An ADA type organic small molecule photosensitizer and its application

Abstract

The present invention discloses an A-D-A type organic small molecule photosensitizer and its application. The present invention obtains a photosensitizer IDT-I with better photothermal-photodynamic synergistic therapy ability by newly designing the side chain structure of the A-D-A type small molecule photosensitizer. The photosensitizer IDT-I has excellent photothermal conversion efficiency and photodynamic activity, and can achieve efficient photothermal and photodynamic synergistic therapy of tumors.

Description

Technical Field

[0001] The present invention relates to the design and synthesis of an organic small molecule photosensitizer, and in particular to the design and synthesis of a novel ADA type organic small molecule photosensitizer IDT-I and its application. Background Art

[0002] Cancer (tumor) is one of the major diseases that threaten the life and health of all mankind. In recent years, some light treatment methods such as photothermal therapy (PTT) and photodynamic therapy (PDT) have attracted more and more attention due to their advantages such as minimal invasiveness and high efficacy (see: Yun, SH; Kwok, SJJ Light in Diagnosis, Therapy and Surgery. Nat. Biomed. Eng. 2017, 1 , 0008). Photothermal therapy kills tumor cells by converting light energy into heat energy through photosensitizers, while photodynamic therapy induces cell death by converting substances such as oxygen into highly reactive oxygen species through photosensitizers. These treatment methods have their own advantages and certain therapeutic effects, but any single treatment method is difficult to achieve the ideal tumor treatment effect, and the disadvantages of a single treatment method are becoming increasingly prominent. Photothermal-photodynamic synergistic treatment methods can make up for the shortcomings of a single treatment method and bring about superimposed or even synergistic tumor treatment effects. Therefore, the development of tumor photothermal-photodynamic synergistic treatment strategies has become a new hot spot in tumor treatment research at home and abroad.

[0003] In recent years, ADA-type organic small molecule photosensitizers have attracted widespread attention due to their advantages such as easy synthesis, variable structure, and strong photoresponsiveness. This type of photosensitizer has the advantages of strong photoresponsiveness in the visible-near infrared region, easy synthesis, variable structure, and stable optical properties. Studies have shown that the lower energy level difference between the singlet and triplet states of this type of photosensitizer can enhance the intersystem crossing (ISC) ability, thereby greatly promoting the generation of highly reactive oxygen species, making this type of photosensitizer exhibit superior photodynamic activity. In terms of photothermal conversion, the high charge migration ability within the molecule can promote the quenching of molecular fluorescence, and the free rotation of the side chain in the molecule can enhance the non-radiative attenuation process, thereby obtaining a higher photothermal conversion efficiency. Since Han et al. first used this type of small molecule photosensitizer in tumor photothermal therapy in 2019 (see: Cai, Y.; Wei, Z.; Song, C.; Tang, C.; Huang, X.; Hu, Q.; Dong, X.; Han, W. Chem. Commun. 2019, 55,8967), this type of molecule has gradually attracted more and more attention in the field of tumor phototherapy. However, there are not many reports of this type of photosensitizers with both high photothermal conversion efficiency and high photodynamic activity. Therefore, it is of great research and practical application value to improve the photothermal-photodynamic synergistic therapy ability of this type of photosensitizers through molecular design.

[0004] ADA-type small molecule photosensitizers are composed of a fused ring electron-donating unit (D) in the middle, cyanoindanone electron-pulling units (A) at both ends, and side chains. Many research results show that the molecular side chains have an important influence on the photophysical properties of photosensitizers. Rigid molecular side chains can reduce molecular stacking, thereby improving molecular motion to improve photothermal conversion capacity. In addition, reduced molecular aggregation can increase the contact between molecules and oxygen, thereby improving photodynamic activity. On the other hand, some heavy atoms can be introduced into the molecular side chains to improve photodynamic activity (see: Lu, B.; Huang, Y.; Quan, H.; Zhang, Z.; Yao, Y. Organic Conjugated Small Molecules with Donor-AcceptorStructures: Design and Application in Phototherapy of Tumors. Mater. Chem. Front. 2022, 6 , 2968-2993). Therefore, it is feasible to design new ADA-type organic small molecule photosensitizers with better photothermal-photodynamic synergistic therapeutic ability by changing the side chain structure of the photosensitizer. Summary of the invention

[0005] The purpose of the present invention is to provide a novel ADA type organic small molecule photosensitizer IDT-I, and use it for tumor photothermal-photodynamic synergistic therapy.

[0006] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions:

[0007] Photosensitizer IDT-I, its structural formula is shown below:

[0008] .

[0009] In one embodiment of the present invention, the synthesis route of photosensitizer IDT-I is shown as follows:

[0010] .

[0011] The use of the above-mentioned photosensitizer IDT-I in the preparation of tumor treatment drugs.

[0012] The present invention makes a new design on the side chain structure of ADA type small molecule photosensitizer. The introduction of asymmetric side chains at both ends of the molecule can weaken the molecular stacking, thereby enhancing the intramolecular motion to improve the photothermal conversion efficiency. At the same time, the introduction of heavy atom iodine at the end of the side chain can improve the intersystem crossing ability of the excited state of the photosensitizer and thus improve the photodynamic activity. Therefore, through the modification of the molecular side chain, a photosensitizer IDT-I with better photothermal-photodynamic synergistic treatment ability can be obtained. The photosensitizer IDT-I of the present invention has excellent photothermal conversion efficiency and photodynamic activity, and can achieve efficient photothermal and photodynamic synergistic treatment of tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is the temperature rise curve of the photosensitizer IDT-I under illumination and the temperature drop curve after the illumination is removed.

[0014] Figure 2 This is the absorbance curve of DPBF 415 nm in the mixture of photosensitizer IDT-I under light conditions.

[0015] Figure 3 This is a fluorescence imaging image of reactive oxygen species detected in cervical cancer cells cultured with photosensitizer IDT-I after illumination.

[0016] Figure 4 The survival rate of cervical cancer cells under different treatments.

[0017] Figure 5 These are fluorescent images of cervical cancer cells alive or dead under different treatments. Implementation

[0018] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0019] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0020] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources. Example

[0021] The photosensitizer IDT-I was synthesized by the following route:

[0022] .

[0023] Synthesis of compound 2: Under nitrogen protection, compound 1 (100 mg, 0.24 mmol) and THF (10 mL) were added to a three-necked round-bottom flask. A THF solution of p-methylphenylmagnesium bromide (1 M, 1.95 mL) was added dropwise to the mixture. o C. The mixture was stirred for 12 h and then cooled to room temperature. Saturated aqueous NH4Cl solution (10 mL) was added and the mixture was extracted with dichloromethane (2 × 25 mL). The organic phase was dried over anhydrous Na2SO4 and filtered. After removing the solvent, the yellow residue was added to a three-necked round-bottom flask, toluene (15 mL) and Amberlyst 15 (150 mg) were added and the mixture was deoxygenated with argon for 30 min. The mixture was refluxed at 110 °C for 12 h, then cooled to room temperature and filtered. After removing the solvent, compound 2 (97 mg, 72%) was purified by column chromatography.

[0024] The product was dissolved in CDCl3 (about 0.4 mL), sealed, and characterized on a Unity Inova-400 NMR instrument: 1 H NMR (400 MHz, CDCl3) δ 7.15 (d, J = 5.2 Hz, 2H), 7.12 (d, J = 5.2 Hz, 2H),7.06 (d, J = 7.9 Hz, 5H), 7.00 (d, J = 7.9 Hz, 5H), 4.76 (s, 2H), 2.25 (s, 6H).

[0025] Synthesis of compound 3: Under nitrogen atmosphere, 50 wt% potassium hydroxide aqueous solution (1.00 mL) was added to a mixture of compound 2 (56 mg, 0.10 mmol), tetrabutylammonium bromide (10 mg, 0.03 mmol) and 1,6-dibromohexane (5 mL). o The reaction mixture was stirred at 40 °C for 12 h. After cooling to room temperature, the organic layer was separated and the aqueous layer was extracted with DCM. The combined organic solution was washed with water several times. After removing the solvent, the residual liquid reagent was removed under vacuum. Purification by column chromatography gave a yellow solid (59 mg, 60%).

[0026] The product was dissolved in CDCl3 (about 0.4 mL), sealed, and characterized on a Unity Inova-400 NMR instrument: 1H NMR (400 MHz, CDCl3) δ 7.32 – 7.28 (m, 6H), 7.24 – 7.19 (m, 4H), 7.13 –7.08 (m, 4H), 3.05 (q, J = 15.0, 7.2 Hz, 4H), 2.62 – 2.52 (m, 2H), 2.46 –2.38(m, 2H), 2.31 (d, J = 8.7 Hz, 6H), 1.66 – 1.61 (m, 4H), 1.26 – 1.22 (m, 12H).

[0027] Synthesis of compound 4: o Vilsmeier reagent prepared by dissolving phosphorus oxychloride (0.30 mL) in DMF (1.50 mL) was added to a solution of compound 3 (60 mg, 0.068 mmol) dissolved in 1,2-dichloroethane (10 mL) under argon protection at 85 °C. o C for 20 h. After cooling to room temperature, the mixture was poured into ice water (50 mL), stirred at room temperature for 1 h, and then extracted with chloroform (2 × 50 mL). After removing the solvent, compound 6 (57 mg, 89%) was obtained by column chromatography. The crude product obtained by column chromatography can be used in the next step without further purification.

[0028] Synthesis of IDT-I: Compound 4 (103 mg, 0.10 mmol), 2FIC (92 mg, 0.40 mmol), pyridine (0.1 mL) and chloroform (10 mL) were added to a three-necked round-bottom flask. The mixture was stirred under reflux for 20 h. After cooling to room temperature, the mixture was poured into methanol (100 mL) and filtered. IDT-I (90 mg, 66%) was obtained by column chromatography using dichloromethane as the eluent.

[0029] The product was dissolved in CDCl3 (about 0.4 mL), sealed, and characterized on a Unity Inova-400 NMR instrument: 1 H NMR (400 MHz, CDCl3) δ 8.90 (s, 2H), 8.54 (dd, J = 9.7, 6.5 Hz, 2H), 8.22(s, 2H), 7.67 (t, J = 7.4 Hz, 2H), 7.46 (d, J = 3.6 Hz, 2H), 7.17 (dt,J = 20.8,8.3 Hz, 8H), 2.91 (q, J = 6.7 Hz, 4H), 2.70 (dd, J = 17.6, 11.6 Hz, 2H), 2.53(dt, J = 14.7, 8.5 Hz, 2H), 2.31 (d, J = 7.9 Hz, 6H), 1.57 (s, 4H), 1.26 (d, J =8.9 Hz, 12H).

[0030] The photothermal activity and photodynamic activity of the photosensitizer IDT-I were tested below.

[0031] 1. Calculation of photothermal efficiency: Measure the photothermal efficiency of 5 μM IDT-I at 660 nm, 1.0 W / cm 2 The photothermal conversion efficiency under .

[0032] like Figure 1 As shown in the figure, the photothermal conversion efficiency η = 40%. This photothermal conversion efficiency is significantly higher than the photothermal conversion efficiency of the FDA-approved photothermal conversion reagent indocyanine green ICG (3.1%).

[0033] 2. Photodynamic activity: Reactive oxygen detection: DPBF was used as the detection reagent. A mixture (3 mL) of DPBF (10 μM) and IDT-I (25 μM) was added to a quartz cuvette and the photoluminescence spectra were measured using a 660 nm 1.0 W / cm 2 The laser was irradiated for 120 s. At the set time point, the absorbance at 415 nm was recorded.

[0034] like Figure 2 As shown in the figure, after irradiation, the absorbance of DPBF in the IDT-I solution continued to decrease, proving that IDT-I has excellent photodynamic activity. The quantum yield of active oxygen reached 54.3%, which exceeds the widely used photosensitizer methylene blue.

[0035] 3. Photodynamic activity of IDI-I in cervical cancer (HeLa) cells: HeLa cells were plated at 20×10 4 The cells were seeded in 6-well plates at a density of 1 mL per well and placed in a 5% CO2 and 37 o C incubator for 24 h. Add 25 μM IDT-I for 5 h, stain with DCFH-DA (10 μM) for 30 min, and rinse three times with PBS buffer. Cells were illuminated at 660 nm, 1.0 W / cm 2After 5 min of laser irradiation, green fluorescence was observed using CLSM with an excitation wavelength of 488 nm and an emission collection wavelength of 500 nm to 550 nm.

[0036] like Figure 3 As shown, only HeLa cells cultured with IDT-I could be observed to have green fluorescence after illumination, confirming that reactive oxygen species could be generated in the cells after illumination.

[0037] 4. Effect of phototherapy for cervical cancer

[0038] Cell viability: HeLa cells were plated at 30 × 10 4 The cells were seeded in 6-well plates at a density of 1 mL per well and placed in a 5% CO2 and 37 o C incubator for 24 h. Two dishes were added with 25 μM IDT-I, and two dishes were added with an equal volume of PBS solution. After 5 h of incubation, one group was exposed to 660 nm, 1.0 W / cm 2 The cells were irradiated with laser for 10 min and cultured for 24 h. The cell survival rate was calculated.

[0039] As shown in FIG4 , only the cells cultured by IDT-I had a greatly reduced survival rate after illumination, confirming that IDT-I has a very good lethality to HeLa cells.

[0040] 5. Cell life and death fluorescence imaging: HeLa cells were plated at 30×10 4 The cells were seeded in 6-well plates at a density of 1 mL per well and placed in a 5% CO2 and 37 o C incubator for 24 h. Add 25 μM IDT-I to two dishes and add an equal volume of PBS solution to two dishes. After 5 h of incubation, one dish in the IDT-I group and one dish in the PBS group were illuminated with 660 nm, 1.0 W / cm 2 The cells were irradiated with laser for 10 min and cultured for 43 h. Calcein / PI cell viability and cytotoxicity detection reagent was used for staining, and CLSM was used to observe cell survival.

[0041] like Figure 5 As shown, only the cells cultured with IDT-I showed large-scale death, confirming that IDT-I had a very good lethality for HeLa cells after illumination.

Claims

1. Photosensitizer IDT-I, the structural formula of which is shown below:

2. The use of photosensitizer IDT-I in the preparation of tumor treatment drugs, characterized in that: The structural formula of the photosensitizer IDT-I is shown below: The tumor is cervical cancer.

3. A tumor treatment drug, characterized in that: The active ingredient is the photosensitizer IDT-I, and the structural formula of the photosensitizer IDT-I is as follows:

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