Novel maleate salts of triazolopyrazine derivatives, compositions, methods of use, and processes of manufacturing the same

Abstract

To provide pharmaceutical compositions and methods of using maleate salts of triazolopyrazine derivatives.SOLUTION: In some aspects, pharmaceutical compositions provided herein have improved storage stability and pharmacokinetic properties. Maleate salts of triazolopyrazine derivatives bind competitively to the phosphorylation site of c-MET kinase and can be used for the treatment or prevention of various hyper proliferative disorders.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] Triazolopyrazine derivatives are described in U.S. Patent No. 9,403,831 as substances used to treat hyperproliferative disorders by inhibiting the activity of c-Met kinase, but such triazolopyrazine derivatives are described in the free base form, not in the salt form.

[0002] Therefore, there is a need for new salt forms of novel triazolopyrazine derivatives that substantially retain the pharmacokinetics and therapeutic efficacy of the free base forms, while possessing additional advantageous properties. [Background technology]

[0003] The present inventors have conducted extensive research to discover compounds having inhibitory activity against tyrosine kinases and develop compositions for effectively preventing or treating various hyperproliferative disorders caused by abnormal tyrosine kinase activity. As a result, the present inventors have discovered that a novel salt compound of the compound of Formula 1, which was hitherto unknown, improves the storage safety and photostability of the compound while maintaining its function of inhibiting c-Met kinase activity, thereby completing the present invention.

[0004] According to the present invention, the novel salt compounds of the compound of Chemical Formula 1 of the present invention significantly inhibit the activity of c-Met kinase, which binds to hepatocyte growth factor (HGF) and activates its phosphorylation, thereby catalyzing cell proliferation, migration, and angiogenesis.

[0005] Therefore, the salt compounds of the present invention can be usefully used to treat or prevent various hyperproliferative diseases mediated by abnormal cell proliferation activation and excessive angiogenesis.

[0006] Furthermore, the novel salt compound of the compound of Formula 1 exhibits physical properties, such as melting point, hygroscopicity, solubility, flow properties, or thermodynamic stability, that are different from those of the pure water compound.

[0007] Therefore, the salt compound of the compound of Formula 1 provided in the present invention can be used to select the most suitable form for pharmacological use in the process of drug production or purification, in the process of producing separate drug dosage forms such as capsules, ointments, suspensions, etc., or in the production of drug forms with optimal pharmacokinetic properties. Summary of the Invention [Problem to be solved by the invention]

[0008] In order to improve the poor storage stability and light stability of the pure substance of the triazolopyrazine derivative represented by Chemical Formula 1, and the poor processability in pharmaceutical manufacturing, the present invention performed salt screening on the compound, and thereby developed a new salt of the compound, thereby solving the above problems. [Means for solving the problem]

[0009] The present invention provides novel salt forms of triazolopyrazine derivatives represented by the following Chemical Formula 1, which have improved solubility, safety, and other properties while maintaining pharmacokinetic properties substantially similar to those of the free base form of the compound.

[0010] In one aspect, there is provided a maleate salt of a triazolopyrazine derivative represented by the following chemical formula 1 (ABN401):

[0011] [ka]

[0012] The present invention provides a maleate salt of the triazolopyrazine derivative represented by Chemical Formula 1, which is represented by Chemical Formula 2 below.

[0013] [ka]

[0014] The present invention also provides a method for preparing a maleate salt of the triazolopyrazine derivative represented by Chemical Formula 2, the method comprising:

[0015] (a) adding a compound represented by the following Formula 1 to a reactor containing a solvent; [ka]

[0016] (b) stirring the compound and the solvent in a reactor; (c) adding maleic acid to the solution prepared in (b) in an equivalent ratio of 1:1 to 1:3 relative to Formula 1; (d) stirring the solution prepared in (c); and (e) cooling the solution prepared in (d) to obtain a precipitate of maleate represented by Formula 2 below.

[0017] [ka]

[0018] The present invention also provides pharmaceutical compositions containing the maleate salt of the triazolopyrazine derivative represented by Chemical Formula 2 and pharmaceutically acceptable salts thereof.

[0019] In another aspect, the present invention provides a method for inhibiting c-Met kinase activity in a subject, comprising administering to a subject in need of treatment for a disorder associated with increased c-Met kinase activity a maleate salt of a triazolopyrazine derivative represented by Formula 2.

[0020] One aspect of the present invention is to develop a novel maleate salt of the compound represented by Chemical Formula 1, thereby improving the storage stability of the triazolopyrazine derivative (ABN401) represented by Chemical Formula 1.

[0021] The maleate salt, which is a novel salt of the compound of Formula 1 provided by the present invention, includes maleates having one or more maleic acid groups bound thereto (eg, dimaleates).

[0022] The present invention also provides a method for producing the salt and a pharmaceutical composition containing the salt as an active ingredient.

[0023] The present invention relates to a novel salt of a triazolopyrazine derivative represented by Chemical Formula 1 and a method for preparing the same. The novel salt of the compound provided by the present invention can improve the storage stability of the compound, thereby improving its pharmaceutical usability and processability.

[0024] The novel salts of the compounds inhibit the activity of c-Met tyrosine kinase, a kinase implicated in a variety of abnormal proliferative diseases associated with excessive cell proliferation and growth due to abnormal kinase activity, such as cancer, psoriasis, rheumatoid arthritis, and diabetic retinopathy.

[0025] The present invention also discloses a pharmaceutical composition for inhibiting the activity of c-Met tyrosine kinase and a pharmaceutical composition for preventing or treating hyperproliferative diseases, each of which contains the novel salt as an active ingredient.

[0026] The present invention also provides a pharmaceutical composition comprising the maleate salt of the triazolopyrazine derivative represented by Chemical Formula 1 and a pharmaceutically acceptable excipient.

[0027] The present invention also provides a method for treating hyperproliferative diseases using the maleate salt of the triazolopyrazine derivative represented by Formula 1 and a pharmaceutically acceptable excipient.

[0028] In yet another aspect, the present invention provides a method of treating a subject or patient, comprising administering to the subject or patient one or more of the maleate or dimaleate forms to treat, ameliorate, or prevent a hyperproliferative disorder.

[0029] The present invention also provides a method for binding hepatocyte growth factor to a salt of the compound of Formula I by administering one or more of the maleate or dimaleate forms to a patient or subject. [Effects of the Invention]

[0030] The present invention relates to a novel salt compound of a triazolopyrazine derivative represented by Chemical Formula 1 and a method for preparing the same. The novel salt compound provided by the present invention improves storage safety and light stability, which have been problems with pure water substances, and improves the pharmaceutical usefulness and processability of the compound.

[0031] The novel salt of the compound effectively inhibits c-Met tyrosine kinase activity and can be usefully used as a therapeutic agent for various hyperproliferative disorders associated with excessive cell proliferation and growth due to abnormal kinase activity, such as cancer, psoriasis, rheumatoid arthritis, diabetic retinopathy, etc. Also provided are pharmaceutical compositions for inhibiting c-Met tyrosine kinase activity and pharmaceutical compositions for preventing or treating hyperproliferative disorders, each of which contains the novel salt compound as an active ingredient. [Brief explanation of the drawings]

[0032] The features and other advantages of the compounds and pharmaceutical compositions provided by the present invention can be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings. [Figure 1] FIG. 2 is a diagram showing the results of DSC analysis of an example of the maleate salt of Chemical Formula 1 of the present invention. [Figure 2] FIG. 1 shows the results of DVS analysis of an example of the maleate salt of Chemical Formula 1 of the present invention. [Figure 3]FIG. 1 shows the results of H-NMR analysis of an example of the maleate salt of Chemical Formula 1 of the present invention. [Figure 4] FIG. 2 is a graph showing the amount of water absorption by TGA for an example of the maleate salt of Chemical Formula 1 of the present invention. [Figure 5] FIG. 1 shows the results of measuring the purity of the maleate salt of the present invention by HPLC. [Figure 6] FIG. 1 shows the results of measuring the solubility of the maleate salt and free base form of Chemical Formula 1 provided by the present invention. [Figure 7] FIG. 1 shows the results of measuring the solubility of the maleate salt and free base form of Chemical Formula 1 provided by the present invention at different pH values. [Figure 8] FIG. 1 shows the pharmacokinetics of the dimaleate salt of Chemical Formula 1 provided by the present invention. [Figure 9] FIG. 1 shows the pharmacokinetic values of the free base form of Chemical Formula 1. [Figure 10] FIG. 1 shows the pharmacokinetics of dimaleate salt beads of Chemical Formula 1 provided by the present invention. [Figure 11] FIG. 1 shows the pharmacokinetic values of the free base form of Chemical Formula 1. DETAILED DESCRIPTION OF THE INVENTION

[0033] In one aspect, the present invention relates to a novel maleate salt of a triazolopyrazine derivative represented by the following Chemical Formula 1, a method for preparing the same, and a pharmaceutical composition containing the same.

[0034] Surprisingly, the maleate salt described in this invention substantially maintains the pharmacokinetic properties of the free base form, while providing improved properties, for example, related to solubility and hygroscopicity.

[0035] [ka]

[0036] The object of the present invention is to find a compound having inhibitory activity against tyrosine kinase and to develop a composition for effectively preventing or treating various hyperproliferative diseases caused by abnormal activity of tyrosine kinase. As a result, the results described herein show that a novel salt of the compound of Formula 1, which was hitherto unknown, improves the storage safety of the compound while maintaining its function of inhibiting c-Met kinase activity.

[0037] One aspect of the present invention provides a maleate salt of a triazolopyrazine derivative represented by the following Chemical Formula 2:

[0038] [ka]

[0039] In one aspect, the maleate salt provided herein has a chemical purity of at least about 4.7% greater than the free base form represented by Formula 1 after about 15 months at 40° C. and 75% relative humidity.

[0040] In one aspect, the maleate salt provided herein has a moisture content at least about 22% lower than the free base form of Formula 1 after about 15 months at 40° C. and 75% relative humidity.

[0041] In one aspect, the maleate salt provided herein is 75% or more less soluble than the free base form of Formula 1 at pH 1-3.

[0042] In one aspect, the maleate salt provided herein is 75% or more less soluble than the free base form of Formula 1 in 0.05M medium at a pH of about 1.2.

[0043] In one aspect, the maleate salt form provided herein is characterized by a differential scanning calorimeter (DSC) thermogram having endothermic peaks at about 55°C and about 145°C and an exothermic peak at 280°C.

[0044] In one aspect, the maleate salt provided herein has a proton nuclear magnetic resonance (NMR) spectrum with peaks at 3.48-3.58 (m, 2H), 3.64 (brs, 3H), 3.87-4.03 (m, 4H), 4.17-4.28 (m, 1H), 4.40 (brd, J=13.43 Hz, 1H), 4.74 (brd, J=12.21 Hz, 1H), 4.88-5.00 (m, 2H), 6.16 (s, 4H), 7.42 (d, J=8.24 Hz, 2H), 7.66 (d, J=8.24 Hz, 2H), 8.33 (d, J=0.61 Hz, 1H), 8.67 (s, 1H), 8.77 (s, 2H), and 9.23 (s, 1H).

[0045] The present invention also provides a method for preparing a maleate salt of a triazolopyrazine derivative represented by Chemical Formula 2, the method comprising:

[0046] (a) adding a compound represented by the following Formula 1 to a reactor containing a solvent; [ka] (b) stirring the compound and the solvent in a reactor; (c) adding maleic acid to the solution prepared in (b) in an equivalent ratio of 1:1 to 1:3 relative to Formula 1; (d) stirring the solution prepared in (c); and (e) cooling the solution prepared in (d) to obtain a precipitate of maleate represented by Formula 2 below.

[0047] [ka]

[0048] In one aspect, the maleate salt provided herein comprises at least one maleic acid molecule.

[0049] In one aspect, the maleate salt provided herein contains at least two maleic acid molecules.

[0050] In one aspect, the solvent is selected from the group consisting of acetonitrile, acetone, and 1,2-dimethoxyethane.

[0051] In one aspect, step (b) is carried out at about 50° C. for a minimum of 1 hour.

[0052] In one aspect, the precipitate is cooled to about 25° C. and then aged for 24 to 48 hours.

[0053] In another aspect, the present invention provides a pharmaceutical composition comprising a maleate salt of a triazolopyrazine derivative represented by the following Chemical Formula 2 and a pharmaceutically acceptable carrier.

[0054] [ka]

[0055] In another aspect, the pharmaceutically acceptable carrier contained in the pharmaceutical composition provided by the present invention is one or more components selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0056] In addition, the pharmaceutical composition provided by the present invention may further contain lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, and the like in addition to the above ingredients.

[0057] In yet another aspect, the invention provides a method of inhibiting the activity of c-Met kinase in a subject.

[0058] The method includes administering a maleate salt of a triazolopyrazine derivative represented by the following Chemical Formula 2 to a subject in need of treatment for a disease associated with increased activity of c-Met kinase.

[0059] [ka]

[0060] In one aspect, the disease is a hyperproliferative disease.

[0061] In one aspect, the hyperproliferative disease is selected from the group consisting of lung cancer, gastric cancer, pancreatic cancer, colon cancer, ovarian cancer, renal cell carcinoma, prostate cancer and brain cancer.

[0062] In one aspect, the maleate salt is administered to a subject in a pharmaceutically effective amount.

[0063] In another aspect, the maleate salt of the present invention competitively binds to the phosphorylation site of c-Met kinase, thereby significantly inhibiting the activity of c-Met kinase, which induces cell proliferation, migration, and angiogenesis.

[0064] Therefore, the maleate salt provided in the present invention can be useful for treating or preventing various hyperproliferative disorders mediated by abnormal cell proliferation activation and excessive angiogenesis.

[0065] The maleate salt provided by the present invention surprisingly exhibits physical properties, such as melting point, hygroscopicity, solubility, flowability, or thermal stability, different from those of the free base form of the triazolopyrazine derivative represented by Chemical Formula 1, while substantially maintaining the pharmacokinetic properties of the free base form.

[0066] As used herein, the term "substantially maintains the pharmacokinetic properties" refers to having at least 50, 60, 70, 80, 90, or 100% of the various pharmacokinetic parameters of the free base form, as described.

[0067] In one aspect, the maleate salts (e.g., maleate, dimaleate) provided herein surprisingly exhibit significantly reduced solubility compared to the free base form of the compound. For example, as shown in Figure 7, the maleate salt (Formula 2) is more than 50% less soluble at upper pH (gastric pH) than the free base form.

[0068] Table 1. Buffers used in pH-dependent solubility measurements JPEG2025116207000012.jpg65168

[0069] In general, salt forms of a compound would be expected to have increased rather than decreased solubility relative to the free base form (see Handbook of Pharmaceutical Salt: Properties, Selection, and Use, Edited by P. Heinrich Stahl and Camile G. Wermuth. VHCA, Verlag Helvetica Chimica Acta, Zurich, Switzerland, and Wiley-VCH, Weinheim, Germany. Introduction, Page 2, Paragraph 2, 2002).

[0070] The maleate salt provided by the present invention surprisingly exhibits reduced solubility and hygroscopicity. As seen in Figure 4, the maleate salt (Mae2) retains significantly less water content over time than the free base form at 40°C and 75% relative humidity (right panel), almost four-fold over 15 months.

[0071] The maleate salts provided herein exhibit greater safety compared to the free base forms of the compounds. As shown in Figure 5, the maleate salts of Formula 1 generally have higher chemical purity over time compared to the free base form at 40°C and 75% relative humidity (right panel). In particular, the maleate salt form exhibits approximately 4.7% higher chemical purity at 15 months.

[0072] In addition to increased safety, lower solubility, and lower hygroscopicity, the maleate salt provided herein maintains pharmacokinetic (PK) properties substantially similar to those of the free base form (see Figures 8 and 9).

[0073] 8 shows the plasma concentration of the dimaleate salt form represented by Formula 2 over time at specific concentrations in Wistar rats after a single administration. The pharmacokinetic (PK) characteristics are shown in Table 2 below.

[0074] (Table 2) PK values of dimaleate expressed by chemical formula 2 after a single administration JPEG2025116207000013.jpg153168

[0075] 9 shows the plasma concentration of the free base form of Formula 1 over time at specific concentrations in Wistar rats after a single dose. The pharmacokinetic (PK) characteristics are shown in Table 3 below.

[0076] Table 3: PK values of the compound represented by formula 1 after a single administration JPEG2025116207000014.jpg160168

[0077] As shown in Figures 8, 9, Tables 2 and 3, the PK values of the free base form of Formula 1 and the dimaleate salt are essentially equivalent.

[0078] FIG. 10 and Table 4 show the PK values of the compound represented by Formula 2 after twice-daily administration (beads).

[0079] Table 4: PK values of the compound of formula 2 after bead administration JPEG2025116207000015.jpg177134

[0080] FIG. 11 and Table 5 show the PK values of the compound represented by Formula 1 after twice-daily administration.

[0081] Table 5: PK values of the compound of Formula 1 after bead administration JPEG2025116207000016.jpg177133

[0082] As shown in Figures 10, 11, Tables 4 and 5, the PK values of the free base form of Formula 1 and the dimaleate salt are substantially equivalent.

[0083] Therefore, the maleate salt provided in the present invention is a drug form with optimal pharmacokinetic properties that can be selected as the most suitable form for pharmacological use in the drug manufacturing process, purification, and manufacturing of other formulations such as capsules, ointments, and suspensions.

[0084] In one aspect, the invention provides a method of treating, ameliorating, or preventing a hyperproliferative disorder by administering to a subject or patient one or more of the salt forms.

[0085] The present invention also provides a method for binding to hepatocyte growth factor by administering one or more of the salts described above to a patient or subject.

[0086] In another aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of the novel salt of the compound of Chemical Formula 1. For example, the present invention provides a pharmaceutical composition for preventing or treating a hyperproliferative disease, comprising the novel salt of the compound of Chemical Formula 1 as an active ingredient.

[0087] As used herein, the term "hyperproliferative disorder" refers to a pathological condition caused by excessive cell growth, division, and migration that is not regulated by normal restrictive measures in the body of a normally developing animal. Examples of hyperproliferative disorders that can be prevented or treated with the compositions of the present invention include, but are not limited to, cancer, diabetic retinopathy, retinopathy of prematurity, corneal graft rejection, neovascular glaucoma, erythrochromia, proliferative retinopathy, psoriasis, rheumatoid arthritis, osteoarthritis, autoimmune diseases, Crohn's disease, restenosis, atherosclerosis, intestinal adhesions, ulcers, liver cirrhosis, glomerulonephritis, diabetic nephropathy, malignant nephrosclerosis, thrombotic microangiopathy, organ transplant rejection, and neoglomerular diseases, and include all hyperproliferative disorders caused by abnormal cell proliferation and excessive new blood vessel formation.

[0088] More preferably, one of the hyperproliferative diseases that can be prevented and treated with the composition of the present invention is lung cancer, gastric cancer, pancreatic cancer, colon cancer, ovarian cancer, renal cell carcinoma, prostate cancer, or brain cancer.

[0089] The term 'therapeutically effective amount' refers to an amount of an active pharmaceutical ingredient sufficient to treat, ameliorate, cure or reduce the symptoms of a disease or condition.

[0090] Pharmaceutically acceptable carriers included in the pharmaceutical compositions of the present invention include, but are not limited to, commonly used carriers for formulation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above ingredients, the pharmaceutical compositions of the present invention may additionally contain lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc. The pharmaceutical compositions may be provided in any suitable dosage form (e.g., oral solutions, emulsions, syrups, elixirs, refinements, capsules, liquid-filled capsules, Soptogels, sustained-release and solid dosage forms, etc.).

[0091] In one aspect, the compound represented by Chemical Formula 1 can be successfully prepared as a general pharmaceutical composition, but the maleate salt represented by Chemical Formula 2 may have superior physical properties, such as solubility and thermal stability, to Chemical Formula 1 in the preparation of a general pharmaceutical composition.

[0092] Therefore, the present inventors have conducted various salt screenings to develop salts suitable for the manufacture of pharmaceutical formulations, such as those that improve the solubility and safety of triazolopyrazine derivatives. As a result, they have developed novel salts that simultaneously have high solubility and excellent safety, and the pharmacological activity of the novel salt compounds is maintained.

[0093] The present invention will be described in more detail below through examples of the present invention, but the scope of the present invention is not limited to the following examples.

[0094] The present invention relates to a novel maleate salt of the compound of Formula 1, which can effectively prevent or treat various hyperproliferative diseases by inhibiting the activity of c-Met kinase, and a method for preparing the same.

[0095] The present invention relates to a novel maleate salt of the compound represented by Chemical Formula 1. A method for preparing the maleate salt of the compound represented by Chemical Formula 1 will be described below.

[0096] The embodiments presented below may be modified in various other ways, and the scope of the present invention is not limited to the embodiments described below. The embodiments of the present disclosure are intended to more completely explain the present disclosure, and are obvious to those skilled in the art.

[0097] The maleate salt of the triazolopyrazine derivative provided in the present invention is represented by the following chemical formula 2:

[0098] [ka]

[0099] The maleate salt provided by the present invention can be formed by binding at least one maleic acid molecule to a triazolopyrazine derivative of the compound of Formula 1. In one aspect, the maleate salt contains two maleic acid molecules (dimaleate).

[0100] DSC (Differential Scanning Calorimetry) analysis was performed to confirm the thermal stability of the novel salt provided by the present invention, and DVS (Dynamic Vapor Sorption) analysis was performed to confirm the hygroscopicity.

[0101] To confirm the thermal fluidity of the salt, DSC analysis was performed using a DSC3+STARe instrument. The salt sample was attached to an aluminum foil and heated at 25 to 300°C using a pinhole at a heating rate of 10°C / min.

[0102] To confirm the hygroscopicity of the salt, DVS analysis was performed using a DVS-1 instrument at 40%, 95%, and 0% relative humidity at 25° C. for 60 minutes.

[0103] Example 1 Preparation of maleate salt of ABN401 (compound of formula 1) 300 mg of the compound of Formula 1 was dissolved in 5 mL of acetonitrile, and 130 mg of maleic acid was added so that the mole ratio of the compound of Formula 1 to maleic acid was 1:2.1. 5 mL of a 20 mg / mL maleic acid solution was added, and the mixture was reacted at 50°C for 1 hour.

[0104] The reaction solution was cooled to 25°C in 5°C intervals and aged for 48 hours to obtain 350 mg of crystals of the compound of Formula 2.

[0105] The crystals were washed with distilled water and dried under vacuum at 10 mbar and 50°C.

[0106] The compound of Formula 2 obtained through the above process was confirmed by H nuclear magnetic resonance spectrum (NMR), and the peaks were as follows:

[0107] 1H NMR (500MHz, DMSO-d6) δppm2.78 (br s, 3H) 3.48-3.58 (m, 2H), 3.64 (brs, 3H), 3.87-4.03 (m, 4H), 4.17-4.28 (m, 1H), 4.40 (br d, J=13.43Hz, 1H), 4.74(br d, J=12.21Hz, 1H), 4.88-5.00(m, 2H), 6.16(s, 4H), 7.42(d, J=8.24Hz, 2H), 7.66( d, J=8.24Hz, 2H), 8.33(d, J=0.61Hz, 1H), 8.67(s, 1H), 8.77(s, 2H), 9.23(s, 1H).

[0108] DSC analysis of the maleate salt showed that it was endothermic between 25°C and 100°C, which appears to be related to the evaporation of water. The DSC results also showed a strong endothermic peak at 145.1°C, indicating that the maleate salt has much better melting and thermal stability than pure water.

[0109] According to DSC analysis, the maleate salt exhibits endothermic peaks at about 55°C and about 145°C, and an exothermic peak at 280°C.

[0110] The DVS analysis result (FIG. 2) for the compound of Formula 2 shows that the mass loss due to humidity change is very low in the range of 80% or less relative humidity, which indicates that the hygroscopicity of the maleate salt is significantly improved compared to pure water and that the storage stability is also excellent.

[0111] Physical and chemical safety tests were performed on the compounds of Formula 2 and Formula 1 (reference). Long-term safety studies were performed at specific temperatures and relative humidity levels (25°C / 60%RH and 40°C / 75%RH), and solid samples were analyzed by XRPD, TGMS, and HPLC at regular intervals.

[0112] The maleate salt does not absorb any significant level of water after 15 months of incubation, indicating that the maleate salt is a less hygroscopic solid form.

[0113] To measure the chemical purity of the compounds of Chemical Formula 1 and Chemical Formula 2, the compounds were exposed to temperatures of 25°C / 60% RH and 40°C / 75% RH for 1 month, 3 months, and 15 months, and then the chemical purity of the compounds of Chemical Formula 1 and Chemical Formula 2 was measured. The results are shown in Figures 4 and 5.

[0114] The free base form represented by formula 1 underwent partial chemical decomposition over time after incubation at 25°C / 60% RH, with further decomposition occurring at 40°C / 75% RH.

[0115] On the other hand, the maleate salt represented by formula 2 did not show any significant chemical decomposition even after exposure to 25°C / 60%RH and was found to be chemically stable for up to 15 months at 40°C / 75%RH.

[0116] Similar to the exemplary experimental results shown in FIGS. 4 and 5, it can be seen that the maleate salt exhibits approximately 4.7% more chemical purity than the free base form of Chemical Formula 1 after approximately 15 months at 40° C. and 75% relative humidity, and that the maleate salt has approximately 22% more moisture content than the free base form of Chemical Formula 1 after approximately 15 months at 40° C. and 75% relative humidity.

[0117] Based on these results, it can be seen that the compound of Chemical Formula 2 is less hygroscopic than the compound of Chemical Formula 1 and has improved chemical safety.

[0118] To develop a dosage form suitable for parenteral administration, the quantitative solubility of the compounds represented by Formula 1 and Formula 2 was measured in a phosphate buffer solution containing solubility-enhancing excipients at pH 7.4. The target concentration for the solubility was 1-1.3 mg / mL.

[0119] Based on the qualitative solubility results determined in a PBS formulation vehicle containing Transcutol (Gattefosse, France) and TPGS (tocopherolethylene glycol succinate), the quantitative solubilities of Compounds of Formula 1 and Formula 2 were measured near their maximum solubility in the vehicle.

[0120] Next, thermodynamic solubility was determined in formulation vehicles containing PBS buffer containing Transcutol (50% and 10%) and TPGS (20% and 10%), and PEG 400 (50%, with and without 2% DMSO). Suspensions of the compound of Formula 1 and the compound of Formula 2 were prepared in the selected vehicle at a compound of Formula 1 concentration of approximately 3.5-5 mg / mL. After incubation at room temperature for 24 hours, the solid and liquid phases were separated. The API concentrations in the solutions were determined by HPLC analysis, and the results are shown in Figure 6.

[0121] The compound of Formula 1 exhibits a solubility of approximately 1 mg / mL or more in formulation vehicles containing 50% Transcutol, 10% TPGS, and 20% TPGS. The solubility in a vehicle containing PEG400 was approximately 0.3 mg / mL, and when 10% Transcutol alone was used, the solubility was measured to be approximately 0.1 mg / mL.

[0122] The compound of Formula 2 exhibits a solubility of 1.3 mg / mL or more in almost all vehicles except for a vehicle containing 10% transcutol. In a vehicle containing 50% transcutol, the salt was completely dissolved and no precipitation occurred even after 24 hours, and the solubility was as high as 3.5 mg / mL or more.

[0123] The pH of the solutions used in the free base form experiments was shifted to a higher pH, while the pH of the samples of Formula 2 was shifted to a lower pH. The solubility of the compound of Formula 2 was higher than that of the compound of Formula 1 in all formulation vehicles tested.

[0124] From the above examples, the maleate salt is 75% less soluble at pH 1 to 3 than the free base form of Chemical Formula 1. Also, the maleate salt is 75% less soluble in 0.05M gastric juice at a pH of approximately 1.2 than the free base form of Chemical Formula 1.

[0125] The present invention also provides a pharmaceutical composition containing the novel salt of the triazolopyrazine derivative represented by Chemical Formula 1 as an active ingredient.

[0126] The active ingredient of the pharmaceutical composition provided by the present invention is the maleate salt of the compound represented by Chemical Formula 2, and the pharmaceutical composition includes any pharmaceutically acceptable carrier.

[0127] To test the activity of the maleate compounds represented by Formula 2 described herein, the following exemplary experimental protocol was performed.

[0128] RPMI 1640 cell culture medium, fetal bovine serum (FBS), and trypsin were purchased from GIBCO Co., Ltd. (Grand Island, NY), and sodium bicarbonate, amphotericin B, and gentamicin were used from Signemic Pharmaceuticals. Reagents used in cytotoxicity assays, such as sulforhodamine (SRB) B, Trisma Base, and trichloroacetic acid (TCA), were purchased from Sigma Chemical Co., Ltd. For MTS analysis, Celltiter 96 Aqueous Non-Radioactive Cell Proliferation Assay products were purchased from Promega Co., Ltd. T-25 culture vessels, 96-well plates, and other disposable glassware used for cell culture were purchased from Folkon (Lincoln Park, NJ).

[0129] Drugs used in the experiments were diluted to the desired concentrations in experimental medium, and the final dimethyl sulfoxide (DMSO) concentration was 0.5% or less.

[0130] The cancer cell lines used in the experiment were a human cancer cell line and the lung cancer cell line EBC-1. The cells were cultured in RPMI1640 medium containing 10% FBS (fetal bovine serum) at 37°C in a 5% CO2 incubator, and subcultured once every 3-4 days.

[0131] Specifically, after incubation with the compounds of the present invention, the culture medium was removed and cold TCA solution was added to each well. The cells were then left at 4°C for 1 hour to fixate. The TCA solution was then removed, and the cells were dried at room temperature. They were then incubated with 0.4% SRB in 1% acetic acid, and then left at room temperature for 30 minutes before staining. Excess SRB not bound to the cells was washed away with 1% acetic acid, and 10mM Tris buffer (unbuffered) at pH 10.3-10.5 was added to the stained cells to release the SRB.

[0132] The results of the cancer cell proliferation inhibition test of the maleate salt are shown in Table 6 below.

[0133] (Table 6) JPEG2025116207000018.jpg22168

[0134] From the above experimental results, it can be seen that the maleate salt of the triazolopyrazine derivative represented by Chemical Formula 2 provided in the present invention has much improved cancer cell growth inhibitory activity compared to the free base compound represented by Chemical Formula 1. INDUSTRIAL APPLICABILITY

[0135] The present invention is industrially applicable.

Claims

1. A maleate salt of a triazolopyrazine derivative represented by the following chemical formula 2: A maleate salt of a triazolopyrazine derivative, having a purity of 4.7% or more compared to the free base compound represented by the following formula 1 after 15 months at 40°C and 75% relative humidity. 【Chemical 1】 【Chemistry 2】

2. A maleate salt of a triazolopyrazine derivative represented by the following chemical formula 2: A maleate salt of a triazolopyrazine derivative having a moisture content of 22% or less after 15 months at 40° C. and 75% relative humidity compared to a free base compound represented by the following formula 1: 【Chemical 1】 【Chemistry 2】

3. A maleate salt of a triazolopyrazine derivative represented by the following chemical formula 2: A maleate salt of a triazolopyrazine derivative, which has a solubility of 75% or less at pH 1 to 3 compared to the free base compound represented by the following formula 1: 【Chemical 1】 【Chemistry 2】

4. A maleate salt of a triazolopyrazine derivative represented by the following chemical formula 2: A maleate salt of a triazolopyrazine derivative having a solubility in 0.05M gastric juice at pH 1.2 of 75% or less compared to the free base compound represented by the following Chemical Formula 1: 【Chemical 1】 【Chemistry 2】

5. A pharmaceutical composition for treating a hyperproliferative disorder, comprising the maleate salt of the triazolopyrazine derivative of claim 1 and a pharmaceutically acceptable carrier.

6. 6. The pharmaceutical composition of claim 5, wherein the pharmaceutically acceptable carrier is selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

7. The pharmaceutical composition according to claim 5, further comprising one or more ingredients selected from the group consisting of lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, and preservatives.

8. 6. The pharmaceutical composition of claim 5, wherein the hyperproliferative disorder is selected from the group consisting of lung cancer, gastric cancer, pancreatic cancer, colon cancer, ovarian cancer, renal cell carcinoma, prostate cancer, and brain cancer.

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