(E)-N-hydroxy-3-(1-(phenylsulfonyl)indolin-5-yl)acrylamide crystal form

Abstract

This invention discloses specific crystalline forms of (E)-N-hydroxy-3-(1-(phenylsulfonyl)indolin-5-yl)acrylamide (ABT-301), pharmaceutical compositions and capsules containing the same, intermediates, and their medical applications. These crystalline forms of ABT-301 exhibit unexpected stability and improved pharmacokinetic properties compared to other forms or salts thereof, making the compound more suitable for pharmaceutical research and development and meeting the requirements for bioavailability and drug efficacy.

Description

[Technical Field]

[0001] [Cross-references to related applications] This application claims priority and interest to U.S. Provisional Applications No. 63 / 523,377 and No. 63 / 523,378, filed on 27 June 2023, the contents of which are incorporated herein by reference.

[0002] The present invention relates to a novel crystalline form of (E)-N-hydroxy-3-(1-(phenylsulfonyl)indolin-5-yl)acrylamide (ABT-301), a pharmaceutical composition and capsules containing the same, and a method for treating cancer using these crystalline forms. [Background technology]

[0003] Histone deacetylases (HDACs) are a type of enzyme that regulates histone acetylation, further modulates gene expression, and can suppress tumor progression. HDACs are divided into four classes: Class I (HDAC1, 2, 3, and 8), Class IIa (HDAC4, 5, 7, and 9), Class IIb (HDAC6 and 10), Class III (SIRT1-7), and Class IV (HDAC11). These are involved in the post-translational modification of core histones and non-histones.

[0004] These are involved in the post-translational modifications of core histones and non-histones. U.S. Patent No. 8,846,748 describes several hydroxamic acid compounds with indolyl or indoline groups as HDAC inhibitors with potent anticancer activity. Masahiro Yoshikawa et al. have shown that HDAC inhibitors can prevent fibrosis of the liver, skin, and lungs, and U.S. Patent No. 11,278,523B2 discloses the efficacy of HDAC inhibitors in treating pulmonary fibrosis, hepatic fibrosis, or renal fibrosis, although their potential mechanisms remain largely unknown. Furthermore, it has been shown that TSA (an HDAC inhibitor) induces several inhibitors of the TGF-beta1 signaling pathway, such as Id2 and BMP-7, in human RPTEC (Masahiro Yoshikawa et al., J Am Soc Nephrol 18:58-65, 2007). Maoyin Pang and Shougang Zhuang have noted that the onset and progression of several chronic diseases, including chronic kidney disease, cardia hypertrophy, and idiopathic pulmonary fibrosis, are characterized by fibrosis (Maoyin Pang and Shougang Zhuang, The Journal of Pharmacology and Experimental Therapeutics, Vol. 355, No. 2, pp. 266-272, 2010).

[0005] Therefore, HDAC inhibitors (HDACi) are promising drugs in cancer treatment. Several HDACi (e.g., SAHA, LBH589, PXD101, MS-275, FK228) are currently being validated in clinical trials to confirm their therapeutic capabilities against various solid tumors and hematological malignancies. Meanwhile, SAHA and FK228 have been approved by the U.S. Food and Drug Administration (FDA) for their therapeutic application in cutaneous T-cell lymphoma.

[0006] U.S. Patent Application No. 8,846,748B2 discloses several hydroxamic acid compounds with indolyl or indoline groups as HDAC inhibitors. However, the differences in each aspect between the crystalline forms of these compounds have been little discussed. [Overview of the project]

[0007] This invention is based on the finding that several crystalline forms of (E)-N-hydroxy-3-(1-(phenylsulfonyl)indolin-5-yl)acrylamide (ABT-301) exhibit unexpected stability and improved pharmacokinetic properties.

[0008] Accordingly, the present invention provides crystalline forms of (E)-N-hydroxy-3-(1-(phenylsulfonyl)indolin-5-yl)acrylamide (ABT-301), the crystalline forms of which include Type A, characterized in that its X-ray powder diffraction pattern has peaks at 9.6°±0.2°, 15.1°±0.2°, 15.7°±0.2°, 16.7°±0.2°, 18.4°±0.2°, 19.0°±0.2° and 20.4°±0.2° at 2θ values, and Type B, characterized in that its X-ray powder diffraction pattern has peaks at 15.7°±0.2°, 16.6°±0.2°, 20.1°±0.2° and 24.1°±0.2° at 2θ values.

[0009] Furthermore, Type A is characterized in that its X-ray powder diffraction pattern has peaks at 10.2°±0.2°, 11.8°±0.2°, 17.4°±0.2°, 21.3°±0.2°, and 21.9°±0.2° in 2θ values.

[0010] Furthermore, Type A is a hydrate.

[0011] Furthermore, Type B is characterized in that its X-ray powder diffraction pattern has peaks at 12.5°±0.2° and 21.7°±0.2° in 2θ values.

[0012] Furthermore, Type B is an anhydrous substance.

[0013] Also, the crystalline form provided in this specification has a melting point temperature of 125°C to 132°C.

[0014] Also, when the Type A is heated to 100°C to 150°C, the TGA weight loss is 2% to 4%.

[0015] Also, the water absorption rate of the Type A at 25°C / 80%RH is 0.13% to 0.14%.

[0016] Also, when the Type B is heated to 150°C to 170°C, the TGA weight loss is 0.7% to 0.8%.

[0017] Also, the water absorption rate of the Type B at 25°C / 80%RH is 0.103% to 0.109%.

[0018] The present invention provides an intermediate for preparing Type A or Type B, the intermediate is a solvate of ABT-301, and the solvate includes an IPA solvate, an acetone solvate, an ACN solvate, a MeOH solvate, a NMP solvate, a THF solvate, an EtOAc solvate, a DMAc solvate, an EtOH solvate, a DCM solvate, a DMSO solvate, a 1,4-dioxane solvate or a MIBK solvate.

[0019] The present invention provides a pharmaceutical composition comprising the crystalline form, a surfactant, and an oil provided in this specification.

[0020] Also, the surfactant is polysorbate 80.

[0021] Also, the oil is castor oil.

[0022] Also, the crystalline form provided in this specification is an HDAC inhibitor.

[0023] Also, the pharmaceutical composition is in the form of a capsule.

[0024] Furthermore, the capsules provided herein contain 25 mg to 100 mg of the crystalline form.

[0025] Furthermore, the pharmaceutical composition is encapsulated in a gelatin shell.

[0026] The pharmaceutical composition further contains a plasticizer.

[0027] Furthermore, the plasticizer is propylene glycol.

[0028] The present invention provides a method for treating cancer or fibrosis, comprising administering a therapeutically effective dose of the crystalline form provided herein to a patient in need of treatment.

[0029] Furthermore, according to the method of the present invention, the cancers are pancreatic cancer, bladder cancer, colorectal cancer, breast cancer, prostate cancer, kidney cancer, hepatocellular carcinoma, lung cancer, non-small cell lung cancer (NSCLC), ovarian cancer, cervical cancer, stomach cancer, esophageal cancer, neuroendocrine cancer, bone cancer, or head and neck cancer.

[0030] Furthermore, according to the method of the present invention, the fibrosis is pulmonary fibrosis, hepatic fibrosis, cutaneous fibrosis, or renal fibrosis.

[0031] Thus, the mechanism of action of the present invention is that a specific crystalline form of ABT-301 exhibits unexpected stability and improved pharmacokinetic properties compared to other forms or salts thereof, making the compound more suitable for pharmaceutical research and development and capable of meeting the requirements for bioavailability and drug efficacy. [Brief explanation of the drawing]

[0032] To further clarify the above-mentioned and other objectives, features, advantages, and embodiments of the present invention, the drawings are described below.

[0033] [Figure 1] This is an overlay diagram of the XRPD patterns of the crystalline form of ABT-301 free base. [Figure 2]This is an overlay diagram of the XRPD patterns of the crystalline form of ABT-301 free base. [Figure 3] This is an overlay diagram of the XRPD patterns of the crystalline form of ABT-301 free base. [Figure 4] This is the XRPD pattern of the crystalline form of ABT-301 Type A free base. [Figure 5] This is the TGA / DSC graph for the ABT-301 TypeA. [Figure 6] This is the 1H NMR spectrum of ABT-301 Type A. [Figure 7] This is an overlay diagram of the XRPD patterns of ABT-301 TypeA before and after heating. [Figure 8] This is an overlay diagram of the TGA (Thermal Gauge Analysis) of ABT-301 Type A before and after heating. [Figure 9] This is an overlay diagram of the VT-XRPD patterns for ABT-301 TypeA. [Figure 10] This is an overlay diagram of the VT-XRPD patterns for ABT-301 TypeA. [Figure 11] This is an overlay diagram of the VT-XRPD patterns for ABT-301 TypeA. [Figure 12] This is an overlay diagram of the VT-XRPD patterns for ABT-301 TypeA. [Figure 13] This is a DVS diagram of the ABT-301 Type A. [Figure 14] This is an overlay diagram of the XRPD patterns of ABT-301 TypeA before and after DVS (Digital Ventilation). [Figure 15] This is the XRPD pattern of the crystalline form of ABT-301 Type B free base. [Figure 16] This is the TGA / DSC graph for the ABT-301 TypeB. [Figure 17] This is the 1H NMR spectrum of ABT-301 Type B. [Figure 18] This is an overlay diagram of the re-prepared ABT-301 Type B XRPD pattern. [Figure 19] This is the TGA / DSC graph for the ABT-301 TypeB. [Figure 20] This is the 1H NMR spectrum of ABT-301 Type B. [Figure 21] This is a DVS diagram of the ABT-301 Type B. [Figure 22] This is an overlay diagram of the XRPD patterns of ABT-301 TypeB before and after DVS (Digital Ventilation). [Figure 23] This is an overlay of XRPD patterns of competing slurry samples at 50°C. [Figure 24] This is an overlay of XRPD patterns of competing slurry samples at room temperature. [Figure 25] This is an overlay of XRPD patterns of competing slurry samples at room temperature. [Figure 26] This is an overlay of XRPD patterns of competing slurry samples in H2O at room temperature. [Figure 27] This is an overlay of XRPD patterns of competing slurry samples in n-heptane at room temperature. [Figure 28] This is an overlay of XRPD patterns of competing slurry samples in toluene at room temperature. [Figure 29] This is an overlay of XRPD patterns at room temperature for competing slurry samples with different water activity levels. [Figure 30] This is an overlay of XRPD patterns at room temperature for competing slurry samples with different water activity levels. [Figure 31] This is a PLM image of an ABT-301 Type A single crystal. [Figure 32] This is an overlay diagram of the XRPD patterns of an ABT-301 Type A single crystal and a Type A reference. [Figure 33] This is a PLM image of an ABT-301 Type B single crystal. [Figure 34] This is an overlay diagram of the XRPD patterns of an ABT-301 Type B single crystal and a Type B reference. [Figure 35] It is an asymmetric unit of the ABT-301 Type A single crystal structure. [Figure 36] This is the asymmetric unit of the ABT-301 Type B single crystal structure. [Figure 37] This is an overlay diagram of the XRPD patterns of the re-prepared amorphous state (826562-02-A). [Figure 38] This is a TGA / mDSC graph of a re-prepared amorphous material (826562-02-A). [Figure 39] This is a TGA / DSC graph of the re-prepared amorphous state (826562-02-A). [Modes for carrying out the invention]

[0034] definition

[0035] The terms used herein generally fall within the scope of the invention, and the specific context of each term has the usual meaning in its relevant art. Specific terms used herein to describe the invention are described below or elsewhere in this specification to help those skilled in the art understand the relevant descriptions of the invention. The same terms have the same scope and meaning in the same context. Furthermore, since there is no single way to express the same thing, terms discussed herein may be replaced by alternative terms or synonyms, and no special significance is given to the definition or discussion of a term herein. While several synonyms are provided herein, the use of one or more synonyms is not intended to exclude other synonyms.

[0036] As used herein, “one” and “the said” may be interpreted as plural unless otherwise explicitly indicated in the context. Furthermore, in this specification and the appended claims, “intermediate” and “inside” include “located within…” unless otherwise explicitly stated in the context, and the bullet tip direction is defined as “upward” or “downward” unless otherwise explicitly stated in the context. Also, headings and subheadings may be included in this specification for readability, but these headings do not affect the scope of the invention.

[0037] As used herein, the term "crystal" may refer to a molecule or an outer plane having a regular repeating arrangement. Crystal forms may differ in thermodynamic stability, physical parameters, X-ray structure, and preparation process.

[0038] As used herein, the term "amorphous state" may refer to a compound, or a form of salt or molecular complex of a compound, that lacks long-range crystalline order and does not exhibit Bragg reflection in its X-ray diffraction pattern.

[0039] As used herein, the term “solid form” may refer to a crystalline solid form or phase including a crystalline free base, a crystalline salt, or a eutectic, and an amorphous phase including an amorphous dispersion.

[0040] As used herein, the term “solvate” may generally refer to a form of compound that has bonded with a solvent through a dissolution reaction. Such physical bonding may include hydrogen bonding. Conventional solvents include water, methanol, ethanol, acetic acid, DMSO, THF, and diethyl ether. The compounds described herein may be prepared in crystalline or other forms, or they may be dissolved. Suitable solvates include pharmaceutically acceptable solvates, as well as stoichiometric and non-stoichiometric solvates. In some cases, solvates can be separated, for example, by incorporating one or more solvent molecules into a crystalline solid lattice. “Solvates” include solution-phase solvates and separable solvates. Representative solvates include hydrates, glycolates, and methanolates.

[0041] As used herein, unless otherwise specified, the term “treatment” means reversing, alleviating, suppressing, or preventing a disorder or condition, or one or more symptoms of such disorder or condition, to which the term applies. Unless otherwise specified, the term “treatment” means a treatment that “is defined above.”

[0042] As used herein, the terms “effective dose” or “therapeutic dose” mean a quantity of a compound or combination of compounds sufficient to achieve the intended use, including but not limited to the treatment of a disease. The therapeutic dose varies depending on the intended use (in vitro or in vivo) or the individual and disease state being treated (e.g., the individual’s weight, age, and sex), the severity of the disease state, the method of administration, etc., all of which can be readily determined by those skilled in the art. The term also applies to doses that induce a specific response in target cells (e.g., reduced platelet adhesion and / or cell migration). The specific dose varies depending on the particular compound selected, the administration plan to be followed, whether the compound is administered in combination with other compounds, the timing of administration, the tissue to which it is administered, and the physical delivery system through which the compound is carried.

[0043] Crystal form of ABT-301

[0044] In one embodiment, the present invention provides a crystalline form of (E)-N-hydroxy-3-(1-(phenylsulfonyl)indolin-5-yl)acrylamide (ABT-301) represented by formula I.

[0045] [ka]

[0046] ABT-301 (formerly known as MPT0E028) is a novel N-hydroxyacrylamide derivative HDAC inhibitor whose crystalline form includes Type A (hereinafter also referred to as "Type A") and / or Type B (hereinafter also referred to as "Type B").

[0047] Type A is a hydrate characterized in that its X-ray powder diffraction pattern contains peaks selected from the group consisting of 2θ values ​​of 9.6°±0.2°, 15.1°±0.2°, 15.7°±0.2°, 16.7°±0.2°, 18.4°±0.2°, 19.0°±0.2°, and 20.4°±0.2°. Preferably, Type A is characterized in that its X-ray powder diffraction pattern contains peaks selected from the group consisting of 2θ values ​​of 9.6°±0.2°, 15.1°±0.2°, 15.7°±0.2°, 16.7°±0.2°, 18.4°±0.2°, 19.0°±0.2°, and 20.4°±0.2°. In another embodiment, type A is characterized in that its X-ray powder diffraction pattern includes peaks selected from the group consisting of 2θ values ​​of 10.2°±0.2°, 11.8°±0.2°, 17.4°±0.2°, 21.3°±0.2°, and 21.9°±0.2°. More preferably, type A is characterized in that its X-ray powder diffraction pattern further includes peaks at 2θ values ​​of 10.2°±0.2°, 11.8°±0.2°, 17.4°±0.2°, 21.3°±0.2°, and 21.9°±0.2°.

[0048] Type B is an anhydrous material characterized in that its X-ray powder diffraction pattern contains peaks selected from the group consisting of 2θ values ​​of 15.7°±0.2°, 16.6°±0.2°, 20.1°±0.2°, and 24.1°±0.2°. Preferably, Type B is characterized in that its X-ray powder diffraction pattern contains peaks at 2θ values ​​of 15.7°±0.2°, 16.6°±0.2°, 20.1°±0.2°, and 24.1°±0.2°. In another embodiment, Type B is characterized in that its X-ray powder diffraction pattern further contains peaks at 2θ values ​​of 12.5°±0.2° and / or 21.7°±0.2°. More preferably, Type B is characterized in that its X-ray powder diffraction pattern further contains peaks at 2θ values ​​of 12.5°±0.2° and 21.7°±0.2°.

[0049] In the art to which the present invention pertains, it is known that X-ray powder diffraction (XPRD) patterns can produce one or more measurement errors depending on different measurement conditions (e.g., equipment used, sample preparation, or instrumentation). In particular, it is well known that the intensity of X-ray powder diffraction patterns can vary depending on the measurement conditions and sample preparation. For example, technicians in the field of X-ray powder diffraction know that the relative intensity of peaks can vary depending on the orientation of the test sample and the type and setup of the equipment used. Those skilled in the art also know that the reflection position can be affected by the precise height of the sample in the diffractometer, the flatness of the sample surface, and the zero calibration of the diffractometer. Therefore, those skilled in the art understand that the diffraction pattern data presented herein should not be interpreted as absolute, and that any crystalline form that provides a powder diffraction pattern substantially identical to the powder diffraction patterns disclosed herein falls within the scope of the present invention. For further details, see Jenkins & Snyder, Introduction to X-Ray Powder Diffractometry, John Wiley & Sons, 1996.

[0050] In one embodiment, the crystalline form provided herein has a melting point temperature of 125°C to 132°C. More specifically, the melting point temperature is, for example, 125, 125.5, 126, 126.5, 127, 127.5, 128, 128.5, 129, 129.5, 130, 130.5, 131, 131.5, or 132°C. In each of the above embodiments, the melting point characteristics of the crystalline form of the present invention are analyzed by differential scanning calorimetry (DSC), including modulated differential scanning calorimetry or temperature-modulated differential scanning calorimetry.

[0051] In one embodiment, type A exhibits a TGA weight loss of 2 to 4% when heated to 100°C to 150°C. For example, the TGA weight loss is 2%, 2.5%, 3%, 3.5%, or 4%. In another embodiment, type B exhibits a TGA weight loss of 0.7% to 0.8% when heated to 150°C to 170°C. For example, the TGA weight loss is 0.7%, 0.75%, or 0.8%.

[0052] In one embodiment, the water absorption rate of type A at 25°C / 80%RH is 0.13 to 0.14%. For example, the water absorption rate is 0.13%, 0.135%, or 0.14%. In another embodiment, the water absorption rate of type B at 25°C / 80%RH is 0.103 to 0.109%. For example, the water absorption rate is 0.103%, 0.105%, 0.107%, or 0.109%.

[0053] On the other hand, the present invention provides intermediates for preparing type A or type B, which are solvates of ABT-301. In one preferred embodiment, the solvates include IPA solvate, acetone solvate, ACN solvate, MeOH solvate, NMP solvate, THF solvate, HCl solvate, DMAc solvate, EtOH solvate, DCM solvate, DMSO solvate, 1,4-dioxane solvate, or MIBK solvate. In some examples, type A is used as a starting material and dissolved in different solvents to obtain the final intermediate from a clear solution. In some examples, type A or type B can be obtained from the intermediate.

[0054] Pharmaceutical composition

[0055] In one embodiment, the present invention provides a pharmaceutical composition comprising a crystalline form provided herein. More specifically, the pharmaceutical composition is an HDAC inhibitor. If necessary, the pharmaceutical composition comprises a surfactant and an oil. In some embodiments, the surfactant is polysorbate 80 and the oil is castor oil.

[0056] In some embodiments, the pharmaceutical composition includes a plasticizer, preferably propylene glycol. In some embodiments, the pharmaceutical composition includes a pharmaceutically acceptable salt thereof and one or more types of pharmaceutically acceptable excipients, carriers, including inert solid diluents and fillers, diluents, penetration enhancers, solubilizers, or auxiliaries.

[0057] On the other hand, the present invention provides capsules containing the pharmaceutical composition. Preferably, the capsules are encapsulated in a gelatin shell. The capsules provided herein contain 25 mg to 100 mg of crystalline form of ABT-301, more specifically, the amount of crystalline form is, for example, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 mg. In one preferred embodiment, the capsule provided herein contains 50 mg of crystalline form of ABT-301.

[0058] The pharmaceutical compositions of the present invention, intended for oral administration, are provided in individual dosage forms such as capsules, sachets, or tablets, or liquids or aerosol sprays, each containing a predetermined amount of the active ingredient, for example, in the form of powder or granules, a solution or suspension in an aqueous or non-aqueous liquid, an oil-in-water emulsion, or an oil-in-water emulsion. The pharmaceutical compositions of the present invention also include restorative powders, orally administered powders, bottles (bottled powders or liquids), orally dissolving films, lozenges, pastes, tubes, gels, and packaging. These dosage forms can be prepared by any pharmaceutical method, all of which involve a step of binding (multiple types) of active ingredients to a carrier comprising one or more types of essential ingredients. Generally, these compositions are prepared by uniformly and densely mixing (multiple types) of active ingredients with a liquid carrier or a fine solid carrier, or both, and then, if necessary, shaping the product into a desired appearance.

[0059] Treatment method

[0060] The present invention provides a method for treating cancer, comprising administering a therapeutically effective dose of the crystalline form of ABT-301 free base to a patient in need of treatment.

[0061] In some examples, the crystalline form of ABT-301 administered is an HDAC inhibitor.

[0062] In the selected examples, the crystalline form of ABT-301 is administered as a single dose. A single dose of the crystalline form of ABT-301 may also be used to treat acute symptoms. In the selected examples, the crystalline form of ABT-301 is administered in multiple doses. Doses may be approximately once, twice, three times, four times, five times, six times, or more than six times per day. Preferably, the crystalline form of ABT-301 is administered once a day. Doses may be approximately once a month, once every two weeks, once a week, or once every other day. In some examples, in multiple doses, the interval between the first and last dose is approximately one to one and a half months. In other examples, the crystalline form of ABT-301 is administered once or multiple times. For example, the crystalline form of ABT-301 is administered once throughout the entire course of treatment. The crystalline form of free ABT-101 base is administered approximately once to six times per day. In another example, administration of the crystalline form of ABT-101 continues for less than seven days. In yet another example, administration continues for approximately six days, ten days, fourteen days, twenty-eight days, two months, six months, or more than one year. In some cases, continuous administration is achieved and maintained as needed.

[0063] In some embodiments, the crystalline form of ABT-301 is administered by any suitable route, including, but not limited to, intestinal, oral, nasal, extragastric (e.g., intratumoral, intramuscular, intravenous, intraarticular, intraarterial, subcutaneous, intraperitoneal, intracerebral, intrathecal, intracellular, intracerebral venous or intrathecal injection) or transmucosal administration. Preferably, the crystalline form of ABT-301 is administered orally.

[0064] Furthermore, according to the method of the present invention, the cancer includes solid tumors of various organs and hematological malignancies. In some embodiments, the cancers include pancreatic cancer, bladder cancer, colorectal cancer, breast cancer (e.g., metastatic breast cancer), prostate cancer (e.g., androgen-dependent and androgen-independent prostate cancer), kidney cancer (e.g., metastatic renal cell carcinoma), hepatocellular carcinoma, lung cancer (e.g., bronchioloalveolar carcinoma (BAC) and lung adenocarcinoma), non-small cell lung cancer (NSCLC), ovarian cancer (e.g., advanced epithelial carcinoma or primary peritoneal cancer), cervical cancer, gastric cancer, esophageal cancer, neuroendocrine cancer (e.g., metastatic neuroendocrine tumors), bone cancer or head and neck cancer (e.g., squamous cell carcinoma of the head and neck), melanoma or brain tumors (e.g., glioblastoma, anaplastic oligodendroglioma, adult glioblastoma multiforme, and adult anaplastic astrocytoma). This is astrocytoma. Here, the fibrosis in question is pulmonary fibrosis, hepatic fibrosis, cutaneous fibrosis, or renal fibrosis.

[0065] The numerical ranges and parameters used to define the present invention are approximations, although the relevant numerical values ​​in specific examples are presented as accurately as possible. However, any numerical value will inevitably include the standard deviation due to the individual test method. Here, "approximately" usually means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a particular value or range. Alternatively, the term "approximately" means that the actual value falls within the acceptable standard error of the mean value as defined by the articulators in the field to which the present invention pertains. Accordingly, unless otherwise specified, the numerical parameters disclosed herein and in the appended patent applications are approximations and may be modified as necessary. At a minimum, these numerical parameters should be understood as values ​​obtained by applying the indicated number of significant figures and the usual rounding method.

[0066] Examples

[0067] The present invention will be described in detail below through the following examples. These examples are provided for illustrative purposes only, and those skilled in the art can easily conceive of various modifications and changes. Therefore, although each example of the present invention will be described in detail below, the present invention is not limited to these examples described herein.

[0068] Example 1

[0069] Preparation of ABT-301 compound

[0070] The ABT-301 compound can be synthesized or prepared by any suitable method known in the art, such as the route and method disclosed in, for example, U.S. Patent No. US8,846,748B2.

[0071] Example 2

[0072] Physical properties of the crystal form of ABT-301

[0073] 1. Screening of the polycrystalline form of ABT-301

[0074] Using ABT-301 Type A (sample name: ABT-301, CP ID: 826543-01-A) as the starting material, a total of 100 polycrystalline forms were screened using methods such as gas-liquid diffusion, gas-solid diffusion, slow evaporation, slurry (room temperature and 50°C), temperature cycling, slow cooling, poor solvent addition, and polishing. Based on the characteristic results and further experiments, a total of 18 polycrystalline forms were obtained, which were analyzed using X-ray powder diffraction (XRPD), thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), and 1 H solution nuclear magnetic resonance ( 1 The compounds were characterized by methods such as 1H NMR. Type A was presumed to be the hydrate, type B the anhydride, types C / D / E / F / G / H / I / J / K / L / M / N / O / R were presumed to be the solvates, and types P / Q could not be identified due to morphological transformation after drying. The characteristic results for the polycrystalline forms are shown in Table 1, and the XRPD results are shown in Figures 1 to 3.

[0075] [Table 1] *: Fever

[0076] 2. Symptoms of Type A

[0077] The XRPD pattern shown in Figure 4 indicates that the obtained starting material was crystalline and was named Type A. Figure 5 shows the TGA / DSC graph of the starting material, which shows a weight loss of 3.77% at 150°C, two endothermic peaks at 118.7°C and 125.5°C, and one exothermic peak at 166.1°C. 1 The results of the 1H NMR spectrum are shown in Figure 6. The approximate solubility of the starting material was measured in 20 different solvents at room temperature. Approximately 2 milligrams of sample were placed in a 3-milliliter glass vial. Next, the solvents listed in Table 2 were added to the glass vial in stages until the solid was visually dissolved or the total volume reached 2 mL. The solubility results are listed in Table 2 as guidance for solvent selection in screening experiment design.

[0078] [Table 2]

[0079] To investigate TGA weight loss and DSC signaling, heating experiments were performed on type A, and the results are shown in Figure 7. According to the XRPD results, there was no change in morphology after heating to 100°C and cooling to room temperature, but after heating to 122°C / 130°C and cooling to room temperature, the morphology was transformed into a new morphology named type B. After heating to 180°C and cooling to room temperature, melting and color change were observed, so XRPD testing was not performed. After heating to 100°C, according to the TGA results (Figure 8), the weight loss was 2.87% when heated to 150°C, indicating that type A did not undergo significant changes before and after heating.

[0080] According to the results of variable temperature XRPD (VT-XRPD) (Figures 9 to 12), there was no change in the morphology of type A after 20 minutes of N2 purging. However, after heating to 90, 95, and 100°C with N2 purging, an additional peak appeared, and after heating to 102 and 104°C with N2 purging, it was almost completely converted to type B. After heating to 106, 108, 110, 112, 114, 116, 118, 120, 125, and 130°C with N2 purging, the sample was converted to type B (the peak shift at high temperatures is presumed to be due to thermal expansion of the lattice with increasing temperature), and after cooling to 30°C with N2 purging, no change in the morphology of type B was observed. Combining the TGA weight loss and the morphological change during heating, it is presumed that type A is a hydrate. The DVS results (Figure 13) show that a water absorption rate of 0.1396% was observed at 25°C / 80%RH. The XRPD results (Figure 14) show that no morphological changes were observed after the DVS test.

[0081] 3. Symptoms of type B

[0082] Type B (826543-03-A2) was obtained by heating Type A to 122°C and then cooling it to room temperature. The XRPD pattern results are shown in Figure 15. As shown in Figure 16, the TGA / DSC results show a weight loss of 0.94% at a maximum of 150°C, with one exothermic point (peak) at 166.0°C. 1 The results of the 1H NMR spectrum are shown in Figure 17. Since the TGA weight loss was small, it is presumed that type B is an anhydrous form.

[0083] For further research, an attempt was made to re-prepare type B using a slurry on a scale of approximately 300 mg. Type B (826543-22-A) was obtained by slurrying approximately 345 milligrams of type A in MTBE at 50°C for 2 days, and then vacuum drying at room temperature for 1 day (325 milligrams of type B were obtained, with a yield of 94%). The XRPD pattern of type B (826543-22-A) is shown in Figure 18. The TGA / DSC graph, as shown in Figure 19, shows a weight loss of 0.74% at a maximum of 150°C and one exothermic point (peak) at 166.2°C. 1The 1H NMR results showed no detection of MTBE residue. The DVS results (Figure 21) showed a water absorption rate of 0.1039% at 25°C / 80%RH. The XRPD results (Figure 22) showed no morphological changes after the DVS test.

[0084] 4. Interconverting Relationship Studies

[0085] To study the interconversion relationship, competitive slurry experiments were conducted for types A and B using MIBK, 2-MeTHF, n-heptane, and toluene at room temperature or 50°C, and in acetone and acetone / H2O systems with different water activity at room temperature. Approximately 10-20 mg of type A was weighed into 1.0 mL of solvent at room temperature / 50°C to prepare a slurry. After filtering the suspension sample, the filtrate was transferred to HPLC vials containing approximately 10 mg of each type A / B, and the samples were stirred at RT / 50°C. The results are summarized in Table 3, and the XRPD results are shown in Figures 23 to 25. The results show that type B was obtained with MIBK, 2-MeTHF, n-heptane, and toluene at 50°C, type O solvate was obtained with MIBK at room temperature, and a mixture of type B and stable type P was obtained with 2-MeTHF at room temperature. In acetone and acetone / H2O systems, different water activity (a w Another D-type solvate with a ratio of 0.2 to 0.8 was obtained.

[0086] [Table 3]

[0087] Based on the results of the competitive slurry experiment, additional competitive slurry experiments were conducted. Approximately 10 mg of type A was weighed into each type of solvent and slurryed at room temperature using a molecular sieve. After filtering the suspension samples, the filtrates were transferred to HPLC vials containing approximately 10 mg of type A / B, respectively, and the samples were stirred at room temperature, with and without the use of a molecular sieve. After the competitive slurry experiment, the suspension samples were filtered, and the filtrate was subjected to a KF test. The results are summarized in Table 4, and the XRPD results are shown in Figures 26 to 28. The results indicate that type A was obtained in H2O at room temperature, type B was obtained in n-heptane and toluene at room temperature for 2 days using a molecular sieve, and type A was obtained in n-heptane and toluene at room temperature for 1 day using a molecular sieve. The KF test performed on the filtrate after the competitive slurry experiment showed that the water content was lower when a molecular sieve was used, resulting in the acquisition of type B. The results indicate that the interconversion relationship between type A and type B is related to water content.

[0088] [Table 4] *: Slurry using molecular sieves #: Slurry without molecular sieves **: Water activity is calculated by COSMOlogic software based on KF results. Since water is immiscible with n-heptane and toluene, the water activity result is for reference only.

[0089] Another solvent system, t-Butanol / H2O, was used for additional competitive slurry experiments. Approximately 10 mg of type A was weighed into 1.0 mL of solvent at room temperature and slurryed in t-Butanol and t-Butanol / H2O with different water contents. After filtering the suspension samples, the filtrates were transferred to HPLC vials containing approximately 10 mg of type A / B, respectively, and the samples were stirred at room temperature. After the competitive slurry, the suspension samples were filtered, and the filtrate was subjected to a KF test. Water activity was calculated using COSMOlogic software based on the KF results. The results are summarized in Table 5, and the XRPD results are shown in Figures 29 and 30. The results indicate that type B was obtained when the water activity was ≤0.17 at room temperature, and type A was obtained when the water activity was ≥0.37.

[0090] [Table 5] *: Water activity is calculated by COSMOlogic software based on KF results. #: To obtain low water activity, t-butanol from a new vial with low water content was used.

[0091] Based on the above results, the interconversion relationship between form A and form B is related to water content (or water activity) and temperature. Therefore, it is recommended to further evaluate the two forms (e.g., solid stability) and select the dominant form for further development. Also, since many solvates of this compound have been observed, it is recommended to pay attention to solvent selection in further crystallization process studies.

[0092] method

[0093] Solvents used in this specification

[0094] The abbreviations for solvents are shown in Table 6 below.

[0095] [Table 6]

[0096] Screening of polycrystalline forms

[0097] A total of 100 polycrystalline form screening experiments were conducted using different crystallization methods or solid-state transition methods. The methods used and results are shown in Table 7 below.

[0098] [Table 7]

[0099] Vapor-solution diffusion

[0100] Gas-liquid diffusion experiments were conducted under 14 different conditions. Approximately 20 mg of the starting material (826543-01-A) was dissolved in 0.5-1.0 mL of a suitable solvent, and a clear solution was obtained in a 3 mL vial and filtered into a new vial (0.45 μm, PTFE). This solution was then placed in a 20 mL vial with 3 mL of volatile solvent. The 20 mL vial was sealed with a lid and allowed sufficient time for the organic gas to react with the solution at room temperature. Finally, the solid was separated and analyzed by XRPD.

[0101] Vapor-solid diffusion

[0102] Gas-solid diffusion experiments were conducted using 12 different solvents. Approximately 20 milligrams of the starting material (826543-01-A) was weighed and placed in a 3-milliliter vial. This vial was then placed in a 20-milliliter vial along with 3 milliliters of volatile solvent. The 20 mL vial was sealed with a lid and left at room temperature for 7 days to allow the solvent vapor to react with the sample.

[0103] Slow evaporation

[0104] Slow evaporation experiments were conducted under six conditions. 20 mg of the starting material (826543-01-A) was dissolved in 1.0-2.0 mL of solvent and placed in a 3 mL glass vial. The resulting solution was slowly evaporated at room temperature, the vial was sealed, and four needle holes were made.

[0105] Slurry at room temperature (Slurry at RT)

[0106] Approximately 20 mg of the starting material (826543-05-A) was suspended in 0.5 mL of solvent and placed in an HPLC glass vial. The suspension was magnetically stirred (1000 rpm) at room temperature for 3 days, and the remaining solid was centrifuged and subjected to XRPD analysis.

[0107] Slurry at 50°C

[0108] Approximately 20 mg of the starting material (826543-01-A) was suspended in 0.5 mL of solvent and placed in an HPLC glass vial. The suspension was stirred at 50°C for 4 days (1000 rpm), and the remaining solid was centrifuged and subjected to XRPD analysis.

[0109] Temperature cycling

[0110] Approximately 20 mg of the starting material (826543-01-A) was suspended in 0.5 mL of solvent and placed in an HPLC glass vial. The suspension was heated and cooled (50°C to 5°C, 0.1°C / min) for two cycles, after which the remaining solid was centrifuged and subjected to XRPD analysis.

[0111] Slow cooling

[0112] Slow cooling experiments were performed using six different solvent systems. At room temperature, 20 mg of the starting material (826543-01-A) was suspended in 1.0–2.0 mL of solvent and placed in a 3 mL glass vial. The suspension was heated to 50°C and equilibrated for 2 hours, after which it was filtered into a new vial (0.45 μm, PTFE). The filtrate was slowly cooled to 5°C at a rate of 0.1°C per minute. The resulting solid was kept at a constant temperature of 5°C and then tested using XRPD.

[0113] Anti-solvent addition

[0114] A total of 20 poor solvent addition experiments were performed. Approximately 20 mg of the starting material (826543-01-A) was dissolved in 0.6–1.2 mL of solvent to obtain a clear solution. If the solid did not dissolve, the solution was filtered into a new vial (0.45 μm, PTFE), and the poor solvent was added while magnetically stirring the solution (~1000 rpm) until a precipitate appeared or the total volume of the poor solvent reached 10.0 mL. Samples without precipitate were transferred to a slurry at 5°C, and then to a slurry at -20°C. The clear samples were allowed to evaporate at room temperature. Finally, the solid was separated and analyzed by XRPD.

[0115] Grinding

[0116] Polishing experiments were conducted with and without the addition of a solvent. Approximately 20 mg of the starting material (826543-01-A) was weighed into a mortar. 20 μL of solvent was added to the mortar, and the solid was polished for 3 to 5 minutes. Finally, the solid was separated and subjected to XRPD analysis.

[0117] XRPD

[0118] XRPD analysis was performed using a PANalytical X-ray powder diffractometer. The XRPD parameters used are shown in Table 8.

[0119] [Table 8]

[0120] TGA and DSC

[0121] TGA data were collected using TA Instruments' TA Discovery 5500TGA. DSC testing was performed using TA Instruments' TA Discovery 2500DSC and TA Discovery DSC250. The detailed parameters used are shown in Table 9 below.

[0122] [Table 9]

[0123] Solution NMR

[0124] Solution NMR was collected using a Bruker 400M NMR spectrometer with DMSO-d6 and MeOD.

[0125] DVS

[0126] DVS was measured using SMS (Surface Measurement Systems) DVS Intrinsic or Intrinsic Plus. Relative humidity at 25°C was calibrated based on the deliquescence points of LiCl, Mg(NO3)2, and KCl. The DVS test parameters are shown in Table 10 below.

[0127] [Table 10]

[0128] Karl Fishcher (KF)

[0129] The instrument (Metrohm 870 KF Titrinoplus) was calibrated with pure water, and the titration reagent was Hydranal® R-Composite 5, supplied by Sigma-aldrich. Methanol (HPLC grade) was used to dissolve the samples.

[0130] Example 3

[0131] Identification of the single crystal structures of ABT-301 type A and type B

[0132] In Example 2, types A and B were identified as hydrate and anhydrous, respectively. For further study, this single-crystal study will investigate the single-crystal structures of types A and B by single-crystal growth and single-crystal X-ray diffraction (SCXRD) analysis.

[0133] 1. Single crystal growth

[0134] The ABT-301 type A single crystal sample (CP ID: 826543-04-A9) was obtained in a previous polycrystalline form screening study using a liquid-vapor diffusion experimental system (solvent: 1,4-Dioxane; poor solvent: toluene). Details of the growth experiment are as follows.

[0135] 20.4 mg of ABT-301 type A starting material was weighed into a 3 mL glass vial, and 1.0 mL of 1,4-dioxane was added to prepare a sample solution. The glass vial was then sealed in a 20 mL glass vial pre-filled with 3 mL of toluene (as a poor solvent), and vapor diffusion was performed at room temperature. After 6 days of evaporation, a plate-like single crystal was obtained as shown in Figure 31, and the single crystal was characterized using XRPD. The superposition of XRPD patterns (Figure 32) shows that the XRPD experiment of the obtained single crystal is consistent with the ABT-301 type A reference material (CP ID: 826543-01-A).

[0136] The ABT-301 type B single crystal sample (CP ID: 826543-07-A7) was obtained from a slow evaporation experimental system at 80°C. Details of the growth experiment are as follows.

[0137] A 5.1 mg sample of ABT-301 type B (CP ID: 826543-39-A) and 0.5 mL of acetone were placed in a 3 mL glass vial. After promoting the dissolution of the solid sample using sonication, the sample suspension was filtered using a syringe and syringe filter (0.45 μm PTFT membrane). The filtrate was transferred to a clean 4 mL shell vial, the vial was sealed with a ta cap, and placed in an 80°C biochemical incubator. The acetone in the sample solution was slowly evaporated at 80°C. After 7 days of evaporation, a plate-like single crystal (CP ID: 8268115-07-A7) was obtained as shown in Figure 33, and this single crystal was further characterized by XRPD. The superposition of XRPD patterns (Figure 34) shows that the XRPD experiment of the obtained single crystal is consistent with that of the ABT-301 type B reference (CP ID: 826543-03-A2).

[0138] 2. Identification of single crystal structure

[0139] Type A single crystal structure

[0140] From the obtained crystal sample (CP ID: 826543-04-A9), an A-type single crystal of appropriate size and good diffraction quality was cut out and selected, and its characteristics were identified by single-crystal X-ray diffraction. This single crystal belongs to the monoclinic system, and its space group is P21 / c. The unit cell dimensions are {a=14.68420(10)Å, b=44.7093(3)Å, c=10.46470(10)Å, α=90°, β=102.5980(10)°, γ=90°, V=6704.88(9)}.

[0141] Other crystallographic data and settlement parameters are shown in Table 11 below. As shown in Figure 35, the asymmetric unit of the single crystal structure consists of four independent ABT-301 molecules and two water molecules, indicating that this single crystal is a hemihydrate (API / water molar ratio 2:1).

[0142] [Table 11]

[0143] Type B single crystal structure

[0144] From the obtained B-type single crystal sample (CP ID: 8268115-07-A7), a B-type single crystal of appropriate size and good diffraction quality was cut out and selected, and its characteristics were identified by single-crystal X-ray diffraction. The structure of this single crystal was determined, and it was found to belong to the tetragonal system and the P212121 space group. The unit cell dimensions are {a=8.13110(10)Å, b=9.21070(10)Å, c=42.5238(3)Å, α=90°, β=90°, γ=90°, V=3184.74(6)Å}. 3}. Other crystallographic data and settlement parameters are shown in Table 12 below. As shown in Figure 36, the asymmetric unit of the type B single crystal structure consists of two independent ABT-301 molecules and does not contain water or other solvent molecules, thus confirming that the type B crystal is anhydrous.

[0145] [Table 12]

[0146] method

[0147] Single-crystal diffraction data collection

[0148] From the obtained single crystals, single crystals of appropriate size and good diffraction quality were cut and selected, and coated with Paratone-N (an oil-based cryoprotectant). The selected single crystals were mounted on a Cryoloop and fixed to a goniometer head in a random orientation. The single crystals were immersed in a 120K nitrogen stream, and preliminary inspection and data acquisition were performed using a 120K Rigaku XtaLAB Synergy R (Cu / Kα X-ray emission, λ=1.54184Å) diffractometer.

[0149] For a type A single crystal sample (CP ID: 826543-04-A9), CrysAlisPro (version: 1.171.42.89a) software was used to acquire and refine unit cell parameters and orientation matrices for data acquisition within the range of 1.9580° < θ < 77.1750° using the T-vector algorithm. The minimum diffraction angle (θ) for data acquisition was 1.976°, and the maximum diffraction angle (θ) was 77.294°. The data acquisition integrity was 99.63%. The mean I / σ of the acquired data was 42.0, and the maximum resolution was truncated at 0.79 Å.

[0150] For the type B single crystal sample (CP ID: 8268115-07-A7), CrysAlisPro (version: 1.171.42.89a) software was used to acquire and refine the unit cell parameters and orientation matrix for data acquisition within the range of 4.1250° < θ < 77.0180° using the T-vector algorithm. The minimum diffraction angle (θ) for data acquisition was 2.078°, and the maximum diffraction angle (θ) was 77.260°. The completeness of data acquisition was 100.00%. The mean I / σ of the acquired data was 43.0, and the maximum resolution was truncated at 0.79 Å.

[0151] Reduction of single-crystal diffraction data

[0152] Frames were merged using CrysAlisPro (version: 1.171.42.89a). Lorentz polarization correction was applied to the data.

[0153] For the α-type single crystal sample (CP ID: 826543-04-A9), a total of 151,407 reflections were collected in the range of 1.976° < θ < 77.294°, of which 14,167 were unique reflections. Using CrysAlisPro (version: 1.171.42.89a), empirical absorption correction was performed using spherical harmonics by SCALE3 ABSPACK. The absorption coefficient μ of this material at this wavelength (λ = 1.54184 Å) is 1.962 mm -1 and the minimum and maximum transmittances were 0.81852 and 1.00000, respectively. The intensities of equivalent reflections were average values. Based on the intensities, the average agreement coefficient (Rint) of all equivalent reflections was 5.47%.

[0154] For the β-type single crystal sample (CP ID: 8268115-07-A7), a total of 20,012 reflections were collected in the range of 2.078° < θ < 77.260°, of which 6,540 were unique reflections. Using CrysAlisPro (version: 1.171.42.89a), empirical absorption correction was performed using spherical harmonics by SCALE3 ABSPACK. The absorption coefficient μ of this material at this wavelength (λ = 1.54184 Å) is 2.027 mm -1 and the minimum and maximum transmittances were 0.71191 and 1.00000, respectively. The intensities of equivalent reflections were average values. Based on the intensities, the average agreement coefficient (Rint) of all equivalent reflections was 2.19%.

[0155] Methods for the analysis and refinement of the single crystal structure

[0156] Using the ShelXT (version: 2018 / 2) structure solution program, two single crystal structures were solved by the Intrinsic Phasing method in the space group P1, refined using ShelXT (version 2018 / 3), and refined using the full matrix least squares method included in Olex2 (version: 1.5). All non-hydrogen atoms were refined isotropically. 2 ​

[0157] For the Type A single crystal sample (CP ID: 826543-04-A9), the hydrogen atoms were calculated geometrically and then refined using a model.

[0158] For the type B single crystal sample (CP ID: 8268115-07-A7), hydrogen atoms in nitrogen or oxygen bonds were identified from the Fourier map and their isotropy and position were calculated. Other hydrogen atoms were calculated geometrically and calculated using the Riding Model.

[0159] XRPD Pattern Layer Calculation Software

[0160] Using the Mercury program (version 4.3.1) and atomic coordinates, space group, and unit cell parameters in the single crystal structure, a calculated XRPD pattern layer was generated for Cu emission.

[0161] Single crystal structure visualization software

[0162] Crystal structure diagrams were created using Olex2 (version 1.5) and Mercury (version 4.3.1). The thermal ellipsoid diagram was generated using ORTEP-III2 (version 2014.1) software.

[0163] Equipment and parameters

[0164] PLM images of single-crystal samples were acquired using an OLYMPUS SZX-7 stereomicroscope. Single-crystal X-ray diffraction data were collected at a temperature of 120K using a Rigaku XtaLAB Synergy R (Cu / Kα emission, λ=1.54184Å) diffractometer. XRPD data were collected using a PANalytical Empyrean X-ray diffractometer. The SCXRD and XRPD instrument parameters are shown in Tables 13 and 14 below, respectively.

[0165] [Table 13]

[0166] [Table 14]

[0167] Example 4

[0168] Solid Stability Evaluation

[0169] In Example 2, a polycrystalline form screening was performed, and hydrated form A and anhydrous form B were discovered. The objective of Example 4 is to evaluate forms A and B of ABT-301 and to find a crystalline form suitable for further development. Based on the results of the polycrystalline form screening, the interconversion relationship between form A and form B is related to water content (or water activity) and temperature. Therefore, these two forms were selected and their solid stability was evaluated to find a leading form for further development. The amorphous state was also selected as a reference for evaluation.

[0170] 1. Notation results

[0171] The characteristic results for types A and B are disclosed in Example 2. The amorphous state was re-prepared by different methods, including polishing, rotational evaporation, reverse addition with antisolvents, and lyophilization. For example, the amorphous state was re-prepared by rotational evaporation under low ambient humidity (20% RH). Approximately 156 mg of type A (826543-01-A) was rotationally evaporated with acetone to obtain the amorphous state (826562-02-A). The XRPD results (Figure 37) show that the amorphous state was obtained, and this sample was used for solid stability evaluation. The TGA / mDSC results are shown in Figure 38. The mDSC results show that the glass transition temperature is 53.5°C (inflection point temperature). The DSC results (Figure 39) show that two exothermic points were observed at 118.9°C and 160.6°C (peak values).

[0172] 2.Solid stability

[0173] Types A and B were stored for 1 / 2 / 4 weeks under conditions of 60°C, 25°C / 60%RH, 25°C / 92.5%RH, and 40°C / 75%RH, and their solid stability was evaluated. The amorphous state was stored for 1 / 2 / 4 weeks under conditions of 25°C / 60%RH and 40°C / 75%RH, and its solid stability was evaluated. Physical and chemical stability were evaluated by X-ray powder diffraction (XRPD), thermogravimetric analysis (TGA) / Karl Fischer method (KF), differential scanning calorimetry (DSC) / modulated differential scanning calorimetry (mDSC), high-performance liquid chromatography (HPLC) purity, and assay methods, respectively.

[0174] The results are summarized in Tables 15 and 16. The solid stability results show that types A and B showed no morphological changes or significant changes in HPLC purity even after 4 weeks of solid stability. After the solid stability evaluation, the amorphous state was converted to type A, and there was no significant change in HPLC purity after 4 weeks of solid stability.

[0175] [Table 15] *: Fever

[0176] [Table 16] *:Inflection point temperature #: Fever

[0177] method

[0178] In this embodiment, XRPD, TGA, DSC, and KF were under the same conditions as in Example 2.

[0179] PLM

[0180] PLM images were acquired using an Axio Lab.A1 upright microscope (purchased from Carl Zeiss, Germany).

[0181] mDSC

[0182] The mDSC test parameters are shown in Table 17 below.

[0183] [Table 17]

[0184] freeze dryer

[0185] Freeze-drying was performed using a SCIENTZ-30FG / A freeze dryer.

[0186] HPLC / IC

[0187] Using the Agilent 1260, the detailed chromatography conditions are shown in Table 18 below.

[0188] [Table 18]

[0189] Furthermore, long-term stability tests and accelerated stability tests were performed on Type A (powder form), and the results are shown in Tables 19 and 20, respectively. Specifically, for the long-term stability test, the packaging and storage conditions were as follows: the sample was packaged in a double PE bag with silica gel inside an aluminum bag inside a paper drum; sample amount: 0.6g; temperature: 25±2℃; relative humidity (%): 60±5%; analysis frequency: 0, 1, 2, 3, 6, 9, 12, 18, 24, 36, 48, 60 months; duration: 60 months. For the accelerated stability test, the packaging and storage conditions were as follows: the sample was packaged in a double PE bag with silica gel inside an aluminum bag inside a paper drum; sample amount: 0.6g; temperature: 40±2℃; relative humidity (%): 75±5%; analysis frequency: 0, 1, 2, 3, 6 months; duration: 6 months.

[0190] [Table 19]

[0191] [Table 20]

[0192] Example 5

[0193] ABT-301 Capsule Ingredients and Stability Study

[0194] prescription

[0195] Here, we will test the stability of the ABT-301 crystalline form as an active pharmaceutical ingredient (API) from various angles. First, Table 21 shows typical formulations of capsules containing 50 mg of ABT-301A as an API in combination with other pharmaceuticals.

[0196] [Table 21] *:qs represents quantum saturation. Medium-chain triglycerides may be removed in this process.

[0197] 2. Manufacturing Process

[0198] The manufacturing process for 50mg ABT-301 capsules is outlined below.

[0199] Step 1: Preparation of germanium

[0200] Add pure water to a colloidal digester, slowly add glycerin, and stir for about 10 minutes. Heat to 65°C while stirring, and add gelatin 160 until the gelatin dissolves. Next, evacuate and maintain a vacuum pressure of less than -0.06 MPa while stirring until the gel becomes a brown solution. Take a sample for viscosity testing.

[0201] The separated titanium dioxide, iron oxide black, and pure water are added to a suitable container and homogenized in a homogenizer for approximately 25-30 minutes. During the stirring process, the mixed suspension of titanium dioxide and iron oxide black is poured into the colloidal digester and vacuumed to less than -0.06 MPa. Samples are taken for viscosity and drying loss tests, and the gelatin solution is kept at 60°C.

[0202] Step 2: Preparation of the contents mixture

[0203] Place the divided polyoxyethylene (35) castor oil, polysorbate 80, and propylene glycol into a jacketed container and protect from light. Stir and mix at approximately 50°C for approximately 25 minutes. Fill a portion of the prepared ABT-301 pharmaceutical substance into a portion of the heated solution and stir in a homogenizer for approximately 2-5 minutes. Repeat the above procedure until all of the prepared ABT-301 pharmaceutical substance is homogenized. After standing overnight, take a sample to check for mixing homogenization.

[0204] Step 3: Encapsulation

[0205] The mixture from Step 2 is filled into the capsule shells from Step 1 using a soft capsule machine. During the packaging process, the capsule shells are lubricated with medium-chain triglycerides (see encapsulation parameters in Table 22). Throughout the encapsulation process, samples are taken at appropriate intervals for individual filling weight and ribbon thickness testing of each soft capsule.

[0206] [Table 22]

[0207] Step 4: Drying

[0208] Under conditions of 15-25°C and less than 35% RH humidity, soft capsules were polished and molded in a rotary dryer for 1.5 hours. Samples were taken for hardness and dryness loss testing.

[0209] Step 5: Packaging

[0210] The 50 mg ABT-301 capsules are packaged in 120 mL HDPE (high-density polyethylene) vials, each vial containing 2 tablets of 1 g desiccant and approximately 5 cm of pharmaceutical coil, and are packaged with a 38 mm PP (polypropylene) cap, with 35 capsules per vial.

[0211] 3. Stability study

[0212] Here, the stability of the above-mentioned 50 mg ABT-301 capsules under long-term storage conditions (5 ± 3°C) is examined, and the results are shown in Tables 23 and 24.

[0213]

Table 23

[0214]

Table 24

[0215] Furthermore, the accelerated stability data of the above-mentioned 50 mg ABT-301 capsules are examined, and the results are shown in Table 25.

[0216]

Table 25

[0217] Example 6

[0218] Pharmacokinetics study

[0219] Here, the pharmacokinetics of different dosage forms of ABT-301 are tested and compared. More specifically, the purpose of this study is to evaluate the pharmacokinetics of different dosage forms of ABT-301 in beagle dogs after single oral and intravenous administrations. The animals administered orally were fasted on the day before administration and for 4 hours after administration, and water was restricted only within 5 minutes after administration, and they were allowed to drink water freely during the study period.

[0220] The experiment was designed as a crossover study (in three stages). Three male beagle dogs were used to test different doses of ABT-301. In the first stage, the animals were administered ABT-301 oral capsules (2 capsules, 100 mg) orally, in the second stage, the animals were administered ABT-301 oral suspension (50 mL, 100 mg) orally, and in the third stage, the animals were injected with ABT-301 IV formulation (10 mL, 15 mg) intravenously. The washout period between each stage was at least 144 hours.

[0221] After treatment, blood samples were collected at appropriate times and then centrifuged. Plasma fractions were collected, and the plasma concentration of ABT-301 was measured by LC / MS / MS method. Using the plasma concentration-time data, the pharmacokinetic parameters of ABT-301 were calculated.

[0222] The average AUC of 100 mg ABT-301 oral capsule (0-t) and AUC (0-∞) were 618.6 ± 8.4 ng×h / mL and 622.9 ± 7.2 ng×h / mL, respectively. The average C max was 538.435 ± 11.352 ng / mL. The mean residence time (MRT), terminal half-life (T 1 / 2 ) and T max were 1.91 ± 0.24 hours, 3.84 ± 3.47 hours and 1.00 ± 0.00 hours, respectively. The mean clearance (CL / F) and volume of distribution (V dThe flow rates ( / F) were 160.6±1.8 L / h and 891.0±809.7 L, respectively.

[0223] Average AUC of ABT301 oral suspension 100 mg (0-t) and AUC (0-∞) The values ​​were 120.8±24.8 ng×h / mL and 132.0±27.2 ng×h / mL, respectively. max The concentration was 41.992 ± 4.872 ng / mL. Mean residence time (MRT), final half-life (T) 1 / 2 ) and T max The mean clearance (CL / F) and volume of distribution (V) were 6.57±3.26 hours, 5.35±2.88 hours, and 0.83±0.29 hours, respectively. d The values ​​for / F were 781.5±176.7 L / h and 5664.5±2567.8 L, respectively.

[0224] Average AUC of ABT301IV preparation 15mg (0-t) and AUC (0-∞) The values ​​were 294.6±22.5 ng×h / mL and 295.6±23.1 ng×h / mL, respectively. Average C max The mean residence time (MRT) and the final half-life (T) were 480.610±82.087 ng / mL and 601.327±135.844 ng / mL, respectively. 1 / 2 ) and T max The mean clearance (CL / F) and volume of distribution (V) were 0.583±0.015 hours, 0.805±0.298 hours, and 0.083±0.00 hours, respectively. d The flow rates (F) were 51.0±4.1 L / h and 58.3±17.8 L, respectively.

[0225] Pharmacokinetic parameters are shown in Table 26. The bioavailability (F%) of ABT-301 oral capsules and oral suspension was 31.7±2.4% and 6.7±1.4%, respectively. These results indicate that the improved oral capsule formulation is far superior to the oral suspension.

[0226] [Table 26]

[0227] method

[0228] Test material

[0229] ABT-301 capsules (50 mg / capsule) and ABT-301 active pharmaceutical ingredient powder were supplied by Anbogen Therapeutics, Inc. An oral suspension was prepared as a 2 mg / mL suspension of 1% (w / v) carboxymethylcellulose sodium / 0.5% (v / v) Tween-80 in water (Water for Injection). An intravenous injection formulation was prepared as an administration solution of polyethylene glycol 400 / N,N-dimethylacetamide (DMA) / physiological saline (30 / 5 / 65, w / v / v) to a final concentration of 1.5 mg / mL. The intravenous injection formulation was filtered through a 0.45 μM filter before injection.

[0230] Animal experiments

[0231] This study used three male beagle dogs. The animals were supplied by Kitayama Labs, Inc., Japan. All animals were raised at the National Medical University. Humidity and temperature were controlled to 50-75% and 20-25°C. The light-dark cycle was set to 12h:12h. The experiment was designed as a crossover study (3 phases). Three male beagle dogs were tested with different dosage forms of ABT-301.

[0232] In the first stage, animals were orally administered ABT-301 oral capsules (2 capsules, 100 mg). In the second stage, animals were orally administered ABT-301 oral suspension (50 mL, 100 mg). In the third stage, animals were intravenously injected with ABT-301IV (10 mL, 15 mg).

[0233] The washout period between each stage was at least 144 hours. Animals administered orally were fasted the day before administration and for 4 hours after administration. In the first stage, within 5 minutes of administering the ABT-301 capsule, the animals were orally administered 50 mL of drinking water via an oral tube.

[0234] Sample collection

[0235] Blood samples (0.7–0.8 mL) were collected from the radial vein of each animal, placed in a 3 mL microcentrifuge tube containing the anticoagulant EDTA, and centrifuged for 10 minutes (3,000 g). The plasma was transferred to a clean 1.5 mL microcentrifuge tube and stored at -80°C for further analysis. The sampling times for the first and second stages were set before administration and 0.25, 0.5, 1, 2, 4, 6, 8, 12, and 24 hours after administration, respectively. The sampling times for the first and third stages were set before administration and 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, and 12 hours after administration, respectively.

[0236] Bioanalytical methods

[0237] The plasma concentration of ABT-301 is measured by LC-MS / MS (LC-10AD VP Pump, SIL-HT A Measurements were performed using an automated sampler (Shimadzu Corporation) and an API 4000 triple quadrupole mass spectrometer. The mobile phase consisted of acetonitrile / formic acid / water (46 / 1.0 / 54) and was entered into the MS / MS at a flow rate of 1.0 mL / min. For separation, a Phenomenex, Luna®, 5 μm, C18(2), 100 Å, 150 x 4.6 mm (Phenomenex, Inc., Taiwan) filter was used.

[0238] All quantitative analyses were performed in Multiple Reaction Monitoring (MRM) mode. ABT-301 was analyzed in Positive Mode. The monitoring ion of the parent ion was m / z345.00, and the monitoring ion of the daughter ion was m / z326.90. The internal standard (Cinacalcet, MW357.41) was analyzed in Positive Mode. The monitoring ion of the parent ion was m / z357.70, and the monitoring ion of the daughter ion was m / z154.90. The Spray Needle was 5500V, the Desolvation Temperature was 600 °C, the Declustering Potential was 40V, and the Collision Energy was 30V.

[0239] Each plasma sample (50 μL) was extracted with 200 μL of 100% CH3CN containing 0.2 ng / μL of the internal standard. After centrifugation, 200 μL of the supernatant was evaporated to dryness and reconstituted with 200 μL of an acetonitrile / formic acid / water mixture (40 / 0.1 / 60). Finally, an equal volume of the sample (50 μL) was injected into LC / MS / MS.

[0240] Calculation of Pharmacokinetic Parameters

[0241] The concentration of ABT-301 was analyzed by LC / MS / MS. Pharmacokinetic parameter analysis using a non-compartmental model was provided, and with the Phoenix program, plasma concentration-time data, the terminal half-life (T 1 / 2 ), and the mean residence time (MRT), the maximum plasma concentration (C max ), the time to peak (T max ), the area under the concentration-time curve from time 0 to infinity (AUC( 0-∞ )), the area under the concentration-time curve from time 0 to the final measurable concentration (AUC( 0-t )), the mean clearance (CL or CL / F), the volume of distribution (V d or V dThe bioavailability (F%) and bioavailability (F%) were calculated. The formula for calculating bioavailability (F%) is as follows: F(%)=Average AUC 0-∞ (oral capsule or suspension) / mean AUC 0-∞ (Intravenous preparation)

[0242] Plasma concentration and pharmacokinetic parameter data for each animal, as well as the "mean ± SD" for each group, are disclosed.

[0243] In summary, the crystalline form of ABT-301 of the present invention exhibits unexpected stability and improved pharmacokinetic properties compared to other forms or salts thereof, thereby making the compound more suitable for drug development and meeting bioavailability and pharmaceutical requirements.

[0244] While certain embodiments of the present invention are disclosed, this does not limit the invention. These will be understandable to those skilled in the art to which the invention pertains. Furthermore, since various modifications and amendments are possible without departing from the principles and spirit of the invention, the scope of protection of the present invention should be based on the scope of protection of the present invention as defined in the appended claims.

Claims

1. Type A is characterized in that its X-ray powder diffraction pattern has peaks at 9.6°±0.2°, 15.1°±0.2°, 15.7°±0.2°, 16.7°±0.2°, 18.4°±0.2°, 19.0°±0.2°, and 20.4°±0.2° in 2θ values. Type B is characterized in that its X-ray powder diffraction pattern has peaks at 15.7°±0.2°, 16.6°±0.2°, 20.1°±0.2°, and 24.1°±0.2° in 2θ values, A crystalline form of (E)-N-hydroxy-3-(1-(phenylsulfonyl)indoline-5-yl)acrylamide (ABT-301) characterized by containing the above.

2. The crystal form according to claim 1, characterized in that Type A has peaks in its X-ray powder diffraction pattern at 10.2°±0.2°, 11.8°±0.2°, 17.4°±0.2°, 21.3°±0.2°, and 21.9°±0.2° at 2θ values.

3. The crystal form according to claim 1, characterized in that Type A is a hydrate.

4. The crystal form according to claim 1, characterized in that Type B has peaks at 12.5°±0.2° and 21.7°±0.2° in its X-ray powder diffraction pattern at 2θ values.

5. The crystal form according to claim 1, wherein Type B is anhydrous.

6. The crystal form according to claim 1, characterized by having a melting point temperature of 125°C to 132°C.

7. The crystal form according to claim 1, characterized in that Type A exhibits a TGA weight loss of 2 to 4% when heated to 100°C to 150°C.

8. The crystal form according to claim 1, characterized in that the water absorption rate of Type A at 25°C / 80%RH is 0.13 to 0.14%.

9. The crystal form according to claim 1, characterized in that Type B exhibits a TGA weight loss of 0.7 to 0.8% when heated to 150°C to 170°C.

10. The crystal form according to claim 1, characterized in that the water absorption rate of Type B at 25°C / 80%RH is 0.103 to 0.109%.

11. An intermediate for preparing Type A or Type B, characterized by being a solvate of ABT-301.

12. The intermediate according to claim 11, characterized in that the solvate includes IPA solvate, acetone solvate, ACN solvate, MeOH solvate, NMP solvate, THF solvate, EtOAc solvate, DMAc solvate, EtOH solvate, DCM solvate, DMSO solvate, 1,4-dioxane solvate, or MIBK solvate.

13. A pharmaceutical composition characterized by comprising the crystalline form, surfactant, and oil described in claim 1.

14. The pharmaceutical composition according to claim 13, characterized in that the surfactant is polysorbate 80.

15. The pharmaceutical composition according to claim 13, characterized in that the oil is castor oil.

16. The pharmaceutical composition according to claim 13, characterized in that it is an HDAC inhibitor.

17. The pharmaceutical composition according to claim 13, characterized in that it is in the form of a capsule.

18. The pharmaceutical composition according to claim 17, characterized by containing 25 mg to 100 mg of the crystalline form described in claim 1.

19. The pharmaceutical composition according to claim 17, characterized in that it is encapsulated in a gelatin shell.

20. The pharmaceutical composition according to claim 13, further comprising a plasticizer.

21. The pharmaceutical composition according to claim 20, characterized in that the plasticizer is propylene glycol.

22. A method for treating cancer or fibrosis, characterized by comprising administering a therapeutically effective amount of the crystalline form described in any one of claims 1 to 10 to a patient in need of treatment.

23. The method according to claim 22, characterized in that the crystalline form described in any one of claims 1 to 10 is an HDAC inhibitor.

24. The method according to 22, characterized in that the cancer is pancreatic cancer, bladder cancer, colorectal cancer, breast cancer, prostate cancer, kidney cancer, hepatocellular carcinoma, lung cancer, non-small cell lung cancer (NSCLC), ovarian cancer, cervical cancer, stomach cancer, esophageal cancer, neuroendocrine cancer, bone cancer, or head and neck cancer.

25. The method according to 22, characterized in that the fibrosis is pulmonary fibrosis, hepatic fibrosis, cutaneous fibrosis, or renal fibrosis.

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