WO2021219071A1

WO2021219071A1 - Process for preparing protac btk degraders

Info

Publication number: WO2021219071A1
Application number: PCT/CN2021/090900
Authority: WO
Inventors: Hexiang Wang; Bailin LEI; Changxin HUO; Dongqing SUN; Jie Chen; Zhiwei Wang
Original assignee: Beigene (Beijing) Co., Ltd.
Priority date: 2020-04-30
Filing date: 2021-04-29
Publication date: 2021-11-04
Also published as: CN115461343A

Abstract

Disclosed herein is a process for preparing novel bifunctional compounds formed by conjugating BTK inhibitor moieties with E3 ligase Ligand moieties, which function to recruit targeted proteins to E3 ubiquitin ligase for degradation.

Description

[Title established by the ISA under Rule 37.2] PROCESS FOR PREPARING PROTAC BTK DEGRADERS

FIELD OF THE INVENTION

Disclosed herein is a process for preparing bifunctional compounds formed by conjugating BTK inhibitor moieties with E3 ligase Ligand moieties, which function to recruit targeted proteins to E3 ubiquitin ligase for degradation.

BACKGROUND OF THE INVENTION

Proteolysis-targeting chimera (PROTAC) is a novel strategy for selective knockdown of target proteins by small molecules (Sakamoto KM et al., Proc Natl Acad Sci 2001, 98: 8554–9.; Sakamoto K.M. et al., Methods Enzymol. 2005; 399: 833‐847. ) . PROTAC utilizes the ubiquitin‐protease system to target a specific protein and induce its degradation in the cell (Zhou P. et al., Mol Cell. 2000; 6 (3) : 751‐756; Neklesa T.K. et al., Pharmacol Ther. 2017; 174: 138‐144; Lu M. et al., Eur J Med Chem. 2018; 146: 251‐259; ) . The normal physiological function of the ubiquitin-protease system is responsible for clearing denatured, mutated, or harmful proteins in cells. The normal physiological function of the ubiquitin-protease system is responsible for clearing denatured, mutated, or harmful proteins in cells. The ubiquitin-proteasome system (UPS) , also known as the ubiquitin-proteasome pathway (UPP) , is a common posttranslational regulation mechanism that is responsible for protein degradation in normal and pathological states (Ardley H. et al., Essays Biochem. 2005, 41, 15-30; Komander D. et al., Biochem. 2012, 81, 203-229; Grice G.L. et al., Cell Rep. 2015, 12, 545-553; Swatek K.N. et al., Cell Res. 2016, 26, 399-422) . Ubiquitin, which is highly conserved in eukaryotic cells, is a modifier molecule, composed of 76 amino acids, that covalently binds to and labels target substrates via a cascade of enzymatic reactions involving E1, E2, and E3 enzymes. Subsequently, the modified substrate is recognized by the 26S proteasome complex for ubiquitination-mediated degradation. So far, two E1 enzymes have been discovered, which are termed UBA1 and UBA6. On the other hand, there are about 40 E2 enzymes and more than 600 E3 enzymes that offer the functional diversity to govern the activity of many downstream protein substrates. However, only a limited number of E3 ubiquitin ligases have been successfully hijacked for use by small molecule PROTAC technology: the Von Hippel-Lindau disease tumor suppressor protein (VHL) , the Mouse Double Minute 2 homologue (MDM2) , the Cellular Inhibitor of Apoptosis (cIAP) , and cereblon (Philipp O. et al., Chem. Biol. 2017, 12, 2570-2578) . Bifunctional compounds composed of a target protein-binding moiety and an E3 ubiquitin ligase-binding moiety have been shown to induce proteasome-mediated degradation of selected proteins. These drug-like molecules offer the possibility of temporal control over protein expression and could be useful as biochemical reagents for the treatment of diseases. In recent years, this newly developed method has been widely used in antitumor studies (Lu J. et al., Chem Biol. 2015; 22 (6) : 755‐763; Ottis P. et al., Chem Biol. 2017; 12 (4) : 892‐898.; Crews C.M. et al., J Med Chem. 2018; 61 (2) : 403‐404; Neklesa T.K. et al., Pharmacol Ther. 2017, 174: 138‐144.; Cermakova K. et al., Molecules, 2018.23 (8) .; An S. et al., EBioMedicine, 2018.; Lebraud H. et al., Essays Biochem. 2017; 61 (5) : 517‐527.; Sun Y.H. et al., Cell Res. 2018; 28: 779–81; Toure M. et al., Angew Chem Int Ed Engl. 2016; 55 (6) : 1966‐1973; Yonghui Sun et al., Leukemia, volume 33, pages2105–2110 (2019) ; Shaodong Liu et al., Medicinal Chemistry Research, volume 29, pages802–808 (2020) ; and has been disclosed or discussed in patent publications, e.g., US20160045607, US20170008904, US20180050021, US20180072711, WO2002020740, WO2014108452, WO2016146985, WO2016149668, WO2016149989, WO2016197032, WO2016197114, WO2017011590, WO2017030814, WO2017079267, WO2017182418, WO2017197036, WO2017197046, WO2017197051, WO2017197056, WO2017201449, WO2017211924, WO2018033556, and WO2018071606.

Bruton’s tyrosine kinase (Btk) belongs to the Tec tyrosine kinase family (Vetrie et al., Nature 361: 226-233, 1993; Bradshaw, Cell Signal. 22: 1175-84, 2010) . Btk is primarily expressed in most hematopoietic cells such as B cells, mast cells and macrophages (Smith et al., J. Immunol. 152: 557-565, 1994) and is localized in bone marrow, spleen and lymph node tissue. Btk plays an important role in B-cell receptor (BCR) and FcR signaling pathways, which involve in B-cell development, differentiation (Khan, Immunol. Res. 23: 147, 2001) . Btk is activated by upstream Src-family kinases. Once activated, Btk, in turn, phosphorylates PLC-gamma, leading to effects on B-cell function and survival (Humphries et al., J. Biol. Chem. 279: 37651, 2004) . These signaling pathways must be precisely regulated. Mutations in the gene encoding Btk cause an inherited B-cell specific immunodeficiency disease in humans, known as X-linked agammaglobulinemia (XLA) (Conley et al., Annu. Rev. Immunol. 27: 199-227, 2009) . Aberrant BCR-mediated signaling may result in dysregulated B-cell activation leading to a number of autoimmune and inflammatory diseases. Preclinical studies show that Btk deficient mice are resistant to developing collagen-induced arthritis. Moreover, clinical studies of Rituxan, a CD20 antibody to deplete mature B-cells, reveal the key role of B-cells in a number of inflammatory diseases such as rheumatoid arthritis, systemic lupus erythematosus and multiple sclerosis (Gurcan et al., Int. Immunopharmacol. 9: 10-25, 2009) . Therefore, Btk inhibitors can be used to treat autoimmune and/or inflammatory diseases.

Inhibition of BTK has been shown to affect cancer development (B cell malignancies) and cell viability, and improve autoimmune diseases (e.g., rheumatoid arthritis and lupus) . Inhibition of BTK has also been reported via alternative strategies, such as through degradation of BTK (Alexandru D. et al., Biochemistry 2018, 57, 26, 3564-3575; Adelajda Z. et al., PNAS 2018 115 (31) ; Dennis D., et al., Blood, 2019, 133: 952-961; Yonghui S. et al., Cell Research, 2018, 28, 779-781; Yonghui S. et al., Leukemia, 2019, Degradation of Bruton’s tyrosine kinase mutants by PROTACs for the potential treatment of ibrutinib-resistant non-Hodgkin lymphomas) and has been disclosed or discussed in patent publications, e.g. US20190276459, WO2019186343, WO2019186358, WO2019148150, WO2019177902, and WO2019127008.

There is a need of new BTK inhibitors or degraders which are more potent than known inhibitors of BTK and inhibit BTK via alternative strategies, such as through degradation of BTK, as well as the process preparing the same. The present application addresses the need and provides a process for preparing PROTAC BTK Degraders.

SUMMARY OF THE INVENTION

One objective of the present invention is to provide a process for preparing of proteolysis targeting chimera (PROTAC) compound by conjugating a BTK inhibitor with an E3 ligase ligand, which function to recruit targeted proteins to E3 ubiquitin ligase for degradation, In particular, the present disclosure provides a preparation method of PROTAC compounds with the Formula (I) .

Aspect 1: A process for preparing the compound of Formula (I) :

wherein: A ₁ and A ₂ each are independently selected from CH and N;

Step 1: A solution of Compound I-1 in 1, 4-dioxane and H ₂O and I-2 are reacted in the presence of K ₂CO ₃ and Pd (dppf) Cl ₂. CH ₂Cl ₂ to form give I-3;

Step 2: A solution of compound I-3 in 1, 4-dioxane and H ₂O and I-4 are reacted in the presence of K ₂CO ₃ and Pd (dppf) Cl ₂. CH ₂Cl ₂ under N ₂ to give I-5;

Step 3: A solution of compound I-5 in THF is then deprotected in the presence of NaOH in MeOH to give I-6; and

Step 4: A solution of compound I-6 in DCM/EtOH and I-7 are reacted in the presence of HOAc and NaOAc to give the compound of formula (I) .

More specifically, the steps are as follows:

Step 1: to a solution of compound I-1 in 1, 4-dioxane and H ₂O was added I-2, K ₂CO ₃ and Pd (dppf) Cl ₂. CH ₂Cl ₂; the mixture was stirred then concentrated, dissolved in H ₂O and extracted with EtOAc; the organic phase was concentrated and purified by flash chromatography to give I-3;

Step 2: to a solution of compound I-3 in 1, 4-dioxane and H ₂O was added I-4, K ₂CO ₃ and Pd (dppf) Cl ₂. CH ₂Cl ₂; the mixture was stirred under N ₂; the solvent was evaporated, diluted with H ₂O and extracted with EtOAc; the organic phase was combined, concentrated and purified by flash chromatography to give I-5;

Step 3: To a solution of compound I-5 in THF was added NaOH in MeOH; the mixture was stirred for 0.5～2 hour, concentrated and slurried with H ₂O; the solid was filtered and washed with H ₂O; the filter cake was dried under reduced pressure, then the solid was transferred into a flask and HCl/MeOH was added; after stirring for 1-6 hours, the solvent was evaporated, slurried with MeOH, filtered and the filter cake was washed with MeOH and MTBE (Methyl tert-butyl ether) ; the filter cake was dried and used for next step directly;

Step 4: To a solution of compound I-6 in DCM/EtOH was added I-7, HOAc and NaOAc; after stirring for 5-120 min, NaBH (OAc) ₃ was added; the mixture was stirred for 1-6 hours; the solvent was evaporated, diluted with H ₂O and extracted with DCM/iPrOH; the organic phase was combined, concentrated and purified by pre-TLC to give the compound of formula (I) .

Aspect 2: The process according to Aspect 1, wherein A ₁ is CH and A ₂ is CH.

Aspect 3: The process according to Aspect 1, wherein A ₁ is N and A ₂ is CH.

Aspect 4: The process according to Aspect 1, wherein A ₁ is N and A ₂ is N.

Aspect 5: The process according to Aspect 1, the compound of Formula (I) is selected from

Aspect 6: The process according to Aspect 1, wherein the reaction in Step 1 is conducted at 80 ℃.

Aspect 7: The process according to Aspect 1, wherein the reaction in Step 2 is conducted at 100 ℃ under N ₂ for 18 hours.

Aspect 8: The process according to Aspect 1, wherein in Step 3, the concentration of NaOH in MeOH is 4% (w/v) .

EXAMPLES

The examples below are intended to be purely exemplary and should not be considered to be limiting in any way. Efforts have been made to ensure accuracy with respect to numbers used (for example, amounts, temperature, etc. ) , but some experimental errors and deviations should be accounted for. Unless indicated otherwise, temperature is in degrees Centigrade. Reagents were purchased from commercial suppliers such as Sigma-Aldrich, Alfa Aesar, or TCI, and were used without further purification unless indicated otherwise. Unless indicated otherwise, the reactions set forth below were performed under a positive pressure of nitrogen or argon or with a drying tube in anhydrous solvents; the reaction flasks were fitted with rubber septa for the introduction of substrates and reagents via syringe; and glassware was oven dried and/or heat dried.

¹H NMR spectra were recorded on a Agilent instrument operating at 400 MHz. ¹HNMR spectra were obtained using CDCl ₃, CD ₂Cl ₂, CD ₃OD, D ₂O, d ₆-DMSO, d ₆-acetone or (CD ₃) ₂CO as solvent and tetramethylsilane (0.00 ppm) or residual solvent (CDCl ₃: 7.25 ppm; CD ₃OD: 3.31 ppm; D ₂O: 4.79 ppm; d ₆-DMSO: 2.50 ppm; d ₆ -acetone: 2.05; (CD ₃) ₃CO: 2.05) as the reference standard. When peak multiplicities are reported, the following abbreviations are used: s (singlet) , d (doublet) , t (triplet) , q (quartet) , qn (quintuplet) , sx (sextuplet) , m (multiplet) , br (broadened) , dd (doublet of doublets) , dt (doublet of triplets) . Coupling constants, when given, are reported in Hertz (Hz) .

LCMS-1: LC-MS spectrometer (Agilent 1260 Infinity) Detector: MWD (190-400 nm) , Mass detector: 6120 SQ Mobile phase: A: water with 0.1%Formic acid, B: acetonitrile with 0.1%Formic acid Column: Poroshell 120 EC-C18, 4.6x50 mm, 2.7pm Gradient method: Flow: 1.8 mL/min Time (min) A (%) B (%)

LCMS, LCMS-3: LC-MS spectrometer (Agilent 1260 Infinity II) Detector: MWD (190-400 nm) , Mass detector: G6125C SQ Mobile phase: A: water with 0.1%Formic acid, B: acetonitrile with 0.1%Formic acid Column: Poroshell 120 EC-C18, 4.6x50 mm, 2.7pm Gradient method: Flow: 1.8 mL/min Time (min) A (%) B (%)

LCMS-2: LC-MS spectrometer (Agilent 1290 Infinity II) Detector: MWD (190-400 nm) , Mass detector: G6125C SQ Mobile phase: A: water with 0.1%Formic acid, B: acetonitrile with 0.1% Formic acid Column: Poroshell 120 EC-C18, 4.6x50 mm, 2.7pm Gradient method: Flow: 1.2 mL/min Time (min) A (%) B (%)

Preparative HPLC was conducted on a column (150 x 21.2 mm ID, 5 pm, Gemini NXC 18) at a flow rate of 20 ml/min, injection volume 2 ml, at room temperature and UV Detection at 214 nm and 254 nm.

In the following examples, the abbreviations below are used:

Example 1: 1- (4- (4- ( (4- (4- (4- (3- (7, 7-dimethyl-1-oxo-1, 3, 4, 6, 7, 8-hexahydro-2H-cyclopenta [4, 5] pyrrolo [1, 2-a] pyrazin-2-yl) -2-methylphenyl) -7H-pyrrolo [2, 3-d] pyrimidin-6-yl) phenyl) piperazin-1-yl) methyl) piperidin-1-yl) phenyl) dihydropyrimidine-2, 4 (1H, 3H) -dione

Step 1: tert-butyl 4- (4- (4-chloro-7- (phenylsulfonyl) -7H-pyrrolo [2, 3-d] pyrimidin-6- yl) phenyl) piperazine-1-carboxylate

To a solution of 4-chloro-6-iodo-7- (phenylsulfonyl) -7H-pyrrolo [2, 3-d] pyrimidine (2.5 g, 14.4 mmol) in dioxane (35 mL) and H ₂O (7 mL) was added tert-butyl 4- (4- (4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) phenyl) piperazine-1-carboxylate (1.6 g, 4.2 mmol) , K ₂CO ₃ (1.6 g, 12 mmol) and Pd (dppf) Cl ₂. CH ₂Cl ₂ (0.3 g, 0.4 mmol) . The mixture was stirred at 80 ℃ for 6 hours. The mixture was concentrated, dissolved in H ₂O (30 mL) and extracted with EtOAc (30 mL*2) . The organic phase was concentrated and purified by flash chromatography with PE/EA (100: 1 to 7: 3) to give the product (1.9 g, 86.4%) .

Step 2: tert-butyl 4- (4- (4- (3- (7, 7-dimethyl-1-oxo-1, 3, 4, 6, 7, 8-hexahydro-2H- cyclopenta [4, 5] pyrrolo [1, 2-a] pyrazin-2-yl) -2-methylphenyl) -7- (phenylsulfonyl) -7H-pyrrolo [2, 3- d] pyrimidin-6-yl) phenyl) piperazine-1-carboxylate

To a solution of tert-butyl 4- (4- (4-chloro-7- (phenylsulfonyl) -7H-pyrrolo [2, 3-d] pyrimidin-6-yl) phenyl) piperazine-1-carboxylate (1.9 g, 3.4 mmol) in dioxane (30 mL) and H ₂O (6 mL) was added 7, 7-dimethyl-2- (2-methyl-3- (4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) phenyl) -3, 4, 7, 8-tetrahydro-2H-cyclopenta [4, 5] pyrrolo [1, 2-a] pyrazin-1 (6H) -one (1.4 g, 3.4 mmol) , K ₂CO ₃ (1.4 g, 10.0 mmol) and Pd (dppf) Cl ₂. CH ₂Cl ₂ (0.3 g, 0.3 mmol) . The mixture was stirred at 100 ℃ under N ₂ for 18 hours. The solvent was evaporated, added H ₂O (30 mL) and extracted with EtOAc (50 mL*2) . The organic phase was combined, concentrated and purified by flash chromatography with PE/EA (100: 1 to 1: 100) to give the product (1.1g, crude) .

Step 3: 7, 7-dimethyl-2- (2-methyl-3- (6- (4- (piperazin-1-yl) phenyl) -7H-pyrrolo [2, 3-d] pyrimidin-4- yl) phenyl) -3, 4, 7, 8-tetrahydro-2H-cyclopenta [4, 5] pyrrolo [1, 2-a] pyrazin-1 (6H) -one hydrochloride

To a solution of tert-butyl 4- (4- (4- (3- (7, 7-dimethyl-1-oxo-1, 3, 4, 6, 7, 8-hexahydro-2H-cyclopenta [4, 5] pyrrolo [1, 2-a] pyrazin-2-yl) -2-methylphenyl) -7- (phenylsulfonyl) -7H-pyrrolo [2, 3- d] pyrimidin-6-yl) phenyl) piperazine-1-carboxylate (1.1 g, 1.4 mmol) in THF (10 mL) was added NaOH in MeOH (4%, 3 mL) . The mixture was stirred at 20-30℃ for 1 hour, concentrated and slurried with H ₂O (30 mL) . The solid was filtered and washed with H ₂O (30 mL) . The filter cake was dried under reduced pressure. The solid was transferred into a flask and added HCl/MeOH (4 N, 30 mL) . The mixture was stirred at 20-30℃ for 3 hours. The solvent was evaporated and slurried with MeOH, filtered and the filter cake was washed with MeOH (30 mL) and MTBE (20 mL) . The filter cake was dried and used for next step directly. ¹H NMR (400 MHz, DMSO) δ _H 12.54 (s, 1H) , 8.77 (s, 1H) , 7.82 (d, J = 8.4 Hz, 2H) , 7.57-7.39 (m, 3H) , 6.99 (d, J = 8.4 Hz, 2H) , 6.62 (s, 1H) , 6.50 (s, 1H) , 4.18 (br, 3H) , 3.84 (br, 1H) , 3.15 (s, 4H) , 2.84 (s, 4H) , 2.56 (s, 2H) , 2.50 (br, 2H) , 2.41 (s, 2H) , 2.11 (s, 3H) , 1.21 (s, 6H) . [M+H] ⁺ = 572.3.

Step 4: 1- (4- (4- ( (4- (4- (4- (3- (7, 7-dimethyl-1-oxo-1, 3, 4, 6, 7, 8-hexahydro-2H- cyclopenta [4, 5] pyrrolo [1, 2-a] pyrazin-2-yl) -2-methylphenyl) -7H-pyrrolo [2, 3-d] pyrimidin-6- yl) phenyl) piperazin-1-yl) methyl) piperidin-1-yl) phenyl) dihydropyrimidine-2, 4 (1H, 3H) -dione

To a solution of 7, 7-dimethyl-2- (2-methyl-3- (6- (4- (piperazin-1-yl) phenyl) -7H-pyrrolo [2, 3-d] pyrimidin-4-yl) phenyl) -3, 4, 7, 8-tetrahydro-2H-cyclopenta [4, 5] pyrrolo [1, 2-a] pyrazin-1 (6H) -one (114 mg, 0.2 mmol) in DCM/EtOH (5: 1, 30 mL) was added 1- (4- (4-oxotetrahydropyrimidin-1 (2H) -yl) phenyl) piperidine-4-carbaldehyde (60 mg, 0.2 mmol) HOAc (1 drop) and NaOAc (32.8 mg, 0.4 mmol) . After stirring at 20-30 ℃ for 60 min, NaBH (OAc) ₃ (127 mg, 0.6 mmol) was added. The mixture was stirred at 20-30 ℃ for 3 hours. The solvent was evaporated, added H ₂O (30 mL) and extracted with DCM/iPrOH (10: 1, 30 mL*3) . The organic phase was combined, concentrated and purified by pre-TLC with DCM/MeOH (10: 1) to give the product (53 mg, 31%) . ¹H NMR (400 MHz, DMSO) δ _H 12.55 (s, 1H) , 10.27 (s, 1H) , 8.77 (s, 1H) , 7.82 (d, J = 8.4 Hz, 2H) , 7.51-7.36 (m, 3H) , 7.13 (d, J = 8.8 Hz, 2H) , 7.01 (d, J = 8.4 Hz, 2H) , 6.93 (d, J = 8.8 Hz, 2H) , 6.63 (s, 1H) , 6.50 (s, 1H) , 4.18 (br, 3H) , 3.84 (br, 1H) , 3.74-3.67 (m, 4H) , 3.24 (br, 4H) , 2.69-2.64 (m, 4H) , 2.56 (s, 2H) , 2.55-2.50 (m, 3H) , 2.41 (s, 2H) , 2.30-2.06 (m, 5H) , 1.87-1.65 (m, 3H) , 1.21 (s, 9H) ; [M+H] ⁺ =857.5.

Example 2: 1- (4- (4- ( (4- (4- (4- (3- (7, 7-dimethyl-1-oxo-1, 3, 4, 6, 7, 8-hexahydro-2H-cyclopenta [4, 5] pyrrolo [1, 2-a] pyrazin-2-yl) -2-methylphenyl) -7H-pyrrolo [2, 3-d] pyrimidin-6-yl) phenyl) piperidin-1-yl) methyl) piperidin-1-yl) phenyl) dihydropyrimidine-2, 4 (1H, 3H) -dione

The titled compound was synthesized in the procedures similar to Example 1. ¹H NMR (400 MHz, DMSO) δ _H 12.71 (s, 1H) , 10.28 (s, 1H) , 8.83 (s, 1H) , 8.20 (s, 1H) , 7.90 (d, J = 6.9 Hz, 2H) , 7.47 (d, J = 17.7 Hz, 3H) , 7.36 (d, J = 6.9 Hz, 2H) , 7.13 (d, J = 7.5 Hz, 2H) , 6.93 (d, J = 7.6 Hz, 2H) , 6.78 (s, 1H) , 6.51 (s, 1H) , 4.19 (s, 3H) , 3.85 (s, 1H) , 3.69 (d, J = 6.7 Hz, 4H) , 2.97 (d, J = 8.6 Hz, 3H) , 2.72-2.62 (m, 5H) , 2.17 (d, J = 19.8 Hz, 6H) , 1.87-2.10 (m, 3H) , 1.85-1.63 (m, 8H) , 1.15-1.25 (m, 9H) ; [M+H] ⁺ = 856.5

Example 3: 1- (4- (4- ( (4- (5- (4- (3- (7, 7-dimethyl-1-oxo-1, 3, 4, 6, 7, 8-hexahydro-2H-cyclopenta [4, 5] pyrrolo [1, 2-a] pyrazin-2-yl) -2-methylphenyl) -7H-pyrrolo [2, 3-d] pyrimidin-6-yl) pyridin-2-yl) piperazin-1-yl) methyl) piperidin-1-yl) phenyl) dihydropyrimidine-2, 4 (1H, 3H) -dione

The titled compound was synthesized in the procedures similar to Example 1. ¹H NMR (400 MHz, DMSO) δ _H 12.62 (s, 1H) , 10.27 (s, 1H) , 8.79 (s, 1H) , 8.73 (s, 1H) , 8.10 (d, J = 9.0 Hz, 1H) , 7.46 (d, J = 18.9 Hz, 3H) , 7.13 (d, J = 8.5 Hz, 2H) , 6.93 (d, J = 7.9 Hz, 3H) , 6.69 (s, 1H) , 6.50 (s, 1H) , 4.17 (d, J = 8.7 Hz, 3H) , 3.85 (s, 1H) , 3.68 (d, J = 6.8 Hz, 4H) , 3.58 (s, 4H) , 2.67 (dd, J = 13.8, 9.1 Hz, 4H) , 2.56 (s, 2H) , 2.46 (s, 4H) , 2.41 (s, 2H) , 2.22 (d, J = 6.3 Hz, 2H) , 2.14 (s, 3H) , 1.82 (d, J = 12.3 Hz, 2H) , 1.73 (s, 1H) , 1.21 (s, 8H) ; [M+H] ⁺ = 858.4.

Cell Degradation

Cell treatment

TMD-8 cells are seeded at 20000 cells/well at a volume of 15μl/well in cell culture medium [RPMI1640 (Gibco, phenol red free, Cat#11835-030) , 10%heat-inactive FBS, 1%PS (Gibco, Cat#10378) ] in Corning 96 well plate (Cat#3799) . TMD-8 cells are treated with compounds diluted in 0.2%DMSO, dilution is done according to the following protocol: (1) make 500× stock solution in DMSO from 1mM by 6-fold dilution, total 8 doses were included; (2) make 2× solution in cell culture medium by transferring 0.5μl 500× stock solution into 125μl medium; (3) 15μl of 2×solution is added to cells and incubate for 6h.

HTFR assay

After 6h treatment, add 10μl 4xlysis buffer to each well ; seal the plate and incubate 30min at room temperature on a plate shaker; Once the cells are lysed, 16 μL of cell lysate are transferred to a PE 384-well HTRF detection plate; 4 μL of pre-mixed HTRF antibodies are added to each well ; Cover the plate with a plate sealer, spin 1000 rpm for 1 min, Incubate overnight at room temperature; Read on BMG PheraStar with HTRF protocol (337nm-665nm-620nm) .

The inhibition (degradation) percentage of the compound was calculated by the following equation: Inhibition percentage of Compound = 100-100 × (Signal-low control) / (High control-low control) , wherein signal = each test compound group

Low control = only lysis buffer without cells, indicating that BTK is completely degraded;

High control = Cell group with added DMSO and without compound, indicating microplate readings without BTK degradation;

Dmax is the maximum percentage of inhibition (degradation) .

The IC ₅₀ (DC ₅₀) value of a compound can be obtained by fitting the following equation

Y = Bottom + (TOP-Bottom) / (1 + ( (IC ₅₀ /X) ^ hillslope) )

Wherein, X and Y are known values, and IC ₅₀, Hillslope, Top and Bottom are the parameters obtained by fitting with software. Y is the inhibition percentage (calculated from the equation) , X is the concentration of the compound; IC ₅₀ is the concentration of the compound when the 50%inhibition is reached. The smaller the IC ₅₀ value is, the stronger the inhibitory ability of the compound is. Vice versa, the higher the IC ₅₀ value is, the weaker the ability the inhibitory ability of the compound is; Hillslope represents the slope of the fitted curve, generally around 1 *; Bottom represents the minimum value of the curve obtained by data fitting, which is generally 0%± 20%; Top represents the maximum value of the curve obtained by data fitting, which is generally 100%±20%. The experimental data were fitted by calculating and analyzing with Dotmatics data analysis software.

Table 1. Degradation result for Example 1 to Example 3

The foregoing examples and description of certain embodiments should be taken as illustrating, rather than as limiting the present invention as defined by the claims. As will be readily appreciated, numerous variations and combinations of the features set forth above can be utilized without departing from the present invention as set forth in the claims. All such variations are intended to be included within the scope of the present invention. All references cited are incorporated herein by reference in their entireties.

It is to be understood that, if any prior art publication is referred to herein, such reference does not constitute an admission that the publication forms a part of the common general knowledge in the art in any country.

Claims

A process for preparing the compound of Formula (I) :

wherein: A ₁ and A ₂ each are independently selected from CH and N;

Step 1: A solution of Compound I-1 in 1, 4-dioxane and H ₂O and I-2 are reacted in the presence of K ₂CO ₃ and Pd (dppf) Cl ₂. CH ₂Cl ₂ to form give I-3;

Step 2: A solution of compound I-3 in 1, 4-dioxane and H ₂O and I-4 are reacted in the presence of K ₂CO ₃ and Pd (dppf) Cl ₂. CH ₂Cl ₂ under N ₂ to give I-5;

Step 3: A solution of compound I-5 in THF is then deprotected in the presence of NaOH in MeOH to give I-6; and

Step 4: A solution of compound I-6 in DCM/EtOH and I-7 are reacted in the presence of HOAc and NaOAc to give the compound of formula (I) .
The process according to Claim 1, wherein A ₁ is CH and A ₂ is CH.
The process according to Claim 1, wherein A ₁ is N and A ₂ is CH.
The process according to Claim 1, wherein A ₁ is N and A ₂ is N.
The process according to Claim 1, the compound of Formula (I) is selected from
The process according to Claim 1, wherein the reaction in Step 1 is conducted at 80 ℃.
The process according to Claim 1, wherein the reaction in Step 2 is conducted at 100 ℃ under N ₂ for 18 hours.
The process according to Claim 1, wherein in Step 3, the concentration of NaOH in MeOH is 4% (w/v) .