PROTEOLYSIS TARGETING CHIMERA (PROTAC) FOR DEGRADATION OF AURORA A-KINASE

Abstract

The invention concerns a Proteolysis Targeting Chimera (PROTAC) or a pharmaceutically acceptable salt thereof for degradation of Aurora A-kinase in cells of a mammal which PROTAC has the chemical structure AAB-L-E3B, wherein AAB is a binding unit for Aurora A-kinase, L is a linker and E3B is a binding unit for E3-ubiquitin ligase Cereblon, wherein E3B comprises the structure of thalidomide or of one of its analogs lenalidomide, pomalidomide, and apremilast, wherein L comprises or consists of an alkyl ether residue or a polyalkyl ether residue or an alkyl ether residue or a polyalkyl ether residue in which at least one C—C bond is replaced by a C═C double bond, which polyalkyl ether residue has at least two ether groups or at least two ether groups in which one O-atom is replaced by an S-atom or a part of the O-atoms is replaced by S-atoms or wherein L comprises or consists of an alkyl thioether residue or a polyalkyl thioether residue or an alkyl thioether residue or a polyalkyl thioether residue in which at least one C—C bond is replaced by a C═C double bond, which polyalkyl thioether residue has at least two thioether groups, wherein L connects AAB and E3B via a chain of atoms having five to thirteen subsequently arranged atoms, wherein atoms of functional groups connecting the linker with AAB and E3B are not considered as part of said chain.

Claims

1. Proteolysis Targeting Chimera (PROTAC) or a pharmaceutically acceptable salt thereof for degradation of Aurora A-kinase in cells of a mammal which PROTAC has a chemical structure AAB-L-E3B, wherein AAB is a binding unit for Aurora A-kinase, L is a linker and E3B is a binding unit for E3-ubiquitin ligase Cereblon, wherein E3B comprises a structure of thalidomide or of one of its analogs lenalidomide, pomalidomide, and apremilast, wherein L comprises or consists of an alkyl ether residue or a polyalkyl ether residue or an alkyl ether residue or a polyalkyl ether residue in which at least one C—C bond is replaced by a C═C double bond, which polyalkyl ether residue has at least two ether groups or at least two ether groups in which one O-atom is replaced by an S-atom or a part of the O-atoms is replaced by S-atoms or wherein L comprises or consists of an alkyl thioether residue or a polyalkyl thioether residue or an alkyl thioether residue or a polyalkyl thioether residue in which at least one C—C bond is replaced by a C═C double bond, which polyalkyl thioether residue has at least two thioether groups, wherein the alkyl ether residue, polyalkyl ether residue, alkyl thioether residue or polyalkyl thioether residue is optionally substituted at least at one position by an amino group, a hydroxyl group or a carbonyl group, wherein L connects AAB and E3B via a chain of atoms having five to thirteen subsequently arranged atoms, wherein atoms of functional groups connecting the linker with AAB and E3B are not considered as part of said chain, wherein functional groups are selected from —NH—, —COO—, —CONH—, —CSO— and —COS—.

2. PROTAC or a pharmaceutically acceptable salt thereof according to claim 1, wherein L is bound to AAB via an amide bond A, in particular a peptide bond A, and/or wherein L is bound to E3B via an amide bond B, in particular a peptide bond B.

3. PROTAC or a pharmaceutically acceptable salt thereof according to claim 2, wherein an H-atom of the amide bond A is substituted by an alkyl residue A, in particular a methyl residue A or an ethyl residue A, and/or wherein an H-atom of the amide bond B is substituted by an alkyl residue B, in particular a methyl residue B or an ethyl residue B.

4. PROTAC or a pharmaceutically acceptable salt thereof according to claim 1, wherein L comprises at least two O-atoms, at least two S-atoms or at least one O-atom and at least one S-atom.

5. PROTAC or a pharmaceutically acceptable salt thereof according to claim 1, wherein L is a linear molecule residue and/or said chain of atoms has 6 to 13 subsequently arranged atoms, in particular 6 to 10 subsequently arranged atoms.

6. PROTAC or a pharmaceutically acceptable salt thereof according to claim 1, wherein L is any of the following moieties: —CH.sub.2—CH.sub.2—O—CH.sub.2—CH.sub.2— and —CH.sub.2—(CH.sub.2—O—CH.sub.2).sub.2—CH.sub.2—.

7. PROTAC or a pharmaceutically acceptable salt thereof according to claim 1, wherein AAB comprises a structure of alisertib, (3-chloro-2-fluorophenyl)[4-[[6-(2-thiazolylamino)-2-pyridinyl]methyl]-1-pi perazinyl]-methanone (MK-8745) or 1-[4-({4-[(5-cyclopentyl-1H-pyrazol-3-yl)amino]pyrimidin-2-yl}amino)phenyl]-3-[3-(trifluoromethyl)phenyl]urea (CD532).

8. PROTAC or a pharmaceutically acceptable salt thereof according to claim 7, wherein AAB comprises the structure of alisertib which is bound to L via a peptide bond formed from alisertib's carboxy group and an amino group of L.

9. PROTAC or a pharmaceutically acceptable salt thereof according to claim 1, wherein the PROTAC is any of the following compounds: ##STR00012## ##STR00013## ##STR00014## ##STR00015##

10. Method for synthesizing the PROTAC or a pharmaceutically acceptable salt thereof according to claim 1, wherein 2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetic acid (compound a) ##STR00016## is dissolved in an aprotic solvent followed by addition of (1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU) and N,N-diisopropylethylamine (DIPEA) or trimethylamine as well as a linker precursor having a structure “NH.sub.2-alkylether residue-NH-amine protecting group”, “NH.sub.2-polyalkylether residue-NH-amine protecting group”, “NH.sub.2-alkyl thioether residue-NH-amine protecting group” or “NH.sub.2-polyalkyl thioether residue-NH-amine protecting group” and further followed by incubation resulting in an intermediate product which is then deprotected to result in compound b ##STR00017## wherein the polyalkyl ether residue has at least two ether groups or at least two ether groups in which at least one O-atom is replaced by an 5-atom and the polyalkyl thioether residue has at least two S-atoms, wherein the alkyl ether residue, the polyalkyl ether residue, alkyl thioether residue, or the polyalkyl thioether residue has a chain of atoms having 5 to 13 subsequently arranged atoms, wherein no linear sequence of atoms of the alkyl ether residue, the polyalkyl ether residue, alkyl thioether residue, or the polyalkyl thioether residue exceeds the number of 17 atoms, wherein compound b is dissolved in an aprotic solvent followed by addition of (1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU), alisertib and N,N-diisopropylethylamine (DIPEA) or trimethylamine and further followed by incubation to result in compound c ##STR00018## and optionally synthesizing a pharmaceutically acceptable salt thereof.

11. Method according to claim 10, wherein the solvent is a polar solvent, in particular Dimethylformamide (DMF), and/or the amine protecting group is tert-butyloxycarbonyl (Boc).

12. Method according to claim 10, wherein the linker precursor is any of following compounds 1 and 2: 1. NH.sub.2—CH.sub.2—CH.sub.2—O—CH.sub.2—CH.sub.2—NH-Boc 2. NH.sub.2—CH.sub.2—(CH.sub.2—O—CH.sub.2).sub.2—CH.sub.2—NH-Boc

13. Method for treating cancer of a human being or another mammal comprising administering the PROTAC or a pharmaceutically acceptable salt thereof of claim 1 to said human being or mammal.

14. Method according to claim 13, wherein the cancer is leukemia, neuroblastoma, hepatocellular carcinoma or osteosarcoma.

Description

EMBODIMENTS OF THE INVENTION

[0030] FIG. 1 shows schematically the general synthesis of compounds JB158, JB159, JB169, JB170, JB171 having a thalidomide moiety and of compounds JB160, JB161 having a VHL-ligand which compounds are linked to alisertib by various (poly)ethylene glycol (PEG) and aliphatic linkers.

[0031] FIG. 2 shows an immunoblot and quantification of endogenous Aurora-A from cells of human leukemia cell line MV4-11 which cells were treated in each case with a single dose of compounds JB158, JB160, JB161, JB169, JB170, JB159 and JB171 in a concentration of 1 μM. Vinculin served as a loading control in the immunoblot.

[0032] FIG. 3 shows immunoblots of endogenous Aurora-A from MV4-11 untreated cells and cells treated with various concentrations of JB170 (upper panel) and JB158 (lower panel) as well as with unconjugated alisertib (uA) for 6 hours with Vinculin as a loading control.

[0033] FIG. 4 shows immunoblots of endogenous Aurora-A from cells of human osteosarcoma cell line U2OS treated with various concentrations of JB170 (upper panel) and JB158 (lower panel) as well as with unconjugated alisertib (uA) for 6 hours with Vinculin as a loading control.

[0034] FIG. 5 shows immunoblots of endogenous Aurora-A from cells of human hepatocellular carcinoma cell line HLE treated with various concentrations of JB170 (upper panel) and JB158 (lower panel) as well as with unconjugated alisertib (uA) for 6 hours with Vinculin as a loading control.

[0035] FIG. 6 shows an immunoblot of endogenous Aurora-A from cells of human neuroblastoma cell line IMR5 treated with JB170 in a concentration of 0.1 μM for the indicated time periods with Vinculin as a loading control.

[0036] FIG. 7 shows an immunoblot of endogenous Aurora-A from cells of HLE cells treated with JB158 in a concentration of 0.1 μM for the indicated time periods with Vinculin as a loading control.

[0037] FIG. 8 is a bar diagram showing cellular viability of MV4-11 cells treated with 1 μM JB170.

[0038] FIG. 9 is a diagram showing apoptosis of MV4-11 cells treated with 0.5 μM JB170 and stained for Annexin and with Propidium Iodide (PI), wherein the amounts of early (Annexin+, PI−) and late (Annexin+, PI+) apoptotic cells were analyzed by FACS.

[0039] FIG. 10 is a diagram showing apoptosis of IMR5 cells (EtOH) and IMR5 cells expressing Aurora-A.sup.T217D upon incubation with doxycycline (Dox (Aurora-A.sup.T217D)) which cells were treated with 0.5 μM JB170 for 72 hours, stained for Annexin and with Propidium Iodide (PI) and analyzed by FACS.

[0040] The inventors synthesized Aurora-A targeting chimeric degraders by linking alisertib to thalidomide as a Cereblon-binding moiety or to a HIF1-derived peptidomimetic as a VHL-binding moiety via a variety of (poly)ethylene glycol (PEG) or aliphatic linkers as schematically shown in FIG. 1.

[0041] In detail, syntheses were performed as follows:

[0042] Unless otherwise stated, all reactions were performed at room temperature (RT). The Structure and purity of the compounds was confirmed by HPLC, mass spectrometry and NMR.

[0043] For thalidomide derivative-linker coupling the thalidomide-derivative 2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetic acid (Compound a) (310 mg, 0.93 mmol, 1.0 eq) was dissolved in 5 ml DMF under argon and HATU (418 mg, 1.02 mmol, 1.1 eq) and DIPEA (324 μl, 1.86 mmol, 2.0 eq) were added.

[0044] After 15 minutes stirring at RT, a solution of the respective linker 1-5 (1.12 mmol, 1.2 eq) in 2 ml DMF was added and the reaction mixture was stirred overnight. The mixture was transferred into a separation funnel and extracted with water (20 ml) and ethyl acetate (30 ml). The layers were separated and the aqueous layer was extracted 3 times with ethyl acetate. The organic layers were combined, dried over MgSO.sub.4 and concentrated. The residue was purified with Flash Chromatography (FC) using DCM/MeOH, 95:5. The resulting intermediate product was obtained as colorless oil. The intermediate product was dissolved in a mixture of DCM and trifluoroacetic acid (6 ml, 40 vol. %). After 30 min the solvent was removed under reduced pressure, to obtain the deprotected thalidomide-linker intermediate as TFA-salt (c-g). The reaction is illustrated in the following reaction scheme:

##STR00009##

[0045] Intermediate c N-(2-(2-aminoethoxy)ethyl)-2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamide (Th-PEG1-NH2) was obtained with overall yield of 69% (339 mg, 0.93 mmol) over two steps (1.sup.st 71%, 2.sup.nd 97%).

[0046] Intermediate d N-(3-(2-(2-(3-Aminopropoxy)ethoxy)ethoxy)propyl)-2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamide (Th-PEG3-NH2) was obtained with overall yield of 57% (285 mg, 0.53 mmol) over two steps (1st 60%, 2.sup.nd 95%).

[0047] Intermediate e N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamide (Th-PEG2-NH.sub.2) was obtained with overall yield of 73% (316 mg, 0.68 mmol) over two steps (1.sup.st 75%, 2.sup.nd 97%).

[0048] Intermediate f N-(3-aminopropyl)-2-[[2-(2,6-dioxo-3-piperidinyl)-2,3-dihydro-1,3-dioxo-1H-isoindol-4-yl]oxy] acetamide was obtained with overall yield of 60% (233 mg, 0.60 mmol) over two steps (1st 63%, 2.sup.nd 95%).

[0049] Intermediate g N-(5-aminopentyl)-2-[[2-(2,6-dioxo-3-piperidinyl)-2,3-dihydro-1,3-dioxo-1H-isoindol-4-yl]oxy] acetamide was obtained with overall yield of 53% (206 mg, 0.50 mmol) over two steps (1st 60%, 2.sup.nd 88%).

[0050] For VHL-ligand-linker coupling VHL ligand Compound b (92 mg, 0.214 mmol, 1.0 eq) was dissolved in 6 ml DMF in a flask (A) (reaction mixture (A)) under argon. The respective linker 6-7 (0.236 mmol, 1.1 eq) and HATU (92 mg, 0.241 mmol, 1.1 eq) were dissolved in 6 ml DMF in a separate flask (B) (reaction mixture (B)) under argon atmosphere. Both flasks were cooled to 0° C. DIPEA (117 μl, 0.65 mmol, 4.0 eq) was added to flask (A), 0.12 ml (117 μl, 0.65 mmol, 4 eq) to flask (B). The ice bath was removed and both solutions were stirred for 20 min, then solution from flask (B) was transferred into flask (A) and reaction mixtures was stirred overnight. The orange mixture was transferred into a separation funnel and brine was added. The layers were separated and the aqueous layer was extracted 5 times with DCM. The organic layers were combined, dried over MgSO.sub.4, filtered and concentrated under reduced pressure. The remaining residue was purified with FC (DCM/MeOH, 100:0-95:5). The resulting intermediate product was obtained as colorless oil. The intermediate product was dissolved in DCM and trifluoroacetic acid (10 vol. %) was added. After 30 min the solvent was removed under reduced pressure, to obtain the deprotected VHL-ligand-linker intermediate as free TFA salt (h-i). The reaction is illustrated in the following reaction scheme:

##STR00010##

[0051] Intermediate h L-Prolinamide, N-[3-[2-(2-aminoethoxy)ethoxy]-1-oxopropyl] methyl-L-valyl-4-hydroxy-N-[[4-(4-methyl-5-thiazolyl)phenyl]methyl]-(4R) (VHL-PEG2-NH.sub.2)) was obtained with overall yield of 59% (75 mg, 0.13 mmol) over two steps (1st 61%, 2.sup.nd 97%).

[0052] Intermediate i N-[3-[2,2[2-aminoethoxy-(2-aminoethoxy)]ethoxy]-1-oxopropyl]-3-methyl-L-valyl-4-hydroxy-N-[[4-(4-methyl-5-thiazolyl)phenyl]methyl]-(4R) (VHL-PEG4-NH.sub.2) was obtained with overall yield of 52% (75 mg, 0.11 mmol) over two steps (1st 51%, 2.sup.nd 98%).

[0053] For coupling intermediate compounds c-g (thalidomide derivatives) and h-i (VHL-ligands), 0.02 mmol (1.0 eq) were dissolved in 1.5 ml DMF. HBTU (7.6 mg, 0.02 mmol, 1.0 eq) was added. After dissolving, alisertib (10 mg, 0.02 mmol, 1.0 eq) was added, followed by DIPEA (14 μl, 0.08 mmol, 4.0 eq). pH was adjusted to 8-10 by adding more equivalents DIPEA. Reaction mixtures were stirred overnight. The solvent was removed using a SpeedVac concentrator. The residues were purified and characterized via preparative and analytical HPLC. All reactions were done with 10 mg alisertib except JB170. This compound was synthesized first with 5 mg and then with 50 mg alisertib. The reaction is illustrated in the following reaction scheme:

##STR00011##

[0054] JB158 thalidomide-PEG3-alisertib was obtained as white solid with a yield of 90% (17 mg, 0.018 mmol).

[0055] JB159 thalidomide-(CH.sub.2).sub.3-alisertib was obtained as white solid with a yield of 70% (12 mg, 0.013 mmol).

[0056] JB160 VHL-PEG2-alisertib was obtained as white solid with a yield of 43% (9 mg, 0.008 mmol).

[0057] JB161 VHL-PEG4-alisertib was obtained as white solid with a yield of 62% (14 mg, 0.012 mmol).

[0058] JB169 thalidomide-(CH.sub.2).sub.5-alisertib was obtained as white solid with a yield of 61% (11 mg, 0.012 mmol).

[0059] JB170 thalidomide-PEG2-alisertib was obtained as white solid with a yield of 48% (9 mg, 0.009 mmol).

[0060] JB171 thalidomide-PEG1-alisertib was obtained as white solid with a yield of 75% (13 mg 0,014 mmol).

Formation of Productive Aurora-A-E3 Ligase Complexes

[0061] For testing which of the synthesized molecules mediate the formation of Aurora-A-E3 ligase complexes resulting in degradation of endogenous Aurora-A, cells of the leukemia cell line MV4-11 were treated with 0.1 μM thalidomide-based PROTACs JB158, JB160, JB161, JB169 or JB170 or 1 μM VHL-ligand-based PROTACs JB159 or JB171 at a single dose of the respective compound. Then, the cells were lysed in RIPA lysis buffer (50 mM HEPES pH 7.9, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.1% sodium deoxycholate) containing protease and phosphatase inhibitors (Sigma) and incubated for 20 minutes at 4° C. in head-over-tail. Lysates were cleared by centrifugation. Protein quantification was done using BCA assay and equal amounts of proteins were separated by BisTris-PAGE and transferred to PVDF membranes (Millipore). The membranes were blocked with 5% (w/v) nonfat dry milk dissolved in TBS-T (20 mM Tris/HCl, pH 7.5, 150 mM NaCl, and 0.1% (v/v) Tween 20) at RT for 1 hour and then incubated with the primary antibody overnight at 4° C. Visualization was done with HRP-labeled secondary antibodies and detected using Chemiluminescent HRP substrate (Millipore) in LAS3000 or LAS4000 Mini (Fuji). The signal was quantified using ImageJ (version 1.52q) or Image Studio Lite (LI-COR Biosciences, Version 5.2.5). The upper part of FIG. 2 shows immunoblots of Aurora-A. Vinculin was used as a loading control. The bar diagram at the lower part of FIG. 2 shows cellular Aurora-A levels of the cells upon degrader treatment compared to the Aurora A level of control cells. Error bars represent standard deviations of four biological replicate experiments.

[0062] While neither of the VHL-ligand-based degraders JB160 and JB161 reduced Aurora-A steady-state levels, thalidomide-based degrader JB158 resulted in a reduction of Aurora-A protein level by 62% and thalidomide-based degrader JB170 in a reduction by 69%. In these two degraders resulting in the strongest Aurora-A degradation the linkage between the structure of thalidomide and that of alisertib is provided by a linker having two (JB170) or three (JB158) ethylene glycol moieties. Both compounds led to a rapid decrease in Aurora-A levels that reached its maximum after about three hours of treatment with JB158 or JB170.

Degradation of Endogenous Aurora-A

[0063] Cells of cell lines MV4-11, U2OS and HLE were treated with indicated concentration of compounds JB170 or JB158 as well as with unconjugated alisertib (uA) for 6 hours. In further experiments cells of cell lines IMR5 and HLE were treated with 0.1 μM of compound JB170 for the indicated time periods. Afterwards, cells were lysed in RIPA lysis buffer (50 mM HEPES pH 7.9, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.1% sodium deoxycholate) containing protease and phosphatase inhibitors (Sigma) and incubated for 20 minutes at 4° C. in head-over-tail. Further processing of resulting cell lysates and immunoblotting was performed as described above. Results for cells of cell lines MV4-11, U2OS and HLE treated with JB170 (upper panel) and JB158 (lower panel) are shown in FIGS. 3 to 5. Results for cells treated with 0.1 μM of JB170 for the indicated time periods are shown for cells of cell lines IMR5 in FIG. 6 and for cells of cell lines HLE in FIG. 7.

[0064] According to FIG. 3, upper panel Aurora-A levels already significantly decreased in MV4-11 cells at concentrations of 10 nM JB170 and almost complete degradation was observed at 100 nM and 1 μM. In agreement with other degraders/PROTACs, a reversal of the Aurora-A depleting activity was observed at high concentrations of JB170. This phenomenon is called “Hook effect” and is expected for degrader molecules, which depend on ternary complex formation. 1 μM of non-conjugated alisertib did not decrease Aurora-A levels, but induced a significant increase, as frequently observed for type-1 kinase inhibitors that stabilize the active state of their targets. Similar results shown in FIG. 3, lower panel were obtained with JB158 and with U2OS cells (FIG. 4, upper panel with JB170 and lower panel with JB158) and HLE cells (FIG. 5, upper panel with JB170 and lower panel with JB158). The degree of depletion and the concentration, at which the Hook effect became visible varied significantly between cell lines, potentially due to different cellular concentrations of Cereblon and Aurora-A.

[0065] FIGS. 6 and 7 show that maximal degradation of endogenous Aurora-A occurred in IMR5 cells and HLE cells already after 3 to 6 hours.

Cell Death Caused by a PROTAC According to the Invention

[0066] To determine the effect of JB170-mediated depletion of Aurora-A on cancer cell survival, MV4-11 cells were treated with JB170 and the intracellular reduction of resazurin to resorufin was measured to assess cell viability via an alamarBlue assay. For alamarBlue assay, 6000 MV4-11 cells per well were seeded into 96 well plate and treated with 1 μM JB170 for various time points (refreshed every 24 h). The alamarBlue assay (Thermo Fisher Scientific) was performed according to manufacturer's instruction using HS Cell Viability Reagent. The fluorescence was measured in Tecan Infinite-200 using a fluorescence excitation wavelength of 550 and an emission of 600 nm. After 72 hours, viable cells were reduced to 32% by JB170 compared to control cells (FIG. 8).

[0067] MV4-11 cells and IMR5 cells and IMR5 cells expressing Aurora-AT.sup.217D upon incubation with doxycycline were treated with 0.5 μM JB170. For Annexin-PI FACS, the medium in which the cells were cultured was combined with the cells.

[0068] The cells were washed once with ice-cold PBS, resuspended in 100 μl Annexin V Binding buffer (10 mM HEPES pH 7.4, 140 mM NaCl, 2.5 mM CaCl.sub.2)) with 2 μl of Annexin V/Pacific Blue dye and incubated for 15 minutes in the dark at room temperature. 400 μl Annexin V Binding buffer with propidium iodide (18.5 μM) was added and the samples were stored cold and in the dark until analysis. FACS experiments were performed on a BD FACSCanto II flow cytometer and analysis was done using BD FACSDIVA Software and FlowJo (version 8.8.6). Results are shown in FIGS. 9 and 10.

[0069] FIG. 9 shows that JB170 increased the fraction of apoptotic cells in the culture over time, culminating in 56% of Annexin-positive MV4-11 cells after 72 hours.

[0070] Aurora-AT.sup.217D is a functional mutant of Aurora-A. IMR5 cells express Aurora-A.sup.T217D upon incubation with doxycycline (Dox) but not upon incubation with ethanol (control-EtOH). Affinity of Aurora-AT.sup.217D to alisertib analogue MLN8054 is reduced vis-à-vis affinity of Aurora-A to that analogue by about a factor of 8.

[0071] FIG. 10 shows that expression of Aurora-AT.sup.217D completely reverted the induction of apoptosis induced by JB170 in IMR5 cells indicating that JB170-induced apoptosis is exclusively caused by depletion of Aurora-A.

[0072] Experiments show that exposure of cancer cells to a PROTAC according to the invention resulted in strong induction of apoptosis and cytotoxicity in these cells.