KRAS small molecule fluorescent probe and preparation method and application thereof

Abstract

The application discloses a KRAS small-molecule fluorescent probe and a preparation method and application thereof, a structural general formula of the KRAS small-molecule fluorescent probe is shown as formula (I), and the fluorescence polarization activity determination method based on the small-molecule fluorescent probe has the advantages of simple test system, less required protein types, low dosage, no need of a complex buffer system, small influence of time and solvent and the like compared with HTRF. The method can be used for screening of KRAS wild type and various mutant inhibitors including but not limited to G12C, G12V, G12D, G12A, G12S and G12R, and has a good application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to a KRAS small molecule fluorescent probe and a preparation method and application thereof, and belongs to the technical field of chemical biology. BACKGROUND

[0002] RAS is the most frequently mutated oncogene in human cancer, with about 30% mutations in all cancers. In 1982, three laboratories discovered the first confirmed human oncogene RAS and cloned three variants: KRAS, HRAS and NRAS, of which KRAS is the most frequently mutated oncogene, occurring in about one-fourth of human tumors. Its mutation rate in lung cancer, pancreatic cancer and colorectal cancer is about 30%, 90% and 40% respectively, and is associated with poor prognosis. Taking lung cancer as an example, 85% of KRAS mutations are G12 mutations. Among them, G12C, G12V, G12D mutations account for about 46%, 23%, and 17% respectively (J. Med. Chem. 2020, 63(23), 14404-14424).

[0003] Currently, research on KRAS G12C mutant has made some achievements, but people soon realized that targeting mutant KRAS is not as simple as initially thought. First, Sotorasib is a specific inhibitor of KRAS G12C, while G12C is only one of many KRAS mutants. Another common mutation, such as G12D, is found in most pancreatic tumors. Compared with the more studied KARS G12C mutant, the study of G12D mutant is relatively less. But G12D also has a very high mutation frequency in various tumors, with a mutation frequency of up to 49%, 35%, and 48% in pancreatic cancer, colorectal cancer, and biliary cancer, respectively, making it a potential target for treating solid tumors. In addition, the MAPK pathway including KRAS, HRAS and NRAS has different repeated signals and feedback mechanisms, which make the human body very easy to produce drug resistance. In clinical trials, the remission rates of Sotorasib and Adagasib are not more than 30-40%, and the median improvement in progression-free survival (PFS) is about 6 months. For specific treatment targeting mutations, this is not an ideal data, nor is it as observed for other targeted drugs such as EGFR and ALK inhibitors.

[0004] The most representative KRAS G12D inhibitor at present is MRTX-1133 which is currently in clinical phase II study. MRTX1133 is a non-covalent, potent and selective KRAS G12D inhibitor. It can prevent SOS1 catalyzed nucleotide exchange and the formation of KRAS G12D / GTP / RAF1 complex, thereby inhibiting mutant KRAS-dependent signal transduction. MRTX1133 has single-digit nanomolar activity in cell experiments, has significant in vivo efficacy in tumor models containing KRAS G12D mutation, and has good selectivity with more than 500-fold selectivity for G12D over wild type (J. Med. Chem. 2021, 65(4), 3123-3133).

[0005] Therefore, the development of in vitro KRAS inhibition activity test methods and high-throughput screening methods is crucial for the development of small molecule inhibitors thereof. Accurate, stable and reliable activity test methods help to discover novel lead compounds, guide the structural optimization of lead compounds to obtain more active candidate drug molecules, and lay the foundation for the development of therapeutic drugs for lung cancer, pancreatic cancer and colorectal cancer and other diseases targeting KRAS.

[0006] The most commonly used in vitro target activity test method for KRAS at present is HTRF (homogeneous time-resolved fluorescence) which is a commonly used method for detecting substances in a homogeneous system and is an ideal platform for studying drug targets (J. Med. Chem. 2021, 65(4), 3123-3133). This technology combines fluorescence resonance energy transfer (FRET) and time-resolved technology (TR). In TR-FRET experiments, when the donor and acceptor are very close, there will be fluorescence resonance energy transfer between the donor and acceptor to generate a signal. Dual-wavelength detection can significantly reduce the interference of buffers and culture media, and the final signal is proportional to the amount of product formed. Although this technology has the advantages of high sensitivity, stable signal, and can be used for high-throughput screening, its test system and operation process are relatively complex, and the test cost is relatively high.

[0007] Fluorescence polarization (FP) assay is a fluorescence-labeled detection technique that labels fluorescent substances on specific substances, converts the originally weak reaction signal into a stronger fluorescent signal, and plays a signal enhancement role. Macromolecules (such as proteins) in solution are relatively slow in rotation due to their large volume, and emit polarized light when excited by polarized light; the rapid rotation of small molecules will lead to signal depolarization. The emission system uses a polarizing filter to analyze the polar of the emitted light, and low polarization level indicates that fluorescent small molecules are free to move in the sample. High polarization level indicates that fluorescent molecules are attached to larger molecular complexes (J. Med. Chem. 2023, 66(16), 10934-10958). Therefore, it is urgent to break through the limitations of the current KRAS inhibition activity test method. SUMMARY

[0008] The first object of the present application is to provide a KRAS small molecule fluorescent probe. The second object of the present application is to provide a preparation method of the KRAS small molecule fluorescent probe. The third object of the present application is to provide the application of the KRAS small molecule fluorescent probe as a fluorescence polarization probe in KRAS protein ligand screening.

[0009] Technical scheme: The KRAS small molecule fluorescent probe or its pharmaceutically acceptable salt or carrier provided by the present application, the structure general formula of the KRAS small molecule fluorescent probe is as shown in formula (I):

[0010]

[0011] Among them, R 1 is H or halogen.

[0012] Further, the KRAS small molecule fluorescent probe is selected from:

[0013]

[0014] The preparation method of the KRAS small molecule fluorescent probe or its pharmaceutically acceptable salt or carrier provided by the present application comprises the following synthesis route:

[0015]

[0016] Among them, R 1 is H or halogen.

[0017] Further, the preparation method comprises the following steps:

[0018] (A1) Compound 1 is deprotected in an organic solvent under acidic conditions to obtain compound 2;

[0019] (A2) Compound 2 and compound 3 are reacted under alkaline conditions to obtain the probe;

[0020] Furthermore, in step (A1), the organic solvent used is methanol or dioxane, and the acid is trifluoroacetic acid or hydrochloric acid.

[0021] Furthermore, in step (A1), the reaction temperature is 60-70℃ and the reaction time is 3-4h.

[0022] Furthermore, in step (A2), the organic solvent used for the addition reaction is methanol or dichloromethane, and the base used is DIPEA or triethylamine.

[0023] Furthermore, in step (A2), the reaction temperature is 20-30℃ and the reaction time is 1-2h.

[0024] Furthermore, in step (A1), compound 1 is synthesized according to the following route:

[0025]

[0026] Among them, R 1 It is H or halogen.

[0027] Furthermore, it includes the following steps:

[0028] (B1)(R)-1-(2,7-dichloro-8-fluoropyridino[4,3-d]pyrimidin-4-yl)-3-methylpiperidin-3-ol, (3R,5S)-5-(hydroxymethyl)-1-methylpyrrolidine-3-yl(3-(1-(2-(((benzyloxy)carbonyl)amino)ethyl)-1H-1,2,3-triazol-4

[0029] 1-(propyl)carbamate, cesium carbonate, dissolved in dioxane, reacted under nitrogen protection by heating, extracted with ethyl acetate, dried over anhydrous sodium sulfate, concentrated, and separated by column chromatography to obtain the intermediate;

[0030] (B2) Intermediate, Cesium carbonate and tetra-triphenylphosphine palladium were dissolved in a mixed solvent of dioxane and water, and reacted under nitrogen protection by heating. The mixture was then extracted with dichloromethane, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography to obtain compound 1.

[0031] Furthermore, in step (B1), the temperature of the heating reaction is 95-105℃, and the heating reaction time is 8-10h.

[0032] Furthermore, in step (B2), the temperature of the heating reaction is 70-80℃, and the heating reaction time is 6-8h.

[0033] The present invention relates to the application of the KRAS small molecule fluorescent probe or its pharmaceutically acceptable salt or carrier as a fluorescent polarization probe in the screening of KRAS protein ligands.

[0034] The present application also includes a method for screening KRAS protein ligands, comprising the following steps:

[0035] (C1) dissolving the KRAS small molecule fluorescent probe or its pharmaceutically acceptable salt or carrier according to the present application, KRAS protein and the test compound in a buffer solution and incubating;

[0036] (C2) using a fluorescence polarization enzyme marker to determine the polarization value of the mixture under the excitation wavelength of 485 nm and the emission wavelength of 535 nm of the KRAS small molecule fluorescent probe or its pharmaceutically acceptable salt or carrier according to the present application, and confirming the affinity of the test compound to the KRAS protein according to the polarization value.

[0037] Further, the buffer solution is a PBS buffer solution, a Tris buffer solution or a Hepes buffer solution.

[0038] Further, the KRAS protein includes but is not limited to KRAS wild type, G12C, G12V, G12D, G12A, G12S, G12R and the like mutants.

[0039] The present application also includes a kit, which comprises the KRAS small molecule fluorescent probe or its pharmaceutically acceptable salt or carrier according to the present application.

[0040] In order to break through the limitations of the current KRAS inhibition activity test method, the present application uses a small molecule pan-inhibitor skeleton as a binding fragment of KRAS protein, and by further introducing a connecting chain and a fluorescent group, a small molecule fluorescent polarization probe based on KRAS inhibitor design is designed and synthesized. Compared with HTRF, the activity determination method based on the small molecule fluorescent probe has the advantages of simple test system, no need for complex buffer system, small influence of time and solvent, etc., and can be used for screening of KRAS wild type and G12C, G12V, G12D, G12A, G12S, G12R and the like mutants inhibitors. Based on the principle of fluorescence polarization, a stable, rapid and inexpensive in vitro KRAS protein inhibition activity detection method is established. The activity determination method based on the small molecule fluorescent polarization probe has the advantages of simple test system, no need for complex buffer system, small influence of time and solvent, etc. At the same time, by testing the reported small molecule inhibitor BI-2865 (Nature 2023, 619, 160-166), the applicability and accuracy of this method are verified. It can be used for screening of KRAS inhibitors.

[0041] Advantages: Compared with the prior art, the present application has the following obvious advantages:

[0042] The application is based on the small molecule fluorescent probe to establish a fluorescence polarization activity determination method, compared with HTRF, the test system is simple: the required protein types are few, the amount is low; no complex buffer system is needed; the influence of time and solvent is small and the like. It can be used for screening of KRAS wild type and G12C, G12V, G12D, G12A, G12S, G12R and various mutant inhibitors, and has good application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0043] Figure 1 The fluorescence polarization value (mP) and the concentration of the fluorescent probe I after incubation of the fluorescent probe I and KRAS protein are shown in the following figure: G12D The fluorescence polarization value (mP) and the concentration of the fluorescent probe I after incubation of the fluorescent probe I and KRAS protein are shown in the following figure:

[0044] Figure 2 The inhibition rate of the positive drug BI-2865 on KRAS protein is shown in the following figure:

[0045] Figure 3 The results of the investigation of the experimental stability Z' factor are shown in the following figure. DETAILED DESCRIPTION

[0046] The technical solutions of the application will be further described below in combination with the drawings.

[0047] Example 1: Preparation of the fluorescent probe I

[0048] The fluorescent probe I is synthesized according to the following route:

[0049]

[0050] Step 1: Preparation of intermediate 2

[0051] Compound 1, (R)-1-(2,7-dichloro-8-fluoropyrido[4,3-d]pyrimidin-4-yl)-3- methylpiperidin-3-ol (66 mg, 0.2 mmol, CAS No.: 2791271-47-9), (3R,5S)-5- (hydroxymethyl)-1-methylpyrrolidin-3-yl (3-(1-(2-(((benzyloxy)carbonyl)amino)ethyl)- 1H-1,2,3-triazol-4-yl)propyl)carbamate (92 mg, 0.2 mmol, CAS No.: 3063554-97-9) and cesium carbonate (130 mg, 0.4 mmol) were dissolved in 3 ml of dioxane, heated to 100 °C under nitrogen protection for 8 h. After the reaction was completed, the reaction solution was concentrated, quenched with water (20 mL), extracted with ethyl acetate (10 mL) for 3 times, the combined organic phase was dried with anhydrous sodium sulfate, concentrated, and separated and purified by column chromatography (dichloromethane:methanol = 20:1) to obtain intermediate 2, (3R,5S)-5-(((7-chloro-8-fluoro-4-((R)-3-hydroxy-3- methylpiperidin-1-yl)pyrido[4,3-d]pyrimidin-2-yl)oxy)methyl)-1-methylpyrrolidin-3-yl (3-(1- (2-(((benzyloxy)carbonyl)amino)ethyl)-1H-1,2,3-triazol-4-yl)propyl)carbamate, white solid powder 65 mg, yield 43.0%.

[0052] Intermediate 2 was analyzed by nuclear magnetic hydrogen spectrum and mass spectrum, and the results were as follows:

[0053] 1H NMR (400 MHz, Chloroform-d) δ 8.59 (s, 2H), 7.61 (s, 2H), 7.38 - 7.26 (m, 10H), 5.80 (t, J = 6.5 Hz, 2H), 5.50 (t, J = 7.1 Hz, 2H), 5.13 - 5.06 (m, 3H), 5.06 - 5.02 (m, 1H), 4.78 - 4.69 (m, 4H), 4.43 (dt, J = 12.5, 7.1 Hz, 2H), 4.31 (dt, J = 12.5, 7.1 Hz, 2H), 4.15 - 4.05 (m, 4H), 3.74 - 3.67 (m, 3H), 3.67 - 3.63 (m, 1H), 3.63 - 3.55 (m, 4H), 3.55 - 3.46 (m, 4H), 3.46 - 3.37 (m, 2H), 3.33 - 3.16 (m, 4H), 2.96 (dd, J = 12.4, 7.0 Hz, 2H), 2.90 - 2.80 (m, 5H), 2.80 - 2.75 (m, 1H), 2.35 (d, J = 1.4 Hz, 6H), 2.10 - 2.01 (m, 2H), 2.01 - 1.92 (m, 6H), 1.92 - 1.85 (m, 4H), 1.85 - 1.76 (m, 2H), 1.70 (dt, J = 12.3, 7.0 Hz, 2H), 1.34 (s, 6H).

[0054] ESI-MS [M+H] + : 755.2

[0055] From the above results, the structure of intermediate 2 is as follows:

[0056]

[0057] Step 2: Preparation of intermediate 3

[0058] Intermediate 2 (151 mg, 0.2 mmol), 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)naphthalen-2-ol (65 mg, 0.24 mmol, CAS No.: 2043962-01-0), cesium carbonate (130 mg, 0.4 mmol) and tetrakis(triphenylphosphine) palladium (7 mg, 0.006 mmol) were dissolved in 5 ml mixed solvent (dioxane: water = 10:1) and heated to 70 °C for 6 h under nitrogen protection. After the reaction was completed, water (20 mL) was added to quench, extracted with dichloromethane (20 mL) for 3 times, the combined organic phase was dried over anhydrous sodium sulfate, concentrated, and separated and purified by column chromatography (dichloromethane:methanol = 15:1) to obtain intermediate 3 (3R,5S)-5-(((8-fluoro-4-((R)-3-hydroxy-3-methylpiperidin-1-yl)-7-(3-hydroxynaphthalen-1-yl)pyrido[4,3-d]pyrimidin-2-yl)oxy)methyl)-1-methylpyrrolidin-3-yl (3-(1-(2-(((benzyloxy)carbonyl)amino)ethyl)-1H-1,2,3-triazol-4-yl)propyl)carbamate, white solid 90 mg, yield 47.5%.

[0059] Intermediate 3 was analyzed by nuclear magnetic hydrogen spectrum and mass spectrum, and the results were as follows:

[0060] 1H NMR (400 MHz, Chloroform-d) δ 9.03 (s, 2H), 8.74 (s, 2H), 7.84 - 7.75 (m, 4H), 7.61 (s, 2H), 7.43 (dtd, J = 20.4, 7.4, 1.6 Hz, 4H), 7.37 - 7.30 (m, 11H), 7.30 - 7.27 (m, 1H), 7.23 (t, J = 1.7 Hz, 2H), 5.80 (t, J = 6.5 Hz, 2H), 5.50 (t, J = 7.1 Hz, 2H), 5.10 (dt, J = 12.3, 0.9 Hz, 2H), 5.05 (dt, J = 12.5, 0.9 Hz, 2H), 4.76 (pd, J = 7.1, 0.9 Hz, 2H), 4.72 (s, 2H), 4.43 (dt, J = 12.5, 7.1 Hz, 2H), 4.31 (dt, J = 12.4, 7.1 Hz, 2H), 4.15 - 4.05 (m, 4H), 3.74 - 3.67 (m, 3H), 3.67 - 3.62 (m, 1H), 3.62 - 3.55 (m, 4H), 3.55 - 3.46 (m, 4H), 3.46 - 3.37 (m, 2H), 3.33 - 3.16 (m, 4H), 2.95 (dd, J = 12.5, 6.9 Hz, 2H), 2.90 - 2.81 (m, 4H), 2.81 - 2.75 (m, 2H), 2.35 (d, J = 1.5 Hz, 6H), 2.10 - 1.76 (m, 14H), 1.70 (dt, J = 12.3, 7.0 Hz, 2H), 1.34 (s, 6H).

[0061] ESI-MS [M+H] + : 863.5

[0062] From the above results, the structure of intermediate 3 is as follows:

[0063]

[0064] Step 3: Preparation of intermediate 4

[0065] Intermediate 3 (86 mg, 0.1 mmol) was dissolved in 5 mL saturated hydrochloric acid in dioxane solution, heated to 60 °C for 4 h, the reaction was filtered, the filter cake was washed with ethyl acetate (20 mL) for 2 times, dried to obtain light yellow solid product intermediate 4 (3R,5S)-5-(((4-((1R,5S)-3,8-diazabicyclo[3.2.1]octan-3-yl)-8-fluoro-7-(3-hydroxynaphthalen-1-yl)pyrido[4,3-d]pyrimidin-2-yl)oxy)methyl)-1-methylpyrrolidin-3-yl (3-(1-(2-aminoethyl)-1H-1,2,3-triazol-4-yl)propyl)carbamate, 68 mg, yield 89.1%.

[0066] The intermediate 4 was analyzed by nuclear magnetic hydrogen spectrum, and the results were as follows:

[0067] 1 H NMR (400 MHz, Chloroform-d) δ 9.03 (s, 1H), 8.74 (s, 1H), 7.96-7.89 (m, 1H), 7.80-7.74 (m, 1H), 7.61 (s, 1H), 7.47-7.39 (m, 2H), 7.37 (d, J = 1.6 Hz, 1H), 7.24 (t, J = 1.6 Hz, 1H), 5.50 (t, J = 7.1 Hz, 1H), 4.80-4.70 (m, 2H), 4.32 (dt, J = 12.4, 7.1 Hz, 1H), 4.24 (dt, J = 12.5, 7.1 Hz, 1H), 4.15-4.05 (m, 2H), 3.74-3.63 (m, 2H), 3.60 (d, J = 12.5 Hz, 1H), 3.50 (d, J = 12.4 Hz, 1H), 3.47-3.37 (m, 1H), 3.34-3.16 (m, 4H), 2.96 (dd, J = 12.5, 7.0 Hz, 1H), 2.90-2.75 (m, 3H), 2.37-2.29 (m, 5H), 2.10-2.02 (m, 1H), 2.02-1.92 (m, 3H), 1.92-1.81 (m, 2H), 1.79 (t, J = 7.0 Hz, 1H), 1.70 (dt, J = 12.3, 7.0 Hz, 1H), 1.34 (s, 3H).

[0068] ESI-MS [M+H] + : 729.3

[0069] From the above results, the structure of intermediate 4 is as follows:

[0070]

[0071] Step 4: Preparation of probe I

[0072] Intermediate 4 (36 mg, 0.05 mmol), 3',6'-dihydroxy-5-isothiocyanato-3H- spiro[isobenzofuran-1,9'-xanthene]-3-one (19 mg, 0.05 mmol, CAS No.: 3326-32-7) and DIPEA (13 mg, 0.1 mmol) were dissolved in 7 ml of methanol and reacted at room temperature for 1 h. After the reaction was completed, the reaction solution was concentrated, and HPLC (MeOH:H2O = 40%-65%) was used to separate to obtain probe I (3R,5S)-5-(((8-fluoro-4-((R)-3-hydroxy-3-methylpiperidin-1-yl)-7-(3-hydroxynaphthalen-1-yl)pyrido[4,3-d]pyrimidin-2-yl)oxy)methyl)-1-methylpyrrolidin-3-yl (3-(1-(2-(3-(3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-5-yl)thioureido)ethyl)-1H-1,2,3-triazol-4-yl)propyl)carbamate, a dark yellow solid 8 mg, yield 14.3%.

[0073] The probe I was analyzed by nuclear magnetic hydrogen spectrum and mass spectrum, and the results were as follows:

[0074] 1H NMR (400 MHz, Chloroform-d) δ 9.41 (s, 1H), 9.03 (s, 1H), 8.99 (s, 2H), 8.74 (s, 1H), 8.22 (d, J = 1.5 Hz, 1H), 7.83 - 7.74 (m, 3H), 7.63 - 7.56 (m, 2H), 7.48 - 7.38 (m, 3H), 7.35 (d, J = 1.4 Hz, 1H), 7.23 (t, J = 1.7 Hz, 1H), 7.15 (d, J = 7.5 Hz, 2H), 6.62 - 6.55 (m, 4H), 5.48 (t, J = 7.2 Hz, 1H), 4.76 (pd, J = 7.1, 0.9 Hz, 1H), 4.72 (s, 1H), 4.44 (dt, J = 12.2, 7.1 Hz, 1H), 4.32 (dt, J = 12.3, 7.1 Hz, 1H), 4.14 - 4.08 (m, 2H), 3.83 (m, 2H), 3.68 (td, J = 7.1, 5.3 Hz, 2H), 3.65 - 3.57 (m, 1H), 3.50 (d, J = 12.5 Hz, 1H), 3.49 - 3.40 (m, 1H), 3.33 - 3.16 (m, 2H), 2.97 (dd, J = 12.3, 7.1 Hz, 1H), 2.90 - 2.81 (m, 2H), 2.81 - 2.75 (m, 1H), 2.37 (d, J = 1.4 Hz, 3H), 2.11 - 2.02 (m, 1H), 2.02 - 1.82 (m, 5H), 1.82 - 1.75 (m, 1H), 1.70 (dt, J = 12.4, 7.1 Hz, 1H), 1.34 (s, 3H).

[0075] ESI-MS: calcd for C 58 H 56 FN 11 O 10 S[M+H] + = 1118.40 found 1118.49.t R = 4.5 min

[0076] (MeOH:H2O = 90:10), HPLC purity: 98.0 %.

[0077] From the above results, the structure of probe I is as follows:

[0078]

[0079] Preparation of fluorescent probe II

[0080] Fluorescent probe II was synthesized according to the following route:

[0081]

[0082] Step 1: Preparation of intermediate 2 was the same as step 1 in example 1.

[0083] Step 2: Preparation of intermediate 5

[0084] Intermediate 2 (151 mg, 0.2 mmol), 5-chloro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)naphthalen-2-ol (61 mg, 0.2 mmol, CAS No.: 2757096-51-6), cesium carbonate (130 mg, 0.4 mmol) and tetrakis(triphenylphosphine) palladium (7 mg, 0.006 mmol) were dissolved in 5 ml mixed solvent (dioxane: water = 10:1), heated to 70 °C under nitrogen protection for 8 h. After the reaction was completed, water (20 mL) was added to quench, extracted with dichloromethane (20 mL) for 3 times, the combined organic phase was dried over anhydrous sodium sulfate, concentrated, column chromatography (dichloromethane:methanol = 15:1) was used for separation and purification, and then (3R,5S)-5-(((7-(8-chloro-3-hydroxynaphthalen-1-yl)-8-fluoro-4-((R)-3-hydroxy-3-methylpiperidin-1-yl)pyrido[4,3-d]pyrimidin-2-yl)oxy)methyl)-1-methylpyrrolidin-3-yl(3-(1-(2-(((benzyloxy)carbonyl)amino)ethyl)-1H-1,2,3-triazol-4-yl)propyl)carbamate, white solid, 70 mg, yield 40.0% was obtained.

[0085] Intermediate 5 was analyzed by nuclear magnetic hydrogen spectrum and mass spectrum, and the results were as follows:

[0086] 1H NMR (400 MHz, Chloroform-d) δ 9.03 (s, 1H), 8.74 (s, 1H), 7.63 - 7.55 (m, 2H), 7.43 - 7.27 (m, 8H), 7.23 (t, J = 1.5 Hz, 1H), 5.80 (t, J = 6.5 Hz, 1H), 5.50 (t, J = 7.1 Hz, 1H), 5.13 - 5.02 (m, 2H), 4.76 (pd, J = 7.1, 0.9 Hz, 1H), 4.72 (s, 1H), 4.43 (dt, J = 12.5, 7.1 Hz, 1H), 4.31 (dt, J = 12.5, 7.1 Hz, 1H), 4.15 - 4.05 (m, 2H), 3.74 - 3.40 (m, 7H), 3.33 - 3.16 (m, 2H), 2.97 (dd, J = 12.3, 7.1 Hz, 1H), 2.90 - 2.75 (m, 3H), 2.37 (d, J = 1.5 Hz, 3H), 2.10 - 1.76 (m, 7H), 1.70 (dt, J = 12.3, 7.0 Hz, 1H), 1.34 (s, 3H).

[0087] ESI-MS [M+H] + : 897.2

[0088] From the above results, the structure of intermediate 5 is as follows:

[0089]

[0090] Step 3: Preparation of intermediate 6

[0091] Intermediate 5 (80 mg, 0.09 mmol) was dissolved in 5 ml saturated hydrochloric acid in dioxane solution, and reacted at room temperature overnight, then filtered, the filter cake was washed with ethyl acetate (20 mL) twice, and dried to obtain a light yellow solid product intermediate 6 (3R,5S)-5-(((7-(8-chloro-3-hydroxynaphthalen-1-yl)-8-fluoro-4-((R)-3-hydroxy-3-methylpiperidin-1-yl)pyrido[4,3-d]pyrimidin-2-yl)oxy)methyl)-1-methylpyrrolidin-3-yl(3-(1-(2-aminoethyl)-1H-1,2,3-triazol-4-yl)propyl)carbamate, 63 mg, yield 91.8%.

[0092] Intermediate 6 was analyzed by nuclear magnetic hydrogen spectrum and mass spectrum, and the results were as follows:

[0093] 1H NMR (400 MHz, Chloroform-d) δ 9.03 (s, 1H), 8.74 (s, 1H), 7.64 - 7.59 (m, 2H), 7.43 - 7.38 (m, 2H), 7.37 (d, J = 7.4 Hz, 1H), 7.32 (d, J = 1.4 Hz, 1H), 7.24 (t, J = 1.7 Hz, 1H), 5.50 (t, J = 7.1 Hz, 1H), 4.80 - 4.70 (m, 2H), 4.32 (dt, J = 12.4, 7.1 Hz, 1H), 4.24 (dt, J = 12.5, 7.1 Hz, 1H), 4.15 - 4.05 (m, 2H), 3.74 - 3.63 (m, 2H), 3.60 (d, J = 12.5 Hz, 1H), 3.50 (d, J = 12.4 Hz, 1H), 3.47 - 3.37 (m, 1H), 3.34 - 3.16 (m, 4H), 2.96 (dd, J = 12.3, 7.0 Hz, 1H), 2.90 - 2.75 (m, 3H), 2.37 - 2.29 (m, 5H), 2.10 - 1.76 (m, 7H), 1.70 (dt, J = 12.3, 7.0 Hz, 1H), 1.34 (s, 3H).

[0094] ESI-MS [M+H] + : 763.5

[0095] From the above results, the structure of intermediate 6 is as follows:

[0096]

[0097] Step 4: Preparation of probe II

[0098] Intermediate 6 (38 mg, 0.05 mmol), 3',6'-dihydroxy-5-isothiocyanato-3H-spiro[isobenzofuran-1,9'-xanthene]-3-one (19 mg, 0.05 mmol, CAS No.: 3326-32-7) and DIPEA (13 mg, 0.1 mmol) were dissolved in 7 ml of methanol and reacted at room temperature for 1 h. After the reaction was completed, the reaction solution was concentrated, and HPLC (MeOH:H2O = 40%-65%) was used for separation to obtain probe II (3R,5S)-5-(((7-(8-chloro-3-hydroxynaphthalen-1-yl)-8-fluoro-4-((R)-3-hydroxy-3-methylpiperidin-1-yl)pyrido[4,3-d]pyrimidin-2-yl)oxy)methyl)-1-methylpyrrolidin-3-yl (3-(1-(2-(3-(3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-5-yl)thioureido)ethyl)-1H-1,2,3-triazol-4-yl)propyl)carbamate, yellow solid 7 mg, yield 12.1%.

[0099] The probe II was subjected to nuclear magnetic hydrogen spectrum and mass spectrum analysis, and the results were as follows:

[0100] 1H NMR (500 MHz, Chloroform-d) δ 9.41 (s, 1H), 9.03 (s, 1H), 8.99 (s, 2H), 8.74 (s, 1H), 8.22 (d, J = 1.6 Hz, 1H), 7.77 (dd, J = 7.5, 1.5 Hz, 1H), 7.63 - 7.55 (m, 3H), 7.45 - 7.38 (m, 2H), 7.37 (t, J = 7.4 Hz, 1H), 7.30 (d, J = 1.4 Hz, 1H), 7.23 (t, J = 1.7 Hz, 1H), 7.15 (d, J = 7.5 Hz, 2H), 6.62 - 6.55 (m, 4H), 5.48 (t, J = 7.2 Hz, 1H), 4.77 (qd, J = 7.0, 0.8 Hz, 1H), 4.72 (s, 1H), 4.44 (dt, J = 12.2, 7.1 Hz, 1H), 4.32 (dt, J = 12.3, 7.1 Hz, 1H), 4.14 - 4.08 (m, 2H), 3.83 (m, 2H), 3.68 (ddt, J = 12.4, 7.0, 5.3 Hz, 2H), 3.60 (d, J = 12.5 Hz, 1H), 3.50 (d, J = 12.5 Hz, 1H), 3.49 - 3.40 (m, 1H), 3.33 - 3.16 (m, 2H), 2.97 (dd, J = 12.3, 7.1 Hz, 1H), 2.90 - 2.81 (m, 2H), 2.80 (dt, J = 12.5, 7.1 Hz, 1H), 2.37 (d, J = 1.4 Hz, 3H), 2.11 - 1.75 (m, 7H), 1.70 (dt, J = 12.4, 7.1 Hz, 1H), 1.34 (s, 2H).

[0101] ESI-MS: calcd for C 58 H 56 ClFN 11 O 10 S[M+H] + = 1152.36 found 1152.23.t R = 4.5 min

[0102] (MeOH:H2O = 90:10), HPLC purity: 98.2%.

[0103] From the above results, the structure of probe II is as follows:

[0104]

[0105] Preparation of Comparative Compound 1

[0106] Comparative Compound 1 was synthesized according to the following route:

[0107]

[0108] (1) The synthesis route of intermediate 7 is referred to CN118930562A.

[0109] (2) Intermediate 7 (36 mg, 0.05 mmol), 3',6'-dihydroxy-5-isothiocyanato-3H- spiro[isobenzofuran-1,9'-xanthene]-3-one (19 mg, 0.05 mmol, CAS No.: 3326-32-7) and DIPEA (13 mg, 0.1 mmol) were dissolved in 6 ml of methanol and reacted at room temperature for 0.5 h. After the reaction was completed, the reaction solution was concentrated, and HPLC (MeOH:H2O = 45%-65%) was used for separation to obtain (3R,5S)-5-(((4-((1R,5S)-3,8-diazabicyclo[3.2.1]octan-3-yl)-8-fluoro-7-(3- hydroxynaphthalen-1-yl)pyrido[4,3-d]pyrimidin-2-yl)oxy)methyl)-1-methylpyrrolidin-3-yl (3-(1-(2-(3-(3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-5-yl)thioureido)ethyl)-1H-1,2,3-triazol-4-yl)propyl)carbamate, orange solid 12 mg, yield 10.8% of the reference compound 1.

[0110] ESI-MS [M+H] + : 1115.5.

[0111] The structure of the reference compound 1 is shown as follows:

[0112]

[0113] Preparation of Reference Compound 2

[0114] Reference Compound 2 was synthesized according to the following route:

[0115]

[0116] To a solution of 3-oxo-2',3,3',5',6',7',11',12',13',15',16',17'-dodecahydro-1'H- spiro[2-benzofuran-1,9'-quinazolo[9',1':6,7,8]carbazole[3,2-g]pyrido[3,2,1- ij]quinoline]-5-carboxylic acid (45 mg, 0.1 mmol, CAS No: 197854-71-0), HATU (38 mg, 0.1 mmol), DIPEA (52 mg, 0.4 mmol) in 3 ml, DMF at room temperature for 0.5 h, then intermediate 4 (73 mg, 0.1 mmol) was added. The reaction was continued at room temperature for 1 h. After the reaction was completed, the reaction solution was concentrated, and HPLC (MeOH:H2O = 40%-65%) was used to separate to give (3R,5S)-5-(((7-(8-chloro-3-hydroxynaphthalen-1-yl)-8-fluoro-4-((R)-3-hydroxy-3- methylpiperidin-1-yl)pyrido[4,3-d]pyrimidin-2-yl)oxy)methyl)-1-methylpyrrolidin-3-yl((3-[1- (2-([(3-oxo-2',3,3',5',6',7',11',12',13',15',16',17'-dodecahydro-1'H-spiro[2-benzofuran-1,9'- quinazolo[9',1':6,7,8]carbazole[3,2-g]pyrido[3,2,1-ij]quinoline]-5-yl)formyl]amino)ethyl)- 1,2,3-triazol-4-yl)propanoic acid amide, dark purple solid 6 mg, yield 4.8%.

[0117] ESI-MS [M+H] + : 1245.5.

[0118] The structure of the reference compound 2 is shown as follows:

[0119]

[0120] Example 3

[0121] To be applied to the activity detection of KRAS inhibitors, the probe molecule first needs to ensure that the probe molecule can stably bind to the KRAS protein and has good affinity. The affinity of the fluorescent probe and the protein can be detected by the fluorescence polarization method (affinity-based fluorescence polarization assay, FP assay).

[0122] For the binding affinity of KRAS, the half effect concentration (EC 50 ) is used to characterize the strength of the affinity. First, the probe is used for KRAS G12DThe affinities of the proteins were investigated to explore the effects of different fragments of the probe ligand and the fluorescent fragment on the affinities, and it was proved that the affinities of the probes I and II to the proteins were optimal. G12D The affinities of the proteins were investigated to explore the effects of different fragments of the probe ligand and the fluorescent fragment on the affinities, and it was proved that the affinities of the probes I and II to the proteins were optimal.

[0123] Specific experimental operation: 10 nM of the probes I and II and the control compound respectively, 10 nM of the fluorescent probe KRAS G12D The initial concentration of the protein was 150 μM, and three-fold dilution was started, 3 replicate wells were set for each probe, 11 concentration gradients, and data reading was performed in a 384-well black plate (#3575, Corning) for 4 O C co-incubation for 1 h. The 384-well black plate was used for data reading of the polarization value at an excitation wavelength of 485 nm and an emission wavelength of 535 nm by a multifunctional enzyme label meter (SpectraMax Paradigm, Molecular Devices), with the solvent as a blank control.

[0124] The fragments in the probes are as follows:

[0125]

[0126] (1) The effect of ligand fragment 1 on the affinity of the probe:

[0127] The ligand fragment 1 of the probe I was replaced with different substituents to investigate the effect of the ligand fragment 1 on the affinity of KRAS G12D protein. The test results are shown in Table 1. The results show that the ligand fragment 1 has a significant effect on the affinity of the fluorescent probe to KRAS G12D protein, and the affinity of the probe I to KRAS G12D protein is optimal at 230 nM, and the affinity decreases after replacing the fragment with other fragments. The affinity of the control compound 1 is referenced to CN118930562A.

[0128] Table 1: Effect of fragment on affinity of probe

[0129]

[0130] (2) The effect of fluorescent fragment on the affinity of the probe:

[0131] The fluorescent fragment of the probe I was replaced with different fluorescent fragments to investigate the effect of the fluorescent fragment on the affinity of KRAS G12D protein. The test results are shown in Table 2. The results show that the fluorescent fragment of the fluorescent probe has a significant effect on the affinity of the probe to KRAS G12D protein, and the affinity decreases after replacing the fragment with other fragments.

[0132] Table 2: Effect of fluorescence on affinity of probe

[0133]

[0134] (3) Effect of hydrophobic fragment on probe affinity:

[0135] The effect of ligand fragment 2 on the affinity of probe KRAS G12D protein was also investigated by the same method. The test results are shown in Table 3, and the results show that the hydrophobic fragment of the probe has a weak effect on its affinity to KRAS G12D protein.

[0136] Table 3: Effect of ligand fragment 2 on probe affinity

[0137]

[0138] (3) Comparison with published KRAS G12D small molecule fluorescent probe:

[0139] The probe I was compared with the published KRAS G12D small molecule fluorescent probe (CN118930562A) for affinity to KRAS G12D protein. The test results are shown in Table 4, and the results show that the affinity of probe I to KRAS G12D protein is significantly better than that of the reported small molecule fluorescent probe.

[0140] Table 4: Affinity comparison

[0141]

[0142] Example 4

[0143] After determining the affinity of the probe to KRAS G12D protein, further affinity experiments were performed on KRAS wild type and G12C, G12V, G12D, G12A, G12S, G12R mutant proteins with high mutation frequency in tumors.

[0144] The EC 50 calculation process of the probe I in Example 1 to different proteins is illustrated. The polarization values at 485 nm excitation wavelength and 535 nm emission wavelength and the corresponding KRAS G12D protein concentrations are shown in 1-7 (Tables 5-1 to 5-7) in Table 5, and the negative control represents only 10 nM of probe I, and the experimental buffer is Hepes buffer. When the probe and the protein are combined, the fluorescence polarization value (mp) becomes larger. Δmp = negative control mp - average mp, and the data is imported into Graphpad prism 9.5 to obtain the EC 50 of probe I, and the results are shown in Table 6 (fitting curve as Figure 1As can be seen, probe I binds well to a variety of mutant proteins, which can be used to further establish activity screening methods for different protein subtypes. Figure 1 This is a graph showing the relationship between the fluorescence polarization value (mP) of fluorescent probe I and the concentration of fluorescent probe I after incubation with different mutant KRAS proteins. Analysis using Graphpad Prism 9.5 revealed the EC50 values ​​of fluorescent probe I for different mutant KRAS proteins. 50 This indicates that probe I is effective against KRAS. G12D The protein exhibits good affinity and the expected binding effect, and the curve fitting shows R0. 2 ≥99.0% indicates that the experimental results are true and reliable.

[0145] Table 5-1: Probe I and KRAS G12D Combined with EC 50 Raw data

[0146]

[0147] Table 5-2: Probe I and KRAS G12V Combined with EC 50 Raw data

[0148]

[0149]

[0150] Table 5-3: Probe I and KRAS WT Combined with EC 50 Raw data

[0151]

[0152] Table 5-4: Probe I and KRAS G12R Combined with EC 50 Raw data

[0153]

[0154]

[0155] Table 5-5: Probe I and KRAS G12A Combined with EC 50 Raw data

[0156]

[0157] Table 5-6: Probe I and KRAS G12C Combined with EC 50 Raw data

[0158]

[0159]

[0160] Table 5-7: EC of probe I with KRAS G12S EC of binding 50 Raw data

[0161]

[0162] Table 6: EC of probe I with different mutant proteins 50

[0163]

[0164] Example 5

[0165] Taking probe I in Example 1 as an example, its application in the determination of the inhibitory activity of a compound on KRAS and the screening of KRAS inhibitors is illustrated.

[0166] Taking probe I as a tool molecule, a high-throughput screening method for KRAS inhibitors based on fluorescence polarization is illustrated:

[0167] (1) Determination of the optimal concentration of probe I;

[0168] Taking the most common PBS buffer as the solvent, the concentration range of probe I is set to 10 μM-0.1 nM. When the concentration of the probe is higher, the fluorescence polarization value is lower. When the fluorescence polarization value reaches the critical value of the lower platform, the corresponding concentration is the optimal concentration of the probe. The data is read by a fluorescence polarimeter, and the experimental data is analyzed by Graphpad prism 9.5, indicating that the optimal concentration of the polarization light of probe I is 10-20 nM.

[0169] (2) Test the inhibitory IC value of the reported positive drug pan-inhibitor BI-2865 on KRAS protein by the established screening method 50 value;

[0170] Taking Hepes as the buffer system, the concentration of probe I is 10 nM, and the initial concentration of different subtypes of KRAS protein is set. The initial concentration of the positive drug BI-2865 is 10 μM, which is diluted by three times, and there are twelve concentration gradients (10.00 μM, 3.30 μM, 1.10 μM, 0.37 μM, 0.12 μM, 0.04 μM, 13.70 nM, 4.57 nM, 1.52 nM, 0.51 nM, 0.17 nM, 0.06 nM), two duplicate wells, and the KRAS protein and probe I are incubated in a 384-well plate at room temperature for 0.5 h. The fluorescence polarization value is read by a microplate reader, and the blank control is only added with the probe buffer, and the negative control is added with the probe and KRAS protein.

[0171] Example 1: BI-2865 50 The calculation process, the fluorescence polarization values of two wells were read by the microplate reader as shown in Table 7-1 to 7-7, and the inhibition rate at specific concentration was obtained by the formula: inhibition rate % = 100*(1-(measured value-blank) / (negative value-blank). The obtained data were imported into Graphpad prism 9.5 for analysis and fitting to obtain IC 50 values.

[0172] Table 7-1: BI-2865 KRAS G12D protein IC 50 Raw data

[0173]

[0174]

[0175] Table 7-2: BI-2865 KRAS G12A protein IC 50 Raw data

[0176]

[0177] Table 7-3: BI-2865 KRAS G12C protein IC 50 Raw data

[0178]

[0179]

[0180] Table 7-4: BI-2865 KRAS WT protein IC 50 Raw data

[0181]

[0182] Table 7-5: BI-2865 KRAS G12V protein IC 50 Raw data

[0183]

[0184] Table 7-6: BI-2865 KRAS G12R protein IC 50 Raw data

[0185]

[0186]

[0187] Table 7-7: BI-2865KRAS G12S Protein IC 50 Raw data

[0188]

[0189] Same as the IC mentioned above 50 The calculation process yields the IC of BI-2865. 50 Value. IC 50 The test results are shown in Table 8 (the fitted curves are shown in Table 8). Figure 2 ):

[0190] Table 8: Control compound BI-2865IC 50 value

[0191]

[0192]

[0193] Figure 2 This is a schematic diagram showing the inhibition rate of the positive control drug BI-2865 against KRAS protein. The IC50 of BI-2865 against KRAS protein was determined using Graphpad Prism 9.5 analysis. 50 And the curve fit R 2 The activity level was ≥99.0%, showing a certain correlation with reported activity data (WO2023099592A1) and (Nature 2023, 619, 160–166), confirming the accuracy and reliability of this screening method. The IC50 of the positive control drug BI-2865... 50 The test results showed a certain correlation with the reported activity data, confirming the accuracy and reliability of this high-throughput screening.

[0194] Example 6: Stability assessment of the screening method

[0195] (1) Investigation of experimental stability Z' factor;

[0196] Z' factorial statistical experiments, based on the high signal group (probe I combined with KRAS) in 50 experiments, G12D The experiment was conducted by polarizing light onto the protein and low-signal groups (probe I only), and the experiment was repeated twice at different time points to analyze the stability of this high-throughput screening method. The experimental results are as follows: Figure 3 As shown, Figure 3For the experimental stability Z' factor investigation results figure, the fluorescence polarization values of 50 experiments were statistically analyzed using GraphPad Prism 9.5, and the Z' factor was calculated to be 0.77 using the formula Z'=1-3(σb-σf) / |μb-μf|, and the signal-to-noise ratio S / N was 28.8, which confirmed that the method had good stability. It is significantly better than the published small molecule fluorescent probe (CN118930562A) whose Z' factor is 0.61 and signal-to-noise ratio S / N is 15.05. This method can be used for screening of KRAS G12D protein inhibitors, with the advantages of low cost, stability, speed, and high efficiency. Among them, σb and σf are the standard deviations of the high signal group and the low signal group, respectively.

Claims

1. A KRAS small molecule fluorescent probe or a pharmaceutically acceptable salt thereof, characterized in that, The structural general formula of the KRAS small molecule fluorescent probe is shown as formula (I): Among them, R 1 It is H or halogen. 2.The KRAS small molecule fluorescent probe or pharmaceutically acceptable salt thereof according to claim 1, characterized in that, The KRAS small molecule fluorescent probe is selected from the group consisting of:

3. The method for preparing the KRAS small molecule fluorescent probe or a pharmaceutically acceptable salt thereof according to claim 1 or 2, characterized in that, The synthesis route comprises the following steps: 。 4. The production method according to claim 3, characterized by, The synthesis route comprises the following steps: (A1) Compound 1 is deprotected in an organic solvent under acidic conditions to obtain compound 2; (A2) Compound 2 and compound 3 are reacted under basic conditions to obtain the probe through an addition reaction. The organic solvent selected for the addition reaction is methanol or dichloromethane, and the base selected for the addition reaction is DIPEA or triethylamine.

5. The preparation method according to claim 4, characterized in that, In step (A1), the organic solvent selected is methanol or dioxane, the acid is trifluoroacetic acid or hydrochloric acid, the reaction temperature is 60-70 DEG C, and the reaction time is 3-4 h.

6. The preparation method according to claim 4, characterized in that, In step (A2), the organic solvent selected for the addition reaction is methanol or dichloromethane, and the base selected is DIPEA or triethylamine, the reaction temperature is 20-30 DEG C, and the reaction time is 1-2 h.

7. The preparation method according to claim 4, characterized in that, In step (A1), compound 1 is synthesized according to the following route:

8. The preparation method according to claim 7, characterized in that, The synthesis route comprises the following steps: (B1) (R)-1-(2,7-dichloro-8-fluoropyrido [4,3-d] pyrimidin-4-yl)-3-methylpiperidin-3-ol, (3R,5S)-5-(hydroxymethyl)-1-methylpyrrolidin-3-yl (3-(1-(2-(((benzyloxy)carbonyl)amino)ethyl)-1H-1,2,3-triazol-4-yl)propyl)carbamate, cesium carbonate, dissolved in dioxane, heated under nitrogen protection, extracted with ethyl acetate, dried with anhydrous sodium sulfate, concentrated, and separated by column chromatography to obtain the intermediate, the heating reaction temperature is 95-105 DEG C, and the heating reaction time is 8-10 h; (B2) intermediate, Cesium carbonate, tetrakis triphenyl phosphine palladium was dissolved in a mixed solvent of dioxane and water, heated under nitrogen protection, dichloromethane extraction, anhydrous sodium sulfate drying, concentration, column chromatography separation and purification to obtain compound 1, the heating reaction temperature was 70-80℃, and the heating reaction time was 6-8h.

9. The KRAS small molecule fluorescent probe or a pharmaceutically acceptable salt thereof according to claim 1 or 2 for use as a fluorescent probe in KRAS protein ligand screening, which is not for the purpose of diagnosis or treatment of a disease.

10. A kit characterized in that, The kit comprises the KRAS small molecule fluorescent probe or a pharmaceutically acceptable salt thereof according to claim 1 or 2.

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