A fluorescent / photoacoustic dual-modal probe responsive to Cu(I) and its preparation method and application

Abstract

The present invention belongs to the field of biochemistry technology and relates to a fluorescent / photoacoustic dual-modal probe responsive to Cu(I), and its preparation method and application. The fluorescent / photoacoustic dual-modal probe responsive to Cu(I) provided by the present invention has high selectivity and sensitivity, has efficient tumor targeting ability, and can be used for dynamic tracing of Cu(I) in tumors. Combined with three-dimensional photoacoustic / in vivo fluorescence imaging technology, the probe HCy Cu 1. Dynamic imaging of Cu(I) in tumors provides a new method for Cu(I) detection and a promising tool for tumor diagnosis. The dual-modal probe preparation method provided by the present invention utilizes readily available raw materials, mild and easily controllable reaction conditions, reduces reaction costs, and ensures target product yield. The structural formula of the probe is: #imgabs0#.

Description

Technical Field

[0001] The present invention belongs to the technical field of biochemistry, and in particular relates to a fluorescence / photoacoustic dual-modal probe responsive to Cu(I), and a preparation method and application thereof. Background Art

[0002] As an essential trace element for the human body, copper plays a crucial role in a variety of physiological and pathological processes through its redox properties. The close association between Cu(I) homeostasis imbalance and cancer has garnered significant attention in recent years, as the molecular mechanisms underlying copper hyperplasia and copper death have been uncovered. The dual role of dysregulated copper metabolism in tumorigenesis and cancer treatment has become a hot topic in cancer therapy research. Therefore, dynamic tracing of Cu(I) in living tumor tissues is crucial for uncovering the pathological role of Cu(I) in tumor development and progression, as well as for early diagnosis and therapeutic intervention.

[0003] In recent years, fluorescence imaging has become an important technology for dynamic imaging of Cu(I) due to its advantages such as non-invasiveness, high sensitivity, and real-time visualization. A variety of excellent fluorescent probes have been developed, enabling precise imaging of Cu(I) in cells and superficial tissues, providing important underlying information for the mechanistic understanding of Cu(I)-related physiological and pathological functions. Of particular note, the Chang group developed a ratiometric Cu(I) probe, FCP-1, which revealed the connection between the intracellular labile Cu(I) pool, glutathione metabolism, and oncogenic transformation, providing a precise detection tool for exploring the association between intracellular labile Cu(I) levels and the development and progression of cancer. However, due to factors such as the poor deep tissue penetration of fluorescent signals and interference from tissue autofluorescence, fluorescent probes have been difficult to achieve high-resolution imaging of Cu(I) in living tumors. Therefore, achieving high-resolution imaging of Cu(I) in living tumor tissues has always been a challenging issue in molecular probe design.

[0004] Photoacoustic imaging (PA), a non-invasive imaging modality, has attracted widespread attention in the field of biomedical imaging due to its advantages in deep-tissue high-resolution imaging. To date, small-molecule photoacoustic probes with varying performance have been developed, enabling the detection and imaging of a variety of life-related species within deep tissues in vivo. Recently, small-molecule photoacoustic probes have demonstrated significant potential for in vivo high-resolution dynamic imaging of Cu(I) / Cu(II). For example, the Chan group developed the first Cu(I) photoacoustic probe, achieving in situ imaging of Cu(I) levels in a Wilson disease model mouse model. Our group designed a ratiometric photoacoustic probe capable of crossing the blood-brain barrier and, in combination with three-dimensional photoacoustic imaging, achieved high-resolution imaging of Cu(II) in the brains of Parkinson's disease model mice and analysis of its spatial distribution within the brain. However, it should be pointed out that compared with fluorescence imaging, photoacoustic probes still face the problems of long signal acquisition time and low sensitivity. The design of Cu(I) photoacoustic probes still faces the following challenges: (1) real-time response capability of Cu(I); (2) ratio imaging of Cu(I); and (3) the difficulty of accurate dynamic tracing of Cu(I). Summary of the Invention

[0005] The present invention aims to address the aforementioned issues of the prior art by providing a Cu(I)-responsive fluorescence / photoacoustic dual-modality probe, its preparation method, and its application. The probe of the present invention simultaneously possesses tumor-targeting capabilities and a real-time response to Cu(I). Combined with three-dimensional photoacoustic / in vivo fluorescence imaging technology, the probe has the potential to be used for dynamic tracking of Cu(I) in living tumors, providing a reliable research tool for elucidating the mechanisms of Cu(I) regulation and intervention on tumors, as well as for the diagnosis and treatment of related diseases.

[0006] In order to achieve the above objectives, the first aspect of the present invention provides a fluorescence / photoacoustic dual-modal probe responsive to Cu(I), the structural formula of the probe being:

[0007] .

[0008] The second aspect of the present invention provides a method for preparing the fluorescent / photoacoustic dual-modal probe responsive to Cu(I), comprising the following steps:

[0009] (1) adding compound (I), compound (II) and anhydrous sodium acetate to a first solvent to carry out a first reaction to obtain compound (III);

[0010] (2) adding compound (III), N-tert-butoxycarbonyl-1,2-ethylenediamine, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to a second solvent, and performing a second reaction to obtain compound (IV);

[0011] (3) adding compound (IV), trifluoroacetic acid, compound (V) and triethylamine to a third solvent to carry out a third reaction to obtain the compound probe;

[0012] The reaction process is:

[0013] .

[0014] The third aspect of the present invention provides the use of the Cu(I)-responsive fluorescence / photoacoustic dual-modality probe in the preparation of a Cu(I) imaging agent for in vivo tumors.

[0015] The present invention has the following beneficial effects:

[0016] 1. The dual-modal probe provided by the present invention has high selectivity and sensitivity. After reacting with Cu(I), the absorption wavelength of the formed complex blue-shifts by about 10 nm, showing a clear ratiometric response. At the same time, the fluorescence is enhanced by about 15 times. It has good stability under physiological pH conditions, which is conducive to real-time in situ imaging of Cu(I). The probe itself has a strong photoacoustic signal. At the same time, ratiometric photoacoustic imaging can significantly reduce the interference of photoacoustic signals of biological molecules in the body (such as hemoglobin and melanin), which is beneficial for in vivo imaging.

[0017] 2. The preparation method of the dual-modal probe provided by the present invention uses readily available raw materials, and the reaction conditions are mild and easy to control, thereby saving reaction costs and ensuring the yield of the target product.

[0018] 3. The dual-modal probe HCy provided by the present invention Cu 1 has high tumor targeting ability and can be used for dynamic tracing of Cu(I) in tumors. Combined with three-dimensional photoacoustic / in vivo fluorescence imaging technology, the probe HCy Cu 1 can dynamically image the dynamics of Cu(I) in tumors, providing a new detection method for Cu(I) and a potential tool for tumor diagnosis.

[0019] Other features and advantages of the present invention will be described in detail in the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The above and other objects, features and advantages of the present invention will become more apparent through a more detailed description of exemplary embodiments of the present invention with reference to the accompanying drawings.

[0021] Figure 1 It shows that HCy Cu NMR spectrum of 1.

[0022] Figure 2 It shows that HCy Cu 1 Spectrum before and after reaction with Cu(I) in Mops buffer solution, where: Figure 2 The ultraviolet absorption spectrum is shown in a. Figure 2 The fluorescence spectrum is shown in b.

[0023] Figure 3 The fluorescence imaging diagram and fluorescence intensity diagram of the present invention are shown, wherein: Figure 3 a shows HCy Cu 1. HCy Cu 2. ATTM+HCy Cu Group 1 and Cu(I) response in vivo fluorescence imaging, Figure 3 HCy is shown in b Cu 1. HCy Cu 2. ATTM+HCy Cu 1 set of graphs showing changes in fluorescence intensity over time.

[0024] Figure 4 The two-dimensional photoacoustic imaging diagram and the photoacoustic signal intensity diagram of the present invention are shown, wherein: Figure 4 a shows HCy Cu 1 and HCy Cu Two groups of two-dimensional photoacoustic imaging in tumor model mice, Figure 4 HCy is shown in b Cu 1 and HCy Cu Two groups of graphs showing changes in photoacoustic signal intensity over time.

[0025] Figure 5 HCy is shown Cu 1 Three-dimensional photoacoustic imaging and photoacoustic signal intensity diagram in tumor model mice, among which, Figure 5 a shows HCy Cu 1 Imaging effect diagram over time, Figure 5 HCy is shown in b Cu 1. Changes of photoacoustic signal intensity over time; Figure 5 HCy is shown in c Cu 1Photoacoustic imaging effects in mice with different tumor sizes. Figure 5 d shows HCy Cu1. Changes in photoacoustic signal intensity in model mice with different tumor sizes. DETAILED DESCRIPTION

[0026] The preferred embodiments of the present invention will be described in more detail below. Although the preferred embodiments of the present invention are described below, it should be understood that the present invention can be implemented in various forms and should not be limited to the embodiments set forth herein.

[0027] The first aspect of the present invention provides a fluorescence / photoacoustic dual-modal probe responsive to Cu(I), the structural formula of the probe being:

[0028] .

[0029] The second aspect of the present invention provides a method for preparing the fluorescent / photoacoustic dual-modal probe responsive to Cu(I), comprising the following steps:

[0030] (1) adding compound (I), compound (II) and anhydrous sodium acetate to a first solvent to carry out a first reaction to obtain compound (III);

[0031] (2) adding compound (III), N-tert-butoxycarbonyl-1,2-ethylenediamine, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to a second solvent, and performing a second reaction to obtain compound (IV);

[0032] (3) Compound (IV), trifluoroacetic acid, compound (V) and triethylamine are added to a third solvent to carry out a third reaction to obtain compound HCy Cu 1;

[0033] The reaction process is:

[0034] .

[0035] According to the present invention, preferably, the first solvent, the second solvent and the third solvent are each independently anhydrous acetic anhydride and / or dichloromethane.

[0036] According to the present invention, preferably, in step (1), the molar ratio of compound (I) to compound (II) is 1:1.1-1.5; based on 1 mol of compound (I), the volume of the first solvent is 500-1000 mL, and the mass of sodium acetate is 300-500 g.

[0037] According to the present invention, preferably, in step (1), the conditions of the first reaction include: a reaction temperature of 10-40° C., and a reaction time of 0.5-50 min.

[0038] According to the present invention, preferably, in step (2), based on 1 mol of compound (III), the amount of 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate added is 300-500 g, the amount of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride added is 100-120 g, and the amount of N-tert-butoxycarbonyl-1,2-ethylenediamine added is 160-200 g.

[0039] According to the present invention, preferably, in step (2), the conditions of the second reaction include: reaction temperature of 10-40°C, and reaction time of 10-24 h.

[0040] According to the present invention, preferably, in step (3), the molar ratio of compound (IV), compound (V), trifluoroacetic acid and triethylamine is 1:2.0~2.4:20~40:1.0~1.2;

[0041] The conditions of the third reaction include: reaction temperature of -10 to 25° C., and reaction time of 1 to 10 h.

[0042] According to the present invention, preferably, in step (3), the third reaction comprises the following steps:

[0043] (a) dissolving compound (IV) in a third solvent, lowering the reaction temperature to -10-5°C, adding trifluoroacetic acid, and raising the reaction temperature to 10-25°C for reaction. After the reaction is completed, the reaction solution is dried by rotary evaporation to obtain a reaction product;

[0044] (b) The reaction product is redissolved in a third solvent, the reaction temperature is lowered to -10-5°C, the compound (V) and triethylamine are added, and the reaction temperature is raised to 10-25°C for reaction. After the reaction is completed, the reaction solution is dried in a spin-drying machine to obtain the probe.

[0045] The third aspect of the present invention provides the use of the Cu(I)-responsive fluorescence / photoacoustic dual-modality probe in the preparation of a Cu(I) imaging agent for in vivo tumors.

[0046] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in this field can be used. If the manufacturer of the reagents or instruments is not specified, they are all commercially available conventional reagents.

[0047] In the examples, the device used for two-dimensional photoacoustic imaging was the Vevo F2 LAZR-X photoacoustic imaging platform, the device used for in vivo fluorescence imaging was the IVIS Lumina K Series III instrument (PerkinElmer), and the device used for three-dimensional photoacoustic imaging was the LOIS-3D, Tomo Wave Laboratories, US.

[0048] Example 1

[0049] Compound III / HCy Cu Synthesis of 2: Under nitrogen, compound I (520 mg, 1 mmol) and compound II (358 mg, 1.1 mmol) dissolved in 10 mL of glacial acetic acid were added to a 50 mL Schlenk tube. Anhydrous sodium acetate (162 mg, 2 mmol) was then added, and the reaction mixture was stirred at room temperature for 0.5 h. The mixture was extracted with dichloromethane, the pH was adjusted to neutral with sodium bicarbonate, and the mixture was washed three times with saturated brine. The organic phase was then dried over anhydrous MgSO₄, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to yield 443 mg of a black solid (eluent: dichloromethane and methanol, dichloromethane:methanol = 40:1). v / v ; Yield: 51%).

[0050] Synthesis of Compound IV: Under nitrogen, Compound III (400 mg, 0.48 mmol), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU, 184 mg, 0.48 mmol), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 48 mg, 0.24 mmol) were dissolved in 20 mL of dry dichloromethane (DCM) in a 50 mL Schlenk tube and stirred at room temperature for 30 minutes. N-tert-Butoxycarbonyl-1,2-ethylenediamine (92 mg, 0.56 mmol) was then added, and the reaction mixture was stirred for 1 hour. The solvent was removed under reduced pressure, and the crude product was purified by silica gel column chromatography to yield 360 mg of a black solid (eluent: dichloromethane and methanol, dichloromethane:methanol = 50:1). v / v ; Yield: 72%).

[0051] Compound HCy Cu Synthesis method of 1: Under nitrogen protection, compound IV (200 mg, 0.27 mmol) was dissolved in 10 mL of dry dichloromethane (DCM), added to a 25 mL two-necked flask, and cooled to 0 oC, trifluoroacetic acid (TFA, 2 mL, 1.1 mmol) was added dropwise, and the reaction was terminated after 1 h. The solvent was removed under reduced pressure, and 10 mL of toluene was added azeotropically to remove the trifluoroacetic acid (TFA). The residue was then dissolved in 10 mL of dry dichloromethane (DCM) and added to a 50 mL Schlenk tube. Triethylamine (200 μL, 0.54 mmol) was added dropwise, and the reaction mixture was stirred for 1 h. The solvent was removed under reduced pressure, and the crude product was purified by silica gel column chromatography to obtain 168 mg of a black solid (eluent: dichloromethane and methanol, dichloromethane:methanol = 50:1, v / v ; Yield: 62%).

[0052] Compound HCy Cu 1 NMR images Figure 1 As shown in the figure, 1 H NMR (600 MHz, CDCl3) δ / ppm8.60 (d, J = 14.2 Hz, 1H), 7.42 (dd, J = 14.1, 7.6 Hz, 2H), 7.35 (d, J = 8.8Hz, 1H), 7.33 (d, J = 8.8 Hz, 1H). 7.27 – 7.29 (m, 2H), 7.23 (d, J = 6.8 Hz, 1H), 7.12 (m, 2H), 7.02 (d, J = 7.7 Hz, 1H), 6.98 (t, J = 5.5 Hz, 1H), 6.75 (d, J = 8.8 Hz, 1H), 6.57 (s, 1H), 6.33 (d, J = 14.4 Hz, 1H), 5.91 (s, 1H), 4.46 (t, J = 7.1 Hz, 2H), 3.74 (t, J = 7.4 Hz, 4H), 3.39 (d, J = 20.2 Hz, 4H), 3.13 (m, J = 14.1 Hz, 1H), 2.82 (t, J = 7.3 Hz, 6H), 2.73 (t, J = 13.1Hz, 8H), 2.62 (t, J = 7.0 Hz, 4H), 2.10 (s, 6H), 1.90 (m, 6H), 1.77 (s, 6H), 1.19 (d, J = 6.9 Hz, 6H). Verifiable HCy Cu 1's structure.

[0053] Take the probe HCy Cu1 was dissolved in dimethyl sulfoxide (DMSO) to prepare a 10 mM stock solution. The test solution was ethanol / Mops buffer solution (10 / 90, 10 mM, pH 7.0), and the probe test concentration was 10 μM. The absorption spectrum test results are shown in Figure 2. Figure 2 As shown in (a), the probe HCy Cu The absorption peaks of 1 are around 715 nm and 680 nm, and the probe HCy Cu After 1 reacted with Cu(I), the absorption intensity at 715 nm and 680 nm gradually decreased, and a new absorption peak appeared at 699 nm and 665 nm, and the absorption intensity gradually increased. The UV absorption spectrum showed that the probe HCy Cu 1 has obvious ratio response characteristics. Figure 2 As shown in (b), after adding Cu(I) to the probe solution, the fluorescence intensity of the probe at 725 nm gradually increases with the increase of Cu(I) concentration. When the content of Cu(I) reaches 1 equivalent, the fluorescence of the probe reaches saturation. Cu 1The fluorescence intensity increased by about 15 times.

[0054] Example 2

[0055] Probe HCy Cu 1 Fluorescence imaging of response to Cu(I) in in vivo fluorescence imaging experiments.

[0056] To further evaluate the probe HCy Cu 1 and HCy Cu 2 Performance of in vivo fluorescence imaging: These probes were administered via tail vein injection to mice bearing 4T1 tumors. In the control group receiving normal saline, no significant fluorescence signal was observed at the tumor site. Figure 3 As shown in middle a, after the probe was injected, HCy Cu 1 and HCy Cu 2. Accumulation in the tumor area. Figure 3 As shown in b, HCy Cu The fluorescence of the tumor site in group 1 gradually increased and reached the maximum value 2 hours after injection. Cu The fluorescence signals of the two groups decreased significantly over time and almost disappeared after 5 hours. These observations indicate that HCy Cu 1 and HCy Cu 2 has excellent tumor targeting and accumulation capabilities. In order to avoid interference from other biological factors, ATTM is Cu 1 was injected into the tumor site 30 minutes before tail vein injection. Fluorescence imaging showed that the tumor fluorescence intensity of the ATTM-treated group was significantly lower than that of the untreated HCy Cu1 group, confirming that the observed fluorescence signal was mainly due to the elevated Cu(I) level in the tumor. These results indicate that HCy Cu 1 can effectively achieve tumor targeting and in vivo imaging of Cu(I) dynamics.

[0057] Example 3

[0058] Probe HCy Cu 1 Photoacoustic imaging of tumors in mice.

[0059] HCy Cu 1 and HCy Cu 2 Mops buffered saline solution (concentration of 100 M, 150 L) was injected into 4T1 tumor-bearing mice. Photoacoustic imaging was performed on the mice 0.5 hours later, and ratiometric photoacoustic imaging images were obtained through dual-channel photoacoustic imaging analysis. Figure 4 As shown in a, in the absence of a targeting group HCy Cu In group 2, due to HCy Cu 2 has no targeting, and the photoacoustic intensity is almost unchanged; while in HCy Cu The probes in group 1 have efficient targeting ability and the photoacoustic signal is significantly enhanced. Figure 4 As shown in b, HCy Cu The photoacoustic signal intensity of group 1 increased with the extension of injection time and reached the maximum at 2 h; while Cu The intensity of the two groups of photoacoustic signals hardly changed.

[0060] Then, three-dimensional photoacoustic imaging was used to further study Cu(I) in tumors, such as Figure 5 As shown in middle a, PA 670 / 730 The ratio signal increases with the injection time, which is consistent with the two-dimensional photoacoustic imaging effect. Figure 5 As shown in Figure b, the photoacoustic signal intensity increases with the extension of injection time and reaches the maximum at 2 h. Cu 1 Imaging effects in mice with different tumor sizes, such as Figure 5 As shown in Figure c, as the tumor volume increases, the imaging effect becomes more and more obvious; Figure 5 As shown in middle d, the photoacoustic signal intensity is at 200 mm 3 The largest tumor in the model mice.

[0061] The above experimental results show that the probe HCy Cu 1 has efficient tumor targeting ability and can be used for dynamic tracing of Cu(I) in living tumors in combination with photoacoustic imaging, which has great application prospects in the biomedical field.

[0062] While various embodiments of the present invention have been described above, the above description is intended to be illustrative, not exhaustive, and not limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments.

Claims

1. A fluorescence / photoacoustic dual-modal probe responsive to Cu(I), characterized in that: The structural formula of the probe is: 。 2. The method for preparing the Cu(I)-responsive fluorescence / photoacoustic dual-modality probe according to claim 1, wherein: The following steps are involved: (1) adding compound (I), compound (II) and anhydrous sodium acetate to a first solvent to carry out a first reaction to obtain compound (III); (2) adding compound (III), N-tert-butoxycarbonyl-1,2-ethylenediamine, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to a second solvent, and performing a second reaction to obtain compound (IV); (3) adding compound (IV), trifluoroacetic acid, compound (V) and triethylamine to a third solvent to carry out a third reaction to obtain the probe; The reaction process is: 。 3. The preparation method according to claim 2, wherein The first solvent, the second solvent and the third solvent are each independently anhydrous acetic anhydride and / or dichloromethane.

4. The preparation method according to claim 2, wherein In step (1), the molar ratio of compound (I) to compound (II) is 1:1.1-1.5; based on 1 mol of compound (I), the volume of the first solvent is 500-1000 mL, and the mass of anhydrous sodium acetate is 300-500 g.

5. The preparation method according to claim 2, wherein In step (1), the conditions of the first reaction include: reaction temperature of 10-40°C, and reaction time of 0.5-50 min.

6. The preparation method according to claim 2, wherein In step (2), based on 1 mol of compound (III), the amount of 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate added is 300-500 g, the amount of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride added is 100-120 g, and the amount of N-tert-butoxycarbonyl-1,2-ethylenediamine added is 160-200 g.

7. The preparation method according to claim 2, wherein In step (2), the conditions of the second reaction include: reaction temperature of 10-40°C, and reaction time of 10-24 h.

8. The preparation method according to claim 2, wherein In step (3), the molar ratio of compound (IV), compound (V), trifluoroacetic acid and triethylamine is 1:2.0-2.4:20-40:1.0-1.2; The conditions of the third reaction include: reaction temperature of -10 to 25° C., and reaction time of 1 to 10 h.

9. The preparation method according to claim 2, wherein In step (3), the third reaction comprises the following steps: (a) dissolving compound (IV) in a third solvent, lowering the reaction temperature to -10-5°C, adding trifluoroacetic acid, and raising the reaction temperature to 10-25°C for reaction. After the reaction is completed, the reaction solution is dried by rotary evaporation to obtain a reaction product; (b) The reaction product is redissolved in a third solvent, the reaction temperature is lowered to -10-5°C, the compound (V) and triethylamine are added, and the reaction temperature is raised to 10-25°C for reaction. After the reaction is completed, the reaction solution is dried in a spin-drying machine to obtain the probe.

10. Use of the Cu(I)-responsive fluorescence / photoacoustic dual-modality probe according to claim 1 in preparing a Cu(I) imaging agent for in vivo tumors.

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