A BRD4 / PLK1 dual-target small molecule fluorescent probe and its preparation method and application

Abstract

The present invention discloses a BRD4 / PLK1 dual-target small molecule fluorescent probe and its preparation method and application, belonging to the field of biomedicine technology. The BRD4 / PLK1 dual-target small molecule fluorescent probe provided by the present invention has a structure shown in Formula I or a structure shown in Formula II. It has strong biological activity, high affinity with BRD4 / PLK1 family proteins, and high sensitivity. It can be used as a probe to identify BRD4 family proteins and for research on related physiological, pathological, and related diseases; it can be used for high-throughput screening of BRD4 / PLK1 dual-target inhibitors and their application in anti-tumor aspects; it can also be used for labeling BRD4 / PLK1 family proteins and tumor cells or tissues in which they are highly expressed. The probe of the present invention can be used as a probe to identify BRD4 / PLK1 dual-target proteins and BRD4 / PLK1 dual-target proteins have broad application prospects in physiological, pathological, and related diseases. In addition, the preparation method of the BRD4 / PLK1 dual-target small molecule fluorescent probe provided by the present invention has mild reaction conditions, cheap and readily available raw materials, and simple operation and post-processing.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a BRD4 / PLK1 dual-target 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] BRD4, a member of the bromodomain and extraterminal (BET) protein family, is a transcriptional and epigenetic regulator that plays a key role in embryogenesis and cancer development. It is widely recognized in cancer for its role in super enhancer (SE) organization and regulation of oncogene expression. BRD4 inhibition shortens communication between SEs and target promoters, subsequently leading to cell-specific repression of oncogenes to which cancer cells are addicted and cell death. PLK1 is a member of the Polo-like kinases (PLKs) family, which consists of five homologs: PLK1, PLK2, PLK3, PLK4, and PLK5. It is a conserved serine / threonine kinase that is an important regulator of mitotic progression and crucial for maintaining genomic stability. Overexpression of PLK1 is a hallmark of many types of human cancer, including melanoma, ovarian cancer, breast cancer, prostate cancer, thyroid cancer, and glioma, and is associated with chemotherapy resistance and poor patient prognosis.

[0004] Targeted selective inhibitors may work well in vitro, but upregulation of compensatory signaling pathways often compromises their efficacy in cells. Rationally designed multidrug therapies, where a single synthetic agent is used to identify several key biological targets, are expected to provide more effective drugs in cells and overcome the shortcomings associated with multidrug regimens. This involves targeting multiple protein targets from different families involved in tumorigenesis. Polo-like kinase 1 (PLK1) and bromodomain 4 (BRD4) both target the MYC pathway. They are also complexly involved in mitosis. Their dual inhibition by a single drug molecule may provide a platform for new cancer strategies. Therefore, research on small molecule inhibitors targeting the dual BRD4 / PLK1 target has become a research hotspot for anti-tumor drugs in recent years.

[0005] Simultaneous inhibition of multiple cancer-driving kinases is an established strategy to improve the durability of clinical responses to targeted therapies. However, the difficulty in discovering kinase inhibitors with appropriate multitarget profiles necessitates the use of combination therapies. Epigenetic reader domains of the bromodomain family have recently emerged as novel targets for cancer therapy. Furthermore, structure-activity relationships and cocrystal structures have identified design features that provide a general platform for the rational design of dual-kinase bromodomain inhibitors. This “dual-targeting single-agent” approach could, in principle, offer the same benefits as combination therapy (e.g., a larger therapeutic window, more durable responses) while also minimizing some of the liabilities of combination approaches, including complex and lengthy clinical studies, the potential for synergistic non-mechanistic toxicities and drug-drug interactions, and high disposal costs.

[0006] Small molecule fluorescent probes have the characteristics of rapidity, sensitivity, high throughput and easy automation, and play an important role in protein labeling and imaging technology. Currently, small molecule fluorescent probes have been widely used in the biological and pharmacological detection of important biomolecules such as proteins and nucleic acids, and are of great significance to the development of disease mechanism exploration, clinical diagnosis and drug screening.

[0007] The inventors discovered that due to a lack of design and synthesis methods, there are currently no small-molecule fluorescent probes for studying the interaction between the BRD4 / PLK1 dual targets. Therefore, to gain a deeper understanding of dual inhibition of the BRD4 / PLK1 dual targets and to discover more effective inhibitors, it is urgent to develop a small-molecule fluorescent probe that can bind to both BRD4 and PLK1 targets. Summary of the Invention

[0008] In response to the needs of the prior art, the present invention aims to provide a BRD4 / PLK1 dual-target small molecule fluorescent probe and a preparation method thereof. The fluorescent probe has significant optical activity and biological activity and is suitable for the study of physiology, pathology and related diseases.

[0009] Specifically, the present invention provides the following technical solutions:

[0010] In a first aspect of the present invention, a BRD4 / PLK1 dual-target small molecule fluorescent probe or a stereoisomer or a pharmaceutically acceptable salt thereof is provided. The BRD4 / PLK1 dual-target small molecule fluorescent probe has a structure shown in Formula I or Formula II:

[0011]

[0012] n=1-3, n can be 1, 2 or 3;

[0013] Wherein, R is selected from the following structures:

[0014]

[0015] Preferably, the pharmaceutically acceptable salts are derivatives modified with groups to improve chemical, physical and chemical properties, generally including salts formed with inorganic salts such as hydrochloric acid, sulfuric acid, nitric acid or hydrobromic acid, and salts formed with organic acids such as methanesulfonic acid, toluenesulfonic acid, citric acid or trifluoroacetic acid.

[0016] In a second aspect of the present invention, a method for preparing the above-mentioned BRD4 / PLK1 dual-target small molecule fluorescent probe is provided, comprising the following steps:

[0017] S1, subjecting the compound represented by Formula III to an amidation reaction with the compound represented by Formula IV to obtain the compound represented by Formula I;

[0018]

[0019] S2, subjecting the compound represented by Formula V to an amidation reaction with the compound represented by Formula VI to obtain the compound represented by Formula II;

[0020]

[0021] Preferably, in step S1, the preparation method of the compound represented by formula III is: reacting (R)-2-chloro-8-cyclopentyl-7-ethyl-5-methyl-7,8-dihydropterin-6(5H)-one with 4-amino-3-methoxybenzoic acid to obtain the compound.

[0022] Preferably, in step S1, the preparation method of the compound represented by formula IV is: reacting dansyl chloride with N-tert-butyloxycarbonyl-1,3-propylenediamine to obtain compound 6, and performing a deprotection reaction on compound 6 to obtain the compound represented by formula IV;

[0023]

[0024] Preferably, in step S2, the preparation method of the compound represented by formula V is: subjecting the compound represented by formula III to an amide condensation reaction with tert-butyl 4-aminopiperidine-1-carboxylate to obtain compound 8, removing the Boc protecting group of compound 8 in an ethyl acetate solution of hydrogen chloride to obtain intermediate 9, subjecting intermediate 9 to an amide condensation reaction with 4-(tert-butoxycarbonyl)aminobutyric acid to obtain compound 10, and subjecting compound 10 to a deprotection reaction to obtain the compound represented by formula V;

[0025]

[0026] n=1~3, n can be 1, 2 or 3.

[0027] The third aspect of the present invention provides use of the BRD4 / PLK1 dual-target small molecule fluorescent probe described in the first aspect in preparing a reagent or kit for recognizing BRD4.

[0028] The fourth aspect of the present invention provides the use of the BRD4 / PLK1 dual-target small molecule fluorescent probe described in the first aspect as a marker for tumor cells or tissues where the BRD4 / PLK1 is highly expressed.

[0029] The fifth aspect of the present invention provides the use of the BRD4 / PLK1 dual-target small molecule fluorescent probe described in the first aspect in high-throughput screening of BRD4 inhibitors.

[0030] Compared with the prior art, the present invention has achieved the following beneficial effects:

[0031] (1) The BRD4 / PLK1 dual-target small molecule fluorescent probe prepared by the present invention has strong biological activity, high affinity with BRD4 / PLK1 family proteins, and high sensitivity. It can be used as a probe to identify BRD4 family proteins and to study related physiology, pathology and related diseases, and has broad application prospects.

[0032] (2) The BRD4 / PLK1 dual-target small molecule fluorescent probe prepared by the present invention can be used for high-throughput screening of BRD4 / PLK1 dual-target inhibitors and their application in anti-tumor; it can also be used to label BRD4 / PLK1 family proteins and their highly expressed tumor cells or tissues.

[0033] (3) The preparation method of the BRD4 / PLK1 dual-target small molecule fluorescent probe provided by the present invention has mild reaction conditions, cheap and easily available raw materials, and simple operation and post-processing. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0035] Figure 1 This is the general structural formula of the BRD4 / PLK1 dual-target small molecule fluorescent probe;

[0036] Figure 2 is the UV absorption spectrum of fluorescent probes L1 to L4;

[0037] Figure 3 is the fluorescence emission spectrum of fluorescent probes L1 to L4;

[0038] Figure 4 is the fluorescence excitation spectrum of fluorescent probes L1 to L4;

[0039] Figure 5This is the cell activity detection diagram of fluorescent probe molecules L1 to L4;

[0040] Figure 6 Confocal cell imaging of fluorescent probes L1 to L4;

[0041] Figure 7 This is the cycle map data of the fluorescent probe L1 in breast cancer cells MDA-MB-231;

[0042] Figure 8 This is the cycle map data of the fluorescent probe L2 in breast cancer cells MDA-MB-231;

[0043] Figure 9 This is the cycle map data of the fluorescent probe L3 in breast cancer cells MDA-MB-231;

[0044] Figure 10 This is the cycle map data of the fluorescent probe L4 in breast cancer cells MDA-MB-231;

[0045] Figure 11 The blank control group is the cycle map data of breast cancer cells MDA-MB-231;

[0046] Figure 12 This is a 2D image of the docking of fluorescent probe L1 and BRD4 / PLK1 protein;

[0047] Figure 13 2D image of the docking of fluorescent probe L2 and BRD4 / PLK1 protein;

[0048] Figure 14 This is a 2D image of the docking of fluorescent probe L3 and BRD4 / PLK1 protein;

[0049] Figure 15 This is a 2D image of the docking of fluorescent probe L4 and BRD4 / PLK1 protein. DETAILED DESCRIPTION

[0050] 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.

[0051] In the following examples, the abbreviations represent the following meanings: DMSO: dimethyl sulfoxide; DIPEA: N,N-diisopropylethylamine; HATU: 2-(7-azabenzotriazole)-N,N,N,N-tetramethyluronium hexafluorophosphate; CDCl3: deuterated chloroform; 1 HNMR: hydrogen nuclear magnetic resonance; 13C NMR: carbon nuclear magnetic resonance spectroscopy; HRMS (ESI): high-resolution mass spectrometry (electrospray ionization);

[0052] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0053] Example 1

[0054] This example provides the preparation process of the compound of formula I and the efficacy verification data.

[0055] (1) Preparation of compound of formula III

[0056]

[0057] (R)-2-chloro-8-cyclopentyl-7-ethyl-5-methyl-7,8-dihydropterin-6(5H)-one (Compound 1, 0.294 g, 1 mmol) and 4-amino-3-methoxybenzoic acid (Compound 2, 0.25 g, 1.5 mmol) were placed in a round-bottom flask. Concentrated hydrochloric acid (0.178 mL, 15 mmol), methanol (0.35 mL, 30 mmol), and water (1.4 mL, 0.12 mol) were added, and the mixture was refluxed at 100°C for 48 h. The solvent was evaporated, and the mixture was extracted with dichloromethane. The mixture was dried, concentrated, and purified by column chromatography (dichloromethane:methanol = 15:1) to afford compound III as a white solid in a 73% yield with an mp (melting point) of 255-258°C. 1 H NMR (400MHz, DMSO) δ8.50 (d, J = 8.4Hz, 1H), 7.86 (s, 1H), 7.69 (s, 1H), 7.57 (d d,J=8.5,1.8Hz,1H),5.75(s,3H),4.35(p,J=8.4Hz,1H),4.25(dd,J=7.6,3. 6Hz,1H),4.10(s,5H),3.94(s,3H),3.25(s,3H),3.17(s,14H),2.02(d,J=7. 6Hz, 1H), 1.95-1.86 (m, 1H), 1.77 (qd, J = 7.2, 3.4Hz, 3H), 1.70-1.57 (m, 3H).

[0058] (2) Preparation of compound of formula IV

[0059]

[0060] 1) Preparation of Intermediate 6

[0061] Dansyl chloride (compound 4, 0.26 g, 1 mmol) and N-tert-butyloxycarbonyl-1,3-propanediamine (compound 5, 0.18 g, 1 mmol) were dissolved in 10 mL of dichloromethane, and 0.1 mL of triethylamine was added. The reaction mixture was stirred at room temperature for 6 h, extracted with dichloromethane, dried, concentrated, and purified by column chromatography (petroleum ether:ethyl acetate = 5:1) to afford 6 as a light yellow oil in 100% yield. 1 H NMR (400MHz, DMSO) δ8.46(dt,J=8.6,1.1Hz,1H),8.30(d,J=8.6Hz,1H),8.10(dd,J=7.3,1.3Hz,1H),7.84(t,J=5.9Hz,1H),7.61(ddd,J=10.9,8 .6,7.4Hz,2H),7.26(dd,J=7.6,0.9Hz,1H),6.69(t,J=5.8Hz,1H),2.83(s,8H),2.76(dt,J=7.9,6.2Hz,2H),1.47(p,J=7.0Hz,2H),1.33(s,9H).

[0062] 2) Compound 6 was added to 10 mL of saturated hydrogen chloride in ethyl acetate and stirred at 0°C in an ice bath for 12 h. After filtration and drying, the compound of formula IV was obtained and directly used in the next reaction without purification.

[0063] (3) Preparation of compound of formula I

[0064]

[0065] The compound of formula III (0.21 g, 0.5 mmol) was dissolved in 10 mL of dry dichloromethane. DIPEA (0.19 g, 1.5 mmol) was added in an ice bath at 0°C. After reacting for 10 min, HATU (0.23 g, 1.2 mmol) was added. After stirring for half an hour after the reaction solution became turbid, the compound of formula IV (0.19 g, 0.55 mmol) was added. The reaction solution was stirred at room temperature for 6 h, extracted with dichloromethane, dried and the solvent was removed by rotary evaporation. The pure final product of formula I (probe molecule L1) was obtained by column chromatography (dichloromethane:methanol = 15:1) as a white solid with a yield of 63% and mp: 133-135°C. 1H NMR (400MHz, DMSO) δ8.43 (dd, J=17.6, 8.4Hz, 2H), 8.34-8.23 (m, 2H), 8.10 (dd, J=7.3, 1. 2Hz,1H),7.94(d,J=5.8Hz,1H),7.84(s,1H),7.64-7.54(m,3H),7.42(d,J=8.6Hz,2H),7 .25(d,J=7.6Hz,1H),4.35(p,J=8.4Hz,1H),4.24(dd,J=7.6,3.6Hz,1H),3.92(s,3H),3. 25(s,3H),3.20(q,J=6.6Hz,2H),2.82(s,8H),2.03-1.59(m,12H),0.76(t,J=7.4Hz,3H). 13 C NMR (100MHz, DMSO) δ166.20,163.39,154.79,151.94,151.80,147.14,138.80,136.42,132.61,129.83,129.52,128.67,128.31,126.90 ,124.04,120.37,119.55,116.53,115.58,109.54,60.26,58.82,56.45,45.52,37.11,30.25,29.18,28.85,28.26,26.95,23.64,9.32. HRMS(ESI)m / z calcd forC 37 H 47 N8O5S([M+H] + )715.3390; found 715.33846.

[0066] Example 2

[0067] This example provides the preparation process of the compound of formula II and the effect verification data.

[0068] (1) Preparation of compound of formula V

[0069]

[0070] 1) Preparation of Intermediate 8

[0071] Compound III (0.269 g, 1 mmol) was dissolved in 10 mL of dry dichloromethane. DIPEA (0.19 g, 1.5 mmol) was added in an ice bath. After 10 min of reaction, HATU (0.23 g, 1.2 mmol) was added. The reaction solution became turbid and continued stirring for half an hour. Tert-butyl 4-aminopiperidine-1-carboxylate (0.261 g, 1.5 mmol) was then added. The reaction solution was stirred overnight, extracted with dichloromethane, dried, and the solvent removed by rotary evaporation. Column chromatography (dichloromethane:methanol = 40:1) afforded 8 as a white solid in 80% yield, mp: 148-152°C. 1 H NMR(400MHz,DMSO)δ8.40(d,J=8.8Hz,1H),8.13(d,J=7.9Hz,1H),7.85(s,1H),7.7 0(s,1H),7.52-7.44(m,2H),4.34(q,J=8.3Hz,1H),4.26(dd,J=7.5,3.5Hz,1H),3. 99(dd,J=7.9,3.8Hz,2H),3.94(s,3H),3.25(s,3H),2.91(s,3H),2.84(s,1H),2.0 7-1.76(m,10H),1.70-1.55(m,4H),1.41(d,J=5.9Hz,12H),0.76(t,J=7.4Hz,3H).

[0072] 2) Preparation of Intermediate 9

[0073] Compound 8 was added to 8 mL of saturated hydrogen chloride in ethyl acetate and stirred for 12 h. The crude product 9 was obtained after filtration and drying, and was directly used for the next reaction without purification.

[0074] 3) Preparation of Intermediate 10

[0075] Compound 9 (0.54 g, 1 mmol) was dissolved in 10 mL of dry dichloromethane. DIPEA (0.19 g, 1.5 mmol) was added in an ice bath. After 10 minutes of reaction, HATU (0.23 g, 1.2 mmol) was added. The reaction mixture became turbid and continued stirring for half an hour. 4-(tert-Butyloxycarbonyl)aminobutyric acid (0.3 g, 1.5 mmol) was then added. The reaction mixture was stirred overnight and extracted with dichloromethane. The solvent was then dried and removed by rotary evaporation. Intermediate 10 was obtained by column chromatography (dichloromethane:methanol = 20:1).

[0076] The product 10 (n=1) is a light yellow solid with a yield of 84% and mp: 136-138°C. 1H NMR (400MHz, DMSO) δ8.42(d,J=8.7Hz,1H),8.10(d,J=7.8Hz,1H),7.85(s,1H),7.60(s,1H),7.48(d, J=7.2Hz,2H),6.83(s,1H),4.42-4.34(m,2H),4.24(dd,J=7.7,3.6Hz,1H),3.94(s,3H),3.25(s,3H), 3.11(t,J=12.8Hz,2H),2.94(q,J=6.6Hz,2H),2.67(t,J=12.4Hz,2H),2.31(t,J=7.5Hz,2H),1.87(d ,J=14.9Hz,4H),1.84-1.71(m,6H),1.62(dp,J=14.1,7.3Hz,6H),1.38(s,9H),0.76(t,J=7.4Hz,3H).

[0077] The product 10 (n=2) is a light yellow solid with a yield of 84% and mp: 139-141°C. 1 H NMR (400MHz, DMSO) δ8.41(d,J=8.4Hz,1H),8.13(d,J=7.8Hz,1H),7.85(s,1H),7.68(s,1H),7.49( d,J=6.4Hz,2H),6.82(t,J=5.8Hz,1H),4.38(t,J=12.5Hz,2H),4.25(dd,J=7.6,3.6Hz,1H),4.05( s,1H),3.94(s,3H),3.25(s,3H),3.11(t,J=12.4Hz,2H),2.92(q,J=6.5Hz,2H),2.66(t,J=12.1Hz ,1H),2.32(t,J=7.0Hz,2H),2.07-1.55(m,14H),1.47(s,4H),1.38(s,9H),0.76(t,J=7.3Hz,3H).

[0078] The product 10 (n=3) is a light yellow solid with a yield of 85% and mp: 233-236°C. 1H NMR (400MHz, DMSO) δ8.36(d,J=8.6Hz,1H),8.14(d,J=7.8Hz,1H),7.84(s,2H),7.49(dq,J=3.5,1.9Hz,2H),6.7 9(t,J=5.7Hz,1H),4.43-4.31(m,2H),4.28(dt,J=7.6,3.8Hz,1H),3.94(s,3H),3.25(s,3H),3.10(qd,J=7.4,4. 7Hz,4H),2.90(q,J=6.6Hz,2H),2.70-2.63(m,1H),2.31(t,J=7.5Hz,2H),2.06-1.74(m,10H),1.66(dt,J=14.6, 7.5Hz,2H),1.61-1.55(m,2H),1.49(t,J=7.6Hz,2H),1.37(s,9H),1.18(t,J=7.3Hz,4H),0.76(t,J=7.4Hz,3H).

[0079] 4) Compound 10 was added to 8 mL of saturated hydrogen chloride in ethyl acetate and stirred for 12 h. After filtration and drying, the compound of formula V was obtained and directly used in the next reaction without purification.

[0080] (2) Preparation of compound of formula II

[0081]

[0082] The compound of formula V (0.61 g, 1 mmol) and dansyl chloride (0.26 g, 1 mmol) were dissolved in 10 mL of dichloromethane solution, 0.1 mL of triethylamine was added, and the mixed reaction solution was reacted for 6 hours. The mixture was extracted with dichloromethane, dried and concentrated, and then purified by column chromatography (dichloromethane: methanol = 80:1) to obtain the final product of formula II (probe molecules L2 to L4).

[0083] The final product L2 (n=1) is a light green solid with a yield of 57% and mp: 140-142°C. 1H NMR (400MHz, DMSO) δ8.45(dd,J=12.5,8.4Hz,2H),8.31(d,J=8.6Hz,1H),8.15–8.05(m,2H),7.95(t,J=5.7Hz,1H),7.85(s,1H),7.67–7 .57(m,3H),7.48(d,J=7.8Hz,2H),7.26(d,J=7.5Hz,1H),4.42-4.26(m,2H),4.24(dd,J=7.6,3.6Hz,1H),4.05-3.96(m,1H),3.94(s,3H) ,3.54(d,J=13.7Hz,1H),3.25(s,3H),2.95-2.84(m,2H),2.83(s,6H),2.58(t,J=12.4Hz,1H),2.14(t,J=7.4Hz,2H),2.02(q,J=9.4Hz,1 H),1.96-1.85(m,2H),1.84-1.70(m,6H),1.63(dq,J=12.4,6.7Hz,3H),1.53(p,J=7.1Hz,2H),1.40-1.27(m,2H),0.76(t,J=7.4Hz,3H). 13 C NMR(100MHz,DMSO)δ170.12,165.54,163.39,154.77,151.96,151.84,147.10,138 .80,136.44,132.68,129.86,129.51,128.88,128.29,127.01,124.09,120.57,119 .55,116.58,116.38,115.57,109.64,60.22,58.77,56.50,46.98,45.53,44.21,4 2.60,32.57,31.70,29.71,29.22,28.90,28.26,26.94,25.36,23.71,23.44,9.34. HRMS(ESI)m / zcalcd for C43H56N9O6S([M+H] + )826.4074; found 826.40687.

[0084] The final product L3 (n=2) is a light green solid with a yield of 59% and mp: 143-145°C. 1H NMR (400MHz, DMSO) δ8.45(t,J=8.6Hz,2H),8.32(d,J=8.6Hz,1H),8.14-8.05(m,2H),7.92(t,J=5.7Hz,1H),7.85(s,1H), 7.67-7.55(m,3H),7.50(d,J=7.7Hz,2H),7.25(d,J=7.5Hz,1H),4.35(p,J=8.4Hz,2H),4.24(dd,J=7.6,3.6Hz,1H),4.04( dtd,J=15.3,7.7,3.2Hz,1H),3.94(s,3H),3.79(d,J=13.0Hz,1H),3.25(s,3H),3.10-2.99(m,1H),2.79(d,J=6.1Hz,2H), 2.68-2.57(m,1H),2.17(t,J=7.0Hz,2H),2.06-1.74(m,9H),1.69-1.55(m,3H),1.48-1.31(m,6H),0.76(t,J=7.4Hz,3H). 13 C NMR(100MHz,DMSO)δ170.57,165.54,163.39,154.77,151.96,151.80,147.09,138.78 ,136.60,132.68,129.79,129.57,129.52,128.67,128.25,127.01,124.06,120.59,11 9.62,116.58,116.38,115.56,109.63,60.22,58.77,56.50,47.04,45.53,44.53,42.6 6,32.76,32.13,31.82,29.31,29.22,28.91,28.25,26.95,23.71,23.43,22.37,9.33. HRMS(ESI)m / z calcd for C44H58N9O6S([M+H] + )840.4231; found 840.42252.

[0085] The final product L4 (n=3) is a light green solid with a yield of 55% and mp: 130-132°C. 11H NMR (400 MHz, DMSO) δ 8.49 - 8.39 (m, 2H), 8.34 - 8.29 (m, 1H), 8.13 - 8.05 (m, 2H), 7.86 (d, J = 11.5 Hz, 2H), 7.60 (ddd, J = 12.2, 8.6, 7.4 Hz, 3H), 7.49 (d, J = 7.6 Hz, 2H), 7.25 (dd, J = 7.6, 0.9 Hz, 1H), 4.35 (dd, J = 10.4, 6.3 Hz, 2H), 4.24 (dd, J = 7.6, 3.6 Hz, 1H), 4.03 (dq, J = 11.1, 5.6 Hz, 1H), 3.94 (s, 3H), 3.79 (d, J = 13.5 Hz, 1H), 3.25 (s, 3H), 3.07 (t, J = 12.7 Hz, 1H), 2.82 (s, 6H), 2.78 (q, J = 6.6 Hz, 2H), 2.69 - 2.58 (m, 1H), 2.18 - 2.08 (m, 2H), 2.01 (dd, J = 10.2, 6.2 Hz, 1H), 1.89 (t, J = 7.2 Hz, 8H), 1.86 - 1.71 (m, 6H), 1.69 - 1.63 (m, 1H), 1.61 (dq, J = 7.4, 3.6 Hz, 2H), 1.44 - 1.26 (m, 6H), 1.15 (qd, J = 8.7, 3.4 Hz, 2H), 0.76 (t, J = 7.4 Hz, 3H). 13 13C NMR (100 MHz, DMSO) δ 1^{70.69}, 165.56, 163.39, 154.75, 151.98, 151.81, 147.13, 138.72, 136.69, 132.68, 129.78, 129.59, 129.53, 128.67, 128.22, 127.07, 124.04, 120.59, 119.65, 116.60, 116.43, 115.53, 109.68, 60.24, 58.80, 56.51, 47.06, 45.53, 44.58, 42.74, 32.76, 32.59, ^{31.82}, 29.38, 29.22, 28.93, 28.26, 26.95, 26.16, 24.82, 23.72, 23.44, 9.33. HRMS (ESI) m / z calcd for C45H60N9O6S ([M + H] + ) 854.4387; found 854.43818.

[0086] Example 3 Determination of enzymatic activity

[0087] In this example, the enzymatic activity of the probes described in Examples 1 and 2 was determined using the Kinase-Glo Plus Luminescent Kinase Detection Kit.

[0088] Experimental Methods: (1) All enzymatic reactions were performed at 30°C for 40 min. A 50 μl reaction mixture contained 40 mM Tris (pH 7.4), 10 mM MgCl2, 0.1 mg / ml BSA, 1 mM DTT, 10 μM ATP, 0.2 μg / ml kinase, and 100 μM lipid substrate. Compounds were diluted with 10% dimethyl sulfoxide (DMSO), and 5 μl of the diluent was added to the 50 μl reaction, resulting in a final DMSO concentration of 1% in all reactions.

[0089] (2) The Kinase-Glo Plus Luminescent Kinase Assay Kit 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 is inversely proportional to the amount of kinase activity. 50 Values ​​were calculated by nonlinear regression of normalized dose-response fits using Prism GraphPad software.

[0090] The enzymatic characteristics of the probe molecules described in Examples 1 and 2 are shown in Table 1 below:

[0091] Table 1

[0092] Compound <![CDATA[PLK1 IC 50 (nM)]]> <![CDATA[BRD4 IC 50 (nM)]]> L1 1.8±0.7 650±218 L2 7.5±2.1 714±203 L3 3.2±0.9 1841±708 L4 6.1±2.1 1956±743

[0093] As can be seen from Table 1, L1 performed best in inhibiting PLK1, IC 50 The value is the lowest (1.8nM); and the probe molecules L1 to L4 have affinity for both PLK1 protein and BRD4 protein, among which L1 has the best affinity, indicating that the shorter the chain length, the more conducive it is to binding.

[0094] Example 4 Determination of optical activity

[0095] This example provides the optical activities of the probe molecules L1 to L4 of Examples 1 to 2 detected by ultraviolet spectrophotometer and fluorescence spectrophotometer.

[0096] Experimental Method: Fluorescent probe concentrates L1-L4 (10 mM in DMSO) were dissolved in 10 mol / L PBS buffer, maintaining a pH of 7.4 to simulate physiological conditions. Four 5 ml EP tubes were added with 3 ml of PBS solution, followed by 3 μM of each fluorescent probe concentrate L1-L4, and the mixture was shaken thoroughly. Subsequently, UV absorption and fluorescence properties were measured using a UV spectrophotometer and a fluorescence spectrophotometer set to appropriate wavelengths. UV absorption and fluorescence spectra were plotted using GraphPad Prism.

[0097] The optical characteristics of the probe molecules described in Examples 1 and 2 are shown in Table 2 below:

[0098] Table 2

[0099]

[0100] Note: All the above optical properties are measured in phosphate buffer at pH 7.4. Figures 2-4 shown.

[0101] From Table 2 and Figures 2 to 4 As can be seen, probe molecules L1–L4 exhibit maximum UV absorption at 350 nm, maximum fluorescence excitation at 352 nm, and maximum fluorescence emission at 488 nm. L1, L2, L3, and L4 have similar UV absorption wavelengths, but slightly different fluorescence excitation and emission wavelengths. These differences may be related to subtle changes in the molecular structure, which affect the molecule's electronic energy levels and non-radiative relaxation processes.

[0102] Example 5 Determination of cell viability

[0103] This embodiment provides the probe molecules L1 to L4 of Examples 1 to 2 for detecting cell activity using an enzyme-labeled instrument.

[0104] Experimental Methods: Logarithmic-phase breast cancer MDA-MB-231 cells were seeded into 96-well plates at 5,000 cells per well and cultured overnight. The next day, different concentrations of probe molecules L1 to L4 from Examples 1 to 2 were added and incubated for 24 and 48 hours, respectively. CCK-8 working solution was then added and incubated for 0.5 hours. OD values ​​were measured at 450 nm using a microplate reader.

[0105] pass Figure 5 From the cell viability data, it can be seen that the probe molecules L1 to L4 have no obvious cytotoxicity, indicating that the addition of this fluorophore and other structures will not cause cytotoxicity, eliminating the possibility that the nature of the drug action will not be changed due to structural changes, and thus predicting that the probe molecules L1 to L4 act consistently on the cell cycle.

[0106] Example 6Probe Molecular Cell Imaging

[0107] This example provides cell imaging of the probe molecules L1 to L4 of Examples 1 to 2 in breast cancer cells MDA-MB-231.

[0108] Experimental Methods: Logarithmic-phase breast cancer MDA-MB-231 cells were seeded into confocal microplates and cultured overnight. The next day, 10 μM probe molecules L1–L4 and 10 μg / mL acridine orange dye were added and stained for 20 minutes before imaging under a confocal microscope.

[0109] It is known from the literature that the PLK1 / BRD4 protein is located in the cell nucleus, and acridine orange (AO) dye can be used to label DNA and RNA. Therefore, most of the acridine orange is also stained in the cell nucleus, which can be used for verification and control with the probe.

[0110] pass Figure 5 It can be seen that the fluorescence activity intensities of probe molecules L1 to L4 in breast cancer cells MDA-MB-231 are similar, and the fluorescence intensity of probe molecules L1 to L4 in the cell nucleus is the highest, which further verifies that the probe can specifically bind to the PLK1 / BRD4 protein.

[0111] Example 7 Periodic testing of probe molecules L1 to L4

[0112] This example provides the cycle status of the probe molecules L1 to L4 of Examples 1 and 2 and a blank control group in breast cancer cells MDA-MB-231.

[0113] Experimental Methods: Logarithmic-phase breast cancer MDA-MB-231 cells were seeded into six-well plates, with 1 million cells per well. Cultured overnight. The next day, probe molecules L1 to L4 were added at concentrations of 0.5, 1, and 5 μM. After incubation for 24 hours, cells were collected and analyzed by flow cytometry after adding PI dye. The results are shown in Table 3 and Figures 6 to 10 shown.

[0114] The results of the cycle status of the probe molecules L1 to L4 in Examples 1 and 2 in breast cancer cells MDA-MB-231 are shown in Table 3 below:

[0115] Table 3

[0116]

[0117]

[0118] Through Table 3 and Figures 6 to 10As can be seen, probe molecules L1-L4 can induce cell cycle arrest at the G2 or mitotic phase. Specifically, by inhibiting PLK1 activity, the cell cycle arrest occurs at the G2 / M phase, followed by DNA degradation and apoptosis. This effect is concentration-dependent. Table 3 shows that the effect of probe molecules L1-L4 on cell cycle arrest becomes more pronounced with increasing fluorescent probe concentration.

[0119] Example 8

[0120] This example provides a verification experiment showing that the probe molecules L1 to L4 can introduce a small molecule fluorophore via a fatty chain into the piperidine ring of the probe molecules without interfering with the binding mode of the BRD4 protein / PLK1 protein.

[0121] Specifically, it can be obtained through protein docking, including the following steps:

[0122] 1. Protein Pretreatment

[0123] The corresponding crystal structures of two proteins were obtained from the RCSB PDB database. The obtained protein crystals were processed using the Protein Preparation Wizard module of Schrödinger software, including protein preprocessing, regenerate states of native ligand, H-bond assignment optimization, protein energy minimization, and water removal.

[0124] 2. Ligand Pretreatment

[0125] The 2D sdf structure files of the four fluorescent probes were processed using the LigPrep module in Schrödinger to generate all their 3D chiral conformations.

[0126] 3. Active Site Identification

[0127] The ReceptorGrid Generation module in Schrödinger was used to set the most appropriate Enclosing box to perfectly encapsulate the ligand molecules in the two protein crystals, and on this basis, the active sites of the two proteins were obtained respectively.

[0128] The test results are shown in the following table and Figures 11 to 14 :

[0129]

[0130] Based on the XP docking and MM-GBSA analysis results, the docking scores of L1, L4, L2, and L3 with PLK1 were -15.023, -14.675, -13.905, and -13.404, respectively. The MM-GBSA analysis results were -100.40, -76.50, -104.47, and -77.14 kcal / mol, respectively. The binding free energy and docking scores were both very low, indicating that the four compounds were very stably bound to PLK1.

[0131]

[0132]

[0133] Combining the XP docking and MM-GBSA analysis results, the docking scores of L4 and L1 with BRD4 were -10.651 and -9.878, respectively, and the MM-GBSA analysis results were -54.64 and -54.61 kcal / mol, respectively. Both the binding free energies and docking scores were very low, indicating that the two compounds bound stably to BRD4. The docking scores of L2 and L3 with BRD4 were -8.572 and -8.532, respectively, and the MM-GBSA analysis results were -48.86 and -46.71 kcal / mol, respectively. Both the binding free energies and docking scores were low, indicating that the two compounds bound stably to BRD4.

[0134] In summary, the fluorescent probes L1 and L4 designed in this invention bind most stably to the BRD4 / PLK1 protein, and L2 and L3 bind stably to the BRD4 / PLK1 protein.

[0135] 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 BRD4 / PLK1 dual-target small molecule fluorescent probe, characterized in that: Its structure is the structure shown in Formula I or a pharmaceutically acceptable salt thereof, or the structure shown in Formula II or a pharmaceutically acceptable salt thereof: Formula I; Formula II; n=1~3; R is selected from the following structures: 。 2. The BRD4 / PLK1 dual-target small molecule fluorescent probe according to claim 1, characterized in that: The pharmaceutically acceptable salt is a salt formed by the structure represented by Formula I or the structure represented by Formula II and an inorganic acid or an organic acid, wherein the inorganic acid is hydrochloric acid, sulfuric acid, nitric acid or hydrobromic acid, and the organic acid is methanesulfonic acid, toluenesulfonic acid or trifluoroacetic acid.

3. The method for preparing the BRD4 / PLK1 dual-target small molecule fluorescent probe according to claim 1, wherein: The steps include: S1, subjecting the compound represented by Formula III to an amidation reaction with the compound represented by Formula IV to obtain the compound represented by Formula I; Alternatively, S2, subjecting the compound represented by Formula V to an amidation reaction with the compound represented by Formula VI to obtain the compound represented by Formula II; 4. The method for preparing the BRD4 / PLK1 dual-target small molecule fluorescent probe according to claim 3, wherein: In step S1, the preparation method of the compound represented by formula III is: reacting (R)-2-chloro-8-cyclopentyl-7-ethyl-5-methyl-7,8-dihydropterin-6(5H)-one with 4-amino-3-methoxybenzoic acid to obtain the compound.

5. The method for preparing the BRD4 / PLK1 dual-target small molecule fluorescent probe according to claim 3, wherein: In step S1, the preparation method of the compound represented by formula IV is: reacting dansyl chloride with N-tert-butyloxycarbonyl-1,3-propylenediamine to obtain compound 6, and performing a deprotection reaction on compound 6 to obtain the compound represented by formula IV; 6. The method for preparing the BRD4 / PLK1 dual-target small molecule fluorescent probe according to claim 3, wherein: In step S2, the preparation method of the compound represented by formula V is as follows: the compound represented by formula III is subjected to an amide condensation reaction with tert-butyl 4-aminopiperidine-1-carboxylate to obtain compound 8, compound 8 is subjected to deprotection of the Boc protecting group in hydrogen chloride ethyl acetate solution to obtain intermediate 9, intermediate 9 and intermediate 9-1 are subjected to an amide condensation reaction to obtain compound 10, and compound 10 is subjected to a deprotection reaction to obtain the compound represented by formula V; ； ； ； ；n=1~3。 7. Use of the BRD4 / PLK1 dual-target small molecule fluorescent probe according to claim 1 in preparing a reagent for identifying BRD4 / PLK1 dual targets.

8. Use of the BRD4 / PLK1 dual-target small molecule fluorescent probe according to claim 1 in preparing a marker for tumor cells with high BRD4 / PLK1 expression.

9. The use according to claim 8, characterized in that The tumor is selected from solid tumors and hematological tumors; the solid tumor is breast cancer.

Images