阅读说明：本技术 Pdt化合物及其制备方法和用途 (PDT compounds, methods of preparation and uses thereof ) 是由 张俊龙 杨字舒 王炳武 张洪 于 2020-05-13 设计创作，主要内容包括：本发明提供了PDT化合物及其制备方法和用途。通过轴向配体、离子配体和结构调整的卟啉类化合物形成稳定的配合物,使化合物的吸收光谱实现红移,得到了光毒性强、组织穿透深度大、生物相容性良好的PDT化合物,同时还兼具光热效应和荧光性能,有助于提高治疗效果和降低光动力治疗对机体的损伤。(The invention provides PDT compounds, methods of preparation and uses thereof. The stable complex is formed by the axial ligand, the ionic ligand and the porphyrin compound with the adjusted structure, so that the absorption spectrum of the compound realizes red shift, the PDT compound with strong phototoxicity, large tissue penetration depth and good biocompatibility is obtained, and the PDT compound also has the photothermal effect and the fluorescence property, and is beneficial to improving the treatment effect and reducing the damage of photodynamic therapy to the organism.)

A PDT compound, or a pharmaceutically acceptable salt, solvate, non-covalent complex or prodrug thereof, comprising the structure:

l is an axial ligand selected fromTripodal stool ligand, acetylacetone-bidentate ligand, H₂O or DMSO solvent ligand;

m is an ionic ligand selected from one of La, Sm, Eu, Gd, Sc, Y, Ce, Pr, Nd, Pm, Tb, Dy, Ho, Er, Tm, Yb, Lu, Zn, Pd, Pt, Rh, Si and Ni;

X₁and X₂Is a five-membered ring residue, and carbon atoms at two ends of the five-membered ring residue are connected with N atoms on a porphyrin ring to form a ring;

X₅、X₆、X₇、X₈each independently selected from one of fluorine, chlorine, bromine, carboxyl, nitrile group, nitro group, aldehyde group, acyl group and amide group;

Ar₁、Ar₂、Ar₃、Ar₄each independently isy₁、y₂、y₃、y₄And y₅Each independently selected from hydrogen, fluorine, chlorine, bromine, hydroxyl, amino, C_1-3Alkyl substituted amino, sulfonic acid group, mercapto and C_1-3Alkyl substituted silicon group.

2. A PDT compound according to claim 1,

the ionic ligand M is a metal ionic ligand, preferably Lu³⁺、Gd³⁺、Eu³⁺、Yb³⁺、Er³⁺、Zn²⁺、Pd²⁺、Pt²⁺、Rh³⁺And Ni²⁺One of (1); more preferably Lu³⁺、Gd³⁺、Eu³⁺、Yb³⁺、Er³⁺One of (1); and/or the presence of a gas in the gas,

X₁and X₂Each independently selected from the group consisting of five-membered rings, which are bonded to the N atom of the porphyrin ring One kind of (1).

3. A PDT compound according to claim 1,

the axial ligand L isA trisodium ligand, preferably one of tris (dimethyl phosphite) cyclopentadienyl cobalt (III) acid-based ligand, deuterated or halogenated tris (dimethyl phosphite) cyclopentadienyl cobalt (III) acid-based ligand, tris (diethyl phosphite) cyclopentadienyl cobalt (III) acid-based ligand, and trispyrazolyl hydroborate ligand; and/or the presence of a gas in the gas,

X₅、X₆、X₇、X₈each independently selected from one of fluorine, chlorine, bromine, carboxyl, nitro and aldehyde group; and/or the presence of a gas in the gas,

Ar₁、Ar₂、Ar₃、Ar₄of (1), y is preferred₁、y₂、y₃、y₄、y₅Each independently selected from one of hydrogen, fluorine, chlorine, bromine, hydroxyl, amino and sulfonic acid groups.

4. A PDT compound as claimed in claim 1, wherein said PDT compound is selected from one or more of the following structures:

5. a process for the preparation of a PDT compound as claimed in any one of claims 1 to 4, comprising:

the method comprises the following steps: under the anaerobic condition, reacting porphyrin and acetylacetone salt of the ionic ligand M for 1-3 h at 200-240 ℃ to obtain an intermediate I; reacting the intermediate I with a sodium salt of an axial ligand L in a mixed solvent of chloroform/methanol-1/1 to obtain an intermediate I;

step two: under the atmosphere of inert gas, dissolving the intermediate product I in an organic solvent I, dripping a reducing agent at the temperature of-100 to-60 ℃, recovering the room temperature, and carrying out a light-resistant reaction until the reaction is finished to obtain the PDT compound.

6. The method of claim 5, wherein, in the first step,

reacting porphyrin and acetylacetone salt of ionic ligand M in trichlorobenzene until the reaction is finished, and separating by silica gel column chromatography to obtain an intermediate I;

reacting the intermediate I with the sodium salt of the axial ligand L at 50-80 ℃ and preferably 50-70 ℃ for 1-3 h, separating by silica gel column chromatography, and performing petroleum ether: ethyl acetate (10-15): 1, to give intermediate i.

7. The process according to claim 5, wherein in the second step, the organic solvent I is one or more selected from the group consisting of tetrahydrofuran, 2-methyltetrahydrofuran, 1, 4-dioxane and dichloromethane, preferably tetrahydrofuran;

the reducing agent is selected from diisobutyl aluminum hydride, lithium triethoxy aluminum hydride or sodium borohydride, and is preferably diisobutyl aluminum hydride;

and (3) removing the reaction solvent after the reaction is finished, and separating the solid substance by using column chromatography to obtain the PDT compound.

8. A pharmaceutical composition comprising as active ingredient a PDT compound as defined in any of claims 1 to 4 or a PDT compound obtainable by a process as defined in any of claims 5 to 7, together with pharmaceutically acceptable excipients;

the pharmaceutical composition is administered by injection;

in unit dosage forms of the pharmaceutical composition, the amount of active ingredient is 0.01mg-10 g.

9. Use of a PDT compound, a pharmaceutically acceptable salt, solvate, non-covalent complex or prodrug as claimed in any one of claims 1 to 4, and a pharmaceutical composition comprising a PDT compound as active ingredient, in photodynamic therapy and/or photothermal therapy;

preferably, the application in preparing the medicine for photodynamic therapy and photothermal therapy of tumors.

10. Use of a PDT compound, pharmaceutically acceptable salt, solvate, non-covalent bond complex or precursor according to any of claims 1 to 4 in the near infrared region 700 and 900nm for fluorescence labeling or fluorescence imaging, photoacoustic imaging.

Technical Field

The present invention relates to the field of photodynamic therapy (PDT), in particular to photosensitive compounds, methods of preparation and uses thereof.

Background

After the photosensitive compound is enriched in the pathological tissue, the photosensitive compound is activated by light irradiation, and the activated photosensitive compound kills the focus to achieve the purpose of treating diseases, namely photodynamic therapy (PDT). Light-sensitive compounds are compounds that play a decisive role in photodynamic therapy.

PDT has the advantages of high selectivity, no wound or tiny wound, repeated treatment, low toxicity and the like, and increasingly plays an important role in the comprehensive treatment of tumors. However, PDT also has some disadvantages: in PDT, photosensitizing compounds generally need to be used in conjunction with nanoparticles with luminescent properties to determine the aggregation of photosensitizing compounds within the body, making the composition of clinical photosensitizers complex; in addition, the clearance rate of the photosensitive compound in normal tissues is roughly judged according to the half-life period of the photosensitive compound (or photosensitizer) in the body in clinic to determine the time of the light therapy, which also results in low treatment selectivity and strong systemic photosensitive side effect of the body.

In addition, in photodynamic therapy, the infrared wavelength of the photosensitive compound can significantly influence the phototoxicity and the tissue penetration depth, and the longer the infrared wavelength is, the deeper the tissue penetration depth of the photosensitive compound is, the more beneficial the phototoxicity is exerted.

Compounds useful in photodynamic therapy are PDT compounds. At present, porphyrin compounds are hot research spots of PDT compounds, but most compounds are only suitable for diagnosis or photodynamic therapy, the diagnosis and the therapy cannot be realized simultaneously, and the tissue penetration depth is also very limited.

Therefore, the research on the PDT compound with both diagnostic and therapeutic effects has practical significance for simplifying photosensitizer, improving the penetration depth of photodynamic therapy tissue and reducing the injury of body tissue.

Disclosure of Invention

In order to solve the above problems, the present inventors have conducted intensive studies and, as a result, have found that: the structure of the porphyrin compound is adjusted, so that the absorption spectrum of the compound realizes red shift, and the PDT compound with strong phototoxicity, large tissue penetration depth and good biocompatibility is obtained. The present invention has the advantages of both the combined therapeutic effect of photothermal therapy and fluorescence imaging, and can realize diagnosis and therapy simultaneously, thus contributing to the improvement of photodynamic therapy effect and the simplification of the composition of photosensitizer, and thus completing the present invention.

The object of the present invention is to provide the following:

in a first aspect, the present invention provides a PDT compound, or a pharmaceutically acceptable salt, solvate, non-covalent complex or prodrug thereof, comprising the structure:

l is an axial ligand selected fromTripodal stool ligand, acetylacetone-bidentate ligand, H₂O or DMSO solvent ligand;

m is an ionic ligand selected from one of La, Sm, Eu, Gd, Sc, Y, Ce, Pr, Nd, Pm, Tb, Dy, Ho, Er, Tm, Yb, Lu, Zn, Pd, Pt, Rh, Si and Ni;

X₁and X₂Is a five-membered ring residue, and carbon atoms at two ends of the five-membered ring residue are connected with N atoms on a porphyrin ring to form a ring;

X₅、X₆、X₇、X₈each independently selected from one of fluorine, chlorine, bromine, carboxyl, nitrile group, nitro group, aldehyde group, acyl group and amide group;

Ar₁、Ar₂、Ar₃、Ar₄each independently isy₁、y₂、y₃、y₄And y₅Each independently selected from hydrogen, fluorine, chlorine, bromine, hydroxyl, amino, C_1-3Alkyl substituted amino, sulfonic acid group, mercapto and C_1-3Alkyl substituted silicon group.

In a second aspect, the present invention provides a process for the preparation of a PDT compound as defined above, comprising:

the method comprises the following steps: under the anaerobic condition, reacting porphyrin and acetylacetone salt of the ionic ligand M for 1-3 h at 200-240 ℃ to obtain an intermediate I; reacting the intermediate I with a sodium salt of an axial ligand L in a mixed solvent of chloroform/methanol-1/1 to obtain an intermediate I;

step two:

under the atmosphere of inert gas, dissolving the intermediate product I in an organic solvent I, dripping a reducing agent at the temperature of-100 to-60 ℃, recovering the room temperature, and carrying out a light-resistant reaction until the reaction is finished to obtain the PDT compound.

In a third aspect, the invention provides a pharmaceutical composition, which comprises the PDT compound as an active ingredient, and a pharmaceutically acceptable excipient;

the pharmaceutical composition is administered by injection;

in unit dosage forms of the pharmaceutical composition, the amount of active ingredient is 0.01mg-10 g.

In a fourth aspect, the present invention provides the use of a PDT compound, a pharmaceutically acceptable salt, solvate, non-covalent complex or prodrug as described above, and a pharmaceutical composition comprising a PDT compound as active ingredient, in photodynamic therapy and photothermal therapy;

preferably, the application in preparing the medicine for photodynamic therapy and/or photothermal therapy of tumors.

In a fifth aspect, the invention provides the use of a PDT compound, pharmaceutically acceptable salt, solvate, non-covalent bond complex or precursor as described above in the near infrared region 700-900nm for fluorescence labeling or fluorescence imaging, photoacoustic imaging.

The PDT compound provided by the invention and the preparation method and the application thereof have the following beneficial effects:

(1) the PDT compound provided by the invention has photophysical properties of bacteriochlorin, not only has second type photodynamic therapy capability (namely generating singlet oxygen), but also can generate active oxygen species such as superoxide anion free radicals, hydroxyl free radicals and the like, and can be used for first type photodynamic therapy, so that the treatment effect is effectively improved, and the damage to an organism is reduced;

(2) the PDT compound provided by the invention has the advantages that the triplet state energy level is reduced, so that molecules do not emit phosphorescence, and non-radiative transition is enhanced, so that the PDT compound has stronger photothermal effect while being used as a triplet state sensitizer to generate active oxygen species, and has combined effects of photodynamic therapy and photothermal therapy;

(3) the absorption spectrum of the PDT compound provided by the invention is in a near infrared region, so that the exciting light has larger tissue penetration depth, and the PDT compound is helpful for reducing the damage of a light source to the tissue during photodynamic therapy;

(4) the PDT compound provided by the invention has stronger fluorescence, and simultaneously realizes the functions of fluorescence imaging, photodynamic therapy and photothermal therapy.

Drawings

FIG. 1 shows the preparation of the compound of example 1¹H NMR spectrum;

FIG. 2 shows the HR-MS spectrum of the compound prepared in example 1;

FIG. 3 shows the FT-IR spectrum of the compound obtained in example 1;

in FIG. 4, (1) shows an absorption spectrum in Experimental example 1, (2) shows an emission spectrum;

fig. 5 (1) shows a singlet oxygen phosphorescence emission curve of experimental example 2; (2) showing a superoxide radical generation curve; (3) shows hydroxyl radical generation curves;

fig. 6 (1) shows a graph of the temperature change of the dispersion; (2) showing the infrared thermographic variation of the dispersion;

FIG. 7 shows cytotoxicity profiles in Experimental example 5;

FIG. 8 shows the results of PCR detection in Experimental example 6;

FIG. 9 shows the flow cytometer detection results in Experimental example 6;

FIG. 10 shows a graph of cellular fluorescence imaging of compound Gd-1 in Experimental example 7;

FIG. 11 is a fluorescence image of a living animal and an organ in Experimental example 8;

FIG. 12 shows a MRI image of the compound of Experimental example 9;

fig. 13 shows a photoacoustic imaging graph of the compound in experimental example 10;

FIG. 14 is a thermal image of a living animal in Experimental example 11;

FIG. 15 (1) shows a tumor volume change curve of a mouse in Experimental example 12; (2) shows a mouse survival rate change curve; (3) the body weight change curve of the mice is shown.

Detailed Description

The features and advantages of the present invention will become more apparent and appreciated from the following detailed description of the invention, as illustrated in the accompanying drawings. The word "exemplary" is used exclusively herein to mean "serving as an example, embodiment, or illustration. Any embodiment described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments.

The present invention is described in detail below.

A PDT compound, or a pharmaceutically acceptable salt, solvate, non-covalent bond complex, complex or prodrug thereof, comprising the structure:

wherein L is an axial ligand selected fromTripodal stool ligand, acac (acetylacetone) -bidentate ligand, H₂O or DMSO solvent ligand;

m is an ion ligand selected from one of lanthanum (La), samarium (Sm), europium (Eu), gadolinium (Gd), scandium (Sc), yttrium (Y), cerium (Ce), praseodymium (Pr), neodymium (Nd), promethium (Pm), terbium (Tb), dysprosium (Dy), holmium (Ho), erbium (Er), thulium (Tm), ytterbium (Yb), lutetium (Lu), zinc (Zn), palladium (Pd), platinum (Pt), rhodium (Rh), silicon (Si) and nickel (Ni) ions;

X₁and X₂Is a five-membered ring residue, and carbon atoms at two ends of the five-membered ring residue are connected with N atoms on a porphyrin ring to form a ring;

X₅、X₆、X₇、X₈each independently selected from one of fluorine, chlorine, bromine, carboxyl, nitrile group, nitro group, aldehyde group, acyl group and amide group;

Ar₁、Ar₂、Ar₃、Ar₄each independently isy₁、y₂、y₃、y₄,y₅Each independently selected from hydrogen, fluorine, chlorine, bromine, hydroxyl, amino, C_1-3Alkyl substituted amino, sulfonic acid group, mercapto and C_1-3Alkyl substituted silicon group. On a single bond(wavy bond) means that the single bond is linked to other groups.

For increasing the stability of the PDT compound, it is preferred that the axial ligand L isThe trisilament ligand is more preferably one of a tris (dimethyl phosphite) -cyclopentadienyl cobalt (III) acid-based ligand, a deuterated or halogenated tris (dimethyl phosphite) -cyclopentadienyl cobalt (III) acid-based ligand, a tris (diethyl phosphite) -cyclopentadienyl cobalt (III) acid-based ligand and a trispyrazolyl hydroborate ligand.

In some preferred embodiments, the axial ligand L is a tris (dimethyl phosphite) -cyclopentadienyl cobalt (iii) acyloxy ligand, a tris (diethyl phosphite) -cyclopentadienyl cobalt (iii) acyloxy ligand or a trispyrazolyl hydroboronic acid ligand.

The ionic ligand M can enhance the stability of compound molecules, and further, the ionic ligand M is a metal ion ligand, preferablyIs lutetium (Lu)³⁺) Gadolinium (Gd)³⁺) Europium (Eu)³⁺) Ytterbium (Yb)³⁺) Erbium (Er)³⁺) Zinc (Zn), zinc (Zn)²⁺) Palladium (Pd)²⁺) Platinum (Pt)^{2 +}) Rhodium (Rh)³⁺) And nickel (Ni)²⁺) One of the ions.

In some preferred embodiments, the ionic ligand M is a rare earth ionic ligand selected from lutetium (Lu)³⁺) Gadolinium (Gd)^{3 +}) Europium (Eu)³⁺) Ytterbium (Yb)³⁺) Erbium (Er)³⁺) One kind of (1). The reason is that after the rare earth ions are coordinated, the cross-over among molecular systems is enhanced due to the heavy atom effect, the capability of generating active oxygen species is enhanced, and the photodynamic therapy capability is stronger; meanwhile, the service life of the molecular excited singlet state is shortened, and the photoacoustic signal is favorably generated.

More preferably, the ionic ligand M is lutetium (Lu)³⁺) Or gadolinium (Gd)³⁺) This is due to the fact that³⁺Plasma phase of rare earth ions, Lu³⁺Has a full 4f orbit, no f-f transition occurs; gd (Gd)³⁺Has higher 4f-4f transition energy level, can not be sensitized by porphyrin compounds, so that the energy transfer process from ligand to metal does not occur in the coordination compound of the two metals, and the coordination compound can be used as PDT and PTT (photo-thermal therapy) reagents.

In addition, the rare earth ion lutetium (Lu)³⁺) Or gadolinium (Gd)³⁺) The method has moderate spin-orbit coupling constant and more reasonable distribution of excited state distribution. After illumination, the rare earth complex can generate more ROS (reactive oxygen species) and simultaneously retain stronger fluorescence, and has the effects of fluorescence imaging, photodynamic therapy and photothermal therapy.

Further, X₁And X₂Each independently selected from the group consisting of five-membered rings, which are bonded to the N atom of the porphyrin ring One kind of (1). Wherein, singly or doubly bound(wavy bond) means that the single bond or double bond is linked to other groups.

More preferably, X₁And X₂Each independently selected from the group consisting of five-membered rings, which are bonded to the N atom of the porphyrin ring

The inventor finds that, at the moment, the absorption spectrum of the PDT compound is red-shifted to a near infrared region (700-900nm), and the extinction coefficient is increased, so that the molecules of the compound have the possibility of photoacoustic imaging; the red-shifted exciting light has larger penetration depth, and the damage of a light source to tissues is also reduced during photodynamic therapy; moreover, the PDT compound also retains strong fluorescence, so that the PDT compound can be used as a fluorescence imaging probe.

X₅、X₆、X₇、X₈Each independently selected from one of fluorine, chlorine, bromine, carboxyl, nitro and aldehyde group; preferably X₅、X₆、X₇、X₈Each independently selected from fluorine, chlorine or bromine.

In a preferred embodiment, X₅、X₆、X₇、X₈The fluorine is a fluorine group, and the fluorine element can improve the reactivity of the porphyrin macrocycle, thereby being beneficial to the preparation of PDT compounds; in addition, the vibration energy of the C-F bond is smaller than that of the C-H bond, so that the heat energy loss caused by vibration relaxation can be reduced, and the fluorescence intensity is improved.

Ar₁、Ar₂、Ar₃、Ar₄Of (1), y is preferred₁、y₂、y₃、y₄、y₅Each independently selected from one of hydrogen, fluorine, chlorine, bromine, hydroxyl, amino and sulfonic acid groups.

In a preferred embodiment, Ar₁、Ar₂、Ar₃、Ar₄Is phenyl or

The PDT compounds provided by the present invention are selected from one or more of the following structures:

the invention also provides a preparation method of the PDT compound, which comprises the following steps:

the method comprises the following steps: under the anaerobic condition, reacting porphyrin and acetylacetone salt of the ionic ligand M for 1-3 h at 200-240 ℃ to obtain an intermediate I; reacting the intermediate I with a sodium salt of an axial ligand L in a mixed solvent of chloroform/methanol-1/1 to obtain an intermediate I;

step two:

under the atmosphere of inert gas, dissolving the intermediate product I in an organic solvent I, dripping a reducing agent at the temperature of-100 to-60 ℃, recovering the room temperature, and carrying out a light-resistant reaction until the reaction is finished to obtain the PDT compound.

In the first step, preferably, the acetylacetone salt of the ionic ligand M is a sodium salt or a potassium salt, and the molar weight of the acetylacetone salt is 1-2 times of that of the porphyrin; porphyrin and acetylacetone salt of the ionic ligand M react in trichlorobenzene till the end, and the intermediate I is obtained by silica gel column chromatography separation.

In the separation of the intermediate I, preferably, petroleum ether is used for removing a reaction solvent, dichloromethane is used for removing an unreacted reactant, and a mixed solution of dichloromethane/methanol (4-6)/1 is used for eluting the intermediate I; more preferably dichloromethane/methanol-5/1.

Furthermore, the molar weight ratio of the intermediate I to the sodium salt of the axial ligand L is 1:1, and the reaction is carried out for 1-3 h at 50-80 ℃, preferably 50-70 ℃.

After the reaction was completed, the reaction solvent was removed, and the solid was separated by silica gel column chromatography, petroleum ether: ethyl acetate (10-15): 1, to give intermediate i.

In order to improve the purity of the intermediate product I, the intermediate product I can be recrystallized. In a preferred embodiment, the intermediate i can be prepared in the presence of (1 to 20) dichloromethane/n-hexane: 10, more preferably (1-20): 10 in a solvent mixture.

In the second step, the inert gas atmosphere refers to a nitrogen atmosphere, a carbon dioxide atmosphere, a helium atmosphere, an argon atmosphere or a hydrogen atmosphere.

The organic solvent I is one or more selected from tetrahydrofuran, 2-methyltetrahydrofuran, 1, 4-dioxane and dichloromethane, and tetrahydrofuran is preferably used as a reaction solvent.

The reducing agent is selected from diisobutylaluminum hydride, lithium triethoxyaluminum hydride or sodium borohydride, preferably diisobutylaluminum hydride as reducing agent to reduce the lactone group on the porphyrin macrocycle of intermediate I in excess relative to intermediate I to allow sufficient reaction of intermediate I.

In a preferred embodiment, the ratio of diisobutylaluminum hydride to intermediate I is 0.2 to 0.35ml/40mg, for example 0.25ml/40 mg.

And after the dropwise addition of the diisobutyl aluminum hydride is finished, recovering the reaction system to room temperature (10-30 ℃), reacting for 1-3 h in a dark place, and then quenching the reaction. The quenching may be carried out by a method commonly used in the art, and it is preferable to quench the reaction by adding water or methanol to the reaction system.

Preferably, after the reaction is finished, the reaction solvent is removed, and the solid substance is separated by silica gel column chromatography to obtain the PDT compound. During elution, one or more of petroleum ether, ethyl acetate, methanol, ethanol, acetone, dichloromethane and chloroform are selected as the eluent. It is preferable to use a mixed solution of petroleum ether and ethyl acetate, and petroleum ether: ethyl acetate ═ (5 to 8):1, for example, petroleum ether: ethyl acetate was 7: 1.

The PDT compound provided by the invention has photophysical properties of bacteriochlorin, not only has second type photodynamic therapy capability (namely generating singlet oxygen), but also can generate active oxygen species such as superoxide anion free radicals, hydroxyl free radicals and the like to perform first type photodynamic therapy. According to literature reports, the first PDT active oxygen species generally have stronger lethality; therefore, the PDT compound provided by the invention not only effectively improves the treatment effect, but also reduces the damage to the body.

In addition, the PDT compound provided by the invention has a reduced triplet energy level, so that molecules do not emit phosphorescence, and nonradiative transition is enhanced, so that the PDT compound has a stronger photothermal effect while being used as a triplet sensitizer to generate active oxygen species, and has combined effects of photodynamic therapy and photothermal therapy.

Further, the absorption spectrum of the PDT compounds provided by the invention is in the near infrared region (700-900nm), so that the excitation light has larger tissue penetration depth, which helps to reduce the damage of the light source to the tissue during the photodynamic therapy.

Accordingly, the present invention provides the use of the PDT compounds described above, and pharmaceutically acceptable salts, solvates, non-covalent complexes, complexes or precursors thereof, as active ingredients or as medicaments in photodynamic therapy or photothermal therapy.

Further, the PDT compounds also have strong fluorescence and can be used for fluorescent labeling, for example, as fluorescent imaging probes or fluorescent imaging agents.

In a preferred embodiment, the PDT compound is used in a near-infrared living cell fluorescence imaging probe, more preferably it is a lysosome-localized near-infrared living cell fluorescence imaging probe.

When the PDT compound is used for photodynamic therapy, the distribution condition of the PDT compound in a body can be observed in real time through fluorescence imaging, and the PDT compound is used for guiding diagnosis and treatment; the combined effects of photodynamic therapy and photothermal therapy contribute to improved therapeutic efficacy, and thus, PDT compounds can simultaneously achieve the effects of fluorescence imaging, photodynamic therapy, and photothermal therapy, which are difficult to achieve simultaneously by other photodynamic therapy compounds in the prior art, and contribute to simplification of photosensitizers for photodynamic therapy, improvement of treatment selectivity, and reduction of body tissue damage.

In particular, the PDT compound has good affinity to tumor cells, can be gathered at a tumor site of a living body, shows high phototoxicity at a cellular level and a living body level, and has an effect of treating tumors.

Therefore, the invention provides the application of the PDT compound in preparing medicaments for treating tumors, in particular the application in photodynamic therapy or photothermal therapy of tumors.

The tumor comprises breast cancer, cervical cancer, liver cancer, lung cancer, malignant melanoma or squamous cell carcinoma.

The invention also provides a pharmaceutical composition, which comprises the PDT compound or the PDT compound obtained by the preparation method as an active ingredient, and pharmaceutically acceptable auxiliary materials. The PDT compounds may also be used as active ingredients in pharmaceutical compositions as pharmaceutically acceptable salts, solvates, non-covalent complexes or prodrugs thereof.

Preferably, the pharmaceutical composition is administered by injection and prepared in various forms of predetermined dosage of the active ingredient, such as common dosage forms of injection solution, injection emulsion, injection sustained-release solution, injection suspension, etc.

The injection administration includes intravenous injection, arterial injection, intramuscular injection and vertebral cavity injection, and the active ingredients in the pharmaceutical composition are gathered to the tumor site through circulation or through targeted administration.

The auxiliary materials in the pharmaceutical composition should be non-active ingredients which have no toxic or harmful effect on human bodies and accord with the medication route or the administration mode. Mixing the active ingredient with adjuvants such as solvent, isotonic regulator, surfactant, antioxidant, etc., and making into sterile solution or dispersion for injection or sterile powder for injection before use.

In a preferred embodiment, the PDT compound may be coated as nanoparticles with a hydrophilic substance to increase the water solubility of the compound, facilitate absorption in the body and enhance the therapeutic effect of the compound.

Preferably, the amount of active ingredient in a unit dosage form of the pharmaceutical composition is from 0.01mg to 10 g.

The specific dosage may vary from patient to patient depending on the age, body weight, health condition, diet, administration route, combination, treatment time, etc. of the patient. In general, the PDT compounds are administered to treat the above conditions at a dosage level of 0.01 to 500mg/kg body weight per day, or 0.1 to 20g per patient per day.

Further, the absorption spectrum of the PDT compound provided by the invention is located in a near infrared region, the extinction coefficient is large, and the PDT compound can be used for photoacoustic imaging.

Therefore, the invention also provides the PDT compound and the pharmaceutically acceptable salt, solvate, non-covalent bond complex or precursor substance thereof, and the application of the PDT compound and the pharmaceutically acceptable salt, solvate, non-covalent bond complex or precursor substance in fluorescence labeling or fluorescence imaging and photoacoustic imaging, and the PDT compound and the pharmaceutically acceptable salt, solvate, non-covalent bond complex or precursor substance are potential fluorescent labeling agents, photoacoustic imaging and fluorescence imaging agents.

Examples

EXAMPLE 1 Synthesis of Compound Lu-1

Step 1: adding Lu (acac). 6H into Schlenk tube₂0.10mmol of O (lutetium acetylacetonate hexahydrate), 0.05mmol of porphyrin a and 8mL of trichlorobenzene as a solvent, degassing, introducing nitrogen, and sealing the tube at 240 ℃ for reaction for 1-3 hours. After monitoring the reaction system until no porphyrin fluorescence is present, the reaction system is cooled to room temperature. Separating by silica gel column chromatography, eluting with petroleum ether, dichloromethane/methanol (5/1), and collecting rare earth porphyrin. A rare earth porphyrin andligand sodium tris (dimethyl phosphite) & cyclopentadienyl cobalt (III) (NaL)_OMe) (0.05mmol) was dissolved in 10mL of CHCl₃And reacting in/MeOH-1/1 at 60 ℃ for 2h, removing the solvent by rotary evaporation, separating the solid by silica gel column chromatography, and recrystallizing with dichloromethane/n-hexane (1-10: 10) to obtain Lu-a.

Step 2: lu-a was dissolved in anhydrous oxygen-free tetrahydrofuran under nitrogen atmosphere, diisobutylaluminum hydride (DIBAL) was added dropwise at-80 ℃ in an amount of 0.25ml/40mg Lu-a. And (5) recovering the temperature to room temperature, reacting for 1h in a dark place, and adding water to quench the reaction. Spin-drying solvent, separating by silica gel column chromatography, and separating by petroleum ether: eluting with ethyl acetate 7:1, collecting, and removing the solvent to obtain the product Lu-1 as a red solid.

Structural characterization data:

¹the H NMR spectrum is shown in FIG. 1:¹H NMR(400MHz,CHCl₃-d):δ8.02(d,J＝4.5Hz,2H),7.95(d,J＝4.5Hz,2H),7.68(d,J＝8.9Hz,2H),4.47(s,5H),4.15(d,J＝9.0Hz,2H),2.77(dd,J＝7.2,3.6Hz,18H).

the HR-MS profile is shown in FIG. 2: HR-MS (ESI)⁺)m/z[M+H]⁺:Calcd for C₅₃H₃₂CoF₂₀LuN₄O₁₃P₃ ⁺1638.95975；found:1638.95527.

The FT-IR spectrum is shown in FIG. 3: molecule 1600-1800cm compared to the unreduced compound (a or Lu-a)^-1The peak of the stretching vibration characteristic of the carbonyl group (C ═ O) of the region disappeared, indicating the successful reduction of the lactone group. At the same time, the length of the groove is 3400cm^-1A stronger O-H bond vibration broad peak appears nearby, which indicates the existence of an-OH structure;

UV/Vis(CH₂Cl₂,25℃):λ_max(nm)(logε):341(5.01),396(5.14),503(3.77),543(4.42),697(4.15),753(5.16).

EXAMPLE 2 Synthesis of the Compound Lu-2

The reaction procedure is substantially similar to example 1, except that:

after Lu-1 was obtained, BF was further used₃·Et₂Catalyzing by O, and etherifying in a methanol solvent to obtain Lu-2.

Structural characterization data:

UV/Vis(CH₂Cl₂,25℃):λ_max(nm)(logε):345(4.99),397(5.15),509(3.98),545(4.50),690(4.16),749(5.10).

EXAMPLE 3 Synthesis of the Compound Lu-3

The reaction procedure is substantially similar to example 1, except that:

in step 1, the porphyrin involved in the reaction is b (5,10,15, 20-tetrakis (pentafluorophenyl) -cis-porphine dilactone).

Structural characterization data:

UV/Vis(CH₂Cl₂,25℃):λ_max(nm)(logε):346(5.04),394(5.19),536(4.52),738(4.99).

EXAMPLE 4 Synthesis of the Compound Lu-4

The reaction procedure is substantially similar to example 1, except that:

in step 1, the porphyrin involved in the reaction is c (7,8,17, 18-tetrafluoro-5, 10,15, 20-tetrakis (pentafluorophenyl) -trans-porphine dilactone).

EXAMPLE 5 Synthesis of Compound Lu-5

The reaction procedure is substantially similar to example 1, except that:

in step 1, the porphyrin involved in the reaction is d (5,10,15, 20-tetraphenyl-trans-porphine dilactone).

EXAMPLE 6 Synthesis of Gd-1 Compound

The reaction procedure is substantially similar to example 1, except that:

in step 1, Gd (acac) is used as a raw material participating in the reaction)·6H₂O (gadolinium acetylacetonate hexahydrate);

finally obtaining the product Gd-1, a red solid.

Structural characterization data:

HR-MS(ESI⁺)m/z[M+H]⁺:Calcd for C₅₃H₃₂CoF₂₀LGdN₄O₁₃P₃ ⁺1621.94445；found:1621.97887.UV/Vis(CH₂Cl₂,25℃):λ_max(nm)(logε):344(5.05),397(5.17),503(3.79),545(4.43),694(4.18),753(5.17).

EXAMPLE 7 Synthesis of Gd-2 Compound

The reaction procedure was substantially similar to example 6, except that:

after Gd-1 was obtained, BF was further used₃·Et₂Catalyzing by O, and etherifying in a methanol solvent to obtain Gd-2.

Structural characterization data:

UV/Vis(CH₂Cl₂,25℃):λ_max(nm)(logε):345(4.99),397(5.15),509(3.98),545(4.50),690(4.16),749(5.10).

EXAMPLE 8 Synthesis of Gd-3 Compound

The reaction procedure was substantially similar to example 6, except that:

in the step 1, the porphyrin participating in the reaction is b (5,10,15, 20-tetra (pentafluorophenyl) -cis-porphine dilactone);

structural characterization data:

UV/Vis(CH₂Cl₂,25℃):λ_max(nm)(logε):346(5.04),394(5.19),536(4.52),738(4.99).

EXAMPLE 9 Compound GdSynthesis of (E) -4

The reaction procedure was substantially similar to example 6, except that:

in step 1, the porphyrin involved in the reaction is c (7,8,17, 18-tetrafluoro-5, 10,15, 20-tetrakis (pentafluorophenyl) -trans-porphine dilactone).

EXAMPLE 10 Synthesis of Gd-5 Compound

The reaction procedure was substantially similar to example 6, except that:

in step 1, the porphyrin involved in the reaction is d (5,10,15, 20-tetraphenyl-trans-porphine dilactone).

Example 11

The reaction procedure is substantially similar to that of example 1 and example 6, except that:

in step 1, the Klaui ligand involved in the reaction is a cyclopentadienyl cobalt (III) tris (diethylphosphite) ligand to obtain Lu-1 'and Gd-1', respectively.

Example 12 nanoparticle coating

Mu. mol of Gd-1 (prepared in example 6) or Lu-1 (prepared in example 1) compound was dissolved in 5mL of tetrahydrofuran, 20mg of Mesoporous Silica Nanoparticles (MSN) were added, and the mixture was stirred open at room temperature until the solvent was completely volatilized. To the resulting solid was added 5mL of water and sonicated for 30 minutes. And centrifuging, washing with water for 3 times, and drying to obtain the compound-loaded silicon nanoparticles.

And (2) taking 20mg of the particles, adding 80mg of polyether F127, adding 2mL of mixed solution of toluene and tetrahydrofuran in a volume ratio of 1:1, evaporating at 35 ℃ until the solvent is completely volatilized, adding 2mL of water, stirring at room temperature for 4 hours, standing for 24 hours, and taking supernatant, namely the aqueous dispersion of Gd/Lu-1-MSN.

Examples of the experiments

Experimental example 1 photo-physical Properties measurement

The Lu-1 obtained in example 1 and Gd-1 obtained in example 6 were subjected to ultraviolet-visible absorption spectroscopy and emission spectroscopy scanning, and the results are shown in FIG. 4.

As can be seen from the absorption spectrum chart, the absorption spectrum of the compounds Lu-1 and Gd-1 can cover the visible region and the near infrared region, and the absorption band is stronger in the near infrared region of 700-800 nm. From the emission spectrum, when the compound is excited by light, the fluorescence spectrum of the compound is also in the near infrared region.

As can be seen, the compounds Lu-1 and Gd-1 have deeper tissue penetration depth in infrared imaging or in vivo fluorescence imaging.

Experimental example 2 detection of reactive oxygen species

1) Phosphorescence emission of singlet oxygen was detected near 1270nm under excitation of 760nm near infrared light by the chloroform solutions of Lu-1 obtained in example 1 and Gd-1 obtained in example 6 under an air atmosphere, and the results are shown in (1) in fig. 5, indicating that the molecule has the ability to generate singlet oxygen under light excitation.

2) Dihydroaethidium as a superoxide anion radical probe was added to the aqueous dispersion of Gd/Lu-1-MSN prepared in example 12 in an air atmosphere, and light irradiation (760nm light emitting diode lamp, 7.5 mW/cm)²) Under the conditions, the fluorescence of ethidium oxide generated by oxidation of ethidium dihydroxide by superoxide radical is detected, and the fluorescence intensity is increased along with the increase of illumination time. The results are shown in (2) in FIG. 5, which shows that the compound has the ability to generate superoxide anion radicals under light irradiation.

3) To the aqueous dispersion of Gd/Lu-1-MSN prepared in example 12 was added 5-methyl-5-tert-butylformyl-pyrroline nitroxide (BPMO) as a hydroxyl radical scavenger under air atmosphere. Under illumination (760nm light emitting diode lamp, 7).5mW/cm²) Under these conditions, the BMPO hydroxyl adduct signal was detected by electron paramagnetic resonance. The results are shown in (3) in FIG. 5, which shows that the compound has the ability to generate hydroxyl radicals under light irradiation.

Experimental example 3 photothermal detection

Photo-thermal conversion experiments were performed on aqueous dispersions of Gd/Lu-1-MSN.

80 μ g/mL (based on the compound molecule) of Gd/Lu-1-MSN in water dispersion illumination (760nm light emitting diode lamp, 100 mW/cm)²) Under the conditions, the temperature change of the dispersion was measured by a thermocouple and an infrared thermal imager, and the results are shown in (1) and (2) of fig. 6.

As can be seen from the figure, Gd/Lu-1-MSN can raise the temperature of the solution under illumination, while blank nanoparticles can hardly raise the temperature, which shows that the compound Gd-1/Lu-1 can obviously raise the water temperature. The compound has stronger photothermal effect and has the potential of photoacoustic imaging and photothermal treatment.

Experimental example 4 cytotoxicity

The compound was adsorbed and supported by the mesoporous silica nanoparticles in the method of example 12, and was well dispersed in the aqueous solution.

The cells used in the experiment comprise HeLa human cervical carcinoma cells and 4T1 mouse breast cancer cells. The cell culture was performed in DMEM complete medium supplemented with 10% inactivated fetal bovine serum and 1% penicillin-streptomycin at 37 ℃ under 5% carbon dioxide.

The subcultured HeLa cells were trypsinized and then dispersed in the medium at an appropriate concentration. The dispersed HeLa cells were seeded into poly-D-lysine modified flat-bottomed 96-well plates at 200 μ L per well medium, with about 104 cells, and a blank was run with a set of cell-free medium. After culturing the cells in a dark environment for 24 hours, the medium was removed, 100. mu.L of fresh medium and 100. mu.L of a medium solution prepared from Lu-1, Gd-1 prepared in advance were added, and the sample was diluted to a gradient concentration of 0.1 to 5. mu.M. After incubation for 24 hours in a dark environment, the medium was removed and each well was rinsed 3 times with PBS at pH 7.4. mu.L of PBS buffer was added to each well and irradiated for 30 minutes under a 760nm light emitting diode lamp of the same intensity (about 7.5mW/cm 2). PBS was removed from each well and replaced with 200 μ L fresh medium and incubation was continued for 24 hours. After completion, the medium was removed and each well was rinsed 3 times with PBS. Then, 10% of CCK-8 reagent (Cell Counting Kit-8) was prepared in the medium, and 100. mu.L of the reagent was added to each well and cultured for 2 hours. In this process, 2- (2-methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2, 4-disulfonic acid phenyl) -2H-tetrazole monosodium salt (WST-8) in CCK-8 reagent was reduced by living cells to a yellow formazan product with the aid of an electron coupling reagent, resulting in a change in the absorbance of the solution at 450nm, which was proportional to the number of living cells. The absorbance change at 450nm was measured for each well using a microplate reader and the cell viability at each incubation concentration was calculated as follows:

CV＝(As–Ab)/(Ac–Ab)×100％

CV refers to cell survival; as, Ac and Ab refer to absorbance of cells of incubated compound, absorbance of blank cells and absorbance of blank control, respectively.

Half-lethal concentration IC of compound in different cell lines was calculated from the cell viability at each incubation concentration₅₀The following are: unit is μ M

Experimental example 5 research on phototoxicity mechanism of Compound

The Gd-1-MSN prepared in example 12 was tested for phototoxicity to 4T1 cells in the presence of hypoxia, a cold water bath, or both hypoxia and cold water baths.

The cells were treated in a cold water bath during light irradiation to maintain the cell temperature at room temperature, eliminating the toxic effect of the photothermal effect, and the increase in cell viability and half-lethal concentration to greater than 16 μ M were detected, as shown in fig. 7, demonstrating that some of the cytotoxicity was due to the photothermal effect.

Cells were subjected to hypoxic treatment with anaerobic gas-producing bags 6 hours prior to illumination, followed by illumination (24 hours of incubation at normal oxygen levels after illumination), and an increase in cell viability was observed, with half-lethal increases to (14.5. + -. 0.4) μ M, as shown in FIG. 7, demonstrating that some cytotoxicity arises from aerobic photodynamic effects.

The increased cell viability was observed in the cytotoxicity experiments with simultaneous anoxic and cold water bath treatments, similar to no light exposure, and the results are shown in fig. 7, indicating that cytotoxicity results from the combined action of photothermal and aerobic photodynamic effects.

Experimental example 6 mechanism study of apoptosis induced by phototoxicity of Compound

The PCR results of HeLa cells treated with Gd-1 at concentrations of 0, 4. mu.M, respectively, are shown in FIG. 8. under light conditions, Gd-1 can activate the expression of clear PARP protein and clear Caspase3 protein. Activation of the two proteins proves that Gd-1 can stimulate an apoptosis signal channel and induce apoptosis of cells. Under the condition of no illumination, Gd-1 can not activate an apoptosis signal channel and has no killing effect on tumor cells.

The apoptosis phenomenon of the cells was detected by the flow cytometry technique, and the result is shown in fig. 9, in the same manner, Gd-1 can induce the increase of the apoptosis cell ratio under the condition of light irradiation; under the condition of no light, Gd-1 can not induce the apoptosis of the cells.

Experimental example 7 cellular fluorescence imaging

The compound was adsorbed and supported by the mesoporous silica nanoparticles in the method of example 12, and was well dispersed in the aqueous solution.

The cells used in the experiment are HeLa human cervical carcinoma cells. The cell culture was performed in DMEM complete medium supplemented with 10% inactivated fetal bovine serum and 1% penicillin-streptomycin at 37 ℃ under 5% carbon dioxide.

The subcultured HeLa cells were trypsinized and then dispersed in the medium at an appropriate concentration. The dispersed HeLa cells were inoculated onto a poly-D-lysine-modified slide glass, and after culturing the cells for 24 hours, 100. mu.L of a previously prepared medium solution of molecular Gd-1 was added to dilute the cells to a concentration of 4. mu.M. After 12 hours of incubation, the lysosome green fluorescent probe was added, and after 15 minutes of co-incubation, the medium was removed and rinsed 3 times with PBS at pH 7.4. Performing fluorescence imaging by using an ISS integrated laser scanning confocal fluorescence lifetime imaging system, exciting at 740nm by using super-continuous laser, using a 760nm high-pass filter, and collecting Gd-1 molecular fluorescence signals; and (3) exciting by a 488nm laser, filtering by an 525/50nm band-pass filter, and collecting a lysosome probe fluorescence signal.

The result is shown in fig. 10, Gd-1 has stronger fluorescence signals in the near infrared region, and can be better overlapped with the signal comparison of a lysosome probe, which indicates that the molecule has the potential of being used as a near infrared living cell fluorescence imaging probe and is positioned on lysosome.

Experimental example 8 in vivo fluorescence imaging in animals

All animal experiments in vivo were performed strictly following the regulations of the Chinese animal experiments, using five-week old BALB/C white mice subcutaneously implanted with 4T1 tumor in the right hind leg.

The imaging instrument used for the in-vivo fluorescence imaging is an IVIS Spectrum fluorescence imaging system, can realize high-sensitivity bioluminescence and fluorescence imaging, and is provided with 28 high-efficiency filters covering the whole waveband of 430-850 nm.

The experimental process comprises the following steps: mice were anesthetized by intravenous injection of 250uL of 80 μ g/mL (compound basis) PBS dispersion of Gd-1-MSN into the tail vein of the mice in an imaging instrument under a mixed gas atmosphere of 2L/min oxygen and 2% isoflurane. Excitation was carried out using an excitation wavelength of 740 nm. The image acquisition wavelength was 820nm and the exposure time was automatic.

Mice were sacrificed and dissected 24 hours after tail vein injection of compounds, and heart, liver, spleen, lung, kidney, pancreas, stomach, bladder muscle, hind limb bone, brain, tumors were removed and imaged under an imager. Other conditions were consistent with in vivo experiments.

The results of the experiment are shown in fig. 11, and the imaging of tumor sites was achieved after the compound was administered to mice, with the signal reaching the highest at 24 hours, with low background interference and weak signal in the surrounding tissues. The results of the dissection experiments were consistent with the results of in vivo imaging, with the compounds localized to the tumor tissue and partially to the liver. This shows that the near infrared luminescence property of the compound effectively reduces background interference in vivo fluorescence imaging, and high resolution fluorescence imaging of tumors can be realized even in a non-anatomical state of a living body.

Experimental example 9 magnetic resonance imaging Properties

The results of T1 magnetic resonance imaging experiments on Gd-1-MSN are shown in FIG. 12, and it can be seen from the figure that the compound has certain T1 magnetic resonance contrast capability and longitudinal relaxation rate of about 1.1s^-1·M^-1。

Experimental example 10 photoacoustic imaging

The imaging instrument used for in vivo photoacoustic imaging is the iThera ultra-high resolution small animal photoacoustic imaging system MOST invision 128.

The experimental process comprises the following steps: mice were anesthetized by tail vein injection of 250uL of 80 μ g/mL (compound basis) of a PBS dispersion of Gd-1/Lu-1-MSN (approximately 1mg/kg) and placed in an imaging apparatus under a mixed gas atmosphere of 2L/min oxygen and 2% isoflurane. Excitation was carried out using an excitation wavelength of 780 nm. The exposure time is automatic.

The experimental results are shown in fig. 13, and the compounds can realize photoacoustic imaging of tumor sites after entering mice, and the signals reach the highest at 24 hours.

Experimental example 11 photothermal Effect of living body

BALB/C mice, 5 weeks old, male, 16-25 g in weight were used. Each mouse was inoculated subcutaneously with 100ul (5X 10) of 4T1 murine mammary carcinoma cells in the right hind limb⁶) The experiment was performed one week later.

The mice were randomly grouped by number, and the experiment was divided into a blank control group (PBS-), a drug administration dark control group (Gd-1-MSN-), a light control group (PBS +) and an experimental group (Gd-1-MSN +, Lu-1-MSN +), with 7 mice per group. Administration of 250. mu.L PBS and Gd-1-MSN aqueous dispersion via tail vein into dark control group and blank control group, respectively, without intervention, and light irradiation (760nm LED lamp, 100 mW/cm) only in light irradiation control group²) (ii) a The experimental group was administered at 1mg/kg based on the body weight of the mouse and the tail vein was injected with about 250. mu.L of a nanoparticle dispersion of 80. mu.g/mL of the compound. After administration, the animals are kept in the dark for 24h, and then the tumor parts are irradiated for 5 min.

Thermal imaging was performed on 3 illumination groups (PBS +, Gd-1-MSN +, and Lu-1-MSN +) with a FLIR E40 thermal infrared imager during the illumination, and the results are shown in FIG. 14, where the compound increased the temperature of the tumor site during the illumination, and the temperature increased with the increase of the illumination time. Indicating that the compound also has a photothermal effect in vivo.

Experimental example 12 example of photo-treatment of drug at animal level

In experimental example 11, the mice were kept in a cage without being shaded after completion of treatment, and the tumor volume and body weight were measured with a vernier caliper every two days. The treatment is carried out for 4 weeks.

The experimental result is shown in fig. 15, the compounds Gd-1 and Lu-1 can effectively inhibit the growth of subcutaneous tumor by the mode of tail vein injection administration, the survival rate of the mouse is increased, and the weight of the mouse is not obviously reduced; whereas the non-dosed control mice showed rapid tumor growth.

In addition, the tumor growth trend of the dosed dark control group was similar to that of the non-dosed group, demonstrating the phototherapy mechanism of the compound, i.e. the therapeutic effect could be achieved by light excitation.

The invention has been described in detail with reference to specific embodiments and illustrative examples, but the description is not intended to be construed in a limiting sense. Those skilled in the art will appreciate that various equivalent substitutions, modifications or improvements may be made to the technical solution of the present invention and its embodiments without departing from the spirit and scope of the present invention, which fall within the scope of the present invention. The scope of the invention is defined by the appended claims.