Monoheterocyclic substituted porphyrin derivatives, preparation methods thereof, and use thereof as photosensitizers

Abstract

The present invention relates to compounds of formula (I) or salts thereof, wherein the group R is as defined in the specification; and methods for preparing the compounds and their use as photosensitizers in photodynamic therapy. The monosubstituted protoporphyrin derivatives disclosed herein all exhibit high permeability, high activity, and good biosafety. Their preparation methods are simple, cost-effective, and capable of obtaining the target product in good yield, making them particularly suitable for industrial production. They can be used as highly effective photosensitizers in photodynamic therapy to treat hyperproliferative diseases such as cancer or precancerous lesions, or to treat oral diseases such as periodontitis, gingivitis, dental plaque, and dental caries caused by Gram-negative anaerobic cocci and other bacteria, such as Porphyromonas gingivalis.

Description

Technical Field

[0001] The present invention belongs to the field of medicinal chemistry, and specifically relates to monosubstituted protoporphyrin derivatives, their preparation methods and their use as photosensitizers, and more specifically to monoheterocyclic substituted porphyrin derivatives, their preparation methods and their use as photosensitizers in photodynamic therapy. Background Art

[0002] Photodynamic therapy (PDT) is a novel treatment that uses photosensitizers, light, and oxygen molecules to produce a photodynamic reaction, selectively targeting diseases such as malignant tumors, vascular lesions, and microbial infections. The photosensitizer is the core of PDT. Currently, the main photosensitizers used clinically include Photofrin from the United States, Photogem from Russia, and Photosan from Germany. However, these photosensitizers have numerous drawbacks. For example, they are often mixed preparations composed of porphyrin derivatives, with unclear active ingredients and uncontrolled quality standards. Furthermore, due to their long-term retention in the skin for several weeks, they can easily cause phototoxic effects such as rashes and blisters. Patients must avoid direct sunlight for one month or even longer after treatment.

[0003] 5-Aminolevulinic acid (5-ALA) is a recently developed second-generation photosensitizer. While not inherently photosensitizing, exogenous 5-ALA is selectively absorbed and accumulated by actively proliferating cells, where it is converted into protoporphyrin IX (PPIX). PPIX within cells is a photosensitizer that, upon exposure to red light of a specific wavelength, undergoes a photodynamic reaction, generating reactive oxygen species such as singlet oxygen, which kills actively proliferating cells.

[0004] Cervical cancer is one of the most common tumors of the female reproductive system. Currently, only 5-aminolevulinic acid and 5-aminolevulinic acid hexyl ester (HAL) are used as photosensitizers for photodynamic therapy of cervical precancerous lesions. These are currently in clinical trials. No photosensitizers for the treatment of cervical cancer or precancerous lesions are currently available on the market. Therefore, the development of new photosensitizers is urgently needed to promote the development of this industry.

[0005] Periodontitis is a chronic inflammatory disease primarily caused by the destruction of periodontal tissues by bacteria in dental plaque. Porphyromonas gingivalis, a Gram-negative coccobacillus, is the primary pathogenic bacteria that causes periodontitis and gingivitis. Currently, treatment options include metronidazole tablets and amoxicillin capsules. However, frequent use of antibiotics can lead to drug resistance, and some patients may even experience adverse reactions such as allergies. Therefore, safer, less toxic, and non-invasive alternative treatments are needed.

[0006] In summary, it is of great significance to develop photosensitizers with high efficiency in killing cancerous cells and / or antibacterial ability. Summary of the Invention

[0007] The present invention aims to provide a class of monoheterocyclic substituted porphyrin derivatives, a preparation method thereof, and use thereof as photosensitizers. More specifically, the derivatives are used in photodynamic therapy to treat hyperproliferative diseases, especially cervical cancer or cervical precancerous lesions, or to inhibit bacteria to treat diseases such as periodontitis, gingivitis, dental plaque or caries.

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

[0009]

[0010] in:

[0011] R represents a 3-10 membered heterocyclic group containing at least one N atom, which is unsubstituted or optionally substituted by one or more substituents selected from the group consisting of (C1-C8)-alkyl and halo(C1-C8)-alkyl,

[0012] The 3-10 membered heterocyclic group containing at least one nitrogen atom optionally further contains one or more heteroatoms selected from N, O and S.

[0013] 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 condensation agent and a condensation activator, wherein the compound having a reactive hydrogen atom is selected from the compounds of general formula (II)

[0014] RH (II)

[0015] wherein R is as defined above.

[0016] The third aspect of the present invention relates to use of the compound or salt thereof according to the first aspect of the present invention as a photosensitizer for photodynamic therapy or use in the preparation of a photosensitizer for photodynamic therapy.

[0017] The fourth aspect of the present invention relates to use of the compound or salt thereof according to the first aspect of the present invention in the preparation of a medicament for treating a hyperproliferative disease.

[0018] The present invention also relates to a method for treating a hyperproliferative disease, which comprises administering a therapeutically effective amount of a compound of formula (I) or a salt thereof as described above to a subject in need thereof, and administering a therapeutically effective amount of light irradiation of a specific wavelength, preferably, wherein the subject is a mammal, more preferably a human.

[0019] The fifth aspect of the present invention relates to use of the compound or salt thereof according to the first aspect of the present invention in the preparation of a medicament for treating diseases or conditions caused by Gram-negative or Gram-positive cocci, bacilli or coccobacilli.

[0020] The present invention also relates to a method for inhibiting Gram-negative or Gram-positive cocci, bacilli, or coccobacilli for non-therapeutic purposes, comprising contacting the compound or a salt thereof with Gram-negative or Gram-positive cocci, bacilli, or coccobacilli and irradiating the compound with an effective inhibitory amount of light of a specific wavelength. The contacting can be performed in vivo or in vitro, for example, in vitro.

[0021] 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 a therapeutically effective amount of the compound or a salt thereof to a subject in need thereof and irradiating with a therapeutically effective amount of light of a specific wavelength, preferably, wherein the subject is a mammal, more preferably a human.

[0022] The sixth aspect of the present invention relates to a pharmaceutical composition comprising the compound or a salt thereof according to the first aspect of the present invention, and optionally further comprising one or more other active compounds.

[0023] Surprisingly, the present invention has the following beneficial effects:

[0024] The present invention has developed a novel class of monoheterocyclic substituted porphyrin derivatives that 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, low-cost, and can obtain the target product in good yield, making them particularly suitable for industrial production. They can be used as highly effective photosensitizers in photodynamic therapy to treat hyperproliferative diseases such as cancer or precancerous lesions, particularly cervical cancer or precancerous lesions. Their efficacy is significantly superior to that of the drug 5-ALA, especially when used in significantly smaller dosages, and is comparable to or even significantly superior to that of the drug HAL. They also have excellent antibacterial effects against 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

[0025] Figure 1 The standard curves of the transdermal diffusion amounts of PPIX and the compound N-01 of the present invention in the in vitro transdermal permeation experiment are shown.

[0026] Figure 2 The standard curves of tissue retention samples of PPIX and the compound N-01 of the present invention for 6 hours in an in vitro transdermal permeation experiment are shown.

[0027] Figure 3The 6-hour tissue retention of PPIX and the compound N-01 of the present invention per unit area is shown. Compared with the PPIX group, the compound N-01 group had a significantly higher retention rate than the PPIX group (P<0.05).

[0028] Figure 4 The weight changes of mice in each treatment group after administration are shown. The data points represent the mean weight within the group, and the error bars represent the standard error (SEM).

[0029] Figure 5 The graph shows the in vivo imaging of mice in the high-dose group of compound N-01 of the present invention.

[0030] Figure 6 The optical density (OD) of each test group in the antibacterial experiment and the antibacterial rate against Porphyromonas gingivalis are shown (compared with the blank light group in Figure ac, compared with the PPIX group in Figure d, **** P <0.0001).

[0031] Figure 7 The UV absorption spectra of compounds N-01, N-02, N-03 and N-04 of the present invention are shown. DETAILED DESCRIPTION

[0032] For a better understanding of the present invention, the present invention will be described in detail below with reference to the embodiments and drawings. However, it should be understood that these embodiments and drawings are merely illustrative of the present invention and are not intended to limit the present invention.

[0033] definition

[0034] Unless otherwise specified, the terms "include" and "comprising" used in this application may be open-ended or closed-ended. For example, "include" and "comprising" may mean that other members, elements, or method steps not listed may also be included or comprised, or may mean that only the other members, elements, or method steps listed may be included or comprised.

[0035] As used herein, the term "optionally" means that the subsequently described event, circumstance or material may or may not occur or exist, and that such description includes instances where said event, circumstance or material occurs or exists and instances where said event, circumstance or material does not occur or exist.

[0036] Unless otherwise expressly stated, herein, when referring to a "heterocyclic group containing at least one N atom", it means that the connection of the specified heterocyclic group containing a N atom to the skeleton or the rest of the molecule is through a nitrogen atom.

[0037] As used herein, the term "alkyl" refers to a saturated, straight-chain or branched hydrocarbon radical having in each case the number of carbon atoms specified, such as (C1-C8)-alkyl, (C1-C6)-alkyl and (C1-C4)-alkyl, examples of which 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.

[0038] Unless otherwise defined, the term "heterocyclyl" refers to a saturated or partially saturated monocyclic ring of carbon atoms and at least one heteroatom in the ring. Preferably, the heterocyclyl is a 3-10 membered heterocyclyl, 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 attached to the parent molecular moiety through any carbon atom or nitrogen atom contained in the heterocyclic ring. If the ring contains more than one oxygen atom, they are not directly adjacent. As used herein, examples of heterocyclic groups containing at least one N atom include, but are not limited to, aziridine; azetidinyl; pyrrolidinyl, pyrazolidinyl, imidazolidinyl, isoxazolidinyl, oxazolidinyl, thiazolidinyl, isothiazolidinyl; piperidinyl, hexahydropyridazinyl, hexahydropyrimidinyl, piperazinyl, morpholinyl, 1,2-oxazepanyl, thiomorpholinyl; azepanyl, 1,4-diazepanyl, 1,4-oxazepanyl; pyrrolinyl, dihydroimidazolyl, dihydropyrazolyl, dihydrooxazolyl, dihydrothiazolyl; and thiazinyl.

[0039] As used herein, the expression "membered heterocycle" refers to a saturated or partially saturated hydrocarbon ring system having the specified number of ring atoms and at least one carbon atom in the ring is replaced by a heteroatom (e.g. selected from N, O and S), for example a ring system containing 3 to 10, more preferably 3 to 8, in particular 5 or 6 ring atoms, and at least one N atom and up to three heteroatoms selected from N, O and S. If the ring contains more than one oxygen atom, they are not directly adjacent.

[0040] In the context of the present invention, reference to a salt of a compound of formula (I) means a pharmaceutically acceptable salt thereof, generally a salt that is considered pharmaceutically safe and suitable for use in pharmaceutical formulations. Unless otherwise expressly stated, the term "pharmaceutically acceptable salt" refers to pharmaceutically acceptable acid and base addition salts and solvates. Such pharmaceutically acceptable salts include, but are not limited to, salts of acids such as 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, and the like, preferably acetate. Non-toxic pharmaceutical base addition salts include salts of bases such as sodium, potassium, calcium, ammonium, and the like. Those skilled in the art will recognize a variety of non-toxic pharmaceutically acceptable addition salts.

[0041] As used herein, the term "cancer" includes, but is not limited to, breast cancer, respiratory tract cancer, brain cancer, reproductive organ cancer, digestive tract cancer, urinary tract cancer, liver cancer, eye cancer, skin cancer, head and neck tumors and distant metastases, and also includes multiple myeloma, lymphoma and sarcoma.

[0042] Unless otherwise clearly defined, "precancerous lesions" herein refer to abnormal cell proliferation with a high potential for canceration, examples of which include but are not limited to cervical precancerous lesions, oral leukoplakia, myelodysplastic diseases, familial intestinal polyps, skin nevi, psoriasis, solar keratosis, etc.

[0043] As used herein, the term "treating" means killing, inhibiting or slowing the growth or increase in size of a hyperproliferative cell mass or group or a tumor or cancerous growth, reducing the number of hyperproliferative cells, or preventing spread to other anatomical sites, as well as methods of reducing the size of a hyperproliferative growth or the number of hyperproliferative cells. However, it should be understood that "treating" does not necessarily mean curing or completely eliminating a hyperproliferative growth.

[0044] As used herein, the term "therapeutically effective amount" or "effective amount" or "effective dose" refers to that amount of an active compound or agent that elicits the biological or medicinal response that is being sought or desired by a researcher, physician, or other clinician in a tissue, system, animal, individual, or human.

[0045] Administration and dosage

[0046] The compounds or medicines of the present invention can work systemically and / or locally. For this purpose, they can be administered with a suitable route of administration, for example, by oral, lung, nose, sublingual, tongue, buccal, rectal, vaginal, dermal, transdermal, conjunctival or ear canal administration, or by subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, intrasternal or intracranial injection or infusion, or as implant or stent administration. For example, oral, spray inhalation, rectal administration, nasal administration, vaginal administration, topical administration, parenteral administration such as subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, intrasternal or intracranial injection or input, or by a kind of explanted reservoir medication.

[0047] Preferred routes of administration include oral administration, oral administration, intramuscular injection, topical administration, vaginal administration, rectal administration, intraperitoneal administration, or intravenous administration. Oral administration includes local injection, local application, local irrigation, oral gargle, local controlled / sustained release, and microneedle device administration.

[0048] 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 it can be a novel delivery system, such as an emulsified drug delivery system, a biomimetic drug delivery system, a living cell delivery system, an exosome drug delivery system, a microneedle drug delivery system, a nanodrug delivery system, or a protein or polypeptide delivery system.

[0049] For these administration routes, the compounds according to the invention can be administered in suitable administration forms.

[0050] The medicament of the present invention can be administered in a unit dosage form. The dosage form can be a liquid dosage form, a semisolid preparation, or a solid dosage form. Liquid dosage forms can be true solutions, colloids, microparticle dosage forms, suspension dosage forms, etc. Semisolid dosage forms can be ointments, creams, pastes, gels, etc. Solid dosage forms can be orally disintegrating films, tablets, capsules, pellets, pills, powders, granules, suppositories, lyophilized powder injections, inclusion compounds, implants, patches, etc.

[0051] The compounds according to the invention can be incorporated into the aforementioned administration forms. This can be achieved in a known manner by mixing with pharmaceutically suitable carriers, excipients and / or other auxiliaries.

[0052] The single administration dose of the medicament of the present invention is 0.01-100 mg active ingredient / kg body weight, preferably 0.02-80 mg active ingredient / kg body weight, more preferably 0.05-50 mg active ingredient / kg body weight, still more preferably 0.08-40 mg active ingredient / kg body weight, particularly preferably 0.1-20 mg active ingredient / kg body weight, further preferably 0.1-15 mg active ingredient / kg body weight, for example 0.1-10 mg active ingredient / kg body weight, 0.1-8 mg active ingredient / kg body weight, most preferably 0.2-6 mg active ingredient / kg body weight.

[0053] 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. Herein, the term "active ingredient" refers to a compound of formula (I) or a salt thereof of the present invention.

[0054] plan

[0055] The first aspect of the present invention relates to a compound of formula (I) or a salt thereof:

[0056]

[0057] in:

[0058] R represents a 3-10 membered heterocyclic group containing at least one N atom, which is unsubstituted or optionally substituted by one or more substituents selected from the group consisting of (C1-C8)-alkyl and halo(C1-C8)-alkyl,

[0059] The 3-10 membered heterocyclic group containing at least one nitrogen atom optionally further contains one or more heteroatoms selected from N, O and S.

[0060] The haloalkyl group may be a mono-, di- or tri-substituted fluoro-, chloro-, bromo- or iodo-alkyl group; preferably a fluoroalkyl group, such as a trifluoroalkyl group.

[0061] In a preferred embodiment, R represents a 3-8 membered heterocyclyl containing at least one N atom, which is unsubstituted or optionally substituted by one or more substituents selected from the group consisting of (C1-C6)-alkyl and halo(C1-C6)-alkyl, wherein the 3-8 membered heterocyclyl containing at least one N atom optionally further comprises 1 to 3 heteroatoms selected from N, O and S.

[0062] In a preferred embodiment, the 3-8 membered heterocyclic group containing at least one N atom is a 3-8 membered heterocyclic group containing 1-3 (preferably 1-2) O atoms, 1-3 (preferably 1-2) N atoms and / or 1-3 (preferably 1-2) S atoms.

[0063] In a further preferred embodiment, R represents morpholinyl (e.g. morpholin-4-yl), piperazinyl (e.g. piperazin-1-yl), imidazolidinyl (e.g. imidazolidin-1-yl) or piperazinyl optionally substituted by halogen-substituted alkyl (e.g. 4-(2,2,2-trifluoroethyl)-piperazin-1-yl).

[0064] The definitions of radicals listed above in general terms or in preferred ranges can be combined with one another as desired, ie including combinations between the preferred ranges given.

[0065] Very particular preference is given to the compounds of the formula (I) according to the invention listed in Table 1 below.

[0066] Table 1: Compounds of formula (I), in which R has the meaning given below.

[0067] .Note:" ” indicates connection with the rest of the compound of formula (I).

[0068] A second aspect of the present invention provides a method for preparing a compound of formula (I) or a salt thereof, the method comprising:

[0069]

[0070] In the presence of a catalyst, a condensation agent and a condensation activator, protoporphyrin (PPIX) is subjected to a condensation reaction with a compound having a reactive hydrogen atom in a polar organic solvent, wherein the compound having a reactive hydrogen atom is selected from the compound of formula (II)

[0071] RH (II)

[0072] wherein R is as defined above.

[0073] The catalyst suitable for 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-lutidine or a mixture thereof, more preferably DIPEA, NMI, and particularly preferably DIPEA.

[0074] The condensing agent suitable for 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.

[0075] Examples of condensation activators suitable for the method of the present invention include, but are not limited to, N,N-dimethylaminopyridine (DMAP), 4-pyrrolidinylpyridine (4-PPY), 1-hydroxy-7-azabenzotriazole (HOAT), 1-hydroxybenzotriazole (HOBT), N-hydroxysuccinimide (NHS), N-hydroxyphthalimide (NHPI), pentafluorophenol (PFP), etc., preferably HOBT.

[0076] The polar organic solvent suitable for the method of the present invention may be preferably selected from N,N-dimethylformamide (DMF), N,N-dimethylacetamide, formamide, dimethyl sulfoxide (DMSO), acetone, pyridine or a mixture thereof, more preferably DMF.

[0077] 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).

[0078] In the method of the present invention, the molar ratio of the compound having reactive hydrogen atoms to the condensing agent or condensation activator is 1:(0.6-1.5), preferably 1:(0.7-1.3), and more preferably 1:(0.8-1.1).

[0079] In a preferred embodiment of the present invention, the compound having a reactive hydrogen atom is selected from morpholine, N-tert-butoxycarbonylpiperazine, N-tert-butoxycarbonylimidazoline, and 1-(2,2,2-trifluoroethyl)piperazine.

[0080] Depending on the reaction requirements, the compound having a reactive hydrogen atom may optionally carry an amino-protecting group commonly used in amidation reactions, such as a tert-butoxycarbonyl group (Boc). Removal of the amino-protecting group can be performed by conventional methods known to those skilled in the art, for example, using a solution of trifluoroacetic acid in a specific organic solvent (e.g., dichloromethane, methanol, or N,N-dimethylformamide), preferably a solution of trifluoroacetic acid in dichloromethane.

[0081] The method of the present invention for preparing the compound of formula (I) uses a combination of a specific organic base catalyst, a condensing agent, and a condensation activator, particularly a combination of DIPEA, EDCI, and HOBT in a specific ratio. The method can obtain the target compound of high purity in good yield through simple post-treatment. Excessively low or high amounts of the catalyst, condensing agent, or condensation activator can adversely affect the target product, such as making post-treatment difficult, increasing the proportion of by-products, and reducing the yield of the target product.

[0082] In the method of the present invention, the condensation reaction time is 0.5-15 hours, preferably 1-10 hours, more preferably 2-7 hours.

[0083] In the above-described method for preparing the compounds of the present invention, the method may optionally include additional post-processing steps. These post-processing steps may include, for example, pH adjustment, crystallization, extraction, filtration, concentration under reduced pressure, and drying, among other conventional purification steps. Each of these steps can be performed in conventional manners known to those skilled in the art. If present, extraction is typically performed using a mixture of dichloromethane and methanol, preferably a mixture of dichloromethane and methanol at a ratio of 10:1 (v / v). Drying is typically performed by freeze drying, infrared drying, vacuum drying, or the like, with freeze drying being preferred.

[0084] In a preferred embodiment of the present invention, the method may further comprise a purification step by chromatography. The purification may be performed using a normal phase silica gel column having a silica gel particle size of 30-100 μm, preferably 40-63 μm, a loading of 20-120 g, preferably 40 g, and elution using dichloromethane / methanol containing 5% acetic acid (v / v) (elution gradient 100% / 0% to 90% / 10%, gradient elution time 15 minutes); or a reverse phase cyano column having a particle size of 10-100 μm, preferably 20-45 μm, a loading of 20-120 g, preferably 40 g, and elution using water containing 5% acetic acid / acetonitrile containing 5% acetic acid (v / v) (elution gradient 95% / 5% to 5% / 95%, gradient elution time 20 minutes).

[0085] 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.

[0086] The third aspect of the present invention relates to use of the compound or salt thereof according to the first aspect of the present invention as a photosensitizer for photodynamic therapy or use in the preparation of a photosensitizer for photodynamic therapy.

[0087] The fourth aspect of the present invention relates to use of the compound or salt thereof according to the first aspect of the present invention in the preparation of a medicament for treating a hyperproliferative disease.

[0088] The present invention also relates to a method for treating a hyperproliferative disease, which comprises administering a therapeutically effective amount of a compound of formula (I) or a salt thereof as described above to a subject in need thereof, and administering a therapeutically effective amount of light irradiation of a specific wavelength, preferably, wherein the subject is a mammal, more preferably a human.

[0089] The compound or salt thereof according to the first aspect of the present invention is used as a photosensitizer in photodynamic therapy, specifically for treating hyperproliferative diseases.

[0090] Preferably, the hyperproliferative disease is cancer or a precancerous lesion, and 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 cancer, thyroid cancer, breast cancer, anal cancer, Kaposi's sarcoma, lung cancer, gastric cancer, bile duct cancer, prostate cancer, melanoma and brain cancer, more preferably cervical cancer and skin cancer, particularly preferably cervical cancer; the precancerous lesion is preferably selected from cervical precancerous lesions, oral leukoplakia, myelodysplastic disease, familial intestinal polyps, skin nevus, psoriasis, solar keratosis, more preferably cervical precancerous lesions and skin nevus, particularly preferably cervical precancerous lesions.

[0091] The fifth aspect of the present invention relates to use of the compound or salt thereof according to the first aspect of the present invention in the preparation of a medicament for treating diseases or conditions caused by Gram-negative or Gram-positive cocci, bacilli or coccobacilli.

[0092] The present invention also relates to a method for inhibiting Gram-negative or Gram-positive cocci, bacilli, or coccobacilli, comprising contacting the compound or a salt thereof with Gram-negative or Gram-positive cocci, bacilli, or coccobacilli and irradiating the compound with an effective inhibitory amount of light of a specific wavelength. Preferably, the inhibition is for non-therapeutic purposes. The contacting can be performed in vivo or in vitro, for example, in vitro.

[0093] 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 a therapeutically effective amount of the compound or a salt thereof to a subject in need thereof and irradiating with a therapeutically effective amount of light of a specific wavelength, preferably, wherein the subject is a mammal, more preferably a human.

[0094] The compound or salt thereof according to the first aspect of the present invention is used as a photosensitizer in photodynamic therapy, specifically for treating diseases or conditions caused by Gram-negative or Gram-positive cocci, bacilli or coccobacilli.

[0095] Preferably, the disease or condition is selected from one or more of the following: periodontitis, gingivitis, dental plaque and caries; preferably, the periodontitis is chronic periodontitis or aggressive periodontitis.

[0096] The Gram-negative or -positive cocci, bacilli or coccobacilli can be selected from Gram-negative bacilli or coccobacilli, or Gram-positive cocci; for example, Gram-negative anaerobic bacilli or coccobacilli, Gram-negative facultative anaerobic cocci, Gram-negative aerobic bacilli or Gram-positive facultative anaerobic cocci.

[0097] In a preferred embodiment, the Gram-negative or Gram-positive cocci, bacilli or coccobacilli is 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 gingivalis); preferably Porphyromonas gingivalis.

[0098] In a preferred embodiment, the disease or condition is selected from one or more of the following: periodontitis, gingivitis, dental plaque and caries; preferably, the periodontitis is chronic periodontitis or aggressive periodontitis;

[0099] Preferably, the disease or condition is selected from one or more of the following: swollen gums, bleeding gums, painful gums, bad breath, periodontal pocket formation, alveolar bone resorption and loose teeth.

[0100] In a preferred embodiment, the compound of formula (I) or a salt thereof is used as a photosensitizer in photodynamic therapy to treat cancer or precancerous lesions, in particular cervical cancer or cervical precancerous lesions, or for treating diseases or conditions caused by Gram-negative or Gram-positive cocci, bacilli or coccobacilli.

[0101] 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.

[0102] 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.

[0103] 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, 405 nm, 505 nm, 540 nm, 575 nm or 630 nm; when used to treat hyperproliferative diseases, it is preferably 630 nm; when used to treat diseases or conditions caused by Gram-negative or Gram-positive cocci, bacilli or coccobacilli, it is preferably 405 nm.

[0104] Preferably, the optical power of the light irradiation is 30 mW-1000 mW, preferably 60 mW-600 mW, more preferably 80 mW-300 mW.

[0105] 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.

[0106] In the context of the present invention, the term "optical power" refers to the actual optical power at the light irradiation site, which is measured by an optical power meter (model: PM100D) purchased from THORLABS to determine the actual power at the treatment site.

[0107] Preferably, in the context of the present invention, the salt is a pharmaceutically acceptable salt.

[0108] A sixth aspect of the present invention relates to a pharmaceutical composition comprising a compound or a salt thereof according to the first aspect of the present invention, optionally further comprising one or more other active compounds. The other active compounds are compounds that are generally effective in treating hyperproliferative diseases, or diseases or conditions caused by Gram-negative or Gram-positive cocci, bacilli or coccobacilli.

[0109] In one embodiment, the pharmaceutical composition is a kit further comprising instructions for using the compound or salt thereof as a photosensitizer in photodynamic therapy.

[0110] 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 a compound of formula (I) or a salt thereof, for example 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 for example 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.

[0111] In a preferred embodiment, the medicine also includes pharmaceutically acceptable carriers, excipients and / or other adjuvants. When comprising pharmaceutically acceptable carriers, excipients and / or other adjuvants, usually the compound of formula (I) or its salt and one or more pharmaceutically acceptable carriers, excipients and / or other adjuvants of effective dose are combined to make suitable application form or dosage form, and this program includes mixing, granulating, compressing, dissolving or lyophilizing components by a suitable method. The content of carrier in the medicine can be 1 to 98 weight %, usually accounting for approximately 80 weight %. For convenience, other adjuvants such as local anesthetics, preservatives, buffers can be directly dissolved in the carrier.

[0112] Example

[0113] The following are detailed synthesis examples of selected compounds of the present invention. However, these examples are merely illustrative and should not be interpreted as limiting the scope of the present invention in any way.

[0114] The NMR peaks reported in the synthesis examples are 1 H NMR spectral data were obtained on a Bruker 400 MHz nuclear magnetic resonance spectrometer, and the signals listed have the meanings given below: s = singlet, d = doublet, t = triplet, dd = doublet of doublet, m = multiplet, br s = broad singlet. The deuterated solvents used in each case are also specified in the table.

[0115] In the present invention, in addition to the above-mentioned NMR peak list 1 In addition to H NMR spectral data, the structures of the compounds prepared in the synthetic examples were characterized by liquid chromatography-mass spectrometry (LC-MS). In the present invention, the liquid chromatography-mass spectrometry (LC-MS) instrument used for mass spectrometry data was purchased from Waters Technology (Shanghai) Co., Ltd., Model: SQD2. The medium-pressure column filtration system used for compound separation and purification was purchased from Santai Technology (Changzhou) Co., Ltd., Model: SepaBean Mechine T.

[0116] Unless otherwise explained or defined, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by one of ordinary skill in the art.

[0117] All reagents and instruments used, unless otherwise specified by manufacturer, are commercially available conventional products commonly used in the art. In the present invention, all operations were performed at room temperature and normal pressure unless otherwise specified. Contents and percentages in this application are all based on weight, unless otherwise specified.

[0118] A. Synthesis Examples

[0119] Example 1: Synthesis of N-01

[0120] Protoporphyrin (200 mg, 0.36 mmol, 1 equ) was dissolved in 10 mL of N,N-dimethylformamide (DMF), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 61 mg, 0.32 mmol, 0.9 equ), 1-hydroxybenzotriazole (HOBt, 43 mg, 0.32 mmol, 0.9 equ), and DIPEA (41 mg, 0.32 mmol, 0.9 equ) were added. The mixture was stirred at room temperature for 10 min, and morpholine (31 mg, 0.36 mmol, 1.0 equ) was added. After further stirring at room temperature for 4 h, the reaction solution was poured into 30 mL of water containing 10% citric acid and extracted three times with a mixture of dichloromethane / methanol (10 / 1, v / v) (30 mL × 40 mL). 3), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by normal phase silica gel column (dichloromethane / methanol containing 5% acetic acid (v / v) 100% / 0% to 90% / 10%, elution time 15 min). After evaporation of the solvent, the product N-01 (180 mg, yield 80%) was obtained by lyophilization.

[0121] LC-MS: 632.7 [M+H] + .

[0122] 1 H NMR (400 MHz, DMSO- d 6): δ 10.18-10.09 (m, 4H), 8.45 (dd, J =17.8,11.6 Hz, 2H), 6.42 (d, J =17.6 Hz, 2H), 6.20 (d, J =11.6 Hz, 2H), 4.30-4.26 (m,4H), 3.68-3.67 (m, 6H), 3.58-3.56 (m, 6H), 3.44-3.40 (m, 2H), 3.26-3.22 (m,6H), 3.18-3.14 (m, 2H), 2.97-2.95 (m, 2H).

[0123] Example 2: Synthesis of N-02

[0124] Protoporphyrin (200 mg, 0.36 mmol, 1 equ) was dissolved in 10 mL of DMF, and 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) were added. The mixture was stirred at room temperature for 10 min, and N-tert-butyloxycarbonylpiperazine (67 mg, 0.36 mmol, 1.0 equ) was added. After further stirring at room temperature for 4 h, the reaction solution was poured into 30 mL of water containing 10% citric acid and extracted three times with a mixture of dichloromethane / methanol (10 / 1, v / v) (30 mL × 3). The product was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by a normal phase silica gel column (dichloromethane / methanol containing 5% acetic acid (v / v) 100% / 0% to 90% / 10%, elution time 15 min). min), the solvent was evaporated and the mixture was lyophilized to obtain intermediate N-02-01 (182 mg, yield 70%).

[0125] At 0°C, 180 mg of intermediate N-02-01 was dissolved in a mixed solution of 3 mL of trifluoroacetic acid and 12 mL of dichloromethane. After stirring at this temperature for 2 h, the solution was concentrated under reduced pressure at room temperature and lyophilized to obtain product N-02 (152 mg, yield 97%).

[0126] LC-MS (m / z): 631.7 [M+H] + .

[0127] 1 H NMR (400 MHz, DMSO- d 6): δ 10.16-10.07 (m, 4H), 8.45 (dd, J = 17.6,11.6 Hz, 2H), 6.42 (d, J = 17.6 Hz, 2H), 6.21 (d, J = 11.6 Hz, 2H), 4.32-4.28 (m,4H), 3.677 (m, 6H) 3.59-3.57 (m, 10H), 3.30 (t, J = 7.6 Hz, 2H), 3.18 (t, J = 7.6Hz, 2H), 2.90-2.82 (m, 2H), 2.69-2.63 (m, 2H).

[0128] Example 3: Synthesis of N-03

[0129] Protoporphyrin (200 mg, 0.36 mmol, 1 equ) was dissolved in 10 mL of DMF, and 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) were added. The mixture was stirred at room temperature for 10 min, and N-tert-butyloxycarbonyl imidazoline (61 mg, 0.36 mmol, 1.0 equ) was added. After further stirring at room temperature for 4 h, the reaction solution was poured into 30 mL of water containing 10% citric acid and extracted three times with a mixed solution of dichloromethane / methanol (10 / 1, v / v) (30 mL × 3). The product was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by a normal phase silica gel column (dichloromethane / methanol containing 5% acetic acid (v / v) 100% / 0% to 90% / 10%, elution time 15 min). min), the solvent was evaporated and the mixture was lyophilized to obtain intermediate N-03-01 (190 mg, yield 75%).

[0130] At 0°C, 190 mg of intermediate N-03-01 was dissolved in a mixed solution of 3 mL of trifluoroacetic acid and 12 mL of dichloromethane. After stirring at this temperature for 2 h, the mixture was concentrated under reduced pressure at room temperature and lyophilized to obtain product N-03 (151 mg, yield 92%).

[0131] LC-MS (m / z): 617.6 [M+H] + .

[0132] 1 H NMR (400 MHz, DMSO- d 6): δ 10.19-10.11 (m, 4H), 8.46 (dd, J = 17.6,11.6 Hz, 2H), 6.43 (d, J = 18.0 Hz, 2H), 6.21 (d, J = 11.6 Hz, 2H), 4.32 (br s,4H), 3.70 (s, 3H), 3.69 (s, 3H), 3.60 (s, 3H), 3.59 (s, 3H), 3.36-3.27 (m,6H), 3.18 (t, J = 7.6 Hz, 2H), 3.11-3.08 (m, 2H).

[0133] Example 4: Synthesis of N-04

[0134] Protoporphyrin (200 mg, 0.36 mmol, 1 equ) was dissolved in 10 mL of N,N-dimethylformamide, and 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) were added. The mixture was stirred at room temperature for 10 min, and 1-(2,2,2-trifluoroethyl)piperazine (61 mg, 0.36 mmol, 1.0 equ) was added. After further stirring at room temperature for 4 h, the reaction solution was poured into 30 mL of water containing 10% citric acid and extracted three times with a mixed solution of dichloromethane / methanol (10 / 1, v / v) (30 mL × 40 mL). 3), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by normal phase silica gel column (dichloromethane / methanol containing 5% acetic acid (v / v) 100% / 0% to 90% / 10%, elution time 15 min). After evaporation of the solvent, the product N-04 (205 mg, yield 81%) was obtained by lyophilization.

[0135] LC-MS: 713.7 [M+H] + .

[0136] 1 H NMR (400 MHz, DMSO- d 6): δ 10.15-10.06 (m, 4H), 8.43 (dd, J = 18.0,11.6 Hz, 2H), 6.41 (d, J = 18.0 Hz, 2H), 6.20 (d, J = 11.6 Hz, 2H), 4.29-4.24 (m,4H), 3.66 (s, 3H), 3.65 (s, 3H), 3.57 (s, 3H), 3.55 (s, 3H), 3.48-3.44(m,4H), 3.25-3.21(m, 2H), 3.16-3.13(m, 2H), 2.85-2.78(m, 2H), 2.38-2.36(m, 2H), 2.17-2.14(m, 2H), -4.24 (s, 2H).

[0137] B. Absorption spectra of the compounds of the present invention

[0138] To explore the optimal excitation wavelength for the drug, the absorption wavelengths of compounds N-01, N-02, N-03, and N-04 of the present invention were tested. The specific steps are as follows:

[0139] Prepare 4 mL of each porphyrin derivative of the present invention in dimethyl sulfoxide (brand: Shanghai Runjie) at a concentration of 20 μg / mL and place in a cuvette. Absorption spectra of the solutions in the cuvettes were measured using a UV spectrophotometer (purchased from Shanghai Yuanxi Instrument Co., Ltd., model: X-8S).

[0140] like Figure 7 As shown in Figure 3, the absorption wavelengths of each compound of the present invention are similar, i.e., they can absorb energy at 405, 505, 540, 575, and 630 nm, especially absorbing the strongest energy near 405 nm. According to the above results, it is determined that the excitation wavelengths of the series of porphyrin derivatives of the present invention are 405 nm and 630 nm. Wherein the protoporphyrin peak shape is not smooth, suggesting that the molecule has the situation of molecular aggregation at this concentration. Simultaneously, the ACQ effect of the compounds of the present invention is weaker than protoporphyrin, and is less likely to occur intermolecular aggregation under the same concentration, and the PDT effect is stronger.

[0141] C. Effect Example

[0142] C.1 Treatment of hyperproliferative diseases

[0143] Information of the drug to be tested:

[0144] Compound N-01 of formula (I) of the present invention shown in Table 1 above.

[0145] Compound: Protoporphyrin (PPIX).

[0146] Positive drug 1: 5-aminolevulinic acid (5-ALA).

[0147] Positive drug 2: 5-aminolevulinic acid hexyl ester (HAL) hydrochloride.

[0148] The mortar matrix used in the present invention is prepared by the following method:

[0149] Add 90 mL of purified water to a 200 mL beaker and place it on a magnetic stirrer. Slowly add 1.5 g of sodium carboxymethyl cellulose while stirring. After stirring at room temperature for 40 minutes, heat to 40°C and continue stirring for 30 minutes. Then, cool to room temperature while stirring. Next, add 0.5 g of laurocapram and adjust the pH to approximately 10 with an appropriate amount (approximately 2 mL) of 1 mol / L sodium hydroxide solution. Add an appropriate amount of purified water to bring the total weight of the beaker contents to 100 g. Stir thoroughly and let stand overnight to obtain a transparent blank mortar matrix.

[0150] Example 1: Transdermal penetration experiment

[0151] In order to evaluate the effectiveness of the compounds of the present invention and provide a basis for in vivo experimental administration, the in vitro transdermal absorption and intradermal retention of the compounds of the present invention were tested. The specific steps are as follows:

[0152] 1.1 Experimental Methods

[0153] A transdermal diffusion instrument with a single-chamber Franz cell was used, and miniature pig skin was used as the in vitro transdermal absorption skin. The concentration of the compound in the receiving solution at different time points was measured using a microplate reader. The cumulative permeation amount was calculated, and the amount of compound retained in the skin at the end of sampling was determined. The specific steps are as follows:

[0154] (1) Preparation of test preparation:

[0155] Accurately weigh 4 mg each of protoporphyrin and N-01, dissolve them in 200 μL of DMSO, and add them to 4 g of the above-prepared slurry matrix. Vortex to mix thoroughly to prepare a test preparation with a concentration of 1 mg / g.

[0156] (2) Fluorescence value measurement of receiving pool sample

[0157] Miniature pig skin was fixed in a Franz diffusion cell. A volume V (approximately 15 mL) of PBS containing 40% PEG 300 was added to the receiving cell. 3 g of a 1 mg / g test formulation was added to the donor cell. The Franz diffusion cell was placed in a TP-6 ​​transdermal diffusion instrument (purchased from Tianjin Jingtuo Instrument Technology Co., Ltd.), and stirring (constant temperature at 32°C, rotation speed at 150 rpm) was initiated and timed. 0.8 mL samples were withdrawn from the receiving cell at 0.5, 1, 2, 3, 4, and 6 hours (hours) (immediately replenished with 0.8 mL of PBS containing 40% PEG 300). Samples at all time points were placed in a 96-well plate, 100 μL per well, with triplicate wells for each sample. Fluorescence values ​​(Ft) were read on a microplate reader (BioTek Synergy H1) using excitation at 402 nm and reception at 631 nm.

[0158] (3) Construction of transdermal diffusion standard curve

[0159] Protoporphyrin and N-01 were dissolved in DMSO to prepare a 1 mg / mL stock solution. Protoporphyrin was diluted to 2, 1, 0.5, 0.25, and 0.125 ng / mL, and the compound of the present invention was diluted to 4, 2, 1, 0.5, and 0.25 ng / mL using 40% PEG 300 in PBS. The fluorescence value F at each concentration was measured on a microplate reader. s1 After regression analysis, record the transdermal diffusion standard curve equation y1 and determination coefficient R 2 .

[0160] (4) Fluorescence value determination of tissue retention samples

[0161] After the experiment, the skin was minced and placed in a dark centrifuge tube. 1 mL of DMSO was added and ultrasonicated for 10 minutes at room temperature. The sample was placed in a 96-well plate, with 100 μL per well and three replicates per sample. Fluorescence values ​​(F) were read on a microplate reader using excitation at 402 nm and reception at 631 nm.

[0162] (5) Construction of standard curve for tissue retention samples

[0163] Protoporphyrin and N-01 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 compound of the present invention was diluted to 200, 100, 50, 25, 12.5, and 6.25 ng / mL. The fluorescence value F at each concentration was measured on a microplate reader. s2 After regression analysis, record the tissue retention sample standard curve equation y2 and determination coefficient R 2 .

[0164] (6) Calculation of cumulative infiltration per unit area

[0165] The fluorescence value Ft of the receiving pool sample at each time point is substituted into the transdermal diffusion standard curve equation y1 to calculate the transdermal diffusion concentration C at each time point. n Calculate the cumulative permeability per unit area Q (ng / cm) according to the following formula 1 2 ):

[0166] (Formula 1)

[0167] Among them, C n represents the drug concentration measured at the nth point (ng / mL), C i represents the drug concentration measured at the i-th point (ng / mL), V represents the total volume of the receiving pool (mL), V0 represents the volume of each sampling (mL), and 1.13 represents the penetration area (cm 2 ).

[0168] (8) Calculation of tissue retention per unit area

[0169] Substitute the fluorescence value F of the tissue retention sample into the tissue retention sample standard curve equation y2 to calculate the tissue retention sample concentration C. Calculate the tissue retention amount per unit area X (ng / cm) according to the following formula 2. 2 ):

[0170] (Formula 2)

[0171] Where C represents the calculated drug concentration (ng / mL), V represents the DMSO extraction volume (mL), and 1.13 represents the permeation area (cm2 ).

[0172] 1.2 Experimental Results

[0173] Figure 1 The standard curves of transdermal diffusion of protoporphyrin and compound N-01 of the present invention are shown. 2 The values ​​were all >0.98, indicating that the test compounds had good linearity within the corresponding concentration range (protoporphyrin: 0-2 ng / mL; compounds of the present invention: 0-4 ng / mL).

[0174] Table 2 shows the cumulative permeation per unit area Q (ng / cm 2 The results showed that the cumulative permeation per unit area of ​​N-01 was about 1.26 times that of PPIX.

[0175] Table 2: Cumulative permeation per unit area of ​​the tested compounds at different time points

[0176] .

[0177] Figure 2 The tissue retention sample standard curves of protoporphyrin and compound N-01 of the present invention are shown, R 2 The values ​​were all >0.99, indicating that the test compounds had good linearity within the corresponding concentration range (protoporphyrin: 15.625-250 ng / mL; compound N-01 of the present invention: 6.25-200 ng / mL).

[0178] Figure 3 The intradermal tissue retention amount per unit area X (ng / cm2) of protoporphyrin and compound N-01 of the present invention after 6 hours is shown. 2 ), indicating that the intradermal tissue retention per unit area of ​​compound N-01 of the present invention is significantly higher than that of protoporphyrin, wherein the unit tissue retention of N-01 after 6 hours is 74 ng / cm 2 (P<0.05).

[0179] The transdermal permeation experiment showed that the compound N-01 of the present invention has better solubility and transdermal absorption than the lead compound PPIX, and can be used for in vivo efficacy testing via topical administration.

[0180] Example 2: Evaluation of drug efficacy in mice with cervical carcinoma in situ

[0181] 2.1 Animal Experimental Methods

[0182] (1) Cell culture

[0183] HeLa-luc human cervical cancer cells were cultured in vitro in RPMI-1640 medium supplemented with 10% fetal bovine serum in a 37°C, 5% CO2 incubator (Phcbi). Routine treatment and passage were performed twice a week. When cell saturation reached 80%-90% and the required number was reached, cells were harvested and counted (Countstar cell counter). The cell density was adjusted to 5 × 10 cells / mL using PBS. 7 The Hela-luc cell suspension was then mixed with the matrix gel at a ratio of 1:1.

[0184] (2) Animal vaccination

[0185] 0.2 mL (5.0 × 10 6 HeLa-luc cells (cells + Matrigel / mouse) were injected into the uterine cervix of Balb / c-nude mice (female, 6-8 weeks old, weighing 18-22 g). The model formation was observed on the 8th day after the injection of cancer cells using a mouse living imager (purchased from Shanghai Tianneng Technology Co., Ltd., model ABL-X5 PRO).

[0186] (3) Experimental groups and drug treatment

[0187] At two weeks, 42 mice with tumors of appropriate size were randomly divided into seven groups of six mice each based on tumor fluorescence intensity. Dosing began on the day of grouping, designated D0, with each group receiving a single vaginal application. The drug solution for each group was prepared according to Table 3 immediately prior to the experiment and refrigerated in the dark until use.

[0188] Drug preparation and animal grouping were performed according to Table 3. Each group received topical vaginal administration. Three hours after administration, treatment was initiated with a 630 nm semiconductor laser photodynamic therapy device (purchased from Guilin Xingda Optoelectronics Medical Devices Co., Ltd., model PDT630-II) at a power of 100 mW, an irradiation time of 10 minutes, and a total energy of 60 J. During phototherapy, the optical fiber was inserted transvaginally to fully irradiate the cervical tumor site.

[0189] Table 3: Experimental group design and drug solution preparation

[0190] (4)Detection indicators

[0191] After group administration, tumor fluorescence intensity was measured twice a week using an imager and mice were weighed. The relative fluorescence intensity of the tumor was calculated according to the following formula 3:

[0192] (Formula 3)

[0193] Among them, F i represents the average fluorescence intensity of the tumor in the experimental group on day i, Fc represents the average fluorescence intensity of the tumor in the blank control group on day i.

[0194] (5) Data processing and statistical analysis

[0195] Graphs were plotted using GraphPad Prism 8, with the number of days after grouping as the horizontal axis and the mouse body weight as the vertical axis. Statistical analysis was performed based on data obtained at the end of the experiment to assess differences between groups. Data were analyzed using ordinary one-way ANOVA in GraphPad Prism 8 software, with p < 0.05 considered significant.

[0196] 2.2 Experimental Results

[0197] (1) Weight changes

[0198] During the experiment, the body weight changes of mice in each treatment group on days 0, 4, 7, 11 and 14 after administration were as follows: Figure 4 As shown, all treatment groups were well tolerated.

[0199] (2) Anti-tumor efficacy evaluation indicators

[0200] The experiment was completed on the 14th day after grouping. Figure 5 The following images show in vivo imaging of mice treated with a high dose (4 mg / kg) of compound N-01 at different days post-dosing. It is clear that at this dose, tumor fluorescence intensity decreases with increasing days post-treatment, demonstrating that the compound has a significant inhibitory effect on tumor growth.

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

[0202] Table 4: Relative fluorescence intensity of tumors in % for each group of test compounds

[0203] .

[0204] These results demonstrate that, on day 14 after dosing, the tumor growth trend in the illumination-only group was consistent with that in the blank control group, indicating that illumination alone had no significant inhibitory effect on HeLa orthotopic tumor growth. Under identical illumination conditions, the relative fluorescence intensity of the tumor decreased with increasing doses of compound N-01 (e.g., at doses of 1 mg / kg, 2 mg / kg, and 4 mg / kg). Compared with the blank control group, the medium-dose (2 mg / kg) and high-dose (4 mg / kg) treatment groups of the present invention, as well as the HAL (150 mg / kg) treatment group, all showed significant inhibitory effects on HeLa orthotopic tumors. The therapeutic efficacy of the present compound (medium-dose group, 2 mg / kg) was comparable to that of HAL (150 mg / kg), with no statistically significant difference (P>0.05). The therapeutic efficacy of the present compound (high-dose group, 4 mg / kg) was significantly superior to that of HAL (150 mg / kg), with a statistically significant difference (P<0.05). Specifically, the relative fluorescence intensity of HAL (150 mg / kg) was 51.5% (P<0.05), compared to 25.2% (P<0.01) of the present compound N-01 (4 mg / kg). Therefore, in terms of overall tumor inhibition, the monosubstituted porphyrin derivatives of the present invention were comparable to or even significantly superior to HAL, even at significantly lower dosages.

[0205] In addition, during the experiment, the overall body weight of the mice was relatively stable and the animals were in good condition, indicating that the mono-substituted porphyrin derivatives of the present invention have good biosafety.

[0206] C.2 Antibacterial effect

[0207] Example 3

[0208] 1. Materials and Methods

[0209] 1.1 Information on the drug to be tested

[0210] Compounds N-01, N-02, N-03 and N-04 of the present invention are shown in Table 1 above.

[0211] Positive drug 1: protoporphyrin

[0212] Positive drug 2: 5-aminolevulinic acid

[0213] Solvent: DMSO.

[0214] 1.2 Experimental methods

[0215] Porphyromonas gingivalis (purchased from Beijing Beinachuanglian Biotechnology Research Institute, batch number BNCC353909) was revived and inoculated into BHI medium (brain heart infusion medium) and cultured under anaerobic conditions of 10% H2, 10% CO2, 80% N2, and 37°C until the logarithmic phase; 1 mL of the bacterial solution was taken, the OD value was measured, and after centrifugation and the supernatant was discarded, a certain amount of BHI medium was added to adjust the bacterial solution concentration to 10 10 CFU / mL(1 OD≈9.52*10 9 CFU / mL) for later use.

[0216] Test drug group: add 100 μL bacterial solution, 898 μL BHI medium and 2 μL DMSO solution of the drug to be tested into the centrifuge tube respectively, shake well (the final concentration of bacterial solution is 10 9 CFU / mL, with a final concentration of 10 μg / mL for each test drug, 10 μg / mL for positive drug 1, and 80 μg / mL for positive drug 2. Protected from light, the cells were cultured under the same anaerobic conditions as described above for 4 h, the supernatant was centrifuged and discarded, and the cells were resuspended in 1 mL of BHI medium. The resuspension was then added to a 96-well plate, with 100 μL of bacterial solution added to each well. Each group was repeated three times, with four replicates set up each time. Illumination was performed using a 405 nm laser with a laser output power of 300 mW for 66.67 s and a light dose of 20 J. After illumination, the cells were cultured under the same anaerobic conditions as described above for another 24 h. The OD value of each bacterial solution at 600 nm was measured, and the inhibition rate was calculated.

[0217] Blank light group (abbreviated as blank light group): the above method was followed, except that 100 μL of bacterial solution and 900 μL of BHI medium were added to the centrifuge tube.

[0218] Blank no-light group (abbreviated as blank group): The above method was followed, except that 100 μL of bacterial solution and 900 μL of BHI medium were added to the centrifuge tube without light treatment.

[0219] Vehicle group: The above method was followed, except that 100 μL of bacterial solution, 898 μL of BHI medium and 2 μL of DMSO were added to the centrifuge tube.

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

[0221]

[0222] Among them: OD 阴性 It represents the average OD value of bacterial solution measured in the blank illumination group; OD 待测 It represents the average 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 Prism8 to compare OD 600 The antibacterial effect was evaluated by the difference in values. One-way ANOVA was used for multiple group comparisons, and post hoc multiple comparisons were performed. P < 0.05 was considered statistically significant, among which * < 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 follows Figure 6 As shown in (a), compared with the blank light group, the optical density of compounds N-01, N-02, N-03 and N-04 and the optical density of positive drug 1 were significantly reduced (p<0.0001), and their inhibition rates were 47.05%, 55.14%, 70.23%, 67.90% and 32.65%, respectively.

[0228] 2.2 Results of the second biological replicate

[0229] The results are as follows Figure 6 As shown in (b), compared with the blank light group, the optical density of compounds N-01, N-02, N-03 and N-04 and the optical density of positive drug 1 were significantly reduced (p<0.0001), and their inhibition rates were 57.7%, 56.0%, 67.9%, 71.9% and 28.2%, respectively.

[0230] 2.3 Results of the third biological replicate

[0231] The results are as follows Figure 6 As shown in (c), compared with the blank light group, the optical density of compounds N-01, N-02, N-03 and N-04 and the optical density of positive drug 1 were significantly reduced (p<0.0001), and their inhibition rates were 49.9%, 58.9%, 61.7%, 66.5% and 32.4%, respectively.

[0232] 2.4 Inhibition rate of three biological replicates

[0233] The results are as follows Figure 6 As shown in (d) and Table 5, compounds N-01, N-02, N-03 and N-04 of the present invention have significant and relatively stable antibacterial effects, and the effects are significantly better than those of the control compounds PPIX and 5-ALA, with significant differences (p<0.0001).

[0234] Table 5: Inhibitory effects of the compounds of the present invention, PPIX, and 5-ALA on Porphyromonas gingivalis

[0235] .

Claims

1. A compound of formula (I) or a salt thereof: (I) in: R represents a 3-8 membered saturated monocyclic heterocyclic group containing at least one N atom, wherein the heterocyclic group is unsubstituted or optionally substituted by one or more substituents selected from the group consisting of (C1-C8)-alkyl and halo(C1-C8)-alkyl, The heterocyclic group optionally further comprises one or more heteroatoms selected from N, O and S.

2. The compound or salt thereof according to claim 1, wherein R represents a 5-6 membered saturated monocyclic heterocyclic group containing at least one N atom, wherein the heterocyclic group is unsubstituted or optionally substituted by one or more substituents selected from the group consisting of (C1-C6)-alkyl and halo(C1-C6)-alkyl, The heterocyclic group optionally further comprises 1 to 3 heteroatoms selected from N, O and S.

3. The compound or salt thereof according to claim 2, wherein R represents morpholin-4-yl, piperazin-1-yl, imidazolidin-1-yl or 4-(2,2,2-trifluoroethyl)-piperazin-1-yl.

4. A method for preparing a compound of formula (I) or a salt thereof according to any one of claims 1 to 3, 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 a compound 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-lutidine 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-pyrrolidinylpyridine, 1-hydroxy-7-azabenzotriazole, 1-hydroxybenzotriazole, N-hydroxysuccinimide, N-hydroxyphthalimide, pentafluorophenol; or The molar ratio of the compound having reactive hydrogen atoms to the condensing agent or condensation activator is 1:(0.6-1.5).

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

8. Use of a compound of formula (I) or a salt thereof according to any one of claims 1 to 3 in the preparation of a medicament for treating 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 caries; or the disease or condition is selected from one or more of the following: swollen gums, bleeding gums, painful gums, bad breath, periodontal pocket formation, alveolar bone resorption and loose teeth.

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

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