Hydroxamic acid double-target compound derivative and application thereof

Abstract

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

Description

Technical Field

[0001] The invention belongs to the technical field of medicinal chemistry, and specifically relates to a class of hydroxamic acid dual-target compound derivatives and applications thereof. Background Art

[0002] WD40 repeat domain proteins are widely expressed in the human proteome. WD40 proteins play an important role 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 different diseases. WDR5 is a member of the WD40 protein family and was first discovered in mouse chondrocytes. One of the most important functions of WDR5 is to participate in gene expression regulation as an epigenetic "reader". A large number of WDR5 interaction partners and their unique role 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 a variety of cancers, such as prostate cancer, breast cancer, leukemia, liver cancer, pancreatic cancer, bile duct cancer, colon cancer, lung cancer, glioblastoma, ovarian cancer, cervical cancer, gastric cancer, etc., and is associated with poor clinical prognosis.

[0003] Studies have shown that rearrangements of the MLL1 gene lead to a unique class of leukemias characterized by poor outcomes, including approximately 70% of infant acute lymphoblastic leukemia (ALL) cases and 70% of adult acute myeloid leukemia (AML) cases. The MLL1 protein, encoded by the MLL1 gene, plays an important role in the development of 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 a motif (WIN) site. Interference with the MLL1-WDR5 protein-protein interaction has emerged as a promising and effective strategy for the treatment of MLL-rearranged leukemias.

[0004] Histone deacetylases (HDACs) are a class of epigenetic regulators that play an important role in various tumorigenesis, neurological diseases, and immune disorders. At the same time, HDACs are involved in various biological processes of cancer cells, including apoptosis, cell growth, and cell differentiation. In addition, 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 an important factor in tumorigenesis. Therefore, HDACs are effective targets for the development of HDAC inhibitors (HDACi) for the treatment of a variety of solid and blood cancers, such as liver cancer, pancreatic cancer, and acute myeloid leukemia. To date, many HDAC inhibitors have been developed and have achieved strong therapeutic effects in preclinical and clinical studies.

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

[0006] In order to solve the above technical problems existing in the prior art, the present invention provides a class of WDR5-HDAC dual-target inhibitors and applications thereof.

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

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

[0009]

[0010] in:

[0011] R 1 Selected from C 1 -C 7 Alkyl or C 1 -C 7 haloalkyl, naphthyl, pyridyl, piperazinyl, piperidinyl, phenyl or substituted phenyl, the substituent of the substituted phenyl is C 1 -C 7 Alkyl, C 1 -C 7 haloalkyl, cyano, hydroxy, mercapto, halogen, nitro, amino, methoxy or trifluoromethyl;

[0012] R 2 -NH-OH

[0013] L 1 , L 2 independently selected from carbonyl, sulfonyl, amino, imino, aminoidene, ether, thioether, methylene or methylidene;

[0014] Furthermore, the L 1 , L 2 are independently selected from carbonyl, imino, aminoidene, sulfonyl, methylene or methylidene.

[0015] Furthermore, the pharmaceutically acceptable salts include acid addition salts formed between the compound of formula I and 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, mandelic acid; and also include basic salts formed between the compound of formula I and inorganic bases: sodium salt, potassium salt, lithium salt, calcium salt, zinc salt, magnesium salt, meglumine salt, tromethamine salt, diethylamine salt or ethanolamine salt.

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

[0017]

[0018]

[0019] The second object of the present invention is to provide a pharmaceutical composition, which comprises the compound represented by the aforementioned formula I or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

[0020] The pharmaceutical composition of the present invention can be administered in various known ways, such as orally, by injection, or by inhalation spray. The pharmaceutical composition of the present invention can be administered alone or in combination with other drugs. The oral composition can be in 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, adhesives, 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 dosage level of the active ingredient in the pharmaceutical composition of the present invention can be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage 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 and previous medical history of the patient being treated, and similar factors well known in the medical field.

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

[0023] The fourth object of the present invention is to provide the use of the compound represented by the aforementioned formula I or its pharmaceutically acceptable salt or the aforementioned pharmaceutical composition in the preparation of therapeutic drugs for diseases related to WDR5 and / or HDAC dysfunction.

[0024] Furthermore, the disease associated with WDR5 and / or HDAC dysfunction is 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 obvious inhibitory activity on WDR5 protein and HDAC, and can be used as a WDR5-HDAC dual-target small molecule inhibitor for treating or enhancing the treatment of diseases related to WDR5 or HDAC. The small molecule has shown good activity in various experiments and is expected to be developed into a specific anti-tumor drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 . In vitro antiproliferative ability of compounds 9 and 16, SAHA and DDO-2074 as positive controls, where:

[0028] (A) Inhibition of cell proliferation of leukemia cells by compounds 9, 16, DDO-2074, and SAHA;

[0029] (B) Compounds 9, 16, and the combination of DDO-2074 and SAHA inhibited cell proliferation of solid cancers;

[0030] Data are shown as mean ± SD of three experiments. DETAILED DESCRIPTION

[0031] The preparation method of the compound of general formula I of the present invention is described below in conjunction with specific examples, but these specific methods do not constitute any limitation to the present invention. The compound 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 a combination can be easily carried out by a technician in the field to which the present invention belongs.

[0032] The starting materials, reaction reagents, etc. used in the specific examples of the present invention are all commercially available.

[0033] The experimental methods in the examples of the present invention without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by raw material or commodity manufacturers. Reagents without specifying specific sources are conventional reagents purchased from the market.

[0034] The structures of the compounds were determined by nuclear magnetic resonance (NMR). 1 H NMR and 13 C NMR spectra were measured by Bruker AV-300 NMR spectrometer, and the solvent was deuterated dimethyl sulfoxide (DMSO-d 6 ), and the internal standard was tetramethylsilane (TMS).

[0035] The chemical reaction was detected using 0.25 mm GF254 thin layer chromatography silica gel plates and observed by a ZF7 three-purpose UV analyzer.

[0036] Unless otherwise specified in the examples, the reactions were carried out under air atmosphere.

[0037] Unless otherwise specified in the examples, the reaction temperature is room temperature and is 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: Synthesis of compound 1-2

[0041] Compound 1-1 (10.0 g, 69.2 mmol) was dissolved in DCM (50 mL), and then anhydrous aluminum chloride (18.4 g, 138.3 mmol) and acetyl chloride (5.4 g, 78.5 mmol) were added. After the addition was completed, the reaction solution was stirred at room temperature for 2 h. After the reaction was completed, the reaction solution was poured into ice water, and then HCl (2 M) was used to adjust the pH to 4. The reaction solution was extracted with dichloromethane, and the organic phase was collected and concentrated to obtain a crude product, which was separated and purified by group chromatography (petroleum ether: ethyl acetate = 200: 1) to obtain compound 1-2 (6 g, 46.5%). 1 H NMR (300 MHz, CDCl 3 )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: Synthesis of compounds 1-3

[0043] Bromine (8.3 g, 2.7 mL, 51.8 mmol) was added dropwise to a NaOH (6 mol / L) solution, the temperature was maintained at 0°C and stirred continuously 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 completed, the reaction solution was poured into water and washed with CHCl 3 Extraction was performed to remove the residual bromine. After the aqueous solution was acidified with HCl (3 mmol / L) and washed with water and ether, the white precipitate was filtered out to obtain white solid compound 1-3 (2.07 g, 63.7%). 1 H NMR (300 MHz, DMSO-d 6)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: Synthesis of compounds 1-4

[0045] Concentrated nitric acid (4.34 g, 2.90 mL, 68.94 mmol) was added dropwise to a concentrated sulfuric acid solution (200 mL) of 1-3 (10 g, 53.03 mmol) at 0°C. The reaction mixture was then reacted at room temperature for 4 hours. A white solid was precipitated while the mixture was poured into ice water, and 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 (300 MHz, DMSO-d 6 )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: Synthesis of compounds 1-6

[0047] 1-5 (10 g, 45.46 mmol) was 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 above mixture in sequence. The reaction was monitored by TLC, and the solvent was removed by distillation under reduced pressure after the reaction was complete to obtain a red oily crude product. The crude product was then dissolved in ethyl acetate, washed with water and precipitated with anhydrous Na 2 SO 4 After drying, red oil compound 1-6 (12 g, 87.8%) was obtained. 1 HNMR (300MHz, CDCl 3 )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: Synthesis of compounds 1-7

[0049] To a solution of compound 1-6 (10 g, 33.32 mmol) in ethyl acetate (400 mL) was added SnCl at room temperature. 2 ·2H 2 O (37.59 g, 166.58 mmol). 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 filtered and washed with ethyl acetate (5 × 100) and washed with NaSO 4 The organic phase was dried and concentrated to obtain the crude product. 2 Cl 2 / MeOH=80:1) to give compound 1-7 (8 g, 88.9%). 1 H NMR (300 MHz, CDCl 3 )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: Synthesis of compounds 1-8

[0051] Compound 1-7 (3 g, 11.10 mmol) was dissolved in anhydrous dichloromethane (150 mL), stirred at 0 ° C for 10 minutes, and then DIPEA (4.31 g, 5.80 mL, 33.31 mmol) was added. Then the dichloromethane solution (4.20 g, 16.66 mmol) of the acid chloride intermediate of 1-4 prepared in the above reaction was added dropwise, and the reaction was kept at room temperature for 4 hours. When the reaction was completed, the reaction was diluted with dichloromethane (100 mL). Then it was washed with water (50 mL), saturated sodium bicarbonate solution (2×40 mL), and then washed with saturated sodium chloride solution (50 mL). The organic phase was separated and dried (Na 2 SO 4 ), concentrated, and purified by column chromatography to obtain intermediate 1-8 (3 g, 55.6%) 1 H NMR (300 MHz, DMSO-d 6)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=8.6H z,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: Synthesis of Compounds 1-9

[0053] To a solution of compound 1-8 (5 g, 10.29 mmol) in ethyl acetate (400 mL) was added SnCl at room temperature. 2 ·2H 2 O (37.59 g, 166.58 mmol). The mixture was then refluxed for 6 h under stirring. After the reaction was completed, the reaction solution was neutralized by adding saturated sodium bicarbonate solution. The filtrate was then diluted with ethyl acetate (5 × 100 mL) and filtered and washed. The filtrate was washed with NaSO 4 The organic phase was dried and concentrated to obtain the crude product. Purification by silica gel column chromatography (CH 2 Cl 2 / MeOH=50:1) to give compound 1-9 (2 g, 42.6%). 1 H NMR (300 MHz, CDCl 3 )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: Synthesis of 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), and Cs 2 CO 3 (285.96 mg, 0.878 mmol) and a catalytic amount of Pd(PPh3 ) 2 Cl 2 (9.24 mg, 0.0131 mmol). Under nitrogen protection, the reaction was heated to 100 °C for 10 h. After the reaction was completed, the mixture was filtered to remove the catalyst and Cs 2 CO 3 The organic phase was separated and concentrated to obtain a crude product, which was then purified by column chromatography using 1.25% to 2% DCM / MeOH to obtain intermediate 1-10 (110 mg, 39%). 1 H NMR (300 MHz, DMSO-d 6 )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: Synthesis of Compounds 1-11

[0057] 6-Methoxy-6-oxohexanoic acid (0.555 mmol) was dissolved in DMF (2 mL) at room temperature, and then a DMF solution (1 mL) of PyBOP (0.444 mg, 0.854 mmol) was added dropwise, followed by DIPEA (165.71 mg, 0.223 mL, 1.28 mmol). After the above solution was stirred for 30 minutes, intermediate 1-10 (200 mg, 0.427 mmol) was added. The resulting mixture was reacted at room temperature for 8 to 10 h. After the reaction was completed, 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 obtain 1-11 (yield: 34.1%). 1 H NMR (300 MHz, DMSO-d 6)δ10.01(s,1H),9.31(s,1H),8.33(s,1H),7.69(d,J=8.3Hz,2H),7.55(d,J=8.4 Hz,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: Synthesis of Compound 1

[0059] Potassium hydroxide (11.2 g, 200 mmol) was dissolved in methanol (30 mL) and added to a methanol solution of hydroxylamine hydrochloride (9.34 g, 134.4 mmol) at 0°C. The mixture was reacted for 1 h under continuous stirring. The precipitate was removed by filtration and the filtrate was collected to obtain a fresh hydroxylamine solution, which was prepared for further reaction. 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 completed, the mixture was neutralized with hydrochloric acid, the precipitate was collected, washed with water, and dried in vacuo to obtain compound 1 (Yield: 24.96%). 1 H NMR (300 MHz, DMSO-d 6 )δ10.43(s,1H),10.03(s,1H),9.30(s,1H),8.73(s,1H),8.37(s,1H),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,2 H), 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 (75 MHz, DMSO-d 6)δ171.36,169.09,165.40,151.29,142.91,139.19,136.32,136.18,135.99,134.84,133.31,133.09,127.11,123.92,123.69,123.1 9,121.55,119.97,119.68,115.99,113.45,55.50,52.00,46.33,36.14,32.09,29.54,21.72,12.59,12.53.HRMS(ESI,positive)m / z Calcd.for C 30 H 34 CIF 6 O 4 [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 -Hydroxyadipamide (Compound 2). Referring to the preparation method of Example 1, Compound 2 was synthesized according to steps 9 and 10 of Example 1, except that 5-methoxy-5-oxopentanoic acid was replaced with 6-methoxy-6-oxohexanoic acid in this example. Compound 2 was obtained as a white solid. Yield: 35.12%. 1 H NMR (300 MHz, DMSO-d 6 )δ10.44(s,1H),10.11(s,1H),9.35(s,1H),8.73(s,1H),8.33(d,J=2.2Hz,1H),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=6.9H z,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 CIF6 O 4 [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 -Hydroxypimelamide (Compound 3). Referring to the preparation method of Example 1, Compound 3 was synthesized according to steps 9 and 10 of Example 1, except that 5-methoxy-5-oxopentanoic acid was replaced with 7-methoxy-7-oxoheptanoic acid in this example. Compound 3 was obtained as a white solid. Yield: 29%. 1 H NMR (300 MHz, DMSO-d 6 )δ10.42(s,1H),10.13(s,1H),9.39(s,1H),8.70(s,1H),8.32(s,1H),7.74(d,J=8.3Hz,2 H),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=2.6 Hz,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 (75 MHz, DMSO-d 6 )δ171.69,169.45,165.67,151.20,148.00,142.61,139.24,136.40,1 36.15,135.96,134.70,133.17,127.12,123.82,123.60,123.34,121. 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 H38 CIF 6 O 4 [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 -Hydroxyoctanamide (Compound 4). Referring to the preparation method of Example 1, Compound 4 was synthesized according to steps 9 and 10 of Example 1, except that 5-methoxy-5-oxopentanoic acid was replaced by 8-methoxy-8-oxooctanoic acid in this example.

[0063] The obtained compound 5 is a white solid. Yield: 31%. 1 H NMR (300 MHz, DMSO-d 6 )δ10.38(s,1H),10.04(s,1H),9.37(s,1H),8.70(s,1H),8.32(s,1H),7.72(d,J=8.3Hz,2 H),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,1 H),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 (75 MHz, DMSO-d 6)δ171.80,169.61,165.52,151.28,148.09,142.64,139.23,136.45,136 .15,135.96,134.78,133.28,133.18,127.12,123.88,123.65,123.23,1 21.55,119.97,116.05,115.99,114.73,113.42,54.99,51.28,45.50,36.90,32.76,29.53,28.93,25.55,12.58,12.51.HRMS(ESI,positive)m / z Calcd.for Chemical Formula:C 32 H 38 CIF 6 O 4 [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 Hydroxyzelaic acid (Compound 5). Referring to the preparation method of Example 1, Compound 5 was synthesized according to steps 9 and 10 of Example 1, except that 5-methoxy-5-oxopentanoic acid was replaced by 9-methoxy-9-oxononanoic acid in this example.

[0065] The obtained compound 5 is a white solid. Yield: 35%. 1 H NMR (300 MHz, DMSO-d 6)δ10.35(s,1H),9.98(s,1H),9.31(s,1H),8.68(s,1H),8.37(s,1H),7.71(d,J=8.3Hz,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),2.9 1(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 CIF 6 O 4 [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-methylbenzamide)-N-(4-(hydroxyamino)-4-oxobutyl)-4'-(4-methylpiperazine-1-yl)-[1,1'-biphenyl]-4-carboxamide (Compound 6). Referring to the preparation method of Example 1, intermediate 1-9 was synthesized according to step 7 in Example 1, except that 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline was replaced with 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoic acid in this example. Then, an ester intermediate was obtained by amide condensation in Example 1, and then converted into a hydroxamic acid fragment by hydroxylamine ester exchange to obtain compound 6-10.

[0067] The obtained compound 6 is a white solid. Yield: 24.96%. 1 H NMR (300 MHz, DMSO-d 6)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=8.0 Hz,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 CIF 6 O 4 [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] The obtained compound 7 is a white solid. Yield: 35.12%. 1 H NMR (300 MHz, DMSO-d 6 )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),2.28(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 C31 H 36 CIF 6 O 4 [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] The obtained compound 8 is a white solid. Yield: 29%. 1 H NMR (300 MHz, DMSO-d 6 )δ10.37(s,1H),9.35(s,1H),8.69(s,1H),8.52(t,J=5.6Hz,1H),8.41(d,J=2.2Hz,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 (75 MHz, DMSO-d 6 )δ169.53,166.23,165.51,151.30,148.10,143.99,142.71,136.15,1 35.96,135.51,133.84,133.27,133.07,133.02,128.40,126.64,123.9 0,123.68,121.57,120.50,116.03,115.97,113.46,55.43,51.88,46.32,32.75,29.44,26.65,25.45,12.58,12.52.HRMS(ESI,positive)m / z Calcd.forC 32 H 38 CIF6 O 4 [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] The obtained compound 9 is a white solid. Yield: 31%. 1 H NMR (300 MHz, DMSO-d 6 )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,2 H),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 CIF 6 O 4 [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] The obtained compound 10 is a white solid. Yield: 35%. 1 H NMR (300 MHz, DMSO-d 6)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=2.6 Hz, 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 (75 MHz, DMSO-d 6 )δ169.53,166.23,165.50,151.30,148.11,143.98,142.70,136.15,135. 96,135.51,133.86,133.27,133.07,133.02,128.38,126.64,123.90,123. 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 CIF 6 O 4 [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] Referring to the preparation method of Example 1, intermediate 1-10 was synthesized according to steps 1 to 8 in Example 1, and then amide condensation was performed with reference to step 9 in Example 1 to obtain intermediate 11-1, except that in this example, 5-methoxy-5-oxopentanoic acid was replaced with 4-(2-methoxy-2-oxoethyl)benzoic acid, and finally ester exchange was performed with hydroxylamine to obtain compound 11.

[0079] The obtained compound 11 is a white solid. Yield: 35%. 1 H NMR (300 MHz, DMSO-d 6 )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 (75 MHz, DMSO-d 6 )δ167.04,165.84,165.60,151.28,148.08,142.78,140.45,139.13,13 6.37,136.17,135.98,135.38,133.54,133.28,129.45,128.36,128.21, 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 CIF 6 O 4 [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, intermediate 1-10 was synthesized according to steps 1 to 8 in Example 1, and then amide condensation was performed according to step 9 in Example 1 to obtain the intermediate, except that 5-methoxy-5-oxopentanoic acid was replaced with 2-(4-(hydroxycarbamoyl)phenyl)acetic acid in this example, and finally ester exchange was performed with hydroxylamine to obtain compound 12.

[0082] The obtained compound 12 was a white solid. Yield: 39%. 1 H NMR (300 MHz, DMSO-d 6 )δ11.22(s,1H),10.46(s,1H),9.38(s,1H),9.08(s,1H),8.32(s,1H),7.74(d,J=8.0Hz,4H),7.58(d,J=8.2Hz,2H),7.44(d,J=7.9Hz,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 (75 MHz, DMSO-d 6 )δ169.27,165.85,164.65,151.25,148.05,142.38,139.77,139.11,136.5 0,136.14,135.94,133.37,133.20,131.63,129.68,128.87,127.42,127.19 ,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 CIF 6 O 4 [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), tri(o-tolyl)phosphine (18.17 mg, 0.06 mmol) and triethylamine (578.6 mg, 0.795 mL, 4.48 mmol) were dissolved in acetonitrile in sequence, and then heated at 100° C. for 12 h under an argon atmosphere. Acetonitrile was removed by distillation under reduced pressure, and purified by column chromatography (DCM: MeOH = 50: 1) to obtain Intermediate 13-1 as a white powder. Then, referring to the preparation method of Example 1, intermediate 1-10 was synthesized according to steps 1 to 8 in Example 1, and then amide condensation was performed with reference to step 9 in Example 1 to obtain intermediate 13-2, except that 5-methoxy-5-oxopentanoic acid was replaced with intermediate 13-1 in this example, and finally ester exchange was performed with hydroxylamine to obtain compound 13.

[0086] The obtained compound 13 was a white solid. Yield: 41%. 1 H NMR (300 MHz, DMSO-d 6)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 CIF 6 O 4 [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-oxoprop-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, the intermediate 1-10 was synthesized according to steps 1 to 8 in Example 1, and then the synthesis steps of the intermediate 13-1 described in Example 9 were followed, and then amide condensation was performed with reference to step 9 in Example 1 to obtain the intermediate, except that in this example, 4-bromobenzoic acid was replaced with 2-(4-bromophenyl)acetic acid, and finally ester exchange was performed with hydroxylamine to obtain compound 14.

[0089] The obtained compound 14 was a white solid. Yield: 36%. 1 H NMR (300 MHz, DMSO-d 6)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 CIF 6 O 4 [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)propionamido)-4-(4-methylpiperazin-1-yl)-[1,1'-biphenyl]-3-yl)-3-methylbenzamide (Compound 15)

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

[0092] The obtained compound 15 was a white solid. Yield: 32%. 1 H NMR (300 MHz, DMSO-d 6)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 CIF 6 O 4 [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-methylbenzamido)-N-(4-(3-(hydroxyamino)-3-oxoprop-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, the intermediate 1-10 was synthesized according to steps 1 to 8 in Example 1, and then the synthesis steps of intermediate 13-1 described in Example 13 were followed, and then amide condensation was performed with reference to step 9 in Example 1 to obtain the intermediate, except that in this example, 4-bromobenzoic acid was replaced with (4-bromophenyl)methylamine, and finally ester exchange was performed with hydroxylamine to obtain compound 16.

[0095] The obtained compound 16 was a white solid. Yield: 39%. 1 H NMR (300 MHz, DMSO-d 6)δ10.84(s,1H),9.51(s,1H),9.23(t,J=5.9Hz,1H),9.07(s,1H),8.35(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 (75 MHz, DMSO-d 6 )δ166.42,165.52,163.23,160.70,151.31,148.11,144.05,143.01,141.73,13 8.53,136.15,135.96,135.44,133.91,133.37,133.27,133.08,129.68,128.54 ,128.23,128.00,126.75,123.93,123.68,121.59,120.57,119.12,115.32,113 .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 CIF 6 O 4 [M+H] + :671.2548; Found 671.2543.HPLC purity:98.94%.mp247.4~248.1℃.

[0096] The present invention adopts the following method to express and purify WDR5 protein.

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

[0098] The recombinant plasmid was used to transfect the E. coli BL21 (DE3) strain, and the cells were revived in sterile LB medium (37°C). A single clone was picked and transferred to 10 mL LB liquid medium (containing 50 μg / mL ampicillin), cultured at 37°C with shaking (220 rpm) overnight, transferred to 1 L LB liquid medium (containing 50 μg / mL ampicillin), cultured at 37°C with shaking (220 rpm) for 6-8 hours until OD 600 was 0.6-0.8, cooled to 12°C, induced expression by adding 1 mM IPTG for 14-16 hours (180 rpm), and then the cells were harvested and stored at -80°C for later use.

[0099] 4 g of bacterial mass was added with 40 mL of bacterial lysis buffer (20 mM Tris-HCl buffer, pH 7.4, 300 mM NaCl, 20 mM β-mereaptoethanol, 1% Triton X-100 and 0.1% PMSF), and ultrasonic lysis was performed for 40 minutes. The lysed mixture was centrifuged at low temperature and high speed (12000 rpm, 20 min, 4°C), and the supernatant was filtered (0.4 μm microporous filter membrane). It was separated and purified by AKTA instrument using His column (balance solution (20 mM Tris-HCl, 300 mM NaCl, 5 mM imidazole, pH = 7.4; eluent: 20 mM Tris-HCl, 75 mM imidazole, 0.3 M NaCl, pH = 7.4), 10% SDS-PAGE was used to confirm the molecular weight and purity of the band, and dialyzed overnight (10% PBS, 300 mM NaCl, pH = 7.4). The obtained protein was stored at -80°C for future use after the concentration was determined by BCA. Experimental Example 1: Determination of the inhibitory activity of the compound on WDR5 based on fluorescence polarization (FP)

[0100] The instrument used in this experiment is SpectraMax Multi-Mode Microplate Reader (Molecular Devices), and the excitation and emission wavelengths of the instrument are 485nm and 535nm respectively. The protein used is WDR5, and the probe is a fluorescent probe FITC-AHx-SEEEIDVVSV-NH constructed based on the dominant sequence of Myc binding, 6-aminoacetic acid as the connecting chain, and the introduction of the fluorescent group fluorescein isothiocyanate (FITC) 2 The buffer solution used in the test system is: 0.1 mM sodium tetraborate, 1.6 mM boric acid, pH = 7.8 and 0.01% Triton X-100. The 384-well black board used in the experiment was produced by Corning.

[0101] Experimental steps:

[0102] The test system uses 60 μL, and 20 μL of compound, protein, and probe solution are added to each black well in turn. Each compound uses a 3-fold gradient dilution of 10 to 14 gradients, the initial concentration of the compound is 100 μM, and each concentration has one replicate well. Correspondingly, 20 μL of probe + 40 μL of buffer solution is used as a blank control, 20 μL of protein (200 nM) + 20 μL of probe (30 nM) + 20 μL of buffer solution is used as a negative control, and the positive drug is 6 days. After adding the sample, cover the 384-well plate with tin foil, shake on a shaker at room temperature for 20 minutes, and use SpectraMax Paradigm Multi-Mode Microplate Reader to read the fluorescence under the conditions of excitation wavelength of 485 nm and emission wavelength of 535 nm, calculate the mP value, calculate the inhibition rate using the following formula, and then use GraphPadPrism 5.0 to calculate the IC 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) x 100%. Experimental Example 2: Inhibitory activity of compounds on HDAC.

[0104] All enzymatic reactions were performed at 37°C for 30 min. The 50 μL reaction mixture contained 25 mM Tris, pH 8.0, 1 mM MgCl 2 , 0.1mg / ml BSA, 37mM NaCl, 2.7mM KCl, HDAC and enzyme substrate. The compound was diluted in 10% DMSO and 5μL of the dilution was added to 50μL of the reaction, so that the final concentration of DMSO was 1% in all reactions. The assay was performed by quantifying the amount of fluorescent product in the test solution after the enzyme reaction. The fluorescence was then analyzed on a SpectraMax M5 microtiter plate reader with an excitation wavelength of 350-360nm and an emission wavelength of 450-460nm. IC 50 Values ​​were calculated using nonlinear regression and normalized dose-response fits were 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 IC values ​​of the compounds of the present invention for WDR5 protein and HDAC 50 value

[0107]

[0108]

[0109] From the results in Table 1, it can be seen that the compounds of the present invention have significant inhibitory activity against WDR5 and HDAC, and can be used as WDR5-HDAC dual-target small molecule inhibitors.

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

[0111]

[0112] Experimental Example 3: Based on CellTiter-Lumi TM Luminescence assay to detect cell antiproliferative activity

[0113] The cell models used in this experiment were: human myelomonocytic leukemia cells MV4-11, human acute myeloid leukemia cells MOLM-13, human monocytic leukemia THP-1, human chronic myeloid leukemia cells K562, human non-small cell lung cancer cell line A549, human hepatoma cell line HuH-1, human ovarian adenocarcinoma cell line SKOV3, and human colon cancer cell line 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), 10% FBS (GiBco, Invitrogen Corp, USA);

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

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

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

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

[0119] Table 3 Antiproliferative activity of compounds

[0120]

[0121] As can be seen from Table 3, compounds 9 and 16 exhibited strong antiproliferative activity against tumor cells, among which compound 16 had the best inhibitory activity against MV4-11 cells.

[0122] The superior compounds 9, 16 and the combination were tested in vitro for their antiproliferative activity in leukemia cells and solid tumor cells, such as Figure 1 As shown, the results showed that compounds 9 and 16 had significant antiproliferative activity against human myelomonocytic leukemia cells MV4-11, human acute myeloid leukemia cells MOLM-13, human monocytic leukemia 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 effects were observed in leukemia cell lines.

[0123] In summary, it can be seen that the co-inhibition of WDR5-MLL1 interaction and HDAC may improve the anti-cancer activity through dual-target inhibitors in MLL-rearranged AML cells. The dual-function inhibitor of WDR5-MLL1 protein-protein interaction (PPI) and HDAC is an effective strategy as a new type of anti-tumor agent, and provides a molecular tool for exploring the potential mechanism between MLL1 and HDAC. The WDR5-HDAC dual-target compound provided by the present invention has obvious inhibitory activity on WDR5 and HDAC, and is an effective WDR5-HDAC dual-target inhibitor. Therefore, the drug containing the above compound as an active ingredient can be used to prepare a drug for treating clinical conditions related to WDR5-HDAC.

[0124] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A compound as shown in formula I or a pharmaceutically acceptable salt thereof: in: R 1 is C1-C7 alkyl or C1-C7 haloalkyl, naphthyl, pyridyl, piperazinyl, piperidinyl, phenyl or substituted phenyl, the substituent of substituted phenyl is C1-C7 alkyl, C1-C7 haloalkyl, cyano, hydroxyl, mercapto, halogen, nitro, amino, methoxy or trifluoromethyl; R 2 -NH-OH L 1 , L 2 are independently selected from carbonyl, sulfonyl, amino, imino, aminoidene, ether, thioether, methylene or methylidene.

2. The compound according to claim 1, characterized in that The L 1 , L 2 are independently selected from carbonyl, imino, sulfonyl or methylene.

3. The compound according to claim 1, characterized in that The pharmaceutically acceptable salts include acid addition salts formed by the compound of general formula I and 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, mandelic acid; and also include basic salts formed by the compound of general formula I and inorganic bases: sodium salt, potassium salt, lithium salt, calcium salt, zinc salt, magnesium salt, meglumine salt, tromethamine salt, diethylamine salt or ethanolamine salt.

4. The compound according to claim 1, characterized in that The compound shown in formula I is one of the following structural formulas:

5. A pharmaceutical composition comprising the compound of formula I or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, and a pharmaceutically acceptable carrier or excipient.

6. Use of the compound of formula I or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4 in the preparation of a WDR5-HDAC dual-target small molecule inhibitor.

7. Use of the compound of formula I according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof or the pharmaceutical composition according to claim 5 in the preparation of a therapeutic drug for diseases associated with WDR5 and / or HDAC dysfunction.

8. The use according to claim 6, characterized in that The disease associated with WDR5 and / or HDAC dysfunction is leukemia, pancreatic cancer, bile duct cancer, colon cancer, lung cancer, glioblastoma, ovarian cancer, cervical cancer, breast cancer or gastric cancer.

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