Crystalline form, manufacturing method, and use of 2,6-piperidinedione compounds

Abstract

The present invention discloses the crystalline form, method of production, and use of 2,6-piperidinedione compounds, and further includes the use of the crystalline form in the production of therapeutic drugs for related diseases, and specifically discloses the A-type crystal of the compound of formula (I) and a method of production therefor. [Formula 1] JPEG2026512830000040.jpg48154

Description

[Technical Field]

[0001] This application claims the following priority:

[0002] This application claims priority and interest in Chinese patent application No. 2023102936424, 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.

[0003] The present invention relates to the crystalline form, method of production, and use of 2,6-piperidinedione compounds, and further includes the use of the crystalline form in the preparation of therapeutic drugs for related diseases, and more specifically to the A-type crystal of the compound of formula (I) and a method of production therefor. [Background technology]

[0004] 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.

[0005] Myeloid differentiation factor (MyD88), a Toll-like receptor (TLR) signaling factor, is frequently mutated in various lymphomas, including Waldenström macroglobulinemia, lymphoplasmacytic lymphoma, immunoblastic 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, especially as a therapeutic target for MyD88-driven lymphoma.

[0006] 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.

[0007] 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-type drugs are called immunomodulatory drugs (IMiDs), and the E3 ubiquitin ligase complex formed with cereblon (CRBN) activates ubiquitination of transcription factors IKZF1 (zinc finger transcription factor 1) and IKZF3 (zinc finger transcription factor 3). Subsequently, they are recognized and degraded by the proteasome, exerting toxic effects on tumors. CRBN has been shown to be an important target for antitumor drugs and immunomodulators, with clear therapeutic effects confirmed in various hematological malignancies, skin diseases such as erythema nodosum, and autoimmune diseases such as systemic lupus erythematosus.

[0008] Therefore, by developing PROTACs and IMiD bifunctional molecules targeting IRAK4, removing IRAK4 by degrading it, more thoroughly blocking all functions of IRAK4, comprehensively suppressing the IRAK4 signaling pathway fundamentally, and at the same time exerting a synergistic therapeutic effect due to excellent CRBN regulatory effects, the anti-tumor effect can be exerted more effectively, and the clinical treatment effect can be enhanced.

Summary of the Invention

[0009] One aspect of the present invention provides an A-type crystal of a compound of formula (I), characterized in that its powder X-ray diffraction pattern has diffraction peaks characteristic of 2θ angles of 8.91±0.20°, 11.15±0.20°, 17.26±0.20°, 21.14±0.20°, 23.38±0.20°.

[0010]

Chemical Formula

[0011] In some embodiments of the present invention, the A-type crystal of the compound of formula (I) has a powder X-ray diffraction pattern comprising diffraction peaks characteristic of at least 6, 7, or 8 2θ angles selected from 5.02±0.20°, 8.91±0.20°, 11.15±0.20°, 15.08±0.20°, 17.26±0.20°, 18.38±0.20°, 21.14±0.20°, 23.38±0.20°, 23.57±0.20°.

[0012] In some embodiments of the present invention, the A-type crystal of the compound of formula (I) has a powder X-ray diffraction pattern with diffraction peaks characteristic of 2θ angles of 5.02±\alpha°, 8.91±\alpha°, 11.15±\alpha°, 15.08±\alpha°, 17.26±\alpha°, 18.38±\alpha°, 21.14±\alpha°, 23.57±\alpha°.

[0013] It should be noted that in the original text, the value of \(\alpha\) in the 23rd item is not clearly given. Here, it is assumed to be 0.20° according to the context of the previous items for translation. If there are other specific requirements, please adjust according to the actual situation.In some embodiments of the present invention, the A-type crystal of the compound of formula (I) has a powder X-ray diffraction pattern that includes characteristic diffraction peaks at at least 10, 11, 12, or 13 2θ angles selected from 5.02±0.20°, 8.91±0.20°, 9.80±0.20°, 11.15±0.20°, 13.87±0.20°, 14.70±0.20°, 15.08±0.20°, 17.26±0.20°, 18.38±0.20°, 21.14±0.20°, 21.57±0.20°, 23.10±0.20°, 23.38±0.20°, and 23.57±0.20°.

[0014] In some embodiments of the present invention, the A-type crystal of the compound of formula (I) has a powder X-ray diffraction pattern characterized by diffraction peaks at 2θ angles of 5.02±0.20°, 8.91±0.20°, 9.80±0.20°, 11.15±0.20°, 13.87±0.20°, 14.70±0.20°, 15.08±0.20°, 17.26±0.20°, 18.38±0.20°, 21.14±0.20°, 21.57±0.20°, and 23.38±0.20°.

[0015] In some embodiments of the present invention, the A-type crystal of the compound of formula (I) has powder X-ray diffraction patterns of 5.02±0.20°, 8.91±0.20°, 9.80±0.20°, 11.15±0.20°, 12.72±0.20°, 13.87±0.20°, 14.70±0.20°, 15.08±0.20°, 16.41±0.20°, and 17.26±0.20°. It exhibits characteristic diffraction peaks at 2θ angles of °, 18.38±0.20°, 19.22±0.20°, 19.86±0.20°, 21.14±0.20°, 21.57±0.20°, 22.29±0.20°, 23.10±0.20°, 23.38±0.20°, 23.57±0.20°, 25.71±0.20°, 26.49±0.20°, and 28.91±0.20°.

[0016] In some embodiments of the present invention, the A-type crystal of the compound of formula (I) has a powder X-ray diffraction pattern characterized by diffraction peaks at 2θ angles of 5.02°, 8.91°, 9.80°, 11.15°, 12.72°, 13.87°, 14.70°, 15.08°, 16.41°, 17.26°, 18.38°, 19.22°, 19.86°, 21.14°, 21.57°, 22.29°, 23.10°, 23.38°, 23.57°, 25.71°, 26.49°, and 28.91°.

[0017] In some embodiments of the present invention, the A-type crystal of the compound of formula (I) has a powder X-ray diffraction (XRPD) pattern substantially as shown in Figure 1.

[0018] In some embodiments of the present invention, the A-type crystals of the compound of formula (I) have the peak positions and relative intensities of the diffraction peaks in their powder X-ray diffraction (XRPD) patterns as shown in Table 1.

[0019] [Table 1]

[0020] In some embodiments of the present invention, the A-type crystal of the compound of formula (I) has a differential scanning calorimetry curve (DSC) with an endothermic peak value at 272.8°C ± 3°C and an exothermic peak value at 275.4°C ± 3°C.

[0021] In some embodiments of the present invention, the A-type crystal of the compound of formula (I) has a DSC pattern substantially as shown in Figure 2.

[0022] In some embodiments of the present invention, the A-type crystal of the compound of formula (I) exhibits a weight loss of 1.00% at 150.0°C ± 3°C in its thermogravimetric analysis (TGA) curve.

[0023] In some embodiments of the present invention, the A-type crystal of the compound of formula (I) has a TGA pattern substantially as shown in Figure 3.

[0024] In some embodiments of the present invention, type A crystals of compound (I) of the present invention can be prepared by the following method:

[0025] [ka]

[0026] (a) Add the compound of formula (I) to the solvent; (b) Stir at 15-30°C for 16-40 hours; (c) Filter and wash the cake with solvent; (d) Collect the cake and vacuum dry it at 50-70°C for 10-200 hours; However, the solvent is ethanol.

[0027] The present invention also provides the use of type A crystals of the compound of formula (I) in the preparation of therapeutic agents for interleukin-1 receptor-related kinase 4, zinc finger transcription factor 1, and zinc finger transcription factor 3 proteolytic targeting chimeric diseases.

[0028] In some embodiments of the present invention, the interleukin-1 receptor-related kinase 4, zinc finger transcription factor 1, and zinc finger transcription factor 3 proteolytic targeting chimeric disease is diffuse large B-cell lymphoma.

[0029] On the other hand, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of type A crystals of the compound of formula (I) according to the present invention. The pharmaceutical composition of the present invention may or may not contain pharmaceutically acceptable adjuvants. Furthermore, the pharmaceutical composition of the present invention may further contain one or more other therapeutic agents.

[0030] The present invention also provides a method for treating diffuse large B-cell lymphoma, comprising administering a therapeutically effective amount of type A crystals of the compound of formula (I) according to the present invention, or the pharmaceutical composition, to an individual in need.

[0031] Technical effects

[0032] The A-type crystals of the compound of formula (I) of the present invention are easy to prepare, have stable properties, exhibit good hygroscopicity, and are resistant to the effects of light and heat. 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 significant 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.

[0033] 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.

[0034] 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 diffraction peak position of 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.

[0035] 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 means 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] The term "target chimera" refers to a bifunctional molecule containing two types of small molecule ligands: the first has high affinity for the target protein, and the second is used to recruit an E3 ligase, which ubiquitinates the protein and targets it for degradation by the 26S proteasome.

[0040] The term "crystalline composition" refers to a mixture of the crystalline form of compound 1 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 compound (I) includes, in addition to type A crystals of compound (I), other crystalline forms of compound (I), amorphous materials, or other impurities.

[0041] The term "pharmaceutical composition" refers to a mixture comprising one or more compounds of the present invention or salts thereof 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.

[0042] The therapeutic dose of the compound of the present invention can be determined based on, for example, 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.

[0043] 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.

[0044] 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.

[0045] 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.”

[0046] Throughout this specification, the terms “one embodiment,” “an embodiment,” “in another embodiment,” or “in several embodiments” mean that at least one embodiment includes the relevant specific reference elements, structures, or features described in that embodiment. Therefore, the phrases “one embodiment,” “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.

[0047] 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.

[0048] The intermediate compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, and include, but are not limited to, 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. Preferred embodiments include, but are not limited to, the examples of the present invention.

[0049] 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.

[0050] [ka]

[0051] The present invention will be specifically described by examples, but these examples do not limit the present invention in any way.

[0052] All solvents used in this invention are commercially available and can be used without further purification.

[0053] Compounds are named manually or using ChemDraw® software, and commercial compounds use their supplier catalog names.

[0054] 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.

[0055] 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. 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 seconds Measurement time: ~5 minutes

[0056] 1.2 Differential Scanning Calorimeter (DSC) Method of the Present Invention Instrument model: TA 2500 Differential Scanning Calorimeter Measurement method: The sample (~1 mg) was placed in a DSC aluminum pan and heated from 30°C (room temperature) to 300°C at a heating rate of 10°C / min under a 50 mL / min N2 atmosphere.

[0057] 1.3 The Thermogravimetric Analysis (TGA) Method of the Present Invention Equipment Model: TA 5500 Thermogravimetric Analyzer Measurement method: Samples (2-5 mg) were placed in a TGA aluminum pan and heated from room temperature to 350°C at a heating rate of 10°C / min under a 25 mL / min N2 atmosphere.

[0058] 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 Measurement conditions: The sample (10-20 mg) was placed on a DVS sample dish and measured. The detailed DVS parameters are as follows: Temperature: 25℃ Equilibrium: dm / dt=0.002% / min (min: 10 min, max: 180 min) Drying: Dry at 0% RH for 120 minutes. RH (%) Test step: 10% RH (%) Test step range: 0%~95%~0%

[0059] The classification of hygroscopic properties is as follows:

[0060] [Table 1-2] [Brief explanation of the drawing]

[0061] [Figure 1] Figure 1 shows the powder X-ray diffraction (XRPD) pattern of a type A crystal of the compound of formula (I) using Cu-Kα rays. [Figure 2] Figure 2 shows the differential scanning calorimetry (DSC) pattern of a type A crystal of the compound of formula (I). [Figure 3] Figure 3 shows the thermogravimetric analysis (TGA) pattern of the type A crystal of the compound of formula (I). [Figure 4] Figure 4 shows the dynamic water vapor adsorption (DVS) spectrum of the A-type crystal of the compound of formula (I). [Modes for carrying out the invention]

[0062] 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.

[0063] Example 1: Preparation of the compound of formula (I)

[0064] [ka]

[0065] 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 layers 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) and stirred at room temperature for 1 hour, causing a solid to gradually precipitate. The mixture was concentrated under reduced pressure to remove most of the dichloromethane, and methyl tert-butyl ether (1.5 L) was added. After stirring at room temperature for 12 hours, the mixture was filtered to collect the cake. 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] + . 1 H 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).

[0066] 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] + .

[0067] 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), triphenylphosphine (82.09 g, 312.97 mmol) and imidazole (26.63 g, 319.22 mmol) were added, and the reaction mixture was cooled to 0°C in an ice bath. Then, elemental iodine (86.06 g, 339.06 mmol) was added in batches, and the reaction mixture was 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 layers 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. 600 mL of methyl tert-butyl ether was added to the residue, and the mixture was stirred at room temperature for 2 hours. After filtration, the cake was washed with methyl tert-butyl ether (50 mL x 2), and the filtrate was collected. 80 g of anhydrous magnesium chloride was added to the filtrate, and the mixture was stirred at room temperature for 8 hours. After filtration, 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] + .

[0068] 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] + .

[0069] 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-ESI m / z: 890.2 [M+H] + . 1H NMR (400 MHz, 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=9.2 Hz, 1H), 8.23-8.12 (m, 2H), 8.06 (d, J=6.0 Hz, 1H), 7.99 (d, J=9.2 Hz, 1H), 7.81-7.73 (m, 2H), 7.70 (s, 1H), 7.29 (d, J=6.8 Hz, 1H), 7.22 (t, J=54.4 Hz, 1H), 5.11 (dd, J=4.0 Hz, 11.2 Hz, 1H), 4.42 (s, 2H), 4.31-4.20 (m, 1H), 3.81 (s, 6H), 3.47 (s, 2H), 3.39 (d, J=6.4 Hz, 2H), 3.11 (s, 2H), 2.94-2.78 (m, 1H), 2.72-2.54 (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).

[0070] 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, and dried under vacuum to obtain the compound of formula (I) (free base).

[0071] Example 2: Preparation of type A crystals of the compound of formula (I) Method 1: At room temperature, 2.9 g of the compound of formula (I) and 30 mL of acetonitrile were added to a reaction flask, stirred at room temperature for 19 hours, filtered, washed with 2 mL of acetonitrile, collected the cake, and dried under a nitrogen stream for 10 to 48 hours to obtain type A crystals of the compound of formula (I).

[0072] Method 2: At room temperature, 1 g of compound (I) and 4 mL of dimethyl sulfoxide were added to a reaction flask. The temperature was raised to 50°C until the compound was completely dissolved. Then, 2.2 mL of ethanol was slowly added dropwise. After the addition was complete, the mixture was kept warm and stirred for 30 minutes. A further 9.8 mL of ethanol was added dropwise, and after the addition was complete, the mixture was stirred at 50°C for 30 minutes. The mixture was then slowly cooled to room temperature, stirred at room temperature for 15 hours, filtered, the cake was washed with 2 mL of ethanol, the cake was collected, and the mixture was purged with nitrogen gas for 10 to 48 hours to obtain type A crystals of compound (I).

[0073] Method 3: At room temperature, 0.5 g of the compound of formula (I) and 2 mL of dimethyl sulfoxide were added to a reaction flask, and the temperature was raised to 40°C. After the compound was completely dissolved, 20 mL of ethyl acetate was slowly added dropwise. After the addition was complete, the mixture was stirred at room temperature for 2 hours, then gradually cooled to room temperature, stirred for 15 hours, filtered, and the cake was washed with ethyl acetate (1.5 mL x 2). The cake was collected and purged with nitrogen gas for 10 to 48 hours to obtain type A crystals of the compound of formula (I).

[0074] Method 4: At room temperature, 2.5 g of the compound of formula (I) and 25 mL of ethanol were added to a reaction flask, stirred at room temperature for 20 hours, filtered, washed with ethanol (10 mL x 2), the cake was collected, and purged with nitrogen gas for 10 to 48 hours to obtain type A crystals of the compound of formula (I).

[0075] Example 3: Study of the hygroscopicity of type A crystals of the compound of formula (I) Test materials: SMS (Surface Measurement Systems)'s DVS Intrinsic Plus dynamic water vapor adsorption system.

[0076] Experimental method: 10-20 mg of type A crystals of the compound of formula (I) were taken and tested on a DVS sample disk.

[0077] Experimental results: The DVS spectrum of the A-type crystal of compound (I) is basically as shown in Figure 4, with ΔW = 3.536%.

[0078] Experimental conclusion: The A-type crystals of compound (I) showed a 3.536% increase in moisture absorption under 25°C and 80%RH conditions, indicating hygroscopicity, and there was no change in crystal form before and after DVS measurement.

[0079] Experimental Example 4: Stability testing and evaluation of the stability of type A crystals of compound (I) in different solvents and against mechanical forces. Addition of poor solvent: Approximately 20 mg of type A crystals of compound (I) was weighed into a 20 mL vial, and the solid was completely dissolved with 1.0 mL of the solvent shown in the table below (undissolved samples were filtered through a 0.45 μm PTFE filter to prepare a clear solution). The poor solvents shown in the table below were added dropwise to the clear solution while stirring (1000 rpm) until the solid precipitated. Alternatively, samples that did not precipitate even when the total amount of poor solvent reached 10 mL were suspended and stirred at 5°C. Clear samples were transferred to -20°C and suspended and stirred. If they remained clear, they were transferred to room temperature and allowed to volatilize. The precipitated solids were separated and XRPD measurements were performed. The results are shown in Table 2.

[0080] Room temperature suspension stirring: Approximately 20 mg of type A crystals of each compound of formula (I) were weighed into an HPLC vial, and 0.5 mL of the solvents listed in the table below were added one by one. The resulting suspension was magnetically stirred (1000 rpm) at room temperature, and after 3 days, the solid was recovered by centrifugation and XRPD measurement was performed. The results are shown in Table 2.

[0081] Temperature cycling stirring: About 20 mg of the A-type crystal of the compound of formula (I) was weighed into an HPLC glass vial, 0.5 mL of each solvent described in the following table was added, and the resulting suspension was magnetically stirred (1000 rpm) under temperature cycling (after heating the sample to 50 °C, cooling to 5 °C at a rate of 0.1 °C / min, repeating this cycle, and finally holding at 5 °C), centrifuged to recover the solid, and then XRPD measurement was performed. The results are shown in Table 2.

[0082] Gentle cooling: About 20 mg of the A-type crystal of the compound of formula (I) was weighed into an HPLC glass vial, 0.5 mL of the solvent described in the following table was added, and the resulting suspension was magnetically stirred (1000 rpm) at 50 °C. After a clear solution was obtained, if it was turbid, it was filtered (0.45 μm PTFE filter), and the filtrate was collected. The obtained clear solution was cooled from 50 °C to 5 °C (0.1 °C / min), and finally the sample was held at 5 °C. The solid was recovered and XRPD measurement was carried out. The results are shown in Table 2.

[0083] Pressure stability: 66.2 mg of the A-type crystal of the compound of formula (I) was added to the mold of a manual hydraulic press, and the solid obtained by compressing the sample in the mold at a pressure of 350 MPa was characterized by XRPD. The results are shown in Table 2.

[0084]

Table 2

[0085] 1 : The sample became transparent after adding the poor solvent and solids precipitated after stirring overnight at room temperature. 2 : The sample was transparent at room temperature and remained transparent after stirring at 5 °C for 3 days and at -20 °C for 2 days. It was transferred to room temperature and volatilized in an open state to precipitate solids. 3 : The sample became transparent after standing and stirring at 5 °C for 3 days, was transferred to room temperature and gently volatilized in an open state to precipitate solids.

[0086] Conclusion: The A-type crystals of compound (I) exhibited excellent stability in solvents such as acetone, tetrahydrofuran, 1,4-dioxane, methanol, ethanol, isopropanol, water, and tetrahydrofuran, 1,4-dioxane, ethanol, acetonitrile, and a mixture of acetone and water, while also maintaining good stability in a compression mold under a pressure of 350 MPa.

[0087] Example 5: Solid stability test of type A crystals of compound (I) under high temperature, high humidity, and high light conditions. Based on the "Guidelines for Stability Testing of Active Pharmaceutical Ingredients and Formulations" (General Rule 9001, Part 4, 2020 Edition of the Chinese Pharmacopoeia), the A-type crystals of compound (I) underwent high temperature (60°C, open), high humidity (room temperature / relative humidity 92.5%, open) and strong light irradiation (5000±500 Lux, 90 μW / cm²). 2 The stability under open conditions was evaluated.

[0088] 1.5 g of type A crystals of compound (I) was weighed and placed in an open weighing bottle, spreading uniformly into a thin layer. Samples left standing under high temperature conditions were tested in an electric hot air dryer, and samples left standing under high humidity conditions were tested in a comprehensive pharmaceutical stability tester. Samples were taken and tested on days 5, 10, and 30, and the test results were compared with the initial test results on day 0. Samples left standing open under strong light irradiation conditions were also taken and tested on days 5 and 10, and the test results were compared with the initial test results on day 0. The test results are shown in Table 3 below.

[0089] [Table 3]

[0090] Conclusion: The A-type crystals of compound (I) showed good stability under high temperature, high humidity, and strong light irradiation conditions.

[0091] Example 6: Solid stability test of type A crystal of compound (I) under accelerated conditions Based on the "Guiding Principles for Stability Testing of Active Pharmaceutical Ingredients and Formulations" (Chinese Pharmacopoeia 2020 Edition, General Principles for Four Parts 9001), the stability of type A crystals of compound (I) under high-temperature, high-humidity accelerated conditions (40°C / 75% relative humidity, sealed + desiccant) was investigated.

[0092] Approximately 1.5 g of type A crystals of compound (I) was weighed and placed in a double layer of low-density polyethylene bags. Each layer of the low-density polyethylene bag was sealed with a clip and then placed in an aluminum foil bag. Samples were taken and analyzed at 1 month, 2 months, and 3 months, and the analysis results were compared with the initial analysis results on day 0. This experiment was repeated twice, using a different batch of type A crystals of compound (I) each time. The test results are shown in Table 4 below.

[0093] [Table 4]

[0094] Conclusion: The A-type crystals of compound (I) showed good stability under accelerated conditions of 40°C / 75% relative humidity.

[0095] Experimental Example 7: Solid Stability Test of Type A Crystals of Compound (I) under Long-Term Conditions Based on the "Guiding Principles for Stability Testing of Active Pharmaceutical Ingredients and Formulations" (Chinese Pharmacopoeia 2020 Edition, General Principles for Four Parts 9001), the long-term stability of type A crystals of compound (I) was investigated under specific conditions (25°C / 60% relative humidity, sealed with desiccant).

[0096] Approximately 1.5 g of type A crystals of compound (I) was weighed and packed into a double layer of low-density polyethylene bags. Each layer of the low-density polyethylene bags was sealed with clips and then placed in an aluminum foil bag. Samples were taken and analyzed at 1 month and 2 months, and the analysis results were compared with the initial analysis results on day 0. This experiment was repeated twice, using different batches of type A crystals of compound (I) each time. The experimental results are shown in Table 5 below.

[0097] [Table 5]

[0098] Conclusion: The A-type crystals of compound (I) showed good stability under long-term conditions of 25°C / 60% relative humidity.

[0099] 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 in K562 IRAK4-HiBiT cells.

[0100] Experimental materials: 1. 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

[0101] [Table 6]

[0102] [Table 7]

[0103] 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. (2) Using a Multidrop Combi, 6 μL of DMSO was added to columns 2-12 and 14-24. (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. (4) Following the plate layout, 25 nL of compound solution (LDV plate, rows 1-24) was transferred to the assay plate using Echo. (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).

[0104] 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. (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. (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.

[0105] 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. (2) The sample was centrifuged at 2000 rpm for 1 minute to remove air bubbles. (3) The plates were read using the Envision and US Luminescence detection methods.

[0106] 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. The test results are shown in Table 8.

[0107] [Table 8]

[0108] Conclusion: The compounds of the present invention showed excellent target protein degradation activity in K562 IRAK4-HiBiT cells.

[0109] Experimental Example 2: Effect of compound (I) on IKZF1 and IKZF3 protein expression levels in MM.1S cells by in-cell Western analysis. 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.

[0110] Experimental materials: Cell line: MM.1S cells (derived from ATCC; catalog number CRL-2974) Negative control: 0.1% DMSO

[0111] [Table 9]

[0112] [Table 10]

[0113] 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. 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. 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. 4) Preparation of permeate: 0.5 mL of 10% Triton X-100 was added to 49.5 mL of PBS and mixed uniformly. 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 in a shaker for 5 minutes. 6) The washing process was repeated four times. 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. 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. 9) 50 μL of mixed antibody was added to each well, and each well was incubated overnight in a 4°C shaker with three double pores. 10) PBST (PBS containing 0.1% Tween-20) was prepared. 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. 12) The washing process was repeated four times. 13) Preparation of secondary antibody diluent: A final concentration of 0.2% tween 20 was added to the Licor INTERCEPT blocking solution. 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). 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. 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. 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.

[0114] 3. 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

[0115] The test results are shown in Table 11.

[0116] [Table 11]

[0117] Conclusion: The compounds of the present invention showed excellent targeted proteolytic activity against IKZF1 and IKZF3 proteins in MM.1S cells.

[0118] Experimental Example 3: Evaluation of the antiproliferative activity of the compound of formula (I) in lymphoma cell lines OCI-LY10 and TMD-8. Experimental Objective: This experiment investigated whether the compound of formula (I) exhibits cell proliferation inhibitory effects in the diffuse large B-cell lymphoma cell lines OCI-LY10 and TMD-8.

[0119] Experimental materials:

[0120] [Table 12]

[0121] [Table 13]

[0122] 1. Porous plate Greiner CELLSTAR® 96-well plate, flat-bottomed white plate (with lid and clear bottom), #3610.

[0123] 2. Reagents and equipment for cell activity experiments (1) Promega CellTiter-Glo Luminescence Cell Activity Detection Kit (Promega-G7573). (2) 2104 EnVision® Plate Reader, PerkinElmer.

[0124] 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.

[0125] 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.

[0126] [Table 14]

[0127] (3) As shown in the table above, 100 μL of cell suspension was added to each well of the culture plate. (4) The culture plates were incubated overnight in an incubator at 37°C, 5% CO2, and 100% relative humidity.

[0128] 3. Preparation of storage plates for compounds Preparation of storage plates for compound stock solutions at 1000 times the initial concentration: The compound was gradient diluted from the highest to the lowest concentration using DMSO. Each solution was prepared before use.

[0129] 4. Preparation of 1000x compound working solution and cell treatment with the compound (1) Preparation of working solution at 5x the starting concentration of the compound: When diluting the compound 3-fold, 30 μL was taken from a 1000-fold stock solution of the starting concentration of the compound, 20 μL of DMSO was added to the subsequent wells, and 10 μL was sequentially taken from the previous concentration to the next concentration. When diluting the compound 5-fold, 30 μL was taken from a 1000-fold stock solution of the starting concentration of the compound, 24 μL of DMSO was added to the subsequent wells, and 6 μL was sequentially taken from the previous concentration to the next concentration. 20 μL of DMSO was added as a solvent control. The 1000-fold compound was diluted 200-fold in the culture medium, that is, 1 μL of the 1000-fold diluted compound was added to 199 μL of the culture medium and homogenized by blowing with a multichannel pipette. (2) Addition of drug: 25 μL of a compound at 5x concentration was added to the cell culture plate. (3) The 96-well cell plates were returned to the incubator, and OCI-LY10 (3-fold or 5-fold dilution, cultured for 5 days after drug addition) and TMD-8 (3-fold or 5-fold dilution, cultured for 5 days after drug addition) were cultured.

[0130] 5. 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). (1) Dissolve CellTiter-Glo buffer and allow to stand at room temperature. (2) The CellTiter-Glo substrate was left standing at room temperature. (3) Add 10 mL of CellTiter-Glo buffer to one vial of CellTiter-Glo substrate to dissolve the substrate and prepare the CellTiter-Glo working solution. (4) Gently vortex-stirred until completely dissolved. (5) Remove the cell culture plate and allow it to stand for 30 minutes to equilibrate to room temperature. (6) 60 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. (7) The culture plate was shaken in an orbital shaker for 2 minutes to induce cell lysis. (8) To stabilize the luminescence signal, the culture plate was left to stand at room temperature for 10 minutes. (9) The light emission signal was detected using a 2104 EnVision plate reader.

[0131] 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. The test results are shown in Table 15.

[0132] [Table 15]

[0133] Conclusion: The compounds of the present invention showed excellent cell proliferation inhibitory activity in both lymphoma cell lines OCI-LY10 and TMD-8.

[0134] 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.

[0135] [Table 16]

[0136] [Table 17]

[0137] 1. Porous plate Greiner CELLSTAR 384-well plate, flat-bottom black plate with lid, #781090

[0138] 2. Reagents and equipment for cell activity experiments (1) Promega CellTiter-Glo Luminescence Cell Activity Detection Kit (Promega-G7573). (2) 2104 EnVision® plate reader, PerkinElmer.

[0139] 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.

[0140] 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.

[0141] [Table 18]

[0142] (3) As shown in the table above, 50 μL of cell suspension was added to each well of the culture plate. (4) The culture plates were incubated overnight in an incubator at 37°C, 5% CO2, and 100% relative humidity.

[0143] 3. Preparation of storage plates for compounds The drug was added using an Echo655 instrument. The added 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.

[0144] 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). (1) Dissolve CellTiter-Glo buffer and allow to stand at room temperature. (2) The CellTiter-Glo substrate was left standing at room temperature. (3) CellTiter-Glo buffer was added to the CellTiter-Glo substrate vial, and the substrate was dissolved to prepare the CellTiter-Glo working solution. (4) Gently vortex-stirred until completely dissolved. (5) Remove the cell culture plate and allow it to stand for 30 minutes to equilibrate to room temperature. (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. (7) The culture plate was shaken in an orbital shaker for 2 minutes to induce cell lysis. (8) To stabilize the luminescence signal, the culture plate was left to stand at room temperature for 10 minutes. (9) The light emission signal was detected using a 2104 EnVision plate reader.

[0145] 5. Data Analysis

[0146] 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.

[0147] [Table 19]

[0148] Conclusion: The hydrochloride salt of the compound of formula (I) of the present invention showed excellent cell proliferation inhibitory activity in the lymphoma cell line SU-DHL-2.

[0149] 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 the plasma drug concentrations at different time points after intravenous injection or oral administration to mice were quantitatively measured using LC / MS / MS to evaluate the pharmacokinetic properties of the compound of the present invention in mice.

[0150] Experimental materials: C57BL / 6 or C57 mouse (male, 20-30g, 7-10 weeks old, Beijing Weitong Lihua). Experimental procedure: A clear solution of the test compound was administered via tail vein injection to fasted C57BL / 6 mice (solvent: 10% DMSO / 10% solutol / 80% H2O), or orally to fed C57 mice. For intravenous injection, 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 post-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. For oral administration, 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 post-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.

[0151] [Table 20]

[0152] Conclusion: The compound of the present invention has a plasma exposure level (AUC) when administered orally. 0-inf The ) was high. In rodent mice, it exhibited superior pharmacokinetic properties.

[0153] Experimental Example 7: Pharmacokinetic Evaluation in Beagle Dogs Experimental objective: In this study, male beagle dogs were used as experimental animals, and plasma drug concentrations were quantitatively measured at different time points after intravenous injection or oral administration to beagle dogs using the LC / MS / MS method to evaluate the pharmacokinetic properties of the compound of the present invention in beagle dogs.

[0154] Experimental materials: Beagle (male, 7-10kg, Beijing Mas Biotechnology Co., Ltd.)

[0155] Experimental procedure: The test compound was administered by slow injection into the peripheral veins of Beagle dogs after feeding (solvent: 5% DMSO / 10% Solutol / 85% H2O) or by oral administration into Beagle dogs after 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 administration, collected in an EDTA-2K anticoagulant tube, and then 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 then 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 or 8.3.5 pharmacokinetic analysis software. The test results are shown in Table 21.

[0156] [Table 21]

[0157] Conclusion: The compound of the present invention has a plasma exposure level (AUC) when administered orally. 0-inf The pharmacokinetic properties were high in beagle dogs, which are non-rodent animals.

[0158] Experimental Example 8: In vivo drug efficacy study in a human B-cell lymphoma OCI-LY10 cell SCID mouse xenograft tumor model Experimental objective: In this study, the antitumor effects of the test compound were evaluated using a human B-cell lymphoma OCI-LY10 cell SCID mouse xenograft tumor model.

[0159] Experimental materials: 1. Experimental animal: SCID mouse, female, 6~8 weeks old, weight 17~20g. Beijing Wetong Lihua Experimental Animal Technology Co., Ltd. 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.

[0160] [Table 22]

[0161] [Table 23]

[0162] Model building: OCI-LY10 cells were cultured in IMDM medium containing 20% ​​FBS under 5% CO2 conditions. o The cells were maintained in a 1C saturated humidity incubator. Logarithmic growth phase OCI-LY10 cells were harvested, resuspended in IMDM basal medium, and then Matrigel was added in a 1:1 ratio to increase the cell concentration to 4 × 10⁻¹⁴. 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. 6The dosage was 0.1 mL per mouse.

[0163] 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. 3 The group assignment day was designated as Day 0, and medication was initiated based on the animal's body weight. The pharmacodynamic experiment lasted 28 days, with oral administration once daily at 24-hour intervals. During the study period, the animals' body weight and tumor size were measured twice a week. Clinical symptoms were observed and recorded daily.

[0164] 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% 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). Relative tumor volume (RTV) is calculated based on tumor measurement results, and the formula is RTV = Vt / V0 (where V0 is the tumor volume measured at the time of group division and drug 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.

[0165] Data analysis: In this study, all experimental data were expressed as Mean ± SEM. 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.

[0166] Experimental results: The test results are shown in Tables 24 and 25.

[0167] [Table 24]

[0168] [Table 25]

[0169] Conclusion: The compounds of the present invention showed a dose-dependent significant tumor-suppressing effect in a human B-cell lymphoma OCI-LY10 cell SCID mouse xenograft tumor model.

[0170] Experimental Example 9: Evaluation of in vivo drug efficacy in a CB17 SCID mouse model of human lymphoma SU-DHL-2 cell subcutaneous xenograft tumor. Experimental objective: In this study, the antitumor effects of the test compound were evaluated using the SU-DHL-2 subcutaneous xenograft tumor CB17 SCID mouse model.

[0171] 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).

[0172] [Table 26]

[0173] [Table 27]

[0174] 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.

[0175] 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.

[0176] 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.

[0177] 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% H2O. Tumor volume (TV) was calculated using the formula: 1 / 2 × a × b 2, where a and b are the major axis diameter and minor axis diameter at the time of tumor measurement, respectively. The calculation formula for the tumor growth inhibition rate TGI(%) is: TGI(%) = [1 - (average tumor volume at the end of administration in a certain treatment group - average tumor volume at the start of administration in the same treatment group) / (average tumor volume at the end of treatment in the solvent control group - average tumor volume at the start of treatment in the solvent control group)] × 100%. Relative tumor growth rate T / C(%): The calculation formula is: T / C% = T RTV / C RTV × 100% (T RTV : average RTV of the treatment group; C RTV : average RTV of the negative control group). Based on the tumor measurement results, the relative tumor volume (RTV) is calculated, and the calculation formula is RTV = Vt / V0 (V0 is the tumor volume measured on the day of grouped dosing (day 0), Vt is the tumor volume at a certain measurement time point). T RTV and C RTV adopted the data of the same day.

[0178] Data analysis: The statistical analysis was performed using SPSS software based on the RTV data at the end of the test. T-test was applied for comparison between two groups, and one-way analysis of variance (one-way ANOVA) was applied for comparison among three or more groups. When the variances were homogeneous (no significant difference in F value), the Tukey method was used; when the variances were non-homogeneous (significant difference in F value), the Games-Howell method was used for testing. p < 0.05 was determined to be a significant difference.

[0179] Experimental results: The test results are shown in Tables 28 and 29.

[0180]

Table 28

[0181] [[ID=3B]]

Table 29

[0182] Conclusion: The compounds of the present invention showed a significant tumor-suppressing effect in a CB17 SCID mouse model of human lymphoma SU-DHL-2 cell subcutaneous xenograft tumor.

[0183] Experimental Example 10: Evaluation of in vivo drug efficacy in a human diffuse large B-cell lymphoma (TMD-8 cell) subcutaneous xenograft tumor (BALB) mouse model. Experimental objective: In this study, the antitumor effects of the test compound were evaluated using a human diffuse large B-cell lymphoma (TMD-8) subcutaneous xenograft tumor BALB / c nude mouse model.

[0184] Experimental materials: 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.).

[0185] [Table 30]

[0186] [Table 31]

[0187] Model building: 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.

[0188] 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. 7A 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.

[0189] 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.

[0190] 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 drug 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.

[0191] Data analysis: The test data were calculated and related statistical processing was performed using Microsoft Office Excel 2007 software. Unless otherwise specified, the data were expressed as mean ± standard error (Mean±SE), and a t-test was adopted for comparison between two groups.

[0192] Experimental results: The test results are shown in Table 32.

[0193]

Table 32

[0194] Conclusion: The compound of the present invention showed a significant tumor-suppressing effect in the subcutaneous xenograft tumor BALB / c nude mouse model of human diffuse large B-cell lymphoma TMD-8 cells. (Note) This disclosure includes the following aspects: Item 1: A type A crystal of the compound of formula (I), characterized in that the powder X-ray diffraction pattern has characteristic diffraction peaks at 2θ angles of 8.91±0.20°, 11.15±0.20°, 17.26±0.20°, 21.14±0.20°, and 23.38±0.20°.

Chemical formula

Claims

1. A type A crystal of the compound of formula (I), characterized in that the powder X-ray diffraction pattern has characteristic diffraction peaks at 2θ angles of 8.91±0.20°, 11.15±0.20°, 17.26±0.20°, 21.14±0.20°, and 23.38±0.20°. 【Chemistry 1】

2. A type A crystal of the compound of formula (I) according to claim 1, characterized in that the powder X-ray diffraction pattern has characteristic diffraction peaks at 6, 7, or 8 2θ angles: 5.02±0.20°, 8.91±0.20°, 11.15±0.20°, 15.08±0.20°, 17.26±0.20°, 18.38±0.20°, 21.14±0.20°, 23.38±0.20°, and 23.57±0.20°.

3. A type A crystal of the compound of formula (I) according to claim 1, characterized in that the powder X-ray diffraction pattern has characteristic diffraction peaks at 2θ angles of 5.02±0.20°, 8.91±0.20°, 11.15±0.20°, 15.08±0.20°, 17.26±0.20°, 18.38±0.20°, 21.14±0.20°, and 23.57±0.20°.

4. A type A crystal of the compound of formula (I) according to claim 1, characterized in that the powder X-ray diffraction pattern has characteristic diffraction peaks at 10, 11, 12 or 13 2θ angles: 5.02±0.20°, 8.91±0.20°, 9.80±0.20°, 11.15±0.20°, 13.87±0.20°, 14.70±0.20°, 15.08±0.20°, 17.26±0.20°, 18.38±0.20°, 21.14±0.20°, 21.57±0.20°, 23.10±0.20°, 23.38±0.20°, and 23.57±0.20°.

5. A type A crystal of the compound of formula (I) according to claim 1, characterized in that the powder X-ray diffraction pattern has characteristic diffraction peaks at 2θ angles of 5.02±0.20°, 8.91±0.20°, 9.80±0.20°, 11.15±0.20°, 13.87±0.20°, 14.70±0.20°, 15.08±0.20°, 17.26±0.20°, 18.38±0.20°, 21.14±0.20°, 21.57±0.20°, and 23.38±0.20°.

6. The powder X-ray diffraction patterns were 5.02±0.20°, 8.91±0.20°, 9.80±0.20°, 11.15±0.20°, 12.72±0.20°, 13.87±0.20°, 14.70±0.20°, 15.08±0.20°, 16.41±0.20°, 17.26±0.20°, 18.38±0.20°, 19.22±0.20°, and 19.86±0. A type A crystal of the compound of formula (I) according to claim 1, characterized by having characteristic diffraction peaks at 2θ angles of 20°, 21.14±0.20°, 21.57±0.20°, 22.29±0.20°, 23.10±0.20°, 23.38±0.20°, 23.57±0.20°, 25.71±0.20°, 26.49±0.20°, and 28.91±0.20°.

7. A type A crystal of the compound of formula (I) according to claim 1, characterized in that the powder X-ray diffraction pattern has characteristic diffraction peaks at 2θ angles of 5.02°, 8.91°, 9.80°, 11.15°, 12.72°, 13.87°, 14.70°, 15.08°, 16.41°, 17.26°, 18.38°, 19.22°, 19.86°, 21.14°, 21.57°, 22.29°, 23.10°, 23.38°, 23.57°, 25.71°, 26.49°, and 28.91°.

8. The differential scanning calorimetry curve of the aforementioned type A crystal has an endothermic peak at 272.8°C ± 3.0°C and an exothermic peak at 275.4°C ± 3.0°C; or The thermogravimetric analysis curve of the aforementioned type A crystal shows a weight loss of 1.00% at 150.0°C ± 3.0°C. A type A crystal of the compound of formula (I) according to claim 1, characterized in that

9. A pharmaceutical agent for treating diffuse large B-cell lymphoma, comprising a type A crystal of the compound of formula (I) described in any one of claims 1 to 8.

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