A KRAS G12D Small-molecule fluorescent probes, methods of making and using same

Abstract

The application discloses a KRAS G12D A small-molecule fluorescent probe, a preparation method and application thereof, and a structure of the small-molecule fluorescent probe is shown as follows. An activity determination method based on the small-molecule fluorescent probe has the advantages of a simple test system, no need of a complex buffer system, small influence of time and solvent, etc., and can be used for screening of KRAS G12D inhibitors.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of chemical biology. In particular, it relates to a class of small molecule fluorescence polarization probes based on KRAS G12D inhibitor design, methods of preparation, and use in screening ligands for KRAS G12D protein. BACKGROUND

[0002] RAS is the most frequently mutated oncogene in human cancer, with approximately 30% of all cancers being mutated. 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 approximately one-quarter 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] Current research on KRAS G12C mutants has achieved certain results, but people soon realized that targeting mutant KRAS is not as simple as initially thought. First, Sotorasib is a specific inhibitor of KRAS G12C , and 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 mutants, the study of G12D mutants is relatively less. However, 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 repetitive signals and feedback mechanisms, which make the human body very susceptible to 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 research. MRTX1133 is a non-covalent, potent and selective KRASG12D 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, and has significant in vivo efficacy in tumor models containing KRAS G12D mutations. It has good selectivity for G12D, which is more than 500 times selective for wild type (J. Med. Chem. 2021, 65(4), 3123-3133).

[0005] Therefore, the development of in vitro KRAS G12D inhibitory activity test methods and high-throughput screening methods are 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 candidate drug molecules with better activity, and lay the foundation for the development of therapeutic drugs for diseases such as lung cancer, pancreatic cancer and colorectal cancer targeting KRAS G12D .

[0006] At present, the most commonly used in vitro target activity test method for KRAS G12D 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) detection technology is a fluorescence-labeled detection technology that labels fluorescent substances on specific substances, converts the weak reaction signal into a stronger fluorescent signal, and plays a signal enhancement role. Macromolecules (such as proteins) in solution rotate relatively slowly due to their large size, 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 polarization level of the emitted light, and a low polarization level indicates that the fluorescent small molecules are free to move in the sample. A high polarization level indicates that the fluorescent molecules are attached to larger molecular complexes (J. Med. Chem. 2023, 66(16), 10934-10958).

[0008] To break through the limitations of the current KRAS G12D inhibitor scaffolds as the binding fragments of KRAS G12D protein, a small molecule fluorescent probe based on KRAS G12D inhibitor design is designed and synthesized by further introducing a connecting chain and a fluorescent group. Compared with HTRF, the fluorescence polarization 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 G12D inhibitors. SUMMARY

[0009] The purpose of the application is to provide a small molecule fluorescent probe based on the affinity of KRAS G12D protein. The probe molecule can be used to study and explore the action characteristics of KRAS G12D protein ligands in vivo and the action mode, active site structure information of the target, and can be applied to the determination of the affinity of compounds to KRAS G12D protein. The second purpose of the application is to provide a preparation method of the small molecule fluorescent probe. The third purpose of the application is to provide an application of the small molecule fluorescent probe.

[0010] Technical scheme: The small molecule fluorescent probe provided by the application has the following structure:

[0011]

[0012] On the other hand, the application provides a preparation method of the above-mentioned small molecule fluorescent probe, and the preparation method comprises the following steps:

[0013]

[0014] (1) Compound 1 is deprotected under acidic conditions to obtain compound 2;

[0015] (2) Compound 2 and compound 3 are subjected to a condensation reaction under alkaline conditions to obtain the target probe I.

[0016] Preferably, in step (1), the organic solvent used for the deprotection reaction of compound 1 is methanol or dioxane.

[0017] Preferably, in step (1), the acid in the acidic condition is trifluoroacetic acid or hydrochloric acid.

[0018] Preferably, in step (2), the organic solvent used for the condensation reaction is N,N-dimethylformamide or dichloromethane.

[0019] Preferably, in step (2), the condensing agent selected for the condensation reaction is HBTU, EDCI or HATU.

[0020] Preferably, in step (2), the base in the basic condition is DIPEA or triethylamine.

[0021] Specifically, the preparation method of the small molecule fluorescent probe comprises the following steps:

[0022] (1) Compound 1 is deprotected under acidic conditions to obtain compound 2;

[0023] The organic solvent selected for the reaction is methanol or dioxane, and the acid is trifluoroacetic acid or hydrochloric acid. Preferably, the acid is hydrochloric acid, the reaction solvent is preferably dioxane, the reaction temperature is preferably room temperature, and the reaction time is 10-12 hours;

[0024] (2) Compound 2 and compound 3 are condensed under basic conditions to obtain probe I;

[0025] The organic solvent selected for the condensation reaction is N,N-dimethylformamide or dichloromethane, the condensing agent selected for the condensation reaction is HBTU, EDCI or HATU, and the base selected for the condensation reaction is DIPEA or triethylamine. Preferably, the base is DIPEA, the condensing agent is HATU, the reaction solvent is DMF, the reaction temperature is room temperature, and the reaction time is 1-2 hours.

[0026] On the other hand, the present application provides a small molecule fluorescent probe as described above in the screening of KRAS G12D protein ligands.

[0027] The present application also provides a small molecule fluorescent probe as described above as a fluorescence polarization probe in the screening of KRAS G12D protein ligands.

[0028] Further, the present application provides a method for rapidly detecting the binding ability of a small molecule compound to KRAS G12D protein, which comprises the following steps: (1) mixing the compound according to claim 1, KRAS G12D protein and the test compound in a buffer to obtain a mixture; (2) measuring the polarization value of the mixture under the excitation wavelength of 585 nm and the emission wavelength of 635 nm of the compound according to claim 1 by using a fluorescence polarization enzyme label instrument, and confirming the affinity of the test compound to KRAS G12D protein according to the polarization value.

[0029] Further, the method for rapidly detecting the binding ability of a small molecule compound to KRAS G12D protein, wherein the buffer comprises Tris buffer and Hepes buffer.

[0030] In another aspect, the present application provides a kit comprising the above-mentioned small-molecule fluorescent probe.

[0031] To break through the limitations of the current KRAS G12D inhibitor design, a class of small-molecule fluorescent polarization probes based on KRAS G12D inhibitor design was designed and synthesized by further introducing a linker and a fluorescent fragment. A stable, rapid, and inexpensive in vitro KRAS G12D protein inhibition activity detection method was established based on the principle of fluorescent polarization. The applicability and accuracy of this method were verified by testing the reported small-molecule enzyme affinity activity. G12D

[0032] Advantages: Compared with the prior art, the present application has the following significant advantages: 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, little influence of time and solvent, etc., and can be used for screening KRAS G12D inhibitors. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 : Relationship between fluorescence polarization value (mP) and concentration of fluorescent probe I after incubation of fluorescent probe I with KRAS G12D protein. Through Graphpad prism 9.5 analysis, the EC G12D of fluorescent probe I for KRAS 50 protein was obtained, indicating that probe I has good affinity and expected binding effect for KRAS G12D protein, and the curve fitting R 2 ≥ 99.0%, indicating that the experimental results are real and reliable.

[0034] Figure 2 : Schematic diagram of inhibition rate of positive drugs MRTX-1133 and BI-2865 for KRAS G12D protein. Through Graphpad prism 9.5 analysis, the IC G12D of MRTX-1133 and BI-2865 for KRAS 50 protein was obtained, and the curve fitting R 2 ≥ 99.0%, which shows a certain correlation with the reported activity data, confirming the accuracy and reliability of this screening method.

[0035] Figure 3 : is the relationship between fluorescence polarization value (mP) and concentration of fluorescent probe I after incubation of fluorescent probe I with KRAS G12D ​The DMSO solvent tolerance of protein binding was investigated, and when the proportion of DMSO (v / v) was 1%, 10%, 20% and 30%, it was analyzed by GraphPad Prism 9.5, and when DMSO (V / V) was less than or equal to 30%, it had little effect on the binding of the probe protein, indicating that this method had good solvent tolerance and was suitable for high-throughput screening of small molecules.

[0036] Figure 4 The experimental stability Z' factor was investigated, and the fluorescence polarization values of 50 experiments were statistically analyzed by GraphPad Prism 9.5, and the formula Z' = 1-3 (σb-σf) / |μb-μf| was used to obtain the Z' factor of 0.61, indicating that this method can be used for KRAS G12D protein inhibitor screening, which has the advantages of low cost, stability, rapidness, high efficiency, etc. Among them, σb and σf are the standard deviations of the high signal group and the low signal group, respectively. DETAILED DESCRIPTION

[0037] The excitation wavelength of the fluorescence polarization enzyme label instrument used in the embodiment of the application is 585 nm, and the emission wavelength is 635 nm.

[0038] Embodiment 1

[0039] In order to be applied to the activity detection of KRAS G12D inhibitors, the probe molecule first needs to be stably combined with KRAS G12D protein and have good affinity. The application detects the binding affinity of the fluorescent probe for KRAS G12D protein by a fluorescence polarization method (affinity-based fluorescence polarization assay, FP assay), and uses the half effect concentration (EC 50 ) to represent the strength of the affinity. The application investigates the affinity of probe I molecule for KRAS G12D protein, and explores the influence of the connecting chain and the fluorescent fragment on the affinity, and proves that probe I has good affinity for KRAS G12D protein.

[0040] Specific experimental operation: the probe 10 nM, the initial concentration of the fluorescent probe KRAS G12D protein is 150 μM, and three-fold dilution is started, 3 replicate wells are set for each probe, 11 concentration gradients are set, and 4℃ incubation is performed for 1 h in a 384-well black plate (#3575, Corning). The 384-well black plate is used for data reading by a multifunctional enzyme label instrument (SpectraMax Paradigm, Molecular Devices), and the solvent is used as a blank control.

[0041] (1) The effect of fluorescent fragment on the affinity of probe:

[0042] The present application replaces the fluorescent fragment of probe I with different fluorescent fragments to investigate the effect of fluorescent fragment on the affinity of KRAS G12D protein. The test results are shown in Table 1. The results show that the fluorescent fragment of the fluorescent probe has a significant effect on its affinity to KRAS G12D protein, among which the affinity of probe I to KRAS G12D protein is best at 415nM, and the affinity is decreased after replacing it with other fragments.

[0043] Table 1: Effect of fluorescent fragment on the affinity of probe

[0044]

[0045]

[0046] (2) The effect of connecting chain on the affinity of probe:

[0047] Subsequently, the present application investigates the effect of connecting chain on the affinity of KRAS G12D protein by the same method. The test results are shown in Table 2, and the results show that the connecting chain of the probe has a greater effect on its affinity to KRAS G12D protein, and the affinity to protein is greatly decreased after replacing it with other fragments.

[0048] Table 2: Effect of connecting chain on the affinity of probe

[0049]

[0050] Example 2

[0051] The compound I with the best affinity to KRAS G12D protein in the present application is used to illustrate its application in determining the inhibitory activity of compounds to KRAS G12D , and screening of KRAS G12D inhibitors.

[0052] The present application takes probe I as an example to illustrate the EC 50 calculation process. The fluorescence polarization value of the sample under excitation wavelength of 585nm and emission wavelength of 635nm and the corresponding concentration of KRAS G12D protein are read by the enzyme marker, as shown in Table 3, and the negative control represents only 10nM of probe I, and the experimental buffer is Hepes buffer. When the probe and 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 as 415nM (the fitting curve is as followsFigure 1 )

[0053] Table 3: Probe I EC 50 Raw data

[0054]

[0055] Example 3

[0056] Using probe I as a tool molecule, the KRAS G12D High-throughput screening of inhibitors:

[0057] (1) Determination of the optimal concentration of probe I;

[0058] Using the most common PBS buffer as the solvent, the probe I concentration range was set at 100 μM-10 nM. Higher probe concentrations resulted in lower fluorescence polarization values. The optimal probe concentration was determined when the fluorescence polarization value reached the critical value of the lower plateau. Fluorescence polarization readings were taken at an excitation wavelength of 585 nm and an emission wavelength of 635 nm using a fluorescence polarization instrument. Graphpad Prism 9.5 analyzed the data, indicating that the optimal concentration of probe I for polarized light was 10-200 nM.

[0059] (2) Testing the reported positive drugs for KRAS by establishing a screening method G12D Protein inhibition IC 50 value;

[0060] Hepes was used as the buffer system, the concentration of probe I was 10 nM, KRAS G12D The protein concentration was 200 nM. The positive drugs MRTX-1133 and BI-2865 were diluted to 10 μM at the initial concentration, three times, fourteen concentration gradients, and three replicates. G12D Protein and probe I were incubated in a 384-well plate at 4°C for 0.5 h. The fluorescence polarization values ​​of the samples were read by a microplate reader at an excitation wavelength of 585 nm and an emission wavelength of 635 nm. The blank control was treated with only probe buffer. G12D Protein was used as negative control.

[0061] MRTX-1133 is used as an example to illustrate this high-throughput method compound IC 50 The fluorescence polarization values ​​of three replicate wells were read by the microplate reader as shown in Table 4. The inhibition rate corresponding to the 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 value.

[0062] Table 4: MRTX-1133IC50 Raw data

[0063]

[0064] IC of MRTX-1133 IC 50 IC of BI-2865 50 IC of BI-2865 50 The results are shown in Table 5 (the fitted curve is shown in Figure 5): Figure 2

[0065] Table 5: IC of control compounds 50

[0066]

[0067]

[0068] IC of positive drug 50 The test results have a certain correlation with the reported activity data, which confirms the accuracy and reliability of this high-throughput screening.

[0069] Example 4

[0070] Stability and solvent tolerance of the screening method:

[0071] (1) DMSO tolerance;

[0072] Dimethyl sulfoxide (DMSO) is a commonly used solvent, which is often used for testing the solubility of compounds. Therefore, the fluorescence polarization assay based on the interaction of probe I and KRAS G12D protein should be able to tolerate a small volume of DMSO. In the binding experiment of probe I and KRAS G12D protein, the maximum content of DMSO in the binding system is 1.00% (v / v). Based on this, the effect of 1%-30% (v / v) DMSO on the competition binding experiment was further tested. The experimental results are shown in Figure 3 When the content of DMSO (v / v) is increased to 30%, the binding affinity of the probe and the protein in the fluorescence polarization assay is still quite stable. This high-throughput screening method has good DMSO tolerance.

[0073] (2) Z' factor investigation of experimental stability;

[0074] Z' factor statistics experiment, according to the polarization value of high signal group (probe I binding KRAS G12D protein) and low signal group (only probe I) samples at excitation wavelength 585 nm and emission wavelength 635 nm in 50 experiments, and repeating this experiment 2 times at different time periods, to analyze the stability of this high-throughput screening method. The experimental results are shown in Figure 4 ​As shown in the figure, the statistical Z' factor is 0.61 and the signal-to-noise ratio S / N is 15.05, which confirms that the method has good stability.

[0075] Example 5:

[0076] Preparation of fluorescent probe I:

[0077]

[0078] Step 1: Preparation of intermediate 2

[0079] (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), (S)-2-(6-(2-hydroxymethylpyrrolidin-1-yl)hexyl)isoindoline-1,3-dione (86 mg, 0.2 mmol), and cesium carbonate (130 mg, 0.4 mmol) were dissolved in 3 ml of dioxane and heated to 100 °C under nitrogen for 8 h. After the reaction, the reaction solution was quenched by adding water (20 mL), extracted with EA (10 mL × 3), dried over anhydrous sodium sulfate, concentrated, and separated and purified by column chromatography (dichloromethane:methanol=20:1) to obtain a pale white solid powder of tert-butyl (1R,5S)-3-(2-(((2S,4R)-4-(((3-(1-(2-(((benzyloxy)carbonyl)amino)ethyl)-1H-1,2,3-triazol-4-yl)propyl)carbamoyl)oxy)-1-methylpyrrolidin-2-yl)methoxy)-7-chloro-8-fluoropyrido[4,3-d]pyrimidin-4-yl)-3,8-diazabicyclo[3.2.1]octane-8-carboxylate, 50 mg, yield 29.3%.

[0080] Step 2: Preparation of Intermediate 3

[0081] Intermediate 2 ((50 mg, 0.06 mmol), 4-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)naphthalen-2-ol (24 mg, 0.09 mmol), cesium carbonate (40 mg, 0.12 mmol), tetrakis(triphenylphosphine) palladium (7 mg, 0.006 mmol) were dissolved in 3 ml mixed solvent of dioxane: water = 10: 1, heated to 70 °C for 6 h under nitrogen protection. After the reaction was completed, water (20 mL) was added to quench, extracted with DCM (20 mL x 3), dried over anhydrous sodium sulfate, concentrated, and separated and purified by column chromatography (dichloromethane:methanol = 20: 1) to obtain a light white solid powder of tert-butyl (lR,5S)-3-(2-(((2S,4R)-4-(((3-(l-(2-(((benzyloxy)carbonyl)amino)ethyl)-lH-l,2,3-triazol-4-yl)propyl)carbamoyl)oxy)-l-methylpyrrolidin-2-yl)methoxy)-8-fluoro-7-(3-hydroxynaphthalen-l-yl)pyrido[4,3-d]pyrimidin-4-yl)-3,8-diazabicyclo[3.2.1]octane-8-carboxylate, 30 mg, yield 52.1%.

[0082] Step 3: Preparation of Intermediate 4

[0083] Intermediate 3 ((96 mg, 0.1 mmol) was dissolved in 5 ml ethyl acetate saturated with hydrochloric acid, and reacted at room temperature overnight, then filtered, and the filter cake was washed with (20 mL x 2) and dried to obtain a light yellow solid product of (3R,5S)-5-(((4-((lR,5S)-3,8-diazabicyclo[3.2.1]octan-3-yl)-8-fluoro-7-(3-hydroxynaphthalen-l-yl)pyrido[4,3-d]pyrimidin-2-yl)oxy)methyl)-l-methylpyrrolidin-3-yl (3-(l-(2-aminoethyl)-lH-l,2,3-triazol-4-yl)propyl)carbamate, 70 mg, yield 96.4%.

[0084] Step 4: Preparation of Probe I

[0085] 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), HATU ((38 mg, 0.1 mmol), DIPEA (52 mg, 0.4 mmol) were dissolved in 3 ml DMF and reacted at room temperature for 0.5 h. Then intermediate 10 ((72 mg, 0.1 mmol) was added and the reaction was continued at room temperature for 1 h. After the reaction was completed, the reaction solution was concentrated, and (3R,5S)-5-(((4-(3,8-diazabicyclo[3.2.1]octanyl)-8-fluoro-7-(3-hydroxy-1- naphthyl)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, dark purple solid 5 mg, yield 3.8% was separated by HPLC ((MeOH:H2O = 40%-65%). 1H NMR (500 MHz, Chloroform-d) δ 9.03 (s, 1H), 8.74 (s, 1H), 8.32 (d, J = 1.5 Hz, 1H), 8.03 (t, J = 6.0 Hz, 1H), 7.89 (dd, J = 7.4, 1.6 Hz, 1H), 7.83 - 7.75 (m, 2H), 7.61 (s, 1H), 7.51 (d, J = 7.5 Hz, 1H), 7.48 - 7.37 (m, 2H), 7.35 (d, J = 1.6 Hz, 1H), 7.23 (t, J = 1.6 Hz, 1H), 6.75 (t, J = 0.9 Hz, 2H), 5.48 (t, J = 7.2 Hz, 1H), 4.76 (pd, J = 7.1, 0.9 Hz, 1H), 4.42 (dt, J = 12.5, 7.1 Hz, 1H), 4.31 (dt, J = 12.3, 7.1 Hz, 1H), 4.13 - 4.08 (m, 2H), 3.70 - 3.54 (m, 2H), 3.52 (dd, J = 12.4, 7.0 Hz, 2H), 3.49 - 3.35 (m, 11H), 3.33 - 3.17 (m, 2H), 3.20 - 3.09 (m, 2H), 3.03 - 2.97 (m, 1H), 3.01 - 2.93 (m, 1H), 2.93 - 2.75 (m, 12H), 2.37 (d, J = 1.5 Hz, 3H), 2.06 (dt, J = 12.7, 6.9 Hz, 1H), 2.02 - 1.93 (m, 2H), 1.96 - 1.83 (m, 10H), 1.80 - 1.65 (m, 4H). LRMS (ESI): calcd for C 62 H 65 FN 13 O8[M+H] + = 1242.57 found 1242.55.t R = 4.18 min (MeOH:H2O = 90:10), HPLC purity: 98.0%.

Claims

1. A small molecule fluorescent probe, characterized in that Has the following structure: 。 2. A method for preparing a small molecule fluorescent probe, characterized in that: The preparation method comprises the following steps: （1） Under acidic conditions, compound 1 is deprotected to obtain compound 2; (2) Under alkaline conditions, compound 2 and compound 3 undergo condensation reaction to obtain target probe I.

3. The method for preparing a small molecule fluorescent probe according to claim 2, wherein: In step (1), the organic solvent used in the deprotection reaction of compound 1 is methanol or dioxane.

4. The method for preparing a small molecule fluorescent probe according to claim 2, wherein: In step (1), the acid in the acidic condition is trifluoroacetic acid or hydrochloric acid.

5. The method for preparing a small molecule fluorescent probe according to claim 2, wherein: In step (2), the organic solvent used for the condensation reaction is N,N-dimethylformamide or dichloromethane.

6. The method for preparing a small molecule fluorescent probe according to claim 2, wherein: In step (2), the condensation agent selected for the condensation reaction is HBTU, EDCI or HATU.

7. A small molecule fluorescent probe according to claim 1 in KRAS G12D Applications in protein ligand screening that are not intended for disease diagnosis and / or treatment.

8. The use according to claim 7, characterized in that In KRAS G12D The method for protein ligand screening is: (1) Small molecule fluorescent probe, KRAS G12D The protein and the test compound are dissolved in a certain buffer, mixed, and incubated for a certain period of time; (2) Fluorescence polarization microplate reader was used to measure the polarization value of the mixture at the excitation wavelength of 585 nm and the emission wavelength of 635 nm of the small molecule fluorescent probe, and the polarization value was used to confirm the effect of the test compound on KRAS. G12D Protein affinity.

9. The use according to claim 8, characterized in that In step (1), the buffer is PBS buffer, Tris buffer or Hepes buffer.

10. A kit, characterized in that The kit comprises the small molecule fluorescent probe according to claim 1.

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