Small-molecule near-infrared fluorescent probe SHP-PP as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and relates to a small-molecule near-infrared fluorescent probe SHP-PP and a preparation method and application thereof.The small-molecule near-infrared fluorescent probe SHP-PP is composed of a targeted SHP2 recognition group, a linking group and a fluorophore Py5, keeps high affinity to SHP2, can specifically mark SHP2 in living cells, and can be used for detecting the SHP2 in the living cells. According to the present invention, the probe has characteristics of high sensitivity, high protein selectivity, maximum reduction of interference of other proteins, excellent optical property, imaging requirement meeting, maximum emission wavelength in the near-infrared region, large Stokes displacement reaching 200 nm, high signal-to-noise ratio, stable fluorescence signal, and low phototoxicity, and can be used for the preparation of the fluorescent probe, such that the probe has characteristics of high signal-to-noise ratio, stable fluorescence signal, and low phototoxicity.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine and relates to a small molecule near-infrared fluorescent probe SHP-PP and a preparation method and application thereof. Background Art

[0002] SHP2 protein is a non-receptor protein tyrosine phosphatase and a key regulator of signal transduction. It interacts with key signaling molecules in the RAS / ERK and PD-1 / PD-L1 pathways, and has a significant impact on cell proliferation, differentiation, and immune regulation. Currently, SHP2 has become a promising target for cancer treatment and plays a role in the progression of a variety of tumors.

[0003] Current methods for detecting SHP2, such as western blot (WB), enzyme-linked immunosorbent assay (ELISA), immunohistochemistry (IHC), and substrate-based assays, have significantly advanced our understanding of the biological functions of SHP2. However, these techniques are often limited in throughput, complex in operation, low in sensitivity, and the inability to monitor SHP2 activity in real time in living cells.

[0004] Fluorescence imaging is a biomedical imaging method that relies on the fluorescence effect. It uses the property of fluorescent molecules to emit longer wavelength light when excited by light of a specific wavelength to image biological samples. This technology can monitor the distribution, dynamic changes, interactions and reaction processes of biomolecules in living cells, tissues and animal models in real time and non-invasively. As the core component of fluorescence imaging technology, fluorescent probes exhibit unique fluorescence properties in the ultraviolet to near-infrared region and are widely used in the quantitative analysis of proteins, metal ions, pesticide residues and biomolecules, as well as the tracking of biomolecules, the labeling of macromolecules and the visualization of cell and subcellular structures, making them powerful tools for imaging biological systems.

[0005] Therefore, it is urgent to develop small molecule near-infrared fluorescent probes for SHP2, so as to use high-sensitivity and high-temporal and spatial resolution imaging technology to monitor intracellular SHP2 in real time, so as to have a deeper understanding of the role of SHP2 in signal transduction and its specific tumor targeting effect, which has important basic research and clinical application prospects. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a near-infrared small molecule fluorescent probe for identifying and labeling SHP2, which exhibits excellent optical properties, good target affinity and high selectivity. Among them, the best performing probe SHP-PP2 has an IC 50 The K in the binding study was 0.86 μmol / L. dThe value was 0.91 μmol / L. This probe effectively realizes the visualization of SHP2 in living cells and promotes the further development of SHP2 research.

[0007] The specific technical solutions of the present invention are as follows:

[0008] The small molecule near-infrared fluorescent probe SHP-PP includes a recognition group targeting SHP2, a linker group and a fluorescent group. The structure of the recognition group targeting SHP2 is as follows:

[0009]

[0010] The fluorescent group is not limited to Py5, and the structure of Py5 is as follows:

[0011]

[0012] Furthermore, the linking group includes but is not limited to an amide group, an alkyl group, a PEG chain or a combination thereof.

[0013] Furthermore, by connecting the recognition group targeting SHP2, the linking group and the fluorescent group, the structure of SHP-PP is as follows:

[0014]

[0015] Wherein, n=1-20, when n is 1, it is named SHP-PP2; when n is 2, it is named SHP-PP3. It can be foreseen that when n>2, the small molecule near-infrared fluorescent probe SHP-PP also plays the role of a fluorescent probe.

[0016] It should be noted that the small molecule near-infrared fluorescent probe SHP-PP described in the present application includes the compound shown in Formula 1 and its analogs, and the analogs refer to compounds with similar chemical structures and properties obtained by equivalently replacing, increasing, decreasing or changing the position of the substituents therein on the basis of the general formula, or introducing similar structures of protective groups for different solvents / environments.

[0017] Furthermore, the structure of SHP-PP is as follows:

[0018]

[0019] The above structure is named SHP-PP1. The compounds protected by the present invention include but are not limited to SHP-PP1, SHP-PP2, SHP-PP3 and compounds with similar structures, wherein structural similarity refers to compounds with similarities in molecular skeleton, functional group type and connection mode, and capable of achieving similar functions or effects.

[0020] The present invention also provides a method for preparing a small molecule near-infrared fluorescent probe SHP-PP, characterized in that the reaction steps are as follows:

[0021]

[0022] (I) using 2-chloro-3-fluoroaniline and tert-butyl mercaptan as raw materials, reacting under alkaline conditions to obtain compound 2;

[0023] (II) heating the compound 2 obtained in (I) in concentrated hydrochloric acid to remove the tert-butyl group to obtain compound 3;

[0024] (III) cross-linking 2-bromo-5-chloro-3-methylpyrazine with the compound 3 obtained in (II) to obtain compound 4;

[0025] (IV) taking tert-butyl carbamate (4-methylpiperidin-4-yl) and reacting it with the compound 4 obtained in (III) to obtain compound 5;

[0026] (V) Compound 6 obtained by reacting compound 5 obtained in (IV) with bromoacetyl chloride.

[0027] (VI) coupling the recognition group targeting SHP2 with the linker group;

[0028] (VII) The fluorescent group was synthesized from 4-(dimethylamino)cinnamaldehyde and 2,4,6-trimethylpyridinium tetrafluoroborate.

[0029] (VIII) The recognition group and fluorescent group targeting SHP2 are connected by nucleophilic reaction, and Boc is removed by hydrochloric acid-ethyl acetate to obtain the target probes SHP-PP1, SHP-PP2, and SHP-PP3.

[0030] Reagents and conditions for each step: (I) 2-methyl-2-propanethiol, Cs2CO3, DMF, 120°C, 24 hours; (II) hydrochloric acid, 80°C, 6 hours; (III) 2-bromo-5-chloro-3-methylpyrazine, CuI, 1,10-phenanthroline, K3PO4, dioxane, 90°C, 10 hours; (IV) tert-butyl (4-methylpiperidin-4-yl)carbamate, DIPEA, DMSO, 100°C, 3 hours; (V) bromoacetyl chloride, DIPEA, DCM, room temperature, 3 hours; (VI) acetonitrile, room temperature, 30 minutes; (VII) 2,4,6-trimethylpyridinium tetrafluoroborate, methanol, 60°C, 10 minutes; (VIII) acetonitrile, room temperature, 20 hours; (IX) hydrochloric acid-ethyl acetate, room temperature, 12 hours. The protection scope of the present invention includes compounds obtained by the above-mentioned specific preparation method / conditions and their analogs. Analogs refer to compounds obtained by using process steps or reaction conditions similar to those of the preparation method, with only appropriate adjustments made in terms of raw materials, reagents, reaction temperature, reaction time, etc.

[0031] The present invention also provides the use of the small molecule near-infrared fluorescent probe SHP-PP for preparing a product having at least one of the following effects:

[0032] (1) SHP2 expression detection products;

[0033] (2) Tumor detection products;

[0034] (3) In vitro pathological staining products for tumor tissue.

[0035] Furthermore, the product includes reagents and / or kits.

[0036] The beneficial effects of the present invention are:

[0037] 1. In this application, the environmentally responsive fluorescent group Py5 is combined with the SHP2 allosteric inhibitor to prepare a targeted SHP2 small molecule near-infrared fluorescent probe, which has excellent optical properties and meets the imaging requirements. The maximum emission wavelength is located in the near-infrared region and the Stokes shift is large, up to 200nm, indicating that the probe has a high signal-to-noise ratio, stable fluorescence signal and low phototoxicity.

[0038] 2. The small molecule near-infrared fluorescent probe SHP-PP has a high affinity for SHP2, can specifically label SHP2 in living cells, and exhibits extremely high protein selectivity, minimizing interference from other proteins. The probe also has better controllability, is easy to operate, and does not require additional washing steps during imaging. Once the probe binds to SHP2, it drives the target molecule into a hydrophobic environment, thereby activating fluorescence and labeling SHP2. Experiments have confirmed that the probe inhibits SHP2 activity in vitro with an IC of 1. 50 The K of SHP2 binding was 0.86 μmol / L. d The value is 0.75μmol / L. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] The accompanying drawings in the specification, 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.

[0040] Figure 1 Schematic diagram of the synthesis of the small molecule near-infrared fluorescent probe SHP-PP;

[0041] Figure 2(A1, A2, A3) are the emission spectra of the probes in different polar solvents (H2O, MeOH, EtOH, n-PrOH, n-BuOH). (B1, B2, B3) are the emission spectra of the probes at different viscosities (a mixture of glycerol and water, the percentage represents the ratio of the volume of glycerol to the total volume);

[0042] Figure 3 The in vitro inhibition curves of SHP2 by different concentrations of probes and SHP-099;

[0043] Figure 4 Bright field (B), RFP (R) and merged images (M) of SHP-PP1 (A), SHP-PP2 (B) and SHP-PP3 (C). 1 represents the probe (1 μmol / L) group, 2 represents the probe (1 μmol / L) + SHP-099 (10 μmol / L) group; scale bar is 50 μm;

[0044] Figure 5 Flow cytometry was used to detect the binding of probes to SHP2 in living cells. Red: control, blue: probe only, yellow: probe containing competitive inhibitor (SHP-099). (A) SHP-PP1 (B) SHP-PP2 (C) SHP-PP3;

[0045] Figure 6 The fluorescence switching properties of SHP-PP2 were evaluated after incubation with different concentrations of SHP2 protein (A, B, C) and living cells (D, E, F) for 30 min;

[0046] Figure 7 The fluorescence intensity of various proteins and probes after incubation for 30 minutes. (A) SHP-PP1 (B) SHP-PP2 (C) SHP-PP3;

[0047] Figure 8 The fluorescence polarization values ​​after incubation of SHP2 protein and probes of different concentrations were detected. DETAILED DESCRIPTION

[0048] The present invention is described below by specific embodiments. Unless otherwise specified, the technical means used in the present invention are methods known to those skilled in the art. In addition, the embodiments should be understood as illustrative rather than limiting the scope of the present invention, and the essence and scope of the present invention are limited only by the claims. For those skilled in the art, various changes or modifications to the material composition and dosage in these embodiments, without departing from the essence and scope of the present invention, also belong to the protection scope of the present invention.

[0049] SHP2 is a valuable target for many diseases, especially cancer. This application combines the environmentally responsive fluorescent group Py5 with the SHP2 allosteric inhibitor to prepare a series of small molecule fluorescent probes with different connection lengths, and systematically evaluates their performance. These probes have excellent optical properties that meet the imaging requirements. The maximum emission wavelength is located in the near-infrared region, and the Stokes shift is large, up to 200nm, which makes the probes have high signal-to-noise ratio, stable fluorescence signal, and low phototoxicity. They maintain high affinity for SHP2, can specifically label SHP2 in living cells, and show extremely high protein selectivity, minimizing interference from other proteins in biological systems. These probes also have sensitive switching functions, are easy to operate, and do not require additional washing steps during imaging. Once bound to SHP2, the probe drives the target molecule into a hydrophobic environment, thereby activating fluorescence and achieving labeling of SHP2. Among these probes, SHP-PP2 is one of the most promising SHP2 visualization tools. It exhibits satisfactory target affinity and excellent fluorescence properties, and the IC of inhibiting SHP2 activity in vitro is 50 The K of binding to SHP2 is 0.86 μmol / L. d The value was 0.75 μmol / L, and cell imaging and tumor slice imaging showed that it could clearly locate SHP2. In summary, we have developed for the first time a small molecule near-infrared fluorescent probe suitable for SHP2 labeling, which has excellent fluorescence properties and strong imaging capabilities. We hope that these environmentally sensitive fluorescent probes can provide new directions for molecular pharmacology research and drug development of SHP2, and provide a reference for the research of other SHP2 fluorescent probes.

[0050] Classic SHP2 inhibitors are mainly divided into hydrophobic region, central region and polar region, and the solvent accessible regions on both sides provide potential binding sites for structural modification of inhibitors. Figure 1 As shown in the figure, this study brought together the advantageous fragments of SHP-099, RWC-4550, and TNO-055 to form the recognition group of the probe molecule. The Py5 fluorescent group was introduced into its hydrophobic region with PEG chains of different lengths as a linker to obtain the near-infrared fluorescent probe series SHP-PP.

[0051] Example 1

[0052] The small molecule near-infrared fluorescent probe SHP-PP was synthesized with the following steps and conditions:

[0053]

[0054] (I) 2-Chloro-3-fluoroaniline (10.0 g, 68.7 mmol), 2-methyl-2-propanethiol (18.6 g, 206 mmol) and Cs2CO3 (67.2 g, 206 mmol) were mixed in DMF (100 ml). The reaction mixture was stirred at 120°C for 24 hours. After cooling to room temperature, ethyl acetate and water were added, stirred, the organic layer was separated, and the aqueous layer was extracted twice with ethyl acetate. The organic phases were combined, dried over Na2SO4, and the solvent was evaporated under reduced pressure to obtain a yellow oil (12.7 g, yield 85.7%), thereby obtaining compound 2 (3-(tert-butylthio)-2-chloroaniline);

[0055] (II) Compound 2 (12.0 g, 55.6 mmol) obtained in (I) was added to hydrochloric acid (100 ml), and the resulting solution was stirred at 80° C. for 6 hours. After the reaction was completed, the mixture was cooled to room temperature, the precipitate was collected by filtration, and then washed with hydrochloric acid (20 ml) and n-hexane (100 ml) in sequence. After drying under vacuum, a white solid product (10.0 g, yield 92.3%) was obtained, i.e., compound 3 (3-amino-2-chlorothiophenol). 1 H NMR(400MHz, DMSO-d6)δ7.06(t,J=8.0Hz,1H),6.78(dd,J=7.6,4.6Hz,2H).ESI-MS:m / z[MH] + Calcd for C6H7ClNS + 157.98,found158.1.Mp 111.5–113.2℃;

[0056] (III) Compound 3 (2.50 g, 15.7 mmol), 2-bromo-5-chloro-3-methylpyrazine (2.60 g, 12.5 mmol), cuprous iodide (597 mg, 3.13 mmol), 1,10-phenanthroline (1.13 g, 6.26 mmol) and potassium phosphate (9.97 g, 47.0 mmol) were added to a solution of 1,4-dioxane (100 ml). The mixture was reacted at 90°C for 10 hours under nitrogen protection. After the reaction was completed, the mixture was filtered and the filtrate was diluted with ethyl acetate (80 ml) and water (80 ml). The organic layer was separated and the aqueous layer was extracted with ethyl acetate. The organic phases were combined and dried over Na2SO4 to remove residual moisture. The solvent was then evaporated under reduced pressure and the obtained residue was purified by silica gel column chromatography to give a white solid product (2.03 g, 45.3% yield), Compound 4 (2-chloro-3-((5-chloro-3-methylpyrazin-2-yl)thio)aniline). 1H NMR (400MHz, DMSO-d6) δ8.42(s,1H),7.07(t,J=7.8Hz,1H),6.90(d,J=8.1Hz,1H),6.81(d,J=7.5Hz,1H),5.59(s,2H).ESI-MS:m / z[M+H] + Calculate for C 11 H 10 Cl2N3S + 286.00,found 286.17.Mp 130.2.2–133.1℃;

[0057] (IV) Compound 4 (1.00 g, 3.49 mmol) was dissolved in DMSO (15 ml), and then tert-butyl (4-methylpiperidin-4-yl) carbamate (1.50 g, 6.99 mmol) and DIPE A (1.87 ml, 10.5 mmol) were added. The reaction mixture was stirred at 100 ° C for 3 hours. After the reaction was completed, the mixture was cooled to room temperature, ethyl acetate (100 ml) and water (100 ml) were added and stirred. The organic layer was separated and the aqueous phase was extracted twice with ethyl acetate. The organic layers were combined, washed six times to ensure complete removal of water, and dried over Na2SO4. After the solvent was removed under reduced pressure, it was purified by silica gel column chromatography to obtain a light yellow solid compound 5, tert-butyl (4-(5-((3-amino-2-chlorophenyl)thio)-6-methylpyrazine-2-yl)-1-methylcyclohexyl) carbamate (1.25 g, 77.2% yield). 1 HNMR (400MHz, DMSO-d6) δ8.19(s,1H),6.86(t,J=7.9Hz,1H),6.67(s,1H),6.60(d,J=7.9Hz,1H),5.89(d,J=7.7Hz,1H),5.47(s,2H) ,3.91(d,J=13.1Hz,2H),3.25(t,2H),2.36(s,3H),2.10(d,2H),1.43(t,J=14.3Hz,2H),1.40(s,9H),1.26(s,3H).ESI-MS:m / z[M+H] + Calculate for C 11 H 10 Cl2N3S + 464.19,found464.08.Mp 187.5–189.8℃;

[0058] (V) Compound 5 (1.00 g, 2.15 mmol) and DIPEA (2.14 ml, 12.9 mmol) were dissolved in dichloromethane (DCM, 20 ml) in an ice bath. Bromoacetyl chloride (1.08 ml, 12.9 mmol) was added dropwise and the mixture was stirred at room temperature for 3 hours. After the reaction, n-hexane (50 ml) was added to the mixture to produce a large amount of precipitation. Filtration gave a yellow solid (1.12 g, 88.9% yield), tert-butyl (1-(5-((3-(2-bromoacetylamino)-2-chlorophenyl)thio)-6-methylpyrazine-2-yl)-4-methylpiperidin-4-yl)carbamate. 1 HNMR(400MHz, DMSO-d6)δ9.96(s,1H),8.23(s,1H),7.52(d,J=7.8Hz,1H),7.22(t,J=8.1Hz,1H),6.68(s,1H),6.62(d,J=8.0Hz,1H),4.39( s,2H),3.94(d,J=13.3Hz,2H),3.27(t,2H),2.39(s,3H),2.10(s,2H),1.43(d,J=12.9Hz,2H),1.40(s,9H),1.26(s,3H).ESI-MS:m / z[M+H] + Calculate for C 11 H 10 Cl2N3S + 585.96,found 586.39.Mp 174.2–176.6℃,.

[0059] (VI) Conjugation of recognition groups targeting SHP2 with different linker groups:

[0060] Tert-butyl (1-(5-((3-(2-((3-aminopropyl)amino)acetylamino)-2-chlorophenyl)thio)-6-methylpyrazin-2-yl)-4-methylpiperidin-4-yl)carbamate (7a)

[0061] 1,3-Propylenediamine (144 μL, 341.8 μmol) and compound 6 (200 mg, 341.8 μmol) were dissolved in acetonitrile (20 mL) and reacted at room temperature for 30 minutes. The solvent was removed under reduced pressure, and dichloromethane (DCM, 50 mL) and water (20 mL) were added. The organic phase was washed twice with saturated sodium chloride solution and dried over anhydrous Na2SO4. Thin layer chromatography was used to obtain a white solid product 7a (170 mg, 86.0% yield). 1H NMR (400MHz, CDCl3) δ10.29–9.75(m,1H),8.18(d,J=8.0Hz,1H),7.98(s,1H),7.05(t,J=8.1H z,1H),6.48(d,J=8.1Hz,1H),4.51(s,1H),3.91(d,J=13.6Hz,2H),3.45(s,2H),3.34(t,J=10. 6Hz,2H),2.96(t,J=6.8Hz,2H),2.78(t,J=6.7Hz,2H),2.44(s,3H),2.17–2.08(m,2H),1.86–1 .77(m,2H),1.63(td,J=10.0Hz,2H),1.44(s,9H),1.39(s,3H),1.25(s,2H).ESI-MS:m / z[M+H] + Calculate for C 27 H 41 ClN7O3S578.27,found 578.85.Mp 84.2–86.6℃;

[0062] Tert-butyl (1-(5-((3-(2-((2-(2-aminoethoxy)ethyl)amino)acetylamino)-2-chlorophenyl)thio)-6-methylpyrazin-2-yl)-4-methylpiperidin-4-yl)carbamate (7b)

[0063] 7b was synthesized similarly to 7a (156 mg, 56.5% yield). 1 H NMR (400MHz, CDCl3) δ10.18(s,1H),8.29(dd,J=8.2,1.3Hz,1H),8.00(s,1H),7.10(t,J=8.1Hz ,1H),6.55(dd,J=8.0,1.4Hz,1H),4.44(s,1H),3.93(d,J=13.7Hz,2H),3.67–3.58(m,2H),3.51 (t,J=5.2Hz,2H),3.46(s,2H),3.36(td,J=10.5Hz,2H),2.89(t,J=4.6Hz,4H),2.47(s,3H),2. 12(s,2H),1.66(dd,J=17.2,7.1Hz,2H),1.43(d,J=13.1Hz,9H),1.41(s,3H).ESI-MS:m / z[M+H] + Calculate for C 28 H 43 ClN7O3S 608.28,found 608.96.Mp 82.5–84.2℃;

[0064] Tert-butyl (1-(5-((3-(2-((2-(2-aminoethoxy)ethoxy)ethyl)amino)acetylamino)-2-chlorophenyl)thio)-6-methylpyrazin-2-yl)-4-methylpiperidin-4-yl)carbamate (7c)

[0065] 7b was synthesized similarly to 7a (168 mg, 75.3% yield). 1 H NMR (400MHz, DMSO-d6) δ10.14 (s, 1H), 8.26 (d, J = 8.2Hz, 1H), 8.02 (s, 1H), 7.10 (t,J=8.1Hz,1H),6.53(d,J=7.9Hz,1H),4.44(s,1H),3.93(dt,J=13.7Hz,2H),3 .66–3.62(m,2H),3.62(s,2H),3.59–3.56(m,2H),3.47(s,2H),3.35(d,J=10.5H z,2H),2.95(t,J=5.0Hz,2H),2.92–2.87(m,2H),2.47(s,3H).ESI-MS:m / z[M+H] + Calculate for C 30 H 47 ClN7O3S 652.30,found 653.02.Mp77.3–78.9℃;

[0066] (VII) 4-Dimethylaminocinnamaldehyde (2 g, 11.4 mmol) and 2,4,6-trimethylpyridinium tetrafluoroborate (2.88 g, 13.7 mmol) were added to methanol (20 ml), and the mixture was refluxed at 60°C for 10 minutes to produce a large amount of precipitate. After filtration, the filter cake was dissolved in dichloromethane, and n-hexane was added to precipitate. The precipitate was filtered and recrystallized from anhydrous ethanol (100 ml) to obtain a dark green solid product 9, i.e., a fluorescent group (2.86 g, yield 68.3%). 1 H NMR (400MHz, CDCl3) δ8.26–8.19(m,1H),7.51(d,J=3.9Hz,2H),7.41–7.38(m,1H),6.99–6.92( m,1H),6.68(d,J=9.0Hz,2H),6.43(d,J=14.4Hz,1H),3.13(s,6H),2.58(s,6H).ESI-MS:m / z[M] + Calculate for C 19 H 23 NO +280.17,found 280.40.Mp>300℃.

[0067] (VIII) Connecting the recognition group and fluorescent group targeting SHP2 through nucleophilic reaction. The reactions of different connecting groups are as follows:

[0068] 1-(3-((2-((3-((5-(4-(tert-Butyloxycarbonylamino)-4-methylpiperidin-1-yl)-3-methylpyrazin-2-yl)thio)-2-chlorophenyl)amino)-2-oxoethyl)amino)propyl)-4-((1E,3E)-4-(4-dimethylaminophenyl)buta-1,3-dien-1-yl)-2,6-dimethylpyridin-1-ium (10a)

[0069] Compound 7a (100 mg, 173 μmol) and compound 9 (72.7 mg, 259 μmol) were added to acetonitrile (ACN, 20 ml) and reacted at room temperature for 20 hours. The solvent was removed under reduced pressure, and dichloromethane and water were added. After stirring, the liquid was separated, and the organic phase was dried over anhydrous Na2SO4. Thin layer chromatography purification gave a dark red solid product 10a (102 mg, yield 70.2%). 1 H NMR (400MHz, CDCl3) δ9.94(s,1H),8.27(d,J=6.9Hz,1H),7.98(s,1H),7.48(q,1H),7.45(d,J=9.6Hz,2H),7.39(d,J=8.9Hz,2H),7. 08(t,J=8.1Hz,1H),6.97(d,J=15.2Hz,1H),6.78(dd,J=14.9,10.6Hz,1H),6.66(d,J=9.0Hz,2H),6.49(dd,J=8.0,1.4Hz,1H),6.38( d,J=15.2Hz,1H),4.48(d,J=16.2Hz,2H),4.46(s,1H),3.91(dt,J=13.6Hz,2H),3.45(s,2H),3.36(td,J=10.4Hz,2H),3.03(s,6H),2 .85(d,J=5.8Hz,2H),2.80(s,6H),2.45(s,3H),2.15–2.01(m,4H),1.65(d,J=10.1Hz,2H),1.44(s,9H),1.40(s,3H).ESI-MS:m / z[M] + Calculate for C 46 H 60 ClN8O3S + 839.42,found 839.67.Mp 146.6–149.0℃.

[0070] 1-(2-(2-((2-((3-((5-(4-(tert-Butyloxycarbonylamino)-4-methylpiperidin-1-yl)-3-methylpyrazin-2-yl)thio)-2-chlorophenyl)amino)-2-oxoethyl)amino)ethoxy)ethyl)-4-((1E,3E)-4-(4-dimethylaminophenyl)buta-1,3-dien-1-yl)-2,6-dimethylpyridin-1-ium (10b)

[0071] The synthesis of 10b was similar to that of 10a (109 mg, 74.3% yield). 1 H NMR(400MHz, CDCl3)δ9.96(s,1H),8.17(dd,J=8.2,1.4Hz,1H),7.99(s,1H),7.45–7.34(m,5H),7.05(t,J=8.1Hz,1H) ,6.92(d,J=15.2Hz,1H),6.75(d,J=4.5Hz,1H),6.66(d,J=8.9Hz,2H),6.51(dd,J=8.0,1.4Hz,1H),6.34(d,J=15.2Hz, 1H),4.72(t,J=4.9Hz,2H),4.45(s,1H),3.96–3.86(m,4H),3.57(t,J=4.9Hz,2H),3.39–3.30(m,4H),3.05–3.00(m,6 H),2.86–2.82(m,2H),2.79(s,6H),2.46(s,3H),2.12(s,2H),1.62(td,2H),1.44(s,9H),1.40(s,3H).ESI-MS:m / z[M] + Calculate for C 47 H 62 ClN8O4S + 869.43,found 869.75.Mp 137.9–140.2℃.

[0072] 1-(2-(2-(2-((2-((3-((5-(4-(tert-Butyloxycarbonylamino)-4-methylpiperidin-1-yl)-3-methylpyrazin-2-yl)thio)-2-chlorophenyl)amino)-2-oxoethyl)amino)ethoxy)ethoxy)ethyl)-4-((1E,3E)-4-(4-dimethylaminophenyl)buta-1,3-dien-1-yl)-2,6-dimethylpyridin-1-ium (10c)

[0073] 10c was synthesized similarly to 10a (105 mg, 74.9% yield). 1H NMR (400MHz, CDCl3) δ10.15(s,1H),8.26(d,J=8.2Hz,1H),8.00(s,1H),7.48–7.33(m,5H),7.07(td,J=8.1Hz,1H),6.92( d,J=15.2Hz,1H),6.79–6.73(m,1H),6.66(dd,J=6.6Hz,2H),6.50(dq,J=7.9Hz,1H),6.33(d,J=15.4Hz,1H),4.77–4.63( m,2H),4.46(s,1H),3.96–3.85(m,4H),3.52(s,4H),3.41(s,2H),3.35(d,J=10.6Hz,2H),3.03(d,J=2.5Hz,6H),2.81(d, J=6.0Hz,6H),2.46(s,3H),2.12(s,2H),1.73(s,4H),1.63(d,J=10.1Hz,2H),1.44(s,9H),1.40(s,3H).ESI-MS:m / z[M+H] + Calculate for C 49 H 66 ClN8O4S + 914.47,found 914.66.Mp 127.2–129.5℃

[0074] (IX) 1-(3-((2-((3-((5-(4-amino-4-methylpiperidin-1-yl)-3-methylpyrazin-2-yl)thio)-2-chlorophenyl)amino)-2-oxoethyl)amino)propyl)-4-((1E,3E)-4-(4-dimethylaminophenyl)buta-1,3-dien-1-yl)-2,6-dimethylpyridin-1-ium (SHP-PP1)

[0075] Compound 10a was dissolved in dichloromethane, and then a hydrochloric acid-acetic acid mixture was added, followed by reaction at room temperature for 3 hours. The solvent was removed under reduced pressure, dichloromethane and a saturated sodium carbonate solution were added, the mixture was stirred and separated, and the organic phase was dried over anhydrous Na2SO4. After purification by thin layer chromatography, a dark red solid product SHP-PP1 (27 mg, yield 61.3%) was obtained. 1H NMR(400MHz, DMSO-d6)δ10.07(s,1H),8.22(s,1H),8.06(d,J=7.4Hz,1H),7.88(s,2H),7.76–7.68(m,1H), 7.47(d,J=8.8Hz,2H),7.23(t,J=8.1Hz,1H),7.01(d,J=10.1Hz,1H),6.95(t,J=15.3Hz,1H),6.73(d,J=8. 9Hz,2H),6.60(d,J=15.3Hz,1H),6.53(dd,J=8.0Hz,1H),4.46(t,2H),3.76–3.63(m,4H),3.42–3.39(m,4H ),2.98(s,6H),2.87–2.71(m,8H),2.38(s,3H),2.03–1.94(m,2H),1.51(s,4H),1.23(s,1H),1.15(s,3H). 13 C NMR(101MHz,DMSO-d6)δ170.89,154.77,154.37,153.51,152.24,151.49,1 43.24,141.94,136.89,135.90,131.48,130.12,129.50,128.26,124.03,12 3.70,123.67,123.53,123.19,121.58,119.36,112.47,53.11,50.35,49.20 ,46.67,40.68,40.23,37.40,28.63,27.98,22.72,20.96.ESI-HRMS:m / z[M] + Calculate for C 41 H 52 C1N8OS + 739.3668,found 739.3665.Mp 172.1–175.0℃.HPLC purity 97.9%,t R =5.140min, 250mm×4.6mm, MeOH:H2O=90:10, 0.5mL / min.

[0076] 1-(2-(2-((2-((3-((5-(4-amino-4-methylpiperidin-1-yl)-3-methylpyrazin-2-yl)thio)-2-chlorophenyl)amino)-2-oxoethyl)amino)ethoxy)ethyl)-4-((1E,3E)-4-(4-dimethylaminophenyl)buta-1,3-dien-1-yl)-2,6-dimethylpyridin-1-ium (SHP-PP2)

[0077] The synthesis method of SHP-PP2 was similar to that of SHP-PP1 (28 mg, yield 63.3%). 1 H NMR (400MHz, DMSO-d6) δ10.26–9.95(m,1H),8.21(s,1H),8.03(d,J=7.2Hz,1H),7.80(d,J=18.2Hz,2H),7.70(dd,J =15.3,10.0Hz,1H),7.44(d,J=8.9Hz,2H),7.19(t,J=8.1Hz,1H),7.04–6.96(m,1H),6.92(d,J=15.2Hz,1H),6.72( d,J=8.9Hz,2H),6.56(d,J=15.2Hz,1H),6.49(d,J=8.0Hz,1H),4.61(s,2H),3.85(s,2H),3.74–3.63(m,4H),3.50( t,J=5.3Hz,2H),3.29(s,4H),2.98(s,6H),2.80–2.70(m,8H),2.38(s,3H),1.51(s,4H),1.23(s,1H),1.15(s,3H). 13 C NMR(101MHz,DMSO-d6)δ171.03,155.13,154.86,153.51,152.52,151.49,1 43.46,142.03,136.95,135.82,131.43,130.12,129.51,128.25,124.01,12 3.66,123.52,123.44,123.05,121.01,118.84,112.44,71.54,68.51,53.36 ,51.72,49.05,40.68,40.23,37.54,28.23,22.74,21.61.ESI-HRMS:m / z[M] + Calculate for C 42 H 54 ClN8O2S + 769.3773,found769.3768.Mp 146.3–148.4℃.HPLC purity 96.2%,t R =4.657min, 250mm×4.6mm, MeO H:H2O=90:10, 0.5mL / min.

[0078] 1-(2-(2-(2-((2-((3-((5-(4-amino-4-methylpiperidin-1-yl)-3-methylpyrazin-2-yl)thio)-2-chlorophenyl)amino)-2-oxoethyl)amino)ethoxy)ethoxy)ethyl)-4-((1E,3E)-4-(4-dimethylaminophenyl)buta-1,3-dien-1-yl)-2,6-dimethylpyridin-1-ium (SHP-PP3)

[0079] The synthesis method of SHP-PP3 was similar to that of SHP-PP1 (31 mg, yield 69.6%). 1 HNMR (400MHz, DMSO-d6) δ10.38–9.95(m,1H),8.21(s,1H),8.09(d,J=8.1Hz,1H),7.84(s,2H),7.70(dd,J=15.4,9.7H z,1H),7.44(d,J=8.9Hz,2H),7.19(t,J=8.1Hz,1H),7.02–6.95(m,1H),6.92(d,J=15.3Hz,1H),6.71(d,J=9.0Hz,2H), 6.55(d,J=15.2Hz,1H),6.49(dd,J=8.0,1.4Hz,1H),4.61(s,2H),3.85(s,2H),3.75–3.62(m,4H),3.46(d,J=7.3Hz,4 H),3.43(d,J=2.7Hz,4H),3.31(s,4H),2.98(s,6H),2.78(s,6H),2.38(s,3H),1.50(s,4H),1.23(s,1H),1.14(s,3H). 13 C NMR(101MHz,DMSO-d6)δ170.89,154.77,154.37,153.51,152.24,151.49,1 43.24,141.94,136.89,135.90,131.48,130.12,129.50,128.26,124.03,12 3.70,123.67,123.53,123.19,121.58,119.36,112.47,53.11,50.35,49.20 ,46.67,40.68,40.23,37.40,28.63,27.98,22.72,20.96.ESI-HRMS:m / z[M] + Calculate for C 42 H 54 ClN8O2S +813.4036,found 813.4030.Mp 140.9–142.5℃.HPLC purity 98.7%,t R =5.173min, 250mm×4.6mm, MeOH:H2O=90:10, 0.5mL / min.

[0080] Example 2

[0081] Small molecule near-infrared fluorescent probe SHP-PP property detection experiment:

[0082] 1. Optical properties

[0083] The UV absorption, fluorescence excitation and emission wavelengths for preliminary testing are shown in Table 1.

[0084] Table 1 Optical properties of fluorescent probes

[0085]

[0086] Φ is the relative quantum yield calculated with reference to sodium fluorescein. max is the maximum UV absorption wavelength, λ ex is the maximum fluorescence excitation wavelength, λ em is the wavelength of maximum fluorescence emission.

[0087] The data shown in Table 1 show that the probe reaches a UV absorption peak at a wavelength of 465nm. The maximum excitation wavelengths of all probes are 485nm and 490nm, respectively, the emission spectrum is at 680nm, and the Stokes shift is 200nm. A large Stokes shift can reduce background noise, reduce phototoxic effects on biological specimens, and increase the sensitivity of detection. In addition, the responsiveness of the probe to environmental changes was evaluated. For the probe, there is a direct relationship between the increase in fluorescence intensity and the decrease in solvent polarity and the increase in viscosity ( Figure 2 ). In addition, as shown in Table 1, the quantum yields of these probes are significantly improved in a low-polarity environment, showing good environmental sensitivity, which meets the design requirements.

[0088] 2. Enzyme Activity Inhibition Test

[0089] One of the key factors in evaluating the activity of a probe is its ability to inhibit the enzymatic activity of SHP1 and SHP2, which is crucial because it reveals the binding strength of the probe to the target protein and the selectivity for homologous proteins. We performed enzyme activity assays by pre-incubating the commercially available enzyme with the probe for 30 min, then adding the phosphatase substrate DiFMUP, and then measuring the fluorescence signal of the product. Related data can be found in Figure 3and Table 2. The probe had a significant inhibitory effect on SHP2 and had no effect on SHP1, indicating that the probe could selectively recognize and label SHP2. Although the inhibitory effect of the probe was reduced compared with SHP-099, the SHP-PP series probes still had good IC in the submicromolar range. 50 Inhibitory activity. The results showed that SHP-PP1 had the best activity, and the activity slightly decreased with increasing linker length. Despite the potential influence of the fluorophore on the inhibitory activity, our probe still had a strong binding ability to SHP2 and maintained good selectivity.

[0090] Table 2 IC of probes inhibiting enzyme activity 50 value

[0091]

[0092] 3. Cytotoxicity Assay

[0093] For probes designed for live cell imaging, ensuring biocompatibility is critical. After confirming that the probes can bind to SHP2, we proceeded to evaluate their potential cytotoxicity. Cells were co-cultured with the probes for 24 h, and the number of viable cells was determined by the SRB assay. 50 The values ​​were all greater than 30 μmol / L, which was significantly higher than the concentration required for imaging, and the imaging incubation time was only 20 min, which was much shorter than the incubation time (24 h) of the cytotoxicity test. These results showed that the probe had sufficient biocompatibility to meet the requirements of imaging.

[0094] Table 3 Cytotoxicity of probes

[0095]

[0096] 4. Live Cell Imaging

[0097] Live cell imaging is a key application for detectors to achieve real-time observation of targets. Figure 4 As shown, after the cells were incubated with the probes for 20 minutes, fluorescence imaging without washing showed that the probes labeled SHP2, and all three probes emitted enhanced fluorescence. In addition, the binding of the probes to SHP2 could be competed by SHP-099, resulting in a decrease in fluorescence, which indicates the specific binding of the probes to SHP2. Under the same conditions, SHP-PP2 showed the best imaging performance while maintaining strong binding to SHP2. In addition, in the environmental sensitivity test, SHP-PP2 also showed the best environmental sensitivity in the optical property test (switching characteristics) ( Figure 2 ). The imaging properties of SHP-PP1 and SHP-PP3 are slightly weaker due to the influence of the linker chain length. In short, cell imaging identified SHP-PP2 as the best.

[0098] 5. Flow Cytometry Analysis

[0099] Flow cytometric analysis was performed to evaluate the ability of the probe to specifically label SHP2 in living cells. Figure 5 As shown, a significant increase in fluorescence intensity was observed after the probe was co-incubated with live cells. In the case of competition with SHP-099, the histogram shifted significantly to the left, indicating a decrease in cellular fluorescence intensity. This shift indicates that SHP-099 effectively competes with the probe for binding to SHP2. Overall, these findings from flow cytometry experiments confirm the specificity of the probe in targeting SHP2 in a cellular context.

[0100] 6. Verify the probe's selectivity switch

[0101] To further confirm the switching properties of the environmentally sensitive fluorescent probe we designed, we conducted experiments at the protein and cell levels. After co-culture with SHP2 protein, the fluorescence intensity of the probe was significantly enhanced, and as the protein concentration gradually increased, the fluorescence intensity tended to stabilize ( Figure 6 A, 6B, 6C). These probes showed excellent switching performance. In addition, we evaluated the switching behavior of these probes in living cells ( Figure 6 D, 6E, 6F), and obtained consistent results.

[0102] 7. Selectivity for SHP2

[0103] The specificity of the probe for the target protein is also critical. We selected bovine serum albumin (BSA), papain, and trypsin as controls to compare with SHP2 protein to evaluate the specificity of the probe and determine whether it binds nonspecifically to other cellular components. The concentration of the control protein was set to twice that of the SHP2 protein. Figure 6 As shown in Figure 2, the fluorescence intensity of the three probes after incubation with bovine serum albumin, papain, and trypsin was significantly lower than that after incubation with SHP2 (P<0.0001) ( Figure 7 ). In the presence of SHP-099, competition for binding sites prevented the probes from entering the hydrophobic environment to activate fluorescence, resulting in a significant decrease in the fluorescence intensity of the system (P<0.001). Therefore, these probes showed high specificity and less nonspecific binding to SHP2.

[0104] 8. Fluorescence Polarization Experiment

[0105] Fluorescence polarization is a technique that uses the change in the polarization state of light emitted by fluorescent molecules after being excited by polarized light to study molecular interactions. It has a wide range of applications in monitoring molecular interactions in solution, including but not limited to receptor-ligand binding, protein-peptide interaction, DNA-protein binding, kinase activity assays, and competitive immunoassays. Fluorescence polarization can not only characterize the affinity of fluorescent molecules for their targets, but can also be further used to establish competitive fluorescence screening methods. Figure 8 As shown in Figure 2, after incubation with the protein, the FP value of the probe increased significantly and was positively correlated with the concentration, gradually reaching a saturated state. The K values ​​of the SHP-PP series probes were calculated based on the saturation curve. d Consistent with the inhibition of SHP2 enzyme activity, the minimum affinity K between SHP-PP1 and SHP2 was calculated by FP method. d value, and as the connection chain lengthens, K d The results showed that all three probes bound to SHP2 protein, and K d The values ​​were 0.53 μmol / L, 0.75 μmol / L and 0.91 μmol / L respectively. Fluorescence polarization further confirmed the specific binding of the probe to SHP2.

Claims

1. Small molecule near-infrared fluorescent probe SHP-PP, characterized in that: It is composed of a recognition group targeting SHP2, a linking group and a fluorescent group. The structure of the recognition group targeting SHP2 is as follows: The structure of the fluorescent group is as follows:

2. The small molecule near-infrared fluorescent probe SHP-PP according to claim 1, characterized in that: The structure of the SHP-PP is as follows: Wherein, n=1 or 2.

3. The small molecule near-infrared fluorescent probe SHP-PP according to claim 1, characterized in that: The structure of the SHP-PP is as follows:

4. A method for preparing the small molecule near-infrared fluorescent probe SHP-PP according to claim 2 or 3, characterized in that: The reaction steps are as follows:

5. Use of the small molecule near-infrared fluorescent probe SHP-PP according to any one of claims 1 to 3 for preparing a product having at least one of the following effects: (1) SHP2 expression detection products; (2) Tumor detection products; (3) In vitro pathological staining products for tumor tissue.

6. The use according to claim 4, characterized in that The products include reagents and / or kits.

Images