Hydroxamic acid-based dual-target compound derivatives and uses thereof

Abstract

The application discloses a kind of hydroxamic acid double-target compound derivatives and application thereof.The double-target derivative designed in the application has obvious inhibitory activity to WDR5 protein and HDAC protein, can be used as WDR5, HDAC double-target inhibitor, is used for treating diseases related to WDR5, HDAC, aims at improving the therapeutic effect of existing HDAC inhibitor.The small molecule shows good activity in various experiments, and is expected to be developed into specific antitumor drugs.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry technology, specifically relating to a class of hydroxamic acid dual-target compound derivatives and their applications. Background Technology

[0002] WD40 repeat domain proteins are widely expressed in the human proteome. WD40 proteins play important roles in a wide range of biological activities, including signal transduction, transcriptional regulation, DNA damage sensing and repair, apoptosis, cell growth and division, epigenetic regulation, immune regulation, and the occurrence and maintenance of various diseases. WDR5, a member of the WD40 protein family, was first discovered in mouse chondrocytes. One of the most important functions of WDR5 is as an epigenetic "reader" involved in gene expression regulation. Numerous WDR5 interacting partners and their unique roles in epigenetics endow WDR5 with a variety of biological functions, including reproduction, development, metabolism, immune and inflammatory responses, and neural and humoral regulation. Overexpression of WDR5 is associated with the occurrence and development of various cancers, such as prostate cancer, breast cancer, leukemia, liver cancer, pancreatic cancer, cholangiocarcinoma, colon cancer, lung cancer, glioblastoma, ovarian cancer, cervical cancer, and gastric cancer, and is also associated with poor clinical prognosis.

[0003] Studies have shown that rearrangements of the MLL1 gene lead to a distinct class of leukemias characterized by adverse outcomes, including approximately 70% of infantile acute lymphoblastic leukemia (ALL) cases and 70% of adult acute myeloid leukemia (AML) cases. The MLL1 protein, encoded by the MLL1 gene, plays a crucial role in the development of both ALL and AML. To enhance its histone methyltransferase activity, the MLL1 protein relies on the formation of its core catalytic complex, including WDR5, RbBP5, Ash2L, and DPY30 via motif (WIN) sites. Interfering with the MLL1-WDR5 protein-protein interaction has emerged as a promising and effective strategy for treating MLL rearrangement leukemia.

[0004] Histone deacetylases (HDACs) are a class of epigenetic regulators that play a crucial role in various tumorigenesis, neurological disorders, and immune dysregulations. HDACs are also involved in various biological processes in cancer cells, including apoptosis, cell growth, and cell differentiation. Furthermore, many non-histone proteins are also regulated by HDACs, such as nuclear transporters, α-tubulin in microtubules, and the tumor suppressor p53. Dysregulation of HDACs is a significant factor in tumorigenesis. Therefore, HDACs are an effective target for developing HDAC inhibitors (HDACi) for the treatment of various solid and hematologic cancers, such as liver cancer, pancreatic cancer, and acute myeloid leukemia. To date, numerous HDAC inhibitors have been developed, demonstrating strong efficacy in preclinical and clinical studies.

[0005] Therefore, it is necessary to provide a small molecule inhibitor that can simultaneously inhibit WDR5 and HDAC, thus providing an effective means to combat the anti-proliferative activity of tumor cells. Summary of the Invention

[0006] To address the aforementioned technical problems in the existing technology, this invention provides a class of WDR5-HDAC dual-target inhibitors and their applications.

[0007] The technical solution of this invention is as follows:

[0008] The first object of this invention is to provide a compound as shown in Formula I or a pharmaceutically acceptable salt thereof:

[0009]

[0010] in:

[0011] R 1 It is selected from C1-C7 alkyl or C1-C7 haloalkyl, naphthyl, pyridyl, piperazinyl, piperidinyl, phenyl or substituted phenyl, wherein the substituent of the substituted phenyl is C1-C7 alkyl, C1-C7 haloalkyl, cyano, hydroxyl, mercapto, halogen, nitro, amino, methoxy or trifluoromethyl;

[0012] R 2 -NH-OH

[0013] L 1 L 2 The group is independently selected from carbonyl, sulfonyl, amino, imino, aminoide, ether, thioether, methylene or methylene;

[0014] Furthermore, the L 1 L 2 The group is independently selected from carbonyl, imino, aminoide, sulfonyl, methylene, or methylene.

[0015] Furthermore, the pharmaceutically acceptable salts include acid addition salts formed by compounds of general formula I with the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, citric acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, maleic acid or succinic acid, fumaric acid, salicylic acid, phenylacetic acid, and mandelic acid; and also include basic salts formed by compounds of general formula I with inorganic bases: sodium salts, potassium salts, lithium salts, calcium salts, zinc salts, magnesium salts, meglumine salts, aminobutanetriol salts, diethylamine salts, or ethanolamine salts.

[0016] Furthermore, the compound represented by Formula I has one of the following structural formulas:

[0017]

[0018]

[0019] A second object of the present invention is to provide a pharmaceutical composition comprising the aforementioned compound of Formula I or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

[0020] The pharmaceutical compositions of the present invention can be administered in various known ways, such as orally, by injection, or by inhalation spray. The pharmaceutical compositions of the present invention can be administered alone or in combination with other drugs. Oral compositions can be any orally acceptable dosage form, including, but not limited to, tablets, capsules, suspensions, and solutions. Commonly used pharmaceutically acceptable carriers or excipients include diluents, surfactants, lubricants, antioxidants, binders, colorants, emulsifiers, etc. Sterile injectable compositions can be formulated using suitable dispersants or wetting agents and suspending agents according to techniques known in the art. Pharmaceutically acceptable carriers and solvents that can be used include water, sodium chloride solution, etc.

[0021] The actual dose level of the active ingredient in the pharmaceutical composition of the present invention can be varied to obtain an amount of active ingredient that is effective in achieving the desired therapeutic response for a particular patient, composition, and route of administration, and is non-toxic to the patient. The selected dose level depends on a variety of factors, including the activity of the specific compound of the present invention or its salt used, the route of administration, the time of administration, the excretion rate of the specific composition used, the duration of treatment, other drugs, compounds, and / or materials used in combination with the specific composition used, the age, sex, weight, general health condition, and medical history of the patient being treated, and similar factors known in the medical field.

[0022] A third object of the present invention is to provide the use of the compound shown in Formula I or a pharmaceutically acceptable salt thereof in the preparation of a WDR5-HDAC dual-target small molecule inhibitor.

[0023] A fourth object of the present invention is to provide the use of the compound of Formula I or a pharmaceutically acceptable salt thereof or the aforementioned pharmaceutical composition in the preparation of a therapeutic remedy for diseases associated with WDR5 and / or HDAC dysfunction.

[0024] Furthermore, the diseases associated with WDR5 and / or HDAC dysfunction are leukemia, pancreatic cancer, bile duct cancer, colon cancer, lung cancer, glioblastoma, ovarian cancer, cervical cancer, breast cancer, or gastric cancer.

[0025] Beneficial effects:

[0026] The compound of the present invention, as shown in Formula I, has significant inhibitory activity against WDR5 protein and HDAC, and can be used as a dual-target small molecule inhibitor of WDR5-HDAC for the treatment or enhancement of diseases related to WDR5 or HDAC. This small molecule has shown good activity in various experiments and is expected to be developed into a specific anti-tumor drug. Attached Figure Description

[0027] Figure 1 The in vitro antiproliferative activity of compounds 9 and 16 was assessed, with SAHA and DDO-2074 serving as positive controls.

[0028] (A) Compounds 9, 16, DDO-2074, and SAHA inhibited the proliferation of leukemia cells;

[0029] (B) Compounds 9 and 16, as well as the combination of DDO-2074 and SAHA, inhibited the proliferation of solid cancer cells;

[0030] The data is the average of the three experiments ± SD. Detailed Implementation

[0031] The preparation method of the compound of general formula I of the present invention is described below with reference to specific embodiments, but these specific methods do not constitute any limitation on the present invention. The compounds of the present invention can also be conveniently prepared by optionally combining various synthetic methods described in this specification or known in the art, and such combinations can be easily performed by those skilled in the art.

[0032] The starting materials and reaction reagents used in the specific embodiments of this invention are all commercially available.

[0033] Experimental methods in the embodiments of this invention that do not specify specific conditions are generally performed under conventional conditions or as recommended by the raw material or product manufacturer. Reagents whose specific source is not specified are commercially available conventional reagents.

[0034] The structure of the compound was determined using nuclear magnetic resonance (NMR). 1 H NMR and 13 C10 NMR spectra were determined using a Bruker AV-300 NMR spectrometer. The solvent used for the determination was deuterated dimethyl sulfoxide (DMSO-d6), and the internal standard was tetramethylsilane (TMS).

[0035] Chemical reactions were detected using a 0.25 mm GF254 thin-layer chromatography silica gel plate and observed using a ZF7 three-way ultraviolet analyzer.

[0036] Unless otherwise specified in the embodiments, the reaction is carried out in an air atmosphere.

[0037] Unless otherwise specified in the examples, the reaction temperature is room temperature, ranging from 20°C to 30°C.

[0038] Example 1: N 1 -(5-amino-2-chloro-4-fluoro-3-methylbenzamido)-4-(4-methylpiperazin-1-yl)-[1,1'-biphenyl]-4-yl)-N 5 -Hydroxyglutaramide (Compound 1)

[0039]

[0040] Step 1: Synthesize compounds 1-2

[0041] Compound 1-1 (10.0 g, 69.2 mmol) was dissolved in DCM (50 mL), followed by the addition of anhydrous aluminum trichloride (18.4 g, 138.3 mmol) and acetyl chloride (5.4 g, 78.5 mmol). After the addition was complete, the reaction mixture was stirred at room temperature for 2 h. After the reaction was complete, the reaction mixture was poured into ice water, and the pH was adjusted to 4 with HCl (2 M). The reaction mixture was then extracted with dichloromethane, and the organic phase was collected and concentrated to obtain the crude product. The crude product was purified by chromatography (petroleum ether: ethyl acetate = 200:1) to obtain compound 1-2 (6 g, 46.5%). 1 H NMR (300MHz, CDCl3) δ7.37 (dd, J=8.6, 6.0Hz, 1H), 7.01 (t, J=8.6Hz, 1H), 2.62 (s, 3H), 2.34 (d, J=2.4Hz, 3H). m / z (ESI-MS): 187.03[M+H]+.

[0042] Step 2: Synthesize compounds 1-3

[0043] Bromine (8.3 g, 2.7 mL, 51.8 mmol) was added dropwise to a NaOH (6 mol / L) solution, and the temperature was maintained at 0 °C with continuous stirring for 10 minutes. Compound 1-2 (3.22 g, 17.2 mmol) was dissolved in 1,4-dioxane (33 mL) and then added dropwise to the above mixture. After the reaction was complete, the reaction solution was poured into water and extracted with CHCl3 to remove residual bromine. After acidifying the aqueous solution with HCl (3 mmol / L) and washing with water and diethyl ether, a white precipitate was filtered off to obtain a white solid compound 1-3 (2.07 g, 63.7%). 1 H NMR (300MHz, DMSO-d6) δ13.42(s,1H),7.70(dd,J=8.6,6.2Hz,1H),7.30(t,J=8.8Hz,1H),2.31(d,J=2.4Hz,3H).m / z(ESI-MS):186.99[MH]- .mp194.2-195.0℃.

[0044] Step 3: Synthesize compounds 1-4

[0045] Concentrated nitric acid (4.34 g, 2.90 mL, 68.94 mmol) was added dropwise to 1–3 (10 g, 53.03 mmol) of concentrated sulfuric acid solution (200 mL) at 0 °C. The reaction mixture was then allowed to react at room temperature for 4 hours. A white solid precipitated while the mixture was poured into ice water. The filter cake was then washed with water (200 mL) and dried to obtain a white solid compound (11.2 g, 90.4%). 1 H NMR(300MHz, DMSO-d6)δ8.40(d,J=8.0Hz,1H),2.42(d,J=3.0Hz,3H).m / z(ESI-MS):231.98[MH] - .mp143-145℃.

[0046] Step 4: Synthesize compounds 1-6

[0047] Compounds 1-5 (10 g, 45.46 mmol) were dissolved in acetonitrile (200 mL), and then N,N-diisopropylethylamine (17.62 g, 23.75 mL, 136.37 mmol) and 1-methylpiperazine (5.92 g, 6.55 mL, 59.09 mmol) were added dropwise to the mixture. The reaction was monitored by TLC, and after the reaction was complete, the solvent was removed by vacuum distillation to give a red oily crude product. The crude product was then dissolved in ethyl acetate, washed with water, and dried over anhydrous Na₂SO₄ to give red oil compound 1-6 (12 g, 87.8%). 1 HNMR (300MHz, CDCl3) δ7.95 (d, J = 2.4Hz, 1H), 7.62 (dd, J = 8.8, 2.4Hz, 1H), 7.09 (d, J = 8. 8Hz,1H),3.19-3.09(m,4H),2.69-2.59(m,4H),2.42(s,3H).m / z(ESI-MS):300.02[M+H] + .

[0048] Step 5: Synthesize compounds 1-7

[0049] SnCl₂·2H₂O (37.59 g, 166.58 mmol) was added to an ethyl acetate solution (400 mL) of compounds 1-6 (10 g, 33.32 mmol) at room temperature. The mixture was then refluxed at 80 °C for 6 h. The mixture was neutralized by adding saturated sodium bicarbonate solution. The residue was then washed with ethyl acetate (5 × 100), the organic phase was dried over Na₂SO₄, and the crude product was concentrated. Column chromatography (CH₂Cl₂ / MeOH = 80:1) was used to purify compounds 1-7 (8 g, 88.9%). 1 H NMR (300MHz, CDCl3) δ6.90-6.88 (m, 2H), 6.88 (d, J = 1.7Hz, 1H), 4.06 (s, 2H), 2.95(t,J=4.8Hz,4H),2.61(s,4H),2.41(s,3H).m / z(ESI-MS):270.05[M+H] + .mp165.6-166.3℃.

[0050] Step 6: Synthesize compounds 1-8

[0051] Compounds 1-7 (3 g, 11.10 mmol) were dissolved in anhydrous dichloromethane (150 mL), stirred at 0 °C for 10 min, and then DIPEA (4.31 g, 5.80 mL, 33.31 mmol) was added. Then, a dichloromethane solution (4.20 g, 16.66 mmol) of the acyl chloride intermediates 1-4 prepared in the above reaction was added dropwise, and the reaction was maintained at room temperature for 4 h. When the reaction was complete, the reaction mixture was diluted with dichloromethane (100 mL). The mixture was then washed with water (50 mL), saturated sodium bicarbonate solution (2 × 40 mL), and then with saturated sodium chloride solution (50 mL). The organic phase was separated, dried (Na₂SO₄), concentrated, and purified by column chromatography to obtain intermediate 1-8 (3 g, 55.6%). 1 H NMR (300MHz, DMSO-d6) δ9.85(s,1H),8.37(d,J=7.8Hz,1H),8.14(d,J=2.4Hz,1H),7.38(dd,J=8.6,2.4Hz,1H),7.16 (d,J=8.6Hz,1H),2.89(t,J=4.6Hz,4H),2.48(s,4H),2.44(d,J=2.8Hz,3H),2.22(s,3H).m / z(ESI-MS):485.03[M+H] + .mp177.3-179.0℃.

[0052] Step 7: Synthesize compounds 1-9

[0053] SnCl₂·2H₂O (37.59 g, 166.58 mmol) was added to an ethyl acetate (400 mL) solution of compounds 1-8 (5 g, 10.29 mmol) at room temperature. The mixture was then refluxed continuously with stirring for 6 h. After the reaction was complete, the reaction solution was neutralized by adding saturated sodium bicarbonate solution. The solution was then diluted with ethyl acetate (5 × 100 mL), filtered, and the filtrate was washed. The organic phase was dried over Na₂SO₄ and concentrated to obtain the crude product. Purification by silica gel column chromatography (CH₂Cl₂ / MeOH = 50:1) yielded compounds 1-9 (2 g, 42.6%). 1 H NMR (300MHz, CDCl3) δ9.29 (s, 1H), 8.82 (d, J = 2.3Hz, 1H), 7.27 (dd, J = 8.5, 2.3Hz, 1H), 7.15 (d, J = 8.5Hz, 1H), 7.08 (d, J = 9. 2Hz,1H),3.93(s,2H),2.96(d,J=4.8Hz,4H),2.64(s,4H),2.43(s,3H),2.40(d,J=2.7Hz,3H).m / z(ESI-MS):455.05[M+H] + .mp207.4-208.1℃.

[0054] Step 8: Synthesize compounds 1-10

[0055] Boric acid (124.99 mg, 0.570 mmol) and intermediate 1-9 (200 mg, 0.439 mmol) were dissolved in DMF (10 mL). Cs₂CO₃ (285.96 mg, 0.878 mmol) and a catalytic amount of Pd(PPh₃)₂Cl₂ (9.24 mg, 0.0131 mmol) were added to the mixture. The reaction was heated to 100 °C for 10 h under nitrogen protection. After the reaction was complete, the mixture was filtered to remove the catalyst and Cs₂CO₃. The organic phase was separated and concentrated to obtain the crude product. Then, intermediate 1-10 (110 mg, 39%) was purified by column chromatography using 1.25%–2% DCM / MeOH. 1H NMR (300MHz, DMSO-d6) δ9.28(s,1H),8.28(d,J=2.1Hz,1H),7.35-7.28(m,3H),7.25(d,J=8.3Hz,1H),6.90(d,J=9.2Hz,1H),6.66(d,J=8 .2Hz,2H),5.55(s,2H),5.27(s,2H),2.90(t,J=4.8Hz,4H),2.57(s,4H),2.30(s,3H),2.27(d,J=2.7Hz,3H).m / z(ESI-MS):468.18[M+H] + .

[0056] Step 9: Synthesize compounds 1-11

[0057] 0.555 mmol of 6-methoxy-6-oxohexanoic acid was dissolved in 2 mL of DMF at room temperature. Then, 1 mL of a DMF solution containing 0.444 mg (0.854 mmol) of PyBOP was added dropwise, followed by DIPEA (165.71 mg, 0.223 mL, 1.28 mmol). After stirring the solution for 30 minutes, intermediate 1-10 (200 mg, 0.427 mmol) was added. The resulting mixture was reacted at room temperature for 8–10 h. After the reaction was complete, the solution was poured into water, and the crude product was extracted from the water with ethyl acetate. The crude product was purified by silica gel column chromatography (CH₂Cl₂ / MeOH = 30:1) to give 1-11 (yield: 34.1%). 1 H NMR (300MHz, DMSO-d6) δ10.01(s,1H),9.31(s,1H),8.33(s,1H),7.69(d,J=8.3Hz,2H ),7.55(d,J=8.4Hz,2H),7.43-7.39(m,1H),7.32-7.25(m,1H),6.89(d,J=9.1Hz,1H), 5.50(s,2H),3.61(s,3H),2.94(t,J=4.6Hz,4H),2.67(s,4H),2.40(d,J=7.5Hz,4H), 2.36(s,3H),2.26(d,J=2.7Hz,3H),1.86(p,J=7.4Hz,2H).m / z(ESI-MS):596.23[M+H] + .

[0058] Step 10: Synthesize compound 1

[0059] Potassium hydroxide (11.2 g, 200 mmol) was dissolved in methanol (30 mL), and hydroxylamine hydrochloride (9.34 g, 134.4 mmol) was added to a methanol solution at 0 °C. The mixture was reacted with continuous stirring for 1 h. The precipitate was removed by filtration, and the filtrate was collected to obtain a fresh hydroxylamine solution, which was prepared for further reactions. Ester 1-11 (0.227 mmol) was dissolved in the above fresh hydroxylamine solution at 0 °C. The reaction mixture was stirred at room temperature for 1 h. After the reaction was complete, the mixture was neutralized with hydrochloric acid, the precipitate was collected, washed with water, and dried under vacuum to give compound 1 (Yield: 24.96%). 1 H NMR(300MHz,DMSO-d6)δ10.43(s,1H),10.03(s,1H),9.30(s,1H),8.73(s,1H),8.37(s,1 H),7.72(d,J=8.3Hz,2H),7.57(d,J=8.3Hz,2H),7.46-7.38(m,1H),7.31(d,J=8.3Hz,1H) ,6.92(d,J=9.2Hz,1H),5.53(s,2H),2.89(q,J=10.6,7.6Hz,4H),2.48(s,4H),2.40-2.33 (m,2H),2.28(d,J=2.7Hz,3H),2.23(s,3H),2.05(t,J=7.4Hz,2H),1.86(q,J=7.3Hz,2H). 13 C NMR(75MHz,DMSO-d6)δ171.36,169.09,165.40,151.29,142.91,139.19,136.32,136.18,135.99,134.84,133.31,133.09,127.11,123.92,123 .69,123.19,121.55,119.97,119.68,115.99,113.45,55.50,52.00,46.33,36.14,32.09,29.54,21.72,12.59,12.53.HRMS(ESI,positive)m / z Calcd.for C 30 H 34 ClFN6O4[M+H] + :597.2386; Found 597.2391.HPLC purity:97.47%.mp248.6~249.1℃.

[0060] Example 2: N 1-(5-amino-2-chloro-4-fluoro-3-methylbenzamide)-4-(4-methylpiperazin-1-yl)-[1,1'-biphenyl]-4-yl)-N 6 -Hydroxyhexadiamide (Compound 2). Compound 2 was synthesized according to steps 9 and 10 of Example 1, following the preparation method described in Example 1, except that 5-methoxy-5-oxopentanoic acid was replaced with 6-methoxy-6-oxohexanoic acid. Compound 2 was obtained as a white solid. Yield: 35.12%. 1 H NMR (300MHz, DMSO-d6) δ10.44(s,1H),10.11(s,1H),9.35(s,1H),8.73(s,1H),8.33(d,J=2.2Hz,1 H),7.73(d,J=8.4Hz,2H),7.56(d,J=8.3Hz,2H),7.43(dd,J=8.3,2.2Hz,1H),7.30(d,J=8.3Hz,1H ),6.91(d,J=9.2Hz,1H),5.55(s,2H),3.00(t,J=4.6Hz,4H),2.76(s,4H),2.42(s,3H),2.34(d,J= 6.9Hz,2H),2.28(d,J=2.6Hz,3H),2.06-1.96(m,2H),1.68-1.50(m,4H).HRMS(ESI,positive)m / z Calcd.for C 31 H 36 ClFN6O4[M+H] + :611.2543; Found 611.2547.HPLC purity:97.75%.mp252.9~254.6℃.

[0061] Example 3: N 1 -(5-amino-2-chloro-4-fluoro-3-methylbenzamide)-4-(4-methylpiperazin-1-yl)-[1,1'-biphenyl]-4-yl)-N 7 -Hydroxyheptanamide (Compound 3). Compound 3 was synthesized according to steps 9 and 10 of Example 1, following the preparation method described in Example 1, except that 5-methoxy-5-oxopentanoic acid was replaced with 7-methoxy-7-oxoheptanoic acid. Compound 3 was obtained as a white solid. Yield: 29%. 1H NMR(300MHz,DMSO-d6)δ10.42(s,1H),10.13(s,1H),9.39(s,1H),8.70(s,1H),8.32(s,1H),7.7 4(d,J=8.3Hz,2H),7.57(d,J=8.3Hz,2H),7.49-7.41(m,1H),7.30(d,J=8.3Hz,1H),6.91(d,J=9 .2Hz,1H),5.55(s,2H),3.10(s,4H),3.00(s,4H),2.58(s,3H),2.35(t,J=7.2Hz,2H),2.28(d,J =2.6Hz, 3H), 1.98 (t, J = 7.3Hz, 2H), 1.57 (dq, J = 22.2, 7.5Hz, 4H), 1.31 (dd, J = 16.1, 8.8Hz, 2H). 13 C NMR(75MHz,DMSO-d6)δ171.69,169.45,165.67,151.20,148.00,142.61,13 9.24,136.40,136.15,135.96,134.70,133.17,127.12,123.82,123.60,123 .34,121.49,120.32,119.92,115.99,115.93,113.29,54.54,50.69,44.89 ,36.66,32.63,29.55,25.36,25.31,12.59,12.53.HRMS(ESI,positive)m / z Calcd.for Chemical Formula:C 32 H 38 ClFN6O4[M+H] + :625.2699; Found 625.2689.HPLC purity:95.41%.mp242.4~243.1℃.

[0062] Example 4: N 1 -(5-amino-2-chloro-4-fluoro-3-methylbenzamide)-4-(4-methylpiperazin-1-yl)-[1,1'-biphenyl]-4-yl)-N 8 -Hydroxyoctanediamide (compound 4). Compound 4 was synthesized according to steps 9 and 10 of Example 1, with the difference that 5-methoxy-5-oxopentanoic acid was replaced with 8-methoxy-8-oxooctanoic acid in this example.

[0063] Compound 5 was obtained as a white solid. Yield: 31%. 1H NMR(300MHz,DMSO-d6)δ10.38(s,1H),10.04(s,1H),9.37(s,1H),8.70(s,1H),8.32(s,1H),7.7 2(d,J=8.3Hz,2H),7.56(d,J=8.1Hz,2H),7.44(d,J=8.0Hz,1H),7.30(d,J=8.1Hz,1H),6.89(d, J=9.2Hz,1H),5.54(s,2H),3.03(s,4H),2.88(s,4H),2.56(s,3H),2.35(d,J=7.0Hz,2H),2.30- 2.23(m,3H),1.96(t,J=7.3Hz,2H),1.61(s,2H),1.51(t,J=6.7Hz,2H),1.29(d,J=12.8Hz,4H). 13 C NMR (75MHz, DMSO-d6) δ171.80,169.61,165.52,151.28,148.09,142.64,139. 23,136.45,136.15,135.96,134.78,133.28,133.18,127.12,123.88,123.65 ,123.23,121.55,119.97,116.05,115.99,114.73,113.42,54.99,51.28,45. 50,36.90,32.76,29.53,28.93,25.55,12.58,12.51.HRMS(ESI,positive)m / z Calcd. for Chemical Formula: C 32 H 38 ClFN6O4[M+H] + :639.2856; Found639.2845.HPLC purity:97.77%.mp249.2~250.1℃.

[0064] Example 5: N 1 -(3'-(5-amino-2-chloro-4-fluoro-3-methylbenzamide)-4'-(4-methylpiperazin-1-yl)-[1,1'-biphenyl]-4-yl)-N 9 Hydroxynonadiamide (compound 5). Compound 5 was synthesized according to steps 9 and 10 of Example 1, with the difference that 5-methoxy-5-oxopentanoic acid was replaced with 9-methoxy-9-oxononanoic acid in this example.

[0065] Compound 5 was obtained as a white solid. Yield: 35%. 1H NMR (300MHz, DMSO-d6) δ10.35(s,1H),9.98(s,1H),9.31(s,1H),8.68(s,1H),8.37(s,1H),7.71(d,J=8.3 Hz,2H),7.56(d,J=8.2Hz,2H),7.42(d,J=8.3Hz,1H),7.31(d,J=8.4Hz,1H),6.92(d,J=9.2Hz,1H),5.53( s,2H),2.91(t,J=4.6Hz,4H),2.34(t,J=7.5Hz,2H),2.28(d,J=2.7Hz,3H),2.25(s,3H),1.96(t,J=7.3Hz ,2H),1.61(t,J=6.9Hz,2H),1.51(t,J=6.9Hz,2H),1.35-1.26(m,6H).HRMS(ESI,positive)m / zCalcd.for Chemical Formula:C 32 H 38 ClFN6O4[M+H] + :653.3013; Found 653.3012.HPLC purity:95.1%.mp249.2~250.1℃.

[0066] Example 6: 3'-(5-amino-2-chloro-4-fluoro-3-methylbenzamido)-N-(4-(hydroxyamino)-4-oxobutyl)-4'-(4-methylpiperazin-1-yl)-[1,1'-biphenyl]-4-carboxamide (compound 6). Following the preparation method of Example 1, intermediates 1-9 were synthesized according to step 7 of Example 1, except that 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)aniline was replaced with 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)benzoic acid. The intermediates were then subjected to amide condensation as described in Example 1 to obtain ester intermediates, which were subsequently converted to hydroxamic acid fragments via hydroxylamine transesterification to yield compounds 6-10.

[0067] Compound 6 was obtained as a white solid. Yield: 24.96%. 1H NMR (300MHz, DMSO-d6) δ10.46(s,1H),9.36(s,1H),8.73(s,1H),8.60(t,J=5.6Hz,1H),8.38(s,1H) ,7.96(d,J=8.0Hz,2H),7.70(d,J=8.0Hz,2H),7.52(dd,J=8.3,2.2Hz,1H),7.33(d,J=8.4Hz,1H),6 .90(d,J=9.2Hz,1H),5.52(s,2H),3.28(q,J=6.6Hz,2H),2.98(t,J=4.5Hz,4H),2.67(s,4H),2.35( s,3H),2.26(d,J=2.6Hz,3H),2.04(t,J=7.5Hz,2H),1.77(p,J=7.2Hz,2H).HRMS(ESI,positive)m / z Calcd.for C 30 H 34 ClFN6O4[M+H] + :597.2386; Found 597.2387.HPLC purity:98.51%.mp242.1~243.6℃.

[0068] Example 7: 3'-(5-amino-2-chloro-4-fluoro-3-methylbenzamido)-N-(5-(hydroxyamino)-5-oxopentyl)-4'-(4-methylpiperazin-1-yl)-[1,1'-biphenyl]-4-carboxamide (compound 7).

[0069] Compound 7 was obtained as a white solid. Yield: 35.12%. 1 H NMR (300MHz, DMSO-d6) δ10.39(s,1H),9.36(s,1H),8.71(s,1H),8.55(t,J=5.6Hz,1H),8.46-8.35( m,1H),7.96(d,J=8.2Hz,2H),7.72(d,J=8.0Hz,2H),7.52(dd,J=8.2,2.3Hz,1H),7.35(d,J=8.4Hz, 1H),6.92(d,J=9.2Hz,1H),5.54(s,2H),3.33-3.24(m,2H),2.93(t,J=4.5Hz,4H),2.49(s,4H),2.2 8(d,J=2.7Hz,3H),2.24(s,3H),2.00(d,J=6.7Hz,2H),1.59-1.52(m,4H).HRMS(ESI,positive)m / z Calcd.for C 31 H36 ClFN6O4[M+H] + :611.2543; Found 611.2547.HPLCpurity:97.75%.mp243.2~244.1℃.

[0070] Example 8: 3'-(5-amino-2-chloro-4-fluoro-3-methylbenzamido)-N-(6-(hydroxyamino)-6-oxohexyl)-4'-(4-methylpiperazin-1-yl)-[1,1'-biphenyl]-4-carboxamide (compound 8).

[0071] Compound 8 was obtained as a white solid. Yield: 29%. 1 H NMR (300MHz, DMSO-d6) δ10.37(s,1H),9.35(s,1H),8.69(s,1H),8.52(t,J=5.6Hz,1H),8.41(d,J=2.2H z,1H),7.96(d,J=8.0Hz,2H),7.71(d,J=8.1Hz,2H),7.52(dd,J=8.3,2.2Hz,1H),7.35(d,J=8.3Hz,1H) ,6.92(d,J=9.2Hz,1H),5.54(s,2H),3.28(q,J=6.5Hz,2H),2.92(t,J=4.6Hz,4H),2.50-2.45(m,4H),2 .28(d,J=2.6Hz,3H),2.23(s,3H),1.98(t,J=7.3Hz,2H),1.55(p,J=7.4Hz,4H),1.32(q,J=7.8Hz,2H). 13 C NMR(75MHz,DMSO-d6)δ169.53,166.23,165.51,151.30,148.10,143.99,14 2.71,136.15,135.96,135.51,133.84,133.27,133.07,133.02,128.40,126 .64,123.90,123.68,121.57,120.50,116.03,115.97,113.46,55.43,51.88 ,46.32,32.75,29.44,26.65,25.45,12.58,12.52.HRMS(ESI,positive)m / z Calcd.forC 32 H 38 ClFN6O4[M+H] +:625.2699; Found 625.2695.HPLC purity:95.40%.mp269.1~270.8℃.

[0072] Example 9: 3'-(5-amino-2-chloro-4-fluoro-3-methylbenzamido)-N-(7-(hydroxyamino)-7-oxohexyl)-4'-(4-methylpiperazin-1-yl)-[1,1'-biphenyl]-4-carboxamide (compound 9).

[0073] Compound 9 was obtained as a white solid. Yield: 31%. 1 H NMR (300MHz, DMSO-d6) δ10.40(s,1H),9.49(s,1H),8.69(s,1H),8.56(t,J=5.6Hz,1H),8.36(s,1H),7. 97(d,J=7.9Hz,2H),7.72(d,J=8.0Hz,2H),7.56(dd,J=8.3,2.2Hz,1H),7.33(d,J=8.3Hz,1H),6.90(d,J =9.1Hz,1H),5.54(s,2H),3.28(d,J=6.8Hz,6H),3.26-3.20(m,4H),2.79(s,3H),2.28(d,J=2.6Hz,3H), 1.97(t,J=7.3Hz,2H),1.52(p,J=7.4Hz,4H),1.31(d,J=6.1Hz,4H).HRMS(ESI,positive)m / zCalcd.for C 33 H 40 ClFN6O4[M+H] + :639.2856; Found 639.2857.HPLCpurity:97.77%.mp252.1~253.3℃.

[0074] Example 10: 3'-(5-amino-2-chloro-4-fluoro-3-methylbenzamido)-N-(8-(hydroxyamino)8-oxohexyl)-4'-(4-methylpiperazin-1-yl)-[1,1'-biphenyl]-4-carboxamide (compound 10).

[0075] Compound 10 was obtained as a white solid. Yield: 35%. 1H NMR (300MHz, DMSO-d6) δ10.33(s,1H),9.33(s,1H),8.66(s,1H),8.49(t,J=5.6Hz,1H),8.42-8.36(m, 1H),7.94(d,J=8.1Hz,2H),7.69(d,J=8.0Hz,2H),7.50(dd,J=8.4,2.2Hz,1H),7.33(d,J=8.4Hz,1H),6 .90(d,J=9.2Hz,1H),5.51(s,2H),3.26(t,J=6.4Hz,2H),2.91(t,J=4.6Hz,4H),2.47(s,4H),2.26(d, J=2.6Hz,3H),2.22(s,3H),1.94(t,J=7.3Hz,2H),1.50(dd,J=13.7,6.9Hz,4H),1.29(d,J=5.1Hz,6H). 13 C NMR(75MHz,DMSO-d6)δ169.53,166.23,165.50,151.30,148.11,143.98,142.7 0,136.15,135.96,135.51,133.86,133.27,133.07,133.02,128.38,126.64,12 3.90,123.68,121.57,120.49,116.04,115.97,113.46,55.43,51.88,46.32,32 .74,29.65,29.09,29.02,26.94,25.62,12.57,12.51.HRMS(ESI,positive)m / z Calcd.forC 34 H 42 ClFN6O4[M+H] + :653.3012; Found 653.3012.HPLC purity:95.27%.mp249.0~250.6℃.

[0076] Example 11: 5-Amino-2-chloro-4-fluoro-N-(4'-(4-(2-(hydroxyamino)-2-oxoethyl)benzamido)-4-(4-methylpiperazin-1-yl)-[1,1'-biphenyl]-3-yl)-3-methylbenzamide (Compound 11)

[0077]

[0078] Following the preparation method of Example 1, intermediates 1-10 were synthesized according to steps 1-8 in Example 1. Then, amide condensation was performed according to step 9 in Example 1 to obtain intermediate 11-1. The difference is that in this example, 5-methoxy-5-oxovaleric acid was replaced with 4-(2-methoxy-2-oxoethyl)benzoic acid. Finally, ester exchange with hydroxylamine was performed to obtain compound 11.

[0079] Compound 11 was obtained as a white solid. Yield: 35%. 1 H NMR (300MHz, DMSO-d6) δ10.83(s,1H),10.38(s,1H),9.37(s,1H),8.93(s,1H),8.39(d,J=1.9Hz,1H),7.96(s,4H),7.65(d,J=8.4Hz,2H),7.45(d,J =7.9Hz,3H),7.34(d,J=8.3Hz,1H),6.93(d,J=9.2Hz,1H),5.56(s,2H),3 .43(s,2H),3.01(s,4H),2.73(s,4H),2.40(s,3H),2.29(d,J=2.7Hz,3H). 13 C NMR(75MHz,DMSO-d6)δ167.04,165.84,165.60,151.28,148.08,142.78,140 .45,139.13,136.37,136.17,135.98,135.38,133.54,133.28,129.45,128.3 6,128.21,127.03,126.18,123.87,123.65,123.37,121.57,121.26,120.16, 116.06,113.40,54.83,51.07,45.28,12.59,12.52.HRMS(ESI,positive)m / z Calcd. for Chemical Formula: C 34 H 34 ClFN6O4[M+H] + 645.2383; Found645.2383.HPLC purity:99.86%.mp245.7~246.4℃.

[0080] Example 12: 5-Amino-2-chloro-4-fluoro-N-(4'-(2-(4-(hydroxycarbamoyl)phenyl)acetamide)-4-(4-methylpiperazin-1-yl)-[1,1'-biphenyl]-3-yl)-3-methylbenzamide (Compound 12)

[0081] Referring to the preparation method of Example 7, intermediates 1-10 were synthesized according to steps 1-8 in Example 1. Then, amide condensation was performed according to step 9 in Example 1 to obtain intermediates. The difference is that in this example, 5-methoxy-5-oxopentanoic acid was replaced with 2-(4-(hydroxycarbamoyl)phenyl)acetic acid. Finally, ester exchange with hydroxylamine was performed to obtain compound 12.

[0082] Compound 12 was obtained as a white solid. Yield: 39%. 1 H NMR(300MHz,DMSO-d6)δ11.22(s,1H),10.46(s,1H),9.38(s,1H),9.08(s,1 H),8.32(s,1H),7.74(d,J=8.0Hz,4H),7.58(d,J=8.2Hz,2H),7.44(d,J=7.9 Hz,3H),7.29(d,J=8.3Hz,1H),6.89(d,J=9.2Hz,1H),5.53(s,2H),3.76(s, 2H),3.06(d,J=5.2Hz,4H),2.95(s,4H),2.55(s,3H),2.27(d,J=2.6Hz,3H). 13 C NMR(75MHz,DMSO-d6)δ169.27,165.85,164.65,151.25,148.05,142.38,139.77, 139.11,136.50,136.14,135.94,133.37,133.20,131.63,129.68,128.87,127.4 2,127.19,127.08,123.82,123.59,123.45,121.56,121.33,120.72,120.10,116 .14,113.33,53.93,49.81,43.87,43.57,12.57,12.51.HRMS(ESI,positive)m / z Calcd.for ChemicalFormula:C 34 H 34 ClFN6O4[M+H] + :645.2375; Found 645.2376.HPLC purity:96.98%.mp255.8~256.5℃.

[0083]

[0084] Example 13: 5-Amino-2-chloro-4-fluoro-N-(4'-(4-(2-(hydroxyamino)-2-oxoethyl)benzamido)-4-(4-methylpiperazin-1-yl)-[1,1'-biphenyl]-3-yl)-3-methylbenzamide (Compound 13)

[0085] Synthesis of Intermediate 13-1: 4-Bromobenzoic acid (1.49 mmol), methyl acrylate (267.11 mg, 0.281 mL, 1.87 mmol), palladium acetate (3.35 mg, 0.0149 mmol), tris(o-tolyl)phosphine (18.17 mg, 0.06 mmol), and triethylamine (578.6 mg, 0.795 mL, 4.48 mmol) were sequentially dissolved in acetonitrile, and then heated at 100 °C for 12 h under an argon atmosphere. Acetonitrile was removed by vacuum distillation, and the solution was purified by (DCM:MeOH = 50:1) column chromatography to obtain intermediate 13-1 as a white powder. Subsequently, referring to the preparation method of Example 1, intermediates 1-10 were synthesized according to steps 1-8 in Example 1. Then, referring to step 9 in Example 1, amide condensation was performed to obtain intermediate 13-2. The difference is that in this example, 5-methoxy-5-oxovaleric acid was replaced with intermediate 13-1. Finally, it was transesterified with hydroxylamine to obtain compound 13.

[0086] Compound 13 was obtained as a white solid. Yield: 41%. 1 H NMR (300MHz, DMSO-d6) δ10.88(s,1H),10.43(s,1H),9.34(s,1H),9.17(s,1H),8.39(s,1H),8.04(d,J=7 .9Hz,2H),7.93(d,J=8.3Hz,2H),7.75(d,J=7.9Hz,2H),7.64(d,J=8.1Hz,2H),7.56(d,J=15.7Hz,1H),7 .47(d,J=8.2Hz,1H),7.33(d,J=8.2Hz,1H),6.92(d,J=9.2Hz,1H),6.62(d,J=15.8Hz,1H),5.55(s,2H), 2.93(t,J=4.4Hz,4H),2.60-2.49(s,4H),2.28(s,3H),2.28(s,3H).HRMS(ESI,positive)m / zCalcd.for Chemical Formula:C 35 H 34 ClFN6O4[M+H] + :657.2386; Found657.2405.HPLC purity:96.826%.mp269.0~270.7℃.

[0087] Example 14: (E)-5-amino-2-chloro-4-fluoro-N-(4'-(2-(4-(3-(hydroxyamino)-3-oxopropyl-1-en-1-yl)phenyl)acetamide)-4-(4-methylpiperazin-1-yl)-[1,1'-biphenyl]-3-yl)-3-methylbenzamide (Compound 14)

[0088] Referring to the preparation method of Example 9, intermediates 1-10 were synthesized according to steps 1-8 in Example 1, and then intermediates 13-1 were synthesized according to the synthesis steps of intermediate 13-1 in Example 9. Subsequently, amide condensation was performed according to step 9 in Example 1 to obtain intermediates. The difference is that in this example, 4-bromobenzoic acid was replaced with 2-(4-bromophenyl)acetic acid, and finally, hydroxylamine was transesterified to obtain compound 14.

[0089] Compound 14 was obtained as a white solid. Yield: 36%. 1 H NMR(300MHz,DMSO-d6)δ10.79(s,1H),10.39(s,1H),9.36(s,1H),9.08(s,1H),8.34(s,1H ),7.73(d,J=8.3Hz,2H),7.62-7.52(m,4H),7.49(s,1H),7.45-7.37(m,3H),7.30(d,J=8. 4Hz,1H),6.90(d,J=9.2Hz,1H),6.47(d,J=15.8Hz,1H),5.53(s,2H),3.72(s,2H),3.02(t ,J=4.6Hz,4H),2.84(s,4H),2.48(s,3H),2.28(d,J=2.6Hz,3H).HRMS(ESI,positive)m / z Calcd.for Chemical Formula:C 36 H 36 ClFN6O4[M+H] + :671.2531; Found 671.2531.HPLC purity:99.877%.mp254.1~255.7℃.

[0090] Example 15: (E)-5-amino-2-chloro-4-fluoro-N-(4'-(3-(4-(3-(hydroxyamino)-3-oxoprop-1-en-1-yl)phenyl)propamido)-4-(4-methylpiperazin-1-yl)-[1,1'-biphenyl]-3-yl)-3-methylbenzamide (Compound 15)

[0091] Referring to the preparation method of Example 9, intermediates 1-10 were synthesized according to steps 1-8 in Example 1, and then intermediates 13-1 were synthesized according to the synthesis steps of intermediate 13-1 in Example 9. Subsequently, amide condensation was performed according to step 9 in Example 1 to obtain intermediates. The difference is that in this example, 4-bromobenzoic acid was replaced with 3-(4-bromophenyl)propionic acid, and finally, hydroxylamine was transesterified to obtain compound 15.

[0092] Compound 15 was obtained as a white solid. Yield: 32%. 1 H NMR(300MHz,DMSO-d6)δ10.82(s,1H),10.18(s,1H),9.45(s,1H),9.07(s,1H),8.29(s,1H), 7.72(d,J=8.2Hz,2H),7.57(d,J=8.4Hz,2H),7.54-7.39(m,4H),7.33(d,J=8.5Hz,3H),6.88( d,J=9.1Hz,1H),6.46(d,J=15.8Hz,1H),5.55(s,2H),3.18(s,4H),2.96(t,J=7.5Hz,2H),2. 76(s,4H),2.72-2.63(m,2H),2.24-2.55(s,3H),2.31-2.24(m,3H).HRMS(ESI,positive)m / z Calcd.for ChemicalFormula:C 37 H 38 ClFN6O4[M+H] + :685.2699; Found 685.2689.HPLC purity:95.88%.mp255.4~256.3℃.

[0093] Example 16: (E)-3'-(5-amino-2-chloro-4-fluoro-3-methylbenzoamide)-N-(4-(3-(hydroxyamino)-3-oxopropyl-1-en-1-yl)benzyl)-4'-(4-methylpiperazin-1-yl)-[1,1'-biphenyl]-4-carboxamide (Compound 16)

[0094] Referring to the preparation method of Example 6, intermediates 1-10 were synthesized according to steps 1-8 in Example 1, and then intermediates 13-1 were synthesized according to the synthesis steps of intermediate 13-1 in Example 13. Subsequently, amide condensation was performed according to step 9 in Example 1 to obtain intermediates. The difference is that in this example, 4-bromobenzoic acid was replaced with (4-bromophenyl)methylamine, and finally, hydroxylamine was transesterified to obtain compound 16.

[0095] Compound 16 was obtained as a white solid. Yield: 39%. 1H NMR (300MHz, DMSO-d6) δ10.84(s,1H),9.51(s,1H),9.23(t,J=5.9Hz,1H),9.07(s,1H),8.3 5(s,1H),8.03(d,J=8.0Hz,2H),7.74(d,J=7.9Hz,2H),7.55(t,J=8.7Hz,3H),7.49-7.35(m, 3H),7.32(d,J=8.4Hz,1H),6.88(d,J=9.1Hz,1H),6.48(d,J=15.8Hz,1H),5.55(s,2H),4.52 (d,J=5.8Hz,2H),3.29-3.16(m,4H),2.79(s,3H),2.57-2.46(m,3H),2.26(d,J=2.6Hz,3H). 13 C NMR(75MHz,DMSO-d6)δ166.42,165.52,163.23,160.70,151.31,148.11,144.05,143 .01,141.73,138.53,136.15,135.96,135.44,133.91,133.37,133.27,133.08,129. 68,128.54,128.23,128.00,126.75,123.93,123.68,121.59,120.57,119.12,115.3 2,113.46,55.40,51.82,46.27,42.96,29.53,12.58,12.52.HRMS(ESI,positive)m / z Calcd.for Chemical Formula:C 36 H 36 ClFN6O4[M+H] + :671.2548; Found 671.2543.HPLC purity:98.94%.mp247.4~248.1℃.

[0096] The following method is used in this invention to express and purify the WDR5 protein.

[0097] The WDR5 gene plasmid was purchased from Biohelpers. The WDR5 gene with a His tag was cloned into the pCzn1 vector and preserved in puncture bacteria containing cryoprotectant.

[0098] Escherichia coli BL21(DE3) strain was transfected with a recombinant plasmid, and the cells were revived in sterile LB medium (37°C). Single colonies were picked and transferred to 10 mL of LB liquid medium (containing 50 μg / mL ampicillin), and cultured overnight at 37°C with shaking (220 rpm). The culture was then transferred to 1 L of LB liquid medium (containing 50 μg / mL ampicillin), and cultured at 37°C with shaking (220 rpm) for 6-8 hours until the OD 600 reached 0.6-0.8. The culture was then cooled to 12°C, and 1 mM IPTG was added to induce expression for 14-16 hours (180 rpm). The cells were then harvested and stored at -80°C for later use.

[0099] 4g of bacterial clumps were added to 40mL of lysis buffer (20mM Tris-HCl buffer, pH 7.4, 300mM NaCl, 20mM β-mereaptoethanol, 1% Triton X-100, and 0.1% PMSF). The mixture was sonicated for 40 minutes. The lysed mixture was centrifuged at low temperature and high speed (12000rpm, 20min, 4℃). The supernatant was filtered (using a 0.4μm microporous membrane) and purified using an AKTA instrument with a His column (equilibration buffer: 20mM Tris-HCl, 300mM NaCl, 5mM imidazole, pH 7.4; eluent: 20mM Tris-HCl, 75mM imidazole, 0.3M NaCl, pH 7.4). The molecular weight and purity of the bands were confirmed by 10% SDS-PAGE. The mixture was dialyzed overnight (10% PBS, 300mM...). NaCl (pH = 7.4). The obtained protein was stored at -80℃ after concentration determination by BCA. Example 1: Determination of the inhibitory activity of the compound on WDR5 based on fluorescence polarization (FP).

[0100] The instrument used in this experiment was a SpectraMax Multi-Mode Microplate Reader (Molecular Devices), with excitation and emission wavelengths of 485 nm and 535 nm, respectively. The protein used was WDR5, and the probe was a fluorescent probe FITC-AHx-SEEEIDVVSV-NH2 constructed based on the dominant Myc-binding sequence, using 6-aminoacetic acid as the linker, and incorporating the fluorescent group fluorescein isothiocyanate (FITC). The buffer formulation used in the test system was: 0.1 mM sodium tetraborate, 1.6 mM boric acid, pH 7.8, and 0.01% Triton X-100. The 384-well blackboard used in the experiment was manufactured by Corning.

[0101] Experimental steps:

[0102] The test system used was 60 μL, with 20 μL of compound, protein, and probe solution added sequentially to each well. Each compound was serially diluted 10–14 times (3-fold) with an initial concentration of 100 μM. One replicate was used for each concentration. Correspondingly, 20 μL of probe + 40 μL of buffer solution served as a blank control, and 20 μL of protein (200 nM) + 20 μL of probe (30 nM) + 20 μL of buffer solution served as a negative control. Positive control was administered for 6 days. After sample addition, the 384-well plate was covered with aluminum foil and shaken on a shaker at room temperature for 20 min. Fluorescence was read using a SpectraMax Paradigm Multi-Mode Microplate Reader at an excitation wavelength of 485 nm and an emission wavelength of 535 nm. The mP value was calculated, and the inhibition rate was calculated using the following formula. Finally, the IC50 was calculated using GraphPadPrism 5.0. 50 value.

[0103] Inhibition rate = (mP value of compound group - mP value of blank group) / (mP value of negative control group - mP value of blank group) × 100%. Example 2: Inhibitory activity of the compound against HDAC.

[0104] All enzymatic reactions were carried out at 37°C for 30 minutes. 50 μL of the reaction mixture contained 25 mM Tris, pH 8.0, 1 mM MgCl2, 0.1 mg / mL BSA, 37 mM NaCl, 2.7 mM KCl, HDAC, and the enzyme substrate. The compounds were diluted in 10% DMSO, and 5 μL of the diluent was added to 50 μL of the reaction mixture to make the final DMSO concentration 1% in all reactions. The fluorescence was determined by quantifying the amount of fluorescent product in the solution after the enzyme reaction. Fluorescence was then analyzed on a SpectraMax M5 microtiter plate reader at excitation wavelengths of 350–360 nm and emission wavelengths of 450–460 nm. IC 50 The values ​​were calculated using nonlinear regression and normalized dose-response fitting was performed using Prism GraphPad software.

[0105] The experimental results of the compounds on HDAC and WDR5 proteins are shown in Table 1.

[0106] Table 1. IC50 of the compounds of the present invention against WDR5 protein and HDAC. 50 value

[0107]

[0108]

[0109] As shown in Table 1, the compounds of the present invention all have significant inhibitory activity against WDR5 and HDAC, and can be used as small molecule inhibitors targeting both WDR5 and HDAC.

[0110] Further assays were performed on the inhibitory activities of the dominant compounds 9 and 16 on other HDAC family subtypes, as shown in Table 2.

[0111]

[0112] Experimental Example 3: Based on CellTiter-Lumi TM Detection of cell antiproliferative activity by luminescence method

[0113] The cell types used in this experiment were: human myeloid monocytic leukemia cells MV4-11, human acute myeloid leukemia cells MOLM-13, human monocytic leukemia cells THP-1, human chronic myeloid leukemia cells K562, human non-small cell lung cancer cell line A549, human liver cancer cells HuH-1, human ovarian adenocarcinoma cells SKOV3, and human colon cancer cells HCT-116, all of which were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences.

[0114] The culture medium used for MV4-11 cells was: RPMI-IMDM medium (GiBco, Invitrogen Corp, USA) and 10% FBS (GiBco, Invitrogen Corp, USA);

[0115] The culture media used for MOLM-13, THP-1, K562, and HCT-116 cells were RPMI-1640 medium (GiBco, Invitrogen Corp, USA) and 10% FBS (GiBco, Invitrogen Corp, USA).

[0116] The culture media used for A549, HuH-1, and SKOV3 cells were DMEM (GiBco, Invitrogen Corp, USA) and 10% FBS (GiBco, Invitrogen Corp, USA), and all were cultured at 37°C under a humid atmosphere of 5% CO2.

[0117] Cells in the logarithmic growth phase were collected into 96-well opaque white cell culture plates (Greiner 655083) and maintained at a density of 5000 cells / well in 100 μL of medium. Different concentrations of the target compound were serially diluted with 100 μL of the appropriate medium and treated for 72 h. SAHA and DDO-2074 were used as positive controls, treated in the same manner as the target compound. Subsequently, CellTiter-Lumi was used... TMCell viability was assessed using a luminescent cell viability assay kit (purchased from Beyotime). 100 μL of CTL stabilizing reagent was mixed with 100 μL of the corresponding treated cells and incubated at 37°C for 15 minutes. The optical density was measured at 450 nm using a Thermo Multiskan Spectrum analyzer. 50 The values ​​were calculated using GraphPad Prism ver.8.0 software.

[0118] The test results for representative compounds are shown in Table 3.

[0119] Table 3 Antiproliferative activity of compounds

[0120]

[0121] As shown in Table 3, compounds 9 and 16 exhibited strong anti-proliferative activity against tumor cells, with compound 16 showing the best inhibitory activity against MV4-11 cells.

[0122] The dominant compounds 9, 16, and combinations were tested in vitro for their antiproliferative activity in leukemia cells and solid tumor cells, such as... Figure 1 As shown, the results indicated that compounds 9 and 16 exhibited significant antiproliferative activity against human myeloid monocytic leukemia cells MV4-11, human acute myeloid leukemia cells MOLM-13, human monocytic leukemia cells THP-1, human chronic myeloid leukemia cells K562, human ovarian adenocarcinoma cells SKOV3, and human colon cancer cells HCT-116. Specifically, compounds 9 and 16 showed excellent inhibitory effects on HCT-116 and SKOV3. Significantly enhanced antiproliferative activity was observed in leukemia cell lines.

[0123] In summary, the co-inhibition of WDR5-MLL1 interaction and HDAC may enhance anti-cancer activity through dual-target inhibitors in MLL-rearranged AML cells. Dual-function inhibitors of WDR5-MLL1 protein-protein interaction (PPI) and HDAC represent an effective strategy as novel antitumor agents and provide molecular tools for exploring the potential mechanisms between MLL1 and HDAC. The WDR5-HDAC dual-target compound provided in this invention exhibits significant inhibitory activity against both WDR5 and HDAC, making it an effective WDR5-HDAC dual-target inhibitor. Therefore, drugs containing the above compound as an active ingredient can be used to prepare medications for treating clinical conditions related to WDR5-HDAC.

[0124] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A compound of formula I or a pharmaceutically acceptable salt thereof: Its characteristics are, The compound represented by Formula I has one of the following structural formulas: , .

2. A pharmaceutical composition comprising the compound of formula I as claimed in claim 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

3. The use of the compound of Formula I as claimed in claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a WDR5-HDAC dual-target small molecule inhibitor.

4. The use of the compound of formula I according to claim 1 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 2, in the preparation of a therapeutic remedy for diseases related to WDR5 and HDAC dysfunction, characterized in that, The diseases associated with WDR5 and HDAC dysfunction are leukemia, colon cancer, lung cancer, and ovarian cancer.

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