Phenothiazine functional precursor, photosensitizer and preparation methods of phenothiazine functional precursor and photosensitizer

Abstract

According to the phenothiazine functional precursor, the photosensitizer and the preparation method of the phenothiazine functional precursor, halogen or thienyl is introduced into R1 and / or R2 sites of a benzophenothiazine structure to serve as heavy atoms on the basis of structure-function relationship research of benzophenothiazine site modification, generation of strong spin-orbital coupling can be promoted, and the photosensitizer has the advantages that the photosensitizer can be used as a photosensitizer; further, intersystem crossing of the phenothiazine photosensitizer from an excited singlet state (S1) to a triplet state (T1) is promoted, so that the photosensitive effects of the phenothiazine functional precursor and the corresponding phenothiazine photosensitizer are effectively improved. Besides, after the phenothiazine photosensitizer is modified by adding a tetrazine group, controllable closing and opening of phenothiazine are realized, so that the dark toxicity of phenothiazine is effectively reduced, the targeting property and the treatment effect of the photosensitizer are improved by accessing a targeting group and a drug group into the phenothiazine photosensitizer, and the phenothiazine photosensitizer has a wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of photosensitizers, in particular to a phenothiazine functional precursor, and a phenothiazine photosensitizer synthesized based on the phenothiazine functional precursor and a preparation method thereof. BACKGROUND

[0002] Phenothiazine photosensitizer is an important photosensitive compound developed from phenothiazine as the mother nucleus structure. Phenothiazine photosensitizer can efficiently convert intracellular oxygen into singlet oxygen with high reactivity, thereby triggering a series of photochemical reactions, leading to cell death or inactivation of biomolecules. They play a crucial role in modern photomedicine, especially in photodynamic therapy and photodynamic antibacterial chemotherapy.

[0003] In the prior art, patent CN107375929B discloses a class of benzophenothiazine photosensitizers, which connects targeting groups and drug molecules by adjusting the functional groups on the side benzene ring of the phenothiazine mother nucleus, thereby increasing the uptake of photosensitizers by tumor tissues and achieving the purpose of synergistic treatment of photodynamic therapy and chemotherapy. Patent CN117486828B also discloses a class of photosensitizers with benzophenothiazine as the skeleton, which also designs functional groups on the side benzene ring of the phenothiazine mother nucleus to improve the ability of the photosensitizer to oxidize and nitrate cells.

[0004] However, the existing benzophenothiazine photosensitizer only focuses on the adjustment of substituents on the side benzene ring of the phenothiazine mother nucleus, and does not focus on the design of substituents on the non-mother nucleus side benzene ring of the benzophenothiazine photosensitizer. Therefore, it is necessary to design the skeleton of the existing benzophenothiazine photosensitizer to further improve its photosensitivity. SUMMARY

[0005] One object of the present application is to provide a phenothiazine functional precursor which can effectively improve the photosensitivity of the phenothiazine functional precursor and the phenothiazine photosensitizer prepared based thereon by introducing specific substituents at specific positions on the non-mother nucleus side benzene ring.

[0006] The present application is realized by the following technical solutions:

[0007] A phenothiazine functional precursor has the structure shown in formula I:

[0008] Formula I:

[0009] In formula I, groups R1 and R2 are each independently selected from H, halogen or thienyl, and groups R1 and R2 are not H at the same time.

[0010] In the technical solution, the skeleton of the phenothiazine functional precursor adopts a benzophenothiazine structure, and based on the structure-activity relationship research of the benzophenothiazine site modification, halogen or thiophene group is introduced as a heavy atom at the R1 and / or R2 site of the benzophenothiazine structure, which can promote the generation of strong spin-orbital coupling, and further promote the intersystem crossing of the phenothiazine photosensitizer from the excited singlet state (S1) to the triplet state (T1), thereby effectively improving the photosensitive effect of the phenothiazine functional precursor. Moreover, the phenothiazine photosensitizer prepared based on the phenothiazine functional precursor can also have excellent photosensitive effect.

[0011] In the technical solution, the groups R1 and R2 are each independently selected from H, halogen or thiophene group. In some embodiments, the groups R1 and R2 are each independently selected from H, Br, I or thiophene group. In some preferred embodiments, R1 is H or Br, and R2 is H, Br, I or thiophene group.

[0012] In the technical solution, the groups R1 and R2 are not both H. That is, when the group R1 is H, the group R2 is halogen or thiophene group; or when the group R2 is H, the group R1 is halogen or thiophene group; or the groups R1 and R2 are both halogen or thiophene group. In some preferred embodiments, the group R1 is H, and the group R2 is halogen or thiophene group; or the group R2 is H, and the group R1 is halogen or thiophene group. In more preferred embodiments, the group R1 is H, and the group R2 is halogen or thiophene group. It is found through experiments that among the two modification sites of the phenothiazine photosensitizer, the photosensitive effect of the site R2 is better than that of the site R1, which can significantly improve the ROS production in vitro and in cells.

[0013] Another object of the present application is to provide a preparation method of any of the aforementioned phenothiazine functional precursors, which specifically comprises the following steps:

[0014] The compound shown in formula II is used as a raw material, and after amino protection, it is reacted with the compound shown in formula III to obtain the compound shown in formula IV. After deprotection of the amino group of the intermediate, the phenothiazine functional precursor shown in formula I is obtained.

[0015] Formula II: Formula III: Formula IV: ;

[0016] In formula II and formula IV, the groups R1 and R2 match the structure of the phenothiazine functional precursor according to claim 1 or 2.

[0017] In the technical solution, an α-naphthylamine with R1 and / or R2 sites substituted is used as a raw material, the amino group thereof is protected, and then the protected compound is reacted with a compound shown in formula III to obtain a compound shown in formula IV, and then the amino protecting group of the compound shown in formula IV is removed to obtain the phenothiazine functional precursor. The overall synthesis path is as follows:

[0018]

[0019] Further, the preparation method of the phenothiazine functional precursor comprises the following steps:

[0020] The compound shown in formula II is reacted with N-tert-butoxycarbonyl-2-aminoacetaldehyde under acidic conditions to obtain a first intermediate, and the first intermediate is ammoniated by sodium cyanoborohydride to obtain a second intermediate;

[0021] The second intermediate is oxidized by an oxidizing agent to obtain a compound shown in formula VI;

[0022] The compound shown in formula VI is deprotected by trifluoroacetic acid to obtain the phenothiazine functional precursor shown in formula I.

[0023] In some embodiments, the compound shown in formula II is dissolved in methanol, and acetic acid is used to provide an acidic environment, and the second intermediate is obtained by reductive amination at room temperature. In one or more embodiments, the solvent of the compound shown in formula II can also be ethanol, propanol, acetonitrile, tetrahydrofuran, dimethyl sulfoxide, N,N-dimethylformamide, N-methyl pyrrolidone, benzene, n-butanol, isopropanol, etc. In one or more embodiments, the acid can also be hydrochloric acid, formic acid, citric acid, propionic acid, etc.

[0024] In some embodiments, the second intermediate is oxidized by an oxidizing agent with DMSO as a solvent to obtain the compound shown in formula VI. In one or more preferred embodiments, the solvent used can also be toluene, xylene, dimethyl sulfoxide, N,N-dimethylformamide. In one or more embodiments, the oxidizing agent can be potassium dichromate, trivalent cobalt salt, persulfate, peroxide, potassium permanganate, oxonate, etc.

[0025] In some embodiments, after the compound shown in formula VI is dissolved, the Boc is removed by trifluoroacetic acid to obtain the phenothiazine functional precursor. In one or more embodiments, the solvent can be ethanol, propanol, acetonitrile, tetrahydrofuran, dimethyl sulfoxide, N,N-dimethylformamide, 1,4-dioxane. In one or more embodiments, the acidic environment can also be provided by hydrochloric acid or formic acid.

[0026] Another object of the present application is to provide a phenothiazine photosensitizer prepared based on the aforementioned phenothiazine functional precursor, which can achieve excellent photosensitive effect based on the design of substituents on the precursor skeleton structure.

[0027] Specifically, a phenothiazine photosensitizer has a structure shown in formula V, formula VI or formula VII:

[0028] Formula V: ;

[0029] Formula VI: ;

[0030] Formula VII: ;

[0031] In formula V, formula VI and formula VII, each of groups R1 and R2 is independently selected from H, halogen or thienyl, and groups R1 and R2 are not H at the same time; in formula V, n = 1-10; in formula VI, group X is selected from phenyl or C1-C8 chain alkyl, group R4 is selected from H or azido, and m = 1-10.

[0032] In the present technical solution, based on the active amino group of the phenothiazine functional precursor, a polyethylene glycol group, a tetrazine functional group, or a drug and a targeting group can be connected to obtain a phenothiazine photosensitizer shown in formula X:

[0033] Formula X:

[0034] In formula X, the selection of groups R1 and R2 is the same as that in formula I, each of the two groups is independently selected from H, halogen or thienyl, and the two groups are not H at the same time, so as to improve the photosensitive performance of the phenothiazine photosensitizer by heavy atom effect. In some preferred embodiments, group R1 is H, and group R2 is halogen or thienyl; or, group R2 is H, and group R1 is halogen or thienyl.

[0035] Further, according to the type of the connected group, three types of phenothiazine photosensitizers of formula V, formula VI and formula VII can be obtained.

[0036] Formula V corresponds to the first type of phenothiazine photosensitizer, which has n PEG segments connected through an amide group, wherein n = 1-10. In some preferred embodiments, n = 1-5, and further preferably, n = 2-4.

[0037] The second type of phenothiazine photosensitizer corresponding to formula VI utilizes a tetrazine functional group to achieve quenching of the phenothiazine photosensitizer through fluorescence resonance energy transfer (FRET) and reduction of water solubility, so that the photodynamic effect is weakened. Then, through a bio-orthogonal click reaction between the tetrazine group and trans-cyclooctene (TCO), the quenching is released and the active photosensitizer is released, so that the controllable closing and opening of the phenothiazine photosensitizer is realized, and the side effects of photodynamic therapy are effectively reduced. In some preferred embodiments, the group X can be a phenyl group or a C1-C8 chain alkyl group, and more preferably, the group X can be a phenyl group, a methyl group, an ethyl group, a n-propyl group, a n-butyl group, etc. More preferably, the R4 group is preferably an azido group, which can efficiently perform a click reaction with the alkyne group in BCN under mild conditions, so as to introduce a water-soluble group into the photosensitizer molecule.

[0038] The third type of phenothiazine photosensitizer corresponding to formula VII simultaneously connects a targeting molecule triphenylphosphine and a drug molecule lonidamide at the R3 site, which can further improve the targeting and therapeutic effect, and expand the application of the photosensitizer.

[0039] Therefore, in the technical solution, the photosensitivity of the phenothiazine is improved based on the R1 and R2 groups of the phenothiazine functional precursor, the controllable closing and opening of the phenothiazine is realized by adding a tetrazine group for modification, the dark toxicity of the phenothiazine is effectively reduced, the targeting and therapeutic effect of the photosensitizer are improved by connecting a targeting group and a drug group, and the photosensitizer has a wide application prospect.

[0040] Further, the phenothiazine photosensitizer is selected from the following compounds:

[0041] 、 、 、 、 、 .

[0042] Another object of the present application is to provide a preparation method of any one of the aforementioned phenothiazine photosensitizers, which specifically comprises the following steps:

[0043] The phenothiazine functional precursor shown in formula I, mPEG-COOH and a condensing agent are dissolved, and then a phenothiazine photosensitizer shown in formula V is prepared by reacting at room temperature in an alkaline environment.

[0044] The phenothiazine functional precursor shown in formula I, the compound shown in formula VIII and a condensing agent are dissolved, and then a phenothiazine photosensitizer shown in formula VI is prepared by reacting at room temperature in an alkaline environment.

[0045] The phenothiazine functional precursor shown in formula I, the compound shown in formula IX, the condensing agent is dissolved, and then the phenothiazine photosensitizer shown in formula VII is prepared by reacting at room temperature in an alkaline environment.

[0046] Formula I: ; formula VIII: ;

[0047] Formula IX ;

[0048] In formula I, groups R1 and R2 are each independently selected from H, halogen or thienyl, and groups R1 and R2 are not H at the same time; in formula VIII, group X is selected from phenyl or C1-C8 linear alkyl, group R4 is selected from H or azido, and m = 1-10.

[0049] In the technical solution, the phenothiazine functional precursor is dehydrated and condensed with mPEG-COOH, the compound of formula VIII and the compound of formula IX to obtain the corresponding phenothiazine photosensitizer.

[0050] In some preferred embodiments, the dehydrated condensation reaction of the phenothiazine functional precursor with mPEG-COOH, the compound of formula VIII or the compound of formula IX uses anhydrous dichloromethane as a solvent, DIPEA provides an alkaline environment, and HOBT and EDCI are condensing agents. In one or more embodiments, the solvent can also be dichloroethane, chloroform, dimethyl sulfoxide, N,N-dimethylformamide, and N-methyl pyrrolidone.

[0051] In one or more embodiments, based on the phenothiazine photosensitizer of formula V in which R2 is halogen-substituted and R1 is H, further reaction with 2-thiophene boronic acid can be carried out to obtain a phenothiazine photosensitizer in which R2 is thienyl-substituted and R1 is H. In some embodiments, the base used in the reaction can be K2CO3, CsCO3, Na2CO3, TiOH, NaOCH2CH3; the catalyst can use Pd(PPh3)4 or Pd(dppf)Cl2; the solvent can be a mixture of toluene, isopropanol and water, a mixture of ethanol and water, a mixture of isopropanol and water, a mixture of ethylene glycol dimethyl ether and water, tetrahydrofuran, toluene, and further preferably, the solvent is a mixture of toluene, isopropanol and water.

[0052] Further, the preparation method of the compound shown in formula IX comprises the following steps:

[0053] After chloronidamine, hydroxysuccinimide and diisopropyl carbodiimide are dissolved, the reaction is carried out in an alkaline environment, then Boc-L-lysine is added to obtain a third intermediate, trifluoroacetic acid is added to the solution of the third intermediate, and the fourth intermediate is prepared by reaction.

[0054] (3-carboxylpropyl)triphenylphosphonium bromide, pentafluorophenol, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride are dissolved, and then the fifth intermediate is obtained by reaction;

[0055] The fourth intermediate and the fifth intermediate are reacted in an alkaline environment to obtain a compound shown in formula IX.

[0056] Compared with the prior art, the present application has the following advantages and beneficial effects:

[0057] 1. In the present application, the study on the structure-activity relationship based on the modification of the benzophenothiazine site introduces halogen or thiophene group as heavy atom at the R1 and / or R2 site of the benzophenothiazine structure, which can promote the generation of strong spin-orbital coupling, and then promote the intersystem crossing of the thiophenazine photosensitizer from the excited singlet state (S1) to the triplet state (T1), thereby effectively improving the photosensitivity of the phenothiazine functional precursor and the corresponding phenothiazine photosensitizer;

[0058] 2. The present application finds through experiments that among the two modified sites of the phenothiazine photosensitizer, the photosensitivity effect of the site R2 is better than that of the site R1, which can significantly improve the ROS production in vitro and in cells;

[0059] 3. The phenothiazine photosensitizer in the present application not only improves the photosensitivity of the phenothiazine based on the R1 and R2 groups of the phenothiazine functional precursor, but also realizes the controllable closing and opening of the phenothiazine by adding a tetrazine group, thereby effectively reducing the dark toxicity of the phenothiazine, and also improving the targeting and therapeutic effect of the photosensitizer by connecting the targeting group and the drug group, which has a wide application prospect;

[0060] 4. The synthesis path of the present application is simple and has mild reaction conditions, which is conducive to large-scale production. BRIEF DESCRIPTION OF DRAWINGS

[0061] The drawings described herein are used to provide further understanding of the embodiments of the present application, constitute a part of the present application, and do not constitute a limitation on the embodiments of the present application. In the drawings:

[0062] Figure 1 It is a flowchart of the preparation method of the phenothiazine functional precursor in the specific embodiments of the present application;

[0063] Figure 2 The figure shows the DPBF absorption decay curve in the singlet oxygen production process of each phenothiazine photosensitizer in ACN-H2O solution under irradiation of 15 mW intensity in the embodiment 10 of the present application, wherein the left column is the absorption curve at each wavelength, and the right column is the absorption curve at the wavelength of 410 nm;

[0064] Figure 3The figure shows the change of fluorescence intensity of each phenothiazine photosensitizer in ACN-H2O solution under irradiation of 15 mW intensity over time in the embodiment 11 of the present application, wherein the left column is the fluorescence intensity at each wavelength, and the right column is the fluorescence intensity at wavelength 525 nm;

[0065] Figure 4 The figure shows the change of fluorescence intensity of each phenothiazine photosensitizer in ACN-H2O solution under irradiation of 15 mW intensity over time in the embodiment 11 of the present application, wherein the left column is the fluorescence intensity at each wavelength, and the right column is the fluorescence intensity at wavelength 525 nm; 2 Cell survival rate after 24 hours under irradiation of 5 min;

[0066] Figure 5 The figure shows the change of fluorescence intensity of phenothiazine photosensitizer E in methanol and PBS solution before and after reaction with BCN in the embodiment 13 of the present application;

[0067] Figure 6 The figure shows the cell survival rate of phenothiazine photosensitizer F of different concentrations under no light (left figure) and the cell survival rate of phenothiazine photosensitizer F and phenothiazine photosensitizer B of different concentrations under light and no light in the embodiment 14 of the present application. DETAILED DESCRIPTION

[0068] In order to make the objectives, technical solutions and advantages of the present application clearer, further detailed description will be given below in combination with embodiments and drawings, and the schematic embodiments of the present application and the description thereof are only used for explaining the present application, and not as a limitation to the present application.

[0069] All raw materials of the present application are not particularly limited in source, and can be purchased on the market or prepared according to conventional methods well known to those skilled in the art. All raw materials of the present application are not particularly limited in purity, and the present application preferably adopts analytical purity or the purity requirement in the field of photosensitizer. The grade and abbreviation of all raw materials of the present application belong to conventional grade and abbreviation in the art, and each grade and abbreviation is clear and explicit in the field of its relevant use, and those skilled in the art can purchase from the market or prepare by conventional methods according to the grade, abbreviation and corresponding use.

[0070] The expression of the substituent group is not particularly limited in the present application, and the expression well known to those skilled in the art is adopted, and those skilled in the art can correctly understand its meaning according to its expression based on common sense.

[0071] The “first”, “second” and the like (such as first intermediate, second intermediate and the like) used herein are only for distinguishing the corresponding components for the sake of clear description, and are not intended to limit any order or emphasize importance, etc. In addition, the term “connection” used herein can be direct connection or indirect connection via other groups without special description.

[0072] I. Preparation of phenothiazine functional precursors

[0073] Example 1

[0074]

[0075] Dissolve LXD-10 (4.2 mmol, 930 mg) in methanol, then add N-Boc-2-aminoacetaldehyde (5 mmol, 810 mg), acetic acid (8.5 mmol, 488 μL). React at room temperature for 3 h. Then add sodium cyanoborohydride (6 mmol, 374 mg), and react at room temperature overnight. Add 50 mL of water, extract with dichloromethane, dry over anhydrous sodium sulfate, remove dichloromethane using a rotary evaporator, and purify the residue by silica gel column chromatography to obtain intermediate W1645 (742 mg, 48 %).

[0076] 1 H NMR (400 MHz, Chloroform-d) δ 8.04 (s, 1H), 7.62 (d, J = 8.7 Hz, 1H), 7.49 (dd, J = 8.8, 1.8 Hz, 1H), 7.32 (t, J = 7.8 Hz, 1H), 7.15 (d, J = 8.2 Hz, 1H), 6.52 (d, J = 7.6 Hz, 1H), 5.00 - 4.93 (m, 1H), 3.54 (t, J = 5.9 Hz, 2H), 3.33 (t, J = 5.4 Hz, 2H), 1.49 (s, 9H).

[0077]

[0078] Dissolve intermediate W1645 (1.4 mmol, 510 mg) and compound ③ (1 mmol, 276 mg) in DMSO, and add potassium dichromate (1.1 mmol, 324 mg). React at room temperature for 30 min. Transfer the mixture into methanol, add 2 M HCl (5 mL), and react at room temperature for 60 min. Collect the solid by suction filtration, and purify the filtrate by silica gel column chromatography to obtain intermediate W1646 (275 mg, 50 %).

[0079]

[0080]

[0081] After intermediate W1646 (0.5 mmol, 275 mg) was dissolved in DCM (4 ml), trifluoroacetic acid (1 ml) was added, and the reaction was allowed to proceed at room temperature for 2 h. DCM was removed using a rotary evaporator and DCM was added once to obtain phenothiazine functional precursor 1 (224 mg, 99%). The theoretical molecular weight of phenothiazine functional precursor 1 was 454.0827, and the molecular weight was 454.1 after mass spectrometry.

[0082] [Example 2]

[0083]

[0084] After LXD-51 (0.82 mmol, 220 mg) was dissolved in methanol, N-Boc-2- aminoacetaldehyde (0.98 mmol, 156 mg) and acetic acid (1.64 mmol, 96 μL) were added. The reaction was allowed to proceed at room temperature for 3 h. Sodium cyanoborohydride (1.15 mmol, 74 mg) was then added, and the reaction was allowed to proceed at room temperature overnight. Water (10 ml) was added, and the mixture was extracted with dichloromethane. The dichloromethane was removed using a rotary evaporator after drying over anhydrous sodium sulfate, and the residue was purified by column chromatography on silica gel to obtain intermediate LXD-52 (117 mg, 35 %).

[0085]

[0086]

[0087] After intermediate LXD-52 (0.28 mmol, 117 mg) and compound ③ (0.2 mmol, 55 mg) were dissolved in DMSO, potassium dichromate (0.22 mmol, 65 mg) was added. The reaction was allowed to proceed at room temperature for 30 min. The mixture was transferred to methanol, and 2 M HCl (1 ml) was added. The reaction was allowed to proceed at room temperature for 60 min. The solid was collected by suction filtration, and the filtrate was purified by column chromatography on silica gel to obtain intermediate LXD-54 (72 mg, 60 %).

[0088]

[0089]

[0090] After LXD-54 (0.12 mmol, 72 mg) was dissolved in DCM (0.8 ml), trifluoroacetic acid (0.2 ml) was added, and the reaction was allowed to proceed at room temperature for 2 h. DCM was removed using a rotary evaporator and DCM was added once to obtain phenothiazine functional precursor 2 (57 mg, 95%). The theoretical molecular weight of phenothiazine functional precursor 2 was 502.0688, and the molecular weight was 502.1 after mass spectrometry.

[0091] Example 3

[0092]

[0093] LXD-14 (3 mmol, 660 mg) was dissolved in methanol, followed by the addition of N-Boc-2-aminoacetaldehyde (3.6 mmol, 573 mg), acetic acid (6 mmol, 345 μL). The reaction was allowed to proceed at room temperature for 3 h. Sodium cyanoborohydride (4.2 mmol, 264 mg) was then added and the reaction was allowed to proceed at room temperature overnight. Water (50 mL) was added and the mixture was extracted with dichloromethane. The dichloromethane was removed using a rotary evaporator after drying over anhydrous sodium sulfate. The residue was purified by column chromatography on silica gel to give intermediate LXD-15 (440 mg, 40%).

[0094]

[0095]

[0096] LXD-15 (1.2 mmol, 440 mg) and compound ③ (0.87 mmol, 240 mg) were dissolved in DMSO and potassium dichromate (1.02 mmol, 300 mg) was added. The reaction was allowed to proceed at room temperature for 30 min. The mixture was transferred to methanol and 2 M HCl (5 mL) was added. The reaction was allowed to proceed at room temperature for 60 min. The solid was collected by suction filtration and the residue was purified by column chromatography on silica gel to give intermediate LXD-16 (189 mg, 40%).

[0097]

[0098]

[0099] LXD-16 (0.1 mmol, 55 mg) was dissolved in DCM (0.8 mL) and trifluoroacetic acid (0.2 mL) was added. The reaction was allowed to proceed at room temperature for 2 h. DCM was removed using a rotary evaporator and DCM was added once to give phenothiazine functional precursor 3 (43 mg, 95%). The theoretical molecular weight of phenothiazine functional precursor 3 is 454.0827 and the molecular weight was identified as 454.1 by mass spectrometry.

[0100] II. Preparation of phenothiazine photosensitizers

[0101] Example 4

[0102] In this example, phenothiazine photosensitizer A having the group R 3 is prepared based on phenothiazine functional precursor 1 prepared in Example 1.

[0103] ​

[0104] Specifically, phenothiazine functional precursor 1 (0.02 mmol, 10.6 mg), m-PEG2-COOH (0.1 mmol, 24 mg), HOBT (1-hydroxybenzotriazole) (0.1 mmol, 14 mg), EDCI (1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride) (0.14 mmol, 28 mg) and DIPEA (N,N-diisopropylethylamine) (0.1 mmol, 18 μL) were dissolved in anhydrous DCM. The reaction was carried out at room temperature for 2 h. Phenothiazine photosensitizer A (10 mg, 86 %) was obtained by purification through a reversed-phase column.

[0105]

[0106] Example 5

[0107] In this example, phenothiazine photosensitizer B was prepared based on phenothiazine functional precursor 2 prepared in Example 2. 3

[0108]

[0109] Specifically, phenothiazine functional precursor 2 (0.12 mmol, 61 mg), m-PEG2-COOH (0.36 mmol, 53 mg), HOBT (0.6 mmol, 81 mg), EDCI (0.88 mmol, 169 mg) and DIPEA (0.6 mmol, 105 μL) were dissolved in anhydrous DCM. The reaction was carried out at room temperature for 2 h. Phenothiazine photosensitizer B (49 mg, 64 %) was obtained by purification through a reversed-phase column.

[0110]

[0111] Example 6

[0112] In this example, phenothiazine photosensitizer C was prepared based on phenothiazine functional precursor 3 prepared in Example 3. 3

[0113]

[0114] ​​​​Specifically, phenothiazine functional precursor 3 (0.095 mmol, 43 mg), m-PEG2-COOH (0.45 mmol, 67 mg), HOBT (0.45 mmol, 61 mg) were dissolved in anhydrous DCM, and EDCI (0.65 mmol, 125 mg) and DIPEA (0.45 mmol, 78 μL) were added. The reaction was carried out at room temperature for 2 h. Phenothiazine photosensitizer C (38 mg, 70%) was obtained by purification through a reversed-phase column.

[0115]

[0116] Although only the case where n = 2 is shown in the examples, it will be understood by those skilled in the art that n can also take other values, such as 1, 3, 4, etc.

[0117]

Example 7

[0118] In this example, phenothiazine photosensitizer D with the group R 2 as a thiophene group was further prepared based on phenothiazine photosensitizer C.

[0119]

[0120] Specifically, 2-thiophene boronic acid (0.2 mmol, 15 mg), phenothiazine photosensitizer C (0.1 mmol, 60 mg), K2CO3(0.3 mmol, 41 mg), Pd(PPh3)4(0.05 mmol, 58 mg) were added under nitrogen protection in 1,4-dioxane and water (operation in a glove box), and the reaction was carried out at 80°C for 10 h. Ice acetic acid (1 mmol, 57 μL) was added for quenching, and phenothiazine photosensitizer D (34 mg, 57%) was obtained by purification through a silica gel column.

[0121]

[0122]

Example 8

[0123] In this example, phenothiazine photosensitizer E with the group R 3 as was prepared based on phenothiazine functional precursor 2 prepared in Example 2, wherein the group X is a phenyl group and the group R4 is an azido group.

[0124]

[0125] ​Take 4-cyanobenzoic acid (5 mmol, 735 mg) under nitrogen protection, use ethanol to dissolve and add Boc-aminopropionitrile (10 mmol, 1.7 g), 3-mercapto propionic acid (5 mmol, 436 μL) and hydrazine hydrate (50 mmol, 2.5 ml). Reaction at room temperature for 15 h. Ice bath under aqueous sodium nitrite solution (50 mmol, 3.45 g) 100 ml reaction for 10 min, adjust pH to 2-3, EA (ethyl acetate) extraction, anhydrous sodium sulfate drying, then use rotary evaporator to remove EA, the residue is purified by silica gel column chromatography to obtain intermediate HX-376 (1.55 g, 91%).

[0126] 1 H NMR (400 MHz, DMSO-d6) δ 8.60 (d, J = 8.2 Hz, 3H), 8.21 (d, J = 8.3Hz, 3H), 3.49 (t, J = 6.1 Hz, 3H), 3.42 (d, J = 6.2 Hz, 3H), 1.27 (s, 13H).

[0127]

[0128] Take intermediate HX-376 (0.3 mmol, 104 mg) and dissolve it in DCM (1 ml), then add 4M HCl dioxane (0.5 ml), react at room temperature for two hours, directly spin dry, and then add DCM once to obtain LXD-80 (73 mg, 99%). The theoretical molecular weight is 245.0913, and the molecular weight is 245.1 after mass spectrometry.

[0129]

[0130] Take LXD-80 (0.3 mmol, 73 mg), HX-302 (0.4 mmol, 100 mg), DIPEA (0.8 mmol, 140 μL) and dissolve them in anhydrous DMF, react at room temperature for 2 h, add 50 ml of water, extract with EA, dry with anhydrous sodium sulfate, then use a rotary evaporator to remove EA, and then purify the residue by silica gel column chromatography to obtain compound 6 (32 mg, 20%).

[0131]

[0132]

[0133] Phenothiazine functional precursor 2 (0.01 mmol, 6 mg), compound ⑥ (0.012 mmol, 5 mg), HOBT (0.015 mmol, 2 mg), after dissolved in anhydrous DCM, EDCI (0.03 mmol, 6 mg) and DIPEA (0.02 mmol, 4 μL) were added, and the reaction was carried out at room temperature for 2 h. Ice acetic acid (0.04 mmol, 2.3 μL) was added to quench, and purified by silica gel column chromatography to obtain phenothiazine photosensitizer E (8.5 mg, 95 %). The theoretical molecular weight of phenothiazine photosensitizer E was 868.2241, and the molecular weight was 868.2 after mass spectrometry.

[0134] In some embodiments, group X can also be a C1-C8 linear alkyl group, for example, methyl, ethyl, n-propyl, n-butyl, t-butyl, and the like. In one or more embodiments, R 4 may also be H.

[0135]

Example 9

[0136] In this embodiment, group R 3 Phenothiazine photosensitizer F with triphenylphosphine group and lonidamide.

[0137]

[0138] Specifically, lonidamide (1.5 mmol, 480 mg), NHS (hydroxysuccinimide) (1.65 mmol, 190 mg), DIC (diisopropyl carbodiimide) (1.65 mmol, 208 mg) were dissolved in DMF (7 mL) and DIPEA (1.8 mmol, 313 μL) was added. The reaction was carried out at room temperature for 1 h. Then Boc-L-lysine (2 mmol, 493 mg) was added. The reaction was carried out at room temperature for 1 h. Purified by silica gel column chromatography to obtain HX-433 (600 mg, 73 %). The theoretical molecular weight was 548.1593, and the molecular weight was 548.2 after mass spectrometry.

[0139] Subsequently, after HX-433 (1.1 mmol, 600 mg) was dissolved in DCM (9 ml), trifluoroacetic acid (3 ml) was added, and the reaction was carried out at room temperature for 2 h. DCM was removed using a rotary evaporator and was taken once with DCM to obtain intermediate LQ-42 (468 mg, 95 %). The theoretical molecular weight was 448.1069, and the molecular weight was 448.1 after mass spectrometry.

[0140]

[0141] (3-carboxypropyl)triphenylphosphonium bromide (1 mmol, 429 mg), pentafluorophenol (1.5 mmol, 276 mg), EDCI (2 mmol, 384 mg) were dissolved in anhydrous DMF (5 mL) and reacted at room temperature for 3 h. The pentafluorophenol intermediate was obtained. Subsequently, LQ-42 (0.15 mmol, 5 mg), the pentafluorophenol intermediate (0.16 mmol, 67 mg) were dissolved in anhydrous DCM (0.2 mL) and DIPEA (0.4 mmol, 70 μL) was added. The reaction was carried out at room temperature for 1 h. Purification by silica gel column chromatography yielded compound ⑦ (23 mg, 20 %). The theoretical molecular weight was 779.2315, and the molecular weight was 779.2 after mass spectrometry.

[0142]

[0143] The phenothiazine functional precursor 2 (0.01 mmol, 6 mg), compound ⑦ (0.012 mmol, 5 mg), HOBT (0.015 mmol, 2 mg) were dissolved in anhydrous DCM, and then EDCI (0.03 mmol, 6 mg) and DIPEA (0.02 mmol, 4 μL) were added. The reaction was carried out at room temperature for 2 h. Ice acetic acid (0.04 mmol, 2.3 μL) was added to quench the reaction, and purification by silica gel column chromatography yielded the phenothiazine photosensitizer F (8.5 mg, 90 %). The theoretical molecular weight was 1263.2698, and the molecular weight was 1263.3 after mass spectrometry.

[0144]

Comparative Example

[0145]

[0146] 1-naphthylamine (3 mmol, 430 mg) was dissolved in methanol, and then N-Boc-2- aminoacetaldehyde (3.6 mmol, 573 mg) and acetic acid (6 mmol, 345 μL) were added. The reaction was carried out at room temperature for 3 h. Subsequently, sodium cyanoborohydride (4.2 mmol, 264 mg) was added, and the reaction was carried out at room temperature overnight. Water (50 mL) was added, and dichloromethane was extracted. After drying with anhydrous sodium sulfate, dichloromethane was removed using a rotary evaporator, and the residue was purified by silica gel column chromatography to obtain the intermediate LXD-31 (450 mg, 50 %).

[0147] 1H NMR (400 MHz, Chloroform-d) δ 7.95 - 7.84 (m, 1H), 7.81 - 7.72 (m, 1H), 7.49 - 7.38 (m, 2H), 7.33 (t, J = 7.8 Hz, 1H), 7.25 (d, J = 8.2 Hz, 1H), 6.62 (d, J = 7.5 Hz, 1H), 5.00 (d, J = 6.5 Hz, 1H), 3.56 (q, J = 5.7 Hz, 2H), 3.39 (t, J = 5.4 Hz, 2H), 1.46 (s, 9H).

[0148]

[0149] DMSO was added to LXD-31 (1.61 mmol, 461 mg) and compound ③ (1.15 mmol, 318 mg), and potassium dichromate (1.26 mmol, 371 mg) was added. The mixture was reacted at room temperature for 30 min. The mixture was transferred into methanol, 2 M HC1 (6 ml) was added, and the mixture was reacted at room temperature for 60 min. The solid was collected by suction filtration, and the filtrate was purified by silica gel column chromatography to obtain intermediate LXD-32 (267 mg, 49 %).

[0150] 1 H NMR (400 MHz, Chloroform-d) δ 9.13 - 9.06 (m, 1H), 8.82 (dt, J = 7.3, 2.7 Hz, 1H), 7.82 (dd, J = 9.5, 2.2 Hz, 1H), 7.70 (t, J = 4.6 Hz, 2H), 7.12 (dd, J = 9.4, 2.5 Hz, 1H), 6.98 (s, 1H), 6.76 (d, J = 2.7 Hz, 1H), 6.46 (s, 1H), 3.88 - 3.78 (m, 2H), 3.76 (t, J = 5.6 Hz, 2H), 3.64 (q, J = 7.1 Hz, 4H), 1.44 (s, 9H), 1.35 (t, J = 7.1 Hz, 6H).

[0151]

[0152] LXD-32 (0.44 mmol, 210 mg) was dissolved in DCM (3 ml), followed by the addition of trifluoroacetic acid (1 ml). The reaction was carried out at room temperature for 2 h. The DCM was removed using a rotary evaporator, and the product (162 mg, 98%) was obtained by repeating the reaction with the DCM. The theoretical molecular weight was 376.1766, and the molecular weight was determined to be 376.2 by mass spectrometry.

[0153]

[0154] LXD-35 (0.1 mmol, 38 mg), m-PEG2-COOH (0.5 mmol, 74 mg), and HOBT (0.5 mmol, 68 mg) were dissolved in anhydrous DCM, and then EDCI (0.65 mmol, 125 mg) and DIPEA (0.5 mmol, 87 μL) were added. The reaction was carried out at room temperature for 2 h. The phenothiazine photosensitizer G (31 mg, 61%) was purified by reverse-phase column chromatography.

[0155] 1 H NMR (400 MHz, Chloroform-d) δ 8.86 (d, J = 7.3 Hz, 1H), 8.59 (d, J= 22.9 Hz, 3H), 7.87 (d, J = 9.3 Hz, 1H), 7.73 (d, J = 6.8 Hz, 2H), 7.11 (d,J = 10.7 Hz, 1H), 6.82 (s, 1H), 3.88 – 3.73 (m, 4H), 3.70 – 3.52 (m, 6H), 3.44 (t, J = 4.4 Hz, 2H), 3.30 (s, 4H), 2.60 (d, J = 6.4 Hz, 2H), 1.36 (d, J= 7.0 Hz, 6H).

[0156] III. Performance testing of phenothiazine photosensitizers

[0157]

Example 10

[0158] In this embodiment, the ability of phenothiazine photosensitizers A, B, C, D, and G to generate reactive oxygen species (ROS) was tested in vitro using characteristic DPBF (1,3-diphenylisobenzofuran).

[0159] Specifically, at 37°C, 36µM of DPBF was added to a 70% ACN-H2O solution containing 5µM phenothiazine photosensitizer. Under irradiation at 15mW, the absorption attenuation of DPBF during singlet oxygen generation was measured. The experimental results are as follows: Figure 2As shown, with the increase of irradiation time, the absorption of the solution at 410 nm gradually decreased, and the trend showed a good linear relationship, indicating that the ROS generation ability of the phenothiazine photosensitizer is stable.

[0160] Furthermore, through comparison Figure 2 The absorption decay curves of each phenothiazine photosensitizer show that, compared to phenothiazine photosensitizer G in the comparative example, at R... 1 Or R 2 After introducing halogen or thiophene groups, phenothiazine photosensitizers A through D all exhibit improved photosensitivity. Among them, phenothiazine photosensitizer B shows the best performance, followed by phenothiazine photosensitizers C and D, and then phenothiazine photosensitizer A, reflecting the effect of R... 2 Introducing halogen or thiophene groups into the position of the radical can achieve better photosensitivity.

[0161]

Example 11

[0162] In this embodiment, the ability of phenothiazine photosensitizers A, B, C, D, and G to generate superoxide anions was detected in DHR123 (dihydrorhodamine 123) cells.

[0163] Specifically, at 37°C, 10 µM of phenothiazine photosensitizer was added to 10 µM of DHR123 in a 50% ACN-H2O solution, and the fluorescence intensity was measured over time under irradiation at 100 mW. The experimental results are as follows: Figure 3 As shown, the fluorescence intensity of the solution at 525 nm gradually increased with the increase of irradiation time, and the change trend showed a good linear relationship, further indicating that the phenothiazine photosensitizer also has a stable ROS generation ability in cells.

[0164] Furthermore, through comparison Figure 3 The fluorescence intensities corresponding to the various phenothiazine photosensitizers in the comparison sample show that phenothiazine photosensitizers A to D, compared to R in the comparative example, exhibit different fluorescence intensities. 1 Or R 2 Unsubstituted phenothiazine photosensitizers G all exhibited better results. As shown in the figure, among the modification sites of phenothiazine photosensitizers, site R2 showed better photosensitivity than site R1. Meanwhile, due to the heavy atom effect, phenothiazine photosensitizer B also showed the best effect in cells.

[0165]

Example 12

[0166] In this embodiment, the phototoxicity of phenothiazine photosensitizers A, B, C, D, and G at different concentrations was measured using CCK8, thereby comparing the ROS generation capacity of different phenothiazine photosensitizers.

[0167] Specifically, different concentrations of phenothiazine photosensitizer A, B, C, D, G were added for incubation for 1h, and 660nm 25mW / cm 2 Light irradiation for 5min. 24h later, cell survival rate was determined by CCK8 analysis, so as to determine the phototoxicity of photosensitizer.

[0168] The test results are shown in Figure 4 As shown, the killing effect of phenothiazine photosensitizer B on cancer cells is the best, and the killing effect of phenothiazine photosensitizer C is the second, and the killing effect of phenothiazine photosensitizer with R 1 or R 2 substituted phenothiazine photosensitizer is better than that of unsubstituted phenothiazine photosensitizer G.

[0169] Further, the IC 50 of the five phenothiazine photosensitizers is calculated according to known data, wherein the IC 50 of phenothiazine photosensitizer G is 470nM, the IC 50 of phenothiazine photosensitizer D is 219nM, the IC 50 of phenothiazine photosensitizer A is 153nM, the IC 50 of phenothiazine photosensitizer C is 120nM, and the IC 50 of phenothiazine photosensitizer B is 52nM. It can be seen that the IC 50 of phenothiazine photosensitizer B is reduced by 9 times compared with unsubstituted phenothiazine photosensitizer G, indicating that the effect of phenothiazine photosensitizer B is the best.

[0170] The above experimental results further show that the modification of the para-position R2 is better, and the ROS production capacity of the phenothiazine photosensitizer B with R2 modified group as iodine is obviously better than that of other phenothiazine photosensitizers due to the heavy atom effect.

[0171] Based on this, in subsequent performance tests, phenothiazine photosensitizer E and phenothiazine photosensitizer F with R2 also being iodine substitution are used for further application expansion.

[0172]

Example 13

[0173] In this embodiment, the fluorescence on-off ratio of phenothiazine photosensitizer E is determined by using a fluorescence spectrometer, so as to determine the effect of the tetrazine group on quenching the phenothiazine photosensitizer, and prove the potential of realizing selective on-off of the phenothiazine photosensitizer.

[0174] Specifically, 2mM tetrazine quenched phenothiazine photosensitizer E in methanol or PBS solution is added with 6mM BCN (bicyclo[6,1,0]nonyne) at 37°C, and the fluorescence change of phenothiazine photosensitizer E before and after the reaction of BCN and phenothiazine photosensitizer E is determined. The reaction formula of BCN and phenothiazine photosensitizer E is as follows:

[0175]

[0176] As can be seen from the reaction formula, the phenothiazine photosensitizer E is quenched by the introduction of a tetrazine group, using fluorescence resonance energy transfer (FRET) and the reduction of water solubility, reducing its photodynamic effect. After the biological orthogonal click reaction between the phenothiazine photosensitizer E and the trans-cyclooctene, such as BCN, the tetrazine group reacts and removes the quenching, releasing the phenothiazine on the right side of the above reaction formula with photosensitizer activity; at the same time, the alkyne and azide groups in BCN undergo a click reaction, introducing a water-soluble group to improve the water solubility of the photosensitizer, and the activated phenothiazine produces ROS, thereby triggering a series of photochemical reactions, leading to cell death or inactivation of biomolecules, and achieving the treatment of cancer or other diseases.

[0177] The test results are shown in Table 1. Figure 5 As shown in Table 1, the fluorescence intensity of the phenothiazine photosensitizer E in methanol solution after adding 6mM BCN is about 1.1 times that before adding BCN; and the fluorescence intensity of the phenothiazine photosensitizer E in PBS solution after adding 6mM BCN is about 30 times that before adding BCN. This result proves that the tetrazine group and the azide group successfully achieve the quenching of the phenothiazine photosensitizer E through photo-induced charge transfer and the reduction of water solubility, and the photodynamic effect is weakened. It proves that the phenothiazine photosensitizer E has the potential of selective opening. This strategy effectively inhibits the side effects of photodynamic therapy, and reduces the damage of the photosensitizer “NO” state to normal tissue cells. In addition, the water solubility of the photosensitizer after the reaction is improved, further improving its photodynamic effect in cells.

[0178]

Example 14

[0179] In this embodiment, the phenothiazine photosensitizer F connected with a targeting and drug group is subjected to phototoxicity analysis.

[0180] Specifically, first, different concentrations of the phenothiazine photosensitizer F are added for incubation for 24h, and then the cell survival rate is analyzed by CCK8 after 24h. Subsequently, 660nm 25mW / cm 2 light irradiation for 5min. The cell survival rate is analyzed by CCK8 after 24h, so as to determine the dark toxicity and phototoxicity of the photosensitizer.

[0181] The test results are shown in Table 1. Figure 6 As shown in Table 1, the phenothiazine photosensitizer F does not show obvious cytotoxicity in the absence of light, while the phenothiazine photosensitizer F shows excellent phototoxicity under light. When the concentration is higher than 50nM, the killing effect of the phenothiazine photosensitizer F on cancer cells is better than that of the phenothiazine photosensitizer B. It can be known that after the triphenylphosphine group and the lonidamide are introduced at the group R 3 , the phenothiazine derivative F has the best treatment effect on tumors, which proves the feasibility of the strategy of introducing a targeting group and a drug group.

[0182] The above detailed description of the specific embodiments of the present application has been given to understand the purpose, technical solutions and beneficial effects of the present application. It should be understood that the above description is only a specific embodiment of the present application and is not used to limit the protection scope of the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A phenothiazine functional precursor, characterized in that, It has the structure shown in Equation I: Formula I: In Formula I, groups R1 and R2 are each independently selected from H, halogen, or thiophene group, and groups R1 and R2 are not both H.

2. The functional precursor of phenothiazine according to claim 1, characterized in that, The group R1 is H, and the group R2 is a halogen or a thiophene group; or, the group R2 is H, and the group R1 is a halogen or a thiophene group.

3. A method of preparing a functionalized phenothiazine, characterized in that, The preparation method for the phenothiazine functional precursor according to claim 1 or 2 includes the following steps: Using the compound shown in Formula II as a raw material, after amino protection, it reacts with the compound shown in Formula III to obtain the compound shown in Formula IV. After deprotection of the amino group of the intermediate, the phenothiazine functional precursor shown in Formula I is obtained. Formula II: , Formula III: , Formula IV: ; In Formulas II and IV, groups R1 and R2 match the structure of the phenothiazine functional precursor as described in claim 1 or 2.

4. The method for preparing a phenothiazine functional precursor according to claim 3, characterized in that, Includes the following steps: The compound shown in Formula II reacts with N-tert-butoxycarbonyl-2-aminoacetaldehyde under acidic conditions to give a first intermediate. The first intermediate is then aminated with sodium cyanoborohydride to give a second intermediate. The second intermediate and the compound shown in Formula III are oxidized with an oxidizing agent to obtain the compound shown in Formula VI; The compound shown in Formula VI was deprotected from Boc by trifluoroacetic acid to obtain the phenothiazine functional precursor shown in Formula I.

5. A phenothiazine photosensitizer characterized in that, It has the structure shown in formula V, formula VI, or formula VII: Formula V: ; Formula VI: ; Equation VII: In formulas V, VI, and VII, groups R1 and R2 are each independently selected from H, halogen, or thiophene, and groups R1 and R2 are not simultaneously H; in formula V, n = 1 to 10; in formula VI, group X is selected from phenyl or C1 to C8 chain alkyl, group R4 is selected from H or azide, and m = 1 to 10.

6. The phenothiazine photosensitizer according to claim 5, wherein In formulas V, VI, and VII, the group R1 is H, and the group R2 is a halogen or a thiophene group; or, the group R2 is H, and the group R1 is a halogen or a thiophene group; in formula VI, the group X is selected from phenyl or a C1-C4 chain alkyl group, and the group R4 is selected from azide group.

7. A phenothiazine photosensitizer according to claim 5 or 6, characterized in that, 、 、 、 、 、 8. A method of preparing a phenothiazine photosensitizer, characterized by, The preparation method for the phenothiazine photosensitizer according to claim 5 includes the following steps: The phenothiazine photosensitizer of formula V was prepared by dissolving the phenothiazine functional precursor, mPEG-COOH, and condensing agent shown in formula I, and reacting them at room temperature under alkaline conditions. The phenothiazine photosensitizer of formula VI is prepared by dissolving the phenothiazine functional precursor of formula I, the compound of formula VIII, and the condensing agent, and reacting them at room temperature under alkaline conditions; or The phenothiazine functional precursor shown in Formula I, the compound shown in Formula IX, and the condensing agent were dissolved and reacted at room temperature under alkaline conditions to prepare the phenothiazine photosensitizer shown in Formula VII. Formula I: #imgpt13#; Formula VIII: #imgpt14#; Formula IX In Formula I, groups R1 and R2 are each independently selected from H, halogen or thiophene group, and groups R1 and R2 are not both H; in Formula VIII, group X is selected from phenyl or C1~C8 chain alkyl group, group R4 is selected from H or azide group, and m=1~10.

9. The method for preparing a phenothiazine photosensitizer according to claim 8, characterized in that, The preparation method of the compound shown in formula IX comprises the following steps: After chloronidamine, hydroxysuccinimide and diisopropyl carbodiimide are dissolved, they are reacted in an alkaline environment, then Boc-L-lysine is added to obtain a third intermediate, trifluoroacetic acid is added to the solution of the third intermediate, and the fourth intermediate is prepared through reaction; After (3-carboxypropyl)triphenylphosphonium bromide, pentafluorophenol and 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride are dissolved, they are reacted to obtain the fifth intermediate; The fourth intermediate and the fifth intermediate are reacted in an alkaline environment to obtain the compound shown in formula IX.

10. The method for preparing a phenothiazine photosensitizer according to claim 8, characterized in that, The condensing agent is 1-hydroxybenzotriazole and 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, and the solvent is anhydrous dichloromethane.

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