CN116135228A - Application of hypocrellin 2-amino substituted or ethylenediamine substituted derivative in preparation of antitumor photodynamic medicine - Google Patents

Application of hypocrellin 2-amino substituted or ethylenediamine substituted derivative in preparation of antitumor photodynamic medicine Download PDF

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CN116135228A
CN116135228A CN202111372435.5A CN202111372435A CN116135228A CN 116135228 A CN116135228 A CN 116135228A CN 202111372435 A CN202111372435 A CN 202111372435A CN 116135228 A CN116135228 A CN 116135228A
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汪鹏飞
吴加胜
刘卫敏
郑秀丽
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Abstract

The invention relates to a 2-amino substituted derivative (formula I) or ethylenediamine substituted derivative (formulas II and III) of hypocrellin as photodynamic medicine for treating the following tumors: esophageal cancer, gastric cancer, lung cancer, liver cancer, bile duct cancer and colon cancer cells related to digestive tract tumor; head and neck tumor-associated brain cancer, head and neck cancer, tongue cancer, nose cancer, oral cancer, brain glioma cells; basal cell carcinoma associated with skin tumor, squamous skin carcinoma, cutaneous T cell lymphoma, melanoma cells; prostate cancer and bladder cancer cells associated with tumors of the genitourinary system. Meanwhile, the derivatives can position the position of tumor tissues by autofluorescence and can be used for fluorescence guided tumor surgical excision.
Figure DDA0003362796910000011

Description

Application of hypocrellin 2-amino substituted or ethylenediamine substituted derivative in preparation of antitumor photodynamic medicine
Technical Field
The invention relates to the technical field of photosensitizer medicaments, in particular to application of hypocrellin derivatives in preparation of photodynamic anti-tumor medicaments.
Background
Photodynamic action is that under the action of light, photosensitizers cause the body cells or biomolecules to change in function or morphology, thereby causing damage and necrosis of the cells. Non-invasive or minimally invasive treatment techniques that exploit photodynamic action to selectively destroy diseased tissue for therapeutic purposes are known as photodynamic therapy (PDT). In photodynamic therapy, a photosensitizer selectively accumulates in a target tissue, and when irradiated with light of an appropriate wavelength, absorbs photon energy, and changes from a ground state to an excited state. Since the excited state of the photosensitive material is extremely unstable, energy is then released back to the ground state through a physical or chemical de-excitation process. Wherein, the chemical de-excitation process generates a large amount of free radicals and singlet Reactive Oxygen Species (ROS), and the ROS oxidize various biological macromolecules such as amino acids, unsaturated fatty acids, adenosine and the like through the action of the ROS and the cells, damage the cell structure, influence the cell functions and further cause cell death. While the photosensitizers absorbed by normal tissues are already metabolically excreted and do not produce photodynamic effects. The photosensitizer, the light source and the oxygen are three elements of photodynamic therapy, the photosensitizer is specifically gathered on a target spot, and the light and the photosensitizer generate chemical action to generate active substances such as singlet oxygen, and the active substances are used for directionally damaging target cells, so that the method is a key link of photodynamic therapy.
Tumors are the most widely used field of photodynamic therapy. At present, photodynamic therapy of more than ten tumors in clinic achieves good effect. PDT can radically cure early in-situ malignant tumor, can be used for palliative treatment of intermediate-stage tumor, can improve symptoms and prolong life, and can prevent recurrence of tumor by laser irradiation for PDT of tumor operation excision part. The malignant tumor at the body surface part is particularly suitable for photodynamic therapy because the lesion part is shallower and the laser can directly penetrate the focus; for malignant tumors in deep parts, the cavity mirror can be used for illumination treatment. In addition, photodynamic therapy can also be used to intervene in the treatment of liver cancer or bone marrow purification, etc., by some special methods. Compared with the traditional three therapeutic means (operation, chemotherapy and radiotherapy), the photodynamic therapy has more pertinence, can directionally eliminate primary and recurrent tumors, has small toxic and side effects, rarely damages normal cells, and has therapeutic effect on various types of tumors. Along with the improvement of photosensitizers and the development of photodynamic therapy technology, the limitation and the deficiency of PDT therapy are gradually improved, but the PDT therapy is applied to clinic and has a certain limitation, and according to different patients, how to formulate individual treatment schemes, determine the treatment range according to the size and the depth of tumors, and further study on the selection of a light source, the size of power and the like. Currently, photodynamic therapy has been reported for treating tumors of body surface, including skin tumor and precancerous lesions (e.g., basal cell carcinoma, squamous skin carcinoma, melanoma, cutaneous T-cell lymphoma, etc.), head and neck tumor (e.g., nasopharyngeal carcinoma, laryngeal carcinoma, tongue carcinoma, oral cancer, etc.), brain tumor (brain glioma), reproductive tumor (prostate cancer, bladder cancer, cervical cancer, etc.), and digestive system tumor (cholangiocarcinoma, gastric cancer, lung cancer, liver cancer, colorectal cancer, etc.). Photodynamic therapy has not only good therapeutic effects on many primary tumors, but also unique technical advantages for many metastatic tumors. Studies have shown that a possible mechanism of action of photodynamic therapy in anti-tumor is direct killing of tumor cells or anti-vascular photodynamic therapy. Direct killing of tumor cells takes advantage of the dual selectivity of photodynamic therapy, selective retention of photosensitizers at tumor cells, and the generation of ROS near lesions upon irradiation with light waves of specific wavelengths results in lesion and extinction of tumor cells. Anti-vascular photodynamic therapy is where the survival of tumor cells is dependent on vascular support, and photodynamic therapy can damage the vasculature associated with the tumor, resulting in ischemic death of the tumor. If light is irradiated in the peak period of the concentration of the photosensitizer in the blood vessel, the damage of the micro blood vessel can be caused, and the blood supply in the focus is insufficient, so that the cells are necrotized or apoptotic.
Photosensitizers are the most critical factor in photodynamic therapy. The first generation photosensitizer developed in the early 70 s and 80 s of the last century is mainly porphyrin derivative (HpD) photosensitizer represented by hematoporphyrin, and is a mixture obtained by extracting pig blood or bovine blood as a raw material, and the effective components of the photosensitizer are mainly bishematoporphyrin ether or ester. The clinical application history is longest and the study is also the most detailed. Including Canadian, united states, germany, russia, belgium, cancer Bo Lin developed by China, cancer optical line, optical porphyrin, etc. Over the past 30 years, there have been a variety of commercial products of HpD available for clinical use, with thousands of patients receiving PDT treatment. The research results on HpD have achieved effects in the treatment of some body surface tumors, and the treatment effects on respiratory tract tumors such as bronchopulmonary carcinoma are quite remarkable. In addition, hpD has good curative effect on upper digestive tract malignant tumor, and even a few cases reach clinical cure. Barrett's esophagus is a very important precancerous lesion of esophageal cancer, and HpD has become one of the most preferred treatments at present. In addition, the HpD has good treatment effect on head and neck tumor, brain tumor, bladder cancer and bile duct cancer. The first generation photosensitizer has definite curative effect in tumor treatment, but has a plurality of defects such as complex components, poor tissue selectivity, slow metabolism, long photophobia time, certain toxicity and the like. The development of the second generation photosensitizer starts in the later 80 s of the last century, and the activity, absorption spectrum and tissue selectivity of the second generation photosensitizer are greatly improved compared with those of the first generation photosensitizer. The second generation photosensitizer is mostly a monomer compound, including porphyrin derivatives, metal phthalocyanine, chlorophyll degradation derivatives, condensed ring quinone compounds, porphins and the like. The endogenous porphyrin photosensitizer ALA has been successfully applied to the treatment of genital condyloma acuminatum diseases in 1990, and ALA-mediated photodynamic therapy has been widely applied to neoplastic skin diseases such as squamous cell carcinoma, basal cell carcinoma and the like nowadays; hypericin photosensitizer has been studied in recent years in terms of anti-tumor, and hypericin-mediated PDT can treat various tumors such as pancreatic cancer, bladder cancer, lymphatic cancer, prostate cancer, basal cell carcinoma and the like; the phthalocyanine photosensitizer is applied to countries and regions such as Russia, has remarkable anti-tumor and anti-infection effects and also has good safety in phase I/II clinical research. The second generation photosensitizer has definite chemical structure, high singlet oxygen yield, short photosensitization period and red shift of maximum absorption wavelength, increases the depth of photodynamic therapy, has very optimistic commercial and clinical application prospects, and still has the defects of high separation and purification difficulty and non-ideal targeting. Third generation photosensitizers have been developed starting at the end of the 20 th century, in order to improve biocompatibility, targeting, and develop photosensitizing drug delivery systems, the third generation photosensitizers have been developed by combining porphyrin or phthalocyanine precursors with certain chemical substances having biological properties such as: amino acid, polymer, egg matter, saccharide, liposome, antigen expressed by tumor tissue, antibody or ligand corresponding to receptor, etc. to constitute photosensitive system with tumor targeting and photodynamic treating effect. For example, hematoporphyrin has a very large killing effect on target cells in combination with monoclonal antibodies; compared with the uncomplexed phthalocyanine complex, the phthalocyanine-lipoprotein complex has greatly improved uptake rate to tumor tissues both in vitro and in vivo experiments. At present, the third generation photosensitizer is still in the preclinical animal research stage and has a distance from the actual clinical application.
Although the above photosensitizers have been developed over several decades in different periods, they have been a great progress in photodynamic therapy of tumors, but the types of these photosensitizers are relatively single and are basically porphyrin derivatives. The metabolism time of porphyrin derivatives in vivo is longer, the metabolism time of early HpD in human body is required to be 1-3 months, and although the later developed metabolism time of Hua Bulin nm in vivo is greatly shortened to one two weeks, the problems that the phototherapy window has weak light absorption capacity, stereoisomers are difficult to chemically separate and the like exist, so that the development of the high-efficiency photosensitizer with a novel structure is urgently needed.
Hypocrellins are natural photosensitizers extracted and separated from hypocrellins (hypocrellins) which are a kind of parasitic fungus of bamboo and are found in the arrow bamboo forests with the altitude of more than 3000 m in Yunnan of China, and belong to perylenequinone compounds. The natural hypocrellin mainly comprises hypocrellin A, hypocrellin B and the like, wherein the hypocrellin A accounts for 95 percent. Under alkaline condition, hypocrellin A can be dehydrated and converted into hypocrellin B, and the highest conversion rate can reach 99%. Hypocrellin has several advantages over the phototherapy drug hematoporphyrin derivative (HpD) used clinically: for example, the composition is simple, the raw materials are easy to purify, the triplet state quantum yield and the singlet state oxygen quantum yield are high, the phototoxicity is high, the dark toxicity is low, the photosensitizer has dual photodynamic mechanisms of Type I and Type II, the excretion is fast, and the like, and is a photosensitizer with very good application prospect. In addition, the structure of hypocrellin is easy to chemically modify, and the modified derivative can meet the requirements of strong light absorption capacity of the wavelength in a phototherapy window (600-900 nm) and clinical intravenous injection due to water solubility, so that the hypocrellin has wide application prospect as a photodynamic medicine. However, hypocrellin photosensitizers are currently in the laboratory research stage for the treatment of tumors, and no related hypocrellin drugs for clinical research exist. Therefore, development of a new hypocrellin photodynamic medicine is urgently needed for photodynamic treatment of tumors clinically.
The inventor has disclosed a 2-amino substituted or ethylenediamine substituted derivative of hypocrellin (CN 201610894129.0) for the first time, and through further research, we have surprisingly found that the derivative can kill certain specific tumor cells, such as esophageal cancer, gastric cancer, lung cancer, liver cancer, cholangiocarcinoma and colon cancer cells related to digestive tract tumor with high efficiency; head and neck cancer, brain cancer, tongue cancer, nose cancer, oral cancer, brain glioma cells associated with head and neck and face tumors; basal cell carcinoma associated with skin tumor, squamous skin carcinoma, cutaneous T cell lymphoma, melanoma cells; prostate cancer and bladder cancer cells associated with tumors of the genitourinary system. In addition, aiming at the problem that no mediated medicine is used for positioning and guiding tumor excision in the operation of some special tumors (such as brain glioma), the inventor finds that the hypocrellin derivative can be specifically gathered in brain glioma cells and tissues, no photosensitizer is gathered in brain areas without tumors, the hypocrellin derivative has good tumor targeting enrichment effect, and at the moment, the tumor tissues are irradiated by light with specific wavelength, detectable fluorescence can be excited to position the positions of the tumor tissues, and the hypocrellin derivative is used for fluorescence guided tumor excision (FGS).
Disclosure of Invention
In order to solve the technical problems, the invention provides a compound shown in a formula (I), a formula (II) or a formula (III), an hypocrellin 2-amino substituted or ethylenediamine substituted derivative, an isomer, an isotope label, a pharmaceutically acceptable salt or solvate thereof, and application in preparing photodynamic anti-tumor medicaments, wherein tumors are esophageal cancer, gastric cancer, lung cancer, liver cancer, cholangiocarcinoma, colon cancer, head and neck cancer, brain cancer, tongue cancer, nasal cancer, oral cancer, brain glioma, basal cell carcinoma, squamous skin cancer, skin T cell lymphoma, melanoma, prostate cancer and bladder cancer.
Figure BDA0003362796890000031
R in formula (I) 1 The structural general formula is shown as formula (IV), R 2 is-H or-COCH 3 ：
Figure BDA0003362796890000032
In the formula (IV), m is more than or equal to 0 and less than or equal to 12, n is more than or equal to 0 and less than or equal to 500, p is more than or equal to 0 and less than or equal to 12, and q is more than or equal to 0 and less than or equal to 12; the m, n, p, q is zero or a positive integer; y is a linking group; z is a terminal group; (OCH) 2 CH 2 ) n Is a polyethylene glycol unit;
the linking group Y in the formula (IV) is O, NH, S, carboxylic ester group, amide group, sulfocarboxylic ester group, phenylene group, alkenylene group of 3-12 carbon atoms or cycloalkyl group of 3-12 carbon atoms;
the cycloalkyl of 3-12 carbon atoms comprises a substituted or unsubstituted cycloalkyl or contains a heteroatom which is an oxygen, nitrogen or sulfur atom; the substituent is alkyl with 1-12 carbon atoms;
The end group Z in the formula (IV) is hydrogen, alkyl of 1-12 carbon atoms, alkoxy of 1-12 carbon atoms, phenyl, hydroxyl, amino, sulfhydryl, carboxylic acid group, sulfonic acid group, pyridyl, quaternary ammonium salt or pyridinium salt;
when the end group Z is quaternary ammonium salt, three substituents on the quaternary ammonium salt are respectively and independently or simultaneously alkyl with 1-12 carbon atoms; the anions in the quaternary ammonium salt are anions allowed by the pharmaceutical preparation;
when the end group Z is pyridine salt, the substituent group on the pyridine ring in the pyridine salt is in ortho-position, meta-position or para-position; the pyridinium is formed by quaternizing pyridine and halogenated hydrocarbon containing 1-12 carbon atoms with different chain lengths; anions in the pyridinium salt are anions allowed by the pharmaceutical preparation;
substituent R in formula (II) 2 is-H or-COCH 3 ：
R in formula (II) 3 ～R 8 Identical or different, as in formula (I) the substituents R 1 Definition of (i.e. R) 3 ～R 8 The same or different is independently shown as a formula (IV).
Preferably, the linking group Y in formula (IV) above is: -O-; -NH-; -S-; -COO-; -O-CO-; -CONH-; -NH-CO-; -SO 3 -；-SO 2 -NH-；-C 6 H 4 - (phenyl); -C 3 H 4 - (cyclopropyl); -C 4 H 6 - (cyclobutyl); -C 5 H 8 - (cyclopentyl); -C 5 H 7 (CH 3 ) - (methylcyclopentyl); -C 6 H 10 - (cyclohexyl); -C 6 H 9 (CH 3 ) - (methylcyclohexyl); -C 7 H 12 - (cycloheptyl);
Figure BDA0003362796890000033
(piperazinyl).
Preferably, the end group Z in formula (IV) above is: -H; -CH 3 ；-C 2 H 5 ；-C 4 H 9 ；-C 6 H 13 ；-OCH 3 ；-OC 2 H 5 ；-OC 4 H 9 ；-OC 6 H 13 ；-C 6 H 5 ；-OH,-NH 2 ；-SH；-COOH；-COOCH 3 ；-SO 3 H；-C 5 H 4 N；-C 5 H 4 N + ；-N + (CH 3 ) 3 ；-N + (C 2 H 5 ) 3 ；-N + (C 6 H 13 ) 3 ；-N + (CH 3 ) 2 (C 2 H 5 )；-N + (CH 3 ) 2 (C 6 H 13 )；-N + (CH 3 ) 2 (C 8 H 17 )。
In one embodiment, m is an integer from 0 to 8, n is an integer from 0 to 200, p is an integer from 0 to 8, and q is an integer from 0 to 8, e.g., 0,1,2,3,4, etc.
Specifically, the derivatives of formula I and formula I' are enol tautomers; the derivatives of formula II and formula II' are enol tautomers; the derivatives of formula III and formula III' are enol tautomers.
Figure BDA0003362796890000041
Preferably, the substituent R 1 Is an alcohol with different chain lengths and a carboxylic ester formed by the alcohol and carboxyl polyethylene glycol: - (CH) 2 ) m -OH；-(CH 2 ) m -OCH 3 ；-(CH 2 ) m -O-CO-CH 2 CH 2 -(OCH 2 CH 2 ) n -OCH 3 [ m is an integer of 1 to 8, n is an integer of 0 to 100]；
Preferably, the substituent R 1 Carboxylic acids of different chain lengths, and carboxylic esters or amides thereof with polyethylene glycol: - (CH) 2 ) m -COOH；-(CH 2 ) m -COOCH 3 ；-(CH 2 ) m -CO-(OCH 2 CH 2 ) n -OH；-(CH 2 ) m -CO-(OCH 2 CH 2 ) n -OCH 3 ；-(CH 2 ) m -CO-NH-CH 2 CH 2 -(OCH 2 CH 2 ) n -OCH 3 [ m is an integer of 1 to 8, n is an integer of 0 to 100]；
Preferably, the substituent R 1 Sulfonate or sulfonamide formed from sulfonate and polyethylene glycol of different chain lengths: - (CH) 2 ) m -SO 3 H；-(CH 2 ) m -SO 2 -(OCH 2 CH 2 ) n -OH；-(CH 2 ) m -SO 2 -(OCH 2 CH 2 ) n -OCH 3 ；-(CH 2 ) m -SO 2 -NH-CH 2 CH 2 -(OCH 2 CH 2 ) n -OH；-(CH 2 ) m -SO 2 -NH-CH 2 CH 2 -(OCH 2 CH 2 ) n -OCH 3 [ m is an integer of 1 to 8, n is an integer of 0 to 100]；
Preferably, the substituent R 1 Is thiapolyethylene glycol: -CH 2 CH 2 -SH；-CH 2 CH 2 -S-CH 2 CH 2 OH；-CH 2 CH 2 -S-CH 2 CH 2 OCH 3 ；-CH 2 CH 2 -S-CH 2 CH 2 -(OCH 2 CH 2 ) n -OH；
Preferably, the substituent R 1 Is alkyl, amino, hydroxyl, or substituent containing phenyl, pyridyl, alkene: -H; -CH 3 ；-C 2 H 5 ；-C 3 H 7 ；-C 4 H 9 ；-C 5 H 11 ；-C 6 H 13 ；-C 8 H 17 ；-NH 2 ；-NHCH 3 ；-NHC 2 H 5 ；-OH；-CH 2 CH＝CH 2 ；-(CH 2 ) 2 CH＝CH 2 ；-(CH 2 ) 3 CH＝CH 2 ；-CH 2 C 6 H 5 ；-C 5 H 4 N；-CH 2 C 5 H 4 N；-(CH 2 ) 2 C 5 H 4 N；-NHC 6 H 5 ；-NHC 5 H 4 N；
Preferably, the substituent R 1 Is a substituent containing cycloalkyl: -C 3 H 5 (cyclopropyl) -C 4 H 7 (cyclobutyl) -C 5 H 9 (cyclopentyl) -C 6 H 11 (cyclohexyl) -C 6 H 10 (CH 3 ) (methylcyclohexyl) -C 6 H 10 (OH) (hydroxycyclohexyl), -C 7 H 13 (cycloheptyl) -CH 2 C 6 H 10 COOH、-CH 2 C 6 H 10 COOCH 3 、-CH 2 C 6 H 10 OH、-C 6 H 10 COOH；
More preferably, the substituent R 1 Is cyclohexane (-C) containing substituent 6 H 10 -OH；-CH 2 C 6 H 10 COOH；-CH 2 C 6 H 10 COOCH 3 ；-CH 2 C 6 H 10 OH；-C 6 H 10 COOH), the substituent being located in the ortho, para, meta position of cyclohexane;
preferably, the substituent R 1 Is a substituent containing quaternary ammonium salt: - (CH) 2 ) m -N + (CH 3 ) 3 ；-(CH 2 ) m -N + (CH 3 ) 2 (C 2 H 5 )；-(CH 2 ) m -N + (CH 3 ) 2 (C 3 H 7 )；-(CH 2 ) m -N + (CH 3 ) 2 (C 4 H 9 )；-(CH 2 ) 3 -N + (CH 3 ) 2 (C 6 H 13 )；-(CH 2 ) m -N + (CH 3 ) 2 (C 8 H 17 )；-(CH 2 ) m -N + (CH 3 ) 2 (C 12 H 25 )；-(CH 2 ) m -O-CO-(CH 2 ) 2 -N + (CH 3 ) 3 ；-(CH 2 ) m -O-CO-(CH 2 ) 3 -N + (CH 3 ) 3 ；-(CH 2 ) m -O-CO-(CH 2 ) 4 -N + (CH 3 ) 3 ；-(CH 2 ) m -O-CO-(CH 2 ) 5 -N + (CH 3 ) 3 ；-(CH 2 ) m -O-CO-(CH 2 ) 6 -N + (CH 3 ) 3 ；-(CH 2 ) m -COO-(CH 2 ) 2 -N + (CH 3 ) 3 ；-(CH 2 ) m -COO-(CH 2 ) 3 -N + (CH 3 ) 3 ；-(CH 2 ) m -COO-(CH 2 ) 4 -N + (CH 3 ) 3 ；-(CH 2 ) m -COO-(CH 2 ) 5 -N + (CH 3 ) 3 ；-(CH 2 ) m -COO-(CH 2 ) 6 -N + (CH 3 ) 3 ；-(CH 2 ) m -CONH-(CH 2 ) 2 -N + (CH 3 ) 3 ；-(CH 2 ) m -CONH-(CH 2 ) 3 -N + (CH 3 ) 3 ；-(CH 2 ) m -CONH-(CH 2 ) 4 -N + (CH 3 ) 3 [ m is an integer of 1 to 8]；
Preferably, the substituent R 1 Is a heterocyclic-containing substituent:
Figure BDA0003362796890000042
according to an embodiment of the invention, the tumor is esophageal cancer, gastric cancer, lung cancer, liver cancer, bile duct cancer, colon cancer, head and neck cancer, brain cancer, tongue cancer, nose cancer, oral cancer, brain glioma, basal cell carcinoma, squamous skin carcinoma, cutaneous T-cell lymphoma, melanoma, prostate cancer, bladder cancer.
According to an embodiment of the invention, the tumor cells are esophageal cancer cells AKR, gastric cancer cells MFC, lung cancer cells a549, liver cancer cells HepG2, cholangiocarcinoma cells MCC, colon cancer cells HCT116, head and neck cancer cells SCC2, brain cancer cells G442, tongue cancer cells TSCCa, nasal cancer cells KB, oral cancer cells CAL27, brain glioma cells C6, basal cell carcinoma cells BCC, squamous skin cancer cells PECA, cutaneous T cell lymphoma HH, melanoma cells B16, prostate cancer cells LNCaP, bladder cancer cells MBT-2.
According to embodiments of the invention, the drug may be a photodynamic drug, a fluorescence-mediated drug.
According to an embodiment of the invention, the drug may be enriched in the tumor cells.
According to an embodiment of the present invention, the pharmaceutically acceptable salts include salts of the compound of formula (I) with organic acids selected from propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid and citric acid or acidic amino acids selected from aspartic acid and glutamic acid, followed by formation with inorganic bases, including sodium, potassium, calcium, aluminum salts and ammonium salts, or salts with organic bases, including methylamine salts, ethylamine salts and ethanolamine salts; or with a basic amino acid selected from lysine, arginine and ornithine, and then with an inorganic acid selected from hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid and phosphoric acid, or with an organic acid selected from formic acid, acetic acid, picric acid and methanesulfonic acid.
The invention also provides an application of the compound shown in the formula (I), the formula (II) or the formula (III), an isomer, an isotope label, a pharmaceutically acceptable salt or a solvate thereof in preparing a fluorescence-mediated medicament for guiding the boundary excision of tumors.
The invention also provides application of the compound shown in the formula (I), the formula (II) or the formula (III), isomer, isotope label or pharmaceutically acceptable salt thereof in treating tumor diseases, wherein the tumor is esophageal cancer, gastric cancer, lung cancer, liver cancer, bile duct cancer, colon cancer, head and neck cancer, brain cancer, tongue cancer, nasal cancer, oral cancer, glioma, basal cell carcinoma, squamous skin cancer, cutaneous T cell lymphoma, melanoma, prostate cancer and bladder cancer.
According to embodiments of the invention, the treatment is photodynamic inactivation of tumor cells or as a fluorescence-mediated drug directed boundary excision of tumors.
The present invention also provides a method for preventing or treating a neoplastic disease, comprising administering to a patient a prophylactically or therapeutically effective amount of at least one of the compounds of formula (I), formula (II), or formula (III), isomers, isotopic labels, pharmaceutically acceptable salts, or solvates thereof.
In some embodiments, the patient is a human.
Advantageous effects
1) The compound of the formula (I), the formula (II) or the formula (III) can be used as a photodynamic medicament to kill the following cancers or tumor cells with high efficiency: esophageal cancer, gastric cancer, lung cancer, liver cancer, bile duct cancer, colon cancer, head and neck cancer, brain cancer, tongue cancer, nose cancer, oral cancer, brain glioma, basal cell carcinoma, squamous skin cancer, cutaneous T-cell lymphoma, melanoma, prostate cancer and bladder cancer, the photodynamic medicine with the concentration of 20-30 nM can kill more than 90% of tumor cells without affecting normal cells basically, and the medicine is basically discharged out of the body after one week;
2) The invention discloses hypocrellin derivatives as mediating drugs in clinical tumor surgery for the first time, which are used for guiding tumor excision. The derivative can be specifically gathered in tumor tissues, the areas without tumors have no photosensitizer gathering, and the derivative has a good tumor targeting gathering effect, and at the moment, the tumor tissues are irradiated by light with specific wavelength, so that detectable fluorescence can be excited to be generated, and the position of the tumor tissues is positioned, so that the derivative is used for fluorescence guided tumor excision operation.
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The following describes the embodiments of the present invention in further detail with reference to the drawings.
FIG. 1 shows the general structural formula of hypocrellin 2-amino substituted or ethylenediamine substituted derivatives.
FIG. 2 shows the synthesis of the 2-position polyethylene glycol-aminopentanol substituted derivative HB-4-PEGn of hypocrellin B.
FIG. 3 shows the reaction products HC-74 and HC-75 of deacetylated hypocrellin HC and methyl ethylenediamine.
FIG. 4 (a) is a graph showing the comparison of absorption spectra of the commercial porphyrin-based photosensitizers PpIX and Ce 6. FIG. 4 (b) is a graph showing the comparison of absorption spectra of hypocrellin HB, HB-6-PEG1 (example 6) as the 2-position substitution product of HB, and HC-74 (example 31) as the ethylenediamine substitution product.
FIG. 5 is an ESR chart showing the effect of derivatives HB-45, HC-45 (example 23) on an active oxygen scavenger: (a) a singlet oxygen scavenger; (b) a superoxide radical scavenger.
FIG. 6 (a) is a photodegradation curve of HB-73 in example 31; FIG. 6 (b) is a photodegradation curve of HC-73 in example 31; FIG. 6 (c) is a photodegradation curve of HC-80 in example 32.
FIG. 7 shows a derivative HB-2-PEG4 (example 2), HC-2-PEG8 (example 2), HC-73 (example 31), HC-80 (example 32) and commercial photosensitizers (Ce 6, hematoporphyrin HpD) at 20mW/cm 2 Light stability contrast plot for a laser light of 30 min.
FIG. 8 is a graph comparing pH stability of derivatives HB-3-PEG4 (example 3), HC-3-PEG8 (example 3), HC-73 (example 31), HB-77 (example 32) and commercial photosensitizer (hematoporphyrin HpD).
FIG. 9 is a confocal fluorescence imaging of the derivative HB-1-PEG12 (example 1) in A549 cells: (a) dark field and bright field superimposed images; (b) dark field images; (c) bright field image.
FIG. 10-1 is a graph (a) showing dark toxicity of HB-1-PEG4 and HC-1-PEG8 (example 1), and commercial photosensitizer HpD on AKR of esophageal cancer cells and a graph (b) showing phototoxicity.
FIG. 10-2 is a graph (a) of dark toxicity and a graph (b) of HB-3-PEG8 and HC-3-PEG16 (example 3), and commercial photosensitizer HpD on lung cancer cell A549.
FIG. 10-3 shows the dark toxicity patterns (a) and phototoxicity patterns (b) of HB-6-PEG2 and HC-6-PEG6 (example 6), and commercial photosensitizer HpD on hepatoma cell HepG 2.
FIGS. 10-4 are dark toxicity patterns (a) and phototoxicity patterns (b) of HB-14-PEG6 and HC-14-PEG12 (example 13), and of commercial photosensitizer HpD for colon cancer cell HCT 116.
FIGS. 10-5 are dark toxicity patterns (a) and phototoxicity patterns (b) of HB-19-PEG4 and HC-19-PEG8 (example 15), and commercial photosensitizer HpD on cholangiocarcinoma cell MCC.
FIGS. 10-6 are dark toxicity patterns (a) and phototoxicity patterns (b) of HB-45 and HC-45 (example 23), and commercial photosensitizer HpD on gastric cancer cell MFCs.
FIG. 11 (a) is a phototoxicity profile of HB-10-PEG4 and HC-10-PEG8 (example 10), and HpD on brain cancer cell G442;
FIG. 11 (b) is a phototoxicity profile of HB-12-PEG8 and HC-12-PEG12 (example 12), hpD for cervical cancer cell SCC 2;
FIG. 11 (c) is a phototoxicity profile of HB-20-PEG6 and HC-20-PEG12 (example 16) and HpD on tongue carcinoma cell TSCCa.
FIG. 12 (a) is a phototoxicity profile of HB-29 and HC-29 (example 19), and commercial photosensitizer HpD on nasal carcinoma cell KB;
FIG. 12 (b) is a phototoxicity profile of HB-61 and HC-61 (example 28), and HpD for oral cancer cell CAL 27;
FIG. 12 (C) is a phototoxicity profile of HB-73 (example 31), HC-80 (example 32), and HpD on glioma cells C6.
FIG. 13 (a) is a chart of the phototoxicity of HB-4-PEG8 and HC-4-PEG16 (example 4), hpD to basal cell carcinoma cell BCC;
FIG. 13 (B) is a phototoxicity profile of HB-9-PEG4 and HC-9-PEG8 (example 9), and HpD for melanoma cell B16;
FIG. 13 (c) is a phototoxicity profile of HB-36 and HC-36 (example 20), and HpD for squamous cell carcinoma PECA.
FIG. 14 (a) is a phototoxicity profile of HB-70 and HC-71 (example 30), and HpD for prostate cancer cells LNCaP;
FIG. 14 (b) is a phototoxicity profile of HB-73 and HC-73 (example 31), and HpD on bladder cancer cell MBT-2;
FIG. 14 (c) is a phototoxicity profile of HC-80 (example 32) and HC-81 (example 33), and agent HpD on skin T cell lymphoma cell HH.
FIG. 15 is an in vivo fluorescence imaging of mice in different tumor models 4h after tail vein injection administration of the derivatives of the invention: (a) HB-1-PEG4 (example 1) was used for esophageal carcinoma tumor AKR cell imaging; (b) HC-3-PEG12 (example 3) was used for gastric cancer MFC cell imaging; (c) HB-6-PEG8 (example 6) was used for lung cancer A549 cell imaging; (d) HB-7-PEG16 (example 7) was used for liver cancer HepG2 cell imaging; (e) HC-9-PEG10 (example 9) was used for cholangiocarcinoma MCC cell imaging; (f) HC-1-PEG50 (example 1) was used for colon cancer HCT116 cell imaging.
FIG. 16 is an in vivo fluorescence imaging of mice in different tumor models 4h after tail vein injection administration of the derivatives of the invention: (a) HB-11-PEG6 (example 11) was used for brain cancer G442 cell imaging; (b) HC-18-PEG8 (example 15) was used for head and neck cancer SCC2 cell imaging; (c) HC-45 (example 23) was used for tongue carcinoma TSCCa cell imaging; (d) HC-57 (example 27) was used for oral cancer CAL27 cell imaging; (e) HB-65 (example 29) was used for nasal carcinoma KB cell imaging; (f) HC-81-PEG16 (example 34) was used for brain glioma C6 cell imaging.
FIG. 17 is an in vivo fluorescence imaging of mice in different tumor models 4h after tail vein injection administration of the derivatives of the invention: (a) HB-5-PEG10 (example 5) was used for basal cell carcinoma BCC cell imaging; (b) HC-8-PEG12 (example 8) was used for squamous skin carcinoma PECA cell imaging; c) HB-64 (example 28) was used for melanoma B16 cell tumor imaging; d) HB-89-PEG16 (example 36) was used for skin T cell lymphoma HH cell imaging; e) HC-90-PEG30 (example 37) was used for prostate cancer LNCaP cell tumor imaging; f) HC-91-PEG16 (example 38) was used for bladder cancer MBT-2 cell imaging.
FIG. 18 is an in vivo fluorescence imaging of mice in different tumor models 4h after intratumoral injection administration of the derivatives of the invention: (a) HB-28 (example 19) was used for basal cell carcinoma BCC cell imaging; (b) HC-36 (example 20) was used for squamous skin carcinoma PECA cell imaging; (c) HB-45 (example 23) was used for melanoma B16 cell imaging; (d) HC-73 (example 31) was used for skin T cell lymphoma HH cell imaging; (e) HC-77 (example 32) was used for lung cancer A549 cell imaging; (f) HC-80 (example 32) was used for cholangiocarcinoma MCC cell imaging.
FIG. 19 is a graph showing fluorescence imaging of HB-3-PEG12 in example 3 after tail vein administration in tumor-bearing mice for 0 to 5 hours.
FIG. 20 is 0.1W/cm 2 Light irradiation for 10min at 635nm of (a), 6 days and 12 days after photodynamic therapy effect profile of mice vaccinated with lung cancer cells (a 549): the first row is not injected with medicine, and only is injected with physiological saline; the second row is for tail vein injection of the drug HB-1-PEG8 (example 1).
FIG. 21 shows the effect of administering a different photosensitizing drug by tail vein injection at 0.1W/cm 2 Is irradiated by a 635nm laser for 10min, and the tumor part picture of a tumor-bearing mouse is inoculated after 6 days of photodynamic therapy: FIG. (a) shows mice vaccinated with brain glioma cells (C6) without drug injection, with saline alone; FIG. (b) is a mouse tail vein injection of drug HB-6-PEG6 (example 6) inoculated with brain glioma cells (C6); FIG. (c) is a mouse tail vein injection of drug HC-9-PEG8 (example 9) inoculated with melanoma cells (B16); FIG. (d) is a mouse tail vein injection inoculated with bladder cancer cells (MBT-2)Drug HB-63 (example 28);
FIG. 22 shows the effect of administering a different photosensitizing drug by intratumoral injection at 0.1W/cm 2 Is irradiated by a 635nm laser for 10min, and the tumor part picture of a tumor-bearing mouse is inoculated after 6 days of photodynamic therapy: FIG. (a) is a intratumoral injection of drug HB-29 (example 19) into a mouse vaccinated with cholangiocarcinoma cells (MCC); FIG. (b) is a diagram showing intratumoral injection of drug HB-45 in mice vaccinated with colon cancer cells (HCT 116) (example 23); FIG. (c) is a mouse intratumoral injection of drug HC-73 (example 31) vaccinated with oral cancer cells (CAL 27); FIG. d shows intratumoral injection of drug HC-80 into basal cell carcinoma cells (BCC) -vaccinated mice (example 32);
FIG. 23 shows the photodynamic effects of the derivatives HB-1-PEG6 (example 1), HB-73 (example 31), and HC-80 (example 32) on HeLa cells.
Detailed Description
The technical scheme of the invention will be further described in detail below with reference to specific embodiments. It is to be understood that the following examples are illustrative only and are not to be construed as limiting the scope of the invention. All techniques implemented based on the above description of the invention are intended to be included within the scope of the invention.
Unless otherwise indicated, the starting materials and reagents used in the following examples were either commercially available or may be prepared by known methods.
In the invention, the experimental methods are all conventional methods unless specifically stated. The starting materials used are available commercially from the public sources unless otherwise specified; the percentages are mass percentages unless otherwise specified; and M is mol/L unless specified otherwise.
The raw materials used in the invention are hypocrellin A HA, hypocrellin B HB and deacetylated hypocrellin HC; all hypocrellin derivatives according to the present invention are shown in examples 1 to 38, and the characterization results of some of them are shown in table 1.
It is further noted that the hypocrellin derivatives to be protected in the present invention contain two enol tautomers, and the chemical structures of the two isomers are shown as formula (I) and (I '), formula (II) and (II '), and formula (III) and (III '), which are all within the protection range. For the sake of simplicity, only one enol tautomer is listed in all examples of the invention, the other enol tautomer and its corresponding structural formula are described in detail in the specification, and the structures thereof are of course within the scope of protection. In addition, the structural general formula of the hypocrellin derivative comprises polyethylene glycol units (PEGn), wherein the number n of the units is any integer between 0 and 100, and the chemical structures corresponding to the hypocrellin derivatives are all within a protection range. For the sake of simplicity, only some integers are listed in all embodiments of the present invention, and the general structural formulas corresponding to the rest are described in detail in the specification, and the structures thereof are of course within the scope of protection. Any range recited in the invention includes any numerical value recited in, and any subrange formed by, the endpoints, or any numerical value recited between the endpoints.
Example 1
Reacting Hypocrellin B (HB) or deacetylated Hypocrellin (HC) with amino ethanol, amino ethanol methyl ether, amino ethanol-polyethylene glycol, wherein the 2-position substitution products of HB are HB-1, HB-1-CH respectively 3 The 2-position substitution products of HB-1-PEGn and HC are HC-1 and HC-1-CH respectively 3 HC-1-PEGn (n is an integer between 1 and 100), and has a structural formula as follows:
Figure BDA0003362796890000081
example 2
HB (or HC) respectively reacts with aminopropanol, aminopropanol methyl ether and aminopropanol-polyethylene glycol, and the 2-position substitution products of HB are HB-2 and HB-2-CH respectively 3 The 2-position substitution products of HB-2-PEGn and HC are HC-2 and HC-2-CH respectively 3 HC-2-PEGn (n is an integer between 1 and 100), and has the following structural formula:
Figure BDA0003362796890000082
example 3
HB (or HC) respectively reacts with the aminobutanol, the aminobutanol methyl ether and the aminobutanol-polyethylene glycol, and the 2-bit substitution products of HB are HB-3 and HB-3-CH respectively 3 The 2-position substitution products of HB-3-PEGn and HC are HC-3 and HC-3-CH respectively 3 HC-3-PEGn (n is an integer between 1 and 100) has the following structural formula:
Figure BDA0003362796890000083
example 4
HB (or HC) respectively reacts with aminopentanol, aminopentanol methyl ether and aminopentanol-polyethylene glycol, and the 2-position substitution products of HB are HB-4 and HB-4-CH respectively 3 The 2-position substitution products of HB-4-PEGn and HC are HC-4 and HC-4-CH respectively 3 HC-4-PEGn (n is an integer between 1 and 100) has the following structural formula:
Figure BDA0003362796890000084
example 5
HB (or HC) respectively reacts with amino-hexanol, amino-hexanol methyl ether and amino-hexanol-polyethylene glycol, and the 2-position substitution products of HB are HB-5 and HB-5-CH respectively 3 The 2-position substitution products of HB-5-PEGn and HC are HC-5 and HC-5-CH respectively 3 HC-5-PEGn (n is an integer between 1 and 100) has the following structural formula:
Figure BDA0003362796890000091
example 6
HB (or HC) respectively reacts with amino polyethylene glycol and amino polyethylene glycol methyl ether, and the 2-bit substitution products of HB are HB-6-PEGn and HB-6-PEGn-CH respectively 3 The 2-position substitution products of HC are HC-6-PEGn and HC-6-PEGn-CH respectively 3 (n is an integer between 1 and 100), and the structural formula is as follows:
Figure BDA0003362796890000092
example 7
HB (or HC) respectively reacts with glycine, methyl glycine and glycine-polyethylene glycol, and the 2-bit substitution products of HB are HB-7 and HB-7-CH respectively 3 The 2-position substitution products of HB-7-PEGn and HC are HC-7 and HC-7-CH respectively 3 HC-7-PEGn (n is an integer between 1 and 100), and has the following structural formula:
Figure BDA0003362796890000093
example 8
HB (or HC) respectively reacts with aminopropionic acid, methyl aminopropionate and aminopropionic acid-polyethylene glycol, and the 2-position substitution products of HB are HB-8 and HB-8-CH respectively 3 The 2-position substitution products of HB-8-PEGn and HC are HC-8 and HC-8-CH respectively 3 HC-8-PEGn (n is an integer between 1 and 100) has the following structural formula:
Figure BDA0003362796890000094
example 9
HB (or HC) respectively reacts with aminobutyric acid, aminobutyric acid methyl ester and aminobutyric acid-polyethylene glycol, and the 2-position substitution products of HB are HB-9 and HB-9-CH respectively 3 The 2-position substitution products of HB-9-PEGn and HC are HC-9 and HC-9-CH respectively 3 HC-9-PEGn (n is an integer between 1 and 100) has the following structural formula:
Figure BDA0003362796890000101
example 10
HB (or HC) respectively reacts with aminopentanoic acid, aminopentanoic acid methyl ester and aminopentanoic acid-polyethylene glycol, and the 2-position substitution products of HB are HB-10 and HB-10-CH respectively 3 、HThe 2-position substitution products of B-10-PEGn and HC are HC-10 and HC-10-CH respectively 3 HC-10-PEGn (n is an integer between 1 and 100), and has the following structural formula:
Figure BDA0003362796890000102
example 11
HB (or HC) respectively reacts with aminocaproic acid, methyl aminocaproate and aminocaproic acid-polyethylene glycol, and the 2-position substitution products of HB are HB-11 and HB-11-CH respectively 3 The 2-position substitution products of HB-11-PEGn and HC are HC-11 and HC-11-CH respectively 3 HC-11-PEGn (n is an integer between 1 and 100), and has the following structural formula:
Figure BDA0003362796890000103
example 12
HB (or HC) respectively reacts with the amino acetic acid amide-polyethylene glycol and the amino propionic acid amide-polyethylene glycol, the 2-position substitution products of HB are HB-12-PEGn and HB-13-PEGn respectively, the 2-position substitution products of HC are HC-12-PEGn and HC-13-PEGn (n is an integer between 1 and 100), and the structural formulas are shown as follows:
Figure BDA0003362796890000104
Example 13
HB (or HC) respectively reacts with aminobutyric acid amide-polyethylene glycol and aminopentanoic acid amide-polyethylene glycol, the 2-position substitution products of HB are HB-14-PEGn and HB-15-PEGn respectively, the 2-position substitution products of HC are HC-14-PEGn and HC-15-PEGn (n is an integer between 1 and 100), and the structural formulas are shown as follows:
Figure BDA0003362796890000111
example 14
HB (or HC) respectively reacts with sulfamic acid-polyethylene glycol and sulfamic acid-polyethylene glycol, the 2-position substitution products of HB are HB-16-PEGn and HB-17-PEGn, the 2-position substitution products of HC are HC-16-PEGn and HC-17-PEGn (n is an integer between 1 and 100), and the structural formulas are shown as follows:
Figure BDA0003362796890000112
example 15
HB (or HC) respectively reacts with the aminopropanesulfonic acid-polyethylene glycol and the aminobutanesulfonic acid-polyethylene glycol, the 2-position substitution products of HB are HB-18-PEGn and HB-19-PEGn respectively, and the 2-position substitution products of HC are HC-18-PEGn and HC-19-PEGn (n is an integer between 1 and 100) respectively, and the structural formulas are shown as follows:
Figure BDA0003362796890000113
example 16
HB (or HC) respectively reacts with sulfamide-polyethylene glycol and aminoethanesulfonamide-polyethylene glycol, the 2-position substitution products of HB are HB-20-PEGn and HB-21-PEGn respectively, and the 2-position substitution products of HC are HC-20-PEGn and HC-21-PEGn (n is an integer between 1 and 100), and the structural formula is shown as follows:
Figure BDA0003362796890000114
Example 17
HB (or HC) respectively reacts with aminopropanesulfonamide-polyethylene glycol and aminobutanesulfonamide-polyethylene glycol, the 2-position substitution products of HB are HB-22-PEGn and HB-23-PEGn respectively, and the 2-position substitution products of HC are HC-22-PEGn and HC-23-PEGn (n is an integer between 1 and 100), and the structural formula is shown as follows:
Figure BDA0003362796890000121
example 18
HB (or HC) respectively reacts with thioamino glycol and thioglycolamine, and the 2-position substitution products of HB are HB-24 and HB-24-CH respectively 3 HB-25, 2-position substitution products of HC are HC-24, HC-24-CH, respectively 3 HC-25, the structural formula is shown as follows:
Figure BDA0003362796890000122
example 19
HB (or HC) respectively reacts with alkylamines (ethylamine, propylamine, butylamine, hexylamine and octylamine) with different chain lengths, the 2-position substitution products of HB are HB-26-HB-30 respectively, the 2-position substitution products of HC are HC-26-HC-30 respectively, and the structural formula is shown as follows:
Figure BDA0003362796890000123
example 20
HB (or HC) respectively reacts with hydrazine, hydroxylamine, cyclopropylamine, cyclobutylamine, cyclopentylamine and cyclohexylamine, the 2-position substitution products of HB are HB-31-HB-36 respectively, the 2-position substitution products of HC are HC-31-HC-36 respectively, and the structural formula is shown as follows:
Figure BDA0003362796890000124
Figure BDA0003362796890000131
example 21
HB (or HC) respectively reacts with acrylamide, butenamine, hexenamine and octenamine, the 2-position substitution products of HB are HB-37-HB-40 respectively, the 2-position substitution products of HC are HC-37-HC-40 respectively, and the structural formula is shown as follows:
Figure BDA0003362796890000132
Example 22
HB (or HC) respectively reacts with benzylamine, aminomethylpyridine, aminobutylpyridine and aminobutylpyridine salt, the 2-position substitution products of HB are HB-41-HB-44 respectively, the 2-position substitution products of HC are HC-41-HC-44 respectively, and the structural formula is shown as follows:
Figure BDA0003362796890000133
example 23
HB (or HC) respectively reacts with para-position, meta-position and ortho-position aminomethyl cyclohexanoic acid, the 2-position substitution products of HB are HB-45-HB-48 respectively, the 2-position substitution products of HC are HC-45-HC-48 respectively, and the structural formula is shown as follows:
Figure BDA0003362796890000134
example 24
HB (or HC) respectively reacts with methyl para, meta and ortho aminomethylcyclohexanoate, and the 2-position substitution products of HB are HB-45-CH respectively 3 ～HB-48-CH 3 The 2-substituted products of HC are HC-45-CH respectively 3 ～HC-48-CH 3 The structural formula is as follows:
Figure BDA0003362796890000135
example 25
HB (or HC) respectively reacts with para-position, meta-position and ortho-position aminomethylcyclohexanol, the 2-position substitution products of HB are HB-49-HB-52 respectively, the 2-position substitution products of HC are HC-49-HC-52 respectively, and the structural formula is shown as follows:
Figure BDA0003362796890000141
example 26
HB (or HC) respectively reacts with amino quaternary ammonium salts with different chain lengths (counter ions are bromide ions or iodide ions), 2-position substitution products of HB are HB-53-HB-56 respectively, 2-position substitution products of HC are HC-53-HC-56 respectively, and the structural formula is shown as follows:
Figure BDA0003362796890000142
Example 27
HB (or HC) respectively reacts with aminoethanol-quaternary ammonium salt with different chain length and aminopropanol-quaternary ammonium salt with different chain length (counter ion is Br) - Or I - ) The 2-position substitution products of HB are HB-57-HB-60 respectively, and the 2-position substitution products of HC are HC-57-HC-60 respectively, and the structural formulas are shown as follows:
Figure BDA0003362796890000143
example 28
HB (or HC) respectively reacts with the aminopropionic acid-quaternary ammonium salt and the aminobutyric acid-quaternary ammonium salt (the counter ion is Br) - Or I - ) The 2-position substitution products of HB are HB-61-HB-64, respectively, and the 2-position substitution products of HC are HC-61-HC-64, respectively, and the structural formulas are shown as follows:
Figure BDA0003362796890000151
example 29
HB (or HC) respectively reacts with amino propionic acid-amino quaternary ammonium salt and amino butyric acid-amino quaternary ammonium salt (counter ion is Br) - Or I - Son), HB 2-position substitution products are HB-65-HB-68 respectively, HC 2-position substitution products are HC-65-HC-68 respectively, the structural formula is shown as follows:
Figure BDA0003362796890000152
example 30
HB (or HC) respectively reacts with aminopiperidine, aminomorpholine and aminopiperidine, the 2-position substitution products of HB are HB-69 to HB-72 respectively, the 2-position substitution products of HC are HC-69 to HC-72 respectively, and the structural formula is shown as follows:
Figure BDA0003362796890000153
example 31
HB (or HC) respectively reacts with ethylenediamine, methyl ethylenediamine and dimethyl ethylenediamine, the 2-position substitution products of HB are HB-73-HB-76 respectively, the 2-position substitution products of HC are HC-73-HC-76 respectively, the structural formula is shown as follows:
Figure BDA0003362796890000154
Example 32
HB (or HC) respectively reacts with dimethylethylenediamine, butylethylenediamine and cyclohexanediamine, the 2-position substitution products of HB are HB-77-HB-80 respectively, the 2-position substitution products of HC are HC-77-HC-80 respectively, and the structural formula is shown as follows:
Figure BDA0003362796890000155
example 33
HB (or HC) respectively reacts with hydroxyethyl ethylenediamine and hydroxyethyl ethylenediamine-polyethylene glycol, the 2-position substitution products of HB are HB-81, HB-81-PEGn, HB-82-PEGn, the 2-position substitution products of HC are HC-81, HC-81-PEGn, HC-82-PEGn (n is an integer between 1 and 100), the structural formula is shown as follows:
Figure BDA0003362796890000161
example 34
HB (or HC) respectively reacts with hydroxybutyl ethylenediamine and hydroxybutyl ethylenediamine-polyethylene glycol, the 2-position substitution products of HB are HB-83, HB-83-PEGn, HB-84-PEGn, the 2-position substitution products of HC are HC-83, HC-83-PEGn, HC-84-PEGn (n is an integer between 1 and 100), the structural formula is shown as follows:
Figure BDA0003362796890000162
example 35:
HB (or HC) respectively reacts with dihydroxyethyl ethylenediamine and trihydroxyethyl ethylenediamine-polyethylene glycol, the 2-position substitution products of HB are HB-85-HB-88 respectively, the 2-position substitution products of HC are HC-85-HC-88 respectively, and the structural formula is shown as follows:
Figure BDA0003362796890000163
example 36
HB (or HC) respectively reacts with hydroxyethyl ethylenediamine, hydroxyethyl ethylenediamine methyl ether and hydroxyethyl ethylenediamine-polyethylene glycol, and the 2-position substitution products of HB are HB-89 and HB-89-CH respectively 3 The 2-position substitution products of HB-89-PEGn and HC are HC-89 and HC-89-CH respectively 3 HC-89-PEGn (n is an integer between 1 and 100), and has the following structural formula:
Figure BDA0003362796890000164
example 37
HB (or HC) respectively reacts with hydroxypropyl ethylenediamine, hydroxypropyl ethylenediamine methyl ether and hydroxypropyl ethylenediamine-polyethylene glycol, and the 2-position substitution products of HB are HB-90 and HB-90-CH respectively 3 The 2-position substitution products of HB-90-PEGn and HC are HC-90 and HC-90-CH respectively 3 HC-90-PEGn (n is an integer between 1 and 100), and has the following structural formula:
Figure BDA0003362796890000171
example 38
HB (or HC) respectively reacts with hydroxybutyl ethylenediamine, hydroxybutyl ethylenediamine methyl ether and hydroxybutyl ethylenediamine-polyethylene glycol, and the 2-position substitution products of HB are HB-91 and HB-91-CH respectively 3 The 2-position substitution products of HB-91-PEGn and HC are HC-91 and HC-91-CH respectively 3 HC-91-PEGn (n is an integer between 1 and 100), and has the following structural formula:
Figure BDA0003362796890000172
example 39
The structural general formula of the hypocrellin derivative is shown in figure 1. Reacting hypocrellin B HB (or deacetylated hypocrellin HC) with amino derivatives to mainly generate hypocrellin 2-amino substituted derivatives shown in formula (I); the hypocrellin and ethylenediamine derivative react to obtain hypocrellin ethylenediamine substituted derivative shown in formula (II) and formula (III). Taking HB and aminopentanol reaction as an example, mainly generating a 2-amino substituted derivative HB-4, and continuously esterifying the obtained product with carboxyl polyethylene glycol to obtain HB-4-PEGn, wherein the synthetic method is shown in figure 2; HC and methyl ethylenediamine react to mainly produce HC-74 and HC-75, and the synthesis method is shown in FIG. 3.
The derivatives of the present invention have maximum absorption wavelength of 580-650 nm and molar extinction coefficient of 15000-40000M -1 cm -1 The light absorption capacity is stronger in the phototherapy window; under the photosensitive condition, the device can not only efficiently generate singlet oxygen, but also generate a small amount of superoxide radical. The derivatives have better photostability and pH stability, and the specific detection results are as follows:
1) Absorption spectrum
FIG. 4 shows the absorption spectrum of the related compound of the present invention. In FIG. 4 (a), the commercial porphyrin-based photosensitizer PpIX is multi-band absorption, both are narrow absorption bands, and the maximum absorption wavelength usable for phototherapy is 570nm, its molar eliminationThe light coefficient is lower than 8000M -1 cm -1 The method comprises the steps of carrying out a first treatment on the surface of the The maximum absorption wavelength of the commercial photosensitizer Ce6 is 650nm, and the molar extinction coefficient is about 15000M -1 cm -1 It is also a narrow absorption in the phototherapy window, so commercial PpIX and Ce6 have limited absorption capacity in the phototherapy window. Whereas the absorption spectrum properties of the derivatives according to the invention are quite different. As shown in FIG. 4 (b), the maximum absorption peak of HB is around 470nm, the derivative HB-6-PEG1 in example 6 of the invention has a wide strong absorption in the phototherapy window, has a wide absorption band between 500 and 750nm, the maximum absorption peak is around 580nm, the maximum absorption peak is red-shifted by about 110nm from the maximum absorption peak of HB, and the molar extinction coefficient is about 20000M -1 cm -1 About, a strong red light absorbing ability is exhibited. Similarly, HC-74 is the reaction product of deacetylated hypocrellin and 2-methyl ethylenediamine in example 31 of the present invention, whose absorption spectrum undergoes a greater degree of red shift, is located in an ideal phototherapy window, has a very broad absorption band between 500 and 750nm, has a main absorption peak at 650nm, and exhibits a strong red light absorption capacity. The absorption spectrum of the other derivatives provided by the invention is similar to HB-6-PEG1 and HC-74, and the derivatives have a very wide absorption band between 500 and 750nm, and the maximum absorption peak is between 580 and 650 nm. Therefore, the hypocrellin derivative disclosed by the invention has better light absorption capacity in a phototherapy window than that of commercial photosensitizers PpIX and Ce6, and shows more outstanding red light absorption capacity.
2) Reactive oxygen species
FIG. 5 shows the measurement of active oxygen species of hypocrellin derivatives HB-45 and HC-45 (example 23) related to the invention by paramagnetic resonance method (ESR). As shown in FIG. 5, HB-45 and HC-45 can both efficiently generate Reactive Oxygen Species (ROS), and the derivatives can efficiently generate photosensitive active species, mainly singlet oxygen, and can also generate a small amount of superoxide radicals, as measured by singlet oxygen and superoxide radical capturing agents respectively. Both active oxygen species are advantageous for photodynamic therapy. The derivatives disclosed in other embodiments of the present invention also have the ability to efficiently generate singlet oxygen, assisting in the generation of small amounts of superoxide radicals.
3) Singlet oxygen efficiency
The compound of the present invention was tested for singlet oxygen efficiency in solution using the DHPA method. FIGS. 6 (a), 6 (b) and 6 (c) show the photodegradation curves of HB-73, HC-73 (example 31) and HC-80 (example 32), respectively. It can be seen that the photosensitizers HB-73, HC-73 and HC-80 obviously generate singlet oxygen under the illumination condition, thereby obviously degrading DHPA. By comparison and calculation with the reference rose bengal RB singlet oxygen efficiency curve, the singlet oxygen efficiencies of HB-73 and HC-73 were 0.38 and 0.40, respectively, while HC-80 produced the highest singlet oxygen efficiencies of up to 0.45. The singlet oxygen efficiency of other derivatives of the invention is between 0.15 and 0.45, and the derivatives have the capability of efficiently generating active oxygen.
4) Water-solubility
Most of the derivatives disclosed by the invention contain hydrophilic groups such as polyethylene glycol, quaternary ammonium salt, sulfonic acid, carboxylic acid and the like, so that the photosensitizer molecule has stronger water solubility under physiological conditions. The photosensitizer molecule can be well dissolved in each milliliter of normal saline or glucose injection, so that the photosensitizing drug can be well transported in blood vessels without causing blood vessel blockage during intravenous injection. Such as HB-1-PEG8 (example 1), containing 8 ethylene glycol units soluble in more than 5 mg per ml of physiological saline; HB-1-PEG16 contains 16 glycol units, and can be dissolved by more than 10 mg per milliliter of physiological saline; HC-3-PEG6 of example 3 was dissolved in 5 mg/ml saline; HB-6-PEG16 of example 6 can be dissolved in 15 mg/ml physiological saline; HC-8-PEG12 in example 8 was dissolved in 10 mg/ml of physiological saline; these photosensitizer molecules all exhibit excellent water solubility. The other derivatives containing the strong water-soluble groups have better water solubility and biocompatibility, and can dissolve more than 1-20 milligrams of photosensitive drug molecules per milliliter of physiological saline.
5) Light stability
The photostability of the derivatives of the present invention and commercial photosensitizers are shown in FIG. 7, for example. As can be seen, a 635nm laser was used at 20mW/cm 2 The absorption spectra of HB-2-PEG4 and HC-2-PEG8 (example 2) are not significantly reduced under light intensity illumination for 30min, and the absorption intensity of the maximum wavelength is reduced by less than 10%The light stability is better; the decrease in absorption intensity at the maximum wavelength of the derivatives HC-73 (example 31) and HC-80 (example 32) was also less than 10%, and also had better photostability. Under the same conditions, a 635nm laser was used at 20mW/cm 2 The maximum absorption of commercial photosensitizers Ce6 and HpD was reduced by 30% and 50% respectively when illuminated at light intensity for 30 min. Other derivatives of the invention also have better photostability, and the decrease of absorption intensity of the maximum wavelength is basically less than 10% under the same conditions. Therefore, the hypocrellin derivatives of the present invention have better photostability than commercial photosensitizers.
6) pH stability
The pH stability of hypocrellin derivatives of the present invention is shown in FIG. 8. The derivative has no obvious change in absorption spectrum within the pH range of 6.2-8.0, which indicates that the derivative has better pH stability under physiological conditions. As shown in the figure, the absorption spectra of HB-3-PEG2 and HC-3-PEG8 (example 3) have no obvious change in the pH range of 6.2-8.0; HC-73 (example 31) and HB-77 (example 32) had no significant change in their absorption spectra in the pH range of 6.2 to 8.0. In this range, the pH stability is good because the two phenolic hydroxyl groups of hypocrellin are less susceptible to deprotonation of the phenolic hydroxyl groups under these conditions. Whereas commercial hematoporphyrin HpD contains two carboxyl groups, which can be deprotonated in the pH range 6.2-8.0, resulting in a significant change in the absorbance spectrum and thus exhibiting instability of the HpD photosensitizer. Other derivatives of the invention have better pH stability under physiological conditions, and the change in pH range of 6.2-8.0 has less influence on the absorption spectrum of the photosensitive medicament. Therefore, the hypocrellin derivative of the present invention has better pH stability than commercial line HpD.
The structure of the partial hypocrellin derivative is shown in examples 1-38, the maximum absorption wavelength of the partial hypocrellin derivative is about 580-630 nm, and the molar extinction coefficient can reach 15000-40000M -1 cm -1 The light absorption capacity is very strong in a phototherapy window; active oxygen species such as singlet oxygen and the like can be efficiently generated under the photosensitive condition, and the singlet oxygen efficiency can reach about 40 percent at the highest;the derivatives have better photostability and pH stability, and partial photophysical data of the derivatives are shown in table 1.
Table 1: photophysical data of part of hypocrellin derivatives of the present invention
Figure BDA0003362796890000191
Example 40
Culturing tumor cells: various cell lines (esophageal cancer cell AKR, gastric cancer cell MFC, lung cancer cell a549, liver cancer cell HepG2, biliary cancer cell MCC, colon cancer cell HCT116, brain cancer cell G442, head and neck cancer cell SCC2, tongue cancer cell TSCCa, nasal cancer cell KB, oral cancer cell CAL27, brain glioma cell C6, basal cell carcinoma cell BCC, squamous skin cancer cell PECA, melanoma cell B16, skin T cell lymphoma cell HH, prostate cancer cell LNCaP, bladder cancer cell MBT-2) were provided by the beige co-ordinates college cell center. The culture conditions of the above cells were: RPMI-1640 medium was supplemented with 10% FBS,1% streptomycin (100. Mu.g/mL) and penicillin (100. Mu.g/mL), and placed at 37℃in 5% CO 2 Culturing in an incubator. Tumor cells were inoculated in a glass-based confocal dish, a solution of hypocrellin-sensitive drug (100 μl) was added to the culture broth, and incubated in an incubator for 4 hours. The cells were carefully washed twice with pre-chilled PBS solution to remove the light sensitive drugs that did not enter the cells and the cultured tumor cells were used for MTT experiments.
Example 41
Cytotoxicity experiments, illustrated by HB-1-PEG8 (example 1): the cultured lung cancer cells (A549 cells) were digested with 0.25% trypsin to prepare a single cell suspension, which was inoculated in 96-well plates and placed in an incubator for culture. After the cells are attached, the culture solution of the supernatant is discarded, photosensitizers (such as HB-1-PEG 8) with different concentrations are added under the dark condition for further incubation for 1h, and the survival rate of the cells is detected by an MTT method. 20uL MTT was added to each well, the culture was continued for 4 hours, the supernatant was discarded, DMSO (150 uL) was added to each well, and shaking was performed with a micro-shaker for 10 minutes to allow the purple crystals to be sufficiently dissolved. OD values (570 nm) of each well were measured on a microplate reader and cell viability was calculated. Cell viability = experimental OD/blank OD x 100%. As shown in FIG. 10-2, cytotoxicity (dark toxicity) studies showed that HB-1-PEG8 was less cytotoxic, similar to commercial photosensitizers HpD, incubated for half an hour with a 20 μm concentration of photosensitizers, and no apparent death of A549 cells was seen, indicating that such photosensitizers were substantially devoid of cytotoxicity. The dark toxicity of other derivatives of the present invention was similar to HB-1-PEG8, and the results are shown in FIGS. 10-14 and tables 2-4, which show that the derivatives are substantially free of cytotoxicity, and the cell viability was over 90% at a concentration of 20. Mu.M.
Example 42
Cytotoxicity experiments were performed using HC-1-PEG8 synthesized in example 1 as an example:
experimental procedure As dark toxicity experiments, semiconductor laser with wavelength of 635nm (20 mW/cm 2 ) Irradiation was performed so that the light beam was uniformly and vertically irradiated onto the 96-well plate for 1000 seconds. The cytotoxicity experiments as shown in FIG. 10-1 indicate that HC-1-PEG8 shows very strong killing power on A549 cells under red light irradiation. The concentration range of 50nM can kill more than 90% of A549 cells, while under the same condition, the commercial photosensitizer can only kill about 20% of A549 cells, which shows that the photodynamic effect of the derivative is obviously better than that of the commercial photosensitizer HpD. The phototoxicity of other derivatives of the invention is similar to HC-1-PEG8, and the results are shown in FIGS. 10-14, the synthesized derivatives can kill more than 80-90% of tumor cells in the 50nM concentration range, and the semi-lethal concentration IC 50 The value is about 20-30 nM. Therefore, the hypocrellin derivatives disclosed by the invention have better photodynamic effect than the commercial photosensitizer HpD.
Example 43
Animal experiment: all animal experiment operations conform to the regulations of animal use and feeding in China animal research ethics committee. Experiments A tumor model was established using 4-6 week female Balb/c nude mice (about 20 g), and 50. Mu.L of each of the suspensions of tumor cells (5X 10) was injected into the rear side of the right thigh of the mice 6 And (c) a). Tumor volume grows to about 200mm 3 In-vivo fluorescence imaging and photodynamic therapy experiments were performed.
Small animal fluorescence imaging experiments: after injecting 40. Mu.L of physiological saline solution (10 mg/mL) of photosensitive drug into tumor-bearing mice by tail vein at mouse dosage (10 mg/kg), tumor fluorescence imaging was respectively acquired for 0, 1, 2, 4, 6 and 8 hours by using living animal imaging. After 8 hours, tumor-bearing mice are killed by cervical removal, tumors and major organs are taken out for in vitro fluorescence imaging, and the average fluorescence intensity in the ROS region is counted for semi-quantitative analysis.
Photodynamic therapy experiments: when the tumor volume is as long as 200mm 3 At this time, tumor-bearing mice were randomly divided into 3 groups (5 per group): without any treatment (group I); only tail vein (100 μl,500 μΜ) of the photosensitive drug (group II); the tail vein was injected with a photosensitizing drug (100. Mu.L, 500. Mu.M) and after 4h the tumor site was irradiated with 635nm laser (10 min,0.1W cm) -2 ) (group III). Tumor volume size and body weight were measured every other day after laser treatment and survival was recorded.
Example 44
As described above, the hypocrellin derivatives of the present invention were tested for pharmaceutical activity, and specific results are as follows:
1) Confocal fluorescence imaging of hypocrellin derivatives in cancer cells
The cytofluorescence imaging of hypocrellin derivatives of the present invention is shown in FIG. 9. The photosensitizer HB-1-PEG12 has better water solubility and biocompatibility, and is incubated with lung cancer cells (A549), the photosensitizer can be found to rapidly enter lysosomes of the cells and generate red fluorescence in the cells, the HB-1-PEG12 can be used for fluorescence imaging of the lung cancer cells, and enrichment, distribution and metabolism conditions of the drugs in vivo can be tracked through fluorescence detection. The DCFH-DA is used for detecting intracellular singlet oxygen, the intracellular co-incubation photosensitizer HB-1-PEG12 and the fluorescent probe DCFH-DA are increased to 120s along with the irradiation time, and green fluorescence is gradually enhanced, which indicates that intracellular singlet oxygen is increased. The other derivatives can well enter lysosomes of lung cancer cells, and can carry out cell fluorescence imaging, and enrichment, distribution and metabolism conditions of the medicines in the body can be tracked through fluorescence detection.
2) Dark toxicity and phototoxicity test of hypocrellin derivatives on digestive tract tumor cells
The digestive tract tumor cells mainly comprise esophageal cancer cells, gastric cancer cells, lung cancer cells, liver cancer cells, bile duct cancer cells and colon cancer cells.
Incubation of HB-1-PEG4 and HC-1-PEG8 (example 1) with esophageal cancer cell AKR cells As shown in FIG. 10-1 (a), studies of cytotoxicity (dark toxicity) of the photosensitizing drug in the absence of light showed that cytotoxicity of HB-1-PEG4 and HC-1-PEG8 was smaller, either with 4 PEG or 8 PEG units, similar to that of commercial photosensitizing drug HpD. Incubation with 20. Mu.M concentrations of photosensitizer HB-1-PEG4 or HC-1-PEG8 for half an hour showed no apparent death of esophageal cancer cells, indicating that such photosensitizers were substantially free of cytotoxicity. Cytotoxicity study as shown in FIG. 10-1 (b) revealed that the photosensitizing drug was irradiated with 635nm red light (10 min,0.1W cm) -2 ) Shows very strong killing power to esophageal cancer cells. HB-1-PEG4 with 50nM concentration can kill more than 90% of esophageal cancer cells, semi-lethal concentration IC 50 The value (drug concentration required to kill half of the tumor cells) was about 25nM; HC-1-PEG8 with 50nM concentration can kill more than 90% of esophageal cancer cells, and IC with semi-lethal concentration 50 A value of about 25nM; the commercial photosensitizer HpD can only kill about 30% of esophageal cancer cells under the same condition, which shows that the photodynamic killing effect of the derivative on esophageal cancer is obviously better than that of the commercial photosensitizer.
Incubation of the derivatives of the present invention with lung cancer cell A549, as shown in FIG. 10-2 (a), showed that HB-3-PEG8 and HC-3-PEG16 (example 3) were less cytotoxic, similar to the commercial photosensitizing drug hematoporphyrin HpD. The lung cancer cells were incubated with 20. Mu.M concentration of photosensitizer HB-3-PEG8 or HC-3-PEG16 for half an hour, and no apparent death of the lung cancer cells was seen, indicating that such photosensitizers were not cytotoxic. The cytotoxicity experiments as shown in FIG. 10-2 (b) show that the photosensitizing drug shows very strong killing power on lung cancer cells under red light irradiation. HB-3-PEG8 or HC-3-PEG16 with 50nM concentration can kill more than 90% of lung cancer cells, semi-lethal concentration IC 50 A value of about 30nM; the commercial photosensitizer HpD can only kill about 20% of lung cancer cells under the same condition, which proves that the photodynamic effect of the hypocrellin derivative is obviously better than that of the hypocrellin derivativeCommercial photosensitizers.
The dark toxicity study of the photosensitizing drug shows that both HB-6-PEG2 and HC-6-PEG6 prepared in example 6 have less cytotoxicity, similar to commercial photosensitizing drug HpD, as shown in FIG. 10-3 (a) when the derivatives of the present invention are incubated with hepatoma cell HepG 2. The liver cancer cells are incubated for half an hour by using the photosensitizer with the concentration of 20 mu M, and no obvious death of the liver cancer cells is seen, which indicates that the photosensitizer basically has no cytotoxicity. The cytotoxicity study shown in FIG. 10-3 (b) shows that the photosensitizing drug shows very strong killing power to liver cancer cells under red light irradiation. HB-6-PEG2 or HC-6-PEG12 with 50nM concentration can kill more than 85% of liver cancer cells, and IC with semi-lethal concentration 50 A value of about 30nM; under the same conditions, the commercial hematoporphyrin derivative HpD only kills about 20% of liver cancer cells, which proves that the photodynamic effect of the hypocrellin derivative is obviously better than that of a commercial photosensitizer.
Incubation of the derivative of the present invention with HCT116, a colon cancer cell, as shown in FIGS. 10-4 (a), showed that the dark toxicity study of the photosensitizing drug showed that either HB-14-PEG6 or HC-14-PEG12 prepared in example 13 was less cytotoxic, similar to the commercial photosensitizing drug HpD. Colon cancer cells were incubated for half an hour with 20 μm concentration of photosensitizer, no apparent death of colon cancer cells was seen, indicating that such photosensitizer was essentially free of cytotoxicity. Cytotoxicity studies as shown in fig. 10-4 (b) indicate that the photosensitizing drug exhibits very strong killing power on colon cancer cells under red light irradiation. HB-14-PEG6 and HC-14-PEG12 with 50nM concentration can kill more than 85% of colon cancer cells, and IC with semi-lethal concentration 50 A value of about 25nM; the commercial photosensitizer HpD can only kill about 20% of colon cancer cells under the same condition, which shows that the photodynamic effect of the derivative is obviously better than that of the commercial photosensitizer HpD.
Incubation of the derivative of the present invention with cholangiocarcinoma cell MCC as shown in FIGS. 10-5 (a), the dark toxicity study of the photosensitizing drug showed that both HB-19-PEG4 and HC-19-PEG8 prepared in example 15 were less cytotoxic, similar to the commercial photosensitizing drug HpD. Bile duct cancer cells were incubated with photosensitizer at 20. Mu.M concentration for half an hour, and no obvious bile duct cancer cells were seenIndicating that such photosensitizers are substantially free of cytotoxicity. The cytotoxicity study experiments shown in fig. 10-5 (b) show that the photosensitizing drug shows very strong killing power to bile duct cancer cells under red light irradiation. HB-19-PEG4 and HC-19-PEG8 with 50nM concentration can kill bile duct cancer cells more than 85%, and IC with semi-lethal concentration 50 A value of about 30nM; under the same conditions, the commercial photosensitizer HpD can only kill about 20% of bile duct cancer cells, which proves that the photodynamic effect of the hypocrellin derivatives is obviously better than that of the commercial photosensitizer HpD.
Incubation of the derivatives of the present invention with gastric cancer cell MFC as shown in FIGS. 10-6 (a) revealed that both HB-45 and HC-45 prepared in example 23 were less cytotoxic and similar to the commercial photosensitizing drug hematoporphyrin HpD. Gastric cancer cells were incubated for half an hour with 20 μm concentration of photosensitizer without significant death of MFC cells, indicating that such photosensitizers were essentially devoid of cytotoxicity. Cytotoxicity studies as shown in fig. 10-6 (b) indicate that the photosensitizing drug exhibits very strong killing power against gastric cancer cells under red light irradiation. HB-45 and HC-45 with 50nM concentration can kill more than 85% of gastric cancer cells, IC 50 A value of about 30nM; the commercial photosensitizer HpD can only kill about 20% of gastric cancer cells under the same condition, which shows that the photodynamic effect of the hypocrellin derivatives is obviously better than that of the commercial photosensitizer.
The above details illustrate that part of hypocrellin derivatives disclosed by the invention can effectively kill digestive tract tumor cells such as esophageal cancer AKR, gastric cancer MFC, lung cancer A549, liver cancer HepG2, cholangiocarcinoma MCC, colon cancer HCT116 and the like. Whether Hypocrellin (HB) or a derivative of deacetylated Hypocrellin (HC), the cells are not damaged basically by no illumination, and the capability of inactivating the digestive tract tumor cells is strong under illumination. In addition, hypocrellin derivatives with different PEG chain lengths, different sulfonic acid group chain lengths and different quaternary ammonium salt chain lengths all show better photodynamic cell inactivation capacity on tumor cells. Therefore, the derivatives can effectively kill digestive tract tumor cells such as esophageal cancer, gastric cancer, lung cancer, liver cancer, cholangiocarcinoma, colon cancer and the like, and related data are shown in table 2.
Table 2: MTT data of partial hypocrellin derivatives of the present invention on digestive tract tumor cells
Figure BDA0003362796890000221
3) Dark toxicity and phototoxicity test of hypocrellin derivatives on tumor cells of head, neck and face
The tumor cells of head and neck and face mainly comprise head and neck cancer cells, brain cancer cells, tongue cancer cells, nose cancer cells, oral cancer cells and brain glioma cells.
The hypocrellin derivative disclosed by the invention can effectively kill head and neck tumor cells such as brain cancer, head and neck cancer, tongue cancer, nose cancer, oral cancer, brain glioma and the like under 635nm laser irradiation. Taking the derivatives of hypocrellin disclosed in the examples as examples, the phototoxic and darktoxic effects on tumor cells of the head, neck and face are illustrated.
Dark toxicity studies of photosensitizing drugs by incubating the derivatives of the invention with brain cancer cells G442 showed that HB-10-PEG4 and HC-10-PEG8 (example 10) were less cytotoxic, either with 4 PEG or 8 PEG units, similar to the commercial photosensitizing drug hematoporphyrin HpD. Brain cancer cells were incubated with 20. Mu.M concentrations of photosensitizers HB-10-PEG4 and HC-10-PEG8 for half an hour, and no apparent death of brain cancer cells was seen, indicating that such photosensitizers were substantially devoid of cytotoxicity. Cytotoxicity studies as shown in fig. 11 (a) indicate that the photosensitizing drug exhibits very strong killing power against brain cancer cells under red light irradiation. HB-10-PEG4 and HC-10-PEG8 with 50nM concentration can kill more than 85% of brain cancer cells, and IC with semi-lethal concentration 50 A value of about 30nM; the commercial photosensitizer HpD can only kill about 20% of brain cancer cells under the same condition, which shows that the photodynamic effect of the hypocrellin derivatives is obviously better than that of the commercial photosensitizer HpD.
The dark toxicity study of the photosensitizing drug by incubating the derivatives of the invention with the head and neck cancer cell SCC2 shows that HB-12-PEG8 and HC-12-PEG12 prepared in example 12 have smaller cytotoxicity, similar to commercial photosensitizing drug HpD. Head and neck cancer cells were incubated with photosensitizer at 20 μm concentration for half an hour, no apparent death of head and neck cancer cells was seen, indicating that such photosensitizer was not cytotoxic. Cytotoxicity studies as shown in fig. 11 (b) indicate that the photosensitizing drug exhibits very strong killing power against the cancer cells of the head and neck under red light irradiation. HB-12-PEG8 and HC-12-PEG12 with 50nM concentration can kill more than 85% of head and neck cancer cells, and IC with semi-lethal concentration 50 A value of about 30nM; the commercial photosensitizer HpD can only kill about 20% of head and neck cancer cells under the same condition, which shows that the photodynamic effect of the hypocrellin derivatives is obviously better than that of the commercial photosensitizer HpD.
The dark toxicity study of the photosensitizing drug by incubating the derivative of the present invention with the tongue cancer cell TSCCa shows that both HB-20-PEG6 and HC-20-PEG12 prepared in example 16 are less cytotoxic, similar to the commercial photosensitizing drug HpD. Tongue cancer cells were incubated for half an hour with 20 μm concentration of photosensitizer, no apparent death of tongue cancer cells was seen, indicating that such photosensitizer was essentially free of cytotoxicity. Cytotoxicity studies as shown in fig. 11 (c) indicate that the photosensitizing drug exhibits very strong killing power against tongue cancer cells under red light irradiation. HB-20-PEG6 and HC-20-PEG12 with 50nM concentration can kill more than 85% of tongue cancer cells, and IC with semi-lethal concentration 50 A value of about 30nM; under the same conditions, the commercial photosensitizer HpD can only kill about 20% of tongue cancer cells, which proves that the photodynamic effect of the derivative is obviously better than that of the commercial photosensitizer.
Dark toxicity studies of photosensitizing drugs by incubating the derivatives of the present invention with nasal cancer cell KB showed that HB-29 and HC-29 prepared in example 19 were both less cytotoxic, similar to the commercial photosensitizing drug hematoporphyrin HpD. The cytotoxicity study experiment shown in fig. 12 (a) shows that the photosensitizing drug shows very strong killing power to the nasal cancer cells under the irradiation of red light. HB-29 and HC-29 with 50nM concentration can kill more than 90% KB cells, and IC with semi-lethal concentration 50 A value of about 30nM; the commercial photosensitizer HpD can only kill about 20% of KB cells under the same condition, which shows that the photodynamic effect of the hypocrellin derivatives is obviously better than that of the commercial photosensitizer HpD.
Incubation of the derivatives of the present invention with CAL27, an oral cancer cell, showed that the cytotoxicity of HB-61 and HC-61 prepared in example 28 was less, similar to that of the commercial photosensitizing drug hematoporphyrin HpD. Cytotoxicity studies as shown in fig. 12 (b) indicate that the photosensitizing drug exhibits very strong killing power against oral cancer cells under red light irradiation. HB-61 and HC-61 with 50nM concentration can kill more than 90% of oral cancer cells, and IC with semi-lethal concentration 50 A value of about 25nM; the commercial photosensitizer HpD can only kill about 25% of oral cancer cells under the same condition, which shows that the photodynamic effect of the hypocrellin derivatives is obviously better than that of the commercial photosensitizer HpD.
The dark toxicity study of the photosensitizing drug shows that hypocrellin derivatives HB-73 prepared in example 31, HC-80 prepared in example 32 and the derivatives have smaller cytotoxicity to the glioma, which is similar to commercial photosensitizing drug hematoporphyrin HpD. Cytotoxicity studies as shown in fig. 12 (c) indicate that the photosensitizing drug exhibits very strong killing power against brain glioma cells under red light irradiation. HB-73 and HC-80 with 50nM concentration can kill more than 85% of brain glioma cells, and IC with semi-lethal concentration 50 A value of about 30nM; the commercial photosensitizer HpD can only kill about 20% of C6 cells under the same condition, which shows that the photodynamic effect of the hypocrellin derivative is obviously better than that of the commercial photosensitizer HpD.
The above details illustrate that part of hypocrellin derivatives disclosed by the invention can effectively kill head and neck tumor cells such as brain cancer G442, head and neck cancer SCC2, tongue cancer TSCCa, nose cancer KB, oral cancer CAL27, brain glioma C6 and the like. Neither HB nor HC derivatives have substantial damage to cells when exposed to light, but have a strong ability to inactivate the tumor cells of the digestive tract when exposed to light. In addition, hypocrellin derivatives with different PEG chain lengths, different sulfonic acid group chain lengths and different quaternary ammonium salt chain lengths all show better photodynamic cell inactivation capacity on tumor cells. Therefore, the derivatives can effectively kill head and neck tumor cells such as brain cancer, head and neck cancer, tongue cancer, nose cancer, oral cancer, brain glioma and the like, and the related data are shown in table 3.
Table 3: MTT data of partial hypocrellin derivatives of the present invention on tumor cells of the head, neck and face
Figure BDA0003362796890000241
4) Dark toxicity and phototoxicity test of hypocrellin derivatives on tumor cells of reproductive and urinary systems
The hypocrellin derivative disclosed by the invention can effectively kill tumor cells of genitourinary systems such as basal cell carcinoma, cutaneous T cell lymphoma, melanoma, squamous skin carcinoma, prostatic cancer, bladder cancer and the like under 635nm laser irradiation. Taking hypocrellin derivatives disclosed in examples as an example, their phototoxic and darktoxic effects on skin tumor and urinary system tumor cells are illustrated.
Incubation of the derivatives of the present invention with basal cell carcinoma cell BCC, the study of dark toxicity of the photosensitizing drug showed that both HB-4-PEG8 and HC-4-PEG16 (example 4) were less cytotoxic, similar to the commercial photosensitizing drug hematoporphyrin HpD. Incubation with photosensitizer at 20 μm concentration for half an hour showed no apparent death of basal cell carcinoma cells, indicating that such photosensitizer was essentially free of cytotoxicity. Cytotoxicity studies as shown in fig. 13 (a) indicate that the photosensitizing drug exhibits very strong killing power against basal cell carcinoma cells under red light irradiation. HB-4-PEG8 and HC-4-PEG16 with 50nM concentration can kill more than 90% of basal cell carcinoma cells, and IC with semi-lethal concentration 50 A value of about 30nM; under the same conditions, the commercial photosensitizer hematoporphyrin derivative HpD can only kill about 20% of basal cell carcinoma cells, which proves that the photodynamic effect of the hypocrellin polyethylene glycol derivative is obviously better than that of the commercial photosensitizer hematoporphyrin HpD.
Incubation of the derivatives of the present invention with melanoma cells B16 showed that the dark toxicity study of the photosensitizing drug showed that HB-9-PEG4 and HC-9-PEG8 prepared in example 9 were less cytotoxic and similar to the commercial photosensitizing drug hematoporphyrin HpD. Cytotoxicity studies as shown in FIG. 13 (b) indicate that the photosensitizing agentThe composition shows very strong killing power on melanoma cells under red light irradiation. HB-9-PEG4 and HC-9-PEG8 with 50nM concentration can kill more than 85% of melanoma cells, and IC with semi-lethal concentration 50 A value of about 30nM; the commercial photosensitizer HpD can only kill about 20% of melanoma cells under the same condition, which shows that the photodynamic effect of the hypocrellin derivatives is obviously better than that of the commercial photosensitizer HpD.
The dark toxicity study of the photosensitizing drug by incubating the derivative of the present invention with squamous skin carcinoma cells PECA shows that both HB-36 and HC-36 prepared in example 20 are less cytotoxic, similar to commercial photosensitizing drug HpD. Cytotoxicity studies as shown in fig. 13 (c) indicate that the photosensitizing drug exhibits very strong killing power on PECA cells under red light irradiation. HB-36 and HC-36 with 50nM concentration can kill more than 85% of skin cancer cells, and IC with semi-lethal concentration 50 A value of about 30nM; the commercial photosensitizer HpD can only kill about 20% of PECA cells under the same conditions, which shows that the photodynamic effect of the hypocrellin derivatives is obviously better than that of the commercial photosensitizer HpD.
Incubation of the derivative of the present invention with prostate cancer cells LNCaP revealed that both HB-70 and HC-71 prepared in example 30 were less cytotoxic and similar to the commercial photosensitizing drug hematoporphyrin HpD. Cytotoxicity studies as shown in fig. 14 (a) indicate that the photosensitizing drug exhibits very strong killing power against prostate cancer cells under red light irradiation. The HB-70 and HC-71 with 50nM concentration can kill more than 85% of prostate cancer cells, and the IC with semi-lethal concentration 50 A value of about 30nM; the commercial photosensitizer HpD can only kill about 20% of prostate cancer cells under the same conditions, which shows that the photodynamic effect of the hypocrellin derivative is obviously better than that of the commercial photosensitizer HpD.
Incubation of the derivatives of the present invention with bladder cancer cell MBT-2, photo-sensitive drug darkness toxicity studies showed that HB-73 and HC-73 prepared in example 31 were less cytotoxic, similar to commercial photo-sensitive drug hematoporphyrin HpD. As shown in FIG. 14 (b), cytotoxicity studies indicate that the photosensitizing drug exhibits very strong effect on bladder cancer cells under red light irradiation Killing power. HB-73 and HC-73 with 50nM concentration can kill more than 85% of bladder cancer cells, and IC with semi-lethal concentration 50 A value of about 25nM; under the same conditions, the commercial photosensitizer HpD can only kill about 20% of bladder cancer cells, which proves that the photodynamic effect of the hypocrellin polyethylene glycol derivative is obviously better than that of the commercial photosensitizer.
The dark toxicity study experiment of the photosensitizing drug shows that the cytotoxicity of HC-80 prepared in example 32 and HC-81 prepared in example 33 is smaller and similar to that of the commercial photosensitizing drug hematoporphyrin HpD when the derivative is incubated with skin T cell lymphoma cell HH. Cytotoxicity studies as shown in fig. 14 (c) indicate that the photosensitizing drug exhibits very strong killing power against cutaneous T cell lymphoma cells under red light irradiation. HC-80 and HC-81 with 50nM concentration can kill more than 85% of HH cells, and IC with semi-lethal concentration 50 A value of about 30nM; the commercial photosensitizer HpD can only kill 20% of HH cells under the same condition, which shows that the photodynamic effect of the hypocrellin derivative is obviously better than that of the commercial photosensitizer.
The above details illustrate that some hypocrellin derivatives disclosed in the present invention can kill tumor cells of the skin and urinary system such as basal cell carcinoma BCC, squamous skin carcinoma PECA, melanoma B16, cutaneous T cell lymphoma HH, prostate cancer LNCaP, bladder cancer MBT-2, etc. with high efficiency. Whether hypocrellin or a derivative of deacetylated hypocrellin, the cells are not damaged basically by no illumination, and the capability of inactivating the digestive tract tumor cells is very strong under illumination. In addition, hypocrellin derivatives with different PEG chain lengths, different sulfonic acid group chain lengths and different quaternary ammonium salt chain lengths all show better photodynamic cell inactivation capacity on tumor cells. Therefore, the derivatives can effectively kill tumor cells of the reproductive and urinary systems such as basal cell carcinoma, squamous skin carcinoma, melanoma, cutaneous T cell lymphoma, prostatic cancer, bladder cancer and the like, and the detailed data are shown in table 4.
Table 4: MTT data of partial hypocrellin derivatives of the present invention on skin tumor and genitourinary tumor cells
Figure BDA0003362796890000261
The above study showed that: the hypocrellin derivative has better photodynamic killing effect on various tumor cells by changing the molecular structure and different PEG chain lengths and end groups: FIGS. 10-14 and tables 2-4 illustrate that such derivatives have little cytotoxicity in the absence of light, and have significant photodynamic effects in the presence of light, and that a 50nM concentration of photosensitizing drug is effective in killing a substantial portion of various tumor cells, a semi-lethal concentration of IC 50 IC having a value of about 20 to 30nM, and being lower than that of commercial photosensitizer HpD under the same conditions 50 The value is 1-2 orders of magnitude lower. Other derivatives of the invention also have dark and phototoxicity as shown in FIGS. 10-14. Therefore, the hypocrellin 2-amino substituted or ethylenediamine substituted derivatives disclosed in the present invention have better dark toxicity and photodynamic effect than commercial photosensitizers HpD.
EXAMPLE 45 animal imaging
The fluorescence imaging of the cells (FIG. 9) shows that the derivatives provided by the invention can well enter tumor cells to generate near infrared fluorescence imaging. In order to study the enrichment and metabolic process of the derivatives at the tumor in vivo, the application takes tumor-bearing mice with various tumor cells as models, and studies the enrichment process of the derivatives at the tumor of the mice through in vivo fluorescence imaging of small animals. The target enrichment of the medicine at the tumor position is realized by tail intravenous injection through the derivatives with better water solubility, such as polyethylene glycol, sulfonic acid group, carboxylic acid group, quaternary ammonium salt and the like; and for the derivatives with slightly poor water solubility, the enrichment of the photosensitive drugs in the tumor area is realized by injecting the photosensitive drugs into the tumor of the tumor-bearing mice.
1) Drug enrichment in digestive tract tumor mice (esophageal cancer, gastric cancer, lung cancer, liver cancer, cholangiocarcinoma, colon cancer)
Subcutaneous tumors were inoculated with esophageal cancer AKR cells to obtain esophageal cancer subcutaneous mouse tumor models. HB-1-PEG4 (example 1) was used as a photosensitizing drug and injected into tumor-bearing mice via tail vein at a dose of 10mg/kg, and fluorescence imaging behavior was observed in tumor-bearing mice with esophageal carcinoma, and fluorescence signals at 4 hours tumor sites were collected by using a multispectral small animal in vivo imaging system, respectively. As shown in FIG. 15 (a), after intravenous injection of the drug molecule HB-1-PEG4 in vivo for several cycles, the drug is enriched at the tumor site for about 4 hours by passive targeting, which is a moderate enrichment because the derivative molecule contains only 4 polyethylene glycol units, and the whole drug molecule has insufficient water solubility, thereby resulting in a moderate enrichment at the tumor site.
And inoculating the subcutaneous tumor with gastric cancer MFC cells to obtain the gastric cancer subcutaneous mouse tumor model. HC-3-PEG12 (example 3) was injected into tumor-bearing mice via tail vein at a dose of 10mg/kg as a photosensitizing drug, and fluorescence imaging behavior was observed to collect fluorescence signals from tumor sites at 4 hours. As shown in FIG. 15 (b), after the drug molecule HC-3-PEG12 is injected intravenously for a plurality of times, the drug is enriched in the tumor site by passive targeting for about 4 hours, because the derivative molecule contains 12 polyethylene glycol units, the water solubility of the drug molecule is better, the hydrophilic-hydrophobic ratio of the whole molecule is proper, and the drug is enriched in the tumor site.
The lung cancer A549 cells are used for inoculating subcutaneous tumor, and the lung cancer subcutaneous mouse tumor model is obtained. HB-6-PEG8 (example 6) was used as a photosensitizing drug and injected into tumor-bearing mice via tail vein at a dose of 10mg/kg, and fluorescence imaging behavior was observed in lung cancer tumor-bearing mice, and fluorescence signals at 4 hours tumor sites were collected. As shown in FIG. 15 (c), after intravenous injection for several times, the drug molecule HB-6-PEG8 has strong enrichment at the tumor site by passive targeting for about 4h, because the derivative molecule contains 8 polyethylene glycol units, so that the water solubility of the molecule is better, the hydrophilic-hydrophobic ratio of the whole molecule is proper, and the tumor site has strong enrichment.
Subcutaneous tumors were inoculated with hepatoma HepG2 cells to obtain a hepatoma subcutaneous mouse tumor model. HB-7-PEG16 (example 7) was used as a photosensitizing drug and injected into tumor-bearing mice via tail vein at a dose of 10mg/kg, and fluorescence imaging behavior in liver cancer tumor-bearing mice was observed, and fluorescence signals at 4 hours tumor sites were collected. As shown in FIG. 15 (d), HB-7-PEG16 has strong enrichment at tumor site after intravenous injection for several times in vivo for about 4h, because the molecule contains 16 polyethylene glycol units, so that the whole drug molecule has good water solubility and proper hydrophilic-hydrophobic ratio, thereby causing strong enrichment at tumor site.
Subcutaneous tumors are inoculated with cholangiocarcinoma MCC cells to obtain a cholangiocarcinoma subcutaneous mouse tumor model. HC-9-PEG10 (example 9) was injected into tumor-bearing mice via tail vein at a dose of 10mg/kg as a photosensitizing drug, and fluorescence imaging behavior in bile duct cancer tumor-bearing mice was observed, and fluorescence signals at 4 hours tumor sites were collected. As shown in FIG. 15 (e), HC-9-PEG10 is strongly enriched in tumor sites by passive targeting for about 4h after several cycles in vivo by intravenous injection, because the molecule contains 10 polyethylene glycol units, so that the whole drug molecule has good water solubility and proper hydrophilic-hydrophobic ratio, thereby leading to strong enrichment in tumor sites.
Subcutaneous tumors were inoculated with colon cancer HCT116 cells to obtain a colon cancer subcutaneous mouse tumor model. HC-1-PEG50 (example 1) was injected into tumor-bearing mice via tail vein at a dose of 10mg/kg as a photosensitizing drug, and fluorescence imaging behavior in colon cancer tumor-bearing mice was observed, and fluorescence signals at 4 hours tumor sites were collected. As shown in FIG. 15 (f), HC-1-PEG50 is strongly enriched in tumor sites by passive targeting for about 4h after intravenous injection for several cycles, because the derivative contains 50 polyethylene glycol units, so that the whole drug molecule has better water solubility and proper hydrophilic-hydrophobic ratio, thereby leading to stronger enrichment in tumor sites.
2) Drug enrichment in head and neck tumor mice (brain, head and neck, tongue, oral, nasal, brain glioma)
Subcutaneous tumors were inoculated with brain cancer G442 cells to obtain a brain cancer subcutaneous mouse tumor model. HB-11-PEG6 (example 11) was used as a photosensitizing drug and injected into tumor-bearing mice via tail vein at a dose of 10mg/kg, and fluorescence imaging behavior in brain cancer tumor-bearing mice was observed, and fluorescence imaging of 4h tumor sites was collected. As shown in FIG. 16 (a), HB-11-PEG6 has a certain enrichment capacity at the tumor site through passive targeting for about 4h after being subjected to intravenous injection for a plurality of times, and is moderately enriched because the derivative molecule only contains 6 polyethylene glycol units, so that the water solubility of the whole drug molecule is insufficient, thereby leading to the medium enrichment at the tumor site.
Subcutaneous tumors were inoculated with head and neck cancer SCC2 cells to obtain a head and neck cancer subcutaneous mouse model. HC-18-PEG8 (example 15) was injected into tumor-bearing mice via tail vein at a dose of 10mg/kg as a photosensitizing drug, and its fluorescence imaging behavior in head and neck cancer tumor-bearing mice was observed, and fluorescence signals at 4 hours tumor sites were collected. As shown in FIG. 16 (b), HC-18-PEG8 is strongly enriched in tumor sites by passive targeting for about 4h after intravenous injection for several cycles, because the derivative contains 8 polyethylene glycol units, so that the whole drug molecule has better water solubility and proper hydrophilic-hydrophobic ratio, thereby leading to stronger enrichment in tumor sites.
Subcutaneous tumors were inoculated with tongue carcinoma TSCCa cells to obtain a tongue carcinoma subcutaneous mouse tumor model. HC-45 (example 23) was injected into tumor-bearing mice via tail vein at a dose of 10mg/kg as a photosensitizing drug, and its fluorescence imaging behavior in tongue cancer tumor-bearing mice was observed, and fluorescence signals at 4 hours tumor sites were collected. As shown in fig. 16 (c), HC-45 has a moderate concentration in the tumor site by passive targeting for about 4 hours after several cycles in vivo by intravenous injection, because the derivative molecule contains no polyethylene glycol unit, only one carboxyl group, and is not sufficiently water-soluble, resulting in a moderate concentration in the tumor site.
Subcutaneous tumors were inoculated with oral cancer CAL27 cells to obtain oral cancer subcutaneous mouse tumor models. HC-57 (example 27) was injected into tumor-bearing mice via tail vein at a dose of 10mg/kg as a photosensitizing drug, and its fluorescence imaging behavior in oral cancer tumor-bearing mice was observed, and fluorescence signals at 4 hours tumor sites were collected. As shown in FIG. 16 (d), HC-57 has strong enrichment capacity at the tumor site through passive targeting for about 4h after a plurality of cycles in vivo by intravenous injection, because the derivative contains water-soluble groups such as carboxyl and quaternary ammonium salt although not containing polyethylene glycol units, so that the water solubility of the whole drug molecule is better, the hydrophilicity and hydrophobicity ratio of the whole molecule is proper, and thus the drug molecule has strong enrichment at the tumor site.
Subcutaneous tumors were inoculated with nasal carcinoma KB cells to give a nasal carcinoma subcutaneous mouse tumor model. HB-65 (example 29) was used as a photosensitizing drug and injected into tumor-bearing mice via tail vein at a dose of 10mg/kg, and its fluorescence imaging behavior in the tumor-bearing mice of nasal carcinoma was observed, and fluorescence signals at 4 hours tumor sites were collected. As shown in FIG. 16 (e), HB-65 has strong enrichment at the tumor site through passive targeting action for about 4h after a plurality of times of circulation in vivo by intravenous injection, because the derivative contains hydrophilic groups such as quaternary ammonium salt, amide group and the like, the water solubility of the whole molecule is better, and the hydrophilic-hydrophobic ratio is proper, thereby leading to strong enrichment at the tumor site.
And inoculating the subcutaneous tumor with the glioma C6 cells to obtain a glioma subcutaneous mouse tumor model. HC-81-PEG16 (example 33) was injected into tumor-bearing mice via tail vein at a dose of 10mg/kg as a photosensitizing drug, and fluorescence imaging behavior in brain glioma-bearing mice was observed, and fluorescence signals at tumor sites were collected for 4 hours, respectively. As shown in FIG. 16 (f), HC-81-PEG16 is subjected to intravenous injection for a plurality of times in vivo, and has strong enrichment at the tumor site by passive targeting for about 4h, because the derivative contains 16 polyethylene glycol units, the water solubility of the whole drug molecule is better, the hydrophilic-hydrophobic ratio is proper, and the drug molecule is enriched at the tumor site strongly.
3) Drug enrichment in tumor mice of the skin and urinary system (basal cell carcinoma, squamous skin carcinoma, melanoma, cutaneous T-cell lymphoma, prostate carcinoma, bladder carcinoma)
Subcutaneous tumors were inoculated with basal cell carcinoma BCC to obtain basal cell carcinoma subcutaneous mouse tumor models. HB-5-PEG10 (example 5) was used as a photosensitizing drug and injected into tumor-bearing mice via tail vein at a dose of 10mg/kg, and fluorescence imaging behavior in basal cell carcinoma tumor-bearing mice was observed, and fluorescence signals at tumor sites were collected for 4 hours. As shown in FIG. 17 (a), HB-5-PEG10 has strong enrichment at tumor site through passive targeting action for about 4h after several cycles in vivo by intravenous injection, because the derivative contains 10 polyethylene glycol units, the water solubility of the whole drug molecule is better, the hydrophilic-hydrophobic ratio is proper, and thus the drug molecule has strong enrichment at tumor site.
Subcutaneous tumors were inoculated with squamous skin carcinoma PECA cells to give a squamous skin carcinoma subcutaneous mouse model. HC-8-PEG12 (example 8) was used as a photosensitizing drug and injected into tumor-bearing mice via tail vein at a dose of 10mg/kg, and fluorescence imaging behavior in squamous skin carcinoma tumor-bearing mice was observed, and fluorescence signals at 4 hours tumor sites were collected. As shown in FIG. 17 (b), HC-8-PEG12 is strongly enriched in tumor sites by passive targeting for about 4h after several cycles in vivo by intravenous injection, because the molecule contains 8 polyethylene glycol units, the whole drug molecule has better water solubility and proper hydrophilic-hydrophobic ratio, thereby leading to stronger enrichment in tumor sites.
Subcutaneous tumors were inoculated with melanoma B16 cells to give a melanoma subcutaneous mouse tumor model. HB-64 (example 28) was used as a photosensitizing drug and injected into tumor-bearing mice via tail vein at a dose of 10mg/kg, and its fluorescence imaging behavior in melanoma-bearing mice was observed, and fluorescence signals at 4 hours tumor sites were collected. As shown in FIG. 17 (c), HB-64 has strong enrichment at tumor site through passive targeting action for about 4h after several cycles in vivo by intravenous injection, because the molecule contains water-soluble groups such as carboxylate and quaternary ammonium salt although it does not contain polyethylene glycol unit, so that the whole drug molecule has good water solubility and proper hydrophilic-hydrophobic ratio, thus resulting in strong enrichment at tumor site.
Subcutaneous tumors were inoculated with cutaneous T cell lymphoma HH cells to give a subcutaneous mouse tumor model of lymphoma. HB-89-PEG16 (example 36) was used as a photosensitizing drug and injected into tumor-bearing mice via tail vein at a dose of 10mg/kg, and its fluorescence imaging behavior in tumor-bearing mice with cutaneous T cell lymphoma was observed, and fluorescence signals at 4 hours tumor sites were collected. As shown in FIG. 17 (d), HB-89-PEG16 has strong enrichment at tumor site through passive targeting action for about 4h after several cycles in vivo by intravenous injection, because the derivative contains 16 polyethylene glycol units, the water solubility of the whole drug molecule is better, the hydrophilic-hydrophobic ratio is proper, and thus, the drug molecule has strong enrichment at tumor site.
Subcutaneous tumors were inoculated with prostate cancer LNCaP cells to obtain a prostate cancer subcutaneous mouse tumor model. HC-90-PEG30 (example 37) was injected into tumor-bearing mice via tail vein at a dose of 10mg/kg as a photosensitizing drug, and its fluorescence imaging behavior in prostate cancer tumor-bearing mice was observed, and fluorescence signals at 4 hours tumor sites were collected. As shown in FIG. 17 (e), HC-90-PEG30 is subjected to intravenous injection and then is subjected to a plurality of circulation in vivo, and is subjected to a passive targeting effect for about 4 hours to achieve strong enrichment at the tumor site, wherein the molecule contains 30 polyethylene glycol units, so that the whole drug molecule has good water solubility and proper hydrophilic-hydrophobic ratio, and the tumor site is enriched strongly.
Subcutaneous tumors were inoculated with bladder cancer MBT-2 cells to obtain a bladder cancer subcutaneous mouse tumor model. HC-91-PEG16 (example 38) was injected into tumor-bearing mice via tail vein at a dose of 10mg/kg as a photosensitizing drug, and its fluorescence imaging behavior in bladder cancer tumor-bearing mice was observed, and fluorescence signals at 4 hours tumor sites were collected, and its animal fluorescence imaging was shown in FIG. 17 (f). It can be seen that the drug molecule HC-91-PEG16 has strong enrichment capacity at the tumor site through passive targeting action for about 4 hours after being subjected to intravenous injection for a plurality of times, and the reason is that the derivative molecule contains 16 polyethylene glycol units, so that the water solubility of the whole drug molecule is better, the hydrophilicity and hydrophobicity ratio of the whole molecule is proper, and the drug molecule has stronger enrichment at the tumor site.
The results of fig. 15-17 show that the hypocrellin derivatives provided by the invention can be used for realizing the administration to tumor-bearing mice by a tail vein injection method, and the medicaments can generate super-strong or moderate fluorescence imaging at the tumors of the mice by the living body imaging of small animals, so that the hypocrellin derivatives can be used as photosensitive medicaments for fluorescence imaging at the tumors of the tumor-bearing mice and can be used for guiding the excision of tumor boundaries in the operation by using fluorescent mediated materials. The tumor comprises: esophageal cancer, gastric cancer, lung cancer, liver cancer, bile duct cancer, colon cancer, head and neck cancer, brain cancer, tongue cancer, nose cancer, oral cancer, brain glioma, basal cell carcinoma, squamous skin carcinoma, melanoma, cutaneous T-cell lymphoma, prostate cancer, and bladder cancer.
Whereas hypocrellin derivatives of the present invention, which are slightly less water-soluble, can be administered by intratumoral injection. Hypocrellin derivatives were dissolved in DMSO (dimethylsulfoxide), diluted 5-10 fold with physiological saline for intratumoral injection. Taking a subcutaneous mouse tumor model of basal cell carcinoma (BCC cells) as an example, the derivative (HB-28 example 19) is used as a photosensitive drug and is injected into a tumor-bearing mouse body by intratumoral injection at a dose of 10mg/kg, the fluorescence imaging behaviors in the tumor-bearing mouse body are observed, and the fluorescence signals of tumor sites are respectively collected for 4 hours by using a small animal imaging system. As shown in FIG. 18 (a), the drug molecule HB-28 was injected intratumorally into tumor cells and tissues, which had a strong enrichment capacity at the tumor.
The invention also discloses the enrichment of other poorly water-soluble derivatives in different tumor cells by intratumoral administration. By a similar administration method, the fluorescence imaging behavior of tumor-bearing mice in tumor cells is observed, and the fluorescence signals of tumor sites at 4 hours are respectively collected by using a multispectral small animal living body imaging system. Such as: 18 Fluorescence imaging of photosensitizing drug HC-36 of (b) (example 20) in squamous skin carcinoma PECA cell mice; 18 Fluorescence imaging of the photosensitizing drug HB-45 of (c) (example 23) in melanoma B16 cell mice; 18 Fluorescence imaging of photosensitizing drug HC-73 in (d) (example 31) in cutaneous T cell lymphoma HH cell mice; 18 Fluorescence imaging of HC-77 (example 32) in (e) in lung cancer cell a549 cell mice; 18 Fluorescence imaging of HC-80 in (f) (example 32) in cholangiocarcinoma cell MCC cell mice. As can be seen from FIG. 18, the photosensitizing drug HB-28 was intratumorally injected into tumor cells and tissues in basal cell carcinoma, HC-36 in squamous skin carcinoma, HB-45 in melanoma, HC-73 in cutaneous T cell lymphoma, HC-77 in lung cancer, HC-80 in cholangiocarcinoma, which had a strong enrichment capacity at the tumor site.
By changing the molecular structure and different substituents, various hypocrellin derivatives of the invention have similar enrichment effect on other different tumors. The derivatives have good drug enrichment on mice with esophageal cancer, gastric cancer, lung cancer, liver cancer, bile duct cancer, colon cancer, head and neck cancer, brain cancer, tongue cancer, nose cancer, oral cancer, brain glioma, basal cell carcinoma, squamous skin cancer, cutaneous T-cell lymphoma, melanoma, prostate cancer and bladder cancer.
The derivatives of the present application have good enrichment in the subcutaneous tumor of mice, and their drug enrichment effect in the in situ tumor of mice? In order to study the in-situ enrichment of the derivatives in tumors, the experimental brain glioma C6 cells are inoculated with in-situ tumors in the brain of mice, and an in-situ model of the brain glioma is obtained for fluorescence imaging. Using the derivative HB-3-PEG12 of example 3 as a photosensitizing drug, the fluorescent imaging behavior in tumor-bearing mice was observed by tail vein injection at a dose of 10mg/kg, and fluorescent signals at tumor sites were collected by a small animal living body imaging system for 0, 2, 3.5 and 5 hours, respectively. As shown in FIG. 19, the fluorescence signal of the tumor region gradually increased with the increase of time and reached the highest at 5h, indicating that the photosensitive drug HB-3-PEG12 was highly enriched at the tumor site, whereas the non-tumor region of the brain was not enriched with the drug, and the photosensitive drug had a very good targeted enrichment effect on glioma. In addition, brains of blank mice without dosing were not fluorescent.
The invention aims at the situation that no mediated medicine is used for positioning to guide the excision of the tumor in the brain glioma operation, and discovers that the derivative can specifically gather in brain glioma tissues, does not gather in brain areas without tumor and does not gather photosensitizer, and has good tumor targeting enrichment effect. At the moment, the tumor tissue is irradiated by light with specific wavelength, and detectable fluorescence is excited so as to position the position of the tumor tissue for fluorescence guided glioma excision operation; on the other hand, the derivative can generate singlet oxygen under the photosensitive condition to cause the damage of tumor cells, and is used for photodynamic therapy of brain glioma without damaging normal tissues basically. The derivatives provided by the application not only have good enrichment effect on brain glioma, but also have good enrichment effect on other tumors, such as: the composition also has good drug enrichment on mice with esophageal cancer, gastric cancer, lung cancer, liver cancer, cholangiocarcinoma, colon cancer, head and neck cancer, brain cancer, tongue cancer, nose cancer, oral cancer, basal cell carcinoma, squamous skin carcinoma, melanoma, prostatic cancer and bladder cancer. The invention discloses a hypocrellin derivative used as a fluorescence-mediated material for guiding the excision of solid tumors for the first time.
The results of FIGS. 15-19 above illustrate the fluorescent imaging of tumor-bearing mice with various derivatives provided in the present invention. It can be seen that these photosensitizers produce a super-strong or moderate fluorescence imaging at the tumor site of mice, indicating that these derivatives can be used as photosensitizers for fluorescence imaging in tumor cells of tumor-bearing mice, and can be used for fluorescence-mediated material-guided resection of tumor boundaries during surgery. The tumor comprises: esophageal cancer, gastric cancer, lung cancer, liver cancer, bile duct cancer, colon cancer, head and neck cancer, brain cancer, tongue cancer, nose cancer, oral cancer, brain glioma, basal cell carcinoma, squamous skin carcinoma, cutaneous T-cell lymphoma, melanoma, prostate cancer, and bladder cancer. Other derivatives in this application also have in situ enrichment of the tumor cells described above, can be used for fluorescence imaging of the tumor cells described above in vivo, and can be used to mediate excision of material-guided tumor boundaries during surgery. The results of fluorescence imaging of the above partial hypocrellin derivatives in tumors are shown in table 5.
Table 5: in vivo enrichment of partial derivatives of the invention on digestive tract tumors, head and neck facial tumors, skin tumors, and genitourinary tumors
Figure BDA0003362796890000301
Figure BDA0003362796890000311
EXAMPLE 46 photodynamic therapy
In view of the fact that the hypocrellin derivatives of the present invention can kill various tumor cells with high efficiency in cell experiments and can be well enriched in mouse tumors as photosensitive drugs, the inventors further tested that the derivatives can kill tumor cells in mice as photodynamic drugs. Taking a mouse as a model, inoculating lung cancer A549 cells into the mouse, and when the tumor volume reaches 200mm 3 When left and right, the lung cancer animal model is obtained for photodynamic therapy. The 10mg/kg dose of the photosensitizing drug HB-1-PEG8 (example 1) was injected into tumor bearing mice by tail vein, the in vivo fluorescence imaging behavior was observed, and the 4h tumor site fluorescence signal was collected using a small animal in vivo imaging system. After 4 hours, 0.1W/cm was used 2 The tumor site was irradiated with 635nm laser light for 10min, and data were recorded. Incrustation appeared on day 2 of photodynamic therapy of lung cancer in mice, gradually shrinking by day 6, and completely disappeared after day 12, indicating that lung cancer in mice was completely inhibited by photodynamic therapy (fig. 20). In contrast, the lung cancer tumor-bearing mice (normal saline alone, no photosensitive drug) of the control group were treated with 0.1W/cm 2 The 635nm laser of the formula (I) irradiates the tumor part for 10min, the tumor is found to grow rapidly after 6 days, and the tumor is serious after 12 days and has no effect of inhibiting the tumor. Therefore, HB-1-PEG8 can be used as photosensitive medicine for photodynamic therapy of lung cancer, has obvious effect of killing tumor cells and can inhibit tumor regeneration and recurrence.
Experiments were also performed with a brain glioma subcutaneous mouse model (C6 cell inoculation) for photodynamic therapy. The light-sensitive drug HB-6-PEG6 (example 6) was injected into tumor-bearing mice at a dose of 10mg/kg by tail vein, and fluorescence imaging behavior was observed in vivo. After 4 hours, 0.1W/cm was used 2 The tumor site was irradiated with 635nm laser light for 10min, and data were recorded. As shown in fig. 21 (b), crusting occurred on day 2 after photodynamic therapy of mouse glioma, gradually shrinking on day 6, completely disappearing after day 14,indicating that the growth of glioma in mice was completely inhibited in photodynamic therapy. In contrast, in the case of brain glioma mice of the control group (saline alone, no photosensitizing agent), tumors grew rapidly after 6 days under the same experimental conditions, without any tumor inhibition, fig. 21 (a). Therefore, HB-6-PEG6 can be used as a photosensitive drug for photodynamic therapy of brain glioma, has obvious effect of killing tumor cells and can inhibit regeneration and recurrence of tumors.
Experiments were also performed with a melanoma subcutaneous mouse model (B16 cell inoculation) for photodynamic therapy. Tail vein injection HC-9-PEG8 (example 9) in tumor bearing mice, fluorescence imaging in vivo was observed. After 4 hours, 0.1W/cm was used 2 The tumor site was irradiated with 635nm laser light for 10min, and data were recorded. As shown in fig. 21 (c), crusting occurred on day 2 after photodynamic therapy of melanoma in mice, gradually shrinking on day 6, and completely disappearing after day 14, indicating that the tumors in mice were completely inhibited by photodynamic therapy. In the tumor mice of the control group, under the same conditions, but only normal saline is injected, no photosensitive drug is injected, and the tumor rapidly grows after 6 days, and has no tumor inhibiting effect. Therefore, HC-9-PEG8 is used as a photosensitive drug for photodynamic therapy of melanoma, has the effect of killing tumors and can inhibit regeneration and recurrence of tumors.
Experiments also used a subcutaneous mouse model of bladder cancer (seeded with MBT-2 cells) for photodynamic therapy. The tail vein was injected with HB-63 (example 28) into tumor-bearing mice and fluorescence imaging was observed in vivo. After 4 hours, 0.1W/cm was used 2 The tumor site was irradiated with 635nm laser light for 10min, and data were recorded. As shown in fig. 21 (d), crusting occurred on day 2 after photodynamic therapy of the bladder cancer in mice, and gradually decreased on day 6, and completely disappeared after day 14, indicating that the tumors of mice were completely inhibited after photodynamic therapy. In the tumor mice of the control group, under the same experimental conditions, but only normal saline is injected, no photosensitive drug is injected, and the tumor is found to grow rapidly after 6 days, and no tumor inhibiting effect is achieved. Therefore, HB-63 can be used as a photosensitive drug for photodynamic therapy of bladder cancer, has obvious tumor killing effect and can inhibit tumor regeneration and recurrence.
H & E staining and pathological analysis were performed on the tumor tissues of mice after different treatments. The study found that none of the tumor cells in the blank group were damaged, and that the tumor cells in the photodynamic therapy group were completely dead. Statistical analysis of the survival rate of mice shows that compared with the control group, the mice subjected to photodynamic therapy do not die within 30 days, which indicates that the photodynamic therapy has better anti-tumor effect on tumor-bearing mice.
The experiment uses a mouse as a model, and also uses cholangiocarcinoma (MCC cells) to inoculate subcutaneous tumors of the mouse, so as to obtain the cholangiocarcinoma model for photodynamic therapy. Intratumoral injection of a photoactive drug HB-29 (example 19) in tumor-bearing mice was observed for its fluorescence imaging behavior in tumor-bearing mice. After 4 hours, 0.1W/cm was used 2 The tumor site was irradiated with 635nm laser light for 10min, and data were recorded (FIG. 22 a). Incrustation appears on day 2 after photodynamic therapy of mouse bile duct cancer, gradually shrinks on day 6, and completely disappears after day 14, indicating that after photodynamic therapy, mouse bile duct cancer is almost completely inhibited. In the mice of the control group, under the same experimental conditions, but with only normal saline injection and no photosensitive drug injection, the tumors are found to grow rapidly after 6 days, and no tumor inhibiting effect is achieved. Therefore, HB-29 serving as a photosensitive drug has obvious effect of killing tumors and can inhibit the regeneration and recurrence of the tumors in photodynamic therapy of bile duct cancer.
Experiments were also performed with a subcutaneous mouse model of colon cancer (vaccinated with HCT116 cells) for photodynamic therapy. Intratumoral injection of a photosensitive drug HB-45 (example 23) in tumor-bearing mice was visualized by in vivo fluorescence imaging. After 4 hours, 0.1W/cm was used 2 The tumor site was irradiated with 635nm laser light for 10min. As shown in fig. 22 (b), crusting occurred on day 2, gradually shrinking on day 6, and completely disappearing on day 14 after photodynamic therapy of colon cancer in mice, indicating that colon cancer in mice was completely inhibited by photodynamic therapy. The mice in the control group are injected with normal saline only and no photosensitive drug, and the mice in the control group find that the tumor grows rapidly after 6 days without any tumor inhibition effect. Therefore, HB-45 can be used as a photosensitive drug for photodynamic therapy of colon cancer, has obvious effect of killing tumor cells and can inhibit tumor regeneration and recurrence.
Experiments were performed in a subcutaneous mouse model of oral cancer (vaccinated with CAL27 cells) for photodynamic therapy. Intratumoral injection of HC-73 (example 31) into tumor bearing mice was observed for in vivo fluorescence imaging. After 4 hours, 0.1W/cm was used 2 The tumor site was irradiated with 635nm laser light for 10min, and data were recorded. As shown in fig. 22 (c), crusting occurred on day 2, gradually shrinking on day 6, and completely disappeared after day 14 of the photodynamic therapy of oral cancer in mice, indicating that oral cancer in mice was completely inhibited after the photodynamic therapy. In the mice of the control group, under the same experimental conditions, but with only physiological saline injection and no photosensitive drug injection, the tumors rapidly grow after 6 days, and no tumor inhibition effect is achieved. Therefore, HC-73 is used as a photosensitive medicament for photodynamic therapy of oral cancer, has obvious tumor killing effect and can inhibit regeneration and recurrence of tumors.
Experiments were performed in a basal cell carcinoma subcutaneous mouse model (BCC cells inoculated) for photodynamic therapy. Intratumoral injection of HC-80 (example 32) into tumor bearing mice was observed for in vivo fluorescence imaging. After 4 hours, 0.1W/cm was used 2 The tumor site was irradiated with 635nm laser light for 10min, and data were recorded. As shown in fig. 22 (d), crusting occurred on day 2 of photodynamic therapy, gradually decreased on day 6, and completely disappeared after 14 days, indicating that the tumor of the mice was completely inhibited after photodynamic therapy. The mice in the control group are injected with normal saline only and no photosensitive drug, and the mice in the control group find that the tumor grows rapidly after 6 days without any tumor inhibition effect. Therefore, HC-80 is used as a photosensitive drug for photodynamic therapy of basal cell carcinoma, has obvious tumor killing effect and can inhibit regeneration and recurrence of tumors.
The hypocrellin derivatives of the present invention are shown in detail as above, and can be used for killing various tumor cells (such as lung cancer, cholangiocarcinoma, colon cancer, oral cancer cells, basal cell carcinoma cells, melanoma cells, brain glioma cells and bladder cancer cells) in tumor-bearing mice with high efficiency, and have good photodynamic therapy effect by injecting a certain dose of photosensitive drugs into the tumor-bearing mice and matching with laser irradiation with certain intensity and wavelength.
Comparative example 1
The photodynamic effect of hypocrellin derivatives in the present invention on HeLa is shown in fig. 23. Under the irradiation of red light, HB-1-PEG6 with 200nM concentration can kill more than 80% of HeLa cells, and IC with semi-lethal concentration 50 A value of about 120nM; similarly, HB-73 at 200nM concentration kills more than 80% of HeLa cells, semi-lethal concentration IC 50 A value of about 80nM; HC-80 at 200nM concentration can kill more than 80% of HeLa cells, semi-lethal concentration IC 50 A value of about 80nM;
under the same conditions, HB-1-PEG6 can kill 80% of esophageal cancer cells AKR, gastric cancer cells MFC, lung cancer cells A549, liver cancer cells HCC, biliary duct cancer cells MCC, colon cancer cells HCT116 and semi-lethal concentration IC only with 50nM concentration 50 Values of about 20 to 30nM (Table 2); under the same conditions, HB-73 can kill 85% of head and neck cancer cells SCC2, brain cancer cells G442, tongue cancer cells TSCCa, nasal cancer cells KB, oral cancer cells CAL27, brain glioma cells C6 and semi-lethal concentration IC only with 50nM concentration 50 Values of about 20 to 30nM (Table 3); under the same conditions, HC-80 can kill 85% of basal cell carcinoma cells BCC, squamous skin carcinoma cells PECA, melanoma cells B16, prostate cancer cells LNCaP, bladder cancer cells MBT-2, and semi-lethal concentration IC at 50nM concentration 50 The values were about 20-30 nM (Table 4). Thus, as can be seen from the comparison of fig. 23 and tables 2 to 4, the phototoxic effect of hypocrellin derivatives disclosed in the present invention on the above tumor cells is significantly higher than that of HeLa cells.
It should be understood that the foregoing examples of the present invention are provided merely for clearly illustrating the present invention and are not intended to limit the embodiments of the present invention, and that various other changes and modifications may be made therein by one skilled in the art without departing from the spirit and scope of the present invention as defined by the appended claims.
It should be noted that the hypocrellin derivatives to be protected in the present invention contain two enol tautomers, and the chemical structures of the two isomers are shown as formula (I) and formula (I'), which are all within the protection scope. For the sake of simplicity, only one enol tautomer is listed in all examples of the invention, the other enol tautomer and its corresponding structural formula are described in detail in the specification, and the structures thereof are of course within the scope of protection. In addition, the structural general formula of the hypocrellin derivative comprises polyethylene glycol units (PEGn), wherein the number n of the units is any integer between 1 and 50, and the chemical structures corresponding to the hypocrellin derivatives are all within a protection range. For the sake of simplicity, only some integers are listed in all embodiments of the present invention, and the general structural formulas corresponding to the rest are described in detail in the specification, and the structures thereof are of course within the scope of protection. Any range recited in the invention includes any numerical value recited in, and any subrange formed by, the endpoints, or any numerical value recited between the endpoints.
The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (9)

1. Use of hypocrellin derivatives or mixtures thereof in the preparation of photodynamic antitumor drugs, wherein the tumors are esophageal cancer, gastric cancer, lung cancer, liver cancer, bile duct cancer, colon cancer, brain cancer, head and neck cancer, tongue cancer, nasal cancer, oral cancer, glioma, basal cell carcinoma, squamous skin cancer, melanoma, cutaneous T-cell lymphoma, prostate cancer, bladder cancer, and the hypocrellin derivatives are compounds represented by formula (I), formula (II) or formula (III), isomers, isotopic markers, pharmaceutically acceptable salts or solvates thereof;
Figure FDA0003362796880000011
r in formula (I) 1 The structural general formula is shown as formula (IV), R 2 is-H or-COCH 3 ：
Figure FDA0003362796880000012
In the formula (IV), m is more than or equal to 0 and less than or equal to 12, n is more than or equal to 0 and less than or equal to 500, p is more than or equal to 0 and less than or equal to 12, and q is more than or equal to 0 and less than or equal to 12; the m, n, p, q is zero or a positive integer; y is a linking group; z is a terminal group; (OCH) 2 CH 2 ) n Is a polyethylene glycol unit;
the linking group Y in the formula (IV) is O, NH, S, carboxylic ester group, amide group, sulfonate group, sulfonamide group, phenylene group, alkenylene group of 3-12 carbon atoms or cycloalkyl group of 3-12 carbon atoms;
The cycloalkyl of 3-12 carbon atoms comprises a substituted or unsubstituted cycloalkyl or contains a heteroatom which is an oxygen, nitrogen or sulfur atom; the substituent is alkyl with 1-12 carbon atoms;
the end group Z in the formula (IV) is hydrogen, alkyl of 1-12 carbon atoms, alkoxy of 1-12 carbon atoms, phenyl, hydroxyl, amino, sulfhydryl, carboxylic acid group, sulfonic acid group, pyridyl, quaternary ammonium salt or pyridinium salt;
when the end group Z is quaternary ammonium salt, three substituents on the quaternary ammonium salt are respectively and independently selected from alkyl groups with 1-12 carbon atoms; the anions in the quaternary ammonium salt are anions allowed by the pharmaceutical preparation;
when the end group Z is pyridine salt, the substituent group on the pyridine ring in the pyridine salt is in ortho-position, meta-position or para-position; the group connected with pyridine N in the pyridine salt is C1-8 alkyl, C1-8 alkyl substituted by hydroxyl, carboxyl and halogen; anions in the pyridinium salt are anions allowed by the pharmaceutical preparation;
r in formula (II) 2 is-H or-COCH 3 ：
R in formula (II) 3 ～R 8 Identical or different, independently of one another, R is as in formula (I) 1 Is defined in (a).
2. The use according to claim 1, wherein the linking group Y in formula (IV) is: -O-; -NH-; -S-; -COO-; -O-CO-; -CONH-; -NH-CO-; -SO 3 -；-C 6 H 4 - (phenyl); -C 3 H 4 - (cyclopropyl); -C 4 H 6 - (cyclobutyl); -C 5 H 8 - (cyclopentyl); -C 5 H 7 (CH 3 ) - (methylcyclopentyl); -C 6 H 10 - (cyclohexyl); -C 6 H 9 (CH 3 ) - (methylcyclohexyl); -C 7 H 12 - (cycloheptyl);
Figure FDA0003362796880000013
(piperazinyl).
3. Use according to claim 1 or 2, wherein the end group Z in formula (IV) above is: -H; -CH 3 ；-C 2 H 5 ；-C 4 H 9 ；-C 6 H 13 ；-OCH 3 ；-OC 2 H 5 ；-OC 4 H 9 ；-OC 6 H 13 ；-C 6 H 5 ；-OH,-NH 2 ；-SH；-COOH；-COOCH 3 ；-SO 3 H；-C 5 H 4 N；-C 5 H 4 N + ；-N + (CH 3 ) 3 ；-N + (C 2 H 5 ) 3 ；-N + (C 6 H 13 ) 3 ；-N + (CH 3 ) 2 (C 2 H 5 )；-N + (CH 3 ) 2 (C 6 H 13 )；-N + (CH 3 ) 2 (C 8 H 17 )。
4. The use according to any one of claims 1 to 3, wherein the derivatives of formula I and formula I' are enol tautomers; the derivatives of formula II and formula II' are enol tautomers; the derivatives of formula III and formula III' are enol tautomers.
Figure FDA0003362796880000021
5. The use according to any one of claims 1-4, wherein R 1 Is an alcohol with different chain lengths and a carboxylic ester formed by the alcohol and carboxyl polyethylene glycol: - (CH) 2 ) m -OH；-(CH 2 ) m -OCH 3 ；-(CH 2 ) m -O-CO-CH 2 CH 2 -(OCH 2 CH 2 ) n -OCH 3 [ m is an integer of 1 to 8, n is an integer of 0 to 100]；
Or R is 1 Carboxylic acids of different chain lengths, and carboxylic esters or amides thereof with polyethylene glycol: - (CH) 2 ) m -COOH；-(CH 2 ) m -COOCH 3 ；-(CH 2 ) m -CO-(OCH 2 CH 2 ) n -OH；-(CH 2 ) m -CO-(OCH 2 CH 2 ) n -OCH 3 ；-(CH 2 ) m -CO-NH-CH 2 CH 2 -(OCH 2 CH 2 ) n -OCH 3 [ m is an integer of 1 to 8, n is an integer of 0 to 100]；
Or R is 1 Sulfonate or sulfonamide formed from sulfonate and polyethylene glycol and sulfonate groups of different chain lengths: - (CH) 2 ) m -SO 3 H；-(CH 2 ) m -SO 2 -(OCH 2 CH 2 ) n -OH；-(CH 2 ) m -SO 2 -(OCH 2 CH 2 ) n -OCH 3 ；-(CH 2 ) m -SO 2 -NH-CH 2 CH 2 -(OCH 2 CH 2 ) n -OH；-(CH 2 ) m -SO 2 -NH-CH 2 CH 2 -(OCH 2 CH 2 ) n -OCH 3 [ m is an integer of 1 to 8, n is an integer of 0 to 100]；
Or R is 1 Is thiapolyethylene glycol: -CH 2 CH 2 -SH；-CH 2 CH 2 -S-CH 2 CH 2 OH；-CH 2 CH 2 -S-CH 2 CH 2 OCH 3 ；-CH 2 CH 2 -S-CH 2 CH 2 -(OCH 2 CH 2 ) n -OH；
Or R is 1 Is alkyl, amino, hydroxyl, or substituent containing phenyl, pyridyl, alkene: -H; -CH 3 ；-C 2 H 5 ；-C 3 H 7 ；-C 4 H 9 ；-C 5 H 11 ；-C 6 H 13 ；-C 8 H 17 ；-NH 2 ；-NHCH 3 ；-NHC 2 H 5 ；-OH；-CH 2 CH＝CH 2 ；-(CH 2 ) 2 CH＝CH 2 ；-(CH 2 ) 3 CH＝CH 2 ；-CH 2 C 6 H 5 ；-C 5 H 4 N；-CH 2 C 5 H 4 N；-(CH 2 ) 2 C 5 H 4 N；-NHC 6 H 5 ；-NHC 5 H 4 N；
Or R is 1 Is a substituent containing cycloalkyl: -C 3 H 5 (cyclopropyl) -C 4 H 7 (cyclobutyl) -C 5 H 9 (cyclopentyl) -C 6 H 11 (cyclohexyl) -C 6 H 10 (CH 3 ) (methylcyclohexyl) -C 6 H 10 (OH) (hydroxycyclohexyl), -C 7 H 13 (cycloheptyl) -CH 2 C 6 H 10 COOH、-CH 2 C 6 H 10 COOCH 3 、-CH 2 C 6 H 10 OH、-C 6 H 10 COOH；
Or R is 1 Is cyclohexane (-C) containing substituent 6 H 10 -OH；-CH 2 C 6 H 10 COOH；-CH 2 C 6 H 10 COOCH 3 ；-CH 2 C 6 H 10 OH；-C 6 H 10 COOH)；
Or R is 1 Is a substituent containing quaternary ammonium salt: - (CH) 2 ) m -N + (CH 3 ) 3 ；-(CH 2 ) m -N + (CH 3 ) 2 (C 2 H 5 )；-(CH 2 ) m -N + (CH 3 ) 2 (C 3 H 7 )；-(CH 2 ) m -N + (CH 3 ) 2 (C 4 H 9 )；-(CH 2 ) m -N + (CH 3 ) 2 (C 5 H 11 )；-(CH 2 ) 3 -N + (CH 3 ) 2 (C 6 H 13 )；-(CH 2 ) m -N + (CH 3 ) 2 (C 8 H 17 )；-(CH 2 ) m -N + (CH 3 ) 2 (C 10 H 21 )；-(CH 2 ) m -N + (CH 3 ) 2 (C 12 H 25 )；-(CH 2 ) m -O-CO-(CH 2 ) 2 -N + (CH 3 ) 3 ；-(CH 2 ) m -O-CO-(CH 2 ) 3 -N + (CH 3 ) 3 ；-(CH 2 ) m -O-CO-(CH 2 ) 4 -N + (CH 3 ) 3 ；-(CH 2 ) m -O-CO-(CH 2 ) 5 -N + (CH 3 ) 3 ；-(CH 2 ) m -O-CO-(CH 2 ) 6 -N + (CH 3 ) 3 ；-(CH 2 ) m -COO-(CH 2 ) 2 -N + (CH 3 ) 3 ；-(CH 2 ) m -COO-(CH 2 ) 3 -N + (CH 3 ) 3 ；-(CH 2 ) m -COO-(CH 2 ) 4 -N + (CH 3 ) 3 ；-(CH 2 ) m -COO-(CH 2 ) 5 -N + (CH 3 ) 3 ；-(CH 2 ) m -COO-(CH 2 ) 6 -N + (CH 3 ) 3 ；-(CH 2 ) m -CONH-(CH 2 ) 2 -N + (CH 3 ) 3 ；-(CH 2 ) m -CONH-(CH 2 ) 3 -N + (CH 3 ) 3 ；-(CH 2 ) m -CONH-(CH 2 ) 4 -N + (CH 3 ) 3 [ m is an integer of 1 to 8]；
Or R is 1 Is a heterocyclic-containing substituent:
Figure FDA0003362796880000031
6. the use according to any one of claims 1 to 5, wherein the tumor is esophageal cancer cells, gastric cancer cells, lung cancer cells, liver cancer cells, bile duct cancer cells, colon cancer cells, head and neck cancer cells, brain cancer cells, tongue cancer cells, nose cancer cells, oral cancer cells, brain glioma cells, basal cell cancer cells, squamous skin cancer cells, cutaneous T cell lymphoma, melanoma cells, prostate cancer cells, bladder cancer cells.
7. The use according to any one of claims 1 to 6, wherein the medicament is a photodynamic medicament, a fluorescence mediated medicament; preferably, the drug may be enriched in the tumor cells.
8. The use according to any one of claims 1 to 7, wherein the pharmaceutically acceptable salts comprise salts of the compounds of formula (I) with organic acids selected from propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid and citric acid or acidic amino acids selected from aspartic acid and glutamic acid, with inorganic bases, including sodium, potassium, calcium, aluminium salts and ammonium salts, or with organic bases, including methylamine salts, ethylamine salts and ethanolamine salts; or with a basic amino acid selected from lysine, arginine and ornithine, followed by an inorganic acid selected from hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid and phosphoric acid, or with an organic acid selected from formic acid, acetic acid, picric acid, methanesulfonic acid and ethanesulfonic acid.
9. Use of a compound of formula (I), formula (II) or formula (III), an isomer, an isotopic label, a pharmaceutically acceptable salt or solvate thereof, as claimed in any one of claims 1 to 8, in the manufacture of a fluorescence-mediated medicament for guiding resection of a tumor boundary.
CN202111372435.5A 2021-11-18 2021-11-18 Application of hypocrellin 2-amino substituted or ethylenediamine substituted derivative in preparation of antitumor photodynamic medicine Pending CN116135228A (en)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1686093A (en) * 2005-04-13 2005-10-26 云南大学 Application of boisynthesized hypocrelline B in preparation of anticancer medicine
WO2007016762A1 (en) * 2005-08-10 2007-02-15 Quest Pharmatech Inc. Perylenequinone derivatives and uses thereof
CN102526055A (en) * 2010-12-24 2012-07-04 北京工业大学 Application of cyclohexanediamine hypocrelline B in photodynamic anti-tumor medicaments
CN107935943A (en) * 2016-10-13 2018-04-20 中国科学院理化技术研究所 Ester-water amphiphilic hypocrellin derivative and preparation method and application thereof

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1686093A (en) * 2005-04-13 2005-10-26 云南大学 Application of boisynthesized hypocrelline B in preparation of anticancer medicine
WO2007016762A1 (en) * 2005-08-10 2007-02-15 Quest Pharmatech Inc. Perylenequinone derivatives and uses thereof
CN102526055A (en) * 2010-12-24 2012-07-04 北京工业大学 Application of cyclohexanediamine hypocrelline B in photodynamic anti-tumor medicaments
CN107935943A (en) * 2016-10-13 2018-04-20 中国科学院理化技术研究所 Ester-water amphiphilic hypocrellin derivative and preparation method and application thereof

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