Near-infrared two-region water-soluble fluorophore, preparation method and application thereof

Abstract

The application aims to provide a water-soluble fluorophore in near-infrared region II and a preparation method and application thereof, and solves the problem of poor water solubility of the existing fluorophore in near-infrared region II. The water-soluble fluorophore in near-infrared region II has the following general structure: wherein n is 0, 1, 2 or 3; and R is H, an ortho-carboxyl phenyl or a phenyl.

Description

Technical Field

[0001] This application relates to the field of compounds, specifically to a water-soluble fluorophore in the near-infrared II region, its preparation method, and its application. Background Technology

[0002] Fluorescence imaging, with its advantages of high sensitivity and non-invasive analysis, is widely used in the detection of biological species. In particular, small organic molecule fluorescent probes in the near-infrared second window (900–1700 nm) have attracted much attention due to their weak background fluorescence, low light scattering, and low tissue absorption. Compared to inorganic materials, small organic molecule fluorescent probes often exhibit good biocompatibility and structural modifiability. Currently, most fluorophores emitting in the near-infrared second window exhibit poor water solubility, failing to emit fluorescence in aqueous solutions. To improve water solubility, liposome encapsulation is commonly used, which increases the difficulty of synthesis and preparation, and the resulting substances are not easily characterized accurately, resulting in poor reproducibility. Therefore, developing a small molecule fluorophore emitting in the near-infrared second window suitable for aqueous solutions is of great significance.

[0003] Therefore, this invention is proposed. Summary of the Invention

[0004] The main objective of this invention is to provide a water-soluble fluorophore in the near-infrared II region, its preparation method, and its application, thereby solving the problem of poor water solubility of existing near-infrared II fluorophores.

[0005] To achieve the above objectives, the present invention provides the following technical solutions.

[0006] A first aspect of the present invention provides a water-soluble fluorophore in the near-infrared II region, the general structural formula of which is shown below:

[0007]

[0008] Where n = 0, 1, 2 or 3; R can be H or ortho-carboxyphenyl or phenyl.

[0009] Furthermore, n = 1 or 2, and R is an ortho-carboxyphenyl.

[0010] A second aspect of the present invention provides a method for preparing the water-soluble near-infrared II fluorophore described above, characterized in that the synthetic route is as follows:

[0011]

[0012] Where n = 0, 1, 2 or 3; R can be H or o-carboxyphenyl or phenyl. The fluorophore 1 shown is obtained by condensation of compound 1 and compound 2 in an organic solvent.

[0013] The reaction conditions (such as the type and amount of raw materials, temperature, and time) of the above synthetic routes can be further optimized to improve the reaction rate, yield, or purity, as shown in the following improvement methods.

[0014] Furthermore, the organic solvent is one or a mixture of several of toluene, n-butanol, N,N-dimethylformamide and dimethyl sulfoxide.

[0015] Furthermore, the organic solvent is a mixture of toluene and n-butanol.

[0016] Furthermore, the volume ratio of toluene to n-butanol is 1:0.8 to 1.2, and more preferably equal in volume.

[0017] Furthermore, the condensation reaction temperature is 80–120°C, and the reaction time is 6–12 h.

[0018] Furthermore, the condensation reaction temperature is 100–120°C, more preferably 100°C.

[0019] Furthermore, the condensation is followed by purification: silica gel column chromatography, eluted with CH2Cl2-MeOH at a volume ratio of 20:1.

[0020] A third aspect of the present invention provides the application of the near-infrared II water-soluble fluorophores described above in vascular imaging.

[0021] In practical applications, the fluorophore is dissolved in water or a water-soluble environment such as a saline solution to adjust its concentration and enable it to emit fluorescence of the required intensity.

[0022] In summary, compared with the prior art, the present invention achieves the following technical effects:

[0023] 1) All fluorophores contain groups with good hydrophilicity, which is beneficial to improve water solubility and avoid the decrease in sensitivity caused by liposome encapsulation;

[0024] 2) The maximum emission wavelength of the fluorophores is basically in the second window of the near-infrared spectrum (900-1700 nm), therefore it has the characteristics of weak background fluorescence, low light scattering and tissue absorption.

[0025] 3) The fluorophore, when injected into mice, exhibits strong fluorescence and provides high resolution for vascular imaging;

[0026] 4) High sensitivity;

[0027] 5) The preparation method is simple, it can be mass-produced, and it is easy to store.

[0028] The above description is only an overview of the technical solution of this application. In order to better understand the technical means of this application and to implement it in accordance with the contents of the specification, and to make the above and other objects, features and advantages of this application more obvious and understandable, the following are specific embodiments of this application. Attached Figure Description

[0029] Various other advantages and benefits will become apparent to those skilled in the art upon reading the detailed description of the preferred embodiments below. The accompanying drawings are for illustrative purposes only and are not intended to limit the scope of this application. Furthermore, the same reference numerals denote the same parts throughout the drawings. In the drawings:

[0030] Figure 1 The absorption and emission spectra of fluorophore 1 in phosphate buffer solution are shown.

[0031] Figure 2 The image shows fluorescence imaging of fluorophore 1 in the blood vessels of a mouse. Detailed Implementation

[0032] The embodiments of the technical solution of this application will now be described in detail with reference to the accompanying drawings. These embodiments are only used to more clearly illustrate the technical solution of this application and are therefore merely examples, and should not be used to limit the scope of protection of this application.

[0033] 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 application pertains; the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the application; the terms “comprising” and “having”, and any variations thereof, in the specification, claims, and foregoing description of the drawings are intended to cover non-exclusive inclusion.

[0034] In this document, the term "embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The appearance of this phrase in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0035] Example 1:

[0036] Preparation of fluorophore 1a, the compound structure is as follows:

[0037]

[0038] The specific synthesis route is as follows:

[0039]

[0040] The specific steps are as follows:

[0041] Compound 3 (0.2 mmol, 95 mg) and compound 2 (0.1 mmol, 15 mg) were dissolved in 5 mL of toluene and 5 mL of n-butanol and stirred at 100 °C for 12 h. After cooling, the reaction solution was evaporated to dryness under reduced pressure. The crude product was separated by column chromatography (silica gel G, 200-300 mesh) using CH2Cl2 / MeOH (20:1, v / v) as eluent to obtain the final fluorophore 1a in 30% yield. The prepared fluorophore 1a was used in Examples 3 and 4.

[0042] 1 H NMR (400MHz, MeOD) δ8.30(d,J=1.6Hz,2H),8.28(s,1H),8.21(s,1H),7.70-7.85(m,5H),7.67(dd,J=8.8,1.6Hz,1H),7.32(t,J=7.6Hz,2H),7. 17-7.28(m,4H),7.06-7.17(m,3H),3.65-3.78(m,8H),2.95-3.10(m,4 H),2.40-2.50(m,4H),1.80-1.95(m,4H),1.32(m,12H).HR-ESI-MS:m / z calcd for C 56 H 53 N2O7[M] + ,865.3847; found,865.3849.

[0043] Example 2:

[0044] Preparation of fluorophore 1b, the compound structure is as follows:

[0045]

[0046] The specific synthesis route is as follows:

[0047]

[0048] The specific steps are as follows:

[0049] Compound 4 (0.2 mmol, 92 mg) and compound 2 (0.1 mmol, 15 mg) were dissolved in 5 mL of toluene and 5 mL of n-butanol and stirred at 100 °C for 12 h. After cooling, the reaction solution was evaporated to dryness under reduced pressure. The crude product was separated by column chromatography (silica gel G, 200-300 mesh) using CH2Cl2 / MeOH (20:1, v / v) as eluent to obtain the final fluorophore 1b in 25% yield. The prepared fluorophore 1b was used in Example 3.

[0050] 1 H NMR(400MHz,DMSO-d6)δ7.91-7.98(m,2H),7.74-7.82(m,2H),7.64-7.72(m,2H) ,7.50(s,1H),7.32(d,J=8.0Hz,1H),7.29(d,J=8.0Hz,2H),7.11(s,1H),6.95(d ,J=8.4Hz,1H),6.78(s,1H),6.45-6.55(m,6H),3.34-3.42(m,8H),2.85-3.05(m ,4H),2.25-2.35(m,2H),1.87-1.97(m,2H),1.05-1.15(m,12H).HR-ESI-MS:m / z calcd for C 54 H 49 N2O7[M] + ,837.3534;found,837.3538.

[0051] Example 3: Absorption and emission spectra of fluorophores 1a and 1b in phosphate buffer solution.

[0052] Add fluorophores 1a or 1b to the test tubes respectively, and then add an appropriate volume of phosphate buffer solution to make the final fluorophore concentration 10 μM. Measure the absorption spectrum of the solution using a UV spectrophotometer and its fluorescence spectrum using a fluorescence spectrophotometer, using an 808 nm laser as the excitation wavelength, to obtain the UV absorption and fluorescence emission spectra (see...). Figure 1 ).Depend on Figure 1 It can be seen that fluorophores 1a and 1b can both emit strong fluorescence in an aqueous environment, with maximum emission wavelengths of 940 nm and 1040 nm, respectively.

[0053] Example 4: Fluorescence imaging of fluorophore 1a in mouse blood vessels.

[0054] 200 μL of fluorophore 1a was injected intravenously into mice at a concentration of 500 μM. The mice were then irradiated with an 808 nm laser using a 1300 nm long-pass filter, resulting in clear and high-resolution vascular images (see [link to original text]). Figure 2 Therefore, fluorophore 1a can be well applied in in vivo analysis.

[0055] Finally, it should be noted that the above embodiments only illustrate the preparation and application of fluorophores 1a and 1b. The preparation methods and testing conditions of other fluorophores and probes are not listed one by one because they have similar structures and properties.

[0056] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and not to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. These modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application, and they should all be covered within the scope of the claims and specification of this application. In particular, as long as there is no structural conflict, the various technical features mentioned in the embodiments can be combined in any way. This application is not limited to the specific embodiments disclosed herein, but includes all technical solutions falling within the scope of the claims.

Claims

1. A water-soluble fluorophore in the near-infrared II region, the general structural formula of which is shown below: ， in, n = 0, 1, 2 or 3; R is an ortho-carboxyphenyl or phenyl.

2. The near-infrared II water-soluble fluorophore according to claim 1, characterized in that, n=1 or 2, R is o-carboxyphenyl.

3. A method for preparing a near-infrared II water-soluble fluorophore as described in claim 1 or 2, characterized in that, The synthesis route is as follows: ， Wherein, n = 0, 1, 2 or 3; R is ortho-carboxyphenyl or phenyl; fluorophore 1 is obtained by condensation reaction of compound 1 and compound 2 in an organic solvent; The organic solvent is one or a mixture of several of toluene, n-butanol, N,N-dimethylformamide and dimethyl sulfoxide.

4. The preparation method according to claim 3, characterized in that, The organic solvent is a mixture of toluene and n-butanol.

5. The preparation method according to claim 4, characterized in that, The volume ratio of toluene to n-butanol is 1:0.8~1.

2.

6. The preparation method according to any one of claims 3-5, characterized in that, The condensation reaction is carried out at a temperature of 80~120℃ for 6h~12h.

7. The preparation method according to claim 6, characterized in that, The condensation reaction is carried out at a temperature of 100~120℃.

8. The preparation method according to claim 3, characterized in that, The condensation reaction was followed by purification: silica gel column chromatography, eluted with CH2Cl2-MeOH at a volume ratio of 20:

1.

9. The application of the near-infrared II water-soluble fluorophore as described in claim 1 or 2 in vascular imaging.

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