KR20210131288A - Benzimidazole thiophene derivative compounds inducing selective degradation of PLK1 - Google Patents

Abstract

The present invention relates to novel compounds that induce selective degradation of PLK1, and more particularly, to 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 above compound, a method for manufacturing the same, and uses thereof. The compounds according to the present invention can be usefully used for the prevention or treatment of PLK1-related diseases.

Description

Benzimidazole thiophene derivative compounds inducing selective degradation of PLK1

The present invention relates to a compound inducing selective degradation of PLK1, a method for preparing the same, and a use thereof.

PLK1 (Polo-like kinase 1) is a serine/threonine kinase and is known to be involved in G2/M phase conversion during cell growth and division. PLK1 is expressed and activated in a pulse form from S phase to G2/M phase, and is rapidly degraded as mitosis is terminated.

PLK1 is overexpressed in various carcinomas such as colorectal cancer, lung cancer, bladder cancer, and melanoma, and PLK1 overexpressed cancer cells tend to show resistance to various types of anticancer drugs. As PLK1 dependence has been revealed in various carcinomas, development of PLK1 inhibitor compounds such as volasertib (also known as BI6727) has been attempted.

However, existing PLK1 inhibitors do not sufficiently inhibit PLK1 activity at clinically safe concentrations. There are problems that have not been addressed (Gheghiani et al. CULM Reports, 2017, etc.). In fact, many pharmaceutical companies, such as Boehringer Ingelheim and GlaxoSmithKline (GSK), have attempted to develop PLK1 inhibitors based on low molecular weight compounds, but most have failed or discontinued in the clinical trial stage. This shows that the pharmacological mechanism of inhibiting enzymatic activity by binding to the active site of PLK1 like a small molecule compound inhibitor is not sufficiently effective for the purpose of developing new drugs to induce anticancer effects by inhibiting the PLK1 activity of cancer cells.

Recently, proteolysis targeting chimera (PROTAC) has been proposed as a platform technology based on a low molecular weight compound capable of inducing proteolysis of a target protein in the body. PROTAC is a bifunctional compound in which a ligand molecule binding to a disease-associated target protein and an E3 ubiquitin ligase binding moiety are linked by a chemical linker. Theoretically, PROTAC compounds can induce degradation of the target protein by locating the disease-associated target protein near the E3 ubiquitin ligase.

It is an object of the present invention to provide compounds that induce selective degradation of PLK1.

Another object of the present invention is to provide a method for preparing a compound that induces selective degradation of PLK1.

Another object of the present invention is to provide the use of compounds that induce selective degradation of PLK1.

PLK1 selective degradation inducing compound

The present invention provides novel compounds that induce selective degradation of PLK1. 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.

One embodiment of the present invention is a compound represented by the following formula (I), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

[Formula I]

In the above formula (I),

ULM is an E3 ubiquitin ligase binding moiety represented by Formula A or Formula B,

[Formula A]

{In Formula A, X ₁ is -CH ₂ - or -CO-}

[Formula B]

는 5원 헤테로아릴 고리이고, Y₁은 수소 또는 C_1-3알킬임}{In Formula B,

is a 5-membered heteroaryl ring, Y ₁ is hydrogen or C _1-3 alkyl}

PTM is a PLK1 binding moiety represented by the following formula (II),

[Formula II]

Linker is a group that chemically connects ULM and PTM.

는 ULM을 링커에 연결하는 공유 결합을 나타낸다.In formulas A and B,

represents the covalent bond connecting the ULM to the linker.

는 PTM을 링커에 연결하는 공유 결합을 나타낸다.In formula II,

represents a covalent bond connecting the PTM to the linker.

(1) E3 ubiquitin ligase binding moiety

According to one embodiment of the present invention, ULM is a CRBN E3 ubiquitin ligase binding moiety represented by formula (A).

In the present invention, CRBN means Cereblon E3 ubiquitin ligase. CRBN together with DDB1, Cul4A and ROC1 constitute the E3 ubiquitin ligase complex, where CRBN is the substrate recognition subunit of the complex. Some compounds capable of binding to CRBN E3 ubiquitin ligase are known in the art. For example, after it was known that thalidomide binds to CRBN E3 ubiquitin ligase (Ito et al. 2010), a number of imide-based small molecule compounds including lenalidomide and pomalidomide (immunomodulatory imide) drug; IMiD) has been reported to have CRBN binding capacity (Chamberlain and Brian. 2019, Akuffo et al. 2018), Burslem et al. 2018], etc.).

According to one embodiment of the present invention, ULM is a VHL E3 ubiquitin ligase binding moiety represented by formula (B).

In the present invention, VHL means von Hippel-Lindau tumor suppressor. VHL together with Elongin B, Elongin C, CUL2 and Rbx1 constitute the VCB E3 ubiquitin ligase complex, where VHL is the substrate recognition subunit of the complex. Some compounds capable of binding to VHL E3 ubiquitin ligase are known in the art. For example, Ala-Leu-Ala-(Hy)Pro-Tyr-Ile-Pro heptapeptide (Schneekloth et al. 2004), Leu-Ala-(Hy)Pro-Tyr-Ile pentapeptide (Rodriguez- After peptides such as Gonzalez et al. 2008) were known, a small molecule VHL E3 ubiquitin ligase-binding compound that improved it was reported (Buckley et al. J. Am. Chem. Soc. 2012), literature [Buckley et al. Ang. Chem. Int. Ed. 2012], Galdeano et al. 2014, Soares et al. 2017, etc.).

(2) target protein ligand (PTM)

In the compound represented by the formula (I) of the present invention, the PTM, which is a moiety performing a target protein ligand function, is a PLK1 binding moiety represented by the aforementioned formula (II).

Formula II constituting some moieties of the compound of formula I of the present invention is solely a benzimidazole thiophene-derived compound, capable of binding to the active site of PLK1 (International Patent Publication Nos. WO2004/014899 and WO2007/030361, etc.)

In one embodiment of formula (I) of the present invention, ULM is represented by formula (A), and formula (II) is represented by:

In one embodiment of formula (I) of the present invention, ULM is represented by formula (B) and formula (II) is represented as follows.

In a specific embodiment, the compound of formula (I) of the present invention is selected from the group consisting of compound 1 and compound 2.

(3) Linker

According to one embodiment of the present invention, Linker is represented by the following formula (L).

[Formula L]

및

는 결합이고,In the above formula L,

and

is a bond,

를 통해 ULM 모이어티와 결합하고,L _ULM is connected to

binds to the ULM moiety through

를 통해 PTM 모이어티와 결합하고,L _PTM is connected to

binds to the PTM moiety via

L _ULM and L _PTM are single bonds, -CH ₂ -, -NH-, -O-, -CO-, -CONH- or -NHCO-,

로 구성된 군에서 선택되고{여기서,

은 3원 내지 10원 사이클로알킬, 4원 내지 10원 헤테로사이클로알킬, 6원 내지 10원 아릴 또는 5원 내지 10원 헤테로아릴로 구성된 군에서 선택된 고리임},L _INT is -CH ₂ -, -CH ₂ CH ₂ -, -CHCH-, -CC-, -CH ₂ CH ₂ O-, -OCH ₂ CH ₂ -, -CH ₂ CH ₂ S-, -SCH ₂ CH ₂ -, -COO-, -CONH-, -NHCO- and

is selected from the group consisting of {here,

is a ring selected from the group consisting of 3-10 membered cycloalkyl, 4-10 membered heterocycloalkyl, 6-10 membered aryl or 5-10 membered heteroaryl},

p is an integer from 1 to 10;

In one embodiment, the Linker is a Linker included in compounds 1-2.

According to a specific embodiment of the present invention, the compound represented by the formula (I) is at least one compound selected from the group consisting of the following compounds 1 and 2, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

In the present invention, the pharmaceutically acceptable salt refers to any organic or inorganic acid addition salt at a concentration having an effective action that is relatively non-toxic and harmless to a patient, and the side effects due to the salt do not reduce the beneficial efficacy of the compound represented by the formula (I). means For example, the pharmaceutically acceptable salt may be hydrochloric acid, phosphoric acid, sulfuric acid, or nitric acid as the inorganic acid, and the organic acid as methanesulfonic acid, p-toluenesulfonic acid, formic acid, acetic acid, trifluoroacetic acid, maleic acid, succinic acid, oxalic acid, Benzoic acid, tartaric acid, fumaric acid, manderic acid, propionic acid, citric acid, lactic acid, glycolic acid, gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanillic acid or hydroiodic acid may be, but is not limited thereto.

Method for preparing PLK1 selective degradation inducing compound

In the present invention, the compound of formula (I), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof is prepared by a synthetic method known in the art of organic chemistry or a modification and derivatization technique obvious to a person skilled in the art. to 3 can be prepared by the same reaction.

[Scheme 1]

[Scheme 2]

[Scheme 3]

In Schemes 1 to 3, PTM, Linker and ULM are groups as defined above or suitable derivatives thereof, and RG ¹ , RG ² , RG ^2a , RG ^2b , RG ³ , RG ^3a , RG ^3b and RG ⁴ are organic synthesis moieties comprising suitable reactive groups capable of linking together the intermediate PROTAC compounds represented by formula (I) via covalent bond formation in the art. The covalent bond formation is amide formation, ester formation, carbamate formation, urea formation, ether formation, amine formation, and various carbon-to-carbon single bonds, double bonds formation, and click chemistry according to specific reactive groups. may be formed, but is not limited thereto.

A variation of each step in the above scheme may include one or multiple synthetic steps. Isolation and purification of the product can be accomplished by standard procedures known to those skilled in the art of organic chemistry.

In the present invention, the preparation method of Example 1 is presented as an example of Scheme 1, and the preparation method of Example 2 is presented as an example of Scheme 3.

In the above scheme, a reactant represented by PTM and a reactant represented by ULM can be easily synthesized by those skilled in the art with reference to literatures known in the field of organic chemistry and descriptions of examples of the present invention.

The present invention includes compounds in the form of PTM-Linker-RG ³ or PTM-Linker 1-RG ^2b , which correspond to intermediates corresponding to the reaction intermediates of formula (I).

Use of PLK1 selective degradation inducing compounds

One embodiment of the present invention is a composition for inducing degradation of PLK1 comprising a compound represented by Formula I or a pharmaceutically acceptable salt thereof. Formula I is as defined above.

Since the PLK1 degradation-inducing PROTAC compound of the present invention can degrade PLK1, a target protein, from the viewpoint of its mechanism of action, it achieves an excellent PLK1 inhibitory effect compared to conventional PLK1 small molecule inhibitors that inhibit simple PLK1 activity.

Accordingly, the composition comprising the compound represented by Formula I of the present invention or a pharmaceutically acceptable salt thereof may be usefully used for selective degradation of PLK1.

One embodiment of the present invention is a pharmaceutical composition for preventing or treating a PLK1-related disease comprising a compound represented by Formula I or a pharmaceutically acceptable salt thereof. Formula I is as defined above.

In the present invention, a PLK1-related disease means any disease or condition that can be treated, alleviated, delayed, inhibited or prevented from inducing degradation or inhibiting the activity of PLK1. In one embodiment, the PLK1-associated disease may be cancer (malignant tumor), benign tumor, neurological disease, or other genetic or non-genetic disease caused by excessive cell division.

The 　 cancer includes all cancers that can exhibit preventive or therapeutic efficacy due to inhibition of PLK1 activity, and may be a solid cancer or a blood cancer. For example, the cancer is squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous cell carcinoma of the lung, peritoneal cancer, skin cancer, skin or intraocular melanoma, rectal cancer, perianal cancer, esophageal cancer, small intestine cancer, endocrine cancer Adenocarcinoma, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, chronic or acute leukemia, lymphocytic lymphoma, hepatocellular carcinoma, gastrointestinal cancer, gastric cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, liver tumor, breast cancer, colon cancer , colon cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, head and neck cancer, brain cancer, may be at least one selected from the group consisting of osteosarcoma, but is not limited thereto. The above 　 cancer includes not only primary 　 cancer but also metastatic 　 cancer.

The benign tumors include all benign tumors capable of exhibiting prophylactic or therapeutic efficacy due to inhibition of PLK1 activity, such as benign tumors at a precancerous stage, and may be solid tumors or hematologic tumors. For example, the tumor may be one or more selected from the group consisting of Barrett's esophagus, colon adenoma and polyp, breast fibroadenoma and cyst, single cell gamma globulinopathy (MGUS), monoclonal lymphocytosis, and the like, but is not limited thereto.

The neurological diseases include all neurological diseases that can exhibit preventive or therapeutic efficacy due to inhibition of PLK1 activity, specifically central nervous system diseases, degenerative neurological diseases, Alzheimer's disease, Parkinson's disease, multiple sclerosis, Huntington's disease, senile dementia, It may be one or more selected from epilepsy, ALS, stroke, and neurological damage following brain or spinal cord injury and axonal degeneration-related disorders, but is not limited thereto.

The pharmaceutical composition of the present invention may further include one or more active ingredients exhibiting the same or similar efficacy in addition to the compound represented by Formula I or a pharmaceutically acceptable salt thereof.

One embodiment of the present invention is a method for degrading PLK1 protein by administering a compound represented by Formula (I) or a pharmaceutically acceptable salt thereof to mammals including humans.

Another embodiment of the present invention is a method for degrading PLK1 protein by administering a compound represented by Formula (I) or a pharmaceutically acceptable salt thereof to a sample in vitro. The sample may be a cell, a cell culture, a body fluid or tissue of a mammal including a human, but is not limited thereto.

The compounds of the present invention exhibit the effect of inducing degradation of PLK1. Therefore, the compound of the present invention can be usefully used for the prevention or treatment of PLK1-related diseases.

Hereinafter, the configuration and effect of the present invention will be described in more detail through Examples and Experimental Examples. These Examples and Experimental Examples are only for illustrating the present invention, and the scope of the present invention is not limited by these Examples.

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

[Table 1]

The compound of the present invention was purified according to the method below and the structure was analyzed.

analytical instrument

LCMS: Shimadzu LCMS-2020

NMR : BRUKER AVANCE III/400MHz

HPLC: Shimadzu LC-20AB and Shimadzu LC-20AD, Agilent 1100 LC, Agilent 1200 LC, Agilent 1290 LC

LCMS analysis method

LCMS data were recorded with a Shimadzu LCMS-2020 equipped with an electron spray ionization apparatus. 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, 5um was used.

HPLC analysis method

HPLC was performed using Shimadzu LC-20AB or Shimadzu LC-20AD or Agilent 1100 LC or Agilent 1200 LC or Agilent 1290 LC, 0.0375% TFA in water (Solvent A) and 0.01875% TFA in acetonitrile (Solvent B) or in water 0.025% NH ₃ ·H 2 O (solvent A) and acetonitrile (solvent B) were used as mobile phases. The column is XBridge C18 (2.1*50)mm, 5um or Kinetex C18 LC column (4.6*50)mm, 5um or Eclipse plus C18 (4.6*150)mm, 3.5um or Waters XBridge® C18 (4.6*150)mm, 3.5 μm was used.

NMR analysis method

¹ H NMR spectra were recorded with a Bruker AVANCE III/400 MHz Probe (BBO).

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

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

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 ₂ (2 M, 36.4 mL). The mixture was stirred at 25 °C for 2 h. TLC (petroleum ether:ethyl acetate=2:1) confirmed that the starting material was consumed and a new spot was detected. The mixture was concentrated under reduced pressure to give the title compound (8 g, crude) as a yellow solid.

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

To a solution of 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) TosCl (9.29 g, 48.71 mmol) was added at 25 °C. The solution was stirred at 25 °C for 12 h. The main peak of the desired molecular weight was confirmed by LCMS. The mixture was concentrated under reduced pressure. The residue was purified by silica gel chromatography (45 g SepaFlash® Silica Flash Column, Eluent of 0-65% Ethyl acetate/Petroleum ether gradient @ 75 mL/min) to obtain the title compound (9.1 g, 20.59 mmol, 84.55) as a colorless oil. % yield, 99% purity).

MS(M+H) ⁺ =438.0.

Step 3: Methyl 4-((3-oxo-1-phenyl-2,7,10-trioxa-4-azadodecan-12-yl)oxy) -2-(trifluoromethyl)benzoate (4 ) synthesis

3-oxo-1-phenyl-2,7,10-trioxa-4-azadodecan-12-yl 4-methylbenzenesulfonate (8.9 g, 20.34 mmol) and methyl 4-hydride in ACN (180 mL) To a solution of hydroxy-2-(trifluoromethyl)benzoate (4.93 g, 22.38 mmol) was added K ₂ CO ₃ (5.62 g, 40.69 mmol) and KI (168.84 mg, 1.02 mmol). The mixture was stirred at 80 °C for 14 h. It was confirmed that the desired molecular weight was detected by LCMS. The mixture was filtered and the filtered cake was washed with EtOAc (100 mL). The filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography (20 g SepaFlash® Silica Flash Column, Eluent of 0-30% Ethyl acetate/Petroleum Ether gradient @ 60 mL/min) to obtain the title compound as a yellow oil (7.4 g, 14.94 mmol, 73.44). % yield, 98% purity).

MS(M+H) ⁺ =486.2.

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

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 methyl 4-((3-oxo-) in toluene (49 mL) A solution of 1-phenyl-2, 7,10-trioxa-4-azadodecan-12-yl)oxy)-2-(trifluoromethyl)benzoate (7.1 g, 14.63 mmol,) was added. The final mixture was stirred at 25 °C for 14 h. It was confirmed that the desired molecular weight was detected by LCMS. _{The mixture was quenched by addition of NH 4} Cl (10 mL) at 0 °C and then extracted with EtOAc (10 mL x 3). The combined organic layers were washed with H ₂ O (10 mL), dried over Na ₂ SO ₄ overnight and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography (40 g SepaFlash® Silica Flash Column, Eluent of 10-60% Ethyl acetate/Petroleum Ether gradient @ 100 mL/min) to obtain the title compound (5.4 g, 10.93 mmol, 74.72) as a yellow oil. % yield, 95% purity).

MS(M+H) ⁺ =470.2

Step 5: Preparation of benzyl (2-(2-(2-(4-(1-hydroxyethyl)-3-(trifluoromethyl)phenoxy)ethoxy)ethoxy)ethyl)carbamate (6)

of benzyl (2-(2-(2-(4-acetyl-3-(trifluoromethyl)phenoxy)ethoxy)ethoxy)ethyl)carbamate (1.8 g, 3.83 mmol) in MeOH (40 mL) To the solution was _{slowly added NaBH 4} (450 mg, 11,90 mmol) and the mixture was stirred at 25 °C for 1 h. It was confirmed that the desired molecular weight was detected by LCMS. The mixture was _{quenched by addition of NH 4} Cl (30 mL) and then concentrated under reduced pressure. The mixture was extracted with EtOAc (20 mL×3). The combined organic layers were washed with NaHCO ₃ (10 mL×2) and H ₂ O (10 mL×2), dried over Na ₂ SO ₄ , and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography (12 g SepaFlash® Silica Flash Column, Eluent of 20-80% Ethyl acetate/Petroleum ether gradient @ 50 mL/min) to obtain the title compound (1.77 g, 3.75 mmol, 97.91) as a yellow oil. % yield, 100% purity).

MS(M+Na) ⁺ =494.2

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

Benzyl (2-(2-(2-(4-(1-hydroxyethyl)-3-(trifluoromethyl)phenoxy)ethoxy)ethoxy)ethyl)carbamate (1.77) in DCM (40 mL) g, 3.75 mmol), methyl 3-hydroxy-5-nitrothiophene-2-carboxylate (915 mg, 4.50 mmol) and DTBAD (1.30 g, 5.63 mmol) in a solution of _{PPh 3 (1.48 g, 5.63 mmol)} was added 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 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 solidified with MTBE (10 mL) and filtered. The filtered cake was collected and dried under reduced pressure to give the yellow title compound (2.06 g, 3.14 mmol, 83.57% yield).

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

Methyl 5-nitro-3-(1-(4-((3-oxo-1-phenyl-2,7,10-trioxa-4-azado) in EtOH (20 mL) and H _{2 O (20 mL))} To a solution of decan-12-yl)oxy)-2-(trifluoromethyl)phenyl)ethoxy)thiophene-2-carboxylate (2.06 g, 3.14 mmol) with NH ₄ Cl (1.01 g, 18.82 mmol) and Fe (1.05 g, 18.82 mmol) was added and the final mixture was stirred at 80 °C for 1 h. It was confirmed that the desired molecular weight was detected by LCMS. The mixture was filtered at 80 °C and the filtered 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 layers were washed with H ₂ O (10 mL×2), dried over Na ₂ SO ₄ , and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography (20 g SepaFlash® Silica Flash Column, Eluent of 20-60% Ethyl acetate/Petroleum ether gradient @ 50 mL/min) to give the yellow title compound (0.9 g, 1.44 mmol, 45.78%). yield) was obtained.

MS(M+H) ⁺ =627.4

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

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) AcOH (10.50 mg, 174.85 μmol, 10 μL) was added 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 final mixture was stirred at 25 °C for 14 h. It was confirmed that the desired molecular weight was detected by LCMS. The mixture was diluted with H ₂ O (20 mL) and citric acid solution (50 mL), then washed with EtOAc (10 mL×2) and the organic layer was removed. The pH of the aqueous layer was adjusted to 9 and extracted with EtOAc (10 mL×2). The combined organic layers were dried over Na ₂ SO ₄ and filtered. The filtrate was concentrated under reduced pressure to afford the title compound (1.2 g, 3.74 mmol, 86.10% yield, 98% purity) as a yellow oil.

MS(M+H) ⁺ =314.1

Step 9: Methyl 5-((5-((4-methylpiperazin-1-yl)methyl)-2-nitrophenyl)amino)-3-(1-(4-((3-oxo-1-phenyl) Synthesis of -2,7,10-trioxa-4-azadodecan-12-yl)oxy)-2-(trifluoromethyl)phenyl)ethoxy)thiophene-2-carboxylate (11)

1-(3-bromo-4-nitrobenzyl)-4-methylpiperazine (355.14 mg, 1.13 mmol) and methyl 5-amino-3-(1-(4-((3-) in toluene (15 mL) 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) were added Cs ₂ CO ₃ (1.10 g, 3.39 mmol), Pd ₂ (dba) ₃ (20.70 mg, 22.61 μmol) and Xantphos (28.78 mg, 49.74 μmol) and the final mixture was added to 80 Stirred at °C for 14 hours. It was confirmed that the reaction was complete by TLC (ethyl acetate:methanol=5:1). The mixture was filtered and the filtered cake was washed with EtOAc (50 mL). The filtrate was concentrated under reduced pressure. The crude product was _{then diluted with H 2} O (10 mL) and extracted with EtOAc (10 mL×2). The combined organic layers were washed with H ₂ O (10 mL×2), dried over Na ₂ SO ₄ and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by 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 give the title compound (510 mg, 593.09 μmol, 52.47% yield) as a yellow oil.

MS(M+H) ⁺ =860.4

Step 10: 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)thiophene-2-carboxylate (12 ) synthesis

Methyl 5-((5-((4-methylpiperazin-1-yl)methyl)-2-nitrophenyl)amino)-3-(1-(4-((3-oxo-) in MeOH (8 mL) 1-phenyl-2,7,10-trioxa-4-azadodecan-12-yl)oxy)-2-(trifluoromethyl)phenyl)ethoxy)thiophene-2-carboxylate (410 mg, 476.80 μmol) 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). Stirred at 70 °C for 6 hours. It was confirmed that the desired molecular weight was detected by LCMS. The mixture was filtered, the filtered cake was washed with hot MeOH (50 mL, 50 °C), and the filtrate was concentrated under reduced pressure. The crude product _{was diluted with H 2} O (10 mL) and the pH was adjusted to 8 with _{NaHCO 3 solution.} The mixture was then extracted with EtOAc (10 mL x 3). The combined organic layers were washed with H ₂ O (10 mL×2), dried over _{Na 2} SO _{4 and filtered.} The filtrate was concentrated under reduced pressure to give the title compound (330 mg, crude) as a yellow oil.

MS(M+H) ⁺ =840.5

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

Methyl 5-(6-((4-methylpiperazin-1-yl)methyl)-1H-benzo[d]imidazol-1-yl)-3-(1-(4-() in THF (3 mL) (3-oxo-1-phenyl-2,7,10-trioxa-4-azadodecan-12-yl)oxy)-2-(trifluoromethyl)phenyl)ethoxy)thiophene-2-carboxyl To a solution of the rate (250.00 mg, 297.65 μmol) was added a solution of _{LiOH·H 2} O (14.39 mg, 342.89 μmol) _{in H 2} O (1.5 mL). The mixture was stirred at 40 °C for 14 h. To the mixture was _{further added LiOH·H 2} O (17.98 mg, 428.61 μmol) and the mixture was stirred at 40° C. for 14 h. LCMS confirmed that the starting material was consumed and the desired molecular weight was detected. The mixture was concentrated under reduced pressure to give the title compound (250 mg, crude) as a yellow oil.

MS(M+H) ⁺ =826.6

Step 12: Benzyl (2-(2-(2-(4-(1-((2-carbamoyl-5-(6-((4-methylpiperazin-1-yl)methyl)-1H-benzo[ Preparation of d]imidazol-1-yl)thiophen-3-yl)oxy)ethyl)-3-(trifluoromethyl)phenoxy)ethoxy)ethoxy)ethyl)carbamate (14)

5-(6-((4-methylpiperazin-1-yl)methyl)-1H-benzo[d]imidazol-1-yl)-3-(1-(4-(() in DMF (4 mL) 3-oxo-1-phenyl-2,7,10-trioxa-4-azadodecan-12-yl)oxy)-2-(trifluoromethyl)phenyl)ethoxy)thiophene-2-carboxylic acid ( To a solution of 250 mg, 300.54 μmol) and NH ₄ Cl (80.38 mg, 1.50 mmol) was added DIPEA (118.72 mg, 918.58 μmol, 160 μL) and HATU (171.41 mg, 450.82 μmol) and the final mixture was stirred at 25 °C for 14 stirred for hours. LCMS confirmed that the starting material remained. Further NH ₄ Cl (80.38 mg, 1.50 mmol) and HATU (171.41 mg, 450.82 μmol) were added and the final mixture was stirred at 25° C. for 1 h. It was confirmed that the desired molecular weight was detected by LCMS. The reaction mixture _{was diluted with H 2} O (10 mL) and extracted with EtOAc (10 mL×3). The combined organic layers were washed with brine (10 mL x 2), dried over Na ₂ SO ₄ and filtered. The filtrate was concentrated under reduced pressure to give the title compound (170 mg, crude) as a yellow oil.

MS(M+H) ⁺ =825.6

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

Benzyl (2-(2-(2-(4-(1-((2-carbamoyl-5-(6-((4-methylpiperazin-1-yl)methyl)-1H) in ACN (0.75 mL)) -benzo[d]imidazol-1-yl)thiophen-3-yl)oxy)ethyl)-3-(trifluoromethyl)phenoxy)ethoxy)ethoxy)ethyl)carbamate (150 mg, 181.84) μmol) was added TMSI (220.50 mg, 1.10 mmol, 150 μL) and the mixture was stirred at 20 °C for 1 h. LCMS confirmed that the starting material was consumed and the desired molecular weight was detected. The mixture was _{quenched with H 2} O (20 mL, 0° C.) and washed with EtOAc (10 mL×3) and the organic layer was removed. The pH was adjusted to 7-8 by adding _{Na 2} CO ₃ solution to the aqueous phase, and then the mixture was lyophilized. The crude product was prep-HPLC (column: Unisil 3-100 C ₁₈ μLtra 150*50 mm*3 um; mobile phase: [water (0.225%FA) -ACN]; B%: 5% - 35%, 10 min) to give the title compound (60 mg, 70.22 umol, 38.62% yield, 97% purity, FA salt) as a yellow solid.

MS(M+H) ⁺ =691.4

Step 14: 3-(1-(4-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4- yl) amino)ethoxy)ethoxy)ethoxy)-2-(trifluoromethyl)phenyl)ethoxy)-5-(6-((4-methylpiperazin-1-yl)methyl)-1H- Synthesis of benzo[d]imidazol-1-yl)thiophene-2-carboxamide (Compound 1)

3-(1-(4-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)-2-(trifluoromethyl)phenyl)ethoxy)-5- in DMSO (1.2 mL) (6-((4-methylpiperazin-1-yl)methyl)-1H-benzo[d]imidazol-1-yl)thiophene-2-carboxamide (60 mg, 72.39 μmol, FA salt) and TEA (43.95 mg, 434.34 μmol, 60.45 μL) in a solution of 2-(2,6-dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione (24 mg, 86.89 μmol) was added and the mixture was stirred at 80 °C for 14 hours. It was confirmed that the desired molecular weight was detected by LCMS. The mixture was filtered and the filtrate was prep-HPLC (column: Phenomenex Gemini-NX C ₁₈ 75*30 mm*3um;mobile phase: [water (10mM NH ₄ HCO ₃ ) -ACN]; B%: 30% - 60% , 8 min). The eluent was lyophilized to give the final title compound (10.2 mg, 9.80 μmol, 13.54% yield, 91% purity) as a yellow solid.

MS(M+H) ⁺ =947.7

¹ H NMR (400 MHz, CD ₃ CN) δ = 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. (2S,4R)-1-((S)-2-(tert-butyl)-14-(4-((1-(5-carbamoyl-4-((R)-1-((R)-1-() 2-(trifluoromethyl)phenyl)ethoxy)thiophen-2-yl)-1H-benzo[d]imidazol-6-yl)methyl)piperazin-1-yl)-4-oxo-6, 9,12-Trioxa-3-acatetradecanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (Compound 2 ) synthesis

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

Methyl 3-hydroxy-5-nitrothiophene-2-carboxylate (1.23 g, 6.05 mmol) in DCM (20 mL), (S)-1-(2-(trifluoromethyl)phenyl)ethanol (1 g, 5.26 mmol, 80.00 μL) 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 h. It was confirmed by TLC (Petroleum ether: Ethyl acetate=3:1; Rf=0.75) that (S)-1-(2-(trifluoromethyl)phenyl)ethanol was completely consumed and a new spot was formed. The mixture was concentrated under reduced pressure. The residue was purified by silica gel chromatography (40 g SepaFlash® Silica Flash Column, Eluent of 0-5% Ethyl acetate/Petroleum ether gradient @ 30 mL/min) to obtain the title compound (1.8 g, 4.51 mmol, 85.73) as a yellow oil. % yield, 94% purity).

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

(R)-methyl 5-nitro-3-(1-(2-(trifluoromethyl)phenyl)ethoxy)thiophene-2-carboxylate in EtOH (15 mL) and H _{2 O (15 mL) (} To a solution of 1.8 g, 4.80 mmol) was added Fe (1.61 g, 28.78 mmol) and NH ₄ Cl (1.54 g, 28.78 mmol) at 25 °C, and the mixture was stirred at 80 °C for 1 h. It was confirmed by TLC (Petroleum ether: Ethyl acetate=3:1; Rf=0.4) that the starting material was completely consumed and a new spot was formed. The mixture was filtered through a pad of celite 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 layers were washed with brine (20 mL x 3), dried over Na ₂ SO ₄ and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography (12 g SepaFlash® Silica Flash Column, Eluent of 0-30% Ethyl acetate/Petroleum ether gradient @ 75 mL/min) to obtain the title compound (1.4 g, 3.81 mmol, 79.46) as a brown oil. % yield, 94% purity) and used directly in the next step.

MS(M+H) ⁺ =345.8

Step 3: (R)-tert-Butyl 4-(3-((5-(methoxycarbonyl)-4-(1-(2-(trifluoromethyl)phenyl)ethoxy)thiophene-2- Synthesis of yl)amino)-4-nitrobenzyl)piperazine-1-carboxylate (6)

(R)-Methyl 5-amino-3-(1-(2-(trifluoromethyl)phenyl)ethoxy)thiophene-2-carboxylate (530 mg, 1.53 mmol) in t-BuOH (10 mL) _{, Pd 2} (dba) in a solution of tert-butyl 4-(3-bromo-4-nitrobenzyl)piperazine-1-carboxylate (675.73 mg, 1.69 mmol) and K ₂ CO ₃ (636.33 mg, 4.60 mmol) ) ₃ (28.11 mg, 30.69 μmol) and XPhos (32.19 mg, 67.53 μmol) _{were added at 25° C. under N 2} conditions and the final mixture was stirred at 60° C. for 12 hours. LCMS confirmed that the peak at 37% was tert-butyl 4-(3-bromo-4-nitrobenzyl)piperazine-1-carboxylate. In addition, Pd ₂ (dba) ₃ (28.11 mg, 30.69 μmol) and XPhos (32.19 mg, 67.53 μmol) were added at _{25° C. under N 2 conditions.} The reaction mixture was stirred at 70 °C for 12 h. The mixture was diluted with ethyl acetate (50 mL), filtered through a pad of celite and the filtrate was concentrated. The residue was purified by 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 ₁₈ 150*50 mm). * 10um; mobile phase: [water (10 mM NH ₄ HCO ₃ )-ACN]; B%: 65%-95%, min). The eluent was lyophilized to give the title compound (400 mg, 601.78 μmol, 39.21% yield) as a brown solid. The crude product was used directly in the next step.

MS(M+H) ⁺ =665.1

¹ H NMR (400 MHz, CDCl 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).

Step 4: (R)-tert-Butyl 4-((1-(5-(methoxycarbonyl)-4-(1-(2-(trifluoromethyl)phenyl)ethoxy)thiophene-2- Synthesis of yl)-1H-benzo[d]imidazol-6-yl)methyl)piperazine-1-carboxylate (7)

(R)-tert-Butyl 4-(3-((5-(methoxycarbonyl)-4-(1-(2-(trifluoromethyl)phenyl)ethoxy)thiophene in MeOH (20 mL) In a solution of -2-yl)amino)-4-nitrobenzyl)piperazine-1-carboxylate (0.4 g, 601.78 μmol) in 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) were added. The reaction mixture was stirred at 70 °C for 12 h. LCMS confirmed complete consumption of the starting material and one main peak with the desired molecular weight. The mixture was filtered and the filtered cake was washed with MeOH (50 mL, 50 °C). The filtrate was concentrated under reduced pressure. The residue _{was diluted with H 2} O (15 mL) and the pH was adjusted to 8 with _{NaHCO 3 solution.} The mixture was then extracted with EtOAc (20 mL x 3). The combined organic layers were washed with H ₂ O (10 mL×2), dried over Na ₂ SO ₄ , and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography (12 g SepaFlash® Silica Flash Column, Eluent of 0-50% Ethyl acetate/Petroleum ether gradient @ 50 mL/min) to obtain the title compound (300 mg, 456.02 μmol, 75.78) as a yellow oil. % yield, 98% purity) and used directly in the next step.

MS(M+H) ⁺ =645.2

Step 5: (R)-5-(6-((4-(tert-butoxycarbonyl)piperazin-1-yl)methyl)-1H-benzo[d]imidazol-1-yl)-3- Synthesis of (1-(2-(trifluoromethyl)phenyl)ethoxy)thiophene-2-carboxylic acid (8)

(R)-tert-Butyl 4-((1-(5-(methoxycarbonyl)-4-(1-(2-(trifluoromethyl)phenyl)ethoxy)thiophene in THF (5 mL) To a solution of -2-yl)-1H-benzo[d]imidazol-6-yl)methyl)piperazine-1-carboxylate (300 mg, 465.33 μmol) in H ₂ O (5 mL) LiOH (33.43 mg, 1.40 mmol) solution was added dropwise at 25 °C. The mixture was stirred at 40 °C for 12 h. LCMS confirmed complete consumption of the starting material and one major peak of the desired molecular weight. The reaction mixture was concentrated under reduced pressure to give the title compound (296 mg, crude) as a brown solid, which was used directly in the next step.

MS (M+H) ⁺ =631.0

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

(R)-5-(6-((4-(tert-butoxycarbonyl)piperazin-1-yl)methyl)-1H-benzo[d]imidazol-1-yl) in DMF (3 mL) HATU (353.59) in a solution of -3-(1-(2-(trifluoromethyl)phenyl)ethoxy)thiophene-2-carboxylic acid (296 mg, 464.96 μmol), NH _{4 Cl (124.36 mg, 2.32 mmol)} mg, 929.93 μmol) and DIPEA (180.28 mg, 1.39 mmol, 242.96 μL) were added at 30°C. The mixture was stirred at 30 °C for 2 h. LCMS confirmed complete consumption of the starting material and one major peak of the desired molecular weight. The mixed solution was concentrated under reduced pressure. The residue was subjected to 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) was purified to give the title compound (400 mg, crude) as a brown solid, which was used directly in the next step.

MS (M+H) ⁺ =630.0

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

(R)-tert-Butyl 4-((1-(5-carbamoyl-4-(1-(2-(trifluoromethyl)phenyl)ethoxy)thiophen-2-yl in DCM (5 mL) )-1H-benzo[d]imidazol-6-yl)methyl)piperazine-1-carboxylate (400 mg, 635.23 μmol) in DCM (5 mL) solution in TFA (1.54 g, 13.51 mmol, 1 mL) was added and the final mixture was stirred at 30 °C for 0.5 h. LCMS confirmed complete consumption of the starting material and one main peak of the desired molecular weight. The mixture was concentrated under reduced pressure at 10 °C. The residue was dissolved in demineralized water (40 mL) and lyophilized to obtain the title compound (0.5 g, crude, TFA salt) as a brown solid, which was used directly in the next step.

MS(M+H) ⁺ =530.0

Step 8: (2S,4R)-1-((S)-2-(tert-butyl)-14-(4-((1-(5-carbamoyl-4-((R)-1-(2) -(trifluoromethyl)phenyl)ethoxy)thiophen-2-yl)-1H-benzo[d]imidazol-6-yl)methyl)piperazin-1-yl)-4-oxo-6,9 ,12-trioxa-3-azatetradecan-1-yl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide ( Synthesis of compound 2)

(R)-5-(6-(piperazin-1-ylmethyl)-1H-benzo[d]imidazol-1-yl)-3-(1-(2-(tri) in dioxane (10 mL) Fluoromethyl)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-aza To a solution of pentadecyl 4-methylbenzenesulfonate (481.63 mg, 621.50 μmol) was 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 at 100 °C for 12 h. LCMS confirmed complete consumption of the reactant and one main peak of the desired molecular weight. The mixture was concentrated under reduced pressure. The crude product was prep-HPLC (column: Waters Xbridge C ₁₈ 150*50 mm* 10um; mobile phase: [water (10 mM NH ₄ HCO ₃ )-ACN]; B%: 33%-63%, 11 min) Purification and lyophilization of the eluent gave the title compound (48.9 mg, 41.46 μmol, 6.67% yield, 96% purity) as a white solid.

MS(M+H) ⁺ =1132.2

¹ H NMR (400 MHz, DMSO- d 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).

<Experimental example>

1. Cultivation of HeLa Cell Lines

The HeLa cell line was purchased from the Korea Cell Line Bank. The passage of cultured cells was maintained at P115 to P125.

For cell counting, Thermo's cell counter (cULM counter) (Catalog # AMQAX1000) and 0.4% Trypan blue solution were used.

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 mm2 cell culture dish (SPL, Cat. No. 20100), 150 mm2 cell culture dish (SPL, Cat. No. 20150), 12 wells Culture plate (SPL, Cat. No. 30012), PBS pH7.4 (Gibco, Cat. No. 10010-023; Lot. No. 2085080), TrypLE ^™ Express (Gibco, Cat. No. 12605-010; Lot. No. 2070638), a counting chamber (Hematocytometer) (Hirschmann, Cat. No. 8100204), and a 0.4% trypan blue solution (DYNEBIO, Cat. No. CBT3710; Lot. No. 20190723) were used.

2. Treatment of compounds of the present invention

^{2Х10 5} cells were seeded in each well of a 12-well plate (SPL), and the cells were cultured in a total culture medium volume of 2 mL.

Example compound was completely dissolved in DMSO and used in the experiment, and thymidine was completely dissolved in DW and used in the experiment. For the thymidine block, thymidine (Sigma-Aldrich Cat. No. T9250-5G) 2 mM treatment was followed by incubation for 24 hours.

For release and chemical treatment, the medium was sucked and washed three times with 1× PBS. Put the complete medium (complete media), CO ₂ Incubated in an incubator for 4 hours. Each compound was treated according to a concentration of 100 nM and then incubated again for 6 hours.

3. Western Blotting

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. PCO50038424) ), 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 solution (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), SuperBlock™ T20 (TBS) blocking buffer (ThermoScientific, Cat. No. 37536; Lot no. UC282578), 1M sodium azide solution (Sigma-Aldrich, Cat. No. 08591-1mL-F; Lot no. BCBV4989), α-Rabbit pAb to Ms IgG (abcam, Cat. No. ab97046; Lot no. GR3252115-1), α-Goat pAb to Rb IgG (CST, Cat. No. 7074S; Lot no. 28), α -GAPDH (abcam, Cat. No. ab8245; Lot no. GR3275542-2), αPlk1 (CST, Cat. No. 208G4), α-BRD4 (CST, Cat. No. 13440S), ECL™ 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), iBlot® 2 NC Regular stacks (Invitrogen, Cat. No. IB23001; Lot no. 2NR110619-02). was used.

For cell harvest, cells were detached from the plate using trypsin and washed with medium and PBS. Specifically, after suctioning the medium, it was washed with 1 mL PBS, and the PBS was sucked. Cells were detached by treatment with 0.5 mL TrypLE™ Express at 37 °C, 7 min, 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 supernatant was removed. After washing with 0.2 mL of PBS, the PBS was removed.

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

Then, a standard curve was obtained using the BCA assay, and the protein mass in the lysate was quantified by substituting it. The mixture was incubated at 37° C. for 30 min using 20 μL of standard or sample solution, 200 μL of BCA or Bradford solution, and measured at 562 nm absorbance. Samples were prepared by adding 4X sample buffer so as to be 15 μg per well.

SDS-PAGE was performed by setting a running time of 100 min at 120 V on 4-15 % Mini-PROTEAN TGX stain-free gel (15　wULM). Transferred to iBlot®　2 NC　Mini stacks using P0 mode of dry blotting system. After staining using Ponceau S solution, blocking was performed with a blocking buffer (Thermo) for 1 hour. After washing with 1X TBS containing 0.05% Tween20, anti-Plk1 (CST) antibody (1:500), anti-BRD4 (CULM signaling) antibody (1:1000) or anti-GAPDH in 1X TBS-T as primary antibody (abcam) was reacted with the antibody (1:10,000) at 4 °C for 16 hours. After washing 3 times for 10 min with 1X TBS with 0.05% Tween20, anti-mouse antibody (abcam) (1:10,000) or anti-rabbit antibody (CST) (1:5,000) in 1X TBS-T as secondary antibody ) and reacted at room temperature for 1 hour. Then, after washing 3 times for 10 minutes with 1X TBS containing 0.05% Tween 20, it was detected with ECL working solution (1:1).

For the analysis of the results, the final blot data was obtained using an image analyzer (GE). 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 resolution.

4. Confirmation of PLK1 resolution of the compound of the present invention

As a result of the above-described experiment, it was confirmed that all of the example compounds of the present invention exhibited PLK1 protein degradation of 40 to 60%.

As the present invention has been described in detail above, for those of ordinary skill in the art, it will be apparent that these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. Accordingly, it is intended that the substantial scope of the present invention be defined by the appended claims and their equivalents.

Claims (3)

A compound represented by the following formula (I), a stereoisomer or a pharmaceutically acceptable salt thereof:
[Formula I]

In the above formula (I),
ULM is an E3 ubiquitin ligase binding moiety represented by Formula A or Formula B,
[Formula A]

{In Formula A, X ₁ is -CH ₂ - or -CO-}
[Formula B]

{In Formula B,

is a 5-membered heteroaryl ring, Y ₁ is hydrogen or C _1-3 alkyl}
PTM is a PLK1 binding moiety represented by the following formula (II),
[Formula II]

indicated within the PTM or ULM.

indicates that it is covalently linked with the Linker,
Linker is a group that chemically connects ULM and PTM, and is represented by the following formula L,
[Formula L]

[In the formula L,

and

is a bond,
L _ULM is connected to

binds to the ULM moiety through
L _PTM is connected to

binds to the PTM moiety via
L _ULM and L _PTM are each independently a single bond, -CH ₂ -, -NH-, -O-, -CO-, -CONH- or -NHCO-,
L _INT is -CH ₂ -, -CH ₂ CH ₂ -, -CHCH-, -CC-, -CH ₂ CH ₂ O-, -OCH ₂ CH ₂ -, -CH ₂ CH ₂ S-, -SCH ₂ CH ₂ -, -COO-, -CONH-, -NHCO- and

is selected from the group consisting of {where,

is a ring selected from the group consisting of 3-10 membered cycloalkyl, 4-10 membered heterocycloalkyl, 6-10 membered aryl or 5-10 membered heteroaryl},
p is an integer from 1 to 10];
The compound represented by the formula (I) is not any of the following compounds 1 and 2:

The compound according to claim 1, wherein ULM is represented by Formula A, and Formula II is represented by the following Formula, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

The compound according to claim 1, wherein ULM is represented by Formula B, and Formula II is represented by the following Formula, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof: