s-Triazine compounds with Aurora kinase inhibitory activity and applications thereof

Abstract

The present invention discloses an s-triazine compound having Aurora kinase inhibitory activity and its application. The s-triazine compound or its pharmaceutically acceptable salts, hydrates, solvates, polymorphs, tautomers, prodrugs, isotopic derivatives, or mixtures thereof, wherein the s-triazine compound is represented by the following general formula (I): The present invention provides a newly synthesized s-triazine compound by introducing different functional groups at the 2, 4, and 6-positions of the s-triazine parent nucleus. This newly synthesized s-triazine compound exhibits good inhibitory activity against Aurora kinase.

Description

Technical Field

[0001] The present invention relates to the technical field of medicinal chemistry, and in particular to an s-triazine compound having Aurora kinase inhibitory activity and application thereof. Background Art

[0002] Aurora kinases are a class of serine / threonine kinases that play a crucial regulatory role in the cell cycle. They regulate spindle formation, centrosome maturation, chromatin differentiation, and cytokinesis, playing a crucial role in maintaining genomic stability. Studies have shown that overexpression of Aurora kinases can lead to abnormal mitosis and is closely associated with tumor formation. Aurora kinases are overexpressed in many cancer cells (such as lung, breast, colorectal, thyroid, and pancreatic cancers). Inhibiting Aurora kinase activity can lead to polyploid aggregation of tumor cells, promote apoptosis, and block cell proliferation. The research and development of anti-tumor drugs targeting Aurora kinases is gaining increasing attention. Aurora kinases are expressed and activated only during mitosis, making them ineffective against non-proliferating cells. Most normal cells in the human body do not proliferate rapidly. Therefore, Aurora kinase inhibitors are targeted anti-tumor drugs and offer significant advantages over other nonspecific cytotoxic drugs.

[0003] Based on their amino acid sequences, human Aurora kinases are divided into three subtypes: Aurora A, Aurora B, and Aurora C. These subtypes share highly conserved C-terminal catalytic and N-terminal variable regions, with highly homologous ATP (adenosine triphosphate) binding sites (C-terminal catalytic regions), but differ significantly in the length and sequence of the N-terminal amino acids.

[0004] Since scientists confirmed in 1998 that tumorigenesis is closely linked to overexpression of Aurora kinases, a wave of research has ignited within academia and the pharmaceutical industry, focusing on Aurora kinases as anti-tumor drug targets. With the advancement of crystal structure studies of Aurora kinases and the application of computer simulations, it has been discovered that most synthetic Aurora kinase inhibitors are ATP-competitive kinase inhibitors, competing for the ATP binding site of the kinase, depriving the kinase of its direct energy source and thereby inhibiting its activity. Studies have shown that the purine ring in the ATP structure binds to the hydrophobic pocket of the Aurora kinase structure and forms hydrogen bonds with amino acid residues in the linker region. The ATP binding pocket is composed of several regions: the kinase hinge region, the hydrophobic pocket binding region, the phosphate groove, the solvent-accessible region, and the ribose moiety. The ATP binding sites of the Aurora kinase family are highly homologous, making the selection of kinase inhibitors a significant challenge. Many reported Aurora kinase inhibitors continue to exhibit good selectivity. One important reason is that, in addition to interacting with the kinase through the ATP binding site, the inhibitors also target regions near the ATP binding pocket, including a hydrophobic pocket at the rear of the kinase. Differences in amino acid residues in all these regions contribute to the differences in kinase inhibitor selectivity. The basic structural features of reported kinase inhibitors include: a planar heterocyclic ring system that competes for binding to the kinase's ATP pocket and mimics the interaction between adenine and the kinase; hydrogen bonding between the inhibitor and the kinase hinge region, forming a "donor-acceptor-donor" interaction mechanism; and functional groups of the inhibitors often have access to the phosphate binding region or selective binding pocket of the kinase.

[0005] Structurally, currently synthesized Aurora kinase inhibitors primarily include pyrimidine rings, pyrrolopyrazoles, indoles, quinazolines, pyrimidobenzoazepines, and other structural classes. Vertex and Merck jointly developed the first Aurora kinase inhibitor, VX-680 (also known as MK-0457) (WO2004000833A1).

[0006]

[0007] The molecular structure is Y-shaped, with the central pyrimidine ring located in the hydrophobic region of the kinase, forming a hydrophobic interaction with the amino acid residues of the kinase; the aminopyrazole-containing arm extends into the hinge region of the kinase, and the nitrogen atom of the pyrazole ring forms a hydrogen bond with the amino acid residues in the hinge region; the phenyl-containing arm extends into the active site pocket of the kinase, and the cyclopropyl group at the end of the phenyl group forms a hydrophobic interaction with the F275 residue of the Aurora A kinase; the piperazine side chain extends from the active region of the enzyme to the solvent-accessible region of the enzyme. Studies have found that VX-680 has a strong inhibitory effect on Aurora A, B, and C, with an IC of 1.50 The values ​​are 0.6, 18, and 4.6 nM, respectively, and are capable of inhibiting the proliferation of numerous tumor cells, including colorectal cancer, breast cancer, prostate cancer, pancreatic cancer, melanoma, neck tumors, and leukemia. In 2006, VX-680 entered Phase II clinical trials, primarily for the treatment of refractory chronic myeloid leukemia and acute lymphoblastic leukemia. Studies have shown that the drug can increase the risk of QTc prolongation in patients (QTc is the time difference between the corrected T wave and Q wave on an electrocardiogram, which can easily lead to arrhythmias). Merck terminated the Phase II clinical trial of VX-680 in November 2007 (Expert Opin. Investing Drugs. 2009, 18, 379).

[0008] Although there are some Aurora kinase inhibitors in the prior art, it is still necessary to further develop Aurora kinase inhibitors to provide more options for the preparation of drugs for Aurora kinase-related diseases. Summary of the Invention

[0009] The technical problem to be solved by the present invention is to provide an s-triazine compound with Aurora kinase inhibitory activity and its application. By introducing different functional groups at the 2, 4, and 6 positions of the s-triazine mother nucleus, a newly synthesized s-triazine compound is provided. This newly synthesized s-triazine compound shows good inhibitory activity against Aurora kinase.

[0010] To solve the above technical problems, the present invention provides an s-triazine compound having Aurora kinase inhibitory activity, or a pharmaceutically acceptable salt, hydrate, solvate, polymorph, tautomer, prodrug, isotopic derivative, or mixture thereof, wherein the s-triazine compound is represented by the following general formula (I):

[0011]

[0012] In formula (I):

[0013] The R1 is methyl, ethyl, propyl, n-butyl, 3-aminopropyl, or propargyl;

[0014] The R2 is methyl, ethyl, propyl, n-butyl, isobutyl, tert-butyl, N-(1H-pyrrolo[2,3-b]pyridin-3-yl)methyl;

[0015] The R3 is N-[(3-chlorophenyl)methyl]-methylamino, N-[(2-chlorophenyl)methyl]-methylamino, N-hydroxyethylanilino, 4-aminomethylbenzylamino, 3-substituent R5 is 1H-pyrazole-5-amino and the substituent R5 is methyl, ethyl, isopropyl, propyl, cyclopropyl, butyl, n-butyl, isopropyl, 5-amino-3-substituent R6 is 1H-pyrazole-1-yl and the substituent R6 is methyl, ethyl, isopropyl, propyl, cyclopropyl, butyl, n-butyl, isopropyl;

[0016] The R4 is N-hydroxyethylaniline, 4-aminomethylbenzylamino, 3-substituent R5 is 1H-pyrazole-5-amino and the substituent R5 is methyl, ethyl, isopropyl, propyl, cyclopropyl, butyl, n-butyl, isopropyl, 5-amino-3-substituent R6 is 1H-pyrazole-1-yl and the substituent R6 is methyl, ethyl, isopropyl, propyl, cyclopropyl, butyl, n-butyl, isopropyl.

[0017] Specifically, the s-triazine compound is selected from any one of the following structural formulas:

[0018]

[0019]

[0020]

[0021]

[0022] The present invention also provides a pharmaceutical composition comprising a compound having the structure of general formula (I), or a pharmaceutically acceptable salt, hydrate, solvate, polymorph, tautomer, prodrug, isotopic derivative or a mixture thereof.

[0023] Specifically, the pharmaceutical composition is an injection, an oral agent or a mucosal administration agent.

[0024] The present invention also provides a compound having the structure of general formula (I), or its pharmaceutically acceptable salts, hydrates, solvates, polymorphs, tautomers, prodrugs, isotopic derivatives or mixtures thereof, or use of said pharmaceutical composition in the preparation of a drug for inhibiting Aurora kinase.

[0025] Specifically, the Aurora kinase is Aurora A kinase.

[0026] The present invention also provides a compound having the structure of general formula (I), or its pharmaceutically acceptable salt, hydrate, solvate, polymorph, tautomer, prodrug, isotope derivative or mixture thereof, or the use of said pharmaceutical composition in the preparation of anti-tumor drugs.

[0027] Specifically, the tumor is an Aurora A high-expressing tumor or an Aurora A inhibitor-resistant tumor.

[0028] The present invention provides an s-triazine compound having Aurora kinase inhibitory activity and its application. By introducing different functional groups at the 2, 4, and 6-positions of the s-triazine parent nucleus, a newly synthesized s-triazine compound is provided. The newly synthesized s-triazine compound shows good inhibitory activity against Aurora kinase, and some compounds have an activity comparable to that of the positive control VX-680. Further optimization can be performed to develop new compounds that inhibit Aurora kinase activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the technical solution of the present invention, the following briefly introduces the drawings required for use in the present invention. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0030] Figure 1 The results are the results of the inhibitory activity of the s-triazine compounds of the present invention on Aurora A kinase determined by fluorescence resonance energy transfer experiments. DETAILED DESCRIPTION

[0031] The following is a clear and complete description of the technical solutions of the present invention in conjunction with the accompanying drawings. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0032] Example 1. Synthesis of 2-((4-((3-chlorobenzyl)(methyl)amino)-6-((3-ethyl-1H-pyrazol-5-yl)amino)-1,3,5-triazin-2-yl)amino)-N-methyl-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)propionamide (Compound I-1)

[0033]

[0034] Step 1: Synthesis of 4,6-dichloro-N-(3-ethyl-1H-pyrazol-5-yl)-1,3,5-triazine-2-amine (Compound 3)

[0035] 2,4,6-Trichloro-1,3,5-triazine (Compound 1, 8.7 g, 47.2 mmol, 1.1 eq) was weighed and dissolved in 120 mL of tetrahydrofuran (THF). N,N-Diisopropylethylamine (DIEA, 18.2 g, 140.8 mmol, 3.3 eq) and 3-ethyl-1H-pyrazol-5-amine (Compound 2, 4.7 g, 42.3 mmol, 1.0 eq) were added to the solution and stirred at -78°C for 2 hours. TLC (dichloromethane:methanol = 10:1, iodine colorimetry, R f =0.26) to monitor the reaction completion. After the reaction was completed, 40 mL of ethyl acetate was added and the mixture was washed three times with water. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated. Purification by column chromatography (dichloromethane:methanol = 100:0 to 97:3) afforded 8.1 g of compound 3 as a white solid in a 66.9% yield.

[0036] 1 H NMR: (400MHz, CD3OD) δppm 6.43 (s, 1H), 2.69 (q, J = 7.6Hz, 2H), 1.28 (t, J = 7.6Hz, 3H).

[0037] Step 2, Synthesis of 6-chloro-N2-(3-chlorobenzyl)-N4-(3-ethyl-1H-pyrazol-5-yl)-N2-methyl-1,3,5-triazine-2,4-diamine (Compound 5)

[0038] 4,6-Dichloro-N-(3-ethyl-1H-pyrazol-5-yl)-1,3,5-triazin-2-amine (Compound 3, 4.0 g, 15.4 mmol, 1.1 eq) was dissolved in 60 mL of tetrahydrofuran (THF). N,N-Diisopropylethylamine (DIEA, 6.0 g, 46.3 mmol, 8.1 mL, 3.3 eq) and 1-(3-chlorophenyl)-N-methylmethanamine (Compound 4, 2.2 g, 13.9 mmol, 2.0 mL, 1.0 eq) were added to the solution and stirred at 0°C for 2 hours. LC-MS results indicated the reaction was complete. After completion of the reaction, 40 mL of ethyl acetate was added, and the mixture was washed three times with water. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated. Purification by column chromatography (dichloromethane:methanol=100:0 to 97:3) gave yellow solid compound 5, 5.4 g, with a yield of 92.1%.

[0039] LC-MS: m / z=378.1 (M+H) + , R t =1.588min.

[0040] Step 3, Synthesis of methyl 2-amino-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)propionate (Compound 6)

[0041] 2-Amino-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)propionic acid (Compound 6-1, 900 mg, 4.4 mmol, 1.0 eq) was dissolved in 10 mL of methanol, and thionyl chloride (2.6 g, 22.1 mmol, 1.6 mL, 5.0 eq) was added dropwise at 0°C, and then stirred at 75°C for 10 hours. TLC (dichloromethane:methanol = 10:1, R f =0.12) to monitor the reaction completion. Concentration gave yellow solid compound 6, 960 mg, with a yield of 99.8%.

[0042] 1 H NMR: (400MHz, CD3OD) δppm 8.81(d,J=7.6Hz,1H),8.50(d,J=6.0Hz,1H),7.74(s,1H),7.68-7.63(m,1H ), 4.50 (t, J = 6.4Hz, 1H), 3.83 (s, 3H), 3.61-3.56 (m, 1H), 3.54-3.50 (m, 1H).

[0043] Step 4. Synthesis of methyl 2-((4-((3-chlorobenzyl)(methyl)amino)-6-((3-ethyl-1H-pyrazol-5-yl)amino)-1,3,5-triazin-2-yl)amino)-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)propyl ester (Compound 7)

[0044] 6-Chloro-N2-(3-chlorobenzyl)-N4-(3-ethyl-1H-pyrazol-5-yl)-N2-methyl-1,3,5-triazine-2,4-diamine (Compound 5, 1.2 g, 3.3 mmol, 1.0 eq) was dissolved in 20 mL of dioxane. N,N-Diisopropylethylamine (DIEA, 2.4 g, 18.2 mmol, 3.2 mL, 5.5 eq) and methyl 2-amino-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)propanoate (Compound 6, 800 mg, 3.6 mmol, 1.1 eq) were added to the solution. The reaction was stirred at 120°C for 10 hours. LC-MS results indicated the reaction was complete. 20 mL of ethyl acetate was added, and the mixture was washed three times with water. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated. Purification by column chromatography (dichloromethane:tetrahydrofuran = 100:0 to 0:100) afforded 802 mg of yellow solid compound 7 in a yield of 39.2%.

[0045] LC-MS: m / z=561.3 (M+H)+ , R t =1.244min.

[0046] Step 5. Synthesis of 2-((4-((3-chlorobenzyl)(methyl)amino)-6-((3-ethyl-1H-pyrazol-5-yl)amino)-1,3,5-triazin-2-yl)amino)-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)propanoic acid (Compound 8)

[0047] Methyl 2-((4-((3-chlorobenzyl)(methyl)amino)-6-((3-ethyl-1H-pyrazol-5-yl)amino)-1,3,5-triazin-2-yl)amino)-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)propanoate (Compound 7, 700 mg, 1.2 mmol, 1.0 eq) was dissolved in 6 mL of tetrahydrofuran (THF). 2 mL of methanol, 2 mL of water, and lithium hydroxide monohydrate (157 mg, 3.7 mmol, 3.0 eq) were added, and the mixture was stirred at 25°C for 10 hours. LC-MS results indicated that the reaction was complete. After completion of the reaction, 10 mL of dichloromethane was added, and the mixture was washed twice with 5 mL of saturated brine. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to afford 300 mg of Compound 8 as a yellow solid in a 44.0% yield.

[0048] LC-MS: m / z=547.2 (M+H) + , R t =1.161min.

[0049] Step 6. Synthesis of 2-((4-((3-chlorobenzyl)(methyl)amino)-6-((3-ethyl-1H-pyrazol-5-yl)amino)-1,3,5-triazin-2-yl)amino)-N-methyl-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)propionamide (Compound I-1)

[0050] 2-((4-((3-chlorobenzyl)(methyl)amino)-6-((3-ethyl-1H-pyrazol-5-yl)amino)-1,3,5-triazin-2-yl)amino)-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)propanoic acid (Compound 8, 26 mg, 47.5 mmol, 1.0 eq) was weighed and dissolved in 0.5 mL of N,N-dimethylformamide (DMF). Methylamine hydrochloride (MeNH) was added to the solution. 2.HCl, 9.82 mg, 142.6 μmol, 3.0 eq), 1-hydroxybenzotriazole (HOBt, 10 mg, 70 μmol, 1.5 eq), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCl, 14 mg, 70 μmol, 1.5 eq) were stirred at 25°C for 2 hours. LC-MS results indicated the reaction was complete. After completion, the product was purified by prep-HPLC (column: C18-6 100*30 mm*5 μm; mobile phase: [water(trifluoroacetic acid)-acetonitrile]; B%: 30%-50%, 11 min) to afford 6 mg of compound I-1 as a white solid in a 22.3% yield.

[0051] 1 H NMR(400MHz,CD3OD)δppm 8.15(d,J=4.4Hz,1H),8.11-7.90(m,1H),7.36-7.15(m,5H),7.15-6.99(m,2H),4.81-4 .72(m,2H),3.28-3.16(m,2H),3.12(s,3H),2.76-2.44(m,6H),1.07(t,J=12.0Hz,3H).

[0052] LC-MS: m / z=560.3 (M+H) + , R t =0.582min.

[0053] Example 2. Synthesis of N-(3-aminopropyl)-2-((4-((3-chlorobenzyl)(methyl)amino)-6-((3-ethyl-1H-pyrazol-5-yl)amino)-1,3,5-triazin-2-yl)amino)-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)propanamide (Compound I-45)

[0054]

[0055] Step 1. Synthesis of N-(3-Boc-aminopropyl)-2-((4-((3-chlorobenzyl)(methyl)amino)-6-((3-ethyl-1H-pyrazol-5-yl)amino)-1,3,5-triazin-2-yl)amino)-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)propionamide (Compound 10)

[0056] Compound 8 (200 mg, 366 μmol, 1.0 eq) was weighed and dissolved in 4 mL of dichloromethane (DCM). 1-Hydroxybenzotriazole (HOBt, 74.1 mg, 548 μmol, 1.5 eq), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCl, 105 mg, 548 μmol, 1.5 eq), N,N-diisopropylethylamine (DIEA, 142 mg, 1.1 mmol, 191 μL, 3.0 eq), and 3-Bocaminopropylamine (63.7 mg, 366 μmol, 63.8 μL, 1.0 eq) were added to the solution. The mixture was stirred at 25°C for 2 hours. LC-MS results indicated that the reaction was complete. After completion of the reaction, 5 mL of dichloromethane was added, and the mixture was washed twice with 4 mL of saturated brine. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a yellow solid compound 10, 100 mg, with a yield of 38.9%.

[0057] LC-MS: m / z=703.2 (M+H) + , R t =1.268min.

[0058] Step 2, Synthesis of N-(3-aminopropyl)-2-((4-((3-chlorobenzyl)(methyl)amino)-6-((3-ethyl-1H-pyrazol-5-yl)amino)-1,3,5-triazin-2-yl)amino)-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)propionamide (Compound I-45)

[0059] Compound 10 (100 mg, 142 μmol) was dissolved in 1 mL of dichloromethane (DCM). A 4 M hydrochloric acid / dioxane solution (0.3 mL) was added. The reaction was stirred at 25°C for 0.5 hours. LC-MS results indicated the reaction was complete. The product was concentrated and purified by prep-HPLC (column: C18-6 100 x 30 mm x 5 μm; mobile phase: [water (formic acid)-acetonitrile]; B%: 6%-46%, 15 min) to afford 55.5 mg of compound I-45 as a white solid in a 64.5% yield.

[0060] 1H NMR(400MHz,CD3OD)δppm 8.43-8.61(m,1H),8.16(d,J=4.0Hz,1H),7.98-8.06(m,1H),7.28-7.32(m,2H),7.26 (d,J=8.0Hz,2H),7.11-7.13(mz,1H),7.05-7.09(m,1H),6.00-6.18(m,1H),4.79(br s,2H),4.70(br s,1H),3.25-3.30(m,2H),3.20(br s,1H),3.06(br s,3H),2.69-2.77(m,2H),2.56-2.61(m,2H),1.63-1.71(m,2H),1.16-1.29(m,3H).

[0061] LC-MS: m / z=603.2 (M+H) + , R t =1.007min.

[0062] Example 3. Synthesis of 2-((4-((3-chlorobenzyl)(methyl)amino)-6-((3-ethyl-1H-pyrazol-5-yl)amino)-1,3,5-triazin-2-yl)amino)-N-(prop-2-yn-1-yl)-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)propionamide (Compound I-46)

[0063]

[0064] Compound 8 (100 mg, 183 μmol, 1.0 eq) was dissolved in 2 mL of dichloromethane (DCM). 1-Hydroxybenzotriazole (HOBt, 37.1 mg, 274 μmol, 1.5 eq), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCl, 52.6 mg, 274 μmol, 1.5 eq), N,N-diisopropylethylamine (DIEA, 70.9 mg, 548 μmol, 95.5 μL, 3.0 eq), and prop-2-yn-1-amine (10.1 mg, 183 μmol, 11.7 μL, 1.0 eq) were added to the solution and stirred at 25°C for 10 hours. LC-MS results indicated the reaction was complete. After completion of the reaction, 5 mL of dichloromethane was added, and the mixture was washed twice with 5 mL of saturated brine. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated. The obtained compound was purified by prep-HPLC (column: C18-6 100*30mm*5μm; mobile phase: [water (formic acid)-acetonitrile]; B%: 18%-48%, 15min) to obtain a white solid compound I-46, 25.9 mg, with a yield of 23.9%.

[0065] 1 H NMR (400MHz, CD3OD) δppm 8.14(d,J=4.4Hz,1H),8.06-7.98(m,1H),7.3-7.27(m,2H),7.24(d,J=6.4Hz,2H),7.13-7.11(m,1H),7.06(br s,1H),6.35-5.85(m,1H),4.79(br s,2H),4.73(br s,1H),3.95-3.77(m,2H),3.28-3.11(m,2H),3.04-3.02(m,3H),2.60-2.56(m,2H),2.47(s,1H),1.25-1.10(m,3H).

[0066] LC-MS: m / z=584.2 (M+H) + , R t =1.148min.

[0067] Example 4. Synthesis of (S)-2-((4-((2-chlorobenzyl)(methyl)amino)-6-((3-cyclopropyl-1H-pyrazol-5-yl)amino)-1,3,5-triazin-2-yl)amino)-N,3,3-trimethylbutanamide (Compound I-13)

[0068]

[0069] Step 1. Synthesis of 4,6-dichloro-N-(3-cyclopropyl-1H-pyrazol-5-yl)-1,3,5-triazine-2-amine (Compound 13)

[0070] 2,4,6-Trichloro-1,3,5-triazine (Compound 1, 6.9 g, 37.4 mmol, 1.0 eq) was weighed and dissolved in 100 mL of tetrahydrofuran (THF). N,N-diisopropylethylamine (DIEA, 14.5 g, 112.2 mmol, 19.5 mL, 3.0 eq) and 3-cyclopropyl-1H-pyrazol-5-amine (4.6 g, 37.5 mmol, 1.0 eq) were added. The reaction was stirred at -78°C for 4 hours. TLC (dichloromethane:methanol = 10:1, R f =0.20) to monitor the reaction completion. The reaction mixture was extracted with 40 mL of ethyl acetate, washed three times with 50 mL of water, and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 8.1 g of yellow solid compound 13 with a yield of 79.5%.

[0071] 1H NMR (400MHz, CD3OD) δppm 6.29 (s, 1H), 1.94-1.88 (m, 1H), 1.02-0.98 (m, 2H), 0.76-0.72 (m, 2H).

[0072] Step 2: Synthesis of 6-chloro-N2-(2-chlorobenzyl)-N4-(3-cyclopropyl-1H-pyrazol-5-yl)-N2-methyl-1,3,5-triazine-2,4-diamine (Compound 15)

[0073] 4,6-Dichloro-N-(3-cyclopropyl-1H-pyrazol-5-yl)-1,3,5-triazin-2-amine (Compound 13, 4.0 g, 14.8 mmol, 1.1 eq) was dissolved in 60 mL of tetrahydrofuran (THF). N,N-Diisopropylethylamine (DIEA, 5.7 g, 44.3 mmol, 7.7 mL, 3.3 eq) and 1-(2-chlorophenyl)-N-methylmethanamine (2.1 g, 13.3 mmol, 1.9 mL, 1.0 eq) were added. The reaction was stirred at 0°C for 1 hour. LC-MS and TLC (dichloromethane:methanol = 10:1, iodine colorimetry) showed the reaction was complete. The product was extracted with 40 mL of ethyl acetate and washed three times with 30 mL of saturated brine. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 5.67 g of crude yellow solid Compound 15.

[0074] LC-MS: m / z=390.1 (M+H) + , R t =1.608min.

[0075] Step 3, Synthesis of (S)-2-((4-((2-chlorobenzyl)(methyl)amino)-6-((3-cyclopropyl-1H-pyrazol-5-yl)amino)-1,3,5-triazin-2-yl)amino)-N,3,3-trimethylbutanamide (Compound I-13)

[0076] (S)-2-Amino-N,3,3-trimethylbutanamide (Compound 16, 0.1 g, 0.7 mmol, 1.0 eq) was dissolved in 1 mL of dimethyl sulfoxide (DMSO). 6-Chloro-N2-(2-chlorobenzyl)-N4-(3-cyclopropyl-1H-pyrazol-5-yl)-N2-methyl-1,3,5-triazine-2,4-diamine (Compound 15, 0.28 g, 0.7 mmol, 3.0 eq) and N,N-diisopropylethylamine (DIEA, 0.28 g, 2.2 mmol, 0.4 mL, 3.0 eq) were added. The mixture was stirred at 100°C for 4 hours. LC-MS analysis indicated the reaction was complete. The residue was filtered and purified by prep-HPLC (mobile phase: [water (0.05% ammonia)-acetonitrile]; B%: 40%-70%, 15 min) to obtain compound I-13 as a white solid, 100 mg, with a yield of 27.3%.

[0077] 1 H NMR (400MHz, CDCl3) δppm 10.65 (br s, 1H), 10.48-10.12 (m, 1H), 9.58 (br s, 1H), 7.42 (t, J = 8.0Hz, 1H), 7.25-7.18 (m, 3H), 6.46 (br s,1H),6.02(m,1H),5.04(m,1H),4.58(m,1H),4.18(d,J=9.2Hz,1H),3.29(m,3H),2.70(m,3H),1.94-1.78(m,1H),1.12-0.68(m,13H).

[0078] LC-MS: m / z=498.2 (M+H) + , R t =0.653min.

[0079] Example 5. Synthesis of (S)-N-(3-aminopropyl)-2-((4-((2-chlorobenzyl)(methyl)amino)-6-((3-cyclopropyl-1H-pyrazol-5-yl)amino)-1,3,5-triazin-2-yl)amino)-3,3-dimethylbutanamide (Compound I-47)

[0080]

[0081] Step 1, Synthesis of (S)-2-amino-3,3-dimethylbutyric acid methyl ester (Compound 17)

[0082] (S)-2-Bocamino-3,3-dimethylbutanoic acid (Compound 17-1, 2.0 g, 8.6 mmol) was dissolved in hydrochloric acid / methanol (4 M, 2.2 mL) and stirred at 80°C for 10 hours. TLC (dichloromethane:methanol = 20:1, R f =0.24) to monitor the reaction completion. After the reaction was completed, the mixture was concentrated to obtain 1.2 g of white solid compound 17 with a yield of 99.6%.

[0083] 1 H NMR (400MHz, CD3OD) δppm (s, 3H), 3.81 (s, 1H), 1.11 (s, 9H).

[0084] Step 2: Synthesis of (S)-methyl 2-((4-((2-chlorobenzyl)(methyl)amino)-6-((3-cyclopropyl-1H-pyrazol-5-yl)amino)-1,3,5-triazin-2-yl)amino)-3,3-dimethylbutanoate (Compound 18)

[0085] Compound 15 (3.4 g, 8.6 mmol, 1.0 eq) was weighed and dissolved in 40 mL of dioxane. N,N-diisopropylethylamine (DIEA, 3.3 g, 25.8 mmol, 4.5 mL, 3.0 eq) and (S)-methyl 2-amino-3,3-dimethylbutanoate (compound 17, 1.2 g, 8.6 mmol, 1.0 eq) were added. The reaction was stirred at 120°C for 10 hours. LC-MS and TLC (dichloromethane:methanol = 10:1, R f =0.18) indicated that the reaction was complete. After completion of the reaction, the mixture was extracted with 40 mL of ethyl acetate and washed twice with 30 mL of saturated brine. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated. Purification by column chromatography (dichloromethane:ethyl acetate = 100:0 to 0:100) afforded 1.3 g of compound 18 as a yellow solid in a 27.6% yield.

[0086] LC-MS: m / z=499.3 (M+H) + , R t =1.516min.

[0087] Step 3. Synthesis of (S)-2-((4-((2-chlorobenzyl)(methyl)amino)-6-((3-cyclopropyl-1H-pyrazol-5-yl)amino)-1,3,5-triazin-2-yl)amino)-3,3-dimethylbutanoic acid (Compound 19)

[0088] Compound 18 (1.0 g, 2.0 mmol) was weighed and dissolved in 2 mL of water. 6 mL of methanol, 6 mL of tetrahydrofuran (THF), and lithium hydroxide monohydrate (255 mg, 6.1 mmol) were added. The reaction was stirred at 25°C for 10 hours. LC-MS and TLC (dichloromethane:methanol = 10:1, R f =0.40) indicated that the reaction was complete. After completion of the reaction, the product was extracted with 10 mL of dichloromethane and washed twice with 8 mL of saturated brine. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated. Purification by column chromatography (dichloromethane:methanol = 100:0 to 95:5) afforded 547 mg of compound 19 as a yellow solid in a 55.8% yield.

[0089] LC-MS: m / z=485.3 (M+H) + , R t =1.323min.

[0090] Step 4. Synthesis of (S)-N-(3-Bocaminopropyl)-2-((4-((2-chlorobenzyl)(methyl)amino)-6-((3-cyclopropyl-1H-pyrazol-5-yl)amino)-1,3,5-triazin-2-yl)amino)-3,3-dimethylbutanamide (Compound 21)

[0091] (S)-2-((4-((2-chlorobenzyl)(methyl)amino)-6-((3-cyclopropyl-1H-pyrazol-5-yl)amino)-1,3,5-triazin-2-yl)amino)-3,3-dimethylbutanoic acid (Compound 19, 200 mg, 412 μmol, 1.0 eq) was dissolved in 4 mL of dichloromethane, and 1-hydroxybenzotriazole (HOBt, 83.6 mg, 619 μmol, 1 .5eq), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCL, 118.6 mg, 619 μmol, 1.5eq) and N,N-diisopropylethylamine (DIEA, 160 mg, 1.2 mmol, 215 μL, 3.0eq), and then 3-Bocaminopropylamine (Compound 20, 71.9 mg, 412 μmol, 72.0 μL, 1.0eq) were added. The reaction was stirred at 25°C for 10 hours. LC-MS and TLC (dichloromethane:methanol = 20:1, R f =0.36) indicated that the reaction was complete. After completion of the reaction, the mixture was extracted with 3 mL of dichloromethane and washed twice with 2 mL of saturated brine. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated. Purification by column chromatography (dichloromethane:methanol = 100:0 to 95:5) afforded 150 mg of compound 21 as a yellow solid in a 56.7% yield.

[0092] LC-MS: m / z=641.3 (M+H)+ , R t =1.520min.

[0093] Step 5, Synthesis of (S)-N-(3-aminopropyl)-2-((4-((2-chlorobenzyl)(methyl)amino)-6-((3-cyclopropyl-1H-pyrazol-5-yl)amino)-1,3,5-triazin-2-yl)amino)-3,3-dimethylbutanamide (Compound I-47)

[0094] Compound 21 (130 mg, 203 μmol) was dissolved in 1.5 mL of dichloromethane, and hydrochloric acid / dioxane (4 M, 0.5 mL) was added. The reaction was stirred at 25°C for 0.5 hours. LC-MS results indicated the reaction was complete. After completion of the reaction, the product was concentrated and purified by prep-HPLC (column: C18-6 100 x 30 mm x 5 μm; mobile phase: [water (formic acid)-acetonitrile]; B%: 15%-45%, 15 min) to afford compound I-47 as a white solid (31.9 mg, 29.1% yield).

[0095] 1 H NMR(400MHz,CD3OD)δppm 7.45-7.07(m,4H),6.30-5.60(m,1H),4.80-4.50(m,2H),4.31-4.18(m,1H),3.33-3.31(m,1H),3.30(s,1H),3.16-3.07( m,3H),2.93-2.87(m,2H),1.90-1.70(m,3H),1.11-0.99(m,9H),0.95-0.88(m,2H),0.72-0.68(m,1H),0.55-0.51(m,1H).

[0096] LC-MS: m / z=541.3 (M+H) + , R t =1.163min.

[0097] Example 6. Synthesis of (S)-2-((4-((2-chlorobenzyl)(methyl)amino)-6-((3-cyclopropyl-1H-pyrazol-5-yl)amino)-1,3,5-triazin-2-yl)amino)-3,3-dimethyl-N-(prop-2-yn-1-yl)butanamide (Compound I-48)

[0098]

[0099] Compound 19 (347 mg, 715 μmol, 1.0 eq) was dissolved in 4 mL of dichloromethane. 1-Hydroxybenzotriazole (HOBt, 145 mg, 1.1 mmol, 1.5 eq), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCl, 206 mg, 1.1 mmol, 1.5 eq), and N,N-diisopropylethylamine (DIEA, 277 mg, 2.2 mmol, 374 μL, 3.0 eq) were added. Prop-2-yn-1-amine (compound 22, 39.4 mg, 715 μmol, 45.8 μL, 1.0 eq) was then added. The reaction was stirred at 25°C for 10 hours. LC-MS results indicated the reaction was complete. After completion of the reaction, the mixture was extracted with 5 mL of dichloromethane and washed twice with 4 mL of saturated brine. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated. The obtained compound was purified by prep-HPLC (column: C18-6 100*30mm*5μm; mobile phase: [water (formic acid)-acetonitrile]; B%: 35%-65%, 15min) to obtain a white solid compound I-48, 124 mg, with a yield of 32.2%.

[0100] 1 H NMR (400MHz, CD3OD) δppm 7.41-7.09(m,4H),6.60-5.25(m,1H),5.06-4.97(m,1H),4.60(br s,1H),4.31-4.14(m,1H),4.05-3.48(m,2H),3.16-3.05(m,3H),2.55-2.50(m,1H),1.87- 1.77(m,1H),1.16-0.99(m,9H),0.94-0.82(m,2H),0.77-0.62(m,1H),0.58-0.41(m,1H).

[0101] LC-MS: m / z=522.3 (M+H) + , Rt=2.337min.

[0102] Example 7. Synthesis of (S)-2-((4-((2-chlorobenzyl)(methyl)amino)-6-((3-isopropyl-1H-pyrazol-5-yl)amino)-1,3,5-triazin-2-yl)amino)-N,3,3-trimethylbutanamide (Compound I-16)

[0103]

[0104] Step 1. Synthesis of 4,6-dichloro-N-(3-isopropyl-1H-pyrazol-5-yl)-1,3,5-triazine-2-amine (Compound 24): 2,4,6-trichloro-1,3,5-triazine (Compound 1, 0.7 mg, 3.6 mmol, 1.0 eq) was dissolved in 5 mL of tetrahydrofuran (THF), followed by the addition of N,N-diisopropylethylamine (DIEA, 1.4 g, 10.8 mmol, 1.9 mL, 3.0 eq). 3-isopropyl-1H-pyrazol-5-amine (Compound 23, 0.4 g, 3.6 mmol, 1.0 eq) was added at -60°C. The reaction was stirred at -60°C for two hours. LC-MS analysis indicated the reaction was complete. 50 mL of water and ethyl acetate (1:1 volume ratio) were added, and the aqueous phase was extracted four times with 30 mL of ethyl acetate. The organic phases were combined, washed twice with 30 mL of saturated brine, then dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated in vacuo to obtain 1.4 g of a crude yellow solid compound 24.

[0105] LC-MS: m / z=273.0 (M+H) + , Rt=0.498min.

[0106] Step 2: Synthesis of 6-chloro-N2-(2-chlorobenzyl)-N4-(3-isopropyl-1H-pyrazol-5-yl)-N2-methyl-1,3,5-triazine-2,4-diamine (Compound 25)

[0107] 4,6-Dichloro-N-(3-isopropyl-1H-pyrazol-5-yl)-1,3,5-triazin-2-amine (Compound 24, 0.2 g, 0.7 mmol, 1.0 eq) was dissolved in 5 mL of tetrahydrofuran (THF). N,N-Diisopropylethylamine (DIEA, 0.3 g, 2.2 mmol, 0.4 mL, 3.0 eq) and 1-(2-chlorophenyl)-N-methylmethanamine (Compound 14, 0.1 g, 0.7 mmol, 0.1 mL, 1.0 eq) were added. The reaction was stirred at 25°C for 2 hours. LC-MS analysis indicated the reaction was complete. 50 mL of water and ethyl acetate (1:1 volume ratio) were added, and the aqueous phase was extracted four times with 30 mL of ethyl acetate. The combined organic phases were washed twice with 15 mL of saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated in vacuo to afford 0.365 g of crude compound 25 as a yellow oil.

[0108] LC-MS: m / z=392.1 (M+H) + , Rt=0.596min.

[0109] Step 3, Synthesis of (S)-2-((4-((2-chlorobenzyl)(methyl)amino)-6-((3-isopropyl-1H-pyrazol-5-yl)amino)-1,3,5-triazin-2-yl)amino)-N,3,3-trimethylbutanamide (Compound I-16)

[0110] 6-Chloro-N2-(2-chlorobenzyl)-N4-(3-isopropyl-1H-pyrazol-5-yl)-N2-methyl-1,3,5-triazine-2,4-diamine (Compound 25, 0.3 g, 0.8 mmol, 1.0 eq) was dissolved in 3 mL of dimethyl sulfoxide (DMSO). N,N-Diisopropylethylamine (DIEA, 0.3 g, 2.4 mmol, 0.4 mL, 3.0 eq) and (S)-2-amino-N,3,3-trimethylbutanamide (Compound 16, 0.1 g, 0.8 mmol, 1.0 eq) were added. The mixture was stirred at 100°C for 12 hours. LC-MS analysis indicated the reaction was complete. The residue was filtered and purified by prep-HPLC (column: C18-6 100*30mm*5μm; mobile phase: [water(trifluoroacetic acid)-acetonitrile]; B%: 30%-50%, 15min) to obtain white solid compound I-16, 152 mg, with a yield of 37.9%.

[0111] 1 H NMR(400MHz,DMSO-d6)δppm 10.58-10.23(m,1H),8.19-7.65(m,1H),7.52-7.43(m,1H),7.37-7.27(m,2H),7.20(s,1H),6.09-5.76(m,1H),4.9 6-4.82(m,2H),4.37(s,1H),3.25-3.03(d,J=1.6Hz,3H),2.99-2.83(m,1H),2.61(s,3H),1.20(m,6H),0.97(s,9H).

[0112] LC-MS: m / z=500.4 (M+H) + , Rt=0.498min.

[0113] In the present invention, other compounds were also prepared. The synthesis methods of these compounds refer to the methods in the above examples. The characterization data of some compounds, including NMR data and mass spectrometry data, are shown in Table 1.

[0114] Table 1 Characterization data of compounds

[0115]

[0116]

[0117]

[0118]

[0119] Example 8. In vitro inhibitory activity test of the compound on the target protein Aurora A kinase

[0120] Test method: The kinase inhibitory activity of the triazine compounds was determined using fluorescence resonance energy transfer experiments. 4 μM serine / threonine was used as a substrate peptide and 3 nM Aurora A kinase was used to determine the kinase inhibitory activity of different concentrations of compounds dissolved in 0.05% DMSO in a buffer containing 50 mM HEPES (pH 7.0), 10 mM MgCl2, 1 mM EGTA, and 0.01% Brij-35. The samples were incubated in a 384-well plate (Perkin Elmer 6007279) for 60 minutes, and the emission signal was measured using a PerkinElmer Envision 2104 plate reader with a second emission filter of 405 / 460 nm for excitation and emission, and 535 nm, respectively. Graphics and IC 50 The values ​​were generated using a four-parameter dose response function in GraphPad Prism. Figure 1 As shown, IC 50 As shown in Table 2 below:

[0121] Table 2 Aurora A kinase inhibitory activity of compounds in fluorescence resonance energy transfer assay

[0122]

[0123]

[0124] **** represents IC 50 ≤0.5μM; *** represents 0.5μM <IC 50 ≤1μM; ** represents 1μM <IC 50 ≤10

[0125] μM; * represents IC 50 >10μM.

[0126] The above experimental results demonstrate that the s-triazine compounds described herein exhibit potent inhibitory activity against Aurora kinase, with some exhibiting comparable activity to the positive control, VX-680. Further optimization is warranted to develop novel compounds that inhibit Aurora kinase activity. Furthermore, compounds I-45, I-46, I-47, and I-48 can be used as BRET probes to develop novel compounds with Aurora kinase inhibitory activity in living cells.

[0127] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A s-triazine compound having Aurora kinase inhibitory activity, or a pharmaceutically acceptable salt thereof, characterized in that: The s-triazine compound is represented by the following general formula (I): In formula (I): The R 1 is methyl, ethyl, propyl, n-butyl, 3-aminopropyl, propargyl; The R 2 is methyl, ethyl, propyl, n-butyl, isobutyl, tert-butyl, N-(1H-pyrrolo[2,3-b]pyridin-3-yl)methyl; The R 3 is N-[(3-chlorophenyl)methyl]-methylamino, N-[(2-chlorophenyl)methyl]-methylamino, N-hydroxyethylanilino, 4-aminomethylbenzylamino, 3-substituent R 5 -1H-pyrazol-5-amine and the substituent R 5 is methyl, ethyl, propyl, cyclopropyl, butyl, 5-amino-3-substituted R 6 -1H-pyrazol-1-yl and the substituent R 6 is methyl, ethyl, propyl, cyclopropyl, butyl; The R 4 is N-hydroxyethylaniline, 3-substituted R 5 -1H-pyrazol-5-amine and the substituent R 5 is methyl, ethyl, propyl, cyclopropyl, butyl, 5-amino-3-substituted R 6 -1H-pyrazol-1-yl and the substituent R 6 It is methyl, ethyl, propyl, cyclopropyl, and butyl.

2. The s-triazine compound according to claim 1, or a pharmaceutically acceptable salt thereof, characterized in that: The s-triazine compound is selected from any one of the following structural formulas: 。 3. A pharmaceutical composition, characterized in that The invention relates to a compound having the general formula (I) as claimed in claim 1, or a pharmaceutically acceptable salt thereof.

4. The pharmaceutical composition according to claim 3, characterized in that The pharmaceutical composition is an injection, an oral preparation or a mucosal administration preparation.

5. Use of the compound of formula (I) as claimed in claim 1, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition as claimed in claim 3 in the preparation of a medicament for inhibiting Aurora kinase.

6. The use according to claim 5, characterized in that The Aurora kinase is Aurora A kinase.

7. Use of the compound of formula (I) as claimed in claim 1, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition as claimed in claim 3 in the preparation of an anti-tumor drug; wherein: The tumor is an Aurora A-high-expressing tumor or an Aurora A inhibitor-resistant tumor.

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