated, ABL1-phosphorylated, BLK, EPHA4, EPHB2, EPHB3, EPHB4, JAK3(JH1domain-catalytic), KIT, KIT(L576P), KIT(V559D), LYN, PDGFRB, SRC, YES, ABL1-nonphosphorylated, ABL1(Y253F)-phosphorylated, ERBB3, FGR, FRK, p38-alpha, ABL1(F317I)-nonphosphorylated, DDR1, EPHA2, ABL1(Q252H)-phosphorylated, MEK5, ABL1(Q252H)-nonphosphorylated, ABL2, FYN, EPHB1, ABL1(E255K)-phosphorylated, ABL1(F317L)-phosphorylated, EPHA1, ABL1(M351T)-phosphorylated, ERBB4, TXK, LCK, EPHA8, SIK, EPHA5, EGFR(L861Q), CSF1R-autoinhibited, BRAF(V600E), BRK, CSK, KIT(D816V), KIT-autoinhibited, EGFR(L747-T751del,Sins), EGFR(L858R), EGFR(L747-E749del, A750P), and CSF1R. In certain embodiments, the additional pharmaceutical agent is an anti-angiogenesis agent, anti-inflammatory agent, immunosuppressant, anti-bacterial agent, anti-viral agent, cardiovascular agent, cholesterol-lowering agent, anti-diabetic agent, anti-allergic agent, pain-relieving agent, or a combination thereof. In certain embodiments, the compounds described herein or pharmaceutical compositions can be administered in combination with an anti-cancer therapy including, but not limited to, transplantation (e.g., bone marrow transplantation, stem cell transplantation), surgery, radiation therapy, immunotherapy, and chemotherapy.

0569Also encompassed by the disclosure are kits (e.g., pharmaceutical packs). The kits provided may comprise a pharmaceutical composition or compound described herein and a container (e.g., a vial, ampule, bottle, syringe, and/or dispenser package, or other suitable container). In some embodiments, provided kits may optionally further include a second container comprising a pharmaceutical excipient for dilution or suspension of a pharmaceutical composition or compound described herein. In some embodiments, the pharmaceutical composition or compound described herein provided in the first container and the second container are combined to form one unit dosage form.

0570In certain embodiments, a kit described herein includes a first container comprising a compound or pharmaceutical composition described herein. In certain embodiments, a kit described herein is useful in treating a proliferative disease (e.g., cancers (e.g., leukemia, acute lymphoblastic leukemia, lymphoma, Burkitt's lymphoma, melanoma, multiple myeloma, breast cancer, Ewing's sarcoma, osteosarcoma, brain cancer, neuroblastoma, lung cancer, colorectal cancer), benign neoplasms, diseases associated with angiogenesis, inflammatory diseases, autoinflammatory diseases, and autoimmune diseases) in a subject in need thereof, preventing a proliferative disease in a subject in need thereof, inhibiting the activity of a protein kinase (e.g., CDK (e.g., CDK7, CDK12, or CDK13)) in a subject, biological sample, tissue, or cell, and/or inducing apoptosis in a cell.

0571In certain embodiments, a kit described herein further includes instructions for using the compound or pharmaceutical composition included in the kit. A kit described herein may also include information as required by a regulatory agency such as the U.S. Food and Drug Administration (FDA). In certain embodiments, the information included in the kits is prescribing information. In certain embodiments, the kits and instructions provide for treating a proliferative disease in a subject in need thereof, preventing a proliferative disease in a subject in need thereof, inhibiting the activity of a protein kinase (e.g., CDK (e.g., CDK7, CDK12, or CDK13)) in a subject, biological sample, tissue, or cell, and/or inducing apoptosis in a cell. A kit described herein may include one or more additional pharmaceutical agents described herein as a separate composition.

0000Methods of Treatment and Uses

0572The present invention also provides methods for the treatment or prevention of a proliferative disease (e.g., cancers (e.g., leukemia, acute lymphoblastic leukemia, lymphoma, Burkitt's lymphoma, melanoma, multiple myeloma, breast cancer, Ewing's sarcoma, osteosarcoma, brain cancer, neuroblastoma, lung cancer, colorectal cancer), benign neoplasms, diseases associated with angiogenesis, inflammatory diseases, autoinflammatory diseases, and autoimmune diseases).

0573The compounds described herein may exhibit kinase inhibitory activity; the ability to inhibit cyclin-dependent kinase (CDK); the ability to inhibit cyclin-dependent kinase 7 (CDK7); the ability to inhibit cyclin-dependent kinase 7 (CDK7), without inhibiting another cyclin-dependent kinase (CDK); the ability to inhibit cyclin-dependent kinase 12 (CDK12); the ability to inhibit cyclin-dependent kinase 12 (CDK12), without inhibiting another cyclin-dependent kinase (CDK); the ability to inhibit cyclin-dependent kinase 13 (CDK13); the ability to inhibit cyclin-dependent kinase 13 (CDK13), without inhibiting another cyclin-dependent kinase (CDK); the ability to inhibit cyclin-dependent kinases 12 and 13 (CDK12 and CDK13); the ability to inhibit cyclin-dependent kinases 12 and 13 (CDK12 and CDK13), without inhibiting another cyclin-dependent kinase (CDK); a therapeutic effect and/or preventative effect in the treatment of cancers; a therapeutic effect and/or preventative effect in the treatment of Myc-dependent cancers; and/or a therapeutic profile (e.g., optimum safety and curative effect) that is superior to existing chemotherapeutic agents.

0574Without wishing to be bound by any particular theory, the compounds described herein are able to bind (e.g., covalently modify) a protein kinase described herein. In certain embodiments, the R²group of a compound described herein is able to bind (e.g., covalently modify) to the protein kinase. In certain embodiments, the R²group of a compound described herein is able to covalently bind a cysteine residue of the protein kinase. In certain embodiments, the R²group of a compound described herein is able to covalently bind Cys312 residue of CDK7. In certain embodiments, the R²group of a compound described herein is able to covalently bind Cys Cys1039 residue of CDK12. In certain embodiments, the R²group of a compound described herein is able to covalently bind Cys1017 residue of CDK13.

0575In another aspect, the present disclosure provides methods of inhibiting the activity of a protein kinase in a subject, the methods comprising administering to the subject an effective amount (e.g., therapeutically effective amount) of a compound, or pharmaceutical composition thereof, as described herein.

0576In another aspect, the present disclosure provides methods of inhibiting the activity of a protein kinase in a biological sample, the methods comprising contacting the biological sample with an effective amount of a compound, or pharmaceutical composition thereof, as described herein.

0577In another aspect, the present disclosure provides methods of inhibiting the activity of a protein kinase in a tissue, the methods comprising contacting the tissue with an effective amount of a compound, or pharmaceutical composition thereof, as described herein.

0578In another aspect, the present disclosure provides methods of inhibiting the activity of a protein kinase in a cell, the methods comprising contacting the cell with an effective amount of a compound, or pharmaceutical composition thereof, as described herein.

0579In certain embodiments, the subject being treated is a mammal. In certain embodiments, the subject is a human. In certain embodiments, the subject is a domesticated animal, such as a dog, cat, cow, pig, horse, sheep, or goat. In certain embodiments, the subject is a companion animal such as a dog or cat. In certain embodiments, the subject is a livestock animal such as a cow, pig, horse, sheep, or goat. In certain embodiments, the subject is a zoo animal. In another embodiment, the subject is a research animal such as a rodent, dog, or non-human primate. In certain embodiments, the subject is a non-human transgenic animal such as a transgenic mouse or transgenic pig.

0580In certain embodiments, a biological sample described herein is a breast tissue, bone marrow, lymph node, spleen, or blood.

0581In certain embodiments, a cell described herein is in vitro. In certain embodiments, a cell described herein is ex vivo. In certain embodiments, a cell described herein is in vivo. In certain embodiments, a cell described herein is a malignant cell (e.g., malignant blood cell). In certain embodiments, a cell described herein is a malignant hematopoietic stem cell (e.g., malignant myeloid cell or malignant lymphoid cell). In certain embodiments, a cell described herein is a malignant lymphocyte (e.g., malignant T-cell or malignant B-cell). In certain embodiments, a cell described herein is a malignant red blood cell, malignant white blood cell, or malignant platelet. In certain embodiments, a cell described herein is a malignant neutrophil, malignant macrophage, or malignant plasma cell. In certain embodiments, a cell described herein is a carcinoma cell. In certain embodiments, a cell described herein is a carcinoma breast cell. In certain embodiments, a cell described herein is a sarcomas cell. In certain embodiments, a cell described herein is a sarcomas breast cell.

0582The proliferative disease to be treated or prevented using the compounds described herein may be associated with overexpression of a kinase, such as cyclin-dependent kinase (CDK). The process of eukaryotic cell division may be broadly divided into a series of sequential phases termed G1, S, G2, and M. Correct progression through the various phases of the cell cycle has been shown to be critically dependent upon the spatial and temporal regulation of a family of proteins known as cyclin dependent kinases (CDKs) and a diverse set of their cognate protein partners termed cyclins. CDKs are CDC2 (also known as CDK1) homologous serine-threonine kinase proteins that are able to utilize ATP as a substrate in the phosphorylation of diverse polypeptides in a sequence-dependent context. Cyclins are a family of proteins characterized by a homology region, containing approximately 100 amino acids, termed the “cyclin box” which is used in binding to, and defining selectivity for, specific CDK partner proteins.

0583Modulation of the expression levels, degradation rates, protein levels, and activity levels of various CDKs and cyclins throughout the cell cycle leads to the cyclical formation of a series of CDK/cyclin complexes, in which the CDKs are enzymatically active. The formation of these complexes controls passage through discrete cell cycle checkpoints and thereby enables the process of cell division to continue. Failure to satisfy the prerequisite biochemical criteria at a given cell cycle checkpoint, i.e., failure to form a required CDK/cyclin complex, can lead to cell cycle arrest and/or cellular apoptosis. Aberrant cellular proliferation can often be attributed to loss of correct cell cycle control. Inhibition of CDK enzymatic activity therefore provides a means by which abnormally dividing cells can have their division arrested and/or be killed. The diversity of CDKs, and CDK complexes, and their critical roles in mediating the cell cycle, provides a broad spectrum of potential therapeutic targets selected on the basis of a defined biochemical rationale.

0584CDK7, a member of the CDK family, was originally isolated as the catalytic subunit of the trimeric CDK-activating kinase (CAK) complex. This complex, consisting of CDK7, cyclin H, and MAT1, is responsible for activation of the mitotic promoting factor in vitro. The discovery that CDK7 was also a component of the basal transcription repair factor IIH (TFIIH) implicated a dual role for CDK7 in transcription as part of TFIIH and in the control of the cell cycle as the trimeric CAK complex. TFIIH is a multi-subunit protein complex identified as a factor required for RNA polymerase II (RNAP II)-catalyzed transcription, and subsequently this complex was found to play a key role in nucleotide excision repair. CDK7 is a component of at least three complexes, i.e., the trimeric CAK complex, the quaternary complex with the XPD (or ERCC2, a protein involved in transcription-coupled nucleotide excision repair), and the nine-subunit TFIIH complex. The two functions of CDK7 in CAK and CTD phosphorylation support critical facets of cellular proliferation, cell cycling, and transcription. Overexpression of CDK7 may inhibit apoptosis, promote transcription and cell proliferation, and/or disrupt DNA repair, and therefore, cause proliferative diseases. In certain embodiments, the proliferative disease to be treated or prevented using the compounds described herein may be associated with overexpression of a CDK (e.g., CDK7).

0585Cdk12 and Cdk13 are Cdc2-related proteins that share 92% identity in their kinase domains (Chen et al., Exp. Neurol., 2014, 261, 10-21). CDK12 plays a critical role in cell processes, for example, regulating transcription and splicing machinery by stabilizing the RNAPII and DNA interaction, and regulating DNA damage response (DDR) and maintenance of genomic stability by modulating the expression of DDR genes. Overexpression of CDK12 has been found to correlate, both at the transcriptional and protein level, with pathological parameters of breast cancer disease.

0586A proliferative disease may be associated with aberrant activity of a CDK (e.g., CDK7, CDK12, and/or CDK13). Aberrant activity of a CDK (e.g., CDK7, CDK12, and/or CDK13) may be an elevated and/or an inappropriate activity of the CDK. Deregulation of cell cycle progression is a characteristic of a proliferative disease, and a majority of proliferative diseases have abnormalities in some component of CDK (e.g., CDK7, CDK12, and/or CDK13) activity, frequently through elevated and/or inappropriate CDK activation. Inhibition of the catalytic activity of CDK7, CDK12, and/or CDK13 would be expected to inhibit cell cycle progression by blocking the phosphorylation of cell cycle CDKs, and would additionally inhibit transcription of effectors of cell division. In certain embodiments, CDK7 is not overexpressed, and the activity of CDK7 is elevated and/or inappropriate. In certain other embodiments, CDK7 is overexpressed, and the activity of CDK7 is elevated and/or inappropriate. In certain embodiments, CDK12 is not overexpressed, and the activity of CDK12 is elevated and/or inappropriate. In certain embodiments, CDK12 is overexpressed, and the activity of CDK12 is elevated and/or inappropriate. In certain other embodiments, CDK13 is not overexpressed, and the activity of CDK13 is elevated and/or inappropriate. In certain other embodiments, CDK13 is overexpressed, and the activity of CDK13 is elevated and/or inappropriate. The compounds described herein, and pharmaceutically acceptable salts, solvates, hydrates, polymorphs, co-crystals, tautomers, stereoisomers, isotopically labeled derivatives, prodrugs, and compositions thereof, may inhibit the activity of CDK7 and be useful in treating and/or preventing proliferative diseases. The compounds described herein, and pharmaceutically acceptable salts, solvates, hydrates, polymorphs, co-crystals, tautomers, stereoisomers, isotopically labeled derivatives, prodrugs, and compositions thereof, may inhibit the activity of CDK12 and/or CDK13 and be useful in treating and/or preventing proliferative diseases.

0587A proliferative disease may also be associated with inhibition of apoptosis of a cell in a biological sample or subject. All types of biological samples described herein or known in the art are contemplated as being within the scope of the invention. Apoptosis is the process of programmed cell death. Inhibition of apoptosis may result in uncontrolled cell proliferation and, therefore, may cause proliferative diseases. The cell cycle CDKs (CDK1, 2, 4, and 6) are activated by phosphorylation by CDK7/cyclin H (also called CAK). Inhibition of CDK7 would therefore result in cell-cycle arrest at multiple points in the cell cycle due to failure to activate the cell cycle CDKs. CDK 7 activates transcription by phosphorylating the CTD of RNAP II. Inhibition of CTD phosphorylation has been shown to inhibit transcription and reduce expression of short lived proteins, including those involved in apoptosis regulation. It is appreciated in the art that stalling of RNA polymerase may activate p53 (also known as protein 53 or tumor protein 53, a tumor suppressor protein that is encoded in humans by the TP53 gene), leading to apoptosis. Thus, inhibition of the activity of CDK7 are expected to cause cytotoxicity by inducing apoptosis. The compounds described herein, and pharmaceutically acceptable salts, solvates, hydrates, polymorphs, co-crystals, tautomers, stereoisomers, isotopically labeled derivatives, prodrugs, and compositions thereof, may induce apoptosis, and therefore, be useful in treating and/or preventing proliferative diseases.

0588The CycK/Cdk12 complex regulates phosphorylation of Ser2 in the C-terminal domain of RNA polymerase II and expression of a small subset of human genes, as revealed in expression microarrays. Through regulation of expression of DNA damage response genes (i.e. oncogenes), CycK/Cdk12 protects cells from genomic instability. In certain embodiments, the DNA damage response genes are BRCA1, BRCA2, HER1, HER2, ATR, FANCI, or FANCD2. In certain embodiments, the DNA damage response genes are BRCA1, HER2, ATR, FANCI, and FANCD2. In certain embodiments, the DNA damage response genes are BRCA1. In certain embodiments, the DNA damage response genes are HER2.

0589In certain embodiments, the proliferative disease to be treated or prevented using the compounds described herein is cancer. All types of cancers disclosed herein or known in the art are contemplated as being within the scope of the invention. In certain embodiments, the proliferative disease is a cancer associated with dependence on BCL-2 anti-apoptotic proteins (e.g., MCL-1 and/or XIAP). In certain embodiments, the proliferative disease is a cancer associated with dependence on BCL-2 anti-apoptotic proteins (e.g., MCL-1 and/or XIAP). In certain embodiments, the proliferative disease is a cancer associated with overexpression of MYC (a gene that codes for a transcription factor). In certain embodiments, the cancer is a MYC-dependent cancer. In certain embodiments, the proliferative disease is a cancer associated with amplification of BRCA1. In certain embodiments, the proliferative disease is a cancer associated with amplification of HER2. In certain embodiments, the proliferative disease is a hematological malignancy. In certain embodiments, the proliferative disease is a blood cancer. In certain embodiments, the proliferative disease is a hematological malignancy. In certain embodiments, the proliferative disease is leukemia. In certain embodiments, the proliferative disease is chronic lymphocytic leukemia (CLL). In certain embodiments, the proliferative disease is acute lymphoblastic leukemia (ALL). In certain embodiments, the proliferative disease is T-cell acute lymphoblastic leukemia (T-ALL). In certain embodiments, the proliferative disease is chronic myelogenous leukemia (CML). In certain embodiments, the proliferative disease is acute myelogenous leukemia (AML). In certain embodiments, the proliferative disease is acute monocytic leukemia (AMoL). In certain embodiments, the proliferative disease is lymphoma. In some embodiments, the proliferative disease is Burkitt's lymphoma. In certain embodiments, the proliferative disease is a Hodgkin's lymphoma. In certain embodiments, the proliferative disease is a non-Hodgkin's lymphoma. In certain embodiments, the proliferative disease is multiple myeloma. In certain embodiments, the proliferative disease is melanoma. In certain embodiments, the proliferative disease is colorectal cancer. In certain embodiments, the proliferative disease is breast cancer. In certain embodiments, the proliferative disease is recurring breast cancer. In certain embodiments, the proliferative disease is mutant breast cancer. In certain embodiments, the proliferative disease is HER2+ breast cancer. In certain embodiments, the proliferative disease is HER2− breast cancer. In certain embodiments, the proliferative disease is triple-negative breast cancer (TNBC). In certain embodiments, the proliferative disease is a bone cancer. In certain embodiments, the proliferative disease is osteosarcoma. In certain embodiments, the proliferative disease is Ewing's sarcoma. In some embodiments, the proliferative disease is a brain cancer. In some embodiments, the proliferative disease is neuroblastoma. In some embodiments, the proliferative disease is a lung cancer. In some embodiments, the proliferative disease is small cell lung cancer (SCLC). In some embodiments, the proliferative disease is non-small cell lung cancer. In some embodiments, the proliferative disease is a benign neoplasm. All types of benign neoplasms disclosed herein or known in the art are contemplated as being within the scope of the invention. In some embodiments, the proliferative disease is associated with angiogenesis. All types of angiogenesis disclosed herein or known in the art are contemplated as being within the scope of the invention. In certain embodiments, the proliferative disease is an inflammatory disease. All types of inflammatory diseases disclosed herein or known in the art are contemplated as being within the scope of the invention. In certain embodiments, the inflammatory disease is rheumatoid arthritis.

0590In certain embodiments, the proliferative disease is an acute inflammatory disease. In certain embodiments, the acute inflammatory disease is rheumatoid arthritis, Crohn's disease, or fibrosis. In some embodiments, the proliferative disease is an autoinflammatory disease. All types of autoinflammatory diseases disclosed herein or known in the art are contemplated as being within the scope of the invention. In some embodiments, the proliferative disease is an autoimmune disease. All types of autoimmune diseases disclosed herein or known in the art are contemplated as being within the scope of the invention.

0591Another aspect of the invention relates to methods of inhibiting the activity of a kinase in a biological sample or subject. In certain embodiments, the kinase is CDK. In certain embodiments, the kinase is CDK7. In certain embodiments, the kinase is CDK12. In certain embodiments, the kinase is CDK13. In certain embodiments, the activity of the kinase is aberrant activity of the kinase. In certain embodiments, the inhibition of the activity of the kinase is irreversible. In other embodiments, the inhibition of the activity of the kinase is reversible. In certain embodiments, the methods of inhibiting the activity of the kinase include attaching a compound described herein to the kinase.

0592Also provided in the present invention are methods of inhibiting transcription of genes in a biological sample or subject. In certain embodiments, the genes which may have their transcription inhibited by the activity of CDK7, CDK12, and/or CDK13 are listed in <figref idref="DRAWINGS">FIG. <b>11</b></figref>. In certain embodiments, the transcription of genes affected by the activity of CDK7 may be inhibited by a compound of the invention. In certain embodiments, the genes which may have their transcription inhibited by the activity of CDK7 are one or more selected from the group consisting of MYC, RUNX1, MYB, TAL1, GATA3, KLF2, HNRPDL, p21, ASCL1, MYCN, INSM1, NEUROD1, NEUROG1, FOXG1, FOXA1, SOX2, SOX4, BCL11A, OTX2, GAT2, PHOX2B, PLK2, TAF1, CTGF, WEE1, SDIM, JUN, PIM1, IL8, and FOS1. In certain embodiments, the genes which may have their transcription inhibited by the activity of CDK7 include MYC, KLF2, E2F2, CDK6, CCND3, E2F3, HNRPDL, TET1, IL7R, BRCA1, BRCA2, HER1, and HER2. In certain embodiments, the transcription of genes affected by the activity of CDK12 may be inhibited by a compound of the invention. In certain embodiments, the genes which may have their transcription inhibited by the activity of CDK12 are one or more selected from the group consisting of BRCA1, FANCI, ATR, FANCD2, APEX1, NEK9, CHEK1, CHEK2, ATM, RAD51C, RAD51D, ORC3L, MDC1, TERF2, ERCC4, FANCF, PARP9, RUNX1, MYB, TAL1, MCL1, MYC, BCL2, ETS1, and EWS-FLI. In certain embodiments, the transcription of genes affected by the activity of CDK13 may be inhibited by a compound of the invention. In certain embodiments, the genes which may have their transcription inhibited by the compounds herein are SNORA38.

0593The present invention also provides methods of inhibiting cell growth in a biological sample or subject.

0594In still another aspect, the present invention provides methods of inducing apoptosis of a cell in a biological sample or a subject.

0595In certain embodiments, the methods described herein include administering to a subject or contacting a biological sample with an effective amount of a compound described herein, or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof, or a pharmaceutical composition thereof. In certain embodiments, the methods described herein include administering to a subject or contacting a biological sample with an effective amount of a compound described herein, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof. In certain embodiments, the compound is contacted with a biological sample. In certain embodiments, the compound is administered to a subject. In certain embodiments, the compound is administered in combination with one or more additional pharmaceutical agents described herein. The additional pharmaceutical agent may be an anti-proliferative agent. In certain embodiments, the additional pharmaceutical agent is an anti-cancer agent. The additional pharmaceutical agent may also be a kinase inhibitor. In certain embodiments, the additional pharmaceutical agent is an inhibitor of a CDK. In certain embodiments, the additional pharmaceutical agent is an inhibitor of CDK7. In certain embodiments, the additional pharmaceutical agent is a selective inhibitor of CDK7. In certain embodiments, the additional pharmaceutical agent is a nonselective inhibitor of CDK7. In certain embodiments, the additional pharmaceutical agent is an inhibitor of CDK12. In certain embodiments, the additional pharmaceutical agent is a selective inhibitor of CDK12. In certain embodiments, the additional pharmaceutical agent is a nonselective inhibitor of CDK12. In certain embodiments, the additional pharmaceutical agent is an inhibitor of CDK13. In certain embodiments, the additional pharmaceutical agent is a selective inhibitor of CDK13. In certain embodiments, the additional pharmaceutical agent is a nonselective inhibitor of CDK13. In certain embodiments, the additional pharmaceutical agent is an inhibitor of another CDK. In certain embodiments, the additional pharmaceutical agent is a selective inhibitor of another CDK. In certain embodiments, the additional pharmaceutical agent is a nonselective inhibitor of another CDK. In certain embodiments, the additional pharmaceutical agent is flavopiridol, triptolide, SNS-032 (BMS-387032), PHA-767491, PHA-793887, BS-181, (S)—CR8, (R)—CR8, or NU6140. In certain embodiments, the additional pharmaceutical agent is an inhibitor of a mitogen-activated protein kinase (MAPK). In certain embodiments, the additional pharmaceutical agent is an inhibitor of a glycogen synthase kinase 3 (GSK3). In certain embodiments, the additional pharmaceutical agent is an inhibitor of an AGC kinase. In certain embodiments, the additional pharmaceutical agent is an inhibitor of a calmodulin-dependent kinase (CaM Kinase). In certain embodiments, the additional pharmaceutical agent is an inhibitor of a casein kinase 1. In certain embodiments, the additional pharmaceutical agent is an inhibitor of a STE kinase. In certain embodiments, the additional pharmaceutical agent is an inhibitor of a tyrosine kinase.

0596In some embodiments, the additional pharmaceutical agent is a topoisomerase inhibitor, a MCL1 inhibitor, a BCL-2 inhibitor, a BCL-xL inhibitor, a BRD4 inhibitor, a BRCA1 inhibitor, BRCA2 inhibitor, HER1 inhibitor, HER2 inhibitor, a CDK9 inhibitor, a Jumonji histone demethylase inhibitor, or a DNA damage inducer. In some embodiments, the additional pharmaceutical agent is etoposide, obatoclax, navitoclax, JQ1, 4-(((5′-chloro-2′-(((1R,4R)-4-(((R)-1-methoxypropan-2-yl)amino)cyclohexyl)amino)-[2,4′-bipyridin]-6-yl)amino)methyl)tetrahydro-2H-pyran-4-carbonitrile, JIB04, or cisplatin. In some embodiments, the additional pharmaceutical agent is etoposide, obatoclax, or navitoclax, and the disease to be treated is breast cancer, e.g., triple-negative breast cancer, HER2 positive breast cancer, HER2 negative breast cancer, ER-positive breast cancer, ER-negative breast cancer, or ER/PR-positive breast cancer. In some embodiments, the additional pharmaceutical agent is etoposide, JIB04, or cisplatin, and the disease to be treated is Ewing's sarcoma. In some embodiments, the additional pharmaceutical agent is JQ1 or NVP2, and the disease to be treated is leukemia, e.g., acute myelogenous leukemia, myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, monoblastic leukemia, or megakaryoblastic leukemia. In certain embodiments, a pharmaceutical composition described herein further comprises a combination of the additional pharmaceutical agents described herein.

0597The inventive compounds or compositions may synergistically augment inhibition of CDK7 induced by the additional pharmaceutical agent(s) in the biological sample or subject. The inventive compounds or compositions may synergistically augment inhibition of CDK12 and/or CDK13 induced by the additional pharmaceutical agent(s) in the biological sample or subject. Thus, the combination of the inventive compounds or compositions and the additional pharmaceutical agent(s) may be useful in treating proliferative diseases resistant to a treatment using the additional pharmaceutical agent(s) without the inventive compounds or compositions.

0598In some embodiments, the activity of a protein kinase is non-selectively inhibited by the compounds or pharmaceutical compositions described herein. In some embodiments, the activity of a protein kinase described herein is selectively inhibited by the compounds or pharmaceutical compositions described herein, compared to the activity of a different protein (e.g., a different protein kinase). In certain embodiments, the activity of CDK (e.g., CDK7, CDK12, or CDK13) is selectively inhibited by a compound or pharmaceutical composition described herein, compared to the activity of a different protein. In certain embodiments, the activity of CDK7 is selectively inhibited by a compound or pharmaceutical composition described herein, compared to the activity of a different CDK protein. In certain embodiments, the activity of CDK7 is selectively inhibited by a compound or pharmaceutical composition described herein, compared to the activity of CDK12. In certain embodiments, the activity of CDK7 is selectively inhibited by a compound or pharmaceutical composition described herein, compared to the activity of CDK13. In certain embodiments, the activity of CDK7 is selectively inhibited by a compound or pharmaceutical composition described herein, compared to the activity of CDK12 and the activity of CDK13. In certain embodiments, the activity of CDK12 is selectively inhibited by a compound or pharmaceutical composition described herein, compared to the activity of CDK7. In certain embodiments, the activity of CDK13 is selectively inhibited by a compound or pharmaceutical composition described herein, compared to the activity of CDK7. In certain embodiments, the activity of CDK12 and the activity of CDK13 are selectively inhibited by a compound or pharmaceutical composition described herein, compared to the activity of CDK7.

0599The selectivity of a compound or pharmaceutical composition described herein in inhibiting the activity of a protein kinase over a different protein (e.g., a different protein kinase) may be measured by the quotient of the IC₅₀value of the compound or pharmaceutical composition in inhibiting the activity of the different protein over the IC₅₀value of the compound or pharmaceutical composition in inhibiting the activity of the protein kinase. The selectivity of a compound or pharmaceutical composition described herein for a protein kinase over a different protein may also be measured by the quotient of the K_dvalue of an adduct of the compound or pharmaceutical composition and the different protein over the K_dvalue of an adduct of the compound or pharmaceutical composition and the protein kinase. In certain embodiments, the selectivity is at least 2-fold, at least 3-fold, at least 5-fold, at least 10-fold, at least 30-fold, at least 100-fold, at least 300-fold, at least 1,000-fold, at least 3,000-fold, at least 10,000-fold, at least 30,000-fold, or at least 100,000-fold. In certain embodiments, the selectivity is not more than 100,000-fold, not more than 10,000-fold, not more than 1,000-fold, not more than 100-fold, not more than 10-fold, or not more than 2-fold. Combinations of the above-referenced ranges (e.g., at least 2-fold and not more than 10,000-fold) are also within the scope of the disclosure.

EXAMPLES

0600In order that the invention described herein may be more fully understood, the following examples are set forth. The synthetic and biological examples described in this application are offered to illustrate the compounds, pharmaceutical compositions, and methods provided herein and are not to be construed in any way as limiting their scope.

0000Synthesis of the Compounds

0601The compounds provided herein can be prepared from readily available starting materials using the following general methods and procedures. Reactions were monitored by thin layer chromatography (TLC) with 0.25 mm E. Merck pre-coated silica gel plates (60 F₂₅₄) and Waters LCMS system (Waters 2489 UV/Visible Detector, Waters 3100 Mass, Waters 515 HPLC pump, Waters 2545 Binary Gradient Module, Waters Reagent Manager, Waters 2767 Sample Manager) using SunFire™ C18 column (4.6×50 mm, 5 μm particle size): solvent gradient=95% A at 0 min, 0% A at 5 min; solvent A=0.5% TFA in Water; solvent B=Methanol; flow rate: 1.5 mL/min Purification of reaction products was carried out by flash chromatography using CombiFlash®Rf with Teledyne Isco RediSep®Rf High Performance Gold or Silicycle SiliaSep™ High Performance columns (4 g, 12 g, 24 g, 40 g, 80 g or 120 g) or by Waters preparative HPLC system with a C18 column: solvent gradient=100% A at 0 min, 0% A at 15 min; solvent A=0.5% TFA in Water; solvent B=Methanol; flow rate: 20 mL/min. The purity of all compounds was over 95% and was analyzed with Waters LCMS system. ¹H NMR and ¹³C NMR spectra were obtained using a Varian Inova-600 or 400 MHz spectrometer. Chemical shifts are reported relative to chloroform (δ=7.24) for ¹H NMR or dimethyl sulfoxide (δ=2.50) for ¹H NMR and dimethyl sulfoxide (δ=39.51) for ¹³C NMR. Data are reported as (br=broad, s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet).

Example 1. 4-Acrylamido-N-(3-((2-(2-(2-hydroxyethyl)piperidin-1-yl)-9-isopropyl-9H-purin-6-yl)amino)phenyl)benzamide (THZ-4-124-1)

0602The synthesis of THZ-4-124-1 follows Synthetic Scheme 1. The reagents and conditions used for the synthesis are: (1) NMP, DIPEA, 100° C. (2) pyridine, 80° C. (3) NMP, 135° C. (4) SnCl₂, Ethyl acetate and Methanol (5) acryl chloride, acetonitrile, 0° C.

0603<chemistry id="CHEM-US-00194" num="00194"><img file="USRE50776E_D0197.tif" /></chemistry><chemistry id="CHEM-US-00195" num="00195"><img file="USRE50776E_D0198.tif" /></chemistry>

N1-(2-Fluoro-9-isopropyl-9H-purin-6-yl)benzene-1,3-diamine

0604<chemistry id="CHEM-US-00196" num="00196"><img file="USRE50776E_D0199.tif" /></chemistry>

0605To a solution of 6-chloro-2-fluoro-9-isopropyl-9H-purine (214 mg) in NMP (N-Methyl-2-pyrrolidone) was added benzene-1,3-diamine (130 mg, 1.2 equiv) and diisopropylethylamine (129 mg, 1.0 equiv). The solution was heated for 2 h at 100° C. The cooled solution was diluted with 100 mL of ethyl acetate and then washed with water. The separation by silica gel with CH₂Cl₂/methanol (10/1) to give of the product (225 mg, 79%).

N-(3-((2-Fluoro-9-isopropyl-9H-purin-6-yl)amino)phenyl)-4-nitrobenzamide

0606<chemistry id="CHEM-US-00197" num="00197"><img file="USRE50776E_D0200.tif" /></chemistry>

0607To a stirred solution of the above product (225 mg, 0.80 mmol) in 10 mL of pyridine was added 4-nitrobenzoyl chloride (219 mg, 1.5 equiv) and resulting solution was heated to 80° C. The reaction mixture was stirred for 2 h and concentrated under reduced pressure. The crude was purified by silica gel with CH₂Cl₂/methanol (10/1) to give of the product (285 mg, 82%).

N-(3-((2-(2-(2-Hydroxyethyl)piperidin-1-yl)-9-isopropyl-9H-purin-6-yl)amino)phenyl)-4-nitrobenzamide

0608<chemistry id="CHEM-US-00198" num="00198"><img file="USRE50776E_D0201.tif" /></chemistry>

0609The nitro compound (285 mg, 0.65 mmol) obtained from above reaction was dissolved in 3 mL of NMP and then was added with 2-(piperidin-2-yl)ethanol (170 mg, 2.0 equiv). The solution was heated to 135° C. for 2h and then cooled down to RT. The product was purified by HPLC to give the product (105 mg, 30%).

4-Amino-N-(3-((2-(2-(2-hydroxyethyl)piperidin-1-yl)-9-isopropyl-9H-purin-6-yl)amino)phenyl)benzamide

0610<chemistry id="CHEM-US-00199" num="00199"><img file="USRE50776E_D0202.tif" /></chemistry>

0611The nitro compound from step 3 (105 mg, 0.19 mmol) was suspended in 5 mL of ethyl acetate/methanol (5:1) and treated with SnCl₂(91 mg, 2.5 equiv). After stirring for 2 h at 80° C., the reaction mixture was cooled to room temperature and poured into saturated aqueous NaHCO₃. The mixture was stirred for 10 min and the aqueous phase was then extracted with 30 mL of chloroform and 2-propanol (4:1). The combined organic layer was washed with water and brine, dried over MgSO₄, filtered through a pad of celite and concentrated under reduced pressure. The resulting crude product was purified by flash column chromatography with CH₂Cl₂/methanol (10/1) to provide the title compound (68 mg, 70%).

4-Acrylamido-N-(3-((2-(2-(2-hydroxyethyl)piperidin-1-yl)-9-isopropyl-9H-purin-6-yl)amino)phenyl)benzamide

0612<chemistry id="CHEM-US-00200" num="00200"><img file="USRE50776E_D0203.tif" /></chemistry>

0613To the solution of the aniline (26 mg, 0.05 mmol)) obtained above in 5 mL of acetonitrile was added diisopropylethylamine (13 mg, 2.0 equiv). The reaction mixture was cooled to 0° C. and then treated with 4-chlorobut-2-enoyl chloride (15 mg, 3.0 equiv) in CH₂Cl₂. After the stirring for 10 min at 0° C., the reaction was then quenched with Sat. NaHCO₃and extracted with 30 mL of chloroform and 2-propanol (4:1). The solvent was removed at reduce pressure and the crude was purified with HPLC to give the final product THZ-4-124-1 (15 mg, 55%) MS 569 (M+1), ¹H NMR (DMSO-d6): 10.42 (s, 1H), 10.15 (s, 1H), 9.53 (s, 1H), 8.40 (d, J=12.0 Hz, 2H), 7.93 (d, J=7.2 Hz, 2H), 7.79 (d, J=7.2 Hz, 2H), 7.52 (d, J=7.2 Hz, 1H), 7.27 (m, 2H), 6.43 (m, 1H), 6.28 (d, J=15 Hz, 1H), 5.78 (d, J=9.6 Hz, 1H), 4.99 (s, 1H), 4.65 (m, 2H), 3.45 (m, 2H), 2.88 (t, J=6.8 Hz, 1H), 1.85 (m, 1H), 1.72 (m, 1H), 1.52 (s, 6H), 1.61-1.30 (m, 12H).

Example 2. 4-Acrylamido-N-(3-((2-((trans-4-(dimethylamino)cyclohexyl)amino)-9-isopropyl-9H-purin-6-yl)amino)phenyl)benzamide (THZ-5-38-1)

0614<chemistry id="CHEM-US-00201" num="00201"><img file="USRE50776E_D0204.tif" /></chemistry><chemistry id="CHEM-US-00202" num="00202"><img file="USRE50776E_D0205.tif" /></chemistry>

0615The synthesis of THZ-4-124-1 follows Synthetic Scheme 2. The reagents and conditions used for the synthesis are: (1) pyridine, 80° C. (2) NMP, 135° C. (3) SnCl₂, Ethyl acetate and Methanol (4) acryl chloride, acetonitrile, 0° C.

N-(3-((2-Fluoro-9-isopropyl-9H-purin-6-yl)amino)phenyl)-4-nitrobenzamide

0616<chemistry id="CHEM-US-00203" num="00203"><img file="USRE50776E_D0206.tif" /></chemistry>

0617To a stirred solution of N1-(2-fluoro-9-isopropyl-9H-purin-6-yl)benzene-1,3-diamine (286 mg, 1.0 mmol) in 10 mL of pyridine was added 4-nitrobenzoyl chloride (277 mg, 1.5 equiv) and resulting solution was heated to 80° C. The reaction mixture was stirred for 2 h and concentrated under reduced pressure. The crude was purified by silica gel with CH₂Cl₂/methanol (10/1) to give of the product (326 mg, 75%)

N-(3-((2-(((1r,4r)-4-(dimethylamino)cyclohexyl)amino)-9-isopropyl-9H-purin-6-yl)amino)phenyl)-4-nitrobenzamide

0618<chemistry id="CHEM-US-00204" num="00204"><img file="USRE50776E_D0207.tif" /></chemistry>

0619The nitro compound (326 mg, 0.75 mmol) obtained from above reaction was dissolved in 3 mL of NMP and then was added with trans-N1,N1-dimethylcyclohexane-1,4-diamine (213 mg, 2.0 equiv). The solution was heated to 135° C. for 2h and then cooled down to RT. The product was purified by HPLC to give the product (208 mg, 50%).

4-Amino-N-(3-((2-((trans-4-(dimethylamino)cyclohexyl)amino)-9-isopropyl-9H-purin-6-yl)amino)phenyl)benzamide

0620<chemistry id="CHEM-US-00205" num="00205"><img file="USRE50776E_D0208.tif" /></chemistry>

0621The nitro compound from step 2 (208 mg, 0.37 mmol) was suspended in 5 mL of ethyl acetate/methanol (5:1) and treated with SnCl₂(177 mg, 2.5 equiv). After stirring for 2 h at 80° C., the reaction mixture was cooled to room temperature and poured into saturated aqueous NaHCO₃. The mixture was stirred for 10 min and the aqueous phase was then extracted with 30 mL of chloroform and 2-propanol (4:1). The combined organic layer was washed with water and brine, dried over MgSO₄, filtered through a pad of celite and concentrated under reduced pressure. The resulting crude product was purified by flash column chromatography with CH₂Cl₂/methanol (10/1) to provide the title compound (126 mg, 65%).

4-Acrylamido-N-(3-((2-((trans-4-(dimethylamino)cyclohexyl)amino)-9-isopropyl-9H-purin-6-yl)amino)phenyl)benzamide

0622<chemistry id="CHEM-US-00206" num="00206"><img file="USRE50776E_D0209.tif" /></chemistry>

0623To the solution of the aniline (26 mg, 0.05 mmol)) obtained above in 5 mL of acetonitrile was added diisopropylethylamine (13 mg, 2.0 equiv). The reaction mixture was cooled to 0° C. and then treated with 4-chlorobut-2-enoyl chloride (15 mg, 3.0 equiv) in CH₂Cl₂. After the stirring for 10 min at 0° C., the reaction was then quenched with Sat. NaHCO₃and extracted with 30 mL of chloroform and 2-propanol (4:1). The solvent was removed at reduce pressure and the crude was purified with HPLC to give the final product (17 mg, 60%) MS 582 (M+1), ¹H NMR (DMSO-d6): 10.45 (s, 1H), 10.17 (s, 1H), 9.63 (br, 1H), 9.43 (br, 1H), 8.26 (br, 1H), 8.25 (s, 1H), 7.98 (d, J=7.2 Hz, 2H), 7.79 (d, J=7.2 Hz, 2H), 7.65 (br, 1H), 7.45 (br, 1H), 7.28 (t, J=7.8 Hz, 1H), 6.48 (m, 1H), 6.28 (d, J=15 Hz, 1H), 5.78 (d, J=9.6 Hz, 1H), 4.62 (m, 1H), 2.75-2.60 (m, 2H), 2.55 (s, 6H), 2.22-1.65 (m, 4H), 1.51 (d, J=6.6 Hz, 6H), 1.55-1.25 (m, 4H).

Example 3. 4-((E)-4-(Dimethylamino)but-2-enamido)-N-(3-((2-(((1r,4r)-4-(dimethylamino)cyclohexyl)amino)-9-isopropyl-9H-purin-6-yl)amino)phenyl)benzamide (THZ-3-49-1)

0624<chemistry id="CHEM-US-00207" num="00207"><img file="USRE50776E_D0210.tif" /></chemistry>

0625To the solution of the aniline (53 mg) obtained above in 10 mL of acetonitrile was added diisopropylethylamine (26 mg, 2.0 equiv). The reaction mixture was cooled to 0° C. and then treated with 4-chlorobut-2-enoyl chloride (54 mg, 3.0 equiv) in CH₂Cl₂. The reaction mixture was stirred for 10 min at 0° C. and then treated with a solution of dimethylamine in THF (3 mL, 1M). The reaction mixture was then warmed to room temperature, stirred for 1 h and concentrated under reduced pressure. The resulting crude product was purified by preparative HPLC to give the final product (28.8 mg, 45%). MS 639 (M+1).

Example 4. (E)-N-(3-((2-(3-Aminopiperidin-1-yl)-9-isopropyl-9H-purin-6-yl)amino)phenyl)-4-(4-(dimethylamino)but-2-enamido)benzamide (THZ-4-119-1)

0626The synthesis of THZ-4-119-1 follows Synthetic Scheme 4. The reagents and conditions used for the synthesis are: (1) NMP, 135° C. (2) SnCl₂, Ethyl acetate and Methanol (3) a) 4-bromobut-2-enoyl chloride, CH₃CN, NHMe₂, 0° C.-RT b) TFA, CHCl₃.

0627<chemistry id="CHEM-US-00208" num="00208"><img file="USRE50776E_D0211.tif" /></chemistry><chemistry id="CHEM-US-00209" num="00209"><img file="USRE50776E_D0212.tif" /></chemistry>

tert-butyl (1-(9-iso-Propyl-6-((3-(4-nitrobenzamido)phenyl)amino)-9H-purin-2-yl)piperidin-3-yl)carbamate

0628<chemistry id="CHEM-US-00210" num="00210"><img file="USRE50776E_D0213.tif" /></chemistry>

0629To a stirred solution of N-(3-((2-fluoro-9-isopropyl-9H-purin-6-yl)amino)phenyl)-4-nitrobenzamide (435 mg) in NMP (3 mL) was added tert-butyl piperidin-3-ylcarbamate (300 mg, 1.5 equiv) and then the resulting solution was heated to 130° C. for 6 h. After cooling down to RT, the solution was then extracted with ethyl acetate, washed with water and brine and then dried on Na₂SO₄. The crude was obtained after removing the solvent and then was used in the next step directly.

tert-butyl (1-(6-((3-(4-Aminobenzamido)phenyl)amino)-9-isopropyl-9H-purin-2-yl)piperidin-3-yl)carbamate

0630<chemistry id="CHEM-US-00211" num="00211"><img file="USRE50776E_D0214.tif" /></chemistry>

0631The crude from step 1 was suspended in 5 mL of ethyl acetate/methanol (5:1) and treated with SnCl₂(562 mg, 2.5 equiv). After stirring for 2 h at 80° C., the reaction mixture was cooled to room temperature and poured into saturated aqueous NaHCO₃. The mixture was stirred for 10 min and the aqueous phase was then extracted with 30 mL of chloroform and 2-propanol (4:1). The combined organic layer was washed with water and brine, dried over MgSO₄, filtered through a pad of celite and concentrated under reduced pressure. The resulting crude product was purified by flash column chromatography with CH₂Cl₂/methanol (10/1) to provide the product (230 mg, 40% from two steps).

(E)-N-(3-((2-(3-Aminopiperidin-1-yl)-9-isopropyl-9H-purin-6-yl)amino)phenyl)-4-(4-(dimethylamino)but-2-enamido)benzamide

0632<chemistry id="CHEM-US-00212" num="00212"><img file="USRE50776E_D0215.tif" /></chemistry>

0633To the solution of the aniline (58 mg) obtained above in 10 mL of acetonitrile was added diisopropylethylamine (26 mg, 2.0 equiv). The reaction mixture was cooled to 0° C. and then treated with 4-chlorobut-2-enoyl chloride (54 mg, 3.0 equiv) in CH₂Cl₂. The reaction mixture was stirred for 10 min at 0° C. and then treated with a solution of dimethylamine in THF (3 mL, 1M). The reaction mixture was then warmed up to room temperature, stirred for 1 h and concentrated under reduced pressure. The resulting crude product was then dissolved in CHCl3 (3 mL) and TFA (1 mL). The solution was then allowed for stirring for 2 h. After removing the solvent, the crude was purified by preparative HPLC to give the final product (24.8 mg, 35%). MS 597 (M+1)¹H NMR (DMSO-d6): 10.64 (s, 1H), 10.20 (s, 1H), 10.01 (br, 1H), 9.56 (s, 1H), 8.60 (s, 1H), 8.17 (s, 1H), 8.02 (m, 3H), 7.95 (d, J=7.2 Hz, 2H), 7.80 (d, J=7.2 Hz, 2H), 7.45 (d, J=8.4 Hz, 1H), 7.26 (t, J=7.8 Hz, 1H), 7.20 (d, J=8.4 Hz, 1H), 6.78 (m, 1H), 6.50 (d, J=15 Hz, 1H), 4.66 (m, 1H), 4.53 (m, 1H), 4.21 (m, 1H), 3.96 (d, J=7.2 Hz, 2H), 3.37-3.25 (m, 4H), 2.83 (s, 6H), 1.97 (m, 1H), 1.76 (m, 1H), 1.59-1.55 (m, 2H), 1.50 (d, J=6.6 Hz, 6H).

Example 5. (E)-N-(3-((5-(3-Aminopiperidin-1-yl)-3-ethylpyrazolo[1,5-a]pyrimidin-7-yl)amino)phenyl)-4-(4-(dimethylamino)but-2-enamido)benzamide (THZ-4-128-1)

0634The synthesis of THZ-4-128-1 follows Synthetic Scheme 5. The reagents and conditions used for the synthesis are: (1) a) NMP, DIPEA, 100° C. b) TFA, CHCl₃(2) Pyridine, 80° C. (3) NMP, DIPEA, 135° C. (4) SnCl₂, Ethyl acetate and Methanol (5) a) 4-bromobut-2-enoyl chloride, CH₃CN, NHMe₂, 0° C.-RT b) TFA, CHCl₃.

0635<chemistry id="CHEM-US-00213" num="00213"><img file="USRE50776E_D0216.tif" /></chemistry><chemistry id="CHEM-US-00214" num="00214"><img file="USRE50776E_D0217.tif" /></chemistry>

N1-(5-Chloro-3-ethylpyrazolo[1,5-a]pyrimidin-7-yl)benzene-1,3-diamine

0636<chemistry id="CHEM-US-00215" num="00215"><img file="USRE50776E_D0218.tif" /></chemistry>

0637To a solution of 5,7-dichloro-3-ethylpyrazolo[1,5-a]pyrimidine (215 mg) in NMP (N-Methyl-2-pyrrolidone) was added tert-butyl (3-aminophenyl)carbamate (230 mg, 1.1 equiv) and diisopropylethylamine (129 mg, 1.0 equiv). The solution was heated for 2 h at 100° C. The cooled solution was diluted with 100 mL of ethyl acetate and then washed with water. After removing the solvent, the crude was obtained which was dissolved in CHCl₃(3 mL) and TFA (1 mL). The resulting solution was stirred for 1 h and then the solvent was removed in the reduced pressure. The crude was then purified by silica gel with CH₂Cl₂/methanol (10/1) to give the product (215 mg, 75%).

N-(3-O-Chloro-3-ethylpyrazolo[1,5-a]pyrimidin-7-yl)amino)phenyl)-4-nitrobenzamide

0638<chemistry id="CHEM-US-00216" num="00216"><img file="USRE50776E_D0219.tif" /></chemistry>

0639To a stirred solution of the above product (215 mg, 0.75 mmol) in 10 mL of pyridine was added 4-nitrobenzoyl chloride (210 mg, 1.5 equiv) and resulting solution was heated to 80° C. The reaction mixture was stirred for 2 h and concentrated under reduced pressure. The crude was purified by silica gel with CH₂Cl₂/methanol (10/1) to give the product (212 mg, 65%).

tert-Butyl (1-(3-ethyl-7-O-(4-nitrobenzamido)phenyl)amino)pyrazolo[1,5-a]pyrimidin-5-yl)piperidin-3-yl)carbamate

0640<chemistry id="CHEM-US-00217" num="00217"><img file="USRE50776E_D0220.tif" /></chemistry>

0641To a stirred solution of the product from step 2 (212 mg) in NMP (3 mL) was added tert-butyl piperidin-3-ylcarbamate (145 mg, 1.5 equiv) and then the resulting solution was heated to 130° C. for 6 h. After cooling down to RT, the solution was then extracted with ethyl acetate washed with water and brine and dried on Na₂SO₄. The crude was obtained after removing the solvent and then was used in the next step directly.

tert-butyl (1-(7-((3-(4-Aminobenzamido)phenyl)amino)-3-ethylpyrazolo[1,5-a]pyrimidin-5-yl)piperidin-3-yl)carbamate

0642<chemistry id="CHEM-US-00218" num="00218"><img file="USRE50776E_D0221.tif" /></chemistry>

0643The crude from step 3 was suspended in 5 mL of ethyl acetate/methanol (5:1) and treated with SnCl₂(281 mg, 2.5 equiv). After stirring for 2 h at 80° C., the reaction mixture was cooled to room temperature and poured into saturated aqueous NaHCO₃. The mixture was stirred for 10 min and the aqueous phase was then extracted with 30 mL of chloroform and 2-propanol (4:1). The combined organic layer was washed with water and brine, dried over MgSO₄, filtered through a pad of celite and concentrated under reduced pressure. The resulting crude product was purified by flash column chromatography with CH₂Cl₂/methanol (10/1) to provide the product (82 mg, 30% from two steps).

(E)-N-(3-((5-(3-Aminopiperidin-1-yl)-3-ethylpyrazolo[1,5-a]pyrimidin-7-yl)amino)phenyl)-4-(4-(dimethylamino)but-2-enamido)benzamide

0644<chemistry id="CHEM-US-00219" num="00219"><img file="USRE50776E_D0222.tif" /></chemistry>

0645To the solution of the aniline (57 mg) obtained above in 10 mL of acetonitrile was added diisopropylethylamine (26 mg, 2.0 equiv). The reaction mixture was cooled to 0° C. and then treated with 4-chlorobut-2-enoyl chloride (54 mg, 3.0 equiv) in CH₂Cl₂. The reaction mixture was stirred for 10 min at 0° C. and then treated with a solution of dimethylamine in THF (3 mL, 1M). The reaction mixture was then warmed to room temperature, stirred for 1 h and concentrated under reduced pressure. The resulting crude product was then dissolved in CHCl3 (3 mL) and TFA (1 mL). The solution was then allowed for stirring for 2 h. After removing the solvent, the crude was purified by preparative HPLC to give the final product (15 mg, 25%). MS 582 (M+1) ¹H NMR (DMSO-d6): 10.64 (s, 1H), 10.32 (s, 1H), 10.09 (br, 1H), 9.44 (s, 1H), 8.03 (m, 1H), 7.98 (br, 2H), 7.95 (d, J=7.2 Hz, 2H), 7.80 (d, J=7.2 Hz, 2H), 7.43 (d, J=8.4 Hz, 1H), 7.37 (t, J=7.8 Hz, 1H), 7.15 (d, J=8.4 Hz, 1H), 6.78 (m, 1H), 6.50 (d, J=15 Hz, 1H), 6.06 (s, 1H), 4.31 (m, 1H), 3.96 (d, J=7.2 Hz, 2H), 3.86 (m, 1H), 3.20-3.10 (m, 3H), 2.79 (s, 6H), 2.59 (q, J=8.4 Hz, 2H), 1.97 (m, 1H), 1.76 (m, 1H), 1.59 (m, 2H), 1.23 (d, J=7.8 Hz, 3H).

Example 6. 4-Acrylamido-N-(3-(3-ethyl-5-(2-(2-hydroxyethyl)piperidin-1-yl)pyrazolo[1,5-a]pyrimidin-7-ylamino)phenyl)benzamide (SB1-E-24)

0646<chemistry id="CHEM-US-00220" num="00220"><img file="USRE50776E_D0223.tif" /></chemistry>

4-Acrylamido-N-(3-(3-ethyl-5-(2-(2-hydroxyethyl)piperidin-1-yl)pyrazolo[1,5-a]pyrimidin-7-ylamino)phenyl)benzamide (SB1-E-24)

0647The mixture of SB1-E-23-1 (100 mg, 0.263 mmol), SB1-E-22-1 (100 mg, 0.523 mmol), HATU (200 mg, 0.526 mmol), DIPEA (0.5 mL) and DMF (5 mL) was stirred at r.t overnight. After completion, the mixture was purified by prep-TLC (DCM/MeOH=15/1) and prep-HPLC (C18 column, CH₃CN/H₂O, containing 0.05% NH₄HCO₃) to obtain SB1-E-24 (off-white solid, 23 mg, 16%). HPLC: 99% (254 nm); LCMS (m/z): 554 [M+H]⁺; ¹H NMR (DMSO-d₆, 500 MHz): δ 10.45 (s, 1H), 10.27 (s, 1H), 9.63 (bs, 1H), 8.01 (s, 1H), 7.97 (d, J=9.0 Hz, 2H), 7.83 (m, 3H), 7.49 (d, J=8.5 Hz, 1H), 7.39 (t, J=8.5 Hz, 1H), 7.19 (d, J=7.5 Hz, 1H), 6.48 (dd, J₁=16.5 Hz, J₂=10.5 Hz, 1H), 6.31 (d, J=16.5 Hz, 1H), 6.03 (s, 1H), 5.81 (d, J=10.5 Hz, 1H), 4.58 (s, 1H), 4.17 (m, 1H), 3.33-3.43 (m, 2H), 2.99 (m, 1H), 2.55-2.60 (m, 2H), 1.18-1.89 (m, 12H).

Example 7. 4-Acrylamido-N-(3-(5-((1r,4r)-4-(dimethylamino)cyclohexylamino)-3-isopropylpyrazolo[1,5-a]pyrimidin-7-ylamino)phenyl)benzamide (SB1-E-25)

0648<chemistry id="CHEM-US-00221" num="00221"><img file="USRE50776E_D0224.tif" /></chemistry><chemistry id="CHEM-US-00222" num="00222"><img file="USRE50776E_D0225.tif" /></chemistry>

2-Formyl-3-methylbutanenitrile (SB1-E-25-1)

0649<chemistry id="CHEM-US-00223" num="00223"><img file="USRE50776E_D0226.tif" /></chemistry>

0650To a solution of SM-25-1 (10.0 g, 120 mmol) in THF (10 mL), LDA (2M, 60 ml, 120 mmol) was added slowly at −78° C. After completion, the mixture was added to a solution of ethyl formate (9.0 g, 121.5 mmol) in THF (40 ml) at −78° C. After completion, the mixture was stirred at 0° C. for 3h. The solvent was removed, then water (100 mL) was added, and the resulting mixture was extracted with Et₂O (200 mL×3). The combined organic layer was washed with brine (100 mL×2), dried over sodium sulfate, filtered through Celite, and concentrated under reduced pressure to give the crude SB1-E-25-1 (colorless oil, 12.5 g, 94% yield). The crude product was used directly for the next step without further purification.

4-iso-Propyl-1H-pyrazol-5-amine (SB1-E-25-2)

0651<chemistry id="CHEM-US-00224" num="00224"><img file="USRE50776E_D0227.tif" /></chemistry>

0652To a solution of SB1-E-25-1 (12.5 g, 112 mmol) in EtOH (200 mL), hydrazine hydrate (28 g, 560 mmol) and AcOH (26.9 g, 448 mmol) was added. The mixture was stirred at 80° C. for 4h. The solvent was removed, then water (200 ml) was added, and the resulting mixture was extracted with DCM (300 mL×3). The combined organic layer was washed with brine (100 mL×2), dried over sodium sulfate, filtered through Celite, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (DCM/MeOH=15/1 to 8/1) to afford SB1-E-25-2 (yellow solid, 13.3 g, 95% yield). LCMS (m/z): 126 [M+H]⁺.

3-iso-Propylpyrazolo[1,5-a]pyrimidine-5,7(4H,6H)-dione (SB1-E-25-3)

0653<chemistry id="CHEM-US-00225" num="00225"><img file="USRE50776E_D0228.tif" /></chemistry>

0654To a solution of SB1-E-25-2 (1.7 g, 13.6 mmol) in CH₃OH (20 ml), SM-25-2 (5.4 g, 40.8 mmol) and sodium methoxide (3.7 g, 68 mmol) was added. The mixture was stirred at 70° C. for 4 h. After completion, the solvent was removed, water (200 ml) was added, then 1M HCl aq. (40 mL) was added to adjust the pH to 3. The mixture was filtered and the filter residue was evaporated to dryness under reduced pressure to give the crude SB1-E-25-3 (off-white solid, 2.5 g, 95% yield). LCMS (m/z): 194 [M+H]⁺.

5,7-Dichloro-3-isopropylpyrazolo[1,5-a]pyrimidine (SB1-E-25-4)

0655<chemistry id="CHEM-US-00226" num="00226"><img file="USRE50776E_D0229.tif" /></chemistry>

0656To a solution of SB1-E-25-3 (2.4 g, 12.4 mmol) in CH₃CN (30 ml), POCl₃(9.4 g, 62 mmol) and N,N-dimethylaniline (4.5 g, 37.2 mmol) was added. The mixture was stirred at 80° C. for 8 h. The mixture was concentrated and water (40 mL) was added. The mixture was filtered and concentrated to remove the solvent. The residue was washed with water (100 ml) and then evaporated to dryness under reduced pressure to give the crude SB1-E-25-4 (brown solid, 2.7 g, 85% yield). LCMS (m/z): 230 [M+H]⁺.

N

1

-(5-Chloro-3-isopropylpyrazolo[1,5-a]pyrimidin-7-yl)benzene-1,3-diamine (SB1-E-25-5)

0657<chemistry id="CHEM-US-00227" num="00227"><img file="USRE50776E_D0230.tif" /></chemistry>

0658To a solution of SB1-E-25-4 (2.6 g, 11.3 mmol), in i-PrOH (20 mL), 1,3-diaminobenzene (1.47 g, 13.6 mmol) and DIPEA (2.92 g, 22.6 mmol) was added. The mixture was stirred at 110° C. for 8 h. The mixture was concentrated, water (20 mL) was added, and the resulting mixture was extracted with DCM (30 mL×3). The combined organic layer was washed with brine (20 mL×2), dried over sodium sulfate, filtered through Celite, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (PE/ethyl acetate=8/1 to 4/1) to afford SB1-E-25-5 (off-white solid, 2.2 g, 65% yield). LCMS (m/z): 302 [M+H]⁺.

N7-(3-Aminophenyl)-N5-((1s,4s)-4-(dimethylamino)cyclohexyl)-3-isopropylpyrazolo[1,5-a]pyrimidine-5,7-diamine (SB1-E-25-6)

0659<chemistry id="CHEM-US-00228" num="00228"><img file="USRE50776E_D0231.tif" /></chemistry>

0660The mixture of SB1-E-25-5 (350 mg, 1.16 mmol), SB1-E-21-3 (200 mg, 1.41 mmol), KF (400 mg, 6.88 mmol) and NMP (2 mL) was stirred at 170° C. for 8 h, after completion, concentrated to remove the solvent, the residue was purified by prep-TLC (DCM/MeOH=10/1) to obtain SB1-E-25-6 (light brown solid, 130 mg, yield 28%). LCMS (m/z): 408 [M+H]⁺.

4-Acrylamido-N-(3-(5-((1r,4r)-4-(dimethylamino)cyclohexylamino)-3-isopropylpyrazolo[1,5-a]pyrimidin-7-ylamino)phenyl)benzamide (SB1-E-25)

0661<chemistry id="CHEM-US-00229" num="00229"><img file="USRE50776E_D0232.tif" /></chemistry>

0662The mixture of SB1-E-25-5 (40 mg, 0.0981 mmol), SB1-E-24-1 (35 mg, 0.183 mmol), HATU (75 mg, 0.197 mmol) and DMF (5 mL) was stirred at r.t overnight. After completion, the mixture was purified by prep-TLC (DCM/MeOH=8/1) and prep-HPLC (C18 column, CH₃CN/H₂O, containing 0.05% NH₄HCO₃) to obtain SB1-E-25 (gray solid, 15 mg, yield 26%). HPLC: 97% (254 nm); LCMS (m/z): 581 [M+H]⁺; ¹H NMR (DMSO-d₆, 500 MHz): δ 10.44 (s, 1H), 10.26 (s, 1H), 9.07 (s, 1H), 7.97 (d, J=8.5 Hz, 2H), 7.85 (s, 1H), 7.82 (d, J=8.5 Hz, 2H), 7.67 (s, 1H), 7.64 (t, J=4.0 Hz, 1H), 7.37 (t, J=8.0 Hz, 1H), 7.10 (d, J=7.5 Hz, 1H), 6.62 (d, J=7.0 Hz, 1H), 6.47 (dd, J₁=17.0 Hz, J₂=10.5 Hz, 1H), 6.31 (d, J=17.0 Hz, 1H), 5.81 (d, J=10.5 Hz, 1H), 3.63 (s, 1H), 3.00 (m, 1H), 1.77-2.17 (m, 11H), 1.09-1.29 (m, 10H).

Example 8. 4-Acrylamido-N-(3-(5-((1r,4r)-4-(dimethylamino)cyclohexylamino)-3-ethylpyrazolo[1,5-a]pyrimidin-7-ylamino)phenyl)benzamide (SB1-E-22)

0663<chemistry id="CHEM-US-00230" num="00230"><img file="USRE50776E_D0233.tif" /></chemistry>

4-Acrylamidobenzoic Acid (SB1-E-22-1)

0664<chemistry id="CHEM-US-00231" num="00231"><img file="USRE50776E_D0234.tif" /></chemistry>

0665To the solution of SM-22-1 (500 mg, 3.65 mmol) in pyridine (20 mL) was added acrylyl chloride (500 mg, 5.52 mmol), the mixture was stirred at r.t overnight, after completion, the mixture was poured to a ice-water (20 mL), filtered, the solid was washed with H₂O, dried to obtain SB1-E-22-1 (light yellow solid, 600 mg, yield 86%). LCMS (m/z): 192 [M+H]⁺.

4-Acrylamido-N-(3-(5-((1r,4r)-4-(dimethylamino)cyclohexylamino)-3-ethylpyrazolo[1,5-a]pyrimidin-7-ylamino)phenyl)benzamide (SB1-E-22)

0666<chemistry id="CHEM-US-00232" num="00232"><img file="USRE50776E_D0235.tif" /></chemistry>

0667The mixture of SB1-E-21-7 (50 mg, 0.127 mmol), SB1-E-22-1 (45 mg, 0.235 mmol), HATU (88 mg, 0.231 mmol), DIPEA (0.3 mL) and DMF (2 mL) was stirred at r.t overnight, after completion, concentrated to remove the solvent, the residue was purified by prep-TLC (DCM/MeOH=10/) and prep-HPLC (C18 column, CH₃CN/H₂O, containing 0.05% NH₄HCO₃) to obtain SB1-E-22 (light gray solid, 7 mg, yield 10%). HPLC: 95% (214 nm); LCMS (m/z): 567 [M+H]⁺; ¹H NMR (DMSO-d₆, 500 MHz): δ 10.44 (s, 1H), 10.25 (s, 1H), 9.06 (s, 1H), 7.97 (d, J=9.0 Hz, 2H), 7.85 (d, J=5.0 Hz, 1H), 7.82 (d, J=9.0 Hz, 2H), 7.70 (s, 1H), 7.63 (d, J=8.0 Hz, 1H), 7.37 (t, J=8.5 Hz, 1H), 7.10 (d, J=7.0 Hz, 1H), 6.61 (d, J=7.5 Hz, 1H), 6.47 (dd, J₁=16.5 Hz, J₂=9.5 Hz, 1H), 6.31 (d, J=16.5 Hz, 1H), 5.81 (d, J=9.5 Hz, 1H), 5.63 (s, 1H), 3.68 (s, 1H), 2.54 (q, J=7.5 Hz, 2H), 2.15 (s, 6H), 1.77-2.15 (m, 5H), 1.11-1.27 (m, 7H).

Example 9. 4-Acrylamido-N-(3-(8-ethyl-2-(2-(2-hydroxyethyl)piperidin-1-yl)pyrazolo[1,5-a][1,3,5]triazin-4-ylamino)phenyl)benzamide (SB1-E-19)

0668<chemistry id="CHEM-US-00233" num="00233"><img file="USRE50776E_D0236.tif" /></chemistry>

4-Acrylamido-N-(3-(8-ethyl-2-(2-(2-hydroxyethyl)piperidin-1-yl)pyrazolo[1,5-a][1,3,5]triazin-4-ylamino)phenyl)benzamide (SB1-E-19)

0669A mixture of SB1-E-18-08 (70 mg, 0.18 mmol), 4-acrylamidobenzoic acid (52 mg, 0.27 mmol), HATU (68 mg, 0.18 mmol) and triethylamine (54 mg, 0.54 mmol) in DCM (3 mL) was stirred at r.t overnight, diluted with dichloromethane (10 ml), washed with water (10 mL) and saturated sodium bicarbonate solution (10 mL×2), dried over anhydrous sodium sulfate filtered and concentrated in vacuo, purified by prep-HPLC (C18 column, CH₃CN/H₂O, containing 0.05% TFA) to get SB1-E-19, also referred to herein as E19 and E-19 (white solid, 25 mg, yield: 25%). HPLC: 100% (254 nm); LCMS (m/z): 555 [M+H]⁺; ¹H NMR (DMSO-d₆, 500 MHz): δ: 10.45 (s, 1H), 10.19 (s, 1H), 8.49 (s, 1H), 7.97 (d, J=8.5 Hz, 2H), 7.88 (s, 1H), 7.82 (d, J=8.5 Hz, 1H), 7.45-7.51 (m, 2H), 7.34 (t, J=8.0 Hz, 1H), 6.48 (dd, J=16.5, 10 Hz, 1H), 6.32 (d, J=17.0 Hz, 1H), 5.82 (d, J=10.0 Hz, 1H), 5.00 (s, 1H), 4.66 (d, J=10.0 Hz, 1H), 2.91 (t, J=13.0 Hz, 1H), 2.50-2.54 (m, 2H), 1.56-1.71 (m, 8H), 1.38-1.40 (m, 1H), 1.20-1.23 (m, 4H).

Example 10. N-(3-(2-((1s,4s)-4-(Dimethylamino)cyclohexylamino)-9-isopropyl-9H-purin-6-ylamino)phenyl)acrylamide (SB1-E-14)

0670<chemistry id="CHEM-US-00234" num="00234"><img file="USRE50776E_D0237.tif" /></chemistry><chemistry id="CHEM-US-00235" num="00235"><img file="USRE50776E_D0238.tif" /></chemistry>

tert-Butyl (1s,4s)-4-(dimethylamino)cyclohexylcarbamate (SB1-E-14-2)

0671<chemistry id="CHEM-US-00236" num="00236"><img file="USRE50776E_D0239.tif" /></chemistry>

0672To the solution of SB1-E-14-1 (1.0 g, 4.67 mmol), MeOH (30 mL) and HCHO (600 mg, 20.0 mmol) was added NaBH₃CN (1.5 g, 23.9 mmol), the mixture was stirred at r.t overnight. After completion, concentrated to remove the solvent, then extracted with ethyl acetate (100 mL×4), the organic phase was washed with H₂O (50 mL), brine (50 mL×2), dried with Na₂SO₄. Filtered, concentrated to remove the solvent, the residue was purified by silica gel (DCM/MeOH=10/1, 4/1) to obtain SB1-E-14-2 (light brown solid, 800 mg, yield 71%). LCMS (m/z): 243 [M+H]⁺.

(1s,4s)-N1,N1-Dimethylcyclohexane-1,4-diamine (SB1-E-14-3)

0673<chemistry id="CHEM-US-00237" num="00237"><img file="USRE50776E_D0240.tif" /></chemistry>

0674The mixture of SB1-E-14-2 (350 mg, 1.44 mmol), DCM (3 mL) and TFA (3 mL) was stirred at r.t for 3 h. after completion, concentrated to remove the solvent to obtain SB1-E-14-3 (light yellow sticky oil, 200 mg, yield 98%). LCMS (m/z): 143 [M+H]⁺.

2,6-Dichloro-9-isopropyl-9H-purine (SB1-E-14-4)

0675<chemistry id="CHEM-US-00238" num="00238"><img file="USRE50776E_D0241.tif" /></chemistry>

0676To a solution of SM-14-2 (3.0 g, 15.3 mmol) and 2-propanol (2.75 g, 45.9 mmol) in THF (30 mL), PPh₃(8.02 g, 30.6 mmol) was added. The mixture was stirred at room temperature for 10 min. Then DIEA (6.18 g, 30.6 mmol) was added. The final mixture was stirred at 70° C. for 3h. after completion, concentrated to remove the solvent, water (30 mL) was added, the resulting mixture was extracted with DCM (50 mL×3). The combined organic layer was washed with brine (50 mL×2), dried over sodium sulfate, filtered through Celite, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (ethyl acetate/acetone=20/1 to 10/1) to afford SB1-E-14-4 (off-white solid, 2.34 g, 66% yield).

N1-(2-Chloro-9-isopropyl-9H-purin-6-yl)benzene-1,3-diamine (SB1-E-14-5)

0677<chemistry id="CHEM-US-00239" num="00239"><img file="USRE50776E_D0242.tif" /></chemistry>

0678The mixture of SB1-E-14-4 (1.2 g, 5.19 mmol), SM-14-3 (850 mg, 7.86 mmol), n-BuOH (30 mL) and DIPEA (2 mL) was stirred at 115° C. for 4 h. after completion, concentrated to remove the solvent, the residue was purified by silica gel (DCM/MeOH=50/1) to obtain SB1-E-14-5 (light brown solid, 1.2 g, yield 76%). LCMS (m/z): 303 [M+H]⁺.

N6-(3-Aminophenyl)-N2-((1s,4s)-4-(dimethylamino)cyclohexyl)-9-isopropyl-9H-purine-2,6-diamine (SB1-E-14-6)

0679<chemistry id="CHEM-US-00240" num="00240"><img file="USRE50776E_D0243.tif" /></chemistry>

0680The mixture of SB1-E-14-5 (370 mg, 1.22 mmol), SB1-E-14-3 (200 mg, 1.41 mmol), KF (360 mg, 6.20 mmol) and NMP (2 mL) was stirred at 172° C. for 8 h. after completion, the mixture was purified by silica gel (DCM/MeOH=10/1, 8/1, 6/1) to obtain SB1-E-14-6 (brown solid, 150 mg, yield 30%). LCMS (m/z): 409 [M+H]⁺.

N-(3-(2-((1s,4s)-4-(Dimethylamino)cyclohexylamino)-9-isopropyl-9H-purin-6-ylamino)phenyl)acrylamide (SB1-E-14)

0681<chemistry id="CHEM-US-00241" num="00241"><img file="USRE50776E_D0244.tif" /></chemistry>

0682To a solution of SB1-E-14-6 (60 mg, 0.147 mmol), DIPEA (0.5 mL) in CH₃CN (4 mL) was added the solution of acryloyl chloride (20 mg, 0.221 mmol) in CH₃CN (1 mL) dropwise, the mixture was stirred at OC for 2 h. after completion, concentrated to remove the solvent, the residue was purified by prep-TLC (DCM/MeOH=30/1) and prep-HPLC (C18 column, CH₃CN/H₂O, containing 0.05% NH₄HCO₃) to obtain SB1-E-14 (off-white solid, 20 mg, yield 29%). HPLC: 100% (254 nm); LCMS (m/z): 463 [M+H]⁺; ¹H NMR (DMSO-d₆, 400 MHz): δ 10.04 (s, 1H), 9.38 (s, 1H), 8.44 (s, 1H), 7.95 (s, 1H), 7.63 (d, J=7.2 Hz, 1H), 7.25 (s, 1H), 7.20 (t, J=8.0 Hz, 1H), 6.47 (dd, J₁=16.8 Hz, J₂=9.6 Hz, 1H), 6.26 (d, J=16.8 Hz, 1H), 5.75 (d, J=9.6 Hz, 1H), 4.59 (m, 1H), 3.95 (m, 1H), 2.20 (s, 6H), 2.07 (m, 1H), 1.48-1.76 (m, 14H).

Example 11. N-(3-(2-((1r,4r)-4-(Dimethylamino)cyclohexylamino)-9-isopropyl-9H-purin-6-ylamino)cyclohexyl)acrylamide (SB1-E-15)

0683<chemistry id="CHEM-US-00242" num="00242"><img file="USRE50776E_D0245.tif" /></chemistry><chemistry id="CHEM-US-00243" num="00243"><img file="USRE50776E_D0246.tif" /></chemistry>

tert-butyl 3-(2-Chloro-9-isopropyl-9H-purin-6-ylamino)cyclohexylcarbamate (SB1-E-15-2)

0684<chemistry id="CHEM-US-00244" num="00244"><img file="USRE50776E_D0247.tif" /></chemistry>

0685The mixture of SB1-E-15-1 (520 mg, 2.25 mmol), SM-15-2 (490 mg, 2.29 mmol), n-BuOH (15 mL) and DIPEA (1 mL) was stirred at 100° C. overnight, after completion, concentrated to remove the solvent, the residue was purified by silica gel (PE/ethyl acetate=2/1) to obtain SB1-E-15-2 (light broen solid, 380 mg, yield 41%). LCMS (m/z): 409 [M+H]⁺.

tert-butyl 3-(2-((1r,4r)-4-(Dimethylamino)cyclohexylamino)-9-isopropyl-9H-purin-6-ylamino)cyclohexylcarbamate (SB1-E-15-3)

0686<chemistry id="CHEM-US-00245" num="00245"><img file="USRE50776E_D0248.tif" /></chemistry>

0687The mixture of SB1-E-15-2 (180 mg, 0.440 mmol), 4 (150 mg, 1.05 mmol), KF (110 mg, 1.89 mmol) and n-BuOH (1.5 mL) was stirred at 135° C. overnight, after completion, concentrated to remove the solvent, the residue was purified by prep-TLC (DCM/MeOH=6/1) to obtain SB1-E-15-3 (light brown solid, 25 mg, yield 11%). LCMS (m/z): 515 [M+H]⁺.

N6-(3-Aminocyclohexyl)-N2-((1r,4r)-4-(dimethylamino)cyclohexyl)-9-isopropyl-9H-purine-2,6-diamine (SB1-E-15-4)

0688<chemistry id="CHEM-US-00246" num="00246"><img file="USRE50776E_D0249.tif" /></chemistry>

0689The mixture of SB1-E-15-3 (25 mg, 0.0486 mmol), DCM (3 mL) and TFA (3 mL) was stirred at r.t overnight. after completion, concentrated to remove the solvent to obtain SB1-E-15-4 (light brown sticky oil, 20 mg, yield 95%). LCMS (m/z): 415 [M+H]⁺.

N-(3-(2-((1r,4r)-4-(Dimethylamino)cyclohexylamino)-9-isopropyl-9H-purin-6-ylamino)cyclohexyl)acrylamide (SB1-E-15)

0690<chemistry id="CHEM-US-00247" num="00247"><img file="USRE50776E_D0250.tif" /></chemistry>

0691To a solution of SB1-E-15-4 (20 mg, 0.0482 mmol), CH₃CN (4 mL) and DIPEA (0.5 mL) was added acryloyl chloride (7 mg, 0.0773 mmol) in CH₃CN (1 mL) dropwise, the mixture was stirred at r.t overnight. After completion, the mixture was purified by prep-TLC (DCM/MeOH=8/1) and prep-HPLC (C18 column, CH₃CN/H₂O, containing 0.05% NH₄HCO₃) to obtain SB1-E-15 (white solid, 5 mg, yield 24%). HPLC: 100% (254 nm); LCMS (m/z): 469 [M+H]⁺; ¹H NMR (DMSO-d₆, 500 MHz): δ 8.03 (m, 1H), 7.79 (s, 1H), 6.79 (s, 1H), 6.34 (dd, J₁=17.0 Hz, J₂=12.0 Hz, 1H), 6.07 (d, J=17.0 Hz, 1H), 5.56 (d, J=12.0 Hz, 1H), 4.51 (m, 1H), 4.07 (s, 1H), 3.61 (s, 1H), 2.22 (s, 7H), 2.00 (s, 2H), 1.81 (m, 5H), 1.19-1.65 (m, 17H).

Example 12. N-(3-(2-((1r,4r)-4-(Dimethylamino)cyclohexylamino)-9-isopropyl-9H-purin-6-ylamino)benzyl)acrylamide (SB1-E-16)

0692<chemistry id="CHEM-US-00248" num="00248"><img file="USRE50776E_D0251.tif" /></chemistry><chemistry id="CHEM-US-00249" num="00249"><img file="USRE50776E_D0252.tif" /></chemistry>

tert-butyl 3-(2-Chloro-9-isopropyl-9H-purin-6-ylamino)benzylcarbamate (SB1-E-16-3)

0693<chemistry id="CHEM-US-00250" num="00250"><img file="USRE50776E_D0253.tif" /></chemistry>

0694The mixture of SB1-E-16-2 (500 mg, 2.16 mmol), SB1-E-16-1 (490 mg, 2.20 mmol), n-BuOH (20 mL) and DIPEA (1 mL) was stirred at 90° C. overnight, after completion, concentrated to remove the solvent, the residue was purified by silica gel (PE/ethyl acetate=2/1) to obtain SB1-E-16-3 (off-white solid, 360 mg, yield 40%). LCMS (m/z): 417 [M+H]⁺.

tert-butyl 3-(2-((1r,4r)-4-(Dimethylamino)cyclohexylamino)-9-isopropyl-9H-purin-6-ylamino)benzylcarbamate (SB1-E-16-4)

0695<chemistry id="CHEM-US-00251" num="00251"><img file="USRE50776E_D0254.tif" /></chemistry>

0696The mixture of SB1-E-16-3 (210 mg, 0.504 mmol), 4 (150 mg, 1.05 mmol), KF (90 mg, 1.55 mmol) and n-BuOH (2 mL) was stirred at 135° C. for 2 days, after completion, concentrated to remove the solvent, the residue was purified by prep-TLC (DCM/MeOH=10/1) to get SB1-E-16-4 (light yellow solid, 90 mg, yield 34%. LCMS (m/z): 523 [M+H]⁺.

N6-(3-(Aminomethyl)phenyl)-N2-((1r,4r)-4-(dimethylamino)cyclohexyl)-9-isopropyl-9H-purine-2,6-diamine (SB1-E-16-5)

0697<chemistry id="CHEM-US-00252" num="00252"><img file="USRE50776E_D0255.tif" /></chemistry>

0698The mixture of SB1-E-16-4 (50 mg, 0.0957 mmol), DCM (5 mL) and TFA (3 mL) was stirred at r.t overnight, after completion, concentrated to remove the solvent to get SB1-E-16-5 (light brown solid, 40 mg, 95%). LCMS (m/z): 423 [M+H]⁺.

N-(3-(2-((1r,4r)-4-(Dimethylamino)cyclohexylamino)-9-isopropyl-9H-purin-6-ylamino)benzyl)acrylamide (SB1-E-16)

0699<chemistry id="CHEM-US-00253" num="00253"><img file="USRE50776E_D0256.tif" /></chemistry>

0700To a solution of SB1-E-16-5 (40 mg, 0.0947 mmol), CH₃CN (9 mL) and DIPEA (1 mL) was added acryloyl chloride (13 mg, 0.144 mmol) in CH₃CN (1 mL) dropwise, the mixture was stirred at r.t overnight, after completion, concentrated to remove the solvent, the residue was purified by prep-TLC (DCM/MeOH=8/1) and prep-HPLC (C18 column, CH₃CN/H₂O, containing 0.05% NH₄HCO₃) to obtain SB1-E-16 (white solid, 4.5 mg, yield 10%). HPLC: 95% (214 nm); LCMS (m/z): 477 [M+H]⁺; ¹H NMR (DMSO-d₆, 500 MHz): δ 9.40 (s, 1H), 8.60 (d, 1H), 7.94 (s, 1H), 7.88 (d, 1H), 7.22 (t, J=8.0 Hz, 1H), 6.87 (d, J=7.5 Hz, 1H), 6.30 (dd, J₁=17.0 Hz, J₂=10.0 Hz, 1H), 6.14 (d, J=17.0 Hz, 1H), 5.62 (d, J=10.0 Hz, 1H), 4.57 (m, 1H), 4.36 (d, J=6.0 Hz, 2H), 1.84-2.36 (m, 10H), 1.49 (s, 3H), 1.46 (s, 3H), 1.23-1.34 (m, 5H).

Example 13. N-(3-((2-(2-(2-Hydroxyethyl)piperidin-1-yl)-9-isopropyl-9H-purin-6-yl)amino)phenyl)acrylamide (THZ-4-134-1)

0701<chemistry id="CHEM-US-00254" num="00254"><img file="USRE50776E_D0257.tif" /></chemistry>

2-(1-(6-((3-Aminophenyl)amino)-9-isopropyl-9H-purin-2-yl)piperidin-2-yl)ethan-1-ol

0702<chemistry id="CHEM-US-00255" num="00255"><img file="USRE50776E_D0258.tif" /></chemistry>

0703The mixture of SB1-E-14-5 (1.1 g, 3.72 mmol), SM-23-1 (800 mg, 6.19 mmol), KF (1.0 g, 17.2 mmol) and NMP (3 mL) was stirred at 168° C. for 12 h, after completion, concentrated to remove the solvent, the residue was purified by silica gel (PE/ethyl acetate=2/1, 1/1) to obtain desired product (1.2 g, yield 81%). LCMS (m/z): 396 [M+H]⁺.

N-(3-((2-(2-(2-Hydroxyethyl)piperidin-1-yl)-9-isopropyl-9H-purin-6-yl)amino)phenyl)acrylamide (THZ-4-134-1)

0704<chemistry id="CHEM-US-00256" num="00256"><img file="USRE50776E_D0259.tif" /></chemistry>

0705To a solution of free amine obtained from above reaction (30 mg, 0.076 mmol), DIPEA (0.7 mL) in CH3CN (12 mL) was added the solution of acryloyl chloride (11 mg, 0.12 mmol) in CH₃CN (2 mL) dropwise, the mixture was stirred at 0° C. for 3 h. after completion, concentrated to remove the solvent, the residue was purified by prep-TLC (DCM/MeOH=30/1) and prep-HPLC (C18 column, CH₃CN/H₂O, containing 0.05% NH₄HCO₃) to obtain THZ-4-134-1 (off-white solid, 23 mg, yield 70%). HPLC: 98% (254 nm); LCMS (m/z): 450 [M+H]⁺; ¹H NMR (DMSO-d₆, 600 MHz): δ 10.42 (s, 1H), 9.50 (s, 1H), 8.34 (s, 1H), 7.25 (s, 1H), 7.52 (d, J=7.8 Hz, 1H), 7.25-7.16 (m, 2H), 6.45 (dd, J₁=17.2 Hz, J₂=10.0 Hz, 1H), 6.27 (d, J=17.2 Hz, 1H), 5.77 (d, J=10.0 Hz, 1H), 4.97 (br, 1H), 4.61 (m, 1H), 3.34-3.38 (m, 3H), 2.87 (t, J=12 Hz, 1H), 1.83 (m, 1H), 1.74 (m, 1H), 1.69 (m, 3H), 1.60-1.50 (m, 9H), 1.34 (m, 1H).

Example 14. N-(3-(2-((1r,4r)-4-(Dimethylamino)cyclohexylamino)-8-ethylpyrazolo[1,5-a][1,3,5]triazin-4-ylamino)phenyl)acrylamide (compound SB1-E-17)

0706<chemistry id="CHEM-US-00257" num="00257"><img file="USRE50776E_D0260.tif" /></chemistry>

N4-(3-Aminophenyl)-N2-((1r,4r)-4-(dimethylamino)cyclohexyl)-8-ethylpyrazolo[1,5-a][1,3,5]triazine-2,4-diamine (SB1-E-17-01)

0707<chemistry id="CHEM-US-00258" num="00258"><img file="USRE50776E_D0261.tif" /></chemistry>

0708To a stirred mixture of SB1-E-18-07 (60 mg, 0.14 mmol) and SB1-E-21-3 (30 mg, 0.21 mmol) in N-methyl-2-pyrrolidone (1 mL) was added KF (24 mg, 0.42 mmol). This mixture was heated at 170° C. for 3 h, cooled to r.t, filtered, purified by prep-TLC (DCM/MeOH=6/1) to get SB1-E-17-01 (white solid, 40 mg, yield: 73%). MS (ESI): m/z 395 [M+H]⁺.

N-(3-(2-((1r,4r)-4-(Dimethylamino)cyclohexylamino)-8-ethylpyrazolo[1,5-a][1,3,5]triazin-4-ylamino)phenyl)acrylamide (compound SB1-E-17)

0709<chemistry id="CHEM-US-00259" num="00259"><img file="USRE50776E_D0262.tif" /></chemistry>

0710To a solution of SB1-E-17-01 (40 mg, 0.10 mmol) in THF (1 mL) was added Acryloyl chloride (14 mg, 0.15 mmol) and Triethylamine (30 mg, 0.30 mmol) was stirred at r.t for 2 h. After completion, the reaction mixture was diluted with dichloromethane (10 ml), washed with water (10 mL) and saturated sodium bicarbonate solution (10 mL×2), dried over anhydrous sodium sulfate, concentrated, purified by prep-HPLC (C18 column, CH₃CN/H₂O, containing 0.05% NH₄HCO₃) to get SB1-E-17, also referred to herein as E17 and E-17 (white solid, 4 mg, yield: 11%). MS (ESI): m/z 449 [M+H]⁺. ¹H NMR (300 MHz, DMSO) δ: 9.98 (s, 1H), 9.65 (s, 1H), 8.14 (s, 1H), 7.76 (s, 1H), 7.52 (d, J=8.1 Hz, 1H), 7.45 (d, J=7.8 Hz, 1H), 7.29 (t, J=8.1 Hz, 1H), 6.64 (s, 1H), 6.42-6.51 (m, 1H), 6.23-6.29 (m, 1H), 5.70-5.74 (m, 1H), 3.69-3.72 (m, 1H), 2.51-2.56 (m, 2H), 2.36 (s, 6H), 2.03-2.06 (m, 2H), 1.86-1.90 (m, 2H), 1.18-1.37 (m, 8H).

Example 15. N-(3-(8-Ethyl-2-(2-(2-hydroxyethyl)piperidin-1-yl)pyrazolo[1,5-a][1,3,5]triazin-4-ylamino)phenyl)acrylamide (SB1-E-18)

0711<chemistry id="CHEM-US-00260" num="00260"><img file="USRE50776E_D0263.tif" /></chemistry><chemistry id="CHEM-US-00261" num="00261"><img file="USRE50776E_D0264.tif" /></chemistry>

ethyl N-[(4-Ethyl-1H-pyrazol-5-yl)carbamothioyl]carbamate (SB1-E-18-01)

0712<chemistry id="CHEM-US-00262" num="00262"><img file="USRE50776E_D0265.tif" /></chemistry>

0713To a solution of SM-18-1 (2.8 g, 25.2 mmol) in ethanol (50.0 mL) was added ethoxycarbonyl isothiocyanate (3.3 g, 25.2 mL) in one portion at r.t. The mixture was stirred at 80° C. overnight. The reaction mixture was concentrated under reduced pressure to afford the residue, purified by flash column chromatography (PE/ethyl acetate=3/1) to get SB1-E-18-01 (white solid, 4 g, yield: 65%). LCMS (m/z): 243 [M+H]⁺.

8-Ethyl-2-thioxo-2,3-dihydropyrazolo[1,5-a][1,3,5]triazin-4(1H)-one (SB1-E-18-02)

0714<chemistry id="CHEM-US-00263" num="00263"><img file="USRE50776E_D0266.tif" /></chemistry>

0715To a solution of SB1-E-18-01 (3.9 g, 16.1 mmol) in acetonitrile (40 mL) was added K₂CO₃(6.67 g, 48.3 mmol) in one portion at r.t. The mixture was heated at 85° C. overnight, cooled, acidified with AcOH. The solid was filtered off to get SB1-E-18-02 (yellow solid, 2.1 g, yield: 66%). LCMS (m/z): 197 [M+H]⁺.

8-Ethyl-2-(methylthio)pyrazolo[1,5-a][1,3,5]triazin-4(3H)-one (SB1-E-18-03)

0716<chemistry id="CHEM-US-00264" num="00264"><img file="USRE50776E_D0267.tif" /></chemistry>

0717To a stirred mixture of SB1-E-18-02 (2.1 g, 10.7 mmol) and NaOH (0.86 g, 21.4 mmol) in Dioxane/H₂O (30/8 mL) was added iodomethane (1.52 g, 10.7 mol). This mixture was stirred at r.t for 1 h, acidified with Hydrochloric acid, concentrated to remove the solvent, the residue was purified by silica gel (DCM/MeOH=30/1) to get SB1-E-18-03 (white solid, 2.0 g, yield: 89%). LCMS (m/z): 211 [M+H]⁺.

4-Chloro-8-ethyl-2-(methylthio)pyrazolo[1,5-a][1,3,5]triazine (SB1-E-18-04)

0718<chemistry id="CHEM-US-00265" num="00265"><img file="USRE50776E_D0268.tif" /></chemistry>

0719To a stirred mixture SB1-E-18-03 (2.0 g, 9.5 mmol) and N,N-dimethyl aniline (2.3 g, 19.0 mmol) in acetonitrile (10 mL) under argon was added POCl₃(20 ml, 219 mol). This mixture was heated at 85° C. overnight, cooled, the reaction mixture was concentrated under reduced pressure to afford the residue SB1-E-18-04 (white solid, 2.2 g, yield: 100% used next step directly). LCMS (m/z): 229 [M+H]⁺.

N1-(8-Ethyl-2-(methylthio)pyrazolo[1,5-a][1,3,5]triazin-4-yl)benzene-1,3-diamine (SB1-E-18-05)

0720<chemistry id="CHEM-US-00266" num="00266"><img file="USRE50776E_D0269.tif" /></chemistry>

0721To a stirred mixture SB1-E-18-04 (2.2 g, 9.5 mmol), benzene-1,3-diamine (1.23 g, 11.4 mmol) and NaHCO₃(130.4 mg, 0.95 mmol) in acetonitrile (25 mL). This mixture was heated at 75° C. overnight, cooled, filtered and concentrated to remove the solvent, the residue was purified by silica gel (PE/ethyl acetate=3/1 to 1/1) to get SB1-E-18-05 (white solid, 1.1 g, yield: 38%). LCMS (m/z): 301 [M+H]⁺.

tert-Butyl 3-(8-ethyl-2-(methylthio)pyrazolo[1,5-a][1,3,5]triazin-4-ylamino) phenylcarbamate (SB1-E-18-06)

0722<chemistry id="CHEM-US-00267" num="00267"><img file="USRE50776E_D0270.tif" /></chemistry>

0723To a solution of SB1-E-18-05 (1.1 g, 3.67 mmol) in MeOH (20.0 mL) was added Di-tert-butyl dicarbonate (1.2 g, 5.5 mmol) and TEA (1.1 g, 11.0 mmol) in one portion at r.t. The reaction was stirred at r.t. overnight. The reaction mixture was concentrated under reduced pressure to afford the residue, purified by flash column chromatography (PE/ethyl acetate=3/1) to get SB1-E-18-06 (white solid, 1.26 g, yield: 86%). LCMS (m/z): 401 [M+H]+.

tert-Butyl 3-(8-ethyl-2-(methylsulfonyl)pyrazolo[1,5-a][1,3,5]triazin-4-ylamino) phenylcarbamate (SB1-E-18-07)

0724<chemistry id="CHEM-US-00268" num="00268"><img file="USRE50776E_D0271.tif" /></chemistry>

0725To a solution of SB1-E-18-06 (600 mg, 1.5 mmol) in DCM (15 mL) was added 3-Chloroperbenzoic acid (776 mg, 4.5 mmol) in one portion at r.t. The mixture was stirred at r.t for 1 h. The reaction mixture was quenched with sat Na₂S₂O₃solution (10 mL), diluted with DCM (50 mL) and washed with saturated aqueous NaCl (3×50 mL), dried over anhydrous Na₂SO₄, filtered and concentrated to give SB1-E-18-07 (yellow solid, 620 mg, yield: 96%), used for next step directly. LCMS (m/z): 433 [M+H]+.

2-(1-(4-(3-Aminophenylamino)-8-ethylpyrazolo[1,5-a][1,3,5]triazin-2-yl) piperidin-2-yl)ethanol (SB1-E-18-08)

0726<chemistry id="CHEM-US-00269" num="00269"><img file="USRE50776E_D0272.tif" /></chemistry>

0727To a stirred mixture SB1-E-18-07 (360 mg, 0.84 mmol) and 2-(piperidin-2-yl)ethanol (215 mg, 1.67 mmol) in N-methyl-2-pyrrolidone (3 mL) was added KF (146 mg, 2.51 mmol). This mixture was heated at 170° C. for 3 h, cooled, filtered, the crude was purified by silica gel (PE/ethyl acetate=2/1) to get SB1-E-18-08 (yellow solid, 150 mg, yield: 47%). LCMS (m/z): 382 [M+H]⁺.

N-(3-(8-Ethyl-2-(2-(2-hydroxyethyl)piperidin-1-yl)pyrazolo[1,5-a][1,3,5] triazin-4-ylamino)phenyl)acrylamide (SB1-E-18)

0728<chemistry id="CHEM-US-00270" num="00270"><img file="USRE50776E_D0273.tif" /></chemistry>

0729To a solution of SB1-E-18-08 (70 mg, 0.18 mmol) in THF (2 mL) was added Acryloyl chloride (18 mg, 0.20 mmol) and triethylamine (54 mg, 0.54 mmol) was stirred at r.t for 2 h. After completion, the reaction mixture was diluted with dichloromethane (10 ml), washed with water (10 mL) and saturated sodium bicarbonate solution (10 mL×2), dried over anhydrous sodium sulfate, concentrated, purified by prep-HPLC (C18 column, CH₃CN/H₂O, containing 0.05% NH₄HCO₃) to get SB1-E-18, also referred to herein as E-18 and E18 (white solid, 13 mg, yield: 16%). HPLC: 100% (254 nm); LCMS (m/z): 555 [M+H]⁺; ¹H NMR (DMSO-d₆, 500 MHz): δ: 10.17 (s, 1H), 10.07 (s, 1H), 8.34 (s, 1H), 7.84 (s, 1H), 7.51 (s, 1H), 7.30-7.34 (m, 2H), 6.46 (dd, J=17, 10.0 Hz, 1H), 6.28 (d, J=17 Hz, 1H), 5.77 (d, J=11.5 Hz, 1H), 4.98 (s, 1H), 4.65 (d, J=13 Hz, 1H), 4.56 (s, 1H), 2.88 (t, J=12.5 Hz, 1H), 2.47-2.50 (m, 2H), 1.56-1.88 (m, 8H), 1.36-1.38 (m, 1H), 1.19-1.23 (m, 4H).

Example 16. N-(3-(3-Ethyl-5-(2-(2-hydroxyethyl)piperidin-1-yl)pyrazolo[1,5-a]pyrimidin-7-ylamino)phenyl)acrylamide (SB1-E-23)

0730<chemistry id="CHEM-US-00271" num="00271"><img file="USRE50776E_D0274.tif" /></chemistry>

2-(1-(7-(3-Aminophenylamino)-3-ethylpyrazolo[1,5-a]pyrimidin-5-yl)piperidin-2-yl)ethanol (SB1-E-23-1)

0731<chemistry id="CHEM-US-00272" num="00272"><img file="USRE50776E_D0275.tif" /></chemistry>

0732The mixture of SB1-E-21-6 (1.1 g, 3.82 mmol), SM-23-1 (800 mg, 6.19 mmol), KF (1.0 g, 17.2 mmol) and NMP (3 mL) was stirred at 168° C. for 12 h, after completion, concentrated to remove the solvent, the residue was purified by silica gel (PE/ethyl acetate=2/1, 1/1) to obtain SB1-E-23-1 (light brown solid, 1.0 g, yield 69%). LCMS (m/z): 381 [M+H]⁺.

N-(3-(3-Ethyl-5-(2-(2-hydroxyethyl)piperidin-1-yl)pyrazolo[1,5-a]pyrimidin-7-ylamino)phenyl)acrylamide (SB1-E-23)

0733<chemistry id="CHEM-US-00273" num="00273"><img file="USRE50776E_D0276.tif" /></chemistry>

0734To a solution of SB1-E-23-1 (150 mg, 0.394 mmol), DIPEA (0.7 mL) in CH3CN (12 mL) was added the solution of acryloyl chloride (54 mg, 0.597 mmol) in CH₃CN (2 mL) dropwise, the mixture was stirred at 0° C. for 3 h. after completion, concentrated to remove the solvent, the residue was purified by prep-TLC (DCM/MeOH=30/1) and prep-HPLC (C18 column, CH₃CN/H₂O, containing 0.05% NH₄HCO₃) to obtain SB1-E-23, also referred to herein as E-9 and E9 (off-white solid, 15 mg, yield 9%). HPLC: 98% (254 nm); LCMS (m/z): 435 [M+H]⁺; ¹H NMR (DMSO-d₆, 500 MHz): δ 10.23 (s, 1H), 9.29 (s, 1H), 7.88 (s, 1H), 7.76 (s, 1H), 7.33-7.36 (m, 2H), 7.16-7.19 (m, 1H), 6.45 (dd, J₁=17.2 Hz, J₂=10.0 Hz, 1H), 6.27 (d, J=17.2 Hz, 1H), 6.01 (s, 1H), 5.77 (d, J=10.0 Hz, 1H), 4.58 (m, 2H), 4.26 (m, 1H), 3.34-3.38 (m, 1H), 2.87 (t, 1H), 2.54 (q, J=7.2 Hz, 2H), 1.28-1.84 (m, 8H), 1.22 (t, J=7.6 Hz, 3H).

Example 17. N-(3-(5-((1r,4r)-4-(Dimethylamino)cyclohexylamino)-3-ethylpyrazolo[1,5-a]pyrimidin-7-ylamino)phenyl)acrylamide (SB1-E-21)

0735<chemistry id="CHEM-US-00274" num="00274"><img file="USRE50776E_D0277.tif" /></chemistry><chemistry id="CHEM-US-00275" num="00275"><img file="USRE50776E_D0278.tif" /></chemistry>

tert-Butyl (1r,4r)-4-aminocyclohexylcarbamate (SB1-E-21-1)

0736<chemistry id="CHEM-US-00276" num="00276"><img file="USRE50776E_D0279.tif" /></chemistry>

0737To a solution of SM-21-1 (2.0 g, 17.5 mmol) in MeOH (100 mL) was added the solution of (Boc)₂O (1.1 g, 5.04 mmol) in MeOH (60 mL) dropwise for 30 min, the mixture was stirred at r.t overnight. After completion, concentrated to remove the solvent, the residue was added H₂O (50 mL), further stirred at r.t for 20 min, then filtered, the filtrate was extracted with ethyl acetate (120 mL×2), the organic phase was washed with brine (50 mL×2), dried with Na₂SO₄. Filtered, concentrated to remove the solvent to obtain SB1-E-21-1 (off-white solid, 900 mg, yield 83%).

tert-Butyl (1r,4r)-4-(dimethylamino)cyclohexylcarbamate (SB1-E-21-2)

0738<chemistry id="CHEM-US-00277" num="00277"><img file="USRE50776E_D0280.tif" /></chemistry>

0739To a solution of SB1-E-21-1 (850 mg, 3.97 mmol) and HCHO (600 mg, 20.0 mmol) in MeOH (30 mL) was added NaBH₃CN (1.1 g, 17.5 mmol), the mixture was stirred at r.t overnight, after completion, concentrated to remove the solvent, the residue was extracted with ethyl acetate (100 mL×4), the organic phase was washed with brine (50 mL×2), dried with Na₂SO₄. Filtered, concentrated to remove the solvent, the residue was purified by silica gel (DCM/MeOH=10/1, 5/1) to obtain SB1-E-21-2 (light brown solid, 800 mg, yield 83%). LCMS (m/z): 243 [M+H]⁺.

(1r,4r)-N1,N1-Dimethylcyclohexane-1,4-diamine (SB1-E-21-3)

0740<chemistry id="CHEM-US-00278" num="00278"><img file="USRE50776E_D0281.tif" /></chemistry>

0741The mixture of SB1-E-21-2 (350 mg, 1.44 mmol), DCM (3 mL) and TFA (3 mL) was stirred at r.t for 3 h, after completion, concentrated to remove the solvent to get SB1-E-21-3 (light yellow sticky oil, 200 mg, yield 98%). LCMS (m/z): 143 [M+H]⁺.

3-Ethylpyrazolo[1,5-a]pyrimidine-5,7(4H,6H)-dione (SB1-E-21-4)

0742<chemistry id="CHEM-US-00279" num="00279"><img file="USRE50776E_D0282.tif" /></chemistry>

0743The mixture of SM-21-2 (5.0 g, 24.8 mmol), dimethyl malonate (10 mL, 87.0 mmol), MeOH (80 mL) and NaOMe (7.0 g, 129.6 mmol) was stirred at 65° C. overnight, after completion, concentrated to remove the solvent, the residue was added H₂O (10 mL), 2M HCl to make PH&lt;7, then filtered, the solid was washed with H₂O (50 mL), dried to obtain SB1-E-21-4 (light yellow solid, 2.6 g, yield 58%). LCMS (m/z): 180 [M+H]⁺.

5,7-Dichloro-3-ethylpyrazolo[1,5-a]pyrimidine (SB1-E-21-5)

0744<chemistry id="CHEM-US-00280" num="00280"><img file="USRE50776E_D0283.tif" /></chemistry>

0745To a suspension of SB1-21-4 (2.6 g, 14.5 mmol) in CH₃CN (20 mL) was added N,N-dimethylaniline (3.6 g, 29.7 mmol), POCl₃(11.5 g, 75.0 mmol) dropwise, the mixture was stirred at 85° C. overnight. After completion, cooled to 0° C., added H₂O (50 mL) to quench the reaction, filtered, the solid was washed with H₂O (50 mL), dried to obtain SB1-E-21-5 (light brown solid, 2.7 g, 86%). LCMS (m/z): 216 [M+H]⁺

N1-(5-Chloro-3-ethylpyrazolo[1,5-a]pyrimidin-7-yl)benzene-1,3-diamine (SB1-E-21-6)

0746<chemistry id="CHEM-US-00281" num="00281"><img file="USRE50776E_D0284.tif" /></chemistry>

0747The mixture of SB1-E-21-5 (2.6 g, 12.0 mmol), SM-21-3 (1.5 g, 13.9 mmol), DIPEA (5 mL) and i-PrOH (30 mL) was stirred at 85° C. for 6 h, after completion, concentrated to remove the solvent, the residue was purified by silica gel (DCM/MeOH=200/1) to obtain SB1-E-21-6 (brown solid, 2.8 g, yield 81%). LCMS (m/z): 288 [M+H]⁺.

N7-(3-Aminophenyl)-N5-((1r,4r)-4-(dimethylamino)cyclohexyl)-3-ethylpyrazolo[1,5-a]pyrimidine-5,7-diamine (SB1-E-21-7)

0748<chemistry id="CHEM-US-00282" num="00282"><img file="USRE50776E_D0285.tif" /></chemistry>

0749The mixture of SB1-E-21-6 (400 mg, 1.39 mmol), SB1-E-21-3 (200 mg, 1.41 mmol), KF (400 mg, 6.88 mmol) and NMP (1.5 mL) was stirred at 145° C. for 10 h in a sealed tube. After completion, concentrated to remove the solvent, the residue was purified by prep-TLC (DCM/MeOH=6/1) to obtain SB1-E-21-7 (light brown solid, 160 mg, yield 29%). LCMS (m/z): 394 [M+H]⁺.

N-(3-(5-((1r,4r)-4-(Dimethylamino)cyclohexylamino)-3-ethylpyrazolo[1,5-a]pyrimidin-7-ylamino)phenyl)acrylamide (SB1-E-21)

0750<chemistry id="CHEM-US-00283" num="00283"><img file="USRE50776E_D0286.tif" /></chemistry>

0751To a solution of SB1-E-21-7 (60 mg, 0.152 mmol) and DIPEA (0.3 mL) in CH₃CN (3 mL) was added acrylyl chloride (25 mg, 0.276 mmol) in CH₃CN (1 mL) dropwise, the mixture was stirred at 0° C. for 3 h. after completion, concentrated to remove the solvent, the residue was purified by prep-TLC (DCM/MeOH=10/1) and prep-HPLC (C18 column, CH₃CN/H₂O, containing 0.05% NH₄HCO₃) to obtain SB1-E-21, also referred to herein as E-21 and E21 (off-white solid, 7 mg, yield 10%). HPLC: 99% (254 nm); LCMS (m/z): 448 [M+H]⁺; ¹H NMR (DMSO-d₆, 500 MHz): δ 10.26 (s, 1H), 9.07 (s, 1H), 7.69 (s, 2H), 7.54 (d, J=9.0 Hz, 1H), 7.36 (t, J=8.5 Hz, 1H), 7.09 (d, J=7.0 Hz, 1H), 6.63 (d, J=7.0 Hz, 1H), 6.46 (dd, J₁=17.5 Hz, J₂=12.0 Hz, 1H), 6.28 (d, J=17.5 Hz, 1H), 5.78 (d, J=12.0 Hz, 1H), 5.62 (s, 1H), 2.55 (m, 4H), 2.17 (s, 6H), 1.78-2.13 (m, 4H), 1.12-1.27 (m, 8H).

Example 18. N-(3-(5-((1r,4r)-4-(Dimethylamino)cyclohexylamino)-3-isopropylpyrazolo[1,5-a]pyrimidin-7-ylamino)phenyl)acrylamide (SB1-E-26)

0752<chemistry id="CHEM-US-00284" num="00284"><img file="USRE50776E_D0287.tif" /></chemistry>

N-(3-(5-((1r,4r)-4-(Dimethylamino)cyclohexylamino)-3-isopropylpyrazolo[1,5-a]pyrimidin-7-ylamino)phenyl)acrylamide (SB1-E-26)

0753To a solution of SB1-E-25-6 (70 mg, 0.172 mmol) and DIPEA (0.5 mL) in CH₃CN (5 mL) was added acrylyl chloride (30 mg, 0.331 mmol) in CH₃CN (1 mL), the mixture was stirred at r.t for 15 h, after completion, concentrated to remove the solvent, the residue was purified by prep-TLC (DCM/MeOH=10/1) and prep-HPLC (C18 column, CH₃CN/H₂O, containing 0.05% NH₄HCO₃) to obtain SB1-E-26, also referred to herein as E-26 and E26 (white solid, 12 mg, yield 15%). HPLC: 100% (254 nm); LCMS (m/z): 462 [M+H]⁺; ¹H NMR (DMSO-d₆, 500 MHz): δ 10.26 (s, 1H), 9.06 (s, 1H), 7.70 (s, 1H), 7.67 (s, H), 7.54 (d, J=6.5 Hz, 1H), 7.36 (t, J=6.5 Hz, 1H), 7.08 (d, J=1.5 Hz, 1H), 6.62 (d, J=6.0 Hz, 1H), 6.45 (dd, J₁=16.0 Hz, J₂=9.5 Hz, 1H), 6.28 (d, J=16.0 Hz, 1H), 5.78 (d, J=9.5 Hz, 1H), 5.62 (s, 1H), 3.00 (m, 1H), 2.18 (s, 7H), 2.06 (d, J=10.5 Hz, 1H), 1.80 (d, J=10.5 Hz, 1H), 1.12-1.29 (m, 11H).

Example 19. N-((1R,4s)-4-((3-ethyl-5-((S)-2-(2-hydroxyethyl)piperidin-1-yl)pyrazolo[1,5-a]pyrimidin-7-yl)amino)cyclohexyl)acrylamide (MFH-1-169-1)

0754<chemistry id="CHEM-US-00285" num="00285"><img file="USRE50776E_D0288.tif" /></chemistry>

0755<chemistry id="CHEM-US-00286" num="00286"><img file="USRE50776E_D0289.tif" /></chemistry>

(1s,4s)-N1-(5-chloro-3-ethylpyrazolo[1,5-a]pyrimidin-7-yl)cyclohexane-1,4-diamine (MFH-1-153-1)

0756The mixture of SM-1-49-1 (300 mg, 1.388 mmol), SM-1-167-1 (182 mg, 1.6 mmol), DIPEA (270 mg) and i-PrOH (8 mL) was stirred at 85° C. for 2 h. After completion, the solvent was removed and the residue was purified by silica gel (NH₃/MeOH (1.75N)/DCM=0-20%) to obtain MFH-1-153-1 (240 g, yield 59%). LCMS (m/z): 294 [M+H]⁺.

2-((S)-1-(7-((1s,4R)-4-aminocyclohexylamino)-3-ethylpyrazolo[1,5-a]pyrimidin-5-yl)piperidin-2-yl)ethanol (MFH-1-159-1)

0757The mixture of MFH-1-153-1 (230 mg, 0.783 mmol), SM-1-49-3 (182 mg, 1.41 mmol), KF (205 mg, 3.5235 mmol), K₂CO₃(194 mg, 1.41 mmol) and NMP (2 mL) was stirred at 170° C. for 48 h. After completion, the residue was extracted with chloroform/i-propanol (4/1) and the organic phase was washed with brine (50 mL×2) and dried with Na₂SO₄. The residue after removal of the solvent was purified by silica gel (NH₃/MeOH (1.75N)/DCM=0-20%) to obtain MFH-1-159-1 (50 mg, yield 16.5%). LCMS (m/z): 387 [M+H]⁺.

N-((1R,4s)-4-(3-ethyl-5-((S)-2-(2-hydroxyethyl)piperidin-1-yl)pyrazolo[1,5-a]pyrimidin-7-ylamino)cyclohexyl)acrylamide (MFH-1-169-1)

0758To a solution of MFH-1-159-1 (25 mg, 0.06468 mmol) and DIPEA (0.2 mL) in CH₃CN (2 mL) was added acryloyl chloride (8 mg, 0.0841 mmol) in DCM (0.5 mL) dropwise. The mixture was stirred at 0° C. for 1 h. After completion, the solvent was removed and the residue was purified by prep-HPLC (C18 column, MeOH/H₂O, containing 0.05% TFA) to obtain MFH-1-169-1 (off-white solid, 4.8 mg, yield 16%). HPLC: 97% (254 nm); LCMS (m/z): 441 [M+H]⁺; ¹H NMR (500 MHz, DMSO) δ 7.97 (d, J=7.2 Hz, 1H), 7.87 (s, 1H), 7.44 (s, 1H), 6.30 (dd, J=17.1, 10.2 Hz, 1H), 6.09 (dd, J=17.1, 2.1 Hz, 1H), 5.78 (s, 1H), 5.59 (dd, J=10.2, 2.1 Hz, 1H), 4.49 (s, 1H), 4.15 (d, J=12.3 Hz, 1H), 3.98-3.79 (m, 3H), 3.08 (t, J=12.4 Hz, 2H), 2.61-2.52 (m, 2H), 1.95 (dd, J=18.6, 10.5 Hz, 1H), 1.84 (dt, J=12.9, 7.4 Hz, 2H), 1.79-1.57 (m, 10H), 1.56-1.44 (m, 1H), 1.18 (t, J=7.5 Hz, 3H).

Example 20. N-((1S,4r)-4-((3-ethyl-5-((S)-2-(2-hydroxyethyl)piperidin-1-yl)pyrazolo[1,5-a]pyrimidin-7-yl)amino)cyclohexyl)acrylamide (MFH-1-175-1)

0759<chemistry id="CHEM-US-00287" num="00287"><img file="USRE50776E_D0290.tif" /></chemistry>

0760<chemistry id="CHEM-US-00288" num="00288"><img file="USRE50776E_D0291.tif" /></chemistry>

(1r,4r)-N1-(5-chloro-3-ethylpyrazolo[1,5-a]pyrimidin-7-yl)cyclohexane-1,4-diamine (MFH-1-161-1)

0761The mixture of SM-1-49-1 (300 mg, 1.388 mmol), SM-1-175-1 (250 mg, 2.19 mmol), DIPEA (270 mg) and i-PrOH (8 mL) was stirred at 85° C. for 1 h. After completion, the solvent was removed and the residue was purified by silica gel (NH₃/MeOH (1.75N)/DCM=0-20%) to obtain MFH-1-161-1 (230 g, yield 56%). LCMS (m/z): 294 [M+H]⁺.

2-((S)-1-(7-((1r,4S)-4-aminocyclohexylamino)-3-ethylpyrazolo[1,5-a]pyrimidin-5-yl)piperidin-2-yl)ethanol (MFH-1-163-1)

0762The mixture of MFH-1-161-1 (230 mg, 0.783 mmol), SM-1-49-3 (182 mg, 1.41 mmol), KF (205 mg, 3.5235 mmol), K₂CO₃(194 mg, 1.41 mmol) and NMP (2 mL) was stirred at 170° C. for 48 h. After completion, the residue was extracted with chloroform and 2-propanol (4:1) and the organic phase was washed with brine (50 mL×2) and dried with Na₂SO₄. The residue after removal of solvent was purified by silica gel (NH₃/MeOH (1.75N)/DCM=0-20%) to obtain MFH-1-163-1(30 mg, yield 10%). LCMS (m/z): 387 [M+H]⁺.

N-((1S,4r)-4-(3-ethyl-5-((S)-2-(2-hydroxyethyl)piperidin-1-yl)pyrazolo[1,5-a]pyrimidin-7-ylamino)cyclohexyl)acrylamide (MFH-1-175-1)

0763To a solution of MFH-1-163-1 (30 mg, 0.07762 mmol) and DIPEA (0.2 mL) in CH₃CN (2 mL) was added acryloyl chloride (9 mg, 0.101 mmol) in DCM (0.5 mL) dropwise. The mixture was stirred at 0° C. for 1 h. After completion, the solvent was removed and the residue was purified by prep-HPLC (C18 column, MeOH/H₂O, containing 0.05% TFA) to obtain MFH-1-175-1 (off-white solid, 12.8 mg, yield 37%). HPLC: 97% (254 nm); LCMS (m/z): 441 [M+H]⁺; ¹H NMR (500 MHz, DMSO) δ 8.03 (d, J=7.5 Hz, 1H), 7.90 (s, 1H), 6.24 (dd, J=17.1, 10.1 Hz, 1H), 6.08 (dd, J=17.1, 2.2 Hz, 1H), 5.77 (d, J=10.8 Hz, 1H), 5.58 (dd, J=10.1, 2.3 Hz, 1H), 4.49 (s, 1H), 4.14 (d, J=12.4 Hz, 2H), 3.74 (s, 2H), 3.62-3.49 (m, 2H), 3.43-3.34 (m, 1H), 3.11 (t, J=13.0 Hz, 1H), 2.57 (dt, J=9.8, 4.9 Hz, 2H), 2.07-1.82 (m, 5H), 1.83-1.56 (m, 8H), 1.53 (d, J=11.8 Hz, 1H), 1.39 (td, J=12.9, 3.6 Hz, 2H), 1.25-1.13 (m, 3H).

Example 21. (S)—N-(4-((3-ethyl-5-(2-(2-hydroxyethyl)piperidin-1-yl)pyrazolo[1,5-a]pyrimidin-7-yl)amino)phenyl)acrylamide (MFH-1-187-1)

0764<chemistry id="CHEM-US-00289" num="00289"><img file="USRE50776E_D0292.tif" /></chemistry>

0765<chemistry id="CHEM-US-00290" num="00290"><img file="USRE50776E_D0293.tif" /></chemistry>

tert-butyl4-(5-chloro-3-ethylpyrazolo[1,5-a]pyrimidin-7-ylamino)phenylcarbamate (MFH-1-177-1)

0766The mixture of SM-1-49-1 (500 mg, 2.314 mmol), SM-1-187-1 (520 mg, 2.5 mmol), DIPEA (898 mg) and i-PrOH (8 mL) was stirred at 85° C. for 6 h. After completion, the solvent was removed and the residue was purified by silica gel chromatography (MeOH/DCM=0-20%) to obtain MFH-1-177-1 (898 g, yield 100%). LCMS (m/z): 388 [M+H]⁺.

(S)-2-(1-(7-(4-aminophenylamino)-3-ethylpyrazolo[1,5-a]pyrimidin-5-yl)piperidin-2-yl)ethanol (MFH-1-183-1)

0767The mixture of MFH-1-177-1 (449 mg, 1.157 mmol), SM-1-49-3 (240 mg, 1.8512 mmol), KF (303 mg, 5.2 mmol) and NMP (2 mL) was stirred at 170° C. for 10 h. After completion, the solution was extracted with chloroform and 2-propanol (4:1) and the organic phase was washed with brine (50 mL×2) and dried with Na₂SO₄. The solvent was removed and the residue was purified by silica gel chromatography (MeOH/DCM=0-20%) to obtain MFH-1-183-1 (400 mg, yield 91%). LCMS (m/z): 381 [M+H]⁺.

(S)—N-(4-(3-ethyl-5-(2-(2-hydroxyethyl)piperidin-1-yl)pyrazolo[1,5-a]pyrimidin-7-ylamino)phenyl)acrylamide (MFH-1-187-1)

0768To a solution of MFH-1-183-1 (80 mg, 0.21 mmol) and DIPEA (0.2 mL) in CH₃CN (2 mL) was added acryloyl chloride (25 mg, 0.273 mmol) in DCM (0.5 mL) dropwise. The mixture was stirred at 0° C. for 1 h. After completion, the solvent was removed and the residue was purified by prep-HPLC (C18 column, MeOH/H₂O, containing 0.05% TFA) to obtain MFH-1-187-1 (off-white solid, 33.6 mg, yield 36.8%). HPLC: 96% (254 nm); LCMS (m/z): 435 [M+H]⁺; ¹H NMR (500 MHz, DMSO) δ 10.26 (s, 1H), 9.83 (s, 1H), 7.91 (s, 1H), 7.76 (t, J=13.5 Hz, 2H), 7.42 (d, J=8.9 Hz, 2H), 6.46 (dd, J=17.0, 10.1 Hz, 1H), 6.28 (dd, J=17.0, 1.9 Hz, 1H), 5.84-5.68 (m, 2H), 4.42 (s, 1H), 4.02 (s, 2H), 3.32 (ddd, J=10.9, 8.2, 4.9 Hz, 2H), 3.00 (dd, J=24.6, 11.8 Hz, 1H), 2.64-2.55 (m, 2H), 1.90 (td, J=13.5, 5.1 Hz, 1H), 1.76-1.51 (m, 6H), 1.49-1.33 (m, 1H), 1.27-1.17 (m, 3H).

Example 22. N-(3-(((2-(((1r,4r)-4-(dimethylamino)cyclohexyl)amino)-8-ethylpyrazolo[1,5-a][1,3,5]triazin-4-yl)amino)methyl)phenyl)acrylamide (MFH-2-67-1)

0769<chemistry id="CHEM-US-00291" num="00291"><img file="USRE50776E_D0294.tif" /></chemistry>

0770<chemistry id="CHEM-US-00292" num="00292"><img file="USRE50776E_D0295.tif" /></chemistry>

tert-butyl (1r,4r)-4-aminocyclohexylcarbamate (MFH-2-61-1)

0771To a solution of SM-2-67-1 (2.0 g, 17.5 mmol) in MeOH (100 mL) was added the solution of (Boc)₂O (1.1 g, 5.04 mmol) in MeOH (60 mL) dropwise for 30 min. The mixture was stirred at room temperature overnight. After completion, the solvent was removed and to the residue was added H₂O (50 mL) and further stirred at room temperature for 20 min and then filtered. The filtrate was extracted with ethyl acetate (120 mL×2) and the organic phase was washed with brine (50 mL×2) and dried with Na₂SO₄. The solution was then concentrated under reduced pressure to obtain MFH-2-61-1 (off-white solid, 900 mg, yield 83%).

tert-butyl (1r,4r)-4-(dimethylamino)cyclohexylcarbamate (MFH-2-62-1)

0772To a solution of MFH-2-61-1 (850 mg, 3.97 mmol) and HCHO (600 mg, 20.0 mmol) in MeOH (30 mL) was added NaBH₃CN (1.1 g, 17.5 mmol) and the mixture was stirred at room temperature overnight. After completion, the solvent was removed and the residue was extracted with ethyl acetate (100 mL×4) and the organic phase was washed with brine (50 mL×2) and dried with Na₂SO₄. The residue after removal of solvent was purified by silica gel chromatography (DCM/MeOH=10/1, 5/1) to obtain MFH-2-62-1 (light brown solid, 800 mg, yield 83%). LCMS (m/z): 243 [M+H]⁺.

(1r,4r)-N1,N1-dimethylcyclohexane-1,4-diamine (MFH-2-64-1)

0773To a mixture of compound MFH-2-62-1 (350 mg, 1.44 mmol) in methanol (5 mL) was added 4N HCl/dioxane (10 mL) and stirred for 3h at room temperature. The mixture was concentrated and the crude mixture was directly used in the next step. LCMS (m/z): 143 [M+H]⁺.

tert-butyl3-((8-ethyl-2-(methylthio)pyrazolo[1,5-a][1,3,5]triazin-4-ylamino)methyl)phenylcarbamate (MFH-2-55-1)

0774A stirred mixture of MFH-2-34-1 (220 mg, 0.962 mmol), tert-butyl 3-(aminomethyl) phenylcarbamate (214 mg, 0.962 mmol) and NaHCO₃(121 mg, 1.443 mmol) in acetonitrile (5 mL) was heated at 75° C. overnight and then was cooled to room temperature. The solution was filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography (PE/ethyl acetate=3:1 to 1:1) to afford MFH-2-55-1 (white solid, 210 mg, yield: 52%). LCMS (m/z): 415 [M+H]⁺.

tert-butyl3-((8-ethyl-2-(methylsulfonyl)pyrazolo[1,5-a][1,3,5]triazin-4-ylamino)methyl)phenylcarbamate (MFH-2-65-1)

0775To a solution of MFH-2-55-1 (110 mg, 0.2654 mmol) in DCM (3 mL) was added 3-chloroperbenzoic acid (137 mg, 0.7961 mmol) in one portion at room temperature and was stirred at for 1 h. The reaction mixture was quenched with a saturated Na₂S₂O₃solution (10 mL), diluted with DCM (50 mL), washed with saturated aqueous NaCl (3×50 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated to give MFH-2-65-1 (yellow solid, 118 mg, yield: 100%), which was used in next step directly. LCMS (m/z): 447 [M+H]⁺.

N4-(3-aminobenzyl)-N2-((1r,4r)-4-(dimethylamino)cyclohexyl)-8-ethylpyrazolo[1,5-a][1,3,5]triazine-2,4-diamine (MFH-2-66-1)

0776To a stirred mixture MFH-2-65-1 (118 mg, 0.2654 mmol) and MFH-2-64-1 (98 mg, 0.4554 mmol) in N-methyl-2-pyrrolidone (3 mL) was added KF (46 mg, 0.7962 mmol). This mixture was heated at 170° C. for 3 h and then was cooled and filtered. The crude mixture was purified by silica gel chromatography (MeOH/DCM=0-20%) to afford MFH-2-66-1 (yellow solid, 30 mg, yield: 28%). LCMS (m/z): 409 [M+H]⁺.

N-(3-((2-((1r,4r)-4-(dimethylamino)cyclohexylamino)-8-ethylpyrazolo[1,5-a][1,3,5]triazin-4-ylamino)methyl)phenyl)acrylamide (MFH-2-67-1)

0777To a solution of MFH-2-66-1 (30 mg, 0.07343 mmol) in CH₃CN (2 mL) were added acryloyl chloride (9 mg, 0.1 mmol) and DIPEA (0.2 mL). The reaction was stirred at room temperature for 2 h. After completion, the reaction mixture was diluted with dichloromethane (10 ml), washed with water (10 mL) and a saturated sodium bicarbonate solution (10 mL×2), and dried over anhydrous sodium sulfate. The residue after removal of the solvent was purified by prep-HPLC (C18 column, MeOH/H₂O, containing 0.05% TFA) to obtain MFH-2-67-1 (off-white solid, 11.3 mg, yield 28%). HPLC: 97% (254 nm); LCMS (m/z): 463 [M+H]⁺; ¹H NMR (500 MHz, DMSO) δ 10.16 (s, 1H), 9.81 (s, 1H), 9.53 (s, 1H), 8.75 (s, 1H), 7.95 (d, J=40.1 Hz, 1H), 7.77 (s, 1H), 7.64-7.48 (m, 1H), 7.30 (s, 1H), 6.42 (dd, J=16.9, 10.1 Hz, 1H), 6.31-6.14 (m, 1H), 5.75 (d, J=11.4 Hz, 1H), 4.64 (d, J=6.1 Hz, 2H), 3.17 (d, J=8.6 Hz, 2H), 2.94-2.56 (m, 6H), 2.48 (s, 1H), 1.99 (dd, J=87.3, 31.8 Hz, 4H), 1.51 (s, 2H), 1.39-0.96 (m, 6H).

Example 23. N-(3-((2-(((1r,4r)-4-(dimethylamino)cyclohexyl)amino)-8-ethylpyrazolo[1,5-a][1,3,5]triazin-4-yl)amino)cyclohexyl)acrylamide (MFH-2-78-1)

0778<chemistry id="CHEM-US-00293" num="00293"><img file="USRE50776E_D0296.tif" /></chemistry>

0779<chemistry id="CHEM-US-00294" num="00294"><img file="USRE50776E_D0297.tif" /></chemistry>

N1-(8-ethyl-2-(methylthio)pyrazolo[1,5-a][1,3,5]triazin-4-yl)cyclohexane-1,3-diamine (MFH-2-56-1)

0780MFH-2-34-1 (220 mg, 0.96 mmol) and cyclohexane-1,3-diamine (330 mg, 2.88 mmol) were dissolved in acetonitrile (5 mL). The mixture was heated at 75° C. overnight. The solvent was then removed under reduced pressure. The crude mixture was purified by silica gel chromatography (NH₃/MeOH (1.75N)/DCM=0-20%) to obtain MFH-2-56-1 (white solid, 193 mg, yield: 65%). LCMS (m/z): 307 [M+H]⁺.

tert-butyl3-(8-ethyl-2-(methylthio)pyrazolo[1,5-a][1,3,5]triazin-4-ylamino)cyclohexylcarbamate (MFH-2-58-1)

0781To a solution of MFH-2-56-1 (193 mg, 0.63 mmol) in MeOH (6 mL) was added di-tert-butyl dicarbonate (206 mg, 0.94 mmol) and TEA (191 mg 1.89 mmol). The reaction mixture was stirred at room temperature overnight. The reaction mixture was concentrated under reduced pressure to afford the residue, which was purified by flash column chromatography (MeOH/DCM=0-20%) to afford MFH-2-58-1 (210 mg, yield: 82%). LCMS (m/z): 407 [M+H]⁺.

tert-butyl3-(8-ethyl-2-(methylsulfonyl)pyrazolo[1,5-a][1,3,5]triazin-4-ylamino)cyclohexylcarbamate (MFH-2-69-1)

0782To a solution of MFH-2-58-1 (210 mg, 0.51 mmol) in DCM (5 mL) was added 3-chloroperbenzoic acid (268 mg, 1.55 mmol). The mixture was stirred at room temperature for 1 h and then was quenched with a saturated Na₂S₂O₃solution (5 mL). The reaction mixture was extracted with DCM (50 mL) and the organic layer was washed with saturated aqueous NaCl (3×50 mL), dried over anhydrous Na₂SO₄, filtered and concentrated to give MFH-2-69-1 (226.6 mg, yield: 100%) as the crude product, which was used for next step directly. LCMS (m/z): 439 [M+H]⁺.

tert-butyl3-(2-((1r,4r)-4-(dimethylamino)cyclohexylamino)-8-ethylpyrazolo[1,5-a][1,3,5]triazin-4-ylamino)cyclohexylcarbamate (MFH-2-70-1)

0783To a stirred mixture MFH-2-69-1 (226.6 mg, 0.51 mmol) and MFH-2-64-1 (118 mg, 0.83 mmol) in N-methyl-2-pyrrolidone (3 mL) was added KF (90 mg, 1.551 mmol). This mixture was heated at 170° C. for 3 h, and then cooled and filtered. The crude mixture was then purified by silica gel chromatography (NH₃/MeOH (1.75N)/DCM=0-20%) to afford MFH-2-70-1 (120 mg, yield: 47%). LCMS (m/z): 501 [M+H]⁺.

N4-(3-aminocyclohexyl)-N2-((1r,4r)-4-(dimethylamino)cyclohexyl)-8-ethylpyrazolo[1,5-a][1,3,5]triazine-2,4-diamine (MFH-2-77-1)

0784To a mixture of compound MFH-2-70-1 (60 mg, xx mmol) in methanol (5 mL) was added 4N HCl/dioxane (5 mL) and the solution was stirred for 3h at room temperature. The mixture was concentrated under reduced pressure and the crude mixture was directly used in next step. LCMS (m/z): 401 [M+H]⁺.

N-(3-(2-((1r,4r)-4-(dimethylamino)cyclohexylamino)-8-ethylpyrazolo[1,5-a][1,3,5]triazin-4-ylamino)cyclohexyl)acrylamide (MFH-2-78-1)

0785To a solution of MFH-2-77-1 (48 mg, 0.12 mmol) in CH₃CN (2 mL) was added acryloyl chloride (14 mg, 0.156 mmol) and DIPEA (0.2 ml). The reaction mixture was stirred at room temperature for 2 h. After completion, the reaction mixture was diluted with dichloromethane (10 ml), washed with a saturated NaHCO₃solution (10 mL×2), and washed with water (10 mL). The organic layer was dried over anhydrous sodium sulfate, concentrated under reduced pressure and the residue was purified by prep-HPLC (C18 column, MeOH/H₂O, containing 0.05% TFA) to afford MFH-2-78-1 (white solid, 25.2 mg, yield: 46%). HPLC: 97% (254 nm); LCMS (m/z): 455 [M+H]⁺; ¹H NMR (500 MHz, DMSO) δ 9.51 (s, 1H), 8.15 (s, 1H), 7.92 (s, 1H), 7.74 (s, 1H), 6.20 (dd, J=16.9, 9.9 Hz, 1H), 6.15-6.03 (m, 1H), 5.58 (d, J=9.8 Hz, 1H), 3.17 (s, 3H), 2.75 (d, J=4.9 Hz, 6H), 2.49 (d, J=2.3 Hz, 1H), 2.47 (dd, J=7.5, 2.1 Hz, 1H), 2.23-1.91 (m, 5H), 1.90-1.71 (m, 3H), 1.45 (ddd, J=45.3, 44.9, 21.3 Hz, 8H), 1.15 (s, 4H).

Example 24. (S)—N-(3-(((3-ethyl-5-(2-(2-hydroxyethyl)piperidin-1-yl)pyrazolo[1,5-a]pyrimidin-7-yl)amino)methyl)phenyl)acrylamide (MFH-1-49-1)

0786<chemistry id="CHEM-US-00295" num="00295"><img file="USRE50776E_D0298.tif" /></chemistry>

0787<chemistry id="CHEM-US-00296" num="00296"><img file="USRE50776E_D0299.tif" /></chemistry>

tert-butyl-3-((5-chloro-3-ethylpyrazolo[1,5-a]pyrimidin-7-ylamino)methyl)phenylcarbamate (MFH-1-39-1)

0788The mixture of SM-1-49-1 (400 mg, 1.85 mmol), SM-1-49-2 (452.6 mg, 2.0364 mmol), DIPEA (718 mg) and i-PrOH (5 mL) was stirred at 85° C. for 6 h. Then the reaction mixture was concentrated under reduced pressure and the residue was purified by silica gel chromatography (PE/EA=0-50%) to obtain MFH-1-39-1 (white solid, 0.66 g, yield 88.7%). LCMS (m/z): 402 [M+H]⁺.

(S)-2-(1-(7-(3-aminobenzylamino)-3-ethylpyrazolo[1,5-a]pyrimidin-5-yl)piperidin-2-yl)ethanol (MFH-1-43-1)

0789The mixture of MFH-1-39-1 (660 mg, 1.6422 mmol), SM-1-49-3 (318 mg, 2.4633 mmol), KF (429 mg, 7.4 mmol) and NMP (2 mL) was stirred at 170° C. for 10 h. After completion, the residue was extracted with chloroform and isopropanol (4:1) and the organic phase was washed with brine (50 mL×2) and dried with Na₂SO₄. The solution was filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography (MeOH/DCM=0-20%) to obtain MFH-1-43-1 (396.3 mg, yield 61%). LCMS (m/z): 395 [M+H]⁺.

(S)—N-(3-(((3-ethyl-5-(2-(2-hydroxyethyl)piperidin-1-yl)pyrazolo[1,5-a]pyrimidin-7-ylamino)methyl)phenyl)acrylamide (MFH-1-49-1)

0790To a solution of MFH-1-43-1 (60 mg, 0.15 mmol) and DIPEA (0.2 mL) in CH₃CN (2 mL) was added acryloyl chloride (18 mg, 0.20 mmol) in DCM (0.5 mL) dropwise. The mixture was then stirred at 0° C. for 1 h. After reaction completion, the reaction was concentrated to remove the solvent and the residue was purified by prep-HPLC (C18 column, MeOH/H₂O, containing 0.05% TFA) to obtain MFH-1-49-1 (off-white solid, 8.6 mg, yield 12.6%). HPLC: 96% (254 nm); LCMS (m/z): 449 [M+H]⁺; ¹H NMR (500 MHz, DMSO) δ 10.14 (s, 1H), 8.94 (s, 1H), 7.91 (s, 1H), 7.72 (s, 1H), 7.53 (d, J=8.1 Hz, 1H), 7.30 (t, J=7.8 Hz, 1H), 7.13 (d, J=7.6 Hz, 1H), 6.41 (dd, J=17.0, 10.1 Hz, 1H), 6.22 (dd, J=17.0, 1.7 Hz, 1H), 5.73 (dd, J=10.1, 1.7 Hz, 1H), 5.66 (s, 1H), 4.62 (d, J=6.3 Hz, 2H), 4.41 (s, 1H), 3.96 (d, J=11.7 Hz, 2H), 3.06 (t, J=12.6 Hz, 1H), 2.60-2.51 (m, 2H), 1.96 (d, J=5.0 Hz, 1H), 1.73-1.58 (m, 5H), 1.53 (s, 1H), 1.43 (d, J=11.6 Hz, 2H), 1.12 (t, J=7.5 Hz, 3H), 1.08 (s, 1H).

Example 25 (R)—N-(3-((3-ethyl-5-(2-(2-hydroxyethyl)piperidin-1-yl)pyrazolo[1,5-a]pyrimidin-7-ylamino)methyl)phenyl)acrylamide (MFH-1-56-1)

0791<chemistry id="CHEM-US-00297" num="00297"><img file="USRE50776E_D0300.tif" /></chemistry>

0792<chemistry id="CHEM-US-00298" num="00298"><img file="USRE50776E_D0301.tif" /></chemistry>

(R)-2-(1-(7-(3-aminobenzylamino)-3-ethylpyrazolo[1,5-a]pyrimidin-5-yl)piperidin-2-yl)ethanol (MFH-1-47-1)

0793The mixture of MFH-1-39-1 (330 mg, 0.8211 mmol), SM-1-56-1 (248.6 mg, 1.3884 mmol), KF (242 mg, 4.1652 mmol) and NMP (1 mL) was stirred at 170° C. for 10 h. After completion, the residue was extracted with chloroform and 2-propanol (4:1). The organic phase was washed with brine (50 mL×2) and dried with Na₂SO₄. The solvent was removed and the residue was purified by silica gel chromatography (MeOH/DCM=0-20%) to obtain MFH-1-56-1 (259 mg, yield 80%). LCMS (m/z): 395 [M+H]⁺.

(R)—N-(3-((3-ethyl-5-(2-(2-hydroxyethyl)piperidin-1-yl)pyrazolo[1,5-a]pyrimidin-7-ylamino)methyl)phenyl)acrylamide (MFH-1-56-1)

0794To a solution of MFH-1-47-1 (60 mg, 0.152 mmol) and DIPEA (0.2 mL) in CH₃CN (2 mL) was added acryloyl chloride (18 mg, 0.198 mmol) in DCM (0.5 mL) dropwise. The mixture was then stirred at 0° C. for 1 h. After completion, the solution was concentrated and the residue was purified by prep-HPLC (C18 column, MeOH/H₂O, containing 0.05% TFA) to obtain MFH-1-56-1 (off-white solid, 10 mg, yield 14.6%). HPLC: 98% (254 nm); LCMS (m/z): 449 [M+H]⁺; ¹H NMR (500 MHz, DMSO) δ 10.16 (s, 1H), 9.04 (s, 1H), 7.93 (s, 1H), 7.76 (s, 1H), 7.54 (d, J=8.1 Hz, 1H), 7.31 (t, J=7.8 Hz, 1H), 7.14 (d, J=7.6 Hz, 1H), 6.42 (dd, J=17.0, 10.1 Hz, 1H), 6.24 (dd, J=17.0, 1.7 Hz, 1H), 5.75 (dd, J=10.1, 1.7 Hz, 1H), 5.68 (s, 1H), 4.65 (d, J=6.3 Hz, 2H), 4.42 (s, 1H), 3.98 (d, J=11.7 Hz, 2H), 3.09 (t, J=12.6 Hz, 1H), 2.61-2.54 (m, 2H), 1.96 (d, J=5.0 Hz, 1H), 1.76-1.60 (m, 5H), 1.58 (s, 1H), 1.44 (d, J=11.6 Hz, 2H), 1.19 (t, J=7.5 Hz, 3H), 1.10 (s, 1H).

Example 26 (R)—N-(3-(3-ethyl-5-(2-(2-hydroxyethyl)piperidin-1-yl)pyrazolo[1,5-a]pyrimidin-7-ylamino)phenyl)acrylamide (MFH-1-143-1)

0795<chemistry id="CHEM-US-00299" num="00299"><img file="USRE50776E_D0302.tif" /></chemistry>

0796<chemistry id="CHEM-US-00300" num="00300"><img file="USRE50776E_D0303.tif" /></chemistry>

(R)-2-(1-(7-(3-aminophenylamino)-3-ethylpyrazolo[1,5-a]pyrimidin-5-yl)piperidin-2-yl)ethanol (MFH-1-121-1)

0797The mixture of SM-1-141-1 (250 mg, 0.869 mmol), SM-1-56-3 (168 mg, 1.3 mmol), KF (227 mg, 3.9 mmol) and NMP (2 mL) was stirred at 170° C. for 10 h. After completion, the residue was extracted with chloroform and 2-propanol (4:1). The organic phase was washed with brine (50 mL×2) and dried with Na₂SO₄. The solvent was removed and the residue was purified by silica gel chromatography (MeOH/DCM=0-20%) to obtain MFH-1-43-1(220 mg, yield 66.5%). LCMS (m/z): 381 [M+H]⁺.

(R)—N-(3-(3-ethyl-5-(2-(2-hydroxyethyl)piperidin-1-yl)pyrazolo[1,5-a]pyrimidin-7-ylamino)phenyl)acrylamide (MFH-1-143-1)

0798To a solution of MFH-1-121-1 (50 mg, 0.13141 mmol) and DIPEA (0.2 mL) in CH₃CN (2 mL) was added acryloyl chloride (15 mg, 0.17 mmol) in DCM (0.5 mL) dropwise. The mixture was stirred at 0° C. for 1 h. After completion, the solvent was removed and the residue was purified by prep-HPLC (C18 column, MeOH/H₂O, containing 0.05% TFA) to obtain MFH-1-143-1 (off-white solid, 18 mg, yield 31.5%). HPLC: 96% (254 nm); LCMS (m/z): 435 [M+H]⁺; ¹H NMR (500 MHz, DMSO) δ 10.29 (s, 1H), 9.89 (s, 1H), 7.92 (d, J=11.7 Hz, 2H), 7.43-7.34 (m, 2H), 7.24-7.15 (m, 1H), 6.46 (dd, J=17.0, 10.1 Hz, 1H), 6.28 (dd, J=17.0, 1.9 Hz, 1H), 5.94 (s, 1H), 5.79 (dd, J=10.1, 1.9 Hz, 1H), 4.51 (s, 1H), 4.07 (s, 1H), 3.48-3.40 (m, 2H), 3.35-3.31 (m, 1H), 3.02 (t, J=12.8 Hz, 1H), 2.63-2.55 (m, 2H), 1.92 (td, J=13.6, 5.2 Hz, 1H), 1.76-1.60 (m, 5H), 1.58 (s, 1H), 1.44 (d, J=11.9 Hz, 1H), 1.28-1.08 (m, 4H).

Example 27 N-(3-(3-ethyl-5-((S)-2-(2-hydroxyethyl)piperidin-1-yl)pyrazolo[1,5-a]pyrimidin-7-ylamino)cyclohexyl)acrylamide (MFH-1-167-1)

0799<chemistry id="CHEM-US-00301" num="00301"><img file="USRE50776E_D0304.tif" /></chemistry>

0800<chemistry id="CHEM-US-00302" num="00302"><img file="USRE50776E_D0305.tif" /></chemistry>

N1-(5-chloro-3-ethylpyrazolo[1,5-a]pyrimidin-7-yl)cyclohexane-1,3-diamine (MFH-1-151-1)

0801The mixture of SM-1-49-1 (300 mg, 1.388 mmol), SM-1-167-1 (500 mg, 4.442 mmol), DIPEA (270 mg) and i-PrOH (5 mL) was stirred at 85° C. for 2 h. After completion, the reaction was concentrated to remove the solvent and the residue was purified by silica gel (NH₃/MeOH (1.75N)/DCM=0-20%) to obtain MFH-1-155-1 (230 g, yield 56%). LCMS (m/z): 294 [M+H]⁺.

2-((S)-1-(7-(3-aminocyclohexylamino)-3-ethylpyrazolo[1,5-a]pyrimidin-5-yl)piperidin-2-yl)ethanol (MFH-1-157-1)

0802The mixture of MFH-1-155-1 (230 mg, 0.783 mmol), SM-1-49-3 (152 mg, 1.1745 mmol), KF (205 mg, 3.5235 mmol) and NMP (2 mL) was stirred at 170° C. for 10 h. After completion, the residue was extracted with chloroform and 2-propanol (4:1), the organic phase was washed with brine (50 mL×2), and dried with Na₂SO₄. The solution was filtered, concentrated to remove the solvent, and the residue was purified by silica gel chromatography (NH₃/MeOH (1.75N)/DCM=0-20%) to obtain MFH-1-157-1(100 mg, yield 33%). LCMS (m/z): 387 [M+H]⁺.

N-(3-(3-ethyl-5-((S)-2-(2-hydroxyethyl)piperidin-1-yl)pyrazolo[1,5-a]pyrimidin-7-ylamino)cyclohexyl)acrylamide (MFH-1-167-1)

0803To a solution of MFH-1-157-1 (30 mg, 0.07762 mmol) and DIPEA (0.2 mL) in CH₃CN (2 mL) was added acrylyl chloride (9 mg, 0.101 mmol) in DCM (0.5 mL) dropwise. The mixture was stirred at 0° C. for 1 h. After completion, the reaction was concentrated to remove the solvent and the residue was purified by prep-HPLC (C18 column, MeOH/H₂O, containing 0.05% TFA) to obtain MFH-1-167-1 (off-white solid, 13 mg, yield 38%). HPLC: 96% (254 nm); LCMS (m/z): 441 [M+H]⁺; ¹H NMR (500 MHz, DMSO) δ 8.26 (s, 1H), 8.14 (dd, J=18.8, 7.9 Hz, 1H), 8.05 (d, J=7.3 Hz, 1H), 7.97-7.83 (m, 2H), 6.41-6.29 (m, 1H), 6.21 (ddd, J=17.1, 10.1, 2.0 Hz, 1H), 6.09 (dddd, J=12.3, 10.2, 3.9, 2.2 Hz, 2H), 5.83 (d, J=9.4 Hz, 1H), 5.69-5.51 (m, 3H), 4.49 (s, 2H), 4.12 (d, J=4.8 Hz, 2H), 3.88-3.81 (m, 2H), 3.11 (dd, J=22.2, 12.4 Hz, 2H), 2.60-2.56 (m, 2H), 2.01-1.96 (m, 2H), 1.69 (d, J=12.3 Hz, 6H), 1.54 (s, 2H), 1.48 (d, J=11.4 Hz, 2H), 1.10 (dd, J=24.9, 12.8 Hz, 1H).

Example 28 (S)—N-(3-(8-ethyl-2-(2-(2-hydroxyethyl)piperidin-1-yl)pyrazolo[1,5-a][1,3,5]triazin-4-ylamino)phenyl)acrylamide (MFH-2-53-1)

0804<chemistry id="CHEM-US-00303" num="00303"><img file="USRE50776E_D0306.tif" /></chemistry>

0805<chemistry id="CHEM-US-00304" num="00304"><img file="USRE50776E_D0307.tif" /></chemistry>

ethyl N-[(4-ethyl-1H-pyrazol-5-yl)carbamothioyl]carbamate (MFH-1-113-1)

0806To a solution of SM-2-53-1 (2.8 g, 25.2 mmol) in ethanol (50.0 mL) was added ethoxycarbonyl isothiocyanate (3.3 g, 25.2 mL) in one portion at room temperature. The mixture was stirred at 80° C. overnight. The reaction mixture was concentrated under reduced pressure to afford the residue, which was purified by flash column chromatography (PE/ethyl acetate=3:1) to afford MFH-1-113-1 (white solid, 4 g, yield: 65%). LCMS (m/z): 243 [M+H]⁺.

8-ethyl-2-thioxo-2,3-dihydropyrazolo[1,5-a][1,3,5]triazin-4(1H)-one (MFH-1-135-1)

0807To a solution of MFH-1-113-1 (3.9 g, 16.1 mmol) in acetonitrile (40 mL) was added K₂CO₃(6.67 g, 48.3 mmol) in one portion at room temperature. The mixture was heated at 85° C. overnight, cooled, and then acidified with AcOH. The resulting solid was filtered off to afford MFH-1-135-1 (yellow solid, 2.1 g, yield: 66%). LCMS (m/z): 197 [M+H]⁺.

8-ethyl-2-(methylthio)pyrazolo[1,5-a][1,3,5]triazin-4(3H)-one (MFH-1-149-1)

0808To a stirred mixture of MFH-1-135-1 (2.1 g, 10.7 mmol) and NaOH (0.86 g, 21.4 mmol) in dioxane/H₂O (30/8 mL) was added iodomethane (1.52 g, 10.7 mol). This mixture was stirred at room temperature for 1 h, acidified with hydrochloric acid, concentrated to remove the solvent, and the residue was purified by silica gel chromatography (DCM/MeOH=30:1) to afford MFH-1-149-1 (white solid, 2.0 g, yield: 89%). LCMS (m/z): 211 [M+H]⁺.

4-chloro-8-ethyl-2-(methylthio)pyrazolo[1,5-a][1,3,5]triazine (MFH-2-34-1)

0809To a stirred mixture MFH-1-149-1 (2.0 g, 9.5 mmol) and N,N-dimethyl aniline (2.3 g, 19.0 mmol) in acetonitrile (10 mL) under argon was added POCl₃(20 ml, 219 mol). This mixture was heated at 85° C. overnight. Then the reaction mixture was concentrated under reduced pressure to afford the residue MFH-2-34-1 (white solid, 2.2 g, yield: 100% used next step directly). LCMS (m/z): 229 [M+H]⁺.

tert-butyl 3-(8-ethyl-2-(methylthio)pyrazolo[1,5-a][1,3,5]triazin-4-ylamino)phenylcarbamate (MFH-2-45-1)

0810A stirred mixture MFH-2-34-1 (2.2 g, 9.5 mmol), tert-butyl 3-aminophenylcarbamate (2.37 g, 11.4 mmol) and NaHCO₃(130.4 mg, 0.95 mmol) in acetonitrile (25 mL) was heated at 75° C. overnight and then cooled to room temperature. The solvent was removed and the residue was purified by silica gel chromatography (PE/ethyl acetate=3/1 to 1/1) to afford MFH-2-45-1 (white solid, 2.52 g, yield: 67%). LCMS (m/z): 401 [M+H]⁺.

tert-butyl 3-(8-ethyl-2-(methylsulfonyl)pyrazolo[1,5-a][1,3,5]triazin-4-ylamino) phenylcarbamate (MFH-2-50-1)

0811To a solution of MFH-2-45-1 (600 mg, 1.5 mmol) in DCM (15 mL) was added 3-chloroperbenzoic acid (776 mg, 4.5 mmol) in one portion at room temperature and stirred for 1 h. The reaction mixture was quenched with a saturated Na₂S₂O₃solution (10 mL), diluted with DCM (50 mL), washed with saturated aqueous NaCl (3×50 mL), dried over anhydrous Na₂SO₄, filtered and concentrated to give MFH-2-50-1 (yellow solid, 620 mg, yield: 96%), which was used in the next step directly. LCMS (m/z): 433 [M+H]⁺.

(S)-2-(1-(4-(3-aminophenylamino)-8-ethylpyrazolo[1,5-a][1,3,5]triazin-2-yl)piperidin-2-yl)ethanol (MFH-2-51-1)

0812To a stirred mixture of MFH-2-50-1 (360 mg, 0.84 mmol) and (S)-2-(piperidin-2-yl)ethanol (215 mg, 1.67 mmol) in N-methyl-2-pyrrolidone (3 mL) was added KF (146 mg, 2.51 mmol). This mixture was heated at 170° C. for 3 h, cooled, and filtered. The crude mixture was purified by silica gel chromatography (MeOH/DCM=0-20%) to afford MFH-2-51-1 (yellow solid, 150 mg, yield: 47%). LCMS (m/z): 382 [M+H]⁺.

(S)—N-(3-(8-ethyl-2-(2-(2-hydroxyethyl)piperidin-1-yl)pyrazolo[1,5-a][1,3,5]triazin-4-ylamino)phenyl)acrylamide (MFH-2-53-1)

0813To a solution of MFH-2-51-1 (70 mg, 0.18 mmol) in THF (2 mL) were added acryloyl chloride (18 mg, 0.20 mmol) and triethylamine (54 mg, 0.54 mmol). The reaction was stirred at room temperature for 2 h. After completion, the reaction mixture was diluted with dichloromethane (10 ml), washed with water (10 mL) and saturated sodium bicarbonate solution (10 mL×2), dried over anhydrous sodium sulfate, concentrated and the crude mixture was purified by prep-HPLC (C18 column, MeOH/H₂O, containing 0.05% TFA) to afford MFH-2-53-1 (white solid, 13 mg, yield: 16%). HPLC: 97% (254 nm); LCMS (m/z): 436 [M+H]⁺; ¹H NMR (DMSO-d₆, 500 MHz): δ: 10.17 (s, 1H), 10.07 (s, 1H), 8.34 (s, ¹H), 7.84 (s, 1H), 7.51 (s, 1H), 7.30-7.34 (m, 2H), 6.46 (dd, J=17, 10.0 Hz, 1H), 6.28 (d, J=17 Hz, 1H), 5.77 (d, J=11.5 Hz, 1H), 4.98 (s, 1H), 4.65 (d, J=13 Hz, 1H), 4.56 (s, 1H), 2.88 (t, J=12.5 Hz, 1H), 2.47-2.50 (m, 2H), 1.56-1.88 (m, 8H), 1.36-1.38 (m, 1H), 1.19-1.23 (m, 4H).

Example 29 N-(3-(5-(3-aminopiperidin-1-yl)-3-ethylpyrazolo[1,5-a]pyrimidin-7-ylamino)phenyl)acrylamide (MFH-3-140-1)

0814<chemistry id="CHEM-US-00305" num="00305"><img file="USRE50776E_D0308.tif" /></chemistry>

0815<chemistry id="CHEM-US-00306" num="00306"><img file="USRE50776E_D0309.tif" /></chemistry>

tert-butyl1-(7-(3-aminophenylamino)-3-ethylpyrazolo[1,5-a]pyrimidin-5-yl)piperidin-3-ylcarbamate (MFH-3-130-1)

0816To a stirred mixture SM-1-141-1 (150 mg, 0.5213 mmol) and SM-3-140-1 (157 mg, 0.782 mmol) in N-methyl-2-pyrrolidone (1 mL) was added KF (136 mg, 2.3458 mmol). After heating at 150° C. for 10 h, the reaction mixture was cooled and filtered. After removal of the solvent, the crude mixture was purified by silica gel chromatography (MeOH/DCM=0-20%) to afford MFH-3-130-1 (110 mg, yield: 47%). LCMS (m/z): 452 [M+H]⁺.

tert-butyl1-(7-(3-acrylamidophenylamino)-3-ethylpyrazolo[1,5-a]pyrimidin-5-yl)piperidin-3-ylcarbamate (MFH-3-139-1)

0817To a solution of MFH-3-130-1 (110 mg, 0.24 mmol) in CH₃CN (2 mL) was added acryloyl chloride (28 mg, 0.3167 mmol) and DIPEA (0.2 ml). The reaction was stirred at room temperature for 2 h and then was diluted with dichloromethane (10 ml). The solution was washed with saturated NaHCO₃(10 mL×2) and water (10 mL). The organic layer was dried over anhydrous sodium sulfate and then concentrated under reduced pressure. The residue was purified by prep-HPLC (C18 column, MeOH/H₂O, containing 0.05% TFA) to afford MFH-3-139-1 (56.6 mg, yield: 46%). LCMS (m/z): 506 [M+H]⁺.

N-(3-(5-(3-aminopiperidin-1-yl)-3-ethylpyrazolo[1,5-a]pyrimidin-7-ylamino)phenyl)acrylamide (MFH-3-140-1)

0818To a mixture of compound MFH-3-139-1 (56.6 mg) in DCM (5 mL) was added TFA (2 mL). The reaction mixture was stirred for 1 h at room temperature. The mixture was concentrated under reduced pressure and the residue was purified by prep-HPLC (C18 column, MeOH/H₂O, containing 0.05% TFA) to afford MFH-3-140-1 (20 mg, yield: 44%). LCMS (m/z): 406 [M+H]⁺. ¹H NMR (500 MHz, DMSO) δ 10.32 (s, 1H), 9.48 (s, 1H), 7.97 (d, J=8.0 Hz, 1H), 7.94 (s, 2H), 7.84 (s, 1H), 7.38 (t, J=8.0 Hz, 1H), 7.32 (d, J=8.2 Hz, 1H), 7.17 (d, J=8.6 Hz, 1H), 6.46 (dd, J=17.0, 10.2 Hz, 1H), 6.28 (dd, J=17.0, 1.8 Hz, 1H), 6.03 (s, 1H), 5.80 (dd, J=10.1, 1.8 Hz, 1H), 4.30 (s, 1H), 3.85 (s, 1H), 3.22 (s, 1H), 3.13 (dd, J=12.6, 9.2 Hz, 2H), 2.66-2.57 (m, 2H), 1.98 (s, 1H), 1.81-1.74 (m, 1H), 1.56 (dd, J=16.8, 9.0 Hz, 2H), 1.25 (t, J=7.5 Hz, 3H).

Example 30 (S)—N-(3-(3-ethyl-5-(2-(2-hydroxyethyl)piperidin-1-yl)pyrazolo[1,5-a]pyrimidin-7-ylamino)phenyl)acrylamide (MFH-1-141-1)

0819<chemistry id="CHEM-US-00307" num="00307"><img file="USRE50776E_D0310.tif" /></chemistry>

0820<chemistry id="CHEM-US-00308" num="00308"><img file="USRE50776E_D0311.tif" /></chemistry>

(S)-2-(1-(7-(3-aminophenylamino)-3-ethylpyrazolo[1,5-a]pyrimidin-5-yl)piperidin-2-yl)ethanol (MFH-1-119-1)

0821The mixture of SM-1-141-1 (250 mg, 0.87 mmol), SM-1-49-3 (168 mg, 1.3 mmol), KF (227 mg, 3.9 mmol) and NMP (2 mL) was stirred at 170° C. for 10 h. After completion, the residue was extracted with chloroform and isopropanol (4:1). The organic phase was washed with brine (50 mL×2) and dried with Na₂SO₄. The mixture was filtered, concentrated to remove the solvent under reduced pressure, and purified by silica gel chromatography (MeOH/DCM=0-20%) to obtain MFH-1-43-1(250 mg, yield 75.6%). LCMS (m/z): 381 [M+H]⁺.

(S)—N-(3-(3-ethyl-5-(2-(2-hydroxyethyl)piperidin-1-yl)pyrazolo[1,5-a]pyrimidin-7-ylamino)phenyl)acrylamide (MFH-1-141-1)

0822To a solution of MFH-1-119-1 (50 mg, 0.13 mmol) and DIPEA (0.2 mL) in CH₃CN (2 mL) was added acryloyl chloride (15 mg, 0.17 mmol) in DCM (0.5 mL) dropwise. The mixture was stirred at 0° C. for 1 h. After completion, the reaction was concentrated under reduced pressure, and the residue was purified by prep-HPLC (C18 column, MeOH/H₂O, containing 0.05% TFA) to obtain MFH-1-141-1 (off-white solid, 33.7 mg, yield 59%). HPLC: 96% (254 nm); LCMS (m/z): 435 [M+H]⁺.

0000Biological Assays of the Compounds

0000Pulldown Assay

0823Jurkat cells were treated with DMSO, 1 uM or 200 nM of compound E9, E17, or dinaciclib (<figref idref="DRAWINGS">FIG. <b>2</b></figref>). 6 hours after treatment, cells were washed and harvested by resuspending in lysis buffer (50 mM Hepes pH 7.4, 150 mM NaCl, 1% NP-40, 5 mM EDTA, protease and phosphatase inhibitors) and lysing on ice 30 minutes. Lysates were cleared by centrifugation at 15,000 rpm 30 minutes. Biotin-labeled THZ1 was added to 1 uM to lysates and rotated at 4° C. overnight. Streptavidin-agarose beads were washed and 30 uL slurry was added to each lysate and rotated for 1 hour at 4° C. Beads were washed 5 times with lysis buffer and 50 uL 2×LDS buffer was added to each sample. Samples were boiled and equal volume of protein was loaded onto gel. Gel was transferred to nitrocellulose and blotted for Cyclin K and Cyclin H.

0824We conclude that pre-treatment of cells with compound E9, E17 or dinaciclib, but not DMSO, blocks biotin-THZ1 from being able to bind to CDK12, which blocks the pulldown of Cyclin K. This indicates that compound E9 is able to engage CDK12 in cells at 1 uM and 200 nM and block binding of bio-THZ1. We do not see a similar loss of pulldown of Cyclin H, indicating that E9 and E17 are not able to bind to CDK7 and block its association with bio-THZ1.

0000Jurkat Proliferation Assay

0825Jurkat cells were seeded at a density of 25,000 cells/well in 96-well plates. Cells were then treated with the indicated compounds in a 10-pt dose escalation format from 1 nM to 10 μM or DMSO control for 72 hrs. After 72 hrs, cells were assayed using CellTiter-Glo Luminescent Cell Viability Assay (Promega) to determine cell viability by measuring the amount of ATP present in each sample cell population, which is an indicator of cell metabolic activity. Results are graphed as fraction of the DMSO control at 72 hrs. All data points were performed in biological triplicate. Error bars are +/−SD.

0826These results suggest that dinaciclib has the most anti-proliferative effect on Jurkat cells with an IC50 below 10 nM. However, the covalent inhibitors E9, E17, and E18 all display potent activity with IC50s in the sub-100 nM range.

0000Growth Assay

0827Jurkat cells were treated with 1 uM of compound E9, E17, Dinaciclib or DMSO control. 4 hours after treatment, cells were either not treated or washed three times with RPMI media to remove all compound. Cells were replated and allowed to grow for 68 hours. Cells were assayed using celltiter glo (Promega) to determine cell viability by measuring the amount of ATP present, which is an indicator of cell metabolic activity. Results are graphed as luminescent values (<figref idref="DRAWINGS">FIG. <b>1</b></figref>).

0828We can conclude that the covalent nature of the compounds E9 and E11 allow for a long term effect in cells, even after compound is removed. Conversely, dinaciclib has a strong effect on cell viability when it is maintained in culture, however, once it is removed, cells are able to reinitiate growth. Taken together, this indicates that the covalent nature of E9 and E11 has the advantage of short dosing periods translating into longer term effects.

0000Washout Expression Assay

0829Jurkat cells were treated with 1 uM of compound E9, E17, Dinaciclib or DMSO control (<figref idref="DRAWINGS">FIG. <b>3</b></figref>). 4 hours after treatment, cells were either not treated or washed three times with RPMI media to remove all compound. Cells were replated and allowed to grow for 6 hours. Cells were harvested, washed 3 times with PBS and resuspended in RIPA buffer (25 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS). Equal amount of protein was loaded onto gel and blotted for Pol II Ser2 (Millipore 04-1571), total pol II (Santa Cruz 17798), Tubulin (Cell Signaling 11H10) or MCL1 (Cell Signaling 4572S)

0830We can see that when we treat with any of the compounds that Ser2 phosphorylation and MCL1 levels are decreased. However, when we washout the compounds, only our covalent compounds are able to have an effect after the washout whereas dinaciclib is unable to continue this effect.

0831<tables id="TABLE-US-00004" num="00004"><table frame="none" colsep="0" rowsep="0" pgwide="1"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="350pt" align="center" /><thead><row><entry namest="1" nameend="1" rowsep="1">TABLE A1</entry></row></thead><tbody valign="top"><row><entry namest="1" nameend="1" align="center" rowsep="1" /></row><row><entry>Biological evaluation of the exemplified compounds (IC50 values/nM)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="6"><colspec colname="1" colwidth="42pt" align="left" /><colspec colname="2" colwidth="154pt" align="center" /><colspec colname="3" colwidth="42pt" align="center" /><colspec colname="4" colwidth="42pt" align="center" /><colspec colname="5" colwidth="28pt" align="center" /><colspec colname="6" colwidth="42pt" align="center" /><tbody valign="top"><row><entry>Compound</entry><entry>Structure</entry><entry>CDK7/CycH</entry><entry>CDK9/CycT</entry><entry>Jurkat</entry><entry>CDK2/CycA</entry></row><row><entry namest="1" nameend="6" align="center" rowsep="1" /></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="6"><colspec colname="1" colwidth="42pt" align="left" /><colspec colname="2" colwidth="154pt" align="center" /><colspec colname="3" colwidth="42pt" align="char" char="." /><colspec colname="4" colwidth="42pt" align="char" char="." /><colspec colname="5" colwidth="28pt" align="center" /><colspec colname="6" colwidth="42pt" align="char" char="." /><tbody valign="top"><row><entry>SB1-E-24</entry><entry><chemistry id="CHEM-US-00309" num="00309"><img file="USRE50776E_D0312.tif" /></chemistry></entry><entry>774</entry><entry>140</entry><entry>—</entry><entry>1260</entry></row><row><entry></entry></row><row><entry>SB1-E-19</entry><entry><chemistry id="CHEM-US-00310" num="00310"><img file="USRE50776E_D0313.tif" /></chemistry></entry><entry>383</entry><entry>123</entry><entry>—</entry><entry>295</entry></row><row><entry></entry></row><row><entry>THZ-4-124-1</entry><entry><chemistry id="CHEM-US-00311" num="00311"><img file="USRE50776E_D0314.tif" /></chemistry></entry><entry>122</entry><entry>373</entry><entry>117</entry><entry>976</entry></row><row><entry></entry></row><row><entry>SB1-E-22</entry><entry><chemistry id="CHEM-US-00312" num="00312"><img file="USRE50776E_D0315.tif" /></chemistry></entry><entry>246</entry><entry>88.9</entry><entry>—</entry><entry>28300</entry></row><row><entry></entry></row><row><entry>SB1-E-25</entry><entry><chemistry id="CHEM-US-00313" num="00313"><img file="USRE50776E_D0316.tif" /></chemistry></entry><entry>410</entry><entry>387</entry><entry>—</entry><entry>33400</entry></row><row><entry></entry></row><row><entry>THZ-5-38-1</entry><entry><chemistry id="CHEM-US-00314" num="00314"><img file="USRE50776E_D0317.tif" /></chemistry></entry><entry>25.9</entry><entry>220</entry><entry>—</entry><entry>563</entry></row><row><entry></entry></row><row><entry>THZ-3-49-1</entry><entry><chemistry id="CHEM-US-00315" num="00315"><img file="USRE50776E_D0318.tif" /></chemistry></entry><entry>9.43</entry><entry>445</entry><entry>4029</entry><entry>—</entry></row><row><entry></entry></row><row><entry>THZ-4-119-1</entry><entry><chemistry id="CHEM-US-00316" num="00316"><img file="USRE50776E_D0319.tif" /></chemistry></entry><entry>11.5</entry><entry>519</entry><entry>1960</entry><entry>—</entry></row><row><entry></entry></row><row><entry>THZ-4-128-1</entry><entry><chemistry id="CHEM-US-00317" num="00317"><img file="USRE50776E_D0320.tif" /></chemistry></entry><entry>46.8</entry><entry>73.4</entry><entry>1037</entry><entry>17100</entry></row><row><entry></entry></row><row><entry>THZ-4-141-1</entry><entry><chemistry id="CHEM-US-00318" num="00318"><img file="USRE50776E_D0321.tif" /></chemistry></entry><entry>3600</entry><entry>3570</entry><entry>—</entry><entry>>1000</entry></row><row><entry></entry></row><row><entry>THZ-4-148-1</entry><entry><chemistry id="CHEM-US-00319" num="00319"><img file="USRE50776E_D0322.tif" /></chemistry></entry><entry>>10000</entry><entry>1710</entry><entry>—</entry><entry>7050</entry></row><row><entry></entry></row><row><entry>THZ-4-149-1</entry><entry><chemistry id="CHEM-US-00320" num="00320"><img file="USRE50776E_D0323.tif" /></chemistry></entry><entry>4430</entry><entry>2290</entry><entry>—</entry><entry>>1000</entry></row><row><entry></entry></row><row><entry>X1</entry><entry><chemistry id="CHEM-US-00321" num="00321"><img file="USRE50776E_D0324.tif" /></chemistry></entry><entry>—</entry><entry>—</entry><entry>—</entry><entry>—</entry></row><row><entry></entry></row><row><entry>X2</entry><entry><chemistry id="CHEM-US-00322" num="00322"><img file="USRE50776E_D0325.tif" /></chemistry></entry><entry>—</entry><entry>—</entry><entry>—</entry><entry>—</entry></row><row><entry></entry></row><row><entry>X3</entry><entry><chemistry id="CHEM-US-00323" num="00323"><img file="USRE50776E_D0326.tif" /></chemistry></entry><entry>—</entry><entry>—</entry><entry>—</entry><entry>—</entry></row><row><entry></entry></row><row><entry>X4</entry><entry><chemistry id="CHEM-US-00324" num="00324"><img file="USRE50776E_D0327.tif" /></chemistry></entry><entry>—</entry><entry>—</entry><entry>—</entry><entry>—</entry></row><row><entry namest="1" nameend="6" align="center" rowsep="1" /></row></tbody></tgroup></table></tables>

0832<tables id="TABLE-US-00005" num="00005"><table frame="none" colsep="0" rowsep="0" pgwide="1"><tgroup align="left" colsep="0" rowsep="0" cols="1"><colspec colname="1" colwidth="357pt" align="center" /><thead><row><entry namest="1" nameend="1" rowsep="1">TABLE A2</entry></row></thead><tbody valign="top"><row><entry namest="1" nameend="1" align="center" rowsep="1" /></row><row><entry>Biological evaluation of additional exemplified compounds (IC50 values/nM)</entry></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="6"><colspec colname="1" colwidth="49pt" align="left" /><colspec colname="2" colwidth="154pt" align="center" /><colspec colname="3" colwidth="42pt" align="center" /><colspec colname="4" colwidth="42pt" align="center" /><colspec colname="5" colwidth="28pt" align="center" /><colspec colname="6" colwidth="42pt" align="center" /><tbody valign="top"><row><entry>Compound</entry><entry>Structure</entry><entry>CDK7/CycH</entry><entry>CDK9/CycT</entry><entry>Jurkat</entry><entry>CDK2/CycA</entry></row><row><entry namest="1" nameend="6" align="center" rowsep="1" /></row></tbody></tgroup><tgroup align="left" colsep="0" rowsep="0" cols="6"><colspec colname="1" colwidth="49pt" align="left" /><colspec colname="2" colwidth="154pt" align="center" /><colspec colname="3" colwidth="42pt" align="char" char="." /><colspec colname="4" colwidth="42pt" align="char" char="." /><colspec colname="5" colwidth="28pt" align="char" char="." /><colspec colname="6" colwidth="42pt" align="char" char="." /><tbody valign="top"><row><entry>THZ-4-134-1</entry><entry><chemistry id="CHEM-US-00325" num="00325"><img file="USRE50776E_D0328.tif" /></chemistry></entry><entry>1180</entry><entry>379</entry><entry>522</entry><entry>163</entry></row><row><entry></entry></row><row><entry>THZ-5-18-1</entry><entry><chemistry id="CHEM-US-00326" num="00326"><img file="USRE50776E_D0329.tif" /></chemistry></entry><entry>121</entry><entry>1530</entry><entry>1336</entry><entry>—</entry></row><row><entry></entry></row><row><entry>E-9(SB1-E-23)</entry><entry><chemistry id="CHEM-US-00327" num="00327"><img file="USRE50776E_D0330.tif" /></chemistry></entry><entry>1090</entry><entry>21.6</entry><entry>78.8</entry><entry>250</entry></row><row><entry></entry></row><row><entry>THZ-5-18-1</entry><entry><chemistry id="CHEM-US-00328" num="00328"><img file="USRE50776E_D0331.tif" /></chemistry></entry><entry>121</entry><entry>1530</entry><entry>1336</entry><entry>—</entry></row><row><entry></entry></row><row><entry>E-1(SB1-E-14)</entry><entry><chemistry id="CHEM-US-00329" num="00329"><img file="USRE50776E_D0332.tif" /></chemistry></entry><entry>254</entry><entry>1360</entry><entry>—</entry><entry>735</entry></row><row><entry></entry></row><row><entry>SB1-E-18</entry><entry><chemistry id="CHEM-US-00330" num="00330"><img file="USRE50776E_D0333.tif" /></chemistry></entry><entry>461</entry><entry>41.8</entry><entry>19.2</entry><entry>15.0</entry></row><row><entry></entry></row><row><entry>SB1-E-21</entry><entry><chemistry id="CHEM-US-00331" num="00331"><img file="USRE50776E_D0334.tif" /></chemistry></entry><entry>1210</entry><entry>226</entry><entry>318</entry><entry>1220</entry></row><row><entry></entry></row><row><entry>SB1-E-26</entry><entry><chemistry id="CHEM-US-00332" num="00332"><img file="USRE50776E_D0335.tif" /></chemistry></entry><entry>1090</entry><entry>429</entry><entry>518</entry><entry>939</entry></row><row><entry></entry></row><row><entry>SB1-E-15</entry><entry><chemistry id="CHEM-US-00333" num="00333"><img file="USRE50776E_D0336.tif" /></chemistry></entry><entry>763</entry><entry>58.9</entry><entry>—</entry><entry>1480</entry></row><row><entry></entry></row><row><entry>SB1-E-16</entry><entry><chemistry id="CHEM-US-00334" num="00334"><img file="USRE50776E_D0337.tif" /></chemistry></entry><entry>255</entry><entry>116</entry><entry>—</entry><entry>30.3</entry></row><row><entry></entry></row><row><entry>SB1-E-17</entry><entry><chemistry id="CHEM-US-00335" num="00335"><img file="USRE50776E_D0338.tif" /></chemistry></entry><entry>1020</entry><entry>171</entry><entry>60.6</entry><entry>247</entry></row><row><entry></entry></row><row><entry>Y1</entry><entry><chemistry id="CHEM-US-00336" num="00336"><img file="USRE50776E_D0339.tif" /></chemistry></entry><entry>—</entry><entry>—</entry><entry>—</entry><entry>—</entry></row><row><entry></entry></row><row><entry>Y2</entry><entry><chemistry id="CHEM-US-00337" num="00337"><img file="USRE50776E_D0340.tif" /></chemistry></entry><entry>—</entry><entry>—</entry><entry>—</entry><entry>—</entry></row><row><entry></entry></row><row><entry>Y3</entry><entry><chemistry id="CHEM-US-00338" num="00338"><img file="USRE50776E_D0341.tif" /></chemistry></entry><entry>—</entry><entry>—</entry><entry>—</entry><entry>—</entry></row><row><entry></entry></row><row><entry>Y4</entry><entry><chemistry id="CHEM-US-00339" num="00339"><img file="USRE50776E_D0342.tif" /></chemistry></entry><entry>—</entry><entry>—</entry><entry>—</entry><entry>—</entry></row><row><entry></entry></row><row><entry>Y5</entry><entry><chemistry id="CHEM-US-00340" num="00340"><img file="USRE50776E_D0343.tif" /></chemistry></entry><entry>—</entry><entry>—</entry><entry>—</entry><entry>—</entry></row><row><entry></entry></row><row><entry>Y6</entry><entry><chemistry id="CHEM-US-00341" num="00341"><img file="USRE50776E_D0344.tif" /></chemistry></entry><entry>—</entry><entry>—</entry><entry>—</entry><entry>—</entry></row><row><entry></entry></row><row><entry>Y7</entry><entry><chemistry id="CHEM-US-00342" num="00342"><img file="USRE50776E_D0345.tif" /></chemistry></entry><entry>—</entry><entry>—</entry><entry>—</entry><entry>—</entry></row><row><entry></entry></row><row><entry>Y8</entry><entry><chemistry id="CHEM-US-00343" num="00343"><img file="USRE50776E_D0346.tif" /></chemistry></entry><entry>—</entry><entry>—</entry><entry>—</entry><entry>—</entry></row><row><entry></entry></row><row><entry>Y9</entry><entry><chemistry id="CHEM-US-00344" num="00344"><img file="USRE50776E_D0347.tif" /></chemistry></entry><entry>—</entry><entry>—</entry><entry>—</entry><entry>—</entry></row><row><entry></entry></row><row><entry>Y10</entry><entry><chemistry id="CHEM-US-00345" num="00345"><img file="USRE50776E_D0348.tif" /></chemistry></entry><entry>—</entry><entry>—</entry><entry>—</entry><entry>—</entry></row><row><entry></entry></row><row><entry>Y11</entry><entry><chemistry id="CHEM-US-00346" num="00346"><img file="USRE50776E_D0349.tif" /></chemistry></entry><entry>—</entry><entry>—</entry><entry>—</entry><entry>—</entry></row><row><entry></entry></row><row><entry>Y12</entry><entry><chemistry id="CHEM-US-00347" num="00347"><img file="USRE50776E_D0350.tif" /></chemistry></entry><entry>—</entry><entry>—</entry><entry>—</entry><entry>—</entry></row><row><entry></entry></row><row><entry>Y13</entry><entry><chemistry id="CHEM-US-00348" num="00348"><img file="USRE50776E_D0351.tif" /></chemistry></entry><entry>—</entry><entry>—</entry><entry>—</entry><entry>—</entry></row><row><entry></entry></row><row><entry>Y14</entry><entry><chemistry id="CHEM-US-00349" num="00349"><img file="USRE50776E_D0352.tif" /></chemistry></entry><entry>—</entry><entry>—</entry><entry>—</entry><entry>—</entry></row><row><entry></entry></row><row><entry>MFH-1-169-1</entry><entry><chemistry id="CHEM-US-00350" num="00350"><img file="USRE50776E_D0353.tif" /></chemistry></entry><entry>7250</entry><entry>135</entry><entry>—</entry><entry>3880</entry></row><row><entry></entry></row><row><entry>MFH-1-175-1</entry><entry><chemistry id="CHEM-US-00351" num="00351"><img file="USRE50776E_D0354.tif" /></chemistry></entry><entry>442</entry><entry>48.2</entry><entry>—</entry><entry>1080</entry></row><row><entry></entry></row><row><entry>MFH-1-187-1</entry><entry><chemistry id="CHEM-US-00352" num="00352"><img file="USRE50776E_D0355.tif" /></chemistry></entry><entry>279</entry><entry>23.5</entry><entry>—</entry><entry>171</entry></row><row><entry></entry></row><row><entry>MFH-2-67-1</entry><entry><chemistry id="CHEM-US-00353" num="00353"><img file="USRE50776E_D0356.tif" /></chemistry></entry><entry>23.7</entry><entry>103</entry><entry>—</entry><entry>1020</entry></row><row><entry></entry></row><row><entry>MFH-2-78-1</entry><entry><chemistry id="CHEM-US-00354" num="00354"><img file="USRE50776E_D0357.tif" /></chemistry></entry><entry>842</entry><entry>68.7</entry><entry>—</entry><entry>>1000</entry></row><row><entry></entry></row><row><entry>MFH-1-49-1</entry><entry><chemistry id="CHEM-US-00355" num="00355"><img file="USRE50776E_D0358.tif" /></chemistry></entry><entry>165</entry><entry>2.8</entry><entry>—</entry><entry>27.6</entry></row><row><entry></entry></row><row><entry>MFH-1-56-1</entry><entry><chemistry id="CHEM-US-00356" num="00356"><img file="USRE50776E_D0359.tif" /></chemistry></entry><entry>345</entry><entry>300</entry><entry /><entry>1530</entry></row><row><entry></entry></row><row><entry>MFH-1-143-1</entry><entry><chemistry id="CHEM-US-00357" num="00357"><img file="USRE50776E_D0360.tif" /></chemistry></entry><entry>2510</entry><entry>859</entry><entry /><entry>>10000</entry></row><row><entry></entry></row><row><entry>MFH-1-167-1</entry><entry><chemistry id="CHEM-US-00358" num="00358"><img file="USRE50776E_D0361.tif" /></chemistry></entry><entry>739</entry><entry>24.7</entry><entry /><entry>328</entry></row><row><entry></entry></row><row><entry>MFH-2-53-1</entry><entry><chemistry id="CHEM-US-00359" num="00359"><img file="USRE50776E_D0362.tif" /></chemistry></entry><entry>1100</entry><entry>61.6</entry><entry /><entry>53.5</entry></row><row><entry></entry></row><row><entry>MFH-3-140-1</entry><entry><chemistry id="CHEM-US-00360" num="00360"><img file="USRE50776E_D0363.tif" /></chemistry></entry><entry>356</entry><entry>465</entry><entry /><entry>4190</entry></row><row><entry></entry></row><row><entry>MFH-1-141-1</entry><entry><chemistry id="CHEM-US-00361" num="00361"><img file="USRE50776E_D0364.tif" /></chemistry></entry><entry>1210</entry><entry>23.9</entry><entry /><entry>932</entry></row><row><entry namest="1" nameend="6" align="center" rowsep="1" /></row></tbody></tgroup></table></tables>

Example 31. KiNativ™ Kinome Profiling

0833Jurkat cells were seeded at a density of 100 million cells/50 mL. Cells were treated with E9 (1 μM) or DMSO control for 6 hrs. Cells were washed twice with cold phosphate—buffered saline (PBS). PBS-washed cell pellets were flash frozen and subjected to KiNativ™ kinome profiling at ActivX Biosciences, Inc. according to their specifications using their desthiobiotin-ATP probe. Peptide sequences shown in Table A4 belong to the indicated kinase(s) and were detected by mass spectrometry (MS) under DMSO control conditions following enrichment for biotinylated proteins by streptavidin pulldown and subsequent proteolysis. Kinases labeled by the reactive desthiobiotin-ATP probe indicate that the kinase was accessible to desthiobiotin-ATP probe binding. Results shown are normalized to these paired DMSO controls and numbers represent the percentage (compared to DMSO control) of MS signal lost for sequences of an indicated kinase, eg—numbers approaching 100% indicate that test compound effectively out-competed the desthiobiotin ATP probe for binding to the kinase, resulting in decreased labeling and enrichment for peptides representing this kinase. This result suggests that E9 binds predominantly CDK12 and CDK13 in Jurkat cells (<figref idref="DRAWINGS">FIG. <b>10</b></figref>)

Example 32. Jurkat Gene Expression Analysis

0834Jurkat cells were seeded at a density of 5 million cells/10 mL. Cells were then treated with 200 nM or 1 μM of the indicated compounds or with DMSO control for 6 hrs. Total RNA was extracted from 5 million cells using the RNeasy Plus Mini Kit (Qiagen) with a gDNA eliminator mini column to remove genomic DNA. mRNA was reverse transcribed into cDNA using the SuperScript III First-Strand Synthesis Kit (Life Technologies) using an oligo-dT primer to capture polyadenylated mRNAs. Quantitative PCR (qPCR) using transcript—specific Taqman probes (Applied Biosystems) was used to assess the effect of compound treatment on the expression of the indicated mRNA transcripts. All experiments shown were performed in biological triplicate. Each individual biological sample was qPCR—amplified in technical duplicate. Error bars are +/−SD. Expression data from drug treatments were normalized to GAPDH probe.

0835These results suggest that the covalent inhibitors E9, E17, and E18 as well as Dinaciclib (<figref idref="DRAWINGS">FIG. <b>6</b></figref>), a reversible inhibitor, potently downregulate the key T-ALL transcription factors TAL1, RUNX1, and MYB at a dose of 200 nM. Inhibitors E21 and E26 downregulate these factors to a lesser degree at 200 nM and only potently reduce these transcripts at 1 μM. Furthermore, the magnitude with which these inhibitors downregulate the expression of these transcription factors at 200 nM correlates with their effective IC50s on Jurkat T-ALL cell proliferation. For example, Dinaciclib and E18 downregulate these transcription factor genes by at least 80% at 200 nM and have IC50s less than 20 nM. E9 and E17 downregulate these transcription factor genes by at least 40% at 200 nM and have IC50s less than 100 nM. Lastly, E21 and E26 downregulate these genes between 0-60% and have IC50s greater than 300 nM. This suggests that the magnitude of the reduction in expression of these transcription factor genes may serve as an indicator of overall phenotypic response to the inhibitor.

Example 33. Mutagenesis Analysis

0836Genome Editing: The CRISPR/Cas9 system was used to mutate the endogenous CDK12 and CDK13 loci to encode for CDK12 C1039S and CDK13 C1017S, both of which are putative CDK12/13 inhibitor-refractory mutants. Target-specific oligonucleotides were cloned into pX330, which carries a codon-optimized version of Cas9 and was further modified to express GFP for identifying transfectants. Cells were co-transfected (X-tremeGENE 9 (Roche)) with 1) pX330 expressing Cas9 and CDK12-targeting guide RNAs and 2) a pUC57-AMP construct bearing 1500 bp of modified CDK12 reference genome* that is centered around the CRISPR targeting site in CDK12. Two days after transfection, cells were sorted using GFP as a marker of transfected cells and cells were re-plated for five days. Cells were then re-plated at low density to facilitate the isolation of individual clones. Individual clones were isolated, expanded, and PCR genotyped using mutant specific PCR primers. Following initial PCR screening, individual clones were Sanger sequenced to confirm the presence of the desired mutation. Western blot confirmed the presence of intact CDK12 kinase. The process was sequentially repeated with Cas9/sgRNA constructs to target and replace the CDK13 genetic loci. Subsequent experiments were conducted using a CDK12 C1039S/CDK13 C1017S clone and a wild type control clone that was carried through the entirety of the CRISPR protocol but that was verified by Sanger sequencing to be wild typeT for CDK12 and CDK13. The genomic sequence complementary to the CDK12-directed guide RNA that was cloned into pX330 and used in the genome editing experiments is: GGCAGGATTGCCATGAGTTG. The genomic sequence complementary to the CDK13-directed guide RNA that was cloned into pX330 and used in the genome editing experiments is: GGCAAGAT<u style="single">TGT</u>CATGAGTTA. * The reference genome sequence used as a repair template for CDK12 and CDK13 CRISPR was edited to 1) introduce DNA coding for serine, 2) introduce mutations to either remove the PAM site (NGG) targeted by CRISPR/Cas9 or introduce sufficient wobble mutations such that the guide RNA could not recognize the repair template and thus could not be cut by CRISPR/Cas9, and 3) introduce mutations that could allow for mutant and wild type-specific PCR amplification.

0837HAP1 cell proliferation assay: HAP1 wild type and double mutants cells were seeded at a density of 12,000 cells/well in 96-well plates. Twenty four hours cells were then treated with the indicated compounds in a 10-pt dose escalation format from 1 nM to 10 μM or DMSO control for 72 hrs. After 72 hrs, cells were assayed using CellTiter-Glo Luminescent Cell Viability Assay (Promega) to determine cell viability by measuring the amount of ATP present in each sample cell population, which is an indicator of cell metabolic activity. Results are graphed as fraction of the DMSO control at 72 hrs. All data points were performed in biological triplicate. Error bars are +/−SD.

0838HAP1 cells expressing inhibitor-refractory mutations in CDK12 (C1039S) and CDK13 (C1017S) were 4-fold less sensitive to E17 compared to control wild type HAP1 cells. This result indicates that a significant portion of intracellular E17 activity comes from covalent inhibition of CDK12 and/or CDK13. Mutation of these targeted cysteines to a less nucleophilic serine is sufficient to rescue a significant portion of anti-prolierative activity at concentrations less than 350 nM.

EQUIVALENTS AND SCOPE

0839In the claims articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

0840Furthermore, the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Where elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements and/or features, certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements and/or features. For purposes of simplicity, those embodiments have not been specifically set forth in haec verba herein. It is also noted that the terms “comprising” and “containing” are intended to be open and permits the inclusion of additional elements or steps. Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or sub-range within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

0841This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. If there is a conflict between any of the incorporated references and the instant specification, the specification shall control. In addition, any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims. Because such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the invention can be excluded from any claim, for any reason, whether or not related to the existence of prior art.

0842Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above Description, but rather is as set forth in the appended claims. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present invention, as defined in the following claims.