A heptamethine indolocyanine photosensitive small molecule releasing ido inhibitor and application thereof

Abstract

The application belongs to the field of pharmaceutical chemistry, and specifically discloses a heptamethine indolenine cyanine photosensitive small molecule releasing an IDO inhibitor and application thereof. The small molecule compound can be specifically accumulated in tumor cell mitochondria, and can generate local high heat and generate ROS under 808nm laser irradiation, so as to induce ICD of a primary tumor and activate an anti-tumor immune effect. Meanwhile, the small molecule releases an IDO inhibitor through ROS response, so as to relieve immunosuppression, synergistically amplify the anti-tumor immune effect, and establish a long-lasting immune memory.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry, specifically relating to a heptamethrin-indocyanine photosensitive small molecule that releases an IDO inhibitor and its applications. Background Technology

[0002] Malignant tumors are a major disease threatening human life and health worldwide, with their incidence and mortality rates rising year by year. Traditional tumor treatments, such as surgical resection, chemotherapy, and radiotherapy, generally have limitations such as poor treatment specificity, significant toxic side effects, easy development of drug resistance, and difficulty in eliminating micrometastases, and cannot fully meet the actual needs of clinical precision treatment and long-term control.

[0003] Phototherapy, including photothermal therapy (PTT) and photodynamic therapy (PDT), has attracted much attention due to its advantages such as being minimally invasive, spatially and temporally controllable, and having low systemic toxicity. Among them, heptamethrin indocyanine near-infrared fluorescent dyes have excellent photostability, high molar extinction coefficient, and good biocompatibility. They can not only be used as near-infrared imaging probes to achieve precise tumor localization, but also efficiently generate reactive oxygen species or convert light energy into heat energy under near-infrared light irradiation. During phototherapy, they induce immunogenic cell death (ICD) in tumor cells, release damage-associated molecular patterns (DAMPs), thereby activating dendritic cells (DCs) and recruiting cytotoxic T lymphocytes (CTLs), inhibiting primary tumor growth to a certain extent and producing anti-tumor immune effects.

[0004] However, the immune response elicited by phototherapy alone is often insufficient to eradicate metastases or achieve a sustained systemic anti-tumor effect. The fundamental reason lies in the complex immunosuppressive mechanisms present in the tumor microenvironment (TME), particularly the tryptophan metabolic pathway mediated by indoleamine-2,3-dioxygenase (IDO). IDO catalyzes the degradation of tryptophan into metabolites such as kynurenine, leading to tryptophan depletion in the tumor microenvironment, inhibiting the proliferation and activation of effector T cells, and simultaneously promoting the infiltration of immunosuppressive cells such as regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs). IDO inhibitors can effectively block the tryptophan metabolic pathway, reverse the immunosuppressive state of the tumor microenvironment, and restore the body's normal anti-tumor immune function, thus becoming an important research direction in the field of tumor immunotherapy.

[0005] Studies have shown that during phototherapy-induced immunogenic cell death, cytotoxic tumor cells (CTLs) secrete IFN-γ (Interferon-γ) to enhance immune infiltration, but conversely, they induce tumor cells to overexpress IDO. This rapidly suppresses the limited immune response activated by phototherapy, resulting in a weak "distant effect" of treatment and a significantly increased risk of tumor recurrence. The immunosuppressive microenvironment formed by tumor overexpression of IDO under CTL stimulation directly weakens the anti-tumor effect of immune cells and hinders their effective infiltration into tumor tissue. This is one of the key bottlenecks hindering the efficacy of phototherapy alone. It is also noteworthy that clinical studies of IDO inhibitors show that the combination of Epacadostat and pembrolizumab did not significantly improve treatment efficacy, suggesting that using IDO inhibitors alone is insufficient to enhance the tumor infiltration capacity of immune cells and cannot fundamentally reverse the rapidly formed immunosuppressive state after phototherapy. Therefore, the dual deficiency of phototherapy-mediated anti-tumor immunity being weakened by IDO overexpression and the inability of IDO inhibitors alone to rebuild effective immune infiltration remains a critical problem that urgently needs to be solved in the field of phototherapy-immunotherapy synergistic therapy for tumors. Summary of the Invention

[0006] The purpose of this invention is to provide a photosensitive small molecule compound of heptamethrin indocyanine that releases an IDO inhibitor in response to ROS. This type of small molecule compound can specifically accumulate in the mitochondria of tumor cells, generating local hyperthermia and ROS under 808 nm laser irradiation, inducing ICD in the primary tumor and activating anti-tumor immune effects. Simultaneously, the small molecule relieves immunosuppression by releasing an IDO inhibitor in response to ROS, synergistically amplifying the anti-tumor immune effect and establishing durable immune memory. Although we have previously synthesized small molecules of heptamethrin indocyanine with different chain lengths, achieving tumor targeting, near-infrared fluorescence imaging, and photothermal and photodynamic synergistic induction of anti-tumor immune effects through self-assembly with albumin in vivo, the complex immunosuppression in the tumor microenvironment significantly weakens the immune effect induced by phototherapy. To address this, this invention further synthesizes the small molecule compounds IR-C6 and IR-IDO, constructed using heptamethrin indocyanine as a backbone, covalently coupled with a small molecule drug with IDO inhibitory activity via ROS-sensitive ketethiocondensation bonds. Near-infrared laser irradiation induces ICD in tumor cells through photothermal / photodynamic effects, releasing tumor antigens and activating anti-tumor immunity. Simultaneously, ROS-responsive release of IDO inhibitors (YY, KUN-1) specifically blocks the tryptophan metabolism pathway, restoring T-cell infiltration and breaking the immunosuppressive state. This synergistic strategy not only enhances phototherapy-induced immune activation but also effectively overcomes immunosuppression in the tumor microenvironment, thereby improving overall therapeutic efficacy. Treatment with this type of small molecule-mediated therapy can establish long-term anti-tumor immune memory, effectively inhibiting tumor recurrence and demonstrating promising clinical translational potential.

[0007] To achieve the objectives of this invention, the following implementation scheme is provided.

[0008] In one embodiment, the present invention provides a heptamethrin-indocyanine-based photosensitizing small molecule, which is a compound represented by Formula I or a pharmaceutically acceptable salt thereof. , In the formula, R is a small molecule compound with IDO inhibitory activity.

[0009] In a preferred embodiment, the small molecule compound having IDO inhibitory activity is selected from the following compounds: and .

[0010] In a preferred embodiment, the aforementioned heptamethrin-indocyanine-based photosensitizing small molecule is a compound represented by formula IR-IDO or its pharmaceutical salt. .

[0011] In a preferred embodiment, the aforementioned heptamethrin-indocyanine-based photosensitizing small molecule is a compound of formula IR-C6 or its pharmaceutical salt. .

[0012] In one embodiment, the present invention provides a method for preparing the above-mentioned heptamethrin indocyanine-based photosensitive small molecule, wherein the heptamethrin indocyanine-based photosensitive small molecule is a compound represented by formula IR-IDO or its pharmaceutical salt, comprising the following steps: 1) Compound KUN-1 reacts with compound A1 in the presence of CDI to prepare compound A4. ; 2) Compound B4 was reacted with aminoethanol in the presence of a coupling agent to prepare compound B5. ; 3) Compound A4 reacts with compound B5 to prepare the compound shown in the IR-IDO formula. .

[0013] In some embodiments, the present invention provides a method for preparing the heptamethrin-indocyanine photosensitive small molecule of the present invention, wherein the heptamethrin-indocyanine photosensitive small molecule is a compound of formula IR-C6 or its pharmaceutical salt, comprising the following steps: 1) Compound A2 reacts with ethylenediamine to prepare compound A3. ; 2) Compounds B1 and B2 react with compound B3 to prepare compound B4. ; 3) Compound A3 and compound B4 were reacted in the presence of HATU to prepare the compound shown in formula IR-C6. .

[0014] In one specific embodiment, the preparation formula of the heptamethrin indolecyanine-based photosensitive small molecule of the present invention is as follows: , .

[0015] The compound is IR-C6, and the method includes the following steps: 1) Dissolve mercaptoacetic acid and acetone in trifluoroacetic acid (TFA) and stir at room temperature for 1.5 h. After the reaction is complete, add a mixture of petroleum ether and ice water, slurry, filter, and dry to obtain intermediate A1; 2) Using tetrahydrofuran (THF) as solvent, intermediate A1 and IDO inhibitor (compound YY) were added, followed by N,N'-carbonyldiimidazole (CDI). The mixture was reacted at 40°C for 2 hours. After the reaction was completed, the mixture was concentrated, extracted with ethyl acetate, dried, and concentrated to obtain intermediate A2. 3) Using THF as solvent, add ethylenediamine to intermediate A2 and continue the reaction at 40°C for 2 hours; after the reaction is completed, concentrate, extract with ethyl acetate, dry, concentrate, and separate by column chromatography to obtain intermediate A3; 4) Using N,N-dimethylformamide (DMF) as solvent, phosphorus oxychloride (POCl3) and cyclohexanone were slowly added dropwise in an ice bath. After the addition was complete, the temperature was slowly raised to 60°C and the reaction was carried out for 6 hours. After the reaction was completed, the mixture was cooled and concentrated under reduced pressure to obtain intermediate B1. 5) Dissolve 6-bromohexanoic acid and 2,3,3-trimethylindole in o-dichlorobenzene (o-DCB) and heat at 120°C for 8 hours under nitrogen protection. After the reaction is complete, cool and recrystallize from acetone to obtain indole quaternary ammonium salt B2; 6) Iodoethane and 2,3,3-trimethylindole were dissolved in o-dichlorobenzene (o-DCB), and heated at 120°C for 8 hours under nitrogen protection. After the reaction was completed, the mixture was cooled and recrystallized from acetone to give indole quaternary ammonium salt B3. 7) Using anhydrous ethanol as solvent and sodium acetate as alkaline additive, intermediate B1, indole quaternary ammonium salt B2, and indole quaternary ammonium salt B3 were fed into the mixture and reacted at 70°C for 1 hour under nitrogen protection. After the reaction was completed, the mixture was concentrated under reduced pressure, and the residue was separated by column chromatography to obtain intermediate B4. 8) Using DMF as solvent, add A3, B4 and HATU, and react at room temperature for 45 min; after the reaction, add dichloromethane for extraction, wash the aqueous phase twice with dichloromethane, combine the organic phases, wash with saturated sodium chloride solution, dry with anhydrous sodium sulfate and concentrate under reduced pressure; the residue is purified by silica gel column chromatography to obtain the target compound IR-C6.

[0016] The preparation method of the present invention described above, wherein the compound is IR-IDO, includes the following steps: 1) Using tetrahydrofuran (THF) as solvent, intermediate A1 and IDO inhibitor (compound KUN-1) were added, followed by N,N'-carbonyldiimidazole (CDI). The mixture was reacted at 40°C for 2 hours. After the reaction was completed, the mixture was concentrated, extracted with ethyl acetate, dried, and concentrated to obtain intermediate A4. 2) Using dichloromethane as a solvent, aminoethanol, B4 and HATU were added and reacted at room temperature for 2 hours. After the reaction was completed, dichloromethane was added for extraction. The aqueous phase was washed twice with dichloromethane. The organic phases were combined, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to obtain intermediate B5. 3) Using dichloromethane as solvent, A4, B5, EDCI and DMAP were added and reacted at room temperature for 2 hours. After the reaction was completed, dichloromethane was added for extraction. The aqueous phase was washed twice with dichloromethane. The organic phases were combined, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to obtain the target compound IR-IDO.

[0017] In some embodiments, the use of the heptamethrin indocyanine photosensitizing compound or its pharmaceutical salt of the present invention is provided in the preparation of a drug for treating or preventing tumors.

[0018] In some embodiments, the invention also includes the synergistic treatment of tumors with near-infrared fluorescence imaging, photothermal therapy, photodynamic therapy, and / or immunotherapy using the heptamethrin indocyanine photosensitive compound or its pharmaceutical salt thereof.

[0019] In some embodiments, a pharmaceutical composition is provided comprising the heptamethrin indocyanine photosensitizing compound or its pharmaceutical salt as described above, and pharmaceutical excipients.

[0020] In some embodiments, the above-described pharmaceutical composition is used in the preparation of drugs for the prevention and treatment of tumors.

[0021] The heptamethrin indolecyanine photosensitive compound of the present invention has low toxicity and side effects, good biocompatibility, and excellent near-infrared fluorescence imaging performance. It can specifically target and efficiently accumulate in the mitochondria of tumor cells, enabling real-time monitoring and precise treatment of tumor lesions.

[0022] The preparation process of the heptamethrin indolecyanine photosensitizing compounds of the present invention is simple, the reaction conditions are mild, the raw materials are readily available, the synthetic route is mature and stable, and it is convenient for large-scale preparation and clinical translation application.

[0023] Previous research by the applicant revealed that heptamethrin indocyanine backbone possesses excellent tumor enrichment capacity and near-infrared optical properties, enabling selective and efficient accumulation in tumor tissue. By introducing mitochondrial targeting structures into the side chain of heptamethrin indocyanine backbone and precisely coupling IDO inhibitors through ROS-responsive linkers—ketal thiocarbamate bonds—the applicant rationally designed and synthesized the functionalized heptamethrin indocyanine small molecules (IR-C6, IR-IDO) described in this invention, which possess low cytotoxicity, tumor mitochondrial targeted accumulation, near-infrared fluorescence imaging, highly efficient PDT / PTT phototherapy efficacy, immunosuppression reversal function, and long-lasting anti-tumor immune memory.

[0024] Studies have shown that heptamethrin indocyanine-based photosensitive small molecule compounds can specifically accumulate in the mitochondria of tumor cells. Under 808nm laser irradiation, they can generate strong photothermal and photodynamic effects, inducing mitochondrial oxidative stress and efficiently inducing immunogenic cell death in tumor cells. At the same time, the ROS generated by phototherapy can trigger the cleavage of ketone thiocondensation bonds, controllably releasing IDO inhibitors, blocking the IDO-mediated immunosuppressive negative feedback pathway, reshaping the tumor immune microenvironment, and thus showing extremely significant inhibitory effects on the growth of both in situ and distant ectopic tumors.

[0025] The heptamethrin-indocyanine photosensitive small molecule compounds of this invention, after intraperitoneal injection, can actively target and accumulate at the tumor site. Under near-infrared light excitation, they emit characteristic fluorescence, achieving precise fluorescence imaging of tumor lesions. After targeted irradiation of in situ tumors with 808nm laser, not only can the in situ tumor be efficiently ablated, but the activated systemic anti-tumor immune effect can also significantly inhibit the growth of distant, un-illuminated tumors. Therefore, heptamethrin-indocyanine photosensitive compounds hold promise for applications in precise tumor imaging diagnosis, in situ tumor phototherapy, systemic anti-tumor immunotherapy, and long-term anti-recurrence immune intervention.

[0026] In vitro and in vivo experiments have confirmed that the heptamethrin indocyanine photosensitizing compounds IR-C6 and IR-IDO possess excellent mitochondrial targeting and accumulation capabilities and fluorescence imaging performance. Under 808nm laser irradiation, they can exert significant photothermal and photodynamic antitumor effects, and can simultaneously achieve the synergistic effect of phototherapy promoting immune activation and IDO inhibiting and relieving immunosuppression. They can effectively activate the systemic antitumor immune response, inhibit the growth of in situ and distant tumors, and build a lasting antitumor immune memory. They can serve as highly efficient small molecule candidate drugs for the integrated precision diagnosis and treatment of tumors. Attached Figure Description

[0027] Figure 1 The 1H and 1C spectra of compound IR-C6 in Example 7 are shown. Figure 2 The proton and carbon spectra of compound IR-IDO in Example 10 are shown. Figure 3 The images show the in vivo and in vitro photothermal effects of compounds IR-C6 and IR-IDO in Example 11, where a is a solution-level infrared thermal imaging image and b is an infrared thermal imaging image of a mouse 4T1 tumor model. Figure 4 This is a solution-level photodynamic effect singlet oxygen (SOSG) level diagram for compounds IR-C6 and IR-IDO in Example 12; Figure 5 This is a diagram showing the targeted accumulation of compound IR-C6 in the mitochondria of 4T1 cells in Example 13. Figure 6 Near-infrared in vivo imaging of tumor-targeting accumulation in a mouse 4T1 unilateral tumor model, showing compounds IR-C6 and IR-IDO from Example 14. Figure 7 The images show the Western Blot diagrams of the inhibitory effect of the compounds in Example 15 on IDO protein expression, where a is the Western Blot diagram of the inhibitory effect of IR-C6 on IDO protein expression, and b is the Western Blot diagram of the inhibitory effect of IR-IDO on IDO protein expression. Figure 8 The image shows the results of in vivo phototherapy activation of the immune system by compound IR-C6 in Example 16. In the image, a and e are the flow cytometry graphs and bar charts of the proportion of CD80+CD86+ DC cells, respectively; b and f are the flow cytometry graphs and bar charts of the proportion of CD3+CD8+ CTL cells, respectively; and c and g are the flow cytometry graphs and bar charts of the proportion of CD25+FoxP3+ Treg cells, respectively. Figure 9 The figure shows the results of animal experiments verifying the photo-immunotherapy of compound IR-IDO against distant metastatic subcutaneous tumors in Example 17. In the figure, a is the experimental flowchart, b is the curve of primary tumor volume versus time, c is the curve of distant tumor volume versus time, d is the bar chart of primary tumor weight, e is the bar chart of distant tumor weight, f is the solid image of primary tumor, and g is the solid image of distant tumor. Detailed Implementation

[0028] The following embodiments are provided to describe the present invention in more detail. However, these embodiments are provided only to help further understand the present invention and are not intended to limit the present invention. Those skilled in the art should understand that equivalent substitutions or corresponding improvements made to the content of the present invention still fall within the protection scope of the present invention.

[0029] Example 1: Synthesis of 1-methyl-L-tryptophan methyl ester (YY)

[0030] 1-methyl- L β-Tryptophan (3.0 g, 13.7 mmol) was dissolved in 10 mL of methanol, and 3 mL of concentrated H₂SO₄ was slowly added dropwise with stirring. The reaction was carried out at 75 °C for 4 h. After the reaction was completed, the mixture was concentrated under reduced pressure to remove MeOH, and saturated Na₂CO₃ was added to adjust the pH to weakly alkaline. Then, 60 mL of ethyl acetate (EA) was added for extraction, and the aqueous phase was washed twice with 20 mL of EA. The combined organic phases were washed with saturated NaCl solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 2.1 g of the target compound YY, a brown oil, with a yield of 65%.

[0031] 1 H NMR (600 MHz, DMSO- d 6)δ7.49 (d, J = 5.2 Hz, 1H), 7.36 (d, J = 5.5Hz, 1H), 7.14 – 7.11 (m, 1H), 7.10 (s, 1H), 7.02 – 6.98 (m, 1H), 3.72 (s,3H), 3.60 (t, 13C NMR (151 MHz, DMSO-d6) δ176.10, 136.97, 128.50, 128.20, 121.45, 118.95, 118.86, 110.01, 109.73, 55.63, 51.81, 32.71, 31.02.

[0032] Example 2: Synthesis of 2,2'-[propane-2,2-diylbis(thio)]diacetic acid (A1)

[0033] The reaction mixture was fed with 26.42 g (286 mmol) of mercaptoacetic acid, 0.80 g (7.0 mmol) of trifluoroacetic acid (TFA), and 7.56 g (130 mmol) of acetone, and reacted under Ar protection at room temperature (RT) for 1.5 h. After the reaction was completed, a mixture of petroleum ether and ice water was added and stirred to give 5.82 g of the white solid target compound A1, with a yield of 20%.

[0034] 1H NMR (600 MHz, DMSO- d 6)δ7.49 (d, J = 5.2 Hz, 1H), 7.36 (d, J = 5.5Hz, 1H), 7.14 – 7.11 (m, 1H), 7.10 (s, 1H), 7.02 – 6.98 (m, 1H), 3.72 (s,3H), 3.60 (t, J = 6.3 Hz, 1H), 3.55 (s, 3H), 3.00 (dd, J = 14.2, 6.1 Hz, 1H), 2.92 (dd, J= 14.2, 6.5 Hz, 1H), 1.76 (s, 2H). 13 C NMR (151 MHz, DMSO-d6) δ176.10, 136.97, 128.50, 128.20, 121.45, 118.95, 118.86, 110.01, 109.73,55.63, 51.81, 32.71, 31.02.

[0035] Example 3: Synthesis of 2-chloro-3-(hydroxymethylene)-cyclohex-1-encarbaldehyde (B1)

[0036] 68 mL of POCl3 (113.4 g, 750 mmol) was slowly added dropwise to 100 mL of anhydrous N,N-dimethylformamide (DMF) under ice bath conditions, and the reaction was carried out for 1 h under ice bath conditions. Then, 20 mL of anhydrous cyclohexanone (19.6 g, 200 mmol) was added in portions to the reaction mixture, and the reaction was carried out at 60 °C for 6 h. After the reaction was completed, the mixture was added in portions to 300 g of crushed ice to quench the POCl3, and the mixture was stirred overnight at RT. The solid obtained the next day was purified by recrystallization from PE / EA to give 22.40 g of the target compound B1 as a yellow solid, with a yield of 49%.

[0037] Example 4: 1-(5-Carboxypentyl)-2,3,3-Trimethyl-3 H Synthesis of 1-indole-1-onium bromide (B2)

[0038] 6-Bromohexanoic acid (6.10 g, 31 mmol) and 2,3,3-trimethylindole (5.00 g, 31 mmol) were dissolved in 10 mL of o-dichlorobenzene, and the reaction was carried out at 120 °C for 8 h under nitrogen protection. The reaction solution was cooled to room temperature, and the solid was washed with acetone to give 4.70 g of the target compound B2 as a white solid, with a yield of 42%.

[0039] Example 5: 1-Ethyl-2,3,3-trimethyl-3 H Synthesis of 1-indole-1-onium iodide (B3)

[0040] 1-Iodoethane (9.80 g, 62 mmol) and 2,3,3-trimethylindole (10.00 g, 62 mmol) were dissolved in 10 mL of o-dichlorobenzene, and the reaction was carried out at 120 °C for 8 h under nitrogen protection. The reaction solution was cooled to room temperature, and the solid was washed with acetone to give 12.6 g of the target compound B3 as a white solid, with a yield of 64%.

[0041] Example 6: Synthesis of Compound B4

[0042] B1 (2.00 g, 11.6 mmol), B2 (4.10 g, 5.8 mmol), B3 (3.66 g, 11.6 mmol), and sodium acetate (1.90 g, 23.2 mmol) were dissolved in 25 mL of anhydrous ethanol. The reaction mixture was reacted at 70 °C for 1 h under nitrogen protection. The reaction solution was extracted with dichloromethane (DCM), concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to give 1.7 g of the target compound B4 as a green solid, with a yield of 20%.

[0043] 1 H NMR (600 MHz, DMSO- d 6)δ12.04 (s, 1H), 8.27 (t, J =13.9Hz 2H), 7.71– 7.58 (m, 2H), 7.53 – 7.38 (m, , 4H), 7.36 – 7.24 (m, 2H), 6.33 (t, J = 15.4Hz, 2H), 4.34 – 4.15 (m, 4H), 2.72 (s, 4H), 2.21 (t, J = 7.3 Hz, 2H), 1.91 –1.82 (m, 2H), 1.79 – 1.72 (m, 2H), 1.68 (s, 12H), 1.60 – 1.53 (m, 2H), 1.44 –1.37 (m, 2H), 1.32 (t, J = 7.2 Hz, 3H). 13 C NMR (151 MHz, DMSO- d6) δ 174.85,172.56, 172.40, 148.46, 143.68, 143.33, 142.57, 142.11, 141.71, 141.52,129.15, 129.11, 126.66, 126.58, 125.72, 125.59, 123.07, 123.01, 111.97,111.85, 101.97, 101.92, 49.52, 49.44, 44.12, 40.54, 39.54, 34.01, 27.97,27.86, 27.20, 26.35, 26.32, 26.15, 24.68, 20.85, 12.73.

[0044] Example 7: Synthesis of compound IR-C6

[0045] Compound A1 (1.64 g, 7.3 mmol) and N,N'-carbonyldiimidazole (CDI) (1.19 g, 7.3 mmol) were dissolved in 10 mL of tetrahydrofuran (THF) and reacted at 40 °C for 1 h. YY (1.7 g, 7.3 mmol) was added, and the reaction was continued at 40 °C for 2 h. After the reaction was complete, the pH was adjusted to weakly acidic with 4 mol / L. Extraction was performed with 60 mL of EA, and the aqueous phase was washed twice with 20 mL of EA. The combined organic phases were washed with saturated NaCl solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give 0.8 g of a colorless oil (A2), with a yield of 25%. A2 (1.4 g, 3.2 mmol) and CDI (0.52 g, 3.2 mmol) were dissolved in 10 mL of THF and reacted at 40 °C for 1 h. Ethylenediamine (0.19 g, 6.4 mmol) was added and the reaction was continued at 40 °C for 2 h. After the reaction was complete, 50 mL of EA was added for extraction. The aqueous phase was washed twice with 15 mL of EA. The combined organic phases were washed with saturated NaCl solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give 0.8 g of a colorless oil (A3), with a yield of 52%. A3 (0.25 g, 0.5 mmol), B4 (0.37 g, 0.5 mmol), and HATU (0.23 g, 0.6 mmol) were dissolved in 10 mL of DMF and reacted at RT for 1 h. After the reaction was complete, 30 mL of DCM was added for extraction. The aqueous phase was washed twice with 10 mL of DCM. The combined organic phases were washed with saturated NaCl solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give 0.12 g of a dark green solid compound IR-C6, with a yield of 22%. The compound IR-C6... 1H-NMR and 13 C-NMR are shown in Figure 1 .

[0046] 1 H NMR (600 MHz, DMSO- d 6) δ 8.47 (d, J = 7.4 Hz, 1H), 8.27 (d, J = 12.6Hz, 2H), 8.06 – 8.02 (m, 1H), 7.81 – 7.77 (m, 1H), 7.64 (t, J = 8.2 Hz, 2H),7.51 – 7.42 (m, 5H), 7.37 (d, J = 8.2 Hz, 1H), 7.32 – 7.27 (m, 2H), 7.15 – 7.11(m, 2H), 7.02 (t, J = 7.4 Hz, 1H), 6.33 (t, J = 13.9 Hz, 2H), 4.53 – 4.47 (m,1H), 4.26 (q, J = 7.2 Hz, 2H), 4.20 (t, J = 7.4 Hz, 2H), 3.72 (s, 3H), 3.58 (s,3H), 3.30 (s, 2H), 3.22 (s, 2H), 3.17 – 3.12 (m, 1H), 3.11 – 3.03 (m, 5H),2.75 – 2.67 (m, 4H), 2.05 (t, J = 7.3 Hz, 2H), 1.89 – 1.83 (m, 2H), 1.76 – 1.71(m, 2H), 1.67 (d, J = 2.0 Hz, 12H), 1.59 – 1.53 (m, 2H), 1.45 (s, 6H), 1.39 –1.34 (m, 2H), 1.32 (t, J = 7.2 Hz, 3H).

[0047] 13 C NMR (151 MHz, DMSO- d6) δ 172.58, 172.52, 172.48, 172.38, 169.20,169.17, 148.50, 143.68, 143.36, 142.54, 142.11, 141.70, 141.51, 136.98,129.15, 129.10, 128.59, 127.84, 126.64, 126.57, 125.71, 125.60, 123.06,122.99, 121.58, 119.02, 118.69, 111.96, 111.83, 110.09, 109.02, 101.95,101.86, 56.71, 55.38, 53.81, 52.32, 49.51, 49.44, 44.13, 40.54, 39.24, 38.55,35.60, 34.67, 34.33, 32.77, 30.57, 30.55, 27.96, 27.85, 27.25, 26.32, 26.21,25.29, 20.85, 12.71. HRMS (ESI): m / z [MI] + Calculated for C 60 H 76 ClN6O5S2 + :1059.5002; Found: 1059.5031.

[0048] Example 8: Synthesis of Compound B5

[0049] HATU (0.45 g, 1.2 mmol) and compound B4 (0.40 g, 0.6 mmol) were dissolved in 15 mL of dichloromethane. The mixture was stirred at room temperature for 1 hour, and then the solution was evaporated under vacuum. 2-Aminoethanol (0.11 g, 1.8 mmol) and 5 mL of DMF were added to the residue, and stirring was continued at room temperature for another hour. The reaction mixture was extracted with ethyl acetate, and the organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography with dichloromethane / methanol as eluent to give compound B5 (0.24 g, 49% yield) as a green solid.

[0050] 1 H NMR (600 MHz, DMSO- d 6) δ 8.27 (dd, J = 14.1, 4.6 Hz, 2H), 7.76 (t, J= 5.7 Hz, 1H), 7.70–7.58 (m, 2H), 7.53–7.37 (m, 4H), 7.30 (t, J = 7.1 Hz, 2H), 6.34 (dd, J = 17.8, 14.1 Hz, 2H), 4.21 (t, J = 7.3 Hz, 4H), 3.08 (q, J = 6.0 Hz, 2H), 2.82–2.70 (m, 4H), 2.69 (s, 1H), 2.07 (t, J = 7.3 Hz, 2H), 1.87 (p, J =6.2 Hz, 2H), 1.79 (p, J = 7.3 Hz, 2H), 1.74 (q, J = 7.4 Hz, 2H), 1.68 (d, J = 4.1Hz, 12H), 1.56 (p, J = 7.5 Hz, 2H), 1.36 (t, J = 7.1 Hz, 2H), 1.28–1.03 (m,2H), 0.97 (t, J = 7.4 Hz, 3H). 13 C NMR (151 MHz, DMSO- d 6) δ 172.89, 172.68,172.45, 148.48, 143.50, 143.44, 142.68, 142.54, 141.54, 129.11, 126.64,125.65, 123.00, 112.08, 112.01, 102.14, 102.07, 60.43, 49.50, 49.47, 45.58,44.18, 41.86, 35.51, 27.99, 27.95, 27.25, 26.31, 26.23, 26.02, 25.35, 20.98, 20.86, 11.57.

[0051] Example 9: Synthesis of Compound A4

[0052] Compounds KUN-1 (0.38 g, 1.7 mmol), A1 (0.86 g, 4.1 mmol), EDCI (0.98 g, 5.1 mmol), and DMAP (0.62 g, 5.1 mmol) were dissolved in 25 mL of dichloromethane. The mixture was stirred at room temperature for 2 h. The reaction mixture was extracted with dichloromethane, and the organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using dichloromethane / methanol as eluent to give compound A4 (0.56 g, 76% yield) as an orange oil.

[0053] 1 H NMR (400 MHz, DMSO- d 6) δ 5.94 (s, 1H), 5.09 (s, 2H), 3.77 (s, 3H), 3.41 (s, 2H), 3.29 (s, 2H), 2.42 (s, 3H), 1.46 (s, 6H).

[0054] Example 10: Synthesis of compound IR-IDO

[0055] Compound A4 (0.31 g, 0.7 mmol), compound B5 (0.2 g, 0.3 mmol), EDCI (0.17 g, 0.8 mmol), and DMAP (0.11 g, 0.8 mmol) were dissolved in 15 mL of dichloromethane. The mixture was stirred at room temperature for 2 h. The reaction mixture was extracted with dichloromethane, the organic layer was dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography with dichloromethane / methanol as eluent to give a green solid, compound IR-IDO (0.08 g, 25% yield). The composition of compound IR-IDO... 1 H-NMR and 13 C-NMR see Figure 2 .

[0056] 1 H NMR (600 MHz, Methanol- d 4) δ 8.46 – 8.39 (m, 2H), 7.55 – 7.50 (m,2H), 7.43 (q, J = 7.3 Hz, 2H), 7.34 (dd, J = 10.7, 8.1 Hz, 2H), 7.29 (dt, J=11.5, 7.5 Hz, 2H), 6.33 – 6.25 (m, 2H), 5.86 (s, 1H), 5.17 (s, 2H), 4.17 (dt, J = 11.9, 7.5 Hz, 4H), 4.12 (t, J = 5.4 Hz, 2H), 3.83 (s, 3H), 3.45 – 3.36 (m,6H), 2.71 (q, J = 5.0 Hz, 4H), 2.46 (s, 3H), 2.22 (t, J = 7.2 Hz, 2H), 2.03(q, J = 6.3 Hz, 2H), 1.98 – 1.93 (m, 2H), 1.89 (dq, J = 12.4, 7.3 Hz, 4H), 1.73(d, J = 5.6 Hz, 11H), 1.71 – 1.67 (m, 2H), 1.60 (s, 2H), 1.52 (s, 4H), 1.50 –1.46 (m, 2H), 1.06 (t, J = 7.4 Hz, 3H). 13 C NMR (151 MHz, CDCl3) δ 177.35, 175.44, 173.16, 170.57, 170.40,159.94, 158.55, 156.38, 150.09, 142.82, 141.64, 141.51, 141.18, 130.93,128.85, 128.69, 126.50, 125.78, 124.98, 114.05, 111.36, 110.75, 107.99,105.53, 65.58, 64.20, 57.15, 56.93, 56.29, 49.89, 46.02, 44.73, 40.83, 38.25, 35.54, 32.91, 30.21, 27.99, 27.83, 27.26, 26.27, 24.91, 24.56, 20.87, 20.24, 12.26, 11.61.

[0057] Example 11: In vivo and in vitro photothermal effects of heptamethrin indocyanine small molecules IR-C6 and IR-IDO, which are ROS-responsive IDO release inhibitors. Compounds IR-C6 and IR-IDO were prepared into 10 mM and 50 mg / mL stock solutions using dimethyl sulfoxide (DMSO) and stored at -20°C protected from light. A certain amount of the stock solution from each of the prepared 10 mM stock solutions was added to phosphate-buffered saline (PBS, 10 mM, pH = 7.4) containing 1% bovine serum albumin (BSA) to prepare 5 μM sample detection solutions, which were then added to 1.5 mL centrifuge tubes. The centrifuge tubes containing the detection solutions were irradiated under an 808 nm near-infrared laser for 5 minutes, and the temperature change was recorded every minute. The results are shown below. Figure 3 a. Prepare a working solution with a concentration of 0.5 mg / mL by diluting 50 mg / mL of stock solution with PBS containing 1% BSA. Administer this solution intraperitoneally to 4T1 tumor-bearing mice at a concentration of 5 mg / kg (200 μL of working solution per 20 g of body weight). 24 h after administration, anesthetize the mice and irradiate the tumor site with an 808 nm near-infrared laser for 5 min. Record temperature changes every minute. See the results of the in vivo photothermal experiment below. Figure 3 b. Experimental results showed that compounds IR-C6 and IR-IDO possessed excellent and significant photothermal conversion capabilities in both in vitro solution systems and in vivo tumor tissues, enabling them to efficiently achieve photothermal effects and exert good in vitro and in vivo antitumor effects.

[0058] Example 12: Singlet oxygen generation of heptamethrin indocyanine small molecule IR-C6 and IR-IDO in response to ROS-responsive release of IDO inhibitors SOSG probes (prepared as 1 mM stock solutions using 100% methanol) were added to 2 mL of 10 μM aqueous solutions of compounds IR-C6 and IR-IDO, respectively, to achieve a SOSG concentration of 1.5 μM and a methanol content of 2%. After thorough mixing, the solutions were then heated at 0.8 W / cm². 2 Irradiation with an 808nm laser for 5 minutes, followed by excitation at a wavelength of 494nm, and immediate measurement of the fluorescence emission spectrum in the 500-600nm range, with the fluorescence intensity of the maximum emission peak at 530nm used to quantify the generation of singlet oxygen. For example... Figure 4 As shown, after irradiation with a near-infrared 808nm laser, compounds IR-C6 and IR-IDO can efficiently generate a large amount of singlet oxygen, thus providing a feasible technical basis and experimental evidence for photodynamic therapy of tumors.

[0059] Example 13: Mitochondrial targeting of the ROS-responsive IDO-releasing small molecule heptamethrin indocyanine Taking IR-C6 as an example, 4T1 cells were seeded at a density of 100,000 cells per dish in 35 mm confocal dishes (culture volume 1 mL), and cultured overnight at 37°C with 5% CO2. The original culture medium was aspirated, and the cells were rinsed once with PBS. 1 mL of fresh DMEM complete medium containing 2 μM IR-C6 was added, and the cells were incubated in the dark for 2.5 hours. The original culture medium was aspirated, and the cells were rinsed twice with DMEM. 1 μM Mito-Tracker working solution (preheated to 37°C) was added, and the cells were incubated in the dark for 40 minutes, followed by rinsing twice with PBS. Then, 1 mL of Hoechst 33342 staining solution (1:100) was added, and the cells were incubated at room temperature in the dark for 15 minutes, rinsed twice with PBS, and then 1 mL of fresh PBS was added. The accumulation of the compound in the mitochondria was observed using a confocal microscope (Leica, TCS SP8), and fluorescence images were acquired. The experimental results are as follows: Figure 5 As shown, compound IR-C6 can effectively accumulate in the mitochondria of 4T1 cells, demonstrating good mitochondrial targeting.

[0060] Example 14: Tumor-targeting activity of the ROS-responsive IDO-releasing inhibitors heptamethrin indocyanine small molecules IR-C6 and IR-IDO A subcutaneous tumor-bearing model was established in female Balb / c mice using 4T1 cells. Compounds IR-C6 and IR-IDO were administered via tail vein at a dose of 0.25 mg / kg. Near-infrared fluorescence real-time imaging was performed using a multimodal animal in vivo imaging system (BLT, #AV100) to observe the metabolic distribution of these compounds in the mice at different time points. Results are as follows: Figure 6 As shown, compounds IR-C6 and IR-IDO exhibit good tumor-selective accumulation properties, which are of great significance for tumor diagnosis and imaging-guided precision optical therapy.

[0061] Example 15: IDO inhibitory effect of heptamethrin indocyanine small molecule IR-C6 and IR-IDO, which are ROS-responsive IDO release inhibitors. 4T1 cells were loaded at 2 × 10⁴ cells per well. 5 Cells were seeded in 6-well plates and cultured overnight to allow cell adhesion. After IFN-γ stimulation for 24 h, different concentrations of compounds IR-C6 and IR-IDO were administered, and the cells were cultured for another 24 h. Lysis buffer was then added to extract total cell protein, and protein concentration was measured and balanced. Protein samples were then separated by SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes. After blocking with 5% BSA, the membranes were incubated overnight at 4°C with IDO primary antibody. After washing with TBST, the membranes were incubated with goat anti-rabbit IgG secondary antibody. Finally, chemiluminescence immunoassay was used to detect the immunoblot bands. Results are as follows: Figure 7 The results showed that compounds IR-C6 and IR-IDO both exhibited IDO inhibitory activity and could significantly reduce the expression level of IDO protein.

[0062] Example 16: Activation of the immune response in tumor-bearing mice after phototherapy with a small molecule of heptamethrin indocyanine, a ROS-responsive IDO-releasing inhibitor. Taking IR-C6 as an example. A subcutaneous tumor model of unilateral thigh of Balb / c mice was established using 4T1 cells and divided into (1) PBS group, (2) PBS plus phototherapy group, (3) compound YY alone group, (4) compound YY plus phototherapy group, (5) compound IR-C6 alone group, and (6) compound IR-C6 plus phototherapy group.

[0063] Mice in groups (1) and (2) were administered 200 μL of PBS via intraperitoneal injection; mice in groups (3) and (4) were administered compound YY at 5 mg / kg; and mice in groups (5) and (6) were administered compound IR-C6 at 5 mg / kg. Twenty-four hours later, mice in groups (2), (4), and (6) were administered 0.8 W / cm² of compound to the tumor site. 2 After irradiation with an 808nm laser for 5 minutes, the mice were fed again. Nine days after phototherapy, the mice were sacrificed, and the proximal lymph nodes of the tumors were collected. The lymph nodes were directly ground and filtered, and the red blood cells were lysed, counted, and the cell concentration was adjusted to prepare a single-cell suspension. Anti-mouse BV421-CD11c, PE-CD86, and APC-CD80 were added to form DCs.

[0064] Mice were sacrificed 15 days after phototherapy, and tumor tissue was collected. The tissue was digested with collagenase, ground, filtered, and red blood cells were lysed, counted, and the cell concentration was adjusted to prepare a single-cell suspension. Anti-mouse FITC-CD3, PE-CD4, and BV510-CD8 labeled CD8 were added. + T cells were added to Treg cells, along with anti-mouse BV421-CD25 and PE-Foxp3 (after fixation and membrane rupture). The percentage of mature dendritic cells (DCs) and T cell differentiation were detected using a BECKMAN CytoFLEX LX flow cytometer. Experimental results are as follows: Figure 8 As shown, IR-C6 combined with phototherapy can significantly promote the maturation of dendritic cells (DCs) in proximal lymph nodes of mouse tumors, while simultaneously increasing CD8 levels in tumor tissue. + The levels of T cells and the number of Treg cells are reduced. DC cell maturation is a crucial prerequisite for initiating adaptive anti-tumor immunity, and the ebb and flow between CTLs and Tregs further improves the immune imbalance in the tumor microenvironment. Therefore, the IR-C6 combined with phototherapy strategy can effectively activate the systemic anti-tumor immune response, laying an experimental foundation for achieving synergistic effects between phototherapy and immunotherapy.

[0065] Example 17: Application of heptamethrin indocyanine, a ROS-responsive IDO release inhibitor, in tumor photoimmunotherapy in tumor-bearing mice. Taking IR-IDO as an example. A subcutaneous tumor model of the right thigh (in situ tumor) of Balb / c mice (female) was established using 4T1 cells. The mice were divided into (1) PBS group, (2) PBS plus phototherapy group, (3) KUN-1 alone group, (4) KUN-1 plus phototherapy group, (5) IR-IDO alone group, and (6) IR-IDO plus phototherapy group, with 6 mice in each group.

[0066] Mice in groups (1) and (2) were administered 200 μL of PBS via intraperitoneal injection; mice in groups (3) and (4) were administered compound KUN-1 at 5 mg / kg; and mice in groups (5) and (6) were administered compound IR-IDO at 5 mg / kg. Twenty-four hours later, the tumor (in situ tumor) in the right thigh of mice in groups (2), (4), and (6) was administered 0.8 W / cm² of the irradiated material. 2 The mice were irradiated with an 808nm laser for 5 minutes, and a distal tumor was simultaneously constructed subcutaneously on the left thigh of each mouse. Changes in body weight and bilateral tumor size were continuously observed and measured in the six groups of mice. Results are as follows: Figure 9 The results showed that only group 6 mice exhibited significant inhibition of both in situ and distal tumor growth, with tumor ablation observed; the other five groups showed no significant inhibition of bilateral tumor growth. This indicates that compound IR-IDO possesses excellent phototherapy and immunotherapy-based tumor-suppressing effects, providing a solid foundation and application potential for the development of highly effective phototherapy-activated antitumor immunotherapies.

Claims

1. A photosensitive small molecule of heptamethrin-indocyanine that releases an IDO inhibitor, which is a compound of formula I or a pharmaceutically acceptable salt thereof. ， In the formula, R is a small molecule compound with IDO inhibitory activity.

2. The heptamethrin-indocyanine-based photosensitizing small molecule as described in claim 1, wherein the small molecule compound with IDO inhibitory activity is selected from the following compounds: and .

3. The heptamethrin-indocyanine photosensitive small molecule as described in claim 1, wherein the small molecule is a compound represented by formula IR-IDO or its pharmaceutical salt. 。 4. The heptamethrin-indocyanine photosensitive small molecule as described in claim 1, wherein the small molecule is a compound of formula IR-C6 or its pharmaceutical salt. 。 5. A method for preparing the heptamethrin-indocyanine photosensitive small molecule of claim 3, wherein the heptamethrin-indocyanine photosensitive small molecule is a compound represented by formula IR-IDO or its pharmaceutical salt, comprising the following steps: 1) Compound KUN-1 reacts with compound A1 in the presence of CDI to prepare compound A4. ； 2) Compound B4 was reacted with aminoethanol in the presence of a coupling agent to prepare compound B5. ； 3) Compound A4 reacts with compound B5 to prepare the compound shown in the IR-IDO formula. 。 6. A method for preparing the heptamethrin-indocyanine photosensitive small molecule of claim 4, wherein the heptamethrin-indocyanine photosensitive small molecule is a compound of formula IR-C6 or its pharmaceutical salt, comprising the following steps: 1) Compound A2 reacts with ethylenediamine to prepare compound A3. ； 2) Compounds B1 and B2 react with compound B3 to prepare compound B4. ； 3) Compound A3 and compound B4 were reacted in the presence of HATU to prepare the compound shown in formula IR-C6. 。 7. The use of the heptamethrin indocyanine photosensitive small molecule or its pharmaceutical salt as claimed in any one of claims 1-4 in the preparation of drugs for treating or preventing tumors.

8. The application as described in claim 7 further includes synergistic treatment of tumors with near-infrared fluorescence imaging, photothermal therapy, photodynamic therapy, and / or immunotherapy.

9. A pharmaceutical composition comprising the heptamethrin indolecyanine photosensitive small molecule of any one of claims 1-4 or its pharmaceutical salt, and pharmaceutical excipients.

10. Use of the pharmaceutical composition of claim 9 in the preparation of a medicament for the prevention and treatment of tumors.

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