Near-infrared dyes, methods of making and using the same

Abstract

The present application relates to the field of fluorescence imaging technology, in particular to a near-infrared dye and a preparation method and application thereof. The preparation method of the near-infrared dye is simple, the molecular structure is simple, the PDT and PTT effects can be fully exerted under the mediation of NIR, active oxygen and heat are generated, and the near-infrared dye can be used for intracellular mitochondrial-targeted optical therapy.

Description

Technical Field

[0001] This invention relates to the field of fluorescence imaging technology, and in particular to an infrared dye, its preparation method, and its application. Background Technology

[0002] Near-infrared fluorescence imaging combined with phototherapy has shown significant potential in precision oncology diagnosis and treatment. Photothermal therapy converts light energy into heat energy through photothermal agents, inducing thermal ablation of tumor cells; while photodynamic therapy generates reactive oxygen species (such as singlet oxygen) under light irradiation through photosensitizers, causing oxidative damage to cells. The combination of the two can overcome the limitations of single therapies and improve the treatment effect of deep tumors.

[0003] However, most near-infrared phototherapy agents currently lack organelle-specific targeting capabilities, resulting in their dispersed distribution within tumor cells and limited efficacy. Mitochondria, as key organelles in cellular energy metabolism and apoptosis regulation, are important targets for tumor therapy. Phototherapy agents targeting mitochondria can accumulate in mitochondria under the drive of mitochondrial membrane potential, enhancing the activation of apoptosis pathways by phototherapy, improving treatment efficiency, and reducing side effects.

[0004] Traditional targeting strategies typically require additional synthetic steps to couple the targeting group (such as triphenylphosphine), which not only increases molecular complexity but may also affect the photophysical properties or pharmacokinetics of the parent dye. Therefore, developing novel near-infrared dyes whose molecular structure inherently possesses targeting capabilities is of great significance for simplifying design and improving performance stability. Summary of the Invention

[0005] Based on this, the objective of the present invention includes providing a near-infrared dye that has strong absorption and high fluorescence emission in the near-infrared region, while also possessing excellent photothermal conversion efficiency and singlet oxygen generation capability, and can specifically accumulate in cell mitochondria to achieve imaging-guided optical therapy.

[0006] A first aspect of the present invention provides a near-infrared dye having a structure as shown in Formula I: I.

[0007] A second aspect of the present invention provides a method for preparing the near-infrared dye described above, comprising the following steps: Under inert gas protection, compounds of formula II and formula III are dissolved in a first organic solvent, and sodium acetate and acetic anhydride are added and mixed. The resulting mixture is stirred at a first temperature for a first time to obtain the near-infrared dye. The first temperature is 70℃-90℃; the first time is 6h-24h. II; III.

[0008] In some embodiments, the preparation method satisfies one or more of the following conditions: (1) The molar ratio of the compound of formula II to the compound of formula III is (2-2.1):1; (2) The molar ratio of the acetic anhydride or the sodium acetate to the compound of formula II is (1.8-2):1; (3) The ratio of the sum of the weights of the compounds of formula II and formula III in the mixture to the volume of the organic solvent is 1-2 g: 20 mL; (4) The first organic solvent includes ethanol; (5) The first temperature is 75℃-85℃; the first time is 8h-12h.

[0009] In some embodiments, the preparation method of the compound of formula II includes the following steps: Methyl 3-iodopropionate and compound IV were dissolved in a second organic solvent and then subjected to a reflux reaction to obtain compound V. The compound of formula V was hydrolyzed in an alkaline environment to obtain the compound of formula II; IV; V.

[0010] In some embodiments, the preparation method satisfies one or more of the following conditions: (1) The molar ratio of the methyl 3-iodopropionate and the compound of formula IV is (2-3):1; (2) The second organic solvent includes acetonitrile; (3) The heating and reflux reaction time is 48h-96h; (4) The alkaline environment is a solution environment in which lithium hydroxide is used as the base and a mixture of methanol and water is used as the solvent; (5) The temperature of the hydrolysis reaction is 35℃-45℃ and the time is 3h-5h.

[0011] A third aspect of the invention provides the use of the aforementioned near-infrared dyes in the preparation of reagents for mitochondrial imaging of cells.

[0012] A fourth aspect of the invention provides the use of the aforementioned near-infrared dyes in the preparation of reagents for tumor diagnosis.

[0013] In some embodiments, the tumor includes osteosarcoma, colon cancer, liver cancer, glioma, breast cancer, prostate cancer, melanoma, esophageal cancer, cervical cancer, and ovarian cancer.

[0014] The fifth aspect of the invention provides the use of the aforementioned near-infrared dye in the preparation of medicaments for photothermal therapy of tumors.

[0015] A sixth aspect of the invention provides the use of the aforementioned near-infrared dyes in the preparation of reagents for imaging in vivo.

[0016] This invention, through the preparation of a near-infrared dye with a specific structure, is the first to be developed for mitochondrial targeting, achieving specific localization and staining of cellular mitochondria. Under near-infrared light irradiation, this compound can fully exert the effects of photodynamic therapy and photothermal therapy, while generating reactive oxygen species and heat. It combines the triple functions of near-infrared fluorescence imaging, photothermal therapy, and photodynamic therapy, making it suitable for intracellular mitochondrial-targeting optical therapy applications. Attached Figure Description

[0017] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0018] Figure 1 This is a synthetic route diagram of the near-infrared dye of the present invention; Figure 2 The ultraviolet-visible absorption spectrum of the near-infrared dye of this invention; Figure 3 The fluorescence absorption spectrum of the near-infrared dye of this invention; Figure 4 To detect the singlet oxygen yield of the near-infrared dye of this invention using a DPBF probe; Figure 5 To detect the singlet oxygen yield of the near-infrared dye of the present invention at different power using an SOSG probe; Figure 6 To detect the singlet oxygen yield of the near-infrared dye of the present invention at different concentrations using an SOSG probe; Figure 7 The photothermal yield of the near-infrared dye of the present invention at different concentrations; Figure 8 Photothermal yield of the near-infrared dye of the present invention at different power levels; Figure 9 This is a scatter plot showing the fluorescence imaging and colocalization analysis of near-infrared dyes and mitochondria in this invention. Figure 10 For the hemolytic activity study of the near-infrared dye of this invention; Figure 11This invention relates to the detection of liver and kidney function indicators using near-infrared dyes. Detailed Implementation

[0019] The present invention will be further described below with reference to embodiments and examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the protection scope of the appended claims.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0021] As used herein, the term "and / or" encompasses any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that, in this application, the technical solution undoubtedly includes solutions connected by "logical AND," and also undoubtedly includes solutions connected by "logical OR."

[0022] In this article, the technical features described in an open-ended manner include both closed technical solutions composed of the listed features and open technical solutions that include the listed features.

[0023] In this document, when referring to numerical intervals (i.e., numerical ranges), unless otherwise specified, the selected numerical distribution within the aforementioned numerical interval is considered continuous and includes the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers to an integer within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. Furthermore, when multiple ranges are provided to describe features or properties, these ranges may be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0024] In this article, weight can be a well-known unit of mass in the pharmaceutical, chemical, or materials fields, such as μg, mg, g, or kg.

[0025] A first aspect of the present invention provides a near-infrared dye having a structure as shown in Formula I: I.

[0026] A second aspect of the present invention provides a method for preparing the near-infrared dye described above, comprising the following steps: Under inert gas protection, compounds of formula II and formula III are dissolved in a first organic solvent, and sodium acetate and acetic anhydride are added and mixed. The resulting mixture is stirred at a first temperature for a first time to prepare a near-infrared dye. II; III.

[0027] In some embodiments, the molar ratio of compound II to compound III is (2-2.1):1, for example, it can be 2:1, 2.1:1, etc.

[0028] In some embodiments, the molar ratio of acetic anhydride or sodium acetate to the compound of formula II is (1.8-2):1, for example, it can be 1.8:1, 1.9:1, 2:1, etc.

[0029] In some embodiments, the ratio of the sum of the weights of compounds of formula II and formula III in the mixture to the volume of the organic solvent is 1-2 g: 20 mL, for example, 1 g: 20 mL, 1.5 g: 20 mL, 2 g: 20 mL, etc.

[0030] In some embodiments, the first organic solvent includes ethanol.

[0031] In some embodiments, the first temperature is 70°C-90°C (e.g., 70°C, 75°C, 80°C, 85°C, 90°C, etc.); the first time is 6h-24h (e.g., 6h, 8h, 12h, 16h, 18h, 20h, 24h, etc.); further, the first temperature is 75°C-85°C (e.g., 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, etc.); the first time is 8h-12h (e.g., 8h, 9h, 10h, 11h, 12h, etc.).

[0032] In some embodiments, the preparation method of the compound of formula II includes the following steps: Methyl 3-iodopropionate and compound IV were dissolved in a second organic solvent and then subjected to a reflux reaction to obtain compound V. Compound V was hydrolyzed in an alkaline environment to obtain compound II; IV; V.

[0033] In some embodiments, the molar ratio of methyl 3-iodopropionate to the compound of formula IV is (2-3):1, for example, it can be 2:1, 2.2:1, 2.3:1, 2.4:1, 2.5:1, 2.6:1, 2.7:1, 2.8:1, 2.9:1, 3:1, etc.

[0034] In some embodiments, the second organic solvent includes acetonitrile.

[0035] In some implementations, the heating reflux reaction time is 48h-96h, for example, 48h, 60h, 72h, 84h, 96h, etc.

[0036] In some embodiments, the alkaline environment is a solution environment in which lithium hydroxide is used as the base and a mixture of methanol and water is used as the solvent.

[0037] In some embodiments, the hydrolysis reaction is carried out at a temperature of 35°C-45°C (e.g., 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, etc.) and for a time of 3h-5h (e.g., 3h, 4h, 5h, etc.).

[0038] A third aspect of the invention provides the use of the aforementioned near-infrared dyes in the preparation of reagents for mitochondrial imaging of cells.

[0039] A fourth aspect of the invention provides the use of the aforementioned near-infrared dyes in the preparation of reagents for tumor diagnosis.

[0040] In some embodiments, the tumor includes osteosarcoma, colon cancer, liver cancer, glioma, breast cancer, prostate cancer, melanoma, esophageal cancer, cervical cancer, and ovarian cancer.

[0041] The fifth aspect of the invention provides the use of the aforementioned near-infrared dye in the preparation of medicaments for photothermal therapy of tumors.

[0042] A sixth aspect of the invention provides the use of the aforementioned near-infrared dyes in the preparation of reagents for imaging in vivo.

[0043] The following are some specific examples.

[0044] For experimental parameters not specified in the following specific embodiments, please refer to the guidelines given in this invention document first, or refer to experimental manuals or other experimental methods known in the art, or refer to the experimental conditions recommended by the manufacturer.

[0045] The raw materials and reagents involved in the following specific embodiments can be obtained commercially or prepared by those skilled in the art using known methods.

[0046] The compounds and their structural formulas involved in the following examples are shown in Table 1.

[0047] Table 1. Compounds and their structural formulas

[0048] Example 1 This embodiment provides a method for preparing the near-infrared dye of the present invention. The synthetic route can be found in [reference needed]. Figure 1 The specific steps are as follows: (1) Under nitrogen protection, compound A (6 g, 28 mmol) and compound B (3 g, 10.5 mmol) were dissolved in acetonitrile (20 mL) and refluxed for 72 h. The reaction solution was concentrated and subjected to column chromatography (dichloromethane:methanol = 10:1, v / v) to obtain compound C (2.1 g, 5.6 mmol).

[0049] (2) Compound C was dispersed in a mixture of methanol and water (volume ratio of methanol:water = 5:1), and lithium hydroxide monohydrate (2 g, 47.7 mmol) was added and mixed. The resulting mixture was stirred at 40 °C for 4 h, and the pH was adjusted to 2 with concentrated hydrochloric acid. The reaction solution was concentrated and subjected to column chromatography (dichloromethane:methanol = 10:1) to obtain compound D (1.5 g, 4.2 mmol).

[0050] (3) Under nitrogen protection, compound D (1.17 g, 3.25 mmol) and compound E (0.5 g, 1.55 mmol) were dissolved in ethanol (20 mL), and AcONa (508 mg, 6.2 mmol) and acetic anhydride (632 mg, 6.2 mmol) were added. The resulting mixture was stirred at 80 °C for 12 h. The reaction solution was concentrated and subjected to column chromatography (dichloromethane:methanol = 10:1, v / v) to obtain compound F.

[0051] (4) Characterization: Mass spectrometry: m / z: 849.0467 (MW: 849.0467). 1H NMR spectrum: 1 H NMR(400MHz,Methonal-d4)δ8.37(d,J=14.1Hz,2H),7.82(d,J=1.7Hz,2H),7.72(dd,J=8.3,1.7Hz,2H),7.19(d,J=8.4Hz,2H),6 .43(d,J=14.1Hz,2H),4.34(t,J=7.3Hz,4H),2.74(t,J=6.2Hz,4H),2.58(t,J=7.2Hz,4H),1.92(t,J=6.1Hz,2H),1.69(s,12H).

[0052] Characterization revealed that compound F is the near-infrared dye of this invention, hereinafter referred to as Mitodine-CY, and corresponding performance tests were performed.

[0053] To determine the near-infrared imaging, reactive oxygen species generation, and photothermal functions of Mitodine-CY, the ultraviolet absorption spectrum of Mitodine-CY was first measured. Figure 2 ) and fluorescence spectrum ( Figure 3 ).like Figure 2 As shown, the maximum ultraviolet absorption wavelength of Mitodine-CY is 796 nm. The fluorescence emission peak of Mitodine-CY is at 810 nm, making it an effective fluorescent probe for in vivo tumor imaging.

[0054] To evaluate the efficiency of Mitodine-CY in generating singlet oxygen, a DPBF probe was used for measurement. DPBF, as a highly efficient singlet oxygen scavenger, generates a colorless internal peroxide via a [4+2] cycloaddition reaction, leading to a sharp decrease in its characteristic absorption peak at a specific wavelength. The amount of singlet oxygen generated can be indirectly reflected by monitoring the rate or extent of the decrease in absorbance (A410) at 410 nm using a UV-Vis spectrophotometer. A faster and greater decrease in absorbance indicates a greater amount of singlet oxygen generated in the system. The singlet oxygen production of the near-infrared dye Mitodine-CY after irradiation with a near-infrared laser for 5 minutes (irradiation wavelength: 808 nm, laser intensity: 1.0 W / cm²) was measured. 2 ), the result is as follows Figure 4 As shown, the absorbance of Mitodine-CY decreased significantly with increasing illumination time, indicating an increase in reactive oxygen species (ROS) production. Furthermore, this application used an SOSG probe to detect the singlet oxygen production of the near-infrared dye Mitodine-CY. SOSG (Ex / Em: 504 / 525nm) emits green fluorescence in the presence of singlet oxygen, and the singlet oxygen production was determined by measuring the fluorescence intensity of the sample using a fluorescence spectrophotometer. The results are as follows... Figure 5 and Figure 6 As shown, the singlet oxygen production of Mitodine-CY increases with increasing light intensity and concentration of the near-infrared dye Mitodine-CY. This indicates that Mitodine-CY has a good photodynamic therapy (PDT) effect and is more suitable for photodynamic therapy.

[0055] The temperature change in Mitodine-CY solution under near-infrared light irradiation was measured using a thermocouple probe to reflect its photothermal properties. The solution was irradiated for 10 minutes under the same laser intensity (irradiation wavelength: 808 nm, laser intensity: 1.0 W / cm²). 2 Record the temperature changes. The results are as follows: Figure 7As shown, the temperature of the Mitodine-CY solution initially increases with near-infrared irradiation time, then tends to remain constant after 4 minutes of irradiation, reaching a maximum of 60°C. Since irreversible thermal damage occurs when the temperature around cells or tissues reaches above 45°C, this invention can serve as an effective photothermal reagent. Furthermore, this application modifies different near-infrared light intensities (0.3, 1.0, or 1.6 W / cm²). 2 The temperature changes in the aqueous solution of Mitodine-CY were investigated. Figure 8 As light intensity increases, the photothermal effect also increases, but at a light intensity of 0.3 W / cm², the effect is most pronounced. 2 At this intensity, the solution temperature reached a maximum of 38℃, indicating that this intensity was insufficient to induce a photothermal effect in Mitodine-CY. However, this power level could effectively generate singlet oxygen, thus allowing it to act as a photosensitizer to produce photodynamic therapy effects. At light intensities of 1.0 or 1.6 W / cm², [further details needed]. 2 At the same time, Mitodine-CY possesses both PDT and PTT functions. Therefore, by adjusting the intensity of near-infrared light, the photodynamic properties of Mitodine-CY can be modulated to achieve both photodynamic and photothermal properties, providing a theoretical basis for the synergistic treatment of PDT and PTT.

[0056] Near-infrared dye Mitodine-CY was added to CT26 cell culture medium and further compared with the mitochondrial probe MitoTracker Green FM to observe the mitochondrial targeting function of Mitodine-CY. The Pearson Correlation Coefficient (PCC) was calculated. In biological colocalization, this application typically focuses on the range of 0 to +1. A high positive PCC value is often interpreted as indicating a significant spatial "colocalization" or interaction tendency between the two target molecules. Figure 9 From left to right, the images show: a cell fluorescence staining image of the mitochondrial probe MitoTracker Green FM; a cell fluorescence staining image of the near-infrared dye Mitodine-CY; a cell imaging overlay image; and a Pearson coefficient image. Figure 9 As shown, the near-infrared dye Mitodine-CY exhibits red fluorescence, which has a high degree of overlap with the green fluorescence of mitochondria, and its PCC is 0.77, indicating that it has good mitochondrial targeting and localization functions.

[0057] Biosafety assessment Hemolytic toxicity study: As an important indicator for evaluating the safety of intravenous administration, the hemolytic toxicity of Mitodine-CY was studied. The cell lysis agent Triton-X was used as the indicator of 100% hemolysis rate. Results are as follows: Figure 10As shown, the hemolysis rate of Mitodine-CY remains below 5% even at the highest concentration. This demonstrates that Mitodine-CY exhibits good biocompatibility within the effective therapeutic concentration range, with negligible hemolytic toxicity, thus greatly enhancing its potential for safe clinical application.

[0058] Animal survival rate: Mitodine-CY solution (0.2 mL) was injected into Balb / C mice via the tail vein. The control group received the same volume of PBS buffer (0.2 mL). After injection, the mice were returned to the animal room and fed freely. The mice's response behavior and survival rate were observed. The results showed that Mitodine-CY did not cause significant abnormal reactions in the experimental mice.

[0059] Animal liver and kidney function tests: Two liver and kidney function parameters were measured using a fully automated biochemical analyzer, including alanine transaminase (ALT), aspartate transaminase (AST), blood urea nitrogen (BUN), and serum creatinine (CREA). Serum samples from mice injected with PBS served as the control group. Figure 11 As shown, the data from the PBS group and the Mitodine-CY group were within the normal range of parameters in healthy mice, suggesting that Mitodine-CY did not cause a burden on the liver and kidneys or induce liver and kidney toxicity (the dashed line in the figure represents the normal range value).

[0060] The technical features of the above-described embodiments and examples can be combined in any suitable manner. For the sake of brevity, not all possible combinations of the technical features in the above-described embodiments and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0061] The embodiments described above merely illustrate several implementations of the present invention and should not be construed as limiting the scope of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Furthermore, it should be understood that after reading the above teachings, those skilled in the art can make various alterations or modifications to the present invention, and the equivalent forms obtained also fall within the protection scope of the present invention. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided by the present invention through logical analysis, reasoning, or limited experimentation are all within the protection scope of the appended claims. Therefore, the protection scope of this patent should be determined by the appended claims, and the specification can be used to interpret the content of the claims.

Claims

1. The application of near-infrared dyes in the preparation of reagents for mitochondrial imaging, characterized in that, It has a structure as shown in Equation I: Ⅰ。

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