Small molecule near-infrared fluorescent probe SHP-PP, its preparation method and application

Abstract

The present invention belongs to the field of biomedical technology and relates to a small molecule near-infrared fluorescent probe SHP-PP and its preparation method and application. The small molecule near-infrared fluorescent probe SHP-PP is composed of a recognition group targeting SHP2, a linking group, and a fluorescent group Py5. It maintains a high affinity for SHP2, can specifically label SHP2 in living cells, and exhibits extremely high protein selectivity, minimizing the interference of other proteins. In addition, the probe also has excellent optical properties, meeting the imaging requirements. The maximum emission wavelength is located in the near-infrared region, with a large Stokes shift of up to 200 nm, high signal-to-noise ratio, stable fluorescent signal, and low phototoxicity.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and relates to a small molecule near-infrared fluorescent probe SHP-PP and its preparation method and application. 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 various tumors.

[0003] Current methods for detecting SHP2, such as immunoblotting (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 usually face limitations such as limited throughput, complex operation, low sensitivity and the inability to monitor SHP2 activity in living cells in real time.

[0004] Fluorescence imaging technology is a biomedical imaging method that relies on the fluorescence effect. It uses the property of fluorescent molecules to emit light with a longer wavelength 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 cells and subcellular structures, making them powerful tools for imaging biological systems.

[0005] Therefore, there is an urgent need to develop small molecule near-infrared fluorescent probes for SHP2, so as to use imaging technology with high sensitivity and high spatio-temporal resolution to monitor SHP2 in cells in real time, so as to more deeply understand 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. The probe exhibits excellent optical properties, has good target affinity and high selectivity. Among them, the best performing probe SHP-PP2 has an IC 50 of 0.86 μmol / L for SHP2 activity and a K dThe value is 0.91 μmol / L. This probe effectively realizes the visualization of SHP2 in live cells, promoting the further development of SHP2 research.

[0007] Specifically, the technical solution of the present invention is as follows:

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

[0009]

[0010] The fluorescent group includes but 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, the structure of SHP-PP obtained by connecting the recognition group targeting SHP2, the linking group, and the fluorescent group is as follows:

[0014]

[0015] Among them, n = 1 - 20. When n is 1, it is named SHP-PP2; when n is 2, it is named SHP-PP3. It can be predicted 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 this application includes the compound shown in Formula 1 and its analogs. The analogs refer to compounds with similar chemical structures and properties obtained by equivalently replacing, increasing or decreasing, or changing the position of substituents on the basis of the general formula, or introducing protecting groups for different solvents / environments and other similar structures.

[0017] Even further, 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 to them. Among them, similar structures refer to compounds with similarity in terms of molecular skeleton, functional group type, and connection method, and can achieve similar functions or effects as them.

[0020] The present invention also provides a preparation method of a small molecule near-infrared fluorescence probe SHP-PP, which is 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) Taking the compound 2 obtained in (I) and heating it in concentrated hydrochloric acid to remove the tert-butyl group to obtain compound 3;

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

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

[0026] (V) Reacting the compound 5 obtained in (IV) with bromoacetyl chloride to obtain compound 6.

[0027] (Ⅵ) Coupling the recognition group targeting SHP2 with the linking group;

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

[0029] (Ⅷ) Connecting the recognition group targeting SHP2 and the fluorescent group through a nucleophilic reaction, removing Boc with 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 the compounds obtained by the above specific preparation methods / conditions and their analogs. The analogs refer to the compounds obtained by appropriately adjusting only the raw materials, reagents, reaction temperature, reaction time, etc. by adopting process steps or reaction conditions similar to those of this preparation method.

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

[0032] (1) A product for detecting SHP2 expression;

[0033] (2) A product for tumor detection;

[0034] (3) An in vitro pathological staining product for tumor tissue.

[0035] Furthermore, the product includes a reagent and / or a kit.

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

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

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

[0039] The accompanying drawings forming a part of this specification are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention.

[0040] Figure 1 It is a schematic diagram of the synthesis of the small molecule near-infrared fluorescent probe SHP-PP;

[0041] Figure 2Emission spectra of the (A1, A2, A3) probe in different polar solvents (H2O, MeOH, EtOH, n-PrOH, n-BuOH). Emission spectra of the probe at different viscosities (mixtures of glycerol and water, with the percentage representing the volume fraction of glycerol in the total volume);

[0042] Figure 3 Inhibitory curves of different concentrations of the probe and SHP-099 against SHP2 in vitro;

[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, and 2 represents the probe (1 μmol / L) + SHP-099 (10 μmol / L) group; the scale bar is 50 μm;

[0044] Figure 5 Binding of the probe to SHP2 in living cells detected by flow cytometry. Red: control, blue: only probe, yellow: probe containing a competitive inhibitor (SHP-099). (A) SHP-PP1 (B) SHP-PP2 (C) SHP-PP3;

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

[0046] Figure 7 Fluorescence intensities after incubating various proteins with the probe for 30 minutes. (A) SHP-PP1 (B) SHP-PP2 (C) SHP-PP3;

[0047] Figure 8 Fluorescence polarization values were detected by fluorescence polarization after incubating SHP2 protein with different concentrations of the probe. Detailed implementation manners

[0048] The present invention will be described below through specific implementation examples. Unless otherwise specified, the technical means used in the present invention are all methods well known to those skilled in the art. In addition, the implementation examples should be understood as illustrative and not limiting the scope of the present invention. The essence and scope of the present invention are only defined by the claims. For those skilled in the art, various changes or modifications to the material components and dosages in these implementation examples also belong to the protection scope of the present invention without departing from the essence and scope of the present invention.

[0049] SHP2 is a valuable target for a variety of diseases, especially cancer. In this application, an environmentally responsive fluorophore Py5 was combined with an allosteric inhibitor of SHP2 to prepare a series of small molecule fluorescent probes with different linker lengths, and their properties were systematically evaluated. These probes have excellent optical properties, meeting the imaging requirements. The maximum emission wavelength is located in the near-infrared region, and the Stokes shift is large, up to 200 nm, which endows the probes with characteristics of high signal-to-noise ratio, stable fluorescence signal, and low phototoxicity. They maintain a high affinity for SHP2, can specifically label SHP2 in living cells, and exhibit extremely high protein selectivity, minimizing the interference of other proteins in the biological system. These probes also have a sensitive switching function, are easy to operate, and do not require additional washing steps during imaging. Once bound to SHP2, the probes drive the target molecule into a hydrophobic environment, thereby activating fluorescence and achieving the 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. The IC 50 for inhibiting SHP2 activity in vitro is 0.86 μmol / L, and the K d value for binding to SHP2 is 0.75 μmol / L. Cell imaging and tumor section imaging indicate that it can clearly localize SHP2. In summary, we have developed small molecule near-infrared fluorescent probes suitable for SHP2 labeling for the first time, with excellent fluorescence properties and strong imaging capabilities. We hope that these environment-sensitive fluorescent probes can provide new directions for the molecular pharmacology research and drug development of SHP2, and provide references for the research of other SHP2 fluorescent probes.

[0050] Classical SHP2 inhibitors are mainly divided into a hydrophobic region, a central region, and a polar region. The solvent-accessible regions on both sides provide potential binding sites for the structural modification of the inhibitor. As shown in Figure 1 the appendix, 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 fluorophore was introduced into its hydrophobic region with PEG chains of different lengths as linkers to obtain a series of near-infrared fluorescent probes SHP-PP.

[0051] Example 1

[0052] Synthesize the small molecule near-infrared fluorescent probe SHP-PP, and the steps and conditions are as follows:

[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 h. After cooling to room temperature, ethyl acetate and water were added, stirred, and the organic layer was separated. The aqueous layer was extracted twice with ethyl acetate. The combined organic phases were dried over Na2SO4 and the solvent was removed under reduced pressure to give a yellow oil (12.7 g, yield 85.7%), giving 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 h. After the reaction was completed, the mixture was cooled to room temperature, and the precipitate was collected by filtration and then washed successively with hydrochloric acid (20 mL) and n-hexane (100 mL). After drying under vacuum, a white solid product (10.0 g, yield 92.3%) was obtained, which was compound 3 (3-amino-2-chlorobenzenethiol). 1 H NMR (400 MHz, DMSO-d6) δ 7.06 (t, J = 8.0 Hz, 1H), 6.78 (dd, J = 7.6, 4.6 Hz, 2H). ESI-MS: m / z [M-H] + calcd for C6H7ClNS + 157.98, found 158.1. Mp 111.5–113.2 °C;

[0056] (III) Compound 3 (2.50 g, 15.7 mmol), 2-bromo-5-chloro-3-methylpyrazine (2.60 g, 12.5 mmol), copper(I) 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). Under nitrogen protection, the reaction was carried out at 90 °C for 10 h. After the reaction was completed, it 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 combined organic phases were dried over Na2SO4 to remove residual moisture. Then the solvent was evaporated under reduced pressure, and the residue obtained was purified by silica gel column chromatography to give a white solid product (2.03 g, yield 45.3%), compound 4 (2-chloro-3-((5-chloro-3-methylpyrazin-2-yl)thio)aniline). 11H NMR (400 MHz, DMSO-d6) δ 8.42 (s, 1H), 7.07 (t, J = 7.8 Hz, 1H), 6.90 (d, J = 8.1 Hz, 1H), 6.81 (d, J = 7.5 Hz, 1H), 5.59 (s, 2H). ESI-MS: m / z [M+H] + calcd for C 11 H 10 Cl2N3S + 286.00, found 286.17. Mp 130.2–133.1 °C;

[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 DIPEA (1.87 mL, 10.5 mmol) were added. The reaction mixture was stirred at 100 °C for 3 h. After completion of the reaction, the mixture was cooled to room temperature, ethyl acetate (100 mL) and water (100 mL) were added, and the mixture was stirred. The organic layer was separated, and the aqueous phase was extracted twice more with ethyl acetate. The combined organic layers were washed six times to ensure complete removal of water and dried over Na2SO4. After removal of the solvent under reduced pressure, purification by silica gel column chromatography gave the pale yellow solid compound 5, tert-butyl (4-(5-((3-amino-2-chlorophenyl)thio)-6-methylpyrazin-2-yl)-1-methylcyclohexyl)carbamate (1.25 g, yield 77.2%). 1 1H NMR (400 MHz, DMSO-d6) δ 8.19 (s, 1H), 6.86 (t, J = 7.9 Hz, 1H), 6.67 (s, 1H), 6.60 (d, J = 7.9 Hz, 1H), 5.89 (d, J = 7.7 Hz, 1H), 5.47 (s, 2H), 3.91 (d, J = 13.1 Hz, 2H), 3.25 (t, 2H), 2.36 (s, 3H), 2.10 (d, 2H), 1.43 (t, J = 14.3 Hz, 2H), 1.40 (s, 9H), 1.26 (s, 3H). ESI-MS: m / z [M+H] + calcd for C 11 H 10 Cl2N3S + 464.19, found 464.08. Mp 187.5–189.8 °C;

[0058] (V) In an ice bath, compound 5 (1.00 g, 2.15 mmol) and DIPEA (2.14 mL, 12.9 mmol) were dissolved in dichloromethane (DCM, 20 mL). Bromoacetyl chloride (1.08 mL, 12.9 mmol) was added dropwise, and the mixture was stirred at room temperature for 3 h. After the reaction, n-hexane (50 mL) was added to the mixture, and a large amount of precipitate was produced. The yellow solid (1.12 g, yield 88.9%) was obtained by filtration, tert-butyl (1-(5-((3-(2-bromoacetamido)-2-chlorophenyl)thio)-6-methylpyrazin-2-yl)-4-methylpiperidin-4-yl)carbamate. 1 HNMR (400 MHz, DMSO-d6) δ 9.96 (s, 1H), 8.23 (s, 1H), 7.52 (d, J = 7.8 Hz, 1H), 7.22 (t, J = 8.1 Hz, 1H), 6.68 (s, 1H), 6.62 (d, J = 8.0 Hz, 1H), 4.39 (s, 2H), 3.94 (d, J = 13.3 Hz, 2H), 3.27 (t, 2H), 2.39 (s, 3H), 2.10 (s, 2H), 1.43 (d, J = 12.9 Hz, 2H), 1.40 (s, 9H), 1.26 (s, 3H). ESI-MS: m / z [M+H] + calcd for C 11 H 10 Cl2N3S + 585.96, found 586.39. Mp 174.2–176.6 °C.

[0059] (VI) The recognition group targeting SHP2 was coupled with different linking groups:

[0060] tert-Butyl (1-(5-((3-(2-((3-aminopropyl)amino)acetamido)-2-chlorophenyl)thio)-6-methylpyrazin-2-yl)-4-methylpiperidin-4-yl)carbamate (7a)

[0061] 1,3-Propanediamine (144 μL, 341.8 μmol) and compound 6 (200 mg, 341.8 μmol) were dissolved in acetonitrile (20 mL), and the reaction was carried out at room temperature for 30 min. 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. Purification by thin layer chromatography gave the white solid product 7a (170 mg, yield 86.0%). 11H NMR (400 MHz, CDCl3) δ 10.29–9.75 (m, 1H), 8.18 (d, J = 8.0 Hz, 1H), 7.98 (s, 1H), 7.05 (t, J = 8.1 Hz, 1H), 6.48 (d, J = 8.1 Hz, 1H), 4.51 (s, 1H), 3.91 (d, J = 13.6 Hz, 2H), 3.45 (s, 2H), 3.34 (t, J = 10.6 Hz, 2H), 2.96 (t, J = 6.8 Hz, 2H), 2.78 (t, J = 6.7 Hz, 2H), 2.44 (s, 3H), 2.17–2.08 (m, 2H), 1.86–1.77 (m, 2H), 1.63 (td, J = 10.0 Hz, 2H), 1.44 (s, 9H), 1.39 (s, 3H), 1.25 (s, 2H). ESI-MS: m / z [M+H] + calcd for C 27 H 41 ClN7O3S 578.27, found 578.85. Mp 84.2–86.6 °C;

[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] The synthetic method of 7b is similar to that of 7a (156 mg, yield 56.5%). 1 1H NMR (400 MHz, CDCl3) δ 10.18 (s, 1H), 8.29 (dd, J = 8.2, 1.3 Hz, 1H), 8.00 (s, 1H), 7.10 (t, J = 8.1 Hz, 1H), 6.55 (dd, J = 8.0, 1.4 Hz, 1H), 4.44 (s, 1H), 3.93 (d, J = 13.7 Hz, 2H), 3.67–3.58 (m, 2H), 3.51 (t, J = 5.2 Hz, 2H), 3.46 (s, 2H), 3.36 (td, J = 10.5 Hz, 2H), 2.89 (t, J = 4.6 Hz, 4H), 2.47 (s, 3H), 2.12 (s, 2H), 1.66 (dd, J = 17.2, 7.1 Hz, 2H), 1.43 (d, J = 13.1 Hz, 9H), 1.41 (s, 3H). ESI-MS: m / z [M+H] + calcd for C 28 H 43 ClN7O3S 608.28, found 608.96. Mp 82.5–84.2 °C;

[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] The synthesis method of 7b is similar to that of 7a (168 mg, yield 75.3%). 1 H NMR (400 MHz, DMSO-d6) δ 10.14 (s, 1H), 8.26 (d, J = 8.2 Hz, 1H), 8.02 (s, 1H), 7.10 (t, J = 8.1 Hz, 1H), 6.53 (d, J = 7.9 Hz, 1H), 4.44 (s, 1H), 3.93 (dt, J = 13.7 Hz, 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 = 10.5 Hz, 2H), 2.95 (t, J = 5.0 Hz, 2H), 2.92–2.87 (m, 2H), 2.47 (s, 3H). ESI-MS: m / z [M+H] + calcd for C 30 H 47 ClN7O3S 652.30, found 653.02. Mp 77.3–78.9 °C;

[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). The mixture was refluxed at 60 °C for 10 minutes, and a large amount of precipitate was formed. After filtration, the filter cake was dissolved in dichloromethane, and n-hexane was added to precipitate the product. The precipitate was filtered and recrystallized from absolute ethanol (100 mL) to obtain a dark green solid product 9, namely the fluorescent group (2.86 g, yield 68.3%). 1 H NMR (400 MHz, CDCl3) δ 8.26–8.19 (m, 1H), 7.51 (d, J = 3.9 Hz, 2H), 7.41–7.38 (m, 1H), 6.99–6.92 (m, 1H), 6.68 (d, J = 9.0 Hz, 2H), 6.43 (d, J = 14.4 Hz, 1H), 3.13 (s, 6H), 2.58 (s, 6H). ESI-MS: m / z [M] + calcd for C 19 H 23 NO +280.17, found 280.40. Mp > 300 °C.

[0067] (VIII) Connect the recognition group targeting SHP2 and the fluorophore through a nucleophilic reaction. The reactions of different linking groups are as follows:

[0068] 1-(3-((2-((3-((5-(4-(tert-Butoxycarbonylamino)-4-methylpiperidin-1-yl)-3-methylpyrazin-2-yl)thio)-2-chlorophenyl)amino)-2-oxoethyl)amino)propyl)-4-((1E,3E)-4-(4-Dimethylaminophenyl)but-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 the reaction was carried out at room temperature for 20 h. The solvent was removed under reduced pressure, and dichloromethane and water were added. After stirring, the layers were separated, and the organic phase was dried over anhydrous Na2SO4. Purification by thin-layer chromatography gave a dark red solid product 10a (102 mg, yield 70.2%). 1 H NMR (400 MHz, CDCl3) δ 9.94 (s, 1H), 8.27 (d, J = 6.9 Hz, 1H), 7.98 (s, 1H), 7.48 (q, 1H), 7.45 (d, J = 9.6 Hz, 2H), 7.39 (d, J = 8.9 Hz, 2H), 7.08 (t, J = 8.1 Hz, 1H), 6.97 (d, J = 15.2 Hz, 1H), 6.78 (dd, J = 14.9, 10.6 Hz, 1H), 6.66 (d, J = 9.0 Hz, 2H), 6.49 (dd, J = 8.0, 1.4 Hz, 1H), 6.38 (d, J = 15.2 Hz, 1H), 4.48 (d, J = 16.2 Hz, 2H), 4.46 (s, 1H), 3.91 (dt, J = 13.6 Hz, 2H), 3.45 (s, 2H), 3.36 (td, J = 10.4 Hz, 2H), 3.03 (s, 6H), 2.85 (d, J = 5.8 Hz, 2H), 2.80 (s, 6H), 2.45 (s, 3H), 2.15–2.01 (m, 4H), 1.65 (d, J = 10.1 Hz, 2H), 1.44 (s, 9H), 1.40 (s, 3H). ESI-MS: m / z [M] + calcd for C 46 H 60 ClN8O3S + 839.42, found 839.67. Mp 146.6–149.0 °C.

[0070] 1-(2-(2-((2-((3-((5-(4-(tert-Butoxycarbonylamino)-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 method of 10b is similar to that of 10a (109 mg, yield 74.3%). 1 H NMR (400 MHz, CDCl3) δ 9.96 (s, 1H), 8.17 (dd, J = 8.2, 1.4 Hz, 1H), 7.99 (s, 1H), 7.45–7.34 (m, 5H), 7.05 (t, J = 8.1 Hz, 1H), 6.92 (d, J = 15.2 Hz, 1H), 6.75 (d, J = 4.5 Hz, 1H), 6.66 (d, J = 8.9 Hz, 2H), 6.51 (dd, J = 8.0, 1.4 Hz, 1H), 6.34 (d, J = 15.2 Hz, 1H), 4.72 (t, J = 4.9 Hz, 2H), 4.45 (s, 1H), 3.96–3.86 (m, 4H), 3.57 (t, J = 4.9 Hz, 2H), 3.39–3.30 (m, 4H), 3.05–3.00 (m, 6H), 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] + calcd for C 47 H 62 ClN8O4S + 869.43, found 869.75. Mp 137.9–140.2 °C.

[0072] 1-(2-(2-(2-((2-((3-((5-(4-(tert-Butoxycarbonylamino)-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] The synthesis method of 10c is similar to that of 10a (105 mg, yield 74.9%). 11H NMR (400 MHz, CDCl3) δ 10.15 (s, 1H), 8.26 (d, J = 8.2 Hz, 1H), 8.00 (s, 1H), 7.48–7.33 (m, 5H), 7.07 (td, J = 8.1 Hz, 1H), 6.92 (d, J = 15.2 Hz, 1H), 6.79–6.73 (m, 1H), 6.66 (dd, J = 6.6 Hz, 2H), 6.50 (dq, J = 7.9 Hz, 1H), 6.33 (d, J = 15.4 Hz, 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 = 10.6 Hz, 2H), 3.03 (d, J = 2.5 Hz, 6H), 2.81 (d, J = 6.0 Hz, 6H), 2.46 (s, 3H), 2.12 (s, 2H), 1.73 (s, 4H), 1.63 (d, J = 10.1 Hz, 2H), 1.44 (s, 9H), 1.40 (s, 3H). ESI-MS: m / z [M+H] + calcd for C 49 H 66 ClN8O4S + 914.47, found 914.66. Mp 127.2–129.5 °C

[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)but-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. Subsequently, the reaction was carried out at room temperature for 3 h. The solvent was removed under reduced pressure. Dichloromethane and saturated sodium carbonate solution were added, and the mixture was stirred and separated. The organic phase was dried over anhydrous Na2SO4. After purification by thin-layer chromatography, the dark red solid product SHP-PP1 (27 mg, yield 61.3%) was obtained. 11H NMR (400 MHz, DMSO-d6) δ 10.07 (s, 1H), 8.22 (s, 1H), 8.06 (d, J = 7.4 Hz, 1H), 7.88 (s, 2H), 7.76–7.68 (m, 1H), 7.47 (d, J = 8.8 Hz, 2H), 7.23 (t, J = 8.1 Hz, 1H), 7.01 (d, J = 10.1 Hz, 1H), 6.95 (t, J = 15.3 Hz, 1H), 6.73 (d, J = 8.9 Hz, 2H), 6.60 (d, J = 15.3 Hz, 1H), 6.53 (dd, J = 8.0 Hz, 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 13C NMR (101 MHz, DMSO-d6) δ 170.89, 154.77, 154.37, 153.51, 152.24, 151.49, 143.24, 141.94, 136.89, 135.90, 131.48, 130.12, 129.50, 128.26, 124.03, 123.70, 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 + calcd for C 41 H 52 ClN8OS + 739.3668, found 739.3665. Mp 172.1–175.0 °C. HPLC purity 97.9%, t R = 5.140 min, 250 mm × 4.6 mm, MeOH:H2O = 90:10, 0.5 mL / 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)but-1,3-dien-1-yl)-2,6-dimethylpyridin-1-ium (SHP-PP2)

[0077] The synthesis method of SHP-PP2 is similar to that of SHP-PP1 (28 mg, yield 63.3%). 1 H NMR (400 MHz, DMSO-d6) δ 10.26–9.95 (m, 1H), 8.21 (s, 1H), 8.03 (d, J = 7.2 Hz, 1H), 7.80 (d, J = 18.2 Hz, 2H), 7.70 (dd, J = 15.3, 10.0 Hz, 1H), 7.44 (d, J = 8.9 Hz, 2H), 7.19 (t, J = 8.1 Hz, 1H), 7.04–6.96 (m, 1H), 6.92 (d, J = 15.2 Hz, 1H), 6.72 (d, J = 8.9 Hz, 2H), 6.56 (d, J = 15.2 Hz, 1H), 6.49 (d, J = 8.0 Hz, 1H), 4.61 (s, 2H), 3.85 (s, 2H), 3.74–3.63 (m, 4H), 3.50 (t, J = 5.3 Hz, 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 (101 MHz, DMSO-d6) δ 171.03, 155.13, 154.86, 153.51, 152.52, 151.49, 143.46, 142.03, 136.95, 135.82, 131.43, 130.12, 129.51, 128.25, 124.01, 123.66, 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] + calcd for C 42 H 54 ClN8O2S + 769.3773, found 769.3768. Mp 146.3–148.4 °C. HPLC purity 96.2%, t R = 4.657 min, 250 mm × 4.6 mm, MeOH:H2O = 90:10, 0.5 mL / 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 is similar to that of SHP-PP1 (31 mg, yield 69.6%). 1 HNMR (400 MHz, DMSO-d6) δ 10.38–9.95 (m, 1H), 8.21 (s, 1H), 8.09 (d, J = 8.1 Hz, 1H), 7.84 (s, 2H), 7.70 (dd, J = 15.4, 9.7 Hz, 1H), 7.44 (d, J = 8.9 Hz, 2H), 7.19 (t, J = 8.1 Hz, 1H), 7.02–6.95 (m, 1H), 6.92 (d, J = 15.3 Hz, 1H), 6.71 (d, J = 9.0 Hz, 2H), 6.55 (d, J = 15.2 Hz, 1H), 6.49 (dd, J = 8.0, 1.4 Hz, 1H), 4.61 (s, 2H), 3.85 (s, 2H), 3.75–3.62 (m, 4H), 3.46 (d, J = 7.3 Hz, 4H), 3.43 (d, J = 2.7 Hz, 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 (101 MHz, DMSO-d6) δ 170.89, 154.77, 154.37, 153.51, 152.24, 151.49, 143.24, 141.94, 136.89, 135.90, 131.48, 130.12, 129.50, 128.26, 124.03, 123.70, 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] + calcd for C 42 H 54 ClN8O2S +813.4036, found 813.4030. Mp 140.9–142.5 °C. HPLC purity 98.7%, t R = 5.173 min, 250 mm × 4.6 mm, MeOH:H2O = 90:10, 0.5 mL / min.

[0080] Example 2

[0081] Detection experiment on the properties of the small molecule near-infrared fluorescent probe SHP-PP:

[0082] 1. Optical properties

[0083] The ultraviolet absorption, fluorescence excitation and emission wavelengths were preliminarily tested as shown in Table 1.

[0084] Table 1 Optical properties of the fluorescent probe

[0085]

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

[0087] The data shown in Table 1 indicate that the probe reaches the ultraviolet absorption peak at a wavelength of 465 nm. The maximum excitation wavelengths of all probes are 485 nm and 490 nm respectively, the emission spectrum is at 680 nm, and the Stokes shift is 200 nm. The large Stokes shift can reduce background noise, reduce the phototoxic effect on biological specimens, and increase the detection sensitivity. In addition, the responsiveness of the probe to environmental changes was also 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, with good environmental sensitivity, meeting the design requirements.

[0088] 2. Enzyme activity inhibition assay

[0089] One of the key factors in evaluating the probe activity is its ability to inhibit the activities of SHP1 and SHP2 enzymes, which is crucial because it reveals the binding strength of the probe to the target protein and the selectivity for homologous proteins. We carried out enzyme activity assays. The commercially available enzyme was pre-incubated with the probe for 30 minutes, then the phosphatase substrate DiFMUP was added, and then the fluorescence signal of the product was measured. The relevant data are shown in Figure 3and Table 2. This probe has an obvious inhibitory effect on SHP2 and has no effect on SHP1, indicating that this probe can selectively recognize and label SH P2. Compared with SHP-099, although the inhibitory effect of the probe is reduced, the SHP-PP series of probes still have good IC 50 inhibitory activity. The results show that SHP-PP1 has the best activity, and the activity decreases slightly with the increase in the length of the linker. Although the fluorescent group has a potential impact on the inhibitory activity, our probe still has a strong binding ability to SHP2 and maintains good selectivity.

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

[0091]

[0092] 3. Cytotoxicity experiment

[0093] For probes designed for live cell imaging, ensuring biocompatibility is crucial. After confirming that the probe can bind to SHP2, we continued to evaluate its potential cytotoxicity. The cells were co-cultured with the probe for 24 h, and the number of viable cells was determined by the SRB method. The IC 50 values of the SHP-PP series of probes inhibiting cell proliferation were all greater than 30 μmol / L, significantly higher than the concentration required for imaging. The imaging incubation time was only 20 min, much shorter than the culture time of the cytotoxicity test (24 hours). These results indicate that the probe has sufficient biocompatibility to meet the requirements of imaging.

[0094] Table 3 Cytotoxicity of probe

[0095]

[0096] 4. Live cell imaging

[0097] Live cell imaging is a key application for the detector to achieve real-time observation of the target. As Figure 4 shown, after the cells were co-incubated with the probe for 20 minutes, fluorescence imaging without washing showed that the probe labeled SHP2, and all three probes could emit enhanced fluorescence. In addition, the binding of the probe to SHP2 could be competed by SHP-099, resulting in a decrease in fluorescence, indicating the specific binding of the probe to SHP2. Under the same conditions, SHP-PP2 showed the best imaging performance while maintaining a 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 property) ( Figure 2 ). Affected by the length of the linker chain, the imaging properties of SHP-PP1 and SHP-PP3 were slightly weaker. In summary, 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 the light emitted by fluorescent molecules excited by polarized light to study intermolecular interactions. It has a wide range of applications in monitoring molecular interactions in solutions, including but not limited to studies of receptor-ligand binding, protein-peptide interactions, DNA-protein binding, kinase activity assays, and competitive immunoassays. Fluorescence polarization can not only characterize the affinity of fluorescent molecules for their targets but also be further used to establish competitive fluorescence screening methods. As Figure 8 shown, after incubation with the protein, the FP value of the probe increased significantly and was positively correlated with the concentration, gradually reaching a saturation state. The K d value of the SHP-PP series of probes was calculated from the saturation curve. Consistent with the inhibition of SHP2 enzyme activity, the minimum affinity K d value of SHP-PP1 for SHP2 was calculated by the FP method, and the K d value gradually increased with the extension of the linker chain. The results showed that all three probes bound to the SHP2 protein, and the K d 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. The small molecule near-infrared fluorescent probe SHP-PP is characterized in that The structure of the SHP-PP is as follows: ， where n = 1 or 2; or, the structure of the SHP-PP is as follows: 。 2. A preparation method of the small molecule near-infrared fluorescence probe SHP-PP according to claim 1, characterized in that, The reaction steps are as follows: 。 3. Use of the small molecule near-infrared fluorescence probe SHP-PP described in claim 1 for preparing a product having at least one of the following functions: (1) A product for detecting SHP2 expression; (2) A product for tumor detection; (3) An in vitro pathological staining product for tumor tissues.

4. The application according to claim 3, characterized in that The product includes a reagent and / or a kit.

Images