Preparation and application of anti-tumor heterocyclic compounds targeting KRAS

Abstract

The preparation and application of anti-tumor heterocyclic compounds with KRAS as a target, the structure is shown in the formula, wherein, R1 is selected from H, CH3, CH2CH3, OCH3, OCH2CH3, F, Cl, Br, CF3; R2 is selected from H, CH3, Cl, Br, CF3; R3 is selected from H, CH3, CH2CH3, OCH3, OCH2CH3, F, Cl, Br, CF3, different substituted phenyl; R4 is selected from H, CH3, Cl, Br, CF3, different substituted phenyl.

Description

Technical Field

[0001] This invention relates to the field of medicinal chemistry, specifically the preparation and application of antitumor heterocyclic compounds targeting KRAS. Background Technology

[0002] Quinolinones are a class of pharmaceutical intermediates with multiple excellent biological activities and have been widely used in the development of anti-tumor drugs in recent years. Quinolinones exhibit excellent results through different mechanisms of action, such as cell cycle arrest, apoptosis, inhibition of angiogenesis, disruption of cell migration, and regulation of growth inhibitors. Furthermore, the chemical structure of quinolinones can be modified to develop novel quinoline derivatives, which not only enhance anti-tumor activity but also reduce toxic side effects.

[0003] In the future, research on quinolinones and their derivatives can further explore their applications in the treatment of other diseases, such as cancer and inflammation. Simultaneously, in the agricultural field, further research can be conducted on their compatibility with other pesticides to optimize application methods, improve control efficacy, and reduce environmental impact.

[0004] In conclusion, quinolinones and their derivatives have significant research value and application potential in the fields of medicine and agriculture. Future research can further expand their application scope and optimize their usage methods.

[0005] The application of the urea skeleton in tumor research mainly lies in its structural properties and its role in tumor cell metabolism. The urea skeleton is a compound composed of carbamate and amino groups. In tumor research, the urea structure is not only used to treat certain types of tumors but also exerts its effects by influencing the metabolic processes of tumor cells. As one of the important components of common pharmacophores, the urea skeleton often effectively improves the water solubility and biological activity of drug molecules. Therefore, related compounds are frequently used in the development of antitumor lead compounds.

[0006] Based on this, the present invention constructs quinolinone and urea in the same molecule and designs and synthesizes a series of antitumor heterocyclic compounds targeting KRAS, with the expectation of better biological activity, higher selectivity and lower toxicity. Summary of the Invention

[0007] Purpose of the invention: 1. To provide a series of antitumor heterocyclic compounds targeting KRAS to address the problems existing in the prior art.

[0008] 2. Provide a method for preparing the above derivatives.

[0009] 3. To provide an application of the above-mentioned derivative in the preparation of antitumor drugs.

[0010] Technical solution: An antitumor heterocyclic compound targeting KRAS, having a structure as shown in Formula X. Figure 1 Wherein, R1 is selected from H, CH3, CH2CH3, OCH3, OCH2CH3, F, Cl, Br, CF3; R2 is selected from H, CH3, Cl, Br, CF3; R3 is selected from H, CH3, CH2CH3, OCH3, OCH2CH3, F, Cl, Br, CF3, and phenyl groups with different substitutions; R4 is selected from H, CH3, Cl, Br, CF3, and phenyl groups with different substitutions. The synthesis process of a class of quinolinone derivatives containing urea fragments has the following general formula:

[0011] Scheme 1. (a) Fe, AcOH, 50 ℃; (b) Dimethyl butynedioic acid, MeOH, reflux, 3 h; (c) PPA, 120 , reflux, 6 h; (d) 1 M LiOH, THF, 6 h; (e) SOCl2, reflux, 6 h; (f) 4-Boc-aminopiperidine, TEA, anhydrous CH2Cl2, rt, 24 h; (g) HCl, CH2Cl2, rt; (h) Phenyl isocyanates with different substitutions, CH2Cl2, rt, 6 h.

[0012] The synthesized molecular formula is as follows:

[0013] The synthesis of the quinolinone derivatives containing the urea fragment described above includes the following steps: Step i. Under stirring, 10 mmol of dinitrobenzene with different substitutions was added to 40 mL of acetic acid containing 60 mmol of Fe powder, followed by dropwise addition of 20 mL of distilled water. The reaction solution was heated at 50 °C for 10 minutes. After TLC confirmation of the reaction completion, the mixture was filtered, and the residue was washed with 10 mL of ethyl acetate and 10 mL of water. The filtrate was extracted with ethyl acetate (3 x 30 mL), and the organic phase was washed successively with 2% sodium hydroxide solution and saturated brine. The mixture was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was crystallized, filtered, and dried to give compounds 1a-1d. Step ii. Compounds 1a-d (6 mmol) and dimethyl butyryneidate (9 mmol) were added sequentially to a 10 mL round-bottom flask and heated under reflux for 3 h. The reaction was monitored by TLC until completion. After removing methanol under reduced pressure, the mixture was allowed to stand to crystallize, filtered, and the filter cake was washed with ice-cold methanol and dried under vacuum to obtain compounds 2a-d.

[0014] Step iii. Add compounds 2a-d (4 mmol) to PPA (10 mL) and stir at 120 °C for 1 hour. After the reaction is complete, cool the solution to room temperature and add distilled water (20 mL). Adjust the pH of the reaction solution to 6-7 with solid sodium hydroxide in an ice bath. Precipitate, filter, and dry under vacuum to obtain compounds 3a-d.

[0015] Step iv. Dissolve compounds 3a-d (3 mmol) in THF (20 mL) and add 1 M LiOH aqueous solution (9 mL). After stirring for 6 hours, monitor the reaction by TLC to indicate completion. Adjust the pH of the reaction solution to 4–6 with HCl in an ice bath. Precipitate, filter, and dry under vacuum to obtain compounds 4a-d.

[0016] Step v. Dissolve compounds 4a-d (2.5 mmol) in thionyl chloride (10 mL) and reflux for 6 h. Remove thionyl chloride under reduced pressure, and use the product directly in the next reaction.

[0017] Step vi. Dissolve the reaction product from the previous step in 6 mL of anhydrous CH2Cl2 containing 1.0 mL of triethylamine. Then, add dropwise 10 mL of anhydrous CH2Cl2 containing 4-Boc-aminopiperidine to the reaction mixture at 0 °C. Allow the reaction to slowly return to room temperature and stir for 12 h. Monitor the reaction by TLC until completion. Extract the solution with CH2Cl2 (3 × 20 mL), wash the organic phase with saturated brine, dry with anhydrous sodium sulfate, and remove the solvent under reduced pressure. Crystallize the residue and dry to give compounds 6a-d.

[0018] Step vii. Dissolve compound 6a-d (1 mmol) in CH2Cl2 (8 mL), add HCl (2 mL) ethyl acetate solution dropwise at low temperature, and stir the reaction at room temperature for 3 h. Monitor the reaction by TLC until completion. Remove the solvent under reduced pressure. The crude product can be used directly in the next step without further purification.

[0019] Step viii. Dissolve 0.5 mmol of the substituted phenyl isocyanates and 0.5 mmol of the crude product from the previous step in anhydrous CH2Cl2 (6 mL) and stir at room temperature for 5 h. Monitor the reaction by TLC until completion. Remove the solvent under vacuum, dry the residue, and obtain the target compounds 8a-8r by column chromatography. Detailed Implementation

[0020] Example 1: Preparation of 1-(1-(6-chloro-7-iodo-4-carbonyl-1,4-quinolinone-2-carbonyl)piperidine)-3-(4-chlorobenzene)urea (compound 8a)

[0021] 4-Chlorophenyl isocyanate (0.15 g, 0.1 mmol) and the crude product 7a (0.43 g, 0.1 mmol) from the previous step were dissolved in anhydrous CH2Cl2 (6 mL), and stirred at room temperature for 5 h. The reaction was monitored by TLC until completion. The solvent was removed under vacuum, the residue was dried, and column chromatography was used to obtain the target compound 8a. Yellow solid, 0.51 g, yield 88.2%, ESI-MS: 606.9 [M+Na] + , 1 H NMR (DMSO- d 6, 600MHz) δ (ppm): 11.97 (s, 1H, NH), 8.45 (s, 1H, NH), 7.93~7.81 (m, 2H, ArH), 7.36~7.30 (m, 4H, ArH), 7.28-7.26 (m, 1H, CH), 6.17 (s, 1H, NH), 4.03-3.97(m, 1H,CH), 3.70-3.62(m, 2H, CH2), 3.15-3.01(m, 2H, CH2), 1.85-1.63(m, 4H, CH2). Example 2: Preparation of 1-(1-(6-chloro-7-iodo-4-carbonyl-1,4-quinolinone-2-carbonyl)piperidine)-3-(3,4-dichlorobenzene)urea (compound 8b)

[0022] Preparation method is the same as in Example 1. Pale yellow solid, 0.47 g, yield 76%, ESI-MS: 640.9 [M+Na] + , 1 HNMR (DMSO- d 6,600 MHz) δ(ppm): 11.20 (s, 1H, NH), 8.51 (s, 1H, NH), 7.86~7.81(m, 2H, ArH), 7.36~7.30 (m, 3H, ArH), 7.19-7.10 (m, 1H, CH), 6.23 (s, 1H,NH), 4.06-3.99(m, 1H, CH), 3.68-3.61(m, 2H, CH2), 3.15-3.01(m, 2H, CH2), 1.82-1.66(m, 4H, CH2). Example 3: Preparation of 1-(1-(6-chloro-7-iodo-4-carbonyl-1,4-quinolinone-2-carbonyl)piperidine)-3-(3-chloro-4-trifluoromethylbenzene)urea (compound 8c)

[0023] Preparation method is the same as in Example 1. Orange-yellow solid, 0.44 g, yield 68%, ESI-MS: 675.0 [M+Na] + , 1 HNMR (DMSO- d 6,600 MHz) δ (ppm): 11.21 (s, 1H, NH), 8.53 (s, 1H, NH), 7.88~7.83(m, 2H, ArH), 7.36~7.30 (m, 3H, ArH), 7.19-7.10 (m, 1H, CH), 6.17 (s, 1H,NH), 4.06-3.98(m, 1H, CH), 3.68-3.60(m, 2H, CH2), 3.17-3.01(m, 2H, CH2), 1.82-1.66(m, 4H, CH2). Example 4: Preparation of 1-(1-(6-chloro-7-iodo-4-carbonyl-1,4-quinolinone-2-carbonyl)piperidine)-3-((4-fluoro-4-methoxybenzene)-4-trifluoromethylbenzene)urea (compound 8d)

[0024] Preparation method is the same as in Example 1. Yellow solid, 0.42 g, yield 57%, ESI-MS: 765.1 [M+Na] + , 1 HNMR (DMSO- d 6,600 MHz) δ(ppm): 11.21 (s, 1H, NH), 8.53 (s, 1H, NH), 7.88~7.83(m, 2H, ArH), 7.36~7.30 (m, 3H, ArH), 7.23 (dd, J =5.6 Hz, 1H, ArH), 7.19-7.10(m, 1H, CH), 6.82 (d, J =11.5 Hz, 1H, ArH), 6.40 (d, J =7.8 Hz, 1H, ArH), 6.17(s, 1H, NH), 4.06-3.98(m, 1H, CH), 3.82(s, 3H, OCH3), 3.68-3.60(m, 2H, CH2), 3.17-3.01(m, 2H, CH2), 1.82-1.66(m, 4H, CH2). Example 5: Preparation of 1-(1-(6-chloro-7-iodo-4-carbonyl-1,4-quinolinone-2-carbonyl)piperidine)-3-((2-fluorobenzene)-4-trifluoromethylbenzene)urea (compound 8e)

[0025] The preparation method is the same as in Example 1. Pale yellow solid, 0.41 g, yield 58%, ESI-MS: 735.0 [M+Na] + , 1 HNMR (DMSO- d 6,600 MHz) δ (ppm): 12.02 (s, 1H, NH), 8.62 (s, 1H, NH), 7.86~7.80(m, 2H, ArH), 7.36~7.31 (m, 3H, ArH), 7.28 (dd, J =5.6 Hz, 1H, ArH), 7.19-7.12(m, 1H, CH), 7.28~6.98 (m, 2H, ArH), 6.40 (d, J =7.8 Hz, 1H, ArH), 6.18 (s, 1H,NH), 4.06-3.99(m, 1H, CH), 3.68-3.62(m, 2H, CH2), 3.19-3.10(m, 2H, CH2), 1.86-1.70(m, 4H, CH2). Example 6: Preparation of 1-(1-(6-chloro-4-carbonyl-1,4-quinolinone-2-carbonyl)piperidine)-3-(4-chlorobenzene)urea (compound 8f)

[0026] Preparation method is the same as in Example 1. Yellow solid, 0.24 g, yield 53%, ESI-MS: 481.1 [M+Na] + , 1 HNMR (DMSO- d 6,600 MHz) δ (ppm): 11.06 (s, 1H, NH), 8.57 (s, 1H, NH), 7.83~7.78(m, 3H, ArH), 7.36~7.30 (m, 4H, ArH), 7.19-7.12 (m, 1H, CH), 6.24 (s, 1H,NH), 4.06-4.01(m, 1H, CH), 3.69-3.61(m, 2H, CH2), 3.20-3.09(m, 2H, CH2), 1.82-1.66(m, 4H, CH2). Example 7: Preparation of 1-(1-(6-chloro-7-iodo-4-carbonyl-1,4-quinolinone-2-carbonyl)piperidine)-3-(3,4-dichlorobenzene)urea (compound 8g)

[0027] Preparation method is the same as in Example 1. Pale yellow solid, 0.47 g, yield 76%, ESI-MS: 640.9 [M+Na] + , 1 HNMR (DMSO- d 6,600 MHz) δ (ppm): 11.22 (s, 1H, NH), 8.50 (s, 1H, NH), 8.04–7.97(m, 1H), 7.95–7.90 (m, 2H), 7.16~7.01 (m, 3H, ArH), 7.19-7.10 (m, 1H, CH),6.23 (s, 1H, NH), 4.06-3.99(m, 1H, CH), 3.68-3.61(m, 2H, CH2), 3.15-3.01(m,2H, CH2), 1.82-1.66(m, 4H, CH2). Example 8: Preparation of 1-(1-(6-chloro-7-iodo-4-carbonyl-1,4-quinolinone-2-carbonyl)piperidine)-3-(3-chloro-4-trifluorotoluene)urea (compound 8h)

[0028] Preparation method is the same as in Example 1. Pale yellow solid, 0.32 g, yield 61%, ESI-MS: 649.1 [M+Na] + ,1 HNMR (DMSO- d 6,600 MHz) δ (ppm): 12.01 (s, 1H, NH), 8.53 (s, 1H, NH), 8.01–7.95 (m, 2H, ArH), 7.98–7.90 (m, 1H, ArH), 7.14~7.00 (m, 3H, ArH), 7.18-7.09 (m,1H, CH), 6.24 (s, 1H, NH), 4.05-3.98(m, 1H, CH), 3.69-3.60(m, 2H, CH2), 3.18-3.03(m, 2H, CH2), 1.83-1.67(m, 4H, CH2). Example 9: Preparation of 1-(1-(6-chloro-4-carbonyl-1,4-quinolinone-2-carbonyl)piperidine)-3-((4-fluoro-4-methoxybenzene)-4-trifluoromethylbenzene)urea (compound 8h)

[0029] The preparation method is the same as in Example 1. The product is a pale yellow solid, 0.30 g, yield 49%, ESI-MS: 639.1 [M+Na]. + , 1 HNMR (DMSO- d 6,600 MHz) δ (ppm): 11.08 (s, 1H, NH), 8.52 (s, 1H, NH), 8.01–7.96 (m, 3H, ArH), 7.98–7.91 (m, 2H, ArH), 7.38~7.32 (m, 3H, ArH), 7.25 (dd, J =5.6Hz, 1H, ArH), 7.21-7.13 (m, 1H, CH), 6.84 (d, J =11.7 Hz, 1H, ArH), 6.40 (d, J =7.7 Hz, 1H, ArH), 6.18(s, 1H, NH), 4.08-4.00(m, 1H, CH), 3.81(s, 3H, OCH3), 3.68-3.62(m, 2H, CH2), 3.16-3.00(m, 2H, CH2), 1.84-1.67(m, 4H, CH2). Example 10: Preparation of 1-(1-(6-chloro-4-carbonyl-1,4-quinolinone-2-carbonyl)piperidine)-3-((2-fluorobenzene)-4-trifluoromethylbenzene)urea (compound 8j)

[0030] Preparation method is the same as in Example 1. Yellow solid, 0.31 g, yield 54%, ESI-MS: 609.1 [M+Na] + , 1 HNMR (DMSO- d 6,600 MHz) δ (ppm): 12.00 (s, 1H, NH), 8.63 (s, 1H, NH), 8.07–7.98(m, 2H, ArH), 7.99–7.91 (m, 1H, ArH), 7.38~7.30 (m, 3H, ArH), 7.28 (dd, J =6.0Hz, 1H, ArH), 7.19-7.12 (m, 1H, CH), 7.28~7.12 (m, 2H, ArH), 6.41 (d, J =7.6Hz, 1H, ArH), 6.18 (s, 1H, NH), 4.08-3.99(m, 1H, CH), 3.69-3.64(m, 2H, CH2), 3.19-3.10(m, 2H, CH2), 1.86-1.71(m, 4H, CH2). Example 11: Preparation of 1-(1-(6-chloro-7-trifluoromethyl-4-carbonyl-1,4-quinolinone-2-carbonyl)piperidine)-3-(3-chloro-benzene)urea (compound 8k)

[0031] The preparation method is the same as in Example 1. Pale yellow solid, 0.33 g, yield 63%, ESI-MS: 649.1 [M+Na] + , 1 HNMR (DMSO- d 6,600 MHz) δ(ppm): 12.02 (s, 1H, NH), 8.54 (s, 1H, NH), 8.98–8.90 (m, 1H, ArH), 8.36–8.16 (m, 2H, ArH), 7.14~7.01 (m, 3H, ArH), 7.19-7.11 (m,1H, CH), 6.22 (s, 1H, NH), 4.06-3.98(m, 1H, CH), 3.71-3.61(m, 2H, CH2), 3.20-3.03(m, 2H, CH2), 1.83-1.68(m, 4H, CH2). Example 12: Preparation of 1-(1-(6-chloro-7-trifluoromethyl-4-carbonyl-1,4-quinolinone-2-carbonyl)piperidine)-3-(3,4-dichlorobenzene)urea (compound 8l)

[0032] The preparation method is the same as in Example 1. Pale yellow solid, 0.40 g, yield 72%, ESI-MS: 583.0 [M+Na] + , 1 HNMR (DMSO- d 6,600 MHz) δ (ppm): 12.01 (s, 1H, NH), 8.55 (s, 1H, NH), 8.97–8.91(m, 1H, ArH), 8.36–8.18 (m, 2H, ArH), 7.16~7.00 (m, 4H, ArH), 7.19-7.10 (m,1H, CH), 6.22 (s, 1H, NH), 4.06-3.98(m, 1H, CH), 3.71-3.61(m, 2H, CH2), 3.21-3.03(m, 2H, CH2), 1.83-1.69(m, 4H, CH2). Example 13: Preparation of 1-(1-(6-chloro-7-trifluoromethyl-4-carbonyl-1,4-quinolinone-2-carbonyl)piperidine)-3-(3-chloro-4-trifluoromethylbenzene)urea (compound 8m)

[0033] Preparation method is the same as in Example 1. Yellow solid, 0.31 g, yield 52%, ESI-MS: 617.1 [M+Na] + , 1 HNMR (DMSO- d 6,600 MHz) δ(ppm): 12.01 (s, 1H, NH), 8.55 (s, 1H, NH), 8.99–8.93(m, 2H, ArH), 8.36–8.20 (m, 2H, ArH), 7.17~7.01 (m, 3H, ArH), 7.20-7.11 (m,1H, CH), 6.23 (s, 1H, NH), 4.06-3.99(m, 1H, CH), 3.71-3.60(m, 2H, CH2), 3.21-3.00(m, 2H, CH2), 1.84-1.69(m, 4H, CH2). Example 14: Preparation of 1-(1-(4-carbonyl-1,4-quinolinone-2-carbonyl)piperidine)-3-(3-chloro-4-trifluoromethylbenzene)urea (compound 8n)

[0034] Preparation method is the same as in Example 1. Pale yellow solid, 0.34 g, yield 69%, ESI-MS: 515.1 [M+Na] + , 1 HNMR (DMSO- d 6,600 MHz) δ (ppm): 12.00 (s, 1H, NH), 8.54 (s, 1H, NH), 8.98–8.90(m, 1H, ArH), 8.36–8.19 (m, 2H, ArH), 7.16~7.01 (m, 4H, ArH), 7.19-7.11 (m,1H, CH), 6.23 (s, 1H, NH), 4.09-3.98(m, 1H, CH), 3.73-3.64(m, 2H, CH2), 3.22-3.02(m, 2H, CH2), 1.85-1.69(m, 4H, CH2). Example 15: Studies on the in vitro antitumor activity of quinolinone derivatives containing urea fragments and their antitumor cell proliferation effects. The half-maximal inhibitory concentration (IC50) of dihydropyrazomorpholine derivatives containing a naphthalene ring skeleton against human non-small cell lung cancer cells (NCI–H23), cervical cancer cells (HeLa), lung cancer cells (A549), and liver cancer cells (HepG2) was determined using the MTT assay [3-(4,5)-bismethyl-2-thiazolyl-(2,5)-phenyltetrazolyl blue bromide]. 50 ).

[0035] (1) Preparation of culture medium ( / L): ① Suspension cells: one packet of DMEM culture powder (10.4 g), 100 mL of newborn calf serum, and penicillin solution (2×10⁻⁶ g / L). -5 0.5 mL of streptomycin solution (U / mL) and 2×10⁻⁶ mL of streptomycin solution. -5 ① Dissolve 0.5 mL of HEPES (U / mL) in triple-distilled water, adjust the pH to 7.2-7.4 with 5.6% NaHCO3 solution, and finally bring the volume to 1000 mL. Filter and sterilize. ② Adherent cells: Same as above, but add 2.00 g of NaHCO3 and 2.38 g of HEPES.

[0036] (2) Preparation of D-Hanks buffer (per liter): NaCl 8.00 g, KCl 0.40 g, Na2HPO4·12H2O 0.06 g, KH2PO4 0.06 g, NaHCO3 0.35 g. Autoclave.

[0037] (3) Preparation of trypsin solution: Prepare a 0.5% trypsin solution using D-Hanks buffer. Filter for sterilization.

[0038] (4) Preparation of experimental solutions: Dissolve the test sample in a small amount of triple-distilled water to prepare a stock solution, i.e., prepare a stock solution at 10 times the highest experimental concentration. Depending on the solubility of the compound, it can be dissolved directly in triple-distilled water, or a small amount of DMSO can be used as a dissolving agent before adding triple-distilled water to dissolve it. Store the stock solution in a -20 ˚C refrigerator for later use.

[0039] (5) Culture of human non-small cell lung cancer cells (NCI–H23), cervical cancer cells (HeLa), lung cancer cells (A549), and liver cancer cells (HepG2): These were adherent cells, routinely cultured in DMEM medium (containing 10% fetal bovine serum and 100 U / mL streptomycin) at 37 ˚C in a 5% CO2 incubator, and passaged every 3-4 days. For passage, the culture medium was transferred from the original flask to a centrifuge tube, centrifuged at 1000 rpm for 5 min, the original culture medium was discarded, an equal volume of fresh culture medium was added, the mixture was thoroughly mixed, and an appropriate amount was transferred to a fresh culture flask. Fresh culture medium was then added to bring the volume to approximately 1 / 10 of the original flask capacity.

[0040] (6) Cell incubation: Take tumor cells in the logarithmic growth phase and adjust the cell suspension concentration to 1-1.5×10⁻⁶. 5 Cells / mL. Add 100 μL of cell suspension to each well of a 96-well culture plate. μ L, incubated at 37 ˚C in a 5% CO2 incubator for 24 h. After 24 h of incubation, the drug solution was added according to the design.

[0041] (7) Drug Addition: The test drug solution was added to each well according to the final concentration gradient, with 6 parallel wells for each concentration. The experiment was divided into a drug test group (different concentrations of test drug were added), a control group (only culture medium and cells were added, without test drug), and a blank group (only culture medium was added, without cells and test drug). The 96-well plates after drug addition were incubated at 37 ˚C in a 5% CO2 incubator for 48 h. The activity of the positive control drug was determined according to the method for the test samples.

[0042] (8) Assay for viable cells: MTT 40 g was added to each well of a 96-well plate after 48 h of culture. μ L (prepared to 4 mg / mL with D-Hanks buffer). After incubation at 37 ˚C for 4 h, remove the supernatant. Add 150 μL to each well. μ Dissolve the formazan crystals by shaking L DMSO for 5 min. Finally, measure the optical density (OD value) of each well at 490 nm using an automated microplate reader.

[0043] Half-maximal inhibitory concentration (IC50) 50 The concentration at which 50% of tumor cells survive is defined as the drug concentration. A standard curve of cell growth inhibition rate is constructed based on the measured optical density (OD value), and the corresponding drug concentration is determined from the standard curve.

[0044] Measured IC 50 See Figure 18 As shown.

[0045] The above experiments demonstrate that the quinolinone derivative containing the urea fragment of the present invention exhibits significant inhibitory effects on human non-small cell lung cancer cells (NCI-H23), cervical cancer cells (HeLa), lung cancer cells (A549), and liver cancer cells (HepG2). In particular, it shows comparable or superior inhibitory activity against the positive control drug SH-9 for non-small cell lung cancer cells (NCI-H23). Therefore, the quinolinone derivative containing the urea fragment of the present invention can be used in the preparation of antitumor drugs.

[0046] Example 16: In vitro KRAS protein inhibitory activity assay and analysis Different concentrations of the compound were mixed with 10 μ MKRAS protein was incubated in glutamate buffer (pH 6.6) at 30 °C, then cooled to 0 °C. After adding 0.4 mM GTP, the mixture was transferred to a cuvette at 0 °C and heated to 30 °C. Finally, the assembly of KRAS protein was observed at 350 nm using turbidimetry. The inhibition of KRAS protein by the compound, like its inhibition of cells, was measured using IC50. 50 It indicates that IC 50Defined as the drug concentration at which KRAS protein assembly is inhibited by 50% after 20 min of incubation.

[0047] To further elucidate whether these compounds exert their antitumor activity by acting on the KRAS protein, an in vitro KRAS protein inhibitory activity test was conducted. The results are as follows: Figure 19 As shown, the IC50 of the KRAS protein inhibitory activity of this series of compounds is... 50 The value is between 9.6 and 140. μ Between M and M. Similar to the in vitro antiproliferative activity results, compound 8m exhibited the strongest inhibitory activity (IC50). 50 =9.6 μ M).

[0048] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details of the above embodiments. Within the scope of the technical concept of the present invention, various equivalent transformations can be made to the technical solutions of the present invention, and these equivalent transformations all fall within the protection scope of the present invention. Furthermore, it should be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. To avoid unnecessary repetition, the present invention will not describe the various possible combinations separately. In addition, various different embodiments of the present invention can also be arbitrarily combined, as long as they do not violate the spirit of the present invention, they should also be considered as the content disclosed by the present invention. Figure 1 The structural diagram shown in equation X Figure 2 It is a synthesis roadmap; Figure 3 The synthesized molecular formula diagram Figure 4 It is a diagram of compound 8a. Figure 5 This is diagram of compound 8b. Figure 6 It is the diagram of compound 8c. Figure 7 It is the 8d diagram of the compound. Figure 8 It is the diagram of compound 8e. Figure 9 It is the 8f diagram of compound. Figure 10 It is a diagram of 8g of compound. Figure 11 It is a graph of compound 8h. Figure 12 It is the diagram of compound 8i. Figure 13 It is the diagram of compound 8j. Figure 14 It is an 8k graph of the compound. Figure 15 It is the diagram of compound 8l. Figure 16 It is the 8m diagram of the compound. Figure 17 It is a diagram of compound 8n. Figure 18 This is a graph showing the IC50 (μM) values ​​of the quinolinone derivatives containing urea fragments listed in this invention against tumor cells; Figure 19 This is a graph showing the IC50 values ​​(μM) of the quinolinone derivatives containing urea fragments listed in this invention against KRAS inhibitory activity.

Claims

1. A class of antitumor heterocyclic compounds targeting KRAS, having the structure shown in Formula X. in, R1 is selected from H, CH3, CH2CH3, OCH3, OCH2CH3, F, Cl, Br, CF3; R2 is selected from H, CH3, Cl, Br, CF3; R3 is selected from H, CH3, CH2CH3, OCH3, OCH2CH3, F, Cl, Br, CF3, and phenyl groups with different substitutions; R4 is selected from H, CH3, Cl, Br, CF3, and phenyl groups with different substitutions.

2. A method for preparing antitumor heterocyclic compounds targeting KRAS, characterized in that, The quinolinone derivative containing the urea fragment has a structure as shown in Formula X. Wherein, R1 is selected from H, CH3, CH2CH3, OCH3, OCH2CH3, F, Cl, Br, CF3; R2 is selected from H, CH3, Cl, Br, CF3; R3 is selected from H, CH3, CH2CH3, OCH3, OCH2CH3, F, Cl, Br, CF3, and phenyl groups with different substitutions; R4 is selected from H, CH3, Cl, Br, CF3, and phenyl groups with different substitutions. Its synthesis process has the following general formula: The method includes the following steps: Step i. Under stirring, 10 mmol of dinitrobenzene with different substitutions was added to 40 mL of acetic acid containing 60 mmol of Fe powder, followed by dropwise addition of 20 mL of distilled water. The reaction solution was heated at 50 °C for 10 minutes. After TLC confirmation of the reaction completion, the mixture was filtered, and the residue was washed with 10 mL of ethyl acetate and 10 mL of water. The filtrate was extracted with ethyl acetate (3 × 30 mL), and the organic phase was washed successively with 2% sodium hydroxide solution and saturated brine. The mixture was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was crystallized, filtered, and dried to give compounds 1a-1d. Step ii. Compounds 1a-d (6 mmol) and dimethyl butyrynethioate (9 mmol) were added sequentially to a 10 mL round-bottom flask and heated to reflux for 3 h. The reaction was monitored by TLC until completion. After removing methanol under reduced pressure, the mixture was allowed to stand to crystallize, filtered, and the filter cake was washed with ice-cold methanol and dried under vacuum to obtain compounds 2a-d. Step iii. Add compounds 2a-d (4 mmol) to PPA (10 mL) and stir at 120 °C for 1 hour. After the reaction is complete, cool the solution to room temperature and add distilled water (20 mL). Adjust the pH of the reaction solution to 6-7 with solid sodium hydroxide in an ice bath. Precipitate, filter, and dry under vacuum to obtain compounds 3a-d. Step iv. Dissolve compound 3a-d (3 mmol) in THF (20 mL) and add 1 M LiOH aqueous solution (9 mL). After stirring for 6 hours, the reaction was monitored by TLC to indicate completion. The reaction solution was adjusted to pH 4–6 with HCl under ice bath conditions. The precipitate was collected, filtered, and dried under vacuum to give compound 4a-d. Step v. Dissolve compound 4a-d (2.5 mmol) in thionyl chloride (10 mL) and reflux for 6 h. Remove thionyl chloride under reduced pressure, and use the product directly in the next reaction. Step vi. Dissolve the reaction product from the previous step in 6 mL of anhydrous CH2Cl2 containing 1.0 mL of triethylamine. Then, add 10 mL of anhydrous CH2Cl2 containing 4-Boc-aminopiperidine dropwise to the reaction mixture at 0 °C. Allow the reaction to slowly return to room temperature and stir for 12 h. Monitor the reaction by TLC until completion. Extract the solution with CH2Cl2 (3 × 20 mL), wash the organic phase with saturated brine, dry with anhydrous sodium sulfate, and remove the solvent under reduced pressure. Crystallize the residue and dry to give compounds 6a-d. Step vii. Dissolve compound 6a-d (1 mmol) in CH2Cl2 (8 mL), add HCl (2 mL) of ethyl acetate solution dropwise at low temperature, and stir the reaction at room temperature for 3 h. Monitor the reaction by TLC until completion. Remove the solvent under reduced pressure. The crude product can be used directly in the next step without further purification. Step viii. Dissolve 0.5 mmol of substituted phenyl isocyanates and 0.5 mmol of the crude product from the previous step in anhydrous CH2Cl2 (6 mL) and stir at room temperature for 5 h. Monitor the reaction by TLC until completion. Remove the solvent under vacuum, dry the residue, and obtain the target compounds 8a-8r by column chromatography.

3. The method for preparing dihydropyrazolomorpholine derivatives containing a naphthalene ring skeleton as described in claim 2, characterized in that, Step 1 further comprises: Under stirring at 0±5℃, the compound with the structure shown in Formula I was dissolved in anhydrous methanol in a round-bottom flask, and SOCl2 was added dropwise. After 10±5 min, the mixture was transferred to 20±10℃ and stirred for another 6±3 h. The mixture was then filtered, dried, and the resulting crude solid product was dissolved in anhydrous ethanol and recrystallized to obtain the compound with the structure shown in Formula II.

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