Monosubstituted amide porphyrin derivatives, their preparation methods and uses as photosensitizers

Abstract

The present invention relates to a compound of formula (I) or a salt thereof, wherein the group R is as defined in the specification; it also relates to a method for preparing the compound and its use as a photosensitizer in photodynamic therapy. All the monosubstituted protoporphyrin derivatives involved in the present invention have high permeability, high activity and good biosafety. The preparation method has simple process, low cost, can obtain the target product in good yield and is particularly suitable for industrial production. It can be used as an efficient photosensitizer for treating hyperplastic diseases such as cancer or precancerous lesions in photodynamic therapy, or treating oral diseases such as periodontitis, gingivitis, dental plaque and dental caries caused by bacteria such as Gram-negative anaerobic coccobacilli like Porphyromonas gingivalis (I).

Description

Technical Field

[0001] The present invention belongs to the field of medicinal chemistry, and particularly relates to monosubstituted protoporphyrin derivatives, a preparation method thereof, and their use as photosensitizers. More specifically, it relates to monosubstituted amide porphyrin derivatives, a preparation method thereof, and their use as photosensitizers in photodynamic therapy. Background Art

[0002] Photodynamic therapy (PDT) is a novel therapy that utilizes photosensitizers, light, and oxygen molecules to generate a photodynamic reaction, and then selectively targets diseases such as malignant tumors, vascular lesions, and microbial infections. Among them, photosensitizers are the core of photodynamic therapy. Currently, the photosensitizers clinically used mainly include Photofrin from the United States, Photogem from Russia, Photosan from Germany, etc. However, these photosensitizers still have many drawbacks. For example, they are mostly mixed preparations composed of porphyrin derivatives, with unclear active ingredients, uncontrollable quality standards, and easy to cause skin phototoxic effects such as rashes and blisters due to the long retention time in the skin for several weeks. Patients need to avoid direct sunlight for 1 month or even longer after medication.

[0003] 5-aminolevulinic acid (5-ALA) is the second-generation photosensitizer developed in recent years. It has no photosensitivity itself. After exogenous 5-ALA enters the body, it can be selectively absorbed and accumulated by proliferating cells and converted into protoporphyrin (Protoporphyrin IX, PPIX) in cells. PPIX in cells is a photosensitive substance. After irradiation with red light of a specific wavelength, a photodynamic reaction occurs, generating reactive oxygen species such as singlet oxygen to kill proliferating cells.

[0004] Cervical cancer is one of the most common tumors in the female reproductive system and the second leading cause of tumor-related deaths among women worldwide. Currently, only 5-aminolevulinic acid and hexaminolevulinate (HAL) are used as photosensitizers for the photodynamic treatment of cervical intraepithelial neoplasia in the world, and they are in the clinical trial stage. There is no photosensitizer drug for the treatment of cervical cancer or precancerous lesions on the market. Therefore, it is urgent to develop such new photosensitizers to promote the development of the industry.

[0005] Periodontitis is a chronic inflammatory disease mainly caused by the destruction of periodontal tissues by bacteria in dental plaque. Porphyromonas gingivalis is a Gram-negative coccobacillus and the main pathogen of periodontitis and gingivitis. Currently, drugs such as metronidazole tablets and amoxicillin capsules can be used for treatment. However, frequent use of antibiotics will produce drug resistance, and some patients even have adverse reactions such as allergies. Therefore, safer, less toxic, and non-invasive alternative therapies are needed.

[0006] In summary, it is of great significance to develop photosensitizers with the ability to efficiently kill cancer cells and / or antibacterial ability. SUMMARY OF THE INVENTION

[0007] The present invention aims to provide a class of mono-substituted amide porphyrin derivatives, a preparation method thereof, and their use as photosensitizers, and more specifically to use them in the treatment of hyperplastic diseases in photodynamic therapy, especially cervical cancer or cervical pre-cancerous lesions, or antibacterial to treat diseases such as periodontitis, gingivitis, dental plaque or dental caries.

[0008] The first aspect of the present invention relates to a compound of formula (I) or a salt thereof:

[0009] (I)

[0010] Wherein:

[0011] R represents -NR 1 R 2 ,

[0012] R 1 and R 2 each independently represents hydrogen, heterocyclic alkyl or heteroaryl alkyl, wherein R 1 and R 2 are not both hydrogen at the same time,

[0013] wherein the heterocyclic group or heteroaryl group contains one or more heteroatoms selected from O, S and N;

[0014] provided that the compounds and are excluded.

[0015] The second aspect of the present invention provides a method for preparing a compound of formula (I) or a salt thereof, the method comprising subjecting protoporphyrin (PPIX) to a condensation reaction with a compound having a reactive hydrogen atom in a polar organic solvent in the presence of a catalyst, a condensing agent and a condensation activator, wherein the compound having a reactive hydrogen atom is selected from compounds of general formula (II)

[0016] RH (II)

[0017] wherein R is defined as above.

[0018] The third aspect of the present invention relates to the use of a compound or a salt thereof according to the first aspect of the present invention as a photosensitizer in photodynamic therapy or in the preparation of a photosensitizer for photodynamic therapy.

[0019] The fourth aspect of the present invention relates to the use of a compound or a salt thereof according to the first aspect of the present invention in the preparation of a drug for the treatment of hyperplastic diseases.

[0020] The present invention also relates to a method for treating hyperproliferative diseases, which method comprises administering to a subject in need thereof a therapeutically effective amount of a compound of formula (I) as described above or a salt thereof, and administering a therapeutically effective amount of light irradiation of a specific wavelength. Preferably, the subject is a mammal, more preferably a human.

[0021] The fifth aspect of the present invention relates to the use of a compound or a salt thereof according to the first aspect of the present invention in the preparation of a medicament for treating a disease or condition caused by Gram-negative or Gram-positive cocci, bacilli or coccobacilli.

[0022] The present invention also relates to a method for inhibiting Gram-negative or Gram-positive cocci, bacilli or coccobacilli for non-therapeutic purposes, which method comprises contacting the compound or a salt thereof with Gram-negative or Gram-positive cocci, bacilli or coccobacilli and administering an inhibitory effective amount of light irradiation of a specific wavelength. The contacting can be carried out in vivo or in vitro, for example, in vitro.

[0023] The present invention also relates to a method for treating a disease or condition caused by Gram-negative or Gram-positive cocci, bacilli or coccobacilli, which method comprises administering to a subject in need thereof a therapeutically effective amount of the compound or a salt thereof and administering a therapeutically effective amount of light irradiation of a specific wavelength. Preferably, the subject is a mammal, more preferably a human.

[0024] The sixth aspect of the present invention relates to a pharmaceutical composition, which comprises a compound or a salt thereof according to the first aspect of the present invention, and optionally further comprises one or more other active compounds.

[0025] Surprisingly, the present invention has the following beneficial effects:

[0026] The present invention has developed a novel class of mono-substituted amide porphyrin derivatives, which can be used as photosensitizers for photodynamic therapy. The compounds of the present invention all have high permeability, high activity and good biosafety. Their preparation methods are simple in process, low in cost, can obtain the target product in good yield and are particularly suitable for industrial production. They can be used as highly efficient photosensitizers for treating hyperproliferative diseases such as cancer or precancerous lesions, especially cervical cancer or cervical precancerous lesions, in photodynamic therapy. Especially when the dosage is significantly smaller, their drug efficacy is significantly better than that of the drug 5-ALA, equivalent to or even significantly better than that of the drug HAL, and they have good antibacterial effects on bacteria such as Porphyromonas gingivalis and have therapeutic effects in oral diseases such as periodontitis, gingivitis, dental plaque and dental caries. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Shows the standard curves of the percutaneous diffusion amounts of PPIX and the compounds of the present invention in in vitro percutaneous penetration experiments.

[0028] Figure 2 Showed the standard curves of tissue retention samples of PPIX and the compound of the present invention in in vitro percutaneous penetration experiments for 6 hours.

[0029] Figure 3 Showed the tissue retention amount per unit area of PPIX and the compound of the present invention for 6 hours. Among them, compared with the PPIX group, *P<0.05 for the compound N-01 group, and ****P<0.0001 for the compound N-02 group.

[0030] Figure 4 Showed the body weight changes of mice in each treatment group after administration. The data points represent the average body weight within the group, and the error bars represent the standard error (SEM).

[0031] Figure 5 Showed the in vivo imaging diagrams of mice in the high-dose groups of the compounds N-01 and N-02 of the present invention.

[0032] Figure 6 Showed the optical density (OD) of each test group in the antibacterial experiment and the antibacterial rate against Porphyromonas gingivalis (in a-c in the figure compared with the blank light group, and in d in the figure compared with the PPIX group, **** P <0.0001).

[0033] Figure 7 Was the ultraviolet absorption spectrogram of the compounds N-01 and N-02 of the present invention. Detailed implementation manners

[0034] For a better understanding of the present invention, the present invention will be described in detail below in combination with examples and drawings. It should be understood, however, that these examples and drawings are only for illustrative purposes of the present invention and are not intended to limit the present invention.

[0035] Definition

[0036] Unless otherwise specified, the "including" and "comprising" mentioned in this application mean open-ended, and can also be closed-ended. For example, the "including" and "comprising" can mean that other members, elements or method steps not listed can also be included or comprised, or can only include or comprise the other members, elements or method steps listed.

[0037] As used herein, the term "optionally" means that the events, circumstances or substances described later may occur or exist, may also not occur or exist, and such description includes 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.

[0038] Unless otherwise defined differently, the names of chemical groups shall 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, for example, heterocyclyl-(C1-C8)-alkyl, through the carbon atom of the alkyl group. In the case of a specified group, for example in the case of NR 1 R 2 , the connection to the skeleton or the rest of the molecule is through the structural element first mentioned, i.e., in the case of, for example, NR 1 R 2 through the nitrogen atom.

[0039] As used herein, the term "alkyl" means a saturated straight-chain or branched hydrocarbon group having the specified number of carbon atoms in each case, such as (C1-C8)-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.

[0040] Unless otherwise defined differently, the term "heteroaryl" means a monocyclic, bicyclic or tricyclic heterocyclic group of carbon atoms and at least one heteroatom selected from oxygen, nitrogen and sulfur, wherein at least one ring is aromatic. Preferably, the heteroaryl is a 5- to 12-membered heteroaryl, for example containing 3, 4, 5, 6, 7 or 8 carbon atoms, which can be linked to the parent molecular moiety through any carbon atom or nitrogen atom contained within the heterocycle. Examples include but are not limited to furyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,3,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,3,4-thiadiazolyl, 1,2,5-thiadiazolyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, 1,2,3-triazinyl, 1,2,4-triazinyl, 1,3,5-triazinyl, tetrazolyl, benzofuryl, benzisofuryl, benzothienyl, benzisothienyl, indolyl, isoindolyl, indazolyl, benzothiazolyl, benzisothiazolyl, benzoxazolyl, benzisoxazolyl, benzimidazolyl, 2,1,3-benzoxadiazole, quinolinyl, isoquinolinyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, naphthyridinyl, benzotriazinyl, purinyl, pteridinyl, imidazopyridyl, thienopyrimidinyl, thienopiperidinyl, etc.

[0041] Unless otherwise defined differently, the term "heterocyclic group" means a saturated or partially saturated monocyclic ring of carbon atoms and at least one heteroatom in the ring. Preferably, the heterocyclic group is a 3- to 10-membered heterocyclic group, for example containing 2, 3, 4, 5 or 6 carbon atoms and 1 or 2 heteroatoms selected from oxygen, sulfur and nitrogen, which can be linked to the parent molecular moiety through any carbon atom or nitrogen atom contained within the heterocycle. If more than one oxygen atom is contained in the ring, they are not directly adjacent. Examples of heterocyclic groups include but are not limited to aziridinyl, oxiranyl; azetidinyl, oxetanyl, thietanyl; tetrahydrofuryl, 1,3-dioxolanyl, tetrahydrothienyl, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, isoxazolidinyl, oxazolidinyl, thiazolidinyl, isothiazolidinyl; piperidinyl, hexahydropyridazinyl, hexahydropyrimidinyl, piperazinyl, tetrahydropyranyl, dioxanyl, tetrahydrothianyl, dithianyl, morpholinyl, 1,2-oxazepanyl, oxathiepanyl, thiomorpholinyl; oxepanyl, azepanyl, 1,4-diazepanyl, 1,4-oxazepanyl; dihydrofuryl, 1,3-dioxolenyl, dihydrothienyl, pyrroline, dihydroimidazolyl, dihydropyrazolyl, dihydrooxazolyl, dihydrothiazolyl; pyranyl, thianyl and thiazinyl.

[0042] Unless otherwise defined differently, the term "heteroarylalkyl" is understood to mean the combination of the groups "heteroaryl" and "alkyl" as defined according to the present invention, wherein said groups are typically linked to the backbone or the remainder via an alkyl group, such as 5- to 12-membered heteroaryl-(C1-C8)-alkyl, 5- to 10-membered heteroaryl-(C1-C6)-alkyl, and 5- to 6-membered heteroaryl-(C1-C4)-alkyl. Examples include but are not limited to pyrrolylmethyl, pyrrolylethyl, benzothienylmethyl, benzothienylethyl, furylmethyl, furoylethyl, benzofurylmethyl, thienylmethyl, thienylethyl, pyridylmethyl, etc.

[0043] Unless otherwise defined differently, the term "heterocycloalkylalkyl" is understood to mean the combination of the groups "heterocycloalkyl" and "alkyl" as defined according to the present invention, wherein said groups are typically linked to the backbone or the remainder via an alkyl group, such as 3- to 10-membered heterocycloalkyl-(C1-C8)-alkyl, 3- to 8-membered heterocycloalkyl-(C1-C6)-alkyl, and 5- to 6-membered heterocycloalkyl-(C1-C4)-alkyl. Examples include but are not limited to 2-(morpholin-4-yl)ethyl, morpholin-3-ylmethyl, etc.

[0044] In the context of the present invention, reference to a salt of a compound of formula (I) means its pharmaceutically acceptable salts, which are generally those salts that are considered safe and suitable for use in pharmaceutical formulations in pharmacy. Unless otherwise expressly stated, the term "pharmaceutically acceptable salts" refers to pharmaceutically acceptable acid and base addition salts as well as solvates. Such pharmaceutically acceptable salts include but are not limited to salts of the following acids: for example, hydrochloric acid, trifluoroacetic acid, phosphoric acid, hydrobromic acid, sulfuric acid, sulfinic acid, formic acid, p-toluenesulfonic acid, methanesulfonic acid, benzoic acid, citric acid, acetic acid, etc., preferably acetate. Non-toxic pharmaceutical base addition salts include bases: such as salts of sodium, potassium, calcium, ammonium, etc. Those skilled in the art will recognize a variety of non-toxic pharmaceutically acceptable addition salts.

[0045] As used herein, the term "cancer" includes but is not limited to breast cancer, respiratory cancer, brain cancer, reproductive organ cancer, digestive tract cancer, urinary tract cancer, liver cancer, eye cancer, skin cancer, head and neck tumors and their distant metastases, and also includes multiple myeloma, lymphoma, and sarcoma.

[0046] Unless otherwise expressly defined, "precancerous lesions" herein refer to abnormal cell proliferation with a high potential for canceration, and examples thereof include but are not limited to cervical precancerous lesions, oral leukoplakia, myelodysplastic diseases, familial intestinal polyps, skin moles, psoriasis, actinic keratosis, etc.

[0047] 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, as well as 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.

[0048] As used herein, the term "therapeutically effective amount" or "effective amount" or "effective dose" refers to the amount of an active compound or agent that elicits the biological or pharmaceutical response sought or desired by a researcher, physician, or other clinician in a tissue, system, animal, individual, or human.

[0049] Administration and Dosage

[0050] The compounds or drugs of the present invention can act systemically and / or locally. For this purpose, they can be administered by a suitable route of administration, such as by oral, pulmonary, nasal, sublingual, lingual, buccal, rectal, vaginal, dermal, transdermal, conjunctival, or otic canal routes, or by subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, intracardiac, or intracranial injection or infusion, or as an implant or stent. For example, oral administration, spray inhalation, rectal administration, nasal administration, vaginal administration, topical administration, parenteral administration such as subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, intracardiac, or intracranial injection or infusion, or administration by means of an implanted reservoir.

[0051] Preferred modes of administration are oral mucosal administration, oral administration, intramuscular injection, topical administration, vaginal administration, rectal administration, intraperitoneal or intravenous administration. Oral mucosal administration includes administration by means of devices such as local injection, topical application, local irrigation, oral gargling, local controlled / sustained release, and microneedles.

[0052] The drug delivery system of the present invention can be a targeted drug delivery system, a controlled drug delivery system, and a regulated drug delivery system, or can also be a novel delivery system, such as an emulsion delivery system, a biomimetic drug delivery system, a live cell delivery system, an exosome drug delivery system, a microneedle drug delivery system, a nano-drug delivery system, or a protein or polypeptide delivery system, etc.

[0053] For these routes of administration, the compounds of the present invention can be administered in a suitable dosage form.

[0054] The drugs of the present invention can be administered in unit dosage form. The dosage forms can be liquid dosage forms, semi-solid preparations, and solid dosage forms. Liquid dosage forms can be true solution types, colloidal types, particulate dosage forms, suspension dosage forms, etc. Semi-solid dosage forms can be ointments, creams, pastes, gels, etc. Solid dosage forms can be oral soluble films, tablets, capsules, dripping pills, pills, powders, granules, suppositories, lyophilized powder injections, inclusion compounds, implants, patches, etc.

[0055] The compounds of the present invention can be incorporated into the said dosage forms. This can be achieved in a known manner by mixing with pharmaceutically suitable carriers, excipients and / or other auxiliaries.

[0056] The single administration dose of the medicament of the present invention is 0.01 - 100 mg of active ingredient / kg body weight, preferably 0.02 - 80 mg of active ingredient / kg body weight, more preferably 0.05 - 50 mg of active ingredient / kg body weight, still more preferably 0.08 - 40 mg of active ingredient / kg body weight, particularly preferably 0.1 - 20 mg of active ingredient / kg body weight, also preferably 0.1 - 15 mg of active ingredient / kg body weight, for example 0.1 - 10 mg of active ingredient / kg body weight, 0.1 - 8 mg of active ingredient / kg body weight, and most preferably 0.2 - 6 mg of active ingredient / kg body weight.

[0057] In one embodiment, the medicament of the present invention is administered at least once a month, for example 1, 2, 3, 4 or 5 times a month. Preferably, the medicament of the present invention is administered 1, 2 or 3 times a month. Here, the term "active ingredient" refers to the compound of formula (I) or its salt in the present invention.

[0058] Regimen

[0059] The first aspect of the present invention relates to a compound of formula (I) or its salt:

[0060] (I)

[0061] wherein:

[0062] R represents -NR 1 R 2 ,

[0063] R 1 and R 2 each independently represents hydrogen, heterocyclic alkyl or heteroaryl alkyl, where R 1 and R 2 are not simultaneously hydrogen;

[0064] wherein the heterocyclic group or heteroaryl group contains one or more heteroatoms selected from O, S and N; preferably wherein the heterocyclic group or heteroaryl group contains one or two of the heteroatoms O, S and N;

[0065] provided that the compounds and are excluded.

[0066] In a preferred embodiment, R 1 and R 2Each independently represents hydrogen, a 3- to 10-membered heterocyclic group (C1-C8)-alkyl or a 5- to 12-membered heteroaryl (C1-C8)-alkyl.

[0067] In a further preferred embodiment, R 1 and R 2 Each independently represents hydrogen, a 3- to 8-membered heterocyclic group (C1-C6)-alkyl or a 5- to 10-membered heteroaryl (C1-C6)-alkyl.

[0068] In a preferred embodiment, the 3- to 8-membered heterocyclic group (C1-C6)-alkyl and the 5- to 10-membered heteroaryl (C1-C6)-alkyl are respectively a 3- to 8-membered heterocyclic group (C1-C2)-alkyl and a 5- to 10-membered heteroaryl (C1-C2)-alkyl containing 1 to 3 (preferably 1 to 2) O atoms, 1 to 3 (preferably 1 to 2) N atoms and / or 1 to 3 (preferably 1 to 2) S atoms.

[0069] In a further preferred embodiment, R 1 and R 2 Each independently represents hydrogen, morpholine (C1-C6)-alkyl or thiophene (C1-C6)-alkyl.

[0070] In a further preferred embodiment, R 1 and R 2 Each independently represents hydrogen, morpholin-4-yl (C1-C6)-alkyl (preferably 2-(morpholin-4-yl)ethyl), morpholin-3-yl (C1-C6)-alkyl (preferably morpholin-3-ylmethyl) or thiophen-2-yl (C1-C6)-alkyl (preferably thiophen-2-ylmethyl).

[0071] The definitions of the groups listed above in general terms or within the preferred ranges can be combined with each other as needed, i.e., including combinations between the given preferred ranges.

[0072] Compounds of formula (I) of the present invention listed in Table 1 below are very particularly preferred.

[0073] Table 1: Compounds of formula (I), wherein R has the meanings given below.

[0074] .

[0075] Note: " " indicates connection to the remainder of the compound of formula (I).

[0076] The second aspect of the present invention provides a method for preparing a compound of formula (I) or a salt thereof, the method comprising:

[0077] .

[0078] In the presence of a catalyst, a condensing agent, and a condensation activator, a condensation reaction is carried out between protoporphyrin (PPIX) and a compound having a reactive hydrogen atom in a polar organic solvent, wherein the compound having a reactive hydrogen atom is selected from the compounds of general formula (II)

[0079] RH (II)

[0080] wherein R is as defined above.

[0081] The catalyst applicable to the method of the present invention is an organic base catalyst, which is preferably selected from N,N-diisopropylethylamine (DIPEA), N,N-dimethylcyclohexylamine, N,N-dimethylbenzylamine, N-methylmorpholine (NMM), N-ethylmorpholine, N-methylimidazole (NMI), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), triethylamine, pyridine, N,N-dimethylaminopyridine (DMAP), 2,6-dimethylpyridine or a mixture thereof, more preferably DIPEA and NMI, and particularly preferably DIPEA.

[0082] The condensing agent applicable to the method of the present invention is a carbodiimide type condensing agent, which is preferably selected from 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), 1,3-dicyclohexylcarbodiimide (DCC), N,N-diisopropylcarbodiimide (DIC) or a mixture thereof, more preferably EDCI.

[0083] Examples of the condensation activator applicable to the method of the present invention include but are not limited to N,N-dimethylaminopyridine (DMAP), 4-pyrrolidinopyridine (4-PPY), 1-hydroxy-7-azabenzotriazole (HOAT), 1-hydroxybenzotriazole (HOBT), N-hydroxysuccinimide (NHS), N-hydroxyphthalimide (NHPI), pentafluorophenol (PFP), etc., and HOBT is preferred.

[0084] The polar organic solvent applicable to the method of the present invention can be preferably selected from N,N-dimethylformamide (DMF), N,N-dimethylacetamide, formamide, dimethyl sulfoxide (DMSO), acetone, pyridine or a mixture thereof, more preferably DMF.

[0085] In the method of the present invention, the molar ratio of the protoporphyrin to the catalyst is 1:(0.6 - 1.5), preferably 1:(0.7 - 1.3), and more preferably 1:(0.8 - 1.1).

[0086] In the method of the present invention, the molar ratio of the compound having a reactive hydrogen atom to the condensing agent or the condensation activator is 1:(0.6 - 1.5), preferably 1:(0.7 - 1.3), and more preferably 1:(0.8 - 1.1).

[0087] In a preferred embodiment of the present invention, the compound having a reactive hydrogen atom is selected from tert-butyl 3-(aminomethyl)morpholine-4-carboxylate or 2-morpholino-N-(thiophen-2-ylmethyl)ethylamine.

[0088] According to the reaction requirements, the compound having a reactive hydrogen atom may optionally carry an amino protecting group commonly used in amidation reactions known to those skilled in the art, such as tert-butoxycarbonyl (Boc). The removal of the amino protecting group can be carried out by conventional methods known to those skilled in the art. For example, it can be removed using a solution of hydrogen chloride in a specific organic solvent (such as dioxane, ethyl acetate, methanol), preferably a hydrogen chloride dioxane solution.

[0089] The method for preparing the compound of formula (I) in the present invention, using a combination of a specific organic base catalyst, a condensing agent and a condensation activator, especially a combination of DIPEA, EDCI and HOBT in a specific ratio, can obtain the target compound with high purity in good yield through simple post-treatment. If the amount of the catalyst, the condensing agent or the condensation activator is too low or too high, it will have an adverse effect on the target product, such as difficult post-treatment, an increase in the proportion of by-products, and a low yield of the target product.

[0090] In the method of the present invention, the time of the condensation reaction is 0.5 - 15 hours, preferably 1 - 10 hours, more preferably 2 - 7 hours.

[0091] In the above method for preparing the compound of the present invention, the method may optionally further 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, drying, etc. 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.

[0092] In a preferred embodiment of the present invention, the method may further include a purification step through a chromatographic column. The purification can be carried out in the following ways: a normal-phase silica gel column with a silica gel particle size of 30 - 100 μm, preferably 40 - 63 μm, and a loading amount of 20 - 120 g, preferably 40 g, eluted with dichloromethane / methanol containing 5% acetic acid (v / v) (elution gradient 100% / 0% to 90% / 10%, gradient elution time 15 min); or a reverse-phase cyano column with a particle size of 10 - 100 μm, preferably 20 - 45 μm, and a loading amount of 20 - 120 g, preferably 40 g, eluted with water containing 5% acetic acid / acetonitrile containing 5% acetic acid (v / v) (elution gradient 95% / 5% to 5% / 95%, gradient elution time 20 min).

[0093] The compounds of the present invention can be prepared according to the above method. However, it should be understood that those skilled in the art can adjust the method according to the specific circumstances of the compounds of the present invention to be synthesized based on their own common knowledge and available publications.

[0094] The third aspect of the present invention relates to the use of the compound or its salt according to the first aspect of the present invention as a photosensitizer for photodynamic therapy or in the preparation of a photosensitizer for photodynamic therapy.

[0095] The fourth aspect of the present invention relates to the use of the compound or its salt according to the first aspect of the present invention in the preparation of a drug for treating hyperproliferative diseases.

[0096] The present invention also relates to a method for treating hyperproliferative diseases, the method comprising administering a therapeutically effective amount of the compound of formula (I) or its salt as described above to a subject in need thereof, and administering a therapeutically effective amount of light irradiation of a specific wavelength. Preferably, the subject is a mammal, more preferably a human.

[0097] The compound or its salt according to the first aspect of the present invention, when used as a photosensitizer in photodynamic therapy, is specifically used for treating hyperproliferative diseases.

[0098] Preferably, the hyperplastic disease is cancer or a pre-cancerous lesion. The cancer is preferably selected from bladder cancer, esophageal cancer, bronchial cancer, oral cancer, nasopharyngeal cancer, liver cancer, pancreatic cancer, skin cancer, penile cancer, cervical cancer, vaginal cancer, endometrial cancer, ovarian cancer, colorectal cancer, kidney cancer, urothelial cell carcinoma, thyroid cancer, breast cancer, anal cancer, Kaposi's sarcoma, lung cancer, gastric cancer, cholangiocarcinoma, prostate cancer, melanoma, and brain cancer, more preferably cervical cancer and skin cancer, and particularly preferably cervical cancer; the pre-cancerous lesion is preferably selected from cervical intraepithelial neoplasia, oral leukoplakia, myelodysplastic diseases, familial intestinal polyps, skin moles, psoriasis, actinic keratosis, more preferably cervical intraepithelial neoplasia and skin moles, and particularly preferably cervical intraepithelial neoplasia.

[0099] The fifth aspect of the present invention relates to the use of a compound or a salt thereof according to the first aspect of the present invention in the preparation of a medicament for treating a disease or condition caused by Gram-negative or Gram-positive cocci, bacilli, or coccobacilli.

[0100] The present invention also relates to a method for inhibiting Gram-negative or Gram-positive cocci, bacilli, or coccobacilli, the method comprising contacting the compound or a salt thereof with Gram-negative or Gram-positive cocci, bacilli, or coccobacilli and irradiating with light of a specific wavelength in an inhibitory effective amount. Preferably, the inhibition is for non-therapeutic purposes. The contacting can be carried out in vivo or in vitro, for example, in vitro.

[0101] The present invention also relates to a method for treating a disease or condition caused by Gram-negative or Gram-positive cocci, bacilli, or coccobacilli, which comprises administering to a subject in need a therapeutically effective amount of the compound or a salt thereof and irradiating with light of a therapeutically effective amount of a specific wavelength. Preferably, the subject is a mammal, more preferably a human.

[0102] The compound or a salt thereof according to the first aspect of the present invention, as a photosensitizer for use in photodynamic therapy, specifically for treating a disease or condition caused by Gram-negative or Gram-positive cocci, bacilli, or coccobacilli.

[0103] Preferably, the disease or condition is selected from one or more of the following: periodontitis, gingivitis, dental plaque, and dental caries; preferably, the periodontitis is chronic periodontitis or aggressive periodontitis.

[0104] The Gram-negative or Gram-positive cocci, bacilli, or coccobacilli may be selected from Gram-negative bacilli or coccobacilli, or Gram-positive cocci; for example, Gram-negative anaerobic bacilli or coccobacilli, Gram-negative facultative anaerobic coccobacilli, Gram-negative aerobic bacilli, or Gram-positive facultative anaerobic cocci.

[0105] In a preferred embodiment, the Gram-negative or Gram-positive cocci, bacilli, or coccobacilli are Porphyromonas gingivalis (Porphyromonas gingivalis ), Actinobacillus actinomycetemcomitans ( Actinobacillus actinomycetemcomitans ), Tannerella forsythia ( Tannerella forsythia ), Fusobacterium nucleatum ( Fusobacterium nucleatum ), Prevotella intermedia ( Prevotella intermedia ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa ), or Staphylococcus aureus ( Staphylococcus aureus ); preferably Porphyromonas gingivalis.

[0106] In a preferred embodiment, the disease or condition is selected from one or more of the following: periodontitis, gingivitis, dental plaque, and dental caries; preferably, the periodontitis is chronic periodontitis or aggressive periodontitis;

[0107] Preferably, the disease or condition is selected from one or more of the following: gingival swelling, gingival bleeding, gingival pain, bad breath, periodontal pocket formation, alveolar bone resorption, and tooth loosening.

[0108] In a preferred embodiment, the compound of formula (I) or a salt thereof is used as a photosensitizer in photodynamic therapy for treating cancer or precancerous lesions, particularly cervical cancer or cervical precancerous lesions, or for treating diseases or conditions caused by Gram-negative or Gram-positive cocci, bacilli, or coccobacilli.

[0109] Preferably, the irradiation time of light irradiation is 10 s - 2000 s, preferably 20 - 1500 s, further preferably 30 - 1200 s, more preferably 60 - 900 s; for example, 20 s, 30 s, 60 s, 120 s, 240 s, 480 s, 600 s, 900 s, 1200 s, 1500 s, etc.

[0110] Preferably, the light dose of light irradiation is 1 - 300 J, preferably 5 - 200 J, more preferably 10 J - 180 J, more preferably 20 - 120 J; for example, 10 J, 20 J, 30 J, 40 J, 60 J, 80 J, 100 J, 120 J, 150 J, 180 J, etc.

[0111] Preferably, the wavelength range of light irradiation is 300 nm - 800 nm, preferably 350 nm - 700 nm, more preferably 400 nm - 650 nm, for example, it can be 405 nm, 505 nm, 540 nm, 575 nm, or 630 nm; when treating hyperplastic diseases, it is preferably 630 nm; when treating diseases or conditions caused by Gram-negative or Gram-positive cocci, bacilli, or coccobacilli, it is preferably 405 nm.

[0112] Preferably, the optical power of the light irradiation is 30 mW - 1000 mW, preferably 60 mW - 600 mW, more preferably 80 mW - 300 mW.

[0113] In the context of the present invention, the term "light dose" refers to the actual light dose at the site of light irradiation, which is obtained by multiplying the optical power by the irradiation time.

[0114] In the context of the present invention, the term "optical power" refers to the actual optical power at the site of light irradiation, which is measured by an optical power meter (model: PM100D) purchased from THORLABS to determine the actual power at the treatment site.

[0115] Preferably, in the context of the present invention, the salt is a pharmaceutically acceptable salt.

[0116] The sixth aspect of the present invention relates to a pharmaceutical composition comprising the compound or its salt according to the first aspect of the present invention, optionally further comprising one or more other active compounds. The other active compounds refer to compounds that are generally equally effective in treating hyperplastic diseases or diseases or conditions caused by Gram-negative or Gram-positive cocci, bacilli, or coccobacilli.

[0117] In one embodiment, the pharmaceutical composition is a kit, which further comprises instructions for using the compound or its salt as a photosensitizer in photodynamic therapy.

[0118] In a preferred embodiment, the drug comprises 1 mg - 600 mg, preferably 1 mg - 400 mg, more preferably 2 mg - 300 mg, further preferably 3 mg - 200 mg, further preferably 4 mg - 100 mg of the compound of formula (I) or its salt, such as 2 mg - 500 mg, 2 mg - 400 mg, 2 mg - 300 mg, 3 mg - 240 mg, 5 mg - 120 mg or 5 mg - 60 mg, more specifically such as 2 mg, 4 mg, 10 mg, 12 mg, 15 mg, 18 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 80 mg, 100 mg, 120 mg, 180 mg, 240 mg, 300 mg, 400 mg or 600 mg.

[0119] In a preferred embodiment, the drug further comprises a pharmaceutically acceptable carrier, excipient and / or other adjuvants. When a pharmaceutically acceptable carrier, excipient and / or other adjuvants are included, an effective dose of the compound of formula (I) or its salt and one or more pharmaceutically acceptable carriers, excipients and / or other adjuvants are usually combined to form a suitable administration form 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, usually about 80% by weight. For convenience, other adjuvants such as local anesthetics, preservatives, buffers, etc. can be directly dissolved in the carrier.

[0120] Example

[0121] Detailed synthetic examples of the selected compounds of the present invention are given below. However, these examples are only exemplary and should not be construed as limiting the scope of the present invention in any way.

[0122] Recorded in the form of an NMR peak list in the synthetic examples 1 1H NMR spectral data were obtained on a Bruker 600 MHz nuclear magnetic resonance spectrometer, and the signals listed have the following meanings: s = singlet, d = doublet, m = multiplet, brs = broad singlet. The deuterated solvents used in each case are also specified in the table.

[0123] In the present invention, in addition to the 1H NMR spectral data recorded in the form of the above NMR peak list 1 the structures of the compounds prepared in the synthetic examples were also characterized by liquid chromatography-mass spectrometry (LC-MS). In the present invention, the liquid chromatography-mass spectrometer (LC-MS) used for detecting mass spectral data was purchased from Waters Technology (Shanghai) Co., Ltd., model: SQD2; the medium-pressure column chromatography machine used for compound separation and purification was purchased from Santai Technology (Changzhou) Co., Ltd., model: SepaBean mechine T.

[0124] Unless otherwise stated or defined, scientific and technical terms used in connection with the present invention will have the meanings commonly understood by those of ordinary skill in the art.

[0125] Reagents or instruments not indicated as manufacturers are all commercially available conventional products commonly used in the art. In the present invention, unless otherwise stated, all operations are carried out at room temperature and normal pressure. Unless otherwise stated, the contents and percentages in the context of this application are based on weight.

[0126] A. Synthetic Example

[0127] Example 1: Synthesis of N-01

[0128] Dissolve protoporphyrin (200 mg, 0.36 mmol, 1 equivalent (equ)) in 10 mL of DMF, add EDCI (61 mg, 0.32 mmol, 0.9 equ), HOBT (43 mg, 0.32 mmol, 0.9 equ), DIPEA (41 mg, 0.32 mmol, 0.9 equ), stir for 10 minutes, then add tert-butyl 3-(aminomethyl)morpholine-4-carboxylate (78 mg, 0.36 mmol, 1 equ), and continue to stir at room temperature for 5 hours. Pour the reaction solution into 30 mL of water and extract with 3 × 30 mL of a mixed solution of dichloromethane / methanol (10 / 1, v / v). The organic phase is dried over anhydrous sodium sulfate, filtered, and concentrated. Purify using a normal-phase silica gel column (dichloromethane / methanol containing 5% acetic acid (v / v) from 100% / 0% to 90% / 10%, elution time 15 min). After evaporating the solvent, lyophilize to obtain intermediate N-01-01 (123 mg, yield 45%).

[0129] Dissolve 123 mg of intermediate N-01-01 in 5 mL of 4 mol / L hydrogen chloride dioxane solution, stir for 2 hours, then concentrate under reduced pressure and lyophilize to obtain the target compound N-01 (101 mg, yield 95%).

[0130] LC-MS: 661.7 [M+H] + 。

[0131] 1 1H NMR (600 MHz, CDCl3): δ 11.02 (s, 1H), 10.69 (s, 1H), 10.64 - 10.63 (m, 2H), 8.20 - 8.13 (m, 2H), 6.48 - 6.44 (m, 4H), 4.47 - 4.40 (m, 4H), 3.74 - 3.70 (m, 6H), 3.67 - 3.62 (m, 6H), 3.61 - 3.60 (m, 1H), 3.28 - 3.10 (m, 10H), 2.69 - 2.62 (m, 2H).

[0132] Example 2: Synthesis of N-02

[0133] Dissolve protoporphyrin (200 mg, 0.36 mmol, 1 equ) in 10 mL of DMF, add EDCI (61 mg, 0.32 mmol, 0.9 equ), HOBt (43 mg, 0.32 mmol, 0.9 equ) and DIPEA (41 mg, 0.32 mmol, 0.9 equ). After stirring for 10 minutes, add 2-morpholino-N-(thiophen-2-ylmethyl)ethylamine (81 mg, 0.36 mmol, 1.0 equ) and continue stirring at room temperature for 2 hours. Purify by reverse-phase cyano column (water containing 5% acetic acid / acetonitrile containing 5% acetic acid (v / v) 95% / 5% to 5% / 95%, elution time 20 min). After concentration under reduced pressure, lyophilize to obtain the target compound N-02 (200 mg, yield 73%).

[0134] LC-MS: 771.8 [M+H] + 。

[0135] 1 H NMR (600 MHz, DMSO- d 6): δ 10.08-9.97 (m, 4H), 8.42-8.35 (m, 2H),7.32-7.28 (m, 0.5H), 7.20 (d, J = 4.8 Hz, 0.5H), 6.90 (s, 0.5H), 6.86 (s, 1H),6.67 (br s, 0.5H), 6.41-6.37 (m, 2H), 6.21-6.17 (m, 2H), 4.68-4.65 (m, 2H),4.34-4.28 (m, 4H), 3.64-3.61 (m, 6H), 3.56 (s, 3H), 3.52 -3.51 (m, 3H), 3.29-3.26 (m, 3H), 3.19-3.17 (m, 3H), 3.10-3.05 (m, 10H),-4.39 (s, 2H).

[0136] B. Absorption Spectrogram of the Compounds of the Invention

[0137] To explore the optimal excitation wavelength of the drug, the absorption wavelength of the compound of the present invention was detected. The specific steps are as follows:

[0138] Prepare a dimethyl sulfoxide (brand: Shanghai Runjie) solution of the porphyrin derivative of the present invention with a concentration of 20 μg / mL, 4 mL each, and place it in a cuvette. Use a UV-visible spectrophotometer (purchased from Shanghai Yuanxi Instrument Co., Ltd., model: X-8S) to test the absorption spectrum of the solution in the cuvette.

[0139] As shown Figure 7 in the figure, the absorption wavelengths of the compounds of the present invention are similar, that is, they can absorb the energy at 405, 505, 540, 575 and 630 nm, and especially absorb the strongest energy near 405 nm. According to the above results, the excitation wavelengths of the series of porphyrin derivatives of the present invention are determined to be 405 nm and 630 nm. Among them, the peak shape of protoporphyrin is not smooth, indicating that molecular aggregation exists at this concentration. At the same time, it indicates that the ACQ effect of the compounds of the present invention is weaker than that of protoporphyrin, and it is less likely to occur intermolecular aggregation at the same concentration, and the PDT effect is stronger.

[0140] C. Efficacy Example

[0141] C.1 Treatment of hyperplastic diseases

[0142] Drug information to be tested:

[0143] Compounds N-01 and N-02 of formula (I) of the present invention shown in Table 1 above.

[0144] Compound: Protoporphyrin (PPIX).

[0145] Positive drug 1: 5-Aminolevulinic acid (5-ALA).

[0146] Positive drug 2: Hexyl 5-aminolevulinate (HAL) hydrochloride.

[0147] The mucilage matrix used in the present invention is prepared by the following method:

[0148] Add 90 mL of pure water to a 200 mL beaker, place it on a magnetic stirrer, slowly add 1.5 g of sodium carboxymethylcellulose while stirring, stir for 40 minutes at room temperature, then heat to 40 °C, continue to stir for 30 minutes, and then cool to room temperature while stirring. Then, add 0.5 g of laurocapram to it, and adjust the pH to about 10 with an appropriate amount (about 2 mL) of 1 mol / L sodium hydroxide solution, supplement an appropriate amount of pure water so that the total weight of the contents in the beaker is 100 g, stir evenly, and let it stand overnight to obtain a transparent blank mucilage matrix.

[0149] Example 1: Percutaneous Penetration Experiment

[0150] In order to evaluate the effectiveness of the compounds of the present invention and provide a basis for the in vivo experimental administration method, the in vitro transdermal absorption and intradermal retention of the compounds of the present invention were tested. The specific steps are as follows:

[0151] 1.1 Experimental method

[0152] Using a single-chamber Franz cell of a transdermal diffusion apparatus, with the skin of a miniature pig as the skin for in vitro transdermal absorption, an enzyme-linked immunosorbent assay (ELISA) reader was used to measure the concentration of the compound in the receiving solution at different time points, calculate the cumulative permeation amount, and measure the amount of the compound retained in the skin at the end point of sampling. The specific steps are as follows:

[0153] (1)Preparation of the test preparation:

[0154] Precisely weigh 4 mg each of protoporphyrin, N-01, and N-02, dissolve them in 200 μL of DMSO, and then add them to 4 g of the above-prepared mucilage matrix. Vortex and mix well to prepare a test preparation with a concentration of 1 mg / g.

[0155] (2)Measurement of the fluorescence value of the sample in the receiving cell

[0156] Fix the skin of the miniature pig on the Franz diffusion cell. Add a volume V (about 15 mL) of PBS containing 40% PEG300 to the receiving cell. Add 3 g of the 1 mg / g test preparation to the supply cell. Place the Franz diffusion cell on a TP-6 transdermal diffusion apparatus (purchased from Tianjin Jingtuo Instrument Technology Co., Ltd.), start stirring (constant temperature 32°C, rotation speed 150 rpm), and start timing. Take 0.8 mL of the sample from the receiving cell at 0.5, 1, 2, 3, 4, and 6 hours (h) respectively (immediately supplement 0.8 mL of PBS containing 40% PEG 300). Place the samples at all time points in a 96-well plate, 100 μL per well, with 3 replicates for each sample, and read the fluorescence value Ft at an excitation wavelength of 402 nm and an emission wavelength of 631 nm on an ELISA reader (BioTek Synergy H1).

[0157] (3)Construction of the standard curve for transdermal diffusion amount

[0158] Protoporphyrin, N-01, and N-02 were dissolved in DMSO to prepare a mother liquor with a concentration of 1 mg / mL. Dilute protoporphyrin to 2, 1, 0.5, 0.25, and 0.125 ng / mL with PBS containing 40% PEG 300, and dilute the compound of the present invention to 4, 2, 1, 0.5, and 0.25 ng / mL. Measure the fluorescence value F of each concentration on an ELISA reader s1 . After performing regression analysis, record the standard curve equation y1 of the transdermal diffusion amount and the determination coefficient R 2 .

[0159] (4)Measurement of the fluorescence value of the tissue retention sample

[0160] After the experiment, cut the skin into small pieces, place them in a light-proof centrifuge tube, add 1 mL of DMSO, and ultrasonicate at room temperature for 10 minutes. Place the samples in a 96-well plate, 100 μL per well, with 3 replicates for each sample, and read the fluorescence value F at an excitation wavelength of 402 nm and an emission wavelength of 631 nm on an ELISA reader.

[0161] (5) Construction of Standard Curve for Tissue Retention Samples

[0162] Protoporphyrin, N-01, and N-02 were dissolved in DMSO to prepare a 1 mg / mL stock solution. Protoporphyrin was diluted to 250, 125, 62.5, 31.25, and 15.625 ng / mL, and the compounds of the present invention were diluted to 200, 100, 50, 25, 12.5, and 6.25 ng / mL. The fluorescence values F of each concentration were measured on an enzyme-linked immunosorbent assay (ELISA) reader. s2 After performing regression analysis, the standard curve equation y2 and the coefficient of determination R for tissue retention samples were recorded. 2 .

[0163] (6) Calculation of Cumulative Permeation Amount per Unit Area

[0164] The fluorescence value Ft of the receiving pool sample at each time point was substituted into the standard curve equation y1 of the transdermal diffusion amount to calculate the transdermal diffusion concentration C at each time point. n , and the cumulative permeation amount Q (ng / cm 2 ) per unit area was calculated according to the following formula 1:

[0165] (Formula 1)

[0166] Where C n represents the drug concentration (ng / mL) measured at the nth point, C i represents the drug concentration (ng / mL) measured at the ith point, V represents the total volume of the receiving pool (mL), V0 represents the volume of each sampling (mL), and 1.13 represents the permeation area (cm 2 ).

[0167] (7) Calculation of Tissue Retention Amount per Unit Area

[0168] The fluorescence value F of the tissue retention sample was substituted into the standard curve equation y2 of the tissue retention sample to calculate the concentration C of the tissue retention sample. The tissue retention amount X (ng / cm 2 ) per unit area was calculated according to the following formula 2:

[0169] (Formula 2)

[0170] Where C represents the calculated drug concentration (ng / mL), V represents the volume of DMSO extraction (mL), and 1.13 represents the permeation area (cm 2 ).

[0171] 1.2 Experimental Results

[0172] Figure 1Shows the standard curves of the transdermal diffusion amounts of protoporphyrin and the compounds of the present invention. The R 2 values are all > 0.98, indicating that each test compound has good linearity within the corresponding concentration ranges (protoporphyrin: 0 - 2 ng / mL; compounds of the present invention: 0 - 4 ng / mL).

[0173] Table 2 shows the cumulative permeation amount Q per unit area (ng / cm 2 ) of protoporphyrin and the compounds of the present invention. The results show that the cumulative permeation amount per unit area of N - 01 is about 4 times that of PPIX, and the cumulative permeation amount per unit area of N - 02 is about 2.5 times that of PPIX.

[0174] Table 2: Cumulative permeation amount per unit area of test compounds at different time points

[0175] .

[0176] Figure 2 Shows the standard curves of the tissue retention samples of protoporphyrin and the compounds of the present invention. The R 2 values are all > 0.99, indicating that each test compound has good linearity within the corresponding concentration ranges (protoporphyrin: 15.625 - 250 ng / mL; compounds of the present invention: 6.25 - 200 ng / mL).

[0177] Figure 3 Shows the tissue retention amount X per unit area in the dermis (ng / cm 2 ) of protoporphyrin and the compounds of the present invention after 6 h, indicating that the tissue retention amount per unit area in the dermis of the compounds of the present invention is significantly higher than that of protoporphyrin. Among them, the unit tissue retention amount of N - 02 after 6 h is the largest, being 130 ng / cm 2 (P < 0.0001).

[0178] The transdermal permeation experiment shows that compared with the lead compound PPIX, the compounds N - 01 and N - 02 of the present invention have good solubility and transdermal absorption amount, and can be used for in - vivo pharmacodynamic experiments by local administration.

[0179] Example 2: Efficacy Evaluation of Mouse Cervical In Situ Carcinoma

[0180] 2.1 Animal experiment methods

[0181] (1) Cell culture

[0182] Hela-luc human cervical cancer cells were cultured adherently in vitro. The culture conditions were RPMI-1640 medium supplemented with 10% fetal bovine serum, and they were cultured in a 5% CO2 constant temperature incubator (Phcbi) at 37°C. Routine subculture was performed twice a week. When the cell confluence reached 80%-90% and the cell number reached the requirement, the cells were collected and counted (using a Countstar cell counter), and the cell density was adjusted to 5×10 7 cells / mL with PBS for standby. Then, the Hela-luc cell suspension was mixed with Matrigel at a ratio of 1:1.

[0183] (2)Animal inoculation

[0184] 0.2 mL (5.0×10 6 cells + Matrigel / mouse) of Hela-luc cells were injected into the cervix of Balb / c-nude mice (female, 6-8 weeks old, weighing 18-22 g). On the 8th day after injecting the cancer cells, the tumor formation was observed using a small animal in vivo imager (purchased from Shanghai Tianneng Technology Co., Ltd., model ABL-X5 PRO).

[0185] (3)Experimental grouping and drug administration

[0186] At 2 weeks, 60 mice with appropriate tumor sizes were selected and randomly divided into 10 groups of 6 mice each according to the tumor fluorescence intensity. The day of grouping was recorded as D0, and drug administration started on the day of grouping. Each group was administered the drug once by vaginal application. The drug solutions for each group were freshly prepared according to Table 3 before the experiment and stored in the dark and refrigerated before use.

[0187] Drug preparation and animal grouping were carried out according to Table 3. Each group was administered the drug by vaginal application, and 3 hours after drug administration, treatment was performed using a 630 nm semiconductor laser photodynamic therapy instrument (purchased from Guilin Xingda Optoelectronic Medical Instrument Co., Ltd., model PDT630-II). The light power was 100 mW, the irradiation time was 10 min, and the total energy was 60 J. During light treatment, the optical fiber was inserted through the vagina to irradiate the cervical tumor site as completely as possible.

[0188] Table 3: Experimental grouping design and drug solution preparation table

[0189] 。

[0190] (4)Detection indexes

[0191] After grouping and drug administration, the tumor fluorescence intensity was measured twice a week using an imager, and the body weight of the mice was weighed at the same time. The relative tumor fluorescence intensity was calculated according to the following formula 3:

[0192] (Formula 3)

[0193] Among them, Fi represents the average tumor fluorescence intensity of the experimental group on the i-th day, F c represents the average tumor fluorescence intensity of the blank control group on the i-th day.

[0194] (5)Data processing and statistical analysis

[0195] Taking the days after grouping as the abscissa and the body weight of the mice 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. Ordinary one-way ANOVA in GraphPad Prism8 software was used for data analysis, and p<0.05 was considered to have significant differences.

[0196] 2.2 Experimental results

[0197] (1)Body weight changes

[0198] During the experiment, the body weight changes of the mice in each treatment group on the 0th, 4th, 7th, 11th, and 14th days after administration were as Figure 4 shown, indicating that the mice in all treatment groups had good tolerance.

[0199] (2)Antitumor efficacy evaluation indicators

[0200] The experiment ended completely on the 14th day after grouping. Figure 5 The in vivo imaging diagrams of the mice in the high-dose (4 mg / kg) treatment group of the compound of the present invention at different days after administration are shown. It can be clearly seen from this that at this dose, with the increase of the number of days after treatment, the tumor fluorescence intensity of N-01 and N-02 decreases, indicating that the compound of the present invention has an obvious inhibitory effect on the growth of tumors.

[0201] The relative tumor fluorescence intensities of each test compound calculated based on the tumor fluorescence intensities measured on the 4th, 7th, and 14th days after grouping are summarized in Table 4.

[0202] Table 4: Relative tumor fluorescence intensities of each test compound in each group in %

[0203] 。

[0204] The above results indicate that on the 14th day after drug administration, the tumor growth trend in the group with only light irradiation was consistent with that in the blank control group, suggesting that only light irradiation has no obvious inhibitory effect on the growth of Hela orthotopic tumors. Under the same light conditions, as the administration doses of the compounds N-01 and N-02 of the present invention increased (for example, at doses of 1 mg / kg, 2 mg / kg, and 4 mg / kg), the relative fluorescence intensity of the tumors decreased. The medium-dose (2 mg / kg) and high-dose (4 mg / kg) treatment groups of the compounds of the present invention and the HAL (150 mg / kg) treatment group all had significant inhibitory effects on Hela orthotopic tumors compared with the blank control group. Among them, the compound of the present invention (medium-dose group 2 mg / kg) had a treatment effect equivalent to that of HAL (150 mg / kg), with no statistical difference (P>0.05); the compound of the present invention (high-dose group 4 mg / kg) had a significantly better treatment effect than HAL (150 mg / kg), with a statistical difference (P<0.05); in particular, the relative fluorescence intensity of the drug HAL (150 mg / kg) was 51.5% (P<0.05). In contrast, the relative fluorescence intensities of the compounds N-01 and N-02 of the present invention (4 mg / kg) were 21.0% (P<0.01) and 24.0% (P<0.01), respectively. Therefore, in terms of the overall tumor inhibitory effect, the monosubstituted porphyrin derivatives of the present invention have a drug efficacy equivalent to or even significantly better than that of HAL when the drug dosage is significantly smaller.

[0205] In addition, during the experiment, the overall body weight of the mice was relatively stable and the animal status was good, indicating that the monosubstituted porphyrin derivatives of the present invention have good biosafety.

[0206] C.2 Bacteriostatic effect

[0207] Example 3

[0208] 1. Materials and methods

[0209] 1.1 Information of the drugs to be tested

[0210] Compound N-02 of formula (I) of the present invention shown in Table 1 above.

[0211] Positive drug 1: Protoporphyrin

[0212] Positive drug 2: 5-Aminolevulinic acid

[0213] Solvent: DMSO.

[0214] 1.2 Experimental method

[0215] After resuscitating Porphyromonas gingivalis (purchased from Beijing Beina Chuanglian Biotechnology Research Institute, batch number BNCC353909), inoculate it into BHI medium (brain heart infusion medium) and culture it to the logarithmic phase under anaerobic conditions of 10% H2, 10% CO2, 80% N2, and 37°C; take 1 mL of the bacterial solution, measure the OD value, centrifuge to discard the supernatant, and then add a certain amount of BHI medium to adjust the bacterial solution concentration to 10 10 CFU / mL (1 OD≈9.52*10 9 CFU / mL) for standby.

[0216] Test drug group: Add 100 μL of the bacterial solution, 898 μL of BHI medium, and 2 μL of the DMSO solution of the test drug to the centrifuge tube respectively, and shake well (so that the final concentration of the bacterial solution is 10 9 CFU / mL, the final concentration of the test drug is 10 μg / mL, the final concentration of positive drug 1 is 10 μg / mL, and the final concentration of positive drug 2 is 80 μg / mL). Avoid light, and after culturing for 4 h under the same anaerobic conditions as above, centrifuge to discard the supernatant, then resuspend with 1 mL of BHI medium, add the resuspended solution to a 96-well plate, add 100 μL of the bacterial solution to each well, repeat 3 times for each group, and set 4 replicate wells each time. Irradiate with a 405 nm laser, the laser output power is 300 mW, irradiate for 66.67 s, and the light dose is 20 J. After the light irradiation is completed, continue to culture for 24 h under the same anaerobic conditions as above, and then measure the OD value of each group of bacterial solutions at 600 nm and calculate the antibacterial rate.

[0217] Blank light irradiation group (abbreviated as blank light group): Carry out according to the above method, the difference is: add 100 μL of the bacterial solution and 900 μL of BHI medium to the centrifuge tube.

[0218] Blank non-light irradiation group (abbreviated as blank non-group): Carry out according to the above method, the difference is: add 100 μL of the bacterial solution and 900 μL of BHI medium to the centrifuge tube, and there is no light irradiation treatment.

[0219] Solvent group: Carry out according to the above method, the difference is: add 100 μL of the bacterial solution, 898 μL of BHI medium, and 2 μL of DMSO to the centrifuge tube.

[0220] The calculation formula for the antibacterial rate is as follows:

[0221]

[0222] Among them: OD 阴性 represents the average value of the OD value of the bacterial solution measured in the blank light irradiation group; OD 待测 represents the average value of the OD value of the bacterial solution measured in the test drug group.

[0223] 1.3 Calculation of statistical differences

[0224] Statistical analysis was performed using GraphPad Prism 8 to compare the OD 600 values to evaluate the antibacterial effect. One-way ANOVA was used for multi-group comparison analysis, and post hoc multiple comparisons were performed. A P value < 0.05 was considered to have statistical significance. Among them, * < 0.05, ** < 0.01, *** ＜ 0.001, **** < 0.0001, indicating a significant decrease.

[0225] 2. Results and Discussion

[0226] 2.1 Results of the first biological replicate

[0227] The results are as shown in Figure 6 (a). Compared with the blank light group, the optical density of compound N-02 and the optical density of positive drug 1 were significantly decreased (p < 0.0001), and their antibacterial rates were 44.53% and 32.65% respectively.

[0228] 2.2 Results of the second biological replicate

[0229] The results are as shown in Figure 6 (b). Compared with the blank light group, the optical density of compound N-02 and the optical density of positive drug 1 were significantly decreased (p < 0.0001). The antibacterial rates of N-02 and positive drug 1 were 47.4% and 28.2% respectively.

[0230] 2.3 Results of the third biological replicate

[0231] The results are as shown in Figure 6 (c). Compared with the blank light group, the optical density of compound N-02 and the optical density of positive drug 1 were significantly decreased (p < 0.0001). The antibacterial rates of N-02 and positive drug 1 were 46.8% and 32.4% respectively.

[0232] 2.4 Antibacterial rates of three biological replicate experiments

[0233] The results are as shown in Figure 6 (d) and Table 5. The compound N-02 of the present invention has a significant and relatively stable antibacterial effect, and the effects are significantly better than the control compounds PPIX and 5-ALA, with significant differences (p < 0.0001).

[0234] Table 5: Antibacterial rates of compound N-02, PPIX and 5-ALA of the present invention against Porphyromonas gingivalis

[0235] .

Claims

1. A compound of formula (I) or a salt thereof: (I) Wherein: R represents -NR 1 R 2 , R 1 and R 2 each independently represents hydrogen, a saturated monocyclic heterocyclic group having 2, 3, 4, 5 or 6 carbon atoms and 1 or 2 heteroatoms selected from oxygen, sulfur and nitrogen, a (C1-C6)-alkyl group or a monocyclic heteroaryl (C1-C6)-alkyl group having 3, 4, 5, 6, 7 or 8 carbon atoms and 1 heteroatom selected from oxygen and sulfur, where R 1 and R 2 are not both hydrogen at the same time; Provided that the compound and 。 2. The compound or its salt according to claim 1, wherein, R 1 and R 2 each independently represents hydrogen, a saturated monocyclic heterocyclic group having 3, 4 or 5 carbon atoms and 1 or 2 heteroatoms selected from oxygen, sulfur and nitrogen, a (C1-C6)-alkyl group or a monocyclic heteroaryl (C1-C6)-alkyl group having 3, 4 or 5 carbon atoms and 1 heteroatom selected from oxygen and sulfur.

3. The compound or a salt thereof according to claim 2, wherein, R 1 and R 2 each independently represents hydrogen, morpholine (C1-C6)-alkyl or thiophene (C1-C6)-alkyl.

4. A method for preparing the compound of formula (I) or a salt thereof according to any one of claims 1 to 3, the method comprising subjecting protoporphyrin to a condensation reaction with a compound having a reactive hydrogen atom in a polar organic solvent in the presence of a catalyst, a condensing agent and a condensation activator, wherein the compound having a reactive hydrogen atom is selected from compounds of general formula (II) RH (II) Wherein R is as defined in any one of claims 1 to 3.

5. The method according to claim 4, wherein: The catalyst is an organic base catalyst selected from N,N - diisopropylethylamine, 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 - dimethylpyridine or a mixture thereof; The condensing agent is a carbodiimide - type condensing agent selected from 1 - ethyl - (3 - dimethylaminopropyl)carbodiimide hydrochloride, 1,3 - dicyclohexylcarbodiimide, N,N - diisopropylcarbodiimide or a mixture thereof; The condensation activator is selected from N,N - dimethylaminopyridine, 4 - pyrrolidinopyridine, 1 - hydroxy - 7 - azabenzotriazole, 1 - hydroxybenzotriazole, N - hydroxysuccinimide, N - hydroxyphthalimide, pentafluorophenol; or The molar ratio of the compound having a reactive hydrogen atom to the condensing agent or the condensation activator is 1:(0.6 - 1.5).

6. Use of the compound of formula (I) or a salt thereof according to any one of claims 1 to 3 in the preparation of a photosensitizer for photodynamic therapy.

7. Use of the compound of formula (I) or a salt thereof according to any one of claims 1 to 3 in the preparation of a medicament for the treatment of cervical cancer.

8. Use of the compound of formula (I) or a salt thereof according to any one of claims 1 to 3 in the preparation of a medicament for the treatment of a disease or condition caused by Porphyromonas gingivalis.

9. The use according to claim 8, wherein the disease or condition is selected from one or more of the following: periodontitis, gingivitis, dental plaque and dental caries; or the disease or condition is selected from one or more of the following: gingival swelling, gingival bleeding, gingival pain, bad breath, periodontal pocket formation, alveolar bone resorption and tooth loosening.

10. A pharmaceutical composition comprising the compound or a salt thereof according to any one of claims 1 - 3, optionally further comprising one or more other active compounds.

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