WO2022055940A1 - Vista inhibitors - Google Patents

Vista inhibitors Download PDF

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Publication number
WO2022055940A1
WO2022055940A1 PCT/US2021/049379 US2021049379W WO2022055940A1 WO 2022055940 A1 WO2022055940 A1 WO 2022055940A1 US 2021049379 W US2021049379 W US 2021049379W WO 2022055940 A1 WO2022055940 A1 WO 2022055940A1
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substituted
unsubstituted
membered
substituent group
compound
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PCT/US2021/049379
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French (fr)
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Moustafa T. GABR
Sanjiv S. Gambhir
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The Board Of Trustees Of The Leland Stanford Junior University
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Publication of WO2022055940A1 publication Critical patent/WO2022055940A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a chain containing hetero atoms as chain links

Definitions

  • Negative immune checkpoints such as cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death-ligand 1 (PD-L1) promote cancer growth by downregulation of T-cell activation. 1 Thus, blocking these immune checkpoints restores the ability of immune system to attack cancer cells.
  • FDA-approved monoclonal antibodies (mAbs) against negative immune checkpoints have revealed remarkable clinical success in different malignancies. 2 ' 4 However, overall response rates to mAbs in cancer immunotherapy are generally lower than 30%.
  • 5,6 V-domain Ig Suppressor of T-cell Activation (VISTA) is a negative immune checkpoint protein that shares significant homology to PD-L1 in its extracellular domain (ECD).
  • VISTA has been identified as a potential mediator of resistance to mAb-based immunotherapies in patients based on elevated VISTA levels in patients after administration of anti-CTLA-4 and anti-PD-Ll treatments. 10,11 Moreover, VISTA has been introduced as a potential immunotherapeutic target in pancreatic cancer due to the engagement of VISTA in diminishing cytokine production in T cells isolated from metastatic pancreatic tumors. 12 Thus, it would be advantageous to identify inhibitors of VISTA. Provided herein are solutions to these and other problems in the art.
  • a compound as described herein including the compound of FIG. 3C.
  • a compound having a structure of (I), or a pharmaceutically acceptable salt thereof is provided.
  • L 1 is a bond, -NH-, -O-, -S-, -C(O)-, -C(O)NH-, -NHC(O)-, -NHC(O)NH-, -C(O)O-, -OC(O)-, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene.
  • R 1 is hydrogen, halogen, -CX’ 3 , -CHX’ 2 , -CH2X 1 , -OCX 1 3, -
  • R 2 is hydrogen, halogen, -CX 2 3 , -CHX 2 2 , -CH2X 2 , -OCX 2 3 , -
  • R 1 and R 2 may optionally be joined to form a substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.
  • R 4 is indendently halogen, -CX 4 3 , -CHX 4 2 , -CH2X 4 , -OCX 4 3 , -
  • R 1A , R 1B , R 1C , R 1D , R 2A , R 2B , R 2C , R 2D , R 4A , R 4B , R 4C , and R 4D are independently hydrogen, -CX 3 , -CHX2, -CH2X, -CN, -OH, -COOH, -CONH2, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; R 1A and R 1B or R 2A and R 2B with substituents bonded to the same nitrogen atom may optionally be joined to form a substituted or unsubstituted heterocycloalkyl or substituted or unsubstituted heteroaryl.
  • Each X, X 1 , X 2 , and X 4 is independently -F, -Cl, -Br, or -I.
  • nl, n2 and n4 is independently an integer from 0 to 4.
  • z is an integer from 0 to 5.
  • Each ml, m2, m4, vl, v2 and v4 are independently 1 or 2.
  • the compound has a structure of
  • a pharmaceutical composition including the compound described herein, or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.
  • a method of treating a cancer in a subject in need thereof comprising administrating an effective amount of the compound described herein, or a pharmaceutically acceptable salt thereof.
  • VISTA V-domain Ig Suppressor of T-cell Activation
  • a method of enhancing T-cell proliferation in a subject in the presence of VISTA-expressing cancer cells comprising administrating the subject an effective amount of the compound described herein, or a pharmaceutically acceptable salt thereof.
  • a method of restoring T-cell activation in a subject in the presence of VISTA-expressing cancer cells comprising administrating the subject an effective amount of the compound described herein, or a pharmaceutically acceptable salt thereof.
  • FIGS. 1A-1D present the structure of the NSC622608 compound and examplary interaction data of said compound.
  • FIG. 1 A is a drawing showing the chemical structure of NSC622608.
  • FIG. IB is a graph showing the dose-response curve for NSC622608 in Fluorescence Resonance Energy Transfer (FRET) assay.
  • FIG. ID is a graph showing binding curves of different NSC622608 concentrations to VISTA in SPR screening.
  • FIGS. 2A-2C present exemplary data related to the interaction between the NSC622608 compound and its target, the V-domain Ig Suppressor of T-cell Activation (VISTA).
  • FIG. 2A is a 3D view of predicted binding pose of NSC622608 to VISTA crys-tal structure.
  • FIG. 2B is an overview of the binding pocket of NSC622608 (highlighted in light blue) on VISTA surface.
  • FIGS. 3A-3C present the selection of a small-molecule ligand for VISTA.
  • FIG. 3 A shows the optimization of the furan-2-ylmethyl fragment of the hit.
  • FIG. 3B shows the optimization of the dimethylamino fragment of the hit.
  • FIG. 3C shows the hybrid compound III.
  • the listed IC 5 0 values (pM) for VISTA binding affinity were derived from FRET screening.
  • FIGS. 4A-4D present exemplary data showing the effect of compound III on different cell lines.
  • FIG. 4A is a graph showing luminescence signals in co-cultures of NanoGloTMIL-2/Jurkat VISTA cells with CHOK1 VISTA cells and CHOK1 parental cells in the presence of compound III.
  • FIG. 4B is a graph showing luminescence signals in cocultures of NanoGloTMIL-2/Jurkat VISTA cells with CHOK1 VISTA cells and CHOK1 parental cells in the presence of CA-170.
  • FIG. 4A is a graph showing luminescence signals in co-cultures of NanoGloTMIL-2/Jurkat VISTA cells with CHOK1 VISTA cells and CHOK1 parental cells in the presence of CA-170.
  • FIG. 4C is a bar graph showing PrestoBlue viability of Jurkat T-cells in the presence of compound III (5 pM) and VISTA Ab (1 pM) upon coculturing with ovarian cancer cell lines (from left to right and for each experiment shown on the bar graph: control experiment (Control), co-culture (Co-culture), compound III (III), and antibody (Ab)).
  • FIG. 4D is a bar graph showing PrestoBlue viability of Jurkat T-cells in the presence of compound III (5 pM) and VISTA Ab (1 pM) upon co-culturing with endometrial cancer cell lines (from left to right and for each experiment shown on the bar graph: control experiment (Control), co-culture (Co-culture), compound III (III), and antibody (Ab)).
  • FIG. 5 presents an overview of the evolution of the IC 5 0 values in VISTA FRET assay throughout the structural optimization of the NSC622608 compound.
  • IFNy Interferon gamma
  • TNFa tumor necrosis factor alpha
  • an “inhibitor” refers to a compound (e.g. compounds described herein) that reduces activity when compared to a control, such as absence of the compound or a compound with known inactivity.
  • Contacting is used in accordance with its plain ordinary meaning and refers to the process of allowing at least two distinct species (e.g. chemical compounds including biomolecules or cells) to become sufficiently proximal to react, interact or physically touch. It should be appreciated; however, the resulting reaction product can be produced directly from a reaction between the added reagents or from an intermediate from one or more of the added reagents that can be produced in the reaction mixture.
  • species e.g. chemical compounds including biomolecules or cells
  • contacting may include allowing two species to react, interact, or physically touch, wherein the two species may be a compound as described herein and a protein or enzyme. In some embodiments contacting includes allowing a compound described herein to interact with a protein or enzyme that is involved in a signaling pathway.
  • activation means positively affecting (e.g. increasing) the activity or fimction of the protein relative to the activity or function of the protein in the absence of the activator.
  • activation means positively affecting (e.g. increasing) the concentration or levels of the protein relative to the concentration or level of the protein in the absence of the activator.
  • the terms may reference activation, or activating, sensitizing, or up-regulating signal transduction or enzymatic activity or the amount of a protein decreased in a disease.
  • activation may include, at least in part, partially or totally increasing stimulation, increasing or enabling activation, or activating, sensitizing, or up-regulating signal transduction or enzymatic activity or the amount of a protein associated with a disease (e.g., a protein which is decreased in a disease relative to a non-diseased control).
  • Activation may include, at least in part, partially or totally increasing stimulation, increasing or enabling activation, or activating, sensitizing, or up-regulating signal transduction or enzymatic activity or the amount of a protein
  • agonist refers to a substance capable of detectably increasing the expression or activity of a given gene or protein.
  • the agonist can increase expression or activity 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more in comparison to a control in the absence of the agonist.
  • expression or activity is 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold or higher than the expression or activity in the absence of the agonist.
  • the term “inhibition”, “inhibit”, “inhibiting” and the like in reference to a protein-inhibitor interaction means negatively affecting (e.g. decreasing) the activity or function of the protein relative to the activity or fimction of the protein in the absence of the inhibitor. In embodiments inhibition means negatively affecting (e.g. decreasing) the concentration or levels of the protein relative to the concentration or level of the protein in the absence of the inhibitor. In embodiments inhibition refers to reduction of a disease or symptoms of disease. In embodiments, inhibition refers to a reduction in the activity of a particular protein target.
  • inhibition includes, at least in part, partially or totally blocking stimulation, decreasing, preventing, or delaying activation, or inactivating, desensitizing, or down-regulating signal transduction or enzymatic activity or the amount of a protein.
  • inhibition refers to a reduction of activity of a target protein resulting from a direct interaction (e.g. an inhibitor binds to the target protein).
  • inhibition refers to a reduction of activity of a target protein from an indirect interaction (e.g. an inhibitor binds to a protein that activates the target protein, thereby preventing target protein activation).
  • inhibitor refers to a substance capable of detectably decreasing the expression or activity of a given gene or protein.
  • the antagonist can decrease expression or activity 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more in comparison to a control in the absence of the antagonist. In certain instances, expression or activity is 1.5-fold, 2-fold, 3- fold, 4-fold, 5 -fold, 10-fold or lower than the expression or activity in the absence of the antagonist.
  • expression includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion. Expression can be detected using conventional techniques for detecting protein (e.g., ELISA, Western blotting, flow cytometry, immunofluorescence, immunohistochemistry, etc.).
  • modulator refers to a composition that increases or decreases the level of a target molecule or the function of a target molecule or the physical state of the target of the molecule relative to the absence of the modulator.
  • modulate is used in accordance with its plain ordinary meaning and refers to the act of changing or varying one or more properties. “Modulation” refers to the process of changing or varying one or more properties. For example, as applied to the effects of a modulator on a target protein, to modulate means to change by increasing or decreasing a property or function of the target molecule or the amount of the target molecule.
  • a disease e.g. a protein associated disease, a cancer (e.g., cancer, inflammatory disease, autoimmune disease, or infectious disease)
  • a disease e.g. cancer, inflammatory disease, autoimmune disease, or infectious disease
  • the disease e.g. cancer, inflammatory disease, autoimmune disease, or infectious disease
  • a symptom of the disease is caused by (in whole or in part) the substance or substance activity or function.
  • a causative agent could be a target for treatment of the disease.
  • aberrant refers to different from normal. When used to describe enzymatic activity or protein function, aberrant refers to activity or function that is greater or less than a normal control or the average of normal non-diseased control samples. Aberrant activity may refer to an amount of activity that results in a disease, wherein returning the aberrant activity to a normal or non-disease-associated amount (e.g. by administering a compound or using a method as described herein), results in reduction of the disease or one or more disease symptoms.
  • signaling pathway refers to a series of interactions between cellular and optionally extra-cellular components (e.g. proteins, nucleic acids, small molecules, ions, lipids) that conveys a change in one component to one or more other components, which in turn may convey a change to additional components, which is optionally propagated to other signaling pathway components.
  • extra-cellular components e.g. proteins, nucleic acids, small molecules, ions, lipids
  • salts are meant to include salts of the active compounds that are prepared with relatively nontoxic acids or bases, depending on the particular substituents found on the compounds described herein.
  • base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent.
  • pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salt, or a similar salt.
  • acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent.
  • Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like, as well as the salts derived from relatively nontoxic organic acids like acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic, p- tolylsulfonic, citric, tartaric, oxalic, methanesulfonic, and the like.
  • inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic,
  • salts of amino acids such as arginate and the like, and salts of organic acids like glucuronic or galactunoric acids and the like (see, for example, Berge et al., “Pharmaceutical Salts”, Journal of Pharmaceutical Science, 1977, 66, 1-19).
  • Certain specific compounds of the present disclosure contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts.
  • the compounds of the present disclosure may exist as salts, such as with pharmaceutically acceptable acids.
  • the present disclosure includes such salts.
  • Non-limiting examples of such salts include hydrochlorides, hydrobromides, phosphates, sulfates, methanesulfonates, nitrates, maleates, acetates, citrates, fumarates, proprionates, tartrates (e.g., (+)-tartrates, (-)-tartrates, or mixtures thereof including racemic mixtures), succinates, benzoates, and salts with amino acids such as glutamic acid, and quaternary ammonium salts (e.g.
  • the neutral forms of the compounds are preferably regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner.
  • the parent form of the compound may differ from the various salt forms in certain physical properties, such as solubility in polar solvents.
  • the present disclosure provides compounds, which are in a prodrug form.
  • Prodrugs of the compounds described herein are those compounds that readily undergo chemical changes under physiological conditions to provide the compounds of the present disclosure.
  • Prodrugs of the compounds described herein may be converted in vivo after administration.
  • prodrugs can be converted to the compounds of the present disclosure by chemical or biochemical methods in an ex vivo environment, such as, for example, when contacted with a suitable enzyme or chemical reagent.
  • Certain compounds of the present disclosure can exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to unsolvated forms and are encompassed within the scope of the present disclosure. Certain compounds of the present disclosure may exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the present disclosure and are intended to be within the scope of the present disclosure.
  • “Pharmaceutically acceptable excipient” and “pharmaceutically acceptable carrier” refer to a substance that aids the administration of an active agent to and absorption by a subject and can be included in the compositions of the present disclosure without causing a significant adverse toxicological effect on the patient.
  • Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, normal saline solutions, lactated Ringer’s, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavors, salt solutions (such as Ringer's solution), alcohols, oils, gelatins, carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethycellulose, polyvinyl pyrrolidine, and colors, and the like.
  • preparations can be sterilized and, if desired, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and/or aromatic substances and the like that do not deleteriously react with the compounds of the disclosure.
  • auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and/or aromatic substances and the like that do not deleteriously react with the compounds of the disclosure.
  • auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and/or aromatic substances and the like that do not deleteriously react with the compounds of the disclosure.
  • auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents,
  • the term "about” means a range of values including the specified value, which a person of ordinary skill in the art would consider reasonably similar to the specified value. In embodiments, about means within a standard deviation using measurements generally acceptable in the art. In embodiments, about means a range extending to +/- 10% of the specified value. In embodiments, about includes the specified value.
  • a “synergistic amount” as used herein refers to the sum of a first amount (e.g., an amount of a compound provided herein) and a second amount (e.g., a therapeutic agent) that results in a synergistic effect (i.e. an effect greater than an additive effect). Therefore, the terms “synergy”, “synergism”, “synergistic”, “combined synergistic amount”, and “synergistic therapeutic effect” which are used herein interchangeably, refer to a measured effect of the compound administered in combination where the measured effect is greater than the sum of the individual effects of each of the compounds provided herein administered alone as a single agent.
  • a synergistic amount may be about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8,
  • a synergistic amount may be about 0.1, 0.2, 0.3, 0.4, 0.5,
  • the term “vaccine” refers to a composition that can provide active acquired immunity to and/or therapeutic effect (e.g. treatment) of a particular disease or a pathogen.
  • a vaccine typically contains one or more agents that can induce an immune response in a subject against a pathogen or disease, i.e. a target pathogen or disease.
  • the immunogenic agent stimulates the body’s immune system to recognize the agent as a threat or indication of the presence of the target pathogen or disease, thereby inducing immunological memory so that the immune system can more easily recognize and destroy any of the pathogen on subsequent exposure.
  • Vaccines can be prophylactic (e.g.
  • a vaccine composition can provide nucleic acid, e.g. mRNA that encodes antigenic molecules (e.g. peptides) to a subject.
  • the nucleic acid that is delivered via the vaccine composition in the subject can be expressed into antigenic molecules and allow the subject to acquire immunity against the antigenic molecules.
  • the vaccine composition can provide mRNA encoding antigenic molecules that are associated with a certain pathogen, e.g.
  • the vaccine composition can provide mRNA encoding certain peptides that are associated with cancer, e.g. peptides that are substantially exclusively or highly expressed in cancer cells as compared to normal cells.
  • the subject after vaccination with the cancer vaccine composition, can have immunity against the peptides that are associated with cancer and kill the cancer cells with specificity.
  • immunological memory encompasses, but is not limited to, an “adaptive immune response”, also known as an “acquired immune response” in which adaptive immunity elicits immunological memory after an initial response to a specific pathogen or a specific type of cells that is targeted by the immune response, and leads to an enhanced response to that target on subsequent encounters.
  • adaptive immune response also known as an “acquired immune response” in which adaptive immunity elicits immunological memory after an initial response to a specific pathogen or a specific type of cells that is targeted by the immune response, and leads to an enhanced response to that target on subsequent encounters.
  • the induction of immunological memory can provide the basis of vaccination.
  • an immunogenic or antigenic composition refers to a compound or composition that induces an immune response, e.g., cytotoxic T lymphocyte (CTL) response, a B cell response (for example, production of antibodies that specifically bind the epitope), an NK cell response or any combinations thereof, when administered to an immunocompetent subject.
  • CTL cytotoxic T lymphocyte
  • B cell response for example, production of antibodies that specifically bind the epitope
  • an NK cell response or any combinations thereof, when administered to an immunocompetent subject.
  • an immunogenic or antigenic composition is a composition capable of eliciting an immune response in an immunocompetent subject.
  • an immunogenic or antigenic composition can include one or more immunogenic epitopes associated with a pathogen or a specific type of cells that is targeted by the immune response.
  • an immunogenic composition can include isolated nucleic acid constructs (such as DNA or RNA) that encode one or more immunogenic epitopes of the antigenic polypeptide that can be used to express the epitope(s) (and thus be used to elicit an immune response against this polypeptide or a related polypeptide associated with the targeted pathogen or type of cells).
  • isolated nucleic acid constructs such as DNA or RNA
  • immunogenic epitopes of the antigenic polypeptide that can be used to express the epitope(s) (and thus be used to elicit an immune response against this polypeptide or a related polypeptide associated with the targeted pathogen or type of cells).
  • EC 5 0 or “half maximal effective concentration” as used herein refers to the concentration of a molecule (e.g., antibody, chimeric antigen receptor or bispecific antibody) capable of inducing a response which is halfway between the baseline response and the maximum response after a specified exposure time.
  • the EC 5 0 is the concentration of a molecule (e.g., antibody, chimeric antigen receptor or bispecific antibody) that produces 50% of the maximal possible effect of that molecule.
  • IC 5 0 half maximal inhibitory concentration refers to the concentration of a molecule (e.g., drug, antibody, chimeric antigen receptor or bispecific antibody) capable of inhibiting a specific biological or biochemical function halfway between the baseline response and the maximum response after a specified exposure time.
  • the IC 5 0 is a quantitative measure that indicates how much of a particular inhibitory substance is needed to inhibit a given biological process or biological component by 50% (e.g. an enzyme, cell, cell receptor or microorganism).
  • VISTA or " V-domain Ig suppressor of T cell activation" as provided herein includes any of the recombinant or naturally-occurring forms of VISTA or variants or homologs thereof that maintain VISTA activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to VISTA).
  • the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring VISTA polypeptide.
  • VISTA is the protein as identified by the NCBI sequence reference NP 071436, homolog or functional fragment thereof.
  • Vista is a type I transmembrane protein that functions as an immune checkpoint and is encoded by the C10orf54 gene.
  • the terms “disease” or “condition” refer to a state of being or health status of a patient or subject capable of being treated with the compounds or methods provided herein.
  • the disease may be a cancer.
  • the disease may be an autoimmune disease.
  • the disease may be an inflammatory disease.
  • the disease may be an infectious disease.
  • cancer refers to human cancers and carcinomas, sarcomas, adenocarcinomas, lymphomas, leukemias, etc., including solid and lymphoid cancers, kidney, breast, lung, bladder, colon, ovarian, prostate, pancreas, stomach, brain, head and neck, skin, uterine, testicular, glioma, esophagus, and liver cancer, including hepatocarcinoma, lymphoma, including B-acute lymphoblastic lymphoma, non-Hodgkin’s lymphomas (e.g., Burkitt’s, Small Cell, and Large Cell lymphomas), Hodgkin’s lymphoma, leukemia (including AML, ALL, and CML), or multiple myeloma.
  • cancer refers to human cancers and carcinomas, sarcomas, adenocarcinomas, lymphomas, leukemias, etc., including solid and lymphoid cancers, kidney, breast,
  • cancer refers to all types of cancer, neoplasm or malignant tumors found in mammals (e.g. humans), including leukemias, lymphomas, carcinomas and sarcomas.
  • exemplary cancers that may be treated with a compound or method provided herein include brain cancer, glioma, glioblastoma, neuroblastoma, prostate cancer, colorectal cancer, pancreatic cancer, Medulloblastoma, melanoma, cervical cancer, gastric cancer, ovarian cancer, lung cancer, cancer of the head, Hodgkin's Disease, and NonHodgkin's Lymphomas.
  • Exemplary cancers that may be treated with a compound or method provided herein include cancer (e.g., solid tumor) of the thyroid, endocrine system, brain, breast, cervix, colon, head & neck, liver, kidney, lung, ovary, pancreas, rectum, stomach, and uterus.
  • cancer e.g., solid tumor
  • Additional examples include, thyroid carcinoma, cholangiocarcinoma, pancreatic adenocarcinoma, skin cutaneous melanoma, colon adenocarcinoma, rectum adenocarcinoma, stomach adenocarcinoma, esophageal carcinoma, head and neck squamous cell carcinoma, breast invasive carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, non-small cell lung carcinoma, mesothelioma, multiple myeloma, neuroblastoma, glioma, glioblastoma multiforme, ovarian cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, primary brain tumors, malignant pancreatic insulanoma, malignant carcinoid, urinary bladder cancer, premalignant skin lesions, testicular cancer, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract
  • the cancer is a solid tumor such as sarcomas and carcinomas, which may include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, nile duct carcinoma, choriocarcino
  • leukemia refers broadly to progressive, malignant diseases of the blood- forming organs and is generally characterized by a distorted proliferation and development of leukocytes and their precursors in the blood and bone marrow. Leukemia is generally clinically classified on the basis of (1) the duration and character of the disease-acute or chronic; (2) the type of cell involved; myeloid (myelogenous), lymphoid (lymphogenous), or monocytic; and (3) the increase or non-increase in the number abnormal cells in the blood- leukemic or aleukemic (subleukemic).
  • Exemplary leukemias that may be treated with a compound or method provided herein include, for example, acute nonlymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, a leukocythemic leukemia, basophylic leukemia, blast cell leukemia, bovine leukemia, chronic myelocytic leukemia, leukemia cutis, embryonal leukemia, eosinophilic leukemia, Gross' leukemia, hairy-cell leukemia, hemoblastic leukemia, hemocytoblastic leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphogenous leukemia,
  • lymphoma refers to a group of cancers affecting hematopoietic and lymphoid tissues. It begins in lymphocytes, the blood cells that are found primarily in lymph nodes, spleen, thymus, and bone marrow. Two main types of lymphoma are non-Hodgkin lymphoma and Hodgkin’s disease. Hodgkin’s disease represents approximately 15% of all diagnosed lymphomas. This is a cancer associated with Reed- Sternberg malignant B lymphocytes. Non-Hodgkin’s lymphomas (NHL) can be classified based on the rate at which cancer grows and the type of cells involved.
  • B-cell lymphomas that may be treated with a compound or method provided herein include, but are not limited to, small lymphocytic lymphoma, Mantle cell lymphoma, follicular lymphoma, marginal zone lymphoma, extranodal (MALT) lymphoma, nodal (monocytoid B-cell) lymphoma, splenic lymphoma, diffuse large cell B-lymphoma, Burkitt’s lymphoma, lymphoblastic lymphoma, immunoblastic large cell lymphoma, or precursor B -lymphoblastic lymphoma.
  • small lymphocytic lymphoma Mantle cell lymphoma
  • follicular lymphoma marginal zone lymphoma
  • MALT extranodal lymphoma
  • nodal lymphoma nodal lymphocytoid B-cell lymphoma
  • splenic lymphoma diffuse large cell B-lymphoma
  • Exemplary T- cell lymphomas that may be treated with a compound or method provided herein include, but are not limited to, cunateous T-cell lymphoma, peripheral T-cell lymphoma, anaplastic large cell lymphoma, mycosis fungoides, and precursor T-lymphoblastic lymphoma.
  • sarcoma generally refers to a tumor which is made up of a substance like the embryonic connective tissue and is generally composed of closely packed cells embedded in a fibrillar or homogeneous substance.
  • Sarcomas that may be treated with a compound or method provided herein include a chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abernethy's sarcoma, adipose sarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, chorio carcinoma, embryonal sarcoma, Wilms' tumor sarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcom
  • melanoma is taken to mean a tumor arising from the melanocytic system of the skin and other organs.
  • Melanomas that may be treated with a compound or method provided herein include, for example, acral-lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman's melanoma, S91 melanoma, Harding-Passey melanoma, juvenile melanoma, lentigo maligna melanoma, malignant melanoma, nodular melanoma, subungal melanoma, or superficial spreading melanoma.
  • carcinoma refers to a malignant new growth made up of epithelial cells tending to infiltrate the surrounding tissues and give rise to metastases.
  • exemplary carcinomas that may be treated with a compound or method provided herein include, for example, medullary thyroid carcinoma, familial medullary thyroid carcinoma, acinar carcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, carcinoma adenomatosum, carcinoma of adrenal cortex, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloid carcinoma, basosquamous cell carcinoma, bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocellular carcinoma, chorionic carcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma, cribriform carcinoma, carcinoma en cuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma, carcinoma durum, embryonal carcinoma, encephaloid
  • the terms “metastasis,” “metastatic,” and “metastatic cancer” can be used interchangeably and refer to the spread of a proliferative disease or disorder, e.g., cancer, from one organ or another non-adjacent organ or body part. “Metastatic cancer” is also called “Stage IV cancer.” Cancer occurs at an originating site, e.g., breast, which site is referred to as a primary tumor, e.g., primary breast cancer. Some cancer cells in the primary tumor or originating site acquire the ability to penetrate and infiltrate surrounding normal tissue in the local area and/or the ability to penetrate the walls of the lymphatic system or vascular system circulating through the system to other sites and tissues in the body.
  • a second clinically detectable tumor formed from cancer cells of a primary tumor is referred to as a metastatic or secondary tumor.
  • the metastatic tumor and its cells are presumed to be similar to those of the original tumor.
  • the secondary tumor in the breast is referred to a metastatic lung cancer.
  • metastatic cancer refers to a disease in which a subject has or had a primary tumor and has one or more secondary tumors.
  • non- metastatic cancer or subjects with cancer that is not metastatic refers to diseases in which subjects have a primary tumor but not one or more secondary tumors.
  • metastatic lung cancer refers to a disease in a subject with or with a history of a primary lung tumor and with one or more secondary tumors at a second location or multiple locations, e.g., in the breast.
  • cutaneous metastasis or “skin metastasis” refer to secondary malignant cell growths in the skin, wherein the malignant cells originate from a primary cancer site (e.g., breast).
  • a primary cancer site e.g., breast
  • cancerous cells from a primary cancer site may migrate to the skin where they divide and cause lesions. Cutaneous metastasis may result from the migration of cancer cells from breast cancer tumors to the skin.
  • visceral metastasis refers to secondary malignant cell growths in the interal organs (e.g., heart, lungs, liver, pancreas, intestines) or body cavities (e.g., pleura, peritoneum), wherein the malignant cells originate from a primary cancer site (e.g., head and neck, liver, breast).
  • a primary cancer site e.g., head and neck, liver, breast.
  • a primary cancer site e.g., head and neck, liver, breast
  • Visceral metastasis may result from the migration of cancer cells from liver cancer tumors or head and neck tumors to internal organs.
  • treating refers to any indicia of success in the therapy or amelioration of an injury, disease, pathology or condition, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the injury, pathology or condition more tolerable to the patient; slowing in the rate of degeneration or decline; making the final point of degeneration less debilitating; improving a patient’s physical or mental well-being.
  • the treatment or amelioration of symptoms can be based on objective or subjective parameters; including the results of a physical examination, neuropsychiatric exams, and/or a psychiatric evaluation.
  • the term "treating" and conjugations thereof, may include prevention of an injury, pathology, condition, or disease.
  • treating is preventing.
  • treating does not include preventing.
  • Treating” or “treatment” as used herein also broadly includes any approach for obtaining beneficial or desired results in a subject’s condition, including clinical results.
  • beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of the extent of a disease, stabilizing (i.e., not worsening) the state of disease, prevention of a disease’s transmission or spread, delay or slowing of disease progression, amelioration or palliation of the disease state, diminishment of the reoccurrence of disease, and remission, whether partial or total and whether detectable or undetectable.
  • treatment includes any cure, amelioration, or prevention of a disease. Treatment may prevent the disease from occurring; inhibit the disease’s spread; relieve the disease’s symptoms, fully or partially remove the disease’s underlying cause, shorten a disease’s duration, or do a combination of these things.
  • Treating” and “treatment” as used herein include prophylactic treatment.
  • Treatment methods include administering to a subject a therapeutically effective amount of an active agent.
  • the administering step may consist of a single administration or may include a series of administrations.
  • the length of the treatment period depends on a variety of factors, such as the severity of the condition, the age of the patient, the concentration of active agent, the activity of the compositions used in the treatment, or a combination thereof.
  • the effective dosage of an agent used for the treatment or prophylaxis may increase or decrease over the course of a particular treatment or prophylaxis regime. Changes in dosage may result and become apparent by standard diagnostic assays known in the art.
  • chronic administration may be required.
  • the compositions are administered to the subject in an amount and for a duration sufficient to treat the patient.
  • the treating or treatment is no prophylactic treatment.
  • prevention refers to a decrease in the occurrence of disease symptoms in a patient. As indicated above, the prevention may be complete (no detectable symptoms) or partial, such that fewer symptoms are observed than would likely occur absent treatment.
  • “Patient” or “subject in need thereof’ refers to a living organism suffering from or prone to a disease or condition that can be treated by administration of a pharmaceutical composition as provided herein.
  • Non-limiting examples include humans, other mammals, bovines, rats, mice, dogs, monkeys, goat, sheep, cows, deer, and other non-mammalian animals.
  • a patient is human.
  • an “effective amount” is an amount sufficient for a compound to accomplish a stated purpose relative to the absence of the compound (e.g. achieve the effect for which it is administered, treat a disease, reduce enzyme activity, increase enzyme activity, reduce a signaling pathway, or reduce one or more symptoms of a disease or condition).
  • An example of an “effective amount” is an amount sufficient to contribute to the treatment, prevention, or reduction of a symptom or symptoms of a disease, which could also be referred to as a “therapeutically effective amount.”
  • a “reduction” of a symptom or symptoms means decreasing of the severity or frequency of the symptom(s), or elimination of the symptom(s).
  • a “prophylactically effective amount” of a drug is an amount of a drug that, when administered to a subject, will have the intended prophylactic effect, e.g., preventing or delaying the onset (or reoccurrence) of an injury, disease, pathology or condition, or reducing the likelihood of the onset (or reoccurrence) of an injury, disease, pathology, or condition, or their symptoms.
  • the full prophylactic effect does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses.
  • a prophylactically effective amount may be administered in one or more administrations.
  • An “activity decreasing amount,” as used herein, refers to an amount of antagonist required to decrease the activity of an enzyme relative to the absence of the antagonist.
  • a “function disrupting amount,” as used herein, refers to the amount of antagonist required to disrupt the function of an enzyme or protein relative to the absence of the antagonist. The exact amounts will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins).
  • the therapeutically effective amount can be initially determined from cell culture assays.
  • Target concentrations will be those concentrations of active compound(s) that are capable of achieving the methods described herein, as measured using the methods described herein or known in the art.
  • therapeutically effective amounts for use in humans can also be determined from animal models.
  • a dose for humans can be formulated to achieve a concentration that has been found to be effective in animals.
  • the dosage in humans can be adjusted by monitoring compounds effectiveness and adjusting the dosage upwards or downwards, as described above. Adjusting the dose to achieve maximal efficacy in humans based on the methods described above and other methods is well within the capabilities of the ordinarily skilled artisan.
  • a therapeutically effective amount refers to that amount of the therapeutic agent sufficient to ameliorate the disorder, as described above.
  • a therapeutically effective amount will show an increase or decrease of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%.
  • Therapeutic efficacy can also be expressed as “-fold” increase or decrease.
  • a therapeutically effective amount can have at least a 1.2-fold, 1.5-fold, 2-fold, 5-fold, or more effect over a control.
  • Dosages may be varied depending upon the requirements of the patient and the compound being employed.
  • the dose administered to a patient should be sufficient to effect a beneficial therapeutic response in the patient over time.
  • the size of the dose also will be determined by the existence, nature, and extent of any adverse side-effects. Determination of the proper dosage for a particular situation is within the skill of the practitioner. Generally, treatment is initiated with smaller dosages which are less than the optimum dose of the compound. Thereafter, the dosage is increased by small increments until the optimum effect under circumstances is reached. Dosage amounts and intervals can be adjusted individually to provide levels of the administered compound effective for the particular clinical indication being treated. This will provide a therapeutic regimen that is commensurate with the severity of the individual's disease state.
  • administering means oral administration, administration as a suppository, topical contact, intravenous, parenteral, intraperitoneal, intramuscular, intralesional, intrathecal, intranasal or subcutaneous administration, or the implantation of a slow-release device, e.g., a mini-osmotic pump, to a subject.
  • Administration is by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal).
  • Parenteral administration includes, e.g., intravenous, intramuscular, intra-arteriole, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial.
  • Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infiision, transdermal patches, etc.
  • the administering does not include administration of any active agent other than the recited active agent.
  • compositions described herein are administered at the same time, just prior to, or just after the administration of one or more additional therapies.
  • the compounds provided herein can be administered alone or can be coadministered to the patient. Coadministration is meant to include simultaneous or sequential administration of the compounds individually or in combination (more than one compound).
  • the preparations can also be combined, when desired, with other active substances (e.g. to reduce metabolic degradation).
  • the compositions of the present disclosure can be delivered transdermally, by a topical route, or formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders, and aerosols.
  • a cell can be identified by well-known methods in the art including, for example, presence of an intact membrane, staining by a particular dye, ability to produce progeny or, in the case of a gamete, ability to combine with a second gamete to produce a viable offspring.
  • Cells may include prokaryotic and eukaroytic cells.
  • Prokaryotic cells include but are not limited to bacteria.
  • Eukaryotic cells include but are not limited to yeast cells and cells derived from plants and animals, for example mammalian, insect (e.g., spodoptera) and human cells. Cells may be usefiil when they are naturally nonadherent or have been treated not to adhere to surfaces, for example by trypsinization.
  • Control or “control experiment” is used in accordance with its plain ordinary meaning and refers to an experiment in which the subjects or reagents of the experiment are treated as in a parallel experiment except for omission of a procedure, reagent, or variable of the experiment. In some instances, the control is used as a standard of comparison in evaluating experimental effects. In some embodiments, a control is the measurement of the activity of a protein in the absence of a compound as described herein (including embodiments and examples).
  • Cancer model organism is an organism exhibiting a phenotype indicative of cancer, or the activity of cancer causing elements, within the organism.
  • the term cancer is defined above.
  • a wide variety of organisms may serve as cancer model organisms, and include for example, cancer cells and mammalian organisms such as rodents (e.g. mouse or rat) and primates (such as humans).
  • Cancer cell lines are widely understood by those skilled in the art as cells exhibiting phenotypes or genotypes similar to in vivo cancers. Cancer cell lines as used herein includes cell lines from animals (e.g. mice) and from humans.
  • an “anticancer agent” as used herein refers to a molecule (e.g. compound, peptide, protein, nucleic acid, 0103) used to treat cancer through destruction or inhibition of cancer cells or tissues. Anticancer agents may be selective for certain cancers or certain tissues. In embodiments, anticancer agents herein may include epigenetic inhibitors and multi-kinase inhibitors.
  • Anti-cancer agent and “anticancer agent” are used in accordance with their plain ordinary meaning and refers to a composition (e.g. compound, drug, antagonist, inhibitor, modulator) having antineoplastic properties or the ability to inhibit the growth or proliferation of cells.
  • an anti-cancer agent is a chemotherapeutic.
  • an anti -cancer agent is an agent identified herein having utility in methods of treating cancer.
  • an anti-cancer agent is an agent approved by the FDA or similar regulatory agency of a country other than the USA, for treating cancer. Examples of anti-cancer agents include, but are not limited to, MEK (e.g.
  • MEK1, MEK2, or MEK1 and MEK2 inhibitors e.g. XL518, CL1040, PD035901, selumetinib/ AZD6244, GSK1120212/ trametinib, GDC-0973, ARRY-162, ARRY-300, AZD8330, PD0325901, U0126, PD98059, TAK-733, PD318088, AS703026, BAY 869766
  • alkylating agents e.g., cyclophosphamide, ifosfamide, chlorambucil, busulfan, melphalan, mechlorethamine, uramustine, thiotepa, nitrosoureas, nitrogen mustards (e.g., mechloroethamine, cyclophosphamide, chlorambucil, meiphalan), ethylenimine and methylmelamines (e.g., hexame
  • Taxol.TM i.e. paclitaxel
  • Taxotere.TM compounds comprising the taxane skeleton, Erbulozole (i.e. R- 55104), Dolastatin 10 (i.e. DLS-10 and NSC-376128), Mivobulin isethionate (i.e. as CI-980), Vincristine, NSC-639829, Discodermolide (i.e. as NVP-XX-A-296), ABT-751 (Abbott, i.e. E-7010), Altorhyrtins (e.g. Altorhyrtin A and Altorhyrtin C), Spongistatins (e.g.
  • Epothilones e.g. Epothilone A, Epothilone B, Epothilone C (i.e. desoxyepothilone A or dEpoA), Epothilone D (i.e.
  • Epothilone E Epothilone F
  • Epothilone B N-oxide Epothilone A N-oxide
  • 16-aza- epothilone B 21-aminoepothilone B (i.e. BMS-310705), 21-hydroxyepothilone D (i.e. Desoxyepothilone F and dEpoF), 26-fluoroepothilone
  • Auristatin PE i.e. NSC-654663
  • Soblidotin i.e. TZT-1027
  • LS-4559-P Pulacia, i.e.
  • LS-4577 LS-4578 (Pharmacia, i.e. LS-477-P), LS-4477 (Pharmacia), LS-4559 (Pharmacia), RPR- 112378 (Aventis), Vincristine sulfate, DZ-3358 (Daiichi), FR-182877 (Fujisawa, i.e. WS-9885B), GS-164 (Takeda), GS- 198 (Takeda), KAR-2 (Hungarian Academy of Sciences), BSF-223651 (BASF, i.e.
  • ILX-651 and LU-223651 SAH-49960 (Lilly/Novartis), SDZ-268970 (Lilly/Novartis), AM-97 (Armad/Kyowa Hakko), AM-132 (Armad), AM-138 (Armad/Kyowa Hakko), IDN-5005 (Indena), Cryptophycin 52 (i.e. LY-355703), AC-7739 (Ajinomoto, i.e. AVE-8063A and CS- 39.HC1), AC-7700 (Ajinomoto, i.e.
  • T-900607 RPR-115781 (Aventis), Eleutherobins (such as Desmethyleleutherobin, Desaetyleleutherobin, Isoeleutherobin A, and Z-Eleutherobin), Caribaeoside, Caribaeolin, Halichondrin B, D-64131 (Asta Medica), D-68144 (Asta Medica), Diazonamide A, A-293620 (Abbott), NPI-2350 (Nereus), Taccalonolide A, TUB-245 (Aventis), A-259754 (Abbott), Diozostatin, (-)-Phenylahistin (i.e.
  • NSCL-96F03-7 D-68838 (Asta Medica), D-68836 (Asta Medica), Myoseverin B, D-43411 (Zentaris, i.e. D-81862), A- 289099 (Abbott), A-318315 (Abbott), HTI-286 (i.e.
  • SPA-110, trifluoroacetate salt) (Wyeth), D-82317 (Zentaris), D-82318 (Zentaris), SC- 12983 (NCI), Resverastatin phosphate sodium, BPR-OY-007 (National Health Research Institutes), and SSR-250411 (Sanofi)), steroids (e.g., dexamethasone), finasteride, aromatase inhibitors, gonadotropin-releasing hormone agonists (GnRH) such as goserelin or leuprolide, adrenocorticosteroids (e.g., prednisone), progestins (e.g., hydroxyprogesterone caproate, megestrol acetate, medroxyprogesterone acetate), estrogens (e.g., diethlystilbestrol, ethinyl estradiol), antiestrogen (e.g., tamoxifen), androgens (e
  • gefitinib Iressa TM
  • erlotinib Tarceva TM
  • cetuximab ErbituxTM
  • lapatinib TykerbTM
  • panitumumab VectibixTM
  • vandetanib CaprelsaTM
  • afatinib/BIBW2992 CI- 1033/canertinib, neratinib/HKI-272, CP-724714, TAK-285, AST-1306, ARRY334543, ARRY-380, AG-1478, dacomitinib/PF299804, OSI-420/desmethyl erlotinib, AZD8931, AEE788, pelitinib/EKB-569, CUDC-101, WZ8040, WZ4002, WZ3146, AG-490, XL647, PD153035, BMS-599626), sorafenib, imatinib, sunitinib
  • “Selective” or “selectivity” or the like of a compound refers to the compound’s ability to discriminate between molecular targets (e.g. a compound having selectivity toward HMT SUV49H1 and/or HMT G9a).
  • “Specific”, “specifically”, “specificity”, or the like of a compound refers to the compound’s ability to cause a particular action, such as inhibition, to a particular molecular target with minimal or no action to other proteins in the cell (e.g. a compound having specificity towards HMT SUV49H1 and/or HMT G9a displays inhibition of the activity of those HMTs whereas the same compound displays little-to-no inhibition of other HMTs such as DOTI, EZH1, EZH2, GLP, MLL1, MLL2, MLL3, MLL4, NSD2, SETlb, SET7/9, SET8, SETMAR, SMYD2, SUV49H2).
  • a compound having specificity towards HMT SUV49H1 and/or HMT G9a displays inhibition of the activity of those HMTs whereas the same compound displays little-to-no inhibition of other HMTs such as DOTI, EZH1, EZH2, GLP, MLL1, MLL2, MLL3, MLL4, NSD2, SETl
  • substituent groups are specified by their conventional chemical formulae, written from left to right, they equally encompass the chemically identical substituents that would result from writing the structure from right to left, e.g., -CH2O- is equivalent to - OCH2-.
  • alkyl by itself or as part of another substituent, means, unless otherwise stated, a straight (i.e., unbranched) or branched carbon chain (or carbon), or combination thereof, which may be fully saturated, mono- or polyunsaturated and can include mono-, di- and multivalent radicals.
  • the alkyl may include a designated number of carbons (e.g., C 1 -C 10 means one to ten carbons).
  • the alkyl is fully saturated.
  • the alkyl is monounsaturated.
  • the alkyl is polyunsaturated.
  • Alkyl is an uncyclized chain.
  • saturated hydrocarbon radicals include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, methyl, homologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like.
  • An unsaturated alkyl group is one having one or more double bonds or triple bonds.
  • Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2- isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(l,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs and isomers.
  • An alkoxy is an alkyl attached to the remainder of the molecule via an oxygen linker (-O-).
  • An alkyl moiety may be an alkenyl moiety.
  • An alkyl moiety may be an alkynyl moiety.
  • An alkenyl includes one or more double bonds.
  • An alkynyl includes one or more triple bonds.
  • alkylene by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from an alkyl, as exemplified, but not limited by, - CH2CH2CH2CH2-.
  • an alkyl (or alkylene) group will have from 1 to 24 carbon atoms, with those groups having 10 or fewer carbon atoms being preferred herein.
  • a “lower alkyl” or “lower alkylene” is a shorter chain alkyl or alkylene group, generally having eight or fewer carbon atoms.
  • alkenylene by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from an alkene.
  • the alkylene is fully saturated. In embodiments, the alkylene is monounsaturated. In embodiments, the alkylene is polyunsaturated.
  • An alkenylene includes one or more double bonds.
  • An alkynylene includes one or more triple bonds.
  • heteroalkyl by itself or in combination with another term, means, unless otherwise stated, a stable straight or branched chain, or combinations thereof, including at least one carbon atom and at least one heteroatom (e.g., O, N, P, Si, and S), and wherein the nitrogen and sulfiir atoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quatemized.
  • the heteroatom(s) e.g., O, N, S, Si, or P
  • Heteroalkyl is an uncyclized chain.
  • a heteroalkyl moiety may include one heteroatom (e.g., O, N, S, Si, or P).
  • a heteroalkyl moiety may include two optionally different heteroatoms (e.g., O, N, S, Si, or P).
  • a heteroalkyl moiety may include three optionally different heteroatoms (e.g., O, N, S, Si, or P).
  • a heteroalkyl moiety may include four optionally different heteroatoms (e.g., O, N, S, Si, or P).
  • a heteroalkyl moiety may include five optionally different heteroatoms (e.g., O, N, S, Si, or P).
  • a heteroalkyl moiety may include up to 8 optionally different heteroatoms (e.g., O, N, S, Si, or P).
  • the term “heteroalkenyl,” by itself or in combination with another term, means, unless otherwise stated, a heteroalkyl including at least one double bond.
  • a heteroalkenyl may optionally include more than one double bond and/or one or more triple bonds in additional to the one or more double bonds.
  • heteroalkynyl by itself or in combination with another term, means, unless otherwise stated, a heteroalkyl including at least one triple bond.
  • a heteroalkynyl may optionally include more than one triple bond and/or one or more double bonds in additional to the one or more triple bonds.
  • the heteroalkyl is fully saturated.
  • the heteroalkyl is monounsaturated.
  • the heteroalkyl is polyunsaturated.
  • heteroalkylene by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from heteroalkyl, as exemplified, but not limited by, -CH2-CH2-S-CH2-CH2- and -CH2-S-CH2-CH2-NH-CH2-.
  • heteroatoms can also occupy either or both of the chain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, and the like).
  • no orientation of the linking group is implied by the direction in which the formula of the linking group is written.
  • heteroalkyl groups include those groups that are attached to the remainder of the molecule through a heteroatom, such as -C(O)R', -C(O)NR', -NR'R", -OR', -SR', and/or -SO 2 R'.
  • heteroalkyl is recited, followed by recitations of specific heteroalkyl groups, such as - NR'R" or the like, it will be understood that the terms heteroalkyl and -NR'R" are not redundant or mutually exclusive.
  • heteroalkyl should not be interpreted herein as excluding specific heteroalkyl groups, such as -NR'R" or the like.
  • heteroalkenylene by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from a heteroalkene.
  • heteroalkynylene by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from an heteroalkyne.
  • the heteroalkylene is fully saturated.
  • the heteroalkylene is monounsaturated.
  • the heteroalkylene is polyunsaturated.
  • a heteroalkenylene inlcudes one or more double bonds.
  • a heteroalkynylene includes one or more triple bonds.
  • cycloalkyl and heterocycloalkyl mean, unless otherwise stated, cyclic versions of “alkyl” and “heteroalkyl,” respectively. Cycloalkyl and heterocycloalkyl are not aromatic. Additionally, for heterocycloalkyl, a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule. Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1 -cyclohexenyl, 3 -cyclohexenyl, cycloheptyl, and the like.
  • heterocycloalkyl examples include, but are not limited to, 1- (1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3- morpholinyl, tetrahydrofuran-2-yl, tetrahydrohiran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl, 2-piperazinyl, and the like.
  • the cycloalkyl is hilly saturated.
  • the cycloalkyl is monounsaturated.
  • the cycloalkyl is polyunsaturated.
  • the heterocycloalkyl is hilly saturated.
  • the heterocycloalkyl is monounsaturated.
  • the heterocycloalkyl is polyunsaturated.
  • cycloalkyl means a monocyclic, bicyclic, or a multicyclic cycloalkyl ring system.
  • monocyclic ring systems are cyclic hydrocarbon groups containing from 3 to 8 carbon atoms, where such groups can be saturated or unsaturated, but not aromatic.
  • cycloalkyl groups are fully saturated. Examples of monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl.
  • Bicyclic cycloalkyl ring systems are bridged monocyclic rings or hised bicyclic rings.
  • bridged monocyclic rings contain a monocyclic cycloalkyl ring where two non adjacent carbon atoms of the monocyclic ring are linked by an alkylene bridge of between one and three additional carbon atoms (i.e., a bridging group of the form (CHijw , where w is 1, 2, or 3).
  • bicyclic ring systems include, but are not limited to, bicyclo[3.1.1]heptane, bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane, bicyclo[3.2.2]nonane, bicyclo[3.3.1]nonane, and bicyclo[4.2.1]nonane.
  • fused bicyclic cycloalkyl ring systems contain a monocyclic cycloalkyl ring fused to either a phenyl, a monocyclic cycloalkyl, a monocyclic cycloalkenyl, a monocyclic heterocyclyl, or a monocyclic heteroaryl.
  • the bridged or fiised bicyclic cycloalkyl is attached to the parent molecular moiety through any carbon atom contained within the monocyclic cycloalkyl ring.
  • cycloalkyl groups are optionally substituted with one or two groups which are independently oxo or thia.
  • the fused bicyclic cycloalkyl is a 5 or 6 membered monocyclic cycloalkyl ring fused to either a phenyl ring, a 5 or 6 membered monocyclic cycloalkyl, a 5 or 6 membered monocyclic cycloalkenyl, a 5 or 6 membered monocyclic heterocyclyl, or a 5 or 6 membered monocyclic heteroaryl, wherein the fiised bicyclic cycloalkyl is optionally substituted by one or two groups which are independently oxo or thia.
  • multicyclic cycloalkyl ring systems are a monocyclic cycloalkyl ring (base ring) fused to either (i) one ring system selected from the group consisting of a bicyclic aryl, a bicyclic heteroaryl, a bicyclic cycloalkyl, a bicyclic cycloalkenyl, and a bicyclic heterocyclyl; or (ii) two other ring systems independently selected from the group consisting of a phenyl, a bicyclic aryl, a monocyclic or bicyclic heteroaryl, a monocyclic or bicyclic cycloalkyl, a monocyclic or bicyclic cycloalkenyl, and a monocyclic or bicyclic heterocyclyl.
  • multicyclic cycloalkyl is attached to the parent molecular moiety through any carbon atom contained within the base ring.
  • multicyclic cycloalkyl ring systems are a monocyclic cycloalkyl ring (base ring) fused to either (i) one ring system selected from the group consisting of a bicyclic aryl, a bicyclic heteroaryl, a bicyclic cycloalkyl, a bicyclic cycloalkenyl, and a bicyclic heterocyclyl; or (ii) two other ring systems independently selected from the group consisting of a phenyl, a monocyclic heteroaryl, a monocyclic cycloalkyl, a monocyclic cycloalkenyl, and a monocyclic heterocyclyl.
  • multicyclic cycloalkyl groups include, but are not limited to tetradecahydrophenanthrenyl, perhydrophenothiazin- 1-yl, and perhydrophenoxazin- 1-yl.
  • a bicyclic or multicyclic cycloalkyl ring system refers to multiple rings fiised together wherein at least one of the fiised rings is a cycloalkyl ring and wherein the multiple rings are attached to the parent molecular moiety through any carbon atom contained within a cycloalkyl ring of the multiple rings.
  • a cycloalkyl is a cycloalkenyl.
  • the term “cycloalkenyl” is used in accordance with its plain ordinary meaning.
  • a cycloalkenyl is a monocyclic, bicyclic, or a multicyclic cycloalkenyl ring system.
  • monocyclic cycloalkenyl ring systems are cyclic hydrocarbon groups containing from 3 to 8 carbon atoms, where such groups are unsaturated (i.e., containing at least one annular carbon carbon double bond), but not aromatic. Examples of monocyclic cycloalkenyl ring systems include cyclopentenyl and cyclohexenyl.
  • bicyclic cycloalkenyl rings are bridged monocyclic rings or a fused bicyclic rings.
  • bridged monocyclic rings contain a monocyclic cycloalkenyl ring where two non adjacent carbon atoms of the monocyclic ring are linked by an alkylene bridge of between one and three additional carbon atoms (i.e., a bridging group of the form (CHijw, where w is 1, 2, or 3).
  • Representative examples of bicyclic cycloalkenyls include, but are not limited to, norbomenyl and bicyclo[2.2.2]oct 2 enyl.
  • fused bicyclic cycloalkenyl ring systems contain a monocyclic cycloalkenyl ring fused to either a phenyl, a monocyclic cycloalkyl, a monocyclic cycloalkenyl, a monocyclic heterocyclyl, or a monocyclic heteroaryl.
  • the bridged or fiised bicyclic cycloalkenyl is attached to the parent molecular moiety through any carbon atom contained within the monocyclic cycloalkenyl ring.
  • cycloalkenyl groups are optionally substituted with one or two groups which are independently oxo or thia.
  • multicyclic cycloalkenyl rings contain a monocyclic cycloalkenyl ring (base ring) fiised to either (i) one ring system selected from the group consisting of a bicyclic aryl, a bicyclic heteroaryl, a bicyclic cycloalkyl, a bicyclic cycloalkenyl, and a bicyclic heterocyclyl; or (ii) two ring systems independently selected from the group consisting of a phenyl, a bicyclic aryl, a monocyclic or bicyclic heteroaryl, a monocyclic or bicyclic cycloalkyl, a monocyclic or bicyclic cycloalkenyl, and a monocyclic or bicyclic heterocyclyl.
  • multicyclic cycloalkenyl is attached to the parent molecular moiety through any carbon atom contained within the base ring.
  • multicyclic cycloalkenyl rings contain a monocyclic cycloalkenyl ring (base ring) fused to either (i) one ring system selected from the group consisting of a bicyclic aryl, a bicyclic heteroaryl, a bicyclic cycloalkyl, a bicyclic cycloalkenyl, and a bicyclic heterocyclyl; or (ii) two ring systems independently selected from the group consisting of a phenyl, a monocyclic heteroaryl, a monocyclic cycloalkyl, a monocyclic cycloalkenyl, and a monocyclic heterocyclyl.
  • a bicyclic or multicyclic cycloalkenyl ring system refers to multiple rings fused together wherein at least one of the fused rings is a cycloalkenyl ring and wherein the multiple rings are attached to the parent molecular moiety through any carbon atom contained within a cycloalkenyl ring of the multiple rings.
  • heterocycloalkyl means a monocyclic, bicyclic, or a multicyclic heterocycloalkyl ring system.
  • heterocycloalkyl groups are fully saturated.
  • a bicyclic or multicyclic heterocycloalkyl ring system refers to multiple rings fused together wherein at least one of the fiised rings is a heterocycloalkyl ring and wherein the multiple rings are attached to the parent molecular moiety through any atom contained within a heterocycloalkyl ring of the multiple rings.
  • a heterocycloalkyl is a heterocyclyl.
  • heterocyclyl as used herein, means a monocyclic, bicyclic, or multicyclic heterocycle.
  • the heterocyclyl monocyclic heterocycle is a 3, 4, 5, 6 or 7 membered ring containing at least one heteroatom independently selected from the group consisting of O, N, and S where the ring is saturated or unsaturated, but not aromatic.
  • the 3 or 4 membered ring contains 1 heteroatom selected from the group consisting of O, N and S.
  • the 5 membered ring can contain zero or one double bond and one, two or three heteroatoms selected from the group consisting of O, N and S.
  • the 6 or 7 membered ring contains zero, one or two double bonds and one, two or three heteroatoms selected from the group consisting of O, N and S.
  • the heterocyclyl monocyclic heterocycle is connected to the parent molecular moiety through any carbon atom or any nitrogen atom contained within the heterocyclyl monocyclic heterocycle.
  • heterocyclyl monocyclic heterocycles include, but are not limited to, azetidinyl, azepanyl, aziridinyl, diazepanyl, 1,3-dioxanyl, 1,3-dioxolanyl, 1,3-dithiolanyl, 1,3-dithianyl, imidazolinyl, imidazolidinyl, isothiazolinyl, isothiazolidinyl, isoxazolinyl, isoxazolidinyl, morpholinyl, oxadiazolinyl, oxadiazolidinyl, oxazolinyl, oxazolidinyl, piperazinyl, piperidinyl, pyranyl, pyrazolinyl, pyrazolidinyl, pyrrolinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydrothienyl
  • the heterocyclyl bicyclic heterocycle is a monocyclic heterocycle fused to either a phenyl, a monocyclic cycloalkyl, a monocyclic cycloalkenyl, a monocyclic heterocycle, or a monocyclic heteroaryl.
  • the heterocyclyl bicyclic heterocycle is connected to the parent molecular moiety through any carbon atom or any nitrogen atom contained within the monocyclic heterocycle portion of the bicyclic ring system.
  • bicyclic heterocyclyls include, but are not limited to, 2,3-dihydrobenzofiiran-2-yl, 2,3- dihydrobenzofuran-3-yl, indolin-l-yl, indolin-2-yl, indolin-3-yl, 2,3-dihydrobenzothien-2-yl, decahydroquinolinyl, decahydroisoquinolinyl, octahydro- IH-indolyl, and octahydrobenzofiiranyl.
  • heterocyclyl groups are optionally substituted with one or two groups which are independently oxo or thia.
  • the bicyclic heterocyclyl is a 5 or 6 membered monocyclic heterocyclyl ring fused to a phenyl ring, a 5 or 6 membered monocyclic cycloalkyl, a 5 or 6 membered monocyclic cycloalkenyl, a 5 or 6 membered monocyclic heterocyclyl, or a 5 or 6 membered monocyclic heteroaryl, wherein the bicyclic heterocyclyl is optionally substituted by one or two groups which are independently oxo or thia.
  • Multicyclic heterocyclyl ring systems are a monocyclic heterocyclyl ring (base ring) fused to either (i) one ring system selected from the group consisting of a bicyclic aryl, a bicyclic heteroaryl, a bicyclic cycloalkyl, a bicyclic cycloalkenyl, and a bicyclic heterocyclyl; or (ii) two other ring systems independently selected from the group consisting of a phenyl, a bicyclic aryl, a monocyclic or bicyclic heteroaryl, a monocyclic or bicyclic cycloalkyl, a monocyclic or bicyclic cycloalkenyl, and a monocyclic or bicyclic heterocyclyl.
  • multicyclic heterocyclyl is attached to the parent molecular moiety through any carbon atom or nitrogen atom contained within the base ring.
  • multicyclic heterocyclyl ring systems are a monocyclic heterocyclyl ring (base ring) fused to either (i) one ring system selected from the group consisting of a bicyclic aryl, a bicyclic heteroaryl, a bicyclic cycloalkyl, a bicyclic cycloalkenyl, and a bicyclic heterocyclyl; or (ii) two other ring systems independently selected from the group consisting of a phenyl, a monocyclic heteroaryl, a monocyclic cycloalkyl, a monocyclic cycloalkenyl, and a monocyclic heterocyclyl.
  • multicyclic heterocyclyl groups include, but are not limited to lOH-phenothiazin- 10-yl, 9,10-dihydroacridin-9-yl, 9,10-dihydroacridin-10-yl, lOH-phenoxazin- 10-yl, 10,1 l-dihydro-5H-dibenzo[b,f
  • halo or “halogen,” by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom. Additionally, terms such as “haloalkyl” are meant to include monohaloalkyl and polyhaloalkyl.
  • halo(C 1 -C 4 )alkyl includes, but is not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3 -bromopropyl, and the like.
  • acyl means, unless otherwise stated, -C(O)R where R is a substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.
  • aryl means, unless otherwise stated, a polyunsaturated, aromatic, hydrocarbon substituent, which can be a single ring or multiple rings (preferably from 1 to 3 rings) that are fiised together (i.e., a fused ring aryl) or linked covalently.
  • a fiised ring aryl refers to multiple rings fused together wherein at least one of the fiised rings is an aryl ring and wherein the multiple rings are attached to the parent molecular moiety through any carbon atom contained within an aryl ring of the multiple rings.
  • heteroaryl refers to aryl groups (or rings) that contain at least one heteroatom such as N, O, or S, wherein the nitrogen and sulfiir atoms are optionally oxidized, and the nitrogen atom(s) are optionally quatemized.
  • heteroaryl includes fused ring heteroaryl groups (i.e., multiple rings fiised together wherein at least one of the fused rings is a heteroaromatic ring and wherein the multiple rings are attached to the parent molecular moiety through any atom contained within a heteroaromatic ring of the multiple rings).
  • a 5,6-fiised ring heteroarylene refers to two rings fused together, wherein one ring has 5 members and the other ring has 6 members, and wherein at least one ring is a heteroaryl ring.
  • a 6,6-fiised ring heteroarylene refers to two rings fused together, wherein one ring has 6 members and the other ring has 6 members, and wherein at least one ring is a heteroaryl ring.
  • a 6,5-fused ring heteroarylene refers to two rings fused together, wherein one ring has 6 members and the other ring has 5 members, and wherein at least one ring is a heteroaryl ring.
  • a heteroaryl group can be attached to the remainder of the molecule through a carbon or heteroatom.
  • aryl and heteroaryl groups include phenyl, naphthyl, pyrrolyl, pyrazolyl, pyridazinyl, triazinyl, pyrimidinyl, imidazolyl, pyrazinyl, purinyl, oxazolyl, isoxazolyl, thiazolyl, furyl, thienyl, pyridyl, pyrimidyl, benzothiazolyl, benzoxazoyl benzimidazolyl, benzofuran, isobenzofuranyl, indolyl, isoindolyl, benzothiophenyl, isoquinolyl, quinoxalinyl, quinolyl, 1 -naphthyl, 2-naphthyl, 4-biphenyl, 1 -pyrrolyl, 2-pyrrolyl
  • arylene and heteroarylene independently or as part of another substituent, mean a divalent radical derived from an aryl and heteroaryl, respectively.
  • a heteroaryl group substituent may be -O- bonded to a ring heteroatom nitrogen.
  • a fused ring heterocyloalkyl-aryl is an aryl fiised to a heterocycloalkyl.
  • a fused ring heterocycloalkyl-heteroaryl is a heteroaryl fused to a heterocycloalkyl.
  • a fiised ring heterocycloalkyl-cycloalkyl is a heterocycloalkyl fiised to a cycloalkyl.
  • a fiised ring heterocycloalkyl-heterocycloalkyl is a heterocycloalkyl fused to another heterocycloalkyl.
  • Fused ring heterocycloalkyl-aryl, fused ring heterocycloalkyl-heteroaryl, fused ring heterocycloalkyl-cycloalkyl, or fiised ring heterocycloalkyl-heterocycloalkyl may each independently be unsubstituted or substituted with one or more of the substitutents described herein.
  • Spirocyclic rings are two or more rings wherein adjacent rings are attached through a single atom.
  • the individual rings within spirocyclic rings may be identical or different.
  • Individual rings in spirocyclic rings may be substituted or unsubstituted and may have different substituents from other individual rings within a set of spirocyclic rings.
  • Possible substituents for individual rings within spirocyclic rings are the possible substituents for the same ring when not part of spirocyclic rings (e.g. substituents for cycloalkyl or heterocycloalkyl rings).
  • Spirocylic rings may be substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkyl or substituted or unsubstituted heterocycloalkylene and individual rings within a spirocyclic ring group may be any of the immediately previous list, including having all rings of one type (e.g. all rings being substituted heterocycloalkylene wherein each ring may be the same or different substituted heterocycloalkylene).
  • heterocyclic spirocyclic rings means a spirocyclic rings wherein at least one ring is a heterocyclic ring and wherein each ring may be a different ring.
  • substituted spirocyclic rings means that at least one ring is substituted and each substituent may optionally be different.
  • oxo means an oxygen that is double bonded to a carbon atom.
  • alkylsulfonyl means a moiety having the formula -S(O2)-R', where R' is a substituted or unsubstituted alkyl group as defined above. R' may have a specified number of carbons (e.g., “C 1 -C 4 alkylsulfonyl”).
  • alkylarylene as an arylene moiety covalently bonded to an alkylene moiety (also referred to herein as an alkylene linker).
  • alkylarylene group has the formula:
  • An alkylarylene moiety may be substituted (e.g. with a substituent group) on the alkylene moiety or the arylene linker (e.g. at carbons 2, 3, 4, or 6) with halogen, oxo, -N3, - CF 3 , -CCI3, -CBr 3 , -CI3, -CN, -CHO, -OH, -NH 2 , -COOH, -CONH2, -NO 2 , -SH, -SO2CH3 - SO3H, , -OSO3H, -SO2NH2, -NHNH2, -ONH2, -NHC(O)NHNH 2 , substituted or unsubstituted C 1 -C 5 alkyl or substituted or unsubstituted 2 to 5 membered heteroalkyl).
  • the alkylarylene is unsubstituted.
  • R, R', R", R'", and R" each preferably independently refer to hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl (e.g., aryl substituted with 1-3 halogens), substituted or unsubstituted heteroaryl, substituted or unsubstituted alkyl, alkoxy, or thioalkoxy groups, or arylalkyl groups.
  • aryl e.g., aryl substituted with 1-3 halogens
  • substituted or unsubstituted heteroaryl substituted or unsubstituted alkyl, alkoxy, or thioalkoxy groups, or arylalkyl groups.
  • each of the R groups is independently selected as are each R', R", R'", and R"" group when more than one of these groups is present.
  • R' and R" are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 4-, 5-, 6-, or 7- membered ring.
  • -NR'R includes, but is not limited to, 1-pyrrolidinyl and 4- morpholinyl.
  • alkyl is meant to include groups including carbon atoms bound to groups other than hydrogen groups, such as haloalkyl (e.g., -CF3 and -CH2CF3) and acyl (e.g., - C(O)CH 3 , -C(O)CF 3 , -C(O)CH 2 OCH3, and the like).
  • haloalkyl e.g., -CF3 and -CH2CF3
  • acyl e.g., - C(O)CH 3 , -C(O)CF 3 , -C(O)CH 2 OCH3, and the like.
  • Substituents for rings may be depicted as substituents on the ring rather than on a specific atom of a ring (commonly referred to as a floating substituent).
  • the substituent may be attached to any of the ring atoms (obeying the rules of chemical valency) and in the case of fused rings or spirocyclic rings, a substituent depicted as associated with one member of the fused rings or spirocyclic rings (a floating substituent on a single ring), may be a substituent on any of the fused rings or spirocyclic rings (a floating substituent on multiple rings).
  • the multiple substituents may be on the same atom, same ring, different atoms, different fused rings, different spirocyclic rings, and each substituent may optionally be different.
  • a point of attachment of a ring to the remainder of a molecule is not limited to a single atom (a floating substituent)
  • the attachment point may be any atom of the ring and in the case of a fused ring or spirocyclic ring, any atom of any of the fused rings or spirocyclic rings while obeying the rules of chemical valency.
  • a ring, fused rings, or spirocyclic rings contain one or more ring heteroatoms and the ring, fused rings, or spirocyclic rings are shown with one more floating substituents (including, but not limited to, points of attachment to the remainder of the molecule), the floating substituents may be bonded to the heteroatoms.
  • the ring heteroatoms are shown bound to one or more hydrogens (e.g. a ring nitrogen with two bonds to ring atoms and a third bond to a hydrogen) in the structure or formula with the floating substituent, when the heteroatom is bonded to the floating substituent, the substituent will be understood to replace the hydrogen, while obeying the rules of chemical valency.
  • Two or more substituents may optionally be joined to form aryl, heteroaryl, cycloalkyl, or heterocycloalkyl groups.
  • Such so-called ring-forming substituents are typically, though not necessarily, found attached to a cyclic base structure.
  • the ring-forming substituents are attached to adjacent members of the base structure.
  • two ring-forming substituents attached to adjacent members of a cyclic base structure create a fused ring structure.
  • the ring-forming substituents are attached to a single member of the base structure.
  • two ring-forming substituents attached to a single member of a cyclic base structure create a spirocyclic structure.
  • the ring-forming substituents are attached to non- adjacent members of the base structure.
  • Two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally form a ring of the formula -T-C(O)-(CRR') q -U-, wherein T and U are independently -NR-, -O-, -CRR'-, or a single bond, and q is an integer of from 0 to 3.
  • two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -A-(CH2) r -B-, wherein A and B are independently -CRR'-, -O-, -NR-, -S-, -S(O) -, -S(O)2-, -S(O)2NR'-, or a single bond, and r is an integer of from 1 to 4.
  • One of the single bonds of the new ring so formed may optionally be replaced with a double bond.
  • two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula - (CRR')s-X'- (C"R"R'")d-, where s and d are independently integers of from 0 to 3, and X' is - O-, -NR'-, -S-, -S(O)-, -S(O) 2 -, or -S(O) 2 NR'-.
  • R, R', R", and R' are preferably independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl.
  • heteroatom or “ring heteroatom” are meant to include oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), and silicon (Si).
  • a “substituent group,” as used herein, means a group selected from the following moieties:
  • alkyl e.g., C 1 -C 8 alkyl, C 1 -C 6 alkyl, or C 1 -C 4 alkyl
  • heteroalkyl e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered heteroalkyl, or 2 to 4 membered heteroalkyl
  • cycloalkyl e.g., C 3 -C 8 cycloalkyl, C 3 -C 6 cycloalkyl, or C 5 -C 6 cycloalkyl
  • heterocycloalkyl e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl
  • aryl e.g., C 6 -C 10 aryl, C 10 aryl, or phenyl
  • heteroaryl e.g., 5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or
  • alkyl e.g., C 1 -C 8 alkyl, C 1 -C 6 alkyl, or C 1 -C 4 alkyl
  • heteroalkyl e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered heteroalkyl, or 2 to 4 membered heteroalkyl
  • cycloalkyl e.g., C 3 -C 8 cycloalkyl, C 3 -C 6 cycloalkyl, or C 5 -C 6 cycloalkyl
  • heterocycloalkyl e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl
  • aryl e.g., C 6 - C 10 aryl, C 10 aryl, or phenyl
  • heteroaryl e.g., 5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or
  • alkyl e.g., C 1 -C 8 alkyl, C 1 -C 6 alkyl, or C 1 -C 4 alkyl
  • heteroalkyl e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered heteroalkyl, or 2 to 4 membered heteroalkyl
  • cycloalkyl e.g., C 3 -C 8 cycloalkyl, C 3 -C 6 cycloalkyl, or C 5 -C 6 cycloalkyl
  • heterocycloalkyl e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl
  • aryl e.g., C 6 - C 10 aryl, C 10 aryl, or phenyl
  • heteroaryl e.g., 5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or
  • a “size-limited substituent” or “ size-limited substituent group,” as used herein, means a group selected from all of the substituents described above for a “substituent group,” wherein each substituted or unsubstituted alkyl is a substituted or unsubstituted C 1 -C20 alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 20 membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C 3 -C 8 cycloalkyl, each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 8 membered heterocycloalkyl, each substituted or unsubstituted aryl is a substituted or unsubstituted C 6 -C 10 aryl, and each substituted or unsubstituted heteroary
  • a “lower substituent” or “ lower substituent group,” as used herein, means a group selected from all of the substituents described above for a “substituent group,” wherein each substituted or unsubstituted alkyl is a substituted or unsubstituted C 1 -C 8 alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8 membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C 3 - C 7 cycloalkyl, each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 7 membered heterocycloalkyl, each substituted or unsubstituted aryl is a substituted or unsubstituted phenyl, and each substituted or unsubstituted heteroaryl is a substituted or un
  • each substituted group described in the compounds herein is substituted with at least one substituent group. More specifically, in some embodiments, each substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and/or substituted heteroarylene described in the compounds herein are substituted with at least one substituent group. In other embodiments, at least one or all of these groups are substituted with at least one size-limited substituent group. In other embodiments, at least one or all of these groups are substituted with at least one lower substituent group.
  • each substituted or unsubstituted alkyl may be a substituted or unsubstituted C 1 -C20 alkyl
  • each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 20 membered heteroalkyl
  • each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C 3 -C 8 cycloalkyl
  • each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 8 membered heterocycloalkyl
  • each substituted or unsubstituted aryl is a substituted or unsubstituted C 6 - C 10 aryl
  • each substituted or unsubstituted heteroaryl is a substituted or unsubstituted 5 to 10 membered heteroaryl.
  • each substituted or unsubstituted alkylene is a substituted or unsubstituted C 1 -C20 alkylene
  • each substituted or unsubstituted heteroalkylene is a substituted or unsubstituted 2 to 20 membered heteroalkylene
  • each substituted or unsubstituted cycloalkylene is a substituted or unsubstituted C 3 -C 8 cycloalkylene
  • each substituted or unsubstituted heterocycloalkylene is a substituted or unsubstituted 3 to 8 membered heterocycloalkylene
  • each substituted or unsubstituted arylene is a substituted or unsubstituted C 6 -C 10 arylene
  • each substituted or unsubstituted heteroarylene is a substituted or unsubstituted 5 to 10 membered heteroarylene.
  • each substituted or unsubstituted alkyl is a substituted or unsubstituted C 1 -C 8 alkyl
  • each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8 membered heteroalkyl
  • each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C 3 -C 7 cycloalkyl
  • each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 7 membered heterocycloalkyl
  • each substituted or unsubstituted aryl is a substituted or unsubstituted C 6 -C 10 aryl
  • each substituted or unsubstituted heteroaryl is a substituted or unsubstituted 5 to 9 membered heteroaryl.
  • each substituted or unsubstituted alkylene is a substituted or unsubstituted C 1 -C 8 alkylene
  • each substituted or unsubstituted heteroalkylene is a substituted or unsubstituted 2 to 8 membered heteroalkylene
  • each substituted or unsubstituted cycloalkylene is a substituted or unsubstituted C 3 -C 7 cycloalkylene
  • each substituted or unsubstituted heterocycloalkylene is a substituted or unsubstituted 3 to 7 membered heterocycloalkylene
  • each substituted or unsubstituted arylene is a substituted or unsubstituted C 6 -C 10 arylene
  • each substituted or unsubstituted heteroarylene is a substituted or unsubstituted 5 to 9 membered heteroarylene.
  • the compound is a chemical species set forth in the Examples section, figures, or tables
  • a substituted or unsubstituted moiety e.g., substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, and/or substituted or unsubstituted heteroarylene) is unsubstituted (e.g., is an unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted
  • a substituted or unsubstituted moiety e.g., substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, and/or substituted or unsubstituted heteroarylene) is substituted (e.g., is a substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alky
  • a substituted moiety e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and/or substituted heteroarylene
  • is substituted with at least one substituent group wherein if the substituted moiety is substituted with a plurality of substituent groups, each substituent group may optionally be different. In embodiments, if the substituted moiety is substituted with a plurality of substituent groups, each substituent group is different.
  • a substituted moiety e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and/or substituted heteroarylene
  • is substituted with at least one size-limited substituent group wherein if the substituted moiety is substituted with a plurality of size-limited substituent groups, each size-limited substituent group may optionally be different. In embodiments, if the substituted moiety is substituted with a plurality of size-limited substituent groups, each size-limited substituent group is different.
  • a substituted moiety e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and/or substituted heteroarylene
  • is substituted with at least one lower substituent group wherein if the substituted moiety is substituted with a plurality of lower substituent groups, each lower substituent group may optionally be different. In embodiments, if the substituted moiety is substituted with a plurality of lower substituent groups, each lower substituent group is different.
  • a substituted moiety e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and/or substituted heteroarylene
  • each substituent group, size-limited substituent group, and/or lower substituent group is different.
  • Certain compounds of the present disclosure possess asymmetric carbon atoms (optical or chiral centers) or double bonds; the enantiomers, racemates, diastereomers, tautomers, geometric isomers, stereoisometric forms that may be defined, in terms of absolute stereochemistry, as (R)-or (S)- or, as (D)- or (L)- for amino acids, and individual isomers are encompassed within the scope of the present disclosure.
  • the compounds of the present disclosure do not include those that are known in art to be too unstable to synthesize and/or isolate.
  • the present disclosure is meant to include compounds in racemic and optically pure forms.
  • Optically active (R)- and (S)-, or (D)- and (L)-isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques.
  • the compounds described herein contain olefinic bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers.
  • isomers refers to compounds having the same number and kind of atoms, and hence the same molecular weight, but differing in respect to the structural arrangement or configuration of the atoms.
  • tautomer refers to one of two or more structural isomers which exist in equilibrium and which are readily converted from one isomeric form to another.
  • structures depicted herein are also meant to include all stereochemical forms of the structure; i.e., the R and S configurations for each asymmetric center. Therefore, single stereochemical isomers as well as enantiomeric and diastereomeric mixtures of the present compounds are within the scope of the disclosure.
  • structures depicted herein are also meant to include compounds which differ only in the presence of one or more isotopically enriched atoms.
  • compounds having the present structures except for the replacement of a hydrogen by a deuterium or tritium, or the replacement of a carbon by 13 C- or 14 C-enriched carbon are within the scope of this disclosure.
  • the compounds of the present disclosure may also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds.
  • the compounds may be radiolabeled with radioactive isotopes, such as for example tritium ( 3 H), iodine-125 ( 125 I), or carbon-14 ( 14 C). All isotopic variations of the compounds of the present disclosure, whether radioactive or not, are encompassed within the scope of the present disclosure.
  • bioconjugate and “bioconjugate linker” refers to the resulting association between atoms or molecules of “bioconjugate reactive groups” or “bioconjugate reactive moieties”. The association can be direct or indirect.
  • a conjugate between a first bioconjugate reactive group e.g., -NH2, -C(O)OH, -N- hydroxysuccinimide, or -maleimide
  • a second bioconjugate reactive group e.g., sulfhydryl, sulfur-containing amino acid, amine, amine sidechain containing amino acid, or carboxylate
  • covalent bond or linker e.g. a first linker of second linker
  • indirect e.g., by non-covalent bond (e.g. electrostatic interactions (e.g. ionic bond, hydrogen bond, halogen bond), van der Waals interactions (e.g.
  • bioconjugates or bioconjugate linkers are formed using bioconjugate chemistry (i.e. the association of two bioconjugate reactive groups) including, but are not limited to nucleophilic substitutions (e.g., reactions of amines and alcohols with acyl halides, active esters), electrophilic substitutions (e.g., enamine reactions) and additions to carbon-carbon and carbon-heteroatom multiple bonds (e.g., Michael reaction, Diels-Alder addition).
  • bioconjugate chemistry i.e. the association of two bioconjugate reactive groups
  • nucleophilic substitutions e.g., reactions of amines and alcohols with acyl halides, active esters
  • electrophilic substitutions e.g., enamine reactions
  • additions to carbon-carbon and carbon-heteroatom multiple bonds e.g., Michael reaction, Diels-Alder addition.
  • the first bioconjugate reactive group e.g., maleimide moiety
  • the second bioconjugate reactive group e.g. a sulfhydryl
  • the first bioconjugate reactive group (e.g., haloacetyl moiety) is covalently attached to the second bioconjugate reactive group (e.g. a sulfhydryl).
  • the first bioconjugate reactive group (e.g., pyridyl moiety) is covalently attached to the second bioconjugate reactive group (e.g. a sulfhydryl).
  • the first bioconjugate reactive group e.g., -N- hydroxysuccinimide moiety
  • is covalently attached to the second bioconjugate reactive group (e.g. an amine).
  • the first bioconjugate reactive group (e.g., maleimide moiety) is covalently attached to the second bioconjugate reactive group (e.g. a sulfhydryl).
  • the first bioconjugate reactive group e.g., -sulfo-N-hydroxy succinimide moiety
  • the second bioconjugate reactive group e.g. an amine
  • bioconjugate reactive moieties used for bioconjugate chemistries herein include, for example:
  • haloalkyl groups wherein the halide can be later displaced with a nucleophilic group such as, for example, an amine, a carboxylate anion, thiol anion, carbanion, or an alkoxide ion, thereby resulting in the covalent attachment of a new group at the site of the halogen atom;
  • a nucleophilic group such as, for example, an amine, a carboxylate anion, thiol anion, carbanion, or an alkoxide ion
  • dienophile groups which are capable of participating in Diels-Alder reactions such as, for example, maleimido or maleimide groups;
  • aldehyde or ketone groups such that subsequent derivatization is possible via formation of carbonyl derivatives such as, for example, imines, hydrazones, semicarbazones or oximes, or via such mechanisms as Grignard addition or alkyllithium addition;
  • amine or sulfhydryl groups e.g., present in cysteine
  • cysteine amine or sulfhydryl groups
  • alkenes which can undergo, for example, cycloadditions, acylation, Michael addition, etc;
  • biotin conjugate can react with avidin or strepavidin to form a avidin-biotin complex or streptavidin-biotin complex.
  • bioconjugate reactive groups can be chosen such that they do not participate in, or interfere with, the chemical stability of the conjugate described herein.
  • a reactive functional group can be protected from participating in the crosslinking reaction by the presence of a protecting group.
  • the bioconjugate comprises a molecular entity derived from the reaction of an unsaturated bond, such as a maleimide, and a sulfhydryl group.
  • an analog is used in accordance with its plain ordinary meaning within Chemistry and Biology and refers to a chemical compound that is structurally similar to another compound (i.e., a so-called “reference” compound) but differs in composition, e.g., in the replacement of one atom by an atom of a different element, or in the presence of a particular functional group, or the replacement of one functional group by another functional group, or the absolute stereochemistry of one or more chiral centers of the reference compound. Accordingly, an analog is a compound that is similar or comparable in function and appearance but not in structure or origin to a reference compound.
  • a or “an,” as used in herein means one or more.
  • substituted with a[n] means the specified group may be substituted with one or more of any or all of the named substituents.
  • a group such as an alkyl or heteroaryl group
  • the group may contain one or more unsubstituted C 1 -C20 alkyls, and/or one or more unsubstituted 2 to 20 membered heteroalkyls.
  • R-substituted where a moiety is substituted with an R substituent, the group may be referred to as “R-substituted.” Where a moiety is R-substituted, the moiety is substituted with at least one R substituent and each R substituent is optionally different. Where a particular R group is present in the description of a chemical genus (such as Formula (I)), a Roman alphabetic symbol may be used to distinguish each appearance of that particular R group. For example, where multiple R 13 substituents are present, each R 13 substituent may be distinguished as R 13 A , R 13 B , R 13 C , R 13 D , etc., wherein each of R 13 A , R 13 B , R 13 C , R 13 D , etc. is defined within the scope of the definition of R 13 and optionally differently.
  • a “detectable agent” or “detectable moiety” is a composition, substance, element, or compound; or moiety thereof; detectable by appropriate means such as spectroscopic, photochemical, biochemical, immunochemical, chemical, magnetic resonance imaging, or other physical means.
  • useful detectable agents include 18 F, 32 P, 33 P, 45 Ti, 47 Sc, Er, Tm, Yb, Lu, 32 P, fluorophore (e.g.
  • fluorescent dyes include fluorescent dyes), electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, paramagnetic molecules, paramagnetic nanoparticles, ultrasmall superparamagnetic iron oxide (“USPIO”) nanoparticles, USPIO nanoparticle aggregates, superparamagnetic iron oxide (“SPIO”) nanoparticles, SPIO nanoparticle aggregates, monochrystalline iron oxide nanoparticles, monochrystalline iron oxide, nanoparticle contrast agents, liposomes or other delivery vehicles containing Gadolinium chelate (“Gd-chelate”) molecules, Gadolinium, radioisotopes, radionuclides (e.g.
  • microbubbles e.g. including microbubble shells including albumin, galactose, lipid, and/or polymers; microbubble gas core including air, heavy gas(es), perfluorcarbon, nitrogen, octafluoropropane, perflexane lipid microsphere, perflutren, etc.
  • iodinated contrast agents e.g.
  • a detectable moiety is a monovalent detectable agent or a detectable agent capable of forming a bond with another composition.
  • Radioactive substances e.g., radioisotopes
  • Paramagnetic ions that may be used as additional imaging agents in accordance with the embodiments of the disclosure include, but are not limited to, ions of transition and lanthanide metals (e.g.
  • the term “leaving group” is used in accordance with its ordinary meaning in chemistry and refers to a moiety (e.g., atom, functional group, molecule) that separates from the molecule following a chemical reaction (e.g., bond formation, reductive elimination, condensation, cross-coupling reaction) involving an atom or chemical moiety to which the leaving group is attached, also referred to herein as the “leaving group reactive moiety”, and a complementary reactive moiety (i.e. a chemical moiety that reacts with the leaving group reactive moiety) to form a new bond between the remnants of the leaving groups reactive moiety and the complementary reactive moiety.
  • a chemical reaction e.g., bond formation, reductive elimination, condensation, cross-coupling reaction
  • a complementary reactive moiety i.e. a chemical moiety that reacts with the leaving group reactive moiety
  • Non limiting examples of leaving groups include hydrogen, hydroxide, organotin moieties (e.g., organotin heteroalkyl), halogen (e.g., Br), perfluoroalkylsulfonates (e.g. triflate), tosylates, mesylates, water, alcohols, nitrate, phosphate, thioether, amines, ammonia, fluoride, carboxylate, phenoxides, boronic acid, boronate esters, and alkoxides.
  • organotin moieties e.g., organotin heteroalkyl
  • halogen e.g., Br
  • perfluoroalkylsulfonates e.g. triflate
  • tosylates mesylates, water, alcohols, nitrate, phosphate, thioether, amines, ammonia, fluoride, carboxylate, phenoxides, boronic
  • two molecules with leaving groups are allowed to contact, and upon a reaction and/or bond formation (e.g., acyloin condensation, aldol condensation, Claisen condensation, Stille reaction) the leaving groups separates from the respective molecule.
  • a leaving group is a bioconjugate reactive moiety.
  • at least two leaving groups e.g., R 1 and R 13 ) are allowed to contact such that the leaving groups are sufficiently proximal to react, interact or physically touch.
  • the leaving groups is designed to facilitate the reaction.
  • protecting group is used in accordance with its ordinary meaning in organic chemistry and refers to a moiety covalently bound to a heteroatom, heterocycloalkyl, or heteroaryl to prevent reactivity of the heteroatom, heterocycloalkyl, or heteroaryl during one or more chemical reactions performed prior to removal of the protecting group.
  • a protecting group is bound to a heteroatom (e.g., O) during a part of a multipart synthesis wherein it is not desired to have the heteroatom react (e.g., a chemical reduction) with the reagent. Following protection the protecting group may be removed (e.g., by modulating the pH).
  • the protecting group is an alcohol protecting group.
  • alcohol protecting groups include acetyl, benzoyl, benzyl, methoxymethyl ether (MOM), tetrahydropyranyl (THP), and silyl ether (e.g., trimethylsilyl (TMS)).
  • the protecting group is an amine protecting group.
  • Non-limiting examples of amine protecting groups include carbobenzyloxy (Cbz), tert-butyloxycarbonyl (BOC), 9-Fluorenylmethyloxycarbonyl (FMOC), acetyl, benzoyl, benzyl, carbamate, p- methoxybenzyl ether (PMB), and tosyl (Ts).
  • variable e.g., moiety or linker
  • a compound or of a compound genus e.g., a genus described herein
  • the unfilled valence(s) of the variable will be dictated by the context in which the variable is used.
  • variable of a compound as described herein when a variable of a compound as described herein is connected (e.g., bonded) to the remainder of the compound through a single bond, that variable is understood to represent a monovalent form (i.e., capable of forming a single bond due to an unfilled valence) of a standalone compound (e.g., if the variable is named “methane” in an embodiment but the variable is known to be attached by a single bond to the remainder of the compound, a person of ordinary skill in the art would understand that the variable is actually a monovalent form of methane, i.e., methyl or -CH3).
  • variable is the divalent form of a standalone compound (e.g., if the variable is assigned to “PEG” or “polyethylene glycol” in an embodiment but the variable is connected by two separate bonds to the remainder of the compound, a person of ordinary skill in the art would understand that the variable is a divalent (i.e., capable of forming two bonds through two unfilled valences) form of PEG instead of the standalone compound PEG).
  • exogenous refers to a molecule or substance (e.g., a compound, nucleic acid or protein) that originates from outside a given cell or organism.
  • an "exogenous promoter” as referred to herein is a promoter that does not originate from the plant it is expressed by.
  • endogenous or endogenous promoter refers to a molecule or substance that is native to, or originates within, a given cell or organism.
  • lipid moiety is used in accordance with its ordinary meaning in chemistry and refers to a hydrophobic molecule which is typically characterized by an aliphatic hydrocarbon chain.
  • the lipid moiety includes a carbon chain of 3 to 100 carbons.
  • the lipid moiety includes a carbon chain of 5 to 50 carbons.
  • the lipid moiety includes a carbon chain of 5 to 25 carbons.
  • the lipid moiety includes a carbon chain of 8 to 525 carbons.
  • Lipid moieties may include saturated or unsaturated carbon chains, and may be optionally substituted.
  • the lipid moiety is optionally substituted with a charged moiety at the terminal end.
  • the lipid moiety is an alkyl or heteroalkyl optionally substituted with a carboxylic acid moiety at the terminal end.
  • a charged moiety refers to a functional group possessing an abundance of electron density (i.e. electronegative) or is deficient in electron density (i.e. electropositive).
  • Nonlimiting examples of a charged moiety includes carboxylic acid, alcohol, phosphate, aldehyde, and sulfonamide.
  • a charged moiety is capable of forming hydrogen bonds.
  • the term “coupling reagent” is used in accordance with its plain ordinary meaning in the arts and refers to a substance (e.g., a compound or solution) which participates in chemical reaction and results in the formation of a covalent bond (e.g., between bioconjugate reactive moieties, between a bioconjugate reactive moiety and the coupling reagent).
  • a covalent bond e.g., between bioconjugate reactive moieties, between a bioconjugate reactive moiety and the coupling reagent.
  • the level of reagent is depleted in the course of a chemical reaction. This is in contrast to a solvent, which typically does not get consumed over the course of the chemical reaction.
  • Non-limiting examples of coupling reagents include benzotriazol- 1-yl- oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), 7-Azabenzotriazol-l- yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP), 6-Chloro-benzotriazole-l- yloxy-tris-pyrrolidinophosphonium hexafluorophosphate (PyClock), 1- [Bis(dimethylamino)methylene] - 1 H- 1 ,2, 3 -triazolo [4, 5 -b]pyridinium 3 -oxid hexafluorophosphate (HATU), or 2-(lH-benzotriazol-l-yl)-l,l,3,3-tetramethyluronium hexafluorophosphate (HBTU).
  • PyBOP benzotriazol- 1-yl-
  • solution is used in accor and refers to a liquid mixture in which the minor component (e.g., a solute or compound) is uniformly distributed within the major component (e.g., a solvent).
  • minor component e.g., a solute or compound
  • organic solvent as used herein is used in accordance with its ordinary meaning in chemistry and refers to a solvent which includes carbon.
  • organic solvents include acetic acid, acetone, acetonitrile, benzene, 1 -butanol, 2-butanol, 2-butanone, t-butyl alcohol, carbon tetrachloride, chlorobenzene, chloroform, cyclohexane, 1,2-dichloroethane, diethylene glycol, diethyl ether, diglyme (diethylene glycol , dimethyl ether), 1,2-dimethoxyethane (glyme, DME), dimethylformamide (DMF), dimethyl sulfoxide (DMSO), 1,4-dioxane, ethanol, ethyl acetate, ethylene glycol, glycerin, heptane, hexamethylphosphoramide (HMPA), hexamethylphosphorous,
  • salt refers to acid or base salts of the compounds used in the methods of the present invention.
  • acceptable salts are mineral acid (hydrochloric acid, hydrobromic acid, phosphoric acid, and the like) salts, organic acid (acetic acid, propionic acid, glutamic acid, citric acid and the like) salts, quaternary ammonium (methyl iodide, ethyl iodide, and the like) salts.
  • bound and bound as used herein is used in accordance with its plain and ordinary meaning and refers to the association between atoms or molecules.
  • the association can be direct or indirect.
  • bound atoms or molecules may be bound, e.g., by covalent bond, linker (e.g. a first linker or second linker), or non-covalent bond (e.g. electrostatic interactions (e.g. ionic bond, hydrogen bond, halogen bond), van der Waals interactions (e.g. dipole-dipole, dipole-induced dipole, London dispersion), ring stacking (pi effects), hydrophobic interactions and the like).
  • linker e.g. a first linker or second linker
  • non-covalent bond e.g. electrostatic interactions (e.g. ionic bond, hydrogen bond, halogen bond), van der Waals interactions (e.g. dipole-dipole, dipole-induced dipole, London dispersion), ring stacking (pi effects), hydrophobic interactions and the
  • the term “capable of binding” as used herein refers to a moiety (e.g. a compound as described herein) that is able to measurably bind to a target (e.g., a NF-KB, a Toll-like receptor protein).
  • a target e.g., a NF-KB, a Toll-like receptor protein.
  • the moiety is capable of binding with a Kd of less than about 10 pM, 5 pM, 1 pM, 500 nM, 250 nM, 100 nM, 75 nM, 50 nM, 25 nM, 15 nM, 10 nM, 5 nM, 1 nM, or about 0.1 nM.
  • conjugated when referring to two moieties means the two moieties are bonded, wherein the bond or bonds connecting the two moieties may be covalent or non-covalent.
  • the two moieties are covalently bonded to each other (e.g. directly or through a covalently bonded intermediary).
  • the two moieties are non-covalently bonded (e.g. through ionic bond(s), van der waal’s bond(s)/interactions, hydrogen bond(s), polar bond(s), or combinations or mixtures thereof).
  • non-nucleophilic base refers to any sterically hindered base that is a poor nucleophile.
  • nucleophile refers to a chemical species that donates an electron pair to an electrophile to form a chemical bond in relation to a reaction. All molecules or ions with a free pair of electrons or at least one pi bond can act as nucleophiles.
  • strong acid refers to an acid that is completely dissociated or ionized in an aqueous solution.
  • strong acids include hydrochloric acid (HC 1 ), nitric acid (HNO3), sulfuric acid (H2SO4), hydrobromic acid (HBr), hydroiodic acid (HI), perchloric acid (HCIO4), or chloric acid (HCIO3).
  • carbocation stabilizing solvent refers to any polar protic solvent capable of forming dipole-dipole interactions with a carbocation, thereby stabilizing the carbocation.
  • a compound as described herein including the compound of FIG. 3C or analog thereof or derivative thereof.
  • the compound has the formula:
  • R 1 and R 2 ' are independently hydrogen or substituted or unsubstituted alkyl. In embodiments, R 1 ' and R 2 ' are independently substituted or unsubstituted alkyl. In embodiments, R 1 ' and R 2 ' are independently substituted or unsubstituted C 1 -C 10 alkyl. In embodiments, R 1 ' and R 2 ' are independently substituted or unsubstituted C 1 -C 5 alkyl. In embodiments, R 1 ' and R 2 ' are independently unsubstituted alkyl. In embodiments, R 1 ' and R 2 ' are independently unsubstituted C 1 -C 10 alkyl.
  • R 1 ' and R 2 ' are independently unsubstituted C 1 -C 5 alkyl. In embodiments R 1 ' and R 2 ' are methyl. In embodiments, where R 1 ' is substituted, R 1 ' is substituted with a substituent group. In embodiments, where R 1 ' is substituted, R 1 ' is substituted with a size limited substituent group. In embodiments, where R 1 ' is substituted, R 1 ' is substituted with a lower substituent group. In embodiments, where R 2 ' is substituted, R 2 ' is substituted with a substituent group. In embodiments, where R 2 ' is substituted, R 2 ' is substituted with a size limited substituent group. In embodiments, where R 2 ' is substituted, R 2 ' is substituted, R 2 ' is substituted with a lower substituent group.
  • L 1 ' is a bond, -O-, -S-, -NH-, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene.
  • L r is -O-, -S-, -NH-, unsubstituted alkylene, unsubstituted heteroalkylene, unsubstituted cycloalkylene, unsubstituted heterocycloalkylene, unsubstituted arylene, or unsubstituted heteroarylene.
  • L 1 is substituted or unsubstituted alkylene.
  • L r is substituted or unsubstituted C 1 -C 10 alkylene.
  • L r is substituted or unsubstituted C 1 -C 5 alkylene.
  • L r is unsubstituted alkylene.
  • L r is unsubstituted C 1 -C 10 alkylene. In embodiments, L r is unsubstituted C 1 -C 5 alkylene. In embodiments, L r is methylene. In embodiments, where L r is substituted, L r is substituted with a substituent group. In embodiments, where L r is substituted, L r is substituted with a size limited substituent group. In embodiments, where L 1 ' is substituted, L 1 ' is substituted with a lower substituent group.
  • R 3 is substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl.
  • R 3 ' is substituted or unsubstituted furanyl.
  • R 3 ' is substituted or unsubstituted pyrrolyl.
  • R 3 ' is substituted or unsubstituted thiophenyl.
  • R 3 ' is substituted or unsubstituted thiophenyl.
  • R 3 ' is substituted with a substituent group. In embodiments, where R 3 ' is substituted, R 3 ' is substituted with a size limited substituent group. In embodiments, where R 3 ' is substituted, R 3 ' is substituted with a lower substituent group. In embodiments, where R 3 ' is substituted, R 3 ' is substituted with a -OH, halogen, alkoxy (e.g.
  • R 3 ' is substituted, R 3 ' is substituted with a -Cl, -F, -OMe, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl.
  • R 3 ' is substituted with a -OH, halogen, alkoxy (e.g. methoxy), trifluromethyl, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl or unsubstituted heteroaryl.
  • alkoxy e.g. methoxy
  • R 3 ' is substituted with a -Cl, -F, -OMe, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl or unsubstituted heteroaryl. In embodiments, where R 3 ' is substituted, R 3 ' is substituted with a -Cl, -F, -OMe or unsubstituted aryl or substituted or unsubstituted heteroaryl. In embodiments, where R 3 ' is substituted, R 3 ' is substituted with a - Cl, -F, -OMe or unsubstituted aryl. In embodiments, where R 3 ' is substituted, R 3 ' is substituted, R 3 ' is substituted with a -Cl, -F, -OMe or unsubstituted phenyl.
  • the structure of the compound is:
  • a compound as described herein including the compound of having a structure of Formula (I): (I), or a pharmaceutically acceptable salt thereof.
  • L 1 is a bond, -NH-, -O-, -S-, -C(O)-, -C(O)NH-, -NHC(O)-, -NHC(O)NH-, -C(O)O-, -OC(O)-, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene.
  • R 1 is hydrogen, halogen
  • R 2 is hydrogen, halogen, -CX 2 3 , -CHX 2 2 , -CH 2 X 2 , -OCX 2 3 , -
  • R 1 and R 2 may optionally be joined to form a substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.
  • R 4 is indendently halogen, -CX 4 3 , -CHX 4 2 , -CH2X 4 , -OCX 4 3 , -
  • R 1A , R 1B , R 1C , R 1D , R 2A , R 2B , R 2C , R 2D , R 4A , R 4B , R 4C , and R 4D are independently hydrogen, -CX 3 , -CHX2, -CH2X, -CN, -OH, -COOH, -CONH2, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; R 1A and R 1B or R 2A and R 2B with substituents bonded to the same nitrogen atom may optionally be joined to form a substituted or unsubstituted heterocycloalkyl or substituted or unsubstituted heteroaryl.
  • Each X, X 1 , X 2 , and X 4 is independently -F, -Cl, -Br, or -I.
  • nl, n2 and n4 is independently an integer from 0 to 4.
  • z is an integer from 0 to 5.
  • Each ml, m2, m4, vl, v2 and v4 are independently 1 or 2.
  • L 1 is a bond, substituted or unsubstituted alkylene, or substituted or unsubstituted heteroalkylene. In embodiments, L 1 is a bond. In embodiments, L 1 is substituted or unsubstituted alkylene. In embodiments, L 1 is substituted or unsubstituted heteroalkylene.
  • z is 0. In embodiments, z is 1. In embodiments, z is 2. In embodiments, z is 3. In embodiments, z is 4. [0180] In embodiments, L 1 is substituted or unsubstituted C 1 -C 3 alkylene. In embodiments, L 1 is substituted C 1 -C 3 alkylene. In embodiments, L 1 is unsubstituted C 1 -C 3 alkylene. In embodiments, L 1 is substituted methylene. In embodiments, L 1 is unsubstituted methylene. In embodiments, L 1 is substituted ethylene. In embodiments, L 1 is unsubstituted ethylene. In embodiments, L 1 is substituted propylene. In embodiments, L 1 is unsubstituted propylene. In embodiments, L 1 is substituted isopropylene. In embodiments, L 1 is unsubstituted isopropylene. In embodiments, L 1 is unsubstituted isopropylene.
  • L 1 is substituted or unsubstituted 2 to 3 membered heteroalkylene. In embodiments, L 1 is substituted 2 to 3 membered heteroalkylene. In embodiments, L 1 is unsubstituted 2 to 3 membered heteroalkylene. In embodiments, L 1 is substituted or unsubstituted 2 membered heteroalkylene. In embodiments, L 1 is substituted 2 membered heteroalkylene. In embodiments, L 1 is unsubstituted 2 membered heteroalkylene. In embodiments, L 1 is substituted or unsubstituted 3 membered heteroalkylene. In embodiments, L 1 is substituted 3 membered heteroalkylene. In embodiments, L 1 is unsubstituted 3 membered heteroalkylene.
  • the compound has a structure of are as described herein.
  • the compound has a structure of are as described herein.
  • each R 1 andR 2 is independently substituted or unsubstituted alkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl.
  • R 1 is substituted or unsubstituted alkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl.
  • R 1 is substituted or unsubstituted alkyl.
  • R 1 is substituted or unsubstituted aryl.
  • R 1 is substituted or unsubstituted heteroaryl.
  • R 2 is substituted or unsubstituted alkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl. In embodiments, R 2 is substituted or unsubstituted alkyl. In embodiments, R 2 is substituted or unsubstituted aryl. In embodiments, R 2 is substituted or unsubstituted heteroaryl.
  • each R 1 andR 2 is independently substituted or unsubstituted C 1 -C 4 alkyl, or substituted or unsubstituted phenyl. In embodiments, each R 1 andR 2 is independently substituted or unsubstituted C 1 -C 4 alkyl.
  • R 1 is substituted or unsubstituted C 1 -C 4 alkyl, or substituted or unsubstituted phenyl. In embodiments, R 1 is substituted or unsubstituted C 1 -C 4 alkyl. In embodiments, R 1 is substituted or unsubstituted phenyl. In embodiments, R 1 is substituted C 1 -C 4 alkyl, or substituted phenyl. In embodiments, R 1 is substituted C 1 -C 4 alkyl. In embodiments, R 1 is substituted phenyl. In embodiments, R 1 is unsubstituted C 1 -C 4 alkyl, or unsubstituted phenyl. In embodiments, R 1 is unsubstituted C 1 -C 4 alkyl. In embodiments, R 1 is unsubstituted phenyl.
  • R 2 is substituted or unsubstituted C 1 -C 4 alkyl, or substituted or unsubstituted phenyl. In embodiments, R 2 is substituted or unsubstituted C 1 -C 4 alkyl. In embodiments, R 2 is substituted or unsubstituted phenyl. In embodiments, R 2 is substituted C 1 -C 4 alkyl, or substituted phenyl. In embodiments, R 2 is substituted C 1 -C 4 alkyl. In embodiments, R 2 is substituted phenyl. In embodiments, R 2 is unsubstituted C 1 -C 4 alkyl, or unsubstituted phenyl. In embodiments, R 2 is unsubstituted C 1 -C 4 alkyl. In embodiments, R 2 is unsubstituted phenyl.
  • R 1 is unsubstituted phenyl
  • R 2 is unsubstituted C 1 -C 4 alkyl.
  • R 2 is unsubstituted methyl.
  • R 2 is unsubstituted ethyl.
  • R 2 is unsubstituted propyl.
  • R 2 is unsubstituted isopropyl.
  • R 2 is unsubstituted butyl.
  • R 2 is unsubstituted isobutyl.
  • R 2 is unsubstituted t-butyl.
  • R 1 is unsubstituted phenyl.
  • R 1 is unsubstituted C 1 -C 4 alkyl
  • R 2 is unsubstituted phenyl.
  • R 1 is unsubstituted methyl.
  • R 1 is unsubstituted ethyl.
  • R 1 is unsubstituted propyl.
  • R 1 is unsubstituted isopropyl.
  • R 1 is unsubstituted butyl.
  • R 1 is unsubstituted isobutyl.
  • R 1 is unsubstituted t-butyl.
  • R 2 is unsubstituted phenyl.
  • z is 0. In embodiments, z is 1 and R 4 is halogen. In embodiments, z is 1 and R 4 is -F. In embodiments, z is 1 and R 4 is -Cl. In embodiments, z is 1 and R 4 is - Br. In embodiments, R 4 is halogen. In embodiments, R 4 is -F. In embodiments, R 4 is -Cl. In embodiments, R 4 is -Br.
  • L 1 is unsubstituted C 1 -C 3 alkylene. In embodiments, L 1 is unsubstituted methylene. In embodiments, L 1 is unsubstituted ethylene. In embodiments, L 1 is a bond.
  • the compound [0193] In embodiments, the compound
  • each R 1 andR 2 is independently substituted or unsubstituted C 1 -C 4 alkyl. In embodiments, each R 1 andR 2 is independently unsubstituted C 1 -C 4 alkyl. In embodiments, R 1 is unsubstituted methyl. In embodiments, R 1 is unsubstituted ethyl. In embodiments, R 1 is unsubstituted propyl. In embodiments, R 1 is unsubstituted isopropyl. In embodiments, R 1 is unsubstituted butyl. In embodiments, R 1 is unsubstituted isobutyl. In embodiments, R 1 is unsubstituted t-butyl.
  • R 2 is unsubstituted methyl. In embodiments, R 2 is unsubstituted ethyl. In embodiments, R 2 is unsubstituted propyl. In embodiments, R 2 is unsubstituted isopropyl. In embodiments, R 2 is unsubstituted butyl. In embodiments, R 2 is unsubstituted isobutyl. In embodiments, R 2 is unsubstituted t-butyl.
  • L 1 is a bond, -NH-, -O-, -S-, -C(O)-, -C(O)NH-, -NHC(O)-, -NHC(O)NH-, -C(O)O-, -OC(O)-, substituted (e.g., substituted with a substituent group, a size-limited substituent group, or lower substituent group) or unsubstituted alkylene (e.g., C 1 -C 8 , C 1 -C 6 , or C 1 -C 4 alkylene), substituted (e.g., substituted with a substituent group, a size-limited substituent group, or lower substituent group) or unsubstituted heteroalkylene (e.g., 2 to 8 membered, 2 to 6 membered, or 2 to 4 membered heteroalkylene), substituted (e.g., substituted with a substituent group, a size-limited substituent group,
  • a substituted L 1 is substituted with at least one substituent group, size-limited substituent group, or lower substituent group; wherein if the substituted L 1 is substituted with a plurality of groups selected from substituent groups, size-limited substituent groups, and lower substituent groups; each substituent group, size-limited substituent group, and/or lower substituent group may optionally be different.
  • when L 1 is substituted it is substituted with at least one substituent group.
  • when L 1 is substituted it is substituted with at least one size-limited substituent group.
  • when L 1 is substituted it is substituted with at least one lower substituent group.
  • L 1 is a bond.
  • L 1 is substituted or unsubstituted alkylene (e.g., C 1 -C20, C 1 -C 1 2, C 1 -C 8 , C 1 -C 6 , C 1 -C 4 , or C 1 -C2).
  • L 1 is substituted alkylene (e.g., C 1 -C20, C 1 -C 1 2, C 1 -C 8 , C 1 -C 6 , C 1 -C 4 , or C 1 -C2).
  • L 1 is unsubstituted alkylene (e.g., C 1 -C20, C 1 -C 1 2, C 1 -C 8 , C 1 -C 6 , C 1 -C 4 , or C 1 -C2).
  • L 1 is substituted or unsubstituted C 1 -C20 alkylene.
  • L 1 is substituted C 1 -C20 alkylene.
  • L 1 is unsubstituted C 1 -C20 alkylene.
  • L 1 is substituted or unsubstituted C 1 -C 1 2 alkylene.
  • L 1 is substituted C 1 -C 1 2 alkylene.
  • L 1 is unsubstituted C 1 -C 1 2 alkylene. In embodiments, L 1 is substituted or unsubstituted C 1 -C 8 alkylene. In embodiments, L 1 is substituted C 1 -C 8 alkylene. In embodiments, L 1 is unsubstituted C 1 -C 8 alkylene. In embodiments, L 1 is substituted or unsubstituted C 1 -C 6 alkylene. In embodiments, L 1 is substituted C 1 -C 6 alkylene. In embodiments, L 1 is unsubstituted C 1 -C 6 alkylene. In embodiments, L 1 is substituted or unsubstituted C 1 -C 4 alkylene.
  • L 1 is substituted C 1 -C 4 alkylene. In embodiments, L 1 is unsubstituted C 1 -C 4 alkylene. In embodiments, L 1 is substituted or unsubstituted C 1 -C 3 alkylene. In embodiments, L 1 is substituted C 1 -C 3 alkylene. In embodiments, L 1 is unsubstituted C 1 -C 3 alkylene. In embodiments, L 1 is substituted or unsubstituted ethylene. In embodiments, L 1 is substituted ethylene. In embodiments, L 1 is unsubstituted ethylene. In embodiments, L 1 is substituted or unsubstituted methylene. In embodiments, L 1 is substituted methylene. In embodiments, L 1 is unsubstituted methylene.
  • L 1 is substituted or unsubstituted heteroalkylene (e.g., 2 to 20 membered, 2 to 12 membered, 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4 to 5 membered).
  • L 1 is substituted heteroalkylene (e.g., 2 to 20 membered, 2 to 12 membered, 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4 to 5 membered).
  • L 1 is unsubstituted heteroalkylene (e.g., 2 to 20 membered, 2 to 12 membered, 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4 to 5 membered).
  • L 1 is substituted or unsubstituted 2 to 20 membered heteroalkylene.
  • L 1 is substituted 2 to 20 membered heteroalkylene.
  • L 1 is unsubstituted 2 to 20 membered heteroalkylene.
  • L 1 is substituted or unsubstituted 2 to 8 membered heteroalkylene.
  • L 1 is substituted 2 to 8 membered heteroalkylene.
  • L 1 is unsubstituted 2 to 8 membered heteroalkylene. In embodiments, L 1 is substituted or unsubstituted 2 to 6 membered heteroalkylene. In embodiments, L 1 is substituted 2 to 6 membered heteroalkylene. In embodiments, L 1 is unsubstituted 2 to 6 membered heteroalkylene. In embodiments, L 1 is substituted or unsubstituted 4 to 6 membered heteroalkylene. In embodiments, L 1 is substituted 4 to 6 membered heteroalkylene. In embodiments, L 1 is unsubstituted 4 to 6 membered heteroalkylene.
  • L 1 is substituted or unsubstituted 2 to 3 membered heteroalkylene. In embodiments, L 1 is substituted 2 to 3 membered heteroalkylene. In embodiments, L 1 is unsubstituted 2 to 3 membered heteroalkylene. In embodiments, L 1 is substituted or unsubstituted 4 to 5 membered heteroalkylene. In embodiments, L 1 is substituted 4 to 5 membered heteroalkylene. In embodiments, L 1 is unsubstituted 4 to 5 membered heteroalkylene.
  • R 1 is hydrogen, halogen (e.g., -F, -Cl, -Br, or -I), -CX 3 (e.g., - CF 3 , -CCl 3 ,-CBr 3 , or -CI 3 ), -CHX’ 2 (e.g., -CHF 2 , -CHCl 2 ,-CHBr 2; or -CHI 2 ), -CH 2 X’ (e.g., -CH 2 F,-CH 2 C 1 , -CH 2 Br, or -CH 2 I), -OCX 1 3 (e.g., -OCF 3 , -OCC 13 , -OCBr 3 , or -OCI 3 ), - OCH 2 X’ (e.g., -OCH 2 F, -OCH 2 C 1 , -OCH 2 Br, or -OCH 2 I), -OCHX’ 2 (e.g., -OC
  • a substituted R 1 is substituted with at least one substituent group, size-limited substituent group, or lower substituent group; wherein if the substituted R 1 is substituted with a plurality of groups selected from substituent groups, size-limited substituent groups, and lower substituent groups; each substituent group, size-limited substituent group, and/or lower substituent group may optionally be different.
  • R 1 when R 1 is substituted, it is substituted with at least one substituent group.
  • R 1 when R 1 is substituted, it is substituted with at least one size-limited substituent group.
  • when R 1 is substituted it is substituted with at least one lower substituent group.
  • a substituted R 1A is substituted with at least one substituent group, size-limited substituent group, or lower substituent group; wherein if the substituted R 1A is substituted with a plurality of groups selected from substituent groups, size-limited substituent groups, and lower substituent groups; each substituent group, size-limited substituent group, and/or lower substituent group may optionally be different.
  • R 1A when R 1A is substituted, it is substituted with at least one substituent group.
  • R 1A when R 1A is substituted, it is substituted with at least one size-limited substituent group.
  • R 1A when R 1A is substituted, it is substituted with at least one lower substituent group.
  • a substituted R 1B is substituted with at least one substituent group, size-limited substituent group, or lower substituent group; wherein if the substituted R 1B is substituted with a plurality of groups selected from substituent groups, size-limited substituent groups, and lower substituent groups; each substituent group, size-limited substituent group, and/or lower substituent group may optionally be different.
  • R 1B when R 1B is substituted, it is substituted with at least one substituent group.
  • R 1B when R 1B is substituted, it is substituted with at least one size-limited substituent group.
  • R 1B when R 1B is substituted, it is substituted with at least one lower substituent group.
  • a substituted ring formed when R 1A and R 1B substituents bonded to the same nitrogen atom are joined is substituted with at least one substituent group, sizelimited substituent group, or lower substituent group; wherein if the substituted ring formed when R 1A and R 1B substituents bonded to the same nitrogen atom are joined is substituted with a plurality of groups selected from substituent groups, size-limited substituent groups, and lower substituent groups; each substituent group, size-limited substituent group, and/or lower substituent group may optionally be different.
  • the substituted ring formed when R 1A and R 1B substituents bonded to the same nitrogen atom are joined is substituted, it is substituted with at least one substituent group.
  • the substituted ring formed when R 1A and R 1B substituents bonded to the same nitrogen atom are joined is substituted, it is substituted with at least one size-limited substituent group. In embodiments, when the substituted ring formed when R 1A and R 1B substituents bonded to the same nitrogen atom are joined is substituted, it is substituted with at least one lower substituent group.
  • a substituted R 1C is substituted with at least one substituent group, size-limited substituent group, or lower substituent group; wherein if the substituted R 1C is substituted with a plurality of groups selected from substituent groups, size-limited substituent groups, and lower substituent groups; each substituent group, size-limited substituent group, and/or lower substituent group may optionally be different.
  • R 1C when R 1C is substituted, it is substituted with at least one substituent group.
  • R 1C when R 1C is substituted, it is substituted with at least one size-limited substituent group.
  • R 1C when R 1C is substituted, it is substituted with at least one lower substituent group.
  • a substituted R 1D is substituted with at least one substituent group, size-limited substituent group, or lower substituent group; wherein if the substituted R 1D is substituted with a plurality of groups selected from substituent groups, size-limited substituent groups, and lower substituent groups; each substituent group, size-limited substituent group, and/or lower substituent group may optionally be different.
  • R 1D when R 1D is substituted, it is substituted with at least one substituent group.
  • R 1D when R 1D is substituted, it is substituted with at least one size-limited substituent group.
  • R 1D when R 1D is substituted, it is substituted with at least one lower substituent group.
  • each R 1 is hydrogen, -F, -Cl, -Br, -I, -CF 3 , -CHF 2 , -CH 2 F, -CC 13 , -CHC 12 , -CH 2 C 1 , -CBr 3 , CHBr 2 , -CH 2 Br, -CI 3 , -CHI 2 , -CH 2 I, -OCF 3 , -OCC 13 , -OCBr 3 , - OCI 3 , -OCHF 2 , -OCHCh, -OCHBr 2 , -OCHI 2 , -OCH 2 F, -OCH 2 C 1 , -OCH 2 Br, -
  • each R 1 is independently -F, -Cl, -Br, -I, -CF 3 , -CHF 2 , -CH 2 F, -CC 13 ,-CHC 12 , -CH 2 C 1 , -CBr 3 , CHBr 2 , -CH 2 Br, -CI 3 , -CHI 2 , -CH 2 I, -OCF 3 , -OCC 13 , - OCBr 3 , -OCI 3 , -OCHF 2 , -OCHCh, -OCHBr 2 , -OCHI 2 , -OCH 2 F, -OCH 2 C 1 , -OCH 2 Br, - OCH 2 I, -N 3 , -CN, -SH, -SCH 3 , -SO 2 H, -SO 2 CH 3 , -SO 2 NH 2 , -SO 2 NHCH 3 , -NHC(O)NH 2J
  • each R 1 is hydrogen, -F, -Cl, -Br, -I, -CF 3 , -CHF 2 , -CH 2 F, -CCI3, -CHC 12 , -CH 2 C 1 , -CBr 3 , - CHBr 2 , -CH 2 Br, -CI 3 , -CHI 2 , -CH 2 I, -OCF3, -OCCI3, -OCBr 3 , -OCI3, -OCHF 2 , -OCHCh, -OCHBr 2 , -OCHI 2 , -OCH 2 F, -OCH 2 C 1 , -OCH 2 Br, -OCH 2 I, -N 3 , -CN, -SH, -SCH3, -SO 2 H, -SO 2 CH 3 , -SO 2 NH 2 , -SO 2 NHCH 3 , -NHC(O)NH 2 ,-NHC(O)NH 2
  • NCH 3 C(O)OH, -NHOH, -NCH3OH, -NCH3OCH3, unsubstituted alkyl e.g., C 1 -C 20 , C 1 -C 1 2, C 1 -C 8 , C 1 -C 6 , C 1 -C 4 , or C 1 -C 2
  • unsubstituted heteroalkyl e.g., 2 to 20 membered, 2 to 12 membered, 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4 to 5 membered
  • unsubstituted cycloalkyl e.g., C 3 -C 10 , C 3 -C 8 , C 3 -C 6 , C 4 -C 6 , or C 8 -C 6
  • unsubstituted heterocycloalkyl e.g., 3 to 10 membered, 3 to 8 membered, 3 to 6 member
  • R 2 is hydrogen, halogen (e.g., -F, -Cl, -Br, or -I), -CX 2 3 (e.g., - CF 3 , -CCI3, -CBr 3 , or -CI 3 ), -CHX 2 2 (e.g., -CHF 2 , -CHCh,-CHBr 2; or -CHI 2 ), -CH 2 X 2 (e.g., -CH 2 F,-CH 2 C 1 , -CH 2 Br, or -CH 2 I), -OCX 2 3 (e.g., -OCF3, -OCCI3, -OCBr 3 , or -OCI3), - OCH 2 X 2 (e.g., -OCH 2 F, -OCH 2 C 1 , -OCH 2 Br, or -OCH 2 I), -OCHX 2 2 (e.g., -OCHF
  • a substituted R 2 is substituted with at least one substituent group, size-limited substituent group, or lower substituent group; wherein if the substituted R 2 is substituted with a plurality of groups selected from substituent groups, size-limited substituent groups, and lower substituent groups; each substituent group, size-limited substituent group, and/or lower substituent group may optionally be different.
  • R 2 when R 2 is substituted, it is substituted with at least one substituent group.
  • R 2 when R 2 is substituted, it is substituted with at least one size-limited substituent group. In embodiments, when R 2 is substituted, it is substituted with at least one lower substituent group.
  • a substituted R 2A is substituted with at least one substituent group, size-limited substituent group, or lower substituent group; wherein if the substituted R 2A is substituted with a plurality of groups selected from substituent groups, size-limited substituent groups, and lower substituent groups; each substituent group, size-limited substituent group, and/or lower substituent group may optionally be different.
  • R 2A when R 2A is substituted, it is substituted with at least one substituent group.
  • R 2A when R 2A is substituted, it is substituted with at least one size-limited substituent group.
  • R 2A when R 2A is substituted, it is substituted with at least one lower substituent group.
  • a substituted R 2B is substituted with at least one substituent group, size-limited substituent group, or lower substituent group; wherein if the substituted R 2B is substituted with a plurality of groups selected from substituent groups, size-limited substituent groups, and lower substituent groups; each substituent group, size-limited substituent group, and/or lower substituent group may optionally be different.
  • R 2B when R 2B is substituted, it is substituted with at least one substituent group.
  • R 2B when R 2B is substituted, it is substituted with at least one size-limited substituent group.
  • R 2B when R 2B is substituted, it is substituted with at least one lower substituent group.
  • a substituted ring formed when R 2A and R 2B substituents bonded to the same nitrogen atom are joined is substituted with at least one substituent group, sizelimited substituent group, or lower substituent group; wherein if the substituted ring formed when R 2A and R 2B substituents bonded to the same nitrogen atom are joined is substituted with a plurality of groups selected from substituent groups, size-limited substituent groups, and lower substituent groups; each substituent group, size-limited substituent group, and/or lower substituent group may optionally be different.
  • the substituted ring formed when R 2A and R 2B substituents bonded to the same nitrogen atom are joined is substituted, it is substituted with at least one substituent group.
  • the substituted ring formed when R 2A and R 2B substituents bonded to the same nitrogen atom are joined is substituted, it is substituted with at least one size-limited substituent group. In embodiments, when the substituted ring formed when R 2A and R 2B substituents bonded to the same nitrogen atom are joined is substituted, it is substituted with at least one lower substituent group.
  • a substituted R 2C is substituted with at least one substituent group, size-limited substituent group, or lower substituent group; wherein if the substituted R 2C is substituted with a plurality of groups selected from substituent groups, size-limited substituent groups, and lower substituent groups; each substituent group, size-limited substituent group, and/or lower substituent group may optionally be different.
  • R 2C when R 2C is substituted, it is substituted with at least one substituent group.
  • R 2C when R 2C is substituted, it is substituted with at least one size-limited substituent group.
  • R 2C when R 2C is substituted, it is substituted with at least one lower substituent group.
  • a substituted R 2D is substituted with at least one substituent group, size-limited substituent group, or lower substituent group; wherein if the substituted R 2D is substituted with a plurality of groups selected from substituent groups, size-limited substituent groups, and lower substituent groups; each substituent group, size-limited substituent group, and/or lower substituent group may optionally be different.
  • R 2D when R 2D is substituted, it is substituted with at least one substituent group.
  • R 2D when R 2D is substituted, it is substituted with at least one size-limited substituent group.
  • R 2D when R 2D is substituted, it is substituted with at least one lower substituent group.
  • R 2 is hydrogen, -F, -Cl, -Br, -I, -CF 3 , -CHF 2 , -CH 2 F, -CC 13 , - CHC 12 , -CH 2 C 1 , -CBr 3 , -CHBr 2 , -CH 2 Br, -CI 3 , -CHI 2 , -CH 2 I, -OCF 3 , -OCC 13 , -OCBr 3 , - OCI 3 , -OCHF 2 , -OCHCh, -OCHBr 2 , -OCHI 2 , -OCH 2 F, -OCH 2 C 1 , -OCH 2 Br, -
  • R 2 is hydrogen, -F, -Cl, -Br, -I, -CF 3 , -CHF 2 , -CH 2 F, -CC 13 , - CHC 12 , -CH 2 C 1 , -CBr 3 , -CHBr 2 , -CH 2 Br, -CI 3 , -CHI 2 , -CH 2 I, -OCF 3 , -OCC 13 , -OCBr 3 , - OCI 3 , -OCHF 2 , -OCHCh, -OCHBr 2 , -OCHI 2 , -OCH 2 F, -OCH 2 C 1 , -OCH 2 Br, - OCH 2 I, -N 3 , -CN, -SH, -SCH 3 , -SO 2 H, -SO 2 CH 3 , -SO 2 NH 2 , -SO 2 NHCH 3 , -NHC(O)NH
  • R 2 is hydrogen, -F, -Cl, -Br, -I, -CF 3 , -CHF 2 , -CH 2 F, -CC 13 , -CHC 12 , -CH 2 C 1 , -CBr 3 , - CHBr 2 , -CH 2 Br, -CI 3 , -CHI 2 , -CH 2 I, -OCF 3 , -OCC 13 , -OCBr 3 , -OCI 3 , -OCHF 2 , -OCHCh, -OCHBr 2 , -OCHI 2 , -OCH 2 F, -OCH 2 C 1 , -OCH 2 Br, -OCH 2 I, -N 3 , -CN, -SH, -SCH 3 , -SO 2 H, -SO 2 CH 3 , -SO 2 NH 2 , -SO 2 NHCH 3 , -NHC(O)NH
  • R 1 and R 2 may optionally be joined to form a substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.
  • a substituted ring formed when R 1 and R 2 are joined is substituted with at least one substituent group, size-limited substituent group, or lower substituent group; wherein if the substituted ring formed when R 1 and R 2 are joined is substituted with a plurality of groups selected from substituent groups, size-limited substituent groups, and lower substituent groups; each substituent group, size-limited substituent group, and/or lower substituent group may optionally be different.
  • when the ring formed when R 1 and R 2 are joined is substituted it is substituted with at least one substituent group.
  • when the ring formed when R 1 and R 2 are joined is substituted, it is substituted with at least one size-limited substituent group.
  • when the ring formed when R 1 and R 2 are joined is substituted, it is substituted with at least one lower substituent group.
  • R 1 and R 2 together with atoms (e.g., nitrogen) attached thereto may be joined to form substituted (e.g., substituted with a substituent group, a size-limited substituent group, or lower substituent group) or unsubstituted heterocycloalkyl (e.g., 3 to 10 membered, 3 to 8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered), or substituted (e.g., substituted with a substituent group, a size-limited substituent group, or lower substituent group) or unsubstituted heteroaryl (e.g., 5 to 12 membered, 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered).
  • substituted e.g., substituted with a substituent group, a size-limited substituent group, or lower substituent group
  • unsubstituted heterocycloalkyl e.g., 3 to 10
  • R 1 and R 2 together with atoms (e.g., nitrogen) attached thereto may be joined to form substituted (e.g., substituted with a substituent group, a size-limited substituent group, or lower substituent group) heterocycloalkyl (e.g., 3 to 10 membered, 3 to 8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered), or substituted (e.g., substituted with a substituent group, a size-limited substituent group, or lower substituent group) heteroaryl (e.g., 5 to 12 membered, 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered).
  • substituted e.g., substituted with a substituent group, a size-limited substituent group, or lower substituent group
  • heterocycloalkyl e.g., 3 to 10 membered, 3 to 8 membered, 3 to 6 membered, 4 to
  • R 1 and R 2 together with atoms (e.g., nitrogen) attached thereto may be joined to form unsubstituted heterocycloalkyl (e.g., 3 to 10 membered, 3 to 8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered), or unsubstituted heteroaryl (e.g., 5 to 12 membered, 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered).
  • unsubstituted heterocycloalkyl e.g., 3 to 10 membered, 3 to 8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered
  • unsubstituted heteroaryl e.g., 5 to 12 membered, 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered.
  • R 1 and R 2 together with atoms (e.g., nitrogen) attached thereto may be joined to form substituted (e.g., substituted with a substituent group, a size-limited substituent group, or lower substituent group) or unsubstituted pyridyl.
  • R 1 and R 2 together with atoms (e.g., nitrogen) attached thereto may be joined to form substituted (e.g., substituted with a substituent group, a size-limited substituent group, or lower substituent group) or unsubstituted piperidinyl.
  • R 1 and R 2 together with atoms attached thereto may be joined to form substituted (e.g., substituted with a substituent group, a size-limited substituent group, or lower substituent group) or unsubstituted morpholinyl.
  • R 1 and R 2 together with atoms attached thereto may be joined to form substituted (e.g., substituted with a substituent group, a size-limited substituent group, or lower substituent group) or unsubstituted pyrimidinyl.
  • R 4 is independently halogen (e.g., -F, -Cl, -Br, or -I), -CX 4 3 (e.g., - CF 3 , -CCI3, -CBr 3 , or -CI 3 ), -CHX 4 2 (e.g., -CHF 2 , -CHCl 2 ,-CHBr 2; or -CHI 2 ), -CH 2 X 4 (e.g., -CH 2 F,-CH 2 C 1 , -CH 2 Br, or -CH 2 I), -OCX 4 3 (e.g., -OCF3, -OCCI3, -OCBr 3 , or -OCI3), - OCH 2 X 4 (e.g., -OCH 2 F, -OCH 2 C 1 , -OCH 2 Br, or -OCH 2 I), -OCHX 4 2 (e.g., -OCHX 4 2 (e
  • a substituted R 4 is substituted with at least one substituent group, size-limited substituent group, or lower substituent group; wherein if the substituted R 4 is substituted with a plurality of groups selected from substituent groups, size-limited substituent groups, and lower substituent groups; each substituent group, size-limited substituent group, and/or lower substituent group may optionally be different.
  • R 4 when R 4 is substituted, it is substituted with at least one substituent group.
  • R 4 when R 4 is substituted, it is substituted with at least one size-limited substituent group. In embodiments, when R 4 is substituted, it is substituted with at least one lower substituent group.
  • a substituted R 4A is substituted with at least one substituent group, size-limited substituent group, or lower substituent group; wherein if the substituted R 4A is substituted with a plurality of groups selected from substituent groups, size-limited substituent groups, and lower substituent groups; each substituent group, size-limited substituent group, and/or lower substituent group may optionally be different.
  • R 4A when R 4A is substituted, it is substituted with at least one substituent group.
  • R 4A when R 4A is substituted, it is substituted with at least one size-limited substituent group.
  • R 4A when R 4A is substituted, it is substituted with at least one lower substituent group.
  • a substituted R 4B is substituted with at least one substituent group, size-limited substituent group, or lower substituent group; wherein if the substituted R 4B is substituted with a plurality of groups selected from substituent groups, size-limited substituent groups, and lower substituent groups; each substituent group, size-limited substituent group, and/or lower substituent group may optionally be different.
  • R 4B when R 4B is substituted, it is substituted with at least one substituent group.
  • R 4B when R 4B is substituted, it is substituted with at least one size-limited substituent group.
  • R 4B when R 4B is substituted, it is substituted with at least one lower substituent group.
  • a substituted ring formed when R 4A and R 4B substituents bonded to the same nitrogen atom are joined is substituted with at least one substituent group, sizelimited substituent group, or lower substituent group; wherein if the substituted ring formed when R 4A and R 4B substituents bonded to the same nitrogen atom are joined is substituted with a plurality of groups selected from substituent groups, size-limited substituent groups, and lower substituent groups; each substituent group, size-limited substituent group, and/or lower substituent group may optionally be different.
  • the substituted ring formed when R 4A and R 4B substituents bonded to the same nitrogen atom are joined is substituted, it is substituted with at least one substituent group.
  • the substituted ring formed when R 4A and R 4B substituents bonded to the same nitrogen atom are joined is substituted, it is substituted with at least one size-limited substituent group. In embodiments, when the substituted ring formed when R 4A and R 4B substituents bonded to the same nitrogen atom are joined is substituted, it is substituted with at least one lower substituent group.
  • a substituted R 4C is substituted with at least one substituent group, size-limited substituent group, or lower substituent group; wherein if the substituted R 4C is substituted with a plurality of groups selected from substituent groups, size-limited substituent groups, and lower substituent groups; each substituent group, size-limited substituent group, and/or lower substituent group may optionally be different.
  • R 4C when R 4C is substituted, it is substituted with at least one substituent group.
  • R 4C when R 4C is substituted, it is substituted with at least one size-limited substituent group.
  • when R 4C is substituted it is substituted with at least one lower substituent group.
  • a substituted R 4D is substituted with at least one substituent group, size-limited substituent group, or lower substituent group; wherein if the substituted R 4D is substituted with a plurality of groups selected from substituent groups, size-limited substituent groups, and lower substituent groups; each substituent group, size-limited substituent group, and/or lower substituent group may optionally be different.
  • R 4D when R 4D is substituted, it is substituted with at least one substituent group.
  • R 4D when R 4D is substituted, it is substituted with at least one size-limited substituent group.
  • R 4D when R 4D is substituted, it is substituted with at least one lower substituent group.
  • R 4 is hydrogen, -F, -Cl, -Br, -I, -CF 3 , -CHF 2 , -CH 2 F, -CC 13 , - CHC 12 , -CH 2 C 1 , -CBr 3 , -CHBr 2 , -CH 2 Br, -CI 3 , -CHI 2 , -CH 2 I, -OCF 3 , -OCC 13 , -OCBr 3 , - OCI 3 , -OCHF 2 , -OCHCh, -OCHBr 2 , -OCHI 2 , -OCH 2 F, -OCH 2 C 1 , -OCH 2 Br, -
  • R 4 is hydrogen, -F, -Cl, -Br, -I, -CF3, -CHF2, -CH2F, -CCI3, - CHCh, -CH2CI, -CBr 3 , -CHBr 2 , -CH 2 Br, -CI 3 , -CHI 2 , -CH 2 I, -OCF3, -OCCI3, -OCBr 3 , - OCI3, -OCHF2, -OCHCh, -OCHBr 2 , -OCHI2, -OCH2F, -OCH2CI, -OCH 2 Br, -
  • R 4 is hydrogen, -F, -Cl, -Br, -I, -CF 3 , -CHF 2 , -CH 2 F, -CCI3, -CHCh, -CH 2 C 1 , -CBr 3 , - CHBr 2 , -CH 2 Br, -CI 3 , -CHI 2 , -CH 2 I, -OCF3, -OCCI3, -OCBr 3 , -OCI3, -OCHF 2 , -OCHCh, -OCHBr 2 , -OCHh, -OCH2F, -OCH2CI, -OCH 2 Br, -OCH 2 I, -N3, -CN, -SH, -SCH3, -SO 2 H, -SO2CH3, -SO2NH2, -SO2NHCH3, -NHC(O)NH2,-NHC(O)NHCH 3 , -NO2, -NH 2 , -
  • Each R 1A , R 1B , R 1C , R 1D , R 1E , R 2A , R 2B , R 2C , R 2D , R 2E , R 4A , R 4B , R 4C , R 4D , and R 4E are independently hydrogen, -CX 3 , -CHX 2 , -CH 2 X (e.g., -CF 3 , -CHF 2 , -CH 2 F, -CCI3,- CHCh, -CH2CI, -CBr 3 , -CHBr 2 , -CH 2 Br, -CI 3 , -CHI 2 , -CH 2 I), -CN, -
  • OH, -COOH, -CONH2 substituted (e.g., substituted with a substituent group, a size-limited substituent group, or lower substituent group) or unsubstituted alkyl (e.g., C 1 -C20, C 1 -C 1 2, C 1 - C 8 , C 1 -C 6 , C 1 -C 4 , or C 1 -C2), substituted (e.g., substituted with a substituent group, a sizelimited substituent group, or lower substituent group) or unsubstituted heteroalkyl (e.g., 2 to 20 membered, 2 to 12 membered, 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4 to 5 membered), substituted (e.g., substituted with a substituent group, a size-limited substituent group, or lower substituent group) or unsubstituted cycloalkyl (e.g
  • X is independently - F, -Cl, -Br, or -I.
  • each R 1A , R 1B , R 1C , R 1D , R 1E , R 2A , R 2B , R 2C , R 2D , R 2E , R 4A , R 4B , R 4C , R 4D , and R 4E are independently hydrogen, -CF 3 , -CHF 2 , -CH 2 F, -CCI3, - CHCh, -CH2CI, -CBr 3 , -CHBr 2 , -CH 2 Br, -CI 3 , -CHI 2 , -CH 2 I, -CN, -
  • unsubstituted alkyl e.g., C 1 -C20, C 1 -C 1 2, C 1 -C 8 , C 1 -C 6 , C 1 -C 4 , or C 1 - C2
  • unsubstituted heteroalkyl e.g., 2 to 20 membered, 2 to 12 membered, 2 to 8 membered
  • unsubstituted cycloalkyl e.g., C 3 -C 10 , C 3 -C 8 , C 3 -C 6 , C 4 -C 6 , or C 8 -C 6
  • unsubstituted heterocycloalkyl e.g.,
  • aryl e.g., C 6 -C 1 2, C 6 -C 10 , or phenyl
  • heteroaryl e.g., 5 to 12 membered, 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered.
  • each R 1A , R 1B , R 1C , R 1D , R 1E , R 2A , R 2B , R 2C , R 2D , R 2E , R 4A , R 4B , R 4C , R 4D , and R 4E are independently hydrogen.
  • Each R 1A and R 1B , R 2A and R 2B , and R 4A and R 4B together with nitrogen attached thereto may be joined to form substituted (e.g., substituted with a substituent group, a sizelimited substituent group, or lower substituent group) or unsubstituted heterocycloalkyl (e.g., 3 to 10 membered, 3 to 8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered), or substituted (e.g., substituted with a substituent group, a size-limited substituent group, or lower substituent group) or unsubstituted heteroaryl (e.g., 5 to 12 membered, 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered).
  • substituted e.g., substituted with a substituent group, a sizelimited substituent group, or lower substituent group
  • Each R 1A and R 1B , R 2A and R 2B , and R 4A and R 4B together with nitrogen attached thereto may be joined to form substituted (e.g., substituted with a substituent group, a size-limited substituent group, or lower substituent group) heterocycloalkyl (e.g., 3 to 10 membered, 3 to 8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered), or substituted (e.g., substituted with a substituent group, a size-limited substituent group, or lower substituent group) heteroaryl (e.g., 5 to 12 membered, 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered).
  • substituted e.g., substituted with a substituent group, a size-limited substituent group, or lower substituent group
  • heterocycloalkyl e.g., 3 to 10 membered, 3 to 8 member
  • Each R 1A and R 1B , R 2A and R 2B , and R 4A and R 4B together with nitrogen attached thereto may be joined to form unsubstituted heterocycloalkyl (e.g., 3 to 10 membered, 3 to 8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered), or unsubstituted heteroaryl (e.g., 5 to 12 membered, 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered).
  • unsubstituted heterocycloalkyl e.g., 3 to 10 membered, 3 to 8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered
  • unsubstituted heteroaryl e.g., 5 to 12 membered, 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered.
  • each R 1A and R 1B , R 2A and R 2B , and R 4A and R 4B together with nitrogen attached thereto may be joined to form substituted (e.g., substituted with a substituent group, a size-limited substituent group, or lower substituent group) or unsubstituted pyridyl.
  • each R 1A and R 1B , R 2A and R 2B , and R 4A and R 4B together with nitrogen attached thereto may be joined to form substituted (e.g., substituted with a substituent group, a size-limited substituent group, or lower substituent group) or unsubstituted piperidinyl.
  • each R 1A and R 1B , R 2A and R 2B , and R 4A and R 4B together with nitrogen attached thereto may be joined to form substituted (e.g., substituted with a substituent group, a size-limited substituent group, or lower substituent group) or unsubstituted morpholinyl.
  • each R 1A and R 1B , R 2A and R 2B , and R 4A and R 4B together with nitrogen attached thereto may be joined to form substituted (e.g., substituted with a substituent group, a size-limited substituent group, or lower substituent group) or unsubstituted pyrimidinyl.
  • a substituted R 1E is substituted with at least one substituent group, size-limited substituent group, or lower substituent group; wherein if the substituted R 1E is substituted with a plurality of groups selected from substituent groups, size-limited substituent groups, and lower substituent groups; each substituent group, size-limited substituent group, and/or lower substituent group may optionally be different.
  • R 1E when R 1E is substituted, it is substituted with at least one substituent group.
  • R 1E when R 1E is substituted, it is substituted with at least one size-limited substituent group.
  • R 1E when R 1E is substituted, it is substituted with at least one lower substituent group.
  • a substituted R 2E is substituted with at least one substituent group, size-limited substituent group, or lower substituent group; wherein if the substituted R 2E is substituted with a plurality of groups selected from substituent groups, size-limited substituent groups, and lower substituent groups; each substituent group, size-limited substituent group, and/or lower substituent group may optionally be different.
  • R 2E when R 2E is substituted, it is substituted with at least one substituent group.
  • R 2E when R 2E is substituted, it is substituted with at least one size-limited substituent group.
  • R 2E when R 2E is substituted, it is substituted with at least one lower substituent group.
  • a substituted R 4E is substituted with at least one substituent group, size-limited substituent group, or lower substituent group; wherein if the substituted R 4E is substituted with a plurality of groups selected from substituent groups, size-limited substituent groups, and lower substituent groups; each substituent group, size-limited substituent group, and/or lower substituent group may optionally be different.
  • R 4E when R 4E is substituted, it is substituted with at least one substituent group.
  • R 4E when R 4E is substituted, it is substituted with at least one size-limited substituent group.
  • R 4E when R 4E is substituted, it is substituted with at least one lower substituent group.
  • X, X 1 , X 2 , and X 4 are independently -F, -Cl, -Br, or -I.
  • X is -F.
  • X is -Cl.
  • X is -Br.
  • X is -I.
  • X 1 is -F.
  • X 1 is -Cl.
  • X 1 is -Br.
  • X 1 is -I.
  • X 2 is -F.
  • X 2 is -Cl.
  • X 2 is -Br.
  • X 2 is -I.
  • X 4 is -F.
  • X 4 is -Cl.
  • X 4 is -Br.
  • X 4 is -I.
  • X 4 is -F.
  • X 4 is -Cl.
  • X 4 is -Br.
  • X 4 is -I.
  • X 4 is -F.
  • nl, n2, and n3 are independently an integer from 0 to 4 (e.g. 0).
  • nl is 0.
  • nl is 1.
  • nl is 2.
  • nl is 3.
  • nl is 4.
  • n2 is 0.
  • n2 is 1.
  • n2 is 2.
  • n2 is 3.
  • n2 is 4.
  • n4 is 0.
  • n4 is 1.
  • n4 is 2.
  • n4 is 3.
  • n4 is 4.
  • the symbols ml, m2, and m4 are independently an integer from 1 to 2.
  • ml is 1.
  • ml is 2.
  • m2 is 1.
  • m2 is 1.
  • m2 is 2.
  • m4 is 1.
  • m4 is 2.
  • the symbols vl, v2, and v4 are independently an integer from 1 to 2.
  • vl is 1.
  • vl is 2.
  • v2 is 1.
  • v2 is 2.
  • v4 is 1.
  • v4 is 2.
  • the compound is usefill as a comparator compound.
  • the comparator compound can be used to assess the activity of a test compound as set forth in an assay described herein (e.g., in the examples section, figures, or tables).
  • the compound is a compound as described herein, including in embodiments.
  • the compound is a compound described herein (e.g., in the examples section, figures, tables, or claims).
  • the compound described herein is administered as a pure chemical.
  • the compound as described herein e.g., Formula (I'), (I), (II), or (III)
  • a pharmaceutically suitable or acceptable carrier also referred to herein as a pharmaceutically suitable (or acceptable) excipient, physiologically suitable (or acceptable) excipient, or physiologically suitable (or acceptable) carrier
  • a pharmaceutically suitable or acceptable carrier also referred to herein as a pharmaceutically suitable (or acceptable) excipient, physiologically suitable (or acceptable) excipient, or physiologically suitable (or acceptable) carrier
  • the compound of Formula (I'), (I), (II), or (III) as described herein is administered as a pure chemical.
  • the compound of Formula (I'), (I), (II), or (III) described herein is combined with a pharmaceutically suitable or acceptable carrier (also referred to herein as a pharmaceutically suitable (or acceptable) excipient, physiologically suitable (or acceptable) excipient, or physiologically suitable (or acceptable) carrier) selected on the basis of a chosen route of administration and standard pharmaceutical practice as described, for example, in Gennaro, A. R., “Remington: The Science and Practice of Pharmacy,” 21 st ed., Easton: Lippincott Williams & Wilkins, 2005.
  • a pharmaceutical composition including at least one compound of Formula (I'), (I), (II), or (III) described herein, or a pharmaceutically acceptable salt or solvate thereof, together with one or more pharmaceutically acceptable carriers.
  • the carrier(s) or excipient(s)
  • the carrier is acceptable or suitable if the carrier is compatible with the other ingredients of the composition and not deleterious to the recipient (i.e., the subject) of the composition.
  • a pharmaceutical composition including at least one compound of Formula (I'), (I), (II), or (III) described herein, or a pharmaceutically acceptable salt or solvate thereof, together with one or more pharmaceutically acceptable carriers.
  • the carrier(s) or excipient(s)
  • the carrier is acceptable or suitable if the carrier is compatible with the other ingredients of the composition and not deleterious to the recipient (i.e., the subject) of the composition.
  • the compound described herein e.g., of Formula (I'), (I), (II), or (III)
  • the compound of Formula (I'), (I), (II), or (III) as described herein is substantially pure, in that it contains less than about 5%, or less than about 1%, or less than about 0.1%, of other organic small molecules, such as contaminating intermediates or by-products that are created, for example, in one or more of the steps of a synthesis method.
  • compositions include those suitable for oral, rectal, topical, buccal, parenteral (e.g., subcutaneous, intramuscular, intradermal, or intravenous), vaginal, ophthalmic, or aerosol administration.
  • parenteral e.g., subcutaneous, intramuscular, intradermal, or intravenous
  • vaginal e.g., vaginal, ophthalmic, or aerosol administration.
  • Exemplary pharmaceutical compositions are used in the form of a pharmaceutical preparation, for example, in solid, semisolid or liquid form, which includes one or more of a disclosed compound, as an active ingredient, in a mixture with an organic or inorganic carrier or excipient suitable for external, enteral or parenteral applications.
  • the active ingredient is compounded, for example, with the usual non-toxic, pharmaceutically acceptable carriers for tablets, pellets, capsules, suppositories, solutions, emulsions, suspensions, and any other form suitable for use.
  • the active compound is included in the pharmaceutical composition in an amount sufficient to produce the desired effect upon the process or condition of the disease.
  • the dose of the composition including at least one compound described herein differs, depending upon the patient’s (e.g., human) condition, that is, stage of the disease, general health status, age, and other factors.
  • the dose of the composition including at least one compound of Formula (I'), (I), (II), or (III) as described herein differs, depending upon the patient’s (e.g., human) condition, that is, stage of the disease, general health status, age, and other factors.
  • patient e.g., human
  • compositions are administered in a manner appropriate to the disease to be treated (or prevented).
  • An appropriate dose and a suitable duration and frequency of administration will be determined by such factors as the condition of the patient, the type and severity of the patient’s disease, the particular form of the active ingredient, and the method of administration.
  • an appropriate dose and treatment regimen provides the composition(s) in an amount sufficient to provide therapeutic and/or prophylactic benefit (e.g., an improved clinical outcome, such as more frequent complete or partial remissions, or longer disease-free and/or overall survival, or a lessening of symptom severity.
  • Optimal doses are generally determined using experimental models and/or clinical trials. In some embodiments, the optimal dose depends upon the body mass, weight, or blood volume of the patient.
  • Oral doses typically range from about 0.1 mg to about 1000 mg, or 1.0 mg to about 1000 mg, one to four times, or more, per day.
  • Disclosed compounds are administered to subjects or patients (animals and humans) in need of such treatment in dosages that will provide optimal pharmaceutical efficacy. It will be appreciated that the dose required for use in any particular application will vary from patient to patient, not only with the particular compound or composition selected, but also with the route of administration, the nature of the condition being treated, the age and condition of the patient, concurrent medication or special diets then being followed by the patient, and other factors, with the appropriate dosage ultimately being at the discretion of the attendant physician.
  • a contemplated compound disclosed herein is administered orally, subcutaneously, topically, parenterally, by inhalation spray or rectally in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles. Parenteral administration include subcutaneous injections, intravenous or intramuscular injections or infusion techniques. Effective Dosages
  • the pharmaceutical composition may include compositions wherein the active ingredient is contained in a therapeutically effective amount, i.e., in an amount effective to achieve its intended purpose.
  • a therapeutically effective amount i.e., in an amount effective to achieve its intended purpose.
  • the actual amount effective for a particular application will depend, inter alia, on the condition being treated.
  • the dosage and frequency (single or multiple doses) of compounds administered can vary depending upon a variety of factors, including route of administration; size, age, sex, health, body weight, body mass index, and diet of the recipient; nature and extent of symptoms of the disease being treated; presence of other diseases or other health-related problems; kind of concurrent treatment; and complications from any disease or treatment regimen.
  • Other therapeutic regimens or agents can be used in conjunction with the methods and compounds disclosed herein.
  • therapeutically effective amounts for use in humans can also be determined from animal models.
  • a dose for humans can be formulated to achieve a concentration that has been found to be effective in animals.
  • the dosage in humans can be adjusted by monitoring compounds effectiveness and adjusting the dosage upwards or downwards, as described above. Adjusting the dose to achieve maximal efficacy in humans based on the methods described above and other methods is well within the capabilities of the ordinarily skilled artisan.
  • Dosages may be varied depending upon the requirements of the subject and the compound being employed.
  • the dose administered to a subject should be sufficient to effect a beneficial therapeutic response in the subject over time.
  • the size of the dose also will be determined by the existence, nature, and extent of any adverse side effects. Generally, treatment is initiated with smaller dosages, which are less than the optimum dose of the compound. Thereafter, the dosage is increased by small increments until the optimum effect under circumstances is reached.
  • Dosage amounts and intervals can be adjusted individually to provide levels of the administered compounds effective for the particular clinical indication being treated. This will provide a therapeutic regimen that is commensurate with the severity of the individual's disease state.
  • an effective prophylactic or therapeutic treatment regimen can be planned that does not cause substantial toxicity and yet is entirely effective to treat the clinical symptoms demonstrated by the particular patient. This planning should involve the careful choice of active compound by considering factors such as compound potency, relative bioavailability, patient body weight, presence and severity of adverse side effects, preferred mode of administration, and the toxicity profile of the selected agent.
  • a method of treating a patient in need thereof comprising the administration of the compound (e.g., of Formula (I'), (I), (II), or (III)) as provided herein including embodiments thereof, a derivative thereof, or a salt thereof.
  • the compound e.g., of Formula (I'), (I), (II), or (III)
  • a method of treating cancer in a subject in need thereof comprising the administration an effective amount of the compound (e.g., of Formula (I'), (I), (II), or (III)) as provided herein including embodiments thereof, a derivative thereof, or a salt thereof.
  • the compound e.g., of Formula (I'), (I), (II), or (III)
  • a method of treating a cancer in a subject in need thereof includes administrating an effective amount of the compound (e.g., of Formula (I'), (I), (II), or (III)) as provided herein including embodiments thereof, or a pharmaceutically acceptable salt thereof.
  • an effective amount of the compound e.g., of Formula (I'), (I), (II), or (III) as provided herein including embodiments thereof, or a pharmaceutically acceptable salt thereof.
  • the cancer is a solid tumor.
  • the solid tumor is in thyroid, endocrine system, brain, breast, cervix, colon, prostate, head and neck, liver, kidney, lung, ovary, pancreas, rectum, stomach, oral cavity, or uterus.
  • the solid tumor is in thyroid.
  • the solid tumor is in endocrine system.
  • the solid tumor is in brain.
  • the solid tumor is in breast.
  • the solid tumor is in cervix.
  • the solid tumor is in colon.
  • the solid tumor is in prostate.
  • the solid tumor is in head and neck.
  • the solid tumor is in liver.
  • the solid tumor is in kidney. In embodiments, the solid tumor is in lung. In embodiments, the solid tumor is in ovary. In embodiments, the solid tumor is in pancreas. In embodiments, the solid tumor is in rectum. In embodiments, the solid tumor is in stomach. In embodiments, the solid tumor is in oral cavity. In embodiments, the solid tumor is in uterus.
  • VISTA V-domain Ig Suppressor of T-cell Activation
  • a method of enhancing T-cell proliferation in a subject in the presence of VISTA-expressing cancer cells includes administrating the subject an effective amount of the compound (e.g., of Formula (I'), (I), (II), or (III)) as provided herein including embodiments thereof, or a pharmaceutically acceptable salt thereof.
  • the compound e.g., of Formula (I'), (I), (II), or (III)
  • a method of restoring T-cell activation in a subject in the presence of VISTA-expressing cancer cells includes administrating the subject an effective amount of the compound (e.g., of Formula (T), (I), (II), or (III)) as provided herein including embodiments thereof, or a pharmaceutically acceptable salt thereof.
  • the compound e.g., of Formula (T), (I), (II), or (III)
  • Embodiment Pl A compound as described herein, comprising the compound of
  • FIG. 3C or analog thereof.
  • Embodiment P2 The compound of Embodiment Pl , wherein the structure is:
  • Embodiment P3 A method of treating cancer in a subject in need thereof, the method comprising the administration of an effective amount of the compound of Embodiment Pl or P2, a derivative, analog thereof, or a salt thereof.
  • Embodiment 1 A compound having a structure of pharmaceutically acceptable salt thereof, wherein: L 1 is a bond, -NH-, -O-, -S-, -C(O)-, -C(O)NH-, -NHC(O)-, -NHC(O)NH-, -C(O)O-, -OC(O)-, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene;
  • R 1 is hydrogen, halogen
  • R 2 is hydrogen, halogen, -CX 2 3 , -CHX 2 2 , -CH2X 2 , -OCX 2 3 , -
  • R 1 and R 2 may optionally be joined to form a substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl;
  • R 4 is indendently halogen, -CX 4 3 , -CHX 4 2 , -CH2X 4 , -OCX 4 3 , -
  • R 1A , R 1B , R 1C , R 1D , R 2A , R 2B , R 2C , R 2D , R 4A , R 4B , R 4C , and R 4D are independently hydrogen, -CX3, -CHX2, -CH2X, -CN, -OH, -COOH, -CONH2, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; R 1A and R 1B or R 2A and R 2B with substituents bonded to the same nitrogen atom may optionally be joined to form a substituted or unsubstituted heterocycloalkyl or substituted or unsubstituted heteroaryl; each X, X
  • Embodiment 2 The compound of Embodiment 1, wherein L 1 is a bond, substituted or unsubstituted alkylene, or substituted or unsubstituted heteroalkylene.
  • Embodiment 3 The compound of Embodiment 2, wherein L 1 is substituted or unsubstituted C 1 -C 3 alkylene, or substituted or unsubstituted 2 to 3 membered heteroalkylene.
  • Embodiment 4 The compound of any one of Embodiments 1 to 3, wherein the compound has a structure of
  • Embodiment 5 The compound of any one of Embodiments 1 to 3, wherein the compound has a structure of
  • Embodiment 6 The compound of any one of Embodiments 1 to 5, wherein each R 1 andR 2 is independently substituted or unsubstituted alkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl.
  • Embodiment 7 The compound of Embodiment 6, wherein each R 1 andR 2 is independently substituted or unsubstituted C 1 -C 4 alkyl, or substituted or unsubstituted phenyl.
  • Embodiment 8 The compound of Embodiment 7, wherein each R 1 andR 2 is independently unsubstituted C 1 -C 4 alkyl, or unsubstituted phenyl.
  • Embodiment 9 The compound of Embodiment 8, wherein R 1 is unsubstituted C 1 -C 4 alkyl, and R 2 is unsubstituted phenyl.
  • Embodiment 10 The compound of any one of Embodiments 1 to 9, wherein z is
  • Embodiment 11 The compound of any one of Embodiments 1 to 9, wherein z is
  • R 4 is halogen
  • Embodiment 12 The compound of Embodiment 1, wherein the compound is
  • Embodiment 13 A pharmaceutical composition comprising a compound of any one of Embodiments 1 to 12, or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.
  • Embodiment 14 A method of treating a cancer in a subject in need thereof, the method comprising administrating an effective amount of the compound of any one of Embodiments 1 to 12, or a pharmaceutically acceptable salt thereof.
  • Embodiment 15 The method of Embodiment 14, wherein the cancer is a solid tumor.
  • Embodiment 16 The method of Embodiment 15, wherein the solid tumor is in thyroid, endocrine system, brain, breast, cervix, colon, prostate, head and neck, liver, kidney, lung, ovary, pancreas, rectum, stomach, oral cavity, or uterus.
  • Embodiment 17 A method of inhibiting V-domain Ig Suppressor of T-cell Activation (VISTA) in a cell, the method comprising contacting the cell with an effective amount of the compound of any one of Embodiments 1 to 12, or a pharmaceutically acceptable salt thereof.
  • VISTA V-domain Ig Suppressor of T-cell Activation
  • Embodiment 18 A method of enhancing T-cell proliferation in a subject in the presence of VISTA-expressing cancer cells, the method comprising administrating the subject an effective amount of the compound of any one of Embodiments 1 to 12, or a pharmaceutically acceptable salt thereof.
  • Embodiments 19 A method of restoring T-cell activation in a subject in the presence of VISTA-expressing cancer cells, the method comprising administrating the subject an effective amount of the compound of any one of Embodiments 1 to 12, or a pharmaceutically acceptable salt thereof.
  • V-domain Ig Suppressor of T-cell Activation is an immune checkpoint that affects the ability of T-cells to effectively attack tumors.
  • a FRET -based high throughput screening identified the first small-molecule ligand for VISTA which was further confirmed by biorthogonal assays.
  • Investigation of the hit interaction with VISTA using molecular modeling and STD NMR enabled the identification of a potential binding site in VISTA for the hit.
  • Screening the hit against a small library of single-point VISTA mutants revealed the key residues in VISTA interacting with the hit. Further structural optimization resulted in a lead with submicromolar VISTA binding affinity.
  • the lead compound blocked VISTA signaling in vitro, enhanced T-cell proliferation, and restored T-cell activation in the presence of VISTA-expressing cancer cell lines. This work would enable future development of small molecules targeting VISTA as immunomodulators and imaging probes.
  • VISTA immunomodulatory mechanism of VISTA is unclear because of its expression on both antigen-presenting cells (APC 8 ) and T cells which reveals a ligand-or-receptor paradigm of VISTA.
  • APC 8 antigen-presenting cells
  • T cells which reveals a ligand-or-receptor paradigm of VISTA.
  • V-Set and Immunoglobulin domain containing 3 (VSIG-3) has been recently proposed as a potential ligand for VISTA.
  • VISTA has been identified as an acidic pH-selective ligand for P-selectin glycoprotein ligand- 1 (PSGL-1).
  • PSGL-1 P-selectin glycoprotein ligand- 1
  • Ablation of VISTA by genetic knockout or blocking antibodies activates T cell immune response against tumors in preclinical models, thus, several VISTA antibodies have advanced recently into human clinical trials.
  • Terbium (Tb) cryptate fimctions as the donor in the developed TR-FRET assay by labeling His-tagged human VISTA with MAb Anti-6His Tb cryptate gold.
  • a human VISTA antibody labeled with far-red emitting fluorescent dye (CF647) functions as an acceptor based on reported compatibility of Tb cryptate and CF647 as donor-acceptor pair in FRET signaling.
  • Compounds that can bind VISTA would perturb the interaction between VISTA and its antibody which would correlate with attenuation of FRET signal.
  • a linear relationship between FRET ratio and the acceptor concentration (0-10 pM) was detected.
  • Addition of VSIG3, a ligand of VISTA, in increasing concentrations resulted in gradual decrease in FRET signal.
  • the mean Z’ factor of the assay was 0.64 indicating a high-quality assay for HTS.
  • CA- 170 (2-(3 -(3 -amino- 1 -(3 -( 1 -amino-2-hydroxyethyl)- 1 ,2,4-oxadiazol-5-yl)-3 - oxopropyl)ureido)-3 -hydroxybutanoic acid), a small molecule developed by Aurigene and Curis, is currently undergoing phase II clinical trials for lung cancer, head and neck/oral cavity cancer. Although CA-170 is reported as dual PD-L1 and VISTA inhibitor, no biophysical data have been reported to confirm binding to PD-L1 or VISTA.
  • the developed TR-FRET assay was employed in HTS of chemical libraries of -4,000 compounds from NIH clinical collection (NIHCC) and National Cancer Institute (NCI) Developmental Therapeutics Program (DTP) in a single-dose assay. Compounds that exhibited more than 40% inhibition of the FRET signal in HTS were subjected to additional 10-dose screening. Further dose-dependent FRET screening identified NSC 6 22608 (FIG. 1A) as a ligand for VISTA with IC 5 0 value of 4.8 ⁇ 0.4 pM (FIG. IB). Moreover, competitive ELISA revealed the ability of the hit to disrupt VISTA-VSIG-3 interaction with IC 5 0 value of 9.7 ⁇ 0.3 pM (FIG. 1C). SPR screening confirmed binding of the hit to VISTA ECD with an estimated KD of 6.2 ⁇ 0.5 pM (FIG. ID).
  • Saturation transfer difference (STD) NMR was employed to study VISTA-hit interaction. Observance of strong STD signals confirmed tight binding of the hit to VISTA. High STD effects % (-80-100%) for NMR peaks of 2-furyl rings of the hit indicated direct interaction between the ring and the potential binding site. However, minimal STD effect % for the dimethylamino moiety revealed no direct interaction with the protein.
  • Trials to identify a potential binding site for NSC 6 22608 in VISTA started with exploring possible binding cavities in recent VISTA crystal structures; structure of VISTA ECD (PDB: 6OIL) and structure of VISTA bound to antibody Fab fragment (PDB: 6MVL).
  • Web-based tools for ligand binding site prediction including geometric-based method (DoGSiteScorer), templatebased method (LIBRA-WA), energetic-based method (FT site), and machine learning-based methods (Prankweb and Deepsite) were used to predict 32 potential binding sites in the two crystal structures (PDB IDs: 6OIL, 6MVL).
  • Glide docking of the ligand to the predicted binding sites in VISTA was performed using Maestro (Schrodinger).
  • GlideScore an empirical scoring fimction that approximates ligand binding free energy, was used to evaluate the binding.
  • Favorable Glide Scores of docked poses of the ligand were obtained upon docking into predicted sites in PDB ID 6MVL sharing Tyr69, Argl59, His98, Hisl58, Phe94, and Glu 157 residues. These results reveal that conformational changes to VISTA upon antibody binding have resulted in formation of favorable binding sites for the hit.
  • the lowest GlideScore was observed for the best docked pose of the hit to the site predicted by Prankweb in PDB ID 6MVL (FIGS.
  • the docked pose of the ligand into this site features interaction of the oxygen atom of the 2-fiiryl ring with 2 hydrogen bonds with Argl59 residue (FIG. 2A). Moreover, the 2-furyl ring further interacts with Glul57 residue via aromatic hydrogen bonding interaction. The dithiazole moiety in the hit engages in hydrophobic interaction with Phe94 residue in VISTA (FIG. 2A).
  • a small library of single-point VISTA mutants was then expressed and purified based on the key interacting resides of VISTA in the binding pose shown in FIG. 2 A and other key residues from the rest of docked poses.
  • Competitive ELISA was then performed between VISTA mutants and VSIG-3 in the presence of the hit.
  • Promega VISTA bioluminescent cell-based T-cell activation assay was used to evaluate the ability of III to block VISTA in a cellular platform.
  • the assay is based on VISTA-VISTA interaction between CHOK1 VISTA cells and NanoGloTMIL-2/Jurkat VISTA cells which promotes the production of interleukin 2 (IL-2) resulting in a luminescent signal.
  • VISTA antibody reduces IL-2 signaling at nanomolar concentration as evident from diminished luminescent signaling.
  • compound III blocks VISTAVISTA interaction resulting in decreased IL-2 signaling.
  • CA-170 is not able to decrease IL-2 signaling which comes in agreement with its minimal VISTA binding affinity (FIG. 4B).
  • VISTA expression in ovarian and endometrial cancers has been directly associated with regulation of T-cell function.
  • ovarian cancer cell lines OLKATE, SKOV4, COV504, and A2780
  • endometrial cancer cell lines RL952, HEC 1 A, AN3CA, and Ishikawa
  • S.D. +ve Standard deviation of the positive control.
  • S.D. -ve Standard deviation of the negative control.
  • Mean +ve mean of the positive controls
  • Mean -ve mean of the negative controls
  • NMR experiments were prepared using la (10 mM) prepared in phosphate buffer solution, pH 7.0. Samples had final concentrations of 1% DMSO-ck and D2O (10% v/v). Each sample was made up to a final volume of 500 pL with PBS buffer (20 mm, pH 7.0) and NaCl (20 mm). The experiments were performed at a final concentration of la (1 mM) on Agilent 400 MHz WB NMR spectrometer at 298 K and pH 7.0 in the presence and absence of VISTA extracellular domain (20 pm).
  • the protein was saturated on-resonance at -0.5 ppm and off- resonance at 33 ppm with a cascade of 40 selective Gaussian shaped pulses, of 50 ms duration with a 100 psec delay between each pulse.
  • the total duration of the saturation time was set to 2 s.
  • a total of 256 scans per STD NMR experiment were acquired and a WATERGATE sequence was used to suppress the residual H2O signal.
  • TR-FRET Time-resolved fluorescence resonance energy transfer
  • VISTA Protein Human recombinant VISTA Protein (His Tag), catalogue number: 13482-H08H, was obtained from Sino biological and labeled with MAb Anti-6HIS Tb cryptate from Cisbio.
  • VISTA antibody MAB71263-SP was obtained from Novus Biologicals and labeled using Mix-n-StainTM CFTM 647 Antibody Labeling Kit (Sigma Aldrich).
  • the assay plates were medium binding white 384-well plates (Greiner #784075).
  • the assay buffer was phosphate buffer solution (PBS, pH 7.0).
  • TR-FRET measurements were done on Tecan Infinite M1000 PRO (top read, Z-Position height: 25500 pm, (Donor Read-620) Ex 340-20 nm, Em 620-5 nm, Gain value: 255, (Acceptor Read-665) Ex 340-20 nm, Em 665-5 nm, Gain value: 255; both with 50 flashes/well. 200 ps integration time, 100Hz, 50 ps lag time).
  • VISTA Tb cryptate (25 pM) and CF647 -labeled VISTA antibody (1.6 pM) stock solutions were prepared in PBS (pH7.0).
  • VISTA Tb cryptate and CF647-labeled VISTA antibody were immediately mixed prior to the assay to a final concentration of: His-tagged VISTA (10 nM), MAb Anti-6HIS Tb cryptate Gold (1 nM), and CF647-labeled VISTA antibody (10 nM).
  • Stock solutions of tested compounds in DMSO (50 nL) from the chemical libraries were applied to 384-well plates. The assay mixture (10 pL) was added to the plated compounds and incubated for 3 hrs at room temperature.
  • TR-FRET signals were calculated as a ratio as follows: (intensity of 665 nm)/(intensity of 615 nm) x 10,000.
  • the IC 5 0 values were calculated by plotting compound concentration versus TR-FRET signal.
  • the dose-response curves were analyzed by nonlinear regression using GraphPad Prism 8.0.2 (GraphPad Software, Inc., La Jolla, CA, USA). Each data point from this assay represents the average of three independent measurements. Error bars represent standard deviation.
  • VISTA-VSIG-3 enzyme-linked Immunosorbent assay Human recombinant VISTA (ECD, Fc Tag), catalogue number: 13482-H02H, was obtained from Sino biological. Biotinylated human VSIG3 protein (Fc, Avitag) was obtained from Aero Biosystems. VISTA ECD (2 pg/mL) in PBS (pH 7.0) was immobilized on 96-well clear polystyrene plates from R&D systems (Part # 991427) by addition of 50 pL of stock solution for 12 hrs at 4 °C.
  • the ELISA plates were washed three times with PBS, then blocked with 1% bovine serum albumin (BSA) in PBS at room temperature for 2 hrs.
  • the blocking buffer was removed and plates were washed by PBS three times.
  • the wells then incubated for 2 hrs at room temperature with 50 pL solutions containing various concentrations of tested compounds (obtained by diluting from a 1 mg/mL stock solution of each molecule dissolved in PBS containing 1% DMSO) and a fixed concentration of biotinylated human VSIG3 protein (2 nM) in protein-free blocking buffer.
  • VISTA-VSIG-3 binding was detected by adding streptavidin-horseradish peroxidase (HRP) followed by substrate color reagents (R&D Systems, Minneapolis, MN). The reactions were stopped after 15 min with 2M sulfiiric acid, and absorbance at 450 nm was measured on microplate reader (BioTek Synergy4).
  • HRP streptavidin-horseradish peroxidase
  • substrate color reagents R&D Systems, Minneapolis, MN
  • SPR Surface plasmon resonance
  • Cell culture SKOV4, RL952, HEC 1 A, AN3CA, and Jurkat cell lines were obtained from American Type Culture Collection (ATCC).
  • COV504 and A2780 cell Hines were obtained from European Collection of Authenticated Cell Cultures (EC ACC).
  • OVKATE and Ishikawa cell lines were obtained from Prof. Wendy Fantl lab, Stanford University.
  • Cells were cultured in RPMI1640 medium with 10% Fetal Bovine Serum (FBS), 100 units/mL penicillin and 100 pg/mL streptomycin. All cells were grown at 37 °C in a humidified atmosphere containing 5% CO2.
  • FBS Fetal Bovine Serum
  • VISTA mutants were expressed and purified using previously reported method (with minor modifications) for expression and purification of VISTA mutants.
  • 2 Expi293 cells were acquired from ThermoFisher Scientific and grown in Expi293 Expression Medium according to the manufacturer’s protocol.
  • the extracellular domain of human VISTA (residues 1 to 161) provided by Integrated DNA Technologies (Coralville, IA) was cloned into a pcDNA3.4 vector (Thermo Fisher) with an N-terminal signal peptide from CRYPa and a C-terminal Mpro protease site and His6-tag.
  • Plasmids were transfected into Expi293 cells (Thermo Fisher) using provider’s protocol but with the addition of 20 pM swainsonine (Cayman Chemical) at the time of transfection. Secreted protein in the supernatant was harvested, dialyzed into Ni-NTA binding buffer (50 mM Tris pH 8.0, 300 mM NaCl), and purified using Ni-NTA affinity chromatography (Qiagen). Binding of the mutants to the VISTA antibody (MAB71263-SP) was retained with no significant difference in binding from WT VISTA.
  • Ni-NTA binding buffer 50 mM Tris pH 8.0, 300 mM NaCl
  • Qiagen Ni-NTA affinity chromatography
  • Flow cytometry analysis For the analysis of the expression of human VISTA on the surface of selected ovarian and endometrial cancer cells, flow cytometry analysis was performed. The cells were seeded on 6-well plates and cultured till sub-confluency. The cells were detached from the plates with Cellstripper (Coming, Cat. No. 25-056-CI), placed on ice, washed 2 times with Flow Cytometry Staining Buffer (eBioscience, Waltham, MA, USA) and stained with Human VISTA/B7-H5/PD-1H Alexa Fluor® 647-conjugated Antibody (R&D systems, Cat. No. FAB71261R). The cells were analyzed with the BD Biosciences LSRII cytometer.
  • Promega VISTA bioluminescent cell-based T-cell activation assay The assay kit including NanoGioTM IL2-NLP/Jurkat VISTA cells and CHOK1 VISTA cells were obtained from Promega, WI. The assay was then performed according to provider’s protocol. Breifly, Jurkat cells (5 x 10 4 cells) and CHOK1 cells (3 x 10 4 cells) were co-cultured overnight in RPMI1640 medium with 10% Fetal Bovine Serum (FBS). Antihuman CD3 antibody (1000 ng/mL) was then added and the co-cultures were incubated for 6 hrs at 37 °C in a humidified atmosphere containing 5% CO2.
  • FBS Fetal Bovine Serum
  • Promega Nano-GioTM Reagent was then reconstituted and used at the end of the incubation time according to the manufacturer’s instructions. Each data point from this assay represents the average of three independent measurements. Error bars represent standard deviation.
  • Prestoblue cell viability assay Briefly, 3 x 10 4 Jurkat T cells were cultured alone or co-cultured with 1.5 x 10 5 of 8 cancer cell lines used in the assay in 24-well plates. After incubation with compound III (5 pM) or VISTA antibody (MAB71263-SP) (1 pM) for 24 h, Jurkat T cells were harvested from the supernatant. Presto Blue® Viability Assay (Thermo Fisher Scientific Inc., Waltham, MA) was then performed. Each data point from this assay represents the average of three independent measurements. Error bars represent standard deviation.
  • cytokines production by ELISA Human T cells were isolated from peripheral blood mononuclear cells (PBMC 8 ) using the Human Pan-T Cell Isolation Kit (Miltenyi Biotec). Human IFN-y, and TNFa ELISA kits (Qiagen, Valencia, CA, USA) were used to analyze cytokine production. Each data point from this assay represents the average of three independent measurements. Error bars represent standard deviation.

Abstract

Provided herein are small molecules targeting V-domain Ig Suppressor of T-cell Activation (VISTA) as immunomodulators and imaging probes.

Description

VISTA INHIBITORS
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional Application No. 63/075,672, filed September 8, 2020, which is incorporated herein by reference in its entirety and for all purposes.
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT
[0002] This invention was made with government support under grant no. CA201719, awarded by the National Institutes of Health. The government has certain rights in this invention.
BACKGROUND
[0003] Negative immune checkpoints, such as cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death-ligand 1 (PD-L1) promote cancer growth by downregulation of T-cell activation.1 Thus, blocking these immune checkpoints restores the ability of immune system to attack cancer cells. FDA-approved monoclonal antibodies (mAbs) against negative immune checkpoints have revealed remarkable clinical success in different malignancies.2'4 However, overall response rates to mAbs in cancer immunotherapy are generally lower than 30%.5,6 V-domain Ig Suppressor of T-cell Activation (VISTA) is a negative immune checkpoint protein that shares significant homology to PD-L1 in its extracellular domain (ECD).7'9 VISTA has been identified as a potential mediator of resistance to mAb-based immunotherapies in patients based on elevated VISTA levels in patients after administration of anti-CTLA-4 and anti-PD-Ll treatments.10,11 Moreover, VISTA has been introduced as a potential immunotherapeutic target in pancreatic cancer due to the engagement of VISTA in diminishing cytokine production in T cells isolated from metastatic pancreatic tumors.12 Thus, it would be advantageous to identify inhibitors of VISTA. Provided herein are solutions to these and other problems in the art.
BRIEF SUMMARY
[0004] In an aspect is provided, inter alia, a compound as described herein, including the compound of FIG. 3C. [0005] In an aspect is provided a compound having a structure of
Figure imgf000004_0001
(I), or a pharmaceutically acceptable salt thereof.
[0006] L1 is a bond, -NH-, -O-, -S-, -C(O)-, -C(O)NH-, -NHC(O)-, -NHC(O)NH-, -C(O)O-, -OC(O)-, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene.
[0007] R1 is hydrogen, halogen, -CX’3, -CHX’2, -CH2X1, -OCX13, -
OCH2X1, -OCHX’2, -CN, -SOniR1D, -SOVINR1AR1B, -NR1CNR1AR1B, -ONR1AR1B, -NHC(O)NR1CNR1AR1B, -NHC(O)NR1AR1B, -N(O)mi, -NR1AR1B, -C(O)R1C, -C(O)-OR1C, -C (O)NR1AR1B, -OR1D, -NR1ASO2R1D, -NR1AC(O)R1C, -NR1AC(O)OR1C, -NR1AOR1C, -N3, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.
[0008] R2 is hydrogen, halogen, -CX2 3, -CHX2 2, -CH2X2, -OCX2 3, -
OCH2X2, -OCHX22, -CN, -SOn2R2D, -SOV2NR2AR2B, -NR2CNR2AR2B, -ONR2AR2B, -NHC(O)NR2CNR2AR2B, -NHC(O)NR2AR2B, -N(O)m2, -NR2AR2B, -C(O)R2C, -C(O)-OR2C, -C (O)NR2AR2B, -OR2D, -NR2ASO2R2D, -NR2AC(O)R2C, -NR2AC(O)OR2C, -NR2AOR2C, -N3, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.
[0009] R1 and R2 may optionally be joined to form a substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl. [0010] R4 is indendently halogen, -CX4 3, -CHX4 2, -CH2X4, -OCX4 3, -
OCH2X4, -OCHX42, -CN, -SOn4R4D, -SOV4NR4AR4B, -NR4CNR4AR4B, -ONR4AR4B, -NHC(O)NR4CNR4AR4B, -NHC(O)NR4AR4B, -N(O)m4, -NR4AR4B, -C(O)R4C, -C(O)-OR4C, -C (O)NR4AR4B, -OR4D, -NR4ASO2R4D, -NR4AC(O)R4C, -NR4AC(O)OR4C, -NR4AOR4C, -N3, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.
[0011] R1A, R1B, R1C, R1D, R2A, R2B, R2C, R2D, R4A, R4B, R4C, and R4D are independently hydrogen, -CX3, -CHX2, -CH2X, -CN, -OH, -COOH, -CONH2, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; R1A and R1B or R2A and R2B with substituents bonded to the same nitrogen atom may optionally be joined to form a substituted or unsubstituted heterocycloalkyl or substituted or unsubstituted heteroaryl.
[0012] Each X, X1, X2, and X4 is independently -F, -Cl, -Br, or -I.
[0013] Each nl, n2 and n4 is independently an integer from 0 to 4.
[0014] z is an integer from 0 to 5.
[0015] Each ml, m2, m4, vl, v2 and v4 are independently 1 or 2.
[0016] In embodiments, the compound has a structure of
Figure imgf000005_0001
[0017] In an aspect is provided a pharmaceutical composition including the compound described herein, or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. [0018] In an aspect is provided a method of treating a cancer in a subject in need thereof, the method comprising administrating an effective amount of the compound described herein, or a pharmaceutically acceptable salt thereof.
[0019] In an aspect is provided a method of inhibiting V-domain Ig Suppressor of T-cell Activation (VISTA) in a cell, the method comprising contacting the cell with an effective amount of the compound of described herein, or a pharmaceutically acceptable salt thereof.
[0020] In an aspect is provided a method of enhancing T-cell proliferation in a subject in the presence of VISTA-expressing cancer cells, the method comprising administrating the subject an effective amount of the compound described herein, or a pharmaceutically acceptable salt thereof.
[0021] In an aspect is provided a method of restoring T-cell activation in a subject in the presence of VISTA-expressing cancer cells, the method comprising administrating the subject an effective amount of the compound described herein, or a pharmaceutically acceptable salt thereof.
[0022] Other aspects of the invention are disclosed infra.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIGS. 1A-1D present the structure of the NSC622608 compound and examplary interaction data of said compound. FIG. 1 A is a drawing showing the chemical structure of NSC622608. FIG. IB is a graph showing the dose-response curve for NSC622608 in Fluorescence Resonance Energy Transfer (FRET) assay. FIG. 1C is a graph showing the dose-response curve for NSC622608 in Enzyme-Linked Immunosorbent Assay (ELISA) assay. Error bars represent standard deviation (n=3). FIG. ID is a graph showing binding curves of different NSC622608 concentrations to VISTA in SPR screening.
[0024] FIGS. 2A-2C present exemplary data related to the interaction between the NSC622608 compound and its target, the V-domain Ig Suppressor of T-cell Activation (VISTA). FIG. 2A is a 3D view of predicted binding pose of NSC622608 to VISTA crys-tal structure. FIG. 2B is an overview of the binding pocket of NSC622608 (highlighted in light blue) on VISTA surface. FIG. 2C is a bar graph showing an exemplary evaluation of VISTA mutants in competitive ELISA assay in the presence of NSC622608 (20 pM). Error bars represent standard deviation (n=6). (** p < 0.05; *** p < 0.005 relative to wild-typ (Wt) VISTA). [0025] FIGS. 3A-3C present the selection of a small-molecule ligand for VISTA. FIG. 3 A shows the optimization of the furan-2-ylmethyl fragment of the hit. FIG. 3B shows the optimization of the dimethylamino fragment of the hit. FIG. 3C shows the hybrid compound III. In FIGS. 3A-3C, the listed IC50 values (pM) for VISTA binding affinity were derived from FRET screening. FIG. 3D presents dose-response curve for compound III in competitive VISTA-VSIG-3 ELISA. Error bars represent standard deviation (n=3).
[0026] FIGS. 4A-4D present exemplary data showing the effect of compound III on different cell lines. FIG. 4A is a graph showing luminescence signals in co-cultures of NanoGlo™IL-2/Jurkat VISTA cells with CHOK1 VISTA cells and CHOK1 parental cells in the presence of compound III. FIG. 4B is a graph showing luminescence signals in cocultures of NanoGlo™IL-2/Jurkat VISTA cells with CHOK1 VISTA cells and CHOK1 parental cells in the presence of CA-170. FIG. 4C is a bar graph showing PrestoBlue viability of Jurkat T-cells in the presence of compound III (5 pM) and VISTA Ab (1 pM) upon coculturing with ovarian cancer cell lines (from left to right and for each experiment shown on the bar graph: control experiment (Control), co-culture (Co-culture), compound III (III), and antibody (Ab)). FIG. 4D is a bar graph showing PrestoBlue viability of Jurkat T-cells in the presence of compound III (5 pM) and VISTA Ab (1 pM) upon co-culturing with endometrial cancer cell lines (from left to right and for each experiment shown on the bar graph: control experiment (Control), co-culture (Co-culture), compound III (III), and antibody (Ab)). In FIGS. 4C-4D, error bars represent standard deviation (n=3). ( p < 0.05; p < 0.005 relative to untreated control).
[0027] FIG. 5 presents an overview of the evolution of the IC50 values in VISTA FRET assay throughout the structural optimization of the NSC622608 compound.
[0028] FIG. 6 presents Interferon gamma (IFNy) concentrations at 24 hours, determined by ELISA, in the supernatant of co-culture of purified T cells from human peripheral blood mononuclear cells (PBMC8) with OVKATE ovarian cancer cell line and RL952 endometrial cancer cell line in the presence of III (5 pM) and VISTA Ab (1 pM). Error bars represent standard deviation (n=3). ( p < 0.05; p < 0.005 relative to untreated control).
[0029] FIG. 7 presents tumor necrosis factor alpha (TNFa) concentrations at 24 hours, determined by ELISA, in the supernatant of co-culture of purified T cells from human peripheral blood mononuclear cells (PBMC8) with OVKATE ovarian cancer cell line and RL952 endometrial cancer cell line in the presence of III (5 pM) and VISTA Ab (1 pM). Error bars represent standard deviation (n=3). ( p < 0.05; p < 0.005 relative to untreated control).
DETAILED DESCRIPTION
Definitions
[0030] In this disclosure, “comprises,” “comprising,” “containing” and “having” and the like can have the meaning ascribed to them in U.S. Patent law and can mean “ includes,” “including,” and the like. “Consisting essentially of or “consists essentially” likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.
[0031] An “inhibitor” refers to a compound (e.g. compounds described herein) that reduces activity when compared to a control, such as absence of the compound or a compound with known inactivity.
[0032] “Contacting” is used in accordance with its plain ordinary meaning and refers to the process of allowing at least two distinct species (e.g. chemical compounds including biomolecules or cells) to become sufficiently proximal to react, interact or physically touch. It should be appreciated; however, the resulting reaction product can be produced directly from a reaction between the added reagents or from an intermediate from one or more of the added reagents that can be produced in the reaction mixture.
[0033] The term “contacting” may include allowing two species to react, interact, or physically touch, wherein the two species may be a compound as described herein and a protein or enzyme. In some embodiments contacting includes allowing a compound described herein to interact with a protein or enzyme that is involved in a signaling pathway.
[0034] As defined herein, the term “activation”, “activate”, “activating”, “activator” and the like in reference to a protein-inhibitor interaction means positively affecting (e.g. increasing) the activity or fimction of the protein relative to the activity or function of the protein in the absence of the activator. In embodiments activation means positively affecting (e.g. increasing) the concentration or levels of the protein relative to the concentration or level of the protein in the absence of the activator. The terms may reference activation, or activating, sensitizing, or up-regulating signal transduction or enzymatic activity or the amount of a protein decreased in a disease. Thus, activation may include, at least in part, partially or totally increasing stimulation, increasing or enabling activation, or activating, sensitizing, or up-regulating signal transduction or enzymatic activity or the amount of a protein associated with a disease (e.g., a protein which is decreased in a disease relative to a non-diseased control). Activation may include, at least in part, partially or totally increasing stimulation, increasing or enabling activation, or activating, sensitizing, or up-regulating signal transduction or enzymatic activity or the amount of a protein
[0035] The terms “agonist,” “activator,” “upregulator,” etc. refer to a substance capable of detectably increasing the expression or activity of a given gene or protein. The agonist can increase expression or activity 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more in comparison to a control in the absence of the agonist. In certain instances, expression or activity is 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold or higher than the expression or activity in the absence of the agonist.
[0036] As defined herein, the term “inhibition”, “inhibit”, “inhibiting” and the like in reference to a protein-inhibitor interaction means negatively affecting (e.g. decreasing) the activity or function of the protein relative to the activity or fimction of the protein in the absence of the inhibitor. In embodiments inhibition means negatively affecting (e.g. decreasing) the concentration or levels of the protein relative to the concentration or level of the protein in the absence of the inhibitor. In embodiments inhibition refers to reduction of a disease or symptoms of disease. In embodiments, inhibition refers to a reduction in the activity of a particular protein target. Thus, inhibition includes, at least in part, partially or totally blocking stimulation, decreasing, preventing, or delaying activation, or inactivating, desensitizing, or down-regulating signal transduction or enzymatic activity or the amount of a protein. In embodiments, inhibition refers to a reduction of activity of a target protein resulting from a direct interaction (e.g. an inhibitor binds to the target protein). In embodiments, inhibition refers to a reduction of activity of a target protein from an indirect interaction (e.g. an inhibitor binds to a protein that activates the target protein, thereby preventing target protein activation).
[0037] The terms “inhibitor,” “repressor” or “antagonist” or “downregulator” interchangeably refer to a substance capable of detectably decreasing the expression or activity of a given gene or protein. The antagonist can decrease expression or activity 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more in comparison to a control in the absence of the antagonist. In certain instances, expression or activity is 1.5-fold, 2-fold, 3- fold, 4-fold, 5 -fold, 10-fold or lower than the expression or activity in the absence of the antagonist.
[0038] The term "expression" includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion. Expression can be detected using conventional techniques for detecting protein (e.g., ELISA, Western blotting, flow cytometry, immunofluorescence, immunohistochemistry, etc.).
[0039] The term “modulator” refers to a composition that increases or decreases the level of a target molecule or the function of a target molecule or the physical state of the target of the molecule relative to the absence of the modulator.
[0040] The term “modulate” is used in accordance with its plain ordinary meaning and refers to the act of changing or varying one or more properties. “Modulation” refers to the process of changing or varying one or more properties. For example, as applied to the effects of a modulator on a target protein, to modulate means to change by increasing or decreasing a property or function of the target molecule or the amount of the target molecule.
[0041] The term “associated” or “associated with” in the context of a substance or substance activity or function associated with a disease (e.g. a protein associated disease, a cancer (e.g., cancer, inflammatory disease, autoimmune disease, or infectious disease)) means that the disease (e.g. cancer, inflammatory disease, autoimmune disease, or infectious disease) is caused by (in whole or in part), or a symptom of the disease is caused by (in whole or in part) the substance or substance activity or function. As used herein, what is described as being associated with a disease, if a causative agent, could be a target for treatment of the disease.
[0042] The term “aberrant” as used herein refers to different from normal. When used to describe enzymatic activity or protein function, aberrant refers to activity or function that is greater or less than a normal control or the average of normal non-diseased control samples. Aberrant activity may refer to an amount of activity that results in a disease, wherein returning the aberrant activity to a normal or non-disease-associated amount (e.g. by administering a compound or using a method as described herein), results in reduction of the disease or one or more disease symptoms.
[0043] The term “signaling pathway” as used herein refers to a series of interactions between cellular and optionally extra-cellular components (e.g. proteins, nucleic acids, small molecules, ions, lipids) that conveys a change in one component to one or more other components, which in turn may convey a change to additional components, which is optionally propagated to other signaling pathway components.
[0044] The term “pharmaceutically acceptable salts” is meant to include salts of the active compounds that are prepared with relatively nontoxic acids or bases, depending on the particular substituents found on the compounds described herein. When compounds of the present disclosure contain relatively acidic functionalities, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salt, or a similar salt. When compounds of the present disclosure contain relatively basic functionalities, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like, as well as the salts derived from relatively nontoxic organic acids like acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic, p- tolylsulfonic, citric, tartaric, oxalic, methanesulfonic, and the like. Also included are salts of amino acids such as arginate and the like, and salts of organic acids like glucuronic or galactunoric acids and the like (see, for example, Berge et al., “Pharmaceutical Salts”, Journal of Pharmaceutical Science, 1977, 66, 1-19). Certain specific compounds of the present disclosure contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts.
[0045] Thus, the compounds of the present disclosure may exist as salts, such as with pharmaceutically acceptable acids. The present disclosure includes such salts. Non-limiting examples of such salts include hydrochlorides, hydrobromides, phosphates, sulfates, methanesulfonates, nitrates, maleates, acetates, citrates, fumarates, proprionates, tartrates (e.g., (+)-tartrates, (-)-tartrates, or mixtures thereof including racemic mixtures), succinates, benzoates, and salts with amino acids such as glutamic acid, and quaternary ammonium salts (e.g. methyl iodide, ethyl iodide, and the like). These salts may be prepared by methods known to those skilled in the art. [0046] The neutral forms of the compounds are preferably regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner. The parent form of the compound may differ from the various salt forms in certain physical properties, such as solubility in polar solvents.
[0047] In addition to salt forms, the present disclosure provides compounds, which are in a prodrug form. Prodrugs of the compounds described herein are those compounds that readily undergo chemical changes under physiological conditions to provide the compounds of the present disclosure. Prodrugs of the compounds described herein may be converted in vivo after administration. Additionally, prodrugs can be converted to the compounds of the present disclosure by chemical or biochemical methods in an ex vivo environment, such as, for example, when contacted with a suitable enzyme or chemical reagent.
[0048] Certain compounds of the present disclosure can exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to unsolvated forms and are encompassed within the scope of the present disclosure. Certain compounds of the present disclosure may exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the present disclosure and are intended to be within the scope of the present disclosure.
[0049] “Pharmaceutically acceptable excipient” and “pharmaceutically acceptable carrier” refer to a substance that aids the administration of an active agent to and absorption by a subject and can be included in the compositions of the present disclosure without causing a significant adverse toxicological effect on the patient. Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, normal saline solutions, lactated Ringer’s, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavors, salt solutions (such as Ringer's solution), alcohols, oils, gelatins, carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethycellulose, polyvinyl pyrrolidine, and colors, and the like. Such preparations can be sterilized and, if desired, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and/or aromatic substances and the like that do not deleteriously react with the compounds of the disclosure. One of skill in the art will recognize that other pharmaceutical excipients are usefiil in the present disclosure. [0050] The term "preparation" is intended to include the formulation of the active compound with encapsulating material as a carrier providing a capsule in which the active component with or without other carriers, is surrounded by a carrier, which is thus in association with it. Similarly, cachets and lozenges are included. Tablets, powders, capsules, pills, cachets, and lozenges can be used as solid dosage forms suitable for oral administration.
[0051] As used herein, the term "about” means a range of values including the specified value, which a person of ordinary skill in the art would consider reasonably similar to the specified value. In embodiments, about means within a standard deviation using measurements generally acceptable in the art. In embodiments, about means a range extending to +/- 10% of the specified value. In embodiments, about includes the specified value.
[0052] A “synergistic amount” as used herein refers to the sum of a first amount (e.g., an amount of a compound provided herein) and a second amount (e.g., a therapeutic agent) that results in a synergistic effect (i.e. an effect greater than an additive effect). Therefore, the terms "synergy", "synergism", "synergistic", "combined synergistic amount", and "synergistic therapeutic effect" which are used herein interchangeably, refer to a measured effect of the compound administered in combination where the measured effect is greater than the sum of the individual effects of each of the compounds provided herein administered alone as a single agent.
[0053] In embodiments, a synergistic amount may be about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8,
2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9,
5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0,
7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1,
9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,
25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% of the amount of the compound provided herein when used separately from the therapeutic agent. In embodiments, a synergistic amount may be about 0.1, 0.2, 0.3, 0.4, 0.5,
0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6,
2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7,
4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,
48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,
73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,
98, or 99% of the amount of the therapeutic agent when used separately from the compound provided herein.
[0054] The term “vaccine” refers to a composition that can provide active acquired immunity to and/or therapeutic effect (e.g. treatment) of a particular disease or a pathogen. A vaccine typically contains one or more agents that can induce an immune response in a subject against a pathogen or disease, i.e. a target pathogen or disease. The immunogenic agent stimulates the body’s immune system to recognize the agent as a threat or indication of the presence of the target pathogen or disease, thereby inducing immunological memory so that the immune system can more easily recognize and destroy any of the pathogen on subsequent exposure. Vaccines can be prophylactic (e.g. preventing or ameliorating the effects of a fiiture infection by any natural or pathogen, or of an anticipated occurrence of cancer in a predisposed subject) or therapeutic (e.g., treating cancer in a subject who has been diagnosed with the cancer). The administration of vaccines is referred to vaccination. In some examples, a vaccine composition can provide nucleic acid, e.g. mRNA that encodes antigenic molecules (e.g. peptides) to a subject. The nucleic acid that is delivered via the vaccine composition in the subject can be expressed into antigenic molecules and allow the subject to acquire immunity against the antigenic molecules. In the context of the vaccination against infectious disease, the vaccine composition can provide mRNA encoding antigenic molecules that are associated with a certain pathogen, e.g. one or more peptides that are known to be expressed in the pathogen (e.g. pathogenic bacterium or virus). In the context of cancer vaccine, the vaccine composition can provide mRNA encoding certain peptides that are associated with cancer, e.g. peptides that are substantially exclusively or highly expressed in cancer cells as compared to normal cells. The subject, after vaccination with the cancer vaccine composition, can have immunity against the peptides that are associated with cancer and kill the cancer cells with specificity.
[0055] The term “immune response” used herein encompasses, but is not limited to, an “adaptive immune response”, also known as an “acquired immune response” in which adaptive immunity elicits immunological memory after an initial response to a specific pathogen or a specific type of cells that is targeted by the immune response, and leads to an enhanced response to that target on subsequent encounters. The induction of immunological memory can provide the basis of vaccination.
[0056] The term “immunogenic” or “antigenic” refers to a compound or composition that induces an immune response, e.g., cytotoxic T lymphocyte (CTL) response, a B cell response (for example, production of antibodies that specifically bind the epitope), an NK cell response or any combinations thereof, when administered to an immunocompetent subject. Thus, an immunogenic or antigenic composition is a composition capable of eliciting an immune response in an immunocompetent subject. For example, an immunogenic or antigenic composition can include one or more immunogenic epitopes associated with a pathogen or a specific type of cells that is targeted by the immune response. In addition, an immunogenic composition can include isolated nucleic acid constructs (such as DNA or RNA) that encode one or more immunogenic epitopes of the antigenic polypeptide that can be used to express the epitope(s) (and thus be used to elicit an immune response against this polypeptide or a related polypeptide associated with the targeted pathogen or type of cells).
[0057] The term “EC50” or “half maximal effective concentration” as used herein refers to the concentration of a molecule (e.g., antibody, chimeric antigen receptor or bispecific antibody) capable of inducing a response which is halfway between the baseline response and the maximum response after a specified exposure time. In embodiments, the EC50 is the concentration of a molecule (e.g., antibody, chimeric antigen receptor or bispecific antibody) that produces 50% of the maximal possible effect of that molecule.
[0058] The term “IC50” or “half maximal inhibitory concentration” as used herein refers to the concentration of a molecule (e.g., drug, antibody, chimeric antigen receptor or bispecific antibody) capable of inhibiting a specific biological or biochemical function halfway between the baseline response and the maximum response after a specified exposure time. In embodiments, the IC50 is a quantitative measure that indicates how much of a particular inhibitory substance is needed to inhibit a given biological process or biological component by 50% (e.g. an enzyme, cell, cell receptor or microorganism).
[0059] The term "VISTA" or " V-domain Ig suppressor of T cell activation " as provided herein includes any of the recombinant or naturally-occurring forms of VISTA or variants or homologs thereof that maintain VISTA activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to VISTA). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring VISTA polypeptide. In embodiments, VISTA is the protein as identified by the NCBI sequence reference NP 071436, homolog or functional fragment thereof. Vista is a type I transmembrane protein that functions as an immune checkpoint and is encoded by the C10orf54 gene.
[0060] The terms “disease” or “condition” refer to a state of being or health status of a patient or subject capable of being treated with the compounds or methods provided herein. The disease may be a cancer. The disease may be an autoimmune disease. The disease may be an inflammatory disease. The disease may be an infectious disease. In some fiirther instances, “cancer” refers to human cancers and carcinomas, sarcomas, adenocarcinomas, lymphomas, leukemias, etc., including solid and lymphoid cancers, kidney, breast, lung, bladder, colon, ovarian, prostate, pancreas, stomach, brain, head and neck, skin, uterine, testicular, glioma, esophagus, and liver cancer, including hepatocarcinoma, lymphoma, including B-acute lymphoblastic lymphoma, non-Hodgkin’s lymphomas (e.g., Burkitt’s, Small Cell, and Large Cell lymphomas), Hodgkin’s lymphoma, leukemia (including AML, ALL, and CML), or multiple myeloma.
[0061] As used herein, the term "cancer" refers to all types of cancer, neoplasm or malignant tumors found in mammals (e.g. humans), including leukemias, lymphomas, carcinomas and sarcomas. Exemplary cancers that may be treated with a compound or method provided herein include brain cancer, glioma, glioblastoma, neuroblastoma, prostate cancer, colorectal cancer, pancreatic cancer, Medulloblastoma, melanoma, cervical cancer, gastric cancer, ovarian cancer, lung cancer, cancer of the head, Hodgkin's Disease, and NonHodgkin's Lymphomas. Exemplary cancers that may be treated with a compound or method provided herein include cancer (e.g., solid tumor) of the thyroid, endocrine system, brain, breast, cervix, colon, head & neck, liver, kidney, lung, ovary, pancreas, rectum, stomach, and uterus. Additional examples include, thyroid carcinoma, cholangiocarcinoma, pancreatic adenocarcinoma, skin cutaneous melanoma, colon adenocarcinoma, rectum adenocarcinoma, stomach adenocarcinoma, esophageal carcinoma, head and neck squamous cell carcinoma, breast invasive carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, non-small cell lung carcinoma, mesothelioma, multiple myeloma, neuroblastoma, glioma, glioblastoma multiforme, ovarian cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, primary brain tumors, malignant pancreatic insulanoma, malignant carcinoid, urinary bladder cancer, premalignant skin lesions, testicular cancer, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, endometrial cancer, adrenal cortical cancer, neoplasms of the endocrine or exocrine pancreas, medullary thyroid cancer, medullary thyroid carcinoma, melanoma, colorectal cancer, papillary thyroid cancer, hepatocellular carcinoma, or prostate cancer. In certain embodiments, the cancer is a solid tumor such as sarcomas and carcinomas, which may include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, nile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, uterine cancer, testicular cancer, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, glioblastoma multiforme, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodenroglioma, schwannoma, meningioma, melanoma, neuroblastoma, or retinoblastoma.
[0062] The term "leukemia" refers broadly to progressive, malignant diseases of the blood- forming organs and is generally characterized by a distorted proliferation and development of leukocytes and their precursors in the blood and bone marrow. Leukemia is generally clinically classified on the basis of (1) the duration and character of the disease-acute or chronic; (2) the type of cell involved; myeloid (myelogenous), lymphoid (lymphogenous), or monocytic; and (3) the increase or non-increase in the number abnormal cells in the blood- leukemic or aleukemic (subleukemic). Exemplary leukemias that may be treated with a compound or method provided herein include, for example, acute nonlymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, a leukocythemic leukemia, basophylic leukemia, blast cell leukemia, bovine leukemia, chronic myelocytic leukemia, leukemia cutis, embryonal leukemia, eosinophilic leukemia, Gross' leukemia, hairy-cell leukemia, hemoblastic leukemia, hemocytoblastic leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphogenous leukemia, lymphoid leukemia, lymphosarcoma cell leukemia, mast cell leukemia, megakaryocytic leukemia, micromyeloblastic leukemia, monocytic leukemia, myeloblastic leukemia, myelocytic leukemia, myeloid granulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasma cell leukemia, multiple myeloma, plasmacytic leukemia, promyelocytic leukemia, Rieder cell leukemia, Schilling's leukemia, stem cell leukemia, subleukemic leukemia, or undifferentiated cell leukemia.
[0063] As used herein, the term “lymphoma” refers to a group of cancers affecting hematopoietic and lymphoid tissues. It begins in lymphocytes, the blood cells that are found primarily in lymph nodes, spleen, thymus, and bone marrow. Two main types of lymphoma are non-Hodgkin lymphoma and Hodgkin’s disease. Hodgkin’s disease represents approximately 15% of all diagnosed lymphomas. This is a cancer associated with Reed- Sternberg malignant B lymphocytes. Non-Hodgkin’s lymphomas (NHL) can be classified based on the rate at which cancer grows and the type of cells involved. There are aggressive (high grade) and indolent (low grade) types of NHL. Based on the type of cells involved, there are B-cell and T-cell NHLs. Exemplary B-cell lymphomas that may be treated with a compound or method provided herein include, but are not limited to, small lymphocytic lymphoma, Mantle cell lymphoma, follicular lymphoma, marginal zone lymphoma, extranodal (MALT) lymphoma, nodal (monocytoid B-cell) lymphoma, splenic lymphoma, diffuse large cell B-lymphoma, Burkitt’s lymphoma, lymphoblastic lymphoma, immunoblastic large cell lymphoma, or precursor B -lymphoblastic lymphoma. Exemplary T- cell lymphomas that may be treated with a compound or method provided herein include, but are not limited to, cunateous T-cell lymphoma, peripheral T-cell lymphoma, anaplastic large cell lymphoma, mycosis fungoides, and precursor T-lymphoblastic lymphoma.
[0064] The term "sarcoma" generally refers to a tumor which is made up of a substance like the embryonic connective tissue and is generally composed of closely packed cells embedded in a fibrillar or homogeneous substance. Sarcomas that may be treated with a compound or method provided herein include a chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abernethy's sarcoma, adipose sarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, chorio carcinoma, embryonal sarcoma, Wilms' tumor sarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmented hemorrhagic sarcoma, immunoblastic sarcoma of B cells, lymphoma, immunoblastic sarcoma of T-cells, Jensen's sarcoma, Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma, leukosarcoma, malignant mesenchymoma sarcoma, parosteal sarcoma, reticulocytic sarcoma, Rous sarcoma, serocystic sarcoma, synovial sarcoma, or telangiectaltic sarcoma.
[0065] The term "melanoma" is taken to mean a tumor arising from the melanocytic system of the skin and other organs. Melanomas that may be treated with a compound or method provided herein include, for example, acral-lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman's melanoma, S91 melanoma, Harding-Passey melanoma, juvenile melanoma, lentigo maligna melanoma, malignant melanoma, nodular melanoma, subungal melanoma, or superficial spreading melanoma.
[0066] The term "carcinoma" refers to a malignant new growth made up of epithelial cells tending to infiltrate the surrounding tissues and give rise to metastases. Exemplary carcinomas that may be treated with a compound or method provided herein include, for example, medullary thyroid carcinoma, familial medullary thyroid carcinoma, acinar carcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, carcinoma adenomatosum, carcinoma of adrenal cortex, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloid carcinoma, basosquamous cell carcinoma, bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocellular carcinoma, chorionic carcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma, cribriform carcinoma, carcinoma en cuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma, carcinoma durum, embryonal carcinoma, encephaloid carcinoma, epiermoid carcinoma, carcinoma epitheliale adenoides, exophytic carcinoma, carcinoma ex ulcere, carcinoma fibrosum, gelatinifomi carcinoma, gelatinous carcinoma, giant cell carcinoma, carcinoma gigantocellulare, glandular carcinoma, granulosa cell carcinoma, hair-matrix carcinoma, hematoid carcinoma, hepatocellular carcinoma, Hurthle cell carcinoma, hyaline carcinoma, hypemephroid carcinoma, infantile embryonal carcinoma, carcinoma in situ, intraepidermal carcinoma, intraepithelial carcinoma, Krompecher's carcinoma, Kulchitzky- cell carcinoma, large-cell carcinoma, lenticular carcinoma, carcinoma lenticulare, lipomatous carcinoma, lymphoepithelial carcinoma, carcinoma medullare, medullary carcinoma, melanotic carcinoma, carcinoma molle, mucinous carcinoma, carcinoma muciparum, carcinoma mucocellulare, mucoepidermoid carcinoma, carcinoma mucosum, mucous carcinoma, carcinoma myxomatodes, nasopharyngeal carcinoma, oat cell carcinoma, carcinoma ossificans, osteoid carcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, prickle cell carcinoma, pultaceous carcinoma, renal cell carcinoma of kidney, reserve cell carcinoma, carcinoma sarcomatodes, Schneiderian carcinoma, scirrhous carcinoma, carcinoma scroti, signet-ring cell carcinoma, carcinoma simplex, small-cell carcinoma, solanoid carcinoma, spheroidal cell carcinoma, spindle cell carcinoma, carcinoma spongiosum, squamous carcinoma, squamous cell carcinoma, string carcinoma, carcinoma telangiectaticum, carcinoma telangiectodes, transitional cell carcinoma, carcinoma tuberosum, tuberous carcinoma, verrucous carcinoma, or carcinoma villosum.
[0067] As used herein, the terms "metastasis," "metastatic," and "metastatic cancer" can be used interchangeably and refer to the spread of a proliferative disease or disorder, e.g., cancer, from one organ or another non-adjacent organ or body part. “Metastatic cancer” is also called “Stage IV cancer.” Cancer occurs at an originating site, e.g., breast, which site is referred to as a primary tumor, e.g., primary breast cancer. Some cancer cells in the primary tumor or originating site acquire the ability to penetrate and infiltrate surrounding normal tissue in the local area and/or the ability to penetrate the walls of the lymphatic system or vascular system circulating through the system to other sites and tissues in the body. A second clinically detectable tumor formed from cancer cells of a primary tumor is referred to as a metastatic or secondary tumor. When cancer cells metastasize, the metastatic tumor and its cells are presumed to be similar to those of the original tumor. Thus, if lung cancer metastasizes to the breast, the secondary tumor at the site of the breast consists of abnormal lung cells and not abnormal breast cells. The secondary tumor in the breast is referred to a metastatic lung cancer. Thus, the phrase metastatic cancer refers to a disease in which a subject has or had a primary tumor and has one or more secondary tumors. The phrases non- metastatic cancer or subjects with cancer that is not metastatic refers to diseases in which subjects have a primary tumor but not one or more secondary tumors. For example, metastatic lung cancer refers to a disease in a subject with or with a history of a primary lung tumor and with one or more secondary tumors at a second location or multiple locations, e.g., in the breast.
[0068] The terms “cutaneous metastasis” or “skin metastasis” refer to secondary malignant cell growths in the skin, wherein the malignant cells originate from a primary cancer site (e.g., breast). In cutaneous metastasis, cancerous cells from a primary cancer site may migrate to the skin where they divide and cause lesions. Cutaneous metastasis may result from the migration of cancer cells from breast cancer tumors to the skin.
[0069] The term “visceral metastasis” refer to secondary malignant cell growths in the interal organs (e.g., heart, lungs, liver, pancreas, intestines) or body cavities (e.g., pleura, peritoneum), wherein the malignant cells originate from a primary cancer site (e.g., head and neck, liver, breast). In visceral metastasis, cancerous cells from a primary cancer site may migrate to the internal organs where they divide and cause lesions. Visceral metastasis may result from the migration of cancer cells from liver cancer tumors or head and neck tumors to internal organs.
[0070] The terms “treating”, or “treatment” refers to any indicia of success in the therapy or amelioration of an injury, disease, pathology or condition, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the injury, pathology or condition more tolerable to the patient; slowing in the rate of degeneration or decline; making the final point of degeneration less debilitating; improving a patient’s physical or mental well-being. The treatment or amelioration of symptoms can be based on objective or subjective parameters; including the results of a physical examination, neuropsychiatric exams, and/or a psychiatric evaluation. The term "treating" and conjugations thereof, may include prevention of an injury, pathology, condition, or disease. In embodiments, treating is preventing. In embodiments, treating does not include preventing.
[0071] “Treating” or “treatment” as used herein (and as well-understood in the art) also broadly includes any approach for obtaining beneficial or desired results in a subject’s condition, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of the extent of a disease, stabilizing (i.e., not worsening) the state of disease, prevention of a disease’s transmission or spread, delay or slowing of disease progression, amelioration or palliation of the disease state, diminishment of the reoccurrence of disease, and remission, whether partial or total and whether detectable or undetectable. In other words, "treatment" as used herein includes any cure, amelioration, or prevention of a disease. Treatment may prevent the disease from occurring; inhibit the disease’s spread; relieve the disease’s symptoms, fully or partially remove the disease’s underlying cause, shorten a disease’s duration, or do a combination of these things.
[0072] "Treating" and "treatment" as used herein include prophylactic treatment. Treatment methods include administering to a subject a therapeutically effective amount of an active agent. The administering step may consist of a single administration or may include a series of administrations. The length of the treatment period depends on a variety of factors, such as the severity of the condition, the age of the patient, the concentration of active agent, the activity of the compositions used in the treatment, or a combination thereof. It will also be appreciated that the effective dosage of an agent used for the treatment or prophylaxis may increase or decrease over the course of a particular treatment or prophylaxis regime. Changes in dosage may result and become apparent by standard diagnostic assays known in the art. In some instances, chronic administration may be required. For example, the compositions are administered to the subject in an amount and for a duration sufficient to treat the patient. In embodiments, the treating or treatment is no prophylactic treatment.
[0073] The term “prevent” refers to a decrease in the occurrence of disease symptoms in a patient. As indicated above, the prevention may be complete (no detectable symptoms) or partial, such that fewer symptoms are observed than would likely occur absent treatment.
[0074] “Patient” or “subject in need thereof’ refers to a living organism suffering from or prone to a disease or condition that can be treated by administration of a pharmaceutical composition as provided herein. Non-limiting examples include humans, other mammals, bovines, rats, mice, dogs, monkeys, goat, sheep, cows, deer, and other non-mammalian animals. In some embodiments, a patient is human.
[0075] An “effective amount” is an amount sufficient for a compound to accomplish a stated purpose relative to the absence of the compound (e.g. achieve the effect for which it is administered, treat a disease, reduce enzyme activity, increase enzyme activity, reduce a signaling pathway, or reduce one or more symptoms of a disease or condition). An example of an “effective amount” is an amount sufficient to contribute to the treatment, prevention, or reduction of a symptom or symptoms of a disease, which could also be referred to as a “therapeutically effective amount.” A “reduction” of a symptom or symptoms (and grammatical equivalents of this phrase) means decreasing of the severity or frequency of the symptom(s), or elimination of the symptom(s). A “prophylactically effective amount” of a drug is an amount of a drug that, when administered to a subject, will have the intended prophylactic effect, e.g., preventing or delaying the onset (or reoccurrence) of an injury, disease, pathology or condition, or reducing the likelihood of the onset (or reoccurrence) of an injury, disease, pathology, or condition, or their symptoms. The full prophylactic effect does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses. Thus, a prophylactically effective amount may be administered in one or more administrations. An “activity decreasing amount,” as used herein, refers to an amount of antagonist required to decrease the activity of an enzyme relative to the absence of the antagonist. A “function disrupting amount,” as used herein, refers to the amount of antagonist required to disrupt the function of an enzyme or protein relative to the absence of the antagonist. The exact amounts will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins).
[0076] For any compound described herein, the therapeutically effective amount can be initially determined from cell culture assays. Target concentrations will be those concentrations of active compound(s) that are capable of achieving the methods described herein, as measured using the methods described herein or known in the art.
[0077] As is well known in the art, therapeutically effective amounts for use in humans can also be determined from animal models. For example, a dose for humans can be formulated to achieve a concentration that has been found to be effective in animals. The dosage in humans can be adjusted by monitoring compounds effectiveness and adjusting the dosage upwards or downwards, as described above. Adjusting the dose to achieve maximal efficacy in humans based on the methods described above and other methods is well within the capabilities of the ordinarily skilled artisan.
[0078] The term “therapeutically effective amount,” as used herein, refers to that amount of the therapeutic agent sufficient to ameliorate the disorder, as described above. For example, for the given parameter, a therapeutically effective amount will show an increase or decrease of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%. Therapeutic efficacy can also be expressed as “-fold” increase or decrease. For example, a therapeutically effective amount can have at least a 1.2-fold, 1.5-fold, 2-fold, 5-fold, or more effect over a control.
[0079] Dosages may be varied depending upon the requirements of the patient and the compound being employed. The dose administered to a patient, in the context of the present disclosure, should be sufficient to effect a beneficial therapeutic response in the patient over time. The size of the dose also will be determined by the existence, nature, and extent of any adverse side-effects. Determination of the proper dosage for a particular situation is within the skill of the practitioner. Generally, treatment is initiated with smaller dosages which are less than the optimum dose of the compound. Thereafter, the dosage is increased by small increments until the optimum effect under circumstances is reached. Dosage amounts and intervals can be adjusted individually to provide levels of the administered compound effective for the particular clinical indication being treated. This will provide a therapeutic regimen that is commensurate with the severity of the individual's disease state.
[0080] As used herein, the term "administering" means oral administration, administration as a suppository, topical contact, intravenous, parenteral, intraperitoneal, intramuscular, intralesional, intrathecal, intranasal or subcutaneous administration, or the implantation of a slow-release device, e.g., a mini-osmotic pump, to a subject. Administration is by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, e.g., intravenous, intramuscular, intra-arteriole, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infiision, transdermal patches, etc. In embodiments, the administering does not include administration of any active agent other than the recited active agent.
[0081] "Co-administer" it is meant that a composition described herein is administered at the same time, just prior to, or just after the administration of one or more additional therapies. The compounds provided herein can be administered alone or can be coadministered to the patient. Coadministration is meant to include simultaneous or sequential administration of the compounds individually or in combination (more than one compound). Thus, the preparations can also be combined, when desired, with other active substances (e.g. to reduce metabolic degradation). The compositions of the present disclosure can be delivered transdermally, by a topical route, or formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders, and aerosols.
[0082] A “cell” as used herein, refers to a cell carrying out metabolic or other function sufficient to preserve or replicate its genomic DNA. A cell can be identified by well-known methods in the art including, for example, presence of an intact membrane, staining by a particular dye, ability to produce progeny or, in the case of a gamete, ability to combine with a second gamete to produce a viable offspring. Cells may include prokaryotic and eukaroytic cells. Prokaryotic cells include but are not limited to bacteria. Eukaryotic cells include but are not limited to yeast cells and cells derived from plants and animals, for example mammalian, insect (e.g., spodoptera) and human cells. Cells may be usefiil when they are naturally nonadherent or have been treated not to adhere to surfaces, for example by trypsinization.
[0083] “Control” or “control experiment” is used in accordance with its plain ordinary meaning and refers to an experiment in which the subjects or reagents of the experiment are treated as in a parallel experiment except for omission of a procedure, reagent, or variable of the experiment. In some instances, the control is used as a standard of comparison in evaluating experimental effects. In some embodiments, a control is the measurement of the activity of a protein in the absence of a compound as described herein (including embodiments and examples).
[0084] Cancer model organism, as used herein, is an organism exhibiting a phenotype indicative of cancer, or the activity of cancer causing elements, within the organism. The term cancer is defined above. A wide variety of organisms may serve as cancer model organisms, and include for example, cancer cells and mammalian organisms such as rodents (e.g. mouse or rat) and primates (such as humans). Cancer cell lines are widely understood by those skilled in the art as cells exhibiting phenotypes or genotypes similar to in vivo cancers. Cancer cell lines as used herein includes cell lines from animals (e.g. mice) and from humans.
[0085] An “anticancer agent” as used herein refers to a molecule (e.g. compound, peptide, protein, nucleic acid, 0103) used to treat cancer through destruction or inhibition of cancer cells or tissues. Anticancer agents may be selective for certain cancers or certain tissues. In embodiments, anticancer agents herein may include epigenetic inhibitors and multi-kinase inhibitors.
[0086] “Anti-cancer agent” and “anticancer agent” are used in accordance with their plain ordinary meaning and refers to a composition (e.g. compound, drug, antagonist, inhibitor, modulator) having antineoplastic properties or the ability to inhibit the growth or proliferation of cells. In some embodiments, an anti-cancer agent is a chemotherapeutic. In some embodiments, an anti -cancer agent is an agent identified herein having utility in methods of treating cancer. In some embodiments, an anti-cancer agent is an agent approved by the FDA or similar regulatory agency of a country other than the USA, for treating cancer. Examples of anti-cancer agents include, but are not limited to, MEK (e.g. MEK1, MEK2, or MEK1 and MEK2) inhibitors (e.g. XL518, CL1040, PD035901, selumetinib/ AZD6244, GSK1120212/ trametinib, GDC-0973, ARRY-162, ARRY-300, AZD8330, PD0325901, U0126, PD98059, TAK-733, PD318088, AS703026, BAY 869766), alkylating agents (e.g., cyclophosphamide, ifosfamide, chlorambucil, busulfan, melphalan, mechlorethamine, uramustine, thiotepa, nitrosoureas, nitrogen mustards (e.g., mechloroethamine, cyclophosphamide, chlorambucil, meiphalan), ethylenimine and methylmelamines (e.g., hexamethlymelamine, thiotepa), alkyl sulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine, lomusitne, semustine, streptozocin), triazenes (decarbazine)), anti -metabolites (e.g., 5- azathioprine, leucovorin, capecitabine, fludarabine, gemcitabine, pemetrexed, raltitrexed, folic acid analog (e.g., methotrexate), or pyrimidine analogs (e.g., fluorouracil, floxouridine, Cytarabine), purine analogs (e.g., mercaptopurine, thioguanine, pentostatin), etc.), plant alkaloids (e.g., vincristine, vinblastine, vinorelbine, vindesine, podophyllotoxin, paclitaxel, docetaxel, etc.), topoisomerase inhibitors (e.g., irinotecan, topotecan, amsacrine, etoposide (VP 16), etoposide phosphate, teniposide, etc.), antitumor antibiotics (e.g., doxorubicin, adriamycin, daunorubicin, epirubicin, actinomycin, bleomycin, mitomycin, mitoxantrone, plicamycin, etc.), platinum-based compounds (e.g. cisplatin, oxaloplatin, carboplatin), anthracenedione (e.g., mitoxantrone), substituted urea (e.g., hydroxyurea), methyl hydrazine derivative (e.g., procarbazine), adrenocortical suppressant (e.g., mitotane, aminoglutethimide), epipodophyllotoxins (e.g., etoposide), antibiotics (e.g., daunorubicin, doxorubicin, bleomycin), enzymes (e.g., L- asparaginase), inhibitors of mitogen-activated protein kinase signaling (e.g. U0126, PD98059, PD184352, PD0325901, ARRY-142886, SB239063, SP600125, BAY 43-9006, wortmannin, or LY294002, Syk inhibitors, mTOR inhibitors, antibodies (e.g., rituxan), gossyphol, genasense, polyphenol E, Chlorofusin, all trans-retinoic acid (ATRA), bryostatin, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), 5-aza-2'-deoxycytidine, all trans retinoic acid, doxorubicin, vincristine, etoposide, gemcitabine, imatinib (Gleevec.RTM.), geldanamycin, 17-N-Allylamino-17-Demethoxygeldanamycin (17-AAG), flavopiridol, LY294002, bortezomib, trastuzumab, BAY 11-7082, PKC412, PD184352, 20- epi-1, 25 dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; aclarubicin; acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TK antagonists; altretamine; ambamustine; amidox; amifostine; aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole; andrographolide; angiogenesis inhibitors; antagonist D; antagonist G; antarelix; anti- dorsalizing morphogenetic protein- 1; antiandrogen, prostatic carcinoma; antiestrogen; antineoplaston; antisense oligonucleotides; aphidicolin glycinate; apoptosis gene modulators; apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA; arginine deaminase; asulacrine; atamestane; atrimustine; axinastatin 1; axinastatin 2; axinastatin 3; azasetron; azatoxin; azatyrosine; baccatin III derivatives; balanol; batimastat; BCR/ABL antagonists; benzochlorins; benzoylstaurosporine; beta lactam derivatives; beta-alethine; betaclamycin B; betulinic acid; bFGF inhibitor; bicalutamide; bisantrene; bisaziridinylspermine; bisnafide; bistratene A; bizelesin; breflate; bropirimine; budotitane; buthionine sulfoximine; calcipotriol; calphostin C; camptothecin derivatives; canarypox IL-2; capecitabine; carboxamide-amino-triazole; carboxyamidotriazole; CaRest M4; CARN 700; cartilage derived inhibitor; carzelesin; casein kinase inhibitors (ICOS); castanospermine; cecropin B; cetrorelix; chlorins; chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin; cladribine; clomifene analogues; clotrimazole; collismycin A; collismycin B; combretastatin A4; combretastatin analogue; conagenin; crambescidin 816; crisnatol; cryptophycin 8; cryptophycin A derivatives; curacin A; cyclopentanthraquinones; cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor; cytostatin; dacliximab; decitabine; dehydrodidemnin B; deslorelin; dexamethasone; dexifosfamide; dexrazoxane; dexverapamil; diaziquone; didemnin B; didox; diethylnorspermine; dihydro-5-azacytidine; 9-dioxamycin; diphenyl spiromustine; docosanol; dolasetron; doxifluridine; droloxifene; dronabinol; duocarmycin SA; ebselen; ecomustine; edelfosine; edrecolomab; eflomithine; elemene; emitefur; epirubicin; epristeride; estramustine analogue; estrogen agonists; estrogen antagonists; etanidazole; etoposide phosphate; exemestane; fadrozole; fazarabine; fenretinide; filgrastim; finasteride; flavopiridol; flezelastine; fluasterone; fludarabine; fluorodaunorunicin hydrochloride; forfenimex; formestane; fostriecin; fotemustine; gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix; gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam; heregulin; hexamethylene bisacetamide; hypericin; ibandronic acid; idarubicin; idoxifene; idramantone; ilmofosine; ilomastat; imidazoacridones; imiquimod; immunostimulant peptides; insulin-like growth factor- 1 receptor inhibitor; interferon agonists; interferons; interleukins; iobenguane; iododoxorubicin; ipomeanol, 4-; iroplact; irsogladine; isobengazole; isohomohalicondrin B; itasetron; jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide; leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole; leukemia inhibiting factor; leukocyte alpha interferon; leuprolide+estrogen+progesterone; leuprorelin; levamisole; liarozole; linear polyamine analogue; lipophilic disaccharide peptide; lipophilic platinum compounds; lissoclinamide 7; lobaplatin; lombricine; lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine; lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides; maitansine; mannostatin A; marimastat; masoprocol; maspin; matrilysin inhibitors; matrix metalloproteinase inhibitors; menogaril; merbarone; meterelin; methioninase; metoclopramide; MIF inhibitor; mifepristone; miltefosine; mirimostim; mismatched double stranded RNA; mitoguazone; mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growth factor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonal antibody, human chorionic gonadotrophin; monophosphoryl lipid A+myobacterium cell wall sk; mopidamol; multiple drug resistance gene inhibitor; multiple tumor suppressor 1 -based therapy; mustard anticancer agent; mycaperoxide B; mycobacterial cell wall extract; myriaporone; N-acetyldinaline; N- substituted benzamides; nafarelin; nagrestip; naloxone+pentazocine; napavin; naphterpin; nartograstim; nedaplatin; nemorubicin; neridronic acid; neutral endopeptidase; nilutamide; nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn; O6-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone; ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin; osaterone; oxaliplatin; oxaunomycin; palauamine; palmitoylrhizoxin; pamidronic acid; panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase; peldesine; pentosan polysulfate sodium; pentostatin; pentrozole; perflubron; perfosfamide; perillyl alcohol; phenazinomycin; phenylacetate; phosphatase inhibitors; picibanil; pilocarpine hydrochloride; pirarubicin; piritrexim; placetin A; placetin B; plasminogen activator inhibitor; platinum complex; platinum compounds; platinum-triamine complex; porfimer sodium; porfiromycin; prednisone; propyl bis-acridone; prostaglandin J2; proteasome inhibitors; protein A-based immune modulator; protein kinase C inhibitor; protein kinase C inhibitors, microalgal; protein tyrosine phosphatase inhibitors; purine nucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine; pyridoxylated hemoglobin polyoxyethylerie conjugate; raf antagonists; raltitrexed; ramosetron; ras famesyl protein transferase inhibitors; ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re 186 etidronate; rhizoxin; ribozymes; RII retinamide; rogletimide; rohitukine; romurtide; roquinimex; rubiginone Bl; ruboxyl; safingol; saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics; semustine; senescence derived inhibitor 1 ; sense oligonucleotides; signal transduction inhibitors; signal transduction modulators; single chain antigen-binding protein; sizofuran; sobuzoxane; sodium borocaptate; sodium phenylacetate; solverol; somatomedin binding protein; sonermin; sparfosic acid; spicamycin D; spiromustine; splenopentin; spongistatin 1 ; squalamine; stem cell inhibitor; stem-cell division inhibitors; stipiamide; stromelysin inhibitors; sulfinosine; superactive vasoactive intestinal peptide antagonist; suradista; suramin; swainsonine; synthetic glycosaminoglycans; tallimustine; tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium; tegafiir; tellurapyrylium; telomerase inhibitors; temoporfin; temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine; thaliblastine; thiocoraline; thrombopoietin; thrombopoietin mimetic; thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroid stimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocene bichloride; topsentin; toremifene; totipotent stem cell factor; translation inhibitors; tretinoin; triacetyluridine; triciribine; trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinase inhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenital sinus-derived growth inhibitory factor; urokinase receptor antagonists; vapreotide; variolin B; vector system, erythrocyte gene therapy; velaresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine; vitaxin; vorozole; zanoterone; zeniplatin; zilascorb; zinostatin stimalamer, Adriamycin, Dactinomycin, Bleomycin, Vinblastine, Cisplatin, acivicin; aclarubicin; acodazole hydrochloride; acronine; adozelesin; aldesleukin; altretamine; ambomycin; ametantrone acetate; aminoglutethimide; amsacrine; anastrozole; anthramycin; asparaginase; asperlin; azacitidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide; bisantrene hydrochloride; bisnafide dimesylate; bizelesin; bleomycin sulfate; brequinar sodium; bropirimine; busulfan; cactinomycin; calusterone; caracemide; carbetimer; carboplatin; carmustine; carubicin hydrochloride; carzelesin; cedefingol; chlorambucil; cirolemycin; cladribine; crisnatol mesylate; cyclophosphamide; cytarabine; dacarbazine; daunorubicin hydrochloride; decitabine; dexormaplatin; dezaguanine; dezaguanine mesylate; diaziquone; doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifene citrate; dromostanolone propionate; duazomycin; edatrexate; eflomithine hydrochloride; elsamitrucin; enloplatin; enpromate; epipropidine; epirubicin hydrochloride; erbulozole; esorubicin hydrochloride; estramustine; estramustine phosphate sodium; etanidazole; etoposide; etoposide phosphate; etoprine; fadrozole hydrochloride; fazarabine; fenretinide; floxuridine; fludarabine phosphate; fluorouracil; fluorocitabine; fosquidone; fostriecin sodium; gemcitabine; gemcitabine hydrochloride; hydroxyurea; idarubicin hydrochloride; ifosfamide; iimofosine; interleukin II (including recombinant interleukin II, or rlL.sub.2), interferon alfa-2a; interferon alfa-2b; interferon alfa-nl; interferon alfa-n3; interferon beta- la; interferon gamma- lb; iproplatin; irinotecan hydrochloride; lanreotide acetate; letrozole; leuprolide acetate; liarozole hydrochloride; lometrexol sodium; lomustine; losoxantrone hydrochloride; masoprocol; maytansine; mechlorethamine hydrochloride; megestrol acetate; melengestrol acetate; melphalan; menogaril; mercaptopurine; methotrexate; methotrexate sodium; metoprine; meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin; mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone hydrochloride; mycophenolic acid; nocodazoie; nogalamycin; ormaplatin; oxisuran; pegaspargase; peliomycin; pentamustine; peplomycin sulfate; perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride; plicamycin; plomestane; porfimer sodium; porfiromycin; prednimustine; procarbazine hydrochloride; puromycin; puromycin hydrochloride; pyrazofurin; riboprine; rogletimide; safingol; safingol hydrochloride; semustine; simtrazene; sparfosate sodium; sparsomycin; spirogermanium hydrochloride; spiromustine; spiroplatin; streptonigrin; streptozocin; sulofenur; talisomycin; tecogalan sodium; tegafur; teloxantrone hydrochloride; temoporfin; teniposide; teroxirone; testolactone; thiamiprine; thioguanine; thiotepa; tiazofurin; tirapazamine; toremifene citrate; trestolone acetate; triciribine phosphate; trimetrexate; trimetrexate glucuronate; triptorelin; tubulozole hydrochloride; uracil mustard; uredepa; vapreotide; verteporfin; vinblastine sulfate; vincristine sulfate; vindesine; vindesine sulfate; vinepidine sulfate; vinglycinate sulfate; vinleurosine sulfate; vinorelbine tartrate; vinrosidine sulfate; vinzolidine sulfate; vorozole; zeniplatin; zinostatin; zorubicin hydrochloride, agents that arrest cells in the G2-M phases and/or modulate the formation or stability of microtubules, (e.g. Taxol.TM (i.e. paclitaxel), Taxotere.TM, compounds comprising the taxane skeleton, Erbulozole (i.e. R- 55104), Dolastatin 10 (i.e. DLS-10 and NSC-376128), Mivobulin isethionate (i.e. as CI-980), Vincristine, NSC-639829, Discodermolide (i.e. as NVP-XX-A-296), ABT-751 (Abbott, i.e. E-7010), Altorhyrtins (e.g. Altorhyrtin A and Altorhyrtin C), Spongistatins (e.g. Spongistatin 1, Spongistatin 2, Spongistatin 3, Spongistatin 4, Spongistatin 5, Spongistatin 6, Spongistatin 7, Spongistatin 8, and Spongistatin 9), Cemadotin hydrochloride (i.e. LU-103793 and NSC- D-669356), Epothilones (e.g. Epothilone A, Epothilone B, Epothilone C (i.e. desoxyepothilone A or dEpoA), Epothilone D (i.e. KOS-862, dEpoB, and desoxyepothilone B), Epothilone E, Epothilone F, Epothilone B N-oxide, Epothilone A N-oxide, 16-aza- epothilone B, 21-aminoepothilone B (i.e. BMS-310705), 21-hydroxyepothilone D (i.e. Desoxyepothilone F and dEpoF), 26-fluoroepothilone, Auristatin PE (i.e. NSC-654663), Soblidotin (i.e. TZT-1027), LS-4559-P (Pharmacia, i.e. LS-4577), LS-4578 (Pharmacia, i.e. LS-477-P), LS-4477 (Pharmacia), LS-4559 (Pharmacia), RPR- 112378 (Aventis), Vincristine sulfate, DZ-3358 (Daiichi), FR-182877 (Fujisawa, i.e. WS-9885B), GS-164 (Takeda), GS- 198 (Takeda), KAR-2 (Hungarian Academy of Sciences), BSF-223651 (BASF, i.e. ILX-651 and LU-223651), SAH-49960 (Lilly/Novartis), SDZ-268970 (Lilly/Novartis), AM-97 (Armad/Kyowa Hakko), AM-132 (Armad), AM-138 (Armad/Kyowa Hakko), IDN-5005 (Indena), Cryptophycin 52 (i.e. LY-355703), AC-7739 (Ajinomoto, i.e. AVE-8063A and CS- 39.HC1), AC-7700 (Ajinomoto, i.e. AVE-8062, AVE-8062A, CS-39-L-Ser.HCl, and RPR- 258062A), Vitilevuamide, Tubulysin A, Canadensol, Centaureidin (i.e. NSC-106969), T- 138067 (Tularik, i.e. T-67, TL-138067 and TI-138067), COBRA-1 (Parker Hughes Institute, i.e. DDE-261 and WHI-261), H10 (Kansas State University), H16 (Kansas State University), Oncocidin Al (i.e. BTO-956 and DIME), DDE-313 (Parker Hughes Institute), Fijianolide B, Laulimalide, SPA-2 (Parker Hughes Institute), SPA-1 (Parker Hughes Institute, i.e. SPIKET- P), 3-IAABU (Cytoskeleton/Mt. Sinai School of Medicine, i.e. MF-569), Narcosine (also known as NSC-5366), Nascapine, D-24851 (Asta Medica), A-105972 (Abbott), Hemiasterlin, 3-BAABU (Cytoskeleton/Mt. Sinai School of Medicine, i.e. MF-191), TMPN (Arizona State University), Vanadocene acetylacetonate, T- 138026 (Tularik), Monsatrol, Inanocine (i.e. NSC-698666), 3-IAABE (Cytoskeleton/Mt. Sinai School of Medicine), A-204197 (Abbott), T-607 (Tuiarik, i.e. T-900607), RPR-115781 (Aventis), Eleutherobins (such as Desmethyleleutherobin, Desaetyleleutherobin, Isoeleutherobin A, and Z-Eleutherobin), Caribaeoside, Caribaeolin, Halichondrin B, D-64131 (Asta Medica), D-68144 (Asta Medica), Diazonamide A, A-293620 (Abbott), NPI-2350 (Nereus), Taccalonolide A, TUB-245 (Aventis), A-259754 (Abbott), Diozostatin, (-)-Phenylahistin (i.e. NSCL-96F037), D-68838 (Asta Medica), D-68836 (Asta Medica), Myoseverin B, D-43411 (Zentaris, i.e. D-81862), A- 289099 (Abbott), A-318315 (Abbott), HTI-286 (i.e. SPA-110, trifluoroacetate salt) (Wyeth), D-82317 (Zentaris), D-82318 (Zentaris), SC- 12983 (NCI), Resverastatin phosphate sodium, BPR-OY-007 (National Health Research Institutes), and SSR-250411 (Sanofi)), steroids (e.g., dexamethasone), finasteride, aromatase inhibitors, gonadotropin-releasing hormone agonists (GnRH) such as goserelin or leuprolide, adrenocorticosteroids (e.g., prednisone), progestins (e.g., hydroxyprogesterone caproate, megestrol acetate, medroxyprogesterone acetate), estrogens (e.g., diethlystilbestrol, ethinyl estradiol), antiestrogen (e.g., tamoxifen), androgens (e.g., testosterone propionate, fluoxymesterone), antiandrogen (e.g., flutamide), immunostimulants (e.g., Bacillus Calmette-Guerin (BCG), levamisole, interleukin-2, alphainterferon, etc.), monoclonal antibodies (e.g., anti-CD20, anti-HER2, anti-CD52, anti-HLA- DR, and anti-VEGF monoclonal antibodies), immunotoxins (e.g., anti-CD33 monoclonal antibody-calicheamicin conjugate, anti-CD22 monoclonal antibody-pseudomonas exotoxin conjugate, etc.), immunotherapy (e.g., cellular immunotherapy, antibody therapy, cytokine therapy, combination immunotherapy, etc.), radioimmunotherapy (e.g., anti-CD20 monoclonal antibody conjugated to inIn, 90Y, or 131I, etc.), immune checkpoint inhibitors (e.g., CTLA4 blockade, PD-1 inhibitors, PD-L1 inhibitors, etc.), triptolide, homoharringtonine, dactinomycin, doxorubicin, epirubicin, topotecan, itraconazole, vindesine, cerivastatin, vincristine, deoxyadenosine, sertraline, pitavastatin, irinotecan, clofazimine, 5 -nonyloxytryptamine, vemurafenib, dabrafenib, erlotinib, gefitinib, EGFR inhibitors, epidermal growth factor receptor (EGFR)-targeted therapy or therapeutic (e.g. gefitinib (Iressa ™), erlotinib (Tarceva ™), cetuximab (Erbitux™), lapatinib (Tykerb™), panitumumab (Vectibix™), vandetanib (Caprelsa™), afatinib/BIBW2992, CI- 1033/canertinib, neratinib/HKI-272, CP-724714, TAK-285, AST-1306, ARRY334543, ARRY-380, AG-1478, dacomitinib/PF299804, OSI-420/desmethyl erlotinib, AZD8931, AEE788, pelitinib/EKB-569, CUDC-101, WZ8040, WZ4002, WZ3146, AG-490, XL647, PD153035, BMS-599626), sorafenib, imatinib, sunitinib, dasatinib, or the like.
[0087] “Selective” or “selectivity” or the like of a compound refers to the compound’s ability to discriminate between molecular targets (e.g. a compound having selectivity toward HMT SUV49H1 and/or HMT G9a).
[0088] “Specific”, “specifically”, “specificity”, or the like of a compound refers to the compound’s ability to cause a particular action, such as inhibition, to a particular molecular target with minimal or no action to other proteins in the cell (e.g. a compound having specificity towards HMT SUV49H1 and/or HMT G9a displays inhibition of the activity of those HMTs whereas the same compound displays little-to-no inhibition of other HMTs such as DOTI, EZH1, EZH2, GLP, MLL1, MLL2, MLL3, MLL4, NSD2, SETlb, SET7/9, SET8, SETMAR, SMYD2, SUV49H2).
[0089] The abbreviations used herein have their conventional meaning within the chemical and biological arts. The chemical structures and formulae set forth herein are constructed according to the standard rules of chemical valency known in the chemical arts.
[0090] Where substituent groups are specified by their conventional chemical formulae, written from left to right, they equally encompass the chemically identical substituents that would result from writing the structure from right to left, e.g., -CH2O- is equivalent to - OCH2-.
[0091] The term “alkyl,” by itself or as part of another substituent, means, unless otherwise stated, a straight (i.e., unbranched) or branched carbon chain (or carbon), or combination thereof, which may be fully saturated, mono- or polyunsaturated and can include mono-, di- and multivalent radicals. The alkyl may include a designated number of carbons (e.g., C1-C10 means one to ten carbons). In embodiments, the alkyl is fully saturated. In embodiments, the alkyl is monounsaturated. In embodiments, the alkyl is polyunsaturated. Alkyl is an uncyclized chain. Examples of saturated hydrocarbon radicals include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, methyl, homologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. An unsaturated alkyl group is one having one or more double bonds or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2- isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(l,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs and isomers. An alkoxy is an alkyl attached to the remainder of the molecule via an oxygen linker (-O-). An alkyl moiety may be an alkenyl moiety. An alkyl moiety may be an alkynyl moiety. An alkenyl includes one or more double bonds. An alkynyl includes one or more triple bonds.
[0092] The term “alkylene,” by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from an alkyl, as exemplified, but not limited by, - CH2CH2CH2CH2-. Typically, an alkyl (or alkylene) group will have from 1 to 24 carbon atoms, with those groups having 10 or fewer carbon atoms being preferred herein. A “lower alkyl” or “lower alkylene” is a shorter chain alkyl or alkylene group, generally having eight or fewer carbon atoms. The term “alkenylene,” by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from an alkene. In embodiments, the alkylene is fully saturated. In embodiments, the alkylene is monounsaturated. In embodiments, the alkylene is polyunsaturated. An alkenylene includes one or more double bonds. An alkynylene includes one or more triple bonds.
[0093] The term “heteroalkyl,” by itself or in combination with another term, means, unless otherwise stated, a stable straight or branched chain, or combinations thereof, including at least one carbon atom and at least one heteroatom (e.g., O, N, P, Si, and S), and wherein the nitrogen and sulfiir atoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quatemized. The heteroatom(s) (e.g., O, N, S, Si, or P) may be placed at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule. Heteroalkyl is an uncyclized chain. Examples include, but are not limited to: -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)- CH3, -CH2-S-CH2-CH3, -CH2-S-CH2, -S(O)-CH3, -CH2-CH2-S(O)2-CH3, -CH=CH-O-CH3, - Si(CH3)3, -CH2-CH=N-OCH3, -CH=CH-N(CH3)-CH3, -O-CH3, -O-CH2-CH3, and -CN. Up to two or three heteroatoms may be consecutive, such as, for example, -CH2-NH-OCH3 and - CH2-O-Si(CH3)3. A heteroalkyl moiety may include one heteroatom (e.g., O, N, S, Si, or P). A heteroalkyl moiety may include two optionally different heteroatoms (e.g., O, N, S, Si, or P). A heteroalkyl moiety may include three optionally different heteroatoms (e.g., O, N, S, Si, or P). A heteroalkyl moiety may include four optionally different heteroatoms (e.g., O, N, S, Si, or P). A heteroalkyl moiety may include five optionally different heteroatoms (e.g., O, N, S, Si, or P). A heteroalkyl moiety may include up to 8 optionally different heteroatoms (e.g., O, N, S, Si, or P). The term “heteroalkenyl,” by itself or in combination with another term, means, unless otherwise stated, a heteroalkyl including at least one double bond. A heteroalkenyl may optionally include more than one double bond and/or one or more triple bonds in additional to the one or more double bonds. The term “heteroalkynyl,” by itself or in combination with another term, means, unless otherwise stated, a heteroalkyl including at least one triple bond. A heteroalkynyl may optionally include more than one triple bond and/or one or more double bonds in additional to the one or more triple bonds. In embodiments, the heteroalkyl is fully saturated. In embodiments, the heteroalkyl is monounsaturated. In embodiments, the heteroalkyl is polyunsaturated.
[0094] Similarly, the term “heteroalkylene,” by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from heteroalkyl, as exemplified, but not limited by, -CH2-CH2-S-CH2-CH2- and -CH2-S-CH2-CH2-NH-CH2-. For heteroalkylene groups, heteroatoms can also occupy either or both of the chain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, and the like). Still further, for alkylene and heteroalkylene linking groups, no orientation of the linking group is implied by the direction in which the formula of the linking group is written. For example, the formula - C(O)2R'- represents both -C(O)2R'- and -R'C(O)2-. As described above, heteroalkyl groups, as used herein, include those groups that are attached to the remainder of the molecule through a heteroatom, such as -C(O)R', -C(O)NR', -NR'R", -OR', -SR', and/or -SO2R'. Where “heteroalkyl” is recited, followed by recitations of specific heteroalkyl groups, such as - NR'R" or the like, it will be understood that the terms heteroalkyl and -NR'R" are not redundant or mutually exclusive. Rather, the specific heteroalkyl groups are recited to add clarity. Thus, the term “heteroalkyl” should not be interpreted herein as excluding specific heteroalkyl groups, such as -NR'R" or the like. The term “heteroalkenylene,” by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from a heteroalkene. The term “heteroalkynylene” by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from an heteroalkyne. In embodiments, the heteroalkylene is fully saturated. In embodiments, the heteroalkylene is monounsaturated. In embodiments, the heteroalkylene is polyunsaturated. A heteroalkenylene inlcudes one or more double bonds. A heteroalkynylene includes one or more triple bonds.
[0095] The terms “cycloalkyl” and “heterocycloalkyl,” by themselves or in combination with other terms, mean, unless otherwise stated, cyclic versions of “alkyl” and “heteroalkyl,” respectively. Cycloalkyl and heterocycloalkyl are not aromatic. Additionally, for heterocycloalkyl, a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule. Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1 -cyclohexenyl, 3 -cyclohexenyl, cycloheptyl, and the like. Examples of heterocycloalkyl include, but are not limited to, 1- (1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3- morpholinyl, tetrahydrofuran-2-yl, tetrahydrohiran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl, 2-piperazinyl, and the like. A “cycloalkylene” and a “heterocycloalkylene,” alone or as part of another substituent, means a divalent radical derived from a cycloalkyl and heterocycloalkyl, respectively. In embodiments, the cycloalkyl is hilly saturated. In embodiments, the cycloalkyl is monounsaturated. In embodiments, the cycloalkyl is polyunsaturated. In embodiments, the heterocycloalkyl is hilly saturated. In embodiments, the heterocycloalkyl is monounsaturated. In embodiments, the heterocycloalkyl is polyunsaturated.
[0096] In embodiments, the term “cycloalkyl” means a monocyclic, bicyclic, or a multicyclic cycloalkyl ring system. In embodiments, monocyclic ring systems are cyclic hydrocarbon groups containing from 3 to 8 carbon atoms, where such groups can be saturated or unsaturated, but not aromatic. In embodiments, cycloalkyl groups are fully saturated. Examples of monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl. Bicyclic cycloalkyl ring systems are bridged monocyclic rings or hised bicyclic rings. In embodiments, bridged monocyclic rings contain a monocyclic cycloalkyl ring where two non adjacent carbon atoms of the monocyclic ring are linked by an alkylene bridge of between one and three additional carbon atoms (i.e., a bridging group of the form (CHijw , where w is 1, 2, or 3). Representative examples of bicyclic ring systems include, but are not limited to, bicyclo[3.1.1]heptane, bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane, bicyclo[3.2.2]nonane, bicyclo[3.3.1]nonane, and bicyclo[4.2.1]nonane. In embodiments, fused bicyclic cycloalkyl ring systems contain a monocyclic cycloalkyl ring fused to either a phenyl, a monocyclic cycloalkyl, a monocyclic cycloalkenyl, a monocyclic heterocyclyl, or a monocyclic heteroaryl. In embodiments, the bridged or fiised bicyclic cycloalkyl is attached to the parent molecular moiety through any carbon atom contained within the monocyclic cycloalkyl ring. In embodiments, cycloalkyl groups are optionally substituted with one or two groups which are independently oxo or thia. In embodiments, the fused bicyclic cycloalkyl is a 5 or 6 membered monocyclic cycloalkyl ring fused to either a phenyl ring, a 5 or 6 membered monocyclic cycloalkyl, a 5 or 6 membered monocyclic cycloalkenyl, a 5 or 6 membered monocyclic heterocyclyl, or a 5 or 6 membered monocyclic heteroaryl, wherein the fiised bicyclic cycloalkyl is optionally substituted by one or two groups which are independently oxo or thia. In embodiments, multicyclic cycloalkyl ring systems are a monocyclic cycloalkyl ring (base ring) fused to either (i) one ring system selected from the group consisting of a bicyclic aryl, a bicyclic heteroaryl, a bicyclic cycloalkyl, a bicyclic cycloalkenyl, and a bicyclic heterocyclyl; or (ii) two other ring systems independently selected from the group consisting of a phenyl, a bicyclic aryl, a monocyclic or bicyclic heteroaryl, a monocyclic or bicyclic cycloalkyl, a monocyclic or bicyclic cycloalkenyl, and a monocyclic or bicyclic heterocyclyl. In embodiments, the multicyclic cycloalkyl is attached to the parent molecular moiety through any carbon atom contained within the base ring. In embodiments, multicyclic cycloalkyl ring systems are a monocyclic cycloalkyl ring (base ring) fused to either (i) one ring system selected from the group consisting of a bicyclic aryl, a bicyclic heteroaryl, a bicyclic cycloalkyl, a bicyclic cycloalkenyl, and a bicyclic heterocyclyl; or (ii) two other ring systems independently selected from the group consisting of a phenyl, a monocyclic heteroaryl, a monocyclic cycloalkyl, a monocyclic cycloalkenyl, and a monocyclic heterocyclyl. Examples of multicyclic cycloalkyl groups include, but are not limited to tetradecahydrophenanthrenyl, perhydrophenothiazin- 1-yl, and perhydrophenoxazin- 1-yl. A bicyclic or multicyclic cycloalkyl ring system refers to multiple rings fiised together wherein at least one of the fiised rings is a cycloalkyl ring and wherein the multiple rings are attached to the parent molecular moiety through any carbon atom contained within a cycloalkyl ring of the multiple rings.
[0097] In embodiments, a cycloalkyl is a cycloalkenyl. The term “cycloalkenyl” is used in accordance with its plain ordinary meaning. In embodiments, a cycloalkenyl is a monocyclic, bicyclic, or a multicyclic cycloalkenyl ring system. In embodiments, monocyclic cycloalkenyl ring systems are cyclic hydrocarbon groups containing from 3 to 8 carbon atoms, where such groups are unsaturated (i.e., containing at least one annular carbon carbon double bond), but not aromatic. Examples of monocyclic cycloalkenyl ring systems include cyclopentenyl and cyclohexenyl. In embodiments, bicyclic cycloalkenyl rings are bridged monocyclic rings or a fused bicyclic rings. In embodiments, bridged monocyclic rings contain a monocyclic cycloalkenyl ring where two non adjacent carbon atoms of the monocyclic ring are linked by an alkylene bridge of between one and three additional carbon atoms (i.e., a bridging group of the form (CHijw, where w is 1, 2, or 3). Representative examples of bicyclic cycloalkenyls include, but are not limited to, norbomenyl and bicyclo[2.2.2]oct 2 enyl. In embodiments, fused bicyclic cycloalkenyl ring systems contain a monocyclic cycloalkenyl ring fused to either a phenyl, a monocyclic cycloalkyl, a monocyclic cycloalkenyl, a monocyclic heterocyclyl, or a monocyclic heteroaryl. In embodiments, the bridged or fiised bicyclic cycloalkenyl is attached to the parent molecular moiety through any carbon atom contained within the monocyclic cycloalkenyl ring. In embodiments, cycloalkenyl groups are optionally substituted with one or two groups which are independently oxo or thia. In embodiments, multicyclic cycloalkenyl rings contain a monocyclic cycloalkenyl ring (base ring) fiised to either (i) one ring system selected from the group consisting of a bicyclic aryl, a bicyclic heteroaryl, a bicyclic cycloalkyl, a bicyclic cycloalkenyl, and a bicyclic heterocyclyl; or (ii) two ring systems independently selected from the group consisting of a phenyl, a bicyclic aryl, a monocyclic or bicyclic heteroaryl, a monocyclic or bicyclic cycloalkyl, a monocyclic or bicyclic cycloalkenyl, and a monocyclic or bicyclic heterocyclyl. In embodiments, the multicyclic cycloalkenyl is attached to the parent molecular moiety through any carbon atom contained within the base ring. In embodiments, multicyclic cycloalkenyl rings contain a monocyclic cycloalkenyl ring (base ring) fused to either (i) one ring system selected from the group consisting of a bicyclic aryl, a bicyclic heteroaryl, a bicyclic cycloalkyl, a bicyclic cycloalkenyl, and a bicyclic heterocyclyl; or (ii) two ring systems independently selected from the group consisting of a phenyl, a monocyclic heteroaryl, a monocyclic cycloalkyl, a monocyclic cycloalkenyl, and a monocyclic heterocyclyl. A bicyclic or multicyclic cycloalkenyl ring system refers to multiple rings fused together wherein at least one of the fused rings is a cycloalkenyl ring and wherein the multiple rings are attached to the parent molecular moiety through any carbon atom contained within a cycloalkenyl ring of the multiple rings.
[0098] In embodiments, the term “heterocycloalkyl” means a monocyclic, bicyclic, or a multicyclic heterocycloalkyl ring system. In embodiments, heterocycloalkyl groups are fully saturated. A bicyclic or multicyclic heterocycloalkyl ring system refers to multiple rings fused together wherein at least one of the fiised rings is a heterocycloalkyl ring and wherein the multiple rings are attached to the parent molecular moiety through any atom contained within a heterocycloalkyl ring of the multiple rings.
[0099] In embodiments, a heterocycloalkyl is a heterocyclyl. The term “heterocyclyl” as used herein, means a monocyclic, bicyclic, or multicyclic heterocycle. The heterocyclyl monocyclic heterocycle is a 3, 4, 5, 6 or 7 membered ring containing at least one heteroatom independently selected from the group consisting of O, N, and S where the ring is saturated or unsaturated, but not aromatic. The 3 or 4 membered ring contains 1 heteroatom selected from the group consisting of O, N and S. The 5 membered ring can contain zero or one double bond and one, two or three heteroatoms selected from the group consisting of O, N and S. The 6 or 7 membered ring contains zero, one or two double bonds and one, two or three heteroatoms selected from the group consisting of O, N and S. The heterocyclyl monocyclic heterocycle is connected to the parent molecular moiety through any carbon atom or any nitrogen atom contained within the heterocyclyl monocyclic heterocycle. Representative examples of heterocyclyl monocyclic heterocycles include, but are not limited to, azetidinyl, azepanyl, aziridinyl, diazepanyl, 1,3-dioxanyl, 1,3-dioxolanyl, 1,3-dithiolanyl, 1,3-dithianyl, imidazolinyl, imidazolidinyl, isothiazolinyl, isothiazolidinyl, isoxazolinyl, isoxazolidinyl, morpholinyl, oxadiazolinyl, oxadiazolidinyl, oxazolinyl, oxazolidinyl, piperazinyl, piperidinyl, pyranyl, pyrazolinyl, pyrazolidinyl, pyrrolinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydrothienyl, thiadiazolinyl, thiadiazolidinyl, thiazolinyl, thiazolidinyl, thiomorpholinyl, 1,1-dioxidothiomorpholinyl (thiomorpholine sulfone), thiopyranyl, and trithianyl. The heterocyclyl bicyclic heterocycle is a monocyclic heterocycle fused to either a phenyl, a monocyclic cycloalkyl, a monocyclic cycloalkenyl, a monocyclic heterocycle, or a monocyclic heteroaryl. The heterocyclyl bicyclic heterocycle is connected to the parent molecular moiety through any carbon atom or any nitrogen atom contained within the monocyclic heterocycle portion of the bicyclic ring system. Representative examples of bicyclic heterocyclyls include, but are not limited to, 2,3-dihydrobenzofiiran-2-yl, 2,3- dihydrobenzofuran-3-yl, indolin-l-yl, indolin-2-yl, indolin-3-yl, 2,3-dihydrobenzothien-2-yl, decahydroquinolinyl, decahydroisoquinolinyl, octahydro- IH-indolyl, and octahydrobenzofiiranyl. In embodiments, heterocyclyl groups are optionally substituted with one or two groups which are independently oxo or thia. In certain embodiments, the bicyclic heterocyclyl is a 5 or 6 membered monocyclic heterocyclyl ring fused to a phenyl ring, a 5 or 6 membered monocyclic cycloalkyl, a 5 or 6 membered monocyclic cycloalkenyl, a 5 or 6 membered monocyclic heterocyclyl, or a 5 or 6 membered monocyclic heteroaryl, wherein the bicyclic heterocyclyl is optionally substituted by one or two groups which are independently oxo or thia. Multicyclic heterocyclyl ring systems are a monocyclic heterocyclyl ring (base ring) fused to either (i) one ring system selected from the group consisting of a bicyclic aryl, a bicyclic heteroaryl, a bicyclic cycloalkyl, a bicyclic cycloalkenyl, and a bicyclic heterocyclyl; or (ii) two other ring systems independently selected from the group consisting of a phenyl, a bicyclic aryl, a monocyclic or bicyclic heteroaryl, a monocyclic or bicyclic cycloalkyl, a monocyclic or bicyclic cycloalkenyl, and a monocyclic or bicyclic heterocyclyl. The multicyclic heterocyclyl is attached to the parent molecular moiety through any carbon atom or nitrogen atom contained within the base ring. In embodiments, multicyclic heterocyclyl ring systems are a monocyclic heterocyclyl ring (base ring) fused to either (i) one ring system selected from the group consisting of a bicyclic aryl, a bicyclic heteroaryl, a bicyclic cycloalkyl, a bicyclic cycloalkenyl, and a bicyclic heterocyclyl; or (ii) two other ring systems independently selected from the group consisting of a phenyl, a monocyclic heteroaryl, a monocyclic cycloalkyl, a monocyclic cycloalkenyl, and a monocyclic heterocyclyl. Examples of multicyclic heterocyclyl groups include, but are not limited to lOH-phenothiazin- 10-yl, 9,10-dihydroacridin-9-yl, 9,10-dihydroacridin-10-yl, lOH-phenoxazin- 10-yl, 10,1 l-dihydro-5H-dibenzo[b,f|azepin-5-yl, 1, 2,3,4- tetrahydropyrido[4,3-g]isoquinolin-2-yl, 12H-benzo[b]phenoxazin-12-yl, and dodecahydro- lH-carbazol-9-yl.
[0100] The terms “halo” or “halogen,” by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom. Additionally, terms such as “haloalkyl” are meant to include monohaloalkyl and polyhaloalkyl. For example, the term “halo(C1-C4)alkyl” includes, but is not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3 -bromopropyl, and the like.
[0101] The term “acyl” means, unless otherwise stated, -C(O)R where R is a substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.
[0102] The term “aryl” means, unless otherwise stated, a polyunsaturated, aromatic, hydrocarbon substituent, which can be a single ring or multiple rings (preferably from 1 to 3 rings) that are fiised together (i.e., a fused ring aryl) or linked covalently. A fiised ring aryl refers to multiple rings fused together wherein at least one of the fiised rings is an aryl ring and wherein the multiple rings are attached to the parent molecular moiety through any carbon atom contained within an aryl ring of the multiple rings. The term “heteroaryl” refers to aryl groups (or rings) that contain at least one heteroatom such as N, O, or S, wherein the nitrogen and sulfiir atoms are optionally oxidized, and the nitrogen atom(s) are optionally quatemized. Thus, the term “heteroaryl” includes fused ring heteroaryl groups (i.e., multiple rings fiised together wherein at least one of the fused rings is a heteroaromatic ring and wherein the multiple rings are attached to the parent molecular moiety through any atom contained within a heteroaromatic ring of the multiple rings). A 5,6-fiised ring heteroarylene refers to two rings fused together, wherein one ring has 5 members and the other ring has 6 members, and wherein at least one ring is a heteroaryl ring. Likewise, a 6,6-fiised ring heteroarylene refers to two rings fused together, wherein one ring has 6 members and the other ring has 6 members, and wherein at least one ring is a heteroaryl ring. And a 6,5-fused ring heteroarylene refers to two rings fused together, wherein one ring has 6 members and the other ring has 5 members, and wherein at least one ring is a heteroaryl ring. A heteroaryl group can be attached to the remainder of the molecule through a carbon or heteroatom. Nonlimiting examples of aryl and heteroaryl groups include phenyl, naphthyl, pyrrolyl, pyrazolyl, pyridazinyl, triazinyl, pyrimidinyl, imidazolyl, pyrazinyl, purinyl, oxazolyl, isoxazolyl, thiazolyl, furyl, thienyl, pyridyl, pyrimidyl, benzothiazolyl, benzoxazoyl benzimidazolyl, benzofuran, isobenzofuranyl, indolyl, isoindolyl, benzothiophenyl, isoquinolyl, quinoxalinyl, quinolyl, 1 -naphthyl, 2-naphthyl, 4-biphenyl, 1 -pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5- oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2- furyl, 3 -furyl, 2-thienyl, 3 -thienyl, 2-pyridyl, 3 -pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5 -benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1 -isoquinolyl, 5 -isoquinolyl, 2- quinoxalinyl, 5 -quinoxalinyl, 3 -quinolyl, and 6-quinolyl. Substituents for each of the above noted aryl and heteroaryl ring systems are selected from the group of acceptable substituents described below. An “arylene” and a “heteroarylene,” alone or as part of another substituent, mean a divalent radical derived from an aryl and heteroaryl, respectively. A heteroaryl group substituent may be -O- bonded to a ring heteroatom nitrogen.
[0103] A fused ring heterocyloalkyl-aryl is an aryl fiised to a heterocycloalkyl. A fused ring heterocycloalkyl-heteroaryl is a heteroaryl fused to a heterocycloalkyl. A fiised ring heterocycloalkyl-cycloalkyl is a heterocycloalkyl fiised to a cycloalkyl. A fiised ring heterocycloalkyl-heterocycloalkyl is a heterocycloalkyl fused to another heterocycloalkyl. Fused ring heterocycloalkyl-aryl, fused ring heterocycloalkyl-heteroaryl, fused ring heterocycloalkyl-cycloalkyl, or fiised ring heterocycloalkyl-heterocycloalkyl may each independently be unsubstituted or substituted with one or more of the substitutents described herein.
[0104] Spirocyclic rings are two or more rings wherein adjacent rings are attached through a single atom. The individual rings within spirocyclic rings may be identical or different. Individual rings in spirocyclic rings may be substituted or unsubstituted and may have different substituents from other individual rings within a set of spirocyclic rings. Possible substituents for individual rings within spirocyclic rings are the possible substituents for the same ring when not part of spirocyclic rings (e.g. substituents for cycloalkyl or heterocycloalkyl rings). Spirocylic rings may be substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkyl or substituted or unsubstituted heterocycloalkylene and individual rings within a spirocyclic ring group may be any of the immediately previous list, including having all rings of one type (e.g. all rings being substituted heterocycloalkylene wherein each ring may be the same or different substituted heterocycloalkylene). When referring to a spirocyclic ring system, heterocyclic spirocyclic rings means a spirocyclic rings wherein at least one ring is a heterocyclic ring and wherein each ring may be a different ring. When referring to a spirocyclic ring system, substituted spirocyclic rings means that at least one ring is substituted and each substituent may optionally be different.
[0105] The symbol ” denotes the point of attachment of a chemical moiety to the remainder of a molecule or chemical formula.
[0106] The term “oxo,” as used herein, means an oxygen that is double bonded to a carbon atom.
[0107] The term “alkylsulfonyl,” as used herein, means a moiety having the formula -S(O2)-R', where R' is a substituted or unsubstituted alkyl group as defined above. R' may have a specified number of carbons (e.g., “C1-C4 alkylsulfonyl”).
[0108] The term “alkylarylene” as an arylene moiety covalently bonded to an alkylene moiety (also referred to herein as an alkylene linker). In embodiments, the alkylarylene group has the formula:
Figure imgf000042_0001
[0109] An alkylarylene moiety may be substituted (e.g. with a substituent group) on the alkylene moiety or the arylene linker (e.g. at carbons 2, 3, 4, or 6) with halogen, oxo, -N3, - CF3, -CCI3, -CBr3, -CI3, -CN, -CHO, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO2CH3 - SO3H, , -OSO3H, -SO2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, substituted or unsubstituted C1-C5 alkyl or substituted or unsubstituted 2 to 5 membered heteroalkyl). In embodiments, the alkylarylene is unsubstituted.
[0110] Each of the above terms (e.g., “alkyl,” “heteroalkyl,” “cycloalkyl,” “heterocycloalkyl,” “aryl,” and “heteroaryl”) includes both substituted and unsubstituted forms of the indicated radical. Preferred substituents for each type of radical are provided below.
[0111] Substituents for the alkyl and heteroalkyl radicals (including those groups often referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) can be one or more of a variety of groups selected from, but not limited to, -OR', =O, =NR', =N-OR', -NR'R", -SR', -halogen, - SiR'R"R'", -OC(O)R', -C(O)R', -CO2R', -CONR'R", -OC(O)NR'R", -NR"C(O)R', -NR'- C(O)NR"R'", -NR"C(O)2R', -NR-C(NR'R"R'")=NR"", -NR-C(NR'R")=NR'", -S(O)R', - S(O)2R', -S(O)2NR'R", -NRSO2R', -NR'NR"R'", -ONR'R", -NR'C(O)NR"NR'"R"", -CN, - NO2, -NR'SO2R", -NR'C(O)R", -NR'C(O)-OR", -NR'OR", in a number ranging from zero to (2m'+l), where m' is the total number of carbon atoms in such radical. R, R', R", R'", and R"" each preferably independently refer to hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl (e.g., aryl substituted with 1-3 halogens), substituted or unsubstituted heteroaryl, substituted or unsubstituted alkyl, alkoxy, or thioalkoxy groups, or arylalkyl groups. When a compound described herein includes more than one R group, for example, each of the R groups is independently selected as are each R', R", R'", and R"" group when more than one of these groups is present. When R' and R" are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 4-, 5-, 6-, or 7- membered ring. For example, -NR'R" includes, but is not limited to, 1-pyrrolidinyl and 4- morpholinyl. From the above discussion of substituents, one of skill in the art will understand that the term “alkyl” is meant to include groups including carbon atoms bound to groups other than hydrogen groups, such as haloalkyl (e.g., -CF3 and -CH2CF3) and acyl (e.g., - C(O)CH3, -C(O)CF3, -C(O)CH2OCH3, and the like).
[0112] Similar to the substituents described for the alkyl radical, substituents for the aryl and heteroaryl groups are varied and are selected from, for example: -OR', -NR'R", -SR', - halogen, -SiR'R"R'", -OC(O)R', -C(O)R', -CO2R', -CONR'R", -OC(O)NR'R", -NR"C(O)R', - NR'-C(O)NR"R'", -NR"C(O)2R', -NR-C(NR'R"R'")=NR"", -NR-C(NR'R")=NR'", -S(O)R', - S(O)2R', -S(O)2NR'R", -NRSO2R', -NR'NR"R'", -ONR'R", -NR'C(O)NR"NR'"R"", -CN, - NO2, -R', -N3, -CH(Ph)2, fluoro(C1-C4)alkoxy, and fluoro(C1-C4)alkyl, -NR'SO2R", - NR'C(O)R", -NR'C(O)-OR", -NR'OR", in a number ranging from zero to the total number of open valences on the aromatic ring system; and where R', R", R'", and R"" are preferably independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl. When a compound described herein includes more than one R group, for example, each of the R groups is independently selected as are each R', R", R'", and R"" groups when more than one of these groups is present.
[0113] Substituents for rings (e.g. cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene) may be depicted as substituents on the ring rather than on a specific atom of a ring (commonly referred to as a floating substituent). In such a case, the substituent may be attached to any of the ring atoms (obeying the rules of chemical valency) and in the case of fused rings or spirocyclic rings, a substituent depicted as associated with one member of the fused rings or spirocyclic rings (a floating substituent on a single ring), may be a substituent on any of the fused rings or spirocyclic rings (a floating substituent on multiple rings). When a substituent is attached to a ring, but not a specific atom (a floating substituent), and a subscript for the substituent is an integer greater than one, the multiple substituents may be on the same atom, same ring, different atoms, different fused rings, different spirocyclic rings, and each substituent may optionally be different. Where a point of attachment of a ring to the remainder of a molecule is not limited to a single atom (a floating substituent), the attachment point may be any atom of the ring and in the case of a fused ring or spirocyclic ring, any atom of any of the fused rings or spirocyclic rings while obeying the rules of chemical valency. Where a ring, fused rings, or spirocyclic rings contain one or more ring heteroatoms and the ring, fused rings, or spirocyclic rings are shown with one more floating substituents (including, but not limited to, points of attachment to the remainder of the molecule), the floating substituents may be bonded to the heteroatoms. Where the ring heteroatoms are shown bound to one or more hydrogens (e.g. a ring nitrogen with two bonds to ring atoms and a third bond to a hydrogen) in the structure or formula with the floating substituent, when the heteroatom is bonded to the floating substituent, the substituent will be understood to replace the hydrogen, while obeying the rules of chemical valency.
[0114] Two or more substituents may optionally be joined to form aryl, heteroaryl, cycloalkyl, or heterocycloalkyl groups. Such so-called ring-forming substituents are typically, though not necessarily, found attached to a cyclic base structure. In one embodiment, the ring-forming substituents are attached to adjacent members of the base structure. For example, two ring-forming substituents attached to adjacent members of a cyclic base structure create a fused ring structure. In another embodiment, the ring-forming substituents are attached to a single member of the base structure. For example, two ring-forming substituents attached to a single member of a cyclic base structure create a spirocyclic structure. In yet another embodiment, the ring-forming substituents are attached to non- adjacent members of the base structure.
[0115] Two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally form a ring of the formula -T-C(O)-(CRR')q-U-, wherein T and U are independently -NR-, -O-, -CRR'-, or a single bond, and q is an integer of from 0 to 3. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -A-(CH2)r-B-, wherein A and B are independently -CRR'-, -O-, -NR-, -S-, -S(O) -, -S(O)2-, -S(O)2NR'-, or a single bond, and r is an integer of from 1 to 4. One of the single bonds of the new ring so formed may optionally be replaced with a double bond. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula - (CRR')s-X'- (C"R"R'")d-, where s and d are independently integers of from 0 to 3, and X' is - O-, -NR'-, -S-, -S(O)-, -S(O)2-, or -S(O)2NR'-. The substituents R, R', R", and R'" are preferably independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl.
[0116] As used herein, the terms “heteroatom” or “ring heteroatom” are meant to include oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), and silicon (Si). [0117] A “substituent group,” as used herein, means a group selected from the following moieties:
(A) oxo, halogen, -CC13, -CBr3, -CF3, -CI3, -CH2C1, -CH2Br, -CH2F, -CH2I, -CHCh, -CHBr2, -CHF2, -CHI2, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCC13, -OCF3, -OCBr3, -OCI3,-OCHC12, -OCHBr2, -OCHI2, -OCHF2, -N3, unsubstituted alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl), unsubstituted heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered heteroalkyl, or 2 to 4 membered heteroalkyl), unsubstituted cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl), unsubstituted heterocycloalkyl (e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl), unsubstituted aryl (e.g., C6- C10 aryl, C10 aryl, or phenyl), or unsubstituted heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered heteroaryl), and
(B) alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl), heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered heteroalkyl, or 2 to 4 membered heteroalkyl), cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl), heterocycloalkyl (e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl), aryl (e.g., C6-C10 aryl, C10 aryl, or phenyl), heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered heteroaryl), substituted with at least one substituent selected from:
(i) oxo, halogen, -CC13, -CBr3, -CF3, -CI3, -CH2C1, -CH2Br, -CH2F, -CH2I, -CHCh, -CHBr2, -CHF2, -CHI2, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCC13, -OCF3, -OCBr3, -OCI3,-OCHC12, -OCHBr2, -OCHI2, -OCHF2, -N3, unsubstituted alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl), unsubstituted heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered heteroalkyl, or 2 to 4 membered heteroalkyl), unsubstituted cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl), unsubstituted heterocycloalkyl (e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl), unsubstituted aryl (e.g., C6- C10 aryl, C10 aryl, or phenyl), or unsubstituted heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered heteroaryl), and
(ii) alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl), heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered heteroalkyl, or 2 to 4 membered heteroalkyl), cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl), heterocycloalkyl (e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl), aryl (e.g., C6- C10 aryl, C10 aryl, or phenyl), heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered heteroaryl), substituted with at least one substituent selected from:
(a) oxo, halogen, -CCI3, -CBr3, -CF3, -CI3, -CH2C1, -CH2Br, -CH2F, -CH2I, -CHCh, -CHBr2, -CHF2, -CHI2, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCC13, -OCF3, -OCBr3, -OCI3, -OCHCh, -OCHBr2, -OCHI2, -OCHF2, -N3, unsubstituted alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl), unsubstituted heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered heteroalkyl, or 2 to 4 membered heteroalkyl), unsubstituted cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl), unsubstituted heterocycloalkyl (e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl), unsubstituted aryl (e.g., C6-C10 aryl, C10 aryl, or phenyl), or unsubstituted heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered heteroaryl), and
(b) alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl), heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered heteroalkyl, or 2 to 4 membered heteroalkyl), cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl), heterocycloalkyl (e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl), aryl (e.g., C6- C10 aryl, C10 aryl, or phenyl), heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered heteroaryl), substituted with at least one substituent selected from: oxo, halogen, -CCI3, -CBn, -CF3, -CI3, -CH2C1, -CH2Br, -CH2F, -CH2I, -CHCh, -CHBr2, -CHF2, -CHI2, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO3H, -SO4H, -SO2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH, -OCCI3, -OCF3, -OCBr3, -OCI3,-OCHC12, -OCHBr2, -OCHI2, -OCHF2, -N3, unsubstituted alkyl (e.g., C1-C8 alkyl, C1-C6 alkyl, or C1-C4 alkyl), unsubstituted heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered heteroalkyl, or 2 to 4 membered heteroalkyl), unsubstituted cycloalkyl (e.g., C3-C8 cycloalkyl, C3-C6 cycloalkyl, or C5-C6 cycloalkyl), unsubstituted heterocycloalkyl (e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl), unsubstituted aryl (e.g., C6-C10 aryl, C10 aryl, or phenyl), or unsubstituted heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered heteroaryl).
[0118] A “size-limited substituent” or “ size-limited substituent group,” as used herein, means a group selected from all of the substituents described above for a “substituent group,” wherein each substituted or unsubstituted alkyl is a substituted or unsubstituted C1-C20 alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 20 membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C3-C8 cycloalkyl, each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 8 membered heterocycloalkyl, each substituted or unsubstituted aryl is a substituted or unsubstituted C6-C10 aryl, and each substituted or unsubstituted heteroaryl is a substituted or unsubstituted 5 to 10 membered heteroaryl.
[0119] A “lower substituent” or “ lower substituent group,” as used herein, means a group selected from all of the substituents described above for a “substituent group,” wherein each substituted or unsubstituted alkyl is a substituted or unsubstituted C1-C8 alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8 membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C3- C7 cycloalkyl, each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 7 membered heterocycloalkyl, each substituted or unsubstituted aryl is a substituted or unsubstituted phenyl, and each substituted or unsubstituted heteroaryl is a substituted or unsubstituted 5 to 6 membered heteroaryl.
[0120] In some embodiments, each substituted group described in the compounds herein is substituted with at least one substituent group. More specifically, in some embodiments, each substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and/or substituted heteroarylene described in the compounds herein are substituted with at least one substituent group. In other embodiments, at least one or all of these groups are substituted with at least one size-limited substituent group. In other embodiments, at least one or all of these groups are substituted with at least one lower substituent group.
[0121] In other embodiments of the compounds herein, each substituted or unsubstituted alkyl may be a substituted or unsubstituted C1-C20 alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 20 membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C3-C8 cycloalkyl, each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 8 membered heterocycloalkyl, each substituted or unsubstituted aryl is a substituted or unsubstituted C6- C10 aryl, and/or each substituted or unsubstituted heteroaryl is a substituted or unsubstituted 5 to 10 membered heteroaryl. In some embodiments of the compounds herein, each substituted or unsubstituted alkylene is a substituted or unsubstituted C1-C20 alkylene, each substituted or unsubstituted heteroalkylene is a substituted or unsubstituted 2 to 20 membered heteroalkylene, each substituted or unsubstituted cycloalkylene is a substituted or unsubstituted C3-C8 cycloalkylene, each substituted or unsubstituted heterocycloalkylene is a substituted or unsubstituted 3 to 8 membered heterocycloalkylene, each substituted or unsubstituted arylene is a substituted or unsubstituted C6-C10 arylene, and/or each substituted or unsubstituted heteroarylene is a substituted or unsubstituted 5 to 10 membered heteroarylene.
[0122] In some embodiments, each substituted or unsubstituted alkyl is a substituted or unsubstituted C1-C8 alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8 membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C3-C7 cycloalkyl, each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 7 membered heterocycloalkyl, each substituted or unsubstituted aryl is a substituted or unsubstituted C6-C10 aryl, and/or each substituted or unsubstituted heteroaryl is a substituted or unsubstituted 5 to 9 membered heteroaryl. In some embodiments, each substituted or unsubstituted alkylene is a substituted or unsubstituted C1-C8 alkylene, each substituted or unsubstituted heteroalkylene is a substituted or unsubstituted 2 to 8 membered heteroalkylene, each substituted or unsubstituted cycloalkylene is a substituted or unsubstituted C3-C7 cycloalkylene, each substituted or unsubstituted heterocycloalkylene is a substituted or unsubstituted 3 to 7 membered heterocycloalkylene, each substituted or unsubstituted arylene is a substituted or unsubstituted C6-C10 arylene, and/or each substituted or unsubstituted heteroarylene is a substituted or unsubstituted 5 to 9 membered heteroarylene. In some embodiments, the compound is a chemical species set forth in the Examples section, figures, or tables below.
[0123] In embodiments, a substituted or unsubstituted moiety (e.g., substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, and/or substituted or unsubstituted heteroarylene) is unsubstituted (e.g., is an unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, unsubstituted alkylene, unsubstituted heteroalkylene, unsubstituted cycloalkylene, unsubstituted heterocycloalkylene, unsubstituted arylene, and/or unsubstituted heteroarylene, respectively). In embodiments, a substituted or unsubstituted moiety (e.g., substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, and/or substituted or unsubstituted heteroarylene) is substituted (e.g., is a substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and/or substituted heteroarylene, respectively).
[0124] In embodiments, a substituted moiety (e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and/or substituted heteroarylene) is substituted with at least one substituent group, wherein if the substituted moiety is substituted with a plurality of substituent groups, each substituent group may optionally be different. In embodiments, if the substituted moiety is substituted with a plurality of substituent groups, each substituent group is different.
[0125] In embodiments, a substituted moiety (e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and/or substituted heteroarylene) is substituted with at least one size-limited substituent group, wherein if the substituted moiety is substituted with a plurality of size-limited substituent groups, each size-limited substituent group may optionally be different. In embodiments, if the substituted moiety is substituted with a plurality of size-limited substituent groups, each size-limited substituent group is different.
[0126] In embodiments, a substituted moiety (e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and/or substituted heteroarylene) is substituted with at least one lower substituent group, wherein if the substituted moiety is substituted with a plurality of lower substituent groups, each lower substituent group may optionally be different. In embodiments, if the substituted moiety is substituted with a plurality of lower substituent groups, each lower substituent group is different.
[0127] In embodiments, a substituted moiety (e.g., substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and/or substituted heteroarylene) is substituted with at least one substituent group, size-limited substituent group, or lower substituent group; wherein if the substituted moiety is substituted with a plurality of groups selected from substituent groups, size-limited substituent groups, and lower substituent groups; each substituent group, size-limited substituent group, and/or lower substituent group may optionally be different. In embodiments, if the substituted moiety is substituted with a plurality of groups selected from substituent groups, size-limited substituent groups, and lower substituent groups; each substituent group, size-limited substituent group, and/or lower substituent group is different. [0128] Certain compounds of the present disclosure possess asymmetric carbon atoms (optical or chiral centers) or double bonds; the enantiomers, racemates, diastereomers, tautomers, geometric isomers, stereoisometric forms that may be defined, in terms of absolute stereochemistry, as (R)-or (S)- or, as (D)- or (L)- for amino acids, and individual isomers are encompassed within the scope of the present disclosure. The compounds of the present disclosure do not include those that are known in art to be too unstable to synthesize and/or isolate. The present disclosure is meant to include compounds in racemic and optically pure forms. Optically active (R)- and (S)-, or (D)- and (L)-isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques. When the compounds described herein contain olefinic bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers.
[0129] As used herein, the term “isomers” refers to compounds having the same number and kind of atoms, and hence the same molecular weight, but differing in respect to the structural arrangement or configuration of the atoms.
[0130] The term “tautomer,” as used herein, refers to one of two or more structural isomers which exist in equilibrium and which are readily converted from one isomeric form to another.
[0131] It will be apparent to one skilled in the art that certain compounds of this disclosure may exist in tautomeric forms, all such tautomeric forms of the compounds being within the scope of the disclosure.
[0132] Unless otherwise stated, structures depicted herein are also meant to include all stereochemical forms of the structure; i.e., the R and S configurations for each asymmetric center. Therefore, single stereochemical isomers as well as enantiomeric and diastereomeric mixtures of the present compounds are within the scope of the disclosure.
[0133] Unless otherwise stated, structures depicted herein are also meant to include compounds which differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures except for the replacement of a hydrogen by a deuterium or tritium, or the replacement of a carbon by 13C- or 14C-enriched carbon are within the scope of this disclosure.
[0134] The compounds of the present disclosure may also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. For example, the compounds may be radiolabeled with radioactive isotopes, such as for example tritium (3H), iodine-125 (125I), or carbon-14 (14C). All isotopic variations of the compounds of the present disclosure, whether radioactive or not, are encompassed within the scope of the present disclosure.
[0135] It should be noted that throughout the application that alternatives are written in Markush groups, for example, each amino acid position that contains more than one possible amino acid. It is specifically contemplated that each member of the Markush group should be considered separately, thereby comprising another embodiment, and the Markush group is not to be read as a single unit.
[0136] As used herein, the terms “bioconjugate” and “bioconjugate linker” refers to the resulting association between atoms or molecules of “bioconjugate reactive groups” or “bioconjugate reactive moieties”. The association can be direct or indirect. For example, a conjugate between a first bioconjugate reactive group (e.g., -NH2, -C(O)OH, -N- hydroxysuccinimide, or -maleimide) and a second bioconjugate reactive group (e.g., sulfhydryl, sulfur-containing amino acid, amine, amine sidechain containing amino acid, or carboxylate) provided herein can be direct, e.g., by covalent bond or linker (e.g. a first linker of second linker), or indirect, e.g., by non-covalent bond (e.g. electrostatic interactions (e.g. ionic bond, hydrogen bond, halogen bond), van der Waals interactions (e.g. dipole-dipole, dipole-induced dipole, London dispersion), ring stacking (pi effects), hydrophobic interactions and the like). In embodiments, bioconjugates or bioconjugate linkers are formed using bioconjugate chemistry (i.e. the association of two bioconjugate reactive groups) including, but are not limited to nucleophilic substitutions (e.g., reactions of amines and alcohols with acyl halides, active esters), electrophilic substitutions (e.g., enamine reactions) and additions to carbon-carbon and carbon-heteroatom multiple bonds (e.g., Michael reaction, Diels-Alder addition). These and other useful reactions are discussed in, for example, March, ADVANCED ORGANIC CHEMISTRY, 3rd Ed., John Wiley & Sons, New York, 1985; Hermanson, BIOCONJUGATE TECHNIQUES, Academic Press, San Diego, 1996; and Feeney et al., MODIFICATION OF PROTEINS; Advances in Chemistry Series, Vol. 198, American Chemical Society, Washington, D.C., 1982. In embodiments, the first bioconjugate reactive group (e.g., maleimide moiety) is covalently attached to the second bioconjugate reactive group (e.g. a sulfhydryl). In embodiments, the first bioconjugate reactive group (e.g., haloacetyl moiety) is covalently attached to the second bioconjugate reactive group (e.g. a sulfhydryl). In embodiments, the first bioconjugate reactive group (e.g., pyridyl moiety) is covalently attached to the second bioconjugate reactive group (e.g. a sulfhydryl). In embodiments, the first bioconjugate reactive group (e.g., -N- hydroxysuccinimide moiety) is covalently attached to the second bioconjugate reactive group (e.g. an amine). In embodiments, the first bioconjugate reactive group (e.g., maleimide moiety) is covalently attached to the second bioconjugate reactive group (e.g. a sulfhydryl). In embodiments, the first bioconjugate reactive group (e.g., -sulfo-N-hydroxy succinimide moiety) is covalently attached to the second bioconjugate reactive group (e.g. an amine).
[0137] Useful bioconjugate reactive moieties used for bioconjugate chemistries herein include, for example:
(a) carboxyl groups and various derivatives thereof including, but not limited to, N-hydroxy succinimide esters, N-hydroxybenztriazole esters, acid halides, acyl imidazoles, thioesters, p-nitrophenyl esters, alkyl, alkenyl, alkynyl and aromatic esters;
(b) hydroxyl groups which can be converted to esters, ethers, aldehydes, etc.
(c) haloalkyl groups wherein the halide can be later displaced with a nucleophilic group such as, for example, an amine, a carboxylate anion, thiol anion, carbanion, or an alkoxide ion, thereby resulting in the covalent attachment of a new group at the site of the halogen atom;
(d) dienophile groups which are capable of participating in Diels-Alder reactions such as, for example, maleimido or maleimide groups;
(e) aldehyde or ketone groups such that subsequent derivatization is possible via formation of carbonyl derivatives such as, for example, imines, hydrazones, semicarbazones or oximes, or via such mechanisms as Grignard addition or alkyllithium addition;
(f) sulfonyl halide groups for subsequent reaction with amines, for example, to form sulfonamides;
(g) thiol groups, which can be converted to disulfides, reacted with acyl halides, or bonded to metals such as gold, or react with maleimides;
(h) amine or sulfhydryl groups (e.g., present in cysteine), which can be, for example, acylated, alkylated or oxidized; (i) alkenes, which can undergo, for example, cycloadditions, acylation, Michael addition, etc;
(j) epoxides, which can react with, for example, amines and hydroxyl compounds;
(k) phosphoramidites and other standard fimctional groups usefiil in nucleic acid synthesis;
(l) metal silicon oxide bonding; and
(m) metal bonding to reactive phosphorus groups (e.g. phosphines) to form, for example, phosphate diester bonds.
(n) azides coupled to alkynes using copper catalyzed cycloaddition click chemistry.
(o) biotin conjugate can react with avidin or strepavidin to form a avidin-biotin complex or streptavidin-biotin complex.
[0138] The bioconjugate reactive groups can be chosen such that they do not participate in, or interfere with, the chemical stability of the conjugate described herein. Alternatively, a reactive functional group can be protected from participating in the crosslinking reaction by the presence of a protecting group. In embodiments, the bioconjugate comprises a molecular entity derived from the reaction of an unsaturated bond, such as a maleimide, and a sulfhydryl group.
[0139] “Analog,” or “analogue” is used in accordance with its plain ordinary meaning within Chemistry and Biology and refers to a chemical compound that is structurally similar to another compound (i.e., a so-called “reference” compound) but differs in composition, e.g., in the replacement of one atom by an atom of a different element, or in the presence of a particular functional group, or the replacement of one functional group by another functional group, or the absolute stereochemistry of one or more chiral centers of the reference compound. Accordingly, an analog is a compound that is similar or comparable in function and appearance but not in structure or origin to a reference compound.
[0140] The terms "a" or "an," as used in herein means one or more. In addition, the phrase "substituted with a[n]," as used herein, means the specified group may be substituted with one or more of any or all of the named substituents. For example, where a group, such as an alkyl or heteroaryl group, is "substituted with an unsubstituted C1-C20 alkyl, or unsubstituted 2 to 20 membered heteroalkyl," the group may contain one or more unsubstituted C1-C20 alkyls, and/or one or more unsubstituted 2 to 20 membered heteroalkyls.
[0141] Moreover, where a moiety is substituted with an R substituent, the group may be referred to as “R-substituted.” Where a moiety is R-substituted, the moiety is substituted with at least one R substituent and each R substituent is optionally different. Where a particular R group is present in the description of a chemical genus (such as Formula (I)), a Roman alphabetic symbol may be used to distinguish each appearance of that particular R group. For example, where multiple R13 substituents are present, each R13 substituent may be distinguished as R13 A, R13 B, R13 C, R13 D, etc., wherein each of R13 A, R13 B, R13 C, R13 D, etc. is defined within the scope of the definition of R13 and optionally differently.
[0142] A “detectable agent” or “detectable moiety” is a composition, substance, element, or compound; or moiety thereof; detectable by appropriate means such as spectroscopic, photochemical, biochemical, immunochemical, chemical, magnetic resonance imaging, or other physical means. For example, useful detectable agents include 18F, 32P, 33P, 45Ti, 47Sc,
Figure imgf000055_0001
Er, Tm, Yb, Lu, 32P, fluorophore (e.g. fluorescent dyes), electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, paramagnetic molecules, paramagnetic nanoparticles, ultrasmall superparamagnetic iron oxide ("USPIO") nanoparticles, USPIO nanoparticle aggregates, superparamagnetic iron oxide ("SPIO") nanoparticles, SPIO nanoparticle aggregates, monochrystalline iron oxide nanoparticles, monochrystalline iron oxide, nanoparticle contrast agents, liposomes or other delivery vehicles containing Gadolinium chelate ("Gd-chelate") molecules, Gadolinium, radioisotopes, radionuclides (e.g. carbon-11, nitrogen-13, oxygen-15, fluorine-18, rubidium- 82), fluorodeoxyglucose (e.g. fluorine-18 labeled), any gamma ray emitting radionuclides, positron-emitting radionuclide, radiolabeled glucose, radiolabeled water, radiolabeled ammonia, biocolloids, microbubbles (e.g. including microbubble shells including albumin, galactose, lipid, and/or polymers; microbubble gas core including air, heavy gas(es), perfluorcarbon, nitrogen, octafluoropropane, perflexane lipid microsphere, perflutren, etc.), iodinated contrast agents (e.g. iohexol, iodixanol, ioversol, iopamidol, ioxilan, iopromide, diatrizoate, metrizoate, ioxaglate), barium sulfate, thorium dioxide, gold, gold nanoparticles, gold nanoparticle aggregates, fluorophores, two-photon fluorophores, or haptens and proteins or other entities which can be made detectable, e.g., by incorporating a radiolabel into a peptide or antibody specifically reactive with a target peptide. A detectable moiety is a monovalent detectable agent or a detectable agent capable of forming a bond with another composition.
[0143] Radioactive substances (e.g., radioisotopes) that may be used as imaging and/or labeling agents in accordance with the embodiments of the disclosure include, but are not limited to, 18F, 32P, 33P, 45Ti, 47Sc, 52Fe, 59Fe, 62Cu, 64Cu, 67Cu, 67Ga, 68Ga, 77As, 86Y, 90Y.
Figure imgf000056_0001
198 Au, 199 Au, 211At, 211Pb, 212Bi, 212Pb, 213Bi, 223Ra and 225 Ac. Paramagnetic ions that may be used as additional imaging agents in accordance with the embodiments of the disclosure include, but are not limited to, ions of transition and lanthanide metals (e.g. metals having atomic numbers of 21-29, 42, 43, 44, or 57-71). These metals include ions of Cr, V, Mn, Fe, Co, Ni, Cu, La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb and Lu.
[0144] Descriptions of compounds of the present disclosure are limited by principles of chemical bonding known to those skilled in the art. Accordingly, where a group may be substituted by one or more of a number of substituents, such substitutions are selected so as to comply with principles of chemical bonding and to give compounds which are not inherently unstable and/or would be known to one of ordinary skill in the art as likely to be unstable under ambient conditions, such as aqueous, neutral, and several known physiological conditions. For example, a heterocycloalkyl or heteroaryl is attached to the remainder of the molecule via a ring heteroatom in compliance with principles of chemical bonding known to those skilled in the art thereby avoiding inherently unstable compounds.
[0145] The term “leaving group” is used in accordance with its ordinary meaning in chemistry and refers to a moiety (e.g., atom, functional group, molecule) that separates from the molecule following a chemical reaction (e.g., bond formation, reductive elimination, condensation, cross-coupling reaction) involving an atom or chemical moiety to which the leaving group is attached, also referred to herein as the “leaving group reactive moiety”, and a complementary reactive moiety (i.e. a chemical moiety that reacts with the leaving group reactive moiety) to form a new bond between the remnants of the leaving groups reactive moiety and the complementary reactive moiety. Thus, the leaving group reactive moiety and the complementary reactive moiety form a complementary reactive group pair. Non limiting examples of leaving groups include hydrogen, hydroxide, organotin moieties (e.g., organotin heteroalkyl), halogen (e.g., Br), perfluoroalkylsulfonates (e.g. triflate), tosylates, mesylates, water, alcohols, nitrate, phosphate, thioether, amines, ammonia, fluoride, carboxylate, phenoxides, boronic acid, boronate esters, and alkoxides. In embodiments, two molecules with leaving groups are allowed to contact, and upon a reaction and/or bond formation (e.g., acyloin condensation, aldol condensation, Claisen condensation, Stille reaction) the leaving groups separates from the respective molecule. In embodiments, a leaving group is a bioconjugate reactive moiety. In embodiments, at least two leaving groups (e.g., R1 and R13) are allowed to contact such that the leaving groups are sufficiently proximal to react, interact or physically touch. In embodiments, the leaving groups is designed to facilitate the reaction.
[0146] The term “protecting group” is used in accordance with its ordinary meaning in organic chemistry and refers to a moiety covalently bound to a heteroatom, heterocycloalkyl, or heteroaryl to prevent reactivity of the heteroatom, heterocycloalkyl, or heteroaryl during one or more chemical reactions performed prior to removal of the protecting group.
Typically a protecting group is bound to a heteroatom (e.g., O) during a part of a multipart synthesis wherein it is not desired to have the heteroatom react (e.g., a chemical reduction) with the reagent. Following protection the protecting group may be removed (e.g., by modulating the pH). In embodiments the protecting group is an alcohol protecting group. Non-limiting examples of alcohol protecting groups include acetyl, benzoyl, benzyl, methoxymethyl ether (MOM), tetrahydropyranyl (THP), and silyl ether (e.g., trimethylsilyl (TMS)). In embodiments the protecting group is an amine protecting group. Non-limiting examples of amine protecting groups include carbobenzyloxy (Cbz), tert-butyloxycarbonyl (BOC), 9-Fluorenylmethyloxycarbonyl (FMOC), acetyl, benzoyl, benzyl, carbamate, p- methoxybenzyl ether (PMB), and tosyl (Ts).
[0147] A person of ordinary skill in the art will understand when a variable (e.g., moiety or linker) of a compound or of a compound genus (e.g., a genus described herein) is described by a name or formula of a standalone compound with all valencies filled, the unfilled valence(s) of the variable will be dictated by the context in which the variable is used. For example, when a variable of a compound as described herein is connected (e.g., bonded) to the remainder of the compound through a single bond, that variable is understood to represent a monovalent form (i.e., capable of forming a single bond due to an unfilled valence) of a standalone compound (e.g., if the variable is named “methane” in an embodiment but the variable is known to be attached by a single bond to the remainder of the compound, a person of ordinary skill in the art would understand that the variable is actually a monovalent form of methane, i.e., methyl or -CH3). Likewise, for a linker variable (e.g., L1, L2, or L3 as described herein), a person of ordinary skill in the art will understand that the variable is the divalent form of a standalone compound (e.g., if the variable is assigned to “PEG” or “polyethylene glycol” in an embodiment but the variable is connected by two separate bonds to the remainder of the compound, a person of ordinary skill in the art would understand that the variable is a divalent (i.e., capable of forming two bonds through two unfilled valences) form of PEG instead of the standalone compound PEG).
[0148] The term “exogenous” refers to a molecule or substance (e.g., a compound, nucleic acid or protein) that originates from outside a given cell or organism. For example, an "exogenous promoter" as referred to herein is a promoter that does not originate from the plant it is expressed by. Conversely, the term "endogenous" or "endogenous promoter" refers to a molecule or substance that is native to, or originates within, a given cell or organism.
[0149] The term “lipid moiety” is used in accordance with its ordinary meaning in chemistry and refers to a hydrophobic molecule which is typically characterized by an aliphatic hydrocarbon chain. In embodiments, the lipid moiety includes a carbon chain of 3 to 100 carbons. In embodiments, the lipid moiety includes a carbon chain of 5 to 50 carbons. In embodiments, the lipid moiety includes a carbon chain of 5 to 25 carbons. In embodiments, the lipid moiety includes a carbon chain of 8 to 525 carbons. Lipid moieties may include saturated or unsaturated carbon chains, and may be optionally substituted. In embodiments, the lipid moiety is optionally substituted with a charged moiety at the terminal end. In embodiments, the lipid moiety is an alkyl or heteroalkyl optionally substituted with a carboxylic acid moiety at the terminal end.
[0150] A charged moiety refers to a functional group possessing an abundance of electron density (i.e. electronegative) or is deficient in electron density (i.e. electropositive). Nonlimiting examples of a charged moiety includes carboxylic acid, alcohol, phosphate, aldehyde, and sulfonamide. In embodiments, a charged moiety is capable of forming hydrogen bonds.
[0151] The term “coupling reagent” is used in accordance with its plain ordinary meaning in the arts and refers to a substance (e.g., a compound or solution) which participates in chemical reaction and results in the formation of a covalent bond (e.g., between bioconjugate reactive moieties, between a bioconjugate reactive moiety and the coupling reagent). In embodiments, the level of reagent is depleted in the course of a chemical reaction. This is in contrast to a solvent, which typically does not get consumed over the course of the chemical reaction. Non-limiting examples of coupling reagents include benzotriazol- 1-yl- oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), 7-Azabenzotriazol-l- yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP), 6-Chloro-benzotriazole-l- yloxy-tris-pyrrolidinophosphonium hexafluorophosphate (PyClock), 1- [Bis(dimethylamino)methylene] - 1 H- 1 ,2, 3 -triazolo [4, 5 -b]pyridinium 3 -oxid hexafluorophosphate (HATU), or 2-(lH-benzotriazol-l-yl)-l,l,3,3-tetramethyluronium hexafluorophosphate (HBTU).
[0152] The term “solution” is used in accor and refers to a liquid mixture in which the minor component (e.g., a solute or compound) is uniformly distributed within the major component (e.g., a solvent).
[0153] The term “organic solvent” as used herein is used in accordance with its ordinary meaning in chemistry and refers to a solvent which includes carbon. Non-limiting examples of organic solvents include acetic acid, acetone, acetonitrile, benzene, 1 -butanol, 2-butanol, 2-butanone, t-butyl alcohol, carbon tetrachloride, chlorobenzene, chloroform, cyclohexane, 1,2-dichloroethane, diethylene glycol, diethyl ether, diglyme (diethylene glycol , dimethyl ether), 1,2-dimethoxyethane (glyme, DME), dimethylformamide (DMF), dimethyl sulfoxide (DMSO), 1,4-dioxane, ethanol, ethyl acetate, ethylene glycol, glycerin, heptane, hexamethylphosphoramide (HMPA), hexamethylphosphorous, triamide (HMPT), hexane, methanol, methyl t-butyl ether (MTBE), methylene chloride, N-methyl-2- pyrrolidinone (NMP), nitromethane, pentane, petroleum ether (ligroine), 1 -propanol, 2- propanol, pyridine, tetrahydrofiiran (THF), toluene, triethyl amine, o-xylene, m-xylene, or p- xylene. In embodiments, the organic solvent is or includes chloroform, dichloromethane, methanol, ethanol, tetrahydrofiiran, or dioxane.
[0154] As used herein, the term “salt” refers to acid or base salts of the compounds used in the methods of the present invention. Illustrative examples of acceptable salts are mineral acid (hydrochloric acid, hydrobromic acid, phosphoric acid, and the like) salts, organic acid (acetic acid, propionic acid, glutamic acid, citric acid and the like) salts, quaternary ammonium (methyl iodide, ethyl iodide, and the like) salts.
[0155] The terms “bind” and “bound” as used herein is used in accordance with its plain and ordinary meaning and refers to the association between atoms or molecules. The association can be direct or indirect. For example, bound atoms or molecules may be bound, e.g., by covalent bond, linker (e.g. a first linker or second linker), or non-covalent bond (e.g. electrostatic interactions (e.g. ionic bond, hydrogen bond, halogen bond), van der Waals interactions (e.g. dipole-dipole, dipole-induced dipole, London dispersion), ring stacking (pi effects), hydrophobic interactions and the like).
[0156] The term “capable of binding” as used herein refers to a moiety (e.g. a compound as described herein) that is able to measurably bind to a target (e.g., a NF-KB, a Toll-like receptor protein). In embodiments, where a moiety is capable of binding a target, the moiety is capable of binding with a Kd of less than about 10 pM, 5 pM, 1 pM, 500 nM, 250 nM, 100 nM, 75 nM, 50 nM, 25 nM, 15 nM, 10 nM, 5 nM, 1 nM, or about 0.1 nM.
[0157] As used herein, the term "conjugated” when referring to two moieties means the two moieties are bonded, wherein the bond or bonds connecting the two moieties may be covalent or non-covalent. In embodiments, the two moieties are covalently bonded to each other (e.g. directly or through a covalently bonded intermediary). In embodiments, the two moieties are non-covalently bonded (e.g. through ionic bond(s), van der waal’s bond(s)/interactions, hydrogen bond(s), polar bond(s), or combinations or mixtures thereof).
[0158] The term “non-nucleophilic base” as used herein refers to any sterically hindered base that is a poor nucleophile.
[0159] The term “nucleophile” as used herein refers to a chemical species that donates an electron pair to an electrophile to form a chemical bond in relation to a reaction. All molecules or ions with a free pair of electrons or at least one pi bond can act as nucleophiles.
[0160] The term “strong acid” as used herein refers to an acid that is completely dissociated or ionized in an aqueous solution. Examples of common strong acids include hydrochloric acid (HC1), nitric acid (HNO3), sulfuric acid (H2SO4), hydrobromic acid (HBr), hydroiodic acid (HI), perchloric acid (HCIO4), or chloric acid (HCIO3).
[0161] The term “carbocation stabilizing solvent” as used herein refers to any polar protic solvent capable of forming dipole-dipole interactions with a carbocation, thereby stabilizing the carbocation.
Compounds
[0162] In an aspect is provided a compound as described herein, including the compound of FIG. 3C or analog thereof or derivative thereof. In an aspect is provided a compound as described herein, including the compound of FIG. 3C or analog thereof. In an aspect is provided a compound as described herein, including the compound of FIG. 3C.
[0163] In embodiments, the compound has the formula:
Figure imgf000061_0001
In formula (I'), R1 and R2' are independently hydrogen or substituted or unsubstituted alkyl. In embodiments, R1' and R2' are independently substituted or unsubstituted alkyl. In embodiments, R1' and R2' are independently substituted or unsubstituted C1-C10 alkyl. In embodiments, R1' and R2' are independently substituted or unsubstituted C1-C5 alkyl. In embodiments, R1' and R2' are independently unsubstituted alkyl. In embodiments, R1' and R2' are independently unsubstituted C1-C10 alkyl. In embodiments, R1' and R2' are independently unsubstituted C1-C5 alkyl. In embodiments R1' and R2' are methyl. In embodiments, where R1' is substituted, R1' is substituted with a substituent group. In embodiments, where R1' is substituted, R1' is substituted with a size limited substituent group. In embodiments, where R1' is substituted, R1' is substituted with a lower substituent group. In embodiments, where R2' is substituted, R2' is substituted with a substituent group. In embodiments, where R2' is substituted, R2' is substituted with a size limited substituent group. In embodiments, where R2' is substituted, R2' is substituted with a lower substituent group.
[0164] In formula (I'), L1' is a bond, -O-, -S-, -NH-, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene. In embodiments, Lris -O-, -S-, -NH-, unsubstituted alkylene, unsubstituted heteroalkylene, unsubstituted cycloalkylene, unsubstituted heterocycloalkylene, unsubstituted arylene, or unsubstituted heteroarylene. In embodiments, L1 is substituted or unsubstituted alkylene. In embodiments, Lris substituted or unsubstituted C1-C10 alkylene. In embodiments, Lris substituted or unsubstituted C1-C5 alkylene. In embodiments, Lris unsubstituted alkylene. In embodiments, Lris unsubstituted C1-C10 alkylene. In embodiments, Lris unsubstituted C1-C5 alkylene. In embodiments, Lris methylene. In embodiments, where Lris substituted, Lris substituted with a substituent group. In embodiments, where Lris substituted, Lris substituted with a size limited substituent group. In embodiments, where L1' is substituted, L1' is substituted with a lower substituent group.
[0165] In formula (I'), R3 is substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl. In embodiments, R3' is substituted or unsubstituted furanyl. In embodiments, R3' is substituted or unsubstituted pyrrolyl. In embodiments, R3' is substituted or unsubstituted thiophenyl. In embodiments, R3' is substituted or unsubstituted thiophenyl. In embodiments, where R3' is substituted, R3' is substituted with a substituent group. In embodiments, where R3' is substituted, R3' is substituted with a size limited substituent group. In embodiments, where R3' is substituted, R3' is substituted with a lower substituent group. In embodiments, where R3' is substituted, R3' is substituted with a -OH, halogen, alkoxy (e.g. methoxy), trifluromethyl, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl. In embodiments, where R3' is substituted, R3' is substituted with a -Cl, -F, -OMe, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl. In embodiments, where R3' is substituted, R3' is substituted with a -OH, halogen, alkoxy (e.g. methoxy), trifluromethyl, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl or unsubstituted heteroaryl. In embodiments, where R3' is substituted, R3' is substituted with a -Cl, -F, -OMe, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl or unsubstituted heteroaryl. In embodiments, where R3' is substituted, R3' is substituted with a -Cl, -F, -OMe or unsubstituted aryl or substituted or unsubstituted heteroaryl. In embodiments, where R3' is substituted, R3' is substituted with a - Cl, -F, -OMe or unsubstituted aryl. In embodiments, where R3' is substituted, R3' is substituted with a -Cl, -F, -OMe or unsubstituted phenyl.
[0166] In embodiments, the structure of the compound is:
Figure imgf000063_0001
[0167] In an aspect is provided a compound as described herein, including the compound of having a structure of Formula (I):
Figure imgf000063_0002
(I), or a pharmaceutically acceptable salt thereof.
[0168] L1 is a bond, -NH-, -O-, -S-, -C(O)-, -C(O)NH-, -NHC(O)-, -NHC(O)NH-, -C(O)O-, -OC(O)-, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene.
[0169] R1 is hydrogen, halogen,
Figure imgf000063_0003
OCH2X1, -OCHXS, -CN, -SOniR1D, -SOVINR1AR1B, -NR1CNR1AR1B, -ONR1AR1B, -NHC(O)NR1CNR1AR1B, -NHC(O)NR1AR1B, -N(O)mi, -NR1AR1B, -C(O)Rlc, -C(O)-ORlc, -C (O)NR1AR1B, -OR1D, -NR1ASO2R1D, -NR1AC(O)R1C, -NR1AC(O)ORIC, -NR1AOR1C, -N3, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.
[0170] R2 is hydrogen, halogen, -CX2 3, -CHX2 2, -CH2X2, -OCX2 3, -
OCH2X2, -OCHX2 2, -CN, -SOn2R2n, -SOV2NR2AR2B, -NR2CNR2AR2B, -ONR2AR2B, -NHC(O)NR2CNR2AR2B, -NHC(O)NR2AR2B, -N(O)m2, -NR2AR2B, -C(O)R2C, -C(O)-OR2C, -C (O)NR2AR2B, -OR2D, -NR2ASO2R2D, -NR2AC(O)R2C, -NR2AC(O)OR2C, -NR2AOR2C, -N3, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.
[0171] R1 and R2 may optionally be joined to form a substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.
[0172] R4 is indendently halogen, -CX4 3, -CHX4 2, -CH2X4, -OCX4 3, -
OCH2X4, -OCHX42, -CN, -SOn4R4D, -SOV4NR4AR4B, -NR4CNR4AR4B, -ONR4AR4B, -NHC(O)NR4CNR4AR4B, -NHC(O)NR4AR4B, -N(O)m4, -NR4AR4B, -C(O)R4C, -C(O)-OR4C, -C (O)NR4AR4B, -OR4D, -NR4ASO2R4D, -NR4AC(O)R4C, -NR4AC(O)OR4C, -NR4AOR4C, -N3, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.
[0173] R1A, R1B, R1C, R1D, R2A, R2B, R2C, R2D, R4A, R4B, R4C, and R4D are independently hydrogen, -CX3, -CHX2, -CH2X, -CN, -OH, -COOH, -CONH2, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; R1A and R1B or R2A and R2B with substituents bonded to the same nitrogen atom may optionally be joined to form a substituted or unsubstituted heterocycloalkyl or substituted or unsubstituted heteroaryl.
[0174] Each X, X1, X2, and X4 is independently -F, -Cl, -Br, or -I.
[0175] Each nl, n2 and n4 is independently an integer from 0 to 4;
[0176] z is an integer from 0 to 5.
[0177] Each ml, m2, m4, vl, v2 and v4 are independently 1 or 2.
[0178] In embodiments, L1 is a bond, substituted or unsubstituted alkylene, or substituted or unsubstituted heteroalkylene. In embodiments, L1 is a bond. In embodiments, L1 is substituted or unsubstituted alkylene. In embodiments, L1 is substituted or unsubstituted heteroalkylene.
[0179] In embodiments, z is 0. In embodiments, z is 1. In embodiments, z is 2. In embodiments, z is 3. In embodiments, z is 4. [0180] In embodiments, L1 is substituted or unsubstituted C1-C3 alkylene. In embodiments, L1 is substituted C1-C3 alkylene. In embodiments, L1 is unsubstituted C1-C3 alkylene. In embodiments, L1 is substituted methylene. In embodiments, L1 is unsubstituted methylene. In embodiments, L1 is substituted ethylene. In embodiments, L1 is unsubstituted ethylene. In embodiments, L1 is substituted propylene. In embodiments, L1 is unsubstituted propylene. In embodiments, L1 is substituted isopropylene. In embodiments, L1 is unsubstituted isopropylene.
[0181] In embodiments, L1 is substituted or unsubstituted 2 to 3 membered heteroalkylene. In embodiments, L1 is substituted 2 to 3 membered heteroalkylene. In embodiments, L1 is unsubstituted 2 to 3 membered heteroalkylene. In embodiments, L1 is substituted or unsubstituted 2 membered heteroalkylene. In embodiments, L1 is substituted 2 membered heteroalkylene. In embodiments, L1 is unsubstituted 2 membered heteroalkylene. In embodiments, L1 is substituted or unsubstituted 3 membered heteroalkylene. In embodiments, L1 is substituted 3 membered heteroalkylene. In embodiments, L1 is unsubstituted 3 membered heteroalkylene.
[0182] In embodiments, the compound has a structure of
Figure imgf000065_0001
are as described herein.
[0183] In embodiments, the compound has a structure of
Figure imgf000065_0002
are as described herein.
[0184] In embodiments, each R1 andR2 is independently substituted or unsubstituted alkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl. In embodiments, R1 is substituted or unsubstituted alkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl. In embodiments, R1 is substituted or unsubstituted alkyl. In embodiments, R1 is substituted or unsubstituted aryl. In embodiments, R1 is substituted or unsubstituted heteroaryl. In embodiments, R2 is substituted or unsubstituted alkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl. In embodiments, R2 is substituted or unsubstituted alkyl. In embodiments, R2 is substituted or unsubstituted aryl. In embodiments, R2 is substituted or unsubstituted heteroaryl.
[0185] In embodiments, each R1 andR2 is independently substituted or unsubstituted C1-C4 alkyl, or substituted or unsubstituted phenyl. In embodiments, each R1 andR2 is independently substituted or unsubstituted C1-C4 alkyl.
[0186] In embodiments, R1 is substituted or unsubstituted C1-C4 alkyl, or substituted or unsubstituted phenyl. In embodiments, R1 is substituted or unsubstituted C1-C4 alkyl. In embodiments, R1 is substituted or unsubstituted phenyl. In embodiments, R1 is substituted C1-C4 alkyl, or substituted phenyl. In embodiments, R1 is substituted C1-C4 alkyl. In embodiments, R1 is substituted phenyl. In embodiments, R1 is unsubstituted C1-C4 alkyl, or unsubstituted phenyl. In embodiments, R1 is unsubstituted C1-C4 alkyl. In embodiments, R1 is unsubstituted phenyl.
[0187] In embodiments, R2 is substituted or unsubstituted C1-C4 alkyl, or substituted or unsubstituted phenyl. In embodiments, R2 is substituted or unsubstituted C1-C4 alkyl. In embodiments, R2 is substituted or unsubstituted phenyl. In embodiments, R2 is substituted C1-C4 alkyl, or substituted phenyl. In embodiments, R2 is substituted C1-C4 alkyl. In embodiments, R2 is substituted phenyl. In embodiments, R2 is unsubstituted C1-C4 alkyl, or unsubstituted phenyl. In embodiments, R2 is unsubstituted C1-C4 alkyl. In embodiments, R2 is unsubstituted phenyl.
[0188] In embodiments, R1 is unsubstituted phenyl, and R2 is unsubstituted C1-C4 alkyl. In embodiments, R2 is unsubstituted methyl. In embodiments, R2 is unsubstituted ethyl. In embodiments, R2 is unsubstituted propyl. In embodiments, R2 is unsubstituted isopropyl. In embodiments, R2 is unsubstituted butyl. In embodiments, R2 is unsubstituted isobutyl. In embodiments, R2 is unsubstituted t-butyl. In embodiments, R1 is unsubstituted phenyl.
[0189] In embodiments, R1 is unsubstituted C1-C4 alkyl, andR2 is unsubstituted phenyl. In embodiments, R1 is unsubstituted methyl. In embodiments, R1 is unsubstituted ethyl. In embodiments, R1 is unsubstituted propyl. In embodiments, R1 is unsubstituted isopropyl. In embodiments, R1 is unsubstituted butyl. In embodiments, R1 is unsubstituted isobutyl. In embodiments, R1 is unsubstituted t-butyl. In embodiments, R2 is unsubstituted phenyl.
[0190] In embodiments, z is 0. In embodiments, z is 1 and R4 is halogen. In embodiments, z is 1 and R4 is -F. In embodiments, z is 1 and R4 is -Cl. In embodiments, z is 1 and R4 is - Br. In embodiments, R4 is halogen. In embodiments, R4 is -F. In embodiments, R4 is -Cl. In embodiments, R4 is -Br.
[0191] In embodiments, L1 is unsubstituted C1-C3 alkylene. In embodiments, L1 is unsubstituted methylene. In embodiments, L1 is unsubstituted ethylene. In embodiments, L1 is a bond.
[0192] In embodiments, the compound
Figure imgf000067_0001
Figure imgf000067_0002
[0193] In embodiments, the compound
Figure imgf000068_0001
Figure imgf000068_0004
[0194] In embodiments, the compound
Figure imgf000068_0002
Figure imgf000068_0003
Figure imgf000069_0001
[0195] In embodiments, each R1 andR2 is independently substituted or unsubstituted C1-C4 alkyl. In embodiments, each R1 andR2 is independently unsubstituted C1-C4 alkyl. In embodiments, R1 is unsubstituted methyl. In embodiments, R1 is unsubstituted ethyl. In embodiments, R1 is unsubstituted propyl. In embodiments, R1 is unsubstituted isopropyl. In embodiments, R1 is unsubstituted butyl. In embodiments, R1 is unsubstituted isobutyl. In embodiments, R1 is unsubstituted t-butyl. In embodiments, R2 is unsubstituted methyl. In embodiments, R2 is unsubstituted ethyl. In embodiments, R2 is unsubstituted propyl. In embodiments, R2 is unsubstituted isopropyl. In embodiments, R2 is unsubstituted butyl. In embodiments, R2 is unsubstituted isobutyl. In embodiments, R2 is unsubstituted t-butyl.
[0196] L1 is a bond, -NH-, -O-, -S-, -C(O)-, -C(O)NH-, -NHC(O)-, -NHC(O)NH-, -C(O)O-, -OC(O)-, substituted (e.g., substituted with a substituent group, a size-limited substituent group, or lower substituent group) or unsubstituted alkylene (e.g., C1-C8, C1-C6, or C1-C4 alkylene), substituted (e.g., substituted with a substituent group, a size-limited substituent group, or lower substituent group) or unsubstituted heteroalkylene (e.g., 2 to 8 membered, 2 to 6 membered, or 2 to 4 membered heteroalkylene), substituted (e.g., substituted with a substituent group, a size-limited substituent group, or lower substituent group) or unsubstituted cycloalkylene (e.g., C3-C8, C3-C6, or C5-C6 cycloalkylene), substituted (e.g., substituted with a substituent group, a size-limited substituent group, or lower substituent group) or unsubstituted heterocycloalkylene (e.g., 3 to 8 membered, 3 to 6 membered, or 5 to 6 membered heterocycloalkylene), substituted (e.g., substituted with a substituent group, a size-limited substituent group, or lower substituent group) or unsubstituted arylene (e.g., C6- C10, C10 aryl, or phenylene), or substituted (e.g., substituted with a substituent group, a size- limited substituent group, or lower substituent group) or unsubstituted heteroarylene (e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered heteroarylene).
[0197] In embodiments, a substituted L1 is substituted with at least one substituent group, size-limited substituent group, or lower substituent group; wherein if the substituted L1 is substituted with a plurality of groups selected from substituent groups, size-limited substituent groups, and lower substituent groups; each substituent group, size-limited substituent group, and/or lower substituent group may optionally be different. In embodiments, when L1 is substituted, it is substituted with at least one substituent group. In embodiments, when L1 is substituted, it is substituted with at least one size-limited substituent group. In embodiments, when L1 is substituted, it is substituted with at least one lower substituent group.
[0198] In embodiments, L1 is a bond. In embodiments, L1 is substituted or unsubstituted alkylene (e.g., C1-C20, C1-C12, C1-C8, C1-C6, C1-C4, or C1-C2). In embodiments, L1 is substituted alkylene (e.g., C1-C20, C1-C12, C1-C8, C1-C6, C1-C4, or C1-C2). In embodiments, L1 is unsubstituted alkylene (e.g., C1-C20, C1-C12, C1-C8, C1-C6, C1-C4, or C1-C2). In embodiments, L1 is substituted or unsubstituted C1-C20 alkylene. In embodiments, L1 is substituted C1-C20 alkylene. In embodiments, L1 is unsubstituted C1-C20 alkylene. In embodiments, L1 is substituted or unsubstituted C1-C12 alkylene. In embodiments, L1 is substituted C1-C12 alkylene. In embodiments, L1 is unsubstituted C1-C12 alkylene. In embodiments, L1 is substituted or unsubstituted C1-C8 alkylene. In embodiments, L1 is substituted C1-C8 alkylene. In embodiments, L1 is unsubstituted C1-C8 alkylene. In embodiments, L1 is substituted or unsubstituted C1-C6 alkylene. In embodiments, L1 is substituted C1-C6 alkylene. In embodiments, L1 is unsubstituted C1-C6 alkylene. In embodiments, L1 is substituted or unsubstituted C1-C4 alkylene. In embodiments, L1 is substituted C1-C4 alkylene. In embodiments, L1 is unsubstituted C1-C4 alkylene. In embodiments, L1 is substituted or unsubstituted C1-C3 alkylene. In embodiments, L1 is substituted C1-C3 alkylene. In embodiments, L1 is unsubstituted C1-C3 alkylene. In embodiments, L1 is substituted or unsubstituted ethylene. In embodiments, L1 is substituted ethylene. In embodiments, L1 is unsubstituted ethylene. In embodiments, L1 is substituted or unsubstituted methylene. In embodiments, L1 is substituted methylene. In embodiments, L1 is unsubstituted methylene.
[0199] In embodiments, L1 is substituted or unsubstituted heteroalkylene (e.g., 2 to 20 membered, 2 to 12 membered, 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4 to 5 membered). In embodiments, L1 is substituted heteroalkylene (e.g., 2 to 20 membered, 2 to 12 membered, 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4 to 5 membered). In embodiments, L1 is unsubstituted heteroalkylene (e.g., 2 to 20 membered, 2 to 12 membered, 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4 to 5 membered). In embodiments, L1 is substituted or unsubstituted 2 to 20 membered heteroalkylene. In embodiments, L1 is substituted 2 to 20 membered heteroalkylene. In embodiments, L1 is unsubstituted 2 to 20 membered heteroalkylene. In embodiments, L1 is substituted or unsubstituted 2 to 8 membered heteroalkylene. In embodiments, L1 is substituted 2 to 8 membered heteroalkylene. In embodiments, L1 is unsubstituted 2 to 8 membered heteroalkylene. In embodiments, L1 is substituted or unsubstituted 2 to 6 membered heteroalkylene. In embodiments, L1 is substituted 2 to 6 membered heteroalkylene. In embodiments, L1 is unsubstituted 2 to 6 membered heteroalkylene. In embodiments, L1 is substituted or unsubstituted 4 to 6 membered heteroalkylene. In embodiments, L1 is substituted 4 to 6 membered heteroalkylene. In embodiments, L1 is unsubstituted 4 to 6 membered heteroalkylene. In embodiments, L1 is substituted or unsubstituted 2 to 3 membered heteroalkylene. In embodiments, L1 is substituted 2 to 3 membered heteroalkylene. In embodiments, L1 is unsubstituted 2 to 3 membered heteroalkylene. In embodiments, L1 is substituted or unsubstituted 4 to 5 membered heteroalkylene. In embodiments, L1 is substituted 4 to 5 membered heteroalkylene. In embodiments, L1 is unsubstituted 4 to 5 membered heteroalkylene.
[0200] In embodiments, R1 is hydrogen, halogen (e.g., -F, -Cl, -Br, or -I), -CX 3 (e.g., - CF3, -CCl3,-CBr3, or -CI3), -CHX’2 (e.g., -CHF2, -CHCl2,-CHBr2; or -CHI2), -CH2X’ (e.g., -CH2F,-CH2C1, -CH2Br, or -CH2I), -OCX13 (e.g., -OCF3, -OCC13, -OCBr3, or -OCI3), - OCH2X’ (e.g., -OCH2F, -OCH2C1, -OCH2Br, or -OCH2I), -OCHX’2 (e.g., -OCHF2, - OCHCh, -OCHBr2, -OCHI2), -CN, -SOniR1D (e.g., -SH, -SCH3, -SO2H, -SO3H, or - SO4H), -SOVINR1AR1B (e.g., -SO2NH2, or -SO2NHCH3), -NR1CNR1AR1B (e.g., NHNH2or NHNHCH3), -ONR1AR1B (e.g., -ONH2, or -ONHCH3), -NHC(O)NRlcNR1AR1B (e.g., -NHC(O)NHNH2, or -NHC(O)NHNHCH3), -NHC(O)NR1AR1B (e.g., -NHC(O)NH2, or -NHC(O)NHCH3), -N(O)mi (e.g., -NO, or -NO2), -NR1AR1B (e.g., -NH2, or - NHCH3), -C(O)Rlc (e.g., -C(O)H or -C(O)CH3), -C(O)-ORlc (e.g., -C(O)OH or - C(O)OCH3), -C(O)NR1AR1B (e.g., -C(O)NH2 or -C(O) NHCH3), -OR1D (e.g., -OH, or - OCH3), -NR1ASO2R1D (e.g., -NHSO2H), -NR1AC(O)R1C (e.g., -NHCOH), -NR1AC(O)ORlc (e.g., -NHC(O)OH), -NR1AOR1C (e.g., -NHOH), -N3, substituted (e.g., substituted with a substituent group, a size-limited substituent group, or lower substituent group) or unsubstituted alkyl (e.g., C1-C8, C1-C6, or C1-C4 alkyl), substituted (e.g., substituted with a substituent group, a size-limited substituent group, or lower substituent group) or unsubstituted heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered, or 2 to 4 membered heteroalkyl), substituted (e.g., substituted with a substituent group, a size-limited substituent group, or lower substituent group) or unsubstituted cycloalkyl (e.g., C3-C8, C3-C6, or C5-C6 cycloalkyl), substituted (e.g., substituted with a substituent group, a size-limited substituent group, or lower substituent group) or unsubstituted heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, or 5 to 6 membered heterocycloalkyl), substituted (e.g., substituted with a substituent group, a size-limited substituent group, or lower substituent group) or unsubstituted aryl (e.g., C6-C10, C10 aryl, or phenyl), or substituted (e.g., substituted with a substituent group, a size-limited substituent group, or lower substituent group) or unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered heteroaryl).
[0201] In embodiments, a substituted R1 is substituted with at least one substituent group, size-limited substituent group, or lower substituent group; wherein if the substituted R1 is substituted with a plurality of groups selected from substituent groups, size-limited substituent groups, and lower substituent groups; each substituent group, size-limited substituent group, and/or lower substituent group may optionally be different. In embodiments, when R1 is substituted, it is substituted with at least one substituent group. In embodiments, when R1 is substituted, it is substituted with at least one size-limited substituent group. In embodiments, when R1 is substituted, it is substituted with at least one lower substituent group.
[0202] In embodiments, a substituted R1A is substituted with at least one substituent group, size-limited substituent group, or lower substituent group; wherein if the substituted R1A is substituted with a plurality of groups selected from substituent groups, size-limited substituent groups, and lower substituent groups; each substituent group, size-limited substituent group, and/or lower substituent group may optionally be different. In embodiments, when R1A is substituted, it is substituted with at least one substituent group. In embodiments, when R1A is substituted, it is substituted with at least one size-limited substituent group. In embodiments, when R1A is substituted, it is substituted with at least one lower substituent group. [0203] In embodiments, a substituted R1B is substituted with at least one substituent group, size-limited substituent group, or lower substituent group; wherein if the substituted R1B is substituted with a plurality of groups selected from substituent groups, size-limited substituent groups, and lower substituent groups; each substituent group, size-limited substituent group, and/or lower substituent group may optionally be different. In embodiments, when R1B is substituted, it is substituted with at least one substituent group. In embodiments, when R1B is substituted, it is substituted with at least one size-limited substituent group. In embodiments, when R1B is substituted, it is substituted with at least one lower substituent group.
[0204] In embodiments, a substituted ring formed when R1A and R1B substituents bonded to the same nitrogen atom are joined is substituted with at least one substituent group, sizelimited substituent group, or lower substituent group; wherein if the substituted ring formed when R1A and R1B substituents bonded to the same nitrogen atom are joined is substituted with a plurality of groups selected from substituent groups, size-limited substituent groups, and lower substituent groups; each substituent group, size-limited substituent group, and/or lower substituent group may optionally be different. In embodiments, when the substituted ring formed when R1A and R1B substituents bonded to the same nitrogen atom are joined is substituted, it is substituted with at least one substituent group. In embodiments, when the substituted ring formed when R1A and R1B substituents bonded to the same nitrogen atom are joined is substituted, it is substituted with at least one size-limited substituent group. In embodiments, when the substituted ring formed when R1A and R1B substituents bonded to the same nitrogen atom are joined is substituted, it is substituted with at least one lower substituent group.
[0205] In embodiments, a substituted R1C is substituted with at least one substituent group, size-limited substituent group, or lower substituent group; wherein if the substituted R1C is substituted with a plurality of groups selected from substituent groups, size-limited substituent groups, and lower substituent groups; each substituent group, size-limited substituent group, and/or lower substituent group may optionally be different. In embodiments, when R1C is substituted, it is substituted with at least one substituent group. In embodiments, when R1C is substituted, it is substituted with at least one size-limited substituent group. In embodiments, when R1C is substituted, it is substituted with at least one lower substituent group. [0206] In embodiments, a substituted R1D is substituted with at least one substituent group, size-limited substituent group, or lower substituent group; wherein if the substituted R1D is substituted with a plurality of groups selected from substituent groups, size-limited substituent groups, and lower substituent groups; each substituent group, size-limited substituent group, and/or lower substituent group may optionally be different. In embodiments, when R1D is substituted, it is substituted with at least one substituent group. In embodiments, when R1D is substituted, it is substituted with at least one size-limited substituent group. In embodiments, when R1D is substituted, it is substituted with at least one lower substituent group.
[0207] In embodiments, each R1 is hydrogen, -F, -Cl, -Br, -I, -CF3, -CHF2, -CH2F, -CC13, -CHC12, -CH2C1, -CBr3, CHBr2, -CH2Br, -CI3, -CHI2, -CH2I, -OCF3, -OCC13, -OCBr3, - OCI3, -OCHF2, -OCHCh, -OCHBr2, -OCHI2, -OCH2F, -OCH2C1, -OCH2Br, -
OCH2I, -N3, -CN, -SH, -SCH3, -SO2H, -SO2CH3, -SO2NH2, -SO2NHCH3, -NHC(O)NH2J -NHC(O)NHCH3J -NO2J -NH2, -NHCH3J -C(O)H, -C(O)CH3J -C(O)OH, -
C(O)OCH3, -C(O)NH2, -C(O)NHCH3, -OH, -OCH3, -NHSO2H, -NHSO2CH3, -NHC(O)H, - NCH3C(O)H, -NHC(O)OH, -NCH3C(O)OH, -NHOH, -NCH3OH, -NCH3OCH3, R1E- substituted or unsubstituted alkyl (e.g., C1-C2o, C1-C12, C1-C8, C1-C6, C1-C4, or C1-C2), R1E- substituted or unsubstituted heteroalkyl (e.g., 2 to 20 membered, 2 to 12 membered, 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4 to 5 membered), R1E- substituted or unsubstituted cycloalkyl (e.g., C3-C10, C3-C8, C3-C6, C4-C6, or C8-C6), R1E- substituted or unsubstituted heterocycloalkyl (e.g., 3 to 10 membered, 3 to 8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered), R1E-substituted or unsubstituted aryl (e.g., C6-C12, C6-C10, or phenyl), or R1E-substituted or unsubstituted heteroaryl (e.g., 5 to 12 membered, 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). In embodiments, each R1 is independently -F, -Cl, -Br, -I, -CF3, -CHF2, -CH2F, -CC13,-CHC12, -CH2C1, -CBr3, CHBr2, -CH2Br, -CI3, -CHI2, -CH2I, -OCF3, -OCC13, - OCBr3, -OCI3, -OCHF2, -OCHCh, -OCHBr2, -OCHI2, -OCH2F, -OCH2C1, -OCH2Br, - OCH2I, -N3, -CN, -SH, -SCH3, -SO2H, -SO2CH3, -SO2NH2, -SO2NHCH3, -NHC(O)NH2J -NHC(O)NHCH3J -NO2J -NH2, -NHCH3J -C(O)H, -C(O)CH3J -C(O)OH, -
C(O)OCH3, -C(O)NH2, -C(O)NHCH3, -OH, -OCH3, -NHSO2H, -NHSO2CH3, -NHC(O)H, - NCH3C(O)H, -NHC(O)OH, -NCH3C(O)OH, -NHOH, -NCH3OH, -NCH3OCH3, R1E- substituted alkyl (e.g., C1-C2o, C1-C12, C1-C8, C1-C6, C1-C4, or C1-C2), R1E-substituted heteroalkyl (e.g., 2 to 20 membered, 2 to 12 membered, 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4 to 5 membered), R1E-substituted cycloalkyl (e.g., C3- C10, C3-C8, C3-C6, C4-C6, or C8-C6), R1E-substituted heterocycloalkyl (e.g., 3 to 10 membered, 3 to 8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered), R1E-substituted aryl (e.g., C6-C12, C6-C10, or phenyl), or R1E-substituted heteroaryl (e.g., 5 to 12 membered, 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). In embodiments, each R1 is hydrogen, -F, -Cl, -Br, -I, -CF3, -CHF2, -CH2F, -CCI3, -CHC12, -CH2C1, -CBr3, - CHBr2, -CH2Br, -CI3, -CHI2, -CH2I, -OCF3, -OCCI3, -OCBr3, -OCI3, -OCHF2, -OCHCh, -OCHBr2, -OCHI2, -OCH2F, -OCH2C1, -OCH2Br, -OCH2I, -N3, -CN, -SH, -SCH3, -SO2H, -SO2CH3, -SO2NH2, -SO2NHCH3, -NHC(O)NH2,-NHC(O)NHCH3, -NO2J -NH2, -NHCH3, - C(O)H, -C(O)CH3, -C(O)OH, -C(O)OCH3, -C(O)NH2, -C(O)NHCH3, -OH, -0CH3, - NHSO2H, -NHSO2CH3, -NHC(O)H, -NCH3C(O)H, -NHC(O)OH, -
NCH3C(O)OH, -NHOH, -NCH3OH, -NCH3OCH3, unsubstituted alkyl (e.g., C1-C20, C1-C12, C1-C8, C1-C6, C1-C4, or C1-C2), unsubstituted heteroalkyl (e.g., 2 to 20 membered, 2 to 12 membered, 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4 to 5 membered), unsubstituted cycloalkyl (e.g., C3-C10, C3-C8, C3-C6, C4-C6, or C8-C6), unsubstituted heterocycloalkyl (e.g., 3 to 10 membered, 3 to 8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered), unsubstituted aryl (e.g., C6-C12, C6- C10, or phenyl), or unsubstituted heteroaryl (e.g., 5 to 12 membered, 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered).
[0208] In embodiments, R2 is hydrogen, halogen (e.g., -F, -Cl, -Br, or -I), -CX23 (e.g., - CF3, -CCI3, -CBr3, or -CI3), -CHX2 2 (e.g., -CHF2, -CHCh,-CHBr2; or -CHI2), -CH2X2 (e.g., -CH2F,-CH2C1, -CH2Br, or -CH2I), -OCX2 3 (e.g., -OCF3, -OCCI3, -OCBr3, or -OCI3), - OCH2X2 (e.g., -OCH2F, -OCH2C1, -OCH2Br, or -OCH2I), -OCHX2 2 (e.g., -OCHF2, - OCHCh, -OCHBr2, -OCHI2), -CN, -SOn2R2n (e.g., -SH, -SCH3, -SO2H, -SO3H, or - SO4H), -SOV2NR2AR2B (e.g., -SO2NH2, or -SO2NHCH3), -NR2CNR2AR2B (e.g., NHNH2or NHNHCH3), -ONR2AR2B (e.g., -ONH2, or -ONHCH3), -NHC(O)NR2CNR2AR2B (e.g., -NHC(O)NHNH2, or -NHC(O)NHNHCH3), -NHC(O)NR2AR2B (e.g., -NHC(O)NH2, or -NHC(O)NHCH3), -N(O)m2 (e.g., -NO, or -NO2), -NR2AR2B (e.g., -NH2, or - NHCH3), -C(O)R2C (e.g., -C(O)H or -C(O)CH3), -C(O)-OR2C (e.g., -C(O)OH or - C(O)OCH3), -C(O)NR2AR2B (e.g., -C(O)NH2 or -C(O) NHCH3), -OR2D (e.g., -OH, or - OCH3), -NR2ASO2R2D (e.g., -NHSO2H), -NR2AC(O)R2C (e.g., -NHCOH), -NR2AC(O)OR2C (e.g., -NHC(O)OH), -NR2AOR2C (e.g., -NHOH), -N3, substituted (e.g., substituted with a substituent group, a size-limited substituent group, or lower substituent group) or unsubstituted alkyl (e.g., C1-C8, C1-C6, or C1-C4 alkyl), substituted (e.g., substituted with a substituent group, a size-limited substituent group, or lower substituent group) or unsubstituted heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered, or 2 to 4 membered heteroalkyl), substituted (e.g., substituted with a substituent group, a size-limited substituent group, or lower substituent group) or unsubstituted cycloalkyl (e.g., C3-C8, C3-C6, or C5-C6 cycloalkyl), substituted (e.g., substituted with a substituent group, a size-limited substituent group, or lower substituent group) or unsubstituted heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, or 5 to 6 membered heterocycloalkyl), substituted (e.g., substituted with a substituent group, a size-limited substituent group, or lower substituent group) or unsubstituted aryl (e.g., C6-C10, C10 aryl, or phenyl), or substituted (e.g., substituted with a substituent group, a size-limited substituent group, or lower substituent group) or unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered heteroaryl).
[0209] In embodiments, a substituted R2 is substituted with at least one substituent group, size-limited substituent group, or lower substituent group; wherein if the substituted R2 is substituted with a plurality of groups selected from substituent groups, size-limited substituent groups, and lower substituent groups; each substituent group, size-limited substituent group, and/or lower substituent group may optionally be different. In embodiments, when R2 is substituted, it is substituted with at least one substituent group. In embodiments, when R2 is substituted, it is substituted with at least one size-limited substituent group. In embodiments, when R2 is substituted, it is substituted with at least one lower substituent group.
[0210] In embodiments, a substituted R2A is substituted with at least one substituent group, size-limited substituent group, or lower substituent group; wherein if the substituted R2A is substituted with a plurality of groups selected from substituent groups, size-limited substituent groups, and lower substituent groups; each substituent group, size-limited substituent group, and/or lower substituent group may optionally be different. In embodiments, when R2A is substituted, it is substituted with at least one substituent group. In embodiments, when R2A is substituted, it is substituted with at least one size-limited substituent group. In embodiments, when R2A is substituted, it is substituted with at least one lower substituent group.
[0211] In embodiments, a substituted R2B is substituted with at least one substituent group, size-limited substituent group, or lower substituent group; wherein if the substituted R2B is substituted with a plurality of groups selected from substituent groups, size-limited substituent groups, and lower substituent groups; each substituent group, size-limited substituent group, and/or lower substituent group may optionally be different. In embodiments, when R2B is substituted, it is substituted with at least one substituent group. In embodiments, when R2B is substituted, it is substituted with at least one size-limited substituent group. In embodiments, when R2B is substituted, it is substituted with at least one lower substituent group.
[0212] In embodiments, a substituted ring formed when R2A and R2B substituents bonded to the same nitrogen atom are joined is substituted with at least one substituent group, sizelimited substituent group, or lower substituent group; wherein if the substituted ring formed when R2A and R2B substituents bonded to the same nitrogen atom are joined is substituted with a plurality of groups selected from substituent groups, size-limited substituent groups, and lower substituent groups; each substituent group, size-limited substituent group, and/or lower substituent group may optionally be different. In embodiments, when the substituted ring formed when R2A and R2B substituents bonded to the same nitrogen atom are joined is substituted, it is substituted with at least one substituent group. In embodiments, when the substituted ring formed when R2A and R2B substituents bonded to the same nitrogen atom are joined is substituted, it is substituted with at least one size-limited substituent group. In embodiments, when the substituted ring formed when R2A and R2B substituents bonded to the same nitrogen atom are joined is substituted, it is substituted with at least one lower substituent group.
[0213] In embodiments, a substituted R2C is substituted with at least one substituent group, size-limited substituent group, or lower substituent group; wherein if the substituted R2C is substituted with a plurality of groups selected from substituent groups, size-limited substituent groups, and lower substituent groups; each substituent group, size-limited substituent group, and/or lower substituent group may optionally be different. In embodiments, when R2C is substituted, it is substituted with at least one substituent group. In embodiments, when R2C is substituted, it is substituted with at least one size-limited substituent group. In embodiments, when R2C is substituted, it is substituted with at least one lower substituent group.
[0214] In embodiments, a substituted R2D is substituted with at least one substituent group, size-limited substituent group, or lower substituent group; wherein if the substituted R2D is substituted with a plurality of groups selected from substituent groups, size-limited substituent groups, and lower substituent groups; each substituent group, size-limited substituent group, and/or lower substituent group may optionally be different. In embodiments, when R2D is substituted, it is substituted with at least one substituent group. In embodiments, when R2D is substituted, it is substituted with at least one size-limited substituent group. In embodiments, when R2D is substituted, it is substituted with at least one lower substituent group.
[0215] In embodiments, R2 is hydrogen, -F, -Cl, -Br, -I, -CF3, -CHF2, -CH2F, -CC13, - CHC12, -CH2C1, -CBr3, -CHBr2, -CH2Br, -CI3, -CHI2, -CH2I, -OCF3, -OCC13, -OCBr3, - OCI3, -OCHF2, -OCHCh, -OCHBr2, -OCHI2, -OCH2F, -OCH2C1, -OCH2Br, -
OCH2I, -N3, -CN, -SH, -SCH3, -SO2H, -SO2CH3, -SO2NH2, -SO2NHCH3, -NHC(O)NH2J -NHC(O)NHCH3J -NO2J -NH2, -NHCH3J -C(O)H, -C(O)CH3J -C(O)OH, -
C(O)OCH3, -C(O)NH2, -C(O)NHCH3, -OH, -OCH3, -NHSO2H, -NHSO2CH3, -NHC(O)H, - NCH3C(O)H, -NHC(O)OH, -NCH3C(O)OH, -NHOH, -NCH3OH, -NCH3OCH3, R2E- substituted or unsubstituted alkyl (e.g., C1-C2o, C1-C12, C1-C8, C1-C6, C1-C4, or C1-C2), R2E- substituted or unsubstituted heteroalkyl (e.g., 2 to 20 membered, 2 to 12 membered, 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4 to 5 membered), R2E- substituted or unsubstituted cycloalkyl (e.g., C3-C10, C3-C8, C3-C6, C4-C6, or C8-C6), R2E- substituted or unsubstituted heterocycloalkyl (e.g., 3 to 10 membered, 3 to 8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered), R2E-substituted or unsubstituted aryl (e.g., C6-C12, C6-C10, or phenyl), or R2E-substituted or unsubstituted heteroaryl (e.g., 5 to 12 membered, 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). In embodiments, R2 is hydrogen, -F, -Cl, -Br, -I, -CF3, -CHF2, -CH2F, -CC13, - CHC12, -CH2C1, -CBr3, -CHBr2, -CH2Br, -CI3, -CHI2, -CH2I, -OCF3, -OCC13, -OCBr3, - OCI3, -OCHF2, -OCHCh, -OCHBr2, -OCHI2, -OCH2F, -OCH2C1, -OCH2Br, - OCH2I, -N3, -CN, -SH, -SCH3, -SO2H, -SO2CH3, -SO2NH2, -SO2NHCH3, -NHC(O)NH2J -NHC(O)NHCH3J -NO2J -NH2, -NHCH3J -C(O)H, -C(O)CH3J -C(O)OH, -
C(O)OCH3, -C(O)NH2, -C(O)NHCH3, -OH, -OCH3, -NHSO2H, -NHSO2CH3, -NHC(O)H, - NCH3C(O)H, -NHC(O)OH, -NCH3C(O)OH, -NHOH, -NCH3OH, -NCH3OCH3, R2E- substituted alkyl (e.g., C1-C2o, C1-C12, C1-C8, C1-C6, C1-C4, or C1-C2), R2E-substituted heteroalkyl (e.g., 2 to 20 membered, 2 to 12 membered, 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4 to 5 membered), R2E-substituted cycloalkyl (e.g., C3- C10, C3-C8, C3-C6, C4-C6, or C8-C6), R2E-substituted heterocycloalkyl (e.g., 3 to 10 membered, 3 to 8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered), R2E-substituted aryl (e.g., C6-C12, C6-C10, or phenyl), or R2E-substituted heteroaryl (e.g., 5 to 12 membered, 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). In embodiments, R2 is hydrogen, -F, -Cl, -Br, -I, -CF3, -CHF2, -CH2F, -CC13, -CHC12, -CH2C1, -CBr3, - CHBr2, -CH2Br, -CI3, -CHI2, -CH2I, -OCF3, -OCC13, -OCBr3, -OCI3, -OCHF2, -OCHCh, -OCHBr2, -OCHI2, -OCH2F, -OCH2C1, -OCH2Br, -OCH2I, -N3, -CN, -SH, -SCH3, -SO2H, -SO2CH3, -SO2NH2, -SO2NHCH3, -NHC(O)NH2, -NHC(O)NHCH3, -NO2, -NH2, -NHCH3, - C(O)H, -C(O)CH3, -C(O)OH, -C(O)OCH3J -C(O)NH2, -C(O)NHCH3, -OH, -OCH3, - NHSO2H, -NHSO2CH3, -NHC(O)H, -NCH3C(O)H, -NHC(O)OH, - NCH3C(O)OH, -NHOH, -NCH3OH, -NCH3OCH3, unsubstituted alkyl (e.g., C1-C20, C1-C12, C1-C8, C1-C6, C1-C4, or C1-C2), unsubstituted heteroalkyl (e.g., 2 to 20 membered, 2 to 12 membered, 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4 to 5 membered), unsubstituted cycloalkyl (e.g., C3-C10, C3-C8, C3-C6, C4-C6, or C8-C6), unsubstituted heterocycloalkyl (e.g., 3 to 10 membered, 3 to 8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered), unsubstituted aryl (e.g., C6-C12, C6- C10, or phenyl), or unsubstituted heteroaryl (e.g., 5 to 12 membered, 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered).
[0216] R1 and R2 may optionally be joined to form a substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.
[0217] In embodiments, a substituted ring formed when R1 and R2 are joined is substituted with at least one substituent group, size-limited substituent group, or lower substituent group; wherein if the substituted ring formed when R1 and R2 are joined is substituted with a plurality of groups selected from substituent groups, size-limited substituent groups, and lower substituent groups; each substituent group, size-limited substituent group, and/or lower substituent group may optionally be different. In embodiments, when the ring formed when R1 and R2 are joined is substituted, it is substituted with at least one substituent group. In embodiments, when the ring formed when R1 and R2 are joined is substituted, it is substituted with at least one size-limited substituent group. In embodiments, when the ring formed when R1 and R2 are joined is substituted, it is substituted with at least one lower substituent group.
[0218] R1 and R2 together with atoms (e.g., nitrogen) attached thereto may be joined to form substituted (e.g., substituted with a substituent group, a size-limited substituent group, or lower substituent group) or unsubstituted heterocycloalkyl (e.g., 3 to 10 membered, 3 to 8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered), or substituted (e.g., substituted with a substituent group, a size-limited substituent group, or lower substituent group) or unsubstituted heteroaryl (e.g., 5 to 12 membered, 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). In embodiments, R1 and R2 together with atoms (e.g., nitrogen) attached thereto may be joined to form substituted (e.g., substituted with a substituent group, a size-limited substituent group, or lower substituent group) heterocycloalkyl (e.g., 3 to 10 membered, 3 to 8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered), or substituted (e.g., substituted with a substituent group, a size-limited substituent group, or lower substituent group) heteroaryl (e.g., 5 to 12 membered, 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). In embodiments, R1 and R2 together with atoms (e.g., nitrogen) attached thereto may be joined to form unsubstituted heterocycloalkyl (e.g., 3 to 10 membered, 3 to 8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered), or unsubstituted heteroaryl (e.g., 5 to 12 membered, 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). In embodiments, R1 and R2 together with atoms (e.g., nitrogen) attached thereto may be joined to form substituted (e.g., substituted with a substituent group, a size-limited substituent group, or lower substituent group) or unsubstituted pyridyl. In embodiments, R1 and R2 together with atoms (e.g., nitrogen) attached thereto may be joined to form substituted (e.g., substituted with a substituent group, a size-limited substituent group, or lower substituent group) or unsubstituted piperidinyl. In embodiments, R1 and R2 together with atoms attached thereto may be joined to form substituted (e.g., substituted with a substituent group, a size-limited substituent group, or lower substituent group) or unsubstituted morpholinyl. In embodiments, R1 and R2 together with atoms attached thereto joined to form substituted (e.g., substituted with a substituent group, a size-limited substituent group, or lower substituent group) or unsubstituted pyrrolyl. In embodiments, R1 and R2 together with atoms attached thereto may be joined to form substituted (e.g., substituted with a substituent group, a size-limited substituent group, or lower substituent group) or unsubstituted pyrimidinyl.
[0219] In embodiments, R4 is independently halogen (e.g., -F, -Cl, -Br, or -I), -CX43 (e.g., - CF3, -CCI3, -CBr3, or -CI3), -CHX4 2 (e.g., -CHF2, -CHCl2,-CHBr2; or -CHI2), -CH2X4 (e.g., -CH2F,-CH2C1, -CH2Br, or -CH2I), -OCX4 3 (e.g., -OCF3, -OCCI3, -OCBr3, or -OCI3), - OCH2X4 (e.g., -OCH2F, -OCH2C1, -OCH2Br, or -OCH2I), -OCHX4 2 (e.g., -OCHF2, - OCHCh, -OCHBr2, -OCHI2), -CN, -SOn4R4D (e.g., -SH, -SCH3, -SO2H, -SO3H, or - SO4H), -SOV4NR4AR4B (e.g., -SO2NH2, or -SO2NHCH3), -NR4CNR4AR4B (e.g., NHNH2or NHNHCH3), -ONR4AR4B (e.g., -ONH2, or -ONHCH3), -NHC(O)NR4CNR4AR4B (e.g., -NHC(O)NHNH2, or -NHC(O)NHNHCH3), -NHC(O)NR4AR4B (e.g., -NHC(O)NH2, or -NHC(O)NHCH3), -N(O)m4 (e.g., -NO, or -NO2), -NR4AR4B (e.g., -NH2, or - NHCH3), -C(O)R4C (e.g., -C(O)H or -C(O)CH3), -C(O)-OR4C (e.g., -C(O)OH or - C(O)OCH3), -C(O)NR4AR4B (e.g., -C(O)NH2 or -C(O) NHCH3), -OR4D (e.g., -OH, or - OCH3), -NR4ASO2R4D (e.g., -NHSO2H), -NR4AC(O)R4C (e.g., -NHCOH), -NR4AC(O)OR4C (e.g., -NHC(O)OH), -NR4AOR4C (e.g., -NHOH), -N3, substituted (e.g., substituted with a substituent group, a size-limited substituent group, or lower substituent group) or unsubstituted alkyl (e.g., C1-C8, C1-C6, or C1-C4 alkyl), substituted (e.g., substituted with a substituent group, a size-limited substituent group, or lower substituent group) or unsubstituted heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered, or 2 to 4 membered heteroalkyl), substituted (e.g., substituted with a substituent group, a size-limited substituent group, or lower substituent group) or unsubstituted cycloalkyl (e.g., C3-C8, C3-C6, or C5-C6 cycloalkyl), substituted (e.g., substituted with a substituent group, a size-limited substituent group, or lower substituent group) or unsubstituted heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, or 5 to 6 membered heterocycloalkyl), substituted (e.g., substituted with a substituent group, a size-limited substituent group, or lower substituent group) or unsubstituted aryl (e.g., C6-C10, C10 aryl, or phenyl), or substituted (e.g., substituted with a substituent group, a size-limited substituent group, or lower substituent group) or unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered heteroaryl).
[0220] In embodiments, a substituted R4 is substituted with at least one substituent group, size-limited substituent group, or lower substituent group; wherein if the substituted R4 is substituted with a plurality of groups selected from substituent groups, size-limited substituent groups, and lower substituent groups; each substituent group, size-limited substituent group, and/or lower substituent group may optionally be different. In embodiments, when R4 is substituted, it is substituted with at least one substituent group. In embodiments, when R4 is substituted, it is substituted with at least one size-limited substituent group. In embodiments, when R4 is substituted, it is substituted with at least one lower substituent group. [0221] In embodiments, a substituted R4A is substituted with at least one substituent group, size-limited substituent group, or lower substituent group; wherein if the substituted R4A is substituted with a plurality of groups selected from substituent groups, size-limited substituent groups, and lower substituent groups; each substituent group, size-limited substituent group, and/or lower substituent group may optionally be different. In embodiments, when R4A is substituted, it is substituted with at least one substituent group. In embodiments, when R4A is substituted, it is substituted with at least one size-limited substituent group. In embodiments, when R4A is substituted, it is substituted with at least one lower substituent group.
[0222] In embodiments, a substituted R4B is substituted with at least one substituent group, size-limited substituent group, or lower substituent group; wherein if the substituted R4B is substituted with a plurality of groups selected from substituent groups, size-limited substituent groups, and lower substituent groups; each substituent group, size-limited substituent group, and/or lower substituent group may optionally be different. In embodiments, when R4B is substituted, it is substituted with at least one substituent group. In embodiments, when R4B is substituted, it is substituted with at least one size-limited substituent group. In embodiments, when R4B is substituted, it is substituted with at least one lower substituent group.
[0223] In embodiments, a substituted ring formed when R4A and R4B substituents bonded to the same nitrogen atom are joined is substituted with at least one substituent group, sizelimited substituent group, or lower substituent group; wherein if the substituted ring formed when R4A and R4B substituents bonded to the same nitrogen atom are joined is substituted with a plurality of groups selected from substituent groups, size-limited substituent groups, and lower substituent groups; each substituent group, size-limited substituent group, and/or lower substituent group may optionally be different. In embodiments, when the substituted ring formed when R4A and R4B substituents bonded to the same nitrogen atom are joined is substituted, it is substituted with at least one substituent group. In embodiments, when the substituted ring formed when R4A and R4B substituents bonded to the same nitrogen atom are joined is substituted, it is substituted with at least one size-limited substituent group. In embodiments, when the substituted ring formed when R4A and R4B substituents bonded to the same nitrogen atom are joined is substituted, it is substituted with at least one lower substituent group. [0224] In embodiments, a substituted R4C is substituted with at least one substituent group, size-limited substituent group, or lower substituent group; wherein if the substituted R4C is substituted with a plurality of groups selected from substituent groups, size-limited substituent groups, and lower substituent groups; each substituent group, size-limited substituent group, and/or lower substituent group may optionally be different. In embodiments, when R4C is substituted, it is substituted with at least one substituent group. In embodiments, when R4C is substituted, it is substituted with at least one size-limited substituent group. In embodiments, when R4C is substituted, it is substituted with at least one lower substituent group.
[0225] In embodiments, a substituted R4D is substituted with at least one substituent group, size-limited substituent group, or lower substituent group; wherein if the substituted R4D is substituted with a plurality of groups selected from substituent groups, size-limited substituent groups, and lower substituent groups; each substituent group, size-limited substituent group, and/or lower substituent group may optionally be different. In embodiments, when R4D is substituted, it is substituted with at least one substituent group. In embodiments, when R4D is substituted, it is substituted with at least one size-limited substituent group. In embodiments, when R4D is substituted, it is substituted with at least one lower substituent group.
[0226] In embodiments, R4 is hydrogen, -F, -Cl, -Br, -I, -CF3, -CHF2, -CH2F, -CC13, - CHC12, -CH2C1, -CBr3, -CHBr2, -CH2Br, -CI3, -CHI2, -CH2I, -OCF3, -OCC13, -OCBr3, - OCI3, -OCHF2, -OCHCh, -OCHBr2, -OCHI2, -OCH2F, -OCH2C1, -OCH2Br, -
OCH2I, -N3, -CN, -SH, -SCH3, -SO2H, -SO2CH3, -SO2NH2, -SO2NHCH3, -NHC(O)NH2J -NHC(O)NHCH3J -NO2J -NH2, -NHCH3J -C(O)H, -C(O)CH3J -C(O)OH, -
C(O)OCH3, -C(O)NH2, -C(O)NHCH3, -OH, -OCH3, -NHSO2H, -NHSO2CH3, -NHC(O)H, - NCH3C(O)H, -NHC(O)OH, -NCH3C(O)OH, -NHOH, -NCH3OH, -NCH3OCH3, R4E- substituted or unsubstituted alkyl (e.g., C1-C2o, C1-C12, C1-C8, C1-C6, C1-C4, or C1-C2), R4E- substituted or unsubstituted heteroalkyl (e.g., 2 to 20 membered, 2 to 12 membered, 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4 to 5 membered), R4E- substituted or unsubstituted cycloalkyl (e.g., C3-C10, C3-C8, C3-C6, C4-C6, or C8-C6), R4E- substituted or unsubstituted heterocycloalkyl (e.g., 3 to 10 membered, 3 to 8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered), R4E-substituted or unsubstituted aryl (e.g., C6-C12, C6-C10, or phenyl), or R4E-substituted or unsubstituted heteroaryl (e.g., 5 to 12 membered, 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). In embodiments, R4 is hydrogen, -F, -Cl, -Br, -I, -CF3, -CHF2, -CH2F, -CCI3, - CHCh, -CH2CI, -CBr3, -CHBr2, -CH2Br, -CI3, -CHI2, -CH2I, -OCF3, -OCCI3, -OCBr3, - OCI3, -OCHF2, -OCHCh, -OCHBr2, -OCHI2, -OCH2F, -OCH2CI, -OCH2Br, -
OCH2I, -N3, -CN, -SH, -SCH3, -SO2H, -SO2CH3, -SO2NH2, -SO2NHCH3, -NHC(O)NH2, -NHC(O)NHCH3, -NO2, -NH2, -NHCH3, -C(O)H, -C(O)CH3, -C(O)OH, -
C(O)OCH3, -C(O)NH2, -C(O)NHCH3, -OH, -0CH3, -NHSO2H, -NHSO2CH3, -NHC(O)H, - NCH3C(O)H, -NHC(O)OH, -NCH3C(O)OH, -NHOH, -NCH3OH, -NCH3OCH3, R4E- substituted alkyl (e.g., C1-C20, C1-C12, C1-C8, C1-C6, C1-C4, or C1-C2), R4E-substituted heteroalkyl (e.g., 2 to 20 membered, 2 to 12 membered, 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4 to 5 membered), R4E-substituted cycloalkyl (e.g., C3- C10, C3-C8, C3-C6, C4-C6, or C8-C6), R4E-substituted heterocycloalkyl (e.g., 3 to 10 membered, 3 to 8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered), R4E-substituted aryl (e.g., C6-C12, C6-C10, or phenyl), or R4E-substituted heteroaryl (e.g., 5 to 12 membered, 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). In embodiments, R4 is hydrogen, -F, -Cl, -Br, -I, -CF3, -CHF2, -CH2F, -CCI3, -CHCh, -CH2C1, -CBr3, - CHBr2, -CH2Br, -CI3, -CHI2, -CH2I, -OCF3, -OCCI3, -OCBr3, -OCI3, -OCHF2, -OCHCh, -OCHBr2, -OCHh, -OCH2F, -OCH2CI, -OCH2Br, -OCH2I, -N3, -CN, -SH, -SCH3, -SO2H, -SO2CH3, -SO2NH2, -SO2NHCH3, -NHC(O)NH2,-NHC(O)NHCH3, -NO2, -NH2, -NHCH3, - C(O)H, -C(O)CH3, -C(O)OH, -C(O)OCH3, -C(O)NH2, -C(O)NHCH3, -OH, -OCH3, - NHSO2H, -NHSO2CH3, -NHC(O)H, -NCH3C(O)H, -NHC(O)OH, -
NCH3C(O)OH, -NHOH, -NCH3OH, -NCH3OCH3, unsubstituted alkyl (e.g., C1-C20, C1-C12, C1-C8, C1-C6, C1-C4, or C1-C2), unsubstituted heteroalkyl (e.g., 2 to 20 membered, 2 to 12 membered, 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4 to 5 membered), unsubstituted cycloalkyl (e.g., C3-C10, C3-C8, C3-C6, C4-C6, or C8-C6), unsubstituted heterocycloalkyl (e.g., 3 to 10 membered, 3 to 8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered), unsubstituted aryl (e.g., C6-C12, C6- C10, or phenyl), or unsubstituted heteroaryl (e.g., 5 to 12 membered, 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered).
[0227] Each R1A, R1B, R1C, R1D, R1E, R2A, R2B, R2C, R2D, R2E, R4A, R4B, R4C, R4D, and R4E are independently hydrogen, -CX3, -CHX2, -CH2X (e.g., -CF3, -CHF2, -CH2F, -CCI3,- CHCh, -CH2CI, -CBr3, -CHBr2, -CH2Br, -CI3, -CHI2, -CH2I), -CN, -
OH, -COOH, -CONH2, substituted (e.g., substituted with a substituent group, a size-limited substituent group, or lower substituent group) or unsubstituted alkyl (e.g., C1-C20, C1-C12, C1- C8, C1-C6, C1-C4, or C1-C2), substituted (e.g., substituted with a substituent group, a sizelimited substituent group, or lower substituent group) or unsubstituted heteroalkyl (e.g., 2 to 20 membered, 2 to 12 membered, 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4 to 5 membered), substituted (e.g., substituted with a substituent group, a size-limited substituent group, or lower substituent group) or unsubstituted cycloalkyl (e.g., C3-C10, C3-C8, C3-C6, C4-C6, or C8-C6), substituted (e.g., substituted with a substituent group, a size-limited substituent group, or lower substituent group) or unsubstituted heterocycloalkyl (e.g., 3 to 10 membered, 3 to 8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered), substituted (e.g., substituted with a substituent group, a sizelimited substituent group, or lower substituent group) or unsubstituted aryl (e.g., C6-C12, C6- C10, or phenyl), or substituted (e.g., substituted with a substituent group, a size-limited substituent group, or lower substituent group) or unsubstituted heteroaryl (e.g., 5 to 12 membered, 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). X is independently - F, -Cl, -Br, or -I. In embodiments, each R1A, R1B, R1C, R1D, R1E, R2A, R2B, R2C, R2D, R2E, R4A, R4B, R4C, R4D, and R4E are independently hydrogen, -CF3, -CHF2, -CH2F, -CCI3, - CHCh, -CH2CI, -CBr3, -CHBr2, -CH2Br, -CI3, -CHI2, -CH2I, -CN, -
OH, -COOH, -CONH2, unsubstituted alkyl (e.g., C1-C20, C1-C12, C1-C8, C1-C6, C1-C4, or C1- C2), unsubstituted heteroalkyl (e.g., 2 to 20 membered, 2 to 12 membered, 2 to 8 membered,
2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4 to 5 membered), unsubstituted cycloalkyl (e.g., C3-C10, C3-C8, C3-C6, C4-C6, or C8-C6), unsubstituted heterocycloalkyl (e.g.,
3 to 10 membered, 3 to 8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered), unsubstituted aryl (e.g., C6-C12, C6-C10, or phenyl), or unsubstituted heteroaryl (e.g., 5 to 12 membered, 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). In embodiments, each R1A, R1B, R1C, R1D, R1E, R2A, R2B, R2C, R2D, R2E, R4A, R4B, R4C, R4D, and R4E are independently hydrogen.
[0228] Each R1A and R1B, R2A and R2B, and R4A and R4B together with nitrogen attached thereto may be joined to form substituted (e.g., substituted with a substituent group, a sizelimited substituent group, or lower substituent group) or unsubstituted heterocycloalkyl (e.g., 3 to 10 membered, 3 to 8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered), or substituted (e.g., substituted with a substituent group, a size-limited substituent group, or lower substituent group) or unsubstituted heteroaryl (e.g., 5 to 12 membered, 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). Each R1A and R1B, R2A and R2B, and R4A and R4B together with nitrogen attached thereto may be joined to form substituted (e.g., substituted with a substituent group, a size-limited substituent group, or lower substituent group) heterocycloalkyl (e.g., 3 to 10 membered, 3 to 8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered), or substituted (e.g., substituted with a substituent group, a size-limited substituent group, or lower substituent group) heteroaryl (e.g., 5 to 12 membered, 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). Each R1A and R1B, R2A and R2B, and R4A and R4B together with nitrogen attached thereto may be joined to form unsubstituted heterocycloalkyl (e.g., 3 to 10 membered, 3 to 8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered), or unsubstituted heteroaryl (e.g., 5 to 12 membered, 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). In embodiments, each R1A and R1B, R2A and R2B, and R4A and R4B together with nitrogen attached thereto may be joined to form substituted (e.g., substituted with a substituent group, a size-limited substituent group, or lower substituent group) or unsubstituted pyridyl. In embodiments, each R1A and R1B, R2A and R2B, and R4A and R4B together with nitrogen attached thereto may be joined to form substituted (e.g., substituted with a substituent group, a size-limited substituent group, or lower substituent group) or unsubstituted piperidinyl. In embodiments, each R1A and R1B, R2A and R2B, and R4A and R4B together with nitrogen attached thereto may be joined to form substituted (e.g., substituted with a substituent group, a size-limited substituent group, or lower substituent group) or unsubstituted morpholinyl. In embodiments, each R1A and R1B, R2A and R2B, and R4A and R4B joined to form substituted (e.g., substituted with a substituent group, a size-limited substituent group, or lower substituent group) or unsubstituted pyrrolyl. In embodiments, each R1A and R1B, R2A and R2B, and R4A and R4B together with nitrogen attached thereto may be joined to form substituted (e.g., substituted with a substituent group, a size-limited substituent group, or lower substituent group) or unsubstituted pyrimidinyl.
[0229] In embodiments, a substituted R1E is substituted with at least one substituent group, size-limited substituent group, or lower substituent group; wherein if the substituted R1E is substituted with a plurality of groups selected from substituent groups, size-limited substituent groups, and lower substituent groups; each substituent group, size-limited substituent group, and/or lower substituent group may optionally be different. In embodiments, when R1E is substituted, it is substituted with at least one substituent group. In embodiments, when R1E is substituted, it is substituted with at least one size-limited substituent group. In embodiments, when R1E is substituted, it is substituted with at least one lower substituent group. [0230] In embodiments, a substituted R2E is substituted with at least one substituent group, size-limited substituent group, or lower substituent group; wherein if the substituted R2E is substituted with a plurality of groups selected from substituent groups, size-limited substituent groups, and lower substituent groups; each substituent group, size-limited substituent group, and/or lower substituent group may optionally be different. In embodiments, when R2E is substituted, it is substituted with at least one substituent group. In embodiments, when R2E is substituted, it is substituted with at least one size-limited substituent group. In embodiments, when R2E is substituted, it is substituted with at least one lower substituent group.
[0231] In embodiments, a substituted R4E is substituted with at least one substituent group, size-limited substituent group, or lower substituent group; wherein if the substituted R4E is substituted with a plurality of groups selected from substituent groups, size-limited substituent groups, and lower substituent groups; each substituent group, size-limited substituent group, and/or lower substituent group may optionally be different. In embodiments, when R4E is substituted, it is substituted with at least one substituent group. In embodiments, when R4E is substituted, it is substituted with at least one size-limited substituent group. In embodiments, when R4E is substituted, it is substituted with at least one lower substituent group.
[0232] X, X1, X2, and X4 are independently -F, -Cl, -Br, or -I. In embodiments, X is -F. In embodiments, X is -Cl. In embodiments, X is -Br. In embodiments, X is -I. In embodiments, X1 is -F. In embodiments, X1 is -Cl. In embodiments, X1 is -Br. In embodiments, X1 is -I. In embodiments, X2 is -F. In embodiments, X2 is -Cl. In embodiments, X2 is -Br. In embodiments, X2 is -I. In embodiments, X4 is -F. In embodiments, X4 is -Cl. In embodiments, X4 is -Br. In embodiments, X4 is -I.
[0233] In embodiments, the symbols nl, n2, and n3 are independently an integer from 0 to 4 (e.g. 0). In embodiments, nl is 0. In embodiments, nl is 1. In embodiments, nl is 2. In embodiments, nl is 3. In embodiments, nl is 4. In embodiments, n2 is 0. In embodiments, n2 is 1. In embodiments, n2 is 2. In embodiments, n2 is 3. In embodiments, n2 is 4. In embodiments, n4 is 0. In embodiments, n4 is 1. In embodiments, n4 is 2. In embodiments, n4 is 3. In embodiments, n4 is 4.
[0234] In embodiments, the symbols ml, m2, and m4 are independently an integer from 1 to 2. In embodiments, ml is 1. In embodiments, ml is 2. In embodiments, m2 is 1. In embodiments, m2 is 1. In embodiments, m2 is 2. In embodiments, m4 is 1. In embodiments, m4 is 2.
[0235] In embodiments, the symbols vl, v2, and v4 are independently an integer from 1 to 2. In embodiments, vl is 1. In embodiments, vl is 2. In embodiments, v2 is 1. In embodiments, v2 is 2. In embodiments, v4 is 1. In embodiments, v4 is 2.
[0236] In embodiments, the compound is usefill as a comparator compound. In embodiments, the comparator compound can be used to assess the activity of a test compound as set forth in an assay described herein (e.g., in the examples section, figures, or tables).
[0237] In embodiments, the compound is a compound as described herein, including in embodiments. In embodiments the compound is a compound described herein (e.g., in the examples section, figures, tables, or claims).
Pharmaceutical Composition
[0238] In embodiments, the compound described herein (e.g., Formula (I'), (I), (II), or (III)) is administered as a pure chemical. In embodiments, the compound as described herein (e.g., Formula (I'), (I), (II), or (III)) is combined with a pharmaceutically suitable or acceptable carrier (also referred to herein as a pharmaceutically suitable (or acceptable) excipient, physiologically suitable (or acceptable) excipient, or physiologically suitable (or acceptable) carrier) selected on the basis of a chosen route of administration and standard pharmaceutical practice as described, for example, in Gennaro, A. R., “Remington: The Science and Practice of Pharmacy,” 21st ed., Easton: Lippincott Williams & Wilkins, 2005.
[0239] In certain embodiments, the compound of Formula (I'), (I), (II), or (III) as described herein is administered as a pure chemical. In some embodiments, the compound of Formula (I'), (I), (II), or (III) described herein is combined with a pharmaceutically suitable or acceptable carrier (also referred to herein as a pharmaceutically suitable (or acceptable) excipient, physiologically suitable (or acceptable) excipient, or physiologically suitable (or acceptable) carrier) selected on the basis of a chosen route of administration and standard pharmaceutical practice as described, for example, in Gennaro, A. R., “Remington: The Science and Practice of Pharmacy,” 21st ed., Easton: Lippincott Williams & Wilkins, 2005.
[0240] Accordingly, provided herein is a pharmaceutical composition including at least one compound of Formula (I'), (I), (II), or (III) described herein, or a pharmaceutically acceptable salt or solvate thereof, together with one or more pharmaceutically acceptable carriers. The carrier(s) (or excipient(s)) is acceptable or suitable if the carrier is compatible with the other ingredients of the composition and not deleterious to the recipient (i.e., the subject) of the composition.
[0241] Accordingly, provided herein is a pharmaceutical composition including at least one compound of Formula (I'), (I), (II), or (III) described herein, or a pharmaceutically acceptable salt or solvate thereof, together with one or more pharmaceutically acceptable carriers. The carrier(s) (or excipient(s)) is acceptable or suitable if the carrier is compatible with the other ingredients of the composition and not deleterious to the recipient (i.e., the subject) of the composition.
[0242] In embodiments, the compound described herein (e.g., of Formula (I'), (I), (II), or (III)) is substantially pure, in that it contains less than about 5%, or less than about 1%, or less than about 0.1%, of other organic small molecules, such as contaminating intermediates or by-products that are created, for example, in one or more of the steps of a synthesis method.
[0243] In certain embodiments, the compound of Formula (I'), (I), (II), or (III) as described herein is substantially pure, in that it contains less than about 5%, or less than about 1%, or less than about 0.1%, of other organic small molecules, such as contaminating intermediates or by-products that are created, for example, in one or more of the steps of a synthesis method.
[0244] These pharmaceutical compositions include those suitable for oral, rectal, topical, buccal, parenteral (e.g., subcutaneous, intramuscular, intradermal, or intravenous), vaginal, ophthalmic, or aerosol administration.
[0245] Exemplary pharmaceutical compositions are used in the form of a pharmaceutical preparation, for example, in solid, semisolid or liquid form, which includes one or more of a disclosed compound, as an active ingredient, in a mixture with an organic or inorganic carrier or excipient suitable for external, enteral or parenteral applications. In some embodiments, the active ingredient is compounded, for example, with the usual non-toxic, pharmaceutically acceptable carriers for tablets, pellets, capsules, suppositories, solutions, emulsions, suspensions, and any other form suitable for use. The active compound is included in the pharmaceutical composition in an amount sufficient to produce the desired effect upon the process or condition of the disease. [0246] The dose of the composition including at least one compound described herein (e.g., of Formula (I'), (I), (II), or (III)) differs, depending upon the patient’s (e.g., human) condition, that is, stage of the disease, general health status, age, and other factors.
[0247] The dose of the composition including at least one compound of Formula (I'), (I), (II), or (III) as described herein differs, depending upon the patient’s (e.g., human) condition, that is, stage of the disease, general health status, age, and other factors.
[0248] Pharmaceutical compositions are administered in a manner appropriate to the disease to be treated (or prevented). An appropriate dose and a suitable duration and frequency of administration will be determined by such factors as the condition of the patient, the type and severity of the patient’s disease, the particular form of the active ingredient, and the method of administration. In general, an appropriate dose and treatment regimen provides the composition(s) in an amount sufficient to provide therapeutic and/or prophylactic benefit (e.g., an improved clinical outcome, such as more frequent complete or partial remissions, or longer disease-free and/or overall survival, or a lessening of symptom severity. Optimal doses are generally determined using experimental models and/or clinical trials. In some embodiments, the optimal dose depends upon the body mass, weight, or blood volume of the patient.
[0249] Oral doses typically range from about 0.1 mg to about 1000 mg, or 1.0 mg to about 1000 mg, one to four times, or more, per day.
[0250] Disclosed compounds are administered to subjects or patients (animals and humans) in need of such treatment in dosages that will provide optimal pharmaceutical efficacy. It will be appreciated that the dose required for use in any particular application will vary from patient to patient, not only with the particular compound or composition selected, but also with the route of administration, the nature of the condition being treated, the age and condition of the patient, concurrent medication or special diets then being followed by the patient, and other factors, with the appropriate dosage ultimately being at the discretion of the attendant physician. For treating clinical conditions and diseases noted above, a contemplated compound disclosed herein is administered orally, subcutaneously, topically, parenterally, by inhalation spray or rectally in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles. Parenteral administration include subcutaneous injections, intravenous or intramuscular injections or infusion techniques. Effective Dosages
[0251] The pharmaceutical composition may include compositions wherein the active ingredient is contained in a therapeutically effective amount, i.e., in an amount effective to achieve its intended purpose. The actual amount effective for a particular application will depend, inter alia, on the condition being treated.
[0252] The dosage and frequency (single or multiple doses) of compounds administered can vary depending upon a variety of factors, including route of administration; size, age, sex, health, body weight, body mass index, and diet of the recipient; nature and extent of symptoms of the disease being treated; presence of other diseases or other health-related problems; kind of concurrent treatment; and complications from any disease or treatment regimen. Other therapeutic regimens or agents can be used in conjunction with the methods and compounds disclosed herein.
[0253] As is well known in the art, therapeutically effective amounts for use in humans can also be determined from animal models. For example, a dose for humans can be formulated to achieve a concentration that has been found to be effective in animals. The dosage in humans can be adjusted by monitoring compounds effectiveness and adjusting the dosage upwards or downwards, as described above. Adjusting the dose to achieve maximal efficacy in humans based on the methods described above and other methods is well within the capabilities of the ordinarily skilled artisan.
[0254] Dosages may be varied depending upon the requirements of the subject and the compound being employed. The dose administered to a subject, in the context of the pharmaceutical compositions presented herein, should be sufficient to effect a beneficial therapeutic response in the subject over time. The size of the dose also will be determined by the existence, nature, and extent of any adverse side effects. Generally, treatment is initiated with smaller dosages, which are less than the optimum dose of the compound. Thereafter, the dosage is increased by small increments until the optimum effect under circumstances is reached.
[0255] Dosage amounts and intervals can be adjusted individually to provide levels of the administered compounds effective for the particular clinical indication being treated. This will provide a therapeutic regimen that is commensurate with the severity of the individual's disease state. [0256] Utilizing the teachings provided herein, an effective prophylactic or therapeutic treatment regimen can be planned that does not cause substantial toxicity and yet is entirely effective to treat the clinical symptoms demonstrated by the particular patient. This planning should involve the careful choice of active compound by considering factors such as compound potency, relative bioavailability, patient body weight, presence and severity of adverse side effects, preferred mode of administration, and the toxicity profile of the selected agent.
Methods of Use
[0257] In an aspect is provided a method of treating a patient in need thereof, the method comprising the administration of the compound (e.g., of Formula (I'), (I), (II), or (III)) as provided herein including embodiments thereof, a derivative thereof, or a salt thereof.
[0258] In an aspect is provided a method of treating cancer in a subject in need thereof, the method comprising the administration an effective amount of the compound (e.g., of Formula (I'), (I), (II), or (III)) as provided herein including embodiments thereof, a derivative thereof, or a salt thereof.
[0259] In an aspect is provided is a method of treating a cancer in a subject in need thereof. The method includes administrating an effective amount of the compound (e.g., of Formula (I'), (I), (II), or (III)) as provided herein including embodiments thereof, or a pharmaceutically acceptable salt thereof.
[0260] In embodiments, the cancer is a solid tumor. In embodiments, the solid tumor is in thyroid, endocrine system, brain, breast, cervix, colon, prostate, head and neck, liver, kidney, lung, ovary, pancreas, rectum, stomach, oral cavity, or uterus. In embodiments, the solid tumor is in thyroid. In embodiments, the solid tumor is in endocrine system. In embodiments, the solid tumor is in brain. In embodiments, the solid tumor is in breast. In embodiments, the solid tumor is in cervix. In embodiments, the solid tumor is in colon. In embodiments, the solid tumor is in prostate. In embodiments, the solid tumor is in head and neck. In embodiments, the solid tumor is in liver. In embodiments, the solid tumor is in kidney. In embodiments, the solid tumor is in lung. In embodiments, the solid tumor is in ovary. In embodiments, the solid tumor is in pancreas. In embodiments, the solid tumor is in rectum. In embodiments, the solid tumor is in stomach. In embodiments, the solid tumor is in oral cavity. In embodiments, the solid tumor is in uterus. [0261] In an aspect is provided a method of inhibiting V-domain Ig Suppressor of T-cell Activation (VISTA) in a cell. The method includes contacting the cell with an effective amount of the compound (e.g., of Formula (I'), (I), (II), or (III)) as provided herein including embodiments thereof, or a pharmaceutically acceptable salt thereof.
[0262] In an aspect is provided a method of enhancing T-cell proliferation in a subject in the presence of VISTA-expressing cancer cells. The method includes administrating the subject an effective amount of the compound (e.g., of Formula (I'), (I), (II), or (III)) as provided herein including embodiments thereof, or a pharmaceutically acceptable salt thereof.
[0263] In an aspect is provided a method of restoring T-cell activation in a subject in the presence of VISTA-expressing cancer cells. The method includes administrating the subject an effective amount of the compound (e.g., of Formula (T), (I), (II), or (III)) as provided herein including embodiments thereof, or a pharmaceutically acceptable salt thereof.
Embodiments
Embodiments P
[0264] Embodiment Pl. A compound as described herein, comprising the compound of
FIG. 3C or analog thereof.
[0265] Embodiment P2. The compound of Embodiment Pl , wherein the structure is:
Figure imgf000093_0001
[0266] Embodiment P3. A method of treating cancer in a subject in need thereof, the method comprising the administration of an effective amount of the compound of Embodiment Pl or P2, a derivative, analog thereof, or a salt thereof.
Embodiments
[0267] Embodiment 1. A compound having a structure of
Figure imgf000094_0001
pharmaceutically acceptable salt thereof, wherein: L1 is a bond, -NH-, -O-, -S-, -C(O)-, -C(O)NH-, -NHC(O)-, -NHC(O)NH-, -C(O)O-, -OC(O)-, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene;
R1 is hydrogen, halogen,
Figure imgf000094_0002
OCH2X1, -OCHX’2, -CN, -SOniR1D, -SOVINR1AR1B, -NR1CNR1AR1B, -ONR1AR1B, -NHC(O)NR1CNR1AR1B, -NHC(O)NR1AR1B, -N(O)mi, -NR1AR1B, -C(O)R1C, -C(O)-OR1C, -C (O)NR1AR1B, -OR1D, -NR1ASO2R1D, -NR1AC(O)R1C, -NR1AC(O)OR1C, -NR1AOR1C, -N3, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl;
R2 is hydrogen, halogen, -CX2 3, -CHX2 2, -CH2X2, -OCX2 3, -
OCH2X2, -OCHX22, -CN, -SOn2R2D, -SOV2NR2AR2B, -NR2CNR2AR2B, -ONR2AR2B, -NHC(O)NR2CNR2AR2B, -NHC(O)NR2AR2B, -N(O)m2, -NR2AR2B, -C(O)R2C, -C(O)-OR2C, -C (O)NR2AR2B, -OR2D, -NR2ASO2R2D, -NR2AC(O)R2C, -NR2AC(O)OR2C, -NR2AOR2C, -N3, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl;
R1 and R2 may optionally be joined to form a substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl;
R4 is indendently halogen, -CX4 3, -CHX4 2, -CH2X4, -OCX4 3, -
OCH2X4, -OCHX42, -CN, -SOn4R4D, -SOV4NR4AR4B, -NR4CNR4AR4B, -ONR4AR4B, -NHC(O)NR4CNR4AR4B, -NHC(O)NR4AR4B, -N(O)m4, -NR4AR4B, -C(O)R4C, -C(O)-OR4C, -C (O)NR4AR4B, -OR4D, -NR4ASO2R4D, -NR4AC(O)R4C, -NR4AC(O)OR4C, -NR4AOR4C, -N3, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl;
R1A, R1B, R1C, R1D, R2A, R2B, R2C, R2D, R4A, R4B, R4C, and R4D are independently hydrogen, -CX3, -CHX2, -CH2X, -CN, -OH, -COOH, -CONH2, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; R1A and R1B or R2A and R2B with substituents bonded to the same nitrogen atom may optionally be joined to form a substituted or unsubstituted heterocycloalkyl or substituted or unsubstituted heteroaryl; each X, X1, X2, and X4 is independently -F, -Cl, -Br, or -I; each nl, n2 and n4 is independently an integer from 0 to 4; z is an integer from 0 to 5; and each ml, m2, m4, vl, v2 and v4 are independently 1 or 2.
[0268] Embodiment 2. The compound of Embodiment 1, wherein L1 is a bond, substituted or unsubstituted alkylene, or substituted or unsubstituted heteroalkylene.
[0269] Embodiment 3. The compound of Embodiment 2, wherein L1 is substituted or unsubstituted C1-C3 alkylene, or substituted or unsubstituted 2 to 3 membered heteroalkylene.
[0270] Embodiment 4. The compound of any one of Embodiments 1 to 3, wherein the compound has a structure of
Figure imgf000095_0001
[0271] Embodiment 5. The compound of any one of Embodiments 1 to 3, wherein the compound has a structure of
Figure imgf000096_0001
[0272] Embodiment 6. The compound of any one of Embodiments 1 to 5, wherein each R1 andR2 is independently substituted or unsubstituted alkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl.
[0273] Embodiment 7. The compound of Embodiment 6, wherein each R1 andR2 is independently substituted or unsubstituted C1-C4 alkyl, or substituted or unsubstituted phenyl.
[0274] Embodiment 8. The compound of Embodiment 7, wherein each R1 andR2 is independently unsubstituted C1-C4 alkyl, or unsubstituted phenyl.
[0275] Embodiment 9. The compound of Embodiment 8, wherein R1 is unsubstituted C1-C4 alkyl, and R2 is unsubstituted phenyl.
[0276] Embodiment 10. The compound of any one of Embodiments 1 to 9, wherein z is
0.
[0277] Embodiment 11. The compound of any one of Embodiments 1 to 9, wherein z is
1 and R4 is halogen.
[0278] Embodiment 12. The compound of Embodiment 1, wherein the compound is
Figure imgf000096_0002
[0279] Embodiment 13. A pharmaceutical composition comprising a compound of any one of Embodiments 1 to 12, or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. [0280] Embodiment 14. A method of treating a cancer in a subject in need thereof, the method comprising administrating an effective amount of the compound of any one of Embodiments 1 to 12, or a pharmaceutically acceptable salt thereof.
[0281] Embodiment 15. The method of Embodiment 14, wherein the cancer is a solid tumor.
[0282] Embodiment 16. The method of Embodiment 15, wherein the solid tumor is in thyroid, endocrine system, brain, breast, cervix, colon, prostate, head and neck, liver, kidney, lung, ovary, pancreas, rectum, stomach, oral cavity, or uterus.
[0283] Embodiment 17. A method of inhibiting V-domain Ig Suppressor of T-cell Activation (VISTA) in a cell, the method comprising contacting the cell with an effective amount of the compound of any one of Embodiments 1 to 12, or a pharmaceutically acceptable salt thereof.
[0284] Embodiment 18. A method of enhancing T-cell proliferation in a subject in the presence of VISTA-expressing cancer cells, the method comprising administrating the subject an effective amount of the compound of any one of Embodiments 1 to 12, or a pharmaceutically acceptable salt thereof.
[0285] Embodiments 19. A method of restoring T-cell activation in a subject in the presence of VISTA-expressing cancer cells, the method comprising administrating the subject an effective amount of the compound of any one of Embodiments 1 to 12, or a pharmaceutically acceptable salt thereof.
EXAMPLES
EXAMPLE 1
[0286] V-domain Ig Suppressor of T-cell Activation (VISTA) is an immune checkpoint that affects the ability of T-cells to effectively attack tumors. A FRET -based high throughput screening identified the first small-molecule ligand for VISTA which was further confirmed by biorthogonal assays. Investigation of the hit interaction with VISTA using molecular modeling and STD NMR enabled the identification of a potential binding site in VISTA for the hit. Screening the hit against a small library of single-point VISTA mutants revealed the key residues in VISTA interacting with the hit. Further structural optimization resulted in a lead with submicromolar VISTA binding affinity. The lead compound blocked VISTA signaling in vitro, enhanced T-cell proliferation, and restored T-cell activation in the presence of VISTA-expressing cancer cell lines. This work would enable future development of small molecules targeting VISTA as immunomodulators and imaging probes.
[0287] The immunomodulatory mechanism of VISTA is unclear because of its expression on both antigen-presenting cells (APC8) and T cells which reveals a ligand-or-receptor paradigm of VISTA. V-Set and Immunoglobulin domain containing 3 (VSIG-3) has been recently proposed as a potential ligand for VISTA.14 Moreover, VISTA has been identified as an acidic pH-selective ligand for P-selectin glycoprotein ligand- 1 (PSGL-1). Ablation of VISTA by genetic knockout or blocking antibodies activates T cell immune response against tumors in preclinical models, thus, several VISTA antibodies have advanced recently into human clinical trials.
[0288] Although crystal structures of human VISTA ECD are recently reported, development of small-molecule ligands for VISTA remains unexplored. In general, mAbs feature poor pharmacokinetic (PK) profiles, ineffective penetration into tumor microenvironment, immune-related side effects, long half-lives in the body, and high manufacturing costs. On the other hand, small molecules with lower production costs possess beneficial PK profiles in comparison to mAbs enabling their oral bioavailability, enhanced tumor penetration, and shorter half-lives' Herein, we report the first time-resolved fluorescence resonance energy transfer (TR-FRET) assay to evaluate ability of small molecules to bind VISTA. Optimization of the assay for high throughput screening (HTS) enables screening large chemical libraries. Terbium (Tb) cryptate fimctions as the donor in the developed TR-FRET assay by labeling His-tagged human VISTA with MAb Anti-6His Tb cryptate gold. A human VISTA antibody labeled with far-red emitting fluorescent dye (CF647) functions as an acceptor based on reported compatibility of Tb cryptate and CF647 as donor-acceptor pair in FRET signaling. Compounds that can bind VISTA would perturb the interaction between VISTA and its antibody which would correlate with attenuation of FRET signal. A linear relationship between FRET ratio and the acceptor concentration (0-10 pM) was detected. Addition of VSIG3, a ligand of VISTA, in increasing concentrations resulted in gradual decrease in FRET signal. The mean Z’ factor of the assay was 0.64 indicating a high-quality assay for HTS.
[0289] CA- 170 (2-(3 -(3 -amino- 1 -(3 -( 1 -amino-2-hydroxyethyl)- 1 ,2,4-oxadiazol-5-yl)-3 - oxopropyl)ureido)-3 -hydroxybutanoic acid), a small molecule developed by Aurigene and Curis, is currently undergoing phase II clinical trials for lung cancer, head and neck/oral cavity cancer. Although CA-170 is reported as dual PD-L1 and VISTA inhibitor, no biophysical data have been reported to confirm binding to PD-L1 or VISTA. Musielak et al., recently demonstrated minimal binding of CA-170 to PD-L1 in NMR binding assay, FRET, and cell-based activation assay. Evaluation of CA-170 for VISTA binding using the developed TR-FRET assay revealed negligible change in the FRET signal. Moreover, a competitive enzyme-linked immunosorbent assay (ELISA) utilizing interaction between immobilized VISTA and biotinylated VSIG3 illustrated limited capability of CA-170 to impede VISTA-VSIG3 interactio). Minimal binding of CA-170 to VISTA was further confirmed using surface plasmon resonance (SPR) by allowing the ligand to flow over VISTA ECD immobilized on a chip. Therefore, we concluded minimal binding affinity of CA-170 to human VISTA.
[0290] The developed TR-FRET assay was employed in HTS of chemical libraries of -4,000 compounds from NIH clinical collection (NIHCC) and National Cancer Institute (NCI) Developmental Therapeutics Program (DTP) in a single-dose assay. Compounds that exhibited more than 40% inhibition of the FRET signal in HTS were subjected to additional 10-dose screening. Further dose-dependent FRET screening identified NSC622608 (FIG. 1A) as a ligand for VISTA with IC50 value of 4.8 ± 0.4 pM (FIG. IB). Moreover, competitive ELISA revealed the ability of the hit to disrupt VISTA-VSIG-3 interaction with IC50 value of 9.7 ± 0.3 pM (FIG. 1C). SPR screening confirmed binding of the hit to VISTA ECD with an estimated KD of 6.2 ± 0.5 pM (FIG. ID).
[0291] Saturation transfer difference (STD) NMR was employed to study VISTA-hit interaction. Observance of strong STD signals confirmed tight binding of the hit to VISTA. High STD effects % (-80-100%) for NMR peaks of 2-furyl rings of the hit indicated direct interaction between the ring and the potential binding site. However, minimal STD effect % for the dimethylamino moiety revealed no direct interaction with the protein. Trials to identify a potential binding site for NSC622608 in VISTA started with exploring possible binding cavities in recent VISTA crystal structures; structure of VISTA ECD (PDB: 6OIL) and structure of VISTA bound to antibody Fab fragment (PDB: 6MVL). Web-based tools for ligand binding site prediction, including geometric-based method (DoGSiteScorer), templatebased method (LIBRA-WA), energetic-based method (FT site), and machine learning-based methods (Prankweb and Deepsite) were used to predict 32 potential binding sites in the two crystal structures (PDB IDs: 6OIL, 6MVL).
[0292] Glide docking of the ligand to the predicted binding sites in VISTA was performed using Maestro (Schrodinger). GlideScore, an empirical scoring fimction that approximates ligand binding free energy, was used to evaluate the binding. Favorable Glide Scores of docked poses of the ligand were obtained upon docking into predicted sites in PDB ID 6MVL sharing Tyr69, Argl59, His98, Hisl58, Phe94, and Glu 157 residues. These results reveal that conformational changes to VISTA upon antibody binding have resulted in formation of favorable binding sites for the hit. The lowest GlideScore was observed for the best docked pose of the hit to the site predicted by Prankweb in PDB ID 6MVL (FIGS. 2 A and 2B). The docked pose of the ligand into this site features interaction of the oxygen atom of the 2-fiiryl ring with 2 hydrogen bonds with Argl59 residue (FIG. 2A). Moreover, the 2-furyl ring further interacts with Glul57 residue via aromatic hydrogen bonding interaction. The dithiazole moiety in the hit engages in hydrophobic interaction with Phe94 residue in VISTA (FIG. 2A). A small library of single-point VISTA mutants was then expressed and purified based on the key interacting resides of VISTA in the binding pose shown in FIG. 2 A and other key residues from the rest of docked poses. Competitive ELISA was then performed between VISTA mutants and VSIG-3 in the presence of the hit. The most remarkable effect on the binding signal was observed in VISTA Argl59 mutant (FIG. 2C). Considerable weak binding of the hit was detected in VISTA Phe94 and Glul57 mutants. Thus, these results are in consistent with proposed binding pose of the hit. A detectable effect in the binding signal using VISTA Tyr69 mutant might reflect interaction of the hit in a secondary binding site in VISTA including Tyr69.
[0293] A two-step synthetic route was developed for the preparation of the hit and analogues (Schemes SI -3). The synthesized compounds were screened for VISTA binding affinity using FRET assay. As shown in FIG. 3 A, compounds lb and Ic with 2-thienyl and ( 1 H-pyrrol -2-yl), respectively, had lower VISTA affinity in comparison to la with 2-furyl ring. Replacement of the 2-furyl ring with tetrahydrofuran-2yl ring in compound Id resulted in minimal VISTA binding affinity. However, incorporation of a phenyl ring to the 5 -position in the 2 furyl ring (compound le) afforded ~3-fold enhancement in VISTA binding affinity. Further substitution of the incorporated phenyl ring in compound If decreased VISTA binding affinity by ~1 -fold (FIG. 3 A). Moreover, replacement of the furan-2-ylmethyl moiety of the hit with substituted and unsubstituted phenyl and benzyl moieties (compounds Ig-Il) resulted in considerable attenuation in VISTA binding affinity. Compound Im bearing 4- phenylbenzyl fragment exhibited ~2-fold higher affinity in comparison to li with a benzyl fragment highlighting the significance of additional hydrophobic interaction similarly to le. We further investigated the effect of replacement of dimethylamino moiety in the hit with hydrophobic fragments in compounds Ila and lib (FIG. 3B). Compound lib with JV-ethyl, N- phenyl-amino fragment displayed ~3-fold enhancement in VISTA binding in comparison to la. Thus, we designed compound III based on the hybridization of 5-phenyl-fiiran-2-yl and JV-ethyl, /V-phenyl-amino fragments in le and lib, respectively (FIG. 3C). In consistence with our design strategy, III displayed ~10-fold enhancement in VISTA binding with IC50 value of 486 ±18 nM in FRET assay.
[0294] The binding affinity of III to VISTA was fiirther confirmed by competitive ELISA with IC50 value of 716 ± 21 nM (FIG. 3D). Kinetic analysis of III- VISTA interaction using SPR indicated an estimated Ko value of 247 nM (not shown). Glide docking of III into the identified binding pocket in VISTA for NSC622608 revealed that III maintained interaction with Phe94, Argl59, and Glul57 residues (FIG. 2B). In comparison to NSC622608, III featured additional hydrophobic interaction with Tyr69 and Hisl53 residues. The interaction of III with predicted key residues was confirmed by evaluating the binding of III to singlepoint VISTA mutants of these residues in competitive ELSIA (FIG. 2C).
[0295] Promega VISTA bioluminescent cell-based T-cell activation assay was used to evaluate the ability of III to block VISTA in a cellular platform. The assay is based on VISTA-VISTA interaction between CHOK1 VISTA cells and NanoGlo™IL-2/Jurkat VISTA cells which promotes the production of interleukin 2 (IL-2) resulting in a luminescent signal. VISTA antibody reduces IL-2 signaling at nanomolar concentration as evident from diminished luminescent signaling. As shown in FIG. 4A, compound III blocks VISTAVISTA interaction resulting in decreased IL-2 signaling. However, CA-170 is not able to decrease IL-2 signaling which comes in agreement with its minimal VISTA binding affinity (FIG. 4B).
[0296] VISTA expression in ovarian and endometrial cancers has been directly associated with regulation of T-cell function. We have selected 4 ovarian cancer cell lines (OVKATE, SKOV4, COV504, and A2780) and 4 endometrial cancer cell lines (RL952, HEC1 A, AN3CA, and Ishikawa) with reported varying VISTA gene expression. The differential VISTA expression on the surface of these cells has been further confirmed with flow cytometry analysis (not shown). Similarly to VISTA antibody (FIGS. 4C and 4D), addition of III to co-cultured Jurkat T cells with 8 cancer cell lines selectively restored the proliferation of T cells in the presence of high VISTA expressing cell lines (OVKATE, SKOV4, COV504, RL952, HEC1 A) while minimal effect was evident for cancer cell lines with low VISTA expression (A2780, AN3CA, Ishikawa). In addition, both interferon gamma (IFNy) and tumor necrosis factor alpha (TNFa), cytokines secreted by activated T cells, were upregulated in the presence of III in the supernatants of co-cultures of purified human T cells with OVKATE and RL952 cell lines with high VISTA expression (FIGS. 6 and 7). These results indicate that III can restore the viability and activation of T cells in cellular platforms based on blocking VISTA expressed on cancer cells. Future work based on optimization of III would enable the discovery of next generation of small molecule-based diagnostics and therapeutics in cancer immunotherapy.
Experiment and Methods
[0297] Validation of the VISTA TR-FRET assay for high-throughput screening (HTS):
Z’ factor was calculated using the following equation:1
(S.D.+ve)+(S.D.-ve)
Z’ factor = 1 -
(mean+ve)-(mean-ve)
Where, S.D. +ve : Standard deviation of the positive control. S.D. -ve: Standard deviation of the negative control.
Mean +ve : mean of the positive controls Mean -ve : mean of the negative controls
[0298] Saturation-transfer difference (STD) NMR spectroscopy: Samples for STD
NMR experiments were prepared using la (10 mM) prepared in phosphate buffer solution, pH 7.0. Samples had final concentrations of 1% DMSO-ck and D2O (10% v/v). Each sample was made up to a final volume of 500 pL with PBS buffer (20 mm, pH 7.0) and NaCl (20 mm). The experiments were performed at a final concentration of la (1 mM) on Agilent 400 MHz WB NMR spectrometer at 298 K and pH 7.0 in the presence and absence of VISTA extracellular domain (20 pm). The protein was saturated on-resonance at -0.5 ppm and off- resonance at 33 ppm with a cascade of 40 selective Gaussian shaped pulses, of 50 ms duration with a 100 psec delay between each pulse. The total duration of the saturation time was set to 2 s. A total of 256 scans per STD NMR experiment were acquired and a WATERGATE sequence was used to suppress the residual H2O signal.
[0299] Time-resolved fluorescence resonance energy transfer (TR-FRET) assay:
Human recombinant VISTA Protein (His Tag), catalogue number: 13482-H08H, was obtained from Sino biological and labeled with MAb Anti-6HIS Tb cryptate from Cisbio. VISTA antibody (MAB71263-SP) was obtained from Novus Biologicals and labeled using Mix-n-Stain™ CF™ 647 Antibody Labeling Kit (Sigma Aldrich). The assay plates were medium binding white 384-well plates (Greiner #784075). The assay buffer was phosphate buffer solution (PBS, pH 7.0). TR-FRET measurements were done on Tecan Infinite M1000 PRO (top read, Z-Position height: 25500 pm, (Donor Read-620) Ex 340-20 nm, Em 620-5 nm, Gain value: 255, (Acceptor Read-665) Ex 340-20 nm, Em 665-5 nm, Gain value: 255; both with 50 flashes/well. 200 ps integration time, 100Hz, 50 ps lag time). VISTA Tb cryptate (25 pM) and CF647 -labeled VISTA antibody (1.6 pM) stock solutions were prepared in PBS (pH7.0).
[0300] VISTA Tb cryptate and CF647-labeled VISTA antibody (assay mixture) were immediately mixed prior to the assay to a final concentration of: His-tagged VISTA (10 nM), MAb Anti-6HIS Tb cryptate Gold (1 nM), and CF647-labeled VISTA antibody (10 nM). Stock solutions of tested compounds in DMSO (50 nL) from the chemical libraries were applied to 384-well plates. The assay mixture (10 pL) was added to the plated compounds and incubated for 3 hrs at room temperature. Measurements were performed as described above and TR-FRET signals were calculated as a ratio as follows: (intensity of 665 nm)/(intensity of 615 nm) x 10,000. The IC50 values were calculated by plotting compound concentration versus TR-FRET signal. The dose-response curves were analyzed by nonlinear regression using GraphPad Prism 8.0.2 (GraphPad Software, Inc., La Jolla, CA, USA). Each data point from this assay represents the average of three independent measurements. Error bars represent standard deviation.
[0301] Competitive VISTA-VSIG-3 enzyme-linked Immunosorbent assay (ELISA): Human recombinant VISTA (ECD, Fc Tag), catalogue number: 13482-H02H, was obtained from Sino biological. Biotinylated human VSIG3 protein (Fc, Avitag) was obtained from Aero Biosystems. VISTA ECD (2 pg/mL) in PBS (pH 7.0) was immobilized on 96-well clear polystyrene plates from R&D systems (Part # 991427) by addition of 50 pL of stock solution for 12 hrs at 4 °C. The ELISA plates were washed three times with PBS, then blocked with 1% bovine serum albumin (BSA) in PBS at room temperature for 2 hrs. The blocking buffer was removed and plates were washed by PBS three times. The wells then incubated for 2 hrs at room temperature with 50 pL solutions containing various concentrations of tested compounds (obtained by diluting from a 1 mg/mL stock solution of each molecule dissolved in PBS containing 1% DMSO) and a fixed concentration of biotinylated human VSIG3 protein (2 nM) in protein-free blocking buffer. VISTA-VSIG-3 binding was detected by adding streptavidin-horseradish peroxidase (HRP) followed by substrate color reagents (R&D Systems, Minneapolis, MN). The reactions were stopped after 15 min with 2M sulfiiric acid, and absorbance at 450 nm was measured on microplate reader (BioTek Synergy4).
Absorbance values of control wells with no coated protein were subtracted from sample wells and corrected values were plotted against compound concentration and fit to a one-site specific binding curve on GraphPad Prism 8.0.2 (GraphPad Software, Inc., La Jolla, CA, USA). Each data point from this assay represents the average of three independent measurements. Error bars represent standard deviation.
[0302] Surface plasmon resonance (SPR) screening: The kinetic analysis for the interaction of tested small molecules with VISTA was performed on a Biacore T200 (GE Healthcare Bio-sciences, Sweden) using Series S Sensor Chip NTA (GE Healthcare Biosciences, Sweden, catalog # 28-9949-51). Human recombinant VISTA Protein (His Tag), catalogue number: 13482-H08H, obtained from Sino biological was captured in PBS (pH 7.0) on sensor chip using NTA reagent kit (GE Healthcare Bio-sciences, Sweden, catalog # 28- 9950-43).
[0303] Stock solution of tested compounds were prepared in DMSO at 1 mM final concentration. Samples were then serially diluted while maintaining final DMSO concentration to 5% in all tested samples. Tested compounds in PBS (pH 7.0) containing 5% DMSO and 0.05% Tween20 were allowed to flow through both ligand-captured flow cells and reference flow cells at the same rate (30 pl/min) and contact time (120 sec). Solvent correction was included to avoid the impact of DMSO on surface plasmon effect during binding analysis. Extra wash of the flow system using 50% DMSO in PBS buffer was then performed following each run. Kinetic analysis was performed at 25 °C. Equilibrium dissociation rate constant (KD) values were calculated using Biacore T200 Evaluation software following the 1 : 1 Langmuir binding model with global fit parameters for solvent- corrected sensograms.
[0304] Cell culture: SKOV4, RL952, HEC1 A, AN3CA, and Jurkat cell lines were obtained from American Type Culture Collection (ATCC). COV504 and A2780 cell Hines were obtained from European Collection of Authenticated Cell Cultures (EC ACC). OVKATE and Ishikawa cell lines were obtained from Prof. Wendy Fantl lab, Stanford University. Cells were cultured in RPMI1640 medium with 10% Fetal Bovine Serum (FBS), 100 units/mL penicillin and 100 pg/mL streptomycin. All cells were grown at 37 °C in a humidified atmosphere containing 5% CO2. [0305] Expression and purification of VISTA mutants: VISTA mutants were expressed and purified using previously reported method (with minor modifications) for expression and purification of VISTA mutants.2 Expi293 cells were acquired from ThermoFisher Scientific and grown in Expi293 Expression Medium according to the manufacturer’s protocol. The extracellular domain of human VISTA (residues 1 to 161) provided by Integrated DNA Technologies (Coralville, IA) was cloned into a pcDNA3.4 vector (Thermo Fisher) with an N-terminal signal peptide from CRYPa and a C-terminal Mpro protease site and His6-tag. Plasmids were transfected into Expi293 cells (Thermo Fisher) using provider’s protocol but with the addition of 20 pM swainsonine (Cayman Chemical) at the time of transfection. Secreted protein in the supernatant was harvested, dialyzed into Ni-NTA binding buffer (50 mM Tris pH 8.0, 300 mM NaCl), and purified using Ni-NTA affinity chromatography (Qiagen). Binding of the mutants to the VISTA antibody (MAB71263-SP) was retained with no significant difference in binding from WT VISTA.
[0306] Flow cytometry analysis: For the analysis of the expression of human VISTA on the surface of selected ovarian and endometrial cancer cells, flow cytometry analysis was performed. The cells were seeded on 6-well plates and cultured till sub-confluency. The cells were detached from the plates with Cellstripper (Coming, Cat. No. 25-056-CI), placed on ice, washed 2 times with Flow Cytometry Staining Buffer (eBioscience, Waltham, MA, USA) and stained with Human VISTA/B7-H5/PD-1H Alexa Fluor® 647-conjugated Antibody (R&D systems, Cat. No. FAB71261R). The cells were analyzed with the BD Biosciences LSRII cytometer.
[0307] Promega VISTA bioluminescent cell-based T-cell activation assay: The assay kit including NanoGio™ IL2-NLP/Jurkat VISTA cells and CHOK1 VISTA cells were obtained from Promega, WI. The assay was then performed according to provider’s protocol. Breifly, Jurkat cells (5 x 104 cells) and CHOK1 cells (3 x 104 cells) were co-cultured overnight in RPMI1640 medium with 10% Fetal Bovine Serum (FBS). Antihuman CD3 antibody (1000 ng/mL) was then added and the co-cultures were incubated for 6 hrs at 37 °C in a humidified atmosphere containing 5% CO2. Promega Nano-Gio™ Reagent was then reconstituted and used at the end of the incubation time according to the manufacturer’s instructions. Each data point from this assay represents the average of three independent measurements. Error bars represent standard deviation. [0308] Prestoblue cell viability assay: Briefly, 3 x 104 Jurkat T cells were cultured alone or co-cultured with 1.5 x 105 of 8 cancer cell lines used in the assay in 24-well plates. After incubation with compound III (5 pM) or VISTA antibody (MAB71263-SP) (1 pM) for 24 h, Jurkat T cells were harvested from the supernatant. Presto Blue® Viability Assay (Thermo Fisher Scientific Inc., Waltham, MA) was then performed. Each data point from this assay represents the average of three independent measurements. Error bars represent standard deviation.
[0309] Assessment of cytokines production by ELISA: Human T cells were isolated from peripheral blood mononuclear cells (PBMC8) using the Human Pan-T Cell Isolation Kit (Miltenyi Biotec). Human IFN-y, and TNFa ELISA kits (Qiagen, Valencia, CA, USA) were used to analyze cytokine production. Each data point from this assay represents the average of three independent measurements. Error bars represent standard deviation.
[0310] Statistical analyses: Statistical significance was determined using a 2-tailed Student t test, with P values of less than 0.05 being considered significant. For analysis across multiple samples, 1- or 2-way ANOVA was used, followed by multiple comparisons of means with Bonferroni adjustment.
[0311] Molecular modeling: The crystal structure of human VISTA ECD (PDB ID: 6OIL) and human VISTA in complex with antibody Fab fragment (PDB ID: 6MVL) were obtained from protein data bank (PDB). Refinement of crude PDB structure of receptor was performed. Polar hydrogens were added, Kollman charges were assigned and atomic solvation parameters were added. The optimized receptors were then used for docking simulation.
[0312] The 2D structures of the compounds were built and then converted into 3D using vLife MDS 3.0 software. The 3D structures were energetically minimized up to the rms gradient of 0.01 using CHARM22 force field. Glide docking was performed using Maestro 12.0 (Schrodinger). 10 poses were generated for each docking run to a given site. GlideScore, an empirical scoring function that approximates ligand binding free energy, was used to evaluate the binding.27 All visualizations of ligand-protein interactions were done using Maestro 12.0 (Schrodinger). References in Example 1
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EXAMPLE 2: Synthesis
[0314] All commercially available starting materials, reagents, and solvents were used as supplied unless otherwise stated. CA-170 was purchased from MedChemExpress (Monmouth Junction, NJ, USA) CAS Number: 1673534-76-3. Proton (’H) and carbon (l3C) NMR were collected on Agilent 400 MHz WB NMR spectrometer. Chemical shifts (6) are reported in parts-per million (ppm) relative to residual undeuterated solvent. High resolution mass spectra were obtained in positive ion mode using electrospray ionization (ESI) on Thermo Exactive Orbitrap LC/MS. The purity of all final compounds was >99% as revealed by HPLC analysis using an Agilent system equipped with 1260 Infinity II quaternary pump and 1260 Infinity II diode array detector. Analytical HPLC reverse phase column (Phenomenex, Gemini, Hesperia, CA, Cl 8, 5 pm, 250 x 4.6 mm) at a flow rate of 1 mL/min was used for HPLC analysis.
Figure imgf000110_0001
Scheme 1. General procedure for the synthesis of compounds la-m.
Figure imgf000111_0001
Scheme 2. Synthesis of compounds IIa,b.
Figure imgf000111_0002
Scheme 3. Synthesis of compound III.
[0315] General procedure for the preparation of compounds 3a-m, 6a, b and 7: A solution of thiocarbamoyl chloride derivative (10.0 mmol) and potassium thiocyanate (0.98 g, 10.0 mmol) in dry acetonitrile (25 mL) was heated under reflux for 15 min. A solution of the corresponding primary amine (9 mmol) in dry acetonitrile (10 mL) was added to the reaction mixture and kept stirring under reflux for 1 hr. The crude reaction mixture was filtered over Celite and concentrated under vacuum. The crude mixture was purified by Teledyne CombiFlash Rf200 Flash Chromatography System (silica) using hexane-ethyl acetate gradient.
[0316] N, N-Dimethyl-N’-(furan-2-ylmethyl)-thioimidodicarbonic diamide (3a) Yield
87%. !H NMR (400 MHz, CDC13) 63.27 (s, 3H), 3.42 (s, 3H), 4.76 (d, J= 5.4 Hz, 2H), 6.26- 6.29 (m, 1H), 6.86 (dd, J= 12.9, 3.2 Hz, 1H), 6.91-6.97 (m, 1H).13C NMR (100 MHz, DMSO-Jo) 546.2, 46.7, 47.8, 127.0, 130.3, 137.1, 140.2, 177.2, 181.0. HRMS (ESI): calcd for C9HI4N3OS2 [M+H]+, 244.0578; found, 244.0579.
[0317] N,N-Dimethyl-N’-( thiophen-2-ylmethyl)-thioimidodicarbonic diamide (3b).
Yield 75%. 3H NMR (400 MHz, CDCh) 63.29 (s, 3H), 3.33 (s, 3H), 5.04 (d, J= 5.2 Hz, 2H), 6.95 (ddd, 7 = 4.8, 3.5, 1.1 Hz, 1H), 7.07 (dt, J= 3.5, 1.1 Hz, 1H), 7.23 (dd, 7 = 5.1, 1.2 Hz, 1H), 8.47 (s, 1H), 12.51 (s, 1H).13C NMR (100 MHz, CDCh) 840.1, 40.6, 44.9, 125.3, 126.4, 126.8, 140.7, 177.9, 182.0. HRMS (ESI): calcd for C9H14N3 S3 [M+H]+, 260.0349; found, 260.0355.
[0318] 7V^V-Dimethyl-7V '-((17f-pyrrol-2-yl)methyl)-thioimidodicarbonic diamide (3c) Yield 79%. ’H NMR (400 MHz, CDCh) 83.21 (s, 3H), 3.25 (s, 3H), 4.81 (d, J= 5.6 Hz, 2H), 5.88-6.18 (m, 2H), 6.69-6.71 (m, 1H), 8.42 (s, 1H), 9.34 (s, 1H), 12.41 (s, 1H).13C NMR (100 MHz, CDCh) 538.3, 40.7, 41.4, 110.0, 112.5, 130.3, 132.4, 175.6, 189.5. HRMS (ESI): calcd for C9H15N4S2 [M+H]+, 243.0738; found, 243.0742.
[0319] 7V^V-Dimethyl-7V-((tetrahydrofuran-2-yl)methyl)-thioimidodicarbonic diamide (3d) Yield 68%. ’H NMR (400 MHz, CDCh) 8 1.54-2.04 (m, 4H), 3.28 (s, 6H), 3.66-3.92 (m, 4H), 4.11 (qd, J= 6.6, 3.9 Hz, 1H), 8.43 (s, 1H), 12.17 (s, 1H).13C NMR (100 MHz, CDCh) 825.9, 28.2, 40.9, 41.9, 49.9, 68.3, 76.3, 175.3, 179.8. HRMS (ESI): calcd for C9H18N3OS2 [M+H]+, 248.0891; found, 248.0894.
[0320] 7V^V-Dimethyl-7V-((5-phenylfuran-2-yl)methyl)-thioimidodicarbonic diamide (3e) Yield 65%. ’H NMR (400 MHz, CDCh) 8 3.12 (s, 3H), 3.32 (s, 3H), 4.48 (s, 2H), 6.44 (d, J= 3.2 Hz, 1H), 6.55 (t, J= 3.0 Hz, 1H), 7.18-7.29 (m, 1H), 7.29-2.47 (m, 2H), 7.60 (dd, J= 2.7, 1.3 Hz, 1H), 7.61-7.73 (m, 1H).13C NMR (100 MHz, CDCh) 8 37.3, 38.9, 53.3, 106.1, 110.7, 123.6, 127.3, 128.6, 130.5, 149.4, 153.6, 168.8, 177.5. HRMS (ESI): calcd for C15H18N3OS2 [M+H]+, 320.0891; found, 320.0896.
[0321] 7V^V-Dimethyl-7V-((5-(4-fluorophenyl)furan-2-yl)methyl)-thioimidodicarbonic diamide (3f) Yield 72%. ’H NMR (400 MHz, CDCh) 8 3.10 (s, 3H), 3.32 (s, 3H), 4.52 (s, 2H), 6.41-6.49 (m, 2H), 7.01-7.06 (m, 2H), 7.57 (ddd, J= 8.8, 5.3, 1.5 Hz, 2H).13C NMR (100 MHz, CDCh) 839.3, 41.4, 42.9, 106.0, 110.4, 115.7, 126.0, 126.9, 148.9, 152.9, 160.8, 175.3, 179.2. HRMS (ESI): calcd for C15H17FN3OS2 [M+H]+, 338.0797; found, 338.0799.
[0322] 7V^V-Dimethyl-7V -(4-chlorophenyl)-thioimidodicarbonic diamide (3g) Yield 94%. ’H NMR (400 MHz, CDCh) 83.14 (s, 3H), 3.24 (s, 3H), 6.96 (d, J= 7.5 Hz, 2H), 7.27 (d, J= 7.5 Hz, 2H).13C NMR (100 MHz, CZ>C13) 839.1, 41.8, 122.7, 128.8, 129.6, 149.2, 168.0, 174.8. HRMS (ESI): calcd for C10H13CIN3S2 [M+H]+, 274.0239; found, 274.0241.
[0323] 7V^V-Dimethyl-7V -(4-methoxyphenyl)-thioimidodicarbonic diamide (3h) Yield 91%. ’H NMR (400 MHz, CDCI3) 63.20 (s, 3H), 3.25 (s, 3H), 3.77 (s, 3H), 6.86 (d, J= 8.3 Hz, 2H), 7.49 (d, J= 8.3 Hz, 2H), 8.54 (s, 1H), 13.82 (s, 1H).13C NMR (100 MHz, CBCh) 8
40.7, 41.8, 55.5, 121.5, 125.7, 130.2, 143.6, 174.9, 177.6. HRMS (ESI): calcd for C11H16N3OS2 [M+H]+, 270.0734; found, 270.0739.
[0324] 7V^V-Dimethyl-7V -benzyl-thioimidodicarbonic diamide (3i) Yield 95%. 1 H NMR (400 MHz, CBCh) 83.22 (s, 3H), 3.26 (s, 3H), 4.89 (d, J= 2.9 Hz, 2H), 7.29-7.41 (m, 5H), 8.50 (s, 1H), 12.53 (s, 1H).13C NMR (100 MHz, CDCI3) 842.1, 42.6, 49.9, 127.7, 127.8,
128.7, 136.2, 175.2, 179.2. HRMS (ESI): calcd for C11H16N3S2 [M+H]+, 254.0785; found, 254.0788.
[0325] 7V^V-Dimethyl-7V -(4-chlorobenzyl)-thioimidodicarbonic diamide (3j) Yield 78%. ’H NMR (400 MHz, CDCI3) 83.26 (s, 3H), 3.29 (s, 3H), 4.64 (d, J= 2.9 Hz, 2H), 7.28 (d, J= 7.9 Hz, 2H), 7.39 (d, J= 7.9 Hz, 2H) 10.75 (s, 1H).13C NMR (100 MHz, CDCh) 8 42.3, 42.8, 49.0, 128.8, 129.2, 133.3, 134.8, 175.1, 179.4. HRMS (ESI): calcd for C11H15CIN3S2 [M+H]+, 288.0395; found, 288.0396.
[0326] 7V^V-Dimethyl-7V -(4-fluorobenzyl)-thioimidodicarbonic diamide (3k) Yield 86%. ’H NMR (400 MHz, CDCh) 83.24 (s, 3H), 3.28 (s, 3H), 4.85 (d, J = 5.3 Hz, 2H), 6.84- 7.05 (m, 2H), 7.26-7.47 (m, 2H), 8.49 (s, 1H), 12.51 (s, 1H).13C NMR (100 MHz, CDCI3) 8 42.8, 43.6, 49.1, 115.4, 129.6, 132.1, 163.4, 175.3, 179.3s. HRMS (ESI): calcd for C11H15FN3S2 [M+H]+, 272.0691; found, 272.0690.
[0327] 7V^V-Dimethyl-7V -(4-methoxybenzyl)-thioimidodicarbonic diamide (31) Yield 82%. ’H NMR (400 MHz, CDCh) 83.21 (s, 3H), 3.24 (s, 3H), 3.78 (s, 3H), 4.80 (d, J= 5.1 Hz, 2H), 6.85 (d, J= 8.9 Hz, 2H), 7.26 (d, J= 8.9 Hz, 2H), 8.46 (s, 1H), 12.31 (s, 1H).13C NMR (100 MHz, CDCh) 840.1, 40.6, 49.5, 55.3, 114.1, 128.2, 129.2, 159.1, 175.3, 178.9. HRMS (ESI): calcd for C12H18N3OS2 [M+H]+, 284.0891; found, 284.0893.
[0328] 7V^V-Dimethyl-7V-([l,l’-Biphenyl]-4-ylmethyl)-thioimidodicarbonic diamide (3m) Yield 70%. ’H NMR (400 MHz, CDCh) 83.28 (s, 3H), 3.31 (s, 3H), 4.98 (d, J = 5.4 Hz, 2H), 7.34-7.49 (m, 5H), 7.56-7.61 (m, 4H), 8.54 (s, 1H), 12.61 (s, 1H).13C NMR (100 MHz, CZ>C13) S 40.6, 41.0, 49.6, 127.0, 127.4, 127.5, 128.2, 128.8, 135.3, 140.5, 140.6, 175.3, 179.4. HRMS (ESI): calcd for C17H20N3S2 [M+H]+, 330.1098; found, 330.1096.
[0329] 7V-(4-Chlorophenyl)-7V-methyl-7V -(furan-2-ylmethyl)-thioimidodicarbonic diamide (6a) Yield 92%. ’H NMR (400 MHz, CDCI3) 63.28 (s, 3H), 4.34 (d, J= 5.2 Hz, 2H), 4.61 (s, 1H), 5.67 (s, 1H), 6.13 (dt, J =2.2, 1.1 Hz, 1H), 6.26 (dt, J= 3.1, 1.5 Hz, 1H), 7.06 (d, J= 8.2 Hz, 1H), 7.16 (d, J= 8.7 Hz, 2H), 7.30 (d, J= 8.7 Hz, 2H).13C NMR (100 MHz, CBCh) 8 37.8, 43.9, 106.8, 110.3, 127.3, 128.6, 130.1, 131.1, 141.9, 152.3, 179.5, 182.6. HRMS (ESI): calcd for C14H15CIN3OS2 [M+H]+, 340.0345; found, 340.0346.
[0330] 7V-Ethyl-7V-phenyl-7V -(furan-2-ylmethyl)-thioimidodicarbonic diamide (6b) Yield 80%. ’H NMR (400 MHz, CBCh) 8 1.24 (t, J= 7.1 Hz, 3H), 3.59 (q, J= 7.2 Hz, 2H), 4.28 (d, J= 5.1 Hz, 2H), 6.75-6.81 (m, 2H), 7.10-7.41 (m, 6H).13C NMR (100 MHz, CBCh) 8 12.9, 46.0, 53.0, 127.1, 128.2, 128.7, 128.9, 129.2, 139.8, 144.9, 162.4, 180.3, 188.1. HRMS (ESI): calcd for C15H18N3OS2 [M+H]+, 320.0891; found, 320.0887.
[0331] 7V-Ethyl-7V-phenyl-7V-((5-phenylfuran-2-yl)methyl)-thioimidodicarbonic diamide (7) Yield 77%. ’H NMR (400 MHz, CDCI3) 8 1.25 (t, J= 7.3 Hz, 3H), 3.59 (q, J= 7.3 Hz, 2H), 4.27 (d, J= 5.4 Hz, 2H), 6.46-6.48 (m, 1H), 6.73-6.84 (m, 2H), 7.15-7.23 (m, 2H), 7.25-7.39 (m, 3H), 7.45-7.65 (m, 4H).13C NMR (100 MHz, CBCh) 8 12.9, 46.0, 53.0, 119.4, 120.5, 127.1, 128.2, 128.7, 128.9, 129.0, 129.2, 139.8, 144.9, 152.0, 162.4, 176.9,
188.1. HRMS (ESI): calcd for C21H22N3OS2 [M+H]+, 396.1204; found, 396.1208.
General procedure for the preparation of compounds la-m, IIa,b and III:
[0332] To a solution of thioimidodicarbonic diamide derivative (1.00 mmol) in absolute ethanol (10 mL), bromine liquid (2.00 mmol) was added dropwise at 0 °C. The reaction mixture was warmed to room temperature and kept stirring for 6 hrs. The crude mixture was evaporated under vacuum and purified by Teledyne CombiFlash Rf200 Flash Chromatography System (C1s silica/reversed phase) using acetonitrile-water gradient.
[0333] 3-((Furan-2-ylmethyl)imino)-/V,/V-dimethyl-3Z7-l,2,4-dithiazol-5-amine hydrogen bromide (la) Yield 55%. ’H NMR (400 MHz, CB3OB) 83.37 (s, 3H), 3.54 (s, 3H), 4.45 (d, J= 5.2 Hz, 2H), 6.40-6.49 (m, 2H), 7.48-7.50 (m, 1H).13C NMR (100 MHz, CB3OB) 840.9,
42.1, 43.0, 108.9, 110.3, 143.1, 148.6, 177.7, 179.9. HRMS (ESI): calcd for C9HI2N3OS2 [M+H]+, 242.0421; found, 242.0425. [0334] 7V^V-Dimethyl-3-((thiophen-2-ylmethyl)imino)-3Z/-l,2,4-dithiazol-5-amine hydrogen bromide (lb) Yield 61%. ’H NMR (400 MHz, DMSO-^) 63.23 (s, 3H), 3.27 (s, 3H), 4.96 (d, J= 5.5 Hz, 2H), 6.96 (ddd, J= 4.9, 3.5, 1.2 Hz, 1H), 7.09 (dd, J= 3.4, 1.5 Hz, 1H), 7.41 (dt, J= 5.2, 1.3 Hz, 1H).13C NMR (100 MHz, DMSO-A) 542.1, 43.1, 43.5, 126.2, 127.0, 127.1, 140.1, 177.3, 179.7. HRMS (ESI): calcd for C9H12N3S3 [M+H]+, 258.0193; found, 258.0191.
[0335] 3-(((l/7-Pyrrol-2-y I) methyl)i mi no)-/V,/V-dimethy 1-377-1,2, 4-dithiazol-5-amine hydrogen bromide (Ic) Yield 59%. ’H NMR (400 MHz, DMSO-^) 6 3.21 (s, 3H), 3.24 (s, 3H), 4.65 (d, J= 4.8 Hz, 2H), 5.95 (t, J= 2.8 Hz, 1H), 5.98-6.09 (m, 1H), 6.61-6.72 (m, 1H), 9.72 (s, 1H).13C NMR (100 MHz, DMSO-A) 543.1, 45.7, 46.4, 107.3, 107.9, 118.1, 126.7, 177.0, 179.0. HRMS (ESI): calcd for C9H13N4S2 [M+H]+, 241.0581; found, 241.0576.
[0336] /V,/V-Dimethyl-3-(((tetrahydrofuran-2-yl)methyl)imino)-377-l,2,4-dithiazol-5- amine hydrogen bromide (Id) Yield 51%. ’H NMR (400 MHz, DMSO-^) 5 1.24-1.65 (m, 1H), 1.67-2.13 (m, 3H), 3.22 (s, 3H), 3.26 (s, 3H), 3.40-3.85 (m, 4H), 3.89-4.09 (m, 1H).13C NMR (100 MHz, DMSO-Jo) 525.8, 28.8, 41.9, 43.6, 49.3, 67.9, 76.4, 176.8, 179.8. HRMS (ESI): calcd for C9H16N3OS2 [M+H]+, 246.0734; found, 246.0735.
[0337] 7V^V-Dimethyl-3-(((5-phenylfuran-2-yl)methyl)imino)-3Zf-l,2,4-dithiazol-5- amine hydrogen bromide (le) Yield 48%. ’H NMR (400 MHz, DMSO-^) 5 3.17 (s, 3H), 3.29 (s, 3H), 4.52 (s, 2H), 6.50 (dd, J= 3.4, 1.3 Hz, 1H), 6.86 (dd, J= 3.4, 1.3 Hz, 1H), 7.22- 7.38 (m, 3H), 7.56-7.67 (m, 2H).13C NMR (100 MHz, DMSO-A) 543.0, 44.1, 44.9, 107.3, 111.1, 123.6, 127.9, 129.3, 130.5, 150.5, 153.0, 168.0, 176.7. HRMS (ESI): calcd for C15H16N3OS2 [M+H]+, 318.0734; found, 318.0737.
[0338] 3-(((5-(4-Fluorophenyl)furan-2-yl)methyl)imino)-/V,/V-dimethyl-377-l,2,4- dithiazol-5-amine hydrogen bromide (If) Yield 58%. H NMR (400 MHz, DMSO-Ju) 5 3.19 (s, 3H), 3.29 (s, 3H), 4.51 (s, 2H), 6.49 (dd, J= 3.4, 1.8 Hz, 1H), 6.83 (t, J= 2.6 Hz, 1H), 7.15-7.26 (m, 2H), 7.59-7.71 (m, 2H).13C NMR (100 MHz, DMSO-A) 543.5, 44.4, 45.7, 107.1, 111.1, 116.4, 125.7, 127.2, 150.5, 152.2, 160.6, 168.0, 176.7. HRMS (ESI): calcd for C15H15FN3OS2 [M+H]+, 336.0640; found, 336.0637.
[0339] 3-((4-Chlorophcnyl)imino)-/V,/V-dimcthyl-377-l,2,4-dithiazol-5-aminc hydrogen bromide (Ig) Yield 71%. ’H NMR (400 MHz, DMSO-Jo) 5 3.25 (s, 3H), 3.53 (s, 3H), 6.95 (d, J= 7.9 Hz, 2H), 7.51 (d, J= 7.9 Hz, 2H).13C NMR (100 MHz, DMSO-Js) 841.8, 42.6, 123.5, 128.8, 130.0, 144.2, 158.9, 174.0. HRMS (ESI): calcd for C10H11CIN3S2 [M+H]+, 272.0082; found, 272.0083.
[0340] 3-((4-Methoxyphenyl)imino)-7V^V-dimethyl-3Zf-l,2,4-dithiazol-5-amine hydrogen bromide (Ih) Yield 66%. ’H NMR (400 MHz, DMSO-^) 63.25 (s, 3H), 3.28 (s, 3H), 3.68 (s, 3H), 6.96 (d, J= 8.1 Hz, 2H), 7.49 (d, J= 8.1 Hz, 2H).13C NMR (100 MHz, DMSO-Jo) 542.1, 43.3, 55.7, 114.2, 115.2, 122.4, 144.3, 166.9, 174.6. HRMS (ESI): calcd for C11H14N3OS2 [M+H]+, 268.0578; found, 268.0573.
[0341] 3-(Benzylimino)-7V^V-dimethyl-3Zf-l,2,4-dithiazol-5-amine hydrogen bromide (li) Yield 62%. ’H NMR (400 MHz, DMSO-Jo) 53.16 (s, 3H), 3.30 (s, 3H), 4.72 (s, 2H), 7.15-7.42 (m, 5H).13C NMR (100 MHz, DMSO-A) 545.0, 45.7, 57.0, 124.4, 125.2, 130.2,
136.7, 165.4, 172.2. HRMS (ESI): calcd for C11H14N3S2 [M+H]+, 252.0629; found, 252.0633.
[0342] 3-((4-Chlorobenzyl)imino)-7V^V-dimethyl-3Zf-l,2,4-dithiazol-5-amine hydrogen bromide (Ij) Yield 78%. ’H NMR (400 MHz, DMSO-Jo) 53.29 (s, 3H), 3.32 (s, 3H), 4.71 (s, 2H) 7.23(d, J= 7.2 Hz, 2H), 7.96 (d, J= 12 Hz, 2H).13C NMR (100 MHz, DMSO-Jo) 5 45.8, 46.9, 53.5, 128.9, 129.8, 132.3, 136.5, 159.8, 178.7. HRMS (ESI): calcd for C11H13CIN3S2 [M+H]+, 286.0239; found, 286.0238.
[0343] 3-((4-Fluorobcnzyl)imino)-/V,/V-dimcthyl-3Z7-l,2,4-dithiazol-5-aminc hydrogen bromide (Ik) Yield 81%. ’H NMR (400 MHz, DMSO-Jo) 5 3.25 (s, 3H), 3.31 (s, 3H), 4.71 (s, 2H), 7.09-7.21 (m, 2H), 7.33-7.46 (m, 2H).13C NMR (100 MHz, DMSO-A) 542.1, 43.7, 52.2, 116.5, 120.2, 130.2, 133.3, 171.5, 177.2. HRMS (ESI): calcd for C11H13FN3S2 [M+H]+, 270.0534; found, 270.0537.
[0344] 3-((4-Methoxybenzyl)imino)-7V^V-dimethyl-3Zf-l,2,4-dithiazol-5-amine hydrogen bromide (II) Yield 85%. ’H NMR (400 MHz, DMSO-56) 53.22 (s, 3H), 3.25 (s, 3H), 3.72 (s, 3H), 4.71 (d, J= 5.3 Hz, 2H), 6.91 (d, J= 8.1 Hz, 2H), 7.28 (d, J= 8.1 Hz, 2H).13C NMR (100 MHz, DMSO-Js) 544.5, 45.4, 48.3, 53.5, 114.3, 129.5, 129.6, 159.0, 177.1, 179.7. HRMS (ESI): calcd for C12H16N3OS2 [M+H]+, 282.0734; found, 282.0735.
[0345] 3-(([l,l ’-Biphenyl] -4-ylmethyl)imino)-7V^V-dimethyl-3Zf-l ,2,4-dithiazol-5-amine hydrogen bromide (Im) Yield 48%. ’H NMR (400 MHz, DMSO-^) 63.23 (s, 3H), 3.26 (s, 3H), 4.88 (s, 2H), 7.28-7.49 (m, 4H), 7.56-7.72 (m, 5H).13C NMR (100 MHz, DMSO-A) 6
42.7, 43.8, 48.4, 127.0, 127.2, 127.8, 128.6, 129.3, 137.1, 139.5, 140.2, 177.2, 180.2. HRMS (ESI): calcd for C17H18N3S2 [M+H]+, 328.0942; found, 328.0939. [0346] 7V-(4-Chlorophenyl)-3-((furan-2-ylmethyl)imino)-7V-methyl-3Zf-l,2,4-dithiazol- 5-amine hydrogen bromide (Ila) Yield 76%. ’H NMR (400 MHz, DMSO-^) 63.16 (s, 3H), 4.17 (d, J= 5.7 Hz, 2H), 6.10-6.14 (m, 1H), 6.34 (dt, J= 3.3, 1.6 Hz, 1H), 6.67 (t, J= 5.8 Hz, 1H), 7.27 (d, J= 8.6 Hz, 2H), 7.72 (d, J= 8.6 Hz, 2H).13C NMR (100 MHz, DMSO- J6) 542.8, 49.3, 106.5, 110.8, 128.5, 129.4, 129.8, 129.9, 143.4, 154.1, 156.8, 182.6. HRMS (ESI): calcd for C14H13CIN3OS2 [M+H]+, 338.0188; found, 338.0191.
[0347] 7V-Ethyl-3-((furan-2-ylmethyl)imino)-7V-phenyl-3Zf-l,2,4-dithiazol-5-amine hydrogen bromide (lib) Yield 63%. ’H NMR (400 MHz, DMSO-^) 6 1.11 (t, J= 7.3 Hz, 3H), 3.51 (q, J= 7.3 Hz, 2H), 4.17 (d, J= 5.5 Hz, 2H), 6.79-6.91 (m, 2H), 7.17-7.47 (m, 6H).13C NMR (100 MHz, DMSO-Js) 8 13.1, 46.0, 52.3, 127.4, 128.9, 129.1, 129.6, 129.8,
139.9, 144.6, 154.6, 161.3, 187.7. HRMS (ESI): calcd for C15H16N3OS2 [M+H]+, 318.0734; found, 318.0737.
[0348] 7V-Ethyl-7V-phenyl-3-(((5-phenylfuran-2-yl)methyl)imino)-3Zf-l,2,4-dithiazol-5- amine hydrogen bromide (III) Yield 73%. ’H NMR (400 MHz, DMSO-^) 6 1.11 (t, J = 7.2 Hz, 3H), 3.48 (q, J= 7.2 Hz, 2H), 4.19 (d, J= 5.3 Hz, 2H), 6.17-6.21 (m, 1H), 6.72-6.79 (m, 2H), 6.98-7.21 (m, 4H), 7.24-7.58 (m, 5H).13C NMR (100 MHz, DMSO-A) 8 13.1, 46.0,
52.9, 125.8, 127.4, 128.0, 128.5, 128.9, 129.1, 129.6, 129.8, 140.0, 144.6, 151.7, 161.3,
166.9, 187.7. HRMS (ESI): calcd for C21H20N3OS2 [M+H]+, 394.1047; found, 394.1049.

Claims

WHAT IS CLAIMED:
1. A compound having a structure of
Figure imgf000118_0001
(I), or a pharmaceutically acceptable salt thereof, wherein:
L1 is a bond, -NH-, -O-, -S-, -C(O)-, -C(O)NH-, -NHC(O)-, -NHC(O)NH-, -C(O)O-, -OC(O)-, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene;
R1 is hydrogen, halogen, -CXS, -CHX'2, -CH2X1, -OCXS, -
OCH2X1, -OCHXS, -CN, -SOniR1D, -SOVINR1AR1B, -NR1CNR1AR1B, -ONR1AR1B, -NHC(O)NR1CNR1AR1B, -NHC(O)NR1AR1B, -N(O)mi, -NR1AR1B, -C(O)R1C, -C(O)-OR1C , -C(O)NR1AR1B, -OR1D, -NR1ASO2R1D, -NR1AC(O)R1C, -NR1AC(O)OR1C, -NR1AOR1C, - N3, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl;
R2 is hydrogen, halogen, -CX2 3, -CHX2 2, -CH2X2, -OCX2 3, -
OCH2X2, -OCHX22, -CN, -SOn2R2D, -SOV2NR2AR2B, -NR2CNR2AR2B, -ONR2AR2B, -NHC(O)NR2CNR2AR2B, -NHC(O)NR2AR2B, -N(O)m2, -NR2AR2B, -C(O)R2C, -C(O)-OR2C , -C(O)NR2AR2B, -OR2D, -NR2ASO2R2D, -NR2AC(O)R2C, -NR2AC(O)OR2C, -NR2AOR2C, - N3, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl;
R1 and R2 may optionally be joined to form a substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl;
R4 is indendently halogen, -CX4 3, -CHX4 2, -CH2X4, -OCX4 3, - OCH2X4, -OCHX4 2, -CN, -SOn4R4D, -SOV4NR4AR4B, -NR4CNR4AR4B, -ONR4AR4B, -NHC(O)NR4CNR4AR4B, -NHC(O)NR4AR4B, -N(O)m4, -NR4AR4B, -C(O)R4C, -C(O)-OR4C , -C(O)NR4AR4B, -OR4D, -NR4ASO2R4D, -NR4AC(O)R4C, -NR4AC(O)OR4C, -NR4AOR4C, - N3, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl;
R1A, R1B, R1C, R1D, R2A, R2B, R2C, R2D, R4A, R4B, R4C, and R4D are independently hydrogen, -CX3, -CHX2, -CH2X, -CN, -OH, -COOH, -CONH2, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; R1A and R1B or R2A and R2B with substituents bonded to the same nitrogen atom may optionally be joined to form a substituted or unsubstituted heterocycloalkyl or substituted or unsubstituted heteroaryl; each X, X1, X2, and X4 is independently -F, -Cl, -Br, or -I; each nl, n2 and n4 is independently an integer from 0 to 4; z is an integer from 0 to 5; and each ml, m2, m4, vl, v2 and v4 are independently 1 or 2.
2. The compound of claim 1 , wherein L1 is a bond, substituted or unsubstituted alkylene, or substituted or unsubstituted heteroalkylene.
3. The compound of claim 2, wherein L1 is substituted or unsubstituted C1-C3 alkylene, or substituted or unsubstituted 2 to 3 membered heteroalkylene.
4. The compound of claim 1, wherein the compound has a structure of
Figure imgf000120_0001
5. The compound of claim 1 , wherein the compound has a structure of
Figure imgf000120_0002
6. The compound of claim 1 , wherein each R1 and R2 is independently substituted or unsubstituted alkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl.
7. The compound of claim 6, wherein each R1 and R2 is independently substituted or unsubstituted C1-C4 alkyl, or substituted or unsubstituted phenyl.
8. The compound of claim 7, wherein each R1 and R2 is independently unsubstituted C1-C4 alkyl, or unsubstituted phenyl.
9. The compound of claim 8, wherein R1 is unsubstituted phenyl, and R2 is unsubstituted C1-C4 alkyl.
10. The compound of claim 1 , wherein z is 0.
11. The compound of claim 1 , wherein z is 1 and R4 is halogen.
118
12. The compound of claim 1 , wherein the compound is
Figure imgf000121_0001
13. A pharmaceutical composition comprising a compound of claim 1, or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.
14. A method of treating a cancer in a subject in need thereof, the method comprising administrating an effective amount of the compound of claim 1, or a pharmaceutically acceptable salt thereof.
15. The method of claim 14, wherein the cancer is a solid tumor.
16. The method of claim 15, wherein the solid tumor is in thyroid, endocrine system, brain, breast, cervix, colon, prostate, head and neck, liver, kidney, lung, ovary, pancreas, rectum, stomach, oral cavity, or uterus.
17. A method of inhibiting V-domain Ig Suppressor of T-cell Activation (VISTA) in a cell, the method comprising contacting the cell with an effective amount of the compound of claim 1, or a pharmaceutically acceptable salt thereof.
18. A method of enhancing T-cell proliferation in a subject in the presence of VISTA- expressing cancer cells, the method comprising administrating the subject an effective amount of the compound of claim 1, or a pharmaceutically acceptable salt thereof.
119
19. A method of restoring T-cell activation in a subject in the presence of VISTA- expressing cancer cells, the method comprising administrating the subject an effective amount of the compound of any one of claim 1, or a pharmaceutically acceptable salt thereof.
120
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011053468A1 (en) * 2009-10-30 2011-05-05 Sanofi-Aventis Amino-benzoic acid derivatives for use in the treatment of dihydrogenase-related disorders
WO2018057989A1 (en) * 2016-09-24 2018-03-29 Washington University INHIBITORS OF SARM1 NADase ACTIVITY AND USES THEREOF

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011053468A1 (en) * 2009-10-30 2011-05-05 Sanofi-Aventis Amino-benzoic acid derivatives for use in the treatment of dihydrogenase-related disorders
WO2018057989A1 (en) * 2016-09-24 2018-03-29 Washington University INHIBITORS OF SARM1 NADase ACTIVITY AND USES THEREOF

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
KHARATMAL SHIVSHARAN, SINGH JITENDRA, SHARMA SHYAM: "Voltage-Gated Sodium Channels as Therapeutic Targets for Treatment of Painful Diabetic Neuropathy", MINI-REVIEWS IN MEDICINAL CHEMISTRY, vol. 15, no. 14, 2015, pages 1134 - 1147, XP055917000 *

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