CRYSTALLINE FORM OF TRICYCLIC DERIVATIVE COMPOUND, METHOD FOR PREPARING SAME, AND PHARMACEUTICAL COMPOSITION COMPRISING SAME
20240246933 ยท 2024-07-25
Assignee
Inventors
- Seong Hyeon JEON (Suwon-si, Gyeonggi-do, KR)
- Jung Gi AN (Yongin-si, Gyeonggi-do, KR)
- John Kim (Seoul, KR)
Cpc classification
A61P29/00
HUMAN NECESSITIES
A61P25/28
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
International classification
Abstract
A novel crystalline form of a citrate of a tricyclic derivative compound represented by Formula 1, a method for preparing the crystalline form of the citrate, and a crystalline form of a tricyclic derivative compound represented by Formula 1, usefully used in the preparation of the crystalline form of the citrate.
##STR00001##
Claims
1-23. (canceled)
24. A crystalline form of a citrate of a tricyclic derivative compound represented by the following formula: ##STR00026##
25. The crystalline form of the citrate of the tricyclic derivative compound according to claim 24, characterized in that the citrate of the tricyclic derivative compound is an anhydride.
26. The crystalline form of the citrate of the tricyclic derivative compound according to claim 25, characterized in that the crystalline form comprises)2?(?0.2? values of 10.01?, 15.86?, 19.62?, and 26.58? in a powder XRD pattern.
27. The crystalline form of the citrate of the tricyclic derivative compound according to claim 26, characterized in that the crystalline form further comprises)2?(?0.2? values of 9.79?, 20.10?, and 27.71? in the powder XRD pattern.
28. The crystalline form of the citrate of the tricyclic derivative compound according to claim 24, characterized in that the citrate of the tricyclic derivative compound is a monohydrate.
29. The crystalline form of the citrate of the tricyclic derivative compound according to claim 28, characterized in that the crystalline form comprises)2?(?0.2? values of 6.94?, 9.99?, 16.57?, 18.17?, 23.68? and 26.39? in the powder XRD pattern.
30. The crystalline form of the citrate of the tricyclic derivative compound according to claim 29, characterized in that the crystalline form further comprises)2?(?0.2? values of 11.89?, 13.35?, 15.07? and 20.90? in the powder XRD pattern.
31. The crystalline form of the citrate of the tricyclic derivative compound according to claim 24, characterized in that the citrate of the tricyclic derivative compound is a dihydrate.
32. The crystalline form of the citrate of the tricyclic derivative compound according to claim 31, characterized in that the crystalline form comprises)2?(?0.2? values of 8.15?, 10.96?, 16.09?, 21.47?, 25.45? and 26.86? in the powder XRD pattern.
33. The crystalline form of the citrate of the tricyclic derivative compound according to claim 32, characterized in that the crystalline form further comprises)2?(?0.2? values of 13.35?, 18.73?, and 28.51? in the powder XRD pattern.
34. A method for preparing a crystalline form of a citrate of a tricyclic derivative compound represented by Formula 1 below, comprising: (a) obtaining a crystalline compound of Formula 1 (i) by reacting a compound of Formula 2 with a compound of Formula 3 in Scheme 1-1 below, or (ii) by reacting a compound of Formula 2 with a compound of Formula 3 in Scheme 1-2 below, and then performing a crystallization process; and (b) reacting the crystalline compound of Formula 1 above with citric acid at 50 to 80? C. in one or more solvents selected from the group consisting of an organic solvent and water: ##STR00027## ##STR00028##
35. The method for preparing the crystalline form of the citrate of the tricyclic derivative compound according to claim 34, characterized in that at least one selected from N,N-dimethylformamide (DMF), dimethylacetamide (DMAC), N-methylpiperidone (NMP), and dimethylsulfoxide (DMSO) are used as a reaction solvent in the reaction of step (a) or step (1).
36. The method for preparing the crystalline form of the citrate of the tricyclic derivative compound according to claim 35, characterized in that methanol or water is used as a crystallization solvent in the crystallization process of step (a) or step (1).
37. The method for preparing the crystalline form of the citrate of the tricyclic derivative compound according to claim 34, characterized in that the crystalline form is an anhydride or a hydrate.
38. The method for preparing the crystalline form of the citrate of the tricyclic derivative compound according to claim 37, characterized in that the crystalline form is an anhydride, and the anhydride is prepared by preparing the citrate of the tricyclic derivative compound represented by Formula 1 in the monohydrate crystalline form in step (b) or step (2) and then converting the monohydrate crystalline form back to the anhydride crystalline form.
39. The method for preparing the crystalline form of the citrate of the compound of Formula 1 according to claim 34, characterized in that a mixed solvent of methanol and ethanol is used as the solvent in step (b) or step (2), and an anhydride crystalline form is prepared by using this solvent.
40. The method for preparing the crystalline form of the citrate of the compound of Formula 1 according to claim 34, characterized in that a mixed solvent of methanol and water is used as the solvent in step (b) or step (2), and a monohydrate crystalline form is prepared by using this solvent.
41. The method for preparing the crystalline form of the citrate of the compound of Formula 1 according to claim 34, characterized in that water is used as the solvent in step (b) or step (2), and a dihydrate crystalline form is prepared by using this solvent.
Description
DESCRIPTION OF DRAWINGS
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BEST MODE
[0132] Hereinafter, the present invention will be described in more detail through examples. It will be apparent to those skilled in the art that these examples are only for illustrating the present invention and the scope of the present invention is not to be construed as being limited to these examples.
Preparative Example 1: Preparation of Compound 2
[0133] ##STR00011##
[0134] 46.7 L of ethanol was added to Compound 5 (4.5 kg, 15.37 mol), and then c-HCl (5.3 L, 61.48 mol) was added thereto. After stirring under reflux for 2 hours and confirming the completion of the reaction, it was cooled and 16 L of ethanol and 29.4 L of purified water were added thereto. The pH was adjusted to 8 to 10 with 6 N aqueous NaOH solution, and then filtered to obtain Compound 2 (3.46 kg, yield of 84.4%).
Preparative Example 2: Preparation of Compound 2
[0135] ##STR00012##
[0136] Isopropanol (6813 mL) was added to Compound 5 (681.3 g, 2.33 mol), and then c-HCl (970 g, 9.32 mol) was added thereto. It was stirred for 2 to 3 hours at 75 to 85? C. After confirming the completion of the reaction, it was cooled and a 6 N aqueous HCl solution was added thereto. The resulting solid was filtered to obtain Compound 2 (600 g, yield of 85.1%).
Example 1: Synthesis of 6-{4-[(5-oxo-1, 2, 3,4,5, 6-hexahydrobenzo[h][1,6]naphthyridin-8-yl) methyl]piperazin-1-yl}nicotinonitrile (Free Form)
(1) Example 1-1
[0137] ##STR00013##
[0138] DMF (65 L) was added to Compound 2 (6.5 kg, 24.37 mol) and Compound 3 (5.73 kg, 30.46 mol). Triethylamine (2.71 kg, 26.8 mol) was added thereto and stirred at 45 to 50? C. for 5 hours. After confirming the completion of the reaction, it was cooled and crystallized by the addition of methanol (130 L). The resulting solid was filtered. 65 L of methanol was added to the wet solid and stirred under reflux for 1.5 hours. After cooling, it was filtered to obtain Compound 1 (7.34 kg, yield of 75.2%).
(2) Example 1-2
[0139] ##STR00014##
[0140] DMF (100 mL) was added to Compound 2 (10 g, 37.49 mmol) and Compound 3 (8.47 g, 44.99 mmol). Triethylamine (5.75 g, 41.24 mmol) was added thereto and stirred at 38 to 42?C for 6 hours. After confirming the completion of the reaction, it was cooled and crystallized by the addition of purified water (100 mL). The resulting solid was filtered to obtain Compound 1 (13.99 g, yield of 93%).
(3) Example 1-3
[0141] ##STR00015##
[0142] DMF (9.96 L) was added to Compound 3 (750.34 g, 2.87 mol), and then triethylamine (1097 g, 10.84 mol) was added thereto and stirred for 2 hours. Compound 2 (664.3 g, 2.19 mol) was added thereto and stirred at 45 to 50? C. for 5.5 hours. After confirming the completion of the reaction, it was cooled and crystallized by the addition of purified water (19.93 L). The resulting solid was filtered to obtain an anhydrous crystal of Compound 1 (788.41 g, yield of 89.9%).
(4) Example 1-4
[0143] Compound 1 (100 g, 249.7 mmol) prepared in Examples 1-1 to 1-3 was added to a mixed solvent of DMAC (1.6 L) and THE (1.6 L), and then dissolved by stirring at 45 to 55? C. After filtering, purified water (3.2 L) was added and cooled to 0 to 10? C. Through a process of stirring and filtering at the same temperature, a dihydrate crystal of Compound 1 was obtained.
[0144] As shown in
[0145] It was confirmed that the dihydrate crystal of Compound 1 further prepared in Example 1-4 above showed peaks at 20 values of 7.95?, 10.25?, 13.25?, 13.78?, 21.12? and 25.22? in the X-ray diffraction (XRD) analysis (
Example 2: Synthesis of Citrate Anhydride of 6-{4-[(5-oxo-1, 2, 3, 4, 5, 6-hexahydrobenzo[h][1,6]naphthyridin-8-yl) methyl]piperazine-1-yl}nicotinonitrile
(1) Example 2-1
[0146] ##STR00016##
[0147] Ethanol (7.2 L) and methanol (2.4 L) were added to citric acid (690.8 g, 3.59 mol). Compound 1 (960 g, 2.39 mol) prepared in Example 1 above was added thereto and stirred at 65 to 70? C. for 2 hours. After cooling to room temperature, it was filtered. Methanol (7.2 L) was added to the wet solid and stirred under reflux for 2 hours. After cooling, it was filtered to obtain the target compound (1.31 kg, 92%).
(2) Example 2-2
[0148] ##STR00017##
[0149] Ethanol (15.5 L), acetone (15.5 L) and isopropanol (15.5 L) were added to citrate of 6-{4-[(5-oxo-1, 2, 3, 4, 5, 6-hexahydrobenzo[h][1,6]naphthyridin-8-yl) methyl]piperazin-1-yl}nicotinonitrile (monohydrate) (4.7 kg, 7.7 mol) prepared in Example 3 below. After elevating the temperature to 55? C., it was stirred at 55 to 70? C. for 4 hours. After cooling to 25? C. or less, it was stirred for 30 minutes. The resulting solid was filtered to obtain a crystalline form of the target compound (4.39 kg, yield of 96.3%).
(3) Example 2-3
[0150] ##STR00018##
[0151] Ethanol (2.5 L), acetone (2.5 L) and isopropanol (2.5 L) were added to citrate of 6-{4-[(5-oxo-1, 2, 3, 4, 5, 6-hexahydrobenzo[h][1,6]naphthyridin-8-yl) methyl]piperazin-1-yl}nicotinonitrile (monohydrate) (500 g, 0.82 mol) prepared in Example 3 below, and purified water (20 mL) was added thereto. After elevating the temperature to 55? C., it was stirred at 55 to 75? C. for 4 hours. After cooling to 25? C. or less, it was stirred for 30 minutes. The resulting solid was filtered to obtain a crystalline form of the target compound (470 g, yield of 96.7%).
[0152] As shown in
[0153] The methods of Example 2-2 and Example 2-3 showed an effect in which related substances were additionally removed in the process of converting from hydrate to anhydride, so that a higher purity anhydrous crystalline compound could be obtained.
Example 3: Synthesis of Citrate Monohydrate of 6-{4-[(5-oxo-1, 2, 3, 4, 5, 6-hexahydrobenzo[h][1,6]naphthyridin-8-yl) methyl]piperazine-1-yl}nicotinonitrile
[0154] ##STR00019##
[0155] Methanol (25.7 L) and purified water (25.7 L) were added to Compound 1 (7.34 kg, 18.32 mol) prepared in Example 1 above. Citric acid (5.28 kg, 27.49 mol) dissolved in a 1:1 mixed solution (22 L) of methanol and purified water was added thereto. After stirring at 15 to 25? C. for 30 minutes, the temperature was elevated to 60? C., and stirred at 60 to 70? C. for 2 hours. After cooling to room temperature, it was filtered to obtain the target compound (10.7 kg, 95.8%).
[0156] As shown in
[0157] In the preparation method, the target compound is obtained in crystalline form as the reaction is completed. Thus, recrystallization to increase purity or an additional process to reduce residual solvent is not required.
Example 4: Synthesis of Citrate Dihydrate of 6-{4-[(5-oxo-1, 2, 3, 4, 5, 6-hexahydrobenzo[h][1,6]naphthyridin-8-yl) methyl]piperazine-1-yl}nicotinonitrile
[0158] ##STR00020##
[0159] Purified water (42 mL) was added to Compound 1 (6 g, 15 mmol) prepared in Example 1 above. Citric acid (3.6 g, 22.5 mmol) dissolved in purified water (18 mL) was added thereto. After stirring at room temperature for 30 minutes, the temperature was elevated to 60? C., and stirred at 60-65? C. for 1.5 hours. After cooling to room temperature, it was filtered to obtain the target compound (8.45 g, yield of 97.8%).
[0160] As shown in
Comparative Examples 1: Synthesis of 6-{4-[(5-oxo-1,2, 3, 4, 5, 6-hexahydrobenzo[h][1,6]naphthyridin-8-yl) methyl]piperazin-1-yl}nicotinonitrile (free form)
(1) Comparative Synthesis Example 1-1
[0161] ##STR00021##
[0162] 35.8 L of methanol was added to Compound 2 (2.56 kg, 9.6 mol) and Compound 3 (1.98 kg, 10.56 mol). Triethylamine (2.67 L, 19.2 mol) was added thereto and stirred under reflux for 24 hours. After confirming the completion of the reaction, DMAC (38.4 L) and THF (38.4 L) were added thereto, and dissolved by elevating the temperature. Activated carbon was added to the solution and stirred. After filtration, it was added dropwise to purified water to crystallize and filtered.
[0163] DMAC and THE were added to the wet solid and dissolved, and then recrystallized to obtain Compound 1 (2.93 kg, 71.1%).
(Step 1)
[0164] Thereafter, an additional purification process was performed to remove related substances to obtain clean Compound 1 (1.59 kg, yield of 39%). (Step 2) [0165] (2) Comparative Synthesis Example 1-2
(Step a)
[0166] ##STR00022##
[0167] Methanol was added to Compound 5 above, and then 6-(piperazin-1-yl) nicotinonitrile and triethylamine were added thereto. After stirring at 80? C. for 24 hours and confirming the completion of the reaction, the reaction solution was concentrated. Thereafter, it was extracted with dichloromethane, the organic solvent layer was dried over anhydrous magnesium sulfate, and the solvent was concentrated under reduced pressure to obtain a residue, which was purified by column chromatography (hexane:ethyl acetate=1: 9) to obtain a compound (yield of 75%) similar to the scheme above.
(Step b)
[0168] ##STR00023##
[0169] The compound prepared in step a was dissolved in dichloromethane, and then trifluoroacetic acid was added thereto and heated for 24 hours using a reflux condenser. After confirming the completion of the reaction, it was extracted with dichloromethane and extracted with dichloromethane once more in a saturated aqueous sodium hydrogen carbonate solution. It was dried over anhydrous magnesium sulfate, and the solvent was concentrated under reduced pressure to obtain a residue, which was purified by column chromatography (dichloromethane:methanol=1: 9) to obtain the compound of Formula 1 (yield of 65%).
Comparative Examples 2: Synthesis of Dichloride Salt of 6-{4-[(5-oxo-1,2, 3, 4, 5, 6-hexahydrobenzo[h][1,6]naphthyridin-8-yl) methyl]piperazine-1-yl}nicotinonitrile
[0170] ##STR00024##
[0171] Compound 1 obtained in Comparative Synthesis Example 1-2 above was dissolved in methanol, and then 1.25 M hydrochloric acid methanol solution was added thereto and stirred for 12 hours. After completion of the reaction, the solvent was concentrated under reduced pressure, and then the solid was filtered with ethyl acetate to obtain the dihydrochloride salt of Compound 1 (yield of 95%).
Analytical Method: Moisture Content, DSC, TGA and XRD Analysis
[0172] Moisture content, DSC, TGA and XRD analysis described in the present invention were conducted in the following manner:
(1) Moisture Content Measurement
[0173] Moisture content was measured using an 870 KF Titrino Plus (Metrohm) Karl-Fisher moisture titrator.
(2) DSC Analysis
[0174] DSC analysis was performed on a DSC 8000 (PerkinElmer) analyzer at 30 to 250 to 350? C. A 0.5 to 2 mg of sample was weighed into an aluminum DSC pan and sealed non-hermetically with a perforated aluminum lid, and then the sample was heated from 30? C. to 250 to 350? C. at a scan rate of 10? C./min, and the resulting heat flow reaction was monitored.
(3) TGA Analysis
[0175] TGA analysis was performed on a TGA 8000 (PerkinElmer) analyzer at 30 to 900? C. A 0.5 to 2 mg of sample was weighed into a ceramic crucible, the sample was heated from 30? C. to 900? C. at a scan rate of 5? C./min, and the resulting mass loss was monitored.
(4) XRD Analysis
[0176] XRD analysis was performed on a D8 Focus (Bruker ASX) analyzer from 2? 2? to 40? 2?. An about 100 mg of sample was gently pressed onto the plastic sample holder so that the sample surface was smooth and just above the level of the sample holder, and then measured under the following conditions.
<Analysis Conditions>
[0177] Anode material (Ka): Cu Ka (1.5406 ?); scan range: 2 to 40?; generator settings: 40 mA, 40 kV; scan rate: 10?/min; divergence slit size: 0.6 mm; temperature: 25? C. ; step size: 0.02? 20; rotation: used
Experimental Example 1: Analysis of Related Substances
[0178] The amounts of related substances included in the compounds prepared in the Examples and Comparative Examples above were measured using HPLC (product name: Agilent 1100; manufacturer: Agilent), and the results are shown in Table 2 below.
<HPLC Operating Conditions>
[0179] Detector: UV detector (detection wavelength: 220 nm) [0180] Column: Watchers 100 ODS-P, 4.6?250 mm, 5 ?m, or equivalent column [0181] Temperature: constant temperature around 25? C. [0182] Mobile phase A: prepared by dissolving 11.503 g of ammonium dihydrophosphate in 1000 mL of water and degassing after filtering, Mobile phase B: acetonitrile [0183] Mobile phase gradient:
TABLE-US-00001 TABLE 1 Analysis time (min) Mobile phase A (%) Mobile phase B (%) 0 85 15 5 80 20 15 80 20 20 75 25 25 75 25 75 30 70 76 85 15 90 85 15 Diluent: mobile phase A and acetonitrile mixed in a ratio of 5:5 are used. Flow rate: 1.0 mL/min; injection volume: 10.0 ?L; analysis time: 90 min * Preparation of test solution: 10 mg of sample dissolved in 50 mL of diluent is used.
TABLE-US-00002 TABLE 2 Analysis of related substances in the crystal (free form) of Compound 1 Compound 1 prepared in Compound 1 prepared in Comparative Synthesis Comparative Synthesis Compound 1 prepared in Example 1-1 (Step 1) Example 1-1 (Step 2) Example 1-2 RT (min) Area (%) RT (min) Area (%) RT (min) Area (%) 43.884 0.2435 44.006 0.0530 49.185 0.2475 48.987 0.1655 48.734 0.0225 Analysis of related substances in the crystal of citrate of Compound 1 Crystal of citrate of Compound 1 Crystal of citrate of Compound 1 prepared in Example 3 prepared in Example 2-3 RT (min) Area (%) RT (min) Area (%) 48.843 0.0122
[0184] As a result of the above experiment, in the case of the crystalline form of citrate of Compound 1 prepared according to the Examples of the present invention, it was confirmed that although no recrystallization process was performed throughout the entire preparation process, major related substances were remarkably reduced or not detected.
[0185] In the case of HPLC analysis, related substances coming out around the retention time of 49 min are predicted to be substances generated due to an overreaction in the coupling process of Formulas 2 and 3 above, or Formulas 5 and 3 above, and it is difficult to separate and remove after generation, and thus, it is very important to reduce the related substances in the preparation process.
[0186] Related substances around RT 49:
##STR00025##
Experimental Example 2: Measurement of Solubility in Water
[0187] The solubility of the addition salt of Compound 1 in water was measured, and the results are shown in Table 4 below.
[0188] Specifically, an excess amount of the compound and solvent (10 mM, in 1 mL of water) were put into a shake-flask and shaken for 24 hours, and then quantitatively analyzed using UPLC.
<Equipment and Conditions Used>
[0189] Equipment name: Acquity UPLC (Waters) [0190] Column: ACQUITY UPLC BEH C18 1.7 ?m?2.1 mm?50 mm [0191] Wavelength: 226, 276, 310 nm (PDA detector) [0192] Column temperature: 40? C.; sample temperature: 25? C.; injection volume: 5 ?l
[0193] Mobile phase and concentration gradient
TABLE-US-00003 TABLE 3 Time (min) Water (0.2% phosphoric acid) Acetonitrile 0 90 10 0.3 90 10 1.5 1 99 1.6 0 100 1.8 90 10 3.0 90 10
[0194] Compounds are dissolved in DMSO and measured by UPLC, and then a calibration curve is drawn.
[0195] The compounds saturated by the Shake-flask method are filtered and then measured by UPLC, and it is substituted into a calibration curve to calculate the concentration.
TABLE-US-00004 TABLE 4 Water Water solubility solubility Salt (mM) Salt (mM) Crystalline 1.067 Crystalline 0.498 anhydride of anhydride of citrate of succinic acid salt Compound 1 of Compound 1 Crystalline 0.241 Crystalline 0.444 anhydride of tartaric anhydride of fumaric acid salt of acid salt of Compound 1 Compound 1 Crystalline 0.392 Crystal (free form) 0.001 anhydride of 2HCl of the compound of salt of Formula 1 in Compound 1 Comparative Example 1-1
[0196] As a result of the above experiment, it was confirmed that the crystalline anhydride of citrate of Compound 1 of the present invention had remarkably excellent solubility in water compared to other salts.
Experimental Example 3: Confirmation of the Stability of the Addition Salt of Compound 1
[0197] The stability of crystals of the addition salt of Compound 1 prepared in the Examples and Comparative Examples above was confirmed, and the results are shown in Table 5 below.
[0198] Experiments according to temperature, humidity and light conditions were carried out by a method of confirming purity by HPLC after storage in a stability chamber under each condition.
<UV 200 Watt Irradiation>
[0199] Each sample was spread thinly in a Petri dish, and then the stability was evaluated after irradiation with UV light having a light intensity of 35 W to a total irradiation amount of 200 watts under conditions of 25? C. and 60% humidity using a photostability test chamber (CARON 6542-2).
<Visible 1200 k Lux Irradiation>
[0200] Each sample was spread thinly in a Petri dish, and then the stability was evaluated after irradiation with visible light having a light intensity of 35 k Lux to a total irradiation amount of 1200 k lux under conditions of 25? C. and 60% humidity using a photostability test chamber (CARON 6542-2).
TABLE-US-00005 TABLE 5 Purity (%) Crystalline anhydride of 2HCl Crystalline salt in Comparative anhydride of citrate Condition Example 2 in Example 2 Initial value 97.89 97.97 Temperature of 50? C., 97.79 97.86 3 days Humidity of 75%, 3 days 97.76 97.81 Temperature of 40? C., 97.83 97.79 humidity of 70%, 3 days UV 200 watt - dark 97.89 98.38 UV 200 watt 86.89 96.94 Visible 1200k lux - dark 97.89 98.39 Visible 1200k lux 93.40 96.73
[0201] As a result of the above experiment, both dihydrochloride and citrate anhydride of Compound 1 were found to be stable in the temperature and humidity. However, the dihydrochloride salt showed remarkably poor photostability at UV200 watt and Visible1200 k lux. If the photostability is low, storage stability may decrease by indoor lighting as well as sunlight, thereby lowering purity, and thus, care should be taken in management such as the need to store raw materials shaded. When the stability was confirmed in this way, the citrate of Compound 1 was found to be the most suitable.
Experimental Example 4: Confirmation of the Melting Point of a Salt
[0202] The melting points of the compounds of the citrate prepared in the Examples above were measured using a B-454 (BUCHI) melting point detector, and the results are shown in Table 6 below.
TABLE-US-00006 TABLE 6 Crystalline Crystalline Crystalline anhydride of monohydrate of dihydrate of citrate in Example 2 citrate in Example 3 citrate in Example 4 206? C. 176? C. 173? C.
[0203] As a result of the above experiment, it was confirmed that all of the crystalline forms of the citrate salt of the present invention had sufficiently high melting points as pharmaceutical raw materials.
Experimental Example 5: Measurement of solubility in various solvents
[0204] The solubility of the crystalline compounds prepared in the Examples above in various solvents was measured, and the results are shown in Table 7 below.
[0205] Specifically, 0.1 g of sample was accurately weighed, put into each solvent, and shaken and mixed vigorously for 30 seconds every 5 minutes at 15 to 25? C., and the amount dissolved within 30 minutes was measured.
TABLE-US-00007 TABLE 7 Solubility (0.1 g/mL) Crystalline Crystalline Crystalline anhydride monohydrate dihydrate of citrate in of citrate in of citrate in Solvent Free form Example 2 Example 3 Example 4 DMF 8 mL 8 mL 2.9 mL 2.5 mL MeOH 300 mL 100 mL 9 mL 9 mL or more Aqueous HC1 350 mL 100 mL 50 mL 55 mL solution or less (pH 1.1) EtOH 1000 mL 1000 mL 350 mL 450 mL or more or more Water 1200 mL 1000 mL 400 mL 400 mL or more or less or more
Experimental Example 6: Evaluation of Purity and Stability of Anhydride and Monohydrate of Citrate of Compound 1
[0206] The purity and stability of the crystalline anhydride of the citrate prepared in Example 2 and the crystalline monohydrate of the citrate prepared in Example 3 according to the present invention were evaluated, and the results are shown in Tables 8 and 9 below.
[0207] In the tables below, stability tests were conducted under accelerated storage conditions of a temperature of 40?2? C. and humidity of 75?5%, and purity and stability tests were conducted under long-term storage conditions of a temperature of 25?2? C. and humidity of 60?5%. Experimental results were measured under the same conditions as in Experimental Example 1 using HPLC (product name: agilent 1290, manufacturer: Agilent).
TABLE-US-00008 TABLE 8 (Standard %) Purity under accelerated Purity under long-term storage conditions storage conditions (period: month, area %) (period: month, area %) Period initial 1 3 6 initial 1 3 6 Crystalline 99.66% 99.64% 99.65% 99.54% 99.66% 99.64% 99.66% 99.58% anhydride of 9 12 18 citrate in 99.67% 99.72% 99.73% Example 2 24 36 (purity) 99.74% 99.73%
TABLE-US-00009 TABLE 9 (Standard %) Accelerated storage Long-term storage conditions (related conditions (related substances) substances) (period: month, area %) (period: month, area %) Period initial 3 6 initial 3 6 9 Crystalline 0.26% 0.24% 0.28% 0.26% 0.28% 0.29% 0.31% anhydride of 12 18 24 citrate in 0.29% 0.33% 0.34% Example 2 30 36 0.34% 0.30% Period initial 3 6 initial 3 6 Crystalline 0.38% 0.34% 0.32% 0.38% 0.29% 0.32% monohydrate of citrate in Example 3
Experimental Example 7: Inhibition Test of Poly (ADP-Ribose) Polymerase [PARP-1]Enzyme
[0208] The PARP-1 enzyme inhibitory activity of the crystalline anhydride of the citrate of the compound of Formula 1 (Example 2) according to the present invention and the crystalline anhydride of the hydrochloride salt of the compound of Formula 1 (Comparative Example 2) was assayed using a kit (cat. 80551) purchased from BPS Bioscience as follows.
[0209] Histones were coated on a 96-well plate provided from BPS Bioscience's kit and left at 4? C. for 16 hours. Thereafter, the plate was washed 4 times with PBST (7.5 mM Na.sub.2HPO.sub.4, 2.5 mM NaH.sub.2 PO.sub.4, 145 mM NaCl, 0.05% Tween 20, pH 7.4), and then blocking buffer (provided from BPS Bioscience's kit) was added thereto to prevent non-specific reactions and left at 25? C. for 1 hour. After standing for 1 hour, the plate was washed 4 times with PBST, and various concentrations of the compounds of Example 2 and Comparative Example 2 were added to a reaction solution containing PARP-1 enzyme (50 ng/well), an assay mixture and an activated DNA, and reacted at 25? C. for 1 hour. After 1 hour, each well was washed 4 times with PBST, and streptavidin-linked peroxidase (Strep-HRP, 1:50 dilution) was added thereto to measure the amount of ribosylation by PARP enzyme, and reacted at 25? C. for 30 minutes. The plate was washed 4 times with PBST, and then HRP chemiluminescent substrate was finally added thereto and reacted. The amount of histone ribosylation formed by each enzyme was quantified using a Synergy? H4 Hybrid Multi-Mode Microplate Reader (BioTek Instruments, Inc., US (A)). The results obtained for each concentration of the compounds of the present invention are the average values obtained in two wells, and the IC50 values of the compounds were calculated using SigmaPlot 10 (Systat Software Inc., US (A)) for analysis of the results. AZD-2281 (Olaparib), which is a representative PARP inhibitor, was used as a control compound.
[0210] The experimental results are shown in Table 10 below, and are shown graphically in
TABLE-US-00010 TABLE 10 IC.sub.50 Crystalline anhydride of dihydrochloride Crystalline anhydride AZD-2281 salt in Comparative of citrate in (Olaparib) Example 2 Example 2 5.48 nM 3.03 nM 2.62 nM
Experimental Example 8: Inhibition Test of Tankyrase-1 and Tankyrase-2 Enzymes
[0211] The tankyrase-1 or tankyrase-2 enzyme inhibitory activity of the crystalline anhydride of the citrate of the compound of Formula 1 (Example 2) according to the present invention and the crystalline anhydride of the hydrochloride salt of the compound of Formula 1 (Comparative Example 2) was assayed as follows using kits (cat. 80573, 80578) purchased from BPS Bioscience:
[0212] Histones were coated on a 96-well plate provided from BPS Bioscience's kit and left at 4?C for 16 hours. Thereafter, the plate was washed 4 times with PBST (7.5 mM Na.sub.2HPO.sub.4, 2.5 mM NaH.sub.2PO.sub.4, 145 mM NaCl, 0.05% Tween 20, pH 7.4), and then blocking buffer (provided from BPS Bioscience's kit) was added thereto to prevent non-specific reactions and left at 25? C. for 1 hour. After standing for 1 hour, the plate was washed 4 times with PBST, and various concentrations of the compounds of the Examples were added to a reaction solution containing tankyrase-1 enzyme (40 ng/well) or tankyrase-2 enzyme (15 ng/well) and an assay mixture, and reacted at 25? C. for 1 hour. After 1 hour, each well was washed 4 times with PBST, and streptavidin-linked peroxidase (Strep-HRP, 1:50 dilution) was added thereto to measure the amount of ribosylation by PARP enzyme, and reacted at 25? C. for 30 minutes. The plate was washed 4 times with PBST, and then HRP chemiluminescent substrate was finally added thereto and reacted. The amount of histone ribosylation formed by each enzyme was quantified using a Synergy? H4 Hybrid Multi-Mode Microplate Reader (BioTek Instruments, Inc., US (A)). The results obtained for each concentration of the compounds of the present invention are the average values obtained in two wells, and the IC50 values of the compounds were calculated using SigmaPlot 10 (Systat Software Inc., US (A)) for analysis of the results.
[0213] XAV-939, which is a representative tankyrase inhibitor, and BMN-673 (Talazoparib), which was developed as a PARP inhibitor but known to be effective in inhibiting tankyrases, were used as control compounds.
[0214] The experimental results are shown in Tables 11 and 12 below, and are shown graphically in
TABLE-US-00011 TABLE 11 IC.sub.50 BMN-673 Anhydride of citrate (Talazoparib) XAV-939 in Example 2 8.29 nM 8.22 nM 4.31 nM
TABLE-US-00012 TABLE 12 IC.sub.50 Crystalline Crystalline anhydride of anhydride of BMN-673 dihydrochloride salt in citrate in (Talazoparib) XAV-939 Comparative Example 2 Example 2 2.53 nM 2.21 nM 1.12 nM 1.06 nM