A KIAA1363 enzyme-targeted near-infrared fluorescent dye and its preparation method and application

Abstract

The present invention belongs to the technical field of biomedical functional dyes, and in particular relates to a KIAA1363 enzyme-targeted near-infrared fluorescent dye, and a preparation method and application thereof. The KIAA1363 enzyme-targeted near-infrared fluorescent dye has an excitation wavelength of about 775 nm and an emission wavelength of about 810 nm, has strong tissue penetration ability, low tissue self-absorption and light scattering, and can selectively illuminate tumor cells by targeting the KIAA1363 enzyme overexpressed in tumors, thereby locating tumor lesions and providing a clear cutting margin for tumor resection surgery.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical functional dyes, and in particular relates to a KIAA1363 enzyme-targeted near-infrared fluorescent dye and a preparation method and application thereof. Background Art

[0002] In recent years, cancer has become one of the common diseases that seriously threaten human life and health. The current treatment methods mainly include surgical resection, radiotherapy, chemotherapy, immunotherapy, targeted therapy, hormone therapy, etc., among which surgical resection is one of the main methods of cancer treatment. At present, during the surgical resection of cancer, surgeons mainly rely on preoperative imaging methods and experience such as B-ultrasound, X-rays and MRI, and intraoperative visual (ordinary white light) and tactile positioning of tumors to determine tumor margins. This intraoperative positioning method greatly affects the accuracy of the operation and will have an adverse effect on the patient's quality of life after surgery. Therefore, it is urgent to develop tumor margin auxiliary identification technology suitable for intraoperative tumor resection.

[0003] Among them, near-infrared fluorescence imaging technology has the characteristics of high sensitivity, high signal-to-noise ratio, high biocompatibility, and easy operation. With the mature near-infrared fluorescence imaging system, it can visualize the lesions in the operating room scene and assist doctors in performing precise tumor resection surgery. For example, the near-infrared fluorescent dye indocyanine green (ICG) has been widely used in clinical fluorescence imaging after being approved by the US FDA, but ICG has no tumor targeting and still has problems such as low selectivity, low signal-to-noise ratio, and rapid metabolism. Therefore, it is not conducive to being used as a dye for fluorescence navigation surgery.

[0004] Tumor marker enzymes are endogenous biological enzymes with abnormally elevated concentration levels in tumor tissues, providing targets for locating tumors. By introducing small molecule inhibitors that are highly affinity for tumor marker enzymes and covalently modifying dyes, the dyes are given the ability to specifically recognize tumor marker enzymes, thereby specifically targeting and imaging tumor tissues. For example, the U.S. FDA approved the first near-infrared fluorescent targeted dye Cytalux for ovarian cancer in November 2021 (achieved by binding to overexpressed folate receptors). The dye can image the surgical margins of ovarian cancer with a high signal-to-noise ratio and help detect tiny tumor lesions.

[0005] Research has found that neutral cholesterol ester hydrolase 1 (KIAA1363) is overexpressed in a variety of tumors, such as breast cancer and ovarian cancer, and is expected to become a universal cancer marker. Based on this, we developed a KIAA1363 enzyme targeting dye that matches the parameters of mainstream fluorescence navigation surgery equipment on the market. By binding to the overexpressed KIAA1363 enzyme, it selectively targets and enriches in tumor tissues, and maintains sufficient intratumoral retention time, achieving high signal-to-noise ratio and long-term imaging of tumor tissues, which is expected to provide a reliable and efficient intraoperative tumor margin auxiliary identification tool for tumor surgery. Summary of the invention

[0006] The purpose of the present invention is to provide a KIAA1363 enzyme-targeted near-infrared fluorescent dye and its preparation method and application in order to provide a reliable and efficient intraoperative tumor margin auxiliary identification tool for tumor surgery.

[0007] In view of this, the present invention provides a KIAA1363 enzyme-targeted near-infrared fluorescent dye having the following structural formula 1:

[0008]

[0009] Furthermore, in the general formula 1:

[0010] R1 is selected from one of an oxygen atom, a sulfur atom, and a selenium atom;

[0011] R2 is selected from one of a sulfonate group, a carboxyl group, and a hydrogen atom;

[0012] R3 is selected from one of a sulfonate group, a carboxyl group, and a hydrogen atom;

[0013] R4 is selected from one of a sulfonate group, a carboxyl group, a quaternary amino group, and a hydrogen atom;

[0014] R5 is selected from one of a sulfonate group, a carboxyl group, a quaternary amino group, and a hydrogen atom;

[0015] R6 is selected from one of methylene, secondary amine, N-methylamino, and N,N-dimethylamino;

[0016] T is selected from one of the small molecule inhibitors of KIAA1363 enzyme;

[0017] L is selected from one of the structures described in the following formulas L1 to L4:

[0018]

[0019] In formula L1, n=2, 4, 6, 8, 10, 12;

[0020] In formula L2, n = 1, 2, 3, 4;

[0021] In formula L3, n=2, 4, 6, 8, 10, 12;

[0022] In formula L4, n=1, 2, 3, 4.

[0023] Furthermore, the KIAA1363 enzyme-targeted near-infrared fluorescent dye has an excitation wavelength of 730 to 790 nm and an emission wavelength of 800 to 860 nm.

[0024] A method for preparing a KIAA1363 enzyme-targeted near-infrared fluorescent dye, the method is used to prepare the above-mentioned KIAA1363 enzyme-targeted near-infrared fluorescent dye, and the synthesis route of the KIAA1363 enzyme-targeted near-infrared fluorescent dye is as follows:

[0025]

[0026] The synthesis process of the KIAA1363 enzyme-targeted near-infrared fluorescent dye is as follows:

[0027] In an organic solvent, under alkaline conditions, the parent core dye shown in general formula 6 and the linker arm shown in general formula 7 undergo a substitution reaction at 25-50°C, and after multiple recrystallizations, the targeting dye shown in general formula 1 is obtained, wherein the molar ratio of the parent core dye to the linker arm is 1:(1.2-1.5).

[0028] Furthermore, the parent core dye has the following structural formula 6-1 or 6-2:

[0029]

[0030] Further, the synthetic route of the parent core dye is as follows:

[0031]

[0032] The synthesis process of the parent core dye is:

[0033] In an organic solvent acetic anhydride solution, with sodium acetate as a catalyst, the intermediate S-1 containing R2 and R4 substituents and the intermediate S-2 containing R6 substituents are reacted at 50-80°C for 6-12 hours, and recrystallized 3-5 times after the reaction to obtain the mother core dye shown in the general formula 6-1;

[0034] Alternatively, in an organic solvent acetic anhydride solution, with sodium acetate as a catalyst, the intermediate S-3 containing R3 and R5 substituents and the intermediate S-2 containing R6 substituents are reacted at 50-80°C for 6-12 hours. After the reaction is completed, recrystallization is performed 3-5 times to obtain the mother core dye shown in the general formula 6-2.

[0035] Further, the connecting arm has the following structural formula 7-1, 7-2, 7-3 or 7-4:

[0036]

[0037] Further, the synthetic route of the connecting arm is as follows:

[0038]

[0039] The synthesis process of the linker shown in the general formula 7-1 is as follows: in an organic solvent, the intermediate L1 containing the R1 atom and the KIAA1363 enzyme inhibitor molecule containing a carboxyl group are subjected to an amidation reaction at room temperature to prepare S-4, and then the triphenylmethane protecting group is removed at room temperature, and the linker shown in the general formula 7-1 is obtained after purification;

[0040] The synthesis process of the linker shown in the general formula 7-2 is as follows: in an organic solvent, the intermediate L2 and the KIAA1363 enzyme inhibitor molecule are subjected to an amidation reaction at room temperature, and after purification, the linker shown in the general formula 7-2 is obtained;

[0041] The synthesis process of the linker shown in the general formula 7-3 is as follows: in an organic solvent, the intermediate L3 containing the R1 atom undergoes an amidation reaction with S-5 at room temperature, and the Fmoc protecting group is removed and purified to obtain S-6, and then S-6 undergoes an amidation reaction with the KIAA1363 enzyme inhibitor molecule, and the triphenylmethane protecting group is removed, and after purification, the linker shown in the general formula 7-3 is obtained;

[0042] The synthesis process of the connecting arm shown in the general formula 7-4 is as follows: in an organic solvent, the intermediate L4 containing the R1 atom undergoes an amidation reaction with S-5 at room temperature, and the Fmoc protecting group is removed and purified to obtain S-7. Thereafter, S-7 undergoes an amidation reaction with the KIAA1363 enzyme inhibitor molecule, and the triphenylmethane protecting group is removed. After purification, the connecting arm shown in the general formula 7-4 is obtained.

[0043] The above-mentioned KIAA1363 enzyme-targeted near-infrared fluorescent dye is used in the field of biological imaging.

[0044] The above-mentioned KIAA1363 enzyme-targeted near-infrared fluorescent dye is used in tumor fluorescence navigation, cancer cell screening, protein labeling, protein molecule docking, protein inhibitor molecule screening, cell imaging or fluorescent dye probes.

[0045] The KIAA1363 enzyme-targeted near-infrared fluorescent dye described in the present invention has an excitation wavelength of about 775 nm and an emission wavelength of about 810 nm. It has strong tissue penetration ability, low tissue self-absorption and light scattering, and can match the parameters of mainstream fluorescence navigation surgery equipment on the market (developed based on ICG, with an effective excitation band of the filter of about 735-785 nm). It can selectively illuminate tumor cells by targeting the KIAA1363 enzyme overexpressed in tumors (such as breast cancer), thereby locating tumor lesions and providing a clear resection margin for tumor resection surgery. It has good clinical transformation application prospects in biomedicine fields such as tumor fluorescence navigation surgery. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 is the nuclear magnetic resonance of the targeting dye I of the present invention 1 H-Figure;

[0047] Figure 2 is the nuclear magnetic resonance of the targeting dye I of the present invention 13 C spectrum;

[0048] Figure 3 is a high-resolution mass spectrum of the targeting dye I of the present invention;

[0049] Figure 4 It is the absorption and emission spectra of the targeting dye I and the parent core dye S0456 in PBS solution of the present invention;

[0050] Figure 5 is the cytotoxicity evaluation result of the targeting dye I of the present invention;

[0051] Figure 6 This is an image of the targeted dye I of the present invention being used for fluorescent screening of breast cancer cells. DETAILED DESCRIPTION

[0052] The following will be combined with the drawings in the embodiments of the present application to clearly describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments in the present application belong to the scope of protection of this application.

[0053] In the description of the present application, it should be noted that the terms used herein are only for describing specific embodiments, and are not intended to limit the exemplary embodiments according to the present application. For ease of description, the sizes of the various parts shown in the drawings are not drawn according to the actual proportional relationship. The technology, methods and equipment known to ordinary technicians in the relevant field may not be discussed in detail, but in appropriate cases, the technology, methods and equipment should be regarded as part of the authorization specification. In all examples shown and discussed here, any specific value should be interpreted as merely exemplary, rather than as a limitation. Therefore, other examples of the exemplary embodiments may have different values. It should be noted that similar numbers and letters represent similar items in the following drawings, so once an item is defined in one drawing, it does not need to be further discussed in subsequent drawings.

[0054] It should be noted that the terms "first", "second", etc. in the specification and claims of the present application are used to distinguish similar objects, and are not used to describe a specific order or sequence. It should be understood that the data used in this way can be interchangeable under appropriate circumstances, so that the embodiments of the present application can be implemented in an order other than those illustrated or described here, and the objects distinguished by "first", "second", etc. are usually one, and the number of objects is not limited. For example, the first object can be one or more. In addition, "and / or" in the specification and claims represents at least one of the connected objects, and the character " / " generally indicates that the objects associated before and after are in an "or" relationship.

[0055] It should be noted that, in the present application, the terms "comprise", "include" or any other variant thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the sentence "comprises one..." does not exclude the presence of other identical elements in the process, method, article or device including the element. In addition, it should be noted that the scope of the method and device in the embodiment of the present application is not limited to performing functions in the order shown or discussed, and may also include performing functions in a substantially simultaneous manner or in reverse order according to the functions involved, for example, the described method may be performed in an order different from that described, and various steps may also be added, omitted, or combined. In addition, the features described with reference to certain examples may be combined in other examples.

[0056] A KIAA1363 enzyme-targeted near-infrared fluorescent dye having the following general structural formula 1:

[0057]

[0058] Wherein, in the general formula 1:

[0059] R1 is selected from one of an oxygen atom, a sulfur atom, and a selenium atom;

[0060] R2 is selected from one of a sulfonate group, a carboxyl group, and a hydrogen atom;

[0061] R3 is selected from one of a sulfonate group, a carboxyl group, and a hydrogen atom;

[0062] R4 is selected from one of a sulfonate group, a carboxyl group, a quaternary amino group, and a hydrogen atom;

[0063] R5 is selected from one of a sulfonate group, a carboxyl group, a quaternary amino group, and a hydrogen atom;

[0064] R6 is selected from one of methylene, secondary amine, N-methylamino, and N,N-dimethylamino;

[0065] T is selected from one of the small molecule inhibitors of KIAA1363 enzyme;

[0066] L is selected from one of the structures described in the following formulas L1 to L4:

[0067]

[0068] In formula L1, n=2, 4, 6, 8, 10, 12;

[0069] In formula L2, n = 1, 2, 3, 4;

[0070] In formula L3, n=2, 4, 6, 8, 10, 12;

[0071] In formula L4, n=1, 2, 3, 4.

[0072] As some embodiments of the present invention, the KIAA1363 enzyme small molecule inhibitor is AX-11890 and the like.

[0073] Furthermore, the KIAA1363 enzyme-targeted near-infrared fluorescent dye has an excitation wavelength of about 730 to 790 nm and an emission wavelength of about 800 to 860 nm.

[0074] Preferably, the KIAA1363 enzyme-targeted near-infrared fluorescent dye has an excitation wavelength of about 775 nm and an emission wavelength of about 810 nm.

[0075] The KIAA1363 enzyme-targeted near-infrared fluorescent dye described in the present invention has strong tissue penetration ability, low tissue self-absorption and light scattering, and can selectively illuminate tumor cells by targeting the tumor-overexpressed KIAA1363 enzyme, thereby locating tumor lesions and providing a clear resection margin for tumor resection surgery.

[0076] Surgical resection is one of the most effective methods for tumor treatment at present, but current surgical procedures lack technical means to clearly display the tumor resection margin during surgery. The KIAA1363 enzyme-targeted near-infrared fluorescent dye disclosed in the present invention has the following advantages in tumor surgical resection:

[0077] First, high specificity: the KIAA1363 enzyme-targeted near-infrared fluorescent dye disclosed in the present invention can achieve targeting by specifically binding to the KIAA1363 enzyme overexpressed in tumors. This highly specific binding can increase the enrichment of the dye in tumor tissues and improve the accuracy of imaging;

[0078] Second, high selectivity: Because the dye selectively targets the KIAA1363 enzyme, it can be more precisely enriched in tumor tissues without accumulating too much in normal tissues, which helps improve the contrast and clarity of imaging;

[0079] Third, high signal-to-noise ratio: Due to the high enrichment of the dye in tumor tissue, its imaging has a high signal-to-noise ratio, which is very important for clearly showing the tumor margin during surgery and helps doctors make more accurate decisions;

[0080] Fourth, sufficient intratumoral residence time: It is critical to maintain sufficient residence time in the tumor tissue, which can ensure that during the operation, the doctor has enough time to observe, locate and remove the tumor tissue, thereby improving the success rate of the operation; at the same time, the long-term imaging of the dye means that during the operation, the doctor can continue to observe the tumor tissue, ensuring that the tumor is completely removed without easily missing potential cancer cells;

[0081] Fifth, reduce the risk of recurrence: by accurately identifying the tumor resection margin, we can ensure that the tumor is completely removed, thereby reducing the risk of postoperative recurrence;

[0082] Sixth, reduce the amount of normal tissue resection: By providing clear imaging of tumor margins, this dye can help surgeons more accurately locate and remove tumors, thereby reducing miscutting of normal tissue, helping patients recover better after surgery and improving their quality of life after surgery.

[0083] In summary, the dye of the present invention can selectively target and enrich in tumor tissue by specifically binding to the KIAA1363 enzyme overexpressed in the tumor, and maintain a sufficient intratumoral retention time, thereby achieving high signal-to-noise ratio and long-term imaging of tumor tissue and accurately identifying tumor resection margins. Accurately identifying tumor resection margins can ensure that the tumor is completely removed and reduce the risk of recurrence; on the other hand, it can reduce the amount of normal tissue removed, facilitate postoperative recovery of patients and improve their quality of life after surgery.

[0084] In addition, the present invention also provides a method for preparing the above-mentioned KIAA1363 enzyme-targeted near-infrared fluorescent dye, which specifically comprises:

[0085] Step 1: synthesize the parent core dye 6 according to the following route. The parent core dye 6 has the following structural formula 6-1 or 6-2:

[0086]

[0087] Step 2: Synthesize the linker arm and covalently link it to the target head according to the following route to obtain intermediate 7. Intermediate 7 has the following general structural formula 7-1, 7-2, 7-3 or 7-4:

[0088]

[0089] Step 3, synthesize the targeting dye 1 according to the following route:

[0090]

[0091] Furthermore, in step 1, the synthesis process of the parent core dye 6 is:

[0092] In an organic solvent acetic anhydride solution, with sodium acetate as a catalyst, the intermediate S-1 containing R2 and R4 substituents and the intermediate S-2 containing R6 substituents are reacted at 50-80°C for 6-12 hours. After the reaction is completed, the mother core dye 6-1 is obtained by recrystallization 3-5 times in the form of a green solid powder.

[0093] Preferably, the molar ratio of S-1 to S-2 is 2:1.

[0094] Similarly, in an organic solvent acetic anhydride solution, with sodium acetate as a catalyst, the intermediate S-3 containing R3 and R5 substituents and the intermediate S-2 containing R6 substituents are reacted at 50-80°C for 6-12 hours. After the reaction is completed, the mother core dye 6-2 is obtained by recrystallization 3-5 times.

[0095] Preferably, the optimal molar ratio of the reaction of S-3 and S-2 is 2:1.

[0096] Preferably, in step 1, the recrystallization solvent is selected from any one or a combination of water, methanol, ether, dichloromethane, ethyl acetate, isopropanol and acetone.

[0097] Furthermore, in step 2, the synthesis process of the connecting arm 7-1 is:

[0098] In an organic solvent, the intermediate L1 containing the R1 atom reacts with the KIAA1363 enzyme inhibitor molecule containing a carboxyl group at room temperature to prepare S-4, and then the triphenylmethane protecting group is removed at room temperature, and the linker 7-1 is obtained after purification.

[0099] Further, in the step 2, the synthesis process of 7-2 is:

[0100] In an organic solvent, the intermediate L2 reacts with the KIAA1363 enzyme inhibitor molecule at room temperature through an amidation reaction, and 7-2 is obtained after purification.

[0101] Further, in the step 2, the synthesis process of 7-3 is:

[0102] In an organic solvent, the intermediate L3 containing the R1 atom undergoes an amidation reaction with S-5 at room temperature, and the Fmoc protecting group is removed and purified to obtain S-6. S-6 then undergoes an amidation reaction with the KIAA1363 enzyme inhibitor molecule, and the triphenylmethane protecting group is removed, and 7-3 is obtained after purification.

[0103] Similarly, in step 2, the synthesis process of 7-4 is:

[0104] In an organic solvent, the intermediate L4 containing the R1 atom undergoes an amidation reaction with S-5 at room temperature, and the Fmoc protecting group is removed and purified to obtain S-7. Then, S-7 undergoes an amidation reaction with the KIAA1363 enzyme inhibitor molecule, and the triphenylmethane protecting group is removed, and 7-4 is obtained after purification.

[0105] Preferably, in step 2, the amidation reaction solvent is selected from anhydrous DMF, anhydrous DMSO, anhydrous acetonitrile, and anhydrous dichloromethane.

[0106] Preferably, in step 2, the solvent for the triphenylmethane protecting group removal reaction is a mixed solution of dichloromethane and trifluoroacetic acid, wherein the volume ratio of dichloromethane to trifluoroacetic acid is 1:1 to 1:4.

[0107] Preferably, in the step 2, the solvent for the Fmoc protecting group removal reaction is a mixed solvent of piperidine and DMF, wherein the volume ratio of piperidine to DMF is 1:4 to 1:1.

[0108] Furthermore, in step 3, the synthesis process of the targeting dye 1 is:

[0109] In an organic solvent, under alkaline conditions, the mother core dye 6 and the connecting arm 7 undergo a substitution reaction at 25-50°C, and the targeting dye 1 can be obtained after multiple recrystallizations.

[0110] Preferably, in step three, the alkali used in the substitution reaction is selected from one of K2CO3, KOH, Na2CO3, NaOH, and triethylamine, and the organic solvent used is selected from one of DMF, DMSO, and methanol or a combination of any two thereof.

[0111] As some embodiments of the present application, in step three, the organic solvent used is DMSO or methanol, and the molar ratio of the dye core 6 to the linker arm 7 is 1:(1.2-1.5).

[0112] In addition, the aforementioned L3 is prepared by amidation reaction of general formula 2 and general formula 3, and L4 is prepared by amidation reaction of general formula 4 and general formula 5, and the reaction solvent is DMF, DMSO, acetonitrile, etc.

[0113] Specifically, the synthetic routes of L3 and L4 are as follows:

[0114]

[0115] In addition, the KIAA1363 enzyme-targeted near-infrared fluorescent dye described in the present invention can be used in the biomedical field, such as the field of biological imaging.

[0116] Preferably, the KIAA1363 enzyme-targeted near-infrared fluorescent dye described in the present invention can be used in biomedical fields such as early tumor diagnosis, tumor fluorescence navigation, cancer cell screening, protein labeling, protein molecule docking and protein inhibitor molecule screening, cell imaging, fluorescent dye probes, tumor light-driven therapy, and tumor fluorescence navigation surgery.

[0117] Compared with the prior art, the dyes proposed in the present invention have the following advantages:

[0118] (1) The KIAA1363 enzyme-targeted near-infrared fluorescent dye of the present invention has high water solubility and good biosafety;

[0119] (2) The KIAA1363 enzyme-targeted near-infrared fluorescent dye described in the present invention has an excitation wavelength of 775 nm and an emission wavelength of about 810 nm, has strong tissue penetration ability, low tissue self-absorption and light scattering, and is suitable for mainstream fluorescent navigation surgery equipment on the market;

[0120] (3) KIAA1363 enzyme is overexpressed in many tumors. Dyes targeting KIAA1363 enzyme can be used as contrast agents for pan-tumor fluorescence navigation surgery.

[0121] The following specific examples illustrate the KIAA1363 enzyme-targeted near-infrared fluorescent dye and its preparation method and application according to the present invention:

[0122] Example 1

[0123] The synthesis of the targeting dye 1, the synthesis route is as follows:

[0124]

[0125] The synthesis process of targeting dye 1 is as follows:

[0126] (1) Synthesis of the parent core dye S0456

[0127] Prepare 20 ml of a mixed solution of acetic anhydride and methanol (the volume ratio of acetic anhydride to methanol is 4:1), add intermediate S-1-1 (826 mg, 2 mmol), 2-chloro-3-(hydroxymethylene)-cyclohex-1-enecarboxaldehyde (172 mg, 1 mmol) and sodium acetate (82 mg, 1 mmol) respectively, and react at 60°C for 6 hours. After the reaction solution is cooled, the inorganic salt is filtered to remove the inorganic salt, and the filtrate is slowly poured into an ether solution (100 mL) to precipitate a solid powder, which is filtered to obtain a crude product. Repeat crystallization 3 times in a mixed solution of water and isopropanol to obtain a dark green solid powder S0456 (698 mg, 71%).

[0128] 1 H NMR(400MHz,D2O)δ8.23(d,J=13.9Hz,2H),7.82(s,2H),7.72(d,J=8.3Hz,2H),7.26(d,J=8.4Hz,2H),6.16(d,J=14.1 Hz,2H),4.10(t,J=6.3Hz,4H),2.92(t,J=7.3Hz,4H),2.46(bs,4H),1.95–1.78(m,8H),1.65(s,14H).HRMS(ESI):m / z 421.1371[M-2K-Na] 3- (calcd for C 38 H 44 ClN2O 12 S4 3- ,294.3827).

[0129] (2) Synthesis of intermediate S-6-1

[0130] N-Boc-6-aminocaproic acid (1.16 g, 5 mmol), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, 2.85 g, 7.5 mmol) and N,N-diisopropylethylamine (DIPEA, 1.74 mL, 10 mmol) were added to anhydrous DMF solution (20 mL). After reacting at room temperature for 20 minutes, a DMF solution (5 mL) of raw material L-3-1 (1.83 g, 5 mmol) was added and reacted at room temperature overnight. After the reaction solution was concentrated, it was dissolved in ethyl acetate (150 mL), extracted with deionized water 3 times, washed with saturated brine once, and the ethyl acetate phase was dried over anhydrous sodium sulfate and concentrated to obtain an amidated intermediate. Further, it was dissolved in a mixed solution of DMF (20 mL) and piperidine (5 mL) and reacted at 40°C for 5 hours. After cooling, the reaction solution was concentrated and dissolved with ethyl acetate (150 mL), extracted with deionized water three times, washed once with saturated brine, and the organic phase was dried over anhydrous sodium sulfate and concentrated to obtain S-6-1 (2.1 g, 72%), which was directly used in the next step.

[0131] (3) Synthesis of intermediate 7-3-1

[0132] To anhydrous DMF solution (5mL), AX-04 (337mg, 1mmol), HATU (570mg, 1.5mmol) and DIPEA (0.35mL, 2mmol) were added successively. After reacting at room temperature for 10 minutes, a DMF solution (1mL) of raw material S-6-1 (578mg, 1mmol) was added and reacted at room temperature for 5 hours. After the reaction solution was concentrated, it was dissolved in ethyl acetate (50mL), extracted with deionized water 3 times, washed with saturated brine once, the ethyl acetate phase was dried over anhydrous sodium sulfate, and concentrated. Further, it was dissolved in a mixed solution of DCM (5mL) and trifluoroacetic acid (TFA, 5mL) and reacted at room temperature for 2 hours. The reaction solution was concentrated, dissolved in ethyl acetate (50mL), extracted with deionized water once, washed with saturated brine once, the organic phase was dried over anhydrous sodium sulfate, and concentrated to obtain a crude product, which was purified by silica gel column chromatography to obtain a white solid 7-3-1 (487mg, 84%).

[0133] 1H NMR(400MHz,DMSO)δ9.27(s,1H),8.72(t,J=5.3Hz,1H),8.53(s,1H),8.22–8.1 5(m,2H),8.11(d,J=8.8Hz,2H),7.53(d,J=8.8Hz,1H),7.03(d,J=8.2Hz,2H),6. 68(d,J=8.3Hz,2H),4.12(d,J=5.7Hz,2H),3.31(dd,J=12.7,6.6Hz,2H),3.16(s ,3H),2.97(s,3H),2.12(t,J=7.3Hz,2H),1.58–1.55(m,2H),1.38–1.25(m,4H). 13 C NMR (101MHz, DMSO) δ171.87,165.54,156.18,152.94,148.09,132.91,132.49,131.12,129.88,129.83,128.54,1 27.93,126.47,126.25,123.91,114.99,114.32,41.56,36.51,36.32,35.33,28.95,26.27,25.15.HRMS(ESI):m / z 580.1262[M+Na] + (calcd for C 27 H 30 BrN3NaO5 + ,580.1241).

[0134] (4) Synthesis of KIAA1363 enzyme-targeted near-infrared fluorescent dye 1

[0135] To the organic solvent DMSO (40mL), the intermediate 7-3-1 (128mg, 0.22mmol) and potassium carbonate (83mg, 0.6mmol) were added in sequence, and after stirring at room temperature for 20 minutes, the parent core dye S0456 (197mg, 0.20mmol) was added, and the reaction was continued overnight at 40°C. After cooling and filtering, the filtrate was introduced into ether (100mL) and filtered to obtain a crude product. Further, the dark green solid powder KIAA1363 enzyme-targeted near-infrared fluorescent dye 1 (246mg, 82%) was obtained by three recrystallizations using methanol and ethyl acetate.

[0136] 1H NMR (400MHz, DMSO) δ8.77(t,J=4.6Hz,1H),8.63(s,1H),8.26(t,J=5.5Hz,1H),8.21–8.11(m,3H),7.79(d,J=1 4.0Hz,2H),7.67–7.58(m,4H),7.51(d,J=8.8Hz,1H),7.32(d,J=8.2Hz,2H),7.27(d,J=8.2Hz,2H),7.09(d,J=8 .2Hz,2H),6.21(d,J=14.1Hz,2H),4.20–4.07(m,6H),3.25(d,J=5.8Hz,2H),3.17(s,3H),2.97(s,3H),2.71(s, 4H), 2.57 (s, 4H), 2.07 (t, J = 7.2Hz, 2H), 1.91 (s, 2H), 1.73 (s, 8H), 1.51 (dd, J = 13.8, 6.9Hz, 4H), 1.26 (s, 14H). 13 C NMR (101MHz, DMSO) δ172.48,172.01,165.81,163.44,158.97,153.40,148.51,145.54,142.5 5,141.27,140.76,134.48,133.34,132.78,131.64,130.49,129.90,128.38,127.06,126.61, 124.22,122.53,120.01,114.94,114.66,110.90,101.11,60.23,51.17,48.94,44.06,41.72, 36.96,36.79,35.86,29.34,27.64,26.79,26.31,25.71,24.26,22.78,21.53.HRMS(ESI):m / z 468.1074[M-2K-Na] 3- (calcd for C 65 H 73 BrN5O 17 S4 3- ,468.1021).

[0137] The obtained KIAA1363 enzyme-targeted near-infrared fluorescent dye 1 was analyzed by nuclear magnetic resonance spectroscopy. 1 H Figure Figure 1 As shown, NMR 13 C spectrum Figure 2 As shown, the high-resolution mass spectrum is as follows Figure 3 shown.

[0138] Example 2

[0139] Absorption and fluorescence spectra of KIAA1363 enzyme-targeted near-infrared fluorescent dye 1 in PBS buffer

[0140] KIAA1363 enzyme-targeted near-infrared fluorescent dye 1 or parent nuclear dye S0456 were dissolved in the organic solvent DMSO to prepare a storage stock solution with a concentration of 10mM and stored at 4°C for later use. Take 1μL of dye I stock solution or S0456 stock solution with a pipette, dilute it in 2mL PBS solution, mix it thoroughly, and use Duetta at room temperature. TM Fluorescence and absorption spectrometers were used to measure the absorption and fluorescence spectra respectively. The test results are as follows: Figure 4 As shown, the absorption spectra of KIAA1363 enzyme-targeted near-infrared fluorescent dye 1 and the parent core dye S0456 are similar, and the molar extinction coefficients are close. Both show the strongest absorption near 775nm, which matches the parameters of mainstream fluorescence navigation surgery equipment on the market (the effective excitation band of the filter is about 735-785nm); S0456-AX and S0456 both show maximum emission near 810nm, but the fluorescence signal of S0456-AX is relatively stronger. The quantum yields of S0456-AX and S0456 were measured by an absolute quantum yield analyzer to be 0.072 and 0.037, respectively.

[0141] Example 3 Evaluation of cytotoxicity of KIAA1363 enzyme-targeted near-infrared fluorescent dye 1

[0142] After MCF-7 cells were incubated with 0-40 μM dye I for 24 h, the cytotoxicity of KIAA1363 enzyme-targeted near-infrared fluorescent dye 1 was determined using the MTT assay. Figure 5 As shown, after incubating MCF-7 cells with 0-20 μM dye I for 24 hours, the survival rate was higher than 90%, proving that S0456-AX had no obvious cytotoxicity within this concentration range, had high biocompatibility, and could be safely used in biomedicine and other fields.

[0143] Example 4 KIAA1363 enzyme-targeted near-infrared fluorescent dye 1 is used for fluorescent screening of breast cancer cells

[0144] First, a mixed culture medium of human breast cancer cell MCF-7 culture medium and human mammary epithelial cell MCF-10A culture medium was prepared (volume ratio of 1:1), and the two types of cells were cultured in laser confocal culture dishes with the mixed culture medium. After the cell abundance reached 60%, MCF-7 cells and MCF-10A cells were incubated with targeted dye I (5μM) for 4 hours, respectively. As a control, MCF-7 cells were incubated with non-targeted nuclear dye S0456 (5μM) for 4 hours. After the above three groups of cells were washed three times with PBS, fresh culture medium was added, and the cell fluorescence signals were collected using a Leica STELLARIS 5 laser scanning confocal fluorescence microscope (excitation wavelength 638nm, emission wavelength receiving range is 780-850nm). The results are as follows Figure 6 As shown, after MCF-7 cells and MCF-10A cells were incubated with targeted dye I respectively, only MCF-7 cells showed bright fluorescence; on the other hand, after MCF-7 cells were incubated with non-targeted dye S0456, the cell fluorescence signal was still very weak, only slightly stronger than that of MCF-10A cells, but significantly weaker than that of MCF-7 cells incubated with dye I, indicating that the targeting performance of targeted dye I to KIAA1363 is conducive to the cellular uptake and intracellular retention of the dye, so that MCF-7 cells overexpressing KIAA1363 enzyme show significantly stronger fluorescence signals, proving that KIAA1363 enzyme targeted near-infrared fluorescent dye 1 can be used to effectively distinguish breast cancer cells from normal cells.

[0145] The embodiments of the present application are described above in conjunction with the accompanying drawings. In the absence of conflict, the embodiments in the present application and the features in the embodiments can be combined with each other. The present application is not limited to the above-mentioned specific implementation methods. The above-mentioned specific implementation methods are merely illustrative and not restrictive. Under the guidance of the present application, ordinary technicians in this field can also make many forms without departing from the purpose of the present application and the scope of protection of the claims, all of which are within the protection of the present application.

Claims

1. A KIAA1363 enzyme-targeted near-infrared fluorescent dye, characterized in that: It has the following structure:

2. The KIAA1363 enzyme-targeted near-infrared fluorescent dye according to claim 1, characterized in that: The KIAA1363 enzyme-targeted near-infrared fluorescent dye has an excitation wavelength of 775 nm and an emission wavelength of 810 nm.

3. Use of the KIAA1363 enzyme-targeted near-infrared fluorescent dye as claimed in any one of claims 1 to 2 in the preparation of cancer cell screening preparations or fluorescent dye probes.

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