salt form of a human ezh2 histone methyltransferase inhibitor

专利摘要:
SALT FORM OF A HUMAN INHIBITOR METHYLTRANSPHERASE HUMAN N - ((4,6-dimethyl-2-oxo-1,2,2-dihydropyridin-3-yl) methyl) -5- (ethyl ( tetrahydro-2H-pyran-4-yl) amino) -4-methyl-4 '- (morpholinomethyl) - [1,1'-biphenyl] -3-carboxamide. A particular polymorphic form of this compound is also provided here.
公开号:BR112014025508B1
申请号:R112014025508-3
申请日:2013-04-11
公开日:2020-11-17
发明作者:Kevin Wayne Kuntz；Kuan-Chun Huang；Hyeong Wook Choi；Kristen Sanders；Steven Mathieu；Arani Chanda；Frank Fang
申请人:Eisai R&D Management Co., Ltd.；Epizyme, Inc.；
IPC主号:

专利说明:

RELATED ORDER
[001] This application claims priority for, and the benefit of, U.S. provisional application No. 61 / 624,215, filed on April 13, 2012, all of the content of which is incorporated herein by reference in its entirety. BACKGROUND OF THE INVENTION
[002] More than 1.6 million people are estimated to be diagnosed with cancer in 2012. For example, the most common type of cancer in women is breast cancer, and this disease is responsible for one of the highest rates of mortality from all cancers affecting women. Current breast cancer treatment is limited to total, or partial, mastectomy, radiation therapy, or chemotherapy. Almost 230,000 of the cancer cases in 2012 will be breast cancer, which will result in an estimated 40,000 deaths. See, Siegel et al., Ca Cancer JClin 2012; 62: 10-29.
[003] Several cancer deaths are caused by blood cancers including leukemias, myelomas and lymphomas. In 2012, almost 80,000 of the cancer cases will be lymphomas, which will result in an estimated 20,000 deaths.
[004] Radiotherapy, chemotherapy and surgery are the main methods of treating cancer. However, these therapies are most successful only when cancer is detected at an early stage. Once the cancer reaches invasive / metastatic stages, invasive cell lines or metastatic cells can escape detection, thus resulting in recurrence, which requires the use of therapy that is highly toxic. At this point, both cancer cells and patients' unaffected cells are exposed to toxic therapy, resulting in, among other complications, a weakening of the immune system.
[005] As such, there remains a need in the art for new methods to treat cancer, such as breast cancer or lymphomas, in a patient. SUMMARY OF THE INVENTION
[006] Accordingly, N - ((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl) methyl) -5- (ethyl (tetrahydro-2H- pyran-4-yl) amino) -4-methyl-4'- (morpholinomethyl) - [1,1 '-biphenyl] -3-carboxamide:

[007] A particular polymorphic form of N - ((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl) methyl) -5- (ethyl (tetra- hydro-2H-pyran-4-yl) amino) -4-methyl-4 '- (morpholinomethyl) - [1,1'-biphenyl] -3-carboxamide (“Polymorph A”, or “N hydrobromide Polymorph A - (((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl) methyl) -5- (ethyl (tetrahydro-2H-pyran-4-yl) amino) -4- methyl-4 '- (morpholinomethyl) - [1,1'-biphenyl] -3-carboxamide ”). As described herein, the hydrobromide salt provided herein, as well as Polymorph A, exhibit physical properties that can be exploited in order to obtain new pharmacological properties and, which can be used in the development of drug substance and drug product.
[008] In one embodiment, the hydrobromide is crystalline. In another embodiment, the hydrobromide is substantially free of impurities. In another embodiment, the hydrobromide is a crystalline solid substantially free of N - ((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl) methyl) -5- ( ethyl (tetrahydro-2H-pyran-4-yl) amino) -4-methyl-4 '- (morpholinomethyl) - [1,1'-biphenyl] -3-amorphous carboxamide.
[009] In another aspect, a pharmaceutical composition comprising the hydrobromide described above and a pharmaceutically acceptable carrier or diluent is provided herein.
[010] In one aspect, the hydrobromide described above is prepared using a method comprising the combination of N - ((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl) methyl) -5 - (ethyl (tetrahydro-2H-pyran-4-yl) amino) -4-methyl-4 '- (morpholinomethyl) - [1,1' -biphenyl] - 3-carboxamide with hydrobromic acid.
[011] Polymorph A of N - ((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl) methyl) -5- (ethyl (tetrahydro-2H-pyran-4 -yl) amino) -4-methyl-4 '- (morpholinomethyl) - [1,1'-biphenyl] -3-carboxamide can be defined according to its X-ray powder diffraction pattern. Consequently, in a one embodiment, the polymorph exhibits an X-ray powder diffraction pattern having one or more characteristic peaks expressed in degrees 2-theta at about 3.9 +/- 0.3 degrees, about 17.5 +/- 0.3 degrees, and about 22.0 +/- 0.3 degrees 2-theta. In another embodiment, the polymorph exhibits an X-ray powder diffraction pattern having characteristic peaks expressed in degrees 2-theta at about 3.9 +/- 0.3 degrees, about 17.5 +/- 0.3 degrees, and about 22.0 +/- 0.3 degrees 2-theta. In yet another embodiment, the polymorph exhibits an X-ray powder diffraction pattern having characteristic peaks expressed in 2-theta degrees at about 3.9 +/- 0.3 degrees, 10.1 +/- 0 , 3 degrees, 14.3 +/- 0.3 degrees, 17.5 +/- 0.3 degrees, 18.7 +/- 0.3 degrees, 20.6 +/- 0.3 degrees, 20, 9 +/- 0.3 degrees, 21.8 +/- 0.3 degrees, 22.0 +/- 0.3 degrees, 23.3 +/- 0.3 degrees and 23.6 +/- 0, 3 degrees 2-theta. In yet another embodiment, the polymorph exhibits an X-ray powder diffraction pattern substantially in accordance with Figure 1. In another embodiment, the polymorph exhibits an X-ray powder diffraction pattern substantially in accordance with Figure 1. with Table 1.
[012] Polymorph A can also be defined according to its differential scanning calorimetry thermogram. In one embodiment, the polymorph exhibits a differential scanning calorimetry thermogram having a characteristic peak expressed in units of ° C at a temperature of 255 +/- 5 ° C. In one embodiment, the polymorph exhibits a differential scanning calorimetry thermogram substantially in accordance with Figure 3.
[013] In one aspect, Polymorph A is prepared using a method comprising the combination of N - ((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl) methyl) - 5- (ethyl (tetrahydro-2H-pyran-4-yl) amino) -4-methyl-4 '- (morpholinomethyl) - [1,1'-biphenyl] -3-carboxamide with hydrobromic acid.
[014] In another aspect, a method of recrystallizing Polymorph A is provided, which comprises the following steps: (a) dissolving Polymorph A in a first solvent, and (b) adding a second solvent, such that said polymorph is recrystallized. In one embodiment, the first solvent is ethanol, and the second solvent is MTBE. In another embodiment, the method comprises (a) dissolving Polymorph A in ethanol, (b) heating the mixture, (c) adding MTBE to the mixture, forming a precipitate comprising said polymorph, and filtering the precipitate such that said polymorph is recrystallized.
[015] In yet another aspect, a pharmaceutical composition comprising Polymorph A, and a pharmaceutically acceptable carrier or diluent is provided herein.
[016] Also provided herein is a method of treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of the hydrobromide compound described above, Polymorph A, or a pharmaceutical composition comprising any of these compounds. A variety of cancers can be treated, including non-Hodgkin's lymphoma or breast cancer.
[017] In another aspect, a method of inhibiting histone methyltransferase activity of EZH2 is provided herein in a subject in need thereof comprising administering to the subject an effective amount of the hydrobromide compound described above, Polymorph A, or a pharmaceutical composition comprising any of these compounds.
[018] In yet another aspect, a method of inhibiting the histone methyltransferase activity of EZH2 in vitrochorizone is provided herein comprising administering the hydrobromide compound described above, or Polymorph A.
[019] Also provided herein is the use of the hydrobromide compound described above, Polymorph A, or a pharmaceutical composition comprising any of these compounds, for the preparation of a medicament for the treatment of cancer in a subject in need thereof. BRIEF DESCRIPTION OF THE DRAWINGS
[020] Figure 1 represents the X-ray powder diffraction pattern of Polymorph A (monohydrobromide).
[021] Figure 2 represents the X-ray powder diffraction pattern of Compound I dihydrobromide.
[022] Figure 3 represents the differential scanning calorimetry thermogram of Polymorph A.
[023] Figure 4 represents the dynamic vapor sorption of Polymorph A, which demonstrates the low hygroscopicity of this compound.
[024] Figure 5 represents HPLC analysis of Polymorph A over three days at an elevated temperature. Polymorph A produced minimal impurities during this time.
[025] Figure 6 represents the dynamic vapor sorption of the sodium salt of Compound I, which demonstrates the significant hygroscopicity of this compound.
[026] Figure 7 represents the dynamic vapor sorption of the hemisulfate salt of Compound I, which demonstrates that this compound has moderately high hygroscopicity.
[027] Figure 8 shows the differential scanning calorimetry data for Compound I mono-hydrochloride salt, which indicates that this compound is deficiently crystalline.
[028] Figure 9 represents the X-ray powder diffraction pattern of synthetic intermediate 5.
[029] Figure 10 represents the Polymorph B powder X-ray powder diffraction pattern.
[030] Figure 11 represents the crystalline X-ray structure of Compound I monohydrobromide.
[031] Figures 12 to 14 show the results of in vivo studies of the Compound I hydrobromide in a human lymphoma cell line.
[032] Figures 15 to 16 show the anti-cancer effect of Compound I hydrobromide in a lymphoma mouse xenograft model.
[033] Figure 17 represents the X-ray powder diffraction pattern of synthetic intermediate 2.
[034] Figures 18A and B represent (A) the X-ray powder diffraction pattern of the Compound I trichloride salt and (B) the dynamic vapor sorption of the Compound I mono-hydrochloride salt, which demonstrates the significant hygroscopicity of this compound. DETAILED DESCRIPTION OF THE INVENTION HBr Salt Form and Polymorph Form A
[035] N - ((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl) methyl) -5- (ethyl (tetrahydro-2H-pyran- 4-yl) amino) -4-methyl-4 '- (morpholinomethyl) - [1,1'-biphenyl] - 3-carboxamide:

[036] As used herein, "Compound I" refers to N - ((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl) methyl) -5- (ethyl ( tetrahydro-2H-pyran-4-yl) amino) -4-methyl-4'- (morpholinomethyl) - [1,1'-biphenyl] -3-carboxamide. The hydrobromide of Compound I can be used to inhibit the histone methyltransferase activity of EZH2, in a subject or in vitro. The compound I hydrobromide can also be used to treat cancer in a subject in need of it.
[037] Compound I can be protonated at one or more of its basic sites, such as portions of morpholine, disubstituted aniline and / or pyridone. Consequently, in certain embodiments, Compound I monohydrobromide, dihydrobromide or trihydrobromide is provided herein. In one embodiment, Compound I monohydrobromide is provided herein. When the compound is mono -hydrobromide, the compound can be protonated in any basic site. In a non-limiting embodiment, Compound I is protonated in the nitrogen of the morpholino substituent, providing a Compound I monohydrobromide having the following structure:

[038] This particular monohydrobromide can be referred to as “4- ((3 '- ((((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl) methyl) carbamoyl bromide ) -5 '- (ethyl (tetrahydro-2H-pyran-4-yl) amino) -4'-methyl- [1,1'-biphenyl] -4-yl) methyl) morpholin-4-io ”. Figure 11 represents the X-ray crystalline structure of this particular salt form.
[039] Compound I hydrobromide has several advantageous physical properties over its free base form, as well as other salts of the free base. In particular, Compound I hydrobromide has low hygroscopicity compared to other forms of Compound I salt. For a compound to be effective in therapy, it is generally necessary that the compound be minimally hygroscopic. Forms of drug that are highly hygroscopic may be unstable, as the rate of drug form dissolution may change as it is stored in environments with varying humidity. Also, hygroscopicity can impact large-scale handling and manufacture of a compound, as it can be difficult to determine the true weight of a hygroscopic active agent when preparing a pharmaceutical composition comprising this agent. The hydrobromide of Compound I has a low hygroscopicity compared to other forms of salt of Compound I. As such, it can be stored for appreciable periods, and does not undergo harmful changes in, for example, solubility, density, or even chemical composition. In addition to the above advantages, Compound I hydrobromide can be produced in a highly crystalline form, which is useful in the preparation of pharmaceutical formulations, and will improve the handling, handling and general storage of the drug compound. In a preferred embodiment, the crystalline form of the Compound I hydrobromide is in a form referred to as "Polymorph A".
[040] The ability of a substance to exist in more than one crystal form is defined as polymorphism; crystal forms other than a particular substance are referred to as "polymorphs". In general, polymorphism is affected by the ability of a molecule of a substance to change its conformation or to form different inter-molecular or intra-molecular interactions, particularly hydrogen bonds, which is reflected in different atom arrangements in the crystal structure of different polymorphs. In contrast, the overall external shape of a substance is known as "morphology", which refers to the external shape of the crystal and the flat surfaces present, without reference to the internal structure. Crystals may exhibit different morphology based on different conditions, such as, for example, growth rate, agitation, and the presence of impurities.
[041] Polymorphs different from a substance can have different energies than the crystal structure and thus, in solid state they can show different physical properties, such as shape, density, melting point, color, stability, solubility, dissolution rate , etc., which can, in turn, affect the stability, dissolution rate and / or bioavailability of a given polymorph and its suitability for use as a pharmaceutical substance and in pharmaceutical compositions.
[042] Polymorph A is highly crystalline, and exhibits low hygroscopicity. Also, this polymorph can be obtained reproductively, and slight changes in crystallization conditions do not result in different crystal forms.
[043] Access to polymorphs other than the Compound I hydrobromide is desirable for several reasons. One reason is that individual polymorphs can incorporate different impurities, or chemical residues, through crystallization. For example, impurities can be removed during the Polymorph A. Compound I coating process.
[044] Without pretending to be limited by theory, the polymorphous form that exhibits compact crystal forms has advantages in terms of ease of filtration and ease of flow. Polymorph A exhibits a compact crystal shape, which therefore has these advantages.
[045] In certain embodiments, Polymorph A is identifiable on the basis of characteristic peaks in an X-ray powder diffraction analysis. X-ray powder diffraction, also referred to as XRPD, is a scientific technique using diffraction in X-ray powder, neutrons or electrons, microcrystalline, or other solid materials for the structural characterization of these materials. In one embodiment, Polymorph A exhibits an X-ray powder diffraction pattern having one or more characteristic peaks expressed in 2-theta degrees at about 3.9 +/- 0.3 degrees, about 17.5 +/- 0.3 degrees, and about 22.0 +/- 0.3 degrees 2-theta. In another embodiment, the polymorph exhibits an X-ray powder diffraction pattern having characteristic peaks expressed in degrees 2-theta at about 3.9 +/- 0.3 degrees, about 17.5 +/- 0.3 degrees, and about 22.0 +/- 0.3 degrees 2-theta.
[046] In one embodiment, Polymorph A exhibits an X-ray powder diffraction pattern having at least 5 characteristic peaks expressed in 2-theta degrees at about 3.9 +/- 0.3 degrees, 10, 1 +/- 0.3 degrees, 14.3 +/- 0.3 degrees, 17.5 +/- 0.3 degrees, 18.7 +/- 0.3 degrees, 20.6 +/- 0, 3 degrees, 20.9 +/- 0.3 degrees, 21.8 +/- 0.3 degrees, 22.0 +/- 0.3 degrees, 23.3 +/- 0.3 degrees and 23.6 +/- 0.3 degrees 2-theta. In another embodiment, Polymorph A exhibits an X-ray powder diffraction pattern having at least 6 characteristic peaks expressed in 2-theta degrees at about 3.9 +/- 0.3 degrees, 10.1 + / - 0.3 degrees, 14.3 +/- 0.3 degrees, 17.5 +/- 0.3 degrees, 18.7 +/- 0.3 degrees, 20.6 +/- 0.3 degrees , 20.9 +/- 0.3 degrees, 21.8 +/- 0.3 degrees, 22.0 +/- 0.3 degrees, 23.3 +/- 0.3 degrees and 23.6 + / - 0.3 degrees 2-theta. In yet another embodiment, Polymorph A exhibits an X-ray powder diffraction pattern having at least 7 characteristic peaks expressed in 2-theta degrees at about 3.9 +/- 0.3 degrees, 10.1 +/- 0.3 degrees, 14.3 +/- 0.3 degrees, 17.5 +/- 0.3 degrees, 18.7 +/- 0.3 degrees, 20.6 +/- 0.3 degrees, 20.9 +/- 0.3 degrees, 21.8 +/- 0.3 degrees, 22.0 +/- 0.3 degrees, 23.3 +/- 0.3 degrees and 23.6 + / - 0.3 degrees 2-theta. In another embodiment, Polymorph A exhibits an X-ray powder diffraction pattern having at least 8 characteristic peaks expressed in 2-theta degrees at about 3.9 +/- 0.3 degrees, 10.1 + / - 0.3 degrees, 14.3 +/- 0.3 degrees, 17.5 +/- 0.3 degrees, 18.7 +/- 0.3 degrees, 20.6 +/- 0.3 degrees , 20.9 +/- 0.3 degrees, 21.8 +/- 0.3 degrees, 22.0 +/- 0.3 degrees, 23.3 +/- 0.3 degrees and 23.6 + / - 0.3 degrees 2-theta. In yet another embodiment, Polymorph A exhibits an X-ray powder diffraction pattern having at least 9 characteristic peaks expressed in 2-theta degrees at about 3.9 +/- 0.3 degrees, 10.1 +/- 0.3 degrees, 14.3 +/- 0.3 degrees, 17.5 +/- 0.3 degrees, 18.7 +/- 0.3 degrees, 20.6 +/- 0.3 degrees, 20.9 +/- 0.3 degrees, 21.8 +/- 0.3 degrees, 22.0 +/- 0.3 degrees, 23.3 +/- 0.3 degrees and 23.6 + / - 0.3 degrees 2-theta. In yet another embodiment, the polymorph exhibits an X-ray powder diffraction pattern having at least 10 characteristic peaks expressed in 2-theta degrees at about 3.9 +/- 0.3 degrees, 10.1 + / - 0.3 degrees, 14.3 +/- 0.3 degrees, 17.5 +/- 0.3 degrees, 18.7 +/- 0.3 degrees, 20.6 +/- 0.3 degrees , 20.9 +/- 0.3 degrees, 21.8 +/- 0.3 degrees, 22.0 +/- 0.3 degrees, 23.3 +/- 0.3 degrees and 23.6 + / - 0.3 degrees 2-theta.
[047] In yet another embodiment, the polymorph exhibits an X-ray powder diffraction pattern having characteristic peaks expressed in 2-theta degrees at about 3.9 +/- 0.3 degrees, about 14, 3 +/- 0.3 degrees, about 18.7 +/- 0.3 degrees, about 23.3 +/- 0.3 degrees, and about 23.6 +/- 0.3 degrees 2- theta.
[048] In yet another embodiment, the polymorph exhibits an X-ray powder diffraction pattern having characteristic peaks expressed in 2-theta degrees at about 3.9 +/- 0.3 degrees, about 10, 1 +/- 0.3 degrees, about 14.3 +/- 0.3 degrees, about 17.5 +/- 0.3 degrees, about 18.7 +/- 0.3 degrees, about 20.6 +/- 0.3 degrees, about 20.9 +/- 0.3 degrees, about 21.8 +/- 0.3 degrees, about 22.0 +/- 0.3 degrees, about 23.3 +/- 0.3 degrees and about 23.6 +/- 0.3 degrees 2-theta. In yet another embodiment, the polymorph exhibits an X-ray powder diffraction pattern substantially in accordance with Figure 1. In another embodiment, the polymorph exhibits an X-ray powder diffraction pattern substantially in accordance with Figure 1. with the 2-theta values listed in Table 1.
[049] As used herein, the term "about", when used in reference to a 2-theta grade value refers to the established value +/- 0.3 degrees 2-theta.
[050] Pharmaceutical compositions comprising Polymorph A can be identified by comparing the X-ray powder diffraction patterns of the compositions to a Polymorph A X-ray powder diffraction pattern. It will be appreciated that pharmaceutical compositions comprising Polymorph A may exhibit non-identical X-ray powder diffraction patterns as compared to a pure Polymorph A X-ray powder diffraction pattern.
[051] In certain embodiments, Polymorph A is identifiable on the basis of a characteristic peak observed in a differential scanning calorimetry thermogram. Differential scanning calorimetry, or DSC, is a thermoanalytical technique, in which the difference in the amount of heat required to increase the temperature of a sample and reference is measured as a function of temperature. In one embodiment, Polymorph A displays a differential scanning calorimetry thermogram having a characteristic peak expressed in units of ° C at a temperature of about 255 +/- 5 ° C. In another embodiment, Polymorph A exhibits a differential scanning calorimetry thermogram having a single endothermic peak observed in the temperature range 250 to 255 ° C. In another embodiment, Polymorph A exhibits a differential scanning calorimetry thermogram substantially in accordance with Figure 3.
[052] In certain embodiments, Polymorph A may contain impurities. Non-limiting examples of impurities include unwanted polymorphous form, or residual organic and inorganic molecules, such as solvents, water or salts. In one embodiment, Polymorph A is substantially free of impurities. In another embodiment, Polymorph A contains less than 10% by weight of total impurities. In another embodiment, Polymorph A contains less than 5% by weight of total impurities. In another embodiment, Polymorph A contains less than 1% by weight of total impurities. In yet another embodiment, Polymorph A contains less than 0.1% by weight of total impurities.
[053] In certain embodiments, Polymorph A is a crystalline solid substantially free of amorphous Compound I hydrobromide. As used herein, the term "substantially free of amorphous Compound I hydrobromide" means that the compound does not contain a significant amount of amorphous Compound I hydrobromide. In certain embodiments, at least about 95% by weight of crystalline Polymorph A is present. In still other embodiments of the invention, at least about 99% by weight of crystalline Polymorph A is present.
[054] In another embodiment, Polymorph A is substantially free of Polymorph B.
[055] The salt of the invention, and its crystal form of Polymorph A, can be found together with other substances or can be isolated. In some embodiments, the salt of the invention, or its crystal form, is substantially isolated. By "substantially isolated" it means that the salt or its crystal form is at least partially or substantially separated from the environment in which it was formed or detected. Partial separation can include, for example, a composition enriched in the salt of the invention. Substantial separation can include compositions containing at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least at least about 97%, or at least about 99% by weight of the Compound I and Polymorph A hydrobromide. Methods for isolating the compounds and their salts are routine in the art.
[056] Both Compound I hydrobromide and Polymorph A can occur as any reasonable tautomer, or a mixture of reasonable tautomers. As used herein, the "tautomer" refers to one of two or more structural isomers that exist in equilibrium and are easily converted from one isomeric form to another. Examples include keto-enol tautomers, such as acetone / propen-2-ol, and the like. The hydrobromide of Compound I and Polymorph A can have one or more tautomers and therefore include several isomers, i.e., pyridin-2 (1H) -one and the corresponding pyridin-2-ol. All such isomeric forms of these compounds are expressly included in the present invention. Preparation of HBr and Polymorph A Salt Form
[057] Compound I hydrobromide, as well as Polymorph A, can be prepared using known techniques. Conventionally, a salt form is prepared by combining the free base compound and an acid containing the anion of the desired salt form in solution and then isolating the solid salt product from the reaction solution (for example, by crystallization, precipitation, evaporation, etc.). Other salt-forming techniques can be used.
[058] Scheme 1 below outlines a particular embodiment for the production of the free base of Compound I, as well as the hydrobromide of Compound I. Briefly, methyl 3-amino-5-bromo-2-methylbenzoate (1) is reacted with dihydro-2H-pyran-4 (3H) -one under reductive amination conditions to form 5-bromo-2-methyl-3 - ((tetrahydro-2H-pyran-4-yl) amino) methyl benzoate (2) in step 1. In step 2, the reductive amination is again used to form 5-bromo-3- (ethyl (tetrahydro-2H-pyran-4-yl) amino) -2-methylbenzoate ( 3). This compound is then reacted under Suzuki coupling conditions in step 3 to form 5- (ethyl (tetrahydro-2H-pyran-4-yl) amino) -4-methyl-4 '- (morpholinomethyl) - [ Methyl 1,1'-biphenyl] -3-carboxylate (4), which is hydrolyzed to the corresponding acid (5) in step 4. In step 5, acid (5) is reacted under amide-hydrochloride coupling reaction conditions 3- (aminomethyl) -4,6-dimethyl-dihydro-pyridin-2 (1H) -one to form Compound I.
[059] As shown, Compound I can then be reacted with aqueous HBr to form Compound I hydrobromide.

[060] The synthesis described above has several advantages. For example, it uses several intermediates that can be prepared in crystalline forms that can be isolated. Using crystalline intermediates, minimal purification techniques (for example, chromatography) are necessary, leading to an improved overall yield of final Compound I.
[061] Consequently, intermediate compound 1 in crystalline form is provided here. In another embodiment, intermediate compound 2 in crystalline form is provided here. Figure 17 shows an X-ray powder diffraction pattern of crystalline compound 2. In yet another embodiment, intermediate compound 5 is crystalline. Figure 9 shows an X-ray powder diffraction pattern of crystalline compound 5. In other embodiments, Compounds 2 and / or 5 are produced in substantially pure form without the use of chromatography. It will be assessed by the skilled technician that the crystallization of intermediates does not necessarily proceed easily or efficiently.
[062] Also provided herein is a method of preparing N - ((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl) methyl) -5- (ethyl (tetrahydro-2H -pyran-4-yl) amino) -4-methyl-4'- (morpholinomethyl) - [1,1'-biphenyl] -3-carboxamide comprising reacting 5- (ethyl (tetrahydro-2H-pyran-4) acid -yl) amino) -4-methyl-4 '- (morpholinomethyl) - [1,1' -biphenyl] -3-carboxylic (5) with a salt of 3- (aminomethyl) -4,6-dimethyl-di- hydro-pyridin-2 (1H) -one. In an embodiment of this method, (5) is in crystalline form.
[063] Compound I can be reacted with aqueous HBr in the presence of an appropriate solvent to form Polymorph A, a particular crystal form of the hydrobromide. In one embodiment, Compound I is reacted with aqueous HBr in the presence of ethanol and ethyl acetate to form Polymorph A.
[064] Once the polymorph is prepared, it can be recrystallized, using the same solvent (or solvents) that were used to prepare the polymorph, or a different solvent (or solvents), to produce a composition that has increased crystallinity. In general, Polymorph A can be recrystallized by dissolving the polymorph in one or more solvents, optionally heating, followed by an optional cooling step and then isolating the crystalline structure, for example, through a filtering step. After the polymorph is initially dissolved in the first solvent (or combination of solvents), an additional different solvent can be added at any point in the process (before or after heating, before or after cooling, etc.) to produce the desired crystalline structure . For example, a first solvent can be used to dissolve the polymorph compound and then a second solvent (for example, an anti-solvent) can be added to make the polymorph precipitate out of the solution. In one embodiment, water is added to the first solvent to assist in dissolving the polymorph.
[065] Non-limiting examples of solvents that can be used for the recrystallization of Polymorph A are as follows: methanol, ethanol, ethyl acetate, tert-butyl methyl ether, water, isopropyl alcohol, tetrahydrofuran, acetone, acetonitrile, and 2-methyltetrahydrofuran, as well as combinations thereof. Non-limiting examples of solvent combinations that are useful for the recrystallization of Polymorph A are (solvent and anti-solvent, where water can be added to the first solvent to aid in the dissolution of the polymorph): methanol / water and ethyl acetate, isopropyl alcohol / water and ethyl acetate, tetrahydrofuran / water and ethyl acetate, acetone and ethyl acetate, acetonitrile / water and ethyl acetate, ethanol / water and tert-butyl methyl ether, isopropyl alcohol / water and methyl ether tert-butyl, ethanol / water and tetrahydrofuran, isopropyl alcohol / water and acetone, and ethanol / water and ethyl acetate. In particular embodiments, the solvent combinations are methanol / water and ethyl acetate, isopropyl alcohol / water and ethyl acetate, ethanol / water and 2-methyltetrahydrofuran, and methanol / 2-methyltetrahydrofuran.
[066] In one aspect, Polymorph A is prepared using a method comprising the combination of N - ((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl) methyl) - 5- (ethyl (tetrahydro-2H-pyran-4-yl) amino) -4-methyl-4 '- (morpholinomethyl) - [1,1'-biphenyl] -3-carboxamide with hydrobromic acid.
[067] In another aspect, a method of recrystallizing Polymorph A is provided, which comprises the following steps: (a) dissolving Polymorph A in a first solvent, and (b) adding a second solvent, such that said polymorph is recrystallized. In one embodiment, the first solvent is ethanol, and the second solvent is MTBE. In another embodiment, the method comprises (a) dissolving Polymorph A in ethanol, (b) heating the mixture, (c) adding MTBE to the mixture, forming a precipitate comprising said polymorph, and filtering the precipitate such that said polymorph is recrystallized. Pharmaceutical Compositions
[068] In another aspect, a pharmaceutical composition comprising the hydrobromide of Compound I, and a pharmaceutically acceptable carrier or diluent is provided herein. Also provided herein is a pharmaceutical composition comprising Polymorph A, and a pharmaceutically acceptable carrier or diluent.
[069] The term "pharmaceutical composition" includes preparations suitable for administration to mammals, for example, humans. When the compounds of the present invention are administered as medicaments for mammals, for example, humans, they can be supplied by themselves or as a pharmaceutical composition containing, for example, 0.1% to 99.9% (more preferably, 0, 5 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.
[070] The compounds described herein (i.e., Compound I hydrobromide and Polymorph A) can be combined with a pharmaceutically acceptable carrier according to conventional pharmaceutical composition techniques. As used herein, "pharmaceutically acceptable carrier" may include any and all solvents, diluents, or other liquid carrier, dispersion or suspending aids, active surface agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and similar, as suitable for the particular desired dosage form. Remington’s Pharmaceutical Sciences, Sixteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980) discloses various vehicles used in the formulation of pharmaceutical compositions and techniques known for the preparation thereof. Except to the extent that any conventional carrier medium is incompatible with the compounds, such as producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component of the pharmaceutical composition, its use is considered to be within the scope of this invention. Some examples of materials that can serve as pharmaceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatine; baby powder; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil, sesame oil; olive oil; corn oil and soy oil; glycols; such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline solution; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants they may also be present in the composition, according to the formulator's judgment.
[071] In addition, the vehicle can take a wide variety of forms depending on the form of the desired preparation for administration, for example, oral, nasal, rectal, vaginal, parenteral (including intravenous injections or infusions). In the preparation of compositions for the oral dosage form any of the usual pharmaceutical means can be used. Usual pharmaceutical means include, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, and the like in the case of liquid oral preparations (such as, for example, suspensions, solutions, emulsions and elixirs); aerosols; or vehicles such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents and the like, in the case of solid oral preparations (such as, for example, powders, capsules and tablets).
[072] Wetting, emulsifying and lubricating agents, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions .
[073] Examples of pharmaceutically acceptable antioxidants include: water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gall, tocopherols, and the like; and metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
[074] Pharmaceutical compositions comprising the compounds can be formulated to have any desired concentration. In some embodiments, the composition is formulated such that it comprises at least a therapeutically effective amount. In some embodiments, the composition is formulated such that it comprises an amount that would not cause one or more unwanted side effects.
[075] As the crystalline form of the Compound I hydrobromide is more easily maintained during its preparation, solid dosage forms are a preferred form for the pharmaceutical composition of the invention. Solid dosage forms for oral administration, such as capsules, tablets, pills, powders, and granules, are particularly preferred. If desired, the tablets can be coated by techniques known to those skilled in the art.
[076] The pharmaceutical compositions include those suitable for oral, sublingual, nasal rectal, vaginal, topical, buccal and parenteral (including subcutaneous, intramuscular and intravenous) administration, although the most appropriate route will depend on the nature and severity of the condition being treated. The compositions can be conveniently presented in unit dosage form, and prepared by any of the methods well known in the pharmacy art. In certain embodiments, the pharmaceutical composition is formulated for oral administration in the form of a pill, capsule, expectorant tablet or tablet. In other embodiments, the pharmaceutical composition is in the form of a suspension.
[077] The compounds provided here are suitable as an active agent in pharmaceutical compositions that are particularly effective in treating disorders associated with EZH2, especially cancer. The pharmaceutical composition in various embodiments has a pharmaceutically effective amount of the Compound I or Polymorph A hydrobromide, along with other pharmaceutically acceptable excipients, vehicles, fillers, diluents and the like.
[078] A therapeutically or pharmaceutically "effective amount" is an amount of a compound (Compound I hydrobromide or Polymorph A), which when administered to a patient, improves a symptom of a disease or condition, for example, avoids the various morphological and somatic symptoms of cancer. In one example, an effective amount of the Compound I or Polymorph A hydrobromide is the amount sufficient to treat cancer in a subject. The amount can vary depending on such factors as the subject's size and weight, the type of disease, or the particular compound of the invention. The amount of the Compound I or Polymorph A hydrobromide that constitutes an "effective amount" will vary depending on the compound, the condition of the disease and its severity, the age of the patient to be treated, and the like. The effective amount can be determined routinely by a person of ordinary skill in the art taking into account their knowledge and this disclosure.
[079] The administration regimen may affect what constitutes a pharmaceutically effective amount. The hydrobromide of Compound I or Polymorph A, and compositions comprising any of these compounds, can be administered to the subject before or after the onset of a disease. In addition, several divided dosages, as well as staggered dosages, can be administered daily or sequentially, or the dose can be continuously infused, or it can be a bolus injection. In addition, dosages can be proportionally increased or decreased as indicated by the requirements of the therapeutic or prophylactic situation. Treatment Methods
[080] The compounds of the present invention (i.e., Compound I hydrobromide, as well as Polymorph A) inhibit the histone methyltransferase activity of EZH2 or a mutant thereof and, consequently, in one aspect of the invention, certain compounds disclosed herein are candidates to treat, or prevent certain conditions and diseases. The present invention provides methods for treating conditions and diseases of the course which can be influenced by modulating the methylation status of histones or other proteins, wherein said methylation status is mediated at least in part by the activity of EZH2. The modulation of the methylation status of histones can, in turn, influence the level of expression of target genes activated by methylation, and / or target genes suppressed by methylation. The method includes administering to a subject in need of such treatment, a therapeutically effective amount of a compound of the present invention.
[081] The disorder, in which EZH2-mediated protein methylation plays a part may be cancer or a precancerous condition. The present invention further provides the use of a compound of the present invention (i.e., Compound I hydrobromide, as well as Polymorph A) in the treatment of cancer or pre-cancer of which course it can be influenced by modulating protein-mediated methylation by EZH2, or, for the preparation of a medicine useful for the treatment of such cancer or pre-cancer. Exemplary cancers that can be treated include lymphomas, including non-Hodgkin's lymphoma, follicular lymphoma (F L) and diffuse large B cell lymphoma (DLBC L); melanoma; and leukemia, including CML. The exemplary precancerous condition includes myelodysplastic syndrome (MDS; formerly known as pre-leukemia).
[082] In yet another embodiment, a method of treating a lymphoma is provided here comprising administering to the subject in need of it an effective amount of the Compound I hydrobromide.
[083] In yet another embodiment, a method of treating a lymphoma is provided here comprising administering to a subject in need thereof an effective amount of Polymorph A.
[084] The present invention also provides methods of protecting against a disorder, in which EZH2-mediated protein methylation plays a part in a subject in need of it by administering a therapeutically effective amount of the compound of the present invention (i.e., the hydrobromide of Compound I, as well as Polymorph A) to a subject in need of such treatment. The disorder can be cancer, for example, cancer in which EZH2-mediated protein methylation plays a role. The present invention also provides the use of the compound of the present invention (i.e., the Compound I hydrobromide, as well as Polymorph A) for the preparation of a medicament useful for the prevention of a cell proliferative disorder associated, at least in part, with EZH2-mediated protein methylation.
[085] The compounds of this invention can be used to modulate protein methylation (for example, histone), for example, to modulate histone methyltransferase or histone demethylase activity. At least some of the compounds of the invention can be used in vivo or in vitro to modulate protein methylation. Histone methylation has been reported to be involved in aberrant expression of certain genes in cancers, and in silencing neuronal genes in non-neuronal cells. At least some compounds described herein are suitable candidates for treating these diseases, that is, decreasing methylation or restoring methylation to approximately the level of, on the other hand, normal cells.
[086] Compounds that are methylation modulators can be used to modulate cell proliferation. For example, in some cases excessive proliferation can be reduced with agents that decrease methylation, while insufficient proliferation can be stimulated with agents that increase methylation. Consequently, diseases that can be treated by the compounds of the invention can include hyperproliferative diseases, such as growth of benign cells and growth of malignant cells.
[087] As used herein, a “subject in need of this” is a subject having a disorder, in which EZH2-mediated protein methylation plays a part, or a subject having an increased risk of developing such a disorder in relation to the population, generally. A subject in need of this may have a precancerous condition. Preferably, a subject in need of this has cancer. A "subject" includes a mammal. The mammal can be, for example, a suitable human or non-human mammal, such as primate, mouse, rat, dog, cat, cow, horse, goat, camel, sheep or pig. The subject can also be a bird or chicken. In one embodiment, the mammal is a human being.
[088] As used herein, the term "cell proliferative disorder" refers to conditions, in which unregulated or abnormal growth, or both, of cells can lead to the development of an unwanted condition or disease, which may or may not be cancerous . Exemplary cell proliferative disorders that can be treated with the compounds of the invention cover a variety of conditions, in which cell division is unregulated. Exemplary cell proliferative disorders include, but are not limited to, neoplasms, benign tumors, malignant tumors, precancerous conditions, tumors in situ, encapsulated tumors, metastatic tumors, liquid tumors, solid tumors, immunological tumors, hematological tumors, cancers, carcinomas, leukemias, lymphomas, sarcomas, and rapidly dividing cells. The term "rapidly dividing cell" as used herein is defined as any cell that divides at a rate that exceeds or is greater than what is expected or observed between neighboring or juxtaposed cells within the same tissue. A cell proliferative disorder includes a precancer or precancerous condition. A cell proliferative disorder includes cancer. In one aspect, the methods provided herein are used to treat or alleviate a symptom of cancer or to identify suitable candidates for such purposes. The term "cancer" includes solid tumors, as well as hematological tumors and / or malignancies. A "precancerous cell" or "precancerous cell" is a cell manifesting a cell proliferative disorder that is either a precancer or a precancerous condition. A "cancer cell" or "cancer cell" is a cell manifesting a cell proliferative disorder that is a cancer. Any reproducible means of measurement can be used to identify cancer cells or pre-cancer cells. Cancer cells or pre-cancer cells can be identified by identification or histological classification of a tissue sample (for example, a biopsy sample). Cancer cells or pre-cancer cells can be identified through the use of appropriate molecular markers.
[089] Exemplary non-cancerous conditions or disorders that can be treated using one or more compounds of the present invention include, but are not limited to, rheumatoid arthritis; inflammation; autoimmune disease; lymphoproliferative conditions; acromegaly; rheumatoid spondylitis; osteoarthritis; gout, other arthritic conditions; sepsis; septic shock; endotoxic shock; sepsis by gram-negatives; toxic shock syndrome; asthma; adult respiratory distress syndrome; chronic obstructive pulmonary disease; chronic lung inflammation; inflammatory bowel disease; Crohn's disease; psoriasis; eczema; ulcerative colitis; pancreatic fibrosis; hepatic fibrosis; acute and chronic kidney disease; irritable bowel syndrome; pyrése; restenosis; cerebral malaria; stroke and ischemic injury; neural trauma; Alzheimer's disease; Huntington's disease; Parkinson's disease; acute and chronic pain; allergic rhinitis; allergic conjunctivitis; Chronic heart failure; acute coronary syndrome; cachexia; malaria; leprosy; leishmaniasis; Lyme disease; Reiter's syndrome; acute synovitis; muscle degeneration, bursitis; tendonitis; tenosynovitis; hernia, ruptures, or prolapsed intervertebral disc syndrome; osteopetrosis; thrombosis; restenosis; silicosis; pulmonary sarcosis; bone resorption diseases, such as osteoporosis; graft-versus-host reaction; Multiple sclerosis; lupus; fibromyalgia; AIDS and other viral diseases such as Herpes Zoster, Herpes Simplex I or II, influenza virus and cytomegalovirus; and diabetes mellitus.
[090] Exemplary cancers that can be treated using one or more compounds of the present invention include, but are not limited to, adrenocortical carcinoma, AIDS-related cancers, AIDS-related lymphoma, anal cancer, anorectal cancer, anal canal cancer, appendix cancer, infantile cerebellar astrocytoma, infantile cerebral astrocytoma, basal cell carcinoma, skin cancer (non-melanoma), bile duct cancer, extrahepatic bile tube cancer, intrahepatic bile tube cancer, bladder cancer , bladder cancer, bone and joint cancer, osteosarcoma and malignant fibrous histiocytoma, brain cancer, brain tumor, brain stem glioma, cerebellar astrocytoma, brain astrocytoma / malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumors, visual glioma of the pathway and hypothalamic, breast cancer, bronchial adenomas / carcinoids, carcinoid, gastrointestinal tumor, cancer of the nervous system, lymphoma of the nervous system cancer, central nervous system cancer, central nervous system lymphoma, cervical cancer, childhood cancers, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorders, colon cancer, colorectal cancer, cutaneous T-cell lymphoma, lymphoid neoplasm, fungal mycosis , Seziary syndrome, endometrial cancer, esophageal cancer, extracranial germ cell tumor, extragonal germ cell tumor, extrahepatic bile tube cancer, eye cancer, intraocular melanoma, retinoblastoma, gallbladder cancer, gastric cancer (stomach ), gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumor, ovarian germ cell tumor, gestational trophoblastic glioma tumor, head and neck cancer, hepatocellular (liver) cancer, Hodgkin's lymphoma, hypopharyngeal cancer, melanoma intraocular, eye cancer, islet cell tumors (endocrine pancreas), Sarcoma Kaposi's disease, kidney cancer, kidney cancer, kidney cancer, larynx cancer, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, hairy cell leukemia, lip and oral cavity cancer, liver cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, AIDS-related lymphoma, non-Hodgkin's lymphoma, primary central nervous system lymphoma, Waldenstram macroglobulinemia, medulloblastoma, melanoma, intraocular melanoma (eye ), merkel cell carcinoma, malignant mesothelioma, mesothelioma, metastatic squamous cell cancer, mouth cancer, tongue cancer, multiple endocrine neoplasia syndrome, mycosis fungoides, myelodysplastic syndromes, myelodysplastic / myeloproliferative diseases, myelogenous leukemia, chronic myelogenous leukemia acute, multiple myeloma, chronic myeloproliferative disorders, nasopharynx cancer, neuroblastoma, oral cancer, cancer of the oral cavity, oropharyngeal cancer, ovarian cancer, ovarian epithelial cancer, ovarian tumor of low malignant potential, pancreatic cancer, pancreatic islet cell cancer, paranasal sinus and nasal cavity cancer, parathyroid cancer, penis cancer, pharynx cancer, pheochromocytoma, pineoblastoma and supratentorial primitive neuroectodermal tumors, pituitary tumor, plasma cell neoplasm / multiple myeloma, pleuropulmonary blastoma, prostate cancer, rectal cancer, renal pelvis and ureter, transitional cell cancer, retinoblastoma, rhabdomycoma, of salivary glands, Ewing family of sarcoma tumors, Kaposi's sarcoma, soft tissue sarcoma, uterine cancer, uterine sarcoma, skin cancer (non-melanoma), skin cancer (melanoma), skin merkel cell carcinoma, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, stomach (gastric) cancer, primitive neuroectodermal tumors supratentorial, testicular cancer, throat cancer, thymoma, thymoma and thymic carcinoma, thyroid cancer, transitional cell cancer of the renal pelvis and uterus and other urinary organs, gestational trophoblastic tumor, urethra cancer, endometrial uterine cancer, uterine sarcoma, uterine body cancer, vaginal cancer, vulvar cancer and Wilm's Tumor.
[091] A "hematological cell proliferative disorder" is a cell proliferative disorder involving cells of the hematological system. A cell proliferative disorder of the hematological system may include lymphoma, leukemia, myeloid neoplasms, mast cell neoplasms, myelodysplasia, benign monoclonal gammopathy, lymphomatoid granulomatosis, lymphoid papulosis, polycythemia vera, chronic myelocytic leukemia, essential agonogenic myeloid metaplasia and thrombogenic myeloid. A cell proliferative disorder of the hematological system can include hyperplasia, dysplasia and metaplasia of cells in the hematological system. In one aspect, the compositions of the present invention can be used to treat a cancer selected from the group consisting of a hematological cancer of the present invention or a hematological cell proliferative disorder of the present invention, or used to identify suitable candidates for such purposes. A hematological cancer of the present invention can include multiple myeloma, lymphoma (including Hodgkin's lymphoma, non-Hodgkin's lymphoma, childhood lymphomas, and lymphomas of lymphocytic and cutaneous origin), leukemia (including childhood leukemia, tricoleukemia, acute lymphocytic leukemia, myelocytic leukemia acute, chronic lymphocytic leukemia, chronic myelocytic leukemia, chronic myelogenous leukemia, and mast cell leukemia), myeloid neoplasms and mast cell neoplasms.
[092] A "cell proliferative disorder of the lung" is a cell proliferative disorder that involves the cells of the lung. Lung cell proliferative disorders can include all forms of cell proliferative disorders that affect lung cells. Cellular proliferative disorders of the lung can include lung cancer, a precancerous or precancerous condition of the lung, benign growths or lesions of the lung, and malignant growths or lesions of the lung, and metastatic lesions in tissue and organs in the body except the lung. In one aspect, the compositions of the present invention can be used to treat lung cancer or cell proliferative disorders of the lung, or used to identify suitable candidates for such purposes. Lung cancer can include all forms of lung cancer. Lung cancer can include malignant lung neoplasms, carcinoma in situ, typical carcinoid tumors, and atypical carcinoid tumors. Lung cancer can include small cell lung cancer (“SCLC”), non-small cell lung cancer (“NSCLC”), squamous cell carcinoma, adenocarcinoma, small cell carcinoma, large cell carcinoma, carcinoma adenosquamous cells and mesothelioma. Lung cancer can include cancer in lung scars “scar carcinoma”, bronchialveolar carcinoma, giant cell carcinoma, spindle cell carcinoma and large cell neuroendocrine carcinoma. Lung cancer can include lung cancers having histological and ultrastructural heterogeneity (for example, mixed cell types).
[093] Lung cell proliferative disorders can include all forms of cell proliferative disorders that affect lung cells. Cellular proliferative disorders of the lung can include lung cancer, precancerous conditions of the lung. Cellular proliferative disorders of the lung can include hyperplasia, metaplasia and dysplasia of the lung. Cellular proliferative disorders of the lung can include asbestos-induced hyperplasia, squamous metaplasia and benign reactive mesothelial metaplasia. Cellular proliferative disorders of the lung may include replacement of columnar epithelium with stratified squamous epithelium and mucosal dysplasia. Individuals exposed to harmful inhaled environmental agents such as cigarette smoke and asbestos may be at increased risk for developing lung cell proliferative disorders. Previous lung diseases that may predispose individuals to the development of cell proliferative disorders of the lung may include chronic interstitial lung disease, necrotizing lung disease, scleroderma, rheumatoid disease, sarcoidosis, interstitial pneumonitis, tuberculosis, repeated pneumonias, idiopathic pulmonary fibrosis, granulomata, asbestosis, fibrosing alveolitis, and Hodgkin's disease.
[094] A "cell proliferative disorder of the colon" is a cell proliferative disorder that involves the cells of the colon. Preferably, the cell proliferative disorder of the colon is colon cancer. In one aspect, the compositions of the present invention can be used to treat colon cancer or cell proliferative disorders of the colon, or used to identify suitable candidates for such purposes. Colon cancer can include all forms of colon cancer. Colon cancer can include sporadic and hereditary colon cancers. Colon cancer can include malignant colon neoplasms, carcinoma in situ, typical carcinoid tumors and atypical carcinoid tumors. Colon cancer can include adenocarcinoma, squamous cell carcinoma and adenosquamous cell carcinoma. Colon cancer may be associated with a hereditary syndrome selected from the group consisting of colorectal cancer without hereditary polyposis, familial adenomatous polyposis, Gardner's syndrome, Peutz-Jeghers syndrome, Turcot syndrome and juvenile polyposis. Colon cancer can be caused by a hereditary syndrome selected from the group consisting of colorectal cancer without hereditary polyposis, familial adenomatous polyposis, Gardner's syndrome, Peutz-Jeghers syndrome, Turcot syndrome and juvenile polyposis.
[095] Colonic cell proliferative disorders can include all forms of cell proliferative disorders that affect colon cells. Colon cell proliferative disorders can include colon cancer, precancerous conditions of the colon, adenomatous polyps of the colon, and metachronic colon lesions. A cell proliferative disorder of the colon can include adenoma. Colonic proliferative disorders of the colon can be characterized by hyperplasia, metaplasia and dysplasia of the colon. Previous colon diseases that can predispose individuals to the development of colon proliferative cell disorders may include anterior colon cancer. The current disease that can predispose individuals to the development of cellular proliferative disorders of the colon can include Crohn's disease and ulcerative colitis. A cell proliferative disorder of the colon may be associated with a mutation in a gene selected from the group consisting of p53, ras, FAPe DCC. An individual may have a high risk of developing a cell proliferative disorder of the colon due to the presence of a mutation in a gene selected from the group consisting of p53, ras, FAP and DCC.
[096] A "cell proliferative disorder of the pancreas" is a cell proliferative disorder that involves cells of the pancreas. Pancreas cell proliferative disorders can include all forms of cell proliferative disorders that affect pancreatic cells. Pancreas cell proliferative disorders can include pancreatic cancer, a precancerous or precancerous condition of the pancreas, pancreas hyperplasia and pancreas dysplasia, benign growths or lesions of the pancreas, and malignant growths or lesions of the pancreas, and metastatic lesions in tissue and organs in the body except the pancreas. Pancreatic cancer includes all forms of pancreatic cancer. Pancreatic cancer can include ductal adenocarcinoma, adenosquamous carcinoma, pleomorphic giant cell carcinoma, mucinous adenocarcinoma, giant cell carcinoma such as osteoclast, mucinous cystadenocarcinoma, acinar carcinoma, unclassified large cell carcinoma, small cell carcinoma, pancreatic tumors mucinous cystadenoma, papillary cystic neoplasia, and serous cystadenoma. Pancreatic cancer can also include pancreatic neoplasms having histological and ultrastructural heterogeneity (for example, mixed cell types).
[097] A "cell proliferative disorder of the prostate" is a cell proliferative disorder that involves prostate cells. Prostate cell proliferative disorders can include all forms of cell proliferative disorders that affect prostate cells. Prostate cell proliferative disorders can include prostate cancer, a precancerous or precancerous condition of the prostate, benign growths or lesions of the prostate, and malignant growths or lesions of the prostate, and metastatic lesions in tissue and organs in the body except prostate. Prostate cell proliferative disorders can include hyperplasia, metaplasia and prostate dysplasia.
[098] A “cell proliferative skin disorder” is a cell proliferative disorder that involves skin cells. Cellular proliferative disorders of the skin can include all forms of cellular proliferative disorders that affect skin cells. Cell proliferative skin disorders may include a pre-cancer or precancerous skin condition, benign growths or lesions of the skin, melanoma, malignant melanoma and other malignant growths or lesions of the skin, and metastatic lesions in tissue and organs in the body except the skin. Cellular proliferative disorders of the skin can include hyperplasia, metaplasia and dysplasia of the skin.
[099] An "ovarian cell proliferative disorder" is a cell proliferative disorder that involves ovarian cells. Ovarian cell proliferative disorders can include all forms of cell proliferative disorders that affect ovary cells. Ovarian cell proliferative disorders can include a precancerous or precancerous condition of the ovary, benign ovarian growths or lesions, ovarian cancer, malignant growths or lesions of the ovary, and metastatic lesions in tissue and organs in the body except the ovary. Cellular proliferative disorders of the skin can include hyperplasia, metaplasia and dysplasia of ovarian cells.
[0100] A "cell proliferative disorder of the breast" is a cell proliferative disorder that involves cells of the breast. Cellular proliferative disorders of the breast can include all forms of cellular proliferative disorders that affect breast cells. Cellular breast proliferative disorders can include breast cancer, a precancerous or precancerous condition of the breast, benign growths or lesions of the breast, and malignant growths or lesions of the breast, and metastatic lesions in tissue and organs in the body except mama. Cellular proliferative disorders of the breast can include hyperplasia, metaplasia and dysplasia of the breast.
[0101] A cell proliferative disorder of the breast can be a precancerous condition of the breast. The compositions of the present invention can be used to treat a precancerous condition of the breast. A precancerous breast condition can include atypical breast hyperplasia, ductal carcinoma in situ (TWO), intraductal carcinoma, lobular carcinoma in situ (LCIS), lobular neoplasia, and stage 0 or grade 0 growth or injury to the breast (for stage 0 or grade 0 of breast cancer or carcinoma in situ). A precancerous condition of the breast can be staged according to the TNM classification scheme as accepted by the American Joint Committee on Cancer (AJCC), where the primary tumor (T) was assigned to a TO or Tis stage; and where the regional (N) lymph nodes were assigned to an NO stage; and where distant metastasis (M) was attributed to an OM stage.
[0102] Breast cell proliferative disorder can be breast cancer. In one aspect, the compositions of the present invention can be used to treat breast cancer, or used to identify suitable candidates for such purposes. Breast cancer can include all forms of breast cancer. Breast cancer can include primary epithelial breast cancers. Breast cancer can include cancers, in which the breast is involved in other tumors, such as lymphoma, sarcoma or melanoma. Breast cancer can include breast carcinoma, breast ductal carcinoma, lobular breast carcinoma, undifferentiated breast carcinoma, phyloid cystosarcoma of the breast, breast angiosarcoma and primary breast lymphoma. Breast cancer can include Stage I, II, IIIA, IIIB, NIC and IV breast cancer. Ductal breast carcinoma can include invasive carcinoma, invasive carcinoma in situ with a predominant intraductal component, inflammatory breast cancer, and a ductal breast carcinoma with a histological type selected from the group consisting of pustule, mucus (colloid), medullary , medullary with lymphocytic, papillary, fibrous and tubular infiltrate. Lobular carcinoma of the breast may include invasive lobular carcinoma with predominant in situ component, invasive lobular carcinoma and infiltrating lobular carcinoma. Breast cancer can include Paget's disease, Paget's disease with intraductal carcinoma and Paget's disease with invasive ductal carcinoma. Breast cancer can include breast neoplasms having histological and ultrastructural heterogeneity (for example, mixed cell types).
[0103] The compounds of the present invention can be used to treat breast cancer, or used to identify suitable candidates for such purposes. Breast cancer that is to be treated can include familial breast cancer. A breast cancer that is to be treated can include sporadic. A breast cancer that is to be treated can arise in a male subject. A breast cancer that is to be treated can arise in a female subject. A breast cancer that is to be treated can arise in a pre-menopausal female subject or a post-menopausal female subject. A breast cancer that is to be treated can arise in a subject equal to or over 30 years of age, or a subject under 30 years of age. Breast cancer that is to be treated has arisen in a subject equal to or over 50 years of age, or a subject under 50 years of age. A breast cancer that is to be treated can arise in a subject equal to or over 70 years of age, or a subject under 70 years of age.
[0104] A breast cancer that is to be treated can be classified to identify a familial or spontaneous mutation in BRCA1, BRCA2 or p53. A breast cancer that is to be treated can be classified as having a HER2 / neu gene amplification, as a HER2 / neu overexpression, or as having a low, intermediate or high level of HER2 / neu expression. A breast cancer that is to be treated can be classified as a marker selected from the group consisting of estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor-2 receptor, Ki-67 , CA15-3, CA 27-29 and c-Met. A breast cancer that is to be treated can be classified as ER-unknown, ER-rich or ER-deficient. A breast cancer that is to be treated can be classified as ER-negative or ER-positive. The classification of ER for breast cancer can be performed by any reproducible means. The classification of ER for breast cancer can be performed as presented in Onkologie 27: 175 - 179 (2004). A breast cancer that is to be treated can be classified as PR-unknown, PR-rich or PR-deficient. A breast cancer that is to be treated can be classified as PR-negative or PR-positive. A breast cancer that is to be treated can be classified as a positive or negative receptor. A breast cancer that is to be treated can be classified as being associated with elevated blood levels of CA 15-3 or CA 27-29, or both.
[0105] A breast cancer that is to be treated can include a localized breast tumor. A breast cancer that is to be treated can include a breast tumor that is associated with a negative sentinel lymph node (SLN) biopsy. A breast cancer that is to be treated can include a breast tumor that is associated with a positive sentinel lymph node biopsy (SLN). A breast cancer that is to be treated can include a breast tumor that is associated with one or more positive auxiliary lymph nodes, where the auxiliary lymph nodes were staged by any applicable method. A breast cancer that is to be treated can include a breast tumor that has been classified as not having a negative nodal state (for example, node-negative) or positive nodal state (for example, node-positive). A breast cancer that is to be treated can include a breast tumor that has metastasized elsewhere in the body. A breast cancer that is to be treated can be classified as having metastasis at a site selected from the group consisting of bone, lung, liver or brain. A breast cancer that is to be treated can be classified according to a characteristic selected from the group consisting of metastatic, localized, regional, local-regional, locally advanced, distant, multicentric, bilateral, ipsilateral, contralateral, newly diagnosed, recurrent and inoperable.
[0106] A compound of the present invention can be used to treat or prevent a cell proliferative disorder of the breast, or to treat or prevent breast cancer, in a subject having an increased risk of developing breast cancer in relation to the general population. , or used to identify suitable candidates for such purposes. A subject with an increased risk of developing breast cancer in relation to the population, in general, is a female subject with a family history or personal history of breast cancer. A subject with an increased risk of developing breast cancer in relation to the population, in general, is a female subject having a germline or spontaneous mutation in BRCA1 or BRCA2, or both. A subject with an increased risk of developing breast cancer in relation to the population, in general, is a female subject with a family history of breast cancer and a germinal or spontaneous mutation in BRCA1 or BRCA2, or both. A subject with an increased risk of developing breast cancer in relation to the population, in general, is a female who is over 30 years old, over 40 years old, over 50 years old, over 60 years old. age, over 70 years of age, over 80 years of age, or over 90 years of age. A subject with an increased risk of developing breast cancer in relation to the population, in general, is a subject with atypical breast hyperplasia, ductal carcinoma in situ (DCIS), intraductal carcinoma, lobular carcinoma in situ (LCIS), lobular neoplasia, or a stage 0 of breast growth or injury (for example, stage 0 or grade 0 of breast cancer or carcinoma in situ).
[0107] A breast cancer that is to be treated can be histologically classified according to the Scarff-Bloom-Richardson system, in which a breast tumor was assigned a mitosis count score of 1.2 or 3; a nuclear pleiomorphism score of 1.2 or 3; a tubule score of 1.2, or 3; and a total Scarff-Bloom-Richardson score between 3 and 9. A breast cancer that is to be treated can be assigned a tumor grade according to the International Consensus Panel on Breast Cancer Treatment selected from the group consisting of grade 1, grade 1 - 2, grade 2, grade 2 - 3 or grade 3.
[0108] In one embodiment, a method of treating breast cancer is provided here comprising administering to a subject in need thereof an effective amount of the Compound I hydrobromide.
[0109] In another embodiment, a method of treating breast cancer is provided here comprising administering to a subject in need thereof an effective amount of Polymorph A.
[0110] A cancer that is to be treated can be organized according to the TNM classification system of the American Joint Committee in Cancer (AJCC), where the tumor (T) has been assigned a stage of TX, T1, T1mic, T1a , T1b, T1c, T2, T3, T4, T4a, T4b, T4c, or T4d; and where the regional lymph nodes (N) were assigned an NX, NO, N1, N2, N2a, N2b, N3, N3a, N3b or N3c stage; and where distant metastasis (M) can be attributed to an MX, MO or M1 stage. A cancer that is to be treated can be organized according to an American Joint Committee in Cancer (AJCC) classification as Stage I, Stage HA, Stage IIB, Stage IIIA, Stage IIIB, Stage NIC or Stage IV. A cancer that is to be treated can be assigned a degree according to an AJCC classification as Grade GX (for example, the grade cannot be assessed), Grade 1, Grade 2, Grade 3 or Grade 4. A cancer that is to be treated can be organized according to a AJCC pathological classification (pN) of pNX, pNO, PNO (I-), PNO (l +), PNO (mol-), PNO (mol +), PN1, PN1 (mi) , PN1a, PN1b, PN1c, pN2, pN2a, pN2b, pN3, pN3a, pN3b or pN3c.
[0111] A cancer that is to be treated may include a tumor that has been determined to be less than or equal to about 2 centimeters in diameter. A cancer that is to be treated can include a tumor that has been determined to be about 2 to about 5 inches in diameter. A cancer that is to be treated may include a tumor that has been determined to be greater than or equal to about 3 centimeters in diameter. A cancer that is to be treated can include a tumor that has been determined to be larger than 5 centimeters in diameter. A cancer that is to be treated can be classified by microscopic appearance as well differentiated, moderately differentiated, poorly differentiated or undifferentiated. A cancer that is to be treated can be classified by microscopic appearance with respect to mitosis count (eg, amount of cell division) or nuclear pleiomorphism (eg, change in cells). A cancer that is to be treated can be classified by microscopic appearance as being associated with areas of necrosis (for example, areas of cells dying or degenerating). A cancer that is to be treated can be classified as having an abnormal karyotype, having an abnormal number of chromosomes, or having one or more chromosomes that are abnormal in appearance. A cancer that is to be treated can be classified as being aneuploid, triploid, tetraploid, or having an altered ploidy. A cancer that is to be treated can be classified as having a chromosomal translocation, or a deletion or duplication of an entire chromosome, or a region of deletion, duplication or amplification of a portion of a chromosome.
[0112] A cancer that is to be treated can be evaluated by DNA cytometry, flow cytometry or image cytometry. A cancer that is to be treated can be classified as having 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of cells in the cell division synthesis stage (for example , in cell division S phase). A cancer that is to be treated can be classified as having a low S-phase fraction or a high S-phase fraction.
[0113] As used herein, a "normal cell" is a cell that cannot be classified as part of a "cell proliferative disorder". A normal cell lacks unregulated or abnormal growth, or both, which can lead to the development of an unwanted condition or disease. Preferably, a normal cell has normally functioning cell cycle checkpoint control mechanisms.
[0114] As used herein, "contacting a cell" refers to a condition, in which a compound or other composition of matter is in direct contact with a cell, or is close enough to induce a desired biological effect on a cell .
[0115] As used herein, "candidate compound" refers to a compound of the present invention (i.e., Compound I hydrobromide, as well as Polymorph A) that has been or will be tested in one or more in vitro or in vivo biological assays , in order to determine whether the compound is likely to evoke a desired biological or medical response in a cell, tissue, system, animal or human being being requested by a researcher or clinician. A candidate compound is a compound of the present invention. The biological or medical response may be cancer treatment. The biological or medical response may be the treatment or prevention of a cell proliferative disorder. The biological response or effect may also include a change in cell proliferation or growth that occurs in vitro or in an animal model, as well as other biological changes that are observable in vitro. In vitro or in vivo biological assays may include, but are not limited to a, enzyme activity assays, electrophoretic mobility deviation assays, reporter gene assays, in vitro cell viability assays, and the assays described here.
[0116] As used herein, "monotherapy" refers to the administration of a single active or therapeutic compound to a subject in need of it. Preferably, monotherapy will involve the administration of a therapeutically effective amount of an active compound. For example, cancer monotherapy with one of the compound of the present invention (i.e., Compound I hydrobromide, as well as Polymorph A) to a subject in need of cancer treatment. Monotherapy can be contrasted with combination therapy, in which a combination of multiple active compounds is administered, preferably with each component of the combination present in a therapeutically effective amount. In one aspect, monotherapy with a compound of the present invention is more effective than combination therapy in inducing a desired biological effect.
[0117] As used herein, "treatment" or "treating" describes the management and care of a patient for the purpose of combating a disease, condition or disorder and includes the administration of a compound of the present invention (i.e., hydrobromide of Compound I, as well as Polymorph A) to relieve the symptoms or complications of a disease, condition or disorder, or to eliminate the disease, condition or disorder. The term "treat" can also include treating a cell in vitro or an animal model.
[0118] A compound of the present invention (i.e., Compound I hydrobromide, as well as Polymorph A) can also be used to prevent a disease, condition or disorder, or used to identify suitable candidates for such purposes. As used herein, "avoiding" or "avoiding" describes reducing or eliminating the onset of symptoms or complications of the disease, condition or disorder.
[0119] As used herein, the term "relieve" means to describe a process by which the severity of a sign or symptom of a disorder is decreased. Importantly, a sign or symptom can be relieved without being eliminated. In a preferred embodiment, administration of pharmaceutical compositions of the invention leads to the elimination of a sign or symptom, however, elimination is not necessary. Effective dosages are expected to decrease the severity of a sign or symptom. For example, a sign or symptom of a disorder, such as cancer, that can occur in multiple locations, is relieved if the severity of the cancer is decreased within at least one of multiple locations.
[0120] As used here, the term "severity" means to describe the potential for cancer to transform from a precancerous, or benign, state into a malignant state. Alternatively, or in addition, severity means describing a cancer stage, for example, according to the TNM system (accepted by the International Union Against Cancer (UICC) and the American Joint Committee on Cancer (AJCC)) or by other recognized methods by technique. Cancer stage refers to the extent or severity of the cancer, based on factors such as the location of the primary tumor, tumor size, number of tumors, and lymph node involvement (cancer spread in lymph nodes). Alternatively, or in addition, severity means describing the degree of tumor by methods recognized by the technique (see, National Cancer Institute, www.cancer.gov). The tumor grade is a system used to classify cancer cells in terms of how abnormal they appear under a microscope and how quickly the tumor tends to grow and spread. Many factors are considered in determining the degree of tumor, including the pattern of cell structure and growth. The specific factors used to determine the degree of tumor vary with each type of cancer. Severity also describes a histological grade, also called differentiation, which refers to how much tumor cells resemble normal cells of the same type of tissue (see, National Cancer Institute, www.cancer.gov). In addition, severity describes a nuclear grade, which refers to the size and shape of the nucleus in tumor cells and the percentage of tumor cells that are dividing (see, National Cancer Institute, www.cancer.gov).
[0121] In another aspect of the invention, severity describes the degree to which a tumor has growth factors secreted, degraded extracellular matrix, becomes vascularized, loss of adhesion to juxtaposed tissues, or metastasis. In addition, severity describes the number of locations that a primary tumor has metastasized. Finally, severity includes the difficulty of treating tumors of various types and locations. For example, inoperable tumors, those cancers that have greater access to multiple systems in the body (hematological and immunological tumors), and those that are the most resistant to traditional treatments are considered more severe. In these situations, prolonging the subject's life expectancy and / or reducing pain, decreasing the proportion of cancer cells or restricting cells to a system, and improving the stage of cancer / tumor grade / histological grade / nuclear grade are considered to relieve a sign or symptom of cancer.
[0122] As used here, the term "symptom" is defined as an indication of illness, injury or that something is not right in the body. Symptoms are felt or perceived by the individual experiencing the symptom, but cannot be easily perceived by others. Others are defined as non-health professionals.
[0123] As used here, the term "signal" is also defined as an indication that something is not right in the body. But signs are defined as things that can be observed by a doctor, nurse or other healthcare professional.
[0124] Cancer is a group of diseases that can cause almost any sign or symptom. The signs and symptoms will depend on where the cancer is, the size of the cancer, and how much it affects neighboring organs or structures. If a cancer spreads (metastases), then the symptoms can appear in different parts of the body.
[0125] As a cancer grows, it starts to push the surrounding organs, blood vessels and nerves. This pressure creates some of the signs and symptoms of cancer. If the cancer is in a critical area, such as certain parts of the brain, even the smallest tumor can cause early symptoms.
[0126] But sometimes cancers start in places where they don't cause any symptoms until the cancer has grown large enough. Pancreatic cancers, for example, do not usually grow large enough to be felt from outside the body. Some pancreatic cancers do not cause symptoms until they start to grow around neighboring nerves (this causes back pain). Others grow around the bile duct, which blocks the flow of bile and leads to a yellowish discoloration of the skin known as jaundice. By the time a pancreatic cancer causes these signs or symptoms, it has usually reached an advanced stage.
[0127] Cancer can also cause symptoms such as fever, fatigue or weight loss. This may be because cancer cells use too much of the body's energy supply or release substances that change the body's metabolism. Or cancer can cause the immune system to react to produce these symptoms.
[0128] Sometimes, cancer cells release substances into the bloodstream that do not cause symptoms usually thought to result from cancers. For example, some cancers of the pancreas can release substances that cause blood clots to develop in the leg veins. Some lung cancers make hormone-like substances that affect blood calcium levels, affecting nerves and muscles and causing weakness and dizziness.
[0129] Cancer has several general signs or symptoms that occur when a variety of cancer cell subtypes are present. More people with cancer will lose weight in time with their disease. An unexplained (unintended) weight loss of 10 pounds or more can be the first sign of cancer, particularly cancers of the pancreas, stomach, esophagus or lung.
[0130] Fever is very common with cancer, but is most often seen in advanced disease. Almost all cancer patients will have a fever at some point, especially if the cancer or its treatment affects the immune system and makes it more difficult for the body to fight infection. Less often, fever can be an early sign of cancer, such as with leukemia or lymphoma.
[0131] Fatigue can be an important symptom as the cancer progresses. It can happen earlier, however, in cancers such as with leukemia, or if the cancer is causing continuous blood loss, as in some types of colon or stomach cancer.
[0132] Pain can be an initial symptom with some cancers such as bone cancer or testicular cancer. But most of the time, pain is a symptom of advanced disease.
[0133] Along with skin cancers (see, next section), some internal cancers can cause signs of skin that can be seen. These changes include darker-looking skin (hyperpigmentation), yellow (jaundice) or red (erythema); itch; or excessive hair growth.
[0134] Alternatively, or in addition, cancer subtypes show specific signs or symptoms. Changes in bowel habits or bladder function can indicate cancer. Long-term constipation, diarrhea, or a change in stool size can be a sign of colon cancer. Pain when urinating, blood in the urine, or a change in bladder function (such as more frequent or less frequent urination) can be related to bladder or prostate cancer.
[0135] Changes in the condition or appearance of the skin of a new skin condition can indicate cancer. Skin cancers can bleed and look like wounds that don't heal. Long-lasting pain in the mouth can be oral cancer, especially in patients who often smoke, chew tobacco or drink alcohol. Sores on the penis or vagina can be signs of infection or early cancer.
[0136] Bleeding or abnormal discharge may indicate cancer. Hemorrhages can happen in any early or advanced cancer. Blood in the sputum (phlegm) can be a sign of lung cancer. Blood in the stool (or dark or black stools) can be a sign of colon or rectal cancer. Cancer of the cervix or endometrium (lining of the uterus) can cause vaginal bleeding. Blood in the urine can be a sign of bladder or kidney cancer. A bloody discharge from the nipple can be a sign of breast cancer.
[0137] A thickening or lump in the breast or other parts of the body may indicate the presence of cancer. Many cancers can be felt through the skin, especially in the breast, testicles, lymph nodes (glands), and the soft tissues of the body. A lump or thickening can be an early or late sign of cancer. Any lump or thickening can be indicative of cancer, especially if the formation is new or has grown in size.
[0138] Indigestion or difficulty swallowing may indicate cancer. Although these symptoms commonly have other causes, problems with indigestion or swallowing can be a sign of cancer of the esophagus, stomach or pharynx (throat).
[0139] Recent changes in a mole or spot may be indicative of cancer. Any mole, spot or freckle that changes color, size or shape, or loses its definitive boundaries indicates the potential development of cancer. For example, the skin lesion can be a melanoma.
[0140] A persistent cough or hoarseness can be indicative of cancer. A cough that won't go away can be a sign of lung cancer. Hoarseness can be a sign of cancer of the larynx (vocal cords) or thyroid.
[0141] Although the signs and symptoms listed above are the most common seen with cancer, there are many others that are less common and are not listed here. However, all signs and symptoms of cancer recognized in the art are considered and covered by the present invention.
[0142] Treating cancer can result in a reduction in the size of a tumor. A reduction in the size of a tumor can also be referred to as "tumor regression". Preferably, after treatment, the tumor size is reduced by 5% or more from its size before treatment; more preferably, the tumor size is reduced by 10% or more; more preferably, reduced by 20% or more; more preferably, reduced by 30% or more; more preferably, reduced by 40% or more; even more preferably, reduced by 50% or more; and most preferably, reduced by 75% or more. The size of a tumor can be measured by any reproducible means of measurement. The size of a tumor can be measured as a diameter of the tumor.
[0143] Treating cancer can result in a reduction in tumor volume. Preferably, after treatment, the tumor volume is reduced by 5% or more in relation to its size before treatment; more preferably, the tumor volume is reduced by 10% or more; more preferably, reduced by 20% or more; more preferably, reduced by 30% or more; more preferably, reduced by 40% or more; even more preferably, reduced by 50% or more; and most preferably, reduced by 75% or more. The tumor volume can be measured by any reproducible measurement means.
[0144] Treating cancer results in a decrease in the number of tumors. Preferably, after treatment, the tumor number is reduced by 5% or more compared to the number before treatment; more preferably, the tumor number is reduced by 10% or more; more preferably, reduced by 20% or more; more preferably, reduced by 30% or more; more preferably, reduced by 40% or more; even more preferably, reduced by 50% or more; and most preferably, reduced by greater than 75%. The number of tumors can be measured by any reproducible means of measurement. The number of tumors can be measured by counting tumors visible to the naked eye or at a specific magnification. Preferably, the specific magnification is 2x, 3x, 4x, 5x, 10x or 50x.
[0145] Treating cancer can result in a decrease in the number of metastatic lesions in other tissues or organs distant from the primary tumor site. Preferably, after treatment, the number of metastatic lesions is reduced by 5% or more compared to the number before treatment; more preferably, the number of metastatic lesions is reduced by 10% or more; more preferably, reduced by 20% or more; more preferably, reduced by 30% or more; more preferably, reduced by 40% or more; even more preferably, reduced by 50% or more; and most preferably, reduced by more than 75%. The number of metastatic lesions can be measured by any reproducible means of measurement. The number of metastatic lesions can be measured by counting metastatic lesions visible to the naked eye or at a specific magnification. Preferably, the specific magnification is 2x, 3x, 4x, 5x, 10x or 50x.
[0146] Treating cancer can result in an increase in the average survival time of a population of treated subjects compared to a population that receives the vehicle alone. Preferably, the average survival time is increased by more than 30 days; more preferably, for more than 60 days; more preferably, for more than 90 days; and most preferably, for more than 120 days. An increase in a population's average survival time can be measured by any reproducible means. An increase in the average survival time of a population can be measured, for example, by calculating the average survival length for a population after initiation of treatment with an active compound. An increase in the average survival time of a population can also be measured, for example, by calculating the average survival length for a population after completing a first round of treatment with an active compound.
[0147] Treating cancer can result in an increase in the average survival time of a population of treated subjects compared to a population of untreated subjects. Preferably, the average survival time is increased by more than 30 days; more preferably, for more than 60 days; more preferably, for more than 90 days; and most preferably, for more than 120 days. An increase in a population's average survival time can be measured by any reproducible means. An increase in the average survival time of a population can be measured, for example, by calculating the average survival length for a population after initiation of treatment with an active compound. An increase in the average survival time of a population can also be measured, for example, by calculating the average survival length for a population after completing a first round of treatment with an active compound.
[0148] Treating cancer can result in an increase in the average survival time of a population of treated subjects compared to a population receiving monotherapy with a drug that is not a compound of the present invention. Preferably, the average survival time is increased by more than 30 days; more preferably, for more than 60 days; more preferably, for more than 90 days; and most preferably, for more than 120 days. An increase in a population's average survival time can be measured by any reproducible means. An increase in the average survival time of a population can be measured, for example, by calculating the average survival length for a population after initiation of treatment with an active compound. An increase in the average survival time of a population can also be measured, for example, by calculating the average survival length for a population after completing a first round of treatment with an active compound.
[0149] Treating cancer can result in a decrease in the mortality rate of a population of treated subjects compared to a population that receives the vehicle alone. Treating cancer can result in a decrease in the mortality rate of a population of treated subjects compared to an untreated population. Treating cancer can result in a decrease in the mortality rate of a population of treated subjects compared to a population receiving monotherapy with a drug that is not a compound of the present invention. Preferably, the mortality rate is decreased by more than 2%; more preferably, by more than 5%; more preferably, by more than 10%; and most preferably, by more than 25%. A decrease in the mortality rate of a population of treated subjects can be measured by any reproducible means. A decrease in a population's mortality rate can be measured, for example, by calculating for a population the average number of illness-related deaths per unit time after starting treatment with an active compound. A decrease in a population's mortality rate can also be measured, for example, by calculating for a population the average number of illness-related deaths per unit of time after completing a first round of treatment with an active compound.
[0150] Treating cancer can result in a decrease in the rate of tumor growth. Preferably, after treatment, the rate of tumor growth is reduced by at least 5% from the number before treatment; more preferably, the tumor growth rate is reduced by at least 10%; more preferably, reduced by at least 20%; more preferably, reduced by at least 30%; more preferably, reduced by at least 40%; more preferably, reduced by at least 50%; even more preferably, reduced by at least 50%; and most preferably, reduced by at least 75%. The rate of tumor growth can be measured by any reproducible means of measurement. The rate of tumor growth can be measured according to a change in tumor diameter per unit time.
[0151] Treating cancer can result in a decrease in tumor regrowth. Preferably, after treatment, tumor regrowth is less than 5%; more preferably, tumor regrowth is less than 10%; more preferably, less than 20%; more preferably, less than 30%; more preferably, less than 40%; more preferably, less than 50%; even more preferably, less than 50%; and most preferably, less than 75%. Tumor regrowth can be measured by any reproducible means of measurement. Tumor regrowth is measured, for example, by measuring an increase in the diameter of a tumor after a tumor contracted before treatment followed. A decrease in tumor regrowth is indicated by the failure of tumors to reappear after treatment has stopped.
[0152] Treating or preventing a cell proliferative disorder can result in a reduction in the rate of cell proliferation. Preferably, after treatment, the rate of cell proliferation is reduced by at least 5%; more preferably, by at least 10%; more preferably, by at least 20%; more preferably, by at least 30%; more preferably, by at least 40%; more preferably, by at least 50%; even more preferably, by at least 50%; and most preferably, by at least 75%. The rate of cell proliferation can be measured by any reproducible means of measurement. The rate of cell proliferation is measured, for example, by measuring the number of dividing cells in a tissue sample per unit time.
[0153] Treating or preventing a cell proliferative disorder can result in a reduction in the proportion of proliferating cells. Preferably, after treatment, the proportion of proliferating cells is reduced by at least 5%; more preferably, by at least 10%; more preferably, by at least 20%; more preferably, by at least 30%; more preferably, by at least 40%; more preferably, by at least 50%; even more preferably, by at least 50%; and most preferably, by at least 75%. The proportion of proliferating cells can be measured by any reproducible means of measurement. Preferably, the proportion of proliferating cells is measured, for example, by quantifying the number of dividing cells in relation to the number of cells without dividing in a tissue sample. The proportion of proliferating cells can be equivalent to the mitotic index.
[0154] Treating or preventing a cell proliferative disorder can result in a decrease in the size of an area or zone of cell proliferation. Preferably, after treatment, the size of an area or zone of cell proliferation is reduced by at least 5% from its size before treatment; more preferably, reduced by at least 10%; more preferably, reduced by at least 20%; more preferably, reduced by at least 30%; more preferably, reduced by at least 40%; more preferably, reduced by at least 50%; even more preferably, reduced by at least 50%; and most preferably, reduced by at least 75%. The size of an area or zone of cell proliferation can be measured by any reproducible means of measurement. The size of an area or zone of cell proliferation can be measured as a diameter or width of an area or zone of cell proliferation.
[0155] Treating or preventing a cell proliferative disorder can result in a decrease in the number or proportion of cells having an abnormal appearance or morphology. Preferably, after treatment, the number of cells having an abnormal morphology is reduced by at least 5% in relation to their size before treatment; more preferably, reduced by at least 10%; more preferably, reduced by at least 20%; more preferably, reduced by at least 30%; more preferably, reduced by at least 40%; more preferably, reduced by at least 50%; even more preferably, reduced by at least 50%; and most preferably, reduced by at least 75%. An abnormal cell appearance or morphology can be measured by any reproducible means of measurement. An abnormal cell morphology can be measured by a microscope, for example, using an inverted tissue culture microscope. An abnormal cell morphology can take the form of nuclear pleiomorphism.
[0156] As used here, the term “selectively” means that it tends to occur at a higher frequency in one population than in another population. The compared populations can be cell populations. Preferably, a compound of the present invention (i.e., Compound I hydrobromide, as well as Polymorph A) acts selectively on a cancer or precancerous cell, but not on a normal cell. Preferably, a compound of the present invention acts selectively to modulate a molecular target (for example, a target methyltransferase protein), but does not significantly modulate another molecular target (for example, a non-target methyltransferase protein). The invention also provides a method for selectively inhibiting the activity of an enzyme, such as a protein methyltransferase. Preferably, an event occurs selectively in population A compared to population B if it occurs more than twice as often in population A as compared to population B. An event occurs selectively if it occurs more than five times more often in population A An event occurs selectively if it occurs more than ten times more frequently in population A; more preferably, more than fifty times; even more preferably, more than 100 times; and most preferably, more than 1000 times more often in population A as compared to population B. For example, cell death would be said to occur selectively in cancer cells if it occurred more than twice as often in cancer cells as compared to normal cells.
[0157] A compound of the present invention can modulate the activity of a molecular target (for example, a target methyltransferase protein). Modular refers to the stimulation or inhibition of an activity of a molecular target. Preferably, a compound of the present invention modulates the activity of a molecular target if it stimulates or inhibits the activity of the molecular target at least 2 times in relation to the activity of the molecular target under the same conditions, but only the presence of said compound is missing. More preferably, a compound of the present invention modulates the activity of a molecular target if it stimulates or inhibits the activity of the molecular target at least 5 times, at least 10 times, at least 20 times, at least 50 times, at least 100 times in relation to the activity of the molecular target under the same conditions, but lacking only the presence of said compound. The activity of a molecular target can be measured by any reproducible means. The activity of a molecular target can be measured in vitro or in vivo. For example, the activity of a molecular target can be measured in vitro by an enzyme activity assay or a DNA binding assay, or the activity of a molecular target can be measured in vivo by evaluating for the expression of a reporter gene.
[0158] A compound of the present invention (i.e., Compound I hydrobromide, as well as Polymorph A) does not significantly modulate the activity of a molecular target if the addition of the compound does not stimulate or inhibit the activity of the molecular target for more than 10% in relation to the activity of the molecular target under the same conditions but missing only the presence of said compound.
[0159] As used herein, the term "selective isozyme" means inhibition or preferential stimulation of a first isoform of an enzyme as compared to a second isoform of an enzyme (for example, inhibition or preferential stimulation of an alpha isozyme methyltransferase protein in comparison with a beta isozyme methyltransferase protein). Preferably, a compound of the present invention demonstrates a minimum of a four-fold spread, preferably a ten-fold spread, more preferably a fifty-fold spread, at the dosage required to achieve a biological effect. Preferably, a compound of the present invention demonstrates this differential over the inhibition range, and the differential is exemplified in ICso, that is, a 50% inhibition, for a molecular target of interest.
[0160] Administering a compound of the present invention to a cell or subject in need of it may result in modulation (i.e., stimulation or inhibition) of an activity of a methyltransferase protein of interest.
[0161] Treating cancer or a cell proliferative disorder can result in cell death, and preferably cell death results in a decrease of at least 10% in the number of cells in a population. More preferably, cell death means a decrease of at least 20%; more preferably, a decrease of at least 30%; more preferably, a decrease of at least 40%; more preferably, a decrease of at least 50%; most preferably, a decrease of at least 75%. Number of cells in a population can be measured by any reproducible means. Several cells in a population can be measured by fluorescence-activated cell separation (FACS), fluorescence immunomicroscopy and light microscope. Methods of measuring cell death are as shown in Li et a!., Proc Natl Acad Sei USA. 100 (5): 2674 - 8, 2003. In one aspect, cell death occurs by apoptosis.
[0162] Preferably, an effective amount of a compound of the present invention is not significantly cytotoxic to normal cells. A therapeutically effective amount of a compound is not significantly cytotoxic to normal cells if administration of the compound in a therapeutically effective amount does not induce cell death in more than 10% of normal cells. A therapeutically effective amount of a compound does not significantly affect the viability of normal cells if administration of the compound in a therapeutically effective amount does not induce cell death in more than 10% of normal cells. In one aspect, cell death occurs by apoptosis.
[0163] Contacting a cell with a compound of the present invention can selectively induce or activate cell death in cancer cells. Administering a subject in need of it a compound of the present invention can selectively induce or activate cell death in cancer cells. Contacting a cell with a compound of the present invention can selectively induce cell death in one or more cells affected by a cell proliferative disorder. Preferably, administering to a subject in need thereof a compound of the present invention selectively induces cell death in one or more cells affected by a cell proliferative disorder.
[0164] The present invention relates to a method of treating or preventing cancer (for example, the course of which can be influenced by modulating EZH2-mediated protein methylation) by administering a compound of the present invention (i.e., the hydrobromide of Compound I, as well as Polymorph A) to a subject in need thereof, where administration of the compound of the present invention results in one or more of the following: prevention of cancer cell proliferation by accumulation of cells in one or more phases of the cell cycle (for example, G1, G1 / S, G2 / M), or inducing cell senescence, or promoting differentiation of tumor cells; promotion of cell death in cancer cells through cytotoxicity, necrosis or apoptosis, without a significant amount of cell death in normal cells, antitumor activity in animals with a therapeutic index of at least 2. As used herein, the “therapeutic index” is the maximum tolerated dose divided by the effective dose. The present invention also relates to a method used to identify suitable candidates to treat or prevent cancer.
[0165] A person skilled in the art can refer to general reference texts for detailed descriptions of known techniques discussed here or equivalent techniques. These texts include Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Inc. (2005); Sambrook et al., Molecular Cloning, A Laboratory Manual (3rd edition), Cold Spring Harbor Press, Cold Spring Harbor, New York (2000); Coligan et al., Current Protocols in Immunology, John Wiley & Sons, N.Y .; Enna et al., Current Protocols in Pharmacology, John Wiley & Sons, N.Y .; Fingl et al., The Pharmacological Basis of Therapeutics (1975), Remington’s Pharmaceutical Sciences, Mack Publishing Co., Easton, PA, 18th edition (1990). These texts can also, of course, be referred to in the production or use of an aspect of the invention. Exemplification Materials and Methods X-Ray Powder Diffraction
[0166] PXRD for all samples was made on a Rigaku MultiFlex (Target: Cu; Tube voltage: 40 kV; Tube current: 30 mA). Differential Scanning Calorimetry
[0167] DSC for all samples was made on a Mettler-Toledo DSC 1/700 (Conducted conditions: Initial temperature 35 ° C, Final temperature 325 ° C, Heating rate 30 ° C / min). X-Ray Crystallography
[0168] A colorless crystal plate with dimensions 0.28 x 0.22 x 0.06 mm was mounted on a nylon mesh using a very small amount of paratone oil. The data were collected using a Bruker CCD diffractometer (load-coupled device) equipped with an Oxford Cryostream low temperature device operating at 173 K (-100 C). The data were measured using omega and phi scans of 0.5 ° per frame for 45 s. The total number of images was based on the results from the COSMO program, where redundancy was expected to be 4.0 and 100% completeness to 0.83 Â. Cellular parameters were recovered using the APEX II program and refined using SAINT in all observed reflections. Data reduction was performed using the SAINT program that corrects for Lp. Scaling and absorption corrections were applied using SADABS multi-scan technique, provided by George Sheldrick. The structures are solved by the direct method using the SHELXS-97 program and refined by the least squares method in F2, SHELXL-97, which are incorporated in SHELXTL-PC V 6,10.
[0169] The structure shown in Figure 11 was solved in the space group P2i / c (# 14). Anything other than hydrogen atoms is refined anisotropically. Hydrogens were calculated by geometric methods and refined as a transport model. The crystal used for the diffraction study showed no decomposition during data collection. All drawings are made with 50% ellipsoids. Dynamic Steam Sorption
[0170] DVS was measured on a VTI Model SGA-100 system. Measurement method: The relative humidity (RH) was changed in a controlled manner, in 5% steps from 5.0% to 95.0%, then back to 5.0% using the vapor sorption system gravimetric, and the change in percentage by weight (% by weight) of the sample at each stage was measured. HPLC
[0171] HPLC was performed on a Quilary HPLC pump Agilent 1200, low pressure mixture, with an in-line degasser. Analytical method conditions: 8 pL of sample (20 mg of ER-581982-06 diluted with 50 ml of methanol to provide approximately 0.4 mg / ml of solution) was injected into an Agilent Zorbax Eclipse XDB-C18 (4, 6 x 150 mm, 3.5 um), Chromatography conditions: mobile phase A, water with 5 mM ammonium formate; mobile phase B, 5 mM ammonium formate in 50/45/5 acetonitrile / methanol / water; flow rate, 1.5 ml / min .; gradient: isocratic at 10% B from 0 to 3 min; linear increase at 70% B for 3 to 7 min; isocratic at 70% B for 7 to 12 min; linear increase to 100% B from 12 to 15 min; isocratic to 100% B from 15 to 20 min; column temperature, 35 ° C; detection, UV 230 nm. Approximate retention time for Compound I = 10.7 min.

[0172] 5-Bromo-2-methyl-3-nitrobenzoic acid. Stirred solution of 2-methyl-3-nitrobenzoic acid (100 g, 552 mmols) in conc. (400 mL), 1,3-dibromo-5,5-dimethyl-2,4-imidazolidinedione (88 g, 308 mmols) was added to the portions at room temperature and the reaction mixture was then stirred at room temperature for 5 h . The reaction mixture was poured into ice water, the precipitated solid was filtered off, washed with water and dried in vacuo to provide the desired compound as a solid (140 g, 98%). The isolated compost was made directly in the next step. 1H NMR (DMSO-cfe, 400 MHz) δ 8.31 (s, 1H), 8.17 (s, 1H), 2.43 (s, 3H).
Methyl 5-bromo-2-methyl-3-nitrobenzoate To a stirred solution of 5-bromo-2-methyl-3-nitrobenzoic acid (285 g, 1105 mmols) in DMF (2.8 L) at room temperature was added sodium carbonate (468 g, 4415 mmols) followed by addition of methyl iodide (626.6 g, 4415 mmols). The resulting reaction mixture was heated at 60 ° C for 8 h. After completion (monitored by TLC), the reaction mixture was filtered (to remove sodium carbonate) and washed with ethyl acetate (1 LX 3). The combined filtrate was washed with water (3 LX 5) and the aqueous phase was back extracted with ethyl acetate (1 LX 3). The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to provide the title compound as a solid (290 g, 97% yield). The isolated compost was made directly in the next step. 1H NMR (CDCh, 400 MHz) δ 8.17 (s, 1H), 7.91 (s, 1H), 3.96 (s, 3H), 2.59 (s, 3H).
Methyl 3-amino-5-bromo-2-methylbenzoate (1) To a stirred solution of methyl 5-bromo-2-methyl-3-nitrobenzoate (290 g, 1058 mmols) in ethanol (1.5 L) aqueous ammonium chloride (283 g, 5290 mmols dissolved in 1.5 L of water) is added. The resulting mixture was stirred at 80 ° C, to which the iron powder (472 g, 8451 mmols) was added to the portions. The resulting reaction mixture was heated at 80 ° C for 12 h. After completion as determined by TLC, the reaction mixture was hot filtered through Celite® and the celite bed was washed with methanol (5 L) followed by washing with 30% MeOH in DOM (5 L). The combined filtrate was concentrated in vacuo, the residue obtained was diluted with aqueous sodium bicarbonate solution (2 L) and extracted with ethyl acetate (5 LX 3). The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to provide the title compound as a solid (220 g, 85%). The compost was made directly in the next step. 1H NMR (CDCb, 400 MHz) δ 7.37 (s, 1H), 6.92 (s, 1H), 3.94 (s, 3H), 3.80 (bs, 2H), 2.31 ( s, 3H).
Methyl 5-bromo-2-methyl-3 - ((tetrahydro-2H-pyran-4-yl) amino) benzoate (2) A reactor was charged with methyl 3-amino-5-bromo-2-methylbenzoate (455.8 g, 1.87 mol), 1,2-Dichloroethane (4.56 L), and acetic acid (535 ml, 9.34 moles). To the mixture were added dihydro-2H-pyran-4 (3H) -one (280 g, 2.80 moles) and sodium triacetoxyborohydride (594 g, 2.80 moles) maintaining the internal temperature below 40 ° Ç. The mixture was stirred at 25 ° C for 2.5 h and then the reaction was cooled quickly with a solution of sodium hydroxide (448 g, 11.20 moles) in water (5.61 L). After stirring for 20 minutes at room temperature, the organic layer was separated and the aqueous layer was extracted with ethyl acetate (3.65 L). The organic layers were combined, washed with brine (1.5 L), and concentrated in vacuo.
[0173] The residue was treated with ethyl acetate (1.8 L) and heated to 65 to 70 ° C. The mixture was stirred at 65 to 70 ° C for 15 minutes to provide a clear solution and then treated with n-heptane (7.3 L) maintaining the temperature between 60 to 70 ° C. Once heptane was completely added to the solution, the mixture was kept at 65 to 70 ° C for 15 minutes and then allowed to cool between 18 to 22 ° C for 3 h. The resulting suspension was stirred at 18 to 22 ° C for 4 h, cooled to 0 to 5 ° C for 1 h, and maintained between 0 to 5 ° C for 2 h. The precipitate was filtered, washed twice with n-heptane (1.4 L), and dried in vacuo to provide the title compound (540 g, 88%). The XRPD pattern of this compound is shown in Figure 17.

[0174] Methyl 5-bromo-3- (ethyl (tetrahydro-2H-pyran-4-yl) amino) -2-methylbenzoate (3) To a stirred solution of 5-bromo-2-methyl-3- Methyl ((tetrahydro-2H-pyran-4-yl) amino) benzoate (14 g, 42.7 mmol) in dichloroethane (150 mL) were added acetaldehyde (3.75 g, 85.2 mmol) and acid acetic (15.3 g, 256 mmols). The resulting reaction mixture was stirred at room temperature for 15 minutes. The mixture was cooled to 0 ° C and sodium triacetoxyborohydride (27 g, 128 mmol) was added. The reaction mixture was stirred at room temperature for 3 hours. After completion of the reaction as determined by TLC, aqueous sodium bicarbonate solution was added to the reaction mixture until a pH 7-8 was obtained, the organic phase was separated and the aqueous phase was extracted with ethyl acetate. The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The crude compound was purified by column chromatography (silica gel, 100 - 200 mesh) eluting with ethyl acetate: hexane to provide the desired compound as a viscous liquid (14 g, 93%). 1H NMR (DMSO-ote, 400 MHz) δ 7.62 (s, 1H), 7.52 (s, 1H), 3.80 (bs, 5H), 3.31 (t, 2H), 2, 97 - 3.05 (m, 2H), 2.87 - 2.96 (m, 1H), 2.38 (s, 3H), 1.52 - 1.61 (m, 2H), 1, 37 - 1.50 (m, 2H), 0.87 (t, 3H, J = 6.8 Hz).

[0175] Methyl 5- (ethyl (tetrahydro-2H-pyran-4-yl) amino) -4-methyl-4 '- (morpholinomethyl) - [1,1' -biphenyl] - 3-carboxylate (4 ): A mixture of methyl 5-bromo-3- (ethyl (tetrahydro-2H-pyran-4-yl) amino) -2-methylbenzoate (580 g, 1.63 mol), 4- (4- ( 4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) benzyl) morpholine (592 g, 1.95 mol), 1,4-dioxane (3.86 L), sodium carbonate (618 g, 5.83 moles), and water (771 ml) was degassed by bubbling nitrogen through the mixture at 20 ° C for 20 minutes and treated with tetracis (triphenylphosphine) palladium (0) (14.11 g, 12.21 mmols ). The resulting mixture was degassed for an additional 20 minutes and then heated at 87 to 89 ° C for 17 h. After cooling to 20 ° C, the mixture was diluted with ethyl acetate (5.80 L) and a solution of (R) -2-amino-3-mercaptopropionic acid (232 g) in water (2,320 L). After stirring for 1 h at 20 ° C, the organic layer was separated and washed again with a solution of (R) -2-amino-3-mercaptopropionic acid (232 g) in water (2.320 L). The aqueous layers were combined and extracted with ethyl acetate (5.80 L). The organic layers were combined, washed with a solution of sodium hydroxide (93 g) in water (2.32 L), and concentrated in vacuo at 35 ° C to provide the title compound as an orange oil (1.21 kg) , 164% yield).

[0176] 5- (Ethyl (tetrahydro-2H-pyran-4-yl) amino) -4-methyl-4 '- (morpholinomethyl) - [1,1 biphenyl] -3-carboxylic acid (5): 5 - Methyl (ethyl (tetrahydro-2H-pyran-4-yl) amino) -4-methyl-4'- (morpholinomethyl) - [1,1'-biphenyl] -3-carboxylate (69.0 g, 152.5 mmols) (based on theoretical yield from the previous step) was suspended in ethanol (380 ml) and treated with a solution of sodium hydroxide (24.84 g, 621.0 mmols) in water ( 207 mL). The mixture was stirred at 40 ° C for 18 h. After cooling to 0 to 5 ° C, the mixture was neutralized to pH 6.5 with 1 N hydrochloric acid (580 ml) keeping the temperature below 25 ° C. Then, the mixture was extracted twice with a mixture of dichloromethane (690 ml) and methanol (69.0 ml). The organic layers were combined and concentrated in vacuo to provide a crude product as a yellow solid (127 g).
[0177] The crude product was dissolved in 2-methyltetrahydrofuran (656 ml) at 70 ° C and then treated with IPA (828 ml). The mixture was allowed to cool to room temperature for 3 to 4 h and then stirred overnight at room temperature. The precipitate was filtered, washed twice with IPA (207 ml), and dried in vacuo to provide the title compound as an off-white solid (53.54 g, 80%). The XRPD pattern of this compound is shown in Figure 9.

[0178] N - ((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl) methyl) -5- (ethyl (tetrahydro-2H-pyran-4-yl) amino ) -4-methyl-4 '- (morpholinomethyl) - [1,1'-biphenyl] -3-carboxamide (Compound I): A mixture of 5- (ethyl (tetrahydro-2H-pyran-4-yl) acid ) amino) -4-methyl-4 '- (morpholinomethyl) - [1,1'-biphenyl] -3-carboxylic (540 g, 1.23 moles) and 3- (aminomethyl) -4,6-dimethyl hydrochloride -dihydro-pyridin-2 (1 H) -one (279 g, 1.48 mol) was suspended in DMSO (2.70 L) and treated with triethylamine (223 ml, 1.60 mol). The mixture was stirred at 25 ° C for 30 min and treated with EDC-HCI (354 g, 1.85 mol) and hydrated HOBT (283 g, 1.85 mol). The reaction mixture was stirred at room temperature for 16 h. After the addition of triethylamine (292 ml, 2.09 moles), the mixture was cooled to 15 ° C, diluted with water (10.1 L) keeping the temperature below 30 ° C, and stirred between 19 to 25 ° C for 4 h. The resulting precipitate was filtered, washed twice with water (2.70 L), and dried under vacuum to provide a crude product (695 g, weight-by-weight analysis = 78%).
[0179] For the other product purification, recrystallization was performed. A crude product (20.00 g, 34.92 mmols) was suspended in a mixture of ethanol (190 ml) and water (10.00 ml) and heated to 75 ° C until a clear solution was obtained. The solution was allowed to stir to room temperature overnight. The precipitate was filtered, washed twice with a mixture of ethanol (30.0 ml) and water (30.0 ml), and dried under vacuum at 35 ° C to provide the title compound as an off-white solid (14, 0 g, 70% recovery from crude and 90% yield based on weight-to-weight testing).

[0180] 4 - ((3 '- ((((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl) methyl) carbamoyl) - 5' - (ethyl (tetra- hydro-2H-pyran-4-yl) amino) -4'-methyl- [1,1'-biphenyl] -4-yl) methyl) morpholin-4-io (Polymorph A): An N - ((4, 6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl) methyl) -5- (ethyl (tetrahydro-2H-pyran-4-yl) amino) -4-methyl-4'- (morpholinomethyl) - Crude [1,1'-biphenyl] -3-carboxamide (595 g, 464 g based on weight-by-weight assay, 810.3 mmols) was suspended in ethanol (3.33 L) . After heating to 70 ° C, the mixture was treated with 48% aqueous HBr (97 ml, 850.8 mmols) and stirred at 70 ° C for 30 min. The resulting orange-red solution was treated with ethyl acetate (3.33 L) keeping the temperature above 60 ° C. The mixture was slowly cooled to room temperature over 18h. The mixture was cooled to 0 ° C for 1 h and stirred at that temperature for 5.5 h. The resulting precipitate was filtered, washed twice with ethyl acetate (1.39 L), and dried in vacuo to provide the title compound as an off-white solid (515 g, 97% yield).
[0181] Recrystallization of Polymorph A: 4 - ((3 '- ((((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl) methyl) carbamoyl) -5' bromide - (ethyl (tetrahydro-2H-pyran-4-yl) amino) -4'-methyl- [1,1'-biphenyl] -4-yl) methyl) morpholin-4-io (0.50 g, 0.77 mmol; 95.6% pure by HPLC) was suspended in ethanol (3.0 mL) and heated to 80 ° C until a clear solution was obtained. To the solution, MTBE (5.0 mL) was added slowly. The resulting solution was allowed to stir at 18 to 22 ° C for 3 h and stirred at 18 to 22 ° C for 15 h. The precipitate was filtered, washed twice with MTBE (2 ml) and dried in vacuo to provide 0.45 g of the title compound (89% recovery, 96.6% pure by HPLC). The powder X-ray diffraction pattern of Polymorph A (monohydrobromide) is shown in Figure 1. Table 1, below, lists the most significant peaks.
Evaluation of Hydrobromide of Compound I and Polymorph A
[0182] Several different salt forms of Compound I were prepared and screened, including hydrochloride, hydrobromide, hemisulfate, sodium, phosphate, nitrate, maleate, malonate and L-tartrate salts. Among them, the hydrobromide salt (HBr) had the most advantageous physico-chemical properties in terms of ease of preparation and hygroscopicity.
[0183] Detailed studies of the free base of Compound I, as well as the HCI salt of this compound were carried out. At least five different crystal forms were detected from the free form of Compound I during the preliminary polymorph scan using XRD and DSC. Due to the high degree of variability observed during the crystallization of the free form, the crystal forms of other salts were sought. Of the screened salts, the mono-hydrochloride, monohydrobromide, hemisulfate, phosphate, maleate, L-tartrate and sodium forms were crystalline. The phosphate and maleate salts were very hygroscopic and L-tartrate had poor crystallinity.
[0184] It was difficult to obtain a high degree of crystallinity from the HCI salt of Compound I. A mixture of crystalline and amorphous material was obtained regardless of crystallization conditions. As shown in Figure 8, DSC data for the Compound I monohydrochloride salt indicates some degree of non-crystallinity with an endotherm at 190.5 ° C. Also, dynamic vapor sorption (DVS) data for the Compound I monohydrochloride salt was obtained to show some hygroscopicity: between 4 to 6% of weight gain was observed at 75% relative humidity (RH) at 25 ° C (Figure 18B). This can be attributed to a certain amount of non-crystalline nature of the monohydrochloride salt. See, for example, Figure 18A, which shows an amorphous Compound I trichloride. As the level of crystallinity was not controllable, the HCI salt was not considered for further development.
[0185] As shown in Figure 6, the DVS analysis of the sodium salt of Compound I showed significant hygroscopicity: approximately 15% weight gain was observed at 75% relative humidity (RH) at 25 ° C. As shown in Figure 7, the Compound I hemisulfate salt showed moderately high hygroscopicity: between 9 to 11% weight gain was observed at 75% relative humidity (RH) at 25 ° C. This can be attributed to the highly non-crystalline nature of the compound, as the DSC data for the hemisulfate salt indicate a very high degree of non-crystallinity without clean endotherm.
[0186] Of these crystalline compounds, the monohydrobromide was the most crystalline and least hydroscopic (see, Figures 1, 3, and 4). In addition, the monohydrobromide is highly stable and resists the generation of impurities (Figure 5 represents the HPLC analysis of Polymorph A for three days at an elevated temperature. Polymorph A produced minimal impurities for three days at 100 ° C) . Interestingly, the di-HBr salt of Compound I was found to be mainly amorphous (Figure 2).
[0187] Two different crystal forms of Compound I monohydrobromide (Polymorphs A and B) were obtained from different solvent systems and characterized using XRD, DSC and TGA-DSC analyzes. The XRD and DSC data for these two different crystal forms from representative lots of Compound I are shown in Figure 1, Figure 3 and Figure 10. Polymorph B is characterized by a powder XRD pattern with peaks at 8.5, 10.9, 16.7, 17.4, 20.9, 22.1 and 25.7 ± 0.2 degrees 2 theta (see, Figure 10). Between these two, Polymorph A was found to be more crystalline in nature. Dynamic vapor adsorption (DVS) studies have shown that polymorph A is non-hygroscopic (Figure 4). In thermal analysis, a single endothermic peak was observed with an initial temperature of approximately 251 ° C. In addition, it became evident from the DSC analysis that the recrystallization of polymorph A significantly increases the crystallinity of the material (see, Figure 3).
[0188] In several rounds on a laboratory scale, Polymorph A was reproducibly obtained, and the slight changes in crystallization conditions did not result in different crystal forms. Wild and Mutant PRC2 Enzyme Assays
[0189] General Materials. S-adenosylmethionine (SAM), S-adenosylomocytein (SAH), bicin, KCI, Tween20, dimethylsulfoxide (DMSO) and bovine skin gelatin (BSG) were purchased from Sigma-Aldrich at the highest possible level of purity. Dithiothreitol (DTT) was purchased from EMD. 3H SAM was purchased from American Radiolabeled Chemicals with a specific activity of 80 Ci / mmol. 384-well streptavidin flashplates were purchased from PerkinElmer.
[0190] Substrates. Representative peptides from human histone H3 residues 21 - 44 containing an unmodified lysine 27 (H3K27meO) or dimethylated lysine 27 (H3K27me2) were synthesized with a C-terminal motif G (K-biotin) linker affinity tag and a capamide C-terminal by 21st Century Biochemicals. The peptides were purified by high performance liquid chromatography (HPLC) to more than 95% purity and confirmed by liquid chromatography coupled with mass spectrometry (LC-MS). The strings are listed below. H3K27meO: ATKAARKSAPATGGVKKPHRYRPGGK (biotin) -amide (SEQ ID NO: 1) H3K27me2: ATKAARK (me2) SAPATGGVKKPHRYRPGGK (biotin) -amide (SEQ ID NO: 2)
[0191] Chicken erythrocyte oligonucleosomes have been purified from chicken blood according to established procedures.
[0192] Recombinant PRC2 complexes. Human PRC2 complexes were purified as 4-component enzyme complexes co-expressed in Spodoptera frugiperda (sf9) cells using a baculovirus expression system. The expressed subunits were EZH2 wild type (NM_004456) or EZH2 Y641F, N, H, S or C mutants generated from the construction of wild type EZH2, EED (NM_003797), Suz12 (NM_015355) and RbAp48 (NM_005610). The EED subunit contained an N-terminal FLAG tag that was used to purify the entire 4-component lysate complex of sf9 cells. The purity of the complexes reached or exceeded 95% as determined by the analysis of SDS-PAGE and Agilent Bioanalyzer. Concentrations of enzyme stock concentrations (usually 0.3 to 1.0 mg / mL) were determined using a Bradford assay against a bovine serum albumin (BSA) standard.
[0193] General Procedure for PRC2 Enzyme Assays on Peptide Substrates. The tests were all carried out in a buffer consisting of 20 mM bicine (pH = 7.6), 0.5 mM DTT, 0.005% BSG and 0.002% Tween20, prepared on the day of use. The compounds in 100% DMSO (1 pL) were stained in 384 well polypropylene V bottom plates (Greiner) using a 2 X 3 Platemate equipped with a 384 channel pipette head (Thermo). DMSO (1 pL) was added to columns 11, 12, 23, 24, rows A - H for maximum signal control, and SAH, a known product and inhibitor of PRC2 (1 pL) were added to columns 11,12, 23, 24, rows I - P for minimum signal control. A cocktail (40 pL) containing the wild type PRC2 enzyme and H3K27meO peptide or any of mutant Y641 enzymes and H3K27me2 peptide was added by Multidrop Combi (Thermo). The compounds were allowed to incubate with PRC2 for 30 min at 25 ° C, then a cocktail (10 pL) containing a mixture of non-radioactive SAM and 3 H was added to start the reaction (final volume = 51 pL). In all cases, the final concentrations were as follows: wild-type or mutant PRC2 enzyme was 4 nM, SAH in the wells with minimal signal control was 1 mM and the DMSO concentration was 1%. The final concentrations of the rest of the components are shown in Table 2, below. The assays were interrupted by the addition of non-radioactive SAM (10 pL) to a final concentration of 600 pM, which dilutes the 3H SAM to a level where its incorporation into the peptide substrate is no longer detectable. 50 pL of the reaction on the 384-well polypropylene plate was then transferred to a 384-well Flashplated and the biotinylated peptides were allowed to bind to the streptavidin surface for at least 1 h before being washed three times with 0.1 Tween20 % in a Biotek ELx405 plate washer. The plates were then read on a TopCount PerkinElmer plate reader to measure the amount of 3H-labeled peptide limited to the surface of the Flashplate, measured as disintegrations per minute (dpm) or alternatively, referred to as count per minute (cpm). Table 2: Final component concentrations for each assay variation based on the identity of EZH2 (wild-type EZH2 or mutant Y641)

[0194] General Procedure for PRC2 Wild Type Enzyme Assay on Oligonucleosome Substrate. The tests were performed in a buffer consisting of 20 mM bicin (pH = 7.6), 0.5 mM DTT, 0.005% BSG, 100 mM KCI and 0.002% Tween20, prepared on the day of use. The compounds in 100% DMSO (1 pL) were stained in 384 well polypropylene V bottom plates (Greiner) using a 2X3 Platemate equipped with a 384 channel pipette head (Thermo). DMSO (1 pL) was added to columns 11, 12, 23, 24, rows A - H for maximum signal control, and SAH, a known product and inhibitor of PRC2 (1 pL) were added to columns 11,12, 23, 24, rows I - P for minimum signal control. A cocktail (40 pL) containing the wild type PRC2 enzyme and chicken erythrocyte oligonucleosome was added by Multidrop Combi (Thermo). The compounds were allowed to incubate with PRC2 for 30 min at 25 ° C, then a cocktail (10 pL) containing a mixture of non-radioactive SAM and 3 H was added to start the reaction (final volume = 51 pL). Final concentrations were as follows: wild type PRC2 enzyme was 4 nM, non-radioactive SAM was 430 nM, 3H SAM was 120 nM, chicken erythrocyte olignonucleosome was 120 nM, SAH in the least signal control wells was 1 mM and the DMSO concentration was 1%. The assay was interrupted by the addition of non-radioactive SAM (10 pL) to a final concentration of 600 pM, which dilutes the 3H SAM to a level where its incorporation into the chicken erythrocyte olignonucleosome substrate is no longer detectable. 50 pL of the reaction in the 384-well polypropylene plate was then transferred to a 384-well Flashplated and the chicken erythrocyte nucleosomes were immobilized on the plate surface, which was then washed three times with 0.1% Tween20 in a washer. ELx405 Biotek plate. The plates were then read on a TopCount PerkinElmer plate reader to measure the amount of 3H-labeled chicken erythrocyte oligonucleosome limited to the surface of the Flashplate, measured as disintegrations per minute (dpm) or alternatively, referred to as count per minute (cpm).

[0195] Where dpm = disintegrations per minute, cmpd = signal in the test well, and min and max are the respective minimum and maximum signal controls. Four parameter ICso adjustment

[0196] Where the top and bottom are normally allowed to float, but can be set to 100 or 0 respectively in a 3-parameter setting. The Hill coefficient is usually allowed to float, but it can also be set to 1 in a 3-parameter setting. Y is the% inhibition and X is the concentration of the compound.
[0197] IC50 values for the PRC2 enzyme assays on peptide substrate (eg, andY641 F wild type EZH2) are shown in Table 3 below. WSU-DLCL2 Methylation Assay
[0198] WSU-DLCL2 suspension cells were purchased from DSMZ (German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany). RPMI / Glutamax medium, Penicillin-Streptomycin, Heat Inactivated Bovine Fetal Serum, and D-PBS were purchased from Life Technologies, Grand Island, NY, USA. Extraction Buffer and Neutralization Buffer (5X) were purchased from Motivo Active, Carlsbad, CA, USA. Rabbit antihistone H3 antibody was purchased from Abeam, Cambridge, MA, USA. Rabbit anti-H3K27me3 and HRP-conjugated anti-rabbit-IgG were purchased from Cell Signaling Technology, Danvers, MA, USA. TMB substrate “Supor sinsitive” was originated from BioFX Laboratories, Owings Mills, MD, USA. IgG-free bovine serum albumin was purchased from Jackson ImmunoResearch, West Grove, PA, USA. PBS with Tween (10X PBST) was purchased from KPL, Gaithersburg, MD, USA. Sulfuric acid was purchased from Ricca Chemical, Arlington, TX, USA. Immulon ELISA plates were purchased from Thermo, Rochester, NY, USA. Bottom V cell culture plates were purchased from Corning Inc., Corning, NY, USA. The V bottom polypropylene plates were purchased from Greiner Bio-one, Monroe, NC, USA.
[0199] WSU-DLCL2 suspension cells were maintained in the growth medium (RPMI 1640 supplemented with 10% (v / v) heat-inactivated fetal bovine serum and 100 units / ml of penicillin-streptomycin) and cultured at 37 ° C under 5% CO2. Under the assay conditions, the cells were incubated in Assay Medium (RPMI 1640 supplemented with 20% (v / v) heat inactivated fetal bovine serum and 100 units / mL of penicillin-streptomycin) at 37 ° C under CO2 at 5 % on a plate shaker.
[0200] WSU-DLCL2 cells were seeded in assay medium at a concentration of 50,000 cells per ml to a 96-well V bottom cell culture plate with 200 pL per well. Compound (1 pL) from 96-well source plates incubated on a titration plate shaker at 37 ° C, 5% CO2 for 96 hours. After four days of incubation, the plates were rotated at 241 x g for five minutes and the medium was gently aspirated into each cell plate well without disturbing the cell pellet. The pellet was resuspended in DPBS 200 pL and the plates were rotated again at 241 x g for five minutes. The supernatant was aspirated and cooled (4 ° C), extraction buffer (100 pL) was added per well. The plates were incubated at 4 ° C on an orbital shaker for two hours. The plates were rotated at 3427 x g x 10 minutes. The supernatant (80 pL per well) was transferred to its respective well in a 96-well V bottom polypropylene plate. 5X Neutralization Buffer (20 pL per well) was added to the bottom V polypropylene plate containing supernatant. The bottom V polypropylene plates containing crude histone preparation (CHP) were incubated on an orbital shaker x five minutes. Crude Histone Preparations were added (2 pL per well) to each respective well in duplicate 96-well ELISA plates containing 100 pL Coating Buffer (1X PBS + 0.05% (w / v) BSA)). The plates were sealed and incubated overnight at 4 ° C. The next day, the plates were washed three times with 1X PBST (300 pL per well). The wells were blocked for two hours with ELISA Diluent (300 pL per well) ((PBS (1X) BSA (2% (w / v)) and Tween20 (0.05% (v / v))). were washed three times with 1X PBST.For the Histone H3 detection plate, 100 pL per well was added with anti-Histone-H3 antibody (Abeam, ab1791) diluted 1: 10,000 in ELISA Diluent. H3K27 trimethylation, 100 pL per well was added with anti-H3K27me3 diluted 1: 2000 in ELISA diluent. The plates were incubated for 90 minutes at room temperature. The plates were washed three times with 1X PBST (300 pL per well). the detection of Histone H3, 100 μl of antibody to anti-rabbit IgG conjugated to HRP diluted 1: 6000 in ELISA diluent were added per well For the detection of H3K27me3, 100 μl of anti-rabbit IgG antibody conjugated to HRP diluted 1: 4000 in ELISA diluent were added per well.The plates were incubated at room temperature for 90 minutes. four times with 1X PBST (300 pL per well). The TMB substrate (100 pL) was added per well. Histone H3 plates were incubated for five minutes at room temperature. The H3K27me3 plates were incubated for 10 minutes at room temperature. The reaction was stopped with 1N sulfuric acid (100 pL per well). Absorbance for each plate was read at 450 nm.
[0201] First, the reason for each well was determined by:

[0202] Each plate included eight DMSO control wells for treatment only (Minimum Inhibition), as well as eight control wells for maximum inhibition (bottom wells).
[0203] The average of the ratio values for each type of control was calculated and used to determine the percentage of inhibition for each test well on the plate. The test compound was serially diluted three times in DMSO to a total of ten test concentrations, starting at 25 pM. The percentage of inhibition was determined and the ICso curves were generated using duplicate wells by compound concentration. The ICso values for this assay are shown in Table 3 below.
Cell Proliferation Analysis
[0204] WSU-DLCL2 suspension cells were purchased from DSMZ (German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany). RPMI / Glutamax medium, Penicillin-Streptomycin, Fetal Heat Inactivated Bovine Serum were purchased from Life Technologies, Grand Island, NY, USA. The 384 well V polypropylene bottom plates were purchased from Greiner Bio-one, Monroe, NC, USA. White opaque plates from 384 cell culture wells were purchased from Perkin Elmer, Waltham, MA, USA. Cell-Titer Glo® was purchased from Promega Corporation, Madison, WI, USA. SpectraMax M5 card reader was purchased from Molecular Devices LLC, Sunnyvale, CA, USA.
[0205] The WSU-DLCL2 suspension cells were maintained in growth medium (RPMI 1640 supplemented with 10% (v / v) fetal heat-inactivated bovine serum and cultured at 37 ° C under 5% CO2. Under test conditions , the cells were incubated in assay medium (RPMI 1640 supplemented with 20% (v / v) inactivated fetal bovine serum by heat and 100 units / ml of penicillin-streptomycin) at 37 ° C under 5% CO2.
[0206] For the evaluation of the effect of compounds on the proliferation of the WSU-DLCL2 cell line, exponentially growing cells were plated on 384-well white opaque plates at a density of 1250 cells / ml in a final volume of 50 pl of test medium. A compound source plate was prepared by performing 3-fold serial dilutions at nine points in triplicate in DMSO, starting at 10 mM (final top compound concentration in the assay was 20 pM and DMSO was 0.2%). A 100 nL aliquot of the compound stock plate was added to its respective well on the cell plate. The 100% inhibition control consisted of cells treated with 200 nM final concentration destaurosporin and the 0% inhibition control consisted of cells treated with DMSO. After adding compounds, the assay plates were incubated for 6 days at 37 ° C, 5% CO2, relative humidity> 90% for 6 days. Cell viability was measured by quantizing the ATP present in the cell cultures, adding 35 µl of Cell Titer Glo® reagent to the cell plates. Luminescence was read on the M5 SpectraMax. The viability of cells that inhibit the concentration by 50% was determined using a 4-parametric adjustment of the normalized dose response curves. IC50 values for this assay are shown in Table 3 below.
In Vivo Study - SUDHL10 Human Lymphoma Cell Line Mice
[0207] Female Fox Chase SCID® mice (CB17 / cr-Prkdcscid / cr c ° Cr , Beijing Vitalriver Laboratory Animal Co., LTD) aged 6 to 8 weeks and a body weight (BW) ranging from 16.0 to 21.1 g in study D1. The animals were fed with water (sterile) and dry granulated foods sterilized by irradiation. The mice were housed in a corn cob bed in static microisolators in a 12-hour light cycle between 20 to 22 ° C (68 to 72 ° F) and 40 to 60% humidity. All procedures in accordance with the Guide for Care and Use of Laboratory Animals recommendations regarding restriction, breeding, surgical procedures, feeding and fluid regulation, and veterinary care. Tumor Cell Culture
[0208] The human lymphoma cell line SUDHL10 was obtained from DSMZ and maintained in the CRO as suspension cultures in RPMI-1640 medium containing 100 units / ml of penicillin G sodium salt, 100 g / ml of streptomycin and 10 % fetal bovine serum. The cells were grown in tissue culture flasks in a humidified incubator at 37 ° C, in an atmosphere of 5% CO2 and 95% air. Only cultures below passage 12 were used for implantation. In vivo Tumor Implantation
[0209] The human lymphoma cell line SUDHL10 was harvested during mid-log phase growth, and resuspended in PBS with 50% Matrigel ™ (BD Biosciences). Each mouse received 1 x 10 7 cells (0.2 ml of cell suspension) subcutaneously on the right flank. The tumors were calibrated in two dimensions to monitor growth as the approximate average volume in the desired 80 to 120 mm3 range. The tumor size, in mm3, was calculated from:
where w = width and / = length, in mm, of the tumor. The weight of the tumor can be estimated with the assumption that 1 mg is equivalent to 1 mm3 of tumor volume. After 10 days, mice with tumors ranging from 72 to 256 mm3 were divided into four groups (n = 16 per group) with an average tumor volume of 173 to 179 mm3. Test Articles
[0210] Compound I hydrobromide was stored at room temperature and protected from light. On each treatment day, formulations of freshly prepared compounds were prepared by suspending the powder in 0.5% sodium carboxymethyl cellulose (NaCMC) and 0.1% Tween® 80 in deionized water. The vehicle, 0.5% NaCMC and 0.1% Tween® 80 in deionized water, was used to treat the control group on the same schedule. The formulations were stored away from light at 4 ° C before administration. Treatment Plan
[0211] The mice were treated with doses of Compound I hydrobromide ranging from 125 to 500 mg / kg and on BID schedules (twice a day every 12 h) for 28 days by oral tube. Each dose was released in a volume of 0.2 mL / 20 g of mouse (10 mL / kg), and adjusted to the last recorded weight of individual animals. On day 25, the 8 mice with the smallest tumors per group were chosen for a tumor growth delay endpoint (60 day observation). The remaining animals were sacrificed on day 28, 3 h after the last dose for tumor collection. Analysis of Mean Tumor Volume (MTV) and Tumor Growth Inhibition (TGI)
[0212] Treatment effectiveness was determined on the last day of treatment. MTV (n), the average tumor volume for the number of animals, n, assessable on the last day, was determined for each group. The percentage of tumor growth inhibition (% TGI) can be defined in several ways. First, the difference between the MTV (n) of the designated control group and the MTV (n) of the drug-treated group is expressed as a percentage of the MTV (n) of the control group:

[0213] Another way to calculate the% of TGI is by taking the change in tumor size from day 1 to day n with account being n being the last day of treatment.

[0214] Eight mice per group were kept alive after the last day of treatment for the analysis of tumor growth delay. The tumors were calibrated twice a week and each test animal was sacrificed when its neoplasm reached the endpoint volume of 2000 mm3 or on the last pre-specified day of the study, whichever came first. The Kaplan Meier survival analysis was performed. Toxicity
[0215] The animals were weighed daily on Days 1 to 5, and then twice weekly until the study was completed. The mice were examined frequently for obvious signs of any treatment-related adverse side effects that have been documented. Acceptable toxicity for the maximum tolerated dose (BAT) was defined as an average group BW loss of less than 20% during the test, and not more than 10% mortality due to RT deaths. A death was to be classified as TR if it was attributable to the side effects of treatment as evidenced by clinical signs and / or necropsy, or due to an unknown cause during the dosing period. A death was to be classified as NTR if there was evidence that the death was not related to treatment side effects. NTR deaths during the dosing interval would typically be categorized as NTRa (due to an accident or human error) or NTRm (due to tumor spread confirmed by necropsy by invasion and / or metastasis). Orally treated animals that die of unknown causes during the dosing period can be classified as NTRu when the performance of the group does not support a TR classification and necropsy, to rule out a dosing error, is not viable. Sampling
[0216] On day 28, eight mice with the largest tumors were tested in a pre-specified way to assess target inhibition in tumors. The tumors were harvested from specified mice under RNAse-free conditions and divided. The total tumor weight was measured. Frozen tumor tissue from each animal was frozen in liquid N2 and sprayed with a mortar. Statistical and Graphical Analysis
[0217] All statistical and graphical analyzes were performed with Prism 3.03 (GraphPad) for Windows. To test the statistical significance between the control and treated groups throughout the course of treatment, an ANOVA test of repeated measurements followed by Dunnets post-multiple comparison test was used. Prism reports results as non-significant (ns) at P> 0.05, significant (symbolized by “*”) at 0.01 <P <0.05, very significant (“**”) at 0.001 <P < 0.01 and extremely significant (“***”) at P <0.001. For the tumor growth delay branch of the study, the percentage of animals in each group remaining in the study versus time was presented in a Kaplan-Meier survival batch. Histone extraction
[0218] For histone isolation, 60 to 90 mg of tumor tissue were homogenized in 1.5 ml of nuclear extraction buffer (10 mM Tris-HCI, 10 mM MgCI2, 25 mM KCI, 1% Triton X-100 , 8.6% sucrose, plus a Roche 1836145 protease inhibitor tablet) and incubated on ice for 5 minutes. The cores were collected by centrifugation at 600 g for 5 minutes at 4o C and washed once in PBS. The supernatant was removed and histones extracted for one hour, vortexing every 15 minutes, with cold 0.4 N sulfuric acid. The extracts were clarified by centrifugation at 10,000 g for 10 minutes at 4 ° C and transferred to a newly microcentrifuge tube. -prepared containing 10x volume of cold acetone. Histones were precipitated at -20 ° C for 2 hours overnight, pelleted by centrifugation at 10,000 g for 10 minutes and resuspended in water. ELISA
[0219] Histones were prepared in equivalent concentrations in coating buffer (PBS + 0.05% BSA) providing 0.5 ng / ul sample, and 100 ul sample or standard was added in duplicate to 2 ELISA plates of 96 wells (Thermo Labsystems, Immulon 4HBX # 3885). The plates were sealed and incubated overnight at 4 ° C. The next day, the plates were washed 3x with PBST 300 µl / well (PBS + 0.05% Tween 20; 10X PBST, KPL # 51-14-02) in a Bio Tek plate washer. The plates were blocked with 300 µl / well of diluent (PBS + 2% BSA + 0.05% Tween 20), incubated at room temperature for 2 hours, and washed 3x with PBST. All antibodies were diluted in diluent. 100 µl / well of anti-H3K27me3 (CST # 9733, 50% glycerol stock 1: 1,000) or total anti-H3 (Abeam ab1791.50% glycerol 1: 10,000) was added to each plate. The plates were incubated for 90 min at room temperature and washed 3x with PBST. 100 µl / well of anti-Rb-IgG-HRP (Cell Signaling Technology, 7074) was added 1: 2,000 to the H3K27Me3 plate and 1: 6,000 to the H3 plate and incubated for 90 min at room temperature. The plates were washed 4X with PBST. For detection, 100 μl / well of TMB substrate (BioFx Laboratories, #TMBS) was added and the plates incubated in the dark at room temperature for 5 min. The reaction was stopped with 100 μl / well of 1N H2SO4. Absorbance at 450 nm was read on a SpectaMax M5 Microplate reader. Results:
[0220] Mice bearing SUDHL10 tumor xenografts were treated with Compound I hydrobromide at the maximum tolerated dose of 500 mg / kg BID and fractions of BAT (1/2 and% of BAT). All doses were well tolerated for 28 days without any significant loss of body weight. There was a non-treatment-related death in the 500 mg / kg group on day 15 due to a dosing error. All doses resulted in tumor growth inhibition when compared to vehicle on day 28 (Table 4), and the BID groups 250 mg / kg and 500 mg / kg induced regressions (TGI> 100%). Table 4: Summary of tumor growth inhibition values induced by Compound I hydrobromide in SUDHL10 xenografts

[0221] Figure 12A shows the growth of SUDHL10 xenograft tumors over time for different treatment groups. The BID 125 mg / kg group was not significantly different from the vehicle group by repeated measurements ANOVA and Dunnett powders, but the mean terminal tumor size on day 28 was significantly smaller than one in the vehicle group (2-way ANOVA with Bonferroni post test, p <0.0001). The dosage of BID 250 mg / kg and BID 500 mg / kg of the Compound I hydrobromide for 28 days induced comparable regression responses as the terminal tumor weights on day 28 were similar for those of 2 groups (Figure 12B).
[0222] Histones isolated from tumors harvested on day 28 (3 h after the last dose) were subjected to ELISA for global levels of H3K27me3. Figure 13 shows a clear dose-dependent sub-regulation of the methyl brand H3K27me3 treated with Compound I hydrobromide. This figure shows the overall methylation of H3K27me3 in SUDHL10 tumors of mice treated with Compound I hydrobromide for the 28 days.
[0223] On day 25, eight mice per group with small tumors were chosen for a tumor growth delay study to assess tumor regrowth after stopping dosing on day 28. The mice were euthanized when their tumors reached a size 2000 mm3 or on day 60 (whichever comes first). These data were used to perform a Kaplan Meier survival analysis. Figure 14A shows that tumor regrowth was clearly dose dependent, and all mice treated with the highest dose of BID 500 mg / kg for 28 days survived until day 60. Only 2 mice had to be sacrificed in the 250 group mg / kg before day 60. Mice in the 125 mg / kg group had a clear survival benefit over vehicle-treated mice with a 15.5-day mean increase in survival (Figure 14B). Anti-cancer effect of Compound I hydrobromide in the mouse model of widespread human large-cell B lymphoma Pfeiffer
[0224] Compound I monohydrobromide was tested for its anti-cancer activity in a mouse Pfeiffer xenograft model, which is a model of widespread human B-cell lymphoma xenograft. Females of 5-week-old NSG mice (Jackson Labs, Bar Harbor, Maine) were implanted subcutaneously with 20 to 25 mg of tumor fragments. The treatment was started approximately 31 days after the implantation of the tumor, when the average tumors reached approximately 365 mm3. The treatment schedule is described in Table 5.
§ Due to the issue of compound tolerability, only 12 daily doses were provided to these groups.
[0225] The tumor volume was followed throughout the experiment. The tumor volume was measured twice weekly after starting treatment. The tumor load (mg = mm3) was calculated from measurements calibrated by the formula for the volume of an elongated ellipsoid (LxW2) / 2 where L and W are the respective orthogonal length and width measurements (mm).
[0226] Day 1 was the day of the first treatment, and Day 28 was the day of the last treatment. This study was completed 36 days after the last dose, thus Day 64 was the day of the study termination. The primary endpoints used to assess effectiveness in this study were complete tumor regressions (CR), tumor sizes between groups, and percentage of tumor inhibition at the end of the study. A complete response was defined as a decrease in tumor size to an undetectable size (<20mm3) at the end of the study. The values for the percentage of tumor inhibition were calculated using the formula [1- (ΔT / ΔC)] x 100, where ΔT and ΔC are changes in mean tumor volume (Δ growth) for each treated group (T) and control (Ç). To and Co (one day before the first dose) were used for the starting tumor volume. In addition, the tumor volumes that were taken one day after the last dose (T29 and C29) were used for the calculation of ΔT and ΔC. When the value was greater than 100%, it was completed as 100%. The formula used for calculating the percentage of tumor inhibition is shown below.

[0227] During the treatment period, animals were found to be able to tolerate the daily treatment of 1142 mg / kg of Compound I hydrobromide and three animals in this group (group E) needed to be euthanized after the first week of treatment due the loss of more than 20% of body weight as a reference value. Consequently, drug administration for this group was stopped after 12 doses. The animals in three other dosage groups, except one animal in group D (342 mg / kg of Compound I hydrobromide), all tolerated the 28-day treatment with minimal body weight loss well. The relative mouse body weight was represented in Figure 15. The animal body weight obtained on Day 0 was used as the body weight as a reference value in the graph.
[0228] Compound I hydrobromide showed potent and long-lasting anti-cancer activity in a Pfeiffer model with a 100% CR rate in three of the four dosing groups (Table 6). In addition, tumor regrowth was not observed even 36 days after treatment ceased. This suggests that all tumor cells were killed during the treatment. Although tumor regrowth was observed in the group with the lowest dose (group B, 34.2 mg / kg), clear tumor stasis activity was observed during the treatment period (Figure 16). The tumor only started to grow after treatment ceased (Figure 16). This result also suggests that the tumor stasis activity observed in group B is, in fact, activity induced by the test article.
€ An analysis of variance (ANOVA) followed by Dunnett's multiple comparison test (Prism, version 5.02, Lake Forest, CA). One animal was sacrificed on day 36 for low body weight. ¥ 3 animals were sacrificed on day 7, 9, and 11, individually, for low body weight.

权利要求:
Claims (33)
[0001]
1. Compound CHARACTERIZED for being N - ((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl) methyl) -5- (ethyl (tetrahydro-2H-pyran-) hydrobromide 4-yl) amino) -4-methyl-4'- (morpholinomethyl) - [1,1 '-biphenyl] -3-carboxamide.
[0002]
2. Compound according to claim 1, CHARACTERIZED by the fact that it is a monohydrobromide.
[0003]
3. Compound according to claim 1 or 2, CHARACTERIZED by the fact that said compound is crystalline.
[0004]
Compound according to any one of claims 1 to 3, CHARACTERIZED by the fact that said compound is substantially free of impurities.
[0005]
Compound according to any one of claims 1 to 4, CHARACTERIZED by the fact that said compound is a crystalline solid substantially free of N - ((4,6-dimethyl-2-oxo-1,2-) hydrobromide dihydropyridin-3-yl) methyl) -5- (ethyl (tetrahydro-2H-pyran-4-yl) amino) -4-methyl-4 '- (morpholinomethyl) - [1,1' -biphenyl] - Amorphous 3-carboxamide.
[0006]
Pharmaceutical composition CHARACTERIZED by comprising the compound, as defined in any one of claims 1 to 5, and a pharmaceutically acceptable carrier or diluent.
[0007]
7. Method for preparing the compound, as defined in claim 1, CHARACTERIZED by comprising the combination of N - ((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl) methyl) -5 - (ethyl (tetrahydro-2H-pyran-4-yl) amino) -4-methyl-4'- (morpholinomethyl) - [1,1'-biphenyl] -3-carboxamide with hydrobromic acid.
[0008]
8. Polymorph A CHARACTERIZED by being N - ((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl) methyl) -5- (ethyl (tetrahydro-2H- pyran-4-yl) amino) -4-methyl-4 '- (morpholinomethyl) - [1,1' -biphenyl] -3-carboxamide.
[0009]
9. Polymorph according to claim 8, CHARACTERIZED by the fact that the polymorph exhibits an X-ray powder diffraction pattern having one or more characteristic peaks expressed in degrees 2-theta at about 3.9 +/- 0 , 3 degrees, about 17.5 +/- 0.3 degrees, and about 22.0 +/- 0.3 degrees 2-theta.
[0010]
10. Polymorph according to claim 8 or 9, CHARACTERIZED by the fact that the polymorph exhibits an X-ray powder diffraction pattern having characteristic peaks expressed in degrees 2-theta at about 3.9 +/- 0, 3 degrees, about 17.5 +/- 0.3 degrees, and about 22.0 +/- 0.3 degrees 2-theta.
[0011]
11. Polymorph according to claim 8, CHARACTERIZED by the fact that the polymorph exhibits an X-ray powder diffraction pattern having one or more characteristic peaks expressed in degrees 2-theta at about 3.9 +/- 0 , 3 degrees, about 14.3 +/- 0.3 degrees, about 18.7 +/- 0.3 degrees, about 23.3 +/- 0.3 degrees, and about 23.6 + / - 0.3 degrees 2-theta.
[0012]
12. Polymorph according to claim 8, CHARACTERIZED by the fact that the polymorph exhibits an X-ray powder diffraction pattern having at least 5 characteristic peaks expressed in degrees 2-theta at about 3.9 +/- 0 , 3 degrees, 10.1 +/- 0.3 degrees, 14.3 +/- 0.3 degrees, 17.5 +/- 0.3 degrees, 18.7 +/- 0.3 degrees, 20, 6 +/- 0.3 degrees, 20.9 +/- 0.3 degrees, 21.8 +/- 0.3 degrees, 22.0 +/- 0.3 degrees, 23.3 +/- 0, 3 degrees and 23.6 +/- 0.3 degrees 2-theta.
[0013]
13. Polymorph according to claim 8, CHARACTERIZED by the fact that the polymorph exhibits an X-ray powder diffraction pattern having at least 6 characteristic peaks expressed in degrees 2-theta at about 3.9 +/- 0 , 3 degrees, 10.1 +/- 0.3 degrees, 14.3 +/- 0.3 degrees, 17.5 +/- 0.3 degrees, 18.7 +/- 0.3 degrees, 20, 6 +/- 0.3 degrees, 20.9 +/- 0.3 degrees, 21.8 +/- 0.3 degrees, 22.0 +/- 0.3 degrees, 23.3 +/- 0, 3 degrees and 23.6 +/- 0.3 degrees 2-theta.
[0014]
14. Polymorph according to claim 8, CHARACTERIZED by the fact that the polymorph exhibits an X-ray powder diffraction pattern having at least 7 characteristic peaks expressed in 2-theta grades at about 3.9 +/- 0 , 3 degrees, 10.1 +/- 0.3 degrees, 14.3 +/- 0.3 degrees, 17.5 +/- 0.3 degrees, 18.7 +/- 0.3 degrees, 20, 6 +/- 0.3 degrees, 20.9 +/- 0.3 degrees, 21.8 +/- 0.3 degrees, 22.0 +/- 0.3 degrees, 23.3 +/- 0, 3 degrees and 23.6 +/- 0.3 degrees 2-theta.
[0015]
15. Polymorph according to claim 8, CHARACTERIZED by the fact that the polymorph exhibits an X-ray powder diffraction pattern having at least 8 characteristic peaks expressed in degrees 2-theta at about 3.9 +/- 0 , 3 degrees, 10.1 +/- 0.3 degrees, 14.3 +/- 0.3 degrees, 17.5 +/- 0.3 degrees, 18.7 +/- 0.3 degrees, 20, 6 +/- 0.3 degrees, 20.9 +/- 0.3 degrees, 21.8 +/- 0.3 degrees, 22.0 +/- 0.3 degrees, 23.3 +/- 0, 3 degrees and 23.6 +/- 0.3 degrees 2-theta.
[0016]
16. Polymorph according to claim 8, CHARACTERIZED by the fact that the polymorph exhibits an X-ray powder diffraction pattern having at least 9 characteristic peaks expressed in degrees 2-theta at about 3.9 +/- 0 , 3 degrees, 10.1 +/- 0.3 degrees, 14.3 +/- 0.3 degrees, 17.5 +/- 0.3 degrees, 18.7 +/- 0.3 degrees, 20, 6 +/- 0.3 degrees, 20.9 +/- 0.3 degrees, 21.8 +/- 0.3 degrees, 22.0 +/- 0.3 degrees, 23.3 +/- 0, 3 degrees and 23.6 +/- 0.3 degrees 2-theta.
[0017]
17. Polymorph according to claim 8, CHARACTERIZED by the fact that the polymorph exhibits an X-ray powder diffraction pattern having at least 10 characteristic peaks expressed in degrees 2-theta at about 3.9 +/- 0 , 3 degrees, 10.1 +/- 0.3 degrees, 14.3 +/- 0.3 degrees, 17.5 +/- 0.3 degrees, 18.7 +/- 0.3 degrees, 20, 6 +/- 0.3 degrees, 20.9 +/- 0.3 degrees, 21.8 +/- 0.3 degrees, 22.0 +/- 0.3 degrees, 23.3 +/- 0, 3 degrees and 23.6 +/- 0.3 degrees 2-theta.
[0018]
18. Polymorph according to claim 8, CHARACTERIZED by the fact that the polymorph exhibits an X-ray powder diffraction pattern having characteristic peaks expressed in degrees 2-theta at about 3.9 +/- 0.3 degrees , 10.1 +/- 0.3 degrees, 14.3 +/- 0.3 degrees, 17.5 +/- 0.3 degrees, 18.7 +/- 0.3 degrees, 20.6 + / - 0.3 degrees, 20.9 +/- 0.3 degrees, 21.8 +/- 0.3 degrees, 22.0 +/- 0.3 degrees, 23.3 +/- 0.3 degrees and 23.6 +/- 0.3 degrees 2-theta.
[0019]
19. Polymorph according to any of claims 8 to 18, CHARACTERIZED by the fact that the polymorph exhibits an X-ray powder diffraction pattern substantially in accordance with Figure 1.
[0020]
20. Polymorph according to any of claims 8 to 19, CHARACTERIZED by the fact that the polymorph exhibits an X-ray powder diffraction pattern substantially in accordance with Table 1.
[0021]
21. Polymorph according to any one of claims 8 to 20, CHARACTERIZED by the fact that the polymorph exhibits a differential scanning calorimetry thermogram having a characteristic peak expressed in units of ° C at a temperature of 255 +/- 5 ° Ç.
[0022]
22. Polymorph according to any of claims 8 to 21, CHARACTERIZED by the fact that the polymorph exhibits a differential scanning calorimetry thermogram substantially in accordance with Figure 3.
[0023]
23. Method for preparing the polymorph, as defined in any one of claims 8 to 22, CHARACTERIZED by comprising the combination of N- ((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl ) methyl) -5- (ethyl (tetrahydro-2H-pyran-4-yl) amino) -4-methyl-4 '- (morpholinomethyl) - [1,1' -biphenyl] -3-carboxamide with hydrobromic acid .
[0024]
24. Method for recrystallizing the polymorph, as defined in any one of claims 8 to 22, CHARACTERIZED by comprising the following steps: (a) dissolving Polymorph A in a first solvent and (b) adding a second solvent, such as said polymorph is recrystallized.
[0025]
25. Method according to claim 24, CHARACTERIZED by the fact that the first solvent is ethanol and the second solvent is MTBE.
[0026]
26. Method according to claim 16, characterized by comprising (a) dissolving Polymorph A in ethanol, (b) heating the mixture, (c) adding MTBE to the mixture, forming a precipitate comprising said polymorph and filtering the precipitate such that said polymorph is recrystallized.
[0027]
27. Pharmaceutical composition CHARACTERIZED by comprising the polymorph as defined in any one of claims 8 to 22, and a pharmaceutically acceptable carrier or diluent.
[0028]
28. Use of a compound, as defined in any one of claims 1 to 5, a polymorph, as defined in any of claims 8 to 22, or a pharmaceutical composition, as defined in claim 6 or 27, CHARACTERIZED because it is in the manufacture of a drug for the treatment of cancer in a subject in need of it.
[0029]
29. Use according to claim 28, CHARACTERIZED by the fact that the cancer is non-Hodgkin's lymphoma or breast cancer.
[0030]
30. Use of a compound, as defined in any of claims 1 to 5, or of a polymorph, as defined in any of claims 8 to 22, CHARACTERIZED in that it is in the manufacture of a medicament to inhibit EZH2 histone methyltransferase activity in a subject in need of this.
[0031]
31. Method for inhibiting EZH2 histone methyltransferase activity in vitro CHARACTERIZED by comprising administering a compound, as defined in any of claims 1 to 5, or a polymorph, as defined in any of claims 8 to 22.
[0032]
32. Method for preparing N - ((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl) methyl) - 5- (ethyl (tetrahydro-2H-pyran-4-yl ) amino) -4-methyl-4 '- (morpholinomethyl) - [1,1'-biphenyl] -3-carboxamide CHARACTERIZED for comprising reacting 5- (ethyl (tetrahydro-2H-pyran-4-yl) amino acid ) -4-methyl-4 '- (morpholinomethyl) - [1,1'-biphenyl] -3-carboxylic (5) with a salt of 3- (aminomethyl) -4,6-dimethyl-dihydro-pyridin- 2 (1 H) -one.
[0033]
33. Method, according to claim 32, CHARACTERIZED by the fact that (5) is in crystalline form.

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法律状态:
2018-01-16| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2018-03-27| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2019-04-24| B07E| Notice of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|Free format text: NOTIFICACAO DE ANUENCIA RELACIONADA COM O ART 229 DA LPI |

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2019-06-11| B06T| Formal requirements before examination [chapter 6.20 patent gazette]|

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2020-06-09| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2020-11-17| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 11/04/2013, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US201261624215P| true| 2012-04-13|2012-04-13|

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US61/624.215|2012-04-13|

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PCT/US2013/036193|WO2013155317A1|2012-04-13|2013-04-11|Salt form of a human hi stone methyltransf erase ezh2 inhibitor|

---