Organic fluorescent compound as well as preparation method and application thereof

Abstract

The invention provides an organic fluorescent compound as well as a preparation method and application thereof, and belongs to the technical field of biochemical materials. The organic fluorescent compound has a structure as shown in a formula I. D-A interaction exists in a molecular structure of the organic fluorescent compound provided by the invention, so that red shift of absorption and emission wavelengths is facilitated, the penetration depth to biological tissues is increased, and the near-infrared two-region imaging resolution is improved. The organic fluorescent compound provided by the invention has excellent II-type active oxygen generation capability and good photo-thermal performance; meanwhile, the organic fluorescent compound provided by the invention can be efficiently taken by HeLa cells, can effectively kill cancer cells under laser irradiation, and keeps high biological safety under a dark condition. The result of the embodiment shows that the organic fluorescent compound provided by the invention is expected to be used for PDT / PTT synergistic treatment guided by near-infrared two-region fluorescence imaging.

Description

Technical Field

[0001] This invention belongs to the field of biochemical materials technology, specifically relating to an organic fluorescent compound, its preparation method, and its application. Background Technology

[0002] Cancer poses a significant threat to global health systems. Furthermore, 70%–80% of cancer patients are diagnosed at an advanced stage, making treatment more difficult and complex, and increasing the risk of recurrence. Therefore, early diagnosis and treatment of cancer are crucial. Currently, traditional cancer treatments such as surgery, chemotherapy, and radiotherapy are widely used clinically, but they still have drawbacks including invasive procedures, drug resistance, high recurrence rates, and significant toxic side effects. Phototherapy, however, stands out due to its advantages of early diagnosis, precise treatment, and low toxicity.

[0003] In phototherapy, photodynamic therapy (PDT) activates photosensitizers enriched at tumor sites using specific light to generate reactive oxygen species, directly inducing apoptosis or necrosis of cancer cells. It can also avoid damage to healthy tissues by adjusting the irradiation area. Photothermal therapy (PTT), on the other hand, uses laser irradiation of photothermal agents, converting absorbed light energy into heat to locally raise the temperature of the tumor or tissue site, thereby inducing cellular heat loss to kill cancer cells. Therefore, the selection of photosensitizers and photothermal agents is crucial, requiring comprehensive consideration of their light absorption capacity, conversion efficiency, and biocompatibility to ensure therapeutic efficacy and minimize damage to normal tissues. Among these, near-infrared II (NIR-II, 1000–1700 nm) phototherapy molecules, which have received considerable attention in recent years, have become an important direction for improving the efficacy of both photodynamic and photothermal therapies due to their deeper tissue penetration and higher spatial resolution.

[0004] In addition, PTT or PDT alone may not be effective for treating large or inoperable lesions, and may also carry the risk of local recurrence and distant metastasis. PTT / PDT combined therapy can combine multiple treatment methods and is expected to enhance the treatment effect through the complementary effects of different mechanisms.

[0005] Currently, most phototherapy molecules still suffer from problems such as short emission wavelengths, poor reactive oxygen species generation capacity, and poor photothermal properties. Summary of the Invention

[0006] In view of this, the purpose of this invention is to provide an organic fluorescent compound, its preparation method, and its applications. The organic fluorescent compound provided by this invention exhibits near-infrared II emission, a large Stokes shift, good type II reactive oxygen species generation capability, and photothermal generation capability; it can be applied to the preparation of antitumor drugs and non-therapeutic cell imaging.

[0007] To achieve the above objectives, the present invention provides the following solution:

[0008] This invention provides an organic fluorescent compound having the structure shown in Formula I;

[0009]

[0010] In Equation I, R1 is Wherein, X includes halogens;

[0011] R2 is Where X includes halogens; and X is the same in R1 and R2.

[0012] Preferably, X includes I or Br.

[0013] Preferably, it has the structure shown in any one of formulas Ia to If;

[0014]

[0015]

[0016] The present invention provides a method for preparing the organic fluorescent compound described above, comprising the following steps: under a first protective atmosphere, mixing compound II, compound III, an alkaline catalyst and a first polar organic solvent to carry out a condensation reaction to obtain compound IV;

[0017] Under a second protective atmosphere, compound IV, a halide, and a second polar organic solvent are mixed and subjected to a salt-forming reaction to obtain the organic fluorescent compound; the halide includes halomethane or benzyl halide;

[0018] Compound II has the structure shown in Formula II, compound III has the structure shown in Formula III, and compound IV has the structure shown in Formula IV;

[0019]

[0020] In Equation II, R3 and R4 are independent.

[0021]

[0022] In Equation IV, R3 and R4 are independently...

[0023] Preferably, the molar ratio of compound II to compound III is (2-3):1; and the molar ratio of compound III to the alkaline catalyst is 1:(0.83-1.66).

[0024] Preferably, the condensation reaction is carried out at a temperature of 40–60°C for 12–24 hours.

[0025] Preferably, the halide includes iodomethane, benzyl bromide, or benzyl iodide; the alkaline catalyst includes an organic amine.

[0026] Preferably, the salt-forming reaction is carried out at a temperature of 80–150°C for a time of 18–32 hours.

[0027] Preferably, the molar ratio of compound IV to the halide is 1:(10-50).

[0028] This invention provides the application of the organic fluorescent compounds described in the above-described scheme or the organic fluorescent compounds prepared by the above-described preparation method in the preparation of anti-tumor drugs, the preparation of tumor diagnostic reagents, or in cell imaging for non-therapeutic purposes.

[0029] This invention provides an organic fluorescent compound having the structure shown in Formula I. The organic fluorescent compound provided by this invention exhibits DA interactions in its molecular structure, which facilitates a redshift in absorption and emission wavelengths, increasing penetration depth into biological tissues and improving near-infrared II imaging resolution. After absorbing light of a specific wavelength, the organic fluorescent compound provided by this invention generates highly cytotoxic singlet oxygen through a triplet-state-mediated type II photosensitization mechanism, while simultaneously generating heat energy efficiently through a non-radiative vibrational relaxation pathway. Therefore, this compound possesses both excellent type II reactive oxygen species generation capacity and good photothermal conversion performance. Furthermore, the organic fluorescent compound provided by this invention can be efficiently taken up by HeLa cells, effectively killing cancer cells under laser irradiation while maintaining high biocompatibility under dark conditions. The results of the examples indicate that the organic fluorescent compound provided by this invention holds promise for use in near-infrared II fluorescence imaging-guided PDT / PTT synergistic therapy.

[0030] This invention provides a method for preparing the organic fluorescent compound described above. The preparation method provided by this invention is simple, the separation and purification process is easy to operate, and it is suitable for industrial production. Attached Figure Description

[0031] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0032] Figure 1The absorption spectra of the organic fluorescent compounds 2PY-I, 2PY-PhBr, 2PY-PhI, 4PY-I, 4PY-PhBr and 4PY-PhI in DMSO solution and deionized water solution are shown.

[0033] Figure 2 The fluorescence emission spectra of the organic fluorescent compounds 2PY-I, 2PY-PhBr, 2PY-PhI, 4PY-I, 4PY-PhBr and 4PY-PhI in DMSO solution and deionized water solution are shown.

[0034] Figure 3 The organic fluorescent compounds 2PY-I, 2PY-PhBr, 2PY-PhI, 4PY-I, 4PY-PhBr, and 4PY-PhI (molar concentration: 1 μM) were mixed with the singlet oxygen scavenger ABDA under 660 nm laser irradiation (optical power: 0.4 W / cm²). 2 The curve showing the change of the ratio of real-time absorbance to initial absorbance at 380 nm over time;

[0035] Figure 4 The photothermal properties and photothermal conversion efficiency of the organic fluorescent compounds 2PY-I, 2PY-PhBr, 2PY-PhI, 4PY-I, 4PY-PhBr, and 4PY-PhI (molar concentration: 60 μM) under 660 nm laser irradiation were calculated (optical power: 0.4 W / cm²). 2 );

[0036] Figure 5 The dark toxicity and phototoxicity of the organic fluorescent compound 2PY-I on mouse breast cancer cells (4T1 cells) and human cervical cancer cells (HeLa cells);

[0037] Figure 6 Inverted fluorescence imaging images of organic fluorescent compounds 2PY-I, 2PY-PhBr, 2PY-PhI, 4PY-I, 4PY-PhBr and 4PY-PhI (molar concentration: 5 μM) after incubation with HeLa cells for 12 h.

[0038] Figure 7 This is a synthetic route diagram for the compound with the structure shown in Formula II-a;

[0039] Figure 8 Synthetic routes for compounds 3 and 5;

[0040] Figure 9 Synthetic routes for the organic fluorescent compounds 2PY-I, 2PY-PhBr and 2PY-PhI;

[0041] Figure 10Synthetic routes for the organofluorescent compounds 4PY-I, 4PY-PhBr, and 4PY-PhI are shown. Detailed Implementation

[0042] This invention provides an organic fluorescent compound having the structure shown in Formula I;

[0043]

[0044] In Equation I, R1 is Wherein, X includes halogens;

[0045] R2 is Where X includes halogens; and X is the same in R1 and R2.

[0046] In this invention, X preferably includes I or Br.

[0047] In this invention, the organic fluorescent compound preferably has the structure shown in any one of formulas Ia to If;

[0048]

[0049]

[0050] The organic fluorescent compound provided by this invention exhibits DA interactions in its molecular structure, which facilitates a redshift in absorption and emission wavelengths, increasing penetration depth into biological tissues and improving near-infrared II imaging resolution. After absorbing light of a specific wavelength, the organic fluorescent compound provides a triplet-mediated type II photosensitization mechanism to generate highly cytotoxic singlet oxygen, while simultaneously generating heat energy efficiently through a non-radiative vibrational relaxation pathway. Therefore, this compound possesses both excellent type II reactive oxygen species generation capacity and good photothermal conversion performance. Furthermore, the organic fluorescent compound provided by this invention can be efficiently taken up by HeLa cells, effectively killing cancer cells under laser irradiation while maintaining high biocompatibility in the dark. The results of the examples indicate that the organic fluorescent compound provided by this invention holds promise for use in near-infrared II fluorescence imaging-guided PDT / PTT synergistic therapy.

[0051] This invention provides a method for preparing the organic fluorescent compound described above, comprising the following steps:

[0052] Under a first protective atmosphere, compounds II, III, a basic catalyst, and a first polar organic solvent were mixed and subjected to a condensation reaction to obtain compound IV;

[0053] Under a second protective atmosphere, compound IV, a halide, and a second polar organic solvent are mixed and subjected to a salt-forming reaction to obtain the organic fluorescent compound; the halide includes halomethane or benzyl halide;

[0054] Compound II has the structure shown in Formula II, compound III has the structure shown in Formula III, and compound IV has the structure shown in Formula IV;

[0055]

[0056] In Equation II, R3 and R4 are independent.

[0057]

[0058] In Equation IV, R3 and R4 are independently...

[0059] Unless otherwise specified, all raw materials and equipment used in this invention are commercially available products well known to those skilled in the art.

[0060] In this invention, under a first protective atmosphere, compound II, compound III, an alkaline catalyst, and a first polar organic solvent are mixed and subjected to a condensation reaction to obtain compound IV.

[0061] In this invention, compound II preferably has the structure shown in formula II-a or formula II-b:

[0062]

[0063] In this invention, the preparation method of formula II-a preferably includes the following steps:

[0064] Under a third protective atmosphere, 4-bromotriphenylamine, phosphorus oxychloride, and a first organic solvent are mixed and subjected to a first reaction to obtain an aldehyde-modified product; the aldehyde-modified product has the structure shown in the formula TPA-CHO.

[0065]

[0066] Under a fourth protective atmosphere, the aldehyde product, pinacol 4-pyridineboronic acid, palladium catalyst, alkaline solution, and second organic solvent are mixed to carry out a second reaction to obtain formula II-a.

[0067] In this invention, the third protective atmosphere preferably comprises nitrogen; the first organic solvent preferably comprises N,N-dimethylformamide. In this invention, the molar ratio of 4-bromotriphenylamine to phosphorus oxychloride is preferably 3:3.6; the molar amount of 4-bromotriphenylamine and the volume ratio of the first organic solvent are preferably 3 mmol:10 mL. In this invention, the first reaction is preferably carried out at 0°C for 30 min, and then at 110°C for 48 h. In this invention, the first reaction is a Vilsmeier reaction.

[0068] After the first reaction is completed, the present invention preferably performs post-treatment on the obtained reaction solution. In the present invention, the post-treatment preferably includes adding an alkaline solution to the obtained reaction solution for neutralization, then adding water and ethyl acetate sequentially for extraction, collecting the organic phase for concentration under reduced pressure, and purifying the crude product by silica gel chromatography.

[0069] In this invention, the fourth protective atmosphere preferably includes nitrogen; the second organic solvent preferably includes tetrahydrofuran; and the palladium catalyst preferably includes tetratetraphenylphosphine palladium. In this invention, the molar ratio of the aldehyde product to pinacol 4-pyridineboronic acid is preferably 1:2; the molar ratio of the aldehyde product to the palladium catalyst is preferably 10:1; the molar amount of the aldehyde product and the volume ratio of the second organic solvent are preferably 1.5 mmol:10 mL; the alkaline solution preferably includes an aqueous potassium carbonate solution; the concentration of the aqueous potassium carbonate solution is preferably 1.59 mol / L; and the molar amount of the aldehyde product and the volume ratio of the alkaline solution are preferably 1.5 mmol:2 mL. In this invention, the temperature of the second reaction is preferably 70°C, and the time is preferably 48 h. In this invention, the second reaction is a Suzuki coupling reaction.

[0070] After the second reaction is completed, the present invention preferably performs post-processing on the resulting reaction solution. In the present invention, the post-processing preferably includes concentrating the resulting reaction solution under reduced pressure, and purifying the crude product by silica gel chromatography.

[0071] In this invention, compound IV preferably has the structure shown in formula IV-a or IV-b:

[0072]

[0073] In this invention, the first protective atmosphere preferably includes nitrogen or argon, more preferably nitrogen; the alkaline catalyst preferably includes an organic amine; the organic amine preferably includes piperidine; the first polar organic solvent preferably includes dichloromethane and ethanol; the volume ratio of dichloromethane to ethanol is preferably 1:5.

[0074] In this invention, the molar ratio of compound II to compound III is preferably (2-3):1, and in specific embodiments, it can be 2:1, 2.5:1, or 3:1; the molar ratio of compound III to the alkaline catalyst is preferably 1:(0.83-1.66), and in specific embodiments, it can be 1:0.83, 1:1, 1:1.5, or 1:1.66. This invention preferably monitors the condensation reaction using a TLC plate.

[0075] In this invention, the temperature of the condensation reaction is preferably 40-60°C, and in specific embodiments of this invention, it can be 40°C, 50°C, or 60°C; the time is preferably 12-24 hours, and in specific embodiments of this invention, it can be 12 hours, 14 hours, 15 hours, 16 hours, 20 hours, 22 hours, or 24 hours.

[0076] After the condensation reaction is completed, the present invention preferably performs post-treatment on the obtained reaction solution, which preferably includes concentrating, column chromatography and recrystallizing the reaction solution in sequence.

[0077] In this invention, the concentration preferably includes vacuum concentration. In this invention, the solvent for column chromatography is preferably dichloromethane and methanol; the volume ratio of dichloromethane to methanol is preferably 20:1. In this invention, the solvent for recrystallization is preferably dichloromethane and n-hexane; the volume ratio of dichloromethane to n-hexane is preferably 1:10.

[0078] After obtaining compound IV, the present invention mixes compound IV, a halide, and a second polar organic solvent under a second protective atmosphere to carry out a salt-forming reaction to obtain the organic fluorescent compound; the halide includes halomethane or benzyl halide.

[0079] In this invention, the second protective atmosphere preferably includes nitrogen. In this invention, the halide preferably includes iodomethane, benzyl bromide, or benzyl iodine; the second polar organic solvent preferably includes N,N-dimethylformamide (DMF). In this invention, the molar ratio of compound IV to the halide is preferably 1:(10-50), and in embodiments of this invention, it can specifically be 1:10, 1:20, 1:30, 1:40, or 1:50. This invention does not have special requirements on the amount of the second polar organic solvent, as long as it is sufficient to dissolve compound IV.

[0080] In this invention, the molar ratio of compound IV to the halide is 1:(10-50). The salt-forming reaction is preferably monitored by a TLC plate. In this invention, the salt-forming reaction is preferably carried out under stirring conditions; the salt-forming reaction is preferably carried out under reflux conditions.

[0081] In this invention, the preferred temperature for the salt formation reaction is 80–150°C. In specific embodiments of this invention, the temperature can be 80, 100, 110, 120, 130, 140, or 150°C. The preferred time is 18–32 hours. In specific embodiments of this invention, the time can be 18 hours, 20 hours, 24 hours, 28 hours, 30 hours, or 32 hours.

[0082] After the salt formation reaction is completed, the present invention preferably performs post-treatment on the obtained reaction solution. The post-treatment preferably includes cooling the obtained reaction solution, adding an organic solvent under ultrasonic conditions, filtering and washing in sequence to obtain a solid product; recrystallizing and washing the obtained solid product in sequence, then repeating recrystallization and washing in sequence, and finally drying the obtained product after washing in sequence.

[0083] In this invention, the organic solvent is preferably ethyl acetate. In this invention, the solvent for recrystallization is preferably N,N-dimethylformamide and ethyl acetate; the volume ratio of N,N-dimethylformamide to ethyl acetate is preferably 1:(5-10), and in embodiments of this invention, it can specifically be 1:10. In this invention, the first and second washings are performed independently using ethyl acetate, toluene, and diethyl ether sequentially, with each wash preferably being 10-20 times the amount of the substance to be washed. In this invention, the number of repetitions is preferably 3-4 times, and in embodiments of this invention, it can specifically be 3 or 4 times.

[0084] The preparation method provided by this invention is simple, the separation and purification process is easy to operate, and it is suitable for industrial production.

[0085] This invention provides the application of the organic fluorescent compounds described in the above-described scheme or the organic fluorescent compounds prepared by the above-described preparation method in the preparation of anti-tumor drugs, the preparation of tumor diagnostic reagents, or in cell imaging for non-therapeutic purposes.

[0086] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, provides an organic fluorescent compound, its preparation method, and its applications, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0087] Example 1

[0088] according to Figure 7 The synthetic route described above is used to prepare compounds with the structure shown in formula II-a:

[0089] 3 mmol of 4-bromotriphenylamine (TPA-Br) was dissolved in 10 mL of ultradry N,N-dimethylformamide. Under nitrogen protection and an ice-water bath at 0 °C, 3.6 mmol of phosphorus oxychloride was slowly injected, and the reaction proceeded for 30 min. The reaction solution was then transferred to 110 °C and the reaction continued for 48 h (the process was monitored by TLC until completion). A saturated aqueous solution of sodium bicarbonate was added to the reaction solution to neutralize the reaction, followed by extraction with water and ethyl acetate. The organic phase was collected and concentrated under reduced pressure. The crude product was purified by silica gel chromatography (petroleum ether / ethyl acetate volume ratio = 20:1) to obtain a yellow oily substance, TPA-CHO.

[0090] Under N2 protection, a mixture of TPA-CHO (1.5 mmol), pinacol 4-pyridineborate (3 mmol), tetraphenylphosphine palladium (0.15 mmol), and tetrahydrofuran (10 mL) was placed in a double-necked round-bottom flask. Then, a pre-prepared potassium carbonate aqueous solution (2 mL, 1.59 mol / L) was added, and the mixture was reacted at 70 °C for 48 h. After the reaction was complete, the reaction solution was concentrated under reduced pressure, and the crude product was purified by silica gel chromatography (petroleum ether / ethyl acetate volume ratio = 1:3) to obtain an orange-yellow oil (formula II-a) in 73% yield.

[0091] The orange-yellow oily substance obtained in this embodiment was characterized, and the specific NMR data are as follows:

[0092] Formula II-a: 1 H NMR(600MHz,DMSO-d6)δ9.81(s,1H),8.62(d,J=5.7Hz,2H),7.85(d,J=8.5Hz,2H),7.78(d,J=8.6Hz,2H),7.7 1(d,J=5.8Hz,2H),7.45(t,J=7.8Hz,2H),7.27(t,J=6.8Hz,3H),7.23(d,J=7.7Hz,2H),7.02(d,J=8.6Hz,2H).

[0093] Based on the characterization data above, the obtained orange-yellow oily substance is a compound with the structure shown in Formula II-a.

[0094] according to Figure 8 The synthetic route shown is used to prepare the compound with the structure shown in compound 3:

[0095] Raw material 2 (i.e., formula III, 1 mmol), raw material 1 (i.e., formula II-a, 2.5 mmol), and piperidine (0.83 mmol) were dissolved in an ethanol-dichloromethane mixed solvent (ethanol to dichloromethane volume ratio of 5:1). The condensation reaction was carried out at 50 °C for 12 h under nitrogen atmosphere. After the condensation reaction was completed, the reaction solution was concentrated under reduced pressure and further purified by silica gel chromatography (dichloromethane to methanol volume ratio = 20:1). The purified crude product was dissolved in a small amount of dichloromethane, and n-hexane was slowly added in an ultrasonic instrument to precipitate a large amount of solid (dichloromethane to n-hexane volume ratio = 1:10), giving a dark blue product with a yield of 40%.

[0096] The dark blue solid product obtained in this embodiment was characterized, and the specific NMR data are as follows:

[0097] Compound 3: 1H NMR (600MHz, DMSO-d6) δ8.61(d,J=5.7Hz,4H),7.81(d,J=8.6Hz,4H),7.77(d,J=12.5Hz,2H),7.75(d,J=5.3Hz,4H),7.70(d,J=5.7Hz,4H),7.42(t ,J=7.8Hz,4H),7.21(t,J=7.4Hz,2H),7.18(d,J=8.4Hz,8H),7.12(d,J=1 6.0Hz,2H),7.08(d,J=8.6Hz,4H),6.95(s,2H),5.89(s,1H),1.60(s,6H).

[0098] Based on the characterization data above, the obtained dark blue powdery solid product is a compound with the structure shown in compound 3 (i.e., formula IV-a).

[0099] according to Figure 9 The synthetic route described above is used to prepare compounds with the structure shown in formula Ia (i.e., 2PY-I):

[0100] Compound 3 (1 mmol) and iodomethane (30 mmol) were dissolved in 3 mL of N,N-dimethylformamide. The mixture was refluxed at 100 °C and stirred for 24 h under nitrogen atmosphere to induce a salt formation reaction (the process was monitored by TLC until completion). The reaction solution was cooled to room temperature, and ethyl acetate was added under sonication. A black solid precipitated out. The precipitate was filtered and washed successively with ethyl acetate, toluene, and diethyl ether, each time using 10–20 times the volume of the washings. The washed precipitate was dissolved in a small amount of N,N-dimethylformamide, and recrystallized again with ethyl acetate (N,N-dimethylformamide to ethyl acetate volume ratio 1:10). The precipitate was filtered and washed successively with ethyl acetate, toluene, and diethyl ether, repeating this step three times. After drying, a blackish-green solid product was obtained, with a yield of 87%.

[0101] The blackish-green solid obtained in this embodiment was characterized, and the specific data are as follows:

[0102] Equation Ia(2PY-I): 1H NMR (600MHz, DMSO-d6) δ8.91(d,J=6.2Hz,4H),8.41(d,J=6.3Hz,4H),8.06(d,J=8.6Hz,4H),7.82(d,J=8.3Hz,4H),7.78(d,J=16.2Hz,2H),7 .47(t,J=7.7Hz,4H),7.29(t,J=7.3Hz,2H),7.22(d,J=7.8Hz,4H),7.18-7.14(m,10H),6.98(s,2H),5.93(s,1H),4.29(s,6H),1.61(s,6H).

[0103] 13 C NMR (151MHz, DMSO) δ179.28,173.11,159.16,153.61,150.72,150.60,148.00,145.85,145.66,136.90,131.14,130.66,130.22,130.03 ,126.89,126.59,126.22,124.58,123.08,122.39,118.86,114.85,114.05,113.61,101.63,97.91,86.16,54.93,48.40,47.23,26.34.

[0104] HRMS (MALDI-TOF): m / z: [MI] + calcd for C 68 H 53 N7O2 2+ :499.7125; found:499.7134,m / z:[I] + calcd for I - :126.9050; found:126.9049.

[0105] Based on the characterization data above, the obtained blackish-green solid is a compound with the structure shown in 2PY-I (i.e., formula Ia).

[0106] Example 2

[0107] Compound 3 was prepared in the same manner as in Example 1.

[0108] according to Figure 9 The synthetic route described above is used to prepare compounds with the structure shown in formula Ib (i.e., 2PY-PhBr):

[0109] The preparation method was the same as in Example 1, except that iodomethane was replaced with benzyl bromide (30 mmol), resulting in a black-green solid product with a yield of 88%.

[0110] Formula Ib(2PY-PhBr): 1 H NMR(600MHz,DMSO-d6)δ9.09(d,J=6.7Hz,4H),8.42(d,J=6.6Hz,4H),8.03(d,J=8.7Hz, 4H),7.80(d,J=8.4Hz,4H),7.77(d,J=16.2Hz,2H),7.54(d,J=7.1Hz,4H),7.47-7.43(m ,10H),7.28(t,J=7.4Hz,2H),7.22(d,J=7.9Hz,4H),7.18(d,J=6.5Hz,4H),7.16(d,J=1 4.0Hz,2H),7.13(d,J=8.6Hz,4H),6.97(s,2H),5.91(s,1H),5.78(s,4H),1.61(s,6H).

[0111] 13 C NMR(151MHz,DMSO)δ179.28,173.16,159.14,154.43,150.91,150.58,147.8 9,145.78,144.82,136.89,135.09,131.31,130.66,130.25,130.18,129.72 ,129.69,129.12,126.95,126.39,126.29,124.76,123.79,122.12,118.93,114.83,114.03,113.60,101.67,97.91,86.24,62.48,48.46,26.33,21.53.

[0112] HRMS (MALDI-TOF): m / z: [M-Br] + calcd for C 80 H 61 N7O2 2+ :575.7438; found:575.7452,m / z:[Br] + calcd forBr - :78.9189; found:78.9183.

[0113] Based on the characterization data above, the obtained blackish-green solid is a compound with the structure shown in 2PY-PhBr (i.e., formula Ib).

[0114] Example 3

[0115] Compound 3 was prepared in the same manner as in Example 1.

[0116] according to Figure 9 The synthetic route described above is used to prepare compounds with the structure shown in formula Ic (i.e., 2PY-PhI):

[0117] The preparation method was the same as in Example 1, except that iodomethane was replaced with benzyl iodine (30 mmol), resulting in a black-green solid product with a yield of 89%.

[0118] Equation Ic(2PY-PhI): 1 H NMR(600MHz,DMSO-d6)δ9.08(d,J=6.6Hz,4H),8.41(d,J=6.6Hz,4H),8.03(d,J=8.6Hz, 4H),7.80(d,J=8.4Hz,4H),7.77(d,J=16.3Hz,2H),7.54(d,J=7.4Hz,4H),7.48-7.43(m ,10H),7.29(t,J=7.4Hz,2H),7.22(d,J=7.8Hz,4H),7.18(d,J=8.2Hz,4H),7.16(d,J=1 4.0Hz,2H),7.13(d,J=8.6Hz,4H),6.97(s,2H),5.91(s,1H),5.77(s,4H),1.61(s,6H).

[0119] 13 C NMR(151MHz,DMSO)δ179.28,173.15,159.12,154.43,150.90,150.53,147.8 9,145.77,144.82,136.87,135.07,131.28,130.67,130.25,130.21,129.73 ,129.71,129.10,126.95,126.38,126.30,124.76,123.80,122.13,118.95,114.84,114.03,113.58,101.64,97.92,86.24,62.57,48.46,26.33,21.54.

[0120] HRMS (MALDI-TOF): m / z: [MI] + calcd for C 80 H 61 N7O2 2+:575.7438; found:575.7451,m / z:[I] + calcd for I - :126.9050; found:126.9047.

[0121] Based on the characterization data above, the obtained blackish-green solid is a compound with the structure shown in 2PY-PhI (i.e., formula Ic).

[0122] Example 4

[0123] according to Figure 8 The synthetic route described above is used to prepare compounds with the structure shown in compound 5:

[0124] Raw material 2 (Formula III, 1 mmol), raw material 4 (Formula II-b, 2.5 mmol), and piperidine (0.83 mmol) were dissolved in an ethanol-dichloromethane mixed solvent (ethanol to dichloromethane volume ratio of 5:1). The condensation reaction was carried out at 50 °C for 12 h under nitrogen atmosphere. After the condensation reaction was completed, the reaction solution was concentrated under reduced pressure and further purified by silica gel chromatography (dichloromethane:methanol volume ratio = 20:1). The purified crude product was dissolved in a small amount of dichloromethane, and n-hexane was slowly added in an ultrasonic instrument to precipitate a large amount of solid (dichloromethane to n-hexane volume ratio = 1:10), giving a dark blue product with a yield of 44%.

[0125] The dark blue solid product obtained in this embodiment was characterized, and the specific NMR data are as follows:

[0126] Compound 5: 1 H NMR (600MHz, DMSO-d6) δ8.62(d,J=5.0Hz,8H),7.85(d,J=8.2Hz,8H),7.80-7.77(m,6H),7.72(d ,J=5.0Hz,8H),7.25(d,J=8.3Hz,8H),7.18-7.14(m,6H),6.96(s,2H),5.90(s,1H),1.61(s,6H).

[0127] Based on the characterization data above, the obtained dark blue powdery solid product is a compound with the structure shown in compound 5 (i.e., formula IV-b).

[0128] according to Figure 10 The synthetic route described above is used to prepare compounds with the structure shown in formula Id (i.e., 4PY-I):

[0129] Compound 5 (1 mmol) and iodomethane (30 mmol) were dissolved in 3 mL of N,N-dimethylformamide. The mixture was refluxed at 100 °C under nitrogen and stirred for 24 h, with the reaction monitored by TLC until completion. The reaction solution was cooled to room temperature, and ethyl acetate was added under sonication. A black solid precipitated out. The precipitate was filtered and washed successively with ethyl acetate, toluene, and diethyl ether, each time using 10–20 times the volume of the washings. The washed precipitate was dissolved in a small amount of N,N-dimethylformamide, and recrystallized with ethyl acetate (N,N-dimethylformamide to ethyl acetate volume ratio 1:10). The precipitate was filtered and washed successively with ethyl acetate, toluene, and diethyl ether, repeating this step three times. After drying, a blackish-green solid product was obtained, with a yield of 88%.

[0130] Formula Id(4PY-I): 1 H NMR (600MHz, DMSO-d6) δ8.95(d,J=6.6Hz,8H),8.46(d,J=6.6Hz,8H),8.13(d,J=8.7Hz,8H),7.89(d,J=8.4Hz,4H),7.82(d,J=16.0 Hz,2H),7.32(d,J=8.6Hz,8H),7.29(d,J=8.4Hz,4H),7.25(d,J=16.0Hz,2H),7.00(s,2H),5.96(s,1H),4.31(s,12H),1.62(s,6H).

[0131] 13 C NMR (151MHz, DMSO) δ179.29,173.45,158.93,153.52,149.75,147.23,145.84,136.58,132.49,130.28,128 .58,126.11,124.65,123.59,119.70,114.77,113.99,113.49,101.81,98.05,86.74,54.92,47.41,26.31.

[0132] HRMS (MALDI-TOF): m / z: [MI] + calcd for C 80 H 65 N9O2 4+ :295.8810; found:295.8818,m / z:[I] + calcd for I - :126.9050; found:126.9049.

[0133] Based on the characterization data above, the obtained blackish-green solid is a compound with the structure shown in 4PY-I (i.e., formula Id).

[0134] Example 5

[0135] Compound 5 was prepared in the same manner as in Example 4.

[0136] according to Figure 10 The synthetic route described above is used to prepare compounds with the structure shown in formula Ie (i.e., 4PY-PhBr):

[0137] The preparation method was the same as in Example 4, except that iodomethane was replaced with benzyl bromide (30 mmol), resulting in a black-green solid product with a yield of 87%.

[0138] Formula Ie(4PY-PhBr): 1 H NMR(600MHz,DMSO-d6)δ9.16(d,J=6.5Hz,8H),8.48(d,J=6.5Hz,8H),8.11( d,J=8.4Hz,8H),7.88(d,J=8.2Hz,4H),7.81(d,J=16.2Hz,2H),7.56(d,J=7. 4Hz,8H),7.48-7.43(m,12H),7.30(d,J=8.4Hz,8H),7.28(d,J=8.3Hz,4H), 7.23(d,J=16.1Hz,2H),6.99(s,2H),5.95(s,1H),5.83(s,8H),1.62(s,6H).

[0139] 13 C NMR (151MHz, DMSO) δ179.29,173.48,158.89,154.32,150.33,149.85,147.09,145.01,135.07,133.63,132.66,130.50,130.39,129.75 ,129.70,129.18,128.50,126.24,124.53,124.37,119.76,114.77,113.97,113.48,101.89,98.06,86.74,62.59,48.71,26.28,21.54.

[0140] HRMS (MALDI-TOF): m / z: [M-Br] + calcd for C 104 H 81 N9O2 4+ :371.9123; found:371.9137,m / z:[Br]+ calcd forBr - :78.9189; found:78.9182.

[0141] Based on the characterization data above, the obtained blackish-green solid is a compound with the structure shown in 4PY-PhBr (i.e., formula Ie).

[0142] Example 6

[0143] Compound 5 was prepared in the same manner as in Example 4.

[0144] according to Figure 10 The synthetic route described above is used to prepare compounds with the structure shown in Formula If (i.e., 4PY-PhI):

[0145] The preparation method was the same as in Example 4, except that iodomethane was replaced with benzyl iodine (30 mmol), resulting in a black-green solid product with a yield of 88%.

[0146] Formula If(4PY-PhI): 1 H NMR(600MHz,DMSO-d6)δ9.14(d,J=6.5Hz,8H),8.47(d,J=6.5Hz,8H),8.10( d,J=8.5Hz,8H),7.87(d,J=8.2Hz,4H),7.80(d,J=16.2Hz,2H),7.55(d,J=7. 1Hz,8H),7.48-7.43(m,12H),7.30(d,J=8.5Hz,8H),7.28(d,J=7.7Hz,4H), 7.22(d,J=16.1Hz,2H),6.99(s,2H),5.94(s,1H),5.82(s,8H),1.62(s,6H).

[0147] 13 C NMR (151MHz, DMSO) δ179.28,173.47,158.87,154.38,150.29,149.87,147.11,145.01,136.53,134.99,132.63,130.48,130.42,129 .78,129.73,129.14,128.50,126.31,124.57,124.39,119.77,114.73,113.94,113.47,101.82,98.06,86.83,62.76,48.79,26.32.

[0148] HRMS (MALDI-TOF): m / z: [MI] + calcd for C104 H 81 N9O2 4+ :371.9123; found:371.9135,m / z:[I] + calcd forI - :126.9050; found:126.9049.

[0149] Based on the characterization data above, the obtained blackish-green solid is a compound with the structure shown in 4PY-PhI (i.e., formula If).

[0150] Performance testing

[0151] (1) The ultraviolet absorption spectra of 2PY-I, 2PY-PhBr, 2PY-PhI, 4PY-I, 4PY-PhBr and 4PY-PhI were measured, and the specific steps are as follows:

[0152] 2PY-I, 2PY-PhBr, 2PY-PhI, 4PY-I, 4PY-PhBr, and 4PY-PhI were each prepared into DMSO stock solutions with a molar concentration of 1 mM. The corresponding stock solutions were transferred to deionized water (or DMSO solution) to prepare 5 μM standard test solutions, and ultraviolet absorption spectra were collected in the 300 nm–900 nm wavelength range using a UV spectrophotometer.

[0153] Figure 1 The absorption spectra of the organic fluorescent compounds 2PY-I, 2PY-PhBr, 2PY-PhI, 4PY-I, 4PY-PhBr, and 4PY-PhI in DMSO solution and deionized water solution are shown. Figure 1 As shown, in the deionized water system, 2PY-I, 2PY-PhI, 4PY-I, and 2PY-PhBr exhibit almost identical maximum absorption peaks at 590 nm, while the maximum absorption peaks of 4PY-PhBr and 4PY-PhI are located at 645 nm. In DMSO solution, 2PY-I, 4PY-I, 2PY-PhBr, 4PY-PhBr, 2PY-PhI, and 4PY-PhI exhibit almost identical maximum absorption peaks at 630 nm.

[0154] (2) The fluorescence spectra of 2PY-I, 2PY-PhBr, 2PY-PhI, 4PY-I, 4PY-PhBr and 4PY-PhI were measured, and the specific steps are as follows:

[0155] 2PY-I, 2PY-PhBr, 2PY-PhI, 4PY-I, 4PY-PhBr, and 4PY-PhI were prepared as 1 mM DMSO stock solutions. The corresponding stock solutions were transferred to deionized water (or DMSO solution) to prepare 5 μM standard test solutions. These solutions were then placed in a fluorescence spectrometer and excited using an external 660 nm laser. Fluorescence emission spectra in the 700 nm–1300 nm wavelength range were collected.

[0156] Figure 2 The fluorescence emission spectra of the organic fluorescent compounds 2PY-I, 2PY-PhBr, 2PY-PhI, 4PY-I, 4PY-PhBr, and 4PY-PhI in DMSO solution and deionized water solution are shown. Figure 2 As shown, in the deionized water system, 2PY-I, 2PY-PhBr, 2PY-PhI, 4PY-I, 4PY-PhBr, and 4PY-PhI all exhibit the largest emission peak at 950 nm. The Stokes shifts for 2PY-I, 2PY-PhI, 4PY-I, and 2PY-PhBr are 360 ​​nm, while the Stokes shifts for 4PY-PhBr and 4PY-PhI are 305 nm. In DMSO solution, 2PY-I, 2PY-PhBr, 2PY-PhI, 4PY-I, 4PY-PhBr, and 4PY-PhI all exhibit the largest emission peak at 955 nm, with a Stokes shift of 325 nm.

[0157] (3) Singlet oxygen atoms of 2PY-I, 2PY-PhBr, 2PY-PhI, 4PY-I, 4PY-PhBr and 4PY-PhI ( 1 The O2 generation capability test involves the following steps:

[0158] The ability to generate 1O2 was assessed using 9,10-anthratridiyl-bis(methylene)dimalonic acid (ABDA) as a fluorescent indicator. When 1O2 is generated in the system, ABDA specifically binds to 1O2 to form the corresponding internal peroxide, which disrupts the structure of ABDA, causing the absorption spectrum in the 300–500 nm range to gradually weaken or disappear. Specifically, ABDA (40 μL, 50 μM) and the sample (1 μM) were added to PBS buffer solution (pH = 7.4) and mixed thoroughly. The mixture was then irradiated under a 660 nm laser (0.4 W / cm²), and the absorbance changes were recorded at regular intervals using a UV spectrophotometer. The real-time absorbance at 380 nm was plotted against the initial absorbance.

[0159] Figure 3The organic fluorescent compounds 2PY-I, 2PY-PhBr, 2PY-PhI, 4PY-I, 4PY-PhBr, and 4PY-PhI (molar concentration: 1 μM) were mixed with the singlet oxygen scavenger ABDA under 660 nm laser irradiation (optical power: 0.4 W / cm²). 2 The curve showing the change in the ratio of real-time absorbance to initial absorbance at 380 nm over time. Figure 3 As shown, ABDA alone did not degrade after 50 seconds of 660nm laser irradiation. However, in the presence of six near-infrared II fluorescent compounds (i.e., 2PY-I, 2PY-PhBr, 2PY-PhI, 4PY-I, 4PY-PhBr, and 4PY-PhI obtained in Examples 1-6), the characteristic absorbance of ABDA at 380nm decreased rapidly with increasing irradiation time, indicating that all six near-infrared II fluorescent compounds have good type II reactive oxygen species (ROS) generation performance. Among them, 2PY-I showed the fastest O2 generation efficiency, with its absorbance almost decreasing to zero after 40 seconds of 660nm laser irradiation.

[0160] (4) The photothermal performance of 2PY-I, 2PY-PhBr, 2PY-PhI, 4PY-I, 4PY-PhBr and 4PY-PhI was tested, and the specific steps are as follows:

[0161] Six near-infrared II fluorescent compounds were irradiated with aqueous solutions (200 μL, 60 μM) under a 660 nm laser (0.4 W / cm2) for 6 min. At this time point, the laser was turned off and the samples were allowed to cool naturally to room temperature (about 10 min). The temperature change during this process was monitored using an infrared thermal imager.

[0162] Figure 4 The photothermal properties and photothermal conversion efficiency of the organic fluorescent compounds 2PY-I, 2PY-PhBr, 2PY-PhI, 4PY-I, 4PY-PhBr, and 4PY-PhI (molar concentration: 60 μM) under 660 nm laser irradiation were calculated (optical power: 0.4 W / cm²). 2 ).like Figure 4 As shown, the photothermal conversion efficiencies of 2PY-I, 2PY-PhBr, 2PY-PhI, 4PY-I, 4PY-PhBr, and 4PY-PhI are 42%, 41%, 37%, 33%, 46%, and 41%, respectively.

[0163] (5) 2PY-I cytotoxicity test, the specific steps are as follows:

[0164] Cells in the logarithmic growth phase were evenly seeded into 96-well plates (approximately 5000 cells / well) and pre-cultured at 37°C in a 5% CO2 incubator for 24 h. Subsequently, the culture medium in the 96-well plates was replaced with fresh medium containing different concentrations of 2PY-I, and the plates were incubated for another 20 h. Next, the plates were placed under 660 nm laser irradiation (0.4 W / cm²) for 10 min, with cells not exposed to light serving as a dark control. After incubation for another 4 h, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetraazolium bromide (MTT solution, 10 μL, 5 mg / mL) was added, and the plates were cultured for another 4 h. Then, the MTT-containing medium was removed, and 100 μL of DMSO was added to each well to dissolve the formazan. Finally, the absorbance at 490 nm was measured using a microplate reader. Cells incubated solely with the culture medium served as a control. Results are expressed as the percentage of viable cells in the 2PY-I treatment relative to the control group. Relative cell viability is calculated using the following formula:

[0165] Cell viability (%) = (OD) sample -OD background ) / (OD control -OD background )×100%(Formula 1);

[0166] In Formula 1, OD sample Indicates the absorbance value of the sample being tested; OD control The absorbance value of the blank control group; OD background This represents the absorbance value of the background control group. Figure 5 The dark toxicity and phototoxicity of the organic fluorescent compound 2PY-I on mouse breast cancer cells (4T1 cells) and human cervical cancer cells (HeLa cells) are presented. Figure 5 Figure A shows the dark toxicity and phototoxicity of different concentrations of 2PY-I on human and mouse breast cancer cells (4T1 cells). Figure 5 Figure B shows the dark toxicity and phototoxicity of different concentrations of 2PY-I on human cervical cancer cells (HeLa cells). Under dark conditions, 2PY-I showed low toxicity to both cancer cell lines (HeLa and 4T1), with cell viability remaining above 90%, indicating that 2PY-I has good biosafety under dark conditions. At 660 nm (0.4 W / cm²), the phototoxicity was significantly lower. 2 After 10 minutes of laser irradiation, 2PY-I exhibited significant concentration-dependent cytotoxicity against 4T1 cells, with cell viability dropping below 20% at a concentration of only 5 μM. Similar results were observed in HeLa cells, further validating the highly effective phototoxicity of 2PY-I against cancer cells.

[0167] (6) The fluorescence imaging capabilities of 2PY-I, 4PY-I, 2PY-PhBr, 4PY-PhBr, 2PY-PhI and 4PY-PhI in HeLa cells were tested, and the specific steps are as follows:

[0168] HeLa cells were seeded in a confocal dish and incubated at 37°C in a 5% CO2 incubator for 12 hours. After the cell count reached the experimental requirements, the above six organic fluorescent compounds (5 μM) were added and incubated for another 12 hours. The old culture medium was removed and the cells were washed three times with PBS. Imaging was then performed using an inverted fluorescence microscope.

[0169] Figure 6 Inverted fluorescence imaging images of HeLa cells after incubation for 12 h with the organic fluorescent compounds 2PY-I, 2PY-PhBr, 2PY-PhI, 4PY-I, 4PY-PhBr, and 4PY-PhI (molar concentration: 5 μM). Scale bar: 30 μm. Figure 6 It can be seen that obvious fluorescence signals can be observed in HeLa cells after incubation with the six near-infrared II fluorescent compounds, indicating that they can all be efficiently taken up by HeLa cells.

[0170] As can be seen from the above embodiments, the near-infrared II fluorescent compound with reactive oxygen species and photothermal generation capabilities provided by the present invention has simple synthesis steps, simple separation and purification operations, can generate near-infrared II fluorescence, has a large Stokes shift, excellent type II reactive oxygen species generation capability, good photothermal conversion performance, can effectively kill cancer cells under laser irradiation, and can be efficiently taken up by HeLa cells, and has broad application prospects.

[0171] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An organic fluorescent compound, characterized in that, It has the structure shown in Equation I; In Equation I, R1 is Wherein, X includes halogens; R2 is Where X includes halogens; and X is the same in R1 and R2.

2. The organic fluorescent compound according to claim 1, characterized in that, X includes I or Br.

3. The organic fluorescent compound according to claim 1 or 2, characterized in that, It has the structure shown in any one of the terms from Ia to If; 4. A method for preparing the organic fluorescent compound according to any one of claims 1 to 3, characterized in that, Includes the following steps: Under a first protective atmosphere, compounds II, III, a basic catalyst, and a first polar organic solvent were mixed and subjected to a condensation reaction to obtain compound IV; Under a second protective atmosphere, compound IV, a halide, and a second polar organic solvent are mixed and subjected to a salt-forming reaction to obtain the organic fluorescent compound; the halide includes halomethane or benzyl halide; Compound II has the structure shown in Formula II, compound III has the structure shown in Formula III, and compound IV has the structure shown in Formula IV; In Equation II, R3 and R4 are independent. In Equation IV, R3 and R4 are independently...

5. The preparation method according to claim 4, characterized in that, The molar ratio of compound II to compound III is (2-3):1; the molar ratio of compound III to the alkaline catalyst is 1:(0.83-1.66).

6. The preparation method according to claim 4 or 5, characterized in that, The condensation reaction is carried out at a temperature of 40–60°C for 12–24 hours.

7. The preparation method according to claim 4, characterized in that, The halide includes iodomethane, benzyl bromide, or benzyl iodide; the alkaline catalyst includes organic amines.

8. The preparation method according to claim 4 or 7, characterized in that, The salt formation reaction is carried out at a temperature of 80–150°C for a time of 18–32 hours.

9. The preparation method according to claim 4, characterized in that, The molar ratio of compound IV to the halide is 1:(10-50).

10. The use of the organic fluorescent compound according to any one of claims 1 to 3 or the organic fluorescent compound prepared by the preparation method according to any one of claims 4 to 9 in the preparation of antitumor drugs, the preparation of tumor diagnostic reagents, or in cell imaging for non-therapeutic purposes.

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