Process for the use and preparation of fluorocyclopentenylcytosine

Abstract

To provide a process for the use and preparation of fluorocyclopentenylcytosine.SOLUTION: The disclosed subject matter provides methods of using and kits comprising compounds of Formula (I): Formula (I) or hydrates, solvates or pharmaceutically acceptable salts thereof. The disclosed subject matter further provides methods for treating one or more symptoms of cancer comprising administering to a subject in need thereof a compound of formula (I), as well as processes for their preparation. The present invention also provides, among other things, an improved process for significantly reducing manufacturing costs by combining multiple steps and without isolating and purifying intermediate materials.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] Priority This application claims priority to U.S. Provisional Application No. 62 / 173,174, filed June 9, 2015; U.S. Provisional Application No. 62 / 210,708, filed August 27, 2015; U.S. Provisional Application No. 62 / 289,801, filed February 1, 2016; and U.S. Provisional Application No. 62 / 319,369, filed April 7, 2016, the contents of which are incorporated herein by reference in their entireties. [Background technology]

[0002] U.S. Patent No. 7,405,214 (issued July 29, 2008) discloses a compound of formula (I), also known as RX-3117, fluorocyclopentenylcytosine, or 4-amino-1-((1S,4R,5S)-2-fluoro-4,5-dihydroxy-3-hydroxymethyl-cyclopent-2-enyl)-1H-pyrimidin-2-one: [ka] US Patent No. 7,405,214 also discloses an 11-step synthesis of RX-3117 from D-ribose, which uses expensive catalysts that pose challenges for implementation in industrial production.

[0003] U.S. Patent No. 9,150,520 (issued October 6, 2015) discloses a short route for the preparation of RX-3117 from (3R,4R,6aR)-tert-butyl-(5-fluoro-2,2-dimethyl-6-trityloxymethyl-4,6a-dihydro-3aH-cyclopenta[1,3]dioxol-4-yloxy)-diphenyl-silane via 4-amino-1-(3aS,4S,6aR)-5-fluoro-2,2-dimethyl-6-((trityloxy)methyl)-4,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)pyrimidin-2(1H)-one. The synthesis requires that intermediates be isolated at each step. Therefore, the process is inadequate for scaled-up production of the final product due to time and cost constraints. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] U.S. Patent No. 7,405,214 [Patent Document 2] U.S. Patent No. 9,150,520 Summary of the Invention [Problem to be solved by the invention]

[0005] Thus, there is a need to provide improved processes, for example, by reducing the number of steps and / or eliminating the need to purify each intermediate. [Means for solving the problem]

[0006] The present invention is directed to new uses of the compound of formula (I) and methods of using the same. Among other things, the present invention also provides an improved process for significantly reducing manufacturing costs by combining multiple steps without isolating and purifying intermediate materials. In addition, the present invention provides dosages and exposure levels for using the compound of formula (I) in subjects.

[0007] One aspect of the present disclosure provides a compound of formula (I) for use in the treatment of tumors, [ka] or a hydrate, solvate, or pharmaceutically acceptable salt thereof, is administered to a subject in need thereof in a dosage of about 300 to 2,000 mg / day.

[0008] In embodiments, the compound of formula (I) is administered as a monohydrate. In other embodiments, the compound of formula (I) is administered free of solvates, hydrates, and salts.

[0009] Method embodiments can include administering the oral dosage 5 to 7 days per week. Method embodiments can include administering the oral dosage 5 to 7 days per week for 4 consecutive weeks, or 5 to 7 days per week for 3 consecutive weeks, followed by a 1-week rest period during which no oral dosage form is administered.

[0010] Embodiments of the method may include dosing cycles consisting of either three consecutive weeks of treatment followed by one week off, or four consecutive weeks of treatment, wherein the oral dosage form is administered for up to 12 dosing cycles.

[0011] Embodiments of the method can include an oral dosage form that provides a Cmax of about 700-1,100 ng / mL after a single dose.Embodiments of the method can include an AUC of about 8,000-10,000 h·ng / mL after a single dose. 0~t It may include an oral dosage form that provides (0 to 24 hours).

[0012] Embodiments of the methods can be used to treat tumors, including treating pancreatic, bladder, or colorectal cancer.

[0013] Method embodiments may include administering the oral dosage form together with a second agent or anti-tumor agent selected from the group consisting of antimetabolites, DNA fragmenting agents, DNA cross-linking agents, intercalating agents, protein synthesis inhibitors, topoisomerase I poisons, topoisomerase II poisons, microtubule-directing agents, kinase inhibitors, polyphenols, hormones, hormone antagonists, death receptor agonists, immune checkpoint inhibitors, anti-programmed cell death 1 (PD-1) receptor antibodies, and anti-programmed cell death ligand 1 (PD-L1) antibodies. Method embodiments may include administering a PD-L1 antibody to the subject. Method embodiments may include administering a PD-1 antibody to the subject. Method embodiments may include administering a solid oral dosage form. The second agent or anti-tumor agent may be administered in the same oral dosage form or in a separate oral dosage form.

[0014] In an embodiment, the subject in need thereof is a human subject.

[0015] Another aspect of the present disclosure is a method for treating a subject in need thereof, comprising administering to said subject a compound of formula (I) [ka] or a hydrate, solvate, or pharmaceutically acceptable salt thereof, comprising the steps of (i) collecting a tumor cell or tissue sample from a subject; and (ii) measuring the level of UCK2 expression in the tumor cells or tissue, wherein the expression level of UCK2 indicates the likely effectiveness of treatment with the compound of formula (I).

[0016] Another aspect of the present disclosure is a kit for testing the potential effectiveness of a compound of formula (I), or a hydrate, solvate, or pharmaceutically acceptable salt thereof, in treating tumors by use of an assay that measures the level of kinase, p53, or UCK2 protein in a tumor cell sample.

[0017] Another aspect of the present disclosure provides a compound of formula (I) for use in treating one or more symptoms of cancer. [ka] wherein the compound of formula (I) or a hydrate, solvate, or pharmaceutically acceptable salt thereof is administered in an amount effective to inhibit methyltransferase and upregulate at least one hypomethylated target in a subject. In embodiments, the compound of formula (I) is administered as a monohydrate. In other embodiments, the compound of formula (I) is administered free of solvates, hydrates, and salts.

[0018] Another aspect of the present disclosure is a process for the preparation of 4-amino-1-((1S,4R,5S)-2-fluoro-4,5-dihydroxy-3-(hydroxymethyl)cyclopent-2-en-1-yl)pyrimidin-2(1H)-one 1H2O (RX-3117-MH), comprising the step of reacting tert-butyl(((3aR,4R,6aR)-5-fluoro-2,2-dimethyl-6-((trityl The present invention provides a process for converting 4-amino-1-((3aS,4S,6aR)-5-fluoro-2,2-dimethyl-6-((trityloxy)methyl)-4,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)oxy)diphenylsilane (ASM11) into 4-amino-1-((3aS,4S,6aR)-5-fluoro-2,2-dimethyl-6-((trityloxy)methyl)-4,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)pyrimidin-2(1H)-one (INT14).

[0019] An embodiment of the method can include dissolving tert-butyl(((3aR,4R,6aR)-5-fluoro-2,2-dimethyl-6-((trityloxy)methyl)-4,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)oxy)diphenylsilane (ASM11) in 2-methyltetrahydrofuran, adding tetra-n-butylammonium fluoride to form ((3aS,4R,6aR)-5-fluoro-2,2-dimethyl-6-((trityloxy)methyl)-4,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-ol) (INT12) in the reaction solution, and recovering INT12 in the organic phase.

[0020] An embodiment of recovering INT12 in the organic phase may include washing the reaction solution with an aqueous solution, separating an aqueous extract from the organic phase containing INT12, washing the aqueous extract with 2-methyltetrahydrofuran to extract INT12 from the aqueous extract, and combining the extracted INT12 with the organic phase containing INT12.

[0021] An embodiment of the method can include adding triethylamine and methanesulfonyl chloride in 2-methyltetrahydrofuran to INT12 in an organic phase to form ((3aR,4R,6aR)-5-fluoro-2,2-dimethyl-6-((trityloxy)methyl)-4,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-ylmethanesulfonate) (INT13) in a second reaction solution, and recovering INT13 in DMSO.

[0022] An embodiment of recovering INT13 in DMSO can include adding DMSO to the second reaction solution with INT13 and removing at least 90% w / w of 2-methyltetrahydrofuran by distillation.

[0023] An embodiment of the method can include adding 2.5 equivalents of cesium carbonate and cytosine to INT13 in DMSO to form 4-amino-1-((3aS,4S,6aR)-5-fluoro-2,2-dimethyl-6-((trityloxy)methyl)-4,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)pyrimidin-2(1H)-one (INT14) in a third reaction solution.

[0024] Embodiments of the method may include maintaining the reaction temperature at about 33 to 37°C.

[0025] In embodiments, INT14 has a ratio of N- to O-isomers of greater than about 95:5.

[0026] An embodiment of the method can include adding an acid to a third reaction solution with INT14 to form 4-amino-1-((1S,4R,5S)-2-fluoro-4,5-dihydroxy-3-(hydroxymethyl)cyclopent-2-en-1-yl)pyrimidin-2(1H)-one (RX-3117), washing the RX-3117 with methyl tert-butyl ether and water to form an organic phase and an aqueous phase having RX-3117, and purifying the RX-3117 to form RX-3117-MH.

[0027] An embodiment of the method may include, prior to the washing step, charging the reaction mixture with methanol and distilling the reaction mixture to remove acetonide until the area of ​​the acetonide detected is less than about 1.0%.

[0028] An embodiment of the washing step can include separating an aqueous phase having RX-3117 from an organic phase, washing the aqueous phase having RX-3117 with methyl tert-butyl ether until less than about 0.5% w / w trityl alcohol is detected in the aqueous phase, adding a basic anion resin to the aqueous phase having RX-3117 to form a slurry, filtering the slurry to retain a mother liquor, concentrating the mother liquor to form a concentrate, and adding acetonitrile to the concentrate to form purified RX-3117-MH.

[0029] Another aspect of the present disclosure is the synthesis of tert-butyl(((3aR,4R,6aR)-5-fluoro-2,2-dimethyl-6-((trityloxy)methyl)-4,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)oxy)diphenylsilane (ASM11) from 4-amino-1-((3aS,4S,6aR)-5-fluoro-2,2-dimethyl-6-((trityloxy)methyl)-4,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)oxy)diphenylsilane. A continuous process for preparing (3aS,4R,6aR)-5-fluoro-2,2-dimethyl-6-((trityloxy)methyl)-4,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)pyrimidin-2(1H)-one (INT14) comprising the steps of dissolving ASM11 in 2-methyltetrahydrofuran; adding tetra-n-butylammonium fluoride to form ((3aS,4R,6aR)-5-fluoro-2,2-dimethyl-6-((trityloxy)methyl)-4,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)pyrimidin-2(1H)-one; adding trimethylamine and methanesulfonyl chloride in 2-methyltetrahydrofuran to INT12 to form ((3aR,4R,6aR)-5-fluoro-2,2-dimethyl-6-((trityloxy)methyl)-4,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-ylmethanesulfonate) (INT13); and adding cesium carbonate and cytosine to to INT13 to form 4-amino-1-((3aS,4S,6aR)-5-fluoro-2,2-dimethyl-6-((trityloxy)methyl)-4,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)pyrimidin-2(1H)-one (INT14), wherein the steps are carried out in one or more fixed reactors without isolating either INT12 or INT13.

[0030] Another aspect of the present disclosure is the preparation of 4-amino-1-((3aS,4S,6aR)-5-fluoro-2,2-dimethyl-6-((trityloxy)methyl)-4,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)pyrimidin-2(1H)-one (INT14) from 4-amino-1-((1S,4R,5S)-2-fluoro-4,5-dihydroxy-3-(hydroxymethyl)cyclopenta- 2-en-1-yl)pyrimidin-2(1H)-one 1H2O (RX-3117-MH), comprising the steps of reacting INT14 with an acid to form 4-amino-1-((1S,4R,5S)-2-fluoro-4,5-dihydroxy-3-(hydroxymethyl)cyclopent-2-en-1-yl)pyrimidin-2(1H)-one (RX-3117); reacting RX-3117 with methyl and separating the aqueous phase having RX-3117 from the organic phase; washing the aqueous phase having RX-3117 with methyl tert-butyl ether until less than about 0.5% w / w trityl alcohol is detected in the aqueous phase; adding a strong base anion resin to the aqueous phase having RX-3117 to form a slurry; filtering the slurry to retain a mother liquor; concentrating the mother liquor to form a concentrate; adding acetonitrile to the concentrate to form purified RX-3117-MH; and isolating the purified RX-3117-MH, wherein the steps are carried out in one or more fixed reactors.

[0031] Another aspect of the present disclosure is a method of inducing apoptosis in a cell, comprising treating the cell with an effective amount of a compound of formula (I) [ka] or a hydrate, solvate, or pharmaceutically acceptable salt thereof.

[0032] Another aspect of the present disclosure is a method of sensitizing a cell to an apoptotic signal, comprising administering to the cell an effective amount of a compound of formula (I) [ka] or a hydrate, solvate, or pharmaceutically acceptable salt thereof.

[0033] Another aspect of the present disclosure provides a method of modulating a protein kinase in a cell by contacting the cell with an effective amount of a compound of formula (I), or a hydrate, solvate, or pharmaceutically acceptable salt thereof.

[0034] Another aspect of the present disclosure is a method of treating non-small cell lung cancer cells, comprising: (i) diagnosing a subject with non-small lung cancer cells; and (ii) administering to the subject an effective amount of a compound of formula (I), or a hydrate, solvate, or pharmaceutically acceptable salt thereof. [Brief explanation of the drawings]

[0035] [Figure 1] Figure 1 is a series of bar graphs showing the effect of RX-3117 (1 μM) on A549, SW1573, SW1573 / G-, and H460 cells in G1 phase after 24 hours. At a dose of 1 μM, RX-3117 induced the accumulation of A549, SW1573, SW1573 / G-, and H460 cells in G1 phase after 24 hours of exposure.

[0036] [Figure 2] Figure 2 is a series of bar graphs showing the effect of RX-3117 (5 x IC50) on A549, SW1573, and SW1573 / G- cells in S phase after 24 and 48 hours. At doses higher than 5 x IC50, RX-3117 induced the accumulation of A549, SW1573, and SW1573 / G- cells in S phase.

[0037] [Figure 3] Figure 3 is a series of Western blots showing the effect of increasing concentrations of RX-3117 on procaspase-9 activation in SW1573 cells. RX-3117 reduced procaspase-9 in SW1573 cells. The reduction in procaspase-9 indicates caspase activation and subsequent apoptosis induction.

[0038] [Figure 4] Figure 4 is a series of Western blots showing the effect of increasing concentrations of RX-3117 on procaspase 9 activation in A549 cells. RX-3117 reduced procaspase 9 in A549 cells.

[0039] [Figure 5] Figure 5 is a series of Western blots showing double-strand breaks (DSBs) induced by RX-3117 as indicated by the biomarker γH2A.X (phospho-S139) in SW1573 cells after 48 hours. RX-3117 induces DSBs after 48 hours, indicated by the marker representing DSB phosphorylated S139 H2A.X. C* denotes DNA damage caused by 50 μM etoposide after 2 days, which served as a positive control.

[0040] [Figure 6] FIG. 6 is a series of Western blots showing cleaved PARP induced by increasing concentrations of RX-3117 after 24 hours.

[0041] [Figure 7] 7 is a series of Western blots showing the effect of 1 μM and 5 μM RX-3117 on p53 expression levels in A549 cells. At 1 μM and 5 μM, RX-3117 increased p53 expression levels in the A549 cell line.

[0042] [Figure 8]Figure 8 is a series of Western blots showing the effect of 10 μM RX-3117 on Chk1, Chk2, Cdk1, Cdk2, and p-Cdc25C expression levels in SW1573 cells after 24 and 48 hours. In SW1573 cells, Chk1 is increased, pCDC25C is decreased, and Cdk2 is increased after 48 hours of exposure to 10 μM RX-3117.

[0043] [Figure 9] 9 is a series of Western blots showing the effect of increasing concentrations of RX-3117 on wee1 expression levels in SW1573 cells after 24 hours. Increasing concentrations of RX-3117 have an effect on wee1, which decreases after 24 hours in the SW1573 cell line.

[0044] [Figure 10] FIG. 10 is a bar graph showing the effect of RX-3117 at 5×IC50 on PI stained, apoptotic A549 and SW1573 cells in sub-G1 phase after 24 hours (d1) and 48 hours (d2).

[0045] [Figure 11] Figure 11 is a bar graph showing the effect of 5 μM (for A549) and 10 μM (for SW1573) RX-3117 on Annexin V staining of apoptotic A549 and SW1573 cells in sub-G1 phase after 24 hours (d1), 48 hours (d2), 72 hours (d3), and 96 hours (d4).

[0046] [Figure 12] FIG. 12 is a graph showing peak plasma concentrations Cmax (ng / mL) after dose 1 and dose 7.

[0047] [Figure 13] FIG. 13 is a graph showing plasma exposure after dose 1 and dose 7 in terms of AUC 0-t (ng·hr / mL).

[0048] [Figure 14] FIG. 14 shows structural formulas representing cytidine, gemcitabine, and the novel cytidine analog RX-3117.

[0049] [Figure 15] Figure 15 is a bar graph showing the radiosensitizing effect of pre- or post-incubation with RX-3117. Gray bars represent the control, where A2780 cells were irradiated with 4 Gy, and black bars represent incubation with 1 μM RX-3117 for 24 hours after 4 Gy irradiation. White / gray bars represent incubation with 1 μM RX-3117 for 24 hours before 4 Gy irradiation. PE represents plating efficiency.

[0050] [Figure 16AB] Figure 16 is a series of graphs showing the radiosensitizing effect of 1 μM RX-3117 (5 μM RX-3117 for SW1573 cells) with different doses of irradiation using a colony formation assay. Cells were preincubated with RX-3117 for 24 hours. A: A2780 cells had a dose modification factor (DMF) of 1.8. B: A549 cells had a DMF of 1.8. C: H460 cells showed poor radiosensitizing effect. D: SW1573 cells had a DMF of 1.5. E: SW1573 / G-cells had a DMF of 1.4. F: Fractionated irradiation of SW1573 cells with 2 Gy over 5 days after 24 hours of incubation with 1 μM RX-3117. [Figure 16CD]Figure 16 is a series of graphs showing the radiosensitizing effect of 1 μM RX-3117 (5 μM RX-3117 for SW1573 cells) with different doses of irradiation using a colony formation assay. Cells were preincubated with RX-3117 for 24 hours. A: A2780 cells had a dose modification factor (DMF) of 1.8. B: A549 cells had a DMF of 1.8. C: H460 cells showed poor radiosensitizing effect. D: SW1573 cells had a DMF of 1.5. E: SW1573 / G-cells had a DMF of 1.4. F: Fractionated irradiation of SW1573 cells with 2 Gy over 5 days after 24 hours of incubation with 1 μM RX-3117. [Figure 16EF] Figure 16 is a series of graphs showing the radiosensitizing effect of 1 μM RX-3117 (5 μM RX-3117 for SW1573 cells) with different doses of irradiation using a colony formation assay. Cells were preincubated with RX-3117 for 24 hours. A: A2780 cells had a dose modification factor (DMF) of 1.8. B: A549 cells had a DMF of 1.8. C: H460 cells showed poor radiosensitizing effect. D: SW1573 cells had a DMF of 1.5. E: SW1573 / G-cells had a DMF of 1.4. F: Fractionated irradiation of SW1573 cells with 2 Gy over 5 days after 24 hours of incubation with 1 μM RX-3117.

[0051] [Figure 17]Figure 17 is a series of graphs showing the radiosensitizing effect of RX-3117 on spheroids. A: SW1573, normalized spheroid volume over 15 days. Control; 1 μM RX-3117 treatment; 2 Gy RT treatment for 5 days; 2 Gy RT treatment for 5 days and pre-incubated with 1 μM RX-3117. B: A549 spheres, normalized spheroid volume over 15 days. Control; 1 μM RX-3117 treatment; 2 Gy RT treatment for 5 days; 2 Gy RT treatment for 5 days and pre-incubated with 1 μM RX-3117 for 24 hours.

[0052] [Figure 18] Figure 18 shows Western blot analysis of DNA damage. A: Expression of the DSB damage marker γH2A.X in A2780 cells exposed to increasing concentrations of RX-3117 starting from 0.1 μM to 10 μM for 48 hours. B: Time-dependent induction of DNA damage in SW1573 cells in combination with irradiation. C* denotes DNA damage caused by 50 μM etoposide after 2 days, which served as a positive control.

[0053] [Figure 19] Figure 19 is a series of histograms (A) bar graphs and Western blots (B) showing the perturbation of cell cycle distribution by RX-3117 and radiation in cell lines. A: Cell phase histograms of cell lines treated with 1 μM RX-3117 for 24 hours with or without 4 Gy of radiation. Cells were harvested 24 hours after treatment. B: Cell cycle protein analysis by Western blot after 24 hours of drug incubation and 30 minutes after irradiation.

[0054] [Figure 20] Figure 20 is a series of Western blots showing the effect of RX-3117 and radiation on the expression of cell cycle proteins in SW1573 cells. Cells were irradiated (RT) in the presence and absence of RX-3117. Expression was measured by Western blotting using the Odyssey system.

[0055] [Figure 21] FIG. 21 shows the outline of the metabolic pathway of RX-3117.

[0056] [Figure 22] FIG. 22 shows a schematic diagram of the mechanism of downregulation of maintenance DNA methyltransferase (DNMT1) by RX-3117.

[0057] [Figure 23] Figure 23 is a series of Western blots showing the effects of 1 μM, 5 μM, 25 μM, and 75 μM RX-3117 on DNMT1, DNMT3A, DNMT3B, and β-actin expression levels in A549 cells. RX-3117 down-regulates DNA methyltransferase 1 maintenance.

[0058] [Figure 24] FIG. 24 is a diagram showing potential effects on cell cycle proteins: regulation of the cell cycle by checkpoint kinases Chk1 and Chk2 after induced damage.

[0059] [Figure 25] Figure 25 is a series of graphs and associated Western blots showing the effects of RX-3117 on A549 and SW1573 cells at 5 μM, 10 μM, 20 μM, and 50 μM for 24 or 48 hours. Cells were harvested and protein expression was measured using Western blotting (A and B). RNA was isolated and gene expression was measured using RT-PCR (C and D). RX-3117 down-regulates DNMT1 protein and gene expression.

[0060] [Figure 26]Figure 26 shows Western blots and graphs showing the effects of RX-3117 (1 μM) and Aza-dC (5 μM) on A2780 ovarian cancer cells over 24 hours. Nuclear extracts were isolated and DNMT1 expression was measured by Western blot (A) and activity (B) using a commercially available kit as described in the methods.

[0061] [Figure 27] Figure 27 shows the effects of RX-3711 and Aza-dC on A549 cells. Global methylation was measured using FACS (A) or immunofluorescence (B) with an antibody against 5-methyl-cytosine. Control cells were set at 100% (A). Western blot (C) shows the expression of MGMT and E-cadherin in A549 cells and p16 in SW1573 cells after exposure to RX-3711 and Aza-dC.

[0062] [Figure 28] Figure 28 shows the effect of RX-3117 on PCFT-mediated transport of MTX over 24 hours. Folic acid (FA) was added to inhibit PCFT and L-LV to inhibit RFC-mediated MTX transport. Aza-CdR and Aza-CR were included as positive controls.

[0063] [Figure 29] FIG. 29 is a 1H NMR of RX-3117 made using the process described in Example 9.

[0064] [Figure 30] FIG. 30 is the 13C NMR of RX-3117 made using the process described in Example 9.

[0065] [Figure 31] FIG. 31 is the 19F NMR of RX-3117 made using the process described in Example 9.

[0066] [Figure 32] FIG. 32 is a mass spectrum of RX-3117 made using the process described in Example 9.

[0067] [Figure 33] FIG. 33 is a mass spectrum (using an ES-filter) of RX-3117 made using the process described in Example 9.

[0068] [Figure 34] Figure 34 is a microscopic comparison of RX-3117 made according to the process of Example 9 (top row) and prepared using a laboratory-scale process (bottom row) under plain polarized light (left column) and cross-polarized light (right column).

[0069] [Figure 35] FIG. 35 is X-ray powder diffraction data comparing RX-3117 made using a laboratory-scale process (top spectrum) and RX-3117 made using the process described in Example 9 (bottom spectrum). DETAILED DESCRIPTION OF THE INVENTION

[0070] definition Unless otherwise defined, the meaning of all scientific and technical terms used herein is that commonly understood by one of ordinary skill in the art to which the disclosed subject matter belongs. Those skilled in the art will recognize that any methods and materials similar or equivalent to those described herein can also be used to practice or test the disclosed subject matter.

[0071] Unless expressly indicated otherwise, the following terms, as used herein, have the meanings indicated below, which are intended to supplement, but not modify, the meaning of these terms as understood in the art.

[0072] "Cmax " refers to the maximum observed plasma concentration.

[0073] "T max " is C max It refers to the time it takes to reach

[0074] "T 1 / 2 "Terminal T" refers to the time required for the plasma concentration of a drug to reach half of its original value. 1 / 2 " is a T in the terminal stage 1 / 2 Refers to...

[0075] "AUC 0~t " refers to the area under the plasma concentration versus time curve (AUC) from time zero to time t, where "t" is the last sample collection time with a measurable concentration. For example, AUC 0~24 or AUC 0~t (0-24 hours) refers to the AUC from time zero to 24 hours.

[0076] "Oral dosage form" refers to a pharmaceutical composition formulated for oral administration. Oral dosage forms can be formulated to provide immediate, sustained, extended, delayed, or controlled release. Examples of oral dosage forms include tablets, capsules, granules, and gelcaps.

[0077] An "effective amount" refers to the amount of a compound or pharmaceutical composition that produces a desired effect, such as treating or preventing a condition, based on its parameters of efficacy and potential toxicity and the knowledge of one of ordinary skill in the art. An effective amount can be administered in one or more doses.

[0078] "Contacting" refers to bringing a compound and a cell into sufficient proximity to produce a desired effect, either directly or indirectly, such as inducing apoptosis or modulating a protein kinase. Contacting can be performed in vitro or in vivo. For example, contacting a cell with a compound can involve delivering the compound directly to the cell using known techniques such as microinjection, administering the compound to a subject bearing the cell, or incubating the cell in a medium containing the compound.

[0079] "Treating" refers to achieving a beneficial or desired result, such as a clinical result. In some embodiments, the beneficial or desired result is any one or more of the following: inhibiting or suppressing the onset or occurrence of a condition, reducing the severity of a condition, reducing the number or severity of symptoms associated with a condition, increasing the quality of life of a subject suffering from a condition, reducing the dose of another medication needed to treat a condition, enhancing the effectiveness of another medication the subject is taking for a condition, and prolonging the survival of a subject with a condition.

[0080] " Preventing " refers to reducing the probability that a subject will develop a condition that the subject does not have but is at risk of developing. " At risk " refers to the subject having one or more risk factors that correlate with the development of a condition and are measurable parameters known in the art. The subject that has one or more risk factors has a higher probability of developing a condition than the subject that does not have such risk factors.

[0081] "Subject" refers to an animal, such as a mammal, including, but not limited to, a human. Thus, the methods disclosed herein may be useful in human therapy and veterinary applications. In one embodiment, the subject is a mammal. In another embodiment, the subject is a human.

[0082] "Fasted" refers to subjects who had fasted for at least 8 hours prior to treatment.

[0083] "Apoptosis" or the "apoptotic process" refers to the programmed cell death process that begins when a cell receives an internal or external signal (the apoptotic signal) and proceeds through a series of biochemical events (the signaling pathway phase) that trigger the execution phase. During the execution phase, effector caspases cleave key cellular proteins, leading to the morphological changes that characterize apoptosis. These changes may include, for example, cell shrinkage, endoplasmic reticulum dilation, cell fragmentation, formation of membrane vesicles (apoptotic bodies), deoxyribonucleic acid (DNA) fragmentation, chromatin condensation, chromosome migration, intranuclear margination, mitochondrial swelling, mitochondrial cristae expansion, opening of the mitochondrial permeability transition pore, dissipation of the mitochondrial proton gradient, and / or plasma membrane blebbing. Exemplary assays used to detect and measure apoptosis include microscopic examination of pyknotic bodies and enzyme assays such as terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), caspase assays, annexin assays, and DNA laddering. Apoptotic cells can be quantified, for example, by FACS analysis of cells stained with propidium iodide for DNA hypoploidy.

[0084] "Inducing apoptosis" refers to directly or indirectly causing apoptosis and may be characterized by an increase in the number of cells in a given cell population that undergo apoptosis, an increase in the rate at which cells undergo apoptosis, or an increase in the intensity, number, or rate of onset of one or more morphological characteristics of apoptosis.

[0085] "Sensitizing" refers to increasing the sensitivity of a cell to an apoptotic signal or decreasing the resistance of the cell to respond to the signal.

[0086] An "apoptotic signal" refers to a stimulus that activates an apoptotic signaling pathway.

[0087] "Apoptosis signaling pathway" refers to a series of molecular signals that initiate apoptotic cell death. The pathway begins with the reception of a signal and ends when the execution phase of apoptosis is initiated.

[0088] "Modulating" refers to altering the expression and / or activity of a biomolecule, such as a protein kinase. In one embodiment, modulating refers to increasing the expression and / or activity of a biomolecule. In other embodiments, modulating refers to decreasing the expression and / or activity of a biomolecule.

[0089] "Protein kinase" refers to a kinase enzyme that modifies other proteins by phosphorylation. Examples of protein kinases include serine / threonine protein kinases (e.g., checkpoint kinase 1, checkpoint kinase 2), tyrosine-specific protein kinases, histidine-specific protein kinases, and mixed kinases (e.g., mitogen-activated protein kinase kinases). In one embodiment, the protein kinase is a serine / threonine protein kinase. In one embodiment, the protein kinase is a serine / threonine protein kinase. In another embodiment, the protein kinase is checkpoint kinase 1 (Chk1) or checkpoint kinase 2 (Chk2). In another embodiment, the protein kinase is Chk1. In another embodiment, the protein kinase is Chk2.

[0090] "p53" refers to the protein encoded by the p53 tumor suppressor gene.

[0091] "UCK2" refers to uridine cytidine kinase 2, which is predominantly expressed in tumor cells or tissues.

[0092] "Tumor cell" refers to a cell derived from a tumor.

[0093] "Tumor" refers to an abnormal growth of tissue or cells, whether benign or malignant. Examples include prostate, lung, brain, breast, kidney, liver, lung, intestine, lymph, muscle, bone, bone marrow, uterus, ovary, vagina, vulva, pancreas, adrenal gland, central nervous system, peripheral nervous system, cervix, bladder, endometrium, throat, esophagus, larynx, thyroid, blood, penis, testicle, thymus, skin, spine, stomach, bile duct, small intestine ... Includes tumors found in the intestine, hepatobiliary tract, colorectum, colon, rectum, anus, endocrine glands, eye, and gallbladder.

[0094] "Cancer" refers to a malignant tumor. Cancer cells may or may not invade surrounding tissues and therefore may or may not metastasize to new body sites. Cancer encompasses carcinomas, which are cancers of epithelial cells; carcinomas include squamous cell carcinoma, adenocarcinoma, melanoma, and hepatocellular carcinoma. Cancer also encompasses sarcomas, which are tumors of mesenchymal origin; sarcomas include osteosarcoma, leukemia, and lymphoma. Cancer may involve one or more neoplastic cell types.

[0095] "Antineoplastic agent" refers to any agent useful for treating or preventing tumors. Examples of antitumor agents include the active agents described in the pharmaceutical compositions below. In embodiments, in addition to RX-3117, the antitumor agent is selected from antimetabolites, DNA fragmenting agents, DNA cross-linking agents, intercalating agents, protein synthesis inhibitors, topoisomerase I poisons, topoisomerase II poisons, microtubule-directing agents, kinase inhibitors, polyphenols, hormones, hormone antagonists, death receptor agonists, immune checkpoint inhibitors, anti-programmed cell death 1 (PD-1) receptor antibodies, and anti-programmed cell death ligand 1 (PD-L1) antibodies. In other embodiments, the additional antitumor agent is a PD-1 receptor antibody. In other embodiments, the additional antitumor agent is pembrolizumab. In other embodiments, the additional antitumor agent is nivolumab. In other embodiments, the additional antitumor agent is duryalumab. In other embodiments, the additional antitumor agent is a combination of nivolumab and pembrolizumab.

[0096] "Radiation" refers to any radiation useful for treating or preventing tumors. Examples of radiation include X-rays, gamma rays, and charged particles. Radiation may be delivered by any form of radiotherapy, such as external beam radiation therapy (EBRT, XBRT, or teletherapy), brachytherapy (internal radiation therapy or sealed source therapy), intraoperative radiation therapy, or systemic radiation therapy.

[0097] "Isolation" refers to any process in which an intermediate is separated from a reaction mixture by purification, such as by chromatography, distillation, filtration, extraction, drying, or recrystallization.

[0098] "Fixed reactor or vessel" refers to a reactor system that is in a fixed location in a plan that cannot be moved.

[0099] "Etc." has the same meaning as "such as, but not limited to." Similarly, "include" has the same meaning as "include, but not limited to," while "including" has the same meaning as "including, but not limited to."

[0100] The singular forms "a," "or," and "the" include plural references unless the context requires otherwise. Thus, for example, reference to "a compound" may include one or more compounds and / or equivalents thereof.

[0101] Any numerical range disclosed herein includes the upper and lower limits and each intervening value, unless otherwise specified.

[0102] Other than in the practical examples, or where otherwise indicated, numerical values ​​(such as amounts of ingredients and numbers expressing reaction conditions) used in the specification and claims are modified by the term "about." Accordingly, unless indicated to the contrary, such numbers are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should be construed in light of the number of significant digits and ordinary rounding techniques.

[0103] Notwithstanding that the numerical parameters setting forth the scope of the disclosed subject matter are approximations, the numerical values ​​set forth in the practical examples are reported as precisely as possible. However, any numerical value inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Methods for inducing apoptosis or sensitizing cells to apoptotic signals - Patent Application 20070122999

[0104] One aspect of the present disclosure is a method for inducing apoptosis, comprising administering an effective amount of a compound of formula (I) (RX-3117) [ka] or a hydrate, solvate, or pharmaceutically acceptable salt thereof. In embodiments, the compound of formula (I) may be administered as a monohydrate or in free form.

[0105] Another aspect of the present disclosure provides a method of inducing apoptosis in a cell by contacting the cell with an effective amount of a compound of formula (I), or a hydrate, solvate, or pharmaceutically acceptable salt thereof.

[0106] In one embodiment, the method induces apoptosis via a single-strand break (SSB) or a double-strand break (DSB). In another embodiment, the method induces apoptosis via an SSB. In another embodiment, the method induces apoptosis via a DSB.

[0107] Another aspect of the present disclosure provides a method of sensitizing a cell to apoptotic signals by contacting the cell with an effective amount of a compound of formula (I), or a hydrate, solvate, or pharmaceutically acceptable salt thereof.

[0108] In one embodiment of any of the methods provided herein, the cell is a tumor cell. In other embodiments, the cell is a malignant tumor cell. In other embodiments, the cell is a lung cancer cell. In other embodiments, the cell is a non-small cell lung cancer cell. In other embodiments, the cell is a pancreatic cancer cell. In other embodiments, the cell is a bladder cancer cell. In other embodiments, the cell is a colorectal cancer cell. In other embodiments, the cell is a mammalian cell or a cell in a mammal. In other embodiments, the cell is a human cell or a cell in a human. Methods for modulating protein kinases

[0109] Another aspect of the present disclosure provides a method of modulating a protein kinase in a cell, the method comprising contacting the cell with an effective amount of a compound of Formula (I), or a hydrate, solvate, or pharmaceutically acceptable salt thereof. In embodiments, the protein kinase is modulated by increasing the protein kinase. The increase may be, for example, by 5% or more, 10% or more, 20% or more, 25% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 75% or more, 80% or more, 90% or more, or 95% or more.

[0110] In embodiments, the protein kinase is a checkpoint protein kinase. In other embodiments, the protein kinase is checkpoint kinase 1 (Chk1) or checkpoint kinase 2 (Chk2). In other embodiments, the protein kinase is Chk1. In other embodiments, the protein kinase is Chk2.

[0111] In embodiments, the cell is in a mammal, hi other embodiments, the cell is in a human.

[0112] In embodiments, the method increases protein kinase expression levels. Methods for treating or preventing non-small cell lung cancer

[0113] Another aspect of the present disclosure is a method of treating or preventing non-small cell lung cancer, comprising: (i) diagnosing a subject with non-small lung cancer cells; (ii) administering to the subject an effective amount of a compound of formula (I), or a hydrate, solvate, or pharmaceutically acceptable salt thereof; A method is provided by Methods for predicting the effectiveness of treatment

[0114] Another aspect of the present disclosure is a method of predicting the effectiveness of treatment of a subject in need thereof with a compound of formula (I), or a hydrate, solvate, or pharmaceutically acceptable salt thereof, comprising the steps of: (i) collecting a tumor cell or tissue sample from the subject; (ii) measuring one of a protein kinase or p53 expression level in the sample; (iii) contacting the tumor cells or tissue with a compound of formula (I); (iv) measuring one of protein kinase or p53 expression levels in the tumor cells or tissue after contact with the compound of formula (I), wherein an increase in protein kinase or p53 expression levels indicates potential efficacy; A method is provided by

[0115] In embodiments, contacting tumor cells or tissue with a compound of Formula (I) is accomplished by contacting a sample of tumor cells or tissue. In such embodiments, measuring one of protein kinase or p53 expression levels in the tumor cells or tissue after contact with the compound of Formula (I) is performed on the sample. In other embodiments, contacting tumor cells or tissue with a compound of Formula (I) is accomplished by administering a compound of Formula (I) to a subject. In such embodiments, measuring one of protein kinase or p53 expression levels in the tumor cells or tissue after contact with the compound of Formula (I) is performed by collecting a second sample of tumor cells or tissue from the subject and measuring one of protein kinase or p53 expression levels in the second sample.

[0116] In embodiments, the method further comprises administering a compound of Formula (I) to the subject if an increase in either the protein kinase or p53 expression level is detected. The protein kinase is considered to be increased if the amount of the protein kinase is increased by a statistically significant amount. Thus, the increase may be 5% or more, 10% or more, 20% or more, or 25% or more. In other embodiments, the method further comprises measuring the expression level of protein Cdc25C or p-Cdc25C in the sample in steps (ii) and (iv), wherein a decrease in the expression level of protein Cdc25C or p-Cdc25C indicates potential efficacy. The expression level of protein Cdc25C or p-Cdc25C is considered to be decreased if the expression level of protein Cdc25C or p-Cdc25C is reduced by a statistically significant amount. Thus, the reduction may be 5% or more, 10% or more, 20% or more, or 25% or more. In other embodiments, the method further comprises administering to the subject a compound of Formula (I) if an increase in one of protein kinase or p53 expression levels and a decrease in protein Cdc25C or p-Cdc25C expression levels are detected. In embodiments, the compound of Formula (I) is administered as a monohydrate. In other embodiments, the compound of Formula (I) is administered free of solvates, hydrates, and salts.

[0117] In embodiments, the method comprises measuring protein kinase expression levels in steps (ii) and (iv). In other embodiments, the protein kinase is a checkpoint kinase. In other embodiments, the protein kinase is Chk1 or Chk2. In other embodiments, the protein kinase is Chk1. In other embodiments, the protein kinase is Chk2.

[0118] Another aspect of the present disclosure is a method of predicting the effectiveness of treatment of a subject in need thereof with a compound of formula (I), or a hydrate, solvate, or pharmaceutically acceptable salt thereof, comprising the steps of: (i) collecting a tumor cell or tissue sample from the subject; (ii) measuring the level of UCK2 expression in the tumor cells or tissue; wherein the expression level of UCK2 indicates the likelihood of efficacy of treatment with a compound of formula (I).

[0119] In embodiments, the method further comprises administering a compound of Formula (I) to a subject when increased expression of UCK2 is measured in tumor cells or tissues. In embodiments, UCK2 expression is measured by immunoblotting UCK2 protein levels normalized to beta-actin. UCK2 is considered to have an increased expression level when the amount of UCK2 expression is statistically significant compared to a predetermined level. Thus, the increase may be 5% or more, 10% or more, 20% or more, or 25% or more. In embodiments, the predetermined level may be the level of UCK2 expression in non-tumor cells. In embodiments, the predetermined level may be the level of UCK2 expression in non-tumor cells of the subject.

[0120] In embodiments, the subject is a mammal, hi other embodiments, the subject is a human.

[0121] In embodiments, the tumor cells are lung cancer cells, hi other embodiments, the tumor cells are non-small cell lung cancer cells. Methods for treating or preventing tumors

[0122] Another aspect of the present disclosure is a method of treating or preventing a tumor, comprising administering to a subject in need thereof an effective amount of a compound of formula (I) [ka] or a hydrate, solvate, or pharmaceutically acceptable salt thereof, in a dosage amount of about 300-2,000 mg / day. In another embodiment, the dosage amount is about 400-800 mg / day. In another embodiment, the dosage amount is about 500-700 mg / day. In another embodiment, the dosage amount is about 300 mg / day. In another embodiment, the dosage amount is about 400 mg / day. In another embodiment, the dosage amount is about 500 mg / day. In another embodiment, the dosage amount is about 600 mg / day. In another embodiment, the dosage amount is about 700 mg / day. In another embodiment, the dosage amount is about 800 mg / day.

[0123] The dosage of approximately 300 to 2,000 mg / day is based on an adult having a body weight or body mass of approximately 60 to 80 kg. Thus, dosages can range from approximately 5 to 33 mg / kg / day. Additional dosages based on the subject's body weight can be easily calculated from these values. Similarly, one skilled in the art would be able to calculate dosages for other species based on known correlations with human dosages.

[0124] In embodiments, the oral dosage form is administered 3 to 7 days per week. In other embodiments, the oral dosage form is administered 4 to 7 days per week. In other embodiments, the oral dosage form is administered 5 to 7 days per week. In other embodiments, the oral dosage form is administered 5 or 7 days per week. In other embodiments, the oral dosage form is administered 3 days per week. In other embodiments, the oral dosage form is administered 4 days per week. In other embodiments, the oral dosage form is administered 5 days per week. In other embodiments, the oral dosage form is administered 6 days per week. In other embodiments, the oral dosage form is administered 7 days per week.

[0125] In embodiments, the total daily dose is administered in one or more doses. In other embodiments, the oral dosage form is administered once daily. In other embodiments, the oral dosage form is administered twice daily. In other embodiments, the oral dosage form is administered three times daily. In other embodiments, the oral dosage form is administered four times daily.

[0126] In embodiments, the oral dosage form is administered at a dosage of up to about 20,000 mg / month. The total monthly dose may be administered either 1-7 days per week for 3 weeks, followed by a 1-week rest (a "rest week"), or for 4 weeks with no rest. For each week of treatment, the oral dosage form may be administered 1-7 days per week. In one embodiment, the oral dosage form is administered for 3 weeks, followed by a 1-week rest. In another embodiment, the oral dosage form is administered 3-7 days per week for 3 weeks, followed by a 1-week rest. In another embodiment, the oral dosage form is administered 5-7 days per week for 3 weeks, followed by a 1-week rest. In another embodiment, the oral dosage form is administered daily for 3 weeks, followed by a 1-week rest. In another embodiment, the oral dosage form is administered daily for 28 days. Each dosing cycle may be 3 weeks of treatment, followed by a 1-week rest, or continuous / consecutive ) 4 weeks of treatment. The dosing cycle may be repeated as often as necessary, as determined by one skilled in the art. In one embodiment, the oral dosage form is administered for up to 12 dosing cycles. In another embodiment, the oral dosage form is administered for up to 6 dosing cycles.

[0127] In one embodiment, the oral dosage form is administered at a dosage of about 300-2,000 mg / day, 5-7 days per week. In another embodiment, the dosage is about 400-800 mg / day, 5-7 days per week. In another embodiment, the dosage is about 500-700 mg / day, 5-7 days per week. In another embodiment, the dosage is about 500-700 mg / day, 5 or 7 days per week. In another embodiment, the dosage is about 500 mg / day, 5 days per week. In another embodiment, the dosage is about 500 mg / day, 7 days per week. In another embodiment, the dosage is about 600 mg / day, 5 days per week. In another embodiment, the dosage is about 600 mg / day, 7 days per week. In another embodiment, the dosage is about 700 mg / day, 5 days per week. In another embodiment, the dosage is about 70 mg / day, 7 days per week.

[0128] In embodiments, the oral dosage form is administered once daily at about 400 mg / day, five days a week. In other embodiments, the oral dosage form is administered once daily at about 500 mg / day, five days a week. In other embodiments, the oral dosage form is administered once daily at about 600 mg / day, five days a week. In other embodiments, the oral dosage form is administered once daily at about 700 mg / day, five days a week. In other embodiments, the oral dosage form is administered once daily at about 800 mg / day, five days a week.

[0129] In embodiments, the oral dosage form is administered once daily at about 400 mg / day, 7 days a week. In other embodiments, the oral dosage form is administered once daily at about 500 mg / day, 7 days a week. In other embodiments, the oral dosage form is administered once daily at about 600 mg / day, 7 days a week. In other embodiments, the oral dosage form is administered once daily at about 700 mg / day, 7 days a week. In other embodiments, the oral dosage form is administered once daily at about 800 mg / day, 7 days a week.

[0130] In embodiments, the oral dosage form is administered at about 3-35 mg / kg / day, 5-7 days per week. In other embodiments, the oral dosage form is administered at about 3-35 mg / kg / day, 5 days per week. In other embodiments, the oral dosage form is administered at about 3-35 mg / kg / day, 6 days per week. In other embodiments, the oral dosage form is administered at about 3-35 mg / kg / day, 7 days per week. In other embodiments, the oral dosage form is administered at about 6-12 mg / kg / day, 5-7 days per week. In other embodiments, the oral dosage form is administered at about 6-12 mg / kg / day, 5 days per week. In other embodiments, the oral dosage form is administered at about 6-12 mg / kg / day, 6 days per week. In other embodiments, the oral dosage form is administered at about 6-12 mg / kg / day, 7 days per week.

[0131] In some embodiments, the oral dosage form is a solid. In other embodiments, the oral dosage form is a tablet. In other embodiments, the oral dosage form is a capsule. In other embodiments, the oral dosage form is immediate release. In other embodiments, the oral dosage form is extended release.

[0132] In some embodiments, the oral dosage form is administered to the subject after the subject has fasted for at least about 8 hours. In other embodiments, the subject continues to fast for at least about 1 hour after administration. In other embodiments, the oral dosage form is administered to the subject with food.

[0133] In embodiments, the compound of formula (I) is administered as a monohydrate. In other embodiments, the compound of formula (I) is administered free of solvates, hydrates, and salts.

[0134] In embodiments, the oral dosage form has a T of about 3 to 20 hours after a single administration. 1 / 2 In other embodiments, the oral dosage form has a T of about 5 to 10 hours after a single administration. 1 / 2 In other embodiments, the oral dosage form provides a T of about 6 to 9 hours after a single administration. 1 / 2 In other embodiments, the oral dosage form has a T of about 9 to 11 hours after a single administration. 1 / 2 In other embodiments, the oral dosage form has a T of about 6 to 7 hours after a single administration. 1 / 2 In other embodiments, the oral dosage form has a T of about 6 hours after a single administration. 1 / 2 In other embodiments, the oral dosage form has a T of about 7 hours after a single administration. 1 / 2 In other embodiments, the oral dosage form has a T of about 8 hours after a single administration. 1 / 2 In other embodiments, the oral dosage form has a T of about 9 hours after a single administration. 1 / 2 In other embodiments, the oral dosage form has a T of about 10 hours after a single administration. 1 / 2 In other embodiments, the oral dosage form has a T of about 11 hours after a single administration. 1 / 2 to provide.

[0135] In embodiments, the oral dosage form has a T of about 2 to 6 hours after a single administration. max In other embodiments, the oral dosage form provides a T of about 4 to 6 hours after a single administration. max In another embodiment, the oral dosage form has a T of about 2 to 4 hours after a single administration. max In other embodiments, the oral dosage form has a T of about 2 hours after a single administration. maxIn other embodiments, the oral dosage form has a T of about 3 hours after a single administration. max In other embodiments, the oral dosage form has a T of about 4 hours after a single administration. max In other embodiments, the oral dosage form is a single dose After about 5 hours of T max In other embodiments, the oral dosage form has a T of about 6 hours after a single administration. max to provide.

[0136] In embodiments, the oral dosage form provides a Cmax of about 30-3,000 ng / mL after a single administration. In other embodiments, the oral dosage form provides a Cmax of about 600-2,000 ng / mL after a single administration. In other embodiments, the oral dosage form provides a Cmax of about 700-1,500 ng / mL after a single administration. In other embodiments, the oral dosage form provides a Cmax of about 600-1,100 ng / mL after a single administration. In other embodiments, the oral dosage form provides a Cmax of about 700-1,100 ng / mL after a single administration. In other embodiments, the oral dosage form provides a Cmax of about 600-700 ng / mL after a single administration. In other embodiments, the oral dosage form provides a Cmax of about 700-800 ng / mL after a single administration. In other embodiments, the oral dosage form provides a Cmax of about 800-900 ng / mL after a single administration. In other embodiments, the oral dosage form provides a Cmax of about 900-1,000 ng / mL after a single dose. In other embodiments, the oral dosage form provides a Cmax of about 1,000-1,100 ng / mL after a single dose.

[0137] In embodiments, the oral dosage form has an AUC of about 200 to 18,000 h·ng / mL after a single dose. 0~t In other embodiments, the oral dosage form provides an AUC of about 7,000 to 14,000 h·ng / mL after a single dose. 0~t In other embodiments, the oral dosage form provides an AUC of about 8,000 to 12,000 h·ng / mL after a single dose. 0~t In other embodiments, the oral dosage form provides an AUC of about 8,000 to 10,000 h·ng / mL after a single dose.0~t In other embodiments, the oral dosage form provides an AUC of about 8,000 to 9,000 h·ng / mL after a single dose. 0~t In other embodiments, the oral dosage form provides an AUC of about 9,000 to 10,000 h·ng / mL after a single dose. 0~t In other embodiments, the oral dosage form provides an AUC of about 10,000 to 11,000 h·ng / mL after a single dose. 0~t In other embodiments, the oral dosage form provides an AUC of about 11,000 to 12,000 h·ng / mL after a single dose. 0~t (0-24 hours).

[0138] In embodiments, the tumor is ovarian cancer; metastatic breast cancer; pancreatic adenocarcinoma; gastrointestinal cancer such as colorectal adenocarcinoma or cancer of the esophagus, stomach, pancreas, small intestine, hepatobiliary tract, colon, rectum, or anus; bladder cancer such as metastatic bladder cancer, muscle-invasive bladder cancer, or non-muscle-invasive bladder cancer; cervical cancer; lung cancer; non-small cell lung cancer; or renal cell carcinoma. In other embodiments, the tumor is pancreatic, bladder, or colorectal cancer. In other embodiments, the tumor is pancreatic cancer. In other embodiments, the cancer is bladder cancer. In other embodiments, the cancer is colorectal cancer. In other embodiments, the cancer is colon cancer. In other embodiments, the cancer is rectal cancer. In other embodiments, the tumor is non-small cell lung cancer, and the compound of Formula (I), or a hydrate, solvate, or pharmaceutically acceptable salt thereof, is administered with cisplatin. In other embodiments, the tumor is resistant to gemcitabine. See Yang et al., Anticancer Research 34:6951-6960 (2014) (demonstrating the efficacy of RX-3117 in various xenograft models, even in tumors resistant to gemcitabine).

[0139] In embodiments, the subject is a mammal, hi other embodiments, the subject is a human. Kits for testing the efficacy of treatments

[0140] Another aspect of the present disclosure is the use of a compound of formula (I) in the treatment of tumors using an assay that measures one of the protein kinase, p53, or UCK2 expression levels in a sample of tumor cells. [ka] Alternatively, a kit for testing the potential effectiveness of a hydrate, solvate or pharmaceutically acceptable salt is provided.

[0141] In an embodiment, the kit further comprises an assay for measuring the level of expression of protein Cdc25C or p-Cdc25C in a sample of tumor cells.

[0142] In some embodiments, the tumor cells are lung cancer cells, in other embodiments, the tumor cells are non-small cell lung cancer cells, in other embodiments, the tumor cells are pancreatic cancer cells or bladder cancer cells. Pharmaceutical Composition

[0143] In any of the methods and kits provided herein, the compound of formula (I) may be in a pharmaceutical composition. Such pharmaceutical compositions may be prepared in any suitable unit dosage form. For example, the pharmaceutical composition may be formulated for administration in solid or liquid forms, including those suitable for: (1) oral administration, such as drenches, tablets (including over-encapsulated tablets, buccal, sublingual, and systemic absorption-targeted tablets), capsules (dry-filled, hard gelatin, soft gelatin, or over-encapsulated capsules), caplets, boluses, powders, sachets, granules, pastes, oral sprays, lozenges, pellets, syrups, suspensions, elixirs, liquids, liposomes, emulsions, and microemulsions; or (2) parenteral administration, such as subcutaneous, intramuscular, intravenous, or epidural injection, for example, as a sterile solution or suspension. In addition, the pharmaceutical composition may be formulated for immediate, sustained, extended, delayed, or controlled release.

[0144] In one embodiment, the pharmaceutical composition is formulated for oral administration. In an embodiment, the pharmaceutical composition is in tablet or capsule form. In another embodiment, the pharmaceutical composition is in tablet form. In another embodiment, the pharmaceutical composition is in capsule form. In another embodiment, the tablet or capsule is formulated for immediate release. In another embodiment, the tablet or capsule is formulated for sustained, extended, delayed, or controlled release.

[0145] Tablets may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing Compound (I) in a free-flowing form, such as a powder or granules, optionally mixed with binders, lubricants, inert excipients, preservatives, surfactants, or dispersing agents, in a suitable machine. Molded tablets may be made by molding a mixture of powdered compound moistened with an inert liquid excipient in a suitable machine. Tablets may optionally be coated or scored and may be formulated to provide sustained, extended, delayed, or controlled release of Compound (I). Methods for formulating such sustained, extended, delayed, or controlled release compositions are known in the art and are disclosed in issued U.S. patents, including, but not limited to, U.S. Pat. Nos. 4,369,174 and 4,842,866, as well as the references cited therein. Coatings can be used for intestinal compound delivery (see, e.g., U.S. Patent Nos. 6,638,534, 5,217,720, 6,569,457, and references cited therein). In addition to tablets, other dosage forms such as capsules, granules, and gelcaps can be formulated to provide sustained, extended, delayed, or controlled release of Compound (I).

[0146] In an embodiment, pharmaceutical compositions are formulated for parenteral administration.Examples of pharmaceutical compositions suitable for parenteral administration include aqueous sterile injection solutions and non-aqueous sterile injection solutions, each containing antioxidants, buffers, bacteriostatic agents and / or solutes, which make the formulation isotonic with the blood of the intended recipient; and aqueous sterile suspensions and non-aqueous sterile suspensions, each containing suspending agents and / or thickening agents.Preparations can be presented in unit-dose or multi-dose containers, for example, sealed ampoules or vials, and can be stored in a freeze-dried (lyophilized) state, which only requires the addition of sterile liquid carriers such as water immediately before use.In one embodiment, pharmaceutical compositions are formulated for intravenous administration.

[0147] In embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient. A pharmaceutically acceptable excipient may be any substance that is not itself a therapeutic agent, but is used as a carrier, excipient, adjuvant, binder and / or vehicle for delivery of a therapeutic agent to a patient, or added to a pharmaceutical composition to improve its handling or storage characteristics, or to enable or facilitate the formation of a compound or pharmaceutical composition into a unit dosage form for administration. Pharmaceutically acceptable excipients are known in the pharmaceutical arts and are described, for example, in Remington: The Science and Practice of Pharmacy, 21st Edition (Lippincott, Williams & Wilkins, Baltimore, MD, 2005). As is known to those skilled in the art, pharmaceutically acceptable excipients can serve a variety of functions and can be described as wetting agents, buffers, suspending agents, lubricants, emulsifiers, disintegrating agents, absorbents, preservatives, surfactants, colorants, flavoring agents, and sweeteners. Examples of pharmaceutically acceptable excipients include, but are not limited to, (1) sugars such as lactose, glucose, and sucrose; (2) starches such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, cellulose acetate, hydroxypropylmethylcellulose, and hydroxypropylcellulose; (4) tragacanth powder; (5) malt; (6) gelatin; (7) talc; (8) excipients such as cocoa butter and suppository wax; and (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil. (11) polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffer solutions; (21) polyesters, polycarbonates, and / or polyanhydrides; and (22) other non-toxic, compatible substances used in pharmaceutical formulations.

[0148] In some embodiments, the pharmaceutical composition further comprises at least one active agent in addition to RX-3117. The active agent may be an anti-neoplastic agent, a chemotherapeutic agent, a cytotoxic agent, radiation therapy (external beam radiation therapy, internal radiation therapy, or systemic radiation therapy), or any other agent capable of inducing apoptosis, sensitizing cells to apoptosis, modulating protein kinases, or treating neoplasms, tumors, or cancer. Examples of active agents include (1) cytarabine, fludarabine, 5-fluoro-2'-deoxyuridine, gemcitabine, hydroxyurea, or methotrexate. (2) DNA fragmenting agents, such as bleomycin; (3) DNA cross-linking agents, such as chlorambucil, cisplatin, cyclophosphamide, and nitrogen mustard; (4) intercalating agents, such as adriamycin (doxorubicin) and mitoxantrone; (5) protein synthesis inhibitors, such as L-asparaginase, cycloheximide, puromycin, and diphtheria toxin; (6) topoisomerase I poisons, such as camptothecin and topotecan; (7) endothelial cell carcinomas; (8) microtubule-directing agents such as colcemid, colchicine, paclitaxel, vinblastine, and vincristine; (9) kinase inhibitors such as flavopiridol, staurosporin, and 7-hydroxystaurosporine; (10) quercetin, resveratrol, piceatannol, epigallocatechin gallate, theaflavin; (11) hormones such as glucocorticoids and fenretinide; (12) hormone antagonists such as tamoxifen, finasteride, and LHRH antagonists; and (13) death receptor agonists, such as tumor necrosis factor alpha (TNF-α), tumor necrosis factor beta (TNF-β), LT-β (lymphotoxin-β), TRAIL (Apo2L, DR4 ligand), CD95 (Fas, APO-1) ligand, TRAMP (DR3, Apo-3) ligand, DR6 ligand, and fragments and derivatives thereof.

[0149] In embodiments, the amount of the compound of Formula (I), or hydrate, solvate, or pharmaceutically acceptable salt thereof, in the pharmaceutical composition is between about 0.1% and about 100% by weight. In other embodiments, the amount is between about 0.5% and about 99.5% by weight. In embodiments, the amount is between about 10% and about 95% by weight. In embodiments, the amount is between about 15% and about 90% by weight. In embodiments, the amount is between about 80% and about 90% by weight. In embodiments, the amount is between about 80% and about 85% by weight. In embodiments, the amount is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80%. In embodiments, the pharmaceutical composition is in an oral dosage form. In embodiments, the pharmaceutical composition is a tablet. Administration method

[0150] In any of the methods provided herein, administration of the compound or pharmaceutical composition may be via accepted modes known in the art, such as orally or parenterally. The term "parenterally" includes, but is not limited to, subcutaneous, intravenous, intramuscular, intraperitoneal, intravesical, intrathecal, intraventricular, intrasternal, intracranial, intraosseous injection, and by infusion techniques. In one embodiment, the compound or pharmaceutical composition is administered orally. In another embodiment, the compound or pharmaceutical composition is administered parenterally. In another embodiment, the compound or pharmaceutical composition is administered intravenously.

[0151] In one embodiment, the compound or pharmaceutical composition is orally administered at a dose or dosage as disclosed herein, such as in the methods for treating or preventing tumors described above. In any of the methods disclosed herein, the compound or pharmaceutical composition is administered on a weight-based basis. In other embodiments, the effective amount is about 0.01 to about 100 mg / kg / day or about 3 to about 35 mg / kg / day. In an embodiment, the effective amount is about 6 to 12 mg / kg / day.

[0152] Dose levels may be adjusted for intravenous administration, in which case the compound or pharmaceutical composition may be administered in an amount between about 0.01 μg / kg / min and about 100 μg / kg / min. Combination Therapy

[0153] In any of the methods for treating or preventing tumors provided herein, the method may further comprise administering RX-3117 to a subject together with one or more additional anti-tumor agents or radiation. In one embodiment, the method further comprises administering radiation to the subject. In another embodiment, the method further comprises administering one or more additional anti-tumor agents to the subject.

[0154] The additional anti-tumor agent or radiation may be administered before, after, or during administration of the compound of Formula (I), or a hydrate, solvate, or pharmaceutically acceptable salt thereof. In one embodiment, the additional anti-tumor agent or radiation is administered before administration of the compound of Formula (I), or a hydrate, solvate, or pharmaceutically acceptable salt thereof. In another embodiment, the additional anti-tumor agent or radiation is administered after administration of the compound of Formula (I), or a hydrate, solvate, or pharmaceutically acceptable salt thereof. In another embodiment, the additional anti-tumor agent or radiation is administered during administration of the compound of Formula (I), or a hydrate, solvate, or pharmaceutically acceptable salt thereof. In another embodiment, the additional anti-tumor agent and the compound of Formula (I), or a hydrate, solvate, or pharmaceutically acceptable salt thereof, are formulated into pharmaceutical compositions for simultaneous administration. Radiation sensitization effect

[0155] The radiosensitizing effect of RX-3117 was investigated in A2780 ovarian cancer cells and NSCLC cell lines. RX-3117 was found to have a schedule-dependent radiosensitizing effect, but only during preincubation (dose modification factor: 1.4-1.8), as observed with pulsed and fractionated irradiation. Radiosensitization was also observed in a 3D spheroid model. At low radiosensitizing concentrations, RX-3117 in combination with radiation led to the accumulation of cells in S phase, which was accompanied by an increase in all cell cycle proteins, including p-Chk2 and p-cdc25C. In addition, RX-3117 caused cell death due to DNA damage. In conclusion, in vitro experiments demonstrated the radiosensitizing effect of RX-3117.

[0156] Lung cancer patients are standardly treated with surgery, and those with advanced disease receive chemotherapy (Baas P, Belderbos JSA, Senan S, Kwa HB, van Bochove A, van Tinteren H, Burgers JA and van Meerbeeck JP: Concurrent chemotherapy (carboplatin, paclitaxel, etoposide) and involved-field radiotherapy in limited Stage small cell lung cancer: a Dutch multicenter phase II study. Br J Cancer 94:625-30, 2006). A combination of the cytidine analog, gemcitabine, and cisplatin is used in the clinic to treat the disease (El-Naggar M, Ebbing E, Bijnsdorp I, van den Berg J, and Peters GJ: Radiosensitization by thymidine phosphorylase inhibitor in thymidine phosphorylase negative and overexpressing bladder cancer cell lines. Nucleosides Nucleotides Nucleic Acids vol. 33:413-21, 2014). deer However, resistance is a limiting factor and therefore new drugs are needed that circumvent resistance mechanisms and ideally exhibit effective combination properties. RX-3117 mechanism of action

[0157] RX-3117 is an analog of cytidine (Figure 14). It has a modification on the ribose molecule consisting of a carbon-fluorine bond instead of an oxygen and double bond (Choi WJ, Chung HJ, Chandra G, Alexander V, Zhao LX, Lee HW, Nayak A, Majik MS, Kim HO, Kim JH, Lee YB, Ahn CH, Lee SK and Jeong LS: Fluorocyclopentenyl-cytosine with broad spectrum and potent antitumor activity. J Med Chem 5 5:4521-4525, 2012). As shown in Figure 21, RX-3117 enters cells via the human equilibrative nucleoside transporter (hENT) (Peters GJ, Smid K, Vecchi L, Kathmann I, Sarkisjan D, Honeywell RJ, Losekoot N, Ohne O, Orbach A, Blaugrund E, Jeong LS, Lee YB, Ahn CH and Kim DJ: Metabolism, Mechanism of action and sensitivity profile of fluorocyclopentenylcytosine (RX-3117). Invest New Drugs, Vol. 31, pp. 1444-57, 2013. In cells, RX-3117 is phosphorylated by uridine / cytidine kinase 2 (UCK2) to its monophosphate form; i.e., RX-3117 is activated by UCK2 to become RX-3117 MP. RX-3117 triphosphate (RX-3117 TP) is incorporated into RNA. RX-3117 diphosphate (RX-3117 DP) is reduced to deoxy-diphosphate (dRX-3117 DP) by ribonucleotide reductase (RR) before being incorporated into DNA. RX-3117 is a poor substrate for cytidine deaminase (CDA) (ibid.). Recently, potent tumor growth inhibition by RX-3117 in a gemcitabine-resistant mouse model has been demonstrated (Yang MY, Lee YB, Ahn CH, Kaye J, Fine T, Kashi R). , Ohne O, Smid K, Peters GJ and Kim DJ: A novel cytidine analog, RX-3117, shows potent efficacy in xenograft models, even in tumors that are resistant to gemcitabine. Anticancer Res 34:6951-9, 201 The radiosensitizing effect of RX-3117 was investigated, and the results are shown below.

[0158] RX-3117 is categorized as a pyrimidine analog, along with other analogs such as gemcitabine and azacitidine, which are widely used in the clinic (Peters GJ: Novel developments in the use of antimetabolites. Nucleosides Nucleic Acids 33:358-74, 2014). Gemcitabine is a potent radiosensitizer that enhances ionization-induced DNA damage repair (Morgan M a, Parsels L a, Maybaum J and Lawrence TS: Improving gemcitabine-mediated radiosensitization using molecularly targeted therapy: a review. Clin Cancer Res 14:6744-50, 2008).

[0159] In addition, the cytidine analogs 5-azacytidine (Aza-C, Vidaza™) and 5-aza-2′-deoxy-cytidine (decitabine, Dacogen®) are used in the clinic for the treatment of myelodysplastic syndromes (MDS) (Peters GJ: Novel developments in the use of antimetabolites. Nucleosides Nucleic Acids 33:358-74, 2014. The two main mechanisms of the antitumor effects of these drugs are DNA methyltransferase (DNMT) inhibition and cytotoxic incorporation into RNA and / or DNA (Kaminskas E, Farrell A, Abraham S, Baird A, Hsieh LS, Lee SL, Leighton JK, Patel H, Rahman A (Sridhara R, Wang YC, and Pazdur R: Approval summary: azacitidine for treatment of myelodysplastic syndrome subtypes. Clin Cancer Res 11:3604-8, 2005). After cellular uptake, Aza-C is phosphorylated by UCK2 to 5-azacytidine monophosphate (Aza-CMP) and by pyrimidine nucleotide kinase to Aza-CDP and Aza-CTP. However, Aza-C is inactivated by deamination by CDA. RR reduces Aza-CDP to Aza-dCDP, which is phosphorylated by nucleoside diphosphate kinase to Aza-dCTP. Aza-dCTP is then incorporated into DNA, resulting in inhibition of DNA synthesis (Vesely J: Mode of action and effects of 5-azacytidine and of its derivatives in eukaryotic cells. Pharmacol Ther 28:227-35). , 1985). Stoichiometric binding of aza-dCTP to DNMTs would result in DNA hypomethylation (Jones PA: Effects of 5-azacytidine and its 2'-deoxyderivative on cell differentiation and DNA methylation. Pharmacol Ther 28:17-27, 1985). Aza-dCTP can also be formed from 5-aza-2'-deoxycytidine by direct phosphorylation catalyzed by deoxycytidine kinase (dCK) and nucleotide kinase. DNA hypermethylation in CpG islands has been described in different malignancies, including MDS (Kaminskas E, Farrell A, Abraham S, Baird A, Hsieh LS, Lee SL, Leighton JK, Patel. H, Rahman A, Sridhara R, Wang YC and Pazdur R: Approval summary: azacitidine for treatment of myelodysplastic syndrome subtypes. Clin Cancer Res 11:3604-8, 2005). On the other hand, aza-CTP is expressed in the cytoplasm and and nuclear RNA, which is incorporated into the RNA-disrupting metabolism of protein synthesis (Glover AB and Leyland-Jones B: Biochemistry of azacitidine: a review. Cancer Treat Rep 7 1:959-64, 1987). One mechanism of resistance to azacitidine is a point mutation in the UCK2 gene, which results in an inactive metabolite (Sripayap P , Nagai T, Uesawa M, Kobayashi H, Tsukahara T, Ohmine K, Muroi K and Ozawa K: Mechanisms of resistance to azacitidine in human leukemia cell lines. Exp Hematol 42:294-306 e2, 2014). Aza-dCTP The underlying mechanism of resistance to antimetabolites is a deficiency of dCK (Peters GJ: Novel developments in the use of antimetabolites. Nucleosides Nucleotides Nucleic Acids 33:358-74, 2014).

[0160] As shown in Figure 22, RX-3117 can also downregulate DNMT1 (Peters GJ, Smid K, Vecchi L, Kathmann I, Sarkisjan D, Honeywell RJ, Losekot N, Ohne O, Orbach A, Blaugrund E, Jeong LS, Lee YB, Ahn CH and Kim DJ: Metabolism, mechanism of action and sensitivity profile of fluorocyclopentenylcytosine (RX-3117). Invest New Drugs 31:1444-57 (p. 2013), but appears to act differently from Aza-C. Furthermore, some, but not all, cytidine analogs exhibit radiosensitizing effects. Therefore, the potential radiosensitizing effects of RX-3117 and potential mechanisms, such as cell cycle effects and cell death, were evaluated. Therefore, RX-3117 was shown to have radiosensitizing effects.

[0161] Preincubation with RX-3117 had the best radiosensitizing effect, and four of the five cell lines tested were sensitized by RX-3117. Gemcitabine-resistant SW1573 / G- was sensitized by RX-3117 with almost the same efficacy as its wild-type counterpart. RX-3117 also showed radiosensitizing effects in two spheroid models.

[0162] Nucleoside analogues have been shown to enhance radiation-induced cell killing (Shewach DS and Lawrence TS: Antimetabolite radiosensitizers. J Clin Oncol 25:4043-50, 2007). Radiosensitizing effects may be due to the lack of deoxyribonucleotide biosynthesis (which is required for DNA replication) or DNA polymerase (Shewach DS and Lawrence TS: Antimetabolite radiosensitizers. J Clin Oncol 25:404 3-50, 2007; Lawrence TS, Blackstock AW and McGinn C: The mechanism of action of radiosensitization of conventional chemotherapeutic agents. Semin Radiat Oncol 13:13-21, 2003) An example of deoxynucleotide pool deregulation is the TS inhibitor 5-fluoro-2'-deoxyuridine (FdUrd). TS inhibitors cause deoxynucleotide pool imbalance, leading to DNA synthesis inhibition and S-phase arrest (Hwang HS, Davis TW, Houghton J a and Kinsella TJ: Radiosensitivity of thymidylate synthase-deficient human tumor cells is affected by progression. through the G1 restriction point into S-phase: implications for fluoropyrimidine radiosensitization. Cancer Res 60:92-100, 2000 Imbalance in the deoxynucleotide pool leads to incorrect nucleotide incorporation (Ingraham HA, Tseng BY and Goulian M: Nucleotide levels and incorporation of 5-fluorouracil and uracil into DNA of cells treated with 5-fluorodeoxyuridine. Mol Pharmacol 21:211-6, 1982). The concentration required to achieve a radiosensitizing effect is not necessarily the concentration required for a cytotoxic effect. Lower concentrations of the drug can establish radiosensitivity (Hwang HS, Davis TW, Houghton J a and Kinsella TJ: Radiosensitivity of thymidylate synthase-deficient human tumor cells is affected by progression through the G1 restriction point into S-phase: implications for fluoropyrimidine radiosensitization. Cancer Res 60:92-100, 2000). Low doses of RX-3117 induced radiosensitizing effects in colony formation assays using a three-dimensional model and a fractionated irradiation schedule. Furthermore, the double-strand DNA breaks induced by RX-3117 were dose-dependent.

[0163] RX-3117 was shown to be a potent schedule-dependent radiosensitizer in four of the five cell lines, and has potential clinical applications in NSCLC where combination therapy may be explored, which may also be applied to other tumor types (such as prostate, skin, head and neck, throat, larynx, breast, brain, colorectal, bone, leukemia, ovarian, and uterine cancers). Improved process for making RX-3117

[0164] US Patent No. 7,405,214 discloses an 11-step synthesis of RX-3117 from D-ribose. The synthesis uses expensive catalysts that pose challenges for implementation in large-scale industrial production. U.S. Patent No. 9,150,520 discloses a shorter route for the preparation of RX-3117 from (3R,4R,6aR)-tert-butyl-(5-fluoro-2,2-dimethyl-6-trityloxymethyl-4,6a-dihydro-3aH-cyclopenta[1,3]dioxol-4-yloxy)-diphenyl-silane (ASM11) via 4-amino-1-(3aS,4S,6aR)-5-fluoro-2,2-dimethyl-6-((trityloxy)methyl)-4,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)pyrimidin-2(1H)-one (INT14). However, the synthesis from ASM11 to INT14 required the isolation of intermediates at each step. Thus, the process is cost and time constrained, especially when scaled up for commercial manufacturing.

[0165] The present invention provides an improved process for preparing RX-3117 that is commercially viable for large-scale production. The process shortens the synthesis from ASM11 to INT14 without the requirement to isolate each of the intermediate materials, thereby improving efficiency and reducing costs. More specifically, the present invention provides a three-step continuous process for shortening the synthesis from ASM11 to INT14. The present invention also provides a process for producing RX-3117 monohydrate (RX-3117-MH) in a fixed vessel to significantly reduce production costs. By shortening three steps to a single step, the process of the present invention eliminates the requirement to concentrate the intermediate to a residue. These improvements are based on unexpected benefits when substituting reagents that are not readily apparent to those skilled in the art.

[0166] Additionally, the present invention provides optimized reaction and isolation conditions for increasing nitrogen-to-oxygen (N / O) selectivity in Step 3, in which cytosine is added to (3aR,4R,6aR)-5-fluoro-2,2-dimethyl-6-((trityloxy)methyl)-4,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-yl methanesulfonate (INT13) to produce INT14. In the improved process, the ratio of N- to O-isomers was improved to 99.03:0.97 from a previously optimized value of 88:12. The fixed-pot production process of the present invention realizes the cost benefits of scaled-up production operations of the desired product in monohydrate form.

[0167] Scheme 1 below illustrates an improved process for preparing RX-3117MH. [ka] Step 1 - Process improvement for deprotection of ASM11 to form INT12

[0168] 2-Methyl-tetrahydrofuran was used as the process solvent in process Step 1. This modification allowed the workup to be performed without the need to concentrate the intermediate (3aS,4R,6aR)-5-fluoro-2,2-dimethyl-6-((trityloxy)methyl)-4,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-ol (INT12), avoiding a solvent exchange of the intermediate to methyl tert-butyl ether (MTBE) used in the previous process.

[0169] In addition, in-process control (IPC) is quantitative due to the use of TLC. 1 The H NMR method was changed. The process was further optimized by using azeotropic removal of water instead of chemical drying. The use of 2-methyl-tetrahydrofuran as the process solvent allowed for the direct use of INT12 in solution in step 2 of the process without further isolation or purification. Step 2 - Process Improvement for Mesylation of INT12 to Form INT13

[0170] A solution of INT12 in 2-methyl-tetrahydrofuran was carried directly to Step 2 to prepare (3aR,4R,6aR)-5-fluoro-2,2-dimethyl-6-((trityloxy)methyl)-4,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-yl methanesulfonate (INT13). This improved process eliminated the use of environmentally undesirable dichloromethane as the reaction solvent. Additionally, the process used an ammonium chloride wash to further control residual triethylamine. The workup volume was reduced to a maximum process volume of 12.5 volumes, a reduction from 14 volumes. Again, the process was further optimized by using azeotropic removal of water instead of chemical drying. The use of 2-methyl-tetrahydrofuran as the process solvent allowed for the direct use of INT13 in solution in Step 3 of the process. Step 3 - Process Improvement for Addition of Cytosine to INT13 to Form INT14

[0171] A solution of INT13 in 2-methyl-tetrahydrofuran was carried directly into Step 3 to produce INT14. Dimethyl sulfoxide (DMSO) was retained as the reaction solvent, and removal of 2-methyl-tetrahydrofuran was carried out by distillation. It was found that specifying 27% w / w of 2-methyl-tetrahydrofuran to product allowed the reaction in Step 3 to proceed successfully. The inventors screened bases (inorganic and amine) and found that cesium carbonate provided the highest chemoselectivity and the most rapid reaction rate. The inventors also screened reaction solvents and found that DMSO was the most suitable solvent for the reaction.

[0172] Furthermore, the inventors investigated the effects of reagent input, temperature, and concentration on the selectivity of the N- to O-isomer of INT14. According to the improved process of the present invention, the N- to O-isomer ratio was improved from a previously optimized 88:12, isolated using SiO column chromatography, to 99.03:0.97 using solvent extraction and recrystallization / precipitation. The process of the present invention eliminates the need for column chromatography and also provides greater than 99% of the desired N-isomer. Notably, the inventors found that chemoselectivity was primarily affected by reaction temperature. In particular, lowering the reaction temperature slowed the conversion rate to product. The reaction conditions were further improved by increasing the input of base and cytosine from 2.0 to 2.5 equivalents. The reaction temperature was reduced from 40°C to 35°C. Additionally, to improve throughput, the workup procedure was modified to reduce the total process volume from 22 to 12.5 volumes. The workup solvent was changed from ethyl acetate to isopropyl acetate, allowing the reaction mixture to proceed directly to isolation without the need for a solvent exchange. Because INT14 was found to be more soluble after treatment with acetic acid, the workup procedure was also modified to start from the tautomer. Isolation of INT14 was modified by first precipitating the product from a high volume of isopropyl acetate, followed by reducing the volume and adding n-heptane. This improvement was found to prevent oil contamination and vessel adhesion prior to isolation. Thus, an overall improved synthesis with increased selectivity was achieved by simultaneously varying multiple reaction parameters. Modifications of temperature and concentration were found to have a favorable effect on the conversion rate and chemoselectivity of the N / O alkylation. Step 4—Process for Deprotecting INT14 to Form RX-3117 Anhydrate

[0173] The original conditions of 2M HCl in ethanol were found to be most favorable for the product and were retained. However, the process was improved by reducing the reaction temperature from 60°C to 50°C to aid solubility. A methyl tert-butyl ether (MTBE) wash was used to remove the trityl alcohol by-product. The product in the aqueous phase was taken directly to isolation in Step 5 after resin salt release. Step 5—Process for Isolating RX-3117 Monohydrate

[0174] The solution of RX-3117MH was carried directly to Step 5. The combined Steps 4 and 5 were optimized for yield and operability at scale with minor procedural modifications. RX-3117-MH was dried on a filter under air, which maintained the water content while controlling the acetonitrile content below ICH guidelines. This improved process eliminated the time-consuming requirement of first drying the product and then rehydrating it to yield a crystalline product. The product isolated using the improved process had a purity of (99.83%), comparable to that of custom synthesis of small quantities of the product. Other improvements in the process for making the starting material for RX-3117

[0175] Other process improvements for the synthesis of the starting material for RX-3117 are possible. The synthesis of ASM11 using different intermediates and protecting groups is shown in Scheme 2 and Scheme 3, respectively. and 3 are shown below. Synthesis of ASM11 via bromo intermediate [ka]

[0176] In U.S. Patent No. 9,150,520, iodoform was used in step 3 of the reaction to convert RXN-2 to RXN-3. In the present invention, the use of bromoform or mixed bromo-iodomethane produces bromo intermediates instead of iodo intermediates. Bromo intermediates may be more stable than their iodo derivatives. Therefore, overall yield and purity may be increased as a result. Synthesis of ASM11Bn by benzyl protection of the 5-hydroxyl group [ka]

[0177] Unexpectedly, changing a protecting group placed in an intermediate early on can have a dramatic effect on reaction steps carried out later in the process, without requiring multiple step modifications along the way. For example, changing the trityl protecting group to benzyl in step 2 can improve the yield of fluorination in step 10 of the reaction. These improvements can be made without further modification of the overall procedure. Synthesis of ASM11 by ring-closing metathesis

[0178] The present inventors have also developed a scheme for the synthesis of ASM11 by employing ring-closing metathesis. In Scheme 4, a ring-closing metathesis reaction is used to form the five-membered ring moiety. The ruthenium in the Grubbs catalyst is recoverable, further improving the scale-up process by reducing waste and costs. [ka] Synthesis of intermediate RXN-6 by ring-closing metathesis

[0179] The synthesis of intermediate RXN-6 can be accomplished by ring-closing metathesis involving RXN-5 to introduce a fluorine atom into the five-membered ring, creating fluorinated RXN-6. As shown in Scheme 5, a ring-closing metathesis reaction is used to form the five-membered ring moiety (fluoro-RXN-6). As in Scheme 4, the ruthenium of the Grubbs catalyst is recoverable. [ka] Synthesis of intermediate RXN-6 via epoxide-mediated nucleophilic fluorination

[0180] The synthesis of intermediate RXN-6 via an alternative nucleophilic fluorination via an epoxide can be provided. Scheme 6 shows the formation of the epoxide ring from the starting material (3aR,6aR)-6-(((tert-butyldiphenylsilyl)oxy)methyl)-2,2-dimethyl-3a,6a-dihydro-4H-cyclopenta[d][1,3]dioxol-4-one. The epoxide is opened by nucleophilic fluorination using, for example, potassium fluoride. Alternatively, other fluoride sources, such as tetrabutylammonium fluoride, can be used to open the epoxide ring. Water removal is a difficult step in this process, and alternative dehydrating agents, such as carbomethoxysulfamoyltriethylammonium salts, can be used to access the modified intermediate fluoro-RXN6-TBDPS. [ka] Synthesis of intermediate RXN6 via aldol condensation

[0181] The synthesis of intermediate RXN6 can also be accomplished using an aldol condensation to introduce a fluorine atom into the five-membered ring by creating fluorinated RXN-6. As shown in Scheme 7, the fluorine atom is introduced initially to form a fluorinated derivative of RXN6 (fluoro-RXN6). An internal aldol condensation can form a five-membered ring with a vinyl fluorine moiety in place. [ka] Alternative synthesis of intermediate fluoro-RXN6

[0182] Additional alternatives for the synthesis of intermediate fluoro-RXN-6 are shown in Schemes 8 and 9. In Scheme 8, D-ribolactone 1 is reacted with phosphonate 2 to form fluoro-RXN-6. A shorter route is obtained by generating intermediate 3. We found that D-ribolactone derivative 1 does not readily react with dimethylfluoroalkylphosphonates, but better reactivity can be obtained using dimethylcarboalkoxymethylsulfonates. Intermediate 3 then undergoes Hundsdiecker iododecarboxylation to form intermediate 4, which can then undergo nucleophilic substitution using tetra-n-butylammonium fluoride (TBAF). [ka]

[0183] Scheme 9 (below) provides an even shorter route by reacting D-ribolactone 1 with alkylphenyl sulfone 7, prepared via an electrophilic fluorination reagent, to form intermediate 8. D-ribolactone derivatives react more readily with lithiofluoroalkyl sulfone 7 than fluoroalkyl phosphonates. Intermediate 8 can be converted to intermediate 9, which undergoes elimination to form F-RXN6. Alternatively, a more efficient but more expensive option is to use fluorinated tetrazolyl sulfone 10 instead of lithiofluoroalkyl sulfone 7. [ka] Synthesis using different chiral sources

[0184] The synthesis of the present invention can also be achieved by using different chiral sources as starting materials.

[0185] In Scheme 10 (below), (2S,3S,4R,5S,6R)-6-(hydroxymethyl)tetrahydro-2H-pyran-2,3,4,5-tetraol is used as an alternative chiral starting material to generate ASM-11. [ka] [Example]

[0186] The following examples are presented for illustrative purposes and do not serve to limit the scope of the disclosed subject matter.

[0187] IC of the cell lines A549, SW1573 and SW1573 / G used herein 50 The values ​​are 8.3 μM, 13.7 μM, and 7.3 μM, respectively, as reported in Peters et al., "Metabolism, mechanism of action and sensitivity profile of fluorocyclopentenylcytosine (RX-3117)," Investigational New Drugs, December 2013, Vol. 31, No. 6, pp. 1444-1457 (available online at http: / / link.springer.com / article / 10.1007 / s10637-013-0025-x). Example 1 Effect of RX-3117 on non-small cell lung cancer cell lines

[0188] The effects of RX-3117 on cell cycle regulation and cell death in human non-small cell lung cancer (NSCLC) cell lines A549 (adenocarcinoma), SW1573 (alveolar carcinoma), and SW1573 / G- (a gemcitabine-resistant SW1573 cell line), as well as H460 (large cell carcinoma), were assessed. The A549 and H460 cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA). The SW1573 cell line, obtained from Dr. Johan van Rijn (see Keiser et al., Cancer Research, 49:2988-2993 (1989)), also served as the parental cell line for SW1573 / G-. A549, SW1573, and SW1573 / G-, as well as H460, were maintained in exponential growth in T25 flasks (Greiner Bio-One GmbH, Frickenhausen, Germany) in Dulbecco's minimum essential medium (DMEM) or RPMI 1640 (A549) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% streptomycin and penicillin, and 20 mM HEPES, and maintained at 37°C in a water-saturated 5% CO atmosphere. For cell cycle distribution, 1.5 × 10 5Cells were seeded in T25 flasks (Greiner Bio-One GmbH) and cultured for 72 hours, after which 1 μM RX-3117 was added and incubated for 24 hours. The cells were then harvested. After treatment, the medium was collected into a 15 ml tube (Greiner Bio-One GmbH). The cells were washed with ice-cold PBS and trypsinized (Lonza) at 37°C. The collected medium was used to inactivate trypsin in the corresponding sample and collected again into a 15 ml tube. The cells were centrifuged at 4°C and 12,000 rpm for 5 minutes using a standard centrifugation program. The medium was removed, and the pellet was washed with 1 ml of PBS / 0.01% BSA and centrifuged. After removing the supernatant, the cells were fixed with 1 ml of 70% ethanol and incubated at -20°C for at least 24 hours. The cells were then centrifuged, washed with 1 ml of PBS / 0.1% BSA, and transferred to a FALCON FACS tube (BD, Franklin Lakes, NJ, USA). The cells were centrifuged, the supernatant removed, and then 0.5 μg of propidium iodide (PI) (Sigma, St. Louis, MO, USA), 0.1% trisodium citrate (Riedel-de Haen, Sigma-Aldrich Laborchemikalien GmbH, St. Louis, MO, USA), 0.1% Triton X-100™ (Merck), and 0.1 mg / ml of RNase (Sigma) (PI solution) were added to the sample. The cells were then incubated with the PI solution on ice for at least 15 minutes to stain DNA before analysis. The PI-stained cells were analyzed using a FACSCalibur™ (BD Biosciences, Mount View, CA, USA). Data were analyzed using CellQuest™ Pro software.

[0189] The mechanism of cell cycle arrest was investigated by measuring cell cycle protein expression using Western blotting. The effect of RX-3117 on protein expression in different treatment conditions was analyzed by Western blot. Cells were lysed for 30 minutes on ice using cell lysis buffer 1x (Cell Signaling, Danvers, MA, USA) containing 4% protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany) and centrifuged at 14,000 rpm for 10 minutes at 4°C. Protein-containing supernatants were collected and Bio-Rad assays were performed to determine the expression of cell cycle proteins as described by Lemos et al., Pharmacogenomics, 12(2):159-70 ( Protein abundance was determined as described in (2011). The following antibodies were used for protein expression: DNMT1 (Cell Signaling, 1:1000 #5032S), DNMT3A (Cell Signaling, 1:1000 #2160S), DNMT3B (Abcam, 1:1000), Chk2 (Cell Signaling, 1:1000 #6334P), Chk1 (Cell Signaling, 1:1000), p-CDC25C (Cell Signaling 1:1000 #4901S), Cdk1 (Cell Signaling 1:1000 #9112S), Cdk2 (Cell Signaling 1:1000 #2546S), wee1 (Cell Signaling 1:1000), S139-γH2A.X (Cell Signaling, 1:1000), β-actin (Sigma, 1:10,000), caspase 9 (Cell Signaling, 1:1000), PARP (Roche 2003, 1:1000), p53 (Cell Signaling, 1:1000 #9282). Antibodies were diluted in a 1:1 solution of Rockland buffer (Rockland Inc., Philadelphia, PA, USA) and PBS supplemented with 0.05% Tween® 20. Proteins were separated in 20% SDS-PAGE and transferred to a PVDF membrane. The fluorescent signal secondary antibodies used were goat anti-mouse infrared dye and goat anti-rabbit infrared dye. Proteins were detected by an Odyssey infrared imager (Li-COR Bioscience, Lincoln, NE, USA). The abbreviations used herein represent the following: BSA = bovine serum albumin HEPES = 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid PBS = phosphate-buffered saline PVDF = Polyvinylidene difluoride RPM = Revolutions per minute SDS-PAGE = sodium dodecyl sulfate polyacrylamide gel electrophoresis cell cycle

[0190] At a dose of 1 μM, RX-3117 induced the accumulation of A549, SW1573, SW1573 / G-, and H460 cells in the G1 phase after 24 h of exposure (Figure 1). 5×IC 50 At higher doses, RX-3117 induced the accumulation of A549, SW1573, and SW1573 / G- cells in S phase (Fig. 2). Caspase activation

[0191] RX-3117 reduced procaspase 9 in SW1573 and A549 cells after 24 h of exposure to increasing concentrations of RX-3117. Reduction of procaspase 9 indicates activation of caspases and subsequent induction of apoptosis (Figures 3 and 4). DNMT proteins

[0192] At higher doses, RX-3117 downregulated the maintenance of DNA methyltransferase 1 (DNMT1) in A549 cells (Figure 23) and increased DNMT3A and DNMT3B expression levels in A549 cells. The proposed mechanism for this downregulation is shown in Figure 22. DNA damage

[0193] RX-3117 induced double-strand breaks (DSBs) in SW1573 cells after 48 hours of exposure, as indicated by the biomarker γH2A.X (phosphorylated S139) (Figure 5). RX-3117 induced cleaved PARP after 24 hours of exposure to increasing concentrations of RX-3117 (Figure 6). Cleaved PARP indicates activated caspase activity in apoptotic cells. At 1 μM and 5 μM, RX-3117 increased p53 expression levels in A549 cells (Figure 7). At 10 μM, RX-3117 increased Chk1 and Cdk2 expression levels, while decreasing p-Cdc25C expression levels in SW1573 cells after 48 hours of exposure (Figure 8). DNA damage induced by RX-3117 triggers the Chk1 pathway. The ATR / Chk1 pathway is induced by DNA replication stress and DSBs. RX-3117 reduced wee1 expression levels in SW1573 cells after 24 hours of exposure to increasing concentrations of RX-3117 (Figure 9). Figure 24 is a diagram showing potential effects on cell cycle proteins and regulation of the cell cycle by checkpoint kinases Chk1 and Chk2 after damage induction; RX-3117 may have activity along several of these pathways. Apoptosis induction

[0194] 5×IC 50 At doses of 10 μM, RX-3117 induced apoptosis in PI-stained A549 and SW1573 cells at the sub-GI phase after 24 and 48 hours of exposure (Figure 10). At 5 μM (for A549) and 10 μM (for SW1573), RX-3117 induced apoptosis in Annexin V-stained A549 and SW1573 cells at the sub-GI phase after 24, 48, 72, and 96 hours of exposure (Figure 11). result

[0195] The results suggest that cell cycle arrest was time-, concentration-, and cell line-dependent. In A549, H460, and SW1573 cells, 24-hour exposure to 1 μM RX-3117 increased cell accumulation in G1 (approximately 20-40%) and S phase (to a lesser extent), but decreased cell accumulation in G2 / M phase. Thus, low doses of RX-3117 induce G1 accumulation, while high doses induce S phase accumulation. No cell death was observed after 24 hours of exposure, but cell death was observed after 48 hours of exposure (15% in SW1573 and 8% in A549 cells), accompanied by γH2AX induction. In A549 cells, the effect of RX-3117 on cell cycle distribution was most pronounced after 48 hours of exposure, with 45% accumulation in S phase. S phase accumulation was time-dependent. Treatment with RX3117 increased the expression levels of p53, Chk1, Chk2, and Cdk2, but decreased the expression levels of Cdc25C and p-Cdc25C. RX-3117 increased the expression level of wee1, mostly after 48 hours. RX-3117 appeared to induce apoptosis via single-stranded and double-stranded subunits (SSBs). Cleaved PARP in SW1573 cells indicates upregulated caspase activity in apoptotic cells. Reduction of procaspase 9 in A549 cells indicates caspase activation and subsequent apoptosis induction. In conclusion, DNA damage induced by RX-3117 triggered apoptosis on the one hand and increased the expression levels of Chk1 and Chk2 on the other hand. Without being limited to any mechanism of action, it is believed that phosphorylated Chk1 and Chk2 may trigger the phosphorylation of Cdc25C, leading to its degradation, which results in a decrease in Cdk1 levels and therefore the accumulation of cells in S phase. Example 2 Efficacy of RX-3117 in a syngeneic MC38 mouse colon cancer xenograft model

[0196] The effect of RX-3117 on tumor growth in a syngeneic model was examined using female C57BL / 6 mice bearing MC38 murine colon carcinoma according to the protocol described below. Tumor growth was measured in the treatment group compared to the control (vehicle-treated) group (see Table 1 below for dosing scheme and treatment regimen). The results of this study (Table 2) demonstrate that the addition of RX-3117 to the programmed death receptor 1 (PD-1) inhibitor, RMP1-14, had an additive effect in inhibiting tumor growth (80% RX-3117 alone, 93% RMP1-14 alone, vs. 99% for the combination of the two agents). The combination of the two agents also resulted in a greater number of mice (9 mice) with partial and complete regressions and 7 animals with tumor progression-free survival compared to 4 animals with partial and complete regressions and 2 animals with tumor progression-free survival in the RMP1-14 alone group. All results were obtained without any adverse effects to the mice in the combination group.

[0197] Briefly, the method is described as follows: Cells were harvested during exponential growth and resuspended in phosphate-buffered saline. Each test animal received 1 x 10 6 They received subcutaneous (sc) injections of tumor cells and had an average tumor size of 60-100 mm 3 Tumor growth was monitored as the tumor approached the target range of 0.01 mg / kg / day. Once each animal reached this target range, dosing was initiated according to Table 1. [Table 1] [Table 2]

[0198] Tumors were measured in two dimensions using calipers and volume was calculated using the formula:

number

[0199] Data from day 45 were used to determine treatment efficacy. The MTV (n), the median tumor volume for the number of animals n, at day 45 was determined for each group. Percent tumor growth inhibition (%TGI) is defined as the difference between the MTV of the designated control group (vehicle-treated) and the MTV of the drug-treated group, expressed as a percentage of the MTV of the control group:

number

[0200] The dataset for TGI analysis includes all animals in the group except those that died from treatment-related (TR) or non-treatment-related (NTR) causes. Agents that produce a TGI of at least 60% in this assay are considered potentially therapeutically active.

[0201] The study protocol specifies a tumor growth delay assay based on the median time to endpoint (TTE) in the treatment group versus the control group. Each animal was assigned a tumor growth rate of 1500 mm 3 Once the volume endpoint of 0.25 mm was reached, the mice were euthanized due to tumor progression (TP). The time to endpoint (TTE) for each mouse was calculated using the following equation:

number

[0202] [where b is the intercept and m is the slope of the line obtained by linear regression of the log-transformed tumor growth dataset]. The dataset consists of the first observation exceeding the study endpoint volume and the three consecutive observations immediately prior to reaching the endpoint volume. Any animals that did not reach the endpoint were euthanized at the end of the study and assigned a TTE value equal to the last day of the study (day 71). In cases where the TTE calculated by log transformation precedes the day the endpoint is reached or exceeds the day the tumor volume endpoint is reached, linear interpolation is performed to approximate the TTE. Any animals determined to have died from treatment-related (TR) causes are assigned a TTE value equal to the day of death. Any animals that died from non-treatment-related (NTR) causes are excluded from the TTE analysis.

[0203] Treatment efficacy was determined from the number of regression responses. Treatment may result in partial tumor regression (PR) or complete tumor regression (CR) in animals. In a PR response, the tumor volume was 50% or less of its D1 volume for three consecutive measurements during the course of the study, and was 13.5 mm for one or more of these three measurements. 3 In a CR response, tumor volume was greater than or equal to 13.5 mm for three consecutive measurements during the course of the study. 3 Any animal with a CR response on the last day of the study was additionally classified as a tumor progression-free survivor.

[0204] For toxicity assessment, animals were weighed daily for the first 5 days of the study and twice weekly thereafter. Mice were frequently observed for overt signs of any adverse treatment-related side effects, and if observed, clinical signs of toxicity were recorded.

[0205] The acceptable toxicity is defined as a group mean body weight loss of less than 20% during the study and no more than 1 treatment-related (TR) death out of 10 treated animals. Any dosing regimen that results in greater toxicity is considered to exceed the maximum tolerated dose (MTD). Deaths are classified as TR if they are due to the side effects of treatment, as evident from clinical signs and / or autopsy, or if they are due to an unknown cause during the dosing period or within 14 days of the final dose. Deaths are classified as non-treatment-related (NTR) if there is no basis to relate them to the side effects of treatment.

[0206] For statistical and graphical analysis, Prism 6.05 (GraphPad) for Windows® was used. MTV values for multiple groups were compared by non-parametric Kruskal–Wallis test and Dunn's post-multiple comparison test. Two-sided statistical analysis was performed at P = 0.05. Prism reports the results as not significant (ns) for P > 0.05, significant (indicated by the symbol " * ") for 0.01 < P ≤ 0.05, very significant ( " ** ") for 0.001 < P ≤ 0.01, and extremely significant ( " *** ") for P ≤ 0.001. Statistical tests are tests of significance and do not provide an estimate of the magnitude of the difference between groups, so all levels of significance are described as either significant or not significant within the body of this report.

[0207] The "box-and-whisker" plots were constructed to show the distribution of individual tumor volumes for each group at D15. The box represents the 25th to 75th percentile of observations, the horizontal line corresponds to the median, and the "whiskers" indicate the maximum and minimum values. The group median tumor volume was plotted as a function of time. The change in group mean BW was graphed as percent change from D1, ±SEM. Animals that died due to NTR causes were excluded from all graphical representations.

[0208] Survival was analyzed by the Kaplan-Meier method based on TTE values. Log-rank (Mantel-Cox) and Gehan-Breslow-Wilcoxon tests were used to determine the significance of differences between the overall survival experiences (survival curves) of two groups based on TTE values. Kaplan-Meier plots and statistical tests used the same data set and excluded any animals recorded as non-transplant deaths. Scatter plots were constructed to show TTE values ​​for individual mice by group, indicating non-transplant deaths excluded from all other figures. Group mean tumor volumes were plotted as a function of time. If an animal was removed from the study due to tumor size or TR death, its last recorded tumor volume was included with the data used to calculate the median volume at subsequent time points. Tumor growth curves were truncated after two TR deaths occurred within the same group. Group mean BW changes over the course of the study were graphed as percent change from day 1 ± SEM. Tumor growth and BW change curves are truncated after more than half of the assessable mice in a group have been removed from the study. Example 3 Pharmacokinetics, safety, and tolerability of RX-3117 in humans

[0209] The pharmacokinetics, safety, and tolerability of RX-3117 were evaluated in a first-in-human, open-label, exploratory study. The study duration was 14-15 days (7-day screening period; 3-day treatment period; 4 (+1)-day safety follow-up period). Nine adult male and female subjects with histologically confirmed solid tumors enrolled and completed the study. Subjects received RX-3117 (n = 3 subjects per dose) as a single oral dose (50 mg or 100 mg) or a single intravenous dose (20 mg). Pharmacokinetics (PK)

[0210] The absolute bioavailability (F) of oral RX-3117 was 55.67% and 33.42% for the 50 and 100 mg doses, respectively. maxwere 2.16 and 2.49 hours for the 50 and 100 mg doses, respectively. max were 303.3 ng / mL and 311.43 ng / mL for the 50 and 100 mg doses, respectively. The greater absolute bioavailability and C max The results suggest that the oral bioavailability of RX3117 in plasma may not be dose-proportional. 1 / 2 were 13.95 hours and 20.92 hours, respectively, indicating that RX-3117 may exhibit dose proportionality for some parameters but not others at the doses tested.

[0211] The plasma PK profile of intravenous RX-3117 differed from that of oral RX-3117. A 20 mg dose of intravenous RX-3117 was administered as a bolus infusion (T max A 20 mg dose of intravenous RX-3117 resulted in a mean C of 1143.63 ng / mL. max which was approximately a four-fold increase over the peak concentration of the oral dose. Safety and Tolerability

[0212] RX-3117 was safe and well tolerated in all subjects. There were no adverse events (AEs), treatment-emergent adverse events (TEAEs), or serious adverse events (SAEs).

[0213] The results indicate that RX-3117 is safe and well-tolerated with oral bioavailability, supporting studies at higher doses. Example 4 Pharmacokinetics, safety and tolerability of RX-3117 at different oral doses

[0214] In an open-label, dose-ranging study, the pharmacokinetics (PK) of RX-3117 at various oral doses was evaluated. Subjects with advanced malignancies were administered capsules containing RX-3117 at daily doses of 30 mg (N=1), 60 mg (N=1), 100 mg (N=3), 150 mg (N=3), 200 mg (N=3), 500 mg (N=3), 1000 mg (N=3), 1500 mg (N=4), and 2000 mg (N=5 to date) three times weekly (TIWK) for 3 weeks, with a one-week break in each 4-week cycle. Based on the ongoing safety profile, more frequent dosing was also implemented to enhance weekly RX-3117 exposure. In addition to the TIWK dosing scheme discussed above, subjects also received 500 mg and 700 mg five times per week and 500 mg seven times per week for three weeks, with one week off each four-week cycle. Dose escalation began with an accelerated design, treating one subject per dose (Simon et al., J. Natl. Cancer Inst., Vol. 89(15)). :1138-47 (1997)), followed by a standard 3 + 3 design using a modified Fibonacci sequence after the occurrence of a single relevant grade 2 or higher adverse event. Table 3 summarizes the dosing schedule. [Table 3] Pharmacokinetics (PK)

[0215] PK data are presented in Table 4.

[0216] RX-3117 demonstrated a median T of 2 to 3 hours, without significant time lag, typically observed. max It was rapidly absorbed by T max After this, the AUC observed in the first 8 hours 0~t Approximately half of the time points (0-24 hours) and over 80% at 24 hours were biphasic. 1 / 2did not exhibit either dose- or time-dependent pharmacokinetics, with mean values ​​for the dose range of 60 to 2000 mg ranging from 11.6 to 16.7 hours after the first dose and 12.3 to 20.2 hours after the seventh dose (day 15 of dosing). max and AUC 0~t (0-24 hours) increased fairly linearly with dose, but not in a proportional manner, presumably reaching a plateau at the 1500 mg dose (Figures 12 and 13). Over the dose range of 30 to 2000 mg, mean C max ranged from 32 to 1858 ng / mL after the first dose and from 99 to 1703 ng / mL after the seventh dose (Figure 12). Over the same dose range, the mean AUC 0~t (0–24 h) ranged from 164 to 20,544 h·ng / mL after the first dose and from 702 to 20,919 h·ng / mL after the seventh dose (Figure 13). Accumulation was generally minimal.

[0217] PK data show a dose-dependent increase in exposure at doses up to 1000 mg TIWK. At doses greater than 500 mg TIWK, the C max and AUC 0~t (0-24 hours) was consistently lower than that measured after the first dose (Figures 12 and 13). max and AUC 0~t Due to the plateau in (0-24 h) values, a more frequent dosing schedule was used to enhance weekly exposure (Table 4). Based on the results of this study, the maximum tolerated dose (MTD) of RX-3117 was determined to be 700 mg daily, 5 days per week, for 3 weeks, given with a 1-week rest period in each 4-week cycle. This MTD was then used in a follow-up efficacy study. [Table 4] Safety and Tolerability

[0218] The most frequently observed adverse events were mild to moderate fatigue and nausea, mild diarrhea, mild vomiting, mild anorexia, and moderate dehydration. Dose-limiting toxicities were limited to grade 3 anemia and thrombocytopenia. Example 5 Efficacy, safety, and tolerability of RX-3117 in humans

[0219] The efficacy, safety, and tolerability of RX-3117 at various doses and frequencies were evaluated (see Example 4 above). Subjects with advanced malignant tumors were administered capsules containing RX-3117 at various doses, seven times weekly from TIWK for three weeks, with one week off treatment every four-week cycle. Dose escalation began with an accelerated design, treating one subject per dose (Simon et al., J. Natl. Cancer Inst., 89(15):1138-4). 7 (1997)), followed by a standard 3 + 3 design using a modified Fibonacci sequence after the occurrence of a single relevant grade 2 or higher adverse event. Table 4 (above) summarizes the dosing schedule.

[0220] Subjects were assessed for efficacy, safety, and tolerability of RX-3117. A total of 48 subjects were enrolled (30 females, 18 males). Seventeen subjects experienced stable disease over 1 to 10 cycles, and 10 subjects received treatment for 82 to 276 days. Reductions in tumor burden were observed in three subjects with pancreatic (tumor volume and CA19-9 biomarker), breast, and mesothelioma cancers. The most common associated adverse events were moderate to severe anemia, mild to moderate fatigue and nausea, mild diarrhea, vomiting, and anorexia.

[0221] In another phase of this study, RX-3117 is being evaluated in a Phase Ib / IIa trial in cancer patients with recurrent or refractory pancreatic cancer or advanced bladder cancer (including muscle-invasive bladder cancer). The Phase Ib / IIa trial is a multicenter study evaluating the safety and efficacy of RX-3117 in these target patient populations. Secondary endpoints include safety and pharmacokinetic analyses. Patients in the trial are receiving a daily oral dose of 700 mg of RX-3117, five times weekly for three weeks, in 28-day cycles and four treatment cycles, or until disease progression. Example 6 Radiosensitizing effects of fluorocyclopentenyl-cytosine (RX-3117) in ovarian and lung cancer cell lines Drugs and chemicals

[0222] A stock solution of RX-3117 was made in deionized water. All other chemicals used were of standard quality and commercially available. cell culture

[0223] Human NSCLC cell lines A549 (adenocarcinoma), H460 (large cell carcinoma), SW1573 (alveolar carcinoma), and SW1573 / G- (a gemcitabine-resistant SW1573 cell line) and ovarian cancer cell line A2780 were maintained in exponential growth in T25 flasks (Greiner Bio-One GmbH, Frickenhausen, Germany) in Dulbecco's minimum essential medium (DMEM) or RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% streptomycin, and penicillin, and maintained at 37°C in a 5% CO2-saturated atmosphere. Cells were harvested using trypsin-EDTA (Invitrogen, Paisley, UK). Cells were counted using a Coulter® Z™ series counter. Colony formation assay

[0224] Exponentially growing A2780, SW1573, A549, H460 and SW1573 / G- cells were exposed to 1 μM RX-3117 for 24 hours or left untreated (control). 60 The cells were irradiated with a single dose of γ-irradiation (0–6 Gy) using a Co source (Gamma Cell 200, Atomic Energy of Canada, Ltd.). 500 cells / T25 flask were then seeded and allowed to form colonies. After 10 days, the colonies were fixed with 100% ethanol and stained with 10% Giemsa stain (Merck Chemicals BV, Amsterdam, the Netherlands) for colony counting. The seeding efficiency (PE) was calculated by dividing the number of colonies formed by the number of cells seeded and normalized to the cytotoxicity induced by the control. To illustrate the effect of RX-3117 on radiation, dose-modifying factors (DMFs) were calculated as previously described (Bijnsdorp IV, van den Berg J, Kuipers GK, Wedekind LE, Slotman BJ, van Rijn J, Lafleur MVM, and Sminia P: Radiosensitizing potential of the selective cyclooxygenase-2 (COX-2) inhibitor meloxicam on human glioma cells. J Neurooncol 85:25-31, 2007). Spheroid assay

[0225] NSCLC cell lines A549 and SW1573 were seeded at a density of 100,000 cells / well in low-attachment 24-well plates (Corning Incorporated, Corning, NY) and allowed to form spheroids. After 3 days, single spheroids were transferred to new 24-well low-attachment plates (one spheroid / well). Treatment began immediately after transfer, and for A549 and SW1573 cells, 1 μM RX-3117 was combined with fractionated 2 Gy irradiation (a single 2 Gy dose over 5 days). Photographs were taken using a phase-contrast microscope (Leica DMI300B Universal Grab 6.3 software, Digital Cell Imaging Labs) on day 0 (before irradiation) and after days 3, 6, 9, and 15. Measurements were performed using spheroid volume calculations (V = 4 / 3π(D / 2)). 3 ) was performed using Image J software (Image J 1.45s, Wayne Rasband, National Institutes of Health, Bethesda, MD) as previously described by Galvani et al. (Galvani E, Giovannetti E, Saccani F, Cavazzoni A, Leon LG, Dekker H, Alfieri R, Carmi C , Mor M, Ardizzoni A, Petroni PG and Peters GJ: Molecular mechanisms underlying the antitumor activity of 3-aminopropanamide irreversible inhibitors of the epidermal growth factor receptor in non-small cell lung cancer. Neoplasia 15:61-72, 2013). Flow cytometry analysis

[0226] Cell cycle distribution and apoptosis were measured by growing cells in flat-bottom 6-well plates (Greiner Cells were analyzed by seeding at a density of 5,000 cells in 5× IC medium (Bio-One GmbH, Frickenhausen, Germany) and allowed to adhere for 24 hours. 50 A cytotoxic concentration of 1000 mg / mL of ATP was added. Exposure times were 24 and 48 hours, and a control group was included for comparison. At each time point, the total amount of adherent and floating cells was harvested into round-bottom FALCON tubes (BD, Franklin Lakes, NJ, USA). After centrifugation, the cell pellet was resuspended in 1.0 ml of propidium iodide (PI) solution (50 μg / ml PI, 0.1% sodium citrate, 0.1% Triton X-100, 0.1 mg / ml RNase A) or 10 μL of Annexin V (Cat. No. 31490014, Immunotools) and placed on ice for 30 minutes. Samples were then analyzed using a FACSCalibur (BD Biosciences, Mount View, CA, USA). For data analysis, CELLQuest™ software was run using a gate on the DNA histogram to estimate the amount of cells in sub-G1 phase (apoptotic cells). Protein expression analysis

[0227] The effect of RX-3117 on protein expression under different treatment conditions was analyzed by Western blot. Cells were lysed for 30 minutes on ice using 1x cell lysis buffer (Cell Signaling, Danvers, MA, USA) containing 4% protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany) and centrifuged at 14,000 rpm for 10 minutes at 4°C. A Bio-Rad assay was performed to determine protein content in the collected supernatants as previously described (Lemos C, Kathmann I, Giovannetti E, Calhau C, Jansen G, and Peters GJ: Impact of cellular folate status and epidermal growth factor receptor expression on BCRP / ABCG2-mediated resistance to gefitinib and erlotinib. Br J Cancer 100:1120-7, 2009). The following antibodies were used for protein expression: γH2A.X (Cat. No. 9718, Cell Signaling, 1:1000), β-actin (Sigma, 1:10,000), Cdc25C Ser216 (Cat. No. 4901, Cell Signaling, 1:1000), Cdk1 Tyr15 (Cat. No. 9111, Cell Signaling, 1:1000), Chk1 Thr68 (Cat. No. 2197S, Cell Signaling, 1:1000), and histone 3 (Cat. No. 4499, Cell Signaling). Antibodies were diluted 1:1 in phosphate-buffered saline (PBS) supplemented with Rockland buffer (Rockland Inc., Philadelphia, PA) and 0.05% Tween 20. Proteins were separated in a 20% SDS-PAGE gel and transferred to a polyvinylidene difluoride (PVDF) membrane. The fluorescent signal secondary antibodies used were goat anti-mouse infrared dye and goat anti-rabbit infrared dye, and proteins were detected using an Odyssey infrared imager (Li-COR Bioscience, Lincoln, NE). result Radiosensitizing effect of RX-3117

[0228] To investigate the effect of RX-3117 on radiation, a colony formation assay was performed. First, we examined whether pre- or post-incubation with RX-3117 enhanced the effect of radiation. A2780 cells were compared before and after treatment with RX-3117 combined with 4 Gy.

[0229] Colony formation assay data showed that preincubation with RX-3117 was the most effective condition for radiosensitization: preincubation with 1 μM RX-3117 had a 5-fold lower seeding efficiency compared to the control (Figure 15).

[0230] Using a preincubation schedule, potential radiosensitizing effects were investigated in A2780 cells and the non-small cell lung cancer cell lines A549, SW1573, and SW1573 / G- and H460. Overall, all cell lines showed radiosensitizing effects when treated with RX-3117 and radiation. The greatest radiosensitization was observed in the A2780 and A549 cell lines, with a DMF of 1.8, and SW1573, with a DMF of 1.5 (Figures 16A, 16B, and 16D). The gemcitabine-resistant cell line SW1573 / G- had a DMF of 1.4 (Figure 16E), whereas H460 cells showed poor radiosensitizing effects. Because fractionated radiation is a standard procedure in the clinic, a 5-day fractionated dose of 2 Gy irradiation in SW1573 cells was also studied. Incubation with 1 μM RX-3117 prior to five 2 Gy fractionated radiation treatments showed the lowest colony growth (FIG. 16F).

[0231] We also investigated the radiosensitizing potential of RX-3117 in combination with irradiation in a three-dimensional model using a spheroid assay. A sphere formation assay revealed the radiosensitizing effect of RX-3117 on sphere formation in SW1573 and A549 spheroids (Figure 17). SW1573 spheres were highly affected by both 1 μM RX-3117 alone and radiation therapy (RT) alone (2 Gy for 5 days), and the combination enhanced the effect. In A549 cells, RX-3117 treatment or irradiation alone had only a small effect on volumetric growth, whereas 1 μM RX-3117 enhanced the effect of 2 Gy irradiation for 5 days (Figure 17). Apoptosis initiation

[0232] The potential of RX-3117 to induce apoptosis was investigated in NSCLC cell lines. The amount of apoptotic cells was measured by Annexin V staining (FIG. 18) after 24, 48, and 72 hours of exposure. Annexin V cell membrane staining showed a gradual increase in apoptotic cells for A549 and SW1573 cells, which was more pronounced in SW1573 cells than in A549. DNA damage begins

[0233] DNA damage is a hallmark of cell death. DNA damage was studied by assessing γH2A.X expression. In the A2780 cell line, RX-3117 induced γH2A.X S139 in a dose-dependent manner with increasing concentrations of RX-3117 starting from 0.1 μM to 10 μM (Figure 18A). In the SW1573 cell line, double-strand break damage markers increased up to 0.3 μM RX-3117 after 48 hours of exposure (Figure 18B). The combination of 0.3 μM RX-3117 and irradiation showed more pronounced γH2A.X S139 protein expression (Figure 18B). As expected, radiation caused an immediate increase in γH2A.X expression, and repair was delayed in the presence of RX-3117. Effects of RX-3117 and radiation treatment on cell cycle distribution and cell death

[0234] Disturbances in cell cycle distribution have been reported to be involved in the radiosensitizing effects of other nucleoside analogs (Shewach DS and Lawrence TS: Antimetabolite radiosensitizers. J Clin Oncol 25:4043-50, 2007), and therefore may be useful in combination with radiation. The combined effects of RX-3117 were investigated in NSCLC cells using FACS analysis. At a relatively low concentration of 1 μM RX-3117, a small but marginally significant (p<0.05) cell line-dependent increase in S phase was observed in three of the four cell lines. An increase in G1 phase was observed in all cell lines (Figure 19A), and a strong decrease in G2 / M phase was observed. 4 Gy of radiation caused a clear decrease in the amount of cells in S phase, an increase in G2 / M phase in both SW1573 cells, no effect in A549 cells, but a decrease in H460 cells (Figure 19A). The combination of radiation and RX-3117 led to an increase in the number of cells in S phase in both SW1573 mutants, but a decrease in H460 cells. Cell death (sub-G1) was clearly increased in A549 cells, but not in the other cells (Figure 19A).

[0235] To understand some of these phenomena, we also investigated the effects of RX-3117 and radiation on the expression of several essential cell cycle proteins (Figures 19B and 20). In SW1573 cells, the effects of both RX-3117 and radiation were examined on various cell cycle checkpoint proteins (Figure 20). Radiation caused interesting decreases in wee1, Chk2, CDC25c, and p-CDC25c after 48 hours. In both SW1573 cells, radiation caused an increase in Chk2 phosphorylation (Figure 19B). In almost all cell lines (except gemcitabine-resistant SW-1573 / G), RX-3117 decreased Cdk1 phosphorylation. Similarly, radiation increased the phosphorylation of cdc-25C (except in SW1573 / G) within 24 hours (Figure 19B). This effect was maintained when combined with RX-3117 (Figure 19B). Example 7 Inhibition of DNA methyltransferases by RX-3117 leads to upregulation of hypomethylated targets

[0236] RX-3117 is similar to azacitidine (aza-CR) and aza-deoxycytidine (aza-CdR). RX-3117 is taken up by human equilibrative nucleoside transporters (hENT) and activated by uridine-cytidine kinase 2 (UCK2) to form RX-3117-MP (Figure 21). RX-3117 is taken up by human equilibrative nucleoside transporters (hENT) and activated by uridine-cytidine kinase 2 (UCK2) to form RX-3117-MP. RX-3117 downregulates DNA methyltransferase 1 (DNMT1) (Choi WJ et al., J. Med. Chem. 55 (2012) 4521-4525; Peters GJ et al., Invest New Drugs 31 (2013) 1444-1457). DNMT1 is a newly expressed protein in the S phase. It is responsible for maintaining methylation in newly synthesized DNA and methylates cytosine residues in hemimethylated DNA. The deamination rate of RX-3117 is much slower than that of gemcitabine.

[0237] RX-3117 is a novel, orally bioavailable cytidine analog currently being evaluated in a Phase I clinical study. The maximum tolerated dose is greater than 2,000 mg / day. Downregulation of DNMT1 by RX-3117 has been demonstrated in various cell lines with different histological backgrounds. Currently, both UCK2 and DNMT1 are being evaluated as potential biomarkers. In this example, we determined the effects of RX-3117 on DNMT1 in DNA, RNA, protein, and enzyme activity, as well as reactivation of repressed target genes, including p16INK4A, methylguanine methyltransferase (MGMT), and proton-coupled folate transporter (PCFT). PCFT transports folate, methotrexate (MTX), and pemetrexed (PMX) at pH 5.5 and 7.4, and the genes are highly methylated. In addition, the functions of proteins known to be regulated by methylation, including proton-coupled folate transporter (PCFT), were investigated. The expression of E-cadherin (an adhesion molecule), p16INK (a tumor suppressor protein), and O-6 methylguanine DNA methyltransferase (MGMT), a DNA repair gene, in the A549 cell line was also investigated. method

[0238] The following cell lines were used in this study: (1) CCRF-CEM cells and their MTX-resistant mutant, CEM-MTX, characterized by a deficiency of the reduced folate carrier (RFC) (Jansen G. et al., JBC Vol. 273 (1998) pp. 30189-30198). The FT gene is highly methylated (Gonen N. et al., BBRC Vol. 376 (2008) (2) CEM cells cultured in RPMI medium supplemented with 10% fetal bovine serum (FBS); and (3) A549 and SW1573 non-small cell lung cancer (NSCLC) and A2780 ovarian cancer cell lines cultured in DMEM medium supplemented with 10% FBS.

[0239] DNMT1 protein expression was measured by Western blotting after exposure to RX-3117 for 24 or 48 hours. DNMT1 RNA expression was measured by real-time PCR after exposure to RX-3117 for 24 and 48 hours. DNMT enzyme activity was measured in isolated nuclei using the ability of the CpG-binding domain to bind methylated DNA after exposure to 1 μM RX-3117 or 5 μM Aza-CdR. The effect of 5 μM RX-3117 on global methylation in A549 cells was measured using a DNA methyltransferase assay kit provided by EpiGentek. The effect of 5 μM RX-3117 on global methylation was measured using a specific antibody against 5-methyl-cytosine in A549 cells. Bands in Western blots were visualized using appropriate infrared dyes using an Odyssey infrared imager.

[0240] MTX transport was measured using radiolabeled MTX in CEM wild-type and CEM-MTX cell lines. CEM cells have high RFC activity. CEM-MTX is completely defective in RFC-mediated transport. CEM cells have highly methylated PCFT transporters and very low PCFT-mediated transport (Gonen N. et al., BBRC 376). (2008) pp. 787-92). MTX transport at pH 7.4 was primarily RFC-mediated, with less than 2% mediated by PCFT. Folic acid was used to inhibit PCFT-mediated transport. L-leucovorin (L-LV) was added to completely inhibit RFC-mediated transport. CEM and CEM-MTX cells were exposed to 29.6 μM RX-3117 and 0.19 μM Aza-CdR as a positive control. After 24 h, MTX transport to the drug was measured in a 3-minute uptake assay using 2 μM [3',5,'7-3H]-MTX.

[0241] Statistics were performed using Student's t-test. result

[0242] In moderately sensitive non-small cell lung cancer (NSCLC) cell lines, such as A549 and SW1573, 5-50 μM RX-3117 downregulated DNMT1 protein expression by 5-20% after 24 h of exposure and by over 90% after 48 h (Fig. 25A and B). DNMT1 mRNA was unaffected after 24 h of exposure but moderately affected after 48 h (Fig. 25C and D).

[0243] In the sensitive ovarian cancer cell line A2780, protein downregulation was observed already after 24 hours with 1 μM RX-3117 (Figures 26A and B). DNMT1 activity was inhibited by 32% by 1 μM RX-3117, which was similar to the percent inhibition by 5 μM of the reference compound 5-aza-2'-deoxycytidine (DAC, 31%).

[0244] In A549 cells, 5 μM RX-3117 reduced global DNA methylation (detected by an antibody against 5-methylcytosine) by 25% after 48 hours of exposure, whereas 5 μM DAC inhibited it by only 9% (Figure 27A). Protein expression and activity of several genes known to be affected by methylation were assessed. A549 cells were exposed to RX-3117 and measured using immunofluorescence with an antibody against 5-methylcytosine (Figure 27B). In A549 and SW1573 cells, 24 hours of exposure to 5 μM RX-3117 increased the expression of the cell cycle protein p16INK4A and the DNA repair enzyme MGMT. Figure 27C shows the expression of MGMT, E-cadherin, and p16INK4 after exposure to RX-3117 and Aza-dC.

[0245] For PCFT, the functional activity of RX-3117 was evaluated in CCRF-CEM leukemia cells, which have a highly methylated PCFT promoter, and in CEM-MTX cells, which are deficient in the reduced folate carrier (RFC). PCFT is a specific folate transporter responsible for the uptake of folic acid and the folate analogs methotrexate (MTX) and pemetrexed. Incubation of both CEM and CEM-MTX cells with either 29.6 μM RX-3117 or DAC as a positive control significantly increased PCFT-mediated transport of MTX. This was more pronounced in CEM-MTX cells, with a 10- to 11-fold increase for both RX-3117 and DAC compared to a 4-fold increase in CEM cells. To inhibit RFC-mediated MTX transport, folic acid (FA) was added to inhibit PCFT and L-LV. Aza-CdR and Aza-CR were included as positive controls (Figure 28A, B, and C). conclusion

[0246] In conclusion, RX-3117 down-regulates DNMT1 protein and RNA expression by reducing DNA methylation. RX-3117-mediated hypomethylation increases the expression of MGMT, E-cadherin, PCFT, and the tumor suppressor gene p16INK4A. PCFT mediated MTX transport. These data demonstrate DNMT1 inhibition as a novel mechanism of action for RX-3117. RX-3117 is a novel epigenetic regulator. Example 8 Evaluation of UCK2 protein expression as a potential predictive biomarker for RX-3117 background

[0247] RX-3117, a novel, orally bioavailable nucleoside analog, is a prodrug that is activated intracellularly by uridine cytidine kinase 2 (UCK2), which is thought to be predominantly expressed in tumor tissue. RX-3117 is currently being evaluated in a Phase Ib / IIa multicenter, open-label clinical study in patients with advanced pancreatic and bladder cancer. The study also investigated the relationship between UCK2 tissue protein expression and RX-3117 efficacy in a mouse xenograft model, as well as UCK2 protein expression in a panel of human cancer tissues compared with normal tissues. method

[0248] UCK2 protein expression in tumor tissues was analyzed by immunoblotting using the clone 22-1 rabbit monoclonal antibody. A validated procedure for immunohistochemistry (IHC) of UCK2 using clone 22-1 was performed on a panel of human formalin-fixed, paraffin-embedded (FFPE) cancer and normal tissues. result

[0249] Immunoblotting of UCK2 protein levels normalized to beta-actin and the corresponding tumor growth inhibition (at a 500 mg / kg oral RX-3117 dose, TIWK) were 57% and 67% in MiaPaCa2, 30% and -5% in BxPC3, 199% and 92% in Colo-205, 21% and 90% in Caki-1, 2% and 39% in A549, and 146% and 79% in H460, respectively. These data indicate a trend toward UCK2-dependent antitumor efficacy. IHC of UCK2 showed that positive staining for UCK2 in cancer tissues was observed in 20 / 20 bladder cancer tissues (100% frequency), 19 / 20 CRC tissues (95% frequency), 18 / 20 NSCLC tissues (90% frequency), and 19 / 20 pancreatic cancer tissues (95% frequency). The mean H-scores for UCK2 in cancerous versus normal tissues were 104 vs. 9 in lung, 97 vs. 20 in bladder, 67 vs. 41 in pancreas, and 39 vs. 21 in colon, respectively. conclusion

[0250] Current data shows a correlation trend between UCK2 protein expression level in xenograft model and the degree of antitumor activity of RX-3117.The data also support the higher UCK2 protein expression level in human cancer tissues compared with their normal tissues.This suggests that RX-3117 activity may be tumor tissue specific, and quantification of UCK2 expression in human cancer tissues may be useful as a predictive biomarker for selecting patients with RX-3117 sensitivity in future clinical studies. Example 9 Synthesis of RX-3117 monohydrate Preparation of INT14 from ASM11 by sequential reactions of steps 1 to 3 in a fixed reactor

[0251] ASM11, tert-butyl(((3aR,4R,6aR)-5-fluoro-2,2-dimethyl-6-((trityloxy)methyl)-4,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)oxy)diphenylsilane (37.65 kg, 1 wt, 1 eq, 55 mol), was dissolved in 2-methyltetrahydrofuran (4.0 vol, 3.4 wt). TBAF (tetra-n-butylammonium fluoride) 1.0 M in THF (tetrahydrofuran, 1.61 vol, 1.45 wt, 1.1 eq) was added in one portion (mildly exothermic controlled addition) to the reaction vessel over 15 to 45 minutes, maintaining the temperature at 18 to 23°C. 2-Methyltetrahydrofuran (1.0 vol, 0.9 wt) was charged to the vessel as a line rinse, maintaining the temperature at 18 to 23°C. The resulting solution was heated at 18 to 23°C. 1The reaction mixture was stirred for 6 hours until complete by H NMR. The reaction mixture was charged with 8% w / w sodium bicarbonate (3.0 vol) and stirred at 18-23°C for 5-10 minutes (Caution: mild exotherm), the phases were allowed to separate, and the lower aqueous phase (2 x 2.0 vol) was removed. The first 8% w / w sodium hydrogen carbonate extraction yielded a milky aqueous layer, and extended settling time did not remove the emulsion. Examination indicated that the emulsion was trapped in the aqueous layer and had a low organic content, so the process continued. Total separation time for the first extraction was 5 hours 29 minutes. The second 8% w / w sodium bicarbonate extraction took only 52 minutes and separated without issue. The aqueous phase was extracted with 2-methyltetrahydrofuran (2.0 vol, 1.7 wt), and the line was rinsed with 2-methyltetrahydrofuran (2.0 vol, 1.7 wt). The combined organic phase containing INT12 was heated to 40-50°C and concentrated to approximately 4 volumes under reduced pressure at 40-50°C. Samples were taken for analysis and analyzed by Karl Fischer until the water content was 0.2% w / w or less. The process yielded 158.8 kg net weight of INT12 ASM11-alcohol in 2-methyltetrahydrofuran containing 15.3% w / w of INT12 ASM11-alcohol, corresponding to an overall yield of 99.0%. 92.83% area purity of INT12 ASM11-alcohol ((3aS,4R,6aR)-5-fluoro-2,2-dimethyl-6-((trityloxy)methyl)-4,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-ol) was determined by HPLC analysis.

[0252] The INT12 solution was returned to the vessel, cooled to 0-5° C., and charged with triethylamine (0.41 vol, 0.30 wt, 2.0 eq). After rinsing the lines with 2-methyltetrahydrofuran (1.0 vol, 0.9 wt), methanesulfonyl chloride (0.17 vol, 0.25 wt, 1.5 eq) diluted in 2-methyltetrahydrofuran (1.0 vol, 0.9 wt) (carefully mixed in the header vessel) was charged to the solution over at least 30 minutes while maintaining the temperature at 0-5° C. (exothermic). Additional 2-methyltetrahydrofuran (0.5 vol, 0.4 wt) was added as a line rinse while maintaining the temperature at 0-5° C. The contents of the vessel were then cooled to 0° C. after 1 hour. 1 The reaction was stirred at 0-5° C. until complete by H NMR. Representative samples were removed from the reaction vessel after 1 hour and approximately every 2 hours thereafter if necessary, and analyzed to confirm remaining INT12. 1After 100% conversion was confirmed by H NMR analysis, water (4.0 vol) was charged while maintaining the temperature at 0-10°C, and the reaction mixture was warmed to 18-23°C and stirred at 18-23°C for 5-10 minutes. The upper organic phase in the vessel was separated and charged with 8% w / w sodium bicarbonate solution (4.0 vol) while maintaining the temperature at 18-23°C. The resulting biphasic solution was stirred at 18-23°C for 1-2 hours, and the separated organic phase was charged with 20% w / w aqueous ammonium chloride solution (2.0 vol) and 2-methyltetrahydrofuran (2.0 vol, 1.7 wt). The temperature was adjusted to 18-23°C as needed. The 20% w / w ammonium chloride wash resulted in vigorous gas evolution, likely due to reaction with residual sodium bicarbonate from the previous step. After stirring for 5 to 10 minutes at 18 to 23° C., the upper organic phase was separated and charged with purified water (2.0 volumes) adjusted to 18 to 23° C. The separated organic phase containing INT13 was concentrated to approximately 2 volumes under reduced pressure at 35 to 45° C. Sampling was performed for analysis. The process yielded 78.4 kg net weight of INT13 ASM11-mesylate in 2-methyltetrahydrofuran containing 34.9% w / w of INT13 ASM11-mesylate, corresponding to an overall yield of 94.9%. INT13 ASM11-mesylate ((3aR,4R,6aR)-5-fluoro-2,2-dimethyl-6-((trityloxy)methyl)-4,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-yl methanesulfonate) of 62.91% area purity was determined by HPLC analysis, with 32.23% area of ​​TBDPS by-product present.

[0253] The INT13 solution was subsequently charged with DMSO (3.8 vol, 4.2 wt), heated to 40-45° C., and the organic phase was concentrated under reduced pressure below 45° C. until no more solvent (2-methyltetrahydrofuran) was distilled. Concentration was continued for 5 hours and 25 minutes. 1IPC by H NMR indicated a 2-methyltetrahydrofuran content of 8.3% w / w. After cooling the solution to 27-33°C, cesium carbonate (1.2 wt) and cytosine (0.41 wt) were charged. The reaction mixture was heated to 33-37°C and stirred until complete by HPLC. Sampling for N / O-alkylation ratio analysis was performed from the reaction vessel after 24 hours and at appropriate time points thereafter. After 33 hours and 47 minutes, the reaction was deemed complete with an IPC result of 99.5% conversion. The ratio of N- to O-isomers was 99.03:0.97. Upon completion, the mixture was charged with isopropyl acetate (2.0 vol, 1.7 wt) and purified water (4.0 vol, 4.0 wt), maintaining the temperature below 50°C (water addition is exothermic). After stirring for 5 to 15 minutes, the biphasic mixture was allowed to settle for 10 minutes, and then the upper organic phase was separated. The aqueous phase was re-extracted twice, stirred at 40 to 50°C for 5 to 15 minutes, and again allowed to settle for 10 minutes before separation to recover all the product along with isopropyl acetate (2.0 vol, 1.7 wt each). The combined organic phases were cooled to 25 to 30°C, charged with 10% v / v acetic acid (3.0 vol) and 26% w / w brine solution (1.0 vol) while maintaining the temperature at 25 to 30°C, and the biphasic solution was stirred at 25 to 30°C for 30 to 60 minutes. The upper organic phase was washed three times with 10% v / v acetic acid (3.0 vol) and 26% w / w brine solution (1.0 vol) while maintaining the temperature at 25 to 30°C. In each wash step, the upper organic solution was 1 The organic phase was washed again with approximately 3% w / w brine solution (3 x 2.0 vol) at 25-30°C and sampled for H NMR analysis. 1The organic phase containing INT14 was heated to 35-45°C and concentrated to approximately 5 volumes under reduced pressure at 35-45°C. The solution was charged with isopropyl acetate (3.0 volumes, 2.6 weights) and concentrated to approximately 5 volumes under reduced pressure at 35-45°C. The solution was again charged with isopropyl acetate (5.0 volumes, 4.4 weights), adjusted to 57-63°C, stirred at 57-63°C for 1.5-3 hours, and checked for crystallization / precipitation by HPLC. The slurry was cooled to 35-45°C and concentrated to approximately 5 volumes under reduced pressure at 35-45°C. The slurry was further cooled to 18-23°C over 1.0-2.0 hours and charged with n-heptane (7.0 volumes, 4.8 weights) over 30-90 minutes while maintaining the temperature at 18-23°C. After 1-2 hours at 18-23°C and 1-2 hours at 0-5°C, the slurry was filtered through a 20 μM cloth and washed with premixed n-heptane / isopropyl acetate (5:1, 2 x 1.0 vol) at 0-5°C. The product containing INT14 was dried under vacuum up to 55°C. 1 The crude product was assayed by H NMR. Acceptance criteria were ≦2.0% w / w isopropyl acetate and ≦2.0% w / w n-heptane. The process yielded 24.27 kg net weight of INT14, 4-amino-1-((3aS,4S,6aR)-5-fluoro-2,2-dimethyl-6-((trityloxy)methyl)-4,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)pyrimidin-2(1H)-one (45 mol, 93.25% purity), corresponding to an overall yield of 82% and 64% w / w. Preparation of RX-3117 monohydrate from INT14 by sequential reactions from steps 4 to 5 in a fixed reactor

[0254] INT14, 4-amino-1-((3aS,4S,6aR)-5-fluoro-2,2-dimethyl-6-((trityloxy)methyl)-4,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)pyrimidin-2(1H)-one (24.27 kg, 45 mol, 1.0 wt) was charged to the vessel, followed by methanol (7.5 vol, 5.9 wt) and the temperature of the reaction mixture was adjusted to 18-23° C. To the reaction vessel was added 2 M HCl (1.1 vol, 1.2 eq) maintaining the temperature below 50° C. The slurry mixture was heated to 45-55° C. and stirred at 45-55° C. (target 50° C.) for 2-2.5 hours. The vessel volume was noted and the reaction mixture was distilled under reduced pressure while maintaining the temperature at 45-55°C and maintaining a constant volume by the addition of MeOH (5.0 vol, 4.0 wt). The mixture was sampled and MeOH (2.5 vol, 2.0 wt) was added while maintaining a constant volume by distillation at 45-50°C until there was less than 1.0% area of ​​the acetonide intermediate by HPLC. Once conversion was complete, the reaction mixture was cooled to 25-30°C. The reaction mixture was concentrated to 5 volumes under reduced pressure while maintaining the temperature at 35-45°C. The reaction mixture was cooled to 25-30°C. TBME (methyl tert-butyl ether) (5.0 vol, 3.7 wt) and water (5.0 vol) were charged to the reaction vessel while maintaining the temperature at 25-30°C. The biphasic solution was stirred at 25-30°C for 10-20 minutes and the phases were separated at 25-30°C, retaining the lower aqueous phase. The retained lower phase was transferred to a vessel and recharged with TBME (5.0 vol, 3.7 wt) while maintaining the temperature at 25-30° C. The biphasic solution was stirred at 25-30° C. for 10-20 minutes, after which the lower aqueous phase was separated. The lower aqueous phase was returned to the vessel and the lines were rinsed with water for injection (0.5 vol, 0.5 wt). 1Removal of trityl alcohol was confirmed by H NMR assay, which showed 0.3% w / w trityl alcohol content. If the assay result was not 0.5% w / w or less trityl alcohol, the aqueous phase was charged with TBME (5.0 vol, 3.7 wt) and stirred at 25-30°C for 10-20 minutes, then the separation was repeated. The combined aqueous solution was adjusted to 18-23°C, pretreated AmberSep 900 (OH form) resin (5 / 6 of the bulk processed material) was charged, stirred for 15 minutes, and the pH was checked. If the pH was less than 8.0, additional AmberSep 900 resin (OH form) was added, and the solution was stirred for 30-45 minutes at 18-23°C. The slurry was filtered and washed with water for injection (2 x 4.0 vol), for 15-30 minutes per wash. The resin filter cake on the filter was further washed with water for injection (3 x 4.0 vol) for 15 to 30 minutes per wash until each wash yielded a result of 1.0% or less by HPLC assay. The mother liquor and any resulting washes containing 1.0% or more were clarified through a 1 μm filter. The solution was heated to 40-45°C and concentrated to 1.5 volumes under reduced pressure at 40-45°C. After the aqueous solution was cooled to 18-23°C over 2-3 hours, the mixture was aged for 60 minutes and then charged with acetonitrile (9.5 vol) at a near-constant rate over 1.5-2 hours, maintaining the temperature at 18-23°C. The slurry was aged for 2 hours at 18-23°C and cooled to 0-5°C over 90 minutes. The solid was filtered through a 20 μm cloth, washed with MeCN / water (5:1, 1.5 vol), and dried under air until the MeCN content was less than 400 ppm by GC. The process yielded 8.20 kg (99.83% purity) net weight of RX-3117 monohydrate, 4-amino-1-((1S,4R,5S)-2-fluoro-4,5-dihydroxy-3-(hydroxymethyl)cyclopent-2-en-1-yl)pyrimidin-2(1H)-one 1H2O (29.8 mol, 99.83% purity), corresponding to an overall yield of 66% and 34% w / w.

[0255] FIG. 29 shows RX-3117 made using the process described in Example 9. 1 H NMR. 1 H- NMR (400 MHz, DMSOd6), δ 7.40ppm, (d, J=7.3Hz, 1H) CH cytosine, δ 7.20ppm, (broad d, J=9.1Hz, 2H) NH2, δ 5.74ppm, (d, J-7.3Hz, 1H) CH cytosine, δ 5.30ppm, Wide s, 1H, CH, δ 5.15ppm, (d, J=7.1Hz, 1H) (OH), δ 5.00ppm, (d, J-6.1Hz, 1H) (OH), δ 4.80ppm, (q, J=5.3Hz, 1H)(OH), δ 4.48ppm, (q, J=5.3Hz, 1H) CH, δ 4.17ppm, (dd, J=9.1Hz, 3.8Hz, 1H) CH, δ 4.13ppm, (dt, J=6.1Hz, 5.8Hz, 1H) CH, δ 3.91ppm, (wide width d, J=12.9Hz, 2.8Hz, 1H) CH .

[0256] Figure 30 shows the results of RX-3117 made using the process described in Example 9. 13 C NMR.

[0257] Figure 31 shows the results of RX-3117 made using the process described in Example 9. 19 F NMR.

[0258] Figure 32 is a mass spectrum of RX-3117 made using the process described in Example 9. The mass spectrum was performed using an ES+ filter showing the protonated species of RX-3117 (M+H) as well as RX-3117 plus sodium adduct (M+sodium) at m / z = 280.0 (a common species seen in this analysis). The sodium is from the analysis and not the manufacturing process.

[0259] Figure 33 is a mass spectrum of RX-3117 produced using the process described in Example 9. The mass spectrum was performed using an ES- filter, which shows the M-H species of RX-3117 during the analytical process. The ES- and ES+ filter methods together provide the basis for the complete mass spectrum of RX-3117.

[0260] To verify the crystalline nature of the material prepared according to the large-scale synthesis process described above, microscopic comparisons were made with crystals prepared by a high-purity laboratory-scale process, as well as comparisons of X-ray powder diffraction patterns. Figure 34 shows a microscopic comparison of RX-3117 made according to the process of Example 9 (top row) and prepared using the laboratory-scale process (bottom row) under plane polarized light (left column) and cross-polarized light (right column). Figure 35 shows X-ray powder diffraction data comparing RX-3117 made using the laboratory-scale process (top spectrum) with RX-3117 made using the process described in Example 9 (bottom spectrum). As can be seen, there are no significant differences in the crystal structure.

[0261] It will be apparent to those skilled in the art that specific embodiments of the disclosed subject matter may be directed to one or more of the embodiments set forth above and below in any combination.

[0262] Although the present invention has been disclosed in some detail by way of illustration and example, it will be apparent to those skilled in the art that changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. Accordingly, the descriptions and examples should not be construed as limiting the scope of the invention.

[0263] All references, publications, patents, and patent applications disclosed herein are hereby incorporated by reference in their entirety. The present invention provides, for example, the following items. (Item 1) A compound of formula (I) for use in the treatment of tumors, comprising: [ka] or a hydrate, solvate, or pharmaceutically acceptable salt thereof, is administered to a subject in need thereof in a dosage of about 300 to 2,000 mg / day. (Item 2) Item 1, wherein the dosage is about 500 to 700 mg / day. (Item 3) 3. The use according to item 1 or 2, wherein the dosage is about 6 to 12 mg / kg / day. (Item 4) 4. The use according to any one of items 1 to 3, wherein the oral dosage form is administered 5 to 7 days per week. (Item 5) 5. The use of items 1 to 4, wherein the oral dosage form is administered 5 to 7 days per week for 4 consecutive weeks, or 5 to 7 days per week for 3 consecutive weeks, followed by a 1-week washout period during which the oral dosage form is not administered. (Item 6) 6. The use of item 5, wherein a dosing cycle consists of either 3 consecutive weeks of treatment followed by 1 week of rest, or 4 consecutive weeks of treatment, and wherein the oral dosage form is administered for up to 12 dosing cycles. (Item 7) 7. The use of any one of items 1 to 6, wherein the oral dosage form provides a Cmax of about 700 to 1,100 ng / mL after a single administration. (Item 8) the oral dosage form has an AUC of about 8,000 to 10,000 h·ng / mL after a single dose 0~t The use according to any one of items 1 to 7, resulting in a serotonin-dependent increase in the serotonin receptor agonist activity (0 to 24 hours). (Item 9) 9. The use according to any one of items 1 to 8, wherein the tumor is pancreatic, bladder or colorectal cancer. (Item 10) 10. The use of any one of items 1 to 9, further comprising administering radiation or an anti-tumor agent to the subject. (Item 11) 11. The use of any one of items 1 to 10, further comprising administering an anti-tumor agent selected from the group consisting of antimetabolites, DNA fragmenting agents, DNA cross-linking agents, intercalating agents, protein synthesis inhibitors, topoisomerase I poisons, topoisomerase II poisons, microtubule-directing agents, kinase inhibitors, polyphenols, hormones, hormone antagonists, death receptor agonists, immune checkpoint inhibitors, anti-programmed death 1 (PD-1) receptor antibodies, and anti-programmed death-ligand 1 (PD-L1) antibodies. (Item 12) 11. The use of any one of items 1 to 10, further comprising administering to the subject a PD-L1 antibody. (Item 13) 11. The use of any one of items 1 to 10, further comprising administering to the subject a PD-1 antibody. (Item 14) 14. The use according to any one of items 1 to 13, wherein the oral dosage form is a solid. (Item 15) 14. The use according to any one of items 1 to 13, wherein the oral dosage form is a tablet. (Item 16) 14. The use according to any one of items 1 to 13, wherein the oral dosage form is a capsule. (Item 17) 14. The use according to any one of items 1 to 13, wherein the subject is a human. (Item 18) a compound of formula (I) in a subject in need of treatment [ka] 1. A method for predicting the efficacy of treatment with a compound of formula (I) or a hydrate, solvate, or pharmaceutically acceptable salt thereof, comprising: (i) collecting a sample of tumor cells or tissue from said subject; (ii) measuring the level of UCK2 expression in the tumor cells or tissue; wherein the expression level of UCK2 indicates the likelihood of efficacy of treatment with the compound of formula (I). (Item 19) A method of sensitizing a cell to an apoptotic signal, comprising treating the cell with an effective amount of a compound of formula (I) [ka] or a hydrate, solvate or pharmaceutically acceptable salt thereof. (Item 20) A compound of formula (I) for use in the treatment of one or more symptoms of cancer [ka] The compound of formula (I), or a hydrate, solvate, or pharmaceutically acceptable salt thereof, is administered in an amount effective to inhibit methyltransferase and upregulate at least one hypomethylated target in a subject. (Item 21) A process for the preparation of 4-amino-1-((1S,4R,5S)-2-fluoro-4,5-dihydroxy-3-(hydroxymethyl)cyclopent-2-en-1-yl)pyrimidin-2(1H)-one 1H2O (RX-3117-MH), comprising the step of reacting tert-butyl(((3aR,4R,6aR)-5-fluoro-2,2-dimethyl-6-((trityloxy)methyl)pyrimidin-2(1H)-one with tert-butyl(((3aR,4R,6aR)-5-fluoro-2,2-dimethyl-6-((trityloxy)methyl)pyrimidin-2(1H)-one) in a continuous process involving more than one step without isolating any intermediate. 1. A process comprising the step of converting 4-amino-1-((3aS,4S,6aR)-5-fluoro-2,2-dimethyl-6-((trityloxy)methyl)-4,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)oxy)diphenylsilane (ASM11) into 4-amino-1-((3aS,4S,6aR)-5-fluoro-2,2-dimethyl-6-((trityloxy)methyl)-4,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)pyrimidin-2(1H)-one (INT14). (Item 22) dissolving tert-butyl(((3aR,4R,6aR)-5-fluoro-2,2-dimethyl-6-((trityloxy)methyl)-4,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)oxy)diphenylsilane (ASM11) in 2-methyltetrahydrofuran; adding tetra-n-butylammonium fluoride to form ((3aS,4R,6aR)-5-fluoro-2,2-dimethyl-6-((trityloxy)methyl)-4,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-ol) (INT12) in the reaction solution; recovering INT12 in the organic phase; 22. The process of claim 21, further comprising: (Item 23) recovering INT12 in the organic phase washing the reaction solution with an aqueous solution; Separating the aqueous extract from the organic phase having INT12; washing the aqueous extract with 2-methyltetrahydrofuran to extract INT12 from the aqueous extract; combining the extracted INT12 with the organic phase containing INT12; 23. The process according to item 22, comprising: (Item 24) adding triethylamine and methanesulfonyl chloride in 2-methyltetrahydrofuran to the INT12 in the organic phase to form ((3aR,4R,6aR)-5-fluoro-2,2-dimethyl-6-((trityloxy)methyl)-4,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-yl methanesulfonate) (INT13) in a second reaction solution; recovering INT13 in DMSO; 23. The process of claim 22, further comprising: (Item 25) recovering INT13 in DMSO adding DMSO to the second reaction solution with INT13; removing at least 90% w / w of the 2-methyltetrahydrofuran by distillation; 25. The process according to item 24, comprising: (Item 26) adding 2.5 equivalents of cesium carbonate and cytosine to the INT13 in DMSO to form 4-amino-1-((3aS,4S,6aR)-5-fluoro-2,2-dimethyl-6-((trityloxy)methyl)-4,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)pyrimidin-2(1H)-one (INT14) in a third reaction solution. 25. The process of claim 24, further comprising: (Item 27) maintaining the reaction temperature at about 33 to 37°C. 27. The process of claim 26, further comprising: (Item 28) 27. The process of claim 26, wherein the INT14 has a ratio of N- to O-isomers of greater than about 95:5. (Item 29) adding an acid to the third reaction solution with INT14 to form 4-amino-1-((1S,4R,5S)-2-fluoro-4,5-dihydroxy-3-(hydroxymethyl)cyclopent-2-en-1-yl)pyrimidin-2(1H)-one (RX-3117); washing the RX-3117 with methyl tert-butyl ether and water to form an organic phase and an aqueous phase having RX-3117; purifying the RX-3117 to form RX-3117-MH; 27. The process of claim 26, further comprising: (Item 30) prior to said washing step, charging the reaction mixture with methanol and distilling said reaction mixture to remove said acetonide until the area of ​​said acetonide detected is less than about 1.0%. 30. The process of item 29, further comprising: (Item 31) The washing step comprises: separating an aqueous phase having RX-3117 from the organic phase; washing the aqueous phase having RX-3117 with methyl tert-butyl ether until trityl alcohol is detected in the aqueous phase at less than about 0.5% w / w; and adding a basic anion resin to the aqueous phase having RX-3117 to form a slurry. filtering the slurry to retain the mother liquor; concentrating the mother liquor to form a concentrate; adding acetonitrile to the concentrate to form purified RX-3117-MH; 30. The process of item 29, further comprising: (Item 32) 1. A continuous process for preparing 4-amino-1-((3aS,4S,6aR)-5-fluoro-2,2-dimethyl-6-((trityloxy)methyl)-4,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)pyrimidin-2(1H)-one (INT14) from tert-butyl(((3aR,4R,6aR)-5-fluoro-2,2-dimethyl-6-((trityloxy)methyl)-4,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)oxy)diphenylsilane (ASM11), comprising: dissolving the ASM11 in 2-methyltetrahydrofuran; adding tetra-n-butylammonium fluoride to form ((3aS,4R,6aR)-5-fluoro-2,2-dimethyl-6-((trityloxy)methyl)-4,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-ol) (INT12); adding trimethylamine and methanesulfonyl chloride in 2-methyltetrahydrofuran to INT12 to form ((3aR,4R,6aR)-5-fluoro-2,2-dimethyl-6-((trityloxy)methyl)-4,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-yl methanesulfonate) (INT13); adding cesium carbonate and cytosine to INT13 to form 4-amino-1-((3aS,4S,6aR)-5-fluoro-2,2-dimethyl-6-((trityloxy)methyl)-4,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)pyrimidin-2(1H)-one (INT14); Including, A continuous process wherein said steps are carried out in one or more fixed reactors without isolating either INT12 or INT13. (Item 33) 1. A continuous process for preparing 4-amino-1-((1S,4R,5S)-2-fluoro-4,5-dihydroxy-3-(hydroxymethyl)cyclopent-2-en-1-yl)pyrimidin-2(1H)-one 1H2O (RX-3117-MH) from 4-amino-1-((3aS,4S,6aR)-5-fluoro-2,2-dimethyl-6-((trityloxy)methyl)-4,6a-dihydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)pyrimidin-2(1H)-one (INT14), comprising: reacting the INT14 with an acid to form 4-amino-1-((1S,4R,5S)-2-fluoro-4,5-dihydroxy-3-(hydroxymethyl)cyclopent-2-en-1-yl)pyrimidin-2(1H)-one (RX-3117); washing the RX-3117 with methyl tert-butyl ether and water to form an organic phase and an aqueous phase having RX-3117; separating an aqueous phase having RX-3117 from the organic phase; washing the aqueous phase containing RX-3117 with methyl tert-butyl ether until less than about 0.5% w / w trityl alcohol is detected in the aqueous phase; and adding a strong base anion resin to the aqueous phase containing RX-3117 to form a slurry. filtering the slurry to retain the mother liquor; concentrating the mother liquor to form a concentrate; adding acetonitrile to the concentrate to form purified RX-3117-MH; isolating the purified RX-3117-MH. Including, A continuous process wherein said steps are carried out in one or more fixed reactors.

Claims

[Claim 1] The invention as set forth in the drawings.

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