Fused ring tin porphyrin photosensitizer with high singlet oxygen sensitization efficiency and photothermal effect

Abstract

The invention discloses a fused ring tin porphyrin photosensitizer with high singlet oxygen sensitization efficiency and photothermal effect, and relates to the technical field of biological medicine. The invention provides a structural general formula and a preparation method of a fused ring tin porphyrin photosensitizer. According to the invention, the photosensitizer is endowed with strong absorption capacity in an NIR region through an isoquinoline fused ring on the periphery of porphyrin, < 1 > O2 sensitization activity and photothermal effect of the photosensitizer under light excitation are promoted through metal tin coordination in a parent nucleus, and an efficient photodynamic / photothermal synergistic killing effect on 4T1 cells of a mouse breast cancer cell line under NIR light irradiation is realized; the problems of poor light stability, reduced photosensitive activity and the like in the prior art are effectively solved.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a condensed ring tin porphyrin photosensitizer having both high singlet oxygen sensitization efficiency and photothermal effect. Background Art

[0002] Compared with traditional therapies, phototherapy (including photodynamic therapy and photothermal therapy) has become a promising cancer treatment due to its controllability and low systemic toxicity. Under irradiation with light of a specific wavelength, photosensitizers can produce highly cytotoxic reactive oxygen species or local hyperthermia, thereby inducing tumor cell death. Ideally, photosensitizers with high photosensitivity in the near-infrared (NIR) phototherapy window (650~850 nm) have greater application potential. Their advantages are that the available therapeutic light sources have stronger tissue penetration and less photodamage to healthy tissues. Unfortunately, the design and synthesis of such NIR photosensitizers are extremely challenging because the conventional synthetic strategy of extending the backbone π conjugation can effectively red-shift the excitation wavelength of the photosensitizer, but it usually results in a significant decrease or even loss of photosensitivity.

[0003] Photosensitizers based on porphyrin structures are characterized by their characteristic Q-band absorption in the red / near-infrared region, singlet oxygen ( 1 O2) sensitizing activity, excellent photostability and low biological toxicity, it is considered to be the first choice for phototherapy. Some porphyrin photosensitizers have been successfully used in clinical trials and treatments for cancer in the oral cavity, skin, bladder and other parts, including hypocretin, hemoporfin and rotapolfin. In view of the bottlenecks that porphyrin photosensitizers still have in the depth and efficacy of tumor treatment, current related research is mainly focused on the following two points. On the one hand, the photosensitivity of porphyrin molecules in the NIR phototherapy window is improved by conjugation expansion or core modification of the periphery of porphyrin molecules. On the other hand, the unique cavity of porphyrin molecules is a good coordination site for various metal ions. Regulating the molecular orbital energy levels of porphyrin photosensitizers through metallation can not only improve 1 O2 sensitization efficiency (radiative transition) can also be used to regulate the process of energy dissipation of excited state electrons through non-radiative transition, thereby achieving a synergistic photothermal therapeutic effect to improve the efficacy of tumor treatment. 1 New metalloporphyrin photosensitizers with O2 sensitization activity and photothermal effect are of great value. Summary of the invention

[0004] In order to solve the above technical problems, the purpose of the present invention is to provide a condensed ring tin porphyrin photosensitizer with high singlet oxygen sensitization efficiency and photothermal effect, which can simultaneously produce 1O2 and photothermal effects can efficiently kill cells, thereby achieving photodynamic / photothermal synergistic therapy, effectively solving the problems of poor photostability and reduced photosensitivity in existing technologies.

[0005] The technical solution of the present invention to solve the above technical problems is as follows: a condensed ring tin porphyrin photosensitizer having both high singlet oxygen sensitization efficiency and photothermal effect is provided, and its general structural formula is: ; Wherein, R is an aryl group or a thienyl group.

[0006] The preparation method of the above-mentioned condensed ring tin porphyrin photosensitizer having both high singlet oxygen sensitization efficiency and photothermal effect comprises the following steps: (1) Add monooxime ether substituted zinc porphyrin, internal alkyne, [Cp*RhCl2]2, AgSbF6 and solvent to a dry reactor, react at -40~140 ℃ under nitrogen protection for 0.1~480 h, cool to room temperature, then add dichloromethane, filter, remove the solvent under reduced pressure, separate and purify by silica gel column chromatography, and dry in vacuum to obtain isoquinoline fused ring zinc porphyrin 1; (2) adding the isoquinoline fused-ring zinc porphyrin 1 obtained in step (1), excess concentrated hydrochloric acid and a solvent into a dry reactor, stirring at a temperature of -40 to 70 °C for 0.1 to 240 h, then adding a saturated sodium bicarbonate aqueous solution for neutralization, and then extracting with dichloromethane, washing with water, drying with anhydrous sodium sulfate and removing the solvent under reduced pressure to obtain isoquinoline fused-ring porphyrin 2; (3) Add the isoquinoline fused-ring porphyrin 2 obtained in step (2), stannous chloride dihydrate and a solvent into a dry reactor, react at a temperature of -40 to 120 °C for 0.1 to 240 h, cool to room temperature, add water, filter and wash with water, and vacuum dry to obtain a fused-ring tin porphyrin photosensitizer having both high singlet oxygen sensitization efficiency and photothermal effect.

[0007] Furthermore, in step (1), the molar ratio of monooxime ether-substituted zinc porphyrin, internal alkyne, [Cp*RhCl2]2 and AgSbF6 is 1:0.01~10:0.001~0.2:0.001~1.

[0008] Furthermore, in step (1), the reaction concentration of the monooxime ether-substituted zinc porphyrin is 0.001-10 mol / L.

[0009] Furthermore, in step (1), the internal alkyne is one of diphenylacetylene, 1,2-bis(4-methoxyphenyl)acetylene, 1,2-bis(4-fluorophenyl)acetylene, 1,2-bis(3-methoxyphenyl)acetylene and 1,2-bis(2-thienyl)acetylene.

[0010] Furthermore, in step (1), the structural formula of isoquinoline fused-ring zinc porphyrin 1 is: ; Wherein, R is an aryl group or a thienyl group.

[0011] Furthermore, in step (2), the reaction concentration of isoquinoline fused-ring zinc porphyrin 1 is 0.01-10 mol / L.

[0012] Further, in step (2), the structural formula of isoquinoline fused-ring porphyrin 2 is: ; Wherein, R is an aryl group or a thienyl group.

[0013] Furthermore, in step (3), the molar ratio of isoquinoline fused-ring porphyrin 2 to stannous chloride dihydrate is 1:0.01-20; and the reaction concentration of isoquinoline fused-ring porphyrin 2 is 0.01-10 mol / L.

[0014] Furthermore, in steps (1) to (3), the solvent is methanol, ethanol, isopropanol, tert-amyl alcohol, acetone, acetonitrile, tetrahydrofuran, dichloromethane, chloroform, 1,2-dichloroethane, pyridine, ethyl ether, dimethyl sulfoxide, benzene, chlorobenzene, o-dichlorobenzene, toluene, xylene, mesitylene, cyclohexane, petroleum ether, 1,4-dioxane, N,N -Dimethylformamide and N,N - at least one of dimethylacetamide.

[0015] The present invention has the following beneficial effects: 1. The present invention endows the photosensitizer with strong absorption ability in the NIR region through the isoquinoline fused ring on the periphery of the porphyrin, and promotes the photosensitizer under light excitation through the coordination of metal tin in the mother core. 1 O2 sensitization activity and photothermal effect achieved efficient photodynamic / photothermal synergistic killing of mouse breast cancer cell line 4T1 cells under NIR light irradiation.

[0016] 2. The condensed ring tin porphyrin photosensitizer with high singlet oxygen sensitization efficiency and photothermal effect of the present invention exhibits strong light absorption ability in the NIR region. The maximum absorption Q peak in the solvent reaches the near-infrared region of 690 nm, and the absorption intensity reaches 46155 M -1 cm -1 , which increases the tissue penetration depth during phototherapy. The DMSO solution of the photosensitizer was exposed to 660 nm laser (20 mW / cm 2 )can be efficiently produced under irradiation 1 O2; at the same time, a 660 nm laser (0.5 W / cm 2 ) exhibits excellent photostability and photothermal effect under irradiation.

[0017] 3. The polycyclic tin porphyrin photosensitizer of the present invention has high1 O2 sensitization efficiency and photothermal effect can simultaneously exert the functions of photodynamic and photothermal therapy, significantly improving the treatment effect.

[0018] 4. The photostability of the condensed ring tin porphyrin photosensitizer of the present invention is better than that of most commercially available photosensitizers. There is no photobleaching effect under long-term high-intensity laser irradiation, and the photosensitivity is not significantly reduced. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 A preparation process of condensed ring tin porphyrin photosensitizers with both high singlet oxygen sensitization efficiency and photothermal effect; Figure 2 The molecular structure and H NMR spectrum of the condensed ring tin porphyrin photosensitizer obtained in Example 1; Figure 3 The UV-vis-NIR absorption spectrum of the condensed ring tin porphyrin photosensitizer obtained in Example 1 in DMSO; Figure 4 The DMSO solution of the polycyclic tin porphyrin photosensitizer obtained in Example 1 under 660 nm light 1 O2 sensitization efficiency test results; Figure 5 The photothermal effect test results of the DMSO solution of the condensed ring tin porphyrin photosensitizer obtained in Example 1 under 660 nm light; Figure 6 The results of the photostability test of the DMSO solution of the condensed ring tin porphyrin photosensitizer obtained in Example 1 under 660 nm light; Figure 7 The results of the cell phototoxicity test of the fused-ring tin porphyrin photosensitizer NPs obtained in Example 1 are shown in FIG. DETAILED DESCRIPTION

[0020] The principles and features of the present invention are described below, and the examples are only used to explain the present invention and are not used to limit the scope of the present invention. If no specific conditions are specified in the embodiments, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0021] By nuclear magnetic resonance spectroscopy ( 1H NMR), MALDI-TOF mass spectrometry and X-ray single crystal diffraction were used to verify the structure of Sn(IV) porphyrin photosensitizer. The instruments used for the detection were: Bruker AV II-400 MHz NMR, in which TMS was used as the internal standard, and the solvents CDCl3 and DMSO-d6 were deuterated solvents; Waters-Q-TOF-Premier (ESI) high-resolution mass spectrometer; BrukerAutoflex III smartbeam MALDI TOF spectrometer; Oxford Xcalibur E single crystal diffractometer.

[0022] The instrument used for spectral characterization was a HITACHI U-2910 UV-visible spectrophotometer (scanning range 250~1100 nm).

[0023] The instrument used for photostability and photothermal effect testing is a FLIR E6 thermal infrared camera.

[0024] Example 1 A method for preparing a condensed ring tin porphyrin photosensitizer having both high singlet oxygen sensitization efficiency and photothermal effect (preparation process as shown in Figure 1 ), including the following steps: (1) Monooxime ether-substituted zinc porphyrin (27.8 mg, 0.05 mmol), diphenylacetylene (17.8 mg, 0.1 mmol), [Cp*RhCl2]2 (1.5 mg, 2.5 µmol), AgSbF6 (3.4 mg, 10 µmol) and tetrahydrofuran (0.5 mL) were added to a dry reactor. The mixture was reacted at 120 °C for 24 h under nitrogen protection. The mixture was cooled to room temperature, filtered and washed with dichloromethane. The solvent was removed under reduced pressure. The product was separated and purified by neutral alumina column chromatography (petroleum ether / ethyl acetate = 10 / 1, v / v) and dried in vacuo to obtain isoquinoline fused-ring zinc porphyrin 1 (27.2 mg, 62% yield). 1 H NMR (400 MHz, CDCl3): δ (ppm) 9.65 (s,1H), 9.17 (d, J = 4.4 Hz, 1H), 8.76 (d, J =4.8 Hz, 1H), 8.71-8.67 (m, 2H), 8.63-8.60 (m, 2H), 8.51-8.47 (m, 2H), 8.19-8.16 (m, 4H), 8.11-8.09 (m, 2H), 7.97-7.93 (m, 1H), 7.74-7.64 (m, 12H), 7.45-7.43 (m, 4H), 7.30-7.24 (m, 3H). HRMS(ESI + ): C 59 H 36 N5Zn [M+H] + , calculated value 878.2257, measured value 878.2259; (2) Add the isoquinoline fused-ring zinc porphyrin 1 (17.6 mg, 0.02 mmol) obtained in step (1), concentrated hydrochloric acid (0.1 mL) and acetone (1 mL) into a dry reactor, stir at room temperature for 2 h, then add saturated sodium bicarbonate aqueous solution to neutralize, and then extract with dichloromethane, wash with water, dry with anhydrous sodium sulfate and remove the solvent under reduced pressure to obtain isoquinoline fused-ring porphyrin 2 (16 mg, 98% yield); 1 H NMR (CDCl3, 400 MHz): δ (ppm) 9.65 (s, 1H), 9.03 (d, J = 4.4 Hz,1H), 8.73 (d, J = 4.4 Hz, 1H), 8.68-8.65 (m, 3H), 8.61 (d, J = 4.8 Hz, 1H), 8.33(d, J = 6.4 Hz, 1H), 8.23-8.21 (m, 2H), 8.19-8.15 (m, 4H), 7.92 (t, J = 8.0 Hz,1H), 7.79-7.74 (m, 10H), 7.67-7.65 (m, 2H), 7.48-7.44 (m, 5H), 7.34-7.30 (m,3H), -1.20 (s, 2H). HRMS (ESI + ): C 59 H 37 N5[M+H] + , calculated value 816.3122, measured value 816.3124; (3) Add the isoquinoline fused-ring porphyrin 2 (16 mg, 0.02 mmol) obtained in step (2), stannous chloride dihydrate (14 mg, 0.06 mmol) and pyridine (2 mL) into a dry reactor, react at 120 °C for 12 h, cool to room temperature, add water, filter and wash with water, and vacuum dry to obtain a fused-ring tin porphyrin photosensitizer 3a-Sn with high singlet oxygen sensitization efficiency and photothermal effect. IV (14.4 mg, 72% yield). 1 H NMR (CDCl3, 400 MHz): δ (ppm) 10.00 (s, 1H), 9.64(d, J = 4.8 Hz, 1H), 9.25 (d, J = 4.8 Hz, 1H), 9.06-9.03 (m, 3H), 8.80 (d, J = 7.6Hz, 1H), 8.37-8.34 (m, 4H), 8.29 (d, J = 7.6 Hz, 2H), 8.15 (t, J = 8.0 Hz, 1H),7.99 (d, J = 8.4 Hz, 1H), 7.87-7.81 (m, 10H), 7.69 (d, J = 8.0 Hz, 2H), 7.50-7.47(m, 5H), 7.37-7.34 (m, 3H) Figure 2 MALDI-TOF MS (m / z): C 59 H 35 N5ClSn [M–Cl] + , calculated value 968.1603, measured value 968.1992.

[0025] Test Example 1 The condensed ring tin porphyrin photosensitizer 3a-Sn obtained in Example 1 was IV Dissolve in DMSO to prepare a 10 mM stock solution. Take 2 µL of the stock solution and dilute it to 2 mL with DMSO. Test the UV-vis-NIR absorption spectrum of the solution. The results are as follows: Figure 3 shown.

[0026] Depend on Figure 3 It can be seen that the condensed ring tin porphyrin photosensitizer 3a-Sn IV It exhibits strong light absorption in the NIR region, with the maximum absorption Q peak at 690 nm and a molar extinction coefficient of 46155 M -1 cm-1 .

[0027] Test Example 2 A certain volume of the polycyclic tin porphyrin photosensitizer 3a-Sn obtained in Example 1 was added. IV The DMSO stock solution (10 mM) was diluted with DMSO to keep the absorbance (Abs.) of the solution at 660 nm at about 0.01. 2 mL of the above solution and 10 µL of the DMSO stock solution (20 mM) of the indicator 1,3-diphenylisobenzofuran (DPBF) were added to the cuvette, and then the 660 nm laser (20 mW / cm 2 ) irradiate the solution at intervals of 6 s and measure the changes in the solution's UV absorption spectrum. The results are as follows Figure 4 shown.

[0028] Depend on Figure 4 It can be seen that the absorbance of DPBF at 416 nm decreases rapidly after illumination, indicating that the photosensitizer 3a-Sn IV The aqueous solution can efficiently produce 1 O2. As a comparison, no 3a-Sn was added IV The absorption spectrum of the single DPBF solution under the same illumination conditions has no obvious change.

[0029] Test Example 3 A certain volume of the polycyclic tin porphyrin photosensitizer 3a-Sn obtained in Example 1 was added. IV The DMSO stock solution (10 mM) was diluted with DMSO to keep the absorbance (Abs.) of the solution at 660 nm about 1. 2 mL of the above solution was added to a cuvette and the solution was illuminated by a 660 nm laser (0.5 W / cm 2 After continuous irradiation for 6 min, the light source was removed and the temperature change of the solution was recorded at 30 s intervals using a thermal infrared camera. The results are shown in Figure 5 shown.

[0030] Depend on Figure 5 It can be seen that the photosensitizer 3a-Sn IV DMSO solution at 0.5 W / cm 2 The temperature can be raised by nearly 40 degrees under the action of laser, showing excellent photothermal effect. IV The temperature of DMSO solution did not change significantly under the same lighting conditions.

[0031] Test Example 4 Take 2 µL of photosensitizer 3a-Sn IV DMSO solution (10 mM) was dissolved in 2 mL of DMSO solution and added to the cuvette, and then the sample was illuminated by a 660 nm laser (0.5 W / cm 2) irradiate the solution at 1 min intervals and measure the changes in the solution absorption spectrum. The results are as follows Figure 6 shown.

[0032] Depend on Figure 6 It can be seen that the condensed ring tin porphyrin photosensitizer 3a-Sn IV DMSO solution at 0.5 W / cm 2 There was no significant decrease in absorbance after laser irradiation, showing excellent photostability.

[0033] Test Example 5 4T1 cells were plated in 96-well plates (5 × 10 3 The cells were incubated for 24 h to allow them to adhere to the wall. Subsequently, the original culture medium was removed and a porphyrin nanosensitizer 3a-Sn was added at different concentration gradients. IV NPs (0, 0.125, 0.25, 0.5, 1.0, 2.0 µM) were added to the fresh culture medium and incubated for another 24 h. The cells were illuminated with a 660 nm laser at 0.3 W / cm 2 The cells were irradiated with a power of 100 nm for 3 min, and then the cells were incubated for another 4 h before the cell survival rate was detected using CCK-8. In this group of experiments, the effects of photodynamic therapy (PDT) and photothermal therapy (PTT) were simulated by controlling the plate temperature (4 °C) and adding a reducing agent (NAC) to treat the cells before laser irradiation. At the same time, a group of cells treated with 3a-SnIV NPs were placed in the dark as a control group. The results are shown in Figure 2. Figure 7 shown.

[0034] Depend on Figure 7 It can be seen that the survival rate of the cells in the control group was above 80%, indicating that the nanosensitizer 3a-Sn IV NPs have good biocompatibility. At the same time, compared with the single PDT or PTT group, the PDT / PTT synergistic treatment group had a significantly improved killing effect on 4T1 cells, with the cell half inhibitory concentration (IC50) as low as 0.3 μM.

[0035] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A condensed ring tin porphyrin photosensitizer having both high singlet oxygen sensitization efficiency and photothermal effect, characterized in that: Its general structural formula is: ； Wherein, R is an aryl group or a thienyl group.

2. The method for preparing the condensed ring tin porphyrin photosensitizer having both high singlet oxygen sensitization efficiency and photothermal effect according to claim 1, characterized in that: The following steps are involved: (1) Add monooxime ether substituted zinc porphyrin, internal alkyne, [Cp*RhCl2]2, AgSbF6 and solvent to a dry reactor, react at -40~140 ℃ under nitrogen protection for 0.1~480 h, cool to room temperature, then add dichloromethane, filter, remove the solvent under reduced pressure, separate and purify by silica gel column chromatography, and dry in vacuum to obtain isoquinoline fused ring zinc porphyrin 1; (2) adding the isoquinoline fused-ring zinc porphyrin 1 obtained in step (1), excess concentrated hydrochloric acid and a solvent into a dry reactor, stirring at a temperature of -40 to 70 °C for 0.1 to 240 h, then adding a saturated sodium bicarbonate aqueous solution for neutralization, and then extracting with dichloromethane, washing with water, drying with anhydrous sodium sulfate and removing the solvent under reduced pressure to obtain isoquinoline fused-ring porphyrin 2; (3) Add the isoquinoline fused-ring porphyrin 2 obtained in step (2), stannous chloride dihydrate and a solvent into a dry reactor, react at a temperature of -40 to 120 °C for 0.1 to 240 h, cool to room temperature, add water, filter and wash with water, and vacuum dry to obtain a fused-ring tin porphyrin photosensitizer having both high singlet oxygen sensitization efficiency and photothermal effect.

3. The method for preparing the condensed ring tin porphyrin photosensitizer having both high singlet oxygen sensitization efficiency and photothermal effect as claimed in claim 2, characterized in that: In step (1), the molar ratio of monooxime ether-substituted zinc porphyrin, internal alkyne, [Cp*RhCl2]2 and AgSbF6 is 1:0.01~10:0.001~0.2:0.001~1.

4. The method for preparing the condensed ring tin porphyrin photosensitizer having both high singlet oxygen sensitization efficiency and photothermal effect as claimed in claim 2, characterized in that: In step (1), the reaction concentration of the monooxime ether-substituted zinc porphyrin is 0.001-10 mol / L.

5. The method for preparing the condensed ring tin porphyrin photosensitizer having both high singlet oxygen sensitization efficiency and photothermal effect as claimed in claim 2, characterized in that: In step (1), the internal alkyne is one of diphenylacetylene, 1,2-bis(4-methoxyphenyl)acetylene, 1,2-bis(4-fluorophenyl)acetylene, 1,2-bis(3-methoxyphenyl)acetylene and 1,2-bis(2-thienyl)acetylene.

6. The method for preparing the condensed ring tin porphyrin photosensitizer having both high singlet oxygen sensitization efficiency and photothermal effect as claimed in claim 2, characterized in that: In step (1), the structural formula of isoquinoline fused-ring zinc porphyrin 1 is: ； Wherein, R is an aryl group or a thienyl group.

7. The method for preparing the condensed ring tin porphyrin photosensitizer having both high singlet oxygen sensitization efficiency and photothermal effect as claimed in claim 2, characterized in that: In step (2), the reaction concentration of isoquinoline fused-ring zinc porphyrin 1 is 0.01-10 mol / L.

8. The method for preparing the condensed ring tin porphyrin photosensitizer having both high singlet oxygen sensitization efficiency and photothermal effect as claimed in claim 2, characterized in that: In step (2), the structural formula of isoquinoline fused-ring porphyrin 2 is: ； Wherein, R is an aryl group or a thienyl group.

9. The method for preparing the condensed ring tin porphyrin photosensitizer having both high singlet oxygen sensitization efficiency and photothermal effect as claimed in claim 2, characterized in that: In step (3), the molar ratio of isoquinoline fused-ring porphyrin 2 to stannous chloride dihydrate is 1:0.01-20; and the reaction concentration of isoquinoline fused-ring porphyrin 2 is 0.01-10 mol / L.

10. The method for preparing the condensed ring tin porphyrin photosensitizer having both high singlet oxygen sensitization efficiency and photothermal effect as claimed in claim 2, characterized in that: In steps (1) to (3), the solvent is methanol, ethanol, isopropanol, tert-amyl alcohol, acetone, acetonitrile, tetrahydrofuran, dichloromethane, chloroform, 1,2-dichloroethane, pyridine, ether, dimethyl sulfoxide, benzene, chlorobenzene, o-dichlorobenzene, toluene, xylene, mesitylene, cyclohexane, petroleum ether, 1,4-dioxane, N,N -Dimethylformamide and N,N - at least one of dimethylacetamide.

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