A fluorescent compound based on new indocyanine green IR820 and its preparation and application

Abstract

The present invention relates to a fluorescent compound based on new indocyanine green IR820 and its preparation and application. The molecular structure of the fluorescent compound is shown in formula (1): Compared with the prior art, the fluorescent probe of the present invention has near-infrared fluorescence emission, good tumor targeting, biocompatibility, low cytotoxicity, and strong biological tissue penetration. It can be used for targeted fluorescence imaging of tumor cells and living tumors, and is expected to be further used in fluorescence surgical navigation.

Description

Technical Field

[0001] The present invention belongs to the technical field of fluorescent compounds and relates to a fluorescent compound based on new indocyanine green IR820 and a preparation and application thereof. Background Art

[0002] Fluorescence imaging has shown broad application prospects in basic biomedical research and clinical translation, including precise intraoperative tumor resection. One of the core technologies of fluorescence imaging is the development of fluorescent probe molecules that can be used for imaging. Fluorescent probe molecules can dynamically track the occurrence and progression of various physiological and pathological processes and diseases at the molecular level. In the field of tumor visualization imaging, in particular, because fluorescent probe molecules can illuminate cancer cells in real time during surgery, they can help doctors more accurately determine tumor boundaries and detect metastases. Although there are numerous reports in academic papers on fluorescent probes for tumor imaging, few probe molecules have been approved by the FDA for clinical tumor imaging. This is primarily due to the biological toxicity of most fluorescent dyes currently used to construct fluorescent probes, which limits their clinical use. To date, the only fluorescent dyes approved by the FDA for clinical use are fluorescein, methylene blue, and indocyanine green (ICG). Fluorescein and methylene blue, however, are limited to a fluorescence emission wavelength of less than 700 nm, making them susceptible to interference from spontaneous background fluorescence signals when used for tumor fluorescence imaging. Furthermore, their tissue penetration is poor. Therefore, fluorescent probes based on fluorescein and methylene blue are only suitable for use during open surgery and are susceptible to interference from background fluorescence signals. ICG, with its emission wavelength reaching approximately 800 nm, is currently the only FDA-approved fluorescent dye for fluorescent surgical navigation. Therefore, the development of fluorescent probe molecules based on ICG dyes has been a focus of tumor fluorescence imaging. However, ICG for in vivo tumor fluorescence imaging has two major limitations: 1) It lacks targeting, requiring intratumoral injection or passive targeting (high permeability and long retention (EPR)) after injection to accumulate in the tumor region and metabolize the free dye to achieve high-contrast fluorescence imaging. This complicates tumor imaging procedures. 2) ICG dyes are susceptible to photobleaching after prolonged excitation. Consequently, ICG's photostability is insufficient, preventing prolonged excitation in tumor fluorescence imaging, which limits its application.

[0003] To solve the above problems, there are reports on directly linking tumor targeting groups to the side chain of ICG to solve the tumor targeting problem. On the other hand, the introduction of a stable six-membered ring structure in the long-chain conjugated part of ICG can greatly improve the photostability of the dye to obtain a series of ICG-derived dyes such as IRDye800CW and ZW800-1 dyes. However, since the IRDye800CW dye introduces two sulfonic acid groups on the benzene ring and the ZW800-1 dye introduces two positively charged quaternary ammonium salts on the side chain, it is quite different from the ICG structure in terms of charging mode (ICG carries two negative charges, which exist on the side chain). Therefore, there may be some changes in biocompatibility, and further clinical verification is required. It cannot be compared with ICG, which has been approved by the FDA for a long time and has been verified to have good biocompatibility. Furthermore, the different charging modes of the dyes will directly affect the size of the background signal (mainly from non-specific adsorption to biological macromolecules). IR820, also known as the new indocyanine green, completely retains the charge and core structure of ICG compared to other fluorescent dyes based on ICG analogs, such as IRDye800CW and ZW800-1. Therefore, its biocompatibility is closest to ICG, which has been approved by the FDA and has long been clinically validated for its excellent biocompatibility. Currently, a number of tumor-targeting fluorescent probes based on IR820 have been designed, but these probes are synthesized by encapsulating IR820 with nanomaterials. While these IR820-based nanoprobes show promise for tumor imaging, their larger size (nanoscale) may lead to greater nephrotoxicity and hepatotoxicity compared to organic small molecule probes. Tumor-targeting small molecule fluorescent probes based on IR820 are relatively rare, and currently, no organic small molecule fluorescent probes based on IR820 have been designed for the highly expressed integrin αvβ3 on tumor cells. On the other hand, in the coupling of the targeting group with IR820, the mid-chlorine substitution of IR820 is the only group that can be used for coupling. However, during coupling, the mid-chlorine group of IR820 is easily replaced by groups such as amino and thiol (Sci.China Chem.2020,63,699-706). Therefore, there are many side reactions in the coupling reaction, which leads to greater challenges in the synthesis of the target probe by directly introducing the targeting group using the substitution reaction of the mid-chlorine of IR820. At the same time, in the subsequent coupling reaction using IR820, since it carries two sulfonic acid groups, purification is difficult. Therefore, each step of the coupling product is often separated by reverse-phase HPLC, which requires large-scale reverse-phase preparative HPLC equipment and consumes a lot of solvent and time. Exploring new and simple synthesis processes is conducive to saving the cost of fluorescent reagents developed based on IR820, and also lays the foundation for its commercialization.

[0004] There are also no reports on methods for targeting IR820 by introducing a cRGD cyclic peptide that targets integrin αvβ3, which is highly expressed on the surface of tumor cells. Furthermore, the emission wavelengths of IRDye800CW and ZW800-1 are around 800 nm. Although they reach the near-infrared region, they are approximately 20 nm shorter than the emission wavelength of the new indocyanine green dye (IR820) (820 nm). Therefore, the tissue penetration ability of bevacizumab-IRDye800CW and cRGD-ZW800-1 developed based on these two dyes is weaker than that of IR820. Summary of the Invention

[0005] The purpose of the present invention is to provide a fluorescent compound based on the new indocyanine green IR820 and its preparation and application.

[0006] The purpose of the present invention can be achieved by the following technical solutions:

[0007] One of the technical solutions of the present invention provides a fluorescent compound based on the new indocyanine green IR820, whose molecular structure is shown in formula (1):

[0008]

[0009] The second technical solution of the present invention provides a method for preparing a fluorescent compound based on the new indocyanine green IR820, wherein the compound represented by formula (2) is reacted with a precursor of the linker arm and cRGD in sequence to obtain the target product;

[0010] Wherein, the molecular structure of the compound represented by formula (2) is as follows:

[0011] wherein R1 is selected from halogen elements;

[0012] The precursor of the connecting arm includes a compound having a structure shown in formula (3):

[0013] Wherein, R2 includes a hydroxyl group, R3 includes a carboxyl group, and n is 1 to 10.

[0014] Furthermore, R1 is Cl.

[0015] Furthermore, R2 is a hydroxyl group, R3 is a carboxyl group, and n=2.

[0016] Furthermore, the molar ratio of the compound represented by formula (2), the precursor of the linker arm, and cRGD is 1:(1-10):(1-3), and can be specifically 1:1:1, or 1:10:3, or 1:5:2, or any intermediate value within this range.

[0017] Furthermore, the compound represented by formula (2) is reacted with the precursor of the linker arm in sequence in an organic solvent system (such as DMF), sodium hydride is also added to the reaction system, the reaction temperature is room temperature, and the reaction time is 3-5 hours.

[0018] Furthermore, the intermediate product obtained by reacting the compound represented by formula (2) with the precursor of the linker arm is first activated by EDC and NHS in NMP or DMF solvent, and then reacted with cRGD at room temperature.

[0019] The third technical solution of the present invention provides an application of a fluorescent compound based on the new indocyanine green IR820 in the preparation of a surgical fluorescent navigation probe.

[0020] A fourth technical solution of the present invention provides a fluorescent composition, which includes the fluorescent compound based on the new indocyanine green IR820 as described above, and a pharmaceutically acceptable carrier.

[0021] A fifth technical solution of the present invention provides a fluorescence imaging system, comprising a fluorescence detection device and a fluorescent probe, wherein the fluorescent probe comprises the fluorescent compound based on the new indocyanine green IR820 as described above.

[0022] In addition, the present invention also provides a fluorescence imaging method for non-diagnostic purposes, which includes: administering a fluorescent probe to a subject, and then performing fluorescence imaging on the subject; wherein the subject includes living cells, active physiological tissues of animals or living animals; and the fluorescent probe includes the fluorescent compound.

[0023] Compared with the prior art, the present invention has the following advantages:

[0024] (1) The fluorescent probe for targeting tumors based on the ICG core structure has better tumor targeting and photostability than the FDA-approved ICG.

[0025] (2) Compared to currently reported fluorescent probes developed based on ICG analogs such as IRDye800CW and ZW800-1 (bevacizumab-IRDye800CW and cRGD-ZW800-1), the fluorescent probe of the present invention fully retains the charge mode and core structure of ICG in terms of charge and chemical structure. Therefore, in terms of biocompatibility, it is closest to ICG, which has been approved by the FDA and has long been clinically verified to have good biocompatibility.

[0026] (3) Currently, there is no tumor-targeting organic small molecule fluorescent probe based on IR820 fluorescent dye and using cRGD as a targeting group. This application provides a new tumor-targeting organic small molecule fluorescent probe based on IR820 fluorescent dye.

[0027] (4) The present invention introduces a linker with a phenolic hydroxyl group into the chlorine group in IR820, and then uses the linker to further couple with the desired targeting group. In this way, the phenolic hydroxyl group replaces the chlorine on IR820, forming a stable phenol-substituted IR820. This avoids the situation where the chlorine atom on IR820 is easily affected by coupling auxiliary reagents such as amino or sulfhydryl groups when directly coupling with the targeting group, resulting in unsuccessful coupling synthesis. Therefore, the present application provides a synthetic method for successfully coupling IR820 with a targeting group.

[0028] (5) After the IR820 coupling group is present in the present invention, each step of the reaction is mostly prepared and purified by reverse phase HPLC. The present invention uses column chromatography (developing solvent, dichloromethane: methanol = 10:1 to 5:1) to obtain pure IR820-COOH, which reduces the loss of instruments, equipment and manpower, and the yield is close to that of reverse phase HPLC preparation. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a mass spectrum of IR820-COOH in an embodiment of the present invention

[0030] Figure 2 This is a mass spectrum of IR820-cRGD in an embodiment of the present invention.

[0031] Figure 3 This is an HPLC chart of IR820-cRGD in an embodiment of the present invention.

[0032] Figure 4 These are fluorescence and bright field photographs of IR820-cRGD and different cells in an embodiment of the present invention.

[0033] Figure 5 This is an in vivo fluorescence imaging diagram of IR820-cRGD in mouse tumors according to an embodiment of the present invention. DETAILED DESCRIPTION

[0034] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments. This embodiment is implemented based on the technical solution of the present invention, and provides a detailed implementation method and specific operation process, but the protection scope of the present invention is not limited to the following embodiments.

[0035] In the following embodiments, the structural formulas of ICG, IR820, IRDye800CW, and ZW800-1 are as follows:

[0036]

[0037] cRGD was purchased from Xi'an Qiyue Biotechnology Co., Ltd.

[0038] Unless otherwise specified, the remaining raw materials or processing techniques are conventional commercially available raw materials or conventional processing techniques in the art.

[0039] Example 1

[0040] The fluorescent compound provided in this embodiment can be named IR820-cRGD, and its synthesis route is as follows:

[0041]

[0042] Specifically, the synthesis method of the fluorescent compound includes the following steps:

[0043] (1) Synthesis of intermediate IR820-COOH:

[0044] 332 mg of p-hydroxyphenylpropionic acid was dissolved in 10 ml of anhydrous N,N-dimethylformamide (DMF) solvent, 47 mg of sodium hydride was added, and the mixture was stirred at room temperature for half an hour under nitrogen protection. 170 mg of IR820 was added and the reaction was continued to stir at room temperature for 4 hours. TLC showed that the raw material IR820 disappeared and new spots appeared. The reaction was stopped and the reaction solution was added to methyl tert-butyl ether, and a large amount of dark green solid appeared. Centrifugation obtained a solid. Washed three times with 10 ml of acetone or ethyl acetate. The obtained solid was dried under vacuum to obtain a crude product. Further column chromatography (developing solvent, dichloromethane: methanol = 10:1 to 5:1) obtained a pure product with a metallic luster and light green color, with a yield of 32%. 1 H NMR(400MHz, DMSO-d6)δ8.87(d,J=14.1Hz,2H),8.26(d,J=8.5Hz,2H),8.07–8.03(m ,4H),7.75(d,J=9.0Hz,2H),7.63(t,8.5,2H),7.49(t,J=7.5Hz,2H),7.11(d,J=8.6H z,2H),6.59(d,J=8.5Hz,2H),6.37(d,J=14.2Hz,2H),4.30(s,4H),3.53(s,2H),2.7 5(d,J=5.9Hz,4H),2.63(d,J=8.8Hz,4H),2.58(t,J=7.3Hz,4H),2.10–1.66(m,22H). 13CNMR(101MHz,DMSO-d6)δ172.24,151.56,146.96,144.03,139.17,132.82,132.6 6,130.69,129.75,129.25,128.06,127.08,126.87,124.23,121.65,120.46,114. 47,111.10,100.52,54.25,50.07,49.87,43.12,39.43,39.38,39.22,39.17,39.01,38.96,38.80,38.76,38.55,38.34,38.13,26.35,25.69,25.26,21.80,21.12.

[0045] HRMS(ES+,m / z):calcd for C55H59N2O9S2+:1001.3452,found:1001.3459. For the mass spectrum, please refer to Figure 1 .

[0046] (2) Synthesis of IR820-cRGD:

[0047] Dissolve 100 mg of IR820-COOH in NMP or DMF, add 10 equivalents of EDC and NHS, activate for half an hour, then add an equivalent amount of cRGD. Continue stirring at room temperature overnight. TLC shows that the starting material IR820-COOH disappears and new spots appear. End the reaction. HPLC separation yields a green liquid, which is freeze-dried to obtain a grass-green solid, the target product IR820-cRGD, with a yield of approximately 20%.

[0048] 1H NMR (400MHz, DMSO-d6) δ8.15(d,J=8.4Hz,2H),8.04-7.95(m,5H),7.74(d,J=8.8Hz),7.62-7.58(m,6H),7.49-7.45(m,2H),7.28(d,J=8.0Hz ,2H),7.16(d,J=8.0Hz,2H),6.71(s,br,1H),6.26(d,J=12.4Hz,2H),4.27(s,4H),4.25-4.17(m,2H),4.12-4.05(q,J1=7.5Hz,J2=13.5Hz,2H ),3.92(s,4H),3.89-3.82(q,J1=6.0Hz,J2=15.0Hz,2H),3.73-3.65(m,4H),3.13(s,6H),3.04(s,2H),2.77(s,4H),2.65-2.64(m,4H),2.46 -2.45(m,6H),2.35-2.21(m,4H),2.10-2.06(m,8H),1.96-1.80(m,6H) ,1.88(d,J=6.0Hz,1H),1.40(s,2H),1.07-0.99(m,6H).1.22(s,12H).

[0049] LC-MS (ES+, m / z): [M+H] 2+ ,772.21; its purity was about 95% as determined by HPLC (buffer A: 0.1% TFA in H2O; buffer B: 0.1% TFA in acetonitrile). For its mass spectrum, please refer to Figure 2 , HPLC Figure 3 shown.

[0050] Example 2

[0051] Fluorescence imaging of IR820-cRGD in living cells

[0052] Cell imaging was performed using a Leica TCS SP5 II confocal laser scanning microscope using a HC×PLAPO 63×oil objective (NA: 1.40).

[0053] The experiment was conducted in three groups: one using normal CHO cells, one using breast cancer cells (MCF-7), and one using HeLa cells. Before imaging, the culture medium was aspirated and washed once with PBS buffer and then once with DMEM or 1640. 10 μL of the prepared probe stock solution (1 mM DMSO) was added to a 2 mL culture dish containing fresh DMEM or 1640 medium. After incubation, excess culture medium was removed, followed by washing with PBS buffer (pH 7.4) to remove excess probe. Confocal fluorescence microscopy was then used to image the cells. Figure 4 The following images show fluorescence imaging of the probe in three cell types. These images demonstrate the probe's highly selective fluorescence signal for tumor cells, demonstrating its potential for tumor imaging.

[0054] Example 3

[0055] Fluorescence imaging of IR820-cRGD in living mice

[0056] Three tumor-bearing mice were injected with a 10 μM IR820-cRGD aqueous solution, a DMSO aqueous solution containing an equivalent probe concentration, and an IR820 aqueous solution through the tail vein. Two hours later, the tumor sites of the mice were observed using a small animal in vivo imaging instrument. Figure 5 As shown, IR820-cRGD displayed a very bright fluorescence signal at the mouse tumor site. In contrast, the pure aqueous solution showed no fluorescence signal, and the non-targeted IR820 had only a weak fluorescence signal. These results indicate that the targeted probe IR820-cRGD greatly improves the imaging effect of the IR820 dye on living tumors.

[0057] Example 4:

[0058] Compared with Example 1, most of the steps are the same, except that the molar ratio of IR820, p-hydroxyphenylpropionic acid, and cRGD is adjusted to 1:1:1.

[0059] Example 5:

[0060] Compared with Example 1, most of the steps are the same, except that the molar ratio of IR820, p-hydroxyphenylpropionic acid, and cRGD is adjusted to 1:10:3.

[0061] The above description of the embodiments is intended to facilitate understanding and use of the invention by those skilled in the art. It will be apparent that those skilled in the art can readily make various modifications to these embodiments and apply the general principles described herein to other embodiments without requiring inventive effort. Therefore, the present invention is not limited to the above-described embodiments. Improvements and modifications made by those skilled in the art based on the disclosure of the present invention, without departing from the scope of the present invention, should be within the scope of protection of the present invention.

Claims

1. A fluorescent compound based on new indocyanine green IR820, characterized in that Its molecular structure is shown in formula (1):

2. The method for preparing a fluorescent compound based on new indocyanine green IR820 according to claim 1, wherein: The compound represented by formula (2) is reacted with the precursor of the linker arm and cRGD in sequence to obtain the target product; Wherein, the molecular structure of the compound represented by formula (2) is as follows: Wherein, R1 is selected from halogen elements; the precursor of the connecting arm includes a compound having a structure shown in formula (3): Wherein, R2 includes a hydroxyl group, R3 includes a carboxyl group, and n is 1 to 10.

3. The method for preparing a fluorescent compound based on new indocyanine green IR820 according to claim 2, characterized in that: R1 is Cl.

4. The method for preparing a fluorescent compound based on new indocyanine green IR820 according to claim 2, characterized in that: R2 is a hydroxyl group, R3 is a carboxyl group, and n=2.

5. The method for preparing a fluorescent compound based on new indocyanine green IR820 according to claim 2, characterized in that: The molar ratio of the compound represented by formula (2), the precursor of the linker arm, and cRGD is (1:(1-10):(1-3).

6. The method for preparing a fluorescent compound based on new indocyanine green IR820 according to claim 2, characterized in that: The compound represented by formula (2) reacts with the precursor of the linker arm in sequence in an organic solvent system. Sodium hydride is also added to the reaction system. The reaction temperature is room temperature and the reaction time is 3-5 hours.

7. The method for preparing a fluorescent compound based on new indocyanine green IR820 according to claim 2, characterized in that: The intermediate product obtained by the reaction of the compound represented by formula (2) with the precursor of the linker arm is first activated by EDC and NHS in NMP or DMF solvent, and then reacted with cRGD at room temperature.

8. Use of the fluorescent compound based on the new indocyanine green IR820 as claimed in claim 1 in the preparation of a surgical fluorescent navigation probe.

9. A fluorescent composition, characterized in that The invention comprises the fluorescent compound based on the novel indocyanine green IR820 as claimed in claim 1 and a pharmaceutically acceptable carrier.

10. A fluorescence imaging system, comprising a fluorescence detection device and a fluorescence probe, characterized in that: The fluorescent probe comprises the fluorescent compound based on the novel indocyanine green IR820 as claimed in claim 1.

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