An ir-780 derivative rapidly targeted to accumulate in tumor tissue and a preparation method and application thereof

Abstract

The application discloses a preparation method of an IR-780 derivative which can be quickly targeted and accumulated in tumor tissues, and comprises the following steps: step 1, adding potassium iodide into a dry round-bottom flask provided with a magnetic stirrer, then replacing air sufficiently, adding substrates A and B under the condition of nitrogen protection, then adding super-dry solvent into the system, heating and refluxing for a certain time, removing excessive potassium iodide by filtering through diatomite after the reaction is completed, adding petroleum ether after the solvent is spun dry, fully stirring and repeatedly washing for several times, repeatedly washing with petroleum ether / ethyl acetate after the petroleum ether washing, and obtaining a pure red powder solid compound C after the washing is completed; it is tested that the long carbon chain substitution containing fluorine obviously accelerates the kinetics of distribution in vivo, but the rare tumor targeting property is reserved; compared with the tumor targeting organic compound IR-780, the derivative can be more quickly targeted and accumulated in tumor tissues after being intravenously injected into tumor-bearing nude mice.

Description

Technical Field

[0001] This invention relates to the field of molecular probe technology, and in particular to an IR-780 derivative that rapidly targets and accumulates in tumor tissue, its preparation method, and its application. Background Technology

[0002] Cyanine (Cy) dyes are an important class of small organic molecule dyes, with a history dating back to the 1850s. Since Greville Williams reported the synthesis of the first cyanine dye, querline blue, in 1856, research on cyanine dyes has developed rapidly, and various cyanine dyes and their derivatives with different structures have been synthesized. By controlling the large π-conjugated system and delocalized electron distribution in cyanine dyes, researchers have designed and prepared organic dyes with special photophysical and photoelectric properties to meet different application needs. Currently, the research and application of cyanine dyes are widely valued for their use in biometric imaging, nucleic acid labeling, DNA sequencing, and tumor photodynamic and photothermal therapy.

[0003] Cyanide dye IR-780 absorbs near-infrared light and emits near-infrared fluorescence. It can selectively accumulate in various tumor cells without the need for conjugated antibodies, peptides, or nucleic acid aptamers, exhibiting broad-spectrum tumor targeting and therapeutic effects including chemical toxicity, photothermal activity, photodynamic activity, and sonodynamic activity. However, in experimental animal models, IR-780 injected via the tail vein of tumor-bearing nude mice takes a considerable amount of time to accumulate at the tumor site. Furthermore, IR-780 present in non-tumor tissues requires a long time to be metabolized and eliminated from the body, resulting in a prolonged waiting period before targeted accumulation is achieved after injection. To overcome these shortcomings, structural modifications to IR-780 are needed to obtain IR-780 derivatives with varying properties, which is crucial for constructing high-performance tumor-targeted diagnostic and therapeutic reagents or drugs. The introduction of fluorine atoms can significantly alter the physical and chemical properties of organic / inorganic compounds. The lipophilic nature of fluorine atoms is highly beneficial for intracellular drug delivery; therefore, fluorinated compounds exhibit higher delivery efficiency and utilization rates when applied in vivo.

[0004] To address the problem of the slow accumulation rate of the tumor-targeting organic compound IR-780 in tumors, this invention proposes an IR-780 derivative that rapidly accumulates in tumor tissue, along with its preparation method and applications. Summary of the Invention

[0005] (a) Technical problems to be solved

[0006] To overcome the shortcomings of existing technologies, this invention provides an IR-780 derivative that can rapidly target and accumulate in tumor tissues, along with its preparation method and applications. By replacing the n-propyl group on the nitrogen atom in the IR-780 structure with a fluorine-containing long carbon chain, the physical properties of the molecule, such as its lipophilicity, are altered, thereby obtaining a compound that can target and accumulate in tumors at a faster rate. This solves the problem mentioned in the background art that the current tumor-targeting organic compound IR-780 accumulates in tumors too slowly.

[0007] (II) Technical Solution

[0008] To address the aforementioned technical problems, this invention provides an IR-780 derivative that rapidly targets and accumulates in tumor tissue, with the following structural formula:

[0009]

[0010] This invention also provides a method for preparing an IR-780 derivative that rapidly targets and accumulates in tumor tissue, characterized by comprising the following steps:

[0011] Step 1: After adding potassium iodide to a dry round-bottom flask equipped with a magnetic stirrer, the gas was completely replaced. Under nitrogen protection, substrates A and B were added, and then ultra-dry solvent was added to the system. The mixture was heated under reflux for a certain period of time. After the reaction was completed, excess potassium iodide was removed by diatomaceous earth filtration. After the solvent was evaporated, petroleum ether was added and stirred thoroughly and washed repeatedly. After washing with petroleum ether, the mixture was washed repeatedly with petroleum ether / ethyl acetate. After washing, a pure red powdery solid compound C was obtained.

[0012] The structural formula of compound A is as follows:

[0013] The structural formula of compound B is:

[0014] Step 2: After adding substrates C and D to a dry round-bottom flask equipped with a magnetic stirrer, the gas was fully purged. The prepared solvent was added to dissolve the substrates, and the reaction was heated for a certain time. The reaction system changed from red to green as the reaction time progressed. After the reaction was completed, the solvent was thoroughly evaporated, and the product was purified by silica gel column chromatography. Small polar impurities were first removed with dichloromethane, and then a dark green solid product was obtained by filtration with a certain volume ratio of dichloromethane / methanol. After obtaining the solid product, it was washed thoroughly with petroleum ether / ethyl acetate several times. After washing, a pure dark green solid product was obtained, which is the IR-780 derivative.

[0015] Wherein: the structural formula of compound C is:

[0016] The structural formula of compound D is:

[0017] In a preferred embodiment of the method for preparing an IR-780 derivative that is rapidly targeted and accumulated in tumor tissue according to the present invention, the molar ratio of compounds A and B in step 1 is 1:1.2.

[0018] In a preferred embodiment of the method for preparing an IR-780 derivative that is rapidly targeted and accumulated in tumor tissue according to the present invention, the reaction temperature in step 1 is 110°C and the reaction time is 48 h.

[0019] In a preferred embodiment of the method for preparing an IR-780 derivative that is rapidly targeted and accumulated in tumor tissue according to the present invention, the ultra-dry solvent in step 1 is acetonitrile.

[0020] In a preferred embodiment of the method for preparing an IR-780 derivative that is rapidly targeted and accumulated in tumor tissue according to the present invention, in step 2, the molar ratio of compounds C and D is 2:1.

[0021] In a preferred embodiment of the method for preparing an IR-780 derivative that is rapidly targeted and accumulated in tumor tissue according to the present invention, in step 2, the solvent is toluene / n-butanol with a volume ratio of 7 / 3.

[0022] In a preferred embodiment of the method for preparing an IR-780 derivative that is rapidly targeted and accumulated in tumor tissue according to the present invention, in step 2, the volume ratio of dichloromethane / methanol is 50 / 1 and the volume ratio of petroleum ether / ethyl acetate is 10 / 1.

[0023] The present invention provides an IR-780 derivative that rapidly targets and accumulates in tumor tissue, which is synthesized by the above-described synthesis method.

[0024] In addition to the above, the present invention also provides an application of an IR-780 derivative that rapidly targets and accumulates in tumor tissue, wherein the IR-780 derivative is used in the preparation of reagents for tumor diagnosis and treatment.

[0025] (III) Beneficial Effects

[0026] Compared with the prior art, the beneficial effects that this invention can achieve are:

[0027] This invention replaces the n-propyl group on the nitrogen atom of the organic structure of IR-780 with a fluorinated long carbon chain without significantly altering the morphology of IR-780. The charge of the cationic structure remains unchanged. Tests show that the fluorinated long carbon chain substitution significantly accelerates the distribution kinetics in vivo while retaining the rare tumor targeting properties. The time required for targeted accumulation in tumors of tumor-bearing nude mice is significantly shortened. Compared with the tumor-targeting organic compound IR-780, the derivative of this invention, when injected intravenously into tumor-bearing nude mice, can more rapidly and targetedly accumulate in tumor tissue. Attached Figure Description

[0028] Figure 1 The 1H NMR spectrum of compound E of this invention;

[0029] Figure 2 The 19F NMR spectrum of compound E of this invention;

[0030] Figure 3 Here is the HRMS chromatogram of compound E of this invention;

[0031] Figure 4 Fluorescence in vivo imaging of tumor-bearing nude mice. (A) shows fluorescence images at different time points after injection of compound E into tumor-bearing mice; (B) shows fluorescence images at different time points after injection of IR-780 into tumor-bearing mice.

[0032] Figure 5 for Figure 4 Fluorescence ratio of the ROI delineated area at different time points (fluorescence intensity of tumor I1 / fluorescence intensity of non-tumor, stronger fluorescence area I2). Detailed Implementation

[0033] To make the technical means, creative features, and achieved objectives and effects of this invention easier to understand, the invention is further described below with reference to specific embodiments. However, the following embodiments are merely preferred embodiments of this invention and not all of them. Other embodiments obtained by those skilled in the art based on the embodiments described herein without creative effort are all within the protection scope of this invention. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0034] Example:

[0035] like Figure 1-5 As shown, the present invention provides:

[0036] Example 1

[0037] An IR-780 derivative that rapidly targets and accumulates in tumor tissue has the following structural formula:

[0038]

[0039] A method for preparing an IR-780 derivative that rapidly targets and accumulates in tumor tissue includes the following steps:

[0040]

[0041] (1) Potassium iodide (50 mmol) was added to a dry round-bottom flask equipped with a magnetic stirrer, and the gas was completely purged. Under nitrogen protection, substrate A (3.4 mmol) and substrate B (4.1 mmol) were added, followed by the addition of ultra-dry solvent acetonitrile (acetonitrile is an organic compound with the chemical formula CH3CN or C2H3N, a colorless and transparent liquid with excellent solvent properties, capable of dissolving a variety of organic, inorganic, and gaseous substances, and infinitely miscible with water and alcohols. Acetonitrile can undergo typical nitrile reactions and is used to prepare many typical nitrogen-containing compounds, making it an important organic intermediate), and the mixture was refluxed at 110 °C for 48 h. After the reaction, excess potassium iodide (KI) was removed by diatomaceous earth filtration. The solvent was then evaporated, and petroleum ether (petroleum ether is a light petroleum product, a mixture of low molecular weight hydrocarbons (mainly pentane and hexane), a colorless, transparent liquid with a kerosene odor. It is insoluble in water but soluble in most organic solvents such as ethanol, benzene, chloroform, and oils. It is mainly used as a solvent and for oil and fat treatment. Although petroleum ether is not gasoline, it is volatile and flammable, so caution must be taken when using it) was added. The mixture was thoroughly stirred and washed several times. After washing with petroleum ether, it was washed repeatedly with petroleum ether / ethyl acetate (>5 times). After washing, a pure red powdery solid C (0.75 g, yield 55%) was obtained.

[0042] (2) After adding substrate C (1.9 mmol) and D (0.94 mmol) to a dry round-bottom flask equipped with a magnetic stirrer, the gas was completely purged. After dissolving in the prepared solvent, the reaction was carried out at 110°C for 12 h, during which time the reaction system changed from red to green. After the reaction was completed, the solvent was thoroughly evaporated, and the mixture was purified by silica gel column chromatography (the principle of silica gel column chromatography is based on the hydrophilicity and lipophilicity of silica gel. Silica gel is a porous solid material with a large number of hydroxyl and methyl groups on its surface, thus exhibiting strong hydrophilicity and lipophilicity. In silica gel column chromatography purification, sample molecules first interact with the hydroxyl or methyl groups on the silica gel surface to form an adsorbed state. Then, through the action of the eluent, molecules with different affinities are adsorbed and eluted to different degrees in the silica gel column, thereby achieving separation and purification). Small polar impurities were first removed with dichloromethane, and then a dark green solid product was obtained by dichloromethane / methanol (50 / 1). After obtaining the solid product, it was thoroughly washed several times with petroleum ether / ethyl acetate (10 / 1) (because petroleum ether is a weakly polar organic solvent, it can be used as a developing solvent for thin-layer chromatography when mixed with the strongly polar solvent ethyl acetate). After washing, a pure dark green solid product was obtained, which is IR-780 derivative E (0.64 g, yield 80%).

[0043] 1H NMR (600MHz, DMSO-D6) δ (ppm): 8.29 (d, 2H, J = 13.8), 7.65 (d, 2H, J = 7.2), 7.54 (d, 2H, J = 7.8), 7.46 (t, 2H, J = 7.5), 7.31 (t, 2H, J =7.5),6.38(d,2H,J=13.8),4.32(t,4H,J=7.5),2.73(t,4H,J=5.7),2.51(t,4H,J=1.8),1.99-1.88(m,6H),1.69(s,12H); 19F NMR (376MHz, DMSO) δ (ppm): 84.64,117.06; HRMS m / z: calc.for C40H42N2F10Cl+, 775.2877, obsd.775.2868.

[0044] Example 2

[0045] 1. Preparation of test solution

[0046] IR-780 derivative E and IR-780 were dissolved separately in DMSO (dimethyl sulfoxide, a sulfur-containing organic compound with the molecular formula C2H6OS, a colorless, odorless, transparent liquid at room temperature, and a hygroscopic, flammable liquid. It is highly polar, has a high boiling point, good thermal stability, is aprotic, miscible with water, and is soluble in most organic compounds such as ethanol, propanol, benzene, and chloroform, earning it the reputation of a "universal solvent") to prepare a stock solution with a concentration of 80 mg / ml. Before use, it was diluted with PBS (pH = 7.4) containing human serum albumin (20 mg / ml).

[0047] 2. Fluorescence imaging experiments and targeting analysis of the compound's distribution in tumor-bearing nude mice.

[0048] The experimental animal model used was BALB / c Nude mice. Mouse breast cancer 4T1 cells were cultured and well-grown cells were dispersed in PBS buffer. (Buffer solution is a solution resistant to pH changes; its pH remains relatively stable even when diluted or when a small amount of acid or base is added. It is typically composed of a weak acid and its conjugate base, or a weak base and its conjugate acid. Phosphate buffer solution...) Buffered Saline (PBS) is a commonly used buffer in biological research to maintain a stable pH environment. PBS buffer is prepared from water, sodium chloride, potassium chloride, the weak acid KH₂PO₄, and the conjugate base Na₂HPO₄, and is typically adjusted to pH 7.4. The pH of the human body ranges from 7.35 to 7.45, with an average of 7.40. PBS buffer also mimics the osmotic pressure and ion concentration of the human body. Because it is non-toxic to cells, it is widely used for washing cells, transporting tissues, and diluting them. Especially in animal cell culture experiments, it is often used for washing cells before trypsin digestion to thoroughly remove serum. In [the study], a tumor model was constructed by subcutaneously injecting 100 μl of cell suspension (1 × 10⁶ cells / 100 μl) into each mouse.

[0049] The stock solutions of IR-780 derivative E and IR-780 were diluted 100-fold with PBS buffer (containing 20 mg / ml human serum albumin, pH = 7.4) to obtain an injection solution with a concentration of 0.8 mg / ml. This solution was injected intravenously (100 μL / mouse) into tumor-bearing nude mice. Immediately after injection, mice were anesthetized for in vivo imaging. The anesthetic gas was removed after imaging, and anesthesia was repeated before the next imaging session. Fluorescence in vivo imaging was performed using a fluorescence in vivo imaging system to obtain fluorescence images of tumor-bearing mice at 0.2 h, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, and 36 h after injection of E or IR-780. Figure 4 ). Figure 4 This indicates that at 8 hours, E can be easily identified by the naked eye as having achieved targeted accumulation in mice, with tumor signals showing significant differences from image signals in other regions, while IR-780 only showed good targeting at 36 hours.

[0050] Two regions of interest (ROIs) were selected for each mouse, namely the tumor region and other highly fluorescent regions, for image analysis. The ratio of the fluorescence intensity of the tumor to that of other highly fluorescent regions in the same mouse over time was used to characterize the accumulation of IR-780 and IR-780 derivative E in vivo in mice over time. Figure 5 The fluorescence signal originated from IR-780 or its derivative E, and the signal intensity in different regions was directly proportional to the molar concentration of IR-780 or derivative E in those regions. Results showed that derivative E exhibited significantly better targeting performance than IR-780 from 8 to 36 hours, with a fluorescence ratio remaining stable around 2.0. In contrast, tumor-bearing mice injected with IR-780 only showed significant fluorescence targeting at 36 hours, and the fluorescence ratio increased over time, reaching only 1.2 at 36 hours. Therefore, the IR-780 derivative of this invention can more rapidly target and accumulate in tumor tissue compared to IR-780.

[0051] In summary, this invention replaces the n-propyl group on the nitrogen atom of the IR-780 organic structure with a fluorine-containing long carbon chain without significantly altering the morphology of IR-780. The charge of the cationic structure remains unchanged. Tests show that the fluorine-containing long carbon chain substitution significantly accelerates the distribution kinetics in vivo while retaining the rare tumor targeting properties. The time required for targeted accumulation in tumors of tumor-bearing nude mice is significantly shortened. Compared with the tumor-targeting organic compound IR-780, the derivative of this invention, when injected intravenously into tumor-bearing nude mice, can more rapidly and effectively accumulate in tumor tissue.

[0052] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0053] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

[0054] Furthermore, various different embodiments of the present invention can be combined in any way, as long as they do not violate the spirit of the present invention, they should also be regarded as the content disclosed by the present invention.

Claims

1. An IR-780 derivative that rapidly targets and accumulates in tumor tissue, characterized in that: Its structural formula is: 。 2. A method for preparing an IR-780 derivative that rapidly targets and accumulates in tumor tissue, characterized in that: Includes the following steps: Step 1: After adding potassium iodide to a dry round-bottom flask equipped with a magnetic stirrer, the gas was completely replaced. Under nitrogen protection, substrates A and B were added, and then ultra-dry solvent was added to the system. The mixture was heated under reflux for a certain period of time. After the reaction was completed, excess potassium iodide was removed by diatomaceous earth filtration. After the solvent was evaporated, petroleum ether was added and stirred thoroughly and washed repeatedly. After washing with petroleum ether, the mixture was washed repeatedly with petroleum ether / ethyl acetate. After washing, a pure red powdery solid compound C was obtained. The structural formula of compound A is as follows: ; The structural formula of compound B is: ; Step 2: After adding substrates C and D to a dry round-bottom flask equipped with a magnetic stirrer, the gas was fully purged. The prepared solvent was added to dissolve the substrates, and the reaction was heated for a certain time. The reaction system changed from red to green as the reaction time progressed. After the reaction was completed, the solvent was thoroughly evaporated, and the product was purified by silica gel column chromatography. Small polar impurities were first removed with dichloromethane, and then a dark green solid product was obtained by filtration with a certain volume ratio of dichloromethane / methanol. After obtaining the solid product, it was washed several times with petroleum ether / ethyl acetate. After washing, a pure dark green solid product was obtained, which is the IR-780 derivative. Wherein: the structural formula of compound C is: ; The structural formula of compound D is: ; In step 1, the molar ratio of compounds A and B is 1:1.2; the reaction temperature in step 1 is 110℃ and the reaction time is 48h. The ultra-dry solvent in step 1 is acetonitrile; In step 2, the molar ratio of compounds C and D is 2:1; In step 2, the solvent is toluene / n-butanol, with a volume ratio of 7 / 3; In step 2, the volume ratio of dichloromethane to methanol is 50 / 1, and the volume ratio of petroleum ether to ethyl acetate is 10 / 1.

3. The application of the IR-780 derivative as described in claim 1, characterized in that: It is used in the preparation of reagents for tumor diagnosis and treatment.

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