Intermediates for the preparation of a KRAS inhibitor compound and methods of synthesis thereof

Abstract

This invention relates to an intermediate compound having the structure shown in formula INT-33 for synthesizing KRAS inhibitor compounds and a method thereof for their synthesis.

Description

Technical Field

[0001] This invention relates to a compound, and more particularly to an intermediate compound for the preparation of a highly active pan-KRAS inhibitor. Background Technology

[0002] RAS is one of the most frequently mutated genes in human tumors, occurring in approximately 30% of cancer patients, with KRAS accounting for about 85% of RAS mutations. KRAS mutations are found in 88% of pancreatic cancers, 50% of colorectal adenocarcinomas, and 32% of lung adenocarcinomas, making the development of KRAS-targeting inhibitors of great clinical significance and value.

[0003] KRAS is a membrane-bound protein with GTPase activity. It acts as a "molecular switch" by cycling between the GDP-binding inactive conformation and the GTP-binding active conformation through nucleotide exchange. In its GTP-bound state, KRAS can activate multiple downstream signaling pathways, including RAF-MEK-ERK and PI3K-AKT, to regulate life processes such as cell growth, proliferation, differentiation, and apoptosis.

[0004] KRAS mutations (such as G12C, G12D, G12V, and G13D) affect GTP hydrolysis mediated by GTPase-activating proteins (GAPs), increasing the number of KRAS in a GTP-bound activated state. This overactivation of downstream signaling pathways ultimately leads to tumor development and progression. However, due to the lack of a suitable hydrophobic pocket for drug binding in the KRAS protein, and its affinity for GTP and GDP being in the picomolar range (~20 pM), the development of inhibitors that competitively bind to KRAS is extremely difficult. For decades, KRAS has been considered an untreatable target.

[0005] In May 2021, AMG510 was approved by the FDA for the treatment of patients with KRAS. G12C The emergence of mutations in locally advanced or metastatic non-small cell lung cancer has broken the historical barrier of KRAS being "untreatable." However, G12C mutations account for only a small fraction of KRAS mutations. For mutations at other KRAS sites, there is currently a lack of satisfactory and effective inhibitory compounds, leaving a large unmet clinical need. Therefore, the development of effective pan-KRAS inhibitory compounds is a necessity given the current technology. Summary of the Invention

[0006] This invention provides a pan-KRAS inhibitor. This structure differs from existing KRAS inhibitors that function through covalent binding. G12CInstead of acting as an inhibitor, KRAS exerts its effects by mediating the formation of a ternary complex between KRAS and ubiquitous intracellular chaperone proteins (such as Cyclophilin A). The formation of this ternary complex can sterically block the binding of KRAS to its downstream effector molecules (such as RAF), inhibiting the activation of the MAPK and PI3K-AKT signaling pathways, thereby suppressing tumor development and progression, and playing a therapeutic role in diseases such as cancer.

[0007] In one aspect, the present invention provides an intermediate compound for synthesizing KRAS inhibitor compounds, having the structure shown in formula INT-33:

[0008]

[0009] In one embodiment, the method for synthesizing the intermediate compound INT-33 includes the following steps:

[0010]

[0011] Step 1: After dissolving compound INT-2, add INT-18, [1,1'-bis(diphenylphosphine)ferrocene]palladium dichloride and potassium phosphate in sequence; after the reaction is complete, filter and purify to obtain INT-33a;

[0012] Step 2: After dissolving compound INT-33a, cesium carbonate and iodoethane were added to it; after the reaction was complete, it was extracted and purified to obtain INT-33b;

[0013] Step 3: After dissolving compound INT-33b, p-toluenesulfonic acid monohydrate was added to it; after the reaction was complete, compound INT-33c was obtained by extraction and purification.

[0014] Step 4: After dissolving compound INT-33c, p-toluenesulfonyl chloride and potassium hydroxide are added to it; after the reaction is complete, INT-33 is obtained by purification.

[0015] The synthesis of compound INT-2 includes the following steps:

[0016]

[0017] Step 1: Dissolve compound INT-1m, add lithium hydroxide monohydrate; after the reaction is complete, extract and purify to obtain compound INT-2a;

[0018] Step 2: After dissolving compounds INT-2a and INT-2b, N,N,N',N'-tetramethylchloromethamidine hexafluorophosphate and 1-methylimidazole were added at 0°C. After the reaction was completed at 0°C, the compounds were extracted and purified to obtain the compounds.

[0019] Step 3: After dissolving compound INT-2c, add lithium hydroxide monohydrate; after the reaction is complete, precipitate and purify to obtain compound INT-2d;

[0020] Step 4: After dissolving compound INT-2d, 1-hydroxybenzotriazole, and 4-dimethylaminopyridine, N,N-diisopropylethylamine was added at 0°C, followed by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; after the reaction was completed, the reaction solution was washed with ammonium chloride aqueous solution to purify and obtain compound INT-2e.

[0021] Step 5: After dissolving compound INT-2e, 2-dicyclohexylphosphine-2′,6′-dimethylbiphenyl, tris(dibenzylideneacetone)dipalladium and potassium acetate, pinacol borane was added under nitrogen protection. After the addition was complete, the reaction was carried out at 50°C under nitrogen protection. After the reaction was completed, the compound INT-2 was obtained by filtration and purification.

[0022] The synthesis of compound INT-18 includes the following steps:

[0023]

[0024] After dissolving (S)-3-bromo-5-iodo-2-(1-methoxyethyl)pyridine INT-5a, cuprous iodide, palladium dichloride of bis(triphenylphosphine), triethylamine and INT-18a were added sequentially; the reaction mixture was carried out under nitrogen protection; after the reaction was complete, the compound INT-5 was obtained by purification.

[0025] In one embodiment, the KRAS inhibitor compound described above is selected from the group consisting of:

[0026]

[0027] Other features of the invention will become apparent in the process of describing exemplary embodiments. The embodiments described are given to illustrate the invention and are not intended to be limiting. The following examples use the methods disclosed in the invention to prepare, separate and characterize.

[0028] The compounds of the present invention can be prepared in a variety of ways known to those skilled in the art of organic synthesis. They can be synthesized using the methods described below, as well as synthetic methods known in the field of organic synthetic chemistry, or by variations thereof understood by those skilled in the art. Preferred methods include, but are not limited to, those described below. The reaction is carried out in a solvent or solvent mixture suitable for the kit materials used and suitable for the transformation achieved. Those skilled in the art of organic synthesis will understand that the functionalities present on the molecule are consistent with the proposed transformation. This sometimes necessitates determining whether to change the order of synthetic steps or the starting materials to obtain the desired compound of the present invention. Detailed Implementation

[0029] the term

[0030] Unless otherwise specified, the terms used in this application, including those in the specification and claims, are defined as follows. Unless otherwise specified, conventional methods such as mass spectrometry, nuclear magnetic resonance, HPLC, protein chemistry, biochemistry, recombinant DNA techniques, and pharmacology are used. In this application, unless otherwise specified, "or" or "and" refers to "and / or".

[0031] All features described in this specification (including any claims or abstracts) and / or all steps involved in any method or process may exist in any combination unless certain features or steps are mutually exclusive in the same combination.

[0032] The features mentioned above in this invention, or the features mentioned in the embodiments, can be combined arbitrarily. All features disclosed in this specification can be used in any compositional form, and each feature disclosed in the specification can be replaced by any alternative feature that provides the same, equivalent, or similar purpose. Therefore, unless otherwise specified, the disclosed features are merely general examples of equivalent or similar features.

[0033] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise stated, all percentages, ratios, proportions, or parts are by weight.

[0034] The units used in weight-volume percentages in this invention are well known to those skilled in the art, for example, referring to the weight (g) of the solute in 100 ml of solution. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as known to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0035] Example

[0036] General process

[0037] When the preparation method is not specified, all raw materials and reagents used in this invention are known products that can be synthesized according to methods known in the art, or can be obtained by purchasing commercially available products. None of the commercially available reagents used require further purification.

[0038] Room temperature refers to 20-30℃.

[0039] Unless otherwise specified in the reaction examples, all reactions were carried out under a nitrogen atmosphere. A nitrogen atmosphere refers to a reaction flask connected to a nitrogen balloon of approximately 1L.

[0040] Hydrogenation reactions are typically carried out under vacuum, filled with hydrogen gas, and repeated three times. A hydrogen atmosphere refers to a reaction flask connected to a hydrogen balloon of approximately 1L.

[0041] Microwave reaction use Initiator + Microwave Reactor.

[0042] The structure of the compounds of this invention was determined by nuclear magnetic resonance (NMR) and mass spectrometry (MS). NMR shifts (δ) were expressed in terms of 10⁻¹⁰. -6 The measurements are given in units of (ppm). NMR determinations are performed using (Bruker Ascend) TM A Model 500 NMR spectrometer was used. The solvents used were deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), and deuterated methanol (CD3OD). The internal standard was tetramethylsilane (TMS). The following abbreviations are used for NMR signal multiplicity: s = singlet, brs = broad peak, d = doublet, t = triplet, m = multiplet. Coupling constants are listed in J values ​​and measured in Hz.

[0043] Reversed-phase preparative chromatography was performed using a Thermo (UltiMate 3000) reversed-phase preparative chromatograph. Rapid column chromatography was performed using an Agilent (FS-9200T) automated column press, and pre-packed silica gel columns were obtained from Sante. Pre-packed column. Thin-layer chromatography silica gel plates are Yantai Huanghai HSGF254 or Qingdao GF254. The thickness used for thin-layer chromatography separation and purification of products is 0.4mm to 0.5mm.

[0044] The LC-MS analysis method is as follows:

[0045] 1) Mass spectrometry method: Thermo Fisher MSQ PLUS mass spectrometer, ESI source, positive ion mode. Ion source parameter settings: drying gas temperature 350℃; drying gas flow rate 10L / min; MS range: 120-1000.

[0046] 2) Liquid chromatography conditions: Column: Waters XBridge (3.5 μm, 50 mm × 4.6 mm); Mobile phase A is an aqueous solution containing 0.1% ammonium bicarbonate, and mobile phase B is an acetonitrile solution, with linear gradient elution according to Table 1; Flow rate: 2 mL / min; Column temperature: 30 ℃; UV detection wavelength: 214 nm, 254 nm, 280 nm; Injection volume: 2 μL.

[0047] Table 1. Gradient elution conditions

[0048]

[0049] The HPLC analysis method is as follows:

[0050] Chromatographic column: Waters XBridge phenyl (3.5 μm, 150 mm × 4.6 mm); mobile phase A was an aqueous solution containing 0.1% ammonium bicarbonate, and mobile phase B was an acetonitrile solution, with linear gradient elution performed according to Table 2; flow rate: 1 mL / min; column temperature: 30 ℃; UV detection wavelengths: 214 nm, 254 nm, 280 nm; injection volume: 2 μL.

[0051] Table 2. Gradient elution conditions

[0052]

[0053] The synthesis methods of some intermediates in the invention are as follows:

[0054] Intermediate 1

[0055]

[0056] Intermediate 1 is prepared by the following steps:

[0057]

[0058] Step 1: Dissolve methyl 2,2-dimethyl-3-hydroxypropionate INT-1a (100 g, 757 mmol) in 1 L of N,N-dimethylformamide, add imidazole (129 g, 1.89 mol), stir to dissolve, and add tert-butyldiphenylchlorosilane (229 g, 832 mmol) dropwise at room temperature. After the addition is complete, continue stirring for 4 hours. After the reaction is complete, pour the reaction solution into 3 L of ice water. Extract the suspension with ethyl acetate (1 L * 2). Wash the organic phase three times with water, concentrate under reduced pressure to obtain a colorless oily substance INT-1b, which can be used directly in the next step without purification. ESI-MS (m / z): 371.2 [M+H] + .

[0059] Step 2: Add the residual INT-1b obtained in the previous step to 2L of methanol, then add 360g of a prepared 33% sodium hydroxide aqueous solution, and stir at room temperature for 17 hours. After the reaction is complete, add 1L of water, remove methanol under reduced pressure, and extract the residual liquid with petroleum ether (1L*5). After extraction, adjust the pH of the aqueous phase to 4-5 with hydrochloric acid, continue stirring for 30 minutes, filter, and dry to obtain a white solid INT-1c (269g, yield 90%). ESI-MS (m / z): 357.8 [M+H] + .

[0060] Step 3: Dissolve INT-1c (130g, 365mmol) in 500mL of dichloromethane, add thionyl chloride (130g, 1.09mol, 79.4mL) at room temperature, stir at 60℃ for 3 hours. After the reaction is complete, remove dichloromethane and the remaining thionyl chloride under reduced pressure to obtain a pale yellow oily substance INT-1d. Without purification, add 200mL of dichloromethane for later use.

[0061] Step 4: Dissolve INT-1e (64.8 g, 331 mmol) in 400 mL of dichloromethane. Add 198 mL of diethylaluminum chloride solution (2 M in hexanes) dropwise at 0 °C, controlling the temperature to not exceed 5 °C during the addition. Stir for 30 minutes after the addition is complete. Add the resulting dichloromethane solution of INT-1d dropwise to the reaction flask. Control the temperature to not exceed 10 °C during the addition. Continue stirring for 2 hours after the addition is complete. After the reaction is complete, pour the reaction solution into 1 L of ice water, stir for 30 minutes, concentrate under reduced pressure to remove dichloromethane, and extract the residue with ethyl acetate (1 L * 2). Wash with water, and rotary evaporate the organic phase to obtain a brown oily substance. Add the oily substance to 2 L of a 10 / 1 mixture of petroleum ether / ethyl acetate, stir to precipitate a solid, filter, and obtain a yellow solid INT-1f (139 g, yield 78%). ESI-MS (m / z): 534.8 [M + H] + .

[0062] Step 5: Dissolve INT-1f (100g, 187mmol) in 500mL of tetrahydrofuran, add lithium borohydride (12.2g, 561mmol), stir overnight at 60°C. After the starting material disappears, quench the reaction solution in 200mL of ice water, extract with ethyl acetate (500mL*3), wash the organic phase with water, dry it, concentrate under reduced pressure, dissolve the residue in 500mL of dichloromethane, add diethyl 2,6-dimethyl-1,4-dihydro-3,5-pyridinedicarboxylate (28.4g, 112mmol) and p-toluenesulfonic acid (21.4g, 112mmol), stir at room temperature for 3 hours, concentrate under reduced pressure after the reaction is complete, remove dichloromethane, dissolve the residue in 500mL of methanol, add 100mL of pre-prepared 14% lithium hydroxide aqueous solution, stir at room temperature for 3 hours, and filter to obtain yellow solid INT-1g (84g, yield 86.3%). ESI-MS (m / z): 520.2 [M+H] + .

[0063] Step 6: Dissolve INT-1 g (50 g, 96 mmol) in 250 mL of tetrahydrofuran, add tetrabutylammonium fluoride (1 M inTHF, 197 mL), stir overnight at 60 °C. After the reaction is complete, add the reaction solution to 300 mL of water, extract with ethyl acetate (200 mL * 3), wash with water, concentrate under reduced pressure to obtain a brown oil. Dissolve the residue in 40 mL of methanol, add 20 mL of water, wash the mixture with petroleum ether (40 mL * 5), concentrate under reduced pressure to remove methanol, extract the residue with ethyl acetate (50 mL * 2), wash the organic phase with water, dry to obtain a pale yellow oil INT-1h (25 g, yield 90.4%). ESI-MS (m / z): 282.8 [M + H] + .

[0064] Step 7: Dissolve compound INT-1h (22 g, 77 mmol) in 100 mL of dichloromethane. Add 4-dimethylaminopyridine (467 mg, 3.82 mmol) and triethylamine (23.2 g, 230 mmol). Add acetic anhydride (7.9 g, 77 mmol) dropwise at 0 °C. After the addition is complete, allow the mixture to heat naturally and stir overnight. Once the reaction is complete, wash the reaction solution with water, dry it, and concentrate it to obtain a brown oil. Purify the oil by silica gel column chromatography (petroleum ether / ethyl acetate = 4 / 1) to obtain a pale yellow oil INT-1i (22.5 g, yield 90.7%). ESI-MS (m / z): 324.2 [M+H] + .

[0065] Step 8: Compound INT-1i (40 g, 123 mmol) was dissolved in dioxane (400 mL), and potassium acetate (30.3 g, 308.4 mmol), [1,1'-bis(diphenylphosphine)ferrocene]palladium dichloride (10 g, 12.3 mmol), and pinacol diboronate (78.3 g, 308 mmol) were added. The reaction was carried out at 90 °C for 3 hours under nitrogen protection. The reaction mixture was monitored by LCMS until the starting material was completely reacted. The reaction solution was directly concentrated under reduced pressure. The residue was dissolved in ethyl acetate (300 mL), washed with water and brine, and the organic phase was purified by silica gel column chromatography to obtain a white solid compound INT-1j (35 g, yield 76.4%). ESI-MS (m / z): 372.5 [M+H] + .

[0066] Step 9: Compound INT-1j (35 g, 94.3 mmol) and compound INT-1k (37.9 g, 104 mmol) were dissolved in dioxane (300 mL) and water (30 mL). Potassium phosphate (50 g, 236 mmol) and [1,1'-bis(diphenylphosphine)ferrocene]palladium dichloride (6.89 g, 9.43 mmol) were added. The reaction was carried out overnight at 90 °C under nitrogen protection. The reaction mixture was monitored by LCMS until the starting material was completely reacted. The reaction solution was directly concentrated under reduced pressure. The residue was dissolved in ethyl acetate (300 mL), washed with water and brine, and the organic phase was purified by silica gel column chromatography to obtain a yellow oily compound INT-1l (28 g, yield 56.1%). ESI-MS (m / z): 530.7 [M+H] + .

[0067] Step 10: Compound INT-1l (28 g, 52.9 mmol) was dissolved in N,N-dimethylformamide (280 mL), and N-iodosuccinimide (11.9 g, 52.9 mmol) was added. The reaction was carried out at 50 °C for 2 hours. The reaction mixture was monitored by LCMS until the starting material was completely reacted. The reaction solution was poured into water (800 mL), extracted with ethyl acetate (200 mL * 2), washed with saturated brine, dried, filtered, and purified by silica gel column chromatography to obtain a yellow solid compound INT-1m (22 g, yield 63.5%). ESI-MS (m / z): 656.6 [M + H] + .

[0068] Step 11: Compound INT-1m (5.0 g, 7.63 mmol), 2-dicyclohexylphosphine-2′,6′-dimethoxy-biphenyl (939 mg, 2.29 mmol), tris(dibenzylacetone)dipalladium (838 mg, 0.915 mmol), and potassium acetate (2.6 g, 26.7 mmol) were dissolved in toluene (100 mL). Pinara-borane (4.9 g, 38.1 mmol) was added dropwise under nitrogen protection. After the addition was complete, the reaction was carried out at 50 °C for 5 hours under nitrogen protection. The reaction mixture was monitored by LC-MS to ensure complete reaction of the starting material. The reaction solution was filtered and purified by silica gel column chromatography to obtain a yellow oily compound INT-1 (4.5 g, 90% yield). ESI-MS (m / z): 656.5 [M+H] + .

[0069] Intermediate 2

[0070]

[0071] Intermediate 2 is prepared by the following steps:

[0072]

[0073] Step 1: Compound INT-1m (12 g, 18.3 mmol) was dissolved in tetrahydrofuran (120 mL) and water (20 mL). Lithium hydroxide monohydrate (3.84 g, 91.5 mmol) was added, and the mixture was reacted overnight at room temperature. The reaction mixture was monitored by LCMS until the starting material was completely reacted. The reaction solution was directly concentrated under reduced pressure. The residue was dissolved in water (100 mL), and the pH was adjusted to 4–5 with 4 M hydrochloric acid. The mixture was extracted with dichloromethane (100 mL * 3), the organic phase was washed with water and brine, dried over anhydrous sodium sulfate, filtered, and concentrated to give a white solid compound INT-2a (10.6 g, yield 96.6%). ESI-MS (m / z): 600.7 [M + H] + .

[0074] Step 2: Compounds INT-2a (9.5 g, 15.9 mmol) and INT-2b (11.7 g, 31.7 mmol) were dissolved in acetonitrile (190 mL). N,N,N',N'-tetramethylchloromethanesulfonyl hexafluorophosphate (6.67 g, 23.8 mmol) and 1-methylimidazole (6.51 g, 79.2 mmol) were added at 0 °C. The reaction was carried out at 0 °C for 1 hour. LC-MS was used to monitor the reaction until complete. The reaction solution was poured into water (200 mL), extracted with dichloromethane (100 mL x 3), and the organic phase was washed with water. The solution was purified by silica gel column chromatography to obtain a yellow solid compound INT-2c (9.6 g, yield 83.5%). ESI-MS (m / z): 726.3 [M+H] + .

[0075] Step 3: Compound INT-2c (9.6 g, 13.2 mmol) was dissolved in tetrahydrofuran (100 mL) and water (10 mL). Lithium hydroxide monohydrate (1.39 g, 33.1 mmol) was added, and the reaction was carried out at room temperature for 4 hours. The reaction mixture was monitored by LCMS until the starting material was completely reacted. The reaction solution was directly concentrated under reduced pressure, and the residue was dissolved in water (100 mL). The pH was adjusted to 4-5 with 4 M hydrochloric acid, and a white solid precipitated. The solid was filtered, washed with water, and dried to obtain a white solid compound INT-2d (8.3 g, yield 88.2%). ESI-MS (m / z): 712.6 [M+H] + .

[0076] Step 4: Compound INT-2d (3.5 g, 4.9 mmol), 1-hydroxybenzotriazole (1.99 g, 14.8 mmol), and 4-dimethylaminopyridine (1.8 g, 14.8 mmol) were dissolved in dichloromethane (170 mL). N,N-diisopropylethylamine (6 mL, 34.4 mmol) was added at 0 °C, followed by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (4.71 g, 24.6 mmol). The reaction was carried out overnight at room temperature. The reaction mixture was monitored by LCMS to ensure complete reaction. The reaction solution was washed with saturated ammonium chloride aqueous solution, dried over sodium sulfate, and purified by silica gel column chromatography to obtain a yellow solid compound INT-2e (2 g, yield 58.6%). ESI-MS (m / z): 694.6 [M+H] + .

[0077] Step 5: Compound INT-2e (500 mg, 0.721 mmol), 2-dicyclohexylphosphine-2′,6′-dimethylbiphenyl (88.8 mg, 0.216 mmol), tris(dibenzylacetone)dipalladium (79 mg, 0.086 mmol), and potassium acetate (247 mg, 2.52 mmol) were dissolved in tetrahydrofuran (20 mL). Pinara-borane (461 mg, 3.6 mmol) was added dropwise under nitrogen protection. After the addition was complete, the reaction was carried out at 50 °C for 3 hours under nitrogen protection. LC-MS was used to monitor the reaction until complete. The reaction solution was filtered and purified by silica gel column chromatography to obtain a yellow solid compound INT-2 (400 mg, 80% yield). ESI-MS (m / z): 694.6 [M+H] + .

[0078] Intermediate 3

[0079]

[0080] Intermediate 3 is prepared by the following steps:

[0081]

[0082] Step 1: Compound INT-2e (1.7 g, 2.45 mmol) was dissolved in dichloromethane (20 mL), and trifluoroacetic acid (5 mL) was added. The reaction was carried out at room temperature for 2 hours. The reaction mixture was monitored by LCMS until the starting material was completely reacted. The reaction solution was directly concentrated under reduced pressure. The residue was dissolved in DCM (50 mL), washed twice with saturated NaHCO3 aqueous solution, washed with water with the organic phase, dried over sodium sulfate, filtered, and concentrated to give a yellow solid compound INT-3a (1.3 g, yield 89.4%). ESI-MS (m / z): 594.7 [M+H] + .

[0083] Step 2: Compounds INT-3a (1.3 g, 2.19 mmol) and INT-3b (0.24 g, 2.41 mmol) were dissolved in acetonitrile (30 mL). N,N,N',N'-tetramethylchloromethanesulfonyl hexafluorophosphate (922 mg, 3.29 mmol) and 1-methylimidazole (414 mg, 5.04 mmol) were added at 0 °C. The reaction was carried out at 0 °C for 1 hour. LC-MS was used to monitor the reaction until complete. The reaction solution was poured into water (50 mL), extracted with dichloromethane (50 mL x 3), and the organic phase was washed with water. The mixture was then purified by column chromatography to obtain a white solid compound INT-3c (1.3 g, yield 87.9%). ESI-MS (m / z): 675.7 [M+H] + .

[0084] Step 3: Compound INT-3c (1.1 g, 1.63 mmol), 2-dicyclohexylphosphine-2′,6′-dimethylbiphenyl (200 mg, 0.188 mmol), tris(dibenzylacetone)dipalladium (179 mg, 0.195 mmol), and potassium acetate (559 mg, 5.7 mmol) were dissolved in toluene (30 mL). Pinara-borane (1.04 g, 8.14 mmol) was added dropwise under nitrogen protection. After the addition was complete, the reaction was carried out at 50 °C for 3 hours under nitrogen protection. LC-MS was used to monitor the reaction until complete. The reaction solution was filtered and purified by silica gel column chromatography to obtain a yellow solid compound INT-3 (990 mg, 90% yield). ESI-MS (m / z): 676.9 [M+H] + .

[0085] Intermediate 4

[0086]

[0087] Intermediate 4 is prepared by the following steps:

[0088]

[0089] Step 1: Compounds INT-3a (2.2 g, 3.71 mmol) and INT-4a (0.47 g, 4.08 mmol) were dissolved in dichloromethane (50 mL). N,N,N',N'-tetramethylchloromethanesulfonyl hexafluorophosphate (1.56 g, 5.56 mmol) and 1-methylimidazole (0.70 g, 8.53 mmol) were added at 0 °C. The reaction was carried out at 0 °C for 1 hour. LC-MS was used to monitor the reaction until complete. The reaction solution was poured into water (50 mL), extracted with dichloromethane (50 mL x 3), and the organic phase was washed with water. The mixture was then purified by column chromatography to obtain a white solid compound INT-4b (2.3 g, yield 90.0%). ESI-MS (m / z): 690.2 [M+H] + .

[0090] Step 2: Compound INT-4b (2.1 g, 3.05 mmol), 2-dicyclohexylphosphine-2′,6′-dimethylbiphenyl (375 mg, 0.91 mmol), tris(dibenzylacetone)dipalladium (335 mg, 0.365 mmol), and potassium acetate (1.05 g, 10.7 mmol) were dissolved in toluene (30 mL). Pinara-borane (1.95 g, 15.2 mmol) was added dropwise under nitrogen protection. After the addition was complete, the reaction was carried out at 50 °C for 3 hours under nitrogen protection. LC-MS was used to monitor the reaction until complete. The reaction solution was filtered and purified by silica gel column chromatography to obtain a yellow solid compound INT-4 (1.8 g, yield 85.7%). ESI-MS (m / z): 690.3 [M+H] + .

[0091] Intermediate 5

[0092]

[0093] Intermediate 5 is prepared by the following steps:

[0094]

[0095] Step 1: (S)-3-bromo-5-iodo-2-(1-methoxyethyl)pyridine INT-5a (2.0 g, 5.85 mmol) was dissolved in tetrahydrofuran (20 mL), followed by the addition of cuprous iodide (111 mg, 0.585 mmol), bis(triphenylphosphine)palladium dichloride (410 mg, 0.585 mmol), triethylamine (1.18 g, 11.7 mmol), and 4-propynyl-1-morpholine INT-5b (878 mg, 7.02 mmol). The reaction mixture was stirred at room temperature for 3 hours under nitrogen protection. After the reaction was complete, the reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane / ethyl acetate = 1 / 1) to give a pale yellow oily compound INT-5 (1.8 g, yield 90.7%). ESI-MS (m / z): 339.4 [M+H] + .

[0096] Intermediate 15

[0097]

[0098] Intermediate 15 is prepared by the following steps:

[0099]

[0100] Step 1: Compound INT-15a (600 mg, 3.0 mmol) was dissolved in methanol (5 mL), and potassium carbonate (1.25 g, 9.0 mmol) and dimethyl (1.16 g, 6 mmol) phosphonate were added at room temperature. The reaction mixture was stirred at room temperature for 12 h. The reaction was stopped by TLC. Saturated brine was added to the reaction system, and the mixture was extracted with dichloromethane. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain crude compound INT-15b (587 mg, 99% yield).

[0101] Step 2: Compound INT-15b (570 mg, 2.92 mmol) and compound INT-5a (1 g, 2.92 mmol) were dissolved in tetrahydrofuran (8 mL), and bis(triphenylphosphine)palladium dichloride (204 mg, 0.29 mmol), cuprous iodide (56 mg, 0.29 mmol), and triethylamine (591 mg, 5.85 mmol) were added. The reaction system was purged with nitrogen and stirred at room temperature for 8 h. The reaction was confirmed by LCMS. Saturated brine was added to the reaction system, and the mixture was extracted with ethyl acetate. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 5 / 1) to give a yellow oily liquid INT-15 (1.08 g, 90% yield). ESI-MS (m / z): 409.6 [M+H] + .

[0102] Intermediate 16

[0103]

[0104] Intermediate 16 is prepared by the following steps:

[0105]

[0106] By replacing INT-15a in intermediate INT-15 with INT-16a, and using a similar method and reaction steps, compound INT-16 can be obtained. ESI-MS (m / z): 409.5 [M+H] + .

[0107] Intermediate 17

[0108]

[0109] Intermediate 17 is prepared by the following steps:

[0110]

[0111] By replacing INT-15a in intermediate INT-15 with INT-17a, and using a similar method and reaction steps, compound INT-17 can be obtained. ESI-MS (m / z): 409.3 [M+H] + .

[0112] Intermediate 18

[0113]

[0114] Intermediate 18 is prepared by the following steps:

[0115]

[0116] By replacing INT-5b in intermediate INT-5 with INT-18a, and using a similar method and reaction steps, compound INT-18 can be obtained. ESI-MS (m / z): 354.3 [M+H] + .

[0117] Intermediate 19

[0118]

[0119] Intermediate 19 is prepared by the following steps

[0120]

[0121] Step 1: Compound INT-3 (300 mg, 0.44 mmol) was dissolved in a mixed solution of 1,4-dioxane (5 mL) and water (1 mL). Then, INT-18 (170 mg, 0.48 mmol), [1,1'-bis(diphenylphosphine)ferrocene]palladium dichloride (32 mg, 0.04 mmol), and potassium phosphate (188 mg, 0.88 mmol) were added sequentially. The reaction mixture was stirred at 70 °C for 16 hours under nitrogen protection. After the reaction was complete, the reaction solution was filtered through diatomaceous earth, and the concentrated residue was purified by silica gel column chromatography (dichloromethane / methanol = 20 / 1) to give a pale yellow oily compound INT-19a (260 mg, yield 71.2%). ESI-MS (m / z): 823.1 [M+H] + .

[0122] Step 2: Compound INT-19a (260 mg, 0.31 mmol) was dissolved in N,N-dimethylformamide (4 mL), and cesium carbonate (205 mg, 0.63 mmol) and iodoethane (145 mg, 0.93 mmol) were added. The reaction mixture was stirred at room temperature for 16 hours. After the reaction was complete, water (20 mL) was added to the reaction system, and the mixture was extracted with ethyl acetate (30 mL * 2). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to give a pale yellow solid compound INT-19b (110 mg, yield 41.0%). ESI-MS (m / z): 851.2 [M+H] + .

[0123] Step 3: Compound INT-19b (110 mg, 0.13 mmol) was dissolved in methanol (3 mL), and p-toluenesulfonic acid monohydrate (123 mg, 0.65 mmol) was added. The reaction mixture was stirred at room temperature for 3 hours. After the reaction was complete, water (30 mL) was added to the reaction system, and the mixture was extracted with ethyl acetate (30 mL * 2). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to give a pale yellow solid compound INT-19c (80 mg, yield 80.8%). ESI-MS (m / z): 767.5 [M + H] + .

[0124] Step 4: Compound INT-19c (80 mg, 0.10 mmol) was dissolved in dichloromethane (4 mL), and methanesulfonic anhydride (54 mg, 0.31 mmol) and diisopropylethylamine (68 mg, 0.53 mmol) were added. The reaction mixture was stirred at room temperature for 2 hours. After the reaction was complete, dichloromethane (30 mL) was added to the reaction system, and the mixture was washed with water (15 mL * 2) and saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to give a pale yellow solid compound INT-19 (70 mg, yield 79.5%). ESI-MS (m / z): 845.5 [M+H] + .

[0125] Intermediate 33

[0126]

[0127] Intermediate 33 is prepared by the following steps:

[0128]

[0129] Step 1: Compound INT-2 (300 mg, 0.43 mmol) was dissolved in a mixed solution of 1,4-dioxane (5 mL) and water (1 mL). Then, INT-18 (170 mg, 0.48 mmol), [1,1'-bis(diphenylphosphine)ferrocene]palladium dichloride (32 mg, 0.04 mmol), and potassium phosphate (188 mg, 0.88 mmol) were added sequentially. The reaction mixture was stirred at 70 °C for 16 hours under nitrogen protection. After the reaction was complete, the reaction solution was filtered through diatomaceous earth, and the concentrated residue was purified by silica gel column chromatography (dichloromethane / methanol = 20 / 1) to give a pale yellow oil solid INT-33a (210 mg, yield 57%). ESI-MS (m / z): 841.6 [M+H] + .

[0130] Step 2: Compound INT-33a (200 mg, 0.28 mmol) was dissolved in N,N-dimethylformamide (3 mL), and cesium carbonate (232 mg, 0.71 mmol) and iodoethane (185 mg, 1.19 mmol) were added. The reaction mixture was stirred at room temperature for 16 hours. After the reaction was complete, water (20 mL) was added to the reaction system, and the mixture was extracted with ethyl acetate (30 mL * 2). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to give a pale yellow solid INT-33b (200 mg, 95% yield). ESI-MS (m / z): 869.8 [M+H] + .

[0131] Step 3: Compound INT-33b (200 mg, 0.23 mmol) was dissolved in methanol (3 mL), and p-toluenesulfonic acid monohydrate (175 mg, 0.92 mmol) was added. The reaction mixture was stirred at room temperature for 3 hours. After the reaction was complete, water (30 mL) was added to the reaction system, and the mixture was extracted with ethyl acetate (30 mL * 2). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to give a yellow oily compound INT-33c (160 mg, yield 89%). ESI-MS (m / z): 785.6 [M + H] + .

[0132] Step 4: Compound INT-33c (140 mg, 0.18 mmol) was dissolved in tetrahydrofuran (3 mL) and diethyl ether (3 mL), and p-toluenesulfonyl chloride (68 mg, 0.36 mmol) and potassium hydroxide (20 mg, 0.36 mmol) were added. The reaction mixture was stirred at 0 °C for 2 hours. After the reaction was complete, dichloromethane (30 mL) was added to the reaction system, and the mixture was washed with water (15 mL * 2) and saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by preparative thin-layer chromatography (dichloromethane / methanol = 20 / 1) to obtain INT-33 (110 mg, yield 66%). ESI-MS (m / z): 939.9 [M+H] + .

[0133] Intermediate 34

[0134]

[0135] Intermediate 34 is prepared by the following steps:

[0136]

[0137] Step 1: Compound INT-18 (500 mg, 1.41 mmol) was dissolved in methanol (5 mL), and p-toluenesulfonic acid monohydrate (537 mg, 2.82 mmol) was added. The reaction mixture was stirred at room temperature for 3 hours. After the reaction was complete, water (30 mL) was added to the reaction system, and the mixture was extracted with ethyl acetate (30 mL * 2). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to give a pale yellow solid compound INT-34a (210 mg, yield 55%). ESI-MS (m / z): 270.3 [M + H] + .

[0138] Step 2: Compound INT-34a (350 mg, 1.30 mmol) was dissolved in dichloromethane (5 mL), and methanesulfonic anhydride (1.13 g, 6.48 mmol) and diisopropylethylamine (1.34 g, 10.37 mmol) were added. The reaction mixture was stirred at room temperature for 2 hours. After the reaction was complete, water (30 mL) was added to the reaction system, and the mixture was extracted with dichloromethane (30 mL * 2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to give a pale yellow solid compound INT-34b (330 mg, yield 73%). ESI-MS (m / z): 348.2 [M + H] + .

[0139] Step 3: Compound INT-34b (200 mg, 0.58 mmol) and (S)-3-hydroxymethylmorpholine (87 mg, 0.75 mmol) were dissolved in dichloromethane (5 mL), and N,N-diisopropylethylamine (148 mg, 1.15 mmol) was added. The reaction mixture was stirred at room temperature for 4 hours. After the reaction was complete, water (30 mL) was added to the reaction system, and the mixture was extracted with dichloromethane (30 mL * 2). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography (dichloromethane / methanol = 10 / 1) to give a colorless oily liquid INT-34 (180 mg, yield 85%). ESI-MS (m / z): 369.3 [M+H] + .

[0140] Intermediate 35

[0141]

[0142] By replacing (S)-3-hydroxymethylmorpholine in intermediate INT-34 with (S)-octahydropyrazine [2,1-c][1,4]oxazine dihydrochloride, compound INT-35 can be obtained using a similar method and reaction steps. ESI-MS (m / z): 394.5 [M+H] + .

[0143] Intermediate 36

[0144]

[0145] By replacing (S)-3-hydroxymethylmorpholine in intermediate INT-34 with 1-acetylpiperazine, compound INT-36 can be obtained using a similar method and reaction steps. ESI-MS (m / z): 380.4 [M+H] + .

[0146] Intermediate 37

[0147]

[0148] By replacing INT-28a in intermediate INT-28 with N-Boc-2-amino-5-bromopyrimidine, compound INT-37 can be obtained using a similar method and reaction steps. ESI-MS (m / z): 433.4 [M+H] + .

[0149] Intermediate 38

[0150]

[0151] By replacing INT-5b in intermediate INT-5 with 4-propargylthiomorpholine-1,1-dioxide, compound INT-38 can be obtained using a similar method and reaction steps. ESI-MS (m / z): 387.5 [M+H] + .

[0152] Intermediate 45

[0153]

[0154] By replacing (S)-3-hydroxymethylmorpholine in intermediate INT-34 with 1-tert-butyloxycarbonylpiperazine, compound INT-45 can be obtained using a similar method and reaction steps. ESI-MS (m / z): 437.4 [M+H] + .

[0155] The synthesis method of the compounds in the embodiments of this invention is as follows:

[0156] Example 15

[0157] (1S,2S)-N-((6 3 S,4S,Z)-1 1 -ethyl-1 2 -(2-((S)-1-methoxyethyl)-5-(3-((R)-3-methylmorpholino)prop-1-yn-1-yl)pyridin-3-yl)-10,10-dimethyl-5,7-dioxo-6 1 6 2 6 3 6 4 6 5 6 6 -hexahydro-1 1 H-8-oxa-2(4,2)-thiazola-1(5,3)-indola-6(1,3)-pyridazinacycloundecaphane-4-yl)-2-methylcyclopropane-1-carboxamide

[0158]

[0159] Example 15 was prepared by the following steps:

[0160]

[0161] Step 1: Compound INT-19 (20 mg, 0.02 mmol) was dissolved in dichloromethane (5 mL), and (R)-3-methylmorpholine hydrochloride (10 mg, 0.07 mmol) and diisopropylethylamine (18 mg, 0.14 mmol) were added. The reaction mixture was stirred at 50 °C for 16 hours. After the reaction was complete, the reaction solution was concentrated, and the residue was purified by preparative liquid chromatography to give a white solid compound 15 (3.0 mg, 15.0% yield) and its epimer 15' (5.0 mg, 25.0% yield). The absolute configurations of the two compounds are assumed based on experience. In existing analytical methods, 15 is a compound with relatively low polarity and relatively long LC-MS and HPLC retention times, while 15' is a compound with relatively high polarity and relatively short LC-MS and HPLC retention times.

[0162] Compound 15:

[0163] ESI-MS (m / z): 850.6 [M+H] + LC-MS retention time RT = 1.90 min. HPLC retention time RT = 13.50 min.

[0164] 1 H NMR(500MHz,DMSO-d6)δ8.80(d,J=2.0Hz,1H),8.54–8.48(m,2H),7.85(d,J=2.0Hz,1H),7.81(s,1H),7.75(dd,J=8.5,2.0Hz,1H),7.58 (d,J=8.5Hz,1H),5.56(t,J=9.0Hz,1H),5.10–5.05(m,1H),4.38–4.15(m,4H),4.12–4.04(m,1H),3.78–3.61(m,4H),3.58(s,2H),3.52 –3.47(m,1H),3.25(s,3H),3.19–3.02(m,3H),3.00–2.95(m,1H),2.80–2.69(m,2H),2.41–2.36(m,1H),2.11–2.05(m,1H),1.84–1.74( m,2H),1.57–1.46(m,2H),1.35(d,J=6.0Hz,3H),1.07(s,3H),0.94–0.91(m,6H),0.88(t,J=7.0Hz,3H),0.58–0.52(m,1H),0.34(s,3H).

[0165] Compound 15':

[0166] ESI-MS (m / z): 850.6 [M+H] + LC-MS retention time RT = 1.86 min. HPLC retention time RT = 13.14 min.

[0167] 1 H NMR (500MHz, DMSO-d6) δ8.82(d,J=2.0Hz,1H),8.58–8.48(m,2H),7.99(d,J=2.0Hz,1H),7.81(s,1H),7.74(dd,J=8.5,2.0Hz,1H),7.54(d,J=8.5H z,1H),5.55(t,J=9.0Hz,1H),5.10–5.05(m,1H),4.28–4.16(m,2H),4.01 –3.89(m,2H),3.87–3.73(m,3H),3.71–3.60(m,3H),3.57–3.45(m,2H),3 .18–3.12(m,1H),3.09(s,3H),3.07–3.02(m,2H),2.83–2.67(m,2H),2.6 3–2.53(m,2H),2.35–2.30(m,1H),2.15–2.0(m,1H),1.85–1.74(m,2H),1 .58–1.45(m,2H),1.22(d,J=6.0Hz,3H),1.11(t,J=7.0Hz,3H),1.07(s,3 H),0.95–0.91(m,6H),0.90–0.84(m,1H),0.58–0.53(m,1H),0.50(s,3H).

[0168] Example 25

[0169] (1r,2R,3S)-N-((63S,4S,Z)-1 1 -ethyl-1 2 -(5-(3-((S)-3-(hydroxymethyl)morpholino)prop-1-yn-1-yl)-2-((S)-1-methoxyethyl)pyridin-3-yl)-10,10-dimethyl-5,7-dioxo-6 1 6 2 6 3 6 4 6 5 6 6 -hexahydro-1 1H-8-oxa-2(4,2)-thiazola-1(5,3)-indola-6(1,3)-pyridazinacycloundecaphane-4-yl)-2,3-dimethylcyclopropane-1-carboxamide

[0170]

[0171] Compound 25 was obtained by replacing (R)-3-methylmorpholine hydrochloride in the synthesis step of compound 15 with compound (S)-3-hydroxymethylmorpholine, and INT-19 with INT-25, using a similar method and reaction steps. ESI-MS (m / z): 880.6 [M+H] + LC-MS retention time RT = 1.68 min.

[0172] 1 H NMR(500MHz,DMSO-d6)δ8.81(d,J=2.0Hz,1H),8.51–8.48(m,1H),8.39(d,J=8.5Hz,1H),7.85(d,J=2.0Hz,1H),7.81(s,1H),7.77–7.73(m,1H) ,7.58(d,J=8.5Hz,1H),5.55(t,J=9.0Hz,1H),5.10–5.02(m,1H),4.60( t,J=5.5Hz,1H),4.38–4.04(m,5H),3.83–3.78(m,2H),3.76–3.71(m,2H) ),3.62–3.56(m,3H),3.25(s,3H),3.17–3.11(m,2H),2.99–2.93(m,1H) ,2.79–2.68(m,2H),2.64–2.59(m,2H),2.42–2.35(m,1H),2.11–2.04(m ,1H),1.83–1.75(m,2H),1.58–1.44(m,2H),1.36(d,J=6.0Hz,3H),1.25 –1.15(m,5H),1.11–1.04(m,6H),0.94–0.87(m,6H),0.39–0.31(s,3H).

[0173] Example 36

[0174] (1r,2R,3S)-N-((6 3 S,4S,Z)-1 1 -ethyl-1 2 -(2-((S)-1-methoxyethyl)-5-(3-

[0175] (methyl(tetrahydro-2H-pyran-4-yl)amino)prop-1-yn-1-yl)pyridin-3-yl)-10,10-dimethyl-5,7-dioxo-6 1 6 2 6 3 6 4 6 5 6 6 -hexahydro-1 1 H-8-oxa-2(4,2)-thiazola-1(5,3)-indola-6(1,3)-

[0176] pyridazinacyclodecaphane-4-yl)-2,3-dimethylcyclopropane-1-carboxamide

[0177]

[0178] Example 36 was prepared by the following steps:

[0179]

[0180] Step 1: Compound INT-33 (90 mg, 0.10 mmol) was dissolved in dichloromethane (5 mL), and N-methyltetrahydro-2H-pyran-4-amine (55 mg, 0.48 mmol) and diisopropylethylamine (62 mg, 0.48 mmol) were added. The reaction mixture was stirred at room temperature for 16 hours. The reaction was confirmed to be complete by LCMS. Water (20 mL) was added to the reaction mixture, and the mixture was extracted with dichloromethane (20 mL x 2). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by preparative thin-layer chromatography (dichloromethane / methanol = 20 / 1) to give compound 36a (60 mg, 71% yield). ESI-MS (m / z): 882.7 [M+H] + ;

[0181] Step 2: Compound 36a (50 mg, 0.06 mmol) was dissolved in dichloromethane (2 mL), and trifluoroacetic acid (0.5 mL) was added. The reaction mixture was stirred at 0 °C for 1 hour. The reaction was confirmed to be complete by LCMS. A saturated sodium bicarbonate solution (10 mL) was added to the reaction mixture under ice bath conditions, followed by extraction with dichloromethane (20 mL x 2). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain crude compound 36b (37 mg, 83% yield). ESI-MS (m / z): 782.8 [M+H] + ;

[0182] Step 3: Compound 36b (37 mg, 0.05 mmol) was dissolved in acetonitrile (2 mL), and INT-4a (5 mg, 0.05 mmol), N,N-diisopropylethylamine (30 mg, 0.24 mmol), N-methylimidazole (6 mg, 0.07 mmol), and (2-oxime-cyanoethyl acetate)-N,N-dimethylmorpholinourea hexafluorophosphate (19 mg, 0.07 mmol) were added. The reaction mixture was stirred in an ice bath for 1 hour. After the reaction was complete, water (20 mL) was added to the reaction system, and the mixture was extracted with dichloromethane (20 mL * 2). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by preparative liquid chromatography to give a white solid compound 36 (3.0 mg, yield 7%). ESI-MS (m / z): 878.6 [M+H] + LC-MS retention time RT = 1.85 min. HPLC retention time RT = 13.67 min.

[0183] 11H NMR (500 MHz, DMSO-d6) δ 8.80 (d, J = 2.0 Hz, 1H), 8.52–8.48 (m, 1H), 8.39 (d, J = 9.0 Hz, 1H), 7.85–7.80 (m, 2H), 7.77–7.73 (m, 1H), 7.58 (d, J = 8.5 Hz, 1H), 5.56 (t, J = 9.0 Hz, 1H), 5.10–5.04 (m, 1H), 4.39–4.02 (m, 5H), 3.91–3.84 (m, 2H), 3.70–3.65 (m, 2H), 3.58 (s, 2H), 3.30–3.29 (m, 2H), 3.25 (s, 3H), 3.18–3.11 (m, 2H), 2.98–2.91 (m, 1H), 2.79–2.71 (m, 1H), 2.42–2.35 (m, 2H), 2.32 (s, 3H), 2.14–2.02 (m, 2H), 1.82–1.74 (m, 4H), 1.55–1.46 (m, 1H), 1.43–1.32 (m, 5H), 1.18–1.1 (m, 2H), 1.10–1.04 (m, 6H), 0.93–0.84 (m, 6H), 0.35 (s, 3H).

[0184] Example 38

[0185] (1r,2R,3S)-N-((6 3 S,4S,Z)-1 1 -ethyl-1 2 -(5-(3-((R)-3-(hydroxymethyl)morpholino)prop-1-yn-1-yl)-2-((S)-1-methoxyethyl)pyridin-3-yl)-10,10-dimethyl-5,7-dioxo-6 1 ,6 2 ,6 3 ,6 4 ,6 5 ,6 6 -hexahydro-1 1 H-8-oxa-2(4,2)-thiazola-1(5,3)-indola-6(1,3)-pyridazinacycloundecaphane-4-yl)-2,3-dimethylcyclopropane-1-carboxamide

[0186]

[0187] By replacing N-methyltetrahydro-2H-pyran-4-amine in Example 36 with (R)-3-hydroxymethylmorpholine, compound 38 can be obtained using a similar method and reaction steps. ESI-MS (m / z): 881.1 [M+H] + LC-MS retention time RT = 1.67 min.

[0188] 1 H NMR(500MHz,DMSO-d6)δ8.81(d,J=2.0Hz,1H),8.51–8.49(m,1H),8.39(d,J=9.0Hz,1H),7.85(d,J=2.0Hz,1H),7.82(s,1H),7.77–7.73(m,1H),7 .58(d,J=8.5Hz,1H),5.56(t,J=9.0Hz,1H),5.09–5.03(m,1H),4.59(t, J=5.5Hz,1H),4.38–4.03(m,5H),3.87–3.66(m,4H),3.63–3.55(m,3H),3 .51–3.43(m,1H),3.31–3.28(m,2H),3.25(s,3H),3.22–3.07(m,2H),2. 99–2.93(m,1H),2.78–2.68(m,2H),2.64–2.57(m,1H),2.56–2.52(m,1H) ,2.42–2.35(m,1H),2.11-2.04(m,1H),1.83–1.74(m,2H),1.57–1.46(m ,1H),1.36(d,J=6.0Hz,3H),1.24–1.15(m,4H),1.10–1.04(m,6H),0.95–

[0189] 0.85 (m, 6H), 0.35 (s, 3H).

[0190] Example 58

[0191] (1r,2R,3S)-N-((6 3 S,4S,Z)-1 2 -(5-(3-(dimethylamino)prop-1-yn-1-yl)-2-((S)-1-

[0192] methoxyethyl)pyridin-3-yl)-1 1 -ethyl-10,10-dimethyl-5,7-dioxo-6 1 6 2 6 3 6 46 5 6 6 -hexahydro-1 1 H-8-oxa-2(4,2)-thiazola-1(5,3)-indola-6(1,3)-pyridazinacycloundecaphane-4-yl)-2,3-dimethylcyclopropane-1-carboxamide

[0193]

[0194] By replacing N-methyltetrahydro-2H-pyran-4-amine in the synthesis of compound 36 with dimethylamine, compound 58 can be obtained using a similar method and reaction steps. ESI-MS (m / z): 808.6 [M+H] + LC-MS retention time RT = 1.96 min. HPLC retention time RT = 14.23 min.

[0195] 1 H NMR (500MHz, DMSO-d6) δ8.81(d,J=2.0Hz,1H),8.53–8.47(m,1H),8.41(d,J=9.0Hz,1H),7.88–7.81(m,2H),7.76(dd,J=8.5,1.5H z,1H),7.59(d,J=8.5Hz,1H),5.55(t,J=9.0Hz,1H),5.13–5.04(m,1H),4.36–4.07(m,5H),3.57(s,2H),3.51(s,2H),3.32–3.29(m ,1H),3.25(s,3H),3.17–3.11(m,1H),2.99–2.93(m,1H),2.80–2.72(m,1H),2.41–2.33(m,2H),2.26(s,6H),2.11–2.03(m,1H),1 .82–1.74(m,2H),1.56–1.46(m,1H),1.35(d,J=6.0Hz,3H),1.17–1.14(m,2H),1.09–1.05(m,6H),0.91–0.83(m,6H),0.34(s,3H).

[0196] Biological screening and results of RAS inhibitors

[0197] Experimental Example 1: In vitro cell proliferation inhibition assay

[0198] Due to the diversity of RAS mutations, and in order to evaluate the activity of compounds in different RAS mutant cell lines, we selected KRAS.WT KRAS G12C KRAS G12D KRAS G12V In vitro activity assessment and screening of compounds were performed using BRAF-mutant cell lines (see table below).

[0199]

[0200]

[0201] Experimental plan: Cell Luminescent Viability Assay(Promega)

[0202] Depending on the doubling time of different cell lines, varying numbers of cells (1000-5000 cells / well) were seeded into 96-well plates containing 180 μl of the corresponding culture medium and cultured overnight in a 37°C cell culture incubator containing 5% CO2. The next day, the test compound was pre-diluted 3-fold serially with the culture medium, with a maximum concentration of 100 μM, for a total of 10 concentration gradients. Then, 20 μl of culture medium containing different concentrations of the compound was added to the cells in the 96-well plates, ensuring the final concentration of the compound was at a maximum of 10 μM, representing 10 concentration gradients of 3-fold dilutions. After co-incubating the cells and compound for 72 h, the 96-well plates were removed from the incubator and equilibrated at room temperature for 30 min. Then, 25 μl of CellTiter- (the culture medium was added to each well) was added. Mix the Reagent thoroughly and incubate at room temperature for 10 min. Then transfer 100 μl of sample to a white 96-well plate (OptiPlate). TM -96, PerkinElmer), using a multi-functional microplate reader ( The i3x (Molecular Devices) were used to read the fluorescence signal values. The signal values ​​were then standardized, and a four-parameter regression equation was used to fit a curve to calculate the half maximal inhibitory concentration (IC50) of the compound on the cell line.

[0203] Table 3: Antiproliferative activity of the compounds of this invention against KRAS cell mutants

[0204]

Claims

1. An intermediate compound for synthesizing a KRAS inhibitor compound, having the structure shown in formula INT-33: INT-33.

2. The method for synthesizing the intermediate compound INT-33 as described in claim 1, comprising the following steps: Step 1: After dissolving compound INT-2, add INT-18, [1,1'-bis(diphenylphosphine)ferrocene]palladium dichloride and potassium phosphate in sequence; after the reaction is complete, filter and purify to obtain INT-33a; Step 2: After dissolving compound INT-33a, cesium carbonate and iodoethane were added to it; after the reaction was complete, it was extracted and purified to obtain INT-33b; Step 3: After dissolving compound INT-33b, p-toluenesulfonic acid monohydrate was added to it; after the reaction was complete, the compound INT-33c was obtained by extraction and purification. Step 4: After dissolving compound INT-33c, p-toluenesulfonyl chloride and potassium hydroxide are added to it; after the reaction is complete, INT-33 is obtained by purification.

3. The synthesis method according to claim 2, wherein the synthesis of compound INT-2 comprises the following steps: Step 1: After dissolving compound INT-1m, lithium hydroxide monohydrate was added; after the reaction was complete, compound INT-2a was obtained by extraction and purification. Step 2: After dissolving compounds INT-2a and INT-2b, N,N,N',N'-tetramethylchloromethamidine hexafluorophosphate and 1-methylimidazole were added at 0 °C. After the reaction was complete at 0 °C, the compounds were extracted and purified to obtain the compounds. Step 3: After dissolving compound INT-2c, add lithium hydroxide monohydrate; after the reaction is complete, precipitate and purify to obtain compound INT-2d; Step 4: After dissolving compound INT-2d, 1-hydroxybenzotriazole, and 4-dimethylaminopyridine, N,N-diisopropylethylamine was added at 0°C, followed by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; after the reaction was completed, the reaction solution was washed with ammonium chloride aqueous solution to purify and obtain compound INT-2e. Step 5: After dissolving compound INT-2e, 2-dicyclohexylphosphine-2′,6′-dimethylbiphenyl, tris(dibenzylacetone)dipalladium and potassium acetate, pinacol borane was added under nitrogen protection. After the addition was complete, the reaction was carried out at 50 °C under nitrogen protection. After the reaction was completed, the compound INT-2 was obtained by filtration and purification.

4. The synthesis method according to claim 2, wherein the synthesis of compound INT-18 comprises the following steps: After dissolving (S)-3-bromo-5-iodo-2-(1-methoxyethyl)pyridine INT-5a, cuprous iodide, palladium dichloride of bis(triphenylphosphine), triethylamine and INT-18a were added sequentially; the reaction mixture was carried out under nitrogen protection; after the reaction was complete, the compound INT-5 was obtained by purification.

5. A KRAS inhibitor compound selected from the group consisting of: 。

Images