CN113717183B - Phthalocyanine modified by pericyclic asymmetric arginine, preparation thereof and application thereof in pharmaceutical field - Google Patents

Phthalocyanine modified by pericyclic asymmetric arginine, preparation thereof and application thereof in pharmaceutical field Download PDF

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CN113717183B
CN113717183B CN202111133916.0A CN202111133916A CN113717183B CN 113717183 B CN113717183 B CN 113717183B CN 202111133916 A CN202111133916 A CN 202111133916A CN 113717183 B CN113717183 B CN 113717183B
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phthalocyanine
arginine
phenoxy
zinc phthalocyanine
aminoethyl
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CN113717183A (en
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黄剑东
柯美荣
马新月
杨丽芳
郑碧远
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Fuzhou University
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Abstract

The invention discloses a peripherial asymmetric arginine modified phthalocyanine, and preparation and application thereof in the field of pharmacy, and belongs to the technical field of photosensitizer and medicine preparation. The compounds of the present invention exhibit a unique type I mechanism photosensitizing response. The compound obtained by the invention shows high photodynamic cancer cell growth inhibition activity under the anoxic condition, has high photodynamic solid tumor growth inhibition capability, simultaneously shows the capability of obviously inhibiting tumor metastasis and resisting metastasis by cooperating with an immune checkpoint inhibitor, and can be used as a novel medicament or photosensitizer for photodynamic therapy and immunophotodynamic therapy of hypoxic tumors.

Description

Phthalocyanine modified by pericyclic asymmetric arginine, preparation thereof and application thereof in pharmaceutical field
Technical Field
The invention belongs to the technical field of medicine preparation, and particularly relates to pericyclic asymmetric arginine modified phthalocyanine, and preparation and application thereof in the field of pharmacy.
Background
Photodynamic therapy (or called photodynamic therapy) is a novel therapy for treating diseases, and the action process of the photodynamic therapy is to inject photosensitizer into a body and enable the photosensitizer to be enriched in a target body, then irradiate the target body with light with specific wavelength, enable the photosensitizer enriched in the target body to generate a series of photophysical and photochemical reactions under the excitation of specific light, generate active oxygen and further generate oxidative damage to cells, tissues and the like of pathological changes. Thus, the key to photodynamic therapy is the photosensitizer. To date, the photosensitizers approved for clinical use have been mainly hematoporphyrin derivatives. Photofrin (formally approved by the FDA in the united states for the clinical treatment of cancer in 1995) is used in the united states, canada, germany, japan, etc., as a mixture of hematoporphyrin oligomers extracted from cow blood and chemically modified. Although hematoporphyrin derivatives show some efficacy, they also expose their serious drawbacks: the maximum absorption wavelength (380-420 nm) is not in the red light region (650-800 nm) with better transmittance for human tissues, the skin phototoxicity is high, the mixture composition is unstable, and the like, so that the clinical application of the composition is limited. Therefore, the development of a new generation of photodynamic drugs (photosensitizers) is an international research focus.
The mechanism of action of photodynamic therapy can be divided into two mechanisms, type i and type ii, depending on the type and mode of ROS production by the photosensitizer. In the type I mechanism, an excited photosensitizer directly performs electron transfer with matrix molecules to generate free radical species (such as superoxide anion free radicals, hydroxyl free radicals and the like); in the type II mechanism, excited stateThe photosensitizer and oxygen generate energy transfer effect to generate singlet oxygen 1 O 2 ）， 1 O 2 It reacts rapidly with many biological substrates, causing oxidative damage thereof, and is considered to be the major cytotoxic agent produced in PDT treatments.
The phthalocyanine metal complex is highly regarded as the application of the novel photosensitizer due to the characteristics of the maximum absorption wavelength in the red light region which is easy to penetrate human tissues, low dark toxicity and the like. However, many of the phthalocyanine complexes having biological activities reported so far are used for photodynamic therapy by a type ii mechanism, and the specific mechanism for photodynamic therapy is not clear. Therefore, it is necessary to research the action mechanism of photodynamic therapy in order to prepare more phthalocyanine complexes with better advantages as candidate drugs.
Meanwhile, the existing research shows that the singlet oxygen generated by the II type photosensitizer has strong cytotoxicity, but the generation of active oxygen by the II type photosensitizer is a process depending on oxygen, and solid tumors are anaerobic tissues, so the photodynamic treatment effect of the II type photosensitizer on the solid tumors is limited. The type I mechanism is an oxygen-independent process, and therefore type I mechanism photosensitizers have advantages in photodynamic therapy of solid tumors. At present, most phthalocyanine photosensitizers are type II photosensitizers, so that the development of phthalocyanine photosensitizers having a type I photosensitizing mechanism or a combination of type I and type II is of great significance.
Disclosure of Invention
The invention aims to provide a peripherical asymmetric arginine modified phthalocyanine, a preparation method thereof and application thereof in the field of pharmacy, and provides a phthalocyanine photosensitizer with a type I photosensitization mechanism on the basis.
In order to achieve the purpose, the invention adopts the following technical scheme.
It is an object of the present invention to provide a peri-asymmetric arginine-modified phthalocyanine which is capable of self-assembly in water.
The peripherical asymmetric arginine modified phthalocyanine specifically comprises the following components:
(1) The phthalocyanine ring substituent contains arginine, and the structural formula is as follows:
Figure DEST_PATH_IMAGE002
(I) Is expressed as ZnPcR1B
Or
Figure DEST_PATH_IMAGE004
(II) is expressed as ZnPcR1.
(2) The phthalocyanine ring substituent contains two arginines, and the structural formula is as follows:
Figure DEST_PATH_IMAGE006
(III) is expressed as ZnPcR2 or
Figure DEST_PATH_IMAGE008
(IV) is expressed as ZnPcR2B.
(3) The phthalocyanine ring substituent contains three or four arginines, and the structural formula is as follows:
Figure DEST_PATH_IMAGE010
(V) is ZnPcR3 or
Figure DEST_PATH_IMAGE012
(VI) is expressed as ZnPcR4.
The invention also aims to protect the preparation method of the peripherical asymmetric arginine modified phthalocyanine, which comprises the following steps:
(1) The preparation method of the compound (I) comprises the following steps: 1- [4- (2-aminoethyl) phenoxy ] zinc phthalocyanine (ZnPcN, the structure of which is shown in the following formula) and N-Boc-L-arginine are used as reactants, N, N-Dimethylformamide (DMF) is used as a solvent, the mixture is stirred and reacted for 10-60min at the temperature of 0-10 ℃ in the presence of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI), 1-hydroxybenzotriazole (HOBt) and N-methylmorpholine (NMM) under the protection of nitrogen, then the mixture is stirred and reacted for 7-36h at the temperature of room temperature to 45 ℃, and the zinc phthalocyanine ZnPcR1B substituted by pericyclic arginine is obtained through column chromatography and molecular exclusion chromatography. Wherein the molar ratio of 1- [4- (2-aminoethyl) phenoxy ] zinc phthalocyanine to N-Boc-L-arginine is 1 to 3; 1-Ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride (EDCI), 1-hydroxybenzotriazole (HOBt) in an amount of 1.5-6mmol per mmol of 1- [4- (2-aminoethyl) phenoxy ] zinc phthalocyanine; the amount of N-methylmorpholine (NMM) is 0.2-1mL per mmol of 1- [4- (2-aminoethyl) phenoxy ] zinc phthalocyanine.
Figure DEST_PATH_IMAGE014
。
(2) The preparation method of the compound (II) comprises the following steps: dispersing ZnPcR1B into a mixed solution of dichloromethane and trifluoroacetic acid, stirring and reacting for 10-60min at 0-10 ℃ under the protection of nitrogen, then continuously stirring and reacting for 7-24h at room temperature-45 ℃, and separating by using size exclusion chromatography to obtain the zinc phthalocyanine ZnPcR1 substituted by cycloarginine. The volume ratio of trifluoroacetic acid to dichloromethane is 1 to 5; a mixture of trifluoroacetic acid and methylene chloride was used in an amount of 1 to 10mL per mg of 1- [4- (2-aminoethyl) phenoxy ] zinc phthalocyanine.
(3) The preparation method of the compound (III) comprises the following steps: 1- [4- (2-aminoethyl) phenoxy ] zinc phthalocyanine (ZnPcN) and N-Boc-L-arginine are used as reactants, N, N-Dimethylformamide (DMF) is used as a solvent, the mixture is stirred and reacted for 10-60min at the temperature of 0-10 ℃ in the presence of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI), 1-hydroxybenzotriazole (HOBt) and N, N-Diisopropylethylamine (DIPEA) under the protection of nitrogen, then the mixture is stirred and reacted for 7-36h at the temperature of room temperature-45 ℃, and the zinc phthalocyanine ZnPcR2 substituted by pericyclic arginine is obtained through column chromatography and molecular exclusion chromatography. Wherein the molar ratio of the 2- [4- (2-aminoethyl) phenoxy ] zinc phthalocyanine to the N-Boc-L-arginine is 1 to 3; the dosage of the 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) and the 1-hydroxybenzotriazole (HOBt) is 1.5 to 6mmol per mmol of 2- [4- (2-aminoethyl) phenoxy ] zinc phthalocyanine; the dosage of the N, N-Diisopropylethylamine (DIPEA) is 0.2 to 1mL per mmol of 1- [4- (2-aminoethyl) phenoxy ] zinc phthalocyanine.
(4) The preparation method of the compound (IV) comprises the following steps: 2,3- [4- (2-aminoethyl) phenoxy ] zinc phthalocyanine (ZnPcBN, the structure is shown in the following formula) and N-Boc-L-arginine are used as reactants, N, N-Dimethylformamide (DMF) is used as a solvent, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI), 1-hydroxybenzotriazole (HOBt) and N, N-Diisopropylethylamine (DIPEA) are stirred and reacted at the temperature of 0-10 ℃ for 10-60min under the protection of nitrogen, then the reaction is continuously stirred and reacted at the temperature of room temperature-45 ℃ for 7-24h, and the zinc phthalocyanine ZnPcR2B substituted by pericyclic arginine is obtained through column chromatography and molecular exclusion chromatography. Wherein the molar ratio of 2,3- [4- (2-aminoethyl) phenoxy ] zinc phthalocyanine to N-Boc-L-arginine is 1; the 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) and 1-hydroxybenzotriazole (HOBt) are used in amounts of 1.5 to 6mmol per mmol of 3,3' - [4- (2-aminoethyl) phenoxy ] zinc phthalocyanine; the dosage of the N, N-Diisopropylethylamine (DIPEA) is 0.2 to 1mL per mmol of 2,3- [4- (2-aminoethyl) phenoxy ] zinc phthalocyanine.
Figure DEST_PATH_IMAGE016
。
(5) The preparation method of the compound (V) comprises the following steps: 1- [4- (2-aminoethyl) phenoxy ] zinc phthalocyanine and N-Boc-L-arginine are used as reactants, N, N-Dimethylformamide (DMF) is used as a solvent, the mixture is stirred and reacted for 10 to 60min under the conditions of 0 to 10 ℃ in the presence of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI), 1-hydroxybenzotriazole (HOBt) and N, N-Diisopropylethylamine (DIPEA) and under the protection of nitrogen, then the mixture is continuously stirred and reacted for 7 to 24h at room temperature to 45 ℃, and the pericyclic arginine-substituted zinc phthalocyanine ZnPcR3 is obtained through column chromatography and molecular exclusion chromatography. Wherein the molar ratio of the 1- [4- (2-aminoethyl) phenoxy ] zinc phthalocyanine to the N-Boc-L-arginine is 1 to 3; the dosage of the 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) and the 1-hydroxybenzotriazole (HOBt) is 1.5 to 6mmol per mmol of 1- [4- (2-aminoethyl) phenoxy ] zinc phthalocyanine; the dosage of the N, N-Diisopropylethylamine (DIPEA) is 0.2 to 1mL per mmol of 1- [4- (2-aminoethyl) phenoxy ] zinc phthalocyanine.
(6) The preparation method of the compound (VI) comprises the following steps: 2,3- [4- (2-aminoethyl) phenoxy ] zinc phthalocyanine and N-Boc-L-arginine are used as reactants, N, N-Dimethylformamide (DMF) is used as a solvent, the mixture is stirred and reacted for 10 to 60min at the temperature of 0 to 10 ℃ in the presence of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI), 1-hydroxybenzotriazole (HOBt) and N, N-Diisopropylethylamine (DIPEA) under the protection of nitrogen, then the mixture is stirred and reacted for 7 to 24h at the temperature of room temperature to 45 ℃, and the pericyclic arginine substituted zinc phthalocyanine PcZnBNR 2 is obtained through column chromatography and molecular exclusion chromatography. Wherein the molar ratio of 2,3- [4- (2-aminoethyl) phenoxy ] zinc phthalocyanine to N-Boc-L-arginine is 1 to 2 to 6; the dosage of the 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) and the 1-hydroxybenzotriazole (HOBt) is 1.5 to 6mmol per mmol of 2,3- [4- (2-aminoethyl) phenoxy ] zinc phthalocyanine; the dosage of the N, N-Diisopropylethylamine (DIPEA) is 0.2 to 1mL per mmol of 2,3- [4- (2-aminoethyl) phenoxy ] zinc phthalocyanine.
The invention also aims to provide application of the phthalocyanine with the asymmetric arginine modification in the peripheral ring, and particularly relates to application of the phthalocyanine or the self-assembly thereof in preparing a photodynamic medicament. The photodynamic medicament has a type I photosensitive mechanism and can perform photodynamic activity in an anoxic environment.
The photodynamic medicaments, or photosensitizing medicinal preparations, also known as photosensitizers, can be used for photodynamic therapy, photodynamic diagnosis or photodynamic disinfection. The photodynamic therapy may be photodynamic therapy of malignancy, or photodynamic purification therapy of leukemia in vitro, or photodynamic therapy of non-cancer diseases, such as fungal infections, bacterial infections, oral diseases, macular degeneration eye diseases, arteriosclerosis, wound infections, skin diseases, viral infections. The photodynamic disinfection can be photodynamic disinfection and purification of blood or blood derivatives, or photodynamic disinfection of water, or photodynamic disinfection of medical or living appliances.
The application of the perinuclear asymmetric arginine-modified phthalocyanine in photodynamic therapy, photodynamic diagnosis, photodynamic disinfection and photodynamic pollutant degradation needs to be matched with a proper light source, the proper light source can be provided by connecting a common light source with a proper optical filter or provided by a laser or an LED lamp or other lamp sources with specific wavelength, and the wavelength range of the light source is 630 to 730 nm.
The invention has the following beneficial effects and outstanding advantages:
(1) The peripherical asymmetric arginine modified phthalocyanine can be self-assembled in water, and the formation of a self-assembly can enhance the I-type photosensitive efficiency of the phthalocyanine.
(2) In water, the peripherical asymmetric arginine modified phthalocyanine generates higher amount of active oxygen under the irradiation of near-infrared laser and under the condition of hypoxia. Wherein the I-type photosensitive mechanism exerted by the phthalocyanine is not possessed by other similar phthalocyanines (such as peripherical asymmetric lysine modified phthalocyanine, peripherical asymmetric histidine modified phthalocyanine, axial symmetric arginine modified phthalocyanine silicon, axial asymmetric arginine modified phthalocyanine silicon and the like).
(3) Cell experiments prove that the photodynamic medicament prepared by modifying phthalocyanine and self-assemblies thereof by using the cyclotomic asymmetric arginine has higher anticancer activity under aerobic and anaerobic conditions, and shows higher photodynamic treatment effect. Animal experiments show that the prepared photodynamic medicament has remarkably high anti-tumor activity which is remarkably higher than that of axially symmetrical arginine modified silicon phthalocyanine, and has excellent application prospect in the field of treatment of hypoxic tumors.
(4) The PDF-L1 antibody combined with the photodynamic therapy using the perinuclear asymmetric arginine modified phthalocyanine as the photosensitizer has obvious anti-tumor metastasis effect.
(5) The peripherical asymmetric arginine modified phthalocyanine has the advantages of definite structure, simple preparation process operation, stable property, contribution to large-batch preparation in industrial production and good industrialization prospect.
Detailed Description
In order to make the present invention more comprehensible, the technical solutions of the present invention are further described below with reference to specific embodiments, but the present invention is not limited thereto.
Example 1
(1) Synthesis of 1- [4- (2-aminoethyl) phenoxy ] zinc phthalocyanine (structure shown below):
Figure DEST_PATH_IMAGE018
the synthesis was carried out by referring to the published patent methods of our topic group (CN 105585571A, published 2016-5-18) and published papers (composite beta amine-terminated phthalocyanines and resin chlorococci conjugates: synthesis, spectroscopic properties, and in vitro anticancer activity, tetrahedron, 2017, 73, 378-384).
The product was characterized as follows: 1 H NMR (400 MHz, DMSO) δ 9.35 – 9.15 (m, 5H), 8.81 (d, J = 10.9 Hz, 1H), 8.28 – 8.08 (m, 6H), 7.88 – 7.74 (m, 3H), 7.49 (d, J = 10.2 Hz, 2H), 7.41 (d, J = 8.5 Hz, 2H), 3.03 (s, 2H), 2.87 (t, 2H).
HRMS (ESI) m/z：calcd for C 40 H 25 N 9 OZn [M+H] + 712.1474, found 712.1547;calcd for C 40 H 25 N 9 OZn [M+2H] 2+ 356.5737, found 356.5811。
(2) Synthesis of 2,3-bis [4- (2-aminoethyl) phenoxy ] zinc phthalocyanine (structure shown in formula below)
Figure DEST_PATH_IMAGE020
First, a phthalonitrile derivative (structure shown below) was synthesized:
Figure DEST_PATH_IMAGE022
the reaction was stirred for 18-36 hours at 20-45 deg.C (preferably 30 deg.C) in the presence of potassium carbonate (5-15 mmol, preferably 10 mmol) and under nitrogen with 4,5-dichlorophthalonitrile (2.54 mmol), 4- (2-N-Boc-amino) ethyl-phenol (5.08 mmol) as a reactant and DMF (20-50 mL, preferably 30 mL) as a solvent, and the end point of the reaction was monitored by thin layer chromatography. After the reaction is finished, adding the reaction solution into ice water of 300 mL, separating out a large amount of reddish precipitate, filtering, washing with water until the filtrate is neutral, and drying to obtain the product with the yield of 66.7%. The characterization data of the product are as follows: 1 H NMR (400 MHz, DMSO-d 6 ) δ 7.60(m, 2H), 7.32 (d, J = 8.0 Hz, 1H), 7.26 (d, J = 8.0 Hz, 3H), 7.13 (d, J = 8.0 Hz, 1H), 7.06 (d, J =8.0 Hz, 3H), 6.87 (s, 2H), 3.16 (d, J = 8.0 Hz, 4H), 2.72 (dd, J = 20.0, 12.0 Hz, 4H), 1.37 (s, 18H)。HRMS (ESI): m/z calcd for C 34 H 38 N 4 O 6 [M + Na] + , 621.2684; found 621.2682。
further, synthesis of 2,3-bis [4- (2-N-Boc-amino) ethyl-phenoxy]Zinc phthalocyanine: the phthalonitrile derivative (0.17 mmol) and phthalonitrile (1.00 mmol) are used as reactants, n-pentanol (20-35 mL, preferably 30 mL) is used as solvent, anhydrous zinc acetate (0.30 mmol) is added, 1,8-diazabicyclo [5.4.0]Undec-7-ene (DBU, 0.4-0.8 mL, preferably 0.5 mL) is used as a catalyst, the reaction is stirred for 12-24 hours at 130-150 ℃, and the reaction end point is monitored by thin layer chromatography to generate a corresponding zinc phthalocyanine complex. And after the reaction is finished, performing rotary evaporation to dryness, dissolving the mixture by using a small amount of ethyl acetate, separating the mixture by using a silica gel column, using the ethyl acetate as an eluent, and gradually increasing the polarity of the eluent until the ethyl acetate: DMF =20 (1 (v/v), phthalocyanine band was washed and collected, organic solvent was removed by rotary evaporation, a small amount of THF was added to dissolve, and further purified by Bio-Beads S-X3 type gel column, and dried in vacuum to obtain blue solid with a yield of 40.5%. The characterization data of the product are as follows: 1 H NMR (400 MHz, DMSO-d 6 ) δ 9.28-8.99 (m, 4H), 8.98-8.70 (m, 2H), 8.62 (s, 1H), 8.38-7.99 (m, 5H), 7.60-7.22(m, 6H), 6.98 (s, 2H), 6.53 (s, 2H), 2.98-2.79 (m, 4H), 2.64(s, 2H), 2.19 (s, 2H), 1.54-1.12 (m, 18H)。HRMS (ESI): m/z calcd for C 34 H 38 N 4 O 6 [M + H] + , 1047.3279; found 1047.3311。
finally, 2,3-bis [4- (2-aminoethyl) phenoxy ] phenoxy was synthesized]Zinc phthalocyanine: the resulting 2,3-bis [4- (2-N-Boc-amino) ethyl-phenoxy]Zinc phthalocyanine (0.048 mmol) is used as a reactant, dichloromethane: trifluoroacetic acid =3:1 (v/v, 5-10 mL) is used as a solvent, the reaction is stirred for 8-24 hours at 0-30 ℃ under the protection of nitrogen, the reaction end point is monitored by thin-layer chromatography, after the reaction is finished, the reaction solvent is removed by rotary evaporation, ethyl acetate (a small amount of DMF) is added for dissolution, the mixture is purified by a silica gel column, impurities are washed away by ethyl acetate as an eluent, then a blue target product is collected by elution of DMF, the solvent is removed by rotary evaporation, and the deep blue target product is obtained by vacuum drying, wherein the yield is 49.4%. The characterization data of the product are as follows: 1 H NMR (400 MHz, DMSO-d 6 ) δ 9.46-9.20(m, 4H), 9.14 (s, 1H), 8.98-8.80 (m, 2H), 8.72 (s, 1H), 8.45-8.13 (m, 5H), 8.11-7.84 (m, 3H), 7.58-7.19 (m, 6H), 2.98 (s, 4H), 2.85 (s, 4H), 1.09 (t, J = 8.0 Hz, 4H)。HRMS (ESI): m/z calcd for C 48 H 34 N 10 O 2 Zn [M + H] + , 847.2230; found 847.2231。
example 2
Synthesis of a peripheral ring asymmetric arginine modified phthalocyanine ZnPcR1B. The structural formula of ZnPcR1B is as follows:
Figure DEST_PATH_IMAGE024
taking 1- [4- (2-aminoethyl) phenoxy ] zinc phthalocyanine (0.055 mmol) synthesized in example 1 and N-Boc-L-arginine (0.055-0.165 mmol, preferably 0.066 mmol) as raw materials, in the presence of 1-hydroxybenzotriazole (HOBt, 0.110 mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCI, 0.110 mmol) and N-methylmorpholine (NMM, 17 μ L), N-dimethylformamide (DMF, 2-6 mL) as a solvent, stirring and reacting for 10-60min at 0-10 ℃ under the protection of nitrogen, then continuing to stir and react for 7-36h at room temperature to 45 ℃, and monitoring the reaction endpoint through thin layer chromatography. After the reaction is finished, removing the reaction solvent by rotary evaporation, adding a small amount of dichloromethane for dissolution, purifying by a silica gel column, and adding dichloromethane: methanol =20 (1 v/v) as eluent, then gradually increasing the polarity of the eluent to dichloromethane: methanol =2:1 (v/v), washed and collected blue phthalocyanine band, rotary evaporated to remove organic solvent, added small amount of DMF, further refined through Bio-Beads S-X3 type gel column, vacuum dried to give dark blue product with 68.3% yield.
The characterization data of the product are as follows: 1 H NMR (400 MHz, DMSO) δ 9.50 – 9.29 (m, 3H), 8.94 (d, J = 6.2 Hz, 1H), 8.32 – 8.11 (m, 1H), 7.96 (d, J = 2.7 Hz, 4H), 7.83 – 7.69 (m, 1H), 7.66 – 7.52 (m, 2H), 7.46 (d, J = 8.0 Hz, 2H), 7.35 (d, J = 8.1 Hz, 2H), 7.28 (d, J = 8.0 Hz, 3H), 7.06 (d, J = 6.2 Hz, 2H), 7.02 – 6.91 (m, 3H), 6.90 – 6.79 (m, 1H), 3.87 (s, 1H), 3.06 (s, 2H), 2.69 (d, J = 19.2 Hz, 2H), 1.35 (d, J = 10.7 Hz, 9H), 1.23 (s, 4H), 0.94 – 0.76 (m, 2H)。HRMS (ESI) m/z：calcd for C 51 H 45 N 13 O 4 Zn [M+H] + 968.3009, found 968.3080。
example 3
The synthesis and physical and chemical properties of the zinc phthalocyanine ZnPcR1 modified by the asymmetric arginine on the peripheral ring are as follows:
Figure DEST_PATH_IMAGE026
the product ZnPcR1B (0.038 mmol) obtained in example 2 is used as a reactant, dichloromethane: trifluoroacetic acid =3:1 (v/v, 40-300 mL, preferably 70 mL) is used as a solvent, the reaction is stirred for 10-60min at 0-10 ℃ under the protection of nitrogen, then the reaction is continuously stirred to 7 36h at room temperature to 45 ℃, and the reaction end point is monitored by thin layer chromatography. And after the reaction is finished, removing the reaction solvent by rotary evaporation, adding a methanol solvent for dissolving, removing the solvent and residual trifluoroacetic acid by rotary evaporation, adding a small amount of DMF, purifying by a Bio-Beads S-X3 type gel column, collecting a blue phthalocyanine band, removing the DMF by rotary evaporation, and drying in vacuum to obtain a dark blue product with the yield of 27.1%.
The characterization data of the product are as follows: 1 H NMR (400 MHz, DMSO) δ 9.55 – 9.23 (m, 6H), 8.89 (d, J = 7.2 Hz, 1H), 8.54 (s, 2H), 8.39 – 8.09 (m, 7H), 7.81 (d, J = 7.8 Hz, 2H), 7.48 (d, J = 8.0 Hz, 2H), 7.39 (d, J = 8.2 Hz, 2H), 7.31 (d, J = 6.3 Hz, 1H), 7.17 – 7.03 (m, 2H), 3.65 (s, 1H), 3.11 (s, 2H), 2.77 (d, J = 6.5 Hz, 2H), 1.65 (d, J = 5.6 Hz, 2H), 1.53 – 1.33 (m, 4H)。HRMS (ESI) m/z：calcd for C 46 H 37 N 13 O 2 Zn [M+H] + 867.2658.2485, found 868.2557。
example 4
The synthesis and physical and chemical properties of the zinc phthalocyanine ZnPcR2 modified by the asymmetric arginine on the peripheral ring are as follows:
Figure DEST_PATH_IMAGE028
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1- [4- (2-aminoethyl) phenoxy ] zinc phthalocyanine (0.055 mmol) and N-Boc-L-arginine (0.055-0.165 mmol, preferably 0.066 mmol) synthesized in example 1 were used as starting materials, DMF (2-5 mL) was used as a solvent in the presence of 1-hydroxybenzotriazole (HOBt, 0.110 mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCI, 0.110 mmol) and N, N-diisopropylethylamine (20. Mu.L), and the reaction was stirred at 0-30 ℃ for 12-36 hours under nitrogen protection, and the end point of the reaction was monitored by thin layer chromatography. And after the reaction is finished, removing the reaction solvent by rotary evaporation, adding a small amount of DMF, purifying by using a Bio-Beads S-X3 type gel column, collecting the blue phthalocyanine band, carrying out rotary evaporation and concentration to a small amount, further refining by using a Bio-Beads S-X1 type gel column, and carrying out vacuum drying to obtain a dark blue product, wherein the yield is 21.2%.
The characterization data of the product are as follows: 1 H NMR (400 MHz, DMSO) δ 9.50-9.30 (m, 4H), 8.95 (d, J = 8.0 Hz, 1H), 8.39-8.09 (m, 6H), 7.96 (s, 1H), 7.90 (d, J = 8.0 Hz, 1H), 7.85-7.71 (m, 1H), 7.57 (s, 2H), 7.42 (dd, J = 36.0, 8.0 Hz, 4H), 7.32-6.61 (m, 8H), 4.22 (s, 1H), 3.93 (s, 1H), 3.08 (s, 4H), 2.90 (s, 2H), 2.74 (s, 2H), 2.53 (s, 2H), 2.34 (s, 2H), 2.04 (s, 1H), 1.64 (s, 2H), 1.48 (s, 4H), 1.42-1.05 (m, 9H)。HRMS (ESI): m/z calcd for C 57 H 57 N 17 O 5 Zn [M+H] + ,1124.4138, found,1124.4093。
example 5
The synthesis and physical and chemical properties of the zinc phthalocyanine ZnPcR3 modified by the asymmetric arginine on the peripheral ring are as follows:
Figure DEST_PATH_IMAGE030
1- [4- (2-aminoethyl) phenoxy ] zinc phthalocyanine (0.030 mmol) and N-Boc-L-arginine (0.030-0.090 mmol, preferably 0.060 mmol) synthesized in example 1 were used as raw materials, and in the presence of HOBt (0.060 mmol), EDCI (0.060 mmol) and N, N-diisopropylethylamine (20. Mu.L), DMF (8-15 mL) was used as a solvent, and the reaction was stirred at 0-10 ℃ for 10-60min under nitrogen protection, and then the reaction was further stirred at room temperature to 45 ℃ for 7-36h, and the end point of the reaction was monitored by thin layer chromatography. After the reaction is finished, the reaction solvent is removed by rotary evaporation. Adding a small amount of DMF, passing through a Bio-Beads S-X3 type DMF gel column, collecting blue phthalocyanine band, concentrating to a small amount by rotary evaporation, refining by a Bio-Beads S-X1 type DMF gel column, and vacuum drying to obtain a dark blue product with a yield of 22.4%.
The characterization data of the product are as follows: 1 H NMR (400 MHz, DMSO-d6) δ 9.46 (s, 3H), 9.07 – 8.88 (m, 2H), 8.29 (s, 3H), 8.24 – 8.11 (m, 2H), 7.97 (s, 2H), 7.88 – 7.71 (m, 2H), 7.52 – 7.36 (m, 3H), 7.20 (s, 7H), 6.99 – 6.79 (m, 3H), 4.25 – 3.91 (m, 6H), 3.09 (s, 8H), 2.13 (s, 3H), 1.79 – 1.65 (m, 3H), 1.55 (s, 5H), 1.43 – 1.18 (m, 9H)。HRMS (ESI): m/z calcd for C 63 H 69 N 21 O 6 Zn [M+H]+,1280.5104, found, 1280.5139。
example 6
The synthesis and physical and chemical properties of the zinc phthalocyanine ZnPcR2B modified by the asymmetric arginine on the peripheral ring are as follows:
Figure DEST_PATH_IMAGE032
2,3-bis [4- (2-aminoethyl) phenoxy ] zinc phthalocyanine (0.020 mmol) and N-Boc-L-arginine (0.040-0.120 mmol, preferably 060 mmol) synthesized in example 1 were used as raw materials, and in the presence of HOBt (0.06 mmol), EDCI (0.06 mmol) and DIPEA (16 μ L), DMF (8-15 mL) was used as a solvent, and the reaction was stirred at 0-10 ℃ for 10-60min, then the reaction was continued to be stirred at room temperature to 45 ℃ for 7-36h, and the end point of the reaction was monitored by thin layer chromatography. And after the reaction is finished, removing the reaction solvent by rotary evaporation. Adding a small amount of DMF, passing through a Bio-Beads S-X3 type gel column, collecting blue phthalocyanine band, concentrating to a small amount by rotary evaporation, further refining by a Bio-Beads S-X1 type gel column, and vacuum drying to obtain a dark blue product with the yield of 31.2%.
The characterization data of the product are as follows: 1 H NMR (400 MHz, DMSO) δ 9.45 (s, 3H), 9.36 (s, 3H), 8.99 (s, 2H), 8.27 (s, 7H), 8.10-7.94 (m, 5H), 7.40 (s, 6H), 7.33 (s, 5H), 6.92-6.83 (m, 3H), 6.58-6.51 (m, 2H), 5.41 (s, 2H), 4.02-3.95 (m, 2H), 3.09 (s, 4H), 2.87-2.78 (m, 4H), 2.38-2.33 (m, 4H), 1.72-1.61 (m, 4H), 1.49 (s, 8H), 1.36 (s, 10H)。HRMS (ESI): m/z calcd for C 70 H 74 N 18 O 8 Zn [M + 2H] 2+ , 680.2687; found 680.2654。
example 7
The synthesis and physical and chemical properties of the zinc phthalocyanine ZnPcR4 modified by the asymmetric arginine on the peripheral ring are as follows:
Figure DEST_PATH_IMAGE034
2,3-bis [4- (2-aminoethyl) phenoxy ] zinc phthalocyanine (0.05 mmol) and N-Boc-L-arginine (0.10-0.30 mmol, preferably 0.150 mmol) synthesized in example 1 were used as raw materials, and in the presence of HOBt (0.20 mmol), EDCI (0.20 mmol) and DIPEA (20. Mu.L), DMF (8-15 mL) was used as a solvent, and the reaction was stirred at 0-10 ℃ for 10-60min under nitrogen protection, and then the reaction was further stirred at room temperature to 45 ℃ for 7-24h, and the end point of the reaction was monitored by thin layer chromatography. And after the reaction is finished, removing the reaction solvent by rotary evaporation. Adding a small amount of DMF, passing through a Bio-Beads S-X3 type gel column, collecting blue phthalocyanine band, concentrating by rotary evaporation to a small amount, further refining by a Bio-Beads S-X1 type gel column, and vacuum drying to obtain a dark blue product with the yield of 19.7%.
The characterization data of the product are as follows: 1H NMR (400 MHz, DMSO-d 6) δ 9.53-9.35 (m, 5H), 8.98 (s, 2H), 8.29 (s, 3H), 8.16 (s, 4H), 7.85 (s, 4H), 7.48 (s, 4H), 7.38 (s, 2H), 7.26-7.05 (m, 5H), 7.04-6.93 (m, 3H), 6.93-6.63 (m, 7H), 6.54 (s, 1H), 4.23 (s, 4H), 4.01-3.84 (m, 4H), 3.08 (s, 8H), 2.69 (s, 8H), 2.35 (s, 8H), 1.42 (d, J = 36.0 Hz, 18H). HRMS (ESI) M/z calcd for C70H74N18O8Zn [ M +2H ] (2 +, 836.3698. Found, 836.3717).
Example 8
Synthesis of pericyclic asymmetric histidine modified zinc phthalocyanine (ZnPcH 1B, structure shown in formula below)
Figure DEST_PATH_IMAGE036
Using N-Boc-L-histidine instead of N-Boc-L-arginine in example 2, the pericyclic asymmetric histidine-modified zinc phthalocyanine as shown in the above formula was obtained with reference to the synthesis and purification method of example 2.
The characterization data of the product are as follows:
1H NMR (400 MHz, DMSO) δ 9.49 – 9.28 (m, 3H), 8.95 (d, J = 7.0 Hz, 1H), 8.37 – 8.14 (m, 4H), 7.91 – 7.78 (m, 2H), 7.57 (d, J = 6.8 Hz, 1 H), 7.45 (d, J = 7.8 Hz, 1H), 7.36 (d, J = 8.1 Hz, 1H), 7.30 (d, J = 8.1 Hz, 1H), 7.07 (d, J = 8.0 Hz, 1H), 6.73 (s, 2H), 3.82 (s, 1H), 2.89 (d, J = 9.6 Hz, 2H), 2.77 – 2.63 (m, 2H), 1.60 – 1.29 (m, 13H), 1.26 (d, J = 11.1 Hz, 9H), 0.94 – 0.76 (m, 4H)。HRMS (ESI) m/z：calcd for C56H54N11O6Zn [M+H-CH3]+ 1040.3550, found 1040.3518。
example 9
Synthesis of pericyclic asymmetric histidine modified zinc phthalocyanine (ZnPcH 1, structure shown in formula below)
Figure DEST_PATH_IMAGE038
Referring to example 3, the objective product can be obtained by using the peripherical asymmetric histidine-modified zinc phthalocyanine ZnPcH1B obtained in example 8 instead of ZnPcR1B in example 3.
The characterization data of the product are as follows:
1 H NMR (400 MHz, DMSO) δ 9.55 – 9.23 (m, 6H), 8.89 (d, J = 7.2 Hz, 1H), 8.54 (s, 2H), 8.39 – 8.09 (m, 7H), δ 7.96 (s, 1H),7.81 (d, J = 7.8 Hz, 2H), 7.48 (d, J = 8.0 Hz, 2H), 7.39 (d, J = 8.2 Hz, 2H), 7.31 (d, J = 6.3 Hz, 1H), 7.17 – 7.03 (m, 2H), 3.65 (s, 1H), 3.11 (s, 2H), 2.77 (d, J = 6.5 Hz, 2H), 1.65 (d, J = 5.6 Hz, 2H), 1.53 – 1.33 (m, 4H)。HRMS (ESI) m/z：calcd for C 41 H 40 N 11 O 2 Zn [M+H] + 855.2658, found 855.2073。
example 10
Synthesis of pericyclic asymmetric lysine modified zinc phthalocyanine (ZnPcK 1B, structure shown as the following formula)
Figure DEST_PATH_IMAGE040
Using N2, N6-di-Boc-L-lysine instead of N-Boc-L-arginine in example 2, the synthesis and purification procedure of example 2 was followed to obtain pericyclic asymmetric histidine-modified zinc phthalocyanine as shown in the above formula.
The product was characterized as follows:
1 H NMR (400 MHz, DMSO) δ 9.49 – 9.28 (m, 3H), 8.95 (d, J = 7.0 Hz, 1H), 8.37 – 8.14 (m, 4H), 7.91 – 7.78 (m, 2H), 7.57 (d, J = 6.8 Hz, 1 H), 7.45 (d, J = 7.8 Hz, 1H), 7.36 (d, J = 8.1 Hz, 1H), 7.30 (d, J = 8.1 Hz, 1H), 7.07 (d, J = 8.0 Hz, 1H), 6.73 (s, 2H), 3.82 (s, 1H), 2.89 (d, J = 9.6 Hz, 2H), 2.77 – 2.63 (m, 2H), 1.60 – 1.29 (m, 13H), 1.26 (d, J = 11.1 Hz, 9H), 0.94 – 0.76 (m, 4H)。HRMS (ESI) m/z：calcd for C 56 H 54 N 11 O 6 Zn [M+H-CH 3 ] + 1040.3550, found 1040.3518。
example 11
Synthesis of pericyclic asymmetric lysine modified zinc phthalocyanine (ZnPcK 1, structure shown as the following formula)
Figure DEST_PATH_IMAGE042
Referring to example 3, the objective product can be obtained by using the peripherical asymmetric histidine-modified zinc phthalocyanine ZnPcK1B obtained in example 9 instead of ZnPcR1B in example 3. The characterization data of the product are as follows:
1 H NMR (400 MHz, DMSO) δ 9.55 – 9.23 (m, 6H), 8.89 (d, J = 7.2 Hz, 1H), 8.54 (s, 2H), 8.39 – 8.09 (m, 7H), δ 7.96 (s, 1H),7.81 (d, J = 7.8 Hz, 2H), 7.48 (d, J = 8.0 Hz, 2H), 7.39 (d, J = 8.2 Hz, 2H), 7.31 (d, J = 6.3 Hz, 1H), 7.17 – 7.03 (m, 2H), 3.65 (s, 1H), 3.11 (s, 2H), 2.77 (d, J = 6.5 Hz, 2H), 1.65 (d, J = 5.6 Hz, 2H), 1.53 – 1.33 (m, 4H)。HRMS (ESI) m/z：calcd for C 41 H 40 N 11 O 2 Zn [M+H] + 855.2658, found 855.2073。
example 12
Synthesis of axial arginine modified silicon phthalocyanine: the following axial Arginine-modified Silicon phthalocyanines (structures shown below) were synthesized by reference to our published papers (synthetic, spectroscopic Properties, and Photocurable amide Activities of Novel Arginine-modified Silicon (IV) phthalocyanines, chinese J. Structure. Chem., 2020, 39 (1), 66-78):
Figure DEST_PATH_IMAGE044
Figure DEST_PATH_IMAGE046
.。
example 13
The phthalocyanines synthesized in examples 2-12 were tested for their electron absorption spectra (concentration 4. Mu.M) in aqueous solutions of DMF, water and castor oil derivative (Cremophor EL). The results show that in DMF, all phthalocyanine compounds show strong and sharp Q band absorption peaks, and the molar absorption coefficient can reach 1.0-2.0 multiplied by 10 5 cm -1 mol -1 L, where the absorption maximum of the zinc phthalocyanines is located between 676 and 672nm and the absorption maximum of the silicon phthalocyanines is located between 684 and 686nm, indicates that these phthalocyanines are present predominantly in monomeric form in DMF. However, in water, different phthalocyanine compounds show different absorption spectra, and the zinc phthalocyanine described in examples 2 to 11 exhibits a weak and broadened Q band absorption in water with an absorption intensity of 1/5 to 1/4 of that in DMF, indicating that these phthalocyanine compounds form aggregates in water; the silicon phthalocyanines described in example 12 still exhibit strong and sharp Q-band absorption in water, comparable to that in DMF, indicating that these silicon phthalocyanines are also present predominantly in monomeric form in water. In a 1% aqueous castor oil derivative solution (Cremophor EL, wt%), both the zinc phthalocyanine and silicon phthalocyanine described in examples 2-12 exhibited strong and sharp Q-band absorption with comparable absorption intensity as in DMF, indicating that the 1% aqueous castor oil derivative solution was present in monomeric form.
Example 14
The particle size distribution of the phthalocyanine compounds prepared in examples 2 to 12 in water and in a 1% aqueous solution of castor oil derivatives was determined using a particle size distribution analyzer, and the detailed experimental procedures were carried out with reference to j. Mater. Chem. B, 2021,9, 2845.
The test results show that the zinc phthalocyanine (4 mu M) prepared in the examples 2 to 11 can be self-assembled in water to form nanoparticles with the particle size of about 100 to 250nm, while the silicon phthalocyanine in the example 12 has no self-assembly behavior in water, and no nanoparticles can be observed. None of the phthalocyanines prepared in examples 2-12 were observed as distinct nanoparticles in a 1% aqueous solution of the castor oil derivative (1% cel), indicating that in the presence of 1% castor oil derivative, the self-assembly of the phthalocyanines prepared in examples 2-12 in an aqueous solution is prevented, mainly in the form of monomers.
Example 15
The phthalocyanine compounds prepared in examples 2-12 were tested for their ability to photolytically generate Reactive Oxygen Species (ROS) in water or in a 1% CEL solution.
Total active oxygen assay hydrolyzed 2,7-dichlorofluorescein diacetate (DCFH-DA) (i.e., 2,7-dichlorofluorescein protein acetate) was used as the fluorescent probe. DCFH was able to be activated by oxygen to form dichlorofluorescein that emitted a fluorescent signal at 520 nm. The preparation method of 2,7-dichlorofluorescein protein acetate (DCFH) comprises the following steps: 2,7-dichlorofluorescein diacetate (DCFH-DA) was dissolved in methanol to make a DCFH-DA solution of 5mM, stored at-20 ℃. In the detection, after mixing DCFH-DA solution with 0.1 mol/L sodium hydroxide solution and reacting for 30 min in a dark place, diluting the mixture with PBS solution with pH 7.4 to obtain mother liquor with the concentration of 200 mM.
A mixed deionized water solution of phthalocyanine (4. Mu.M aqueous solution or 1% CEL solution) in a total amount of 2 mL and the activated active oxygen probe (5. Mu.M) was placed in a quartz cuvette, and red light (15 mW/cm) of 610 or more and nm was used 2 ) The cuvette was irradiated and the change in fluorescence intensity of the oxygen probe was measured at different illumination times (488 nm excitation, scan range 500 nm-600 nm fluorescence). Plotting the relative fluorescence intensity at 522 nm (Ft-F0/F0) against the illumination time (T) yields a slope of the linear relationship, with a larger slope indicating a greater ability to generate ROS. The relative ROS-generating ability of the test phthalocyanine was obtained by dividing the slope of the experimental group by the slope of the control group, using the common photosensitizer methylene blue (MB,) as the control group (methylene blue has no significant difference in its ability to generate ROS in water and 1% CEL).
The red light with the wavelength being equal to or more than 610nm is provided by connecting a halogen lamp of 500W with a heat-insulating water tank and a filter with the wavelength being more than 610 nm.
TABLE 1 comparison of relative capacities of different samples to generate reactive oxygen species
Figure DEST_PATH_IMAGE048
The results are shown in Table 1. As can be seen from the table, in water, the ability of peripheral asymmetric arginine-modified zinc phthalocyanine (phthalocyanine obtained in examples 2-7) to generate ROS is significantly higher than that of peripheral asymmetric histidine or lysine-modified zinc phthalocyanine (phthalocyanine obtained in examples 8-11) and control methylene blue, and also higher than that of axial arginine-modified silicon phthalocyanine (phthalocyanine obtained in example 12); 1% CEL, ROS production by the peri-asymmetric arginine-modified zinc phthalocyanine (example 2-7 phthalocyanine) and the peri-asymmetric histidine or lysine-modified zinc phthalocyanine (example 8-11 phthalocyanine) was inferior to that of the control methylene blue; the ability of pericyclic asymmetric arginine-modified zinc phthalocyanine (phthalocyanine obtained in examples 2-7) to generate ROS in water is significantly higher than that (6-46 times higher) in 1%CEL, and pericyclic asymmetric lysine-modified zinc phthalocyanine (phthalocyanine obtained in examples 10-11) to generate ROS in water is higher than that in 1%CEL, but this is not the case for pericyclic asymmetric histidine-zinc phthalocyanine and axial arginine-modified silicon phthalocyanine. As described in examples 13-14, peri-cyclic asymmetric arginine-modified zinc phthalocyanine (phthalocyanine obtained in examples 2-7) and peri-cyclic asymmetric histidine or lysine-modified zinc phthalocyanine (phthalocyanine obtained in examples 8-11) present in water as aggregates and in monomeric form in 1% cel, it can be seen that peri-cyclic asymmetric arginine-modified zinc phthalocyanine exhibits a unique and significant ability to enhance aggregation to generate ROS, which is significantly different from axial arginine-modified silicon phthalocyanine and peri-cyclic asymmetric histidine or lysine-modified zinc phthalocyanine, which are unique properties that other common phthalocyanine photosensitizers do not have.
Example 16
Determination of photo-sensitive production of superoxide anion (O) in Water of the peripherical asymmetric arginine-modified Zinc phthalocyanine prepared in examples 2-7 2 ·- ) The ability of the cell to perform. The assay was performed using ethidium Dihydrogenate (DHE) as a fluorescent probe for detection of superoxide anion. In the presence of superoxide anion, DHE is oxidized to ethidium bromide, which, after binding to surrounding DNA, emits bright red fluorescence (excitation wavelength around 500-510 nm, emission wavelength around 600 nm). The stronger the fluorescence, the more the amount of superoxide anion produced. The method comprises the following specific steps: to deionized water were added the phthalocyanine compound to be measured (phthalocyanine concentration 4. Mu.M), DHE probe (25. Mu.M), and calf thymus DNA (250. Mu.g/mL), and mixed well. The fluorescence intensity at the beginning was measured, and then red light having a wavelength of 610nm or more (light density of 1 mW/cm) was used 2 ) The generation of superoxide anion was judged by illuminating for 25min and recording the change in fluorescence intensity of the probe using a fluorescence gradiometer. The stronger the fluorescence intensity, the greater the superoxide anion generation ability. Common photosensitizer Methylene Blue (MB) is used as a control group, and the increase of the fluorescence intensity of the probe of the experimental group (the increase before and after illumination) is divided by the increase of the fluorescence intensity of the probe of the control group MB (the increase before and after illumination), so that the relative superoxide anion generating capability of the phthalocyanine to be tested is obtained.
TABLE 2 comparison of relative capacities of different samples for photosensitization of superoxide anions
Figure DEST_PATH_IMAGE050
The experimental result is shown in table 2, and it can be seen from table 2 that the peripherical asymmetric arginine modified zinc phthalocyanine can effectively generate superoxide anions in water, and the generation capacity is 3-16 times higher than that of methylene blue which is a common photosensitizer. Photosensitization produced superoxide anion, suggesting that cyclotomic asymmetric arginine-modified zinc phthalocyanine can generate reactive oxygen species via type I photosensitization mechanism.
Under the same test conditions, the production of superoxide anion was not clearly observed in water for the pericyclic asymmetric histidine or lysine-modified zinc phthalocyanine (phthalocyanine obtained in examples 8-11) and the axial arginine-modified silicon phthalocyanine (phthalocyanine obtained in example 12).
Example 17
The ability of the peripherical asymmetric arginine-modified zinc phthalocyanine prepared in examples 2-7 to generate active oxygen in water by photosensitization under anoxic conditions was tested.
Before the test, the used deionized water is boiled for 20 min and subjected to ultrasonic treatment to remove oxygen, then a large amount of nitrogen is introduced to prevent oxygen from entering, and a Shanghai Lei Ci JPB607A portable dissolved oxygen tester is used for detecting the oxygen content so as to check the oxygen deficiency state of the water (the oxygen content is not higher than 2.5 mg/L). Deoxygenated water prepared in advance was selected, a sample to be measured and a probe were added, and the other steps were performed as described in reference example 15. The test results are shown in Table 3.
TABLE 3 comparison of the relative ability of different samples to photosensitize ROS production under hypoxic conditions
Figure DEST_PATH_IMAGE052
It can be seen that the above-mentioned peripherical asymmetric arginine modified zinc phthalocyanine (phthalocyanine compound obtained in examples 2-7) can effectively generate active oxygen under the condition of oxygen deficiency, and particularly the phthalocyanine obtained in examples 2-4 still has high active oxygen generating capability, which is very beneficial for the photodynamic therapy of hypoxic solid tumor.
Example 18
The method for preparing the photosensitizer or the phototherapy medicament by utilizing the phthalocyanine comprises the following steps: firstly, dissolving a phthalocyanine compound by using DMF (dimethyl formamide), DMSO (dimethyl sulfoxide) or ethanol to prepare a mother solution of 1-2 mM, and then diluting the mother solution by using water to prepare a medicinal aqueous solution with a certain concentration.
The phthalocyanine photosensitizer or the 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 common light source with a proper light filter or by laser with a specific wavelength or LED light with a corresponding wavelength range, and the wavelength range of the light source is 600 to 800nm, preferably 620 to 700 nm.
Example 19
Partial circumambient asymmetric arginine modified zinc phthalocyanine is tested for photodynamic inhibition of cancer cell growth activity under normoxic and hypoxic conditions.Dissolving the phthalocyanine compound to be detected in DMF to prepare a mother solution of 1 mM, and further diluting the mother solution into a cell culture solution to prepare the cell culture solution containing the phthalocyanines with different concentrations. HepG2 cells in logarithmic growth phase (approximately 1X 10 cells per plate) were seeded in culture plates on which clean sterile coverslips were placed 5 Respectively) at 37 deg.C, 95% air, 5% CO 2 (normoxic condition) or 3% O 2 、5% CO 2 And 92% N 2 (hypoxic condition), 24h, then the culture medium is removed, cancer cells are cultured in culture medium containing phthalocyanine of different concentrations for 2 hours, then the culture medium is discarded, the cells are washed with PBS buffer solution, and then new culture medium (containing no phthalocyanine) is added. The light experiment group irradiates cells with red light (the exciting light source is red light with wavelength of more than 610nm, the irradiation time is 30 min, and the irradiation power is 15mw cm -2 ) (ii) a The group was left unlit and the cells were left in the dark for 20 minutes. After the cells were exposed to light or not, the viability of the cells was examined by the MTT method. The specific experimental procedures are described in Bioorganic& Medicinal Chemistry Letters》，2006，16，2450-2453。
The experimental results show that: the peripherical asymmetric arginine-modified zinc phthalocyanine obtained in examples 2-7 had no killing and growth inhibitory effect on HepG2 cells in the absence of light, indicating that the phthalocyanine had no dark toxicity. However, after light irradiation, no matter under the condition of normal oxygen or hypoxia, the prepared peripherical asymmetric arginine modified zinc phthalocyanine shows high photodynamic anticancer activity and presents dose-effect relationship, and corresponding IC 50 The values (half-lethal concentration, i.e., the concentration of drug required to kill 50% of the cancer cells) are shown in table 4. In particular, the zinc phthalocyanine asymmetrically modified by the cyclol di-arginine prepared in the example 4 has the best photodynamic anticancer activity, and IC is IC under the hypoxic condition 50 The value was still as low as 0.47. Mu.M. In contrast, the aqueous axial arginine-modified silicon phthalocyanine solution described in example 12 did not exhibit significant photodynamic anti-cancer activity under hypoxic conditions.
TABLE 4 IC of different samples under normoxic and hypoxic light conditions 50 Value comparison
Figure DEST_PATH_IMAGE054
Example 20
The photodynamic anti-tumor effect of the phthalocyanine compound obtained by the invention is tested. KM mice with subcutaneously transplanted H22 hepatoma cells were established by the literature reference method (ACS appl. Mater. Interfaces 2019, 11, 36435-36443). H22 tumor-bearing KM mice were divided into the following experimental groups: the single dosing group, the single PBS group, the single laser group, the dosing + laser group, 5 per group. The administration and the laser irradiation are carried out until the tumor grows to 60-100 mm 3 In a small scale, 100. Mu.L of phthalocyanine aqueous solution with a concentration of 100. Mu.M (dose of 0.5 nmol/g) was intravenously injected, and the tumor site was irradiated with 685nm laser at an illumination intensity of 15mW/cm for 8 minutes 8 hours after the administration 2 The light group was illuminated once more on day 4 after the first illumination. After each group of mice are treated according to requirements, the mice are continuously raised, the conditions of the mice are 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 (ACS appl. Mater. Interfaces 2019, 11, 36435-36443).
The experimental results show that: (1) The tumor growth inhibition effect of the mouse tumor is not generated in the single administration group and the single laser group (the tumor is increased by about 14 times). (2) The body weight of the mice of the single administration group and the administration and laser group is increased within 14 days, which shows that the measured phthalocyanine has no obvious toxicity to the mice and has good biocompatibility. (3) The peripherical asymmetric arginine modified zinc phthalocyanine (ZnPcNR 2) + laser group obtained in example 4 shows very high tumor growth inhibition effect, and the tumor inhibition rate is as high as 100% (tumor growth is completely inhibited). Under the same conditions, the axial arginine modified silicon phthalocyanine (SiPcR 1B or SiPcR 2B) obtained in example 11 and the laser group do not show obvious capacity of inhibiting tumor growth, and the tumor inhibition rate is less than 35%. Therefore, the peripherical asymmetric arginine modified zinc phthalocyanine provided by the invention has remarkably high photodynamic anti-solid tumor activity, and particularly has the stress that the medicament amount required by ZnPcNR2 for obtaining 100% tumor inhibition rate is only 0.5 nmol/g, thereby showing good clinical application prospect.
Example 21
A bilateral tumor-bearing mouse model (a near end and a far end) of melanoma cells B16-F10 is established, and the anti-tumor effect of the combination of photodynamic therapy using the peripherical asymmetric arginine modified zinc phthalocyanine ZnPcNR2 provided by the invention as a photosensitizer and immunotherapy based on a PD-L1 antibody is tested. Proximal refers to the illuminated side of photodynamic therapy (PDT) and distal is the non-illuminated side. The following 5 sets of experiments were set up, 5 per set: a PBS control group; anti-PD-L1 antibody treatment group; the phthalocyanine only (no light) treatment group; phthalocyanine + light treatment group, i.e. PDT treatment group; phthalocyanine + light + anti-PD-L1 antibody treatment group, i.e., combination treatment group. Among them, anti-PD-L1 antibody was purchased from BioX cell.
The combination treatment group, photodynamic treatment group and photosensitizer only group were injected with 100. Mu.L of phthalocyanine compound (concentration 200. Mu.M) per mouse tail vein. The combination treatment group and the photodynamic treatment group are irradiated by laser with the wavelength of 685nm (the irradiation power is 15 mW/cm) after 8 hours after the administration 2 5min exposure time) right (i.e., proximal) tumor. The combined treatment group was subjected to intraperitoneal injection of 50. Mu.g of PD-L1 antibody per mouse immediately after laser treatment, and the antibody treatment group was subjected to intraperitoneal injection of 50. Mu.g of PD-L1 antibody per mouse at the same time. Treatment was performed once on day one and day four, respectively.
The body weights and tumor volumes of all mice were measured every other day from the start of the first treatment, and the tumor sizes were calculated according to the formula tumor volume = tumor length × width × height × pi/6, and the tumor inhibition rates of the respective experimental groups were calculated by continuous observation for 14 days.
The experimental results show that the peripherical asymmetric arginine-modified zinc phthalocyanine (ZnPcNR 2) described in example 4 has a tumor inhibition rate of 92.8% for proximal tumors (with light-irradiated tumors) and almost no inhibition effect (tumor inhibition rate of 5.6%) for distal tumors (without light-irradiated tumors) in PDT treatment groups (phthalocyanine + light-irradiated treatment groups), indicating that tumor metastasis and metastasis cannot be inhibited by solely using phthalocyanine photodynamic therapy. On the other hand, the inhibition effect of pure PD-L1 antibody treatment on proximal and distal tumors is very limited, and the tumor inhibition rate is 12% and 13%, respectively. For the combination treatment group, the zinc phthalocyanine (ZnPcNR 2) + light + anti-PD-L1 antibody treatment group described in example 4 showed a tumor inhibition rate of up to 99.4% for distal tumors (non-light tumors) and up to 99.9% for proximal tumors (light tumors) in B16-F10 tumor-bearing mice. This demonstrates that the photodynamic therapy with zinc phthalocyanine described in example 4 in combination with PD-L1 antibody has a significant anti-tumor metastatic effect.
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.

Claims (6)

1. A peri-asymmetric arginine-modified phthalocyanine characterized by: the phthalocyanine ring substituent contains arginine, and the structural formula is as follows:
Figure 753582DEST_PATH_IMAGE001
(I) Is represented by ZnPcR1B or
Figure 139564DEST_PATH_IMAGE002
(II) is recorded as ZnPcR1;
the preparation method of the peripherical asymmetric arginine modified phthalocyanine comprises the following steps:
the preparation method of the compound (I) comprises the following steps: taking 1- [4- (2-aminoethyl) phenoxy ] zinc phthalocyanine and N-Boc-L-arginine as reactants, taking N, N-Dimethylformamide (DMF) as a solvent, stirring and reacting for 10-60min at the temperature of 0-10 ℃ in the presence of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI), 1-hydroxybenzotriazole (HOBt) and N-methylmorpholine (NMM) and under the protection of nitrogen, then continuing stirring and reacting for 7-24h at the temperature of room temperature-45 ℃, and separating by column chromatography and molecular exclusion chromatography to obtain the pericyclic arginine substituted zinc phthalocyanine ZnPcR1B;
the preparation method of the compound (II) comprises the following steps: dispersing ZnPcR1B into a mixed solution of dichloromethane and trifluoroacetic acid, stirring and reacting for 10-60min at 0-10 ℃ under the protection of nitrogen, then continuously stirring and reacting for 7-24h at room temperature-45 ℃, and separating by using size exclusion chromatography to obtain the pericyclic arginine substituted zinc phthalocyanine ZnPcR1;
wherein the molar ratio of the 1- [4- (2-aminoethyl) phenoxy ] zinc phthalocyanine to the N-Boc-L-arginine is 1 to 3;
1-Ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride (EDCI), 1-hydroxybenzotriazole (HOBt) in an amount of 1.5-6mmol per mmol of 1- [4- (2-aminoethyl) phenoxy ] zinc phthalocyanine; the dosage of N-methylmorpholine (NMM) is 0.2-1mL per mmol of 1- [4- (2-aminoethyl) phenoxy ] zinc phthalocyanine;
the volume ratio of trifluoroacetic acid to dichloromethane is 1 to 5; a mixture of trifluoroacetic acid and methylene chloride was used in an amount of 1 to 10mL per mg of 1- [4- (2-aminoethyl) phenoxy ] zinc phthalocyanine.
2. A peri-asymmetric arginine-modified phthalocyanine characterized by: the phthalocyanine ring substituent contains two arginines, and the structural formula is as follows:
Figure 184881DEST_PATH_IMAGE003
(III) is expressed as ZnPcR2 or
Figure 48931DEST_PATH_IMAGE004
(IV) is expressed as ZnPcR2B;
the preparation method of the phthalocyanine with the asymmetric arginine modification in the peripheral ring comprises the following steps:
the preparation method of the compound (III) comprises the following steps: taking 1- [4- (2-aminoethyl) phenoxy ] zinc phthalocyanine and N-Boc-L-arginine as reactants, taking N, N-Dimethylformamide (DMF) as a solvent, stirring and reacting for 10-60min at 0-10 ℃ in the presence of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI), 1-hydroxybenzotriazole (HOBt) and N, N-Diisopropylethylamine (DIPEA) under the protection of nitrogen, then continuously stirring and reacting for 7-24h at room temperature-45 ℃, and separating by column chromatography and molecular exclusion chromatography to obtain the pericyclic arginine-substituted zinc phthalocyanine ZnPcR2;
the preparation method of the compound (IV) comprises the following steps: using 2,3- [4- (2-aminoethyl) phenoxy ] zinc phthalocyanine and N-Boc-L-arginine as reactants, using N, N-Dimethylformamide (DMF) as a solvent, stirring and reacting for 10-60min at 0-10 ℃ in the presence of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI), 1-hydroxybenzotriazole (HOBt) and N, N-Diisopropylethylamine (DIPEA) under the protection of nitrogen, then continuously stirring and reacting for 7-24h at room temperature to 45 ℃, and separating by column chromatography and molecular exclusion chromatography to obtain the zinc phthalocyanine ZnPcR2B substituted by pericyclic arginine;
wherein the molar ratio of the 1- [4- (2-aminoethyl) phenoxy ] zinc phthalocyanine to the N-Boc-L-arginine is 1 to 3;
5363 a molar ratio of 2,3- [4- (2-aminoethyl) phenoxy ] zinc phthalocyanine to N-Boc-L-arginine of 1;
the 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) and 1-hydroxybenzotriazole (HOBt) are used in amounts of 1.5 to 6mmol per mmol of 1- [4- (2-aminoethyl) phenoxy ] zinc phthalocyanine or 2,3- [4- (2-aminoethyl) phenoxy ] zinc phthalocyanine; the dosage of the N, N-Diisopropylethylamine (DIPEA) is 0.2 to 1mL per mmol of 1- [4- (2-aminoethyl) phenoxy ] zinc phthalocyanine or 2,3- [4- (2-aminoethyl) phenoxy ] zinc phthalocyanine.
3. A peri-asymmetric arginine-modified phthalocyanine characterized by: the phthalocyanine ring substituent contains three or four arginines, and the structural formula is as follows:
Figure 584342DEST_PATH_IMAGE005
(V) is ZnPcR3 or
Figure 610067DEST_PATH_IMAGE006
(VI) is expressed as ZnPcR4;
the preparation method of the peripherical asymmetric arginine modified phthalocyanine comprises the following steps:
the preparation method of the compound (V) comprises the following steps: taking 1- [4- (2-aminoethyl) phenoxy ] zinc phthalocyanine and N-Boc-L-arginine as reactants, taking N, N-Dimethylformamide (DMF) as a solvent, in the presence of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI), 1-hydroxybenzotriazole (HOBt) and N, N-Diisopropylethylamine (DIPEA) and under the protection of nitrogen, stirring for reaction for 10-60min at the temperature of 0 to 10 ℃, then continuously stirring for reaction for 7 to 36h at the temperature of room temperature to 45 ℃, and separating by column chromatography and molecular exclusion chromatography to obtain the pericyclic arginine substituted zinc phthalocyanine ZnPcR3;
the preparation method of the compound (VI) comprises the following steps: using 2,3- [4- (2-aminoethyl) phenoxy ] zinc phthalocyanine and N-Boc-L-arginine as reactants, using N, N-Dimethylformamide (DMF) as a solvent, in the presence of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI), 1-hydroxybenzotriazole (HOBt) and N, N-Diisopropylethylamine (DIPEA), stirring and reacting for 10-60min at 0 to 10 ℃ under the protection of nitrogen, then continuing stirring and reacting for 7 to 36h at room temperature to 45 ℃, and separating by column chromatography and molecular exclusion chromatography to obtain the pericyclic arginine substituted zinc phthalocyanine PcZnBNR 2;
wherein the molar ratio of the 1- [4- (2-aminoethyl) phenoxy ] zinc phthalocyanine to the N-Boc-L-arginine is 1 to 3;
5363 a molar ratio of 2,3- [4- (2-aminoethyl) phenoxy ] zinc phthalocyanine and N-Boc-L-arginine of 1;
the 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) and 1-hydroxybenzotriazole (HOBt) are used in an amount of 1.5 to 6mmol per mmol of 2- [4- (2-aminoethyl) phenoxy ] zinc phthalocyanine or 3,3' - [4- (2-aminoethyl) phenoxy ] zinc phthalocyanine; the dosage of the N, N-Diisopropylethylamine (DIPEA) is 0.2 to 1mL per mmol of 1- [4- (2-aminoethyl) phenoxy ] zinc phthalocyanine or 2,3- [4- (2-aminoethyl) phenoxy ] zinc phthalocyanine.
4. Use of a peripherical asymmetric arginine-modified phthalocyanine according to any one of claims 1-3 wherein: the phthalocyanine or the self-assembly thereof is used for preparing the photodynamic therapy and the drug with the adjustable and controllable performance and the preparation thereof.
5. Use of the peri-cyclic asymmetric arginine-modified phthalocyanine according to claim 4, wherein: the photodynamic therapy medicament is an I-type mechanism photosensitizer.
6. Use of a peripherical asymmetric arginine-modified phthalocyanine according to any one of claims 1-3 wherein: the phthalocyanine or the self-assembly thereof is used for preparing a photosensitive drug which is synergistic with immunotherapy.
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