2-thiazolylamino phenol derivatives and use thereof

Abstract

The application discloses a 2-thiazole amino phenol derivative and application thereof. The 2-thiazole amino phenol derivative has obvious inhibitory activity on WDR5 protein, can be used as a WDR5 small molecule inhibitor, and is used for treating diseases related to WDR5. The small molecule has good activity in various experiments, and is hopeful to be developed into a specific anti-tumor drug.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry technology, specifically relating to a class of 2-thiazolaminophenol 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. In summary, as a member of the WD40 protein family, WDR5 plays an important biological role in the initiation, development, and maintenance of disease. Therefore, developing small molecule inhibitors of WDR5 is an effective strategy for treating cancer.

[0003] Currently, there are no commercially available small molecule inhibitors of WDR5. Therefore, designing and synthesizing safe and efficient small molecule inhibitors of WDR5 has extremely high application value and is expected to become a promising candidate anti-tumor drug. Summary of the Invention

[0004] One object of the present invention is to provide a class of 2-thiazolaminophenol derivatives or pharmaceutically acceptable salts thereof, said derivatives having a structure as shown in Formula I:

[0005]

[0006] in:

[0007] R1 and R2 may be the same as or different from each other, and are each independently selected from H, cyano, hydroxyl, mercapto, halogen, nitro, amino, C1-C4 alkyl, C1-C4 alkoxy, -COOR a -CONR b R c -CONHOH, or C1-C4 alkylamino; or R1 and R2 are each independently selected from unsubstituted or substituted by any of the R groups. A The following groups are substituted: C3-C10 cycloalkyl, C3-C10 aryl, 5-8 membered heterocyclic or 5-8 membered heteroaryl; RA Selected from H, cyano, hydroxy, mercapto, halogen, nitro, amino, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 alkoxy, C1-C4 alkylacyl or C1-C4 alkylsulfonyl;

[0008] R a R b R c Each independently represents hydrogen, C1-C4 alkyl, unsubstituted or with any number of R A Substituted aryl or heteroaryl;

[0009] R3 can be H, hydroxyl, amino, nitro, carboxyl, cyano, halogen, C1-C4 alkyl, C1-C3 alkoxy, C1-C4 alkylsulfonyl, C1-C4 alkylsulfonylamino, or -C(O)-XR. d ;

[0010] X is selected from covalent bonds, -O-, or -NH-;

[0011] R d Selected from C1-C4 alkyl groups, or unsubstituted or with any number of R groups. B The following groups are substituted: C3-C10 cycloalkyl, C3-C10 aryl, 5-8 membered heterocyclic or 5-8 membered heteroaryl; R B It is selected from H, cyano, hydroxy, mercapto, halogen, nitro, amino, C1-C4 alkyl, C1-C4 haloalkyl, C1-C4 alkoxy, C1-C4 alkyl acyl or C1-C4 alkyl sulfonyl.

[0012] In a specific instance, R1 is H, halogen, methyl, ethyl, nitro, or unsubstituted or modified by any number of Rs. 1A The following groups are substituted: C3-C10 aryl or 5-8 membered heteroaryl. In a specific example, R 1A It is selected from H, cyano, hydroxy, mercapto, halogen, nitro, amino, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 alkyl acyl or C1-C3 alkyl sulfonyl.

[0013] In a specific instance, R2 is H, halogen, methyl, ethyl, n-propyl, isopropyl, or unsubstituted or modified by any number of R. 2A The following groups are substituted: C3-C8 cycloalkyl, C3-C10 aryl, 5-8 membered heterocyclic or 5-8 membered heteroaryl; in a specific example, R 2A Selected from H, cyano, hydroxy, mercapto, halogen, nitro, amino, C1-C3 alkyl, C1-C3 haloalkyl, C1-C3 alkoxy, C1-C3 alkyl acyl or C1-C3 alkyl sulfonyl.

[0014] In a specific instance, R3 is H, hydroxyl, amino, nitro, carboxyl, cyano, halogen, methyl, ethyl, n-propyl, isopropyl, -C(O)-XR d In a specific instance, X is selected from covalent bonds, -O-, or -NH-; R d Selected from methyl, ethyl, n-propyl, isopropyl, or unsubstituted or modified by any number of R B Substitute the following groups: C3-C10 cycloalkyl or 5-8 membered heterocyclic groups; R B It is selected from H, cyano, hydroxy, mercapto, halogen, nitro, amino, C1-C3 alkyl, C1-C3 haloalkyl, C1-C3 alkoxy, C1-C3 alkylacyl, or C1-C3 alkylsulfonyl. In one specific example, R3 is a carboxyl group.

[0015] In some embodiments, the cycloalkyl group described in this invention is cyclopropane, cyclobutane, cyclopentane, or cyclohexane.

[0016] In some embodiments, the aryl group of the present invention is benzene or naphthalene, preferably phenyl.

[0017] In some embodiments, the heteroaryl group of the present invention is a 4-10 member heteroaryl group containing 1-4 heteroatoms selected from O, N or S, preferably a 4-10 member heteroaryl group containing 1-3 heteroatoms selected from O, N or S, and more preferably a 4-10 member heteroaryl group containing 1 or 2 heteroatoms selected from O, N or S; in some specific examples, the heteroaryl group is selected from pyridinyl, oxazolyl, isoxazolyl, indolyl, thiazolyl, pyrazolyl, imidazolyl, pyrroleyl, furanyl, thiophene, isothiazolyl, pyrimidinyl, triazolyl, 1,3,4-oxadiazolyl, 1,3,4-thiadiazolyl, 1,2,4-oxadiazolyl or 1,2,4-thiadiazolyl.

[0018] In some embodiments, the heterocyclic group is a 4-10 membered heterocyclic group containing 1-4 heteroatoms selected from O, N or S, preferably a 4-10 membered heterocyclic group containing 1-3 heteroatoms selected from O, N or S; in some specific examples, the heterocyclic group includes piperidinyl, piperazineyl, morpholinyl, thiomorpholinyl, 1,1-thiomorpholinyl dioxide, pyrrolidinyl, 1,3-oxopentacyclic, tetrahydropyranyl, tetrahydropyranyl, tetrahydropyrazolyl or 1,4-dioxohexacyclic.

[0019] In some specific instances, the present invention provides a class of 2-thiazolamine derivatives or pharmaceutically acceptable salts thereof, said derivatives having a structure as shown in formula I-a or I-b:

[0020]

[0021] R1, R2, and R3 are as described above.

[0022] Another object of the present invention is to provide a pharmaceutical composition comprising the 2-thiazolaminophenol derivative of the present invention or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

[0023] 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.

[0024] 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.

[0025] Another object of the present invention is to provide the use of compounds of Formula I or pharmaceutically acceptable salts thereof in the preparation of therapeutic remedies for diseases associated with WDR5 dysfunction.

[0026] The diseases associated with WDR5 dysfunction are leukemia, pancreatic cancer, bile duct cancer, colon cancer, lung cancer, liver cancer, glioblastoma, ovarian cancer, cervical cancer, prostate cancer, breast cancer, or stomach cancer.

[0027] Beneficial effects:

[0028] The compound of the present invention, as shown in Formula I, has significant inhibitory activity against WDR5 protein and can be used as a small molecule inhibitor of WDR5 for the treatment of diseases related to WDR5. This small molecule has shown good activity in various experiments and is expected to be developed into a specific anti-tumor drug. Detailed Implementation

[0029] 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.

[0030] The starting materials and reaction reagents used in the specific embodiments of this invention are all commercially available.

[0031] 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.

[0032] The structure of the compound was determined by nuclear magnetic resonance (NMR). ¹H NMR and ¹³C NMR spectra were measured using a Bruker AV-300 NMR spectrometer with deuterated dimethyl sulfoxide (DMSO-d6) as the solvent and tetramethylsilane (TMS) as the internal standard.

[0033] 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.

[0034] Unless otherwise specified in the embodiments, the reaction is carried out in an air atmosphere.

[0035] Unless otherwise specified in the examples, the reaction temperature is room temperature, ranging from 20°C to 30°C.

[0036] Example 1: Preparation of Compound 1

[0037]

[0038] Step 1: Synthesize compounds 1-3

[0039] Compound 1-1 (3 g, 24.36 mmol) was dissolved in THF (50 mL), followed by the slow dropwise addition of compound 1-2 (3.98 g, 24.36 mmol) under ice bath conditions. After the addition was complete, the reaction was allowed to proceed at room temperature for 6 h. The reaction was confirmed to be complete by TLC. The solvent was then removed by vacuum distillation to obtain a pale yellow crude solid. Anhydrous ethanol (50 mL) was added to the crude solid for recrystallization. Filtration yielded 4 g of a white solid, with a yield of 57.31%. 1H NMR (300MHz, DMSO-d6) δ11.28(s,1H),10.65(s,1H),7.91(dd,J=7.3,1.6Hz,1H),7.82(m,2H ),7.63-7.51(m,1H),7.56-7.43(m,2H),7.23-7.11(m,1H),7.03-6.91(m,2H),3.85(s,2H).

[0040] Step 2: Synthesize compounds 1-4

[0041] Compounds 1-3 (3 g, 10.48 mmol) were dissolved in anhydrous ethanol, followed by the addition of anhydrous potassium carbonate (1.74 g, 12.57 mmol). The mixture was refluxed at 80 °C for 8 h. After the reaction was complete, the reaction solution was removed by vacuum distillation. The crude compound was then dissolved in EA and washed three times with water. The organic phase was concentrated to give 1.5 g of a pale yellow solid, with a yield of 78.95%. This can be directly used in the next step. 1 H NMR (300MHz, DMSO-d6) δ9.63(s,1H),7.98(dd,J=7.9,1.3Hz,1H),7.72(s,2H),7.18(td,J=8.1,1.4Hz,1H),7.07-6.92(m,2H).

[0042] Step 3: Synthesize compounds 1-6

[0043] Compounds 1-4 (400 mg, 2 mmol) were dissolved in ethanol, followed by the addition of triethylamine (809 mg, 7.99 mmol), and finally compound 1-5 (1.08 mg, 5.99 mmol) was slowly added dropwise. The solution changed from yellow to dark brown. The mixture was then refluxed at 80 °C for 8 h. After the reaction was complete, the reaction solution was concentrated, and the solution was purified by column chromatography to obtain a pale yellow key intermediate 1-6 (120 mg, 21.28%). 1 H NMR (300MHz, DMSO-d6) δ9.79 (s, 1H), 7.67 (s, 1H), 7.61 (dd, J = 7.7, 1.4Hz, 1H), 7.20-6.92 (m, 3H), 3.90 (s, 3H), 3.84 (s, 3H).

[0044] Step 4: Synthesize compounds 1-7

[0045] Compounds 1-6 (150 mg) were dissolved in methanol, and 5 mol / L sodium hydroxide was added. The mixture was stirred at room temperature for 4 h until the reaction was complete. The pH was adjusted to 5 with HCl, and a pale yellow solid precipitated out. The solid was then filtered to obtain compounds 1-7 (100 mg, 70.63%). 1H NMR (300MHz, DMSO-d6) δ10.44(s,1H),9.79(s,1H),7.86(s,1H),7.61(dd,J=7.7,1.4Hz,1H),7.19-6.93(m,3H),3.84(s,3H).

[0046] Step 5: Synthesize compound 1

[0047] Compounds 1-7 (150 mg) were dissolved in DCM, and BBr3 (1 mL, 2 mol / L DCM) was added dropwise at -20 °C, and the reaction was continued overnight. After the reaction was complete, the reaction was quenched with methanol, and the reaction solution was removed by vacuum distillation and purified by silica gel column chromatography (PE:EA = 10:1). A pale yellow solid, compound 1 (100 mg, 70.63%), was obtained. 1 H NMR (300MHz, DMSO-d6) δ12.68(s,1H),9.92(s,1H),9.61(s,1H),8.26-8.16(m,1H),7.64(s,1H),6.86(d,J=4.4Hz,2H),6.81(dd,J=7.5,3.8Hz,1H).

[0048] Example 2: Preparation of Compound 2

[0049]

[0050] Compound 2 was synthesized according to steps 3 and 5 of Example 1, except that in step 3 of this example, compounds 1-5 were replaced with bromoacetone.

[0051] Compound 2 is a pale yellow solid with a yield of 48.06%. 1 H NMR(300MHz,DMSO-d6)δ11.16(s,1H),7.37(dd,J=7.9,1.6Hz,1H),7.23(m,1H),7.0 6(dd,J=8.2,1.4Hz,1H),6.91(m,1H),6.66(d,J=1.5Hz,1H),2.20(d,J=1.3Hz,3H).

[0052] Example 3: Preparation of Compound 3

[0053]

[0054] The synthesis steps are the same as in Example 2, except that bromoacetone is replaced with 1-bromo-2,3-butanedione.

[0055] Compound 3 is a pale yellow solid with a yield of 53%. 1H NMR (300MHz, DMSO-d6) δ9.90(s,1H),9.63(s,1H),8.29-8.16(m,1H),7.73(s,1H),6.88-6.84(m,2H),6.84-6.78(m,1H),2.49-2.48(m,3H).

[0056] Example 4: Preparation of Compound 4

[0057]

[0058] Step 1: Synthesize compound 4-3

[0059] Compound 4-1 (1 g, 4.31 mmol) was dissolved in 1,4-dioxane, followed by compound 4-2 (555.29 mg, 6.46 mmol), and then cesium carbonate aqueous solution (2.81 g, 8.62 mmol) and palladium catalyst (151.25 mg, 0.216 mmol) were added sequentially. The reaction was carried out at 135 °C for 10 h under nitrogen protection. The reaction was then confirmed to be complete by TLC. The reaction mixture was evaporated to dryness, dissolved in DCM, and filtered to remove undissolved cesium carbonate and palladium catalyst. The combined organic phases were then purified by column chromatography to give compound 4-3 in 58% yield. 1 H NMR (300MHz, DMSO-d6) δ7.98 (dd, J=2.2, 0.8Hz, 1H), 7.34 (ddd, J=8.2, 2.1, 0.7Hz, 1H), 7. 01(d,J=8.2Hz,1H),3.98(s,3H),3.04-2.90(m,1H),1.64-1.48(m,2H),1.38-1.24(m,2H).

[0060] Step 2: Synthesize compound 4-4

[0061] Compound 4-3 was dissolved in ethyl acetate, and stannous chloride was added. The mixture was heated to 80°C and reacted for 4 hours. The reaction was then confirmed to be complete by TLC. The pH was adjusted to 8 with sodium bicarbonate and 5 mol / L NaOH, and the upper organic phase was concentrated under reduced pressure to obtain compound 4-4 (200 mg). 1 H NMR (300MHz, DMSO-d6) δ6.93-6.79(m,3H),4.69(s,2H),3.87(s,3H),2.44-2.30(m,1H),1.76-1.62(m,2H),1.40-1.46(m,2H).

[0062] Step 3: Synthesize compounds 4-5

[0063] Compound 4-4 (2 g, 14.17 mmol) was dissolved in tetrahydrofuran (50 mL), followed by the slow dropwise addition of 1-2 (2.31 g, 14.17 mmol) under ice bath conditions. After the addition was complete, the reaction was allowed to proceed at room temperature for 6 h. The reaction was confirmed to be complete by TLC. The solvent was then removed by vacuum distillation to obtain a pale yellow crude solid. Anhydrous ethanol (50 mL) was then added to the crude solid and the mixture was stirred. Filtration yielded 2.62 g of a yellow solid, with a yield of 65.51%. 1 H NMR (300MHz, DMSO-d6) δ11.57(s,1H),11.42(s,1H),7.69(m,3H),7.67-7.49(m,3H),7. 15-7.02(m,2H),3.85(s,3H),3.56-3.42(m,1H),1.06-0.86(m,2H),0.74-0.54(m,2H).

[0064] Step 4: Synthesize compounds 4-6

[0065] Compound 4-5 (2 g, 6.13 mmol) was dissolved in anhydrous ethanol, followed by the addition of anhydrous potassium carbonate (817.4 mg, 5.91 mmol). The mixture was refluxed at 80 °C for 8 h. After the reaction was complete, the reaction solution was removed by vacuum distillation. The crude compound was then dissolved in EA and washed three times with water. The organic phase was concentrated to give 958 mg of a brown solid, with a yield of 70.44%. This can be directly added to the next step. 1 H NMR (300MHz, DMSO-d6) δ9.81(s,1H),7.72(s,2H),7.63(dd,J=2.1,0.8Hz,1H),7.07(ddd,J=8.8, 2.1,0.7Hz,1H),6.95(d,J=8.8Hz,1H),2.62-2.48(m,1H),1.76-1.60(m,2H),1.41-1.47(m,2H).

[0066] Step 5: Synthesize compounds 4-7

[0067] Compounds 4-6 (400 mg, 1.80 mmol) were dissolved in ethanol, followed by the addition of triethylamine (728.32 mg, 1.00 mL, 7.20 mmol), and finally, compounds 1-5 (976.95 mg, 0.575 mL, 5.40 mmol) were slowly added dropwise. The solution changed from yellow to dark brown. The mixture was then refluxed at 80 °C for 6 h. After the reaction was complete, the reaction solution was concentrated, and the solution was purified by column chromatography to obtain 214 mg of a pale yellow solid, with a yield of 39.08%. 1H NMR (300MHz, DMSO-d6) δ9.79 (s, 1H), 7.67 (s, 1H), 7.33 (dd, J = 2.3, 0.7Hz, 1H), 6.99 (d, J = 8.8Hz, 1H), 6.92-6.82(m,1H),3.90(s,3H),3.84(s,3H),3.56-3.42(m,1H),1.06-0.86(m,2H),0.74-0.54(m,2H).

[0068] Step 6: Synthesize compounds 4-8

[0069] Compound 4-7 (150 mg, 0.493 mmol) was dissolved in methanol, and 5 mol / L sodium hydroxide was added. The mixture was stirred at room temperature for 4 h until the reaction was complete. The pH was adjusted to 3 with HCl, and a pale yellow solid precipitated. The solid was filtered to obtain 103 mg of pale yellow solid, with a yield of 71.98%. 1 H NMR (300MHz, DMSO-d6) δ10.44(s,1H),9.79(s,1H),7.86(s,1H),7.33(dd,J=2.3,0.7Hz,1H),6.99(d,J =8.8Hz,1H),6.92-6.82(m,1H),3.84(s,3H),3.56-3.42(m,1H),1.06-0.86(m,2H),0.74-0.54(m,2H).

[0070] Step 7: Synthesize compound 4

[0071] Compounds 4-8 (100 mg, 0.344 mmol) were dissolved in DCM, and BBr3 (1 mL, 2 mol / L in DCM) was added dropwise at -20 °C, and the reaction was continued overnight. After the reaction was complete, the reaction was quenched with methanol, and the reaction solution was removed by vacuum distillation. The solution was then purified by silica gel column chromatography (PE:EA = 10:1). 48 mg of a pale yellow solid was given, with a yield of 50.44%. 1 H NMR (300MHz, DMSO-d6) δ9.54(s,1H),7.86(d,J=2.0Hz,1H),7.62(s,1H),6.77(d,J=8.1Hz,1H),6.7 0(dd,J=8.1,2.0Hz,1H),2.44(d,J=7.6Hz,1H),1.56(m,J=7.3Hz,2H),0.89(td,J=7.3,2.4Hz,2H).

[0072] Example 5: Preparation of Compound 5

[0073]

[0074] Compound 5 was synthesized according to steps 3-7 of Example 4, except that compounds 4-4 were replaced with 2-methoxy-5-fluoroaniline.

[0075] Compound 5 is a pale yellow solid with a yield of 54.87%. 1 H NMR (300MHz, DMSO-d6) δ12.74(s,1H),9.92(s,1H),9.87(s,1H),8.39(dd,J=11.7, 3.1Hz,1H),7.73(s,1H),6.82(dd,J=8.7,5.6Hz,1H),6.63(td,J=8.5,3.1Hz,1H). 13 C NMR (75MHz, DMSO-d6) δ163.41,162.71,145.09,143.26,130.72,123.00,121.59,119.85,118.02,115.91.

[0076] Example 6: Preparation of Compound 6

[0077]

[0078] Compound 6 was synthesized according to steps 3-7 of Example 4, except that compounds 4-4 were replaced with 5-chloro-2-methoxyaniline.

[0079] Compound 6 is a pale yellow solid with a yield of 45.37%. 1 H NMR (300MHz, DMSO-d6) δ10.33(s,1H),9.92(s,1H),8.62(t,J=1.4Hz,1H),7.79(s,1H),6.91(d,J=1.5Hz,2H).

[0080] Example 7: Preparation of Compound 7

[0081]

[0082] Compound 7 was synthesized according to steps 3-7 of Example 4, except that compounds 4-4 were replaced with 5-bromo-2-methoxyaniline.

[0083] Compound 7 is a pale yellow solid with a yield of 53.81%. 1 H NMR (300MHz, DMSO-d6) δ10.44(s,1H),9.79(s,1H),9.75(s,1H),7.86(s,1H),7.30(dd,J=8.2,2.3Hz,1H),7.07(d,J=2.2Hz,1H),6.83(d,J=8.2Hz,1H).

[0084] Example 8: Preparation of Compound 8

[0085]

[0086] Compound 8 was synthesized according to steps 3-7 of Example 4, except that compounds 4-4 were replaced with 2-methoxy-5-methylaniline.

[0087] Compound 8 is a pale yellow solid with a yield of 49.59%. 1 H NMR (300MHz, DMSO-d6) δ9.56(s,1H),7.93(d,J=2.0Hz,1H),7.65(s,1H),6.77(d,J=8.0Hz,1H),6.69(dd,J=8.1,2.1Hz,1H),2.23(s,3H).

[0088] Example 9: Preparation of Compound 9

[0089]

[0090] Compound 9 was synthesized according to steps 3-7 of Example 4, except that compounds 4-4 were replaced with 2-methoxy-5-ethylaniline.

[0091] Compound 9 is a pale yellow solid with a yield of 43.18%. 1 H NMR (300MHz, DMSO-d6) δ10.44(s,1H),9.79(s,1H),9.75(s,1H),7.86(s,1H),7.04(dt,J=2. 1,1.0Hz,1H),6.93(d,J=8.5Hz,1H),6.62(m,1H),2.81-2.67(m,2H),1.28(t,J=7.2Hz,3H).

[0092] Example 10: Preparation of Compound 10

[0093]

[0094] Compound 10 was synthesized according to steps 3-7 of Example 4, except that compounds 4-4 were replaced with 4,5-difluoro-2-methoxyaniline.

[0095] Compound 10 is a pale yellow solid with a yield of 68.98%. 1H NMR (300MHz, DMSO-d6) δ10.40(s,4H),9.94(s,1H),8.52(dd,J=13.6,9.0Hz,1H),7.72(s,1H),6.88(dd,J=11.8,7.9Hz,1H).

[0096] Example 11: Preparation of Compound 11

[0097]

[0098] Compound 11 was synthesized according to steps 3-7 of Example 4, except that compounds 4-4 were replaced with 5-fluoro-4-bromo-2-methoxyaniline.

[0099] Compound 11 is a pale yellow solid with a yield of 68.98%. 1 H NMR (300 MHz, DMSO-d6) δ10.40(s,4H),9.94(s,1H),8.52(dd,J=13.6,9.0 Hz,1H),7.72(s,1H),6.88(dd,J=11.8,7.9 Hz,1H).

[0100] Example 12: Preparation of Compound 12

[0101]

[0102] Compound 12 was synthesized according to steps 3-7 of Example 4, except that compounds 4-4 were replaced with 5-fluoro-3-bromo-2-methoxyaniline.

[0103] Compound 12 is a pale yellow solid with a yield of 47.52%. 1 H NMR (300 MHz, DMSO-d6) δ10.17(s,1H),9.54(s,1H),8.38(dd,J=11.4,3.1 Hz,1H),7.78(s,1H),7.07(dd,J=8.0,3.0 Hz,1H).

[0104] Example 13: Preparation of Compound 13

[0105]

[0106] Compound 13 was synthesized according to steps 3-7 of Example 4, except that compounds 4-4 were replaced with 3,5-difluoro-2-methoxyaniline.

[0107] Compound 13 is a pale yellow solid with a yield of 75.29%. 1H NMR (300 MHz, DMSO-d6) δ10.17 (d, J = 14.5 Hz, 1H), 8.33-8.21 (m, 1H), 7.76 (s, 1H), 6.93-6.67 (m, 1H).

[0108] Example 14: Preparation of Compound 14

[0109]

[0110] Compound 14 was synthesized according to steps 3-7 of Example 4, except that compounds 4-4 were replaced with 3-chloro-5-bromo-2-methoxyaniline.

[0111] Compound 14 is a yellow solid with a yield of 68.02%. 1 H NMR (300 MHz, DMSO-d6) δ10.15 (d, J = 13.6 Hz, 2H), 8.63 (dd, J = 4.1, 2.4 Hz, 1H), 7.78 (s, 1H), 7.23 (t, J = 2.4 Hz, 1H).

[0112] Example 15: Preparation of Compound 15

[0113]

[0114] Compound 15 was synthesized according to steps 3-7 of Example 4, except that compounds 4-4 were replaced with 5-chloro-3-bromo-2-methoxyaniline.

[0115] Compound 15 is a yellow solid with a yield of 54.82%. 1 H NMR (300MHz, DMSO-d6) δ10.15(s,1H),10.03(s,1H),8.48(d,J=2.5Hz,1H),7.78(s,1H),7.25(d,J=2.5Hz,1H).

[0116] Example 16: Preparation of Compound 16

[0117]

[0118] Compound 16 was synthesized according to steps 3-7 of Example 4, except that compounds 4-4 were replaced with 3,5-dibromo-2-methoxyaniline.

[0119] Compound 16 is a pale yellow solid with a yield of 42.31%. 1H NMR (300MHz, DMSO-d6) δ12.77(s,1H),10.08(s,1H),10.04(s,1H),8.53(d,J=2.4Hz,1H),7.76(s,1H),7.35(d,J=2.4Hz,1H).

[0120] Example 17: Preparation of Compound 17

[0121]

[0122] Compound 17 was synthesized according to steps 3-7 of Example 4, except that compounds 4-4 were replaced with 5-bromo-3-fluoro-2-methoxyaniline.

[0123] Compound 17 is a yellow solid with a yield of 46.82%. 1 H NMR (300MHz, DMSO) δ10.08 (s, 1H), 8.51 (t, J = 2.0Hz, 1H), 7.76 (s, 1H), 7.11 (dd, J = 9.9, 2.4Hz, 1H).

[0124] Example 18: Preparation of Compound 18

[0125]

[0126] Step 1: Synthesize compound 18-1

[0127] Compound 5-5 (400 mg) was dissolved in DCM, followed by the addition of triethylamine (150.89 mg, 0.207 mL, 1.49 mmol), then HOBT (151 mg, 1.12 mmol) and EDCI (214.38 mg, 1.12 mmol) were added sequentially. The reaction was carried out at room temperature for 15 minutes, and finally methanol (120.94 mg, 0.895 mmol) was added. The reaction was continued for 6 hours, and the reaction was confirmed to be complete by TLC. The solvent was then evaporated, and the product was dissolved in EA and washed three times with water. The organic phases were combined and purified by sand separation to obtain 260 mg of a pale yellow solid, with a yield of 62.15%. 1 H NMR (300MHz, DMSO-d6) δ9.79(s,1H),7.67(s,1H),7.19-7.09(m,1H),6.98-6.84(m,2H),3.90(s,3H),3.84(s,3H).

[0128] Step 2: Synthesize compound 18

[0129] Compound 18-2 (150 mg) was dissolved in DCM, and BBr3 (1 mL, 2 mol / L DCM) was added dropwise at -20 °C, and the reaction was continued overnight. After the reaction was complete, the reaction was quenched with methanol / ether, and the solvent was removed by vacuum distillation. The solution was then purified by silica gel column chromatography (PE:EA = 10:1). 54 mg of a pale yellow solid was given, with a yield of 38.28%. 1 H NMR (300MHz, DMSO-d6) δ9.94(s,2H),8.36(dd,J=11.6,3.1Hz,1H),7.82(s,1H),6.84(dd,J=8.7,5.6Hz,1H),6.64(td,J=8.5,3.1Hz,1H),3.82(s,3H).

[0130] Example 19: Preparation of Compound 19

[0131]

[0132] Compound 19 was synthesized according to steps 1-2 of Example 18, except that methylamine was used instead of methanol.

[0133] Compound 19 is a pale yellow solid with a yield of 39.99%. 1 H NMR(300MHz, DMSO-d6)δ9.76(s,1H),8.30(dd,J=11.2,3.1Hz,1H),8.09(d,J=4.5Hz,1H),7 .46(s,1H),6.81(dd,J=8.7,5.5Hz,1H),6.61(td,J=8.5,3.1Hz,1H),2.79(d,J=4.4Hz,3H).

[0134] Example 20: Preparation of Compound 20

[0135]

[0136] Compound 20 was synthesized according to steps 1-2 of Example 18, except that methanol was replaced with thiazoline 1,1-dioxide hydrochloride.

[0137] Compound 20 is a pale yellow solid with a yield of 48%. 1H NMR (300MHz, DMSO-d6) δ9.94(s,1H),9.81(s,1H),8.42(d,J=8.3Hz,1H),8.31(dd,J=11.3,3.1Hz,1H),7.58(s,1H),6.84(dd,J=8.7, 5.5Hz,1H),6.65(td,J=8.5,3.1Hz,1H),4.72(h,J=8.4Hz,1H),3.55-3.34(m,2H),3.25(m,2H),2.50-2.40(m,1H),2.39-2.24(m,1H).

[0138] Example 21: Preparation of compound 21

[0139]

[0140] Compound 21 was synthesized according to steps 1-2 of Example 18, except that methanol was replaced with thiomorphon dioxide.

[0141] Compound 21 is a yellow solid with a yield of 56.87%. 1 H NMR (300MHz, DMSO-d6) δ9.94(s,1H),9.81(s,1H),8.42(d,J=8.3Hz,1H),8.31(dd,J=11.3,3.1Hz,1H),7.58(s,1H),6.84(dd,J=8.7,5.5 Hz,1H),6.65(td,J=8.5,3.1Hz,1H),4.72(h,J=8.4Hz,1H),3.55-3.34(m,2H),3.32-3.14(m,2H),2.50-2.40(m,1H),2.39-2.24(m,1H).

[0142] Example 22: Preparation of compound 22

[0143]

[0144] Compound 22 was synthesized according to steps 1-2 of Example 18, except that methanol was replaced with 1-methylsulfonyl-4-aminopiperidine.

[0145] Compound 22 is a yellow solid with a yield of 62.8%. 1H NMR(300MHz,DMSO-d6)δ9.79(s,1H),8.24(dd,J=11.4,3.0Hz,1H),7.94(d,J=8.2Hz,1H),7.51(s,1H),6.95-6 .76(m,1H),6.64(td,J=8.5,3.0Hz,1H),3.99-3.68(m,1H),3.55(d,J=11.9Hz,2H),2.88(s,3H),2.85(s,2H).

[0146] Example 23: Preparation of compound 23

[0147]

[0148] Compound 23 was synthesized according to steps 1-2 of Example 18, except that compound 18-1 was replaced with cyclopentylamine.

[0149] Compound 23 is a yellow solid with a yield of 43.49%. 1 H NMR (300MHz, DMSO-d6) δ9.92(s,1H),9.78(s,1H),8.23(dd,J=11.5,3.1Hz,1H),7.71(d,J=7.9Hz,1H),7.48(s,1H),6.83(dd,J=8.8,5 .5Hz,1H),6.64(td,J=8.5,3.1Hz,1H),4.18(p,J=6.9Hz,1H),1.89(dd,J=11.0,6.3Hz,2H),1.71(t,J=6.6Hz,2H),1.64-1.46(m,4H).

[0150] Example 24: Preparation of compound 24

[0151]

[0152] Step 1: Synthesize compound 24-3

[0153] Compound 24-1 (3.00 g, 12.71 mmol) was dissolved in acetonitrile (50 mL), followed by the addition of anhydrous potassium carbonate (5.27 g, 38.14 mmol), and finally compound 24-2 (2.17 g, 12.71 mmol) was added dropwise. After the addition was complete, the mixture was refluxed at 80 °C for 6 h, and the reaction was confirmed to be complete by TLC. The solvent was then removed by vacuum distillation to give 4.10 g of a brown solid product, with a yield of 98.80%. 1HNMR (300MHz, DMSO-d6) δ7.67-7.63(dd,1H),7.62-7.58(dd,1H),7.48-7.38(m,2H),7.39-7.26(m,3H),5.03(s,2H).

[0154] Step 2: Synthesize compound 24-4

[0155] Compound 24-3 (1.10 g, 3.37 mmol) was dissolved in anhydrous ethanol (30 ml), followed by the addition of water (10 ml), reduced iron powder (753.46 mg, 13.49 mmol), and ammonium chloride solid (1.80 g, 33.73 mmol). The mixture was refluxed at 80 °C for 4 h. After the reaction was complete, the iron powder was removed by filtration. Then, ethanol and water were removed by vacuum distillation. Finally, the crude compound was dissolved in EA and washed three times with water. The organic phase was concentrated to give 990 mg of a pale yellow oily product, with a yield of 90.10%. This product can be directly used in the next step. 1 H NMR (300MHz, DMSO-d6) δ7.48-7.23(m,5H),7.00(dd,J=12.1,2.1Hz,1H),6.67(d,J=2.1Hz,1H),5.14(s,2H),4.36(s,2H).

[0156] Step 3: Synthesize compound 24-5

[0157] Compound 24-4 (990 mg, 3.34 mmol) was dissolved in THF (20 mL), followed by the slow dropwise addition of compound 1-2 (545.56 mg, 3.34 mmol) under ice bath conditions. After the addition was complete, the reaction was allowed to proceed at room temperature for 6 h. The reaction was confirmed to be complete by TLC. The solvent was then removed by vacuum distillation to obtain a pale yellow crude solid. Anhydrous ethanol (30 mL) was then added to the crude solid and the mixture was stirred. Filtration yielded 1.24 g of a white solid, with a yield of 88.57%. 1 H NMR (300MHz, DMSO-d6) δ7.53-7.40(m,2H),7.30-7.20(m,4H),7.15(ddq,J=6.3,1.5,0.8Hz,2H),7.11-7.00(m,3H),6.99-6.92(m,1H),4.87(s,2H).

[0158] Step 4: Synthesize compound 24-6

[0159] Compound 24-5 (1.24 g, 2.7 mmol) was dissolved in anhydrous ethanol (30 mL), followed by the addition of anhydrous potassium carbonate (746.18 mg, 5.40 mmol). The mixture was refluxed at 80 °C for 4 h. After the reaction was complete, the solvent was removed by vacuum distillation. The crude compound was then dissolved in EA and washed three times with water. The organic phase was concentrated to give 745 mg of a white solid, with a yield of 77.68%. This can be directly added to the next step. 1 H NMR (300MHz, DMSO-d6) δ7.95 (d, J = 2.2Hz, 1H), 7.72 (s, 2H), 7.48-7.16 (m, 6H), 5.15 (s, 2H).

[0160] Step 5: Synthesize compound 24-7

[0161] Compound 24-6 (745 mg, 2.10 mmol) was dissolved in ethanol, followed by the addition of triethylamine (848.91 mg, 8.39 mmol), and finally the slow addition of compound 1-5 (1.14 g, 6.29 mmol). The solution changed from light yellow to dark brown. The mixture was then refluxed at 80 °C for 4 h. After the reaction was complete, the reaction solution was concentrated, and the solution was purified by column chromatography to obtain the pale yellow key intermediate 24-7 (225 mg, 24.53%). 1 H NMR(300MHz,DMSO-d6)δ9.69(s,1H),7.61(s,1H),7.48-7.38(m,2H),7.39-7.32( m,2H),7.32-7.25(m,2H),7.16(dd,J=12.1,2.2Hz,1H),5.15(s,2H),3.89(s,3H).

[0162] Step 6: Synthesize compound 24-9

[0163] Compound 24-7 (400 mg, 914.74 μmol) was dissolved in 1,4-dioxane, followed by compound 24-8 (167.30 mg, 1.37 mmol), and then aqueous cesium carbonate solution (596.08 mg, 1.83 mmol) and palladium dichloride bis(triphenylphosphine) (32.10 mg, 45.74 μmol). The reaction was carried out at 105 °C for 12 h under nitrogen protection. The reaction was then confirmed to be complete by TLC. After the solvent was evaporated, the compound was dissolved in DCM, and the undissolved cesium carbonate and palladium catalyst were removed by filtration. The combined organic phases were then purified by column chromatography to give a pale yellow solid, compound 24-9, in 40.26% yield. 1H NMR (300MHz, DMSO-d6) δ10.14(s,1H),8.45(dd,J=11.4,3.1Hz,1H),7.90(s,1H),7.62(d,J=7.3Hz,2H),7.50( t,J=7.2Hz,3H),7.25-7.21(m,3H),7.13-7.05(m,2H),6.86(dd,J=9.0,3.1Hz,1H),4.51(s,2H),3.86(s,3H).

[0164] Step 7: Synthesize compound 24-10

[0165] Compound 24-9 (160 mg, 401.55 μmol) was dissolved in methanol, and 1.5 mL of 5 mol / L sodium hydroxide solution was added. The mixture was stirred at room temperature for 6 h until the reaction was complete. The pH was adjusted to 5 with HCl, and a pale yellow solid precipitated out. The solid was filtered to obtain compound 24-10 (130 mg, 84.21%). This compound can be directly added to the next step. 1 H NMR (300MHz, DMSO-d6) δ9.72 (s, 1H), 7.57-7.43 (m, 4H), 7.48-7.36 (m, 2H), 7.36 (ddt, J = 7.0, 5.9, 1.1Hz, 2H), 7. 36-7.24(m,2H),7.19(dd,J=12.1,2.2Hz,1H),7.08(s,1H),7.07(dd,J=12.1,2.1Hz,1H),5.17(t,J=0.8Hz,2H).

[0166] Step 8: Synthesize compound 24

[0167] Compound 24-10 (130 mg, 332.97 μmol) was dissolved in DCM, and BBr3 (320.83 μL, 3.33 mmol) was added dropwise at -20 °C, and the reaction was continued overnight. After the reaction was complete, the reaction was quenched with methanol, and the reaction solution was removed by vacuum distillation and purified by slurrying with PE and EA. A pale yellow solid, compound 24 (100 mg, 50.00%), was obtained. 1 H NMR(300MHz,DMSO-d6)δ10.07(s,1H),8.25(dd,J=11.1,2.8Hz,1H),7.74(s,1H),7.6 2-7.52(m,2H),7.45(t,J=7.4Hz,2H),7.40-7.33(m,1H),6.74(dd,J=9.3,3.1Hz,1H).

[0168] Example 25: Preparation of Compound 25

[0169]

[0170] Compound 25 was synthesized according to steps 6-8 of Example 24, except that compounds 24-8 were replaced with pinacol 4-pyridineboronic acid ester.

[0171] Compound 25 is a yellow solid with a yield of 48.42%. 1 H NMR (300MHz, DMSO-d6) δ10.19 (s, 1H), 9.23 (s, 1H), 8.68 (d, J = 5.1Hz, 2H), 8.39 (dd ,J=11.1,3.2Hz,1H),7.78(s,1H),7.71-7.64(m,2H),6.88(dd,J=9.1,3.2Hz,1H).

[0172] Example 26: Preparation of Compound 26

[0173]

[0174] Compound 26 was synthesized according to steps 6-8 of Example 24, except that compounds 24-8 were replaced with 2-(3-chlorophenyl)-4,4,5,5-tetramethyl-1,3,2-dioxoborane.

[0175] Compound 26 is a pale yellow solid with a yield of 58.79%. 1 H NMR (300MHz, DMSO-d6) δ10.10(s,1H),8.30(dd,J=11.1,3.1Hz,1H),7.76(s,1H),7.62(t,J=1.8Hz,1H),7.56-7.39(m,4H),6.79(dd,J=9.2,3.2Hz,1H).

[0176] Example 27: Preparation of Compound 27

[0177]

[0178] Compound 27 was synthesized according to steps 6-8 of Example 24, except that compounds 24-8 were replaced with pinacol 3-nitrophenylboronic acid ester.

[0179] Compound 27 is a yellow solid with a yield of 49.28%. 1H NMR (300MHz, DMSO-d6) δ10.14(s,1H),8.42(t,J=2.0Hz,1H),8.33(dd,J=11.1,3.2Hz,1H),8.24(d d,J=8.4,2.3Hz,1H),8.02(dt,J=7.8,1.4Hz,1H),7.82-7.71(m,2H),6.90(dd,J=9.1,3.2Hz,1H).

[0180] Example 28: Preparation of compound 28

[0181]

[0182] Compound 28 was synthesized according to steps 6-8 of Example 24, except that 4-(methanesulfonyl)phenylboronic acid was used to replace compounds 24-8.

[0183] Compound 28 is a yellow solid with a yield of 53.26%. 1 H NMR(300MHz,DMSO-d6)δ10.14(s,1H),8.33(dd,J=11.1,3.1Hz,1H),8.05-7.95( m,2H),7.88-7.79(m,2H),7.76(s,1H),6.83(dd,J=9.0,3.1Hz,1H),3.27(s,3H).

[0184] Example 29: Preparation of compound 29

[0185]

[0186] Compound 29 was synthesized according to steps 6-8 of Example 24, except that it was synthesized using 3,5-dimethylisoxazole-4-boronic acid pinacol ester compounds 24-8.

[0187] Compound 29 is a pale yellow solid with a yield of 49.87%. 1 H NMR (300MHz, DMSO-d6) δ8.33(dd,J=11.3,3.2Hz,1H),7.75(s,1H),6.68(dd,J=8.9,3.2Hz,1H),2.29(s,3H),2.13(s,3H).

[0188] Example 30: Preparation of compound 30

[0189]

[0190] Compound 30 was synthesized according to steps 6-8 of Example 24, except that compounds 24-8 were replaced with 4-methoxyphenylboronic acid pinacol ester.

[0191] Compound 30 is a pale yellow solid with a yield of 46.83%. 1 H NMR(300MHz,DMSO-d6)δ10.00(s,1H),8.17(dd,J=11.0,3.2Hz,1H),7.73(s,1 H),7.38(d,J=8.2Hz,2H),6.83(d,J=8.3Hz,2H),6.67(dd,J=9.5,3.2Hz,1H).

[0192] Example 31: Preparation of compound 31

[0193]

[0194] Compound 31 was synthesized according to steps 1-8 of Example 24, except that compound 24-1 was replaced with compound 31-1.

[0195] Compound 31-2 is an orange-red solid with a yield of 98.83%. 1 H NMR (300MHz, DMSO-d6) δ8.02(d,J=2.2Hz,1H),7.68(d,J=2.1Hz,1H),7.28-7.21(m,2H),7.20-7.07(m,3H),4.96(s,2H).

[0196] Compound 31-3 is a yellow solid with a yield of 90.13%. 1 H NMR (300MHz, DMSO-d6) δ7.50(d,J=2.2Hz,1H),7.45(ddq,J=6.3,1.6,0.8Hz,2H),7.41-7.28(m,3H),7.03(d,J=2.2Hz,1H),5.14(s,2H),4.60(s,2H).

[0197] Compound 31-4 is a white solid with a yield of 72.50%. 1 H NMR (300MHz, DMSO-d6) δ7.78 (d, J = 2.1Hz, 1H), 7.70-7.58 (m, 2H), 7.51-7.38 (m, 4H), 7.35-7.14 (m, 5H), 5.05 (s, 2H).

[0198] Compound 31-5 is a white solid with a yield of 82.77%. 1H NMR (300MHz, DMSO-d6) δ9.90 (s, 1H), 7.73 (s, 1H), 7.44 (ddq, J = 6.4, 1.7, 0.9Hz, 1H), 7.40-7.27 (m, 2H), 5.19 (s, 2H).

[0199] Compound 31-6 is a pale yellow solid with a yield of 28.40%. 1 H NMR (300MHz, DMSO-d6) δ10.20(s,1H),8.76(d,J=2.4Hz,1H),7.89(s,1H),7.57-7.46(m,2H),7.41-7.26(m,4H),5.02(s,2H),3.80(s,3H).

[0200] Compound 31-7 is a white solid with a yield of 53.31%. 1 H NMR (300MHz, DMSO-d6) δ9.47 (s, 1H), 7.69 (d, J = 2.2Hz, 1H), 7.65-7.54 (m, 3H), 7. 49-7.43(m,2H),7.43-7.35(m,4H),7.35-7.23(m,3H),5.13(s,2H),3.86(s,3H).

[0201] Compound 31-8 is a yellow solid with a yield of 75.89%. 1 H NMR (300MHz, DMSO-d6) δ9.58 (s, 1H), 7.74 (d, J = 2.2Hz, 1H), 7.67-7.62 (m, 2H), 7.54-7.47 (m ,3H),7.45(dddd,J=4.8,3.9,2.4,1.6Hz,3H),7.41-7.28(m,3H),7.10(s,1H),5.18(s,2H).

[0202] Compounds 31-9 are yellow solids with a yield of 48.58%. 1 H NMR (300MHz, DMSO-d6) δ12.78(s,1H),10.23(s,1H),10.07(s,1H),8.48(d,J=2.2 Hz,1H),7.74(s,1H),7.67-7.61(m,2H),7.45(t,J=7.4Hz,2H),7.39-7.33(m,2H).

[0203] Example 32: Preparation of compound 32

[0204]

[0205] Compound 32 was synthesized according to steps 6-8 of Example 24, except that compound 24-7 was replaced by compound 31-6 and 24-8 was replaced by 3-nitrophenylboronic acid pinacol ester.

[0206] Compound 30 is a yellow solid with a yield of 43.78%. 1 H NMR (300MHz, DMSO-d6) δ10.14(s,1H),8.60(d,J=2.3Hz,1H),8.42(t,J=2.1Hz,1H),8. 19(dd,J=7.9,2.3Hz,1H),8.16-8.06(m,1H),7.79-7.69(m,2H),7.54(d,J=2.3Hz,1H).

[0207] Example 33: Preparation of compound 33

[0208]

[0209] Compound 32 was synthesized according to steps 6-8 of Example 24, except that compound 24-7 was replaced by compound 31-6 and 24-8 was replaced by 4-(methanesulfonyl)phenylboronic acid.

[0210] Compound 33 is a white solid with a yield of 45.39%. 1 H NMR (300MHz, DMSO-d6) δ10.14(s,1H),8.59(d,J=2.2Hz,1H),7.94(q,J=8.2Hz,4H),7.75(s,1H),7.50(d,J=2.1Hz,1H),3.26(s,3H).

[0211] Example 34: Preparation of compound 34

[0212]

[0213] Compound 32 was synthesized according to steps 6-8 of Example 24, except that compound 24-7 was replaced by compound 31-6 and 24-8 was replaced by 3-(methanesulfonyl)phenylboronic acid.

[0214] Compound 34 is a yellow solid with a yield of 52.38%. 1H NMR (300MHz, DMSO-d6) δ10.12(s,1H),8.60(d,J=2.3Hz,1H),8.11(d,J=1.9Hz,1H),8.00(dt,J=7.9 ,1.5Hz,1H),7.88(dt,J=7.8,1.3Hz,1H),7.73(d,J=7.7Hz,2H),7.53(d,J=2.2Hz,1H),3.31(s,3H).

[0215] Example 35: Preparation of compound 35

[0216]

[0217] Compound 32 was synthesized according to steps 6-8 of Example 24, except that compound 24-7 was replaced by compound 31-6 and 24-8 was replaced by 3-methoxyphenylboronic acid pinacol ester.

[0218] Compound 35 is an orange solid with a yield of 54.61%. 1 H NMR (300MHz, DMSO-d6) δ10.09(s,1H),8.25(d,J=2.3Hz,1H),7.73(s,1H),7.29(d,J=2.2Hz,1H), 7.23(t,J=7.9Hz,1H), 7.03(d,J=7.8Hz,1H), 6.97(t,J=2.1Hz,1H), 6.75(dd,J=8.2,2.3Hz,1H).

[0219] Example 36: Preparation of compound 36

[0220]

[0221] Compound 32 was synthesized according to steps 6-8 of Example 24, except that compound 24-7 was replaced by compound 31-6 and 24-8 was replaced by pinacol 2-methylphenylboronic acid ester.

[0222] Compound 36 is a yellow solid with a yield of 51.47%. 1 H NMR (300MHz, DMSO-d6) δ10.09(s,1H),10.02(s,1H),8.19(d,J=2.1Hz,1H),7.71(s,1H),7.29-7.20(m,4H),6.99(d,J=2.1Hz,1H),2.31(s,3H).

[0223] Example 37: Preparation of compound 37

[0224]

[0225] Compound 32 was synthesized according to steps 6-8 of Example 24, except that compound 24-7 was replaced by compound 31-6 and 24-8 was replaced by 3-acetylphenylboronic acid pinacol ester.

[0226] Compound 37 is a yellow solid with a yield of 48.92%. 1 H NMR (300MHz, DMSO-d6) δ10.12(s,1H),8.61(d,J=2.3Hz,1H),8.19(t,J=1.9Hz,1H),7.9 6-7.85(m,2H),7.75(s,1H),7.59(t,J=7.7Hz,1H),7.48(d,J=2.2Hz,1H),2.68(s,3H).

[0227] Example 38: Preparation of compound 38

[0228]

[0229] Compound 32 was synthesized according to steps 6-8 of Example 24, except that compound 24-7 was replaced by compound 31-6 and 24-8 was replaced by 4-chlorophenylboronic acid pinacol ester.

[0230] Compound 38 is a yellow solid with a yield of 51.57%. 1 H NMR (300MHz, DMSO-d6) δ10.10(s,1H),8.44(d,J=2.3Hz,1H),7.73(s,1H),7.69-7.61(m,2H),7.53-7.45(m,2H),7.39(d,J=2.3Hz,1H).

[0231] Example 39: Preparation of compound 39

[0232]

[0233] Compound 32 was synthesized according to steps 6-8 of Example 24, except that compound 24-7 was replaced by compound 31-6 and 24-8 was replaced by 4-aminophenylboronic acid pinacol ester.

[0234] Compound 39 is a brown solid with a yield of 53.38%. 1 H NMR (300MHz, DMSO-d6) δ10.01(s,1H),8.33(d,J=2.2Hz,1H),7.72(s,1H),7.46(d,J=8.1Hz,2H),7.27(d,J=2.2Hz,1H),6.89(d,J=8.1Hz,2H).

[0235] Example 40: Preparation of Compound 40

[0236]

[0237] Compound 32 was synthesized according to steps 6-8 of Example 24, except that compound 24-7 was replaced by compound 31-6 and 24-8 was replaced by 3-trifluoromethylphenylboronic acid pinacol ester.

[0238] Compound 40 is a yellow solid with a yield of 46.50%. 1 H NMR (300MHz, DMSO-d6) δ10.10(s,1H),8.65(d,J=2.3Hz,1H),7.97(t,J=4.1Hz,2H),7.74(s,1H),7.68(d,J=6.7Hz,2H),7.50(d,J=2.3Hz,1H).

[0239] Example 41: Preparation of compound 41

[0240]

[0241] Step 1: Synthesize compound 41-2

[0242] Compound 31-6 (750 mg, 1.65 mmol) was dissolved in toluene (30 mL), followed by compound 41-1 (390.73 mg, 2.48 mmol), then cesium carbonate (1.62 g, 4.96 mmol) and BINAP (102.76 mg, 165.29 μmol), and finally palladium acetate (18.55 mg, 82.65 μmol). The reaction was carried out at 85 °C for 12 h under nitrogen protection. The reaction was then confirmed to be complete by TLC. After the solvent was evaporated, the mixture was dissolved in DCM, and the undissolved cesium carbonate and palladium catalyst were removed by filtration. The combined organic phases were then purified by column chromatography to give 220 mg of a pale yellow solid, compound 41-2, in 26.94% yield. 1 H NMR (300MHz, DMSO-d6) δ9.99(s,1H),8.35(d,J=2.9Hz,1H),7.83(s,1H),7.53(m,J=5.7,1.6Hz,2H),7.36(m,J=8.3,6.8,2.5Hz,3H),6. 74(d,J=2.9Hz,1H),4.91(s,2H),3.79(s,3H),3.48(t,J=11.9Hz,2H),3.24(t,J=5.3Hz,2H),2.04(m,J=12.5,5.3Hz,2H),1.80(s,2H).

[0243] Steps 2-3 refer to steps 7-8 in Example 31.

[0244] Compound 41-3 is a yellow solid with a yield of 82.63%. 1 H NMR (300MHz, DMSO-d6) δ9.57(s,1H),7.45-7.24(m,5H),7.08(s,1H),6.88(d,J=2.1Hz,1H),6.50(d,J=2 .2Hz,1H),5.24(s,2H),3.83(t,J=12.8Hz,2H),3.63-3.41(m,2H),2.08-1.93(m,2H),1.83-1.70(m,2H).

[0245] Compound 41 is a yellow-green solid with a yield of 49.02%. 1 H NMR (300MHz, DMSO-d6) δ10.01(s,1H),7.87(d,J=2.9Hz,1H),7.71(s,1H),6.71(d,J=2. 8Hz,1H),3.43-3.27(m,2H),3.13(t,J=5.4Hz,2H),1.99(m,J=7.0Hz,2H),1.78(s,2H).

[0246] Example 42: Preparation of compound 42

[0247]

[0248] Compound 42 was synthesized according to steps 1-3 of Example 41, except that compound 41-1 was replaced with 1,1-thiomorpholine dioxide.

[0249] Compound 42 is a yellow solid with a yield of 47.74%. 1 H NMR (300MHz, DMSO-d6) δ9.98(s,1H),7.97(d,J=2.9Hz,1H),7.70(s,1H),6.78(d,J=2.9Hz,1H),3.68(t,J=4.9Hz,4H),3.19-3.10(t,4H).

[0250] Example 43: Preparation of compound 43

[0251]

[0252] Compound 43 was synthesized according to steps 1-3 of Example 41, except that compound 41-1 was replaced with N-methylpiperazine.

[0253] Compound 43 is a gray solid with a yield of 44.29%. 1H NMR (300MHz, DMSO-d6) δ7.69(d,J=2.8Hz,1H),7.60(s,1H),6.65(d,J=2.8Hz,1H),3.14-3.07(t,4H),2.65(t,J=4.9Hz,4H),2.36(s,3H).

[0254] Example 44: Preparation of compound 44

[0255]

[0256] Compound 44 was synthesized according to steps 1-3 of Example 41, except that morpholine was used to replace compound 41-1.

[0257] Compound 44 is a brown solid with a yield of 47.61%. 1 H NMR (300MHz, DMSO-d6) δ9.94(s,1H),7.87(d,J=2.9Hz,1H),7.70(s,1H),6.69(d,J=2.8Hz,1H),3.74(t,J=4.7Hz,4H),3.05(t,J=5.7,3.5Hz,4H).

[0258] Example 45: Preparation of compound 45

[0259]

[0260] Compound 45 was synthesized according to steps 1-3 of Example 41, except that compound 41-1 was replaced with piperidine.

[0261] Compound 45 is a pale yellow solid with a yield of 45.91%. 1 H NMR (300MHz, DMSO-d6) δ10.22(s,1H),8.45(s,1H),7.80(s,1H),7.43(s,1H),3.47(s,4H),1.87(s,4H),1.63(s,2H).

[0262] Example 46: Preparation of Compound 46

[0263]

[0264] Compound 46 was synthesized according to steps 1-3 of Example 41, except that (R)-2-methylmorpholine was used to replace compound 41-1.

[0265] Compound 46 is a brownish-yellow solid with a yield of 49.82%. 1H NMR (300MHz, DMSO-d6) δ12.74(s,1H),9.91(s,1H),9.28(s,1H),7.94(d,J=2.8Hz,1H),7.69(s,1H),6.62(d,J=2.8Hz,1H),3.92 -3.84(m,1H),3.68-3.56(m,2H),3.51-3.36(m,2H),2.59(td,J=11.7,3.4Hz,1H),2.28(t,J=10.9Hz,1H),1.14(d,J=6.2Hz,3H).

[0266] Example 47: Preparation of Compound 47

[0267]

[0268] Compound 47 was synthesized according to steps 1-3 of Example 41, except that (S)-2-methylmorpholine was used to replace compound 41-1.

[0269] Compound 47 is a brownish-yellow solid with a yield of 52.38%. 1 H NMR (300MHz, DMSO-d6) δ12.71(s,1H),9.89(s,1H),9.28(s,1H),7.92(d,J=2.9Hz,1H),7.69(s,1H),6.62(d,J=2.8Hz,1H),3.88(dd,J=11.8,3 .1Hz,1H),3.67-3.57(m,2H),3.46(d,J=11.8Hz,1H),3.33(s,1H),2.59(td,J=11.7,3.3Hz,1H),2.28(t,J=10.9Hz,1H),1.14(d,J=6.2Hz,3H).

[0270] Example 48: Preparation of Compound 48

[0271]

[0272] Compound 48 was synthesized according to steps 1-3 of Example 41, except that compound 41-1 was replaced with cis-2,6-dimethylmorpholine.

[0273] Compound 48 is a grayish-white solid with a yield of 49.83%. 1H NMR (300MHz, DMSO-d6) δ12.77(s,1H),9.93(s,1H),9.26(s,1H),8.03(d,J=2.9Hz,1H),7.69(s,1H),6. 61(d,J=2.8Hz,1H),3.68(t,J=8.4Hz,2H),3.49(s,2H),2.20(t,J=11.0Hz,2H),1.14(d,J=6.1Hz,6H).

[0274] The following method is used in this invention to express and purify the WDR5 protein.

[0275] 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.

[0276] 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.

[0277] 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 its concentration was determined by BCA.

[0278] Experimental Example 1: Determination of the inhibitory activity of a compound against WDR5 based on fluorescence polarization (FP)

[0279] 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.

[0280] Experimental steps:

[0281] 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.

[0282] Inhibition rate = (mP value of compound group - mP value of blank group) / (mP value of negative control group - mP value of blank group) × 100%.

[0283] The experimental results are shown in Table 1.

[0284] Table 1 shows the IC50 of some compounds of this invention against WDR5 protein. 50 value

[0285]

[0286] As shown in Table 1, the compounds of the present invention have significant inhibitory activity against WDR5 and can be used as small molecule inhibitors of WDR5.

[0287] Experimental Example 2: Thermal shift assay to test the thermal stability of proteins with WDR5 binding activity.

[0288] The Thermal Shift assay utilizes the autofluorescence of tyrosine and tryptophan in proteins to detect changes in fluorescence intensity and peak value during protein unfolding. Melting curves are plotted using fluorescence intensities at two wavelengths (350 nm and 330 nm). The melting temperature (Tm) on the curve, i.e., the temperature at which half of the protein unfolds, measures the protein's stability. If an added compound alters protein stability, the melting curve shifts; the shift value ΔTm reflects whether the test compound binds to the protein.

[0289] The assay was performed using a 96-well plate (Life Technologies, CF98PV02), with a final test volume of 20 μL per well. 1 μL of WDR5 protein (final concentration 1 μM), 1 μL of compound dilution buffer, and 16 μL of assay buffer were added to each well. After incubation at room temperature for 30 min, 2 μL of 8× fluorescent dye (SYPRO Orange dye, Thermo Fisher Scientific) was added and thoroughly mixed. Three replicates were prepared for each compound. A negative control (1 μL His-WDR5 protein, negative compound, and 19 μL assay buffer) and a blank control (2 μL 8× fluorescent dye and 18 μL assay buffer) were included in each assay. After sealing the plate, a gradient temperature program was used on a PCR instrument (Step One Plus, Applied Biosystems), uniformly increasing the temperature from 37°C to 99°C at a rate of 2°C per minute. Data analysis was performed using Protein Thermal Shift software v1.3 (Thermo Fisher Scientific) after the program was completed. ΔTm represents the shift in the melting curve of the WDR5 protein after binding with the compound compared to the melting curve of the blank control.

[0290] The test results for representative compounds are shown in Table 2.

[0291] Table 2 Thermal shift assay ΔTm of representative compounds

[0292]

[0293] Experimental Example 3: Based on CellTiter-Lumi TM Detection of cell antiproliferative activity by luminescence method

[0294] The cell types used in this experiment were: human myeloid monocytic leukemia cells MV4-11, human acute myeloid leukemia cells MOLM-13, and human chronic myeloid leukemia cells K562, all purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. The culture medium used for MV4-11 cells was RPMI-IMDM medium (GiBco, Invitrogen Corp, USA) and 10% FBS (GiBco, Invitrogen Corp, USA); the culture medium used for MOLM-13 and K562 cells was RPMI-1640 medium (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.

[0295] 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 medium and treated for 72 h. Subsequently, CellTiter-Lumi was used. TM Cell 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.

[0296] The test results for representative compounds are shown in Table 3.

[0297] Table 3 Antiproliferative activity of representative compounds

[0298]

[0299] As can be seen from Table 3, compounds 5 and 14 exhibited broad anti-proliferative activity against tumor cells, with the best activity against MV4-11 cells.

[0300] In summary, the 2-thiazolaminophenol derivative provided by this invention exhibits significant inhibitory activity against WDR5, making it an effective WDR5 inhibitor. Therefore, drugs containing the above-mentioned compound as an active ingredient can be used to prepare remedies for clinical conditions related to WDR5.

[0301] As described above, although the invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the invention itself. Various changes in form and detail may be made without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. A class of 2-thiazolaminophenol derivatives or pharmaceutically acceptable salts thereof, said derivatives having a structure as shown in Formula I: ； in: R1 is H, halogen, methyl, ethyl, nitro, or unsubstituted or modified by any of the following R groups. 1A Substituted groups include: C3-C10 aryl or 5-8 heteroaryl; R 1A Selected from H, cyano, hydroxy, mercapto, halogen, nitro, amino, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 alkyl acyl, or C1-C3 alkyl sulfonyl; R2 is unsubstituted or has been substituted by any of the R groups. 2A The following groups are substituted: C3-C8 cycloalkyl, C3-C10 aryl, 5-8 membered heterocyclic or 5-8 membered heteroaryl; R 2A Selected from H, cyano, hydroxy, mercapto, halogen, nitro, amino, C1-C3 alkyl, C1-C3 haloalkyl, C1-C3 alkoxy, C1-C3 alkylacyl, or C1-C3 alkylsulfonyl; R3 is carboxyl or -C(O)-XR d X is selected from covalent bonds, -O-, or -NH-; R d Selected from methyl, ethyl, n-propyl, isopropyl, or unsubstituted or modified by any number of R B Substitute the following groups: C3-C10 cycloalkyl or 5-8 membered heterocyclic groups; R B Selected from H, cyano, hydroxy, mercapto, halogen, nitro, amino, C1-C3 alkyl, C1-C3 haloalkyl, C1-C3 alkoxy, C1-C3 alkyl acyl or C1-C3 alkyl sulfonyl.

2. The 2-thiazolaminophenol derivative or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, R3 is a carboxyl group.

3. The 2-thiazolaminophenol derivative or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, The cycloalkyl group is cyclopropane, cyclobutane, cyclopentane, or cyclohexane.

4. The 2-thiazolaminophenol derivative or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, The aryl group is benzene or naphthalene.

5. The 2-thiazolaminophenol derivative or a pharmaceutically acceptable salt thereof according to claim 4, characterized in that, The aryl group is phenyl.

6. The 2-thiazolaminophenol derivative according to claim 1, or a pharmaceutically acceptable salt thereof, characterized in that, The heteroaryl group is selected from pyridinyl, oxazolyl, isoxazolyl, indolyl, thiazolyl, pyrazolyl, imidazolyl, pyrroleyl, furanyl, thiophenyl, isothiazolyl, pyrimidinyl, triazolyl, 1,3,4-oxadiazolyl, 1,3,4-thiadiazolyl, 1,2,4-oxadiazolyl or 1,2,4-thiadiazolyl.

7. The 2-thiazolaminophenol derivative or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, The heterocyclic group is selected from piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, 1,1-thiomorpholinyl dioxide, pyrrolidinyl, 1,3-oxopentacycloyl, tetrahydropyranyl, tetrahydropyranyl, tetrahydropyrazolyl or 1,4-dioxohexacycloyl.

8. A class of 2-thiazolaminophenol derivatives or pharmaceutically acceptable salts thereof, said derivatives having a structure as shown in formula I-a or I-b: ； R1, R2, and R3 are as described in any one of claims 1 to 6.

9. Any of the following 2-thiazolaminophenol derivatives or a pharmaceutically acceptable salt thereof: 。 10. A pharmaceutical composition comprising a 2-thiazolamine derivative or a pharmaceutically acceptable salt thereof as described in any one of claims 1 to 9, and a pharmaceutically acceptable excipient.

11. The pharmaceutical composition according to claim 10, characterized in that, The composition is in the form of tablets, capsules, suspensions, or solutions.

12. Use of the 2-thiazolaminophenol derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 9 in the preparation of a therapeutic remedy for diseases associated with WDR5 dysfunction, wherein the diseases associated with WDR5 dysfunction are leukemia, pancreatic cancer, cholangiocarcinoma, colon cancer, lung cancer, liver cancer, glioblastoma, ovarian cancer, cervical cancer, prostate cancer, breast cancer, or gastric cancer.

13. Use of a 2-thiazolaminophenol derivative in the preparation of a therapeutic agent for diseases associated with WDR5 dysfunction, wherein the 2-thiazolaminophenol derivative is selected from any of the following: ； The diseases associated with WDR5 dysfunction are leukemia, pancreatic cancer, bile duct cancer, colon cancer, lung cancer, liver cancer, glioblastoma, ovarian cancer, cervical cancer, prostate cancer, breast cancer, or stomach cancer.

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