A condensed-ring tin porphyrin photosensitizer with both high singlet oxygen sensitization efficiency and photothermal effect

Abstract

The present invention discloses a condensed ring tin porphyrin photosensitizer with high singlet oxygen sensitization efficiency and photothermal effect, which relates to the field of biomedicine technology. The present invention provides a structural formula and preparation method of the condensed ring tin porphyrin photosensitizer. The present invention gives the photosensitizer a strong absorption ability in the NIR region through the isoquinoline condensed ring on the periphery of the porphyrin, and promotes the photosensitizer to absorb light 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, effectively solving the problems of poor photostability and reduced photosensitivity in existing technologies.

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 controllable nature and low systemic toxicity. Under irradiation with light of specific wavelengths, photosensitizers can generate highly cytotoxic reactive oxygen species or local hyperthermia, thereby inducing tumor cell death. Ideally, photosensitizers with high photosensitivity within the near-infrared (NIR) phototherapy window (650–850 nm) have greater potential for application. Their advantages lie in the enhanced tissue penetration of available therapeutic light sources and reduced photodamage to healthy tissue. Unfortunately, the design and synthesis of such NIR photosensitizers is extremely challenging. Conventional synthetic strategies, such as extending the backbone π conjugation degree, can effectively red-shift the excitation wavelength of the photosensitizer, but this often results in a significant decrease or even loss of photosensitivity.

[0003] Photosensitizers based on porphyrin structures are characterized by their 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 xifen, hemoporfin and rotapol. In view of the bottlenecks that porphyrin photosensitizers still have in the depth and efficacy of tumor treatment, current research mainly focuses 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. On the other hand, the unique cavity of porphyrin molecules is a good coordination site for various metal ions. By regulating the molecular orbital energy level of porphyrin photosensitizers through metallation, it 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 transitions, 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 effectively kill cells and thus achieve photodynamic / photothermal synergistic therapy, effectively solving the problems of poor photostability and reduced photosensitivity in existing technologies.

[0005] The present invention solves the above technical problems by providing a condensed ring tin porphyrin photosensitizer having both high singlet oxygen sensitization efficiency and photothermal effect, the general structural formula of which is:

[0006] ;

[0007] Wherein, R is an aryl group or a thienyl group.

[0008] The preparation method of the above-mentioned fused-ring tin porphyrin photosensitizer having both high singlet oxygen sensitization efficiency and photothermal effect comprises the following steps:

[0009] (1) Add monooxime ether-substituted zinc porphyrin, internal alkyne, [Cp*RhCl2]2, AgSbF6 and solvent to a dry reactor. Under nitrogen protection, react at -40~140℃ for 0.1~480h, 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 vacuo to obtain isoquinoline fused-ring zinc porphyrin 1.

[0010] (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;

[0011] (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.

[0012] Furthermore, in step (1), the molar ratio of the monooxime ether-substituted zinc porphyrin, the internal alkyne, [Cp*RhCl2]2 and AgSbF6 is 1:0.01~10:0.001~0.2:0.001~1.

[0013] Furthermore, in step (1), the reaction concentration of the monooxime ether-substituted zinc porphyrin is 0.001-10 mol / L.

[0014] 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.

[0015] Furthermore, in step (1), the structural formula of isoquinoline fused-ring zinc porphyrin 1 is:

[0016] ;

[0017] Wherein, R is an aryl group or a thienyl group.

[0018] Furthermore, in step (2), the reaction concentration of isoquinoline fused-ring zinc porphyrin 1 is 0.01-10 mol / L.

[0019] Furthermore, in step (2), the structural formula of isoquinoline fused-ring porphyrin 2 is:

[0020] ;

[0021] Wherein, R is an aryl group or a thienyl group.

[0022] 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.

[0023] Furthermore, 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.

[0024] The present invention has the following beneficial effects:

[0025] 1. The present invention endows the photosensitizer with strong absorption ability in the NIR region through the isoquinoline fused ring on the periphery of porphyrin, and promotes the photosensitizer to absorb light 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.

[0026] 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 improves 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 irradiation1 O2; at the same time, a 660 nm laser (0.5 W / cm 2 ) exhibits excellent photostability and photothermal effect under irradiation.

[0027] 3. The condensed ring tin porphyrin photosensitizer of the present invention has high 1 The O2 sensitization efficiency and photothermal effect can simultaneously exert the functions of photodynamic and photothermal therapy, significantly improving the treatment effect.

[0028] 4. The photostability of the fused-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 does not decrease significantly. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 A preparation process for condensed-ring tin porphyrin photosensitizers with both high singlet oxygen sensitization efficiency and photothermal effect;

[0030] Figure 2 The molecular structure and H NMR spectrum of the condensed ring tin porphyrin photosensitizer obtained in Example 1 are shown;

[0031] Figure 3 The UV-vis-NIR absorption spectrum of the condensed ring tin porphyrin photosensitizer obtained in Example 1 in DMSO;

[0032] Figure 4 The DMSO solution of the condensed ring tin porphyrin photosensitizer obtained in Example 1 was irradiated at 660 nm. 1 O2 sensitization efficiency test results;

[0033] 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;

[0034] 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 are shown;

[0035] Figure 7 The results of the cell phototoxicity test of the fused-ring tin porphyrin photosensitizer NPs obtained in Example 1 are shown. DETAILED DESCRIPTION

[0036] The principles and features of the present invention are described below. The examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. In the examples, where specific conditions are not specified, conventional conditions or manufacturer-recommended conditions were used. Reagents or instruments used where the manufacturer is not specified are conventional products that can be purchased commercially.

[0037] H NMR spectroscopy ( 1 The structure of the Sn(IV) porphyrin photosensitizer was verified by H NMR, MALDI-TOF mass spectrometry, and single-crystal X-ray diffraction. The instruments used for the analysis included a Bruker AV II-400 MHz NMR spectrometer, with TMS as the internal standard and deuterated solvents CDCl₃ and DMSO-d₆; a Waters-Q-TOF-Premier (ESI) high-resolution mass spectrometer; a Bruker Autoflex III smartbeam MALDI-TOF spectrometer; and an Oxford Xcalibur E single-crystal diffractometer.

[0038] The instrument used for spectral characterization was a HITACHI U-2910 UV-visible spectrophotometer (scanning range 250-1100 nm).

[0039] The instrument used for photostability and photothermal effect testing is a FLIR E6 thermal infrared camera.

[0040] Example 1

[0041] A method for preparing a condensed ring tin porphyrin photosensitizer with high singlet oxygen sensitization efficiency and photothermal effect (preparation process as shown in the following example) Figure 1 ), including the following steps:

[0042] (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, and the product was separated and purified by neutral alumina column chromatography (petroleum ether / ethyl acetate = 10 / 1, v / v). The product was 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;

[0043] (2) Add isoquinoline fused-ring zinc porphyrin 1 (17.6 mg, 0.02 mmol), concentrated hydrochloric acid (0.1 mL) and acetone (1 mL) obtained in step (1) to a dry reactor, stir at room temperature for 2 h, then add saturated sodium bicarbonate aqueous solution for neutralization, and then extract with dichloromethane, wash with water, dry over 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;

[0044] (3) Add the isoquinoline fused-ring porphyrin 2 (16 mg, 0.02 mmol), stannous chloride dihydrate (14 mg, 0.06 mmol) and pyridine (2 mL) obtained in step (2) 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 both 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.

[0045] Test Example 1

[0046] 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.

[0047] 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 .

[0048] Test Example 2

[0049] A certain volume of the condensed ring tin porphyrin photosensitizer 3a-Sn obtained in Example 1 was added. IV The DMSO stock solution (10 mM) was diluted with DMSO to maintain the absorbance (Abs.) of the solution at 660 nm at approximately 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 a cuvette and then illuminated with a 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.

[0050] 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 did not change significantly.

[0051] Test Example 3

[0052] A certain volume of the condensed ring 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 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 the figure. Figure 5 shown.

[0053] 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 increased by nearly 40 degrees under the action of laser, showing an excellent photothermal effect. IV The temperature of DMSO solution under the same lighting conditions did not change significantly.

[0054] Test Example 4

[0055] Take 2 μL of photosensitizer 3a-Sn IVDMSO 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 intervals of 1 min and measure the changes in the solution absorption spectrum. The results are as follows Figure 6 shown.

[0056] 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, demonstrating excellent photostability.

[0057] Test Example 5

[0058] 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 24 h. 2 The cells were irradiated with a power of 100 nm for 3 minutes, and then the cells were incubated for another 4 hours 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.

[0059] Depend on Figure 7 It can be seen that the survival rate of the cells in the control group is above 80%, indicating that the nanosensitizer 3a-Sn IV NPs have good biocompatibility. Furthermore, compared with the PDT or PTT groups alone, the PDT / PTT synergistic treatment group significantly enhanced the killing effect on 4T1 cells, with the cell half-inhibitory concentration (IC50) as low as 0.3 μM.

[0060] 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 principles of the present invention should be included in the scope of protection 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 phenyl, 4-methoxyphenyl, 4-fluorophenyl, 3-methoxyphenyl or 2-thienyl, and two Rs are the same 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. Under nitrogen protection, react at -40~140℃ for 0.1~480h, 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 vacuo to obtain isoquinoline fused-ring zinc porphyrin 1. Among them, the structural formula of isoquinoline fused-ring zinc porphyrin 1 is: ； (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; Among them, the structural formula of isoquinoline fused ring porphyrin 2 is: ； (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 a condensed-ring tin porphyrin photosensitizer having both high singlet oxygen sensitization efficiency and photothermal effect according to claim 2, wherein: 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 a condensed-ring tin porphyrin photosensitizer having both high singlet oxygen sensitization efficiency and photothermal effect according to claim 2, wherein: In step (1), the reaction concentration of the monooxime ether-substituted zinc porphyrin is 0.001-10 mol / L.

5. The method for preparing a condensed-ring tin porphyrin photosensitizer having both high singlet oxygen sensitization efficiency and photothermal effect according to claim 2, wherein: In step (2), the reaction concentration of isoquinoline fused-ring zinc porphyrin 1 is 0.01~10 mol / L.

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

7. The method for preparing the condensed-ring tin porphyrin photosensitizer having both high singlet oxygen sensitization efficiency and photothermal effect according to claim 2, wherein: 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.

Images