Novel salt of bicyclic heterocyclyl compound and crystalline form thereof

Abstract

The present invention relates to a novel salt of a bicyclic heterocyclyl compound and a crystalline form thereof. The novel salt of a bicyclic heterocyclyl compound is N-((R)-1-(3-(difluoromethyl)-2-fluorophenyl)ethyl)-6-((S)-hexahydropyrazino[2,1-c][1,4]oxazin-8(1H)-yl)cinnolin-4-amine hydrochloride, has excellent photostability, heat / moisture stability, bioavailability, and the like, can be effectively used in pharmaceutical formulation, and can exhibit an excellent effect on the prevention or treatment of cancer.

Description

[0001]

Description of the Invention

[0002]

Title of Invention

[0003] Novel salts of bicyclic heterocyclil compounds and their crystalline forms

[0004]

Technology Field

[0005] The present invention relates to a novel salt of a bicyclic heterocyclile compound and a crystalline form thereof.

[0006] This research was conducted with funding from the Ministry of Science and ICT, the Ministry of Trade, Industry and Energy, and the Ministry of Health and Welfare, through the National New Drug Development Project of the National New Drug Development Project Group (Project No.: RS-2023-00217674).

[0007]

Background Techniques

[0008] The Renin-Angiotensin System (RAS) family of proteins includes KRAS, NRAS, and HRAS, and their mutations exist in cells in either a Guanosine Triphosphate (GTP)-bound or Guanosine Diphosphate (GDP)-bound state. RAS family proteins inherently possess weak GTPase activity and slow nucleotide exchange rates. Binding of guanine nucleotide exchange factors (GEFs), such as Son of Seven Less 1 (SOSl), to RAS proteins promotes the release of GDP from the RAS proteins and enables GTP binding. When in the GTP-bound state, RAS family proteins are activated and interact with downstream effector proteins such as C-RAF and PI3K (Phosphatidyl Inositol-3-kinase). These pathways influence various cellular processes such as proliferation, survival, metabolism, motility, angiogenesis, immunity, and growth. Cancer-associated mutations in RAS family proteins inhibit GAP-induced GTPase activity to increase GTP-bound active RAS family proteins. This leads to the sustained activation of effector pathways downstream of RAS family proteins (e.g., MEK / ERK, PI3K / AKT / mT0R, RalGDS pathways). KRAS (Kirsten Rat Sarcoma 2 Viral Oncogene Homolog) is a small GTPase and one of the Ras family oncogenes. KRAS acts as a molecular switch circulating between inactive (GDP-bound) and active (GTP-bound) states, regulating various processes including cell proliferation by transmitting upstream cellular signals received from various tyrosine kinases to downstream pathways. The role of activated KRAS in malignant tumors was observed 30 years ago.KRAS mutations (e.g., amino acids G12, G13, Q61, A146) are found in various human cancers, including lung, colorectal, and pancreatic cancers. Abnormal expression of KRAS accounts for up to 20% of all cancers. Oncogenic KRAS mutations that stabilize GTP binding and induce activation of KRAS and downstream signaling have been reported in 25–30% of lung adenocarcinomas. Single nucleotide substitutions resulting in missense mutations at codons 12 and 13 of the KRAS primary amino acid sequence account for approximately 40% of lung adenocarcinomas.

[0009] The role of KRAS in malignant tumors is well known, and with the discovery of frequent KRAS mutations (KRAS G12C, KRAS G12D, KRAS G12V) in various tumor types, KRAS has become a highly promising target for anticancer therapy in the pharmaceutical industry. Despite research efforts to develop KRAS inhibitors as cancer treatments, no KRAS inhibitor has demonstrated sufficient efficacy to receive regulatory approval. Consequently, there is still a need to develop alternative approaches to maximize the efficacy and effectiveness of KRAS inhibitors as cancer treatments.

[0010] The SOS (Son of Seven less) protein exists in two isomorphic states, S0S1 and S0S2, but only S0S1 is phosphorylated by Dokke. Growth factor-induced phosphorylation of S0S1 is mostly mediated by Dokke, which phosphorylates at least four serine residues in the C-terminal region of S0S1. This suggests that S0S1 plays an important role in the negative feedback regulation of the KRAS pathway.

[0011] S0S1 is the human homolog of the first discovered Drosophila protein, SOS. S0S1 has two binding sites for RAS family proteins: a catalytic site that binds to GDP-binding RAS family proteins and an allosteric site that binds to GTP-binding RAS family proteins. KRAS proteins exchange GDP for GTP for activation, and S0S1, a GEF, activates KRAS by increasing the conversion to GTP. Therefore, the activity of S0S1 regulates the active state of KRAS.

[0012] Since S0S1 inhibitors significantly reduce pERK activity in cells with various KRAS mutations (KRAS G12C, KRAS G12D, KRAS G12V), S0S1 inhibitors exhibit pan-KRAS inhibitory activity regardless of the KRAS mutation type, and thus are receiving attention as excellent cancer treatments. Meanwhile, in order to be considered as a candidate substance for drug development, a compound must possess not only excellent biological properties but also physical properties that enable its use in the manufacture of pharmaceutical compositions. Furthermore, such a compound must be easy to manufacture and stable to formulate.

[0013] Accordingly, the inventors of the present invention completed the present invention by identifying a novel salt that exhibits unexpected and significant effects, as a result of diligent research efforts to discover a form that is capable of industrial application while exhibiting excellent bioavailability and stability in various aspects, in order to use a bicyclic heterocyclile compound as a S0S1 inhibitor in pharmaceutical formulation.

[0014] Prior art literature

[0015] Patent literature

[0016] (Patent Document 001) Republic of Korea Published Patent No. 10-2024-0049772

Description of the Invention

[0017]

Technical Challenges

[0018] One objective of the present invention is to provide a novel salt of a bicyclic heterocyclyl compound.

[0019] Another objective of the present invention is to provide a crystalline form of a novel salt of a bicyclic heterocyclile compound.

[0020]

Technical Solution

[0021] Novel salts of bicyclic heterocyclile compounds

[0022] A novel salt of a bicyclic heterocyclile compound according to the present invention is N-((R)-1-(3-(difluoromethyl)-2-fluorphenyl)ethyl)-6-((S)-hexahydropyrazino[2,1-c][1,4]oxazine-8(1H)-yl)cinnoline-4-amine hydrochloride salt.

[0023] In one embodiment, in a novel salt of a bicyclic heterocyclile compound according to the present invention, the molar ratio of N-((R)-1-(3-(difluoromethyl)-2-fluorphenyl)ethyl)-6-((S)-hexahydropyrazino[2,1-c][l,4]oxazine-8(1H)-yl)cinnoline-4-amine to hydrochloric acid may be 1:1 to 1:2. For example, the molar ratio may be 1:1 or 1:2.

[0024] In one embodiment, the novel salt of the bicyclic heterocyclile compound according to the present invention may be N-((R)-1-(3-(difluoromethyl)-2-fluorphenyl)ethyl)-6-((S)-hexahydropyrazino[2,1-c][l,4]oxazine-8(1H)-yl)cinnoline-4-amine monohydrochloride or dihydrochloride.

[0025] The N-((R)-1-(3-(difluoromethyl)-2-fluorphenyl)ethyl)-6-((S)-hexahydropyrazino [2,1-c] [l,4]oxazine-8(1H)-yl)cinnoline-4-amine of the present invention is represented by the following chemical formula I. <Chemical Formula I>

[0026]

[0027] The N-((R)-1-(3-(difluoromethyl)-2-fluorphenyl)ethyl)-6-((S)-hexahydropyrazino [2,1-c][1,4]oxazine-8(1H)-yl)cinnoline-4-amine hydrochloride salt of the present invention is represented by the following chemical formula II.

[0028] <Chemical Formula 11>

[0029]

[0030] In the above chemical formula II, n can represent 1 to 2. For example, in the above chemical formula II, n can represent 1 or 2.

[0031] In one embodiment, N-((R)-1-(3-(difluoromethyl)-2-fluorphenyl)ethyl)-6-((S)-hexahydropyrazino[2,1-c][1,4]oxazine-8(1H)-yl)cinnoline-4-amine dihydrochloride according to the present invention is represented by the following chemical formula III.

[0032] <Chemical Formula 111>

[0033]

[0034] In one embodiment, the N-((R)-1-(3-(difluoromethyl)-2-fluorphenyl)ethyl)-6-((S)-hexahydropyrazino[2,1-c][1,4]oxazine-8(1H)-yl)cinnoline-4-amine hydrochloride salt according to the present invention may be anhydrous.

[0035] In one embodiment, the N-((R)-1-(3-(difluoromethyl)-2-fluorphenyl)ethyl)-6-((S)-hexahydropyrazino[2,1-c][1,4]oxazine-8(1H)-yl)cinnoline-4-amine monohydrochloride or dihydrochloride according to the present invention may be anhydrous.

[0036] In one embodiment, N-((R)-1-(3-(difluoromethyl)-2-fluorphenyl)ethyl)-6-((S)-hexahydropyrazino[2,1-c][l,4]oxazine-8(1H)-yl)cinnoline-4-amine hydrochloride salt may be in a crystalline form.

[0037] In one embodiment, the N-((R)-1-(3-(difluoromethyl)-2-fluorphenyl)ethyl)-6-((S)-hexahydropyrazino[2,1-c][1,4]oxazine-8(1H)-yl)cinnoline-4-amine monohydrochloride or dihydrochloride according to the present invention may be in a crystalline form.

[0038] In one embodiment, N-((R)-1-(3-(difluoromethyl)-2-fluorphenyl)ethyl)-6-((S)-hexahydropyrazino[2,1-c][1,4]oxazine-8(1H)-yl)cinnoline-4-amine dihydrochloride according to the present invention may be a crystalline form (crystalline form 1) having a powder X-ray diffraction pattern including diffraction peaks with values ​​of 10.59°, 15.25°, and 21.95°.

[0039] In one embodiment, the powder X-ray diffraction pattern of the crystalline form (crystalline form 1) may further include at least one, at least two, or at least three diffraction peaks selected from the group consisting of diffraction peaks with 20 (0.2°) values ​​of 7.68°, 11.00°, 16.76°, 19.06°, 19.27°, 22.59°, 23.86°, and 28.67°.

[0040] In one embodiment, N-((R)-1-(3-(difluoromethyl)-2-fluorphenyl)ethyl)-6-((S)-hexahydropyrazino[2,1-c][1,4]oxazine-8(1H)-yl)cinnoline-4-amine dihydrochloride according to the present invention has 20 (0.2°) values ​​of 5.34°, 7.68°, 10.59°, 11.00°, 12.21°, 15.25°, 15.48°, 15.82°, 16.76°, 19.06°, 19.27°, 20.53°, 21.95°, and 22.59° It may be a crystalline form (crystalline form 1) having a powder X-ray diffraction pattern including at least 3, at least 4, at least 5, or at least 6 selected from the group of diffraction peaks at , 23.86°, 28.04°, and 28.67°.

[0041] In one embodiment, crystalline form 1 according to the present invention may have a thermal gravitational analysis (TGA) profile in which decomposition occurs at 147.36 °C, 261.99 °C, and 436.50 °C. At this time, the heating rate may be 10 °C / min.

[0042] In one embodiment, N-((R)-1-(3-(difluoromethyl)-2-fluorphenyl)ethyl)-6-((S)-hexahydropyrazino[2,1-c][1,4]oxazine-8(1H)-yl)cinnoline-4-amine monochloride according to the present invention may be a crystalline form (crystalline form 2) having a powder X-ray diffraction pattern comprising at least three peaks selected from the group consisting of diffraction peaks with values ​​of 9.97°, 13.65°, 19.02°, 20.70°, and 24.19°.

[0043] In one embodiment, N-((R)-1-(3-(difluoromethyl)-2-fluorphenyl)ethyl)-6-((S)-hexahydropyrazino[2,1-c][1,4]oxazine-8(1H)-yl)cinnoline-4-amine monohydrochloride according to the present invention may be a crystalline form (crystalline form 2) having a powder X-ray diffraction pattern including diffraction peaks with values ​​of 9.97°, 13.65°, 19.02°, 20.70°, and 24.19°. In one embodiment, the powder X-ray diffraction pattern of the crystalline form (crystalline form 2) may further include at least one, at least two, or at least three selected from the group consisting of diffraction peaks with 20 (0.2°) values ​​of 7.22°, 16.52°, 16.99°, 21.23°, and 22.97°.

[0044] In one embodiment, N-((R)-1-(3-(difluoromethyl)-2-fluorphenyl)ethyl)-6-((S)-hexahydropyrazino[2,1-c][1,4]oxazine-8(1H)-yl)cinnoline-4-amine monohydrochloride according to the present invention has 20 (0.2°) values ​​of 7.22°, 9.53°, 9.97°, 10.38°, 13.65°, 13.99°, 15.05°, 16.10°, 16.52°, 16.99°, 19.02°, 19.95°, 20.70°, and 21.23°. It may be a crystalline form (crystalline form 2) having a powder X-ray diffraction pattern including at least 3, at least 4, at least 5, or at least 6 selected from the group of diffraction peaks at 22.97°, 24.19°, 24.62°, and 26.14°.

[0045] In one embodiment, the crystal form 2 according to the present invention may have a differential scanning calorimetry (DSC) endothermic peak at 292.10 °C (3 °C) when the heating rate is 5 °C / min.

[0046] In one embodiment, the crystalline form 2 according to the present invention may have a thermal weight analysis (TGA) profile in which decomposition occurs at 275.57 °C to 533.16 °C. At this time, the heating rate may be 10 °C / min.

[0047] In one embodiment, the N-((R)-1-(3-(difluoromethyl)-2-fluorphenyl)ethyl)-6-((S)-hexahydropyrazino[2,1-c][1,4]oxazine-8(1H)-yl)cinnoline-4-amine hydrochloride salt according to the present invention may be in an anhydrous crystalline form.

[0048] In one embodiment, the N-((R)-1-(3-(difluoromethyl)-2-fluorphenyl)ethyl)-6-((S)-hexahydropyrazino[2,1-c][1,4]oxazine-8(1H)-yl)cinnoline-4-amine monohydrochloride or dihydrochloride according to the present invention may be in an anhydrous crystalline form. The N-((R)-1-(3-(difluoromethyl)-2-fluorphenyl)ethyl)-6-((S)-hexahydropyrazino[2,1-c][1,4]oxazine-8(1H)-yl)cinnoline-4-amine hydrochloride according to the present invention exhibits excellent bioavailability, thereby demonstrating excellent effects in the prevention or treatment of cancer.

[0049] In addition, the N-((R)-1-(3-(difluoromethyl)-2-fluorphenyl)ethyl)-6-((S)-hexahydropyrazino [2,1-c][1,4]oxazine-8(1H)-yl)cinnoline-4-amine hydrochloride salt according to the present invention can be easily prepared by a simple process and obtained with high purity and high yield. In addition, the N-((R)-1-(3-(difluoromethyl)-2-fluorphenyl)ethyl)-6-((S)-hexahydropyrazino [2,1-c][1,4]oxazine-8(1H)-yl)cinnoline-4-amine hydrochloride salt according to the present invention is in crystalline form and has excellent photostability, thermal / moisture stability, and bioavailability, and can be usefully utilized in the formulation of pharmaceuticals.

[0050] N-((R)-1-(3-(difluoromethyl)-2-fluorphenyl)ethyl)-6-((S)-hexahydropyrazino [2,1-c][1,4]oxazine-8(1H)-yl)cinnoline-4-amine monohydrochloride or dihydrochloride according to the present invention exhibits excellent bioavailability, thereby demonstrating excellent effects in the prevention or treatment of cancer, can be easily manufactured through a simple process, can be obtained with high purity and high yield, and as a crystalline form, has excellent light stability, thermal / moisture stability, and bioavailability, and can be usefully utilized in the formulation of pharmaceuticals.

[0051] Method for manufacturing a new salt

[0052] A method for preparing N-((R)-1-(3-(difluoromethyl)-2-fluorphenyl)ethyl)-6-((S)-hexahydropyrazino[2,1-c][l,4]oxazine-8(1H)-yl)cinnoline-4-amine hydrochloride salt according to the present invention comprises the step of preparing a hydrochloride salt by reacting N-((R)-1-(3-(difluoromethyl)-2-fluorphenyl)ethyl)-6-((S)-hexahydropyrazino[2,1-c][l,4]oxazine-8(1H)-yl)cinnoline-4-amine (hereinafter referred to as compound of formula I) with hydrochloric acid. At this time, the hydrochloride salt may be a monohydrochloride or a dihydrochloride.

[0053] In the step of preparing the above hydrochloric acid salt, the compound of Formula I and hydrochloric acid can be reacted under a solvent containing any one selected from acetone, 1-propanol, and ethyl acetate.

[0054] In one embodiment, when reacting a compound of formula I with hydrochloric acid under conditions of acetone alone, an aqueous hydrochloric acid solution or a solution containing hydrochloric acid in a diethyl ether may be used.

[0055] In one embodiment, the step of preparing the hydrochloric acid salt may include a crystallization step.

[0056] In one embodiment, the crystallization step may be performed at 0 to 5°C, 20 to 25°C, or 40 to 50°C, for example, 40 to 45°C or 40 to 50°C.

[0057] In one embodiment, the step of preparing the hydrochloric acid salt may include a cooling step and / or a drying step performed after the crystallization step.

[0058] In one embodiment, the step of preparing the hydrochloric acid salt may include reacting a compound of formula I with hydrochloric acid at 0 to 5°C, 20 to 25°C, or 40 to 50°C, cooling the solid obtained by cooling at 0 to 5°C or 20 to 25°C, and then vacuum drying or room temperature drying at 40 to 60°C.

[0059] In one embodiment, the step of preparing the hydrochloric acid salt may include reacting a compound of formula I with hydrochloric acid at 40 to 50°C to vacuum dry a solid obtained at 20 to 25°C at 40 to 60°C, or reacting at 0 to 5°C to vacuum dry a solid obtained at the same temperature at room temperature.

[0060] In one embodiment, the hydrochloric acid salt obtained in the step of manufacturing the hydrochloric acid salt may be anhydrous.

[0061] In one embodiment, the hydrochloric acid salt obtained in the step of preparing the hydrochloric acid salt may be in a crystalline form. In this case, the crystalline form may be crystalline form 1 or crystalline form 2, and each of crystalline forms 1 and 2 may be substantially the same as described above.

[0062] In one embodiment, the hydrochloric acid salt obtained in the step of preparing the hydrochloric acid salt may be in an anhydrous crystalline form. In this case, the anhydrous crystalline form may be an anhydrous form of crystalline form 1 or an anhydrous form of crystalline form 2, and each of crystalline forms 1 and 2 may be substantially the same as described above.

[0063] In one embodiment, the hydrochloric acid salt obtained in the step of preparing the hydrochloric acid salt may be an anhydrous substance containing a compound of formula I and hydrochloric acid in a molar ratio of 1:1 to 1:2. In one embodiment, the hydrochloric acid salt obtained in the step of preparing the hydrochloric acid salt may be an anhydrous substance containing a compound of formula I and hydrochloric acid in a molar ratio of 1:1 or 1:2.

[0064] In one embodiment, the hydrochloric acid salt obtained in the step of preparing the hydrochloric acid salt may be in a crystalline form containing a compound of formula I and hydrochloric acid in a molar ratio of 1:2. At this time, the crystalline form may be crystalline form 1 as described above.

[0065] In one embodiment, the hydrochloric acid salt obtained in the step of preparing the hydrochloric acid salt may be an anhydrous crystalline form containing a compound of formula I and hydrochloric acid in a molar ratio of 1:2. At this time, the anhydrous crystalline form may be the anhydrous form of crystal form 1 described above.

[0066] The pharmaceutical composition of the present invention may contain N-((R)-1-(3-(difluoromethyl)-2-fluorphenyl)ethyl)-6-((S)-hexahydropyrazino [2,1-c][1,4]oxazine-8(1H)-yl)cinnoline-4-amine hydrochloride in a therapeutically effective amount. The hydrochloride may be a monohydrochloride or a dihydrochloride, may be anhydrous, or may be in a crystalline form. For example, the pharmaceutical composition may contain an anhydrous crystalline form of the hydrochloride in a therapeutically effective amount. Accordingly, the pharmaceutical composition according to the present invention may exhibit excellent effects in the prevention or treatment of cancer.

[0067] In addition, the present invention includes the following items (1) to (18) regarding novel salts of bicyclic heterocyclile compounds and their crystalline forms.

[0068] In the present invention, the term “about” means having a value that falls within the allowable standard error of the average as considered by a person skilled in the art.

[0069] (1) The present invention provides a novel salt of a bicyclic heterocyclile compound, N-((R)-1-(3-(difluoromethyl)-2-fluorphenyl)-ethyl)-6-((S)-hexahydropyrazino[2,1-c][1,4]oxazine-8(1H)-yl)cinnoline-4-amine hydrochloride.

[0070] (2) In the above (1), the molar ratio of N-((R)-1-(3-(difluoromethyl)-2-fluorphenyl)ethyl)-6-((S)-hexahydropyrazino [2,1-c][1,4]oxazine-8(1H)-yl)cinnoline-4-amine to hydrochloric acid may be about 1:1 to about 1:2.

[0071] (3) In the above (1) or (2), the hydrochloric acid salt may be a monochloride or a dichloride.

[0072] (4) In any one of (1) to (3) above, the monohydrochloride or dihydrochloride may be anhydrous.

[0073] (5) In any one of (1) to (4) above, the hydrochloric acid salt is a monochloric acid salt and may be a crystalline form having a powder X-ray diffraction pattern including at least three selected from the group of diffraction peaks with 20 (0.2°) values ​​of 9.97°, 13.65°, 19.02°, 20.70° and 24.19°.

[0074] (6) In any one of (1) to (5) above, the powder X-ray diffraction pattern of the hydrochloric acid salt may further include at least one selected from the group consisting of diffraction peaks with 20 (0.2°) values ​​of 7.22°, 16.52°, 16.99°, 21.23° and 22.97°.

[0075] (7) In any one of (1) to (6) above, the hydrochloric acid salt is a monochloric acid salt and may be a crystalline form having a powder X-ray diffraction pattern comprising at least three selected from the group of diffraction peaks with 20 (0.2°) values ​​of 7.22°, 9.53°, 9.97°, 10.38°, 13.65°, 13.99°, 15.05°, 16.10°, 16.52°, 16.99°, 19.02°, 19.95°, 20.70°, 21.23°, 22.97°, 24.19°, 24.62°, and 26.14°.

[0076] (8) In any one of (1) to (4) above, the hydrochloric acid salt is a dihydrochloride and may be in a crystalline form having a powder X-ray diffraction pattern including diffraction peaks with values ​​of 10.59°, 15.25° and 21.95°.

[0077] (9) In any one of (1) to (4) and (8), the powder X-ray diffraction pattern of the hydrochloric acid salt may further include at least one selected from the group consisting of diffraction peaks with 20 (0.2°) values ​​of 7.68°, 11.00°, 16.76°, 19.06°, 19.27°, 22.59°, 23.86° and 28.67°.

[0078] (10) In any one of (1) to (4), (8) and (9), the hydrochloride salt is a dihydrochloride and may be a crystalline form having a powder X-ray diffraction pattern including at least three diffraction peaks selected from the group consisting of diffraction peaks with values ​​of 5.34°, 7.68°, 10.59°, 11.00°, 12.21°, 15.25°, 15.48°, 15.82°, 16.76°, 19.06°, 19.27°, 20.53°, 21.95°, 22.59°, 23.86°, 28.04° and 28.67°.

[0079] (11) In any one of (1) to (10) above, the hydrochloric acid salt may be anhydrous.

[0080] (12) In any one of (1) to (4) and (8) to (10) above, the hydrochloride may have a thermal gravitational analysis (TGA) profile in which decomposition occurs at about 147.36°C, about 261.99°C, and about 436.50°C. At this time, the heating rate may be 10°C / min.

[0081] (13) In any one of (1) to (7) above, the hydrochloric acid salt may have a differential scanning calorimetry (DSC) endothermic peak at 292.10 °C (3 °C) when the heating rate is 5 °C / min.

[0082] (14) In any one of (1) to (7) and (13) above, the hydrochloric acid salt may have a thermal gravitational analysis (TGA) profile in which decomposition occurs at about 275.57 °C to about 533.16 °C. At this time, the heating rate may be 10 °C / min.

[0083] (15) The present invention provides N- ((R)- 1-(3- (difluoromethyl)- 2-fluorphenyl) ethyl)- 6- ((S)- hexahydropyrazino [2,1- c] [l,4]oxazine- 8(1H)-yl) cinnoline- 4-amine monohydrochloride anhydride.

[0084] (16) In the above (15), the monohydrochloride anhydride is a crystal having a powder X-ray diffraction pattern comprising at least three selected from the group of diffraction peaks having 20 (±0.2°) values ​​of about 7.22°, about 9.53°, about 9.97°, about 10.38°, about 13.65°, about 13.99°, about 15.05°, 16.10°, about 16.52°, about 16.99°, about 19.02°, about 19.95°, about 20.70°, about 21.23°, about 22.97°, about 24.19°, about 24.62° and about 26.14°. It could be a brother.

[0085] (17) The present invention provides N- ((R)- 1-(3- (difluoromethyl)- 2-fluorphenyl) ethyl)- 6- ((S)- hexahydropyrazino [2,1- c] [1,4]oxazine- 8(1H)-yl) cinnoline- 4-amine dichloride anhydride.

[0086] (18) In the above (17), the dihydrochloride may be in a crystalline form having a powder X-ray diffraction pattern comprising at least three selected from the group of diffraction peaks with 20 (0.2°) values ​​of about 5.34°, about 7.68°, about 10.59°, about 11.00°, about 12.21°, about 15.25°, about 15.48°, 15.82°, about 16.76°, about 19.06°, about 19.27°, about 20.53°, about 21.95°, about 22.59°, about 23.86°, about 28.04° and about 28.67°.

[0087] In addition, the present invention provides a method for preventing or treating cancer comprising administering N-((R)-1-(3-(difluoromethyl)-2-fluorphenyl)ethyl)-6-((S)-hexahydropyrazino[2,1-c][l,4]oxazine-8(1H)-yl)cinnoline-4-amine hydrochloride. In this case, N-((R)-l-(3-(difluoromethyl)-2-fluorphenyl)ethyl)-6-((S)-hexahydropyrazino[2,1-c][1,4]oxazine-8(1H)-yl)cinnoline-4-amine hydrochloride may be according to any one of (2) to (18).

[0088] The present invention provides a use of N-((R)-1-(3-(difluoromethyl)-2-fluorophenyl)ethyl)-6-((S)-hexahydropyrazino[2,1-c][1,4]oxazine-8(1H)-yl)cinnoline-4-amine hydrochloride salt for the prevention or treatment of cancer. In this case, N-((R)-1-(3-(difluoromethyl)-2-fluorophenyl)ethyl)-6-((S)-hexahydropyrazino[2,1-c][1,4]oxazine-8(1H)-yl)cinnoline-4-amine hydrochloride salt may be according to any one of (2) to (18) above.

[0089] The present invention provides a use of N-((R)-1-(3-(difluoromethyl)-2-fluorphenyl)ethyl)-6-((S)-hexahydropyrazino[2,1-c][1,4]oxazine-8(1H)-yl)cinnoline-4-amine hydrochloride salt for the manufacture of a drug for the prevention or treatment of cancer. In this case, N-((R)-1-(3-(difluoromethyl)-2-fluorphenyl)ethyl)-6-((S)-hexahydropyrazino[2,1-c][1,4]oxazine-8(1H)-yl)cinnoline-4-amine hydrochloride salt may be according to any one of (2) to (18) above.

[0090]

Effects of the Invention

[0091] According to the novel salt of the bicyclic heterocyclile compound of the present invention and its crystal form, a hydrochloric acid salt containing N-((R)-1-(3-(difluoromethyl)-2-fluorphenyl)ethyl)-6-((S)-hexahydropyrazino[2,1-c][l,4]oxazine-8(1H)-yl)cinnoline-4-amine monohydrochloride or dihydrochloride as an S0S1 inhibitor can improve bioavailability. Accordingly, N-((R)-1-(3-(difluoromethyl)-2-fluorphenyl)-ethyl)-6-((S)-hexahydropyrazino[2,1-c][1,4]oxazine-8(1H)-yl)cinnoline-4-amine hydrochloride can also exhibit excellent effects in the prevention or treatment of cancer. In addition, N-((R)-1-(3-(difluoromethyl)-2-fluorphenyl)-ethyl)-6-((S)-hexahydropyrazino[2,1-c][1,4]oxazine-8(1H)-yl)cinnoline-4-amine hydrochloride exhibits excellent stability against light, heat, and / or moisture. Accordingly, it has excellent light stability, heat / moisture stability, and bioavailability, so it can be usefully utilized in the formulation of pharmaceuticals.

[0092]

Brief Description of the Drawing

[0093] FIG. 1 is a diagram showing the XRD analysis results of crystal form 1 according to the present invention. FIG. 2 is a diagram showing the TGA analysis results of crystal form 1 according to the present invention. FIG. 3 to 5 are diagrams showing the XRD analysis results of crystal form 2 according to the present invention.

[0094] FIG. 6 is a diagram showing the TGA analysis results of crystal form 2 according to the present invention. FIG. 7 is a diagram showing the DSC analysis results of crystal form 2 according to the present invention.

[0095]

Form for carrying out the invention

[0096] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as generally understood by those skilled in the art to which the present invention pertains. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and should not be interpreted in an ideal or overly formal sense unless explicitly defined in this application.

[0097] The measurement method used for characteristic evaluation and / or analysis of the sample obtained in the present invention is as follows.

[0098] <Measurement Method>

[0099] 1. Moisture content, i.e., determination / analysis

[0100] The moisture content of the obtained samples was measured using an 870KF Titrino Plus (Metrohm) Karl-Fischer moisture meter.

[0101] 2. HPLC Purity Analysis

[0102] Purity analysis was performed on the obtained samples using the Agi lent 1260 series.

[0103] 3. pH measurement

[0104] The pH of the obtained sample was determined using a 913 pH meter (Metrohm).

[0105] 4. X-ray Powder Diffraction (XPRD)

[0106] The five-line powder diffraction pattern of the obtained sample was measured using a solid-state detector with a D8 Focus (Bruker ASX) in a step size of 0.02° over a diffraction angle range of 2° to 40°.

[0107] 5. Thermal Weight Analysis (TGA)

[0108] Thermal weight analysis was performed using a TGA 8000 (PerkinElmer) by weighing 0.5 mg to 2 mg of a sample into a ceramic crucible at temperatures from 30 °C to 1,000 °C and measuring under conditions of 10 °C / min.

[0109] 6. Differential Scanning Calorimetry (DSC) Analysis

[0110] DSC analysis was performed on the obtained samples using DSC 8000 (PerkinElmer). The samples were evaluated using a linear heating ramp at a heating rate of 5°C / min in the range of 30 °C to 400 °C.

[0111] Preparation Example 1: Chemical Formula I

[0112]

[0113] Synthesis of compounds

[0114] 50 g of 6-bromo-4-chlorocinnoline, 46.6 g of (R)-1-(3-(difluoromethyl)-2-fluorphenyl) ethanamine, and 107 mL of thylamine were added to diisopropyl and stirred at 140°C for 3 hours. The reaction was terminated by adding 1 L of an aqueous NH4Cl1 solution. The product was extracted using 1 L of methyl chloride (MC). This same extraction process was then performed two more times. Subsequently, g of MgS0410 was added to the MC layer, stirred, and filtered. After concentrating the organic layer, 33.4 g of (R)-6-bromo-N-(1-(3-(difluoromethyl)-2-fluorphenyl)ethyl)cinnoline-4-amine was obtained by column chromatography (yield 41.1%, MS (ESI+) m / z 396, 398 (M+H)+).

[0115] Subsequently, 3.2 g of the obtained (R)-6-bromo-N-(1-(3-(difluoromethyl)-2-fluorphenyl)ethyl)cinnoline-4-amine, 0.74 g of Pd2(dba)3, 0.77 g of xphos, and 24 mL of tetrahydrofuran (THF) were added and stirred. 1.38 g of (S)-octahydropyrazino [2,1-c][l,4]oxazine was diluted in 8 mL of THF and added, followed by the addition of 32 mL of Li HMDS (1.0 M in THF). The mixture was stirred at 80 °C for 2 hours. The reaction was terminated using 200 mL of an aqueous NH4Cl solution. The product was extracted using 200 mL of a mixed organic solvent (MC:isopropyl alcohol (IPA) = 3:1). Subsequently, the same extraction process was performed two more times. 40.6 g of MgS0 was added to the organic layer, stirred, and filtered. After concentrating the organic layer, column chromatography yielded 1.48 g of the target compound, N-((R)-1-(3-(difluoromethyl)-2-fluorphenyl)ethyl)-6-((S)-hexahydropyrazino[2,1-c][1,4]oxazine-8(1H)-yl)cinnoline-4-amine (yield 40.1%).

[0116]

[0117] NMR (400 MHz, DMSO—d6) 5 8.10 (s, 1H) , 7.96 (d, J = 9.2 Hz, 1H) , 7.72 (dd, J = 2.4 Hz, 6.8 Hz, 1H) , 7.59 (t , J = 7.2 Hz, 1H) , 7.54 (t, J = 7.2 Hz, 1H) , 7.46 (d, J = 2.4 Hz, 1H) , 7.42 (s, 0.25H) , 7.34 (s, 0.25H) , 7.31 (d, J = 3.2 Hz, 1H) , 7.28 (d, J = 4.0 Hz, 1H) , 7.27 (s, 0.25H) , 7.14 (s, 0.25 H) , 5.25-5.18 (m, 1H) , 4.02 (d, J = 11.6 Hz, 1H) , 3.85-3.79 (m, 3H) , 3.61-3.55 (m, 1H) , 3.25 (t, J = 10.4 Hz, 2H) , 3.00-2.92 (m, 2H) , 2.74 (d, J = 11.2 Hz, 1H) , 2.41- 2.24 (m, 3H) , 1.70 (d, J = 6.8 Hz, 3H);

[0118] MS (ESI+) m / z 458 (M+H) + ,

[0119] Preparation Example 2: Preparation of Example (Chemical Formula I compound hydrochloride salt)

[0120]

[0121] (1) Preparation Example 2-1 - Preparation of Example 1 (Dihydrochloride)

[0122] 200 mL of acetone and 10.0 g of N-((R)-1-(3-(difluoromethyl)-2-fluorphenyl)ethyl)-6-((S)-hexahydropyrazino [2,1-c][1,4]oxazine-8(1H)-yl)cinnoline-4-amine were added to a reactor, and the mixture was heated to 40–45°C. A solution of 4.13 mL of a 35–37% aqueous hydrochloric acid solution diluted with 200 mL of acetone was slowly added dropwise over 30 minutes at the same temperature, stirred for 1 hour, cooled to 20–25°C, and stirred for an additional 1 hour. Subsequently, the resulting solid was filtered and vacuum dried at 40-60 °C to obtain 9.51 g of N-((R)-1-(3-(difluoromethyl)-2-fluorphenyl)ethyl)-6-((S)-hexahydropyrazino[2,1-c][1,4]oxazine-8(1H)-yl)cinnoline-4-amine dichloride anhydride (Form 1) (yield 82%).

[0123] (2) Preparation Example 2-2 - Preparation of Example 2 (Dihydrochloride)

[0124] After adding 9 mL of acetone and 0.62 g of N-((R)-1-(3-(difluoromethyl)-2-fluorphenyl)ethyl)-6-((S)-hexahydropyrazino [2,1-c][1,4]oxazine-8(1H)-yl)cinnoline-4-amine to the reactor, 0.4 mL of a 35-37% aqueous hydrochloric acid solution diluted with 1 mL of acetone was added dropwise to the solution at the same temperature at 0-5°C, and stirred for 1 hour. Subsequently, the resulting solid was filtered and vacuum dried at room temperature to obtain 0.65 g of N-((R)-1-(3-(difluoromethyl)-2-fluorphenyl)ethyl)-6-((S)-hexahydropyrazino[2,1-c][1,4]oxazine-8(1H)-yl)cinnoline-4-amine dihydrochloride anhydride (Form 1) (yield 91%).

[0125] (3) Preparation Examples 2-3 - Preparation of Example 3 (Dihydrochloride)

[0126] 80 mL of acetone and 3.0 g of N-((R)-1-(3-(difluoromethyl)-2-fluorphenyl)ethyl)-6-((S)-hexahydropyrazino [2,1-c][l,4]oxazine-8(1H)-yl)cinnoline-4-amine were added to a reactor, and the mixture was heated to 40–50°C while stirring. A solution diluted by adding 15 mL of acetone to 14.43 mL of 1 M diethyl ether hydrochloride solution (a solution containing 1 M hydrochloric acid in diethyl ether) was slowly added dropwise over 30 minutes and stirred for 1 hour at the same temperature. Subsequently, the mixture was slowly cooled to 0–5°C and stirred for 1 hour before filtration. The filter cake was washed with 12 mL of acetone and vacuum dried at 45°C to obtain 2.88 g of N-((R)-1-(3-(difluoromethyl)-2-fluorophenyl)ethyl)-6-((S)-hexahydropyrazino[2,1-c][1,4]oxazine-8(1H)-yl)cinnoline-4-amine dichloride anhydride (Form 1) (yield 83%). (4) Preparation Examples 2-4 - Preparation of Example 4 (dichloride)

[0127] 16 mL of 1-propanol and 2.0 g of N-((R)-1-(3-(difluoromethyl)-2-fluorphenyl)ethyl)-6-((S)-hexahydropyrazino [2,1-c][1,4]oxazine-8(1H)-yl)cinnoline-4-amine were added to a reactor and heated to 40–45°C. Subsequently, a solution of 1.2 mL of 35–37% aqueous hydrochloric acid diluted with 4 mL of 1-propanol was added dropwise at the same temperature, stirred at the same temperature for 1 hour, cooled to 0–5°C, and stirred for an additional 1 hour. Subsequently, the resulting solid was filtered and vacuum dried at 40-45 °C to obtain 1.43 g of N-((R)-1-(3-(difluoromethyl)-2-fluorphenyl)ethyl)-6-((S)-hexahydropyrazino[2,1-c][1,4]oxazine-8(1H)-yl)cinnoline-4-amine dihydrochloride anhydride (Form 1) (yield 61%).

[0128] (5) Preparation Examples 2-5 - Preparation of Example 5 (Dihydrochloride)

[0129] 60 mL of ethyl acetate and 3.0 g of N-((R)-1-(3-(difluoromethyl)-2-fluorophenyl)ethyl)-6-((S)-hexahydropyrazino[2,1-c][1,4]oxazine-8(1H)-yl)cinnoline-4-amine were added to a reactor, and the mixture was heated to 40–50°C while stirring. A solution diluted by adding 15 mL of ethyl acetate to 14.43 mL of 1 M diethyl ether hydrochloride solution (a solution containing 1 M hydrochloric acid in diethyl ether) was slowly added dropwise over 1 hour and stirred at the same temperature for 1 hour. Subsequently, the mixture was slowly cooled to 20–25°C and stirred for 1 hour before filtration. The filter cake was washed with 12 mL of ethyl acetate and vacuum dried at 45°C to obtain 2.99 g of N-((R)-1-(3-(difluoromethyl)-2-fluorphenyl)ethyl)-6-((S)-hexahydropyrazino[2,1-c][1,4]oxazine-8(1H)-yl)cinnoline-4-amine dichloride anhydride (Form 1) (yield 86%). (6) Preparation Examples 2-6 - Preparation of Example 6 (monochloride)

[0130] After adding 100 mL of acetone and 5.0 g of N-((R)-1-(3-(difluoromethyl)-2-fluorphenyl)ethyl)-6-((S)-hexahydropyrazino [2,1-c][1,4]oxazine-8(1H)-yl)cinnoline-4-amine to a reactor, a solution of 0.985 mL of 35-37% aqueous hydrochloric acid diluted with 2.0 mL of acetone was added dropwise at 20-25 °C for 5 minutes, and stirred at the same temperature for 1 hour. Subsequently, the resulting solid was filtered and vacuum dried at 45°C to obtain 3.93 g of N-((R)-1-(3-(difluoromethyl)-2-fluorphenyl)ethyl)-6-((S)-hexahydropyrazino [2,1-c][1,4]oxazine-8(1H)-yl)cinnoline-4-amine monochloride anhydride (Form 2) (yield 73%).

[0131] Manufacturing Example 3 - Preparation / Manufacturing of Comparative Example

[0132] (1) Comparative Example 1 - Preparation of Free Base

[0133] N-((R)-1-(3-(difluoromethyl)-2-fluorphenyl)ethyl)-6-((S)-hexahydropyrazino[2,1-c][1,4]oxazine-8(1H)-yl)cinnoline-4-amine (free base) obtained in Preparation Example 1 was prepared as Comparative Example 1.

[0134] (2) Comparative Example 2 - Preparation of Igusanic Salt

[0135] 34.8 mL of acetone and 2.0 g of N-((R)-1-(3-(difluoromethyl)-2-fluorphenyl)ethyl)-6-((S)-hexahydropyrazino [2,1-c][1,4]oxazine-8(1H)-yl)cinnoline-4-amine were added to a reactor, and the mixture was heated to 40–50°C while stirring. A solution diluted by adding 8.7 mL of acetone to 1.53 g of citric acid was slowly added dropwise over 30 minutes, and the mixture was stirred at the same temperature for 1 hour. The mixture was slowly cooled to 0–5°C and stirred for 1 hour before being filtered. The filter cake was washed with 7 mL of acetone and vacuum dried at 45°C to obtain 2.59 g of N-((R)-l-(3-(difluoromethyl)-2-fluorphenyl)ethyl)-6-((S)-hexahydropyrazino[2,1-c][1,4]oxazine-8(1H)-yl)cinnoline-4-amine icurate salt (yield 81%).

[0136] (2) Comparative Example 3 - Preparation of Jujube Stone Salt

[0137] 34.8 mL of acetone and 2.0 g of N-((R)-1-(3-(difluoromethyl)-2-fluorphenyl)ethyl)-6-((S)-hexahydropyrazino[2,1-c][1,4]oxazine-8(1H)-yl)cinnoline-4-amine were added to a reactor, and the mixture was heated to 40–50°C while stirring. A solution diluted by adding 8.7 mL of acetone to 1.20 g of L-tartaric acid was slowly added dropwise over 30 minutes, and the mixture was stirred at the same temperature for 1 hour. The mixture was slowly cooled to 0–5°C and stirred for 1 hour before filtration. The filtered cake was washed with 7 mL of acetone and vacuum dried at 45 °C to obtain 2.45 g of N-((R)-1-(3-(difluoromethyl)-2-fluorphenyl)ethyl)-6-((S)-hexahydropyrazino[2,1-c][1,4]oxazine-8(1H)-yl)cinnoline-4-amine citrate (yield 85%).

[0138] Analysis 1 - Moisture Analysis Results (Confirmation of Anhydrous)

[0139] Moisture content analysis was performed on each of the samples according to Examples 1 to 6 of the present invention and the samples according to Comparative Examples 2 and 3. As a result, it was confirmed that all prepared samples were anhydrous.

[0140] Analysis 2 - XRD, TGA, and DSC Measurement Results

[0141] Referring to FIG. 1 and Table 1 below, the XRD pattern and diffraction angle (20, unit: °) for each diffraction peak of crystal form 1 according to the present invention can be seen. In FIG. 1, the x-axis represents the diffraction angle (20), and the y-axis represents the diffraction peak intensity (Lin, unit: counts).

[0142] The XRD pattern of crystal form 1 according to the present invention includes diffraction peaks with diffraction angles (20) of 5.34°, 7.68°, 10.59°, 11.00°, 12.21°, 15.25°, 15.48°, 15.82°, 16.76°, 19.06°, 19.27°, 20.53°, 21.95°, 22.59°, 23.86°, 28.04°, and 28.67°.

[0143] Referring to FIG. 2, as a result of TGA measurement, it is confirmed that the crystal form 1 according to the present invention undergoes decomposition in three stages. In FIG. 2, the x-axis represents temperature (unit: °C), the left side represents y dead weight % (unit: %), and the right side represents y dead derivative weight % (unit: % / min). Specifically, the crystal form 1 according to the present invention undergoes decomposition in the first stage at 147.36 °C, in the second stage at 261.99 °C, and in the third stage at 436.50 °C, and the maximum inflection points of decomposition are confirmed at 247.66 °C, 296.79 °C, and 563.42 °C.

[0144] It was confirmed that the crystalline form 1 according to the present invention is a compound of formula I and hydrochloric acid combined in a molar ratio of 1:2.

[0145] [Table 1]

[0146] Diffraction angle (20, unit: °) Relative intensity

[0147] 5.34 Weakness

[0148] 7.68 Medium

[0149] 10.59 Strength

[0150] 11.00 Intermediate

[0151] 12.21 Weakness

[0152] 15.25 Strength

[0153] 15.48 Weakness

[0154]

[0155] 15.82 Weak 16.76 Medium

[0156] 19.06 Midterm

[0157] 19.27 Midterm

[0158] 20.53 Weakness

[0159] 21.95 Strength

[0160] 22.59 Medium

[0161] 23.86 Medium

[0162] 28.04 Weakness

[0163]

[0164] 28.67 Medium

[0165] Referring to FIGS. 3 to 5 and Table 2 below, the XRD pattern and diffraction angle (20, unit: °) for each diffraction peak of crystal form 2 according to the present invention can be observed. In FIGS. 3 to 5, the x-axis represents the diffraction angle (20), and the y-axis represents the diffraction peak intensity (Lin, unit: counts). Specifically, FIG. 3 shows the XRD pattern at a diffraction angle from 4° to 40°, FIG. 4 shows the diffraction peaks in the range of 4° to 22.5° in FIG. 3, and FIG. 5 shows the diffraction peaks in the range of 22.5° to 40° in FIG. 3.

[0166] The XRD pattern of crystal form 2 according to the present invention includes diffraction peaks with diffraction angles (20) of 7.22°, 9.53°, 9.97°, 10.38°, 13.65°, 13.99°, 15.05°, 16.10°, 16.52°, 16.99°, 19.02°, 19.95°, 20.70°, 21.23°, 22.97°, 24.19°, 24.62°, and 26.14°.

[0167] Referring to FIG. 6, as a result of TGA measurement, it is confirmed that the decomposition of crystalline form 2 according to the present invention occurs in one stage. In FIG. 6, the x-axis represents temperature (unit: °C), the left side represents y-weight% (unit: %), and the right side represents y-derived weight (unit: mg / min). Specifically, the decomposition of crystalline form 2 according to the present invention occurred between 275.57 °C and 533.16 °C, and the maximum inflection point of decomposition was confirmed at 291.22 °C.

[0168] It was confirmed that the crystalline form 2 according to the present invention is formed by combining the compound of formula I and hydrochloric acid in a molar ratio of 1:1.

[0169] Referring to FIG. 7, the crystalline form 2 according to the present invention exhibits a DSC endothermic peak at 292.10°C. In FIG. 7, the x-axis represents temperature (unit: °C), and the y-axis represents heat flow (endothermic) (Normalized heat flow, unit: W / g).

[0170] [Table 2]

[0171] Diffraction angle (20, unit: °) Relative intensity

[0172] July 22 Midterm

[0173] 9.53 Weakness

[0174] 9.97 Strength

[0175] 10.38 Weakness

[0176] 13.65 Strength

[0177] 13.99 Weak

[0178] 15.05 Weak

[0179] 16.10 Weakness

[0180] 16.52 medium

[0181] 16.99 Medium

[0182] 19.02 Strength

[0183] 19.95 Weak

[0184] 20.70 Strength

[0185]

[0186] 21.23 Intermediate 22.97 Intermediate

[0187] 24.19 Strength

[0188] 24.62 Yaha

[0189]

[0190] 26.14 Yaha

[0191] Experimental Example 1: Evaluation of Photostability and Thermal Stability

[0192] As samples, the initial purity was measured for N-((R)-1-(3-(difluoromethyl)-2-fluorphenyl)ethyl)-6-((S)-hexahydropyrazino[2,1-c][1,4]oxazine-8(1H)-yl)cinnoline-4-amine (free base), N-((R)-1-(3-(difluoromethyl)-2-fluorphenyl)ethyl)-6-((S)-hexahydropyrazino[2,1-c][1,4]oxazine-8(1H)-yl)cinnoline-4-amine monohydrochloride and dihydrochloride, respectively.

[0193] As samples, free base, monochloride, and dichloride were each placed thinly in a Petri dish, and then irradiated with visible light of 35 k lux at a total irradiation amount of 1200 k lux using a light stability chamber (CARON 6542-2) at 25 °C and 60% humidity, and then HPLC was performed to measure purity. In addition, as samples, free base, monochloride, and dichloride were each placed thinly in a Petri dish, and then irradiated with ultraviolet light of 35 W at a total irradiation amount of 200 watts using a light stability chamber (CARON 6542-2) at 25 °C and 60% humidity, and then HPLC was performed to measure purity.

[0194] In addition, after measuring the initial purity of each of the free base, monochloride, and dichloride, the purity was measured again after leaving them at 60 °C for one week.

[0195] The results are shown in Table 3.

[0196] [Table 3] Ultraviolet Visible Light Temperature 60°C, 1

[0197] Initial value

[0198] Classification (200 Watt (1.2 million hours / m 2 ) lux hours)

[0199] Purity Purity Purity Purity Free Base 98.38% 98.08% 97.52% 98.38% (Comparison Example 1)

[0200] Monohydrochloride 99.58% 99.56% 99.46% 99.54% Dihydrochloride 97.46% 97.23% 97.35% 97.29%

[0201]

[0202] Referring to Table 3, it can be seen that the purity of the free base decreased by more than 0.3 point% after light was applied compared to the initial purity. In particular, it was confirmed that the purity decreased by more than 0.8 point% under visible light conditions.

[0203] On the other hand, it can be confirmed that the purity of the hydrochloric acid salt according to the present invention is substantially the same before and after the experiment. That is, both the monohydrochloride and dihydrochloride according to the present invention are confirmed to have excellent photostability and excellent thermal stability.

[0204] Experimental Example 2: Moisture Stability Evaluation

[0205] For each of the free base, monochloride, dichloride, dicitric acid, and oligosaccharide samples, the initial purity and moisture content were measured, and then the purity and moisture content were measured after leaving them for one week at a relative humidity (RH) of 80% and a temperature of 60°C. The results are shown in Table 4.

[0206] [Table 4]

[0207] Humidity conditions

[0208] Classification initial value

[0209]

[0210] (RH 80%, 60°C, 1 week) Purity Purity Free Base 1.40% 98.38% 13.28% 98.03% (Comparative Example 1)

[0211] Monohydrochloride 1.66% 99.58% 2.10% 99.52% Dihydrochloride 0.66% 97.46% 3.37% 97.19% Diciphosphate 0.57% 97.09% Viscous liquid 96.48% (Comparative Example 2)

[0212] Iju stone quartz salt 0.50% 97.33% sticky liquid 96.75%

[0213]

[0214] (Comparison Example 3)

[0215] Referring to Table 4, it is confirmed that citrate salts and tartrate salts have very high hygroscopicity and turned into sticky liquids, whereas monohydrochloride salts and dihydrochloride salts according to the present invention show very little change in moisture content. In addition, it is confirmed that free bases do not undergo a phase change like citrate salts and tartrate salts, but show a significant increase in moisture content of 10% or more.

[0216] In addition, it is confirmed that while the hydrochloric acid salt according to the present invention shows no substantial change in purity before and after testing, the purity of the free base changes by more than 0.3 point%, and the purity of the citrate salt and tartrate salt changes by more than 0.5 point%. Accordingly, it can be confirmed that the hydrochloric acid salt according to the present invention has excellent moisture stability.

[0217] Experimental Example 3: Evaluation of Bioavailability - Bioavailability of Free Bases and Dihydrochloride

[0218] The bioavailability of N-((R)-1-(3-(difluoromethyl)-2-fluorphenyl)ethyl)-6-((S)-hexahydropyrazino[2,1-c][l,4]oxazine-8(1H)-yl)cinnoline-4-amine (free base) and the hydrochloride according to the present invention, N-((R)-1-(3-(difluoromethyl)-2-fluorphenyl)ethyl)-6-((S)-hexahydropyrazino[2,1-c][l,4]oxazine-8(1H)-yl)cinnoline-4-amine dihydrochloride was evaluated according to the following method.

[0219] After orally administering free base and dihydrochloride to male SD rats at a dose of 10 mg / kg based on free base, blood samples were collected at 5, 15, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 8 hours, and 24 hours, and plasma was separated. The test substances in the plasma were pretreated by protein precipitation, and quantitative analysis was performed using an LC-MS / MS system. Pharmacokinetic analysis was performed by non-compartmental analysis using PK Solution, and parameters such as time to peak blood concentration (Tmax), peak blood concentration (Cmax), and area under the blood curve (AUCo-t, AUCo-mf) were determined. The results are shown in Table 5.

[0220] [Table 5]

[0221] Classification Tmax(hr) Cmax(ng / ml) AUCo-t(hr.ng / ml) AUCo-inf(hr.ng / ml) Free Base 1.4 14.7 75.5 84.0 (Comparison Example 1)

[0222] Dihydrochloride 2.2 104.9 504.4 581.7

[0223]

[0224] Referring to Table 5, it can be confirmed that the dihydrochloride according to the present invention has a Cmax that is more than 7 times higher than that of a free base, and an AUC that is also more than 6 times superior to that of a free base. In other words, it can be confirmed that the dihydrochloride according to the present invention has significantly superior bioavailability compared to a free base.

[0225] Experimental Example 4: Evaluation of the Inhibitory Activity of Biochemical Interactions between KRAS(G12C, G12D, G12V)-SOS1 and KRAS(G12C)-S0S2 To confirm the inhibitory activity of the interaction between KRAS(G12C, G12D, G12V) proteins and S0S1 proteins and the interaction between KRAS(G12C, G12D, G12V) proteins and S0S2 proteins by N- ( (R)-1-(3-(difluoromethyl)-2-fluorphenyl)ethyl)-6- ( (S)-hexahydropyrazino [2,1-c] [l,4]oxazine-8(1H)-yl)cinnoline-4-amine (free base) and the hydrochloride according to the present invention, HTRF-based KRAS(G12C, G12D, G12V)-SOS1 and It was evaluated using the KRAS(G12C)-S0S2 interaction inhibition efficacy evaluation method.

[0226] Specifically, to evaluate the inhibitory effect of the interaction between KRAS (G12C, G12D, G12V) proteins and S0S1 proteins, KRAS (G12C, G12D, G12V) proteins were reacted with Q-GST Tb antibody for 1 hour, then a 1.5-fold solution of S0S1 was prepared in buffer and 10 µL was dispensed into the reaction plate. Subsequently, GTP was added to the KRAS / α-GST Tb antibody. Afterward, 5 µL of the mixture of S0S1 and the compound (free base or dihydrochloride) reacted for 15 minutes was dispensed to start the reaction. The exchange reaction of GDP to GTP-DY-647P1 via SOS1 was measured using an HTRF® compatible reader (PHERAstar, BMG Labtech, Germany) with Ex / Em=(337 / 665; 337 / 620), and the compound concentration with no SOS1 reaction or the highest concentration was used as a blank. IC50 was measured using a sigmoidal dose response curve (variation slope).

[0227] The inhibitory effect of the interaction between KRAS(G12C) protein and S0S2 protein was measured using the same method as above, except that KRAS(G12C) was used instead of KRAS(G12C, G12D, G12V) protein and S0S2 protein was used instead of S0S1 protein.

[0228] The results are shown in Table 6.

[0229] [Table 6]

[0230] Target inhibition

[0231]

[0232] Classification (Target inhibition) (ICso, nM) KRAS(G12C)- KRAS(G12D)- KRAS(G12V)- KRAS(G12C)- SOS1 SOS1 SOS1 SOS2 Free bases 25.28 23.83 53.31 >10,000 (Comparison Example 1)

[0233] Dihydrochloride 24.48 16.39 33.48 >10,000

[0234]

[0235] Referring to Table 6, it was confirmed that the dihydrochloride according to the present invention exhibits inhibitory activity against the interaction between KRAS (G12C, G12D, G12V) proteins and S0S1 proteins, but does not inhibit the interaction between KRASCG12C) and S0S2 (IC50 >10,000 nM). Through the above results, it was confirmed that the hydrochloride according to the present invention is a selective inhibitor of S0S1 and exhibits pan-KRAS inhibitory activity. In addition, it was confirmed that the hydrochloride according to the present invention has significantly superior inhibitory and selective inhibitory activity against S0S1 proteins compared to free bases.

[0236] Experimental Example 5: Evaluation of in vitro CYPs inhibition

[0237] To predict drug interactions between free bases and hydrochloric acid salts according to the present invention, the inhibitory activity of CYP 7 type isoforms (3A4, 2D6, 2C9, 2C19, 1A2, 2B6, 2C8) was evaluated using a Vivid P450 screening kit.

[0238] [Table 7]

[0239] % inhibition at 10 uM

[0240] Classification CYP isoforms

[0241] 3A4 2D6 2C9 2C19 1A2 2B6 2C8 Free bases 72.9 22.4 31.8 64.3 27.1 18.4 47.9 (Comparison Example 1)

[0242] Dihydrochloride 59.8 11.4 27.7 38.0 5.3 4.0 38.2

[0243]

[0244] Referring to Table 7, it can be seen that the CYP enzyme inhibition rate of the dihydrochloride according to the present invention is significantly lower compared to free bases. Accordingly, it has been confirmed that the dihydrochloride according to the present invention has a low likelihood of drug interactions.

[0245] Although the present invention has been described above with reference to preferred embodiments, those skilled in the art will understand that various modifications and changes can be made to the invention without departing from the spirit and scope of the invention as described in the following claims.

Claims

【Scope of Claim】 【Claim 11 N- ((R)- 1-(3- (difluoromethyl)- 2-fluorphenyl) ethyl)- 6- ((S)-hexahydropyrazino [2,1- c] [1,4]oxazine- 8(1H)-yl) cinnoline- 4-amine hydrochloride .

2. In Paragraph 1, N- ((R)- 1-(3- (difluoromethyl)- 2-fluorphenyl) ethyl)- 6- ((S)-hexahydropyrazino [2,1- c][1,4] oxazine- 8(1H)-yl) cinnoline- 4-amine hydrochloride salt, in which the molar ratio of cinnoline- 4-amine to hydrochloride is 1:1 to 1:

2.

3. In Paragraph 1, The above hydrochloric acid salt is a monochloride or dichloride, N- ((R)- 1-(3- (difluoromethyl)- 2-fluorphenyl) ethyl)- 6- ((S)-hexahydropyrazino [2,1- c] [1,4]oxazine- 8(1H)-yl) cinnoline- 4-amine hydrochloride .

4. In Paragraph 3, The above monohydrochloride or dihydrochloride is an anhydrous, N- ((R)- 1-(3- (difluoromethyl)- 2-fluorphenyl) ethyl)- 6- ((S)-hexahydropyra Gino [2,1-c] [1,4]oxazine-8(1H)-yl)cinnoline-4-amine hydrochloride.

5. In Paragraph 1, The above hydrochloric acid salt is a monochloride, and A crystalline form having a powder X-ray diffraction pattern comprising at least three selected from the group consisting of diffraction peaks with 2θ (±0.2°) values of 9.97°, 13.65°, 19.02°, 20.70°, and 24.19°, N-((R)-1-(3-(Difluoromethyl)-2-fluorophenyl)ethyl)-6-((S)-hexahydropyridazino[2,1-c][1,4]oxazin-8(1H)-yl)cinnolin-4-amine hydrochloride.

6. According to claim 5, The powder X-ray diffraction pattern is Further comprising at least one selected from the group consisting of diffraction peaks with 2θ (±0.2°) values of 7.22°, 16.52°, 16.99°, 21.23°, and 22.97°, N-((R)-1-(3-(Difluoromethyl)-2-fluorophenyl)ethyl)-6-((S)-hexahydropyridazino[2,1-c][1,4]oxazin-8(1H)-yl)cinnolin-4-amine hydrochloride.

7. According to claim 1, The hydrochloride is a monohydrochloride, A crystalline form having a powder X-ray diffraction pattern comprising at least three selected from the group consisting of diffraction peaks with 2θ (±0.2°) values of 7.22°, 9.53°, 9.97°, 10.38°, 13.65°, 13.99°, 15.05°, 16.10°, 16.52°, 16.99°, 19.02°, 19.95°, 20.70°, 21.23°, 22.97°, 24.19°, 24.62°, and 26.14°, N-((R)-1-(3-(difluoromethyl)-2-fluorophenyl)ethyl)-6-((S)-hexahydropyridazino[2,1-c][1,4]oxazin-8(1H)-yl)cinnolin-4-amine hydrochloride.

8. According to claim 1,[[]] The hydrochloride is a dihydrochloride,[[]] and is a crystalline form having a powder X-ray diffraction pattern including diffraction peaks with 2θ (±0.2°) values of 10.59°, 15.25°, and 21.95°. N-((R)-1-(3-(difluoromethyl)-2-fluorophenyl)ethyl)-6-((S)-hexahydropyridazino[2,1-c][1,4]oxazin-8(1H)-yl)cinnolin-4-amine hydrochloride. 【Claim According to claim 8,[[]] the powder X-ray diffraction pattern[[]] further includes at least one selected from the group consisting of diffraction peaks with 2θ (±0.2°) values of 7.68°, 11.00°, 16.76°, 19.06°, 19.27°, 22.59°, 23.86°, and 28.67°. N-((R)-1-(3-(difluoromethyl)-2-fluorophenyl)ethyl)-6-((S)-hexahydropyridazino [2,1-c][1,4]oxazin-8(1H)-yl)cinnolin-4-amine hydrochloride.

10. According to claim 1,[[]] The hydrochloride is a dihydrochloride,[[]] A crystalline form having a powder X-ray diffraction pattern comprising at least three selected from the group of diffraction peaks with values ​​of 5.34°, 7.68°, 10.59°, 11.00°, 12.21°, 15.25°, 15.48°, 15.82°, 16.76°, 19.06°, 19.27°, 20.53°, 21.95°, 22.59°, 23.86°, 28.04°, and 28.67°, N- ((R)- 1-(3- (difluoromethyl)- 2-fluorphenyl) ethyl)- 6- ((S)-hexahydropyrazino [2,1- c] [1,4]oxazine- 8(1H)-yl) cinnoline- 4-amine hydrochloride .

11. In Paragraph 1, The above hydrochloric acid salt is an anhydrous, N- ((R)- 1-(3- (difluoromethyl)- 2-fluorphenyl) ethyl)- 6- ((S)-hexahydropyrazino [2,1- c] [1,4]oxazine- 8(1H)-yl) cinnoline- 4-amine hydrochloride .

12. N- ((R)- 1-(3- (difluoromethyl)- 2-fluorphenyl) ethyl)- 6- ((S)-hexahydropyrazino [2,1- c] [1,4]oxazine- 8(1H)-yl) cinnoline- 4-amine monohydrochloride anhydride.

13. In Paragraph 12, A crystalline form having a powder X-ray diffraction pattern comprising at least three selected from the group consisting of diffraction peaks where the 2θ (±0.2°) values are 7.22°, 9.53°, 9.97°, 10.38°, 13.65°, 13.99°, 15.05°, 16.10°, 16.52°, 16.99°, 19.02°, 19.95°, 20.70°, 21.23°, 22.97°, 24.19°, 24.62° and 26.14°. N-((R)-1-(3-(difluoromethyl)-2-fluorophenyl)ethyl)-6-((S)-hexahydropyridazino[2,1-c][1,4]oxazin-8(1H)-yl)cinnolin-4-amine monohydrochloride anhydride.

14. N-((R)-1-(3-(difluoromethyl)-2-fluorophenyl)ethyl)-6-((S)-hexahydropyridazino[2,1-c][1,4]oxazin-8(1H)-yl)cinnolin-4-amine hydrochloride anhydride.

15. According to claim 14, where the 2θ (±0.2°) values are 5.34°, 7.68°, 10.59°, 11.00°, 12.21°, 15.25°, 15.48°, 15.82°, 16.76°, 19.06°, 19.27°, 20.53°, 21.95°, 22.59°, 23.86°, 28.04° and 28. 67 ° A crystalline form having a powder X-ray diffraction pattern comprising at least three selected from the group of diffraction peaks, N- ((R)- 1-(3- (difluoromethyl)- 2-fluorphenyl) ethyl)- 6- ((S)-hexahydropyrazino [2,1- c] [1,4]oxazine- 8(1H)-yl) cinnoline- 4-amine dichloride anhydride.

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