BENZIMIDAZOLE THIOPHENE DERIVATIVE COMPOUNDS INDUCING SELECTIVE DEGRADATION OF PLK1

Abstract

The present invention provides novel compounds that induce selective polo-like kinase 1 (PLK1) degradation. Specifically, the present invention provides a bifunctional compound in which a PLK1 binding moiety and an E3 ubiquitin ligase-binding moiety are linked by a chemical linker. The present invention provides the compound, a method for preparing the same, and the use thereof. The compounds may be effectively utilized for preventing or treating PLK1 related diseases.

Claims

1. A compound represented by the following Formula I, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:
ULM—Linker—PTM  [Formula I] wherein in the Formula I above, ULM is E3 ubiquitin ligase binding moiety represented by the following Formula A or B: ##STR00022## wherein X.sub.1 is —CH.sub.2— or —CO—; ##STR00023## wherein ##STR00024## is a 5-membered heteroaryl; and Y.sub.1 is hydrogen or C.sub.1-3 alkyl; PTM is PLK1 binding moiety represented by the following Formula II: ##STR00025## and Linker is a chemical group that links ULM and PTM.

2. The compound of claim 1, wherein ULM is represented by Formula A and Formula II is represented by the following formula: ##STR00026##

3. The compound of claim 1, wherein ULM is represented by Formula B and Formula II is represented by the following formula: ##STR00027##

4. The compound of claim 1, wherein the compound is compound 1 or compound 2.

5. The compound of claim 1, wherein the compound is a bifunctional compound that induces PLK1 protein degradation.

6. A composition for prevention or treatment of a PLK1 related disease, the composition comprising the compound according to claim 1, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

7. The composition of claim 6, wherein the PLK1 related disease is a cancer, a benign tumor, or a neurological disease.

Description

BEST MODE FOR CARRYING OUT THE INVENTION

[0064] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The terminology used in the description is for describing particular embodiments only and is not intended to be limiting of the disclosure.

[0065] The present invention provides synthetic methods for compounds 1 and 2 shown in the table below.

TABLE-US-00001 TABLE 1 Compound Structure 1 [00013] 2 [00014]

[0066] The compounds of the present invention were purified according to the following method and the structure was analyzed.

[0067] Instruments

[0068] LCMS: Shimadzu LCMS-2020

[0069] NMR: BRUKER AVANCE III 400 MHz

[0070] HPLC: Shimadzu LC-20AB, Shimadzu LC-20AD, Agilent 1100 LC, Agilent 1200 LC, Agilent 1290 LC

[0071] LCSM Analysis

[0072] LCMS data were recorded with Shimadzu LCMS-2020 equipped with an electron spray ionization device. 0.0375% TFA in water (solvent A) and 0.01875% TFA in acetonitrile (solvent B) were used as mobile phases. As a column, Kinetex EVO C18 (2.1*30)mm, 5 um was used.

[0073] HPLC Analysis

[0074] In HPLC analysis, Shimadzu LC-20AB, Shimadzu LC-20AD, Agilent 1100 LC, Agilent 1200 LC or Agilent 1290 LC was used. 0.0375% TFA in water (solvent A) and 0.01875% TFA in acetonitrile (solvent B) or 0.025% NH.sub.3.Math.H2O in water (solvent A) and acetonitrile (Solvent B) was used as the mobile phase. As a column, XBridge C18 (2.1*50)mm, Sum or Kinetex C18 LC column (4.6*50)mm, Sum or Eclipse plus C18 (4.6*150)mm, 3.5 um or Waters XBridge® C18 (4.6*150)mm, 3.5 μm was used.

[0075] NMR Analysis

[0076] .sup.1H NMR spectrum was recorded with Bruker AVANCE III 400 MHz/5 mm Probe (BBO).

Example 1. Synthesis of 3-(1-(4-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethox y)ethoxy)ethoxy)-2-(trifluoromethyl)phenyl)ethoxy)-5-(6-((4-methylpiperazin-1-yl)methyl)-1H-benzo[d]imidazol-1-yl)thiophene-2-carboxamide (Coumpound 1)

[0077] ##STR00015## ##STR00016## ##STR00017## ##STR00018##

[0078] Step 1: Synthesis of methyl 4-hydroxy-2-(trifluoromethyl)benzoate (2)

[0079] To a solution of 4-hydroxy-2-(trifluoromethyl)benzoic acid (7.5 g, 36.39 mmol) in toluene (75 mL) and MeOH (37 mL) was added TMSCHN.sub.2 (2 M, 36.4 mL) and the mixture was stirred at 25° C. for 2 h. TLC (Petroleum ether:Ethyl acetate=2:1) showed the starting material was consumed and new spot was detected. The mixture was concentrated under reduced pressure to afford methyl 4-hydroxy-2-(trifluoromethyl)benzoate (8 g, crude) was obtained as yellow solid.

[0080] Step 2: Synthesis of 3-oxo-1-phenyl-2,7,10-trioxa-4-azadodecan-12-yl 4-methylbenzenesulfonate (3)

[0081] To a solution benzyl (2-(2-(2-hydroxyethoxy)ethoxy)ethyl)carbamate (6.9 g, 24.35 mmol), TEA (7.39 g, 73.06 mmol, 10.17 mL) in DCM (50 mL) was added TosCl (9.29 g, 48.71 mmol) at 25° C. The solution was stirred at 25° C. for 12 h. LCMS showed a main peak with desired mass. The mixture was concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (45 g SepaFlash® Silica Flash Column, Eluent of 0˜65% Ethylacetate/Petroleum ether gradient @ 75 mL/min) to afford 3-oxo-1-phenyl-2,7,10-trioxa-4-azadodecan-12-yl 4-methylbenzenesulfonate (9.1 g, 20.59 mmol, 84.55% yield, 99% purity) as colorless oil. MS(M+H)+=438.0.

[0082] Step 3: Synthesis of methyl 4-((3-oxo-1-phenyl-2,7,10-trioxa-4-azadodecan-12-yl)oxy)-2-(trifluoromethyl)benzoate (4)

[0083] To a solution of 3-oxo-1-phenyl-2,7,10-trioxa-4-azadodecan-12-yl 4-methylbenzenesulfonate (8.9 g, 20.34 mmol) and methyl 4-hydroxy-2-(trifluoromethyl)benzoate (4.93 g, 22.38 mmol) in ACN (180 mL) was added K .sub.2CO.sub.3 (5.62 g, 40.69 mmol) and KI (168.84 mg, 1.02 mmol) and the mixture was stirred at 80° C. for 14 h. LCMS showed the desired mass was detected. The mixture was filtered and the filter cake was washed with EtOAc (100 mL). The filtrate was concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (20 g SepaFlash® Silica Flash Column, Eluent of 0˜30% Ethyl acetate/Petroleum ether gradient @ 60 mL/min) to afford methyl 4-((3-oxo-1-phenyl-2,7,10-trioxa-4-azadodecan-12-yl)oxy)-2-(trifluoromethyl)benzoate (7.4 g, 14.94 mmol, 73.44% yield, 98% purity) as yellow oil. MS(M+H)+=486.2.

[0084] Step 4: Synthesis of benzyl (2-(2-(2-(4-acetyl-3-(trifluoromethyl)phenoxy)ethoxy)ethoxy) ethyl)carbamate (5)

[0085] To a solution of MeMgBr (3 M, 35.50 mL) in toluene (49 mL) was added TEA (7.74 g, 76.52 mmol, 10.65 mL) at 0° C. and a solution of methyl 4-((3-oxo-1-phenyl-2, 7,10-trioxa-4-azadodecan-12-yl)oxy)-2-(trifluoromethyl)benzoate (7.1 g, 14.63 mmol,) in toluene (49 mL) was added and the resulting mixture was stirred at 25° C. for 14 h. LCMS showed the desired mass was detected. The mixture was quenched with NH .sub.4C1 (10 mL) at 0° C. and extracted with EtOAc (10 mL×3). The combined organic layers was washed with H.sub.20 (10 mL) and dried over Na .sub.2SO 4, filtered. The filtrate was concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (40 g SepaFlash® Silica Flash Column, Eluent of 10˜60% Ethyl acetate/Petroleum ether gradient @ 100 mL/min) to afford benzyl (2-(2-(2-(4-acetyl-3-(trifluoromethyl)phenoxy)ethoxy)ethoxy)ethyl)carbamate (5.4 g, 10.93 mmol, 74.72% yield, 95% purity) as yellow oil. MS(M+H)+=470.2

[0086] Step 5: Synthesis of benzyl (2-(2-(2-(4-(1-hydroxyethyl)-3-(trifluoromethyl)phenoxy)ethoxy) ethoxyl)ethyl)carbamate (6)

[0087] To a solution of benzyl (2-(2-(2-(4-acetyl-3-(trifluoromethyl)phenoxy)ethoxy)ethoxy)ethyl)carbamate (1.8 g, 3.83 mmol) in MeOH (40 mL) was added NaBH 4 (450 mg, 11.90 mmol) slowly and the mixture was stirred at 25° C. for 1 h. LCMS showed the desired mass was detected. The mixture was quenched with NH .sub.4C1 (30 mL) and concentrated under reduced pressure. The mixture was extracted with EtOAc (20 mL×3). The combined organic layers was washed with NaHCO3 (10 mL×2) and H.sub.20 (10 mL×2), dried over Na 2 SO 4, filtered. The filtrate was concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (12 g SepaFlash® Silica Flash Column, Eluent of 20˜80% Ethyl acetate/Petroleum ether gradient @ 50 mL/min) to afford benzyl (2-(2-(2-(4-(1-hydroxyethyl)-3-(trifluoromethyl)phenoxy)ethoxy)ethoxy)ethyl)carbam ate (1.77 g, 3.75 mmol, 97.91% yield, 100% purity) as yellow oil. MS(M+Na)+=494.2

[0088] Step 6: Synthesis of methyl 5-nitro-3-(1-(4-((3-oxo-1-phenyl-2,7,10-trioxa-4-azadodecan-12-yl) oxy)-2-(trifluoromethyl)phenyl)ethoxy)thiophene-2-carboxylate (8)

[0089] To a solution of benzyl (2-(2-(2-(4-(1-hydroxyethyl)-3-(trifluoromethyl)phenoxy)ethoxy)ethoxy) ethyl)carbamate (1.77 g, 3.75 mmol), methyl 3-hydroxy-5-nitrothiophene-2-carboxylate (915 mg, 4.50 mmol) and PPh 3 (1.48 g, 5.63 mmol) in DCM (40 mL) was added DTBAD (1.30 g, 5.63 mmol) at 25° C. The mixture was stirred at 25° C. for 14 h. The mixture was concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (20 g SepaFlash® Silica Flash Column, Eluent of 10-100% Ethyl acetate/Petroleum ether gradient @ 50 mL/min). The crude product was triturated with MTBE (10 mL) and filtered. The filter cake was collected and dried under reduced pressure to afford methyl 5-nitro-3-(1-(4-((3-oxo-1-phenyl-2,7,10-trioxa-4-azadodecan-12-yl) oxy)-2-(trifluoromethyl) phenyl)ethoxy)thiophene-2-carboxylate (2.06 g, 3.14 mmol, 83.57% yield) as yellow oil.

[0090] Step 7: Synthesis of methyl 5-amino-3-(1-(4-((3-oxo-1-phenyl-2,7,10-trioxa-4-azadodecan-12-yl) oxy)-2-(trifluoromethyl)phenyl)ethoxy)thiophene-2-carboxylate (9)

[0091] To a solution of methyl 5-nitro-3-(1-(4-((3-oxo-1-phenyl-2,7,10-trioxa-4-azadodecan-12-yl)oxy)-2-(trifluoromethyl)phenyl)ethoxy)thiophene-2-carboxylate (2.06 g, 3.14 mmol) in EtOH (20 mL) and H.sub.20 (20 mL) was added NH .sub.4C1 (1.01 g, 18.82 mmol) and Fe (1.05 g, 18.82 mmol) and the resulting mixture was stirred at 80° C. for 1 h. LCMS showed the desired mass was detected. The mixture was filtered at 80° C. and the filter cake was washed with MeOH (50 mL). The filtrate was concentrated under reduced pressure to remove MeOH and extracted with EtOAc (20 mL×3). The combined organic layer was washed with H.sub.20 (10 mL×2), dried over Na .sub.2SO 4, filtered. The filtrate was concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (20 g SepaFlash® Silica Flash Column, Eluent of 20˜60% Ethyl acetate/Petroleum ether gradient @ 50 mL/min) to afford methyl 5-amino-3-(1-(4-((3-oxo-1-phenyl-2,7,10-trioxa-4-azadodecan-12-yl)oxy)-2-(trifluoro methyl)phenyl)ethoxy)thiophene-2-carboxylate (0.9 g, 1.44 mmol, 45.78% yield) as yellow oil. MS(M+H)+=627.4

[0092] Step 8: Synthesis of 1-(3-bromo-4-nitrobenzyl)-4-methylpiperazine (10)

[0093] To a solution of 1-methylpiperazine (866.88 mg, 8.65 mmol, 960 μL) and 3-bromo-4-nitrobenzaldehyde (1 g, 4.35 mmol) in DCE (30 mL) was added AcOH (10.50 mg, 174.85 μmol, 10 μL) at 0° C. and the mixture was stirred at 0° C. for 30 min, then NaBH(OAc) 3 (2.77 g, 13.05 mmol) was added and the resulting mixture was stirred at 25° C. for 14 h. LCMS showed the desired mass was detected. The mixture was diluted with H.sub.20 (20 mL) and citric acid solution (50 mL), then washed with EtOAc (10 mL×2), the organic layer was discarded. The aqueous phase was adjusted the pH=9 and extracted with EtOAc (10 mL×2). The combined organic layers was dried over Na .sub.2SO 4, filtered. The filtrate was concentrated under reduced pressure to afford 1-(3-bromo-4-nitrobenzyl)-4-methylpiperazine (1.2 g, 3.74 mmol, 86.10% yield, 98% purity) as yellow oil. MS(M+H)+=314.1

[0094] Step 9: Synthesis of methyl 5-((5-((4-methylpiperazin-1-yl)methyl)-2-nitrophenyl)amino)-3-(1-(4-((3-oxo-1-phenyl-2.7.10-trioxa-4-azadodecan-12-yl)oxy)-2-(trifluoromethyl)phenyl)ethoxy)thiophene-2-carboxylate (11)

[0095] To a solution of 1-(3-bromo-4-nitrobenzyl)-4-methylpiperazine (355.14 mg, 1.13 mmol) and methyl 5-amino-3-(1-(4-((3-oxo-1-phenyl-2,7,10-trioxa-4-azadodecan-12-yl)oxy)-2-(trifluoro-methyl)phenyl)ethoxy)thiophene-2-carboxylate (850.00 mg, 1.36 mmol) in toluene (15 mL) were added Cs .sub.2CO.sub.3 (1.10 g, 3.39 mmol), Pd.sub.2(dba) 3 (20.70 mg, 22.61 μmol) and Xantphos (28.78 mg, 49.74 μmol) and the resulting mixture was stirred at 80° C. for 14 h. TLC (Ethyl acetate: Methanol=5:1) showed the desired spot was detected. The mixture was filtered and the filter cake was washed with EtOAc (50 mL). The filtrate was concentrated under reduced pressure. Then the crude product was diluted with H .sub.2O (10 mL) and extracted with EtOAc (10 mL×2). The combined organic was washed with H.sub.20 (10 mL×2), dried over Na .sub.2SO 4, filtered. The filtrate was concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (5 g SepaFlash® Silica Flash Column, Eluent of 50-100% Ethyl acetate/Petroleum ether to 0˜20% Methanol/Ethyl acetate gradient @ 50 mL/min) to afford methyl 5-((5-((4-methylpiperazin-1-yl)methyl)-2-nitrophenyl)amino)-3-(1-(4-((3-oxo-1-phenyl-2,7,10-trioxa-4-azadodecan-12-yl)oxy)-2-(trifluoromethyl)ph enyl)ethoxy)thiophene-2-carboxylate (510 mg, 593.09 μmol, 52.47% yield) as yellow oil. MS(M+H)+=860.4

[0096] Step 10: Synthesis of methyl 5-(6-((4-methylpiperazin-1-yl)methyl)-1H-benzo[d]imidazol-1-yl)-3-(1-(4-((3-oxo-1-phenyl-2,7,10-trioxa-4-azadodecan-12-yl)oxy)-2-(trifluoromethyl)ph enyl)ethoxy)thiophene-2-carboxylate (12)

[0097] To a solution of methyl 5-((5-((4-methylpiperazin-1-yl)methyl)-2-nitrophenyl)amino)-3-(1-(4-((3-oxo-1-phenyl-2,7,10-trioxa-4-azadodecan-12-yl)oxy)-2-(trifluoromethyl)p henyl)ethoxy)thiophene-2-carboxylate (410 mg, 476.80 μmol) in MeOH (8 mL) was added trimethoxymethane (1.27 g, 11.97 mmol, 1.31 mL), formic acid (200.09 mg, 4.35 mmol, 164.00 μL) and Zn (156 mg, 2.39 mmol). The mixture was stirred at 70° C. for 6 h. LCMS showed the desired mass was detected. The mixture was filtered and the filter cake was washed with hot MeOH (50 mL, 50° C.) and the filtrate was concentrated under reduced pressure. The crude was diluted with H.sub.20 (10 mL) and adjusted the pH=8 with NaHCO 3 solution. Then the mixture was extracted with EtOAc (10 mL×3). The combined organic layers was washed with H.sub.20 (10 mL×2), dried over Na .sub.2SO 4, filtered. The filtrate was concentrated under reduced pressure to afford methyl 5-(6-((4-methylpiperazin-1-yl)methyl)-1H-benzo[d]imidazol-1-yl)-3-(1-(4-((3-oxo-1-phenyl-2,7,10-trioxa-4-azadodecan-12-yl)oxy)-2-(trifluoromethyl)phenyl)ethoxy)thiop hene-2-carboxylate (330 mg, crude) as yellow oil. MS(M+H)+=840.5

[0098] Step 11: Synthesis of 5-(6-((4-methylpiperazin-1-yl)methyl)-1H-benzo[d]imidazol-1-yl)-3-(1-(4-((3-oxo-1-p henyl-2,7,10-trioxa-4-azadodecan-12-yl)oxy)-2-(trifluoromethyl)phenyl)ethoxy)thioph ene-2-carboxylic acid (13)

[0099] To a solution of methyl 5-(6-((4-methylpiperazin-1-yl)methyl)-1H-benzo[d]imidazol-1-yl)-3-(1-(4-((3-oxo-1-phenyl-2,7,10-trioxa-4-azadodecan-12-yl)oxy)-2-(trifluoromethyl)ph enyl)ethoxy)thiophene-2-carboxylate (250.00 mg, 297.65 μmol) in THF (3 mL) was added a solution of LiOH.sub.20 (14.39 mg, 342.89 μmol) in H.sub.20 (1.5 mL) and the mixture was stirred at 40° C. for 14 h. Additional LiOH .sub.20 (17.98 mg, 428.61 μmol) was added and the mixture was stirred at 40° C. for another 14 h. LCMS showed the starting material was consumed and the desired mass was detected. The mixture was concentrated under reduced pressure to afford 5-(6-((4-methylpiperazin-1-yl)methyl)-1H-benzo[d]imidazol-1-yl)-3-(1-(4-((3-oxo-1-p henyl-2,7,10-trioxa-4-azadodecan-12-yl)oxy)-2-(trifluoromethyl)phenyl)ethoxy)thioph ene-2-carboxylic acid (250 mg, crude) as yellow oil. MS(M+H)+=826.6

[0100] Step 12: Synthesis of benzyl (2-(2-(2-(4-(1-((2-carbamoyl-5-(6-((4-methvlninerazin-1-yl)methyl)-1H-benzo[d]imidazol-1-yl)thiophen-3-yl)oxy)ethyl)-3-(trifluoromethyl)phenoxy)ethox y)ethoxy)ethyl)carbamate (14)

[0101] To a solution of 5-(6-((4-methylpiperazin-1-yl)methyl)-1H-benzo[d]imidazol-1-yl)-3-(1-(4-((3-oxo-1-p henyl-2,7,10-trioxa-4-azadodecan-12-yl)oxy)-2-(trifluoromethyl)phenyl)ethoxy)thioph ene-2-carboxylic acid (250 mg, 300.54 μmol) and NH .sub.4C1 (80.38 mg, 1.50 mmol) in DMF (4 mL) were added DIPEA (118.72 mg, 918.58 μmol, 160 μL) and HATU (171.41 mg, 450.82 μmol) and the resulting mixture was stirred at 25° C. for 14 h. LCMS showed the starting material remained. Additional NH .sub.4C1 (80.38 mg, 1.50 mmol) and HATU (171.41 mg, 450.82 μmol) were added and the resulting mixture was stirred at 25° C. for 1 h. LCMS showed the desired mass was detected. The reaction mixture was diluted with H.sub.20 (10 mL) and extracted with EtOAc (10 mL×3). The combined organic layer was washed with brine (10 mL×2), dried over Na .sub.2SO 4, filtered. The filtrate was concentrated under reduced pressure to afford benzyl (2-(2-(2-(4-(1-((2-carbamoyl-5-(6-((4-methylpiperazin-1-yl)methyl)-1H-benzo[d]imida zol-1-yl)thiophen-3-yl)oxy)ethyl)-3-(trifluoromethyl)phenoxy)ethoxy)ethoxy)ethyl)car bamate (170 mg, crude) as yellow oil. MS(M+H)+=825.6

[0102] Step 13: Synthesis of 3-(1-(4-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)-2-(trifluoromethyl)phenvl) ethoxy)-5-(6-((4-methylpiperazin-1-yl)methyl)-1H-benzo[d]imidazol-1-yl)thiophene-2-carboxamide (15)

[0103] To a solution of benzyl (2-(2-(2-(4-(1-((2-carbamoyl-5-(6-((4-methylpiperazin-1-yl)methyl)-1H-benzo[d]imidazol-1-yl)thiophen-3-yl)oxy)ethyl)-3-(trifluoromethyl)phenoxy)ethox y)ethoxy)ethyl)carbamate (150 mg, 181.84 μmol) in ACN (0.75 mL) was added TMSI (220.50 mg, 1.10 mmol, 150 μL) and the mixture was stirred at 20° C. for 1 h. LCMS showed the starting material was consumed and the desired mass was detected. The mixture was quenched with H.sub.20 (20 mL, 0° C.) and washed with EtOAc (10 mL×3), the organic layer was discarded. The aqueous phase was adjusted the pH=7-8 with Na .sub.2CO.sub.3 solution, then the mixture was lyophilized. The crude product was purified by prep-HPLC (column: Unisil 3˜100 C.sub.18 μLtra 150*50 mm*3 um; mobile phase: [water (0.225% FA)-ACN]; B %: 5%-35%, 10 min) to afford 3-(1-(4-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)-2-(trifluoromethyl)phenyl)ethoxy)-5-(6-((4-methylpiperazin-1-yl)methyl)-1H-benzo[d]imidazol-1-yl)thiophene-2-carboxamid e (60 mg, 70.22 umol, 38.62% yield, 97% purity, FA salt) as yellow solid. MS(M+H)+=691.4

[0104] Step 14: Synthesis of 3-(1-(4-(2-(2-(2-((2-(2.6 dioxopiperidin-3-yl)-1.3 dioxoisoindolin-4-yl) amino)ethoxy)ethoxy)ethoxy)-2-(trifluoromethyl)phenyl)ethoxy)-5-(6-((4-methylpiper azin-1-yl)methyl)-1H-benzo[d]imidazol-1-yl)thiophene-2-carboxamide(Compound 1)

[0105] To a solution of 3-(1-(4-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)-2-(trifluoromethyl)phenyl)ethoxy)-5-(6-((4-methylpiperazin-1-yl)methyl)-1H-benzo[d]imidazol-1-yl)thiophene-2-carboxa mide (60 mg, 72.39 μmol, FA salt) and 2-(2,6-dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione (24 mg, 86.89 μmol) in DMSO (1.2 mL) was added TEA (43.95 mg, 434.34 μmol, 60.45 μL) and the mixture was stirred at 80° C. for 14 h. LCMS showed the desired mass was detected. The mixture was filtered and the filtrate was purified by prep-HPLC (column: Phenomenex Gemini-NX C.sub.18 75*30 mm*3 um;mobile phase: [water (10 mM NH .sub.4HCO 3)-ACN]; B %: 30%-60%, 8 min) and the eluent was lyophilized to afford 3-(1-(4-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1, 3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy) ethoxy)-2-(trifluoromethyl)phenyl)ethoxy)-5-(6-((4-methylpiperazin-1-yl)methyl)-1H-benzo[d]imidazol-1-yl)thiophene-2-carboxamide (10.2 mg, 9.80 μmol, 13.54% yield, 91% purity) as yellow solid. MS(M+H)+=947.7

[0106] .sup.1H NMR (400 MHz, CD .sub.3CN) S=8.06 (d, J=2.5 Hz, 1H), 7.69 (d, J=8.7 Hz, 1H), 7.64 (d, J=8.3 Hz, 1H), 7.51˜7.44 (m, 1H), 7.33 (d, J=5.6 Hz, 1H), 7.30-7.27 (m, 1H), 7.24-7.17 (m, 2H), 7.11 (br s, 1H), 7.0-6.94 (m, 2H), 6.85 (d, J=1.0 Hz, 1H), 6.48-6.43 (m, 1H), 6.29 (br s, 1H), 5.88-5.82 (m, 1H), 4.91-4.86 (m, 1H), 4.14-4.09 (m, 2H), 3.78-3.74 (m, 2H), 3.66-3.57 (m, 6H), 3.56-3.52 (m, 2H), 3.42-3.37 (m, 2H), 2.74-2.61 (m, 4H), 2.45-2.30 (m, 8H), 2.06-2.00 (m, 2H), 1.78-1.71 (m, 4H).

Example 2. Synthesis of

[0107] (2S,4R)-1-((S)-2-(tert-butyl)-14-(4-((1-(5-carbamoyl-4-((R)-1-(2-(trifluoromethyl)p henyl)ethoxy)thiophen-2-yl)-1H-benzo[d]imidazol-6-yl)methyl)piperazin-1-yl)-4-o xo-6,9,12-trioxa-3-azatetradecanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (Compound 2)

##STR00019## ##STR00020## ##STR00021##

[0108] Step 1: Synthesis of (R)-methyl 5-nitro- 3-(1-(2-(trifluoromethyl)1phenyl)ethoxy)thiophene- 2-carboxylate (3)

[0109] To a solution of methyl 3-hydroxy-5-nitrothiophene-2-carboxylate (1.23 g, 6.05 mmol), (S)-1-(2-(trifluoromethyl)phenyl)ethanol (1 g, 5.26 mmol, 80.00 μL) in DCM (20 mL) was added PPh 3 (2.07 g, 7.89 mmol), DTBAD (1.82 g, 7.89 mmol) at 25° C. The mixture was stirred at 25° C. for 12 hr. TLC (petroleum ether: ethyl acetate=3:1; Rf=0.75) showed the (S)-1-(2-(trifluoromethyl)phenyl)ethanol was consumed completely and new spots were formed. The mixture was concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (40 g SepaFlash@ Silica Flash Column, Eluent of 0˜5% Ethyl acetate/Petroleum ether gradient @ 30 mLmi) to afford (R)-methyl 5-nitro-3-(1-(2-(trifluoromethyl)phenyl)ethoxy)thiophene-2-carboxylate (1.8 g, 4.51 mmol, 85.73% yield, 94% purity) as yellow oil.

[0110] Step 2: Synthesis of (R)-methyl 5-amino-3-(1-(2-(trifluoromethyl)phenyl)ethoxy)thiophene- 2-carboxylate (4)

[0111] To a solution of (R)-methyl 5-nitro-3-(1-(2-(trifluoromethyl)phenyl)ethoxy)thiophene-2-carboxylate (1.8 g, 4.80 mmol) in EtOH (15 mL) and H.sub.20 (15 mL) were added Fe (1.61 g, 28.78 mmol) and NH .sub.4C1 (1.54 g, 28.78 mmol) at 25° C., the mixture was stirred at 80° C. for 1 hr. TLC (petroleum ether: ethyl acetate=3:1; Rf=0.4) showed the starting material was consumed completely and new spot was formed. The mixture was filtered through a celite pad and the filtrate was concentrated to remove the organic solvent. The solution was diluted with water (50 mL) and extracted with ethyl acetate (20 mL×3). The combined organic phase was washed with brine (20 mL×3), dried over Na .sub.2SO 4, filtered. The filtrate was concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (12 g SepaFlash® Silica Flash Column, Eluent of 0˜30% Ethyl acetate/Petroleum ether gradient @ 75 mL/min) to afford (R)-methyl 5-amino-3-(1-(2-(trifluoromethyl)phenyl)ethoxy)thiophene-2-carboxylate (1.4 g, 3.81 mmol, 79.46% yield, 94% purity) was obtained as brown oil which was used for the next step directly. MS(M+H)+=345.8

[0112] Step 3: Synthesis of (R)-tert-butyl 4-(3-((5-(methoxycarbonll)-4-(1-(2-(trifluoromethyl)phenyl)ethoxy)thiophen-2-yl)amino)-4-nitrobenzyl)piperazine-1-carboxylate (6)

[0113] To a solution of (R)-methyl 5-amino-3-(1-(2-(trifluoromethyl)phenyl)ethoxy)thiophene-2-carboxylate (530 mg, 1.53 mmol), tert-butyl 4-(3-bromo-4-nitrobenzyl)piperazine-1-carboxylate (675.73 mg, 1.69 mmol) and K .sub.2CO.sub.3 (636.33 mg, 4.60 mmol) in t-BuOH (10 mL) were added and Pd.sub.2(dba) 3 (28.11 mg, 30.69 μmol) and XPhos (32.19 mg, 67.53 μmol) under N .sub.2 at 25° C. and the resulting mixture was stirred at 60° C. for 12 h. LCMS showed 37% peak of tert-butyl 4-(3-bromo-4-nitrobenzyl)piperazine-1-carboxylate. Additional Pd.sub.2(dba) 3 (28.11 mg, 30.69 μmol) and XPhos (32.19 mg, 67.53 μmol) were added under N .sub.2 at 25° C. The reaction mixture was stirred at 70° C. for another 12 hrs. The mixture was diluted with ethyl acetate (50 mL) filtered through a celite pad and the filtrate was concentrated. The residue was purified by flash silica gel chromatography (10 g SepaFlash® Silica Flash Column, Eluent of 5˜50% Ethyl acetate/Petroleum ether gradient @ 60 mL/min) followed by prep-HPLC (column: Waters Xbridge C.sub.18 150*50 mm* 10 um; mobile phase: [water (10 mM NH .sub.4HCO 3)-ACN]; B %: 65%-95%, min) and the eluent was lyophilized to afford (R)-tert-butyl 4-(3-((5-(methoxycarbonyl)-4-(1-(2-(trifluoromethyl)phenyl)ethoxy)thiophen-2-yl)ami no)-4-nitrobenzyl)piperazine-1-carboxylate (400 mg, 601.78 μmol, 39.21% yield) as brown solid. The crude product was used for the next step directly. MS(M+H)+=665.1 .sup.1H NMR (400 MHz, CDCl.sub.3)δ=9.71 (s, 1H), 8.16 (br d, J=8.6 Hz, 1H), 7.93 (d, J=7.9 Hz, 1H), 7.68-7.56 (m, 2H), 7.41 (s, 1H), 7.20-7.16 (m, 1H), 6.93 (br d, J=8.4 Hz, 1H), 6.43 (s, 1H), 5.74 (q, J=5.9 Hz, 1H), 3.88 (s, 3H), 3.44 (br s, 6H), 2.38 (br s, 2H), 2.36 (s, 2H), 1.73 (d, J=6.3 Hz, 3H), 1.47 (s, 9H).

[0114] Step 4: Synthesis of (R)-tert-butyl 4-((1-(5-(methoxycarbonyll)-4-(1-(2-(trifluoromethyl)phenyl)ethoxylthiophen-2-yl)-1H-benzo[d]imidazol-6-yl)methyl)piperazine-1-carboxylate (7)

[0115] To a solution of (R)-tert-butyl 4-(3-((5-(methoxycarbonyl)-4-(1-(2-(trifluoromethyl)phenyl)ethoxy)thiophen-2-yl)ami no)-4-nitrobenzyl)piperazine-1-carboxylate (0.4 g, 601.78 μmol) in MeOH (20 mL) were added trimethoxymethane (1.60 g, 15.04 mmol, 1.65 mL), formic acid (249.28 mg, 5.42 mmol, 204.32 μL) and Zn (196.75 mg, 3.01 mmol). The reaction mixture was stirred at 70° C. for 12 h. LCMS showed the starting material was consumed completely and one main peak with desired mass. The mixture was filtered and the filter cake was washed with MeOH (50 mL, 50° C.). The filtrate was concentrated under reduced pressure. The residue was diluted with H.sub.20 (15 mL) and adjusted the pH=8 with NaHCO 3 solution. Then the mixture was extracted with EtOAc (20 mL×3). The combined organic layer was washed with H.sub.20 (10 mL×2), dried over Na .sub.2SO 4, filtered. The filtrate was concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (12 g SepaFlash® Silica Flash Column, Eluent of 0˜50% Ethyl acetate/Petroleum ether gradient @ 50 mL/min) to afford (R)-tert-butyl 4-((1-(5-(methoxycarbonyl)-4-(1-(2-(trifluoromethyl)phenyl)ethoxy)thiophen-2-yl)-1H-benzo[d]imidazol-6-yl)methyl)piperazine-1-carboxylate (300 mg, 456.02 μmol, 75.78% yield, 98% purity) as yellow oil, which was used for the next step directly. MS(M+H)+=645.2

[0116] Step 5: Synthesis of (R)-5-(6-((4-(tert-butoxycarbonyl)piperazin-1-yl)methyll)-1H-benzo[d]imidazol-1-yl)-.sup.3-(1-(2-(trifluoromethyl)phenyl)ethoxylthiophene-2-carboxyli c acid (8)

[0117] To a solution of (R)-tert-butyl 4-((1-(5-(methoxycarbonyl)-4-(1-(2-(trifluoromethyl)phenyl)ethoxy)thiophen-2-yl)-1H-benzo[d]imidazol-6-yl)methyl)piperazine-1-carboxylate (300 mg, 465.33 μmol) in THF (5 mL) was added a solution of LiGH (33.43 mg, 1.40 mmol) in H.sub.20 (5 mL) drop-wise at 25° C. The mixture was stirred at 40° C. for 12 hr. LCMS showed the starting material was consumed completely and one main peak with desired mass. The reaction mixture was concentrated under reduced pressure to afford (R)-5-(6-((4-(tert-butoxycarbonyl)piperazin-1-yl)methyl)-1H-benzo[d]imidazole-1-yl)-3-(1-(2-(trifluoromethyl)phenyl)ethoxy)thiophene-2-carboxylic acid (296 mg, crude) as a brown solid, which was used for the next step directly. MS (M+H)+=631.0

[0118] Step 6: Synthesis of (R)-tert-butyl 4-((1-(5-carbamoyl-4-(1-(2-(trifluoromethyl)phenyl)ethoxy)thiophen-2-yl)-1H-benzo[d]imidazol-6-yl)methyl)piperazine-1-carboxylate (9)

[0119] To solution of (R)-5-(6-((4-(tert-butoxycarbonyl)piperazin-1-yl)methyl)-1H-benzo[d]imidazole-1-yl)-3-(1-(2-(trifluoromethyl)phenyl)ethoxy)thiophene-2-carboxylic acid (296 mg, 464.96 μmol), NH .sub.4C1 (124.36 mg, 2.32 mmol) in DMF (3 mL) were added HATU (353.59 mg, 929.93 μmol) and DIPEA (180.28 mg, 1.39 mmol, 242.96 μL) at 30° C. The mixture was stirred at 30° C. for 2 hr. LCMS showed the starting material was consumed completely and one main peak with desired mass. The mixture solution was concentrated under reduced pressure. The residue was purified by flash silica gel chromatography (4 g SepaFlash® Silica Flash Column, Eluent of 0˜100% Ethyl acetate/Petroleum ether gradient @ 60 mL/min; Eluent of 0˜50% Methanol/Ethyl acetate @60 mL/min) to afford (R)-tert-butyl 4-((1-(5-carbamoyl-4-(1-(2-(trifluoromethyl)phenyl)ethoxy)thiophen-2-yl)-1H-benzo[d]imidazol-6-yl)methyl)piperazine-1-carboxylate (400 mg, crude) as brown solid, which was used for the next step directly. MS (M+H)+=630.0

[0120] Step 7: Synthesis of (R)-5-(6-(piperazin-1-ylmethyl)-1H-benzo[d]imidazol-1-yl)-3-(1-(2-(trifluoromethyl)phenyl)ethoxy)thiophene-2-carboxamide (10)

[0121] To a solution of (R)-tert-butyl 4-((1-(5-carbamoyl-4-(1-(2-(trifluoromethyl)phenyl)ethoxy)thiophen-2-yl)-1H-benzo[d]imidazol-6-yl)methyl)piperazine-1-carboxylate (400 mg, 635.23 μmol) in DCM (5 mL) was added TFA (1.54 g, 13.51 mmol, 1 mL) and the resulting mixture was stirred at 30° C. for 0.5 hr. LCMS showed the starting material was consumed completely and one main peak with desired mass. The mixture was concentrated under reduced pressure at 10° C. The residue was dissolved with deionized water (40 mL) and lyophilized to afford (R)-5-(6-(piperazin-1-ylmethyl)-1H-benzo[d]imidazol-1-yl)-3-(1-(2-(trifluoromethyl)p henyl)ethoxy)thiophene-2-carboxamide (0.5 g, crude, TFA salt) as a brown solid, which was used for the next step directly. MS(M+H)+=530.0

[0122] Step 8: Synthesis of (2S.4R)-1-((S)-2-(tert-butvll)-14-(4-((1-(5-carbamovl-4-((R)-1-(2-(trifluoromethyl)phenvl)ethoxylthiophen-2-yl)-1H-benzo[d]imidazol-6-yl)methyllpip erazin-1-yl)-4-oxo-6.9.12 trioxa-3-azatetradecan-1-ovll)-4-hydroxy-N-(4-(4-methylthia zol-5-vll)benzyll)1vrrolidine-2-carboxamide (Compound 2)

[0123] To a solution of (R)-5-(6-(piperazin-1-ylmethyl)-1H-benzo[d]imidazol-1-yl)-3-(1-(2-(trifluoromethyl)phenyl)ethoxy)thiophene-2-carboxamide (400 mg, 621.50 μmol, TFA salt) and (S)-13-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-carbonyl)-14,14-dimethyl-11-oxo-3,6,9-trioxa-12-azapentadecyl 4-methylbenzenesulfonate (481.63 mg, 621.50 μmol) in dioxane (10 mL) were added DIPEA (240.97 mg, 1.86 mmol, 324.76 μL) and NaI (18.63 mg, 124.30 μmol, 0.2 eq) at 25° C. The reaction mixture was heated to 100° C. for 12 hours. LCMS showed reagent was consumed completely and one main peak with desired mass. The mixture was concentrated under reduced pressure. The crude product was purified by prep-HPLC (column: Waters Xbridge C.sub.18 150*50 mm* 10 um; mobile phase: [water (10 mM NH .sub.4HCO 3)-ACN]; B %: 33%-63%, 11 min) and the eluent was lyophilized to afford (2S,4R)-1-((S)-2-(tert-butyl)-14-(4-((1-(5-carbamoyl-4-((R)-1-(2-(trifluoromethyl)phen yl)ethoxy)thiophen-2-yl)-1H-benzo[d]imidazol-6-yl)methyl)piperazin-1-yl)-4-oxo-6,9, 12-trioxa-3-azatetradecan-1-oyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrroli dine-2-carboxamide (48.9 mg, 41.46 μmol, 6.67% yield, 96% purity) as a white solid. MS(M+H)+=1132.2

[0124] .sup.1H NMR (400 MHz, DMSO-d.sub.6)δ=8.97-8.96 (m, 1H), 8.59 (br t, J=6.0 Hz, 1H), 8.49 (s, 1H), 7.94 (d, J=7.8 Hz, 1H), 7.85 (br s, 1H), 7.82-7.74 (m, 2H), 7.68 (d, J=8.3 Hz, 1H), 7.61-7.52 (m, 1H), 7.45-7.35 (m, 5H), 7.33 (s, 1H), 7.28 (dd, J=1.2, 8.3 Hz, 1H), 7.22-7.11 (m, 1H), 7.06 (s, 1H), 5.96-5.92 (m, 1H), 5.15 (d, J=3.5 Hz, 1H), 4.55 (d, J=9.5 Hz, 1H), 4.46-4.41 (m, 1H), 4.40-4.34 (m, 1H), 4.29-4.19 (m, 1H), 3.95 (s, 2H), 3.73-3.39 (m, 16H), 2.44 (br s, 1H), 2.43 (s, 3H), 2.42-2.22 (m, 8H), 2.11-2.00 (m, 1H), 1.95-1.85 (m, 1H), 1.75 (d, J=6.2 Hz, 3H), 0.93-0.91 (m, 9H).

[0125] Experimental Examples

[0126] 1. Culture of HeLa Cell Line

[0127] The HeLa cell line was purchased from Korea Cell Line Bank (KCLB), Seoul, Korea. The passage in cell culture was maintained at P115 to P125.

[0128] For cell counting, cell counter (Thermo Fisher Scientific Inc., Catalog #AMQAX1000) and 0.4% trypan blue solution were used.

[0129] For cell culture, DMEM (Gibco, Cat. No. 1195-65; Lot. No. 2085318), FBS (Gibco, Cat. No. 16000-044; Lot. No. 2097593), Penicillin/Streptomycin (PS) (Gibco, Cat. No. 15140-122; Lot. No. 2058855), 100 mm.sup.2 cell culture dish (SPL, Cat. No. 20100), 150 mm.sup.2 cell culture dish (SPL, Cat. No. 20150), 12-well culture plate (SPL, Cat. No. 30012), PBS pH 7.4 (Gibco, Cat. No. 10010-023; Lot. No. 2085080), TrypLE™ Express (Gibco, Cat. No. 12605-010; Lot No. 2070638), Counting Chamber (Hematocytometer) (Hirschmann, Cat. No. 8100204), and 0.4% Trypan Blue Solution (DYNEBIO, Cat. No. CBT3710; Lot. No. 20190723) were used.

[0130] 2. Treatment of Compounds of the Present Invention

[0131] 2×10.sup.5 cells were seeded for each well of a 12-well plate (SPL), and the cells were cultured in the culture medium in a total volume of 2 ml.

[0132] The compounds of were completely dissolved in DMSO and used in the experiment, and thymidine was completely dissolved in DW and used in the experiment. For thymidine block, the products were treated with 2 mM of thymidine (Sigma-Aldrich Cat. No. T9250-5G) and then incubated for 24 hours.

[0133] For release and chemical treatment, the medium was suctioned and washed 3 times with 1XPBS. Complete media was added, followed by incubation for 4 hours in a CO.sub.2 incubator. Each compound was treated according to the concentration of 100 nM and then incubated for 6 hours again.

[0134] 3. Western Blotting

[0135] For SDS-PAGE and Western blotting, 1X RIPA lysis buffer (Rockland, Cat. No. MB-030-0050; Lot no. 39751), 100X Protease Inhibitor Cocktail (Quartett, Cat. No. PPI1015; Lot no. PC050038424), Pierce BCA protein assay kit (ThermoScientific, Cat. No. 23225; Lot no. UC276876), albumin standard (ThermoScientific, Cat. No. 23209; Lot no. UB269561), 4-15% Mini-PROTEAN TGX stain-free gel (Bio-rad, Cat. No. 4568085; Lot no. L007041B), 10X Tris/Glycine/SDS buffer (Bio-rad, Cat. No. 1610732; Lot no. 10000044375B); 10X TBS (Bio-rad, Cat. No. 1706435; Lot no. 1000045140B), 10% Tween 20 (Cat. No. 1610781; Lot no. L004152B), Color protein standard broad range (NEB, Cat. No. P7719S; Lot no. 10040349), 4X Laemmli sample buffer (Bio-rad, Cat. No. 1610747; Lot no. L004133B), β-mercaptoethanol (Sigma-Aldrich, Cat. No. M3148; Lot no. 60-24-2), SuperBloc T20 (TBS) blocking buffer (ThermoScientific, Cat. No. 37536; Lot no. UC282578), 1M sodium azide solution (Sigma-Aldrich, Cat. No. 08591-1 mL-F; Lot no. BCBV4989), aI-Rabbit pAb to Ms IgG (abcam, Cat. No. ab97046; Lot no. GR3252115-1), aGoat pAb to Rb IgG (CST, Cat. No. 7074S; Lot no. 28), α-GAPDH (abeam, Cat. No. ab8245; Lot no. GR3275542-2), α-Plkl (CST, Cat. No. 208G4), α-BRD4 (CST, Cat. No. 13440S), ECLA Prime western blotting reagents (GE Healthcare, Cat. No. RPN2232; Lot no. 17001655), Ponceau S solution (Sigma-Aldrich, Cat. No. P7170; Lot no. SLBV4112), Difco ]Skim milk (BD, Cat. No. 232100; Lot no. 8346795), and iBlot © 2 NC Regular stacks (Invitrogen, Cat. No. IB23001; Lot no. 2NR110619-02) were used.

[0136] For cell harvesting, the cells were first separated from the plate using trypsin and then washed with the medium and PBS. Specifically, the medium was suctioned off and washed with 1 mL of PBS, and PBS was suctioned off. The cells were treated with 0.5 mL TrypLEA Express at 37° C. for 7 minutes to separate the cells, and then 0.5 mL of complete medium was added to collect 1 mL of cell culture solution. Then, 1 mL of the cell collection solution was centrifuged at 8,000 rpm for 120 seconds, and the su-pernatant was removed. After washing with 0.2 mL of PBS, the PBS was removed.

[0137] For cell lysis, a lysis buffer was added and cell debris was removed to obtain a cell lysate. Specifically, the cells were treated with 70 μL of 1X RIPA buffer containing a protease inhibitor and incubated for 30 minutes on ice. Then, the cells were centrifuged at 4° C. and 15,000 rpm for 10 minutes to obtain a cell lysate.

[0138] Then, a standard curve was obtained using the BCA assay, and the protein mass in the lysate was quantified by substituting the curve equation. The mixture was incubated at 37° C. for 30 minutes using 20 μL of standard or sample solution, and 200 μL of BCA or Bradford solution, and measured at 562 nm absorbance. Samples were prepared by adding 4X sample buffer so that the quantity of protein added to each well was 15 μg.

[0139] Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed by setting a running time of 100 minutes at 120 V on a 4-15% Mini-PROTEAN TGX stain-free gel (15 well). Transferring was performed on iBlot © 2 NC Mini stacks at PO mode of the dry blotting system. After staining using Ponceau S solution, blocking was performed for 1 hour with a blocking buffer (Thermo). After washing with 1X TBS containing 0.05% Tween 20, the product was reacted at 4° C. for 16 hours with anti-Plkl(CST) antibody (1:500), anti-BRD4 (Cell signaling) antibody (1:1000) or anti-GAPDH(abcam) antibody (1:10,000) in 1X TBS-T as a primary antibody. After washing three times for 10 minutes with 1X TBS containing 0.05% Tween20, the product was reacted at room temperature for 1 hour with anti-mouse antibody (abcam) (1:10000) or anti-rabbit antibody (CST) (1:5000) in 1X TBS-T as a secondary antibody. Then, after washing three times for 10 minutes with 1X TBS containing 0.05% Tween 20, the product was detected with an ECL working solution (1:1).

[0140] To analyze the results, an image analyzer (GE) was used to obtain final blot data. The ratio of PLK1 to GAPDH for each sample was calculated using the ImageQuant TL (ver.8.2.0) program. Each calculated value was entered into each cell of the Graphpad Prism 9 program, and the graph was automatically calculated to confirm the Dmax value corresponding to the protein degradation ability.

[0141] 4. Confirmation of PLK1 Degradability of the Compounds of the Present Invention

[0142] As a result of the experiment, it was confirmed that all the compounds of the examples of the present invention exhibited a PLK1 degradability of 40 to 60%.

[0143] The contents of all references, patents, pending patent applications and published patents, cited throughout this application are hereby expressly incorporated by reference.

[0144] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein. Such equivalents are intended to be encompassed by the following claims. It is understood that the detailed examples and embodiments described herein are given by way of example for illustrative purposes only, and are in no way considered to be limiting to the disclosure. Various modifications or changes in light thereof will be suggested to persons skilled in the art and are included within the spirit and purview of this application and are considered within the scope of the appended claims. For example, the relative quantities of the ingredients may be varied to optimize the desired effects, additional ingredients may be added, and/or similar ingredients may be substituted for one or more of the ingredients described. Additional advantageous features and functionalities associated with the systems, methods, and processes of the present disclosure will be apparent from the appended claims. Moreover, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein. Such equivalents are intended to be encompassed by the following claims.