Cyanine structure compound as well as preparation method and application thereof

Abstract

The invention provides a compound with a cyanine structure and a preparation method and application thereof, the compound has a structure as shown in formula I. The compound with the cyanine structure has the characteristic of performing double recognition on nitroreductase (NTR) highly expressed in a tumor anoxic microenvironment and a tumor acidic microenvironment, and can be used as a fluorescent probe for performing specific recognition on tumors; the accuracy and precision of tumor recognition are improved, and the false positive rate in the tumor recognition process is reduced.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of organic synthesis and imaging technology, in particular to a corrole structured compound and a preparation method and application thereof. BACKGROUND

[0002] Endometrial cancer is one of the three major gynecological malignancies, and its incidence rate ranks the second in China (only next to cervical cancer), and shows a rising trend year by year. In developed countries in Europe and America, the incidence rate of endometrial cancer ranks the first among gynecological malignancies. Surgery is the most important treatment, including total hysterectomy, bilateral salpingo-oophorectomy and systematic lymph node dissection. The most common metastasis pathway of endometrial cancer is through the pathway beside the uterus to the pelvic cavity and abdominal para-aortic lymph nodes, so intraoperative pelvic and abdominal para-aortic lymph node resection is to accurately assess the lymph node status. The lymph node metastasis status is the most important prognostic factor of endometrial cancer. Once the patient has lymph node metastasis, the tumor stage is IIIC, and the 5-year survival rate of the patient decreases from 88.6% to 48.7%; at the same time, once there is lymph node metastasis, it indicates that the patient needs to receive postoperative adjuvant radiotherapy. Therefore, for patients with endometrial cancer, the assessment of lymph node status is of great significance for the development of treatment plan and the judgment of prognosis.

[0003] At present, the main methods for evaluating lymph node metastasis in clinical practice include preoperative imaging evaluation, intraoperative visual and tactile exploration and sentinel lymph node biopsy. Imaging evaluation mainly includes MRI, CT and PET / CT. The sensitivity of MRI for evaluating lymph node metastasis is only 44%; the sensitivity of CT for metastatic lymph nodes of 5-9mm is only 45.1%; and the sensitivity of PET / CT for diagnosing metastatic lymph nodes is only 72%, and it is difficult to distinguish inflammation from cancer. Therefore, imaging examination is usually only used as a reference and cannot guide intraoperative lymph node dissection. Intraoperative visual and tactile exploration can only evaluate suspicious lymph nodes with obvious enlargement or obvious changes in texture. In fact, most of the pathologically confirmed metastatic lymph nodes have no obvious morphological changes, and the average maximum diameter is close to that of non-metastatic lymph nodes (14.1mm vs 13mm), so intraoperative visual and tactile exploration cannot guide intraoperative lymph node resection.

[0004] In addition, the gold standard for diagnosing metastatic lymph nodes is pathological diagnosis, but it usually requires the patient to undergo systematic lymph node dissection to remove lymph nodes during surgery, and pathological diagnosis after surgery to determine. But systematic lymph node dissection can cause a series of complications, such as vascular and nerve damage, lymphedema, lymph cysts, lymph leakage and bleeding, etc., which seriously reduce the postoperative quality of life of patients. Studies have shown that about 60-80% of patients do not have tumor metastasis in the relevant lymphatic drainage area, and these patients can not undergo lymph node dissection; that is, most of the patients with endometrial cancer only receive systematic lymph node dissection in order to assess the status of lymph nodes, resulting in a huge "injury". Because postoperative pathological detection has obvious lag, there is an urgent need in clinical practice for a practical method to early judge metastatic lymph nodes during surgery, and then selectively remove metastatic lymph nodes, avoid systematic lymph node dissection, and thus reduce the damage to patients.

[0005] As an important tool for biomedical research and clinical diagnosis, fluorescence imaging has the advantages of high spatial and temporal resolution, real-time dynamic imaging, high sensitivity, non-invasiveness and low cost. At present, fluorescence probes including fluorescein sodium, 5-aminolevulinic acid (5-ALA), methylene blue (MB) and indocyanine green (ICG) have been successfully developed and approved by the US Food and Drug Administration (FDA), and are widely used in clinical fields such as human angiography, sentinel lymph node biopsy and fluorescence navigation surgery. However, the excitation wavelength of these probes is relatively short (<900 nm), and with the increase of tissue penetration depth, it will be severely scattered / absorbed by photons and high spontaneous fluorescence in vivo, thereby limiting its application in in vivo biological imaging.

[0006] Recent studies have revealed that fluorescence imaging technology in the second near-infrared window (1000-1700 nm) has unique advantages, including lower photon absorption and scattering, deep tissue penetration ability, and extremely low background fluorescence. These factors collectively enhance the spatial resolution and fluorescence signal intensity of the imaging, making the second near-infrared imaging exhibit great potential in the field of biological in vivo imaging. In recent years, some fluorescence probes capable of imaging tumors in the second near-infrared window have been developed. However, most of these probes belong to targeted "fluorescent constant" probes, which means that they will continuously emit fluorescence signals after combining with tumor recognition groups, resulting in high background fluorescence and false positive rates. To solve this problem, some single-target responsive activatable fluorescence probes have also been developed. Unlike targeted "fluorescent constant" probes, these probes emit almost no fluorescence in normal cells, and fluorescence is only activated when they combine with tumor tissue pathological parameters (such as pH value, viscosity, polarity, active oxygen, oxygen content, and other tumor microenvironment parameters or highly expressed biological enzymes). Therefore, the background fluorescence of this type of probe is significantly reduced. However, since some targets may also be highly expressed in other diseases, and non-target cells or healthy tissues may express a certain level of corresponding biomarkers in complex biological environments, this type of probe also faces the risk of false positives. To solve this problem, it is necessary to further develop multi-target activated fluorescence probes that can detect two or more targets simultaneously to improve the detection specificity of fluorescence probes in distinguishing between tumor tissues and normal tissues. SUMMARY

[0007] The purpose of the present application is to solve the false positive problem existing in the prior art targeted fluorescent constant probes and single-target responsive activatable fluorescence probes, and to provide a phycobilin structure compound and a preparation method and application thereof.

[0008] To achieve the above technical purposes, the technical solutions adopted by the present application are as follows:

[0009] In a first aspect, the present application provides a phycobilin structure compound having a structure as shown in formula I:

[0010]

[0011] In formula I, R1-R4 are each independently selected from H, CN, halogen, phenyl, C1-C6 alkyl, C1-C6 haloalkyl, or C1-C6 alkoxy;

[0012] Y1-Y4 are each independently selected from a nitro group;

[0013] The dashed line between Y1-Y4 and the benzene ring indicates that Y1-Y4 are not simultaneously connected to the benzene ring;

[0014] A is selected from the group consisting of C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 alkylsulfonyl, or C1-C6 hydroxyalkyl;

[0015] n is 0, 2 or 3;

[0016] m is 0 or 1;

[0017] the dotted line between X and the benzene ring indicates that the chemical bond between X and the benzene ring is a single bond or a double bond;

[0018] X is selected from the group consisting of C(R)2, CR, S, O, N, NR;

[0019] Z is selected from the group consisting of H, C1-C3 alkyl, halogen, phenyl;

[0020] each R is independently selected from the group consisting of H, C1-C3 alkyl.

[0021] In some embodiments of the present application, in formula I, R1 is H; R2-R4 are each independently selected from the group consisting of H, CN, halogen, phenyl, C1-C6 alkyl, C1-C6 haloalkyl, or C1-C6 alkoxy; A is selected from the group consisting of C1-C3 alkyl; Z is selected from halogen.

[0022] Further, based on the above-mentioned embodiments, in formula I, R2 is H; R3, R4 are each independently selected from the group consisting of H, CN, halogen, phenyl, C1-C6 alkyl, C1-C6 haloalkyl, or C1-C6 alkoxy. Alternatively, in formula I, R3 is H, R2, R4 are each independently selected from the group consisting of H, CN, halogen, phenyl, C1-C6 alkyl, C1-C6 haloalkyl, or C1-C6 alkoxy. Alternatively, in formula I, R2 is H, R3 is H, R4 is selected from the group consisting of H, CN, halogen, phenyl, C1-C6 alkyl, C1-C6 haloalkyl, or C1-C6 alkoxy.

[0023] In some embodiments of the present application, in formula I, R2-R4 are H, R1 is selected from the group consisting of H, CN, halogen, phenyl, C1-C6 alkyl, C1-C6 haloalkyl, or C1-C6 alkoxy; A is selected from the group consisting of C1-C3 alkyl; Z is selected from halogen.

[0024] In a second aspect, the present application provides a derivative of the cyanine structured compound of the first aspect, having a structure as shown in formula II: In formula II, R1-R4 are each independently selected from the group consisting of H, CN, halogen, phenyl, C1-C6 alkyl, C1-C6 haloalkyl, or C1-C6 alkoxy;

[0025] Y'1-Y'4 are each independently selected from the group consisting of NH2;

[0026] The dotted line between Y'1-Y'4 and the benzene ring means that Y1-Y4 are not simultaneously connected to the benzene ring;

[0027] A is selected from the group consisting of C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 alkylsulfonyl, and C1-C6 hydroxyalkyl;

[0028] n is 0, 2, or 3;

[0029] m is 0 or 1;

[0030] The dotted line between X and the benzene ring means that the chemical bond between X and the benzene ring is a single bond or a double bond;

[0031] X is selected from the group consisting of C(R)2, CR, S, O, N, and NR;

[0032] Z is selected from the group consisting of H, C1-C3 alkyl, halogen, and phenyl;

[0033] Each R is independently selected from the group consisting of H and C1-C3 alkyl.

[0034] In a third aspect, the present application provides another derivative of the cyanine compound of the first aspect, having a structure as shown in Formula III:

[0035] In Formula III, R1-R4 are each independently selected from the group consisting of H, CN, halogen, phenyl, C1-C6 alkyl, C1-C6 haloalkyl, and C1-C6 alkoxy;

[0036] Y'1-Y'4 are each independently selected from the group consisting of NH2;

[0037] The dotted line between Y'1-Y'4 and the benzene ring means that Y1-Y4 are not simultaneously connected to the benzene ring;

[0038] A is selected from the group consisting of C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 alkylsulfonyl, and C1-C6 hydroxyalkyl;

[0039] n is 0, 2, or 3;

[0040] m is 0 or 1;

[0041] The dotted line between X and the benzene ring means that the chemical bond between X and the benzene ring is a single bond or a double bond;

[0042] X is selected from the group consisting of C(R)2, CR, S, O, N, and NR;

[0043] Z is selected from the group consisting of H, C1-C3 alkyl, halogen, and phenyl;

[0044] Each R is independently selected from the group consisting of H and C1-C3 alkyl.

[0045] In some embodiments of the present application, in formula II or III, R1 is H; R2-R4 are each independently selected from H, CN, halogen, phenyl, C1-C6 alkyl, C1-C6 haloalkyl or C1-C6 alkoxy; A is selected from C1-C3 alkyl; and Z is selected from halogen.

[0046] Further, based on the above-mentioned embodiments, in formula II or III, R2 is H; R3, R4 are each independently selected from H, CN, halogen, phenyl, C1-C6 alkyl, C1-C6 haloalkyl or C1-C6 alkoxy. Alternatively, in formula II or III, R3 is H, R2, R4 are each independently selected from H, CN, halogen, phenyl, C1-C6 alkyl, C1-C6 haloalkyl or C1-C6 alkoxy. Alternatively, in formula II or III, R2 is H, R3 is H, and R4 is selected from H, CN, halogen, phenyl, C1-C6 alkyl, C1-C6 haloalkyl or C1-C6 alkoxy.

[0047] In some embodiments of the present application, in formula II or III, R2-R4 are H, and R1 is selected from H, CN, halogen, phenyl, C1-C6 alkyl, C1-C6 haloalkyl or C1-C6 alkoxy; A is selected from C1-C3 alkyl; and Z is selected from halogen.

[0048] In the structure of formula I, due to the presence of the nitro substituent on the indole ring, the compound has a dual photo-induced electron transfer (PET) effect. In the original state, the compound of formula I has no fluorescence, when nitroreductase is present in the environment, the nitro group in the indole is reduced by the nitroreductase to convert into the structure of formula II, the PET effect of the nitro group disappears, at this time there is also almost no fluorescence, and with the decrease of pH, the N atom on the indole ring is protonated to convert into the structure of formula III.

[0049] In a fourth aspect, the present application provides the use of the cyanine structure compound of the first aspect and / or the cyanine structure compound derivative of the second aspect and / or the cyanine structure compound derivative of the third aspect in the preparation of a tumor fluorescence imaging reagent.

[0050] Optionally, the tumor is a tumor with high expression of nitroreductase and a pH not higher than 7.0, and specifically, the tumor includes but is not limited to lung cancer, liver cancer, lymphoma, endometrial cancer, cervical cancer, leukemia, bone marrow cancer, melanoma, adrenal cortex cancer, bile duct cancer, colon cancer, colorectal cancer, esophageal cancer, glioma, head and neck cancer, renal chromophobe cancer, renal clear cell carcinoma, renal papillary cell carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, gastric cancer, testicular cancer, thymus cancer, thyroid cancer, thymus cancer.

[0051] In a fifth aspect, the present application provides a tumor fluorescent imaging reagent, comprising: the cyanine compound of the first aspect and / or the cyanine compound derivative of the second aspect and / or the cyanine compound derivative of the third aspect and / or a substance for forming the cyanine compound derivative of the second aspect or the third aspect.

[0052] In a sixth aspect, the present application provides a method for tumor fluorescent imaging for non-diagnostic or non-therapeutic purposes, wherein the cyanine compound of the first aspect is formulated into a solution by a solvent, and is injected into a tumor site or a vein of an individual; preferably, the concentration of the solution is 10-30 μM; and / or, the injection amount of the solution is 0.3-0.7 mg / kg.

[0053] The present application has the following beneficial effects:

[0054] Firstly, the cyanine compound provided by the present application has the characteristics of dual recognition of the tumor hypoxic microenvironment and the tumor acidic microenvironment with high expression of nitroreductase (NTR), and can be used as a fluorescent probe to specifically recognize tumors, thereby improving the accuracy and precision of tumor recognition and reducing the false positive rate in the tumor recognition process.

[0055] Secondly, the cyanine compound provided by the present application has a maximum emission wavelength in the near-infrared II region, has strong light stability, has a better penetration depth compared with existing targeted fluorescent constant probes and single-target response activatable fluorescent probes, and can perform long-time near-infrared II region fluorescent imaging on tumors.

[0056] Thirdly, the cyanine compound provided by the present application has easily available raw materials, a simple preparation process, controllable conditions, and development potential for large-scale production. BRIEF DESCRIPTION OF DRAWINGS

[0057] Figure 1 It is a hydrogen nuclear magnetic spectrum of compound 1 in the embodiment 1 of the present application.

[0058] Figure 2 It is a hydrogen nuclear magnetic spectrum of compound 2 in the embodiment 1 of the present application.

[0059] Figure 3 It is a hydrogen nuclear magnetic spectrum of Cy-pH-NTR in the embodiment 1 of the present application.

[0060] Figure 4 It is the absorption spectrum determination result of the Cy-pH-NTR solution in the embodiment 2 of the present application under different solution environments.

[0061] Figure 5 It is the fluorescence spectrum determination result of the Cy-pH-NTR solution in the embodiment 2 of the present application under different solution environments.

[0062] Figure 6 Molar Absorption Coefficient Correlation Diagram of Cy-pH-NTR Solution in Example 2 of the Invention, Wherein A is the Wavelength- Absorbance Curve of Cy-pH-NTR Solution, B is the Standard Curve of Absorbance and Molar Concentration of Cy-pH-NTR Solution.

[0063] Figure 7 Quantum Efficiency Test Results of Cy-pH-NTR in Example 2 of the Invention.

[0064] Figure 8 Recognition Test Results of Cy-pH-NTR to Acidic Environment in Example 2 of the Invention, Wherein A is the Fluorescence Spectrum of Cy-pH-NTR in Different Acidic Environment; B is the Fluorescence Intensity Curve of Cy-pH-NTR in Different Acidic Environment.

[0065] Figure 9 Recognition Test Results of Cy-pH-NTR to Nitroreductase in Example 2 of the Invention, Wherein A is the Fluorescence Spectrum of Cy-pH-NTR in pH 5.0 DPBS Solution Containing NADH with Different Concentrations; B is the Fitting Standard Curve of A.

[0066] Figure 10 Fluorescence Intensity of Cy-pH-NTR Probe in Example 2 of the Invention after Adding Different Bioactive Molecules in Acidic and Weak Alkaline DPBS Solution Respectively, Wherein A is the Fluorescence Intensity in Acidic Environment; B is the Fluorescence Intensity in Weak Alkaline Environment.

[0067] Figure 11 Cytotoxicity of Cy-pH-NTR Probe in Example 2 of the Invention in Human Endometrial Cancer Cells HEC-1-A and USPC-ARK-1 Respectively.

[0068] Figure 12 Blood Routine Results of Normal Mice after Injection of Cy-pH-NTR in Example 2 of the Invention.

[0069] Figure 13 Blood Biochemistry Results of Normal Mice after Injection of Cy-pH-NTR in Example 2 of the Invention.

[0070] Figure 14 Body Weight Change Results of Normal Mice after Injection of Cy-pH-NTR in Example 2 of the Invention.

[0071] Figure 15 H&E Staining Results of Different Tissues of Normal Mice after Injection of Cy-pH-NTR in Example 2 of the Invention.

[0072] Figure 16The tumor imaging effect of Cy-pH-NTR in the tumor-bearing mouse model in Example 3 of the present application.

[0073] Figure 17 The tumor imaging effect of Cy-pH-N and single-target pH probe in the tumor-bearing mouse model in Example 3 of the present application.

[0074] Figure 18 The fluorescence imaging effect of each organ after 48h of Cy-pH-NTR in the tumor-bearing mouse model in Example 3 of the present application.

[0075] Figure 19 The tumor metastasis lymph node imaging effect of Cy-pH-NTR in the mouse foot pad at different times in Example 3 of the present application.

[0076] Figure 20 The near-infrared two-zone imaging results of ICG and Cy-pH-NTR probe in clinical endometrial cancer tumor (TT) and normal tissue (NT) in Example 3 of the present application.

[0077] Figure 21 The near-infrared two-zone imaging results of ICG and Cy-pH-NTR probe in endometrial cancer metastasis lymph node (MLN) and normal lymph node (NLN) samples in Example 3 of the present application. DETAILED DESCRIPTION

[0078] In the specific embodiment of the present application, the following specific structural forms of the phthalocyanine structured compound are listed:

[0079] 1) R1is selected from H, CN, Cl, Br, I, CH3, CH2CH3, OCH3, OCH2CH3;

[0080] 2) R2is selected from CN, Cl, Br, I, CH3, CH2CH3, OCH3, OCH2CH3;

[0081] 3) R1is selected from H, CN, Cl, Br, I, CH3, CH2CH3, OCH3, OCH2CH3;

[0082] 4) R2is selected from CN, Cl, Br, I, CH3, CH2CH3, OCH3, OCH2CH3;

[0083] 5) R3is selected from CN, Cl, Br, I, CH3, CH2CH3, OCH3, OCH2CH3;

[0084] 6) R4is selected from: CN, CI, Br, I;

[0085] 7) R1is selected from: H, CN, CI, Br, I, CH3, CH2CH3, OCH3, OCH2CH3;

[0086] 8) R1is selected from: H, CN, CI, Br, I, CH3, CH2CH3, OCH3, OCH2CH3;

[0087] 9) R1is selected from: H, CN, CI, Br, I, CH3, CH2CH3, OCH3, OCH2CH3;

[0088] 10) R1is selected from: H, CN, CI, Br, I, CH3, CH2CH3, OCH3, OCH2CH3;

[0089] 11) R1is selected from: H, CN, CI, Br, I, CH3, CH2CH3, OCH3, OCH2CH3;

[0090] 12) R1is selected from: H, CN, CI, Br, I, CH3, CH2CH3, OCH3, OCH2CH3;

[0091] 13) R1is selected from: H, CN, CI, Br, I, CH3, CH2CH3, OCH3, OCH2CH3;

[0092] 14) R1is selected from: H, CN, CI, Br, I, CH3, CH2CH3, OCH3, OCH2CH3;

[0093] 15) R1is selected from: H, CN, CI, Br, I, CH3, CH2CH3, OCH3, OCH2CH3;

[0094] 16) R1is selected from: H, CN, CI, Br, I, CH3, CH2CH3, OCH3, OCH2CH3;

[0095] 17) R1is selected from: H, CN, CI, Br, I, CH3, CH2CH3, OCH3, OCH2CH3;

[0096] 18) R1is selected from: H, CN, Cl, Br, I, CH3, CH2CH3, OCH3, OCH2CH3;

[0097] The calixcyanine structural compounds of the present application are all targeted tumor imaging according to the light-emitting principle shown in Reaction Formula One, and thus can all be used as fluorescent probes for specific recognition of tumors, improving tumor recognition accuracy and precision, and reducing false positive rate in the tumor recognition process.

[0098] Further, a specific calixcyanine structural compound is taken as an example to illustrate the function and preparation process of the embodiment of the present application, which is hereinafter referred to as Cy-pH-NTR and has the following structural formula:

[0099]

[0100] The mechanism of the fluorescence effect of Cy-pH-NTR is as follows: due to the presence of 5-nitro-2,3,3-trimethylindole, Cy-pH-NTR has a dual photo-induced electron transfer (PET) effect. In the original state, Cy-pH-NTR has no fluorescence, when nitroreductase exists in the environment, the nitro group in 5-nitro-2,3,3-trimethylindole is reduced by nitroreductase, the PET effect of the nitro group disappears, at this time there is also almost no fluorescence, and with the decrease of pH, the N atom on the indole ring is protonated, thereby the fluorescence is significantly enhanced. Therefore, Cy-pH-NTR has good imaging effect on tumors with acidic microenvironment and high expression of nitroreductase. The chemical reaction involved is shown in Reaction Formula One:

[0101] Reaction Formula One:

[0102]

[0103] Further, the preparation method of Cy-pH-NTR is through compound 2 Further preferably, the calixcyanine structural compound is prepared by Reaction Formula 1):

[0104] Reaction Formula 1):

[0105]

[0106] More preferably, in the reaction formula 1), an organic alcohol is used as a reaction solvent, and further more preferably, the organic alcohol is methanol and / or ethanol; and / or, the reaction temperature is 70-100℃, preferably 85-95℃; and / or, the reaction time is 1-5h, preferably 2-3h.

[0107] Preferably, the reaction equation 1) further comprises: after the reaction is completed, the reaction mixture is concentrated and then purified by column chromatography to obtain the cyanine compound. The concentration is a conventional operation, which can be reduced pressure concentration or normal pressure concentration, and the purpose is to remove the solvent and water in the reaction mixture. The column chromatography is also conventional, and the purpose is to purify the Cy-pH-NTR obtained in the reaction mixture, and in one embodiment, the purification step is: by silica gel chromatography, using ethyl acetate / petroleum ether (1 / 4, v / v) as the eluent, to obtain a Cy-pH-NTR solid product.

[0108] Preferably, the compound 2 is obtained by the compound 1 Further preferably, the compound 2 is prepared by the following reaction equation 2):

[0109] Reaction equation 2):

[0110]

[0111] More preferably, in the reaction equation 2), methyl magnesium chloride and an acid are used as the catalyst; and / or, the reaction temperature is 50-70°C, preferably 60±3°C.

[0112] Preferably, the reaction equation 2) further comprises: the process of separating, washing and drying the reaction product from the system. The means for separating the reaction product from the system is conventional, and normal pressure filtration, reduced pressure filtration, centrifugation and the like can be used. Then, water and ethyl acetate are used for washing in sequence, and then dried in a drying box to obtain the compound 2.

[0113] Preferably, the compound 1 is obtained by benzo[cd]indol-2(1H)-1-one; further preferably, the compound 1 is prepared by the following reaction equation 3):

[0114] Reaction equation 3):

[0115]

[0116] Preferably, in the reaction equation 3), sodium hydride is used as the catalyst; and / or, the reaction temperature is -5-30°C, preferably 15-25°C.

[0117] Preferably, the reaction equation 3) further comprises: the process of extracting, washing, concentrating and purifying by column chromatography of the reaction mixture.

[0118] Further preferably, the preparation method of the cyanine compound comprises:

[0119] Step 1, mixing NaH, benz[cd]indol-2(1H)-1-one, anhydrous DMF; then cooling the mixture to -5-5℃, adding iodoethane, and reacting at -5-30℃ for 1-3h; after the reaction is completed, sequentially performing ethyl acetate extraction, salt water washing, concentration, and purifying the crude product by column chromatography to obtain compound 1;

[0120] Step 2, mixing compound 1 with anhydrous tetrahydrofuran, adding methyl magnesium chloride under a protective atmosphere, cooling after reacting at 50-70℃ for 0.5-3h, adding hydrochloric acid to the reaction solution, removing the reaction solvent, and then adding KI solution to obtain a crude product, which is separated by solid-liquid separation, the solid is washed with water and ethyl acetate, and then dried to obtain compound 2;

[0121] Step 3, mixing compound 3, compound 2, 5-nitro-2,3,3-trimethylindole, anhydrous ethanol under a protective atmosphere, and reacting at 70-100℃ for 2-4h; after the reaction is cooled, the crude product is purified by column chromatography to obtain the said cyanine structure compound.

[0122] More preferably, in the step 1, the molar ratio of benz[cd]indol-2(1H)-1-one, iodoethane, and NaH is 1:1-1.2:2-4; and / or, in the step 2, the molar ratio of compound 1 and methyl magnesium chloride is 1:1-1.2; and / or, in the step 3, the molar ratio of compound 3, compound 2, and 5-nitro-2,3,3-trimethylindole is 1:1-1.1:1-1.1.

[0123] In a preferred embodiment of the present application, the Cy-pH-NTR is obtained as shown in Reaction Formula Two:

[0124] Reaction Formula Two:

[0125]

[0126] Further, in the description of the present application, it should be noted that, in the examples, the specific conditions not specified are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments not specified by the manufacturer are all conventional products that can be obtained by market purchase.

[0127] The present application will be further described in detail below in conjunction with the accompanying drawings and specific examples, which are an explanation of the present application rather than a limitation.

[0128] Example 1

[0129] The present embodiment provides a preparation method of Cy-pH-NTR, which is specifically as follows:

[0130] (1) Synthesis of compound 1

[0131] NaH (15 mmol) was added to 15 mL of anhydrous DMF containing benzo[cd]indol-2(lH)-l-one (5 mmol) under argon. The mixture was then cooled to 0 °C, and iodoethane (6 mmol) was added gradually. Stirring at room temperature for 3 h, ethyl acetate extraction, brine washing, and concentration. The crude product was purified by column chromatography to obtain compound 1 (81%) as a yellow solid. The hydrogen nuclear magnetic spectrum of compound 1 is shown in Figure 1 1 H NMR (400 MHz, CDC13-dl) δ 8.06 (d, J = 7.0 Hz, 1H), 8.01 (d, J = 8.1 Hz, 1H), 7.71 (dd, J = 8.1, 7.0 Hz, 1H), 7.53 (d, J = 8.4 Hz, 1H), 7.47 (dd, J = 8.4, 6.9 Hz, 1H), 6.92 (d, J = 6.9 Hz, 1H), 3.99 (q, J = 7.2 Hz, 2H), 1.38 (t, J = 7.2 Hz, 3H).

[0132] (2) Synthesis of compound 2

[0133] Compound 1 (5 mmol) was added in 20 ml of anhydrous tetrahydrofuran, and methyl magnesium chloride (3M THF solution, 2 mL, 6 mmol) was added dropwise under Ar atmosphere. After stirring at 60 °C for 1 h, it was cooled, and hydrochloric acid (2 m, 10 mL) was added. THF was removed by vacuum distillation, and KI solution (1M, 5 mL) was added to obtain a red precipitate. The crude product was filtered, washed with water and ethyl acetate, and dried to obtain compound 2. Yield: 1.36 g (84%). The hydrogen nuclear magnetic spectrum of compound 2 is shown in Figure 2 1 H NMR (400 MHz, DMSO-d6) δ 9.00 (d, J = 7.2 Hz, 1H), 8.80 (d, J = 7.8 Hz, 1H), 8.55 (d, 1H), 8.46 (d, 1H), 8.17 (t, 1H), 8.01 (t, 1H), 4.73 (q, 2H), 3.25 (s, 3H), 1.57 (t, 3H).

[0134] (3) Synthesis of Cy-pH-NTR

[0135] Compound 3 (0.76 mmol), compound 2 (0.76 mm), and 5-nitro-2,3,3- trimethylindole (compound 4, 0.79 mmol) were dissolved in anhydrous ethanol (10.0 mL) under N2 atmosphere. The reaction mixture was stirred at 90 °C for 3 hours. After cooling, the reaction mixture was concentrated by evaporation under reduced pressure. The crude product was purified by column chromatography to obtain Cy-pH NTR as a black purple solid. The hydrogen nuclear magnetic spectrum of Cy-pH-NTR is shown in​​Figure 3 as shown, 1 H NMR (400 MHz, CDC13) δ 8.30 (dd, 2H), 8.18 (s, 1H), 8.01 (m, 1H), 7.68 (m, 3H), 7.38 (t, 1H), 7.23 (d, 1H), 6.63 (m, 2H), 6.11 (d, 1H), 3.95 (q, 1H), 2.77 (t, 2H), 2.66 (t, 2H), 1.96 (m, 2H), 1.65 (s, 6H), 1.37 (s, 3H).

[0136] Example 2

[0137] This example is directed to the relevant performance test of Cy-pH-NTR obtained in Example 1, as follows:

[0138] Cy-pH-NTR fluorescent probe solution preparation:

[0139] A certain amount of synthetic probe Cy-pH-NTR was dissolved in dimethyl sulfoxide (DMSO) to prepare a 5 x 10 -3 M stock solution. 4 ul of the stock solution was taken in diphosphate buffer (DPBS) to obtain a final volume of 2 mL, and the probe concentration was 1 x 10 -5 M solution to be tested.

[0140] (1) Determination of Cy-pH-NTR solution absorption spectrum

[0141] Figure 4 Cy-pH-NTR solution absorption spectrum was determined. The absorption spectrum of Cy-pH-NTR in DPBS buffer was investigated, and the solutions used were: 1) Cy-pH-NTR (10 μM) containing no NTR in pH 8.0 DPBS, 2) Cy-pH-NTR (10 μM) and NTR (10 μM) containing reduced nicotinamide adenine dinucleotide (NADH) (500 μM) in pH 8.0 DPBS, 3) Cy-pH-NTR containing no NTR in pH 2.0 DPBS, and Cy-pH-NTR (10 μM) and NTR (10 μM) containing NADH (500 μM) in pH 2.0 DPBS. The instrument for ultraviolet-visible absorption spectrum determination was PerkinElmer LAMBDA1050+, and quartz cuvette (1 cm) was used for absorbance measurement. As can be seen from the figure, the fluorescent probe synthesized in the present application has almost no absorption in weakly alkaline PBS buffer, but has a clear absorption peak in acidic PBS buffer containing NTR at pH 2.0, with a maximum at 880 nm.

[0142] (2) Determination of Cy-pH-NTR solution fluorescence spectrum

[0143] Figure 5 The fluorescence spectrum of Cy-pH-NTR solution was determined. The fluorescence spectrum of Cy-pH-NTR in DPBS buffer was investigated, and the solutions used were pH 8.0 DPBS containing Cy-pH-NTR (10 μM) without NTR, pH 8.0 DPBS containing Cy-pH-NTR (10 μM) and NTR (10 μM), NADH (500 μM), pH 2.0 DPBS containing Cy-pH-NTR without NTR, and pH 2.0 DPBS containing Cy-pH-NTR (10 μM) and NTR (10 μM), NADH (500 μM). The fluorescence excitation wavelength was 808 nm, and the emission wavelength range was 950 nm-1150 nm. The emission slit width was 1.5 nm, and the fluorescence measuring instrument used was an Edinburgh FLS1000 fluorescence spectrophotometer, an external 808 nm semiconductor laser (Changchun Xin Industrial Optoelectronic Technology Co., Ltd.) was used as the excitation light source, and a quartz cuvette (1 cm) was used for emission measurement. As can be seen from the figure, the fluorescent probe Cy-pH-NTR synthesized in the present application has almost no fluorescence emission in weakly alkaline PBS buffer, but has a clear near-infrared two-region fluorescence emission peak in acidic PBS buffer containing NTR at pH 2.0, with a maximum emission wavelength of 980 nm.

[0144] (3) Fluorescent probe Cy-pH-NTR for molar absorption coefficient comparison test

[0145] Figure 6 The probe molar absorption coefficient correlation graph. According to the Lambert-Beer law, the absorbance of the solution and the molar concentration were linearly fitted, and the molar extinction coefficient was calculated as follows:

[0146] A = εcl (equation S1);

[0147] In equation S1, A is the maximum absorption wavelength or the absorbance at 808 nm, ε is the molar extinction coefficient related to the inherent properties of the compound, l is the light path absorption thickness, and c is the solution concentration. Cy-pH-NTR stock solution was diluted with DPBS buffer to obtain solutions of different concentrations, and the absorbance was measured using solutions of different concentrations. The final calculation showed that the molar absorption coefficient in PBS buffer containing NTR (10 μM) and NADH (500 μM) at pH 5.0 was 2.1 x 10 4 L·mol-1·cm -1 .

[0148] (4) Fluorescent probe Cy-pH-NTR for quantum efficiency test

[0149] Figure 7The related graphs for the quantum efficiency test of the probe. The relative strategy was used to measure the quantum yield. The standard dye IR-26 was used as a reference to measure QY, which is the conventional method for measuring quantum yield, and the operation process is not described in detail here. The formula involved is:

[0150]

[0151] Φ X is the quantum efficiency of the fluorescent probe in the PBS solution, Φ ST is the quantum efficiency of IR-26 in 1,2-dichloroethane is 0.5%, Grad X is the slope of the intensity integral area of the fluorescence spectrum of different concentrations of Cy-pH-NTR probe solution in the 950-1150 nm region relative to its absorbance at 808 nm, Grad X is the slope of the intensity integral area of the fluorescence spectrum of different concentrations (0, 1, 5, 10, 20 μM) of IR26 probe solution in the 950-1150 nm region relative to its absorbance at 808 nm, η X and η ST is the refractive index of the respective solvent (PBS: 1.333, 1,2-dichloroethane: 1.4448), and different concentrations of Cy-pH-NTR probe solution is diluted with PBS buffer solution to obtain Cy-pH-NTR mother liquor. The final Cy-pH-NTR quantum yield is calculated to be 0.94%.

[0152] (5) Recognition test of fluorescent probe Cy-pH-NTR to acidic environment

[0153] Figure 8 The fluorescence spectrum of Cy-pH-NTR probe in different pH (2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0) DPBS solution containing NTR (10 μM) and NADH (500 μM) was determined. The fluorescence excitation wavelength was 808 nm, and the emission wavelength range was 950 nm-1150 nm. The emission slit width was 1.5 nm, and the fluorescence measuring instrument used was Edinburgh FLS1000 fluorescence spectrophotometer, the excitation light source was an external 808 nm semiconductor laser (Changchun Xin Industrial Optoelectronic Technology Co., Ltd.), and a quartz cuvette (1 cm) was used for emission measurement. As can be seen from the figure, in the DPBS solution containing excess NTR and NADH, the fluorescence emission spectrum intensity of Cy-pH-NTR probe gradually decreases with the gradual decrease of the pH of the PBS solution, and the pKa of the probe is calculated to be 5.2.

[0154] (6) Recognition test of fluorescent probe Cy-pH-NTR to nitroreductase

[0155] Figure 9 The fluorescence spectra of Cy-pH-NTR probe in pH 5.0 DPBS solution containing NADH (500 μM) were measured with the change of NTR concentration (0, 0.5, 1, 2, 4, 6, 8 and 10 μM). The fluorescence excitation wavelength was 808 nm, and the emission wavelength range was 950 nm-1150 nm. The emission slit width was 1.5 nm, and the fluorescence measuring instrument used was an Edinburgh FLS1000 fluorescence spectrophotometer, the excitation light source was an external 808 nm semiconductor laser (Changchun New Industrial Optoelectronic Technology Co., Ltd.), and a quartz cuvette (1 cm) was used for emission measurement. As can be seen from the figure, in the pH 5.0 DPBS solution containing NADH, the fluorescence emission spectrum intensity of Cy-pH-NTR probe gradually increased with the gradual increase of NTR concentration, and the detection limit of the probe for NTR detection in 5.0 DPBS solution was calculated to be 0.027 μg / mL.

[0156] (7) Test of specific recognition of fluorescence probe Cy-pH-NTR to nitroreductase and acidic environment

[0157] Figure 10 The fluorescence intensity of Cy-pH-NTR probe (10 μM) in acidic (pH 5.0) and weakly alkaline (pH 8.0) DPBS solution after adding different bioactive molecules (100 μM) (Blank, NTR, Glu, VC, GSH, Cys, ATP, L-Lys, L-Try, Na + , Fe 2+ , Cu 2+ , Ca 2+ , K + , H2O2, ONOO - , NO, HClO) was measured. The fluorescence excitation wavelength was 808 nm, and the emission wavelength range was 950 nm-1150 nm. The emission slit width was 1.5 nm, and the fluorescence measuring instrument used was an Edinburgh FLS1000 fluorescence spectrophotometer, the excitation light source was an external 808 nm semiconductor laser (Changchun New Industrial Optoelectronic Technology Co., Ltd.), and a quartz cuvette (1 cm) was used for emission measurement. As can be seen from the figure, in the acidic DPBS solution, only after adding NTR, the fluorescence increased sharply, while adding other active molecules, while in the weakly alkaline DPBS solution, no matter what bioactive molecule was added, the fluorescence intensity of the probe hardly changed, indicating that the recognition of the probe to NTR and acidic environment was specific.

[0158] (8) Test of cytotoxicity of fluorescence probe Cy-pH-NTR

[0159] Figure 11The cytotoxicity of Cy-pH-NTR probe in human endometrial cancer cells HEC-1-A and USPC-ARK-1, respectively, can be seen from the figure, and the effect on the viability of HEC-1-A and USPC-ARK-1 cells is not great with the increase of Cy-pH-NTR concentration. The operation method is as follows: HEC-1-A and USPC-ARK-1 cells are inoculated in a 96-well plate at an average density of 1 × 10 5 cells / well. After the cells adhere, 0, 5, 10, 20, and 50 μM of Cy-pH-NTR are added, respectively, and incubated for 48 hours. Then, 10% MTT solution is added to each well and incubated for 4 hours. In addition, 200 μL of DMSO is added, shaken well, and the absorbance at 490 nm is recorded by an enzyme label instrument (TECAN, Infinite M200, Germany). The cell viability determination formula is:

[0160] Vr = (A-A0) / (A S -A0) × 100% (equation S3);

[0161] Wherein A is the absorbance of the experimental group, A S is the absorbance of the control group, and A0is the absorbance of the blank group (no cells).

[0162] (9) Biological safety test of fluorescent probe Cy-pH-NTR

[0163] Figures 12 to 15 The figure is related to the blood routine, blood biochemical parameters and H&E staining of different tissues of normal mice (BLAB / c mice, Jisui Yaoke) at different times after tail vein injection of 200 μL of Cy-pH-NTR probe with a concentration of 50 μM, and the change of mouse body weight. It can be seen that the blood routine parameters and blood biochemical parameters of normal mice injected with Cy-pH-NTR probe fluctuate within the normal range within 28 days, and there is no obvious lesion in each tissue organ of the mice after H&E staining. The change trend of the body weight of the mice after injection of the probe is consistent with the change trend of the body weight of the normal mice, indicating that the probe has good biological safety.

[0164] Example 3

[0165] This embodiment is directed to animal model test of the fluorescent probe Cy-pH-NTR obtained in Example 1, which is as follows:

[0166] Construction of tumor-bearing mouse model: for female BLAB / c nude mice (Jisui Yaoke), the USPC-ARK-1 cells after canceling the chemical were made into a cell suspension with a concentration of 1 × 10 7 cells / mL. 200 μL of cell suspension was inoculated subcutaneously in the right of the nude mice, and about a month later, the tumor diameter was lengthened to 5-10 mm to obtain a tumor-bearing mouse model.

[0167] Construction of tumor metastatic lymph node mouse model: for female BLAB / c nude mice (Ji Cui Yao Kang), the complete USPC-ARK-1 cells were made into cell suspension, the concentration was 1 x 10 7 After about a month, the tumor diameter was 5-10 mm, and the metastatic lymph node mouse model was obtained.

[0168] (1) The tumor imaging effect of fluorescent probe Cy-pH-NTR in tumor-bearing mouse model

[0169] Figure 16 The related atlas of the tumor imaging effect of the probe in the tumor-bearing mouse model. By injecting 200 μL of ICG and Cy-pH-NTR fluorescent probe with a concentration of 20 μM through the tail vein, it can be seen that the Cy-pH-NTR probe has good tumor targeting effect. At 48 h, ICG still has fluorescence in the mouse body, but it has disappeared in the tumor site of the mouse. Cy-pH-NTR probe still has sustained fluorescence in the tumor site, indicating that Cy-pH-NTR can be retained and enriched in the tumor site for a long time.

[0170] (2) Comparison of the tumor imaging effect of fluorescent probe Cy-pH-NTR and single-target pH probe NSCy-975 (Guangzhou Yunsan Biochemical Technology Co., Ltd.) in tumor-bearing mouse model

[0171] Figure 17 The related atlas of the tumor imaging effect of Cy-pH-NTR and single-target pH probe NSCy-975 in the tumor-bearing mouse model. The mouse model used USPC-ARK-1 cell endometrial cancer subcutaneous model, and 200 μL of pH probe NSCy-975 and Cy-pH-NTR fluorescent probe with a concentration of 20 μM were injected through the tail vein. It can be seen that compared with single-target pH probe NSCy-975, Cy-pH-NTR probe has better tumor targeting effect and retention and enrichment.

[0172] (3) The imaging effect of fluorescent probe Cy-pH-NTR in each organ of tumor-bearing mouse

[0173] Figure 18The relevant atlas of fluorescence of each organ in a tumor-bearing mouse model 48 h after tail vein injection of the probe. The mouse model used a USPC-ARK-1 cell endometrial cancer subcutaneous model (tumor-bearing mice), and 200 μL of Cy-pH-NTR fluorescent probe with a concentration of 20 μM was injected into the tail vein. After 48 h, the mouse was dissected, and each organ of the mouse was removed, including the heart, liver, spleen, lung, kidney, and tumor. Near-infrared two-zone fluorescence imaging was used to image each organ, and it was observed that only the tumor had fluorescence, and the other organs did not have fluorescence.

[0174] (4) Fluorescent probe Cy-pH-NTR imaging effect on metastatic lymph nodes in a mouse model of tumor metastatic lymph nodes

[0175] Figure 19 The relevant atlas of the imaging effect of tumor metastatic lymph nodes at different times after injection of the probe into the mouse footpad. The mouse model used USPC-ARK-1 cell endometrial cancer (injection volume of 200 μL) to inject the mouse footpad to construct a tumor metastatic lymph node model, and then 50 μL of ICG and Cy-pH-NTR fluorescent probe with a concentration of 20 μM was injected into the tumor metastatic lymph node model through the footpad. It can be seen that the Cy-pH-NTR probe has a good tumor targeting effect on metastatic lymph nodes and can be lit by metastatic lymph nodes, and the lighting effect and residence time are better than ICG.

[0176] (5) Fluorescent probe Cy-pH-NTR imaging effect on tumors in clinical samples

[0177] Figure 20 and Figure 21 The relevant atlas of near-infrared two-zone imaging of clinical endometrial cancer tumor (TT) and normal endometrial tissue (NT), as well as endometrial cancer metastatic lymph nodes (MLN) and normal lymph nodes (NLN) samples after spraying 50 μL of ICG and Cy-pH-NTR probe with a concentration of 20 μM. It can be seen that the probe successfully turns on the near-infrared two-zone fluorescence only in the tumor and metastatic lymph nodes, and there is almost no fluorescence in normal tissues and normal lymph node samples, but IGC has fluorescence in each tissue, which has greater interference. In addition, after continuous irradiation with 808 nm laser for 30 minutes, the fluorescence intensity of the tumor metastatic lymph node tissue almost did not decrease significantly, indicating that the probe can be used for a long time in the near-infrared two-zone fluorescence imaging of clinical tumor samples.

[0178] The above embodiments only express several implementation manners of the present application, and the description is more specific and detailed, but it should not be understood as a limitation on the patent scope of the present application. It should be noted that for ordinary skilled persons in the art, without departing from the concept of the present application, several modifications and improvements can be made, which are all within the protection scope of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims.

Claims

1. A cyanin-structured compound, characterized in that, It has the structure shown in Equation I: In Formula I, R1-R4 are each independently selected from: H, CN, halogen, phenyl, C1-C6 alkyl, C1-C6 haloalkyl or C1-C6 alkoxy; Y1-Y4 are each independently selected from: nitro; The dashed line between Y1-Y4 and the benzene ring indicates that Y1-Y4 are not simultaneously attached to the benzene ring; A is selected from: C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 alkylsulfonic acid or C1-C6 hydroxyalkyl; n is 0, 2, or 3; m is 0 or 1; The dashed line between X and the benzene ring indicates that the chemical bond between X and the benzene ring is either a single bond or a double bond. X is selected from: C(R)2, CR, S, O, N, NR; Z is selected from: H, C1-C3 alkyl, halogen, phenyl; Each R is independently selected from: H, C1-C3 alkyl groups.

2. The cyanine-structured compound according to claim 1, characterized in that, In Formula I, R1 is H; R2-R4 are each independently selected from: H, CN, halogen, phenyl, C1-C6 alkyl, C1-C6 haloalkyl or C1-C6 alkoxy; A is selected from: C1-C3 alkyl; Z is selected from halogen.

3. The cyanine-structured compound according to claim 2, characterized in that, In Formula I, R2 is H; R3 and R4 are each independently selected from: H, CN, halogen, phenyl, C1-C6 alkyl, C1-C6 haloalkyl or C1-C6 alkoxy.

4. The cyanin-structured compound according to claim 2, characterized in that, In Formula I, R3 is H, and R2 and R4 are each independently selected from: H, CN, halogen, phenyl, C1-C6 alkyl, C1-C6 haloalkyl or C1-C6 alkoxy.

5. The cyanine-structured compound according to claim 2, characterized in that, In Formula I, R2 is H, R3 is H, and R4 is selected from: H, CN, halogen, phenyl, C1-C6 alkyl, C1-C6 haloalkyl or C1-C6 alkoxy.

6. The cyanine-structured compound according to claim 1, characterized in that, In Formula I, R2-R4 are H, R1 is selected from: H, CN, halogen, phenyl, C1-C6 alkyl, C1-C6 haloalkyl or C1-C6 alkoxy; A is selected from: C1-C3 alkyl; Z is selected from halogen.

7. The derivative of the cyanine-structured compound according to claim 1, characterized in that, It has the structure shown in Equation II: In Formula II, R1-R4 are each independently selected from: H, CN, halogen, phenyl, C1-C6 alkyl, C1-C6 haloalkyl or C1-C6 alkoxy; Y'1-Y'4 are each independently selected from: NH2; The dashed line between Y'1-Y'4 and the benzene ring indicates that Y1-Y4 are not simultaneously attached to the benzene ring; A is selected from: C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 alkylsulfonic acid or C1-C6 hydroxyalkyl; n is 0, 2, or 3; m is 0 or 1; The dashed line between X and the benzene ring indicates that the chemical bond between X and the benzene ring is either a single bond or a double bond. X is selected from: C(R)2, CR, S, O, N, NR; Z is selected from: H, C1-C3 alkyl, halogen, phenyl; Each R is independently selected from: H, C1-C3 alkyl groups.

8. The derivative of the cyanine-structured compound according to claim 1, characterized in that, It has the structure shown in Equation III: In Formula III, R1-R4 are each independently selected from: H, CN, halogen, phenyl, C1-C6 alkyl, C1-C6 haloalkyl or C1-C6 alkoxy; Y'1-Y'4 are each independently selected from: NH2; The dashed line between Y'1-Y'4 and the benzene ring indicates that Y1-Y4 are not simultaneously attached to the benzene ring; A is selected from: C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 alkylsulfonic acid or C1-C6 hydroxyalkyl; n is 0, 2, or 3; m is 0 or 1; The dashed line between X and the benzene ring indicates that the chemical bond between X and the benzene ring is either a single bond or a double bond. X is selected from: C(R)2, CR, S, O, N, NR; Z is selected from: H, C1-C3 alkyl, halogen, phenyl; Each R is independently selected from: H, C1-C3 alkyl groups.

9. The derivative according to claim 7 or 8, characterized in that, In formula II or formula III, R1 is H; R2-R4 are each independently selected from: H, CN, halogen, phenyl, C1-C6 alkyl, C1-C6 haloalkyl or C1-C6 alkoxy; A is selected from: C1-C3 alkyl; Z is selected from halogen.

10. The derivative according to claim 9, characterized in that, In formula II or formula III, R2 is H; R3 and R4 are each independently selected from: H, CN, halogen, phenyl, C1-C6 alkyl, C1-C6 haloalkyl or C1-C6 alkoxy.

11. The derivative according to claim 9, characterized in that, In formula II or formula III, R3 is H, and R2 and R4 are each independently selected from: H, CN, halogen, phenyl, C1-C6 alkyl, C1-C6 haloalkyl or C1-C6 alkoxy.

12. The derivative according to claim 9, characterized in that, In formula II or formula III, R2 is H, R3 is H, and R4 is selected from: H, CN, halogen, phenyl, C1-C6 alkyl, C1-C6 haloalkyl or C1-C6 alkoxy.

13. The cyanine-structured compound according to claim 7 or 8, characterized in that, In formula II or formula III, R2-R4 are H, R1 is selected from: H, CN, halogen, phenyl, C1-C6 alkyl, C1-C6 haloalkyl or C1-C6 alkoxy; A is selected from: C1-C3 alkyl; Z is selected from halogen.

14. The use of the cyanine compound according to any one of claims 1 to 6 and / or the derivative of the cyanine compound according to any one of claims 7 to 13 in the preparation of tumor fluorescence imaging reagents.

15. The application according to claim 14, characterized in that, The tumor is a tumor with high expression of nitroreductase and a pH not higher than 7.

0.

16. A tumor fluorescence imaging reagent, characterized in that, include: The cyanine compound according to any one of claims 1 to 6 and / or a derivative of the cyanine compound according to any one of claims 7 to 13 and / or a substance for forming a derivative of the cyanine compound according to any one of claims 7 to 13.

17. A method for tumor fluorescence imaging that is not for diagnostic or therapeutic purposes, characterized in that, The cyanine compound according to any one of claims 1 to 6 is prepared into a solution using a solvent and injected into an individual tumor site or intravenously; preferably, the concentration of the solution is 10 to 30 μM; and / or, the injection volume of the solution is 0.3 to 0.7 mg / kg of individual.

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