CN117700438A - Silicon phthalocyanine capable of generating hydroxyl free radical through high-efficiency photosensitization and preparation and application thereof - Google Patents
Silicon phthalocyanine capable of generating hydroxyl free radical through high-efficiency photosensitization and preparation and application thereof Download PDFInfo
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- CN117700438A CN117700438A CN202311706475.8A CN202311706475A CN117700438A CN 117700438 A CN117700438 A CN 117700438A CN 202311706475 A CN202311706475 A CN 202311706475A CN 117700438 A CN117700438 A CN 117700438A
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- silicon
- phthalocyanine
- dimethylamino
- acid
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Abstract
The invention belongs to the field of photodynamic therapy medicaments, and particularly relates to silicon phthalocyanine with high-efficiency photosensitization for generating hydroxyl free radicals, and preparation and application thereof. The phthalocyanine silicon is amine oxide symmetrically substituted phthalocyanine silicon, amino asymmetrically substituted phthalocyanine silicon or amine oxide asymmetrically substituted phthalocyanine silicon. Under the irradiation of near infrared light, the silicon phthalocyanine can generate a large amount of hydroxyl free radicals, so that the silicon phthalocyanine can be used as a photosensitizer or a photodynamic medicine or a photosensitizing agent, has higher anticancer activity under aerobic and anaerobic conditions when being used as a photodynamic medicine, and has remarkable application prospect in the field of treating hypoxic tumors. In addition, living animal experiments prove that the phthalocyanine silicon has the capability of precisely targeting tumor tissues, and the damage to normal tissues caused by the off-target of the phthalocyanine silicon can be reduced.
Description
Technical Field
The invention belongs to the field of photodynamic therapy medicaments, and in particular relates to silicon phthalocyanine with high-efficiency photosensitization for generating hydroxyl free radicals, and preparation and application thereof in the field of medicaments.
Background
At present, malignant tumor is one of main diseases affecting human health and threatening human life, but the traditional tumor treatment methods comprise surgery, chemotherapy and the like, which have great defects and shortcomings, so that the prevention and treatment of tumor are the problems which are needed to be solved currently. With the development of technology, new treatments for malignant tumors are emerging, which include photodynamic therapy (Photodynamic therapy, PDT). PDT is a non-invasive treatment means, and is attracting attention and expected because of its advantages of small toxic and side effects, fast effect, no drug resistance under repeated application, etc.
Photosensitizers (or photodynamic drugs) are key factors in determining the efficacy of photodynamic therapy. Photodynamic therapy is the photosensitization of photosensitizers under light excitation to generate reactive oxygen species, thereby destroying tumor cells and tissues. Among the mechanisms for generating active oxygen are two types, type I and type II. In the ground state (S) 0 ) The energy of the photon absorbed by the photosensitizer transitions to an excited singlet state (S 1 ) The excited singlet state is converted to an excited triplet state (T) having a relatively long lifetime by intersystem crossing 1 ) Thereafter, the photosensitizer in the excited triplet state generates superoxide anions (O) by electron transfer and substrate interaction 2 ·－ ) And hydroxyl radicals (. OH) and the like, this process is known as type I reaction. The photosensitizer in the excited triplet state can also directly transfer energy to the molecular oxygen in the ground state to generate singlet oxygen with higher activity 1 O 2 ) This process is known as type II reaction. The active oxygen generated by the reaction can kill both the I-type reaction and the II-type reactionCancer cells, and tumor tissue destruction. Most of the reported photosensitizers generate active oxygen through type II reactions, and few photosensitizers with efficient type I photosensitization reactions are available. Type II reactions are highly dependent on oxygen in the environment, while type I reactions are less dependent on oxygen. Because hypoxia is one of the main characteristics of solid tumors, the photosensitizer with the I-type reaction mechanism has better application value in the aspect of photodynamic anti-solid tumors.
The phthalocyanine has been attracting attention as a new generation photosensitizer due to its strong absorption in phototherapy window (600-900 nm), low dark toxicity, easy modification of structure, etc. The phthalocyanine belongs to benzo aza porphyrin derivatives and is a macrocyclic conjugated system consisting of 18 pi electrons. Phthalocyanines of different structures and functions can be synthesized by introducing different pericyclic and axial substitutions, and by replacing different central ions. One phthalocyanine (photosense) has been approved for clinical use and three phthalocyanines (Pc 4, CGP55847, foddaline) have entered clinical trials. However, the above phthalocyanine photosensitizers all act through a type II photoreaction mechanism with high oxygen dependence, and have limited photodynamic therapy effect on solid tumors. The polyethylene glycol coupled with the small molecular targeting group and the reductase-responsive amine oxide are covalently connected to the axial direction of the silicon dichlorophthalocyanine, so that the photosensitizer with more excellent performance is expected to be obtained. At present, the application of phthalocyanine silicon capable of efficiently generating hydroxyl radicals in the field of photodynamic therapy is not reported.
Disclosure of Invention
The invention aims to provide the phthalocyanine silicon capable of generating hydroxyl free radicals through high-efficiency photosensitization, and the preparation method and the application thereof. In addition, the raw materials are easy to obtain, the preparation is simple and convenient, and the silicon phthalocyanine has certain water solubility.
In order to achieve the above purpose, the invention adopts the following technical scheme:
the phthalocyanine silicon provided by the invention is amine oxide symmetrically substituted phthalocyanine silicon, amino asymmetrically substituted phthalocyanine silicon or amine oxide asymmetrically substituted phthalocyanine silicon.
The structural formula of the amine oxide symmetrically substituted phthalocyanine silicon is as follows:the structural formula of the amino-asymmetrically substituted silicon phthalocyanine is as follows: />The structural formula of the amine oxide asymmetrically substituted phthalocyanine silicon is as follows:
in the structural formula, R1 is an axial substituent, and the structural formula of the substituent R1 is as follows:
in the structural formula, R2 is an axial substituent, and the structural formula of the substituent R2 is as follows:
in the structural formula, R3 is an axial substituent, and the structural formula of the substituent R3 is as follows: H. and (2)>
In the above structuren is the polymerization degree of the polyethylene glycol with the axial substituent group, and the polymerization degree n is 1-45.
The preparation method of the amine oxide symmetrical substituted phthalocyanine silicon comprises the following steps:
(1) Preparing amino symmetrical substituted phthalocyanine silicon: silicon dichlorophthalocyanine and N, N-dimethylamino-N-hexanol, N-dimethylamino-N-propanol, 2- [ [2- (dimethylamino) ethyl ] methylamino ] ethanol, 4-dimethylamino-phenol, 3-dimethylamino-phenol, 2,4, 6-tris (dimethylaminomethyl) phenol, 2- [ (dimethylamino) methyl ] phenol, 4- [ (dimethylamino) methyl ] phenol or 8-dimethylamino-1-octanol are used as reactants, toluene purified by a molecular sieve is used as a solvent, the reaction is stirred for 18-72 hours under the presence of sodium hydride and the protection of nitrogen, the reaction progress is monitored by thin layer chromatography, and when the silicon dichlorophthalocyanine is basically consumed, the reaction is terminated, and the target product is purified by a solvent method and a column chromatography respectively.
The molar ratio of the silicon dichlorophthalocyanine to the N, N-dimethylamino N-hexanol, the N, N-dimethylamino N-propanol, the 2- [ [2- (dimethylamino) ethyl ] methylamino ] ethanol, the 4-dimethylamino phenol, the 3-dimethylamino phenol, the 2,4, 6-tris (dimethylaminomethyl) phenol, the 2- [ (dimethylamino) methyl ] phenol, the 4- [ (dimethylamino) methyl ] phenol or the 8-dimethylamino-1-octanol is 1:3-6, the solvent dosage is 20-30 mL for each mmol of the reactant silicon dichlorophthalocyanine, and the sodium hydride dosage is 0.5-1 mmol for each mmol of the reactant silicon dichlorophthalocyanine.
(2) Preparation of amine oxide symmetrically substituted silicon phthalocyanine: and (3) taking the amino symmetry substituted silicon phthalocyanine and the m-chloroperoxybenzoic acid obtained in the step (1) as reactants, using chloroform, methanol or N, N-dimethylformamide as a reaction solvent, stirring for 1-2 hours under the protection of argon and at normal temperature, monitoring the reaction progress through a thin layer chromatography, stopping the reaction when the amino symmetry substituted silicon phthalocyanine is basically consumed, and purifying a target product through a solvent method and a column chromatography respectively.
The molar ratio of the amino symmetrical substituted phthalocyanine silicon to the m-chloroperoxybenzoic acid used in the reaction is 1:2-3, and the solvent dosage is 5-10 mL for each mmol of reactant amino symmetrical substituted phthalocyanine silicon.
The preparation method of the amino asymmetric substituted phthalocyanine silicon comprises the following steps:
(1) Preparation of amino-asymmetrically substituted silicon phthalocyanines with R3 substituents H: silicon dichlorophthalocyanine, polyethylene glycol (polymerization degree is 1-45) and N, N-dimethylamino N-hexanol, N-dimethylamino N-propanol, 2- [ [2- (dimethylamino) ethyl ] methylamino ] ethanol, 4-dimethylamino phenol, 3-dimethylamino phenol, 2,4, 6-tris (dimethylamino methyl) phenol, 2- [ (dimethylamino) methyl ] phenol, 4- [ (dimethylamino) methyl ] phenol or 8-dimethylamino-1-octanol are used as reactants, toluene purified by molecular sieve is used as solvent, the reaction is stirred for 18-48 hours under the presence of sodium hydride and nitrogen protection, the reaction progress is monitored by thin layer chromatography, and when the silicon dichlorophthalocyanine is basically consumed, the reaction is stopped, and the target product is purified by a solvent method and a column chromatography method respectively.
The molar ratio of the silicon dichlorophthalocyanine, polyethylene glycol (polymerization degree is 1-45) and N, N-dimethylamino N-hexanol, N-dimethylamino N-propanol, 2- [ [2- (dimethylamino) ethyl ] methylamino ] ethanol, 4-dimethylamino phenol, 3-dimethylamino phenol, 2,4, 6-tris (dimethylaminomethyl) phenol, 2- [ (dimethylamino) methyl ] phenol, 4- [ (dimethylamino) methyl ] phenol or 8-dimethylamino-1-octanol used is 1:1.5-3:1.5-3, the solvent is 10-15 mL per mmol of reactant silicon dichlorophthalocyanine, and the amount of sodium hydride is 0.5-1 mmol per mmol of reactant silicon dichlorophthalocyanine.
(2) Preparation of amino-asymmetrically substituted silicon phthalocyanine with R3 substituent of biotin, folic acid, phenylboronic acid, glucose, glycyrrhetinic acid, hyaluronic acid, (2- [3- (1, 3-dicarboxypropyl) -ureido ] glutaric acid or arginyl-glycyl-aspartic acid (RGD), wherein the amino-asymmetrically substituted silicon phthalocyanine with R3 substituent of H obtained in the step (1) and biotin, folic acid, phenylboronic acid, glucose, glycyrrhetinic acid, hyaluronic acid, (2- [3- (1, 3-dicarboxypropyl) -ureido ] glutaric acid or arginyl-aspartic acid (RGD) are used as reactants, N-dimethylformamide is used as a solvent, the corresponding 4-dimethylaminopyridine, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is added, the reaction is monitored for 12-72 hours at room temperature by thin layer chromatography, and the target product is purified by a solvent method or chromatography.
The molar ratio of the 4-dimethylaminopyridine to the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is 1:1.5-4:4-8:4-8, and the solvent is 10-20 mL for the amino-asymmetrically substituted phthalocyanine silicon with H as R3 substituent per mmol of the reactant.
The preparation method of the amine oxide asymmetrically substituted phthalocyanine silicon comprises the following steps:
when the R3 substituent is H, the amine oxide asymmetrically substitutes the preparation of the phthalocyanine silicon:
taking the amino-asymmetrically substituted phthalocyanine silicon with the R3 substituent being H and m-chloroperoxybenzoic acid as reactants, using chloroform, methanol or N, N-dimethylformamide as a reaction solvent, stirring for 1-4 hours under the protection of argon and at normal temperature, monitoring the reaction progress by a thin layer chromatography, stopping the reaction when the amino-asymmetrically substituted phthalocyanine silicon with the R3 substituent being H is basically consumed, and purifying a target product by a solvent method and a column chromatography respectively. The molar ratio of the amino-asymmetrically substituted phthalocyanine silicon with the R3 substituent being H to the m-chloroperoxybenzoic acid used in the reaction is 1:2-3, and the solvent dosage is 5-10 mL for each mmol of the amino-asymmetrically substituted phthalocyanine silicon with the R3 substituent being H.
When the R3 substituent is biotin, folic acid, phenylboronic acid, glucose, glycyrrhetinic acid, hyaluronic acid, (2- [3- (1, 3-dicarboxypropyl) -ureido ] glutaric acid or arginyl-glycyl-aspartic acid (RGD), the preparation of amine oxide asymmetrically substituted silicon phthalocyanine:
the above obtained R3 substituent is used as reactants of biotin, folic acid, phenylboronic acid, glucose, glycyrrhetinic acid, hyaluronic acid, (2- [3- (1, 3-dicarboxypropyl) -ureido ] glutaric acid or arginyl-glycyl-aspartic acid (RGD) amino asymmetrically substituted silicon phthalocyanine and m-chloroperoxybenzoic acid, chloroform, methanol or N, N-dimethylformamide are used as reaction solvents, stirring is carried out for 1-8 hours under the protection of argon gas at normal temperature, the reaction progress is monitored by thin layer chromatography, and when the R3 substituent is used as biotin, folic acid, phenylboronic acid, glucose, glycyrrhetinic acid, hyaluronic acid, (2- [3- (1, 3-dicarboxypropyl) -ureido ] glutaric acid or arginyl-glycyl-aspartic acid (RGD) amino asymmetrically substituted silicon phthalocyanine is completely consumed, the reaction is terminated, and the target product is purified by a solvent method and a column chromatography respectively.
The R3 substituent used in the reaction is biotin, folic acid, phenylboronic acid, glucose, glycyrrhetinic acid, hyaluronic acid, and the molar ratio of the amino-asymmetrically substituted silicon phthalocyanine of (2- [3- (1, 3-dicarboxypropyl) -ureido ] glutaric acid or arginyl-glycyl-aspartic acid (RGD) to the m-chloroperoxybenzoic acid is 1:2-3, and the solvent is 5-10 mL for each mmol of the R3 substituent of the reactant, namely biotin, folic acid, phenylboronic acid, glucose, glycyrrhetinic acid, hyaluronic acid, and the amino-asymmetrically substituted silicon phthalocyanine of (2- [3- (1, 3-dicarboxypropyl) -ureido ] glutaric acid or arginyl-glycyl-aspartic acid (RGD).
The silicon phthalocyanine provided by the invention can be used for photodynamic medicaments, photosensitive medicaments, fluorescent imaging or photoacoustic imaging reagents. The photosensitive agent, or simply photosensitive agent, or photosensitive pharmaceutical preparation, is also called photodynamic agent. The prepared photodynamic medicine or photosensitizing agent can be used for photodynamic therapy, photodynamic diagnosis or photodynamic disinfection. The treatment can be the treatment of malignant tumor, benign tumor, bone marrow external purification treatment of leukemia, or the treatment of non-cancer diseases. The non-cancer disease may be a bacterial infection, or an oral disease, or a macular degeneration eye disease, or arteriosclerosis, or a traumatic infection, or a dermatological disease, or a viral infection. The disinfection can be the disinfection and purification of blood or blood derivatives, or the disinfection and purification of water, or the disinfection of medical or domestic appliances.
The method for preparing the photosensitive medicament by utilizing the phthalocyanine silicon provided by the invention comprises the following steps: the water or the mixed solution of water and other substances, wherein the mass fraction of the other substances is not higher than 10 percent, and the phthalocyanine silicon is dissolved as a solvent to prepare a photosensitive medicament containing a certain concentration, and the concentration of the phthalocyanine silicon is not higher than the saturation concentration; antioxidants, buffers and isotonic agents are added as additives to the prepared solution to maintain chemical stability and biocompatibility of the photosensitizing agent. The other substances are one or a mixture of more of castor oil polyoxyethylene 35 ether, dimethyl sulfoxide, ethanol, glycerol, N-dimethylformamide, polyethylene glycol 50-5000, cyclodextrin, glucose, tween and polyethylene glycol monostearate. For formulations for topical administration, the silicon phthalocyanines of the present invention may be dissolved in an osmotic solvent or injected into ointments, lotions or gels. The penetrating solvent is preferably an aqueous solution of 5-35% (wt%) dimethyl sulfoxide.
The application of the phthalocyanine silicon in photodynamic therapy, photodynamic diagnosis, photodynamic disinfection and photodynamic degradation of pollutants needs to be matched with a proper light source, the proper light source can be provided by connecting a proper optical filter with a common light source or by connecting a laser or an LED lamp or other lamp sources with specific wavelengths, and the wavelength range of the light source is 600-900 nm.
The beneficial effects and outstanding advantages of the invention are:
(1) The structural feature of the invention is that the stituted group is stituted axially, the large space structure and certain water solubility can effectively prevent the phthalocyanine ring from gathering in the water-containing system, thus greatly improving the photodynamic activity.
(2) The phthalocyanine silicon can be efficiently photosensitized in an aqueous system to generate hydroxyl free radicals, so that the phthalocyanine silicon can be used as a photosensitizer and a photodynamic medicine. When the compound is used as a photodynamic medicine, the generated hydroxyl free radical has low dependence on oxygen content, and has good treatment effect on deep hypoxic tumors. In addition, its photosensitizing mechanism for generating hydroxyl radicals is not possessed by other types of phthalocyanine photosensitizers.
(3) Cell experiments prove that the photodynamic medicine prepared from the phthalocyanine silicon has higher anticancer activity under aerobic and anaerobic conditions, and shows lower oxygen dependence. Animal experiments show that the phthalocyanine silicon or the self-assembly body thereof has good tumor targeting and photodynamic treatment effects, and has remarkable application prospect in the field of treatment of hypoxic tumors.
(4) The phthalocyanine silicon can be directly dissolved in water for local administration, or can be assembled into nano dispersion with proper particle size in water for local administration under the action of external force. Therefore, in practical application, the preparation mode of the medicament can be selected according to the requirement.
(5) The preparation process of the phthalocyanine silicon has the advantages of simple operation, stable property, convenient storage, contribution to mass preparation in industrial production and good industrialization prospect.
Drawings
FIG. 1 is an electron absorption spectrum of the asymmetric silicon phthalocyanine (4. Mu.M) obtained in examples 1 and 2 in N, N-dimethylformamide, water and 1% CEL aqueous solution.
FIG. 2 is a graph showing the particle size distribution of the asymmetric silicon phthalocyanine (10. Mu.M) obtained in examples 1 and 2 in water.
FIG. 3 is a synthetic route diagram of the asymmetric silicon phthalocyanine obtained in examples 1 to 5.
Detailed Description
In order to make the contents of the present invention more easily understood, the technical scheme of the present invention will be further described with reference to the specific embodiments, but the present invention is not limited thereto.
Example 1
Asymmetrically substituted silicon phthalocyanines containing tetraethylene glycol and amino groupsIs synthesized by the following steps:
the reaction was stirred for 17 to 24 hours with silicon dichlorophthalocyanine (0.5 mmol), N, N-dimethylamino-N-hexanol (2 to 4 mmol) and tetraethylene glycol (2 to 4 mmol) as reactants, toluene (20 to 100mL, preferably 50 mL) as solvent, in the presence of sodium hydride (0.5 to 2mmol, preferably 1 mmol) and under nitrogen protection at 90 to 120 ℃ (preferably 115 ℃) and the reaction endpoint was monitored by thin layer chromatography. After the reaction, the reaction solution is filtered by a sand core funnel and steamed in a rotary way. Dissolving dichloromethane in silica gel column, eluting with dichloromethane and methanol at volume ratio of dichloromethane to methanol of 100:1-10, collecting, rotary evaporating to remove eluent, dissolving in DMF, passing through Bio-Beads S-X1 type DMF gel column, collecting second band of phthalocyanine band, rotary evaporating to remove eluent, adding ethyl acetate, settling, suction filtering, and drying to obtain product with yield of 55%.
Characterization data: 1 H NMR(500MHz,Chloroform-d)δ9.66-9.64(m,8H),8.37-8.35(m,8H),3.43-3.41(m,2H),3.28-3.27(m,2H),3.20-3.18(m,2H),2.93-2.92(m,2H),2.46-2.44(m,2H),2.41(s,6H),1.67-1.65(m,2H),0.88(t,J＝15.0Hz,2H),0.66-0.60(m,2H),0.42(t,J＝10.0Hz,2H),-0.51--0.56(m,2H),-1.39--1.45(m,2H),-1.58--1.62(m,2H),-1.92(t,J＝10.0Hz,2H),-2.11(t,J＝10.0Hz,2H).HRMS(ESI):m/z calcd for C48H51N9O6Si[M]+,900.3631；found 900.3632.Relative error:0.55ppm.
example 2
Asymmetrically substituted silicon phthalocyanines containing tetraethylene glycol and amine oxideIs synthesized by the following steps:
the reaction was stirred under nitrogen for 1 to 4 hours at 20 to 45 ℃ (preferably 35 ℃) with tetraethylene glycol and amino-containing asymmetrically substituted silicon phthalocyanine (0.5 mmol) and m-chloroperoxybenzoic acid (2.5 to 5.0mmol, preferably 3 mmol) as reactants, chloroform (20 to 50mL, preferably 30 mL) as solvent, and the reaction end point was monitored by thin layer chromatography. After the completion of the reaction, chloroform was removed from the reaction mixture by rotary evaporation using a diaphragm pump. Dissolving dichloromethane, passing through a silica gel column, eluting with dichloromethane and methanol, collecting blue component, removing eluent by rotary evaporation, adding ethyl acetate for sedimentation, suction filtering, and drying to obtain the product with the yield of 38%.
Characterization data: 1 H NMR(500MHz,Chloroform-d)δ:9.63-9.62(m,8H),8.35-8.33(m,8H),3.42-3.40(m,2H),3.28-3.26(m,2H),3.20-3.18(m,2H),3.01(s,6H),2.93-2.91(m,2H),2.49-2.47(m,2H),2.46-2.43(m,2H),1.66-1.67(m,2H),0.65-0.59(m,2H),0.40(t,J＝10.0Hz,2H),-0.50--0.56(m,2H),-1.38--1.45(m,2H),-1.58--1.63(m,2H),-1.93(t,J＝10.0Hz,2H),-2.12(t,J＝10.0Hz,2H).HRMS(ESI):m/z calcd for C48H51N9O7Si[M]+,894.3761；found 894.3743.Relative error:1.12ppm.
example 3
Asymmetrically substituted silicon phthalocyanines containing biotin-conjugated tetraethylene glycol and amino groupsIs synthesized by the following steps:
the above-mentioned asymmetric substituted silicon phthalocyanine (1.0 mmol) containing tetraethylene glycol and an amino group and biotin (4 to 6mmol, preferably 5.0 mmol), 4-dimethylaminopyridine (5 to 50mmol, preferably 20 mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (5 to 50mmol, preferably 20 mmol) were reacted as a reactant with N, N-dimethylformamide (DMF, 20 to 40mL, preferably 30 mL) as a solvent, stirred at 25 to 80℃for 12 to 72 hours, and the end point of the reaction was monitored by thin layer chromatography to produce the corresponding asymmetric silicon phthalocyanine. After the reaction is finished, the mixture is distilled to dryness by rotating, dissolved by N, N-dimethylformamide and passed through a silica gel column, and dichloromethane is respectively used according to the volume ratio: collecting blue color band with methanol as eluent in the ratio of 100:1-10, removing organic solvent by rotary evaporation, adding DMF for dissolution, passing through Bio-Beads S-X1 type DMF gel column, collecting first band phthalocyanine band, removing eluent by rotary evaporation, adding ethyl acetate for sedimentation, suction filtration and drying to obtain the product with the yield of 41%.
Characterization data: 1 H NMR(500MHz,DMSO-d6)δ:9.68-9.66(m,8H),8.52-8.50(m,8H),4.27-4.24(m,1H),4.08-4.05(m,1H),3.95-3.93(m,2H),3.58(s,1H),3.12-3.10(m,3H),3.02-2.98(m,1H),2.84-2.80(m,3H),2.77-2.73(m,1H),2.32-2.29(m,3H),2.16(t,J＝15.0Hz,2H),1.82(s,6H),1.58-1.56(m,3H),1.55-1.50(m,2H),1.47-1.40(m,3H),1.37-1.33(m,3H),0.32(t,J＝10.0Hz,2H),0.25-0.19(m,2H),-0.62--0.68(m,2H),-1.62--1.68(m,4H),-2.06(t,J＝15.0Hz,2H),-2.17(t,J＝10.0Hz,2H).HRMS(ESI):m/z calcd for C58H65N11O8SSi[M]+,1104.4516；found 1104.4596.Relative error:1.45ppm.
example 4
Asymmetrically substituted silicon phthalocyanines containing biotin-conjugated tetraethylene glycol and amine oxidesIs synthesized by the following steps:
the reaction was stirred under nitrogen for 1 to 4 hours at 20 to 45 ℃ (preferably 35 ℃) with the reaction product of asymmetric substituted silicon phthalocyanine (0.5 mmol) containing biotin-conjugated tetraethylene glycol and amino groups, m-chloroperoxybenzoic acid (2.5 to 5mmol, preferably 3 mmol) as a reactant, chloroform (20 to 50mL, preferably 30 mL) as a solvent, and the reaction end point was monitored by thin layer chromatography. After the completion of the reaction, chloroform was removed from the reaction mixture by rotary evaporation using a diaphragm pump. Dissolving dichloromethane, passing through silica gel, eluting with dichloromethane and methanol, collecting blue component, removing eluent by rotary evaporation, adding DMF for dissolving, passing through Bio-Beads S-X1 type DMF gel column, collecting first band phthalocyanine band, removing eluent by rotary evaporation, adding ethyl acetate for sedimentation, suction filtering, and drying to obtain the product with 36% yield.
Characterization data: 1 H NMR(500MHz,DMSO-d6)δ:9.70-9.68(m,8H),8.54-8.52(m,8H),4.46-4.41(m,1H),4.34-4.32(m,1H),4.13(s,2H),3.95(t,J＝10.0Hz,1H),3.63-3.59(m,2H),3.54-3.51(m,2H),3.12-3.10(m,2H),2.85-2.82(m,2H),2.72(s,2H),2.42(s,6H),2.35-2.31(m,2H),2.23-2.19(m,2H),2.15(t,J＝10.0Hz,2H),1.78-1.71(m,2H),1.62-1.55(m,4H),1.23(s,2H),0.50-0.44(m,2H),0.32(t,J＝10.0Hz,2H),-0.54--0.60(m,2H),-1.48--1.54(m,2H),-1.64--1.69(m,2H),-2.05(t,J＝10.0Hz,2H),-2.16(t,J＝15.0Hz,2H).HRMS(ESI):m/z calcd for C58H65N11O9SSi[M]+,1142.4357；found 1142.4307.Relative error:3.67ppm.
example 5
Amine oxide symmetrically substituted silicon phthalocyanineIs synthesized by the following steps:
amino symmetry substituted silicon phthalocyanine (0.5 mmol), m-chloroperoxybenzoic acid (2.5-5 mmol, preferably 3 mmol) as reactant, chloroform (20-50 mL, preferably 40 mL) as solvent, stirring under nitrogen protection at 20-45deg.C (preferably 35deg.C) for 1-4 hr, and monitoring the reaction end point by thin layer chromatography. After the completion of the reaction, chloroform was removed from the reaction mixture by rotary evaporation using a diaphragm pump. Dissolving dichloromethane, passing through silica gel, eluting with dichloromethane and methanol, collecting blue component, removing eluent by rotary evaporation, adding DMF for dissolving, passing through Bio-Beads S-X1 type DMF gel column, collecting first band phthalocyanine band, removing eluent by rotary evaporation, adding ethyl acetate for sedimentation, suction filtering, and drying to obtain the product with 20% yield.
Characterization data: 1 H NMR(500MHz,Chloroform-d)δ:9.70-9.54(m,8H),8.39-8.25(m,8H),2.71(s,12H),1.66-1.67(m,4H),0.40(t,J＝10.0Hz,4H),-0.37--0.59(m,4H),-1.58--1.63(m,4H),-1.93(t,J＝10.0Hz,4H),-2.12(t,J＝10.0Hz,4H).HRMS(ESI):m/z calcd for C48H52N10O4Si[M]+,861.3942；found 861.3943.Relative error:1.16ppm.
example 6
Synthesis of other asymmetric substituted silicon phthalocyanines containing polyethylene glycol or polyethylene glycol coupled with small molecule targeting groups and amino groups:
the N, N-dimethylamino N-hexanol is replaced by equimolar polyethylene glycol with different polymerization degrees or polyethylene glycol with different polymerization degrees coupled by small molecule targeting groups, and N, N-dimethylamino N-propanol, 2- [ [2- (dimethylamino) ethyl ] methylamino ] ethanol, 4-dimethylamino phenol, 3-dimethylamino phenol, 2,4, 6-tris (dimethylamino methyl) phenol, 2- [ (dimethylamino) methyl ] phenol, 4- [ (dimethylamino) methyl ] phenol or 8-dimethylamino-1-octanol which are respectively used for replacing N, N-dimethylamino N-hexanol is replaced by equimolar polyethylene glycol or small molecule targeting group-coupled polyethylene glycol and amino-containing asymmetric substituted silicon phthalocyanine can be obtained. The structure of the resulting product was identical to that of the silicon phthalocyanine product described in example 1 or example 3 (the reaction mechanism was identical to that of example 1 or example 3), except that the polyethylene glycol length in the substituent, the small molecule targeting group and the amino parent structure were replaced.
Example 7
Synthesis of other asymmetric substituted silicon phthalocyanines containing polyethylene glycol or polyethylene glycol coupled with small molecule targeting groups and amine oxide:
And (3) taking other polyethylene glycol or micromolecular targeting group coupled polyethylene glycol and amino group-containing asymmetric substituted silicon phthalocyanine obtained in the embodiment 5 as reactants, adding equimolar m-chloroperoxybenzoic acid, and respectively obtaining other polyethylene glycol or micromolecular targeting group coupled polyethylene glycol and amine oxide-containing asymmetric substituted silicon phthalocyanine by a solvent method and a column chromatography method. The structure of the resulting product is identical to the silicon phthalocyanine product described in example 2 or example 4, except that the polyethylene glycol length in the substituents, the small molecule targeting group and the amine oxide parent structure are replaced.
Example 8
Other amine oxide-containing symmetrical substitution of silicon phthalocyanine:
other amino symmetrically substituted silicon phthalocyanines can be obtained by substituting equimolar amounts of N, N-dimethylamino N-propanol, 2- [ [2- (dimethylamino) ethyl ] methylamino ] ethanol, 4-dimethylaminophenol, 3-dimethylaminophenol, 2,4, 6-tris (dimethylaminomethyl) phenol, 2- [ (dimethylamino) methyl ] phenol, 4- [ (dimethylamino) methyl ] phenol, or 8-dimethylamino-1-octanol for N, N-dimethylamino-hexanol. Then taking other amino symmetrical substituted phthalocyanine silicon and m-chloroperoxybenzoic acid as reactants, taking chloroform as a solvent, stirring and reacting for 1-4 hours at room temperature, monitoring the reaction end point through thin layer chromatography, and respectively obtaining other amine oxide symmetrical substituted phthalocyanine silicon through a solvent method and a column chromatography. The resulting product structure is identical to the silicon phthalocyanine product described in example 5, except that the amine oxide parent structure in the substituent is replaced.
Application example 1
The method for preparing photodynamic medicaments (namely photosensitive medicaments) by utilizing the phthalocyanine silicon provided by the invention comprises the following steps: the phthalocyanine silicon of the present invention is dissolved by using water or a mixed solution of water and other substances (the content of other substances is not more than 10% (wt%) as a solvent to prepare a blue uniform solution (i.e., a photosensitizing agent) in which the concentration of the phthalocyanine silicon is 0.2mM. The other substances can be one or a mixture of the following substances: castor oil derivative (Cremophor EL), dimethyl sulfoxide, ethanol, glycerol, N-dimethylformamide, polyethylene glycol 300-3000, cyclodextrin, glucose, tween, polyethylene glycol monostearate. Antioxidants, buffers and isotonic agents may be added to the resulting solution as additives to maintain the chemical stability and biocompatibility of the photosensitizing agent.
The phthalocyanine silicon can be prepared into a photosensitive medicament in two ways, and the phthalocyanine silicon is dissolved in 5-35% (wt%) of water solution of dimethyl sulfoxide, so that the phthalocyanine silicon can be used as a preparation for local administration. Or the phthalocyanine silicon is prepared into nano dispersion aqueous solution with a certain size by an external force means, and can also be used as a preparation for intravenous injection.
The preparation method of the nano dispersion aqueous solution comprises the following steps: dissolving the asymmetric silicon phthalocyanine of examples 1-4 in chloroform respectively, adding pure water, emulsifying the system by using an ultrasonic crusher, and slowly steaming by a diaphragm pump to remove the chloroform, thereby obtaining the asymmetric silicon phthalocyanine nano dispersion aqueous solution with proper particle size.
Application example 2
The asymmetrically substituted silicon phthalocyanines prepared as described in examples 1-4 were dissolved in DMSO to prepare a 1mM stock solution, and their dispersion in various solutions including DMF, 1% cremophor EL and pure water was tested using an ultraviolet-visible spectrophotometer. In addition, the electron absorption spectrum of the nano-dispersion prepared by external force means in pure water was also tested.
The results of the tests show that the Q bands of the asymmetrically substituted silicon phthalocyanines obtained in examples 1-4 are strong and sharp peaks in DMF and 1% cremophor EL, indicating that the asymmetrically substituted silicon phthalocyanines are present in monomeric form in DMF and 1% cremophor EL. But exhibits a broad and short Q-band in pure water, and the absorption wavelength is red-shifted by 3-5nm, in the form of J aggregates. However, the absorption wavelength of the Q-band of the asymmetrically substituted silicon phthalocyanine nanodispersion in pure water solution is blue-shifted by 5-10nm, and exists in the form of H aggregates. See fig. 1, respectively.
Application example 3
The particle size distribution of the asymmetrically substituted silicon phthalocyanine nanodispersion prepared in examples 1 to 4 in water was measured by means of a particle size distribution meter. For detailed experimental procedures reference is made to j.
Test results show that the asymmetric substituted silicon phthalocyanine (10 mu M) prepared in the examples 1-4 can have a certain size in water by means of external force to form uniform nano particles, and the formed nano particles have good stability, and the particle size of the nano particles is about 120 nm. See fig. 2, respectively.
Application example 4
The asymmetrically substituted silicon phthalocyanines prepared in examples 1-4 were each dissolved in DMF to prepare 1mM stock solution, which was diluted into deionized water, respectively, and tested for their total Reactive Oxygen Species (ROS).
Total active oxygen assay hydrolyzed 2, 7-dichlorofluorescein diacetate (DCFH-DA) was used as a fluorescent probe. DCFH-DA is a probe commonly used to detect active oxygen, and can be activated by oxygen to form dichlorofluorescein which emits a fluorescent signal at a wavelength of 520 nm. The preparation method of the 2, 7-dichloro fluorescein protein acetate (DCF) comprises the following steps: 2, 7-dichlorofluorescein diacetate (DCFH-DA) was dissolved in methanol to prepare a 5mM DCFH-DA solution, which was stored at-20 ℃. When the reaction was carried out, the DCFH-DA solution was mixed with 0.1mol/L sodium hydroxide solution and reacted for 30 minutes in the dark, and then diluted with PBS solution having pH of 7.4 to give a mother solution having a concentration of 200. Mu.M.
Mixing deionized water solution of silicon phthalocyanine (4 μm) and activated active oxygen probe (5 μm) with red light (wavelength of more than 610nm,1 mW/cm) 2 ) The irradiation was performed to measure the change in fluorescence intensity of the active oxygen probe (488 nm excitation, fluorescence at 500nm-600nm in the scanning range) at different irradiation times. Plotting the relative fluorescence intensity at 522nm (Ft-F0) versus the time of illumination (T), a larger value of Ft-F0 per unit time indicates a stronger ROS generating capability.
The experimental results are shown in Table 1.
TABLE 1 comparison of total amount of active oxygen produced by different samples
It can be seen from the table that examples 1-4 can all produce stronger ROS, significantly higher than the common photosensitizer methylene blue.
Application example 5
The asymmetrically substituted silicon phthalocyanines prepared in examples 1 to 4 were dissolved in DMF respectively to prepare mother liquor of 1mM, and each diluted into deionized water, and their ability to generate hydroxyl radicals (. OH) under normoxic conditions was tested. In the test, methylene Blue (MB), a common photosensitizer, was used as a control group.
By usingAminophenyl fluorescein (APF) is used as a probe for detecting hydroxyl radicals. 1mg of the solid powder of APF stored at low temperature was added to 472. Mu.L of DMF to prepare an APF mother liquor at a concentration of 5 mM. In the presence of hydroxyl radicals, APF emits bright green fluorescence (excitation wavelength 490nm, emission wavelength 515 nm). The more intense the fluorescence indicates the greater the amount of superoxide anions generated. The method comprises the following specific steps: the phthalocyanine to be measured (phthalocyanine concentration 4. Mu.M) and APF probe (20. Mu.M) were added to deionized water and mixed well. The fluorescence intensity at the beginning of the test was then measured using red light (wavelength greater than 610nm, luminous intensity 1mW/cm 2 ) The light was irradiated for 5min, the change in fluorescence intensity of the probe was recorded using a fluorescence gradiometer, and the relative fluorescence intensity at 522nm (Ft-F0) was plotted against the time of irradiation with light (T), with a larger value of Ft-F0 per unit time indicating a stronger ability to generate hydroxyl radicals. The experimental results are shown in Table 2.
TABLE 2 comparison of fluorescence intensities of hydroxyl radical generated by different samples
As can be seen from Table 2, the silicon phthalocyanine obtained in example 3 has the strongest relative fluorescence intensity, 7.5 times that of examples 1 and 2, 6.0 times that of example 4, and 15.0 times that of the control photosensitizer methylene blue. This result demonstrates that hydroxyl radicals can be generated in large amounts when the silicon phthalocyanine obtained in example 4 is reduced to the silicon phthalocyanine obtained in example 3 by the reductase in tumor tissue, and thus the silicon phthalocyanine obtained in example 3 of the present invention can be used as a type I photosensitizer or a photosensitizing drug or a photodynamic drug, and can be used for photodynamic therapy against hypoxic tumors or hypoxic tumors.
Application example 6
The method for preparing the photosensitizer or phototherapy medicine by utilizing the phthalocyanine silicon provided by the invention comprises the following steps: directly dissolving silicon phthalocyanine in water to prepare a drug aqueous solution with a certain concentration; or dissolving silicon phthalocyanine into 1-2mM mother liquor by using DMSO or DMF, and diluting with water to prepare a drug aqueous solution with a certain concentration.
The phthalocyanine silicon photosensitizer or phototherapy medicine prepared by the invention needs to be matched with a proper light source in the application process, the proper light source can be provided by connecting a proper optical filter with a common light source or provided by laser with specific wavelength, and the wavelength range of the light source is 300-800nm, preferably 680-704nm.
Application example 7
The phthalocyanine silicon disclosed by the invention is dissolved in water to prepare the 0.08mM photosensitive medicament. The silicon phthalocyanines obtained in example 2, example 3 and example 4 were tested for their cell uptake capacity in human liver cancer HepG2 cells, mouse breast cancer 4T1 cells, mouse melanoma B16 cells and human normal liver L02 cells.
HepG2, 4T1, B16 or L02 cells in logarithmic growth phase (number of cells per plate about 1X 10) were inoculated into culture plates with clean sterile cover slips, respectively 5 And 5% CO at 37deg.C 2 (normoxic) culture was carried out, after 24h the broth was removed and medium (400 μl) mixed with silicon phthalocyanine photosensitizer was added and culture continued for 2h. And then adding PBS buffer solution for twice cleaning, cleaning the culture medium containing the medicine, detecting by using a fluorescence confocal instrument, and collecting fluorescent signals of 640-750nm, wherein the excitation wavelength is 635 nm.
The asymmetrically substituted silicon phthalocyanines obtained in examples 2,3 and 4 show obvious fluorescence intensity in human liver cancer HepG2 cells and mouse breast cancer 4T1 cells with high expression of biotin receptors. Fluorescent signals were also generated in murine melanoma B16 cells with low expression of biotin receptors, but with a fluorescence intensity 2-fold lower than HepG2 cells. In addition, the fluorescent signal was the weakest in L02 normal cells, with 4-fold lower fluorescent intensity than HepG2 cells.
Application example 8
The asymmetric substituted silicon phthalocyanine obtained in the present invention was dissolved in water to prepare a 0.08mM photosensitizing agent. Examples 3 and 4 were tested for the generation of Reactive Oxygen Species (ROS) and hydroxyl radicals (. OH) in human liver cancer HepG2 cells under normoxic and hypoxic conditions.
Determination of total intracellular active oxygen under normoxic and hypoxic conditions: detection of total amount of active oxygen in cells Using 2, 7-dichlorofluorescein diacetate (DCFH-DA) as a ProbeCreating a situation. HepG2 cells in logarithmic growth phase (number of cells per plate approximately 1X 10) were seeded in culture plates with clean sterile coverslips 5 And 5% CO at 37deg.C, respectively 2 (normoxic) or 3%O 2 、5％CO 2 And 92% N 2 (hypoxia) culture was performed, and after 24 hours the culture broth was removed and added to a medium (400. Mu.L) mixed with the asymmetrically substituted silicon phthalocyanine photosensitizer obtained in example 3 or example 4, and culture was continued for 2 hours. Then adding PBS buffer solution for two times of cleaning, cleaning the culture medium containing the medicine, adding prepared probe DCFH-DA for continuous incubation for 30min, using red light (wavelength is more than 610nm, illumination density is 15 mW/cm) 2 ) The cells are illuminated for 5min, a fluorescence confocal instrument is used for detection, the excitation wavelength is 435nm, and fluorescence signals of 500-600nm are collected.
Determination of intracellular hydroxyl radical under normoxic and hypoxic conditions: the production of hydroxyl radicals in cells was detected using aminophenyl fluorescein (APF) as a probe. HepG2 cells in logarithmic growth phase (number of cells per plate approximately 1X 10) were seeded in culture plates with clean sterile coverslips 5 And 5% CO at 37deg.C, respectively 2 (normoxic) or 3%O 2 、5％CO 2 And 92% N 2 (hypoxia) culture was performed, and after 24 hours the culture broth was removed and added to a medium (400. Mu.L) mixed with the asymmetrically substituted silicon phthalocyanine photosensitizer obtained in example 3 or example 4, and culture was continued for 2 hours. Then adding PBS buffer solution for two times of cleaning, cleaning the culture medium containing the medicine, adding prepared probe APF, and further incubating for 30min, using red light (wavelength is greater than 610nm, and illumination density is 15 mW/cm) 2 ) The cells are illuminated for 5min, a fluorescence confocal instrument is used for detection, the excitation wavelength is 435nm, and fluorescence signals of 500-600nm are collected.
The asymmetrically substituted silicon phthalocyanines prepared in examples 3 and 4 resulted in the response of the total amount of active oxygen fluorescent probe and hydroxyl radical probe in HepG2 cells, both under normoxic and hypoxic conditions, and the fluorescent probe intensities remained consistent under normoxic and hypoxic conditions. The asymmetric substituted silicon phthalocyanines obtained in the examples 3 and 4 have strong capability of generating active oxygen and hydroxyl free radicals, and the photosensitive activity is independent of the oxygen content, so that the asymmetric substituted silicon phthalocyanines have great significance in the treatment of clinical hypoxic tumors.
Application example 9
The asymmetric substituted silicon phthalocyanine obtained in the present invention was dissolved in water to prepare a 0.08mM photosensitizing agent. Under normoxic conditions and hypoxic conditions, the silicon phthalocyanine obtained in example 2 or example 4 was tested for photodynamic inhibitory activity on human liver cancer HepG2 cells.
Determination of anticancer Activity under normoxic and hypoxic conditions: the asymmetrically substituted silicon phthalocyanines of examples 2 and 4 were dissolved in DMF to prepare a 1mM stock solution, which was then diluted into a cell culture broth to prepare cell culture broths containing different concentrations of silicon phthalocyanines. HepG2 cells in logarithmic growth phase (number of cells per plate approximately 1X 10) were seeded in culture plates with clean sterile coverslips 5 And respectively at 37 ℃ and 5 percent CO 2 (normoxic) or 3%O 2 ，5％CO 2 And 92% N 2 (hypoxia) and removing the culture solution after 24 hours, respectively culturing cancer cells in the culture solution containing the phthalocyanine silicon with different concentrations for 2 hours, discarding the culture solution, washing the cells with PBS buffer solution, and adding a new culture solution (without the phthalocyanine silicon photosensitizer). For the light experimental group, red light (wavelength longer than 610nm,15mW/cm was used 2 ) Respectively irradiating the cells with light for 30 minutes; for the no-light group, the cells were placed in the dark for 30 minutes. Finally, the viability of the cells for both experiments was investigated using the MTT method. See eur.j. Med. Chem.,2018,155,24-33 for detailed experimental procedures.
The experimental results show that: the asymmetric substituted silicon phthalocyanines obtained in examples 2 and 4 have no killing and growth inhibition effect on HepG2 cells when no light irradiation is performed, indicating that the silicon phthalocyanines have no dark toxicity. However, after light irradiation, the asymmetric substituted silicon phthalocyanines obtained in examples 2 and 4 all show high photodynamic anticancer activity and show dose-effect relationship. The above results demonstrate that the asymmetrically substituted silicon phthalocyanine prepared by the invention has higher photodynamic anticancer activity to HepG2 cells under normoxic and hypoxic conditions, wherein the IC of the silicon phthalocyanine obtained in example 4 50 The value (half lethal concentration, i.e. the concentration of drug required to kill 50% of cancer cells) is as low as 25±2nM and the killing power is 3 times that of example 2.
Application example 10
The obtained asymmetric substituted silicon phthalocyanine was dissolved in DMSO to prepare a mother solution of 1mM, and diluted with water to prepare an aqueous solution of a photosensitizing agent having a concentration of 200. Mu.M. The silicon phthalocyanines obtained in example 2 and example 4 were tested for fluorescence imaging of ICR mice bearing solid tumors of hepatoma cells (H22).
The tail of tumor-bearing mice bearing H22 was injected intravenously with 100. Mu.L of the aqueous solution of the above-mentioned photosensitizing agent at a concentration of 200. Mu.M. The enrichment condition of the drugs at the tumor part of the mice is monitored by using a small animal fluorescence imager, the mice are dissected after 24 hours, and the distribution condition of the drugs at each tissue organ of the mice is monitored by using the small animal fluorescence imager. See ACS appl. Mate. Interfaces 2019,11,36435-36443 for detailed experimental procedures.
The in-vivo fluorescence imaging experimental results show that: the fluorescence signal intensity of the phthalocyanine silicon obtained in example 4 at the tumor site is twice as high as that of the phthalocyanine silicon obtained in example 2. After 2 hours of intravenous photosensitizer injection, fluorescence was observed at the tumor site, peaking at 36 hours and fluorescence was only observed at the tumor site. After 72 hours, the mice were dissected to obtain organs and observed for their fluorescence imaging, which showed that more pronounced fluorescence was observed at the tumor sites of the mice, with the fluorescence intensities in the other organs being relatively weak.
Application example 11
The asymmetric substituted silicon phthalocyanine photodynamic therapy obtained in example 4 was tested for its antitumor effect. KM mice subcutaneously transplanted with H22 hepatoma cells were established by reference to the literature method (ACS appl. Mater. Interfaces 2019,11,36435-36443). H22 tumor-bearing KM mice were divided into 4 experimental groups: a single dose group, a single PBS group, a PBS+680nm light group, a dose +680nm light group, 5 each; administration + laser irradiation is carried out until tumor grows to 60-100mm 3 In size, 100. Mu.L of an aqueous solution of asymmetric silicon phthalocyanine having a concentration of 200. Mu.M was intravenously injected, and then 36 hours after administration, the tumor site was irradiated with light of 680nm for 5 minutes, light of 680nm Illumination intensity 0.1W/cm 2 . After each group of mice is treated according to the requirements, the mice are continuously fed, the condition of the mice is observed every other day, the weight of the mice is measured, and the long diameter and the short diameter of the tumor are measured by a vernier caliper for 14 days. Tumor inhibition was calculated according to literature methods (ACSAppl. Mater. Interfaces 2019,11,36435-36443).
The experimental results show that: (1) The single administration group, the single PBS group and the PBS+680nm illumination group have no tumor growth inhibition effect on the tumor of the mice (the tumor is increased by about 12 times). (2) The +680nm light group shows good effect of inhibiting tumor growth, and the tumor inhibition rate can reach 83% (p < 0.001). It was further demonstrated that the photodynamic effect can be effectively induced under 680nm light. (3) The mice of the pure administration group and the administration +680nm light irradiation group have an increasing trend of weight within 14 days, which indicates that the asymmetrically substituted silicon phthalocyanine obtained in the example 4 has no obvious toxicity to the mice and has good biocompatibility.
The foregoing description is only of the preferred embodiments of the invention, and all changes and modifications that come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Claims (10)
1. A silicon phthalocyanine having a high efficiency of photosensitization to generate hydroxyl radicals, characterized by: the phthalocyanine silicon is one of amine oxide symmetrically substituted phthalocyanine silicon, amino asymmetrically substituted phthalocyanine silicon and amine oxide asymmetrically substituted phthalocyanine silicon.
2. The silicon phthalocyanine having high efficiency photosensitization for generating hydroxyl radicals as claimed in claim 1, wherein: the structural formula of the amine oxide symmetrically substituted phthalocyanine silicon is as follows:the structural formula of the amino asymmetric substituted phthalocyanine silicon is as follows: />The structural formula of the amine oxide asymmetrically substituted phthalocyanine silicon is as follows:
in the structural formula, R1 is an axial substituent, and the structural formula of the substituent R1 is as follows: any one of them; in the structural formula, R2 is an axial substituent, and the structural formula of the substituent R2 is as follows: /> Any one of them; in the structural formula, R3 is an axial substituent, and the structural formula of the substituent R3 is as follows: H. and (2)>
Any one of them; in the structural formula, n is the polymerization degree of polyethylene glycol with an axial substituent group, and the polymerization degree n is 1-45.
3. A method for preparing silicon phthalocyanine having a high efficiency of photosensitization for generating hydroxyl radicals as claimed in claim 2, wherein: the preparation method of the amine oxide symmetrical substituted phthalocyanine silicon comprises the following steps:
(1) Preparing amino symmetrical substituted phthalocyanine silicon: silicon dichlorophthalocyanine and N, N-dimethylamino N-hexanol, N-dimethylamino N-propanol, 2- [ [2- (dimethylamino) ethyl ] methylamino ] ethanol, 4-dimethylamino phenol, 3-dimethylamino phenol, 2,4, 6-tris (dimethylamino methyl) phenol, 2- [ (dimethylamino) methyl ] phenol, 4- [ (dimethylamino) methyl ] phenol or 8-dimethylamino-1-octanol are used as reactants, toluene purified by a molecular sieve is used as a solvent, the reaction is stirred for 18-72 hours under the presence of sodium hydride and the protection of nitrogen, the reaction progress is monitored by thin layer chromatography, the reaction is stopped when the silicon dichlorophthalocyanine is basically consumed, and the target product is purified by a solvent method and a column chromatography respectively;
(2) Preparation of amine oxide symmetrically substituted silicon phthalocyanine: and (3) taking the amino symmetry substituted silicon phthalocyanine and the m-chloroperoxybenzoic acid obtained in the step (1) as reactants, using chloroform, methanol or N, N-dimethylformamide as a reaction solvent, stirring for 1-2 hours under the protection of argon and at normal temperature, monitoring the reaction progress through a thin layer chromatography, stopping the reaction when the amino symmetry substituted silicon phthalocyanine is basically consumed, and purifying a target product through a solvent method and a column chromatography respectively.
4. A method of preparation according to claim 3, characterized in that:
the molar ratio of the silicon dichlorophthalocyanine to the N, N-dimethylamino N-hexanol, the N, N-dimethylamino N-propanol, the 2- [ [2- (dimethylamino) ethyl ] methylamino ] ethanol, the 4-dimethylamino phenol, the 3-dimethylamino phenol, the 2,4, 6-tris (dimethylamino methyl) phenol, the 2- [ (dimethylamino) methyl ] phenol, the 4- [ (dimethylamino) methyl ] phenol or the 8-dimethylamino-1-octanol used in the step (1) is 1:3-6, the solvent dosage is 20-30 mL per mmol of the reactant silicon dichlorophthalocyanine, and the sodium hydride dosage is 0.5-1 mmol per mmol of the reactant silicon dichlorophthalocyanine;
the molar ratio of the amino symmetrical substituted phthalocyanine silicon to the m-chloroperoxybenzoic acid used in the reaction of the step (2) is 1:2-3, and the solvent dosage is 5-10 mL for amino symmetrical substituted phthalocyanine silicon per mmol reactant.
5. A method for preparing silicon phthalocyanine having a high efficiency of photosensitization for generating hydroxyl radicals as claimed in claim 2, wherein: the preparation method of the amino asymmetric substituted phthalocyanine silicon comprises the following steps:
(1) Preparation of amino-asymmetrically substituted silicon phthalocyanines with R3 substituents H: silicon dichlorophthalocyanine, polyethylene glycol (polymerization degree is 1-45) and N, N-dimethylamino N-hexanol, N-dimethylamino N-propanol, 2- [ [2- (dimethylamino) ethyl ] methylamino ] ethanol, 4-dimethylamino phenol, 3-dimethylamino phenol, 2,4, 6-tris (dimethylamino methyl) phenol, 2- [ (dimethylamino) methyl ] phenol, 4- [ (dimethylamino) methyl ] phenol or 8-dimethylamino-1-octanol are used as reactants, toluene purified by molecular sieve is used as solvent, the reaction is stirred for 18-48 hours under the presence of sodium hydride and the protection of nitrogen, the reaction progress is monitored by thin layer chromatography, and when the silicon dichlorophthalocyanine is basically consumed, the reaction is stopped, and the target product is purified by a solvent method and a column chromatography respectively;
(2) Preparation of amino-asymmetrically substituted silicon phthalocyanine with R3 substituent of biotin, folic acid, phenylboronic acid, glucose, glycyrrhetinic acid, hyaluronic acid, (2- [3- (1, 3-dicarboxypropyl) -ureido ] glutaric acid or arginyl-glycyl-aspartic acid (RGD), wherein the amino-asymmetrically substituted silicon phthalocyanine with R3 substituent of H obtained in the step (1) and biotin, folic acid, phenylboronic acid, glucose, glycyrrhetinic acid, hyaluronic acid, (2- [3- (1, 3-dicarboxypropyl) -ureido ] glutaric acid or arginyl-aspartic acid (RGD) are used as reactants, N-dimethylformamide is used as a solvent, the corresponding 4-dimethylaminopyridine, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is added, the reaction is monitored for 12-72 hours at room temperature by thin layer chromatography, and the target product is purified by a solvent method or chromatography.
6. The method of manufacturing according to claim 5, wherein:
the molar ratio of the silicon dichlorophthalocyanine, polyethylene glycol (polymerization degree is 1-45) and N, N-dimethylamino N-hexanol, N-dimethylamino N-propanol, 2- [ [2- (dimethylamino) ethyl ] methylamino ] ethanol, 4-dimethylamino phenol, 3-dimethylamino phenol, 2,4, 6-tris (dimethylamino methyl) phenol, 2- [ (dimethylamino) methyl ] phenol, 4- [ (dimethylamino) methyl ] phenol or 8-dimethylamino-1-octanol used in the step (1:1.5-3:1.5-3, the solvent dosage is 10-15 mL for each mmol of reactant silicon dichlorophthalocyanine, and the sodium hydride dosage is 0.5-1 mmol for each mmol of reactant silicon dichlorophthalocyanine;
the molar ratio of the 4-dimethylaminopyridine to the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is 1:1.5-4:4-8:4-8, and the solvent dosage is 10-20 mL for the amino-asymmetrically substituted phthalocyanine silicon with H as R3 substituent per mmol reactant.
7. A method for preparing silicon phthalocyanine having a high efficiency of photosensitization for generating hydroxyl radicals as claimed in claim 2, wherein: the preparation method of the amine oxide asymmetrically substituted phthalocyanine silicon comprises the following steps:
when the R3 substituent is H, the preparation method of the amine oxide asymmetrically substituted phthalocyanine silicon comprises the following steps:
step (1), taking amino-asymmetrically substituted phthalocyanine silicon with an R3 substituent as H and m-chloroperoxybenzoic acid as reactants, using chloroform, methanol or N, N-dimethylformamide as a reaction solvent, stirring for 1-4 hours under the protection of argon and at normal temperature, monitoring the reaction progress through a thin layer chromatography, stopping the reaction when the amino-asymmetrically substituted phthalocyanine silicon with the R3 substituent as H is basically consumed, and purifying a target product through a solvent method and a column chromatography respectively;
in the step (2), when R3 substituent is biotin, folic acid, phenylboronic acid, glucose, glycyrrhetinic acid, hyaluronic acid, (2- [3- (1, 3-dicarboxypropyl) -ureido ] glutaric acid or arginyl-glycyl-aspartic acid RGD), the preparation method of the amine oxide asymmetric substituted phthalocyanine silicon comprises the following steps:
taking the R3 substituent obtained in the step (1) as biotin, folic acid, phenylboronic acid, glucose, glycyrrhetinic acid, hyaluronic acid, amino-asymmetrically substituted silicon phthalocyanine of (2- [3- (1, 3-dicarboxypropyl) -ureido ] glutaric acid or arginyl-glycyl-aspartic acid RGD and m-chloroperoxybenzoic acid as reactants, using chloroform, methanol or N, N-dimethylformamide as a reaction solvent, stirring for 1-8 hours under the protection of argon gas at normal temperature, monitoring the reaction progress through a thin layer chromatography, and stopping the reaction when the R3 substituent is biotin, folic acid, phenylboronic acid, glucose, glycyrrhetinic acid, hyaluronic acid, and amino-asymmetrically substituted silicon phthalocyanine of (2- [3- (1, 3-dicarboxypropyl) -ureido ] glutaric acid or arginyl-glycyl-aspartic acid RGD is basically consumed, and purifying the target product through a solvent method and a column chromatography respectively.
8. The method of manufacturing according to claim 7, wherein: the molar ratio of the amino-asymmetrically substituted phthalocyanine silicon with the R3 substituent of H to the m-chloroperoxybenzoic acid used in the reaction in the step (1) is 1:2-3, and the solvent dosage is 5-10 mL for each mmol of the amino-asymmetrically substituted phthalocyanine silicon with the R3 substituent of H.
9. The method of manufacturing according to claim 7, wherein: the R3 substituent used in the reaction of the step (2) is biotin, folic acid, phenylboronic acid, glucose, glycyrrhetinic acid, hyaluronic acid, and the molar ratio of the amino-asymmetrically substituted silicon phthalocyanine of (2- [3- (1, 3-dicarboxypropyl) -ureido ] glutaric acid or arginyl-glycyl-aspartic acid RGD to the m-chloroperoxybenzoic acid is 1:2-3, and the solvent is 5-10 mL for each mmol of the R3 substituent of the reactant, namely biotin, folic acid, phenylboronic acid, glucose, glycyrrhetinic acid, hyaluronic acid, and the amino-asymmetrically substituted silicon phthalocyanine of (2- [3- (1, 3-dicarboxypropyl) -ureido ] glutaric acid or arginyl-glycyl-aspartic acid RGD.
10. Use of the silicon phthalocyanine according to claim 1, characterized in that: the silicon phthalocyanine is used for preparing a photosensitizer or photodynamic medicine or a photosensitive medicament or a fluorescence imaging reagent or a photoacoustic imaging reagent.
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