A cyanine probe compound, preparation method and application thereof

Abstract

This invention discloses an anthocyanin-based probe compound, its preparation method, and its applications. The invention provides compound I, its tautomers, or pharmaceutically acceptable salts thereof. The anthocyanin-based probe compound of this invention exhibits one or more of the following advantages: good tumor targeting activity, stable and continuous fluorescence generation, low instrument requirements, clear visualization of anatomical structures, applicability to glioma fluorescence imaging and surgical navigation, effective reduction of background fluorescence effects, rapid response, high sensitivity, high specificity, long in vivo metabolic time, and good biocompatibility.

Description

Technical Field

[0001] The present invention relates to a cyanine probe compound, a preparation method and application thereof. Background Art

[0002] Gliomas are the most common primary intracranial malignant tumors, and surgical resection is the mainstay of clinical treatment for gliomas. The completeness of tumor resection is positively correlated with patient survival, and the patient's prognosis is highly dependent on the effective identification and resection of glioma tissue. However, due to the lack of real-time, high-resolution, and high-accuracy intraoperative imaging technology, surgeons face enormous challenges in accurately identifying tumor infiltration areas and residual tumor tissue during surgery. Tumor cells remaining at the surgical margin are the root cause of glioma recurrence. Therefore, there is an urgent need to establish molecular imaging technology that can accurately trace the margins of gliomas during surgery to guide precise glioma resection and improve the prognosis of glioma patients.

[0003] Neuronavigation and magnetic resonance imaging are currently widely used in clinical practice to locate gliomas by delineating areas of blood-brain barrier disruption. However, due to the infiltrative nature of gliomas within the brain, these techniques are unable to accurately delineate the tumor's infiltrated areas, leading to rapid tumor recurrence after surgery. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the defects of existing tumor immune boundary targeting probes in the body, such as being easily metabolized in the body or having difficulty accurately outlining the tumor infiltration area, resulting in rapid recurrence of tumors after surgery. A cyanine probe compound, a preparation method thereof, and its application are provided. The cyanine probe compound of the present invention has good tumor targeting effect, can stably and continuously generate fluorescence, has low instrumentation requirements, can clearly display anatomical structures, can be used for glioma fluorescence imaging and surgical navigation, can effectively reduce the impact of background fluorescence, has rapid response, high sensitivity, high specificity, long in vivo metabolism time, and good biocompatibility.

[0005] The present invention solves the above technical problems through the following technical solutions.

[0006] The present invention provides a compound I, a tautomer thereof, or a pharmaceutically acceptable salt thereof:

[0007]

[0008] Among them, R 2 C6~C 10 Aryl, C1-C6 straight chain or branched alkyl or 1, 2 or 3 R 2-1 Substituted C1-C6 straight-chain or branched alkyl;

[0009] R 2-1 is phenyl;

[0010] L 1 is a C1-C6 alkylene group;

[0011] m (degree of polymerization) is 20-120;

[0012] X is halogen.

[0013] In certain preferred embodiments of the present invention, certain groups in the compound I, its tautomers or pharmaceutically acceptable salts thereof are defined as follows (unmentioned groups are the same as those described in any embodiment of the present application, hereinafter referred to as "in some embodiments"), R 2 In the definition of C6~C 10 The aryl group is preferably a phenyl group or a naphthyl group, more preferably a phenyl group.

[0014] In some embodiments, R 2 In the definition, the C1-C6 straight or branched alkyl group and the alkyl group are separated by 1, 2 or 3 R 2-1 The C1-C6 straight-chain or branched-chain alkyl group in the substituted C1-C6 straight-chain or branched-chain alkyl group is preferably a C1-C3 straight-chain or branched-chain alkyl group; more preferably a methyl group, an ethyl group, a n-propyl group or an isopropyl group.

[0015] In some embodiments, R 2 In the definition of , the C1-C6 straight-chain or branched alkyl group is preferably an ethyl group.

[0016] In some embodiments, R 2 In the definition of 2-1 The substituted C1-C6 linear or branched alkyl group is preferably a benzyl group.

[0017] In some embodiments, Preferably

[0018] In some embodiments, L 1 In the definition, the C1~C6 alkylene is preferably C1~C3 alkylene; more preferably -CH2-, -CH2CH2-, -CH(CH3)-, -CH2CH2CH2-, -CH(CH3)CH2- or -C(CH3)2-; for example -CH2-.

[0019] In some embodiments, the m is preferably 20-80; more preferably 35-62, such as 44.

[0020] In some embodiments, the The corresponding polyethylene glycol is PEG2000.

[0021] In some embodiments, the molecular weight of Compound I is preferably 2077-3359, for example 2631.

[0022] In some embodiments, in the definition of X, the halogen is preferably fluorine, chlorine, bromine or iodine; more preferably bromine or iodine.

[0023] In the present invention, the compound I can be selected from any one of the following compounds:

[0024]

[0025] Wherein, the m is preferably 20-80; more preferably 35-62, for example 44;

[0026] and / or, the The corresponding polyethylene glycol is PEG2000;

[0027] And / or, the molecular weight of the compound I is preferably 2077-3359, for example 2631.

[0028] In some embodiments, the tautomers of Compound 1 as described above are as follows: Among them, R 2 , L 1 , m and X are defined as above;

[0029] Preferably, the tautomer of compound I is

[0030] wherein m is preferably 20-80; more preferably 35-62, for example 44;

[0031] and / or, the The corresponding polyethylene glycol is PEG2000;

[0032] And / or, the molecular weight range of the tautomer of Compound I is preferably 2077-3359, for example 2631.

[0033] The present invention also provides a method for preparing the compound I or its tautomer as described above, comprising the steps of: subjecting compound II and compound III to a condensation reaction as shown below in a solvent in the presence of a condensing agent and a base to obtain the compound I or its tautomer;

[0034]

[0035] Among them, R 2 , L 1 , m and X are defined as above.

[0036] The reaction operation and conditions of the condensation reaction are conventional operations and conditions in this field of this type of reaction. The preferred reaction conditions of the present invention are as follows:

[0037] In the condensation reaction, the solvent may be one or more of water, a nitrile solvent (such as acetonitrile) and an amide solvent (such as N,N-dimethylformamide); the solvent is preferably a nitrile solvent; the nitrile solvent is preferably acetonitrile.

[0038] In the condensation reaction, the condensing agent may be N,N'-dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS), or 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU); preferably N,N'-dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS).

[0039] In the condensation reaction, when the condensing agents are N,N'-dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS), the molar ratio of N,N'-dicyclohexylcarbodiimide (DCC) to N-hydroxysuccinimide (NHS) can be 1:0.5-1:5, preferably 1:0.8-1:1.5, for example 1:1.

[0040] In the condensation reaction, the molar ratio of the compound II to the N,N'-dicyclohexylcarbodiimide (DCC) can be 1:1-1:10, preferably 1:4-1:6, for example 1:5.

[0041] In the condensation reaction, the base can be an organic base (e.g., triethylamine or N,N-diisopropylethylamine) or an inorganic base (e.g., potassium carbonate or sodium bicarbonate). The base can be in the form of an alkaline aqueous solution; preferably, an alkaline aqueous solution of an inorganic base, more preferably, an aqueous solution of sodium bicarbonate.

[0042] In the condensation reaction, the pH value of the alkaline aqueous solution may be 7.4-9.5, preferably 7.5-9.0, for example 8.

[0043] In the condensation reaction, the volume ratio of the alkaline aqueous solution to the nitriles may be 1:0.8-1:5, preferably 1:1-1:2, for example 1:1.

[0044] In the condensation reaction, the molar ratio of the compound II to the compound III may be 1:1-1:4, preferably 1:1.5-1:2.5, for example 1:1.9.

[0045] In the condensation reaction, the molar volume ratio of the compound II to the nitrile solvent can be 0.0025-0.025 mol / L, preferably 0.005-0.01 mol / L, for example 0.0065 mol / L.

[0046] In the condensation reaction, the temperature of the condensation reaction may be 10-60°C, preferably 20-50°C, such as 25°C, 30 or 40°C.

[0047] The condensation reaction can be monitored by conventional monitoring methods in the art (e.g., HPLC, TLC, or GC). The reaction endpoint is generally determined when the starting material disappears or no longer reacts, or when the product no longer increases. The condensation reaction time is 0.5-6 hours, preferably 1 hour.

[0048] In some embodiments, the method for preparing the compound I may further comprise the following steps: in a solvent, in the presence of a base, subjecting the compound IV to a hydrolysis reaction as shown below to obtain the compound II;

[0049]

[0050] Among them, R 2 、L 1 and X is defined as above;

[0051] R 1 It is a C1-C6 straight-chain or branched-chain alkyl group.

[0052] In some embodiments, R 1 In the definition, the C1-C6 straight-chain or branched alkyl group is preferably a C1-C3 straight-chain or branched alkyl group; more preferably a methyl group, an ethyl group, a n-propyl group or an isopropyl group; for example, a methyl group.

[0053] The reaction operation and conditions of the hydrolysis reaction are conventional operations and conditions in this field of this type of reaction. The present invention preferably performs the following steps:

[0054] In the hydrolysis reaction, the solvent may be an alcohol solvent and / or water. The solvent is preferably water or a mixed solvent of an alcohol solvent and water. The alcohol solvent is preferably methanol or ethanol, such as methanol.

[0055] In the hydrolysis reaction, when the solvent is a mixed solvent of an alcohol solvent and water, the volume ratio of the alcohol solvent to the water in the mixed solvent can be 1:1-3:1, preferably 1.5:1.

[0056] In the hydrolysis reaction, the base may be an inorganic base, preferably potassium hydroxide and / or sodium hydroxide, such as potassium hydroxide.

[0057] In the hydrolysis reaction, the molar ratio of the compound IV to the base may be 1:1-1:8; preferably 1:2-1:6, for example 1:2.1 or 1:5.2.

[0058] In the hydrolysis reaction, the molar volume ratio of the compound IV to the solvent may be 0.0025-0.025 mol / L, preferably 0.005 mol / L-0.015 mol / L, for example 0.0064 mol / L or 0.013 mol / L.

[0059] In the hydrolysis reaction, the temperature of the hydrolysis reaction may be 0 to 60°C, preferably 15-40°C, such as 25°C.

[0060] The hydrolysis reaction can be monitored by conventional monitoring methods in the art (e.g., HPLC, TLC, or GC), and the reaction endpoint is generally when the starting materials disappear or no longer react, or when the product no longer increases. The hydrolysis reaction time can be 0.5-8 h, preferably 2-6 h, for example 4 h or 8 h.

[0061] In some embodiments, the compound I can be prepared by the following route 1 or route 2:

[0062] Route 1:

[0063]

[0064] R 1 、R 2 、L 1 The definitions of , m and X are the same as above. Route 2:

[0065]

[0066] R 2 、L 1 , m and X are as defined above. The present invention also provides a compound II:

[0067]

[0068] R 2 、L 1 and X are defined as above.

[0069] In some embodiments, the compound II is

[0070] The present invention also provides a method for preparing the compound II, which is prepared using the above-mentioned preparation method.

[0071] The present invention also provides a compound IV:

[0072]

[0073] R 1 、R 2 、L 1 and X are defined as above.

[0074] In some embodiments, the compound IV is

[0075] The present invention further provides a detection reagent or diagnostic kit comprising Compound I, a tautomer thereof, or a pharmaceutically acceptable salt thereof as described above. The detection reagent or diagnostic kit can be used to detect tumor-associated macrophages or for glioma surgical navigation. Detection of tumor-associated macrophages can be performed by tumor-associated macrophage-targeted imaging.

[0076] The present invention further provides a use of Compound I, a tautomer thereof, or a pharmaceutically acceptable salt thereof, as described above, in the preparation of a detection reagent or diagnostic kit. In such use, the detection reagent or diagnostic kit can be used to detect tumor-associated macrophages or for glioma surgical navigation. The detection of tumor-associated macrophages can be performed by tumor-associated macrophage-targeted imaging.

[0077] The present invention further provides a method for targeted imaging of glioma infiltration margin or tumor-associated macrophages, which comprises administering an effective amount of the above-described compound I, its tautomer or a pharmaceutically acceptable salt thereof to a patient in need of such diagnosis.

[0078] In the present invention, unless otherwise specified, the terms are defined as follows:

[0079] The term "alkylene" refers to a saturated divalent hydrocarbon group obtained by removing two hydrogen atoms from a saturated straight or branched hydrocarbon group. Examples of alkylene groups include methylene (-CH2-), ethylene (including -CH2CH2- or -CH(CH3)-), isopropylene (including -CH(CH3)CH2- or -C(CH3)2-), and the like.

[0080] The term "tautomer" or "tautomeric form" refers to structural isomers of different energies that are interconvertible via a low energy barrier. If tautomerism is possible (e.g., in solution), a chemical equilibrium of the tautomers can be achieved. For example, proton tautomers (also known as prototropic tautomers) include interconversions via proton migration, such as keto-enol isomerization and imine-enamine isomerization. Unless otherwise indicated, all tautomeric forms of the compounds of the invention are within the scope of the invention.

[0081] The term "pharmaceutically acceptable" means relatively non-toxic, safe, and suitable for use by patients.

[0082] The term "pharmaceutically acceptable salt" refers to a salt obtained by reacting a compound with a pharmaceutically acceptable acid or base. When the compound contains a relatively acidic functional group, a base addition salt can be obtained by contacting the compound with a sufficient amount of a pharmaceutically acceptable base in a suitable inert solvent. Pharmaceutically acceptable base addition salts include, but are not limited to, sodium salts, potassium salts, calcium salts, aluminum salts, magnesium salts, bismuth salts, and ammonium salts. When the compound contains a relatively basic functional group, an acid addition salt can be obtained by contacting the compound with a sufficient amount of a pharmaceutically acceptable acid in a suitable inert solvent. Pharmaceutically acceptable acid addition salts include, but are not limited to, hydrochlorides, sulfates, and methanesulfonates. For details, see Handbook of Pharmaceutical Salts: Properties, Selection, and Use (P. Heinrich Stahl, Camille G. Wermuth, 2011, 2nd Revised Edition).

[0083] Without violating the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain preferred embodiments of the present invention.

[0084] Unless otherwise specified, all reagents and raw materials used in the present invention are commercially available.

[0085] The positive progress of the present invention is to provide a cyanine probe compound, its preparation method and application. The cyanine probe compound of the present invention has good tumor targeting effect, high tumor imaging signal-to-noise ratio, low instrument and equipment requirements, high fluorescence intensity, strong tissue penetration, can be used for glioma fluorescence imaging and surgical navigation, can effectively reduce the impact of background fluorescence, rapid response, high sensitivity, high specificity, long in vivo metabolism time and good biocompatibility. One or more of the advantages. For example, compared with the approved 5-ALA probe for glioma surgical navigation, the maximum emission wavelength of the probe of the present invention reaches 800nm, which reduces the background interference caused by the organism's own fluorescence, which is beneficial to practical application. For another example, by imaging the glioma area with a high content of tumor-associated macrophages, the precise positioning of the glioma infiltration boundary can be improved, providing a new idea for the treatment of solid tumors. The preparation method of the cyanine probe compound of the present invention has the advantages of high yield or high purity. BRIEF DESCRIPTION OF THE DRAWINGS

[0086] Figure 1 The pH ratio responses of the probe CP2-M were analyzed by UV and fluorescence spectra.

[0087] Figure 2 Evaluation of the specific response of probe CP2-M.

[0088] Figure 3 To evaluate the cytotoxicity of probe CP2-M to M2-macrophages.

[0089] Figure 4 The probe CP2-M specifically recognizes M2-macrophages.

[0090] Figure 5 Pharmacokinetic study. A is a schematic diagram of the fluorescent probe pharmacokinetic model. B is the pharmacokinetic curves of the three probes. The simulated probe concentrations show the same trend and similar changes as the experimental data. The gray dots are experimental data, the red solid line represents the predicted median, and the blue area represents the 90% prediction interval of the final model simulation. C is a schematic diagram of in vivo imaging of intracranial glioblastoma. D is an intraoperative fluorescence image of the intracranial tumor area under 740nm laser irradiation after intravenous injection of CP2, CP2-P, or CP2-M (5.0μmol / kg / rat). The tumor is delineated by a white dashed line. E is a pharmacodynamic model showing that the simulated signal-to-noise ratio (S / N) shows the same trend and similar changes as the experimental data. F is the optimal imaging time window after a single dose of CP2-M (5.0μmol / kg); the black dashed line represents the ideal signal-to-noise ratio value (S / N ≥ 2), and the time period between the two red lines is the recommended imaging time window.

[0091] Figure 6To accurately locate the infiltration boundary of glioma. A is a schematic diagram of in vivo fluorescence imaging of the same GBM rat model using three fluorescent probes in succession. B is a white light image of the brain tumor of a rat bearing C6 glioma. C is a fluorescence imaging image of the brain after oral administration of 5-ALA. D is a fluorescence imaging image after intravenous injection of ICG. E is a pH topography map of the tumor depicted by CP2-M. The pH topography is generated by the ratio of fluorescence intensity after CP2-M is excited by lasers with wavelengths of 740 and 670 nanometers. The white dotted line represents the tumor boundary. The color bar represents the pH value. F is Figure 6 B shows H&E-stained images of glioma, G and H correspond to magnified images of the areas in the red and yellow boxes in F, respectively.

[0092] Figure 7 CP2-M-guided surgical resection of an orthotopic glioma in a rat. A is a schematic diagram of the CP2-M-guided surgical resection of a rat glioma. B is a magnified image of real-time fluorescence imaging of the brain after craniotomy in a rat model bearing a C6 glioma. C shows intraoperative glioma resection guided by CP2-M fluorescence imaging of the brain tumor and its ratiometric image at 740 nm and 670 nm laser wavelengths, respectively. The procedure was terminated only after all areas with a pH below 7.0 were resected.

[0093] Figure 8 Preoperative and postoperative magnetic resonance imaging images of the brain of rats bearing C6 glioma. A is the timeline of surgical resection of rats bearing C6 glioma xenografts guided by fluorescence imaging and the evaluation of prognosis by magnetic resonance imaging. B is the recurrence of tumor-bearing mice monitored by magnetic resonance imaging before and after surgery guided by ICG, 5-ALA, and CP2-M, respectively. The dotted line represents the tumor outline. C is the change in tumor volume in the rat model after surgery. The unpaired t-test was used to compare the differences in tumor volume among the groups. D is the Kaplan-Meier survival curve (n = 5 rats / group). The Gehan-Breslow-Wilcoxon test was used to compare the differences in survival rate. DETAILED DESCRIPTION

[0094] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples.

[0095] The amino-polyethylene glycol (PEG2000) used in the examples is methoxy-PEG2000-amine (CAS: 80506-64-5), purchased from Xi'an Ruixi Biotechnology Co., Ltd., with a molecular weight range of 1493.86-2155.26.

[0096] The amino-polyethylene glycol (PEG2000)-mannose used in the examples was homemade. Boc-aminoPEG2000-carboxylic acid and mannose monomers were coupled by esterification, followed by removal of the Boc protection to obtain the compound. The preparation method is based on the literature references AAPSPharmSciTech 20, 188 (2019); International Journal of Pharmaceutics 427, 1, 123-133 (2012); European Journal of Pharmaceutics and Biopharmaceutics 94, 83-93, (2015).

[0097] Example 1

[0098]

[0099]

[0100] In the structural formula The corresponding polyethylene glycol is PEG2000.

[0101] Step 1: Preparation of compound 1

[0102] N, N-dimethylformamide (20 g, 275 mmol) and dichloromethane (20 mL) were uniformly mixed in a three-necked flask. Phosphorus oxychloride (17.2 g, 112 mmol) was added dropwise under ice bath conditions and stirred at 0°C for 0.5 h. Finally, cyclohexanone (5 g, 51 mmol) was slowly added to the above solution. After the reaction solution stabilized, it was transferred to a 70°C oil bath and refluxed under argon for 3 h. Heating was stopped, and the reaction solution was returned to room temperature. It was poured into 200 g of crushed ice, filtered, and freeze-dried to obtain 6.3 g of a yellow powdery solid (Compound 1). No purification was required and it could be used directly in the next step.

[0103] Step 2: Preparation of compound 2

[0104] Dissolve 2,3,3-trimethyl-3H-indole (10 g, 62.8 mmol) and methyl 3-bromopropionate (11.8 g, 70.9 mmol) in anhydrous acetonitrile (30 mL). Heat to 80°C and reflux with vigorous stirring for 12 h. Cool to room temperature. The precipitated solid was filtered to obtain 13.7 g of a light red solid powder (Compound 2). This was carried on to the next step without purification.

[0105] Step 3: Preparation of compound 3

[0106] Compound 1 (1 g, 5.8 mmol), compound 2 (3.6 g, 11 mmol), and sodium acetate (1 g, 12.2 mmol) were dissolved in acetic anhydride (20 mL) under argon. The mixture was heated to 80°C, protected from light, and stirred for 1 h. After the reaction was complete, the mixture was cooled to room temperature. The mixture was added dropwise to glacial ether to precipitate a green solid. The resulting crude green solid was filtered and purified by column chromatography on silica gel (methanol:dichloromethane = 1:15 to 1:5, V:V) with a gradient elution to obtain 2.6 g of a green solid product (compound 3) in a 63.4% yield.

[0107] 1 H NMR (400MHz, MeOD) δ=8.40(d,J=13.9Hz,2H),7.49(d,J=7.2Hz,2H),7.42–7.19(m,6H),6.36(d,J=14.0Hz ,2H),4.47(t,J=6.2Hz,4H),3.57(s,6H),2.85(t,J=6.4Hz,4H),2.72(t,4H),1.94(m,2H),1.69(s,12H).

[0108] Step 4: Preparation of compound 4

[0109] Compound 3 (1 g, 1.4 mmol) and N-ethylpiperazine (0.48 g, 4.2 mmol) were dissolved in 20 mL of acetonitrile and reacted at room temperature for 6 h with sufficient stirring. The organic phase was removed by distillation under reduced pressure, and the remaining blue residue was purified by column chromatography on silica gel (methanol:dichloromethane = 1:15 to 1:4, V:V) with a gradient elution rate to obtain 0.7 g of a blue solid product (Compound 4) in a 63.2% yield.

[0110] 1 H NMR (400MHz, MeOD) δ7.67(d,J=13.2Hz,2H),7.38(t,J=7.1Hz,2H),7.30(t,J=7.5Hz,2H),7.11(d,J=7.3Hz,4H),5.98(d,J=13.5Hz,2H),4. 29(t,J=5.6Hz,4H),3.82(t,4H),3.56(s,6H),2.79–2.69(m,8H),2.51(t,J=6.0Hz,6H),1.78(t,J=6.6Hz,2H),1.63(s,12H),1.18(t,3H).

[0111] Step 5: Preparation of compound CP2

[0112] Compound 4 (0.5 g, 0.64 mmol) was dissolved in methanol (30 mL). Aqueous potassium hydroxide (67 mM, 20 mL) was slowly added to the solution at 0°C and allowed to react at room temperature for 4 h. Methanol was then removed by distillation under reduced pressure. The remaining aqueous solution was adjusted to pH 7 with saturated aqueous ammonium chloride and extracted with dichloromethane (3×). The aqueous layer was discarded, and the organic layer was dried over anhydrous magnesium sulfate and distilled under reduced pressure to obtain a solid powder. The crude product was then purified by column chromatography on silica gel (methanol:dichloromethane = 1:10 to 1:3, v:v) with a gradient elution to obtain 228 mg of a sapphire-colored pure product (Compound CP2) in a 47.3% yield.

[0113] 1 H NMR (400MHz, CDCl3) δ7.76-7.72(d,J=13.4Hz,2H),7.40–7.35(d,J=7.3Hz,2H),7.33–7.27(t,J =7.4Hz,2H),7.24–7.16(d,J=7.8Hz,2H),7.15–7.06(t,J=7.2Hz,2H),6.13-6.03(d,J=13.4Hz,2 H), 4.28-4.18 (t, J = 7.0 Hz, 4H), 3.80-3.71 (m, 4H), 3.15-3.08 (t, J = 4.0 Hz, 4H), 2.86-2.75 (t, 4H), 2.70-2.60 (m, 6H), 1.85-1.74 (m, 2H), 1.65 (s, 12H), 1.13-1.04 (t, J = 7.1 Hz, 3H). TOF-MS calculated value C 42 H 53 N4O4 + [MH + ]:677.4061(100%), measured value 677.4070.

[0114] Step 6: Preparation of compound CP2-M

[0115] Compound CP2 (100 mg, 0.13 mmol), N-hydroxysuccinimide (76 mg, 0.66 mmol), and N,N'-dicyclohexylcarbodiimide (136 mg, 0.66 mmol) were dissolved in 20 mL of acetonitrile and reacted at 25°C for 1 h. Then, amino-polyethylene glycol (PEG2000)-mannose (MS (m / z): 1971.82 (most intense peak on mass spectrum) [1663.20-2685.38] (molecular weight range indicated by mass spectrum)) (500 mg, 0.25 mmol) was dissolved in 20 mL of sodium bicarbonate aqueous solution at pH 8 and added dropwise to the reaction mixture. The reaction was continued at room temperature for 2 h. The acetonitrile was removed by distillation under reduced pressure, and the aqueous layer was dialyzed overnight and lyophilized to obtain a blue solid powder. The crude product was then purified by preparative liquid phase separation to obtain 162 mg of pure product (Compound CP2-M) in a 46.7% yield.

[0116] 1 H NMR(400MHz,D2O)δ8.54–8.51(t,1H),7.71(d,J=14.4Hz,2H),7.47(d,J=7.5Hz,2H),7.41-7.37(t ,2H),7.23(d,J=7.0Hz,2H),7.19–7.14(t,2H),6.07(d,J=13.5Hz,2H),4.34-4.31(t,4H),3.84-3. 83 (m, 9H), 3.59 (s, 187H), 3.18-3.16 (m, 8H), 2.69-2.66 (t, 4H), 2.51-2.48 (m, 6H), 1.95 (s, 12H), 1.81–1.75 (m, 2H), 1.12 (s, 3H). MS (ESI, m / z): 2630.96 (most intense peak in the mass spectrum) [2076.80-3359.37] (molecular weight range displayed by the mass spectrum).

[0117] Step 7: Preparation of compound CP2-P

[0118] Compound CP2 (100 mg, 0.13 mmol), N-hydroxysuccinimide (76 mg, 0.66 mmol), and N,N'-dicyclohexylcarbodiimide (136 mg, 0.66 mmol) were dissolved in 20 mL of acetonitrile and reacted at 25°C for 1 h. Then, amino-polyethylene glycol (PEG2000) (MS (m / z): 1713.99 (most intense peak on mass spectrometry) [1493.86-2155.26] (molecular weight range indicated by mass spectrometry)) (435 mg, 0.25 mmol) was dissolved in 20 mL of sodium bicarbonate aqueous solution at pH 8 and added dropwise to the reaction mixture. The reaction was continued at room temperature for 2 h. The acetonitrile was removed by distillation under reduced pressure, and the aqueous layer was dialyzed overnight and lyophilized to obtain a blue solid powder. The crude product was then purified by preparative liquid phase separation to obtain 171 mg of the pure product (Compound CP2-P) in a 54.6% yield. MS (ESI, m / z): 2375.39 (strongest peak on mass spectrum) [2111.23-2551.49] (molecular weight range shown by mass spectrum).

[0119] Example 2

[0120] Step 1: Preparation of compound 1

[0121] Add N,N-dimethylformamide (40g, 550mmol) to a three-necked flask, add phosphorus oxychloride (17.2g, 112mmol) dropwise under ice bath conditions, and stir at 0°C for 0.5h. Finally, slowly add cyclohexanone (5g, 51mmol) to the above solution. After the reaction solution stabilizes, transfer it to a 70°C oil bath and reflux under argon for 3h. Stop heating, wait for the reaction solution to return to room temperature, pour it into 200g of crushed ice, filter it with suction, and lyophilize it to obtain 3.6g of yellow powdery solid (Compound 1). No purification is required and it can be used directly in the next step.

[0122] Step 2: Preparation of compound 2

[0123] Dissolve 2,3,3-trimethyl-3H-indole (10 g, 62.8 mmol) and methyl 3-bromopropionate (11.8 g, 70.9 mmol) in toluene (50 mL), heat to 100°C, and reflux with vigorous stirring for 12 h. Cool to room temperature, remove toluene by distillation under reduced pressure, and add to a large amount of anhydrous ether to precipitate a solid. Filter with suction to obtain 8.3 g of a light red solid powder (Compound 2). This was carried on to the next step without purification.

[0124] Step 3: Preparation of compound 3

[0125] Compound 1 (1 g, 5.8 mmol), compound 2 (3.6 g, 11 mmol), and sodium acetate (1 g, 12.2 mmol) were dissolved in acetic anhydride (20 mL) under argon. The mixture was heated to 100°C, protected from light, and stirred for 1 h. After the reaction was complete, the mixture was cooled to room temperature. The mixture was added dropwise to glacial ether to precipitate a green solid. The resulting crude green solid was filtered and purified by column chromatography on silica gel (methanol:dichloromethane = 1:15 to 1:5, V:V) with a gradient elution to obtain 2.5 g of a green solid product (compound 3) in a 60.5% yield.

[0126] 1 H NMR (400MHz, MeOD) δ=8.40(d,J=13.9Hz,2H),7.49(d,J=7.2Hz,2H),7.42–7.19(m,6H),6.36(d,J=14.0Hz ,2H),4.47(t,J=6.2Hz,4H),3.57(s,6H),2.85(t,J=6.4Hz,4H),2.72(t,4H),1.94(m,2H),1.69(s,12H).

[0127] Step 4: Preparation of compound 4

[0128] Compound 3 (1 g, 1.4 mmol) and N-ethylpiperazine (0.48 g, 4.2 mmol) were dissolved in 50 mL of dichloromethane and reacted at 40°C for 2 h with vigorous stirring. The organic phase was removed by distillation under reduced pressure, and the remaining blue residue was purified by column chromatography on silica gel (methanol:dichloromethane = 1:15 to 1:4, V:V) with a gradient elution rate to obtain 0.64 g of a blue solid product (Compound 4) in a 57.7% yield.

[0129] 1 H NMR (400MHz, MeOD) δ7.67(d,J=13.2Hz,2H),7.38(t,J=7.1Hz,2H),7.30(t,J=7.5Hz,2H),7.11(d,J=7.3Hz,4H),5.98(d,J=13.5Hz,2H),4. 29(t,J=5.6Hz,4H),3.82(t,4H),3.56(s,6H),2.79–2.69(m,8H),2.51(t,J=6.0Hz,6H),1.78(t,J=6.6Hz,2H),1.63(s,12H),1.18(t,3H).

[0130] Step 5: Preparation of compound CP2

[0131] Compound 4 (0.5 g, 0.64 mmol) was dissolved in water (50 mL). A 67 mM aqueous solution of potassium hydroxide (50 mL) was slowly added to the solution at 0°C and allowed to react at room temperature for 8 h. Methanol was then removed by distillation under reduced pressure. The remaining aqueous solution was adjusted to pH 7 with saturated aqueous ammonium chloride and extracted with dichloromethane (3×). The aqueous layer was discarded, and the organic layer was dried over anhydrous magnesium sulfate and distilled under reduced pressure to obtain a solid powder. The crude product was then purified by column chromatography on silica gel (methanol:dichloromethane = 1:10 to 1:3, v:v) with a gradient elution to obtain 158 mg of a sapphire-colored pure product (Compound CP2) in a 32.7% yield.

[0132] 1 H NMR (400MHz, CDCl3) δ7.76-7.72(d,J=13.4Hz,2H),7.40–7.35(d,J=7.3Hz,2H),7.33–7.27(t,J =7.4Hz,2H),7.24–7.16(d,J=7.8Hz,2H),7.15–7.06(t,J=7.2Hz,2H),6.13-6.03(d,J=13.4Hz,2 H), 4.28-4.18 (t, J = 7.0 Hz, 4H), 3.80-3.71 (m, 4H), 3.15-3.08 (t, J = 4.0 Hz, 4H), 2.86-2.75 (t, 4H), 2.70-2.60 (m, 6H), 1.85-1.74 (m, 2H), 1.65 (s, 12H), 1.13-1.04 (t, J = 7.1 Hz, 3H). TOF-MS calculated value C 42 H 53 N4O4 + [MH + ]:677.4061(100%), measured value 677.4070.

[0133] Step 6: Preparation of compound CP2-M

[0134] Compound CP2 (100 mg, 0.13 mmol), N-hydroxysuccinimide (76 mg, 0.66 mmol), and N,N'-dicyclohexylcarbodiimide (136 mg, 0.66 mmol) were dissolved in 20 mL of acetonitrile and reacted at 40°C for 1 h. Then, amino-polyethylene glycol (PEG2000)-mannose (MS (m / z): 1971.82 (most intense peak on mass spectrum) [1663.20-2685.38] (molecular weight range indicated by mass spectrum)) (500 mg, 0.25 mmol) was dissolved in 20 mL of sodium bicarbonate aqueous solution at pH 8 and added dropwise to the reaction mixture. The reaction was continued at room temperature for 2 h. The acetonitrile was removed by distillation under reduced pressure, and the aqueous layer was dialyzed overnight and lyophilized to obtain a blue solid powder. The crude product was then purified by preparative liquid phase separation to obtain 168 mg of pure product (Compound CP2-M) in a 48.4% yield.

[0135] 1 H NMR(400MHz,D2O)δ8.54–8.51(t,1H),7.71(d,J=14.4Hz,2H),7.47(d,J=7 .5Hz,2H),7.41-7.37(t,2H),7.23(d,J=7.0Hz,2H),7.19–7.14(t,2H),6.0 7(d,J=13.5Hz,2H),4.34-4.31(t,4H),3.84-3.83(m,9H),3.59(s,187H),3 .18-3.16(m,8H),2.69-2.66(t,4H),2.51-2.48(m,6H),1.95(s,12H),1.81 -1.75 (m, 2H), 1.12 (s, 3H). MS (ESI, m / z): 2630.96 (strongest peak in mass spectrum) [2076.80-3359.37] (molecular weight range displayed by mass spectrum).

[0136] Step 7: Preparation of compound CP2-P

[0137] Compound CP2 (100 mg, 0.13 mmol), N-hydroxysuccinimide (76 mg, 0.66 mmol), and N,N'-dicyclohexylcarbodiimide (136 mg, 0.66 mmol) were dissolved in 20 mL of acetonitrile and reacted at 40°C for 1 h. Amino-polyethylene glycol (PEG2000) (MS (m / z): 1713.99 (most intense peak on mass spectrometry) [1493.86-2155.26] (molecular weight range indicated by mass spectrometry)) (435 mg, 0.25 mmol) was then dissolved in 20 mL of sodium bicarbonate aqueous solution at pH 8 and added dropwise to the reaction mixture. The reaction was continued at room temperature for 2 h. The acetonitrile was removed by distillation under reduced pressure, and the aqueous layer was dialyzed overnight and lyophilized to obtain a blue solid powder. The crude product was then purified by preparative liquid phase separation to obtain 176 mg of the pure product (Compound CP2-P) in a 56.2% yield. MS (ESI, m / z): 2375.39 (strongest peak on mass spectrum) [2111.23-2551.49] (molecular weight range shown by mass spectrum).

[0138] Example 3 Optical properties, selectivity, cellular uptake, animal model experiments and results of the compounds of the present invention

[0139] 1. Compound absorption and fluorescence response experiments

[0140] The pH-responsive absorption and fluorescence spectra of the compounds were measured using a Shimadzu UV-2550 ultraviolet spectrophotometer and a Shimadzu RF-5301PC fluorescence spectrophotometer, respectively. Probe CP2-M was dissolved in DMSO to prepare 10 mM stock solutions. These stock solutions were then added to citric acid-sodium hydrogen phosphate buffer solutions at different pH values. The probe was tested at a concentration of 2 μM at pH values ​​of 4.0, 5.0, 5.5, 6.0, 6.5, 6.8, 7.0, 7.2, 7.5, and 8.0. Figure 1 The pH-responsive absorption and fluorescence spectra of the probe are shown. The results show that probe CP2-M exhibits excellent pH ratiometric responsiveness, providing an ideal readout of the acidity of the tumor microenvironment. The maximum absorption peak of probe CP2-M occurs at 740 nm, and the maximum fluorescence emission peak occurs at 790 nm, preliminarily demonstrating that the probe possesses near-infrared fluorescence imaging capabilities at the animal level.

[0141] 2. Probe Selectivity Experiment

[0142] Figure 2This probe selectivity experiment, also known as a specific response experiment, involved dissolving appropriate amounts of potassium chloride, calcium chloride, magnesium chloride, sodium chloride, ammonium chloride, lysine (Lys), arginine (Arg), threonine (Thr), histidine (His), leucine (Leu), alanine (Ala), serine (Ser), valine (Val), and glutamic acid (Glu) in a pH 7.4 citric acid-sodium hydrogen phosphate buffer solution to a 200 mM saline solution. The CP2-M probe stock solution was then diluted to a final concentration of 1.0 μM in the prepared saline solution and a blank PBS buffer. The excitation wavelength was 740 nm, the detection wavelength range was 760–900 nm, the excitation / emission slit widths were 5 / 5 nm, the scan speed was medium, and the sensitivity was low. The maximum fluorescence intensity of each fluorescence spectrum was collected, and the experimental data were processed and fitted using OriginPro 9.1.

[0143] The results showed that common metal ions and amino acids in the body had no effect on the pH ratio response of the probe CP2-M (fluorescence ratio signal fluctuation ≤ 10%), indicating that the compound has specificity for pH response and has high anti-interference ability, and can accurately respond to the pH in the tumor microenvironment in the body. To prove the specificity of the pH response of the probe, we studied the common endogenous cations (K + , Na + , Mg 2+ , Ca 2+ , NH4 + ) and amino acids (Leu, Lys, Ala, Arg, Ser, Thr, Val, Glu, His) (200 mM) were tested to determine if there was potential interference with the pH response of the probe. Figure 2 The results showed that the probe has good pH sensitivity. Whether at pH 4.0 or pH 8.0, the common endogenous ions or amino acids in the body did not produce significant fluorescence response interference (fluorescence ratio signal fluctuation ≤ 10%). This indicates that the compound has specificity for pH response and has high anti-interference ability, and can accurately respond to the pH of the tumor microenvironment in the body.

[0144] 3. Cytotoxicity Assay

[0145] The mononuclear macrophages were cultured in DMEM medium supplemented with 10% (v / v) fetal bovine serum, penicillin (100 U / mL) and streptomycin (100 mg / mL) in a 5% CO2, 37°C constant temperature incubator.

[0146] (1) Mononuclear macrophages in the logarithmic growth phase were digested with 0.25% trypsin, processed into a single-cell suspension with a concentration of 1.0×104 cells / well, and inoculated into 96-well cell culture plates for culture.

[0147] (2) After cells adhered, a concentration gradient of probe CP2-M (6.25-50 μM) was added. 100 μL of probe CP2-M was prepared in culture medium at twice the desired concentration and added to the corresponding wells. The pure culture medium group served as a negative control. The 96-well cell culture plate with probes was placed in a 5% CO2, 37°C incubator and incubated for 12, 24, and 48 h, respectively.

[0148] (3) The 96-well cell culture plate was removed and the cell status was observed under an inverted microscope. 10 μL of CCK-8 reagent was added to each well and incubated for 4 h.

[0149] (4) Take out the 96-well cell culture plate, aspirate the culture medium from the wells, then add 150M DMSO solution to each well and shake at low speed on a shaker for 10 minutes.

[0150] (5) Place the 96-well cell culture plate into the iMark microplate reader and detect and record the absorbance of each well at 450 nm.

[0151] Figure 3 The cytotoxicity test results of the probe showed that it still exhibited good biocompatibility at a concentration of up to 50 μM, and the cell survival rate was higher than 80%, laying the foundation for its further biological application.

[0152] 4. Cell Targeting Experiment

[0153] C6, M0, M1 and M2 macrophages in the logarithmic growth phase were treated with 0.25% trypsin digestion solution and the cell suspension concentration was adjusted to 5×10 5 Cells were added to a confocal dish and cultured overnight in 50 μL of the solution. Observation was performed using an inverted microscope. When the cells occupied 70% to 80% of the dish, the probe CP2-M was added and incubated for 4 hours. The cells were then washed three times with PBS and the probe fluorescence signal was detected using LCFM (LSM 880, Zeiss, Germany). Figure 4 This is the confocal imaging result of the CP2-M probe. M2 macrophages exhibit the strongest fluorescence signal. Due to overexpression of CD206, M2 macrophages specifically recognize the probe's mannose ligand, leading to increased uptake and accumulation of the probe, emitting a fluorescence signal at 800 nm. This demonstrates that the CP2-M probe has good targeting properties for M2 macrophages.

[0154] Establishment of a C6 glioma-bearing rat model

[0155] Take a dish of C6 cells in the logarithmic growth phase in the above-mentioned incubator, digest them with trypsin, and centrifuge them at 2000r / min for 2min. The supernatant is discarded by suction, and 30μL PBS (1×) is added to make a cell suspension, which is placed in an ice box for later use. Take a male SD rat weighing about 160-180g and anesthetize it with 10% (W / W) chloral hydrate by intraperitoneal injection. After about 5min, carefully cut the hair on the upper surface of the anesthetized rat's head with scissors. After alcohol disinfection, make an incision along the line connecting the middle of the rat's brain near the back of the eyes to the middle of the two ears. Use a cotton swab dipped in 10% (V / V) H2O2 to wipe the exposed skull until the anterior fontanelle is exposed. Then fix the rat's head on a brain stereotaxic instrument to ensure that its head is placed horizontally and stably. The rat skull was located 4 mm to the right and 0.5 mm behind the bregma, and a pre-prepared C6 rat glioblastoma cell suspension (5×105 cells, 6 μL PBS) was injected into the parietal bone 4 mm deep through a 10 μL Hamilton syringe using a brain stereotaxic apparatus. The injection speed was 3.0 μL / min, and the syringe stayed at the injection site for 3 minutes to avoid backflow after injection. Then it was slowly lifted up 1 mm and waited for another 1 minute. After slowly withdrawing the needle, the needle hole was finally sealed with bone wax, and the incision was sutured with surgical sutures. About 8-10 days after inoculation, the successful establishment of the in situ glioma model was verified at the in vivo level by magnetic resonance T2-weighted imaging. After one week of modeling and feeding, the rats weighed approximately 180 to 220 g.

[0156] 5. Pharmacokinetics of CP2-M

[0157] Rats bearing C6 gliomas were randomly divided into three groups, with three rats in each group. The rats bearing C6 gliomas were anesthetized by intraperitoneal injection of 0.8 mL of 10% chloral hydrate, and then craniotomy was performed to expose the orthotopic brain tumors of the rats bearing C6 gliomas. Then, a medical syringe was used to inject 60 μL of the probes CP2 (0.5 mg / μL) and CP2-P (2 mg / μL) into the tail vein of the non-targeted group of C6 glioma-bearing rats, and 60 μL of the probe CP2-M (2 mg / μL) was injected into the targeted group of C6 glioma-bearing rats. After administration, the craniotomy sites of the C6 glioma-bearing rats were placed under a stereofluorescence microscope, and time-dependent fluorescence imaging was performed at different time points. The acquisition time points were 30 minutes, 1 hour, 2 hours, 4 hours, and 6 hours after administration for in vivo brain fluorescence imaging. Specific acquisition parameters: stereo fluorescence microscope (MVX10, Olympus), light source PE4000, excitation filter center wavelengths of 670 nm and 740 nm, emission filter range 745–835 nm, exposure time 1 s.

[0158] Figure 5Figure A is a schematic diagram of the pharmacokinetic model for fluorescent probes. Figure B shows the pharmacokinetic curves for the three probes. The simulated probe concentrations show the same trends and similar variations as the experimental data. The gray dots represent experimental data, the solid red line represents the predicted median, and the blue area represents the 90% prediction interval of the final model simulation. Figure C is a schematic diagram of in vivo imaging of intracranial glioblastoma. Figure D is an intraoperative fluorescence image of the intracranial tumor area under 740 nm laser irradiation after intravenous injection of CP2, CP2-P, or CP2-M (5.0 μmol / kg / rat). The tumor is demarcated by a white dashed line. Figure E shows the pharmacodynamic model showing that the simulated signal-to-noise ratio (S / N) shows the same trends and similar variations as the experimental data. Figure F shows the optimal imaging time window after a single dose of CP2-M (5.0 μmol / kg); the black dashed line represents the ideal signal-to-noise ratio (S / N ≥ 2), and the time period between the two red lines is the recommended imaging time window.

[0159] 6. Comparative In Vivo Tumor Imaging Experiments with Clinically Applied Probes

[0160] Fluorescence imaging experiments with the three probes were performed sequentially in randomly selected rats bearing C6 gliomas (n=3 / group). Rats bearing C6 gliomas were anesthetized with an intraperitoneal injection of 0.8 mL of 10% chloral hydrate, followed by craniotomy to expose the tumor. First, rats bearing C6 gliomas were orally administered 5-ALA (5-aminolevulinic acid) (20 mg / kg) by gavage according to FDA standards. Approximately 3 hours later, craniotomy was performed to expose the in situ brain tumor of rats bearing C6 glioma, and fluorescence imaging of the brain tumor was performed; then the probe ICG was injected into the rats bearing C6 glioma via the tail vein, and fluorescence imaging of the brain tumor in the same position was performed again; approximately 0.5 hours later, ICG (indocyanine green) was basically metabolized in the body, and CP2-M (10 mg / kg) was administered via the tail vein. Two corresponding fluorescence images were captured using dual-wavelength excitation of 670 nm and 740 nm, and the two fluorescence maps were compared to obtain the pH topography of the brain tumor.

[0161] Figure 6 Center A is a schematic diagram of in vivo fluorescence imaging of the same GBM rat model using three fluorescent probes in succession. Figure 6 Figure B is a white light image of a brain tumor in a rat bearing C6 glioma. The fluorescence imaging results of 5-ALA are shown in Figure 4. Figure 6 In the middle C, it has certain targeting effect on tumors, but there are also some non-specific distributions, such as obvious false positive fluorescence signals around the tumor, and some false negative areas within the tumor (yellow box). The fluorescence of ICG in the brain shows non-specific distribution, and no obvious fluorescence enhancement signal is seen at the tumor, indicating that ICG has no tumor targeting effect (such as Figure 6 D in the figure). CP2-M (10 mg / kg) is metabolized and cleared in normal brain tissue because there are no binding receptors in non-tumor areas. CP2-M in brain tumor areas specifically binds to the mannose receptors of tumor-associated macrophages, is phagocytosed by M2-macrophages, and is temporarily retained in the tumor tissue area and enriched. Two corresponding fluorescence images are obtained by excitation at 670 nm and 740 nm wavelengths, respectively. By importing the two fluorescence images into the program, the corresponding brain tumor fluorescence ratio image pH topography (such as Figure 6 In E), red represents the more acidic tumor infiltration area, and blue represents the normal brain tissue area. It can be clearly seen that the pH topography map locates the infiltration boundary of glioma more accurately than 5-ALA and ICG fluorescence imaging. Figure 6 H&E-stained images of glioma in B, G and H correspond to magnified images of the areas in the red and yellow boxes in F, respectively. Scale bars: 500 μm and 50 μm (magnified images).

[0162] 7. CP2-M guided glioma surgical resection experiment

[0163] C6 glioma cells (~5×10 6 A rat model bearing C6 glioma was established using a MRI scan of the rat. After MRI confirmed that the tumor had grown to a diameter of 4-5 mm, a craniotomy was performed to expose the brain tumor for intraoperative fluorescence imaging of the tumor boundary. The probe (10 mg / kg) was injected into the mouse tail vein. Using 670 nm and 740 nm near-infrared light as excitation, the stereomicroscope's wide-field imaging enabled real-time imaging of the tumor window in rats bearing C6 gliomas. A pH map calculated using an intelligent automatic processing program was used to determine the tumor immune boundary, and all acidic tissue with a pH below 6.8 was removed. The pH map showed that higher acidity indicated a greater density of M2 macrophages, indicating a more aggressive tumor. The pH map revealed that the most acidic region was located at the edge of the tumor (shown as the upper surface of the tumor in the figure), with the pH reaching as low as 5.5, indicating a large concentration of M2 macrophages at the edge of the tumor. The core of the tumor, on the other hand, was slightly less acidic, with a pH range of 6.2-6.8. Brain tumor imaging experiments on rats bearing C6 gliomas showed that the probe has good tumor targeting, accurate positioning and high resolution. Under the guidance of pH topography, surgical resection is performed along the acidic boundary, such as Figure 7 The procedure is shown as three cuts from left to right. Real-time imaging is used to monitor residual lesions after tumor resection, and the remaining tumor tissue is removed sequentially until the acid-free area (far right) on the pH topography is reached, allowing for precise guidance of the tumor's immune infiltration boundary.

[0164] Figure 7Middle A is a schematic diagram of the CP2-M-guided surgical resection process of rat glioma. Figure 7 Middle B is an enlarged image of real-time fluorescence imaging of the brain after craniotomy in a rat model bearing C6 glioma. Figure 7 Intraoperative glioma resection was guided by real-time fluorescence imaging of brain tumors using CP2-M and ratiometric images at 740 nm and 670 nm laser wavelengths, respectively. The surgery was not terminated until all areas with a pH below 7.0 were removed.

[0165] 8. Magnetic resonance imaging to evaluate surgical prognosis

[0166] Preoperative and postoperative magnetic resonance images of the brain of rats bearing C6 glioma Figure 8 As shown in the figure, rats bearing C6 glioma were randomly divided into 3 groups, with 5 rats in each group. The orthotopic glioma rat models in the 3 groups were successfully established by magnetic resonance T2 imaging before surgery (T2W). The tumor sizes were similar and randomized, and all were located in the right cerebral cortex and striatum. The day of surgical resection was designated as postoperative day 0. MRI was performed every 1 or 2 weeks to monitor postoperative recurrence. Gadolinium was injected as a magnetic resonance T1 contrast enhancer before each scan. Figure 8 Figure B shows representative magnetic resonance images of three tumor-bearing mice from each group. The CP2-M group underwent surgical resection guided by tumor immunosuppression margins. Tumor recurrence was not observed in one rat until the 75th day after surgery (one rat died of postoperative infection), while the other three rats showed no signs of recurrence until the 90th day. The ICG group underwent surgical resection guided by the near-infrared fluorescent probe ICG. Rats in this group showed signs of tumor recurrence one week after surgery. All four rats developed white, brightly colored residual tumor lesions in the tumor bed within one or two weeks (one rat died of postoperative infection). Tumors rapidly grew after recurrence, with one rat's tumor occupying half of the cranial cavity by the third week after surgery. The 5-ALA group underwent surgical resection guided by the fluorescent imaging agent 5-ALA, used for clinical surgical navigation. Rats in this group experienced recurrences starting in the third week, and by approximately the fifth week, the tumor volume exceeded half of the cranial cavity. Rats whose recurrent tumor volume exceeded half of the cranial cavity, compromising survival, were considered endangered and euthanized for ethical reasons.

[0167] Figure 8Figure A shows the timeline of fluorescence imaging-guided surgical resection of rats bearing C6 glioma xenografts, followed by prognostic evaluation using magnetic resonance imaging. Figure B shows recurrence in tumor-bearing mice monitored by magnetic resonance imaging before and after ICG-, 5-ALA-, and CP2-M-guided surgery, respectively. The dotted line represents the tumor outline. Figure C shows the change in tumor volume after surgery in the rat model. Unpaired t-tests were used to compare differences in tumor volume among groups. Figure D shows Kaplan-Meier survival curves (n = 5 rats / group). The Gehan-Breslow-Wilcoxon test was used to compare differences in survival rates.

[0168] Although specific embodiments of the present invention have been described above, those skilled in the art will appreciate that these are merely illustrative and that the scope of the present invention is defined by the appended claims. Those skilled in the art may make various changes or modifications to these embodiments without departing from the principles and essence of the present invention, and such changes and modifications are intended to fall within the scope of the present invention.

Claims

1. A compound I, a tautomer thereof or a pharmaceutically acceptable salt thereof: in, R 2 C6~C 10 Aryl, C1-C6 straight or branched alkyl or 1, 2 or 3 R 2-1 Substituted C1-C6 straight chain or branched alkyl; R 2-1 is phenyl; L 1 is C1-C6 alkylene; m is 20-120; X is halogen.

2. The compound I, its tautomer or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: It meets one or more of the following conditions: (1)R 2 In the definition of C6~C 10 Aryl is phenyl or naphthyl; (2)R 2 In the definition, the C1-C6 straight or branched alkyl group and 1, 2 or 3 R 2-1 The C1-C6 straight chain or branched chain alkyl in the substituted C1-C6 straight chain or branched chain alkyl is a C1-C3 straight chain or branched chain alkyl; (3)L 1 In the definition, the C1-C6 alkylene group is a C1-C3 alkylene group; (4) m is 20-80; (5) In the definition of X, the halogen is fluorine, chlorine, bromine or iodine; (6) The molecular weight of compound I is 2077-3359.

3. The compound I, its tautomer or a pharmaceutically acceptable salt thereof according to claim 2, characterized in that: It meets one or more of the following conditions: (1)R 2 In the definition of C6~C 10 Aryl is phenyl; (2)R 2 In the definition, the C1-C6 straight or branched alkyl group and 1, 2 or 3 R 2-1 The C1-C6 straight-chain or branched-chain alkyl group in the substituted C1-C6 straight-chain or branched-chain alkyl group is methyl, ethyl, n-propyl or isopropyl; (3)L 1 In the definition, the C1-C6 alkylene group is -CH2-, -CH2CH2-, -CH(CH3)-, -CH2CH2CH2-, -CH(CH3)CH2- or -C(CH3)2-; (4) m is 35-62; (5) In the definition of X, the halogen is bromine or iodine.

4. The compound I, its tautomer or a pharmaceutically acceptable salt thereof according to claim 3, characterized in that: It meets one or more of the following conditions: (1)L 1 In the definition, the C1-C6 alkylene group is -CH2-; (2) m is 44; (3) The molecular weight of compound I is 2631.

5. The compound I, its tautomer or a pharmaceutically acceptable salt thereof according to claim 2, characterized in that: It meets one or more of the following conditions: (1)R 2 In the definition, the C1-C6 straight-chain or branched-chain alkyl group is an ethyl group; (2)R 2 In the definition of 2-1 The substituted C1-C6 straight-chain or branched-chain alkyl group is benzyl; (3) The corresponding polyethylene glycol is PEG2000.

6. The compound I, its tautomer or a pharmaceutically acceptable salt thereof according to claim 5, characterized in that: for 7. The compound I, its tautomer or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: It meets one or more of the following conditions: (1) The compound I is selected from any of the following compounds: Wherein, m is 20-80; and / or, The corresponding polyethylene glycol is PEG2000; and / or, the molecular weight of the compound I is 2077-3359; (2) The tautomers of the compound I are as follows: Among them, R 2 , L 1 , m and X are as defined in claim 1.

8. The compound I, its tautomer or a pharmaceutically acceptable salt thereof according to claim 7, characterized in that: m is 35-62.

9. The compound I, its tautomer or a pharmaceutically acceptable salt thereof according to claim 8, characterized in that: m is 44.

10. The compound I, its tautomer or a pharmaceutically acceptable salt thereof according to claim 7, characterized in that: The molecular weight of the compound I is 2631.

11. A method for preparing the compound I or its tautomer according to any one of claims 1 to 10, characterized in that: The method comprises the following steps: in a solvent, in the presence of a condensing agent and a base, subjecting compound II and compound III to a condensation reaction as shown below to obtain the compound I or its tautomer; Among them, R 2 , L 1 , m and X are as defined in any one of claims 1-10.

12. The method for preparing Compound I according to claim 11, characterized in that: It meets one or more of the following conditions: (1) In the condensation reaction, the solvent is one or more of water, a nitrile solvent and an amide solvent; (2) In the condensation reaction, the condensing agent is N,N'-dicyclohexylcarbodiimide and N-hydroxysuccinimide, or 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate; (3) In the condensation reaction, the base is an organic base or an inorganic base; (4) In the condensation reaction, the molar ratio of the compound II to the compound III is 1:1-1:4; (5) In the condensation reaction, the temperature of the condensation reaction is 10-60°C.

13. The method for preparing compound I according to claim 12, characterized in that: It meets one or more of the following conditions: (1) In the condensation reaction, the solvent is a nitrile solvent; (2) In the condensation reaction, the condensing agent is N,N'-dicyclohexylcarbodiimide and N-hydroxysuccinimide; (3) In the condensation reaction, when the base is an inorganic base, the inorganic base is potassium carbonate or sodium bicarbonate; (4) In the condensation reaction, the base is present in the form of an alkaline aqueous solution; (5) In the condensation reaction, the molar ratio of the compound II to the compound III is 1:1.5-1:2.5; (6) In the condensation reaction, the temperature of the condensation reaction is 20-50°C.

14. The method for preparing Compound I according to claim 13, characterized in that: It meets one or more of the following conditions: (1) In the condensation reaction, when the condensing agent is N,N'-dicyclohexylcarbodiimide and N-hydroxysuccinimide, the molar ratio of the N,N'-dicyclohexylcarbodiimide to the N-hydroxysuccinimide is 1:0.5-1:5; (2) In the condensation reaction, when the condensing agent is N,N'-dicyclohexylcarbodiimide and N-hydroxysuccinimide, the molar ratio of the compound II to the N,N'-dicyclohexylcarbodiimide is 1:1-1:10; (3) In the condensation reaction, the base is an alkaline aqueous solution of an inorganic base; (4) In the condensation reaction, when the base is present in the form of an alkaline aqueous solution, the volume ratio of the alkaline aqueous solution to the nitrile solvent is 1:0.8-1:5; (5) In the condensation reaction, the molar ratio of the compound II to the compound III is 1:1.9; (6) In the condensation reaction, when the solvent is a nitrile solvent, the molar volume ratio of the compound II to the nitrile solvent is 0.0025-0.025 mol / L; (7) In the condensation reaction, the temperature of the condensation reaction is 25°C, 30°C or 40°C.

15. The method for preparing Compound I according to claim 14, characterized in that: It meets one or more of the following conditions: (1) In the condensation reaction, when the condensing agent is N,N'-dicyclohexylcarbodiimide and N-hydroxysuccinimide, the molar ratio of the N,N'-dicyclohexylcarbodiimide to the N-hydroxysuccinimide is 1:0.8-1:1.5; (2) In the condensation reaction, when the condensing agent is N,N'-dicyclohexylcarbodiimide and N-hydroxysuccinimide, the molar ratio of the compound II to the N,N'-dicyclohexylcarbodiimide is 1:4-1:6; (3) In the condensation reaction, the base is an aqueous solution of sodium bicarbonate; (4) When the base is present in the form of an alkaline aqueous solution, the pH value of the alkaline aqueous solution is 7.4-9.5; (5) In the condensation reaction, when the base is present in the form of an alkaline aqueous solution, the volume ratio of the alkaline aqueous solution to the nitrile solvent is 1:1-1:2; (6) In the condensation reaction, when the solvent is a nitrile solvent, the molar volume ratio of the compound II to the nitrile solvent is 0.005-0.01 mol / L.

16. The method for preparing Compound I according to claim 15, characterized in that: It meets one or more of the following conditions: (1) In the condensation reaction, when the condensing agent is N,N'-dicyclohexylcarbodiimide and N-hydroxysuccinimide, the molar ratio of the N,N'-dicyclohexylcarbodiimide to the N-hydroxysuccinimide is 1:1; (2) In the condensation reaction, when the condensing agent is N,N'-dicyclohexylcarbodiimide and N-hydroxysuccinimide, the molar ratio of the compound II to the N,N'-dicyclohexylcarbodiimide is 1:5; (3) When the base is present in the form of an alkaline aqueous solution, the pH value of the alkaline aqueous solution is 7.5-9.0; (4) In the condensation reaction, when the base is present in the form of an alkaline aqueous solution, the volume ratio of the alkaline aqueous solution to the nitrile solvent is 1:1; (5) In the condensation reaction, when the solvent is a nitrile solvent, the molar volume ratio of the compound II to the nitrile solvent is 0.0065 mol / L.

17. The method for preparing Compound I according to claim 16, characterized in that: When the alkali is present in the form of an alkaline aqueous solution, the pH value of the alkaline aqueous solution is 8.

18. The method for preparing Compound I according to claim 11, characterized in that: It further comprises the following steps: in a solvent, in the presence of a base, compound IV undergoes a hydrolysis reaction as shown below to obtain the compound II; Among them, R 2 , L 1 and X is defined as in any one of claims 1 to 10; R 1 It is a C1-C6 straight chain or branched chain alkyl group.

19. The method for preparing Compound I according to claim 18, characterized in that: It meets one or more of the following conditions: (1)R 1 In the definition, the C1-C6 straight chain or branched chain alkyl group is a C1-C3 straight chain or branched chain alkyl group; (2) In the hydrolysis reaction, the solvent is an alcohol solvent and / or water; (3) In the hydrolysis reaction, the base is an inorganic base; (4) In the hydrolysis reaction, the molar ratio of the compound IV to the base is 1:1-1:8; (5) In the hydrolysis reaction, the molar volume ratio of the compound IV to the solvent is 0.0025-0.025 mol / L; (6) In the hydrolysis reaction, the temperature of the hydrolysis reaction is 0 to 60°C.

20. The method for preparing Compound I according to claim 19, characterized in that: It meets one or more of the following conditions: (1)R 1 In the definition, the C1-C6 straight-chain or branched alkyl group is methyl, ethyl, n-propyl or isopropyl; (2) In the hydrolysis reaction, the solvent is water, or a mixed solvent of an alcohol solvent and water; (3) The alcohol solvent is methanol or ethanol; (4) In the hydrolysis reaction, when the solvent is a mixed solvent of an alcohol solvent and water, the volume ratio of the alcohol solvent to the water in the mixed solvent is 1:1-3:1; (5) In the hydrolysis reaction, the inorganic base is potassium hydroxide and / or sodium hydroxide; (6) In the hydrolysis reaction, the molar ratio of the compound IV to the base is 1:2-1:6; (7) In the hydrolysis reaction, the molar volume ratio of the compound IV to the solvent is 0.005 mol / L-0.015 mol / L; (8) In the hydrolysis reaction, the temperature of the hydrolysis reaction is 15-40°C.

21. The method for preparing Compound I according to claim 20, characterized in that: It meets one or more of the following conditions: (1)R 1 In the definition, the C1-C6 straight-chain or branched alkyl group is a methyl group; (2) In the hydrolysis reaction, the alcohol solvent is methanol; (3) In the hydrolysis reaction, when the solvent is a mixed solvent of an alcohol solvent and water, the volume ratio of the alcohol solvent to the water in the mixed solvent is 1.5:1; (4) In the hydrolysis reaction, the inorganic base is potassium hydroxide; (5) In the hydrolysis reaction, the molar ratio of the compound IV to the base is 1:2.1 or 1:5.2; (6) In the hydrolysis reaction, the molar volume ratio of the compound IV to the solvent is 0.0064 mol / L or 0.013 mol / L; (7) In the hydrolysis reaction, the temperature of the hydrolysis reaction is 25°C.

22. The method for preparing Compound I according to claim 11, characterized in that: The compound I is prepared by the following route 1 or route 2: Route 1: R 2 , L 1 , m and X are defined as in any one of claims 1 to 10; R 1 The definition as in claim 18; Route 2: R 2 , L 1 , m and X are as defined in any one of claims 1-10.

23. A compound represented by formula II or formula IV: in, R 2 , L 1 and X is defined as in any one of claims 1 to 10; R 1 The definition is as stated in claim 18.

24. The compound according to claim 23, characterized in that The compound II is The compound IV is 25. A detection reagent or diagnostic kit, characterized in that: The detection reagent and diagnostic kit comprise Compound I, a tautomer thereof, or a pharmaceutically acceptable salt thereof as described in any one of claims 1 to 10.

26. The detection reagent or diagnostic kit according to claim 25, characterized in that The detection reagent or diagnostic kit is used for detecting tumor-associated macrophages or glioma surgical navigation.

27. The detection reagent or diagnostic kit according to claim 26, characterized in that The detection of tumor-associated macrophages is performed by targeted imaging of tumor-associated macrophages.

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