CN112062768A

CN112062768A - Micromolecule with Aurora kinase degradation activity and preparation method and application thereof

Info

Publication number: CN112062768A
Application number: CN202010699560.6A
Authority: CN
Inventors: 刘强; 王自峰; 文石军; 段剑礼; 伍洲明; 王璇; 雷汉祺; 刘芳
Original assignee: Sun Yat Sen University Cancer Center
Current assignee: Sun Yat Sen University Cancer Center
Priority date: 2020-07-20
Filing date: 2020-07-20
Publication date: 2020-12-11
Anticipated expiration: 2040-07-20
Also published as: CN112062768B

Abstract

The invention discloses a small molecule with Aurora kinase degradation activity, a preparation method and application thereof. The inventor utilizes three E3 ubiquitin ligases of CRBN, cIAP and VHL to recruit ligands and a known Aurora-A inhibitor MLN8237 to obtain 12 Aurora-A protein degradation agents based on PROTAC technology through the self-owned Linker design technology. The inventor proves the degradation effect of the Aurora-A degrading agent on Aurora-A through a Western Blot experiment. CCK8 cell proliferation experiments and plate clone formation experiments prove that the Aurora-A degrading agent can effectively inhibit cell proliferation and clone formation. SPR experiments prove that the Aurora-A degradation agent is directly combined with Aurora-A protein.

Description

Micromolecule with Aurora kinase degradation activity and preparation method and application thereof

Technical Field

The invention relates to a preparation method and application of a small molecule-protein degradation targeting chimera with Aurora-A degradation activity. The degrading agent can effectively degrade Aurora-A protein in a targeted mode and inhibit the growth of tumor cells.

Background

Aurora kinase (Aurora kinase) is a type of serine/threonine kinase, including three subtypes, Aurora-a, Aurora-B and Aurora-C. Studies have shown that Aurora kinases promote centrosome maturation and chromosome segregation during cell mitosis. Among them, Aurora-a shows high expression in various tumors, and suggests that poor prognosis is one of the important drivers of tumorigenesis and tumor progression. A plurality of previous research works of the inventor prove that the Aurora-A kinase activity plays an important role in malignant transformation of various tumors such as breast Cancer, leukemia, nasopharyngeal carcinoma and the like (Cancer Res, 2007; Blood, 2008; Cancer Res, 2010; Med Res Rev, 2016). The targeted anticancer drug designed aiming at the Aurora-A kinase activity has important clinical value. Therefore, the inventors also developed a plurality of Aurora-a kinase inhibitors or drug combinations with proprietary intellectual property rights in the past (CN103191120B, CN 103202843B). The corresponding medicine shows better cancer inhibition effect on cell models and animal models.

Meanwhile, anticancer drug manufacturers such as merck and astrazen in the world also devote themselves on the research and development of Aurora-A targeted drugs. Aiming at indications such as lymphoma, breast cancer and the like, a plurality of compounds targeting Aurora-A kinase, such as MLN8237(alisertib), PHA-739358 and the like, have been subjected to phase II or phase III clinical tests in multiple centers around the world. For example, information published by the U.S. clinical trials database (clinical trials) shows: MLN8237 carried out a phase iii clinical trial in Peripheral T Cell Lymphoma (PTCL) (NCT 01482962); MLN8237 in combination with paclitaxel or in combination with fulvestrant is conducting phase II clinical trials in breast cancer (NCT02187991, NCT 02860000). However, with the end of early clinical trials, published clinical trial data indicate that inhibitors such as MLN8237 exhibit only limited anti-cancer effects in some clinical trials, fail to achieve the goal, and still face the difficulties of drug resistance and relapse.

Recent research of the inventor discovers that Aurora-A kinase can generate nuclear translocation in tumor cells, and transcriptionally activates xerosis genes such as FOXM1 and Myc to promote xerosis of the tumor stem cells through a non-kinase activity dependent transcriptional activation function, thereby causing tumor resistance (Oncogene 2017; Nat Commun 2016). These findings reveal the mechanism of development of resistance to traditional Aurora-a kinase activity inhibitors, suggesting that simultaneous targeting of Aurora-a kinase and non-kinase activities may be a key strategy to address Aurora-a inhibitor resistance. The induction of Aurora-a protein degradation is the fundamental method for blocking both its kinase activity and non-kinase activity: not only eliminates the kinase activity of the protein, but also eliminates the non-kinase function of the protein, can thoroughly eliminate Aurora protein, and achieves the best treatment effect.

In recent years, people successfully realize targeted degradation of different proteins by using a proteolytic targeting chimera (PROTAC) technology. PROTAC is a bifunctional molecule, consisting of three key components: the Protein ligase comprises a Protein of interest (POI) binding ligand, an E3 ubiquitin ligase recruitment ligand and a Linker connecting the two. PROTAC promotes ubiquitination of target proteins by recruiting E3 ubiquitinases, followed by degradation of target proteins through the ubiquitin-proteasome pathway. ProTAC seems to be simpler to understand in principle, however, it is not easy to develop a PROTAC that efficiently degrades a target protein. The following problems are mainly to be overcome:

1) selection of target protein binding partner: the most common solution at present is to select or improve from known inhibitors of the target protein. However, in the case of kinases, the kinases that are currently common often have multiple, or up to several, structurally disparate inhibitors. In addition to some examples of crystal structure implications, it is often necessary to perform experimental screening to find a target protein binding partner suitable for PROTAC.

2) Selection of E3 ubiquitin ligase recruited ligands: there are over 600 known enzymes E3 in human body, but only MDM2, cIAP, VHL and CRBN are the key components of E3 enzyme or E3 enzyme complex that are successful as E3 ubiquitin enzyme linking ligands of PROTAC at present. How to select a proper E3 ubiquitin ligase recruitment ligand does not have good design guiding principles at present, and screening and experimental attempts are also needed to find the E3 ubiquitin ligase recruitment ligand suitable for a target protein.

3) Optimization of Linker connecting the two parts: this aspect includes the linking position of Linker to the protein binding ligand (whether it interferes with the binding of the target protein linking ligand to the target protein), the molecular structure, molecular weight, length of Linker (whether the spatial structure of Linker itself affects the binding of bifunctional molecules to the target protein and the E3 enzyme), etc., and has a great influence on the effect of the final PROTAC bifunctional molecule. The currently used linkers are mainly PEG linkers, Alkyl linkers and "click chemistry" linkers. Each Linker varied in molecular length from 12 carbons to 20 carbons. How to design and select an effective Linker is not a relatively mature and reliable design method, mainly based on experimental screening and testing, except for some consideration based on the structures of known proteins and compounds when designing the Linker.

4) Solubility of the PROTAC molecule and its ability to penetrate the cell membrane into the cell: this is important to the ultimate ability of the ProTAC to enter cells. Reducing the molecular weight of PROTAC is a known solution, but is difficult to achieve in reality for certain specific target proteins. Therefore, experimental screening and attempts are also required.

Disclosure of Invention

The invention aims to overcome at least one defect in the prior art and provides a small molecule with Aurora kinase degradation activity, a preparation method and application thereof.

Based on the existing PROTAC design concept and the problems to be overcome, the inventor utilizes three E3 ubiquitin ligases of CRBN, cIAP and VHL to recruit ligands and a known Aurora-A inhibitor MLN8237 to obtain 12 Aurora-A protein degradation agents based on the PROTAC technology through the self-owned Linker design technology. And finally obtaining a plurality of effective Aurora protein degrading agents by screening through a Western blot technology, and naming the series of compounds as dAura.

The technical scheme adopted by the invention is as follows:

in a first aspect of the present invention, there is provided:

a compound of targeted ubiquitination degradation Aurora-A, which comprises Aurora-A binding ligand, recruitment ligand of E3 ubiquitin ligase and Linker connecting the Aurora-A binding ligand and the E3 ubiquitin ligase, and the structural formula of the compound is as follows:

in some examples, the compounds also include pharmaceutically acceptable salts, solvates, polymorphs, tautomers, or prodrugs thereof.

In a second aspect of the present invention, there is provided:

the synthesis method of the compound of the first aspect of the invention has the following synthetic route:

in a third aspect of the present invention, there is provided:

a composition comprising an active molecule selected from a compound according to the first aspect of the invention and an acceptable carrier.

In some examples, the composition is used to treat a tumor.

In some examples, the tumor is a tumor with high Aurora-A expression, or a tumor resistant to Aurora-A inhibitor.

In some examples, the tumor is selected from breast cancer, leukemia, lung cancer, liver cancer, esophageal cancer, pancreatic cancer, colorectal cancer, stomach cancer, cervical cancer, brain cancer, nasopharyngeal cancer.

In some examples, the composition is an injectable, oral, mucosal administration.

In a fourth aspect of the present invention, there is provided:

use of a compound as described in the first aspect of the invention in the manufacture of an anti-tumour medicament.

In some examples, the medicament is in the form of injection, oral agent, or mucosal agent.

The invention has the beneficial effects that:

the inventor confirms the degradation effect of the Aurora-A degrading agent on Aurora-A through Western Blot experiments (figure 1). Further, CCK8 cell proliferation experiments and plate clone formation experiments prove that the Aurora-A degrading agent can effectively inhibit cell proliferation and clone formation (figure 2 and figure 3). In addition, the inventors also demonstrated that this class of Aurora-a degradants binds directly to Aurora-a proteins by Surface Plasmon Resonance (SPR) experiments (fig. 4).

Drawings

FIG. 1 shows that Western blot detects the degradation effect of Aurora-A degrading agent on intracellular Aurora-A. Aurora-A degradation agent with the final concentration of 1uM is added into HEK293T cells (A picture) and MDA-MB-231 cells (B picture), and after 24 hours, proteins are collected, and the degradation effect of the compounds on Aurora-A proteins is detected by using a Western Blot technology.

FIG. 2 is a CCK8 experiment for detecting the killing effect of Aurora-A degradation agent on tumor cells. CCK8 experiment detects the killing effect of Aurora-A degradation agent on tumor cell MDA-MB-231.

FIG. 3 is a plate clone formation experiment for examining the inhibitory effect of Aurora-A degradant on the clonogenic capacity of tumor cells. Plate cloning formation test the cloning formation inhibiting effect of Aurora-A degrading agent on tumor cell MDA-MB-231.

FIG. 4 is an SPR experiment for detecting the binding capacity of Aurora-A degrading agent and Aurora-A kinase protein. Surface Plasmon Resonance (SPR) was used to detect the binding capacity of Aurora-A degradants to Aurora-A kinase.

Detailed Description

The embodiments of the invention will now be described in detail with reference to the drawings in the following examples, but the following examples and drawings are only for the understanding of the invention and are not intended to limit the invention, which can be embodied in many different ways as defined and covered by the claims.

Chinese-English comparison:

fetal Bovine Serum (FBS), Penicillin (Penicillin), Streptomycin (Streptomycin).

Primary reagent

Fetal bovine serum was purchased from Invitrogen; basal medium DMEM was purchased from Invitrogen.

Cell line and culture medium

Three cell cultures are mentioned in the examples. The media and formulation methods used are described below.

1) MDA-MB-231 cell (breast cancer cell line)

Culture medium: DMEM was supplemented with 10% Fetal Bovine Serum (FBS), 50U/ml penicillin, 50mg/ml streptomycin.

37℃，5％CO₂Culture, 2One passage for-3 days.

2) HEK293T cell (human embryo kidney cell line)

Culture medium: DMEM was supplemented with 10% fetal bovine serum, 50U/ml penicillin, 50mg/ml streptomycin.

37℃，5％CO₂Culturing and carrying out passage once in 2-3 days.

Firstly, the method comprises the following steps: synthesis procedure for Compounds of the invention

The raw materials used for synthesis comprise:

aurora-align (MLN8237, formula C)₂₇H₂₀ClFN₄O₄The alias 4- [ [9-chloro-7- (2-fluoro-6-methoxyphenyl) -5H-pyrimido [5,4-d ]][2]benzazepin-2-yl]amino]-2-method xy benzoic acid, CAS number 1028486-01-2);

CRBN ligand (Pomalidomide, chemical formula C)₁₃H₁₁N₃O₄Alias 3-AMINO-N- (2,6-DIOXO-3-PIPERIDYL) No. PHTHALIMIDE, CAS 19171-19-8); CRBN ligand1 (chemical formula C19H23N3O7, alias 4- (2- (2-aminoethoxy) ethoxy) ethoxy) -2- (2, 6-dioxyperidin-3-yl) isoindoline-1, 3-dione);

CRBN ligand 2 (chemical formula C)₁₉H₂₂N₄O₇The alias N- (2- (2-aminoethoxy) ethyl) -2- ((2- (2, 6-dioopioridin-3-yl) -1, 3-dioxoindolin-4-yl) oxy) acetamidine, CAS No. 2022182-59-6);

CRBN ligand 3 (chemical formula C)₂₁H₂₇N₃O₈The alias name 4- (2- (2- (2- (2-aminoethoxy) ethoxy) ethoxy) ethoxy) -2- (2, 6-dioxoperidin-3-yl) isoindolone-1, 3-dione);

CRBN ligand 4 (chemical formula C)₁₇H₂₀N₄O₅Alias 4- ((2- (2-aminoethoxy) ethyl) amino) -2- (2, 6-diopoperidin-3-yl) isoindoline-1,3-dione, CAS number 2138439-12-8);

VHL ligand1 (chemical formula C)₃₀H₄₅N₅O₄S, the alias (2S,4R) -1- ((S) -2- (8-aminoctanamidino) -3,3-dimethylbutanoyl) -4-hydroxy-N- (4- (4-methylisozol-5-yl) benzyl) pyrrolidone-2-carboxamide);

VHL ligand 2 (chemical formula C)₂₈H₄₁N₅O₆S, the alias (2S,4R) -1- ((S) -2- (3- (2- (aminomethoxy) ethoxy) propanaminodo) -3,3-dimethylbutanoyl) -4-hydroxy-N- (4- (4-methylthiazolid-5-yl) benzyl) pyrolidine-2-carboxamide);

cIAPeligand 0(N-Bocbestatin, chemical formula C₂₁H₃₂N₂O₆The alias name (S) -2- [ (2S,3R) -3- (tert-butoxyarylamino) -2-hydroxy-4-phenylbutanamido]-4-methylpentanoic acid, CAS number 87304-15-2). The structural formula is as follows:

synthesis of target protein Aurora-A binding ligand

The synthesis of Auroa-A ligand (MLN8237) is carried out step by step from S1-S7 intermediate, wherein:

synthesis of S1 was carried out by dissolving 2-amino-5-chloro-2, 6-difluorobenzophenone (2.5g) in tetrahydrofuran (THF,25mL), adding hydrochloric acid (4M, 24mL), cooling in ice, dissolving sodium nitrite in water, and adding dropwise to the reaction system. After 20 minutes, the reaction mixture was added dropwise to the reaction system with water to dissolve potassium iodide. After one and a half hour, the plate is spotted, and after the reaction is finished, the plate is directly extracted by ethyl acetate. And (6) spin-drying. Purifying with silica gel column chromatography, eluting with ethyl acetate/petroleum ether (1/20). 2.9g of a yellow solid are obtained, yield 82%.

Synthesis of S2 to a reaction system containing anhydrous methanol (12mL) and argon shield was added sodium blocks (280mg, added in 5 portions). After the sodium cake had completely disappeared, it was heated to 60 ℃ and Compound S1(2.8g) and methanol (15mL) were added. And (3) timing the reaction for 3 hours, and after the reaction is finished, spin-drying, extracting and spin-drying. Purifying with silica gel column chromatography, eluting with ethyl acetate/petroleum ether (1/15). 1.97g of a yellow solid was obtained in 68% yield.

Synthesis of S3A flask was charged with Compound S2(1.97g), N-Boc-aminopropyne (1.17g), cuprous iodide (28.82mg) and bis-triphenylphosphine palladium dichloride (106.21mg), and a mixed solution of triethylamine (TEA,20mL) and tetrahydrofuran (20mL) was added. Argon was purged 3 times and stirred at room temperature. The reaction was run overnight, spotted, extracted and spun dry. Purifying with silica gel column chromatography, eluting with ethyl acetate/petroleum ether (1/10). 1.95g of a brown oily substance was obtained in a yield of 93%.

Synthesis of S4 acetyl chloride (3mL) was added dropwise to methanol (18mL), and after 20 minutes, compound S3(1.95g) was dissolved in methanol and added. Half an hour later the plate was spotted and the reaction was complete, spun dry and a mixed solution of trifluoroacetic acid (TFA,7mL) and water (700uL) was added. The reaction was allowed to proceed overnight, the next day, spin-dried, dichloromethane (20mL) added, made basic with potassium carbonate, and stirred for 30 min. And (4) spin-drying, extracting and spin-drying. Purifying with silica gel column chromatography, eluting with ethyl acetate/petroleum ether (1/15). 933mg of brown solid is obtained, yield 65%.

Synthesis of S5 Compound S4(933mg), N-dimethylformamide dimethyl acetal (3.90mL), N-diisopropylethylamine (DMF,729.0uL) and dichloromethane (10mL) were added to a flask, and reacted at 40 ℃ overnight. Spotting, spin-drying, extracting and spin-drying. Purifying with silica gel column chromatography, eluting with ethyl acetate/petroleum ether (1/10). 1.02g of a yellow solid was obtained in 93% yield.

Synthesis of S6 methyl 2-methoxy-4-aminobenzoate (2.00g), methanol (10mL) and cyanamide (1.09mL) were added to a flask, stirred, dropwise added with concentrated nitric acid (2.06mL), and reacted at 80 ℃ under reflux for 4 hours. The plate was spotted, spun dry, 5mL of methanol was added, stirred and filtered when all the solids had broken up. 700mg of a yellow solid are obtained in 22% yield.

Synthesis of S7 Compound S6 and water (5mL) were added to a flask, stirred, and hydrochloric acid (12M, 1mL) was added to react at 78 ℃. The plates were spotted after 4 hours. And (5) filtering. 354mg of a red solid was obtained in 69%.

Synthesis of Aurora-Aliland Compound S5(300.00mg), S7(197.68mg), potassium carbonate (278.02mg) and methanol (5mL) were added to a flask and refluxed at 70 ℃ overnight. The next day, the plate is spotted, and after the reaction is finished, the pH value is adjusted to 1-3 by hydrochloric acid (2M). Extracting with ethyl acetate, and spin-drying. Purification by column chromatography on silica eluting with methanol/dichloromethane (1/50). Yield 190mg of yellow solidThe rate was 47%.¹H NMR(400MHz,DMSO-d6)12.09(s,1H),10.23(s,1H),8.72(s,1H),8.29(d,J＝8.5Hz,1H),7.96(s,1H),7.80(dd,J＝8.5,2.1Hz,1H),7.72(d,J＝8.6Hz,1H),7.46–7.33(m,2H),7.21(s,1H),6.88(s,2H),4.87(s,1H),3.83(s,4H),3.36(s,3H)；LRMS(ESI⁺，E/Z):519.1(M+H)⁺。

Synthesis of Aurora-A degradant dAura379

CRBN ligand 1(35mg) was dissolved in N, N-dimethylformamide (DMF, 1mL) and N, N-diisopropylethylamine (DIPEA, 63.7uL) was added. After stirring for 20 minutes, Aurora-A ligand (40mg) and 2- (7-azobenzotriazol) -N, N, N ', N' -tetramethyluronium hexafluorophosphate (HATU, 29.3mg) were added and the reaction was allowed to proceed overnight. The next day, the plates were spotted, extracted and spin dried. Purification by column chromatography on silica eluting with methanol/dichloromethane (1/30). 40mg of a yellow solid (dAura379) are obtained in 56% yield.¹H NMR(400MHz,CDCl3)10.39(s,1H),8.52(s,2H),8.35(s,1H),8.23(d,J＝8.4Hz,1H),8.11(d,J＝8.6Hz,1H),7.65(t,J＝7.8Hz,1H),7.58(d,J＝8.4Hz,1H),7.53(s,1H),7.43(d,J＝7.3Hz,2H),7.32(s,1H),7.29(d,J＝7.7Hz,1H),7.24(s,1H),6.75(br,2H),4.99(dd,J＝12.5,5.0Hz,1H),4.87(br,1H),4.34(s,2H),3.99–3.87(m,4H),3.84(s,3H),3.78(s,2H),3.69(d,J＝11.5Hz,7H),3.34(br,1H),2.88(d,J＝13.9Hz,2H),2.75(d,J＝14.3Hz,1H),2.13(dd,J＝9.7,5.3Hz,1H)；LRMS(ESI^-，E/Z):904.1(M-H)^-。

Synthesis of Aurora-A degradant dAura380

CRBN ligand 2(38.7mg) was dissolved in DMF (1mL) and DIPEA (63.7uL) was added. After stirring for 20 minutes, Aurora-A ligand (40mg) and HATU (29.3mg) were added to the solution, and the reaction was allowed to proceed overnight. The next day, the plates were spotted, extracted and spin dried. Purification by column chromatography on silica eluting with methanol/dichloromethane (1/30). 33mg of a pale yellow solid (dAura380) are obtained, yieldThe rate was 45%.¹H NMR(400MHz,CDCl3)10.21(s,1H),8.44(s,1H),8.40(s,1H),8.23(d,J＝8.4Hz,2H),8.12(d,J＝8.5Hz,1H),7.89(s,1H),7.68(t,J＝7.8Hz,1H),7.56(d,J＝8.4Hz,1H),7.49(d,J＝7.3Hz,1H),7.28(d,J＝7.2Hz,2H),7.16(d,J＝8.5Hz,1H),7.12(s,1H),6.67(d,J＝35.9Hz,2H),4.88(dd,J＝11.8,5.1Hz,1H),4.72–4.58(m,2H),3.88(s,3H),3.85–3.39(m,11H),2.82–2.61(m,3H),2.07(d,J＝7.4Hz,1H)；LRMS(ESI^-，E/Z):917.0(M-H)^-。

Synthesis of Aurora-A degradation agent dAura383

CRBN ligand 3(41.6mg) was dissolved in DMF (1mL), DIPEA (63.7uL) was added, and after stirring for 20 minutes, Aurora-A ligand (40mg) and HATU (29.3mg) were added and the reaction was allowed to proceed overnight. The next day, the plates were spotted, extracted and spin dried. Purification by column chromatography on silica eluting with methanol/dichloromethane (1/20). 39mg of a yellow solid (dAura383) were obtained in 52% yield.¹H NMR(400MHz,CDCl3)10.59(s,1H),8.62(s,1H),8.53(s,1H),8.23(t,J＝5.9Hz,2H),8.14(d,J＝8.7Hz,1H),7.69–7.54(m,3H),7.45(d,J＝7.3Hz,1H),7.35(s,1H),7.34–7.27(m,3H),6.76(br,2H),5.00(dd,J＝12.5,5.4Hz,1H),4.94–4.72(br,1H),4.39–4.31(m,2H),4.09–3.46(m,20H),2.90(m,J＝13.4Hz,2H),2.76(m,J＝14.8Hz,1H),2.20–2.09(m,1H)。

Synthesis of Aurora-A degradant dAura393

CRBN ligand 4(23mg) was dissolved in DMF (1mL), and DIPEA (52.6uL) was added and stirred. After 20 min, Aurora-A ligand (33mg) and HATU (24.18mg) were added and the reaction was allowed to proceed overnight. The next day, the plates were spotted, extracted and spin dried. Purification by column chromatography on silica eluting with methanol/dichloromethane (1/30). 21mg of a yellow solid (dAura393) was obtained in 38% yield. 1H NMR (400MHz, CDCl3)9.60(s,1H),8.49(s,1H), 8.26-8.08 (m,4H),7.82(s,1H),7.55(dd, J ═ 8.5,2.0Hz,1H), 7.50-7.42 (H) ((s, 1H))m,1H),7.30(t,J＝7.5Hz,2H),7.20(d,J＝8.4Hz,1H),7.07(d,J＝7.1Hz,1H),6.91(d,J＝8.6Hz,1H),6.70(s,2H),6.55(t,J＝5.2Hz,1H),4.81(m,1H),4.00–3.22(m,14H),2.73(dd,J＝25.2,21.2Hz,3H),2.10–2.02(m,1H)；LRMS(ESI⁺，E/Z):861.2(M+H)⁺。

Synthesis of Aurora-A degradant dAura408

VHL ligand 1(27.55mg) was dissolved in DMF (0.5mL) and DIPEA (40.0uL) was added and stirred. After 20 min, Aurora-A ligand (25mg) and HATU (18.32mg) were added, reacted for 2h, spotted, extracted and spin-dried. Purification by column chromatography on silica eluting with methanol/dichloromethane (1/15). 30mg of a white solid (dAura408) were obtained in 58% yield. 1H NMR (400MHz, CDCl3)8.83(s,1H),8.60(s,1H),8.43(s,1H),8.14(d, J ═ 8.4Hz,1H),8.01(d, J ═ 8.5Hz,1H), 7.93-7.79 (m,3H),7.49(d, J ═ 7.7Hz,2H),7.22(dd, J ═ 17.1,7.9Hz,4H),7.04(d, J ═ 8.4Hz,1H),6.62(d, J ═ 71.4Hz,2H),6.33(d, J ═ 8.6Hz,1H),4.83(br,1H),4.65(t, J ═ 7.9, 1H),4.47(dd, J ═ 8.47, 15, 7.6 Hz,1H),4.83(br,1H),4.65(t, J ═ 7.9, 1H),4.47(dd, 15, 7.6H, 3.9 Hz,3H), 3.9H, 1H, 7.9H, 3.9H, 7.9H, 6H, 1H, 7.9H, 6H, 1H, 7.9H, 6H, 7.9H, 1H, 6H, 1H, 7H, 6H, 1H, 7H, 6H, 1H, 3H) 2.37-2.29 (m,1H), 2.16-2.04 (m,3H),1.50(d, J ═ 6.1Hz,4H),0.88(s, 9H); LRMS (ESI)⁺，E/Z):1072.4(M+H)⁺。

Synthesis of Aurora-A degradant dAura425

VHL ligand 2(20mg) was dissolved in DMF (0.5mL) and DIPEA (17.22uL) was added and stirred. After 20 min, Aurora-A ligand (18.03mg) and HATU (13.21mg) were added, reacted for 2h, spotted, extracted and spin-dried. Purification by column chromatography on silica eluting with methanol/dichloromethane (1/20). This gave 5mg of a white solid (dAura425) in 13% yield. 1H NMR (400MHz, CDCl3)8.66(s,1H),8.51(s,1H),8.20(d, J ═ 7.5Hz,2H),8.09(d, J ═ 8.2Hz,1H),7.88(s,1H),7.61–7.50(m,2H),7.41(d,J＝8.4Hz,1H),7.37–7.27(m,6H),7.05(d,J＝7.3Hz,1H),6.79(m,3H),4.87(br,1H),4.60–4.45(m,4H),4.28(d,J＝15.5Hz,1H),4.10–3.96(m,4H),3.93(s,3H),3.69(dd,J＝18.4,13.0Hz,7H),3.57(d,J＝10.2Hz,1H),3.21(dd,J＝14.5,7.2Hz,2H),2.49(d,J＝12.0Hz,3H),2.04(t,J＝10.6Hz,1H),1.39(t,J＝7.2Hz,3H),0.92(s,9H)；LRMS(ESI⁺，E/Z):1076.5(M+H)⁺。

Synthesis of Aurora-A degradant dAura427

Synthesis of 7a starting material S8(18.89mg) was dissolved in DMF (0.5mL), and DIPEA (34uL), Aurora-A ligand (40mg) and HATU (29.31mg) were added to react for 2 hours, spotted and spun dry. Purification by column chromatography on silica eluting with methanol/dichloromethane (1/15). Synthesis of 7b Compound 7a (65mg) was dissolved in dichloromethane (1mL), trifluoroacetic acid (100uL) was added and the reaction was run for 2.5h, spotted. After the reaction, the mixture was concentrated and then dried by a diaphragm pump. And (4) fully harvesting.

Compound 7b (55mg) was dissolved in DMF (0.5mL), DIPEA (40.2uL) was added, and the mixture was stirred. After 20 min, cIAP ligand 0(44.6mg) and HATU (34.6mg) were added, reacted for 2h, spotted, extracted and spin dried. Purification by column chromatography on silica eluting with methanol/dichloromethane (1/20) afforded dAurA 427. The yield was 40%. 1H NMR (400MHz, CDCl3)8.50(s,1H),8.22(d, J ═ 8.5Hz,1H),8.12(t, J ═ 5.9Hz,1H),8.06(d, J ═ 8.6Hz,1H),7.98(t, J ═ 12.5Hz,1H),7.74(s,1H),7.56(dd, J ═ 8.5,2.1Hz,1H), 7.34-7.27 (m,2H),7.22(dd, J ═ 9.2,3.1Hz,4H),7.06(d, J ═ 53.4Hz,2H),6.68(br,2H),4.52(br,1H),4.17(m,3H),3.99(s,3H),3.80 (s,3H), 23.80 (s,3H), 6.68(br,2H),4.52(br,1H),4.17(m,3H), 3.75 (d, 3.87H); LRMS (ESI)⁺，E/Z):995.4(M+H)⁺。

Synthesis of Aurora-A degradant dAura442

Synthesis of 8a YM67(20.0mg) was dissolved in DMF (0.5mL), DIPEA (34.1uL), Aurora-A ligand (40mg) and HATU (29.3mg) were added, and the reaction was carried out for 2 hours, spotted and spun dry. Purification by column chromatography on silica eluting with methanol/dichloromethane (1/15). The yield was 67%. Synthesis of 8b Compound 8a (37mg) was dissolved in dichloromethane (1mL), trifluoroacetic acid (100uL) was added and the reaction was run for 2.5 hours, spotted. After the reaction, the mixture was concentrated and then dried by a diaphragm pump. And (4) fully harvesting.

Compound 8b (30mg) was dissolved in DMF (0.5mL), DIPEA (21.5uL) was added, and the mixture was stirred. After 20 min, cIAP ligand 0(23.8mg) and HATU (18.5mg) were added, reacted for 2h, spotted, extracted and spin dried. Purification by column chromatography on silica eluting with methanol/dichloromethane (1/30). The yield was 69%. 1H NMR (400MHz, CDCl3)8.49(s,1H),8.22(d, J ═ 8.5Hz,1H),8.06(d, J ═ 8.5Hz,1H), 8.02-7.79 (m,3H),7.56(dd, J ═ 8.5,1.9Hz,1H),7.32(d, J ═ 1.7Hz,1H),7.29(d, J ═ 6.7Hz,1H),7.18(m,6H),6.63(br,2H),5.12(d, J ═ 9.1Hz,1H),4.86(br,1H),4.47(m,1H),4.18(m,3H), 4.02-3.93 (m,3H),3.34(m,5H), 3.04-2.88 (m,2H), 2.81-3.81 (m, 0.94H), 0.73-0.7 (m, 3H); LRMS (ESI)⁺，E/Z):1007.4(M+H)⁺。

Synthesis of Aurora-A degradant dAura446

Synthesis of 9a YM74(17.8mg) was dissolved in DMF (0.5mL), DIPEA (34.1uL), Aurora-A ligand (40mg) and HATU (29.3mg) were added, and the reaction was carried out for 2 hours, spotted on plates, and pure spin-dried. Purification by column chromatography on silica eluting with methanol/dichloromethane (1/20). The yield was 96%. Synthesis of 9b Compound 9a (55mg) was dissolved in dichloromethane (1mL), trifluoroacetic acid (TFA,100uL) was added and the reaction was run for 2.5h, and the plates were spotted. After the reaction, the mixture was concentrated and then dried by a diaphragm pump. And (4) fully harvesting.

Compound 9b (47mg) was dissolved in DMF (0.5mL), and DIPEA (33.0uL) was added thereto and stirred. After 20 min, cIAP ligand 0(36.5mg) and HATU (34.0mg) were added, reacted for 2h, spotted, extracted and spin dried. Purification by column chromatography on silica gel eluting with methanol/dichloromethane (1/30) afforded dAurA 446. The yield was 42%. 1H NMR (400MHz, CDCl3)8.49(d, J ═ d6.2Hz,1H),8.22(d,J＝8.5Hz,1H),8.14(d,J＝8.5Hz,1H),7.96(s,1H),7.71(s,1H),7.56(dd,J＝8.5,2.0Hz,1H),7.34–7.31(m,1H),7.29(d,J＝6.7Hz,1H),7.23(t,J＝7.2Hz,4H),7.18(d,J＝6.8Hz,1H),7.14(d,J＝8.4Hz,1H),6.74(br,2H),4.86(br,1H),4.49–4.36(m,1H),4.13(dd,J＝18.5,10.2Hz,2H),3.98(s,3H),3.47–2.92(m,7H),1.64–1.52(m,4H),1.41–1.18(m,22H),0.89(dd,J＝17.7,6.0Hz,6H)；LRMS(ESI⁺，E/Z):1035.3(M+H)⁺。

Synthesis of Aurora-A degradant dAura448

Compound dAURA427(6.6mg) was dissolved in hydrogen chloride in methanol (1M,1ml) and reacted for 5 hours, and spotted. After the reaction was completed, the reaction mixture was concentrated and then dried by a diaphragm pump to obtain dAura 448. And (4) fully harvesting. LRMS (ESI)⁺，E/Z):931.2(M+H)⁺。

Synthesis of Aurora-A degradant dAura449

Compound dAurA442(6.1mg) was dissolved in hydrogen chloride in methanol (1M,1ml) and reacted for 4.5 hours, and spotted. After the reaction, the mixture was concentrated and then dried by a diaphragm pump to obtain dAura 449. And (4) fully harvesting. LRMS (ESI)^-，E/Z):941.2(M-H)^-。

Synthesis of Aurora-A degradant dAura450

Compound dAurA446(5.2mg) was dissolved in hydrogen chloride in methanol (1M,1ml) and reacted for 5.5 hours, and spotted on a plate. After the reaction, the mixture was concentrated and then dried by a diaphragm pump to obtain dAurA 450. And (4) fully harvesting. LRMS (ESI)^-，E/Z):969.1(M-H)^-。

II, secondly: detection of degradation effect of compound of the invention on Aurora-A kinase

1) The breast cancer cell line MDA-MD-231 cells and the human embryonic kidney cell line HEK293T were plated on 6-well plates.

2) After the cells are completely attached to the wall for 6 hours, Aurora-A degradation agents with the final concentration of 1uM are respectively added.

3) Total protein was harvested and quantified from each well after 24 hours and the effect of these compounds on Aurora-A protein degradation was verified using Western Blot technique.

The results are shown in FIG. 1. The Aurora-A degradation agents synthesized by the inventor show different degrees of degradation effect on intracellular Aurora-A in umol grade.

Thirdly, the method comprises the following steps: experiment on inhibition of tumor cell proliferation by the Compound of the present invention

1) Paving the breast cancer cell line MDA-MD-231 cells on a 96-well plate according to 1000/well;

2) after 24 hours, 10uM, 5uM, 2.5uM, 1.25uM, 625nM, 312.5nM, 156.25nM, 78.125nM, 39nM concentrations of dAura degradant were added to 96-well plates, respectively;

3) after 4 days 20uL of CCK8 reagent was added and absorbance was calculated after 3 hours of standing.

As shown in FIG. 2, the Aurora-A degrading agents synthesized by the inventors have half Inhibitory Concentration (IC) 50 on tumor cells₅₀) On the umol scale.

Fourthly, the method comprises the following steps: experiment of inhibiting ability of compound of the present invention to clone tumor cells

1) Paving the breast cancer cell line MDA-MD-231 cells on a 6-well plate according to 1000/well;

2) respectively adding dAura degradation agent according to 8uM, 4uM, 2uM, 1uM and 500 nM;

3) after 1 week, the medium was washed off with PBS, fixed with 4% paraformaldehyde, stained with crystal violet for 15 minutes, and the color development was analyzed by photography.

The result is shown in figure 3, the Aurora-A degradation agent synthesized by the inventor has better inhibiting effect on the clonal formation capability of the tumor cell plate.

Fifthly: detection of binding Capacity of Compounds of the invention to Aurora-A kinase

1) Aurora-A protein was purified and immobilized on a CM5 chip

2) Buffer preparation and solvent Curve calibration Using Biacore 2000

3) Different concentrations of dAura (100uM, 50uM, 25uM, 12.5uM, 6.25uM, 3.125uM, 1.56uM) were incubated with the Aurora-A protein solution, which in turn was coupled to the Aurora-A protein. The known Aurora-A inhibitor MLM8237 was used as a control.

The results are shown in FIG. 4, the Aurora-A degradation agent synthesized by the inventor is directly combined with Aurora-A protein, and the binding force constants are respectively as follows:

dAurA383 Kd＝8.825x10^-6；

dAurA425 Kd＝4.13x10^-5；

dAurA450 Kd＝8.32x10^-5。

therefore, summarizing, the inventors confirmed the degradation effect of this class of Aurora-A degrading agents on Aurora-A by Western Blot experiments, CCK8 cell proliferation experiments and plate clone formation experiments. Further, CCK8 cell proliferation experiments and plate clone formation experiments prove that the Aurora-A degrading agent can effectively inhibit cell proliferation and clone formation. In addition, the inventor also proves that the Aurora-A degradation agent is directly combined with Aurora-A protein through a surface plasmon resonance experiment.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims

1. A compound of targeted ubiquitination degradation Aurora-A, which comprises Aurora-A binding ligand, recruitment ligand of E3 ubiquitin ligase and Linker connecting the Aurora-A binding ligand and the E3 ubiquitin ligase, and the structural formula of the compound is as follows:

2. the compound of claim 1, wherein: also included are pharmaceutically acceptable salts, solvates, polymorphs, tautomers or prodrugs thereof.

3. The compound of claim 1, which is synthesized by the following steps:

4. a composition consisting of an active molecule and an acceptable carrier, wherein: the active molecule is selected from the compounds of claim 1 or 2.

5. The composition of claim 4, wherein: it can be used for treating tumor.

6. The composition of claim 5, wherein: the tumor is a tumor with high Aurora-A expression or an Aurora-A inhibitor-resistant tumor.

7. The composition of claim 6, wherein: the tumor is selected from breast cancer, leukemia, lung cancer, hepatocarcinoma, esophageal cancer, pancreatic cancer, colorectal cancer, gastric cancer, cervical cancer, brain cancer, and nasopharyngeal carcinoma.

8. The composition according to any one of claims 4 to 7, characterized in that: the composition is injection, oral preparation, and mucosa administration preparation.

9. The use of a compound according to claim 1 or 2 in the preparation of an anti-neoplastic medicament, said tumour being a tumour highly expressed in Aurora-a or a tumour resistant to Aurora-a inhibitor.

10. Use according to claim 9, characterized in that: the tumor is selected from breast cancer, leukemia, lung cancer, hepatocarcinoma, esophageal cancer, pancreatic cancer, colorectal cancer, gastric cancer, cervical cancer, brain cancer, and nasopharyngeal carcinoma.