Preparation method and application of salt form and crystal form of 2, 6-piperidinedione compound

Abstract

The invention discloses a preparation method of a salt form and a crystal form of a 2, 6-piperidinedione compound as shown in a formula (I) and application of the salt form and the crystal form of the 2, 6-piperidinedione compound.

Description

Preparation method and application of salt and crystal forms of 2,6-piperidinedione compounds

[0001] This application claims the following priority

[0002] This application claims priority to and the benefits of Chinese Patent Application No. 2023102949509 filed with the State Intellectual Property Office of China on March 24, 2023, and the contents disclosed in said application are incorporated herein by reference in their entirety. Technical Field

[0003] The present invention relates to a preparation method of a salt form and a crystal form of a 2,6-piperidinedione compound and application thereof. Background Art

[0004] Interleukin-1 Receptor-Associated Kinase 4 (IRAK4) plays a connecting role in the signal transduction pathways of the Toll-like receptor family (TLRs) and the interleukin type 1 receptor family (IL-1R), receiving upstream signals to activate the downstream JNK and NF-κB signaling pathways, and is closely related to the development and progression of human inflammatory immune diseases and tumors.

[0005] The Toll-like receptor (TLR) signaling protein myeloid differentiation factor (MyD88) is frequently mutated in various lymphomas, including Waldenstrom's macroglobulinemia, lymphoplasmacytoid lymphoma, immune-resistant large B-cell lymphoma, and marginal zone lymphoma, with mutation rates ranging from 95-97%, 79%, 50-80%, 15-29%, and 6-10%, respectively. IRAK4 participates in nearly all of MyD88's biological functions, making it a highly attractive and promising drug target, particularly for the treatment of MyD88-driven lymphomas.

[0006] Research has found that IRAK4 not only phosphorylates proteins but also forms a complex with MyD88 to exert its biological functions. IRAK4 phosphorylation is required for activation of the JNK signaling pathway, but not for activation of the NF-κB signaling pathway. This suggests that IRAK4 functions as both a protein kinase and a scaffolding protein, playing a role in signaling pathways. Therefore, traditional small molecule kinase inhibitors targeting IRAK4 are unable to completely block all of IRAK4's biological functions.

[0007] Proteolysis Targeting Chimera (PROTAC) is a technology that uses the ubiquitin-proteasome system to target specific proteins and induce their degradation within cells. The ubiquitin-proteasome system is the main pathway for protein degradation within cells. Its normal physiological function is mainly responsible for clearing denatured, mutated, or harmful proteins within cells. The degradation of more than 80% of proteins within cells depends on the ubiquitin-proteasome system. PROTAC uses the cell's own protein destruction mechanism to eliminate specific targeted proteins from cells. To date, PROTAC technology has become increasingly mature and can be used to target a variety of proteins, including scaffold proteins, transcription factors, enzymes, and regulatory proteins. In addition, amides are known as immunomodulatory drugs (IMiDs). They activate the E3 ubiquitin ligase complex formed with Cereblon (CRBN) to ubiquitinate the transcription factors IKZF1 and IKZF3, which are then recognized and degraded by the proteasome, resulting in toxic effects on tumors. As an important target for anti-tumor and immunomodulatory drugs, CRBN has been shown to have clear therapeutic effects in a variety of hematological malignancies, skin diseases such as erythema nodosum leprosum, and autoimmune diseases such as systemic lupus erythematosus.

[0008] Therefore, we develop bifunctional PROTAC and IMiD molecules targeting IRAK4, which can degrade IRAK4, remove IRAK4, and more thoroughly block all functions of IRAK4, thereby fundamentally inhibiting the IRAK4 signaling pathway. At the same time, it has a good CRBN regulatory effect and plays a synergistic therapeutic role, thereby better exerting anti-tumor effects and improving clinical treatment effects.

[0009] Summary of the Invention

[0010] The present invention provides maleate, citrate, p-toluenesulfonate, oxalate and hydrobromide of the compound of formula (I); preferably, the maleate, citrate, p-toluenesulfonate, oxalate and hydrobromide are in crystalline form.

[0011] In some embodiments of the present invention, the maleate salt of the compound of formula (I) is a compound of formula (II),

[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, the maleate salt of the compound of formula (I) above, wherein 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; more preferably 1:2.0.

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

[0015] In some embodiments of the present application, the crystals of the compound of formula (II) may be in the form of solvate crystals.

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

[0018] In some embodiments of the present invention, the X-ray powder diffraction (XRPD) spectrum of Form A of the compound of formula (II) is shown in FIG1 .

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

[0020] Table 1 XRPD spectrum analysis data of Form A of the compound of formula (II)

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

[0022] In some embodiments of the present invention, the DSC spectrum of Form A of the compound of formula (II) is shown in FIG2 .

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

[0024] In some embodiments of the present invention, the TGA spectrum of the crystal form A of the compound of formula (II) is shown in FIG3 .

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

[0026] 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.

[0027] In some embodiments of the present invention, the p-toluenesulfonate salt of the compound of formula (I) above, wherein the molar ratio of the compound of formula (I) to 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; more preferably 1:1.3.

[0028] In some embodiments of the present application, the compound of formula (III) above is in crystalline form.

[0029] In some embodiments of the present application, the crystals of the compound of formula (III) may be in the form of solvate crystals.

[0030] The present invention provides a crystal form A of a compound of formula (III), whose X-ray powder diffraction spectrum has characteristic diffraction peaks at the following 2θ angles: 10.61±0.20°, 12.06±0.20°, 15.79±0.20° and 17.96±0.20°.

[0031] In some embodiments of the present invention, the crystal form A of the compound of formula (III) has an X-ray powder diffraction spectrum with characteristic diffraction peaks at the following 2θ angles: 10.61°, 12.06°, 15.79° and 17.96°.

[0032] In some embodiments of the present invention, the X-ray powder diffraction (XRPD) spectrum of Form A of the compound of formula (III) is shown in FIG4 .

[0033] In some embodiments of the present invention, the X-ray powder diffraction (XRPD) spectrum analysis data of Form A of the compound of formula (III) are shown in Table 2:

[0034] Table 2 XRPD spectrum analysis data of Form A of the compound of formula (III)

[0035] In some embodiments of the present invention, the crystal form A of the compound of formula (III) has a differential scanning calorimetry curve with endothermic peaks at 78.5°C, 158.7°C and 169.0°C.

[0036] In some embodiments of the present invention, the DSC spectrum of Form A of the compound of formula (III) is shown in FIG5 .

[0037] In some embodiments of the present invention, the thermogravimetric analysis curve of the crystal form A of the compound of formula (III) shows a weight loss of 5.16% at 150.0°C.

[0038] In some embodiments of the present invention, the TGA spectrum of Form A of the compound of formula (III) is shown in FIG6 .

[0039] In some embodiments of the present invention, the oxalate salt of the compound of formula (I) is a compound of formula (IV),

[0040] Among them, r is 2.1 to 2.6; preferably 2.1, 2.2, 2.3, 2.4, 2.5 and 2.6; more preferably 2.4.

[0041] In some embodiments of the present invention, the oxalate salt of the compound of formula (I) above, wherein 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; more preferably 1:2.4.

[0042] In some embodiments of the present application, the compound of formula (IV) above is in crystalline form.

[0043] In some embodiments of the present application, the crystals of the compound of formula (IV) may be in the form of solvate crystals.

[0044] The present invention provides a crystal form A of a compound of formula (IV), whose X-ray powder diffraction spectrum has characteristic diffraction peaks at the following 2θ angles: 5.65±0.20°, 11.16±0.20° and 19.49±0.20°.

[0045] In some embodiments of the present invention, the above-mentioned crystal form A of the compound of formula (IV) has an X-ray powder diffraction spectrum having characteristic diffraction peaks at the following 2θ angles: 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°.

[0046] In some embodiments of the present invention, the above-mentioned crystal form A of the compound of formula (IV) has an X-ray powder diffraction spectrum with characteristic diffraction peaks at the following 2θ angles: 5.65°, 8.39°, 11.16°, 17.03°, 19.49°, 22.26° and 23.09°.

[0047] In some embodiments of the present invention, the X-ray powder diffraction (XRPD) spectrum of Form A of the compound of formula (IV) is shown in FIG7 .

[0048] In some embodiments of the present invention, the X-ray powder diffraction (XRPD) spectrum analysis data of Form A of the compound of formula (IV) are shown in Table 3:

[0049] Table 3 XRPD spectrum analysis data of Form A of the compound of formula (IV)

[0050] In some embodiments of the present invention, the crystal form A of the compound of formula (IV) has an endothermic peak at 216.9° C. in its differential scanning calorimetry curve.

[0051] In some embodiments of the present invention, the DSC spectrum of Form A of the compound of formula (IV) is shown in FIG8 .

[0052] In some embodiments of the present invention, the thermogravimetric analysis curve of the crystal form A of the compound of formula (IV) above shows a weight loss of 6.40% at 150.0°C.

[0053] In some embodiments of the present invention, the TGA spectrum of Form A of the compound of formula (IV) is shown in FIG9 .

[0054] In some embodiments of the present invention, the hydrobromide salt of the compound of formula (I) is a compound of formula (V),

[0055] Here, p is 1.0 to 1.5, preferably 1.0, 1.1, 1.2, 1.3, 1.4 and 1.5, and more preferably 1.2.

[0056] In some embodiments of the present invention, the hydrobromide salt of the compound of formula (I) above, wherein 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; more preferably 1:1.2.

[0057] In some embodiments of the present application, the compound of formula (V) above is in crystalline form.

[0058] In some embodiments of the present application, the crystals of the compound of formula (V) may be in the form of solvate crystals.

[0059] The present invention provides a crystal form A of a compound of formula (V), whose X-ray powder diffraction spectrum has characteristic diffraction peaks at the following 2θ angles: 6.51±0.20°, 8.94±0.20°, 17.87±0.20°, 19.88±0.20° and 21.51±0.20°.

[0060] In some embodiments of the present invention, the crystal form A of the compound of formula (V) has an X-ray powder diffraction spectrum having characteristic diffraction peaks at the following 2θ angles: 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°.

[0061] In some embodiments of the present invention, the crystal form A of the compound of formula (V) has an X-ray powder diffraction spectrum having characteristic diffraction peaks at the following 2θ angles: 6.51°, 8.94°, 9.76°, 13.09°, 17.87°, 19.88°, 21.51°, 27.11° and 29.37°.

[0062] In some embodiments of the present invention, the X-ray powder diffraction (XRPD) spectrum of Form A of the compound of formula (V) is shown in FIG10 .

[0063] In some embodiments of the present invention, the X-ray powder diffraction (XRPD) spectrum analysis data of Form A of the compound of formula (V) are shown in Table 4:

[0064] Table 4 XRPD spectrum analysis data of Form A of the compound of formula (V)

[0065] In some embodiments of the present invention, the crystal form A of the compound of formula (V) has endothermic peaks at 64.7°C and 217.5°C in its differential scanning calorimetry curve.

[0066] In some embodiments of the present invention, the DSC spectrum of Form A of the compound of formula (V) is shown in FIG11 .

[0067] In some embodiments of the present invention, the thermogravimetric analysis curve of the crystal form A of the compound of formula (V) shows a weight loss of 4.06% at 150.0°C.

[0068] In some embodiments of the present invention, the TGA spectrum of Form A of the compound of formula (V) is shown in FIG12 .

[0069] In some embodiments of the present invention, the citrate salt of the compound of formula (I) is a compound of formula (VI),

[0070] Among them, q is 2.1 to 2.3; preferably 2.1, 2.2 and 2.3; more preferably 2.2.

[0071] In some embodiments of the present invention, the citrate salt of the compound of formula (I) has a molar ratio of the compound of formula (I) to citric acid of 1:2.1 to 1:2.3; preferably 1:2.1, 1:2.2 or 1:2.3; more preferably 1:2.2.

[0072] In some embodiments of the present application, the compound of formula (VI) above is in crystalline form.

[0073] In some embodiments of the present application, the crystals of the compound of formula (VI) may be in the form of solvate crystals.

[0074] The present invention provides a crystal form A of a compound of formula (VI), whose X-ray powder diffraction spectrum has characteristic diffraction peaks at the following 2θ angles: 7.63±0.20° and 18.46±0.20°.

[0075] In some embodiments of the present invention, the crystal form A of the compound of formula (VI) has an X-ray powder diffraction spectrum with characteristic diffraction peaks at the following 2θ angles: 7.63° and 18.46°.

[0076] In some embodiments of the present invention, the X-ray powder diffraction (XRPD) spectrum of Form A of the compound of formula (VI) is shown in FIG13 .

[0077] In some embodiments of the present invention, the X-ray powder diffraction (XRPD) spectrum analysis data of Form A of the compound of formula (VI) are shown in Table 5:

[0078] Table 5 XRPD spectrum analysis data of Form A of the compound of formula (VI)

[0079] In some embodiments of the present invention, the differential scanning calorimetry curve of the crystal form A of the compound of formula (VI) has endothermic peaks at 77.6°C, 159.4°C and 184.2°C.

[0080] In some embodiments of the present invention, the DSC spectrum of Form A of the compound of formula (VI) is shown in FIG14 .

[0081] In some embodiments of the present invention, the thermogravimetric analysis curve of the crystal form A of the compound of formula (VI) above shows a weight loss of 7.05% at 150.0°C.

[0082] In some embodiments of the present invention, the TGA spectrum of Form A of the compound of formula (VI) is shown in FIG15 .

[0083] The present invention also provides 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 crystal form A of the compound of formula (II), the crystal form A of the compound of formula (III), the crystal form A of the compound of formula (IV), the crystal form A of the compound of formula (V), or the crystal form A of the compound of formula (VI) in the preparation of a medicament for treating diffuse large B-cell lymphoma.

[0084] Technical Effects

[0085] The compound of the present invention exhibits excellent degradation effects on target proteins IRAK4, IKZF1 and IKZF3, and exhibits excellent cell proliferation inhibitory effects in lymphoma cell lines OCI-LY10, TMD-8 and SU-DHL-2; has significant tumor inhibition effects and is dose-dependent; and has good pharmacokinetic properties and oral absorption rate, and is suitable for use as a drug.

[0086] Definition and Description

[0087] Unless otherwise indicated, the following terms and phrases used herein are intended to have the following meanings. A particular phrase or term should not be construed as ambiguous or unclear unless specifically defined, but rather should be understood in accordance with its ordinary meaning. When a trade name appears herein, it is intended to refer to the corresponding commercial product or its active ingredient.

[0088] For any given crystalline form, the relative intensity of the diffraction peaks can change due to factors such as the preferred orientation caused by factors such as the crystal morphology, which is well known in the art of crystallography. Where there is a preferred orientation effect, the peak intensity is changed, but the position of the diffraction peak of the crystal form cannot be changed. In addition, for any given crystal form, there may be slight errors in the position of the peaks, which is also well known in the art of crystallography. For example, due to changes in temperature during sample analysis, movement of the sample, or calibration of the instrument, the position of the peaks can move, and the measurement error of the 2θ value is sometimes about ± 0.20 °. Therefore, it is well known to those skilled in the art that this error should be taken into account when determining each crystalline structure.

[0089] DSC measures the transition temperatures of crystals when they absorb or release heat due to changes in their crystalline structure or melting. For the same crystalline form of the same compound, the error in thermal transition temperatures and melting points in consecutive analyses is typically within about 5°C or 3°C. When we say that a compound has a given DSC peak or melting point, this refers to the DSC peak or melting point ±5°C or ±3°C. DSC provides an auxiliary method for distinguishing different crystalline forms. Different crystalline forms can be identified by their different transition temperature characteristics. It should be noted that for mixtures, their DSC peaks or melting points may vary over a wider range. In addition, since decomposition is associated with the melting process of a substance, the melting temperature is related to the heating rate.

[0090] Unless otherwise specified, DSC spectra are exothermic upwards.

[0091] For the same crystal form, the TGA weight loss temperature may vary due to factors such as the measuring instrument, measuring method / conditions, etc. For any specific crystal form, the weight loss temperature may have an error of about ±5°C, or about ±3°C.

[0092] It should be noted that during the preparation of drug crystal forms, during the contact between drug molecules and solvent molecules, external conditions and internal factors may cause the solvent molecules to form a co-crystal with the compound molecules and remain in the solid material, which is difficult to avoid, thereby forming solvates, specifically including stoichiometric solvates and non-stoichiometric solvates. All such solvates are included within the scope of the present invention.

[0093] The "pharmaceutically acceptable excipients" mentioned above refer to inert substances that are administered together with the active ingredient and facilitate the administration of the active ingredient, including but not limited to any glidants, sweeteners, diluents, preservatives, dyes / colorants, flavor enhancers, surfactants, wetting agents, dispersants, disintegrants, suspending agents, stabilizers, isotonic agents, solvents or emulsifiers that are acceptable for use in humans or animals (e.g., livestock) and approved by the State Food and Drug Administration.

[0094] The term "crystalline composition" refers to a mixture of a crystalline form of the compound of formula (I) of the present invention and other crystalline forms or amorphous forms of the compound or other impurities. For example, a crystalline composition of the compound of formula (I) may contain, in addition to the crystals of the compound of formula (I), other crystalline forms or amorphous forms of compound 1 or other impurities.

[0095] The term "pharmaceutical composition" refers to a mixture of one or more compounds of the present invention or their salts and pharmaceutically acceptable excipients. The purpose of a pharmaceutical composition is to facilitate administration of the compounds of the present invention to an organism.

[0096] The therapeutic dosage of the compounds of the present invention may be determined, for example, based on the specific therapeutic application, the manner in which the compound is administered, the patient's health and condition, and the judgment of the prescribing physician. The proportion or concentration of the compounds of the present invention in a pharmaceutical composition may not be fixed and depends on a variety of factors, including dosage, chemical properties (e.g., hydrophobicity), and route of administration.

[0097] The term "treatment" means administering the compounds or formulations of the present invention to improve or eliminate a disease or one or more symptoms associated with the disease, and includes:

[0098] (i) inhibiting a disease or disease state, i.e., arresting its development;

[0099] (ii) ameliorating the disease or condition, i.e., causing regression of the disease or condition.

[0100] The term "therapeutically effective amount" means an amount of a compound of the present invention that (i) treats a particular disease, condition, or disorder, (ii) alleviates, ameliorates, 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 a compound of the present invention that constitutes a "therapeutically effective amount" varies depending on the compound, the disease state and its severity, the mode of administration, and the age of the mammal to be treated, but can be routinely determined by one skilled in the art based on their knowledge and this disclosure.

[0101] Unless otherwise required herein, throughout the specification and the claims that follow, the word "comprise" and its English variations such as "comprises" and "comprising" should be interpreted as having an open, inclusive meaning, that is, "including but not limited to".

[0102] Reference throughout this specification to "some embodiments" or "an embodiment" or "in another embodiment" or "in certain embodiments" means that at least one embodiment includes the specific referenced elements, structures, or features described in connection with that embodiment. Thus, appearances of the phrases "in some embodiments" or "in an embodiment" or "in another embodiment" or "in certain embodiments" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the specific elements, structures, or features may be combined in any suitable manner in one or more embodiments.

[0103] It should be understood that the singular article "a," "an," and "the," as used in the present specification and the appended claims, includes plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to a reaction including "a catalyst" includes one catalyst, or two or more catalysts. It should also be understood that the term "or" is generally used in its sense including "and / or," unless the context clearly dictates otherwise.

[0104] 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 combining them with other chemical synthesis methods, and equivalent replacement methods well known to those skilled in the art. Preferred embodiments include but are not limited to the examples of the present invention.

[0105] The chemical reactions of the present invention are carried out in suitable solvents that are compatible with the chemical transformations of the present invention and the reagents and materials required. To obtain the compounds of the present invention, it may sometimes be necessary for those skilled in the art to modify or select synthetic steps or reaction schemes based on existing embodiments.

[0106] Unless otherwise specified, use a solid wedge key. and dotted wedge key To indicate the absolute configuration of a stereocenter, use a straight solid bond and straight dashed bond To express relative configuration, for example, Represents trans-1,4-disubstituted cyclohexane, using represents a cis-1,4-disubstituted cyclohexane.

[0107] The present invention will be described in detail below through examples, which are not intended to limit the present invention in any way.

[0108] All solvents used in the present invention were commercially available and used without further purification.

[0109] Compounds are manually or Software naming, commercially available compounds use supplier catalog names.

[0110] The present invention uses the following abbreviations: RT stands for room temperature; MeOH stands for methanol; EtOH stands for ethanol; IPA stands for isopropanol; Acetone stands for acetone; MIBK stands for methyl isobutyl ketone; EtOAc stands for ethyl acetate; IPAc stands for isopropyl acetate; MTBE stands for methyl tert-butyl ether; THF stands for tetrahydrofuran; 2-MeTHF stands for 2-methyltetrahydrofuran; DCM stands for dichloromethane; CHCl3 stands for chloroform; Toluene stands for toluene; n-Heptane stands for n-heptane; DMSO stands for dimethyl sulfoxide; DMAc stands for N,N-dimethylacetamide; NMP stands for N-methylpyrrolidone; H2O stands for water; 1,4-Dioxane stands for 1,4-dioxane; and ACN stands for acetonitrile. BRIEF DESCRIPTION OF THE DRAWINGS

[0111] FIG1 is an X-ray powder diffraction (XRPD) spectrum of Form A of the compound of formula (II);

[0112] FIG2 is a DSC spectrum of Form A of the compound of formula (II);

[0113] FIG3 is a TGA spectrum of crystal form A of the compound of formula (II);

[0114] FIG4 is an X-ray powder diffraction (XRPD) spectrum of Form A of the compound of formula (III);

[0115] FIG5 is a DSC spectrum of Form A of the compound of formula (III);

[0116] FIG6 is a TGA spectrum of Form A of the compound of formula (III);

[0117] FIG7 is an X-ray powder diffraction (XRPD) spectrum of Form A of the compound of formula (IV);

[0118] FIG8 is a DSC spectrum of Form A of the compound of formula (IV);

[0119] FIG9 is a TGA spectrum of Form A of the compound of formula (IV);

[0120] FIG10 is an X-ray powder diffraction (XRPD) spectrum of Form A of the compound of formula (V);

[0121] FIG11 is a DSC spectrum of Form A of the compound of formula (V);

[0122] FIG12 is a TGA spectrum of Form A of the compound of formula (V);

[0123] FIG13 is an X-ray powder diffraction (XRPD) spectrum of Form A of the compound of formula (VI);

[0124] FIG14 is a DSC spectrum of Form A of the compound of formula (VI);

[0125] FIG15 is a TGA spectrum of Form A of the compound of formula (VI);

[0126] FIG16 is a solution NMR spectrum of the crystal of the compound of formula (II);

[0127] FIG17 is a solution NMR spectrum of Form A of the compound of formula (III);

[0128] FIG18 is a solution NMR spectrum of Form A of the compound of formula (IV);

[0129] FIG19 is a solution NMR spectrum of Form A of the compound of formula (V);

[0130] FIG20 is a solution NMR spectrum of the crystals of the compound of formula (VI).

[0131] Instruments and analytical methods

[0132] 1.1 X-ray powder diffractometer (XRPD) method of the present invention

[0133] Instrument model: PANalytacal X-ray powder diffraction analyzer

[0134] Test method: Approximately 10-20 mg of sample was used for XRPD detection.

[0135] The detailed XRPD parameters are as follows:

[0136] Light pipe: Cu, kα,

[0137] Light tube voltage: 45kV, light tube current: 40mA

[0138] Divergence slit: 1 / 8°

[0139] Scan mode: continuous

[0140] Scanning range (°2Theta): 3-40

[0141] Scan time per step (s): 46.7

[0142] Scan step (°2Theta): 0.0263

[0143] Test time: 5 minutes

[0144] 1.2 Differential Scanning Calorimeter (DSC) Method of the Present Invention

[0145] Instrument model: TA 2500 differential scanning calorimeter

[0146] Test method: Take a sample (1 mg) and place it in a DSC aluminum pan for testing. Under 50 mL / min N2 conditions, heat the sample from 25°C (room temperature) to 300°C (or 350°C) at a heating rate of 10°C / min.

[0147] 1.3 Thermogravimetric Analysis (TGA) Method of the Present Invention

[0148] Instrument model: TA5500 thermogravimetric analyzer

[0149] Test method: Take a sample (2-5 mg) and place it in a TGA platinum pan for testing. Under 25 mL / min N2 conditions, heat the sample from room temperature to 350°C or 20% weight loss at a heating rate of 10°C / min.

[0150] 1.4 Dynamic Vapor Sorption (DVS) Method of the Present Invention

[0151] Instrument model: SMS (Surface Measurement Systems) DVS Intrinsic Plus dynamic vapor sorption instrument

[0152] Test conditions: Take a sample (10-20 mg) and place it in the DVS sample tray for testing.

[0153] The detailed DVS parameters are as follows:

[0154] Protective gas and flow rate: N2, 200mL / min

[0155] Temperature: 25℃

[0156] Balance: dm / dt = 0.002% / min (minimum: 10 min, maximum: 180 min)

[0157] Drying: 120 min at 0% RH

[0158] RH (%) range: 0%-95%

[0159] RH (%) gradient: 10% (0% RH-90% RH, 90% RH-0% RH); 5% (90% RH-95% RH, 95% RH-90% RH)

[0160] The classification of moisture absorption evaluation is shown in Table 6 below:

[0161] Table 6 Evaluation of moisture absorption

[0162] Note: ΔW% indicates the weight gain of the test sample due to moisture absorption at 25±1℃ and 80±2% RH.

[0163] 1.5 Ultra-High Performance Liquid Chromatography / Ion Chromatography (UPLC / IC) Method of the Present Invention

[0164] Instrument model: Waters H-Class ultra-high performance liquid chromatograph and ion chromatograph

[0165] The detailed UPLC parameters are as follows:

[0166] Chromatographic column: Xbridge C18, 4.6×150mm, 5μm

[0167] Mobile phase: A: 0.1% TFA in H2O; B: 0.1% TFA in Acetonitrile

[0168] 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

[0169] Run time: 15.0 min

[0170] Mobile phase flow rate: 0.0min

[0171] Injection volume: 1.0 mL / min

[0172] Detection wavelength: 3μL

[0173] Column temperature: UV at 226nm

[0174] Injector temperature: 25°C

[0175] Diluent: ACN / H2O=1:1 (v / v)

[0176] The detailed IC parameters are as follows:

[0177] Column: Dionex IonPac TM AS18 RFIC TM 4×250mm Analytical

[0178] Mobile phase: 25 mM NaOH

[0179] Injection volume: 25 μL

[0180] Flow rate: 1.0 mL / min

[0181] Temperature: 35℃

[0182] Column temperature: 35°C

[0183] Current: 80mA

[0184] Run time: 16 minutes

[0185] 1.6 Solution NMR method of the present invention

[0186] Instrument model: Bruker 400M NMR

[0187] Solvent: DMSO-d6 or deuterated methanol DETAILED DESCRIPTION

[0188] The present invention is described in detail below by way of examples, but this is not intended to limit the present invention in any way. The present invention has been described in detail herein, and specific embodiments thereof have been disclosed. It will be apparent to those skilled in the art that various changes and modifications can be made to the specific embodiments of the present invention without departing from the spirit and scope of the present invention.

[0189] Example 1: Preparation of compound of formula (I)

[0190] Step 1: Synthesis of compound 1-2

[0191] Under nitrogen 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). N,N-diisopropylethylamine (163.62 g, 1.27 mol) and 2-(7-azabenzotriazole)-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 hour. The hydrochloride salt of compound 1-1 (105 g, 316.5 mmol) was then added, and the reaction mixture was stirred at room temperature for 12 hours. After completion of the reaction, the reaction mixture was poured into ice water (5 L) and extracted with ethyl acetate (4 × 1 L). The combined organic phases were 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) and stirred at room temperature for 1 hour. Solids gradually precipitated and were concentrated under reduced pressure to remove most of the dichloromethane. Methyl tert-butyl ether (1.5 L) was added and stirred at room temperature for 12 hours. The mixture was filtered and the filter cake was collected. Ethyl acetate (1.5 L) was added to the filter cake and stirred at room temperature for 2 hours. The filter cake was then filtered and rinsed with ethyl acetate (200 mL x 2). The filter cake was collected and dried under vacuum to obtain compound 1-2. MS-ESI m / z: 522.3 [M+H] + . 1 H NMR(400MHz, DMSO_d6)δ:11.14(s,1H),10.13(s,1H),8.19(d,J=9.6Hz,1H),8.11(d,J=8.0Hz,1H),8.00(d,J=9.26Hz,1H),7.80-7.68(m,2 H),5.09(dd,J=4.8Hz,11.2Hz,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).

[0192] Step 2: Synthesis of trifluoroacetate salt of compound 1-3

[0193] Compound 1-2 (153 g, 293.35 mmol) was dissolved in dichloromethane (1.2 L) at room temperature, and trifluoroacetic acid (462 g, 4.05 mol) was slowly added dropwise. The reaction mixture was stirred at room temperature for 12 hours. After completion of the reaction, the solvent was removed by concentration under reduced pressure to obtain the trifluoroacetate salt of compound 1-3. MS-ESI m / z: 422.1 [M+H] + .

[0194] Step 3: Synthesis of Compounds 1-4

[0195] Under nitrogen at room temperature, compound 1-4-1 (153 g, 260.81 mmol) was dissolved in dichloromethane (3 L). 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. Elemental iodine (86.06 g, 339.06 mmol) was added portionwise. The reaction mixture was allowed to warm to room temperature and stirred for 16 hours. After completion of the reaction, saturated sodium sulfite solution (1 L) was slowly added to the reaction mixture. The mixture was stirred for 20 minutes and the layers were separated. The aqueous phase was extracted with dichloromethane (3 × 3 L). The combined organic phases were 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. To the residue was added methyl tert-butyl ether (600 mL), stirred at room temperature for 2 hours, filtered, and the filter cake was rinsed with methyl tert-butyl ether (50 mL × 2), and the filtrate was collected. To the filtrate was added 80 g of anhydrous magnesium chloride, stirred at room temperature for 8 hours, filtered, and the filter cake was rinsed with methyl tert-butyl ether (50 mL × 2), and the filtrate was collected. The filtrate was concentrated under reduced pressure to obtain compound 1-4. MS-ESI m / z: 697.0 [M+H] + .

[0196] Step 4: Synthesis of Compounds 1-5

[0197] Under nitrogen at room temperature, the trifluoroacetic acid salt of compound 1-3 (129.35 g, 157.64 mmol) was dissolved in acetonitrile (1.3 L). N,N-diisopropylethylamine (158.46 g, 1.23 mol) and compound 1-4 (122 g, 175.16 mmol) were then added. The reaction mixture was heated to 80°C and stirred for 12 hours. After completion of the reaction, the solvent was partially removed by concentration under reduced pressure. Ethyl acetate (500 mL) and water (500 mL) were added, and the layers were separated. The aqueous phase was extracted with ethyl acetate (500 mL x 3). The combined organic phases were 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 filter cake was rinsed with a mixed solvent of ethyl acetate and acetonitrile (volume ratio 9:1, 30 mL×3). The filter cake was collected and dried under vacuum to obtain compound 1-5. MS-ESI m / z: 990.4 [M+H] + .

[0198] Step 5: Synthesis of the hydrochloride salt of the compound of formula (I)

[0199] Under nitrogen protection at room temperature, compound 1-5 (80 g, 80.8 mmol) was dissolved in ethyl acetate (100 mL). A 4 M solution of hydrochloric acid in ethyl acetate (500 mL) was slowly added, and the reaction mixture was stirred at room temperature for 16 hours. After the reaction was complete, the mixture was filtered, and the filter cake was rinsed with ethyl acetate (50 mL x 3). The filter cake was collected. Acetonitrile (350 mL) was added to the filter cake, stirred at room temperature for 2 hours, filtered, and the filter cake was rinsed with acetonitrile (30 mL x 3). The filter cake was collected and dried under vacuum to obtain the hydrochloride salt of compound (I). MS-ESI m / z: 890.2 [M+H] + . 1 H NMR(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).

[0200] Step 6: Synthesis of compound of formula (I)

[0201] Under nitrogen protection at room temperature, the hydrochloride salt (10 g) of the compound of formula (I) was dissolved in a mixed solvent of isopropanol (75 mL) and water (300 mL). The mixed solution was added dropwise to a solution of imidazole (3.43 g, 50.4 mmol) in water (300 mL). The mixture was stirred at room temperature for 15 hours, filtered, and the filter cake was rinsed with water (50 mL × 2) and isopropanol (50 mL). The filter cake was collected and dried in vacuo to obtain the compound of formula (I) (free base).

[0202] Example 2: Preparation of Crystal Form A of the Compound of Formula (II)

[0203] 20 mg of the compound of formula (I) was added to 0.5 mL of tetrahydrofuran, and then 2.0 equivalents of maleic acid were added under stirring. The reaction mixture was stirred at 25° C. for 72 hours to obtain a solid. The sample was centrifuged to obtain Form A of the compound of formula (II). 1 The H NMR results are shown in FIG16 , indicating that the acid-base molar ratio in the sample is 2.0, i.e., n=2.0.

[0204] Example 3: Preparation of Form A of the Compound of Formula (III)

[0205] 20 mg of the compound of formula (I) was added to 0.5 mL of tetrahydrofuran, and then 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 to obtain Form A of the compound of formula (III). 1 The H NMR results are shown in FIG17 , indicating that the acid-base molar ratio in the sample is 1.3, i.e., m=1.3.

[0206] Example 4: Preparation of Crystal Form A of the Compound of Formula (IV)

[0207] Take 20 mg of the compound of formula (I), add 0.5 mL of tetrahydrofuran, and then add 2.0 equivalents of oxalic acid under stirring. The reaction mixture is stirred at 25° C. for 48 hours to obtain a solid. The sample is centrifuged to obtain crystals of the compound of formula (IV). 1 The H NMR results are shown in FIG18 . The UPLC / IC results showed that the acid-base molar ratio was 2.4, i.e., r=2.4.

[0208] Example 5: Preparation of Crystal Form A of the Compound of Formula (V)

[0209] 20 mg of the compound of formula (I) was added to 0.5 mL of tetrahydrofuran, and then 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. The sample was centrifuged to obtain Form A of the compound of formula (V). 1 The H NMR results are shown in FIG19 , and the UPLC / IC results showed that the acid-base molar ratio was 1.2, i.e., p=1.2.

[0210] Example 6: Preparation of Crystal Form A of the Compound of Formula (VI)

[0211] 20 mg of the compound of formula (I) was added to 0.5 mL of tetrahydrofuran, and then 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. The sample was centrifuged to obtain Form A of the compound of formula (VI). 1 The H NMR results are shown in FIG20 , indicating that the acid-base molar ratio in the sample is 2.2, i.e., q=2.2.

[0212] Biological testing

[0213] Experimental Example 1: Evaluation of the target protein degradation effect of compound (I) in K562 IRAK4-HiBiT cells

[0214] Experimental purpose: This experiment detects the degradation effect of the compound of formula (I) on the target protein IRAK4 in K562 IRAK4-HiBiT cells.

[0215] Experimental Materials:

[0216] 1. Cells and Culture Medium

[0217] Cells: K562 IRAK4-HiBiT cells

[0218] Culture medium: RPMI 1640 + 10% fetal bovine serum + 2mM glutamine + 1mM sodium pyruvate + penicillin / streptomycin

[0219] Positive control: 1000 nM; negative control: 0.1% DMSO

[0220] Table 7 Reagents and consumables

[0221] Table 8 Instruments

[0222] Experimental plan:

[0223] Day 1

[0224] 1. Preparation of Compounds

[0225] (1) Dissolve the powder of the test compound in DMSO to 10 mM as the stock concentration. Manually pipette 9 μL of 10 mM test compound into columns 1 and 13 of the LDV plate.

[0226] (2) Use a multidrop combi to add 6 μL of DMSO to columns 2-12 and 14-24;

[0227] (3) Use Bravo to dilute the test compound 3-fold (3 μL + 6 μL) from columns 1-11 and columns 13-23;

[0228] (4) According to the plate layout, use Echo to transfer 25nL of compound solution (columns 1-24 of the LDV plate) to the experimental plate;

[0229] (5) Use Echo to transfer 25 nL of 1 mM positive control solution to the assay plate as a 100% degradation control (ie, LC, HPE), and 25 nL of DMSO to the assay plate as a 0% control (ie, HC, ZPE).

[0230] 2. Cell Plating

[0231] (1) Discard the cell culture medium, wash once with DPBS, digest the cells with trypsin, count the cells, and prepare a cell suspension of 2×10 -5 cells / mL;

[0232] (2) Use a MultiDrop Combi to add 25 μL / well of the cell suspension to the experimental plate containing the test compound at medium speed;

[0233] (3) Place the experimental plate containing cells back into the incubator at 37°C and 5% CO2 for 16 to 18 hours.

[0234] Day 2

[0235] (1) Use MultiDrop Combi to add 25 μL / well of detection reagent (NanoGlo lysis solution + substrate + LgBit protein) to the assay plate at high speed and shake for 10 minutes;

[0236] (2) Centrifuge at 2000 rpm for 1 min to remove air bubbles;

[0237] (3). Read the plate using Envision, US Luminescence detection method.

[0238] 3. Data Analysis

[0239] The degradation rate (DR) of the test compound was calculated using the following formula: DR (%) = (RLU vehicle control - RLU compound) / (RLU vehicle control - RLU positive control) * 100%, where vehicle control is the blank control. The degradation rates of the compounds at different concentrations were calculated in Excel, and then the inhibition curve was plotted using XLFit software and related parameters were calculated, including the minimum degradation rate, maximum degradation rate, and DC. 50 .

[0240] The test results are shown in Table 9.

[0241] Table 9 Target protein degradation effect of the compound of formula (I) of the present invention in K562 IRAK4-HiBiT cells

[0242] Conclusion: The compounds of the present invention exhibit excellent target protein degradation in K562 IRAK4-HiBiT cells.

[0243] Experimental Example 2: In Cell Western analysis of the effect of compound (I) on the expression levels of IKZF1 and IKZF3 proteins in MM.1S cells

[0244] Experimental purpose: This experiment evaluates the degradation effect of the test compound on IKZF1 and IKZF3 proteins in MM.1S cells by detecting the effect of the compound of formula (I) on the expression levels of IKZF1 and IKZF3 proteins in MM.1S cells.

[0245] Experimental Materials:

[0246] Cell line: MM.1S cell (from ATCC; catalog number CRL-2974)

[0247] Negative control: 0.1% DMSO

[0248] Table 10 Reagents and consumables

[0249] Table 11 Instruments

[0250] Experimental plan:

[0251] 1) MM.1S cells in the logarithmic growth phase were plated in a 96-well plate, with 1.2×10 cells per well. 5 , overnight culture;

[0252] 2) On the second day, drug treatment was performed, starting at 300 nM, with three-fold dilutions, triplicate wells, and 10 concentration gradients (containing DMSO), and incubated in the incubator for 24 h;

[0253] 3) Centrifuge, carefully remove the cell supernatant, add 150 μL of 4% paraformaldehyde fixative along the well wall, avoiding touching the cells at the bottom, and incubate at room temperature for 20 minutes;

[0254] 4) Prepare permeabilization solution: Add 0.5 mL of 10% Triton X-100 to 49.5 mL of PBS and mix thoroughly.

[0255] 5) Add 200 μL of permeabilization buffer along the well wall, avoiding touching the cells at the bottom, and incubate on a shaker at room temperature for 5 minutes.

[0256] 6) Repeat washing 4 times;

[0257] 7) Add 150 μL of Licor INERCEPT blocking buffer (intertent blacking buffer) along the well wall, avoiding touching the bottom cells, and incubate on a shaker at room temperature for 1.5 h.

[0258] 8) Ikaros (D6N9Y) Rabbit mAb, Aiolos (D1C1E) Rabbit mAb, and GAPDH mouse mAb (Proteintech, 60004-1-Ig) were diluted 1:100 in antibody diluent.

[0259] 9) Add 50 μL of mixed antibodies to each well, replicate in triplicate, and incubate overnight at 4°C on a shaker.

[0260] 10) Prepare PBST (PBS containing 0.1% Tween 20);

[0261] 11) Remove the primary antibody and add 200 μL of PBST along the well wall, avoiding touching the cells at the bottom. Incubate on a shaker at room temperature for 5 minutes.

[0262] 12) Repeat washing 4 times;

[0263] 13) Prepare secondary antibody diluent: Add tween 20 to a final concentration of 0.2% to Licor INTERCEPT blocking buffer;

[0264] 14) Dilute the fluorescent secondary antibody in the dark (1:800 dilution). Take 400 μL of secondary antibody dilution solution and add 0.5 μL each of IRDye 800CW and IRDye 680CW. (The fluorescent secondary antibody corresponding to the primary antibody)

[0265] 15) Add 50 μL of diluted fluorescent secondary antibody to each well and incubate at room temperature on a shaker in the dark for 60 minutes;

[0266] 16) Remove the secondary antibody and add 200 μL of PBST along the well wall, avoiding touching the cells at the bottom. Incubate at room temperature on a shaker in the dark for 5 minutes.

[0267] 17) Repeat washing 4 times; immediately after washing, use the Odyssey Gel Imaging System for dual-color detection at 700 nm and 800 nm.

[0268] Data Analysis

[0269] GraphPad Prism 6 software was used to fit the inhibition rate data and calculate DC 50 value.

[0270] Protein inhibition rate = (1-RLs / RLv)*100%

[0271] RR (Raw Ratio) = 700nm / 800nm

[0272] RLs = RR of sample-treated cells

[0273] RLv = RR of solvent-treated cells

[0274] The test results are shown in Table 12.

[0275] Table 12 Degradation effect of the compound of formula (I) of the present invention on IKZF1 and IKZF3 proteins in MM.1S cells

[0276] Conclusion: The compounds of the present invention exhibit excellent target protein degradation effects on IKZF1 and IKZF3 proteins in MM.1S cells.

[0277] Experimental Example 3: Evaluation of the antiproliferative effect of the compound of formula (I) in lymphoma cell lines OCI-LY10 and TMD-8

[0278] Experimental purpose: This experiment was conducted to detect the inhibitory effect of the compound of formula (I) on cell proliferation in diffuse large B-cell lymphoma cell lines OCI-LY10 and TMD-8.

[0279] Experimental Materials:

[0280] Table 13 Cell lines and culture methods

[0281] Table 14 Culture medium and reagents

[0282] 1. Multi-well plate

[0283] Greiner 96-well plate, flat-bottom white plate (with lid and clear bottom), #3610.

[0284] 2. Reagents and Instruments Used in Cell Viability Experiments

[0285] (1) Promega CellTiter-Glo luminescent cell viability assay kit (Promega-G7573).

[0286] (2)2104 Plate reader, PerkinElmer.

[0287] Experimental plan:

[0288] 1. Cell Culture

[0289] Tumor cell lines were cultured in an incubator at 37°C and 5% CO2 according to the above-mentioned culture conditions. Cells were passaged regularly and cells in the logarithmic growth phase were used for plating.

[0290] 2. Cell Plating

[0291] (1) Stain cells with trypan blue and count live cells.

[0292] (2) Adjust the cell concentration to an appropriate level.

[0293] Table 15 Cell lines and plate densities

[0294] (3) Add 100 μL of cell suspension to each well of the culture plate as shown in the figure above.

[0295] (4) Incubate the culture plate in an incubator at 37°C, 5% CO2, and 100% relative humidity overnight.

[0296] 3. Compound Storage Plate Preparation

[0297] Prepare a stock plate of compound stocks at 1000 times the starting concentration: dilute the compound in DMSO from the highest concentration to the lowest concentration. Prepare the stocks freshly each time.

[0298] 4. Preparation of 1000x compound working solution and compound treatment of cells

[0299] (1) Preparation of working solution with a compound concentration 5 times the starting concentration: When diluting the compound 3-fold, pipette 30 μL of the compound from a stock solution 1000 times the starting concentration, add 20 μL of DMSO to the next few wells, and pipette 10 μL from the previous concentration to the next concentration. When diluting the compound 5-fold, pipette 30 μL of the compound from a stock solution 1000 times the starting concentration, add 24 μL of DMSO to the next few wells, and pipette 6 μL from the previous concentration to the next concentration. Add 20 μL of DMSO to the vehicle control. Dilute the 1000-fold compound 200-fold with culture medium, i.e., add 1 μL of the 1000-fold diluted compound to 199 μL of culture medium and mix thoroughly by pipetting.

[0300] (2) Drug addition: Take 25 μL of 5-fold compound and add it to the cell culture plate.

[0301] (3) Place the 96-well cell plate back into the incubator and culture OCI-LY10 (3-fold or 5-fold dilution, add drugs and incubate for 5 days) and TMD-8 (3-fold or 5-fold dilution, add drugs and incubate for 5 days).

[0302] 5. CellTiter-Glo luminescence cell activity detection

[0303] The following steps were performed according to the instructions of the Promega CellTiter-Glo Luminescent Cell Viability Assay Kit (Promega-G7573).

[0304] (1) Thaw CellTiter-Glo buffer and allow to cool to room temperature.

[0305] (2) Allow CellTiter-Glo substrate to cool to room temperature.

[0306] (3) Prepare CellTiter-Glo working solution by adding 10 mL of CellTiter-Glo buffer to a bottle of CellTiter-Glo substrate to dissolve the substrate.

[0307] (4) Slowly vortex to fully dissolve.

[0308] (5) Take out the cell culture plate and let it stand for 30 minutes to equilibrate to room temperature.

[0309] (6) Add 60 μL (equal to half the volume of the cell culture medium in each well) of CellTiter-Glo working solution to each well. Wrap the cell plate with aluminum foil to protect it from light.

[0310] (7) Shake the culture plate on an orbital shaker for 2 minutes to induce cell lysis.

[0311] (8) Place the culture plate at room temperature for 10 minutes to stabilize the luminescent signal.

[0312] (9) Detect luminescent signals on a 2104 EnVision plate reader.

[0313] 6. Data Analysis

[0314] The inhibition rate (IR) of the test compound was calculated using the following formula: IR (%) = (1-RLU compound / RLU vehicle control) * 100%. The inhibition rates of different compound concentrations were calculated in Excel, and then the inhibition curves were plotted and relevant parameters, including minimum inhibition rate, maximum inhibition rate, and IC, were calculated using GraphPad Prism software. 50 .

[0315] The test results are shown in Table 16.

[0316] 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

[0317] “ / ” means not detected

[0318] Conclusion: The compounds of the present invention exhibit excellent inhibitory effects on cell proliferation in lymphoma cell lines OCI-LY10 and TMD-8.

[0319] Experimental Example 4: Evaluation of the antiproliferative effect of the compound of formula (I) in the lymphoma cell line SU-DHL-2

[0320] Experimental purpose: This experiment was conducted to detect the inhibitory effect of the compound of formula (I) on cell proliferation in the lymphoma cell line SU-DHL-2.

[0321] Table 17 Cell lines and culture methods

[0322] Table 18 Culture medium and reagents

[0323] 1. Multi-well plate

[0324] Greiner CELLSTAR 384-well plates, flat bottom black plates (with lid), #781090.

[0325] 2. Reagents and Instruments Used in Cell Viability Experiments

[0326] (1) Promega CellTiter-Glo luminescent cell viability assay kit (Promega-G7573).

[0327] (2)2104 Plate reader, PerkinElmer.

[0328] Experimental plan:

[0329] 1. Cell Culture

[0330] Tumor cell lines were cultured in an incubator at 37°C and 5% CO2 according to the above-mentioned culture conditions. Cells were passaged regularly and cells in the logarithmic growth phase were used for plating.

[0331] 2. Cell Plating

[0332] (1) Stain cells with trypan blue and count live cells.

[0333] (2) Adjust the cell concentration to an appropriate level.

[0334] Table 19 Cell lines and plate densities

[0335] (3) Add 50 μL of cell suspension to each well of the culture plate as shown in the figure above.

[0336] (4) Incubate the culture plate in an incubator at 37°C, 5% CO2, and 100% relative humidity overnight.

[0337] 3. Compound Storage Plate Preparation

[0338] Drugs were added using an Echo 655 instrument at a volume of 50 nL and a final DMSO concentration of 0.1%. The culture plate was centrifuged at 1000 rpm for 1 minute and incubated in an incubator at 37°C, 5% CO2, and 100% relative humidity for 4 days.

[0339] 4. CellTiter-Glo luminescent cell viability assay

[0340] The following steps were performed according to the instructions of the Promega CellTiter-Glo Luminescent Cell Viability Assay Kit (Promega-G7573).

[0341] (1) Thaw CellTiter-Glo buffer and allow to cool to room temperature.

[0342] (2) Allow CellTiter-Glo Substrate to cool to room temperature.

[0343] (3) Prepare CellTiter-Glo working solution by adding CellTiter-Glo buffer to one bottle of CellTiter-Glo substrate to dissolve the substrate.

[0344] (4) Slowly vortex to fully dissolve.

[0345] (5) Remove the cell culture plate and let it stand for 30 minutes to equilibrate to room temperature.

[0346] (6) Add 25 μL (equal to half the volume of the cell culture medium in each well) of CellTiter-Glo working solution to each well. Wrap the cell plate with aluminum foil to protect it from light.

[0347] (7) Shake the culture plate on an orbital shaker for 2 minutes to induce cell lysis.

[0348] (8) The culture plate was placed at room temperature for 10 minutes to stabilize the luminescence signal.

[0349] (9) Detection of luminescent signal on 2104 EnVision plate reader

[0350] 5. Data Analysis

[0351] The inhibition rate (IR) of the test compound was calculated using the following formula: IR (%) = (1 – (RLU compound – RLU blank control) / (RLU vehicle control – RLU blank control)) * 100%. The inhibition rate of the compound at different concentrations was calculated in Excel, and then the inhibition curve was plotted and relevant parameters, including minimum inhibition rate, maximum inhibition rate, and IC, were calculated using GraphPad Prism software. 50 .

[0352] Table 20 Cell proliferation inhibition effect of the compound of formula (I) of the present invention in SU-DHL-2 cell line

[0353] Conclusion: The compounds of the present invention exhibit excellent inhibitory effects on cell proliferation in the lymphoma cell line SU-DHL-2.

[0354] Experimental Example 5: Pharmacokinetic evaluation of the compound of formula (I) in mice

[0355] Purpose of the experiment:

[0356] In this study, C57BL / 6 or C57 male mice were used as test animals. The LC / MS / MS method was used to quantitatively determine the drug concentration in the plasma of mice at different time points after intravenous injection or oral administration of the test compound to evaluate the pharmacokinetic characteristics of the compound of formula (I) of the present invention in mice.

[0357] Experimental Materials:

[0358] C57BL / 6 or C57 mice (male, 20-30 g, 7-10 weeks old, purchased from Beijing Weitonglihua).

[0359] Experimental operation:

[0360] Clarified solutions of the test compounds were injected into C57BL / 6 mice (fasted overnight) via the tail vein (vehicle: 10% DMSO / 10% solutol / 80% H2O) or administered orally to C57 mice (fed). Blood was collected by cheek puncture at 0 h (before dosing) and 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 h after intravenous administration. Blood was placed in a sodium heparinized tube, vortexed thoroughly, and centrifuged at 6000 g for 3 minutes at 2-8°C. Blood was collected by cheek puncture at 0 h (before dosing) and 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 h after oral administration. Blood was placed in a sodium heparinized tube, vortexed thoroughly, and centrifuged at 6000 g for 3 minutes at 2-8°C. Plasma drug concentrations were determined by LC-MS / MS, and relevant pharmacokinetic parameters were calculated using Phoenix WinNonlin8.2.0 pharmacokinetic software and the non-compartmental linear-log trapezoidal method.

[0361] Table 21 Pharmacokinetic parameters of the compound of formula (I) of the present invention in mice

[0362] “ / ” means not detected.

[0363] Conclusion: The oral plasma systemic exposure (AUC 0-inf ) is higher. In rodent mice, its pharmacokinetic properties are better.

[0364] Experimental Example 7: Pharmacokinetic Evaluation of the Compound of Formula (I) in Beagle Dogs

[0365] Purpose of the experiment:

[0366] Male beagle dogs were used as test animals in this study. LC / MS / MS was used to quantitatively determine the drug concentration in the plasma of beagle dogs at different time points after intravenous injection or oral administration of the test compound to evaluate the pharmacokinetic characteristics of the compound of formula (I) of the present invention in beagle dogs.

[0367] Experimental Materials:

[0368] Beagle dogs (male, 7-10 kg, Beijing Masi Biotechnology Co., Ltd.).

[0369] Experimental operation:

[0370] A clear solution of the test compound was slowly injected into a peripheral vein (with food) or administered orally to beagle dogs (with food). For intravenous administration, 0.5 mL of blood was collected from a peripheral vein at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours after administration. The blood was placed in an EDTA-2K anticoagulant tube and centrifuged at 3200 g for 10 minutes at 2-8°C. The supernatant was separated. For oral administration, 0.5 mL of blood was collected from a peripheral vein at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours after administration. The blood was placed in an EDTA-2K anticoagulant tube and centrifuged at 3200 g for 10 minutes at 2-8°C. The supernatant was separated. Plasma drug concentrations were determined by LC-MS / MS, and relevant pharmacokinetic parameters were calculated using Phoenix WinNonlin 6.3 pharmacokinetic software with the non-compartmental linear-log trapezoidal method.

[0371] The test results are shown in Table 22.

[0372] Table 22 Pharmacokinetic parameters of the compound of formula (I) of the present invention in beagle dogs

[0373] Conclusion: The oral plasma systemic exposure (AUC 0-inf ) is higher. In non-rodent beagle dogs, its pharmacokinetic properties are better.

[0374] Experimental Example 8: In vivo pharmacodynamic study of the compound of formula (I) in a human B cell lymphoma OCI-LY10 cell SCID mouse xenograft tumor model

[0375] Purpose of the experiment:

[0376] This study used a human B-cell lymphoma OCI-LY10 cell SCID mouse xenograft tumor model to evaluate the anti-tumor effect of the compound of formula (I).

[0377] Experimental Materials:

[0378] 1. Experimental animals: SCID mice, female, 6–8 weeks old, weighing 17–20 g. Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.

[0379] 2. Cell line: Human B-cell lymphoma OCI-LY10 cell line was purchased from Nanjing Kebai Biotechnology Co., Ltd. with the catalog number CBP60558.

[0380] Table 23 Reagent information

[0381] Table 24 Instruments

[0382] Model building:

[0383] OCI-LY10 cells were cultured in IMDM medium containing 20% ​​FBS and maintained in a humidified incubator at 37°C with 5% CO2. OCI-LY10 cells in the logarithmic growth phase were collected, resuspended in IMDM basal medium, and Matrigel was added at a 1:1 ratio to adjust the cell density to 4 × 10 7 Under sterile conditions, 0.1 mL of cell suspension was inoculated subcutaneously on the right side of the back of SCID mice at a concentration of 4 × 10 6 / 0.1mL / mouse.

[0384] Experimental plan:

[0385] When the tumors of pharmacodynamic experimental animals grow to a certain size, animals with tumors that are too large, too small, or with irregular shapes are eliminated, and animals with tumors with a size of 167.65 to 231.29 mm are selected. 3 The animals were randomly divided into groups according to the tumor volume, with 6 mice in each group and an average tumor volume of about 201.15 mm 3 The day of grouping was designated Day 0, and dosing began according to animal body weight. The pharmacodynamic study lasted 28 days, with dosing once daily, 24 hours between doses, and administered by gavage. Animal body weight and tumor size were measured twice weekly during the experiment. Clinical symptoms were observed and recorded daily.

[0386] The doses of the test compound were 10 mg / kg, 30 mg / kg and 100 mg / kg, respectively, and the solvent was 10% DMSO / 10% Solutol / 80% H2O. The tumor volume (TV) was calculated as follows: 1 / 2×a×b 2 , where a and b are the length and width of the tumor, respectively. The formula for calculating the tumor inhibition rate TGI (%) is: TGI (%) = [1-(average tumor volume of a treatment group at the end of drug administration - average tumor volume of the treatment group at the beginning of drug administration) / (average tumor volume of the solvent control group at the end of treatment - average tumor volume of the solvent control group at the beginning of treatment)] × 100%. Relative tumor growth rate T / C (%): The calculation formula is as follows: T / C% = T RTV / C RTV ×100%(T RTV : Average RTV of treatment group; C RTV : Average RTV of negative control group). Relative tumor volume (RTV) was calculated based on the results of tumor measurement. The calculation formula is RTV=Vt / V0, where V0 is the tumor volume measured at the time of group administration (i.e., Day 0), Vt is the tumor volume at a certain measurement, and TRTV with C RTV Get data for the same day.

[0387] Data Analysis:

[0388] In this study, experimental data were expressed as Mean ± SEM.

[0389] Statistical analysis was performed using IBM SPSS Statistics software based on RTV data at the end of the trial. Comparisons between two groups were analyzed using the t test, and comparisons between three or more groups were analyzed using one-way ANOVA. If the variances were equal (F values ​​were not significantly different), Tukey's method was used for analysis. If the variances were unequal (F values ​​were significantly different), the Games-Howell method was used for analysis. A p value of < 0.05 was considered significant.

[0390] Experimental results: The test results are shown in Tables 25 and 26.

[0391] Table 25 Evaluation of the antitumor efficacy of the compound of formula (I) of the present invention in the human B cell lymphoma OCI-LY10 cell subcutaneous xenograft tumor model

[0392] Note: a. Mean ± SEM.

[0393] Table 26 p-values ​​for comparison of relative tumor volumes among groups of the compound of formula (I) of the present invention in the human B-cell lymphoma OCI-LY10 xenograft tumor model

[0394] Note: p-values ​​were analyzed using IBM SPSS Statistics software.

[0395] Conclusion: The compound of the present invention has a significant tumor inhibitory effect in a human B cell lymphoma OCI-LY10 cell SCID mouse xenograft tumor model in a dose-dependent manner.

[0396] Experimental Example 9: In vivo pharmacodynamic study of the compound of formula (I) on a human lymphoma SU-DHL-2 cell subcutaneous xenograft tumor CB17 SCID mouse model

[0397] Purpose of the experiment:

[0398] This study used the SU-DHL-2 subcutaneous xenograft tumor CB17 SCID mouse model to evaluate the anti-tumor effects of the compound of formula (I).

[0399] Experimental Materials:

[0400] 1. Experimental animals: CB17 SCID mice, female, 6–8 weeks old, weighing 18–22 g. Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.

[0401] 2. Cell line: Human lymphoma SU-DHL-2 cells (Cat. No.: ATCC-CRL-2956).

[0402] Table 27 Reagent information

[0403] Table 28 Instruments

[0404] Model building:

[0405] Cell Culture: Human lymphoma SU-DHL-2 cells (ATCC-CRL-2956) were cultured in suspension in RPMI 1640 medium supplemented with 10% inactivated fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin in a 37°C, 5% CO2 incubator. Routine manipulation and passage were performed twice weekly. When cell saturation reached 80%-90% and the desired number of cells was reached, cells were harvested, counted, and plated.

[0406] Tumor cell inoculation and grouping: 0.2 mL (10×10 6 SU-DHL-2 cells (PBS:matrigel=1:1) were subcutaneously inoculated on the right back of each mouse, and the average tumor volume reached approximately 139 mm 3 The day of grouping is designated as Day 0, and dosing begins according to the animal's body weight.

[0407] Experimental plan:

[0408] The pharmacodynamics study consisted of three seven-day dosing cycles, with the test compound administered orally once daily with a 24-hour interval. Animal body weight and tumor size were measured twice weekly during the experiment, and clinical symptoms were observed and recorded daily.

[0409] The doses of the test compound were 10 mg / kg, 30 mg / kg and 100 mg / kg, respectively, and the solvent was 10% DMSO / 10% Solutol / 80% water. The tumor volume (TV) was calculated as follows: 1 / 2×a×b 2, where a and b are the length and width of the tumor, respectively. The formula for calculating the tumor inhibition rate TGI (%) is: TGI (%) = [1-(average tumor volume of a treatment group at the end of drug administration - average tumor volume of the treatment group at the beginning of drug administration) / (average tumor volume of the solvent control group at the end of treatment - average tumor volume of the solvent control group at the beginning of treatment)] × 100%. Relative tumor growth rate T / C (%): The calculation formula is as follows: T / C% = T RTV / C RTV ×100%(T RTV : Average RTV of treatment group; C RTV : Average RTV of negative control group). Relative tumor volume (RTV) was calculated based on the results of tumor measurement. The calculation formula is RTV=Vt / V0, where V0 is the tumor volume measured at the time of group administration (i.e., Day 0), Vt is the tumor volume at a certain measurement, and T RTV with C RTV Get data for the same day.

[0410] Data Analysis:

[0411] Statistical analysis was performed using SPSS software based on RTV data at the end of the study. Comparisons between two groups were analyzed using the T test, and comparisons between three or more groups were analyzed using one-way ANOVA. If the variances were equal (F values ​​were not significantly different), Tukey's method was used for analysis. If the variances were unequal (F values ​​were significantly different), the Games-Howell method was used for analysis. A p value of < 0.05 was considered significant.

[0412] Experimental results: The test results are shown in Tables 29 and 30.

[0413] Table 29 Evaluation of the anti-tumor efficacy of the compound of formula (I) of the present invention in the lymphoma SU-DHL-2 cell subcutaneous xenograft tumor model

[0414] Note: a. Mean ± SEM.

[0415] Table 30 p values ​​for the comparison of relative tumor volumes among the groups of the compound of formula (I) of the present invention in the lymphoma SU-DHL-2 xenograft tumor model

[0416] Note: p values ​​were obtained by analyzing relative tumor volume (RTV) using one-way ANOVA. Data with heterogeneous variance were analyzed using Games-Howell.

[0417] Conclusion: The compound of the present invention has a significant tumor inhibitory effect in the human lymphoma SU-DHL-2 cell subcutaneous xenograft tumor CB17 SCID mouse model.

[0418] Experimental Example 10: In vivo pharmacodynamic study of the compound of formula (I) on a BALB / c nude mouse model of subcutaneous xenograft tumors of human diffuse large B-cell lymphoma TMD-8 cells

[0419] Purpose of the experiment:

[0420] This study used a BALB / c nude mouse model of human diffuse large B-cell lymphoma TMD-8 subcutaneous xenograft tumor to evaluate the antitumor effect of the compound of formula (I).

[0421] Experimental Materials:

[0422] 1. Experimental animals: BALB / c nude mice, female, 6–8 weeks old. Weitonglihua Laboratory Animal Technology Co., Ltd.

[0423] 2. Cell line: human diffuse large B-cell lymphoma TMD-8 cells (purchased from Shanghai Huzhen Industrial Co., Ltd.).

[0424] Table 31 Main reagent information

[0425] Table 32 Main instrument information

[0426] Model building:

[0427] Cell culture: Routine cell culture was performed in RPMI-1640 medium containing 10% fetal bovine serum at 37°C and 5% CO2. Cells were subcultured according to their growth conditions at a subculture ratio of 1:3 to 1:4.

[0428] Tumor cell inoculation and grouping: TMD-8 cells in the logarithmic growth phase were collected, counted, and resuspended in 50% serum-free RPMI-1640 culture medium and 50% Matrigel. The cell concentration was adjusted to 4.0×10 7 cells / mL; the cells were placed in an ice box, and the cell suspension was drawn up with a 1-mL syringe and injected into the subcutaneous tissue of the right axilla of the nude mice. Each animal was inoculated with 200 μL (0.8×10 7 cells / mouse) to establish a TMD-8 transplanted tumor model. The average tumor volume reached approximately 160 mm 3 The day of grouping was the first day of the experiment (D1), and drug administration began according to the animal's body weight.

[0429] Experimental plan:

[0430] The pharmacodynamics study consisted of three seven-day dosing cycles, with the test compound administered orally once daily with a 24-hour interval. Animal body weight and tumor size were measured twice weekly during the experiment, and clinical symptoms were observed and recorded daily.

[0431] The doses of the test compound were 10 mg / kg, 30 mg / kg and 100 mg / kg, respectively, and the solvent was 10% DMSO / 10% Solutol HS15 / 80% water. The tumor volume (TV) was calculated as follows: 1 / 2×a×b 2 , where a and b are the length and width of the tumor, respectively. The formula for calculating the tumor inhibition rate TGI (%) is: TGI (%) = [1-(average tumor volume of a treatment group at the end of drug administration - average tumor volume of the treatment group at the beginning of drug administration) / (average tumor volume of the solvent control group at the end of treatment - average tumor volume of the solvent control group at the beginning of treatment)] × 100%. Relative tumor growth rate T / C (%): The calculation formula is as follows: T / C% = T RTV / C RTV ×100%(T RTV : Average RTV of treatment group; C RTV : Average RTV of negative control group). Relative tumor volume (RTV) was calculated based on the results of tumor measurement. The calculation formula is RTV = Vt / V0, where V0 is the tumor volume measured at the time of group administration (i.e., Day 1), Vt is the tumor volume at a certain measurement, and T RTV with C RTV Get data for the same day.

[0432] Data Analysis:

[0433] The experimental data were calculated and statistically processed using Microsoft Office Excel 2007. Unless otherwise specified, the data were expressed as mean ± standard error (mean ± SE), and the t-test was used for comparison between the two groups.

[0434] Experimental results: The test results are shown in Table 33.

[0435] Table 33 Evaluation of the antitumor efficacy of the compound of formula (I) of the present invention in the human diffuse large B-cell lymphoma TMD-8 cell nude mouse xenograft tumor model

[0436] Note: “*” indicates that the tumor volume is significantly different from that of the solvent control group (P<0.05); “**” indicates that the tumor volume is extremely significantly different from that of the solvent control group (P<0.01); a ” indicates mean ± SEM.

[0437] Conclusion: The compound of the present invention has a significant tumor inhibitory effect in a BALB / c nude mouse model of subcutaneous xenograft tumor of human diffuse large B-cell lymphoma TMD-8 cells.

Claims

1. A compound of formula (II) or a crystal thereof, in, n is 1.9 to 2.1; preferably 1.9, 2.0 and 2.

1.

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

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

4. The crystal of the compound of formula (II) according to claim 2, whose X-ray powder diffraction spectrum is shown in Figure 1.

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

6. The crystal of the compound of formula (II) according to claim 2, whose differential scanning calorimetry curve spectrum is shown in Figure 2.

7. The crystal of the compound of formula (II) according to claim 2, wherein the weight loss of the crystal at 150.0°C is 6.42%.

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

9. A compound of formula (III) or a crystal thereof, in, m is 1.1 to 1.5; preferably 1.1, 1.2, 1.3, 1.4 and 1.

5.

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

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

12. The crystal of the compound of formula (III) according to claim 9, whose X-ray powder diffraction spectrum is shown in FIG4.

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

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

15. The crystal of the compound of formula (III) according to claim 9, wherein the thermogravimetric analysis curve thereof shows a weight loss of 5.16% at 150.0°C.

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

17. A compound of formula (IV) or a crystal thereof, in, r is 2.1 to 2.6; preferably 2.1, 2.2, 2.3, 2.4, 2.5 and 2.

6.

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

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

21. The crystal of the compound of formula (IV) according to claim 19, whose X-ray powder diffraction spectrum is shown in FIG7.

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

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

24. The crystal of the compound of formula (IV) according to claim 19, wherein the thermogravimetric analysis curve thereof shows a weight loss of 6.40% at 150.0°C.

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

26. A compound of formula (V) or a crystal thereof, in, p is 1.0 to 1.5; preferably 1.0, 1.1, 1.2, 1.3, 1.4 and 1.

5.

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

28. The crystal of the compound of formula (V) according to claim 27, whose X-ray powder diffraction spectrum has characteristic diffraction peaks at the following 2θ angles: 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. The crystal of the compound of formula (V) according to claim 28, whose X-ray powder diffraction spectrum has characteristic diffraction peaks at the following 2θ angles: 6.51°, 8.94°, 9.76°, 13.09°, 17.87°, 19.88°, 21.51°, 27.11° and 29.37°.

30. The crystal of the compound of formula (V) according to claim 29, whose X-ray powder diffraction spectrum is shown in Figure 10.

31. The crystal of the compound of formula (V) according to claim 29, which has endothermic peaks at 64.7°C and 217.5°C in its differential scanning calorimetry curve.

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

33. The crystal of the compound of formula (V) according to claim 29, wherein the thermogravimetric analysis curve thereof shows a weight loss of 4.06% at 150.0°C.

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

35. A compound of formula (VI) or a crystal thereof, in, q is 2.1 to 2.3; preferably 2.1, 2.2 and 2.

3.

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

37. The crystal of the compound of formula (VI) according to claim 35, whose X-ray powder diffraction spectrum has characteristic diffraction peaks at the following 2θ angles: 7.63° and 18.46°.

38. The crystal of the compound of formula (VI) according to claim 35, whose X-ray powder diffraction spectrum is shown in Figure 13.

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

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

41. The crystal of the compound of formula (VI) according to claim 35, wherein the thermogravimetric analysis curve thereof shows a weight loss of 7.05% at 150.0°C.

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

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

Images