An imidazo[1,2-a]pyridine-based IRAK4 degrading agent and its application

Abstract

This invention provides an imidazo[1,2-a]pyridine IRAK4 degrading agent and its application, wherein the imidazo[1,2-a]pyridine IRAK4 degrading agent has the structure shown in Formula I. The imidazo[1,2-a]pyridine IRAK4 degrading agent exhibits significant degradation activity against intracellular IRAK4 and can be used to prepare drugs for the treatment and / or prevention of IRAK4-mediated diseases or conditions.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical synthesis technology and relates to an imidazo[1,2-a]pyridine-based IRAK4 degrading agent and its application. Background Technology

[0002] Interleukin-1 receptor kinase 4 (IRAK4) is an isoenzyme of the IRAK protein family of intracellular serine-threonine kinases. It is a key node in the downstream signaling pathways of Toll-like receptors (TLRs) and interleukin-1 receptors (IL-1Rs) and plays an important role in the immune system. Its overactivation, or abnormal activation due to mutations, is involved in the occurrence and development of inflammatory diseases and tumors.

[0003] TLR and IL-1R share the conserved TLR / IL-1R (TIR) ​​domain, recruiting the adaptor protein MyD88 within the cell. MyD88 then recruits IRAK4, forming a myddosome. Within the myddosome, IRAK4 is activated via trans-autophosphorylation, subsequently activating a series of downstream transcription factors, such as NF-κB, CREB, AP-1, and IRF, promoting the secretion of pro-inflammatory cytokines and the proliferation and differentiation of immune cells. Therefore, IRAK4 plays a crucial role in the pathogenesis and progression of inflammatory diseases.

[0004] Dysregulation of the TLR / IL-1R signaling pathway is also closely related to the occurrence and progression of cancer. Mutations in proteins in the TLR / IL-1R pathway may lead to overactive signaling and increased NF-κB activity, thus promoting cancer.

[0005] IRAK4 mediates the downstream signaling pathway of IL-1R / TLR and participates in immune surveillance through two pathways: on the one hand, IRAK4 has kinase activity and can phosphorylate downstream proteins IRF5 / 7; on the other hand, IRAK4 acts as a scaffold structure, responsible for the formation of the myddosome protein complex. Although there are many small molecule kinase inhibitors of IRAK4 in existing technologies, they can only inhibit the activity of the kinase itself, showing moderate efficacy, and cannot completely block the formation of the complex to completely block the generation of inflammatory response signals.

[0006] Targeted protein degradation (TPD) is an emerging therapeutic approach that has attracted significant attention due to its ability to modulate proteins that are difficult to target with traditional small molecules. Protein degradation-targeting chimeras (PROTACs) utilize the ubiquitin-proteasome system (UPS), a natural intracellular protein degradation system, to achieve targeted degradation of a point of interest (POI). PROTACs are heterobifunctional small molecules; one end targets the POI, while the other end recruits an E3 ubiquitin ligase to form a ternary complex. The POI is then tagged with ubiquitin, leading to its recognition and degradation by the proteasome.

[0007] Compared with traditional small molecule drugs, PROTAC molecules have the following advantages: (1) They have catalytic degradation function. Unlike each small molecule inhibitor that can only act on one protein molecule, each PROTAC molecule can degrade many protein molecules, so a very low dose can have a good therapeutic effect; (2) They work by degrading the target protein, thus overcoming the problem of small molecule drug resistance caused by increased target protein expression, and can also block the non-enzymatic function of the target. Therefore, taking advantage of the fact that IRAK4 has both kinase activity and scaffold function, PROTAC technology can degrade the IRAK4 protein, thereby blocking kinase activity and destroying the target protein scaffold function. Compared with conventional kinase inhibitors, this achieves complete inhibition of the pathway, resulting in better anti-inflammatory response or anti-tumor activity. Therefore, further development of such molecules is of great significance in this field. Summary of the Invention

[0008] To address the shortcomings of existing technologies, the present invention aims to provide an imidazo[1,2-a]pyridine-based IRAK4 degrading agent and its application.

[0009] To achieve this objective, the present invention adopts the following technical solution:

[0010] On one hand, the present invention provides an imidazo[1,2-a]pyridine-based IRAK4 degrading agent, wherein the imidazo[1,2-a]pyridine-based IRAK4 degrading agent has the structure shown in Formula I:

[0011]

[0012] Wherein: ring A is a 6-10 aryl or a 5-10 heteroaryl;

[0013] Ring B is a substituted or unsubstituted 4-10 membered cycloalkyl or a substituted or unsubstituted 4-10 membered heterocycloalkyl;

[0014] The ring C is a substituted or unsubstituted 4-10 membered cycloalkyl or a substituted or unsubstituted 4-10 membered heterocycloalkyl;

[0015] X is CH2 or C=O;

[0016] R a Each is independently selected from halogen, cyano, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C4 alkoxy, di(C1-C4 alkyl)amino, 5-10 membered cycloalkyl, 5-10 membered heterocycloalkyl;

[0017] m is an integer between 0 and 5;

[0018] R bSelected from C1-C4 alkoxy, C3-C6 cycloalkyloxy, or hydroxydi(C1-C4 alkyl)methyl;

[0019] Rc is hydrogen, C1-C4 alkyl, C1-C4 alkoxy, or halogen;

[0020] L stands for (CH2) n (CH2) n CO, 4-8 membered cycloalkyl or 4-8 membered heterocycloalkyl, where n is an integer from 0 to 5 (e.g., 0, 1, 2, 3, 4 or 5).

[0021] In this invention, the substituents in the substituted groups as described above are selected from halogens, oxo, cyano, amino, hydroxyl, C1-C6 alkyl or -O-(C1-C6 alkyl).

[0022] Preferably, ring A is The wavy line represents the connection site of the functional group.

[0023] Preferably, the ring B is The wavy line represents the connection site of the functional group.

[0024] Preferably, the ring C is The wavy line represents the connection site of the functional group.

[0025] Preferably, R a Each is independently selected from fluorine, chlorine, cyano, trifluoromethyl, trifluoromethoxy, The wavy line represents the connection site of the functional group.

[0026] Preferably, R b Selected from methoxy, ethoxy, isopropoxy, cyclopropyloxy or Any of the following, the wavy line represents the connection site of the group.

[0027] Preferably, R c It is selected from hydrogen, fluorine, chlorine or bromine.

[0028] Preferably, L is -CH2-, -CH2CH2-, -CH2CH2CH2-, -COCH2-, -COCH2CH2-, -COCH2CH2-, or

[0029] Preferably, the imidazo[1,2-a]pyridine IRAK4 degrading agent is selected from any one of the following compounds:

[0030]

[0031]

[0032]

[0033]

[0034]

[0035] On the other hand, the present invention provides pharmaceutically acceptable salts of the imidazo[1,2-a]pyridine IRAK4 degraders as described above.

[0036] Preferably, the pharmaceutically acceptable salts include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, phosphates, monohydrophosphates, dihydrophosphates, metaphosphates, pyrophosphates, hydrochlorides, hydrobromates, hydroiodates, acetates, propionates, decanoates, caprylates, acrylates, formates, isobutyrates, hexanoates, heptadates, propynylates, oxalates, malonates, succinates, caprylates, sebacic acid, fumarates, maleates, and butyrates. Any one of the following: -1,4-diacidate, hexyn-1,6-diacidate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, γ-hydroxybutyrate, glycolate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, or mandelate.

[0037] On the other hand, the present invention provides stereoisomers, geometric isomers or tautomers of the imidazo[1,2-a]pyridine IRAK4 degrading agents as described above.

[0038] On the other hand, the present invention provides a pharmaceutical composition comprising an imidazo[1,2-a]pyridine IRAK4 degrader as described above, its stereoisomer, geometric isomer, tautomer, or pharmaceutically acceptable salt.

[0039] On the other hand, the present invention provides the use of the imidazo[1,2-a]pyridine IRAK4 degrading agents, their stereoisomers, geometric isomers, tautomers, or pharmaceutically acceptable salts, or the pharmaceutical compositions thereof, as described above, in the preparation of medicaments for treating diseases or conditions mediated by IRAK4.

[0040] Preferably, the disease includes inflammatory diseases, autoimmune diseases, or tumors.

[0041] Preferably, the inflammatory disease is selected from Crohn's disease, ulcerative colitis, asthma, graft-versus-host disease, chronic obstructive pulmonary disease, atopic dermatitis, gout, gouty arthritis, conjunctivitis, hepatitis, chronic pulmonary inflammatory disease, thyroiditis, or interstitial cystitis.

[0042] Preferably, the autoimmune disease is selected from Graves' disease, rheumatoid arthritis, systemic lupus erythematosus, lupus nephritis, cutaneous lupus, psoriasis, psoriatic arthritis, cryptothermal protein-related periodic syndrome, TNF receptor-related periodic syndrome, multiple sclerosis, allergic rhinitis, scleroderma, dermatomyositis, vasculitis, or nephritis.

[0043] Preferably, the tumor is selected from lung cancer, breast cancer, prostate cancer, pancreatic cancer, kidney cancer, liver cancer, gastrointestinal cancer, cervical cancer, endometrial cancer, testicular cancer, urogenital tract cancer, colorectal cancer, laryngeal cancer, skin cancer, bone cancer, head and neck tumors, sarcoma, brain tumor, glioblastoma, melanoma, multiple myeloma, lymphoma, or leukemia.

[0044] Compared with the prior art, the present invention has the following beneficial effects:

[0045] The compounds of this invention exhibit significant degradation ability against IRAK4 in THP1 cells, with some compounds showing a half-maximal concentration (DC) of [missing value]. 50 The degradation activity can reach the nanomolar level; Western blotting results also show that the compounds of this invention have strong degradation activity against IRAK4 protein in OCI-LY10 cells at concentrations of 100 nM and 1000 nM.

[0046] In in vitro and in vivo metabolic stability studies, the compounds of this invention exhibit high metabolic stability to liver microsomes of different species, and in in vivo pharmacokinetic studies in mice, they demonstrate favorable pharmacokinetic characteristics such as low clearance, high plasma exposure, and high oral bioavailability. Attached Figure Description

[0047] Figures 1-26 The figures show the Western blot results of the degradation of IRAK4 protein in OCI-LY10 cells by the compounds of Examples 1-26 at concentrations of 100 nM and 1000 nM. Detailed Implementation

[0048] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0049] In the following embodiments, unless otherwise noted in the structural formula or chemical name, molecules with a single chiral center exist as a racemic mixture. Unless otherwise noted in the structural formula or chemical name, molecules with two or more chiral centers exist as a racemic mixture of diastereomers. The single enantiomer / diastereomer can be obtained by methods known to those skilled in the art.

[0050] Preparation method

[0051] The compounds described in this invention can be synthesized according to the synthetic schemes described herein and / or techniques well known in the art. For example, the compounds provided by this invention can be prepared according to the following general synthetic methods.

[0052] General Synthesis Method 1

[0053]

[0054] Specifically, in general synthetic method 1, intermediate Int-a undergoes a substitution reaction with HOOC-L1-Y under basic conditions to obtain intermediate Int-b; alternatively, intermediate Int-a undergoes a substitution reaction with t-BuOOC-L1-Y under basic conditions, and the resulting product is then de-tert-butylated with trifluoroacetic acid or hydrochloric acid to obtain intermediate Int-b; intermediate Int-b then undergoes a condensation reaction with intermediate Int-c in the presence of a condensing agent and a base to obtain compound of formula I. The base is an inorganic or organic base, including but not limited to triethylamine, N,N-diisopropylethylamine, potassium carbonate, sodium carbonate, and sodium bicarbonate. Y is a halogen, preferably bromine or iodine. Ra, Rb, Rc, m, ring A, ring B, ring C, L, and X are as defined above.

[0055] General Synthesis Method 2:

[0056]

[0057] Specifically, in general synthetic method 2, intermediate Int-a undergoes a substitution reaction with (RdO)2CH-L1-Y under basic conditions to obtain intermediate Int-d; intermediate Int-d reacts with trifluoroacetic acid or hydrochloric acid to obtain intermediate Int-e; intermediate Int-e then undergoes a reductive amination reaction with intermediate Int-c to obtain compound of formula I. The base is an inorganic or organic base, including but not limited to triethylamine, N,N-diisopropylethylamine, potassium carbonate, sodium carbonate, and sodium bicarbonate. Y is a halogen, preferably bromine or iodine. The reducing agent for the reductive amination reaction includes but is not limited to Pd / C, sodium borohydride, sodium cyanoborohydride, borane, and sodium triacetoxyborohydride. Rd is methyl or ethyl. Ra, Rb, Rc, m, ring A, ring B, ring C, L, and X are as defined above.

[0058] General Synthesis Method 3:

[0059]

[0060] Specifically, in general synthetic method 3, intermediate Int-c undergoes a substitution reaction with (RdO)2CH-L1-Y under basic conditions to obtain intermediate Int-f; intermediate Int-f reacts with trifluoroacetic acid or hydrochloric acid to obtain intermediate Int-g; intermediate Int-g then undergoes a reductive amination reaction with intermediate Int-a to obtain compound of formula I. The base is an inorganic or organic base, including but not limited to triethylamine, N,N-diisopropylethylamine, potassium carbonate, sodium carbonate, and sodium bicarbonate. Y is a halogen, preferably bromine or iodine. The reducing agent for the reductive amination reaction includes but is not limited to Pd / C, sodium borohydride, sodium cyanoborohydride, borane, and sodium triacetoxyborohydride. Rd is methyl or ethyl. Ra, Rb, Rc, m, ring A, ring B, ring C, L, and X are as defined above.

[0061] General Synthesis Method 4:

[0062]

[0063] Specifically, in general synthetic method 4, intermediates Int-h and Int-i undergo a substitution reaction under basic conditions to obtain intermediate Int-f; intermediate Int-f reacts with trifluoroacetic acid or hydrochloric acid to obtain intermediate Int-g; intermediate Int-g then undergoes a reductive amination reaction with intermediate Int-a to obtain compound I. The base is an inorganic or organic base, including but not limited to triethylamine, N,N-diisopropylethylamine, potassium carbonate, sodium carbonate, and sodium bicarbonate. The reducing agent for the reductive amination reaction includes but is not limited to Pd / C, sodium borohydride, sodium cyanoborohydride, borane, and sodium triacetoxyborohydride. Rd is methyl or ethyl. Ra, Rb, Rc, m, ring A, ring B, ring C, L, and X are as defined above.

[0064] General Synthesis Method 5:

[0065]

[0066] Specifically, in general synthetic method 5, intermediates Int-h and Int-j undergo a substitution reaction under basic conditions to obtain intermediate Int-k; intermediate Int-k reacts with an oxidizing agent to obtain intermediate Int-g; intermediate Int-g then undergoes a reductive amination reaction with intermediate Int-a to obtain compound I. The base is an inorganic or organic base, including but not limited to triethylamine, N,N-diisopropylethylamine, potassium carbonate, sodium carbonate, and sodium bicarbonate. The reducing agent for the reductive amination reaction includes but is not limited to Pd / C, sodium borohydride, sodium cyanoborohydride, borane, and sodium triacetoxyborohydride. Ra, Rb, Rc, m, ring A, ring B, ring C, L, and X are as defined above.

[0067] General Synthesis Method 6:

[0068]

[0069] Specifically, in general synthetic method 6, intermediate Int-k reacts with methanesulfonyl chloride under alkaline conditions to obtain intermediate Int-l; intermediate Int-l then undergoes a substitution reaction with intermediate Int-a under alkaline conditions to obtain compound of formula I. The base is an inorganic or organic base, including but not limited to triethylamine, N,N-diisopropylethylamine, potassium carbonate, sodium carbonate, and sodium bicarbonate. Ra, Rb, Rc, m, ring A, ring B, ring C, L, and X are as defined above.

[0070] General Synthesis Method 7:

[0071]

[0072] Specifically, in general synthetic method 7, intermediate Int-a undergoes a reductive amination reaction with intermediate Int-m, or intermediate Int-a undergoes a substitution reaction with intermediate Int-n under basic conditions to obtain intermediate Int-o; intermediate Int-o reacts with trifluoroacetic acid or hydrochloric acid to obtain intermediate Int-p; intermediate Int-p then undergoes a reductive amination reaction with intermediate Int-q to obtain compound of formula I. The base is an inorganic or organic base, including but not limited to triethylamine, N,N-diisopropylethylamine, potassium carbonate, sodium carbonate, and sodium bicarbonate. The reducing agent for the reductive amination reaction includes but is not limited to Pd / C, sodium borohydride, sodium cyanoborohydride, borane, and sodium triacetoxyborohydride. Ra, Rb, Rc, m, ring A, ring B, ring C, L, and X are as defined above.

[0073] Preparation Examples

[0074] The compounds described in this invention can be synthesized according to one or more synthetic schemes described herein and / or techniques well known in the art. Those skilled in the art will recognize that the synthetic methods of some embodiments described in detail herein can be readily applied to the synthesis of other embodiments. In some embodiments, the compounds described herein can be prepared by appropriate combinations of synthetic methods well known in the art. Many starting materials and other reagents are available from commercial suppliers, such as Alfaisa (China) Chemical Co., Ltd., or can be readily prepared using synthetic methods commonly used in the art.

[0075] 1HNMR spectra were recorded on an instrument operating at 400 MHz. HNMR spectra were obtained in solution form (reported in ppm). When reporting peak multiplicity, the following abbreviations were used: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), br (broad peak), dd (doublet), dt (doubletuplet). Coupling constants are given in Hertz (Hz).

[0076] When necessary, the (R)- and (S)-isomers of the non-limiting exemplary compounds, if present, can be separated by methods known to those skilled in the art, such as by forming diastereomeric salts or complexes, which can be separated by, for example, crystallization; by forming diastereomeric derivatives, which can be separated by, for example, crystallization or chromatography; by selectively reacting one enantiomer with an enantiomer-specific reagent, thereby separating the modified and unmodified enantiomers; or by chromatographic separation in a chiral environment, for example, a chiral chromatographic column. Alternatively, specific enantiomers can be prepared by asymmetric synthesis using optically active reagents, substrates, catalysts, or solvents, or by converting one enantiomer to another via asymmetric transformation.

[0077] Preparation of compounds:

[0078] Intermediate 1: N-(7-(2-hydroxypropyl-2-yl)-2-(piperidin-4-yl)imidazo[1,2-a]pyridin-6-yl)-6-(trifluoromethyl)pyridine-2-carboxamide hydrochloride (IM-1)

[0079]

[0080] Step 1: Preparation of methyl 6-bromo-2-(1-(tert-butoxycarboxyl)piperidin-4-yl)imidazo[1,2-a]pyridine-7-carboxylate (IM-1a)

[0081]

[0082] 4-(2-bromoacetyl)piperidin-1-carboxylic acid tert-butyl ester (25.0 g, 81.6 mmol) and 2-amino-5-bromoisonicotinic acid methyl ester (18.8 g, 81.6 mmol) were dissolved in acetonitrile (150 mL) and toluene (100 mL). Sodium bicarbonate (13.6 g, 163 mmol) was added to the mixture at 25 °C. The reaction mixture was heated to 90 °C and stirred at 90 °C for 12 hours. Thin-layer chromatography showed that the reaction was complete. The reaction mixture was poured into ice water (500 mL) and stirred for 10 minutes. The aqueous phase was extracted with ethyl acetate (300 mL × 3). The combined organic phases were washed with brine (500 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. Purification by column chromatography (silica gel, petroleum ether / ethyl acetate v / v = 100 / 1 to 0 / 1) gave compound IM-1a as a dark brown solid (20.0 g).

[0083] Step 2: Preparation of methyl 2-(1-(tert-butoxycarboxyloyl)piperidin-4-yl)-6-(6-(trifluoromethyl)pyridin-2-carboxamido)imidazo[1,2-a]pyridine-7-carboxylate (IM-1b)

[0084]

[0085] Compound IM-1a (20.0 g, 45.6 mmol) and 6-(trifluoromethyl)pyridine-2-carboxamide (26.0 g, 136 mmol) were dissolved in dioxane (200.0 mL). Cesium carbonate (29.7 g, 90.7 mmol), 4,5-bis(diphenylphosphine)-9,9-dimethyloxanthracene (10.5 g, 18.2 mmol), and palladium acetate (2.04 g, 9.13 mmol) were added to the reaction mixture under nitrogen protection at 25 °C. The reaction mixture was heated to 100 °C and stirred at 100 °C for 12 hours. The reaction mixture was cooled to 25 °C and concentrated under reduced pressure. The residue was poured into ice water (300 mL) and stirred for 5 minutes. The aqueous phase was extracted with ethyl acetate (300 mL × 3), the combined organic phases were washed with brine (500 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The residue was purified by column chromatography (silica gel, petroleum ether / ethyl acetate volume ratio = 100 / 1 to 0 / 1) to give compound IM-1b as a yellow solid (10.0 g).

[0086] Step 3: Preparation of tert-butyl 4-(7-(2-hydroxypropyl-2-yl)-6-(6-(trifluoromethyl)pyridin-2-carboxamido)imidazo[1,2-a]pyridin-2-yl)piperidine-1-carboxylate (IM-1c)

[0087]

[0088] Compound IM-1b (10.0 g, 18.1 mmol) was dissolved in tetrahydrofuran (100 mL). The reaction mixture was cooled to 0 °C, and methylmagnesium bromide (3 M, 36.5 mL) was slowly added dropwise. After the addition was complete, the system was slowly heated to 25 °C and stirred at 25 °C for 3 hours. The reaction was quenched with an aqueous solution of ammonium chloride (200 mL), extracted with ethyl acetate (3 × 100 mL), and the combined organic layers were dried over anhydrous magnesium sulfate, filtered, and concentrated under vacuum. The residue was purified by column chromatography (silica gel, petroleum ether / ethyl acetate v / v = 100 / 1 to 0 / 1) to give compound IM-1c as a white solid (4.00 g, crude).

[0089] Step 4: Preparation of N-(7-(2-hydroxypropyl-2-yl)-2-(piperidin-4-yl)imidazo[1,2-a]pyridin-6-yl)-6-(trifluoromethyl)pyridine-2-carboxamide hydrochloride (IM-1)

[0090]

[0091] Compound IM-1c (4.00 g, 7.32 mmol) was dissolved in dioxane (40.0 mL), and the system was cooled to 0 °C. Dioxane hydrochloride (4.00 M, 2.00 equivalents) was slowly added to the system. After the addition was complete, the system was slowly heated to 25 °C and stirred at 25 °C for 3 hours. The reaction mixture was concentrated under vacuum. Compound IM-1 was given as a white solid (3.00 g, crude product, hydrochloride).

[0092] Intermediate 2: N-(7-methoxy-2-(piperidin-4-yl)imidazo[1,2-a]pyridin-6-yl)-6-(trifluoromethyl)pyridine-2-carboxamide

[0093]

[0094] Step 1: Preparation of tert-butyl 4-(6-bromo-7-methoxyimidazo[1,2-a]pyridin-2-yl)piperidine-1-carboxylate (IM-2a)

[0095]

[0096] 4-(2-bromoacetyl)piperidine-1-carboxylic acid tert-butyl ester (6.00 g, 19.6 mmol) was dissolved in toluene (13.9 mL) and acetonitrile (27.9 mL). Sodium bicarbonate (3.29 g, 39.2 mmol) and 5-bromo-4-methoxy-2-aminopyridine (3.98 g, 19.6 mmol) were added at room temperature, and the reaction mixture was heated to 90 °C and stirred for 12 hours. Water (50 mL) was added to the reaction mixture, and the mixture was extracted with ethyl acetate (50 mL x 3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography (silica gel, petroleum ether / ethyl acetate v / v = 100 / 1 to 10 / 1) to give intermediate IM-2a as a yellow solid (4.00 g).

[0097] Step 2: Preparation of tert-butyl 4-(7-methoxy-6-(6-(trifluoromethyl)pyridin-2-carbamate)imidazo[1,2-a]pyridin-2-yl)piperidine-1-carboxylic acid (IM-2b)

[0098]

[0099] Compound IM-2a (4.40 g, 10.7 mmol) and 6-(trifluoromethyl)pyridine-2-carboxamide (4.08 g, 21.4 mmol) were dissolved in 1,4-dioxane (40.0 mL). Under nitrogen protection at room temperature, 4,5-bis(diphenylphosphine)-9,9-dimethyloxanthracene (2.48 g, 4.29 mmol), cesium carbonate (6.99 g, 21.4 mmol), and palladium acetate (481 mg, 2.14 mmol) were added sequentially. The reaction mixture was then heated to 100 °C and stirred for 12 hours. The reaction mixture was cooled to room temperature, diluted with water (40.0 mL), and extracted with ethyl acetate (50.0 mL x 3). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (silica gel, petroleum ether / ethyl acetate volume ratio = 100 / 1 to 10 / 1) to give IM-2b as a yellow solid (3.00 g).

[0100] 1 HNMR: (400MHz, DMSO-d6) δ10.19(s,1H),9.34(s,1H),8.36-8.48(m,2H),8.12-8.27(m,1H),7.66(s, 1H),7.08(s,1H),3.98(s,5H),2.71-2.97(m,3H),1.90-2.01(m,2H),1.56-1.46(m,2H),1.41(s,9H).

[0101] Step 3: Preparation of N-(7-methoxy-2-(piperidin-4-yl)imidazo[1,2-a]pyridin-6-yl)-6-(trifluoromethyl)pyridine-2-carboxamide (IM-2)

[0102]

[0103] Compound IM-2b (500 mg, 1.06 mmol) was dissolved in 1,4-dioxane (5.00 mL), and hydrochloric acid / 1,4-dioxane (4 M, 12.0 mL) was added. The reaction mixture was stirred at 25 °C for 3 hours. The reaction mixture was concentrated to dryness under reduced pressure to give intermediate IM-2 as a yellow solid (400 mg, crude product).

[0104] Intermediate 3: 2-(4-(7-(2-hydroxypropyl-2-yl)-6-(6-(trifluoromethyl)pyridin-2-carboxamido)imidazo[1,2-a]pyridin-2-yl)piperidin-1-yl)acetic acid (IM-3)

[0105]

[0106] Compound IM-1 (500 mg, 1.03 mmol) and 2-bromoacetic acid (215 mg, 1.55 mmol) were dissolved in N,N-dimethylformamide (5.00 mL). Triethylamine (313 mg, 3.10 mmol) was added to the reaction mixture at 25 °C. The reaction mixture was stirred at 25 °C for 2 hours. The reaction mixture was concentrated under reduced pressure to give compound IM-3 as a yellow solid (550 mg, crude product), which was used directly without further purification.

[0107] Intermediate 4: 3-(4-(7-(2-hydroxypropyl-2-yl)-6-(6-(trifluoromethyl)pyridin-2-carboxamido)imidazo[1,2-a]pyridin-2-yl)piperidin-1-yl)propionic acid (IM-4)

[0108]

[0109] Intermediate IM-1 (150 mg, 309 μmol) and 3-bromopropionic acid (71.1 mg, 464 μmol) were dissolved in dichloromethane (2.00 mL). Triethylamine (94.1 mg, 929 μmol) was added to the reaction mixture at 25 °C. The reaction mixture was stirred at 25 °C for 12 hours. The reaction mixture was concentrated under reduced pressure to give compound IM-4 as a yellow solid (210 mg, crude product), which was used directly without further purification.

[0110] Intermediate 5: 4-(4-(7-(2-hydroxypropyl-2-yl)-6-(6-(trifluoromethyl)pyridin-2-carboxamido)imidazo[1,2-a]pyridin-2-yl)piperidin-1-yl)butyric acid (IM-5)

[0111]

[0112] Step 1: Preparation of tert-butyl butyrate (IM-5a) of 4-(4-(7-(2-hydroxypropyl-2-yl)-6-(6-(trifluoromethyl)pyridin-2-carboxamido)imidazo[1,2-a]pyridin-2-yl)piperidin-1-yl)butyrate

[0113]

[0114] Compound 1 (300 mg, 670 μmol) was dissolved in acetone (3.00 mL), and then tert-butyl 4-bromobutyrate (299 mg, 1.34 mmol) and potassium carbonate (277 mg, 2.01 mmol) were added at 25 °C. The mixture was heated to 60 °C and stirred for 12 hours. The reaction mixture was concentrated to dryness under reduced pressure. The residue was purified by preparative HPLC (column: Waters Xbridge BEH C18 100*30 mm*10 μm; mobile phase: [water (NH4HCO3)-ACN]; B%: 35%-65%, 8 min) to obtain intermediate IM-5a as a crude off-white solid (150 mg).

[0115] Step 2: Preparation of 4-(4-(7-(2-hydroxypropyl-2-yl)-6-(6-(trifluoromethyl)pyridin-2-carboxamido)imidazo[1,2-a]pyridin-2-yl)piperidin-1-yl)butyric acid (IM-5)

[0116]

[0117] Intermediate IM-5a (120 mg, 203 μmol) was dissolved in dichloromethane (2.00 mL), followed by the addition of trifluoroacetic acid (1.30 g, 11.4 mmol), and the mixture was stirred at 25 °C for 12 hours. The reaction mixture was concentrated to dryness under reduced pressure to obtain crude intermediate IM-5 as a yellow oily compound (100 mg), which was used directly without further purification.

[0118] Intermediate 6: N-(7-(2-hydroxypropyl-2-yl)-2-(1-(2-oxoethyl)piperidin-4-yl)imidazo[1,2-a]pyridin-6-yl)-6-(trifluoromethyl)pyridine-2-carboxamide (IM-6)

[0119]

[0120] Step 1: Preparation of N-(2-(1-(2,2-diethoxyethyl)piperidin-4-yl)-7-(2-hydroxypropyl-2-yl)imidazo[1,2-a]pyridin-6-yl)-6-(trifluoromethyl)pyridine-2-carboxamide (IM-6a)

[0121]

[0122] Compound IM-1 (1.00 g, 2.23 mmol) and 2-bromo-1,1-diethoxyethane (880 mg, 4.47 mmol) were dissolved in acetone (2.00 mL), followed by the sequential addition of potassium carbonate (926 mg, 6.70 mmol) and potassium iodide (185 mg, 1.12 mmol). The resulting mixture was heated to 80 °C and stirred for 12 hours. The mixture was concentrated to dryness under reduced pressure to give intermediate IM-6a as a yellow solid (300 mg).

[0123] Step 2: Preparation of N-(7-(2-hydroxypropyl-2-yl)-2-(1-(2-oxoethyl)piperidin-4-yl)imidazo[1,2-a]pyridin-6-yl)-6-(trifluoromethyl)pyridine-2-carboxamide (IM-6)

[0124]

[0125] Compound IM-6a (300 mg, 532 μmol) was dissolved in water (3.00 mL), and hydrochloric acid / 1,4-dioxane (4 M, 3.00 mL) was slowly added dropwise to the system at 25 °C. The mixture was stirred at 60 °C for 12 hours, and then concentrated to dryness under vacuum to give intermediate IM-6 as a white solid (430 mg).

[0126] Intermediate 7: N-(7-methoxy-2-(1-(2-oxoethyl)piperidin-4-yl)imidazo[1,2-a]pyridin-6-yl)-6-(trifluoromethyl)pyridine-2-carboxamide (IM-7)

[0127]

[0128] Step 1: Preparation of N-(2-(1-(2,2-diethoxyethyl)piperidin-4-yl)-7-methoxyimidazo[1,2-a]pyridin-6-yl)-6-(trifluoromethyl)pyridine-2-carboxamide (IM-7a)

[0129]

[0130] Intermediate IM-1 (500 mg, 1.19 mmol) was dissolved in N,N-dimethylformamide (5.00 mL) at room temperature, followed by the addition of 2-bromo-1,1-diethoxyethane (469 mg, 2.38 mmol) and N,N-diisopropylethylamine (462 mg, 3.58 mmol). The mixture was microwaved at 100 °C for 2 hours. The reaction mixture was concentrated to dryness under reduced pressure and purified by preparative HPLC (column: Phenomenex C18 75*30 mm*3 μm; mobile phase: [water (NH4HCO3)-ACN]; B%: 35%-65%, 8 min) to obtain intermediate IM-7a as a white solid (150 mg).

[0131] Step 2: Preparation of N-(7-methoxy-2-(1-(2-oxoethyl)piperidin-4-yl)imidazo[1,2-a]pyridin-6-yl)-6-(trifluoromethyl)pyridine-2-carboxamide (IM-7)

[0132]

[0133] Compound 2 (150 mg, 280 μmol) was added to water (2.00 mL), followed by the slow addition of HCl / dioxane (4.00 M, 2.00 mL) at room temperature. The reaction mixture was heated to 50 °C and stirred for 12 hours. The reaction mixture was concentrated to dryness to give intermediate IM-7 (140 mg, crude product), which was used directly in the next step without further purification.

[0134] Intermediate 8: N-(2-(1-(azacyclobutan-3-yl)piperidin-4-yl)-7-(2-hydroxypropyl-2-yl)imidazo[1,2-a]pyridin-6-yl)-6-(trifluoromethyl)pyridine-2-carboxamide (IM-8)

[0135]

[0136] Step 1: Preparation of tert-butyl 3-(4-(7-(2-hydroxypropyl-2-yl)-6-(6-(trifluoromethyl)pyridin-2-carboxamido)imidazo[1,2-a]pyridin-2-yl)piperidin-1-yl)azacyclobutane-1-carboxylate (IM-8a)

[0137]

[0138] Intermediate IM-1 (300 mg, 670 μmol) and tert-butyl 3-oxozycyclobutane-1-carboxylate (114 mg, 670 μmol) were dissolved in 1,2-dichloroethane (3.00 mL) and N,N-dimethylformamide (3.00 mL). Triethylamine (203 mg, 2.01 mmol) was added to the reaction mixture under nitrogen protection at 25 °C. The reaction mixture was stirred at 25 °C for 30 min. Then, sodium triacetoxyborohydride (284 mg, 1.34 mmol) and acetic acid (40.2 mg, 670 μmol) were added to the reaction mixture, and the reaction mixture was stirred at 25 °C for 11.5 h. The reaction was quenched with water (5.00 mL), and then extracted with ethyl acetate (10.0 mL * 3). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by high performance liquid chromatography (formic acid system) to obtain intermediate IM-8a as a yellow solid (100 mg).

[0139] Step 2: Preparation of N-(2-(1-(azacyclobutan-3-yl)piperidin-4-yl)-7-(2-hydroxypropyl-2-yl)imidazo[1,2-a]pyridin-6-yl)-6-(trifluoromethyl)pyridine-2-carboxamide (IM-8)

[0140]

[0141] Compound IM-8a (100 mg, 165 μmol) was dissolved in dioxane (1.00 mL), and hydrochloric acid / dioxane (4.00 M, 166 μL) was added to the reaction mixture at 25 °C. The reaction mixture was stirred at 25 °C for 12 hours. The reaction mixture was concentrated to dryness under reduced pressure to give intermediate IM-8 as a white solid (100.0 mg, hydrochloride).

[0142] Intermediate 9: 1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)piperidin-4-carboxaldehyde (IM-9)

[0143]

[0144] Step 1: Preparation of 5-(4-(dimethoxymethyl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (IM-9a)

[0145]

[0146] 2-(2,6-dioxopiperidin-3-yl)-5-fluoroisoindoline-1,3-dione (4.00 g, 14.4 mmol) was dissolved in dimethyl sulfoxide (40.0 mL) at 25 °C. Then, N,N-diisopropylethylamine (3.74 g, 28.9 mmol) and 4-(dimethoxymethyl)piperidine (2.53 g, 15.9 mmol) were added to the reaction mixture. The reaction mixture was heated to 90 °C and stirred at 90 °C for 15 hours. The reaction mixture was quenched with water (50.0 mL) at 25 °C. The resulting mixture was extracted with ethyl acetate (50.0 mL x 3), washed with brine (50.0 mL x 2), dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The residue was purified by column chromatography (silica gel, petroleum ether / ethyl acetate volume ratio = 100 / 1 to 10 / 1) to obtain intermediate IM-9a as a yellow solid (3.10 g, crude product).

[0147] Step 2: Preparation of 1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)piperidin-4-carboxaldehyde (IM-9)

[0148]

[0149] Intermediate IM-9a (500 mg, 1.20 mmol) was added to formic acid (5.00 mL), and the reaction mixture was stirred at 25 °C for 12 hours. The reaction mixture was concentrated to give intermediate IM-9 as a yellow solid (400 mg, crude product), which was used directly without further purification.

[0150] Intermediate 10: 2-(2,6-dioxopiperidin-3-yl)-5-(piperazin-1-yl)isoindoline-1,3-dione (IM-10)

[0151]

[0152] Step 1: Preparation of tert-butyl piperazine-1-carboxylate (IM-10a)

[0153]

[0154] 2-(2,6-dioxadiazin-3-yl)-5-fluoroisoindoline-1,3-dione (7.00 g, 25.3 mmol) was dissolved in dimethyl sulfoxide (60.0 mL) at 25 °C. Tert-butyl piperazine-1-carboxylate (5.62 g, 30.1 mmol) and N,N-diisopropylethylamine (6.55 g, 50.6 mmol) were added to the reaction mixture. The resulting reaction mixture was heated to 95 °C and stirred for 12 hours. Water (60.0 mL) was added to the reaction mixture. The mixture was extracted with ethyl acetate (20.0 mL x 3). The combined organic phases were washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The residue was purified by column chromatography (silica gel, petroleum ether / ethyl acetate v / v = 100 / 1 to 10 / 1) to give intermediate IM-10a as a yellow solid (8.00 g).

[0155] Step 2: Preparation of 2-(2,6-dioxadiazin-3-yl)-5-(piperazin-1-yl)isoindoline-1,3-dione (IM-10)

[0156]

[0157] Intermediate IM-10a (2.00 g, 4.52 mmol) was dissolved in dichloromethane (28.0 mL), and then trifluoroacetic acid (21.5 g, 189 mmol) was added. The reaction mixture was stirred at 25 °C for 12 hours, and then concentrated to dryness under vacuum to give intermediate IM-10 as a yellow solid (2.00 g, trifluoroacetate, crude product), which was used directly without further purification.

[0158] 1 HNMR (400MHz, DMSO-d6) δ11.10(s,1H),7.76(d,J=8.5Hz,1H),7.46(d,J=2.1Hz,1H),7.31-7.35(m,1H),5.03-5.16(m,1H),3.65(br s,1H),2.89(s,8H),2.72-2.73(m,1H),2.52-2.64(m,2H),1.99–2.06(m,1H).

[0159] Intermediate 11: 2-(2,6-dioxopiperidin-3-yl)-5-(2,7-diazaspiro[3.5]nonane-2-yl)isoindoline-1,3-dione

[0160]

[0161] Step 1: Preparation of tert-butyl 2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)-2,7-diazaspiro[3.5]nonane-7-carboxylate (IM-11a)

[0162]

[0163] 2-(2,6-dioxadiazin-3-yl)-5-fluoroisoindoline-1,3-dione (200 mg, 0.724 mmol) was dissolved in dimethyl sulfoxide (2.00 mL) at 25 °C, followed by the addition of tert-butyl 2,7-diazaspiro[3.5]nonane-7-carboxylate (195 mg, 0.861 mmol) and N,N-diisopropylethylamine (187 mg, 1.45 mmol). The reaction mixture was heated to 95 °C and stirred for 12 hours. The reaction mixture was diluted with water (10 mL) and extracted with ethyl acetate (10 mL x 3). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (silica gel, petroleum ether / ethyl acetate v / v = 100 / 1 to 10 / 1) to give intermediate IM-11a as a yellow solid (300 mg, crude).

[0164] Step 2: Preparation of 2-(2,6-dioxadiazin-3-yl)-5-(2,7-diazaspiro[3.5]nonane-2-yl)isoindoline-1,3-dione (IM-11)

[0165]

[0166] Compound IM-11a (300 mg, 0.621 mmol) was dissolved in dichloromethane (5.00 mL), and then trifluoroacetic acid (2.91 g, 25.4 mmol) was added. The reaction mixture was stirred at 25 °C for 12 hours, and then concentrated under reduced pressure to give intermediate IM-11 as a yellow solid (300 mg, crude product), which was used directly without further purification.

[0167] Intermediate 12: 2-(2,6-dioxopiperidin-3-yl)-5-(2,7-diazaspiro[3.5]nonane-7-yl)isoindoline-1,3-dione (IM-12, hydrochloride)

[0168]

[0169] Step 1: Preparation of tert-butyl 7-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)-2,7-diazaspiro[3.5]nonane-2-carboxylate (IM-12a)

[0170]

[0171] 2-(2,6-dioxadiazin-3-yl)-5-fluoroisoindoline-1,3-dione (500 mg, 1.81 mmol) was dissolved in dimethyl sulfoxide (5.00 mL), and tert-butyl 2,7-diazaspiro[3.5]nonane-2-carboxylate (450 mg, 1.99 mmol) and N,N-diisopropylethylamine (467 mg, 3.62 mmol) were added sequentially. The resulting mixture was stirred at 95 °C for 12 hours. The reaction mixture was concentrated under reduced pressure to give intermediate IM-12a as a yellow solid (800 mg), which was used directly without further purification.

[0172] Step 2: Preparation of 2-(2,6-dioxadiazin-3-yl)-5-(2,7-diazaspiro[3.5]nonane-7-yl)isoindoline-1,3-dione (IM-12)

[0173]

[0174] Compound 2 (400 mg, 0.828 mmol) was dissolved in 1,4-dioxane (5.00 mL) solution, and then hydrochloric acid / 1,4-dioxane (5.00 g, 4.00 M) was added. The reaction mixture was stirred at 25 °C for 2 hours, and then concentrated to dryness under reduced pressure to give intermediate IM-12 as a white solid (250 mg, hydrochloride).

[0175] 1 HNMR(400MHz,DMSO-d6)δ11.07(s,1H),9.28(br s,2H),7.66(d,J=8.6Hz,1H),7.18-7.43(m,2H),5.06(dd,J=5.4,12.9Hz,1H),3.73(br t,J=6.1Hz,4H),3.41-3.52(m,4H),2.80-2.94(m,1H),2.58(br d,J=16.1Hz,1H),2.96-1.06(m,1H),1.79-1.89(m,4H).

[0176] Intermediate 13: 1-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)piperidin-4-carboxaldehyde (IM-13)

[0177]

[0178] Step 1: Preparation of methyl 2-cyano-4-(4-(hydroxymethyl)piperidin-1-yl)benzoate (IM-13a)

[0179]

[0180] Methyl 2-cyano-4-fluorobenzoate (10.0 g, 55.8 mmol) and 4-piperidinemethanol (9.64 g, 83.7 mmol) were dissolved in dimethyl sulfoxide (100 mL), and N,N-diisopropylethylamine (20.4 g, 158 mmol) was added under nitrogen atmosphere at 25 °C. The reaction was heated to 110 °C and stirred at 110 °C for 16 hours. The residue was poured into ice water (200 mL) and stirred for 10 minutes. The aqueous phase was extracted with ethyl acetate (200 mL x 3). The combined organic phases were washed with brine (500 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The residue was purified by column chromatography (silica gel, petroleum ether / ethyl acetate v / v = 100 / 1 to 0 / 1) to give intermediate IM-13a as a yellow oil (18.0 g, crude).

[0181] Step 2: Preparation of methyl 2-formyl-4-(4-(hydroxymethyl)piperidin-1-yl)benzoate (IM-13b)

[0182]

[0183] Intermediate IM-13a (10.0 g, 36.4 mmol) was dissolved in a mixed solvent of glacial acetic acid (10.0 mL), pyridine (20.0 mL), and water (10.0 mL). Sodium hypophosphite monohydrate (50.3 g, 364 mmol) and Raney nickel (4.37 g, 51.0 mmol) were added under nitrogen atmosphere at 25 °C. The reaction mixture was purged three times with hydrogen, and then heated to 65 °C. The reaction mixture was stirred at 65 °C under hydrogen atmosphere for 16 hours. The mixture was vacuum filtered and concentrated to obtain intermediate IM-13b as a yellow oil (4.50 g, crude product).

[0184] Step 3: Preparation of 3-(5-(4-(hydroxymethyl)piperidin-1-yl)-1-oxoisoindoline-2-yl)piperidin-2,6-dione (IM-13c)

[0185]

[0186] Compound IM-13b (4.20 g, 15.1 mmol) and 3-aminopiperidin-2,6-dione (2.99 g, 18.1 mmol) were dissolved in dichloromethane (30.0 mL), and N,N-diisopropylethylamine (5.87 g, 45.4 mmol), glacial acetic acid (8.82 g, 146 mmol), and sodium cyanoborohydride (2.86 g, 45.4 mmol) were added under nitrogen atmosphere at 25 °C. The reaction mixture was stirred at 25 °C for 12 hours. The reaction was then quenched with water (100 mL), and the pH was adjusted to 8 with a saturated sodium bicarbonate aqueous solution. The mixture obtained from the extraction was extracted with dichloromethane / methanol (10 / 1, 100 mL * 6). The combined organic phases were dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was purified by column chromatography (silica gel, petroleum ether / ethyl acetate volume ratio = 100 / 1 to 0 / 1) to give compound IM-13c as a white solid (2.00 g).

[0187] Step 4: Preparation of 1-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)piperidin-4-carboxaldehyde (IM-13)

[0188]

[0189] Compound IM-13c (0.50 g, 1.40 mmol) was dissolved in dichloromethane (20.0 mL), and Dys-Martin oxidant (593 mg, 1.40 mmol) was added at 25 °C under nitrogen atmosphere. The reaction mixture was stirred at 35 °C for 1 hour. The reaction mixture was filtered, and the filtrate was concentrated under vacuum. The residue was purified by high performance liquid chromatography (formic acid conditions) to give compound IM-13 as a yellow solid (1.00 g, crude product).

[0190] Intermediate 14: 2-(4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)piperazin-1-yl)acetaldehyde (IM-14)

[0191]

[0192] Step 1: Preparation of 5-(4-(2,2-diethoxyethyl)piperazin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (IM-14a)

[0193]

[0194] Intermediate IM-10 (1.00 g, 2.92 mmol) was dissolved in N,N-dimethylformamide (8.00 mL) at room temperature, followed by the addition of 2-bromo-1,1-diethoxyethane (1.15 g, 5.84 mmol) and N,N-diisopropylethylamine (1.13 g, 8.76 mmol). The reaction mixture was heated to 100 °C and stirred for 12 hours. The reaction mixture was diluted with water (30.0 mL) and extracted with ethyl acetate (30.0 mL x 3). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was subjected to preparative HPLC (column: Phenomenex luna C18 80*40 mm*3 μm; mobile phase: [water (FA)-ACN]; B%: 1%-35%, 8 min) to give intermediate IM-14a as a yellow solid (200 mg).

[0195] Step 2: Preparation of 2-(4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)piperazin-1-yl)acetaldehyde (IM-14)

[0196]

[0197] Intermediate IM-14a (100 mg, 218.10 μmol) was dissolved in formic acid (2.00 mL), and the reaction mixture was stirred at 65 °C for 12 hours. The reaction mixture was concentrated to dryness to give intermediate IM-14 as a yellow solid (40.0 mg, crude product).

[0198] Intermediate 15: 1-(2-(2,6-dioxopiperidin-3-yl)-6-fluoro-1,3-dioxoisoindoline-5-yl)piperidin-4-carboxaldehyde (IM-15)

[0199]

[0200] Step 1: Preparation of 2-(2,6-dioxopiperidin-3-yl)-5,6-difluoroisoindoline-1,3-dione (IM-15a)

[0201]

[0202] 3-Aminopiperidine-2,6-dione hydrochloride (9.83 g, 59.7 mmol) was dissolved in toluene (100 mL), and triethylamine (16.4 g, 162 mmol) and 5,6-difluoroisobenzofuran-1,3-dione (10.0 g, 54.3 mmol) were added at room temperature. The reaction mixture was stirred at 60 °C for 12 hours. The reaction mixture was diluted with water (100 mL) and extracted with ethyl acetate (100 mL x 3). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (silica gel, petroleum ether / ethyl acetate v / v = 100 / 1 to 10 / 1) to give intermediate IM-15a as a yellow solid (4.50 g, crude).

[0203] Step 2: Preparation of 5-(4-(dimethoxymethyl)piperidin-1-yl)-2-(2,6-oxopiperidin-3-yl)-6-fluoroisoindoline-1,3-dione (IM-15b)

[0204]

[0205] IM-15a (4.50 g, 13.6 mmol) was dissolved in dimethyl sulfoxide (40.0 mL) at room temperature, followed by the addition of N,N-diisopropylethylamine (3.51 g, 27.1 mmol) and 4-(dimethoxymethyl)piperidine (2.16 g, 13.6 mmol). The reaction mixture was stirred at 90 °C for 12 hours. The reaction mixture was diluted with water (40.0 mL) and extracted with ethyl acetate (40.0 mL x 3). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography (silica gel, petroleum ether / ethyl acetate v / v = 100 / 1 to 10 / 1) to give intermediate IM-15b as a yellow solid (2.50 g, crude).

[0206] Step 3: Preparation of 1-(2-(2,6-dioxopiperidin-3-yl)-6-fluoro-1,3-dioxoisoindoline-5-yl)piperidin-4-carboxaldehyde (IM-15)

[0207]

[0208] Intermediate IM-15b (1.00 g, 2.31 mmol) was dissolved in tetrahydrofuran (10.0 mL) and water (2.00 mL) at room temperature, followed by the addition of pyridine p-toluenesulfonate (1.16 g, 4.61 mmol). The reaction mixture was stirred at 70 °C for 12 hours and then concentrated to give intermediate IM-15 as a yellow solid (500 mg), which was used directly without further purification.

[0209] Intermediate 16: 2-(1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)piperidin-4-yl)acetaldehyde (IM-16)

[0210]

[0211] Step 1: Preparation of 2-(2,6-dioxopiperidin-3-yl)-5-(4-(2-hydroxyethyl)piperidin-1-yl)isoindoline-1,3-dione (IM-16a)

[0212]

[0213] 2-(2,6-dioxadiazin-3-yl)-5-fluoroisoindoline-1,3-dione (2.00 g, 7.24 mmol) was dissolved in dimethyl sulfoxide (10.0 mL), followed by the addition of N,N-diisopropylethylamine (1.87 g, 14.4 mmol) and 2-(piperidin-4-yl)ethanol-1-ol (1.03 g, 7.96 mmol). The reaction mixture was stirred at 90 °C for 12 hours. The reaction was quenched by the slow addition of water (10.0 mL), and the mixture was extracted with ethyl acetate (10.0 mL x 3). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to give intermediate IM-16a as a yellow solid (1.10 g).

[0214] Step 2: 2-(1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)piperidin-4-yl)acetaldehyde

[0215]

[0216] Intermediate IM-16a (1.10 g, 2.85 mmol) was dissolved in dichloromethane (10.0 mL) at 25 °C, followed by the addition of Dysmartin periodide (1.21 g, 2.85 mmol). The reaction mixture was stirred at 25 °C for 2 hours. The reaction was quenched by the slow addition of water (10.0 mL), and the mixture was extracted with ethyl acetate (30 mL x 3). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give intermediate IM-16 as a yellow solid (600 mg), which was used directly without further purification.

[0217] Intermediate 17: 3-(4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)piperazin-1-yl)propyl methanesulfonate (IM-17)

[0218]

[0219] Step 1: Preparation of 2-(2,6-dioxopiperidin-3-yl)-5-(4-(3-hydroxypropyl)piperazin-1-yl)isoindoline-1,3-dione (IM-17a)

[0220]

[0221] 2-(2,6-dioxadiazin-3-yl)-5-fluoroisoindoline-1,3-dione (1.72 g, 6.24 mmol) was dissolved in dimethyl sulfoxide (10.0 mL), followed by the addition of N,N-diisopropylethylamine (1.61 g, 12.4 mmol) and 3-(piperazin-1-yl)prop-1-ol (900 mg, 6.24 mmol). The reaction mixture was stirred at 90 °C for 12 hours. The reaction mixture was diluted with water (10.0 mL), and the aqueous layer was extracted with ethyl acetate (10 mL x 3). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (formic acid system) to give intermediate IM-17a as a yellow solid compound (1.50 g).

[0222] Step 2: Preparation of 3-(4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)piperazin-1-yl)propyl mesylate (IM-17)

[0223]

[0224] Compound IM-17a (1.00 g, 2.50 mmol) was dissolved in tetrahydrofuran (10.0 mL), followed by the addition of triethylamine (631 mg, 6.24 mmol) and methanesulfonic anhydride (870 mg, 4.99 mmol). The reaction mixture was stirred at 25 °C for 12 hours. The reaction mixture was diluted with water (10 mL) and extracted with ethyl acetate (10 mL x 3). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give intermediate IM-17 as a yellow solid (600 mg, crude product).

[0225] Intermediate 18: 2-(2,6-dioxopiperidin-3-yl)-5-(4-oxopiperidin-1-yl)isoindoline-1,3-dione (IM-18)

[0226]

[0227] Piperidin-4-one (490 mg, 3.62 mmol) was dissolved in dimethyl sulfoxide (10.0 mL) at room temperature, followed by the addition of N,N-diisopropylethylamine (935 mg, 7.24 mmol) and 2-(2,6-dioxopiperidin-3-yl)-5-fluoroisoindoline-1,3-dione (1.00 g, 3.62 mmol). The reaction mixture was heated to 90 °C and stirred for 12 hours. The reaction mixture was cooled to 25 °C, water (10.0 mL) was added, and the mixture was extracted with ethyl acetate (10.0 mL x 3). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to give intermediate IM-18 as a yellow oil (310 mg).

[0228] Intermediate 19: 2-(2,6-dioxopiperidin-3-yl)-5-(2,6-diazaspiro[3.3]hept-2-yl)isoindoline-1,3-dione trifluoroacetate (IM-19)

[0229]

[0230] Step 1: Preparation of tert-butyl 6-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)-2,6-diazaspiro[3.3]heptane-2-carboxylate (IM-19a)

[0231]

[0232] 2-(2,6-dioxadiazin-3-yl)-5-fluoroisoindoline-1,3-dione (3.00 g, 10.8 mmol) and tert-butyl 2,6-diazaspiro[3.3]heptane-2-carboxylate (2.15 g, 10.8 mmol) were dissolved in dimethyl sulfoxide (30.0 mL), followed by the addition of N,N-diisopropylethylamine (4.21 g, 32.5 mmol). The reaction mixture was heated to 95 °C and stirred for 12 hours. The reaction was quenched with 20.0 mL of water at 5 °C, and the resulting mixture was extracted with 45.0 mL (15.0 mL x 3) of dichloromethane. The organic layer was washed with an aqueous sodium chloride solution (5.00 mL x 3), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (silica gel, petroleum ether / ethyl acetate volume ratio = 100 / 1 to 10 / 1) to give intermediate IM-19a as a yellow oil (1.20 g).

[0233] Step 2: Preparation of 2-(2,6-dioxadiazin-3-yl)-5-(2,6-diazaspiro[3.3]hept-2-yl)isoindoline-1,3-dione trifluoroacetate (IM-19)

[0234]

[0235] Intermediate IM-19a (200 mg, 440 μmol) was dissolved in dichloromethane (2.00 mL), and then trifluoroacetic acid (3.07 g, 26.9 mmol) was added. The reaction mixture was stirred at 25 °C for 12 hours, and then concentrated under reduced pressure to give intermediate IM-19a as a white solid (150 mg).

[0236] Intermediate 20: Ethyl 2-(4-(2-(2,6-dioxopiperidin-3-yl)-6-fluoro-1,3-dioxoisoindoline-5-yl)piperazin-1-yl)methanesulfonic acid (IM-20)

[0237]

[0238] Step 1: Preparation of 2-(2,6-dioxopiperidin-3-yl)-5-fluoro-6-(4-(2-hydroxyethyl)piperazin-1-yl)isoindoline-1,3-dione (IM-20a)

[0239]

[0240] Intermediate IM-15a (1.00 g, 3.40 mmol) and 2-(piperazin-1-yl)ethanol-1-ol (486 mg, 3.74 mmol) were dissolved in dimethyl sulfoxide (10.0 mL) at 25 °C. N,N-diisopropylethylamine (439 mg, 3.40 mmol) was then added to the reaction mixture. The reaction mixture was heated to 90 °C and stirred for 12 hours. The reaction was quenched with 20.0 mL of water at 25 °C, followed by extraction with ethyl acetate (30.0 mL x 3). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (silica gel, petroleum ether / ethyl acetate v / v = 100 / 1 to 0 / 1) to give intermediate IM-20a as a yellow solid (1.00 g, 2.97 mmol).

[0241] Step 2: Preparation of 2-(4-(2-(2,6-dioxopiperidin-3-yl)-6-fluoro-1,3-dioxoisoindoline-5-yl)piperazin-1-yl)ethyl mesylate (IM-20)

[0242]

[0243] Intermediate IM-20a (200 mg, 494 μmol) was dissolved in dichloromethane (2.00 mL), and triethylamine (125 mg, 1.24 mmol) was added to the reaction mixture at 25 °C. The reaction mixture was stirred at 25 °C for 30 min, and then methanesulfonic anhydride (129 mg, 741 μmol) was added. The reaction mixture was stirred at 25 °C for 11.5 h. The reaction mixture was slowly poured into water (10 mL), and then extracted with dichloromethane (5 mL x 3). The combined organic phases were washed with (5.00 mL x 2) brine, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to give intermediate IM-20 as a yellow solid (200 mg, crude product), which was used directly without further purification.

[0244] Intermediate 21: 3-(1-oxo-5-(piperazin-1-yl)isoindoline-2-yl)piperidine-2,6-dione hydrochloride (IM-21)

[0245]

[0246] Step 1: Preparation of methyl 4-fluoro-2-formylbenzoate (IM-21a)

[0247]

[0248] Pyridine (15.0 g, 190 mmol) was added to water (50.0 mL) and acetic acid (50 mL). Raney nickel (7.72 g, 90.1 mmol) was added under nitrogen atmosphere, followed by a tetrahydrofuran (50.0 mL) solution of methyl 2-cyano-4-fluorobenzoate (5.00 g, 27.9 mmol). The reaction mixture was heated to 65 °C and stirred for 16 hours. The reaction was quenched by slow addition of water (50.0 mL), and the aqueous layer was extracted with ethyl acetate (50 mL x 3). The combined organic layers were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (silica gel, petroleum ether / ethyl acetate v / v = 100 / 1 to 10 / 1) to give intermediate IM-21a as a white solid (3.00 g, crude product).

[0249] Step 2: Preparation of tert-butyl 4-(3-formyl-4-(methoxyformyl)phenyl)piperazine-1-carboxylate (IM-21b)

[0250]

[0251] Boc-piperazine (2.61 g, 14.0 mmol) was dissolved in dimethyl sulfoxide (30.0 mL) at room temperature, followed by the addition of N,N-diisopropylethylamine (6.39 g, 49.4 mmol) and intermediate IM-21a (3.00 g, 16.4 mmol). The reaction mixture was heated to 100 °C and stirred for 16 hours. The reaction was quenched by the slow addition of water (30.0 mL), and the aqueous layer was extracted with ethyl acetate (30 mL x 3). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (silica gel, petroleum ether / ethyl acetate v / v = 100 / 1 to 10 / 1) to give intermediate IM-21b as a yellow solid (1.40 g).

[0252] 1 HNMR(400MHz,DMSO-d6)δ10.49(s,1H),7.84(d,J=8.6Hz,1H),7.12-7.26(m,2H),3.8 4(s,2H),3.64-3.70(m,1H),3.42-3.50(m,4H),3.34-3.42(m,4H),1.44-1.41(m,9H)

[0253] Step 3: Preparation of tert-butyl 4-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)piperazine-1-carboxylate (IM-21c)

[0254]

[0255] 3-Aminopiperidine-2,6-dione hydrochloride (992 mg, 6.03 mmol) was dissolved in dimethyl sulfoxide (10.0 mL), and N,N-diisopropylethylamine (1.56 g, 12.0 mmol) was added, followed by acetic acid (1.54 g, 25.6 mmol), sodium borohydride acetate (1.70 g, 8.04 mmol), and intermediate IM-21b (1.40 g, 4.02 mmol). The reaction mixture was stirred at 25 °C for 12 hours. The reaction was quenched by the slow addition of water (30 mL), and the aqueous layer was extracted with ethyl acetate (20 mL x 3). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (silica gel, petroleum ether / ethyl acetate v / v = 100 / 1 to 10 / 1) to give intermediate IM-21c as a yellow solid (900 mg).

[0256] Step 4: Preparation of 3-(1-oxo-5-(piperazin-1-yl)isoindoline-2-yl)piperidine-2,6-dione hydrochloride (IM-21)

[0257]

[0258] Intermediate IM-21c (182 mg, 424 μmol) was dissolved in dioxane (2.00 mL), and hydrochloric acid / dioxane (4 M, 1.82 mL) was added. The reaction mixture was stirred at 25 °C for 12 hours. The reaction mixture was concentrated to give intermediate IM-21 as an off-white solid (160 mg).

[0259] Intermediate 22: 3-(1-oxo-6-(piperazin-1-yl)isoindoline-2-yl)piperidine-2,6-dione hydrochloride (IM-22)

[0260]

[0261] Step 1: Preparation of tert-butyl 4-(4-formyl-3-(methoxyformyl)phenyl)piperazine-1-carboxylate (IM-22a)

[0262]

[0263] Boc-piperazine (869 mg, 4.67 mmol) was dissolved in dimethyl sulfoxide (8.00 mL) at room temperature, followed by the addition of N,N-diisopropylethylamine (2.13 g, 16.4 mmol) and methyl 5-fluoro-2-carboxybenzoate (1.00 g, 5.49 mmol). The reaction mixture was heated to 80 °C and stirred for 16 hours. The reaction was quenched by the slow addition of water (30 mL), and the aqueous layer was extracted with ethyl acetate (10 mL x 3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give intermediate IM-22a as a yellow oil (1.80 g, crude product).

[0264] Step 2: Preparation of tert-butyl 4-(2-(2,6-dioxopiperidin-3-yl)-3-oxoisoindoline-5-yl)piperazine-1-carboxylate (IM-22b)

[0265]

[0266] 3-Aminopiperidine-2,6-dione hydrochloride (935 mg, 5.68 mmol) was dissolved in N,N-dimethylformamide (5 mL), and N,N-diisopropylethylamine (1.34 g, 10.3 mmol) was added at room temperature, followed by acetic acid (3.10 g, 51.6 mmol), sodium borohydride acetate (3.29 g, 15.5 mmol), and intermediate IM-22a (1.80 g, 5.17 mmol). The reaction mixture was stirred at 25 °C for 12 hours. The reaction was quenched by the slow addition of water (20 mL), and the aqueous layer was extracted with ethyl acetate (10 mL x 3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give intermediate IM-22b as a yellow oil (650 mg, crude product).

[0267] Step 3: Preparation of 3-(1-oxo-6-(piperazin-1-yl)isoindoline-2-yl)piperidine-2,6-dione hydrochloride (IM-22)

[0268]

[0269] Intermediate IM-22b (650 mg, 1.52 mmol) was dissolved in ethyl acetate (2.00 mL). Hydrochloric acid / ethyl acetate (4 M, 2.00 mL) was added at room temperature and the mixture was stirred for 2 hours. The reaction mixture was concentrated to give intermediate IM-22 as a yellow solid (220 mg).

[0270] Intermediate 23: 1-(2-(2,6-dioxopiperidin-3-yl)-6-fluoro-1-oxoisoindoline-5-yl)piperidin-4-carboxaldehyde (IM-23)

[0271]

[0272] Step 1: Preparation of methyl 4-bromo-2-(bromomethyl)-5-fluorobenzoate (IM-23a)

[0273]

[0274] Methyl 4-bromo-5-fluoro-2-methylbenzoate (4.50 g, 18.2 mmol) was dissolved in dichloroethane (25.0 mL). N-bromosuccinimide (3.53 g, 19.8 mmol) and azobisisobutyronitrile (149 mg, 910 μmol) were added sequentially at room temperature, and the reaction mixture was stirred at 80 °C for 12 hours. The reaction mixture was diluted with water (30.0 mL) and extracted with ethyl acetate (30.0 mL x 3). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (silica gel) to give intermediate IM-23a as a yellow solid (2.50 g, crude).

[0275] Step 2: Preparation of 3-(5-bromo-6-fluoro-1-oxoisoindololin-2-yl)piperidine-2,6-dione (IM-23b)

[0276]

[0277] Compound IM-23a (2.30 g, 7.06 mmol) was dissolved in acetonitrile (15.0 mL), and 3-aminopiperidine-2,6-dione hydrochloride (1.39 g, 8.47 mmol) and N,N-diisopropylethylamine (2.74 g, 21.1 mmol) were added sequentially at 25 °C. The reaction mixture was stirred at 80 °C for 12 hours. The reaction mixture was diluted with water (30.0 mL) and extracted with ethyl acetate (30.0 mL x 3). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (silica gel) to give intermediate IM-23b as a brownish-black solid (1.00 g).

[0278] MS(ESI+)m / z 342.0[M+H] +

[0279] Step 3: Preparation of 3-(5-(4-(dimethoxymethyl)piperidin-1-yl)-6-fluoro-1-oxoisoindololin-2-yl)piperidin-2,6-dione (IM-23c)

[0280]

[0281] Compound IM-23b (1.00 g, 2.93 mmol) was dissolved in dimethyl sulfoxide (10.0 mL), and 4-(dimethoxymethyl)piperidine, cesium carbonate (2.87 g, 8.79 mmol), and Pd-PEPPSI-IPentCl (285 mg, 293 μmol) were added sequentially at room temperature. The reaction mixture was stirred at 80 °C for 12 hours. The reaction mixture was diluted with water (20.0 mL) and extracted with ethyl acetate (20.0 mL x 3). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (silica gel) to give intermediate IM-23c as a yellow solid (200 mg, crude).

[0282] MS(ESI+)m / z 420.1[M+H] +

[0283] Step 4: Preparation of 1-(2-(2,6-dioxopiperidin-3-yl)-6-fluoro-1-oxoisoindoline-5-yl)piperidin-4-carboxaldehyde (IM-23)

[0284]

[0285] Intermediate 23c (200 mg, 47.6 μmol) was dissolved in formic acid (2.00 mL) at room temperature. The reaction mixture was stirred at 55 °C for 12 hours, and then concentrated under reduced pressure to give intermediate IM-23 as a yellow oil (150 mg), which was used directly in the next step without purification.

[0286] MS(ESI+)m / z 374.1[M+H] +

[0287] Intermediate 24: 3-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)-3-azabicyclo[3.1.0]hexane-6-carboxaldehyde (IM-24)

[0288]

[0289] Step 1: Preparation of 6-ethyl 3-tert-butyl 3-(tert-butyl)-3,6-dicarboxylic acid (IM-24a) from 3-azabicyclo[3.1.0]hexane-3,6-dicarboxylic acid

[0290]

[0291] Ethyl diazonate (10.0 g, 59.0 mmol) and rhodium acetate dimer (261 mg, 590 μmol) were dissolved in dichloromethane (100 mL), and tert-butyl 2,5-dihydro-1H-pyrrole-1-carboxylate (10.1 g, 88.6 mmol) was slowly added dropwise. After the addition was complete, the resulting reaction mixture was stirred at 25 °C for 12 hours, and then concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate volume ratio = 100 / 1 to 10 / 1), and then purified by preparative HPLC (column: Welch Xtimate C18 250*70 mm #10 μm; mobile phase: [H2O (10 mM NH4HCO3)-ACN]; gradient: 40%-70% B, 18.0 min) to give intermediate IM-24a as a yellow oily compound (500 mg).

[0292] Step 2: Preparation of tert-butyl 6-(hydroxymethyl)-3-azabicyclo[3.1.0]hexane-3-carboxylate (IM-24b)

[0293]

[0294] At 0 °C, intermediate IM-24a (500 mg, 1.96 mmol) was dissolved in tetrahydrofuran (10.0 mL), and lithium aluminum hydride (2.50 M, 1.96 mL) was slowly added dropwise. The reaction mixture was stirred at 25 °C for 2 hours, then quenched with water (20 mL) at 0 °C, and extracted with dichloromethane (90 mL). The organic layer was washed with an aqueous sodium chloride solution (30 mL), dried over sodium sulfate, and concentrated by filtration under reduced pressure. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate v / v = 100 / 1 to 10 / 1) to give intermediate IM-24b as a yellow solid compound (250 mg).

[0295] Step 3: Preparation of (3-azabicyclo[3.1.0]hex-6-yl)methanol (IM-24c)

[0296]

[0297] Intermediate IM-24b (240 mg, 1.13 mmol) was dissolved in 1,4-dioxane (10.0 mL) at 25 °C, followed by the addition of HCl / dioxane (4 M, 8.44 mL). The reaction mixture was stirred for 2 hours and then concentrated under reduced pressure to obtain intermediate IM-24c as a white solid (200 mg), which was used directly in the next step without further purification.

[0298] 1 HNMR: (400MHz, CDCl3) δ3.49(brd,J=6.9Hz,2H),2.99(brd,J=11.3Hz,2H),2.87(brd,J=11.0Hz,2H),2.33(brs,2H),1.34(brs,2H),0.80-1.00(m,1H)

[0299] Step 4: Preparation of 2-(2,6-dioxadiazin-3-yl)-5-(6-(hydroxymethyl)-3-azabicyclo[3.1.0]hexane-3-yl)isoindoline-1,3-dione (IM-24d)

[0300]

[0301] Intermediate IM-24c (200 mg, 883 μmol) and 2-(2,6-dioxopiperidin-3-yl)-5-fluoroisoindoline-1,3-dione (244.10 mg, 883 μmol) were dissolved in N-methylpyrrolidone (5.00 mL) at room temperature, followed by the addition of diisopropylethylamine (342 mg, 2.65 mmol). The resulting reaction mixture was heated to 80 °C and stirred for 12 hours. The reaction mixture was concentrated under reduced pressure, and the residue was subjected to preparative HPLC (column: Waters Xbridge Prep OBD C18 150*40 mm*10 μm; mobile phase: [H2O(10 mM NH4HCO3)-ACN]; gradient: 2%-40% B, 8.0 min) to obtain intermediate IM-24d as a yellow solid (150 mg).

[0302] Step 5: Preparation of 3-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)-3-azabicyclo[3.1.0]hexane-6-carboxaldehyde (IM-24)

[0303]

[0304] Intermediate IM-24d (150 mg, 391 μmol) was dissolved in dichloromethane (5.00 mL) at room temperature, followed by the addition of Dys-Martin oxidant (342 mg, 810 μmol). The reaction mixture was stirred at 25 °C for 12 hours, then quenched with water (20 mL) at 0 °C, and extracted with dichloromethane (90.0 mL). The organic layer was washed with an aqueous sodium chloride solution (30 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure to give intermediate IM-24d as a yellow solid (100 mg).

[0305] Example 1:

[0306] N-(2-(1-((1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)piperidin-4-yl)methyl)piperidin-4-yl)-7-(2-hydroxypropyl-2-yl)imidazo[1,2-a]pyridin-6-yl)-6-(trifluoromethyl)pyridine-2-carboxamide

[0307]

[0308] IM-1 (100 mg, 206 μmol) and compound IM-9 (76.3 mg, 206 μmol) were dissolved in N,N-dimethylformamide (1.00 mL) and tetrahydrofuran (1.00 mL) at 25 °C, followed by the addition of sodium acetate (25.4 mg, 309 μmol). After stirring for 30 minutes, sodium triacetoxyborohydride (131 mg, 619 μmol) and glacial acetic acid (37.2 mg, 619 μmol) were added. The reaction mixture was stirred at 25 °C for 2 hours. The reaction mixture was filtered and concentrated. The residue was purified by preparative high-performance liquid chromatography (HPLC) (column: Waters Xbridge BEH C18 100*30 mm*10 μm; mobile phase: [water (NH4HCO3)-ACN]; B%: 30%-60%, 8 min) to give compound 1 as a yellow solid (30.0 mg).

[0309] 1 HNMR(400MHz,DMSO-d6)δ12.22(s,1H),11.08(s,1H),9.54(s,1H),8.34-8.50(m,2H),8.1 9(d,J=7.6Hz,1H),7.79(s,1H),7.65(d,J=8.6Hz,1H),7.41(s,1H),7.31(s,1H),7.23(br d,J=7.7Hz,1H),6.16(s,1H),5.06(dd,J=5.4,12.8Hz,1H),4.05(br d,J=12.0Hz,2H),2.78-3.08(m,5H),2.51-2.73(m,4H),2.20(br s,1H),1.92-2.02(m,4H),1.81(br d,J=13.6Hz,2H),1.69(br d,J=10.5Hz,2H),1.60(s,6H),1.41-1.55(m,1H),1.06-1.28(m,3H).

[0310] MS(ESI+)m / z 801.3[M+H] + .

[0311] Example 2:

[0312] N-(2-(1-(2-(4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)piperazin-1-yl)-2-oxoethyl)piperidin-4-yl)-7-(2-hydroxypropyl-2-yl)imidazo[1,2-a]pyridin-6-yl)-6-(trifluoromethyl)pyridine-2-carboxamide

[0313]

[0314] Intermediate IM-3 (100 mg, 197 μmol) and intermediate IM-10 (54.1 mg, 158 μmol) were dissolved in N,N-dimethylformamide (2.00 mL) at 25 °C, followed by the addition of N,N-diisopropylethylamine (102 mg, 791 μmol). The reaction mixture was stirred at 25 °C for 30 minutes. Then, O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethylurea hexafluorophosphine salt (112 mg, 296 μmol) was added to the reaction mixture, and the mixture was stirred at 25 °C for 2.5 hours. The reaction mixture was then concentrated under reduced pressure. The residue was purified by high performance liquid chromatography (column: Waters Xbridge BEH C18 100*30mm*10um; mobile phase: [water (NH4HCO3)-ACN]; B%: 40%-60%, 8min) to obtain compound 2 as a yellow solid (28.0mg).

[0315] 1 HNMR (400MHz, DMSO-d6) δ12.01-12.40(m,1H),9.54(s,1H),8.36-8.47(m,2H),8.19(d,J=7.6Hz,1H),7.79(s,1H),7.71(d,J=8.5Hz,1H),7.40(br d,J=9.9Hz,2H),7.29(br d,J=8.7Hz,1H),5.08(br dd,J=5.2,12.9Hz,1H),3.78(br s,2H),3.47-3.65(m,6H),3.23(br s,4H),2.84-2.96(m,3H),2.58-2.70(m,2H),2.12-2.21(m,2H),1.93–2.05(m,3H),1.65-1.74(m,2H),1.60(s,6H),1.22(br d,J=7.2Hz,1H).

[0316] MS(ESI+)m / z 830.3[M+H] +

[0317] Example 3:

[0318] N-(2-(1-(3-(4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)piperazin-1-yl)-3-oxopropyl)piperidin-4-yl)-7-(2-hydroxypropyl-2-yl)imidazo[1,2-a]pyridin-6-yl)-6-(trifluoromethyl)pyridine-2-carboxamide

[0319]

[0320] Compounds IM-4 (210 mg, 577 μmol) and IM-10 (158 mg, 461 μmol) were dissolved in N,N-dimethylformamide (4.00 mL) at 25 °C, followed by the addition of N,N-diisopropylethylamine (298 mg, 2.31 mmol). The reaction mixture was stirred at 25 °C for 30 min, and then O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethylurea hexafluorophosphine salt (329 mg, 866 μmol) was added. The reaction mixture was stirred at 25 °C for 2.5 h, followed by concentration under reduced pressure. The residue was purified by high-performance liquid chromatography (formic acid system) to give compound 3 as a yellow solid (30.0 mg).

[0321] 1 HNMR(400MHz,DMSO-d6)δ12.22(s,1H),11.08(s,1H),9.54(s,1H),8.34-8.50(m,2H),8.14-8.22(m,1H),7.64- 7.87(m,2H),7.33-7.45(m,2H),7.26(dd,J=2.0,8.4Hz,1H),6.16(s,1H),5.08(dd,J=5.3,12.8Hz,1H),3.64(br d,J=4.4Hz,5H),3.04-3.13(m,3H),2.81-2.94(m,2H),2.54-2.80(m,8H),2.34(br d,J=9.4Hz,2H),1.96-2.07(m,3H),1.68-1.79(m,2H),1.60(s,6H).

[0322] MS(ESI+)m / z 844.4[M+H] +

[0323] Example 4:

[0324] N-(2-(1-(4-(4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)piperazin-1-yl)-4-oxobutyl)piperidin-4-yl)-7-(2-hydroxypropyl-2-yl)imidazo[1,2-a]pyridin-6-yl)-6-(trifluoromethyl)pyridine-2-carboxamide

[0325]

[0326] Intermediate IM-5 (100 mg, 187 μmol) was dissolved in dimethylformamide (2.00 mL) at 25 °C, followed by the addition of O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethylurea hexafluorophosphine salt (142 mg, 374 μmol) and N,N-diisopropylethylamine (121 mg, 937 μmol). After stirring for 30 min, intermediate IM-10 (77.00 mg, 224 μmol) was added, and the reaction mixture was stirred at 25 °C for 11.5 h. The reaction mixture was concentrated under reduced pressure, and the residue was purified by preparative HPLC (column: Waters Xbridge BEH C18 100*30 mm*10 μm; mobile phase: [water (NH4HCO3)-ACN]; B%: 25%-55%, 8 min) to give compound 4 as a yellow solid (8.00 mg).

[0327] 1 HNMR(400MHz,DMSO-d6)δ12.23(s,1H),11.09(s,1H),9.56(s,1H),8.33-8.50( m,2H),8.14-8.21(m,1H),7.85(s,1H),7.71(d,J=8.4Hz,1H),7.24-7.42(m,2H ),6.19(s,1H),4.94–5.19(m,1H),3.58-3.68(m,8H),2.70-2.97(m,8H),2.54- 2.64(m,2H),2.29-2.46(m,2H),2.08-2.16(m,2H),1.98–2.05(m,1H),1.85(br s,5H),1.60(s,6H).

[0328] MS(ESI+)m / z 858.3[M+H] +

[0329] Example 5:

[0330] N-(2-(1-(2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)-2,7-diazaspiro[3.5]nonane-7-yl)-2-oxoethyl)piperidin-4-yl)-7-(2-hydroxypropyl-2-yl)imidazo[1,2-a]pyridin-6-yl)-6-(trifluoromethyl)pyridine-2-carboxamide

[0331]

[0332] Intermediate IM-3 (100 mg, 197 μmol) was dissolved in dimethylformamide (5.00 mL) at room temperature, followed by the addition of O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethylurea hexafluorophosphine salt (107 mg, 282 μmol) and N,N-diisopropylethylamine (109 mg, 847 μmol). The reaction mixture was stirred for 15 min, followed by the addition of intermediate IM-11 (54.0 mg, 141 μmol). The reaction mixture was stirred at room temperature for 12 h, followed by concentration under reduced pressure. The residue was purified by preparative HPLC (column: Waters Xbridge BEH C18 250*50 mm*10 μm; mobile phase: [water (NH4HCO3)-ACN]; B%: 25%-55%, 10 min) to give compound 5 as a yellow solid (14.7 mg).

[0333] 1 HNMR(400MHz,DMSO-d6)δ12.22(s,1H),11.06(s,1H),9.54(s,1H),8.50-8.33(m,2H),8.1 9(d,J=7.8Hz,1H),7.79(s,1H),7.65(d,J=8.3Hz,1H),7.41(s,1H),6.77(s,1H),6.65(br d,J=8.4Hz,1H),6.14(s,1H),5.05(dd,J=5.3,12.9Hz,1H),3.82(s,4H),3.42- 3.59(m,2H),2.81-2.96(m,4H),2.52-2.71(m,6H),2.09-2.19(m,2H),1.97(br d,J=10.8Hz,3H),1.81(br s,2H),1.65-1.75(m,4H),1.60(s,6H).

[0334] MS(ESI+)m / z 870.3[M+H] + .

[0335] Example 6:

[0336] N-(2-(1-(2-(7-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)-2,7-diazaspiro[3.5]nonane-2-yl)-2-oxoethyl)piperidin-4-yl)-7-(2-hydroxypropyl-2-yl)imidazo[1,2-a]pyridin-6-yl)-6-(trifluoromethyl)pyridine-2-carboxamide

[0337]

[0338] Intermediate IM-3 (200 mg, 395 μmol) and O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethylurea hexafluorophosphine salt (90.7 mg, 238 μmol) were dissolved in dichloromethane (3.00 mL), and the reaction mixture was stirred at 25 °C for 1 hour. Intermediate IM-12 (100 mg, 238 μmol) and N,N-diisopropylethylamine (30.8 mg, 238 μmol) were added sequentially, and the resulting reaction mixture was stirred at 25 °C for 2 hours. The reaction mixture was concentrated under reduced pressure, and the residue was purified by preparative HPLC (column: Waters Xbridge BEH C18 250*50 mm*10 μm; mobile phase: [water (NH4HCO3)-ACN]; B%: 25%-55%, 10 min) to give compound 6 as a yellow solid (17.8 mg).

[0339] 1 HNMR(400MHz,DMSO-d6)δ12.22(s,1H),11.08(br s,1H),9.53(s,1H),8.31-8.51(m,2H),8.18(d,J=7.7Hz,1H),7.79(s,1H),7.65(d,J=8. 4Hz,1H),7.21-7.46(m,3H),6.16(s,1H),5.05(s,1H),3.98(s,2H),3.63(s,2H),3.47(br s,4H),2.94–3.07(m,2H),2.79-2.93(m,3H),2.60(br d,J=2.7Hz,3H),2.16(br s,2H),1.96(br d,J=13.7Hz,3H),1.53-1.82(m,12H).

[0340] MS(ESI+)m / z 870.3[M+H] + .

[0341] Example 7:

[0342] N-(2-(1-((1-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)piperidin-4-yl)methyl)piperidin-4-yl)-7-(2-hydroxypropyl-2-yl)imidazo[1,2-a]pyridin-6-yl)-6-(trifluoromethyl)pyridin-2-carboxamide

[0343]

[0344] Intermediate IM-1 (100 mg, 223 μmol) and intermediate IM-13 (79.4 mg, 223 μmol) were dissolved in N,N-dimethylformamide (2.00 mL) and tetrahydrofuran (2.00 mL) at 25 °C. Sodium acetate (27.5 mg, 335 μmol) was then added, and the reaction mixture was stirred at 25 °C for 30 minutes. Sodium triacetoxyborohydride (27.5 mg, 335 μmol) and glacial acetic acid (40.2 mg, 670 μmol) were then added, and the reaction mixture was stirred at 25 °C for 11.5 hours. The reaction mixture was concentrated under reduced pressure, and the residue was purified by preparative high performance liquid chromatography (column: Waters Xbridge BEH C18 100*30mm*10um; mobile phase: [water (NH4HCO3)-ACN]; B%: 35%-65%, 8min) to give compound 7 as a white solid (42.0 mg).

[0345] 1 HNMR(400MHz,DMSO-d6)δ12.22(s,1H),10.93(br s,1H),9.54(s,1H),8.48-8.35(m,2H),8.19(br d,J=7.8Hz,1H),7.78(s,1H),7.50(br d,J=8.6Hz,1H),7.41(s,1H),7.10-6.98(m,2H),6.15(s,1H),5.04(br dd,J=4.9,13.1Hz,1H),4.32(br d,J=17.0Hz,1H),4.19(br d,J=16.6Hz,1H),3.87(br d,J=11.9Hz,2H),2.97-2.77(m,5H),2.70-2.54(m,2H),2.42-2.31(m,1H),2.22-2.14(m,2H),2.06-1.91(m,5H),1.80(br d,J=10.9Hz,3H),1.72-1.65(m,2H),1.63-1.54(m,6H),1.23(br s,1H),1.19-1.10(m,1H).

[0346] MS(ESI+)m / z 787.4[M+H] + .

[0347] Example 8:

[0348] N-(2-(1-((1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)piperidin-4-yl)methyl)piperidin-4-yl)-7-methoxyimidazo[1,2-a]pyridin-6-yl)-6-(trifluoromethyl)pyridine-2-carboxamide

[0349]

[0350] Intermediate IM-2 (400 mg, 953 μmol) and intermediate IM-9 (422 mg, 1.14 mmol) were dissolved in N,N-dimethylformamide (4.00 mL), followed by the addition of sodium acetate (117 mg, 1.43 mmol). The reaction mixture was stirred for 30 minutes, followed by the addition of sodium triacetoxyborohydride (606 mg, 2.86 mmol) and acetic acid (171 mg, 2.86 mmol). The reaction mixture was stirred at 25 °C for 12 hours, followed by dilution with water (10.0 mL). The resulting mixture was extracted with ethyl acetate (10 mL x 3), the combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by preparative HPLC (column: Phenomenex luna C18 80*40 mm*3 μm; mobile phase: [water (FA)-ACN]; B%: 1%-35%, 8 min) to give compound 8 as a yellow solid (12.0 mg).

[0351] 1 HNMR (400MHz, DMSO-d6) δ11.07(s,1H),10.19(s,1H),9.35(s,1H),8.49-8.37(m,2H),8.21-8.26(m,1H),7.65(t,J=4.2Hz,2H),7.31( d,J=1.6Hz,1H),7.21-7.27(m,1H),7.07(s,1H),5.02-5.11(m,1H),3.97–4.07(m,5H),2.89–3.03(m,5H),2.54-2.66(m,3H),2.19(br d,J=6.6Hz,2H),1.93–2.07(m,5H),1.77-1.86(m,3H),1.61-1.72(m,2H),1.12-1.26(m,2H).

[0352] MS(ESI+)m / z 773.2[M+H] + .

[0353] Example 9:

[0354] N-(2-(1-(2-(4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)piperazin-1-yl)ethyl)piperidin-4-yl)-7-(2-hydroxypropyl-2-yl)imidazo[1,2-a]pyridin-6-yl)-6-(trifluoromethyl)pyridine-2-carboxamide

[0355]

[0356] Intermediate IM-1 (23.2 mg, 52.0 μmol) and intermediate IM-14 (20.0 mg, 52.0 μmol) were dissolved in N,N-dimethylformamide (1.00 mL) and 1,2-dichloroethane (1.00 mL) at 25 °C. N,N-diisopropylethylamine (20.1 mg, 156 μmol) was added, and the reaction mixture was stirred at 25 °C for 30 min. Glacial acetic acid (9.37 mg, 156 μmol) and sodium triacetoxyborohydride (16.5 mg, 78.0 μmol) were added, and the reaction mixture was stirred at 25 °C for 11.5 h. Water (3.00 mL) was added, and the mixture was extracted with ethyl acetate (5.00 mL x 3). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by preparative high-performance liquid chromatography (formic acid system) to give compound 9 as a yellow solid (2.00 mg).

[0357] 1 HNMR(400MHz,DMSO-d6)δ12.23(s,1H),11.08(br s,1H),9.54(s,1H),8.32-8.52(m,2H),8.10-8.29(m,2H),7.79(s,1H),7.68(br d,J=8.4Hz,1H),7.20-7.47(m,3H),6.15(br s,1H),5.08(br dd,J=5.0,12.6Hz,1H),2.75–3.07(m,5H),2.67-2.67(m,1H),2.53-2.69(m,10H),1.86–2.24(m,6H),1.65-1.76(m,2H),1.61(s,6H).

[0358] MS(ESI+)m / z 816.3[M+H] + .

[0359] Example 10:

[0360] N-(2-(1-(2-(4-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)piperazin-1-yl)ethyl)piperidin-4-yl)-7-(2-hydroxypropyl-2-yl)imidazo[1,2-a]pyridin-6-yl)-6-(trifluoromethyl)pyridine-2-carboxamide

[0361]

[0362] Intermediate IM-6 (40.2 mg, 122 μmol) was dissolved in 1,2-dichloroethane (1.00 mL) and N,N-dimethylformamide (1.00 mL) at 25 °C, followed by the addition of triethylamine (31.0 mg, 306 μmol), and the reaction mixture was stirred at 25 °C for 0.5 h. Acetic acid (6.13 mg, 102 μmol), sodium triacetoxyborohydride (32.4 mg, 153 μmol), and intermediate IM-21 (50.0 mg, 102 μmol) were added, and the reaction mixture was stirred at 25 °C for 11.5 h, followed by concentration under reduced pressure. The residue was purified by preparative HPLC (column: Phenomenex Luna C1875*30 mm*3 μm; mobile phase: [water (FA)-ACN]; B%: 5%-35%, 8 min) to give compound 10 as a white solid (12.0 mg).

[0363] 1 HNMR(400MHz,DMSO-d6)δ12.22(s,1H),10.49-11.50(m,1H),9.54(s,1H),8.36-8.47(m,2H),8.19(d,J= 8.1Hz,1H),7.78(s,1H),7.44-7.58(m,1H),7.41(s,1H),7.01-7.12(m,2H),6.15(s,1H),5.05(dd,J=5.0 ,13.3Hz,1H),4.33(d,J=17.0Hz,1H),4.20(d,J=16.8Hz,1H),3.22-3.32(m,8H),2.84-3.02(m,3H),2.5 3-2.68(m,6H),2.28-2.46(m,2H),2.04-2.13(m,2H),1.93-1.98(m,2H),1.63-1.72(m,2H),1.60(s,6H).

[0364] MS(ESI+)m / z 802.3[M+H] + .

[0365] Example 11:

[0366] N-(2-(1-(2-(4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)piperazin-1-yl)ethyl)piperidin-4-yl)-7-methoxyimidazo[1,2-a]pyridin-6-yl)-6-(trifluoromethyl)pyridine-2-carboxamide

[0367]

[0368] Intermediate IM-2 (50.0 mg, 119 μmol) was dissolved in N,N-dimethylformamide (1.00 mL) and 1,2-dichloroethane (1.00 mL) at room temperature, followed by the addition of N,N-diisopropylethylamine (61.0 mg, 476 μmol). The reaction mixture was stirred for 15 minutes, and then sodium triacetoxyborohydride (75.8 mg, 357 μmol), glacial acetic acid (7.16 mg, 119 μmol), and intermediate IM-14 (45.8 mg, 119 μmol) were added. The reaction mixture was stirred at room temperature for 12 hours, followed by concentration under reduced pressure. The residue was purified by preparative HPLC (column: Phenomenex luna C1880*40 mm*3 μm; mobile phase: [water (FA)-ACN]; B%: 1%-35%, 8 min) to give compound 11 as a yellow solid (7.50 mg).

[0369] 1 HNMR(400MHz,DMSO-d6)δ11.08(br s,1H),10.19(s,1H),9.35(s,1H),8.37-8.49(m,2H),8.23(br d,J=7.5Hz,1H),7.57-7.75(m,2H),7.19-7.43(m,2H),7.07(s,1H),5.01-5.15(m,1H),3.98(s,3H ),3.46-3.49(m,4H),2.82-3.03(m,4H),2.52-2.71(m,9H),1.89-2.32(m,6H),1.59-1.73(m,2H).

[0370] MS(ESI+)m / z 788.3[M+H] + .

[0371] Example 12:

[0372] N-(2-(1-(2-(4-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)piperazin-1-yl)ethyl)piperidin-4-yl)-7-methoxyimidazo[1,2-a]pyridin-6-yl)-6-(trifluoromethyl)pyridine-2-carboxamide

[0373]

[0374] Intermediate IM-21 (39.1 mg, 119 μmol) was dissolved in N,N-dimethylformamide (1.00 mL) and dichloroethane (1.00 mL), followed by the addition of N,N-diisopropylethylamine (70.0 mg, 541 μmol). The reaction mixture was stirred at 25 °C for 30 minutes, and then sodium borohydride acetate (68.9 mg, 325 μmol), acetic acid (6.51 mg, 108 μmol), and intermediate IM-7 (50.0 mg, 108 μmol) were added. The reaction mixture was stirred at 25 °C for 11.5 hours, followed by concentration under reduced pressure. The residue was purified by preparative HPLC (column: Waters Xbridge BEH C18 100*30mm*10um; mobile phase: [H2O(10mMNH4HCO3)-ACN]; gradient: 20%-50% B, 8.0min) to obtain compound 12 as a white solid (8.00mg).

[0375] 1 HNMR(400MHz,DMSO-d6)δ10.93(br s,1H),10.19(s,1H),9.35(s,1H),8.37-8.49(m,2H),8.23(br d,J=7.8Hz,1H),7.64(s,1H),7.52(d,J=8.6Hz,1H),7.07(s,2H),4.98–5.11(m,1H),4.17-4.36(m,2H),3.98(s,3H),3.29(br d,J=3.6Hz,6H),2.84–3.02(m,4H),2.56-2.68(m,8H),2.33-2.43(m,1H),2.08-2.20(m,2H),1.89–2.02(m,3H),1.58-1.74(m,2H).

[0376] MS(ESI+)m / z 774.3[M+H] + .

[0377] Example 13:

[0378] N-(2-(1-(2-(4-(2-(2,6-dioxopiperidin-3-yl)-3-oxoisoindolin-5-yl)piperazin-1-yl)ethyl)piperidin-4-yl)-7-(2-hydroxypropyl-2-yl)imidazo[1,2-a]pyridin-6-yl)-6-(trifluoromethyl)pyridine-2-carboxamide

[0379]

[0380] Intermediate IM-6 (161 mg, 328 μmol) was dissolved in 1,2-dichloroethane (5.00 mL) and N,N-dimethylformamide (5.00 mL) at 25 °C, followed by the addition of triethylamine (106 mg, 822 μmol). The reaction mixture was stirred for 0.5 h, and then acetic acid (16.4 mg, 274 μmol), sodium triacetoxyborohydride (87.0 mg, 410 μmol), and intermediate IM-22 (100 mg, 274 μmol) were added. The reaction mixture was stirred at 25 °C for 12 h, followed by concentration under reduced pressure. The residue was purified by preparative HPLC (column: Phenomenex Gemini NX-C18 (75*30mm*3um); mobile phase: [H2O(0.05% NH3H2O+10mM NH4HCO3)-ACN]; gradient: 20%-50% B, 8.0 min) to obtain compound 13 as an off-white solid (10.2 mg).

[0381] 1 HNMR(400MHz,DMSO-d6)δ12.22(s,1H),10.97(br s,1H),9.54(s,1H),8.36-8.48(m,2H),8.19(d,J=7.7Hz,1H),7.78(s,1H),7.38-7.46(m,2H),7.21-7.31(m,1H),7.11-7.20(m,1H),6. 15(s,1H),5.10(dd,J=5.1,13.3Hz,1H),4.33(d,J=16.8Hz,1H),4.20(d,J=16.8Hz,1H),3.11-3.26(m,4H),2.84-3.04(m,3H),2.67(br d,J=1.5Hz,1H),2.58(brs,4H),2.50(br s,4H),2.29-2.46(m,2H),2.07-2.20(m,2H),1.88-2.02(m,3H),1.63-1.77(m,2H),1.60(s,6H).

[0382] MS(ESI+)m / z 802.3[M+H] + .

[0383] Example 14:

[0384] N-(2-(1-(2-(4-(2-(2,6-dioxopiperidin-3-yl)-3-oxoisoindolin-5-yl)piperazin-1-yl)ethyl)piperidin-4-yl)-7-methoxyimidazo[1,2-a]pyridin-6-yl)-6-(trifluoromethyl)pyridine-2-carboxamide

[0385]

[0386] Intermediate IM-22 (39.1 mg, 119 μmol) was dissolved in N,N-dimethylformamide (0.50 mL) and 1,2-dichloroethane (0.5 mL), followed by the addition of N,N-diisopropylethylamine (56.0 mg, 433 μmol), intermediate IM-7 (50.0 mg, 108 μmol), sodium triacetoxyborohydride (68.9 mg, 325 μmol), and glacial acetic acid (6.51 mg, 108 μmol). The reaction mixture was stirred at 25 °C for 12 hours, followed by concentration under reduced pressure. The residue was purified by preparative HPLC (column: Phenomenexluna C18 80*40 mm*3 μm; mobile phase: [water (FA)-ACN]; gradient: 1%-35% B, 8 min) to give compound 14 as a yellow solid (8.00 mg).

[0387] 1 HNMR(400MHz,DMSO-d6)δ10.97(s,1H),10.19(s,1H),9.35(s,1H),8.38-8.47(m,2H),8.21-8.26(m,1H),7.65(s,1H),7.43(d,J=8.5Hz,1 H),7.24-7.29(m,1H),7.16(d,J=2.0Hz,1H),7.07(s,1H),5.05-5.13(m,1H),4.30-4.38(m,1H),4.17-4.24(m,1H),3.98(s,3H),3.19(br s,4H),2.85–3.02(m,4H),2.58-2.61(m,4H),2.53(br s,4H),2.32-2.43(m,2H),2.16(br t,J=10.8Hz,2H),1.93–2.01(m,3H),1.62-1.73(m,2H).

[0388] MS(ESI+)m / z 774.3[M+H] + .

[0389] Example 15:

[0390] N-(2-(1-((1-(2-(2,6-dioxopiperidin-3-yl)-6-fluoro-1,3-dioxoisoindolin-5-yl)piperidin-4-yl)methyl)piperidin-4-yl)-7-(2-hydroxypropyl-2-yl)imidazo[1,2-a]pyridin-6-yl)-6-(trifluoromethyl)pyridin-2-carboxamide

[0391]

[0392] Intermediate IM-1 (173 mg, 387 μmol) was dissolved in N,N-dimethylformamide (1.00 mL) and tetrahydrofuran (1.00 mL) at room temperature, followed by the addition of sodium acetate (47.6 mg, 580 μmol), intermediate IM-15 (150 mg, 387 μmol), sodium borohydride acetate (246 mg, 1.16 mmol), and sodium acetate (69.7 mg, 1.16 mmol). The reaction mixture was stirred at 25 °C for 12 hours and concentrated under reduced pressure. The resulting residue was purified by preparative HPLC (column: Waters Xbridge BEH C18 250*50 mm*10 μm; mobile phase: [water (NH4HCO3)-ACN]; B%: 25%-55%, 10 min) to give compound 15 as a yellow solid (11.0 mg).

[0393] 1 HNMR(400MHz,DMSO-d6)δ12.23(s,1H),11.12(br s,1H),9.54(s,1H),8.29-8.53(m,2H),8.18(br d,J=7.5Hz,1H),7.63-7.83(m,2H),7.38-7.50(m,2H),6.17(s,1H),5.00-5.18(m,1H),3.60(br d,J=11.2Hz,2H),2.81-2.97(m,5H),2.52-2.67(m,3H),2.19(br d,J=5.4Hz,2H),1.92-2.06(m,5H),1.65-1.87(m,5H),1.60(br s, 6H), 1.25 (br d, J = 10.4Hz, 2H).

[0394] MS(ESI+)m / z 819.3[M+H] + .

[0395] Example 16:

[0396] N-(2-(1-(2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)-2,7-diazaspiro[3.5]nonane-7-yl)ethyl)piperidin-4-yl)-7-(2-hydroxypropyl-2-yl)imidazo[1,2-a]pyridin-6-yl)-6-(trifluoromethyl)pyridine-2-carboxamide

[0397]

[0398] Compound IM-11 (200 mg, 402 μmol) was dissolved in N,N-dimethylformamide (2.00 mL) and 1,2-dichloroethane (2.00 mL) at room temperature, followed by the addition of N,N-diisopropylethylamine (208 mg, 1.61 mmol). After stirring for 15 minutes, intermediate IM-6 (197 mg, 402 μmol), sodium borohydride acetate (256 mg, 1.21 mmol), and acetic acid (24.19 mg, 402 μmol) were added. The reaction mixture was stirred at room temperature for 12 hours, then diluted with water (10 mL). The aqueous phase was extracted with ethyl acetate (10 mL x 3), the combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by preparative HPLC (column: Waters Xbridge Prep OBD C18 150*40mm*10um; mobile phase: [H2O(10mM NH4HCO3)-ACN]; gradient: 30%-65% B, 8.0min) to obtain compound 16 as a yellow solid (18.0mg).

[0399] 1 HNMR(400MHz,DMSO-d6)δ12.22(s,1H),11.06(br s,1H),9.53(s,1H),8.35-8.48(m,2H),8.19(d,J=7.8Hz,1H),7.78(s,1H),7 .63(d,J=8.3Hz,1H),7.41(s,1H),6.77(d,J=1.6Hz,1H),6.61-6.69(m,1H), 6.15(s,1H),4.99–5.11(m,1H),3.74(s,3H),2.82-2.59(m,3H),2.54-2.68( m,3H),2.33-2.46(m,8H),1.90-2.14(m,6H),1.63-1.77(m,6H),1.60(s,6H).

[0400] Example 17:

[0401] N-(2-(1-(2-(7-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)-2,7-diazaspiro[3.5]nonane-2-yl)ethyl)piperidin-4-yl)-7-(2-hydroxypropyl-2-yl)imidazo[1,2-a]pyridin-6-yl)-6-(trifluoromethyl)pyridine-2-carboxamide

[0402]

[0403] Compound IM-12 (150 mg, 302 μmol) and DIEA (156 mg, 1.21 mmol) were dissolved in 1,2-dichloroethane (2.00 mL) and N,N-dimethylformamide (2.00 mL) at 25 °C. The resulting mixture was stirred for 0.5 h, and then sodium triacetoxyborohydride (192 mg, 906 μmol), compound IM-6 (158 mg, 302 μmol), and acetic acid (18.1 mg, 302 μmol) were added sequentially. The reaction mixture was stirred at 25 °C for 12 h, and then concentrated under reduced pressure. The residue was purified by preparative HPLC (column: Phenomenex Gemini NX-C18 (75*30mm*3um); mobile phase: [H2O(0.05% NH3H2O+10mM NH4HCO3)-ACN]; gradient: 20%-40% B, 8.0min) to obtain compound 17 as an off-white solid (6.00mg).

[0404] 1 HNMR(400MHz,DMSO-d6)δ12.22(s,1H),9.54(s,1H),8.33-8.52(m,2H),8.25(s,1H),8.1 9(d,J=7.6Hz,1H),7.78(s,1H),7.65(d,J=8.6Hz,1H),7.40(s,1H),7.32(s,1H),7.24(br d,J=8.8H z,1H),5.93-6.34(m,1H),5.06(dd,J=5.4,12.9Hz,1H),3.44(br s,5H),3.13(s,4H),2.84-2.98(m,3H),2.53-2.72(m,5H),2.34(br t,J=6.2Hz,2H),2.10(brt,J=10.9Hz,2H),1.91-2.03(m,3H),1.76(br s,4H),1.67(br d,J=10.4Hz,1H),1.60(s,6H).

[0405] Example 18: N-(2-(1-(2-(1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)piperidin-4-yl)ethyl)piperidin-4-yl)-7-(2-hydroxypropyl-2-yl)imidazo[1,2-a]pyridin-6-yl)-6-(trifluoromethyl)pyridine-2-carboxamide

[0406]

[0407] Compound IM-1 (200 mg, 413 μmol) was dissolved in dichloroethane (2.00 mL) and N,N-dimethylformamide (2.00 mL), followed by the addition of N,N-diisopropylethylamine (213 mg, 1.65 mmol). After stirring for 5 minutes, intermediate IM-16 (290 mg, 454 μmol), sodium borohydride acetate (262 mg, 1.24 mmol), and acetic acid (24.8 mg, 413 μmol) were added. The reaction mixture was stirred at 25 °C for 12 hours, and then concentrated to dryness under reduced pressure. The residue was purified by high-performance liquid chromatography (formic acid system) to give compound 18 as a yellow solid (21.0 mg).

[0408] 1 HNMR(400MHz,DMSO-d6)δ12.22(s,1H),11.07(s,1H),9.54(s,1H),8.32-8.50(m,2H),8.17-8.19(m,1H),7.78(s, 1H),7.65(d,J=8.5Hz,1H),7.41(s,1H),7.30(s,1H),7.21-7.25(m,1H),6.14(s,1H),4.98–5.13(m,1H),4.04(br d,J=12.8Hz,2H),2.82–3.00(m,5H),2.55-2.68(m,3H),2.32-2.44(m,2H),1.93–2 .12(m,5H),1.62-1.82(m,5H),1.60(s,6H),1.38-1.48(m,2H),1.13-1.26(m,2H).

[0409] MS(ESI+)m / z 815.3[M+H] + .

[0410] Example 19:

[0411] N-(2-(1-(3-(4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)piperazin-1-yl)propyl)piperidin-4-yl)-7-(2-hydroxypropyl-2-yl)imidazo[1,2-a]pyridin-6-yl)-6-(trifluoromethyl)pyridine-2-carboxamide

[0412]

[0413] Intermediate IM-1 (85.0 mg, 189 μmol) was dissolved in N,N-dimethylformamide (3.00 mL) at room temperature, followed by the addition of N,N-diisopropylethylamine (73.6 mg, 569 μmol), sodium iodide (85.43 mg, 569 μmol), and intermediate IM-17 (200 mg, 208 μmol). The reaction mixture was heated to 80 °C and stirred for 12 hours, then concentrated under reduced pressure. The resulting residue was purified by high-performance liquid chromatography (ammonium bicarbonate system) to give compound 19 (15.0 mg).

[0414] 1 HNMR(400MHz,DMSO-d6)δ12.22(s,1H),11.07(br s,1H),9.54(s,1H),8.35-8.48(m,2H),8.19(d,J=7.7Hz,1H),7.62-7.85(m,2H),7.24-7.43(m,3H),6.13(s,1H),4.99–5.15(m,1H),3.44(br s,4H),2.83-2.95(m,3H),2.52-2.69(m,6H),2.31-2.38(m,4H),1.94–2.05(m,5H),1.62-1.74(m,5H),1.60(s,6H).

[0415] MS(ESI+) m / z 830.4 [M+H] + .

[0416] Example 20:

[0417] N-(2-(1-(1-(1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)piperidin-4-yl)azacyclobutane-3-yl)piperidin-4-yl)-7-(2-hydroxypropyl-2-yl)imidazo[1,2-a]pyridin-6-yl)-6-(trifluoromethyl)pyridine-2-carboxamide

[0418]

[0419] Intermediate IM-8 (100 mg, 185 μmol) was dissolved in N,N-dimethylformamide (2.00 mL) and tetrahydrofuran (2.00 mL), followed by the addition of sodium acetate (30.4 mg, 371 μmol). After stirring for 15 minutes, sodium triacetoxyborohydride (117 mg, 556 μmol), intermediate IM-18 (82.4 mg, 185 μmol), and acetic acid (33.4 mg, 556 μmol) were added. The reaction mixture was stirred at 25 °C for 12 hours, then filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by high-performance liquid chromatography (ammonium bicarbonate system) to give compound 20 as a yellow solid (25 mg).

[0420] 1 HNMR (400MHz, DMSO-d6) δ12.22(s,1H),11.07(s,1H),9.54(s,1H),8.34-8.52(m,2H),8.19(d,J=7.7Hz,1H),7.78(s,1H),7. 65(d,J=8.6Hz,1H),7.40(s,1H),7.31(s,1H),7.19-7.26(m,1H),6.14(s,1H),5.00-5.12(m,1H),3.79-3.88(m,2H),3.40(br t,J=4.8Hz,2H),3.07-3.18(m,2H),2.75-2.94(m,6H),2.54-2.68(m,3H),2.25-2 .33(m,1H),1.82–2.07(m,6H),1.64-1.73(m,3H),1.60(s,6H),1.18-1.27(m,2H).

[0421] MS(ESI+)m / z 842.4[M+H] + .

[0422] Example 21:

[0423] N-(2-(1-(2-(6-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)-2,6-diazaspiro[3.3]heptane-2-yl)ethyl)piperidin-4-yl)-7-(2-hydroxypropyl-2-yl)imidazo[1,2-a]pyridin-6-yl)-6-(trifluoromethyl)pyridine-2-carboxamide

[0424]

[0425] Intermediate IM-19 (100 mg, 282 μmol), N,N-diisopropylethylamine (100 mg, 282 μmol), and N,N-dimethylformamide (2.00 mL) were dissolved in 1,2-dichloroethane (2.00 mL) and N,N-dimethylformamide (2.00 mL) at 25 °C. After stirring for 0.5 hours, sodium triacetoxyborohydride (179 mg, 846 μmol), compound IM-6 (100 mg, 282 μmol), and acetic acid (16.9 mg, 282 μmol) were added sequentially. The reaction mixture was stirred at 25 °C for 12 hours and then concentrated under reduced pressure. The residue was purified by preparative HPLC (column: Waters Xbridge BEH C18 100*30mm*10um; mobile phase: [H2O(10mM NH4HCO3)-ACN]; gradient: 20%-65%B, 8.0min). The crude product was then purified again by preparative HPLC (column: Phenomenexluna C18 100*40mm*5um; mobile phase: [H2O(0.2%FA)-ACN]; gradient: 5%-45%B, 8.0min) to obtain compound 21 as a white solid (10.4mg).

[0426] 1 HNMR(400MHz,DMSO-d6)δ12.22(s,1H),11.06(s,1H),9.53(s,1H),8.36-8.47(m,2H),8 .16-8.24(m,1H),7.77(s,1H),7.64(d,J=8.2Hz,1H),7.40(s,1H),6.79(s,1H),6.65(br d,J=8.2Hz,1H),6.13(s,1H),5.05(dd,J=5.4,12.7Hz,1H),4.10(s,4H),3.33-3.34(m,4H),2.82-2. 97(m,3H),2.52-2.69(m,5H),2.27-2.35(m,2H),1.89-2.11(m,5H),1.62-1.74(m,2H),1.60(s,6H).

[0427] MS(ESI+)m / z 828.4[M+H] + .

[0428] Example 22:

[0429] N-(2-(1-(2-(4-(2-(2,6-dioxopiperidin-3-yl)-6-fluoro-1,3-dioxoisoindolin-5-yl)piperazin-1-yl)ethyl)piperidin-4-yl)-7-(2-hydroxypropyl-2-yl)imidazo[1,2-a]pyridin-6-yl)-6-(trifluoromethyl)pyridine-2-carboxamide

[0430]

[0431] Intermediate IM-20 (100 mg, 207 μmol) and intermediate IM-1 (92.7 mg, 207 μmol) were dissolved in dimethylformamide (2.00 mL) at 25 °C, followed by the addition of N,N-diisopropylethylamine (80.3 mg, 621 μmol) and sodium iodide (93.2 mg, 621 μmol). The reaction mixture was stirred at 80 °C for 12 hours, followed by concentration under reduced pressure. The residue was purified by high-performance liquid chromatography (ammonium bicarbonate system) to give compound 22 as a yellow solid (5.30 mg).

[0432] 1 HNMR(400MHz,DMSO-d6)δ12.22(s,1H),11.11(br s,1H),9.53(s,1H),8.32-8.50(m,2H),8.13-8.28(m,1H),7.69-7.82(m,2H),7.38-7.48(m,2H),6.15(br s,1H),5.03-5.17(m,1H),3.25(br s,6H),2.81-3.04(m,4H),2.55-2.70(m,7H),1.90(br s,6H),1.64-1.74(m,2H),1.60(s,6H).

[0433] MS(ESI+)m / z 834.3[M+H] + .

[0434] Example 23:

[0435] N-(2-(1-((1-(2-(2,6-dioxopiperidin-3-yl)-6-fluoro-1-oxoisoindolin-5-yl)piperidin-4-yl)methyl)piperidin-4-yl)-7-(2-hydroxypropyl-2-yl)imidazo[1,2-a]pyridin-6-yl)-6-(trifluoromethyl)pyridine-2-carboxamide

[0436]

[0437] Intermediate IM-1 (155 mg, 321 μmol) was dissolved in N,N-dimethylformamide (2.00 mL) and tetrahydrofuran (2.00 mL) at room temperature, followed by the addition of sodium acetate (79 mg, 964 μmol). The mixture was stirred for 15 minutes, and then intermediate IM-23 (150 mg, 321 μmol), sodium acetate (102 mg, 482 μmol), and acetic acid (57.9 mg, 964 μmol) were added. The reaction mixture was stirred at 25 °C for 12 hours, and then concentrated to dryness under reduced pressure. The residue was purified by high performance liquid chromatography (HPLC) (column: Waters Xbridge BEH C18 250*50 mm*10 μm; mobile phase: [water (NH4HCO3)-ACN]; B%: 25%-55%, 10 min) to give compound 23 as a white solid (20.0 mg).

[0438] 1 HNMR(400MHz,DMSO-d6)δ12.22(s,1H),10.96(br s,1H),9.54(s,1H),8.43-8.49(m,1H),8.36-8.41(m,1H),8.16-8.23(m,1H),7.78(s,1H),7.37-7.43(m,2H),7.23(br d,J=7.6Hz,1H),6.14(s,1H),5.02-5.11(m,1H),4.32-4.40(m,1H),4.20-4.28(m,1H),3.48(br d,J=2.1Hz,2H),2.86-2.96(m,3H),2.73-2.79(m,2H),2.61-2.68(m,2H),2.32-2.40(m,1H),2.22(br d,J=7.0Hz,2H),1.94–2.07(m,5H),1.83(br d,J=11.6Hz,2H),1.75-1.66(m,3H),1.60(s,6H),1.35-1.24(m,2H).

[0439] MS(ESI+)m / z 805.4[M+H] + .

[0440] Example 24:

[0441] N-(2-(1-((3-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)-3-azabicyclo[3.1.0]hexane-6-yl)methyl)piperidin-4-yl)-7-(2-hydroxypropyl-2-yl)imidazo[1,2-a]pyridin-6-yl)-6-(trifluoromethyl)pyridine-2-carboxamide

[0442]

[0443] Intermediate IM-1 (131 mg, 272 μmol) was dissolved in N,N-dimethylformamide (0.50 mL) and tetrahydrofuran (0.50 mL), and sodium acetate (33.5 mg, 0.406 mmol) was added. The mixture was stirred at 25 °C for 0.5 h. Sodium triacetoxyborohydride (173 mg, 0.816 mmol), intermediate IM-24 (100 mg, 272 μmol), and acetic acid (49.0 mg, 0.816 mmol) were added sequentially, and the resulting reaction mixture was stirred at 25 °C for 12 h. The reaction mixture was concentrated under reduced pressure, and the residue was purified by preparative HPLC (column: Waters Xbridge Prep OBD C18 150*40 mm*10 μm; mobile phase: [H2O (10 mM NH4HCO3)-ACN]; gradient: 30%-70% B, 8.0 min) to give compound 24 as a yellow solid (11.0 mg).

[0444] 1 HNMR(400MHz,DMSO-d6)δ12.22(s,1H),11.06(br s,1H),9.54(s,1H),8.36-8.48(m,2H),8.19(d,J=7.7Hz,1H),7.79(s,1H),7.63(d,J=8.4Hz,1H),7.41(s,1H),6.92(s,1H),6.83(br d,J=8.7Hz,1H),6.14(s,1H),5.05(dd,J=5.4,13.0Hz,1H),3.68(br d,J=10.3Hz,2H),3.42-3.50(m,2H),3.02(br d,J=10.9Hz,2H),2.81-2.94(m,1H),2.58-2.68(m,2H),2.54-2.58(m,1H),2.32(br d,J=6.0Hz,2H),2.10(br t,J=10.5Hz,2H),1.93-2.04(m,3H),1.62-1.72(m,4H),1.60(s,6H),0.49-1.05(m,1H).

[0445] MS(ESI+)m / z 799.4 [M+H] + .

[0446] Example 25:

[0447] (R)-N-(2-(1-((1-(2-(2,6-dioxopiperidin-3-yl)-6-fluoro-1,3-dioxoisoindolin-5-yl)piperidin-4-yl)methyl)piperidin-4-yl)-7-(2-hydroxypropyl-2-yl)imidazo[1,2-a]pyridin-6-yl)-6-(trifluoromethyl)pyridin-2-carboxamide

[0448]

[0449] Step 1: Preparation of (R)-5-amino-4-(5,6-difluoro-1,3-dioxoisoindoline-2-yl)-5-oxovalerate tert-butyl ester (IM-25a)

[0450]

[0451] 5,6-Difluoroisobenzofuran-1,3-dione (5.00 g, 27.1 mmol) and (R)-4,5-diamino-5-oxovalerate tert-butyl ester (6.59 g, 32.5 mmol) were dissolved in toluene (50.0 mL). Triethylamine (8.24 g, 81.4 mmol) was added to the reaction mixture at 25 °C. After the addition was complete, the resulting reaction mixture was heated to 80 °C and stirred for 12 hours. The reaction mixture was quenched with 50 mL of water at 25 °C and then extracted with ethyl acetate (50 mL x 3). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate volume ratio = 100 / 1 to 0 / 1) to give intermediate IM-25a as a white solid (5.00 g).

[0452] MS(ESI+) m / z 313.2 [M-55] + .

[0453] Step 2: Preparation of (R)-5-amino-4-(5-(4-(dimethoxymethyl)piperidin-1-yl)-6-fluoro-1,3-dioxoisoindoline-2-yl)-5-oxovalerate tert-butyl ester (IM-25b)

[0454]

[0455] Intermediate IM-25a (2.00 g, 5.43 mmol) and 4-(dimethoxymethyl)piperidine (1.04 g, 6.52 mmol) were dissolved in dimethyl sulfoxide (20.0 mL), and N,N-diisopropylethylamine (1.40 g, 10.8 mmol) was added at 25 °C. The resulting reaction mixture was stirred at 90 °C for 15 hours. The reaction was quenched with 20 mL of water at 25 °C, and then extracted with ethyl acetate (30 mL x 3). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate v / v = 100 / 1 to 0 / 1) to give intermediate IM-25b as a yellow solid (2.30 g).

[0456] MS(ESI+)m / z 508.2 [M+H] + .

[0457] Step 3: Preparation of (R)-5-amino-4-(5-fluoro-6-(4-formylpiperidin-1-yl)-1,3-dioxoisoindoline-2-yl)-5-oxovalerate tert-butyl ester (IM-25c)

[0458]

[0459] Intermediate IM-25b (1.00 g, 1.97 mmol) was dissolved in tetrahydrofuran (10.0 mL) and water (2.00 mL). Under nitrogen protection at 25 °C, pyridinium p-toluenesulfonate (1.49 g, 5.91 mmol) was added to the reaction mixture. The reaction was heated to 70 °C and stirred at 70 °C for 12 hours. The reaction mixture was concentrated under reduced pressure to obtain intermediate IM-25c as a yellow solid (700 mg, crude product), which was used directly without further purification.

[0460] MS(ESI+)m / z 406.3[M-55].

[0461] Step 4: Preparation of (R)-5-amino-4-(5-fluoro-6-(4-((4-(7-(2-hydroxypropyl-2-yl)-6-(6-(trifluoromethyl)pyridin-2-carbamoyl)imidazo[1,2-a]pyridin-2-yl)piperidin-1-yl)methyl)piperidin-1-yl)-1,3-dioxoisoindoline-2-yl)-5-oxovalerate tert-butyl ester (IM-25d)

[0462]

[0463] Intermediate IM-1 (678 mg, 1.52 mmol) was dissolved in N,N-dimethylformamide (7.00 mL) and tetrahydrofuran (7.00 mL). Anhydrous sodium acetate (248 mg, 3.03 mmol) was added to the reaction system at 25 °C, and the reaction was stirred at 25 °C for 30 minutes. Then, intermediate IM-25c (700 mg, 1.52 mmol), sodium triacetoxyborohydride (964 mg, 4.55 mmol), and glacial acetic acid (273 mg, 4.55 mmol) were added to the reaction system. The reaction mixture was stirred at 25 °C for 3.5 hours, then filtered and concentrated under reduced pressure to give intermediate IM-25d as a yellow solid (1.20 g, crude product).

[0464] MS(ESI+)m / z 893.6 [M+H] + .

[0465] Step 5: Preparation of (R)-N-(2-(1-((1-(2-(2,6-dioxopiperidin-3-yl)-6-fluoro-1,3-dioxoisoindoline-5-yl)piperidin-4-yl)methyl)piperidin-4-yl)-7-(2-hydroxypropyl-2-yl)imidazo[1,2-a]pyridin-6-yl)-6-(trifluoromethyl)pyridine-2-carboxamide (25)

[0466]

[0467] Intermediate IM-25d (100 mg, 111.99 μmol) was dissolved in acetonitrile (1.00 mL). Anhydrous benzenesulfonic acid (35.4 mg, 223 μmol) was added to the reaction system at 25 °C. The reaction was heated to 80 °C and stirred at 80 °C for 12 hours. The mixture was filtered and concentrated under reduced pressure to obtain a residue. The residue was purified by pre-high performance liquid chromatography (column: Phenomenex luna C18 100*40 mm*5 μm; mobile phase: [H2O (0.2% FA)-ACN]; gradient: 1%-40% B, time 8.0 min) to obtain compound 25 as a yellow solid (12.0 mg).

[0468] 1HNMR(400MHz,DMSO-d6)δ12.22(s,1H),11.10(br s,1H),9.54(s,1H),8.34-8.49(m,2H),8.18(s,1H),7.79(s,1H),7.70(d,J=11.4Hz,1H),7.37-7.47(m,2H),6.15(br s,1H),5.10(dd,J=5.4,12.8Hz,1H),3.62(br d,J=12.0Hz,2H),2.83-2.96(m,5H),2.54-2.72(m,3H),2.23(br d,J=6.6Hz,2H),1.93–2.11(m,5H),1.65-1.90(m,5H),1.60(s,6H),1.20-1.35(m,2H).

[0469] MS(ESI+)m / z 819.4 [M+H] + .

[0470] Example 26:

[0471] (S)-N-(2-(1-((1-(2-(2,6-dioxopiperidin-3-yl)-6-fluoro-1,3-dioxoisoindolin-5-yl)piperidin-4-yl)methyl)piperidin-4-yl)-7-(2-hydroxypropyl-2-yl)imidazo[1,2-a]pyridin-6-yl)-6-(trifluoromethyl)pyridin-2-carboxamide

[0472]

[0473] Step 1: Preparation of tert-butyl (S)-5-amino-4-(5,6-difluoro-1,3-dioxoisoindoline-2-yl)-5-oxovalerate (IM-26a)

[0474]

[0475] 5,6-Difluoroisobenzofuran-1,3-dione (10.0 g, 54.3 mmol) and (S)-4,5-diamino-5-oxovalerate tert-butyl ester (15.5 g, 65.1 mmol) were dissolved in toluene (50.0 mL). Triethylamine (16.4 g, 162 mmol) was added to the reaction system at 25 °C. After the addition was complete, the resulting mixture was heated to 80 °C and stirred for 12 hours. The reaction mixture was quenched with water (30 mL) at 25 °C and then extracted with ethyl acetate (40 mL * 3). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate volume ratio = 100 / 1 to 0 / 1) to give intermediate IM-26a as a white solid (1.50 g).

[0476] MS(ESI+)m / z 313.2[M-55].

[0477] Step 2: Preparation of tert-butyl (S)-5-amino-4-(5-(4-(dimethoxymethyl)piperidin-1-yl)-6-fluoro-1,3-dioxoisoindoline-2-yl)-5-oxovalerate (IM-26b)

[0478]

[0479] Intermediate IM-26a (1.50 g, 4.07 mmol) and 4-(dimethoxymethyl)piperidine (778 mg, 4.89 mmol) were dissolved in dimethyl sulfoxide (15.0 mL), and N,N-diisopropylethylamine (1.05 g, 8.14 mmol) was added at 25 °C. The reaction mixture was stirred at 90 °C for 15 hours. The reaction was quenched with 20 mL of water at 25 °C, and then extracted with ethyl acetate (30 mL x 3). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate v / v = 100 / 1 to 0 / 1) to give intermediate IM-26b as a yellow solid (1.50 g, 2.96 mmol).

[0480] MS(ESI+)m / z 508.2 [M+H] + .

[0481] Step 3: Preparation of tert-butyl (S)-5-amino-4-(5-fluoro-6-(4-formylpiperidin-1-yl)-1,3-dioxoisoindoline-2-yl)-5-oxovalerate (IM-26c)

[0482]

[0483] Intermediate IM-26b (700 mg, 1.38 mmol) was dissolved in tetrahydrofuran (5.00 mL) and water (1.00 mL). Pyridinium p-toluenesulfonate (1.04 g, 4.14 mmol) was added under nitrogen protection at 25 °C. The reaction was heated to 70 °C and stirred at 70 °C for 12 hours. The mixture was concentrated under reduced pressure to give intermediate IM-26c as a yellow solid (600 mg, crude product), which was used directly without further purification.

[0484] MS(ESI+)m / z 462.2[M+H] + .

[0485] Step 4: Preparation of (S)-5-amino-4-(5-fluoro-6-(4-((4-(7-(2-hydroxypropyl-2-yl)-6-(6-(trifluoromethyl)pyridin-2-carbamoyl)imidazo[1,2-a]pyridin-2-yl)piperidin-1-yl)methyl)piperidin-1-yl)-1,3-dioxoisoindoline-2-yl)-5-oxovalerate tert-butyl ester (IM-26d)

[0486]

[0487] Intermediate IM-1 (484 mg, 1.08 mmol) was dissolved in N,N-dimethylformamide (5.00 mL) and tetrahydrofuran (5.00 mL). Anhydrous sodium acetate (266 mg, 3.25 mmol) was added at 25 °C, and the reaction mixture was stirred at 25 °C for 30 minutes. Then, intermediate IM-26c (500 mg, 1.08 mmol), sodium triacetoxyborohydride (688 mg, 3.25 mmol), and glacial acetic acid (65.0 mg, 1.08 mmol) were added to the reaction mixture. The reaction mixture was stirred at 25 °C for 3.5 hours, then filtered and concentrated under reduced pressure to give intermediate IM-26d as a yellow solid (500 mg, crude product).

[0488] MS(ESI+) 893.3 [M+H] + .

[0489] Step 5: Preparation of (S)-N-(2-(1-((1-(2-(2,6-dioxopiperidin-3-yl)-6-fluoro-1,3-dioxoisoindoline-5-yl)piperidin-4-yl)methyl)piperidin-4-yl)-7-(2-hydroxypropyl-2-yl)imidazo[1,2-a]pyridin-6-yl)-6-(trifluoromethyl)pyridine-2-carboxamide (26)

[0490]

[0491] Compound IM-26d (50.0 mg, 56.0 μmol) was dissolved in acetonitrile (1.00 mL). Anhydrous benzenesulfonic acid (17.7 mg, 111 μmol) was added to the reaction system at 25 °C. The reaction was heated to 80 °C and stirred at 80 °C for 12 hours. The mixture was filtered and then concentrated under reduced pressure. The residue was purified by high performance liquid chromatography (HPLC) (column: Phenomenex luna C18 100*40 mm*5 μm; mobile phase: [H2O (0.2% FA)-ACN]; gradient: 10%-50% B, time: 8.0 min) to give compound 26 as a yellow solid (11.0 mg).

[0492] 1 HNMR(400MHz,DMSO-d6)δ12.22(s,1H),11.10(br s,1H),9.54(s,1H),8.31-8.51(m,2H),8.09-8.25(m,1H),7.63-7.86(m,2H),7.35-7.51(m,2H),6.15(s,1H),5.10(br dd,J=4.7,12.3Hz,1H),2.81–3.06(m,6H),2.67(br s,2H),2.31(br d,J=17.3Hz,3H),2.00(br d,J=9.4Hz,4H),1.69-1.89(m,5H),1.60(br (s, 6H), 1.19-1.35 (m, 4H)

[0493] MS(ESI+)m / z 819.3[M+H] + .

[0494] Comparative Example 1:

[0495] N-(2-(1-(2-(2-((2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethyl)piperidin-4-yl)-7-(2-hydroxypropyl-2-yl)imidazo[1,2-a]pyridin-6-yl)-6-(trifluoromethyl)pyridine-2-carboxamide

[0496]

[0497] Step 1: Preparation of 2-(2,6-dioxopiperidin-3-yl)-4-((2-(2-hydroxyethoxy)ethyl)amino)isoindoline-1,3-dione (IM-25a)

[0498]

[0499] 2-(2,6-dioxadiazin-3-yl)-4-fluoroisoindoline-1,3-dione (5.00 g, 26.8 mmol) and 2-(2-aminoethoxy)ethanol (2.82 g, 26.8 mmol) were dissolved in dimethyl sulfoxide (10.0 mL), and then N,N-diisopropylethylamine (3.47 g, 26.8 mmol) was added. The resulting reaction mixture was heated to 70 °C and stirred for 12 hours. The reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (SiO2, petroleum ether / ethyl acetate volume ratio = 100 / 1 to 10 / 1) to give intermediate IM-27a as a yellow oil (7.70 g).

[0500] 1 HNMR (400MHz, DMSO-d6) δ11.05(s,1H),7.55(dd,J=7.3,8.3Hz,1H),7.11(d,J=8.6Hz,1H),7.00(d,J=7.0Hz,1H),6.57(t,J=5.7H z,1H),5.02(dd,J=5.4,12.9Hz,1H),4.56(t,J=5.2Hz,1H),3.53-3.65(m, 2H),3.39-3.51(m,6H),2.77-3.02(m,1H),2.67(s,1H),2.60-2.55(m,2H).

[0501] Step 2: Preparation of 2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl)amino)ethoxy)ethyl ester of mesylate (IM-27b)

[0502]

[0503] Intermediate IM-27a (1.00 g, 2.77 mmol) was dissolved in dichloromethane (10 mL), and then triethylamine (1.12 g, 11.0 mmol) and methanesulfonic anhydride (2.23 g, 19.4 mmol) were added at 0 °C. The resulting reaction mixture was stirred at 25 °C for 3 hours. The reaction mixture was quenched with water (20 mL) at 0 °C, and then extracted with dichloromethane (30.0 mL x 3). The combined organic layers were washed with an aqueous sodium chloride solution (10.0 mL x 3), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (SiO2, petroleum ether / ethyl acetate v / v = 100 / 1 to 10 / 1) to give intermediate IM-27b as a yellow solid (500 mg).

[0504] 1HNMR: (400MHz, DMSO-d6) δ11.10(s,1H),7.59(dd,J=7.2,8.4Hz,1H),7.16(d,J=8.6Hz,1H),7.05(d,J=7.0Hz,1H),6.63(br t,J=5.9Hz,1H),5.05(dd,J=5.4,12.8Hz,1H),4.31(dd,J=3.5,5.3Hz,2H),3.61-3 .77(m,4H),3.45-3.55(m,3H),3.16(s,3H),2.82-2.94(m,1H),2.52-2.63(m,2H).

[0505] Step 3: Preparation of N-(2-(1-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl)amino)ethoxy)ethyl)piperidin-4-yl)-7-(2-hydroxypropyl-2-yl)imidazo[1,2-a]pyridin-6-yl)-6-(trifluoromethyl)pyridine-2-carboxamide (Comparative Example 1)

[0506]

[0507] Intermediates IM-27b (162 mg, 368 μmol) and IM-1 (150 mg, 335 μmol) were dissolved in N,N-dimethylformamide (2.00 mL), followed by the sequential addition of N,N-diisopropylethylamine (129 mg, 1.01 mmol) and sodium iodide (100 mg, 670 μmol). The resulting mixture was heated to 80 °C and stirred for 4 hours. The reaction mixture was concentrated under reduced pressure, and the residue was purified by preparative HPLC (column: Phenomenex luna C18 80*40 mm*3 μm; mobile phase: [water (FA)-ACN]; B%: 5%-45%, 8 min) to obtain a yellow solid (27.0 mg) as in Comparative Example 1.

[0508] 1HNMR(400MHz,DMSO-d6)δ12.23(s,1H),11.21(br s,1H),9.54(s,1H),8.36-8.47(m,2H),8.18-8.20(m,1H),7.74(s,1H),7.56-7 .62(m,1H),7.42(s,1H),7.17(d,J=8.7Hz,1H),7.05(d,J=7.1Hz,1H),6.62(br t,J=5.6Hz,1H),6.15(s,1H),5.05(dd,J=5.4,12.4Hz,1H),3.56-3.64(m,4H),3.44-3.52(m,3H),2.74-3.00(m,4H),2.52-2.68(m,5H),2.12(br t,J=11.3Hz,2H),1.85-2.03(m,3H),1.60(s,6H).

[0509] MS(ESI+)m / z 791.4[M+H] + .

[0510] Comparative Example 2:

[0511] N-(2-(1-(6-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)hex-5-yn-1-yl)piperidin-4-yl)-7-(2-hydroxypropyl-2-yl)imidazo[1,2-a]pyridin-6-yl)-6-(trifluoromethyl)pyridine-2-carboxamide

[0512]

[0513] Step 1: Preparation of 3-(4-(6-hydroxyhex-1-yn-1-yl)-1-oxoisoindololin-2-yl)piperidine-2,6-dione (IM-28a)

[0514]

[0515] 3-(4-bromo-1-oxoisoindolin-2-yl)piperidine-2,6-dione (1.00 g, 3.09 mmol) and 5-hexyn-1-ol (607 mg, 6.19 mmol) were dissolved in dimethylformamide (20.0 mL), and cuprous iodide (353 mg, 1.86 mmol), dichlorobis(triphenylphosphine)palladium(II) (217 mg, 309 μmol), and triethylamine (7.27 g, 71.8 mmol) were added sequentially at 25 °C. The reaction mixture was heated to 60 °C and stirred for 12 hours. The reaction mixture was diluted with water (20 mL) and extracted with ethyl acetate (20 mL x 3). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was subjected to column chromatography (silica gel, petroleum ether / ethyl acetate v / v = 100 / 1 to 10 / 1) to give intermediate IM-28a as a yellow solid (200 mg).

[0516] 1 HNMR (400MHz, DMSO-d6) δ11.00(s,1H),7.70(d,J=7.3Hz,1H),7.63(br d,J=7.5Hz,1H),7.53(br d,J=7.6Hz,1H),5.09-5.19(m,1H),4.41-4.48(m,2H),4.27-4.34(m,1H),3.41-3.48(m,4H),2.88-2.98(m,1H),2.59(br d,J=17.7Hz,2H),1.97–2.07(m,1H),1.53-1.63(m,4H).

[0517] Step 2: Preparation of 6-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-4-yl)hex-5-yn-1-yl mesylate (IM-28b)

[0518]

[0519] Intermediate IM-28a (200 mg, 587 μmol) was dissolved in tetrahydrofuran (1.00 mL) at room temperature, followed by the addition of triethylamine (148 mg, 1.47 mmol) and methanesulfonic anhydride (178 mg, 881 μmol). The resulting reaction mixture was stirred at room temperature for 12 hours. The reaction mixture was diluted with water (10 mL) and extracted with ethyl acetate (10 mL x 3). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (silica gel, petroleum ether / ethyl acetate v / v = 100 / 1 to 10 / 1) to give intermediate IM-28b as a white solid (200 mg).

[0520] Step 3: Preparation of N-(2-(1-(6-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-4-yl)hex-5-yn-1-yl)piperidin-4-yl)-7-(2-hydroxypropyl-2-yl)imidazo[1,2-a]pyridin-6-yl)-6-(trifluoromethyl)pyridine-2-carboxamide (Comparative Example 2)

[0521]

[0522] Intermediate IM-28b (110 mg, 210 μmol) was dissolved in acetonitrile (2.00 mL), and sodium bicarbonate (35.3 mg, 420 μmol) and intermediate IM-1 (94.1 mg, 210 μmol) were added at room temperature. The reaction mixture was then heated to 80 °C and stirred for 12 hours. The reaction mixture was concentrated under reduced pressure, and the residue was purified by preparative HPLC (column: Phenomenex luna C1880*40 mm*3 μm; mobile phase: [water (FA)-ACN]; B%: 10%-50%, 8 min) to obtain the compound of Comparative Example 2 as a white solid (16.1 mg).

[0523] 1 HNMR(400MHz,DMSO-d6)δ12.22(s,1H),11.04(br s,1H),9.54(s,1H),8.43-8.47(m,1H),8.35-8.41(m,1H),8.17-8.23(m,2H),7.78(s,1H),7. 71(d,J=7.5Hz,1H),7.64(d,J=7.5Hz,1H),7.50-7.55(m,1H),7.41(s,1H),5.11-5.18(m,1H) ,4.44-4.50(m,1H),4.29-4.35(m,1H),2.84–3.12(m,4H),2.60-2.79(m,2H),2.53-2.60(m,2 H),2.38-2.48(m,3H),2.11-2.33(m,3H),1.96–2.05(m,3H),1.65-1.78(m,4H),1.60(s,6H).

[0524] MS(ESI+)m / z 770.3[M+H] + .

[0525] The compounds prepared in the examples and comparative examples were subjected to IRAK4 protein degradation experiments. The experimental procedures are as follows:

[0526] Intracellular degradation assay of THP1-HIBiT-IRAK4 based on HiBiT

[0527] THP1-HIBiT-IRAK4 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (PS) (37°C, 5% CO2). THP1-HIBiT-IRAK4 cells were seeded at 5000 cells / 40 μL / well in 384-well plates. Different concentrations of compound solutions were added to the wells at 40 nmL / well, and the plates were incubated at 37°C, 5% CO2 for 24 h. HiBit lysis detection reagent was added to the wells, and the plates were incubated at room temperature for 30 min. The values ​​were then read on an Envision microscope.

[0528] Degradation rate (%) = (Average luminescence value of compound pores / Average luminescence value of DMSO pores) × 100

[0529] The DC of the compound was obtained using the following nonlinear fitting formula with XLFIT software. 50 (Half-maximum degradation concentration):

[0530] Y = bottom + (top - bottom) / (1 + 10^(LogDC)) 50 -X)*Hill slope))

[0531] X: Log value of compound concentration;

[0532] Y: Degradation rate

[0533] IRAK4 protein degradation DC 50 The results are shown in Table 1. The experimental results show that the compounds in the examples have significant degradation activity against IRAK4 protein, while the compounds in the comparative examples have no IRAK4 protein degradation activity (DC). 50 >10000nM).

[0534] Table 1

[0535]

[0536]

[0537]

[0538]

[0539]

[0540] Degradation of IRAK4 was determined by Western blotting.

[0541] (1) Compound processing and pyrolysis product preparation

[0542] a) On day 1, OCI-LY10 cells were administered at a rate of 1.5 × 10⁻⁶. 6 Cells / mL / well were seeded into 12-well plates, and the test compound was diluted with DMSO. 1 μL of the diluted compound solution was added to the cell supernatant and incubated overnight in a 37°C, 5% CO2 incubator.

[0543] b) On day 2, harvest the cells and transfer them to 1.5 mL centrifuge tubes, and centrifuge at 3000 rpm for 5 minutes.

[0544] c) Wash the cells once with ice-cold PBS and centrifuge at 3000 rpm for 5 minutes at 4°C.

[0545] d) Add one tablet of protease inhibitor and one tablet of phosphatase inhibitor to 20 mL of cell lysate, and gently mix until completely dissolved.

[0546] Take the prepared cell lysis buffer and add 50 μL / tube to a centrifuge tube. Lyse the cells on ice for 30 minutes.

[0547] e) Centrifuge at 13,000 rpm for 15 minutes at 4°C.

[0548] f) Transfer the supernatant to a new tube and determine the protein concentration using a BCA kit.

[0549] (2) Protein blotting experiment

[0550] a) Add 20 μg / well to a 4-12% BTMidi protein gel (1 mm, 26 wells) and electrophoresis at a constant voltage of 120 V for 5 minutes, followed by electrophoresis at a constant voltage of 150 V for 55 minutes.

[0551] b) Use the Bio-Rad wet transfer apparatus to transfer the sample onto the PVDF membrane at 300mA for 90 minutes.

[0552] c) Seal the membrane with 1xTBS-T containing 5% BSA for 1 hour at room temperature.

[0553] d) Incubate the membrane with the corresponding primary antibody solutions (rabbit anti-IRAK4 and mouse anti-GAPDH) overnight at 4°C.

[0554] e) Wash the membrane with 1×TBST for 3×5 minutes, and then mix the membrane with secondary antibody solution (HRP-labeled anti-mouse antibody and HRP-labeled anti-rabbit antibody).

[0555] Incubate at room temperature for 90 minutes.

[0556] f) Wash the membrane with 1×TBST for 3×5 minutes.

[0557] g) Add an enhanced chemiluminescence (ECL) substrate and scan it using an IBright CL1500 imaging system.

[0558] Experimental results are as follows Figures 1-26 As shown in Table 2, the corresponding tables are listed. Figures 1-26 The correlation between the compounds showed that all compounds in the examples exhibited significant IRAK4 protein degradation activity at concentrations of 100 nM and 1000 nM.

[0559] Table 2

[0560]

[0561]

[0562]

[0563]

[0564]

[0565] Liver microsomal stability test

[0566] (1) Materials

[0567] Microparticles were stored at -80°C and thawed in a 37°C water bath before use, then placed on ice. All other reagents were purchased from local suppliers.

[0568] (2) Experimental Design

[0569] Step 1: Configure the reaction system as follows

[0570] reagents Storage liquid concentration volume Final concentration microparticles 20mg / mL 10μL 0.5 mg / mL Phosphate buffer 200mM 200μL 100mM Magnesium chloride 50mM 40μL 5mM Promethazine 5mg / mL 2μL 25μg / mL water - 64μL -

[0571] Step 2: Add 40 μL of 10 mM NADPH solution and 40 μL of 20 mM UDPGA solution to the above reaction system. The final concentrations of NADPH and UDPGA are 1 mM and 2 mM, respectively. Use 80 μL of ultrapure water instead of NADPH and UDPGA solutions as a negative control. Incubate the reaction system in a 37°C water bath for 10 minutes.

[0572] Step 3: Add 4 μL of 100 μM of the test compound and control drug (verapamil and 7-OH coumarin) to the reaction to start the reaction. The final concentration of the drug is 1 μM.

[0573] Step 4: At 0, 15, 30, 45, and 60 minutes, 50 μL of the reaction sample was collected and quenched with 4 times the volume of cold acetonitrile containing internal standards (100 nM alprazolam, 200 nM labetalol, 2 μM ketoprofen, and 200 nM caffeine). The sample was centrifuged at 3,220 g for 40 minutes. After centrifugation, 100 μL of the supernatant was mixed with 100 μL of ultrapure water for LC-MS / MS analysis.

[0574] (3) Data Analysis

[0575] All data calculations were performed using Microsoft Excel software. Peak areas were determined from the extracted ion chromatograms and used to calculate the remaining percentage at each time point.

[0576] The remaining percentage is calculated using the following formula:

[0577] Remaining percentage (%) = (Peak area ratio) 时间点 / peak area ratio 0分钟 )×100

[0578] The elimination rate constant (k) was determined by linearly fitting the natural logarithm of the remaining percentage of parent drug with time.

[0579] The half-life (t1 / 2) is calculated using the elimination rate constant: t1 / 2 = 0.693 / k

[0580] In vitro intrinsic clearance (unit: μL / min / mg) is calculated using the following formula:

[0581] invitroCLint=kV / N

[0582] In the formula: V = incubation volume per well (0.4 mL); N = microparticle content per well (0.2 mg)

[0583] The experimental results are shown in Table 3. Example 15 showed good metabolic stability in liver microsomal metabolism in different species.

[0584] Table 3

[0585] Species <![CDATA[T 1 / 2 (minutes) Clint (μL / min / mg) people 400.73 3.46 monkey 191.84 7.22 dog 259.72 5.34 rats 133.68 10.37 mice 143.40 9.67

[0586] In vivo pharmacokinetic studies

[0587] The following experimental protocol was used to study the pharmacokinetic behavior of Examples 1 and 15 in mice and to evaluate their pharmacokinetic characteristics.

[0588] Experimental plan:

[0589] Three healthy male CD1 mice, weighing 20-25g, were administered the compound at a dose of 2mg / kg via intravenous injection, in a volume of 10mL / kg, prepared with 5% DMSO / 15% Solutol / 80% PBS (w / v). Free access to water and food was provided before the experiment.

[0590] Three healthy male CD1 mice, weighing 20-25g, were orally administered a compound at a dose of 20mg / kg, in a volume of 10mL / kg, prepared with 5% DMSO / 15% solubilized PBS (w / v). Free access to water and food was provided before the experiment.

[0591] At 0.033h, 0.083h, 0.25h, 0.5h, 1h, 2h, 4h, 8h, and 24h after intravenous administration, approximately 0.03mL of venous blood was collected from the dorsal metatarsal vein of mice. The blood was placed in EDTA-K2 tubes and centrifuged at 4000g for 5min at 4℃ to separate the plasma. The concentration of compounds in the plasma was determined by liquid chromatography-tandem mass spectrometry.

[0592] At 0.083 h, 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, and 24 h after oral administration, approximately 0.03 mL of venous blood was collected from the dorsal metatarsal vein of mice. The blood was placed in EDTA-K2 tubes and centrifuged at 4000 g for 5 min at 4 °C to separate the plasma. The concentration of compounds in the plasma was determined by liquid chromatography-tandem mass spectrometry.

[0593] As shown in Table 4, the experimental results show that the compounds of the present invention have low clearance rates, high plasma exposure, and good oral absorption. In particular, Example 15 has an oral bioavailability of up to 61.7% (in Table 4, Cl is clearance rate, F is bioavailability, and AUC is area under the curve).

[0594] Table 4

[0595]

[0596] The applicant declares that the above embodiments illustrate the compounds of the present invention, their preparation, and applications; however, the present invention is not limited to the above embodiments, i.e., it does not mean that the present invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials used in the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. An imidazo[1,2-a]pyridine IRAK4 degrading agent, and its pharmaceutically acceptable salt, characterized in that, The imidazo[1,2-a]pyridine IRAK degrading agent is selected from any one of the following compounds:

2. The imidazo[1,2-a]pyridine IRAK4 degrading agent according to claim 1, and its pharmaceutically acceptable salt, characterized in that, The pharmaceutically acceptable salts are any one of the following: sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, phosphates, monohydrophosphates, dihydrophosphates, metaphosphates, pyrophosphates, hydrochlorides, hydrobromates, hydroiodates, acetates, propionates, decanoates, octanoates, acrylates, formates, isobutyrates, hexanoates, heptanates, propynates, oxalates, malonates, succinates, octanoates, sebacic acid salts, fumarates, maleates, butyn-1,4-dicitates, hexyn-1,6-dicitates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, xylenesulfonates, phenylacetates, phenylpropionates, phenylbutyrates, citrates, lactates, γ-hydroxybutyrates, glycolate, tartrates, methanesulfonates, propanesulfonates, naphthalene-1-sulfonates, naphthalene-2-sulfonates, or mandelates.

3. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises an imidazo[1,2-a]pyridine IRAK4 degrader as described in claim 1 or 2, or a pharmaceutically acceptable salt thereof.

4. The use of the imidazo[1,2-a]pyridine IRAK4 degrading agent according to claim 1 or 2, a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 3 in the preparation of a medicament for treating diseases or conditions mediated by IRAK4.

5. The application according to claim 4, characterized in that, The disease in question is an inflammatory disease, an autoimmune disease, or a tumor.

6. The application according to claim 5, characterized in that, The inflammatory diseases mentioned are selected from Crohn's disease, ulcerative colitis, asthma, graft-versus-host disease, chronic obstructive pulmonary disease, atopic dermatitis, gout, conjunctivitis, hepatitis, chronic inflammatory lung disease, thyroiditis, or interstitial cystitis.

7. The application according to claim 5, characterized in that, The autoimmune diseases mentioned are selected from Graves' disease, rheumatoid arthritis, systemic lupus erythematosus, cutaneous lupus, psoriasis, cryptothermal protein-related periodic syndrome, TNF receptor-related periodic syndrome, multiple sclerosis, allergic rhinitis, scleroderma, dermatomyositis, vasculitis, or nephritis.

8. The application according to claim 5, characterized in that, The tumor is selected from lung cancer, breast cancer, pancreatic cancer, kidney cancer, liver cancer, gastrointestinal cancer, urogenital tract cancer, skin cancer, bone cancer, head and neck tumors, sarcoma, glioblastoma, multiple myeloma, lymphoma, or leukemia.

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