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WO2025085756A1 - Composés macrocycliques et méthodes de traitement - Google Patents

Composés macrocycliques et méthodes de traitement Download PDF

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Publication number
WO2025085756A1
WO2025085756A1 PCT/US2024/051984 US2024051984W WO2025085756A1 WO 2025085756 A1 WO2025085756 A1 WO 2025085756A1 US 2024051984 W US2024051984 W US 2024051984W WO 2025085756 A1 WO2025085756 A1 WO 2025085756A1
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Prior art keywords
tubulin
compound
subject
pharmaceutically acceptable
acceptable salt
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Hendrik Luesch
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University of Florida
University of Florida Research Foundation Inc
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University of Florida
University of Florida Research Foundation Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1021—Tetrapeptides with the first amino acid being acidic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • Microtubules consist of a/p-tubulin heterodimers that are assembled into longitudinal protofilaments, held together through lateral interactions. 1 They are critical and highly dynamic components of the cytoskeleton of eukaryotic cells, form mitotic spindles, and serve for intracellular trafficking, 2 but also regulate inflammatory and oncogenic signaling. 3,4 Modulation of tubulin dynamics, either stabilizing or destabilizing microtubules, has been highly rewarding for drug discovery. Specifically, tubulin is a validated target for anticancer therapeutic discovery. 5 Seven druggable sites targeted by natural products have been identified, three of which already led to FDA-approved cancer drugs, including the maytansine site, vinca site, and the taxane site. 5,6 The natural product ligand targeting the colchicine site is approved for the prevention and treatment of attacks of gout. There is a demand for tubulin agents that exert their agents through distinct binding to tubulin at a- or P-tubulin or interfaces.
  • Marine cyanobacteria have previously yielded tubulin-targeting dolastatin 10, which provided the starting point for /four FDA-approved antibody-drug conjugates.
  • 7 The seventh pharmacological site targeted by gatorbulin-1 (GB1, la, FIG. 1A), a marine natural product from a Floridian cyanobacterium that destabilizes microtubule, was recently discovered presumably through a new mechanism.
  • 6 GB1 possesses pharmacological and structural novelty, distinguished by being a highly modified depsipeptide (FIG. 1A) without any standard proteinogenic amino acid.
  • the structure was validated by total synthesis, probed the mechanism of action, performed biochemical experiments to determine the inhibitory effects on tubulin polymerization and indirect effects on the adjacent colchicine site, and visualized the a/p-tubulin-GBl complex at high resolution by X-ray analysis (FIGs. IB and 1C), demonstrating the novelty on multiple levels. 6
  • the cellular activity was also assessed against breast and ovarian cancer cell lines, which are generally susceptible to microtubule targeting agents.
  • Gatorbulin-1 (GB1, la) is a recently described marine natural product that targets tubulin at a new, seventh pharmacological site at the tubulin intradimer interface.
  • first-generation gatorbulins GB2-7 (Ib-lg, FIG. 2), were synthesized based on the highly modified cyclodep sipep tide (GB1) that does not contain any proteinogenic amino acid.
  • the first-generation gatorbulins were systematically investigated the structure-activity relationship on the biochemical and cellular level using GB1- susceptible ovarian cancer cells. It was validated that the hydroxamate moiety in the N-mcthyl- alanine residue is important for activity.
  • GB 1 promotes proteasome-mediated tubulin degradation characteristic for the pharmacological targeting of the gatorbulin site.
  • Cevipabulin binds to the same tubulin region as GB 1 (la), although the binding mode is distinct and cevipabulin almost exclusively interacts with a- tubulin, including nonexchangeable GTP.
  • GB 1 (la) makes extensive contact and hydrogen bonds with both a- and //-chains of tubulin.
  • the present disclosure is directed towards macrocyclic compounds (z.e., any delineated herein), methods of modulating cell proliferation and/or tubulin polymerization, and methods of treating disease and disorders.
  • compounds of Formula (I): or pharmaceutically acceptable salt thereof wherein, R 1 , R 2 , R 3 , R 4 , and R 5 are defined herein, provided that the compound is not:
  • the compound of Formula (I) is of Formula (IA): or pharmaceutically acceptable salt thereof.
  • the compound of Formula (I) is of the formula:
  • compositions comprising a compound provided herein (e.g., Formula (I)), or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
  • kits comprising an effective amount of a compound provided herein (e.g., Formula (I)), or pharmaceutically acceptable salt thereof, in unit dosage form, together with instructions for administering the compound to a subject suffering from or susceptible to a cell proliferation disorder.
  • a compound provided herein e.g., Formula (I)
  • pharmaceutically acceptable salt thereof in unit dosage form, together with instructions for administering the compound to a subject suffering from or susceptible to a cell proliferation disorder.
  • a compound provided herein e.g., Formula (I)
  • the modulation is inhibition (i.e., inhibiting cell proliferation).
  • kits for modulating tubulin polymerization in a subject comprising contacting the subject with an effective amount of a compound provided herein (e.g., compound of Formula (I)), or pharmaceutically acceptable salt thereof, or composition described herein.
  • the modulation is inhibition (i.e., inhibiting tubulin polymerization).
  • a subject suffering from or susceptible to a cell proliferation related disorder or disease comprising administering to the subject an effective amount of a compound provided herein (e.g., Formula (I)), or pharmaceutically acceptable salt thereof, or pharmaceutical composition as provided herein.
  • disease or disorder is cancer.
  • kits for treating cancer or tumors comprising administering to a subject in need thereof, an effective amount of a compound provided herein (e.g., Formula (I)), or pharmaceutically acceptable salt thereof, or pharmaceutical composition as provided herein.
  • kits for modulating tubulin polymerization in a subject comprising administering to the subject with an effective amount of a compound provided here (e.g. Formula (I)), or pharmaceutically acceptable salt thereof, or pharmaceutical composition described herein.
  • kits for reducing protein levels in a subject comprising administering to the subject an effective amount of a compound provided here (e.g. Formula (I), GB1, GB2), or pharmaceutically acceptable salt thereof, or pharmaceutical composition described herein.
  • FIGs. 1A-1B shows gatorbulin-1 (GB1, la) structure and target interaction.
  • FIG. 1A shows the chemical structure of GB1
  • FIG. IB shows the crystal structure of a/p- tubulin in complex with GB1 from PDBID 7ALR 6 and shows GTP visible in the upper left region.
  • FIG 2 shows the chemical structures of gatorbulin-2 (GB2, lb) and newly designed simplified analogues GB3-7 (Ic-lg).
  • FIG. 3 shows the general retro synthetic analysis of GB2-7 (Ib-lg).
  • FIG. 4 shows the synthesis of GB4-7 (Id-lg).
  • FIGs. 5A-5F show the effect of gatorbulins on tubulin assembly and ovarian cancer cells proliferation.
  • FIG. 5A shows time course polymerization of 25 pM tubulin in GAB buffer, in the absence (gray line) or the presence of vehicle (DMSO, black line) or 27.5 pM of gatorbulins studied (blue gradient colors lines) or tubulin inhibitor control drug podophyllotoxin (red line);
  • FIG. 5B shows binding affinity of gartorbulins studied (blue gradient colors lines) compared to podophyllotoxin (red line, 1.5 x 107 M-l), measured from MTC displacement assays;
  • 5C-5D shows the percent cell viability normalized to vehicle control (0.5% DMSO) measured by MTT assay of HeLa cervical cells (FIG. 5C) and, SK-OV-3 ovarian cells (FIG. 5D) treated with increasing concentrations of gatorbulins for 48 h;
  • FIG. 5E shows Western blot analysis of a- and P-tubulin levels in SK-OV-3 ovarian cells after treatment with GB1 (la) at different concentrations for 16 h with (+) or without (-) pre-incubation with proteasome inhibitor (MG132, 10 pM final concentration).
  • FIG. 5F shows Tubulin quantification using densitometry with samples normalized to GAPDH loading control and further normalized to DMSO without MG 132.
  • FIG. 6 shows computationally modeled docked poses for GB1-6 (la-lf).
  • FIG. 7 shows the representative structures for the two clusters obtained from the simulated annealing calculations for GB 1.
  • FIGs. 8A-8D show computationally modeled comparisons between the crystal structure of tubulin complexed with cevipabulin (PDBID 7DP8) 9 and GB1(PDBID:7ALR). 6
  • the P2 chain is in light grey, a2 in dark grey, and cevipabulin in depicted in yellow carbons.
  • the P2 is orange, a2 in red, and GB1 with green carbons. In both cases, GTP is depicted with purple carbons.
  • FIG 8A shows the full tubulin protein with cevipabulin and GB1.
  • FIG. 8B shows a zoned in version of the site were cevipabulin and GB 1 complex to tubulin.
  • FIG. 8C shows a zoned in version in 7DP8 of the P2 chain is in light grey, a2 in dark grey, and cevipabulin in depicted in yellow carbons.
  • FIG. 8D shows a zoned in version in 7ALR of the P2 is orange, a2 in red, and GB1 with green carbons.
  • pharmaceutically acceptable salt refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, and the like, and are commensurate with a reasonable benefit/risk ratio.
  • Pharmaceutically acceptable salts are well known in the art. For example, Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, incorporated herein by reference.
  • Pharmaceutically acceptable salts of the compounds of this disclosure include those derived from suitable inorganic and organic acids and bases.
  • Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids, such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid or with organic acids, such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid or by using other methods known in the art such as ion exchange.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid
  • organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid or by using other methods known in the art such as ion exchange.
  • salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2- naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate
  • Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium, and N + (CI-4 alkyl)4 salts.
  • Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like.
  • Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate, and aryl sulfonate.
  • tautomers or “tautomeric” refers to two or more interconvertible compounds resulting from at least one formal migration of a hydrogen atom and at least one change in valency (e.g., a single bond to a double bond, a triple bond to a single bond, or vice versa).
  • the exact ratio of the tautomers depends on several factors, including temperature, solvent, and pH. Tautomerizations (z.e., the reaction providing a tautomeric pair) may catalyzed by acid or base.
  • Exemplary tautomerizations include keto-to-enol, amide-to-imide, lactam-to-lactim, enamine-to-imine, and enamine-to-(a different enamine) tautomerizations .
  • stereoisomers that are not mirror images of one another are termed “diastereomers” and those that are non- superimposable mirror images of each other are termed “enantiomers”.
  • enantiomers When a compound has an asymmetric center, for example, it is bonded to four different groups, a pair of enantiomers is possible.
  • An enantiomer can be characterized by the absolute configuration of its asymmetric center and is described by the R- and S- sequencing rules of Cahn and Prelog, or by the manner in which the molecule rotates the plane of polarized light and designated as dextrorotatory or levorotatory (z.e., as (+) or (-)- isomers respectively).
  • a chiral compound can exist as either individual enantiomer or as a mixture thereof. A mixture containing equal proportions of the enantiomers is called a “racemic mixture”.
  • composition and “formulation” are used interchangeably.
  • a “subject” to which administration is contemplated refers to a human (z.e., male or female of any age group, e.g., pediatric subject (e.g., infant, child, or adolescent) or adult subject (e.g., young adult, middle-aged adult, or senior adult)) or non-human animal.
  • the non-human animal is a mammal (e.g., primate (e.g., cynomolgus monkey or rhesus monkey), commercially relevant mammal (e.g., cattle, pig, horse, sheep, goat, cat, or dog), or bird (e.g., commercially relevant bird, such as chicken, duck, goose, or turkey)).
  • primate e.g., cynomolgus monkey or rhesus monkey
  • commercially relevant mammal e.g., cattle, pig, horse, sheep, goat, cat, or dog
  • bird e.g., commercially relevant bird, such as
  • the non-human animal is a fish, reptile, or amphibian.
  • the non-human animal may be a male or female at any stage of development.
  • the non- human animal may be a transgenic animal or genetically engineered animal.
  • patient refers to a human subject in need of treatment of a disease.
  • administer refers to implanting, absorbing, ingesting, injecting, inhaling, or otherwise introducing a compound described herein, or a composition thereof, in or on a subject.
  • treatment refers to reversing, alleviating, delaying the onset of, or inhibiting the progress of a disease described herein.
  • treatment may be administered after one or more signs or symptoms of the disease have developed or have been observed.
  • treatment may be administered in the absence of signs or symptoms of the disease.
  • treatment may be administered to a susceptible subject prior to the onset of symptoms (e.g., in light of a history of symptoms and/or in light of exposure to a pathogen). Treatment may also be continued after symptoms have resolved, for example, to delay or prevent recurrence.
  • an “effective amount” of a compound described herein refers to an amount sufficient to elicit the desired biological response.
  • An effective amount of a compound described herein may vary depending on such factors as the desired biological endpoint, severity of side effects, disease, or disorder, the identity, pharmacokinetics, and pharmacodynamics of the particular compound, the condition being treated, the mode, route, and desired or required frequency of administration, the species, age and health or general condition of the subject.
  • an effective amount is a therapeutically effective amount.
  • an effective amount is a prophylactic treatment.
  • an effective amount is the amount of a compound described herein in a single dose. In certain embodiments, an effective amount is the combined amounts of a compound described herein in multiple doses. In certain embodiments, the desired dosage is delivered three times a day, two times a day, once a day, every other day, every third day, every week, every two weeks, every three weeks, or every four weeks. In certain embodiments, the desired dosage is delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations).
  • an effective amount of a compound for administration one or more times a day to a 70 kg adult human comprises about 0.0001 mg to about 3000 mg, about 0.0001 mg to about 2000 mg, about 0.0001 mg to about 1000 mg, about 0.001 mg to about 1000 mg, about 0.01 mg to about 1000 mg, about 0.1 mg to about 1000 mg, about 1 mg to about 1000 mg, about 1 mg to about 100 mg, about 10 mg to about 1000 mg, or about 100 mg to about 1000 mg, of a compound per unit dosage form.
  • the compounds provided herein may be administered orally or parenterally at dosage levels sufficient to deliver from about 0.001 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 50 mg/kg, preferably from about 0.1 mg/kg to about 40 mg/kg, preferably from about 0.5 mg/kg to about 30 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, and more preferably from about 1 mg/kg to about 25 mg/kg, of subject body weight per day, one or more times a day, to obtain the desired therapeutic effect.
  • dose ranges as described herein provide guidance for the administration of provided pharmaceutical compositions to an adult.
  • the amount to be administered to, for example, a child or an adolescent can be determined by a medical practitioner or person skilled in the art and can be lower or the same as that administered to an adult.
  • a “therapeutically effective amount” of a compound described herein is an amount sufficient to provide a therapeutic benefit in the treatment of a condition or to delay or minimize one or more symptoms associated with the condition.
  • a therapeutically effective amount of a compound means an amount of therapeutic agent, alone or in combination with other therapies, which provides a therapeutic benefit in the treatment of the condition.
  • the term “therapeutically effective amount” can encompass an amount that improves overall therapy, reduces or avoids symptoms, signs, or causes of the condition, and/or enhances the therapeutic efficacy of another therapeutic agent.
  • a therapeutically effective amount is an amount sufficient for inhibiting cell proliferation.
  • a therapeutically effective amount is an amount sufficient for treating cancer.
  • a therapeutically effective amount is an amount sufficient for inhibiting cell proliferation and cancer. In certain embodiments, a therapeutically effective amount is an amount sufficient for treating a proliferative disease. In certain embodiments, a therapeutically effective amount is an amount sufficient for inhibiting cell proliferation and a proliferative disease. In certain embodiments, a therapeutically effective amount is an amount sufficient for inhibiting tubulin polymerization. In certain embodiments, a therapeutically effective amount is an amount sufficient for inhibiting tubulin polymerization and treating a proliferative disease. In certain embodiments, a therapeutically effective amount is an amount sufficient for inhibiting tubulin polymerization and treating cancer.
  • a “prophylactically effective amount” of a compound described herein is an amount sufficient to prevent a condition, or one or more symptoms associated with the condition or prevent its recurrence.
  • a prophylactically effective amount of a compound means an amount of a therapeutic agent, alone or in combination with other agents, which provides a prophylactic benefit in the prevention of the condition.
  • the term “prophylactically effective amount” can encompass an amount that improves overall prophylaxis or enhances the prophylactic efficacy of another prophylactic agent.
  • a prophylactically effective amount is an amount sufficient for inhibiting cell proliferation.
  • a prophylactically effective amount is an amount sufficient for treating cancer.
  • a prophylactically effective amount is an amount sufficient for inhibiting cell proliferation and cancer. In certain embodiments, a prophylactically effective amount is an amount sufficient for treating a proliferative disease. In certain embodiments, a prophylactically effective amount is an amount sufficient for inhibiting cell proliferation and a proliferative disease. In certain embodiments, a prophylactically effective amount is an amount sufficient for inhibiting tubulin polymerization. In certain embodiments, a prophylactically effective amount is an amount sufficient for inhibiting tubulin polymerization and treating a proliferative disease. In certain embodiments, a prophylactically effective amount is an amount sufficient for inhibiting tubulin polymerization and treating cancer.
  • the term “prevent,” “preventing,” or “prevention” refers to a prophylactic treatment of a subject who is not and was not with a disease but is at risk of developing the disease or who was with a disease, is not with the disease, but is at risk of regression of the disease. In certain embodiments, the subject is at a higher risk of developing the disease or at a higher risk of regression of the disease than an average healthy member of a population.
  • the term “inhibit” or “inhibition” in the context of proliferation for example, refers to a reduction in the proliferation of cells and/or tubulin polymerization.
  • the term refers to a reduction of the level of cell proliferation to a level that is statistically significantly lower than an initial level, which may, for example, be a baseline level of normal cells. In some embodiments, the term refers to a reduction of cell proliferation, to a level that is less than 75%, less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.1%, less than 0.01%, less than 0.001%, or less than 0.0001% of an initial level, which may, for example, be a baseline level of cell proliferation.
  • the term refers to a reduction of the level of tubulin polymerization to a level that is statistically significantly lower than an initial level, which may, for example, be a baseline level of tubulin polymerization. In some embodiments, the term refers to a reduction of tubulin polymerization, to a level that is less than 75%, less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.1%, less than 0.01%, less than 0.001%, or less than 0.0001% of an initial level, which may, for example, be a baseline level of tubulin polymerization.
  • modulate refers to increases or decreases in the activity of or in a cell in response to exposure to a compound or composition disclosed herein. In some embodiments, modulate refers to modulating tubulin polymerization. In some embodiments, modulate refers to decreasing the activity of inhibiting tubulin polymerization. In some embodiments, modulate refers to modulating cell proliferation. In some embodiments, modulate refers to decreasing cell proliferation.
  • isolated refers to material that is substantially or essentially free from components that normally accompany it as found in its native state. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. Particularly, in embodiments the compound is at least 85% pure, more preferably at least 90% pure, more preferably at least 95% pure, and most preferably at least 99% pure.
  • systemic administration means the administration of a compound(s), drug or other material, such that it enters the patient's system and, thus, is subject to metabolism and other like processes.
  • a proliferative disease refers to a disease that occurs due to abnormal growth or extension by the multiplication of cells (Walker, Cambridge Dictionary of Biology, Cambridge University Press: Cambridge, UK, 1990).
  • a proliferative disease may be associated with: 1) the pathological proliferation of normally quiescent cells; 2) the pathological migration of cells from their normal location (e.g., metastasis of neoplastic cells); 3) the pathological expression of proteolytic enzymes such as the matrix metalloproteinases e.g., collagenases, gelatinases, and elastases); or 4) the pathological angiogenesis as in proliferative retinopathy and tumor metastasis.
  • Exemplary proliferative diseases include cancers (z.e., “malignant neoplasms”), benign neoplasms, angiogenesis, inflammatory diseases, and autoimmune diseases.
  • angiogenesis refers to the physiological process through which new blood vessels form from pre-existing vessels.
  • Angiogenesis is distinct from vasculogenesis, which is the de novo formation of endothelial cells from mesoderm cell precursors. The first vessels in a developing embryo form through vasculogenesis, after which angiogenesis is responsible for most blood vessel growth during normal or abnormal development.
  • Angiogenesis is a vital process in growth and development, as well as in wound healing and in the formation of granulation tissue.
  • angiogenesis is also a fundamental step in the transition of tumors from a benign state to a malignant one, leading to the use of angiogenesis inhibitors in the treatment of cancer.
  • Angiogenesis may be chemically stimulated by angiogenic proteins, such as growth factors (e.g.. VEGF).
  • angiogenic proteins such as growth factors (e.g.. VEGF).
  • VEGF growth factors
  • “Pathological angiogenesis” refers to abnormal (e.g.. excessive or insufficient) angiogenesis that amounts to and/or is associated with a disease.
  • neoplasm and “tumor” are used herein interchangeably and refer to an abnormal mass of tissue wherein the growth of the mass surpasses and is not coordinated with the growth of a normal tissue.
  • a neoplasm or tumor may be “benign” or “malignant,” depending on the following characteristics: degree of cellular differentiation (including morphology and functionality), rate of growth, local invasion, and metastasis.
  • a “benign neoplasm” is generally well differentiated, has characteristically slower growth than a malignant neoplasm, and remains localized to the site of origin.
  • a benign neoplasm does not have the capacity to infiltrate, invade, or metastasize to distant sites.
  • Exemplary benign neoplasms include, but are not limited to, lipoma, chondroma, adenomas, acrochordon, senile angiomas, seborrheic keratoses, lentigos, and sebaceous hyperplasias.
  • certain “benign” tumors may later give rise to malignant neoplasms, which may result from additional genetic changes in a subpopulation of the tumor’s neoplastic cells, and these tumors are referred to as “pre-malignant neoplasms.”
  • An exemplary pre-malignant neoplasm is a teratoma.
  • a “malignant neoplasm” is generally poorly differentiated (anaplasia) and has characteristically rapid growth accompanied by progressive infiltration, invasion, and destruction of the surrounding tissue. Furthermore, a malignant neoplasm generally has the capacity to metastasize to distant sites.
  • the term “metastasis,” “metastatic,” or “metastasize” refers to the spread or migration of cancerous cells from a primary or original tumor to another organ or tissue and is typically identifiable by the presence of a “secondary tumor” or “secondary cell mass” of the tissue type of the primary or original tumor and not of that of the organ or tissue in which the secondary (metastatic) tumor is located.
  • a prostate cancer that has migrated to bone is said to be metastasized prostate cancer and includes cancerous prostate cancer cells growing in bone tissue.
  • cancer refers to a class of diseases characterized by the development of abnormal cells that proliferate uncontrollably and have the ability to infiltrate and destroy normal body tissues. See e.g., Stedman’s Medical Dictionary, 25th ed.; Hensyl ed.;
  • Exemplary cancers include, but are not limited to, acoustic neuroma; adenocarcinoma; adrenal gland cancer; anal cancer; angiosarcoma (e.g., lymphangiosarcoma, lymphangioendotheliosarcoma, hemangiosarcoma); appendix cancer; benign monoclonal gammopathy; biliary cancer (e.g., cholangiocarcinoma); bladder cancer; breast cancer (e.g., adenocarcinoma of the breast, papillary carcinoma of the breast, mammary cancer, medullary carcinoma of the breast); brain cancer (e.g., meningioma, glioblastomas, glioma (e.g., astrocytoma, oligodendroglioma), medulloblastoma); bronchus cancer; carcinoid tumor; cervical cancer (e.g., cervical adenocarcinoma; adrenal gland cancer; an
  • Wilms tumor, renal cell carcinoma); liver cancer (e.g., hepatocellular cancer (HCC), malignant hepatoma); lung cancer (e.g., bronchogenic carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung); leiomyosarcoma (LMS); mastocytosis (e.g., systemic mastocytosis); muscle cancer; myelodysplastic syndrome (MDS); mesothelioma; myeloproliferative disorder (MPD) (e.g., polycythemia vera (PV), essential thrombocytosis (ET), agnogenic myeloid metaplasia (AMM) a.k.a.
  • HCC hepatocellular cancer
  • lung cancer e.g., bronchogenic carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung
  • myelofibrosis MF
  • chronic idiopathic myelofibrosis chronic myelocytic leukemia (CML), chronic neutrophilic leukemia (CNL), hypereosinophilic syndrome (HES)
  • neuroblastoma e.g., neurofibromatosis (NF) type 1 or type 2, schwannomatosis
  • neuroendocrine cancer e.g., gastroenteropancreatic neuroendoctrine tumor (GEP-NET), carcinoid tumor
  • osteosarcoma e.g., bone cancer
  • ovarian cancer e.g., cystadenocarcinoma, ovarian embryonal carcinoma, ovarian adenocarcinoma
  • papillary adenocarcinoma pancreatic cancer
  • pancreatic cancer e.g., pancreatic andenocarcinoma, intraductal papillary mucinous neoplasm (IPMN), Islet cell tumors
  • a compound, pharmaceutical composition, method, use, or kit When a compound, pharmaceutical composition, method, use, or kit is referred to as “selectively,” “specifically,” or “competitively” inhibiting a target, the compound, pharmaceutical composition, method, use, or kit inhibits the target to a greater extent (e.g., not less than 2-fold, not less than 5-fold, not less than 10-fold, not less than 30-fold, not less than 100-fold, not less than 1,000-fold, or not less than 10,000-fold; and/or: not more than 2-fold, not more than 5-fold, not more than 10-fold, not more than 30-fold, not more than 100-fold, not more than 1,000-fold, or not more than 10,000-fold) than inhibiting a different potential target.
  • a greater extent e.g., not less than 2-fold, not less than 5-fold, not less than 10-fold, not less than 30-fold, not less than 100-fold, not less than 1,000-fold, or not more than 10,000-fold
  • R 1 is -NH2, -OMe, -OH, -NH(nitrogen protecting group), or -O(oxygen protecting group);
  • R 2 is hydrogen, -OH, or -O(oxygen protecting group);
  • R 3 is hydrogen, -OH, or -O(oxygen protecting group);
  • a compound of Formula (I) is not of the formula: provided herein, a compound of Formula (I) is not of the formula: .
  • the compound of Formula (I) is of Formula (IA): or pharmaceutically acceptable salt thereof.
  • R 1 is -Nth, -OMe, -OH, -NH(nitrogen protecting group), or - O(oxygen protecting group).
  • R 1 is -NH2, -OMe, or -OH.
  • R 1 is -NH2.
  • R 1 is -OMe.
  • R 1 is -OH.
  • R 1 is -NH(nitrogen protecting group).
  • R 1 is -O(oxygen protecting group).
  • R 1 is -OTBS.
  • R 1 is - NH(Trt).
  • R 4 is hydrogen or methyl. In some embodiments, R 4 is hydrogen. In some embodiments, R 4 is methyl.
  • the compound of Formula (I) is of the formula: pharmaceutically acceptable salt thereof. In some embodiments, the compound of Formula (I) is of the formula: embodiments, the compound of Formula (I) is of the formula: pharmaceutically acceptable salt thereof.
  • Additional reaction schemes and protocols may be determined by the skilled artesian by use of commercially available structure-searchable database software, for instance, SciFinder® (CAS division of the American Chemical Society) and CrossFire Beilstein® (Elsevier MDL), or by appropriate keyword searching using an internet search engine such as Google® or keyword databases such as the US Patent and Trademark Office text database.
  • SciFinder® CAS division of the American Chemical Society
  • CrossFire Beilstein® Elsevier MDL
  • Compounds described herein can comprise one or more asymmetric centers, and thus can exist in various stereoisomeric forms, e.g., enantiomers and/or diastereomers.
  • the compounds described herein can be in the form of an individual enantiomer, diastereomer or geometric isomer, or can be in the form of a mixture of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomer.
  • Isomers can be isolated from mixtures by methods known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred isomers can be prepared by asymmetric syntheses.
  • HPLC high pressure liquid chromatography
  • the compounds herein may also contain linkages e.g., carbon-carbon bonds) wherein bond rotation is restricted about that particular linkage, e.g. restriction resulting from the presence of a ring or double bond. Accordingly, all cisltrans and EIZ isomers are expressly included in the present disclosure.
  • While compounds may be depicted as racemic or as one or more diastereoisomers, enantiomers, or other isomers, all such racemic diastereoisomer, enantiomer, or other isomer forms of that depicted are included in the present disclosure. All such isomeric forms of such compounds herein are expressly included in the present disclosure. All crystal forms and polymorphs of the compounds described herein are expressly included in the present disclosure. Also embodied are extracts and fractions comprising compounds of the disclosure. The term isomers is intended to include diastereoisomers, enantiomers, regioisomers, structural isomers, rotational isomers, tautomers, and the like. For compounds which contain one or more stereogenic centers, e.g., chiral compounds, the methods of the disclosure may be carried out with an enantiomerically enriched compound, a racemate, or a mixture of diastereomers.
  • Preferred enantiomerically enriched compounds have an enantiomeric excess of 50% or more, more preferably the compound has an enantiomeric excess of 60%, 70%, 80%, 90%, 95%, 98%, or 99% or more.
  • only one enantiomer or diastereomer of a chiral compound of the disclosure is administered to cells or a subject.
  • compositions comprising a compound provided herein (e.g. a compound of Formula (I)), or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable excipient.
  • the pharmaceutical composition described herein comprises a compound provided herein (e.g. a compound of Formula (I)), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.
  • the compound described herein is provided in an effective amount in the pharmaceutical composition.
  • the effective amount is a therapeutically effective amount.
  • the effective amount is a prophylactically effective amount.
  • the effective amount is an amount effective for treating a proliferative disease in a subject in need thereof.
  • the effective amount is an amount effective for preventing a proliferative disease in a subject in need thereof.
  • the effective amount is an amount effective for treating cancer in a subject in need thereof.
  • the effective amount is an amount effective for preventing cancer in a subject in need thereof.
  • the effective amount is an amount effective for reducing the risk of developing a disease (e.g., proliferative disease, (e.g., cancer)) in a subject in need thereof.
  • the subject is an animal.
  • the animal may be of either sex, and may be at any stage of development.
  • the subject described herein is a human.
  • the subject is a non-human animal.
  • the subject is a mammal.
  • the subject is a non-human mammal.
  • the subject is a domesticated animal, such as a dog, cat, cow, pig, horse, sheep, or goat.
  • the subject is a companion animal, such as a dog or cat.
  • the subject is a livestock animal, such as a cow, pig, horse, sheep, or goat.
  • the subject is a zoo animal.
  • the subject is a research animal, such as a rodent (e.g., mouse, rat), dog, pig, or non-human primate.
  • the animal is a genetically engineered animal.
  • the animal is a transgenic animal (e.g., transgenic mice and transgenic pigs).
  • the subject is a fish or reptile.
  • the cell is present in vitro. In certain embodiments, the cell is present in vivo.
  • the effective amount is an amount effective for inhibiting cell proliferation by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 98%.
  • the effective amount is an amount effective for inhibiting tubulin polymerization by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 98%.
  • compositions described herein can be prepared by any method known in the art of pharmaceutics. In general, such preparatory methods include bringing the compound described herein (z.e., the “active ingredient”) into association with a carrier or excipient, and/or one or more other accessory ingredients, and then, if necessary and/or desirable, shaping, and/or packaging the product into a desired single- or multi-dose unit.
  • Pharmaceutical compositions can be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses.
  • a “unit dose” is a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage, such as one-half or one-third of such a dosage.
  • compositions described herein will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered.
  • the composition may comprise between 0.1% and 100% (w/w) active ingredient.
  • compositions include inert diluents, dispersing and/or granulating agents, surface active agents and/or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and/or oils. Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and perfuming agents may also be present in the composition.
  • Exemplary diluents include calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, cornstarch, powdered sugar, and mixtures thereof.
  • Exemplary granulating and/or dispersing agents include potato starch, corn starch, tapioca starch, sodium starch glycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose, and wood products, natural sponge, cation-exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, crosslinked sodium carboxymethyl cellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (Veegum), sodium lauryl sulfate, quaternary ammonium compounds, and mixtures thereof.
  • cross-linked poly(vinyl-pyrrolidone) crospovidone
  • sodium carboxymethyl starch sodium starch glycolate
  • Exemplary surface active agents and/or emulsifiers include natural emulsifiers (e.g., acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g., bentonite (aluminum silicate) and Veegum (magnesium aluminum silicate)), long chain amino acid derivatives, high molecular weight alcohols (e.g., stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g., carboxy polymethylene, poly aery lie acid, acrylic acid polymer, and carboxy vinyl polymer), carrageenan, cell
  • Exemplary binding agents include starch (e.g., cornstarch and starch paste), gelatin, sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol, etc.), natural and synthetic gums (e.g., acacia, sodium alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage of isapol husks, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, microcrystalline cellulose, cellulose acetate, poly (vinyl-pyrrolidone), magnesium aluminum silicate (Veegum®), and larch arabogalactan), alginates, polyethylene oxide, polyethylene glycol, inorganic calcium salts, silicic acid, polymethacrylates, waxes, water, alcohol, and/
  • Exemplary preservatives include antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, antiprotozoan preservatives, alcohol preservatives, acidic preservatives, and other preservatives.
  • the preservative is an antioxidant.
  • the preservative is a chelating agent.
  • antioxidants include alpha tocopherol, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and sodium sulfite.
  • Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA) and salts and hydrates thereof (e.g., sodium edetate, disodium edetate, trisodium edetate, calcium disodium edetate, dipotassium edetate, and the like), citric acid and salts and hydrates thereof e.g., citric acid monohydrate), fumaric acid and salts and hydrates thereof, malic acid and salts and hydrates thereof, phosphoric acid and salts and hydrates thereof, and tartaric acid and salts and hydrates thereof.
  • EDTA ethylenediaminetetraacetic acid
  • salts and hydrates thereof e.g., sodium edetate, disodium edetate, trisodium edetate, calcium disodium edetate, dipotassium edetate, and the like
  • citric acid and salts and hydrates thereof e.g., citric acid mono
  • antimicrobial preservatives include benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and thimerosal.
  • Exemplary antifungal preservatives include butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and sorbic acid.
  • Exemplary alcohol preservatives include ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and phenylethyl alcohol.
  • Exemplary acidic preservatives include vitamin A, vitamin C, vitamin E, betacarotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and phytic acid.
  • preservatives include tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisol (BHA), butylated hydroxytoluened (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, Glydant® Plus, Phenonip®, methylparaben, Germall® 115, Germaben® II, NeoIone®, Kathon®, and Euxyl®.
  • Exemplary buffering agents include citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline
  • Exemplary lubricating agents include magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behanate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, and mixtures thereof.
  • Exemplary natural oils include almond, apricot kernel, avocado, babassu, bergamot, black current seed, borage, cade, camomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, com, cotton seed, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon, litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, sasquana, savoury,
  • Exemplary synthetic oils include, but are not limited to, butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone oil, and mixtures thereof.
  • Liquid dosage forms for oral and parenteral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.
  • the liquid dosage forms may comprise inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (e.g., cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydro furfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • inert diluents commonly used in the art such as, for example, water or other solvents, so
  • the oral compositions can include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.
  • adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.
  • the conjugates described herein are mixed with solubilizing agents such as Cremophor®, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and mixtures thereof.
  • Injectable preparations for example, sterile injectable aqueous or oleaginous suspensions can be formulated according to the known art using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation can be a sterile injectable solution, suspension, or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol.
  • the acceptable vehicles and solvents that can be employed are water, Ringer’s solution, U.S.P., and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil can be employed including synthetic mono- or di-glycerides.
  • fatty acids such as oleic acid are used in the preparation of injectables.
  • the injectable formulations can be sterilized, for example, by filtration through a bacterial -retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.
  • compositions for rectal or vaginal administration are typically suppositories which can be prepared by mixing the conjugates described herein with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol, or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active ingredient.
  • suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol, or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active ingredient.
  • Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules.
  • the active ingredient is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or (a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, (b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, (c) humectants such as glycerol, (d) disintegrating agents such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, (e) solution retarding agents such as paraffin, (f) absorption accelerators such as quaternary ammonium compounds, (g) wetting agents such as, for example, cetyl alcohol and g
  • Solid compositions of a similar type can be employed as fillers in soft and hard- filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.
  • the solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the art of pharmacology. They may optionally comprise opacifying agents and can be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner.
  • encapsulating compositions which can be used include polymeric substances and waxes.
  • Solid compositions of a similar type can be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.
  • the active ingredient can be in a micro-encapsulated form with one or more excipients as noted above.
  • the solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings, and other coatings well known in the pharmaceutical formulating art.
  • the active ingredient can be admixed with at least one inert diluent such as sucrose, lactose, or starch.
  • Such dosage forms may comprise, as is normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such a magnesium stearate and microcrystalline cellulose.
  • the dosage forms may comprise buffering agents. They may optionally comprise opacifying agents and can be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner.
  • encapsulating agents which can be used include polymeric substances and waxes.
  • Suitable devices for use in delivering intradermal pharmaceutical compositions described herein include short needle devices.
  • Intradermal compositions can be administered by devices which limit the effective penetration length of a needle into the skin.
  • conventional syringes can be used in the classical mantoux method of intradermal administration.
  • Jet injection devices which deliver liquid formulations to the dermis via a liquid jet injector and/or via a needle which pierces the stratum corneum and produces a jet which reaches the dermis are suitable.
  • Ballistic powder/particle delivery devices which use compressed gas to accelerate the compound in powder form through the outer layers of the skin to the dermis are suitable.
  • Formulations suitable for topical administration include, but are not limited to, liquid and/or semi-liquid preparations such as liniments, lotions, oil-in-water and/or water- in-oil emulsions such as creams, ointments, and/or pastes, and/or solutions and/or suspensions.
  • Topically administrable formulations may, for example, comprise from about 1% to about 10% (w/w) active ingredient, although the concentration of the active ingredient can be as high as the solubility limit of the active ingredient in the solvent.
  • Formulations for topical administration may further comprise one or more of the additional ingredients described herein.
  • a pharmaceutical composition described herein can be prepared, packaged, and/or sold in a formulation suitable for pulmonary administration via the buccal cavity.
  • a formulation may comprise dry particles which comprise the active ingredient and which have a diameter in the range from about 0.5 to about 7 nanometers, or from about 1 to about 6 nanometers.
  • Such compositions are conveniently in the form of dry powders for administration using a device comprising a dry powder reservoir to which a stream of propellant can be directed to disperse the powder and/or using a self-propelling solvent/powder dispensing container such as a device comprising the active ingredient dissolved and/or suspended in a low-boiling propellant in a sealed container.
  • Such powders comprise particles wherein at least 98% of the particles by weight have a diameter greater than 0.5 nanometers and at least 95% of the particles by number have a diameter less than 7 nanometers. Alternatively, at least 95% of the particles by weight have a diameter greater than 1 nanometer and at least 90% of the particles by number have a diameter less than 6 nanometers.
  • Dry powder compositions may include a solid fine powder diluent such as sugar and are conveniently provided in a unit dose form.
  • Low boiling propellants generally include liquid propellants having a boiling point of below 65 °F at atmospheric pressure.
  • the propellant may constitute 50 to 99.9% (w/w) of the composition, and the active ingredient may constitute 0.1 to 20% (w/w) of the composition.
  • the propellant may further comprise additional ingredients such as a liquid non-ionic and/or solid anionic surfactant and/or a solid diluent (which may have a particle size of the same order as particles comprising the active ingredient).
  • compositions described herein formulated for pulmonary delivery may provide the active ingredient in the form of droplets of a solution and/or suspension.
  • Such formulations can be prepared, packaged, and/or sold as aqueous and/or dilute alcoholic solutions and/or suspensions, optionally sterile, comprising the active ingredient, and may conveniently be administered using any nebulization and/or atomization device.
  • Such formulations may further comprise one or more additional ingredients including, but not limited to, a flavoring agent such as saccharin sodium, a volatile oil, a buffering agent, a surface active agent, and/or a preservative such as methylhydroxybenzoate.
  • the droplets provided by this route of administration may have an average diameter in the range from about 0.1 to about 200 nanometers.
  • Formulations described herein as being useful for pulmonary delivery are useful for intranasal delivery of a pharmaceutical composition described herein.
  • Another formulation suitable for intranasal administration is a coarse powder comprising the active ingredient and having an average particle from about 0.2 to 500 micrometers. Such a formulation is administered by rapid inhalation through the nasal passage from a container of the powder held close to the nares.
  • Formulations for nasal administration may, for example, comprise from about as little as 0.1% (w/w) to as much as 100% (w/w) of the active ingredient, and may comprise one or more of the additional ingredients described herein.
  • a pharmaceutical composition described herein can be prepared, packaged, and/or sold in a formulation for buccal administration.
  • Such formulations may, for example, be in the form of tablets and/or lozenges made using conventional methods, and may contain, for example, 0.1 to 20% (w/w) active ingredient, the balance comprising an orally dissolvable and/or degradable composition and, optionally, one or more of the additional ingredients described herein.
  • formulations for buccal administration may comprise a powder and/or an aerosolized and/or atomized solution and/or suspension comprising the active ingredient.
  • Such powdered, aerosolized, and/or aerosolized formulations when dispersed, may have an average particle and/or droplet size in the range from about 0.1 to about 200 nanometers, and may further comprise one or more of the additional ingredients described herein.
  • a pharmaceutical composition described herein can be prepared, packaged, and/or sold in a formulation for ophthalmic administration.
  • Such formulations may, for example, be in the form of eye drops including, for example, a 0.1-1.0% (w/w) solution and/or suspension of the active ingredient in an aqueous or oily liquid carrier or excipient.
  • Such drops may further comprise buffering agents, salts, and/or one or more other of the additional ingredients described herein.
  • Other ophthalmically-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form and/or in a liposomal preparation. Ear drops and/or eye drops are also contemplated as being within the scope of this disclosure.
  • compositions suitable for administration to humans are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and/or perform such modification with ordinary experimentation .
  • compositions described herein are typically formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the compositions described herein will be decided by a physician within the scope of sound medical judgment.
  • the specific therapeutically effective dose level for any particular subject or organism will depend upon a variety of factors including the disease being treated and the severity of the disorder; the activity of the specific active ingredient employed; the specific composition employed; the age, body weight, general health, sex, and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific active ingredient employed; the duration of the treatment; drugs used in combination or coincidental with the specific active ingredient employed; and like factors well known in the medical arts.
  • the compounds and compositions provided herein can be administered by any route, including enteral (e.g., oral), parenteral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, interdermal, rectal, intravaginal, intraperitoneal, topical (as by powders, ointments, creams, and/or drops), mucosal, nasal, bucal, sublingual; by intratracheal instillation, bronchial instillation, and/or inhalation; and/or as an oral spray, nasal spray, and/or aerosol.
  • enteral e.g., oral
  • parenteral intravenous, intramuscular, intra-arterial, intramedullary
  • intrathecal subcutaneous, intraventricular, transdermal, interdermal, rectal, intravaginal, intraperitoneal
  • topical as by powders, ointments, creams, and/or drops
  • mucosal nasal,
  • Specifically contemplated routes are oral administration, intravenous administration (e.g., systemic intravenous injection), regional administration via blood and/or lymph supply, and/or direct administration to an affected site.
  • intravenous administration e.g., systemic intravenous injection
  • regional administration via blood and/or lymph supply e.g., via blood and/or lymph supply
  • direct administration e.g., direct administration to an affected site.
  • the most appropriate route of administration will depend upon a variety of factors including the nature of the agent (e.g., its stability in the environment of the gastrointestinal tract), and/or the condition of the subject (e.g., whether the subject is able to tolerate oral administration).
  • the compound or pharmaceutical composition described herein is suitable for topical administration to the eye of a subject.
  • any two doses of the multiple doses include different or substantially the same amounts of a compound described herein.
  • the frequency of administering the multiple doses to the subject or applying the multiple doses to the tissue or cell is three doses a day, two doses a day, one dose a day, one dose every other day, one dose every third day, one dose every week, one dose every two weeks, one dose every three weeks, or one dose every four weeks.
  • the frequency of administering the multiple doses to the subject or applying the multiple doses to the tissue or cell is one dose per day. In certain embodiments, the frequency of administering the multiple doses to the subject or applying the multiple doses to the tissue or cell is two doses per day.
  • the frequency of administering the multiple doses to the subject or applying the multiple doses to the tissue or cell is three doses per day.
  • the duration between the first dose and last dose of the multiple doses is one day, two days, four days, one week, two weeks, three weeks, one month, two months, three months, four months, six months, nine months, one year, two years, three years, four years, five years, seven years, ten years, fifteen years, twenty years, or the lifetime of the subject, tissue, or cell.
  • the duration between the first dose and last dose of the multiple doses is three months, six months, or one year.
  • the duration between the first dose and last dose of the multiple doses is the lifetime of the subject, tissue, or cell.
  • a dose (e.g., a single dose, or any dose of multiple doses) described herein includes independently between 0.1 pg and 1 pg, between 0.001 mg and 0.01 mg, between 0.01 mg and 0.1 mg, between 0.1 mg and 1 mg, between 1 mg and 3 mg, between 3 mg and 10 mg, between 10 mg and 30 mg, between 30 mg and 100 mg, between 100 mg and 300 mg, between 300 mg and 1,000 mg, or between 1 g and 10 g, inclusive, of a compound described herein.
  • a dose described herein includes independently between 1 mg and 3 mg, inclusive, of a compound described herein. In certain embodiments, a dose described herein includes independently between 3 mg and 10 mg, inclusive, of a compound described herein. In certain embodiments, a dose described herein includes independently between 10 mg and 30 mg, inclusive, of a compound described herein. In certain embodiments, a dose described herein includes independently between 30 mg and 100 mg, inclusive, of a compound described herein.
  • Dose ranges as described herein provide guidance for the administration of provided pharmaceutical compositions to an adult.
  • the amount to be administered to, for example, a child or an adolescent can be determined by a medical practitioner or person skilled in the art and can be lower or the same as that administered to an adult.
  • a compound or composition, as described herein, can be administered in combination with one or more additional pharmaceutical agents (e.g., therapeutically and/or prophylactically active agents).
  • the compounds or compositions can be administered in combination with additional pharmaceutical agents that improve their activity (e.g., activity (e.g., potency and/or efficacy) in treating a disease in a subject in need thereof, in preventing a disease in a subject in need thereof, in reducing the risk to develop a disease in a subject in need thereof, and/or inhibiting tubulin polymerization and/or cell proliferation in a subject or cell), improve bioavailability, improve safety, reduce drug resistance, reduce and/or modify metabolism, inhibit excretion, and/or modify distribution in a subject or cell.
  • additional pharmaceutical agents e.g., therapeutically and/or prophylactically active agents.
  • additional pharmaceutical agents that improve their activity (e.g., activity (e.g., potency and/or efficacy) in treating a disease in a subject in need thereof,
  • a pharmaceutical composition described herein including a compound described herein and an additional pharmaceutical agent shows a synergistic effect that is absent in a pharmaceutical composition including one of the compound and the additional pharmaceutical agent, but not both.
  • the additional pharmaceutical agent achieves a desired effect for the same disorder.
  • the additional pharmaceutical agent achieves different effects.
  • the compound or composition can be administered concurrently with, prior to, or subsequent to one or more additional pharmaceutical agents, which may be useful as, e.g., combination therapies.
  • Pharmaceutical agents include therapeutically active agents.
  • Pharmaceutical agents also include prophylactically active agents.
  • Pharmaceutical agents include small organic molecules such as drug compounds (e.g., compounds approved for human or veterinary use by the U.S.
  • CFR Code of Federal Regulations
  • proteins proteins, carbohydrates, monosaccharides, oligosaccharides, polysaccharides, nucleoproteins, mucoproteins, lipoproteins, synthetic polypeptides or proteins, small molecules linked to proteins, glycoproteins, steroids, nucleic acids, DNAs, RNAs, nucleotides, nucleosides, oligonucleotides, antisense oligonucleotides, lipids, hormones, vitamins, and cells.
  • CFR Code of Federal Regulations
  • the additional pharmaceutical agents include, but are not limited to, antiproliferative agents, anti-cancer agents, anti-angiogenesis agents, steroidal or non-steroidal anti-inflammatory agents, immunosuppressants, anti-bacterial agents, anti-viral agents, cardiovascular agents, cholesterol-lowering agents, anti-diabetic agents, anti-allergic agents, contraceptive agents, pain-relieving agents, anesthetics, anti-coagulants, inhibitors of an enzyme, steroidal agents, steroidal or antihistamine, antigens, vaccines, antibodies, decongestant, sedatives, opioids, analgesics, anti-pyretic s, hormones, and prostaglandins.
  • the additional pharmaceutical agent is an anti-proliferative agent. In certain embodiments, the additional pharmaceutical agent is an anti-cancer agent. In certain embodiments, the additional pharmaceutical agent is an anti-viral agent. In certain embodiments, the additional pharmaceutical agent is a binder or inhibitor of a protein kinase.
  • the additional pharmaceutical agent is selected from the group consisting of epigenetic or transcriptional modulators (e.g., DNA methyltransferase inhibitors, histone deacetylase inhibitors (HD AC inhibitors), lysine methyltransferase inhibitors), antimitotic drugs (e.g., taxanes and vinca alkaloids), hormone receptor modulators (e.g., estrogen receptor modulators and androgen receptor modulators), cell signaling pathway inhibitors (e.g., tyrosine protein kinase inhibitors), modulators of protein stability (e.g., proteasome inhibitors), Hsp90 inhibitors, glucocorticoids, all-trans retinoic acids, and other agents that promote differentiation.
  • epigenetic or transcriptional modulators e.g., DNA methyltransferase inhibitors, histone deacetylase inhibitors (HD AC inhibitors), lysine methyltransferase inhibitors
  • antimitotic drugs e.g., taxanes and vinca
  • the compounds described herein or pharmaceutical compositions can be administered in combination with an anti-cancer therapy including, but not limited to, surgery, radiation therapy, transplantation (e.g., stem cell transplantation, bone marrow transplantation), immunotherapy, and chemotherapy.
  • an anti-cancer therapy including, but not limited to, surgery, radiation therapy, transplantation (e.g., stem cell transplantation, bone marrow transplantation), immunotherapy, and chemotherapy.
  • Additional pharmaceutical agents include small organic molecules such as drug compounds (e.g., compounds approved by the US Food and Drug Administration as provided in the Code of Federal Regulations (CFR)), peptides, proteins, carbohydrates, monosaccharides, oligosaccharides, polysaccharides, nucleoproteins, mucoproteins, lipoproteins, synthetic polypeptides or proteins, small molecules linked to proteins, glycoproteins, steroids, nucleic acids, DNAs, RNAs, nucleotides, nucleosides, oligonucleotides, antisense oligonucleotides, lipids, hormones, vitamins and cells.
  • drug compounds e.g., compounds approved by the US Food and Drug Administration as provided in the Code of Federal Regulations (CFR)
  • CFR Code of Federal Regulations
  • kits e.g., pharmaceutical packs
  • the kits provided may comprise a pharmaceutical composition or compound described herein and a container (e.g., a vial, ampule, bottle, syringe, and/or dispenser package, or other suitable container).
  • a container e.g., a vial, ampule, bottle, syringe, and/or dispenser package, or other suitable container.
  • provided kits may optionally further include a second container comprising a pharmaceutical excipient for dilution or suspension of a pharmaceutical composition or compound described herein.
  • the pharmaceutical composition or compound described herein provided in the first container and the second container are combined to form one unit dosage form.
  • kits including a first container comprising a compound or pharmaceutical composition described herein.
  • the kits are useful for treating a disease (e.g., proliferative disease, hematological disease, neurological disease, painful condition, psychiatric disorder, or metabolic disorder) in a subject in need thereof.
  • the kits are useful for preventing a disease (e.g., proliferative disease, hematological disease, neurological disease, painful condition, psychiatric disorder, or metabolic disorder) in a subject in need thereof.
  • kits are useful for reducing the risk of developing a disease (e.g., proliferative disease, hematological disease, neurological disease, painful condition, psychiatric disorder, or metabolic disorder) in a subject in need thereof.
  • a disease e.g., proliferative disease, hematological disease, neurological disease, painful condition, psychiatric disorder, or metabolic disorder
  • the kits are useful for inhibiting tubulin polymerization and/or cell proliferation in a subject or cell.
  • kits described herein further includes instructions for using the kit.
  • a kit described herein may also include information as required by a regulatory agency such as the U.S. Food and Drug Administration (FDA).
  • the information included in the kits is prescribing information.
  • the kits and instructions provide for treating a disease (e.g., proliferative disease, hematological disease, neurological disease, painful condition, psychiatric disorder, or metabolic disorder) in a subject in need thereof.
  • the kits and instructions provide for preventing a disease (e.g., proliferative disease, hematological disease, neurological disease, painful condition, psychiatric disorder, or metabolic disorder) in a subject in need thereof.
  • kits and instructions provide for reducing the risk of developing a disease (e.g., proliferative disease, hematological disease, neurological disease, painful condition, psychiatric disorder, or metabolic disorder) in a subject in need thereof.
  • a disease e.g., proliferative disease, hematological disease, neurological disease, painful condition, psychiatric disorder, or metabolic disorder
  • the kits and instructions provide for inhibiting tubulin polymerization and/or cell proliferation in a subject or cell.
  • a kit described herein may include one or more additional pharmaceutical agents described herein as a separate composition.
  • the cell is a cancer cell.
  • the cell is a tumor cell.
  • the modulation is inhibition (i.e., inhibiting cell proliferation).
  • methods of treating a subject suffering from or susceptible to a proliferative disease comprising administering to the subject an effective amount of a compound provided herein (e.g., Formula (I)), or pharmaceutically acceptable salt thereof, or composition described herein.
  • the disease or disorder is cancer.
  • the disease or disorder is a tumor, colon cancer, breast cancer, bone cancer, or brain cancer.
  • the disease or disorder is osteosarcoma, neuroblastoma, colon adenocarcinoma.
  • kits for cancer or tumors comprising administering to a subject in need thereof, an effective amount of a compound provided herein (e.g., Formula (I)), or pharmaceutically acceptable salt thereof, or composition described herein.
  • methods of modulating tubulin polymerization comprising contacting a sample with an effective amount of a compound provided herein (e.g., Formula (I)), or pharmaceutically acceptable salt thereof, or composition described herein.
  • the modulation is inhibition (i.e., inhibiting tubulin polymerization).
  • the tubulin is a-tubulin.
  • the tubulin is P-tubulin.
  • Also provided herein are methods of modulating tubulin polymerization in a cell comprising contacting a cell with an effective amount of a compound provided herein (e.g., Formula (I)), or pharmaceutically acceptable salt thereof, or composition described herein.
  • the modulation is inhibition (i.e., inhibiting tubulin polymerization).
  • the tubulin is a-tubulin.
  • the tubulin is P- tubulin.
  • tubulin polymerization in a subject, comprising administering to the subject an effective amount of a compound provided herein (e.g., Formula (I)), or pharmaceutically acceptable salt thereof, or composition described herein.
  • the modulation is inhibition (i.e., inhibiting tubulin polymerization).
  • the tubulin is a-tubulin.
  • the tubulin is P-tubulin.
  • the protein is tubulin.
  • the tubulin is a-tubulin.
  • the tubulin is P-tubulin.
  • a compound provided herein e.g., Formula (I)
  • the protein is tubulin.
  • the tubulin is a-tubulin.
  • the tubulin is P-tubulin.
  • the protein is tubulin.
  • the tubulin is a-tubulin.
  • the tubulin is P-tubulin.
  • methods of reducing protein levels comprising contacting a sample with an effective amount of GB 1 or GB2, or pharmaceutically acceptable salt thereof, or pharmaceutical composition comprising GB 1 or GB2, or pharmaceutically acceptable salt thereof.
  • the protein is tubulin.
  • the tubulin is a-tubulin. In some embodiments, the tubulin is P-tubulin.
  • methods of reducing protein levels in a cell comprising contacting a cell with an effective amount of GB 1 or GB2, or pharmaceutically acceptable salt thereof, or pharmaceutical composition comprising GB1 or GB2, or pharmaceutically acceptable salt thereof.
  • the protein is tubulin. In some embodiments, the tubulin is a-tubulin. In some embodiments, the tubulin is P-tubulin.
  • GB 1 or GB2 or pharmaceutically acceptable salt thereof, or pharmaceutical composition comprising GB 1 or GB2, or pharmaceutically acceptable salt thereof.
  • the compound provided herein (e.g., of Formula (I)) or composition provided herein selectively binds to tubulin. In some embodiments, the compound provided herein (e.g., of Formula (I)) or composition provided herein selectively binds to tubulin over P-glycoprotein 1 (P-gp).
  • P-gp P-glycoprotein 1
  • the compound provided herein (e.g., of Formula (I)) or composition provided herein selectively inhibits tubulin. In some embodiments, the compound provided herein (e.g., of Formula (I)) or composition provided herein selectively inhibits tubulin over P-glycoprotein 1 (P-gp).
  • P-glycoprotein 1 P-gp
  • the compound provided herein (e.g., of Formula (I)) or composition provided herein selectively inhibits tubulin polymerization. In some embodiments, the compound provided herein (e.g., of Formula (I)) or composition provided herein selectively inhibits tubulin polymerization over P-glycoprotein 1 (P-gp).
  • GB 1 or GB2, or pharmaceutically acceptable salt thereof, or composition comprising GB1 or GB2, or pharmaceutically acceptable salt thereof selectively binds to tubulin. In some embodiments, GB1 or GB2, or pharmaceutically acceptable salt thereof, or composition comprising GB 1 or GB2, or pharmaceutically acceptable salt thereof, selectively binds to tubulin over P-glycoprotein 1 (P-gp).
  • GB 1 or GB2, or pharmaceutically acceptable salt thereof, or composition comprising GB1 or GB2, or pharmaceutically acceptable salt thereof selectively inhibits tubulin.
  • GB1 or GB2, or pharmaceutically acceptable salt thereof, or composition comprising GB 1 or GB2, or pharmaceutically acceptable salt thereof selectively inhibits tubulin over P-glycoprotein 1 (P-gp).
  • GB 1 or GB2, or pharmaceutically acceptable salt thereof, or composition comprising GB1 or GB2, or pharmaceutically acceptable salt thereof selectively inhibits tubulin polymerization.
  • GB1 or GB2, or pharmaceutically acceptable salt thereof, or composition comprising GB 1 or GB2, or pharmaceutically acceptable salt thereof selectively inhibits tubulin polymerization over P-glycoprotein 1 (P-gp).
  • the compound herein e.g., of Formula (I), GB1, GB2 is selective for tubulin over P-glycoprotein 1 (P-gp).
  • the compound provided herein e.g., of Formula (I), GB1, GB2 does not engage P-glycoprotein 1 (P-gp).
  • the method is selective for tubulin over P-glycoprotein 1 (P- gP)-
  • the method does not engage P-glycoprotein 1 (P-gp).
  • the method selectively engages tubulin. In some embodiments, the method selectively engages tubulin over P-glycoprotein 1 (P-gp).
  • P-gp P-glycoprotein 1
  • the method selectively inhibits tubulin. In some embodiments, the method selectively inhibits tubulin over P-glycoprotein 1 (P-gp). [0151] In some embodiments, the method inhibits tubulin polymerization. In some embodiments, the method selectively inhibits tubulin polymerization over P-glycoprotein 1 (P-gp)-
  • the method is selective for tubulin over P-glycoprotein 1 (P- gP)-
  • the method does not engage P-glycoprotein 1 (P-gp).
  • P-gp P-glycoprotein 1
  • methods of modulating tubulin polymerization in a subject comprising administering to the subject an effective amount of GB 1 or GB2, or pharmaceutically acceptable salt thereof, or pharmaceutical composition comprising GB 1 or GB2, or pharmaceutically acceptable salt thereof, wherein the method is selective for tubulin over P-glycoprotein 1 (P-gp).
  • the subject of the methods and uses is a mammal.
  • the mammal is a primate or human.
  • the effective amount of a compound provided herein (e.g., Formula (I)), or pharmaceutically acceptable salt thereof, or composition described herein is about 0.005 pg/kg to about 200 mg/kg. In some embodiments, the effective amount ranges from about 0.1 mg/kg to about 200 mg/kg. In certain embodiments, the effective amount ranges from about 10 mg/kg to 100 mg/kg.
  • the effective amount of a compound provided herein (e.g., Formula (I)), or pharmaceutically acceptable salt thereof, or composition described herein is in the range from about 1.0 pM to about 500 pM.
  • a compound provided herein e.g., Formula (I)
  • pharmaceutically acceptable salt thereof, or composition described herein is administered intravenously, intramuscularly, subcutaneously, intracerebroventricularly, orally or topically.
  • a compound provided herein (e.g., Formula (I)), or pharmaceutically acceptable salt thereof, or composition described herein is administered alone or in combination with one or more additional therapeutic agents.
  • the additional therapeutic agent is an anti-cancer agent, chemotherapeutic agent, an antiangiogenesis agent, cytotoxic agent, or an anti-proliferation agent.
  • the additional therapeutic agent is a tubulin-interactive anticancer agent.
  • the disclosure provides a method of modulating cell proliferation activity in a subject, comprising contacting the subject with an effective amount of a compound provided herein (e.g., Formula (I)).
  • the disclosure provides a method of treating a subject suffering from or susceptible to a proliferation related disorder or disease, comprising administering to the subject an effective amount of a compound or pharmaceutical composition provided herein (e.g., Formula (I)).
  • a compound or pharmaceutical composition provided herein (e.g., Formula (I)).
  • the disclosure provides a method of treating a subject suffering from or susceptible to a proliferation related activity related disorder or disease, wherein the subject has been identified as in need of treatment for a proliferation related disorder or disease, comprising administering to a subject in need thereof, an effective amount of a compound or pharmaceutical composition provided herein (e.g., Formula (I)), such that the subject is treated for said disorder.
  • a compound or pharmaceutical composition provided herein (e.g., Formula (I)
  • the disclosure provides a method of treating a subject suffering from or susceptible to a cell proliferation related disorder or disease, wherein the subject has been identified as in need of treatment for a cell proliferation related disorder or disease, comprising administering to a subject in need thereof, an effective amount of a compound or pharmaceutical composition provided herein (e.g., Formula (I)), such that cell proliferation in the subject is modulated (e.g. , down regulated).
  • a compound or pharmaceutical composition provided herein (e.g., Formula (I)
  • the compounds delineated herein preferentially target cancer cells over nontransformed cells.
  • the disclosure provides a method of treating cancer, tumor growth, cancer of the colon, breast, bone, brain and others (e.g., osteosarcoma, neuroblastoma, colon adenocarcinoma), comprising administering to a subject in need thereof, an effective amount of a compound provided herein (e.g., Formula (I)), and pharmaceutically acceptable salts thereof.
  • a compound provided herein e.g., Formula (I)
  • cardiac cancer e.g., sarcoma, myxoma, rhabdomyoma, fibroma, lipoma and teratoma
  • lung cancer e.g., bronchogenic carcinoma, alveolar carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatous hamartoma, mesothelioma
  • various gastrointestinal cancer e.g., cancers of esophagus, stomach, pancreas, small bowel, and large bowel
  • genitourinary tract cancer e.g., kidney, bladder and urethra, prostate, testis
  • liver cancer e.g., hepatoma, cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma
  • bone cancer e.g., osteogenic s
  • Methods delineated herein include those wherein the subject is identified as in need of a particular stated treatment. Identifying a subject in need of such treatment can be in the judgment of a subject or a health care professional and can be subjective (e.g. opinion) or objective (e.g. measurable by a test or diagnostic method).
  • the disclosure provides a method of modulating the proliferation activity of a cell in a subject, comprising contacting the subject with an effective amount of a compound provided herein (e.g., Formula (I)).
  • the modulation is inhibition.
  • the disclosure provides a method of treating a subject suffering from or susceptible to a cell proliferation related disorder or disease, comprising administering to the subject an effective amount of a compound or pharmaceutical composition provided herein (e.g., Formula (I)).
  • a compound or pharmaceutical composition provided herein (e.g., Formula (I)).
  • the disclosure provides a method of treating a subject suffering from or susceptible to a cell proliferation related disorder or disease, wherein the subject has been identified as in need of treatment for a cell proliferation related disorder or disease, comprising administering to a subject in need thereof, an effective amount of a compound or pharmaceutical composition provided herein (e.g., Formula (I)), such that the subject is treated for said disorder.
  • a compound or pharmaceutical composition provided herein (e.g., Formula (I)
  • the disclosure provides a method as described above, wherein the compound of Formula (I) is GB 1.
  • the disclosure provides a method of treating a disorder, wherein the disorder is cancer (e.g., breast, colon) or solid tumor.
  • the subject is a mammal, preferably a primate or human.
  • the disclosure provides a method as described above, wherein the effective amount of the compound provided herein (e.g. a compound of Formula (I)) ranges from about 0.005 pg/kg to about 200 mg/kg. In certain embodiments, the effective amount of the compound provided herein (e.g. a compound of Formula (I)) ranges from about 0.1 mg/kg to about 200 mg/kg. In a further embodiment, the effective amount of compound provided herein (e.g. a compound of Formula (I)) ranges from about 10 mg/kg to 100 mg/kg.
  • the disclosure provides a method as described above wherein the effective amount of the compound provided herein (e.g. a compound of Formula (I)) ranges from about 1.0 pM to about 500 pM. In other embodiments, the disclosure provides a method as described above wherein the effective amount of the compound provided herein (e.g. a compound of Formula (I)) ranges from about 1.0 pM to about 500 nM. In certain embodiments, the effective amount ranges from about 10.0 pM to about 1000 pM. In another embodiment, the effective amount ranges from about 1.0 nM to about 10 nM. In certain embodiments, the effective amount ranges from about 10.0 pM to about 50 pM.
  • the effective amount ranges from about 1.0 nM to about 50 pM. In certain embodiments, the effective amount ranges from about 10.0 pM to about 10 pM. In another embodiment, the effective amount ranges from about 1.0 nM to about 10 pM. In certain embodiments, the effective amount ranges from about 0.01 pM to about 10 pM. In another embodiment, the effective amount ranges from about 0.1 pM to about 10 pM.
  • the disclosure provides a method as described above, wherein the compound provided herein (e.g., Formula (I)) is administered intravenously, intramuscularly, subcutaneously, intracerebroventricularly, orally or topically.
  • the compound provided herein e.g., Formula (I)
  • chemotherapeutic agents include but are not limited to daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosine arabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D, mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea, nitrogen mustards, melphalan, cyclophosphamide, 6- mercaptopurine, 6-thioguanine, cytarabine (CA), 5-azacytidine, hydroxy
  • Another object of the present disclosure is the use of a compound as described herein (e.g., of any formulae herein) in the manufacture of a medicament for use in the treatment of a cell proliferation disorder or disease.
  • Another object of the present disclosure is the use of a compound as described herein (e.g., of any formulae herein) for use in the treatment of a cell proliferation disorder or disease.
  • BOP (benzotriazol- l-yloxy)tris-(dimethylamino)phosphonium hexafluorophosphate
  • BOP-CI bis(2-oxo-3-oxazolidinyl)phosphinic chloride
  • DCC A-dicyclohcxylcarbodi imide
  • DEPBT 3-(diethoxyphosphoryloxy)-l,2,3-benzotriazin-4(3H)-one
  • DIEA A,A-diisopropylethylamine
  • GTP Guanosine-5'-triphosphate
  • HATU bis(dimethylamino)methylene]-lH-l,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate
  • HBTU 2-( IH-benzo triazol- 1 -yl)- 1 , 1 ,3,3-tetramethyluronium hexafluorophosphate
  • MM/GBSA Molecular Mechanics Generalized Bom Surface Area
  • SAMD Simulated Annealing Molecular Dynamics
  • TBS tert-butyldimethylsilyl
  • TIPS triisopropylsilyl
  • Trt trityl/triphenylmethyl
  • the gatorbulin site was probed with simplified gatorbulins, which were synthesized and tested alongside GB 1 for effects on tubulin polymerization, binding affinity and cancer cell viability, and interrogated the binding site using molecular docking approaches.
  • the metabolic and physicochemical properties of gatorbulins were assessed and also compared the pharmacological consequences with respect to tubulin stability in ovarian cancer cells. [0214] Through chemical synthesis and medicinal chemistry, the gatorbulin structure was systematically simplified to probe the importance of the modifying elements that confer structural uniqueness to GB1 with respect to the biochemical and cellular activity and mechanism.
  • the first-generation analogues focused on the modulation of current groups as highlighted in FIG.
  • N-deoxy analogue of hydroxamic acid (GB2, lb, natural product) 6 , Asn analog (GB3, 1c), methyl ester instead of primary amide (GB4, Id), Pro analog (GB5, le), replacement of dehydroAla (dhAla) with S-Ala (GB6, If) and R-Ala (GB7, 1g).
  • FIG. 3 depicts the general retrosynthetic analysis of the simplified analogues. Similar to the synthetic strategy of GB1 (la), the proposed analogs Ib-lg (FIGs. 2, 3, and 4) resulted from the global unmasking of cyclized precursors 2b-g. The site between of (Me)Pro and (SePh)Ala was chosen for macrolactamization. Fmoc-Fm pair was chosen as the protection groups of amino and carboxy termini, respectively, thus they could be cleaved simultaneously by the mediation of secondary amines 10 .
  • the linear compounds 3b- g were disconnected into four units: Asp/Asn 4, Ala derivatives 5, proline ester acid 6 and Ala derivative 7; and ester acid 6 were disassembled into two commercially available subunits lactate 8 and proline derivative 9. Allyl instead of benzyl as N-OH protecting group was chosen since allyl could be removed selectively by Pd(Ph3P)4 in the presence of dehydropeptide; however, when benzyl was used, the hydrogenation at the debenzylation stage reduced dehydro-Ala simultaneously, irrespective of if hydrogen gas or transfer hydrogen donor (e.g. 1,4-cyclohexadiene, ammonium formate, etc.) was used in the reactions.
  • hydrogen gas or transfer hydrogen donor e.g. 1,4-cyclohexadiene, ammonium formate, etc.
  • GB2 (lb) was co-isolated with GB1 from marine cyanobacteria as a minor constituent, insufficient for rigorous biological testing 6 .
  • the hydroxamic acid group (NOH) of GB1 (la) plays a role in binding with tubulin based on cocrystal structure analysis 6 .
  • the general synthetic strategy (FIG. 4) and the synthesis of GB1 (la) was followed to synthesize GB2 (lb).
  • the synthetic protocols of building blocks 4a, 6a and 7a were reported in previous work 6 .
  • the Fmoc group of compound 10b was cleaved by Et2NH in MeCN and the released corresponding amine then coupled with 6a using 2-bromo-l -ethylpyridinium tetrafluoroborate-mediated coupling (BEP) 13 to produce 11b in 93% yield for two steps.
  • BEP 2-bromo-l -ethylpyridinium tetrafluoroborate-mediated coupling
  • GB3 (1c, Figure 2) was designed in which
  • Acid A-Me-A-Fmoc-Asp(OMe) (4b) was activated to the acid chloride by (COC1)2 and then subjected to amidation by 5a in the presence of AgCN to afford product 16 in 78% yield 6 16 17 .
  • the methyl ester group of 16 was hydrolyzed with McrSnOH in 1,2-dichloroethane (DCE) under reflux to give acid 17 18 .
  • TrtNH triphenylmethylamine
  • FIG. 4 outlines the optimized synthetic route and strategy for the synthesis of linear precursors 3d-g, cyclized precursors 2d-g and final targets Id-g.
  • Compound 5a, 6a and 7a were synthesized following the original protocol for GB1 6 and 6b was synthesized by another published protocol 21 ; compound 5b,7b,c are commercially available ( Figure 4). With the building blocks in hand, they were sequentially fused into linear precursors 3d-g, cyclized precursor 2d-g and final products Id-g.
  • the Fmoc group of compounds lOd and 10a was cleaved by Et2NH in MeCN and the resulting corresponding free amines then coupled with 6a and 6b, respectively, using BEP as coupling reagent to produce products lid (from lOd, 6a), lie (from 10a, 6b), and Ila (from 10a, 6a) in good yields (78-90%).
  • the tert-butyl groups of lid, lie, and Ila were removed by the buffer of TMSOTf /2,6-lutidine to provide corresponding acids 12d, 12e, and 12a.
  • TBS groups of 2d-2g were mediated by buffer of TBAF/HOAc (10 eq.: 12 eq.) to provide the corresponding free alcohols 13d-13g.
  • Subsequent oxidative elimination of SePh group of 13d and 13e with NaICU (4 eq.) yielded the corresponding dehydro-peptides 14d and 14e.
  • Exposure of 14e, 13f, and 13g to the solution of TFA in CH2Q2 (1:5, v/v) without scavenger cleaved the trityl groups and yielded the corresponding primary amides 15e-15g, respectively.
  • GB1 (la) showed an affinity of 2.2 ⁇ 0.7 x 10 6 M’ 1 , which is close to the 1.01 x 10 6 M' 1 we reported previously 6 .
  • GB2 (lb), GB3 (1c) and GB7 (1g) showed no MTC displacement while GB4-6 (Id-lf) have an affinity in the 10 4 M' 1 range (Table 4). This can be rationalized by the crystal structure previously obtained of the a/p -tubulin- GB1 complex (PDB 7ALR) 6 .
  • Cevipabulin is known to bind simultaneously to the vinblastine and the gatorbulin sites 9 , while tubulin cevipabulin-induced degradation is known to occur through binding to the new site, which is located near the non-exchangeable GTP site on the cx-tubulin subunit 9 .
  • GB1 (la) also promotes proteasomal degradation of ex- and P-tubulin.
  • GB1 treatment for 16 h reduced levels of both proteins in a concentration-dependent manner ( Figures 5E, F).
  • cevipabulin and GB 1 are biochemically distinct because cevipabulin exerts dual targeting (vinca and gatorbulin sites) and promotes in vitro tubulin polymerization (as vinblastine does) 24 , while this mixed pharmacology is not known for gatorbulins. The mechanism of tubulin degradation by GB 1 remains to be determined.
  • Table 5 shows the results for the best poses obtained from the dockings, and the docking modes are shown in Figure 6.
  • the RMSD is calculated using only the nonhydrogen atoms that are present in all gatorbulins. Glide was unable to generate a docked conformation for GB7 (1g), indicating that the methyl group in the R configuration forces a large distortion, such that the compound no longer fits in the pocket. Other gatorbulins fit well into the intradimer binding pocket, with poses similar to GB 1 (la) binding pose from the crystal structure.
  • GB1 (la) is in favorable position to establish H-bonds with Serl78 (2 H-bonds), Glnl76, Prol75, and Arg221 from a-tubulin, and Asp329 and Val353 from ?- tubulin. Most of those are conserved in GB2-GB6 (Ib-lf) ligands. However, in GB2 (lb) the hydrogen bond to pAsp329 seems weaker, and in GB3 (1c) the removal of the OH group leads to loss of the double hydrogen bond to aSerl78. The removal of the NH2 group in GB4 (Id) leads to loss of the hydrogen bond to the aProl75 backbone.
  • FIG. 7 shows the GB 1 representative structures obtained for each cluster SAMD calculations (other molecules are in Figure S97).
  • a common difference between the conformations in the two clusters is a 180° inversion around the HN-N-Ca-C dihedral angle in the DhAla residue, again except for GB6 (If) and GB7 (1g), which allows extra ring flexibility.
  • GB6 (If) the methyl in the .S' configuration points towards the solvent, and the distortion with respect to GB 1 is due to the angle change from sp 2 to sp 3 hybridization states in the DhAla-C a .
  • Cevipabulin targeting the vinca site, has additionally been determined to also bind to the same tubulin region as GB 1 (la), although the binding mode is distinct.
  • FIGs 8A-8D compare the structures for the tubulin complexes with cevipabulin (PDB 7DP8) 9 and GB 1 (la) (PDB 7ALR) 6 . Both ligands present a similar total (unbound) solvent accessible surface area (676 A 2 for cevipabulin, 629 A 2 for GB1).
  • TT-TT in the case of cevipabulin, about 70% of this area is in contact with the protein, and almost exclusively with the al chain (68%), where the triazolopyrimidinyl moiety is engaged in stacking with ⁇ zTyr224 and GTP.
  • GB1 (la) makes extensive contact with both a (53%) and [J (47%) chains and makes H-bonds to aArg221 and aProl75, and bAsp329.
  • GB7 showed lower permeability than would be anticipated given the results from the other six compounds.
  • GB1 was further evaluated using a cellular model with MDCK cells stably transduced with the human efflux transporter MDRl/P-gp.
  • GB1 showed similar permeability as determined in the PAMPA assay.
  • GB1 also showed equivalent permeability with and against the transporter gradient indicating that GB1 is not a P-gp substrate or is a poor P-gp substrate.
  • Standard pharmacological agents unrelated to tubulin, nadolol served as a negative control and quinidine served as the P-gp positive control.
  • Quinidine had an efflux ratio of 7.1, which was abolished with the addition of the P-gp inhibitor cyclosporin A, demonstrating the assay could identify P-gp substrates.
  • GB1 was less effective in the engineered SK-OV-3-MDR1-M6/6 cell line 6 which was credited to P-gp mediated efflux 25 . This is likely caused by the massive overexpression of P-gp, where levels were reported to be lOOOx higher than in the parental cell line. With such high transporter expression, even poor substrates may be excluded. Overall, in the context of previous findings, the data suggest that GB1 is a poor P-gp substrate.
  • Pac paclitaxel; Sun, sunitinib; Fur, furafylline (40 pM); Sul, sulfaphenazole (10 pM); Qui, quinidine (10 pM); Ket, ketoconazole (1 pM); Rit, ritonavir; Car, carbamazepine; Pro, propranolol; Ran, ranitidine; QiC, quinidine+CsA (cyclosporin A); Nad, nadolol. Table 7 Continued. Metabolic and physicochemical properties of gatorbulins GB1-7 (la- lg)
  • Pac paclitaxel; Sun, sunitinib; Fur, furafylline (40 pM); Sul, sulfaphenazole (10 p M); Qui, quinidine (10 pM); Ket, ketoconazole (1 pM); Rit, ritonavir; Car, carbamazepine; Pro, propranolol; Ran, ranitidine; QiC, quinidine+CsA (cyclosporin A); Nad, nadolol.
  • NMR spectra were recorded on a Bruker Avance III 600 MHz spectrometer or a Bruker Avance Neo-600 spectrometer with a broadband Prodigy cryogenic probe. Chemical shifts for 1 H and 13 C NMR spectra are reported in parts per million relative to the signal residual signal (CDCh: 7.26 ppm/77.16 ppm; DMF-c/7 8.03 ppm/163.15 ppm). Optical rotation was measured on a Perkin-Elmer 341 polarimeter (Na D line) using a microcell of 1 dm path length.
  • HRMS was conducted using a Thermo Fisher Q Exactive Focus mass spectrometer equipped with UltiMateTM 3000 RSECnano System and electrospray probe on Universal Ion Max API source.
  • the abbreviations s, d, dd, ddd, dddd, t, q, p, br, and m stand for the NMR multiplicity singlet, doublet, doublet of doublets, doublet of doublet of doublets, doublet of doublet of doublet of doublets, triplet, quartet, pentet, broad and multiplet, respectively.
  • the reaction mixture was purified by a preparative column (Alltech, 1 gram, silica). The column was eluted sequentially by CH2CI2, EtOAc/hexane (1:1) and CtECh/MeOH (4:1). The fraction eluted by CthCh/MeOH (4:1) was collected, evaporated, dried and used in next step directly. DIEA (128 pL, 0.734 mmol, 9 eq.) was added to the solution of the above residue, PyBOP (127.4 mg, 0.245 mmol, 3 eq.) and HOAt (34.4 mg, 0.253 mmol, 3.1 eq.) in dry CH2CI2 (150 mL) at room temperature.
  • Eyophilized tubulin was suspended in GAB buffer (10 mM NaPi, 30% glycerol, 1 mM EGTA, 0.1 mM GTP, pH 6.7) in the cold for 20 min. Then, the sample was centrifuged in Optima XPMax Ultracentrifuge at 50 000 rpm, 4°C and 10 min to remove aggregates. Tubulin concentration was spectrometrically measured and the protein was supplemented with 6 mM MgCh and ImM GTP.
  • GAB buffer 10 mM NaPi, 30% glycerol, 1 mM EGTA, 0.1 mM GTP, pH 6.7
  • the 96 well plates were prepared by adding 100 pL of GAB buffer containing 25 p M tubulin followed by adding the compounds (podophyllotoxin, GB1-7) at 27.5 pM. Control including 0.5% DMSO (vehicle) was also included. The absorbance at a wavelength of 350 nm was measured in a Multiskan plate reader.
  • SK-OV-3 (HTB-77) provided by April Risinger (University of Texas Health Science Center, San Antonio) and HeLa (CCL-2) were from American Type Culture Collection (ATCC; Manassas, VA).
  • ATCC American Type Culture Collection
  • SK-OV-3 and HeLa cells were cultured in Earle’s modified Eagle’s Minimum Essential Medium (EMEM) and Dulbecco’s modified Eagle medium (DMEM) respectively, supplemented with 10% Fetal Bovine Serum (FBS, Sigma, USA) and maintained in 5% CO2 at 37 °C in a humidified incubator.
  • EMEM Minimum Essential Medium
  • DMEM Dulbecco’s modified Eagle medium
  • Lysates containing equal amounts of protein were separated by SDS polyacrylamide gel electrophoresis (4-12%), transferred to polyvinylidene difluoride (PVDF) membranes, probed with primary and secondary antibodies, and detected with the SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific).
  • PVDF polyvinylidene difluoride
  • Anti- ex- tubulin (#2144S), P-tubulin (#2146S), GAPDH (#5174T) and secondary anti-rabbit (#7074S) antibodies were obtained from Cell Signaling Technology, Inc (Danvers, MA).
  • Solubility Kinetic solubility was tested from a 10 mM DMSO stock solution by spiking into pre-warmed pH 7.4 phosphate buffered saline in a 96- well plate. The final concentration was 100 pM (1% DMSO). The plate was maintained at ambient temperature for 24 hours on an orbital shaker. Samples were centrifuged through a Millipore Multiscreen Solvinter 0.45 micron low binding PTFE hydrophilic filter plate and were analyzed by HPLC with UV detection at wavelength equals 254 nm. Peak area was compared to standards of known concentration.
  • Microsome stability was evaluated by incubating 1 pM test compound with 1 mg/mL hepatic microsomes in 100 mM KPi, pH 7.4. The reaction was initiated by adding NADPH (1 mM final concentration). Aliquots were removed at 0, 5, 10, 20, 40, and 60 minutes and added to acetonitrile (5X, v:v) to stop the reaction and precipitate the protein. NADPH dependence of the reaction was evaluated by setting up incubations without NADPH. At the end of the assay, the samples were centrifuged through a Millipore Multiscreen Solvinter 0.45 micron low-binding PTFE hydrophilic filter plate and analyzed by LC-MS/MS. Data were log-transformed and represented as half-life.
  • P450 inhibition for four major human isoforms were evaluated in human hepatic microsomes by following the metabolism of specific marker substrates (CYP1A2 phenaceten demethylation to acetaminophen; CYP2C9, tolbutamide hydroxylation to hydroxy tolbutamide; CYP2D6, bufuralol hydroxylation to 4'-hydroxybufuralol; and CYP3A4, midazolam hydroxylation to l'-hydroxymidazolam) in the presence or absence of 10 pM probe compound.
  • the concentration of each marker substrate is approximately its Km. Specific inhibitors for each isoform are included in each run to validate the system.
  • Plasma Protein binding Plasma protein binding was determined using equilibrium dialysis. All samples were tested in triplicate using the RED Rapid Equilibrium Dialysis Device (Thermo Scientific). The plasma sample was prepared with an initial drug concentration of 2 pM in mouse or human plasma with 0.2% DMSO. The remaining plasma not loaded into the dialysis chamber was immediately frozen to evaluate the stability of the compound in plasma. Phosphate buffered saline was added to the receiver chamber. The plate was covered with gas permeable film and allowed to shake at 300 RPM at 37°C for 6 h in a humidified incubator with 5% CO2 to maintain pH.
  • PAMPA Permeability Assay
  • a commercial PAMPA (Parallel Artificial Membrane Permeability Assay) plate from BD Biosciences (Cat# 353015).
  • Compounds were prepared as DMSO stocks and added to the bottom donor plate containing phosphate-buffered saline, pH 7.4, at a concentration of 5 pM and 1% final DMSO.
  • Preliminary experiments using phosphate-buffered saline in the top receiver plate showed high non-specific binding.
  • 0.4% w:v lyophilized bovine albumin dissolved in phosphate-buffered saline was used in the top receiver plate.
  • MDCK-MDR1 The MDR1-MDCK cells were provided by Dr Michael Gottesman at the United States National Institutes of Health. Cell Culture. Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, #11995-065) supplemented with 10% FBS (Gibco, #10082-147), 50 U/ml Penicillin-Streptomycin (Gibco, #15070-063), and 80 ng/ml colchicine (Sigma- Aldrich, #C9754) in a humidified atmosphere of 5% CO2 at 37 °C. Permeability Assay. Cells were seeded on Greiner Bio-One ThinCertTM inserts at a density of 50000 cells/well.
  • DMEM Modified Eagle Medium
  • FBS Gibco, #10082-147
  • Penicillin-Streptomycin Gibco, #15070-063
  • 80 ng/ml colchicine Sigma- Aldrich, #C9754
  • Test compound solutions for the donor chambers were prepared at 2 pM in the above buffer, containing 100 pM Lucifer Yellow (LY) (Sigma-Aldrich, #L0144) as the monolayer-integrity marker. The final DMSO concentration was 0.5 % for each test compound.
  • the HBSS buffer with 10 mM HEPES was supplemented with 0.5 % BSA in the receiver compartments.
  • the permeability was examined in apical to basolateral (A-B) and basolateral to apical (B-A) directions. Samples were taken at the beginning of the incubation from the donor side, and after 90 minutes from the donor and receiver compartments and the concentration of drug was determined by LC-MS.
  • LY fluorescence was measured at 430/535 nm using a BioTek Synergy Neo2 microplate reader (Agilent, Santa Clara, CA). LY transport of ⁇ 5 % was considered acceptable. Nadolol was used as the negative control and quinidine as positive control for MDR1.
  • the present disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim.
  • any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim.
  • elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the present disclosure, or aspects of the present disclosure, is/are referred to as comprising particular elements and/or features, certain embodiments of the present disclosure or aspects of the present disclosure consist, or consist essentially of, such elements and/or features.

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Abstract

La présente invention concerne des composés macrocycliques présentant une activité anti-proliférative, et des méthodes de traitement de troubles tels que le cancer, les tumeurs et les troubles liés à la prolifération cellulaire.
PCT/US2024/051984 2023-10-18 2024-10-18 Composés macrocycliques et méthodes de traitement Pending WO2025085756A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140200319A1 (en) * 2011-02-14 2014-07-17 Arizona Board of Regents, a body corporate of the State of Arizona Acting for and on behalf of Arizo Fluorescent potassium ion sensors
US20190194166A1 (en) * 2016-06-13 2019-06-27 Glaxosmithkline Intellectual Property Development Limited Substituted pyridines as inhibitors of dnmt1
US20200121607A1 (en) * 2017-07-05 2020-04-23 Novartis Ag Novel pharmaceutical composition
WO2022076563A1 (fr) * 2020-10-07 2022-04-14 University Of Florida Research Foundation, Incorporated Composés macrocycliques et méthodes de traitement

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140200319A1 (en) * 2011-02-14 2014-07-17 Arizona Board of Regents, a body corporate of the State of Arizona Acting for and on behalf of Arizo Fluorescent potassium ion sensors
US20190194166A1 (en) * 2016-06-13 2019-06-27 Glaxosmithkline Intellectual Property Development Limited Substituted pyridines as inhibitors of dnmt1
US20200121607A1 (en) * 2017-07-05 2020-04-23 Novartis Ag Novel pharmaceutical composition
WO2022076563A1 (fr) * 2020-10-07 2022-04-14 University Of Florida Research Foundation, Incorporated Composés macrocycliques et méthodes de traitement

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