Alpha-acyloxy alkenyl amide electrophilic warheads and uses thereof

Abstract

The invention provides an alpha-acyloxy alkenyl amide electrophilic warhead and application of the alpha-acyloxy alkenyl amide electrophilic warhead. The alpha-acyloxy alkenyl amide electrophilic warhead has a structure as shown in a formula (I). The alpha-acyloxy alkenyl amide electrophilic warhead can selectively covalently modify lysine residues in proteins and polypeptides, can be used for developing covalent kinase inhibitors, and can be used for discovering and identifying drug targets. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the fields of chemical biology molecular probes and chemical medicine technology, and particularly relates to an α-acyloxy acrylamide electrophilic warhead and its applications in the selective modification of protein amino acid residues, in the development of covalent kinase inhibitors, and in drug target discovery and identification. Background Art

[0002] Among nearly 20,000 proteins in the human proteome, nearly 74% of the proteins are non-druggable targets, 3% are known therapeutic drug targets, and 7% of the proteins are potential drug targets. Drug target discovery is the most critical link in the process of disease treatment and new drug research and development. Currently, drug target discovery still faces a difficult problem: the development of new druggable targets. Chemical proteomics technology is an efficient research means for drug target discovery and is widely used in the fields of medicinal chemistry, natural product chemistry, pharmacology, and modernization of traditional Chinese medicine. Fragment-based drug design is a new technology for drug research and development. It first screens low-affinity ligands that bind to the sub-active sites of biological target molecules and then obtains the desired high-affinity ligands through optimization and assembly. Natural product fragments with certain activities are ideal fragment compounds for fragment-based drug research and development and new drug target discovery. The main point is to develop a new type of small molecule covalent warhead to introduce the natural product fragment into the target protein pocket and bind to specific reaction sites, thereby detecting the reaction activity and discovering some unknown and important druggable targets in human proteins.

[0003] Small molecule covalent warheads are powerful tools for studying protein functions. They can regulate protein functions by forming covalent bonds, providing benefits for the treatment of some diseases, such as enhancing and sustaining the inhibitory effects of drugs. However, most human proteins cannot be applied to drug design due to the lack of small molecule ligands. Selective modification of specific protein sites is one of the effective ways to solve the above problems. Currently, the most extensive selective modification is the research on small molecules based on cysteine modification. Most of the applied covalent warheads contain α,β-unsaturated amide structures. However, among the 19,613 proteins in the human proteome, especially more than 500 protein kinases, there are only about 200 protein kinases with cysteine residues near the ATP pocket, and less than 50 have been proven to covalently bind to inhibitors. Therefore, it is necessary to expand small molecule covalent warheads that can be covalently modified other than cysteine. The conserved catalytic lysine in protein kinases is a candidate for developing covalent drugs. Lysine residues account for about 5.9% of the abundance in the human proteome. Although its nucleophilicity is not as strong as that of cysteine, it is essential for the activity of protein kinases. In addition, lysine exists in many functional sites, including enzyme active sites and interfaces that mediate protein-protein interactions. Lysine is also often used as a site for post-translational regulation of protein structure and function, such as acetylation, methylation, and ubiquitination. Therefore, developing covalent warheads that specifically target lysine is crucial for chemical proteomics applications and the development of covalent inhibitors. Similarly, using the reaction specificity of small molecules with lysine in proteins is also important for developing natural products to target other affinity targets besides known targets, or for identifying the action targets of known drug active molecules. Summary of the Invention

[0004] Based on this, the present invention provides an α-acyloxy enamide electrophilic warhead, which can selectively covalently modify lysine residues in proteins and polypeptides, and can be used for the development of covalent kinase inhibitors, as well as for discovering and identifying drug targets.

[0005] The present invention includes the following technical solutions.

[0006] On the one hand, the present invention provides an α-acyloxy enamide electrophilic warhead having the structure shown in formula (I),

[0007]

[0008] wherein, R1 is selected from: C1-C6 alkyl, C1-C6 alkyl-substituted C6-C 10 aryl.

[0009] In some embodiments, R1 is selected from: C1-C3 alkyl, phenyl substituted by C1-C3 alkyl.

[0010] In some of these embodiments, R1 is methyl or p-tolyl.

[0011] In a second aspect, the present invention provides the use of the α-acyloxyacrylamide electrophilic warhead in selectively covalently modifying lysine residues in proteins or polypeptides.

[0012] In a third aspect, the present invention provides the use of the α-acyloxyacrylamide electrophilic warhead in the identification and characterization of drug molecule targets.

[0013] In a fourth aspect, the present invention provides the use of the α-acyloxyacrylamide electrophilic warhead in the discovery and analysis of drug targets based on natural product molecules.

[0014] In a fifth aspect, the present invention provides a method for identifying and characterizing drug molecule targets, comprising the following steps:

[0015] (1) Introduce a carboxyl group into the drug molecule to obtain a drug molecule containing a carboxyl group;

[0016] (2) React the drug molecule containing a carboxyl group with a sulfonylacetyleneamine of the structure shown in formula (II) to obtain a drug molecule containing an α-acyloxyacrylamide electrophilic warhead of the structure shown in formula (III);

[0017] (3) Incubate the drug molecule containing an α-acyloxyacrylamide electrophilic warhead with live cells or cell lysates, and then perform post-treatment and analysis on the incubated reaction solution;

[0018]

[0019] wherein, R1 is as described in any one of claims 1-3;

[0020] R2 is the residue of the drug molecule with an alkynyl substituent after carboxyl reaction;

[0021] If the drug molecule itself contains a carboxyl group, step (1) is omitted.

[0022] In some of these embodiments, the drug molecule is a drug molecule having kinase inhibitory activity and / or having tumor cell proliferation inhibitory activity.

[0023] In some of these embodiments, the live cells are tumor cells and the cell lysate is a lysate of tumor cells.

[0024] In some of these embodiments, the incubation includes the following reaction conditions: the concentration of the drug molecule containing an α-acyloxyacrylamide electrophilic warhead is 5 μM - 500 μM, preferably 10 μM - 100 μM.

[0025] In some of these embodiments, the incubation further includes the following reaction conditions: the incubation temperature is 30°C - 40°C, and the incubation time is 30 min - 5 h.

[0026] In some of these embodiments, the drug molecule containing an α - acyloxyacrylamide electrophilic warhead is selected from the following compounds:

[0027]

[0028] In a sixth aspect, the present invention provides the drug molecule containing an α - acyloxyacrylamide electrophilic warhead as described above, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof.

[0029] In a seventh aspect, the present invention provides the use of the drug molecule containing an α - acyloxyacrylamide electrophilic warhead as described above, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, in the preparation of a kinase inhibitor.

[0030] In some of these embodiments, the kinase is EGFR L858R protein kinase, BTK protein kinase, CDK1 protein kinase, CDK2 protein kinase, CDK5 protein kinase, AURKA protein kinase, MEK2 (MP2K2) protein kinase, CHK2 protein kinase, STK38 protein kinase.

[0031] In an eighth aspect, the present invention provides the use of the drug molecule containing an α - acyloxyacrylamide electrophilic warhead as described above, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, in the preparation of an anti - tumor drug.

[0032] In some of these embodiments, the tumor is lung cancer, breast cancer, leukemia.

[0033] In a ninth aspect, the present invention provides an anti - tumor drug, which is prepared from an active ingredient and a pharmaceutically acceptable carrier or excipient, and the active ingredient includes the drug molecule containing an α - acyloxyacrylamide electrophilic warhead as described in the present invention, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof.

[0034] In a tenth aspect, the present invention provides a method for the discovery and analysis of drug targets based on natural product molecules, including the following steps:

[0035] (1) Introduce a carboxyl group into the natural product molecule to obtain a natural product molecule containing a carboxyl group;

[0036] (2) React the natural product molecule containing a carboxyl group with a sulfonylethynylamine having the structure shown in formula (II) to obtain a natural product molecule containing an α - acyloxyacrylamide electrophilic warhead having the structure shown in formula (IV);

[0037] (3) Incubate the natural product molecule containing the α - acyloxyacrylamide electrophilic warhead with live cells or cell lysates, and then perform post - treatment and analysis on the incubated reaction solution;

[0038]

[0039] Among them, R1 is as described in any one of claims 1 - 3;

[0040] R3 is the residue after the reaction of the natural product molecule at its carboxyl group;

[0041] If the natural product molecule itself contains a carboxyl group, step (1) is omitted.

[0042] In some embodiments, the live cells are tumor cells, and the cell lysate is the lysate of tumor cells.

[0043] In some embodiments, the incubation includes the following reaction conditions: the concentration of the natural product molecule containing the α - acyloxyacrylamide electrophilic warhead is 5 μM - 500 μM, preferably 10 μM - 100 μM.

[0044] In some embodiments, the incubation further includes the following reaction conditions: the incubation temperature is 20 °C - 40 °C, and the incubation time is 30 min - 5 h.

[0045] In some embodiments, the natural product molecule containing the α - acyloxyacrylamide electrophilic warhead is selected from the following compounds:

[0046]

[0047] The present invention provides an α - acyloxyacrylamide electrophilic warhead, which can selectively covalently modify lysine residues in proteins or polypeptides, has high selectivity and high efficiency for the modification of lysine residues, can be used for the study of protein activity, and has easily available raw materials and a simple preparation method, which is of great significance for chemical proteomics applications.

[0048] The α - acyloxyacrylamide electrophilic warhead of the present invention can be used for the development of covalent kinase inhibitors. The covalent inhibitor designed and synthesized with α - acyloxyacrylamide as the covalent warhead has good kinase inhibitory activity and good anti - tumor cell proliferation activity, and can be applied to the development of covalent inhibitors for treating tumors.

[0049] The α - acyloxyacrylamide electrophilic warhead of the present invention can be used to discover and identify drug targets. Introducing α - acyloxyacrylamide as a covalent warhead into drug molecules can be used to identify and determine the action targets and their lysine sites of active drug molecules; introducing α - acyloxyacrylamide as a covalent warhead into the structure of natural products can construct a large number of fragment libraries containing α - acyloxyacrylamide electrophilic warheads with natural products as the parent nucleus. Further, using mass spectrometry means to globally analyze the targets and their lysine residues bound by various natural product fragments can be used for the discovery and analysis of drug targets based on natural products. Description of the Drawings

[0050] Figure 1 Test results of covalent labeling of bovine serum albumin by compounds A1 / 2 / 3 / 4.

[0051] Figure 2 Covalent modification results of compounds A1 / 2 / 3 / 4 on the protein amino acid residues of tumor cells.

[0052] Figure 3 Selective modification results of compound A1 on the lysine residues of living cells.

[0053] Figure 4 Inhibitory activities of compounds E2 / 3 / 4 / 5 against EGFR L858R protein kinase.

[0054] Figure 5 Analysis results of the bindable targets of compound E3 in H3255 cells.

[0055] Figure 6 Verification results of the bindable targets of compound E3 in H3255 cells.

[0056] Figure 7 Modification of lysine residues in EGFR L858R protein kinase by compound E3.

[0057] Figure 8 Analysis results of the potential bindable targets of compounds X1 / 2 / 3 / 4 in K562 cells.

[0058] Figure 9 Verification results of the potential bindable targets of compounds X1 / 2 / 3 / 4 in K562 cells.

[0059] Figure 10 、 Figure 11 、 Figure 12 Selective modification results of compounds X1 / 2 / 3 / 4 on the lysine residues of K562 cells.

[0060] Figure 13Verification results of the selective modification of lysine residues in HEK 293T cells by compound X1 / 3.

[0061] Figure 14 Identification results of the target and its lysine site in the cell lysate of tumor cells MDA-MB-231 for natural product fragments containing α-acyloxyacrylamide electrophilic warheads.

[0062] Figure 15 Identification results of the target and its lysine site of natural product fragment MA-3 containing α-acyloxyacrylamide electrophilic warhead in MAD-MB-231 cells. Detailed implementation manners

[0063] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meanings as commonly understood by those skilled in the technical field to which the present invention belongs. The terms used in the description of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0064] The terms "comprising" and "having" and any variations thereof in the present invention are intended to cover non-exclusive inclusion. For example, a process, method, device, product or equipment that includes a series of steps is not limited to the listed steps or modules, but may optionally further include steps not listed, or may optionally further include other steps inherent to these processes, methods, products or equipment.

[0065] As used in the present invention, "a plurality of" means two or more. "And / or" describes the association relationship of associated objects and indicates that there can be three relationships. For example, A and / or B can represent: A exists alone, A and B exist simultaneously, and B exists alone. The character " / " generally represents an "or" relationship between the associated objects before and after.

[0066] As used herein, the term "alkyl" means a branched and straight-chain saturated aliphatic hydrocarbon group having a specific number of carbon atoms. For example, the definition of "C1-C6 alkyl" for "C1-C6" includes groups having 1, 2, 3, 4, 5 or 6 carbon atoms arranged in a straight chain or a branched chain. For example, "C1-C6 alkyl" specifically includes methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, pentyl, and hexyl.

[0067] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions in the following embodiments are usually carried out under conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight. The raw materials and reagents used in the following embodiments are all commercially available.

[0068] Example 1

[0069]

[0070] N-Methyl-N-p-toluenesulfonyl ethynylamine S1 (50 mg, 0.24 mmol), 4-ethynylbenzoic acid S2 (70 mg, 0.48 mmol) and 5 mL of DCM were added to a round-bottom flask. After reacting at room temperature for 5 minutes, the solvent was dried under vacuum. The crude product was eluted on a SiO2 column with petroleum ether / acetone at a ratio of 3:1 to obtain the desired product A1 as a white solid (68 mg, yield 54%). 1 HNMR (400 MHz, DMSO) δ 7.80 (m, 2H), 7.72 - 7.66 (m, 2H), 7.63 (d, J = 7.9 Hz, 2H), 7.39 (d, J = 7.9 Hz, 2H), 5.05 (t, J = 2.6 Hz, 1H), 4.88 (d, J = 2.6 Hz, 1H), 4.55 (d, J = 2.2 Hz, 1H). 13 C NMR (151 MHz, DMSO) δ 163.1, 146.7, 144.7, 133.8, 132.5, 130.3, 130.3, 128.6, 128.0, 127.7, 101.5, 85.1, 82.9, 37.8, 21.5. ESI-MS calcd. for C 19 H 17 NO4S [M+Na] + m / z = 378.07701; Found 378.0773.

[0071] Example 2

[0072]

[0073] The product A2 was obtained by referring to the synthesis method of A1 as a white solid (39 mg, yield 69%). 11H NMR (400 MHz, DMSO) δ 8.03 (d, J = 8.0 Hz, 2H), 7.68 (d, J = 7.9 Hz, 2H), 5.18 (d, J = 2.5 Hz, 1H), 5.07 (d, J = 2.5 Hz, 1H), 4.55 (s, 1H), 3.11 (m, 6H). 13 13C NMR (151 MHz, DMSO) δ 163.5, 146.9, 132.7, 130.5, 128.8, 127.8, 100.6, 85.1, 82.9, 37.2, 37.0. ESI-MS calcd. for C 13 H 13 NO4S [M+Na]+ m / z =

[0074] 302.0457; Found 302.0443.

[0075] Example 3

[0076]

[0077] The product A3 was obtained by referring to the synthesis method of A1, which was a white solid (40 mg, yield 60%). 1 1H NMR (400 MHz, DMSO) δ 7.68 (d, J = 7.9 Hz, 2H), 7.46 (d, J = 7.7 Hz, 2H), 4.78 (d, J = 2.4 Hz, 1H), 4.69 (d, J = 2.7 Hz, 1H), 2.95 (t, J = 1.4 Hz, 3H), 2.78 (d, J = 2.4 Hz, 1H), 2.42 (s, 3H), 2.32 (t, J = 7.3 Hz, 2H), 2.18 - 2.11 (m, 2H), 1.53 (m, 2H), 1.41 (m, 2H). 13 13C NMR (151 MHz, DMSO) δ 170.9, 146.7, 144.6, 134.1, 130.3, 128.0, 100.0, 84.6, 71.9, 37.5, 33.0, 27.6, 23.5, 21.5, 17.8. ESI-MS calcd. for C 17 H 21 NO4S [M+Na]+ m / z = 358.1084; Found 358.1074.

[0078] Example 4

[0079]

[0080] The product A4 was obtained by referring to the synthesis method of A1, which was a white solid (20 mg, yield 35%). 11H NMR (400 MHz, DMSO) δ 8.22 (t, J = 5.5 Hz, 1H), 4.84 (d, J = 2.4 Hz, 1H), 4.66 (d, J = 2.4 Hz, 1H), 3.69 (m, 2H), 2.89 (s, 3H), 2.84 (s, 3H), 2.50 (t, J = 6.7 Hz, 2H), 2.30 - 2.25 (m, 2H), 2.16 (m, 1H). 13 13C NMR (151 MHz, DMSO) δ 174.2, 171.2, 170.9, 170.8, 100.2, 81.6, 73.5, 37.5, 36.4, 29.8, 29.2, 28.3. ESI-MS calcd. for C 11 H 16 N2O5S [M+Na]+ m / z = 311.0672; Found 311.0672.

[0081] Example 5

[0082]

[0083] First step: Add S6 (210 mg, 1.0 mmol), 3-bromopropene (178 mg, 1.5 mmol), potassium carbonate (276 mg, 2.0 mmol) and 5 mL of DMF into a round-bottom flask. After stirring overnight at 50 °C, add 20 mL of water and extract with ethyl acetate (3 × 15 mL). Combine the organic layers, wash with water (3 × 10 mL). Finally, dry with Na2SO4 and evaporate to dryness under reduced pressure. The crude product is purified by eluting with 1:3 ethyl acetate / petroleum ether to obtain S7 as a white solid (156 mg, yield 63%). 1 1H NMR (400 MHz, CDCl3) δ 8.88 (s, 1H), 7.61 (s, 1H), 7.36 (s, 1H), 4.96 (d, J = 2.4 Hz, 2H), 4.07 (s, 3H), 2.63 (t, J = 2.4 Hz, 1H).

[0084] Second step: Add S7 (150 mg, 0.6 mmol), methyl 3-hydroxybenzoate (137 mg, 0.9 mmol), potassium carbonate (166 mg, 1.2 mmol) and 3 mL of DMF into a round-bottom flask. After stirring overnight at 50 °C, add 15 ml of water and extract with ethyl acetate (3 × 15 ml). Combine the organic layers, wash with water (3 × 10 mL). Finally, dry with Na2SO4 and evaporate to dryness under reduced pressure. The crude product is purified by eluting with 10:1 ethyl acetate / DCM to obtain S8 as a white solid (206 mg, yield 94%). 11H NMR (400 MHz, CDCl3) δ 8.65 (s, 1H), 8.02 (m, 1H), 7.96 (t, J = 2.0 Hz, 1H), 7.74 (s, 1H), 7.59 (t, J = 7.9 Hz, 1H), 7.51 (m, 1H), 7.37 (s, 1H), 4.98 (d, J = 2.4 Hz, 2H), 4.09 (s, 3H), 3.95 (s, 3H), 2.63 (t, J = 2.4 Hz, 1H). 13 13C NMR (151 MHz, CDCl3) δ 166.2, 165.3, 156.2, 153.1, 152.5, 149.8, 148.0, 132.0, 129.7, 127.1, 126.7, 123.3, 110.4, 107.2, 103.2, 56.9, 56.4, 52.4.

[0085] Step 3: Add S8 (146 mg, 0.4 mmol) and THF / H2O (3:1, 4 mL) to a 25 mL round-bottom flask. Add LiOH·H2O (34 mg, 0.8 mmol) at 0 °C. After stirring at room temperature for 3 h, concentrate the reaction solution until the volatile solvents are removed. Then acidify the residue to pH -5 with 1 M HCl. Extract the mixture with DCM. Dry over Na2SO4 and evaporate the solvent under reduced pressure to obtain product S9 as a white solid (110 mg, yield 79%). 1 1H NMR (400 MHz, DMSO) δ 13.21 (s, 1H), 8.58 (s, 1H), 7.90 (dt, J = 6.7, 1.8 Hz, 1H), 7.81 (d, J = 2.3 Hz, 1H), 7.74 (s, 1H), 7.63 (dd, J = 4.9, 2.0 Hz, 2H), 7.43 (s, 1H), 5.06 (d, J = 2.4 Hz, 2H), 4.01 (s, 3H), 3.69 (t, J = 2.4 Hz, 1H). 13 13C NMR (151 MHz, DMSO) δ 167.0, 165.2, 156.4, 152.9, 152.8, 149.7, 148.3, 133.0, 130.6, 127.3, 127.0, 123.2, 109.9, 107.5, 103.3, 79.6, 79.0, 56.9, 56.7.

[0086]

[0087] Step 4: Dissolve S9 (35 mg, 0.1 mmol) in a mixed solvent of 0.5 mL hexafluoroisopropanol (HFIP) and 1.5 mL DCM, then add N-methyl ethynyl tosylamide (MYTsA) (21 mg, 0.1 mmol). After 5 minutes, remove the solvent under vacuum, and elute the crude product on a SiO2 column with petroleum ether / acetone at a ratio of 3:1 to obtain the desired white solid product E1 (30 mg, yield 54%). 1 1H NMR (400 MHz, CDCl3) δ 8.65 (s, 1H), 7.87 (dd, J = 4.7, 2.4 Hz, 2H), 7.81 - 7.70 (m, 3H), 7.59 - 7.52 (m, 2H), 7.39 (s, 1H), 7.30 - 7.23 (m, 3H), 5.04 (d, J = 2.6 Hz, 1H), 5.00 (d, J = 2.4 Hz, 2H), 4.86 (d, J = 2.6 Hz, 1H), 4.10 (s, 3H), 3.12 (s, 3H), 2.65 (t, J = 2.4 Hz, 1H), 2.35 (s, 3H). 13 13C NMR (151 MHz, CDCl3) δ 165.2, 163.3, 156.3, 153.0, 152.5, 149.9, 148.1, 147.0, 144.1, 134.1, 130.5, 129.8, 129.6, 128.0, 127.7, 127.6, 123.8, 110.3, 107.3, 103.2, 101.8, 56.9, 56.5, 37.3, 21.5. ESI-MS calcd. for C 29 H 26 N3O7S [M + H]+ m / z = 560.1491; Found 560.1486.

[0088] Example 6

[0089]

[0090] Dissolve S9 (35 mg, 0.1 mmol) in a mixed solvent of 0.5 mL hexafluoroisopropanol (HFIP) and 1.5 mL DCM, then add N-methyl ethynyl methanesulfonamide (MYMsA) (14 mg, 0.1 mmol). After 5 minutes, remove the solvent under vacuum, and elute the crude product on a SiO2 column with petroleum ether / acetone at a ratio of 3:1 to obtain the desired white solid product E2 (31 mg, yield 65%). 11H NMR (400 MHz, CDCl3) δ 8.67 (s, 1H), 8.08 (m, 1H), 8.02 (t, J = 1.9 Hz, 1H), 7.75 (s, 1H), 7.65 (t, J = 7.9 Hz, 1H), 7.59 (m, 1H), 7.46 (s, 1H), 5.16 (d, J = 2.7 Hz, 1H), 5.03 (d, J = 2.7 Hz, 1H), 4.99 (d, J = 2.4 Hz, 2H), 4.10 (s, 3H), 3.21 (s, 3H), 3.05 (s, 3H), 2.65 (t, J = 2.4 Hz, 1H). 13 13C NMR (151 MHz, CDCl3) δ 165.1, 163.4, 156.3, 152.9, 152.7, 149.9, 148.1, 146.2, 130.2, 130.1, 128.1, 127.6, 123.8, 110.3, 107.2, 103.1, 100.4, 56.9, 56.4, 37.7, 36.2. ESI-MS calcd. for C 23 H 22 N3O7S [M+H]+ m / z = 484.1178; Found 484.1173.

[0091] Example 7

[0092]

[0093] Step 1, S10 was synthesized according to the literature [Xiaodong, Yongzhou, Zhu, et al. Design, synthesis and biological evaluation of 6-(nitroimidazole-1H-alkyloxyl)-4-anilinoquinazolines as efficient EGFR inhibitors exerting cytotoxic effects both under normoxia and hypoxia. European Journal of Medicinal Chemistry: Chimie Therapeutique, 89, 826-834 (2015)]. S10 (291 mg, 1.0 mmol), methyl 5-bromovalerate (293 mg, 1.5 mmol), potassium carbonate (166 mg, 1.2 mmol), potassium iodide (50 mg, 0.3 mmol) and 5 mL of DMF were added to a round-bottom flask. After stirring at 100 °C overnight, 20 mL of water was added, and the mixture was extracted with ethyl acetate (3 × 20 mL). The organic layers were combined and washed with water (3 × 15 mL). Finally, it was dried over Na2SO4 and evaporated under reduced pressure. The crude product was purified by elution with 1:2 ethyl acetate / petroleum ether to obtain S11 as a white solid (237 mg, yield 59%). 1 H NMR (400 MHz, CDCl3) δ 8.68 (s, 1H), 8.06 (s, 1H), 7.93 (t, J = 1.8 Hz, 1H), 7.87 (m, 1H), 7.40 - 7.30 (m, 2H), 7.30 - 7.22 (m, 2H), 4.15 (t, J = 7.0 Hz, 2H), 3.98 (s, 3H), 3.68 (s, 3H), 3.09 (s, 1H), 2.47 (t, J = 6.9 Hz, 2H), 1.98 - 1.88 (m, 2H), 1.88 - 1.78 (m, 2H). 13 C NMR (151 MHz, CDCl3) δ 169.8, 151.8, 150.4, 148.8, 143.7, 142.6, 134.4, 124.1, 122.9, 120.4, 117.9, 117.7, 117.7, 102.9, 96.9, 78.7, 63.9, 51.4, 47.2, 28.6, 22.6, 16.4.

[0094] Step 2: Charge S11 (217 mg, 0.5 mmol) and THF / H2O (1:1, 6 mL) into a 25 mL round-bottom flask. Add LiOH·H2O (42 mg, 1.0 mmol) at 0 °C. After stirring at room temperature for 3 hours, concentrate the reaction solution until the volatile solvents are removed. Then acidify the residue to pH -5 with 1 M HCl. Extract the mixture with DCM. Dry the organic layer with Na2SO4. Remove the solvent in vacuo to obtain product S12 as a white solid (147 mg, yield 71%). 1 1H NMR (400 MHz, DMSO) δ 12.05 (s, 1H), 9.65 (s, 1H), 8.53 (s, 1H), 7.99 (s, 1H), 7.90 (d, J = 8.0 Hz, 2H), 7.42 (t, J = 7.9 Hz, 1H), 7.23 (d, J = 10.0 Hz, 2H), 4.18 (dd, J = 13.1, 6.8 Hz, 3H), 3.95 (s, 3H), 2.35 (t, J = 7.3 Hz, 2H), 1.90 - 1.81 (m, 2H), 1.73 (m, 2H). 13 13C NMR (151 MHz, DMSO) δ 174.9, 156.6, 154.9, 153.2, 148.8, 147.5, 140.3, 129.4, 126.8, 125.3, 123.1, 122.2, 109.4, 107.8, 103.0, 81.1, 69.0, 56.4, 56.4, 33.8, 28.5, 21.8.

[0095]

[0096] Step 3: The synthesis process of E3 is similar to that of E1. The product is a white solid with a yield of 49%. 1 1H NMR (400 MHz, CDCl3) δ 8.67 (s, 1H), 8.10 (s, 1H), 7.88 (t, J = 1.8 Hz, 1H), 7.81 - 7.75 (m, 1H), 7.70 - 7.63 (m, 2H), 7.36 - 7.20 (m, 7H), 4.79 (d, J = 2.8 Hz, 1H), 4.43 (d, J = 2.8 Hz, 1H), 4.11 (t, J = 6.5 Hz, 2H), 3.96 (s, 3H), 3.07 (s, 1H), 2.96 (s, 3H), 2.49 (t, J = 6.9 Hz, 2H), 2.42 (s, 3H), 1.94 (m, 3H), 1.86 (m, 2H). 1313C NMR (151 MHz, CDCl3) δ 171.3, 156.5, 155.2, 153.5, 148.8, 147.5, 147.4, 144.4, 139.0, 132.9, 129.7, 129.6, 128.8, 128.0, 127.7, 125.5, 125.5, 122.9, 122.8, 122.6, 109.3, 107.7, 107.6, 107.6, 101.6, 101.5, 100.1, 83.5, 68.5, 56.2, 37.9, 37.9, 33.4, 27.7, 21.6, 20.8. ESI-MS calcd. for C 32 H 32 N4O6SNa [M+Na]+ m / z = 623.1940; Found 623.1935.

[0097] Example 8

[0098]

[0099] The synthesis process of E4 is similar to that of E2. The product is a white solid with a yield of 68%. 1 1H NMR (400 MHz, CDCl3) δ 8.68 (s, 1H), 7.90 - 7.78 (m, 3H), 7.36 (m, 1H), 7.30 - 7.26 (m, 2H), 7.25 (d, J = 2.1 Hz, 2H), 4.90 (d, J = 2.8 Hz, 1H), 4.84 (d, J = 2.8 Hz, 1H), 4.15 (t, J = 6.4 Hz, 2H), 3.99 (s, 3H), 3.10 (m, 4H), 2.97 (s, 3H), 2.58 (t, J = 6.8 Hz, 2H), 2.04 - 1.95 (m, 2H), 1.95 - 1.85 (m, 2H). 13 13C NMR (151 MHz, CDCl3) δ 166.5, 151.6, 150.4, 148.8, 148.8, 144.0, 142.7, 141.8, 134.3, 124.3, 124.3, 124.2, 123.0, 123.0, 123.0, 120.5, 120.4, 118.0, 117.9, 117.8, 104.5, 103.1, 96.7, 94.4, 94.3, 78.7, 63.8, 51.4, 32.3, 32.2, 32.0, 28.7, 28.6, 22.9, 16.2, 16.1. ESI-MS calcd. for C 26 H 29N4O6S[M+H]+ m / z = 525.1808; Found 525.1802.

[0100] Example 9

[0101]

[0102] First step: A mixture of S10 (1 g, 3.4 mmol), methyl bromoacetate (788 mg, 5.1 mmol), potassium carbonate (948 mg, 6.8 mmol) and potassium iodide (170 mg, 1.0 mmol) dissolved in DMF (15 mL) was stirred at room temperature for 3 hours. Water was added to the reaction mixture and filtered. Then ethyl acetate was added to the liquid to form a precipitate. The precipitate was filtered and dried to obtain compound S13 as a gray powder (447 mg, yield 73%). 1 H NMR (400 MHz, DMSO) δ 9.47 (s, 1H), 8.51 (s, 1H), 7.95 (t, J = 1.9 Hz, 1H), 7.89 - 7.81 (m, 2H), 7.42 (t, J = 7.9 Hz, 1H), 7.27 - 7.20 (m, 2H), 4.98 (s, 2H), 4.21 (s, 1H), 3.97 (s, 3H), 3.75 (s, 3H). 13 C NMR (151 MHz, DMSO) δ 169.0, 156.7, 154.8, 153.6, 147.8, 147.5, 140.1, 129.4, 127.0, 125.4, 123.3, 122.2, 109.0, 108.1, 103.6, 83.9, 81.1, 65.7, 56.4, 52.4.

[0103] Second step: S14 was synthesized in the same manner as S12 as a pale yellow solid (474 mg, yield 80%). 1 H NMR (400 MHz, DMSO) δ 9.51 (s, 1H), 8.51 (s, 1H), 7.95 (t, J = 1.8 Hz, 1H), 7.91 - 7.80 (m, 2H), 7.41 (t, J = 7.9 Hz, 1H), 7.32 - 7.17 (m, 2H), 4.88 (s, 2H), 4.21 (s, 1H), 3.96 (s, 3H).

[0104]

[0105] The synthesis process of E5 is similar to that of E1. The product is a white solid with a yield of 68%. 1HNMR(400MHz, DMSO) δ 9.43(s, 1H), 8.54(s, 1H), 7.97(s, 1H), 7.86(d, J = 7.6Hz, 2H), 7.70(d, J = 7.9Hz, 2H), 7.45 - 7.35(m, 3H), 7.22(d, J = 7.7Hz, 1H), 5.03(s, 2H), 4.94(d, J = 2.7Hz, 1H), 4.77(d, J = 2.7Hz, 1H), 4.21(d, J = 1.9Hz, 1H), 3.99(s, 3H), 2.98(s, 3H), 2.36(s, 3H). 13 C NMR(151MHz, DMSO) δ 166.3, 156.7, 154.7, 153.7, 148.0, 147.1, 146.1, 144.8, 140.1, 133.6, 130.3, 130.3, 129.4, 128.1, 127.9, 127.0, 125.2, 123.0, 122.3, 109.0, 108.2, 104.1, 100.4, 83.9, 81.1, 65.7, 56.5, 37.4, 31.2, 21.5. ESI - MS calcd. for C 29 H 27 N4O6S [M + H]+ m / z = 559.1651; Found 559.1652.

[0106] Example 10

[0107]

[0108] In the first step, S15 was synthesized according to the literature [Yang, T., Cuesta, A., Wan, X. et al. Reversible lysine - targeted probes reveal residence time - based kinase selectivity. Nat Chem Biol 18, 934 - 941 (2022)]. S15 (102 mg, 0.28 mmol) and succinyl chloride (44 mg, 0.28 mmol) were added to a mixed solvent of 5 mL DCM and 5 mL DMF, and Et3N (28 mg, 0.28 mmol) was added. After stirring overnight at 50 °C, 0.2 mL of acetic acid was added to the mixture, and the solvent was removed under vacuum. Then DCM was added to the residue to form a precipitate. The precipitate was filtered and dried to obtain compound S16 as a white powder (81 mg, yield 62%). 11H NMR (400 MHz, DMSO) δ 12.11 - 12.00 (m, 2H), 9.81 (br, 1H), 8.95 (t, J = 6.1 Hz, 1H), 6.85 (br, 1H), 6.16 (br, 1H), 4.02 (m, 2H), 3.81 (d, J = 23.7 Hz, 4H), 3.55 (m, 4H), 3.10 (p, J = 2.6 Hz, 1H), 2.62 (dd, J = 7.4, 5.6 Hz, 2H), 2.47 (dd, J = 7.3, 5.6 Hz, 2H), 1.90 (tt, J = 8.4, 5.1 Hz, 1H), 1.01 - 0.90 (m, 2H), 0.76 - 0.61 (m, 2H).

[0109]

[0110] The synthesis process of X1 is similar to that of E1. The reaction was purified by elution on a silica column with DCM / MeOH 30:1 to obtain the desired white solid product X1 (36 mg, yield 53%). 1 1H NMR (400 MHz, CDCl3) δ 8.37 (s, 1H), 7.98 (t, J = 5.7 Hz, 1H), 7.75 (d, J = 8.2 Hz, 2H), 7.35 (d, J = 8.0 Hz, 2H), 6.96 (s, 1H), 6.13 (s, 1H), 4.87 (d, J = 2.7 Hz, 1H), 4.59 (d, J = 2.6 Hz, 1H), 4.25 (m, 2H), 3.85 (m, 4H), 3.74 (m, 2H), 3.61 (m, 2H), 3.03 (s, 3H), 2.76 (m, 4H), 2.30 (t, J = 2.5 Hz, 1H), 1.90 (m, 1H), 1.05 - 0.92 (m, 2H), 0.76 (m, 2H). 13 13C NMR (151 MHz, CDCl3) δ 170.7, 169.6, 164.1, 160.9, 147.0, 144.2, 133.6, 130.0, 129.6, 129.6, 128.0, 127.3, 100.0, 79.3, 71.7, 45.1, 43.9, 43.8, 41.6, 37.3, 29.3, 29.1, 27.9, 21.6, 7.9. ESI - MS calcd. for C 32 H 38 N9O6S [M + H]+ m / z = 676.2666; Found 676.2670.

[0111] Example 11

[0112]

[0113] The synthesis process of X2 is similar to that of E2. The reaction was purified by elution on a silica column with DCM / MeOH 30:1 to obtain the desired white solid product X2 (46 mg, yield 77%). 1 H NMR (400 MHz, DMSO) δ 12.04 (s, 1H), 9.82 (br, 1H), 8.95 (t, J = 6.2 Hz, 1H), 6.74 (br, 1H), 6.22 (br, 1H), 5.02 (d, J = 2.4 Hz, 1H), 4.82 (d, J = 2.4 Hz, 1H), 4.02 (d, J = 6.0 Hz, 2H), 3.82 (m, 4H), 3.56 (m, 4H), 3.10 (d, J = 2.5 Hz, 1H), 3.07 (d, J = 2.0 Hz, 3H), 3.01 (d, J = 2.0 Hz, 3H), 2.83 - 2.63 (m, 4H), 1.89 (m, 1H), 0.93 (m, 2H), 0.69 (m, 2H). 13 C NMR (151 MHz, DMSO) δ 171.5, 171.1, 170.4, 170.3, 169.9, 169.5, 164.1, 163.8, 161.2, 161.0, 152.4, 146.4, 100.3, 93.2, 93.2, 81.7, 73.1, 55.4, 41.0, 40.7, 37.6, 36.4, 33.0, 29.4, 28.6, 28.0, 25.0, 8.3. ESI-MS calcd. for C 26 H 33 N9O6SNa [M + Na]+ m / z = 622.2172; Found 622.2169.

[0114] Example 12

[0115]

[0116] In the first step, methyl terephthalate (180 mg, 1.0 mmol), HATU (380 mg, 1.0 mmol), DIEA (323 mg, 2.5 mmol), and 6 mL of DMF were added to a round-bottom flask and stirred for 15 minutes. Then S15 (366 mg, 1.0 mmol) was added to the mixture and stirred for another 3 h. 20 mL of water was added to the mixture, and it was extracted with ethyl acetate (3 × 10 mL). The combined organic layers were washed with water (3 × 10 mL). Finally, it was dried with Na2SO4 and evaporated under reduced pressure. The crude product was purified by chromatographic elution with 30:1 DCM / MeOH to obtain the white solid S17 (119 mg, yield 23%). 11H NMR (400 MHz, DMSO) δ 12.09 (s, 1H), 9.83 (s, 1H), 8.95 (t, J = 6.2 Hz, 1H), 8.05 (d, J = 8.2 Hz, 2H), 7.61 (d, J = 7.9 Hz, 2H), 6.83 (br, 1H), 6.17 (br, 1H), 4.01 (d, J = 6.0 Hz, 2H), 3.82 (m, 9H), 3.38 (s, 2H), 3.09 (s, 1H), 1.88 (m, 1H), 0.90 (m, 2H), 0.67 (m, 2H).

[0117] In the second step, S18 was synthesized in the same manner as S9 and was obtained as a white solid (150 mg, yield 73%).

[0118]

[0119] In the third step, the synthesis process of X3 was similar to that of E1. The reaction was purified by elution on a silica column with DCM / MeOH 30:1 to obtain the desired white solid product X3 (28 mg, yield 38%). 1 1H NMR (400 MHz, DMSO) δ 12.02 (s, 1H), 9.85 (s, 1H), 8.94 (s, 1H), 7.90 (d, J = 7.8 Hz, 2H), 7.72 (d, J = 7.9 Hz, 2H), 7.62 (d, J = 7.9 Hz, 2H), 7.41 (d, J = 7.9 Hz, 2H), 6.80 (br, 1H), 6.17 (br, 1H), 5.06 (s, 1H), 4.90 (s, 1H), 4.09 - 3.64 (m, 8H), 3.47 - 3.35 (m, 4H) 3.09 (s, 1H), 3.05 (s, 3H), 2.38 (s, 3H), 1.88 (m, 1H), 0.87 (m, 2H), 0.67 (m, 2H). 13 13C NMR (151 MHz, DMSO) δ 168.5, 164.0, 163.3, 161.2, 146.8, 144.7, 141.7, 133.8, 130.4, 130.3, 130.3, 129.3, 128.0, 127.9, 101.4, 93.4, 81.7, 73.1, 37.8, 32.0, 31.6, 30.8, 28.6, 22.6, 21.5, 14.4, 8.3. ESI-MS calcd. for C36H38N9O6S [M+H]+ m / z = 724.2666; Found 724.2663.

[0120] Example 13

[0121]

[0122] The synthesis process of X4 is similar to that of E2. On a silica column, the reaction was eluted and purified with DCM / MeOH 30:1 to obtain the desired white solid product X4 (47 mg, yield 49%). 1 HNMR(400MHz,DMSO)δ12.02(s,1H),9.85(s,1H),8.94(s,1H),8.12(d,J=7.9Hz,2H),7.67(d,J=7.9Hz,2H),6.89(br,1H),6.16(br,1H),5.20(d,J=2.5Hz,1H),5.09(d,J=2.5Hz,1H),4.12 - 3.59(m,8H),3.48 - 3.34(m,4H),3.20 - 2.96(m,7H),1.89(m,1H),0.89(m,2H),0.67(m,2H). 13 C NMR(151MHz,DMSO)δ168.5,164.0,163.6,161.2,147.0,141.8,130.6,129.5,128.0,100.6,81.7,73.1,49.1,44.2,43.7,42.0,37.2,37.0,28.6,25.0,22.6,14.4,8.3.ESI-MS calcd.for C 30 H 34 N9O6S[M + H]+m / z=648.2353;Found 648.2358.

[0123] Example 14

[0124]

[0125] For the synthesis of BA-2, the purchased BA (408 mg, 1 mmol) was dissolved in dichloromethane and methanol (DCM:MeOH = 1:1), then N-methylethynylmethanesulfonamide (MYMsA) (140 mg, 1 mmol) was added and the reaction was carried out at room temperature for 5 min. The solvent was dried under vacuum, and the crude product was eluted and purified on a silica column with PE:EA = 3:1 to obtain the desired white solid product BA-2 (265 mg, yield 65%). 11H NMR (400 MHz, DMSO-d6) δ 5.00 (s, 1H), 4.81 (s, 1H), 4.31 (s, 1H), 4.12 (s, 1H), 4.01 (s, 1H), 3.79 (s, 1H), 3.61 (s, 1H), 3.06 (d, J = 2.6 Hz, 3H), 3.01 (d, J = 2.6 Hz, 3H), 2.47 (d, J = 15.9 Hz, 1H), 2.41 - 2.27 (m, 1H), 2.26 - 2.09 (m, 2H), 1.99 (q, J = 11.2 Hz, 1H), 1.72 (ddd, J = 39.9, 18.9, 10.3 Hz, 6H), 1.52 - 1.09 (m, 13H), 0.94 (d, J = 6.0 Hz, 3H), 0.88 - 0.78 (m, 3H), 0.59 (d, J = 2.6 Hz, 3H). 13 13C NMR (151 MHz, DMSO-d6) δ 171.75, 146.79, 99.49, 71.47, 70.92, 66.72, 46.45, 46.27, 42.01, 41.86, 37.26, 36.76, 35.34, 34.86, 30.89, 30.84, 30.76, 29.00, 27.70, 26.69, 23.26, 23.09, 17.37, 12.79. ESI-MS calcd. for C 28 H 47 NO7S [M + H]+ m / z = 542.3146, found 542.3195.

[0126] Example 15

[0127]

[0128] The synthesis process of BA-3 was to dissolve the purchased BA (408 mg, 1 mmol) in dichloromethane and methanol (DCM:MeOH = 1:1), then add N-methyl-N-p-toluenesulfonyl ethynylamine (208 mg, 1 mmol), and react at room temperature for 5 min. The solvent was dried under vacuum, and the crude product was purified by elution on a silica column with PE:EA = 3:1 to obtain the desired white solid product BA-3. 11H NMR (400 MHz, DMSO-d6) δ 7.68 (d, J = 7.8 Hz, 2H), 7.45 (d, J = 7.9 Hz, 2H). δ 5.00 (s, 1H), 4.81 (s, 1H), 4.31 (s, 1H), 4.12 (s, 1H), 4.01 (s, 1H), 3.79 (s, 1H), 3.61 (s, 1H), 3.06 (d, J = 2.6 Hz, 3H), 3.01 (d, J = 2.6 Hz, 3H), 2.47 (d, J = 15.9 Hz, 1H), 2.41 - 2.27 (m, 1H), 2.26 - 2.09 (m, 2H), 1.99 (q, J = 11.2 Hz, 1H), 1.72 (ddd, J = 39.9, 18.9, 10.3 Hz, 6H), 1.52 - 1.09 (m, 13H), 0.94 (d, J = 6.0 Hz, 3H), 0.88 - 0.78 (m, 3H), 0.59 (d, J = 2.6 Hz, 3H). 13 13C NMR (151 MHz, DMSO-d6) δ 171.45, 146.73, 144.59, 134.26, 130.24, 127.97, 100.08, 71.47, 70.93, 66.73, 55.36, 46.42, 46.25, 42.02, 41.85, 37.42, 35.79, 35.36, 35.29, 34.86, 30.89, 30.79, 30.66, 29.00, 27.69, 26.70, 23.27, 23.09, 21.55, 17.32, 12.77. ESI-MS calcd. for C 34 H 51 NO7S [M+H]+ m / z = 618.3459, found 618.3486.

[0129] Example 16

[0130]

[0131] The synthesis process of Ar-2 is similar to that of BA-2. On a silica column, the reaction was eluted and purified with DCM:MeOH = 20:1 to obtain the desired white solid product Ar-2 in a yield of 60%. 11H NMR (400 MHz, DMSO-d6) δ 5.68 (d, J = 9.6 Hz, 1H), 5.57 (d, J = 4.3 Hz, 1H), 5.03 (d, J = 2.9 Hz, 1H), 4.84 (d, J = 3.0 Hz, 1H), 3.06 (d, J = 2.0 Hz, 3H), 3.00 (d, J = 2.1 Hz, 3H), 2.75 (p, J = 3.3 Hz, 4H), 2.35 - 2.25 (m, 1H), 2.19 (t, J = 13.5 Hz, 1H), 2.00 (d, J = 9.4 Hz, 2H), 1.89 - 1.75 (m, 1H), 1.60 (d, J = 14.4 Hz, 3H), 1.44 (t, J = 11.8 Hz, 2H), 1.29 (s, 3H), 1.24 (s, 2H), 0.89 (d, J = 6.5 Hz, 3H), 0.77 (d, J = 7.1 Hz, 3H). 13 13C NMR (151 MHz, DMSO-d6) δ 171.20, 170.40, 146.32, 104.06, 100.33, 92.44, 91.09, 80.33, 51.59, 45.03, 37.48, 36.46, 36.41, 36.36, 34.17, 32.09, 28.88, 25.97, 24.66, 21.46, 20.53, 12.21. ESI-MS calcd. for C 23 H 35 NO 10 S [M + H]+ m / z = 540.1874, found 540.1878.

[0132] Example 17

[0133]

[0134] The synthesis process of Ar-3 is similar to that of BA-3. On a silica column, the reaction was eluted and purified with DCM:MeOH = 20:1 to obtain the desired white solid product Ar-3 in a yield of 60%. 11H NMR (400 MHz, DMSO-d6) δ 7.69 (d, J = 7.9 Hz, 2H), 7.47 (d, J = 7.9 Hz, 2H), 5.67 (d, J = 9.6 Hz, 1H), 5.57 (d, J = 2.7 Hz, 1H), 4.80 (d, J = 2.8 Hz, 1H), 4.74 (d, J = 2.8 Hz, 1H), 2.93 (d, J = 2.6 Hz, 3H), 2.60 (s, 3H), 2.54 - 2.47 (m, 1H), 2.42 (d, J = 2.6 Hz, 3H), 2.37 - 2.28 (m, 2H), 2.19 (t, J = 14.1 Hz, 1H), 2.06 - 1.96 (m, 1H), 1.87 - 1.76 (m, 3H), 1.58 (dt, J = 20.5, 7.3 Hz, 3H), 1.44 (t, J = 11.7 Hz, 2H), 1.29 (d, J = 2.6 Hz, 3H), 1.19 (qd, J = 7.3, 5.0, 3.5 Hz, 2H), 0.95 (d, J = 12.5 Hz, 1H), 0.89 (d, J = 6.2 Hz, 3H), 0.76 (d, J = 7.1 Hz, 3H). 13 13C NMR (151 MHz, DMSO-d6) δ 170.99, 170.03, 146.43, 144.67, 134.05, 130.30, 128.04, 104.06, 100.45, 92.41, 91.09, 80.32, 55.38, 51.58, 45.03, 37.20, 36.42, 36.36, 34.17, 32.08, 28.81, 28.77, 25.96, 24.66, 21.54, 21.46, 20.52, 12.20. ESI-MS calcd. for C 29 H 39 NO 10 S [M + H]+ m / z = 616.2187, found 616.2178.

[0135] Example 18

[0136]

[0137] The synthesis process of MA-2 is similar to that of BA-2. On a silica column, the reaction was eluted and purified with PE:EA = 3:1 to obtain the desired white solid product MA-2 with a yield of 55%. 11H NMR (400 MHz, Chloroform-d) δ 7.67 (s, 1H), 5.25 (t, J = 7.1 Hz, 1H), 5.19 (s, 2H), 4.95 (d, J = 2.5 Hz, 1H), 4.72 (d, J = 2.6 Hz, 1H), 3.75 (s, 3H), 3.38 (d, J = 7.0 Hz, 2H), 3.02 (s, 3H), 2.94 (s, 3H), 2.54 (t, J = 7.6 Hz, 2H), 2.39 - 2.28 (m, 3H), 1.81 (s, 3H). 13 13C NMR (151 MHz, Chloroform-d) δ 172.91, 168.96, 163.65, 153.54, 144.13, 133.31, 123.28, 121.87, 116.81, 106.34, 70.08, 61.02, 41.28, 33.93, 33.74, 32.85, 24.69, 22.58, 16.13, 11.56. ESI-MS calcd. for C 21 H 27 NO8S [M+H]+ m / z = 454.1530, found 454.1535.

[0138] Example 19

[0139]

[0140] The synthesis process of MA-3 is similar to that of BA-3. On a silica column, the reaction was eluted and purified with PE:EA = 3:1 to obtain the desired white solid product MA-3 with a yield of 55%. 1 1H NMR (400 MHz, Chloroform-d) δ 7.73 - 7.67 (m, 2H), 7.32 (d, J = 8.1 Hz, 2H), 5.31 (s, 1H), 5.20 (s, 2H), 4.74 (d, J = 2.5 Hz, 1H), 4.60 (d, J = 2.5 Hz, 3H), 3.77 (s, 2H), 3.39 (d, J = 7.0 Hz, 3H), 2.97 (s, 5H), 2.42 (d, J = 4.3 Hz, 2H), 2.26 (d, J = 7.9 Hz, 3H), 2.16 (s, 1H), 2.05 (s, 1H), 1.79 (d, J = 1.4 Hz, 3H). 1313C NMR (151 MHz, Chloroform-d) δ 172.93, 170.71, 163.67, 153.57, 146.92, 144.10 (d, J=2.5 Hz), 133.87, 133.64, 129.51, 122.99, 121.97, 116.81, 100.41, 70.08, 61.01, 37.17, 34.03, 32.66, 22.58, 21.55, 16.13, 11.58. ESI-MS calcd. for C 27 H 31 NO8S [M+H]+ m / z=552.1663, found 552.1666.

[0141] Example 20

[0142]

[0143] The synthesis process of UrA-2 is similar to that of BA-2. On a silica column, the reaction was eluted and purified with PE:EA = 3:1 to obtain the desired white solid product UrA-2 with a yield of 50%. 1 1H NMR (400 MHz, Chloroform-d) δ 5.31 (s, 2H), 5.08 (s, 1H), 4.81 (s, 1H), 3.31 - 3.17 (m, 1H), 3.08 (s, 3H), 3.00 (s, 3H), 2.29 - 1.29 (m, 23H), 1.12 (s, 3H), 1.07 - 0.93 (m, 9H), 0.88 (d, J=6.2 Hz, 3H), 0.84 (s, 3H), 0.80 (s, 3H). 13 13C NMR (151 MHz, Chloroform-d) δ 175.26, 146.37, 137.56, 126.25, 101.05, 79.00, 55.22, 53.46, 52.94, 48.78, 47.51, 42.25, 39.69, 39.13, 38.80, 38.66, 37.12, 36.98, 36.46, 35.77, 33.22, 30.56, 28.16, 27.99, 27.22, 24.31, 23.38, 23.31, 21.12, 18.30, 17.60, 16.96, 15.65, 15.52. ESI-MS calcd. for C 34 H 55 NO5S [M+H]+ m / z=590.3874, found 590.3885.

[0144] Example 21

[0145]

[0146] The synthesis process of UrA-3 is similar to that of BA-3. On a silica column, the reaction was eluted and purified with PE:EA = 3:1 to obtain the desired white solid product UrA-3 with a yield of 50%. 1 HNMR(400MHz,Chloroform-d)δ7.73(d,J = 8.3Hz,2H),7.32(d,J = 8.1Hz,2H),5.21(t,J = 3.7Hz,1H),4.81(d,J = 2.2Hz,1H),4.75(d,J = 2.2Hz,1H),3.26 - 3.17(m,2H),2.94(s,3H),2.43(s,3H),1.97 - 1.88(m,3H),1.68 - 1.42(m,17H),1.37 - 1.22(m,13H),1.07(s,3H),0.99(s,3H),0.95 - 0.91(m,6H),0.83(d,J = 6.5Hz,3H),0.78(d,J = 2.0Hz,6H). 13 C NMR(151MHz,Chloroform-d)δ175.12,146.88,143.91,137.61,134.05,130.01,129.53,128.17,127.34,126.08,102.92,79.04,55.23,52.72,48.63,47.53,42.17,39.65,39.06,38.76,38.70,38.66,36.98,36.82,36.04,33.15,30.52,28.16,27.98,27.23,24.04,23.39,23.30,21.60,21.13,18.31,17.45,16.91,15.64(d,J = 2.2Hz),15.51.ESI-MS calcd.for C 40 H 59 NO5S[M + H]+m / z = 688.4006,found 688.4014.

[0147] Example 22 Covalent Modification of Pure Protein Amino Acid Residues by Compounds A1 / 2 / 3 / 4

[0148] 50.0 μM of Compound A1 / 2 / 3 / 4 was incubated with bovine serum albumin (10 μL, 1 mg / mL) at 37 °C for 3 h. Then, click chemistry reagents [TBTA (0.1 mmol), sodium ascorbate (1 mmol), CuSO4 (1 mmol)] and the fluorescent dye TARMA-Azide (0.1 mmol) were added to carry out the click chemical reaction, and the reaction was carried out at room temperature for 2 h. 2 μL of protein loading buffer (5×) was added and separated by polyacrylamide gel electrophoresis. Finally, it was tested by the multi-functional laser scanning imager Typhoon FLA9500.

[0149] As Figure 1 shown, Compounds A1 / 2 / 3 containing an α-acyloxyacrylamide electrophilic warhead were able to covalently label bovine serum albumin, while Compound A4 with aliphatic side chains introduced on both sides of the α-acyloxyacrylamide electrophilic warhead showed no obvious labeling of BSA, indicating that aromatic groups are beneficial to the binding of compounds to proteins.

[0150] Example 23 Covalent Modification of Protein Amino Acid Residues in Tumor Cells by Compounds A1 / 2 / 3 / 4

[0151] Human breast cancer cells MDA-MB-231 were cultured to the logarithmic growth phase at 37 °C under 5% CO2, and the cells were evenly distributed into 6-well plates. After 24 h of adhesion, 100 μM of Compounds A1 / 2 / 3 / 4 were added to the 6-well plates respectively and incubated at 37 °C for 3 h. The culture medium was removed and the cells were washed twice with phosphate buffer (PBS). Then, the cells were lysed with RIPA cell lysis buffer containing 1% protease and phosphatase inhibitors. The protein concentration was quantified to 1 mg / mL using a BCA protein quantification kit. A certain amount of cell lysate was taken, and click chemistry reagents [TBTA (0.1 mmol), sodium ascorbate (1 mmol), CuSO4 (1 mmol)] and the fluorescent dye TARMA-Azide (0.1 mmol) were added to carry out the click chemical reaction, and the reaction was carried out at room temperature for 2 h. Subsequently, frozen acetone solution was added to precipitate the protein and centrifuged to remove the organic solvent. The obtained protein solid was added with protein loading buffer and boiled at 95 °C for 10 min for protein denaturation, and then separated by polyacrylamide gel electrophoresis. Finally, it was tested by the multi-functional laser scanning imager Typhoon FLA 9500.

[0152] As Figure 2 shown, compared with Compounds A3 / 4 containing aliphatic side chains, Compounds A1 / 2 containing benzene rings had stronger ability to covalently label proteins at the live cell level. In particular, Compound A1 with benzene rings on both sides of the α-acyloxyacrylamide electrophilic warhead showed the strongest protein labeling ability, indicating that small molecule compounds containing α-acyloxyacrylamide electrophilic warheads can bind multiple proteins in live cells.

[0153] Example 24 Selective Modification of Lysine Residues of Living Cells by Compound A1

[0154] 100 μM of compound A1 was incubated with MDA-MB-231 cells at 37 °C for 4 h. Then, click chemistry reagents [TBTA (0.1 mmol), sodium ascorbate (1 mmol), CuSO4 (1 mmol)] and PC-Biotion-N3 (100 μM) were added to carry out the click chemical reaction, and the reaction was carried out at room temperature for 2 h. Subsequently, pre-cooled acetone solution was added to precipitate proteins and the organic solvents were removed by centrifugation. The protein sample was dissolved in 1% SDS and sonicated to be fully dissolved. The supernatant was taken and added to streptavidin magnetic beads High Capacity Streptavidin Agarose to enrich the proteins. After incubating at room temperature on a rotator for 4 h, the supernatant was removed by centrifugation, and washed successively with 1% SDS, 0.1% SDS and PBS. The above streptavidin magnetic beads were dissolved in 500 μL of PBS solution containing 6 M urea, and 25 μL of NH4HCO3 (25 mM) buffer containing 200 mM DTT was added, and incubated at 37 °C for 30 min. Then, 25 μL of NH4HCO3 (25 mM) buffer containing 400 mM IAA was added, and the reaction was carried out at room temperature in the dark for 1 h. The supernatant was removed by centrifugation and washed 3 times with PBS. 150 μL of PBS containing 2 M urea and 150 μL of NH4HCO3 (50 mM) buffer containing 1 mM CaCl2 were added, and 5.0 μL of trypsin was incubated at 37 °C overnight. The supernatant was removed by centrifugation and washed 3 times with double-distilled water. 200 μL of 0.1% formic acid aqueous solution was added and irradiated with 365 nm UV for 1 h, centrifuged and washed 3 times with 60% acetonitrile aqueous solution. The eluates were combined and dried by rotation, and the peptide segments were purified by C18 column. The obtained peptide segments were dried by rotation for biomass spectrometry analysis.

[0155] The mass spectrometry results showed that compound A1 could selectively modify 122 lysine residues of different proteins in MDA-MB-231 living cells, Figure 3 and some of the binding sites were listed, such as protein ATP binding sites and non-binding sites like NDKA (K12), OSBL5 (K446), PFKAP (K688), etc. Among the proteins modified by A1 at lysine, 46 druggable targets were found in DrugBank, indicating that small molecule compounds containing α-acyloxyacrylamide electrophilic warheads can bind to the lysine sites of multiple druggable protein targets in living cells and may contain some new potential druggable targets.

[0156] Example 25 Inhibitory Activity of Compounds E2 / 3 / 4 / 5 against EGFR L858R Protein Kinase

[0157] Compound E3 / 4 / 5 is a compound containing an α - acyloxy enamide electrophilic warhead, which is obtained by introducing a fatty carboxyl group into the parent nucleus structure of the EGFR protein kinase inhibitor Erlotinib and then reacting it with N - methyl - N - p - toluenesulfonyl acetylene amine or N - methyl - N - methanesulfonyl acetylene amine.

[0158] Using Z‘ - LYTE TM The inhibitory effect of EGFR kinase was evaluated by the fluorescence resonance energy transfer (FRET) method, with the parental inhibitor as the reference compound. Z‘ - LYTE TM Biochemical analysis was performed using a fret - based coupled enzyme format and based on the differential sensitivity of phosphorylated and non - phosphorylated peptides to protein cleavage. The peptide substrate was labeled with two fluorophores, one for each fluorophore, to form a FRET pair. The reaction was carried out in a 384 - well plate with a 5 μL reaction volume in 50 mM HEPES (pH 7.5), 10 mM MgCl2, 1 mM EGTA and 0.01% Brij - 35 with an appropriate amount of kinase. The reaction solution was incubated at room temperature for 1.5 hours in the presence of 2 μM substrate and the corresponding ATP concentration of the kinase (EGFR (L858R): 50 μM) and in the presence of various concentrations of the compound, then 2.5 μL of developer was added and incubated at room temperature for 1 hour, and then 2.5 μL of termination solution was added. The fluorescence signal ratio of 445 nm (coumarin) / 520 nm (fluorescein) was examined using an EnVision MultilabelReader (Perkin Elmer, Inc.). The data was analyzed using Graphpad Prism5 (Graphpad Software, Inc).

[0159] From Table 1 and Figure 4 the test results, compounds E3 and E5 showed inhibitory activities comparable to Erlotinib against the EGFR L858R kinase, with IC 50 values all below 10 nM.

[0160] Table 1 Inhibitory activities of compounds against EGFR L858R protein kinase

[0161]

[0162] Example 26 Growth inhibitory activities of compounds E1 / 2 / 3 / 4 / 5 in EGFR - overexpressing tumor cells

[0163] Cell viability was detected by CCK-8 assay: 4000 cells per well were seeded in 96-well plates and cultured in an incubator for 24 hours to allow adhesion. Compounds E1 / 2 / 3 / 4 (0 μM - 30 μM) were dissolved in DMSO and added to each well of the cells, keeping the final concentration of DMSO at 0.1%. After incubation for 72 h, 30 μL of CCK-8 reagent was added to each well and incubated for 2 h. Then, the absorbance was measured using a plate reader at wavelengths of 450 nm and 650 nm. The cell survival rate was determined as VR = (A - A0) / (As - A0) × 100%, where A is the absorbance of the experimental group, As is the absorbance of the control group (using DMSO as the control), and A0 is the absorbance of the blank group (without cells). The IC50 value was calculated using Graphpad Pris.

[0164] The results are shown in Table 2: Compounds E3 and E5 showed excellent anti-proliferative activity against H3255 cells containing the EGFR L858R mutant, but did not show significantly stronger anti-proliferative activity against A431 cells containing EGFR WT, which was consistent with the trend of Erlotinib; the inhibitory activity of Compound E4 was weaker than that of Compounds E3 and E5. Combining the results in Table 1 and Figure 4 indicated that the introduction of the α-acyloxyacrylamide electrophilic warhead generated by the reaction of the carboxyl group with N-methyl-N-p-toluenesulfonyl ethynylamine could maintain inhibitory activity comparable to that of the parent nucleus.

[0165] Table 2 Proliferation inhibitory activities of compounds against different EGFR cells

[0166]

[0167]

[0168] Analysis and verification of the target that Compound E3 can bind to in H3255 cells in Example 27

[0169] The compound E3 at 10 μM was incubated with H3255 cells at 37 °C for 2 h. Then, the click chemistry reagents [TBTA (0.1 mmol), sodium ascorbate (1 mmol), CuSO4 (1 mmol)] and Biotion-N3 (100 μM) were added to conduct the click chemical reaction, and the reaction was carried out at room temperature for 2 h. Subsequently, pre-cooled acetone solution was added to precipitate proteins and the organic solvents were removed by centrifugation. The protein samples were dissolved with 1% SDS and sonicated to be fully dissolved. The supernatant was taken and added to streptavidin magnetic beads High Capacity Streptavidin Agarose to enrich the proteins. After incubating at room temperature on a rotator for 4 h, the supernatant was removed by centrifugation, and washed successively with 1% SDS, 0.1% SDS and PBS. The above-mentioned avidin protein agar was dissolved in 500 μL of PBS solution containing 6 M urea, and 25 μL of NH4HCO3 (25 mM) buffer containing 200 mM DTT was added and incubated at 37 °C for 30 min. Subsequently, 25 μL of NH4HCO3 (25 mM) buffer containing 400 mM IAA was added and the reaction was carried out at room temperature in the dark for 30 min. The supernatant was removed by centrifugation and washed 3 times with PBS. 150 μL of PBS containing 2 M urea and 150 μL of NH4HCO3 (50 mM) buffer containing 1 mM CaCl2 were added, and 3.0 μL of trypsin was added and incubated at 37 °C overnight. The supernatant was taken by centrifugation and dried by rotation. The peptide segments were purified with a C18 column, and the obtained peptide segments were dried by rotation for biomass spectrometry analysis.

[0170] As Figure 5 shown, the compound E3 can target multiple target proteins including the EGFR protein kinase.

[0171] The binding of E3 to the EGFR kinase was verified by immunoblotting experiments. In the presence or absence of a competitor, 10 μM of the molecular probe E3 was incubated with H3255 cells at 37 °C for 2 h. Then, click chemistry reagents [TBTA (0.1 mmol), sodium ascorbate (1 mmol), CuSO4 (1 mmol)] and Biotion-N3 (100 μM) were added to carry out the click chemical reaction, and the reaction was carried out at room temperature for 2 h. Subsequently, pre-cooled acetone solution was added to precipitate the protein and centrifuged to remove the organic solvent. The protein sample was dissolved in 1% SDS and sonicated to dissolve it completely. The supernatant was taken and the protein was enriched with Neutravidin agarose resin. After incubating at room temperature on a rotator for 4 h, the supernatant was removed by centrifugation and washed successively with 1% SDS, 0.1% SDS and PBS. The above Neutravidin agarose was dissolved in 50 μL of 2× loading buffer, heated at 95 °C for 30 min, centrifuged at 14,000 rpm for 10 min in a high-speed centrifuge, and 20 μL of the supernatant was loaded. The protein was separated by polyacrylamide gel electrophoresis and then the target protein was verified by Western blotting, and imaging was performed with a hypersensitive multifunctional imager.

[0172] As Figure 6 shown, compounds E1 / 2 / 3 / 4 / 5 can all target and bind to the EGFR protein kinase. In the presence of the competitors afatinib or Erlotinib, EGFR can be completely competed, indicating that compound E3 has the same binding as the parent molecule in living cells. By introducing an α-acyloxyacrylamide electrophilic warhead into Erlotinib, it can be used to identify and characterize the action targets and new action sites of Erlotinib.

[0173] Example 28 Modification of Lysine Residue in EGFR L858R Protein Kinase by Compound E3

[0174] Aspirate the medium from the confluent BaF3 EGFR L858R cells, wash twice with PBS, add 2 ml of trypsin for digestion, discard the supernatant after centrifugation, wash the cells with PBS again, and discard the supernatant. Add 1 ml of PBS containing phosphatase inhibitor and protease inhibitor to resuspend the cells, perform ultrasonic lysis and then quantify with BCA, and dilute to a cell suspension of 1 mg / ml for standby. Take 1 ml of the above cell suspension and add 100 μM compound E3, incubate at 37 °C for 2 h, add click chemistry reagents [TBTA (0.1 mmol), sodium ascorbate (1 mmol), CuSO4 (1 mmol)] and PC-Biotion-N3 (100 μM) to carry out click chemical reaction, and react at room temperature for 2 h. Subsequently, add pre-cooled acetone solution to precipitate the protein and centrifuge to remove the organic solvent. Dissolve the protein sample with 1% SDS, sonicate to fully dissolve it, take the supernatant and add streptavidin magnetic beads High Capacity Streptavidin Agarose to enrich the protein. Incubate at room temperature on a rotator for 4 h, then centrifuge to remove the supernatant, and wash successively with 1% SDS, 0.1% SDS and PBS. Dissolve the above streptavidin magnetic beads in 500 μL of PBS solution containing 6 M urea, add 25 μL of NH4HCO3 (25 mM) buffer containing 200 mM DTT, incubate at 37 °C for 30 min, then add 25 μL of NH4HCO3 (25 mM) buffer containing 400 mM IAA, react at room temperature in the dark for 1 h, centrifuge to remove the supernatant and wash 3 times with PBS. Add 150 μL of PBS containing 2 M urea, 150 μL of NH4HCO3 (50 mM) buffer containing 1 mM CaCl2, and 5.0 μL of trypsin, incubate overnight at 37 °C. Centrifuge to remove the supernatant and wash 3 times with double-distilled water. Add 200 μL of 0.1% formic acid aqueous solution, irradiate with 365 nm UV for 1 h, centrifuge and wash 3 times with 60% acetonitrile aqueous solution, combine the eluates and dry by rotation, purify the peptide segments with a C18 column, and dry the obtained peptide segments for biomass spectrometry analysis.

[0175] As Figure 7 shown, introducing a carboxyl group into the EGFR kinase inhibitor molecule Erlotinib and then transforming it through a one-step reaction to obtain the α-acyloxyacrylamide kinase inhibitor molecule E3 can bind to the lysine residue at position 728 in the EGFR L858R kinase. Compound E3 may play a role in the kinase function and structure by binding to the lysine at position 728. By introducing an α-acyloxyacrylamide electrophilic warhead, the kinase non-covalent inhibitor is transformed into a covalent inhibitor, which may optimize some defects of the original non-covalent inhibitors.

[0176] Example 29 Potential Targets Bound by Compounds X1 / 2 / 3 / 4 in K562 Cells and Verification

[0177] Compound X1 / 2 / 3 / 4 is a compound containing an α - acyloxyenamide electrophilic warhead, which is obtained by introducing a carboxyl group into the parent nucleus structure of the following broad - spectrum kinase inhibitor molecule and then reacting it with N - methyl - N - p - toluenesulfonyl ethynylamine or N - methyl - N - methanesulfonyl ethynylamine.

[0178]

[0179] The potential targets of compound X1 / 2 / 3 / 4 were identified by LC - MS proteomics technology. The cells used for identification were K562, and the specific experimental method was the same as that in Example 27. The experimental results are as Figure 8 shown. Compound X1 / 2 / 3 / 4 has multiple potential binding targets in cells, including multiple protein kinases such as CDK1, CDK2, AURKA, MEK2 (MP2K2), etc.

[0180] These binding proteins were verified by immunoblotting experiments. The cells used for identification were K562, and the specific experimental method was the same as that in Example 27. The experimental results are as Figure 9 shown. The results indicate that the α - acyloxyenamide - type kinase inhibitor molecules X1 / 2 / 3 / 4, which are obtained by introducing a carboxyl group into the broad - spectrum kinase inhibitor molecule and then converting it through a one - step reaction, can all target - bind to the CDK1 / 2 and MEK2 protein kinases.

[0181] Example 30 Selective Modification and Verification of Lysine Residues of Compound X1 / 2 / 3 / 4 in Cells

[0182] The binding sites of compound X1 / 2 / 3 / 4 to proteins were identified by LC - MS proteomics technology. The cells used for identification were K562, and the specific experimental method was the same as that in Example 28. The experimental results are as Figures 10 - 12 shown. Compound X1 / 2 / 3 / 4 can bind to the lysine - binding sites in the ATP pockets of multiple protein kinases, such as the lysine - binding sites of CDK1 (K33), CDK2 (K33), CDK5 (K33), MEK2 (K101), CHK2 (K249), STK38 (K118), etc. This fully demonstrates that the α - acyloxyenamide - type molecular compounds obtained by converting kinase inhibitor molecules containing carboxyl groups through a one - step reaction can be used to analyze the conserved lysine residues of kinases in the whole proteome, and may provide new action sites other than cysteine for the development of covalent drugs for these protein kinases, thus providing a new direction for new drug research and development.

[0183] The full lengths of MEK2, CDK2, CDK5, STK38, CHK2, and CDK1 were cloned into the pReceiver-M14 vector with a C-terminal 3×Flag tag. According to the manufacturer's protocol, the plasmids of the above proteins and the empty vector were transiently transfected using Lipofectamine 2000, respectively. HEK 293T cells were transfected with 3 μg of plasmid / 10 μL of Lipofectamine 2000 per well in Opti-MEM. After culturing for 8 h, the cells were changed with fresh medium and cultured for another 40 h. Then, immunoblot analysis and labeling of the probes were performed. The experimental results are as Figure 13 shown. To verify the labeling of the lysine residues identified by LC-MS / MS, the compound showed good labeling for its wild type. When the modified lysine was mutated to arginine, the labeling of the compound was significantly weakened or completely disappeared. This result indicates that the targeting of the compound to the target protein is achieved by binding to its lysine residues.

[0184] The experimental results of Examples 25 - 30 demonstrated that a kinase inhibitor molecule containing a carboxyl group, or a carboxyl group was first introduced into a kinase inhibitor molecule without a carboxyl group, and then a kinase inhibitor molecule containing an α-acyloxyacrylamide electrophilic warhead could be formed by reacting with sulfonyl acetylene amine. It can be used to identify related targets and their binding lysine sites in living cells or cell lysates.

[0185] Example 31 Identification of Targets and Their Lysine Sites in the Cell Lysate of Tumor Cells MDA-MB-231 by a Natural Product Fragment Containing an α-acyloxyacrylamide Electrophilic Warhead

[0186] MDA-MB-231 cells were lysed in pre-chilled NETN buffer (50 mM HEPES, pH 7.6, 150 mM NaCl and 1% IGEPAL), and 1× protease and phosphatase inhibitor (Thermo Scientific, A32961) was added. Then the cell lysate was incubated with 7 natural product fragments Ar-2 / Ar-3 / BA-3 / MA-2 / MA-3 / UrA-2 / UrA-3 containing α-acyloxyacrylamide electrophilic warheads (at a concentration of 100 μM) or DMSO by rotation at room temperature for 1 hour. Subsequently, it was incubated with purchased NHSyne (100 μM) at room temperature for 1 hour. The protein samples labeled with NHSyne probe were processed. Primarily, the probe-labeled samples were incubated with 10 mM DTT at room temperature for 30 minutes, and then 40 mM IAA was added and incubated in the dark at room temperature for 30 minutes. After that, the proteins were precipitated with a methanol-chloroform system (aqueous phase / methanol / chloroform, 4:4:1 (v / v / v)). After standing on ice for 15 minutes, centrifuged at 4°C and 2000 rpm for 15 minutes. The supernatant was discarded, and the proteins were washed twice with pre-chilled methanol / chloroform (1:1, v / v), and then centrifuged at 12,000 g at 4°C for 5 minutes. The precipitated proteins were resuspended by sonication in 50 mM ammonium bicarbonate (10 seconds, 20% output, repeated 3 times). Then the protein concentration of the lysate samples was determined using BCA (Pierce Thermo Fisher), and 1 mg of protein was used and digested with trypsin at a ratio of 1:50 at 37°C for 16 hours. After the polypeptide system dissolved in ammonium bicarbonate was dried to 200 μl by rotation, the tryptic digest was desalted using an HLB extraction column (Waters), and the dried peptides were resuspended in 30 μL of solution buffer containing 30% acetonitrile. Then by adding 1 mM light or heavy azido-UV-biotin, 10 mM sodium ascorbate, 1 mM TBTA and 10 mM CuSO4. After incubation at room temperature for 2 hours. In the dark, the light isotope-labeled samples and the heavy isotope-labeled samples were immediately mixed. The peptides were diluted with sodium acetate buffer (NaAc, pH 4.5), and then treated with streptavidin agarose beads (GE Cat. No. 17-5113-01). After incubation at room temperature for 2 hours, washed 2-3 times with 50 mM NaAc (pH 4.5), 50 mM NaAc (pH 4.5) containing 2 M NaCl and deionized water, and then resuspended in 25 mM ammonium bicarbonate. Then the samples were transferred to glass tubes (VWR), and irradiated with 365 nm ultraviolet light (UVP, product number UVL-28EL series) at room temperature with magnetic stirring for 2 hours. The supernatant was collected, concentrated under vacuum, and desalted using an HLB extraction column. The supernatant was collected, dried under vacuum, and stored at -20°C until LC-MS / MS analysis.

[0187] The analysis results are asFigure 14 As shown, these 7 natural product fragments containing α-acyloxyacrylamide electrophilic warheads, namely Ar-2 / Ar-3 / BA-3 / MA-2 / MA-3 / UrA-2 / UrA-3, can identify the relevant lysine targets and their lysine sites in the figure, such as OAT (K66), GSR (K430), ERO1A (K455), PLS1 (K543), etc. This fully demonstrates that the natural product containing a carboxyl group forms a natural product fragment containing an α-acyloxyacrylamide electrophilic warhead through a one-step reaction with sulfonylacetyleneamine, and the relevant natural product fragments can be used to identify the relevant targets and their lysine sites in cells or cell lysates by quantitative proteomics methods. This also fully indicates that a large number of fragment libraries containing α-acyloxyacrylamide electrophilic warheads with natural products as the core can be constructed, and these fragment libraries can globally analyze the targets and their lysine residues bound by various natural product fragments by mass spectrometry.

[0188] Example 32 Identification of Targets and Their Lysine Sites of Natural Product Fragment MA-3 Containing α-Acyloxyacrylamide Electrophilic Warhead in MAD-MB-231 Cells

[0189] MDA-MB-231 cells cultured in 10 cm dishes were incubated with the natural product fragment MA-3 (at a concentration of 20 μM) for 4 h. DMSO was added to the control group. Subsequently, MDA-MB-231 cells were lysed in pre-cooled NETN buffer (50 mM HEPES, pH 7.6, 150 mM NaCl, and 1% IGEPAL). After lysis of the control group and the cells treated with the MA-3 compound, purchased NHSyne (100 μM) was added and incubated at room temperature for 1 h. Then, the probe-labeled samples were incubated with 10 mM DTT at room temperature for 30 min, and then 40 mM IAA was added and incubated in the dark at room temperature for 30 min. Thereafter, proteins were precipitated using a methanol-chloroform system (aqueous phase / methanol / chloroform, 4:4:1 (v / v / v)). After standing on ice for 15 min, centrifugation was performed at 4 °C and 2000 rpm for 15 min. The supernatant was discarded, and the proteins were washed twice with pre-cooled methanol / chloroform (1:1, v / v), and then centrifuged at 12,000 g at 4 °C for 5 min. The precipitated proteins were resuspended by sonication in 50 mM ammonium bicarbonate (10 s, 20% output, repeated 3 times). Then, the protein concentration of the lysate samples was determined using BCA (Pierce Thermo Fisher). 1 mg of protein was used and digested with trypsin at a ratio of 1:50 at 37 °C for 16 h. As described above, the tryptic digest was desalted using an HLB extraction column (Waters), and the dried peptides were resuspended in 30 μL of solution buffer containing 30% acetonitrile. Then, by adding 1 mM light or heavy azido-UV-biotin, 10 mM sodium ascorbate, 1 mM TBTA, and 10 mM CuSO4. After incubation at room temperature for 2 h. In the dark, the light isotope-labeled samples and the heavy isotope-labeled samples were immediately mixed. The peptides were diluted with sodium acetate buffer (NaAc, pH 4.5), and then treated with streptavidin agarose beads (GE Cat. No. 17-5113-01). After incubation at room temperature for 2 h, they were washed 2-3 times with 50 mM NaAc (pH 4.5), 50 mM NaAc (pH 4.5) containing 2 M NaCl, and deionized water, and then resuspended in 25 mM ammonium bicarbonate. Then, the samples were transferred to glass tubes (VWR) and irradiated with 365 nm ultraviolet light (UVP, product number UVL-28EL series) at room temperature under magnetic stirring for 2 h. The supernatant was collected, concentrated under vacuum, and desalted using an HLB extraction column. The supernatant was collected, dried under vacuum, and stored at -20 °C until LC-MS / MS analysis was performed.

[0190] The results of the analysis are as Figure 15As shown, through mass spectrometry, the target IMPDH (K438) bound by MA-3 was identified, and at the same time, the target sites of other proteins such as TPR (K364), TM9SF4 (K72), and USP5 (K163) were also found. The mass spectrometry data of MA-3 demonstrated that natural products containing carboxyl groups can react with sulfonyl acetylene amines to synthesize natural products with an α-acyloxy acrylamide electrophilic head, and the covalent natural product MA-3 containing α-acyloxy acrylamide can be used for the identification of tumor cell targets and their lysine sites.

[0191] The above experimental results fully demonstrate the wide application of the α-acyloxy acrylamide electrophilic warhead of the present invention. It can not only be used for the development of covalent kinase inhibitors for target recognition and identification of drug molecules, but also be used to construct covalent compounds containing α-acyloxy acrylamide electrophilic warheads based on natural products for studying the action targets of natural products. At the same time, this type of α-acyloxy acrylamide electrophilic warhead can be widely used not only in polypeptides and proteins, but also in cell lysates and living cells (especially tumor cells).

[0192] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the following embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope described in this specification.

[0193] The above-described embodiments only represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the appended claims.

Claims

1. An α - acyloxyacrylamide electrophilic warhead having the structure shown in formula (I), Among them, R1 is selected from: C1-C6 alkyl, C1-C6 alkyl-substituted C6-C 10 aryl.

2. The α-acyloxyacrylamide electrophilic warhead according to claim 1, wherein R1 is selected from: C1 - C3 alkyl, phenyl substituted by C1 - C3 alkyl.

3. The α-acyloxyacrylamide electrophilic warhead according to claim 2, wherein R1 is methyl or p - tolyl.

4. Use of the α - acyloxyacrylamide electrophilic warhead according to any one of claims 1 - 3 for selectively covalently modifying lysine residues in proteins or polypeptides.

5. Use of the α - acyloxyacrylamide electrophilic warhead according to any one of claims 1 - 3 for the identification and determination of drug molecule targets.

6. Use of the α - acyloxyacrylamide electrophilic warhead according to any one of claims 1 - 3 for the discovery and analysis of drug targets based on natural product molecules.

7. A method for identifying and characterizing a drug molecule's target, characterized in that, Comprising the following steps: (1) Introduce a carboxyl group into the drug molecule to obtain a drug molecule containing a carboxyl group; (2) React the drug molecule containing a carboxyl group with a sulfonylacetyleneamine having the structure shown in formula (II) to obtain a drug molecule containing an α - acyloxyacrylamide electrophilic warhead having the structure shown in formula (III); (3) Incubate the drug molecule containing an α - acyloxyacrylamide electrophilic warhead with living cells or cell lysates; then perform post - treatment and analysis on the incubated reaction solution; wherein, R1 is as described in any one of claims 1 - 3; R2 is the residue after the reaction of the drug molecule with an alkynyl substituent at its carboxyl group; If the drug molecule itself contains a carboxyl group, step (1) is omitted.

8. The method for identifying and determining a drug molecule action target according to claim 7, characterized in that, The drug molecule is a drug molecule having kinase inhibitory activity and / or having tumor cell proliferation inhibitory activity; and / or, The living cells are tumor cells, and the cell lysate is a lysate of tumor cells.

9. The method for identifying and determining a drug molecule action target according to claim 7, wherein The incubation includes the following reaction conditions: the concentration of the drug molecule containing an α - acyloxyacrylamide electrophilic warhead is 5 μM - 500 μM, preferably 10 μM - 100 μM; and / or, The incubation temperature is 30 °C - 40 °C, and the incubation time is 30 min - 5 h.

10. The method for identifying and determining the drug molecule action target according to any one of claims 7-9, characterized in that, The drug molecule containing an α - acyloxyacrylamide electrophilic warhead is selected from the following compounds:

11. The drug molecule containing an α - acyloxyacrylamide electrophilic warhead according to any one of claims 7 - 9, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof.

12. Use of the drug molecule containing an α - acyloxyacrylamide electrophilic warhead according to claim 11, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, in the preparation of a kinase inhibitor; preferably, the kinase is EGFR L858R protein kinase, BTK protein kinase, CDK1 protein kinase, CDK2 protein kinase, CDK5 protein kinase, AURKA protein kinase, MEK2 protein kinase, CHK2 protein kinase, STK38 protein kinase.

13. Use of the drug molecule containing an α - acyloxyacrylamide electrophilic warhead according to claim 11, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, in the preparation of an anti - tumor drug.

14. The use according to claim 13, characterized in that, The tumors are lung cancer, breast cancer, leukemia.

15. An anti-tumor drug, characterized in that, Prepared from an active ingredient and a pharmaceutically acceptable carrier or excipient, wherein the active ingredient comprises the drug molecule containing an α-acyloxyenamide electrophilic warhead described in claim 11, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof.

16. A method for the discovery and analysis of drug targets based on natural product molecules, characterized in that, Comprising the following steps: (1) Introduce a carboxyl group into the natural product molecule to obtain a natural product molecule containing a carboxyl group; (2) React the natural product molecule containing a carboxyl group with a sulfonylethynylamine having the structure shown in formula (II) to obtain a natural product molecule containing an α-acyloxyenamide electrophilic warhead having the structure shown in formula (IV); (3) Incubate the natural product molecule containing an α-acyloxyenamide electrophilic warhead with live cells or cell lysates; then perform post-treatment and analysis on the incubated reaction solution; wherein, R1 is as described in any one of claims 1-3; R3 is the residue of the natural product molecule after carboxyl reaction; If the natural product molecule itself contains a carboxyl group, step (1) is omitted.

17. The method for discovering and analyzing a drug target based on a natural product molecule according to claim 16, wherein The live cells are tumor cells, and the cell lysate is a lysate of tumor cells.

18. The method for discovering and analyzing a drug target based on a natural product molecule according to claim 16, wherein The incubation includes the following reaction conditions: the concentration of the natural product molecule containing an α-acyloxyenamide electrophilic warhead is 5 μM - 500 μM, preferably 10 μM - 100 μM; and / or, the incubation temperature is 20°C - 40°C, and the incubation time is 30 min - 5 h.

19. The method for discovering and analyzing a drug target based on a natural product molecule according to any one of claims 16-18, characterized in that, The natural product molecules containing an α-acyloxyenamide electrophilic warhead are selected from the following compounds:

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