Application of a proximate and 2-position simultaneously substituted derivative of hypocrellin in the preparation of a photodynamic anti-tumor drug

Abstract

The present invention discloses the application of a derivative with simultaneous substitution at the meso-position and 2-position of hypocrellin as a photodynamic drug in the photodynamic therapy of various tumors. The maximum absorption wavelength of such derivatives is around 630 nm, and the molar extinction coefficient can reach 20,000 - 40,000 M-1 cm-1, showing strong light absorption ability in the phototherapy window; under photosensitizing conditions, it can efficiently generate reactive oxygen species such as singlet oxygen. Such derivatives can efficiently kill a variety of tumor cells, such as esophageal cancer, gastric cancer, lung cancer, liver cancer, cholangiocarcinoma, and colon cancer cells related to digestive tract tumors; brain cancer, head and neck cancer, tongue cancer, nasal cancer, oral cancer, and glioblastoma cells related to head and neck tumors; basal cell carcinoma, squamous cell carcinoma, cutaneous T-cell lymphoma, and melanoma cells related to skin tumors; prostate cancer and bladder cancer cells related to genitourinary tumors. At the same time, such derivatives have good enrichment effects in various tumor cells, can specifically accumulate in tumor cells or tissues, and can locate the position of tumor tissues through the self-fluorescence of the photosensitizer, for fluorescence-guided surgical resection of tumors. Under the same conditions, the derivatives in the present invention have higher photodynamic effects in treating tumors than commercial porphyrin-based photosensitizing drugs.

Description

Technical Field

[0001] The present invention relates to the technical field of photosensitizer drugs. More specifically, it relates to the application of a phylloerythrin derivative with simultaneous substitution at the 1-position and 2-position in the preparation of a photodynamic anti-tumor drug. Background Art

[0002] According to statistics from the World Health Organization, cancer is the deadliest disease affecting human life globally. In 2020, it was estimated that there were 19.29 million new cancer cases and 9.96 million cancer deaths worldwide. Among them, digestive tract tumors, such as lung cancer, esophageal cancer, gastric cancer, liver cancer, cholangiocarcinoma, colon cancer, etc., are generally highly prevalent. Lung cancer is one of the malignant tumors with the fastest growing incidence and mortality rates and the greatest threat to human health and life. In the past 50 years, the incidence and mortality rates of lung cancer have increased significantly in many countries. The incidence and mortality rates of lung cancer in men rank first among all malignant tumors, and the incidence rate in women ranks second, and the mortality rate ranks second. A large amount of data shows that the probability of long-term smokers getting lung cancer is 10 to 20 times that of non-smokers. In addition to lung cancer, digestive tract tumors such as esophageal cancer, gastric cancer, liver cancer, cholangiocarcinoma, and colon cancer are all cancer cell lesions with relatively high incidence rates, which are closely related to factors such as unhealthy lifestyles of humans and environmental pollution. Therefore, digestive tract tumors seriously threaten human health and life safety.

[0003] The incidence of head, neck and facial tumors (such as gliomas, oral cancers, head and neck cancers, tongue cancers, nasal cancers, brain cancers) has increased significantly in recent years. For example, gliomas are one of the most challenging tumors in neurosurgery, with an incidence of about seven per 100,000, accounting for about 40% of intracranial tumors. Gliomas are characterized by infiltrative growth and multiple in situ. If surgery can indicate the maximum safe resection of the tumor and be supplemented with effective local treatment, it can improve the prognosis of patients. At present, the treatment method for gliomas is to first surgically remove the tumor, and then supplement it with basic treatments such as radiotherapy, chemotherapy, and traditional Chinese medicine. Although the surgical techniques, radiation equipment, chemotherapy regimens, etc. for treating gliomas have advanced rapidly in recent years, the treatment effect is still not good, and the survival period of patients has not been significantly improved. The survival rate of patients with high-grade gliomas is generally about 11 months, and there has been no major breakthrough in 30 years, posing a great challenge to human survival. The main reason is that there has always been a lack of effective targeted drugs for gliomas. The only chemotherapy drug used clinically is temozolomide, with almost no alternatives. However, the effect of temozolomide is also very limited, at most only able to extend the survival period of patients by 2 months. Other treatment methods such as molecular targeted therapy, immunotherapy, and electric field therapy are still in the research stage, and no effective method for treating gliomas has been found. Therefore, there is an urgent need to develop new drugs and treatment methods to improve the postoperative prognosis and survival period of patients with gliomas. In addition, the incidence of head and neck and facial tumors such as brain cancers, oral cancers, head and neck cancers, tongue cancers, and nasal cancers is also relatively high and shows a trend of getting younger. Although there is a certain survival period after surgical resection of tongue cancers and nasal cancers, the surgery causes great difficulties to the patients' lives and poses a great threat to human healthy life.

[0004] Skin tumors (such as basal cell carcinoma, squamous cell carcinoma, cutaneous T-cell lymphoma, melanoma) also have varying degrees of incidence and mortality, threatening people's lives and health. Basal cell carcinoma, also known as basal cell epithelioma, is highly destructive, mostly occurring in the elderly, and commonly found in the head, face, neck, and back of the hand, especially prominent parts of the face. Melanoma is a highly malignant tumor of melanocytes, mostly occurring in the skin, but also visible in mucous membranes and internal organs. Malignant melanoma evolves from congenital or benign melanocytic nevi, or can also occur newly. In recent years, the incidence and mortality of melanoma have been increasing year by year. Compared with other solid tumors, its lethal age is lower. In addition to early surgical resection, malignant melanoma lacks effective treatment and has a poor prognosis. Therefore, the early diagnosis and treatment of basal cell carcinoma, squamous cell carcinoma, cutaneous T-cell lymphoma, and melanoma are extremely important.

[0005] The high incidence of genitourinary system tumors (such as prostate cancer and bladder cancer) has seriously affected people's physical and mental health. Prostate cancer is an epithelial malignant tumor that occurs in the prostate. It has a high incidence in middle-aged and elderly men. Its incidence is mainly related to family inheritance, diet, and lifestyle habits. Bladder cancer is also a cell lesion with a high incidence rate, posing a serious threat to human life. Traditional treatments for the above tumors mainly include surgery, radiotherapy, chemotherapy and other treatment methods, but these methods have little effect on the treatment of some tumors, and produce serious toxic side effects, and cause serious damage to the human body after surgery or radiotherapy and chemotherapy. Therefore, there is an urgent need to develop new treatments and drugs to improve the prognosis and survival of tumor patients after surgery.

[0006] Photodynamic therapy (PDT) is a new method for treating tumors that appeared in the late 1970s. It has been officially approved in the United States, Europe, Japan, and China, and has become a new technology for treating tumors. PDT is a method in which a photosensitizer is taken into the human body. Within a certain period of time, the photosensitizer can be more enriched in tumor cells and tissues. The tumor lesion is irradiated with a light source of a specific wavelength. The photosensitizer taken into the human body is excited to produce reactive oxygen species, which can damage and destroy tumor cells and tissues through photochemical reactions, thereby achieving the purpose of treating tumors. PDT has been used to treat a variety of tumors and has a certain effect on controlling local growth of tumors. Compared with other therapies, PDT can selectively destroy tumor tissues and has little damage to normal cells and tissues. PDT has a good therapeutic effect on tumor tissues with invasive margins, and invasive tissues are usually not suitable for surgical resection. The photosensitizer used in photodynamic therapy for tumors needs to meet the following conditions: 1) The chemical composition of the photosensitizer is single and has no obvious toxic effect on the human body; 2) It can be targeted and absorbed by tumor cells and tissues and accumulate, and rarely enter normal cells; 3) The maximum absorption wavelength corresponding to the photosensitizer has strong penetration in tumor tissue; 4) It can selectively damage tumor cells under the action of light, while causing less damage to adjacent normal cells; 5) It can be quickly excreted from the body after treatment without other toxic side effects. PDT has become the first choice for some vascular diseases such as port wine stains. PDT has been tried many times in the clinical treatment of cancer, or combined with surgical treatment to remove the invasive tumor boundary and retain normal cells and tissues to the maximum extent. Head, neck, facial and skin tumors, such as basal cell carcinoma, head and neck cancer, tongue cancer, nasal cancer, oral cancer, melanoma, etc. are particularly suitable for photodynamic therapy because they are suitable for light and tissue oxygen supply. Especially with the development of modern medicine and the increasing maturity of endoscopic technology, almost all cavity-type tumors (such as esophageal cancer, gastric cancer, lung cancer, liver cancer, bile duct cancer, colon cancer, nasal cancer, prostate cancer, and bladder cancer) can be treated by introducing light sources through optical fibers, assisted by the enrichment of photodynamic drugs in tumors and tissues, to achieve efficient photodynamic therapy.

[0007] Photodynamic therapy has unique therapeutic advantages for high-risk malignant tumors, especially for tumors that are poorly treated by surgery and radiotherapy. For example, gliomas are very prone to recurrence due to their invasive margins, with extremely high mortality and extremely short postoperative survival. If surgery for gliomas is supplemented with photodynamic therapy, tumor cells at the border can be killed, effectively delaying tumor recurrence and prolonging postoperative survival. Currently, photosensitizers used in tumor research and treatment mainly include: hematoporphyrin derivatives (HpD), talaporfin, temoporfin, 5-aminolevulinic acid (5-ALA), etc. However, these compounds all belong to porphyrin compounds (5-aminolevulinic acid is converted into protoporphyrin through biosynthesis in the body). The main problem is that it is difficult to separate geometric isomers and it is difficult to obtain photosensitizers with a single component. The relatively complex components of these mixed photosensitizers are not conducive to the evaluation of later drug metabolism and toxicological analysis. In order to better play the role of photodynamic therapy in the treatment of tumors, it is necessary to develop efficient, low-toxic and stable photosensitizers with strong light absorption ability in the phototherapy window.

[0008] Hypocrellin belongs to the perylenquinone class of compounds and is a natural plant photosensitizer. It is produced by a parasitic fungus, Hypocrellin, on the arrow bamboo at an altitude of more than 3,000 meters on the Yunnan Plateau in my country. Natural hypocrellin mainly exists in the form of Hypocrellin A (HA) and Hypocrellin B (HB). Hypocrellin has strong absorption in the visible light region and a large molar extinction coefficient, and can efficiently produce reactive oxygen under photosensitizing conditions. Past studies have shown that hypocrellin has the advantages of good phototoxicity, low dark toxicity, clear structure, fast metabolism in the body, and no toxic side effects in the body. In addition, the structure of hypocrellin is easy to modify, and the modified derivatives can meet the requirements of strong light absorption in the phototherapy window (600-900nm) and water solubility to meet the needs of clinical intravenous injection. Therefore, hypocrellin has broad application prospects as a photodynamic drug. However, there are still very few studies on the use of hypocrellin photosensitizers for the treatment of tumors. For example, for photodynamic therapy of brain glioma, the use of photodynamic drugs for clinical tumor treatment is still in the preclinical research stage. The photosensitizers studied mainly include: hematoporphyrin derivatives, talaporfin, temoporfin, and 5-ALA. From the existing literature reports, these drugs have limited effects in treating brain glioma. Therefore, it is urgent to develop new photodynamic drugs to treat tumors.

[0009] We first disclosed a derivative with simultaneous substitution at the 3-position and 2-position of hypocrellin (ZL201811020381.4). Through further research, we surprisingly found that such derivatives can efficiently kill certain specific tumor cells, such as esophageal cancer, gastric cancer, lung cancer, liver cancer, cholangiocarcinoma, and colon cancer cells related to digestive tract tumors; brain cancer, head and neck cancer, tongue cancer, nasal cancer, oral cancer, and glioblastoma cells related to head, neck, and facial tumors; basal cell carcinoma, squamous cell carcinoma, cutaneous T-cell lymphoma, and melanoma cells related to skin tumors; prostate cancer and bladder cancer cells related to genitourinary tumors.

[0010] In addition, regarding the problem that there is no drug to mediate and locate for guiding tumor resection during surgery for some tumors (such as glioblastoma), we also surprisingly found that such hypocrellin derivatives can specifically accumulate in tumor cells and tissues, and there is no photosensitizer accumulation in the brain area without tumors, showing good tumor-targeted enrichment. At this time, when irradiating the tumor tissue with light of a specific wavelength, detectable fluorescence can be excited to locate the position of the tumor tissue, which can be used for fluorescence-guided surgery (FGS) of glioblastoma. Summary of the Invention

[0011] The present invention provides compounds shown in Formulae I-a to I-d, derivatives with simultaneous amino substitution at the 3-position and 2-position of hypocrellin, and their isomers, isotope-labeled substances, pharmaceutically acceptable salts or solvates for use in the preparation of photodynamic anti-tumor drugs, wherein the tumors are esophageal cancer, gastric cancer, lung cancer, liver cancer, cholangiocarcinoma, colon cancer, brain cancer, head and neck cancer, tongue cancer, nasal cancer, oral cancer, glioblastoma, basal cell carcinoma, squamous cell carcinoma, melanoma, cutaneous T-cell lymphoma, prostate cancer, and bladder cancer.

[0012]

[0013] Among them, the 3-position, 4-position, 9-position, or 10-position marked in Formulae I-a to I-d is the 3-position of hypocrellin; derivatives of the above four structural general formulas will be generated simultaneously, and the proportions in the final products obtained under different preparation conditions are different.

[0014] R3 is -COCH3 or -H; R4 is -H, -F, -Cl, -Br, -I, or -S-R5, where R5 is an alkyl group with 2 to 12 carbon atoms, an alkyl group with 2 to 12 carbon atoms having a hydroxyl group at the end, or an alkyl group with 2 to 12 carbon atoms having a carboxyl group at the end;

[0015] R1 and R2 are each independently connected to an amino group; R1 and R2 can be the same or different; the structural general formulas of R1 and R2 are each independently as shown in Formula II:

[0016]

[0017] In formula II, 0 ≤ m ≤ 8, 0 ≤ n ≤ 50, 0 ≤ p ≤ 8, 0 ≤ q ≤ 8, 0 ≤ r ≤ 1, 0 ≤ s ≤ 8; m, n, p, q, r, and s are each independently zero or a positive integer; X and Y are each independently a linking group; Z is a terminal group; (OCH2CH2) n is a polyethylene glycol unit;

[0018] In formula II, the linking groups X and Y are each independently -NH-, -O-, -S-, a carboxylate group, an amide group, a sulfonate group, a sulfonamide group, a carbonyl group, a phosphate group, an unsaturated hydrocarbon group having 3 to 12 carbon atoms, a cycloalkyl group having 3 to 12 carbon atoms, an aryl group having 6 to 12 carbon atoms, or a heterocyclic group having 3 to 12 carbon atoms;

[0019] The unsaturated hydrocarbon group having 3 to 12 carbon atoms is a substituted or unsubstituted alkenyl or alkynyl group or an alkenyl or alkynyl group containing a heteroatom; the cycloalkyl group having 3 to 12 carbon atoms is a substituted or unsubstituted cycloalkyl group, cycloalkenyl group, or cycloalkynyl group, or a cycloalkyl group, cycloalkenyl group, or cycloalkynyl group containing a heteroatom, and the heteroatom is an oxygen, nitrogen, or sulfur atom; the aryl group having 6 to 12 carbon atoms is a substituted or unsubstituted aryl group, and the substituted aryl group is a mono-substituted or multi-substituted aryl group, and the substitution position is the ortho, meta, or para position of the aryl group; the heterocyclic group having 3 to 12 carbon atoms is a substituted or unsubstituted heterocyclic group, and the substituted heterocyclic group is a mono-substituted or multi-substituted heterocyclic group, and the substitution position is the ortho, meta, or para position of the heterocyclic ring; the heterocyclic group is a furyl group, pyrrolyl group, thienyl group, pyrazolyl group, imidazolyl group, oxazolyl group, thiazolyl group, pyridyl group, piperidyl group, pyrimidinyl group, pyrazinyl group, piperazinyl group, indolyl group, quinolinyl group, isoquinolinyl group, purinyl group, pyrimidinyl group, or acridinyl group;

[0020] The substituents on the above cycloalkyl group, cycloalkenyl group, aryl group, or heterocyclic group are each independently an alkyl group having 1 to 8 carbon atoms, an alkenyl group having 2 to 8 carbon atoms, an alkynyl group having 2 to 8 carbon atoms, a cycloalkyl group having 3 to 8 carbon atoms, an aryl group, an aralkyl group having 6 to 12 carbon atoms; or an alkyl group having a terminal group containing a hydroxyl group, a carboxyl group, a sulfonic acid group, or a carboxylate group;

[0021] The terminal group Z in formula II is selected from the group consisting of hydrogen, an alkyl group having 1 to 8 carbon atoms, an alkoxy group having 1 to 8 carbon atoms, a cycloalkyl group having 3 to 8 carbon atoms, a phenyl group, a pyridyl group, a hydroxyl group, an amino group, a mercapto group, a carboxyl group, a carboxylate group, a sulfonic acid group, a sulfonate group, a phosphate group, a phosphate ester group, an amino acid, triphenylphosphine, a quaternary ammonium salt, a pyridinium salt, and a carboxylate salt, sulfonate salt, and amino acid salt formed by a cation allowed in a pharmaceutical preparation;

[0022] When the terminal group Z in formula II is a quaternary ammonium salt, the three substituents of the quaternary ammonium salt are each independently: an alkyl group having 1 to 8 carbon atoms, an alkenyl group having 2 to 8 carbon atoms, an alkynyl group having 2 to 8 carbon atoms, a cycloalkyl group having 3 to 8 carbon atoms, a cycloalkenyl group having 3 to 8 carbon atoms, an aryl group, an aralkyl group having 6 to 12 carbon atoms, or an alkyl group having a terminal group containing a hydroxyl group, a carboxyl group, a sulfonic acid group, or a carboxylate group; the anion in the quaternary ammonium salt is an anion allowed in a pharmaceutical preparation;

[0023] When the end group Z in formula II is a pyridinium salt, the substituents on the pyridine ring in the pyridinium salt are in the ortho, meta or para position; the pyridinium salt is formed by quaternizing pyridine with halogenated hydrocarbons containing 1-8 carbon atoms with different chain lengths; the anion in the pyridinium salt is an anion permitted in pharmaceutical preparations.

[0024] The linking groups X and Y in formula II are each independently: -NH-, -O-, -S-, -COO-, -OC(=O)-, -CONH-, -NHC(=O)-, -SO3-, -SO2NH-, -C(=O)-, -PO3-, vinylene, ethynylene, -cyclopropyl-, -cyclobutyl-, -cyclopentyl-, -cyclohexyl-, -cycloheptyl-, -phenyl-, -pyridyl-, -piperidyl-, -furyl-, -pyrrolyl-, -thienyl-, -pyrazolyl-, -imidazolyl-, -oxazolyl-, -thiazolyl-, -pyrimidinyl-, -indolyl-, -quinolyl-, -isoquinolyl-, -purinyl-, -pyrimidinyl-, -acridinyl-, -morpholinyl- The above groups are optionally substituted with 0 to 4 of the following substituents: methyl, ethyl, propyl, methoxy, ethoxy, propoxy, carboxyl, amino, hydroxyl, hydroxymethyl, hydroxyethyl, carboxymethyl, carboxyethyl, aminomethyl, aminoethyl, halogen (F, Cl, Br, I).

[0025] Preferably, the linking groups X and Y in formula II are each independently: -NH-, -O-, -S-, -COO-, -OC(=O)-, -CONH-, -NHC(=O)-, -SO3-, -SO2NH-, -C(=O)-, -PO3-, -CH=CH-, -C(CH3)=CH-, -C(CH3)=C(CH3)-, -C(COOH)=CH-, -C(CH2COOH)=CH-, -C≡C-, -cyclopropyl-, -(methyl)cyclopropyl-, -(hydroxy)cyclopropyl-, -(carboxy)cyclopropyl-, -cyclobutyl-, -(methyl)cyclobutyl-, -(hydroxy)cyclobutyl-, -(carboxy)cyclobutyl-, -cyclopentyl-, -(methyl)cyclopentyl-, -(hydroxy)cyclopentyl-, -(amino)cyclopentyl-, -(carboxy)cyclopentyl-, -cyclohexyl-, -(methyl)cyclohexyl-, -(ethyl)cyclohexyl-, -(propyl)cyclohexyl-, -(hydroxy)cyclohexyl-, -(amino)cyclohexyl-, -(carboxy)cyclohexyl-, -(carboxymethyl)cyclohexyl-, -(dicarboxy)cyclohexyl-, -cycloheptyl-, -(carboxy)cycloheptyl-, -(hydroxy)cycloheptyl-, -(methyl)cycloheptyl-, -phenyl-, -(methyl)phenyl-, -(ethyl)phenyl-, -(dimethyl)phenyl-, -(hydroxy)phenyl-, -(methoxy)phenyl-, -(ethoxy)phenyl-, -(hydroxymethyl)phenyl-, -(amino)phenyl-, -(aminomethyl)phenyl-, -(fluoro)phenyl-, -(chloro)phenyl-, -(bromo)phenyl-, -(iodo)phenyl-, -(carboxy)phenyl-, -(dicarboxy)phenyl-, -(sulfo)phenyl-, -(carboxymethyl)phenyl-, -(carboxyethyl)phenyl-, -pyridyl-, -(methyl)pyridyl-, -(hydroxy)pyridyl-, -(amino)pyridyl-, -(aminomethyl)pyridyl-, -(carboxy)pyridyl-, -(carboxymethyl)pyridyl-, -piperidyl-, furyl, pyrrolyl, thienyl, pyrazolyl, imidazolyl, oxazolyl, thiazolyl, pyridyl, piperidyl, pyrimidinyl, indolyl, quinolinyl, isoquinolinyl, purinyl, pyrimidinyl, acridinyl, morpholinyl,

[0026] Preferably, the end group Z in formula II is: -H, -CH3, -C2H5, -C3H7, -C4H9, -C5H 11 、-C6H 13 、-C 12 H 25 、-OCH3, -OC2H5, -OC3H7, -OC4H9, -OC5H 11 、-OC6H 13 、-OC 12 H 25 、-C3H5, -C4H7, -C5H9, -C6H 11, -C7H 13 , -C6H5, -C5H4N, -OH, -NH2, -SH, -COOH, -COOCH3, -COOC2H5, -SO3H, -SO3CH3, -SO3C2H5, -PPh3 + (triphenylphosphine), glycine group, alanine group, valine group, leucine group, isoleucine group, phenylalanine group, proline group, tryptophan group, tyrosine group, serine group, cysteine group, methionine group, aspartic acid group, glutamic acid group, threonine group, aspartic acid group, glutamic acid group, lysine group, arginine group, histidine group, cystine group, glutathione group, -C5H4N + , -C5H4N + (CH3), -C5H4N + (C2H5), -C5H4N + (C 12 H 25 ), -N + (CH3)3, -N + (C2H5)3, -N + (C3H7)3, -N + (C4H9)3, -N + (C6H 13 ), -N + (CH3)2(C2H5), -N + (CH3)2(C3H7), -N + (CH3)2(C4H9), -N + (CH3)2(C6H 13 ), -N + (CH3)2(C 12 H 25 ), -N + (C2H5)2(C3H7), -N + (C2H5)2(C6H 13 ), or a quaternary ammonium salt whose end group contains a hydroxyl group, a carboxylic acid group, a sulfonic acid group or a carboxylic acid ester.

[0027] In the above formulas I-a to I-d, the substituents R1 and R2 independently connected to the amino group can be the same or different.

[0028] Specifically, the substituents R1 and R2 can independently be: hydrogen, alkyl, phenyl, substituted phenyl, phenylalkyl, substituted phenylalkyl, such as: -H, -CH3, -C2H5, -C3H7, -C4H9, -C5H 11 , -C6H 13 , -C 12 H 25, -C6H5, -CH2C6H5, -CH2CH2C6H5, -C6H4(COOH), -CH2C6H4(COOH), -CH2C6H4(OH), -C6H4(CH2COOH), -CH2C6H4(CH2COOH).

[0029] Preferably, each of the substituents R1 and R2 is independently: a cycloalkyl group or a cycloalkyl group with a substituent, such as: cyclopropyl, methylcyclopropyl, hydroxycyclopropyl, hydroxymethylcyclopropyl, carboxycyclopropyl, cyclobutyl, methylcyclobutyl, hydroxycyclobutyl, carboxycyclobutyl, -CH2C4H6(COOH), cyclopentyl, methylcyclopentyl, hydroxycyclopentyl, aminocyclopentyl, carboxycyclopentyl, cyclohexyl, methylcyclohexyl, ethylcyclohexyl, propylcyclohexyl, hydroxycyclohexyl, aminocyclohexyl, carboxycyclohexyl, carboxymethylcyclohexyl, dicarboxycyclohexyl, -CH2C6H 10 (COOH), -CH2C6H 10 (OH), cycloheptyl, carboxycycloheptyl, hydroxycycloheptyl, methylcycloheptyl.

[0030] Preferably, each of the substituents R1 and R2 is independently: a carboxylic acid, a carboxylic acid ester, a carboxylate salt, such as: -CH2COOH, -CH2CH2COOH, -CH2(CH2)2COOH, -CH2(CH2)3COOH, -CH2(CH2)4COOH, -CH2(CH2)5COOH, -CH2(CH2)6COOH, -CH2(CH2) 10 COOH, -CH2COOCH3, -CH2CH2COOC6H 13 , -CH2(CH2)2COOCH3, -CH2(CH2)2COOC6H 13 , -CH2(CH2)4COOCH3, -CH2(CH2)6COOC6H 13 , -CH2COONa + , -CH2(CH2)2COONa + , -CH2(CH2)4COONa + .

[0031] Preferably, each of the substituents R1 and R2 is independently: a sulfonic acid, a sulfonic acid ester, a sulfonate salt, such as: -CH2SO3H, -CH2CH2SO3H, -CH2(CH2)2SO3H, -CH2(CH2)3SO3H, -CH2(CH2)4SO3H, -CH2(CH2)5SO3H, -CH2(CH2) 11 SO3H, -CH2SO3CH3, -CH2SO3C6H 13, -CH2CH2SO3CH3, -CH2(CH2)2SO3CH3, -CH2(CH2)2SO3C6H 13 , -CH2(CH2)4SO3C4H9, -CH2(CH2) 11 SO3C6H 13 , -CH2SO3Na, -CH2CH2SO3K.

[0032] Preferably, the substituents R1 and R2 are each independently: hydroxy, alkoxy, substituted and unsubstituted amino, substituted and unsubstituted pyridyl, such as: -OH, -OCH3, -OC2H5, -OC6H 13 , -NH2, -NHC2H5, -NHC6H 13 , -NHC 12 H 25 , -NHC6H5, -NHC5H4N, -C5H4N, -CH2C5H4N, -(CH2)2C5H4N, -(CH2)6C5H4N, -C5H3N(CH3), -C5H3N(OH), -C5H3N(NH2), -C5H3N(COOH), -C5H3N(CH2COOH), -CH2C5H3N(CH2COOH).

[0033] Preferably, the substituents R1 and R2 are each independently: polyethylene glycol, polyethylene glycol ether, polyethylene glycol ester, such as: -CH2CH2-(OCH2CH2) n -OH, -CH2CH2-(OCH2CH2) n -OCH3, -CH2CH2-(OCH2CH2) n -OC6H 13 , -CH2CH2-(OCH2CH2) n -O-COCH3, -CH2CH2-(OCH2CH2) n -O-COC6H 13 (where n is an integer between 0 and 50 for each above).

[0034] Preferably, R1 and R2 are each independently: two polyethylene glycols with different chain lengths connected by a carboxylic acid ester group, such as: -CH2CH2-O-CO-CH2CH2-(OCH2CH2) n -OH, -CH2CH2-O-CO-CH2CH2-(OCH2CH2) n -OCH3, -(CH2CH2-O)2-CO-CH2CH2-(OCH2CH2) n -OH, -(CH2CH2-O)2-CO-CH2CH2-(OCH2CH2) n-OCH3, -(CH2CH2-O)3-O-CO-CH2CH2-(OCH2CH2) n -OH, -(CH2CH2-O)3-CO-CH2CH2-(OCH2CH2) n -OCH3 (where n is an integer between 0 and 50 for all above).

[0035] Preferably, the substituents R1 and R2 in formula II are each independently: polyethylene glycols of different chain lengths and triphenylphosphine linked by a carboxylic acid ester group, such as: -CH2CH2-O-CO-CH2CH2-PPh3 + 、-CH2CH2-O-CO-(CH2)3-PPh3 + 、-CH2CH2-O-CO-(CH2)5-PPh3 + 、-(CH2CH2-O)2-CO-CH2CH2-PPh3 + 、-(CH2CH2-O)2-CO-(CH2)3-PPh3 + 、-(CH2CH2-O)2-CO-(CH2)5-PPh3 + 。

[0036] Preferably, the substituents R1 and R2 are each independently: -C 1-10 alkyl-OH, -C 1-10 alkyl-O-C 1-10 alkyl, -C 1-10 alkyl-O-CO-C 1-10 alkyl, -C 1-10 alkyl-O-CO-polyethylene glycol-OCH3,, such as: -(CH2)3-OH, -(CH2)3-OCH3, -(CH2)3-OC2H5, -(CH2)3-OCOCH3, -(CH2)3-OCOC2H5, -(CH2)3-O-COCH2CH2-(OCH2CH2) n -OCH3; -(CH2)4-OH, -(CH2)4-OCH3, -(CH2)4-OCOCH3, -(CH2)4-OCOC2H5, -(CH2)4-O-COCH2CH2-(OCH2CH2) n -OCH3; -(CH2)6-OH, -(CH2)6-OCH3, -(CH2)6-OCOCH3, -(CH2)6-O-COCH2CH2-(OCH2CH2) n -OCH3 (where n is an integer between 0 and 50 for all above).

[0037] Preferably, substituents R1 and R2 are each independently: azapolyethylene glycol, thia polyethylene glycol, and polyethylene diamine and polyethylene glycol linked by amide, such as: -CH2CH2-NH-CH2CH2-(OCH2CH2) n -OH, -CH2CH2-NH-CH2CH2-(OCH2CH2) n -OCH3, -CH2CH2-(NHCH2CH2) n -NH2, -CH2CH2-(NHCH2CH2) n -N(CH3)2, -CH2CH2-NHCH2CH2-NH-COCH2CH2-(OCH2CH2) n -OCH3, -CH2CH2-S-CH2CH2-(OCH2CH2) n -OH (where n is an integer between 0 and 50 for all above).

[0038] Preferably, substituents R1 and R2 are each independently: aminocarboxylic acid, amino acid ester, amino acid salt, such as: -CH(CH3)-COOH, -CH(CH(CH3)2)-COOH, -CHCH2(CH(CH3)2)-COOH, -CH(CH2CH2SCH3)-COOH, -CHCH(CH3)(C2H5)-COOH, -CH(CH2OH)-COOH, -CHCH(OH)(CH3)-COOH, -CH(CH2SH)-COOH, -CH(CH2CONH2)-COOH, -CH(CH2CH2CONH2)-COOH, -CH(CH2C6H5)-COOH, -CH(CH2C6H5OH)-COOH, -CH(CH2CH2CH2CH2NH3 + )-COOH, -CH(COOH)-CH2COOH, -CH(COOH)-CH2CH2COOH, -CH(CH3)-COOCH3, -CH(CH(CH3)2)-COOCH3, -CHCH2(CH(CH3)2)-COOCH3, -CH(CH2CH2SCH3)-COOCH3, -CH(CH3)-COONa + , -CH(CH(CH3)2)-COONa + , -CHCH2(CH(CH3)2)-COOK + , -CH(CH2CH2SCH3)-COOK + .

[0039] Preferably, substituents R1 and R2 are each independently: -CH2CO-(OCH2CH2)n -OH, -CH2CO-(OCH2CH2) n -OCH3, -CH2CH2CO-(OCH2CH2) n -OH, -CH2CH2CO-(OCH2CH2) n -OCH3, -CH2(CH2)2CO-(OCH2CH2) n -OH, -CH2(CH2)2CO-(OCH2CH2) n -OCH3, -CH2(CH2)4CO-(OCH2CH2) n -OH, -CH2(CH2)4CO-(OCH2CH2) n -OCH3 (where n is an integer between 0 and 50 for all above).

[0040] Preferably, the substituents R1 and R2 are each independently: -C 1-10 alkyl-CO-NH-CH2CH2-(OCH2CH2) n -OH, -C 1-10 alkyl-CO-NH-CH2CH2-(OCH2CH2) n -OCH3, such as: -CH2-CO-NH-CH2CH2-(OCH2CH2) n -OH, -CH2-CO-NH-CH2CH2-(OCH2CH2) n -OCH3, -(CH2)2-CO-NH-CH2CH2-(OCH2CH2) n -OH, -(CH2)2-CO-NH-CH2CH2-(OCH2CH2) n -OCH3, -(CH2)3-CO-NH-CH2CH2-(OCH2CH2) n -OH, -(CH2)3-CO-NH-CH2CH2-(OCH2CH2) n -OCH3, -(CH2)4-CO-NH-CH2CH2-(OCH2CH2) n -OH, -(CH2)4-CO-NH-CH2CH2-(OCH2CH2) n -OCH3, -(CH2)5-CO-NH-CH2CH2-(OCH2CH2) n -OH, -(CH2)5-CO-NH-CH2CH2-(OCH2CH2) n -OCH3 (where n is an integer between 0 and 50 for all above).

[0041] Preferably, the substituents R1 and R2 are each independently: -C1-10 alkyl-SO2-(OCH2CH2) n -OH, -C 1-10 alkyl-SO2-(OCH2CH2) n -OCH3, e.g., -CH2-SO2-(OCH2CH2) n -OH, -CH2-SO2-(OCH2CH2) n -OCH3, -(CH2)2-SO2-(OCH2CH2) n -OH, -(CH2)2-SO2-(OCH2CH2) n -OCH3, -(CH2)3-SO2-(OCH2CH2) n -OH, -(CH2)3-SO2-(OCH2CH2) n -OCH3, -(CH2)4-SO2-(OCH2CH2) n -OH, -(CH2)4-SO2-(OCH2CH2) n -OCH3, -(CH2)5-SO2-(OCH2CH2) n -OH, -(CH2)5-SO2-(OCH2CH2) n -OCH3, -(CH2)6-SO2-(OCH2CH2) n -OH, -(CH2)6-SO2-(OCH2CH2) n -OCH3 (where n is an integer between 0 and 50 for all above).

[0042] Preferably, substituents R1 and R2 are each independently: -C 1-10 alkyl-SO2-NHCH2CH2-(OCH2CH2) n -OH, -C 1-10 alkyl-SO2-NHCH2CH2-(OCH2CH2) n -OCH3, e.g., -CH2-SO2-NHCH2CH2-(OCH2CH2) n -OH, -CH2-SO2-NHCH2CH2-(OCH2CH2) n -OCH3, -(CH2)2-SO2-NHCH2CH2-(OCH2CH2) n -OH, -(CH2)2-SO2-NHCH2CH2-(OCH2CH2) n -OCH3, -(CH2)3-SO2-NHCH2CH2-(OCH2CH2) n-OCH3, -(CH2)4-SO2-NHCH2CH2-(OCH2CH2) n -OCH3, -(CH2)5-SO2-NHCH2CH2-(OCH2CH2) n -OCH3, -(CH2)6-SO2-NHCH2CH2-(OCH2CH2) n -OCH3 (where n is an integer between 0 and 50 for all of the above).

[0043] Preferably, the substituents R1 and R2 are each independently: quaternary ammonium salts of different chain lengths, such as: -CH2CH2-N + (CH3)3, -(CH2)3-N + (CH3)3, -(CH2)4-N + (CH3)3, -(CH2)5-N + (CH3)3, -(CH2)6-N + (CH3)3, -(CH2) 12 -N + (CH3)3, -CH2CH2-N + (C2H5)3, -(CH2)4-N + (C2H5)3, -(CH2)6-N + (C2H5)3, -(CH2) 12 -N + (C2H5)3, -CH2CH2-N + (C3H7)3, (CH2)4-N + (C3H7)3, -(CH2)6-N + (C3H7)3, -CH2CH2-N + (C4H9)3, -(CH2)6-N + (C4H9)3, -CH2CH2-N + (CH3)2(C2H5), -CH2CH2-N + (CH3)2(C4H9), -CH2CH2-N + (CH3)2(C6H 13 )、-CH2CH2-N + (CH3)2(C 12 H 25 )、-(CH2)3-N + (CH3)2(C4H9), -(CH2)3-N + (CH3)2(C6H 13 )、-(CH2)3-N + (CH3)2(C 12 H25 )、-(CH2)4-N + (CH3)2(C6H 13 )、-(CH2)4-N + (CH3)2(C 12 H 25 )、-(CH2)5-N + (CH3)2(C2H5)、-(CH2)5-N + (CH3)2(C6H 13 )、-(CH2)5-N + (CH3)2(C 12 H 25 )、-(CH2)6-N + (CH3)2(C2H5)、-(CH2)6-N + (CH3)2(C6H 13 )、-(CH2)6-N + (CH3)2(C 12 H 25 )。

[0044] Preferably, each of the substituents R1 and R2 is independently: -C 1-10 alkyl-CO-O-C 1-20 alkyl-N + (C 1-10 alkyl)3, such as: -CH2CO-OCH2CH2-N + (CH3)3, -CH2CH2CO-OCH2CH2-N + (CH3)3, -CH2(CH2)2CO-OCH2CH2-N + (CH3)3, -CH2(CH2)6CO-OCH2CH2-N + (CH3)3, -CH2CO-O-(CH3)3-N + (CH3)3, -CH2(CH2)2CO-O-(CH3)3-N + (CH3)3, -CH2COOCH2CH2-N + (CH3)2(C6H 13 )。

[0045] Preferably, each of the substituents R1 and R2 is independently: -C 1-10 alkyl-CO-NH-C 1-20 alkyl-N + (C 1-10 alkyl)3, such as: -CH2CONH-CH2CH2-N + (CH3)3, -CH2CH2CONH-CH2CH2-N+ (CH3)3, -CH2(CH2)4CONH-CH2CH2-N + (CH3)3, -CH2CONH-(CH2)3-N + (CH3)3, -CH2CH2CONH-(CH2)3-N + (CH3)3, -CH2(CH2)4CONH-(CH2)3-N + (CH3)3, -CH2CONH-(CH2)4-N + (CH3)3, -CH2CH2CONH-(CH2)4-N + (CH3)3, -CH2(CH2)4CONH-(CH2)4-N + (CH3)3, -CH2CONH-(CH2)5-N + (CH3)3, -CH2CH2CONH-(CH2)5-N + (CH3)3, -CH2(CH2)4CONH-(CH2)5-N + (CH3)3, -CH2CONH-(CH2)6-N + (CH3)3, -CH2CH2CONH-(CH2)6-N + (CH3)3, -CH2(CH2)4CONH-(CH2)6-N + (CH3)3, -CH2CONH-CH2CH2-N + (CH3)2(C6H 13 )、-CH2CONH-CH2CH2-N + (CH3)2(C 12 H 25 )。

[0046] Preferably, the substituents R1 and R2 are each independently: a substituted or unsubstituted pyridinium salt, such as: -C5H4N + (CH3), -CH2C5H4N + (CH3), -CH2C5H4N + (C6H 13 )、-CH2C5H4N + (CH2COOH), -CH2CH2C5H4N + (CH3), -CH2CH2C5H4N + (C6H 13 )、-CH2CH2C5H4N + (CH2COOH)。

[0047] Preferably, each of R1 and R2 is independently: methylene cyclohexanoic acid and polyethylene glycols with different chain lengths linked by a carboxylic acid ester bond or an amide bond, such as:

[0048] Preferably, each of R1 and R2 is independently: cyclohexanoic acid (ortho, para, meta) and polyethylene glycols with different chain lengths linked by a carboxylic acid ester bond, such as:

[0049] Preferably, the substituents R1 and R2 are each independently: cyclohexylacetic acid (or cyclohexylpropionic acid) and polyethylene glycols with different chain lengths linked by a carboxylic acid ester bond, such as:

[0050] Preferably, the substituents R1 and R2 are each independently: cyclopentanoic acid at the meta or ortho position and polyethylene glycols with different chain lengths linked by a carboxylic acid ester bond, with the end groups being alkoxy or hydroxyl, such as:

[0051] Preferably, the substituents R1 and R2 are each independently: methylene piperidine directly linked to polyethylene glycols with different chain lengths or linked by an amide bond, such as:

[0052] Preferably, each of R1 and R2 is independently: cyclopropane linked to polyethylene glycols with different chain lengths by a carboxylic acid ester or an amide bond, such as:

[0053] In the above, n is a positive integer between 0 and 50;

[0054] Preferably, the substituents R1 and R2 are each independently: methylene cyclohexanoic acid linked to triphenylphosphine by a carboxylic acid ester or an amide bond, such as:

[0055] In addition, the substituents R1 and R2 can also each independently be various heterocyclic substituents, such as:

[0056]

[0057] According to the embodiments of the present invention, the compounds shown in I-a to I-d have enol tautomers; wherein I-a and I-a' are the enol tautomers at the 9th and 10th positions in the structural formula; I-b and I-b' are the enol tautomers at the 3rd and 4th positions in the structural formula; I-c and I-c' are the enol tautomers at the 9th and 10th positions in the structural formula; I-d and I-d' are the enol tautomers at the 3rd and 4th positions in the structural formula, as specifically shown below.

[0058]

[0059] According to an embodiment of the present invention, the tumor may be esophageal cancer cell AKR, gastric cancer cell MFC, lung cancer cell A549, liver cancer cell HCC, cholangiocarcinoma 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, glioblastoma cell C6, basal cell carcinoma cell BCC, squamous skin cancer cell PECA, melanoma cell B16, cutaneous T-cell lymphoma cell HH, prostate cancer cell LNCaP, bladder cancer cell MBT-2.

[0060] According to an embodiment of the present invention, the drug may be a photosensitizing drug, a fluorescence-mediated drug.

[0061] According to an embodiment of the present invention, the drug may be enriched in the tumor cells.

[0062] According to an embodiment of the present invention, the pharmaceutically acceptable salts include salts formed by the compounds of formula I-a to I-d 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 reaction with inorganic bases, including sodium, potassium, calcium, aluminum salts and ammonium salts, or salts formed with organic bases, including methylamine salts, ethylamine salts and ethanolamine salts; or salts formed by reaction with basic amino acids selected from lysine, arginine and ornithine, followed by reaction with inorganic acids selected from hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid and phosphoric acid, or salts formed with organic acids selected from formic acid, acetic acid, picric acid, methanesulfonic acid and ethanesulfonic acid.

[0063] The present invention also provides the use of a compound represented by formula I-a to I-d, its isomers, isotope-labeled compounds, pharmaceutically acceptable salts or solvates in the preparation of a fluorescence-mediated drug for guiding the resection of the tumor boundary.

[0064] The present invention also provides the use of a compound represented by formula I-a to I-d, its isomers, isotope-labeled compounds or pharmaceutically acceptable salts in the treatment of tumor diseases, wherein the tumors are esophageal cancer, gastric cancer, lung cancer, liver cancer, cholangiocarcinoma, colon cancer, brain cancer, head and neck cancer, tongue cancer, nasal cancer, oral cancer, glioblastoma, basal cell carcinoma, squamous skin cancer, melanoma, cutaneous T-cell lymphoma, prostate cancer, bladder cancer.

[0065] According to an embodiment of the present invention, the treatment is photodynamic inactivation of tumor cells or guiding the resection of the tumor boundary as a fluorescence-mediated drug.

[0066] The present invention also provides a method for preventing or treating tumor diseases, which includes administering to a patient a prophylactically or therapeutically effective amount of at least one of the compounds represented by Formula I-a to I-d, their isomers, isotope-labeled substances, pharmaceutically acceptable salts or solvates.

[0067] In some embodiments, the patient is a human.

[0068] Beneficial effects

[0069] 1) The compounds represented by Formula I-a to I-d of the present invention, which are derivatives with simultaneous amino substitutions at the ortho-position and 2-position of hypocrellin, can efficiently kill the following cancer or tumor cells as anti-tumor drugs: cholangiocarcinoma, gastric cancer, lung cancer, liver cancer, cholangiocarcinoma, colon cancer, brain cancer, head and neck cancer, tongue cancer, nasal cancer, oral cancer, glioblastoma, basal cell carcinoma, squamous cell carcinoma, melanoma, cutaneous T-cell lymphoma, prostate cancer, bladder cancer. Such drugs at a concentration of 50 nM can kill more than 90% of the above-mentioned specific tumor cells, with an IC50 value of 20 - 30 nM, which is 1 - 2 orders of magnitude lower than that of commercially available porphyrin-based photosensitizers. Such drugs have basically no effect on normal cells and are basically excreted from the body after one week.

[0070] 2) The present invention for the first time discloses a derivative with simultaneous amino substitutions at the ortho-position and 2-position of hypocrellin as a mediating drug in clinical tumor surgery to guide tumor resection. Such hypocrellin derivatives can specifically accumulate in tumor tissues, and there is no accumulation of photosensitizer in areas without tumors, showing good tumor-targeted enrichment. At this time, when the tumor tissue is irradiated with light of a specific wavelength, detectable fluorescence can be excited to locate the position of the tumor tissue for fluorescence-guided tumor resection surgery. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] The following further details the specific embodiments of the present invention with reference to the accompanying drawings.

[0072] Figure 1 It is the general structural formula of the water-soluble derivative with polyethylene glycol substitution at the 2-position of hypocrellin of the present invention.

[0073] Figure 2 It is the synthetic route diagram of HB-1a-PEGn to HB-1d-PEGn (n is the number of units of polyethylene glycol).

[0074] Figure 3 It is the synthetic route diagram of HC-1a-PEGn to HC-1d-PEGn (n is the number of units of polyethylene glycol).

[0075] Figure 4 (a) It is the absorption spectrum comparison diagram of the commercially available porphyrin-based photosensitizer PpIX and Ce6; Figure 4(b) Absorption spectrum comparison diagrams of hypocrellin B (HB), HB-1c-PEG6-H and HC-1c-PEG6-H of Examples 1-2, respectively.

[0076] Figure 5 (a) Diagram of the interaction between HB-13a-PEG8 of Example 13-1 and singlet oxygen scavenger; Figure 5 (b) Diagram of the interaction between HB-13a-PEG8 and superoxide radical scavenger.

[0077] Figure 6 (a), 6(b) and 6(c) are the photodegradation curves of HB-51c-PEG16, HC-51c-PEG16 (Example 51-1), and the standard reference rose bengal (RB), respectively; Figure 6 (d) Diagram of the comparison of photodegradation curves.

[0078] Figure 7 (a) is the photo-stability comparison diagram of HB-1c-PEG6, HC-1c-PEG6 (Example 1-1) and commercial photosensitizers (HpD, Ce6) under laser irradiation of 20 mW / cm 2 for 30 min; Figure 7 (b) is the photo-stability comparison diagram of HB-13c-PEG8, HC-13c-PEG8 (Example 13-2) and commercial photosensitizers (HpD, Ce6) under laser irradiation of 20 mW / cm 2 for 30 min.

[0079] Figure 8 (a) is the pH-stability comparison diagram of HB-1a-PEG4-H, HC-1c-PEG6-H (Example 1-2) and commercial HpD; Figure 8 (b) is the pH-stability comparison diagram of HB-51b-PEG16, HC-51c-PEG16 (Example 51-1) and commercial HpD.

[0080] Figure 9 is the confocal fluorescence imaging diagram of HC-1c-PEG6 in A549 lung cancer cells; among them Figure 9 (a) is the superimposed image of dark field and bright field, Figure 9 (b) is the dark field image, Figure 9 (c) is the bright field image.

[0081] Figure 10-1 are the dark toxicity diagram (a) and photo-toxicity diagram (b) of commercial HpD, and HB-1c-PEG6 and HC-1c-PEG6 in Example 1-1 against esophageal cancer cell AKR.

[0082] Figure 10-2Dark toxicity graphs (a) and phototoxicity graphs (b) of commercial HpD, as well as HB-1a-PEG4-H and HC-1c-PEG8-H in Examples 1-2 against lung cancer cell A549.

[0083] Figure 10-3 Dark toxicity graphs (a) and phototoxicity graphs (b) of commercial HpD, as well as HB-2b-PEG8 and HC-2d-PEG12 in Example 2-1 against liver cancer cell HCC.

[0084] Figure 10-4 Dark toxicity graphs (a) and phototoxicity graphs (b) of commercial HpD, as well as HB-8a or HC-8c in Example 8 against colon cancer cell HCT116.

[0085] Figure 10-5 Dark toxicity graphs (a) and phototoxicity graphs (b) of commercial HpD, as well as HB-8b-PEG4 and HC-8d-PEG8 in Example 8-1 against cholangiocarcinoma cell MCC.

[0086] Figure 10-6 Dark toxicity graphs (a) and phototoxicity graphs (b) of commercial HpD, as well as HB-13a and HC-13c in Example 13 against gastric cancer cell MFC.

[0087] Figure 11 : (a) Phototoxicity graph of commercial HpD, as well as HB-13a-PEG4 and HC-13c-PEG8 in Example 13-2 against brain cancer cell G442; (b) Phototoxicity graph of commercial HpD, as well as HB-13a-NH-PEG12 and HC-13c-NH-PEG6 in Example 13-3 against head and neck cancer cell SCC2; (c) Phototoxicity graph of commercial HpD, as well as HB-13b-C4-N + and HC-13c-C6-N + against tongue cancer cell TSCCa; (d) Phototoxicity graph of commercial HpD, as well as HB-48c-PEG8 and HC-48d-PEG12 in Example 48 against nasal cancer cell KB; (e) Phototoxicity graph of commercial HpD, as well as HB-51a-PEG12 and HC-51c-PEG18 in Example 51-1 against oral cancer cell CAL27; (f) Phototoxicity graph of commercial HpD, as well as HB-52c-PEG12 and HC-52c-PEG16 in Example 52-2 against glioma cell C6.

[0088] Figure 12 : (a) Commercial HpD, as well as HB-1a-C4-N + and HC-1c-C6-N +Phototoxicity graphs of basal cell carcinoma cells BCC; (b) Phototoxicity graphs of commercial HpD, HB-4a-PEG4 in Example 4, and HC-4c-PEG6 against melanoma cells B16; (c) Phototoxicity graphs of commercial HpD, HB-10a-NH-PEG6 in Example 10-1, and HC-10c-NH-PEG8 against squamous skin cancer cells PECA; (d) Phototoxicity graphs of commercial HpD, HB-13b in Example 13, and HC-13d against prostate cancer cells LNCaP; (e) Phototoxicity graphs of commercial HpD, HB-11b-C4-SO3H in Example 11, and HC-11d-C6-SO3H against bladder cancer cells MBT-2; (f) Phototoxicity graphs of commercial HpD, HB-53a-PEG10 in Example 53-1, and HC-53c-PEG16 against cutaneous T-cell lymphoma HH cells.

[0089] Figure 13 Fluorescence imaging graphs of the drug in tumor-bearing mice within 0 - 24 h after tail vein injection of HC-1c-PEG6 in Example 1-1.

[0090] Figure 14 In-situ tumor enrichment comparison graphs of glioma-bearing mice at different times and doses: (a) Fluorescence imaging comparison graphs of drug enrichment for 4 hours: The left mouse is the reference without drug; the middle mouse is injected with 40 μL of HB-1c-PEG8 (10 mg / kg) via tail vein; the right mouse is injected with 40 μL of HB-1c-PEG8 (5 mg / kg) via tail vein; (b) Comparison graphs of drug enrichment for 6 hours: The left mouse is the reference without drug; the middle mouse is injected with 40 μL of HB-1c-PEG8 (10 mg / kg) via tail vein; the right mouse is injected with 40 μL of HB-1c-PEG8 (5 mg / kg) via tail vein.

[0091] Figure 15 Fluorescence imaging comparison graphs after 4-hour in-situ tumor enrichment of different photosensitizing drugs in glioma-bearing mice: (a) HC-1a-PEG8 in Example 1-1; (b) HB-1c-PEG8-H in Example 1-2; (c) HC-51c-PEG18 in Example 51-1; The drug concentration is 10 mg / kg for all.

[0092] Figure 16 (a) Fluorescence imaging graph of the drug in tumor-bearing mice (esophageal cancer) at 4 h after tail vein injection of HC-13c-PEG4 in Example 13-2; Figure 16 (b) Fluorescence imaging graph of the drug in tumor-bearing mice (lung cancer) at 4 h after tail vein injection of HB-13c-NH-PEG6 in Example 13-3; Figure 16(c) Fluorescence imaging of the drug in tumor-bearing mice (cutaneous T-cell lymphoma) 4 h after tail vein injection of HC-51c-PEG12-H in Example 51-2; Figure 16 (d) Fluorescence imaging of the drug in tumor-bearing mice (gastric cancer) 4 h after tail vein injection of HB-2a-PEG1 in Example 2-1.

[0093] Figure 17 (a) Fluorescence imaging of the drug in tumor-bearing mice 4 h after tail vein injection of HB-1a-PEG6-COOH (left), HB-1c-PEG8-COOH (middle), and HC-1c-C4-N + (right) in Examples 1-3 and 1-5;

[0094] Figure 17 (b) Fluorescence imaging of the drug in tumor-bearing mice 4 h after tail vein injection of HB-13a (left), HB-13c (middle), and HC-13c (right) in Example 13;

[0095] Figure 17 (c) Fluorescence imaging of the drug in tumor-bearing mice 4 h after tail vein injection of HB-51a-PEG6-H (left), HB-51c-PEG12-H (middle), and HC-51c-NH-PEG12 (right) in Examples 51-2 and 51-3.

[0096] Figure 18 Images of the tumor sites in mice inoculated with different tumor cells 6 days after photodynamic therapy by irradiating with a 635 nm laser at 0.1 W / cm 2 for 10 min after tail vein injection of different drugs: (a) Injected with only normal saline without drug, and inoculated with A549 lung cancer cells; (b) Injected with HC-1c-PEG8 (Example 1-1) and inoculated with A549 lung cancer cells; (c) Injected with HB-1c-PEG6-H (Example 1-2) and inoculated with MCC cholangiocarcinoma cells; (d) Injected with HC-1a-C4-N + (Example 1-5) and inoculated with B16 melanoma cells.

[0097] Figure 19 Images of the tumor sites in mice inoculated with different tumor cells 6 days after photodynamic therapy by irradiating with a 635 nm laser at 0.1 W / cm 2635 nm laser illumination for 10 min, pictures of tumor sites in mice inoculated with different tumor cells after 6 days of photodynamic therapy: (a) inoculated with C6 glioma cells after injecting only normal saline without injecting drugs; (b) injected with HB-50a-PEG12 (Example 50-1) and inoculated with C6 cells; (c) injected with HC-54c-PEG12 (Example 54-1) and inoculated with cutaneous T-cell lymphoma cell line HH; (d) injected with HC-52c-PEG12-H (Example 52-2) and inoculated with MBT-2 bladder cancer cells.

[0098] Figure 20 Show the photodynamic effect diagrams of HB-1c-PEG6, HC-1c-PEG6 and HC-1c-PEG12 (Example 1-1) on HeLa cells. Detailed implementation manners

[0099] The technical solutions of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the embodiments are only used to illustrate and explain the present invention exemplarily, and should not be construed as limiting the protection scope of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention. The raw materials and reagents used in the following embodiments are all commercially available products or can be prepared by known methods.

[0100] In the present invention, the experimental methods are all conventional methods unless otherwise specified. The raw materials used can be obtained from public commercial channels unless otherwise specified; the percentages are all mass percentages unless otherwise specified; the M is mol / L unless otherwise specified.

[0101] The initial raw materials involved in the present invention: the structural formulas of hypocrellin A (HA), hypocrellin B (HB), deacetylhypocrellin (HC), and 5-bromohypocrellin derivatives HB-Br and HC-Br are shown as follows:

[0102]

[0103] All hypocrellin derivatives involved in the present invention are shown in Examples 1 to 53, wherein Examples 1 to 42 are derivatives with the same substituents at the ortho and 2-positions, and Examples 43 to 53 are derivatives with different substituents at the ortho and 2-positions; the synthesis methods and characterization data of all derivatives can be found in the previous Chinese patent ("A derivative of hypocrellin with simultaneous amino substitution at the ortho and 2-positions, its preparation method and application", authorization number 201811020381.4), and the property results of some derivatives in Examples 1 to 55 are shown in Table 1.

[0104] Example 1

[0105] The reaction products of hypocrellin B (HB) or deacetylhypocrellin (HC) and glycosyldiolamine are HB-1a to HB-1d, HC-1a to HC-1d, and their structural formulas are as follows:

[0106]

[0107] Example 1-1

[0108] The reaction products of HB (or HC) and glycosyldiolamine - polyethylene glycol with different chain lengths are HB-1a-PEGn to HB-1d-PEGn and HC-1a-PEGn to HC-1d-PEGn (n = 4, 6, 8, 10, 12), and their structural formulas are as follows:

[0109]

[0110] Example 1-2

[0111] The reaction products of HB (or HC) and glycosyldiolamine - polyethylene glycol with different chain lengths (terminal group -H) are HB-1a-PEGn-H to HB-1d-PEGn-H and HC-1a-PEGn-H to HC-1d-PEGn-H (n = 4, 6, 8), and their structural formulas are as follows:

[0112]

[0113] Example 1-3

[0114] The reaction products of HB (or HC) and glycosyldiolamine - polyethylene glycol with different chain lengths (terminal group carboxyl) are HB-1a-PEGn-COOH to HB-1d-PEGn-COOH and HC-1a-PEGn-COOH to HC-1d-PEGn-COOH (n = 1, 6, 8), and their structural formulas are as follows:

[0115]

[0116] Example 1-4

[0117] The reaction products of HB (or HC) and glycosyldiolamine - polyethylene glycol with different chain lengths (terminal group sulfonic acid) are HB-1a-PEGn-SO3H to HB-1d-PEGn-SO3H and HC-1a-PEGn-SO3H to HC-1d-PEGn-SO3H (n = 1, 6, 12), and their structural formulas are as follows:

[0118] Example 1-5

[0119] The reaction products of HB (or HC) and glycosyl diol amine with quaternary ammonium salts of different chain lengths are HB-1a-Cn-N + ~HB-1d-Cn-N + 、HC-1a-Cn-N + ~HC-1d-Cn-N + (n = 2, 4, 6), and their structural formulas are as follows:

[0120]

[0121] Example 2

[0122] The reaction products of hypocrellin B and ethanolamine are HB-2a~HB-2d, and the reaction products of deacetylhypocrellin and ethanolamine are HC-2a~HC-2d. Their structural formulas are as follows:

[0123]

[0124] Example 2-1

[0125] The reaction products of HB (or HC) and ethanolamine with polyethylene glycols of different chain lengths are HB-2a-PEGn~HB-2d-PEGn, HC-2a-PEGn~HC-2d-PEGn (n = 1, 4, 6, 8, 12). Their structural formulas are as follows:

[0126]

[0127] Example 3

[0128] The reaction products of HB (or HC) and aminoethyl-polyethylene glycol (with a hydrogen end group) are HB-3a-PEGn~HB-3d-PEGn, HC-3a-PEGn~HC-3d-PEGn (n = 4, 8, 12). Their structural formulas are as follows:

[0129]

[0130] Example 4

[0131] The reaction products of HB (or HC) and aminoethyl-polyethylene glycols of different chain lengths are HB-4a-PEGn~HB-4d-PEGn, HC-4a-PEGn~HC-4d-PEGn (n = 4, 6, 16). Their structural formulas are as follows:

[0132]

[0133] Example 5

[0134] The reaction products of hypocrellin B (or bromohypocrellin B) and azido polyethyleneglycol are HB-5a-PEGn to HB-5d-PEGn, HB-5a-Br-PEGn to HB-5d-Br-PEGn (n = 1, 4, 6), and their structural formulas are as follows:

[0135]

[0136] Example 6

[0137] The reaction products of HB (or HC) and amino thio polyethyleneglycol are HB-6a-PEGn to HB-6d-PEGn, HC-6a-PEGn to HC-6d-PEGn (n = 1, 4, 6), and their structural formulas are as follows:

[0138]

[0139] Example 7

[0140] The reaction products of HB (or HC) and aminoacetic acid are HB-7a to HB-7d, HC-7a to HC-7d, and their structural formulas are as follows:

[0141]

[0142] Example 8

[0143] The reaction products of HB (or HC) and aminobutyric acid are HB-8a to HB-8d, HC-8a to HC-8d, and their structural formulas are as follows:

[0144] Example 8-1

[0145] The reaction products of HB (or HC) and aminobutyric acid - amino polyethyleneglycol with different chain lengths are HB-8a-PEGn to HB-8d-PEGn, HC-8a-PEGn to HC-8d-PEGn (n = 1, 4, 8), and their structural formulas are as follows:

[0146]

[0147] Example 8-2

[0148] The reaction products of HB (or HC) and aminobutyric acid - amino polyethyleneglycol with different chain lengths are HB-8a-NH-PEGn to HB-8d-NH-PEGn, HC-8a-NH-PEGn to HC-8d-NH-PEGn (n = 1, 6, 12), and their structural formulas are as follows:

[0149]

[0150] Example 8-3

[0151] The reaction products of HB (or HC) and aminobutyric acid - sulfonic acids with different chain lengths are HB-8a-Cn-SO3H to HB-8d-Cn-SO3H, HC-8a-Cn-SO3H to HC-8d-Cn-SO3H (n = 2, 4, 6), and their structural formulas are as follows:

[0152]

[0153]

[0154] Example 8-4

[0155] The reaction products of HB (or HC) and aminobutyric acid - quaternary ammonium salts with different chain lengths are HB-8a-Cn-N + ~HB-8d-Cn-N + 、HC-8a-Cn-N + ~HC-8d-Cn-N + (n = 2, 4, 6), and their structural formulas are as follows:

[0156]

[0157] Example 9

[0158] The reaction products of HB (or HC) and aminohexanoic acid are HB-9a to HB-9d, HC-9a to HC-9d, and their structural formulas are as follows:

[0159] Example 9-1

[0160] The reaction products of HB (or HC) and aminohexanoic acid - polyethylene glycols with different chain lengths are HB-9a-PEGn to HB-9d-PEGn, HC-9a-PEGn to HC-9d-PEGn (n = 1, 6, 12), and their structural formulas are as follows:

[0161]

[0162] Example 9-2

[0163] The reaction products of HB (or HC) and aminohexanoic acid - quaternary ammonium salts with different chain lengths are HB-9a-Cn-N + ~HB-9d-Cn-N + 、HC-9a-Cn-N + ~HC-9d-Cn-N + (n = 2, 4, 6), and their structural formulas are as follows:

[0164]

[0165] Example 10

[0166] The reaction products of HB (or HC) and aminopropionic acid are HB-10a to HB-10d, HC-10a to HC-10d, and their structural formulas are as follows:

[0167] Example 10-1

[0168] The reaction products of HB (or HC) and aminopropionic acid - aminopolyethylene glycol with different chain lengths are HB-10a-NH-PEGn to HB-10d-NH-PEGn, HC-10a-NH-PEGn to HC-10d-NH-PEGn (n = 1, 6, 8), and their structural formulas are as follows:

[0169]

[0170] Example 11

[0171] The reaction products of HB (or HC) and sulfamic acids with different chain lengths are HB-11a-Cn-SO3H to HB-11d-Cn-SO3H, HC-11a-Cn-SO3H to HC-11d-Cn-SO3H (n = 2, 4, 6), and their structural formulas are as follows:

[0172]

[0173] Example 12

[0174] The reaction products of HB (or HC) and aminovaleric acid are HB-12a to HB-12d, HC-12a to HC-12d, and the structural formulas are as follows:

[0175]

[0176] Example 13

[0177] The reaction products of HB (or HC) and tranexamic acid are HB-13a to HB-13d, HC-13a to HC-13d, and their structural formulas are as follows:

[0178] Example 13-1

[0179] The reaction products of HB (or HC) and methyl 4-(aminomethyl)cyclohexanecarboxylate are HB-13a-AcE to HB-13d-AcE, HC-13a-AcE to HC-13d-AcE, and their structural formulas are as follows:

[0180]

[0181] Example 13-2

[0182] The reaction products of HB (or HC) and 4-aminomethylcyclohexanecarboxylic acid - PEGs with different chain lengths (methyl-terminated) are HB-13a-PEGn to HB-13d-PEGn, HC-13a-PEGn to HC-13d-PEGn (n = 1, 4, 8, 16), and their structural formulas are shown as follows:

[0183]

[0184] Example 13-3

[0185] The reaction products of HB (or HC) and 4-aminomethylcyclohexanecarboxylic acid - amino PEGs with different chain lengths (methyl-terminated) are HB-13a-NH-PEGn to HB-13d-NH-PEGn, HC-13a-NH-PEGn to HC-13d-NH-PEGn (n = 4, 6, 12), and their structural formulas are shown as follows:

[0186]

[0187] Example 13-4

[0188] The reaction products of HB (or HC) and 4-aminomethylcyclohexanecarboxylic acid - amino PEGs with different chain lengths (hydrogen-terminated) are HB-13a-NH-PEGn-H to HB-H-13d-NH-PEGn-H, HC-13a-NH-PEGn-H to HC-13d-NH-PEGn-H (n = 4, 6, 12), and their structural formulas are shown as follows:

[0189]

[0190] Example 13-5

[0191] The reaction products of HB (or HC) and 4-aminomethylcyclohexanecarboxylic acid - PEGs with different chain lengths (hydrogen-terminated) are HB-13a-PEGn-H to HB-13d-PEGn-H, HC-13a-PEGn-H to HC-13d-PEGn-H (n = 1, 4, 8), and their structural formulas are shown as follows:

[0192]

[0193] Example 13-6

[0194] The reaction products of HB (or HC) and 4-aminomethylcyclohexanecarboxylic acid with PEGs of different chain lengths (with a carboxyl group at the end) are HB-13a-PEGn-COOH to HB-13d-PEGn-COOH, HC-13a-PEGn-COOH to HC-13d-PEGn-COOH (n = 1, 6, 12), and their structural formulas are as follows:

[0195]

[0196] Example 13-7

[0197] The reaction products of HB (or HC) and 4-aminomethylcyclohexanecarboxylic acid with PEGs of different chain lengths (with a sulfonic acid group at the end) are HB-13a-PEGn-SO3H to HB-13d-PEGn-SO3H, HC-13a-PEGn-SO3H to HC-13d-PEGn-SO3H (n = 4, 6, 12), and their structural formulas are as follows:

[0198]

[0199] Example 13-8

[0200] The reaction products of HB (or HC) and 4-aminomethylcyclohexanecarboxylic acid with quaternary ammonium salts of different chain lengths are HB-13a-Cn-N + ~HB-13d-Cn-N + 、HC-13a-Cn-N + ~HC-13d-Cn-N + (n = 2, 4, 6), and their structural formulas are as follows:

[0201]

[0202] Example 13-9

[0203] The reaction products of HB (or HC) and 4-aminomethylcyclohexanecarboxylic acid with sulfonic acids of different chain lengths are HB-13a-NH-Cn-SO3H to HB-13d-NH-Cn-SO3H, HC-13a-NH-Cn-SO3H to HC-13d-NH-Cn-SO3H (n = 4, 6), and their structural formulas are as follows:

[0204]

[0205]

[0206] Example 14

[0207] The reaction products of HB (or HC) and 4-aminocyclohexanoic acid are HB-14a to HB-14d, HC-14a to HC-14d, and their structural formulas are as follows:

[0208]

[0209] Example 14-1

[0210] The reaction products of HB (or HC) and 4-aminocyclohexanecarboxylic acid with PEGs of different chain lengths are HB-14a-PEGn to HB-14d-PEGn, HC-14a-PEGn to HC-14d-PEGn (n = 4, 6, 12), and their structural formulas are as follows:

[0211]

[0212] Example 15

[0213] The products of HB (or HC) and 3-aminocyclohexanecarboxylic acid are HB-15a to HB-15d, HC-15a to HC-15d, and the structural formulas are as follows:

[0214]

[0215] Example 16

[0216] The products of HB (or HC) and 2-aminocyclohexanecarboxylic acid are HB-16a to HB-16d, HC-16a to HC-16d, and the structural formulas are as follows:

[0217]

[0218] Example 16-1

[0219] The reaction products of HB (or HC) and 2-aminocyclohexanecarboxylic acid with PEGs of different chain lengths are HB-16a-PEGn to HB-16d-PEGn, HC-16a-PEGn to HC-16d-PEGn (n = 4, 6, 12), and their structural formulas are as follows:

[0220]

[0221] Example 17

[0222] The reaction products of HB (or HC) and 4-hydroxycyclohexylamine are HB-17a to HB-17d, HC-17a to HC-17d, and the structural formulas are as follows:

[0223]

[0224] Example 17-1

[0225] The reaction products of HB (or HC) and 4-aminocyclohexanol with carboxyl PEGs of different chain lengths are HB-17a-PEGn to HB-17d-PEGn, HC-17a-PEGn to HC-17d-PEGn (n = 4, 6, 12), and their structural formulas are as follows:

[0226]

[0227] Example 18

[0228] The reaction products of HB (or HC) and 3-aminocyclobutyric acid are HB-18a to HB-18d, HC-18a to HC-18d, and the structural formulas are as follows:

[0229]

[0230] Example 19

[0231] The reaction products of HB (or HC) and 3-aminocyclopentanoic acid are HB-19a to HB-19d, HC-19a to HC-19d, and the structural formulas are as follows:

[0232]

[0233] Example 19-1

[0234] The reaction products of HB (or HC) and 3-aminocyclopentanoic acid with carboxyl PEGs of different chain lengths are HB-19a-PEGn to HB-19d-PEGn, HC-19a-PEGn to HC-19d-PEGn (n = 4, 6, 12), and their structural formulas are as follows:

[0235]

[0236] Example 20

[0237] The reaction products of HB (or HC) and 3-aminocyclopentanol are HB-20a to HB-20d, HC-20a to HC-20d, and the structural formulas are as follows:

[0238]

[0239] Example 20-1

[0240] The reaction products of HB (or HC) and 3-aminocyclopentanol with carboxyl PEGs of different chain lengths are HB-20a-PEGn to HB-20d-PEGn, HC-20a-PEGn to HC-20d-PEGn (n = 4, 6, 12), and their structural formulas are as follows:

[0241]

[0242] Example 21

[0243] The reaction products of HB (or HC) and 2-aminocyclic carboxylic acid are HB-21a to HB-21d, HC-21a to HC-21d, and their structural formulas are as follows:

[0244]

[0245] Example 21-1

[0246] The reaction products of HB (or HC) and 2-aminocyclopentanecarboxylic acid with different chain lengths of PEG are HB-21a-PEGn to HB-21d-PEGn, HC-21a-PEGn to HC-21d-PEGn (n = 4, 6, 12), and their structural formulas are as follows:

[0247]

[0248] Example 22

[0249] The reaction products of HB (or HC) and valine are HB-22a to HB-22d, HC-22a to HC-22d, and their structural formulas are as follows:

[0250]

[0251] Example 23

[0252] The reaction products of HB (or HC) and serine are HB-23a to HB-23d, HC-23a to HC-23d, and their structural formulas are as follows:

[0253]

[0254] Example 24

[0255] The reaction products of HB (or HC) and serine methyl ester are HB-24a to HB-24d, HC-24a to HC-24d, and their structural formulas are as follows:

[0256]

[0257] Example 25

[0258] The reaction products of HB (or HC) and cysteine are HB-25a to HB-25d, HC-25a to HC-25d, and their structural formulas are as follows:

[0259]

[0260] Example 26

[0261] The reaction products of bromo-HB (or HC) and cysteine are HB-26a to HB-26d, HC-26a to HC-26d, and their structural formulas are as follows:

[0262]

[0263] Example 27

[0264] The reaction products of HB (or HC) and aspartic acid are HB-27a to HB-27d, HC-27a to HC-27d, and their structural formulas are as follows:

[0265]

[0266] Example 28

[0267] The reaction products of HB (or HC) and glutamic acid are HB-28a to HB-28d, HC-28a to HC-28d, and their structural formulas are as follows:

[0268]

[0269] Example 29

[0270] The reaction products of HB (or HC) and amino quaternary ammonium salts with different chain lengths are HB-29a-Cn-N + ~HB-29d-Cn-N + 、HC-29a-Cn-N + ~HC-29d-Cn-N + (n = 2, 4, 6), and their structural formulas are as follows:

[0271]

[0272] Example 30

[0273] The reaction products of mercaptoethanol-substituted HB (or HC) and aminoethanol are HB-30a to HB-30d, HC-30a to HC-30d, and their structural formulas are as follows:

[0274]

[0275] Example 31

[0276] The reaction products of hypocrellin B (or bromo-hypocrellin B) and 4-aminomethylpiperidine - PEG with different chain lengths are HB-31a-PEGn to HB-31d-PEGn, HB-31a-Br-PEGn to HB-31d-Br-PEGn (n = 4, 6, 12), and their structural formulas are as follows:

[0277]

[0278] Example 32

[0279] The reaction products of HB (or HC) and 4-aminomethylpiperidine with different chain lengths of PEG are HB-32a-PEGn to HB-32d-PEGn, HC-32a-PEGn to HC-32d-PEGn (n = 4, 6, 12), and their structural formulas are as follows:

[0280]

[0281] Example 33

[0282] The reaction products of HB (or HC) and n-propylamine are HB-33a to HB-33d, HC-33a to HC-33d, and their structural formulas are as follows:

[0283]

[0284] Example 34

[0285] The reaction products of HB (or HC) and n-hexylamine are HB-34a to HB-34d, HC-34a to HC-34d, and their structural formulas are as follows:

[0286] Example 35

[0287] The reaction products of HB (or HC) and hydroxymethylcyclopropylamine are HB-35a to HB-35d, HC-35a to HC-35d, and the structural formulas are as follows:

[0288] Example 35-1

[0289] The reaction products of HB (or HC) and hydroxymethylcyclopropylamine with different chain lengths of polyethylene glycol are HB-35a-PEGn to HB-35d-PEGn, HC-35a-PEGn to HC-35d-PEGn (n = 4, 6, 12), and their structural formulas are as follows:

[0290]

[0291] Example 36

[0292] The reaction products of HB (or HC) and ethylhydrazine are HB-36a to HB-36d, HC-36a to HC-36d, and their structural formulas are as follows:

[0293]

[0294] Example 37

[0295] The reaction products of HB (or HC) and hydroxylamine are HB-37a to HB-37d, HC-37a to HC-37d, and their structural formulas are as follows:

[0296]

[0297] Example 38

[0298] The reaction products of HB (or HC) and benzylaminopyridine are HB-38a to HB-38d, HC-38a to HC-38d, and their structural formulas are as follows:

[0299]

[0300] Example 38-1

[0301] The reaction products of HB (or HC) and amino quaternary ammonium salts are HB-38a-N-COOH to HB-38d-N-COOH, HC-38a-N-COOH to HC-38d-N-COOH, and their structural formulas are as follows:

[0302]

[0303] Example 39

[0304] The reaction products of HB (or HC) and piperazine derivatives are HB-39a to HB-39d, HC-39a to HC-39d, and their structural formulas are as follows:

[0305]

[0306] Example 40

[0307] The reaction products of HB (or HC) and aminoethylpiperazine dione are HB-40a to HB-40d, HC-40a to HC-40d, and their structural formulas are as follows:

[0308]

[0309] Example 41

[0310] The reaction products of HB (or HC) and DABACO are HB-41a to HB-41d, HC-41a to HC-41d, and their structural formulas are as follows:

[0311]

[0312] Example 42

[0313] The reaction products of HB (or HC) and aminomorpholine are HB-42a to HB-42d, HC-42a to HC-42d, and their structural formulas are as follows:

[0314]

[0315] Example 43

[0316] The reaction products of HB (or HC) and glycine / aminobutyric acid are HB-43a to HB-43d, HC-43a to HC-43d, and their structural formulas are as follows:

[0317]

[0318] Example 44

[0319] The reaction products of hypocrellin B (or mercapto-substituted hypocrellin B) and ethylamine / cyclopentylamine are HB-44a to HB-44d, HB-S-44a to HB-S-44d, and their structural formulas are as shown below:

[0320]

[0321] Example 45

[0322] The reaction products of HB (or HC) and ethanesulfonic acid / propanesulfonic acid are HB-45a to HB-45d, HC-45a to HC-45d, and their structural formulas are as shown below:

[0323]

[0324] Example 46

[0325] The reaction products of HB (or HC) and ethylhydrazine / aspartic acid are HB-46a to HB-46d, HC-46a to HC-46d, and their structural formulas are as follows:

[0326]

[0327] Example 47

[0328] The reaction products of HB (or HC) and aminobutyric acid / aminopolyethylene glycol are HB-47a-PEGn to HB-47d-PEGn, HC-47a-PEGn to HC-47d-PEGn (n = 4, 6, 12), and their structural formulas are as shown below:

[0329]

[0330] Example 48

[0331] The reaction products of HB (or HC) and aminobutyric acid / glycolamine-polyethylene glycol are HB-48a-PEGn to HB-48d-PEGn, HC-48a-PEGn to HC-48d-PEGn (n = 6, 8, 12), and their structural formulas are as shown below:

[0332]

[0333] Example 49

[0334] The reaction products of HB (or HC) and aminobutyric acid / 4-aminomethylcyclohexanecarboxylic acid - polyethylene glycol are HB-49a-PEGn to HB-49d-PEGn, HC-49a-PEGn to HC-49d-PEGn (n = 1, 6, 12), and their structural formulas are as follows:

[0335]

[0336] Example 50

[0337] The reaction products of HB (or HC) and glycine / 4-aminomethylcyclohexanoic acid are HB-50a to HB-50d, HC-50a to HC-50d, and their structural formulas are as follows:

[0338]

[0339] Example 50-1

[0340] The reaction products of HB (or HC) and glycine / 4-aminomethylcyclohexanoic acid - polyethylene glycol are HB-50a-PEGn to HB-50d-PEGn, HC-50a-PEGn to HC-50d-PEGn (n = 4, 6, 12), and their structural formulas are as follows:

[0341]

[0342] Example 51

[0343] The reaction products of HB (or HC) and aminomethylcyclohexane / 4-aminomethylcyclohexanoic acid are HB-51a to HB-51d, HC-51a to HC-51d, and their structural formulas are as follows:

[0344]

[0345] Example 51-1

[0346] The reaction products of HB (or HC) and aminomethylcyclohexane / 4-aminomethylcyclohexanoic acid - polyethylene glycol (with a methyl end group) are HB-51a-PEGn to HB-51d-PEGn, HC-51a-PEGn to HC-51d-PEGn (n = 6, 12, 18), and their structural formulas are as follows:

[0347]

[0348] Example 51-2

[0349] The reaction products of HB (or HC) and aminomethylcyclohexane / aminomethylcyclohexanoic acid - polyethylene glycol (with hydrogen at the end) are HB-51a-PEGn-H to HB-51d-PEGn-H, HC-51a-PEGn-H to HC-51d-PEGn-H (n = 1, 6, 12), and their structural formulas are as follows:

[0350]

[0351] Example 51-3

[0352] The reaction products of HB (or HC) and aminomethylcyclohexane / aminocyclohexanecarboxamide - polyethylene glycol (with methyl at the end) are HB-51a-NH-PEGn to HB-51d-NH-PEGn, HC-51a-NH-PEGn to HC-51d-NH-PEGn (n = 1, 6, 12), and their structural formulas are as follows:

[0353]

[0354] Example 51-4

[0355] The reaction products of HB (or HC) and aminomethylcyclohexane / aminocyclohexanecarboxamide - polyethylene glycol (with hydrogen at the end) are HB-51a-NH-PEGn-H to HB-51d-NH-PEGn-H, HC-51a-NH-PEGn-H to HC-51d-NH-PEGn-H (n = 1, 6, 12), and their structural formulas are as follows:

[0356]

[0357] Example 52

[0358] The reaction products of HB (or HC) and butylamine / glycolamine are HB-52a to HB-52d, HC-52a to HC-52d, and the structural formulas are as follows:

[0359]

[0360] Example 52-1

[0361] The reaction products of HB (or HC) and butylamine / glycolamine - polyethylene glycol (with methyl at the end) are HB-52a-PEGn to HB-52d-PEGn, HC-52a-PEGn to HC-52d-PEGn (n = 6, 12, 16), and their structural formulas are as follows:

[0362]

[0363] Example 52-2

[0364] The reaction products of HB (or HC) and butylamine / glycolamine - polyethylene glycol (with hydrogen at the end) are HB - 52a - PEGn - H to HB - 52d - PEGn - H, HC - 52a - PEGn - H to HC - 52d - PEGn - H (n = 4, 6, 12), and their structural formulas are as follows:

[0365]

[0366] Example 53

[0367] The reaction products of HB (or HC) and cyclohexylamine / glycolamine are HB - 53a to HB - 53d, HC - 53a to HC - 53d, and their structural formulas are as follows:

[0368]

[0369] Example 53 - 1

[0370] The reaction products of HB (or HC) and cyclohexylamine / glycolamine - polyethylene glycol (with methyl at the end) are HB - 53a - PEGn to HB - 53d - PEGn, HC - 53a - PEGn to HC - 53d - PEGn (n = 6, 10, 16), and their structural formulas are as follows:

[0371]

[0372] Example 53 - 2

[0373] The reaction products of HB (or HC) and cyclohexylamine / glycolamine - polyethylene glycol (with hydrogen at the end) are HB - 53a - PEGn - H to HB - 53d - PEGn - H, HC - 53a - PEGn - H to HC - 53d - PEGn - H (n = 4, 6, 12), and their structural formulas are as follows:

[0374]

[0375] Example 53 - 3

[0376] The reaction products of HB (or HC) and cyclohexylamine / glycolamine - polyethylene glycol (with carboxyl at the end) are HB - 53a - PEGn - COOH to HB - 53d - PEGn - COOH, HC - 53a - PEGn - COOH to HC - 53d - PEGn - COOH (n = 4, 6, 8), and the structures are as follows:

[0377]

[0378] Example 54

[0379] The reaction products of HB (or HC) and aminopropyl methyl ether / aminomethyl cyclohexanoic acid are HB-54a to HB-54d, HC-54a to HC-54d, and their structural formulas are as follows:

[0380]

[0381] Example 54-1

[0382] The reaction products of HB (or HC) and aminopropyl methyl ether / aminomethyl cyclohexanoic acid - polyethylene glycol are HB-54a-PEGn to HB-54d-PEGn, HC-54a-PEGn to HC-54d-PEGn (n = 6, 8, 12), and their structural formulas are as follows:

[0383]

[0384] Example 55

[0385] The reaction products of HB (or HC) and aminoethyl methyl ether / aminomethyl cyclohexanoic acid - polyethylene glycol are HB-55a-PEGn to HB-55d-PEGn, HC-55a-PEGn to HC-55d-PEGn (n = 6, 8, 12), and their structural formulas are as follows:

[0386]

[0387] Example 56

[0388] The general structural formulas of Formula I-a to I-d involved in the present invention are as Figure 1 shown. Hypocrellin B (or deacetylhypocrellin C) and amino compounds undergo substitution reactions. In addition to amino substitution at the 2-position of hypocrellin, one of the four vicinal positions of hypocrellin ( Figure 1 the 3-position, 4-position, 9-position or 10-position in) can also undergo amino substitution, and finally derivatives with amino substitution at both the 2-position and the vicinal position of hypocrellin are obtained. By controlling different reaction conditions (such as: the acidity and alkalinity of the solution, reaction temperature, reaction time, steric hindrance of the starting amine, feed molar ratio, etc.), the ratios of different vicinal amino substitution products I-a to I-d of hypocrellin can be adjusted. For example, under weak alkaline conditions such as potassium carbonate and ammonia water, vicinal amine-substituted derivatives I-b and I-d mainly formed at the 9-position or 10-position; while under strong alkaline conditions such as sodium hydroxide, vicinal amino-substituted derivatives I-a and I-c mainly formed at the 3-position or 4-position; if the starting amine has a large steric hindrance, since the 2-position of hypocrellin has already undergone amino substitution, its 3-position is not easily subjected to vicinal amino substitution ( Figure 1 ).

[0389] Taking the reaction of hypocrellin B and glucosaminediol as an example, hypocrellin derivatives HB-1a to HB-1d with amino substitutions at the ortho and 2-positions are mainly formed. The obtained products are further esterified with carboxyl-terminated polyethylene glycol to obtain HB-1a-PEGn to HB-1d-PEGn. The synthesis method is as follows Figure 2 ; The reaction of deacetylhypocrellin and glucosaminediol mainly produces deacetylhypocrellin derivatives HC-1a to HC-1d with amino substitutions at the ortho and 2-positions. The obtained products are further esterified with carboxyl-terminated polyethylene glycol to obtain HC-1a-PEGn to HC-1d-PEGn. The synthesis method and the corresponding products are as follows Figure 3 shown. For the specific synthesis methods above, please refer to the patent document CN109456210A.

[0390] For the compounds shown in the above formulas I-a to I-d, their maximum absorption wavelength is around 630 nm, and the molar extinction coefficient can reach 20000 - 40000 M -1 cm -1 , and they have strong light absorption ability in the phototherapy window; under photosensitizing conditions, they can efficiently generate reactive oxygen species such as singlet oxygen, and the singlet oxygen efficiency can reach up to about 40%; such derivatives have good photo-stability and pH-stability. The specific detection results are as follows:

[0391] 1) Absorption spectrum

[0392] Figure 4 shows the comparison of the absorption spectra of the derivatives disclosed in the present invention and commercial photosensitizers. As Figure 4 (a), the commercial porphyrin photosensitizer PpIX has multi-band absorption, all of which are narrow absorption bands. The maximum absorption wavelength available for phototherapy is 570 nm, and the molar extinction coefficient is lower than 8000 M -1 cm -1 ; The maximum absorption wavelength of the commercialized porphycene photosensitizer Ce6 is 650 nm, and the molar extinction coefficient is about 15000 M -1 cm -1 , and it also has narrow absorption in the phototherapy window. Therefore, the light absorption ability of commercial PpIX and Ce6 in the phototherapy window is limited, while the absorption spectral properties of the hypocrellin derivatives disclosed in the present invention are completely different. As Figure 4 (b) shows, the maximum absorption peak of HB is around 470 nm. HB-1c-PEG6-H (Example 1-2) has a wide and strong absorption in the phototherapy window. It has a very wide absorption band between 500 - 750 nm, and the maximum absorption peak is around 630 nm, which is red-shifted by 160 nm compared to the maximum absorption peak of HB. The molar extinction coefficient is about 31500 M -1 cm -1Around, it exhibits extremely strong red light absorption ability. Similarly, the absorption spectrum of the derivative HC-1c-PEG6-H (Example 1-2) is located in the ideal phototherapy window, with a very broad absorption band between 500 and 750 nm, and the maximum absorption peak is around 630 nm, showing extremely strong red light absorption ability. For other hypocrellin derivatives provided by the present invention, their absorption spectra are also similar to those of HB-1c-PEG6-H and HC-1c-PEG6-H, with a very broad absorption band between 500 - 750 nm and the maximum absorption peak around 630 nm. Therefore, the absorption wavelength of the hypocrellin derivatives provided by the present invention and their light absorption ability in the phototherapy window are far superior to those of the commercial photosensitizers PpIX and Ce6, showing more prominent red light absorption ability.

[0393] 2) Reactive oxygen species

[0394] Figure 5 It shows the reactive oxygen species of the hypocrellin derivatives disclosed in the present invention tested by electron spin resonance (ESR). The results show that HB-13a-PEG8 (Example 13) can efficiently produce reactive oxygen (ROS). Measured by singlet oxygen and superoxide radical scavengers respectively, such derivatives of hypocrellin can efficiently produce photosensitizing active species, mainly producing singlet oxygen ( Figure 5 a), and can also produce a small amount of superoxide radicals ( Figure 5 b). Both of these two reactive oxygen species are beneficial to the damage of tumor cells in photodynamic therapy. In addition to HB-13a-PEG8, the hypocrellin derivatives disclosed in other examples of the present invention also have the ability to efficiently produce singlet oxygen and assist in producing a small amount of superoxide radicals.

[0395] 3) Singlet oxygen efficiency

[0396] The singlet oxygen efficiency of the derivatives described in the present invention in solution is detected by the ABDA method. Figure 6 (a) and 6(b) respectively show the photodegradation curves of the derivatives HB-51c-PEG16 and HC-51c-PEG16 prepared in Example 51-1 of the present invention. It can be seen that the photosensitizers HB-51c-PEG16 or HC-51c-PEG16 significantly produce singlet oxygen under light illumination, and then significantly degrade ABDA. Through comparison and calculation with the singlet oxygen efficiency curve of the reference rose bengal RB ( Figure 6 c and Figure 6 d), the singlet oxygen efficiencies of HB-51c-PEG16 or HC-51c-PEG16 are 0.32 and 0.35 respectively. The singlet oxygen production efficiencies of the hypocrellin derivatives disclosed in other examples of the present invention are between 0.2 and 0.4, and they also have the ability to efficiently produce reactive oxygen.

[0397] 4) Water solubility

[0398] Most of the hypocrellin derivatives provided by the present invention contain hydrophilic groups such as polyethylene glycol, quaternary ammonium salt, carboxyl group, sulfonic acid group, etc., making the photosensitizer molecules have strong water solubility under physiological conditions. Experiments show that more than 5 mg of the compound molecules represented by Formula I-a to I-d of the present invention can be dissolved in each milliliter of normal saline or glucose injection solution, showing excellent water solubility, so that the compounds represented by Formula I-a to I-d can be well transported in blood vessels during intravenous injection without causing blood vessel blockage. For example, HB-1c-PEG8 (Example 1-1) contains 16 ethylene glycol units and can dissolve more than 10 mg in each milliliter of normal saline; HC-1c-PEG12 contains 24 ethylene glycol units and can dissolve more than 20 mg in each milliliter of normal saline; HB-1c-C2-N + (Example 1-5) contains two quaternary ammonium salts and can dissolve more than 10 mg in each milliliter of normal saline; HB-8c-PEG6 and HC-8b-PEG6 (Example 8-1) both contain 12 ethylene glycol units and can dissolve more than 10 mg in each milliliter of normal saline; HB-13c prepared in Example 13 contains two carboxyl groups and can dissolve more than 10 mg in each milliliter of normal saline; these derivatives all show excellent water solubility. The derivatives disclosed in other examples of the present invention also have good water solubility and biocompatibility, and 1 to 20 mg or more of the compound molecules represented by Formula I-a to I-d of the present invention can be dissolved in each milliliter of normal saline.

[0399] 5) Photostability

[0400] The present invention compares the photostability of the provided hypocrellin derivatives and commercial photosensitizers. The derivatives of the present invention are irradiated with a 635 nm laser at a light intensity of 20 mW / cm 2 for 30 min, and their absorption spectra are detected. As shown in Figure 7 (a), the absorption spectra of HB-1a-PEG6 and HC-1a-PEG6 (Example 1-1) do not show an obvious decrease, and the absorption intensity at the maximum wavelength decreases by less than 10%; under the same conditions, when irradiated with a 635 nm laser at a light intensity of 20 mW / cm 2 for 30 min, the maximum absorption of the commercial porphyrin photosensitizer Ce6 decreases by 30%; while the absorption spectrum of the commercial hematoporphyrin photosensitizer HpD decreases more, reaching about 50%. As shown in Figure 7As shown in (b), under the same conditions, the absorption intensity at the maximum wavelength of HB-13a-PEG8 and HC-13a-PEG8 (Example 13-2) also decreased by less than 10%, and they also had good photostability. Other derivatives of the present invention also had similar photostability, and under the same conditions, the absorption intensity at their maximum wavelength decreased basically by less than 10%. Therefore, the hypocrellin derivatives prepared by the present invention had better photostability than commercial photosensitizers.

[0401] 6) pH stability

[0402] The pH stability of the derivatives of the present invention was detected in the range of pH 6.2 - 8.0. As Figure 8 shown, for such hypocrellin derivatives, in the range of pH 6.2 - 8.0, the absorption spectrum had no obvious change, indicating that such derivatives had good pH stability under physiological conditions. As Figure 8 shown in (a), HB-1a-PEG4-H (Example 1-2) contained two groups of 4 PEG units and had a hydroxyl group at the end. In the range of pH 6.2 - 8.0, its absorption spectrum had no obvious change; HC-1c-PEG6-H (Example 1-2) contained PEG with two groups of 6 ethylene glycol units and had a hydroxyl group at the end. In the range of pH 6.2 - 8.0, its absorption spectrum had no obvious change; they all had good pH stability in this range. The reason was that the two phenolic hydroxyl groups of hypocrellin were not easily deprotonated under these acid-base conditions. While the commercial hematoporphyrin HpD contained two carboxyl groups, which could be deprotonated in the range of pH 6.2 - 8.0, resulting in an obvious change in the absorption spectrum, thus showing the instability of the HpD photosensitizer. Similarly, as Figure 8 shown in (b), HB-51b-PEG16 and HC-51c-PEG16 (Example 51-1) had no obvious change in their absorption spectra in the range of pH 6.2 - 8.0 and had good pH stability. The reason was also that the two phenolic hydroxyl groups of hypocrellin were not easily deprotonated under these acid-base conditions. Other derivatives of the present invention also had good pH stability under physiological conditions.

[0403] The structures of some hypocrellin derivatives involved in the present invention are as shown in Examples 1 - 55. Their maximum absorption wavelength was about 630 nm, and the molar extinction coefficient could reach 20000 - 40000 M -1 cm -1, it has strong light absorption ability at the phototherapy window; under photosensitive conditions, it can not only efficiently generate singlet oxygen reactive oxygen species (the efficiency of singlet oxygen can reach up to about 40%), but also generate a small amount of superoxide radicals. Such derivatives have good photo-stability and pH-stability. The test results of the properties of more similar derivatives are summarized in Table 1. The above results show that the hypocrellin derivatives described in the present invention have very ideal photophysical properties and can be used as photodynamic drugs for the photodynamic therapy of tumors.

[0404] Table 1: Showing the photophysical data of some hypocrellin derivatives of the present invention

[0405]

[0406]

[0407]

[0408] Example 57

[0409] Culture of tumor cells: Various cell lines (esophageal cancer cell AKR, gastric cancer cell MFC, lung cancer cell A549, liver cancer cell HCC, cholangiocarcinoma 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, glioblastoma cell C6, basal cell carcinoma cell BCC, squamous skin cancer cell PECA, melanoma cell B16, cutaneous T cell lymphoma cell HH, prostate cancer cell LNCaP, bladder cancer cell MBT-2) were provided by the Cell Center of Peking Union Medical College. The culture conditions of the above cells are: RPMI-1640 medium supplemented with 10% FBS, 1% streptomycin (100 μg / mL) and penicillin (100 μg / mL), and cultured in an incubator at 37 °C and 5% CO2. The tumor cells were inoculated in a 96-well plate, and the solution of the hypocrellin derivative (100 μL) was added to the culture medium and incubated in the incubator for 4 h. Then, the cells were carefully washed twice with pre-cooled PBS solution to remove the hypocrellin derivative that did not enter the cells, and the cultured tumor cells were used for the MTT experiment.

[0410] Example 58

[0411] Cell imaging: Confocal fluorescence imaging of the hypocrellin derivatives of the present invention in cancer cells.

[0412] The cell fluorescence imaging of the hypocrellin derivatives prepared by the present invention is as Figure 9As shown. HC-1c-PEG6 (Example 1-1) contains two groups of six PEG units, which not only has good water solubility but also has good lipid solubility, enabling it to enter cells better. The results of confocal fluorescence imaging experiments show that HC-1c-PEG6 has good water solubility and biocompatibility. When incubated with lung cancer cells (A549 cells), it is found that the photosensitizer can quickly enter the lysosomes of A549 cells and can produce good red fluorescence imaging in the cells, indicating that HC-1c-PEG6 can be used for fluorescence imaging of lung cancer cells, and the enrichment, distribution, and metabolism of drugs in vivo can be tracked by fluorescence detection. The intracellular singlet oxygen was detected with DCFH-DA. The photosensitizer HC-1c-PEG6 and the fluorescent probe DCFH-DA were co-incubated in cells. As the irradiation time increased to 120 s, the green fluorescence intensity gradually increased, indicating an increase in intracellular singlet oxygen. When other derivatives of the present invention were used for cell phototoxicity and dark toxicity experiments, it was found that they could also enter the lysosomes of lung cancer cells well, could perform cell fluorescence imaging, and the enrichment, distribution, and metabolism of drugs in vivo could be tracked by fluorescence detection. In addition, a large number of cell phototoxicity and dark toxicity experiments have proved that such derivatives can not only enter A549 lung cancer cells but also enter AKR esophageal cancer cells, MFC gastric cancer cells, HCC liver cancer cells, MCC bile duct cancer cells, HCT116 colon cancer cells, SCC2 head and neck cancer cells, G442 brain cancer cells, TSCCa tongue cancer cells, KB nasal cancer cells, CAL27 oral cancer cells, C6 glioma cells, BCC basal cell carcinoma cells, PECA squamous skin cancer cells, B16 melanoma cells, HH cutaneous T-cell lymphoma cells, LNCaP prostate cancer cells, and MBT-2 bladder cancer cells well.

[0413] Example 59

[0414] Cell dark toxicity experiment, taking HB-1a-PEG4-H synthesized in Example 1-2 as an example:

[0415] The cultured lung cancer cells (A549 cells) were digested with 0.25% trypsin to make a single-cell suspension and placed in an incubator at 37°C containing 5% CO2 for culture. Different concentrations of photosensitizers (hematoporphyrin derivative HpD, HB-1a-PEG4-H in Example 1-2) were added under light-shielded conditions and incubated for 1 hour. The cell viability was detected by the MTT method. 20 μL of MTT was added to each well and incubated in the incubator for another 4 hours before termination. The supernatant was discarded, and 150 μL of DMSO was added to each well and shaken with a micro shaker for 10 minutes to fully dissolve the purple crystals. Selecting a wavelength of 570 nm, the OD values of each well were detected on an enzyme-linked immunosorbent assay (ELISA) reader, and the cell viability was calculated: Cell viability = OD value of the experimental group / OD value of the blank group × 100%. As Figure 10-2As shown, the cytotoxicity (dark toxicity) study test shows that HB-1a-PEG4-H has low cytotoxicity, similar to the commercial photosensitizing drug hematoporphyrin HpD. A549 cells were incubated with the photosensitizing drug HB-1a-PEG4-H at a concentration of 20 μM for half an hour, and no obvious death of A549 cells was observed, indicating that such photosensitizing drugs basically have no cytotoxicity.

[0416] The dark toxicity experimental methods of other compounds described in the present invention are similar to those of HB-1a-PEG4-H, and the results are shown in Figures 10-12 and Tables 2-4. The synthesized hypocrellin derivatives basically have no cytotoxicity, and the cell survival rate is above 90% within the concentration range of 20 μM.

[0417] Example 60

[0418] The cell phototoxicity experiment is illustrated by taking HB-1a-PEG4-H synthesized in Examples 1-2 as an example:

[0419] The experimental procedure is the same as that of the dark toxicity experiment. Irradiation is carried out with a 635 nm semiconductor laser, and the power density is adjusted to 20 mW / cm 2 , so that the light beam is evenly and perpendicularly irradiated onto the 96-well culture plate, and the irradiation time is 1000 s. As Figure 10-2 shown, the cell phototoxicity study experiment shows that HB-1a-PEG4-H exhibits very strong lethality to A549 cells under red light irradiation. More than 90% of A549 cells can be killed within the concentration range of 50 nM, while the commercial photosensitizer HpD can only kill about 20% of A549 cells under the same conditions, indicating that the photodynamic effect of such derivatives is significantly better than that of the commercial photosensitizer. The phototoxicity experimental methods of other derivatives in the present invention are similar to those of HB-1a-PEG4-H, and the results are shown in Figures 10-12. Such derivatives can kill 80-90% or more of tumor cells within the concentration range of 50 nM, and the half-lethal concentration IC 50 value is about 20-30 nM. Therefore, the hypocrellin derivatives described in the present invention have a better photodynamic effect than the commercial photosensitizer hematoporphyrin HpD.

[0420] Example 61

[0421] The drug activities of the hypocrellin derivatives of the present invention were tested, and the specific results are as follows:

[0422] 1) Dark toxicity and phototoxicity tests of hypocrellin derivatives on digestive tract tumor cells

[0423] The hypocrellin derivatives described in the present invention can efficiently kill digestive tract tumor cells such as esophageal cancer AKR, gastric cancer MFC, lung cancer A549, liver cancer HCC, cholangiocarcinoma MCC, and colon cancer HCT116 under 635 nm laser irradiation. Taking the derivatives HB-1a-PEGn to HB-1d-PEGn and HC-1a-PEGn to HC-1d-PEGn in Example 1-1 as examples, their phototoxicity and dark toxicity effects on digestive tract tumor cells are illustrated.

[0424] The derivatives HB-1c-PEG6 and HC-1c-PEG6 (Example 1-1) were incubated with esophageal cancer AKR cells. As Figure 10-1 (a) shows, under the condition of no light, the cytotoxicity (dark toxicity) study experiment of the photosensitizing drug indicates that the derivatives with 6 PEGs have good biocompatibility, and the cytotoxicities of HB-1c-PEG6 and HC-1c-PEG6 are both small, similar to the commercial photosensitizing drug hematoporphyrin HpD. Esophageal cancer cells were incubated with a 20 μM concentration of the photosensitizer HB-1c-PEG6 or HC-1c-PEG6 for half an hour, and no obvious death of esophageal cancer cells was observed, indicating that such photosensitizers basically have no cytotoxicity. As Figure 10-1 (b) shows, the cell phototoxicity study experiment indicates that the photosensitizing drug shows very strong killing power against esophageal cancer cells under the irradiation of 635 nm red light. A 50 nM concentration of HB-1c-PEG6 can kill more than 90% of esophageal cancer cells, and the half-lethal concentration IC 50 value (the drug concentration required to kill half of the tumor cells) is about 25 nM; while a 50 nM concentration of HC-1c-PEG6 can kill more than 90% of esophageal cancer cells, and the half-lethal concentration IC 50 value is about 25 nM; while under the same conditions, the commercial HpD can only kill 20% of esophageal cancer cells, indicating that the photodynamic killing effect of such hypocrellin derivatives on esophageal cancer is significantly better than that of the commercial photosensitizer HpD.

[0425] The derivatives in the present invention were incubated with lung cancer cells A549. As Figure 10-2 (a) shows, the dark toxicity experiment of the photosensitizing drug indicates that whether it is connected with 4 PEGs or 8 PEG units, the cytotoxicities of HB-1a-PEG4-H and HC-1c-PEG8-H prepared in Example 1-2 are both small. Lung cancer cells were incubated with a 20 μM concentration of the photosensitizer for half an hour, and no obvious death of lung cancer cells was observed, indicating that such photosensitizers basically have no cytotoxicity. As Figure 10-2 (b) shows, the cell phototoxicity study indicates that the photosensitizing drug shows very strong killing power against lung cancer cells under red light irradiation. A 50 nM concentration of HB-1a-PEG4-H and HC-1c-PEG8-H can respectively kill more than 85% of lung cancer cells, and the half-lethal concentration IC50 is about 30 nM; while under the same conditions, the commercial photosensitizer HpD can only kill about 20% of lung cancer cells, indicating that the photodynamic effect of such hypocrellin derivatives is significantly better than that of the commercial photosensitizer HpD.

[0426] The derivative in the present invention was incubated with liver cancer cells HCC, as Figure 10-3 (a) shows that the dark toxicity study of the photosensitizing drug indicates that the cytotoxicities of HB-2b-PEG8 and HC-2d-PEG12 prepared in Example 2-1 are both small, similar to that of the commercial HpD. The liver cancer cells were incubated with a 20 μM concentration of the photosensitizer for half an hour, and no obvious death of the liver cancer cells was observed, indicating that such photosensitizers basically have no cytotoxicity. As Figure 10-3 (b) shows that the cell phototoxicity study indicates that the photosensitizing drug shows very strong killing power to liver cancer cells under red light irradiation. HB-2b-PEG8 and HC-2d-PEG12 at a concentration of 50 nM can both kill more than 90% of liver cancer cells, and the half-lethal concentration is about 25 nM; while under the same conditions, the commercial HpD can only kill 20% of liver cancer cells, indicating that the photodynamic effect of such derivatives is significantly better than that of the commercial HpD.

[0427] The derivative in the present invention was incubated with colon cancer cells HCT116, as Figure 10-4 (a) shows that the dark toxicity study of the photosensitizing drug indicates that HB-8a or HC-8c prepared in Example 8 contains two carboxyl groups and has good biocompatibility; the colon cancer cells were incubated with a 20 μM concentration of the photosensitizer for half an hour, and no obvious death of the colon cancer cells was observed, indicating that such photosensitizers basically have no cytotoxicity. As Figure 10-4 (b) shows that the cell phototoxicity study indicates that the photosensitizing drug shows very strong killing power to colon cancer cells under red light irradiation. HB-8a at a concentration of 50 nM can kill more than 85% of colon cancer cells, and the half-lethal concentration IC 50 is about 25 nM; HC-8c at a concentration of 50 nM can kill more than 90% of colon cancer cells, and the half-lethal concentration IC 50 value is about 25 nM; while under the same conditions, the commercial photosensitizer HpD can only kill about 30% of colon cancer cells, indicating that the photodynamic effect of such hypocrellin dicarboxylic acid derivatives is significantly better than that of the commercial photosensitizer HpD.

[0428] The hypocrellin derivative prepared in the present invention was incubated with cholangiocarcinoma cells MCC, as Figure 10-5 (a) shows that the dark toxicity study of the photosensitizing drug indicates that the cytotoxicities of HB-8b-PEG4 and HC-8d-PEG8 prepared in Example 8-1 are both small. The cholangiocarcinoma cells were incubated with a 20 μM concentration of the photosensitizer for half an hour, and no obvious death of the cholangiocarcinoma cells was observed, indicating that such photosensitizers basically have no cytotoxicity. AsFigure 10-5 (b) The cell phototoxicity study shows that under red light irradiation, the photosensitizing drugs exhibit very strong lethality to cholangiocarcinoma cells. HB-8b-PEG4 or HC-8d-PEG8 at a concentration of 50 nM can kill more than 90% of cholangiocarcinoma cells, and the half-lethal concentration IC 50 is about 25 nM; while under the same conditions, the commercial HpD can only kill 20% of cholangiocarcinoma cells, indicating that the photodynamic effect of such derivatives is significantly better than that of the commercial photosensitizer HpD.

[0429] The derivatives in the present invention are incubated with gastric cancer cells MFC. As Figure 10-6 (a) shows, the dark toxicity study of the photosensitizing drugs shows that HB-13a and HC-13c prepared in Example 13 both contain two carboxyl groups and have good water solubility; their cytotoxicities are relatively small. Incubating with a photosensitizer at a concentration of 20 μM for half an hour, no obvious death of MFC cells is observed, indicating that such photosensitizers basically have no cytotoxicity. As Figure 10-6 (b) shows, the cell phototoxicity study shows that under red light irradiation, the photosensitizing drugs exhibit very strong lethality to MFC cells. HB-13a at a concentration of 50 nM can kill more than 85% of MFC cells, and the half-lethal concentration IC 50 is about 30 nM; while HC-13c at a concentration of 50 nM can kill more than 90% of MFC cells, and the half-lethal concentration IC 50 is about 30 nM; while under the same conditions, the commercial photosensitizer HpD can only kill about 20% of MFC cells, indicating that the photodynamic effect of such derivatives is significantly better than that of the commercial photosensitizer HpD.

[0430] The above details illustrate by examples that some of the hypocrellin derivatives disclosed in the present invention can efficiently kill digestive tract tumor cells such as esophageal cancer AKR, gastric cancer MFC, lung cancer A549, liver cancer HCC, cholangiocarcinoma MCC, and colon cancer HCT116. Whether it is a derivative of hypocrellin or deacetyled hypocrellin, without light irradiation, it basically has no damage to cells, while under light irradiation, it has a strong ability to inactivate the above-mentioned digestive tract tumor cells. The detailed data of other derivatives in the present invention for efficiently killing digestive tract tumor cells such as esophageal cancer, gastric cancer, lung cancer, liver cancer, cholangiocarcinoma, and colon cancer are shown in Table 2.

[0431] Table 2: Shows the MTT data of some hypocrellin derivatives of the present invention on digestive tract tumor cells

[0432]

[0433]

[0434]

[0435] 3) Dark toxicity and phototoxicity tests of hypocrellin derivatives on head and neck tumor cells

[0436] The hypocrellin derivatives disclosed in the present invention can efficiently kill head and neck tumor cells such as brain cancer, head and neck cancer, tongue cancer, nasal cancer, oral cancer, and glioblastoma multiforme under 635 nm laser irradiation. Taking the hypocrellin derivatives disclosed in the examples as an example, their phototoxicity and dark toxicity effects on head and neck tumor cells are described.

[0437] The hypocrellin derivatives prepared in the present invention were incubated with the brain cancer cell line G442. The study on the dark toxicity of the photosensitizing drug showed that whether it was linked with 4 PEG or 8 PEG units, the cytotoxicities of HB-13a-PEG4 and HC-13c-PEG8 prepared in Example 13-2 were relatively small. Incubating with a 20 μM concentration of the photosensitizer for half an hour, no obvious death of brain cancer cells was observed, indicating that such photosensitizers basically have no cytotoxicity. As Figure 11 (a) shows that the study on the phototoxicity of the photosensitizing drug showed that the photosensitizing drug showed very strong killing power against brain cancer cells under red light irradiation. HB-13a-PEG4 at a concentration of 50 nM could kill more than 85% of brain cancer cells, and the half-lethal concentration IC 50 was approximately 25 nM; while HC-13c-PEG8 at a concentration of 50 nM could kill more than 90% of the brain cancer cell line G442, and the half-lethal concentration IC 50 was approximately 25 nM; while under the same conditions, the commercial photosensitizer HpD could only kill about 20% of brain cancer cells, indicating that the photodynamic effect of such derivatives was significantly better than that of the commercial photosensitizer HpD.

[0438] The hypocrellin derivatives prepared in the present invention were incubated with the head and neck cancer cell line SCC2. The cytotoxicities of HB-13a-NH-PEG12 and HC-13c-NH-PEG6 prepared in Example 13-3 were relatively small. Incubating with a 20 μM concentration of the photosensitizer for half an hour, no obvious death of the head and neck cancer cell line SCC2 was observed, indicating that such photosensitizers basically have no cytotoxicity. As Figure 11 (b) shows that the study on the phototoxicity of the photosensitizing drug showed that the photosensitizing drug showed very strong killing power against head and neck cancer cells under red light irradiation. HB-13a-NH-PEG12 at a concentration of 50 nM could kill more than 85% of head and neck cancer cells, and the half-lethal concentration IC 50 was approximately 30 nM; while HC-13c-NH-PEG6 at a concentration of 50 nM could kill more than 90% of head and neck cancer cells, and the half-lethal concentration IC 50 was approximately 30 nM; while under the same conditions, the commercial photosensitizer HpD could only kill about 20% of the head and neck cancer cell line SCC2, indicating that the photodynamic effect of such derivatives was significantly better than that of the commercial photosensitizer HpD.

[0439] The hypocrellin derivatives prepared in the present invention were incubated with tongue cancer cells TSCCa, and HB-13b-C4-N prepared in Example 13-8 + and HC-13c-C6-N + showed low cytotoxicity. Incubating with a photosensitizer at a concentration of 20 μM for half an hour, no obvious death of tongue cancer cells was observed, indicating that such photosensitizers basically have no cytotoxicity. As Figure 11 (c) shows, the cell phototoxicity study indicated that the photosensitizing drugs showed very strong killing power against tongue cancer cells TSCCa under red light irradiation. HB-13b-C4-N at a concentration of 50 nM + or HC-13c-C6-N + could kill more than 90% of tongue cancer cells, and the half-lethal concentration IC 50 was about 30 nM; while under the same conditions, the commercial photosensitizer HpD could only kill about 20% of tongue cancer cells, indicating that the photodynamic effect of such derivatives was significantly better than that of the commercial photosensitizer HpD.

[0440] The hypocrellin derivatives prepared in the present invention were incubated with nasal cancer cells KB, and HB-48c-PEG8 and HC-48d-PEG12 prepared in Example 48 showed low cytotoxicity. Incubating with a photosensitizer at a concentration of 20 μM for half an hour, no obvious death of nasal cancer cells was observed, indicating that such photosensitizers basically have no cytotoxicity. As Figure 11 (d) shows, the cell phototoxicity study indicated that the photosensitizing drugs showed very strong killing power against KB cells under red light irradiation. HB-48c-PEG8 at a concentration of 50 nM could kill more than 85% of KB cells, and the half-lethal concentration IC 50 value was about 30 nM; while HC-48d-PEG12 at a concentration of 50 nM could kill more than 90% of KB cells, and the half-lethal concentration IC 50 value was about 30 nM; while under the same conditions, the commercial photosensitizer HpD could only kill about 20% of nasal cancer cells, indicating that the photodynamic effect of such derivatives was significantly better than that of the commercial photosensitizer HpD.

[0441] The hypocrellin derivatives prepared in the present invention were incubated with oral cancer cells CAL27, and HB-51a-PEG12 and HC-51c-PEG18 prepared in Example 51-1 showed low cytotoxicity. Incubating with a photosensitizer at a concentration of 20 μM for half an hour, no obvious death of oral cancer cells was observed, indicating that such photosensitizers basically have no cytotoxicity. As Figure 11 (e) shows, the cell phototoxicity study indicated that the photosensitizing drugs showed very strong killing power against oral cancer cells under red light irradiation. HB-51a-PEG12 at a concentration of 50 nM could kill more than 85% of CAL27 cells, and the half-lethal concentration IC 50The value is approximately 30 nM; while HC-51c-PEG18 at a concentration of 50 nM can kill more than 90% of CAL27 cells, and the half-lethal concentration IC 50 The value is approximately 30 nM; while the commercial photosensitizer HpD under the same conditions can only kill 20% of oral cancer cells, indicating that the photodynamic effect of such derivatives is significantly better than that of the commercial photosensitizer HpD.

[0442] The hypocrellin derivatives prepared in the present invention were incubated with glioma C6 cells. The cytotoxicities of HB-52c-PEG12 and HC-52c-PEG16 prepared in Example 52-1 were both small. When incubated with a photosensitizer at a concentration of 20 μM for half an hour, no obvious death of C6 cells was observed, indicating that such photosensitizers basically have no cytotoxicity. As Figure 11 The cell phototoxicity study shown in (f) indicates that the photosensitizing drug shows very strong killing power against C6 cells under red light irradiation. HB-52c-PEG12 at a concentration of 50 nM can kill more than 85% of C6 cells, and the half-lethal concentration IC 50 The value is approximately 30 nM; while HC-52c-PEG16 at a concentration of 50 nM can kill more than 90% of C6 cells, and the half-lethal concentration IC 50 The value is approximately 30 nM; while the commercial photosensitizer HpD under the same conditions can only kill about 20% of C6 cells, indicating that the photodynamic effect of such derivatives is significantly better than that of the commercial photosensitizer HpD.

[0443] The above detailed examples illustrate that some of the hypocrellin derivatives described in the present invention can efficiently kill head and neck tumor cells such as brain cancer G442, head and neck cancer SCC2, tongue cancer TSCCa, nasal cancer KB, oral cancer CAL27, and glioma C6. Whether it is a derivative of hypocrellin or deacetyldehydrohypocrellin, it basically has no damage to cells without light irradiation, while it has a strong ability to inactivate the above-mentioned head and neck tumor cells under light irradiation. The detailed data of other derivatives in the present invention for efficiently killing head and neck tumor cells such as brain cancer, head and neck cancer, tongue cancer, nasal cancer, oral cancer, and glioma are shown in Table 3.

[0444] Table 3: Shows the MTT data of some hypocrellin derivatives of the present invention against head and neck tumor cells

[0445]

[0446]

[0447] 4) Dark toxicity and phototoxicity tests of hypocrellin derivatives on skin and genitourinary system tumor cells

[0448] The hypocrellin derivatives disclosed in the present invention can efficiently kill skin and genitourinary tumor cells such as basal cell carcinoma, melanoma, squamous cell carcinoma, cutaneous T-cell lymphoma, prostate cancer, and bladder cancer under 635 nm laser irradiation. Taking the hypocrellin derivatives prepared in the examples as an example, their phototoxic and dark toxic effects on genitourinary tumor cells are described.

[0449] The hypocrellin derivatives prepared in the present invention were incubated with basal cell carcinoma cells BCC. Whether it is the quaternary ammonium salt with 4 carbon chains or 6 carbon chains, HB-1a-C4-N prepared in Examples 1-5 + and HC-1c-C6-N + showed relatively low cytotoxicity, similar to the commercial photosensitizing drug hematoporphyrin HpD. The basal cell carcinoma cells were incubated with the photosensitizer HB-1a-C4-N + and HC-1c-C6-N + at a concentration of 20 μM for half an hour, and no obvious death of the basal cell carcinoma cells was observed, indicating that such photosensitizers basically have no cytotoxicity. As shown in Figure 12 (a), the cell phototoxicity study experiment showed that the photosensitizing drug showed very strong killing power against basal cell carcinoma cells under red light irradiation. HB-1a-C4-N at a concentration of 50 nM + could kill more than 85% of the basal cell carcinoma cells, and the half-lethal concentration IC 50 value was about 30 nM; while HC-1c-C6-N at a concentration of 50 nM + could kill more than 90% of the basal cell carcinoma cells, and the half-lethal concentration IC 50 value was about 30 nM; while under the same conditions, the commercial photosensitizing drug hematoporphyrin derivative HpD could only kill about 20% of the basal cell carcinoma cells, indicating that the photodynamic effect of such hypocrellin polyethylene glycol derivatives is significantly better than that of the commercial photosensitizing drug hematoporphyrin HpD.

[0450] The hypocrellin derivatives prepared in the present invention were incubated with melanoma cells B16. The cytotoxicity of HB-4a-PEG4 and HC-4c-PEG6 prepared in Example 4 was relatively low. The melanoma cells were incubated with the photosensitizer at a concentration of 20 μM for half an hour, and no obvious death of the melanoma cells was observed, indicating that such photosensitizers basically have no cytotoxicity. As shown in Figure 12 (b), the cell phototoxicity study showed that the photosensitizing drug showed very strong killing power against melanoma cells under red light irradiation. HB-4a-PEG4 at a concentration of 50 nM could kill more than 85% of the melanoma cells, and the half-lethal concentration IC 50 value was about 30 nM; while HC-4c-PEG6 at a concentration of 50 nM could kill more than 90% of the melanoma cells, and the half-lethal concentration IC 50The value is about 30 nM; under the same conditions, the commercial photosensitizer HpD can only kill 20% of melanoma cells, indicating that the photodynamic effect of such derivatives is significantly better than that of the commercial photosensitizer HpD.

[0451] The hypocrellin derivatives prepared in the present invention were incubated with squamous skin cancer cells PECA. The cytotoxicities of HB-10a-NH-PEG6 and HC-10c-NH-PEG8 prepared in Example 10-1 were both small. When incubated with a photosensitizer at a concentration of 20 μM for half an hour, no obvious death of PECA cells was observed, indicating that such photosensitizers basically have no cytotoxicity. As Figure 12 (c) The cell phototoxicity study showed that the photosensitizing drug showed very strong killing power against squamous skin cancer cells under red light irradiation. HB-10a-NH-PEG6 at a concentration of 50 nM could kill more than 85% of PECA cells, and the half-lethal concentration IC 50 value was about 30 nM; while HC-10c-NH-PEG8 at a concentration of 50 nM could kill more than 90% of PECA cells, and the half-lethal concentration IC 50 value was about 30 nM; while under the same conditions, the commercial photosensitizer HpD could only kill about 20% of squamous skin cancer cells, indicating that the photodynamic effect of such derivatives is significantly better than that of the commercial photosensitizer HpD.

[0452] The hypocrellin derivatives prepared in the present invention were incubated with prostate cancer cells LNCaP. HB-13b and HC-13d prepared in Example 13 contain two carboxyl groups and have good water solubility. Their cytotoxicities were both small. When incubated with a photosensitizer at a concentration of 20 μM for half an hour, no obvious death of prostate cancer cells was observed, indicating that such photosensitizers basically have no cytotoxicity. As Figure 12 (d) The cell phototoxicity study showed that the photosensitizing drug showed very strong killing power against prostate cancer cells under red light irradiation. HB-13b at a concentration of 50 nM could kill more than 85% of prostate cancer cells, and the half-lethal concentration IC 50 value was about 30 nM; while HC-13d at a concentration of 50 nM could kill more than 90% of prostate cancer cells, and the half-lethal concentration IC 50 value was about 30 nM; while under the same conditions, the commercial photosensitizer HpD could only kill about 20% of prostate cancer cells, indicating that the photodynamic effect of such derivatives is significantly better than that of the commercial photosensitizer HpD.

[0453] The hypocrellin derivatives prepared in the present invention were incubated with bladder cancer cells MBT-2. HB-11b-C4-SO3H and HC-11d-C6-SO3H prepared in Example 11 contain two sulfonic acid groups and have good water solubility. Their cytotoxicities are both small. When incubated with a photosensitizer at a concentration of 20 μM for half an hour, no obvious death of bladder cancer cells was observed, indicating that such photosensitizers basically have no cytotoxicity. As Figure 12 (e) shows that the cell phototoxicity study indicates that the photosensitizing drug shows very strong killing power against bladder cancer cells under red light irradiation. HB-11b-C4-SO3H at a concentration of 50 nM can kill more than 85% of bladder cancer cells, and the half-lethal concentration IC 50 value is about 30 nM; while HC-11d-C6-SO3H at a concentration of 50 nM can kill more than 90% of bladder cancer cells, and the half-lethal concentration IC 50 value is about 30 nM; while under the same conditions, the commercial photosensitizer HpD can only kill about 20% of bladder cancer cells, indicating that the photodynamic effect of such derivatives is significantly better than that of the commercial photosensitizer HpD.

[0454] The hypocrellin derivatives prepared in the present invention were incubated with cutaneous T-cell lymphoma HH cells. The cytotoxicities of HB-53a-PEG10 and HC-53c-PEG16 prepared in Example 53-1 are both small. When incubated with a photosensitizer at a concentration of 20 μM for half an hour, no obvious death of HH cells was observed, indicating that such photosensitizers basically have no cytotoxicity. As Figure 12 (f) shows that the cell phototoxicity study indicates that the photosensitizing drug shows very strong killing power against HH cells under red light irradiation. HB-53a-PEG10 at a concentration of 50 nM can kill more than 85% of HH cells, and the half-lethal concentration IC 50 value is about 30 nM; while HC-53c-PEG16 at a concentration of 50 nM can kill more than 90% of HH cells, and the half-lethal concentration IC 50 value is about 30 nM; while under the same conditions, the commercial photosensitizer HpD can only kill 20% of HH cells, indicating that the photodynamic effect of such hypocrellin derivatives is significantly better than that of the commercial photosensitizer HpD.

[0455] The above has detailedly illustrated that some derivatives disclosed by the present invention can efficiently kill skin and urinary system tumor cells such as basal cell carcinoma BCC, melanoma B16, squamous cell carcinoma of the skin PECA, prostate cancer LNCaP, bladder cancer MBT-2, cutaneous T-cell lymphoma HH, etc. Whether it is the derivative of hypocrellin or deacylated hypocrellin, without light irradiation, it basically has no damage to cells, while under light irradiation, it has a strong ability to inactivate the above-mentioned skin and urinary system tumor cells. Other derivatives in the present invention can also efficiently kill skin and urinary system tumor cells such as basal cell carcinoma, melanoma, squamous cell carcinoma of the skin, prostate cancer, bladder cancer, cutaneous T-cell lymphoma, etc., and the detailed data are shown in Table 4.

[0456] Table 4: MTT data of some hypocrellin derivatives of the present invention on skin tumors and genitourinary tumors cells

[0457]

[0458]

[0459]

[0460] The above Figures 10-12 and Tables 2-4 illustrate that such hypocrellin derivatives have almost no cytotoxicity without light irradiation and have a strong photodynamic effect under light irradiation. The photosensitizing drug at a concentration of 50 nM can efficiently kill most various tumor cells, and the half-lethal concentration IC 50 value is about 20-30 nM, which is 1-2 orders of magnitude lower than the IC 50 value of the commercial photosensitizer hematoporphyrin HpD under the same conditions. The hypocrellin derivatives described in other embodiments of the present invention also have the dark toxicity and phototoxicity as shown in Figures 10-12 and Tables 2-4. Therefore, the derivatives described in the present invention have better dark toxicity, phototoxicity and photodynamic effect than the commercial photosensitizer hematoporphyrin HpD.

[0461] Example 62

[0462] Small animal fluorescence imaging experiment: All animal experiment operations comply with the regulations on animal use and breeding stipulated by the Chinese Animal Research Ethics Committee. Female Balb / c nude mice (about 20 g) at 4-6 weeks old were used for establishing tumor models, and 50 μL of suspension of various tumor cells (5×10 6 cells) were respectively injected into the posterior side of the right thigh of the mice. When the tumor volume grew to about 200 mm 3At this time, in vivo fluorescence imaging experiments were carried out. After injecting 40 μL of a physiological saline solution (10 mg / mL) of the photosensitizing drug into the tumor-bearing mice via the tail vein at the mouse dose (20 mg / kg), tumor fluorescence imaging was collected at 0, 2, 4, 6, 8, 12, and 24 hours using a multispectral small animal in vivo imaging system. After observing for 8 h, the tumor-bearing mice were sacrificed by cervical dislocation, and the tumors and main organs were removed for in vitro use. Using a Maestro 2 multispectral small animal fluorescence imaging system, fluorescence images of the tumor-bearing mice were collected at an excitation wavelength of 660 nm. The average fluorescence intensity within the ROS region was statistically analyzed for semi-quantitative analysis.

[0463] The fluorescence imaging of the aforementioned cells ( Figure 9 ) results showed that the derivatives in the present invention could enter tumor cells well, mainly enter the lysosomes of tumor cells, and produce near-infrared fluorescence imaging. To study the enrichment and metabolism processes of such derivatives at the tumor site in vivo, the present invention used mice as a model, inoculated subcutaneous tumors with C6 glioma cells, and obtained a series of subcutaneous animal models of glioma. The derivative HC-1c-PEG6 (Example 1-1) was used as the photosensitizing drug and injected into the tumor-bearing mice via the tail vein at a dose of 10 mg / kg. First, its fluorescence imaging behavior in C6-tumor-bearing mice was observed, and fluorescence signals at the tumor site were collected at 0, 2, 4, 8, 12, and 24 hours using a PerkinElmer multispectral small animal in vivo imaging system. As Figure 13 can be seen, as time extended, the fluorescence signal in the tumor region gradually increased and reached the highest at 4 h, indicating that the photosensitizing drug HC-1c-PEG6 could be highly enriched at the tumor site well. Except for a small amount of drug in the skin in the non-tumor area, there was almost no drug enrichment in the other parts of the mice. After 12 h, the photosensitizing drug HC-1c-PEG6 was basically metabolized out of the body. After 24 h, the tumors and main organs such as the heart, liver, spleen, lungs, and kidneys of the tumor-bearing mice were removed for fluorescence imaging, and it was found that HC-1c-PEG6 was mainly distributed in the liver and tumor, further proving its enrichment in the tumor and mainly metabolized out of the body through the liver.

[0464] To study the pharmacokinetics of the photosensitizing drug in vivo, at different time intervals after tail vein injection, the main organs (heart, liver, spleen, lungs, kidneys) and tumors of the glioma-bearing mice were removed for ex vivo fluorescence imaging. The study found that the fluorescence intensity in the tumor had a maximum value after 4 h, was mainly metabolized out of the body through the kidneys and liver, had a half-life of 8 h in the tumor, and was almost completely metabolized out of the body after one week.

[0465] To study the in-situ enrichment process of the derivatives of the present invention in in-vivo tumors, in this experiment, mice were used as models, and C6 glioma cells were inoculated into the brains of mice to establish in-situ tumors, obtaining an in-situ animal model of glioma for fluorescence imaging. Different doses of the photosensitizing drug HB-1c-PEG8 were injected into the tail veins of tumor-bearing mice, and its fluorescence imaging behavior in C6-tumor-bearing mice was observed. The fluorescence signals at the tumor sites were collected at 4 h and 6 h respectively. As Figure 14 (a), HB-1c-PEG8 was injected into the tail vein of tumor-bearing mice. After 4 h, the photosensitizing drug was well enriched at the glioma site. The enrichment of the mice with a drug dose of 10 mg / kg was significantly more than that of the mice with a dose of 5 mg / kg, and there was no enrichment of the photosensitizing drug in the non-tumor areas of the brain, indicating that the photosensitizing drug has a good targeting enrichment effect on glioma. In addition, there was no fluorescence in the brains of the blank mice without the drug. As Figure 14 (b) shows the drug enrichment of HB-1c-PEG8 in the glioma of tumor-bearing mice after being injected into the tail vein for 6 h. It can be seen that the enrichment situation at 6 h after drug injection is very similar to that at 4 h, indicating that when the photosensitizing drug is injected into mice intravenously, there is a good targeting enrichment of glioma in about 4 h. Therefore, 4 h was selected as the drug enrichment time for the following animal experiments to observe the fluorescence imaging of tumors.

[0466] Figure 15 Shows the comparative diagram of the in-situ tumor enrichment of different photosensitizing drugs in mice bearing glioma. In the following animal experiments, 4 h was selected as the drug enrichment time to observe the fluorescence imaging of tumors, and the intravenous drug administration dose was calculated according to 10 mg / kg of the mouse body weight. Figure 15 (a) is the comparative fluorescence imaging diagram of the drug HC-1a-PEG8 (Example 1-1) enriched for 4 hours. It can be seen that there is no fluorescence at the in-situ glioma without the drug, and there is very strong fluorescence enrichment at the glioma of the mice after 4 h of drug administration. Figure 15 (b) is the comparative fluorescence imaging diagram of HB-1c-PEG8-H (Example 1-2) in enriched for 4 hours; Figure 15 (c) is the comparative fluorescence imaging diagram of the drug HC-51c-PEG18 (Example 51-1) enriched for 4 h. It can be seen that there is no fluorescence at the tumor without drug administration, and there is very strong fluorescence at the in-situ glioma of the mice after 4 h of intravenous drug administration. The above results show that the hypocrellin derivatives of the present invention, as photosensitizing drugs, can be well enriched in the in-situ glioma of mice, and there is almost no enrichment of the photosensitizing drug in the non-tumor areas of the brain. This indicates that the photosensitizing drug has a very good targeting enrichment effect on glioma and can be used as a fluorescence-mediated material to guide the resection of glioma.

[0467] In the operation of glioma, there is no mediated drug for positioning to guide the resection of tumors. In experiments, it was found that such derivatives can specifically accumulate in glioma tissues, and there is no accumulation of photosensitizer in the brain regions without tumors, showing good tumor-targeted enrichment. At this time, when the tumor tissue is irradiated with light of a specific wavelength, detectable fluorescence can be excited to locate the position of the tumor tissue, which is used for fluorescence-guided glioma resection surgery. On the other hand, such hypocrellin derivatives can generate singlet oxygen under photosensitive conditions, causing damage to tumor cells and being used for the photodynamic therapy of glioma with basically no damage to normal tissues. The present invention discloses for the first time a derivative with amino substitution at the 1-position and 2-position of hypocrellin as a fluorescence-mediated material for guiding the resection and photodynamic therapy of solid tumors.

[0468] In addition to glioma cells, using mice as a model, AKR esophageal cancer cells were also used to inoculate subcutaneous tumors in mice to obtain a series of subcutaneous tumor models of esophageal cancer. HC-13c-PEG4 (Example 13-2) was used as a photosensitive drug and injected into tumor-bearing mice via the tail vein at a dose of 10 mg / kg, and its fluorescence imaging behavior in AKR esophageal cancer-bearing mice was observed. The fluorescence signals at the tumor sites at 4 h were collected using a PerkinElmer multispectral small animal in vivo imaging system, and the animal fluorescence imaging is as Figure 16 (a) shown. It can be seen that after several cycles in vivo by intravenous injection, the prepared photosensitive drug HC-13c-PEG4 produces very strong enrichment at the tumor after about 4 h through passive targeting. In addition to the enrichment ability of the hypocrellin photosensitizer, the presence of 8 polyethylene glycol units in the molecule makes the ester-water compatibility of the whole drug molecule particularly suitable, resulting in its very strong enrichment in the tumor.

[0469] A549 lung cancer cells were also used to inoculate subcutaneous tumors in mice to obtain a subcutaneous tumor model of lung cancer. HB-13c-NH-PEG6 (Example 13-3) was used as a photosensitive drug and injected into tumor-bearing mice via the tail vein at a dose of 10 mg / kg, and its fluorescence imaging behavior in A549 lung cancer-bearing mice was observed. The fluorescence signals at the tumor sites at 4 hours were collected, and the animal fluorescence imaging is as Figure 16 (b) shown. It can be seen that after several cycles in vivo by intravenous injection, the photosensitive drug HB-13c-NH-PEG6 produces very strong enrichment at the tumor after about 4 h through passive targeting. In addition to the enrichment ability of the hypocrellin photosensitizer, the presence of 12 polyethylene glycol units in the molecule makes the ester-water compatibility of the whole drug molecule particularly suitable, resulting in its very strong enrichment in the tumor.

[0470] The present invention also inoculated subcutaneous tumors in mice with skin T cell lymphoma HH cells to obtain a subcutaneous tumor model of HH. HC-51c-PEG12-H (Example 51-2) was used as a photosensitizing drug and injected into the tumor-bearing mice via the tail vein at a dose of 10 mg / kg. The fluorescence imaging behavior of the drug in the lymphoma-bearing mice was observed, and the fluorescence signal at the tumor site was collected at 4 h. The animal fluorescence imaging is as shown in Figure 16 (c). It can be seen that after several cycles in the body by intravenous injection, HC-51c-PEG12-H shows very strong accumulation at the tumor site after about 4 h through passive targeting. In addition to the enrichment ability of hypocrellin, the molecule contains 12 polyethylene glycol units with a hydroxyl group at the end, making the ester-water compatibility of the whole drug molecule particularly suitable, resulting in very strong accumulation in the tumor.

[0471] The present invention also inoculated subcutaneous tumors in mice with MFC gastric cancer cells to obtain a subcutaneous tumor model of gastric cancer. HB-2a-PEG1 (Example 2-1) was used as a photosensitizing drug and injected into the tumor-bearing mice via the tail vein at a dose of 10 mg / kg. The fluorescence imaging behavior of the drug in the MFC gastric cancer-bearing mice was observed, and the fluorescence signal at the tumor site was collected at 4 h. The animal fluorescence imaging is as shown in Figure 16 (d). It can be seen that after several cycles in the body by intravenous injection, HB-2a-PEG1 shows a certain accumulation ability at the tumor site after about 4 h through passive targeting, which belongs to medium-level accumulation. The reason is that this derivative molecule only contains two ethylene glycol units, making the water solubility of the whole drug molecule insufficient, resulting in medium-level accumulation at the tumor site.

[0472] Figure 17 (a), using mice as a model, subcutaneous tumors were inoculated in mice with tongue cancer cells TSCCa (left), liver cancer cells HCC (middle), and cholangiocarcinoma cells MCC (right) respectively to obtain a series of subcutaneous tumor models of lung cancer. The derivatives HB-1a-PEG6-COOH (left), HB-1c-PEG8-COOH (middle) of Example 1-3, and the derivative HC-1c-C4-N of Example 1-5 + (right) were used as photosensitizing drugs. After tail vein administration, the fluorescence imaging map of the photosensitizing drugs in the tumor-bearing mice at 4 h was obtained. They were respectively injected into the tumor-bearing mice via the tail vein at a dose of 10 mg / kg. The fluorescence imaging behavior of the drugs in each tumor-bearing mouse was observed, and the fluorescence signal at the tumor site was collected at 4 h. The animal fluorescence imaging is as shown in Figure 17 (a). It can be seen that HB-1a-PEG6-COOH shows very strong accumulation in tongue cancer cells TSCCa (left); HB-1c-PEG8-COOH shows very strong accumulation in liver cancer cells HCC (middle); HC-1c-C4-N+ There is very strong enrichment in cholangiocarcinoma cell MCC (right). The reason is that the photosensitizer molecules all carry water-soluble groups such as long-chain polyethylene glycol, water-soluble carboxyl groups, or quaternary ammonium salts, making the ester-water compatibility of the drug molecules particularly suitable, thus resulting in very strong enrichment ability in tumors.

[0473] Figure 17 (b) Using mice as a model, subcutaneous tumors were inoculated in mice with head and neck cancer cells SCC2 (left), brain cancer cells G442 (middle), and oral cancer cells CAL27 (right) respectively, to obtain a series of subcutaneous tumor models of lung cancer. After the derivatives HB-13a (left), HB-13c (middle), and HC-13c (right) of Example 13 were used as photosensitizing drugs and administered via the tail vein, fluorescence imaging maps of the photosensitizing drugs in the tumor-bearing mice at 4 h were made. They were injected into the tumor-bearing mice via the tail vein at a dose of 10 mg / kg respectively, and their fluorescence imaging behaviors in each tumor-bearing mouse were observed. The fluorescence signals at the tumor sites at 4 h were collected, and their animal fluorescence imaging is as Figure 17 (b) shown. It can be seen that HB-13a has medium-strength enrichment in head and neck cancer cells SCC2 (left); HB-13c has medium-strength enrichment in brain cancer cells G442 (middle); HC-13c has medium-strength enrichment in oral cancer cells CAL27 (right). After several cycles in the body through intravenous injection, such photosensitizer molecules have a certain enrichment ability at the tumor site after about 4 h through passive targeting, belonging to medium-level enrichment. The reason is that this derivative molecule only contains two carboxyl groups, making the water solubility of the whole drug molecule insufficient, thus resulting in only medium-level enrichment at the tumor site.

[0474] Figure 17 (c) Using mice as a model, subcutaneous tumors were inoculated in mice with colon cancer cells HCT116 (left), melanoma cells B16 (middle), and bladder cancer cells MBT-2 (right) respectively, to obtain a series of subcutaneous tumor models of lung cancer. After the derivatives HB-51a-PEG6-H (left), HB-51c-PEG12-H (middle), and HC-51c-PEG12 (right) prepared in Examples 51-2 and 51-3 were used as photosensitizing drugs and administered via the tail vein, fluorescence imaging maps of the photosensitizing drugs in the tumor-bearing mice at 4 h were made. They were injected into the tumor-bearing mice via the tail vein at a dose of 10 mg / kg respectively, and their fluorescence imaging behaviors in each tumor-bearing mouse were observed. The fluorescence signals at the tumor sites at 4 h were collected, and their animal fluorescence imaging is as Figure 17As shown in (d), it can be seen that HB-51a-PEG6-H has very strong enrichment in colon cancer cell line HCT116 (left); HB-51c-PEG12-H has very strong enrichment in melanoma cell line B16 (middle); HC-51c-NH-PEG12 has very strong enrichment in bladder cancer cell line MBT-2 (right). The reason is that the photosensitizer molecules all carry 6 or 12 long-chain polyethylene glycols, making the ester-water compatibility of the drug molecules particularly suitable, thus resulting in very strong enrichment ability in tumors.

[0475] The above Figure 17 The results described above indicate that the various hypocrellin derivatives provided in the present invention show fluorescence imaging in tumor-bearing mice, indicating that such derivatives produce ultra-strong or moderate fluorescence imaging at the tumor sites in mice and can be used for fluorescence-mediated materials to guide the resection of tumor boundaries during surgery. The tumors include: esophageal cancer, gastric cancer, lung cancer, liver cancer, cholangiocarcinoma, colon cancer, head and neck cancer, brain cancer, tongue cancer, nasal cancer, oral cancer, glioblastoma, basal cell carcinoma, squamous cell carcinoma of the skin, cutaneous T-cell lymphoma, melanoma, prostate cancer, bladder cancer. Other derivatives in the present invention also have the in-situ enrichment effect on the above-mentioned tumor cells, can be used for in-vivo fluorescence imaging of the above-mentioned tumor cells, and are used to mediate materials to guide the resection of tumor boundaries during surgery. The derivatives described in the present invention have similar parent structures. Hypocrellin derivatives with different PEG chain lengths, different sulfonic acid chain lengths, and different quaternary ammonium salt chain lengths in the substituents all show good enrichment effects on tumor cells and tissues. The fluorescence imaging results of some of the above-mentioned hypocrellin derivatives in different tumor cells and tissues are shown in Table 5.

[0476] Table 5: In-vivo enrichment degrees of some derivatives for digestive tract tumors, head and neck tumors, skin tumors, and genitourinary tumors

[0477]

[0478]

[0479] Example 63

[0480] In-vivo photodynamic therapy experiment: Tumor models were established using 4-6 week-old female Balb / c nude mice (about 20 g). 50 μL of suspensions of various tumor cells (5×10 6 cells) were respectively injected into the posterior side of the right thigh of the mice. The tumor volume grew to about 200 mm 3When the time is right, in vivo photodynamic therapy experiments are carried out. The tumor-bearing mice are randomly divided into 5 groups (5 mice in each group): without any treatment (Group I); only injecting the solution of photosensitizing drug into the tail vein (100 μL, 500 μM) (Group II); injecting the photosensitizing drug into the tail vein (100 μL, 500 μM), and irradiating the tumor site with a 635 nm laser 4 h later (10 min, 0.1 W cm -2 )(Group III). After laser treatment, the tumor volume and body weight are measured every other day, and the survival rate is recorded. The tumor volume (V) is calculated by the formula (V = a × b 2 / 2), where a is the length of the tumor and b is the width of the tumor.

[0481] In view of the fact that the hypocrellin derivatives prepared by the present invention can efficiently kill various in vitro tumor cells, and as photosensitizing drugs, they can be well enriched in the subcutaneous tumors of mice. The inventors further tested such derivatives as photodynamic drugs to kill tumor cells in mice. Mice inoculated with A549 lung cancer tumor cells were injected with 10 mg / kg dose of photosensitizing drug HC-1c-PEG8 (Example 1-1) into the tail vein in the tumor-bearing mice. 4 h later, the tumor site was irradiated with a 635 nm laser (0.1 W / cm 2 ) for 10 min, and the data were recorded. Figure 18 (As shown in (b)), scabs appeared on the 2nd day after photodynamic therapy of mouse lung cancer tumors. The tumors gradually shrank from the 4th day to the 6th day and completely disappeared after the 14th day, indicating that after photodynamic therapy, the subcutaneous tumors of mice were almost completely inhibited. For the control group of lung cancer tumor mice, only normal saline was injected, and no photosensitizing drug was injected. As Figure 18 (As shown in (a)), the tumor site was irradiated with a 635 nm laser (0.1 W / cm 2 ) for 10 min. After 6 days, it was found that the tumor was growing rapidly and there was no effect of inhibiting the tumor. Therefore, HC-1c-PEG8, as a photosensitizing drug, has an obvious effect of killing tumors and can inhibit the regeneration and recurrence of tumors in photodynamic therapy of lung cancer.

[0482] Mice inoculated with cholangiocarcinoma cells (MCC) were injected with 10 mg / kg dose of photosensitizing drug HB-1c-PEG6-H (Example 1-2) into the tail vein. 4 h later, the tumor site was irradiated with a 635 nm laser of 0.1 W / cm 2 for 10 min. As Figure 18As shown in (c), scabs appeared on the second day after photodynamic therapy for murine cholangiocarcinoma. The tumor gradually shrank from the fourth day to the sixth day and completely disappeared after the fourteenth day, indicating that after photodynamic therapy, the tumor in the mice was almost completely inhibited. Therefore, HB-1c-PEG6-H, as a photosensitizing drug, has an obvious tumor-killing effect in photodynamic therapy for lung cancer and can inhibit the regeneration and recurrence of cholangiocarcinoma tumor cells.

[0483] For the mice inoculated with melanoma cells (B16), 10 mg / kg dose of the photosensitizing drug HC-1a-C4-N was injected via the tail vein. + (Examples 1-5) In tumor-bearing mice, the fluorescence imaging behavior in the tumor-bearing mice was observed. After 4 h, the tumor site was irradiated with a 635 nm laser at 0.1 W / cm 2 for 10 min. As Figure 18 shown in (d), scabs appeared on the second day after photodynamic therapy for murine melanoma. The tumor gradually shrank from the fourth day to the sixth day and completely disappeared after the fourteenth day, indicating that after photodynamic therapy, the tumor in the mice was almost completely inhibited. Therefore, HC-1a-C4-N + as a photosensitizing drug has an obvious tumor-killing effect in photodynamic therapy for melanoma and can inhibit the regeneration and recurrence of melanoma cells.

[0484] H&E staining and pathological analysis were performed on the tumor tissues of nude mice after different treatments. It was found that the tumor cells in the blank control group were not damaged, and the tumor cells in the photodynamic therapy group were completely dead. Statistical analysis of the survival rate of the mice showed that compared with the control group, none of the mice treated with photodynamic therapy died within 50 days, indicating that photodynamic therapy has a good anti-tumor effect on tumor-bearing mice.

[0485] For the mice inoculated with glioma cells (C6), 10 mg / kg dose of the photosensitizing drug HB-50a-PEG12 (Example 50-1) was injected via the tail vein. The fluorescence imaging behavior in the tumor-bearing mice was observed. After 4 h, the tumor site was irradiated with a 635 nm laser at 0.1 W / cm 2 for 10 min. As Figure 19 shown in (b), scabs appeared on the second day after photodynamic therapy for murine glioma. The tumor gradually shrank from the fourth day to the sixth day and completely disappeared after the fourteenth day, indicating that after photodynamic therapy, the tumor in the mice was almost completely inhibited. For the control group of mice with glioma, only normal saline was injected without injecting the photosensitizing drug. As Figure 19 shown in (a), when irradiated with an excitation light source under the same conditions, it was found that the tumor was growing rapidly after 6 days and there was no effect of inhibiting the tumor. Therefore, HB-50a-PEG12, as a photosensitizing drug, has an obvious tumor-killing effect in photodynamic therapy for glioma and can inhibit the regeneration and recurrence of glioma cells.

[0486] Mice inoculated with cutaneous T cell lymphoma cells (HH) were injected via the tail vein with a dose of 10 mg / kg of the photosensitizing drug HC-54c-PEG12 (Example 54-1) into the tumor-bearing mice, and the fluorescence imaging behavior in the tumor-bearing mice was observed. After 4 h, the tumor site was irradiated with a 635 nm laser at 0.1 W / cm 2 for 10 min. As Figure 19 (c) shows, scabbing occurred on the second day after photodynamic therapy for murine lymphoma, gradually shrank from the fourth day to the sixth day, and completely disappeared after the 14th day, indicating that after photodynamic therapy, the tumors in the mice were almost completely inhibited. Therefore, HC-54c-PEG12, as a photosensitizing drug, has an obvious tumor-killing effect in photodynamic therapy for cutaneous T cell lymphoma and can inhibit the regeneration and recurrence of lymphoma cells.

[0487] Mice inoculated with bladder cancer cells (MBT-2) were injected via the tail vein with a dose of 10 mg / kg of the photosensitizing drug HC-52c-PEG12-H (Example 52-2) into the tumor-bearing mice, and the fluorescence imaging behavior in the tumor-bearing mice was observed. After 4 h, the tumor site was irradiated with a 635 nm laser at 0.1 W / cm 2 for 10 min. As Figure 19 (d) shows, scabbing occurred on the second day after photodynamic therapy for murine bladder cancer, gradually shrank from the fourth day to the sixth day, and completely disappeared after the 14th day, indicating that after photodynamic therapy, the tumors in the mice were almost completely inhibited. Therefore, HC-52c-PEG12-H, as a photosensitizing drug, has an obvious tumor-killing effect in photodynamic therapy for bladder cancer and can inhibit the regeneration and recurrence of bladder cancer cells.

[0488] The above has been described in detail by way of examples that some of the hypocrellin derivatives described in the present invention can efficiently kill lung cancer cells, cholangiocarcinoma cells, melanoma cells, glioma cells, cutaneous T cell lymphoma, and bladder cancer cells in tumor-bearing mice. By injecting a certain dose of the photosensitizing drug into the tumor-bearing mice and combining with laser irradiation of a certain intensity and wavelength, good photodynamic therapy effects have been achieved. Since such photosensitizing drugs have a similar killing effect on tumor cells, it is believed that for other tumor-bearing mice, such as those with esophageal cancer, gastric cancer, liver cancer, colon cancer, brain cancer, head and neck cancer, tongue cancer, nasal cancer, oral cancer, basal cell carcinoma, squamous cell carcinoma of the skin, prostate cancer, etc., good photodynamic therapy effects can also be obtained. Other derivatives disclosed in the present invention, whether they are derivatives of hypocrellin or deacetyhypocrellin, linked with different substituted amino groups, all show good ability to photodynamically inactivate tumor cells under photodynamic conditions. Therefore, the hypocrellin derivatives disclosed in the present invention can all photodynamically treat various tumor cells in mice.

[0489] Comparative Example 1

[0490] The photodynamic effect of the derivatives of the present invention on HeLa is as Figure 20 shown. Under red light irradiation, the photosensitizing drug shows good inactivation ability against HeLa cells. The derivative HB-1c-PEG6 (concentration 200 nM) of Example 1-1 can kill more than 80% of HeLa cells, and the half-lethal concentration IC 50 value is about 120 nM; similarly, the HC-1c-PEG6 at a concentration of 200 nM can kill more than 80% of HeLa cells, and the half-lethal concentration is about 80 nM; the HC-1c-PEG12 at a concentration of 200 nM can kill more than 80% of HeLa cells, and the half-lethal concentration is about 80 nM;

[0491] Under the same conditions, HB-1c-PEG6 can kill 80% of esophageal cancer cells AKR, gastric cancer cells MFC, lung cancer cells A549, liver cancer cells HCC, cholangiocarcinoma cells MCC, and colon cancer cells HCT116 only at a concentration of 50 nM, and the half-lethal concentration IC 50 value is about 20 - 30 nM (Table 2); under the same conditions, HC-1c-PEG6 can kill 80% of head and neck cancer cells SCC2, brain cancer cells G442, tongue cancer cells TSCCa, nasal cancer cells KB, oral cancer cells CAL27, and glioma cells C6 only at a concentration of 50 nM, and the half-lethal concentration IC 50 value is about 20 - 30 nM (Table 3); under the same conditions, HC-1c-PEG12 can kill 80% of basal cell carcinoma cells BCC, squamous skin cancer cells PECA, melanoma cells B16, prostate cancer cells LNCaP, and bladder cancer cells MBT-2 only at a concentration of 50 nM, and the half-lethal concentration IC 50 value is about 20 - 30 nM (Table 4). Therefore, from Figure 20 the comparison with Table 2 - 4, it can be seen that the phototoxic effect of the hypocrellin derivatives disclosed in the present invention on the above-mentioned tumor cells is significantly higher than that on HeLa cells.

[0492] Obviously, the above-mentioned embodiments of the present invention are merely examples for clearly illustrating the present invention, rather than limitations on the implementation manners of the present invention. For those of ordinary skill in the art, based on the above description, other different forms of changes or modifications can be made. It is impossible to list all the implementation manners here. Any obvious changes or modifications derived from the technical solutions of the present invention still fall within the protection scope of the present invention.

[0493] It should be noted that the hypocrellin derivatives involved in the present invention all contain two enol tautomers, and the chemical structures of the two isomers are as shown in Formula (I) and Formula (I'), which are of course within the scope of protection. For the sake of simplicity, only one of the enol tautomers is listed in all the embodiments of the present invention, and the other enol tautomer and its corresponding general structural formula are described in detail in the specification, and its structure is of course within the scope of protection. In addition, the general structural formula of the hypocrellin derivatives involved in the present invention contains a polyethylene glycol unit (PEGn), and the unit number n is any integer between 1 and 50, and the corresponding chemical structures are of course within the scope of protection. For the sake of simplicity, only some integers are listed in all the embodiments of the present invention, and the general structural formulas corresponding to the remaining parts are described in detail in the specification, and their structures are of course within the scope of protection. Any range described in the present invention includes the end values and any numerical values between the end values, as well as any sub-ranges formed by the end values or any numerical values between the end values.

[0494] The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention shall be included in the scope of protection of the present invention.

Claims

1. Use of a derivative with simultaneous substitution at the ortho-position and 2-position of hypocrellin or a mixture thereof in the preparation of a photodynamic anti-tumor drug; wherein, The tumor is esophageal cancer, gastric cancer, lung cancer, liver cancer, cholangiocarcinoma, colon cancer, brain cancer, head and neck cancer, tongue cancer, nasal cancer, oral cancer, glioblastoma, basal cell carcinoma, squamous cell carcinoma of the skin, melanoma, cutaneous T-cell lymphoma, prostate cancer, bladder cancer; the derivative is a compound represented by formula I-a, formula I-b, formula I-c or formula I-d, its isotope-labeled substance, pharmaceutically acceptable salt; Among them, the ortho-position of hypocrellin is the 3rd, 4th, 9th or 10th position marked by formula I-a to I-d; R3 is -COCH3 or -H; R1 and R2 are each independently linked to an amino group; R1 and R2 are the same or different; R1 and R2 are each independently: -H, -CH3, -C2H5, -C3H7, -C4H9, -C5H 11 , -C6H 13 ; Or each of R1 and R2 independently is: cyclopropyl, hydroxycyclopropyl, carboxycyclopropyl, cyclobutyl, hydroxycyclobutyl, carboxycyclobutyl, -CH2C4H6(COOH), cyclopentyl, hydroxycyclopentyl, carboxycyclopentyl, cyclohexyl, hydroxycyclohexyl, carboxycyclohexyl, -CH2C6H 10 (COOH), -CH2C6H 10 (OH); Or R1 and R2 are each independently: -CH2COOH, -CH2CH2COOH, -CH2(CH2)2COOH, -CH2(CH2)3COOH, -CH2(CH2)4COOH, -CH2(CH2)5COOH, -CH2(CH2)6COOH, -CH2(CH2) 10 COOH, -CH2COOCH3, -CH2CH2COOC6H 13 、-CH2(CH2)2COOCH3, -CH2(CH2)2COOC2H5, -CH2(CH2)2COOC6H 13 、-CH2(CH2)4COOCH3, -CH2(CH2)6COOC6H 13 ; Or R1 and R2 are each independently: -CH2SO3H, -CH2CH2SO3H, -CH2(CH2)2SO3H, -CH2(CH2)3SO3H, -CH2(CH2)4SO3H, -CH2(CH2)5SO3H, -CH2(CH2) 11 SO3H; Or R1 and R2 are each independently: -OH, -OCH3, -OC2H5, -OC6H 13 , -NH2, -NHC2H5, -NHC6H 13 ; or each of R1 and R2 is independently: -CH2CH2-(OCH2CH2) n -OH, -CH2CH2-(OCH2CH2) n -OCH3, -CH2CH2-(OCH2CH2) n -OC6H 13 , -CH2CH2-(OCH2CH2) n -O-COCH3, -CH2CH2-(OCH2CH2) n -O-COC6H 13 ; or R1 and R2 are each independently: -CH2CH2-O-CO-CH2CH2-(OCH2CH2) n -OH, -CH2CH2-O-CO-CH2CH2-(OCH2CH2) n -OCH3, -CH2CH2-OCH2CH2-O-CO-CH2CH2-(OCH2CH2) n -OH, -CH2CH2-OCH2CH2-O-CO-CH2CH2-(OCH2CH2) n -OCH3, -CH2CH2-OCH2CH2-OCH2CH2-O-CO-CH2CH2-(OCH2CH2) n -OH, -CH2CH2-OCH2CH2-OCH2CH2-O-CO-CH2CH2-(OCH2CH2) n -OCH3; Alternatively, each of R1 and R2 independently is: -(CH2)3-OH, -(CH2)3-OCH3, -(CH2)3-OC2H5, -(CH2)3-OCOCH3, -(CH2)3-OCOC2H5, -(CH2)3-O-COCH2CH2-(OCH2CH2) n -OCH3; -(CH2)4-OH, -(CH2)4-OCH3, -(CH2)4-OCOCH3, -(CH2)4-OCOC2H5, -(CH2)4-O-COCH2CH2-(OCH2CH2) n -OCH3; -(CH2)6-OH, -(CH2)6-OCH3, -(CH2)6-OCOCH3, -(CH2)6-O-COCH2CH2-(OCH2CH2) n -OCH3; Or R1 and R2 are each independently: -CH2CH2-NH-CH2CH2-(OCH2CH2) n -OH, -CH2CH2-NH-CH2CH2-(OCH2CH2) n -OCH3, -CH2CH2-NHCH2CH2-NH-COCH2CH2-(OCH2CH2) n -OCH3; Or each of R1 and R2 is independently: -CH(CH3)-COOH, -CH(CH(CH3)2)-COOH, -CHCH2(CH(CH3)2)-COOH, -CH(CH2CH2SCH3)-COOH, -CHCH(CH3)(C2H5)-COOH, -CH(CH2OH)-COOH, -CHCH(OH)(CH3)-COOH, -CH(CH2SH)-COOH, -CH(CH2CONH2)-COOH, -CH(CH2CH2CONH2)-COOH, -CH(CH2CH2CH2CH2NH3 + )-COOH, -CH(COOH)-CH2COOH, -CH(COOH)-CH2CH2COOH, -CH(CH3)-COOCH3, -CH(CH(CH3)2)-COOCH3, -CHCH2(CH(CH3)2)-COOCH3, -CH(CH2CH2SCH3)-COOCH3; or R1 and R2 are each independently: -CH2CO-(OCH2CH2) n -OH, -CH2CO-(OCH2CH2) n -OCH3, -CH2CH2CO-(OCH2CH2) n -OH, -CH2CH2CO-(OCH2CH2) n -OCH3, -CH2(CH2)2CO-(OCH2CH2) n -OH, -CH2(CH2)2CO-(OCH2CH2) n -OCH3, -CH2(CH2)4CO-(OCH2CH2) n -OH, -CH2(CH2)4CO-(OCH2CH2) n -OCH3; or R1 and R2 are each independently: -CH2-CO-NH-CH2CH2-(OCH2CH2) n -OH, -CH2-CO-NH-CH2CH2-(OCH2CH2) n -OCH3, -(CH2)2-CO-NH-CH2CH2-(OCH2CH2) n -OH, -(CH2)2-CO-NH-CH2CH2-(OCH2CH2) n -OCH3, -(CH2)3-CO-NH-CH2CH2-(OCH2CH2) n -OH, -(CH2)3-CO-NH-CH2CH2-(OCH2CH2) n -OCH3, -(CH2)4-CO-NH-CH2CH2-(OCH2CH2) n -OH, -(CH2)4-CO-NH-CH2CH2-(OCH2CH2) n -OCH3, -(CH2)5-CO-NH-CH2CH2-(OCH2CH2) n -OH, -(CH2)5-CO-NH-CH2CH2-(OCH2CH2) n -OCH3; or R1 and R2 are each independently: -CH2CH2-N + (CH3)3, -(CH2)3-N + (CH3)3, -(CH2)4-N + (CH3)3, -(CH2)5-N + (CH3)3, -(CH2)6-N + (CH3)3, -(CH2) 12 -N + (CH3)3, -CH2CH2-N + (CH3)2(C4H9), -CH2CH2-N + (CH3)2(C6H 13 )、-(CH2)3-N + (CH3)2(C4H9), -(CH2)3-N + (CH3)2(C6H 13 )、-(CH2)4-N + (CH3)2(C6H 13 )、-(CH2)5-N + (CH3)2(C6H 13 )、-(CH2)6-N + (CH3)2(C6H 13 ) or each of R1 and R2 independently is: -CH2CO-OCH2CH2-N + (CH3)3, -CH2CH2CO-OCH2CH2-N + (CH3)3, -CH2(CH2)2CO-OCH2CH2-N + (CH3)3, -CH2(CH2)6CO-OCH2CH2-N + (CH3)3, -CH2CO-O-(CH3)3-N + (CH3)3, -CH2(CH2)2CO-O-(CH3)3-N + (CH3)3, -CH2COOCH2CH2-N + (CH3)2(C6H 13 ) Or R1 and R2 are each independently: -CH2CONH-CH2CH2-N + (CH3)3, -CH2CH2CONH-CH2CH2-N + (CH3)3, -CH2(CH2)4CONH-CH2CH2-N + (CH3)3, -CH2CONH-(CH2)3-N + (CH3)3, -CH2CH2CONH-(CH2)3-N + (CH3)3, -CH2(CH2)4CONH-(CH2)3-N + (CH3)3, -CH2CONH-(CH2)4-N + (CH3)3, -CH2CH2CONH-(CH2)4-N + (CH3)3, -CH2(CH2)4CONH-(CH2)4-N + (CH3)3, -CH2CONH-(CH2)5-N + (CH3)3, -CH2CH2CONH-(CH2)5-N + (CH3)3, -CH2(CH2)4CONH-(CH2)5-N + (CH3)3, -CH2CONH-(CH2)6-N + (CH3)3, -CH2CH2CONH-(CH2)6-N + (CH3)3, -CH2(CH2)4CONH-(CH2)6-N + (CH3)3, -CH2CONH-CH2CH2-N + (CH3)2(C6H 13 ); Or R1 and R2 are each independently: Or R1 and R2 are each independently: Or R1 and R2 are each independently: Or R1 and R2 are each independently: Or R1 and R2 are each independently: In the above, n is a positive integer between 0 and 50.

2. The application according to claim 1, characterized in that The derivatives represented by I-a to I-d have keto-enol tautomers; among them, formula I-a and formula I-a' are the keto-enol tautomers at the 9th and 10th positions in the structural formula; formula I-b and formula I-b' are the keto-enol tautomers at the 3rd and 4th positions in the structural formula; formula I-c and formula I-c' are the keto-enol tautomers at the 9th and 10th positions in the structural formula; formula I-d and formula I-d' are the keto-enol tautomers at the 3rd and 4th positions in the structural formula, specifically as follows: Among them, R1, R2 and R3 independently have the definitions described in claim 1.

3. Use of a derivative with simultaneous substitution at the ortho and 2-positions of hypocrellin as shown below in the preparation of a photodynamic anti-tumor drug; wherein, The tumor is esophageal cancer, gastric cancer, lung cancer, liver cancer, cholangiocarcinoma, colon cancer, brain cancer, head and neck cancer, tongue cancer, nasal cancer, oral cancer, glioblastoma, basal cell carcinoma, squamous cell carcinoma of the skin, melanoma, cutaneous T-cell lymphoma, prostate cancer, bladder cancer; in the following, n is a positive integer between 0 and 50:

4. The application according to any one of claims 1 to 3, characterized in that The drug is a photosensitizing drug, a fluorescence-mediated drug.

5. Use of the following compound, its isotope-labeled substance, pharmaceutically acceptable salt in the preparation of a fluorescence-mediated drug for guiding tumor margin resection; the tumor is esophageal cancer, gastric cancer, lung cancer, liver cancer, cholangiocarcinoma, colon cancer, brain cancer, head and neck cancer, tongue cancer, nasal cancer, oral cancer, glioblastoma, basal cell carcinoma, squamous cell carcinoma of the skin, melanoma, cutaneous T-cell lymphoma, prostate cancer, bladder cancer; n is selected from integers between 2 and 50.

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