7-Azaindole compounds, their synthesis methods and uses

Abstract

This disclosure relates to the field of anticancer drug technology, and discloses a 7-azaindole compound, its synthesis method, and its uses. The structure of the 7-azaindole compound is as follows: This 7-azaindole compound can be used as a fluorescent polarization probe or a degradation chemical probe targeting CSN5; wherein, the fluorescent polarization chemical probe has a good binding affinity to the metalloproteinase CSN5, laying an important foundation for the discovery of novel CSN5 inhibitors; at the same time, the degradation chemical probe exhibits good enzyme-level activity and cell-inhibitory activity, and has the potential to degrade CSN5 protein and its variants, providing a material basis for the development of related drugs targeting CSN5 and the study of its biological functions, and also has great potential in the discovery of cancer immunotherapy drugs and the treatment of other related diseases.

Description

Technical Field

[0001] This disclosure relates to the field of anticancer drug technology, for example to a 7-azaindole compound and its synthesis method and use. Background Technology

[0002] CSN5, initially discovered as a coactivator of the C-Jun protein (also known as Jab1), was later found to be the core catalytic subunit of the COP9 signaling body. Its primary biological function is to remove the NEDD8 molecule bound to the Cullin protein in the active Cullin-RINGE3 ubiquitin ligase complex via the COP9 complex, leading to CRL disintegration and inactivation, thus losing its ubiquitination ability. NEDD8 is a ubiquitin-like protein, therefore CSN5 is considered a deubiquitinating enzyme. Currently, CSN5 has been found to be associated with the development of various cancers. The main reason is that aberrantly expressed CSN5 affects the deNEDDing process, thereby disrupting the balance between oncogenes and tumor suppressors regulated by CRLs. This includes promoting SCF-mediated degradation of tumor suppressors such as p27, p53, and p57, enhancing the stability of the oncogene HIF-1α, and acting through CUL4. DCAF CSN5 negatively regulates proteins related to DNA damage repair. These aberrant regulatory processes are also closely related to poor prognosis and resistance to antitumor drugs. Experiments have shown that knocking out the CSN5 gene inhibits tumor cell growth. Therefore, CSN5 is considered a potential new target for cancer therapy. To date, only a few classes of CSN5 inhibitors have been reported, including natural products such as curcumin and berberine, as well as tetrahydroimidazole and 7-azaindole inhibitors developed by Novartis, and none of these inhibitors have entered clinical trials. Furthermore, some believe that the CSN complex may serve as a "docking platform" for its various subunits, and its core catalytic subunit CSN5 may also exist in monomeric form, with each subunit or subcomplex potentially possessing a unique biological function. CSN5 has been found in different oligomeric states in vitro and in cell lysates, exhibiting two distinct dimer crystals, which also provides evidence for the possible existence of smaller complexes. Therefore, the CSN complex may exist as subcomplexes composed of various subunits, and its core catalytic subunit CSN5 may also exist in monomeric form, with each subunit or subcomplex potentially possessing a unique biological function. Meanwhile, due to the large molecular weight, complex composition, and difficulty in expression and purification of the CSN5 complex and its catalytic substrate CRLs-NEDD8 complex, it is difficult to establish a testing system based on the CSN5-catalyzed removal of NEDD8 bioactivity, which greatly limits the discovery of novel CSN5 inhibitors. Therefore, there is currently a lack of fluorescent chemical probes to establish a rapid experimental platform for CSN5 inhibitor discovery, as well as a lack of degradation probes to study the biological functions and disease relevance of CSN5 monomers or subcomplexes formed with other subunits. Summary of the Invention

[0003] The purpose of this disclosure is to overcome the shortcomings of the prior art and provide a 7-azaindole compound, its synthesis method and uses, which can provide a material basis for the development of drugs targeting CSN5 and the study of their biological functions, and has great potential in the discovery of cancer immunotherapy drugs and the treatment of other related diseases.

[0004] The purpose of this disclosure is achieved through the following technical solution:

[0005] On the one hand, a 7-azaindole compound is provided. The 7-azaindole compound has a structure as shown in Formula I:

[0006]

[0007] In the formula, R1 is selected from phenyl, pyridinyl, or thiophene;

[0008] L is selected from

[0009] as well as One of them; wherein X is selected from a hydrogen atom, an alkyl group, 5-isothiocyanate fluorescein, or a protein ligand of the E3 ubiquitin ligase complex.

[0010] In some embodiments, X is selected from the protein ligand, which is selected from... as well as One of them.

[0011] In some embodiments, L is selected from

[0012] as well as One of them.

[0013] In some embodiments, R1 is selected from the phenyl group.

[0014] In some embodiments, X is selected from the hydrogen atom.

[0015] In some embodiments, L is selected from as well as One of them.

[0016] In some embodiments, the 7-azaindole compound has a structure as shown in any one of formulas L1 to L41:

[0017]

[0018]

[0019]

[0020] In some examples, the 7-azaindole compounds have structures as shown in any one of formulas L20, L22, L27 to L31, L34, L36, L37 and L40.

[0021] On the other hand, a method for synthesizing 7-azaindole compounds as described in any of the above embodiments is provided. The method includes the following steps:

[0022] S1.

[0023] Compound 1 and compound 2 were subjected to a substitution reaction to yield compound 3;

[0024] S2.

[0025] The 7-azaindole compound I was synthesized based on compound 3.

[0026] In some examples, the synthesis of the 7-azaindole compound I based on compound 3 in S2 includes the following steps:

[0027] S11.

[0028] Compound 3 was subjected to a desulfonylation reaction to give compound 4;

[0029] S12.

[0030] Compound 4 was subjected to the Duff reaction to obtain compound 5;

[0031] S13.

[0032] Based on compound 5, the 7-azaindole compound I was synthesized.

[0033] In other examples, the synthesis of the 7-azaindole compound I based on compound 3 in S2 includes the following steps:

[0034] S21.

[0035] Compound 3 was subjected to an iodination reaction to yield compound 7;

[0036] S22.

[0037] Compound 7 was subjected to a Sonogashira coupling reaction to give compound 8;

[0038] S23.

[0039] Based on compound 8, the 7-azaindole compound I was synthesized.

[0040] In another aspect, the use of a 7-azaindole compound as described in any of the above embodiments or the method described in any of the above embodiments in the preparation of an anticancer drug is provided.

[0041] In some embodiments, the 7-azaindole compounds are used for screening and / or CSN5 inhibitors.

[0042] For example, the 7-azaindole compound is used to screen for CSN5 inhibitors. For instance, when X is selected from fluorescein 5-isothiocyanate, the 7-azaindole compound is used to prepare a fluorescent polarization probe targeting CSN5.

[0043] For example, the 7-azaindole compound is used to prepare a CSN5 inhibitor. For instance, when X is a protein ligand selected from the E3 ubiquitin ligase complex, the 7-azaindole compound is used to prepare a degradation chemical probe targeting CSN5.

[0044] The beneficial effects of this disclosure are:

[0045] 1. This disclosure establishes a screening system for CSN5 inhibitors, which has high accuracy and ease of operation, and solves the problem of the difficulty in establishing an in vitro activity system for CSN5.

[0046] 2. The 7-azaindole compounds disclosed herein exhibit good enzymatic activity and cellular inhibitory activity when used as chemical probes for the degradation of CSN5, and have the potential to degrade CSN5 protein and its variants.

[0047] 3. This disclosure can provide a material basis for the development of drugs targeting CSN5 and the study of their biological functions, and also has great potential in the discovery of cancer immunotherapy drugs and the treatment of other related diseases. Attached Figure Description

[0048] To more clearly illustrate the technical solutions in this disclosure, the accompanying drawings used in some embodiments of this disclosure will be briefly described below. Obviously, the drawings described below are merely drawings of some embodiments of this disclosure, and those skilled in the art can obtain other drawings based on these drawings. Furthermore, the drawings described below can be regarded as schematic diagrams and are not intended to limit the actual process of the methods involved in the embodiments of this disclosure.

[0049] Figure 1 This is a diagram showing the effect of some of the potential degrading agents in Experiment Example 3 of this disclosure on HCT116 cells. Detailed Implementation

[0050] The technical solutions in some embodiments of this disclosure will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this disclosure, and not all embodiments. Based on the embodiments provided in this disclosure, all other embodiments obtained by those skilled in the art are within the scope of protection of this disclosure.

[0051] In describing some embodiments, the expression "A and / or B" may be used. It is readily understood that "A and / or B" includes the following three combinations: A only, B only, and a combination of A and B.

[0052] In describing some embodiments, the expressions "at least one of A, B and C" and "at least one of A, B or C" may be used, both of which have the same meaning and include the following combinations of A, B and C: only A, only B, only C, a combination of A and B, a combination of A and C, a combination of B and C, and a combination of A, B and C.

[0053] It should be noted that the raw materials and equipment used in the embodiments of this disclosure are all known products, obtained by purchasing commercially available products.

[0054] 1. Intermediate 3: 4,4,5,5-Tetramethyl-2-(5-methyl-[1,1'-biphenyl]-2-yl)-1,3,2-dioxaboranecyclopentane

[0055]

[0056] Step 1: Synthesis of 2-chloro-5-methyl-1,1'-biphenyl (2)

[0057] Compound 1 (781 mg, 3.8 mmol), phenylboronic acid (927 mg, 7.6 mmol), Pd(Ph3P)4 (219 mg, 0.19 mmol), 2M Na2CO3 (5.7 mL, 11.4 mmol), and 15 mL DME were added to a 100 mL double-necked flask. The mixture was reacted at 100 °C for 16 h under an Ar atmosphere. The reaction was monitored by TLC. After the reaction was completed, the mixture was cooled to ambient temperature, and water (30 mL) and ethyl acetate (20 mL) were added to the mixture. The organic layer was separated, extracted three times, and the organic layers were combined. The mixture was dried over anhydrous Na2SO4, filtered, evaporated to dryness, and purified by column chromatography to obtain intermediate 2 (629 mg, 83%).

[0058] 1H NMR (400MHz, DMSO-d6) δ8.38(dd,J=4.8,1.6Hz,1H),8.12(dd,J=8.5,1.3Hz,2H),8.06(dd,J=7.9,1.7Hz,1H),7.93( d,J=4.0Hz,1H),7.74–7.69(m,1H),7.62(dd,J=8.6,7.1Hz,2H),7.31(dd,J=7.9,4.8Hz,1H),6.85(d,J=4.0Hz,1H).

[0059] Step 2: Synthesis of 4,4,5,5-tetramethyl-2-(5-methyl-[1,1'-biphenyl]-2-yl)-1,3,2-dioxaboranecyclopentane (3)

[0060] Intermediate 2 (629 mg, 3.2 mmol), bis-pinacolborate (1.63 g, 6.4 mmol), Pd2(dba)3 (147 mg, 0.16 mmol), X-Phos (153 mg, 0.032 mmol), 2M KOAc (4.8 mL, 9.6 mmol), and 1,4-dioxane (8 mL) were added to a 50 mL double-necked flask. The mixture was reacted at 110 °C for 1 h under an Ar atmosphere. The reaction was monitored by TLC. After the reaction was complete, the mixture was cooled to ambient temperature, and water (30 mL) and ethyl acetate (20 mL) were added. The organic layer was separated, extracted three times, and the organic layers were combined, dried over anhydrous Na2SO4, filtered, and evaporated to dryness. The mixture was purified by column chromatography (PE / EA = 100:1, v / v) to give intermediate 3 (593 mg, 63%).

[0061] 1 H NMR (400MHz, DMSO-d6) δ7.52(d,J=7.4Hz,1H),7.42–7.29(m,5H),7.19(d,J=3.5Hz,2H),2.36(s,3H),1.15(s,12H).

[0062] 2. Intermediate 6: 2-Iodo-1-(benzenesulfonyl)-1H-pyrrolo[2,3-b]pyridine

[0063]

[0064] Step 1: Synthesis of 1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridine (5)

[0065] Under an Ar atmosphere, 7-azaindole 4 (5.92 g, 50 mmol), TBAB (484 mg, 1.5 mmol), finely ground sodium hydroxide (6 g, 150 mmol), and ultra-dry DCM (115 mL) were added to a dried 250 mL double-necked flask. The flask was cooled to 0 °C in an ice bath, and then benzenesulfonyl chloride (8 mL, 62.5 mmol) was slowly added dropwise. After the addition was complete, the ice bath was removed, and the mixture was stirred at room temperature for 1 h. The reaction was monitored by TLC. After the reaction was complete, water (50 mL) and DCM (50 mL) were added, the organic layer was separated, extracted three times, and the organic layers were combined, dried over anhydrous Na2SO4, filtered, and evaporated to dryness. The mixture was purified by column chromatography (PE / EA = 5:1, v / v) to give intermediate 5 (12 g, 93%) as a bright white solid.

[0066] 1 H NMR (400MHz, DMSO-d6) δ8.38(dd,J=4.8,1.6Hz,1H),8.12(dd,J=8.5,1.3Hz,2H),8.06(dd,J=7.9,1.7Hz,1H),7.93( d,J=4.0Hz,1H),7.74–7.69(m,1H),7.62(dd,J=8.6,7.1Hz,2H),7.31(dd,J=7.9,4.8Hz,1H),6.85(d,J=4.0Hz,1H).

[0067] Step 2: Synthesis of 2-iodo-1-(benzenesulfonyl)-1H-pyrrolo[2,3-b]pyridine (6)

[0068] Under an Ar atmosphere, compound 5 (10 g, 39 mmol) was added to a dried 500 mL double-necked flask, followed by 200 mL of ultra-dry tetrahydrofuran to dissolve it. The reaction solution was stirred at -60 °C for 30 min, and LDA (39 mL 2 M in THF, 78 mmol) was slowly added dropwise. During the addition, the reaction solution gradually changed from colorless to orange-red. After the addition was complete, the mixture was stirred at -60 °C for 15 min. Then, 100 mL of ultra-dry tetrahydrofuran solution of iodine (15 g, 58.5 mmol) was added dropwise. During the addition, the color of the reaction solution continuously deepened, eventually turning brownish-red. After the addition was complete, the mixture was moved to room temperature and allowed to react overnight. The reaction was monitored by TLC. After the reaction was complete, 50 mL of water was added to quench the reaction, followed by 100 mL of ethyl acetate to dilute the reaction solution. The mixture was washed with sodium thiosulfate solution (w / w, 5%) and saturated sodium chloride to separate the organic phase. The organic phase was extracted three times, combined, dried over anhydrous Na₂SO₄, filtered, and evaporated to dryness. Column chromatography purification (PE / EA = 5:1, v / v) yielded intermediate 6 (10 g, 66%), a pale yellow solid.

[0069] 1H NMR(400MHz, DMSO-d6)δ8.32(dd,J=4.8,1.7Hz,1H),8.09–8.02(m,2H),7.92(dd,J=7.8,1.7 Hz,1H),7.72(dd,J=8.6,6.4Hz,1H),7.64(t,J=7.8Hz,2H),7.30–7.26(m,1H),7.25(s,1H).

[0070] Example 1: Ethyl (E)-3-(2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)acrylate (L1)

[0071]

[0072] Step 1: Synthesis of 1-(benzenesulfonyl)-2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrrolo[2,3-b]pyridine (7)

[0073] Intermediate 3 (3.3 g, 11 mmol), intermediate 6 (3.9 g, 10 mmol), Pd(Ph3P)4 (578 mg, 0.5 mmol), 2 M Na2CO3 (35 mL, 70 mmol), and a mixed solvent of 1,4-dioxane and water (v / v = 1:3, 140 mL) were added to a 250 mL double-necked flask. The reaction was carried out at 100 °C for 12 h under an Ar atmosphere. The reaction was monitored by TLC. After the reaction was completed, the mixture was cooled to ambient temperature, and water (50 mL) and ethyl acetate (50 mL) were added. The mixture was extracted three times, and the organic layers were combined, dried over anhydrous Na2SO4, filtered, and evaporated to dryness. The residue was then subjected to column chromatography (PE / EA = 10:1, v / v) to give intermediate 7 (3.3 g, 78%), a pale yellow solid.

[0074] Step 2: Synthesis of 2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrrolo[2,3-b]pyridine (8)

[0075] Intermediate 7 (3.3 g, 7.8 mmol), sodium tert-butoxide (3 g, 31.2 mmol), and 1,4-dioxane (78 mL) were added to a 250 mL flask and stirred under reflux at 100 °C for 2 h. The reaction was monitored by TLC. After the reaction was complete, the mixture was cooled to ambient temperature, the reaction was quenched with water, and then water (30 mL) and ethyl acetate (20 mL) were added. The organic phase was separated, extracted three times, and the organic layers were combined. The mixture was dried over anhydrous Na₂SO₄, filtered, and evaporated to dryness. The solution was purified by column chromatography (PE / EA = 2:1, v / v) to give intermediate 8 (1.2 g, 55%) as a yellow solid.

[0076] 1H NMR (400MHz, DMSO-d6) δ11.69(s,1H),8.12(dd,J=4.7,1.6Hz,1H),7.71(dd,J=7.9,1.6Hz,1H),7.59(d,J=7 .8Hz,1H),7.30(m,4H),7.26–7.19(m,3H),6.96(dd,J=7.8,4.7Hz,1H),5.78(d,J=2.1Hz,1H),2.40(s,3H).

[0077] Step 3: Synthesis of 2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrrolo[2,3-b]pyridine-3-carboxaldehyde (9)

[0078] Intermediate 8 (1.2 g, 4 mmol), HMTA (842 mg, 1.5 mmol), and glacial acetic acid (21 mL) were added to a 100 mL flask and stirred under reflux for 6 h. The reaction was monitored by TLC. After the reaction was complete, the mixture was cooled to ambient temperature and diluted with water. A large amount of white solid precipitated out. The mixture was stirred overnight. The mixture was filtered, the filter cake was washed with ice water, dried, and recrystallized from n-hexane to give intermediate 9 (951 mg, 76%), a white solid.

[0079] 1 H NMR (400MHz, DMSO-d6) δ12.69(s,1H),9.47(s,1H),8.33–8.25(m,2H),7.61–7.55(m,1H),7.44–7.36(m,2H),7.29–7.14(m,6H),2.47(s,3H).

[0080] Step 4: Synthesis of ethyl (E)-3-(2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)acrylate (L1)

[0081] Intermediate 24 (951 mg, 3.1 mmol), ethoxyformylmethylenetriphenylphosphine (4.3 g, 12.4 mmol), and ultra-dry toluene (12 mL) were added to a 100 mL flask. The mixture was stirred and refluxed for 4 h, and the reaction was monitored by TLC. After the reaction was complete, the mixture was concentrated by rotary evaporation and column chromatography (PE / EA = 1:1, v / v) to give L1 (650 mg, 54%), a white solid.

[0082] Example 2: Synthesis of ethyl 3-(2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)propionate (L2)

[0083]

[0084] Compound L1 (132 mg, 0.35 mmol), Pd / C (13.2 mg), and methanol (4 mL) were added to a 25 mL double-necked flask. The mixture was stirred overnight under a hydrogen atmosphere. The reaction was monitored by TLC. After the reaction was complete, the solvent was evaporated and the mixture was subjected to column chromatography (DCM / EA = 10:1, v / v) to give L2 (110 mg, 83%), a white solid.

[0085] Example 3: Synthesis of 3-(2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)propionic acid (L3)

[0086]

[0087] A mixture of compound L2 (80 g, 0.2 mmol), sodium hydroxide (128 mg, 3.2 mmol), ethanol, and water (v / v = 5:1, 4 mL) was added to a 25 mL flask. The mixture was stirred and refluxed for 5 h. The reaction was monitored by TLC. After the reaction was complete, water was added to the reaction solution, and the resulting solution was acidified to pH 4-5 with 1 M hydrochloric acid. A solid precipitated out. After drying, L3 (40 mg, 57%) was obtained as a pale yellow solid.

[0088] Example 4: Synthesis of (E)-3-(2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)acrylic acid (L4)

[0089]

[0090] Referring to the method for synthesizing L3 from L2 in Example 3, compound L4 (112 mg, 60%) was synthesized using L1 (200 mg, 0.53 mmol) as the starting material.

[0091] It should be understood that the "method of synthesizing L3 from L2 in Example 3" mentioned above means that the method of synthesizing L3 from L2 in Example 3 has the same reaction principle and reaction conditions as the method of synthesizing L4 from L1 in this example. Therefore, the detailed synthesis steps will not be repeated here.

[0092] It should be noted that the descriptions mentioned below, which are similar to those in "the method of synthesizing L3 from L2 in Reference Example 3", have similar meanings and will not be repeated below.

[0093] Example 5: Synthesis of 2-(5-methyl-[1,1'-biphenyl]-2-yl)-3-(phenylethynyl)-1H-pyrrolo[2,3-b]pyridine (L5)

[0094]

[0095] Step 1: Synthesis of 3-iodo-2-(5-methyl-[1,1'-biphenyl]-2-yl)-1-(benzenesulfonyl)-1H-pyrrolo[2,3-b]pyridine (10)

[0096] Compound 7 (637 mg, 1.5 mmol), NIS (372 mg, 1.65 mmol), and 7.5 mL of DCM were added to a flask, and the mixture was stirred at room temperature for 12 h. The reaction was monitored by TLC. After the reaction was complete, the solvent was evaporated, and the mixture was purified by column chromatography (PE / EA = 5:1, v / v) to give 10 (437 mg, 53%), a white solid.

[0097] Step 2: Synthesis of 2-(5-methyl-[1,1'-biphenyl]-2-yl)-3-(phenylethynyl)-1-(benzenesulfonyl)-1H-pyrrolo[2,3-b]pyridine (11)

[0098] Under an Ar atmosphere, 10 (300 mg, 0.55 mmol), Et3N (229 μL, 1.65 mmol), and 2 mL of ultra-dry tetrahydrofuran were added to a dried 25 mL flask. Then, Pd(PPh3)2Cl2 (6 mg, 0.005 mmol) and CuI (3 mg, 0.01 mmol) were added, followed by dropwise addition of a tetrahydrofuran solution of phenylacetylene (74 mg, 0.55 mmol). The mixture was stirred at room temperature for 12 h. The reaction was monitored by TLC. After completion, the solvent was evaporated, and the mixture was subjected to column chromatography (PE / EA = 5:1, v / v) to give 11 (100 mg, 35%), a white solid.

[0099] 1 H NMR (400MHz, DMSO-d6) δ8.37(dd,J=4.8,1.6Hz,1H),8.07(dd,J=7.8,1.7Hz,1H),7.83–7.77(m,2H),7.73–7. 67(m,1H),7.57(t,J=7.9Hz,2H),7.47(d,J=7.7Hz,1H),7.42–7.31(m,10H),7.28–7.22(m,3H),2.49(s,3H).

[0100] Step 3: Synthesis of 2-(5-methyl-[1,1'-biphenyl]-2-yl)-3-(phenethynyl)-1H-pyrrolo[2,3-b]pyridine (L5)

[0101] Intermediate 11 (100 mg, 0.2 mmol), sodium tert-butoxide (77 mg, 0.8 mmol), and 5 mL of 1,4-dioxane were added to a flask, and the mixture was refluxed at 100 °C for 2 h. The reaction was monitored by TLC. After the reaction was complete, the mixture was cooled to ambient temperature, and water (5 mL) and ethyl acetate (5 mL) were added. The organic phase was separated, extracted three times, and the organic layers were combined. The mixture was dried over anhydrous Na₂SO₄, filtered, and evaporated to dryness. The solution was purified by column chromatography (PE / EA = 1:1, v / v) to give compound L5 (26 mg, 35%) as a white solid.

[0102] Example 6: Synthesis of 3-((1-methyl-1H-pyrazol-4-yl)ethynyl)-2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrrolo[2,3-b]pyridine (L6)

[0103]

[0104] Step 1: Synthesis of 3-((1-methyl-1H-pyrazol-4-yl)ethynyl)-2-(5-methyl-[1,1'-biphenyl]-2-yl)-1-(benzenesulfonyl)-1H-pyrrolo[2,3-b]pyridine (12)

[0105] Referring to the method for synthesizing 11 from 10 in Example 5, intermediate 12 (120 mg, 41%) was synthesized from intermediate 10 (300 mg, 0.55 mmol) and N-methyl-4-ynylpyrazole (59 mg, 0.55 mmol).

[0106] Step 2: Synthesis of 3-((1-methyl-1H-pyrazol-4-yl)ethynyl)-2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrrolo[2,3-b]pyridine (L6)

[0107] Referring to the method for synthesizing L5 from 11 in Example 5, intermediate L6 (44 mg, 57%) was synthesized from intermediate 12 (120 mg, 0.2 mmol) as a starting material.

[0108] Example 7: Synthesis of ethyl (E)-3-(2-(4-methyl-2-(thiophen-3-yl)phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)acrylate (L7)

[0109]

[0110] Step 1: Synthesis of 3-(2-chloro-5-methylphenyl)thiophene (14)

[0111] Under an Ar atmosphere, Pd(OAc)₂ (25 mg, 0.1 mmol), Sphos (83 mg, 0.2 mmol), K₃PO₄ (6.4 g, 30 mmol), and 20 mL of toluene were added to a dried 100 mL double-necked flask. Then, 3-bromothiophene (938 μL, 10 mmol) and compound 13 (2.6 g, 15 mmol) were added, and the mixture was refluxed at 90 °C for 2 h. The reaction was monitored by TLC until completion. The solvent was evaporated, and the mixture was purified by column chromatography to give intermediate 14 (1.9 g, 91%), a colorless oily liquid.

[0112] 1 H NMR(400MHz, DMSO-d6)δ7.70(dd,J=2.9,1.4Hz,1H),7.63(dd,J=5.0,3.0Hz,1H),7.41( d,J=8.1Hz,1H),7.34(dd,J=5.1,1.5Hz,2H),7.17(dd,J=8.2,2.2Hz,1H),2.32(s,3H).

[0113] Step 2: Synthesis of 4,4,5,5-tetramethyl-2-(4-methyl-2-(thiophen-3-yl)phenyl)-1,3,2-dioxaborhecyclopentane (15)

[0114] Following the method for synthesizing intermediate 3 from intermediate 2, intermediate 15 (1.3 g, 48%) was synthesized from intermediate 14 (1.9 g, 9.2 mmol) and bis-pinacol boronic acid ester (1.9 g, 18.4 mmol).

[0115] 1 H NMR (400MHz, DMSO-d6) δ7.53(dd,J=4.9,2.9Hz,1H),7.47(d,J=7.6Hz,1H),7.41(dd,J=3. 0,1.4Hz,1H),7.23(s,1H),7.15(ddd,J=9.1,6.2,1.5Hz,2H),2.34(s,3H),1.20(s,12H).

[0116] Step 3: Synthesis of 2-(4-methyl-2-(thiophen-3-yl)phenyl)-1-(phenylsulfonyl)-1H-pyrrole[2,3-b]pyridine (16)

[0117] Referring to the method for synthesizing intermediate 7 from intermediate 3 and intermediate 6 in Example 1, intermediate 16 (1.4g, 83%) was synthesized from intermediate 15 (1.3g, 4.4mmol) and intermediate 6 (1.5g, 4mmol) as raw materials.

[0118] Step 4: Synthesis of 2-(4-methyl-2-(thiophen-3-yl)phenyl)-1H-pyrrole[2,3-b]pyridine (17)

[0119] Referring to the method for synthesizing intermediate 8 from intermediate 7 in Example 1, intermediate 17 (263 mg, 30%) was synthesized from intermediate 16 (1.4 g, 3.3 mmol) as a raw material.

[0120] Step 5: Synthesis of 2-(4-methyl-2-(thiophen-3-yl)phenyl)-1H-pyrrole[2,3-b]pyridine-3-carboxaldehyde (18)

[0121] Referring to the method for synthesizing intermediate 9 from intermediate 8 in Example 1, intermediate 18 (100 mg, 34%) was synthesized from intermediate 17 (263 mg, 0.9 mmol) as a raw material.

[0122] Step 6: Synthesis of ethyl (E)-3-(2-(4-methyl-2-(thiophen-3-yl)phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)acrylate (L7)

[0123] Referring to the method for synthesizing intermediate 10 from intermediate 9 in Example 1, L7 (28 mg, 24%) was synthesized from intermediate 18 (100 mg, 0.3 mmol) as a starting material.

[0124] Example 8: Synthesis of ethyl (E)-3-(2-(4-methyl-2-(pyridin-3-yl)phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)acrylate (L8)

[0125]

[0126] Step 1: Synthesis of 2-chloro-5-methylphenyl)pyridine (19)

[0127] Under an Ar atmosphere, 3-bromopyridine (773 mg, 4.9 mmol), 2-chloro-5-methylphenylboronic acid 13 (1.2 g, 5.8 mmol), Pd(PPh3)4 (567 mg, 0.49 mmol), K2CO3 (4.1 g, 30 mmol), and 50 mL of a toluene / EtOH / H2O (3:2:1, v / v) mixed solvent were added to a 100 mL flask, and the mixture was refluxed at 100 °C for 2 h. The reaction was monitored by TLC. After the reaction was complete, the mixture was cooled to ambient temperature, and water (30 mL) and ethyl acetate (20 mL) were added to the mixture. The organic phase was separated, extracted three times, and the organic layers were combined, dried over anhydrous Na2SO4, filtered, and evaporated to dryness. The mixture was purified by column chromatography (PE / EA = 1:1, v / v) to give compound 19 (950 mg, 95%) as a colorless oily liquid.

[0128] 1 H NMR (400MHz, DMSO-d6) δ8.65–8.60(m,2H),7.87(dt,J=7.9,2.0Hz,1H),7.54–7.46(m,2H),7.32–7.24(m,2H),2.35(s,3H).

[0129] Step 2: Synthesis of 3-(5-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaboron-2-yl)phenyl)pyridine (20)

[0130] Following the method for synthesizing intermediate 3 from intermediate 2, intermediate 20 (778 mg, 56%) was synthesized from intermediate 19 (950 mg, 4.7 mmol) and bis-pinacol boronic acid ester (2.4 g, 9.4 mmol).

[0131] Step 3: Synthesis of 2-(4-methyl-2-(pyridin-3-yl)phenyl)-1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridine (21)

[0132] Referring to the method for synthesizing intermediate 7 from intermediate 3 and intermediate 6 in Example 1, intermediate 21 (563 mg, 55%) was synthesized using intermediate 20 (778 mg, 2.64 mmol) and intermediate 6 (920 mg, 2.4 mmol) as raw materials.

[0133] Step 4: Synthesis of 2-(4-methyl-2-(pyridin-3-yl)phenyl)-1H-pyrrolo[2,3-b]pyridine (22)

[0134] Referring to the method for synthesizing intermediate 8 from intermediate 7 in Example 1, intermediate 22 (274 mg, 75%) was synthesized from intermediate 21 (563 mg, 1.33 mmol) as a raw material.

[0135] Step 5: Synthesis of 2-(4-methyl-2-(pyridin-3-yl)phenyl)-1H-pyrrolo[2,3-b]pyridine-3-carboxaldehyde (23)

[0136] Referring to the method for synthesizing intermediate 9 from intermediate 8 in Example 1, intermediate 23 (140 mg, 45%) was synthesized using intermediate 22 (274 mg, 1 mmol) as a raw material.

[0137] Step 6: Synthesis of ethyl (E)-3-(2-(4-methyl-2-(pyridin-3-yl)phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)acrylate (L8)

[0138] Referring to the method for synthesizing intermediate 10 from intermediate 9 in Example 1, L8 (100 mg, 60%) was synthesized from intermediate 23 (140 mg, 0.45 mmol) as a starting material.

[0139] Example 9: Synthesis of ethyl 3-(2-(4-methyl-2-(pyridin-3-yl)phenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)propionate (L9)

[0140]

[0141] Referring to the method for synthesizing L2 from L1 in Example 1, L9 (24 mg, 48%) was synthesized using L8 (50 mg, 0.13 mmol) as the starting material.

[0142] Example 10: Synthesis of N-(2-(3-(3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-flavoside]-5-yl)thioureaformyl)ethyl)-3-(2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrazolo[2,3-b]pyridin-3-yl)propionamide (L10)

[0143]

[0144] Step 1: Synthesis of 1-(2-aminoethyl)-3-(3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-flavonoid]-5-yl)thiourea (25)

[0145] Ethylenediamine (42 μL, 0.64 mmol) was dissolved in 21 mL of methanol. Then, a methanol solution (12.5 mL) of fluorescein isothiocyanate isomer I 24 (250 mg, 0.64 mmol) was slowly added dropwise to the ethylenediamine system. After the addition was complete, the mixture was stirred at room temperature for 3 h. The reaction was monitored by TLC. After the reaction was complete, the solvent was evaporated under reduced pressure, methanol was added to the flask, the mixture was filtered, and the filter cake was dried to give intermediate 25 (127 mg, 46%), an orange-red powder.

[0146] Step 2: Synthesis of N-(2-(3-(3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-flavoside]-5-yl)thioureaformyl)ethyl)-3-(2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrazolo[2,3-b]pyridin-3-yl)propionamide (L10)

[0147] L3 (68 mg, 0.19 mmol) and 2 mL DMF were added to a flask and stirred for 10 min. HATU (181 mg, 0.48 mmol) was then added, and the mixture was stirred at room temperature for 30 min. Intermediate 25 (127 mg, 0.29 mmol) and DIPEA (100 μL, 0.57 mmol) were added, and the mixture was stirred at room temperature for 18 h. The reaction was monitored by TLC. After the reaction was complete, a small amount of water was added to precipitate the product. The precipitate was filtered, and the filter cake was subjected to preparative HPLC to obtain the final product L10 (28 mg, 19%).

[0148] Example 11: Synthesis of N-(3-(3-(3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-flavonoid]-5-yl)thiourea)propyl)-3-(2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrazolo[2,3-b]pyridin-3-yl)propionamide (L11)

[0149]

[0150] Step 1: Synthesis of 1-(3-aminopropyl)-3-(3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-flavonoid]-5-yl)thiourea (26)

[0151] Referring to the method for synthesizing intermediate 25 from compound 24 in Example 10, intermediate 26 (400 mg, 79%) was synthesized from propylene diamine (80 μL, 1.1 mmol) and compound 24 (420 mg, 1.1 mmol).

[0152] Step 2: Synthesis of N-(3-(3-(3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-flavonoid]-5-yl)thiourea)propyl)-3-(2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrazolo[2,3-b]pyridin-3-yl)propionamide (L11)

[0153] Referring to the method for synthesizing L10 from L3 and intermediate 25 in Example 10, L11 (70 mg, 16%) was synthesized from L3 (200 mg, 0.56 mmol) and intermediate 26 (400 mg, 0.84 mmol).

[0154] Example 12: Synthesis of N-(4-(3-(3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-flavonoid]-5-yl)thiourea)butyl)-3-(2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrazolo[2,3-b]pyridin-3-yl)propionamide (L12)

[0155]

[0156] Step 1: Synthesis of 1-(4-aminobutyl)-3-(3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-flavonoid]-5-yl)thiourea (27)

[0157] Referring to the method for synthesizing intermediate 25 from compound 24 in Example 10, intermediate 27 (200 mg, 50%) was synthesized from butanediamine (120 μL, 0.9 mmol) and compound 24 (360 mg, 0.9 mmol).

[0158] Step 2: Synthesis of N-(4-(3-(3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-flavonoid]-5-yl)thiourea)butyl)-3-(2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrazolo[2,3-b]pyridin-3-yl)propionamide (L12)

[0159] Referring to the method for synthesizing L10 from L3 and intermediate 25 in Example 10, L12 (74 mg, 10%) was synthesized from L3 (100 mg, 0.3 mmol) and intermediate 27 (200 mg, 0.4 mmol).

[0160] Example 13: Synthesis of N-((1R,4R)-4-(3-(3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-flavonoid]-5-yl)thiourea)cyclohexyl)-3-(2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrazolo[2,3-b]pyridin-3-yl)propionamide (L13)

[0161]

[0162] Step 1: Synthesis of 1-((1R,4R)-4-aminocyclohexyl)-3-(3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-flavonoid]-5-yl)thiourea (28)

[0163] Referring to the method for synthesizing intermediate 25 from compound 24 in Example 10, intermediate 28 (220 mg, 57%) was synthesized from 1,4-cyclohexanediamine (90 mg, 0.77 mmol) and compound 24 (300 mg, 0.77 mmol).

[0164] Step 2: Synthesis of N-((1R,4R)-4-(3-(3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-flavonoid]-5-yl)thiourea)cyclohexyl)-3-(2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrazolo[2,3-b]pyridin-3-yl)propionamide (L13)

[0165] Referring to the method for synthesizing L10 from L3 and intermediate 25 in Example 10, L13 (52 mg, 22%) was synthesized from L3 (100 mg, 0.28 mmol) and intermediate 28 (200 mg, 0.4 mmol).

[0166] Example 14: Synthesis of 1-(3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-flavonoid]-5-yl)-3-(2-(4-(3-(2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrazolo[2,3-b]pyridin-3-yl)propionyl)piperazin-1-yl)ethyl)thiourea (L14)

[0167]

[0168] Step 1: Synthesis of tert-butyl(2-(4-(3-(2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrazolo[2,3-b]pyridin-3-yl)propionyl)piperazin-1-yl)ethyl)tert-butyl carbamate (29)

[0169] Compound L3 (100 mg, 0.3 mmol), HBTU (180 mg, 0.45 mmol), and 2 mL of DMF were added to a flask and stirred at room temperature for 10 min. Then, 1-[2-(BOC-amino)ethyl]piperazine (100 mg, 0.39 mmol) and DIPEA (480 μL, 0.9 mmol) were added, and the mixture was stirred at room temperature for 1 h. The reaction was monitored by TLC. After the reaction was complete, water (5 mL) and ethyl acetate (5 mL) were added to the mixture, the organic phase was separated, extracted three times, the organic layers were combined, dried over anhydrous Na2SO4, filtered, and evaporated to dryness. The mixture was purified by column chromatography to give intermediate 29 (136 mg, 82%), a white solid.

[0170] Step 2: Synthesis of 1-(4-(2-aminoethylpiperazin-1-yl)-3-(2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrazolo[2,3-b]pyridin-3-yl)acetone (30)

[0171] Add intermediate 29 (100 mg, 0.18 mmol), 2 mL TFA and 3 mL DCM to a flask, and stir at room temperature for 1 h. Monitor the reaction by TLC. After the reaction is complete, evaporate the solvent to obtain intermediate 30 (80 mg, 0.17 mmol), a white oily liquid, which can be used directly in the next step of the reaction.

[0172] Step 3: Synthesis of 1-(3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-flavonoid]-5-yl)-3-(2-(4-(3-(2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrazolo[2,3-b]pyridin-3-yl)propionyl)piperazin-1-yl)ethyl)thiourea (L14)

[0173] Intermediate 30 (80 mg, 0.17 mmol) and 2 mL DMF were added to a flask, followed by FITC (86 mg, 0.17 mmol) and DIPEA (187 μL, 0.85 mmol). The mixture was stirred overnight at room temperature. The reaction was monitored by TLC. After the reaction was complete, the solvent was evaporated under reduced pressure, and the mixture was separated by preparative HPLC to obtain compound L14 (83 mg, 24%).

[0174] Example 15: Synthesis of N-(4-((3-(3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-flavoside)methyl)phenyl)-3-(2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrido[2,3-b]pyridin-3-yl)propionamide (L15)

[0175]

[0176] Step 1: Synthesis of tert-butyl(4-((3-(2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrido[2,3-b]pyridin-3-yl)propamido)methylbenzyl)carbamate (31)

[0177] Referring to the method for synthesizing intermediate 29 from compound L3 in Example 14, intermediate 31 (109 mg, 78%) was synthesized from 1,4-phenylenediamine (81 mg, 0.34 mmol) and compound L3 (90 mg, 0.25 mmol).

[0178] Step 2: Synthesis of N-(4-aminomethylphenyl)-3-(2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrido[2,3-b]pyridin-3-yl)propionamide (32)

[0179] Referring to the method for synthesizing intermediate 30 from intermediate 29 in Example 14, intermediate 32 (100 mg, 0.19 mmol) was synthesized from intermediate 31 (109 mg, 0.2 mmol) as a raw material.

[0180] Step 3: Synthesis of N-(4-((3-(3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-flavoside)methyl)phenyl)-3-(2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrido[2,3-b]pyridin-3-yl)propionamide (L15)

[0181] Referring to the method for synthesizing compound L14 from intermediate 30 in Example 14, compound L15 (57 mg, 35%) was synthesized from intermediate 32 (100 mg, 0.19 mmol) as a starting material.

[0182] Example 16: Synthesis of N-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl)amino)ethyl)-3-(2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)propionamide (L16)

[0183]

[0184] Step 1: Synthesis of tert-butyl(2-((2-(2,6-dioxoperidin-3-yl)-1,3-dioxo-isoindoline-4-yl)amino)ethyl)carbamate (34)

[0185] Compound 33 (80 mg, 0.3 mmol) and 2 mL DMSO were added under ice bath conditions, followed by N-BOC-ethylenediamine (48 mg, 0.3 mmol) and DIPEA (58 μL, 0.33 mmol). The mixture was stirred at 90 °C for 4 h. The reaction was monitored by TLC. After the reaction was complete, water (5 mL) and ethyl acetate (5 mL) were added to the reaction solution. The organic phase was separated, extracted three times, and the organic layers were combined. The mixture was dried over anhydrous Na₂SO₄, filtered, and evaporated to dryness. The solution was purified by column chromatography (PE / EA = 2:1, v / v) to give intermediate 34 (100 mg, 80%) as a yellow solid.

[0186] Step 2: Synthesis of 1,3-dioxo-isoindoline-2-(2,6-dioxopiperidin-3-yl)-4-di(2-aminoethyl)amine (35)

[0187] Intermediate 34 (100 mg, 0.24 mmol), TFA (2 mL / mmol), and DCM (4 mL / mmol) were added to a flask and stirred at room temperature for 3 h. The reaction was monitored by TLC. After the reaction was complete, the solvent was evaporated to obtain intermediate 35, a yellow oily liquid, which was used directly in the next reaction.

[0188] Step 3: Synthesis of N-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl)amino)ethyl)-3-(2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)propionamide (L16)

[0189] Compound L3 (40 mg, 0.13 mmol), HBTU (92 mg, 0.19 mmol), and 2 mL of DMF were added to a flask and stirred at room temperature for 10 min. Then, a mixture of intermediate 35 (70 mg, 0.17 mmol), DIPEA (84 μL, 0.39 mmol), and 2 mL of DMF was added dropwise, and the mixture was stirred at room temperature for 1 h. The reaction was monitored by TLC. After the reaction was complete, water (5 mL) and ethyl acetate (5 mL) were added to the mixture, the organic phase was separated, extracted three times, and the organic layers were combined. The organic layer was dried over anhydrous Na2SO4, filtered, and evaporated to dryness. Column chromatography yielded the final product L16 (43 mg, 51%), a yellow solid.

[0190] Example 17: Synthesis of 2-(2,6-dioxopiperidin-3-yl)-4-(4-(3-(2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)propionyl)piperazin-1-yl)isoindoline-1,3-dione (L17)

[0191]

[0192] Step 1: Synthesis of tert-butyl 4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl)piperazine-1-carboxylic acid tert-butyl ester (36)

[0193] Referring to the method for synthesizing intermediate 34 from compound 33 in Example 16, intermediate 36 (203 mg, 63%) was synthesized from compound 33 (200 mg, 0.73 mmol) and N-Boc piperazine (135 mg, 0.73 mmol).

[0194] Step 2: Synthesis of 2-(2,6-dioxopiperidin-3-yl)-4-(piperazin-1-yl)isoindoline-1,3-dione (37)

[0195] Referring to the method for synthesizing intermediate 35 from intermediate 34 in Example 16, intermediate 37 (130 mg, 0.73 mmol) was synthesized from intermediate 36 (135 mg, 0.73 mmol) as a starting material.

[0196] Step 3: Synthesis of 2-(2,6-dioxopiperidin-3-yl)-4-(4-(3-(2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)propionyl)piperazin-1-yl)isoindoline-1,3-dione (L17)

[0197] Referring to the method for synthesizing L16 from intermediates L3 and 35 in Example 16, L17 (48 mg, 62%) was synthesized using intermediates L3 (40 mg, 0.13 mmol) and intermediates 37 (75 mg, 0.17 mmol) as raw materials.

[0198] Example 18: Synthesis of N-(2-(4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisobenziridine-4-yl)piperazin-1-yl)ethyl)-3-(2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)propionamide (L18)

[0199]

[0200] Step 1: Synthesis of tert-butyl (2-(4-(2-(2,6-dioxopiridine-3-yl)-1,3-dioxoisoindoline-4-yl)piperazin-1-yl)ethyl)carbamate tert-butyl ester (38)

[0201] Referring to the method for synthesizing intermediate 34 from compound 33 in Example 16, intermediate 38 (438 mg, 82%) was synthesized from compound 33 (300 mg, 1.1 mmol) and 1-[2-(BOC-amino)ethyl]piperazine (250 mg, 1.1 mmol).

[0202] Step 2: Synthesis of 4-(4-(2-aminoethyl)piperazin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoquinoline-1,3-dione (39)

[0203] Referring to the method for synthesizing intermediate 35 from intermediate 34 in Example 16, intermediate 39 (434 mg, 0.9 mmol) was synthesized from intermediate 38 (438 mg, 0.9 mmol) as a starting material.

[0204] Step 3: Synthesis of N-(2-(4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisobenziridine-4-yl)piperazin-1-yl)ethyl)-3-(2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)propionamide (L18)

[0205] Referring to the method for synthesizing L16 from intermediate L3 and intermediate 35 in Example 16, L18 (37 mg, 51%) was synthesized from intermediate L3 (36 mg, 0.1 mmol) and intermediate 39 (62 mg, 0.13 mmol).

[0206] Example 19: Synthesis of N-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-5-yl)amino)ethyl)-3-(2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)-propionamide (L19)

[0207]

[0208] Step 1: Synthesis of tert-butyl(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-5-yl)amino)ethyl)carbamate (41)

[0209] Referring to the method for synthesizing intermediate 34 from compound 33 in Example 16, intermediate 41 (98 mg, 80%) was synthesized from compound 40 (80 mg, 0.3 mmol) and N-BOC-ethylenediamine (48 mg, 0.3 mmol).

[0210] Step 2: Synthesis of 5-((2-aminoethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (42)

[0211] Referring to the method for synthesizing intermediate 35 from intermediate 34 in Example 16, intermediate 42 (96 mg, 0.8 mmol) was synthesized from intermediate 41 (98 mg, 0.8 mmol).

[0212] Step 3: Synthesis of N-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-5-yl)amino)ethyl)-3-(2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)-propionamide (L19)

[0213] Referring to the method for synthesizing L16 from intermediate L3 and intermediate 35 in Example 16, L19 (50 mg, 77%) was synthesized from intermediate L3 (40 mg, 0.1 mmol) and intermediate 42 (54 mg, 0.13 mmol).

[0214] Example 20: Synthesis of 2-(2,6-dioxopiperidin-3-yl)-5-(4-(3-(2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)propionyl)piperazin-1-yl)isoindoline-1,3-dione (L20)

[0215]

[0216] Step 1: Synthesis of tert-butyl 4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-5-yl)piperazine-1-carboxylic acid ester (43)

[0217] Referring to the method for synthesizing intermediate 34 from compound 33 in Example 16, intermediate 43 (205 mg, 64%) was synthesized using compound 40 (200 mg, 0.73 mmol) and N-Boc piperazine (135 mg, 0.73 mmol) as raw materials.

[0218] Step 2: Synthesis of 2-(2,6-dioxadiazin-3-yl)-5-(piperazin-1-yl)isoindoline-1,3-dione (44)

[0219] Referring to the method for synthesizing intermediate 35 from intermediate 34 in Example 16, intermediate 44 (200 mg, 0.46 mmol) was synthesized from intermediate 43 (205 mg, 0.46 mmol) as a raw material.

[0220] Step 3: Synthesis of 2-(2,6-dioxopiperidin-3-yl)-5-(4-(3-(2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)propionyl)piperazin-1-yl)isoindoline-1,3-dione (L20)

[0221] Referring to the method for synthesizing L16 from intermediate L3 and intermediate 35 in Example 16, L20 (40 mg, 46%) was synthesized from intermediate L3 (40 mg, 0.1 mmol) and intermediate 44 (75 mg, 0.17 mmol).

[0222] Example 21: Synthesis of 2-(2,6-dioxopiperidin-3-yl)-5-((2-(4-(3-[(2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)propionyl)piperazin-1-yl)ethyl)amino)isoindoline-1,3-dione (L21)

[0223]

[0224] Step 1: Synthesis of tert-butyl 4-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-5-yl)amino)ethyl)piperazine-1-carboxylate (45)

[0225] Referring to the method for synthesizing intermediate 34 from compound 33 in Example 16, intermediate 45 (400 mg, 75%) was synthesized from compound 40 (300 mg, 1.1 mmol) and 4-N-(2-aminoethyl)-1-N-BOC-piperazine (250 mg, 1.1 mmol).

[0226] Step 2: Synthesis of 2-(2,6-dioxadiazin-3-yl)-5-((2-(piperazin-1-yl)ethyl)amino)isoindoline-1,3-dione (46)

[0227] Referring to the method for synthesizing intermediate 35 from intermediate 34 in Example 16, intermediate 46 (400 mg, 0.8 mmol) was synthesized from intermediate 45 (400 mg, 0.8 mmol) as a starting material.

[0228] Step 3: Synthesis of 2-(2,6-dioxopiperidin-3-yl)-5-((2-(4-(3-[(2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)propionyl)piperazin-1-yl)ethyl)amino)isoindoline-1,3-dione (L21)

[0229] Referring to the method for synthesizing L16 from intermediate L3 and intermediate 35 in Example 16, L21 (26 mg, 36%) was synthesized from intermediate L3 (36 mg, 0.1 mmol) and intermediate 46 (62 mg, 0.13 mmol).

[0230] Example 22: Synthesis of 2-(2,6-dioxopiperidin-3-yl)-4-(4-(1-(3-(2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrido[2,3-b]pyrolin-3-yl)propionyl)azacyclobutane-3-yl)piperazin-1-yl)isoindoline-1,3-dione (L22)

[0231]

[0232] Step 1: Synthesis of 2-(2,6-dioxopiperidin-3-yl)-4-(piperazin-1-yl)isoindoline-1,3-dione (37)

[0233] Referring to the method for synthesizing intermediate 35 from intermediate 34 in Example 16, intermediate 37 (490 mg, 1.44 mmol) was synthesized from intermediate 36 (490 mg, 1.44 mmol).

[0234] Step 2: Synthesis of tert-butyl 3-(4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl)piperazin-1-yl)azacyclobutane-1-carboxylic acid ester (47)

[0235] Intermediate 37 (490 mg, 1.44 mmol) was dissolved in 4 mL of DMF, and DIPEA (251 μL, 1.44 mmol) and 1-Boc-3-azacyclobutanone (494 mg, 2.88 mmol) were added. The mixture was stirred at room temperature for 30 min. Glacial acetic acid (290 μL) and NaBH3CN (181 mg, 2.88 mmol) were added to the reaction system, and the mixture was stirred overnight at 40 °C. The reaction was monitored by TLC. After the reaction was completed, saturated sodium bicarbonate solution was added to quench the reaction. Water (5 mL) and ethyl acetate (5 mL) were added to the mixture, the organic phase was separated, extracted three times, and the organic layers were combined. The mixture was dried over anhydrous Na2SO4, filtered, and evaporated to dryness. Column chromatography was performed to give intermediate 47 (365 mg, 51%) as a yellow solid.

[0236] Step 3: Synthesis of 4-(4-(azacyclobutane-3-yl)piperazin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (48)

[0237] Referring to the method for synthesizing intermediate 35 from intermediate 34 in Example 16, intermediate 48 (365 mg, 0.73 mmol) was synthesized from intermediate 47 (365 mg, 0.73 mmol) as a starting material.

[0238] Step 4: Synthesis of 2-(2,6-dioxopiperidin-3-yl)-4-(4-(1-(3-(2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrido[2,3-b]pyrolin-3-yl)propionyl)azacyclobutane-3-yl)piperazin-1-yl)isoindoline-1,3-dione (L22)

[0239] Referring to the method for synthesizing L16 from intermediate L3 and intermediate 35 in Example 16, L22 (34 mg, 36%) was synthesized from intermediate L3 (45 mg, 0.13 mmol) and intermediate 48 (66 mg, 0.17 mmol).

[0240] Example 23: Synthesis of 2-(2,6-dioxopiperidin-3-yl)-4-(4-((1-(3-(2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)propionyl)piperidin-4-ylmethyl)piperazin-1-yl)isoindoline-1,3-dione (L23)

[0241]

[0242] Step 1: Synthesis of 2-(2,6-dioxopiperidin-3-yl)-4-(piperazin-1-yl)isoindoline-1,3-dione (37)

[0243] Referring to the method for synthesizing intermediate 35 from intermediate 34 in Example 16, intermediate 37 (400 mg, 1.2 mmol) was synthesized from intermediate 36 (400 mg, 1.2 mmol).

[0244] Step 2: Synthesis of tert-butyl 4-((4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl)piperazin-1-yl)methyl)piperidin-1-carboxylic acid tert-butyl ester (49)

[0245] Referring to the method for synthesizing intermediate 47 from intermediate 37 in Example 22, intermediate 49 (317 mg, 48%) was synthesized from intermediate 37 (400 mg, 1.2 mmol) and 1-tert-butoxycarbonylpiperidine-4-carboxaldehyde (512 mg, 2.4 mmol).

[0246] Step 3: Synthesis of 2-(2,6-dioxopiperidin-3-yl)-4-(4-(piperidin-4-ylmethyl)piperazin-1-yl)isoindoline-1,3-dione (50)

[0247] Referring to the method for synthesizing intermediate 35 from intermediate 34 in Example 16, intermediate 50 (317 mg, 0.6 mmol) was synthesized from intermediate 49 (317 mg, 0.6 mmol) as a starting material.

[0248] Step 4: Synthesis of 2-(2,6-dioxopiperidin-3-yl)-4-(4-((1-(3-(2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)propionyl)piperidin-4-ylmethyl)piperazin-1-yl)isoindoline-1,3-dione (L23)

[0249] Referring to the method for synthesizing L16 from intermediate L3 and intermediate 35 in Example 16, L23 (30 mg, 11%) was synthesized from intermediate L3 (120 mg, 0.34 mmol) and intermediate 50 (194 mg, 0.44 mmol).

[0250] Example 24: Synthesis of 2-(2,6-dioxopiperidin-3-yl)-5-(4-(1-(3-(2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)propionyl)azacyclobutane-3-yl)piperazin-1-yl)isoquinoline-1,3-dione (L24)

[0251]

[0252] Step 1: Synthesis of 2-(2,6-dioxopiperidin-3-yl)-5-(piperazin-1-yl)isoindoline-1,3-dione (44)

[0253] Referring to the method for synthesizing intermediate 35 from intermediate 34 in Example 16, intermediate 44 (470 mg, 1.4 mmol) was synthesized from intermediate 43 (470 mg, 1.4 mmol).

[0254] Step 2: Synthesis of tert-butyl 3-(4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)piperazin-1-yl)azacyclobutane-1-carboxylic acid ester (51)

[0255] Referring to the method for synthesizing intermediate 47 from intermediate 37 in Example 22, intermediate 51 (424 mg, 61%) was synthesized from intermediate 44 (470 mg, 1.4 mmol) and 1-Boc-3-azacyclobutanone (480 mg, 2.8 mmol).

[0256] Step 3: Synthesis of 5-(4-(azacyclobutane-3-yl)piperazin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoquinoline-1,3-dione (52)

[0257] Referring to the method for synthesizing intermediate 35 from intermediate 34 in Example 16, intermediate 52 (424 mg, 0.8 mmol) was synthesized from intermediate 51 (424 mg, 0.8 mmol).

[0258] Step 4: Synthesis of 2-(2,6-dioxopiperidin-3-yl)-5-(4-(1-(3-(2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)propionyl)azacyclobutane-3-yl)piperazin-1-yl)isoquinoline-1,3-dione (L24)

[0259] Referring to the method for synthesizing L16 from intermediate L3 and intermediate 35 in Example 16, L24 (26 mg, 32%) was synthesized from intermediate L3 (45 mg, 0.13 mmol) and intermediate 52 (84 mg, 0.17 mmol).

[0260] Example 25: Synthesis of 2-(2,6-dioxopiperidin-3-yl)-5-(4-((1-(3-(2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)propionyl)piperidin-4-yl)methyl)piperazin-1-yl)isoindoline-1,3-dione (L25)

[0261]

[0262] Step 1: Synthesis of 2-(2,6-dioxopiperidin-3-yl)-5-(piperazin-1-yl)isoindoline-1,3-dione (44)

[0263] Referring to the method for synthesizing intermediate 35 from intermediate 34 in Example 16, intermediate 44 (400 mg, 0.9 mmol) was synthesized from intermediate 43 (400 mg, 0.9 mmol).

[0264] Step 2: Synthesis of tert-butyl 4-((4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)piperazin-1-yl)methyl)piperidin-1-carboxylic acid tert-butyl ester (53)

[0265] Referring to the method for synthesizing intermediate 47 from intermediate 37 in Example 22, intermediate 53 (420 mg, 85%) was synthesized from intermediate 44 (400 mg, 0.9 mmol) and 1-tert-butyloxycarbonylpiperidine-4-carboxaldehyde (383 mg, 1.8 mmol).

[0266] Step 3: Synthesis of 2-(2,6-dioxopiperidin-3-yl)-5-(4-(piperidin-4-methyl)piperazin-1-yl)isoquinoline-1,3-dione (54)

[0267] Referring to the method for synthesizing intermediate 35 from intermediate 34 in Example 16, intermediate 54 (420 mg, 0.8 mmol) was synthesized from intermediate 53 (420 mg, 0.8 mmol).

[0268] Step 4: Synthesis of 2-(2,6-dioxopiperidin-3-yl)-5-(4-((1-(3-(2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)propionyl)piperidin-4-yl)methyl)piperazin-1-yl)isoindoline-1,3-dione (L25)

[0269] Referring to the method for synthesizing L16 from intermediate L3 and intermediate 35 in Example 16, L25 (46 mg, 30%) was synthesized from intermediate L3 (75 mg, 0.2 mmol) and intermediate 54 (140 mg, 0.26 mmol).

[0270] Example 26: Synthesis of N-(2-(3-(4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl)piperazin-1-yl)piperidin-1-yl)ethyl)-3-(2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)propionamide (L26)

[0271]

[0272] Step 1: Synthesis of 4-(4-(azacyclobutane-3-yl)piperazin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (48)

[0273] Referring to the method for synthesizing intermediate 35 from intermediate 34 in Example 16, intermediate 48 (600 mg, 1.2 mmol) was synthesized from intermediate 47 (600 mg, 1.2 mmol) as a starting material.

[0274] Step 2: Synthesis of tert-butyl(2-(3-(4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoquinoline-4-yl)piperazin-1-yl)azapropylcyclo-1-yl)ethyl)carbamate (55)

[0275] Add 48 (600 mg, 1.2 mmol), N-tert-butoxycarbonyl-2-aminoacetaldehyde (287 mg, 1.8 mmol), and 10 mL of DCM to a 25 mL flask, then add 9 drops of glacial acetic acid. Stir at room temperature for 1 h, then add EtOH and NaBH3CN (170 mg, 2.7 mmol), and stir overnight. Monitor the reaction by TLC. After the reaction is complete, evaporate the solvent under reduced pressure, and perform column chromatography (PE / EA = 1:1, v / v) to give intermediate 55 (337 mg, 52%) as a white solid.

[0276] Step 3: Synthesis of 4-(4-(1-(2-aminoethyl)indololin-3-yl)piperazin-1-yl)-2-[2,6-dioxopiperidin-3-yl]indol-1,3-dione (56)

[0277] Referring to the method for synthesizing intermediate 35 from intermediate 34 in Example 16, intermediate 56 (337 mg, 0.6 mmol) was synthesized from intermediate 55 (337 mg, 0.6 mmol) as a starting material.

[0278] Step 4: Synthesis of N-(2-(3-(4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl)piperazin-1-yl)piperidin-1-yl)ethyl)-3-(2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)propionamide (L26)

[0279] Referring to the method for synthesizing L16 from intermediates L3 and 35 in Example 16, L26 (47 mg, 18%) was synthesized using intermediates L3 (120 mg, 0.34 mmol) and intermediate 56 (245 mg, 0.44 mmol) as raw materials.

[0280] Example 27: Synthesis of (E)-4-(4-methoxyphenyl)-N-(2-(3-(2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)propamido)ethyl)-4-oxobut-2-enamide (L27)

[0281]

[0282] Step 1: Synthesis of (E)-4-(4-methoxyphenyl)-4-oxobut-2-enoic acid (58)

[0283] Under an Ar atmosphere, maleic anhydride (5 g, 51 mmol), anisole 57 (5.6 mL, 51 mmol), and 100 mL of ultra-dry DCM were added to a dried 250 mL flask. Anhydrous aluminum chloride (13.6 g, 102 mmol) was added slowly in portions. The reaction solution changed from colorless to orange-red, and the mixture was stirred at room temperature for 4 h. The reaction was monitored by TLC. After the reaction was complete, the reaction solution was poured into a solution of ice-cold hydrochloric acid and stirred for 10 min. Water (50 mL) and DCM (50 mL) were added to the mixture. The organic layer was separated, extracted three times, and the organic layers were combined. The mixture was dried over anhydrous Na2SO4, filtered, and evaporated to dryness to obtain intermediate 58 (9.6 g, 91%), a pale yellow solid.

[0284] Step 2: Synthesis of tert-butyl(E)-(2-(4-(4-methoxyphenyl)-4-oxobut-2-enamido)ethyl)carbamate (59)

[0285] Referring to the method for synthesizing L16 from intermediates L3 and 35 in Example 16, intermediate 59 (2.8g, 82%) was synthesized from intermediate 58 (2g, 9.8mmol) and N-BOC-ethylenediamine (2.1g, 13mmol).

[0286] Step 3: Synthesis of (E)-N-(2-aminoethyl)-4-(4-methoxyphenyl)-4-oxobut-2-enamide (60)

[0287] Referring to the method for synthesizing intermediate 35 from intermediate 34 in Example 16, intermediate 60 (700 mg, 1.8 mmol) was synthesized from intermediate 59 (700 mg, 1.8 mmol).

[0288] Step 4: Synthesis of (E)-4-(4-methoxyphenyl)-N-(2-(3-(2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)propamido)ethyl)-4-oxobut-2-enamide (L27)

[0289] Referring to the method for synthesizing L16 from intermediates L3 and 35 in Example 16, L27 (34 mg, 21%) was synthesized from L3 (100 mg, 0.28 mmol) and intermediate 60 (126 mg, 0.36 mmol).

[0290] Example 28: Synthesis of (E)-1-(4-methoxyphenyl)-4-(4-(3-(2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)propionyl)piperazin-1-yl)but-2-en-1,4-dione (L28)

[0291]

[0292] Step 1: Synthesis of tert-butyl(E)-4-(4-methoxyphenyl)-4-oxobut-2-enoylpiperazine-1-carboxylic acid tert-butyl ester (61)

[0293] Referring to the method for synthesizing L16 from intermediates L3 and 35 in Example 16, intermediate 61 (1.6g, 86%) was synthesized from intermediate 58 (1g, 4.9mmol) and N-BOC-piperazine (1.2g, 6.4mmol).

[0294] Step 2: Synthesis of (E)-1-(4-methoxyphenyl)-4-(piperazin-1-yl)but-2-ene-1,4-dione (62)

[0295] Referring to the method for synthesizing intermediate 35 from intermediate 34 in Example 16, intermediate 62 (350 mg, 0.9 mmol) was synthesized from intermediate 61 (350 mg, 0.9 mmol) as a starting material.

[0296] Step 3: Synthesis of (E)-1-(4-methoxyphenyl)-4-(4-(3-(2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)propionyl)piperazin-1-yl)but-2-en-1,4-dione (L28)

[0297] Referring to the method for synthesizing L16 from intermediates L3 and 35 in Example 16, L28 (80 mg, 40%) was synthesized from L3 (120 mg, 0.34 mmol) and intermediate 62 (164 mg, 0.44 mmol).

[0298] Example 29: Synthesis of (E)-N-(2-(4-(4-(4-methoxyphenyl)-4-oxybut-2-enoyl)piperazin-1-yl)ethyl)-3-(2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)propionamide (L29)

[0299]

[0300] Step 1: Synthesis of tert-butyl(E)-2-(4-(4-(4-methoxyphenyl)-4-oxybut-2-enoyl)piperazin-1-yl)ethylcarbamate (63)

[0301] Referring to the method for synthesizing L16 from intermediates L3 and 35 in Example 16, intermediate 63 (900 mg, 56%) was synthesized from intermediate 58 (800 mg, 3.9 mmol) and 1-[2-(BOC-amino)ethyl]piperazine (1.2 g, 5 mmol).

[0302] Step 2: Synthesis of (E)-1-(4-(2-aminoethyl)piperazin-1-yl)-4-(4-methoxyphenyl)but-2-ene-1,4-dione (64)

[0303] Referring to the method for synthesizing intermediate 35 from intermediate 34 in Example 16, intermediate 64 (900 mg, 2.2 mmol) was synthesized from intermediate 63 (900 mg, 2.2 mmol).

[0304] Step 3: Synthesis of (E)-N-(2-(4-(4-(4-methoxyphenyl)-4-oxybut-2-enoyl)piperazin-1-yl)ethyl)-3-(2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)propionamide (L29)

[0305] Referring to the method for synthesizing L16 from intermediates L3 and 35 in Example 16, L29 (34 mg, 19%) was synthesized from L3 (100 mg, 0.28 mmol) and intermediate 64 (151 mg, 0.37 mmol).

[0306] Example 30: Synthesis of (E)-1-(4-methoxyphenyl)-4-(4-(1-(3-(2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrido[2,3-b]pyridin-3-yl)propionyl)azacyclobutane-3-yl)piperazin-1-yl)but-2-en-1,4-dione (L30)

[0307]

[0308] Step 1: Synthesis of (E)-1-(4-methoxyphenyl)-4-(piperazin-1-yl)but-2-ene-1,4-dione (62)

[0309] Referring to the method for synthesizing intermediate 35 from intermediate 34 in Example 16, intermediate 62 (3g, 7.8mmol) was synthesized from intermediate 61 (3g, 7.8mmol) as a raw material.

[0310] Step 2: Synthesis of tert-butyl(E)-3-(4-(4-(4-methoxyphenyl)-4-oxybut-2-enoyl)piperazin-1-yl)azacyclobutane-1-carboxylic acid tert-butyl ester (65)

[0311] Referring to the method for synthesizing intermediate 47 from intermediate 37 in Example 22, intermediate 65 (2g, 60%) was synthesized from intermediate 62 (3g, 7.8mmol) and 1-Boc-3-azacyclobutanone (2.7g, 15.6mmol).

[0312] Step 3: Synthesis of (E)-1-(4-azacyclobutan-3-yl)piperazin-1-yl)-4-(4-methoxyphenyl)but-2-en-1,4-dione (66)

[0313] Referring to the method for synthesizing intermediate 35 from intermediate 34 in Example 16, intermediate 66 (800 mg, 1.9 mmol) was synthesized from intermediate 65 (800 mg, 1.9 mmol) as a raw material.

[0314] Step 4: Synthesis of (E)-1-(4-methoxyphenyl)-4-(4-(1-(3-(2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrido[2,3-b]pyridin-3-yl)propionyl)azacyclobutane-3-yl)piperazin-1-yl)but-2-en-1,4-dione (L30)

[0315] Referring to the method for synthesizing L16 from intermediates L3 and 35 in Example 16, L30 (37 mg, 17%) was synthesized from L3 (120 mg, 0.34 mmol) and intermediate 66 (189 mg, 0.44 mmol).

[0316] Example 31: Synthesis of (E)-1-(4-methoxyphenyl)-4-(4-(1-(3-(2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrido[2,3-b]pyrolin-3-yl)propionyl)indol-3-yl)piperazinyl)but-2-en-1,4-dione (L31)

[0317]

[0318] Step 1: Synthesis of (E)-1-(4-methoxyphenyl)-4-(piperazin-1-yl)but-2-ene-1,4-dione (62)

[0319] Referring to the method for synthesizing intermediate 35 from intermediate 34 in Example 16, intermediate 62 (3g, 7.8mmol) was synthesized from intermediate 61 (3g, 7.8mmol) as a raw material.

[0320] Step 2: Synthesis of tert-butyl(E)-4-((4-(4-methoxyphenyl)-4-oxobut-2-enoyl)piperazin-1-ylmethyl)-1-piperidinecarboxylic acid tert-butyl ester (67)

[0321] Referring to the method for synthesizing intermediate 47 from intermediate 37 in Example 22, intermediate 67 (481 mg, 73%) was synthesized from intermediate 62 (500 mg, 1.4 mmol) and 1-tert-butoxycarbonylpiperidine-4-carboxaldehyde (600 mg, 2.8 mmol).

[0322] Step 3: Synthesis of (E)-1-(4-methoxyphenyl)-4-(4-(piperidin-4-ylmethyl)piperazin-1-yl)but-2-en-1,4-dione (68)

[0323] Referring to the method for synthesizing intermediate 35 from intermediate 34 in Example 16, intermediate 68 (481 mg, 1 mmol) was synthesized from intermediate 67 (481 mg, 1 mmol).

[0324] Step 4: Synthesis of (E)-1-(4-methoxyphenyl)-4-(4-(1-(3-(2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrido[2,3-b]pyrolin-3-yl)propionyl)indol-3-yl)piperazinyl)but-2-en-1,4-dione (L31)

[0325] Referring to the method for synthesizing L16 from intermediates L3 and 35 in Example 16, L31 (34 mg, 21%) was synthesized from L3 (80 mg, 0.23 mmol) and intermediate 68 (140 mg, 0.3 mmol).

[0326] Example 32: Synthesis of (E)-2-cyano-3-cyclopropyl-N-(2-(3-(2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)propamido)ethyl)acrylamide (L32)

[0327]

[0328] Step 1: Synthesis of tert-butyl(2-(3-(2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)propamido)ethyl)tert-butyl carbonate (69)

[0329] Referring to the method for synthesizing L16 from intermediates L3 and 35 in Example 16, intermediate 69 (60 mg, 53%) was synthesized from L3 (80 mg, 0.23 mmol) and N-BOC-ethylenediamine (48 mg, 0.3 mmol).

[0330] Step 2: Synthesis of N-(2-aminoethyl)-3-(2-(5-methyl[1,1'-biphenyl]-2-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)propionamide (70)

[0331] Referring to the method for synthesizing intermediate 35 from intermediate 34 in Example 16, intermediate 70 (60 mg, 0.12 mmol) was synthesized from intermediate 69 (60 mg, 0.12 mmol) as a starting material.

[0332] Step 3: Synthesis of (E)-2-cyano-3-cyclopropyl-N-(2-(3-(2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)propamido)ethyl)acrylamide (L32)

[0333] Referring to the method for synthesizing L16 from intermediates L3 and 35 in Example 16, L32 (17 mg, 27%) was synthesized from intermediate 70 (60 mg, 0.12 mmol) and (E)-2-cyano-3-cyclopropylacrylic acid (21 mg, 0.15 mmol).

[0334] Example 33: Synthesis of (E)-3-cyclopropyl-2-(4-(3-(2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)propionyl)piperazine-1-formyl)acrylonitrile (L33)

[0335]

[0336] Step 1: Synthesis of tert-butyl 4-(3-(2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)propionyl)piperazine-1-carboxylic acid tert-butyl ester (71)

[0337] Referring to the method for synthesizing L16 from intermediates L3 and 35 in Example 16, intermediate 71 (100 mg, 83%) was synthesized from L3 (80 mg, 0.23 mmol) and N-BOC-piperazine (34 mg, 0.18 mmol).

[0338] Step 2: Synthesis of 1-(piperidin-1-yl)-3-(2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)-acetone (72)

[0339] Referring to the method for synthesizing intermediate 35 from intermediate 34 in Example 16, intermediate 72 (100 mg, 0.2 mmol) was synthesized from intermediate 71 (100 mg, 0.2 mmol) as a raw material.

[0340] Step 3: Synthesis of (E)-3-cyclopropyl-2-(4-(3-(2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)propionyl)piperazine-1-formyl)acrylonitrile (L33)

[0341] Referring to the method for synthesizing L16 from intermediates L3 and 35 in Example 16, L33 (17 mg, 21%) was synthesized from intermediate 72 (100 mg, 0.2 mmol) and (E)-2-cyano-3-cyclopropylacrylic acid (21 mg, 0.15 mmol).

[0342] Example 34: Synthesis of (E)-2-cyano-3-cyclopropyl-N-(2-(4-(3-(2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)propionyl)piperazin-1-yl)ethyl)acrylamide (L34)

[0343]

[0344] Step 1: Synthesis of 1-(4-(2-aminoethyl)piperazin-1-yl)-3-(2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)acetone (30)

[0345] Referring to the method for synthesizing intermediate 35 from intermediate 34 in Example 16, intermediate 30 (100 mg, 0.18 mmol) was synthesized from intermediate 29 (100 mg, 0.18 mmol).

[0346] Step 2: Synthesis of (E)-2-cyano-3-cyclopropyl-N-(2-(4-(3-(2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)propionyl)piperazin-1-yl)ethyl)acrylamide (L34)

[0347] Referring to the method for synthesizing L16 from intermediates L3 and 35 in Example 16, L34 (20 mg, 19%) was synthesized from intermediate 30 (100 mg, 0.18 mmol) and (E)-2-cyano-3-cyclopropylacrylic acid (25 mg, 0.18 mmol).

[0348] Example 35: Synthesis of (E)-3-cyclopropyl-2-(4-(1-(3-(2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)propionyl)azacyclopropane-3-yl)piperazine-1-carbonyl)acrylonitrile (L35)

[0349]

[0350] Step 1: Synthesis of (E)-2-cyano-3-cyclopropylacrylic acid (74)

[0351] Add cyanoacetic acid (1 g, 12 mmol) and 12 mL of pyridine to a flask, then add cyclopropaneformaldehyde 73 (925 mg, 13.2 mmol) and tetrahydropyrrole (240 μL, 2.9 mmol), and stir at room temperature for 3 h. Monitor the reaction by TLC. After the reaction is complete, add 10 mL of 6 M hydrochloric acid solution to the reaction mixture, stir for 10 min, then add water (15 mL) and ethyl acetate (15 mL), extract three times, combine the organic layers, dry over anhydrous Na2SO4, filter and evaporate to dryness to obtain intermediate 74 (1.4 g, 88%), a white solid.

[0352] Step 2: Synthesis of tert-butyl(E)-4-(2-cyano-3-cyclopropylacryloyl)piperazine-1-carboxylate (75)

[0353] Referring to the method for synthesizing L16 from intermediates L3 and 35 in Example 16, intermediate 75 (3.4 g, 76%) was synthesized from intermediate 74 (3.6 g, 19 mmol) and N-BOC-piperazine (2 g, 14.6 mmol).

[0354] Step 3: Synthesis of (E)-3-cyclopropyl-2-piperazine-1-carbonylacrylonitrile (76)

[0355] Referring to the method for synthesizing intermediate 35 from intermediate 34 in Example 16, intermediate 76 (700 mg, 2.3 mmol) was synthesized from intermediate 75 (700 mg, 2.3 mmol) as a starting material.

[0356] Step 4: Synthesis of tert-butyl(E)-3-(4-(2-cyano-3-cyclopropylacryloyl)piperazin-1-yl)azacyclopropane-1-carboxylic acid tert-butyl ester (77)

[0357] Referring to the method for synthesizing intermediate 47 from intermediate 37 in Example 22, intermediate 77 (422 mg, 51%) was synthesized from intermediate 76 (700 mg, 2.3 mmol) and 1-Boc-3-azacyclobutanone (788 mg, 4.6 mmol).

[0358] Step 5: Synthesis of (E)-2-(4-(azacyclopropane-3-yl)piperazine-1-carbonyl)-3-cyclopropylacrylonitrile (78)

[0359] Referring to the method for synthesizing intermediate 35 from intermediate 34 in Example 16, intermediate 78 (422 mg, 1.2 mmol) was synthesized from intermediate 77 (422 mg, 1.2 mmol) as a raw material.

[0360] Step 6: Synthesis of (E)-3-cyclopropyl-2-(4-(1-(3-(2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)propionyl)azacyclopropane-3-yl)piperazine-1-carbonyl)acrylonitrile (L35)

[0361] Referring to the method for synthesizing L16 from intermediate L3 and intermediate 35 in Example 16, L35 (15 mg, 11%) was synthesized from L3 (100 mg, 0.3 mmol) and intermediate 78 (83 mg, 0.23 mmol).

[0362] Example 36: Synthesis of (E)-3-cyclopropyl-2-(4-((1-(3-(2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)propionyl)piperazin-4-yl)methyl)piperazin-1-carbonyl)acrylonitrile (L36)

[0363]

[0364] Step 1: Synthesis of (E)-3-cyclopropyl-2-piperazine-1-carbonylacrylonitrile (76)

[0365] Referring to the method for synthesizing intermediate 35 from intermediate 34 in Example 16, intermediate 76 (700 mg, 2.3 mmol) was synthesized from intermediate 75 (700 mg, 2.3 mmol) as a starting material.

[0366] Step 2: Synthesis of tert-butyl(E)-4-((4-(2-cyano-3-cyclopropylacryloyl)piperazin-1-yl)methyl)piperidine-1-carboxylic acid ester (79)

[0367] Referring to the method for synthesizing intermediate 47 from intermediate 37 in Example 22, intermediate 79 (398 mg, 43%) was synthesized from intermediate 76 (700 mg, 2.3 mmol) and 1-tert-butyloxycarbonylpiperidine-4-carboxaldehyde (289 mg, 4.6 mmol).

[0368] Step 3: Synthesis of (E)-3-cyclopropyl-2-(4-(piperidin-4-ylmethyl)piperazine-1-carbonyl)acrylonitrile (80)

[0369] Referring to the method for synthesizing intermediate 35 from intermediate 34 in Example 16, intermediate 80 (398 mg, 1 mmol) was synthesized from intermediate 79 (398 mg, 1 mmol).

[0370] Step 4: Synthesis of (E)-3-cyclopropyl-2-(4-((1-(3-(2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)propionyl)piperazin-4-yl)methyl)piperazin-1-carbonyl)acrylonitrile (L36)

[0371] Referring to the method for synthesizing L16 from intermediates L3 and 35 in Example 16, L36 (13 mg, 10%) was synthesized from L3 (90 mg, 0.25 mmol) and intermediate 80 (80 mg, 0.2 mmol).

[0372] Example 37: Synthesis of N-(2-(2-(adamantane-1-yl)acetamityl)ethyl)-3-(2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)propionamide (L37)

[0373]

[0374] Step 1: Synthesis of tert-butyl (2-(2-(adamantane-1-yl)acetamityl)ethyl)carbamate (82)

[0375] Referring to the method for synthesizing L16 from intermediates L3 and 35 in Example 16, intermediate 82 (205 mg, 61%) was synthesized from 81 (194 mg, 1 mmol) and N-BOC-ethylenediamine (208 mg, 1.3 mmol).

[0376] Step 2: Synthesis of adamantane-1-yl-2-aminoethyl)acetamide (83)

[0377] Referring to the method for synthesizing intermediate 35 from intermediate 34 in Example 16, intermediate 83 (205 mg, 0.61 mmol) was synthesized from intermediate 82 (205 mg, 0.61 mmol) as a starting material.

[0378] Step 3: Synthesis of N-(2-(2-(adamantane-1-yl)acetamyl)ethyl)-3-(2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)propionamide (L37)

[0379] Referring to the method for synthesizing L16 from intermediate L3 and intermediate 35 in Example 16, L37 (36 mg, 27%) was synthesized from L3 (80 mg, 0.23 mmol) and intermediate 83 (98 mg, 0.3 mmol).

[0380] Example 38: Synthesis of 1-(4-(2-adamantane-1-yl)acetyl)piperazin-1-yl)-3-(2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)acetone (L38)

[0381]

[0382] Step 1: Synthesis of tert-butyl 4-(2-(adamantane-1-yl)acetyl)piperidine-1-carboxylate (84)

[0383] Referring to the method for synthesizing L16 from intermediates L3 and 35 in Example 16, intermediate 84 (2.5g, 67%) was synthesized using 81 (2g, 10.3mmol) and N-BOC-piperazine (2.5g, 13.4mmol) as raw materials.

[0384] Step 2: Synthesis of 1-(piperazin-1-yl)-2-(adamantyl)acetone (85)

[0385] Referring to the method for synthesizing intermediate 35 from intermediate 34 in Example 16, intermediate 85 (150 mg, 0.42 mmol) was synthesized from intermediate 84 (150 mg, 0.42 mmol) as a raw material.

[0386] Step 3: Synthesis of 1-(4-(2-adamantane-1-yl)acetyl)piperazin-1-yl)-3-(2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)acetone (L38)

[0387] Referring to the method for synthesizing L16 from intermediate L3 and intermediate 35 in Example 16, L38 (37 mg, 31%) was synthesized from L3 (80 mg, 0.23 mmol) and intermediate 85 (108 mg, 0.3 mmol).

[0388] Example 39: Synthesis of N-(2-(4-(2-(adamantane-1-yl)acetyl)piperazin-1-yl)ethyl)-3-(2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)propionamide (L39)

[0389]

[0390] Step 1: Synthesis of tert-butyl(2-(4-(2-(adamantane-1-yl)acetyl)piperazin-1-yl)ethyl)formate (86)

[0391] Referring to the method for synthesizing L16 from intermediates L3 and 35 in Example 16, intermediate 86 (230 mg, 57%) was synthesized from 81 (195 mg, 1 mmol) and 1-[2-(BOC-amino)ethyl]piperazine (299 mg, 1.3 mmol).

[0392] Step 2: Synthesis of 1-(4-(2-aminoethyl)piperazin-1-yl)-2-(adamantane-1-yl)acetone (87)

[0393] Referring to the method for synthesizing intermediate 35 from intermediate 34 in Example 16, intermediate 87 (230 mg, 0.57 mmol) was synthesized from intermediate 86 (230 mg, 0.57 mmol) as a starting material.

[0394] Step 3: Synthesis of N-(2-(4-(2-(adamantane-1-yl)acetyl)piperazin-1-yl)ethyl)-3-(2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)propionamide (L39)

[0395] Referring to the method for synthesizing L16 from intermediates L3 and 35 in Example 16, L39 (24 mg, 16%) was synthesized from L3 (80 mg, 0.23 mmol) and intermediate 87 (121 mg, 0.3 mmol).

[0396] Example 40: Synthesis of adamantyl-1-yl-2-(4-(adamantyl-1-yl-2-acetylpiperazinyl)-3-azacyclobutane-1-yl-1-yl-3-2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)acetone (L40)

[0397]

[0398] Step 1: Synthesis of 1-(piperazin-1-yl)-2-(adamantyl)acetone (85)

[0399] Referring to the method for synthesizing intermediate 35 from intermediate 34 in Example 16, intermediate 85 (3g, 8mmol) was synthesized from intermediate 84 (3g, 8mmol).

[0400] Step 2: Synthesis of tert-butyl 3-(4-(2-(adamantyl-1-yl)acetylpiperazin-1-yl)azacyclobutane-1-carboxylic acid ester (88)

[0401] Referring to the method for synthesizing intermediate 47 from intermediate 37 in Example 22, intermediate 88 (451 mg, 47%) was synthesized from intermediate 85 (800 mg, 2.3 mmol) and 1-Boc-3-azacyclobutanone (768 mg, 4.6 mmol).

[0402] Step 3: Synthesis of adamantyl-1-yl-2-(3-azacyclobutyl-1-yl-4-piperazin-1-yl)acetone (89)

[0403] Referring to the method for synthesizing intermediate 35 from intermediate 34 in Example 16, intermediate 89 (451 mg, 1.1 mmol) was synthesized from intermediate 88 (451 mg, 1.1 mmol) as a starting material.

[0404] Step 4: Synthesis of adamantyl-1-yl-2-(4-(adamantyl-1-yl-2-acetylpiperazinyl)-3-azacyclobutane-1-yl-1-yl-3-2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)acetone (L40)

[0405] Referring to the method for synthesizing L16 from intermediates L3 and 35 in Example 16, L40 (41 mg, 27%) was synthesized from L3 (80 mg, 0.23 mmol) and intermediate 89 (162 mg, 0.3 mmol).

[0406] Example 41: Synthesis of 1-(4-((4-(2-(adamantane-1-yl)acetylpiperazin-1-yl)methyl)piperidin-1-yl)-3-(2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)-acetone (L41)

[0407]

[0408] Step 1: Synthesis of 1-(piperazin-1-yl)-2-(adamantyl)acetone (85)

[0409] Referring to the method for synthesizing intermediate 35 from intermediate 34 in Example 16, intermediate 85 (3g, 8mmol) was synthesized from intermediate 84 (3g, 8mmol).

[0410] Step 2: Synthesis of tert-butyl 4-((4-(2-(adamantane-1-yl)-acetylpiperazin-1-yl)methylpiperidin-1-yl)carboxylic acid tert-butyl ester (90)

[0411] Referring to the method for synthesizing intermediate 47 from intermediate 37 in Example 22, intermediate 90 (463 mg, 42%) was synthesized from intermediate 85 (800 mg, 2.3 mmol) and 1-tert-butyloxycarbonylpiperidine-4-carboxaldehyde (1.1 g, 4.8 mmol).

[0412] Step 3: Synthesis of 1-piperidine-4-methylpiperazine-1-ethyl-2-adamantyl-1-ethyl ketone (91)

[0413] Referring to the method for synthesizing intermediate 35 from intermediate 34 in Example 16, intermediate 91 (463 mg, 1 mmol) was synthesized from intermediate 90 (463 mg, 1 mmol) as a raw material.

[0414] Step 4: Synthesis of 1-(4-((4-(2-(adamantane-1-yl)acetylpiperazin-1-yl)methyl)piperidin-1-yl)-3-(2-(5-methyl-[1,1'-biphenyl]-2-yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)-acetone (L41)

[0415] Referring to the method for synthesizing L16 from intermediates L3 and 35 in Example 16, L41 (40 mg, 23%) was synthesized from L3 (90 mg, 0.25 mmol) and intermediate 91 (148 mg, 0.32 mmol).

[0416] The following experimental examples demonstrate the beneficial effects of the 7-azaindole compounds provided in the embodiments of this disclosure.

[0417] Experimental Example 1: Evaluation of the binding constants of the fluorescent polarization probes L10-L15 provided in this disclosure with CSN5 enzyme.

[0418] 1. Experimental materials: WHB all-black 96-well flat-bottom luminescent plate (specification: WHB-96-02), WHB 96-well cell culture plate (specification: WHB-96), Ni-NTA column, low-temperature high-pressure continuous flow cell disruptor, ELISA reader.

[0419] 2. Experimental Methods:

[0420] The protein expression and purification steps were as follows: The recombinant plasmid was transformed into *E. coli* Transetta (DE3) for expression, and cultured at 37°C until the OD600 reached 0.6–0.8. The temperature was then lowered to 18°C ​​(CSN5), and 0.3 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to induce the expression of the target protein. Culture was continued for 18–20 hours. The next day, bacterial cells were collected by centrifugation at 4000 rpm for 30 minutes and resuspended in lysis buffer A (Buffer A consisted of 20 mM Tris, 200 mM NaCl, pH 8.0). Bacteria were lysed using a low-temperature, high-pressure continuous flow cell disruptor (JNBIO), and cell debris was removed by centrifugation at 12000 rpm for 30 minutes. The supernatant was collected and purified using a Ni-NTA affinity chromatography column (Roche). Before protein purification, the Ni-NTA column was pretreated: residual protein was eluted with 10 mL of Buffer B (the original Buffer A components remained unchanged, but now containing 500 mM imidazole), followed by rinsing with 20 mL of MilliQ water, and finally equilibration of the affinity column with 20 mL of Buffer A. The collected supernatant was added to the Ni-NTA column, and after all the supernatant had passed through the Ni-NTA column, elution was performed using a gradient of Buffer C with different concentrations of imidazole to remove non-specifically bound and weakly bound proteins. Finally, the target protein was eluted with Buffer D containing 250 mM imidazole (the original Buffer A components remained unchanged, but now containing 250 mM imidazole). The eluted target protein was collected and concentrated using an Amicon Ultra 10K (Millipore) filter, and then the target protein was transferred to the appropriate buffer using a Hi Trap Desalting column (GE Healthcare). All steps in the purification process were monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAG), and protein concentration was determined using a Nanno Drop 2000 spectrophotometer (Thermo Scientific). The protein was then flash-frozen in liquid nitrogen and stored at -80°C for subsequent enzyme kinetic experiments.

[0421] K d The experimental steps for value testing are as follows:

[0422] The CSN5 enzyme solution was diluted three-fold to ten concentrations using a test buffer (20 mM Tris, 200 mM NaCl, pH 7.5, 0.01% Triton). Then, 40 μL of buffer solution and 10 μL of the diluted enzyme solution were added to each well, followed by 10 μL of the pre-prepared fluorescent substrate solution (final concentration 0.25 μM). Three replicates were set up for each group, with a total reaction volume of 60 μL. The mixture was incubated at room temperature for 1 h, and then detected using a microplate reader. The changes in fluorescence polarization values ​​were fitted using GraphPadPrism software to obtain the K... d value.

[0423] 3. Experimental Results:

[0424] The 7-azaindole compounds L10–L15 disclosed herein are fluorescent compounds. We used these fluorescent compounds to establish an enzyme activity screening system based on fluorescence polarization technology. Compounds containing fluorescent groups that strongly bind to the CSN5 active site were selected as fluorescent polarization substrates. These substrates exhibited strong fluorescence polarization signals when binding to CSN5. Inhibitors, fluorescent substrates, and CSN5 protein were co-incubated; the two ligands competitively bound to the CSN5 protein. Higher affinity between the inhibitor and protein resulted in weaker polarization signals; conversely, weaker polarization signals resulted in stronger polarization signals. We determined the binding constants of these six fluorescent substrates, and the results are shown in Table 1. It can be seen that L10–L15 all have strong binding affinity to the CSN5 subunit, with the compound whose linker is ethylenediamine showing the strongest affinity. d The concentration was 0.13 μM. Based on optimized fluorescence polarization experimental conditions, we used the fluorescent substrate L10 to test the IC50 of the reported CSN5 inhibitor I-12 and the CSN5 subunit, respectively. 50 The value was 1.04 μM, which verified the sensitivity and accuracy of the fluorescent polarization substrate L10 for screening CSN5 inhibitors.

[0425] Table 1. Determination of the binding constants of L10 to L15 with CSN5 (K d )

[0426]

[0427] Experimental Example 2: Tests of the in vitro inhibitory activity, cell proliferation inhibitory activity, or degradation activity of the 7-azaindole compounds provided in this disclosure against CSN5 enzyme.

[0428] 1. Experimental materials: WHB all-black 96-well flat-bottom luminescent plate (specification: WHB-96-02), WHB 96-well cell culture plate (specification: WHB-96), 6-well cell culture plate, sterile pipette tips, sterile centrifuge tubes.

[0429] 2. Experimental Methods

[0430] The experimental steps for in vitro enzyme activity testing are as follows:

[0431] All inhibitor compounds were dissolved in fresh DMSO to prepare a 100 mM stock solution. At room temperature (25°C), the inhibition rate of single-concentration target compounds (100 mM and 10 mM) against CSN5 was tested in a 96-well black ELISA plate. Three replicates were initially set up. 10 μL of the compound working solution, 30 μL of buffer solution, and 10 μL of enzyme solution were added to each well sequentially, and the plate was incubated for 10 min. Then, 10 μL of the pre-prepared fluorescent substrate solution (final concentration 0.25 μM) was added, bringing the total reaction volume to 60 μL. The plate was incubated for another 1 h, and the fluorescence polarization was detected using a fluorescence polarization detector. The inhibition rate of the tested compound at a single concentration was calculated using Excel software based on the fluorescence polarization change. Compounds with an inhibition rate greater than 50% were then selected for IC50 assay. 50 The IC50 of the inhibitor compound was obtained by plotting the relationship between activity and inhibitor concentration using software. 50 For each concentration, three parallel groups were set up.

[0432] The experimental steps for testing the HCT116 cell proliferation inhibition activity were as follows:

[0433] Collect cells in the logarithmic growth phase, adjust the cell suspension concentration, add 100 μL to each well, and seed the cells. Incubate at 37°C with 5% CO2 until a monolayer of cells forms at the bottom of the wells (96-well flat-bottom plate). After adhesion, remove the 96-well plate, discard the original culture medium, add the drug, and place the cells in an incubator. Incubate at 37°C with 5% CO2 for 24 hours, and observe under an inverted microscope. After the drug treatment is complete, add 20 μL MTT to each well and continue culturing for 3 hours. Terminate the culture and carefully aspirate the culture medium from the wells. Add 100 μL DMSO to each well, and shake on a shaker at low speed for 10 minutes to fully dissolve the crystals. Measure the absorbance of each well using a microplate reader.

[0434] The experimental steps for the degradation activity test are as follows:

[0435] HCT116 cells were cultured in 6-well cell culture plates. Different concentrations of the drug were added to the culture plates and incubated. The old culture medium was discarded, and the cells were washed twice with PBS. The PBS was discarded, and RIPA was added. After lysis, the cells were transferred from the wells to 1.5 mL EP tubes using a cell scraper and further disrupted using a cell wall disruptor. After disruption, the cells were centrifuged (12000 rpm, 4 °C, 10 min), and 10 μL of the supernatant was collected and diluted to 100 μL with PBS (10-fold dilution) for BCR quantification. A large volume of the supernatant was labeled, and SDS loading buffer was added. The mixture was then transferred to 1.5 mL EP tubes and incubated at 95 °C for 20 min to denature the protein and bind it to SDS. 20 μL of different concentrations of protein standard solutions were added sequentially, followed by 20 μL of sample. Then, 200 μL of BCA working solution was added to each well, and the mixture was shaken for 5 min and incubated at 37 °C for 30 min. Finally, the absorbance was measured at 562 nm using a microplate reader to obtain the standard protein concentration curve and the absorbance of each well. The original protein concentration was then calculated by dilution factor, followed by protein quantification to ensure a consistent total protein load in each well. After loading, electrophoresis, membrane transfer, membrane cutting, blocking, incubation with primary antibody, incubation with secondary antibody, and finally, color development were performed.

[0436] To further evaluate the activity of compounds L16–L41 at the cellular level, we tested the effects of these 26 compounds on the proliferation activity of the CSN5-overexpressing cell line HCT116 using the MTT assay.

[0437] 3. Experimental Results:

[0438] The above experimental methods were used to test the inhibitory activity of L3-L4, L9, and L16-L41 on CSN5 enzyme and the effect of L16-L41 on HCT116 proliferation activity. IC50 was used to evaluate the inhibitory activity of L3-L4, L9, and L16-L41 on CSN5 enzyme and the effect of L16-L41 on HCT116 proliferation activity. 50 The values ​​are shown in Table 2 (three parallel experiments were conducted, and the average value of the experimental results was taken, with an error between 5% and 10%).

[0439] Table 2. Effects of L3-L4, L9, L16-L41 on CSN5 enzyme inhibitory activity and L16-L41 on HCT116 inhibitory activity (IC50). 50 ) a

[0440]

[0441]

[0442] The in vitro CSN5 enzyme activity test results in Table 2 show that most of the compounds provided in this disclosure have good CSN5 inhibitory activity. Compounds L1 to L9 are small molecule compounds, and initially, L3 (IC) appears to be the most active. 50=14.97 μM) showed good activity. Compounds L16–L41 were potential degradative agents, exhibiting good inhibitory activity in preliminary enzyme activity evaluation experiments; among compounds L16–L26 containing the pomalidomide structure, those with C4 as the linking site generally showed higher enzyme activity than those with C5 as the linking site; compounds L27–L31 containing the p-methoxyphenyl fumarate structure generally showed good enzyme activity, IC50 = 14.97 μM. 50 All values ​​were less than 4 μM, with compound L27 showing the best enzyme activity (IC50). 50 =1.63 μM); compounds L27-L31 containing the cyanoacrylamide fragment also showed good enzyme activity, and the IC50 of compounds was 1.63 μM. 50 The values ​​were all less than 10 μM; the enzyme activities of compounds containing adamantane structure L37 to L41 varied considerably.

[0443] To further evaluate the activity of the degrading agents at the cellular level, we used an MTT assay to test the effects of 26 potential degrading agents, compounds L16–L41, on the proliferation activity of the CSN5-overexpressing cell line HCT116. Table 2 shows that compounds containing the p-methoxyphenyl fumarate structure exhibited better inhibitory activity, consistent with the enzyme activity results, including compound L28 (IC50). 50 =0.54μM), L29(IC) 50 =2.8μM), L30 (IC) 50 =3.88μM) and L31(IC 50 =1.57 μM); while compounds containing pomalidomide showed weaker inhibitory activity against HCT116 cells, IC50 = 1.57 μM; 50 The values ​​are all above 30 μM; in compounds containing cyanoacrylamide, L36 (IC) 50 (23.21 μM) showed weak inhibitory activity. Some compounds containing the adamantane structure showed moderate inhibitory activity.

[0444] Experimental Example 3: Preliminary evaluation of the degradation potential of the 7-azaindole compounds provided in this disclosure on cellular CSN5.

[0445] Based on enzyme and cell activity, we selected a number of compounds (L20, L22, L27, L28, L29, L31, L34, L36, L37, L40) and used Western blotting to detect their effects on the catalytic activity of the CSN complex and its degradation activity on the CSN5 subunit in HCT116 cells treated at a concentration of 10 μM for 16 h. Figure 1As shown, compared with the DMSO control group, positive compounds I-12, L20, and L22 exhibited significant inhibitory effects on the catalytic activity of the COP9 complex, blocking the deNEDD8ization of Cullin1 protein and resulting in a significant increase in Cullin1-NEDD8 protein. This is consistent with the cell proliferation inhibitory activity (IC50) exhibited by compounds L20 and L22. 50 There are some differences in the levels above 100 μM. It is speculated that L20 and L22 have weak inhibitory effects on the catalytic activity of the COP9 complex, insufficient to kill cells, and therefore did not show any inhibitory effect on cell proliferation. The compound L28 (HCT116, IC50) has a stronger inhibitory effect on cell proliferation. 50 =0.56μM) and L31(HCT116,IC 50 At 1.57 μM, no significant increase in Cullin1-NEDD8 protein was observed, leading us to speculate that this series of compounds likely exhibits strong cell proliferation inhibition through interaction with other targets via the p-methoxyphenyl fumarate structure, rather than through interaction with COP9. We then investigated the effects of these compounds on CSN5 levels; none of the selected compounds showed significant potential for degrading CSN5 subunits. Given the significant differences in CSN5 subunits and variants across other cell lines, these potential degraders may play a role in CSN5 degradation.

[0446] In summary, this disclosure provides a 7-azaindole compound that can be used as a fluorescent polarization probe or a degradation chemical probe targeting CSN5. The fluorescent polarization chemical probe exhibits a good binding affinity to the metalloproteinase CSN5, and based on this, we have established a CSN5 inhibitor screening system with high accuracy and ease of operation, solving the problem of difficulty in establishing in vitro CSN5 activity systems and laying an important foundation for the discovery of novel CSN5 inhibitors. Simultaneously, the degradation chemical probe demonstrates good enzymatic and cellular inhibitory activity, showing potential for degrading CSN5 protein and its variants. This provides a material basis for the development of CSN5-targeting drugs and the study of their biological functions, and also has great potential in the discovery of cancer immunotherapy drugs and the treatment of other related diseases.

[0447] The above description is merely a preferred embodiment of this disclosure. It should be understood that this disclosure is not limited to the forms disclosed herein and should not be construed as excluding other embodiments. It can be used in various other combinations, modifications, and environments, and can be altered within the scope of the concept described herein through the above teachings or techniques or knowledge in related fields. Any modifications and variations made by those skilled in the art that do not depart from the spirit and scope of this disclosure should be within the protection scope of the appended claims.

Claims

1. A 7-azaindole compound, characterized in that, The 7-azaindole compounds have the structure shown in Formula I: Ⅰ。 2. The method for synthesizing the 7-azaindole compound as described in claim 1, characterized in that, Includes the following steps: 。 3. Use of the 7-azaindole compound as described in claim 1 in the preparation of CSN5 inhibitors.

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