Method for producing naphthisoxazole compounds

Abstract

The present invention relates to a method for producing naphthalene isoxazole compounds, and more specifically, to a method for producing the compound of formula (I) and its intermediates. [Formula 1] JPEG2026513531000038.jpg43136

Description

[Technical Field]

[0001] This application claims the following priority:

[0002] This application claims priority and rights to Chinese Patent Application No. 2023102949123, filed with the State Intellectual Property Administration of China on March 24, 2023, the contents disclosed in said application are incorporated herein by reference.

[0003] This invention relates to a method for producing naphthalene isoxazole compounds, and more specifically, to a method for producing H and its intermediates. [Background technology]

[0004] Interleukin-1 receptor-associated kinase (IRAK4) is a serine / threonine kinase that plays a crucial role in Toll / IL-1 receptor (TIR) ​​signaling. Multiple IRAK enzymes are major components in signaling pathways mediated by interleukin-1 receptors (IL-1R) and Toll-like receptors (TLRs). The mammalian IRAK family consists of four members: IRAK-1, IRAK-2, IRAK-3, and IRAK-4. These proteins are characterized by having a typical N-terminal death domain and a central kinase domain that mediates interaction with MyD88 family adapter proteins. When MyD88 and IRAK4 are recruited to the Toll-interleukin domain, activation is triggered, leading to phosphorylation of IRAK-1 and / or IRAK-2, which in turn promotes TRAF6 binding. Subsequent signaling results in NF-κB activation and the release of various cytokines and chemokines. The MyD88 L265P mutation is found in 91% of Waldenström macroglobulinemia, 29% of ABC-DLBCL, 9% of MALT lymphoma, and 3% of CLL, and is associated with constitutively active signalosomes. In this mutation, IRAK-4-mediated phosphorylation of IRAK-1 promotes the assembly of other signaling proteins that contribute to the survival of these cancers.

[0005] IRAK4 possesses kinase-independent scaffolding function. Similar scaffolding function has been observed in human fibroblasts, where kinase-inactive IRAK4 has been shown to restore IL-1-induced NF-κB signaling to levels comparable to that of WT IRAK4. Because IRAK4 can be targeted for degradation, it may offer therapeutic opportunities for autoimmune, inflammatory, and neoplastic diseases. Specific degradation of IRAK4 can be achieved by recruiting IRAK4 to ubiquitin ligases using heterofunctional small molecules, thereby promoting IRAK4 ubiquitination and proteasomal degradation. For example, thalidomide derivatives such as lenalidomide and pomalidomide have been reported to recruit potential protein substrates to cereblon (CRBN), a component of the ubiquitin ligase complex. Cereblon is a protein encoded by the CRBN gene in humans. The high conservation of CRBN orthologues from plants to humans highlights its physiological importance. Cereblon, along with damaged DNA-binding protein 1 (DDB1) and Cullin-4A, forms the E3 ubiquitin ligase complex (CUL4A) and the cullins regulator (ROC1). CRBNs are important target molecules for antitumor and immunomodulatory drugs, and their therapeutic effects have been clearly demonstrated in various hematological malignancies, skin diseases such as erythema nodosum, and autoimmune diseases such as systemic lupus erythematosus.

[0006] Proteolysis Targeting Chimeras (PROTACs) are a recent focus of research in the field of drug discovery. This technology uses small molecules to recruit specific ubiquitin ligases, inducing proteolysis through ubiquitination of target proteins. PROTACs are dual-targeting chimeric molecules consisting of three components: a ligand that binds to the target protein, a ligand that recruits E3 ubiquitin ligases, and a linker that connects the two. Compared to conventional small molecule inhibitors, PROTACs have potential advantages in terms of dosage, selectivity, tolerance, and the regulation of "drug-untargetable targets," and are currently moving from basic research to clinical trials.

[0007] To further promote the accessibility improvement and scale-up production of pharmaceuticals, the development of new manufacturing processes is essential.

Summary of the Invention

[0008] The present invention provides a method for preparing compound 6 using compound 4 and compound 5, wherein the reaction steps are as follows.

[0009]

Chemical Formula

[0010] The invention also provides a method for preparing a compound of formula (I) using compound 1, wherein the reaction steps are as follows.

[0011]

Chemical Formula

[0012] In some embodiments of the present invention, in the method for preparing compound 6 from compound 4 and compound 5,

[0013]

Chemical Formula

[0014] the equivalent ratio of compound 4 to compound 5 is 0.8:1 to 1:1; base 2 is selected from TEA and DIEA; solvent 3 is selected from acetonitrile.

[0015] In some embodiments of the present invention, the reaction temperature is 70-80 °C, and other variables are as defined in the present invention.

[0016] In some embodiments of the present invention, the equivalent ratio of compound 4 to compound 5 is 0.85:1 to 0.95:1, and other variables are as defined in the present invention.

[0017] In some embodiments of the present invention, the equivalent ratio of compound 4 to compound 5 is 0.88:1, and the other variables are as defined in the present invention.

[0018] In some embodiments of the present invention, a method for preparing a compound of formula (I) using compound 1,

[0019] [ka]

[0020] The condensing agent is selected from HATU, benzenesulfonyl chloride, or p-toluenesulfonyl chloride; The aforementioned base 1 is selected from TEA or DIEA; The solvent 1 is selected from DMF, DMSO, or NMP; The acidic system 1 is selected from ethyl acetate hydrochloride solution or TFA; The aforementioned acidic system 2 is selected from ethyl acetate hydrochloride solution or TFA; The aforementioned base 2 is selected from TEA, DIEA, or sodium bicarbonate; The solvent 3 is selected from acetonitrile; The iodinating reagent is selected from PPh3 / Im / I2; The solvent 2 is selected from DCM.

[0021] In some embodiments of the present invention, the condensing agent is selected from HATU, acid system 1 is selected from TFA, acid system 2 is selected from ethyl acetate hydrochloride solution, the concentration of the ethyl acetate hydrochloride solution is 4 mol / L, base 1 is selected from DIEA, and base 2 is selected from DIEA.

[0022] In some embodiments of the present invention, the reaction temperature in step 5 is 40-50°C, and the other variables are as defined in the present invention.

[0023] In some embodiments of the present invention, the reaction time in step 5 is 0.5 to 3 hours, and the other variables are as defined in the present invention.

[0024] Technological effects The synthesis process of the compound of formula (I) provided by the present invention offers a novel method for synthesizing naphthisoxazole compounds, and its beneficial effects include mild reaction conditions throughout the entire process, simple operation, a simple and effective purification method, and suitability for industrial production.

[0025] in particular: 1) In the synthesis process for producing the compound of formula (I) of the present invention, the purification means mainly consists of slurrying or crystallization and extraction operations, which are simple to operate and avoid column chromatography, making them more suitable for industrial production. 2) The process in step 3 can prevent the formation of configuration isomers, and the purification process is further simplified. 3) No highly toxic, flammable, or explosive reagents are used in any of the manufacturing processes, ensuring the safety of the process production.

[0026] Definition and explanation: Unless otherwise specified, the following terms and phrases used in this document shall have the meanings set forth below. Where a particular phrase or term is not specifically defined, it should not be considered uncertain or ambiguous, but rather understood according to its general meaning. Where a product name is mentioned in this document, it is intended to refer to the corresponding product or its active ingredient.

[0027] The intermediate compounds of the present invention can be prepared by a variety of synthetic methods familiar to those skilled in the art, including the specific embodiments listed below, embodiments formed by combinations with other chemical synthesis methods, and equivalent alternative methods familiar to those skilled in the art. Preferred embodiments include, but are not limited to, the examples of the present invention. Those skilled in the art can achieve other corresponding objectives by appropriately modifying elements such as raw materials and process conditions, taking the present invention into consideration. Such related modifications do not depart from the scope of the present invention, and all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included within the scope of the present invention.

[0028] The chemical reactions in specific embodiments of the present invention are carried out in a suitable solvent, which must be compatible with the chemical changes and required reagents and materials of the present invention. To obtain the compounds of the present invention, those skilled in the art may need to modify or select synthesis steps or reaction processes based on existing embodiments.

[0029] A key consideration in the design of any synthetic route in this field is the selection of an appropriate protecting group for the reactive functional group (such as the amino group in this invention).

[0030] All solvents used in this invention are commercially available.

[0031] In this invention, the following abbreviations are used: Boc represents tert-butoxycarbonyl, SiO represents ethyl acetate, HATU represents O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, ACN represents acetonitrile, DIEA represents N,N-diisopropylethylamine, Im represents imidazole, DCM represents dichloromethane, DMF represents N,N-dimethylformamide, and PPh3 represents triphenylphosphine.

[0032] Compounds are named using the nomenclature commonly used in the field or by ChemDraw® software, and commercially available compounds are named using the supplier catalog name. [Modes for carrying out the invention]

[0033] The present invention will be described in detail below through examples, but this will not disadvantageously limit the invention. This specification describes the present invention in detail and discloses specific embodiments thereof, and it will be readily apparent to those skilled in the art that various modifications and improvements can be made to specific embodiments of the present invention without departing from the spirit and scope of the invention.

[0034] Example 1: Preparation of the compound of formula (I)

[0035] [ka]

[0036] Step 1: Synthesis of Compound 3 At 10-15°C under a weak nitrogen stream, N,N-dimethylformamide (4.8 L) was added to the reaction vessel, followed by the addition of compound 2 (702.94 g), the hydrochloride salt of compound 1 (800 g), and N,N-diisopropylethylamine (739.62 g), while maintaining a temperature of 10-20°C throughout the entire process. Finally, O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (1.31 kg) was added in 10 portions. The reaction system became exothermic upon addition, and the temperature was maintained at 15-30°C throughout the entire process. After the addition was complete, the mixture was stirred at 20-30°C for 4-24 hours. After the reaction was complete, acetic acid (137.46 g) was added to the reaction vessel while maintaining the temperature at 20-30°C, and the mixture was stirred for 20-40 minutes. Then methanol (110.02 g) was added, and the mixture was stirred for 1.5-3 hours while maintaining the temperature. Methyl tert-butyl ether (12 L) was transferred to the reaction vessel using a peristaltic pump, and the mixture was stirred for 9-12 hours while controlling the temperature at 10-20°C. The mixture was then filtered, the cake was washed with methyl tert-butyl ether (2 L x 2), and the cake was recovered.

[0037] At 10-20°C under a nitrogen gas atmosphere, water (4.8 L) and isopropanol (480 mL) were transferred to a 10 L reaction flask, the cake was added, and the mixture was stirred for 1.5-2.5 hours. The mixture was filtered, and the cake was washed with a mixed solvent of water and isopropanol (volume ratio: water / isopropanol = 10 / 1, 500 mL x 2). The cake was recovered, dried, and compound 3 (953.2 g, purity: 98.7%, yield: 75.8%) was obtained. 1 H NMR (400MHz, DMSO_d6) δ: 11.14 (s, 1H), 10.13 (s, 1H), 8.19 (d, J=9.6 Hz, 1H), 8.11 (d, J=7.2 Hz, 1H), 8.00 (d, J=9.2 Hz, 1H), 7.82-7.65 (m, 2H), 5.09 (dd, J=4.8, 11.2 Hz, 1H), 3.53-3.40 (m, 4H), 3.32 (s, 2H), 2.87-2.80 (m, 1H), 2.70-2.66 (m, 1H), 2.65-2.55 (m, 5H), 2.44-2.34 (m, 1H), 2.44-2.34 (m, 1H).

[0038] Step 2: Synthesis of trifluoroacetate of compound 4 The temperature was controlled to 15-25°C, and under a weak nitrogen stream, dichloromethane (9.3 L) was transferred to the reaction vessel. Compound 3 (930 g) and trifluoroacetic acid (2.14 kg) were added, and after the addition was complete, the mixture was stirred for 0.5-1 hour while maintaining the temperature. The reaction mixture was gradually heated to 35-42°C and stirred for 3-5 hours. After the reaction was complete, the reaction mixture was cooled to 15-25°C, stirred while maintaining the temperature for 1-3 hours, filtered, and the cake was washed with dichloromethane (930 mL x 3) and the cake was recovered.

[0039] At 15-25°C under a weak nitrogen stream, water (7.4 L) and isopropanol (1.5 L) were transferred to a reaction vessel, imidazole (971.1 g) was added and stirred until dissolved, then the cake was added and stirred for 0.5-1 hour while maintaining the temperature. The reaction mixture was cooled to 0-10°C and stirred for 10-16 hours while maintaining the temperature. The mixture was filtered, the cake was washed with water (500 mL x 2), the cake was collected, and the cake was transferred to a 10 L reaction bottle. Water (4.18 L) was added and stirred at 5-15°C for 0.5-1 hour while maintaining the temperature. The mixture was filtered, the cake was sequentially washed with water (500 mL x 3) and isopropanol (500 mL x 2), the cake was collected and dried to obtain trifluoroacetate of compound 4 (722 g, purity: 99.9%, yield: 77.9%). 1 H NMR (400MHz, DMSO_d6) δ: 11.15 (s, 1H), 10.15 (s, 1H), 8.73 (s, 2H), 8.21 (d, J=9.6 Hz, 1H), 8.14 (d, J=7.6 Hz, 1H), 7.97 (d, J=9.2 Hz, 1H), 7.79-7.61 (m, 2H), 5.09 (dd, J=4.8, 11.6 Hz, 1H), 3.42 (s, 2H), 3.28-3.16 (m, 4H), 2.94-2.78 (m, 5H), 2.72-2.54 (m, 2H), 2.45-2.34 (m, 1H).

[0040] Step 3: Synthesis of Compound 5 At 10-15°C under a weak nitrogen stream, dichloromethane (25 L) was added to the reaction vessel, followed by the addition of compound 5-1 (2.5 kg, 4.26 mol), triphenylphosphine (1.34 kg, 5.11 mol), and imidazole (435.18 g, 6.39 mol). The reaction mixture was cooled to 0-5°C, and elemental iodine (1.41 kg, 5.54 mol) was added in six portions, while maintaining the temperature at 0-5°C. After the addition was complete, the temperature was returned to 15-25°C and the mixture was stirred for 16 hours. After the reaction was complete, saturated sodium sulfite solution (3.7 L) and water (12.5 L) were added to the reaction mixture, stirred for 30 minutes, allowed to stand, and then separated. The aqueous layer was re-extracted with dichloromethane (5 L x 2) to combine the organic phases. The organic phase was sequentially washed with saturated saline solution (12.5 L) and water (12.5 L), dried over anhydrous sodium sulfate, filtered, and the two batches of filtrate were combined and concentrated under reduced pressure to obtain the crude product.

[0041] Ethanol (25 L) was added to the crude product, heated to 70°C to completely dissolve, then kept warm and stirred for 1 hour, and gradually cooled to room temperature while stirring for 16 hours. The two batches were combined and filtered, the cake was washed with ethanol (5 L x 2), and the cake was recovered. Methanol (25 L) was added to this cake, stirred at room temperature for 1 hour, filtered, and the cake was washed with methanol (2.5 L x 2, 2 L x 2, 1.5 L x 2) and the cake was recovered. After drying, compound 5 (4.8 kg, purity: 99.0%, yield: 80.8%) was obtained. 1H NMR (400MHz, DMSO_d6) δ: 9.77 (s, 1H), 9.01 (s, 1H), 8.59 (d, J=5.6 Hz, 1H), 8.29 (s, 1H), 8.19 (s, 1H), 7.68 (dd, J=1.6, 9.2 Hz, 1H), 7.15 (t, J=54.4 Hz, 1H), 4.21 (tt, J=4.0, 12.0 Hz, 1H), 3.86 (d, J=6.8 Hz, 1H), 3.17 (d, J=6.0 Hz, 2H), 2.10-1.91 (m, 4H), 1.88-1.74 (m, 2H), 1.51 (s, 9H), 1.49-1.43 (m, 1H), 1.26-1.12 (m, 3H), 0.44-0.37 (m, 2H), 0.27-0.20 (m, 2H).

[0042] Step 4: Synthesis of Compound 6 At 15-25°C, under a weak nitrogen stream, acetonitrile (8 L), trifluoroacetate of compound 4 (679.63 g), and compound 5 (1.005 kg) were added to the reaction vessel. Then, N,N-diisopropylethylamine (356.26 g) was added. After the addition was complete, the reaction mixture was heated to 75-80°C and allowed to react for 22-24 hours while maintaining the temperature. After the reaction was complete, the mixture was slowly cooled to 15-25°C (cooling rate of 10-20°C / hour when temperature > 50°C, and 5-10°C / hour when temperature ≤ 50°C), and stirred for 1-2 hours while maintaining the temperature. During this time, the solid gradually precipitated, and the stirring speed was controlled at 100-150 rpm. The mixture was filtered, the cake was washed with acetonitrile (600 mL x 2), and the cake was recovered.

[0043] At 15-25°C, acetonitrile (8L) was added to the reaction vessel under a weak nitrogen stream, followed by the addition of the above-mentioned cake. The reaction mixture was heated to 70-80°C, and after the solid was completely dissolved, stirring was continued for a further 0.5-1 hour. The stirring speed was controlled to 100-150 revolutions per minute, and gradient cooling was started to 15-25°C (cooling rate was 10-20°C / hour when temperature > 65°C, and 5-10°C / hour when temperature ≤ 65°C), and stirring was continued for 1-2 hours while maintaining the temperature. The mixture was filtered, the cake was washed with acetonitrile (600mL x 2), the cake was recovered, and after drying, compound 6 (1.03kg, purity: 98.5%, yield: 79.5%) was obtained. 1 H NMR (400MHz, DMSO_d6) δ: 11.17 (s, 1H), 10.13 (s, 1H), 9.79 (s, 1H), 9.01 (s, 1H), 8.59 (d, J=4.8 Hz, 1H), 8.30 (s, 1H), 8.21-8.15 (m, 2H), 8.14-8.06 (m, 1H), 8.01 (d, J=9.2 Hz, 1H), 7.79 (d, J=7.6 Hz, 1H), 7.73 (d, J=8.0 Hz, 1H), 7.70-7.66 (m, 1H), 7.16 (t, J=54.4 Hz, 1H), 5.09 (dd, J=4.4, 11.2 Hz, 1H), 4.28-4.15 (m, 1H), 3.86 (d, J=6.8 Hz, 1H), 3.35 (s, 4H), 3.29 (s, 2H), 2.91-2.80 (m, 1H), 2.74-2.57 (m, 6H), 2.43-2.36 (m, 1H), 2.22-2.14 (m, 2H), 2.10-2.01 (m, 3H), 1.97-1.87 (m, 2H), 1.84-1.70 (m, 2H), 1.68-1.57 (m, 1H), 1.51 (s, 9H), 1.24-0.98 (m, 3H), 0.44-0.37 (m, 2H), 0.27-0.20 (m, 2H).

[0044] Step 5: Synthesis of the compound of formula (I) At 15-25°C under a weak nitrogen stream, ethyl acetate hydrochloride solution (4M, 10L) was transferred to the reaction vessel, and compound 6 (1.01kg) was added in batches. After the addition was complete, the mixture was stirred for 20-40 minutes while maintaining the temperature. Next, the reaction mixture was gradually heated to 40-50°C and stirred for 1-3 hours while maintaining the temperature. After the reaction was complete, the reaction mixture was cooled to 20-25°C, filtered, the cake was washed with ethyl acetate (1L), and the cake was collected and dried under a nitrogen stream for 18-24 hours.

[0045] At 15-25°C, under the protection of a weak nitrogen stream, dimethyl sulfoxide (3.5 L) was transferred to the reactor, the dried cake was added and stirred for 0.5 hours until completely dissolved (dissolution was accelerated by heating to 40-50°C if necessary), filtered, and the cake was washed with dimethyl sulfoxide (120 mL) before the filtrate was collected.

[0046] At 15-25°C under a fine nitrogen stream, water (40 L) was transferred to the reaction vessel, imidazole (661.73 g) was added, and the mixture was stirred for 10 minutes to dissolve it. Then the above filtrate was added, and the addition was completed over approximately 0.5 hours. After that, the mixture was stirred for 2 hours while maintaining the temperature. The mixture was filtered, the cake was washed with water (1 L), and the cake was recovered. Water (10 L) was transferred to the reaction vessel, the cake was added, and the mixture was stirred for 0.5 hours. The mixture was filtered, the cake was sequentially washed with water (1 L x 2) and ethanol (1 L x 2), the cake was recovered, and after drying, the compound of formula (I) (873.9 g, purity: 98.4%, yield: 95.8%) was obtained. 1H NMR (400MHz, DMSO_d6) δ: 11.15 (s, 1H), 10.12 (s, 1H), 9.70 (s, 1H), 8.93 (s, 1H), 8.19 (t, J=4.4 Hz, 2H), 8.15 (d, J=5.6 Hz, 1H), 8.13-8.07 (m, 1H), 8.01 (d, J=9.2 Hz, 1H), 7.80 (d, J=7.2 Hz, 1H), 7.72 (t, J=8.0 Hz, 1H), 7.31-7.03 (m, 3H), 7.01 (dd, J=1.2, 5.2 Hz, 1H), 5.09 (dd, J=4.8, 11.6 Hz, 1H), 4.27-4.15 (m, 1H), 3.37-3.23 (m, 4H), 3.18 (t, J=6.4 Hz, 2H), 2.91-2.80 (m, 1H), 2.73-2.53 (m, 7H), 2.44-2.33 (m, 2H), 2.23-2.14 (m, 2H), 2.10-2.02 (m, 2H), 1.97-1.87 (m, 2H), 1.84-1.70 (m, 2H), 1.68-1.55 (m, 1H), 1.14-0.98 (m, 3H), 0.49-0.41 (m, 2H), 0.25-0.18 (m, 2H).

[0047] Biological Tests Experimental Example 1: Evaluation of target protein degradation activity in K562 IRAK4-HiBiT cells

[0048] Experimental Objective: In this experiment, we detected the degradative effect of the test compound on the target protein IRAK4 in K562 IRAK4-HiBiT cells.

[0049] 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

[0050] [Table 1]

[0051] [Table 2]

[0052] Experimental protocol: Day 1 1. Preparation of the compound (1) The powder of the test compound was dissolved in DMSO to make a stock concentration of 10 mM. Using a pipette, 9 μL of the 10 mM test compound was manually collected and added to the 1st and 13th columns of the LDV plate. (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 distributed to columns 1-11 and 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 n LDMSO was transferred as a 0% control (HC, ZPE).

[0053] 2. Cell seeding (1) Remove the cell culture medium, wash once with DPBS, digest the cells with trypsin, and count the number of cells, 2 × 10 -5 A cell suspension was prepared at a concentration of cells / mL. (2) Using MultiDrop Combi, 25 μL / well of the cell suspension was added to the experimental plate containing the test compound at a medium speed. (3) The cell-containing experimental plates were incubated at 37°C under 5% CO2 conditions for 16-18 hours.

[0054] Day 2 (1) Using MultiDrop Combi, 25 μL / well of the detection reagent (NanoGlo lysis solution + substrate + LgBit protein) was added to the assay plate at high speed and shaken for 10 minutes. (2) Centrifuge at 2000 rpm for 1 minute to remove air bubbles. (3) The plates were measured using the Envision and US Luminescence detection methods.

[0055] 3. Data Analysis The degradation rate (DR) of the test compound was calculated using the following formula: DR (%) = (RLU solvent control - RLU compound) / (RLU solvent control - RLU positive control) * 100%, where the solvent control refers to the blank control. After calculating the degradation rates of the compound at different concentrations in Excel, an inhibition curve plot was created using XLFit software, and the minimum degradation rate, maximum degradation rate, and DC were determined. 50 Related parameters, including [the specified parameter], were calculated. The test results are shown in Table 3.

[0056] [Table 3]

[0057] Conclusion: The compound of the present invention showed excellent target protein degradation activity in K562 IRAK4-HiBiT cells.

[0058] Experimental Example 2: Expression levels of IKZF1 and IKZF3 proteins in MM.1S cells as determined by In-Cell Western Analysis Experimental Objective: This experiment aimed to detect the effects of the test compound 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. Experimental materials: Cell line: MM.1S cell (derived from ATCC; product number CRL-2974) Negative control: 0.1% DMSO

[0059] [Table 4]

[0060] [Table 5]

[0061] 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, and a 3-fold dilution series was created starting from a concentration of 300 nM. Ten concentration gradients (including DMSO) were created by repeating this process three times, and incubated in an incubator for 24 hours. 3) After centrifugation, the cell supernatant was carefully removed, 150 μL of 4% paraformaldehyde fixative was added along the pore wall, taking care not to touch the bottom cells, and incubated at room temperature for 20 minutes. 4) The permeation solution was prepared: 0.5 mL of 10% Triton X-100 was added to 49.5 mL of PBS and mixed uniformly. 5) Add 200 μL of membrane rupture solution along the pore wall, taking care not to touch the cells at the bottom, and incubate on a shaker at room temperature for 5 minutes. 6) The washing process was repeated four times. 7) Add 150 μL of Licor INERCEPT blocking solution (intertent blacking buffer) along the pore wall, taking care not to touch the cells at the bottom, and incubate on a stirrer at room temperature for 1.5 hours. 8) Icarus (D6N9Y) rabbit monoclonal antibody b, Aeolus (D1C1E) rabbit monoclonal antibody, and GAPDH mouse monoclonal antibody (proteintech, 60004-1-Ig) were all diluted with antibody diluent at a ratio of 1:100. 9) 50 μL of mixed antibody was added to each well, creating three overlapping wells, and incubated overnight on a shaker at 4°C. 10) PBST (containing 0.1% Tween 20 in PBS) was prepared. 11) The primary antibody was removed, 200 μL of PBST was added along the well wall, taking care not to touch the cells at the bottom, and incubated for 5 minutes at room temperature on a shaker. 12) Washing was repeated 4 times. 13) A secondary antibody diluent was prepared: 0.2% Tween 20 was added to Licor INTERCEPT blocking buffer to a final concentration. 14) The fluorescently labeled secondary antibody was diluted (diluted 1:800) under light protection, and 0.5 μL each of IRDye 800CW and IRDye 680CW was added to 400 μL of the secondary antibody diluent (fluorescent secondary antibody corresponding to the primary antibody). 15) 50 μL of the diluted fluorescent secondary antibody was added to each well and incubated for 60 minutes at room temperature on a shaker in the dark. 16) The secondary antibody was removed, 200 μL of PBST was added along the well wall, taking care not to touch the cells at the bottom, and incubated for 5 minutes at room temperature in the dark on a shaker. 17) Washing was repeated 4 times, and immediately after washing, detection was performed at two-color wavelengths of 700 nm and 800 nm using an Odyssey Gel Imaging System.

[0062] 3. Data analysis Using GraphPad Prism 6 software, after inputting the inhibition rate data, the curve was fitted to calculate the DC 50 value. Protein inhibition rate = (1 - RLs / RLv) * 100% RR (Raw Ratio) = 700 nm / 800 nm RLs = RR of cells treated with the sample RLv = RR of cells treated with the solvent The test results are shown in Table 6.

[0063]

Table 6

[0064] Conclusion: The compounds of the present invention showed excellent degradative activity against IKZF1 and IKZF3 proteins in MM.1S cells.

[0065] Example 3: Evaluation of antiproliferative activity in lymphoma cell lines OCI-LY10 and TMD-8 Experimental Objective: This experiment investigated the cell proliferation inhibitory effect of the test compound in diffuse large B-cell lymphoma cell lines OCI-LY10 and TMD-8. Experimental materials:

[0066] [Table 7]

[0067] [Table 8]

[0068] 1. Porous plate Greiner CELLSTAR® 96-well plate, flat-bottomed white plate (with lid, transparent bottom), #3610

[0069] 2. Reagents and equipment used in cell activity testing (1) Promega CellTiter-Glo Luminescence Cell Activity Detection Kit (Promega-G7573). (2) 2104 EnVision® Plate Reader, PerkinElmer.

[0070] 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 harvested for seeding onto plates.

[0071] 2. Cell seeding (1) Cells were stained with trypan blue and the number of viable cells was counted. (2) The cell concentration was adjusted to an appropriate level.

[0072] [Table 9]

[0073] (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.

[0074] 3. Preparation of compound stock plates A stock solution storage plate with a starting concentration 1000 times was prepared, and the compound was gradually diluted from the highest to the lowest concentration using DMSO. The solution was prepared each time it was used.

[0075] 4. Preparation of 1000x compound working solution and cell treatment with the compound (1) Preparation of working solution at 5 times the initial concentration of the compound: When diluting the compound 3 times, 30 μL of the compound was taken from the stock solution at 1000 times the initial concentration, then 20 μL of DMSO was added to several wells, and 10 μL was taken from the previous concentration and transferred to the next concentration sequentially. When diluting the compound 5 times, 30 μL of the compound was taken from the stock solution at 1000 times the initial concentration, then 24 μL of DMSO was added to several wells, and 6 μL was taken from the previous concentration and transferred to the next concentration sequentially. 20 μL of DMSO was added as a solvent control. The 1000-fold diluted compound was diluted 200 times using culture medium, that is, 1 μL of the 1000-fold diluted compound was added to 199 μL of culture medium and mixed uniformly using a multichannel pipette. (2) Addition of drug: 25 μL of the compound diluted 5 times 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, co-cultured for 5 days after drug addition) and TMD-8 (3-fold or 5-fold dilution, co-cultured for 5 days after drug addition) were cultured.

[0076] 5. Cell activity measurement using the CellTiter-Glo luminescence method The following steps were performed according to the instructions for the Promega CellTiter-Glo luminescence cell activity measurement kit (Promega-G7573). (1) Dissolve the CellTiter-Glo buffer solution and leave it at room temperature. (2) The CellTiter-Glo substrate was left at room temperature. (3) CellTiter-Glo buffer was added to 10 mL of CellTiter-Glo substrate to dissolve the substrate and prepare CellTiter-Glo working solution. (4) The mixture was slowly shaken in a vortex motion until it was completely dissolved. (5) Remove the cell culture plate and leave it for 30 minutes to equilibrate to room temperature. (6) 60 μL of CellTiter-Glo working solution (equivalent to half the volume of cell culture medium in each well) 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)2104 An illumination signal was detected by the EnVision plate reader.

[0077] 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%. The inhibition rates of the compound at different concentrations were calculated in Excel, and then inhibition curves were created using GraphPad Prism software to determine the minimum inhibition rate, maximum inhibition rate, and IC. 50 Related parameters, including [specific parameter], were calculated. The test results are shown in Table 10.

[0078] [Table 10]

[0079] Conclusion: The compounds of the present invention showed excellent cell proliferation inhibitory effects in both the lymphoma cell lines OCI-LY10 and TMD-8.

[0080] Example 4: Evaluation of antiproliferative activity in the lymphoma cell line SU-DHL-2 Experimental Objective: This experiment aimed to detect the cell proliferation inhibitory effect of the test compound on the lymphoma cell line SU-DHL-2.

[0081] [Table 11]

[0082] [Table 12]

[0083] 1. Porous plate Greiner CELLSTAR 384-well plate, flat-bottomed chalkboard (with lid), #781090

[0084] 2. Reagents and equipment used in cell activity testing (1) Promega CellTiter-Glo Luminescence Cell Activity Detection Reagent Kit (Promega-G7573). (2) 2104 EnVision® Plate Reader, PerkinElmer.

[0085] 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 harvested for seeding onto plates.

[0086] 2. Cell seeding (1) Cells were stained with trypan blue and the number of viable cells was counted. (2) The cell concentration was adjusted to an appropriate level.

[0087] [Table 13]

[0088] (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.

[0089] 3. Preparation of compound stock plates The drug was added using an Echo655 instrument. The amount of drug added was 50 nL, and the final concentration of DMSO was 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.

[0090] 4. Cell activity measurement using the CellTiter-Glo luminescence method The following steps were performed according to the instructions for the Promega CellTiter-Glo luminescence cell activity measurement kit (Promega-G7573). (1) Dissolve the CellTiter-Glo buffer solution and leave it at room temperature. (2) The CellTiter-Glo substrate was left at room temperature. (3) CellTiter-Glo buffer solution was added to the CellTiter-Glo substrate vial and the substrate was dissolved to prepare the CellTiter-Glo working solution. (4) The mixture was slowly shaken in a vortex motion until it was completely dissolved. (5) Remove the cell culture plate and leave it for 30 minutes to equilibrate to room temperature. (6) 25 μL of CellTiter-Glo working solution (equivalent to half the volume of cell culture medium in each well) 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) 2104 An illumination signal was detected by the EnVision plate reader.

[0091] 5. Data Analysis The inhibition rate (IR) of the test compound was calculated using the following formula: IR (%) = (1 - (RLU compound - RLU blank control) / (RLU solvent control - RLU blank control)) * 100%. The inhibition rates of the compound at different concentrations were calculated in Excel, and inhibition curves were created using GraphPad Prism software to determine the minimum inhibition rate, maximum inhibition rate, and IC50. 50 Related parameters, including [specific parameter], were calculated.

[0092] [Table 14]

[0093] Conclusion: The compound of the present invention showed excellent cell proliferation inhibitory activity in the lymphoma cell line SU-DHL-2.

[0094] Experimental Example 5: Evaluation of pharmacokinetics of compounds in mice Experimental objective: In this study, C57BL / 6 or C57 male mice were selected as experimental animals, and the plasma drug concentrations of the test compound administered intravenously or orally to the mice were quantitatively measured at different time points using LC / MS / MS, thereby evaluating the pharmacokinetic properties of the compound in the mouse body.

[0095] Experimental materials: C57BL / 6 or C57 mouse (male, 20-30g, 7-10 weeks old, Beijing Vital River Laboratory Animal Technology Co.,Ltd.).

[0096] A clear solution of the test compound was injected into the tail vein of C57BL / 6 mice (fasted) using a solvent (10% DMSO / 10% solutol / 80% H2O), or administered orally to C57 mice (feeding). For intravenous injection, 50 μL of blood was collected by cheek puncture at 0 h (before administration) and at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 h after administration, and collected in an anticoagulant tube containing heparin sodium. After thoroughly vortex-mixing the mixture, it was centrifuged at 6000 g for 3 minutes at 2-8°C. For oral administration, blood was collected by cheek puncture at 0 h (before administration) and at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 h after administration, collected in an anticoagulant tube containing heparin sodium, and after thoroughly vortex-mixing the mixture, it was centrifuged at 6000 g for 3 minutes at 2-8°C. Blood 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 8.2.0 pharmacokinetic software. The test results are shown in Table 15.

[0097] [Table 15]

[0098] Conclusion: The present invention's compound receives a plasma exposure (AUC) upon oral administration. 0-inf The ) was high. In rodent mice, its pharmacokinetic properties were excellent.

[0099] Example 7: Pharmacokinetic evaluation of the compound in beagle dogs Experimental objective: In this study, male beagle dogs were selected as experimental animals, and the plasma concentrations of the test compound were quantitatively measured at different time points after intravenous injection or oral administration using LC / MS / MS. This allowed for the evaluation of the pharmacokinetic properties of the compound in beagle dogs.

[0100] Experimental materials: Beagle dog (male, 7~10kg, Beijing Masi Biological Technology Co., Ltd.).

[0101] Experimental procedure: A clear solution of the test compound was slowly injected into the peripheral veins of Beagle dogs after feeding (solvent: 5% DMSO / 10% Solutol / 85% H2O), or administered orally into the Beagle dogs after feeding. For intravenous administration, 0.5 mL of blood was collected from the peripheral vein at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours after 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. For oral administration, 0.5 mL of blood was collected from the peripheral vein at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours after 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-compartmental model linear log-trapezoidal method with Phoenix WinNonlin 6.3 pharmacokinetic software. The test results are shown in Table 16.

[0102] [Table 16]

[0103] Conclusion: The present invention's compound receives a plasma exposure (AUC) upon oral administration. 0-inf The ) was high. Its pharmacokinetic properties were excellent in non-rodent animals such as beagle dogs.

[0104] Experimental Example 8: In vivo pharmacokinetic studies of compounds 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.

[0105] 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. (Product number: CBP60558).

[0106] [Table 17]

[0107] [Table 18]

[0108] Model construction: OCI-LY10 cells were cultured in IMDM medium containing 20% ​​FBS and maintained in a 37°C saturated humidity incubator under 5% CO2 conditions. Logarithmic OCI-LY10 cells were harvested, resuspended in IMDM basal medium, and Matrigel was added in a 1:1 ratio to increase the cell concentration to 4 × 10⁶. 7 The concentration was adjusted to 4 × 10 / mL. Under sterile conditions, 0.1 mL of the cell suspension was inoculated subcutaneously into the right dorsal region of SCID mice, with an inoculation concentration of 4 × 10⁻¹⁴. 6 The value was / 0.1 mL / mouse.

[0109] Experimental protocol: In pharmacodynamic experiments, when the tumor reached a certain size, animals with tumor volumes that were too large or too small, or with irregular shapes, were excluded, and the tumor volume was between 167.65 and 231.29 mm. 3 Individuals were selected and randomly divided into groups based on tumor volume, with 6 mice in each group, and the average tumor volume was approximately 201.15 mm². 3 The day of group assignment was recorded as Day 0, and medication was started according to the animal's body weight. The pharmacodynamic experiment lasted 28 days, with oral administration once daily at 24-hour intervals. During the experiment, the animals' body weight and tumor size were measured twice a week. Clinical symptoms were observed and recorded daily.

[0110] The test compounds were administered at doses of 10 mg / kg, 30 mg / kg, and 100 mg / kg, respectively, using 10% DMSO / 10% Solutol / 80% H2O as the solvent. The formula for calculating tumor volume (TV) was 1 / 2 × a × b 2 Here, a and b represent the major and minor diameters of the tumor at the time of measurement. 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 : Mean RTV of the treatment group; C RTV :This is the 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 administration (i.e., 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.

[0111] 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.

[0112] Experimental results: The test results are shown in Tables 19 and 20.

[0113] [Table 19]

[0114] [Table 20]

[0115] Conclusion: The compound of the present invention showed significant tumor suppressive activity in a human B-cell lymphoma OCI-LY10 cell SCID mouse xenograft tumor model, and the effect was dose-dependent.

[0116] Experimental Example 9: In vivo pharmacokinetic studies of compounds 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 a SU-DHL-2 subcutaneous xenograft tumor CB17 SCID mouse model.

[0117] Experimental materials: 1. Experimental animal: CB17 SCID mouse, female, 6~8 weeks old, weight 18~22g. Beijing Wetong Lihua Experimental Animal Technology Co., Ltd. 2. Cell line: Human lymphoma SU-DHL-2 cells (product number: ATCC-CRL-2956).

[0118] [Table 21]

[0119] [Table 22]

[0120] Model construction: 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 in a 37°C, 5% CO2 incubator. Regular subculturing was performed twice a week. When the cell saturation reached 80% to 90% and the predetermined cell count was reached, the cells were harvested, counted, and seeded.

[0121] 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 average tumor volume was approximately 139 mm². 3 Grouping and medication administration were initiated once the target was reached. The day of grouping was recorded as Day 0, and medication was started according to the animal's body weight.

[0122] Experimental protocol: In the pharmacodynamic experiments, 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.

[0123] The dosages of the test compounds were 10 mg / kg, 30 mg / kg, and 100 mg / kg, respectively, and the solvent used was 10% DMSO / 10% Solutol / 80% water. The formula for calculating tumor volume (TV) was 1 / 2 × a × b 2 Here, a and b represent the major and minor diameters of the tumor at the time of measurement. 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 : Mean RTV of the treatment group; C RTV:This is the 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 administration (i.e., 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.

[0124] Data analysis: Statistical analysis was performed using SPSS software based on RTV data at the end of the study. T-tests were used for comparisons between two groups, and one-way ANOVA was used for comparisons between three or more groups. Tukey's method was used when variances were homogeneous (no significant difference in F-scores), and Games-Howell's method was used when variances were heterogeneous (significant difference in F-scores). A p-value of <0.05 was considered statistically significant.

[0125] Experimental results: The test results are shown in Tables 23 and 24.

[0126] [Table 23]

[0127] [Table 24]

[0128] Conclusion: The compounds of the present invention showed significant tumor-suppressing effects in a CB17 SCID mouse model of human lymphoma SU-DHL-2 cell subcutaneous xenograft tumor.

[0129] Experimental Example 10: In vivo pharmacodynamic studies in a BALB / c nude mouse model of human diffuse large B-cell lymphoma (TMD-8 cell subcutaneous xenograft tumor). Pharmacological research Experimental objective: In this study, the antitumor effect of compound (I) was evaluated using a human diffuse large B-cell lymphoma (TMD-8) subcutaneous xenograft tumor BALB / c nude mouse model.

[0130] 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.).

[0131] [Table 25]

[0132] [Table 26]

[0133] Model construction: Cell culture: Standard cell culture was performed using RPMI-1640 medium containing 10% fetal bovine serum under 5% CO2 and 37°C conditions. Passaging was performed according to the cell proliferation status, with a passaging ratio of 1:3 to 1:4.

[0134] Tumor cell inoculation and grouping: After harvesting TMD-8 cells in the logarithmic growth phase, cell counting was performed, and the cells were resuspended in a mixture of 50% serum-free RPMI-1640 medium and 50% Matrigel to a cell concentration of 4.0 × 10⁶. 7 Prepare the cell suspension to the required concentration (0.8 × 10⁴ cells / mL), store it in an ice box, and aspirate it with a 1 mL syringe. Then, inject 200 μL (0.8 × 10⁴) subcutaneously into the right forelimb axilla of a nude mouse. 7 A TMD-8 transplanted tumor model was established by inoculating cells (per animal). The average tumor volume was approximately 160 mm². 3 Group division and administration were initiated when the target was reached. The day of group division was recorded as day 1 (D1), and medication was started according to the animal's body weight.

[0135] Experimental protocol: In the pharmacodynamic experiments, 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.

[0136] The dosages of the test compounds were 10 mg / kg, 30 mg / kg, and 100 mg / kg, respectively, and the solvent used was 10% DMSO / 10% Solutol / 80% water. The formula for calculating tumor volume (TV) was 1 / 2 × a × b 2 Here, a and b represent the major and minor diameters of the tumor at the time of measurement. 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 as follows: T / C% = T RTV / C RTV ×100%(T RTV : Mean 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 using the formula RTV = Vt / V0, where V0 is the tumor volume measured at the time of group administration (i.e., day 1), and Vt is the tumor volume at a single measurement point. RTV and C RTV The data used was from the same day.

[0137] Data analysis: The test data were calculated using Microsoft Office Excel 2007 software, and relevant statistical analysis was performed. Unless otherwise specified, the data are expressed as mean ± standard error (Mean ± SE), and t-tests were used for comparisons between two groups.

[0138] Experimental results: The test results are shown in Table 27.

[0139] [Table 27]

[0140] Conclusion: The compounds of the present invention showed remarkable tumor-suppressing effects in a BALB / c nude mouse model of human diffuse large B-cell lymphoma (TMD-8 cell subcutaneous xenograft tumor).

Claims

1. A method for preparing compound 6 using compound 4 and compound 5, wherein the reaction steps are as follows. 【Chemistry 1】

2. A method for preparing the compound of formula (I) using compound 1, wherein the reaction steps are as follows. 【Chemistry 2】

3. The equivalent ratio of compound 4 to compound 5 is 0.8:1 to 1:1; The aforementioned base 2 is selected from TEA and DIEA; The method according to claim 1, wherein the solvent 3 is selected from acetonitrile. 【Transformation 3】

4. The method according to claim 3, wherein the reaction temperature is 70 to 80°C.

5. The method according to claim 3, wherein the equivalent ratio of compound 4 to compound 5 is 0.85:1 to 0.95:1, preferably 0.88:

1.

6. The condensing agent is selected from HATU, benzenesulfonyl chloride, or p-toluenesulfonyl chloride; The aforementioned base 1 is selected from TEA or DIEA. The solvent 1 is selected from DMF, DMSO, or NMP. The acidic system 1 is selected from ethyl acetate hydrochloride solution or TFA. The acidic system 2 is selected from ethyl acetate hydrochloride solution or TFA. The aforementioned base 2 is selected from TEA, DIEA, or sodium bicarbonate. The solvent 3 is selected from acetonitrile, The iodinating reagent is PPh ３ / Im / I ２ Selected from, The method according to claim 2, wherein the solvent 2 is selected from DCM. 【Chemistry 4】

7. The method according to claim 6, wherein the coupling agent is selected from HATU, the acid system 1 is selected from TFA, the acid system 2 is selected from ethyl acetate hydrochloride solution, the concentration of the ethyl acetate hydrochloride solution is 4 mol / L, the base 1 is selected from DIEA, and the base 2 is selected from DIEA.

8. The method according to claim 6, wherein the reaction temperature of step 5 is 40 to 50°C.

9. The method according to claim 8, wherein the reaction time of step 5 is 0.5 to 3 hours.

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