A CDK4 / 6 small molecule fluorescent probe and its preparation method and application

Abstract

The present invention discloses a small-molecule fluorescent probe for CDK4 / 6, its preparation method, and applications, belonging to the field of biomedicine. The CDK4 / 6 small-molecule fluorescent probe provided by the present invention has a structure represented by Formula (I), a novel probe response group, and good selectivity. The preparation method also features mild reaction conditions, readily available and inexpensive raw materials, and simple operation and post-processing. The probe exhibits high affinity for the CDK4 / 6 protein and high sensitivity.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a CDK4 / 6 small molecule fluorescent probe and a preparation method and application thereof. Background Art

[0002] The information disclosed in the background of the invention is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] The cell cycle consists of four phases: S phase (primarily DNA replication), M phase (primarily mitosis), and two intervals between S and M phases (G1 and G2). Non-proliferating cells also experience a quiescent state called G0. The cell cycle is controlled and regulated by various specific proteins, such as cyclin-dependent kinases (CDKs), cyclins, and the retinoblastoma protein (pRb). Cyclin-dependent kinases (CDKs) are a family of serine / threonine protein kinases. Dysregulation of CDKs leads to cell cycle imbalance and unlimited cell proliferation, thereby promoting the initiation and progression of malignant tumors. CDKs bind to their corresponding cyclins to form functional heterodimeric complexes. To date, 20 CDKs and 29 cyclins have been discovered. CDKs include cell cycle-related CDKs (CDK1, CDK2, CDK4, and CDK6) and transcriptional CDKs (CDK7-9, CDK11-13, and CDK19). Based on their distinct functions, they can be divided into two subfamilies: CDKs 1–6, 11, and 14–18 regulate the cell cycle, and CDKs 7–13, 19, and 20 regulate transcription. CDK activation is controlled by association with their regulatory subunits (cyclins) and phosphorylated inhibitory proteins. CDK4, when coordinated with cyclin D by phosphorylating pRb, and its closely related cousin, CDK6, play a crucial role in the G1 to S phase transition. pRb, in its unphosphorylated state, inhibits the G1 to S phase transition. Aberrations in the cyclin D–CDK4 / 6–INK4–pRb pathway are frequently observed in tumors, and therefore tumors are also considered cell cycle disorders. With significant advances in understanding the mechanisms of cell cycle regulation, CDKs have become increasingly prominent in this field. Therefore, small molecule CDK inhibitors are a hot area of ​​research in anticancer chemotherapy.

[0004] A key area of ​​CDK inhibitor research is the testing and identification of combination therapies involving CDK4 / 6 inhibitors for the treatment of diverse tumor types. CDK4 / 6 inhibitors trigger cell cycle arrest in tumor cells and, in some cases, can trigger senescence. Palbociclib, a specific CDK4 / 6 inhibitor, has recently become a star drug for the treatment of breast cancer, particularly estrogen receptor-positive breast cancer. Identifying combination therapies that convert CDK4 / 6 inhibitors from cytostatic compounds to cytotoxic compounds and unleash tumor cell killing is crucial. Genome-wide high-throughput screening and analysis of mouse cancer models and PDX will help address this issue. Another unexplored area of ​​cyclin D-CDK4 / 6 biology is the potential involvement of these proteins in other pathologies, such as metabolic disorders. Research in this area may expand the use of CDK4 / 6 inhibitors to treat other diseases. These questions ensure that CDK4 / 6 biology will remain an active area of ​​basic, translational, and clinical research for years to come.

[0005] Small molecule fluorescent probes, characterized by rapidity, sensitivity, high throughput, and ease of automation, have important applications in protein labeling and imaging. Currently, small molecule fluorescent probes are widely used in the biological and pharmacological detection of important biomolecules such as proteins and nucleic acids, and are of great significance for the development of fields such as disease mechanisms, clinical diagnosis, and drug screening. However, the number of small molecule probes that can specifically bind to target proteins remains limited, primarily due to a lack of design and synthesis methods. In particular, there are currently no small molecule fluorescent probes for studying CDK4 / 6 protein-protein interactions. Summary of the Invention

[0006] Based on the deficiencies of the existing technology, the present invention provides a CDK4 / 6 small molecule fluorescent probe and its preparation method and application. The probe can specifically bind to the CDK4 / 6 protein. The application of this small molecule probe can provide a deeper understanding of the mechanism of action between CDK4 / 6 proteins and the discovery of more effective inhibitors.

[0007] In order to achieve the above object, the technical solution of the present invention is:

[0008] In the first aspect, a CDK4 / 6 small molecule fluorescent probe has the structure shown in formula (I):

[0009]

[0010] Wherein, R1 is selected from any one of the following structures:

[0011] or n=1, 2, 3 or 5.

[0012] In a second aspect, a CDK4 / 6 small molecule fluorescent probe has a structure shown in formula (I):

[0013]

[0014] Wherein, R1 is selected from any one of the following structures:

[0015] or

[0016] m=1 or 2.

[0017] In a third aspect, the preparation method of the above-mentioned CDK4 / 6 small molecule fluorescent probe comprises the following steps:

[0018] Palbociclib is subjected to an amidation reaction with a compound represented by formula (II) to generate a compound represented by formula (III), and the compound represented by formula (III) is subjected to an amide condensation reaction with dansyl chloride after deprotection to obtain a compound represented by formula (I);

[0019] n=1, 2, 3 or 5, formula (II);

[0020] n=1, 2, 3 or 5, formula (III);

[0021] Alternatively, Palbociclib is subjected to an amidation reaction with 3-(2-((tert-butoxycarbonyl)amino)ethoxy)propionic acid to generate a compound, and the resulting compound is deprotected and then subjected to an amide condensation reaction with dansyl chloride to obtain a compound represented by formula (I);

[0022] Alternatively, 7-(diethylamino)-2-oxo-2H-chromone-3-carboxylic acid reacts with sulfuryl chloride to generate a carboxylic acid derivative, which is then reacted with the compound represented by formula (III) through a nucleophilic substitution reaction to obtain the compound represented by formula (I);

[0023] Alternatively, 4-(3-phenoxypropyl)-1,3-dioxane-2-en-2-one is reacted with chlorosulfonic acid through a sulfonation reaction to obtain an intermediate, the intermediate is subjected to an amidation reaction, and then reacted with the compound represented by formula (III) through a nucleophilic substitution reaction to obtain the compound represented by formula (I);

[0024] Alternatively, 4-(1,2,2-triphenylvinyl)benzoic acid and the compound represented by formula (III) undergo an amide condensation reaction to produce the compound represented by formula (I);

[0025] Alternatively, 4-chloro-7-nitrobenzo[c][1,2,5]oxadiazole and the compound represented by formula (III) undergo an amide condensation reaction to produce the compound represented by formula (I);

[0026] Alternatively, the compound represented by formula (IV) is subjected to a halogenation reaction and then reacted with 2,3,3-trimethylindole. The reaction product is reacted with the compound represented by formula (V), and then reacted with the compound represented by formula (III) or Palbociclib through an amide condensation reaction to produce the compound represented by formula (I); the compound represented by formula (V) is obtained by alkylation of 2,3,3-trimethylindole;

[0027] n=1 or 2, formula (IV);

[0028] Optionally, the compound represented by formula (II) reacts with Palbociclib in the presence of DIEA and HATU to produce the compound represented by formula (III), the compound represented by formula (III) reacts with trifluoroacetic acid to remove the protecting group, and then undergoes an amide condensation reaction with dansyl chloride in the presence of DIEA to produce the compound represented by formula (I).

[0029] Optionally, SOCl2 is added to 7-(diethylamino)-2-oxo-2H-chromone-3-carboxylic acid under nitrogen protection, and DMF is added to react to obtain a carboxylic acid derivative, and the carboxylic acid derivative is sequentially added with DIEA and HATU to react, and then the compound represented by formula (III) is added to carry out a nucleophilic substitution reaction to generate the compound represented by formula (I).

[0030] Optionally, chlorosulfonic acid is added to 4-(3-phenoxypropyl)-1,3-dioxane-2-en-2-one under ice bath conditions, and then heated under reflux to obtain an intermediate, triethylamine is added to the intermediate under ice bath conditions to react, and then dimethylamine hydrochloride is added to react, and N,N-diisopropylethylamine is added to react, and then the compound represented by formula (III) is added to carry out a nucleophilic substitution reaction to generate the compound represented by formula (I).

[0031] Optionally, N,N-diisopropylethylamine and HATU are added to 4-(1,2,2-triphenylvinyl)benzoic acid in sequence for reaction, and then a nucleophilic substitution reaction is carried out with the compound represented by formula (III) to generate the compound represented by formula (I).

[0032] Optionally, 4-chloro-7-nitrobenzo[c][1,2,5]oxadiazole is added to N,N-diisopropylethylamine and then reacted with the compound represented by formula (III) to undergo a nucleophilic substitution reaction to generate the compound represented by formula (I).

[0033] Optionally, the compound represented by formula (IV) undergoes a halogenation reaction with NaI under N2 reaction; ethyl acetate is then added for reaction, and then 2,3,3-trimethylindole is added for reaction to obtain compound 04; 2,3,3-trimethylindole is refluxed with methyl iodide to precipitate crystals of compound 05; malondialdehyde diphenylamine hydrochloride is added to the reaction product 04 and mixed, and acetic acid and acetic anhydride are added under nitrogen protection for reflux reaction; then compound 05, pyridine and acetic acid are added for reflux reaction to obtain compound 06; DIEA and HATU are added to compound 06 in sequence, and then the compound represented by formula (III) is added for nucleophilic substitution reaction to generate the compound represented by formula (I).

[0034]

[0035] Alternatively, after obtaining compound 06, DIEA and HATU are sequentially added to compound 06, and then Palbociclib is added to carry out a nucleophilic substitution reaction to generate the compound represented by formula (I).

[0036] In a fourth aspect, the application of the CDK4 / 6 small molecule fluorescent probe described in the first and second aspects includes: application in identifying CDK4 / 6 protein and / or preparing products for identifying CDK4 / 6 protein.

[0037] In a fifth aspect, the application of the CDK4 / 6 small molecule fluorescent probe described in the first aspect includes: application in cell cycle arrest and / or preparation of cell cycle arrest products;

[0038] Alternatively, the invention can be used in cancer cell fluorescence imaging and / or in the preparation of cancer cell fluorescence imaging products.

[0039] The beneficial effects of the present invention are:

[0040] 1. The probe response group of the present invention is novel and has good selectivity; the preparation method has mild reaction conditions, cheap and easily available raw materials, and simple operation and post-processing.

[0041] 2. The probe molecule of the present invention has strong biological activity, high affinity with CDK4 / 6 protein, and high sensitivity; it can be used to mark CDK4 / 6 protein and its highly expressed tumor cells or tissues; it can be used as a probe to identify CDK4 / 6 protein and CDK4 / 6 protein has broad application prospects in physiology, pathology and related diseases.

[0042] 3. The probe of the present invention can directly react to the inhibitory activity of the compound on CDK4 / 6 protein, and can be used for high-throughput screening of CDK4 / 6 protein inhibitors and their application in anti-tumor evaluation. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] The drawings, which constitute part of the present invention, are intended to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are intended to explain the present invention and do not constitute undue limitations thereon. It is obvious that one of ordinary skill in the art could derive other drawings based on these drawings without inventive effort.

[0044] Figure 1 A diagram showing the results of optical activity measurement in Example 4;

[0045] Figure 2 A diagram showing the results of cell activity assay in Example 6;

[0046] Figure 3 A diagram showing the cell imaging results of the probe molecule in Example 8. DETAILED DESCRIPTION

[0047] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.

[0048] In the following embodiments, the abbreviations have the following meanings:

[0049] DMSO: dimethyl sulfoxide;

[0050] DIEA: N,N-diisopropylethylamine;

[0051] HATU: 2-(7-azabenzotriazole)-N,N,N,N-tetramethyluronium hexafluorophosphate;

[0052] DMF: N,N-dimethylformamide;

[0053] TEA: triethylamine;

[0054] CDCl3: deuterated chloroform;

[0055] SOCl2: thionyl chloride, thionyl chloride;

[0056] Tris: tris(hydroxymethyl)aminomethane;

[0057] BSA: bovine serum albumin;

[0058] DTT: dithiothreitol;

[0059] ATP: adenosine triphosphate;

[0060] 1 H NMR: proton nuclear magnetic resonance;

[0061] 13C NMR: carbon nuclear magnetic resonance;

[0062] HRMS(ESI): high resolution mass spectrometry (electrospray ionization).

[0063] The technical solution of the present invention is further described below with reference to specific embodiments.

[0064] Example 1

[0065] Preparation method of CDK4 / 6 small molecule fluorescent probe,

[0066] The reaction route is shown below:

[0067]

[0068] n=1, 2, 3 or 5.

[0069] The reaction steps include:

[0070] S1, reacting compound (1) and Palbociclib as raw materials to generate intermediate (3);

[0071] S2, adding trifluoroacetic acid to convert intermediate (3) into intermediate (4);

[0072] S3. Dansyl chloride is added to react with the intermediate (4) to obtain compound (6), which is the small molecule fluorescent probe targeting CDK4 / 6 protein.

[0073] Wherein, when n=1, 2, 3 or 5, compound (1) is compound (1a), (1b), (1c), and (1d), respectively; compound (3) is compound (3a), (3b), (3c), and (3d), respectively; compound (4) is compound (4a), (4b), (4c), and (4d), respectively; and compound (6) is compound (6a), (6b), (6c), and (6d), respectively.

[0074] When n=1, compound (1a) is Boc-beta-alanine, and the steps include:

[0075] S1. Boc-beta-alanine (compound (1a), 51 mg, 0.268 mmol) was dissolved in dichloromethane (5 mL), and DIEA (55 μM, 0.334 mmol) and HATU (165 mg, 0.446 mmol) were added and reacted at room temperature for one hour. Palbociclib (compound (2), 100 mg, 0.223 mmol) was then added and stirred at room temperature for 12 hours. The mixture was then extracted with dichloromethane, washed with 1 M NaOH solution, and the solvent was removed by vortexing to obtain a yellow solid. The resulting compound was purified by silica gel column chromatography. After vortexing to remove the solvent, a yellow solid was obtained. The yellow solid was dissolved in a solution of petroleum ether and ethyl acetate (6:1) and filtered to obtain compound (3a) as a yellow solid with a yield of 94.3%. Compound (3a), mp: 160-176°C. 1 H-NMR (400MHz, DMSO-d6) δ10.13(s,1H),8.96(s,1H),8.08(d,J=3.0Hz,1H),7.88(d,J=9. 0Hz,1H),7.51(dd,J=9.1,3.1Hz,1H),6.73(s,1H),5.83(p,J=8.7Hz,1H),3.61(d,J=5.5H z,5H),3.22-3.10(m,6H),2.42(s,3H),2.31(s,3H),2.25(s,2H),2.00(q,J=7.0,6.6Hz,1 H), 1.89 (s, 1H), 1.82-1.72 (m, 1H), 1.58 (d, J = 5.3Hz, 2H), 1.37 (s, 9H), 0.95-0.79 (m, 1H).

[0076] S2. Compound (3a) (130 mg, 0.21 mmol), 5 mL of trifluoroacetic acid and 5 mL of dichloromethane were placed in a 25 mL eggplant-shaped flask and stirred to dissolve. The reaction was completed after 2 h, and the solvent was removed by rotary evaporation. The pH was adjusted to 7-8 with saturated sodium bicarbonate, and water and dichloromethane were added for extraction. The mixture was dried to obtain compound (4a) as a yellow solid (120 mg). The mixture was directly used for the next step without secondary purification.

[0077] S3. Compound (4a) (100 mg, 0.193 mmol) was dissolved in dichloromethane, and DIEA (21 mg, 0.164 mmol) and dansyl chloride (compound (5), 72 mg, 0.231 mmol) were added. The mixture was allowed to react at room temperature for 24 h. The reaction solution was extracted with dichloromethane, washed with 1M citric acid, dried and the solvent removed, and purified by column chromatography. After removal of the solvent, a yellow solid was obtained. The yellow solid was dissolved in a solution of n-hexane and dichloromethane (7:1) and filtered to obtain compound (6a), a small molecule fluorescent probe TYZJ-2202 targeting CDK4 / 6 protein, as a yellow solid with a yield of 48%. Small molecule fluorescent probe TYZJ-2202, mp: 160-167°C. 1 H-NMR (400MHz, DMSO-d6) δ10.14(s,1H),8.96(s,1H),8.47(d,J=8.5Hz,1H),8.28(d,J=8.7Hz,1H),8.15(dd,J=7. 3,1.2Hz,1H),8.05(d,J=3.0Hz,1H),7.95-7.85(m,2H),7.71-7.56(m,2H),7.47(dd,J=9.1,3.0Hz,1H),5.83(p,J= 8.8Hz,1H),3.51(t,J=5.1Hz,2H),3.40(d,J=4.6Hz,2H),3.05(dd,J=7.9,4.7Hz,6H),2.81(s,6H),2.45(d,J=7.0H z,2H),2.43(s,4H),2.31(s,3H),2.28-2.19(m,2H),1.89(s,2H),1.83-1.72(m,2H),1.60(dd,J=12.7,6.8Hz,2H). 13 C-NMR(100MHz,DMSO-d6)δ202.90,168.91,161.22,158.97,158.88,158.71,155.23 ,151.84,145.25,145.16,143.55,142.54,136.36,129.95,129.78,129.54,128.83 ,128.36,125.81,124.09,119.56,115.62,115.46,115.37,107.11,53.42,49.11,48.70,45.52,44.83,41.06,32.99,31.77,31.43,28.04,25.61,22.53,14.43,14.10.

[0078] When n=2, compound (1b) is N-BOC-GAMMA-aminobutyric acid, and the steps include:

[0079] S1, according to the above synthesis step S1 n = 1, compound (1b) as a raw material to produce a yellow solid (3b), the yield is 85.1%, mp: 202-214 ℃. 1 H-NMR (400MHz, DMSO-d6) δ10.13(s,1H),8.95(d,J=10.2Hz,1H),8.08(d,J=3.1Hz,1H),7. 88(d,J=9.1Hz,1H),7.58–7.44(m,1H),6.81(s,1H),5.83(t,J=8.9Hz,1H),3.60(s,6H),3. 17(s,2H),3.11(s,6H),2.95(d,J=6.3Hz,2H),2.42(s,3H),2.31(s,2H),2.25(s,2H),1.99 (s,1H),1.89(s,2H),1.77(s,2H),1.67–1.53(m,7H),1.36(d,J=10.3Hz,1H),0.85(s,1H).

[0080] S2. Compound (3b) (120 mg, 0.190 mmol), 5 mL of trifluoroacetic acid and 5 mL of dichloromethane were placed in a 25 mL eggplant-shaped flask and stirred to dissolve. The reaction was completed after 2 h, and the solvent was removed by rotary evaporation. The pH was adjusted to 7-8 with saturated sodium bicarbonate, and the mixture was extracted with water and dichloromethane and dried to obtain compound (4b) as a yellow solid (120 mg). The mixture was directly subjected to the next step without secondary purification. Compound (4b) is also referred to as intermediate 2203-04 in Examples 2 and 3.

[0081] S3. Compound (4b) (120 mg, 0.225 mmol) was dissolved in dichloromethane and DIEA (58 mg, 0.45 mmol) and dansyl chloride (compound (5), 84 mg, 0.27 mmol) were added. The reaction was allowed to react at room temperature for 24 h. The reaction solution was extracted with dichloromethane, washed with 1M citric acid, dried and the solvent removed, and purified by column chromatography. The solvent was removed to obtain a yellow solid. The yellow solid was dissolved in a 7:1 ratio of n-hexane to dichloromethane and filtered to obtain compound 6b, a small molecule fluorescent probe TYZJ-2203 targeting CDK4 / 6 protein, as a yellow solid with a yield of 46.5%. mp: 151-162°C. 1H-NMR (400MHz, DMSO-d6) δ10.14(s,1H),8.96(s,1H),8.46(d,J=8.5Hz,1H),8.31(d,J=8.6Hz,1H),8.17-8.0 4(m,2H),7.98-7.86(m,1H),7.61(dt,J=8.7,7.3Hz,2H),7.49(dd,J=9.1,3.0Hz,1H),7.25(d,J=7.5Hz,1H),5 .84(p,J=8.9Hz,1H),3.51(t,J=5.1Hz,2H),3.05(d,J=5.4Hz,4H),2.82(s,8H),2.43(s,3H),2.31(s,3H),2.3 0-2.23(m,5H),2.18(t,J=7.3Hz,2H),1.90(d,J=8.6Hz,2H),1.82-1.74(m,2H),1.57(dt,J=21.7,7.7Hz,4H). 13 C-NMR(100MHz,DMSO-d6)δ202.91,170.37,161.22,158.99,158.73,155.23,151.84 ,145.25,143.61,142.54,129.87,129.53,129.51,128.88,128.29,125.83,124.08, 119.55,115.55,107.12,60.23,53.40,49.18,48.84,45.53,44.74,42.52,42.42,41.08,31.78,31.42,29.55,28.04,25.60,25.16,22.53,21.23,14.56,14.43,14.10.

[0082] When n=3, compound (1c) is Boc-5-aminovaleric acid, and the steps include:

[0083] S1, according to the synthesis steps of S1 of this example, compound (1c) as a raw material, and Palbociclib (compound (2)) synthesized a yellow solid (3c), the yield was 74.7%, mp165-177 ℃. 1H-NMR (400MHz, DMSO-d6) δ10.12(s,1H),8.96(s,1H),8.08(d,J=3.0Hz,1H),7.88(d,J=9.0Hz,1H),7 .50(dd,J=9.1,3.1Hz,1H),6.76(s,1H),5.90–5.67(m,1H),3.61(s,4H),3.14(d,J=21.3Hz,6H),2.8 9(q,J=6.6Hz,2H),2.42(s,3H),2.34(t,J=7.5Hz,2H),2.31(s,3H),2.24(d,J=10.1Hz,2H),1.99(p, J=7.1Hz,1H),1.89(s,2H),1.84–1.68(m,1H),1.65–1.55(m,2H),1.49(q,J=7.4Hz,2H),1.37(s,9H).

[0084] S2. The product (3c) (115 mg, 0.178 mmol), 5 mL of trifluoroacetic acid and 5 mL of dichloromethane were placed in a 25 mL eggplant-shaped flask and stirred to dissolve. The reaction was completed after 2 h, and the solvent was removed by rotary evaporation. The pH was adjusted to 7-8 with saturated sodium bicarbonate, and water and dichloromethane were added for extraction. The mixture was dried to obtain compound (4c), a yellow solid (115 mg). The mixture was directly used for the next step without secondary purification.

[0085] S3. Compound (4c) (115 mg, 0.211 mmol) was dissolved in dichloromethane. DIEA (137 mg, 1.055 mmol) and dansyl chloride (compound (5), 78 mg, 0.252 mmol) were added sequentially. The mixture was allowed to react at room temperature for 24 h. The reaction solution was extracted with dichloromethane, washed with 1M citric acid, dried and the solvent removed, and purified by column chromatography. The solvent was removed to obtain a yellow solid. The yellow solid was dissolved in a 7:1 ratio of n-hexane to dichloromethane and filtered to obtain compound (6c), a small molecule fluorescent probe TYZJ-2201 targeting CDK4 / 6 protein. The yield was 48%. mp: 135-142°C. 1H-NMR (400MHz, DMSO-d6) δ10.15(s,1H),8.96(s,1H),8.46(d,J=8.5Hz,1H),8.31(d,J=8.6Hz,1H),8.15-8.06(m ,2H),7.93-7.85(m,2H),7.60(ddd,J=14.2,8.6,7.4Hz,2H),7.50(dd,J=9.1,3.0Hz,1H),7.25(d,J=7.5Hz,1H), 5.96-5.76(m,1H),3.57(s,2H),3.51(s,2H),3.18-3.06(m,7H),2.82(s,1H),2.43(s,3H),2.32(s,3H),2.20(t, J=7.0Hz,4H),1.89(s,3H),1.83-1.73(m,4H),1.66-1.55(m,2H),1.39(dq,J=12.0,5.7,4.7Hz,4H),1.23(s,1H). 13 C-NMR(100MHz,DMSO-d6)δ202.91,170.83,161.22,158.99,158.72,155.23,151.80,1 45.25,143.65,142.54,136.65,136.36,129.79,129.58,129.53,128.67,128.25,125. 86,124.04,119.63,115.55,115.52,107.11,53.41,49.37,48.95,45.53,45.02,42.68,42.58,41.12,32.01,31.78,31.42,29.27,28.03,25.60,22.53,22.24,14.43,14.10.

[0086] When n=5, compound (1d) is 7-(N-tert-butyloxycarbonylamino)heptanoic acid, and the steps include:

[0087] S1, according to the reaction steps of compound (3a) in this example, compound (1d) and Palbociclib (compound (2)) were synthesized into a yellow solid (3d) with a yield of 68.3%, mp170-177 ℃. 1H-NMR (400MHz, DMSO-d6) δ10.13(s,1H),8.95(d,J=9.2Hz,1H),8.08(d,J=3.1Hz,1H),7.88(d,J=9.1Hz, 1H),7.59–7.36(m,1H),6.81(s,1H),6.01–5.71(m,1H),3.60(s,4H),3.17(s,2H),3.11(s,4H),2.95(d, J=6.3Hz,3H),2.45-2.39(m,3H),2.36(d,J=7.3Hz,2H),2.34-2.26(m,3H),2.25(s,2H),2.03-1.94(m,2 H),1.96-1.84(m,2H),1.83-1.72(m,2H),1.68-1.55(m,4H),1.36(d,J=10.3Hz,9H),0.94-0.73(m,1H).

[0088] S2. Compound (3d) (101 mg, 0.150 mmol), 5 mL of trifluoroacetic acid and 5 mL of dichloromethane were placed in a 25 mL eggplant-shaped flask and stirred to dissolve. The reaction was completed after 2 h, and the solvent was removed by rotary evaporation. The pH was adjusted to 7-8 with saturated sodium bicarbonate, and water and dichloromethane were added for extraction. The mixture was dried to obtain compound (4d) as a yellow solid (101 mg). The mixture was directly used for the next step without secondary purification.

[0089] S3. Compound (4d) (81 mg, 0.131 mmol) was dissolved in dichloromethane, and DIEA (34 mg, 0.212 mmol) and dansyl chloride ((5), 49 mg, 0.157 mmol) were added sequentially. The mixture was allowed to react at room temperature for 24 h. The reaction solution was extracted with dichloromethane, washed with 1 M citric acid, dried and the solvent removed, and purified by column chromatography. After removal of the solvent, a yellow solid was obtained. The yellow solid was dissolved in a solution of n-hexane and dichloromethane (7:1) and filtered to obtain compound (6d), a small molecule fluorescent probe TYZJ-2204 targeting CDK4 / 6 protein, as a yellow solid with a yield of 28.6%. mp: 156-160°C. 1H-NMR (400MHz, DMSO-d6) δ10.13(s,1H),8.95(d,J=10.2Hz,1H),8.08(d,J=3.1Hz,1H),7.88(d,J=9.1Hz,1H ),7.56-7.46(m,1H),6.87-6.76(m,1H),5.91-5.77(m,1H),3.60(s,4H),3.23-3.14(m,2H),3.14-3.09(m,6 H),3.00-2.89(m,3H),2.45-2.39(m,3H),2.36(d,J=7.3Hz,3H),2.31(s,3H),2.27-2.19(m,2H),2.03-1.94 (m,2H),1.94-1.85(m,2H),1.83-1.72(m,2H),1.66-1.55(m,4H),1.36(d,J=10.3Hz,9H),0.94-0.74(m,1H). 13 C-NMR(100MHz,DMSO-d6)δ202.91,171.03,161.20,158.88,158.66,155.20,151.77,14 5.24,143.59,142.57,136.64,136.38,129.76,129.73,129.58,129.50,128.71,128.2 1,125.81,124.01,119.65,115.51,115.29,107.04,53.42,49.38,48.96,45.51,45.06,42.75,41.11,39.41,32.51,31.76,29.38,28.61,28.02,26.16,25.61,25.00,14.09.

[0090] Alternatively, the reaction scheme is shown below:

[0091]

[0092] The difference from the above case where n=1, 2, 3 or 5 is that: compound (1) is compound (1e), i.e., 3-(2-((tert-butoxycarbonyl)amino)ethoxy)propanoic acid; compound (3) is compound (3e); compound (4) is compound (4e); and compound (6) is compound (6e); and the steps include:

[0093] S1, according to the reaction steps of compound (3a) in this example, compound (1e) and Palbociclib (compound (2)) were synthesized into a yellow solid (3e) with a yield of 85.6%, mp: 197-199 ℃. 1H-NMR (400MHz, DMSO-d6) δ10.22(s,1H),8.97(s,1H),8.08(d,J=3.0Hz,1H),7.89(d,J=9.0Hz,1H),7.5 1(dd,J=9.1,3.0Hz,1H),6.81(t,J=5.8Hz,1H),5.91–5.72(m,1H),3.62(d,J=6.2Hz,5H),3.37(d,J=7. 6Hz,7H),3.18(d,J=5.0Hz,2H),3.12(d,J=4.9Hz,1H),3.09–3.04(m,2H),2.63(t,J=6.6Hz,2H),2.43( s,2H),2.31(s,2H),2.28–2.19(m,2H),1.89(s,2H),1.83–1.74(m,2H),1.63–1.54(m,2H),1.37(s,8H).

[0094] S2. The product (3e) (120 mg, 0.181 mmol) was added to 5 mL of trifluoroacetic acid and 5 mL of dichloromethane in a 25 mL eggplant-shaped flask and stirred to dissolve. The reaction was completed after 2 h, and the solvent was removed by rotary evaporation. The pH was adjusted to 7-8 with saturated sodium bicarbonate, and the product was extracted with water and dichloromethane. The product was dried to obtain 4e as a yellow solid (118 mg). The product was directly used for the next step without secondary purification.

[0095] S3. Product 4e (118 mg, 0.209 mmol) was dissolved in dichloromethane. DIEA (54 mg, 0.418 mmol) and starting material 5 (67.6 mg, 0.258 mmol) were added sequentially. The mixture was allowed to react at room temperature for 24 hours. The reaction solution was extracted with dichloromethane, washed with 1M citric acid, dried and freed from the solvent, and purified by column chromatography. The solvent was removed by swirl to yield a yellow solid. This solid was dissolved in a 7:1 ratio of hexane to dichloromethane and filtered to obtain TYZJ-2205, a small molecule fluorescent probe targeting CDK4 / 6 proteins. The yield was 78.6%, mp: 167-170°C. 1H-NMR (400MHz, DMSO-d6) δ10.18(s,1H),8.97(s,1H),8.46(d,J=8.5Hz,1H),8.32(d,J=8.6Hz,1H),8.17–8.08(m,2H),7.99(t,J=5.8Hz,1H), 7.91(d,J=9.0Hz,1H),7.61(ddd,J=11.2,8.6,7.4Hz,2H),7.50(dd,J=9.1,3.0Hz,1H),7.25(d,J=7.5Hz,1H),5.90–5.52(m,1H),3.65–3.49( m,4H),3.42(t,J=6.5Hz,2H),3.31(t,J=5.8Hz,2H),3.20–3.09(m,4H),2.98(q,J=5.8Hz,2H),2.82(s,5H),2.51(p,J=1.9Hz,1H),2.43(d,J= 4.4Hz,5H),2.32(s,4H),2.27–2.20(m,2H),2.00(s,1H),1.94–1.88(m,1H),1.82–1.74(m,1H),1.59(q,J=6.0Hz,2H),1.18(t,J=7.1Hz,1H). 13 C-NMR (101MHz, DMSO-d6) δ202.88,169.25,161.22,158.95,158.69,155.23,151.79,145. 27,143.61,142.55,136.84,136.42,129.77,129.58,129.52,128.54,128.21,125.81,124 .03,119.73,115.53,115.42,107.09,69.19,66.83,60.22,55.37,53.43,49.33,48.90,45 .52,42.72,41.13,40.63,39.37,33.00,31.76,28.03,25.60,21.22,14.09.HRMS(ESI)m / z calcd forC 41 H 50 N9O6S([M+H] + )793.3600; found 793.3599.

[0096] Example 2

[0097] The preparation method of CDK4 / 6 small molecule fluorescent probe, the reaction route is shown as follows:

[0098]

[0099] .

[0100] The reaction steps include:

[0101] S1, 7-(diethylamino)-2-oxo-2H-chromone-3-carboxylic acid reacts with sulfuryl chloride to generate a carboxylic acid derivative, which is then reacted with the compound represented by formula (III) through nucleophilic substitution to obtain the compound represented by formula (I).

[0102] Alternatively, S2, 4-(3-phenoxypropyl)-1,3-dioxane-2-en-2-one and chlorosulfonic acid are reacted by sulfonation to obtain an intermediate, the intermediate is subjected to an amidation reaction, and then reacted with the compound represented by formula (III) by nucleophilic substitution to obtain the compound represented by formula (I).

[0103] Alternatively, S3, 4-(1,2,2-triphenylvinyl)benzoic acid and the compound represented by formula (III) undergo amide condensation reaction to generate the compound represented by formula (I).

[0104] Alternatively, S4, 4-chloro-7-nitrobenzo[c][1,2,5]oxadiazole and the compound represented by formula (III) undergo an amide condensation reaction to generate the compound represented by formula (I).

[0105] Specifically, the specific steps of S1 are:

[0106] In a 50 ml round-bottom flask, 7-(diethylamino)-2-oxo-2H-chromone-3-carboxylic acid (compound 2306-01, 1.913 mmol, 500 mg) was added, followed by SOCl2 (1.913 mmol, 0.14 ml) under nitrogen and two drops of DMF. The mixture was stirred at room temperature for 1 hour. The reaction mass was then concentrated under vacuum. 5 ml of petroleum ether was added, stirred, and filtered to obtain compound 2306-02. The reaction mixture was used directly in the next step without purification. Compound 2306-02 (0.536 mmol, 135 mg) was dissolved in DMF, and after 5 minutes, DIEA (0.894 mmol, 115.5 mg) was added. After 15 minutes, HATU (0.536 mmol, 150 mg) was added, and the reaction mixture was allowed to react at room temperature for 50 minutes. Intermediate 2203-04 (0.447 mmol, 238 mg) was added to the reaction solution; the final mixture was allowed to react at room temperature for 24 hours. The product was washed with citric acid and extracted with water and dichloromethane several times. The organic layer was collected and concentrated in vacuo. The crude product was purified by silica gel column chromatography (methanol: dichloromethane = 1:30) to obtain a yellow solid TYZJ-2306, a CDK4 / 6 molecular fluorescent protein.

[0107] Probe. Yield 27.4%, mp: 215-218°C. 1 H-NMR (400MHz, DMSO-d6) δ10.20(s,1H),8.96(s,1H),8.72-8.58(m,2H),8.07(d,J=3.0Hz,1H),7.87(d,J=9.0Hz,1H) ,7.66(d,J=9.0Hz,1H),7.49(dd,J=9.1,3.1Hz,1H),6.78(dd,J=9.1,2.4Hz,1H),6.61(d,J=2.4Hz,1H),5.91-5.74(m, 1H),3.68-3.58(m,4H),3.51-3.44(m,3H),3.20-3.15(m,2H),3.10(d,J=5.8Hz,2H),2.42(s,5H),2.31(s,3H),2.27- 2.18(m,2H),1.88(s,2H),1.82-1.72(m,3H),1.58(d,J=5.6Hz,2H),1.22(s,2H),1.12(t,J=7.0Hz,6H).HRMS(ESI)m / z calcd forC 42 H 50 N9O6([M+H] + )776.3879;found 776.3875.

[0108] The specific steps of S2 are:

[0109] 4-(3-Phenoxypropyl)-1,3-dioxane-2-en-2-one (compound 2307-01, 6.47 mmol, 1 g) was slowly added to chlorosulfonic acid (6 ml) in an ice bath. After 1 hour, the solution was heated to 80°C and refluxed for 6 hours. The resulting mixture was slowly poured into ice water, filtered, and washed twice with water. The precipitate was dissolved in dichloromethane, washed with a dichloromethane:petroleum ether (7:1) solution, and dried to give 940 mg of gray crystals (compound 2307-02). Yield: 80.34%, mp: 73-76°C. 1 H-NMR (400MHz, CDCl3) δ8.21 (d, J = 7.5 Hz, 1H), 7.68 (d, J = 7.5 Hz, 1H).

[0110] The resulting intermediate 2307-02 (2.05 mmol, 520 mg) was dissolved in 10 ml of acetonitrile in an ice bath. Triethylamine (3.32 mmol, 135.34 mg) was added. After half an hour of reaction, dimethylamine hydrochloride (2.05 mmol, 135.34 mg) was added. The mixture was brought to room temperature and allowed to react for 2 hours. The reaction solution was concentrated in vacuo, and the crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 30:1) to afford 2307-03 as a white solid. Yield: 28%, mp: 90-93°C. 1 H-NMR (400MHz, CDCl3) δ7.90 (d, J = 7.4Hz, 1H), 7.49 (d, J = 7.3Hz, 1H), 2.90 (s, 6H).

[0111] Intermediate 2307-03 (0.213 mmol, 58 mg) was placed in a 50 ml glass jar and dissolved in dichloromethane. After 5 minutes, N,N-diisopropylethylamine (0.3192 mmol, 72 mg) was added. Intermediate 2203-04 (0.1912 mmol, 100 mg) was added to the reaction solution. The final mixture was reacted at room temperature for 24 hours. The mixture was extracted with dichloromethane and washed with 1 M citric acid solution. The organic layer was collected and concentrated in vacuo. The crude product was purified by silica gel column chromatography (methanol:dichloromethane = 1:30) to obtain TYZJ-2307 as a yellow solid, a CDK4 / 6 molecular fluorescent probe. Yield: 56.8%, mp: 187-191°C. 1 H-NMR (400MHz, DMSO-d6) δ10.15(s,1H),8.96(s,1H),8.46(t,J=5.6Hz,1H),8.09(d,J=2.9Hz,1H),7.89(d,J= 9.0Hz,1H),7.83(d,J=8.1Hz,1H),7.50(dd,J=9.1,3.0Hz,1H),6.37(d,J=8.2Hz,1H),5.92-5.69(m,1H),3.73- 3.57(m,4H),3.48-3.41(m,2H),3.38(d,J=7.0Hz,0H),3.22-3.09(m,4H),2.69(s,6H),2.59-2.49(m,4H),2.32 (s,3H),2.28-2.20(m,2H),2.03-1.84(m,4H),1.80-1.71(m,2H),1.59(t,J=6.0Hz,2H),1.09(t,J=7.0Hz,1H). 13C-NMR(101MHz,DMSO-d6)δ202.91,170.78,161.21,158.97,158.72,155.22,1 46.99,145.27,144.81,143.62,142.55,141.88,140.95,136.39,129.76,125. 81,115.48,107.09,105.36,99.09,65.39,53.39,49.29,48.94,44.98,43.09, 41.25,37.93,31.77,30.00,28.03,25.60,23.69,15.63,14.10.HRMS(ESI)m / z calcd for C 36 H 44 N 11 O6S([M+H] + )758.3192;found 758.3191.

[0112] The specific steps of S3 are:

[0113] 4-(1,2,2-Triphenylvinyl)benzoic acid (reactant 2308-01, 0.267 mmol, 100 mg) was placed in a 50 ml Erlenmeyer flask and dissolved in dichloromethane. After 5 minutes, DIEA (0.448 mmol, 58 mg) was added. After 15 minutes, HATU (0.336 mmol, 127 mg) was added. The reaction mixture was allowed to react at room temperature for 50 minutes. Intermediate 2203-04 (0.224 mmol, 119.31 mg) was added to the reaction solution. The final mixture was allowed to react at room temperature for 24 hours, then extracted with dichloromethane and washed with 1 M citric acid solution. The organic layer was collected and concentrated in vacuo. The crude product was purified by silica gel column chromatography (methanol:dichloromethane = 1:30) to afford TYZJ-2308 as a yellow solid, a CDK4 / 6 molecular fluorescent probe. Yield: 72.14%, mp: 178-181°C. 1H-NMR (400MHz, DMSO-d6) δ10.17(s,1H),8.96(s,1H),8.39(t,J=5.6Hz,1H),8.08(d,J=3.0Hz,1H),7.90(d,J=9.0Hz,1H),7.65–7.5 7(m,2H),7.55–7.46(m,1H),7.18-7.10(m,9H),7.04(d,J=1.8Hz,1H),7.02(d,J=1.8Hz,1H),6.99(d,J=1.9Hz,1H),6.98(d,J=2.3Hz ,2H),6.96-6.95(m,1H),5.94-5.72(m,1H),3.60(q,J=6.1Hz,4H),3.34(s,3H),3.26(q,J=6.6Hz,2H),3.16(d,J=5.0Hz,2H),3.11( d,J=5.3Hz,2H),2.43(s,3H),2.41(s,2H),2.31(s,3H),2.28-2.20(m,3H),1.96-1.84(m,2H),1.81-1.71(m,4H),1.64-1.53(m,2H). 13 C-NMR(101MHz,DMSO-d6)δ202.92,170.86,166.20,161.21,158.97,158.73,155.23,146.48,14 5.25,143.66,143.36,143.25,142.55,141.85,140.30,136.42,132.92,131.10,131.05,130.95 ,129.76,128.40,128.36,128.30,127.26,127.22,127.16,125.80,115.45,107.08,65.40,53. 39,49.26,48.95,45.03,41.21,31.78,30.21,28.03,25.61,25.16,15.64,14.11.HRMS(ESI)m / z calcd for C 55 H 55 N8O4([M+H] + )891.4341;found 891.4340.

[0114] The specific steps of S4 are:

[0115] 4-Chloro-7-nitrobenz[c][1,2,5]oxadiazole (reactant 2309-01, 0.226 mmol, 45 mg) was placed in a 50 ml glass jar and dissolved in dichloromethane. After 5 minutes, DIEA (0.376 mmol, 49 mg) was added. Intermediate 2203-04 (0.188 mmol, 100 mg) was added to the reaction solution. The resulting mixture was reacted at room temperature for 24 hours, extracted with dichloromethane, and washed with 1 M citric acid solution. The organic layer was collected and concentrated in vacuo. The crude product was purified by silica gel column chromatography (methanol:dichloromethane = 1:30) to afford TYZJ-2309 as a yellow solid, a CDK4 / 6 molecular fluorescent probe. Yield: 56.8%, mp: 207-209°C. 1 H-NMR (400MHz, DMSO-d6) δ10.14(s,1H),9.57(s,1H),8.95(s,1H),8.52(d,J=8.9Hz,1H),8.0 8(s,1H),7.89(d,J=9.0Hz,1H),7.50(d,J=9.1Hz,1H),6.46(d,J=9.0Hz,1H),5.89-5.76(m,1H ),3.62(dd,J=12.9,6.5Hz,4H),3.52(s,2H),3.24-3.08(m,4H),2.53(d,J=11.3Hz,4H),2.31( s,3H),2.24(q,J=8.8Hz,2H),2.00-1.91(m,2H),1.89(s,2H),1.78(s,2H),1.67-1.55(m,2H). 13 C-NMR(101MHz,DMSO-d6)δ202.93,170.66,161.21,158.96,158.71,155.21,145.26,144.94,144.62,143.62,142.54,138.44,136.40,129.75 ,125.80,121.10,115.47,107.08,99.62,53.40,49.26,48.93,44.96, 43.49,41.27,31.77,29.83,28.03,25.60,23.65,14.09.HRMS(ESI)m / z calcd for C 34 H 38 N 11 O6([M+H] + )694.2855;found 694.2857.

[0116] Example 3

[0117] The preparation method of CDK4 / 6 small molecule fluorescent probe, the reaction route is shown as follows:

[0118]

[0119] The reaction steps include:

[0120] S1 and compound 01 undergo substitution reaction to obtain intermediate 04.

[0121] S2 and compound 03 react to form intermediate 05, which then reacts with intermediate 04 to form intermediate 06.

[0122] S3, intermediate 06 and compound 2203-04 undergo amide condensation reaction to generate compounds TYZJ-2310-2311;

[0123] Alternatively, S4, intermediate 06 and compound 08 undergo amide condensation reaction to generate compound TYZJ-2312.

[0124] When n=2, the steps include:

[0125] S1. Dissolve 7-bromoheptanoic acid (Compound 01-a) (584 mg, 2.793 mmol) and NaI (935 mg, 6.425 mmol) in anhydrous acetone and stir overnight under N2. Remove the solvent, then dissolve the product in dichloromethane. Remove inorganic salts by filtration. Concentrate the reaction mixture and vacuum dry to obtain Compound 03-a as a yellow-white solid. The reaction mixture is used in the next reaction without further purification. Yield: 84.8%, mp: 39-41°C. 1 H-NMR (400MHz, DMSO-d6) δ12.03(s,1H),3.29(s,2H),2.22(s,2H),1.72(s,2H),1.50(s,2H),1.32(dt,J=13.1,7.3Hz,4H)

[0126] 7-Iodoheptanoic acid (02-a) (02) (10.743 mmol, 2.0166 g) was added to a 50 mL round-bottom flask, and 2,3,3-trimethylindole (compound 03, 7.16 mmol, 1.1394 g) was added;

[0127] Then, under nitrogen, 15 mL of acetonitrile was added and the mixture was refluxed at 80°C for 24 h. The acetonitrile was removed by vortexing, and a large amount of ethyl acetate was added. The reaction was stirred vigorously for 1 h to complete. The white solid was filtered to obtain product 04-a. Yield: 78.5%, mp: 175-177°C

[0128] S2. In a 50 mL eggplant-shaped flask, add 2,3,3-trimethylindole (Compound 03, 12.46 mmol, 2.0 g). Dissolve the raw material in 25 mL of acetonitrile, then add iodomethane (1.3 mmol, 2.3 g). Reflux at 80°C for 12 h. Upon completion of the reaction, a large amount of crystals precipitated from the flask. These were washed three times with acetonitrile to obtain purple crystals, Compound 05, with a yield of 94.5%. Melting point: 257-259°C. Yield: 94.5%. MP: 257-259°C. 1 H-NMR (400MHz, DMSO-d6) δ7.92-7.90(m,1H),7.85-7.82(m,1H),7.66-7.59(m,2H),3.97(s,3H),2.76(s,3H),1.53(s,6H).

[0129] 1-(6-Carboxyhexyl)-2,3,3-trimethyl-3H-indole-1-ium iodide (Compound 04-a, 6.043 mmol, 2.51 g) and malondialdehyde diphenylamine hydrochloride (5.49 mmol, 1.142 g) were added to a 50 mL two-necked flask. Under nitrogen, 5 mL of acetic acid and 5 mL of acetic anhydride were injected. The mixture was refluxed at 80°C with 5 mL of acetic acid and 5 mL of acetic anhydride. After 4 hours, the solvent was evaporated, and 1,2,3,3-tetramethyl-3H-indole-1-ium iodide (Compound 05, 5.49 mmol, 1.653 g), 4 mL of pyridine, and 4 mL of acetic acid were added to the flask. The mixture was refluxed for 2 hours. After the reaction was complete, the solvent was removed, the mixture was washed with 1M citric acid, and extracted with dichloromethane. The reaction solution was concentrated to dryness under vacuum and then purified by column chromatography using neutral alumina (petroleum ether:dichloromethane = 10:1) to obtain a dark gold solid, compound 06-a, with a yield of 92.7%. mp: 289-290°C. 1 H-NMR (400MHz, DMSO-d6) δ9.16 (s, 1H), 8.34 (t, J = 13.1Hz, 2H), 7.62 (d, J = 7. 4Hz,2H),7.45–7.36(m,4H),7.30–7.20(m,2H),6.54(t,J=12.3Hz,1H),6.29 (s,1H),6.26(s,1H),5.77(s,1H),3.60(s,5H),3.30(t,J=7.0Hz,3H),3.09( m,6H),2.70(s,2H),2.18(t,J=8.0Hz,2H),1.97-1.85(m,2H),1.68(s,12H).

[0130] S3. In a 50 mL round-bottom flask, intermediate compound 06-a (0.204 mmol, 127 mg) was dissolved in 10 mL of dry dichloromethane. DIEA (0.408 mmol, 53 mg) was added and stirred for 10 minutes. HATU (0.306 mmol, 85.7 mg) was then added to the reaction mixture. After stirring for half an hour, compound 2203-04 (0.245 mmol, 130.5 mg) was added. After reacting at room temperature for 24 hours, the compound was washed twice with citric acid and saturated brine, dried over anhydrous sodium sulfate, and filtered. The concentrate was purified by column chromatography (dichloromethane:methanol = 5:1) to obtain compound TYZJ-2310 as a blue solid powder, a CDK4 / 6 molecular fluorescent probe. Yield: 17.8%. MP: 355-357°C. 1 H-NMR (400MHz, CDCl3) δ8.79(s,1H),8.09(d,J=9.1Hz,1H),8.01(d,J=2.9Hz,1H),7.75(td,J=13.0,3.3Hz,2H),7.34–7.24(m,5H),7.1 8–7.10(m,2H),7.07–6.98(m,2H),6.68(t,J=12.5Hz,1H),6.49(d,J=5.9Hz,1H),6.20-6.07(m,2H),5.89-5.74(m,1H),5.23(s,2H),3.9 1(t,J=7.8Hz,2H),3.68(dt,J=20.7,5.1Hz,4H),3.50(s,3H),3.27(q,J=6.1Hz,2H),3.14(t,J=5.0Hz,2H),2.45(d,J=16.5Hz,5H),2.3 1-2.16(m,8H),2.05-1.92(m,2H),1.86-1.78(m,4H),1.76-1.68(m,19H),1.61(d,J=4.0Hz,16H),1.49-1.30(m,5H),1.25-1.09(m,1H). 13C-NMR (101MHz, CDCl3) δ202.79,173.74,173.04,172.95,171.63,161.42,158.03,157.24,155.55,153.18,152.90,145.24,143 .39,142.74,141.90,141.83,141.10,140.79,130.82,128.84,128.70,126.38,126.04,125.35,125.13,122.20,122.10,113.8 0,110.81,110.38,103.88,103.80,54.12,53.47,49.79,49.57,49.35,49.10,46.06,45.26,44.18,41.36,39.11,35.84,31.57 ,31.22,30.75,29.71,28.34,28.10,28.01,27.96,27.11,26.00,25.78,25.16,24.86,22.71,14.14,14.00,8.68.HRMS(ESI)m / z calcd for C 61 H 75 N 10 O4([M+H] + )1011.5969;found 1011.5972.

[0131] When n=1, the steps include:

[0132] S1. Dissolve 6-bromohexanoic acid (Compound 01-b, 2.2094 g, 11.327 mmol) and NaI (3.0999 g, 20.681 mmol) in 10 mL of anhydrous acetone and stir under N2 for 5 hours. Remove the solvent by vortexing, then dissolve the product in dichloromethane. Remove the inorganic salts by filtration, concentrate the reaction solution, and dry it in vacuo to obtain Compound 03-b as a yellow-white solid. Yield: 96%, mp: 40-41°C.

[0133] 6-Iodohexanoic acid (Compound 02-b, 10.32 mmol, 2.5 g) was added to a 50 mL round-bottom flask, along with iodopropionic acid (Compound 03) (6.885 mmol, 1.096 g). Under nitrogen, 15 mL of acetonitrile was added and the mixture was refluxed at 80°C for 24 hours. The acetonitrile was removed by vortexing, and a large amount of ethyl acetate was added. The reaction was stirred vigorously for 1 hour to complete. A white solid was obtained, which was filtered to obtain the intermediate compound 04-b. Yield: 72.8%, mp: 167-171°C.

[0134] S2. In a 50 mL eggplant-shaped flask, add 2,3,3-trimethylindole (Compound 03, 12.46 mmol, 2.0 g). Dissolve the raw material in 25 mL of acetonitrile, then add iodomethane (1.3 mmol, 2.3 g). Reflux at 80°C for 12 h. Upon completion of the reaction, a large amount of crystals precipitated from the flask and were washed three times with acetonitrile to obtain purple Compound 05. Yield: 94.5%. Melting point: 257-259°C. Yield: 94.5%. MP: 257-259°C. 1 H-NMR (400MHz, DMSO-d6) δ7.92-7.90(m,1H),7.85-7.82(m,1H),7.66-7.59(m,2H),3.97(s,3H),2.76(s,3H),1.53(s,6H).

[0135] 1-(5-Carboxypentyl)-2,3,3-trimethyl-3H-indol-1-ium iodide (Compound 04-b, 4.485 mmol, 1.8 g) and malondialdehyde diphenylamine hydrochloride (4.077 mmol, 1.052 g) were added to a 50 mL two-necked flask. Under nitrogen, 5 mL of acetic acid and 5 mL of acetic anhydride were injected. The mixture was refluxed at 80°C with 5 mL of acetic acid and 5 mL of acetic anhydride. After 4 h, the solvent was removed by vortexing, and 1,2,3,3-tetramethyl-3H-indol-1-ium iodide (Compound 05, 4.077 mmol, 1.227 g), 5 mL of pyridine, and 5 mL of acetic acid were added to the flask and refluxed for 2 h. The solvent was removed, the product was washed with 1M citric acid, and extracted with dichloromethane. The reaction solution was concentrated to dryness under vacuum and then purified by column chromatography using neutral alumina (petroleum ether:dichloromethane = 10:1) to afford a dark gold solid, compound 06-b. Yield: 87.6%. MP: 278-281°C. 1 H-NMR (400MHz, DMSO-d6) δ8.34(t,J=13.1Hz,2H),7.63(d,J=7.4Hz,2H),7.41–7.34(m,4H),7.41–7.18(m,2H),6.58(t,J=12.4Hz,1H),6.30( dd,J=22.4,13.8Hz,2H),4.10(t,J=7.3Hz,2H),3.45-3.28(m,3H),2.20(t,J=7.3Hz,2H),1.69(s,14H),1.55-1.43(m,2H),1.40-1.30(m,2H).

[0136] S3. In a 50 mL round-bottom flask, intermediate compound 06-b (0.172 mmol, 105 mg) was dissolved in 10 mL of dry dichloromethane. DIEA (0.258 mmol, 33 mg) was added and stirred for 10 minutes. HATU (0.258 mmol, 73 mg) was then added to the reaction mixture. After stirring for half an hour, compound 2203-04 (0.2065 mmol, 110 mg) was added. After reacting at room temperature for 12 hours, the compound was washed twice with citric acid and saturated brine, dried over anhydrous sodium sulfate, and filtered. The concentrate was purified by column chromatography (dichloromethane:methanol = 5:1) to obtain product TYZJ-2311 as a blue solid powder, a CDK4 / 6 molecular fluorescent probe. Yield: 42.8%. MP: 346-349°C. 1 H-NMR (400MHz, CDCl3) δ8.79(s,1H),8.09(d,J=9.1Hz,1H),8.01(s,1H),7.76(t,J=13.1Hz,2H),7.35–7.24(m,4H),7.20(s,1H),7.18– 7.11(m,2H),7.01(t,J=8.7Hz,2H),6.67(t,J=12.5Hz,1H),6.47(t,J=5.8Hz,1H),6.20-6.08(m,2H),5.84-5.75(m,1H),3.91(t,J=7.6 Hz,2H),3.74-3.58(m,4H),3.50(s,2H),3.30-3.19(m,2H),3.14(t,J=5.1Hz,2H),3.05(t,J=5.2Hz,2H),2.41(t,J=7.5Hz,5H),2.32-2 .11(m,7H),2.04-1.93(m,2H),1.87-1.77(m,4H),1.73(d,J=7.9Hz,3H),1.61(d,J=2.4Hz,14H),1.50-1.39(m,2H),1.21-1.16(m,1H). 13C-NMR (101MHz, CDCl3) δ198.02,168.69,168.29,166.81,156.67,153.30,152.51,150.79,148.38,148.12,140.5 1,138.65,137.97,137.15,137.08,136.30,136.02,126.01,124.08,123.94,121.70,120.63,120.39,117.45,117 .36,108.93,106.00,105.59,99.19,99.02,72.61,72.30,71.98,49.36,45.09,44.87,44.61,44.36,40.53,36.61 ,34.36,31.42,26.81,26.45,26.04,23.35,23.25,23.21,22.29,21.71,21.03,20.50,20.11,9.24.HRMS(ESI)m / z calcd for C 60 H 73 N 10 O4([M+H] + )997.5811; found 997.5810.

[0137] Preferably, when n=1, step S3 is not used to react with the intermediate compound 2203-04; instead, step S4 is used to react with palbociclib:

[0138] S4. Dissolve compound 06-b (0.285 mmol, 0.127 g) in 10 mL of dry dichloromethane in a 50 mL round-bottom flask. Add DIEA (0.356 mmol, 0.046 g) and stir for 10 minutes. Add HATU (0.308 mmol, 0.086 g) and stir for half an hour. Then, add palbociclib (compound 08, 0.2374 mmol, 0.145 g). After reacting at room temperature for 12 hours, wash the compound twice with citric acid and saturated brine, dry over anhydrous sodium sulfate, and filter. The concentrate is purified by column chromatography (dichloromethane:methanol = 20:1) to obtain TYZJ-2312 as a blue solid powder, a CDK4 / 6 molecular fluorescent probe. Yield: 32%. MP: 340-345°C. 1H-NMR (400MHz, DMSO-d6) δ10.09(s,1H),8.94(s,1H),8.32(td,J=13.1,5.7Hz,2H),8.06(d,J=3.1Hz,1H),7.85(d,J=9.0Hz,1H),7.60(dd,J=12.0 ,7.3Hz,2H),7.51–7.44(m,1H),7.39(d,J=4.1Hz,2H),7.36-7.33(m,2H) ,7.25-7.17(m,2H),6.55(t,J=12.3Hz,1H),6.35-6.17(m,2H),5.88-5.7 1(m,1H),4.18-4.04(m,2H),3.58(d,J=5.8Hz,7H),3.10(dt,J=10.2,4.8 Hz,4H),2.43(s,2H),2.36(t,J=7.2Hz,2H),2.31(s,2H),2.29-2.19(m,2 H),1.89(s,2H),1.76(dd,J=11.9,6.6Hz,4H),1.67(d,J=10.6Hz,11H),1.59(dd,J=9.9,4.2Hz,4H),1.41(d,J=7.5Hz,2H),1.24(d,J=10.9Hz,1H). 13 C-NMR(101MHz,DMSO-d6)δ203.03,173.67,173.01,171.03,161.17,158.74,158.55,155.18,154.46,14 4.95,143.52,143.12,142.52,142.49,141.54,141.41,129.92,128.90,128.73,125.75,125.11,122.91 ,122.71,115.51,111.53,111.43,107.26,103.72,103.48,53.36,49.32,49.29,48.87,45.09,43.70,4 1.15,32.25,31.78,31.47,28.04,27.63,27.40,27.24,26.17,25.63,24.98,14.13.HRMS(ESI)m / zcalcd for C 56 H 66 N9O3([M+H] + )912.5284; found 912.5288.

[0139] Example 4

[0140] Optical activity assay

[0141] This example provides spectral data of the probe molecule.

[0142] The three series of CDK4 / 6 molecular fluorescent probes obtained in Examples 1 to 3 were dissolved in PBS buffer at pH = 7.4 to obtain solutions with a concentration of 10 μM. The fluorescence excitation spectra, fluorescence emission spectra and maximum ultraviolet absorption wavelength of the three series of probes were obtained using a microplate reader and an ultraviolet spectrophotometer. The results are shown in Tables 1 and Figure 1 As shown, Figure 1 In the figure, (a) shows the fluorescence excitation spectrum (EX) of the fluorescent probes TYZJ-2201 to 2205, (b) shows the fluorescence emission spectrum (EM) of the fluorescent probes TYZJ-2201 to 2205, (c) shows the fluorescence excitation spectrum (EX) of the fluorescent probe TYZJ-2306, (d) shows the fluorescence emission spectrum (EM) of the fluorescent probe TYZJ-2306, (e) shows the fluorescence excitation spectrum (EX) of the fluorescent probe TYZJ-2307, and (f) shows the fluorescence emission spectrum (EM) of the fluorescent probe TYZJ-2307. , (g) represents the fluorescence excitation spectrum (EX) diagram of the fluorescent probe TYZJ-2308, (h) represents the fluorescence emission spectrum (EM) diagram of the fluorescent probe TYZJ-2308, (i) represents the fluorescence excitation spectrum (EX) diagram of the fluorescent probe TYZJ-2309, (j) represents the fluorescence emission spectrum (EM) diagram of the fluorescent probe TYZJ-2309, (k) represents the fluorescence excitation spectrum (EX) diagram of the fluorescent probe TYZJ-2310~2312, and (l) represents the fluorescence emission spectrum (EM) diagram of the fluorescent probe TYZJ-2310~2310.

[0143] Table 1

[0144] Probe molecules UV absorption wavelength (nm) Fluorescence excitation wavelength (nm) Fluorescence emission wavelength (nm) TYZJ-2201 384.5 371.4 527.8 TYZJ-2202 375.5 370.0 526.2 TYZJ-2203 379.0 371.2 530.0 TYZJ-2204 382.0 369.0 529.0 TYZJ-2205 378.5 368.4 530.0 TYZJ-2306 428.0 415.6 481.8 TYZJ-2307 388.0 371.2 532.0 TYZJ-2308 370.5 637.8 528.0 TYZJ-2309 360.0 483.8 536.6 TYZJ-2310 610.0 / 645.0 641.4 658.0 TYZJ-2311 610.0 / 647.0 642.8 659.0 TYZJ-2312 615.0 / 647.0 642.4 657.8

[0145] Example 5

[0146] Enzyme activity assay

[0147] This example demonstrates the affinity of the probe molecule for the two kinases.

[0148] All enzymatic reactions in this example were carried out at 30°C for 40 minutes. A reaction mixture was prepared containing 40 mM Tris (tromethamine, pH 7.4), 10 mM MgCl2 (magnesium chloride), 0.1 mg / ml BSA (bovine serum albumin), 1 mM DTT (dithiothreitol), 10 μM ATP (adenosine triphosphate), 0.2 μg / ml kinase (one of CDK4 or CDK6), and 100 μM lipid substrate (in this example, the lipid substrate was a commercially available lipid substrate for PI-3 kinase activity assay). A dilution solution was prepared by diluting the compound (selected from one of the various fluorescent probes prepared in Examples 1-3) with 10% dimethyl sulfoxide. 5 μl of the dilution solution was then added to 50 μl of the reaction mixture, resulting in a final dimethyl sulfoxide concentration of 1% in all reactions.

[0149] The assay is performed using the Kinase-Glo Plus Luminescent Kinase Assay Kit, which measures kinase activity by quantifying the amount of ATP remaining in the solution after the kinase reaction. The luminescent signal generated by the assay is related to the amount of ATP present and inversely proportional to the amount of kinase activity. 50 Values ​​were calculated by normalized dose-response fit nonlinear regression using Prism GraphPad software.

[0150] Table 2 shows the affinity results of the target compounds in Example 4 for cell cycle-dependent kinase 4 / 6; among them, there are three series: TYZJ-2201 to TYZJ-2205 are prepared in Example 1, TYZJ-2306 to TYZJ-2309 are prepared in Example 2, and TYZJ-2310 to TYZJ-2312 are prepared in Example 3.

[0151] Table 2

[0152] Probe molecules <![CDATA[CDK4 IC 50 (nM)]]> <![CDATA[CDK6 IC 50 (nM)]]> TYZJ-2201 324 548 TYZJ-2202 1595 1812 TYZJ-2203 151 193 TYZJ-2204 2040 3611 TYZJ-2205 2008 3613 TYZJ-2306 284 874 TYZJ-2307 658 1055 TYZJ-2308 1936 5135 TYZJ-2309 90 156 TYZJ-2310 445 1067 TYZJ-2311 170 267 TYZJ-2312 99 184 Palbociclib 11 17

[0153] As can be seen, these three series of probes have affinity for CDK4 / 6 proteins. Within each series, there is a group of compounds with good enzymatic activity. Among the three series of probes, TYZJ-2203 from Series 1 outperforms the probes in the same series, indicating that shortening the carbon chain of the linker group may facilitate the binding of the probe molecule to CDK4 / 6. TYZJ-2309 from Series 2 has a better affinity for CDK4 / 6 than the probes in the same series. We believe that the introduction of a fluorophore with a nitro group may facilitate binding to CDK4 / 6, but the toxicity of the nitro group cannot be ruled out. TYZJ-2312 from Series 3 has a stronger affinity for CDK4 / 6 than the probes in the same series, indicating that reducing the carbon chain of the linker group may facilitate the binding of the probe molecule to CDK4 / 6. This has important guiding significance for the further development of CDK4 / 6 probes with better activity.

[0154] Example 6

[0155] Cell viability assay

[0156] This example shows the effects of the probe molecules TYZJ-2201 to TYZJ-2205 obtained in Example 1 on the cell activity of human breast cancer cells MCF-7 and MDA-MB-231.

[0157] Logarithmic phase breast cancer cells MCF-7 and MDA-MB-231 were inoculated into 96-well plates respectively. After the cells adhered to the wall, the probe molecules TYZJ-2201 to TYZJ-2205 obtained in Example 1 were added in a concentration gradient. Three replicate wells were set for each concentration of each probe molecule. In addition, at least six wells were left in each 96-well plate as a blank control group. Because the evaporation of the outermost circle of the 96-well plate was too fast, in order to avoid the experimental error caused by it, we added 100 μL PBS to the outermost circle of the 96-well plate and then cultured it for another 72 hours. After that, 10 μL CCK-8 (cell counting reagent) working solution was added to each well, incubated for 30 minutes, and the OD value was read at a wavelength of 450 nm with an enzyme reader. The data are shown as follows. Figure 2 As shown, (a) shows the results of the cell activity effect on breast cancer cells MDA-MB-231, and (b) shows the results of the cell activity effect on breast cancer cells MCF-7.

[0158] pass Figure 2 It can be seen from the data that the probe molecules TYZJ-2201~TYZJ-2205 have no obvious cytotoxicity to MCF-7 and MDA-MB-231, which is conducive to the subsequent biological evaluation experiments.

[0159] Example 7

[0160] Cell cycle assay

[0161] This example shows the effects of the probe molecules TYZJ-2201 to TYZJ-2205 obtained in Example 1 on the cell cycles of breast cancer cells MCF-7 and MDA-MB-231.

[0162] Logarithmic-phase breast cancer cells, MCF-7 and MDA-MB-231, were seeded into 6-well plates. After the cells adhered, the probes obtained in Example 1 were added to the wells at a concentration of 5 μM per well. After 24 hours of incubation, the cells were harvested, washed with PBS, centrifuged, and fixed with 75% ethanol for at least 2 hours. Following fixation, the cells were washed with PBS, centrifuged, and incubated in the dark for 30 minutes with RNase inhibitors and PI dye before analysis on a flow cytometer. The resulting data are shown in Tables 3 and 4. Table 3 shows the cell cycle arrest of MDA-MB-231 cells by probe molecules TYZJ-2201 to TYZJ-2205, while Table 4 shows the cell cycle arrest of MCF-7 cells by probe molecules TYZJ-2201 to TYZJ-2205.

[0163] Table 3

[0164]

[0165]

[0166] Table 4

[0167]

[0168] As shown in Tables 3 and 4, the experimental groups containing probe molecules TYZJ-2201 to TYZJ-2205 showed G1 / S phase arrest in both breast cancer cell lines compared to the control group of normally growing tumor cells. This indicates that the probe molecules obtained in Example 1 can inhibit the transition from G1 to S phase, with a more pronounced arresting effect in MDA-MB-231 cells. This cycle arrest experiment was conducted to initially verify whether the positive drug Palbociclib, after structural modification, still possesses cycle arrest properties, and to select suitable compounds and cell lines for subsequent experiments.

[0169] Example 8

[0170] Cell Imaging Studies Using Probe Molecules

[0171] This example provides the cell imaging effects of the probe molecules TYZJ-2201 to TYZJ-2205 obtained in Example 1 on breast cancer cells MDA-MB-231.

[0172] Logarithmic phase breast cancer cells MDA-MB-231 were inoculated into a confocal microplate, and after the cells adhered to the wall, one of the probe molecules TYZJ-2201 to TYZJ-2205 was added at a concentration of 10 μM ( Figure 3 denoted as probe in the figure), and acridine orange dye ( Figure 3 After staining for 20 minutes, the cells were photographed under a confocal microscope. Figure 3shown.

[0173] pass Figure 3 It can be seen that the probe molecules TYZJ-2201 to TYZJ-2205 and acridine orange dye all showed strong fluorescence signals, indicating that they can be used to mark breast cancer cells.

[0174] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A CDK4 / 6 small molecule fluorescent probe, characterized in that: It has the structure shown in formula (I): Formula (I); Wherein, R1 is selected from any one of the following structures: or ; n=1, 2, 3, or 5.

2. A CDK4 / 6 small molecule fluorescent probe, characterized in that: It has the structure shown in formula (I): Formula (I); Wherein, R1 is selected from any one of the following structures: 、 、 、 or ; m=1 or 2.

3. A method for preparing a CDK4 / 6 small molecule fluorescent probe according to claim 1 or 2, characterized in that: The reaction route is shown below: , n=1,2,3 or 5; Alternatively, the reaction scheme is shown below: ； Alternatively, the reaction scheme is shown below: ； Alternatively, the reaction scheme is shown below: ； Alternatively, the reaction scheme is shown below: ； Alternatively, the reaction scheme is shown below: ； Alternatively, the reaction scheme is shown below: , m=1 or 2.

4. The method for preparing the CDK4 / 6 small molecule fluorescent probe according to claim 3, wherein: Compound (1) reacts with Palbociclib in the presence of DIEA and HATU to generate compound (3). Compound (3) reacts with trifluoroacetic acid to remove the protecting group, and then undergoes an amide condensation reaction with dansyl chloride in the presence of DIEA to generate compound (6).

5. The method for preparing the CDK4 / 6 small molecule fluorescent probe according to claim 3, wherein: Compound 2306-01 was reacted with SOCl2 and DMF under nitrogen to obtain carboxylic acid derivative 2306-02. Carboxylic acid derivative 2306-02 was sequentially added with DIEA, HATU and compound 2203-04 to carry out nucleophilic substitution reaction to generate compound TYZJ-2306.

6. The method for preparing the CDK4 / 6 small molecule fluorescent probe according to claim 3, wherein: Compound 2307-01 was added with chlorosulfonic acid in an ice bath, followed by heating under reflux to obtain intermediate 2307-02. Triethylamine was added to intermediate 2307-02 in an ice bath, followed by the addition of dimethylamine hydrochloride and N,N-diisopropylethylamine to obtain compound 2307-03. Compound 2203-04 was then added for nucleophilic substitution to produce compound TYZJ-2307.

7. The method for preparing the CDK4 / 6 small molecule fluorescent probe according to claim 3, wherein: Compound 2308-01 was sequentially added with N, N-diisopropylethylamine and HATU to carry out nucleophilic substitution reaction with compound 2203-04 to generate compound TYZJ-2308.

8. The method for preparing the CDK4 / 6 small molecule fluorescent probe according to claim 3, wherein: Compound 2309-01 was added with N, N-diisopropylethylamine to undergo a nucleophilic substitution reaction with compound 2203-04 to generate compound TYZJ-2309.

9. Use of the CDK4 / 6 small molecule fluorescent probe according to any one of claims 1 to 2 in the preparation of a product for identifying CDK4 / 6 protein.

10. Use of the CDK4 / 6 small molecule fluorescent probe according to any one of claims 1 to 2 in the preparation of a cell cycle arrest product.

11. Use of the CDK4 / 6 small molecule fluorescent probe according to any one of claims 1 to 2 in the preparation of a cancer cell fluorescence imaging product.

Images