Covalent inhibitor of targeted KRAS protein carcinogenic mutant and application of covalent inhibitor

Abstract

The invention relates to the technical field of RAS inhibitors, in particular to a covalent inhibitor of a targeted KRAS protein carcinogenic mutant and application of the covalent inhibitor. The covalent inhibitor for targeting KRAS protein G12C and G12D mutants contains an alkyl halogen warhead, and the warhead can covalently target cysteine and aspartic acid and is covalently bound with 12-site Cys or Asp in KRAS G12C / D, so that the active or inactive state of KRAS protein is locked, the protein is enabled to lose a conversion function between the active state and the inactive state, and the KRAS protein G12C and G12D mutants can be effectively inhibited. And finally, hydrolyzing ATP (adenosine triphosphate) of the KRAS in an active state by utilizing proteins such as SOS1 in cells, so that the KRAS protein is in an inactive state, a downstream continuously activated signal channel is closed, and cancer cell proliferation is inhibited. The covalent inhibitor disclosed by the invention has a relatively good inhibition effect on KRAS protein G12C and G12D mutants, and can be used for treating diseases mediated by the KRAS protein G12C and G12D mutants.

Description

[0001] The present invention claims priority to a prior application with application number CN202311445853.1, entitled Covalent Inhibitors Targeting KRAS Protein G12D Mutant, filed on November 2, 2023, the entire contents of which are incorporated herein. Technical Field

[0002] The present invention relates to the technical field of RAS inhibitors, and in particular to a covalent inhibitor simultaneously targeting KRAS protein G12C and G12D mutants and an application thereof. Background Art

[0003] The RAS (rat sarcoma) gene is an important oncogene, and its mutations are present in about 30% of human tumors. KRAS (kirsten rat sarcoma viral oncogene) is one of the three subtypes of RAS, and is more prone to mutations than the other two RAS subtypes. KRAS is a common oncogene that is involved in at least one-fifth of all human cancers. Despite its important role in cancer treatment, there is no FDA-approved effective drug for the treatment of KRAS mutant cancers. In early studies, because RAS proteins are generally globular domains and do not have significant deep hydrophobic pockets on the surface, they are difficult to bind to small molecules. RAS has a very strong affinity for guanosine triphosphate (GTP), making it difficult to develop competitive active pocket inhibitors, so RAS was once considered an "undruggable" protein.

[0004] The vast majority of RAS oncogenic mutations are concentrated on the three amino acid residues of Gly12, Gly13, and Gln61. Among them, KRAS G12D accounts for 41% of all KRAS mutation-driven cancers and is the most likely type of cancer. Figure 1 As shown, G12C and G12D indicate that the glycine (G) at the 12th position of RAS mutates to cysteine ​​(C) and aspartic acid (D), respectively. These mutations can cause the mutants to accumulate in a continuous GTP-bound state, breaking the balance between RAS-GTP and RAS-GDP, making the RAS activation state dominant, ignoring the continuation of upstream signals, leading to excessive activation of RAS downstream signals in cells, and ultimately causing cancer.

[0005] Although the protein structures of KRAS G12C and G12D are relatively similar, due to the strong nucleophilicity of the amino acid side chain thiol group at the mutation site, KRAS G12C is more susceptible to covalent attack than other mutations, and it is also easier to develop inhibitors that bind to it.

[0006] KRAS G12C covalent inhibitors were first discovered in 2013. Two lead compounds were screened out from 480 compounds, showing the greatest degree of modification of mutant KRAS G12C. They were named 6H05 and 2E07. Subsequently, the structure-activity relationship of 6H05 was studied, and two effective compounds ( Figure 2 ). The 6H05 series of compounds bind to KRASG12C and discovered a new binding site called Switch-II Pocket (S-IIP). The crystal structure shows that S-IIP is located between the central β-fold of KRAS and Switch-II (SW2), and is formed by inhibitor binding and inducing SW2 rearrangement. The results showed that the 6H05 series made the first attempt at a covalent inhibitor of KRAS G12C successful. The discovery of S-IIP means that there is an additional binding site in the KRAS protein, which became a key point in subsequent drug design.

[0007] Another breakthrough in the development of KRAS G12C covalent inhibitors is the discovery of the quinazoline series of compounds. The researchers designed a group of quinazoline compounds that can bind to the S-IIP cavity and further optimized the structure on this basis. The results showed that the addition of amide substituents can improve drug activity: in addition to enhancing anti-proliferation and apoptosis-inducing abilities, amide modification also enhances selectivity for KRAS G12C cancer cells. The discovery of S-IIP and the discovery and optimization of quinazoline structures have laid a solid foundation for the subsequent development of KRAS G12C inhibitors.

[0008] Based on these findings, covalent inhibitors targeting KRASG12C, such as Wellspring's ARS-1620 and Amgen's AMG510, were subsequently developed and marketed ( Figure 3 ), the structures of these drugs on the market have not changed, and they can specifically covalently bind to the acryloyl group at the mutant amino acid site. They are just optimized and improved on the basis of the quinazoline group, so that the newly developed inhibitors can achieve high therapeutic effects at low doses. However, if the covalent warhead lacks acrylamide and only retains the structure of quinazoline, these inhibitors cannot play the role of covalent inhibition, so the choice of warhead is crucial to the efficacy of the drug.

[0009] Small molecule inhibitors targeting KRAS G12C mutants developed in recent years have shown significant effects in clinical trials, but patients with KRAS G12C mutations only account for a small proportion of the total number of KRAS mutation patients. In order to address KRAS G12D, the largest cancer mutation, researchers have continuously optimized the KRAS G12C inhibitors that have been marketed and developed a non-covalent inhibitor MRTX 1133 ( Figure 4 ). MRTX 1133 utilizes the core skeleton of the previous KRASG12C inhibitor and removes the warhead, interacting with G12D through a salt bridge to produce an inhibitory function. However, this interaction is not a covalent connection and there are certain off-target side reactions.

[0010] Therefore, the prior art still needs to be improved and developed. Summary of the invention

[0011] In view of the above-mentioned deficiencies in the prior art, the object of the present invention is to provide a covalent inhibitor that simultaneously targets KRAS protein G12C and G12D mutants and its application, aiming to discover a new reaction warhead targeting KRAS G12C and solve the problem of off-target side effects of existing non-covalent inhibitors targeting KRAS G12D.

[0012] The present invention provides a covalent inhibitor specific for KRAS G12C and G12D. Specifically based on the property that alkyl halides can perform nucleophilic attacks on cysteine ​​(Cys) and aspartic acid (Asp), covalent inhibitors that simultaneously target G12C and G12D are developed. However, the functional groups that react with the side chains of cysteine ​​and aspartic acid residues cannot be too active, otherwise they will react with other unrelated sites and fail to reach or accurately target the target; the designed functional groups must also not be too weak in reactivity. Therefore, the present invention designs a functional group that has certain reactivity with thiol and carboxyl groups, but is less reactive. Ultimately, the functional group is determined to be an alkyl halogen (as a warhead).

[0013] The present invention changes the warhead based on the structure of the KRAS inhibitor that has been screened and optimized. Specifically, the core of the non-covalent inhibitor of KRAS G12D (MRTX 1133) is used to replace the non-covalent inhibitory part with an alkyl halogen ( Figure 5), so that it can covalently bind to Cys or Asp at position 12 in KRAS G12C / D, thereby locking a certain state of nucleotide exchange of KRAS G12C / D protein, making the protein lose the function of switching between activity and inactivity, thereby inhibiting the continuous activation of downstream signaling pathways, and then inhibiting the proliferation of cancer cells. Compared with the existing non-covalent inhibitors targeting KRAS G12D, the covalent inhibitors of the present invention inhibit the proliferation of cancer cells more persistently through covalent action, achieving a better inhibitory effect. Compared with the existing non-covalent inhibitors targeting KRAS G12D, the covalent inhibitors of the present invention have the characteristics of longer-lasting and irreversible inhibition of cancer cells.

[0014] The technical solution of the present invention is as follows:

[0015] In a first aspect of the present invention, a covalent inhibitor is provided that simultaneously targets KRAS protein G12C and G12D mutants, wherein the covalent inhibitor has one or more of the structures shown in the following general formula:

[0016]

[0017] Wherein: m=0 or 1; n1=1 or 2; n2=0 or 1; R1=H, ethynyl or ethyl; R2=H or F; R3=H or OH; X=Cl or Br.

[0018] The present invention discloses a covalent inhibitor that simultaneously targets KRAS protein G12C and G12D mutants. Its alkyl halogen warhead can covalently target cysteine ​​and aspartic acid, and covalently bind to Cys or Asp at position 12 in KRAS G12C / D, thereby locking a certain state of nucleotide exchange of KRAS G12C / D protein, making the protein lose the function of switching between activity and inactivity, thereby inhibiting the continuous activation of downstream signaling pathways, and further inhibiting the proliferation of cancer cells.

[0019] Therefore, the covalent inhibitor provided by the present invention has a good inhibitory effect on KRAS protein G12C and G12D mutations, and can be used to treat diseases mediated by KRAS protein G12C and G12D mutations.

[0020] Compared with the existing non-covalent inhibitors targeting KRAS G12D, the covalent inhibitors of the present invention inhibit the proliferation of cancer cells more persistently through covalent action, achieving a better inhibitory effect. Compared with the existing non-covalent inhibitors targeting KRAS G12D, the covalent inhibitors of the present invention have the characteristics of longer-lasting and irreversible inhibition of cancer cells.

[0021] Optionally, the covalent inhibitor is selected from one or more of the following structures:

[0022]

[0023] Wherein: X = Cl or Br.

[0024] Optionally, the covalent inhibitor is selected from one or more of the following structures:

[0025]

[0026] The covalent inhibitors provided by the present invention that simultaneously target KRAS protein G12C and G12D mutants all contain alkyl halogen warheads, which covalently bind to cysteine ​​and aspartic acid at the mutation site to achieve targeting of KRASG12C / D protein, rendering KRAS G12C / D protein in an inactive state, thereby inhibiting the proliferation of cancer cells.

[0027] Compared with the existing non-covalent inhibitors targeting KRAS G12D, the covalent inhibitors of the present invention inhibit the proliferation of cancer cells more persistently through covalent action, achieving a better inhibitory effect. Compared with the existing non-covalent inhibitors targeting KRAS G12D, the covalent inhibitors of the present invention have the characteristics of longer-lasting and irreversible inhibition of cancer cells.

[0028] In a second aspect of the present invention, there is provided a use of a covalent inhibitor of the present invention that simultaneously targets KRAS protein G12C and G12D mutants in the preparation of a drug for treating diseases mediated by KRAS G12C and / or KRAS G12D mutations. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Figure 2 shows the mutation of glycine to aspartic acid or cysteine.

[0030] Figure 2 This is the structural diagram of the 6H05 series of compounds.

[0031] Figure 3 This is the structural diagram of the quinazoline series of compounds.

[0032] Figure 4 Optimization pathway diagram for the MRTX 1133 structure.

[0033] Figure 5 Based on the covalent reactivity of the halogen warhead and the backbone structure of MRTX, an inhibitor molecule was designed that can covalently target both KRAS-G12C and KRAS-G12D. DETAILED DESCRIPTION

[0034] The present invention provides a covalent inhibitor that simultaneously targets KRAS protein G12C and G12D mutants and its application. In order to make the purpose, technical scheme and effect of the present invention clearer and more specific, the present invention is further described in detail below. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0035] Example 1: Synthesis of covalent inhibitor N-1

[0036]

[0037] (1) To a solution of 2,4,7-trichloro-8-fluoropyrido[4,3-d]pyrimidine III (i.e., compound a in the reaction formula, 2.5 g, 10 mmol, 1 equivalent) and K2CO3 (2.76 g, 20 mmol, 2 equivalent) in acetone (50 ml) was added 2,2,2-trifluoroethanol (note that 2,2,2-trifluoroethanol should be added dropwise under N2 at -40°C, a total of 950 mg, 684 μl, i.e., 0.95 equivalent). The solution was then warmed to room temperature and stirred overnight. The reaction system was concentrated under vacuum, and the residue was purified by silica gel column chromatography (PE / EA=10 / 1, in the presence of 0.1% Et3N) to obtain 2,7-dichloro-8-fluoro-4-(2,2,2-trifluoroethoxy)pyrido[4,3-d]pyrimidine (i.e., compound b in the reaction formula, 2.55 g, 8.1 mmol, 81% yield) as a white solid.

[0038] (2) Diisopropylethylamine (387 mg, 3 mmol, 522 μl, 3 eq.) was added to compound b (315 mg, 1 mmol, 1 eq.), ((2R)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methanol (1.5 mmol, 1.5 eq.) and dioxane with 4A molecular sieves (5 ml). The mixture was then stirred at 65°C until compound b disappeared. The solution was then filtered and concentrated in vacuo. The crude product was purified by silica gel column chromatography (PE / EA=1 / 1 to dichloromethane / methanol=20:1, containing 0.1% Et3N) to obtain compound c as a white solid.

[0039] (3) Under an inert atmosphere, compound c (200 mg), 1-naphthalene-boronic acid (180 mg), tetrahydrofuran (2.5 ml), potassium phosphate (300 mg) and [(di(1-adamantyl)butylphosphino)-2-(2′-amino-1,1′-biphenyl)]palladium(II) methanesulfonate (29.6 mg) were added to a test tube in sequence. The mixture was then stirred at 60° C. for 4 hours. After cooling to room temperature, the reaction mixture was directly filtered and concentrated in vacuo to obtain a residue. The residue was purified by silica gel column chromatography (PE / EA=1 / 1 to dichloromethane / methanol=20:1 in the presence of 0.1% Et3N) to obtain a white solid d.

[0040] (4) Under an inert atmosphere, compound d (50 mg), compound e (100 mg), dioxane with 4A molecular sieves (3 ml), and then diisopropylethylamine (330 μl) were added to the test tube in sequence. The mixture was then stirred at 80°C, the reaction was concentrated in vacuo, and the crude product was purified by silica gel column chromatography (PE / EA=1 / 1 to dichloromethane / methanol=20:1, in the presence of 0.1% Et3N) to obtain a white solid compound covalent inhibitor N-1 (20 mg).

[0041] The characterization data of covalent inhibitor N-1 are as follows:

[0042]

[0043] 4-(3-bromoazetidin-1-yl)-8-fluoro-2-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl)methoxy)-7-(naphthalen-1-yl)pyrido[4,3-d]pyrimidine:

[0044] 1H NMR (600MHz, CDCl3) δ8.91 (s, 1H), 7.97 (d, J = 8.2Hz, 1H), 7.93 (d, J = 8.1Hz, 1H), 7.75 (d, J = 8.5Hz, 1H ), 7.65 (d, J=7.1Hz, 1H), 7.59 (t, J=7.6Hz, 1H), 7.51 (t, J=7.5Hz, 1H), 7.45 (t, J=7.6Hz, 1H), 5.35-5. 26 (m, 1H), 5.24-5.15 (m, 2H), 4.88-4.79 (m, 3H), 4.38-4.30 (m, 2H), 3.41-3.36 (m, 1H), 3.32-3.20 (m , 2H), 3.04-2.98(m, 1H), 2.32-2.30(m, 1H), 2.28-2.25(m, 1H), 2.22-2.18(m, 1H), 2.00-1.94(m, 3H).

[0045] 13 C NMR (151MHz, CDCl3) δ164.83, 161.62, 150.71 (d, J=263.5Hz), 148.13 (d, J=12.3Hz), 146.00 (d, J=13.1Hz), 142.79 (d, J=6.8Hz), 133.78, 132.48 (d, J=3.2Hz), 131.12, 129.78, 128.46, 128.20 (d, J=1.9Hz), 126.60, 126.03, 125.36, 125.19, 110.61-110.56 (m), 97.91 (d, J=176.3Hz), 73.7 2, 72.63, 60.43 (d, J=19.4Hz), 57.10, 45.91, 42.76 (d, J=20.2Hz), 36.09, 33.08, 29.71, 25.60.

[0046] Example 2: Synthesis of covalent inhibitor N-2

[0047] The synthesis of covalent inhibitor N-2 is consistent with the synthesis route and reaction conditions of N-1 in Example 1, with the only difference being that the raw material of compound d (English name: 3-bromoazetidine, Chinese name: 3-bromoazetidine, CAS number: 760159-34-0) in step (4) is replaced by 3-bromopyrrolidine (CAS number: 952797-35-2).

[0048] The characterization data of the covalent inhibitor N-2 are as follows:

[0049]

[0050] 4-(3-bromopyrrolidin-1-yl)-8-fluoro-2-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl)methoxy)-7-(naphthalen-1-yl)pyrido[4,3-d]pyrimidine:

[0051] 1 H NMR(600MHz,CDCl3)δ9.29(s,1H),7.96(d,J=8.1Hz,1H),7.91(d,J=8.1Hz,1H),7.79(d,J=8.3Hz,1H),7.67(d,J=6.9Hz,1H),7.59(t,J=7.6Hz,1H),7.50(t,J=7.4Hz,1H),7.44(t,J=7.5Hz,1H),5.27(d,J=54.0Hz,1H),4.68(p,J=3.5Hz,1H),4.43-4.30(m,4H),4.27-4.20(m,1H),4.16-4.09(m,1H),3.34-3.26(m,2H),3.23-3.19(m,1H),3.01-2.95(m,1H),2.51-2.45(m,2H),2.30-2.26(m,1H),2.26-2.21(m,1H),2.20-2.16(m,1H),1.98-1.93(m,2H),1.93-1.87(m,1H)。

[0052] 13 C NMR(151MHz,CDCl3)δ164.31,160.88,150.61(d,J=262.3Hz),149.28(d,J=11.7Hz),145.12(d,J=13.1Hz),143.99(d,J=6.3Hz),133.76,132.56(d,J=3.3Hz),131.14,129.69,128.40,128.20(d,J=1.7Hz),126.54,125.99,125.44,125.19,111.33,97.68(dd,J=176.9,3.8Hz),73.37,73.02,60.38(d,J=19.5Hz),57.12,49.21,42.76(d,J=20.4Hz),36.06,25.55。

[0053] Example 3: Synthesis of covalent inhibitors N-3, N-4, N-5, N-6, N-7, and N-8, the synthesis routes are the same. Taking N-3 as an example, the synthesis steps are shown as follows:

[0054]

[0055] N-3 synthesis steps:

[0056] (1) Preparation of compound b: The synthesis steps of compound b are the same as those in Example 1;

[0057] (2) Preparation of compound c: The synthesis steps of compound c are the same as those in Example 1;

[0058] (3) Preparation of Compound d: The synthesis steps of Compound d are the same as those in Example 1;

[0059] (4) Under an inert atmosphere, compound d (50 mg), 4-bromocyclohexylamine (compound f, 100 mg), dioxane with 4A molecular sieves (3 ml), and then diisopropylethylamine (330 μl) were added to the test tube in sequence. The mixture was then stirred at 80°C, the reaction was concentrated in vacuo, and the crude product was purified by silica gel column chromatography (PE / EA=1 / 1 to dichloromethane / methanol=20:1, in the presence of 0.1% Et3N) to obtain a white solid compound covalent inhibitor N-3 (27 mg).

[0060] The characterization data of covalent inhibitor N-3 are as follows:

[0061]

[0062] 4-(4-bromopiperidin-1-yl)-8-fluoro-2-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl)methoxy)-7-(naphthalen-1-yl)pyrido[4,3-d]pyrimidine:

[0063] 1H NMR (600MHz, CDCl3) δ9.09 (s, 1H), 7.98 (d, J = 8.2Hz, 1H), 7.93 (d, J = 8.1Hz, 1H), 7.81 (d, J = 8.4Hz, 1H), 7.68 (d, J=7.0Hz, 1H), 7.62-7.58 (m, 1H), 7.51 (t, J=7.3Hz, 1H), 7.46 (t, J=7.4Hz, 1H), 5.34-5.22 (m, 1H), 4.55 (t t, J=7.2, 3.8Hz, 1H), 4.32 (d, J=10.4Hz, 1H), 4.24-4.17 (m, 3H), 4.00-3.94 (m, 2H), 3.32-3.27 (m, 1H), 3.27- 3.18(m, 2H), 3.01-2.96(m, 1H), 2.39-2.33(m, 2H), 2.29-2.26(m, 1H), 2.24-2.15(m, 4H), 1.98-1.87(m, 3H).

[0064] 13 C NMR (151MHz, CDCl3) δ164.94 (d, J=1.7Hz), 164.50, 150.81 (d, J=263.4Hz), 149.45 (d, J=12.2Hz), 145. 29 (d, J=13.2Hz), 143.95 (d, J=6.6Hz), 133.77, 132.45 (d, J=3.3Hz), 131.11, 129.77, 128.44, 128.24 (d , J=1.8Hz), 126.56, 126.00, 125.37, 125.18, 110.71-110.57 (m), 97.91 (d, J=176.4Hz), 73.88, 72.52, 6 0.44 (d, J=19.4Hz), 57.10, 48.30, 47.72 (d, J=7.1Hz), 42.83, 42.70, 36.10, 35.58 (d, J=1.8Hz), 25.60.

[0065] The synthesis steps of N-4, N-5, N-6, N-7, N-8, and N-9 are the same, except that the secondary amine raw materials used in the last step are different. The f raw materials in the synthesis route are used and different secondary amine compounds containing halogen warheads are added, which will not be repeated here.

[0066] The characterization data of covalent inhibitor N-4 are as follows:

[0067]

[0068] 4-(3-(bromomethyl)azetidin-1-yl)-8-fluoro-2-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl)methoxy)-7-(naphthalen-1-yl)pyrido[4,3-d]pyrimidine:

[0069] 1 H NMR(600MHz,CDCl3)δ9.00(s,1H),7.97(d,J=8.1Hz,1H),7.92(d,J=8.1Hz,1H),7.77(d,J=8.4Hz,1H),7.65(d,J=6.9Hz,1H),7.61-7.57(m,1H),7.50(t,J=7.4Hz,1H),7.47-7.43(m,1H),5.33-5.20(m,1H),4.82-4.54(m,2H),4.53-4.33(m,2H),4.31(d,J=10.5Hz,1H),4.20(d,J=10.5Hz,1H),3.81(d,J=6.6Hz,1H),3.66(d,J=7.1Hz,1H),3.34-3.25(m,2H),3.24-3.17(m,2H),3.00-2.94(m,1H),2.28-2.24(m,1H),2.24-2.19(m,1H),2.18-2.15(m,1H),1.97-1.91(m,2H),1.91-1.85(m,2H)。

[0070] 13 C NMR(151MHz,CDCl3)δ164.94,161.66-161.49(m),150.73(d,J=263.0Hz),148.22(d,J=12.2Hz),145.85(dd,J=13.2,1.8Hz),143.03(d,J=6.8Hz),133.80,132.61(d,J=3.1Hz),131.18,129.73,128.45,128.19(d,J=2.1Hz),126.57,126.02,125.43,125.21,110.79(d,J=1.6Hz),97.95(d,J=176.3Hz),73.63,72.68,60.44(d,J=19.4Hz),57.12,45.90,42.80(d,J=20.2Hz),36.10,34.35,32.59,32.49,25.62。

[0071] The characterization data of the covalent inhibitor N-5 are as follows:

[0072]

[0073] 4-(3-(bromomethyl)pyrrolidin-1-yl)-8-fluoro-2-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl)methoxy)-7-(naphthalen-1-yl)pyrido[4,3-d]pyrimidine:

[0074] 1 H NMR (600MHz, CDCl3) δ9.28 (s, 1H), 7.95 (d, J=8.2Hz, 1H), 7.90 (d, J=8.2Hz, 1H), 7.79 (d, J=8.5Hz, 1H), 7.66 (d, J=7. 0Hz, 1H), 7.60-7.56 (m, 1H), 7.50-7.46 (m, 1H), 7.45-7.41 (m, 1H), 5.32-5.20 (m, 1H), 4.32 (d, J=10.5Hz, 1H), 4.25 ( d, J=10.4Hz, 1H), 4.18 (dd, J=11.5, 7.5Hz, 1H), 4.15-4.09 (m, 1H), 4.02-3.94 (m, 1H), 3.80-3.72 (m, 1H), 3.52-3.45 (m, 2H), 3.35-3.16 (m, 3H), 3.00-2.94 (m, 1H), 2.75 (m, 1H), 2.31-2.22 (m, 3H), 2.22-2.16 (m, 1H), 1.98-1.86 (m, 4H).

[0075] 13C NMR (151MHz, CDCl3) δ164.28, 160.56 (d, J=1.6Hz), 150.54 (d, J=262.0Hz), 149.27 (d, J=11.8H z), 144.92 (d, J = 13.1Hz), 144.13 (d, J = 6.7Hz), 133.72, 132.69-132.49 (m), 131.12, 129.63, 12 8.36, 128.28-128.07 (m), 126.50, 125.95, 125.44, 125.17, 111.39, 97.56 (d, J=176.4Hz), 73.2 1, 73.13, 60.34 (d, J=19.6Hz), 57.12, 55.30, 50.73, 42.73 (d, J=20.2Hz), 36.02, 33.64, 25.51.

[0076] The characterization data of the covalent inhibitor N-6 are as follows:

[0077]

[0078] 4-(4-(bromomethyl)piperidin-1-yl)-8-fluoro-2-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl)methoxy)-7-(naphthalen-1-yl)pyrido[4,3-d]pyrimidine:

[0079] 1H NMR (600MHz, CDCl3) δ9.07 (s, 1H), 7.96 (d, J = 8.2Hz, 1H), 7.91 (d, J = 8.1Hz, 1H), 7.80 (d, J = 8.3Hz, 1H), 7.66 (d, J = 6.9Hz , 1H), 7.58 (t, J=7.6Hz, 1H), 7.49 (t, J=7.4Hz, 1H), 7.44 (t, J=7.6Hz, 1H), 5.29 (dt, J=54.1, 3.1Hz, 1H), 4.66 (d, J=13.3H z, 2H), 4.34 (d, J = 10.5Hz, 1H), 4.28 (d, J = 10.6Hz, 1H), 3.54 (d, J = 6.0Hz, 2H), 3.37 (q, J = 10.5Hz, 2H), 3.31-3.20 (m, 5H) , 3.06 (q, J=7.3Hz, 1H), 3.00 (td, J=9.3, 5.2Hz, 1H), 2.34-2.17 (m, 4H), 2.01-1.86 (m, 8H), 1.48 (qd, J=12.5, 4.1Hz, 3H).

[0080] 13 C NMR (151MHz, CDCl3) δ164.52, 164.51, 164.40, 149.90, 149.60, 149.52, 145.08, 144. 24, 144.20, 133.76, 132.56, 132.54, 131.16, 129.72, 128.42, 128.22, 128.21, 126.56 , 126.00, 125.44, 125.20, 110.81, 110.80, 97.98, 96.81, 73.54, 73.07, 66.91, 60.42, 60.29, 57.21, 49.71, 49.68, 45.85, 42.71, 42.58, 38.67, 36.03, 28.79, 25.47, 8.66.

[0081] The characterization data of the covalent inhibitor N-7 are as follows:

[0082]

[0083] 4-(3-chloroazetidin-1-yl)-8-fluoro-2-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl)methoxy)-7-(naphthalen-1-yl)pyrido[4,3-d]pyrimidine:

[0084] 1 H NMR (600MHz, CDCl3) δ8.92 (s, 1H), 7.97 (d, J = 8.2Hz, 1H), 7.93 (d, J = 8.1Hz, 1H), 7.76 (d, J = 8.4Hz, 1H) , 7.65 (d, J=7.0Hz, 1H), 7.59 (t, J=7.6Hz, 1H), 7.52-7.49 (m, 1H), 7.47-7.43 (m, 1H), 5.33-5.21 (m, 1H) ), 5.11-5.01(m, 2H), 4.85-4.78(m, 1H), 4.74-4.64(m, 2H), 4.32(d, J=10.5Hz, 1H), 4.22(d, J=10.5Hz , 1H), 3.31-3.25(m, 1H), 3.25-3.17(m, 2H), 3.00-2.94(m, 1H), 2.28-2.14(m, 3H), 1.96-1.86(m, 3H).

[0085] 13 C NMR (151MHz, CDCl3) δ164.82, 161.65 (d, J=2.1Hz), 150.73 (d, J=263.3Hz), 148.14 (d, J=12.1Hz) , 146.04 (d, J=13.2Hz), 142.80 (d, J=6.9Hz), 133.79, 132.47 (d, J=3.3Hz), 131.13, 129.81, 128.4 7, 128.21 (d, J = 2.2Hz), 126.62, 126.04, 125.36, 125.20, 110.60 (d, J = 2.1Hz), 97.90 (d, J = 176.3 Hz), 73.69, 72.70, 68.00, 60.44 (d, J=19.4Hz), 57.13, 45.92, 42.75 (d, J=20.3Hz), 36.09, 25.62.

[0086] The characterization data of the covalent inhibitor N-8 are as follows:

[0087]

[0088] 4-(4-chloropiperidin-1-yl)-8-fluoro-2-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl)methoxy)-7-(naphthalen-1-yl)pyrido[4,3-d]pyrimidine:

[0089] 1 H NMR (600MHz, CDCl3) δ9.09 (s, 1H), 7.97 (d, J=8.2Hz, 1H), 7.93 (d, J=8.1Hz, 1H), 7.81 (d, J=8.3Hz, 1H), 7.68 (d, J=7. 0Hz, 1H), 7.60 (t, J=7.6Hz, 1H), 7.51 (t, J=7.4Hz, 1H), 7.46 (t, J=7.6Hz, 1H), 5.33-5.21 (m, 1H), 4.45-4.41 (m, 1H), 4 .30(d, J=10.3Hz, 1H), 4.21-4.17(m, 3H), 4.00-3.94(m, 2H), 3.29-3.24(m, 1H), 3.23-3.17(m, 2H), 3.00-2.94(m, 1H ), 2.32-2.27(m, 2H), 2.26-2.22(m, 1H), 2.20-2.13(m, 2H), 2.11-2.06(m, 2H), 1.96-1.92(m, 2H), 1.92-1.87(m, 1H).

[0090] 13 C NMR (151MHz, CDCl3) δ164.93 (d, J=1.5Hz), 164.52, 150.82 (d, J=263.4Hz), 149.47 (d, J=12.3Hz), 145. 31 (d, J=13.2Hz), 143.97 (d, J=6.6Hz), 133.78, 132.45 (d, J=3.4Hz), 131.12, 129.80, 128.47, 128.26 (d , J=1.7Hz), 126.59, 126.02, 125.37, 125.21, 110.88-110.53 (m), 97.94 (d, J=176.3Hz), 73.87, 72.56, 6 0.44 (d, J=19.4Hz), 57.13, 56.05, 46.78 (d, J=7.1Hz), 42.83, 42.70, 36.11, 34.91 (d, J=2.0Hz), 25.62.

[0091] Example 4: Synthesis of covalent inhibitor A-1

[0092] Synthesis of intermediate compounds

[0093]

[0094] Intermediate compound synthesis steps:

[0095] (1) Preparation of Compound 2: The synthesis steps of Compound 2 are the same as those in Example 1;

[0096] (2) Preparation of Compound 4: The synthesis steps of Compound 4 are the same as those in Example 1;

[0097] (3) Under an inert atmosphere, compound 4 (15 g, 30.43 mmol, 1 eq), compound 5 (23.39 g, 1.5 eq), tetrahydrofuran (90 ml), water (12 ml), potassium phosphate (300 mg, 3 eq) and [(di(1-adamantyl)butylphosphino)-2-(2′-amino-1,1′-biphenyl)]palladium(II) methanesulfonate (2.22 g, 0.1 eq) were added to a round-bottom flask in sequence. The mixture was then stirred at 60° C. for 4 h. After cooling to room temperature, the reaction mixture was directly filtered and concentrated in vacuo to obtain a residue. The residue was purified by silica gel column chromatography (PE / EA=1 / 1 to dichloromethane / methanol=20:1 in the presence of 0.1% Et3N) to obtain compound 6.

[0098] Synthesis of covalent inhibitor A-1

[0099]

[0100] (1) Compound 6 (1.5 g, 1.71 mmol, 1 equivalent), compound 7 (1.49 g, 10 equivalents), N,N-dimethylformamide (15 ml) and dioxane with 4A molecular sieves (500 mg) were added to a round-bottom flask in sequence. The mixture was then stirred at 80°C for 2 h. After cooling to room temperature, the reaction system was directly filtered and concentrated in vacuo to obtain a residue. The residue was purified by silica gel column chromatography (PE / EA=1 / 1 to dichloromethane / methanol=20:1, in the presence of 0.1% Et3N) to obtain compound 8.

[0101] (2) Compound 8 (820 mg, 811.33 μmol, 1 eq.), cesium fluoride (616.21 mg, 5 eq.) and N,N-diformamide (8 ml) were added to a round-bottom flask in sequence. The mixture was then stirred at 30°C for 1.5 h. After the reaction was completed, the reaction system was directly filtered and concentrated in vacuo to obtain a residue. The residue was purified by silica gel column chromatography (PE / EA=1 / 1 to dichloromethane / methanol=20:1, in the presence of 0.1% Et3N) to obtain compound 9.

[0102] (3) Compound 9 (550 mg, 813.15 μmol, 1 equivalent), dichloromethane (4 ml) and trifluoroacetic acid (2 ml) were added to a round-bottom flask in sequence. Then stirred at room temperature for 0.5 h. After the reaction was completed, the reaction system was directly filtered and concentrated in vacuo to obtain a residue. The residue was purified by silica gel column chromatography (PE / EA=1 / 1 to dichloromethane / methanol=20:1, in the presence of 0.1% Et3N) to obtain compound A-1.

[0103] The characterization data of covalent inhibitor A-1 are as follows:

[0104]

[0105] 1 H NMR (400MHz, DMSO-d6) δ=10.88-10.68(m,1H),10.37-9.96(m,1H),8.96(s,1H),7.99(dd,J= 6.0,9.6Hz,1H),7.47(t,J=9.2Hz,1H),7.41(d,J=2.4Hz,1H),7.17(d,J=2.4Hz,1H),5.72-5 .46(m,1H),5.46-4.66(m,4H),4.65-4.31(m,3H),4.00-3.90(m,3H),3.44-3.13(m,2H),2.7 0-2.52(m,1H),2.47-2.45(m,1H),2.34-2.28(m,1H),2.22-2.12(m,2H),2.07-1.99(m,1H);

[0106] The synthesis steps of A-1, A-2, A-3, A-4, A-5, A-6, A-7, A-8, A-9, A-10, and A-11 are the same, except that the secondary amine raw material used in the first step is different. Compound 7 in the synthesis route is used, and different secondary amine compounds containing halogen warheads are added, which will not be repeated here.

[0107] The characterization data of covalent inhibitor A-2 are as follows:

[0108]

[0109] 1H NMR (400MHz, DMSO-d6) δ = 10.87 (s, 1H), 10.47-9.92 (m, 1H), 9.50-9.11 (m, 1H), 7.99 (dd, J = 6.0, 9. 2Hz,1H),7.47(t,J=9.2Hz,1H),7.41(d,J=2.4Hz,1H),7.19(dd,J=2.4,12.0Hz,1H),5.68-5.46(m ,1H),5.10-4.97(m,1H),4.68-4.55(m,2H),4.32-4.08(m,3H),3.98-3.81(m,5H),3.35-3.26(m,1 H),2.62-2.53(m,2H),2.48-2.44(m,1H),2.43-2.28(m,2H),2.24-2.12(m,2H),2.11-1.98(m,1H);

[0110] The characterization data of covalent inhibitor A-3 are as follows:

[0111]

[0112] 1 H NMR (400MHz, DMSO-d6) δ = 10.90 (s, 1H), 10.48-10.01 (m, 1H), 9.10 (s, 1H), 7.99 (dd, J = 6.0, 9.2 Hz,1H),7.47(t,J=9.2Hz,1H),7.41(d,J=2.4Hz,1H),7.20(d,J=2.4Hz,1H),5.67-5.47(m,1H), 4.67-4.55(m,3H),4.29-4.20(m,2H),3.96-3.91(m,2H),3.90-3.85(m,4H),3.35-3.27(m,1H), 2.62-2.53(m,1H),2.48-2.45(m,1H),2.37-2.28(m,3H),2.23-2.11(m,2H),2.09-1.97(m,3H);

[0113] The characterization data of covalent inhibitor A-4 are as follows:

[0114]

[0115] 1H NMR (400MHz, DMSO-d6) δ = 10.87 (s, 1H), 10.49-9.93 (m, 1H), 8.98 (s, 1H), 7.99 (dd, J = 6.0, 9.2Hz, 1H), 7. 47(t,J=9.2Hz,1H),7.40(d,J=2.4Hz,1H),7.17(d,J=2.4Hz,1H),5.68-5.44(m,1H),5.15-4.90(m,1H),4 .66-4.51(m,3H),4.49-4.40(m,1H),4.14-4.11(m,1H),4.02-3.98(m,4H),3.91-3.87(m,2H),3.35-3.2 4(m,2H),2.57-2.53(m,1H),2.48-2.45(m,1H),2.34-2.27(m,1H),2.24-2.11(m,2H),2.09-1.97(m,1H);

[0116] The characterization data of covalent inhibitor A-5 are as follows:

[0117]

[0118] 1 H NMR (400MHz, DMSO-d6) δ = 10.93 (s, 1H), 10.60-9.82 (m, 1H), 9.28 (d, J = 11.2Hz, 1H), 7.99 (dd, J = 6.0, 9.2Hz ,1H),7.47(t,J=9.2Hz,1H),7.41(d,J=2.4Hz,1H),7.18(dd,J=2.4,7.6Hz,1H),5.65-5.48(m,1H),4.70-4. 50(m,3H),4.24-4.04(m,2H),3.97(s,1H),3.91-3.82(m,4H),3.81-3.71(m,2H),3.37-3.26(m,1H),2.89-2 .69(m,1H),2.57-2.52(m,1H),2.48-2.44(m,1H),2.38-2.23(m,2H),2.23-2.12(m,2H),2.09-1.87(m,2H);

[0119] The characterization data of covalent inhibitor A-6 are as follows:

[0120]

[0121] 1H NMR (400MHz, DMSO-d6) δ = 10.86 (s, 1H), 10.45-9.99 (m, 1H), 9.06 (s, 1H), 7.99 (dd, J = 6.0, 9.2Hz, 1H), 7.4 7(t,J=9.2Hz,1H),7.41(d,J=2.4Hz,1H),7.19(d,J=2.4Hz,1H),5.68-5.43(m,1H),4.65-4.53(m,4H),3. 95-3.94(m,1H),3.88-3.78(m,3H),3.66(d,J=6.2Hz,2H),3.46-3.36(m,2H),3.35-3.26(m,1H),2.59-2. 52(m,1H),2.48-2.46(m,1H),2.36-2.28(m,1H),2.24-2.09(m,3H),2.08-1.95(m,3H),1.58-1.44(m,2H).

[0122] The characterization data of covalent inhibitor A-7 are as follows:

[0123]

[0124] 1 H NMR (400MHz, DMSO-d6) δ=11.02-10.76(m,1H),10.50-9.98(m,1H),9.52-9.15(m,1H),7.99(dd,J=6 .0,9.2Hz,1H),7.47(t,J=9.2Hz,1H),7.41(d,J=2.4Hz,1H),7.19(dd,J=2.4,11.7Hz,1H),5.68-5.4 7(m,1H),5.09-5.01(m,1H),4.68-4.55(m,3H),4.35-4.27(m,2H),4.00-3.67(m,5H),3.36-3.24(m, 1H),2.65-2.52(m,2H),2.48-2.39(m,2H),2.35-2.29(m,1H),2.25-2.12(m,2H),2.10-1.99(m,1H);

[0125] The characterization data of covalent inhibitor A-8 are as follows:

[0126]

[0127] 1H NMR (400MHz, DMSO-d6) δ=10.92-10.69(m,1H),10.39-10.02(m,1H),9.11(s,1H),7.99(dd,J=6.0 ,9.2Hz,1H),7.47(t,J=9.2Hz,1H),7.41(d,J=2.4Hz,1H),7.20(d,J=2.4Hz,1H),5.68-5.46(m,1 H),4.83-4.74(m,1H),4.66-4.54(m,2H),4.26-4.20(m,2H),3.97-3.94(m,2H),3.91-3.84(m,4H ),3.35-3.26(m,1H),2.58-2.54(m,1H),2.46-2.28(m,4H),2.23-2.11(m,4H),2.09-2.01(m,1H);

[0128] The characterization data of covalent inhibitor A-9 are as follows:

[0129]

[0130] 1 H NMR(400MHz, DMSO-d6)δ=10.94-10.74(m,1H),10.42-10.01(m,1H),8.98(s,1H),7.99(dd,J=6.0,9.2H z,1H),7.47(t,J=9.2Hz,1H),7.40(d,J=2.4Hz,1H),7.17(d,J=2.4Hz,1H),5.68-5.45(m,1H),5.06-4.9 2(m,1H),4.64-4.53(m,3H),4.46-4.40(m,1H),4.12-4.05(m,1H),3.91-3.82(m,4H),3.81-3.67(m,2H) ,3.38-3.24(m,2H),2.53(s,1H),2.47(s,1H),2.34-2.27(m,1H),2.24-2.10(m,2H),2.09-1.98(m,1H);

[0131] The characterization data of covalent inhibitor A-10 are as follows:

[0132]

[0133] 1H NMR (400MHz, DMSO-d6) δ = 10.97-10.74 (m, 1H), 10.39-10.00 (m, 1H), 9.27 (d, J = 12.0Hz, 1H), 7.99 (dd, J = 6.0, 9.2Hz,1H),7.47(t,J=9.2Hz,1H),7.41(d,J=2.4Hz,1H),7.18(dd,J=2.4,8.0Hz,1H),5.68-5.46(m,1H),4.68 -4.53(m,3H),4.26-4.07(m,2H),3.97(s,1H),3.90-3.82(m,2H),3.81-3.69(m,4H),3.37-3.25(m,1H),2.90 -2.70(m,1H),2.57-2.52(m,1H),2.48-2.46(m,1H),2.34-2.24(m,2H),2.23-2.12(m,2H),2.08-1.87(m,2H);

[0134] The characterization data of covalent inhibitor A-11 are as follows:

[0135]

[0136] 1 H NMR(400MHz,DMSO-d6)δ=10.85(br s,1H),10.42-10.00(m,1H),9.06(s,1H),7.99(dd,J=6.0,9.2Hz,1H),7.47(t,J=9.2Hz,1H),7.4 1(d,J=2.4Hz,1H),7.19(d,J=2.4Hz,1H),5.69-5.46(m,1H),4.66-4.53(m,4H),3.95-3.94(m,1H) ,3.88-3.78(m,3H),3.57(d,J=6.4Hz,2H),3.46-3.36(m,2H),3.35-3.26(m,1H),2.61-2.53(m,1H ),2.48-2.46(m,1H),2.36-2.29(m,1H),2.24-2.09(m,3H),2.08-1.97(m,3H),1.58-1.41(m,2H);

[0137] The structures of control compounds A, B, C, and D are shown below.

[0138]

[0139] The core structures of the control compounds [A, B, C, D] and all the compounds in the present invention [A-1 to A-11] are consistent, with only the warhead structures being different. Moreover, [A, B, C, D] are all F-substituted compounds, which are similar in structure to the compounds A-1 to A-11 in the present invention, respectively. The difference is that the halogen warhead in the control compounds is F while the halogen warhead in the present invention is Cl or Br substituted. This difference is the key to the inhibitors in the present invention being able to covalently bind to KRAS mutants.

[0140] The synthetic route of the control compounds [A, B, C, D] (reference patents WO2022256459A1, WO2023020523A1 and WO2023173017A1) is the same as the synthetic steps in Example 4, except that the secondary amine raw materials used for the covalent warhead are different. Compound 7 in the synthetic route is used, and different secondary amine compounds containing halogen warheads are added, which will not be repeated here.

[0141] The results of H NMR characterization of the control compounds [A, B, C, D] are as follows:

[0142]

[0143] 1 H NMR (400 MHz, CD3OD) δ = 9.05 (d, J = 12.0 Hz,1H),8.03-7.91(m,1H),7.53-7.41(m,1H),7.40-7.36(m,1H),7.25-7.16 (s,1H),5.42-5.19(s,1H),5.10-4.90(m,1H),4.41-4.32(m,1H),4.26-4.17( m,1H),4.16-4.08(m,1H),4.06-3.98(m,2H),3.96-3.83(m,2H),3.61-3.55( m,2H),3.10-3.07(m,2H),3.05(s,1H),2.11-1.97(m,5H),1.87-1.75(m,4H).

[0144]

[0145] 1H NMR (400 MHz, CD3OD), δ = 9.11 (s, 1H), 7.98 (dd, J = 6.0, 9.0 Hz, 1H), 7.56-7.35 (m, 2H), 7.21 (d, J = 2.1 Hz,1H),5.75-5.43(m,1H),5.21-4.96(m,1H),4.71-4.51(m,2H),4.05(br d,J=4.9 Hz,2H),3.96(s,2H),3.87(d,J=3.5 Hz,2H),3.86(br s,2H),3.34(d,J=5.0 Hz,1H),2.58-2.53(m,1H),2.49(br s,1H),2.36-2.28(m,1H),2.22-1.90(m,7H).

[0146]

[0147] 1 H NMR (400 MHz, CD3OD), δ = 9.15 (s, 1H), 7.86 (dd, J = 5.2 Hz, 8.8Hz, 1H), 7.38-7.35 (m, 2H), 7.23 (s, 1H), 5.65 (d, J = 5.3 Hz,1H),4.88-4.68(m,3H),4.42-4.40(m,2H),4.15-4.06(m,3H),3.95-3.83(m, 3H),3.52-3.45(m,2H),2.63-2.58(m,2H),2.45-2.30(m,3H),2.15-2.10(m,1H).

[0148]

[0149] 1 H NMR (400 MHz, CD3OD), δ9.06 (s, 1H), 7.91 (dd, J=5.1 Hz, 8.6Hz, 1H), 7.41-7.39 (m, 2H), 7.25 (s, 1H), 5.61 (d, J=5.3 Hz,1H),4.80-4.72(m,3H),4.45-4.39(m,2H),4.09-3.99(m,3H),3.96-3.85(m,3H),3.51 -3.42(m,2H),2.63-2.58(m,2H),2.46-2.41(m,2H),2.37-2.31(m,2H),2.19-1.99(m,1H).

[0150] Example 5: Evaluation of inhibitor covalent cross-linking efficiency

[0151] Purified KRAS G12C and KRAS G12D proteins were adjusted to 10 μM in a buffer (12.5 mM hydroxyethylpiperazineethanesulfonic acid, 75 mM NaCl and 1 mM MgCl2, pH 7.4). 20 μL of aliquoted protein solution was added to each well of a 96-well microplate, each well containing 20 μL of detection buffer. The initial compound stock solution was prepared with DMSO at a concentration of 1 mM. The compound was then diluted 20-fold into the above mixture and reacted at room temperature for 24 hours. After the reaction, the sample was centrifuged at high speed.

[0152] After high-speed centrifugation, the samples were analyzed by waters SQD2, and the chromatographic column was a C4 reverse phase column to separate the protein and buffer components. The acetonitrile fraction was added to the mobile phase to elute the protein from the chromatographic column and then directly sent to the mass spectrometer. The preliminary analysis of the raw data was performed in the waste masslynx software, and the charge states of various proteins were deconvoluted with a mass step of 1Da. The cross-linking efficiency of each compound after the test is shown in Table 1:

[0153] Table 1. Cross-linking efficiency of each compound

[0154]

[0155]

[0156]

[0157]

[0158] As shown in Table 1, compared with other compounds, compounds A-7, A-9 and A-10 have higher covalent cross-linking efficiency on KRAS G12C and KRAS G12D proteins, and control compounds [A, B, C, D] have no covalent cross-linking effect on KRAS G12C and KRAS G12D proteins. Among them, no cross-linking is indicated by ND, less than 20% is indicated by *, less than 50% is indicated by **, and 50%-90% is indicated by ***.

[0159] Example 6: Inhibitory effect of covalent inhibitors on downstream phosphorylation signals in living cells

[0160] The inhibitory effects of the compounds [A-7, A-9, A-10] synthesized in Example 4 and the control compounds [A, B, C, D] on the downstream phosphorylation signaling pathway:

[0161] The specific implementation plan of the experiment is as follows: HEK 293T cells were cultured in a 12-well plate, and the cells were transfected when the coating rate reached 50%. After transfection, KRAS G12D protein was expressed for 12 hours, and then 5uM inhibitor was added to treat the cells for 12 hours. After that, the cells were treated with a change of medium every 6 hours. After three changes of medium, the downstream pERK phosphorylation signaling pathway was detected by immunoblotting. By comparing the grayscale values ​​of the pERK bands of cells that were not treated with medium and cells that were treated with medium, the inhibitory efficiency of the downstream phosphorylation signaling pathway of KRAS oncogenic mutants under the conditions of medium change and washing was obtained. The results are shown in Table 2 below:

[0162] Table 2. pERK signal inhibition efficiency of each compound

[0163]

[0164] Based on the above results, it can be concluded that the inhibitory effect of non-covalent inhibitors, i.e., control compounds [A, B, C, D], disappears after the inhibitors in the culture medium are removed; while the inhibitory effect of covalent inhibitors, i.e., compounds [A-7, A-9, A-10], can persist for a period of time after the inhibitors in the culture medium are removed.

[0165] Example 7: Effect of covalent inhibitors on tumor cell proliferation under rinsing conditions

[0166] The inhibitory effect of covalent inhibitors on cells is long-lasting and irreversible. In order to confirm this effect, the following model was used: after the cells were treated with inhibition for a period of time, the process of gradual metabolism of drugs in the blood was simulated by changing the medium and washing.

[0167] Miapaca-2 cells (carrying KRAS G12C mutation) or LS-174-T (carrying KRAS G12D mutation) were treated with the compound for 12 hours, and then the cells were treated with a medium change every 6 hours. After the medium was changed three times, the cells were cultured for another 12 hours, and then the cell proliferation was tested, and the inhibitory effect of the inhibitor on the cells was calculated. The results are shown in Table 3 below:

[0168] Table 3. Inhibitory effects of inhibitors on tumor cell proliferation under rinsing conditions

[0169]

[0170]

[0171] The present invention confirms that after fluid replacement, washing and subsequent culture, cells treated with compounds [A-7, A-9, A-10] have better inhibitory effects than cells treated with control compounds [A, B, C, D], proving that the proliferation of tumor cells is more persistently inhibited by covalent action.

[0172] It should be understood that the application of the present invention is not limited to the above examples. For ordinary technicians in this field, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. A covalent inhibitor that simultaneously targets KRAS protein G12C and G12D mutants, characterized in that: The covalent inhibitor has one or more of the following structures: Wherein: m=0 or 1; n1=1 or 2; n2=0 or 1; R1=H, ethynyl or ethyl; R2=H or F; R3=H or OH; X=Cl or Br.

2. The covalent inhibitor targeting KRAS protein G12C and G12D mutants simultaneously according to claim 1, characterized in that: The covalent inhibitor is selected from one or more of the following structures: Wherein: X = Cl or Br.

3. The covalent inhibitor simultaneously targeting KRAS protein G12C and G12D mutants according to claim 1, characterized in that: The covalent inhibitor is selected from one or more of the following structures:

4. Use of a covalent inhibitor targeting both KRAS protein G12C and G12D mutants according to any one of claims 1 to 3 in the preparation of a medicament for treating diseases mediated by KRAS G12C and / or KRAS G12D mutations.

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