Method for producing salt and crystalline forms of 2,6-piperidinedione compounds and their use

Abstract

This invention discloses methods for producing salt and crystalline forms of 2,6-piperidinedione compounds and their uses. JPEG2026511603000053.jpg41143

Description

[Technical Field]

[0001] This application claims the following priority: This application claims priority and interest in Chinese patent application No. 2023102949509, filed with the State Intellectual Property Administration of China on March 24, 2023, the disclosures of which are incorporated herein by reference in their entirety.

[0002] This invention relates to a method for producing salt and crystalline forms of 2,6-piperidinedione compounds and to the use of such compounds. [Background technology]

[0003] Interleukin-1 receptor-associated kinase 4 (IRAK4) plays a relay role in the signaling pathways between the Toll-like receptor family (TLRs) and the interleukin-1 receptor family (IL-1Rs), receiving upstream signals and activating downstream JNK and NF-κB signaling pathways, and is closely related to the development and progression of inflammatory immune diseases and tumors in humans.

[0004] Myeloid differentiation factor (MyD88), a Toll-like receptor (TLR) signaling protein, is frequently mutated in various lymphomas, including Waldenström macroglobulinemia, lymphoplasmacytic lymphoma, diffuse large B-cell lymphoma, and marginal zone lymphoma, with mutation rates of 95-97%, 79%, 50-80%, 15-29%, and 6-10%, respectively. IRAK4 is involved in almost all biological functions of MyD88 and is a drug target with extremely high appeal and unlimited potential, particularly as a therapeutic target for MyD88-driven lymphomas.

[0005] Research has revealed that IRAK4 exerts its biological function not only through protein phosphorylation but also through complex formation with MyD88. While IRAK4 phosphorylation is necessary for activation of the JNK signaling pathway, it is not required for activation of the NF-κB signaling pathway. This suggests that IRAK4 possesses a dual function as both a protein kinase and a scaffolding protein, acting in signaling pathways. Therefore, conventional small molecule kinase inhibitors targeting IRAK4 cannot completely inhibit all of its biological functions.

[0006] Proteolysis Targeting Chimera (PROTAC) is a technology that utilizes the ubiquitin-proteasome system to target specific proteins and induce their degradation within cells. The ubiquitin-proteasome system is the major pathway for intracellular protein degradation, and its normal physiological function is primarily to remove denatured, mutated, or harmful proteins within the cell. More than 80% of intracellular proteins are degraded through this system. PROTAC utilizes cell-specific protein degradation mechanisms to remove specific target proteins within the cell. PROTAC technology has matured over time, making it possible to target a wide variety of proteins, including scaffold proteins, transcription factors, enzymes, and regulatory proteins. Furthermore, thalidomide-based drugs, also known as immunomodulatory drugs (IMiDs), activate the E3 ubiquitin ligase complex formed with cereblon (CRBN), promoting the ubiquitination of transcription factors IKZF1 and IKZF3, and then exerting toxic effects on tumors through recognition and degradation by the proteasome. CRBN has been shown to be an important target for antitumor drugs and immunomodulators, demonstrably demonstrating therapeutic efficacy in various hematological malignancies, skin diseases such as erythema nodosum, and autoimmune diseases such as systemic lupus erythematosus. [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] Therefore, by developing PROTACs and IMiD dual-functional molecules targeting IRAK4, removing IRAK4 by degrading it, more thoroughly blocking all functions of IRAK4, comprehensively suppressing the IRAK4 signaling pathway fundamentally, and simultaneously exerting a synergistic therapeutic effect due to the excellent CRBN regulatory effect, the anti-tumor effect can be exerted more effectively, and the clinical treatment effect can be enhanced.

Means for Solving the Problems

[0008] The present invention provides maleate, citrate, p-toluenesulfonate, oxalate, and hydrobromide salts of the compound of formula (I), and preferably, the maleate, citrate, p-toluenesulfonate, oxalate, and hydrobromide salts are in crystalline form.

[0009]

Chemical formula

[0010] In some embodiments of the present invention, the maleate salt of the compound of formula (I) is the compound of formula (II),

[0011]

Chemical formula

[0012] Here, n is 1.9 to 2.1, preferably 1.9, 2.0, and 2.1, and more preferably 2.0.

[0013] In some embodiments of the present invention, for the maleate salt of the compound of formula (I), the molar ratio of the compound of formula (I) to maleic acid is 1:1.9 to 1:2.1, preferably 1:1.9, 1:2.0, 1:2.1, and more preferably 1:2.0.

[0014] In some embodiments of the present invention, the compound of formula (II) is in crystalline form.

[0015] In some embodiments of the present application, the crystalline form of the compound of formula (II) exists as a solvate crystalline form.

[0016] The present invention provides a type A crystal of the compound of formula (II) whose powder X-ray diffraction pattern has characteristic diffraction peaks at 2θ angles of 6.67±0.20°, 9.52±0.20°, 17.03±0.20°, and 19.31±0.20°.

[0017] In some embodiments of the present invention, the powder X-ray diffraction pattern of the A-type crystal of the compound of formula (II) has characteristic diffraction peaks at 2θ angles of 6.67°, 9.52°, 17.03°, and 19.31°.

[0018] In some embodiments of the present invention, the A-type crystal of the compound of formula (II) has a powder X-ray diffraction (XRPD) pattern substantially as shown in Figure 1.

[0019] In some embodiments of the present invention, the powder X-ray diffraction (XRPD) pattern analysis data of the A-type crystal of the compound of formula (II) is shown in Table 1:

[0020] Table 1: XRPD pattern analysis data of type A crystals of compound (II) [Table 1]

[0021] In some embodiments of the present invention, the A-type crystal of the compound of formula (II) has endothermic peak values ​​at 153.3°C and 172.3°C in its differential scanning calorimetry (DSC) curve.

[0022] In some embodiments of the present invention, the A-type crystal of the compound of formula (II) has a DSC spectrum as shown in Figure 2.

[0023] In some embodiments of the present invention, the A-type crystal of the compound of formula (II) exhibits a weight loss of 6.42% at 150.0°C in its thermogravimetric analysis (TGA) curve.

[0024] In some embodiments of the present invention, the TGA spectrum of the A-type crystal of the compound of formula (II) is as shown in Figure 3.

[0025] In some embodiments of the present invention, the p-toluenesulfonate of the compound of formula (I) is the compound of formula (III),

[0026] [ka]

[0027] Here, m is 1.1 to 1.5; preferably 1.1, 1.2, 1.3, 1.4 and 1.5; and more preferably 1.3.

[0028] In some embodiments of the present invention, the p-toluenesulfonate salt of the compound of formula (I) is used, where the molar ratio of the compound of formula (I) to p-toluenesulfonic acid is 1:1.1 to 1:1.5, preferably 1:1.1, 1:1.2, 1:1.3, 1:1.4, and 1:1.5, and more preferably 1:1.3.

[0029] In some embodiments of the present invention, the compound of formula (III) is in crystalline form.

[0030] In some embodiments of the present application, the crystalline form of the compound of formula (III) exists as a solvate crystalline form.

[0031] The present invention provides a type A crystal of the compound of formula (III) whose powder X-ray diffraction pattern has characteristic diffraction peaks at 2θ angles of 10.61±0.20°, 12.06±0.20°, 15.79±0.20°, and 17.96±0.20°.

[0032] In some embodiments of the present invention, the A-type crystal of the compound of formula (III) has a powder X-ray diffraction pattern characterized by diffraction peaks at 2θ angles of 10.61°, 12.06°, 15.79°, and 17.96°.

[0033] In some embodiments of the present invention, the A-type crystal of the compound of formula (III) has a powder X-ray diffraction (XRPD) pattern as shown in Figure 4.

[0034] In some embodiments of the present invention, the powder X-ray diffraction (XRPD) pattern analysis data of the A-type crystal of the compound of formula (III) is shown in Table 2:

[0035] Table 2: XRPD pattern analysis data of type A crystals of compound (III) [Table 2]

[0036] In some embodiments of the present invention, the A-type crystal of the compound of formula (III) has endothermic peak values ​​at 78.5°C, 158.7°C, and 169.0°C in its differential scanning calorimetry curve.

[0037] In some embodiments of the present invention, the A-type crystal of the compound of formula (III) has a DSC spectrum as shown in Figure 5.

[0038] In some embodiments of the present invention, the A-type crystal of the compound of formula (III) exhibits a weight loss of 5.16% at 150.0°C in its thermogravimetric analysis curve.

[0039] In some embodiments of the present invention, the A-type crystal of the compound of formula (III) has a TGA spectrum as shown in Figure 6.

[0040] In some embodiments of the present invention, the oxalate of the compound of formula (I) is the compound of formula (IV),

[0041] [ka]

[0042] Here, r is 2.1 to 2.6, preferably 2.1, 2.2, 2.3, 2.4, 2.5, and 2.6, and more preferably 2.4.

[0043] In some embodiments of the present invention, the oxalate salt of the compound of formula (I) is used, where the molar ratio of the compound of formula (I) to oxalic acid is 1:2.1 to 1:2.6, preferably 1:2.1, 1:2.2, 1:2.3, 1:2.4, 1:2.5 and 1:2.6, and more preferably 1:2.4.

[0044] In some embodiments of the present invention, the compound of formula (IV) is in crystalline form.

[0045] In some embodiments of the present application, the crystalline form of the compound of formula (IV) may exist as a solvate crystalline form.

[0046] The present invention provides a type A crystal of the compound of formula (IV) whose powder X-ray diffraction pattern has characteristic diffraction peaks at 2θ angles of 5.65±0.20°, 11.16±0.20°, and 19.49±0.20°.

[0047] In some embodiments of the present invention, the A-type crystal of the compound of formula (IV) has a powder X-ray diffraction pattern characterized by diffraction peaks at 2θ angles of 5.65±0.20°, 8.39±0.20°, 11.16±0.20°, 17.03±0.20°, 19.49±0.20°, 22.26±0.20°, and 23.09±0.20°.

[0048] In some embodiments of the present invention, the A-type crystal of the compound of formula (IV) has a powder X-ray diffraction pattern characterized by diffraction peaks at 2θ angles of 5.65°, 8.39°, 11.16°, 17.03°, 19.49°, 22.26°, and 23.09°.

[0049] In some embodiments of the present invention, the A-type crystal of the compound of formula (IV) has a powder X-ray diffraction (XRPD) pattern as shown in Figure 7.

[0050] In some embodiments of the present invention, the powder X-ray diffraction (XRPD) pattern analysis data of the A-type crystal of the compound of formula (IV) is shown in Table 3:

[0051] Table 3 XRPD pattern analysis data of type A crystals of compound (IV) [Table 3]

[0052] In some embodiments of the present invention, the A-type crystal of the compound of formula (IV) has an endothermic peak value at 216.9°C in its differential scanning calorimetry curve.

[0053] In some embodiments of the present invention, the A-type crystal of the compound of formula (IV) has a DSC spectrum as shown in Figure 8.

[0054] In some embodiments of the present invention, the A-type crystal of the compound of formula (IV) exhibits a weight loss of 6.40% at 150.0°C in its thermogravimetric analysis curve.

[0055] In some embodiments of the present invention, the A-type crystal of the compound of formula (IV) has a TGA spectrum as shown in Figure 9.

[0056] In some embodiments of the present invention, the hydrobromide salt of the compound of formula (I) is the compound of formula (V),

[0057] [ka]

[0058] Here, p is 1.0 to 1.5, preferably 1.0, 1.1, 1.2, 1.3, 1.4, and 1.5; more preferably 1.2.

[0059] In some embodiments of the present invention, the hydrobromide salt of the compound of formula (I) is used, where the molar ratio of the compound of formula (I) to hydrobromic acid is 1:1.1 to 1:1.5, preferably 1:1.0, 1:1.1, 1:1.2, 1:1.3, 1:1.4 and 1:1.5, and more preferably 1:1.2.

[0060] In some embodiments of the present invention, the compound of formula (V) is in crystalline form.

[0061] In some embodiments of the present application, the crystalline form of the compound of formula (V) may exist as a solvate crystalline form.

[0062] The present invention provides a type A crystal of the compound of formula (V) whose powder X-ray diffraction pattern has characteristic diffraction peaks at 2θ angles of 6.51±0.20°, 8.94±0.20°, 17.87±0.20°, 19.88±0.20°, and 21.51±0.20°.

[0063] In some embodiments of the present invention, the A-type crystal of the compound of formula (V) has a powder X-ray diffraction pattern characterized by diffraction peaks at 2θ angles of 6.51±0.20°, 8.94±0.20°, 9.76±0.20°, 13.09±0.20°, 17.87±0.20°, 19.88±0.20°, 21.51±0.20°, and 27.11±0.20°.

[0064] In some embodiments of the present invention, the A-type crystal of the compound of formula (V) has a powder X-ray diffraction pattern characterized by diffraction peaks at 2θ angles of 6.51°, 8.94°, 9.76°, 13.09°, 17.87°, 19.88°, 21.51°, 27.11°, and 29.37°.

[0065] In some embodiments of the present invention, the A-type crystal of the compound of formula (V) has a powder X-ray diffraction (XRPD) pattern as shown in Figure 10.

[0066] In some embodiments of the present invention, the A-type crystal of the compound of formula (V) has the following powder X-ray diffraction (XRPD) pattern analysis data, as shown in Table 4:

[0067] Table 4 XRPD pattern analysis data of type A crystals of compound (V) [Table 4]

[0068] In some embodiments of the present invention, the A-type crystal of the compound of formula (V) has endothermic peak values ​​at 64.7°C and 217.5°C in its differential scanning calorimetry curve.

[0069] In some embodiments of the present invention, the A-type crystal of the compound of formula (V) has a DSC spectrum as shown in Figure 11.

[0070] In some embodiments of the present invention, the A-type crystal of the compound of formula (V) exhibits a weight loss of 4.06% at 150.0°C in its thermogravimetric analysis curve.

[0071] In some embodiments of the present invention, the A-type crystal of the compound of formula (V) has a TGA spectrum as shown in Figure 12.

[0072] In some embodiments of the present invention, the citrate of the compound of formula (I) is the compound of formula (VI),

[0073] [ka]

[0074] Here, q is between 2.1 and 2.3, preferably 2.1, 2.2, and 2.3, and more preferably 2.2.

[0075] In some embodiments of the present invention, the citrate of the compound of formula (I) is used, where the molar ratio of the compound of formula (I) to citric acid is 1:2.1 to 1:2.3, preferably 1:2.1, 1:2.2, 1:2.3, and more preferably 1:2.2.

[0076] In some embodiments of the present invention, the compound of formula (VI) is in crystalline form.

[0077] In some embodiments of the present application, the crystalline form of the compound of formula (VI) may exist as a solvate crystalline form.

[0078] The present invention provides a type A crystal of the compound of formula (VI) whose powder X-ray diffraction pattern has characteristic diffraction peaks at 2θ angles of 7.63±0.20° and 18.46±0.20°.

[0079] In some embodiments of the present invention, the A-type crystal of the compound of formula (VI) has a powder X-ray diffraction pattern characterized by diffraction peaks at 2θ angles of 7.63° and 18.46°.

[0080] In some embodiments of the present invention, the A-type crystal of the compound of formula (VI) has a powder X-ray diffraction (XRPD) pattern as shown in Figure 13.

[0081] In some embodiments of the present invention, the powder X-ray diffraction (XRPD) pattern analysis data of the A-type crystal of the compound of formula (VI) is shown in Table 5:

[0082] Table 5: XRPD pattern analysis data of type A crystals of compound (VI). [Table 5]

[0083] In some embodiments of the present invention, the A-type crystal of the compound of formula (VI) has a differential scanning calorimetry curve with endothermic peaks at 77.6°C, 159.4°C, and 184.2°C.

[0084] In some embodiments of the present invention, the A-type crystal of the compound of formula (VI) has a DSC spectrum as shown in Figure 14.

[0085] In some embodiments of the present invention, the A-type crystal of the compound of formula (VI) exhibits a weight loss of 7.05% at 150.0°C in its thermogravimetric analysis curve.

[0086] In some embodiments of the present invention, the A-type crystal of the compound of formula (VI) has a TGA spectrum as shown in Figure 15.

[0087] The present invention further provides the use of the compound of formula (II), the compound of formula (III), the compound of formula (IV), the compound of formula (V), the compound of formula (VI), the A-type crystal of the compound of formula (II), the A-type crystal of the compound of formula (III), the A-type crystal of the compound of formula (IV), the A-type crystal of the compound of formula (V), or the A-type crystal of the compound of formula (VI) in the manufacture of a therapeutic agent for diffuse large B-cell lymphoma.

[0088] Technical effects

[0089] The compound of the present invention exhibits excellent degradative activity against target proteins IRAK4, IKZF1, and IKZF3, exerts excellent cell proliferation inhibitory activity in lymphoma cell lines OCI-LY10, TMD-8, and SU-DHL-2, has a remarkable tumor suppressor effect, is dose-dependent, and simultaneously possesses good pharmacokinetic properties and oral absorption rate, making it suitable for use as a pharmaceutical product.

[0090] Definition and explanation Unless otherwise specified, the following terms and phrases used herein shall have the meanings set forth below. Where no particular phrase or term is specifically defined, it should not be interpreted as uncertain or unclear, but rather understood in its ordinary sense. Where a trademark name is mentioned herein, it is intended to refer to the corresponding product or its active ingredient.

[0091] It is well known in crystallography that the relative intensity of diffraction peaks can change due to preferred orientation, which is influenced by factors such as the crystal morphology, for any given crystal form. While the peak intensity changes when preferred orientation is present, the position of the diffraction peaks in relation to the crystal form remains unchanged. Furthermore, it is a well-known fact in crystallography that slight errors may occur in the peak position for any given crystal form. For example, factors such as temperature changes during analysis, sample movement, or instrument calibration can cause fluctuations in the peak position, resulting in a measurement error of approximately ±0.20° for the 2θ value. Therefore, those skilled in the art know that this error should be considered when identifying each crystal structure.

[0092] DSC measurement measures the transition temperature at which a crystal absorbs or releases heat due to a change in its crystal structure or crystal melting. For identical compounds in the same crystal form, the error between the thermal transition temperature and melting point in continuous analysis is typically within approximately 5°C or 3°C. When a compound is said to have a given DSC peak or melting point, this refers to a range of ±5°C or ±3°C from that DSC peak or melting point. DSC provides an auxiliary method for identifying different crystal forms. Different crystal forms can be distinguished based on different transition temperature characteristics. It should be noted that in the case of mixtures, the DSC peak and melting point can vary over a wider range. Furthermore, because decomposition is involved in the melting process of a substance, the melting temperature depends on the heating rate.

[0093] Unless otherwise specified, DSC spectra indicate exothermic activity with an upward curve.

[0094] Even within the same crystal form, the weight loss temperature due to TGA can vary depending on factors such as the measuring device, measurement method / conditions, etc. For any given specific crystal form, there may be an error in the weight loss temperature, which can be approximately ±5°C or approximately ±3°C.

[0095] Furthermore, during the preparation of the crystalline form of pharmaceuticals, it is unavoidable that, due to external conditions and internal factors, the solvent molecules and compound molecules will form a eutectic during the process of contact between the drug molecules and solvent molecules, and remain in the solid material. This results in the formation of solvates, which specifically include stoichiometric solvates and non-stoichiometric solvates. All solvates described are included within the scope of the present invention.

[0096] The aforementioned “pharmaceutically acceptable adjuvants” refer to inert substances administered together with the active ingredient to facilitate the administration of the active ingredient, and include, but are not limited to, lubricants, sweeteners, diluents, preservatives, dyes / colorants, flavor enhancers, surfactants, wetting agents, dispersants, disintegrants, suspending agents, stabilizers, isotonic agents, solvents, or emulsifiers that are permitted for use in humans or animals (e.g., livestock) as approved by the National Food and Drug Administration.

[0097] The term "crystalline composition" refers to a mixture of the crystalline form of the compound of formula (I) of the present invention with other crystalline forms of the compound, amorphous materials, or other impurities. For example, a crystalline composition of type A crystals of the compound of formula (I) includes, in addition to the type A crystals of the compound of formula (I), other crystalline forms of the compound of formula (I), amorphous materials, or other impurities.

[0098] The term "pharmaceutical composition" refers to a mixture comprising one or more compounds of the present invention or their salts, and pharmaceutically acceptable adjuvants. The purpose of the pharmaceutical composition is to facilitate the administration of the compounds of the present invention to an organism.

[0099] The therapeutic dose of the compound of the present invention can be determined, for example, based on the specific therapeutic use, the method of administration of the compound, the patient's health condition, and the judgment of the prescribing physician. The ratio or concentration of the compound of the present invention in a pharmaceutical composition may not be fixed due to various factors, including dosage, chemical properties (e.g., hydrophobicity), and route of administration.

[0100] The term "treatment" means administering one or more of the compounds or formulations described in the present invention to improve or eliminate a disease or one or more symptoms associated with that disease, and includes: (i) Suppression of a disease or condition, i.e., prevention of its progression; (ii) Relief of the disease or condition, that is, to reduce the disease or condition.

[0101] The term “therapeutic dose” means an amount of the compound of the present invention that (i) treats a particular disease, condition, or disorder; (ii) reduces, improves, or eliminates one or more symptoms of a particular disease, condition, or disorder; or (iii) prevents or delays the onset of one or more symptoms of a particular disease, condition, or disorder as described herein. The amount of the compound of the present invention constituting a “therapeutic dose” varies depending on the compound, the disease condition and its severity, the route of administration, and the age of the mammal being treated, but can be routinely determined by those skilled in the art based on their own knowledge and this disclosure.

[0102] Unless otherwise specified herein, the terms “comprise,” and its English variants “comprises,” and “comprising” shall be interpreted in an open, inclusive sense, that is, “including but not limited to these.”

[0103] Throughout this specification, the terms “several embodiments,” “embodiments,” “in another embodiment,” or “in several embodiments” refer to at least one embodiment that includes the relevant specific reference elements, structures, or features described in that embodiment. Therefore, the phrases “several embodiments,” “in an embodiment,” “in another embodiment,” or “in several embodiments” appearing in various places throughout this specification do not necessarily all refer to the same embodiment. Furthermore, specific elements, structures, or features can be combined in appropriate ways in one or more embodiments.

[0104] As used herein and in the appended claims, the singular article "one" (equivalent to "a," "an," and "the" in English) should be understood to include multiple subjects unless the context explicitly indicates otherwise. Therefore, for example, a reaction involving a "catalyst" as mentioned includes one catalyst, or two or more catalysts. Furthermore, the term "or" should be understood to generally include the meaning of "and / or" unless explicitly specified in the context.

[0105] The intermediate compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combinations with other chemical synthesis methods, and equivalent alternatives well known to those skilled in the art, but preferred embodiments are not limited to the examples of the present invention.

[0106] The chemical reactions in specific embodiments of the present invention are completed in a suitable solvent, which should be suitable for the chemical changes and required reagents and materials of the present invention. Those skilled in the art may need to modify or select synthesis steps or reaction processes based on existing embodiments to obtain the compounds of the present invention.

[0107] [ka]

[0108] The present invention will be specifically described by examples, but these examples do not limit the present invention in any way.

[0109] All solvents used in this invention are commercially available and can be used without further purification.

[0110] Compounds were named manually or using ChemDraw® software, and commercially available compounds were named using their supplier catalog names.

[0111] The abbreviations used in this invention are as follows: RT = room temperature, MeOH = methanol, EtOH = ethanol, IPA = isopropanol, Acetone = acetone, MIBK = methyl isobutyl ketone, siRNA = ethyl acetate, IPAc = isopropyl acetate, MTBE = methyl tert-butyl ether, THF = tetrahydrofuran, 2-Me THF = 2-methyltetrahydrofuran, DCM = dichloromethane, CHCl3 = chloroform, Toluene = toluene, n-Heptane = n-heptane, DMSO = dimethyl sulfoxide, DMAc = N,N-dimethylacetamide, NMP = N-methylpyrrolidone, H2O = water, 1,4-Dioxane = 1,4-dioxane, and ACN = acetonitrile. [Brief explanation of the drawing]

[0112] [Figure 1] Figure 1 shows the powder X-ray diffraction (XRPD) pattern of the A-type crystal of the compound of formula (II). [Figure 2] Figure 2 shows the DSC spectrum of the A-type crystal of the compound of formula (II). [Figure 3] Figure 3 shows the TGA spectrum of the A-type crystal of the compound of formula (II). [Figure 4] Figure 4 shows the powder X-ray diffraction (XRPD) pattern of the A-type crystal of the compound of formula (III). [Figure 5] Figure 5 shows the DSC spectrum of the A-type crystal of the compound of formula (III); [Figure 6] Figure 6 shows the TGA spectrum of the A-type crystal of the compound of formula (III). [Figure 7] Figure 7 shows the powder X-ray diffraction (XRPD) pattern of the A-type crystal of the compound of formula (IV). [Figure 8] Figure 8 shows the DSC spectrum of the A-type crystal of the compound of formula (IV); [Figure 9] Figure 9 shows the TGA spectrum of the A-type crystal of the compound of formula (IV). [Figure 10] Figure 10 shows the powder X-ray diffraction (XRPD) pattern of the A-type crystal of the compound of formula (V). [Figure 11] Figure 11 shows the DSC spectrum of the A-type crystal of the compound of formula (V). [Figure 12] Figure 12 shows the TGA spectrum of the A-type crystal of the compound of formula (V). [Figure 13] Figure 13 shows the powder X-ray diffraction (XRPD) pattern of the A-type crystal of the compound of formula (VI). [Figure 14] Figure 14 shows the DSC spectrum of the A-type crystal of the compound of formula (VI). [Figure 15] Figure 15 shows the TGA spectrum of the A-type crystal of the compound of formula (VI). [Figure 16] Figure 16 shows the solution nuclear magnetic resonance (Solution NMR) spectrum of the crystalline compound of formula (II). [Figure 17] Figure 17 shows the solution nuclear magnetic resonance (Solution NMR) spectrum of the type A crystal of the compound of formula (III). [Figure 18] Figure 18 shows the solution nuclear magnetic resonance (Solution NMR) spectrum of the A-type crystal of the compound of formula (IV). [Figure 19] Figure 19 shows the solution nuclear magnetic resonance (Solution NMR) spectrum of the A-type crystal of the compound of formula (V). [Figure 20] Figure 20 shows the solution nuclear magnetic resonance (Solution NMR) spectrum of the crystalline compound of formula (VI). [Modes for carrying out the invention]

[0113] Equipment and analytical methods 1.1 The Powder X-ray Diffraction (X-ray powder diffractometer, XRPD) Method of the Present Invention Instrument model: PANalytical powder X-ray diffraction analyzer Test method: Approximately 10-20 mg of sample was used for XRPD measurement.

[0114] The detailed measurement parameters are as follows: X-ray source: Cu, Kα, (λ=1.540598Å, λ=1.544426Å) Tube voltage: 45kV, Tube current: 40mA Divergence slit: 1 / 8° Scanning mode: Continuous Scanning range (°2Theta): 3~40 Step scan time (s): 46.7 Scanning step size (°2Theta): 0.0263 Measurement time: 5 minutes

[0115] 1.2 Differential Scanning Calorimeter (DSC) Method of the Present Invention Instrument model: TA 2500 Differential Scanning Calorimeter

[0116] Measurement method: The sample (1 mg) was placed in a DSC aluminum pan and heated from 25°C (room temperature) to 300°C (or 350°C) at a heating rate of 10°C / min under a 50 mL / min N2 atmosphere.

[0117] 1.3 The Thermogravimetric Analysis (TGA) Method of the Present Invention Equipment Model: TA 5500 Thermogravimetric Analyzer

[0118] Test method: The sample (2-5 mg) is placed in a TGA platinum container for measurement and heated from room temperature to 350°C at a heating rate of 10°C / min under N2 conditions of 25 mL / min, or until the weight loss reaches 20%.

[0119] 1.4 Dynamic Vapor Soaping (DVS) Method of the Present Invention Equipment Model: SMS (Surface Measurement Systems) DVS Intrinsic Plus Dynamic Water Vapor Adsorption System

[0120] Measurement conditions: The sample (10-20 mg) was placed on a DVS sample dish and measured.

[0121] The detailed DVS parameters are as follows: Protective gas and flow rate: N2, 200 mL / min Temperature: 25℃ Equilibrium: dm / dt=0.002% / min (Min: 10 minutes, Max: 180 minutes) Drying: Dry at 0% RH for 120 minutes. RH (%) range: 0%~95% RH (%) gradient: 10% (0%RH~90%RH, 90%RH~0%RH); 5% (90%RH~95%RH, 95%RH~90%RH)

[0122] The classification of hygroscopic properties is as shown in Table 6 below:

[0123] Table 6 Evaluation of hygroscopic properties [Table 6]

[0124] Note: ΔW% indicates the rate of increase in moisture absorption of the test sample under conditions of 25±1℃ and 80±2%RH.

[0125] 1.5 The Ultra-High Performance Liquid Chromatography / Ion Chromatography (UPLC / IC) Method of the Present Invention Instrument model: Waters H-Class ultrafast liquid chromatograph and ion chromatograph

[0126] The detailed ULC parameters are as follows: Column: Xbridge C18, 4.6 × 150 mm, 5 μm Mobile phase: A: H2O containing 0.1% TFA; B: Acetonitrile containing 0.1% TFA Gradient: 0.0 min, 10% B; 6.0 min, 90% B; 10.0 min, 90% B; 10.1 min, 10% B; 15.0 min, 10% B Runtime: 15.0 minutes Mobile phase flow rate: 0.0 min Injection volume: 1.0mL / min Detection wavelength: 3 μL Column temperature: UV at 226nm Sampler temperature: 25℃ Diluent: ACN / H2O = 1:1 (v / v)

[0127] The detailed IC parameters are as follows: Column: Dionex IonPac (trademark) AS18 RFC (trademark) 4×250mm Analytical Mobile phase: 25mM NaOH Injection volume: 25μL Flow rate: 1.0mL / min Temperature: 35℃ Column temperature: 35℃ Current: 80mA Operating time: 16 minutes

[0128] 1.6 Solution Nuclear Magnetic Resonance (Solution NMR) Method of the Present Invention Instrument model: Bruker 400M Nuclear Magnetic Resonance Spectrometer Solvent: DMSO-d6 or deuterated methanol

[0129] Specific Embodiments The present application will be described in detail below through examples, but this will not constitute any unfavorable limitation of the present application. The present application has already been described in detail and specific examples have been disclosed, and it will be readily apparent to those skilled in the art that various modifications and improvements can be made to specific embodiments without departing from the spirit and scope of the present application.

[0130] Example 1: Preparation of the compound of formula (I) [ka]

[0131] Step 1: Synthesis of Compounds 1-2 Under a nitrogen atmosphere at room temperature, 2-(4-(tert-butoxycarbonyl)piperazin-1-yl)acetic acid (115.98 g, 474.75 mmol) was dissolved in N,N-dimethylformamide (1 L), and N,N-diisopropylethylamine (163.62 g, 1.27 mol) and O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (180.51 g, 474.75 mmol) were added. The reaction mixture was stirred at room temperature for 0.5 hours, then the hydrochloride salt of compound 1-1 (105 g, 316.5 mmol) was added, and the reaction mixture was stirred at room temperature for 12 hours. After the reaction was complete, the reaction solution was poured into ice water (5 L) and extracted with ethyl acetate (4 × 1 L). The organic phases were combined, washed with saturated brine (3 × 1 L), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to remove the solvent. The residue was dissolved in a mixed solvent of ethyl acetate and dichloromethane (volume ratio 1:1, 1.5 L), stirred at room temperature for 1 hour, and a solid gradually precipitated. The mixture was concentrated under reduced pressure to remove most of the dichloromethane, methyl tert-butyl ether (1.5 L) was added, and the mixture was stirred at room temperature for 12 hours. The mixture was then filtered to collect the filtrate. Ethyl acetate (1.5 L) was added to the cake, stirred at room temperature for 2 hours, filtered, washed with ethyl acetate (200 mL × 2), and the collected cake was vacuum-dried to obtain compounds 1-2. MS-ESI m / z: 522.3 [M+H] + . 1H NMR (400 MHz, DMSO_d6) δ: 11.14 (s, 1H), 10.13 (s, 1H), 8.19 (d, J=9.6 Hz, 1H), 8.11 (d, J=8.0 Hz, 1H), 8.00 (d, J=9.26 Hz, 1H), 7.80-7.68 (m, 2H), 5.09 (dd, J=4.8 Hz, 11.2 Hz, 1H), 3.52-3.39 (m, 4H), 3.32 (s, 2H), 2.93-2.78 (m, 1H), 2.70-2.55 (m, 6H), 2.45-2.35 (m, 1H), 1.42 (s, 9H).

[0132] Step 2: Synthesis of trifluoroacetates of compounds 1-3 At room temperature, compounds 1-2 (153 g, 293.35 mmol) were dissolved in dichloromethane (1.2 L), and trifluoroacetic acid (462 g, 4.05 mol) was slowly added dropwise. The reaction mixture was stirred at room temperature for 12 hours. After the reaction was complete, the mixture was concentrated under reduced pressure to remove the solvent and obtain the trifluoroacetate salt of compound 1-3. MS-ESI m / z: 422.1 [M+H] + .

[0133] Step 3: Synthesis of compounds 1-4 Under a nitrogen atmosphere at room temperature, compound 1-4-1 (153 g, 260.81 mmol) was dissolved in dichloromethane (3 L), and triphenylphosphine (82.09 g, 312.97 mmol) and imidazole (26.63 g, 319.22 mmol) were added. The reaction mixture was cooled to 0°C in an ice bath, and elemental iodine (86.06 g, 339.06 mmol) was added in batches. The reaction mixture was then heated to room temperature and stirred for 16 hours. After the reaction was complete, saturated sodium sulfite aqueous solution (1 L) was slowly added to the reaction mixture, stirred for 20 minutes, and then separated. The aqueous phase was extracted with dichloromethane (3 × 3 L). The organic phases were combined, washed with saturated brine (2 × 2 L), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to remove the solvent. Methyl tert-butyl ether (600 mL) was added to the residue, stirred at room temperature for 2 hours, filtered, and the cake was washed with methyl tert-butyl ether (50 mL x 2) and the filtrate was collected. Anhydrous magnesium chloride (80 g) was added to the filtrate, stirred at room temperature for 8 hours, filtered, and the cake was washed with methyl tert-butyl ether (50 mL x 2) and the filtrate was collected. The filtrate was concentrated under reduced pressure to obtain compounds 1-4. MS-ESI m / z: 697.0 [M+H] + .

[0134] Step 4: Synthesis of compounds 1-5 Under a nitrogen atmosphere at room temperature, the trifluoroacetate salt of compound 1-3 (129.35 g, 157.64 mmol) was dissolved in acetonitrile (1.3 L). Then, N,N-diisopropylethylamine (158.46 g, 1.23 mol) and compound 1-4 (122 g, 175.16 mmol) were added, and the reaction mixture was heated to 80°C and stirred for 12 hours. After the reaction was complete, a portion of the solvent was removed by distillation under reduced pressure, and ethyl acetate (500 mL) and water (500 mL) were added for liquid-liquid separation. The aqueous phase was extracted with ethyl acetate (500 mL x 3 times). The organic phases were combined, washed with saturated brine (300 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to remove the solvent. The residue was dissolved in a mixed solvent of ethyl acetate and acetonitrile (volume ratio 9:1, 700 mL), stirred at room temperature for 2 hours, filtered, and the cake was washed with a mixed solvent of ethyl acetate and acetonitrile (volume ratio 9:1, 30 mL x 3). The cake was recovered and vacuum-dried to obtain compounds 1-5. MS-ESI m / z: 990.4 [M+H] + .

[0135] Step 5: Synthesis of the hydrochloride salt of the compound of formula (I) Under a nitrogen atmosphere at room temperature, compounds 1-5 (80 g, 80.8 mmol) were dissolved in ethyl acetate (100 mL). A 4 M solution of hydrochloric acid and ethyl acetate (500 mL) was slowly added dropwise, and the reaction mixture was stirred at room temperature for 16 hours. After the reaction was complete, the mixture was filtered, and the cake was washed with ethyl acetate (50 mL x 3) and recovered. Ethyl acetate (350 mL) was added to the cake, and the mixture was stirred at room temperature for 2 hours. After filtering, the cake was washed with ethyl acetate (30 mL x 3), and the recovered cake was vacuum-dried to obtain the compound of formula (I). MS-ESIm / z: 890.2[M+H] + . 1HNMR(400MHz, DMSO_d6)δ:11.59(s,1H),11.14(s,1H),11.05(s,1H),9.97(s,1H),9.59(s,1H),9.15(s,1H),8.45(d,J=9.2Hz,1H),8.23-8.12( m,2H),8.06(d,J=6.0Hz,1H),7.99(d,J=9.2Hz,1H),7.81-7.73(m,2H),7.70(s,1H),7.29(d,J=6.8Hz,1H),7.22(t,J=54.4Hz,1H),5.11(dd,J= 4.0Hz,11.2Hz,1H),4.42(s,2H),4.31-4.20(m,1H),3.81(s,6H),3.47( s,2H),3.39(d,J=6.4Hz,2H),3.11(s,2H),2.94-2.78(m,1H),2.72-2.5 4(m,2H),2.45-2.35(m,1H),2.17-2.02(m,5H),2.01-1.90(m,1H),1.88 -1.74(m,2H),1.29-1.09(m,2H),0.61-0.50(m,2H),0.39-0.30(m,2H).

[0136] Step 6: Synthesis of the compound of formula (I) Under room temperature and a nitrogen atmosphere, 10 g of the hydrochloride salt of the compound of formula (I) was dissolved in a mixed solvent of isopropanol (75 mL) and water (300 mL). This mixed solution was added dropwise to a solution of imidazole (3.43 g, 50.4 mmol) in water (300 mL), stirred at room temperature for 15 hours, filtered, washed the cake with water (50 mL x 2) and isopropanol (50 mL), collected the cake, and dried under vacuum to obtain the compound of formula (I) (free base).

[0137] Example 2: Preparation of compound A crystals of formula (II) [ka]

[0138] 20 mg of the compound of formula (I) was taken, 0.5 mL of tetrahydrofuran was added, 2.0 equivalents of maleic acid was added under stirring, the reaction mixture was stirred at 25 °C for 72 hours to obtain a solid, the sample was centrifuged, and A-type crystals of the compound of formula (II) were obtained. 1 The 1H NMR results are shown in Figure 16, and the molar ratio of acid to base in the sample is 2.0, that is, n = 2.0.

[0139] Example 3: Preparation of A-type crystals of the compound of formula (III)

Chemical formula

[0140] 2,0 mg of the compound of formula (I) was taken, 0.5 mL of tetrahydrofuran was added, 1.0 equivalent of p-toluenesulfonic acid was added under stirring, the reaction mixture was stirred at 25 °C for 48 hours to obtain a solid, the sample was centrifuged, and A-type crystals of the compound of formula (III) were obtained. 1 The 1H NMR results are shown in Figure 17, and the molar ratio of acid to base in the sample is 1.3, that is, n = 1.3.

[0141] Example 4: Preparation of A-type crystals of the compound of formula (IV)

Chemical formula

[0142] 20 mg of the compound of formula (I) was taken, 0.5 mL of tetrahydrofuran was added, 2.0 equivalents of oxalic acid was added under stirring, the reaction mixture was stirred at 25 °C for 48 hours to obtain a solid, the sample was centrifuged, and A-type crystals of the compound of formula (IV) were obtained. 1 The 1H NMR results are shown in Figure 18, and it was shown from the UPLC / IC results that the molar ratio of acid to base is 2.4, that is, r = 2.4.

[0143] Example 5: Preparation of A-type crystals of the compound of formula (V)

Chemical formula

[0144] 20 mg of compound (I) was taken, 0.5 mL of tetrahydrofuran was added, and 1.0 equivalent of hydrobromic acid was added under stirring. The reaction mixture was stirred at 25°C for 48 hours to obtain a solid, and the sample was centrifuged to obtain type A crystals of compound (V). 1 The 1H NMR results are shown in Figure 19, and the UPLC / IC results indicate that the acid-to-base molar ratio is 1.2, i.e., p = 1.2.

[0145] Example 6: Preparation of compound A crystals of formula (VI) [ka]

[0146] 20 mg of compound (I) was taken, 0.5 mL of tetrahydrofuran was added, and 2.0 equivalents of citric acid were added under stirring. The reaction mixture was stirred at 25°C for 48 hours to obtain a solid, and the sample was centrifuged to obtain type A crystals of compound (VI). 1 The 1H NMR results are shown in Figure 20, where the acid-to-base molar ratio in the sample is 2.2, i.e., q = 2.2.

[0147] Biological tests Example 1: Evaluation of the degradation activity of the compound of formula (I) against target protein in K562 IRAK4-HiBiT cells. Experimental Objective: This experiment detected the degrading effect of the compound of formula (I) on the target protein IRAK4 within the K562 IRAK4-HiBiT cell.

[0148] Experimental materials: Cells and culture medium Cells: K562 IRAK4-HiBiT cells Culture medium: RPMI 1640 + 10% fetal bovine serum + 2 mM glutamine + 1 mM sodium pyruvate + penicillin / streptomycin Positive control: 1000 nM; Negative control: 0.1% DMSO

[0149] Table 7 Reagents and Consumables [Table 7]

[0150] Table 8 Equipment [Table 8]

[0151] Experimental protocol: Day 1 1. Preparation of the compound (1) The powder of the test compound was dissolved in DMSO to a stock concentration of 10 mM, and 9 μL of the 10 mM test compound was manually dispensed into the first and thirteenth columns of the LDV plate using a pipette.

[0152] (2) Using a Multidrop Combi, 6 μL of DMSO was added to columns 2-12 and 14-24.

[0153] (3) Using Bravo, the test compound was diluted threefold (3 μL + 6 μL), and the samples were taken from rows 1-11 and rows 13-23.

[0154] (4) Following the plate layout, 25 nL of compound solution (LDV plate rows 1-24) was transferred to the assay plate using Echo.

[0155] (5) Using Echo, 25 nL of 1 mM positive control solution was transferred to the assay plate as a 100% decomposition control (LC, HPE), and 25 nL of DMSO was transferred to the assay plate as a 0% control (HC, ZPE).

[0156] 2. Cell seeding (1) Remove the culture medium, wash once with DPBS, digest the cells with trypsin, count the cells, and prepare a cell suspension of 2 × 10⁻⁶ -5 Prepared in cells / mL.

[0157] (2) Using MultiDrop Combi, 25 μL / well of cell suspension was dispensed at a medium speed into experimental plates that had been treated with the test compound.

[0158] (3) The experimental plates containing the added cells were returned to an incubator at 37°C and 5% CO2 and cultured for 16-18 hours.

[0159] Day 2 (1) Using MultiDrop Combi, the detection reagent (NanoGlo lysis solution + substrate + LgBit protein) was rapidly added to the measurement plate at a rate of 25 μL / well and shaken for 10 minutes.

[0160] (2) The sample was centrifuged at 2000 rpm for 1 minute to remove air bubbles.

[0161] (3) The plates were read using the Envision and US Luminescence detection methods.

[0162] 3. Data Analysis The degradation rate (DR) of the test compound is calculated using the following formula: DR (%) = (RLU solvent control - RLU compound) / (RLU solvent control - RLU positive control) * 100%, where the solvent control is a blank control. After calculating the degradation rates of the compound at different concentrations in Excel, an inhibition curve diagram is created using XLFit software, and the minimum degradation rate, maximum degradation rate, and DC are determined. 50 Related parameters, including [specific parameter], were calculated.

[0163] The test results are shown in Table 9.

[0164] Table 9. Degradation effect of the compound of formula (I) of the present invention on target proteins in cells K562 IRAK4-HiBiT. [Table 9]

[0165] Conclusion: The compound of the present invention showed excellent target protein degradation activity in K562 IRAK4-HiBiT cells.

[0166] Experimental Example 2: In-Cell Western Analysis of the Equation (I) Compound's Effect on IKZF1 and IKZF3 Protein Expression Levels in MM.1S Cells

[0167] Experimental Objective: This experiment aimed to detect the effects of the compound of formula (I) on the expression levels of IKZF1 and IKZF3 proteins in MM.1S cells and to evaluate the degradative effects of the test compound on IKZF1 and IKZF3 proteins in MM.1S cells.

[0168] Experimental materials: Cell line: MM.1S cells (derived from ATCC; product number CRL-2974) Negative control: 0.1% DMSO

[0169] Table 10 Reagents and Consumables [Table 10]

[0170] Table 11 Equipment [Table 11]

[0171] Experimental protocol: 1) Seed MM.1S cells in the logarithmic growth phase into a 96-well plate, with 1.2 × 10⁶ cells in each well. 5 Each cell was cultured overnight.

[0172] 2) The following day, the drug was added, diluted three times to an initial concentration of 300 nM, and incubated in a 24-hour incubator with 10 concentration gradients (including DMSO) in 3 multi-wells.

[0173] 3) After centrifugation, the cell supernatant was carefully removed, and 150 μL of 4% paraformaldehyde fixative was added along the well wall, taking care not to touch the cells at the bottom, and incubated at room temperature for 20 minutes.

[0174] 4) Preparation of permeate: 0.5 mL of 10% Triton X-100 was added to 49.5 mL of PBS and mixed uniformly.

[0175] 5) Add 200 μL of permeate along the well wall, taking care not to touch the cells at the bottom, and incubate at room temperature on a shaker for 5 minutes.

[0176] 6) The washing process was repeated four times.

[0177] 7) Add 150 μL of Licor INERCEPT blocking solution along the well wall, taking care not to touch the bottom cells, and incubate at room temperature in a shaker for 1.5 hours.

[0178] 8) Ikaros (D6N9Y) rabbit monoclonal antibody (Ikaros(D6N9Y)Rabbit mAb), Aiolos (D1C1E) rabbit monoclonal antibody (Aiolos(D1C1E)Rabbit mAb), and GAPDH mouse mAb (proteintech, 60004-1-Ig) were all used after being diluted 1:100 with antibody diluent.

[0179] 9) 50 μL of mixed antibody was added to each well, and each well was incubated overnight in a shaker at 4°C with three double pores.

[0180] 10) PBST (PBS containing 0.1% Tween-20) was prepared.

[0181] 11) Remove the primary antibody, add 200 μL of PBST along the well wall, taking care not to touch the cells at the bottom, and incubate at room temperature in a shaker for 5 minutes.

[0182] 12) The washing process was repeated four times.

[0183] 13) Preparation of secondary antibody diluent: A final concentration of 0.2% tween 20 was added to the Licor INTERCEPT blocking solution.

[0184] 14) Under light-shielding conditions, the fluorescently labeled secondary antibody was diluted (1:800 dilution), and 0.5 μL each of IRDye 800CW and IRDye 680CW were added to 400 μL of the secondary antibody dilution (fluorescently labeled secondary antibody corresponding to the primary antibody).

[0185] 15) 50 μL of diluted fluorescent secondary antibody was added to each well and incubated at room temperature in a shaker under light-shielding conditions for 60 minutes.

[0186] 16) Remove the secondary antibody, add 200 μL of PBST along the well wall, taking care not to touch the cells at the bottom, and incubate at room temperature in a shaker for 5 minutes.

[0187] 17) The washing process was repeated four times, and immediately after washing, detection was performed using the Odyssey Gel Imaging System at two wavelengths of 700 nm and 800 nm.

[0188] Data Analysis Using GraphPad Prism 6 software, after inputting inhibition rate data, the curve is fitted to DC. 50 The value was calculated. Protein inhibition rate = (1 - RLs / RLv) × 100% RR (Raw Ratio) = 700nm / 800nm RLs = RR of sample-treated cells RLv = RR of solvent-treated cells

[0189] The test results are shown in Table 12.

[0190] Table 12 Degradation effect of the compound of formula (I) of the present invention on IKZF1 and IKZF3 proteins in MM.1S cells [Table 12]

[0191] Conclusion: The compound of the present invention showed excellent proteolytic activity against IKZF1 and IKZF3 proteins in MM.1S cells.

[0192] Experimental Example 3: Evaluation of the anti-proliferative effect of the compound of formula (I) in lymphoma cell lines OCI-LY10 and TMD-8 Experimental objective: This experiment verified whether the compound of formula (I) exhibits a cell growth inhibitory effect in diffuse large B-cell lymphoma cell lines OCI-LY10 and TMD-8.

[0193] Experimental materials: Table 13 Cell lines and culture methods [Table 13]

[0194] Table 14 Media and reagents [Table 14]

[0195] 1. Multi-well plate Greiner CELLSTAR® 96-well plate, flat bottom white plate (with lid, transparent bottom), #3610. <00oo894> 2. Reagents and equipment for cell viability experiments (I) Promega CellTiter-Glo luminescence cell viability detection kit (Promega-G7573).

[0197] (2) 2104 EnVision® plate reader, PerkinElmer.

[0198] Experimental protocol: 1. Cell culture The tumor cell lines were cultured in an incubator at 37°C and 5% CO2 according to the above-mentioned culture conditions. Subculture was performed regularly, and cells in the logarithmic growth phase were used for plate seeding.

[0199] 2. Cell seeding (1) Cell staining with trypan blue was performed to measure the number of viable cells.

[0200] (2) The cell concentration was adjusted to an appropriate concentration.

[0201] Table 15 Cell lines and seeding density

Table 15

[0202] (3) As shown in the above table, 100 μL of the cell suspension was added to each well of the culture plate.

[0203] (4) The culture plate was cultured overnight in an incubator at 37 °C, 5% CO2, and 100% relative humidity.

[0204] 3. Preparation of the compound storage plate Preparation of the compound stock solution storage plate at 1000 times the starting concentration: The compound was serially diluted from the highest concentration to the lowest concentration using DMSO. It was prepared each time it was used.

[0205] 4. Preparation of the 1000-fold compound working solution and cell treatment with the compound (1) Preparation of the 5-fold concentration working solution of the starting concentration of the compound: When diluting the compound 3-fold, 30 μL was aliquoted from the 1000-fold concentration stock solution of the starting concentration of the compound, 20 μL of DMSO was added to the subsequent wells, and 10 μL was sequentially aliquoted from the previous concentration to the next concentration. When diluting the compound 5-fold, 30 μL was aliquoted from the 1000-fold concentration stock solution of the starting concentration of the compound, 24 μL of DMSO was added to the subsequent wells, and 6 μL was sequentially aliquoted from the previous concentration to the next concentration. 20 μL of DMSO was added to the solvent control. The 1000-fold compound was diluted 200-fold with the medium, that is, 1 μL of the 1000-fold diluted compound was added to 199 μL of the medium, and mixed well with a multi-channel pipette.

[0206] (2) Addition of the drug: 25 μL of the 5-fold concentration compound was added to the cell culture plate.

[0207] (3) Return the 96-well cell plate to the incubator and culture OCI-LY10 (diluted 3-fold or 5-fold and cultured for 5 days after the addition of the drug), and TMD-8 (diluted 3-fold or 5-fold and cultured for 5 days after the addition of the drug).

[0208] 5. Measurement of cell viability by CellTiter-Glo luminescence assay The following procedures were carried out according to the instructions of the Promega CellTiter-Glo luminescence assay cell viability kit (Promega-G7573).

[0209] (1) Dissolve the CellTiter-Glo buffer and leave it at room temperature.

[0210] (2) Leave the CellTiter-Glo substrate at room temperature.

[0211] (3) Add 10 mL of the CellTiter-Glo buffer to the CellTiter-Glo substrate vial to dissolve the substrate and prepare the CellTiter-Glo working solution.

[0212] (4) Vortex gently to dissolve completely.

[0213] (5) Remove the cell culture plate and leave it for 30 minutes to equilibrate to room temperature.

[0214] (6) Add 60 μL (equivalent to half the volume of the cell culture medium) of the CellTiter-Glo working solution to each well. Wrap the cell plate with aluminum foil for light shielding.

[0215] (7) Shake the culture plate on an orbital shaker for 2 minutes to induce cell lysis.

[0216] (8) Leave the culture plate at room temperature for 10 minutes to stabilize the luminescence signal.

[0217] (9) The light emission signal was detected using a 2104 EnVision plate reader.

[0218] 6. Data Analysis The inhibition rate (IR) of the test compound was calculated using the following formula: IR (%) = (1 - RLU compound / RLU solvent control) × 100%. After calculating the inhibition rates of the compound at different concentrations in Excel, an inhibition curve was created using GraphPad Prism software, and the minimum inhibition rate, maximum inhibition rate, and IC were calculated. 50 Related parameters, including [specific parameter], were calculated.

[0219] The test results are shown in Table 16.

[0220] Table 16 Inhibitory effect of the compound of formula (I) of the present invention on cell proliferation in OCI-LY10 and TMD-8 cell lines [Table 16]

[0221] " / " indicates that it was not detected.

[0222] Conclusion: The compound of the present invention showed excellent cell proliferation inhibitory activity in both lymphoma cell lines OCI-LY10 and TMD-8.

[0223] Experimental Example 4: Evaluation of the antiproliferative activity of the compound of formula (I) in lymphoma cell lines and SU-DHL-2. Experimental Objective: This experiment aimed to detect the cell proliferation inhibitory effect of the compound of formula (I) in the lymphoma cell line SU-DHL-2.

[0224] Table 17 Cell lines and culture methods [Table 17]

[0225] Table 18 Culture media and reagents [Table 18]

[0226] 1. Porous plate Greiner CELLSTAR 384-, flat-bottomed black plate (with lid), # 781090

[0227] 2. Reagents and equipment for cell activity experiments (1) Promega CellTiter-Glo Luminescence Cell Activity Detection Kit (Promega-G7573).

[0228] (2) 2104 EnVision® plate reader, PerkinElmer.

[0229] Experimental protocol: 1.Cell culture Tumor cell lines were cultured in an incubator at 37°C and 5% CO2 according to the culture conditions described above. Cells were periodically subcultured, and cells in the logarithmic growth phase were used for plate seeding.

[0230] 2. Cell seeding (1) Cell staining was performed with trypan blue, and the number of viable cells was counted. (2) The cell concentration was adjusted to an appropriate level.

[0231] Table 19 Cell lines and seeding density [Table 19]

[0232] (3) As shown in the table above, 50 μL of cell suspension was added to each well of the culture plate.

[0233] (4) The culture plates were incubated overnight in an incubator at 37°C, 5% CO2, and 100% relative humidity.

[0234] 3. Preparation of storage plates for compounds The drug was added using an Echo655 instrument. The drug addition volume was 50 nL, with a final DMSO concentration of 0.1%. The culture plate was centrifuged at 1000 rpm for 1 minute, and then incubated for 4 days at 37°C, 5% CO2, and 100% relative humidity.

[0235] 4. Cell activity measurement using the CellTiter-Glo luminescence method The following procedure was performed according to the instructions for the Promega CellTiter-Glo luminescence cell activity measurement kit (Promega-G7573).

[0236] (1) Dissolve CellTiter-Glo buffer and allow to stand at room temperature.

[0237] (2) The CellTiter-Glo substrate was left standing at room temperature.

[0238] (3) CellTiter-Glo buffer was added to the CellTiter-Glo substrate vial, and the substrate was dissolved to prepare the CellTiter-Glo working solution.

[0239] (4) Gently vortex-stirred until completely dissolved.

[0240] (5) Remove the cell culture plate and allow it to stand for 30 minutes to equilibrate to room temperature.

[0241] (6) 25 μL (equivalent to half the volume of cell culture medium) of CellTiter-Glo working solution was added to each well. The cell plate was wrapped in aluminum foil to protect it from light.

[0242] (7) The culture plate was shaken in an orbital shaker for 2 minutes to induce cell lysis.

[0243] (8) To stabilize the luminescence signal, the culture plate was left to stand at room temperature for 10 minutes.

[0244] (9) The light emission signal was detected using a 2104 EnVision plate reader.

[0245] 5. Data Analysis The inhibition rate (IR) of the test compound was calculated using the following formula: IR (%) = (1 - (RLU compound - RLU blank control) / (RLU solvent control - RLU blank control)) × 100%. The inhibition rates of the compound at different concentrations were calculated in Excel, and inhibition curves were created using GraphPad Prism software. The minimum inhibition rate, maximum inhibition rate, and IC were then determined. 50 Related parameters, including [specific parameter], were calculated.

[0246] Table 20 Cell proliferation inhibitory effect of the compound of formula (I) of the present invention in the SU-DHL-2 cell line [Table 20]

[0247] Conclusion: The compound of the present invention showed excellent cell proliferation inhibitory activity in the lymphoma cell line SU-DHL-2.

[0248] Experimental Example 5: Evaluation of the pharmacokinetics of the compound of formula (I) in mice Experimental objective: In this study, C57BL / 6 or C57 male mice were used as experimental animals, and plasma drug concentrations were quantitatively measured at different time points after intravenous injection or oral administration to mice using LC / MS / MS to evaluate the pharmacokinetic properties of the compound of formula (I) of the present invention in mice.

[0249] Experimental materials:

[0250] flower).

[0251] Experimental procedure: A clear solution of the test compound was administered via tail vein injection (solvent: 10% DMSO / 10% solutol / 80% H2O) to C57BL / 6 mice (fasted), or orally into the body (feeding) of C57 mice. 50 μL of blood was collected by buccal vascular puncture at 0 hours (before administration) and at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours after intravenous injection. The blood was transferred to an anticoagulant tube containing heparin sodium, thoroughly vortex-mixed, and centrifuged at 6000 g for 3 minutes at 2-8°C. In the oral administration group, blood was collected by buccal vascular puncture at 0 hours (before administration) and at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours after administration. The blood was transferred to an anticoagulant tube containing heparin sodium, thoroughly vortex-mixed, and centrifuged at 6000 g for 3 minutes at 2-8°C. Blood concentrations were measured using LC-MS / MS, and relevant pharmacokinetic parameters were calculated using the non-compartmental model linear logarithmic trapezoidal method with Phoenix WinNonlin 8.2.0 pharmacokinetic analysis software.

[0252] Table 21 Pharmacokinetic parameters of the compound of formula (I) of the present invention in mice [Table 21]

[0253] " / " indicates that it was not detected.

[0254] Conclusion: The present invention provides plasma exposure (AUC) of the compound after oral administration. 0-inf The ) was high. In rodent mice, it exhibited superior pharmacokinetic properties.

[0255] Experimental Example 7: Evaluation of the pharmacokinetics of the compound of formula (I) in mice Experimental objective: In this study, male beagle dogs were used as experimental animals, and the plasma drug concentrations at different time points after intravenous injection or oral administration to beagle dogs were quantitatively measured using the LC / MS / MS method to evaluate the pharmacokinetic properties of the compound of formula (I) of the present invention in beagle dogs.

[0256] Experimental materials: Beagle (male, 7-10kg, Beijing Mas Biotechnology Co., Ltd.)

[0257] Experimental procedure: A clear solution of the test compound was slowly injected into the peripheral veins of Beagle dogs after feeding (solvent: 5% DMSO / 10% Solutol / 85% H2O), or administered orally to Beagle dogs (by feeding). 0.5 mL of blood was collected from the peripheral veins at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours after intravenous injection, collected in an EDTA-2K anticoagulant tube, and centrifuged at 3200 g for 10 minutes at 2-8°C to separate the supernatant. Similarly, 0.5 mL of blood was collected from the peripheral veins at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours after oral administration, collected in an EDTA-2K anticoagulant tube, and centrifuged at 3200 g for 10 minutes at 2-8°C to separate the supernatant. Plasma drug concentrations were measured using LC-MS / MS, and related pharmacokinetic parameters were calculated using the non-atrioventricular model linear log-trapezoidal plotting method with Phoenix WinNonlin 6.3 pharmacokinetic analysis software.

[0258] The test results are shown in Table 22.

[0259] Table 22 Pharmacokinetic parameters of the compound of formula (I) of the present invention in Beagle dogs [Table 22] Conclusion: The compounds of the present invention showed high plasma exposure (AUC0-inf) after oral administration. They exhibited superior pharmacokinetic properties in beagle dogs, a non-rodent animal.

[0260] Experimental Example 8: In vivo efficacy study of the compound of formula (I) in a human B-cell lymphoma OCI-LY10 cell SCID mouse xenograft tumor model. Experimental objective: In this study, the antitumor effect of the compound of formula (I) was evaluated using a human B-cell lymphoma OCI-LY10 cell SCID mouse xenograft tumor model.

[0261] Experimental materials: 1. Experimental animal: SCID mouse, female, 6~8 weeks old, weight 17~20g. Beijing Wetong Lihua Experimental Animal Technology Co., Ltd.

[0262] 2. Cell line: The human B-cell lymphoma OCI-LY10 cell line was purchased from Nanjing Kebai Biotechnology Co., Ltd., and its product number is CBP60558.

[0263] Table 23 Reagent Information [Table 23]

[0264] Table 24 Equipment [Table 24]

[0265] Model construction: OCI-LY10 cells were cultured in IMDM medium containing 20% ​​FBS and maintained in a 37°C saturated humidity incubator under 5% CO2 conditions. Logarithmic OCI-LY10 cells were harvested, resuspended in IMDM basal medium, and Matrigel was added in a 1:1 ratio to increase the cell concentration to 4 × 10⁶. 7 The concentration was adjusted to 4 × 10 / mL. Under sterile conditions, 0.1 mL of cell suspension was inoculated subcutaneously into the right dorsal region of SCID mice, with an inoculation concentration of 4 × 10⁻¹⁶. 6 The dosage was 0.1 mL per mouse.

[0266] Experimental protocol: In pharmacodynamic experiments, when the tumor reached a certain size, animals with excessively large or small tumor volume or irregular shape were excluded, and those with a tumor volume of 167.65 to 231.29 mm² were selected. 3 Individual mice were selected and divided into groups based on tumor volume using a randomization method, with each group consisting of 6 mice. The average tumor volume was approximately 201.15 mm². 3The day of group assignment was designated as Day 0, and medication was initiated based on animal body weight. The pharmacodynamic experiment lasted 28 days, with oral administration once daily at 24-hour intervals. During the study period, animal body weight and tumor size were measured twice a week. Clinical symptoms were observed and recorded daily.

[0267] The dosages of the test compounds were 10 mg / kg, 30 mg / kg, and 100 mg / kg, respectively, and the solvent used was 10% DMSO / 10% Solutol / 80% H2O. Tumor volume (TV) was calculated using the formula: 1 / 2 × a × b 2 Here, a and b are the major and minor diameters at the time of tumor measurement, respectively. The formula for calculating the tumor growth inhibition rate TGI (%) is: TGI (%) = [1 - (average tumor volume at the end of administration for a certain treatment group - average tumor volume at the start of administration for that treatment group) / (average tumor volume at the end of treatment for the solvent control group - average tumor volume at the start of treatment for the solvent control group)] × 100%. The formula for calculating the relative tumor growth rate T / C (%) is: T / C % = T RTV / C RTV × 100%(T RTV :Treatment group average RTV;C RTV : (mean RTV of the negative control group). Based on the tumor measurement results, the relative tumor volume (RTV) is calculated using the formula RTV = Vt / V0 (where V0 is the tumor volume measured at the time of group division and administration (day 0), Vt is the tumor volume at a single measurement point, and T RTV and C RTV The data used was from the same day.

[0268] Data analysis: In this study, all experimental data were expressed as Mean±SEM.

[0269] Statistical analysis was performed using IBM SPSS Statistics software based on RTV data at the end of the study. T-tests were used for comparisons between two groups, and one-way ANOVA was used for comparisons between three or more groups. Tukey's method was used when variances were homogeneous (no significant difference in F-scores), and Games-Howell's method was used when variances were heterogeneous (significant difference in F-scores). A p-value of <0.05 was considered statistically significant.

[0270] Experimental results: The test results are shown in Tables 25 and 26.

[0271] Table 25 Evaluation of tumor suppressive effects of the compound of formula (I) of the present invention in a human B-cell lymphoma OCI-LY10 cell subcutaneous xenograft tumor model. [Table 25]

[0272] Note: a. Mean ± SEM.

[0273] Table 26. p-values ​​of relative tumor volume comparisons between groups in a human B-cell lymphoma OCI-LY10 xenograft tumor model of the compound of formula (I) of the present invention. [Table 26]

[0274] Note: p-values ​​were analyzed using IBM SPSS Statistics software.

[0275] Conclusion: The compounds of the present invention showed a dose-dependent and significant tumor-suppressing effect in a human B-cell lymphoma OCI-LY10 cell SCID mouse xenograft tumor model.

[0276] Experimental Example 9: Evaluation of the in vivo efficacy of the compound of formula (I) in a human lymphoma SU-DHL-2 cell subcutaneous xenograft tumor CB17 SCID mouse model. Experimental objective: In this study, the antitumor effect of the compound shown in formula (I) was evaluated using the SU-DHL-2 subcutaneous xenograft tumor CB17 SCID mouse model.

[0277] Experimental materials: 1. Experimental animals: CB17, SCID mice, female, 6-8 weeks old, weight 18-22 g. Beijing Wetong Lihua Experimental Animal Technology Co., Ltd. 2. Cell line: Human lymphoma SU-DHL-2 cells (product number: ATCC-CRL-2956).

[0278] Table 27 Reagent Information [Table 27]

[0279] Table 28 Equipment [Table 28]

[0280] Model building: Cell Culture: Human lymphoma SU-DHL-2 cells (ATCC-CRL-2956) were cultured in vitro in suspension. The culture conditions were RPMI 1640 medium supplemented with 10% inactivated fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin, and cultured at 37°C in a 5% CO2 incubator. Regular subculturing was performed twice a week. When the cell density reached 80% to 90% and the cell count met the requirements, the cells were harvested, counted, and seeded.

[0281] Tumor cell inoculation and group assignment: 0.2 mL (10 × 10 6 SU-DHL-2 cells (PBS:Matrigel = 1:1) were subcutaneously inoculated into the right dorsal region of each mouse, and the tumor volume averaged approximately 139 mm². 3 Grouping and medication administration were initiated when the grouping was reached. The day of grouping was designated as Day 0, and medication was initiated based on the animal's body weight.

[0282] Experimental protocol: In the pharmacodynamic study, the test compound was administered orally once a day at 24-hour intervals, with each cycle lasting 7 days, for a total of three cycles. During the experiment, the animals' body weight and tumor size were measured twice a week, and clinical symptoms were observed and recorded daily.

[0283] The dosages of the test compound were 10 mg / kg, 30 mg / kg, and 100 mg / kg, respectively, and the solvent used was 10% DMSO / 10% Solutol / 80% water. Tumor volume (TV) was calculated using the formula: 1 / 2 × a × b 2 Here, a and b are the major and minor diameters at the time of tumor measurement, respectively. The formula for calculating the tumor growth inhibition rate TGI (%) is: TGI (%) = [1 - (average tumor volume at the end of administration for a certain treatment group - average tumor volume at the start of administration for that treatment group) / (average tumor volume at the end of treatment for the solvent control group - average tumor volume at the start of treatment for the solvent control group)] × 100%. The formula for calculating the relative tumor growth rate T / C (%) is: T / C % = T RTV / C RTV × 100%(T RTV :Treatment group average RTV;C RTV : (mean RTV of the negative control group). Based on the tumor measurement results, the relative tumor volume (RTV) is calculated using the formula RTV = Vt / V0 (where V0 is the tumor volume measured at the time of group division and administration (day 0), Vt is the tumor volume at a single measurement point, and T RTV and C RTV The data used was from the same day.

[0284] Data analysis: Statistical analysis was performed using SPSS software based on RTV data at the end of the study. T-tests were used for comparisons between two groups, and one-way ANOVA was used for comparisons between three or more groups. Tukey's method was used when variances were homogeneous (no significant difference in F-scores), and Games-Howell's method was used when variances were heterogeneous (significant difference in F-scores). A p-value of <0.05 was considered statistically significant.

[0285] Experimental results: The test results are shown in Tables 29 and 30.

[0286] Table 29 Evaluation of tumor suppressive effects of the compound of formula (I) of the present invention in a human B-cell lymphoma SU-DHL-2 cell subcutaneous xenograft tumor model. [Table 29]

[0287] Note: a. Mean ± SEM.

[0288] Table 30. p-values ​​of relative tumor volume comparisons between groups in a SU-DHL-2 lymphoma xenograft tumor model of the compound of formula (I) of the present invention. [Table 30]

[0289] Note: The p-value was obtained by analyzing the relative tumor volume (RTV) using one-way ANOVA, and since the data variance was heterogeneous, the analysis was performed using the Games-Howell method.

[0290] Conclusion: The compound of the present invention showed a remarkable tumor-suppressing effect in a CB17 SCID mouse model of human lymphoma SU-DHL-2 cell subcutaneous xenograft tumor.

[0291] Experimental Example 10: Evaluation of the in vivo efficacy of the compound of formula (I) in a human diffuse large B-cell lymphoma (TMD-8 cell subcutaneous xenograft tumor B ALB) mouse model. Experimental objective: In this study, the antitumor effect of the compound of formula (I) was evaluated using a human diffuse large B-cell lymphoma (TMD-8) subcutaneous xenograft tumor BALB / c nude mouse model.

[0292] Experimental materials:

[0293] 1. Experimental animals: BALB / c nude mice, female, 6-8 weeks old. Victor Lihua Laboratory Animal Technology Co., Ltd. 2. Cell line: Human diffuse large B-cell lymphoma TMD-8 cells (purchased from Shanghai Huzhen Industrial Co., Ltd.).

[0294] Table 31 Main Reagent Information [Table 31]

[0295] Table 32 Main Equipment Information [Table 32]

[0296] Model construction: Cell culture: Standard cell culture was performed using RPMI-1640 medium containing 10% fetal bovine serum under 5% CO2 and 37°C conditions. Passaging was performed according to the cell proliferation status, with a passaging ratio of 1:3 to 1:4.

[0297] Tumor cell inoculation and grouping: After harvesting TMD-8 cells in the logarithmic growth phase, the cells were counted and resuspended in a mixture of RPMI-1640 medium containing 50% serum and 50% Matrigel to a cell concentration of 4.0 × 10⁶. 7 Prepare the cell / mL solution, store the cells on ice, aspirate the cell suspension with a 1mL syringe, and then inject 200μL (0.8×10) subcutaneously into the right forelimb axilla of nude mice. 7 A TMD-8 transplanted tumor model was established by injecting cells (per animal). The average tumor volume was approximately 160 mm². 3 When the target was reached, group division and medication administration were started. The day of group division was designated as day 1 of the experiment (D1), and medication administration was started based on the animal's body weight.

[0298] Experimental protocol: In the pharmacodynamic study, the test compound was administered orally once a day at 24-hour intervals, with each cycle lasting 7 days, for a total of three cycles. During the experiment, the animals' body weight and tumor size were measured twice a week, and clinical symptoms were observed and recorded daily.

[0299] The dosages of the test compound were 10 mg / kg, 30 mg / kg, and 100 mg / kg, respectively, and the solvent used was 10% DMSO / 10% Solutol HS15 / 80% water. Tumor volume (TV) was calculated using the formula: 1 / 2 × a × b 2 Here, a and b are the major and minor diameters at the time of tumor measurement, respectively. The formula for calculating the tumor growth inhibition rate TGI (%) is: TGI (%) = [1 - (average tumor volume at the end of administration for a certain treatment group - average tumor volume at the start of administration for that treatment group) / (average tumor volume at the end of treatment for the solvent control group - average tumor volume at the start of treatment for the solvent control group)] × 100%. The formula for calculating the relative tumor growth rate T / C (%) is: T / C% = T RTV / C RTV ×100%(T RTV :Treatment group average RTV;C RTV : (mean RTV of the negative control group). Based on the tumor measurement results, the relative tumor volume (RTV) is calculated using the formula RTV = Vt / V0 (where V0 is the tumor volume measured at the time of group division and administration (day 0), and Vt is the tumor volume at a single measurement point). RTV and C RTV The data used was from the same day.

[0300] Data analysis: The test data were calculated and statistically processed using Microsoft Office Excel 2007 software. Unless otherwise specified, data are expressed as mean ± standard error (Mean ± SE), and t-tests were used for comparisons between two groups.

[0301] Experimental results: The test results are shown in Table 33.

[0302] Table 33 Evaluation of the tumor-suppressing effect of the compound of the present invention (I) in a human diffuse large B-cell lymphoma TMD-8 cell nude mouse xenograft tumor model. [Table 33]

[0303] Note: "*" indicates a statistically significant difference in tumor volume compared to the solvent control group (P<0.05); "**" indicates a very statistically significant difference in tumor volume compared to the solvent control group (P<0.01); a The value shown is the mean ± SEM value.

[0304] Conclusion: The compounds of the present invention showed a significant tumor-suppressing effect in a BALB / c nude mouse model of human diffuse large B-cell lymphoma (TMD-8 cell subcutaneous xenograft tumor).

Claims

1. A compound or crystal of formula (II). 【Chemistry 1】 (Here, n is between 1.9 and 2.1, preferably 1.9, 2.0, and 2.1.)

2. A crystal of the compound of formula (II) according to claim 1, characterized in that the powder X-ray diffraction pattern has characteristic diffraction peaks at 2θ angles of 6.67±0.20°, 9.52±0.20°, 17.03±0.20°, and 19.31±0.20°.

3. A crystal of the compound of formula (II) according to claim 2, wherein the powder X-ray diffraction pattern has characteristic diffraction peaks at 2θ angles of 6.67°, 9.52°, 17.03°, and 19.31°.

4. A crystal of the compound of formula (II) according to claim 2, wherein the powder X-ray diffraction pattern is as shown in Figure 1.

5. A crystal of the compound of formula (II) according to claim 2, wherein the differential scanning calorimetry curve has endothermic peak values ​​at 153.3°C and 172.3°C.

6. A crystal of the compound of formula (II) according to claim 2, wherein the differential scanning calorimetry spectrum is as shown in Figure 2.

7. A crystal of the compound of formula (II) according to claim 2, exhibiting a weight loss of 6.42% at 150.0°C in a thermogravimetric analysis curve.

8. A crystal of the compound of formula (II) according to claim 2, wherein the thermogravimetric analysis spectrum is as shown in Figure 3.

9. The compound or crystal of formula (III). 【Chemistry 2】 (Here, m is between 1.1 and 1.5, preferably 1.1, 1.2, 1.3, 1.4, and 1.5.)

10. A crystal of the compound of formula (III) according to claim 9, characterized in that the powder X-ray diffraction pattern has characteristic diffraction peaks at 2θ angles of 10.61±0.20°, 12.06±0.20°, 15.79±0.20°, and 17.96±0.20°.

11. A crystal of the compound of formula (III) according to claim 9, wherein the powder X-ray diffraction pattern has characteristic diffraction peaks at 2θ angles of 10.61°, 12.06°, 15.79°, and 17.96°.

12. A crystal of the compound of formula (III) according to claim 9, wherein the powder X-ray diffraction pattern is as shown in Figure 4.

13. A crystal of the compound of formula (III) according to claim 9, wherein the differential scanning calorimetry curve has endothermic peak values ​​at 78.5°C, 158.7°C, and 169.0°C.

14. A crystal of the compound of formula (III) according to claim 9, wherein the differential scanning calorimetry spectrum is as shown in Figure 5.

15. A crystal of the compound of formula (III) according to claim 9, exhibiting a weight loss of 5.16% at 150.0°C in a thermogravimetric analysis curve.

16. A crystal of the compound of formula (III) according to claim 9, wherein the thermogravimetric analysis spectrum is as shown in Figure 6.

17. The compound or crystal of formula (IV). 【Transformation 3】 (Here, r is 2.1 to 2.6, preferably 2.1, 2.2, 2.3, 2.4, 2.5, and 2.6.)

18. A crystal of the compound of formula (IV) according to claim 17, characterized in that the powder X-ray diffraction pattern has characteristic diffraction peaks at 2θ angles of 5.65±0.20°, 11.16±0.20°, and 19.49±0.20°.

19. A crystal of the compound of formula (IV) according to claim 18, wherein the powder X-ray diffraction pattern has characteristic diffraction peaks at 2θ angles of 5.65±0.20°, 8.39±0.20°, 11.16±0.20°, 17.03±0.20°, 19.49±0.20°, 22.26±0.20°, and 23.09±0.20°.

20. A crystal of the compound of formula (IV) according to claim 19, wherein the powder X-ray diffraction pattern has characteristic diffraction peaks at 2θ angles of 5.65°, 8.39°, 11.16°, 17.03°, 19.49°, 22.26°, and 23.09°.

21. A crystal of the compound of formula (IV) according to claim 19, wherein the powder X-ray diffraction pattern is as shown in Figure 7.

22. A crystal of the compound of formula (IV) according to claim 19, wherein the differential scanning calorimetry curve has an endothermic peak value at 216.9°C.

23. A crystal of the compound of formula (IV) according to claim 19, wherein the differential scanning calorimetry spectrum is as shown in Figure 8.

24. A crystal of the compound of formula (IV) according to claim 19, exhibiting a weight loss of 6.40% at 150.0°C in a thermogravimetric analysis curve.

25. A crystal of the compound of formula (IV) according to claim 19, wherein the thermogravimetric analysis spectrum is as shown in Figure 9.

26. A compound or crystal of formula (V). 【Chemistry 4】 (Here, p is between 1.0 and 1.5, preferably 1.0, 1.1, 1.2, 1.3, 1.4, and 1.5.)

27. A crystal of the compound of formula (V) according to claim 26, characterized in that the powder X-ray diffraction pattern has characteristic diffraction peaks at 2θ angles of 6.51±0.20°, 8.94±0.20°, 17.87±0.20°, 19.88±0.20°, and 21.51±0.20°.

28. A crystal of the compound of formula (V) according to claim 27, wherein the powder X-ray diffraction pattern has characteristic diffraction peaks at 2θ angles of 6.51±0.20°, 8.94±0.20°, 9.76±0.20°, 13.09±0.20°, 17.87±0.20°, 19.88±0.20°, 21.51±0.20°, and 27.11±0.20°.

29. A crystal of the compound of formula (V) according to claim 28, wherein the powder X-ray diffraction pattern has characteristic diffraction peaks at 2θ angles of 6.51°, 8.94°, 9.76°, 13.09°, 17.87°, 19.88°, 21.51°, 27.11°, and 29.37°.

30. A crystal of the compound of formula (V) according to claim 29, wherein the powder X-ray diffraction pattern is as shown in Figure 10.

31. A crystal of the compound of formula (V) according to claim 29, wherein the differential scanning calorimetry curve has endothermic peak values ​​at 64.7°C and 217.5°C.

32. A crystal of the compound of formula (V) according to claim 29, wherein the differential scanning calorimetry spectrum is as shown in Figure 11.

33. A crystal of the compound of formula (V) according to claim 29, exhibiting a weight loss of 4.06% at 150.0°C in a thermogravimetric analysis curve.

34. A crystal of the compound of formula (V) according to claim 29, wherein the thermogravimetric analysis spectrum is as shown in Figure 12.

35. A compound or crystal of formula (VI). 【Transformation 5】 (Here, q is 2.1 to 2.3, preferably 2.1, 2.2, and 2.3.)

36. A crystal of the compound of formula (VI) according to claim 35, characterized in that the powder X-ray diffraction pattern has characteristic diffraction peaks at 2θ angles of 7.63 ± 0.20° and 18.46 ± 0.20°.

37. A crystal of the compound of formula (VI) according to claim 35, wherein the powder X-ray diffraction pattern has characteristic diffraction peaks at 2θ angles of 7.63° and 18.46°.

38. A crystal of the compound of formula (VI) according to claim 35, wherein the powder X-ray diffraction pattern is as shown in Figure 13.

39. A crystal of the compound of formula (VI) according to claim 35, wherein the differential scanning calorimetry curve has endothermic peak values ​​at 77.6°C, 159.4°C, and 184.2°C.

40. A crystal of the compound of formula (VI) according to claim 35, wherein the differential scanning calorimetry spectrum is as shown in Figure 14.

41. A crystal of the compound of formula (VI) according to claim 35, exhibiting a weight loss of 7.05% at 150.0°C in a thermogravimetric analysis curve.

42. A crystal of the compound of formula (VI) according to claim 35, wherein the thermogravimetric analysis spectrum is as shown in Figure 15.

43. Use of the compound of formula (II) or its crystals according to claim 1, the compound of formula (II) or its crystals according to any one of claims 2 to 8, the compound of formula (III) or its crystals according to claim 9, the compound of formula (III) or its crystals according to any one of claims 10 to 16, the compound of formula (IV) or its crystals according to claim 17, the compound of formula (IV) or its crystals according to any one of claims 18 to 25, the compound of formula (V) or its crystals according to claim 26, the compound of formula (V) or its crystals according to any one of claims 27 to 34, the compound of formula (VI) or its crystals according to claim 35, or the compound of formula (VI) or its crystals according to any one of claims 36 to 42 in the manufacture of a therapeutic agent for diffuse large B cell lymphoma.

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