Mitochondria-targeted photosensitizer, preparation method and use thereof, and kit containing the same

Abstract

The present invention relates to a mitochondrion-targeted photosensitizer or a pharmaceutically acceptable salt thereof, wherein the mitochondrion-targeted photosensitizer has a structure of the following general formula (I):, where the groups R each independently have the definitions in the specification. The present invention also relates to the preparation and use of the mitochondrion-targeted photosensitizer and a kit containing the photosensitizer.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical chemistry, and particularly relates to a mitochondrion-targeted photosensitizer, a preparation method and use thereof, and a kit containing the same. Background Art

[0002] Targeted drugs refer to drugs or their preparations endued with targeting ability, aiming to enable the drug or its carrier to target specific diseased sites and accumulate or release active ingredients at the target site. Targeted preparations can make the drug form a relatively high concentration at the target local area, so as to improve the drug efficacy while inhibiting toxic and side effects and reducing the harm to normal tissues and cells. According to different targeting mechanisms, drug targeting can be divided into passive targeting, active targeting and physical targeting. Among them, physical targeting uses physical signals such as light, heat, magnetic field, electric field, ultrasonic wave, etc. to artificially regulate the distribution and drug release characteristics of the drug in the body and achieve targeting of diseased sites. Photosensitizers are used as targeted drugs that play a role in physical targeting in photodynamic therapy (PDT) to effectively treat various diseases. Photodynamic therapy is a new type of therapy for treating diseases. The principle is a photochemical reaction, and its basic elements are oxygen, photosensitizer and visible light. First, the target site selectively takes up the photosensitizer, and then after local irradiation with light of an appropriate wavelength, the photosensitizer is activated to produce a photosensitizing effect.

[0003] Currently, sodium hyaloporphyrin for injection clinically applied is a type of new photosensitizer, which is a single effective chemical component separated from PHOTOFRIN (sodium porfimer). Preclinical studies on esophageal cancer show that the mechanism of action of sodium hyaloporphyrin for injection is the same as that of PHOTOFRIN, but to achieve the same intensity of anti-tumor activity, its dosage is only 10% of that of PHOTOFRIN, and the phototoxic reaction is low, having obvious advantages in terms of effectiveness, safety and quality control. However, in previous studies, it has been found that sodium hyaloporphyrin can selectively accumulate in tumor tissues, but the drug will be distributed throughout the body and there will also be a certain degree of accumulation in normal tissues. Patients need to avoid light for about 7 days after treatment. To reduce the phototoxicity of sodium hyaloporphyrin, it is still necessary to improve the targeting accuracy of sodium hyaloporphyrin. This study designed a mitochondrion-targeted photosensitizer to enhance the PDT effect in anoxic environments, thereby obtaining a photosensitizer with high activity and low toxicity. Summary of the Invention

[0004] In view of the state of the prior art, the present invention aims to provide a photosensitizer that can significantly improve the targeting of tumors, thereby improving the drug efficacy and having lower toxicity, and its preparation method has a high yield and a simple synthesis process. The mitochondrion photosensitizer of the present invention can be excited under light of different wavelengths and doses, and has high biosafety, and is a mitochondrion photosensitizer with potential clinical application value and can be produced on a large scale.

[0005] On the one hand, the present invention provides a mitochondrion-targeted photosensitizer or a pharmaceutically acceptable salt thereof, wherein the mitochondrion-targeted photosensitizer has the structure of the following general formula (I):

[0006] ,

[0007] wherein each group R independently represents C 3-9 heterocyclic group -O- or C 3-9 heterocyclic group -C 1-10 alkylene -O-,

[0008] wherein the C 3-9 heterocyclic group is unsubstituted or substituted with one or more identical or different groups selected from the following: cyano, halogen, alkyl, alkoxy, alkylthio, cycloalkyl, alkylcarbonyl, dialkylamino, haloalkyl or haloalkoxy;

[0009] the C 1-10 alkylene is straight-chain or branched-chain, unsubstituted or substituted with one or more identical or different groups selected from the following: cyano, halogen, alkyl, alkoxy, alkylthio, alkylcarbonyl, dialkylamino, haloalkyl or haloalkoxy, and the alkylene may optionally be interrupted by one or more heteroatoms selected from S, N and O or interrupted by one or more 3- to 6-membered nitrogen-containing heterocyclic groups;

[0010] wherein the 3- to 6-membered nitrogen-containing heterocycle is unsubstituted or substituted with one or more identical or different groups selected from the following: cyano, halogen, alkyl, alkoxy, alkylthio, cycloalkyl, haloalkyl or haloalkoxy.

[0011] On the other hand, the present invention provides a method for preparing the mitochondrion-targeted photosensitizer of formula (I), which comprises the following steps: reacting hematoporphyrin monomethyl ether with a G-OH compound in the presence of a catalyst and a condensing agent in the presence of a solvent to obtain a product;

[0012] The G-O part in the G-OH compound has the same definition as the above R group.

[0013] On the other hand, the present invention provides the use of a mitochondrion-targeted photosensitizer or a pharmaceutically acceptable salt thereof for the preparation of a medicament for treating the following diseases: cancer, which includes reproductive organ cancers (such as cervical cancer, ovarian cancer, endometrial cancer, vaginal cancer, vulvar cancer, uterine sarcoma, prostate cancer, and testicular cancer), leukemia, mast cell tumor, breast cancer, kidney cancer, respiratory tract cancers (such as lung cancer, especially small cell and non-small cell lung cancer, as well as bronchial cancer and pleuropulmonary blastoma), brain cancer, digestive tract cancers (such as esophageal cancer, anal cancer, colon cancer, colorectal cancer, gallbladder cancer, gastric cancer, pancreatic cancer, rectal cancer, small intestine cancer, and salivary gland cancer), urinary tract cancer, liver cancer, eye cancer (such as intraocular melanoma and retinoblastoma), skin cancer, head and neck cancers (such as thyroid cancer, parathyroid cancer, laryngeal cancer, hypopharyngeal cancer, nasopharyngeal cancer, oropharyngeal cancer, lip cancer, and oral cancer, as well as squamous cell carcinoma), multiple myeloma, sarcoma, or lymphoma; and pre-cancerous lesions of the above-mentioned diseases.

[0014] In yet another aspect, the present invention provides a method for treating the following diseases, the method comprising administering to a subject in need thereof a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof; wherein the diseases are selected from cancer, which includes reproductive organ cancers (such as cervical cancer, ovarian cancer, endometrial cancer, vaginal cancer, vulvar cancer, uterine sarcoma, prostate cancer, and testicular cancer), leukemia, mast cell tumor, breast cancer, kidney cancer, respiratory tract cancers (such as lung cancer, especially small cell and non-small cell lung cancer, as well as bronchial cancer and pleuropulmonary blastoma), brain cancer, digestive tract cancers (such as esophageal cancer, anal cancer, colon cancer, colorectal cancer, gallbladder cancer, gastric cancer, pancreatic cancer, rectal cancer, small intestine cancer, and salivary gland cancer), urinary tract cancer, liver cancer, eye cancer (such as intraocular melanoma and retinoblastoma), skin cancer, head and neck cancers (such as thyroid cancer, parathyroid cancer, laryngeal cancer, hypopharyngeal cancer, nasopharyngeal cancer, oropharyngeal cancer, lip cancer, and oral cancer, as well as squamous cell carcinoma), multiple myeloma, sarcoma, or lymphoma; and pre-cancerous lesions of the above-mentioned diseases.

[0015] In yet another aspect, the present invention provides a kit, which comprises a therapeutically effective amount of a mitochondrion-targeted photosensitizer of formula (I) of the present invention or a pharmaceutically acceptable salt thereof; and instructions for using the same in photodynamic therapy.

[0016] Surprisingly, the inventors found that the compound of formula (I) of the present invention is a photosensitizer that can significantly improve the targeting of tumors, thereby improving the drug efficacy and having lower toxicity. Its preparation method has a high yield and a simple synthesis process, and can be used as a highly efficient photosensitizer with potential clinical application value and scalable production. Description of the Drawings

[0017] Figure 1 It is a scatter plot for fluorescence imaging and co-localization analysis of DVDMS and MTDR.

[0018] Figure 2Scatter plot of fluorescence imaging and co-localization analysis of compound X-1 and MTDR.

[0019] Figure 3 Shows the body weight changes of A549 tumor-bearing mice after administration of drugs.

[0020] Figure 4 Shows the tumor volume changes of A549 tumor-bearing mice after administration of drugs.

[0021] Figure 5 Shows the tumor weights of A549 tumor-bearing mice at each experimental endpoint. Detailed implementation manners

[0022] In the following, the present invention will be described in more detail.

[0023] Unless otherwise specified, the terms "compound of formula (I) of the present invention", "compound of formula (I)", "compound of the present invention" and "mitochondria-targeted photosensitizer of the present invention (hereinafter referred to as photosensitizer)" are used synonymously.

[0024] As used herein, the term "comprising" and its synonyms "including" and "containing" mean "including but not limited to", and are not intended to exclude, for example, other additives, components, integers or steps.

[0025] Unless otherwise defined differently, the names of chemical groups should generally be understood such that the connection to the skeleton or the rest of the molecule is through the structural element of the relevant chemical group last mentioned, i.e., in the case of a heterocyclic alkylene group, through the carbon atoms of the alkylene group.

[0026] As used herein, the terms "optionally", "optional" or "optionally" mean that the events, circumstances or substances described later may occur or exist, may also not occur or exist, and such descriptions include the cases where the events, circumstances or substances occur or exist and the cases where the events, circumstances or substances do not occur or exist.

[0027] Unless otherwise specified, the following definitions apply to the groups or substituents used throughout the specification and claims. Within the scope of the present invention, the meanings of all groups that appear repeatedly are independent of each other.

[0028] As used herein, the term "cyano" refers to a functional group formed by a carbon atom and a nitrogen atom connected by a triple bond.

[0029] As used herein, the term "halogen" means, for example, fluorine, chlorine, bromine or iodine. If the term is used for a group, "halogen" means, for example, a fluorine, chlorine, bromine or iodine atom.

[0030] As used herein, the term "alkyl" refers to a saturated straight-chain or branched-chain hydrocarbon group having a specified number of carbon atoms in each case, for example (C1-C10 )-alkyl, (C1-C6)-alkyl, and (C1-C4)-alkyl, examples include but are not limited to methyl, ethyl, propyl, 1-methylethyl, butyl, 1-methylpropyl, 2-methylpropyl, 1,1-dimethylethyl, pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, 2,2-dimethylpropyl, 1-ethylpropyl, hexyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 3,3-dimethylbutyl, 1-ethylbutyl, 2-ethylbutyl, 1,1,2-trimethylpropyl, 1,2,2-trimethylpropyl, 1-ethyl-1-methylpropyl, 1-ethyl-2-methylpropyl, n-heptyl, 1-methylhexyl, 2-methylhexyl, 1,1-dimethylpentyl, 1-ethylpentyl, 2-ethylpentyl, 1-propylbutyl, n-octyl, 1-methylheptyl, 2-ethylhexyl, 1,3-dimethylhexyl, and 1-ethyl-2-methylpentyl.

[0031] As used herein, the term "haloalkyl" means that one or more hydrogen atoms in the alkyl as defined above are replaced by one or more identical or different halogen atoms, for example, (C1-C 10 )-haloalkyl, (C1-C6)-haloalkyl, and (C1-C4)-haloalkyl, examples include but are not limited to chloromethyl, bromomethyl, dichloromethyl, trichloromethyl, fluoromethyl, difluoromethyl, trifluoromethyl, chlorofluoromethyl, dichlorofluoromethyl, chlorodifluoromethyl, 1-chloroethyl, 1-bromoethyl, 1-fluoroethyl, 2-fluoroethyl, 2,2-difluoroethyl, 2,2,2-trifluoroethyl, 2-chloro-2-fluoroethyl, 2-chloro-2,2-difluoroethyl, 2,2-dichloro-2-fluoroethyl, 2,2,2-trichloroethyl, pentafluoroethyl, and 1,1,1-trifluoropropan-2-yl. Fluoromethyl, difluoromethyl, trifluoromethyl, 1-fluoroethyl, 2-fluoroethyl, 2,2-difluoroethyl, 2,2,2-trifluoroethyl, pentafluoroethyl, and 1,1,1-trifluoropropan-2-yl are preferred.

[0032] According to the present invention, "cycloalkyl" - by itself or as part of a chemical group - represents a monocyclic hydrocarbon, bicyclic hydrocarbon, or tricyclic hydrocarbon preferably having 3 to 10 carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, bicyclo[2.2.1]heptyl, bicyclo[2.2.2]octyl, or adamantyl. Cycloalkyls having 3, 4, 5, 6, or 7 carbon atoms are also preferred, such as especially cyclopropyl or cyclobutyl. The cycloalkyls of the present invention can be substituted by one or more identical or different groups.

[0033] According to the present invention, "alkoxy" represents a straight-chain or branched-chain alkyl-O- having preferably 1 to 6 carbon atoms, such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, and tert-butoxy. An alkoxy having 1 to 4 carbon atoms is also preferred. The alkoxy of the present invention may be substituted with one or more identical or different groups.

[0034] As used herein, the term "haloalkoxy" means that one or more hydrogen atoms of the alkyl moiety in the alkoxy as defined above are replaced by one or more identical or different halogen atoms, such as (C1-C 10 )-haloalkoxy, (C1-C6)-haloalkoxy, and (C1-C4)-haloalkoxy. Examples include but are not limited to chloromethoxy, bromomethoxy, dichloromethoxy, fluoromethoxy, difluoromethoxy, trifluoromethoxy, chlorofluoromethoxy, 1-chloroethoxy, 1-bromoethoxy, 1-fluoroethoxy, 2-fluoroethoxy, 2,2-difluoroethoxy, 2-chloro-2-fluoroethoxy, and pentafluoroethoxy. Fluoromethoxy, difluoromethoxy, trifluoromethoxy, 1-fluoroethoxy, 2-fluoroethoxy, 2,2-difluoroethoxy, and pentafluoroethoxy are preferred.

[0035] According to the present invention, "alkylthio" represents a straight-chain or branched-chain alkyl-S- having preferably 1 to 6 carbon atoms, such as methylthio, ethylthio, n-propylthio, isopropylthio, n-butylthio, isobutylthio, sec-butylthio, and tert-butylthio. An alkylthio having 1 to 4 carbon atoms is also preferred. The alkylthio of the present invention may be substituted with one or more identical or different groups.

[0036] According to the present invention, "alkylcarbonyl" represents a straight-chain or branched-chain alkyl-C(=O)- having preferably 2 to 7 carbon atoms, such as methylcarbonyl, ethylcarbonyl, n-propylcarbonyl, isopropylcarbonyl, sec-butylcarbonyl, and tert-butylcarbonyl. An alkylcarbonyl having 1 to 4 carbon atoms is also preferred. The alkylcarbonyl of the present invention may be substituted with one or more identical or different groups.

[0037] According to the present invention, "heterocyclic group" represents a carbocyclic system having at least one ring, in which at least one carbon atom is replaced by a heteroatom, preferably a heteroatom selected from N, O, S, P, B, Si, Se, and which is saturated, unsaturated, or heteroaromatic, and may be unsubstituted or substituted, where the bonding site is on a ring atom. Unless otherwise differently defined, the heterocycle contains 3 to 9 ring atoms (i.e., C 3-9 heterocyclic group), especially 3 to 6 ring atoms (i.e., C 3-6heterocyclic group), and one or more, preferably 1 to 4, especially 1, 2 or 3 heteroatoms in the heterocycle are preferably selected from N, O and S, provided that two oxygen atoms should not be directly adjacent. The heterocycle usually contains no more than 4 nitrogen atoms and / or no more than 2 oxygen atoms and / or no more than 2 sulfur atoms. In the case of an optionally substituted heterocyclic group, the present invention also includes polycyclic ring systems, such as 8-azabicyclo[3.2.1]octyl, 1-azabicyclo[2.2.1]heptyl, 1-oxa-5-azaspiro[2.3]hexyl or 2,3-dihydro-1 H -indole.

[0038] The heterocyclic groups of the present invention are, for example: pyrrolyl, pyrazolyl, imidazolyl, triazolyl, tetrazolyl, isoxazolyl, oxazolyl, oxadiazolyl, oxatriazolyl, furazanyl, dioxazolyl, isothiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, piperidinyl, triazinyl, tetrazinyl, oxazinyl, isoxazinyl, piperazinyl, morpholinyl, thiomorpholinyl, dihydropyranyl, tetrahydropyranyl, dioxanyl, pyrrolinyl, pyrrolidinyl, imidazolinyl, imidazolidinyl, thiazolidinyl, oxazolidinyl, dioxolanyl, dioxoleneyl, pyrazolidinyl, tetrahydrofuranyl, dihydrofuranyl, oxetanyl, oxiranyl, azetidinyl, aziridinyl, oxazetidinyl, oxaziridinyl, oxazepanyl, oxazepinyl, azepanyl, oxopyrrolidinyl, dioxopyrrolidinyl, oxomorpholinyl, oxopiperazinyl and oxepanyl.

[0039] According to the present invention, "C 3-9 heterocyclic group" and "C 3-9 heterocyclic group C 1-10 alkylene" in "heterocyclic group" has the meaning represented above; "C 1-10 alkylene" in "alkylene" represents methylene, ethylene, propylene, butylene, pentylene, hexylene, heptylene, octylene, nonylene and decylene, and their isomers.

[0040] According to the present invention, "3- to 6-membered nitrogen-containing heterocyclic group" is selected from the following groups: pyrrolyl, pyrazolyl, imidazolyl, triazolyl, tetrazolyl, isoxazolyl, oxazolyl, oxadiazolyl, oxatriazolyl, furazanyl, dioxazolyl, isothiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, piperidinyl, triazinyl, tetrazinyl, oxazine, isoxazinyl or morpholinyl; preferably pyrazolyl, imidazolyl, triazolyl (such as sym-triazole, 1,2,4-triazole, 1,2,5-triazole, 1,3,4-triazole), oxazolyl, oxadiazolyl or furazanyl.

[0041] Compounds of formula (I) recited anywhere herein also cover any diastereoisomers or enantiomers and E / Z isomers present of the compounds of formula (I), as well as their salts.

[0042] Depending on the nature of the substituents, the compounds of formula (I) described at any position herein may also be in the form of stereoisomers, i.e., geometric and / or optical isomers or mixtures of isomers of different compositions. The present invention provides pure stereoisomers and any desired mixtures of these isomers, although only the compounds of formula (I) are generally discussed herein.

[0043] If appropriate, the compounds of formula (I) may exist in various polymorphic forms or as mixtures of various polymorphic forms. Both pure polymorphs and mixtures of polymorphs are provided by the present invention and can be used according to the present invention.

[0044] Compounds obtained by combinations that are contrary to the laws of nature and that would thus be excluded by a person skilled in the art based on his / her expertise are not covered herein. For example, ring structures having three or more adjacent oxygen atoms are excluded.

[0045] According to one aspect of the present invention, the present invention provides a mitochondrion-targeting photosensitizer or a pharmaceutically acceptable salt thereof, wherein the mitochondrion-targeting photosensitizer has the structure of the following general formula (I):

[0046] ,

[0047] wherein the groups R each independently represent C 3-9 heterocyclic group -O- or C 3-9 heterocyclic group C 1-10 alkylene -O-,

[0048] wherein said C 3-9 heterocyclic group is unsubstituted or substituted by one or more identical or different groups selected from: cyano, halogen, alkyl, alkoxy, alkylthio, cycloalkyl, alkylcarbonyl, dialkylamino, haloalkyl or haloalkoxy;

[0049] said C 1-10 alkylene is straight-chain or branched-chain, unsubstituted or substituted by one or more identical or different groups selected from: cyano, halogen, alkyl, alkoxy, alkylthio, alkylcarbonyl, dialkylamino, haloalkyl or haloalkoxy, and said alkylene may optionally be interrupted by one or more heteroatoms selected from S, N and O or interrupted by one or more 3- to 6-membered nitrogen-containing heterocyclic groups;

[0050] wherein said 3- to 6-membered nitrogen-containing heterocycle is unsubstituted or substituted by one or more identical or different groups selected from: cyano, halogen, alkyl, alkoxy, alkylthio, cycloalkyl, haloalkyl or haloalkoxy.

[0051] The pharmaceutically acceptable salts of the compounds of formula (I) of the present invention refer to those salts that are considered safe and suitable for use in pharmaceutical formulations in the pharmaceutical field. These salts are usually formed by reacting the compounds of formula (I) with acids or bases, with the aim of improving the solubility, stability, and bioavailability of the drugs.

[0052] The pharmaceutically acceptable salts of the compounds of the present invention are, for example, the products obtained by reacting the compounds of formula (I) with acids. Preferably, the acids include, but are not limited to, hydrofluoric acid, hydrochloric acid, hydrobromic acid, phosphoric acid, acetic acid, oxalic acid, sulfuric acid, methanesulfonic acid, salicylic acid, trifluoroacetic acid, trifluoromethanesulfonic acid, naphthalenesulfonic acid, maleic acid, fumaric acid, citric acid, tartaric acid, succinic acid, malic acid, or glutamic acid. The non-toxic pharmaceutical base addition salts of the compounds of formula (I) of the present invention include salts of bases such as sodium, potassium, calcium, ammonium, etc. Those skilled in the art will recognize a variety of non-toxic pharmaceutically acceptable addition salts.

[0053] In a preferred embodiment of the present invention, in formula (I), each group R has the same definition.

[0054] In a preferred embodiment of the present invention, in formula (I), the C 3-9 The heterocyclic group is selected from the following groups: pyrrolyl, pyrazolyl, imidazolyl, triazolyl, tetrazolyl, isoxazolyl, oxazolyl, oxadiazolyl, oxatriazolyl, furazanyl, dioxazolyl, isothiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, piperidinyl, triazinyl, tetrazinyl, oxazinyl, isoxazinyl, piperazinyl, morpholinyl, thiomorpholinyl, dihydropyranyl, tetrahydropyranyl, dioxanyl, pyrrolinyl, pyrrolidinyl, imidazolinyl, imidazolidinyl, thiazolidinyl, oxazolidinyl, dioxolanyl, dioxoleneyl, pyrazolidinyl, tetrahydrofuranyl, dihydrofuranyl, oxetanyl, oxiranyl, azetidinyl, aziridinyl, oxazetidinyl, oxaziridinyl, oxazepanyl, oxazepinyl, azepanyl, oxopyrrolidinyl, dioxopyrrolidinyl, oxomorpholinyl, oxopiperazinyl, and oxepanyl.

[0055] In a preferred embodiment of the present invention, in formula (I), the C 1-10 The alkyl group is straight-chain and unsubstituted.

[0056] In a preferred embodiment of the present invention, in formula (I), the C 3-9The heterocyclic group contains at least one nitrogen atom and is selected from the following groups: pyrrolyl, pyrazolyl, imidazolyl, triazolyl, tetrazolyl, isoxazolyl, oxazolyl, oxadiazolyl, oxatriazolyl, furazanyl, dioxazolyl, isothiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, tetrazinyl, oxazine, isoxazinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, pyrrolinyl, pyrrolidinyl, imidazolinyl, imidazolidinyl, thiazolidinyl, oxazolidinyl, pyrazolidinyl, azetidinyl, aziridinyl, oxazetidinyl, oxaziridinyl, oxazepanyl, oxazepinyl, azepanyl, oxopyrrolidinyl, dioxopyrrolidinyl, oxomorpholinyl, and oxopiperazinyl.

[0057] In a preferred embodiment of the present invention, in formula (I), the group R is C 3-6 Heterocyclic group C 1-6 Alkylene - O -, where C 3-6 The heterocyclic group is selected from the following groups: pyrrolyl, pyrazolyl, imidazolyl, triazolyl, tetrazolyl, isoxazolyl, oxazolyl, oxadiazolyl, oxatriazolyl, furazanyl, dioxazolyl, isothiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, tetrazinyl, oxazine, isoxazinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, pyrrolinyl, pyrrolidinyl, imidazolinyl, imidazolidinyl, thiazolidinyl, oxazolidinyl, pyrazolidinyl, azetidinyl, aziridinyl, oxazetidinyl, oxaziridinyl, oxazepanyl, oxopyrrolidinyl, dioxopyrrolidinyl, oxomorpholinyl, and oxopiperazinyl, preferably piperazinyl, morpholinyl, thiomorpholinyl, pyrrolinyl, pyrrolidinyl, imidazolinyl, imidazolidinyl, thiazolidinyl, oxazolidinyl, pyrazolidinyl; C 1-6 The alkylene is selected from methylene, ethylene, propylene, n - butylene, n - pentylene or n - hexylene.

[0058] In a preferred embodiment of the present invention, in formula (I), the group R is C 3-6 Heterocyclic group C 1-4 Alkylene - O -, where C 3-6 The heterocyclic group is selected from the following groups: pyrazolyl, imidazolyl, isoxazolyl, oxazolyl, oxadiazolyl, furazanyl, dioxazolyl, isothiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, oxazine, isoxazinyl, piperazinyl, morpholinyl, thiomorpholinyl, imidazolinyl, imidazolidinyl, thiazolidinyl, oxazolidinyl, pyrazolidinyl, azetidinyl, oxazetidinyl, oxaziridinyl, oxazepanyl, oxomorpholinyl and oxopiperazinyl, preferably morpholinyl, oxazine, isoxazinyl, oxazetidinyl, oxaziridinyl and oxazepanyl; C 1-4The alkylene group is selected from methylene, ethylene, propylene or n-butylene. In a preferred embodiment of the present invention, in formula (I), the group R is, for example, morpholinyl-N-ethyl.

[0059] In a preferred embodiment of the present invention, the 3- to 6-membered nitrogen-containing heterocyclic group is selected from the following groups: pyrrolyl, pyrazolyl, imidazolyl, triazolyl, tetrazolyl, isoxazolyl, oxazolyl, oxadiazolyl, oxatriazolyl, furazanyl, dioxazolyl, isothiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, piperidinyl, triazinyl, tetrazinyl, oxazine, isoxazinyl or morpholinyl; preferably pyrazolyl, imidazolyl, triazolyl (such as vic-triazole, 1,2,4-triazole, 1,2,5-triazole, 1,3,4-triazole), oxazolyl, oxadiazolyl or furazanyl.

[0060] In a preferred embodiment of the present invention, in formula (I), the mitochondrial-targeted photosensitizer or a pharmaceutically acceptable salt thereof is used for treating the following diseases: cancer, which includes reproductive organ cancers (such as cervical cancer, ovarian cancer, endometrial cancer, vaginal cancer, vulvar cancer, uterine sarcoma, prostate cancer and testicular cancer), leukemia, mast cell tumor, breast cancer, kidney cancer, respiratory tract cancers (such as lung cancer, especially small cell and non-small cell lung cancer, as well as bronchial cancer and pleuropulmonary blastoma), brain cancer, digestive tract cancers (such as esophageal cancer, anal cancer, colon cancer, colorectal cancer, gallbladder cancer, gastric cancer, pancreatic cancer, rectal cancer, small intestine cancer and salivary gland cancer), urinary tract cancer, liver cancer, eye cancer (such as intraocular melanoma and retinoblastoma), skin cancer, head and neck cancers (such as thyroid cancer, parathyroid cancer, laryngeal cancer, hypopharyngeal cancer, nasopharyngeal cancer, oropharyngeal cancer, lip cancer and oral cancer, as well as squamous cell carcinoma), multiple myeloma, sarcoma or lymphoma; and pre-cancerous lesions of the above-mentioned diseases.

[0061] As used herein, "pre-cancerous lesion" refers to abnormal or excessive cell proliferation with a high potential for canceration.

[0062] The definitions of the groups listed above in general terms or within the preferred ranges can be combined with each other as needed, that is, including combinations between the given preferred ranges.

[0063] Very particularly preferred are the compounds of formula (I) of the present invention,

[0064]

[0065] wherein the R group is morpholinoethyl-O-.

[0066] According to another aspect of the present invention, the present invention also provides a method for preparing the compound of formula (I), which comprises the following steps:

[0067] In the presence of a catalyst and a condensing agent, hematoporphyrin acid is reacted with a G-OH compound in the presence of a solvent to obtain a product;

[0068] The G-O moiety in the G-OH compound has the same definition as the above-mentioned R group.

[0069] In the method of the present invention, the catalyst used is an organic base catalyst, which is selected from N,N-diisopropylethylamine (DIPEA), N,N-dimethylcyclohexylamine, N,N-dimethylbenzylamine, N-methylmorpholine, N-ethylmorpholine, N-methylimidazole (NMI), 1,8-diazabicyclo[5.4.0]undec-7-ene, triethylamine, pyridine, N,N-dimethylaminopyridine, 2,6-dimethylpyridine or a mixture thereof, more preferably N-methylimidazole; the organic solvent used is selected from N,N-dimethylformamide (DMF), N,N-dimethylacetamide, formamide, dimethyl sulfoxide, acetone, pyridine or a mixture thereof, more preferably N,N-dimethylformamide.

[0070] In the method of the present invention, the condensing agent used is a ureonium ion type condensing agent, which is selected from O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TATU), O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU), O-(1,2-dihydro-2-oxo-pyridin-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TPTU); a carbodiimide type condensing agent, such as 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), 1,3-dicyclohexylcarbodiimide (DCC); or a carbonylimidazole type condensing agent, such as N,N-carbonyldiimidazole (CDI); or a mixture thereof, preferably selected from HATU, EDCI, CDI or a mixture thereof.

[0071] In the method of the present invention, the molar ratio of the catalyst to the condensing agent is (0.7-2.0):1, preferably (0.8-1.8):1, more preferably (0.9-1.5):1.

[0072] The method for preparing the compound of formula (I) in the present invention uses a combination of a specific organic base catalyst and a specific condensing agent, especially a specific ratio of N-methylimidazole and HATU, and can obtain a target compound with high purity in good yield through simple post-treatment. Too low or too high dosage of the catalyst or the condensing agent will have an adverse effect on the target product, such as an increase in the proportion of by-products and a low yield of the target product.

[0073] In the method of the present invention, the molar ratio of the tin ethyl etiopurpurin to the R-OH compound is 1:(4.0 - 10.0), preferably 1:(5.0 - 8.0), more preferably 1:(6.0 - 7.0).

[0074] The molar ratio of the tin ethyl etiopurpurin to the catalyst is 1:(4.0 - 12.0), preferably 1:(6.0 - 10.0), more preferably 1:(8.0 - 9.0).

[0075] In the method of the present invention, the condensation reaction time is 0.5 - 24 hours, preferably 1 - 20 hours, more preferably 1 - 18 hours.

[0076] On the other hand, the present invention provides the use of the mitochondrial-targeted photosensitizer or a pharmaceutically acceptable salt thereof for the preparation of a medicament for treating the following diseases: cancer, which includes reproductive organ cancers (such as cervical cancer, ovarian cancer, endometrial cancer, vaginal cancer, vulvar cancer, uterine sarcoma, prostate cancer, and testicular cancer), leukemia, mast cell tumor, breast cancer, kidney cancer, respiratory tract cancers (such as lung cancer, especially small cell and non-small cell lung cancer, as well as bronchial cancer and pleuropulmonary blastoma), brain cancer, digestive tract cancers (such as esophageal cancer, anal cancer, colon cancer, colorectal cancer, gallbladder cancer, gastric cancer, pancreatic cancer, rectal cancer, small intestine cancer, and salivary gland cancer), urinary tract cancer, liver cancer, eye cancer (such as intraocular melanoma and retinoblastoma), skin cancer, head and neck cancers (such as thyroid cancer, parathyroid cancer, laryngeal cancer, hypopharyngeal cancer, nasopharyngeal cancer, oropharyngeal cancer, lip cancer, and oral cancer, as well as squamous cell carcinoma), multiple myeloma, sarcoma, or lymphoma; and hyperplastic diseases of the above-mentioned diseases, such as precancerous lesions.

[0077] On the other hand, the present invention provides a method for treating the following diseases, the method comprising administering to a subject in need a therapeutically effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof as described above; wherein the diseases to be treated are selected from: cancer, which includes reproductive organ cancers (such as cervical cancer, ovarian cancer, endometrial cancer, vaginal cancer, vulvar cancer, uterine sarcoma, prostate cancer, and testicular cancer), leukemia, mast cell tumor, breast cancer, kidney cancer, respiratory tract cancers (such as lung cancer, especially small cell and non-small cell lung cancer, as well as bronchial cancer and pleuropulmonary blastoma), brain cancer, digestive tract cancers (such as esophageal cancer, anal cancer, colon cancer, colorectal cancer, gallbladder cancer, gastric cancer, pancreatic cancer, rectal cancer, small intestine cancer, and salivary gland cancer), urinary tract cancer, liver cancer, eye cancer (such as intraocular melanoma and retinoblastoma), skin cancer, head and neck cancers (such as thyroid cancer, parathyroid cancer, laryngeal cancer, hypopharyngeal cancer, nasopharyngeal cancer, oropharyngeal cancer, lip cancer, and oral cancer, as well as squamous cell carcinoma), multiple myeloma, sarcoma, or lymphoma; and hyperplastic diseases of the above-mentioned diseases, such as precancerous lesions.

[0078] As used herein, the term "therapeutically effective amount" refers to the amount of an active compound or agent that elicits a biological or pharmaceutical response sought or desired by a researcher, physician, or other clinician in a tissue, system, animal, individual, or human.

[0079] As used herein, the term "treatment" means a method of killing, inhibiting, or slowing the growth or increase in size of an overproliferating cell mass or population, or a tumor or cancerous growth, reducing the number of overproliferating cells, or preventing spread to other anatomical sites, and reducing the size of an overproliferative growth or the number of overproliferating cells. However, it should be understood that "treatment" does not necessarily mean a cure or complete elimination of the overproliferative growth.

[0080] Another aspect of the present invention provides a kit comprising the therapeutically effective amount of the mitochondrially targeted photosensitizer or a pharmaceutically acceptable salt thereof; and instructions for using the same in photodynamic therapy.

[0081] As used herein, the term "kit" means any commercial packaging that contains a container for holding the compound of the present invention or a pharmaceutical formulation containing the same, and also optionally contains separate containers such as separate vials or separate foil packages, for example, for holding a reconstitution dissolution matrix. The container can be any conventional shape or form known in the art and is made of a pharmaceutically acceptable material.

[0082] In a preferred embodiment, the kit provided by the present invention comprises a lyophilized preparation of the compound of the present invention in a therapeutically effective amount for treating the diseases described above; a dissolution matrix (such as water for injection containing cosolvents and other excipients) for reconstituting the lyophilized preparation for administration; and instructions for using the compound of the present invention as a photosensitizer in photodynamic therapy.

[0083] Each component of the kit, such as the compound of the present invention or a pharmaceutically acceptable salt thereof, a pharmaceutical formulation containing the same, a dissolution matrix, and other active ingredients for treating cancer or precancerous lesions, etc., can be packaged in separate containers. Regardless of the number or type of containers, the kit may further include a device for facilitating the administration of the drug to a patient. The device can be a patch, inhaler, syringe, pipette, spoon with measuring units, or any approved medical delivery device.

[0084] The photosensitizer of the present invention can be administered in any of the following ways: oral, buccal, spray inhalation, rectal, nasal, vaginal, topical, parenteral such as subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, intrasternal, or intracranial injection or infusion, or via an implanted reservoir, wherein local, intraperitoneal, or intravenous administration is preferred.

[0085] The photosensitizer of the present invention can be administered in unit dosage forms. The dosage forms can be liquid dosage forms, semi-solid dosage forms, and solid dosage forms. The liquid dosage forms can be true solution types, colloid types, particulate dosage forms, and suspension dosage forms. The semi-solid dosage forms can be ointments, creams, pastes, gels, etc. Other dosage forms can be, for example, tablets, capsules, dropping pills, aerosols, pills, powders, solutions, emulsions, granules, suppositories, freeze-dried powder injections, inclusion compounds, implants, patches, liniments, sustained-release agents.

[0086] For oral preparations, oral tablets and capsules can be prepared by methods well-known in the pharmaceutical art. Tablets can also be coated; oral liquids can be made into suspensions, solutions, emulsions, syrups of water and oil, or can also be made into dry products and supplemented with water or other suitable media before use. Flavoring agents or coloring agents can be added if needed.

[0087] For extra-gastric administration, the liquid dosage form is usually made from the drug of the present invention and a sterilized carrier. The preferred carrier is water. Depending on the selected carrier and drug concentration, the drug can either be dissolved in the carrier or made into a suspension solution. When making an injection solution, the drug is first dissolved in water, filtered and sterilized, and then filled into sealed bottles or ampoules.

[0088] It can also be made into sterile injection preparations, including crystalline powder injections, freeze-dried powder injections, etc.

[0089] When topically applied to the skin and mucosa, the drug of the present invention can be made into appropriate forms of ointments, lotions, gels or pastes, in which the active ingredient is suspended or dissolved in one or more carriers. Alternatively, it can also be made into forms such as microneedles for use with instruments.

[0090] The photosensitizer of the present invention also contains a pharmaceutically acceptable carrier, excipient and / or other adjuvants. When containing a pharmaceutically acceptable carrier, excipient and / or other adjuvants, usually an effective dose of the photosensitizer of the present invention or its pharmaceutically acceptable salt and one or more pharmaceutically acceptable carriers, excipients and / or other adjuvants are combined to form an appropriate dosage form for administration or dosage form. This procedure includes mixing, granulating, compressing, dissolving or freeze-drying the components by suitable methods. The content of the carrier in the drug can be 1 to 98% by weight. For convenience, other adjuvants such as local anesthetics, preservatives, buffers, etc. can be directly dissolved in the carrier.

[0091] The photosensitizer of the present invention can be prepared into injection preparations, topical preparations for external use or oral preparations, etc. for use by methods known to those skilled in the art. The photosensitizer of the present invention is preferably an injection preparation and is administered by intravenous injection.

[0092] The pharmaceutically acceptable carriers, excipients and / or other adjuvants that can be used to prepare the photosensitizer of the present invention or its dosage form for administration are all conventional pharmaceutically acceptable carriers, excipients and / or other adjuvants known to those skilled in the art for this purpose.

[0093] The optimal dosage and interval of administration of the photosensitizer of the present invention are determined by the properties of the compound and external conditions such as the form, route and site of administration, the specific mammal being treated, the wavelength, light power and irradiation time of the light used for treatment, and the type and severity of the disease being treated. This optimal dosage can be determined by conventional techniques. The optimal treatment course, i.e., the daily dosage of the compound of the present invention or the medicament of the present invention within a rated time, can be determined by methods well known in the art.

[0094] The dosage of the photosensitizer of the present invention is 0.01 - 100.0 mg / kg of the subject, such as 0.01 - 50.0 mg / kg of the subject, preferably 0.05 - 10.0 mg / kg of the subject, especially 0.1 - 6.0 mg / kg of the subject, 0.2 - 5.0 mg / kg of the subject; and laser light irradiation is carried out at a wavelength of 300 - 800 nm, preferably 600 nm - 650 nm, especially 630 nm, usually with one irradiation; the light dose is 1 - 300 J, preferably 20 - 200 J, more preferably 50 - 150 J; the light power of the laser used as the light source is usually 50 - 800 mW, preferably 100 - 500 mW; the irradiation time is 10 - 2400 seconds, preferably 60 - 1800 seconds, more preferably 100 - 1500 seconds; the irradiation delay is usually 4 - 48 hours, preferably 10 - 25 hours.

[0095] In the context of the present invention, the term "light power" refers to the actual light power at the light irradiation site, which is measured by a light power meter to determine the actual power at the treatment site.

[0096] In the context of the present invention, the term "light dose" refers to the actual light dose at the light irradiation site, which is obtained by multiplying the light power by the irradiation time.

[0097] In the present invention, unless otherwise clearly stated, the contents and percentages in the context of the present application are based on weight; unless otherwise clearly stated, the method steps of the present invention are carried out at normal temperature and pressure. Unless otherwise clearly stated, the reagents used in the following synthesis examples are commercially available conventional products.

[0098] In the method for preparing the compound of formula (I) of the present invention, in the method for preparing the compound of formula (I) of the present invention, normal-phase silica gel thin-layer chromatography (Thin Layer Chromatography, TLC) is used to monitor the reaction process, and the completion of the reaction is judged by observing the gradual disappearance of the raw material spots. This detection is carried out on a thin-layer chromatograph. The thin-layer chromatograph that can be used in the method of the present invention is a commercially available conventional thin-layer chromatograph, such as the three-purpose ultraviolet analyzer purchased from Shanghai Heqi Glass Instrument Co., Ltd., model: WHF - 204B.

[0099] In the above method for preparing the compound of the present invention, the method may optionally include other post-treatment steps. The post-treatment steps may include conventional purification steps such as adjusting the pH value, crystallization, extraction, filtration, concentration under reduced pressure, and drying. Each of the above steps can be carried out in a conventional manner known to those skilled in the art. If present, extraction is usually carried out using a mixed solution of dichloromethane and methanol, preferably a mixed solution of dichloromethane / methanol = 10:1 (v / v); drying is usually carried out by freeze drying, infrared drying, vacuum drying, etc., preferably freeze drying.

[0100] In a preferred embodiment of the present invention, the method may further include a purification step by a chromatographic column. The purification may be performed by a normal phase silica gel column, the silica gel particle size is 30-100 μm, preferably 40-63 μm, the loading amount is 20-120 g, preferably 40 g, and elution is performed using dichloromethane / methanol (v / v) (elution gradient 100% / 0% to 90% / 10%, gradient elution time 15 min).

[0101] Unless explicitly stated otherwise, all operations were performed at room temperature; reagents used were either commercially available or prepared by methods known to those skilled in the art.

[0102] The compounds of the present invention can be prepared according to the above methods. However, it should be understood that a person skilled in the art, based on his own general knowledge and available publications, can adjust the methods according to the specific circumstances of the respective compounds of the present invention that he wishes to synthesize.

[0103] Detailed synthesis examples of selected compounds of the present invention are given below. However, these examples are merely illustrative and should not be interpreted as limiting the scope of the present invention in any way.

[0104] The NMR peaks reported in the synthetic examples are 1 H NMR spectral data were obtained on a Bruker 400 MHz NMR spectrometer and the signals listed have the meanings given below: s = singlet, d = doublet, dd = doublet of doublets, m = multiplet, br s = broad singlet. The deuterated solvents used in each case are also listed in 1 H NMR spectral data are specified.

[0105] In the present invention, in addition to the above-mentioned NMR peak list form 1 In addition to the H NMR spectral data, the structures of the compounds prepared in the synthetic examples were also characterized by liquid chromatography-mass spectrometry (LC-MS). The LC-MS data were obtained using a Waters instrument (model: SQD2).

[0106] Synthesis Example

[0107] Table 1 Synthetic Reagents

[0108]

[0109] Table 2: Synthetic Equipment

[0110]

[0111] Synthesis of Compound X-1

[0112]

[0113]

[0114] At room temperature (20 °C), hematoporphyrin acid (50 mg, 0.044 mmol, 1.0 equ) was dissolved in 5 mL of DMF. Then, HATU (133 mg, 0.350 mmol, 8.0 equ) and N-methylimidazole (29 mg, 0.350 mmol, 8.0 equ) were added sequentially at 20 °C. After reacting at room temperature for 1 hour, N-(2-hydroxyethyl)morpholine (34 mg, 0.263 mmol, 6.0 equ) was added, and the reaction was carried out at room temperature for 12 hours. The reaction was monitored by normal-phase thin-layer chromatography until the raw materials were completely reacted. The reaction solution was poured into 100 mL of water and extracted three times (50 mL × 3) with a mixed solution of dichloromethane / methanol (10 / 1, V / V). The organic phases were combined, then washed with 200 mL of saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered and concentrated, and purified by a normal-phase silica gel column with a particle size of 40 - 63 μm and a loading of 40 g (dichloromethane / methanol (V / V) from 100% / 0% to 90% / 10%, 15 min) to obtain the target compound X-1 (61 mg, yield 87%).

[0115] LC-MS (m / z): 1597.1 [M+H] +

[0116] 1 H NMR (400 MHz, DMSO- d 6 ): δ 10.36 (s, 2H), 10.25 (s, 2H), 9.90 (s, 2H), 9.29 (br s, 2H), 8.48 (dd, J = 18.0, 11.6 Hz, 2H), 6.44(d, J= 18.0Hz, 2H)), 6.22 - 6.18(m, 4H), 4.46–4.40 (m, 4H), 3.93–3.87 (m, 4H), 3.83 (s, 6H), 3.73(s, 6H), 3.24 - 3.19 (m, 4H), 3.10 - 3.08 (m, 12H), 2.94 - 2.90 (m, 10H), 2.82 - 2.72(m, 4H), 2.34 - 2.30(m, 7H), 2.26 - 2.21(m, 4H), 2.09 - 2.06(m, 4H), 2.03 - 1.95 (m,14H), 1.82 - 1.80(m, 8H), 1.74 (s, 3H), -3.97 (br s, 4H).

[0117] Targeting ability evaluation

[0118] Using human lung cancer cell line A549 as the research object, the subcellular localization (mitochondrial targeting) and distribution of mitochondrial hematoporphyrin monosodium (DVDMS) and targeting photosensitizer X-1 in tumor cells were detected.

[0119] 1. Materials and equipment

[0120] Table 3 Material list

[0121]

[0122] Table 4 Equipment list

[0123]

[0124] 2. Experimental system

[0125] Cell information: A549 cells (Cat. No.: FH0045), purchased from Fuheng Biology.

[0126] Cell culture: Take out the cells from the -80°C refrigerator and resuscitate them, and incubate them at 37°C until they grow well. The cells were grown in RPMI-1640 complete medium containing 10% fetal bovine serum, 1% penicillin (10,000 U / mL)-streptomycin (10,000 μg / mL) in a CO2 incubator at 37°C, saturated humidity, and 5% CO2.

[0127] 3. Experimental methods

[0128] Table 5 Drug preparation table

[0129]

[0130] 3.2 Cell administration and staining

[0131] Prepare the working solutions of DVDMS and X-1 (4 μg / mL) by diluting the DVDMS and X-1 stock solutions (4 mg / mL) with RPMI-1640 medium for later use.

[0132] After cell digestion and resuspension, dilute the cells to 1.5×10 5 cells / mL with complete RPMI-1640 medium. Add 1 mL of the cell suspension to a glass-bottom culture dish and incubate at 37°C for more than 20 hours to allow the cells to adhere to the dish.

[0133] Use a pipette to aspirate the old medium, and then add 1 mL of the 4 μg / mL DVDMS working solution and 1 mL of the X-1 working solution to the two culture dishes respectively. Incubate at 37°C in the dark for 4 hours.

[0134] After incubation, aspirate the old medium and add 1 mL of serum-free and phenol red-free RPMI-1640 medium. Repeat this operation twice.

[0135] Prepare the MTDR working solution.

[0136] Aspirate the old medium and add the diluted 250 nM MTDR working solution. Incubate and stain at 37°C for 15 minutes.

[0137] Aspirate the old medium and add 1 mL of serum-free and phenol red-free RPMI-1640 medium. Repeat this operation twice.

[0138] Aspirate the old medium and add 1 mL of universal tissue fixative (neutral) to fix the cells at room temperature for 10 minutes.

[0139] Aspirate the fixative and add 1 mL of PBS. Repeat this operation twice.

[0140] 3.3 Image the cells using a fluorescence microscope

[0141] Locate the cells under bright field, adjust the objective lens to 40×, and adjust the focus to obtain clear mitochondrial imaging.

[0142] Observe the fluorescence of MTDR and capture images using the fluorescence channel with a 640 nm excitation light source and a 690 nm emission light source.

[0143] Observe the fluorescence of DVDMS and X-1 and capture images using the fluorescence channel with a 405 nm excitation light source and a 630 nm emission light source.

[0144] 3.4 Experimental results

[0145] After excitation with a 405 nm light source for DVDMS or X-1, good fluorescence imaging can be observed in the 630 nm emission channel. The imaging results and the scatter plots of the co-localization analysis for the two are respectively asFigure 1 and Figure 2 as shown

[0146] As Figure 1 , the mitochondrial fluorescent probe MTDR clearly labeled the structure of mitochondria in cells. DVDMS was mainly enriched around the nucleus, where the distribution of mitochondria was also relatively concentrated. However, the co-localization of DVDMS and MTDR was weak, and the Pearson correlation coefficient was 0.376, indicating its poor mitochondrial targeting ability.

[0147] As Figure 2 , X-1 had good co-localization with MTDR, and both could clearly show the structure of mitochondria. The Pearson correlation coefficient was 0.901, suggesting that X-1 had good mitochondrial targeting ability.

[0148] As shown by Figure 1 and 2 , compared with DVDMS, X-1 had better mitochondrial targeting ability, and was expected to improve its ability to target and photodynamically kill tumors in vivo, improve the therapeutic effect and reduce the toxic and side effects.

[0149] 4.1 Experimental design

[0150] Table 6 Experimental design

[0151]

[0152] Note: Administration volume: 10 μL / g according to the body weight of mice

[0153] 4.2 Materials and equipment

[0154] Table 7 Material list

[0155]

[0156] Table 8 Equipment list

[0157]

[0158] 4.3 Experimental animals and feeding management

[0159] Experimental animals:

[0160] Strain: Balb / c-nude mice;

[0161] Week age: 6 - 8 weeks old;

[0162] Body weight: 18 - 20 g;

[0163] Gender: Female;

[0164] Quantity: 24;

[0165] Supplier: Shanghai Model Organisms Center, Inc.

[0166] Animal Production License Number: SCXK (Shanghai) 2019-0002

[0167] Animal Use License Number: SYXK (Shanghai) 2018-0002

[0168] Feeding and Management:

[0169] Animals are raised in IVC (Independent Ventilation Cage) cages in the SPF animal facility of Qishang Biology (5 animals per cage). The information card for each cage indicates the number of animals, gender, strain, receiving date, dosing regimen, experiment number, group, and start date of the experiment. All cages, bedding, and drinking water are sterilized before use. Cages, feed, and drinking water are changed once a week. The breeding environment and lighting conditions are as follows:

[0170] Temperature: 20~26°C

[0171] Humidity: 30~70%

[0172] Lighting Cycle: 12 hours of light and 12 hours of darkness

[0173] Cages: Made of polycarbonate. The bedding is corncob and is changed once a week.

[0174] Food: Laboratory animals can freely access irradiated and sterilized dry granular food throughout the experiment.

[0175] Drinking Water: Laboratory animals can freely drink sterilized water.

[0176] Cage Identification: The information card for each cage should indicate the number of animals, gender, strain, receiving date, dosing regimen, experiment number, group, and start date of the experiment.

[0177] Animal Identification: Ear clipping method.

[0178] 4.4 Experimental Methods

[0179] 1. Cell Culture

[0180] A549 cells are cultured adherently in vitro. The culture conditions are as follows: cultured in F12K medium supplemented with 10% fetal bovine serum in a 5% CO2 cell incubator at 37°C. Routine subculture is performed twice a week. When the cell confluence reaches 80%-90% and the cell number meets the requirement, the cells are harvested, counted, and the cell density is adjusted to 5×10 7 cells / ml with PBS for standby.

[0181] 2. Animal Inoculation

[0182] Animals are transferred to the animal house for 7 days of acclimation feeding, and experiments can only be conducted after the acclimation period. 0.1 mL (5.0×10 6 cells / mouse) of A549 cells are subcutaneously inoculated into the right hind back near the thigh of each mouse.

[0183] 3. Daily Observation of Experimental Animals

[0184] The use and welfare of experimental animals comply with the regulations of the International Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). The health status and mortality of animals are monitored daily. Routine examinations include observing tumor growth and the effects of drug treatment on the daily behavior of animals, such as behavioral activities, food and water intake (only visually estimated), appearance signs, or other abnormal conditions.

[0185] 4. Compound Preparation

[0186] Table 9 Compound Preparation Table

[0187]

[0188] 4.5. Random Grouping

[0189] After cell inoculation, the tumor growth is observed regularly. When the tumors have grown for 21 days and the average tumor volume reaches 158 mm 3 , 24 animals with appropriately sized tumors are randomly selected and divided into 4 groups of 6 animals each according to the tumor volume. Administration starts on the day of grouping, denoted as D0. Except for the erlotinib group which is given gavage daily, the other groups are given a single intravenous tail injection. The specific administration scheme is shown in Table 6. The medicinal solutions for each group are freshly prepared before the experiment and stored in the dark and refrigerated before use. Except for the light exposure to the tumors, the tumor-bearing animals are strictly kept in the dark for 3 days after intravenous tail injection and then transferred to a dim light environment for rearing.

[0190] 4.6. Light Exposure

[0191] Groups G3 - G4 receive a single light therapy 19 h after drug administration. A 630 nm semiconductor laser is used. The optical fiber with a micro-lens at the end is directed towards the target site, and the light beam is perpendicularly projected to completely cover the tumor. The following steps are used for parameter setting. The mice are anesthetized by inhalation using an isoflurane small animal anesthesia machine. After anesthesia, they are fixed with medical tape, and the tumor site is fully exposed. The non-tumor sites are covered with black plastic bags.

[0192] a. Turn on the laser therapy instrument for calibration and set the parameters.

[0193] b. Measure the laser treatment distance to ensure that the diameter of the laser spot can cover the size of the tumor.

[0194] c. At the laser treatment distance given in the previous step, measure the laser power value per unit area with a powermeter.

[0195] d. Adjust the laser power value to 318 mW.

[0196] e. Set the laser treatment time and perform PDT treatment on the experimental animals.

[0197] Table 10 Photodose Design Table

[0198]

[0199] 4.7. Data Collection

[0200] After grouping and administering drugs, measure the length and width of the tumor twice a week with a vernier caliper (purchased from Mitutoyo Precision Measuring Instruments (Shanghai) Co., Ltd.), and weigh the mice at the same time. Calculate the tumor volume (TV), tumor growth inhibition rate TGI 体积 (%), relative tumor inhibition rate (T / C%). The specific calculation formulas are as follows:

[0201]

[0202] where V0 is the average tumor volume at the start of drug administration for a certain treatment group, and V t is the average tumor volume at the end of drug administration for this treatment group.

[0203] At the end of the experiment, euthanize the mice in each group, dissect the tumor tissue and weigh and record it. Calculate T / C 重量 percentage and tumor growth inhibition rate TGI 重量 (%). The specific calculation formulas are as follows:

[0204]

[0205] where TW 给药组 represents the tumor weight of the drug administration group, and TW 阴性对照组 represents the tumor weight of the negative control group.

[0206] 4.8. Sample Collection and Processing

[0207] At the end of the experiment, euthanize the mice in each group, dissect the tumor tissue, and weigh the tumor tissue.

[0208] 4.9. Data Processing and Statistical Analysis

[0209] All experimental results were expressed as "mean ± standard error". The days after grouping were used as the abscissa, and the body weight or tumor volume of the mice was used as the ordinate. GraphPad Prism 8 was used for chart drawing. Statistical analysis was performed based on the data obtained at the end of the experiment to evaluate the differences between groups. Data analysis was performed using Ordinary one-way ANOVA in GraphPad Prism 8 software, and a p < 0.05 was considered to indicate a significant difference.

[0210] 5. Results

[0211] Body weight changes

[0212] During the experiment, the mice had good tolerance. The body weight changes of the mice in each group are shown in Figure 3 (The data points represent the average body weight within the group).

[0213] Tumor volume

[0214] The changes in the average tumor volume of the subcutaneous xenograft tumor model of A549 non-small cell lung cancer over time are shown in Table 11.

[0215] Table 11 Tumor volumes at different time points in each group

[0216]

[0217] Note: a. Mean ± standard error; b. Days after starting drug administration

[0218] Tumor growth curve and terminal tumor weight

[0219] The tumor growth curve and tumor weight are shown in Figure 4 and Figure 5 as shown. The data points represent the average body weight within the group, and the error bars represent the standard error (SEM).

[0220] Antitumor efficacy evaluation indicators

[0221] Based on the tumor volume on the 22nd day after drug administration, the growth inhibition rate of the test substance on the subcutaneous xenograft tumor model of A549 non-small cell lung cancer was calculated (Table 12). The tumor weights of all groups on the 22nd day are shown in Table 13.

[0222] Table 12 Growth inhibition rate of the test substance on the subcutaneous xenograft tumor model of A549 non-small cell lung cancer

[0223]

[0224] Note: a. Mean ± standard error; b. p Values were analyzed using one-way ANOVA

[0225] Table 13 Tumor weight and statistical analysis at the end of the experiment (Day 22)

[0226]

[0227] Note: a. Mean ± standard error; b. p Values were analyzed by one-way ANOVA.

[0228] 6. Conclusion

[0229] In the above experiment, the in vivo antitumor effects of sodium hyproporphyrin and the targeted photosensitizer X-1 were evaluated in a subcutaneous xenograft model of A549 non-small cell lung cancer. The body weights and tumor volumes of the animals measured at different time points in each experimental group are as Figure 3 and Figure 4 shown, and the terminal tumor weights are as Figure 5 shown.

[0230] The above experiment was grouped when the average tumor volume reached 158 mm 3 , and the drug was administered on the day of grouping. A single light irradiation at a wavelength of 630 nm was performed 19 hours after drug administration. The experiment was all completed on D22 after grouping. From the above results, it can be seen that at D22, the tumor volume of the negative control group reached 674 mm 3 . Compared with the control group, erlotinib at a dose of 25 mg / kg (TV = 518 mm 3 , TGI = 30.1%, P > 0.05) had no obvious inhibitory effect on the tumor growth of the A549 model. The positive drug sodium hyproporphyrin at a dose of 2 mg / kg (TV = 289 mm 3 , TGI = 74.7%, P <0.001) had a significant inhibitory effect on the tumor growth of the A549 model. Compared with erlotinib and sodium hyproporphyrin, the mitochondrial-targeted photosensitizer X-1 of the present invention (TV = 169 mm 3 , TGI = 97.8%, P <0.001) showed better drug efficacy and small within-group differences, proving enhanced targeting ability and significantly improved drug efficacy.

Claims

1. A mitochondrial targeted photosensitizer or a pharmaceutically acceptable salt thereof, wherein the mitochondrial targeted photosensitizer has the following general formula (I): ， Wherein the groups R each independently represent C 3-9 Heterocyclyl C 1-6 Alkylene-O-, Wherein C 3-9 The heterocyclyl group is unsubstituted or substituted by one or more identical or different groups selected from the group consisting of halogen or (C1-C6)-alkyl; 3-9 The heterocyclic group contains at least one nitrogen atom selected from the group consisting of morpholinyl, thiomorpholinyl, thiazolidinyl, oxazolidinyl, oxazetidinyl, and oxazepanyl; The C 1-6 Alkylene is linear or branched, unsubstituted or substituted by one or more identical or different groups selected from halogen or (C1-C6)-alkyl.

2. The mitochondrial targeting photosensitizer according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the C 1-6 Alkylene is straight chain and unsubstituted.

3. The mitochondrial targeted photosensitizer according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the group R is C 3-6 Heterocyclyl C 1-6 Alkylene-O-, where C 3-6 The heterocyclic group is selected from the group consisting of morpholinyl, thiomorpholinyl, thiazolidinyl, oxazolidinyl and oxazetidinyl; C 1-6 The alkyl group is selected from methylene, ethylene, propylene, n-butylene, n-pentylene or n-hexylene.

4. The mitochondrial targeted photosensitizer according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the group R is C 3-6 Heterocyclyl C 1-4 Alkylene-O-, where C 3-6 The heterocyclic group is selected from the group consisting of morpholinyl, thiomorpholinyl, thiazolidinyl, oxazolidinyl and oxazetidinyl; C 1-4 The alkylene group is selected from methylene, ethylene, n-propylene or n-butylene.

5. A method for preparing a mitochondrial targeted photosensitizer, comprising the following steps: In the presence of a catalyst and a condensing agent, porphyrinic acid is reacted with a G-OH compound in the presence of a solvent to obtain a product; The GO part in the G-OH compound is defined the same as the R group in the mitochondrial targeting photosensitizer or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4.

6. The method according to claim 5, wherein the catalyst is an organic base catalyst selected from N,N-diisopropylethylamine (DIPEA), N,N-dimethylcyclohexylamine, N,N-dimethylbenzylamine, N-methylmorpholine, N-ethylmorpholine, N-methylimidazole, 1,8-diazabicyclo[5.4.0]undec-7-ene, triethylamine, pyridine, N,N'-dimethylaminopyridine, 2,6-lutidine or a mixture thereof; The condensing agent is a urea cation type condensing agent, which is selected from O-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), O-benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), O-(7-azabenzotriazole-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TATU), O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate (T BTU), O-(1,2-dihydro-2-oxy-pyridyl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TPTU); carbodiimide type condensing agent: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), 1,3-dicyclohexylcarbodiimide (DCC); or carbonyl imidazole type condensing agent: N,N-carbonyldiimidazole (CDI); or a mixture thereof, and the molar ratio of the catalyst to the condensing agent is (0.7-3):1; The solvent is selected from N,N-dimethylformamide (DMF), N,N-dimethylacetamide, formamide, dimethyl sulfoxide, acetone, pyridine or a mixture thereof.

7. Use of the mitochondrial targeted photosensitizer or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4 for preparing a medicament for treating the following diseases: lung cancer and precancerous lesions of the disease.

8. Use of the mitochondrial targeted photosensitizer or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4 for preparing a medicament for treating the following diseases: small cell and non-small cell lung cancer, and precancerous lesions of the diseases.

9. A kit comprising the mitochondrial targeting photosensitizer or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, which is present in a therapeutically effective amount; and instructions for using the same for photodynamic therapy.

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