Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D261/00—Heterocyclic compounds containing 1,2-oxazole or hydrogenated 1,2-oxazole rings
  • C07D261/02—Heterocyclic compounds containing 1,2-oxazole or hydrogenated 1,2-oxazole rings not condensed with other rings
  • C07D261/06—Heterocyclic compounds containing 1,2-oxazole or hydrogenated 1,2-oxazole rings not condensed with other rings having two or more double bonds between ring members or between ring members and non-ring members
  • C07D261/10—Heterocyclic compounds containing 1,2-oxazole or hydrogenated 1,2-oxazole rings not condensed with other rings having two or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
  • C07D261/18—Carbon atoms having three bonds to hetero atoms, with at the most one bond to halogen
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/02—Antineoplastic agents specific for leukemia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/04—Antineoplastic agents specific for metastasis
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
  • C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
  • C07D413/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links

Definitions

• SMYD protein inhibitors such as SMYD3 and SMYD2 inhibitors, and therapeutic methods of treating conditions and diseases wherein inhibition of SMYD proteins such as SMYD3 and SMYD2 provides a benefit.
• Epigenetic regulation of gene expression is an important biological determinant of protein production and cellular differentiation and plays a significant pathogenic role in a number of human diseases.
• Epigenetic regulation involves heritable modification of genetic material without changing its nucleotide sequence.
• epigenetic regulation is mediated by selective and reversible modification (e.g., methylation) of DNA and proteins (e.g., histones) that control the conformational transition between transcriptionally active and inactive states of chromatin.
• methyltransferases e.g., SMYD proteins such as SMYD3 and SMYD2
• the present disclosure provides substituted isoxazole carboxamide compounds represented by Formulae I-XII below, and the pharmaceutically acceptable salts and solvates thereof, collectively referred to herein as "Compounds of the Disclosure.”
• the present disclosure provides a Compound of the
• the present disclosure provides a method of inhibiting
• SMYD proteins such as SMYD3 or SMYD2, or both, in a mammal, comprising administering to the mammal an effective amount of at least one Compound of the Disclosure.
• the present disclosure provides a method of irreversibly inhibiting SMYD proteins, such as SMYD3 or SMYD2, or both, in a mammal, comprising administering to the mammal an effective amount of at least one Compound of the Disclosure.
• the present disclosure provides methods for treating a disease, disorder, or condition, e.g., cancer, responsive to inhibition of SMYD proteins, such as SMYD3 or SMYD2, or both, comprising administering a therapeutically effective amount of a Compound of the Disclosure.
• a disease, disorder, or condition e.g., cancer
• SMYD proteins such as SMYD3 or SMYD2
• the present disclosure provides a pharmaceutical composition for treating a disease, disorder, or condition responsive to inhibition of SMYD proteins, such as SMYD3 or SMYD2, or both, wherein the pharmaceutical composition comprises a therapeutically effective amount of a Compound of the Disclosure in a mixture with one or more pharmaceutically acceptable carriers.
• the present disclosure provides Compounds of the Disclosure for use in treating cancer in a mammal, e.g., breast, cervical, colon, kidney, liver, head and neck, skin, pancreatic, ovary, esophageal, lung, and prostate cancer.
• a mammal e.g., breast, cervical, colon, kidney, liver, head and neck, skin, pancreatic, ovary, esophageal, lung, and prostate cancer.
• the present disclosure provides a Compound of the
• Disclosure for use in the manufacture of a medicament for treating cancer in a mammal.
• the present disclosure provides kit comprising a Compound of the Disclosure.
• the Compounds of the Disclosure are useful for treating diseases, disorders, or conditions, e.g., cancer, responsive to inhibition of SMYD proteins.
• the Compounds of the Disclosure are useful for treating diseases, disorders, or conditions, e.g., cancer, responsive to inhibition of SMYD3.
• the Compounds of the Disclosure are useful for treating diseases, disorders, or conditions, e.g., cancer, responsive to inhibition of SMYD2.
• Compounds of the Disclosure are compounds having
• R 1 is selected from the group consisting of ethyl and cyclopropyl
• R 2a , R 2b , R 3a , and R 3b are selected from the group consisting of hydrogen and
• X is selected from the group consisting of -N(-Y-Z)- and -CH[N(H)-Y-Z]-;
• n is O or l
• m is O or l .
• Compounds of the Disclosure are compounds having
• R 1 , R 2a , R 2b , R 3a , R 3b , Y, Z, m, and n are as defined above in connection with Formula I.
• R 2a , R 2b , R 3a , and R 3b are hydrogen
• Compounds of the Disclosure are compounds having
• Compounds of the Disclosure are compounds having
• Compounds of the Disclosure are compounds having
• Compounds of the Disclosure are compounds having
• Compounds of the Disclosure are compounds having any one of Formulae I-XII, and the pharmaceutically acceptable salts or solvates, e.g., hydrates, thereof, wherein R 1 is ethyl.
• Compounds of the Disclosure are compounds having any one of Formulae I-XII, and the pharmaceutically acceptable salts or solvates, e.g., hydrates, thereof, wherein R 1 is cyclopropyl.
• Compounds of the Disclosure are compounds of
• Tables 1 and 2 and the pharmaceutically acceptable salts or solvates, e.g., hydrates, thereof, or different pharmaceutically acceptable salt thereof.
• alkyl refers to a straight- or branched-chain aliphatic hydrocarbon containing one to twelve carbon atoms (i.e. , Ci_i 2 alkyl) or the number of carbon atoms designated (i.e., a Ci alkyl such as methyl, a C 2 alkyl such as ethyl, a C 3 alkyl such as propyl or isopropyl, etc.).
• the alkyl group is chosen from a straight chain Ci_ 4 alkyl group.
• the alkyl group is chosen from a branched chain C 3 _ 4 alkyl group.
• the alkyl group is chosen from a straight or branched chain C 3 _ 4 alkyl group.
• the alkyl group is partially or completely deuterated, i.e., one or more hydrogen atoms of the alkyl group are replaced with deuterium atoms.
• Non-limiting exemplary Ci_ 4 alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, and zso-butyl.
• cycloalkyl as used by itself or as part of another group refers to saturated cyclic aliphatic hydrocarbons containing one to three rings having from three to twelve carbon atoms (i.e., C 3 _i 2 cycloalkyl) or the number of carbons designated.
• the cycloalkyl group is cyclopropyl.
• the present disclosure encompasses any of the Compounds of the Disclosure being isotopically-labelled (i.e., radiolabeled) by having one or more atoms replaced by an atom having a different atomic mass or mass number.
• isotopes that can be incorporated into the disclosed compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine and chlorine, such as 2 H (or deuterium (D)), 3 H, U C, 13 C, 14 C, 15 N, 18 0, 17 0, 31 P, 32 P, 35 S, 18 F, and 36 C1, respectively, e.g., 3 H, UC, and 14 C.
• compositions wherein substantially all of the atoms at a position within the Compound of the Disclosure are replaced by an atom having a different atomic mass or mass number.
• Isotopically-labelled Compounds of the Disclosure can be prepared by methods known in the art.
• Compounds of the Disclosure may contain one or more asymmetric centers and may thus give rise to enantiomers, diastereomers, and other stereoisomeric forms.
• the present disclosure is meant to encompass the use of all such possible forms, as well as their racemic and resolved forms and mixtures thereof.
• the individual enantiomers can be separated according to methods known in the art in view of the present disclosure.
• the compounds described herein contain olefmic double bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that they include both E and Z geometric isomers. All tautomers are intended to be encompassed by the present disclosure as well.
• stereoisomers is a general term for all isomers of individual molecules that differ only in the orientation of their atoms in space. It includes enantiomers and isomers of compounds with more than one chiral center that are not mirror images of one another (diastereomers).
• chiral center or "asymmetric carbon atom” refers to a carbon atom to which four different groups are attached.
• enantiomer and “enantiomeric” refer to a molecule that cannot be superimposed on its mirror image and hence is optically active wherein the enantiomer rotates the plane of polarized light in one direction and its mirror image compound rotates the plane of polarized light in the opposite direction.
• racemic refers to a mixture of equal parts of enantiomers and which mixture is optically inactive.
• absolute configuration refers to the spatial arrangement of the atoms of a chiral molecular entity (or group) and its stereochemical description, e.g., R or S.
• enantiomeric excess refers to a measure for how much of one enantiomer is present compared to the other.
• percent enantiomeric excess is defined as
• R - S ⁇ * 100, where R and S are the respective mole or weight fractions of enantiomers in a mixture such that R + S 1.
• the percent enantiomeric excess is defined as ([a] obs /[a.] max )* 100, where [a] 0bs is the optical rotation of the mixture of enantiomers and [a] max is the optical rotation of the pure enantiomer. Determination of enantiomeric excess is possible using a variety of analytical techniques, including NMR spectroscopy, chiral column chromatography or optical polarimetry.
• enantiomerically pure or “enantiopure” refer to a sample of a chiral substance all of whose molecules (within the limits of detection) have the same chirality sense.
• enantiomerically enriched or “enantioenriched” refer to a sample of a chiral substance whose enantiomeric ratio is greater than 50:50. Enantiomerically enriched compounds may be enantiomerically pure.
• compositions of the Disclosure including non-toxic pharmaceutically acceptable salts.
• pharmaceutically acceptable addition salts include inorganic and organic acid addition salts and basic salts.
• the pharmaceutically acceptable salts include, but are not limited to, metal salts such as sodium salt, potassium salt, cesium salt and the like; alkaline earth metals such as calcium salt, magnesium salt and the like; organic amine salts such as triethylamine salt, pyridine salt, picoline salt, ethanolamine salt, triethanolamine salt, dicyclohexylamine salt, ⁇ , ⁇ '-dibenzylethylenediamine salt and the like; inorganic acid salts such as hydrochloride, hydrobromide, phosphate, sulphate and the like; organic acid salts such as citrate, lactate, tartrate, maleate, fumarate, mandelate, acetate, dichloroacetate, trifluoroacetate, oxalate, formate and the like; sulfonates such as methanesulfon
• pharmaceutically acceptable salt refers to any salt, e.g., obtained by reaction with an acid or a base, of a Compound of the Disclosure that is physiologically tolerated in the target patient (e.g., a mammal, e.g., a human).
• Acid addition salts can be formed by mixing a solution of the particular
• Compound of the Disclosure with a solution of a pharmaceutically acceptable nontoxic acid such as hydrochloric acid, fumaric acid, maleic acid, succinic acid, acetic acid, citric acid, tartaric acid, carbonic acid, phosphoric acid, oxalic acid, dichloroacetic acid, or the like.
• Basic salts can be formed by mixing a solution of the compound of the present disclosure with a solution of a pharmaceutically acceptable non-toxic base such as sodium hydroxide, potassium hydroxide, choline hydroxide, sodium carbonate and the like.
• Solvates typically do not significantly alter the physiological activity or toxicity of the compounds, and as such may function as pharmacological equivalents.
• the term "solvate” as used herein is a combination, physical association and/or solvation of a compound of the present disclosure with a solvent molecule such as, e.g. a disolvate, monosolvate or hemisolvate, where the ratio of solvent molecule to compound of the present disclosure is about 2: 1, about 1 : 1 or about 1 :2, respectively.
• This physical association involves varying degrees of ionic and covalent bonding, including hydrogen bonding.
• the solvate can be isolated, such as when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid.
• solvate encompasses both solution-phase and isolatable solvates.
• Compounds of the Disclosure can be present as solvated forms with a pharmaceutically acceptable solvent, such as water, methanol, ethanol, and the like, and it is intended that the disclosure includes both solvated and unsolvated forms of Compounds of the Disclosure.
• a pharmaceutically acceptable solvent such as water, methanol, ethanol, and the like
• solvate is a hydrate.
• a "hydrate” relates to a particular subgroup of solvates where the solvent molecule is water.
• Solvates typically can function as pharmacological equivalents. Preparation of solvates is known in the art. See, for example, M. Caira et al, J. Pharmaceut.
• a typical, non- limiting, process of preparing a solvate would involve dissolving a Compound of the Disclosure in a desired solvent (organic, water, or a mixture thereof) at temperatures above 20°C to about 25°C, then cooling the solution at a rate sufficient to form crystals, and isolating the crystals by known methods, e.g., filtration.
• Analytical techniques such as infrared spectroscopy can be used to confirm the presence of the solvent in a crystal of the solvate.
• SMYD3 and SMYD2 a number of diseases, conditions, or disorders mediated by SMYD proteins, such as SMYD3 and SMYD2, can be treated by employing these compounds.
• the present disclosure is thus directed generally to a method for treating a disease, condition, or disorder responsive to the inhibition of SMYD proteins, such as SMYD3 and SMYD2, in an animal suffering from, or at risk of suffering from, the disorder, the method comprising administering to the animal an effective amount of one or more Compounds of the Disclosure.
• the Compounds of the Disclosure are therapeutically effective inhibitors of SMYD proteins by irreversibly binding one or more SMYD proteins such as SMYD3 or SMYD2. In some embodiments, the Compounds of the Disclosure are therapeutically effective inhibitors of SMYD proteins by forming covalent bonds with one or more SMYD proteins such as SMYD3 or SMYD2.
• the present disclosure is further directed to a method of inhibiting SMYD proteins in an animal in need thereof, the method comprising administering to the animal a therapeutically effective amount of at least one Compound of the Disclosure.
• the present disclosure is further directed to a method of inhibiting SMYD3 in an animal in need thereof, the method comprising administering to the animal a therapeutically effective amount of at least one Compound of the Disclosure.
• the present disclosure is further directed to a method of inhibiting SMYD2 in an animal in need thereof, the method comprising administering to the animal a therapeutically effective amount of at least one Compound of the Disclosure.
• the terms “treat,” “treating,” “treatment,” and the like refer to eliminating, reducing, or ameliorating a disease or condition, and/or symptoms associated therewith. Although not precluded, treating a disease or condition does not require that the disease, condition, or symptoms associated therewith be completely eliminated.
• the terms “treat,” “treating,” “treatment,” and the like may include “prophylactic treatment,” which refers to reducing the probability of redeveloping a disease or condition, or of a recurrence of a previously-controlled disease or condition, in a subject who does not have, but is at risk of or is susceptible to, redeveloping a disease or condition or a recurrence of the disease or condition.
• the term “treat” and synonyms contemplate administering a therapeutically effective amount of a Compound of the Disclosure to an individual in need of such treatment.
• treatment also includes relapse prophylaxis or phase prophylaxis, as well as the treatment of acute or chronic signs, symptoms and/or malfunctions.
• the treatment can be orientated symptomatically, for example, to suppress symptoms. It can be effected over a short period, be oriented over a medium term, or can be a long-term treatment, for example within the context of a maintenance therapy.
• terapéuticaally effective amount refers to an amount of the active ingredient(s) that is(are) sufficient, when administered by a method of the disclosure, to efficaciously deliver the active ingredient(s) for the treatment of condition or disease of interest to an individual in need thereof.
• the therapeutically effective amount of the agent may reduce (i.e., retard to some extent and preferably stop) unwanted cellular proliferation; reduce the number of cancer cells; reduce the tumor size; inhibit (i.e., retard to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., retard to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; modulate protein methylation in the target cells; and/or relieve, to some extent, one or more of the symptoms associated with the cancer.
• the administered compound or composition prevents growth and/or kills existing cancer cells, it may be cytostatic and/or cytotoxic.
• container means any receptacle and closure therefore suitable for storing, shipping, dispensing, and/or handling a pharmaceutical product.
• the term "insert” means information accompanying a pharmaceutical product that provides a description of how to administer the product, along with the safety and efficacy data required to allow the physician, pharmacist, and patient to make an informed decision regarding use of the product.
• the package insert generally is regarded as the "label" for a pharmaceutical product.
• disease or "condition” or “disorder” denotes disturbances and/or anomalies that as a rule are regarded as being pathological conditions or functions, and that can manifest themselves in the form of particular signs, symptoms, and/or malfunctions.
• Compounds of the Disclosure inhibit SMYD proteins, such as SMYD3 and SMYD2 and can be used in treating diseases and conditions such as proliferative diseases, wherein inhibition of SMYD proteins, such as SMYD3 and SMYD2 provides a benefit.
• the Compounds of the Disclosure can be used to treat a
• SMSD protein mediated disorder e.g., a SMYD3 -mediated disorder or a SMYD2- mediated disorder.
• a SMYD protein mediated disorder is any pathological condition in which a SMYD protein is know to play a role.
• a SMYD- mediated disorder is a proliferative disease.
• SMYD proteins such as SMYD3 and
• SMYD2 is the inhibition of the activity of one or more activities of SMYD proteins such as SMYD3 and SMYD2.
• the activity of the SMYD proteins such as SMYD3 and SMYD2 is the ability of the SMYD protein such as SMY3 or SMYD2 to transfer a methylgroup to a target protein (e.g., histone).
• a target protein e.g., histone
• Examplary levels of inhibition of the activity one or more SMYD proteins such as SMYD3 and SMYD2 include at least 10% inhibiton, at least 20% inhibition, at least 30% inhibiton, at least 40% inhibition, at least 50% inhibiton, at least 60% inhibition, at least 70% inhibiton, at least 80% inhibition, at least 90% inhibiton, and up to 100% inhibition.
• KMTs plays pivotal roles in various cellular processes, including gene expression regulation and DNA damage response.
• the family of human SMYD proteins consists of SMYD1, SMYD2, SMYD3, SMYD4 and SMYD5.
• SMYD1, SMYD2, and SMYD3 share a high degree of sequence homology and, with the exception of SMYD5, human SMYD proteins harbor at least one C-terminal tetratrico peptide repeat (TPR) domain.
• TPR C-terminal tetratrico peptide repeat
• SMYD proteins have been found to be linked to various cancers (See e.g., Hamamoto et al. Nat Cell. Biol. 2004, 6: 731-740), Hu et al. Canncer Research 2009, 4067-4072, and Komatsu et al. Carcinogenesis 2009, 301139-1146.;
• SMYD3 is a protein methyltransferase found to be expressed at high levels in a number of different cancers (Hamamoto, R., et al, Nat. Cell Biol., 6(8):731-40 (2004)). SMYD3 likely plays a role in the regulation of gene transcription and signal transduction pathways critical for survival of breast, liver, prostate and lung cancer cell lines (Hamamoto, R., et al, Nat. Cell Biol, 6(8):731-40 (2004); Hamamoto, R., et al, Cancer Sci., 97(2): 113-8 (2006); Van Aller, G.S., et al, Epigenetics, 7(4):340-3
• RNAi-based technologies have shown that ablation of SMYD3 in hepatocellular carcinoma cell lines greatly reduces cell viability and that its pro- survival role is dependent on its catalytic activity (Hamamoto, R., et al, Nat. Cell Biol, 6(8):731-40 (2004); Van Aller, G.S., et al, Epigenetics, 7(4):340-3 (2012)).
• SMYD3 has also been shown to be a critical mediator of transformation resulting from gain of function mutations in the oncogene, KRAS for both pancreatic and lung adenocarcinoma in mouse models. The dependence of KRAS on SMYD3 was also shown to be dependent on its catalytic activity (Mazur, P.K., et al., Nature, 510(7504):283-7 (2014)).
• SMYD2 (SET and MYND domain-containing protein 2) was first characterized as protein that is a member of a sub-family of SET domain containing proteins which catalyze the site-specific transfer of methyl groups onto substrate proteins.
• SMYD2 was initially shown to have methyltransferase activity towards lysine 36 on histone H3 (H3K36) but has subsequently been shown to have both histone and non-histone methyltrasferase activity.
• SMYD2 has been implicated in the pathogenesis of multiple cancers. It has been shown to be over-expressed, compared to matched normal samples, in tumors of the breast, cervix, colon, kidney, liver, head and neck, skin, pancreas, ovary, esophagus and prostate, as well as hematologic malignancies such as AML, B- and T-ALL, CLL and MCL, suggesting a role for SMYD2 in the biology of these cancers. More specifically, studies using genetic knock-down of SMYD2 have demonstrated antiproliferative effects in esophageal squamous cell carcinoma (ESCC), bladder carcinoma and cervical carcinoma cell lines. Moreover, high expression of SMYD2 has been shown to be a poor prognostic factor in both ESCC and pediatric ALL.
• ESCC esophageal squamous cell carcinoma
• cervical carcinoma a poor prognostic factor in both ESCC and pediatric ALL.
• the present disclosure provides a method of treating cancer in a patient comprising administering a therapeutically effective amount of a Compound of the Disclosure. While not being limited to a specific mechanism, in some embodiemtns, Compounds of the Disclorure can treat cancer by inhibiting SMYD proteins, such as SMYD3 and SMYD2.
• treatable cancers include, but are not limited to, adrenal cancer, acinic cell carcinoma, acoustic neuroma, acral lentigious melanoma, acrospiroma, acute eosinophilic leukemia, acute erythroid leukemia, acute lymphoblastic leukemia, acute megakaryoblastic leukemia, acute monocytic leukemia, acute promyelocytic leukemia, adenocarcinoma, adenoid cystic carcinoma, adenoma, adenomatoid odontogenic tumor, adenosquamous carcinoma, adipose tissue neoplasm, adrenocortical carcinoma, adult T-cell leukemia/lymphoma, aggressive NK-cell leukemia, AIDS-related lymphoma, alveolar rhabdomyosarcoma, alveolar soft part sarcoma, ameloblastic fibroma, anaplastic large cell
• the cancer is breast, cervix, colon, kidney, liver, head and neck, skin, pancreas, ovary, esophagus, or prostate cancer.
• the cancer is a hematologic malignancy such as acute myeloid leukemia (AML), B- and T-acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), or mantle cell lymphoma (MCL).
• AML acute myeloid leukemia
• ALL B- and T-acute lymphoblastic leukemia
• CLL chronic lymphocytic leukemia
• MCL mantle cell lymphoma
• the cancer is esophageal squamous cell carcinoma
• the cancer is a leukemia, for example a leukemia selected from acute monocytic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia and mixed lineage leukemia (MLL).
• the cancer is NUT-midline carcinoma.
• the cancer is multiple myeloma.
• the cancer is a lung cancer such as small cell lung cancer (SCLC).
• SCLC small cell lung cancer
• the cancer is a neuroblastoma.
• the cancer is Burkitt's lymphoma.
• the cancer is cervical cancer.
• the cancer is esophageal cancer.
• the cancer is ovarian cancer.
• the cancer is colorectal cancer.
• the cancer is prostate cancer.
• the cancer is breast cancer.
• the present disclosure provides a therapeutic method of modulating protein methylation, gene expression, cell proliferation, cell differentiation and/or apoptosis in vivo in the cancers mentioned above by administering a therapeutically effective amount of a Compound of the Disclosure to a subject in need of such therapy.
• Compounds of the Disclosure can be administered to a mammal in the form of a raw chemical without any other components present.
• Compounds of the Disclosure can also be administered to a mammal as part of a pharmaceutical composition containing the compound combined with a suitable pharmaceutically acceptable carrier.
• a suitable pharmaceutically acceptable carrier can be selected from pharmaceutically acceptable excipients and auxiliaries.
• pharmaceutically acceptable carrier or “pharmaceutically acceptable vehicle” encompasses any of the standard pharmaceutical carriers, solvents, surfactants, or vehicles. Suitable pharmaceutically acceptable vehicles include aqueous vehicles and nonaqueous vehicles. Standard pharmaceutical carriers and their formulations are described in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA, 19th ed. 1995.
• compositions within the scope of the present disclosure include all compositions where a Compound of the Disclosure is combined with one or more pharmaceutically acceptable carriers.
• the Compound of the Disclosure is present in the composition in an amount that is effective to achieve its intended therapeutic purpose. While individual needs may vary, a determination of optimal ranges of effective amounts of each compound is within the skill of the art.
• a Compound of the Disclosure can be administered to a mammal, e.g., a human, orally at a dose of from about 0.0025 to about 1500 mg per kg body weight of the mammal, or an equivalent amount of a pharmaceutically acceptable salt or solvate thereof, per day to treat the particular disorder.
• a useful oral dose of a Compound of the Disclosure administered to a mammal is from about 0.0025 to about 50 mg per kg body weight of the mammal, or an equivalent amount of the pharmaceutically acceptable salt or solvate thereof.
• the dose is typically about one-half of the oral dose.
• a unit oral dose may comprise from about 0.01 mg to about 1 g of the
• a pharmaceutical composition of the present disclosure can be administered to any patient that may experience the beneficial effects of a Compound of the Disclosure. Foremost among such patients are mammals, e.g., humans and companion animals, although the disclosure is not intended to be so limited. In one embodiment, the patient is a human.
• a pharmaceutical composition of the present disclosure can be administered by any means that achieves its intended purpose.
• administration can be by the oral, parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, intranasal, transmucosal, rectal, intravaginal or buccal route, or by inhalation.
• the dosage administered and route of administration will vary, depending upon the circumstances of the particular subject, and taking into account such factors as age, gender, health, and weight of the recipient, condition or disorder to be treated, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.
• a pharmaceutical composition of the present disclosure can be administered orally.
• a pharmaceutical composition of the present disclosure can be administered orally and is formulated into tablets, dragees, capsules, or an oral liquid preparation.
• the oral formulation comprises extruded multiparticulates comprising the Compound of the Disclosure.
• a pharmaceutical composition of the present disclosure can be administered rectally, and is formulated in suppositories.
• composition of the present disclosure can be administered by injection.
• composition of the present disclosure can be administered transdermally.
• composition of the present disclosure can be administered by inhalation or by intranasal or transmucosal administration.
• composition of the present disclosure can be administered by the intravaginal route.
• a pharmaceutical composition of the present disclosure can contain from about
• a pharmaceutical composition of the present disclosure is manufactured in a manner which itself will be known in view of the instant disclosure, for example, by means of conventional mixing, granulating, dragee-making, dissolving, extrusion, or lyophilizing processes.
• pharmaceutical compositions for oral use can be obtained by combining the active compound with solid excipients, optionally grinding the resulting mixture and processing the mixture of granules, after adding suitable auxiliaries, if desired or necessary, to obtain tablets or dragee cores.
• Suitable excipients include fillers such as saccharides (for example, lactose, sucrose, mannitol or sorbitol), cellulose preparations, calcium phosphates (for example, tricalcium phosphate or calcium hydrogen phosphate), as well as binders such as starch paste (using, for example, maize starch, wheat starch, rice starch, or potato starch), gelatin, tragacanth, methyl cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and/or polyvinyl pyrrolidone.
• saccharides for example, lactose, sucrose, mannitol or sorbitol
• cellulose preparations for example, calcium phosphates (for example, tricalcium phosphate or calcium hydrogen phosphate)
• binders such as starch paste (using, for example, maize starch, wheat starch, rice starch, or potato starch), gelatin, tragacanth, methyl cellulose, hydroxypropylmethyl
• one or more disintegrating agents can be added, such as the above-mentioned starches and also carboxymethyl-starch, cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate.
• Auxiliaries are typically flow-regulating agents and lubricants such as, for example, silica, talc, stearic acid or salts thereof (e.g., magnesium stearate or calcium stearate), and polyethylene glycol.
• Dragee cores are provided with suitable coatings that are resistant to gastric juices.
• concentrated saccharide solutions can be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, polyethylene glycol and/or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures.
• cellulose preparations such as acetylcellulose phthalate or hydroxypropylmethyl-cellulose phthalate
• Dye stuffs or pigments can be added to the tablets or dragee coatings, for example, for identification or in order to characterize combinations of active compound doses.
• Examples of other pharmaceutical preparations that can be used orally include push-fit capsules made of gelatin, or soft, sealed capsules made of gelatin and a plasticizer such as glycerol or sorbitol.
• the push-fit capsules can contain a compound in the form of granules, which can be mixed with fillers such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers, or in the form of extruded multiparticulates.
• the active compounds are preferably dissolved or suspended in suitable liquids, such as fatty oils or liquid paraffin.
• stabilizers can be added.
• Possible pharmaceutical preparations for rectal administration include, for example, suppositories, which consist of a combination of one or more active compounds with a suppository base.
• Suitable suppository bases include natural and synthetic triglycerides, and paraffin hydrocarbons, among others. It is also possible to use gelatin rectal capsules consisting of a combination of active compound with a base material such as, for example, a liquid triglyceride, polyethylene glycol, or paraffin hydrocarbon.
• Suitable formulations for parenteral administration include aqueous solutions of the active compound in a water-soluble form such as, for example, a water-soluble salt, alkaline solution, or acidic solution.
• a suspension of the active compound can be prepared as an oily suspension.
• Suitable lipophilic solvents or vehicles for such as suspension may include fatty oils (for example, sesame oil), synthetic fatty acid esters (for example, ethyl oleate), triglycerides, or a polyethylene glycol such as polyethylene glycol-400 (PEG-400).
• An aqueous suspension may contain one or more substances to increase the viscosity of the suspension, including, for example, sodium carboxymethyl cellulose, sorbitol, and/or dextran.
• the suspension may optionally contain stabilizers.
• kits which comprise a
• the kit includes a Compound of the Disclosure (or a composition comprising a Compound of the Disclosure) packaged in a manner that facilitates their use to practice methods of the present disclosure.
• the kit includes a Compound of the Disclosure (or a composition comprising a Compound of the Disclosure) packaged in a container, such as a sealed bottle or vessel, with a label affixed to the container or included in the kit that describes use of the compound or composition to practice the method of the disclosure.
• the compound or composition is packaged in a unit dosage form.
• the kit further can include a device suitable for administering the composition according to the intended route of administration.
• a suitable base such as TEA or DIPEA
• a suitable solvent such as dichloromethane, acetonitrile, or DMF.
• a suitable acide chloride Z-COC1
• a suitable base such as TEA or DIPEA
• a suitable solvent such as dichloromethane, acetonitrile, or DMF
• Step 1 Synthesis of ethyl 4-cyclopropyl-2,4-dioxobutanoate
• Step 2 Synthesis of ethyl 5-cyclopropylisoxazole-3-carboxylate [0115] Into a 10 L round-bottom flask, was placed a solution of ethyl 4-cyclopropyl-2,4- dioxobutanoate (177 g) in ethanol (1.1 L) and NH 2 OH-HCl (200 g). The resulting solution was stirred for 1 h at 20-30°C. The resulting solution was allowed to react, with stirring, for an additional 1 h at 80°C. The resulting mixture was concentrated under vacuum. The residue was purified on a silica gel column with ethyl acetate/petroleum ether (1/10).
• Step 1 Synthesis of tert-butyl ( 1 r,4r)-4-(5 -cyclopropylisoxazole-3 - carboxamido)cyclohexylcarbamate
• Step 2 Synthesis of N-((lr,4r)-4-aminocyclohexyl)-5 -cyclopropylisoxazole-3 - carboxamide hydrochloride
• Step 1 Synthesis of tert-butyl 3-(5-cyclopropylisoxazole-3-carboxamido)azetidine-l- carboxylate
• Step 3 Synthesis of 5-cyclopropyl-N-(l-(vinylsulfonyl)azetidin-3-yl)isoxazole-3- carboxamide
• SAM S-adenosylmethionine
• SAH S-adenosylhomocysteine
• Tris Tween20, dimethylsulfoxide (DMSO), bovine skin gelatin (BSG), and Tris(2- carboxyethyl)phosphine hydrochloride solution (TCEP)
• SAM S-adenosylmethionine
• SAH S-adenosylhomocysteine
• Tris Tween20
• BSG bovine skin gelatin
• TCEP Tris(2- carboxyethyl)phosphine hydrochloride solution
• 3 H-SAM was purchase from American Radiolabeled Chemicals with a specific activity of 80 Ci/mmol.
• 384-well opaque white OptiPlates and SPA beads (Perkin Elmer, catalog # RPNQ0013) were purchased from PerkinElmer.
• N-terminally GST-tagged MEKK2 (MAP3K2) protein corresponding to reference sequence AAF63496.3 was purchased from Life Technologies (catalog # PV4010). This protein was expressed in High Five insect cells and purified to >85 % purity. Protein identity was confirmed by MS/MS analysis after proteolytic digestion. The protein sequence used was: MAPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNK
• Full-length human SMYD3 isoform 1 (BAB86333) was inserted into a modified pET21b plasmid containing a His6 tag and TEV and SUMO cleavage sites. Because two common variants of SMYD3 exist in the population, site directed mutagenesis was subsequently performed to change amino acid 13 from an asparagine to a lysine, resulting in plasmid pEPZ533. A lysine at position 13 conforms to the more commonly occurring sequence (NP 001161212).
• E. coli (BL21 codonplus RIL strain, Stratagene) were transformed with plasmid pEPZ553 by mixing competent cells and plasmid DNA and incubating on ice for 30 minutes followed by heat shock at 42 °C for 1 minute and cooling on ice for 2 minutes. Transformed cells were grown and selected on LB agar with 100 ⁇ g/mL ampicillin and 17 ⁇ g/mL chloramphenicol at 37 °C overnight. A single clone was used to inoculate 200 mL of LB medium with 100 ⁇ g/mL ampicillin and 17 ⁇ g/mL chloramphenicol and incubated at 37 °C on an orbital shaker at 180 rpm.
• the culture was diluted 1 : 100 into 2 L of LB medium and grown until OD 60 o was about 0.3 after which the culture was incubated at 15 °C and 160 rpm. Once OD 6 oo reached about 0.4, IPTG was added to a final concentration of 0.1 mM and the cells were grown overnight at 15 °C and 160 rpm. Cells were harvested by centrifugation at 8000 rpm, for 4 minutes at 4 °C and stored at -80 °C for purification.
• the His tag, TEV and SUMO cleavage sites were removed generating native SMYD3 by addition of ULP1 protein at a ratio of 1 :200 (ULP1 :SMYD3).
• Imidazole was removed by dialysis overnight in Buffer A.
• the dialyzed solution was applied to a second Nickel column and the native SMYD3 protein was collected from the column flow-through.
• the flow- through was dialyzed in Buffer D (25 mM Tris, 5% glycerol, 5 mM ⁇ -mercaptoethanol, 50 mM NaCl, pH7.8) and ULP1 was removed using a Q sepharose fast flow column.
• SMYD3 was eluted in Buffer A and further purified using an S200 size-exclusion column equilibrated with Buffer A.
• SMYD3 was concentrated to 2 mg/mL with a final purity of 89%.
• the assays were all performed in a buffer consisting of 25 mM Tris-Cl pH 8.0, 1 mM TCEP, 0.005% BSG, and 0.005% Tween 20, prepared on the day of use.
• Compounds in 100% DMSO (lul) were spotted into a 384-well white opaque OptiPlate using a Bravo automated liquid handling platform outfitted with a 384-channel head (Agilent Technologies).
• DMSO (lul) was added to Columns 1 1 , 12, 23, 24, rows A-H for the maximum signal control and lul of SAH, a known product and inhibitor of SMYD3, was added to columns 1 1 , 12, 23, 24, rows I-P for the minimum signal control.
• the assays were stopped by the addition of non-radiolabeled SAM (lOul) to a final concentration of 100 uM, which dilutes the 3 H-SAM to a level where its incorporation into MEKK2 is no longer detectable.
• Radiolabeled MEKK2 was detected using a scintillation proximity assay (SPA). 10 uL of a 10 mg/mL solution of SPA beads in 0.5 M citric acid was added and the plates centrifuged at 600 rpm for 1 min to precipitate the radiolabeled MEKK2 onto the SPA beads. The plates were then read in a PerkinElmer TopCount plate reader to measure the quantity of 3 H-labeled MEKK2 as disintegrations per minute (dpm) or alternatively, referred to as counts per minute (cpm).
• dpm disintegrations per minute
• cpm counts per minute
• top and bottom are the normally allowed to float, but may be fixed at 100 or 0 respectively in a 3 -parameter fit.
• the Hill Coefficient normally allowed to float but may also be fixed at 1 in a 3 -parameter fit.
• Y is the % inhibition and X is the compound concentration.
• MEM/Glutamax medium, Optimem Reduced Serum medium, penicillin-streptomycin, 0.05% trypsin and lx D-PBS were purchased from Life Technologies, Grand Island, NY, USA.
• PBS- 1 OX was purchased from Ambion, Life Technologies, Grand Island, New York, USA.
• PBS with Tween 20 was purchased from KPL, Gaithersburg, Maryland,USA.
• Tet System FBS- approved FBS US Source was purchased from Clontech, Mountain View, California, USA.
• Licor Biosciences Lincoln, NE, USA.
• Tri-methyl-Lysine [A260]-MEKK2 antibody, MEKK2 and SMYD3 plasmids were made at Epizyme.
• Anti-flag monoclonal mouse antibody was purchased from Sigma, St. Louis, MO, USA.
• Methanol was purchased from VWR, Franklin, MA, USA.
• 10% Tween 20 was purchased from KPL, Inc., Gaithersburg, Maryland, USA. Fugene was purchased from Promega, Madison, WI, USA.
• the Biotek ELx405 was purchased from BioTek, Winooski, Vermont, USA.
• the multidrop combi was purchased from Thermo Scientific, Waltham, Massachusetts, USA. [0137] 293T/17 adherent cells were maintained in growth medium (MEM/Glutamax medium supplemented with 10% v/v Tet System FBS and cultured at 37 °C under 5% C0 2 .
• Plasmids were prepared for delivery to cells by first mixing 1350 Opti-MEM with Fugene (81 ⁇ ) in a sterile Eppendorf and incubated for five minutes at room temperature (RT).
• MEK 2-f ag (13.6 ug/T150) MEKK2 p3XFlag-CMV-14 with C-3XFlag and SMYD3 (0.151 ug/T150) SMYD3 p3XFlag-CMV-14 without C-3XFlag plasmids were aliquotted to a 1.7 mL sterile microfuge tube.
• the gene ID for MEKK2 and SMYD3 is NM 006609.3 and Q9H7B4, respectively. Entire volume of Opti-MEM/Fugene mixture was then added to a microfuge tube containing DNA plasmid, mixed and then incubated x 15 minutes at RT.
• the medium on the 293T/17 cells was refreshed, and the DNA/Fugene complex is added aseptically to each flask, rocked gently, and incubated at 37 C for 5 hours. Medium was then removed, and cells were washed once with PBS in the flask. Trypsin 0.05%> (3mL) was added and cells incubated for three minutes. Room temperature MEM+10%> Tet system FBS was added and cells were mixed gently, and counted using the Vi-cell. Cells were seeded at 100,000 cells/mL in 50 MEM/10%Tet FBS/Pen/Strep to a 384 well black/clear poly-D-lysine coated plate containing test agent diluted in DMSO.
• test compound was 40 ⁇ .
• the total concentration of DMSO did not exceed 0.2% (v/v).
• Plates were incubated x 30 minutes at RT in low-airflow area, followed by incubation at 37 °C under 5% C0 2 for 24 hours. Medium was aspirated from all wells of assay plates prior to fixation and permeabilization with ice cold (-20 °C) methanol (90 ⁇ ) for ten minutes. Plates were rinsed with PBS three times on BioTek ELx405. PBS was removed with a final aspiration, and Odyssey blocking buffer (50 ⁇ ) was added to each well and incubated for one hour at RT.
• Detection antibody solution (IRDye 800 CW goat anti-rabbit IgG diluted 1 :400 in diluent (Odyssey Blocking buffer + 0.1% Tween 20), plus IRDye 680CW goat anti-mouse IgG at 1 :500 in diluent (Odyssey Blocking buffer + 0.1 % Tween 20) was added (20 ⁇ , ⁇ ) and incubated in dark for one hour at RT. Plates were then washed four times with PBS-T (IX) on ELx405. A final rinse with water was performed (115 ⁇ x three washes on the ELx405).
• IX PBS-T
• Each plate included fourteen control wells of DMSO only treatment (Minimum
• SAM S-adenosylmethionine
• SAH S-adenosylhomocysteine
• bicine Tween20
• dimethylsulfoxide DMSO
• bovine skin gelatin BSG
• Tris(2- carboxyethyl)phosphine hydrochloride TCEP
• Peptide was synthesized with a N-terminal linker-affinity tag motif and a C- terminal amide cap by 21 st Century Biochemicals.
• the peptide was high high-perfomance liquid chromatography (HPLC) purified to greater than 95% purity and confirmed by liquid chromatography mass spectrometry (LC-MS).
• HPLC high-perfomance liquid chromatography
• LC-MS liquid chromatography mass spectrometry
• Full length SMYD2 (NP 064582.2) was cloned into a pFastbac-Htb-lic vector with an N-terminal His6 tag and FLAG tag, preceded by a TEV protease cleavage site.
• the protein was expressed in Sf9 insect cells. Cells were resuspended in lysis buffer (25 mM HEPES-NaOH, pH 7.5, 200 mM NaCl, 5% glycerol, and 5 mM ⁇ - ⁇ ) and lysed by sonication.
• the protein was purified by Ni-NTA (Qiagen), followed by TEV cleavage to remove the His6 tag, subtractive Ni-NTA (Qiagen), and gel filtration chromatography using an S200 column (GE Healthcare). Purified protein was stored in 20 mM Tris-HCl, pH 8.0, 100 mM NaCl, and 1 mM TCEP.
• ImM TCEP 0.005% Bovine Skin Gelatin, and 0.002% Tween20, prepared on the day of use.
• Compounds in 100%) DMSO (lul) were spotted into a polypropylene 384-well V- bottom plates (Greiner) using a Platemate Plus outfitted with a 384-channel head (Thermo Scientific).
• DMSO (lul) was added to Columns 11, 12, 23, 24, rows A-H for the maximum signal control and lul of SAH, a known product and inhibitor of SMYD2, was added to columns 11, 12, 23, 24, rows I-P for the minimum signal control.
• a cocktail (40ul) containing the SMYD2 enzyme was added by Multidrop Combi (Thermo-Fisher).
• the final concentrations of the components were as follows: SMYD2 was 1.5nM, 3 H-SAM was lOnM, and peptide was 60nM, SAH in the minimum signal control wells was lOOOuM, and the DMSO concentration was 2%.
• the assays were stopped by the addition of non-radioactive SAM (lOul) to a final concentration of 600uM, which dilutes the 3 H-SAM to a level where its incorporation into the peptide substrate is no longer detectable.
• top and bottom are the normally allowed to float, but may be fixed at 100 or 0 respectively in a 3 -parameter fit.
• the Hill Coefficient normally allowed to float but may also be fixed at 1 in a 3 -parameter fit. / is the compound concentration.

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