Fluorescent probe compounds for tumor targeting imaging, and synthesis method therefor and use thereof

Abstract

The present invention relates to fluorescent probe compounds for tumor-targeted imaging, and methods of synthesis and use thereof. The compounds utilize, for the first time, an antitumor drug comprising a pyrrolo[2,3-d]pyrimidine core structure as a targeting ligand, which is conjugated to a fluorescent dye through a linker moiety. The structure of the compound is shown in Formula I. The compound demonstrates high affinity and selectivity for tumor cells expressing target receptors, enabling localization, qualitative and quantitative analysis through in vitro and in vivo tracking, receptor affinity studies, and mechanism of action investigations, thereby exhibiting high specificity, sensitivity, and visualization capability. The fluorescent probe of the present invention achieves rapid clearance from normal tissues while maintaining prolonged retention at tumor sites, thereby enabling in vivo diagnostic functionality. It demonstrates substantial clinical application potential for clinical intraoperative navigation.

Description

Fluorescent probe compound for tumor targeted imaging and its synthesis method and application Technical Field

[0001] The present invention belongs to the technical field of specific molecular targeted diagnostic reagents, and relates to a fluorescent probe compound for tumor targeted imaging, a synthesis method and an application thereof. Background Art

[0002] Cancer has been a century-old problem plaguing humanity, necessitating the development of a variety of treatments, including surgical resection, chemotherapy, radiotherapy, and biological therapy. Although surgical resection of malignant tumor tissue cannot completely eliminate it, approximately 50% of cancer patients who have detectable malignant tumor tissue removed experience no recurrence of the disease, and surgical resection can extend life expectancy or reduce the incidence of cancer recurrence. Therefore, surgical resection remains the most commonly used and effective treatment. Precisely because of the importance of complete resection of malignant tumor tissue, diagnostic methods that can ensure accurate and complete identification of malignant tumor tissue are of great application value.

[0003] Although people have recognized the importance of completely removing tumors and the availability of some identification technologies in visualizing tumor masses, many malignant tumor tissues still escape detection, leading to disease recurrence and ultimately death. Therefore, how to achieve more accurate and complete tumor identification is a core issue that needs to be urgently addressed in this field. With the discovery and application of fluorescent dyes, an intraoperative navigation technology called "fluorescence-guided surgery" has gradually become a "darling" in surgical operations. This technology uses excitation light of a specific wavelength to stimulate the tumor's own fluorescence, retained fluorescent molecules, or exogenous fluorescent substances taken up by cells to emit fluorescence, thereby guiding doctors to accurately remove the tumor. However, since the fluorescent dye itself is not targeted, it not only accumulates in tumor tissue, but also accumulates in normal tissue, which may lead to excessive resection and damage surrounding normal tissues. Therefore, it is necessary to develop a targeted fluorescent probe.

[0004] Due to its highly disease-restricted expression pattern, the α isoform of the folate receptor (FR-α) is recognized as a promising receptor for the development of targeted diagnostic and therapeutic approaches for cancer. At the same time, recent studies have reported that FR-β is specifically and highly expressed on the surface of tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs), and has been identified as a marker of immunosuppressive MDSCs and TAMs, providing a useful tool for precision immunotherapy against tumors. Since the folate receptor shows roughly equal binding affinity (K) to folic acid and folic acid conjugates, d =10 -9M) and is internalized via receptor-mediated endocytosis, making the folate receptor an excellent target for tumor-targeted fluorescent probes. Folate antagonists with a pyrrolo[2,3-d]pyrimidine core structure have excellent anti-tumor activity and potential selectivity for the folate receptor, but their application as ligands in targeted fluorescent probes has not yet been reported.

[0005] Fluorescein 5-isothiocyanate (FITC) is a fluorescein derivative. It is a functionalized fluorescein molecule created by replacing the hydrogen atoms on the base ring of the original fluorescein structure with an isothiocyanate reactive group (-N=C=S). It exhibits high absorptivity, excellent fluorescence quantum yield, and good water solubility, making it suitable for a wide range of applications, including flow cytometry, and is currently the most commonly used fluorescent marker. Compared to cyanine dyes in the near-infrared region, FITC has shorter excitation and emission wavelengths. While not an optimal imaging agent for fluorescence-guided surgery, it has broader applicability as a fluorescent visualization tool. For example, the folate-conjugated FITC molecular fluorescent probe EC17 is a classic FR-targeted fluorescent probe first used in clinical practice. It has been shown to selectively accumulate in cancers such as breast cancer, lung adenocarcinoma, and ovarian cancer. However, due to its visible emission wavelength, EC17 still has the problem of not being able to successfully visualize buried cancer nodules, leading to the undetection of many occult lesions. The latest research findings (J.Med.Chem.2018, 61, 9637-9646) use folic acid molecules as recognition groups and synthesize a new near-infrared fluorescent probe OTL38 by coupling with a cyanine dye in the near-infrared region. While illuminating tumor tissue at a significant depth below the surface, it can achieve the aggregation of fluorescent dyes in tumor tissues with high expression of folate receptors, including ovarian cancer and non-small cell lung cancer, and has been initially applied in clinical trials. However, the above related studies all use traditional folic acid molecules as targeting ligands conjugated to fluorescent dyes, which inevitably leads to a decrease in receptor affinity, resulting in undesirable accumulation in normal tissues.

[0006] In view of this, the present invention applies a folic acid antagonist with a pyrrolo[2,3-d]pyrimidine core structure to the design of tumor-targeted fluorescent probes, develops a series of molecular fluorescent probes that can achieve specific targeting of tumor tissues and in vivo tissue imaging with higher affinity, and applies them to surgical navigation.

[0007] Summary of the Invention

[0008] The purpose of the present invention is to invent a molecular fluorescent probe compound that can achieve specific imaging in targeted tumor tissues and in vivo tissues with higher affinity, and at the same time provide a preparation method and application thereof.

[0009] To achieve the above object, the technical solution adopted by the present invention is:

[0010] In a first aspect, the present invention provides a fluorescent probe compound for tumor-targeted imaging or a pharmaceutically acceptable salt thereof. This compound, for the first time, employs an anti-tumor drug having a pyrrolo[2,3-d]pyrimidine core structure as a targeting ligand, and is modified by a linker molecule and then connected to a fluorescent dye. The structure of the compound is shown in Formula I:

[0011] Wherein, W and Q are selected from the following structures:

[0012] (1) W is —C(O)NH—, Q is —(CH2) j —, wherein j is selected from 1, 2, 3, 4 or 5;

[0013] (2)W is —(CH2) n —, Q is Said n is selected from 1, 2, 3, 4 or 5;

[0014] X is an amino group or an amidated derivative group, wherein the amino group is independently selected from tyrosine, cysteine, glutamic acid, methionine, threonine, serine,

[0015] The amide derivative group represents two or more amino groups independently selected from the following: tyrosine, cysteine, glutamic acid, methionine, threonine, serine, Amidated derivatives formed by condensation of the structures in

[0016] The k is selected from 0, 1, 2, 3, 4 or 5;

[0017] Said p is selected from 1, 2, 3 or 4;

[0018] Y is independently selected from dyes having fluorescence excitation in the visible spectrum and emission spectra in the near infrared range, and the compound maintains or enhances the fluorescence of the dyes.

[0019] As a further improvement of the present invention, the amidated derivative group is selected from:

[0020] The k is selected from 0, 1, 2, 3, 4 or 5.

[0021] As a further improvement of the present invention, the Y is selected from fluorescein isothiocyanate FITC, dye IR-783 and S0456.

[0022] As a further improvement of the present invention, the amino group on one side of the amino group or amidated derivative group forms an amide bond with the carbonyl group bonded to Q, and the amino group, sulfhydryl group or oxy group on the other side is bonded to Y.

[0023] As a further improvement of the present invention, the compound is selected from:

[0024] The "pharmaceutically acceptable salts" of the present invention refer to salts of the compounds of the present invention, prepared by reacting the compounds of the present invention with relatively nontoxic acids or bases. When the compounds of the present invention contain relatively acidic functional groups, base addition salts can be obtained by contacting the neutral form of the compounds of the present invention with a sufficient amount of base in a pure solution or a suitable inert solvent. Pharmaceutically acceptable bases include salts prepared from inorganic bases and organic bases. The inorganic base salts include aluminum salts, ammonium salts, calcium salts, copper salts, iron salts, ferrous salts, lithium salts, magnesium salts, manganic salts, manganous salts, potassium salts, sodium salts, zinc salts, and the like. The organic non-toxic base salts include salts of primary, secondary, and tertiary amines, including substituted amines and cyclic amines. For example: N, N'-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, aminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucosamine, glucosamine, histidine, hydroxocobalamin, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, guaiac, polyamine resins, procaine, purine, theobromine, triethylamine, trimethylamine, tripropylamine, etc. When the compounds of the present invention contain relatively basic functional groups, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of acid in a neat solution or a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, bisulfate, hydroiodic acid, phosphorous acid, and the like; and organic acid salts such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, and methanesulfonic acid, and the like.

[0025] The second aspect of the present invention provides a composition comprising the above-mentioned compound, which further comprises at least one pharmaceutically acceptable carrier or excipient.

[0026] The third aspect of the present invention provides a use of the above compound in preparing a tumor diagnostic agent for tumor-targeted imaging.

[0027] As some preferred embodiments of the present invention, the tumor is one or more of liver cancer, breast cancer, lung cancer, pancreatic cancer, and colorectal cancer.

[0028] A fourth aspect of the present invention provides use of a compound in the preparation of an in vivo fluorescent imaging agent for precise tumor surgical navigation.

[0029] The beneficial effects of adopting the above technical solution are:

[0030] The fluorescent probe compounds provided by the present invention for tumor-targeted imaging exhibit high affinity and selectivity for tumor cells expressing target receptors. They enable in vitro and in vivo tracking of target receptor-expressing tumor cells, as well as localization, qualitative, and quantitative analysis of receptor affinity and mechanism of action, with high specificity, sensitivity, and intuitiveness. The fluorescent probes of the present invention are rapidly cleared from normal tissues but remain at the tumor site for extended periods, enabling in vivo diagnosis. They have promising clinical applications, including intraoperative navigation.

[0031] Figures in the specification

[0032] FIG1 is an absorption spectrum of the GT-NIR series compounds prepared in Example;

[0033] FIG2 is the excitation spectrum of the GT-NIR series compounds prepared in Example;

[0034] FIG3 is the emission spectrum of the GT-NIR series compounds prepared in Example;

[0035] Figure 4 shows an in vitro intracellular fluorescence imaging localization test (KB cells) of the compound GT-NIR-1 prepared in the Example; wherein: (a) the upper and lower figures are, respectively, a fluorescence imaging image of GT-NIR-1 in KB cells and a blank image in KB cells; (b) the upper and lower figures are, respectively, a fluorescence imaging image of GT-NIR-1 and 100 times folic acid in KB cells and a blank image in KB cells; (c) the upper and lower figures are, respectively, a fluorescence imaging image of GT-NIR-1 in A549 cells and a blank image in A549 cells;

[0036] Figure 5 shows the in vitro intracellular fluorescence imaging localization test (M2) of the compound GT-NIR-1 prepared in the Example; wherein: (a) the upper and lower figures are the fluorescence imaging of GT-NIR-1 in M2 macrophages and a blank image in M2 macrophages, respectively; (b) the upper and lower figures are the fluorescence imaging of GT-NIR-1 and 100 times folic acid in M2 macrophages and a blank image in M2 macrophages, respectively; (c) the upper and lower figures are the fluorescence imaging of GT-NIR-1 in M1 macrophages and a blank image in M1 macrophages, respectively;

[0037] FIG6 shows the binding affinity of the GT-NIR series compounds prepared in the examples to KB cells as determined by flow cytometry;

[0038] FIG7 shows the binding affinity of the GT-NIR series compounds prepared in the examples to M2 macrophages as determined by flow cytometry;

[0039] FIG8 shows the equilibrium dissociation constant K of the GT-NIR series compounds prepared in the examples with KB cells measured by flow cytometer. d ;

[0040] FIG9 shows the equilibrium dissociation constant K of the GT-NIR series compounds prepared in the examples with M2 macrophages measured by flow cytometer. d ;

[0041] FIG10 shows the absorption spectra of the GT-FITC series of compounds prepared in the examples; wherein: (a) absorption spectra of FITC, GT-FITC-1 to 4; (b) absorption spectra of GT-FITC-5 to 9;

[0042] FIG11 shows the excitation spectra of the GT-FITC series of compounds prepared in the Examples; wherein: (a) excitation spectra of FITC, GT-FITC-1 to 4; (b) excitation spectra of GT-FITC-5 to 9;

[0043] FIG12 shows the emission spectra of the GT-FITC series of compounds prepared in the Examples; wherein: (a) emission spectra of FITC, GT-FITC-1 to 4; (b) emission spectra of GT-FITC-5 to 9;

[0044] FIG13 shows the binding affinity of the GT-FITC series compounds prepared in the Examples to KB cells as determined by flow cytometry; wherein: (a) flow cytometry results for FITC and GT-FITC-1 to 4; (b) flow cytometry results for GT-FITC-5 to 9;

[0045] FIG14 is a fluorescence quantitative analysis graph of the GT-NIR series compounds (a) and GT-IR series compounds (b) prepared in the Example in an in vitro intracellular fluorescence imaging localization assay; wherein KB represents folate receptor-positive KB cells, A549 represents folate receptor-negative A549 cells, and Blocked represents the presence of 100% excess folic acid in KB cells and co-incubation with the probe;

[0046] FIG15 is a whole-body fluorescence imaging image of the GT-NIR series compounds prepared in Example after injection into a KB cell xenograft mouse model;

[0047] FIG16 is a whole-body fluorescence imaging image of the compound GT-NIR-4 prepared in the Example after injection into an A549 cell xenograft mouse model (A549), and co-injection of GT-NIR-4 and the compound into a KB cell xenograft mouse model (100×FA) using 100×FA;

[0048] Figure 17 shows the in vitro tissue biodistribution of the GT-NIR series compounds prepared in the Examples after injection into a KB cell xenograft mouse model. The in vitro tissues are, from top to bottom, tumor, heart, lung, liver, spleen, stomach, kidney, and intestine. Among them: (a) GT-NIR-1; (b) GT-NIR-2; (c) GT-NIR-3; (d) GT-NIR-4; (e) GT-NIR-5; (f) 100×FA+GT-NIR-4;

[0049] FIG18 shows the in vitro tissue biodistribution of the compound GT-NIR-4 prepared in the Example after injection into an A549 cell xenograft mouse model, wherein the in vitro tissues are, from top to bottom, tumor, heart, lung, liver, spleen, stomach, kidney, and intestine;

[0050] FIG19 is a whole-body fluorescence imaging image and corresponding in vitro tissue biodistribution image of the GT-IR series compounds prepared in Example after injection into a KB cell xenograft mouse model;

[0051] FIG20 shows the effects of the GT-NIR series compounds prepared in Examples on HUVEC cell viability. DETAILED DESCRIPTION

[0052] In order to make the objectives, technical solutions and advantages of the present invention more clear, the invention is clearly and completely described below in conjunction with specific embodiments.

[0053] Example 1 Synthesis of Substrate I-1

[0054] 2,6-Diaminopyrimidin-4(1H)-one (50 g, 0.4 mol) and sodium acetate (33.6 g, 0.4 mol) were added to water (150 ml) and heated to reflux. Chloroacetaldehyde (37.6 g, 0.48 mol) was then added dropwise. The reaction mixture was refluxed for 18 hours, cooled to room temperature, and the precipitate was filtered, washed with water (2 × 50 ml) and acetone (2 × 50 ml), and dried to obtain substrate I-1. ESI-MS (m / z): 151.1 [M+H] + .

[0055] Example 2 Synthesis of substrate I-2

[0056] Substrate I-1 (1.5 g, 0.01 mol) and iodine (5.1 g, 0.02 mol) were dissolved in ethanol / water (2:1, 100 ml) and heated to reflux for 2 hours. The precipitate was cooled and filtered, washed with 1N sodium thiosulfate solution (2 × 30 ml) and water (2 × 50 ml), respectively, and dried to obtain substrate I-2. ESI-MS (m / z): 276.9 [M+H] + .

[0057] Example 3 Synthesis of substrate I-3

[0058] Substrate I-2 (2.7 g, 0.01 mol) and acetylene alcohol (0.43 g, 0.01 mol) were dissolved in anhydrous DMF (30 ml). Palladium chloride (71 mg, 0.40 mmol), triphenylphosphine (131 mg, 0.40 mmol), triethylamine (10.1 g, 0.1 mol), and cuprous iodide (304 mg, 1.60 mmol) were added. The mixture was heated to 100°C and reacted for 12 h. The solvent was evaporated under reduced pressure, and the substrate I-3 was obtained after column chromatography. ESI-MS (m / z): 191.1 [M+H] + .

[0059] Example 4 Synthesis of Substrate I-4

[0060] Substrate I-3 (1.9 g, 0.01 mol) was dissolved in methanol (50 ml), and 5% palladium on carbon (200 mg) was added. Catalytic hydrogenation (50 psi) was carried out at room temperature for 12 h. The mixture was filtered and the solvent was evaporated under reduced pressure to obtain substrate I-4. ESI-MS (m / z): 195.1 [M+H] + .

[0061] Example 5 Synthesis of Substrate I-5

[0062] Substrate I-4 (2 g, 0.01 mol) was dissolved in acetone (20 ml) and a mixture of chromium trioxide (6 g, 0.06 mol) and sulfuric acid (30 ml) / water (90 ml) was added dropwise under ice-cooling. The reaction was continued under ice-cooling for 2 h, then warmed to room temperature and allowed to react overnight. The reaction solution was extracted with ethyl acetate (5 x 30 ml), dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. Substrate I-5 was obtained after column chromatography. ESI-MS (m / z): 207.1 [M+H] + .

[0063] Example 6 Synthesis of Substrate I-6

[0064] Substrate I-5 (1.0 g, 5 mmol) and 1-hydroxybenzotriazole (0.8 g, 6 mmol) were dissolved in anhydrous DMF (20 ml) at room temperature and stirred for 2 h. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.2 g, 6 mmol), glycine methyl ester hydrochloride (1.3 g, 10 mmol), and triethylamine (1.0 g, 10 mmol) were then added. The reaction was continued at room temperature for 5 h. The solvent was evaporated under reduced pressure, and Na2CO3 solution (50 ml) was added to precipitate. The precipitate was filtered and dissolved in 1N NaOH (100 ml). The reaction was continued at room temperature for 1.5 h. Under ice cooling, the pH was adjusted to 2-3 with 1N HCl. The precipitate was filtered, washed with cold water (30 ml), and dried to afford substrate I-6 (860 mg, 65%). 1 H NMR (600MHz, DMSO-d6): 12.24 (br, 1H), 10.81 (s, 1H), 10.18 (s, 1H), 8.10-8.13 (t, J=5.6Hz, 1H) , 6.03 (s, 2H), 6.02 (s, 1H), 3.75-3.77 (d, J=5.6Hz, 2H), δ=3.41 (s, 2H); ESI-MS (m / z): 264.1[MH] - .

[0065] Example 7 Synthesis of substrate I-7

[0066] Substrate I-6 (670 mg, 1.0 eq), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (1.15 g, 1.2 eq), O-tert-butyl-L-tyrosine tert-butyl ester hydrochloride (1.0 g, 1.2 eq), and N,N-diisopropylethylamine (1.3 g, 4 eq) were sequentially dissolved in anhydrous DMF (30 ml) and reacted at room temperature for 4 hours. The solvent was evaporated under reduced pressure, and the product (480 mg, 1.0 eq) was separated by column chromatography and dissolved in anhydrous dichloromethane. Trifluoroacetic acid (3 ml, 50 eq) and triethylsilane (0.8 ml, 5.0 eq) were added sequentially, and the reaction was allowed to proceed at room temperature for 3 hours. The solvent was evaporated under reduced pressure to obtain substrate I-7 (240 mg, 63.1%). 1H NMR (600MHz, DMSO-d6): δ11.19 (s, 1H), 8.13 (d, J=8.0Hz, 1H), 8.06 (t, J=5.6Hz, 2H), 6.99 (d, J=8.3Hz, 2H), 6.64 (d, J=8. 3Hz, 2H), 6.08 (s, 1H), 4.35 (td, J=8.4, 5.4Hz, 1H), 3.70 (ddd, J=52.2, 16.8, 5.7Hz, 2H), 3.44 (s, 2H), 2.96-2.67 (m, 2H); 13 C NMR (151MHz, DMSO-d6) δ174.40, 169.85, 168.24, 159.24, 155.96, 152.88, 151.74, 130.69 , 129.26, 125.14, 115.17, 100.78, 100.29, 55.85, 42.91, 37.24, 35.32; HRMS (APCI): Calcd for C 19 H 20 N6O6[M+H] + :429.1517, found 429.1498.

[0067] Example 8 Synthesis of Substrate II-1

[0068] A similar method to Example 7 was used to obtain substrate II-1 (180 mg, 58.9%). 1 H NMR (600MHz, DMSO-d6) δ10.84 (s, 1H), 8.25 (t, J=5.9Hz, 1H), 7.76 (d, J=6.6Hz, 1H), 6.21 (s, 2H), 6.02 (s, 1 H), 4.07 (q, J=5.4Hz, 1H), 3.73 (dd, J=5.9, 1.9Hz, 2H), 3.45 (s, 2H), 2.83 (ddd, J=48.5, 13.1, 5.0Hz, 2H).; 13 C NMR (151MHz, DMSO-d6) δ172.54, 172.06, 168.87, 168.73, 159.11, 152.72, 151.72, 125.18, 125.10, 100.82 , 100.35, 55.69, 42.97, 40.40, 40.27, 40.13, 39.99, 39.85, 39.71, 39.57, 35.32, 27.10; HRMS (APCI): Calcd for C 13 H 16 N6O5S[M+H] +:369.0976, found 369.0968.

[0069] Example 9 Synthesis of Substrate III-1

[0070] A similar method to Example 7 was used to obtain substrate III-1 (120 mg, 41.5%). 1 H NMR (600MHz, DMSO-d6) δ10.80 (s, 1H), 10.19 (s, 1H), 8.34 (d, J=7.8Hz, 1H), 8.24 (d, J=7.6Hz, 1H), 8.03 (t, J=5.7Hz, 1H), 6.05 (s, 2H), 6.00 (s, 1H), 4.1 3(q, J=7.3Hz, 1H),, 4.05-3.92(m, 1H), 3.84-3.62(m, 2H), 3.41(s, 2H), 2.4 6-2.27 (m, 2H), 2.19 (t, J=8.0Hz, 2H), 1.97-1.71 (m, 2H); HRMS (APCI): Calcd for C 18 H 23 N7O8S[M+H] + :498.1402, found 498.1390.

[0071] Example 10 Synthesis of Substrate IV-1

[0072] To a suspension of 2,6-diaminopyrimidin-4-one (1.26 g, 10 mol) in anhydrous DMF (25 ml) was added 1-bromo-5-hexyn-2-one (0.98 g, 10 mmol). The resulting mixture was stirred at room temperature under N₂ for 3 days. After evaporation of the solvent under reduced pressure, column chromatography afforded substrate IV-1 (1.4 g, 74.0%). 1 H NMR (600MHz, DMSO-d6)10.81(s, 1H), 10.13(s, 1H), 5.98(s, 2H), 5.93(s, 1H), 2.77 (t, J=2Hz, 1H), 2.64-2.67 (m, 2H), δ2.41-2.45 (m, 2H); HRMS (APCI): Calcd for C 10 H 10 N4O[M+H] + :203.0927, found, 203.0935.

[0073] Example 11 Synthesis of Substrate IV-2

[0074] In a 250 ml round-bottom flask equipped with a magnetic stirrer and a gas inlet, tetrakis(triphenylphosphine)palladium (185 mg, 0.16 mmol), triethylamine (1.01 g, 10 mmol), methyl p-iodobenzoate (393 mg, 1.5 mmol), and anhydrous DMF (20 ml) were added. To the stirred mixture, cuprous iodide (30 mg, 0.16 mmol) and substrate IV-1 (202 mg, 1 mmol) were added under N₂, and the reaction mixture was stirred at room temperature overnight. After evaporation of the solvent under reduced pressure, the precipitate was separated by column chromatography and filtered, dissolved in 1N NaOH (100 ml), and allowed to react at room temperature for 1.5 h. Under ice cooling, the pH was adjusted to 2-3 with 1N HCl. The precipitate was filtered, washed with cold water (30 ml), and dried to yield substrate IV-2 (238 mg, 74.0%). 1 H NMR (600MHz, DMSO-d6) δ10.79 (s, 1H), 10.21 (s, 1H), 7.86 (d, J = 8.4Hz, 2H), 7.32 (d, J = 8.4Hz, 2 H), 6.02 (s, 2H), 5.85 (s, 1H), 2.85 (t, J = 7.0Hz, 2H), 1.62 (t, J = 6.4Hz, 2H); HRMS (APCI): Calcd for C 17 H 14 N4O3[M+H] + :323.1139, found, 323.1152.

[0075] Example 12 Synthesis of Substrate IV-3

[0076] Substrate IV-2 (322 mg, 1 mmol) was dissolved in methanol (30 ml), and 5% palladium on carbon (100 mg) was added. Catalytic hydrogenation (50 psi) was carried out at room temperature for 12 h. Filtering and evaporation of the solvent under reduced pressure gave a white solid. Substrate IV-3 (190 mg, 80.6%) was then obtained using a method similar to Example 7. 1H NMR (600MHz, DMSO-d6) δ10.78 (s, 1H), 10.23 (s, 1H), 9.18 (s, 1H), 8.38 (d, J= 8.0Hz, 1H), 7.69 (d, J=8.0Hz, 2H), 7.25 (d, J=8.0Hz, 2H), 7.06 (d, J=8.3Hz, 2H ), 6.67-6.59 (m, 2H), 6.01 (s, 2H), 5.85 (s, 1H), 4.46 (ddd, J=10.0, 7.9, 4.5H z, 1H), 3.08-2.90 (m, 2H), 2.62 (d, J=7.1Hz, 2H), 1.62-1.51 (m, J=3.7Hz, 4H); 13 C NMR (151MHz, DMSO-d6) δ173.98, 166.51, 158.96, 156.18, 152.36, 151.56, 146.25, 132.21, 131.49, 130.48, 1 28.90, 128.62, 127.78, 115.37, 100.12, 98.31, 55.29, 36.16, 35.16, 30.76, 28.81, 27.50; HRMS (APCI): Calcd for C 26 H 27 N5O5[M+H] + :490.2085, found, 490.2067.

[0077] Example 13 Synthesis of Substrate IV-4

[0078] A similar method to Example 12 was used to obtain substrate IV-4 (160 mg, 74.3%). 1 H NMR (600MHz, DMSO-d6) δ10.79 (s, 1H), 7.94 (d, J=7.8Hz, 1H), 7.51 (d, J=3.7Hz, 1H), 6.99 (d, J=8.0Hz, 2H), 6.81 (d, J=3.7Hz, 1H), 6.58 (d, J=8.0H z, 2H), 6.24 (s, 2H), 5.86 (s, 1H), 4.26 (td, J=8.1, 4.5Hz, 1H), 2.96 (ddd, J=113.8, 13.7, 6.4Hz, 2H), 2.77 (d, J=7.0Hz, 2H), 1.61 (p, J=3.7Hz, 4H); 13C NMR (151MHz, DMSO-d6) δ174.59, 160.80, 159.19, 155.96, 152.63, 151.71, 150.06, 137.96, 131.34, 130.54, 129.65, 128.28, 125.71, 115.20, 100.06, 98.33, 56.35, 49.07, 45.72, 36.96, 31.04, 29.72, 28.61, 27.38; HRMS (APCI): Calcd for C 24 H 25 N5O5S[M+H] + :496.1649, found, 496.1633.

[0079] Example 14 Synthesis of Compound GT-NIR-1

[0080] Substrate I-7 (42 mg, 0.1 mmol) was dissolved in 1N NaOH (10 ml) and stirred at room temperature for 30 min. IR-783 (75 mg, 0.1 mmol) was added and reacted at 85°C. The reaction was monitored by HPLC. After completion, the product was purified by preparative liquid chromatography. The target fraction was lyophilized to obtain GT-NIR-1 (34 mg, 28.9%) as a dark green solid. 1 H NMR (600MHz, DMSO-d6) δ10.85 (s, 1H), 8.19 (s, 1H), 7.81 (d, J = 13.9Hz, 2H), 7.43 (d, J = 7.5Hz, 2H), 7.35 (t, J = 8.5Hz, 3H), 7.32 (d, J=7.7Hz, 2H), 7.16 (d, J=8.4Hz, 2H), 7.13 (t, J=7.2Hz, 2H), 6.97 (d, J=8.4Hz, 2H), 6.27 (t, J=19.0Hz, 2 H), 6.19 (d, J=14.0Hz, 2H), 5.97 (s, 1H), 4.11 (t, J=7.4Hz, 4H), 4.05-4.00 (m, 1H), 3.57 (ddd, J=58.4, 17.8, 6.8Hz, 2 H), 3.37-3.27 (m, 2H), 3.03-2.80 (m, 2H), 2.70 (t, J=6.5Hz, 4H), 1.78-1.66 (m, 10H), 1.24 (dd, J=15.6, 4.7Hz, 12H); 13C NMR (151MHz, DMSO-d6) δ174.82, 172.58, 169.79, 163.23, 159.05, 158.53, 152.65, 151.66, 14 2.52, 141.45, 131.50, 130.12, 128.87, 125.14, 122.81, 122.05, 114.43, 111.67, 100.67, 100. 36, 51.19, 48.99, 43.98, 35.58, 35.24, 31.75, 29.55, 29.49, 29.44, 29.30, 29.21, 29.16, 29. 04, 27.74, 27.69, 27.02, 26.47, 25.58, 24.15, 22.93, 22.56, 21.62, 14.42; HRMS (APCI): Calcd for C 57 H 66 N8O 12 S2[MH] - :1117.4169, found, 1117.4174.

[0081] Example 15 Synthesis of Compound GT-NIR-2

[0082] A similar method to Example 14 was used to obtain GT-NIR-2 (22 mg, 34.3%) as a dark green solid. 1 H NMR (600MHz, DMSO-d6) δ10.85 (s, 1H), 8.67 (d, J = 13.9Hz, 2H), 8.11 (d, J = 5.7Hz, 1H), 7.70 (d, J = 6.2Hz, 1H), 7.53 (d, J =7.4Hz, 2H), 7.40 (d, J = 8.0Hz, 2H), 7.37 (t, J = 7.6Hz, 2H), 7.21 (t, J = 7.2Hz, 2H), 6.26 (d, J = 14.2Hz, 2H), 6.23 (s, 2H), 6.03 (s, 1H), 4.16 (t, J=7.7Hz, 4H), 4.05 (q, J=5.6Hz, 1H), 3.68 (qd, J=16.7, 5.6Hz, 2H), 3.44 (d, J=3.0Hz, 2H) , 3.34-3.11 (m, 2H), 2.54 (t, J=7.4Hz, 4H), 1.81 (q, J=7.4Hz, 4H), 1.74 (hept, J=6.5, 6.1Hz, 6H), 1.67 (s, 12H); 13C NMR (151MHz, DMSO-d6) δ172.70, 171.99, 169.83, 168.58, 159.11, 158.69, 152.75 , 151.66, 145.18, 142.76, 141.49, 133.09, 128.90, 125.17, 125.04, 122.83, 111. 58, 101.52, 100.79, 100.38, 55.41, 51.23, 49.21, 43.99, 42.85, 41.28, 35.33, 29 .49, 28.11, 27.97, 27.02, 26.47, 26.13, 22.99, 21.79, 21.00; HRMS (APCI): Calcd for C 51 H 62 N8O 11 S3[MH] - :1057.3627, found, 1057.3590.

[0083] Example 16 Synthesis of Compound GT-NIR-3

[0084] A similar method to Example 14 was used to obtain GT-NIR-3 (22 mg, 31.2%) as a dark green solid. 1 H NMR (600MHz, DMSO-d6) δ11.00 (s, 1H), 8.67 (d, J = 13.7Hz, 2H), 8.36 (t, J = 6.0Hz, 1H), 7.65 (d, J = 6.9Hz, 1H), 7.58 (d, J = 7.3Hz, 2H) , 7.53 (d, J = 6.3Hz, 1H), 7.39 (d, J = 8.1Hz, 2H), 7.37 (t, J = 7.5Hz, 2H), 7.20 (t, J = 7.2Hz, 2H), 6.40 (s, 2H), 6.27 (d, J = 13.9Hz, 2H), 6.02 (s, 1H), 4.17 (d, J = 8.8Hz, 4H), 4.11 (d, J = 6.8Hz, 1H), 3.91 (q, J = 6.4Hz, 1H), 3.71-3.58 (m, 2H), 3.45 (s, 2H), 3.33-3.12 (m, 2 H), 2.56 (t, J=7.4Hz, 5H), 2.16-2.03 (m, 2H), 1.95-1.89 (m, 2H), 1.80 (q, J=7.4Hz, 4H), 1.75 (q, J=7.8, 7.2Hz, 6H), 1.67 (s, 12H); 13C NMR (151MHz, DMSO-d6) δ174.61, 172.86, 172.64, 172.06, 171.95, 170.07, 168.51, 159. 28, 152.91, 151.72, 145.19, 142.73, 141.48, 133.12, 128.89, 125.26, 125.05, 122.92, 111.53, 101.52, 100.86, 100.29, 55.47, 53.96, 51.22, 49.20, 43.96, 43.09, 41.21, 35. 35, 32.86, 28.81, 28.07, 27.99, 26.46, 26.17, 22.96, 22.01, 20.98; HRMS (APCI): Calcd for C 56 H 69 N9O 14 S3[MH] - :1186.4053, found, 1186.4015.

[0085] Example 17 Synthesis of Compound GT-NIR-4

[0086] A similar method to Example 14 was used to obtain GT-NIR-4 (20 mg, 24.3%) as a dark green solid. 1 H NMR (600MHz, DMSO-d6) δ10.82 (s, 1H), 7.91 (s, 1H), 7.76 (d, J = 13.9Hz, 2H), 7.57 (d, J = 7.4Hz, 2H), 7.33 (s, 2H), 7 .28 (d, J=8.0Hz, 4H), 7.20 (t, J=7.3Hz, 2H), 7.12 (t, J=6.8Hz, 2H), 7.08 (d, J=7.8Hz, 2H), 6.98 (d, J=7.8Hz, 2H), 6 .24 (s, 2H), 6.15 (d, J=14.1Hz, 2H), 5.83 (s, 1H), 4.29 (s, 1H), 4.08 (s, 4H), 3.20-2.93 (m, 2H), 2.67 (s, 4H), 2.57 -2.52 (m, 5H), 1.74 (d, J=23.7Hz, 8H), 1.61-1.51 (m, 2H), 1.48 (q, J=7.7Hz, 2H), 1.26-1.20 (m, 2H), 1.12 (s, 12H); 13C NMR (151MHz, DMSO-d6) δ172.09, 171.86, 165.87, 163.30, 158.33, 152.58, 151.70, 145.89, 142.42, 141.44, 1 41.28, 133.58, 132.74, 131.45, 128.88, 128.39, 127.51, 125.05, 122.61, 122.06, 114.21, 111.60, 100.57, 10 0.05, 98.29, 51.18, 48.83, 43.90, 40.39, 40.12, 39.98, 39.84, 39.70, 39.56, 36.61, 35.10, 30.60, 29.49, 28 .78, 27.69, 27.59, 27.44, 26.43, 25.94, 24.10, 22.96, 22.91, 22.87, 22.56, 22.20, 21.19; HRMS (APCI): Calcd for C 64 H 73 N7O 11 S2[MH] - :1178.4737, found, 1178.4705.

[0087] Example 18 Synthesis of Compound GT-NIR-5

[0088] A similar method to Example 14 was used to obtain GT-NIR-5 (22 mg, 23.3%) as a dark green solid. 1 H NMR (600MHz, DMSO-d6) δ10.82 (s, 1H), 8.29-8.25 (m, 1H), 7.76 (d, J = 13.9Hz, 2H), 7.49 (d, J = 3.9Hz, 1H), 7.42- 7.20 (m, 8H), 7.15 (t, J=7.0Hz, 2H), 7.02 (d, J=8.0Hz, 2H), 6.71 (d, J=3.9Hz, 1H), 6.17 (d, J=14.1Hz, 2H), 6.09 (s, 2H), 5.85 (s, 1H), 4.37 (t, J=10.3Hz, 1H), 4.09 (s, 4H), 3.15-2.89 (m, 2H), 2.69 (s, 4H), 2.65-2.57 (m, 2H), 2.48 (s, 4H), 1.71 (d, J = 17.2Hz, 8H), 1.57 (p, J = 7.1Hz, 2H), 1.47 (q, J = 7.7Hz, 2H), 1.23 (s, 4H), 1.14 (s, 12H); 13C NMR (151MHz, DMSO-d6) δ172.58, 171.89, 163.21, 161.37, 159.01, 158.49, 152.43, 151.63, 150.46, 142.43, 141.41 , 141.31, 137.33, 132.93, 131.38, 131.31, 130.12, 128.88, 128.64, 125.44, 125.05, 122.66, 122.03, 114.41, 111.6 2, 100.61, 100.12, 98.35, 55.54, 51.19, 49.07, 48.86, 43.93, 36.18, 35.59, 31.75, 30.73, 29.64, 29.49, 29.29, 29. 16, 28.53, 27.61, 27.53, 27.26, 27.02, 26.46, 24.87, 24.11, 22.92, 22.56, 21.64, 21.20, 14.42; HRMS (APCI): Calcd for C 62 H 71 N7O 11 S3[MH] - :1184.4301, found, 1184.4268.

[0089] Example 19 Synthesis of Substrate V-1

[0090] Substrate I-6 (530 mg, 2 mmol), benzotriazol-1-yl-oxytripyrrolidinophosphine hexafluorophosphate (1.2 g, 2.4 mmol), N-tert-butoxycarbonylethylenediamine (320 mg, 2 mmol), and N,N-diisopropylethylamine (516 mg, 4 mmol) were sequentially dissolved in anhydrous DMF (30 ml) and reacted at room temperature for 4 hours. The solvent was evaporated under reduced pressure, and the product (407 mg, 1 mmol) was separated by column chromatography and dissolved in anhydrous dichloromethane (5 ml). Trifluoroacetic acid (5 ml) was added and the reaction was allowed to proceed at room temperature for 3 hours. The solvent was evaporated under reduced pressure to obtain substrate V-1 (210 mg, 68.4%). 1 H NMR (600MHz, DMSO-d6) δ8.26 (t, J=5.6Hz, 1H), 8.19 (t, J=5.8Hz, 1H), 6.14 (s, 2H), 6.00 ( s, 1H), 3.68 (d, J=5.7Hz, 2H), 3.42 (s, 2H), 3.18 (q, J=6.0Hz, 2H), 2.69 (t, J=6.2Hz, 2H); 13C NMR (151MHz, DMSO-d6) δ174.18, 169.89, 169.56, 129.70, 125.15, 110.55, 102.76, 100.72, 100.32, 42.82, 38.39, 35.46, 23.46, 22.95; HRMS (APCI): Calcd for C 12 H 17 N7O3[M+H] + :308.1466, found308.1478.

[0091] Example 20 Synthesis of Substrate V-2

[0092] A similar method to Example 19 was used to obtain substrate V-2 (250 mg, 57.3%). 1 H NMR (600MHz, DMSO-d6) δ10.90 (s, 1H), 8.31 (t, J=5.9Hz, 1H), 8.02 (t, J=5.8Hz, 1H), 7.60 (d, J=6.8Hz, 1H), 6.20 (s, 2H), 6.03 (s, 1H), 3.86 (q, J=6.0Hz, 1H), 3.69 (d, J=5.1Hz, 2H), 3.45 (s, 2H), 3.33-3.15 (m, 2H), 2.84 (t, J=5.7, 4.6Hz, 2H), 2.11-2.01 (m, 2H), 1.92-1.90 (m, 2H); 13 C NMR (151MHz, DMSO-d6) δ174.54, 173.14, 172.79, 169.99, 168.06, 159.13, 152.72, 151.72, 1 25.14, 100.80, 100.37, 53.70, 43.07, 37.66, 35.37, 31.97, 28.11, 22.04; HRMS (APCI): Calcd for C 17 H 24 N8O6[M+H] + :437.1892, found 437.1875.

[0093] Example 21 Synthesis of Substrate V-3

[0094] Substrate IV-2 (322 mg, 1 mmol) was dissolved in methanol (30 ml), and 5% palladium on carbon (100 mg) was added. Catalytic hydrogenation (50 psi) was carried out at room temperature for 12 h. Filtering and evaporation of the solvent under reduced pressure gave a white solid. Substrate V-3 (300 mg, 81.5%) was then obtained by a similar method as in Example 1. 1 H NMR (600MHz, DMSO-d6) δ10.78 (s, 1H), 8.72 (t, J = 5.5Hz, 1H), 7.78 (d, J = 7.8Hz, 2H), 7.25 (d, J = 7.8Hz, 2H) , 6.11 (s, 2H), 5.82 (s, 1H), 3.40 (q, J=6.0Hz, 2H), 2.84 (t, 2H), 2.64 (t, J=6.4Hz, 2H), 1.66-1.49 (m, 4H); 13 C NMR (151MHz, DMSO-d6) δ173.82, 166.98, 159.05, 152.50, 151.61, 146.15, 132.39, 131.4 1, 128.58, 127.80, 100.10, 98.26, 35.14, 30.76, 28.71, 27.45, 23.06; HRMS (APCI): Calcd for C 19 H 24 N6O2[M+H] + :369.2034, found 369.2039.

[0095] Example 22 Synthesis of Substrate V-4

[0096] A similar method to Example 21 was used to obtain substrate V-4 (260 mg, 69.5%). 1 H NMR (600MHz, DMSO-d6) δ10.79 (s, 1H), 8.83 (t, J = 4.8Hz, 1H), 7.59 (d, J = 3.6Hz, 1H), 6.85 (d, J = 3.6Hz, 1H ), 6.14 (s, 2H), 5.84 (s, 1H), 3.34 (q, J=11.6, 5.8Hz, 2H), 2.80 (t, J=13.1, 6.5Hz, 4H), 1.65-1.55 (m, 4H); 13C NMR (151MHz, DMSO-d6) δ173.82, 161.98, 159.07, 152.55, 151.63, 150.51, 137.62, 131.2 6, 128.73, 125.77, 100.09, 98.32, 31.03, 29.71, 28.47, 27.30, 23.15; HRMS (APCI): Calcd for C 17 H 22 N6O2S[M+H] + :375.1598, found 375.1511.

[0097] Example 23 Synthesis of Substrate V-5

[0098] A similar method to Example 19 was used to obtain substrate V-5 (40 mg, 80%). 1 H NMR (600MHz, DMSO-d6) δ = 10.81 (s, 1H), 8.34 (t, J = 5.8Hz, 1H), 7.91 (d, J = 7.0Hz, 1H ),7.56(d,J=3.7Hz,1H),6.82(d,J=3.6Hz,1H),6.16(s,2H),5.86(s,1H),4.07(q, J=6.1Hz,2H),3.37-3.14(m,2H),2.84(q,J=6.2Hz,2H),2.78(d,J=7.0Hz,2H),2.1 5(dt,J=13.7,7.1Hz,2H),2.00(p,J=7.4,7.0Hz,2H),1.61(dd,J=7.8,3.9Hz,4H); 13 C NMR (151MHz, DMSO-d6)δ=172.96,171.19,170.96,158.50,157.01,150.42,149.55,148.07,135.70,129.23,126.29 ,123.65,116.43,97.96,96.23,52.09,35.34,30.15,28.90,27.59,26.40,26.08,25.21,20.10; HRMS(APCI):Calcd for C 22 H 29 N7O5S[M+H] + :504.2018, found 504.2021.

[0099] Example 24 Synthesis of Substrate V-6

[0100] A similar method to Example 19 was used to obtain substrate V-6 (32 mg, 75%) with tert-butyl N-(6-aminohexyl)carbamate. 1 H NMR (600MHz, DMSO-d6) δ = 10.80 (s, 1H), 8.37 (t, J = 5.7Hz, 1H), 7.55 (d, J = 3.7Hz, 1H), 6.84 (d, J = 3.7Hz, 1H), 6.22 (s, 2H), 5.85 (s, 1H), 3.19 (q, J=6.6Hz,2H),2.79(d,J=6.9Hz,2H),2.65(t,J=7.4Hz,2H),1.62(p,J=3.7Hz,4H),1.47(dt,J=14.2,7.1Hz,4H),1.29(dq,J=11.3,5.7Hz,4H); 13 C NMR (151MHz, DMSO-d6)δ=174.49,161.51,159.14,152.64,151.67,150.24,137.93,131.23,128.25,125.69 ,100.08,98.32,31.01,29.84,29.70,29.56,29.49,28.50,27.31,26.58,26.26,24.19; HRMS(APCI):Calcd for C 21 H 30 N6O2S[M+H] + :431.2224, found 431.2231.

[0101] Example 25 Synthesis of Substrate V-7

[0102] A similar method to Example 19 was used to obtain substrate V-7 (34 mg, 75%) with tert-butyl N-[4-(aminomethyl)benzyl]carbamate. 1 H NMR (600MHz, DMSO-d6) δ = 10.78 (s, 1H), 8.89 (t, J = 6.1Hz, 1H), 7.60 (d, J = 3.7Hz, 1H), 7.29 (d, J = 8.1Hz, 2H), 7.23 (d, J = 8.1Hz, 2H), 6. 86(d,J=3.7Hz,1H),6.01(s,2H),5.85(d,J=2.1Hz,1H),4.39(d,J=6.0Hz,2H),3.73(s,2H),2.80(t,J=6.7Hz,2H),1.67-1.55(m,4H); 13C NMR (151MHz, DMSO-d6) δ=173.67,161.62,159.07,152.52,151.63,150.68,139.34,138.87,137.54,131.29,128. 64,128.18,127.66,125.84,100.11,98.36,44.42,42.66,31.02,29.72,28.52,27.32,23.01; HRMS(APCI):Calcd for C 23 H 26 N6O2S[M+H] + :451.1911, found 451.1906.

[0103] Example 26 Synthesis of Substrate V-8

[0104] A similar method to Example 19 was used to obtain substrate V-8 (41 mg, 77%). 1 H NMR (600MHz, DMSO-d6) δ = 10.77 (s, 1H), 7.89 -7.72(m,1H),7.61(d,J=6.8Hz,2H),7.23(d,J=7.9Hz,2H),7.09(d,J=8.2Hz,2H) ,6.76(d,J=7.2Hz,2H),6.08(s,2H),5.84(d,J=2.1Hz,1H),4.26(q,J=5.9Hz,1H), 4.00(dtd,J=15.4,10.3,5.3Hz,2H),3.14(dd,J=13.5,5.0Hz,1H),3.07(t,J=5.6H z,2H),3.02(dd,J=13.4,6.6Hz,1H),2.62(p,J=4.0Hz,2H),1.57(q,J=3.7Hz,4H); 13 C NMR (151MHz, DMSO-d6)δ=174.24,172.71,165.62,159.12,156.78,152.57,151.65,145.97,132.83,131.83,131.40,130 .85,128.72,127.35,114.36,100.08,98.30,65.74,56.00,36.49,35.13,30.73,28.76,27.49,21.83; HRMS(APCI):Calcd for C 28 H 32 N6O5[M+H] +:533.2507, found 533.2518.

[0105] Example 27 Synthesis of Substrate V-9

[0106] A similar method to Example 19 was used to obtain substrate V-9 (40 mg, 74%). 1 H NMR (600MHz, DMSO-d6) δ = 10.80 (s, 1H), 7.83 (d, J = 7.2Hz, 1H), 7.45 (dd, J = 3.7, 1.9Hz, 1H),7.10(d,J=8.3Hz,2H),6.79(d,J=3.7Hz,1H),6.76(d,J=8.5Hz,2H),6.20(s,2H), 5.85(s,1H),4.25(td,J=7.1,4.8Hz,1H),4.01(hept,J=5.2Hz,2H),3.10(dt,J=14.0, 5.3Hz, 3H), 2.96 (dd, J=13.5, 7.3Hz, 1H), 2.77 (d, J=6.8Hz, 2H), 1.60 (p, J=3.5Hz, 4H); 13 C NMR (151MHz, DMSO-d6)δ=174.17,172.73,160.66,159.13,156.81,152.60,151.67,150.08,137.82,131.90,131.28,130 .78,128.29,125.77,114.40,100.08,98.34,66.03,56.14,36.77,31.01,29.69,28.53,27.33,21.87; HRMS(APCI):Calcd for C 26 H 30 N6O5S[M+H] + :539.2071, found 539.2075.

[0107] Example 28 Synthesis of Compound GT-FITC-1

[0108] Substrate V-1 (31 mg, 0.1 mmol) was dissolved in DMSO (1 ml), and fluorescein 5-isothiocyanate (31 mg, 0.1 mmol) was added, followed by N,N-diisopropylethylamine (26 mg, 0.2 mmol). The reaction was monitored by HPLC. After completion, the product was purified by preparative liquid chromatography, and the target fraction was lyophilized to obtain GT-FITC-1 (21 mg, 30.1%) as an orange-yellow solid. 1H NMR (600MHz, DMSO-d6) δ10.88 (s, 2H), 9.01 (s, 1H), 8.30 (s, 1H), 8.12 (t, J=15.3Hz, 2H ), 7.80 (dd, 1H), 7.15 (dd, J = 8.3Hz, 1H), 6.64 (d, J = 2.2Hz, 2H), 6.63 (d, J = 8.7Hz, 2H), 6.55 (dd, J=8.8, 2.3Hz, 2H), 6.07 (s, 2H), 6.00 (s, 1H), 3.70 (d, J=5.5Hz, 2H), 3.58 (q, 2H), 3.43 (s, 2H), 3.30 (q, J=6.1Hz, 2H); 13 C NMR (151MHz, DMSO-d6) δ174.18, 173.55, 169.89, 169.56, 153.26, 151.81, 142.70, 129.69, 125.15, 110.5 4, 102.75, 100.71, 100.34, 100.29, 42.81, 40.52, 40.40, 38.39, 35.45, 23.43, 22.93; HRMS (APCI): Calcd for C 33 H 28 N8O8S[M+H] + :697.1824, found 697.1811.

[0109] Example 29 Synthesis of Compound GT-FITC-2

[0110] A similar method to Example 28 was used to obtain GT-FITC-2 (20 mg, 24.2%) as an orange-yellow solid. 1H NMR (600MHz, DMSO-d6) δ12.04 (s, 1H), 10.82 (s, 1H), 9.96 (s, 1H), 8.50 (t, J=14.9Hz, 1H), 8.15 (dt, J=30.1, 5.9Hz , 1H), 7.95 (dd, J=13.6, 7.9Hz, 1H), 7.77-7.65 (m, 1H), 7.62 (d, J=6.9Hz, 1H), 7.12 (d, J=2.5Hz, 1H), 6.66 (d, 2H), 6 .61 (d, 2H), 6.56 (dd, J=8.6, 1.9Hz, 2H), 6.14 (s, 1H), 6.07 (s, 1H), 6.01 (s, 1H), 3.96 (q, J=6.0Hz, 1H), 3.69 (d, J= 5.1Hz, 2H),, 3.59 (m, 2H), 3.45 (s, 2H), 3.23 (m, 2H), 2.14 (q, J=7.7, 7.2Hz, 2H), 1.96 (dq, J=18.9, 9.8, 8.2Hz, 2H); 13 C NMR (151MHz, DMSO-d6) δ181.05, 173.64, 172.69, 169.95, 169.38, 168.21, 159.17, 152.74, 151.70, 129.70, 1 10.72, 102.74, 100.74, 100.34, 54.22, 43.80, 42.92, 38.28, 35.38, 32.22, 22.94, 22.84; HRMS (APCI): Calcd for C 38 H 35 N9O 11 S[M+H] + :826.2250, found 826.2251.

[0111] Example 30 Synthesis of Compound GT-FITC-3

[0112] A similar method to Example 28 was used to obtain GT-FITC-3 (25 mg, 33.1%) as an orange-yellow solid. 1H NMR (600MHz, DMSO-d6) δ10.79 (d, J=2.3Hz, 1H), 8.55 (t, J=6.5Hz, 1H), 8.33 (t, 1H), 8. 23 (s, 1H), 7.77 (d, J = 7.9Hz, 2H), 7.72 (s, 1H), 7.24 (d, J = 7.9Hz, 2H), 7.13 (d, J = 8.3Hz, 1H), 6.65 (d, 2H), 6.62 (d, J=8.7Hz, 2H), 6.54 (dd, J=8.7, 2.4Hz, 2H), 5.96 (s, 2H), 5.8 4(s, 1H), 3.71(q, 2H), 3.49(q, J=6.0Hz, 2H), 2.62(t, J=6.7Hz, 2H), 1.67-1.45(m, 4H); 13 C NMR (151MHz, DMSO-d6) δ158.93, 152.31, 151.54, 146.21, 132.33, 131.51, 129.63, 128.6 2, 127.78, 102.74, 100.12, 98.30, 39.00, 35.15, 30.74, 28.79, 27.46; HRMS (APCI): Calcd for C 40 H 35 N7O7S[M+H] + :758.2391, found 758.2381.

[0113] Example 31 Synthesis of Compound GT-FITC-4

[0114] A similar method to Example 28 was used to obtain GT-FITC-4 (23 mg, 30.2%) as an orange-yellow solid. 1 H NMR (600MHz, DMSO-d6) δ10.80 (s, 1H), 10.15 (s, 2H), 8.55 (t, J=5.6Hz, 1H), 8.23 ​​(s, 2H) ), 7.74 (d, J = 8.2Hz, 1H), 7.59 (d, J = 3.7Hz, 1H), 7.15 (d, J = 8.3Hz, 1H), 6.84 (d, J = 3.7Hz , 1H), 6.68 (d, 2H), 6.61 (d, J=8.6Hz, 2H), 6.56 (dd, J=8.7, 2.3Hz, 2H), 5.98 (s, 2H), 5.8 6 (s, 1H), 3.69 (q, 2H), 3.46 (q, J=6.1Hz, 2H), 2.80 (t, J=6.5Hz, 2H), 1.65-1.49 (m, 4H); 13C NMR (151MHz, DMSO-d6) δ168.98, 162.11, 160.00, 158.94, 152.37, 152.32, 151.57, 150.67, 147.73, 141 .64, 137.47, 131.39, 129.52, 128.77, 125.80, 124.58, 113.08, 110.19, 102.72, 100.12, 98.36, 44.01, 38.79, 31.00, 29.71, 28.51, 27.29; HRMS (APCI): Calcd for C 38 H 33 N7O7S2[M+H] + :764.1956, found 764.1932.

[0115] Example 32 Synthesis of Compound GT-FITC-5

[0116] A similar method to Example 28 was used to obtain GT-FITC-5 (16 mg, 36%) as an orange-yellow solid. 1 H NMR (600MHz, DMSO-d6) δ = 11.60 (s, 1H), 10.80 (s, 1H), 9.59 (s, 2H), 8.47 (s, 1H), 8.03-7.93 (m, 1H) ,7.93-7.83(m,2H),7.58(s,1H),7.10(d,J=8.2Hz,1H),6.82(s,1H),6.67(s,2H),6.60(d,J=8.7Hz ,2H),6.56(d,J=9.1Hz,2H),6.01(s,2H),5.86(s,1H),4.14(d,J=7.5Hz,1H),3.63(d,J=11.4Hz,2 H),3.23(s,2H),3.17(s,1H),2.78(s,2H),2.28-2.12(m,2H),2.08(d,J=13.0Hz,2H),1.60(s,4H); 13C NMR (151MHz, DMSO-d6)δ=181.05,175.50,172.74,169.24,160.74,160.11,159.00,152 .45,152.35,151.61,150.31,142.91,137.74,131.43,129.56,128.50,126.88,125.75, 124.14,116.35,113.15,110.42,102.68,100.12,98.36,54.25,49.07,43.68,38.23,3 2.29,31.02,29.71,29.49,29.05,28.58,27.80,27.33,21.75,1.63; HRMS(APCI):Calcd for C 43 H 39 N8O 10 S2[MH] - :891.2236, found 891.2246.

[0117] Example 33 Synthesis of Compound GT-FITC-6

[0118] A similar method to Example 28 was used to obtain GT-FITC-6 (14 mg, 34%) as an orange-yellow solid. 1 H NMR (600MHz, DMSO-d6) δ = 10.81 (s, 1H), 10.12 (s, 2H), 8.34 (t, J = 5.8Hz, 1H), 8.31 (s, 1H), 8.26 (s, 1H), 7.74(d,J=8.3Hz,1H),7.55(d,J=3.7Hz,1H),7.15(d,J=8.2Hz,1H),6.83(d,J=3.7Hz,1H),6.65(s,2H) ,6.63(s,1H),6.56(d,J=8.8Hz,2H),5.98(s,2H),5.87(s,1H),3.51-3.47(m,2H),3.21(q,J=6.8Hz,2H ),3.18(s,1H),2.79(d,J=6.9Hz,2H),1.62(d,J=4.4Hz,4H),1.54(dt,J=28.2,6.9Hz,4H),1.34(s,4H); 13C NMR (151MHz, DMSO-d6)δ=180.82,169.18,161.52,158.96,152.82,152.32,151.57,150.26,141.89,137.93,131.40,129.67,128.24 ,125.68,110.62,102.76,100.13,98.36,49.07,44.26,31.01,29.71,29.68,28.77,28.53,27.31,26.70,26.68; HRMS(APCI):Calcd for C 42 H 40 N7O7S2[MH] - :818.2436, found 818.2443.

[0119] Example 34 Synthesis of Compound GT-FITC-7

[0120] A similar method to Example 28 was used to obtain GT-FITC-7 (12 mg, 30%) as an orange-yellow solid. 1 H NMR (600MHz, DMSO-d6) δ = 10.79 (s, 1H), 10.14 (s, 2H), 8.90 (t, J = 6.0Hz, 1H), 8.58 (s, 1H), 8.26 (s, 1H), 7.75(d,J=8.3Hz,1H),7.60(d,J=3.7Hz,1H),7.32(d,J=8.0Hz,2H),7.28(d,J=7.9Hz,2H),7.17(d,J=8. 2Hz,1H),6.86(d,J=3.7Hz,1H),6.64(s,1H),6.62(d,J=8.8Hz,2H),6.57-6.52(m,2H),5.96(s,2H),5. 85(d,J=2.2Hz,1H),4.80-4.72(m,2H),4.41(d,J=6.0Hz,2H),2.80(d,J=7.1Hz,2H),1.65-1.58(m,4H); 13C NMR (151MHz, DMSO-d6) δ=161.60,158.92,152.32,151.56,150.68,138.83,137.53,131.35,129.62,128.62,127.93, 127.76,125.83,102.74,100.13,98.36,42.68,31.01,29.72,28.52,27.30,22.98,22.56,14.43; HRMS(APCI):Calcd for C 44 H 36 N7O7S2[MH] - :838.2123, found 838.2122.

[0121] Example 35 Synthesis of Compound GT-FITC-8

[0122] A similar method to Example 28 was used to obtain GT-FITC-8 (15 mg, 32%) as an orange-yellow solid. 1 H NMR (600MHz, DMSO-d6) δ = 10.78 (s, 1H), 8.29 (s, 1H), 7.94 (s, 1H), 7.79 (d, J = 8.3Hz, 1H), 7.63 (d, J = 7.8Hz, 2H), 7. 23(d,J=7.9Hz,2H),7.18(d,J=8.3Hz,1H),7.10(d,J=8.1Hz,2H),6.78(d,J=8.0Hz,2H),6.66(d,J=2.1Hz,2H),6. 60(s,2H),6.58(s,1H),6.57(dd,J=8.6,2.2Hz,1H),5.98(s,2H),5.85(d,J=2.2Hz,1H),4.36(d,J=6.7Hz,1H),4. 10-3.97(m,2H),3.83(s,2H),3.08(dt,J=59.2,7.6Hz,2H),2.61(t,J=6.3Hz,2H),2.01(m,1H),1.69-1.50(m,4H); 13C NMR(151MHz, DMSO-d6)δ=181.25,174.49,172.64,169.05,166.06,160.16,158.97,157.16,152.43,152.34, 151.58,146.12,142.05,132.55,131.52,131.33,130.76,130.12,129.50,128.67,127.54,124.49,114.46, 113.16,110.22,110.19,102.71,100.12,98.31,65.91,55.67,43.77,36.39,35.59,35.16,31.74,30.76,29 .50,29.29,29.19,29.16,29.05,29.03,28.85,27.49,27.03,22.98,22.56,21.67,14.43; HRMS(APCI):Calcd for C 49 H 42 N7O 10 S[MH] - :920.2719, found 920.2716.

[0123] Example 36 Synthesis of Compound GT-FITC-9

[0124] A similar method to Example 28 was used to obtain GT-FITC-9 (16 mg, 35%) as an orange-yellow solid. 1 H NMR (600MHz, DMSO-d6) δ=10.78(s,1H),8.69(s,1H),8.29(s,1H),8.16(s,1H),7.77(d,J=8.2Hz,1H),7.52 (d,J=3.6Hz,1H),7.17(d,J=8.3Hz,1H),7.14(d,J=8.1Hz,3H),6.83(s,2H),6.82(d,J=4.6Hz,2H),6.68-6. 65(m,3H),5.97(s,2H),5.85(s,1H),4.37(p,J=10.6,7.1Hz,1H),4.08(tt,J=10.2,5.0Hz,2H),3.85(s,2H ),3.09(dd,J=13.7,4.6Hz,1H),2.96(dd,J=13.8,8.5Hz,1H),2.78(d,J=6.3Hz,2H),1.61(d,J=6.0Hz,4H); 13C NMR (151MHz, DMSO-d6)δ=181.26,174.78,174.46,169.04,161.16,160.09,158.97,157.20,152.40,152.35,151.60 ,150.51,142.00,137.48,131.38,131.28,130.69,130.13,129.50,128.66,127.05,125.76,124.48,117.02,114.5 4,113.13,110.21,102.71,100.12,98.36,65.94,55.58,43.77,36.49,35.60,31.76,31.02,29.71,29.56,29.50,2 9.45,29.34,29.30,29.21,29.17,29.05,28.58,27.33,27.03,25.59,22.57,21.65,14.42,1.63; HRMS(APCI):Calcd for C 47 H 42 N7O 10 S2[M+H] + :928.2429, found 928.2420.

[0125] Example 37 Synthesis of Compound GT-IR-1

[0126] A similar method to Example 14 was used to obtain GT-IR-1 (33 mg, 29%) as a dark green solid. 1 H NMR (600MHz, DMSO-d6) δ10.83 (s, 1H), 8.17 (s, 1H), 7.81 (d, J=14.0Hz, 2H), 7.53 (d, J =7.6Hz, 2H), 7.38 (t, J = 8.5Hz, 3H), 7.32 (d, J = 7.7Hz, 2H), 7.16 (d, J = 8.4Hz, 2H), 6.96 (d, J=8.4Hz, 2H), 6.26 (t, J=19.0Hz, 2H), 6.18 (d, J=14.0Hz, 2H), 5.95 (s, 1H), 4.14 (t , J=7.4Hz, 4H), 4.05-4.00 (m, 1H), 3.59 (ddd, J=58.4, 17.8, 6.8Hz, 2H), 3.37-3.27 (m, 2H), 3.03-2.80 (m, 2H), 2.72 (t, J=6.5Hz, 4H), 1.78-1.66 (m, 10H), 1.34 (dd, J=15.6, 4.7Hz, 12H);13 C NMR (151MHz, DMSO-d6) δ174.92, 172.68, 169.71, 162.23, 159.15, 157.53, 152.65, 150.66, 1 42.52, 140.45, 132.81, 132.05, 131.52, 130.12, 128.87, 125.14, 114.43, 111.67, 100.67, 1 00.36, 51.19, 48.99, 43.98, 35.58, 35.24, 32.75, 29.55, 29.49, 29.46, 29.30, 29.21, 29.16 , 29.04, 27.74, 27.69, 27.22, 26.47, 25.48, 24.15, 22.93, 22.56, 21.52; HRMS (APCI): Calcd for C 57 H 66 N8O 18 S4[MH] - :1277.3305, found, 1277.3314.

[0127] Example 38 Synthesis of Compound GT-IR-2

[0128] A similar method to Example 14 was used to obtain GT-IR-2 (23 mg, 37.3%) as a dark green solid. 1 H NMR (600MHz, DMSO-d6) δ10.88 (s, 1H), 8.47 (d, J = 13.9Hz, 2H), 8.21 (d, J = 5.7Hz, 1H), 7.73 (d, J = 6.2Hz, 1H), 7.51 (d, J=7.4Hz, 2H), 7.44 (d, J=8.0Hz, 2H), 7.34 (t, J=7.6Hz, 2H), 6.36 (d, J=14.2Hz, 2H), 6.33 (s, 2H), 6.0 3 (s, 1H), 4.26 (t, J = 7.7Hz, 4H), 4.02 (q, J = 5.6Hz, 1H), 3.69 (qd, J = 16.7, 5.6Hz, 2H), 3.47 (d, J = 3.0Hz, 2H), 3 .34-3.11 (m, 2H), 2.51 (t, J=7.4Hz, 4H), 1.71 (q, J=7.4Hz, 4H), 1.64 (hept, J=6.5, 6.1Hz, 6H), 1.57 (s, 12H); 13C NMR (151MHz, DMSO-d6) δ174.70, 172.99, 167.83, 168.51, 159.41, 158.59, 152.72, 151.56, 145.78, 142.71, 141.49, 135.34, 133.09, 132.83, 128.93 ,125.19,111.68,101.58,100.74,100.33,55.48,51.28,49.28,43.94,42 .85, 35.36, 28.11, 27.92, 26.45, 26.18, 22.99, 21.59; HRMS (APCI): Calcd for C 51 H 62 N8O 17 S5[MH] - :1217.2763, found, 1217.2760.

[0129] Example 39 Synthesis of Compound GT-IR-3

[0130] A similar method to Example 14 was used to obtain GT-IR-3 (22 mg, 34%) as a dark green solid. 1 H NMR (600MHz, DMSO-d6) δ11.02 (s, 1H), 8.57 (d, J = 13.7Hz, 2H), 8.26 (t, J = 6.0Hz, 1H), 7.66 (d, J = 6.9Hz, 1H), 7.56 (d, J = 7.3H z, 2H), 7.54 (d, J=6.3Hz, 1H), 7.37 (d, J=8.1Hz, 2H), 7.31 (t, J=7.5Hz, 2H), 6.44 (s, 2H), 6.26 (d, J=13.9Hz, 2H), 6.12 (s, 1H ), 4.16 (d, J = 8.8Hz, 4H), 4.01 (d, J = 6.8Hz, 1H), 3.93 (q, J = 6.4Hz, 1H), 3.71-3.58 (m, 2H), 3.48 (s, 2H), 3.33-3.12 (m, 2H), 2 .56 (t, J=7.4Hz, 5H), 2.16-2.03 (m, 2H), 1.95-1.89 (m, 2H), 1.85 (q, J=7.4Hz, 4H), 1.72 (q, J=7.8, 7.2Hz, 6H), 1.67 (s, 12H); 13C NMR (151MHz, DMSO-d6) δ174.81, 172.88, 172.74, 172.26, 171.95, 170.17, 167.51, 159. 28, 152.71, 151.76, 144.19, 143.73, 141.58, 135.26, 135.05, 133.22, 128.79, 122.42, 112.58, 101.57, 100.86, 100.29, 55.47, 53.96, 51.22, 49.28, 43.96, 43.04, 41.21, 35. 35, 32.81, 28.88, 28.07, 27.92, 26.44, 25.17, 22.86, 22.21, 20.78; HRMS (APCI): Calcd for C 56 H 69 N9O 20 S5[MH] - :1346.3190, found, 1346.3199.

[0131] Example 40 Synthesis of Compound GT-IR-4

[0132] A similar method to Example 14 was used to obtain GT-IR-4 (26 mg, 28.3%) as a dark green solid. 1 H NMR (600MHz, DMSO-d6) δ10.88 (s, 1H), 7.98 (s, 1H), 7.71 (d, J = 13.9Hz, 2H), 7.59 (d, J = 7.4Hz, 2H), 7.39 (s, 2H), 7.28 (d, J=8.0Hz, 4H), 7.10 (t, J=7.3Hz, 2H), 7.04 (d, J=7.8Hz, 2H), 6.98 (d, J=7.8Hz, 2H), 6.24 (s, 2H) , 6.35 (d, J=14.1Hz, 2H), 5.73 (s, 1H), 4.29 (s, 1H), 4.08 (s, 4H), 3.24-2.98 (m, 2H), 2.67 (s, 4H), 2.57-2.52 (m, 5H), 1.77 (d, J=23.7Hz, 8H), 1.61-1.51 (m, 2H), 1.46 (q, J=7.7Hz, 2H), 1.26-1.20 (m, 2H), 1.14 (s, 12H); 13C NMR (151MHz, DMSO-d6) δ174.09, 173.86, 166.87, 163.30, 158.33, 153.58, 151.70, 145.89, 143.42, 141.4 6, 141.28, 134.69, 134.06, 133.52, 132.74, 131.45, 128.82, 128.44, 127.51, 125.75, 114.21, 112.67, 100 .57, 100.25, 98.22, 50.18, 48.83, 43.98, 41.39, 40.18, 39.91, 39.84, 39.53, 36.68, 35.10, 30.68, 29.41 , 28.72, 27.69, 27.49, 26.43, 25.93, 24.16, 22.99, 22.98, 22.83, 22.51, 22.26, 21.49; HRMS (APCI): Calcd for C 64 H 73 N7O 17 S4[MH] - :1338.3873, found, 1338.3889.

[0133] Example 41 Synthesis of Compound GT-IR-5

[0134] A similar method to Example 14 was used to obtain GT-IR-5 (24 mg, 25%) as a dark green solid. 1 H NMR (600MHz, DMSO-d6) δ10.89 (s, 1H), 8.39-8.29 (m, 1H), 7.73 (d, J = 13.9Hz, 2H), 7.59 (d, J = 3.9Hz, 1H), 7.42-7.20 (m, 8H), 7.08 (d, J=8.0Hz, 2H), 6.75 (d, J=3.9Hz, 1H), 6.12 (d, J=14.1Hz, 2H), 6.03 (s, 2H), 5. 82 (s, 1H), 4.33 (t, J=10.3Hz, 1H), 4.24 (s, 4H), 3.15-2.89 (m, 2H), 2.69 (s, 4H), 2.65-2.57 (m, 2H), 2.44 (s, 4H), 1.71 (d, J=17.2Hz, 8H), 1.57 (p, J=7.1Hz, 2H), 1.47 (q, J=7.7Hz, 2H), 1.26 (s, 4H), 1.12 (s, 12H); 13C NMR (151MHz, DMSO-d6) δ173.58, 171.89, 164.21, 161.37, 159.01, 157.49, 152.43, 150.46, 1 42.77, 141.51, 137.33, 132.92, 131.34, 130.12, 128.84, 128.68, 125.34, 125.05, 114.41, 1 11.62, 100.68, 98.35, 55.58, 51.12, 49.37, 48.85, 36.14, 35.51, 31.73, 30.77, 29.42, 29.1 6, 28.58, 27.69, 27.02, 26.41, 24.82, 24.41, 22.99, 22.54, 21.68, 21.24; HRMS (APCI): Calcd for C 62 H 71 N7O 17 S5[MH] - :1344.3437, found, 1344.3451.

[0135] Example 42 Optical Property Measurement

[0136] Absorption spectra of all compounds (10 μM) were measured in PBS using a T9S UV-Vis spectrophotometer within the wavelength range of 200-900 nm. Fluorescence spectra of the GT-NIR series compounds (1 μM) and the GT-FITC series compounds (100 nM) were measured in PBS using an RF-6000 fluorescence spectrophotometer. The results are shown in Figures 1-3 and 10-12. The results show that both the GT-NIR series compounds and the GT-FITC series probe compounds exhibit similar optical properties to their associated fluorophores, with minimal variation.

[0137] Example 43 In vitro fluorescence microscopy test

[0138] In order to observe whether the compound GT-NIR-1 can target and enter cells expressing folate receptors and the location of its entry into cells, FR + KB and FR - A549 cells, FR + M2 and FR - M1 macrophages were seeded in confocal microplates at a density of 3 × 10 5cells, grown at 37°C and 5% CO2 for 24 hours. The cells were then incubated with GT-NIR-1 (1 μM) for 30 minutes. In order to conduct a competitive binding experiment, 100-fold folic acid (100 μM) and GT-NIR-1 (1 μM) were co-administered to KB cells and M2 macrophages and incubated for 30 minutes. After incubation, the cells were washed three times with PBS (pH = 7.4), fluorescence was excited at 775 nm, and fluorescence imaging of the cells was observed by confocal fluorescence microscopy (Figures 4 and 5). The experimental results showed that the probe GT-NIR-1 was able to enter the cell interior by targeting folate receptors including FRα (KB cells) and FR-β (M2 macrophages), while no accumulation occurred in cells that were negative for folate receptors (A549 cells and M1 macrophages). This result indicates that the probe GT-NIR-1 has the ability to target tumor cells with high FR expression and achieve fluorescence imaging.

[0139] Example 44 In vitro affinity test of GT-NIR series probes

[0140] In order to compare the targeting affinity of different compounds in the GT-NIR series to FR, FR + KB cells and M2 macrophages were cultured at 1×10 cells per well. 6 The cells were seeded in six-well plates at a density of 100 cells / mL. After culturing for 24 hours, the culture medium was replaced with fresh culture medium without folic acid as a blank control; IR783 (500 nM) was added to eliminate the influence of false positives; culture medium containing GT-NIR series compounds (500 nM) was added to different well plates and incubated for 1 hour. The cells were trypsinized and washed three times with PBS (pH = 7.4), followed by centrifugation (2000 rpm, 5 minutes) to remove unbound probes. The cells were then resuspended in 500 mL PBS and stored in a BD Falcon tube in the dark and analyzed by flow cytometry. FlowJo software was used to analyze and present statistical data, see Figures 6 and 7. The experimental results showed that GT-NIR-5 had the highest binding affinity, whether it was FRα or FR-β; followed by GT-NIR-4, and GT-NIR-1.

[0141] Example 45 In vitro affinity test of GT-FITC series probes

[0142] In order to compare the targeting affinity of different compounds in the GT-FITC series to FR, KB cells were plated at 1×10 6The cells were seeded in six-well plates at a density of 100 cells / mL. After culturing for 24 hours, the culture medium was replaced with fresh culture medium without folic acid as a blank control; FITC (500 nM) was added to eliminate the influence of false positives; culture medium containing GT-FITC series compounds (500 nM) was added to different well plates and incubated for 1 hour. The cells were trypsinized and washed three times with PBS (pH = 7.4), followed by centrifugation (2000 rpm, 5 minutes) to remove unbound probes. The cells were then resuspended in 500 mL PBS and stored in a BD Falcon tube in the dark and analyzed by flow cytometry. FlowJo software was used to analyze and present statistical data, see Figure 13. The experimental results in Figure 13 (a) show that among the compounds with different targeting ligands, GT-FITC-4 has the highest binding affinity for KB cells, followed by GT-FITC-3 and GT-FITC-1. The results in Figure 13(b) show that, given the same targeting ligand, tyrosine is the best performing linker, although other linkers do not weaken the affinity of the targeting ligand for the folate receptor and even slightly enhance it. These results demonstrate that the GT-FITC series of probes are capable of targeting tumor cells that overexpress FR.

[0143] Example 46 Equilibrium dissociation constant K d Determination of

[0144] The GT-NIR series compounds were serially diluted in fresh culture medium at concentrations ranging from 1 nM to 400 nM and mixed with 10 6 KB cells and M2 macrophages were incubated at 4°C for 0.5 h, washed three times with cold PBS (pH = 7.4) to remove unbound probes, and then the cells were resuspended in 500 mL PBS and stored in a BD Falcon tube in the dark, and the fluorescence intensity was quantified using a flow cytometer. Equilibrium dissociation constant K d The nonlinear equation curve was drawn using GraphPad Prism 8.0 software for calculation, and the results are shown in Figures 8 and 9.

[0145] Example 47 In vitro cell fluorescence quantitative analysis

[0146] It is well known that the selectivity of targeted fluorescent probes is determined by the targeting ligand portion. Since the GT-NIR series of compounds and the GT-IR series of compounds differ only in the fluorescent group portion, it can be preliminarily inferred that the two have similar abilities to target tumor cells that highly express FR. Subsequently, using the same experimental steps as in Example 43, all GT-NIR series of compounds and GT-IR series of compounds were subjected to in vitro cell fluorescence quantitative analysis. The results in Figure 14 (ab) show that both the GT-NIR series of compounds and the GT-IR series of compounds are able to target and enter tumor cells that highly express FR and achieve fluorescence imaging in the near-infrared region.

[0147] Example 48 In vivo small animal imaging test

[0148] Construct subcutaneous tumor FR + KB tumor-bearing mouse model and FR - A549 tumor-bearing mouse model was used, in which A549 tumor-bearing mice served as the negative control group and were used together with the subsequent competition group to verify the in vivo targeting of the probe to tissues with high FR expression. When the tumor volume reached 300-400mm 3 At the same time, the blank group was injected with 100 μL of normal saline through the tail vein; the experimental group was administered with each probe (10 nmol, dissolved in 100 μL of normal saline for injection) through the tail vein; the competition group was first injected with 100x folic acid (1 μmol, dissolved in 100 μL of normal saline) through the tail vein, and 15 minutes later, the probe (10 nmol, dissolved in 100 μL of normal saline) was injected. Imaging was performed under 750-800 nm excitation light using the small animal in vivo imaging instrument IVIS, and fluorescence imaging was taken at different time points, namely 2 h, 4 h, 8 h, 12 h, 24 h, and 48 h after administration. After 8 h of whole-body imaging, the mice were euthanized, their organs were removed, and their tissue biodistribution was analyzed using the small animal in vivo imaging instrument IVIS. The results showed that in KB tumor-bearing mice with high expression of folate receptors, the GT-NIR series of compounds had the effect of specific targeted tumor imaging. However, in the negative control group of A549 tumor-bearing mice that did not express folate receptors and the competitive group of KB tumor-bearing mice with excessive folate, the probe GT-NIR-4 did not achieve targeted tumor imaging but was quickly cleared from the body, as shown in Figures 15-18.

[0149] The same steps as above were used to inoculate subcutaneous tumors. +The in vivo imaging capabilities of the GT-IR series of compounds were evaluated in a KB tumor-bearing mouse model. Images were acquired using an IVIS small animal in vivo imaging system under 750-800 nm excitation light, with images taken 8 hours after administration. Mice were euthanized, and their organs were removed and analyzed for tissue biodistribution using an IVIS small animal in vivo imaging system. Figure 19 shows that the GT-IR series of compounds selectively accumulated at the tumor site and achieved tumor fluorescence imaging, demonstrating that these compounds can also serve as tumor-targeted fluorescent probes for in vivo imaging.

[0150] Example 49 Cytotoxicity of Fluorescent Probe

[0151] Because the targeting ligands (i.e., folate antagonists) are cytotoxic, we evaluated the safety of the GT-NIR series of probes. HUVEC cells were routinely cultured in high-glucose DMEM supplemented with 10% FBS. The culture medium contained 100 IU / mL penicillin and streptomycin and was cultured in a 5% CO2, 37°C cell culture incubator. HUVEC cells were seeded at a density of 4000 cells / well in 96-well plates and cultured overnight. These cells were then cultured with culture medium containing different concentrations of each GT-NIR series probe for 24 hours, followed by the addition of 20 μL of Cell Counting Kit-8 reagent to assess cell viability. Optical density was analyzed using a microplate reader at a 450 nm filter. The experimental results were calculated using GraphPad Prism software and are the average of three independent experiments. The cytotoxicity results are shown in Figure 20. All GT-NIR series probes had little toxicity to normal tissue cells, HUVEC.

[0152] Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art may still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A fluorescent probe compound for tumor targeting imaging or a pharmaceutically acceptable salt thereof, characterized in that: The structure of the compound is shown in Formula I: Wherein, W and Q are selected from the following structures: (1) W is -C(O)NH-, Q is -(CH2) j —, wherein j is selected from 1, 2, 3, 4 or 5; (2)W is —(CH2) n —, Q is Said n is selected from 1, 2, 3, 4 or 5; X is an amino group or an amidated derivative group, wherein the amino group is independently selected from tyrosine, cysteine, glutamic acid, methionine, threonine, serine, The amide derivative group represents an amidated derivative formed by condensation of two or more structures independently selected from the amino group; The k is selected from 0, 1, 2, 3, 4 or 5; Said p is selected from 1, 2, 3 or 4; Y is independently selected from dyes having fluorescence excitation in the visible spectrum and emission spectra in the near infrared range, and the compound maintains or enhances the fluorescence of the dyes.

2. According to claim 1, a fluorescent probe compound for tumor targeting imaging or a pharmaceutically acceptable salt thereof, wherein the amidated derivative group is selected from: The k is selected from 0, 1, 2, 3, 4 or 5.

3. The fluorescent probe compound for tumor targeting imaging according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that: The Y is selected from fluorescein isothiocyanate FITC, dye IR-783 and S0456.

4. The fluorescent probe compound for tumor targeting imaging according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that: The compound is selected from:

5. A composition comprising the fluorescent probe compound for tumor targeting imaging according to any one of claims 1 to 4, characterized in that: It also includes at least one pharmaceutically acceptable carrier or excipient.

6. Use of the fluorescent probe compound for tumor targeting imaging according to any one of claims 1 to 4 in the preparation of a tumor diagnostic agent for tumor targeting imaging.

7. The use according to claim 6, characterized in that The tumor is one or more of liver cancer, breast cancer, lung cancer, pancreatic cancer, ovarian cancer, and colorectal cancer.

8. Use of the fluorescent probe compound for tumor targeted imaging as claimed in any one of claims 1 to 4 in the preparation of an in vivo fluorescent imaging agent for precise tumor surgical navigation.