A semi-cyanine icd activator and its application in tumor immunotherapy

Abstract

The application belongs to the field of tumor immunotherapy, and particularly relates to a semi-cyanine ICD activator and application thereof in tumor immunotherapy. The ICD activator has a structure shown in formula I: wherein X is halogen, and R1 and R2 are each independently selected from H,

Description

Technical Field

[0001] This invention belongs to the field of tumor immunotherapy, specifically relating to a hemicyanine ICD activator and its application in tumor immunotherapy. Background Technology

[0002] Immunotherapy controls and eliminates tumors by reactivating and maintaining the tumor-immune cycle, restoring the body's immune system's anti-tumor response. Immunotherapy is favored in cancer treatment due to its unique advantages, such as accurate tumor cell identification, stimulation of long-term immune memory, and fewer adverse reactions. Since the approval of ipilimumab, the first antibody targeting cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), immune checkpoint blockade (ICB) therapy has achieved significant results in the treatment of various cancers. Programmed death-ligand 1 (PD-L1) inhibitors can block the binding of PD-L1 on tumor cells to PD-1 on immune cells, restoring the immune cells' attack on tumor cells and preventing immune escape. However, the immunosuppressive tumor microenvironment (ITM) lacking cytotoxic T-lymphocyte (CTL) infiltration limits the application of existing cancer immunotherapies. Therefore, combining it with other therapies to reverse the ITM and compensate for the shortcomings of existing single immunotherapies is particularly important. Photodynamic therapy (PDT) is considered a promising cancer treatment strategy due to its non-invasiveness, high spatiotemporal resolution, and low toxicity. PDT can induce immunogenic cell death (ICD) in tumor cells. ICD recruits and activates immune cells, such as dendritic cells (DCs), by releasing various damage-associated molecular patterns (DAMPs), such as cell surface-exposed calreticulin (CRT) and heat shock proteins (HSPs), extracellularly released high-mobility group box 1 (HMGB1), and adenosine triphosphate (ATP), thereby promoting antigen presentation and stimulating anti-tumor immune responses. PDT can not only generate reactive oxygen species (ROS) to kill tumor cells, but also remodel the tumor microenvironment and initiate tumor immune responses, thus enhancing the efficacy of immunotherapy. In this PDT-induced ICD pathway, how ROS triggers intense endoplasmic reticulum stress has become a key question.

[0003] The endoplasmic reticulum (ER) is rich in a negatively charged phospholipid bilayer, and anthocyanin photosensitizers, due to their quaternary ammonium cations, can bind to the ER membrane through electrostatic interactions, thereby achieving targeted delivery. Therefore, modifying anthocyanin photosensitizers to achieve efficient ROS generation plays a crucial role in promoting ICD (intracellular photodynamic therapy). Hemicyanin photosensitizers possess advantages such as high molar extinction coefficients, good stability, and ease of structural modification. This invention designs a class of hemicyanin-based ICD activators for highly efficient photodynamic immunotherapy. Summary of the Invention

[0004] This invention provides an ICD activator for photodynamic therapy, a pharmaceutically acceptable salt thereof, or a tautomer thereof, characterized in that the ICD activator has the structure shown in Formula I:

[0005] Where X is a halogen, and R1 and R2 are each independently selected from H, .

[0006] Another embodiment of the present invention provides a method for preparing an ICD activator of the above formula I selected from the following compounds:

[0007] , , X is a halogen.

[0008] Another embodiment of the present invention provides an intermediate for preparing an ICD activator with the structure of Formula I above, characterized in that the intermediate has the structure shown in Formula II:

[0009] R3 is selected from , X is a halogen.

[0010] Another embodiment of the present invention provides the use of the intermediate of Formula II in the preparation of the ICD activator of Formula I.

[0011] Another embodiment of the present invention provides the use of the above-described ICD activator, its pharmaceutically acceptable salt, and its tautomers in the preparation of photodynamic tumor immunotherapy drugs. Preferably, it is used for immunotherapy of breast cancer.

[0012] Another embodiment of the present invention provides a pharmaceutical composition characterized in that the pharmaceutical composition comprises an ICD activator of the above-described formula I, a pharmaceutically acceptable salt thereof, and its tautomer as active ingredients. The pharmaceutical composition may also include other antitumor drugs. The pharmaceutical composition may also include pharmaceutically acceptable excipients. The dosage form is preferably a solid dosage form, a liquid dosage form, or a semi-solid dosage form.

[0013] The halogens used in this invention are preferably fluorine, chlorine, bromine, iodine, etc.

[0014] Compared with existing technologies, the advantages of this invention are as follows: This invention uses positively charged indole salts and 9-phenyl-9h-carbazole as electron acceptors and electron donors to regulate the construction of π-A Hcy, π-AD Hcy-Car, and D-π-AD Hcy-2Car, respectively. The improved construction of Hcy-Car and Hcy-2Car optimizes photophysical properties and promotes ROS generation by optimizing excited-state properties. Specifically, the ΔE of Hcy-2Car... STIt is the smallest and possesses the most efficient ROS generation capacity. Furthermore, in subcellular co-localization experiments, Hcy-2Car exhibits excellent endoplasmic reticulum (ER) targeting ability. Combining abundant ROS generation and ER targeting, Hcy-2Car induces strong ER stress, promotes DAMPs release, and ultimately activates immune cells, enhancing the anti-tumor immune response. This method promotes dendritic cell (DC) activation by improving antigen presentation, thereby enhancing cytotoxic T lymphocyte (CTL) recruitment. This method enables successful immunotherapy by overcoming the immunosuppressive tumor microenvironment (ITM). Therefore, in mouse models, tumor growth is inhibited both locally and distantly, highlighting the promising application of this molecular-electronic engineering in creating photodynamic immunogenic cell death (ICD) inducers and novel immunotherapies against breast cancer. Attached Figure Description

[0015] Figure 1 A shows the optimized geometry and HOMO / LUMO diagrams of Hcy, Hcy-Car, and Hcy-2Car in the ground state; B is a summary chart of the photophysical and DFT calculations of Hcy, Hcy-Car, and Hcy-2Car.

[0016] Figure 2 A is the UV absorption spectrum of Hcy-2Car, Hcy-Car, and Hcy (2 μM) in water; B is the fluorescence spectrum; C, D, and E are the UV absorption spectra of Hcy-2Car, Hcy-Car, and Hcy in water at 660 nm (20 mW / cm²). 2 Under illumination, the ultraviolet absorption spectrum of ABDA; F is the change in ABDA absorption at 380 nm; G is... 1 O2 quantum yield calculation; H is at 660 nm (20 mW / cm²). 2 Hcy-2Car, Hcy-Car, and Hcy were measured under illumination. 1 O2 generation; I is the Hcy-2Carb content measured under SOSG. 1 O2 generation; J is the Hcy-Carb content measured under SOSG. 1 O2 generation amount; K is the ROS generation detected using SOSG; L is the ROS generation detected using DHR123.

[0017] Figure 3Image A shows a co-stained image of MCF7 cells treated with ER-Tracker Green (0.5 μM) and Hcy-2Car, Hcy-Car, and Hcy, scale bar = 50 μm; Image B shows the overall ROS production of Hcy-2Car and Hcy-Car in MCF-7 cells using DCFH-DA as an indicator, scale bar = 50 μm; Image C shows the ROS production of Hcy-2Car and Hcy-Car in MCF-7 cells using SOSG as an indicator. 1 O2 conditions, scale bar = 50 μm; D and E are the quantitative fluorescence results of DCFH-DA and SOSG, respectively; F and G are the fluorescence intensities of DCFH-DA and SOSG detected by flow cytometry, respectively; H is the expression of 53BP1 protein analyzed under laser confocal microscopy, scale bar = 50 μm; I is the expression of γ-H2AX protein analyzed under laser confocal microscopy, scale bar = 50 μm; J is the qualitative analysis using calcein AM and PI probes to evaluate the expression at 660 nm (20 mW / cm²). 2 The killing effect of Hcy-2Car and Hcy-Car on MCF7 cells under 10 min light conditions, scale bar = 200 μm; K is at 660 nm (20 mW / cm²). 2 Phototoxicity of Hcy-2Car, Hcy-Car and Hcy to MCF7 cells under 10 min light conditions; *p< 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.

[0018] Figure 4 Image A shows the co-localization analysis of MCF7 cells after co-incubation with ER green fluorescent probe and Hcy-2Car; Image B shows the Pearson correlation coefficient between the two channels; Images C, D, E, and F show the co-localization of HMGB1, GRP78, CRT, and HSP70 in MCF7 cells after treatment with Hcy-2Car and Hcy-Car, and after near-infrared irradiation (660 nm, 20 mW / cm²), respectively. 2 Immunofluorescence staining images after 10 min; G is the fluorescence intensity of HMGB1 detected by flow cytometry; H is the ATP content after different treatments (660 nm, 20 mW / cm²). 2 (10 min); I is the detection of Hcy-2Car and Hcy-Car by flow cytometry after irradiation with 660 nm light (20 mW / cm).2 Annexin v-FITC / PI staining was performed 10 min later; J, K and L were the fluorescence quantitative PCR results for GRP78, CRT and HSP70, respectively, scale bar = 50 μm, *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.

[0019] Figure 5 Figure A shows the establishment of a 4T1 subcutaneous tumor transplantation mouse model and the detection of immunomolecular expression. Figures B, D, F, and H are flow cytometry plots of the proportions of CD80 / CD86, CD3 / CD4, CD3 / CD8, and CD25 / FOXP3 molecules in the tumors of Balb / c mice (n = 3) with 4T1 tumor transplantation. Figures C, E, G, and I are the corresponding quantitative analyses of the cell proportions in figures B, D, F, and H. Figure J shows the immunofluorescence staining of HMGB1 and CRT in tumor tissues under different conditions. Scale bar = 100 μm, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

[0020] Figure 6 Image A shows the establishment and treatment regimen of the 4T1 subcutaneous tumor transplantation mouse model; images B and E show the proximal and distal tumor volumes during the anti-tumor process after different treatments, respectively; images C and F show the weights of the proximal and distal tumors during treatment, respectively; images D and G show the proximal and distal tumors in mice, respectively; images I, J, K, and L show the serum concentrations of TNF-α, IFN-γ, IL-12, and IL-6 after different treatments, respectively; image H shows H&E staining of tumor tissue and immunofluorescence staining of mouse tumor tissue, including CD3, CD4, and CD8 molecules; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, scale bar = 100 μm.

[0021] Figure 7 The compounds Hcy-2Car and Hcy-Car are at 660 nm (20 mW / cm²). 2 Phototoxicity of 4T1 and HCT-15 cells under 10 min light exposure, **** p < 0.05, p < 0.01, **** p < 0.001, **** p < 0.0001.

[0022] Figure 8 It is the compound Hcy-2Car 11H NMR (400 MHz, DMSO-d6) spectrum.

[0023] Figure 9 It is the compound Hcy-2Car 13 C10 NMR (100 MHz, DMSO-d6) spectrum.

[0024] Figure 10 This is the high-resolution mass spectrum of the compound Hcy-2Car.

[0025] Figure 11 It is the compound Hcy-Car 1 1H NMR (400 MHz, DMSO-d6) spectrum.

[0026] Figure 12 It is the compound Hcy-Car 13 C10 NMR (100 MHz, DMSO-d6) spectrum.

[0027] Figure 13 This is a high-resolution mass spectrum of the compound Hcy-Car.

[0028] Figure 14 It is the compound Hcy 1 1H NMR (400 MHz, DMSO-d6) spectrum.

[0029] Figure 15 It is the compound Hcy 13 C10 NMR (100 MHz, DMSO-d6) spectrum.

[0030] Figure 16 This is a high-resolution mass spectrum of the compound Hcy. Detailed Implementation

[0031] To facilitate a further understanding of the present invention, the following embodiments are provided for more detailed description. However, these embodiments are only for a better understanding of the invention and are not intended to limit the scope or implementation principles of the invention. The implementation of the present invention is not limited to the following.

[0032] Materials: Unless otherwise specified, general chemical reagents were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. All solvents used were analytical grade. Bengal roselic acid (RB) and 9,10-anthratrium dibis(methylene)dimalonic acid (ABDA) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; 1,3-diphenylisobenzofuran (DPBF) was purchased from Beyotime Pharmaceuticals (Shanghai) Co., Ltd. ER-Tracker Green, Lyso-Tracker Green, Golgi-Tracker Green, Hoechst 33342, Annexin V-FITC apoptosis assay kit, and cytotoxicity assay kit were purchased from Beyotime Biotechnology Co., Ltd. Mouse IL-10 ELISA kit, mouse IL-12 ELISA kit, TNF-α ELISA kit, and IFN-γ ELISA kit were purchased from Shanghai Sorapi Biotechnology Co., Ltd. PE anti-mouse CD86 antibody [GL-1], FITC anti-mouse CD80 antibody [16-10A1], FITC anti-mouse CD3 antibody [17A2], PE anti-mouse CD8 antibody [53-6.7], PE anti-mouse CD4 antibody [RM4-5], FITC anti-mouse CD25 antibody [PC-61.5.3], PE anti-mouse Foxp3 antibody [3G3], and FITC anti-mouse CD206 / MMR antibody [C068C2] were provided by Proteintech Biotechnology Co., Ltd. The ATP assay kit was purchased from Beyotime Biotechnology Co., Ltd. MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-bromotetrazole) was purchased from Energy Chemicals Co., Ltd. Singlet oxygen green fluorescent probe (SOSG) was purchased from Meilun Biotechnology (China). Dulbecco modified Eagle medium (DMEM) and PBS buffer were purchased from Wuhan Pusai Life Science Technology Co., Ltd. Human colorectal adenocarcinoma cells (HCT-15), mouse breast cancer cells (4T1), and human breast cancer cells (MCF-7) were purchased from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (IBMS).

[0033] Methods: ¹H-NMR and ¹³C-NMR spectra were measured using a Bruker Avance II 400 spectrometer. Mass spectrometry (ESI-MS) data were detected using an Ultimate 3000 (Thermo Scientific) instrument. Fluorescence images were acquired using a TCS SP8 laser scanning confocal microscope (Lecia). Cell apoptosis was detected using flow cytometry (BECKMAN COULTER), and cytotoxicity experiments were performed using a multi-functional microwell detector (SpectraMax i3x). UV-Vis-NIR absorption spectra of different samples were recorded using a Japanese Evolution 220 UV-Vis spectrophotometer (UV-2700i).

[0034] Example 1

[0035]

[0036] Compound 1 (1.5 mmol) and Compound 2 (1 mmol) were dissolved in DMF (5 mL), and cesium carbonate (1.5 mmol), 4-dimethylaminopyridine (DMAP, 0.05 mmol), and Et3N (0.1 mL) were added. The mixture was stirred at room temperature until the reaction was complete as detected by TLC. The reaction was terminated by adding water, resulting in a yellow flocculent precipitate. The precipitate was separated by column chromatography (petroleum ether / ethyl acetate = 25:1) to obtain a yellow solid (i.e., Compound 3, yield 46.5%). HRMS (ESI): m / zcalc. for [C 14 H 10 BrO2] - 288.98697, found 288.98512 [MH] - .

[0037]

[0038] Following the synthetic method for compound 3, replacing compound 1 in the reactants with salicylaldehyde yielded compound 4 in 43.2% yield. HRMS (ESI): m / zcalc. for [C 14 H 11 O2] - 211.07645, found 211.07506[MH] - .

[0039] Example 2

[0040]

[0041] 4-(9H-carbazole-9-yl)phenylboronic acid pinacol ester (2 mmol) and compound 3 (1 mmol) were dissolved in an ethanol / water mixture (3:1, v / v). Potassium phosphate (2 mmol), potassium fluoride dihydrate (1 mmol), 1,1'-bis(di-tert-butylphosphino)ferrocene dichloropalladium (0.05 mmol), and 2-dicyclohexylphosphine-2',4',6'-triisopropylbiphenyl (0.1 mmol) were added. The mixture was heated to 78 °C and reacted until complete as detected by TLC. The filtrate was collected, concentrated, and then separated by silica gel column chromatography (petroleum ether / ethyl acetate = 25:1) to obtain compound 5 (yield approximately 93.2%). HRMS (ESI): m / zcalc. for [C 32 H 22 NO2] - 452.16560, found 452.16442[MH] - .

[0042]

[0043] Following the synthetic method for compound 5, replacing compound 3 with compound 6 in the reactants yielded compound 7 in 91.5% yield. HRMS (ESI): m / zcalc. for [C 29 H 23 N2] - 399.18667, found 399.18523[MH] - .

[0044] Example 3

[0045]

[0046] Compound 7 (1.0 mmol) was dissolved in acetonitrile (4 mL), and benzyl bromide (1.0 mmol) was added. The mixture was heated to 80 °C and reacted for 48 h. The reaction solution was concentrated under reduced pressure, and the residue was collected and dissolved in a small amount of dichloromethane. Subsequently, a large amount of n-hexane was rapidly added and the mixture was stirred vigorously. The mixture was then filtered to obtain the solid product, which was compound 8 (yield approximately 61.2%). HRMS (ESI): m / zcalc. for [C 36 H 31 N2] + 491.24871, found 491.24674 [M] + .

[0047]

[0048] Following the synthetic method for compound 8, replacing compound 7 with compound 9 in the reactants yielded compound 10 in 59.6% yield. HRMS (ESI): m / zcalc. for [C 18 H 20 N] + 250.15902, found 250.15785 [M] + .

[0049] Example 4

[0050]

[0051] Compound 5 (1.0 mmol) and compound 8 (1.0 mmol) were dissolved in 5 mL of acetonitrile, 0.2 mL of pyridine was added, and the mixture was heated to reflux temperature. After reacting for 12 hours, the reaction solution was concentrated and separated by silica gel column chromatography (dichloromethane / methanol = 15:1) to obtain the blue solid, which was compound Hcy-2Car (yield approximately 53.2%). 1 H NMR (400 MHz, DMSO) δ 8.70 (d, J= 14.8 Hz, 1H), 8.27 (s, 4H), 8.03 (s, 2H), 7.77 (s, 2H), 7.46 (s, 9H), 7.32(s, 6H), 7.18 (s, 1H), 6.89 – 6.76 (m, 2H), 5.88 (s, 2H), 5.76 (s, 2H), 2.76(s, 2H), 2.62 (s, 2H), 2.14 (s, 1H), 1.95 (s, 6H), 1.21 (d, J = 6.8 Hz, 7H),0.89 (d, J = 6.8 Hz, 3H). 13C NMR (101 MHz, DMSO) δ 179.11, 160.76, 152.54,146.60, 143.59, 141.85, 140.49, 140.45, 139.25, 138.76, 137.95, 137.07,136.99, 136.78, 134.79, 131.09, 130.80, 129.74, 129.57, 129.26, 128.82,127.62, 127.27, 126.82, 123.34, 122.60, 121.87, 121.10, 120.70, 115.60,110.14, 55.42, 51.46, 34.18, 31.07, 27.96, 26.39, 26.11, 25.07, 24.32, 22.05.HRMS (ESI): m / zcalc. for [C 68 H 52 N3O] + 926.41049, found 926.40869 [M] + .

[0052]

[0053]

[0054] Following the synthetic method for compound Hcy-2Car, replacing compound 5 with compound 4 in the reactants yielded compound Hcy-Car in 51.6% yield. 1 H NMR (400 MHz, DMSO) δ 8.66 (d, J = 14.8 Hz,1H), 8.34 – 8.20 (m, 3H), 8.08 (d, J = 8.4 Hz, 2H), 7.92 (s, 1H), 7.77 (s,3H), 7.59 (d, J = 7.8 Hz, 2H), 7.54 (s, 2H), 7.42 (d, J = 15.9 Hz, 7H), 7.30(s, 2H), 6.75 (d, J = 15.1 Hz, 1H), 5.80 (d, J = 39.4 Hz, 4H), 2.71 (s, 2H),2.58 (s, 2H), 1.85 (d, J = 47.3 Hz, 9H). 13C NMR (101 MHz, DMSO) δ 179.01,161.11, 152.76, 143.51, 141.87, 140.49, 139.15, 138.77, 137.05, 134.80,132.37, 129.72, 129.24, 127.58, 127.25, 126.82, 123.33, 122.08, 121.11,120.69, 116.62, 115.35, 114.58, 110.13, 106.18, 55.43, 51.38, 29.04, 27.97,24.04, 20.21. HRMS (ESI): m / zcalc. for [C 50 H 41 N2O] + 685.32134, found 685.32086 [M] + .

[0055] Following the synthetic method for compound Hcy-2Car, replacing compound 5 with compound 4 and compound 8 with compound 10 in the reactants yielded compound Hcy in 54.1% yield. 1 H NMR (400 MHz, DMSO) δ 8.64 (d,J = 14.9 Hz, 1H), 7.83 (d, J = 7.1 Hz, 1H), 7.67 (d, J = 7.6 Hz, 1H), 7.58(d, J = 7.3 Hz, 2H), 7.53 (s, 1H), 7.49 (s, 1H), 7.41 (d, J = 7.5 Hz, 2H), 7.35 (d, J = 7.4 Hz, 4H), 6.73 (d, J = 15.0 Hz, 1H), 5.78 (d, J = 15.9 Hz, 3H), 2.72 (s, 2H), 2.57 (s, 2H), 1.82 (s, 9H). 13C NMR (101 MHz, DMSO) δ179.26, 160.91, 152.70, 146.57, 142.55, 142.13, 134.76, 133.24, 132.28,130.61, 129.67, 129.48, 128.64, 128.23, 127.90, 127.21, 126.00, 123.46,122.02, 116.51, 115.08, 114.13, 106.05, 55.44, 51.24, 48.66, 29.03, 27.92,23.97, 20.19. HRMS (ESI): m / zcalc. for [C 32 H 30 NO] + 444.23219, found 444.23096 [M] + .

[0056] Example 5

[0057] Following the methods described in Examples 3 and 4, replacing the reaction reagent BnBr in Example 3 with benzyl halides such as BnCl and BnI yields anion Cl. - I - The corresponding compounds (which have properties similar to Hcy-2Car, Hcy-Car, and Hcy).

[0058] Example 6 Analysis and Testing Procedure

[0059] Singlet oxygen ( 1 O2) detection

[0060] Using SOSG / ABDA as 1 O2 indicator for assessment 1 O2 generation efficiency. Soluble solutions of Hcy-2Car, Hcy-Car, and Hcy at concentrations of 10 μM were prepared, followed by the addition of SOSG / ABDA. The mixtures were thoroughly stirred and allowed to stand for 10 min. The samples were then irradiated at a wavelength of 660 nm (20 mW / cm²). 2 After different laser irradiation times, the fluorescence spectrum and ultraviolet absorption spectrum were recorded.

[0061] 1 Determination of O2 quantum yield

[0062] Prepare 10 μM soluble solutions of Hcy-2Car, Hcy-Car, and Hcy, let stand for 10 min, and adjust the absorbance of ABDA at 380 nm to approximately 1.0. Then, at 660 nm (20 mW / cm²), [the absorbance was measured]. 2 The UV-Vis absorption spectra were recorded after irradiation for different durations. Bengal rose red (RB) was used as a reference photosensitizer, and measurements were taken. 1 The quantum yield of O2 is calculated using the following formula:

[0063]

[0064] Among them, K probe and K RB Ф represents the decomposition constant of ABDA when the probe and RB coexist. RB The quantum yield of singlet oxygen in RB (Ф in ultrapure water) is... RB = 0.75). Plot the natural logarithm of the absorbance of ABDA at 380 nm against the irradiation time; the slope is the decomposition constant.

[0065] Cell culture

[0066] MCF7 cells, 4T1 cells, and HCT-15 cells were in a solution containing 10% fetal bovine serum and 1% antibiotics (penicillin / streptomycin, 100 U / mL). -1 The cells were cultured in DMEM medium. All three cell types were cultured at 37°C with a CO2 / air ratio of 5% / 95%.

[0067] Cytotoxicity test

[0068] MCF7 cells were seeded in 96-well plates at a density of 1 × 10⁻⁶ cells / well. 4 Cells / well, 100 μL, incubated at 37℃ for 24 h, then different concentrations of Hcy-2Car, Hcy-Car, and Hcy were added in the range of 0-5 μM, and incubated in an incubator for 6 h. Then irradiated with 660 nm (20 mW / cm²). 2 The cells were treated for 10 minutes. The absorbance at 570 nm and 630 nm was measured using a microplate reader (SpectraMax i3x). The formula for calculating cell viability is shown below.

[0069]

[0070] The same method was used to detect the cytotoxicity of 4T1 cells and HCT-15 cells.

[0071] Cell uptake assay

[0072] Cellular uptake of Hcy-2Car, Hcy-Car, and Hcy compounds was assessed at different time points. Cells were seeded in laser confocal culture dishes, and cultured until the cell density reached 1×10⁶ cells / year. 5 At a concentration of 100 μL / mL, 2 μM Hcy-2Car, Hcy-Car, and Hcy were added respectively. After treatment, the samples were observed under a laser confocal microscope.

[0073] Subcellular localization

[0074] After incubation for 6 h in cell culture dishes containing Hcy-2Car (1 μM) and Hcy-Car (1 μM), a subcellular localization probe was added and incubation continued for another 1 h. The cells were observed under a laser confocal microscope (CLSM) at the end of the treatment.

[0075] Intracellular singlet oxygen ( 1 O2) detection

[0076] The seeded cell culture dishes were divided into 6 groups: PBS group, PBS + NIR group, Hcy-2Car group, Hcy-2Car + NIR group, Hcy-Car group, and Hcy-Car + NIR group. After incubation in a cell culture incubator for 6 h, SOSG (10 μM) or DCFH-DA (10 μM) green fluorescent probe was added for another 1 h of incubation. Cells in the PBS + NIR group, Hcy-2Car + NIR group, and Hcy-Car + NIR group were irradiated with near-infrared light for 10 min. At the end of the treatment, the cells were observed under CLSM.

[0077] Cell viability test

[0078] The culture plates were divided into four groups: PBS group, PBS + NIR group, Hcy-2Car group, Hcy-2Car + NIR group, Hcy-Car group, and Hcy-Car + NIR group. The PBS + NIR group, Hcy-2Car + NIR group, and Hcy-Car + NIR group were treated with near-infrared light (660 nm, 20 mW / cm²). 2 Irradiate for 10 minutes. After incubation for 6 hours, add the corresponding reagent from the cytotoxicity assay kit and continue incubation for 2 hours. At the end of the treatment, observe under CLSM.

[0079] Apoptosis detection

[0080] After seeding cells into 6-well plates, they were incubated for 24 h until the cells were fully adherent. Subsequently, according to the experimental protocol, the cells were divided into six groups: PBS group, PBS + NIR group, Hcy-2Car group, Hcy-2Car + NIR group, Hcy-Car group, and Hcy-Car + NIR group. Hcy-2Car and Hcy-Car were added accordingly, and the cells were incubated for another 6 h. The PBS + NIR group, Hcy-2Car + NIR group, and Hcy-Car + NIR group were irradiated with near-infrared light (660 nm, 20 mW / cm²). 2 Incubate for 10 minutes, then continue incubation for 12 hours. Add appropriate detection reagents.

[0081] GRP78 protein expression

[0082] According to the experimental requirements, the cells were divided into 6 groups. After treating the cells with Hcy-2Car and Hcy-Car, they were incubated in an incubator for another 6 h, and then exposed to 660 nm near-infrared light at 20 mW / cm². 2 Irradiate for 10 min. Fix cells with GRP78 primary antibody (1:100) and incubate for 4 h. After removing the primary antibody, wash the culture dish and incubate with fluorescent secondary antibody for 4 h. Then observe the samples under CLSM.

[0083] HSP70 protein expression

[0084] The procedure is the same as described above, with the only exception being that the GRP78 primary antibody is replaced with the HSP70 primary antibody.

[0085] HMGB1 protein expression

[0086] The procedure is the same as described above, with the only exception being that the GRP78 primary antibody is replaced with the HMGB1 primary antibody.

[0087] CRT protein expression

[0088] The procedure is the same as described above, with the only exception being that the GRP78 primary antibody is replaced with the CRT primary antibody.

[0089] In vivo antitumor activity

[0090] All procedures were performed in accordance with the "Guidelines for the Protection and Utilization of Laboratory Animal Resources" and the requirements of the National Research Council, and were approved by the Laboratory Animal Ethics Committee of Shandong Second Medical University. Approval ethics number: 2023SDL238. Mice were randomly divided into six groups (n = 5 per group): PBS group, PBS + NIR group, Hcy-2Car group, Hcy-2Car + αPD-L1 group, Hcy-2Car + NIR group, and Hcy-2Car + αPD-L1 + NIR group. A bilateral tumor model was established. When the left tumor volume reached 100 mm³, Hcy-2Car (1 mg / kg) was injected in situ, followed by irradiation 24 h later using NIR light (660 nm, 20 mW / cm²). 2 Irradiation was performed. αPD-L1 was administered intravenously 24 hours later. Tumor volume and body weight were measured in all experiments. Tumor volume was calculated using the formula: Tumor volume = (Length × Width²) / 2. For mice fed with 4T1 tumors, tumor volume and body weight were measured every 2 days.

[0091] Detection of molecular expression on mouse tumor surface

[0092] After extracting mouse tumors, the tumor tissue was minced using surgical scissors. The minced tissue was then digested with collagenase at 37°C for 30 minutes. After digestion, the cells were resuspended in PBS, and the cell suspension was filtered through a 300-mesh filter. The resulting cell suspension was centrifuged at 1500 rpm for 5 minutes. The precipitate was initially resuspended in buffer, with 600 μL of buffer added to each tube. Subsequently, 30 μL was taken for staining, and the staining process was carried out for one hour prior to machine testing.

[0093] Example 7

[0094] 1. Photophysical and photodynamic properties of compounds Hcy-2Car, Hcy-Car, and Hcy

[0095] The photophysical properties of Hcy-2Car, Hcy-Car, and Hcy were investigated using absorption spectroscopy and photoluminescence (PL) spectroscopy. The maximum absorption peaks of Hcy-2Car, Hcy-Car, and Hcy were located at 625 nm, 615 nm, and 558 nm, respectively. Figure 2 A). The fluorescence peaks of Hcy-2Car, Hcy-Car, and Hcy appeared near 729 nm, 724 nm, and 654 nm, respectively. Figure 2B). Compared to Hcy, the absorption peaks of Hcy-2Car and Hcy-Car exhibit a redshift due to their larger conjugated structures. This redshift further validates the influence of the conjugated structure introduced in molecular design on photophysical properties. Furthermore, this redshift opens up possibilities for the application of Hcy-2Car and Hcy-Car in deep tissue therapy.

[0096] Density functional theory (DFT) was used to investigate Hcy-2Car, Hcy-Car, and Hcy to gain a deeper understanding of their photophysical properties. Hcy-2Car exhibits delocalized HOMO and LUMO distributions on the D-π-AD backbone, with a small HOMO-LUMO band gap of 0.998 eV, consistent with the redshift in the absorption spectrum. This is likely due to the introduction of 9-phenyl-9H-carbazole, which lengthens the conjugated system, enhances electronic delocalization, and promotes intramolecular charge transfer (ICT), leading to a reduction in the energy polarity difference between HOMO and LUMO. Next, the values ​​of ΔEST and SOC were calculated to explore the ISC process, which plays a crucial role in ROS generation. A smaller ΔEST corresponds to a larger SOC. 1 The higher the O2 generation efficiency, the better. As shown in Figure 1, the ΔEST values ​​of Hcy-2Car, Hcy-Car, and Hcy are 0.827 eV, 0.929 eV, and 1.154 eV, respectively. Among them, Hcy-2Car has the smallest ΔEST value and the largest SOC, at 0.046 cm⁻¹. -1 This helps to accelerate the ISC process and improve... 1 The quantum yield of O2. Therefore, this strategy of adjusting the D-π-A system holds promise for extending the absorption and emission wavelengths of Hcy photosensitizer and increasing the amount of ROS generated.

[0097] Subsequently, we investigated the formation of three compounds (Hcy-2Car, Hcy-Car, and Hcy) using 9,10-anthratridimyl-bis(methylene)dicarboxylic acid (ABDA). 1 The ability of O2 (Figure 2C-E). Among them, the curve of Hcy changes the least. 1 O2 production is almost negligible, while Hcy-2Car's 1 The O2 production was 1.3 times that of Hcy-Car (Figure 2F). Furthermore, we used rose red (RB) dye as a standard to evaluate the formation of these three compounds in aqueous solution. 1 The quantum yield Φ of O2 Δ(Figure 2G). Hcy-2Car, Hcy-Car, and Hcy 1 The O2 quantum yields were 0.65, 0.42, and 0.06, respectively. Subsequently, it was detected by 1,3-diphenylisobenzofuran (DPBF). 1 O2 generation was observed, and consistent with the results of ABDA, Hcy-2Car and Hcy-Car exhibited high ROS generation capabilities (Figure 2H). ROS detection results using dihydrorhodamine (DHR123) and the singlet oxygen green fluorescent probe (SOSG) are shown in Figures 2I-L, indicating that Hcy-2Car produced the highest ROS generation efficiency. These results collectively demonstrate that Hcy-2Car and Hcy-Car possess excellent ROS generation capabilities, leading to more effective photodynamic therapy.

[0098] 2. Internalization and photodynamic-induced DNA damage

[0099] The accumulation of Hcy-2Car, Hcy-Car, and Hcy in MCF7 cells was detected using confocal laser scanning microscopy (CLSM). Intracellular fluorescence intensity increased in a time-dependent manner. Hcy reached its maximum fluorescence intensity at 1 h, while Hcy-2Car and Hcy-Car reached their maximum intensities at 6 h and 2 h, respectively. This may be related to the introduction of numerous conjugated groups into the molecular structures of Hcy-2Car and Hcy-Car, which alter the efficiency of molecule entry into the cell. Subsequently, co-localization imaging was performed using commercially available probes (ER-Tracker Green, Golgi-Tracker Green, and Lyso-Tracker Green) to analyze the subcellular localization of Hcy-2Car, Hcy-Car, and Hcy. The results showed that the co-localization coefficients of Hcy-2Car, Hcy-Car, and Hcy in the endoplasmic reticulum were 0.89, 0.92, and 0.93, respectively. Figure 3 A). However, the colocation coefficients with the Golgi apparatus are only 0.57, 0.53, and 0.65, and with lysosomes, they are only 0.46, 0.42, and 0.50. Therefore, Hcy-2Car, Hcy-Car, and Hcy are mainly located in the endoplasmic reticulum.

[0100] Given the good reactive oxygen species (ROS) production capacity of Hcy-2Car and Hcy-Car, their dark toxicity to normal cells was then assessed using the MTT assay. After culturing COS7 cells with different concentrations of Hcy-2Car and Hcy-Car for 12 h, the cell viability of both Hcy-2Car and Hcy-Car remained above 85%, indicating good biocompatibility. Next, using HCT-15, MCF7, and 4T1 cell lines as models, the cytotoxicity of Hcy-2Car and Hcy-Car was evaluated using the MTT assay to determine their feasibility for biological applications. Cell viability was measured after different treatments. Under dark conditions, even at a concentration of 5 μM Hcy-2Car and Hcy-Car, the survival rate of MCF7 cells exceeded 80%, indicating low cytotoxicity of Hcy-2Car and Hcy-Car. Figure 3 K). Similarly, similar results were obtained in the other two cell types. Under laser irradiation, the half-maximal inhibitory concentration (IC50) of Hcy-2Car in 4T1 cells was... 50 The IC50 of Hcy-Car was 2.30 μM, and that of Hcy-Car was 2.49 μM. Figure 7 Hcy-2Car IC in HCT-15 and MCF7 50 The concentrations were 1.60 μM and 2.39 μM, respectively. Figure 3 Hcy-2Car and Hcy-Car exhibited lower cytotoxicity in the dark than in the light, indicating that they are selective and controllable for PDT.

[0101] Given the favorable reactive oxygen species (ROS) generation capabilities of Hcy-2Car and Hcy-Car in water, their intracellular photodynamic therapy properties were further evaluated. First, the total intracellular ROS generation was detected using 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) (Figure 3B). This substance can be oxidized by ROS to 2',7'-dichlorofluorescein (DCF), which emits green fluorescence upon entering the cell. Consistent with the ROS generation trend in water, the fluorescence signal of Hcy-2Car + NIR was superior to that of Hcy-Car + NIR. Subsequently, the intracellular photodynamic therapy properties were assessed using the singlet oxygen fluorescent probe SOSG. 1 Regarding O2 generation, Hcy-2Car exhibited the strongest fluorescence intensity. 1 O2 generation characteristics were superior to Hcy-Car (Figure 3C). Quantitative fluorescence analyses of both are shown in Figures 3D and 3E, respectively. Furthermore, flow cytometry (FCM) detection of DCFH-DA and SOSG also showed the same trend (Figures 3F and 3G).

[0102] Next, we assessed the status of DNA damage by detecting the expression of phosphorylation-associated histone H2AX (γH2AX) and p53-binding protein 1 (53BP1). 53BP1 is an important regulator in response to DNA double-strand breaks. After incubation with fluorescent secondary antibody, Hcy-2Car + NIR emitted the strongest fluorescence, followed by Hcy-Car + NIR (Figure 3H). γH2AX is an important DNA damage marker that marks DNA damage sites by forming unique nuclear spots within the cell nucleus. The strongest fluorescence intensity of γ-H2AX after Hcy-2Car + NIR treatment indicates that strong DNA damage has been induced (Figure 3I). These results suggest that the compound Hcy-2Car generates a large amount of ROS under laser irradiation, thereby inducing stress in the ER, which in turn activates the ICD pathway and causes DNA damage. Furthermore, simultaneous staining with Calcein-AM and propidium iodide (PI) was used to distinguish between live cells (green) and dead cells (red) for a more intuitive evaluation of the phototherapy effects of Hcy-2Car and Hcy-Car (Figure 3J). Cells treated with Hcy-Car were subjected to light (wavelength 660 nm, power 20 mW / cm²). 2 After 10 minutes, large areas of red fluorescence appeared, and almost all cells treated with Hcy-2Car showed red fluorescence, with a cell death rate approaching 100%. The control group, however, showed almost no red fluorescence. This indicates that the large amount of ROS produced intracellularly led to significant cell death.

[0103] 3. In vitro immunogenic cell death

[0104] Hcy-2Car maintained precise ER localization under near-infrared light irradiation of varying intensities, highlighting its powerful targeting ability (Figures 4A and 4B). Both Hcy-2Car and Hcy-Car exhibit good ER localization capabilities and generate ROS on the ER, with in situ ROS directly inducing strong ER stress. Glucose regulatory protein 78 (GRP78) maintains endoplasmic reticulum stability and is a major regulator of the UPR (unfolded protein response). Loss of ER homeostasis triggers the unfolded protein response, leading to upregulation of GRP78 expression. The highest fluorescence intensity was observed in Hcy-2Car+NIR after near-infrared light irradiation (Figures 4D and 4J), emphasizing the high expression of GRP78 protein.

[0105] Under light conditions, ROS generated by Hcy-2Car and Hcy-Car induces ER stress, which in turn activates ICDs by triggering the release of a large number of damage-associated molecular patterns (DAMPs). We evaluated the in vitro effects of ICDs by detecting the expression levels of CRT, HMGB1, and HSP70 using confocal laser scanning microscopy (CLSM). CRT, acting as a signal to "eat me," migrates from the endoplasmic reticulum lumen to the cell membrane, and the Hcy-2Car + NIR treatment group showed the strongest green fluorescence intensity (Figs. 4E and 4K), indicating the strongest CRT expression. Subsequently, HMGB1, acting as a signal to "find me," migrates from the nucleus to the extracellular region to trigger inflammation and promote antitumor immune responses, and the Hcy-2Car + NIR group showed the weakest green fluorescence, indicating a large amount of HMGB1 exocytosis (Figs. 4C and 4G). In addition, HSP70, a protein involved in synergistic immunity, is overexpressed during cellular stress responses. The Hcy-2Car + NIR group showed the brightest fluorescence (Figs. 4F and 4L), indicating a significant upregulation of HSP70 expression. ATP release, as a chemotactic signal, recruits immune cells. In the Hcy-2Car + NIR group, ATP efflux was approximately 4.7-fold higher than in the control group and 1.8-fold higher than in the Hcy-Car + NIR group (Fig. 4H). These results suggest that photodynamic therapy induces ER stress through the generated ROS and triggers the ICD pathway. Subsequently, we used Annexin V-FITC and PI to assess the drug's effect on apoptosis. Apoptosis was almost zero in the PBS, PBS + NIR, Hcy-2Car, and Hcy-Car groups. However, under NIR light irradiation, the apoptosis rates in the Hcy-2Car and Hcy-Car groups reached 54.03% and 25.65%, respectively (Fig. 4I).

[0106] 4. In vivo drug-induced immune response

[0107] RAW264.7 macrophages were induced with interleukin-4 (IL-4) to establish an M2 macrophage population, which was then co-cultured with 4T1 cells after different treatments in a Transwell insertion system. The ability of Hcy-2Car to induce tumor-associated macrophage (TAM) polarization was assessed by flow cytometry. Compared with the control group, the proportion of M1 macrophages in the Hcy-2Car + NIR group increased from 21.62% to 31.86%, while the proportion of M2 macrophages significantly decreased from 42.51% to 31.59%. Subsequently, the levels of interleukin-10 (IL-10) and interleukin-12 (IL-12) were quantitatively detected using an ELISA kit. The results showed that the trends of IL-10 and IL-12 in different groups were consistent with those in M2 and M1 macrophages. These results indicate that Hcy-2Car effectively transforms M2 macrophages into M1 macrophages, thereby reversing the immunosuppressive tumor microenvironment.

[0108] Subsequently, a bilateral 4T1 tumor model was established to validate the ability of Hcy-2Car to activate a systemic immune response. Furthermore, αPD-L1 (a commonly used immune checkpoint inhibitor) was used as an adjunct to Hcy-2Car immunotherapy. Following intratumoral Hcy-2Car treatment, αPD-L1 was administered intravenously. At the end of treatment, tumor cells were extracted for immune cell analysis. The release of damage-associated molecular patterns (DAMPs) recruited and activated dendritic cells (DCS) and promoted the release of T cells from lymph nodes. Compared to the control group, the proportion of mature DCS at the primary tumor site increased to 34.53% and 44.30% in the Hcy-2Car+NIR group and the Hcy-2Car+αPD-L1+NIR group, respectively (Figures 5B and 5C), with similar trends observed in distant tumors. Cytotoxic T lymphocytes (CTLS) were able to secrete perforin and other cytotoxic mediators to kill cancer cells. Compared with the control group, the Hcy-2Car + NIR group and the Hcy-2Car + αPD-L1 + NIR group showed significantly increased levels of CD3+CD4+ T cells (from 5.26% to 24.91% and 31.29%, respectively) and CD3+CD8+ T cells in the primary tumors (from 18.73% to 30.07% and 42.42%, respectively) (Figure 5D-G). Expression in distant tumors was also assessed, showing a similar trend. Regulatory T cells (Tregs), as a subset of CD4+ T cells with low proliferative capacity, primarily exert immunosuppressive effects. Compared with the control group, the proportion of CD25+ FOXP3+ T cells in primary tumors decreased from 46.48% to 28.41% and 13.48% in the Hcy-2Car + NIR group and the Hcy-2Car + αPD-L1 + NIR group, respectively (Figures 5H and I), and a similar decreasing trend was observed in distant tumors. Compared with the control group, the primary tumor group treated with Hcy-2Car + αPD-L1 + NIR showed a significant increase in M1 macrophages and a significant decrease in M2 macrophages. These results indicate that photodynamic therapy combined with immune checkpoint inhibitors has the potential to induce a systemic antitumor immune response. In addition, immunohistochemical analysis of HMGB1 and CRT proteins in tissue samples showed that in the Hcy-2Car + αPD-L1 + NIR group, HMGB1 protein was extensively effluxed and CRT protein expression was increased (…). Figure 5 This further validates the application potential of this combination therapy (J), which in turn confirms its potential.

[0109] 5. In vivo anti-tumor effects

[0110] Given the remarkable efficacy of the Hcy-2Car + αPD-L1 + NIR group in inducing a systemic anti-tumor immune response, we further evaluated its ability to treat primary and distant tumors. Mice were randomly assigned to four groups: PBS, PBS + NIR, Hcy-2Car, Hcy-2Car + αPD-L1, Hcy-2Car + NIR, and Hcy-2Car + αPD-L1 + NIR. A bilateral 4 T1 tumor model was established, and the treatment regimens are shown in Figure 6A.

[0111] In the PBS group, the mean volume of primary tumors increased by 11.2-fold, and the mean volume of distal tumors increased by 8.6-fold (Figs. 6B and 6E). Compared with the PBS group, the Hcy-2Car + αPD-L1 + NIR group showed significant tumor suppression in both primary and distal tumors (Figs. 6B and 6E). The tumor weight of both primary and distal tumors in the Hcy-2Car + αPD-L1 + NIR group was significantly lower than that in the other groups (Figs. 6C and 6F). Throughout the treatment, the mice showed almost no increase in body weight and no significant changes, indicating a high safety profile. Histological analysis using hematoxylin and eosin (H&E) staining showed no significant organ damage among the groups, further emphasizing the biocompatibility of the drug. H&E staining of tumor tissue sections showed significant nuclear pyknosis and cellular vacuolation in the Hcy-2Car + αPD-L1 + NIR group, indicating severe damage and necrosis of tumor cells (Fig. 6H). Next, we assessed the infiltration of CD3+ T cells, CD4+ T cells, and CD8+ T cells in distant tumors using immunofluorescence staining. The Hcy-2Car + αPD-L1 + NIR group showed significantly enhanced fluorescence, indicating a large influx of CD3+ T cells, CD4+ T cells, and CD8+ T cells into the tumor region (Fig. 6H). Furthermore, we measured the expression levels of pro-inflammatory cytokines (including TNF-α (tumor necrosis factor-α), IFN-γ (interferon-γ), IL-6 (interleukin-6), and IL-12 (interleukin-12)) in mouse serum samples. Significant production of these immune-related cytokines was observed in the Hcy-2Car + αPD-L1 + NIR group (Fig. 6I-L). These findings suggest that Hcy-2Car-mediated PDT, together with αPD-L1-induced immune checkpoint blockade, activates a systemic, durable, and potent anti-tumor immune response, thus exerting an anti-tumor effect.

[0112] 6. Conclusion

[0113] This invention successfully developed a group of Hcy-derived photodynamic immune cell death (ICD) inducers using molecular-electronic engineering techniques, thereby achieving effective immunotherapy for breast cancer. Through optimization of electronic and molecular structure, Hcy-2Car exhibits better cellular oxidative stress (SOC) levels and lower ΔEST values, leading to increased ROS production. By specifically targeting the ER and generating ROS in breast cancer cells, Hcy-2Car induces significant ER stress, thereby promoting DAMP secretion and a robust ICD effect. Increased DAMP levels promote DC maturation and CTL recruitment. Furthermore, the synergistic effect of Hcy-2Car with α-PD-L1 antibodies shows superior therapeutic efficacy. A significant shift in macrophage polarization from M2 to M1 contributes to a robust immune response. Therefore, this method effectively inhibits the progression of both primary and metastatic 4T1 tumors. The success of the molecular-electronic engineering of Hcy-2Car and its working principle provide valuable insights for designing modified ICD inducers for breast cancer immunotherapy.

Claims

1. An ICD activator for use in photodynamic therapy, a pharmaceutically acceptable salt thereof, or a tautomer thereof, characterized in that The ICD activator is selected from the following compounds: , X is bromo.

2. An intermediate for preparing the ICD activator, a pharmaceutically acceptable salt thereof, or a tautomer thereof according to claim 1, characterized by The intermediate has a structure shown in Formula II: wherein R3is selected from , X is bromo.

3. Use of the intermediate of Formula II according to claim 2 in the preparation of the ICD activator, a pharmaceutically acceptable salt thereof, or a tautomer thereof according to claim 1.

4. Use of the ICD activator, a pharmaceutically acceptable salt thereof, or a tautomer thereof according to claim 1 in the preparation of a photodynamic tumor immunotherapy drug.

5. A pharmaceutical composition, characterized by The pharmaceutical composition comprises the ICD activator, a pharmaceutically acceptable salt thereof, or a tautomer thereof according to claim 1 as an effective ingredient.

6. The pharmaceutical composition of claim 5, wherein The pharmaceutical composition further comprises other anti-tumor drugs.

7. The pharmaceutical composition according to any one of claims 5-6, characterized in that The pharmaceutical composition further comprises pharmaceutically acceptable excipients.

8. The pharmaceutical composition of claim 7, wherein The pharmaceutical composition is in a dosage form selected from a solid preparation, a liquid preparation, or a semi-solid preparation.

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