CN117164500A - Tumor tissue rapid pathological detection method based on near infrared cyanine fluorescent probe - Google Patents

Abstract

The present invention is applicable to the technical field of molecular probes and provides a rapid pathological detection method of tumor tissue based on a near-infrared cyanine fluorescent probe. The near-infrared cyanine fluorescent probe can realize various cancers such as squamous cell carcinoma, adenocarcinoma, and small cell carcinoma. Preoperative/during/postoperative diagnosis of cell carcinoma, signet ring cell carcinoma, head and neck cancer, etc. For example, for breast cancer, thyroid cancer, lung cancer, cervical cancer, ovarian cancer, gastrointestinal cancer, prostate cancer, oral cancer, skin cancer, liver cancer, pancreatic cancer, bladder cancer, esophageal cancer, lymphoma, blood cancer, brain tumor, nasal cancer , tongue cancer, gingival cancer and other tumors, rapid intraoperative marking, fluorescence pathological analysis, tumor contour determination, tumor malignancy analysis, tumor staging and classification diagnosis, and patient survival prediction, etc.

Description

Rapid pathological detection method of tumor tissue based on near-infrared cyanine fluorescent probe

Technical field

The invention belongs to the technical field of molecular probes, and in particular relates to a rapid pathological detection method of tumor tissue based on near-infrared cyanine fluorescent probes.

Background technique

Optical imaging technology is widely used in the detection of clinical diseases due to its easy operation and visualization advantages. Rapid immunofluorescence analysis can not only visualize pathological structural features, but also reveal the expression levels of different disease markers, further improving the refined classification of disease types. Therefore, using fluorescence analysis technology to achieve higher sensitivity, simpler operation and analysis, and higher-throughput visual integration capabilities is one of the current challenges and development trends faced by rapid intraoperative pathological analysis. But there are still several problems. First, molecular probes for in vivo optical imaging have been limited by translational limitations of in vivo biosafety. Only a handful of imaging probes have achieved clinical application. Secondly, in order to achieve rapid intraoperative pathological analysis, we still rely on professional pathologists to perform intraoperative rapid frozen section pathological analysis. This technology cannot simultaneously achieve rapid analysis of large batches of pathological sections. Third, intraoperative pathological analysis (such as rapid immunohistochemistry) cannot simultaneously detect the expression levels of various tumor-related markers in tissues, which is very important for tumor staging and disease pathogenesis.

Current rapid immunofluorescence detection relies on the specific combination of antigens and antibodies of disease (tumor) markers, which is costly, complex to operate, has poor stability, and lacks sufficient antibody selectivity to detect multiple disease (tumor) subtypes. . Based on the above technical limitations, there is an urgent need to develop a fluorescent molecular probe that is simple, fast, and can directly target disease (tumor)-related markers to achieve early detection, intraoperative rapid detection, and prognosis monitoring of such diseases.

Contents of the invention

The purpose of the present invention is to provide a rapid pathological detection method of tumor tissue based on a near-infrared cyanine fluorescent probe, aiming to solve the problems raised in the above background technology.

In order to achieve the above objects, the present invention provides the following technical solutions:

A rapid pathological detection method of tumor tissue based on a near-infrared cyanine fluorescent probe, which has the following structural formula I:

In the general formula I, X ^- is selected from Cl ^- , Br ^- , I ^- , PF ₆ ^- , BF ₄ ^- , ClO ₄ ^- , CH ₃ COO ^- , CF ₃ COO ^- or OTs ^- ; Oxygen or sulfur; Z is selected from C _1-8 alkyl or aryl groups, the methylene bridge is modified with alkyl, five-membered ring, six-membered ring, nitrogen-containing six-membered ring, oxygen-containing six-membered ring, sulfur-containing Six-membered ring, benzene ring or seven-membered ring; heterocyclic salt is selected from indole, benzindole, benzoxazole, benzothiazole, benzoselenazole, quinoline, acridine, benzothiopyranium, Benzopyranium or silyl rhodamine; n and m are any integers greater than 0 (can be the same or different); R ₁ and R ₂ (can be the same or different) are each independently selected from sulfonic acid, arylsulfonic acid, Carboxylic acid, sulfonate, carboxylate, alkyl, aryl, alkenyl, alkynyl, nitro, cyano, azide, amine, hydroxyl, halogen, alkoxy, ester, amide, poly Ethylene glycol chain, piperidine, morpholine, pyrrole, imidazole, pyridine salt or quaternary ammonium salt; p is an integer from 0 to 6; R is selected from alkyl chain, halogen, activated ester, maleimide, sulfoxide , sulfone, sulfonate, thioether, acrylamide, acrylate, vinyl sulfonamide, nitrogen or benzene ring.

Furthermore, the near-infrared cyanine fluorescent probe binds to protein molecules through covalent/non-covalent forces, and the labeled protein molecules are tumors or normal tissues.

Further steps include:

a. Immerse the tumor tissue sections from surgical resection or biopsy into paraformaldehyde fixative for 0 to 30 minutes; the excess fixative is eluted with phosphate Tween buffer, and the elution time is 0 to 30 minutes;

b. Immerse the tumor tissue sections obtained in step a into the Triton X-100 surfactant solution for membrane rupture treatment. The mass concentration of Triton X-100 is 0.1%~30%, and the processing time is 0~30 min; excess Triton X- 100 solution is eluted by PBST, and the elution time is 0~30 minutes;

c. Immerse the tumor tissue sections obtained in step b into H ₂ O ₂ solution. The mass concentration of H ₂ O ₂ is 0.1%~30%, and the processing time is 0~60 min. The excess hydrogen peroxide solution is eluted with PBST. The removal time is 0~30 minutes;

d. Immerse the tumor tissue sections obtained in step c into the near-infrared cyanine fluorescent probe solution and incubate. The incubation temperature is 4~70°C. The molar concentration of the cyanine fluorescent probe solution is not less than 1 nM, and the incubation time is not less than 1 min. ; The excess cyanine fluorescent probe solution is first eluted with DMSO solution, the mass concentration of DMSO is 1~100%, and the elution time is 0~30 min; the remaining DMSO is then eluted with PBST, and the elution time is 0~ 30 minutes;

e. Place the tumor tissue slice obtained in step d on a scanning imager or fluorescence camera with a near-infrared excitation light source for scanning to generate a fluorescent pattern of the tumor tissue; analyze the fluorescent pattern obtained by scanning to determine the tumor outline, tumor staging and classification, Predict malignancy and survival.

Furthermore, one or more near-infrared cyanine fluorescent probes are used simultaneously for multi-index and multi-color labeling.

Further, for the tumor tissue section in step a, the detection method is also applicable to cell smears, tissue fluid smears, frozen/paraffin tissue sections, and the substrate of the smear or tissue section is a positive ion anti-detachment slide. Smears or tissue sections contain near-infrared cyanine fluorescent probes.

Further, the thickness of the tissue section is not less than 1 µm.

Further, the glass slide is made of glass, silicon or silicon dioxide.

Compared with the prior art, the beneficial effects of the present invention are:

This rapid pathological detection method for tumor tissue is based on a near-infrared cyanine fluorescent probe. The near-infrared cyanine fluorescent probe can use covalent/non-covalent binding capabilities to specifically label abnormally expressed protein molecules/cytokines in tissues. , based on the spatial position of cells and related molecules that respond, lesions can be patterned with prominent fluorescent signals, that is, areas with strong fluorescence signals are lesion areas; near-infrared cyanine fluorescent probes can image detailed pathological structures and achieve intensity signals , dual analysis of morphological characteristics; the near-infrared cyanine fluorescent probe can help improve the accuracy of rapid intraoperative pathology and achieve highly sensitive, specific, fast and efficient intraoperative pathological analysis.

Description of drawings

Figure 1 is a schematic diagram of the covalent/non-covalent labeling capabilities of 25 selected near-infrared cyanine fluorescent probes on mouse tumor (SGC-7901) tissue sections.

Figure 2 is a schematic diagram of the operation flow for rapid fluorescence pathological analysis.

Figure 3 shows the effects of cyanine fluorescent probes (IR-6B1, IR-6B3, IR-6B3C, IR-6B3S, IR-6B4S, and IR-6B5C) on human breast cancer tumor tissue (T) and its paracancerous tissue (P) respectively. ) Two-dimensional electrophoresis diagram (a) and fluorescence intensity ratio diagram (b) of the protein labeling ability in the lysate.

Figure 4 shows the effects of cyanine fluorescent probes (IR-6B1, IR-6B3, IR-6B3C, IR-6B3S, IR-6B4S, IR-6B5C) on human breast cancer tumor tissue (T) and paracancerous tissue (P). Fluorescence scanning image of frozen section staining (a) and corresponding H&E pathological analysis image (b).

Figure 5 is a statistical diagram of the tissue average fluorescence intensity signal ratio (T/P) after staining frozen sections of human breast cancer tumor tissue (T) and adjacent tissue (P) with 25 selected cyanine fluorescent probes.

Figure 6 shows the fluorescence pathological analysis pictures (a, c) and tissue average fluorescence intensity statistical diagram (b) of IR-6B3S probe (a, b) and IR-6B3 probe (c, d) used in human thyroid cancer tissue sections. ,d). The gray areas in Figures a and c are tumor tissues, and the light gray areas are normal tissues.

Figure 7 shows the fluorescence pathological analysis chart (a) and the tissue average fluorescence intensity statistical chart (b) of the IR-6B3S probe used in human lung cancer tissue sections. The gray area in picture a is the tumor tissue, and the light gray area is the normal tissue.

Figure 8 shows the relationship between the average fluorescence signal ratio of tumor (T) and adjacent tissue (P) of IR-6B3S labeled human breast cancer tissue to tumor T stage (a), patient 5-year survival (b) and overall survival (c) ) prediction chart.

Figure 9 shows the carcinoma in situ (a, b), paracancerous tissue and invasive cancer (c, d) of human breast cancer tissue labeled with IR-6B3S, the fluorescence pictures (a, c) and H&E picture (b) of the paracancerous tissue ,d).

Figure 10 is the hydrogen nuclear magnetic spectrum of the near-infrared cyanine fluorescent probe D1 of the present invention.

Figure 11 is the hydrogen nuclear magnetic spectrum of the near-infrared cyanine fluorescent probe D2 of the present invention.

Figure 12 is the hydrogen nuclear magnetic spectrum of the near-infrared cyanine fluorescent probe D3 of the present invention.

Figure 13 is the hydrogen nuclear magnetic spectrum of the near-infrared cyanine fluorescent probe D4 of the present invention.

Figure 14 is the hydrogen nuclear magnetic spectrum of the near-infrared cyanine fluorescent probe D5 of the present invention.

Figure 15 is the hydrogen nuclear magnetic spectrum of the near-infrared cyanine fluorescent probe D6 of the present invention.

Figure 16 is the hydrogen nuclear magnetic spectrum of the near-infrared cyanine fluorescent probe D7 of the present invention.

Figure 17 is the hydrogen nuclear magnetic spectrum of the near-infrared cyanine fluorescent probe D8 of the present invention.

Figure 18 is the hydrogen nuclear magnetic spectrum of the near-infrared cyanine fluorescent probe D9 of the present invention.

Figure 19 is the hydrogen nuclear magnetic spectrum of the near-infrared cyanine fluorescent probe D10 of the present invention.

Figure 20 is the hydrogen nuclear magnetic spectrum of the near-infrared cyanine fluorescent probe D11 of the present invention.

Figure 21 is the hydrogen nuclear magnetic spectrum of the near-infrared cyanine fluorescent probe D12 of the present invention.

Figure 22 is the hydrogen nuclear magnetic spectrum of the near-infrared cyanine fluorescent probe D13 of the present invention.

Figure 23 is the hydrogen nuclear magnetic spectrum of the near-infrared cyanine fluorescent probe D14 of the present invention.

Figure 24 is the hydrogen nuclear magnetic spectrum of the near-infrared cyanine fluorescent probe D15 of the present invention.

Figure 25 is the hydrogen nuclear magnetic spectrum of the near-infrared cyanine fluorescent probe D16 of the present invention.

Figure 26 is the hydrogen nuclear magnetic spectrum of the near-infrared cyanine fluorescent probe D17 of the present invention.

Figure 27 is the hydrogen nuclear magnetic spectrum of the near-infrared cyanine fluorescent probe D18 of the present invention.

Figure 28 is the hydrogen nuclear magnetic spectrum of the near-infrared cyanine fluorescent probe D19 of the present invention.

Figure 29 is the hydrogen nuclear magnetic spectrum of the near-infrared cyanine fluorescent probe D20 of the present invention.

Figure 30 is the hydrogen nuclear magnetic spectrum of the near-infrared cyanine fluorescent probe D21 of the present invention.

Figure 31 is the hydrogen nuclear magnetic spectrum of the near-infrared cyanine fluorescent probe D22 of the present invention.

Figure 32 is the hydrogen nuclear magnetic spectrum of the near-infrared cyanine fluorescent probe D23 of the present invention.

Figure 33 is the hydrogen nuclear magnetic spectrum of the near-infrared cyanine fluorescent probe DD1 of the present invention.

Detailed ways

In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described here are only used to explain the present invention and are not intended to limit the present invention.

The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

One embodiment of the present invention provides a rapid pathological detection method of tumor tissue based on a near-infrared cyanine fluorescent probe, which has the following structural formula I:

In the general formula I, X ^- is selected from Cl ^- , Br ^- , I ^- , PF ₆ ^- , BF ₄ ^- , ClO ₄ ^- , CH ₃ COO ^- , CF ₃ COO ^- or OTs ^- ; Oxygen or sulfur; Z is selected from C _1-8 alkyl or aryl groups, the methylene bridge is modified with alkyl, five-membered ring, six-membered ring, nitrogen-containing six-membered ring, oxygen-containing six-membered ring, sulfur-containing Six-membered ring, benzene ring or seven-membered ring; heterocyclic salt is selected from indole, benzindole, benzoxazole, benzothiazole, benzoselenazole, quinoline, acridine, benzothiopyranium, Benzopyranium or silyl rhodamine; n and m are any integers greater than 0 (can be the same or different); R ₁ and R ₂ (can be the same or different) are each independently selected from sulfonic acid, arylsulfonic acid, Carboxylic acid, sulfonate, carboxylate, alkyl, aryl, alkenyl, alkynyl, nitro, cyano, azide, amine, hydroxyl, halogen, alkoxy, ester, amide, poly Ethylene glycol chain, piperidine, morpholine, pyrrole, imidazole, pyridine salt or quaternary ammonium salt; p is an integer from 0 to 6; R is selected from alkyl chain, halogen, activated ester, maleimide, sulfoxide , sulfone, sulfonate, thioether, acrylamide, acrylate, vinyl sulfonamide, nitrogen or benzene ring.

In the embodiment of the present invention, further,

Wherein, R ₃ is selected from amine group, carboxyl group, sulfonic acid group, cyano group, nitro group, alkyl group or halogen. R ₄ and R ₅ are each independently selected from a C _1-8 alkyl chain, alkyl carboxylic acid, alkyl sulfonic acid, alkyl azide or alkyl halohydrocarbon. R ₆ and R ₇ are selected from C _1-8 alkyl, aryl, amine or alkoxy. R ₈ and R ₉ are each independently selected from a C _1-8 alkyl chain, alkyl carboxylic acid, alkyl sulfonic acid, alkyl azide or alkyl halohydrocarbon. R ₁₀ is selected from C _1-8 alkyl chain, alkoxy group or polyethylene glycol chain. R ₁₁ and R ₁₂ are each independently selected from a C _1-5 alkyl chain, alkyl amine, cyano group or nitro group. R ₁₃ and R ₁₄ are each independently selected from a C _1-8 alkyl chain, alkyl carboxylic acid, alkyl sulfonic acid, alkyl azide or alkyl halohydrocarbon. R ₁₅ is selected from a C _1-6 alkyl chain, alkoxy group or aryl group.

The preparation method of cyanine fluorescent probe, the synthesis route is as follows:

In the above preparation method, a condensing agent C and a heterocyclic salt H with an alkyl side chain are used to construct a near-infrared cyanine fluorescent probe D through Knoevenagel condensation reaction. In this preparation method, the alkyl side chains R ₁ and R ₂ may be the same or different, and n and m may be the same or different. Through different substituent combinations, we can prepare symmetrical or asymmetric cyanine fluorescent probes.

In the preferred technical solution, any one or a combination of methanol, ethanol, isopropyl alcohol, n-butanol, acetonitrile, toluene, xylene, acetic acid, acetic anhydride, and n-hexane is selected as the reaction solvent; triethyl is selected. Use any one or a combination of amine, diisopropylethylamine, tetrahydropyrrole, piperidine, ammonium acetate, potassium acetate, sodium acetate, zinc acetate as a weak base catalyst, select 25 ℃ to a single solvent or The reflux temperature of the mixed solvent is the reaction temperature. Including but not limited to combinations of the above.

As a preferred embodiment of the present invention, the near-infrared cyanine fluorescent probe binds to protein molecules through covalent/non-covalent forces, and the labeled protein molecules are tumors or normal tissues.

In the embodiment of the present invention, preferably, as shown in Figure 1, after elution with DMSO, some probes with non-covalent/weak binding effects are eluted again. Since DMSO can elute probes with weak binding effects, by comparing the effects of PBST and DMSO elution methods on fluorescence intensity, probes with stronger binding effects on proteins in tissues can be screened out. As can be seen from Figure 1, some probes still have strong labeling ability for tissues after DMSO elution.

As a preferred embodiment of the present invention, it includes the following steps (0 min means that this step can be omitted, for example, abc is omitted, and only steps d and e are used for detection):

a. Immerse the tumor tissue sections from surgical resection or biopsy into paraformaldehyde fixative for 0 to 30 minutes; the excess fixative is eluted with phosphate-Tween buffer saline (PBST) for 0 to 30 minutes. ;

b. Immerse the tumor tissue sections obtained in step a into Triton X-100 surfactant solution for membrane rupture treatment. The mass concentration of Triton X-100 is 0.1%~30%, and the processing time is 0~30 min; excess Triton X-100 solution is eluted by PBST, and the elution time is 0~30 minutes;

c. Immerse the tumor tissue sections obtained in step b into H ₂ O ₂ solution. The mass concentration of H ₂ O ₂ is 0.1%~30%, and the processing time is 0~60 min; the excess hydrogen peroxide solution is eluted with PBST , the elution time is 0~30 min;

d. Immerse the tumor tissue sections obtained in step c into the near-infrared cyanine fluorescent probe solution and incubate it. The incubation temperature is 4~70°C. The molar concentration of the cyanine fluorescent probe solution is not less than 1 nM, and the incubation time is not less than 1 nM. 1 min; excess cyanine fluorescent probe solution is first eluted with DMSO solution, the mass concentration of DMSO is 1~100%, and the elution time is 0~30 min; the remaining DMSO is then eluted with PBST, and the elution time is 0~30 minutes;

e. Place the tumor tissue slices obtained in step d on a scanning imager or fluorescence camera with a near-infrared excitation light source for scanning to generate a fluorescent pattern of the tumor tissue; analyze the fluorescent pattern obtained by scanning to determine the tumor outline (lesion area), Tumor staging and typing, predicting malignancy and survival, and morphological analysis to assist pathological monitoring.

In embodiments of the present invention, preferably, this method can assist in the survival prediction of clinical tumors and other diseases; can assist in the study of tumor staging and other disease mechanisms; and can be expanded to other biological detection for wide application. The stained sections obtained in step d and stored at low temperature or normal temperature (-80~25°C) can be labeled according to step c multiple times.

As shown in Figure 3, the cyanine fluorescent probe can label related proteins in the total protein of human breast cancer tumor tissue (T) and paracancerous tissue (P) lysate. The intensity of the fluorescence signal can significantly distinguish between tumor tissue and paracancerous tissue. . Specifically, the cyanine fluorescent probe solution and the tissue lysate were pre-reacted at a molar ratio of 1:1 for 2 hours at 37°C to obtain probe@protein complexes (such as IR-6B1@T, IR-6B1@P, etc.) , and then analyze the protein bands in the tissue labeled by the cyanine fluorescent probe through two-dimensional electrophoresis (picture a). It can be seen that a variety of proteins of different molecular weights in tumor tissue lysates can be labeled by cyanine fluorescent probes, while very few proteins in adjacent tissues can be labeled. Further comparative analysis of the total fluorescence intensity ratio of labeled proteins in the lysate of tumor tissue (T) and adjacent tissue (P) (picture b) indirectly proves the high specificity of the cyanine fluorescent probe for labeling tumor-related proteins in tumor tissue. ability.

As shown in Figure 4, the high fluorescence signal in some areas of the tumor tissue (T) can be clearly seen in Figure a, and there is an obvious distribution of cancer cells in the tissue corresponding to H&E in Figure B. However, the cancer-free paracancerous tissue (P) has almost no fluorescence and shows normal cell characteristics under H&E staining. The sharp fluorescence contrast between tumor tissue and adjacent tissue proves the high specificity of cyanine fluorescent probes for labeling tumor-related proteins and the ability to distinguish tumor edges.

As shown in Figure 5, by comparing the labeling and distinguishing abilities of cyanine fluorescent probes with different structures on tumor tissues, it is proved that these probes can clearly distinguish tumor tissues and adjacent tissues, and have 1-20 times the average fluorescence. The signal difference indicates that these probes can be selectively used for qualitative and quantitative tumor detection in biopsy tissues. The number of samples tested by each probe was greater than 12, and each sample was tested at least three times.

As shown in Figure 6, the cyanine fluorescent probe can be applied to the detection of human thyroid cancer tissue, and can accurately distinguish thyroid cancer tissue and para-cancerous tissue through fluorescence. Specifically, the selected human thyroid sample numbers are 948609, 948745, 949790, 945532, 95132, and 948135. By comparing the tumor labeling abilities of IR-6B3, which has a strong binding effect, and IR-6B3S, which has a weak binding effect, on human thyroid cancer, it can be seen that both can completely and correctly distinguish tumor tissues (Figures a and c). And it has passed the dual certification of H&E pathology slide proofreading and professional pathologists. Comparing the fluorescence intensity of the tumor area and normal area labeled by the two probes, it can be seen that the average fluorescence signal intensity of tumor tissue is significantly higher than the fluorescence signal intensity of normal tissue (Figures b and d), proving that IR-6B3 and IR- 6B3S can both qualitatively and quantitatively distinguish tumor tissues, which also proves that this type of cyanine fluorescent probe can be applied to tumor detection.

As shown in Figure 7, the cyanine fluorescent probe can be applied to fluorescence detection of human lung cancer tissue, and can accurately distinguish lung cancer tissue and its adjacent tissues through fluorescence differences. Specifically, the selected human lung cancer sample numbers are 956779A, 957515A, and 957511. By adding tumor types, we found that the IR-6B3S probe can also detect lung cancer. The detection results were confirmed by pathologists, indicating that this type of cyanine fluorescent probe has a variety of tumor detection capabilities.

As shown in Figure 8, by analyzing the changes in fluorescence signals of tumor tissue on tissue sections, changes in the expression of lesion sites and lesion microenvironment-related proteins at different stages of disease development can be revealed, either directly through fluorescence intensity or through tumor tissue (T) and cancer. The signal ratio of the adjacent tissue (P) indirectly further predicts tumor stage and patient survival. Specifically, by analyzing tumor tissue samples of 20 breast cancer patients from different patients (sample numbers are: R193, R377, R498, R366, R391, R403, R418, R445, R488, R491, R590, R713, R069, R249, R401, R429, R510, R531, R091, R223) The results of fluorescence staining of tissue sections can be summarized as follows: as the tumor stage worsens, the expression of tumor-related markers increases, and even causes changes in the tumor microenvironment, leading to cancer. Certain tumor markers of related infiltrating cells in the adjacent tissues increase, thereby increasing the labeling of relevant tumor markers by cyanine fluorescent probes in the adjacent tissues, resulting in a decrease in the T/P ratio, which in turn changes the fluorescence pattern and T/P signal. The two aspects of the ratio assist in analyzing tumor staging (Figure a); similarly, a decrease in the T/P ratio indicates further progression of the disease and reduces five-year survival (Figure b); as the tumor further worsens, the increase in tumor-related markers increases. Expression will also increase the amount of cyanine fluorescent probe bound in tumor tissue, and the increased fluorescence signal also indirectly reflects the impact of disease progression on overall survival. The intensity of the fluorescence signal in tumor tissue is negatively correlated with overall survival (Figure c).

As shown in Figure 9, by observing the distribution characteristics and pattern details of the fluorescence pattern on the tissue section, the types of cancer in situ and invasive cancer can be distinguished. Specifically, the selected human breast cancer sample numbers are R590 and R438. Compared with the more diffuse and scattered invasive carcinoma (R590/T), carcinoma in situ (R438/T) has the characteristics of breast lobular distribution as a whole. By comparing the adjacent tissue, it can be seen that because the adjacent tissue of R590/P contains fewer nuclei and cells, the labeled fluorescence is lower, resulting in very obvious differences between the tumor tissue (R590/T) and the adjacent tissue (R590/ P) differentiation, and for the para-cancerous tissue (R438/P) with a large distribution of breast lobules, the probe can also accurately mark the lobular tissue with dense nuclear distribution, and the normal tissue in the tissue can be clearly distinguished through high-resolution fluorescence analysis. The cells were thus able to differentiate between carcinoma in situ cancer tissue (R438/T) and its paracancerous tissue (R438/P) with similar lobular characteristics. This shows that it is feasible for this type of probe to be used in pathological detection.

As a preferred embodiment of the present invention, one or more near-infrared cyanine fluorescent probes can be used simultaneously for multi-index and multi-color labeling.

As a preferred embodiment of the present invention, for the tumor tissue section in step a), the detection method is also applicable to cell smears, tissue fluid smears, frozen/paraffin tissue sections, and the base sheet of the smear or tissue section is Positive ion-resistant slides, smears or tissue sections contain near-infrared cyanine fluorescent probes.

In the embodiment of the present invention, preferably, each slide contains a tissue section or a liquid/solid-liquid mixed smear, and the tissue section or smear contains a near-infrared cyanine fluorescent probe.

As a preferred embodiment of the present invention, the thickness of the tissue section is not less than 1 μm.

As a preferred embodiment of the present invention, the glass slide is glass, silicon or silicon dioxide.

Example 1. Preparation of heterocyclic salt H

The reaction equation for synthesizing heterocyclic salt H is shown in the following formula (II):

The structure of the cyanine fluorescent probe heterocyclic salt H7 in this embodiment is selected from indole, and R ₁ in the corresponding heterocyclic salt is methyl, n is 15, R ₃ is hydrogen, and X is iodine. The specific steps for synthesizing heterocyclic salt H7 are as follows: Compound A and Compound B are dissolved in acetonitrile at a molar ratio of 1:5, and heated at 70°C for 24 hours under an argon atmosphere. The mixture was cooled to room temperature, and a large amount of brown solid precipitated. The solvent was removed by filtration under reduced pressure. The filter cake was washed with ethyl acetate and dried under vacuum to obtain a white solid heterocyclic salt H7 (1.42 g, 55.67%). The synthesis methods of other heterocyclic salts H are similar.

The structural formulas and characterization data of heterocyclic salts H1~H16 are as follows:

Heterocyclic salt H1: pink solid H1 (2.08 g, 98.48%). ¹ H NMR (400 MHz, DMSO- d ₆ ) δ 7.94– 7.88 (m, 1H), 7.85 – 7.79 (m, 1H), 7.67 – 7.58 (m, 2H), 3.97 (s, 3H), 2.77(s , 3H), 1.53 (s, 6H).

Heterocyclic salt H2: reddish brown solid heterocyclic salt H2 (3.89 g, 98.48%). ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.73 – 7.70 (m, 1H), 7.62 – 7.57 (m, 3H), 4.70 (t, J = 7.6 Hz, 2H), 3.15 (s,3H), 2.05 ( m, 2H), 1.68 (s, 6H), 1.11 (t, J = 7.6 Hz, 3H).

Heterocyclic salt H3: Purple solid heterocyclic salt H3 (1.99 g, 70.3%). ¹ H NMR (400 MHz, DMSO- d ₆ ) δ 8.02 – 7.93 (m, 1H), 7.89 – 7.80 (m, 1H), 7.68 – 7.58 (m, 2H), 4.45 (d, J =8.0 Hz, 2H ), 2.84 (s, 3H), 1.90 – 1.78 (m, 2H), 1.54 (s, 6H), 1.46 – 1.34 (m,4H), 0.89 (t, J = 7.0 Hz, 3H).

Heterocyclic salt H4: reddish brown solid heterocyclic salt H4 (3.26 g, 84.6%). ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.66 – 7.62 (m, 1H), 7.61 – 7.56 (m, 3H), 4.69 (t, J = 8.0 Hz, 2H), 3.14 (s,3H), 1.99 – 1.87 (m, 2H), 1.67 (s, 6H), 1.52 – 1.42 (m, 2H), 1.42 – 1.34 (m,2H), 1.32 – 1.17 (m, 4H), 0.88 (t, J = 6.8 Hz, 3H).

Heterocyclic salt H5: reddish brown solid heterocyclic salt H5 (2.14 g, 51.66%). ¹ H NMR (400 MHz, DMSO- d ₆ ) δ 8.03 – 7.94 (m, 1H), 7.90 – 7.81 (m, 1H), 7.68 – 7.58 (m, 2H), 4.45 (t, J =7.8 Hz, 2H ), 2.85 (s, 3H), 1.87 – 1.77 (m, 2H), 1.54 (s, 6H), 1.46 – 1.37 (m,2H), 1.36 – 1.19 (m, 10H), 0.85 (d, J = 6.8 Hz, 3H).

Heterocyclic salt H6: white solid heterocyclic salt H6 (0.93 g, 60.10%). ¹ H NMR (400 MHz, DMSO- d ₆ ) δ 8.02 – 7.93 (m, 1H), 7.89 – 7.80 (m, 1H), 7.68 – 7.58 (m, 2H), 4.45 (t, J =7.8 Hz, 2H ), 2.84 (s, 3H), 1.89 – 1.77 (m, 2H), 1.54 (s, 6H), 1.44 – 1.36 (m,2H), 1.36 – 1.17 (m, 16H), 0.86 (d, J = 6.8 Hz, 3H).

Heterocyclic salt H7: White solid heterocyclic salt H7 (1.42 g, 55.67%). ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.73 – 7.49 (m, 4H), 4.69 (t, J = 7.8 Hz, 2H), 3.13 (s, 3H), 1.98 – 1.88 (m,2H), 1.67 ( s, 6H), 1.51 – 1.41 (m, 2H), 1.41 -1.34 (m, 2H), 1.33 – 1.18 (m, 22H), 0.88 (t, J = 6.7 Hz, 3H).

Heterocyclic salt H8: reddish brown solid heterocyclic salt H8 (2.31g, 64.20%). ¹ H NMR (400 MHz, DMSO- d ₆ ) δ 8.03 – 7.94 (m, 1H), 7.88 – 7.80 (m, 1H), 7.62 (dd, J = 6.4, 3.2 Hz, 2H), 4.65 (t, J = 7.0 Hz, 2H), 2.98 (t, J = 7.0 Hz, 2H), 2.86 (s, 3H), 1.53 (s, 6H).

Heterocyclic salt H9: Pink solid heterocyclic salt H9 (1.56 g, 43.90%). ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.74 (d, J = 7.6 Hz, 1H), 7.65 – 7.53 (m, 3H), 4.73 (t, J = 7.9 Hz, 2H), 3.15(s, 3H) , 2.46 (t, J = 6.9 Hz, 2H), 2.09 – 1.93 (m, 2H), 1.74 (p, J = 7.1 Hz, 2H), 1.64 (s, 6H), 1.64 – 1.52 (m, 2H).

Heterocyclic salt H10: purple-red solid heterocyclic salt H10 (1.25 g, 55.44%). ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.96 (dd, J = 6.8, 1.6 Hz, 1H), 7.62 – 7.44 (m, 3H), 4.99 (t, J = 8.1 Hz, 2H), 2.42 – 2.33 ( m, 2H), 1.60 (s, 6H).

Heterocyclic salt H11: Purple solid heterocyclic salt H11 (0.55 g, 35.81%). ¹ H NMR (400 MHz, D ₂ O) δ 7.74 – 7.68 (m, 1H), 7.67 – 7.62 (m, 1H), 7.60 – 7.50 (m, 2H), 4.44 (t, J =7.7 Hz, 2H) , 2.89 (t, J = 7.5 Hz, 2H), 2.10 – 1.96 (m, 2H), 1.87 – 1.67 (m,2H), 1.84 (s, 6H).

Heterocyclic salt H12: Blue solid heterocyclic salt H12 (1.45 g, 45.44%). ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.11 (dd, J = 7.6 Hz, 2H), 8.06 (d, J = 7.6 Hz, 1H), 7.83 (d, J = 8.9 Hz, 1H), 7.75 (t , J = 7.7 Hz, 1H), 7.67 (t, J = 8.2 Hz, 1H), 4.81 (t, J = 7.5 Hz, 2H), 3.23 (s, 3H), 2.15 – 2.04 (m, 2H), 1.89 (s, 6H), 1.12 (t, J = 7.4 Hz, 3H).

Heterocyclic salt H13: black solid heterocyclic salt H13 (0.90 g, 54.28%). ¹ H NMR (400 MHz, D ₂ O) δ 8.08 (d, J = 8.5 Hz, 1H), 7.97 (d, J = 9.0 Hz, 1H), 7.91 (d, J = 8.3 Hz, 1H), 7.72 ( d, J = 9.0 Hz, 1H), 7.59 (t, J = 7.6 Hz, 1H), 7.51 (t, J = 7.6 Hz, 1H), 4.40 (m, 2H), 2.83 (t, J = 7.5 Hz, 2H), 2.11 – 1.89 (m, 2H), 1.86 – 1.69 (m,2H), 1.57 (s, 6H).

Heterocyclic salt H14: Yellow solid heterocyclic salt H14 (0.5 g, 35.2%). ¹ H NMR (400 MHz, DMSO- d ₆ ) δ 9.37 (d, J = 6.0 Hz, 1H), 8.56 (d, J = 8.8, 1H), 8.51 (d, J = 8.9 Hz, 1H), 8.29 ( t, J = 7.2, 1H), 8.13 – 8.04 (m, 2H), 4.59 (s, 3H), 3.02 (s, 3H).

Heterocyclic salt H15: Orange-red solid heterocyclic salt H15 (1.35 g, 97%). ¹ H NMR (400 MHz, DMSO- d ₆ ) δ 8.99 (d, J = 6.8 Hz, 1H), 8.81 (d, J = 8.0 Hz, 1H), 8.55 (d, J = 6.8 Hz, 1H), 8.46 (d, J = 8.4 Hz, 1H), 8.18 (t, J = 7.6 Hz, 1H), 8.02 (t, J = 7.8 Hz, 1H), 4.72 (q, J = 7.2 Hz, 2H), 3.24 (s , 3H), 1.55 (t, J = 7.3 Hz, 3H).

Heterocyclic salt H16: Yellow solid heterocyclic salt H16 (0.86 g, 60%). ¹ H NMR (400 MHz, CD ₃ OD) δ 8.85 (d, J = 7.3 Hz, 1H), 8.74 (d, J = 8.1 Hz, 1H), 8.43 – 8.37 (m, 2H), 8.20 – 8.12 (m , 1H), 8.00 – 7.98 (m, 1H), 4.70 (t, J = 7.7 Hz, 2H), 2.32 (t, J = 7.2 Hz, 2H), 2.12 – 2.05 (m, 2H), 1.82 – 1.60 ( m,2H) , 1.65 – 1.54 (m,2H).

Example 2. Preparation of symmetrical near-infrared cyanine fluorescent probe D

The reaction equation for synthesizing near-infrared cyanine fluorescent probe D is shown in the following formula (III):

The structure of the near-infrared cyanine fluorescent probe D1 in this embodiment is selected from indole. R ₁ in the corresponding probe D1 is methyl, n is 4, R ₃ is hydrogen, X is iodine, R is chlorine, and p is 0. , Y is carbon, Z is hydrogen. The specific steps for synthesizing the symmetrical near-infrared cyanine fluorescent probe D1 are as follows: the heterocyclic salt H and compound C are dissolved in an ethanol solvent at a molar ratio of 2:1, and then 3 times the equivalent of sodium acetate relative to compound C is added as a weak base. Catalyst, the mixed solution was heated at 70°C in an argon atmosphere for 24 h. After TLC detected that the reaction was complete, the solvent was removed under reduced pressure, and the residue was purified by column chromatography (dichloromethane: methanol = 50: 1, v/v) to obtain green cyanine fluorescent probe D1 (33.24 mg, 43.33%). The preparation method of near-infrared cyanine fluorescent probes D2~D22 is similar. The structural formula and characterization data of near-infrared cyanine fluorescent probes D1~D22 are as follows:

Green solid D1 (33.24 mg, 43.33%). ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.34 (d, J =14.1 Hz, 2H), 7.44 – 7.36 (m, 4H), 7.28 – 7.22 (m, 2H), 7.16 (d, J = 7.9 Hz, 2H), 6.24 (d, J = 14.1 Hz, 2H), 4.20 (t, J = 7.5 Hz, 4H), 2.75 (t, J = 6.2Hz, 4H), 2.04 -1.96 (m, 2H), 1.90 – 1.81 (m, 4H), 1.73 (s, 12H), 1.497 – 1.36 (m, 8H), 0.93 (t, J = 6.9 Hz, 6H), as shown in Figure 10. HRMS (ESI-TOF) m/z : calcd. for[M] ⁺ C ₄₀ H ₅₂ ClN ₂ = 595.3814; found 595.3806.

Green solid D2 (23.14mg, 29.69%). ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.34 (d, J =14.1 Hz, 2H), 7.44 – 7.36 (m, 3H), 7.31 – 7.21 (m, 3H), 7.16 (d, J = 8.0 Hz, 2H), 6.24 (d, J = 14.1 Hz, 2H), 4.20 (t, J = 7.4 Hz, 4H), 2.75 (t, J = 6.2Hz, 4H), 2.03 – 1.96 (m, 2H), 1.91 – 1.79 (m, 4H), 1.73 (s, 12H), 1.52 – 1.42 (m, 4H), 1.42 – 1.34 (m, 4H), 1.31 – 1.25 (m, 8H), 0.88 (t, J = 7.2 Hz, 6H), as shown in Figure 11. HRMS (ESI-TOF) m/z: calcd. for [M] ⁺ C ₄₄ H ₆₀ ClN ₂ = 651.4440; found651.4416.

Green solid D3 (25.41mg, 30.42%). ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.34 (d, J =14.1 Hz, 2H), 7.44 – 7.36 (m, 4H), 7.29 – 7.21 (m, 2H), 7.16 (d, J = 8.0 Hz, 2H), 6.24 (d, J = 14.1 Hz, 2H), 4.20 (t, J = 7.4 Hz, 4H), 2.75 (t, J = 6.3Hz, 4H), 2.03 – 1.96 (m, 2H), 1.91 – 1.79 (m, 4H), 1.73 (s, 12H), 1.52 – 1.41 (m, 4H), 1.41 – 1.32 (m, 4H), 1.31 – 1.24 (m, 16H), 0.87 (t, J = 6.8 Hz, 6H), as shown in Figure 12. HRMS (ESI-TOF) m/z: calcd. for [M] ⁺ C ₄₈ H ₆₈ ClN ₂ = 707.5066; found707.5065.

Green solid D4 (29.88mg, 32.49%). ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.34 (d, J =14.1 Hz, 2H), 7.44 – 7.36 (m, 4H), 7.30 – 7.21 (m, 2H), 7.16 (d, J = 7.7 Hz, 2H), 6.24 (d, J = 14.1 Hz, 2H), 4.20 (t, J = 7.4 Hz, 4H), 2.75 (t, J = 6.2Hz, 4H), 2.03 – 1.96 (m, 2H), 1.91 – 1.79 (m, 4H), 1.73 (s, 12H), 1.51 – 1.41 (m, 4H), 1.41 – 1.33 (m, 4H), 1.32 – 1.19 (m, 28H), 0.91 – 0.83 (m, 6H), As shown in Figure 13. HRMS (ESI-TOF) m/z: calcd. for [M] ⁺ C ₅₄ H ₈₀ ClN ₂ = 791.6005; found791.6014.

Green solid D5 (32.44 mg, 31.44%). ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.34 (d, J =14.1 Hz, 2H), 7.44 – 7.36 (m, 4H), 7.26 (d, J = 7.2 Hz, 2H), 7.16 (d, J = 7.7Hz, 2H), 6.24 (d, J = 14.1 Hz, 2H), 4.20 (t, J = 7.4 Hz, 4H), 2.75 (t, J =6.2 Hz, 4H), 2.03 – 1.96 (m, 2H) , 1.91 – 1.80 (m, 4H), 1.73 (s, 12H), 1.52 –1.41 (m, 4H), 1.40 – 1.33 (m, 4H), 1.32 – 1.20 (m, 44H), 0.87 (d, J = 6.8 Hz, 6H), as shown in Figure 14. HRMS (ESI-TOF) m/z: calcd. for [M] ⁺ C ₆₂ H ₉₆ ClN ₂ = 903.7257; found 903.7238.

Blue solid D6 (33.24 mg, 43.33%). ¹ H NMR (400 MHz, DMSO- d ₆ ) δ 8.38 (d, J =14.0 Hz, 2H), 8.33 (d, J = 8.8 Hz, 2H), 8.11 (dd, J = 11.7, 8.5 Hz, 4H) , 7.82(d, J = 8.8 Hz, 2H), 7.69 (t, J = 7.7 Hz, 2H), 7.55 (t, J = 7.6 Hz, 2H), 6.41(d, J = 14.3 Hz, 2H), 4.35 (t, J = 7.6 Hz, 4H), 2.78 (t, J = 8.4 Hz, 4H), 1.98 (s, 12H), 1.96 – 1.90 (m, 2H), 1.86 (q, J = 7.6 Hz, 4H) , 1.02 (t, J =7.4 Hz, 6H), as shown in Figure 15. HRMS (ESI-TOF) m/z: calcd. for [M] ⁺ C ₄₄ H ₄₈ ClN ₂ =639.3501; found 639.3501.

Green solid D7 (17.86 mg, 24.56%). ¹ H NMR (400 MHz, DMSO- d ₆ ) δ 8.25 (d, J =14.1 Hz, 2H), 7.63 (d, J = 7.5 Hz, 2H), 7.46 – 7.41 (m, 4H), 7.29 (t, J = 7.8Hz, 2H), 6.43 (d, J = 14.1 Hz, 2H), 4.43 (t, J = 7.1 Hz, 4H), 2.78 – 2.62 (m,8H), 1.87 – 1.84 (m, 2H), 1.67 (s, 6H), as shown in Figure 16. HRMS (ESI-TOF) m/z:calcd. for [M + H] ⁺ C ₃₆ H ₄₀ ClN ₂ O ₄ =599.2671; found 599.2662.

Red solid D8 (143.00mg, 81.10%). ¹ H NMR (400 MHz, Chloroform- d ) δ 8.34(d, J = 14.0 Hz, 2H), 7.40 (dd, J = 8.0 Hz, 4H), 7.24 (d, J = 7.2 Hz, 2H),7.17 ( d, J = 8.0 Hz, 2H), 6.22 (d, J = 14.1 Hz, 2H), 4.14 (t, J = 7.5 Hz, 4H), 2.74 (t, J = 6.3 Hz, 4H), 2.52 (t, J = 7.2 Hz, 4H), 2.04 – 2.00 (m, 2H), 1.93 – 1.83 (m, 4H), 1.83 – 1.74 (m, 4H), 1.72 (s, 12H), 1.63 – 1.53 (m, 4H) , as shown in Figure 17. HRMS (ESI-TOF) m/z: calcd. for [M + H] ⁺ C ₄₂ H ₅₂ ClN ₂ O ₄ = 683.3610; found 683.3570.

Green solid D9 (18.67 mg, 25.88%). ¹ H NMR (400 MHz, DMSO- d ₆ ) δ 8.26 (d, J =14.1 Hz, 2H), 7.62 (d, J = 7.5 Hz, 2H), 7.54 (d, J = 8.0 Hz, 2H), 7.42 (t, J = 7.8 Hz, 2H), 7.27 (t, J = 7.5 Hz, 2H), 6.53 (d, J = 14.1 Hz, 2H), 4.38 (t, J = 7.6 Hz, 4H), 2.75 (t , J = 6.2 Hz, 4H), 2.57 (t, J = 6.7 Hz, 4H), 2.03 (p, J = 7.4 Hz, 4H), 1.87 – 1.80 (m, 2H), 1.68 (s, 12H), as Shown in Figure 18. HRMS (ESI-TOF) m/z: calcd. for [M + 2H] ⁺ C ₃₆ H ₄₄ ClN ₂ O ₆ S ₂ = 699.2324; found 699.2326.

Green solid D10 (127.48 mg, 38.90%). ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.35 (d, J =14.0 Hz, 2H), 7.52 – 7.34 (m, 4H), 7.31 – 7.23 (m, 2H), 7.20 (d, J = 7.9 Hz, 2H), 6.26 (d, J = 14.0 Hz, 2H), 3.77 (s, 6H), 2.78 (t, J = 6.2 Hz, 4H), 2.03– 1.92 (m, 2H), 1.74 (s, 12H), As shown in Figure 19. HRMS (ESI-TOF) m/z: calcd. for [M] ⁺ C ₃₂ H ₃₆ BrN ₂ = 527.2056; found 527.2032.

Green solid D11 (37.00 mg, 51.99%). ¹ H NMR (400 MHz, DMSO- d ₆ ) δ 8.28 (d, J =14.1 Hz, 2H), 7.65 (d, J = 7.3 Hz, 2H), 7.50 – 7.40 (m, 4H), 7.30 (t, J = 7.3Hz, 2H), 6.36 (d, J = 14.2 Hz, 2H), 4.21 (t, J = 7.6 Hz, 4H), 2.73 (t, J =5.9 Hz, 4H), 1.90 – 1.83 (m, 2H), 1.78 (q, J = 7.3 Hz, 4H), 1.69 (s, 12H), 0.97 (t, J = 7.4 Hz, 6H), as shown in Figure 20. HRMS (ESI-TOF) m/z: calcd. for [M] ⁺ C ₃₆ H ₄₄ BrN ₂ = 583.2682; found 583.2667.

Red solid D12 (19.00mg, 21.92%). ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.35 (d, J =14.0 Hz, 2H), 7.51 – 7.36 (m, 2H), 7.29 – 7.23 (m, 2H), 7.17 (d, J = 7.7 Hz, 2H), 6.25 (d, J = 14.0 Hz, 2H), 4.21 (t, J = 7.4 Hz, 4H), 2.77 (t, J = 6.2Hz, 4H), 2.04 – 1.96 (m, 2H), 1.92 – 1.80 (m, 4H), 1.74 (s, 12H), 1.50 – 1.35 (m, 8H), 0.93 (t, J = 6.8 Hz, 6H), as shown in Figure 21. HRMS (ESI-TOF) m/z : calcd. for[M] ⁺ C ₄₀ H ₅₂ BrN ₂ = 639.3308; found 639.3297.

Green solid D13 (147.68 mg, 17.93%). ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.35 (d, J =14.0 Hz, 2H), 7.43 – 7.37 (m, 4H), 7.30 – 7.23 (m, 2H), 7.19 – 7.15 (m, 2H), 6.25 (d, J = 14.1 Hz, 2H), 4.20 (t, J = 7.5 Hz, 4H), 2.76 (t, J = 6.2 Hz, 4H), 2.05 – 1.94 (m, 2H), 1.93 – 1.81 (m , 4H), 1.74 (s, 12H), 1.51 – 1.42 (m,4H), 1.42 – 1.35 (m, 4H), 1.35 – 1.22 (m, 8H), 0.88 (t, J = 7.8 Hz, 6H), As shown in Figure 22. HRMS (ESI-TOF) m/z: calcd. for [M] ⁺ C ₄₄ H ₆₀ BrN ₂ = 695.3934; found695.3941.

Green solid D14 (55.00 mg, 71.26%). ¹ H NMR (400 MHz, DMSO- d ₆ ) δ 8.27 (d, J =14.0 Hz, 2H), 7.63 (d, J = 7.4 Hz, 2H), 7.50 (d, J = 8.0 Hz, 2H), 7.43 (t, J = 7.6 Hz, 2H), 7.28 (t, J = 7.4 Hz, 2H), 6.39 (d, J = 14.1 Hz, 2H), 4.22 (t, J = 7.4 Hz, 4H), 2.75 (t , J = 6.2 Hz, 4H), 1.82 (d, J = 6.5 Hz, 6H), 1.79 –1.70 (m, 4H), 1.69 (s, 12H), as shown in Figure 23. HRMS (ESI-TOF) m /z: calcd. forC ₃₈ H ₄₈ BrN ₂ O ₆ S ₂ = [M + H] ⁺ 771.2137; found 771.2114.

Green solid D15 (58.00 mg, 70%). ¹ H NMR (400 MHz, DMSO- d ₆ ) δ 8.39 (d, J =14.0 Hz, 2H), 8.31 (d, J = 8.5 Hz, 2H), 8.08 (t, J = 9.1 Hz, 4H), 7.82 (d, J = 9.0 Hz, 2H), 7.65 (d, J = 7.2 Hz, 2H), 7.52 (t, J = 7.6 Hz, 2H), 6.43 (d, J = 14.2 Hz, 2H), 4.36 (t , J = 6.9 Hz, 4H), 2.79 (t, J = 5.7 Hz, 4H), 2.55 (d, J = 7.2 Hz, 4H), 1.97 (s, 12H), 1.93 – 1.86 (m, 6H), 1.85 – 1.73 (m, 4H), as shown in Figure 24. HRMS (ESI-TOF) m/z: calcd. for [M + H] ⁺ C ₄₆ H ₅₂ BrN ₂ O ₆ S ₂ = 871.2445; found 871.2443.

Red solid D16 (15.00 mg, 20.25%). ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.88 (t, J =14.4 Hz, 2H), 7.79 – 7.70 (m, 1H), 7.41 – 7.36 (m, 4H), 7.251 – 7.214 (m,2H), 7.09 (d, J = 8.0 Hz, 2H), 6.75 (t, J = 12.4 Hz, 2H), 6.31 (d, J = 13.4Hz, 2H), 4.04 (t, J = 8.8 Hz, 4H), 1.90 ( q, J = 7.5 Hz, 4H), 1.74 (s, 12H), 1.10 (t, J = 7.4 Hz, 6H), as shown in Figure 25. HRMS (ESI-TOF) m/z: calcd. for [M ] ⁺ C ₃₃ H ₄₁ N ₂ = 465.3264; found 465.3249.

Green solid D17 (25.48 mg, 39.40%). ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.05 (d, J =13.7 Hz, 2H), 7.47 – 7.33 (m, 4H), 7.22 (t, J = 7.6 Hz, 2H), 7.12 (d, J = 8.0Hz, 2H), 6.15 (d, J = 13.7 Hz, 2H), 4.09 (t, J = 7.3 Hz, 4H), 2.58 (t, J =6.2 Hz, 4H), 2.42 (s, 3H), 1.98 – 1.83 (m, 6H), 1.72 (s, 12H), 1.07 (t, J =7.4 Hz, 6H), as shown in Figure 26. HRMS (ESI-TOF) m/z: calcd. For [M] ⁺ C ₃₇ H ₄₇ N ₂ =519.3734; found 519.3747.

Green solid D18 (15.56 mg, 19.18%). ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.31 (d, J =14.7 Hz, 2H), 7.48 – 7.38 (m, 4H), 7.36 – 7.30 (m, 2H), 7.27 – 7.23 (m, 2H), 6.76 (d, J = 14.7 Hz, 2H), 5.35 – 5.26 (m, 4H), 4.58 (t, J = 7.4 Hz, 4H), 3.87 (s, 6H), 2.04 – 1.91 (m, 4H), 1.74 (s, 12H), 1.15 (t, J = 7.4 Hz, 6H), as shown in Figure 27. HRMS (ESI-TOF) m/z: calcd. for [M] ²⁺ C ₃₇ H ₄₈ ClN ₃ = 284.6763 ; found284.6746.

Red solid D19 (14.21 mg, 21.69%). ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.82 (d, J =14.0 Hz, 2H), 7.42 – 7.34 (m, 4H), 7.28 – 7.19 (m, 2H), 7.12 (d, J = 7.9 Hz, 2H), 6.09 (d, J = 14.0 Hz, 2H), 4.09 (t, J = 7.3 Hz, 4H), 3.00 (s, 4H), 1.97– 1.84 (m, 4H), 1.71 (s, 12H), 1.06 (t, J = 7.4 Hz, 6H), as shown in Figure 28. HRMS(ESI-TOF) m/z: calcd. for [M] ⁺ C ₃₅ H ₄₂ ClN ₂ = 525.3031; found 525.3027.

D20 as black solid (98 mg, 74%). ¹ H NMR (400 MHz, DMSO) δ 8.54 (d, J = 13.6Hz, 2H), 8.24 (d, J = 7.2 Hz, 2H), 8.12 (d, J = 8.0 Hz, 2H), 7.87 (t, J = 7.7Hz, 2H), 7.65 – 7.40 (m, 6H), 6.72 (d, J = 14.0 Hz, 2H), 4.30 (q, J = 7.2 Hz,4H), 2.85 (m, 4H), 1.99 – 1.88 (m, 2H), 1.33 (t, J = 7.1 Hz, 6H), as shown in Figure 29. HRMS (ESI-TOF) m/z: calcd. For [M] ⁺ C ₃₆ H ₃₂ ClN ₂ = 527.2249 ; found 527.2245.

Green solid D21 (36 mg, 38.3%). ¹ H NMR (400 MHz, DMSO- d ₆ ) δ 8.49 (d, J = 8.6Hz, 2H), 8.10 (d, J = 7.2 Hz, 2H), 8.02 (d, J = 13.3 Hz, 2H), 7.90 – 7.79 (m,4H), 7.61 (t, J = 8.0 Hz, 2H), 7.38 (d, J = 7.1 Hz, 2H), 7.04 (d, J = 13.8Hz, 2H), 4.00 (s, 6H) , 2.87 – 2.76 (m, 4H), 1.92 – 1.84 (m, 2H), as shown in Figure 30. HRMS (ESI-TOF) m/z: calcd. For [M] ⁺ C ₃₀ H ₂₈ ClN ₂ = 451.1936 ; Found 451.1934.

Purple black solid D22 (33 mg, 31%) ¹ H NMR (500 MHz, CD ₂ Cl ₂ ) δ 7.49 – 7.08 (m,14H), 6.71 (d, J = 8.5 Hz, 4H), 3.84 (br. s , 12H), 3.55 (br. s, 8H), 2.88 (br. s, 4H), 2.03 (br. s, 2H), 1.36 – 1.23 (m, 12H), as shown in Figure 31. HRMS (ESI- TOF) m/z: calcd. For [M] ⁺ C ₅₀ H ₅₂ ClN ₂ O ₄ = 779.3610; found 779.3602.

Example 3. Preparation of asymmetric near-infrared cyanine fluorescent probe D

The reaction equation for synthesizing near-infrared cyanine fluorescent probe D is shown in the following formula (IV):

The structure of the asymmetric near-infrared cyanine fluorescent probe D23 in this embodiment is selected from benzindole. R ₁ in the corresponding probe D23 is a carboxyl group, n is 5, R ₂ is a methyl group, m is 1, and R ₃ is Hydrogen, X is iodine, R is chlorine, p is 0, Y is carbon, and Z is hydrogen. Specific steps for synthesizing asymmetric near-infrared cyanine fluorescent probe D23: Dissolve the heterocyclic salt H15 and compound C in toluene/n-butanol (5:5, v/v) at a molar ratio of 1:1, and the mixture is 80 °C under argon atmosphere for 24 h. After the heterocyclic salt H15 is consumed, an equivalent amount of the heterocyclic salt H16 is added to the mixed solution, and the mixture is heated for 24 hours under an argon atmosphere at 80 ^° C. After TLC detection of complete reaction, the solvent was removed under reduced pressure, and the residue was purified by column chromatography (dichloromethane: methanol = 30: 1, v/v) to obtain dark green cyanine fluorescent probe D23 (21 mg, 31%).

The characterization data is as follows:

¹ H NMR (400 MHz, DMSO- d ₆ ) δ 12.04 (s, 1H), 8.60 – 8.45 (m, 2H), 8.30 –8.18 (m, 2H), 8.17 – 8.05 (m, 2H), 7.93 – 7.84 (m, 2H), 7.65 – 7.57 (m, 2H), 7.55 – 7.44 (m, 4H), 6.78 – 6.62 (m, 2H), 4.35 – 4.27 (m, 2H), 4.26 – 4.17(m, 2H) , 2.90 – 2.76 (m, 4H), 2.21 (t, J = 7.2 Hz, 2H), 1.94 (dt, J = 14.0,7.2 Hz, 2H), 1.80 – 1.68 (m, 2H), 1.63 – 1.52 (m , 2H), 1.47 – 1.37 (m, 2H),1.33 (t, J = 6.8 Hz, 3H), as shown in Figure 32. HRMS (ESI-TOF): calcd. for C ₄₀ H ₃₈ ClN ₂ O ₂ ⁺ [M] ⁺ 613.2616; found 613.2631.

Example 4. Preparation of near-infrared cyanine fluorescent probe derivative DD

The reaction equation for synthesizing the near-infrared cyanine fluorescent probe derivative DD is shown in the following formula (V):

The structure of the near-infrared cyanine fluorescent probe derivative DD in this embodiment is selected from indole. R ₁ in the corresponding probe DD is methyl, n is 0, R ₃ is hydrogen, X is iodine, and R is trifluoromethyl. Sulfonate ester, p is 0, Y is carbon, and Z is hydrogen. Specific steps for synthesizing near-infrared cyanine fluorescent probe derivative DD:

(1) Synthesis of intermediate D-OH

Dissolve probe D and sodium acetate in the solvent DMF at a molar ratio of 1:3, and heat the mixture at 80°C under an argon atmosphere for 8 hours. After the reaction was monitored by TLC, the reaction solution was cooled to room temperature, extracted with dichloromethane (3×25 mL), backwashed with saturated brine (15 mL), dried over anhydrous magnesium sulfate, filtered, the filtrate was evaporated to dryness, and the residue was Purification by column chromatography (petroleum ether: ethyl acetate = 30:1, v/v) gave a red powdery solid (66 mg, 25%).

The characterization data is as follows:

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.17 (d, J = 13.3 Hz, 2H), 7.167 – 7.207 (m,4H), 6.91 (t, J = 7.6 Hz, 2H), 6.68 (d, J = 7.7 Hz, 2H), 5.41 (d, J = 13.2Hz, 2H), 3.21 (s, 6H), 2.62 (t, J = 5.5 Hz, 4H), 1.91 – 1.83 (m, 2H), 1.67(s, 12H).

(2) Synthesis of near-infrared cyanine fluorescent probe derivative DD1

Compound D-OH and triethylamine were dissolved in the solvent DMF at a molar ratio of 1:3. 3 equivalents of trifluoromethanesulfonic anhydride (relative to D-OH) was slowly added dropwise in an ice-water bath and stirred for 30 minutes. The mixture was 60°C. Heated for 8 h under argon atmosphere. After the reaction was monitored by TLC, the reaction solution was cooled to room temperature, extracted with dichloromethane (3×25 mL), backwashed with saturated brine (15 mL), dried over anhydrous magnesium sulfate, filtered with suction, and the filtrate was evaporated to dryness. The residue was passed through a column. Purification by chromatography (petroleum ether: ethyl acetate = 50:1, v/v) gave a green powder (68 mg, 55%).

The characterization data is as follows:

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.87 (d, J = 14.0 Hz, 2H), 7.44 – 7.38 (m,2H), 7.36 (d, J = 6.8 Hz, 2H), 7.30 – 7.27 (m, 2H), 7.18 (d, J = 8.0 Hz, 2H), 6.25 (d, J = 14.0 Hz, 2H), 3.72 (s, 6H), 2.77 (t, J = 6.0 Hz, 4H), 2.05 –1.97 ( m, 2H), 1.68 (s, 12H), as shown in Figure 33. HRMS (ESI-TOF): calcd. forC ₃₃ H ₃₆ F ₃ N ₂ O ₃ S ⁺ [M] ⁺ 597.2393; found 597.2342.

The above are only the preferred embodiments of the present invention. It should be pointed out that those skilled in the art can also make several modifications and improvements without departing from the concept of the present invention, and these should also be regarded as the protection scope of the present invention. , none of these will affect the effect of the present invention and the practicality of the patent.

Claims (6)

1. The method for rapidly detecting the pathology of the tumor tissue based on the near infrared cyanine fluorescent probe is characterized in that the near infrared cyanine fluorescent probe has the following structural general formula I:

；

in the general formula I, X ^- Selected from Cl ^- 、Br ^- 、I ^- 、PF ₆ ^- 、BF ₄ ^- 、ClO ₄ ^- 、CH ₃ COO ^- 、CF ₃ COO ^- Or OTs ^- The method comprises the steps of carrying out a first treatment on the surface of the Y is selected from carbon, nitrogen, oxygen or sulfur; z is selected from C _1–8 The methylene bridge is modified with alkyl, five-membered ring, six-membered ring, nitrogen-containing six-membered ring, oxygen-containing six-membered ring, sulfur-containing six-membered ring, benzene ring or seven-membered ring; the heterocyclic salt is selected from indole, benzindole, benzoxazole, benzothiazole, benzoselenazole, quinoline, acridine, benzothiopyylium, benzopyrylium or silicon-based rhodamine; n and m are integers which are arbitrarily more than 0; r is R ₁ And R is ₂ Each independently selected from the group consisting of sulfonic acid, arylsulfonic acid, carboxylic acid, sulfonate, carboxylate, alkyl, aryl, alkenyl, alkynyl, nitro, cyano, azide, amine, hydroxyl, halogen, alkoxy, ester, amide, polyethylene glycol chainsPiperidine, morpholine, pyrrole, imidazole, pyridinium or quaternary ammonium salts; p is an integer of 0 to 6; r is selected from alkyl chain, halogen, activated ester, maleimide, sulfoxide, sulfone, sulfonate, thioether, acrylamide, acrylate, vinyl sulfonamide, nitrogen or benzene ring.

2. The method for rapid pathological detection of tumor tissue based on near infrared cyanine fluorescent probe according to claim 1, wherein the near infrared cyanine fluorescent probe is combined with protein molecules through covalent/non-covalent acting force, and the labeled protein molecules are tumor or normal tissues.

3. The method for rapid pathological detection of tumor tissue based on near infrared cyanine fluorescent probe according to claim 1, comprising the following steps:

a. immersing the tumor tissue slice subjected to surgical excision or biopsy into paraformaldehyde fixing solution for fixing for 0-30 min; eluting the redundant fixing solution by using phosphate Tween buffer for 0-30 min;

b. c, immersing the tumor tissue slice obtained in the step a into a Triton X-100 surfactant solution for membrane rupture treatment, wherein the mass concentration of Triton X-100 is 0.1% -30%, and the treatment time is 0-30 min; eluting the redundant Triton X-100 solution by PBST for 0-30 min;

c. immersing the tumor tissue slice obtained in the step b into H ₂ O ₂ In solution, H ₂ O ₂ The mass concentration of the wastewater is 0.1% -30%, and the treatment time is 0-60 min; eluting the redundant hydrogen peroxide solution by PBST for 0-30 min;

d. c, immersing the tumor tissue slice obtained in the step c into a near infrared cyanine fluorescent probe solution for incubation, wherein the incubation temperature is 4-70 ℃, the molar concentration of the cyanine fluorescent probe solution is not lower than 1 nM, and the incubation time is not lower than 1 min; the redundant cyanine fluorescent probe solution is eluted by a DMSO solution, the mass concentration of the DMSO is 1-100%, and the eluting time is 0-30 min; eluting the residual DMSO by PBST for 0-30 min;

e. placing the tumor tissue slice obtained in the step d on a scanning imager or a fluorescence camera with a near infrared excitation light source for scanning to generate a fluorescence pattern of the tumor tissue; and judging the outline, stage and type of the tumor by analyzing the fluorescent patterns obtained by scanning, and predicting the malignancy degree and survival time.

4. The method for rapid pathological detection of tumor tissue based on near infrared cyanine fluorescent probes according to claim 3, wherein one or more near infrared cyanine fluorescent probes are simultaneously used for multi-index multi-color labeling.

5. The method for rapid pathological detection of tumor tissue based on near infrared cyanine fluorescent probe according to claim 3, wherein for the tumor tissue section in step a, the detection method is also suitable for cell smear, tissue fluid smear, frozen/paraffin tissue section, the substrate of the smear or tissue section is positive ion anti-drop slide, and the smear or tissue section contains near infrared cyanine fluorescent probe.

6. The method for rapid pathological detection of tumor tissue based on a near infrared cyanine fluorescent probe according to claim 5, wherein the thickness of the tissue slice is not less than 1 [ mu ] m.