Heptamethine cyanine near-infrared fluorescent dye, preparation method therefor, and use thereof

Abstract

The present invention relates to the technical field of organic fluorescent molecules, and provides a heptamethine cyanine near-infrared fluorescent dye, a preparation method therefor, and a use thereof. The heptamethine cyanine near-infrared fluorescent dye has a structure as shown in formula I. The heptamethine cyanine near-infrared fluorescent dye provided by the present invention has better in-vivo metabolic characteristics, has the advantages of no accumulation in normal tissues such as liver and long tumor tissue imaging window time, is more suitable for precise diagnosis of tumors, has certain clinical application prospects, and is applied to fluorescence imaging of clinical tumors and the like.

Description

A heptamethine cyanine near-infrared fluorescent dye and a preparation method and application thereof TECHNICAL FIELD

[0001] The present application relates to the technical field of organic fluorescent molecules, in particular to a heptamethine cyanine near-infrared fluorescent dye and a preparation method and application thereof. BACKGROUND

[0002] Cancer seriously threatens human life and health, and with the deepening of population aging and the change of people's lifestyle, the challenge brought by cancer is becoming more and more severe. At present, the treatment methods for cancer mainly include chemotherapy, radiotherapy, ablation, surgical resection, etc. For solid tumors, early screening, early diagnosis and early surgery can effectively improve the cure rate of the disease and prolong the survival period of patients.

[0003] With the emergence of various advanced medical equipment, such as computed tomography (CT), magnetic resonance imaging (MRI), positron emission tomography (PET), near-infrared fluorescence imaging system and ultrasound (US), etc., the early detection rate of tumors has been greatly improved, providing an important reference for precise diagnosis of tumors. Compared with CT, MRI and other optical molecular imaging surgical navigation technologies, it has the characteristics of real-time, non-invasive and high resolution, providing a new image-based auxiliary means for precise surgery of tumors. Near-infrared fluorescence imaging diagnosis has advantages in tumor diagnosis (early, medium and late), intraoperative navigation, prognosis and recurrence monitoring diagnosis, etc. This diagnostic method mainly uses near-infrared fluorescent probes (650-1000 nm) for specific detection, which can provide high-resolution tissue and organ images, has low biological toxicity and low spontaneous fluorescence, and is beneficial to minimizing background interference.

[0004] Indocyanine green is a near-infrared fluorescent dye approved by FDA, which is easy to bind to plasma proteins after intravenous injection, metabolized into bile by the liver, and can be retained in tumor tissue through the EPR effect. It has high safety, broad-spectrum tumor targeting (all solid tumors), but its tumor targeting ability is weak, and the clinical human dose is ≤2.0 mg / kg, so its application range is limited. Therefore, it is of great clinical significance to develop a targeted contrast agent with broad-spectrum, higher fluorescence efficiency and stronger tumor targeting ability.

[0005] CN111196896A discloses a water-soluble heptamethine cyanine near-infrared dye with tumor targeting property and its application. The near-infrared dye of structural formula I is widely distributed in the whole body tissue with hemoglobin, and its metabolism in normal tissue is faster than that in tumor tissue, so it can be retained in tumor tissue in large quantities, thereby playing a function of in vivo diagnosis. Through tumor targeting ability verification, it is found that NIR-04 has stronger tumor targeting ability than ICG, and the tumor retention time is more than 48 h; but NIR-04 has the disadvantage of long-term accumulation in the liver.

[0006] Therefore, it is urgent to develop a near-infrared fluorescent dye with strong tumor targeting ability and long tumor imaging time.

[0007] In view of this, the present application is proposed.

[0008] SUMMARY

[0009] One of the purposes of the present application is to provide a heptamethine cyanine near-infrared fluorescent dye, which is modified based on NIR-04, effectively improves the problem of long-time accumulation of NIR-04 in the liver, while retaining the tumor targeting ability; that is, the development of the heptamethine cyanine near-infrared fluorescent dye has the advantages of strong tumor targeting ability and long tumor imaging time.

[0010] The second purpose of the present application is to provide a preparation method of the heptamethine cyanine near-infrared fluorescent dye, which prepares a series of heptamethine cyanine near-infrared fluorescent dyes through further modification of NIR-04, through amide condensation under the action of a condensing agent, trifluoroacetic acid de-trityl protecting group, nucleophilic substitution, and further deprotection.

[0011] The third purpose of the present application is to provide an application of the heptamethine cyanine near-infrared fluorescent dye in preparing a fluorescent contrast agent.

[0012] The fourth purpose of the present application is to provide an application of the heptamethine cyanine near-infrared fluorescent dye in preparing a tumor diagnosis drug; also an application in preparing a liver cancer or colorectal cancer diagnosis drug.

[0013] In order to achieve the above-mentioned purposes of the present application, the following technical solutions are adopted:

[0014] In a first aspect, the present application provides a heptamethine cyanine near-infrared fluorescent dye, which has the structure shown in the following formula I:

[0015] Formula I;

[0016] In the formula, X or Y is independently a hydrogen ion or a salt-forming positive ion; m or n is independently 3 or 4; g is an integer from 0 to 20, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.

[0017] In the present application, the heptamethine cyanine near-infrared fluorescent dye is modified based on NIR-04, obtaining a water-soluble, non-liver-accumulating, long-time-retained passive-targeting broad-spectrum fluorescent contrast agent, which is applied to clinical tumor diagnosis and intraoperative navigation.

[0018] Preferably, the salt-forming positive ion comprises a positive alkali metal ion and / or NH4 + .

[0019] Preferably, the positive alkali metal ion is Na + and / or K + .

[0020] In a second aspect, the present application provides a preparation method of the heptamethine cyanine near-infrared fluorescent dye according to the first aspect, the preparation method comprising the following steps:

[0021] (1) mixing ((9H-fluoren-9-yl)methoxy)carbonyl tripeptide, 2-(tritylthio)ethylamine, a condensation agent, a base and a reaction solvent, and then performing post-treatment after condensation reaction to obtain intermediate a;

[0022] (2) mixing intermediate a, trifluoroacetic acid, triisopropylsilane and water, and then performing post-treatment after reaction to obtain intermediate b;

[0023] (3) mixing a dye molecule, intermediate b, a base and a reaction solvent, and then performing post-treatment after reaction to obtain intermediate c;

[0024] The dye molecule has the structure shown in the following formula II:

[0025] Formula II

[0026] wherein X or Y is independently hydrogen ion or a salt-forming positive ion; and m or n is independently 3 or 4;

[0027] (4) mixing intermediate c, a secondary amine and a reaction solvent, and then performing post-treatment after reaction to obtain the heptamethine cyanine near-infrared fluorescent dye.

[0028] Preferably, in step (1), the molar ratio of ((9H-fluoren-9-yl)methoxy)carbonyl tripeptide, 2-(tritylthio)ethylamine, the condensation agent and the base is 1:(0.8-1.2):(1.5-3):(2-4);

[0029] For example, “0.8-1.2” can be 0.8, 0.9, 1, 1.1, 1.2, etc.

[0030] For example, “1.5-3” can be 1.5, 1.8, 2, 2.2, 2.5, 3, etc.

[0031] For example, “2-4” can be 2, 2.5, 3, 3.5, 4, etc.

[0032] Preferably, in step (1), the mass ratio of the reaction solvent to the ((9H-fluoren-9-yl)methoxy)carbonyl tripeptide is (5-20): 1, which can be 5:1, 6:1, 8:1, 10:1, 12:1, 14:1, 16:1, 18:1, 20:1, etc.

[0033] Preferably, in step (1), the condensing agent is selected from any one or a combination of at least two of HATU, HBTU, HCTU, HOAT or HOBT.

[0034] Preferably, in step (1), the base is selected from triethylamine and / or diisopropylethylamine.

[0035] Preferably, in step (1), the reaction solvent is a polar solvent.

[0036] Preferably, in step (1), the polar solvent is selected from any one or a combination of at least two of dimethylformamide, dimethyl sulfoxide or N-methyl pyrrolidone.

[0037] Preferably, in step (1), the temperature of the condensation reaction is 10-40℃, which can be 10℃, 15℃, 20℃, 25℃, 30℃, 35℃, 40℃, etc., and the time of the condensation reaction is 0.5-2h, which can be 0.5h, 0.6h, 0.8h, 1h, 1.2h, 1.4h, 1.6h, 1.8h, 2h, etc.

[0038] Preferably, in step (1), the post-treatment specifically includes the following steps: adding water to the reaction liquid obtained after the condensation reaction in step (1) to form a suspension; extracting the suspension with an organic solvent, collecting the organic phase, and then drying and concentrating to obtain the intermediate a.

[0039] Preferably, in step (1), the organic solvent used for extraction is ethyl acetate.

[0040] Preferably, in step (1), the volume ratio of the reaction liquid, water and organic solvent is 1:(0.8-1.2):(1-5).

[0041] For example, "0.8-1.2" can be 0.8, 0.9, 1, 1.1, 1.2, etc.

[0042] For example, "1-5" can be 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, etc.

[0043] Preferably, in step (2), the volume ratio of trifluoroacetic acid, triisopropylsilane and water is (94-96):(2-3):(2-3).

[0044] Wherein, "94-96" may be 94, 94.5, 95, 95.5, 96, etc.

[0045] Wherein, "2-3" may be 2, 2.2, 2.4, 2.6, 2.8, 3, etc.

[0046] Preferably, in step (2), the volume ratio of the mixture of trifluoroacetic acid, triisopropylsilane and water to the organic solvent used for extraction is 1:(0.8-1.2), which may be 1:0.8, 1:0.9, 1:1, 1:1.1, 1:1.2, etc.

[0047] Preferably, in step (2), the reaction temperature is 10-40℃, which may be 10℃, 15℃, 20℃, 25℃, 30℃, 35℃, 40℃, etc., and the reaction time is 0.5-2h, which may be 0.5h, 0.6h, 0.8h, 1h, 1.2h, 1.4h, 1.6h, 1.8h, 2h, etc.

[0048] Preferably, in step (2), the post-treatment specifically comprises the following steps: after adjusting the pH of the reaction solution obtained after step (2) to neutral, filtering, concentrating and column chromatography are performed to obtain intermediate b.

[0049] Preferably, in step (2), the reagent for adjusting pH is sodium carbonate solution.

[0050] Preferably, in step (3), the molar ratio of the dye molecule, intermediate b and base is 1:(0.8-1.2):(1.5-4).

[0051] Wherein, "0.8-1.2" may be 0.8, 0.9, 1, 1.1, 1.2, etc.

[0052] Wherein, "1.5-4" may be 1.5, 2, 2.5, 3, 3.5, 4, etc.

[0053] Preferably, in step (3), the mass ratio of the reaction solvent to intermediate b is (5-20):1, which may be 5:1, 6:1, 8:1, 10:1, 12:1, 14:1, 16:1, 18:1, 20:1, etc.

[0054] Preferably, in step (3), the base is selected from triethylamine and / or diisopropylethylamine.

[0055] Preferably, in step (3), the reaction solvent is a polar solvent.

[0056] Preferably, the polar solvent is selected from any one or a combination of at least two of dimethylformamide, dimethyl sulfoxide or N-methyl pyrrolidone.

[0057] Preferably, in step (3), the temperature of the reaction is 10-40℃, for example, it can be 10℃, 15℃, 20℃, 25℃, 30℃, 35℃, 40℃, etc., and the reaction time is 0.5-2h, for example, it can be 0.5h, 0.6h, 0.8h, 1h, 1.2h, 1.4h, 1.6h, 1.8h, 2h, etc.

[0058] Preferably, in step (3), the post-treatment specifically comprises the following steps: adding an organic solvent to the reaction liquid obtained after the reaction in step (3), and then centrifuging and filtering to obtain the intermediate c.

[0059] Preferably, in step (3), the organic solvent is ethyl acetate and / or methyl tert-butyl ether.

[0060] Preferably, in step (4), the molar ratio of the intermediate c to the secondary amine is 1:(1-2), for example, it can be 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, etc.

[0061] Preferably, in step (4), the mass ratio of the reaction solvent to the intermediate c is (5-20):1, for example, it can be 5:1, 6:1, 8:1, 10:1, 12:1, 14:1, 16:1, 18:1, 20:1, etc.

[0062] Preferably, in step (4), the secondary amine is selected from any one or a combination of at least two of piperidine, morpholine, or diethylamine.

[0063] Preferably, in step (4), the reaction solvent is a polar solvent.

[0064] Preferably, in step (4), the polar solvent is selected from any one or a combination of at least two of dimethylformamide, dimethyl sulfoxide, or N-methyl pyrrolidone.

[0065] Preferably, in step (4), the temperature of the reaction is 10-40℃, for example, it can be 10℃, 15℃, 20℃, 25℃, 30℃, 35℃, 40℃, etc., and the reaction time is 2-6h, for example, it can be 2h, 2.5h, 3h, 3.5h, 4h, 4.5h, 5h, 5.5h, 6h, etc.

[0066] Preferably, in step (4), the post-treatment specifically comprises the following steps: adding an organic solvent to the reaction liquid obtained after the reaction in step (4), filtering to obtain a crude product, and then purifying by liquid chromatography to obtain the heptamethine cyanine near-infrared fluorescent dye.

[0067] Preferably, in step (4), the organic solvent is ethyl acetate and / or methyl tert-butyl ether.

[0068] More specifically, the heptamethine cyanine near-infrared fluorescent dye obtained in step (4) has a structure as shown in Formula I, and g is 2.

[0069] As an optional technical solution of the present application, the preparation method of the heptamethine cyanine near-infrared fluorescent dye further comprises the following steps:

[0070] (5) mixing the heptamethine cyanine near-infrared fluorescent dye prepared in step (4), raw material 2, condensing agent, base and reaction solvent, and after condensation reaction, carrying out post-treatment to obtain intermediate e; wherein the raw material 2 is selected from ((9H-fluoren-9-yl)methoxy)carbonyl tri-glycine or (tert-butoxycarbonyl)glycylglycylglycine;

[0071] (6) carrying out deprotection group reaction on the intermediate e, and then carrying out post-treatment to obtain the heptamethine cyanine near-infrared fluorescent dye.

[0072] More specifically, the heptamethine cyanine near-infrared fluorescent dye obtained in step (6) has a structure as shown in Formula I, and g is 5.

[0073] Preferably, in step (5), the molar ratio of the raw material 2, the heptamethine cyanine near-infrared fluorescent dye prepared in step (4), the condensing agent and the base is 1:(0.8-1.2):(1.5-3):(2-4).

[0074] For example, "0.8-1.2" can be 0.8, 0.9, 1, 1.1, 1.2, etc.

[0075] For example, "1.5-3" can be 1.5, 1.8, 2, 2.2, 2.5, 3, etc.

[0076] For example, "2-4" can be 2, 2.5, 3, 3.5, 4, etc.

[0077] Preferably, in step (5), the mass ratio of the reaction solvent to the raw material 2 is (5-20):1, for example, it can be 5:1, 6:1, 8:1, 10:1, 12:1, 14:1, 16:1, 18:1, 20:1, etc.

[0078] Preferably, in step (5), the condensing agent is selected from any one or a combination of at least two of HATU, HBTU, HCTU, HOAT or HOBT.

[0079] Preferably, in step (5), the base is selected from triethylamine and / or diisopropylethylamine.

[0080] Preferably, in step (5), the reaction solvent is a polar solvent.

[0081] Preferably, in step (5), the polar solvent is selected from any one or a combination of at least two of dimethylformamide, dimethyl sulfoxide or N-methyl pyrrolidone.

[0082] Preferably, in step (5), the temperature of the condensation reaction is 10-40℃, for example, it can be 10℃, 15℃, 20℃, 25℃, 30℃, 35℃, 40℃, etc., and the time of the condensation reaction is 0.5-2h, for example, it can be 0.5h, 0.6h, 0.8h, 1h, 1.2h, 1.4h, 1.6h, 1.8h, 2h, etc.

[0083] Preferably, in step (5), the post-treatment specifically comprises the following steps: adding an organic solvent to the reaction solution obtained after the condensation reaction in step (5), and then filtering and drying to obtain the intermediate e.

[0084] Preferably, in step (5), the organic solvent added to the reaction solution is ethyl acetate and / or methyl tert-butyl ether.

[0085] Preferably, in step (6), the reagent used for the deprotection group is a secondary amine and / or trifluoroacetic acid.

[0086] Preferably, in step (6), the secondary amine used for the deprotection group is selected from any one or a combination of at least two of piperidine, morpholine or diethylamine.

[0087] Preferably, steps (5) and (6) are repeated until a seven-methine cyanine near-infrared fluorescent dye of formula I is prepared.

[0088] In a third aspect, the present application provides a use of the seven-methine cyanine near-infrared fluorescent dye according to the first aspect in the preparation of a fluorescent contrast agent.

[0089] In a fourth aspect, the present application provides a use of the seven-methine cyanine near-infrared fluorescent dye according to the first aspect in the preparation of a tumor diagnosis drug.

[0090] Preferably, the tumor is a liver cancer or colorectal cancer tumor.

[0091] Compared with the prior art, the present application has the following beneficial effects:

[0092] (1) The present application further modifies NIR-04, and through amide condensation, de-trityl protecting group, nucleophilic substitution, deprotection and other reactions, a seven-methine cyanine near-infrared fluorescent dye is prepared, which improves the in vivo metabolic characteristics of NIR-04, such as no liver accumulation, and the fluorescent dye can be used for surgical navigation to resect tumors.

[0093] (2) The heptamethine cyanine near-infrared fluorescent dye prepared by the application has a broad spectrum of passive tumor targeting, and has the advantages of stronger tumor targeting ability, better water solubility, longer tumor retention time, lower dosage, and no accumulation in normal tissues under the same dosage, and can be applied not only to the preparation of fluorescent contrast agents or tumor diagnosis drugs, but also has application potential in the field of fluorescent guided tumor resection in clinical surgery. BRIEF DESCRIPTION OF DRAWINGS

[0094] In order to more clearly illustrate the specific embodiments of the application or the technical solutions in the prior art, the drawings needed in the specific embodiments or the prior art description will be briefly introduced below. Obviously, the drawings in the following description are some embodiments of the application, and other drawings can also be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0095] Fig. 1 is an absorption spectrum diagram of NY-ICG-04-01-04 prepared in Examples 1-4.

[0096] Fig. 2 is a fluorescence spectrum diagram of NY-ICG-04-01-04 prepared in Examples 1-4.

[0097] Fig. 3 is an imaging result diagram of the distribution of NY-ICG-01-03 prepared in Examples 1-3 and NY-ICG-04 provided by Comparative Example 1 in each organ of normal BCR mice.

[0098] Fig. 4 is an in vivo imaging result diagram of NY-ICG-04-01-04 prepared in Examples 1-4 and ICG provided by Comparative Example 2 in hepatocarcinoma HepG2 tumor-bearing mice.

[0099] Fig. 5 is an in vivo imaging result diagram of NY-ICG-04-03 prepared in Example 3 in colorectal cancer HCT116 tumor-bearing mice. DETAILED DESCRIPTION

[0100] Unless otherwise defined herein, scientific and technical terms used in connection with the application shall have the meanings that are commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear; however, in the event of any latent ambiguity, the definitions provided herein take precedence. In this application, the use of "or" means "and / or" unless otherwise stated. In addition, the use of the term "including" and other forms is non-limiting.

[0101] It is to be understood that the embodiments described herein are merely exemplary of the application and should not be considered limiting. Without further restriction, this application can be practiced with other embodiments constructed according to the same principles. Therefore, no limitation is intended. Any and all such modifications are intended to be included within the scope of the present application.

[0102] The technical solutions of the present application will be described clearly and completely in connection with the embodiments. Obviously, the described embodiments are part of the embodiments of the present application, but not all. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of the present application.

[0103] The present application will be further described by the following examples. Unless otherwise specified, the materials in the examples are prepared according to the existing methods or directly purchased from the market.

[0104] The materials and sources of the following examples and comparative examples are shown as follows:

[0105] Example 1

[0106] The present embodiment provides a heptamethine cyanine near-infrared fluorescent dye, which is NY-ICG-04-01 of the following formula:

[0107] The synthesis route of the heptamethine cyanine near-infrared fluorescent dye NY-ICG-04-01 is shown as follows:

[0108] The preparation method of the heptamethine cyanine near-infrared fluorescent dye NY-ICG-04-01 specifically includes the following steps:

[0109] (1) After (9H-fluoren-9-yl)methoxycarbonyl tripeptide (1 g, 1.0 eq), 2-(tritylthio)ethylamine (776 mg, 1.0 eq), HATU (1.85 g, 2.0 eq), N,N-diisopropyl ethylamine (941 mg, 3.0 eq) and DMSO (10 mL, 10V) were mixed, the reaction was carried out at room temperature for 1 h, the reaction was monitored by TLC (DCM:MeOH=50:1), after the reaction was completed, 10 mL of water was added, then extracted with ethyl acetate (10 mL x 3) for three times, washed with saturated brine (20 mL x 2) for two times, dried with anhydrous sodium sulfate and concentrated to obtain the intermediate a.

[0110] (2) To the intermediate a, add a mixture of trifluoroacetic acid, triisopropylsilane and water (10 mL), wherein the volume ratio of trifluoroacetic acid, triisopropylsilane and water is 95:2.5:2.5; react at room temperature for 1 h, TLC monitoring shows that after the reaction is complete, a white solid is precipitated after adjusting the pH to neutral with a sodium carbonate solution, filter and dry, column chromatography, to obtain intermediate b.

[0111] The structure is characterized by mass spectrometry and nuclear magnetic hydrogen spectrum, and the structure determination results are as follows:

[0112] LCMS (ESI): m / z: C 23 H 26 N4O S , [M+H] + calcd for 470.16; found, 470.8.

[0113] 1 H NMR (600 MHz, DMSO-d6): δ 8.16 (ddt, J = 17.4, 11.5, 5.8 Hz, 2H), 8.01-7.84 (m, 3H), 7.73 (dt, J = 13.4, 7.4 Hz, 2H), 7.59 (t, J = 6.2 Hz, 1H), 7.45-7.29 (m, 4H), 4.30 (t, J = 6.6 Hz, 1H), 4.26-4.15 (m, 1H), 3.78-3.72 (m, 2H), 3.68 (t, J = 6.1 Hz, 3H), 3.34 (s, 7H), 3.26-3.19 (m, 2H), 2.58-2.52 (m, 1H), 1.36-1.17 (m, 1H).

[0114] (3) Mix NY-ICG-04 (100 mg, 1.0 eq), intermediate b (66 mg, 1.1 eq), DIPEA (50 mg, 3.0 eq) and DMSO (1 mL, 10V), and react at room temperature for 1 h, HPLC monitoring shows that the reaction of NY-ICG-04 is complete, drop the reaction solution into ethyl acetate (10 mL), precipitate green solid, centrifuge and precipitate to obtain intermediate c.

[0115] (4) After adding DMF (1 mL) and morpholine (17 mg, 1.5 eq) to intermediate c, react at room temperature for 4 h, HPLC monitoring shows that the reaction is complete, drop the reaction solution into ethyl acetate (10 mL) to precipitate green solid, purify by preparative liquid chromatography to obtain a target fraction solution, and freeze-dry to obtain near-infrared fluorescent probe NY-ICG-04-01.

[0116] The structure is characterized by mass spectrometry and nuclear magnetic hydrogen spectrum, and the structure determination results are as follows:

[0117] LCMS (ESI): m / z: [M-H] - C 44 H 59 N6O 12 S4, calcd for 989.3; found, 989.3.

[0118] 1 H NMR (600 MHz, DMSO-d6): δ 8.79 (d, J = 14.1 Hz, 1H), 8.69 (d, J = 13.9 Hz, 1H), 8.60 (t, J = 5.7 Hz, 1H), 8.23 (t, J = 5.9 Hz, 1H), 8.12 (t, J = 5.7 Hz, 1H), 7.98 (t, J = 5.9 Hz, 3H), 7.81 (d, J = 1.6 Hz, 1H), 7.66 - 7.61 (m, 1H), 7.54 (d, J = 8.0 Hz, 1H), 7.46 - 7.37 (m, 2H), 7.28 (t, J = 7.4 Hz, 1H), 6.56 (d, J = 14.3 Hz, 1H), 6.48 (d, J = 14.1 Hz, 1H), 4.41 - 4.36 (m, 2H), 4.34 - 4.29 (m, 2H), 3.82 (d, J = 5.7 Hz, 2H), 3.67 (d, J = 5.8 Hz, 2H), 3.61 (q, J = 5.9 Hz, 2H), 3.27 (q, J = 6.7 Hz, 2H), 2.86 (dd, J = 8.8, 6.2 Hz, 2H), 2.69 (d, J = 5.3 Hz, 3H), 2.61 (t, J = 6.8 Hz, 4H), 2.03 (dt, J = 14.9, 7.5 Hz, 3H), 1.81 (p, J = 7.0, 6.5 Hz, 2H), 1.70 (d, J = 14.4 Hz, 12H).

[0119] 13CNMR (151 MHz, DMSO-d6): δ 173.06, 171.82, 169.21, 168.98, 166.74, 159.22, 158.96, 158.71, 158.45, 154.91, 146.08, 145.15, 144.68, 142.90, 142.63, 141.59, 140.75, 134.31, 133.75, 129.09, 126.58, 125.56, 122.96, 120.34, 116.47, 114.56, 112.65, 112.04, 110.49, 102.82, 101.93, 49.46, 49.01, 48.31, 43.33, 43.14, 42.33, 36.37, 27.91, 27.84, 26.35, 26.27, 23.95, 23.75, 21.13.

[0120] Example 2

[0121] The present example provides a heptamethine cyanine near-infrared fluorescent dye, which is NY-ICG-04-02 of the following formula:

[0122] The synthesis route of the heptamethine cyanine near-infrared fluorescent dye NY-ICG-04-02 is as follows:

[0123] The preparation method of the heptamethine cyanine near-infrared fluorescent dye NY-ICG-04-02 specifically comprises the following steps:

[0124] (5) After NY-ICG-04-01 (110 mg, 1.0 eq), BOC-glycine 3-COOH (65 mg, 2.0 eq), HATU (127 mg, 3.0 eq), DIPEA (72 mg, 5.0 eq) and DMSO (1.1 mL) are mixed, reaction is carried out at room temperature for 1 h, the reaction is monitored by HPLC, and after NY-ICG-04-01 is completely reacted, the reaction solution is dropped into ethyl acetate (11 mL), green flocculent precipitates, which are filtered and dried, to obtain intermediate e.

[0125] (6) After TFA (100 μL) is added to intermediate e, reaction is carried out at room temperature for 15 min, after removing TFA, NY-ICG-04-02 is obtained by preparative liquid chromatography purification.

[0126] The structure is characterized by mass spectrometry and nuclear magnetic hydrogen spectrum and nuclear magnetic carbon spectrum, and the structure determination results are as follows:

[0127] LCMS (ESI): m / z: [M] + calcd for C50 H 68 N9O 15 S4,1163.4;found,1163.6.

[0128] 1 H NMR (600 MHz, DMSO-d6): δ 8.80 (d, J = 14.1 Hz, 1H), 8.73-8.61 (m, 2H), 8.31 (t, J = 5.9 Hz, 1H), 8.16 (t, J = 5.8 Hz, 1H), 8.08 (dt, J = 15.9, 5.9 Hz, 2H), 8.01 (q, J = 6.3 Hz, 4H), 7.78 (s, 1H), 7.66-7.61 (m, 2H), 7.54 (d, J = 8.0 Hz, 1H), 7.43 (t, J = 8.3 Hz, 1H), 7.38 (d, J = 8.4 Hz, 1H), 7.29 (t, J = 7.4 Hz, 1H), 6.56 (d, J = 14.3 Hz, 1H), 6.47 (d, J = 14.0 Hz, 1H), 5.57 (s, 1H), 5.52 (s, 1H), 4.39 (t, J = 7.7 Hz, 2H), 4.34-4.28 (m, 2H), 3.84 (d, J = 5.7 Hz, 2H), 3.77-3.68 (m, 6H), 3.63 (q, J = 6.0 Hz, 4H), 3.25 (dt, J = 8.7, 5.2 Hz, 2H), 2.85 (dd, J = 8.8, 6.5 Hz, 2H), 2.69 (d, J = 5.3 Hz, 4H), 2.62 (td, J = 6.8, 3.7 Hz, 4H), 2.04 (dq, J = 14.5, 7.0 Hz, 4H), 1.81 (p, J = 6.4 Hz, 2H), 1.70 (d, J = 13.6 Hz, 12H).

[0129] 13 CNMR (151 MHz, DMSO-d6): δ 173.15, 171.71, 169.74, 169.54, 169.27, 166.90, 158.96, 158.70, 154.83, 146.14, 145.08, 144.58, 142.92, 142.61, 141.62, 140.69, 134.43, 133.73, 129.09, 126.58, 125.60, 122.96, 120.30, 116.49, 114.57, 112.06, 110.46, 102.89, 101.85, 49.49, 48.99, 48.29, 43.35, 43.13, 42.53, 36.15, 27.90, 27.83, 26.35, 23.94, 23.70, 21.12.

[0130] Example 3

[0131] The present example provides a heptamethine cyanine near-infrared fluorescent dye, which is NY-ICG-04-03 of the following formula:

[0132] Referring to Example 2, NY-ICG-04-03 was obtained after preparation and purification.

[0133] The structure was characterized by mass spectrometry and nuclear magnetic hydrogen spectrum, nuclear magnetic carbon spectrum, and the structure determination results are as follows:

[0134] LCMS (ESI): m / z: [M-2] 2- C 56 H 77 N 12 O 18 S4, calcd for 665.2; found, 664.9.

[0135] 1 H NMR (600 MHz, DMSO-d6): δ 8.80 (d, J = 14.1 Hz, 1H), 8.69 (d, J = 13.8 Hz, 1H), 8.65 (t, J = 5.8 Hz, 1H), 8.31 (t, J = 5.8 Hz, 1H), 8.20-8.12 (m, 4H), 8.07 (dt, J = 12.0, 5.8 Hz, 2H), 8.03-7.95 (m, 4H), 7.78 (s, 1H), 7.64 (td, J = 8.2, 1.3 Hz, 2H), 7.54 (d, J = 8.1 Hz, 1H), 7.45-7.41 (m, 1H), 7.38 (d, J = 8.3 Hz, 1H), 7.29 (t, J = 7.4 Hz, 1H), 6.56 (d, J = 14.3 Hz, 1H), 6.46 (d, J = 13.9 Hz, 1H), 5.55 (s, 1H), 5.33 (s, 1H), 4.39 (t, J = 7.7 Hz, 2H), 4.30 (d, J = 7.7 Hz, 2H), 3.85 (d, J = 5.7 Hz, 2H), 3.78-3.68 (m, 12H), 3.64 (t, J = 5.7 Hz, 4H), 3.25 (dt, J = 10.5, 5.8 Hz, 2H), 2.87-2.82 (m, 2H), 2.69 (q, J = 5.4 Hz, 4H), 2.63 (q, J = 6.4 Hz, 4H), 2.04 (dp, J = 14.7, 7.3 Hz, 4H), 1.81 (p, J = 6.1 Hz, 2H), 1.70 (d, J = 14.5 Hz, 12H).

[0136] 13 CNMR (151 MHz, DMSO-d6): δ 173.21, 171.67, 169.83, 169.56, 169.28, 166.92, 158.96, 158.71, 154.86, 146.19, 144.99, 144.54, 142.96, 142.60, 141.64, 140.70, 134.45, 133.71, 129.09, 126.61, 125.62, 122.97, 120.30, 116.50, 114.59, 112.07, 110.46, 102.92, 101.82, 49.50, 48.99, 48.29, 43.36, 43.12, 42.54, 42.37, 36.16, 27.89, 27.82, 26.35, 26.26, 23.93, 23.68, 21.11.

[0137] Example 4

[0138] The present example provides a heptamethine cyanine near-infrared fluorescent dye, which is NY-ICG-04-04 of the following formula:

[0139] Referring to Example 2, NY-ICG-04-04 was obtained after preparation and purification.

[0140] The structure was characterized by mass spectrometry and nuclear magnetic hydrogen spectrum, nuclear magnetic carbon spectrum, and the structure determination results are as follows:

[0141] LCMS (ESI): m / z: [M-2] 2- C 56 H 77 N 12 O 18 S4, calcd for 750.7; found, 751.1.

[0142] 1H NMR (600 MHz, DMSO-d6): δ 8.80 (d, J = 14.1 Hz, 1H), 8.31 (t, J = 5.6 Hz, 1H), 8.16 (dq, J = 9.6, 5.3 Hz, 6H), 8.06 (dd, J = 9.7, 5.6 Hz, 2H), 8.00 (d, J = 14.3 Hz, 4H), 7.78 (s, 1H), 7.67-7.60 (m, 2H), 7.54 (d, J = 8.0 Hz, 1H), 7.43 (t, J = 7.7 Hz, 1H), 7.38 (d, J = 8.3 Hz, 1H), 7.29 (t, J = 7.4 Hz, 1H), 6.55 (d, J = 14.2 Hz, 1H), 6.47 (d, J = 14.0 Hz, 1H), 4.35 (d, J = 43.6 Hz, 9H), 3.64 (t, J = 6.1 Hz, 10H), 3.25 (d, J = 8.1 Hz, 3H), 2.85 (t, J = 7.7 Hz, 2H), 2.69 (s, 4H), 2.64-2.58 (m, 5H), 2.39 (p, J = 1.9 Hz, 1H), 2.07-1.99 (m, 4H), 1.81 (s, 2H), 1.70 (d, J = 15.0 Hz, 13H), 1.24 (s, 1H), 0.01 (s, 1H).

[0143] Comparative Example 1

[0144] The present comparative example provides a heptamethine cyanine near-infrared fluorescent dye, which is NY-ICG-04 of the following formula:

[0145] Comparative Example 2

[0146] The present comparative example provides a heptamethine cyanine near-infrared fluorescent dye, which is ICG of the following formula:

[0147] Comparative Example 3

[0148] The present comparative example provides a heptamethine cyanine near-infrared fluorescent dye, which is YQ-04-SCH2CH2CONH(CH2CH2O)2CH2CH2NH2 of the following formula (originated from patent CN202110626918.7):

[0149] Test Example 1

[0150] Spectral Test

[0151] Test Sample: The heptamethine cyanine near-infrared fluorescent dyes provided in Examples 1-4.

[0152] Test method: each sample was configured into a 1 nmol aqueous solution; the absorption spectrum of each probe in the range of 500-900 nm was measured under an ultraviolet spectrophotometer (HITACHI, 3J1-0015); the fluorescence emission spectrum of each probe in the range of 750-850 nm was measured under an enzyme marker instrument (Molecule devices, D1524R).

[0153] Test results:

[0154] The absorption spectrum results of each sample are shown in Figure 1, and the maximum absorption of the heptamethine cyanine near-infrared fluorescent dye provided in Examples 1-4 is about 780 nm.

[0155] The emission spectrum results of each sample are shown in Figure 2, and the maximum emission spectrum of the heptamethine cyanine near-infrared fluorescent dye provided in Examples 1-4 is about 810 nm.

[0156] Test Example 2

[0157] Normal mouse in vivo metabolic distribution imaging

[0158] Test sample: heptamethine cyanine near-infrared fluorescent dyes provided in Examples 1-4, heptamethine cyanine near-infrared fluorescent dyes provided in Comparative Examples 1-3.

[0159] Test method: in normal ICR mice, each sample was administered by tail vein (at a dose of 0.5 mg / kg, 100 μL of glucose injection per mouse), and the mice were dissected at 4 h, 8 h, 12 h, and 24 h, respectively, and the main organs (heart, liver, spleen, lung, kidney, intestine, stomach, bone, fat, etc.) of the mice were taken for fluorescence imaging using a surgical fluorescence imaging system (Nanjing Nuoyuan Medical Instrument Co., Ltd., 10B).

[0160] Test results:

[0161] The fluorescence imaging results of each sample are shown in Figure 3, and the in vivo metabolism of the heptamethine cyanine near-infrared fluorescent dye provided in Examples 1-3 is basically consistent with that of Comparative Example 3 within 24 h after administration, has a certain lung fluorescence signal, and is basically excreted from the liver at 8 h after administration; compared with Comparative Example 1 (NY-ICG-04), the liver accumulation is reduced, and the heptamethine cyanine near-infrared fluorescent dye can be further developed and applied to liver disease detection. The results show that the in vivo clearance rate of the heptamethine cyanine near-infrared fluorescent dye provided in each example of the present application increases with the increase in the number of amino acids introduced; compared with Comparative Example 2 (ICG), the in vivo clearance rate shows a more significant increasing trend, and therefore the heptamethine cyanine near-infrared fluorescent dye has more application advantages.

[0162] Test Example 3

[0163] Subcutaneous tumor HepG2 tumor-bearing mouse model (human liver cancer cells) in vivo imaging

[0164] Test sample: The heptamethine cyanine near-infrared fluorescent dye provided in Examples 1-4 and the heptamethine cyanine near-infrared fluorescent dye provided in Comparative Examples 1-3.

[0165] Test method: In a subcutaneous tumor HepG2 tumor-bearing mouse model, each of the above samples was administered (0.5 mg / kg, 100 μL of glucose injection) via tail vein, with indocyanine green (0.5 mg / kg, 100 μL of glucose injection) as a control. After administration, fluorescence imaging was performed using a surgical fluorescence imaging system (Nanjing Nuoyuan Medical Instrument Co., Ltd., 10B), in the order of 0 h (before administration), 6 h, 12 h, 24 h, and 48 h.

[0166] Test results:

[0167] The fluorescence imaging results of each of the above samples are shown in FIG. 4. Compared with Comparative Example 2 (ICG), the heptamethine cyanine near-infrared fluorescent dye provided in the examples has stronger tumor targeting ability in liver cancer and longer tumor imaging time.

[0168] Compared with Comparative Example 1 (NY-ICG-04), the tumor targeting ability of the compound remains essentially unchanged, and effectively improves the deficiency of liver accumulation of non-metabolized Comparative Example 1.

[0169] Compared with Comparative Example 3 (YQ-04-SCH2CH2CONH(CH2CH2O)2CH2CH2NH2), the compound of the present application has comparable or stronger tumor targeting ability, and the normal tissue excretion rate is rapid, the normal tissue background signal is rapidly reduced, and has potential tumor detection ability and clinical application prospect.

[0170] Test Example 4

[0171] Test sample: The heptamethine cyanine near-infrared fluorescent dye provided in Example 3.

[0172] Test method: In a colorectal cancer (HCT116) tumor-bearing mouse model, the probe NY-ICG-04-03 (0.5 mg / kg, 100 μL of glucose injection) was administered via tail vein. After administration, fluorescence imaging was performed using a surgical fluorescence imaging system (Nanjing Nuoyuan Medical Instrument Co., Ltd., 10B), in the order of 0 h (before administration), 6 h, 12 h, 24 h, and 48 h.

[0173] Test results:

[0174] The fluorescence imaging results of the above probe NY-ICG-04-03 are shown in FIG. 5. The probe NY-ICG-04-03 also has good tumor targeting ability on a colorectal cancer (HCT116) tumor-bearing mouse, and has potential clinical application prospect, which needs to be further researched and developed for application in clinical surgery.

[0175] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, and are not intended to limit the present application; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that the technical solutions described in the foregoing embodiments can still be modified, or some or all of the technical features can be replaced by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A heptamethine cyanine near-infrared fluorescent dye, characterized in that The heptamethine cyanine near-infrared fluorescent dye has a structure shown in the following formula I: wherein X or Y is independently hydrogen ion or a salt-forming positive ion; m or n is independently 3 or 4; and g is an integer from 0 to 20.

2. The septamethine cyanine near-infrared fluorescent dye according to claim 1, characterized in that, The salt-forming positive ions include positive valent alkali metal ions and / or NH4 + ; wherein the positive valent alkali metal ion is Na + and / or K + .

3. The method of preparing a septamethine cyanine near-infrared fluorescent dye according to claim 1 or 2, characterized in that, The preparation method comprises the following steps: (1) mixing ((9H-fluoren-9-yl)methoxy)carbonyl tri-glycine, 2-(tritylthio)ethylamine, a condensing agent, a base and a reaction solvent, and then performing condensation reaction and post-treatment to obtain intermediate a; (2) mixing intermediate a, trifluoroacetic acid, triisopropylsilane and water, and then performing reaction and post-treatment to obtain intermediate b; (3) mixing a dye molecule, intermediate b, a base and a reaction solvent, and then performing reaction and post-treatment to obtain intermediate c; The dye molecule has a structure as shown in the following formula II: wherein X or Y is independently hydrogen ion or a salt-forming positive ion; m or n is independently 3 or 4; (4) mixing intermediate c, a secondary amine and a reaction solvent, and then performing reaction and post-treatment to obtain the heptamethine cyanine near-infrared fluorescent dye.

4. The method for preparing the heptamethine cyanine near-infrared fluorescent dye according to claim 3, wherein In step (1), the molar ratio of ((9H-fluoren-9-yl)methoxy)carbonyl tri-glycine, 2-(tritylthio)ethylamine, the condensing agent and the base is 1:(0.8-1.2):(1.5-3):(2-4); And / or, in step (1), the mass ratio of the reaction solvent to the ((9H-fluoren-9-yl)methoxy)carbonyl tri-glycine is (5-20):1; And / or, in step (1), the temperature of the condensation reaction is 10-40℃, and the condensation reaction time is 0.5-2h; And / or, in step (1), the post-treatment specifically comprises the following steps: adding water to the reaction solution obtained after the condensation reaction in step (1) to form a suspension; extracting the suspension with an organic solvent, collecting the organic phase, and then drying and concentrating to obtain intermediate a. In step (2), the volume ratio of trifluoroacetic acid, triisopropylsilane and water is (94-96):(2-3):(2-3); 5. The method of preparing a septamethine cyanine near-infrared fluorescent dye according to claim 3 or 4, characterized in that, And / or, in step (2), the volume ratio of the total volume of the mixture of trifluoroacetic acid, triisopropylsilane and water to the volume of the organic solvent for extraction is 1:(0.8-1.2); And / or, in step (2), the temperature of the reaction is 10-40℃, and the reaction time is 0.5-2h; And / or, in step (2), the post-treatment specifically comprises the following steps: adjusting the pH of the reaction solution obtained after the reaction in step (2) to neutral, and then performing filtration, concentration and column chromatography to obtain intermediate b. In step (3), the molar ratio of the dye molecule, intermediate b and the base is 1:(0.8-1.2):(1.5-4); 6. The method for preparing the heptamethine cyanine near-infrared fluorescent dye according to claim 3, wherein And / or, in step (3), the mass ratio of the reaction solvent to intermediate b is (5-20):1; And / or, in step (3), the temperature of the reaction is 10-40℃, and the reaction time is 0.5-2h; And / or, in step (3), the post-treatment specifically comprises the following steps: adding an organic solvent to the reaction solution obtained after the reaction in step (3), and then performing centrifugation and filtration to obtain intermediate c; wherein the organic solvent is ethyl acetate and / or methyl tert-butyl ether. ​ 7. The method for preparing the heptamethine cyanine near-infrared fluorescent dye according to claim 6, wherein In step (4), the molar ratio of the intermediate c to the secondary amine is 1:(1-2); In step (4), the mass ratio of the reaction solvent to the intermediate c is (5-20):1; In step (4), the reaction temperature is 10-40℃, and the reaction time is 2-6h; In step (4), the post-treatment specifically comprises the following steps: adding an organic solvent to the reaction liquid obtained after the reaction in step (4), filtering to obtain a crude product, and then purifying by liquid chromatography to obtain the heptamethine cyanine near-infrared fluorescent dye; wherein the organic solvent is ethyl acetate and / or methyl tert-butyl ether; In step (4), the heptamethine cyanine near-infrared fluorescent dye has a structure as shown in formula I, and g is 2.

8. The method for preparing the heptamethine cyanine near-infrared fluorescent dye according to claim 7, wherein: The preparation method of the heptamethine cyanine near-infrared fluorescent dye further comprises the following steps: (5) mixing the heptamethine cyanine near-infrared fluorescent dye prepared in step (4), a raw material 2, a condensation agent, a base and a reaction solvent, and then performing post-treatment after condensation reaction to obtain an intermediate e; wherein the raw material 2 is selected from ((9H-fluoren-9-yl)methoxy)carbonyl tri-glycine or (tert-butoxycarbonyl)glycylglycylglycine; (6) performing a deprotection group reaction on the intermediate e, and then performing post-treatment to obtain the heptamethine cyanine near-infrared fluorescent dye; In step (6), the heptamethine cyanine near-infrared fluorescent dye has a structure as shown in formula I, and g is 5; In step (6), the heptamethine cyanine near-infrared fluorescent dye has a structure as shown in formula I, and g is 5; 9. Use of the heptamethine cyanine near-infrared fluorescent dye according to claim 1 or 2 in the preparation of a fluorescent contrast agent.

10. Use of the heptamethine cyanine near-infrared fluorescent dye according to claim 1 or 2 in the preparation of a tumor diagnosis drug.

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