CRYSTALLINE FORM OF TRICYCLIC DERIVATIVE COMPOUNDS, METHODS OF PREPARATION, AND PHARMACEUTICALS CONTAINING THEM.

Abstract

The invention relates to the crystalline form of citrate of a tricyclic derivative compound expressed by formula 1, a method for preparing the crystalline form of citrate, and the crystalline form of the tricyclic derivative compound expressed by formula 1, which is effective in preparing the crystalline form of citrate (I).

Description

Crystalline form of tricyclic derivative compound and preparation method thereof and pharmaceutical composition comprising same

[0001] This application claims the benefit of priority to Korean Patent Application No. 10-2021-0064416, filed May 18, 2021, the entire contents of which are incorporated herein by reference.

[0002] The present invention relates to a novel salt crystalline form of a tricyclic derivative compound having excellent PARP and tankyrase inhibitory activity, a method for preparing the same, a transformation of the crystalline form, and a pharmaceutical composition comprising the same.

[0003] Poly(ADP-ribose) polymerases (PARPs) are composed of approximately 17 proteins, including PARP-1, PARP-2, PARP-3, PARP-4 (vPARP), PARP-5 (Tankyrase-1, Tankyrase-2), PARP-7, and PARP-10 [Curr Pharm Des., 13(9), 933-962, 2007]. They all share a certain level of homology in their catalytic domains, but their functions in cells differ [BioEssays., 26(8), 882-893, 2004].

[0004] The primary role of PARP-1 and PARP-2 is to facilitate DNA repair by ADP-ribosylation and regulate the number of many DNA repair proteins. When the DNA of cancer cells is damaged, PARP is activated and repairs single-strand breaks in DNA, which can induce resistance in various types of cancer treatment. Therefore, anticancer efficacy can be expected through PARP inhibitors, and in particular, they have been reported to be useful for the specific killing of tumors deficient in double-strand break repair factors of DNA, such as BRCA-1 and BRCA-2. Therefore, they are being developed as personalized anticancer agents for various cancers such as breast cancer, ovarian cancer, and prostate cancer that have abnormalities in DNA double-strand break repair factors [Nature, 434, 913-916, 2005; Cancer Biology & Therapy, 4, 934-936, 2005].

[0005] Furthermore, PARP inhibitors can be used to treat various neurological and cardiovascular diseases, including ischemic brain damage, and are also useful in treating inflammatory symptoms. Recently, the therapeutic potential of PARP inhibitors in the treatment of diabetic neuropathy has been raised [Diabetes. 54(12), 3435-3441, 2005].

[0006] Meanwhile, tankyrase-1 and tankyrase-2, which are PARP-5, are known to be involved in mitosis, which is highly related to the Wnt / β-catenin signaling pathway, DNA repair process, and cell cycle [Biochimica et Biophysica Acta, 1846, 201-205, 2014]. In addition, they function as positive regulators of telomere length. Therefore, inhibition of tankyrase-1 and tankyrase-2 may exert anticancer effects through a different mechanism from PARP-1 by inhibiting the Wnt / β-catenin signaling pathway, DNA repair process, and telomere elongation [Nature Reviews Drug Discovery, 11, 923-936, 2012].

[0007] International Patent Publication No. WO2016-200101 discloses various tricyclic derivative compounds that are PARP-1 and tankyrase inhibitors for the treatment of diseases caused by the activity of poly(ADP-ribose) polymerase (PARP). These compounds are prepared as free bases or 1-3 hydrochloride salts, most of which are hydrochloride salts. The present inventors have found that the compounds prepared in WO2016-200101 have low aqueous solubility, making them difficult to use as pharmaceuticals. Furthermore, the multi-step process required to prepare these compounds makes them unsuitable for commercial use. Therefore, the inventors have developed a novel salt crystal form that exhibits aqueous solubility suitable for pharmaceutical applications, while also ensuring photostability and other stability, and a novel manufacturing method for commercial use, thereby completing the present invention. The manufacturing method of the present invention can produce a high-purity compound in a stable crystalline form without additional purification after the reaction is complete, providing a commercially useful method that is capable of mass production.

[0008] The purpose of the present invention is to provide a crystalline form of a novel salt of a tricyclic derivative compound having excellent PARP-1, tankyrase-1 or tankyrase-2 inhibitory activity, and an effective method for preparing the same.

[0009] This aims to solve the problems of conventional technology, especially low water solubility that makes it unsuitable for pharmaceutical applications, improve the inefficiency of having to go through multiple processes to increase purity, and reduce flexible substances that are difficult to separate and remove after production.

[0010] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating various diseases caused by the activity of PARP-1, tankyrase-1 or tankyrase-2, which comprises a crystalline form of a citrate salt of the tricyclic derivative compound as an active ingredient.

[0011] Citrate salt crystalline form of tricyclic derivative compound

[0012] The present invention provides a crystalline form of a citric acid salt of a tricyclic derivative compound represented by the following chemical formula 1:

[0013] [Chemical Formula 1]

[0014]

[0015] The citric acid salt crystal form of the above tricyclic derivative compound may be an anhydrous form.

[0016] The anhydrous crystalline form may contain up to about 1 wt% moisture, preferably up to 0.5 wt%.

[0017] The above anhydrous crystalline form is characterized by 2θ (± 0.2°) values ​​of 10.01°, 15.86°, 19.62° and 26.58° in the powder XRD pattern.

[0018] Additionally, the anhydrous crystalline form may further include 2θ (± 0.2°) values ​​of 9.79°, 20.10°, and 21.71° in the powder XRD pattern.

[0019] In addition, the anhydrous crystalline form exhibits an XRD pattern substantially as shown in Figure 7 below.

[0020]

[0021] The citric acid salt crystal form of the above tricyclic derivative compound may be a monohydrate.

[0022] The monohydrate crystal form may contain moisture in the range of about 2.0 to 4.5 wt%, preferably about 3 wt%.

[0023] The above monohydrate crystal form is characterized by having 2θ (± 0.2°) values ​​of 6.94°, 9.99°, 16.57°, 18.17°, 23.68°, and 26.39° in the powder XRD pattern.

[0024] Additionally, the monohydrate crystal form may further include 2θ (± 0.2°) values ​​of 11.89°, 13.35°, 15.07°, and 20.90° in the powder XRD pattern.

[0025] In addition, the crystal form of the above citric acid salt monohydrate exhibits an XRD pattern substantially as shown in the form of Figure 10 below.

[0026]

[0027] The citric acid salt of the above tricyclic derivative compound may be a dihydrate.

[0028] The dihydrate crystal form may contain moisture in the range of about 5.0 to 7.5 wt%, and preferably about 5.7 wt%. The dihydrate crystal form is characterized by having 2θ (± 0.2°) values ​​of 8.15°, 10.96°, 16.09°, 21.47°, 25.45°, and 26.86° in a powder XRD pattern.

[0029] Additionally, the dihydrate crystal form may further include 2θ (± 0.2°) values ​​of 13.35°, 18.73°, and 28.51° in the powder XRD pattern.

[0030] In addition, the crystal form of the above citric acid salt dihydrate exhibits an XRD pattern substantially as shown in the form of Figure 13 below.

[0031]

[0032] The citric acid salt crystal form of the tricyclic derivative compound of the present invention is in a photostability chamber,

[0033] Under conditions of 25℃ and 60% humidity, the purity can be maintained at 95% or more, preferably 96% or more, after irradiating with UV light of 35W so that the total irradiation amount becomes 200 watts.

[0034] In addition, it can have the characteristic that the purity is maintained at 95% or more, preferably 96% or more, after irradiating visible light with a light quantity of 35k Lux so that the total irradiation amount becomes 1200k Lux under the conditions of 25℃ and 60% humidity.

[0035] That is, the citric acid salt crystal form of the tricyclic derivative compound of the present invention provides very excellent photostability compared to conventional hydrochloric acid salts, etc.

[0036]

[0037] The citric acid salt crystalline form of the above tricyclic derivative compound may have a characteristic in which the area (%) of a related substance peak is less than 0.05% at RT 43 min to 45 min in HPLC analysis, more preferably, no related substance peak is observed (does not exist) at RT 43 min to 45 min.

[0038] In addition, the citric acid salt crystalline form of the tricyclic derivative compound may have a characteristic in which the related substance peak area (%) at RT 48 min to 50 min in HPLC analysis is less than 0.15%, preferably less than 0.1%, more preferably less than 0.05%, even more preferably less than 0.015%, and most preferably no related substance peak is observed (non-existent).

[0039] In addition, the citric acid salt crystal form of the tricyclic derivative compound may have a characteristic in which no relevant material peak is observed (does not exist) at RT 43 min to 45 min and RT 48 min to 50 min in HPLC analysis.

[0040] The flexible substance generated at RT 48 min to 50 min above has the following chemical structural formula.

[0041]

[0042] These flexible substances are difficult to separate and remove after production. They are harmful to the human body, and if they are generated above standards, additional safety testing is required. Therefore, reducing these flexible substances during the manufacturing process is crucial.

[0043]

[0044] The citric acid salt crystal form of the above tricyclic derivative compound may have a purity of 99.6% or more.

[0045] In addition, it may have the characteristic of having a purity of 99.6% or higher when stored for 3 months under long-term storage conditions (storage at a temperature of 25±2℃ and a humidity of 60±5%). In addition, it may have the characteristic of having a purity of 99.5% or higher, preferably 99.55% or higher, when stored for 6 months. In addition, it may have the characteristic of having a purity of 99.7% or higher when stored for 12 months. In addition, it may have the characteristic of having a purity of 99.7% or higher when stored for 24 months. In addition, it may have the characteristic of having a purity of 99.7% or higher when stored for 36 months.

[0046] In addition, it can have the characteristic that the purity does not decrease compared to the initial state when stored for more than 9 months under long-term storage conditions (storage at temperature 25±2℃, humidity 60±5%).

[0047] In addition, it can have the characteristics of having a purity of 99.6% or higher when stored for 3 months under accelerated storage conditions (storage at a temperature of 40±2℃ and a humidity of 75±5%), and a purity of 99.5% or higher when stored for 6 months.

[0048]

[0049] The citric acid salt crystalline form of the tricyclic derivative compound may have a characteristic of a peak area (%) of related substances of 0.30% or less, more preferably 0.29% or less, in HPLC analysis after 3 months of storage under long-term storage conditions (storage at a temperature of 25±2℃ and a humidity of 60±5%). In addition, it may have a characteristic of a peak area (%) of related substances of 0.32% or less, more preferably 0.30% or less, in HPLC analysis after 6 months of storage. In addition, it may have a characteristic of a peak area (%) of related substances of 0.32% or less, more preferably 0.30% or less, in HPLC analysis after 12 months of storage. In addition, it may have a characteristic of a peak area (%) of related substances of 0.35% or less, in HPLC analysis after 24 months of storage. In addition, it may have a characteristic of a peak area (%) of related substances of 0.32% or less, in HPLC analysis after 36 months of storage.

[0050] The citric acid salt crystalline form of the above tricyclic derivative compound may have a characteristic in that the peak area (%) of the related substances is 0.35% or less, more preferably 0.25% or less, in HPLC analysis after storage for 3 months under accelerated storage conditions (storage at a temperature of 40±2°C and a humidity of 75±5%). In addition, the peak area (%) of the related substances is 0.33% or less, more preferably 0.30% or less, in HPLC analysis after storage for 6 months.

[0051]

[0052] Crystalline form of tricyclic derivative compound

[0053] In addition, the present invention

[0054] Provided is a crystalline form of a tricyclic derivative compound represented by the following chemical formula 1:

[0055] [Chemical Formula 1]

[0056]

[0057] The tricyclic derivative compound (free base) of the above chemical formula 1 is an effective ingredient that exhibits the effect of a PARP and tankyrase inhibitor in vivo, and is also very useful as an intermediate for manufacturing a crystalline form of a citrate salt, which is an acid addition salt thereof.

[0058] The crystalline form of the above tricyclic derivative compound may be an anhydride.

[0059] The anhydrous crystalline form may contain less than about 1 wt% moisture, and is preferably characterized by containing less than 0.5 wt% moisture.

[0060] The crystalline form of the above tricyclic derivative compound is characterized by having 2θ (± 0.2°) values ​​of 7.88°, 10.23°, 15.16°, 19.27°, 22.79° and 23.94° in the powder XRD pattern.

[0061] Additionally, the crystalline form of the tricyclic derivative compound may further include 2θ (± 0.2°) values ​​of 13.89°, 15.78°, 18.05°, and 19.51° in the powder XRD pattern.

[0062] In addition, the crystal form of the tricyclic derivative compound exhibits an XRD pattern substantially as shown in the following Figure 1.

[0063]

[0064] The crystal form of the above tricyclic derivative compound may be a dihydrate.

[0065] The dihydrate crystal form can contain moisture in the range of about 7.0 to 9.5 wt%, and is preferably characterized by containing about 8.3 wt%.

[0066] The above dihydrate crystal form is characterized by having 2θ (± 0.2°) values ​​of 7.95°, 10.25°, 13.25°, 13.78°, 21.12°, and 25.22° in the powder XRD pattern.

[0067] Additionally, the dihydrate crystal form may further include 2θ (± 0.2°) values ​​of 11.93°, 12.50°, 15.98°, 19.12°, 22.80°, and 26.72° in the powder XRD pattern.

[0068] In addition, the above dihydrate crystal form exhibits an XRD pattern substantially as shown in the form of Figure 4 below.

[0069]

[0070] Method for preparing crystalline form of citrate salt of tricyclic derivative compound

[0071] Method 1

[0072] The present invention provides a method for preparing a crystalline form of a tricyclic derivative compound represented by the following reaction scheme 1-1. The manufacturing method of the present invention provides excellent yield and purity, and is characterized by significantly improving manufacturing efficiency through simplification of the manufacturing process.

[0073] The citric acid salt crystal form of the tricyclic derivative compound represented by the above chemical formula 1 of the present invention can be prepared by including the following steps.

[0074] (a) a step of reacting a compound of chemical formula 2 and a compound of chemical formula 3 in the following reaction scheme 1, and then performing a crystallization process to obtain a crystal compound of chemical formula 1; and

[0075] (b) a step of reacting the crystal compound of the above chemical formula 1 with citric acid in at least one solvent selected from the group consisting of organic solvents and water at 50 to 80°C:

[0076] [Reaction Formula 1-1]

[0077]

[0078] Below, the manufacturing method is described in detail step by step.

[0079] < (a) Step >

[0080] The reaction in step (a) above can be carried out in the presence of an organic amine such as pyridine, triethylamine, diethylisopropylamine, or a base such as potassium carbonate.

[0081] The above reaction temperature is not particularly limited, but can generally be performed at a low temperature or at a high temperature, and is preferably performed at room temperature or at a high temperature.

[0082] In particular, in the step (a), at least one selected from N,N-dimethylformamide (DMF), dimethylacetamide (DMAC), N-methylpiperidone (NMP), dimethyl sulfoxide (DMSO), etc. can be used as the reaction solvent, and when these solvents are used, the reaction time is drastically shortened (reaction time shortened from 24 hours to 6 hours) compared to the case of using a conventional organic solvent, reflux stirring is unnecessary, and additional processes such as additional recrystallization and chromatography are unnecessary due to the reduction of flexible substances, thereby providing the effect of greatly simplifying the process.

[0083]

[0084] In the crystallization process of step (a) above, at least one selected from methanol, ethanol, isopropanol, butanol, acetone, acetonitrile, ethyl acetate, tetrahydrofuran, and water may be used as the crystallization solvent. Purified water or distilled water may be used as the water.

[0085] In particular, when water is used as the crystallization solvent, high-yield, high-purity crystals can be produced without additional processes such as recrystallization, and the process can be greatly simplified, making it suitable for mass synthesis processes.

[0086] Furthermore, when dimethylformamide (DMF) is used as the reaction solvent and water is used as the crystallization solvent in step (a), even better results are achieved. That is, high-yield, high-purity crystals can be produced without additional processes such as recrystallization, significantly simplifying the process and making it suitable for mass synthesis.

[0087] The reaction of step (a) above can be performed at 35 to 55°C, and a filtration process can be further performed after the crystallization process. In particular, step (a) of the present invention has the characteristic of providing a purity so excellent that recrystallization or the like is unnecessary.

[0088]

[0089] < (b) Step >

[0090] The citric acid salt of the tricyclic derivative compound manufactured by the above manufacturing method may be an anhydride or a hydrate, and the hydrate may be a monohydrate or a dihydrate.

[0091] Among the solvents in step (b), at least one organic solvent selected from among methanol, ethanol, acetone, isopropanol, acetonitrile, butanol, propanol, and tetrahydrofuran may be used.

[0092] Throughout the present invention, purified water or distilled water may be used as the water.

[0093] The citric acid salt of the tricyclic derivative compound manufactured by the above manufacturing method can be manufactured in a crystalline form, and when it is in the citric anhydride crystalline form, an organic solvent among the solvents described above can be used as the solvent in the step (b), and a mixed solvent of methanol and ethanol can be preferably used. At this time, in the mixed solvent, methanol and ethanol can be mixed and used in a volume ratio of 1:9 to 9:1, and preferably can be used in a volume ratio of 4:9 to 2:7.

[0094] In addition, when the citric acid salt of the tricyclic derivative compound is a monohydrate, in the step (b), the organic solvent described above and water can be mixed and used in a volume ratio of 1:9 to 9:1, and a mixed solvent of methanol and water can be preferably used. At this time, methanol and water in the mixed solvent can be used in a volume ratio of 3:7 to 7:3.

[0095] In addition, when the citric acid salt of the tricyclic derivative compound is a dihydrate, the organic solvent described above and water may be mixed and used as a solvent in a volume ratio of 2:8 to 0:10 in the step (b), and it may be preferable to use water.

[0096] After the reaction in step (b) above is completed, a further filtration process can be performed on the reaction product. At this time, a crystalline compound can be obtained with excellent purity and yield by filtration and drying after the completion of the reaction without additional processes such as separate recrystallization or chromatography.

[0097]

[0098] Method 2

[0099] The present invention provides a method for preparing a crystalline form of a tricyclic derivative compound represented by the following reaction scheme 1-2. The manufacturing method of the present invention provides excellent yield and purity, and is characterized by significantly improving manufacturing efficiency through simplification of the manufacturing process.

[0100] The citric acid salt crystal form of the tricyclic derivative compound represented by the above chemical formula 1 of the present invention can be prepared by including the following steps.

[0101] (1) A step of reacting a compound of chemical formula 2' and a compound of chemical formula 3' of the following reaction formula 1-2, and then performing a crystallization process to obtain a crystal compound of chemical formula 1; and

[0102] (2) A step of reacting the crystal compound of the above chemical formula 1 with citric acid in at least one solvent selected from the group consisting of organic solvents and water at 50 to 80°C:

[0103] [Reaction Formula 1-2]

[0104]

[0105] Steps (1) and (2) above can be performed in the same manner as steps (a) and (b) of the first method described above, except that a changed starting material is used.

[0106]

[0107] Meanwhile, when preparing the crystal form of the citric acid salt of the tricyclic derivative compound as an anhydride in the first or second method, the anhydride can be prepared as an anhydride crystal form by adding citrate to the tricyclic derivative compound represented by chemical formula 1 in step (b) or step (2), but as shown in the following reaction scheme 2, the compound of chemical formula 1 can be prepared as a citric acid salt monohydrate crystal form, and then the hydrate crystal can be converted back into an anhydride crystal.

[0108] [Reaction Formula 2]

[0109]

[0110] In the above reaction scheme 2, the process of converting the monohydrate into the anhydride can be performed by reacting the monohydrate in an organic solvent or a mixed solvent of an organic solvent and water at a temperature of 50 to 80°C. At this time, a filtration process can be further performed.

[0111] At this time, at least one selected from ethanol, acetone, isopropanol, acetonitrile, propanol, butanol, ethyl acetate, and tetrahydrofuran may be used as the organic solvent.

[0112] Additionally, when using the above mixed solvent, the beneficial effect of reducing the residual solvent level can be provided by including a small amount of water.

[0113] The water contained in the above mixed solvent may be used in an amount of 2 to 15 parts by weight, preferably 4 to 8 parts by weight, based on 100 parts by weight of the monohydrate. If the water is contained in an amount of less than 2 parts by weight, it is difficult to achieve the above-described effect, and if it exceeds 15 parts by weight, conversion to an anhydride may not occur, which is not preferred.

[0114] In this way, when preparing citric acid salt anhydride from citric acid salt hydrate, the effect of additionally removing flexible substances during the crystal conversion process from hydrate to anhydride can be achieved, so that a compound of superior purity can be obtained.

[0115]

[0116] In the method for producing a citric acid salt crystal form of the tricyclic derivative compound of the present invention,

[0117] The reaction of step (a) above may be performed without performing a reflux process, and after the crystallization process in step (a), a recrystallization process may not be further performed, and after the crystallization process in step (a), a column chromatography purification process may not be performed.

[0118] According to the manufacturing method of the present invention, even if the reflux process, recrystallization process, and column chromatography purification process are not all performed, excellent yield and purity can be provided, and the number of manufacturing processes can also be significantly reduced.

[0119]

[0120] In the above manufacturing method, the compound of chemical formula 2 of the above reaction scheme 1-1

[0121] (a) A step of adding an organic solvent to the compound of chemical formula 4 of the following reaction scheme 3-1 and then reacting with hydrochloric acid;

[0122] (b) a step of adding an organic solvent and water to the above reactants in a volume ratio of 1:1 to 1:3; and

[0123] (c) a step of adjusting the pH of the reactant of step (b) to 8 to 10, and then filtering; can be manufactured including:

[0124] [Reaction Formula 3-1]

[0125]

[0126] In the above compound 4, pro may be a protecting group selected from the group consisting of an aryl group, a benzyl group, a benzyloxymethyl group, a paramethoxybenzyl group (PMB), and a methoxymethyl group (MOM), and preferably a methoxymethyl group.

[0127] In the above steps (a) and (b), at least one selected from among ethanol, acetone, isopropanol, acetonitrile, propanol, butanol, ethyl acetate, and tetrahydrofuran may be used as the organic solvent.

[0128] In the above step (c), pH control can be performed using a base, and as the base, at least one selected from the group consisting of NaOH, KOH, K2CO3, Na2CO3, NaHCO3, etc. can be used.

[0129]

[0130] In addition, in the above manufacturing method, the compound of chemical formula 2' of the above reaction scheme 1-2

[0131] (1) A step of adding an organic solvent to the compound of chemical formula 4 of the following reaction formula 3-2 and then reacting with hydrochloric acid; and

[0132] (2) After the above reaction is completed, a step of adding an aqueous hydrochloric acid solution to the reaction solution and filtering the reaction solution can be prepared, including:

[0133] [Reaction Formula 3-2]

[0134]

[0135] In the above step (1), the reaction can be carried out at 75 to 85°C. In addition, the reaction can be carried out for 1 to 4 hours, preferably 2 to 3 hours, and stirring can also be performed at this time.

[0136] In the above step (2), the addition of the hydrochloric acid aqueous solution may be performed after cooling the reaction solution, and the cooling temperature may be 10 to 40°C. Preferably, it may be 15 to 25°C.

[0137] The above hydrochloric acid aqueous solution may be used having a hydrochloric acid volume concentration of 5 to 25%, preferably 10 to 20%. The hydrochloric acid aqueous solution may be added in an amount of 0.1 to 6.5 parts by weight, preferably 1.5 to 4.5 parts by weight, based on 100 parts by weight of the starting material.

[0138] In the above step (1), at least one selected from among ethanol, acetone, isopropanol, acetonitrile, propanol, butanol, ethyl acetate, and tetrahydrofuran may be used as the organic solvent.

[0139] After the above step (2), at least one selected from isopropyl alcohol, purified water, etc. may be preferably used for washing.

[0140]

[0141] Pharmaceutical composition comprising crystalline form of citrate salt of tricyclic derivative compound, use thereof, and therapeutic method using same

[0142] The present invention provides a pharmaceutical composition for preventing or treating a disease caused by PARP-1, tankyrase-1 or tankyrase-2 activity, which comprises a crystalline form of a citrate salt of a tricyclic derivative compound represented by the following chemical formula 1 as an active ingredient.

[0143] [Chemical Formula 1]

[0144]

[0145] Diseases caused by the above PARP-1, tankyrase-1 or tankyrase-2 activity may include neuropathic pain, epilepsy, stroke, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, schizophrenia, chronic or acute pain, ischemic brain injury, neuronal loss after hypoxia, trauma and nerve damage, neurodegenerative diseases, atherosclerosis, hyperlipidemia, cardiovascular tissue damage, coronary artery disease, myocardial infarction, angina pectoris, cardiogenic shock and other cardiovascular diseases, diabetic neuropathy, osteoarthritis, osteoporosis or cancer.

[0146] The citrate salt crystalline form of the tricyclic derivative compound of the present invention can be usefully used for the prevention or treatment of diseases caused by PARP-1, tankyrase-1 or tankyrase-2 activity, particularly neuropathic pain, neurodegenerative diseases, cardiovascular diseases, diabetic neuropathy, inflammatory diseases, osteoporosis or cancer, by inhibiting the activity of poly(ADP-ribose) polymerase.

[0147] The above cancer may be an advanced solid cancer, a recurrent solid cancer, or a metastatic solid cancer. The above solid cancer may be, but is not limited to, breast cancer, prostate cancer, pancreatic cancer, ovarian cancer, advanced ovarian cancer, high-grade serous ovarian cancer (including fallopian tube cancer or primary peritoneal cancer), metastatic cancer from a primary ovarian cancer, breast cancer, prostate cancer, or pancreatic cancer.

[0148] The composition containing the citric acid salt of the tricyclic derivative compound of the present invention can be formulated and used in the form of oral formulations such as tablets, powders, granules, pills, capsules, suspensions, emulsions, liquids, emulsions, syrups, external preparations, suppositories, or sterile injection solutions, each according to a conventional method.

[0149] The pharmaceutical composition according to the present invention may additionally comprise one or more pharmaceutically acceptable carriers, one or more excipients and / or diluents.

[0150] Non-limiting examples of pharmaceutically suitable carriers include solids and / or liquids, such as ethanol, glycerol, water, and the like. The amount of carrier in the therapeutic composition can range from about 5 to about 99 weight percent, based on the total weight of the therapeutic composition or therapeutic combination. Non-limiting examples of suitable pharmaceutically acceptable excipients and diluents include non-toxic compatible fillers, binders, disintegrants, buffers, preservatives, wetting agents, bulking agents, antioxidants, lubricants, flavoring agents, thickening agents, coloring agents, surfactants, emulsifiers, thickening agents, and the like. Such excipients and diluents include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil, and it will be apparent to those skilled in the art that any other pharmaceutically acceptable carrier, excipient and diluent may be used.

[0151] In the pharmaceutical composition of the present invention, the citrate salt crystalline form of the tricyclic derivative compound is contained in a therapeutically effective amount or a prophylactically effective amount. The preferred dosage of the citrate salt of the tricyclic derivative compound according to the present invention varies depending on the patient's condition and weight, the degree of the disease, the drug form, the administration route, and the period, but can be appropriately selected by those skilled in the art. However, for a desirable effect, the citrate salt crystalline form of the tricyclic derivative compound of the present invention can be administered once or several times a day in an amount of 0.0001 mg to 1000 mg, 0.01 mg to 500 mg, 0.1 mg to 300 mg, or 1 mg to 200 mg, or 50 mg to 200 mg. In the composition of the present invention, the compound of Chemical Formula 1 can be incorporated in an amount of 0.0001 to 50 wt% based on the total weight of the entire composition.

[0152] In addition, the present invention provides a use of the crystalline form of the tricyclic derivative compound, the crystalline form of the citrate salt of the tricyclic derivative compound, or the pharmaceutical composition for the manufacture of a drug for preventing or treating various diseases caused by the activity of PARP-1, tankyrase-1, or tankyrase-2.

[0153] The present invention also provides a method for treating an animal having the diseases described above, comprising administering to the animal an effective amount of a crystalline form of the tricyclic derivative compound, a crystalline form of a citrate salt of the tricyclic derivative compound, or the pharmaceutical composition.

[0154] The citrate salt crystalline form of the tricyclic derivative compound according to the present invention has an excellent inhibitory effect on the activity of PARP-1, tankyrase-1 or tankyrase-2, and can be usefully used for the prevention or treatment of neuropathic pain, neurodegenerative disease, cardiovascular disease, diabetic neuropathy, inflammatory disease, osteoporosis or cancer.

[0155] In addition, the citric acid salt crystal form of the tricyclic derivative compound according to the present invention has improved purity, solubility, and stability, facilitating the manufacture of pharmaceutical formulations and providing the effect of improving storage stability.

[0156] The manufacturing method of the present invention provides a compound of excellent yield and purity, and significantly improves manufacturing efficiency by simplifying the manufacturing process, thereby providing a method that is commercially very useful while enabling mass production.

[0157] Figure 1 is a graph showing the XRD analysis peaks of the compound of chemical formula 1 (anhydride) manufactured in Example 1.

[0158] Figure 2 is a graph showing the DSC analysis endothermic peak of the compound of chemical formula 1 (anhydride) manufactured in Example 1.

[0159] Figure 3 is a graph showing the TGA analysis results of the compound of chemical formula 1 (anhydride) manufactured in Example 1.

[0160] Figure 4 is a graph showing the XRD analysis peaks of the compound of chemical formula 1 (dihydrate) manufactured in Example 1-4.

[0161] Figure 5 is a graph showing the DSC analysis endothermic peak of the chemical formula 1 compound (dihydrate) prepared in Example 1-4.

[0162] Figure 6 is a graph showing the TGA analysis results of the compound of chemical formula 1 (dihydrate) manufactured in Example 1-4.

[0163] Figure 7 is a graph showing the XRD analysis peaks of the citric acid salt (anhydride) of the compound of chemical formula 1 manufactured in Example 2.

[0164] Figure 8 is a graph showing the DSC analysis endothermic peak of the citric acid salt (anhydride) of the compound of formula 1 prepared in Example 2.

[0165] Figure 9 is a graph showing the TGA analysis results of the citric acid salt (anhydride) of the compound of chemical formula 1 manufactured in Example 2.

[0166] Figure 10 is a graph showing the XRD analysis peaks of the citric acid salt (monohydrate) of the compound of chemical formula 1 manufactured in Example 3.

[0167] Figure 11 is a graph showing the DSC analysis endothermic peak of the citric acid salt (monohydrate) of the compound of formula 1 prepared in Example 3.

[0168] Figure 12 is a graph showing the TGA analysis results of the citric acid salt (monohydrate) of the compound of chemical formula 1 manufactured in Example 3.

[0169] Figure 13 is a graph showing the XRD analysis peaks of the citric acid salt (dihydrate) of the compound of chemical formula 1 manufactured in Example 4.

[0170] Figure 14 is a graph showing the DSC analysis endothermic peak of the citric acid salt (dihydrate) of the compound of formula 1 prepared in Example 4.

[0171] Figure 15 is a graph showing the TGA analysis results of the citric acid salt (dihydrate) of the compound of chemical formula 1 manufactured in Example 4.

[0172] Figure 16 is a graph showing the PARP-1 enzyme inhibitory activity of the citric acid salt anhydride crystalline form of the chemical formula 1 compound of the present invention and conventional drugs.

[0173] Figure 17 is a graph showing the results of measuring the tankyrase-1 enzyme inhibitory activity of the citric acid anhydride of the chemical formula 1 compound of the present invention and conventional drugs.

[0174] Figure 18 is a graph showing the results of measuring the tankyrase-2 enzyme inhibitory activity of the citric acid salt anhydride of the chemical formula 1 compound of the present invention and conventional drugs.

[0175] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0176]

[0177] Manufacturing Example 1: Preparation of Compound 2

[0178]

[0179] To the above compound 5 (4.5 kg, 15.37 mol) was added 46.7 L of ethanol, followed by the addition of c-HCl (5.3 L, 61.48 mol). The mixture was stirred under reflux for 2 hours, and the completion of the reaction was confirmed. After cooling, 16 L of ethanol and 29.4 L of purified water were added. After adjusting the pH to 8-10 with a 6 N NaOH aqueous solution, the mixture was filtered to obtain compound 2 (3.46 kg, yield 84.4%).

[0180]

[0181] Manufacturing Example 2: Manufacturing of Compound 2'

[0182]

[0183] Isopropanol (6813 mL) was added to the above compound 5 (681.3 g, 2.33 mol), followed by c-HCl (970 g, 9.32 mol). The mixture was stirred at 75–85°C for 2–3 hours. After confirming the completion of the reaction, cooling was performed, and 6N-HCl aqueous solution was added. The resulting solid was filtered to obtain compound 2' (600 g, yield 85.1%).

[0184]

[0185] 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)

[0186]

[0187] (1) Example 1-1

[0188]

[0189] DMF (65 L) was added to the above 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 and stirred at 45-50°C for 5 hours. After confirming the completion of the reaction, the mixture was cooled and methanol (130 L) was added to crystallize. 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 and filtering, compound 1 (7.34 kg, yield 75.2%) was obtained.

[0190]

[0191] (2) Example 1-2

[0192]

[0193] DMF (100 mL) was added to the above 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 and stirred at 38-42°C for 6 hours. After confirming the completion of the reaction, the mixture was cooled and purified water (100 mL) was added to crystallize. The resulting solid was filtered to obtain compound 1 (13.99 g, yield 93%).

[0194]

[0195] (3) Example 1-3

[0196]

[0197] DMF (9.96 L) was added to the above compound 3` (750.34 g, 2.87 mol), triethylamine (1097 g, 10.84 mol) was added, and the mixture was stirred for 2 hours. Compound 2` (664.3 g, 2.19 mol) was added and stirred at 45-50°C for 5.5 hours. After confirming the completion of the reaction, the mixture was cooled and purified water (19.93 L) was added to crystallize. The resulting solid was filtered to obtain anhydrous crystals of compound 1 (788.41 g, yield 89.9%).

[0198]

[0199] (4) Example 1-4

[0200] 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 THF (1.6 L) and stirred at 45 to 55°C to dissolve. After filtration, purified water (3.2 L) was added and cooled to 0 to 10°C. Through the process of stirring and filtering at the same temperature, dihydrate crystals of compound 1 were obtained.

[0201]

[0202] Compound 1 prepared in Examples 1-1 to 1-3, as shown in FIGS. 1 to 3, showed peaks with 2θ values ​​of 7.88°, 10.23°, 13.89°, 15.16°, 15.78°, 18.05°, 19.27°, 19.51°, 22.79°, and 23.94° in XRD (X-Ray Diffraction) analysis ( FIG. 1 ), and an endothermic peak in DSC (Differential Scanning Calorimetry) analysis was confirmed to appear at 272°C ( FIG. 2 ). In addition, it was confirmed to be an anhydrous crystalline form as no mass loss was observed in the initial TGA (Thermogravimetric Analysis) analysis result ( FIG. 3 ).

[0203] The dihydrate crystals of compound 1 additionally manufactured in the above Examples 1-4 showed peaks with 2θ values ​​of 7.95°, 10.25°, 13.25°, 13.78°, 21.12°, and 25.22° in XRD (X-Ray Diffraction) analysis (Fig. 4), and endothermic peaks in DSC (Differential Scanning Calorimetry) analysis were confirmed to appear at 117°C and 272°C (Fig. 5). In addition, the TGA (Thermogravimetric Analysis) analysis results showed a mass loss of approximately 7.7% around 94°C, confirming that it was a dihydrate (Fig. 6).

[0204]

[0205] Example 2: Synthesis of 6-{4-[(5-oxo-1,2,3,4,5,6-hexahydrobenzo[h][1,6]naphthyridin-8-yl)methyl]piperazin-1-yl}nicotinonitrile citrate anhydride

[0206] (1) Example 2-1

[0207]

[0208] 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 was added and stirred at 65-70°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%).

[0209]

[0210] (2) Example 2-2

[0211]

[0212] Ethanol (15.5 L), acetone (15.5 L), and isopropanol (15.5 L) were added to 6-{4-[(5-oxo-1,2,3,4,5,6-hexahydrobenzo[h][1,6]naphthyridin-8-yl)methyl]piperazin-1-yl}nicotinonitrile citrate (monohydrate) (4.7 kg, 7.7 mol) prepared in Example 3 below. After heating to 55°C, the mixture was stirred at 55 to 70°C for 4 hours. After cooling to 25°C or below, the mixture was stirred for 30 minutes. The resulting solid was filtered to obtain the crystalline form of the target compound (4.39 kg, yield 96.3%).

[0213]

[0214] (3) Example 2-3

[0215]

[0216] To 6-{4-[(5-oxo-1,2,3,4,5,6-hexahydrobenzo[h][1,6]naphthyridin-8-yl)methyl]piperazin-1-yl}nicotinonitrile citrate (monohydrate) (500 g, 0.82 mol) prepared in Example 3 below, ethanol (2.5 L), acetone (2.5 L), and isopropanol (2.5 L) were added, and purified water (20 mL) was added. After heating to 55°C, the mixture was stirred at 55 to 75°C for 4 hours. After cooling to 25°C or lower, the mixture was stirred for 30 minutes. The resulting solid was filtered to obtain the crystalline form of the target compound (470 g, yield 96.7%).

[0217]

[0218] As shown in FIGS. 7 to 9, the citric acid salt anhydride crystal compound of the above chemical formula 1 exhibited peaks with 2θ values ​​of 9.79°, 10.01°, 15.86°, 19.62°, 20.10°, 21.71°, and 26.58 in XRD analysis (FIG. 7), and endothermic peaks in DSC analysis appeared at 230°C and 270°C (FIG. 8). In addition, it was confirmed to be an anhydride as no mass loss was observed in the initial TGA analysis (FIG. 9).

[0219]

[0220] The methods of the above Examples 2-2 and 2-3 showed the effect of additionally removing flexible substances in the process of converting from a hydrate to an anhydride, thereby enabling the acquisition of an anhydrous crystalline compound of higher purity.

[0221]

[0222] Example 3: Synthesis of 6-{4-[(5-oxo-1,2,3,4,5,6-hexahydrobenzo[h][1,6]naphthyridin-8-yl)methyl]piperazin-1-yl}nicotinonitrile citrate monohydrate

[0223]

[0224] 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. Citric acid (5.28 kg, 27.49 mol) was dissolved in a 1:1 mixture of methanol and purified water (22 L) and then added. After stirring at 15-25°C for 30 minutes, the temperature was increased to 60°C and stirred at 60-70°C for 2 hours. After cooling to room temperature and filtering, the target compound (10.7 kg, 95.8%) was obtained.

[0225] As shown in FIGS. 10 to 12, the citric acid salt monohydrate crystal compound of the above chemical formula 1 exhibited peaks with 2θ values ​​of 6.94°, 9.99°, 11.89°, 13.35°, 15.07°, 16.57°, 18.17°, 20.90°, 23.68°, and 26.39 in XRD analysis ( FIG. 10 ), and endothermic peaks in DSC analysis appeared at 120°C and 193°C ( FIG. 11 ). In addition, it was confirmed to be a monohydrate from the TGA analysis result showing a mass loss of approximately 2.4% at 80 to 120°C ( FIG. 12 ).

[0226] The above manufacturing method produces the target compound in crystalline form upon completion of the reaction. Therefore, no additional steps are required to increase purity, such as recrystallization, or to reduce residual solvent.

[0227]

[0228] Example 4: Synthesis of 6-{4-[(5-oxo-1,2,3,4,5,6-hexahydrobenzo[h][1,6]naphthyridin-8-yl)methyl]piperazin-1-yl}nicotinonitrile citrate dihydrate

[0229]

[0230] Purified water (42 mL) was added to compound 1 (6 g, 15 mmol) prepared in Example 1. Citric acid (3.6 g, 22.5 mmol) was dissolved in purified water (18 mL) and added. After stirring at room temperature for 30 minutes, the temperature was increased to 60°C and stirred at 60-65°C for 1.5 hours. After cooling to room temperature, the mixture was filtered to obtain the target compound (8.45 g, yield 97.8%).

[0231] As shown in FIGS. 13 to 15, the citric acid salt dihydrate crystal compound of the above chemical formula 1 exhibited peaks with 2θ values ​​of 8.15°, 10.96°, 13.35°, 16.09°, 18.73°, 21.47°, 25.45°, 26.86°, and 28.51° in XRD analysis (FIG. 13), and endothermic peaks in DSC analysis appeared at 140°C, 176°C, and 266°C (FIG. 14). In addition, it was confirmed to be a dihydrate from the TGA analysis result that a mass loss of approximately 5.6% was observed at 100 to 150°C (FIG. 15).

[0232]

[0233] Comparative 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)

[0234] (1) Comparative synthesis example 1-1

[0235]

[0236] 35.8 L of methanol was added to the above compounds 2 (2.56 kg, 9.6 mol) and 3 (1.98 kg, 10.56 mol). Triethylamine (2.67 L, 19.2 mol) was added and refluxed and stirred for 24 hours. After confirming the completion of the reaction, DMAC (38.4 L) and THF (38.4 L) were added and the temperature was increased to dissolve. Activated carbon was added to the solution and stirred. After filtration, it was added dropwise to purified water to crystallize and filtered.

[0237] DMAC and THF were added to the wet solid, dissolved, and recrystallized to obtain compound 1 (2.93 kg, 71.1%). (Step 1)

[0238] Afterwards, an additional purification process was performed to remove flexible substances, obtaining clean compound 1 (1.59 kg, yield 39%). (Step 2)

[0239]

[0240] (2) Comparative synthesis example 1-2

[0241] (Step a)

[0242]

[0243] After adding methanol to the above compound 5, 6-(piperazin-1-yl)nicotinonitrile and triethylamine were added. The mixture was stirred at 80°C for 24 hours, and the reaction solution was concentrated after confirming the completion of the reaction. After extraction with dichloromethane, the organic solvent layer was dried over anhydrous magnesium sulfate, and the solvent was concentrated under reduced pressure. The residue obtained was purified by column chromatography (hexane:ethyl acetate = 1:9) to obtain a compound (yield: 75%) according to the above reaction scheme.

[0244] (Step b)

[0245]

[0246] The compound prepared in step a was dissolved in dichloromethane, trifluoroacetic acid was added, and the mixture was heated using a reflux condenser for 24 hours. After confirming the completion of the reaction, the mixture was extracted with dichloromethane and then extracted once more with dichloromethane in a saturated aqueous solution of sodium bicarbonate. The residue was dried over anhydrous magnesium sulfate, and the solvent was concentrated under reduced pressure. The residue was purified by column chromatography (dichloromethane: methanol = 1:9) to obtain the compound of chemical formula 1 (yield: 65%).

[0247]

[0248] Comparative Example 2: Synthesis of 6-{4-[(5-oxo-1,2,3,4,5,6-hexahydrobenzo[h][1,6]naphthyridin-8-yl)methyl]piperazin-1-yl}nicotinonitrile dihydrochloride

[0249]

[0250] Compound 1 obtained in the above comparative synthesis example 1-2 was dissolved in methanol, a 1.25 M methanol hydrochloric acid solution was added, and the mixture was stirred for 12 hours. After completion of the reaction, the solvent was concentrated under reduced pressure, and the solid was filtered with ethyl acetate to obtain the dihydrochloride salt (yield: 95%) of the above compound 1.

[0251]

[0252] Analysis methods: moisture content, DSC, TGA, and XRD analysis

[0253] The moisture content, DSC, TGA, and XRD analyses described in the present invention were performed using the following methods.

[0254] (1) Moisture content measurement

[0255] Moisture content was measured using an 870KF Titrino Plus (Metrohm) Karl-Fischer moisture meter.

[0256] (2) DSC analysis

[0257] DSC analysis was performed on a DSC 8000 (PerkinElmer) analyzer at 30–250–350°C. 0.5–2 mg of sample was weighed into an aluminum DSC pan, sealed non-hermetically with a perforated aluminum lid, and the sample was heated from 30°C to 250–350°C at a scan rate of 10°C / min to monitor the resulting heat flow response.

[0258] (3) TGA analysis

[0259] TGA analysis was performed on a TGA 8000 (PerkinElmer) analyzer at 30–900°C. 0.5–2 mg of sample was weighed into a ceramic crucible, and the mass loss was monitored by heating the sample from 30 to 900°C at a scan rate of 5°C / min.

[0260] (4) XRD analysis

[0261] XRD analysis was performed on a D8 Focus (Bruker ASX) analyzer from 2˚ 2θ to 40˚ 2θ. Approximately 100 mg of sample was gently pressed into a plastic sample holder to ensure that the sample surface was smooth and just above the level of the sample holder, and measurements were taken under the following conditions.

[0262] <Analysis Conditions>

[0263] Anode material (Ka): Cu Ka (1.5406Å); Scan range: 2–40°; Genie settings: 40 mA, 40 kV; Scan speed: 10° / min; Divergence slit size: 0.6 mm; Temperature: 25°C; Step size: 0.02° 2θ; Rotation: Enabled

[0264]

[0265] Experimental Example 1: Analysis of Flexible Materials

[0266] Using HPLC (product name: Agilent 1100, manufacturer: Agilent), the amount of flexible substances contained in the compounds prepared in the above examples and comparative examples was measured, and the results are shown in Table 2 below.

[0267] <HPLC 조작 조건>

[0268] Detector: UV detector (detection wavelength: 220 nm)

[0269] Column: Watchers 100 ODS-P, 4.6 X 250mm, 5㎛ or equivalent column

[0270] Temperature: Constant temperature around 25℃

[0271] Mobile phase A: Dissolve 11.503g of ammonium phosphate monobasic in 1000mL of water, filter, and degas to prepare. Mobile phase B: Acetonitrile.

[0272] Mobile phase concentration gradient:

[0273] Analysis time (min)Mobile phase A (%)Mobile phase B (%)0851558020158020207525257525753070768515908515

[0274] Diluent: Mix mobile phase A and acetonitrile in a 5:5 ratio.

[0275] Flow rate: 1.0 mL / min, injection volume: 10.0 μL, analysis time: 90 min

[0276] * Preparation of test solution: Dissolve 10 mg of sample in 50 mL of diluent and use.

[0277] Comparative analysis of the free form of compound 1 crystals Comparative synthesis example 1-1 Manufactured compound 1 (step 1) Comparative synthesis example 1-1 Manufactured compound 1 (step 2) Example 1-2 Manufactured compound 1 RT (min) Area (%) RT (min) Area (%) RT (min) Area (%) 43.8840.243544.0060.0530--49.1850.247548.9870.165548.7340.0225 Analysis of the free form of compound 1 citrate crystals Example 3 Manufactured compound 1 citrate crystals Example 2-3 Manufactured compound 1 citrate crystals RT (min) Area (%) RT (min) Area (%) ---- 48.8430.0122--

[0278]

[0279] As a result of the above experiment, in the case of the citric acid salt crystal form of compound 1 manufactured by the example of the present invention, it was confirmed that the major flexible substances were significantly reduced or not detected even though no recrystallization process was performed throughout the entire manufacturing process.

[0280] The flexible substances that appear around the retention time of 49 minutes during HPLC analysis are predicted to be substances that are produced by excessive reaction of the coupling process of the chemical formulas 2 and 3, or 5 and 3, and are difficult to separate and remove after production. Therefore, it is very important to reduce the flexible substances during the manufacturing process.

[0281] Flexible materials near RT 49:

[0282]

[0283] Experimental Example 2: Measurement of solubility in water

[0284] The solubility of compound 1 in water was measured, and the results are shown in Table 4 below.

[0285] Specifically, an excess of compound and solvent (10 mM, in 1 mL water) were added to a shake flask, shaken for 24 hours, and then quantitatively analyzed using UPLC.

[0286] <Equipment and Conditions>

[0287] Equipment name: Acquity UPLC (Waters)

[0288] Column: ACQUITY UPLC BEH C18 1.7um

[0289] Wavelength: 226, 276, 310nm (PDA detector)

[0290] Column temperature: 40℃, sample temperature: 25℃, injection volume: 5ul

[0291] Mobile phase and concentration gradient

[0292] Time(min)Water(0.2% phosphoric acid)Acetonitrile090100.390101.51991.601001.890103.09010

[0293] The compound is dissolved in DMSO, measured using UPLC, and a calibration curve is drawn.

[0294] The compound saturated by the Schke-flask method is filtered, measured by UPLC, and the concentration is calculated by substituting it into the calibration curve.

[0295] Salt Water solubility (mM) Salt Water solubility (mM) Compound 1 citric acid salt crystal anhydrous 1.067 Compound 1 succinic acid salt crystal anhydrous 0.498 Compound 1 tartrate salt crystal anhydrous 0.241 Compound 1 fumarate salt crystal anhydrous 0.444 Compound 1 2HCl salt crystal anhydrous 0.392 Comparative Example 1-1 Compound of Chemical Formula 1 crystal (Free form) 0.001

[0296] As a result of the above experiment, it was confirmed that the citrate crystal anhydride of compound 1 of the present invention has significantly superior solubility in water compared to other salts.

[0297]

[0298] Experimental Example 3: Confirmation of the stability of the addition salt of compound 1

[0299] The stability of the addition salt crystals of compound 1 manufactured in the above examples and comparative examples was confirmed, and the results are shown in Table 5 below.

[0300] Experiments according to temperature, humidity, and light conditions were conducted by storing the samples under each condition in a stability chamber and then confirming the purity using HPLC.

[0301] <UV 200 watt 조사>

[0302] After spreading each sample thinly in a petri dish, the stability was evaluated by irradiating it with UV light of 35 W at a total irradiation dose of 200 watts under conditions of 25°C and 60% humidity using a photostability test chamber (CARON 6542-2).

[0303] <Visible 1200k lux 조사>

[0304] After spreading each sample thinly in a petri dish, the stability was evaluated by irradiating the sample with visible light of 35k lux at a total irradiance of 1200k lux using a light stability test chamber (CARON 6542-2) under conditions of 25℃ and 60% humidity.

[0305] Conditional Purity (%) Comparative Example 2 2HCl salt crystal anhydrous Example 2 Citric acid salt crystal anhydrous Initial value 97.89 97.97 Temperature 50℃, 3 days 97.79 97.86 Humidity 75%, 3 days 97.76 97.81 Temperature 40℃, humidity 70%, 3 days 97.83 97.79 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

[0306] The above experimental results showed that both the dihydrochloride and the citrate anhydride of compound 1 were stable under temperature and humidity. However, the dihydrochloride showed significantly poor photostability under UV 200 watts and Visible 1200k lux. If photostability is low, storage stability may be reduced not only by sunlight but also by indoor lighting, which may lower purity. Therefore, care must be taken when managing the raw material, such as storing it in a light-shielded storage area. When stability was confirmed in this way, the citrate salt of compound 1 was confirmed to be the most suitable.

[0307]

[0308] Experimental Example 4: Determining the Melting Point of Salt

[0309] The melting points of the citric acid salt compounds prepared in the above examples were measured using a B-454 (BUCHI) melting point measuring device, and the results are shown in Table 6 below.

[0310] Example 2 Citrate salt crystal anhydrous Example 3 Citrate salt crystal monohydrate Example 4 Citrate salt crystal dihydrate 206°C 176°C 173°C

[0311] As a result of the above experiment, it was confirmed that all crystal forms of the citric acid salt of the present invention have a sufficiently high melting point as a pharmaceutical raw material.

[0312]

[0313] Experimental Example 5: Solubility Measurement in Various Solvents

[0314] The solubility of the crystal compounds manufactured in the above examples in various solvents was measured, and the results are shown in Table 7 below.

[0315] Specifically, 0.1 g of the sample was precisely weighed and placed in each solvent, shaken vigorously for 30 seconds every 5 minutes at 15 to 25°C, and the amount dissolved within 30 minutes was measured.

[0316] Solvent solubility (0.1 g / mL) Free form Example 2 Citrate crystal anhydrous Example 3 Citrate crystal monohydrate Example 4 Citrate crystal dihydrate DMF 8 mL 8 mL 2.9 mL 2.5 mL MeOH 300 mL 100 mL or more 9 mL 9 mL HCl aqueous solution (pH 1.1) 350 mL 100 mL or less 50 mL 55 mL LEtOH 1000 mL or more 1000 mL or more 350 mL 450 mL water 1200 mL or more 1000 mL or less 400 mL 400 mL or more

[0317]

[0318] Experimental Example 6: Purity and Stability Evaluation of the Anhydrous and Monohydrate Citrate of Compound 1

[0319] The purity and stability of the citric acid salt crystal anhydrous prepared in Example 2 of the present invention and the citric acid salt crystal monohydrate prepared in Example 3 were evaluated, and the results are shown in Tables 8 and 9 below.

[0320] In the table below, the accelerated storage conditions were 40±2℃ and 75±5% humidity for stability tests, and the long-term storage conditions were 25±2℃ and 60±5% humidity for purity and stability tests. The experimental results were measured under the same conditions as Experimental Example 1 using HPLC (product name: agilent 1290, manufacturer: Agilent).

[0321] (Base %) Accelerated storage condition Purity (Period: month, Area %) Long-term storage condition Purity (Period: month, Area %) Period Initial 136 Initial 136 Example 2 Citrate salt crystals Anhydrous (Purity) 99.66% 99.64% 99.65% 99.54% 99.66% 99.64% 99.66% 99.58% 9121899.67% 99.72% 99.73% 243699.74% 99.73%

[0322] (Base %) Accelerated storage conditions (flexible material) (Period: month, Area %) Long-term storage conditions (flexible material) (Period: month, Area %) Period initial 36 initial 36 9 Example 2 Citrate crystals anhydrous 0.26% 0.24% 0.28% 0.26% 0.28% 0.29% 0.31% 12 18 24 0.29% 0.33% 0.34% 30 36 0.34% 0.30% Period initial 36 initial 36 Example 3 Citrate crystals monohydrate 0.38% 0.34% 0.32% 0.38% 0.29% 0.32% -

[0323]

[0324] Experimental Example 7: Poly(ADP-ribose) polymerase [PARP-1] enzyme inhibition test

[0325] The PARP-1 enzyme inhibitory activity of the citric acid salt crystalline anhydride of the compound of formula 1 of the present invention (Example 2) and the hydrochloride crystalline anhydride of the compound of formula 1 (Comparative Example 2) was assayed as follows using a kit purchased from BPS Bioscience (cat. 80551).

[0326] Histones were coated on a 96-well plate provided in the kit from BPS Bioscience and left at 4°C for 16 hours. After that, the plate was washed four times with PBST (7.5 mM Na2HPO4, 2.5 mM NaH2PO4, 145 mM NaCl, 0.05% Tween 20, pH 7.4), and then blocking buffer (provided in the kit from BPS Bioscience) was added to prevent nonspecific reactions and left at 25°C for one hour. After one hour, the plate was washed again four times with PBST, and various concentrations of compounds of Example 2 and Comparative Example 2 were added to the reaction solution containing PARP-1 enzyme (50 ng / well), assay mixture, and activated DNA, and reacted at 25°C for one hour. After one hour, each well was washed four times with PBST, and streptavidin-linked peroxidase (Strep-HRP, 1:50 dilution) was added to measure the amount of ribosylation by PARP enzyme and reacted at 25°C for 30 minutes. After washing the plate four times with PBST, HRP chemiluminescent substrate was finally added and reacted. The amount of histone ribosylation formed by each enzyme was quantified using Synergy™ H4 Hybrid Multi-Mode Microplate Reader (BioTek Instruments, Inc., US(A). The results obtained for each concentration of the compound of the present invention are the average values ​​obtained from two wells, and the results were analyzed using SigmaPlot 10 (Systat Software Inc., US(A)) to determine the IC of the compound.50 The value was calculated.

[0327] AZD-2281 (Olaparib), a representative PARP inhibitor, was used as a control compound.

[0328] The results of the above experiment are shown in Table 10 below and are graphically depicted in Fig. 16.

[0329] IC 50 AZD-2281 (Olaparib) Comparative Example 2 Hydrochloride Crystal Anhydrous Example 2 Citrate Crystal Anhydrous 5.48 nM 3.03 nM 2.62 nM

[0330]

[0331] Experimental Example 8: Tankyrase-1, 2 Enzyme Inhibition Test

[0332] The tankyrase-1 or tankyrase-2 enzyme inhibitory activity of the citric acid salt crystalline anhydride of the compound of chemical formula 1 of the present invention (Example 2) and the hydrochloride crystalline anhydride of the compound of chemical formula 1 (Comparative Example 2) was assayed as follows using a kit purchased from BPS Bioscience (cat. 80573, 80578).

[0333] Histones were coated on a 96-well plate provided in the kit from BPS Bioscience and incubated at 4°C for 16 hours. Afterwards, the plate was washed four times with PBST (7.5 mM Na2HPO4, 2.5 mM NaH2PO4, 145 mM NaCl, 0.05% Tween 20, pH 7.4), and blocking buffer (provided in the kit from BPS Bioscience) was added to prevent nonspecific reactions and incubated at 25°C for one hour. After one hour, the plate was washed again four times with PBST, and various concentrations of the compounds of the examples were added to the reaction solution containing the assay mixture and tankyrase-1 enzyme (40 ng / well) or tankyrase-2 enzyme (15 ng / well) and reacted at 25°C for one hour. After one hour, each well was washed four times with PBST, and streptavidin-linked peroxidase (Strep-HRP, 1:50 dilution) was added to measure the amount of ribosylation by PARP enzyme and reacted at 25°C for 30 minutes. After washing the plate four times with PBST, HRP chemiluminescent substrate was finally added and reacted. The amount of histone ribosylation formed by each enzyme was quantified using Synergy™ H4 Hybrid Multi-Mode Microplate Reader (BioTek Instruments, Inc., US(A). The results obtained for each concentration of the compound of the present invention are the average values ​​obtained from two wells, and the results were analyzed using SigmaPlot 10 (Systat Software Inc., US(A)) to determine the IC of the compound. 50 The value was calculated.

[0334] As control compounds, XAV-939, a representative tankyrase inhibitor, and BMN-673 (Talazoparib), which was developed as a PARP inhibitor but is also known to be effective in inhibiting tankyrase, were used.

[0335] The above experimental results are shown in Tables 11 and 12 below and are graphically depicted in Figures 17 and 18.

[0336] IC 50 BMN-673 (Talazoparib) XAV-939 Example 2 Citrate anhydrous 8.29 nM 8.22 nM 4.31 nM

[0337] IC 50 BMN-673 (Talazoparib) XAV-939 Comparative Example 2 Dihydrochloride Crystal Anhydrous Example 2 Citrate Crystal Anhydrous 2.53 nM 2.21 nM 1.12 nM 1.06 nM

Claims

1. A citric acid salt crystal form of a tricyclic derivative compound represented by the following chemical formula:

2. In paragraph 1, A citric acid salt crystal form of a tricyclic derivative compound, characterized in that the citric acid salt of the above tricyclic derivative compound is an anhydride.

3. In paragraph 2, A citric acid salt crystal form of a tricyclic derivative compound, characterized in that the above crystal form comprises 2θ (± 0.2°) values ​​of 10.01°, 15.86°, 19.62° and 26.58° in a powder XRD pattern.

4. In paragraph 3, A citric acid salt crystal form of a tricyclic derivative compound, characterized in that the above crystal form further comprises 2θ (± 0.2°) values ​​of 9.79°, 20.10° and 21.71° in a powder XRD pattern.

5. In paragraph 1, A citric acid salt crystal form of a tricyclic derivative compound, characterized in that the citric acid salt of the above tricyclic derivative compound is a monohydrate.

6. In paragraph 5, A citric acid salt crystal form of a tricyclic derivative compound, characterized in that the above crystal form comprises 2θ (± 0.2°) values ​​of 6.94°, 9.99°, 16.57°, 18.17°, 23.68°, and 26.39° in a powder XRD pattern.

7. In paragraph 6, A citric acid salt crystalline form of a tricyclic derivative compound, characterized in that the above crystalline form further comprises 2θ (± 0.2°) values ​​of 11.89°, 13.35°, 15.07°, and 20.90° in a powder XRD pattern.

8. In paragraph 1, A citric acid salt crystal form of a tricyclic derivative compound, characterized in that the citric acid salt of the above tricyclic derivative compound is a dihydrate.

9. In paragraph 8, A citric acid salt crystal form of a tricyclic derivative compound, characterized in that the above crystal form comprises 2θ (± 0.2°) values ​​of 8.15°, 10.96°, 16.09°, 21.47°, 25.45°, and 26.86° in a powder XRD pattern.

10. In paragraph 9, A citric acid salt crystalline form of a tricyclic derivative compound, characterized in that the above crystalline form further comprises 2θ (± 0.2°) values ​​of 13.35°, 18.73°, and 28.51° in a powder XRD pattern.

11. Crystal form of a tricyclic derivative compound represented by the following chemical formula 1: [Chemical Formula 1] 12. In paragraph 11, A crystalline form of a tricyclic derivative compound, characterized in that the above crystalline form comprises 2θ (± 0.2°) values ​​of 7.88°, 10.23°, 15.16°, 19.27°, 22.79° and 23.94° in a powder XRD pattern.

13. In paragraph 11, A crystalline form of a tricyclic derivative compound, characterized in that the above crystalline form comprises 2θ (± 0.2°) values ​​of 7.95°, 10.25°, 13.25°, 13.78°, 21.12°, and 25.22° in a powder XRD pattern. 14.(a) A step of reacting the compound of chemical formula 2 of the following reaction scheme 1-1 with the compound of chemical formula 3, and then performing a crystallization process to obtain a crystal compound of chemical formula 1; and (b) a step of reacting the crystal compound of the above chemical formula 1 with citric acid in at least one solvent selected from the group consisting of organic solvents and water at 50 to 80°C; a method for producing a citric acid salt crystal form of a tricyclic derivative compound represented by the following chemical formula 1: [Reaction Formula 1-1] 15.(1) A step of reacting the compound of chemical formula 2' of the following reaction scheme 1-2 with the compound of chemical formula 3', and then performing a crystallization process to obtain a crystal compound of chemical formula 1; and (2) A method for producing a citric acid salt crystal form of a tricyclic derivative compound represented by the following chemical formula 1, comprising the step of reacting the crystal compound of the above chemical formula 1 with citric acid in at least one solvent selected from the group consisting of organic solvents and water at 50 to 80°C: [Reaction Formula 1-2] 16. In paragraph 14 or 15, A method for producing a citric acid salt crystal form of a tricyclic derivative compound, characterized in that the reaction of step (a) of paragraph 14 or step (1) of paragraph 15 uses at least one selected from N,N-dimethylformamide (DMF), dimethylacetamide (DMAC), N-methylpiperidone (NMP), and dimethyl sulfoxide (DMSO) as a reaction solvent.

17. In paragraph 16, A method for producing a citric acid salt crystal form of a tricyclic derivative compound, characterized in that the crystallization process of step (a) of paragraph 14 or step (1) of paragraph 15 uses methanol or water as a crystallization solvent.

18. In paragraph 14 or 15, A method for producing a citric acid salt crystal form of a tricyclic derivative compound, characterized in that the above crystal form is an anhydride or a hydrate.

19. In Article 18, A method for producing a citric acid salt crystal form of a tricyclic derivative compound, characterized in that the above crystal form is an anhydride, and the anhydride is produced by producing a citric acid salt of a tricyclic derivative compound represented by the chemical formula 1 in a monohydrate crystal form in step (b) of paragraph 14 or step (2) of paragraph 15, and then converting the monohydrate crystal into an anhydride crystal again.

20. In paragraph 14 or 15, A method for producing a citric acid salt crystal form of a compound of formula 1, characterized in that a mixed solvent of methanol and ethanol is used as the solvent in step (b) of paragraph 14 or step (2) of paragraph 15, and an anhydrous crystal form is produced by use of such a solvent.

21. In paragraph 14 or 15, A method for producing a citric acid salt crystal form of a compound of formula 1, characterized in that a mixed solvent of methanol and water is used as the solvent in step (b) of paragraph 14 or step (2) of paragraph 15, and a monohydrate crystal form is produced by use of such a solvent.

22. In paragraph 14 or 15, A method for producing a citric acid salt crystal form of a compound of formula 1, characterized in that water is used as a solvent in step (b) of paragraph 14 or step (2) of paragraph 15, and a dihydrate crystal form is produced by use of such a solvent.

23. A pharmaceutical composition comprising a crystalline form of any one of claims 1 to 13 and at least one pharmaceutically acceptable carrier or diluent.

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