An induced activated self-delivery cyclic dinucleoside derivative and its preparation method and use in anti-tumor immunotherapy

Abstract

This invention provides an inducible, self-delivering cyclic dinucleotide derivative, its preparation method, and its use in antitumor immunotherapy. The structure of this cyclic dinucleotide derivative is shown in Formula (I). This invention provides a novel, inducible, self-delivering cyclic dinucleotide-like STING agonist, iCDN, by introducing a specific group at the phosphothiodiester site of the CDN, achieving on-demand activation of STING. The iCDN provided by this invention can improve cell internalization efficiency and cell permeability. Furthermore, in the poorly immunogenic 4T1 tumor model, the iCDN provided by this invention can initiate a strong immune response under bioorthogonal induction, effectively inhibiting tumor growth.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine. Specifically, this invention relates to an induced-activation self-delivering cyclic dinucleotide derivative, its preparation method, and its use in anti-tumor immunotherapy. Background Technology

[0002] STING plays a crucial role in DNA sensing, initiating innate immune responses and being essential for the treatment of various cancers. Cytoplasmic DNA derived from tumors or microorganisms activates cyclic GMP-AMP synthase (cGAS) to synthesize the second messenger cyclic dinucleotide 2',3'-cGAMP. 2',3'-cGAMP binds to STING in the endoplasmic reticulum and is transported to the Golgi apparatus, further inducing phosphorylation of TANK-binding kinase 1 (TBK1) / interferon regulatory factor 3 (IRF3) and the inhibitory factor kinase (IKK) / nuclear factor-KB (NF-KB) signaling pathway, leading to the release of type I interferon (IFN-I) and other inflammatory cytokines. Currently, STING agonists are primarily its natural ligands, cyclic dinucleotide compounds (CDNs) and their analogues.

[0003] However, CDNs suffer from drawbacks such as poor cell membrane permeability, low cellular uptake, susceptibility to enzymatic degradation, and metabolic instability, which significantly limit their administration methods, typically resulting in intratumoral administration. Furthermore, since STING proteins are not differentially expressed in normal and tumor cells, these CDN-based agonists induce inflammatory factors in both tumor and normal tissues, leading to systemic inflammatory responses. Currently, two main strategies are commonly used: one is to encapsulate CDNs using materials such as nanoparticles and liposomes to address the delivery problem, but this method is limited by the long-term stability, efficacy, and safety of the materials themselves; the other is to modify the amino, hydroxyl, or phosphate ester bonds of CDN through total chemical synthesis, but this often involves lengthy synthesis steps and low overall yield.

[0004] Therefore, developing CDNs and their analogues that are convenient, effective, have high intracellular efficiency, and can controllably activate the STING pathway remains a challenge. Summary of the Invention

[0005] To address the above problems, the purpose of this invention is to provide an inducibly activated self-delivering cyclic dinucleotide derivative, its preparation method, and its use in anti-tumor immunotherapy. The inventors have discovered that by introducing a specific group into the phosphothiodiester site of CDN, a novel inducibly activated self-delivering STING agonist, iCDN, can be obtained. This iCDN exhibits high cell permeability, improves cell internalization efficiency, and significantly inhibits tumor growth. Therefore, this invention provides a cyclic dinucleotide derivative, its preparation method, and its uses.

[0006] The above-mentioned objective of the present invention is achieved by providing the following technical solution:

[0007] In a first aspect, the present invention provides a cyclic dinucleotide derivative (iCDN) or its stereoisomers, tautomers, hydrates, solvates, nitrides, or pharmaceutically acceptable salts, wherein the structure of the cyclic dinucleotide derivative is shown in formula (I).

[0008]

[0009] in,

[0010] R1 and R2 are each an independent purine nucleobase;

[0011] R3 and R4 are each independently selected from hydrogen, halogen, hydroxyl, and -OC. 1-10 One or more of alkyl groups;

[0012] LG is selected from one or more of the following:

[0013]

[0014] In this invention, the LG group can be eliminated or removed spontaneously under activation conditions, such as chemical orthogonal activation, photoactivation, or enzyme activation.

[0015] Preferably, R1 and R2 are each independently selected from adenine, guanine, xanthine, and hypoxanthine, and more preferably, each is independently adenine or guanine. The structures of adenine and guanine are as follows:

[0016]

[0017] Preferably, both R3 and R4 are hydroxyl groups.

[0018] According to some specific embodiments of the present invention, the structure of the cyclic dinucleotide derivative is shown in formula (Ia):

[0019]

[0020] In a second aspect, the present invention provides a method for preparing the cyclic dinucleotide derivative or its stereoisomers, tautomers, hydrates, solvates, nitrides or pharmaceutically acceptable salts according to the first aspect of the present invention, comprising the following steps:

[0021]

[0022] The compound represented by formula (II) or its stereoisomer, tautomer, hydrate, solvate, nitride, or pharmaceutically acceptable salt is reacted with compound Br-LG to obtain the compound represented by formula (I) or its stereoisomer, tautomer, hydrate, solvate, nitride, or pharmaceutically acceptable salt; wherein the compound Br-LG is selected from one or more of the following:

[0023]

[0024]

[0025] According to some embodiments of the present invention, the method includes the following steps:

[0026] (1) Dissolve compound Br-LG in a solvent to obtain the first solution;

[0027] (2) Dissolve the compound of formula (II) or its stereoisomers, tautomers, hydrates, solvates, nitrogen oxides or pharmaceutically acceptable salts in a buffer solution to obtain a second solution;

[0028] (3) Mix the first solution and the second solution and stir at 5-45°C, preferably 25-35°C, for 24-72 hours, preferably 36-48 hours.

[0029] Preferably, in step (1), the concentration of the compound Br-LG in the first solution is 40-100 mM, more preferably 45-55 mM.

[0030] Preferably, in step (1), the solvent is selected from dimethyl sulfoxide and / or N,N-dimethylformamide.

[0031] Preferably, in step (2), the concentration of the compound represented by formula (II) or its stereoisomers, tautomers, hydrates, solvates, nitrides or pharmaceutically acceptable salts in the second solution is 80-150 μM, preferably 95-105 μM.

[0032] Preferably, in step (2), the buffer solution is a phosphate (PB) buffer solution; more preferably, the concentration of phosphate in the phosphate buffer solution is 40-60 mM, more preferably 45-55 mM; and even more preferably, the pH of the phosphate buffer solution is 6-7.

[0033] Preferably, in step (3), the volume ratio of the first solution and the second solution is 1:0.5-2, and more preferably 1:0.8-1.2.

[0034] According to some embodiments of the present invention, the method further includes: freeze-drying and purifying the reaction product obtained in step (3) in sequence.

[0035] Thirdly, the present invention provides a pharmaceutical composition for antitumor purposes, comprising a cyclic dinucleotide derivative or its stereoisomer, tautomer, hydrate, solvate, nitride, or pharmaceutically acceptable salt as described in the first aspect of the present invention.

[0036] Preferably, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.

[0037] Fourthly, the present invention provides the use of the cyclic dinucleotide derivative or its stereoisomers, tautomers, hydrates, solvates, nitrides or pharmaceutically acceptable salts according to the first aspect of the present invention in the preparation of a medicament for use in antitumor purposes.

[0038] Preferably, the antitumor drug is a drug that inhibits tumor growth.

[0039] The present invention has at least the following beneficial effects:

[0040] This invention utilizes a CDN, such as 2′, 3′-cG S A S Introducing an LG group into the phosphothiodiester site of MP provides a novel, inducibly activated, self-delivered STING agonist, iCDN, enabling on-demand activation of STING and minimizing systemic side effects associated with non-specific STING activation. Specifically, without activation conditions, LG-containing iCDN cannot release CDN, thus failing to activate the STING pathway; under activation conditions, LG-containing iCDN deprotects and releases CDN via an orthogonal reaction, thereby activating the STING pathway.

[0041] This invention demonstrates the feasibility of orthogonal activation of iCDN through changes in liquid phase retention time and mass spectrometry of the target product peak. Furthermore, this invention demonstrates at the cellular level that iCDN improves cell internalization efficiency; compared to cells treated with free CDN, iCDN-treated cells showed significantly increased CDN levels. In addition, iCDN exhibits high cell permeability, increasing the relative mRNA levels of IFNβ and CXCL10, as well as the phosphorylation levels of STING, TBK1, and NF-κB in cells.

[0042] In the poorly immunogenic 4T1 tumor model, the iCDN provided by this invention can initiate a strong immune response under bioorthogonal induction, effectively transforming "cold" tumors into "hot" tumors, significantly increasing the levels of pro-inflammatory cytokines CXCL10, IFNβ, TNFα, IFNγ, and IL6, and significantly inhibiting tumor growth. These results indicate that the inducible cyclic dinucleotide derivative provided by this invention has great potential for development into a class of STING agonist drugs with anti-tumor efficacy.

[0043] Currently, free CDN requires intratumoral administration. If intravenous administration is used, the CDN needs to be encapsulated using materials such as nanoparticles or liposomes. However, the iCDN preparation method provided by this invention is simple and convenient to administer. It can be administered intravenously without the need for encapsulation with materials such as nanoparticles or liposomes, and is released under specific conditions, thereby achieving controllable activation of the STING pathway. Attached Figure Description

[0044] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings, wherein:

[0045] Figure 1 This is a schematic diagram illustrating the synthesis and mechanism of action of the cyclic dinucleotide derivative of the present invention;

[0046] Figure 2 The HPLC and MS chromatograms of the iCDN-N3 reaction solution in Example 1 are shown below.

[0047] Figure 3 In Example 1, iCDN-N3 releases 2′3′-cG after activation by tris(hydroxypropyl)phosphine (THPP). s A s HPLC and MS chromatograms of MP;

[0048] Figure 4 The graph shows the internalization efficiency of HEK293 cells in each group in Example 1. Among them, compared with the 2′3′-cGsAsMP group, ****P<0.0001;

[0049] Figure 5 The values ​​represent the changes in the relative mRNA levels of IFNβ and CXCL10 in each group of HEK293 cells as detected by real-time quantitative PCR in Example 1. ***P < 0.001, ****P < 0.0001.

[0050] Figure 6 The changes in phosphorylation levels of STING, TBK1, and NF-κB in CT26 cells of each group were detected by Western blotting (WB) in Example 1.

[0051] Figure 7The tumor volume growth curves of each group of 4T1 tumor-bearing mice in Example 1 are shown. **P<0.01;

[0052] Figure 8 For the RT-PCR analysis of CXCL10, IFNβ, TNFα, IFNγ and IL6 in each group of tumors in Example 1, **P<0.01, ***P<0.001, ****P<0.0001;

[0053] Figure 9 The graph shows the body weight curves of each group of 4T1 tumor-bearing mice in Example 1;

[0054] Figure 10 The above are blood biochemical analysis diagrams of 4T1 tumor-bearing mice in each group in Example 1. ns indicates no significance.

[0055] Figure 11 The HPLC and MS chromatograms of the iCDN-TCO reaction solution in Example 2 are shown below.

[0056] Figure 12 In Example 2, iCDN-TCO releases 2′3′-cG after activation with 3,6-dimethyl-1,2,4,5-tetraazine. s A s HPLC and MS chromatograms of MP;

[0057] Figure 13 The graph shows the internalization efficiency of HEK293 cells in each group in Example 2. Among them, compared with the 2′3′-cGsAsMP group, ***P<0.001;

[0058] Figure 14 The changes in the relative mRNA levels of IFNβ and CXCL10 in each group of HEK293 cells were detected by RT-PCR in Example 2, where ****P<0.0001;

[0059] Figure 15 The HPLC and MS chromatograms of the iCDN-NB reaction solution in Example 3 are shown below.

[0060] Figure 16 In Example 3, the iCDN-NB releases 2′3′-cG after activation by 365nm light. s A s HPLC and MS chromatograms of MP;

[0061] Figure 17 The values ​​are the changes in the relative mRNA content of IFNβ and CXCL10 in each group of HEK293 cells as detected by RT-PCR in Example 3. Among them, ***P<0.001, ****P<0.0001;

[0062] Figure 18 The HPLC and MS chromatograms of the iCDN-QP reaction solution in Example 4 are shown below.

[0063] Figure 19 In Example 4, iCDN-QP releases 2′3′-cG after activation by the NQO1 enzyme. s A s HPLC and MS chromatograms of MP;

[0064] Figure 20 The changes in the relative mRNA content of NQO1 in BMDM, CT26, and 4T1 cells were detected by RT-PCR in Example 4, where P < 0.0001.

[0065] Figure 21 The changes in the relative mRNA content of iCDN-QP in BMDM, CT26, and 4T1 cells under the activation of endogenous NQO1 enzyme, as detected by RT-PCR in Example 4, are shown in the figures. *P<0.05, **P<0.01, ***P<0.001. Detailed Implementation

[0066] The present invention will be further described in detail below with reference to specific embodiments. The embodiments given are only for illustrating the present invention and are not intended to limit the scope of the present invention.

[0067] The 2′3′-cG used in the following examples s A s MP was provided by Invivogen; mouse breast cancer cells (4T1), human embryonic kidney cells 293 (HEK293), and mouse colon cancer cells (CT26) were provided by the Cell Bank of the Chinese Academy of Sciences Type Culture Collection Committee; mouse bone marrow-derived macrophages (BMDM) were freshly extracted from the bone marrow of 6-8 week old C57 mice; female Balb / c and C57 mice were provided by Guangzhou Jicui Pharmaceutical Co., Ltd.

[0068] The phosphate buffer used in the following examples is prepared as follows:

[0069] (1) Prepare solution A (sodium dihydrogen phosphate aqueous solution): Weigh 27.6g NaH2PO4·H2O and dissolve it in distilled water, then dilute with water to 1000mL;

[0070] (2) Prepare solution B (sodium hydrogen phosphate aqueous solution): Weigh 53.6g Na2HPO4·7H2O and dissolve it in distilled water, then dilute with water to 1000mL;

[0071] (3) Mix 87.7 mL of solution A and 12.3 mL of solution B, then dilute with distilled water to 400 mL to obtain PB buffer, in which the final concentration of phosphate is 50 mM and the pH is about 6.0-7.0.

[0072] Example 1: Preparation of iCDN-N3 induced and activated by phosphorylation reagent and its application in antitumor immunotherapy

[0073] 1. Synthesis of small molecule compounds with brominated protecting groups 8

[0074] 2-Methylnicotinic acid methyl ester (1, 2.0 g, 2.0 eq, 13.23 mmol) was dissolved in 20 mL of anhydrous dichloromethane (DCM), and then trichloroisocyanuric acid (3.05 g, 3.0 eq, 16.54 mmol) was added to initiate the reaction. The resulting reaction mixture was stirred overnight at room temperature, and the resulting solution was purified by silica gel column chromatography to give compound 2 (1.0 g). Compound 2 (1.0 g, 1.0 eq, 5.4 mmol) was dissolved in 10 mL of N,N-dimethylformamide (DMF), and then sodium azide (0.52 g, 1.5 eq, 8.1 mmol) was added to initiate the reaction. The mixture was stirred overnight at room temperature to initiate the reaction. The resulting reaction mixture was washed with saturated sodium bicarbonate aqueous solution and extracted with ethyl acetate. The organic layer was then purified by silica gel column chromatography to give a yellow oily compound 3 (0.95 g). Compound 3 (0.95 g, 5 mmol) was dissolved in 10 mL of methanol, followed by the addition of 10 mL of 10% (w / w) NaOH aqueous solution. After 1 hour, the reaction was monitored by thin-layer chromatography to confirm complete conversion of the starting material. The resulting reaction solution was neutralized to give compound 4, which could be used in the next step without purification.

[0075] 4-hydroxybenzyl alcohol (5, 5.0 g, 40.3 mmol), imidazole (3.29 g, 1.2 eq, 48.2 mmol), and tert-butyldimethylchlorosilane (TBSCl, 6.65 g, 44.3 mmol) were added to 50 mL of N,N-dimethylformamide and stirred overnight at room temperature to carry out the reaction. The resulting reaction mixture was then subjected to silica gel column chromatography (eluent: petroleum ether / ethyl acetate, v / v) to give an oily compound 6 (8.0 g, 33.6 mmol).

[0076] Compound 4 (0.8 g, 1.0 eq, 4.49 mmol), 4-dimethylaminopyridine (DMAP, 0.82 g, 1.5 eq, 6.74 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl, 1.72 g, 2.0 eq, 8.98 mmol), and compound 6 (1.61 g, 1.5 eq, 6.74 mmol) were dissolved in 20 mL of dichloromethane. After reacting for 18 hours, the resulting reaction solution was subjected to silica gel column chromatography (eluent: petroleum ether / ethyl acetate, v / v) to give an oily compound 7 (1.2 g). Compound 7 (1.2 g, 1.0 eq, 3.01 mmol) was added to 10 mL of dichloromethane, and phosphorus tribromide (0.57 g, 0.7 eq, 2.11 mmol) was added dropwise at 0 °C. The mixture was stirred at room temperature for 3 hours to allow the reaction to proceed. After quenching the reaction, dichloromethane and saturated brine (volume ratio of approximately 1:1) were added to the reaction solution for extraction. The combined organic layers were subjected to silica gel chromatography (eluent: petroleum ether / ethyl acetate, volume ratio 3:1) to give a white solid 8 (0.8 g, 2.3 mmol).

[0077] The synthesis route is shown below:

[0078]

[0079] 2. Small molecule compounds 8 and 2′3′-cG s A s MP crosslinking

[0080]

[0081] Compound 8 was dissolved in DMSO to obtain a first solution, wherein the concentration of compound 8 was 50 mM; 2′3′-cG s A s MP is dissolved in phosphate buffer to obtain a second solution, in which 2'3'-cG s A s The concentration of MP was 100 μM. The two solutions were mixed at a volume ratio of 1:1 and stirred at room temperature (30°C) for 48 hours. The resulting solution was collected, freeze-dried under vacuum to remove the organic solvent. The crude product was then dissolved in sterile water and purified by high-performance liquid chromatography (HPLC). The HPLC purification method is as follows:

[0082] Phase A was 0.1 M triethylamine-acetic acid (TEAA) buffer, and phase B was chromatographic acetonitrile, with a flow rate of 1 mL / min; elution was performed according to the following gradient elution program:

[0083] 0-5 minutes: 5% by volume of phase B, the rest is phase A;

[0084] 5-10 minutes: Phase B with a volume fraction of 5-25%, the rest is Phase A;

[0085] 10-25 minutes: Phase B with a volume fraction of 25-45%, the remainder being Phase A;

[0086] 25-80 minutes: Phase B with a volume fraction of 45-95%, the remainder being Phase A.

[0087] Because the polarity of the cross-linked target product iCDN-N3 decreases, its retention time in the reversed-phase column is delayed to 22.62 minutes. The main peak (retention time: 22.62 minutes) was collected and analyzed by mass spectrometry. The highest abundance peak, [MH]-, was observed in negative ion mode at 1237.1, with a calculated value of 1237.2, which is within the normal error range. Therefore, it can be confirmed as the target product iCDN-N3. Figure 2 Concentrations were determined using a NanoDrop micro spectrophotometer and stored in a refrigerator at -80°C.

[0088] 3. Extracellular verification of iCDN-N3 deprotection and release of 2'3'-cG upon THPP activation. s A s MP

[0089] iCDN-N3 (2 μM) and THPP (5 mM) were incubated at room temperature for 5 hours, and the resulting reaction solution was analyzed by HPLC (HPLC conditions as above). After activation with THPP, the retention time of the reaction product was advanced from 22.62 minutes with iCDN-N3 to 13.37 minutes. The main peak (retention time: 13.37 minutes) was collected and prepared, and the target peak was analyzed by mass spectrometry. The peak with the highest abundance, [MH], was observed in negative ion mode. - The peak was 705.1, and it was related to 2′3′-cG. s A s The calculated MP value was consistent with 705.1. The HPLC and mass spectrometry results of the reaction product were consistent with those of the standard 2′3′-cG. s A s MP is consistent, which indicates that iCDN-N3 can deprotect and release 2′3′-cG under the activation of THPP. s A s MP( Figure 3 ).

[0090] 4. Intracellular validation shows that iCDN-N3 can improve internalization efficiency.

[0091] Under cell culture conditions of 37℃ and 5% carbon dioxide, equimolar concentrations of iCDN-N3 and 2′3′-cG were directly added to HEK293 cells.s A s MP, without transfection, after 5 hours of incubation, cells were collected and lysed. The iCDN-N3 group was deprotected by adding THPP and then measured by cGAMP enzyme-linked immunosorbent assay (ELISA).

[0092] like Figure 4 As shown, the measurement results indicate that, compared with free 2′3′-cG s A s Compared to MP-treated cells, iCDN-N3-treated cells showed a significant increase in 2'3'-cG. s A s MP level.

[0093] 5. iCDN-N3 can increase the relative mRNA levels of IFNβ and CXCL10 in cells.

[0094] HEK293 cells were seeded in 12-well plates (200,000 cells per well) and incubated overnight at 37°C. The cells were then treated with iCDN-N3 (2 μM) for 5 hours, followed by incubation with THPP (5 mM) for another 8 hours before analysis. This was done to allow for the direct delivery of 2'3'-cG using Lipofectamine 3000 (Thermo Fisher Scientific). s A s MP (i.e., 2'3'-cG encapsulated in liposome nanoparticles of Lipofectamine 3000) s A s MP), using equimolar concentrations of 2'3'-cG s A s MP transfected cells and encapsulated them in Lipofectamine 3000 for 5 hours (2'3'-cG). s A s MP@Lipo was then incubated for another 8 hours in fresh DMEM medium (purchased from CEAM Cell Technology (Beijing) Co., Ltd.). Additionally, blank control, iCDN-N3 alone, THPP alone, and free 2'3'-cG were also included. s A s MP group (2'3'-cG) s A s MP) was used as a control. RNA was extracted from cells in each group and reverse transcribed for RT-PCR analysis.

[0095] The results are as follows Figure 5 As shown, free 2'3'-cG s As MP mildly induced the expression of IFNβ and CXCL10 in HEK293 cells, primarily due to its low cell permeability. In cells treated with iCDN-N3 or THPP alone, the expression changes of IFNβ and CXCL10 were very low. In contrast, co-treatment with iCDN-N3 and THPP (i.e., iCDN-N3+THPP) resulted in approximately 10-fold and 15-fold increases in IFNβ and CXCL10 expression, respectively, compared to the positive control 2'3'-cG. s A s MP@Lipo delivery 2'3'-cG s A s The activity of MP is comparable.

[0096] 6. iCDN-N3 can increase the phosphorylation levels of STING, TBK1, and NF-κB in cells.

[0097] CT26 cells were seeded into 6-well plates (400,000 cells per well), treated with iCDN-N3 (2 μM) for 5 hours, and then incubated in DMEM medium containing THPP (5 mM) (purchased from SAIMA Cell Technology (Beijing) Co., Ltd.) for 8 hours. Except for the blank control group, the other experimental groups (i.e., iCDN-N3 alone, THPP alone, and free 2'3'-cG) were used. s A s MP treatment group and positive control group 2'3'-cG s A s MP@Lipo) was used at the same molar concentration as iCDN-N3. Total protein was extracted by lysing cells with RIPA buffer containing protease inhibitors and phosphatase inhibitors.

[0098] WB analysis showed ( Figure 6 In CT26 cells co-treated with iCDN-N3 and THPP, the phosphorylation levels of STING, TBK1, and NF-κB were significantly increased, validating the activation of the STING signaling pathway at the protein level.

[0099] 7. iCDN-N3 can significantly inhibit the growth of 4T1 tumor models.

[0100] Female Balb / c mice were placed under pathogen-free conditions at 25±2℃ and 40% relative humidity. Then, 4T1 cells were subcutaneously injected into the mice (1.5 × 10⁻⁶ cells in 100 μL PBS buffer). 6 (cells). When the tumor volume reaches approximately 100 mm... 3Mice were randomly divided into different groups (n=4): blank group, iCDN-N3 group, THPP group, and iCDN-N3 and THPP co-treatment group (iCDN-N3+THPP).

[0101] In the iCDN-N3+THPP group, iCDN-N3 (20 μg / mouse, 16 nmol) was injected intravenously via the tail vein on days 0, 3, 6, 9, and 12, followed by intraperitoneal injection of THPP (1 mg per mouse). The iCDN-N3 group was replaced with an equal volume of PBS buffer instead of THPP; the THPP group was replaced with an equal volume of PBS buffer instead of iCDN-N3; and the blank group was replaced with an equal volume of PBS buffer instead of both iCDN-N3 and THPP.

[0102] At the end of the experiment, mice were euthanized, and major organs and tumors were collected and washed with 1×PBS. Tumors were prepared, their volumes were measured, and RT-PCR analysis was performed. Additionally, blood samples were collected, and serum was extracted for biochemical analysis. Tumor volume was measured using calipers, and the calculation formula was: Tumor volume (mm²) 3 = 0.5 × length × width 2 .

[0103] like Figure 7 As shown, the tumor volume was reduced by approximately 70% in the iCDN-N3 and THPP co-treatment group compared to the blank group, while the tumor volume in the groups treated with iCDN-N3 or THPP alone showed almost no change compared to the blank group. RT-PCR analysis of the tumors yielded the following results: Figure 8 As shown, compared with the blank group, the levels of pro-inflammatory cytokines CXCL10, IFNβ, TNFα, IFNγ and IL6 were significantly increased in tumors of the iCDN-N3 and THPP co-treatment group.

[0104] To evaluate the biotoxicity of iCDN-N3 in combination with THPP, the body weight of mice was monitored during treatment. Results are as follows: Figure 9 As shown, no significant weight loss was observed in any group of mice over the course of treatment. Analysis of the liver and kidney functions of the treated mice yielded the following results: Figure 10 As shown, the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), triglycerides (TG), and creatine kinase (CK) in mice co-treated with iCDN-N3 and THPP were not significantly different from those in the control group.

[0105] Example 2: Preparation of iCDN-TCO induced by tetrazine reagents and activation of the STING pathway

[0106] 1. Synthesis of small molecule compounds with brominated protecting groups 11

[0107] (2E)-TCO-PNB ester (9, 60 mg, 1.0 eq, 0.21 mmol) was dissolved in 10 mL of dichloromethane, and then N,N-diisopropylethylamine (DIPEA, 82.72 mg, 3.0 eq, 0.64 mmol), 1-hydroxybenzotriazole (HOBt, 11 mg, 2.6 eq, 0.08 mmol), and 4-aminobenzoylethanol (42.3 mg, 1.6 eq, 0.34 mmol) were added. The mixture was stirred at 30 °C for 6 days. After the reaction, column chromatography was performed to give product 10, which was a colorless oily liquid (10, 45 mg, yield 77.8%). Compound 10 (45 mg, 1.0 eq, 0.16 mmol) was dissolved in 10 mL of dichloromethane, and phosphorus tribromide (32.5 mg, 0.7 eq, 0.12 mmol) was added dropwise at 0 °C. The resulting reaction solution was cooled in an ice bath for 30 minutes and then stirred at room temperature for 2 hours. The reaction solution was quenched with saturated sodium bicarbonate and extracted multiple times. The organic phases were combined and dried under vacuum to obtain compound 11, which did not require further purification.

[0108] The synthesis route is shown below:

[0109]

[0110] 2. Small molecule compound 11 and 2′3′-cG s A s MP crosslinking

[0111]

[0112] Similar to Example 1, compound 11 was dissolved in DMSO to obtain a first solution, wherein the concentration of compound 11 was 50 mM; 2′3′-cG s A s MP is dissolved in phosphate buffer to obtain a second solution, in which 2′3′-cG s A s The concentration of MP was 100 μM. The two solutions were mixed at a volume ratio of 1:1 and stirred at room temperature (30°C) for 48 hours. The resulting solution was collected, freeze-dried under vacuum to remove the organic solvent. The obtained sample was then reconstituted in sterile water and purified by high-performance liquid chromatography (HPLC) (purification method as in Example 1). Due to the reduced polarity of the cross-linked target product iCDN-TCO, the retention time in the reversed-phase column was delayed to 21.4 minutes. The main peak (retention time: 21.4 minutes) was prepared and collected for mass spectrometry analysis. The peak with the highest abundance, i.e., the [MH]- peak, was observed at 1218.7 in negative ion mode, with a calculated value of 1219.3, which is within the normal error range. This peak can be confirmed as the target product iCDN-TCO. Figure 11Concentrations were determined using a NanoDrop micro spectrophotometer and stored in a refrigerator at -80°C.

[0113] 3. Extracellular verification of iCDN-TCO deprotection and release of 2′3′-cG upon tetrazine activation. s A s MP

[0114] iCDN-TCO (2 μM) was incubated with 3,6-dimethyl-1,2,4,5-tetraazine (Me2Tz, 80 μM) at room temperature for 2 hours. The resulting reaction solution was then analyzed by high-performance liquid chromatography (HPLC) (analysis method as in Example 1). After activation with Me2Tz, the retention time of the reaction product was advanced from 21.4 minutes for iCDN-TCO to 13.37 minutes. The main peak (retention time: 13.37 minutes) was collected and prepared, and the target peak was analyzed by mass spectrometry. The peak with the highest abundance, i.e., the [MH]- peak, was observed at 705.3 (2′3′-cG) in negative ion mode. s A s The calculated [MH]- value of MP is 705.1 (with a normal error range of 705.1 ± 0.3). It can be considered that the HPLC and mass spectrometry results of the reaction product are consistent with those of the standard 2′3′-cG. s A s MP is consistent, which indicates that iCDN-TCO can deprotect and release 2′3′-cG under Me2Tz activation. s A s MP( Figure 12 ).

[0115] 4. Intracellular validation shows that iCDN-TCO can improve internalization efficiency.

[0116] Under cell culture conditions of 37℃ and 5% carbon dioxide, equimolar concentrations of iCDN-TCO and 2′3′-cG were directly added to HEK293 cells. s A s MP, without transfection, after 5 hours of incubation, cells were collected and lysed. The iCDN-TCO group was deprotected with 3,6-dimethyl-1,2,4,5-tetraazine and then detected by cGAMP ELISA kit.

[0117] like Figure 13 As shown, the measurement results indicate that, compared with free 2′3′-cG s A s Compared to MP-treated cells, iCDN-TCO-treated cells showed a significant increase in 2'3'-cG. s A s MP level.

[0118] 5. iCDN-TCo can increase the relative mRNA levels of IFNβ and CXCL10 in cells.

[0119] HEK293 cells were seeded in 12-well plates (200,000 cells per well) and incubated overnight at 37°C. The cells were then treated with iCDN-TCO (2 μM) for 5 hours, followed by incubation with 3,6-dimethyl-1,2,4,5-tetraazine (80 μM) for another 8 hours, and then analyzed. This was done to facilitate the direct delivery of 2'3'-cG using Lipofectamine 3000. s A s MP, using equimolar concentrations of 2'3'-cG s A s MP transfected cells and encapsulated them in Lipofectamine 3000 for 5 hours (2'3'-cG). s A s MP@Lipo was then incubated for another 8 hours in fresh DMEM medium (purchased from CEAM Cell Technology (Beijing) Co., Ltd.). Additionally, a blank control group, a group using iCDN-TCO alone, a group using Me2Tz alone, and a group using free 2'3'-cG were also included. s A s MP group (2'3'-cG) s A s MP) was used as a control. RNA was extracted from cells in each group and reverse transcribed for RT-PCR analysis.

[0120] The results are as follows Figure 14 As shown, free 2'3'-cG s A s MP mildly induced the expression of IFNβ and CXCL10 in HEK293 cells, primarily due to its low cell permeability. Changes in IFNβ and CXCL10 expression were very low in cells treated with iCDN-TCO or 3,6-dimethyl-1,2,4,5-tetraazine alone. In contrast, co-treatment with iCDN-TCO and 3,6-dimethyl-1,2,4,5-tetraazine resulted in approximately 8-fold and 20-fold increases in IFNβ and CXCL10 expression, respectively, compared to the positive control 2'3'-cG. s A s MP@Lipo is equivalent.

[0121] Example 3: Preparation of iCDN-NB induced by specific wavelength light and activation of the STING pathway

[0122] 1. Synthesis of small molecule compounds with brominated protecting groups 13

[0123] Commercially available compound 12 (42.5 mg, 1.0 eq, 0.25 mmol), triphenylphosphine (98.4 mg, 1.5 eq, 0.38 mmol), and carbon tetrabromide (125 mg, 1.5 eq, 0.38 mmol) were dissolved in tetrahydrofuran and stirred at room temperature for 2 hours. The resulting reaction solution was then extracted, and the concentrated organic phase was separated using a silica gel column (petroleum ether / ethyl acetate, v / v 4:1) to give compound 13 as a pale yellow oil (45 mg, yield 78.2%).

[0124] The synthesis route is shown below:

[0125]

[0126] 2. Small molecule compound 13 and 2′3′-cG s A s MP crosslinking

[0127]

[0128] Similar to Example 1, compound 13 was dissolved in DMSO to obtain a first solution, wherein the concentration of compound 13 was 50 mM; 2′3′-cG s A s MP is dissolved in phosphate buffer to obtain a second solution, in which 2′3′-cG s A s The concentration of MP was 100 μM. The two solutions were mixed at a volume ratio of 1:1 and stirred at room temperature (30°C) for 48 hours. The resulting solution was collected, freeze-dried under vacuum to remove the organic solvent. The obtained sample was then reconstituted in sterile water and purified using high-performance liquid chromatography (HPLC) (purification method as in Example 1). Due to the reduced polarity of the cross-linked target product iCDN-NB, its retention time in the reversed-phase column was delayed to 17.67 minutes. The main peak (retention time: 17.67 minutes) was prepared and collected for mass spectrometry analysis. The peak with the highest abundance, [MH]-, was observed in negative ion mode at 1002.9, with a calculated value of 1003.1, within the normal error range. This peak can be confirmed as the target product iCDN-NB. Figure 15 The concentration was determined using a NanoDrop micro spectrophotometer and stored in a refrigerator at -80°C.

[0129] 3. Extracellular verification of iCDN-NB deprotection and release of 2'3'-cG upon light activation. s A s MP

[0130] The iCDN-NB (2 μM) was irradiated at 365 nm for 50 minutes. The reaction solution was analyzed by HPLC (analysis method as in Example 1) to observe the change in the retention time of the target product, and the target peak was analyzed by mass spectrometry. The results showed that after activation by irradiation at 365 nm, the retention time of the reaction product was reduced from 17.67 minutes for iCDN-NB to 13.35 minutes. The main peak (retention time: 13.35 minutes) was collected and prepared, and the target peak was analyzed by mass spectrometry. The peak with the highest abundance, i.e., the [MH]- peak, was observed at 705.2 (2′3′-cG) in negative ion mode. s A s The calculated [MH]- value of MP is 705.1 (with a normal error range of 705.1 ± 0.3). It can be considered that the HPLC and mass spectrometry results of the reaction product are consistent with those of the standard 2′3′-cG. s A s MP is consistent, which indicates that iCDN-NB can deprotect and release 2′3′-cG under light activation. s A s MP( Figure 16 ).

[0131] 4. iCDN-NB can increase the relative mRNA levels of IFNβ and CXCL10 in cells.

[0132] HEK293 cells were seeded in 12-well plates (200,000 cells per well) and incubated overnight at 37°C. The cells were then treated with iCDN-NB (2 μM) for 5 hours, followed by illumination at 365 nm for 50 minutes before analysis. Additionally, a blank control group (Blank), a light-only group (Blank+Light), and a control group (iCDN-NB only without illumination) were set up. RNA was extracted from cells in each group and reverse transcribed for RT-PCR analysis.

[0133] The results are as follows Figure 17 As shown, the changes in IFNβ and CXCL10 expression were very low in cells treated with iCDN-NB alone or by light. In contrast, iCDN-NB treatment under light resulted in an approximately 10-fold and 15-fold increase in IFNβ and CXCL10 expression, respectively.

[0134] Example 4: Preparation of iCDN-QP induced by endogenous NQO1 enzyme and activation of the STING pathway

[0135] 1. Synthesis of small molecule compounds with brominated protecting groups 16

[0136] 3-(2,3,5-trimethyl-1,4-benzoquinone)-3-methylbutyric acid (14,250 mg, 1.0 eq, 1 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl, 384 mg, 2.0 eq, 2 mmol), and 4-dimethylaminopyridine (DMAP, 122.2 mg, 1.0 eq, 1 mmol) were stirred in a dichloromethane solution. Then, compound 6 (358 mg, 1.5 eq, 1.5 mmol) was added to the reaction solution. The resulting reaction solution was stirred overnight at room temperature. After the reaction, the crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate, v / v ratio 8:2) to give compound 15, which was a yellow oily liquid. Compound 15 (350 mg, 1.0 eq, 0.75 mmol) was added to 15 mL of dichloromethane, and phosphorus tribromide (140.6 mg, 0.7 eq, 0.52 mmol) was added dropwise at 0 °C. The resulting reaction mixture was reacted in an ice bath for 30 minutes, and then stirred at room temperature for 3 hours. A saturated sodium bicarbonate solution and dichloromethane (v / v) were added to the reaction mixture for extraction. The extracted organic phases were combined and purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate, v / v 9:1) to give compound 16 (300 mg, 96% yield).

[0137] The synthesis route is shown below:

[0138]

[0139] 2. Small molecule compound 16 and 2′3′-cG s A s MP crosslinking

[0140]

[0141] Similar to Example 1, compound 16 was dissolved in DMSO to obtain a first solution, wherein the concentration of compound 16 was 50 mM; 2′3′-cG s A s MP is dissolved in phosphate buffer to obtain a second solution, in which 2′3′-cG s A sThe concentration of MP was 100 μM. The two solutions were mixed at a volume ratio of 1:1 and stirred at room temperature (30°C) for 48 hours. The resulting solution was collected, freeze-dried under vacuum to remove the organic solvent. The obtained sample was then reconstituted in sterile water and purified by high-performance liquid chromatography (HPLC) to obtain iCDN-QP (purification method as in Example 1). Due to the reduced polarity of the cross-linked target product iCDN-QP, the retention time in the reversed-phase column was delayed to 16.7 minutes. The main peak (retention time: 16.7 minutes) was prepared and collected for mass spectrometry analysis. The peak with the highest abundance, i.e., the [MH]- peak, was observed at 1381.6 in negative ion mode, with a calculated value of 1381.4, which is within the normal error range. This peak can be confirmed as the target product iCDN-QP. Figure 18 The concentration was determined using a NanoDrop micro spectrophotometer and stored in a refrigerator at -80°C.

[0142] 3. Extracellular verification of iCDN-QP deprotection and release of 2′3′-cG upon activation by NQO1 enzyme. s A s MP

[0143] 2.5 μg / mL NQO1 enzyme and 100 μM reduced coenzyme II (NAD(P)H) were added to iCDN-QP (2 μM) (NQO1 enzyme and (NAD(P)H) were used for activation), and the mixture was incubated at 37 °C for 5 hours. The resulting reaction solution was analyzed by HPLC (analysis method as in Example 1) to observe the change in the retention time of the target product, and the target peak was analyzed by mass spectrometry. The results showed that after activation with NQO1 enzyme and reduced coenzyme II (NAD(P)H), the retention time of the reaction product was advanced from 16.7 minutes in iCDN-QP to 13.37 minutes. The main peak (retention time: 13.37 minutes) was collected and prepared, and the target peak was analyzed by mass spectrometry. The peak with the highest abundance, namely the [MH]- peak, was observed at 705.2 (2′3′-cG) in negative ion mode. s A s MP of [MH] - The calculated value is 705.1, with a normal error range of 705.1 ± 0.3. It can be considered that the HPLC and mass spectrometry results of the reaction product are consistent with those of the standard 2′3′-cG. s A s MP is consistent, which indicates that iCDN-QP can be deprotected and release 2′3′-cG upon activation by NQO1 enzyme. s A s MP( Figure 19 ).

[0144] 4. iCDN-QP can increase the relative mRNA levels of IFNβ and CXCL10 in cells.

[0145] BMDM, CT26, and 4T1 cells were seeded into 12-well plates (200,000 cells per well) and incubated overnight at 37°C. Then, iCDN-QP (2 μM) was added to each well for 8 hours (these cells contain NQO1 enzyme and reduced coenzyme II (NAD(P)H), so no additional addition is needed), and then the cells were analyzed.

[0146] RNA was extracted from cells in each group and reverse transcribed for RT-PCR analysis. It can be seen that the relative NOQ1 content in BMDM, CT26, and 4T1 cells gradually increased (…). Figure 20 ), under the activation of endogenous NOQ1 enzyme, the relative mRNA content of IFNβ and CXCL10 in iCDN-QP also increases sequentially. Figure 21 ).

[0147] The above descriptions are merely several exemplary embodiments of the present invention and are not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any equivalent or similar implementation schemes obtained by those skilled in the art by making some modifications or alterations to the above-disclosed technical content without departing from the scope of the present invention are within the scope of the present invention.

Claims

1. A cyclic dinucleotide derivative or a pharmaceutically acceptable salt thereof, wherein, The structure of the cyclic dinucleotide derivative is shown in formula (I). in, R1 and R2 are each an independent purine nucleobase; R3 and R4 are each independently selected from hydrogen, halogen, hydroxyl, and -OC. 1-10 One or more of alkyl groups; LG is selected from one or more of the following: , , and .

2. The cyclic dinucleotide derivative or a pharmaceutically acceptable salt thereof according to claim 1, wherein, R1 and R2 are each independently selected from adenine, guanine, xanthine, and hypoxanthine.

3. The cyclic dinucleotide derivative or a pharmaceutically acceptable salt thereof according to claim 2, wherein, R1 and R2 are either adenine or guanine, respectively.

4. The cyclic dinucleotide derivative according to claim 1, or a pharmaceutically acceptable salt thereof, wherein, Both R3 and R4 are hydroxyl groups.

5. A method for preparing a cyclic dinucleotide derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, comprising the following steps: The compound represented by formula (II) or a pharmaceutically acceptable salt thereof is reacted with compound Br-LG to obtain the compound represented by formula (I) or a pharmaceutically acceptable salt thereof; wherein, compound Br-LG is selected from one or more of the following: , , and 。 6. The method according to claim 5, wherein, The method includes the following steps: (1) Dissolve compound Br-LG in a solvent to obtain the first solution; (2) Dissolve the compound represented by formula (II) or its pharmaceutically acceptable salt in a buffer solution to obtain a second solution; (3) Mix the first solution and the second solution and stir at 5-45℃ for 24-72 hours.

7. The method according to claim 6, wherein, In step (3), stir at 25-35℃ for 36-48 hours.

8. The method according to claim 6, wherein, In step (1), the concentration of the compound Br-LG in the first solution is 40-100 mM.

9. The method according to claim 8, wherein, The concentration of the compound Br-LG in the first solution is 45-55 mM.

10. The method according to claim 6, wherein, In step (1), the solvent is dimethyl sulfoxide and / or N,N-dimethylformamide.

11. The method according to claim 6, wherein, In step (2), the concentration of the compound represented by formula (II) or its pharmaceutically acceptable salt in the second solution is 80-150 μM.

12. The method according to claim 11, wherein, The concentration of the compound represented by formula (II) or its pharmaceutically acceptable salt in the second solution is 95-105 μM.

13. The method according to claim 6, wherein, In step (2), the buffer solution is a phosphate buffer.

14. The method according to claim 13, wherein, The phosphate buffer solution contains 40-60 mM phosphate.

15. The method according to claim 14, wherein, The phosphate buffer solution contains 45-55 mM phosphate.

16. The method according to claim 13, wherein, The pH of the phosphate buffer solution is 6-7.

17. The method according to claim 6, wherein, In step (3), the volume ratio of the first solution and the second solution is 1:0.5-2.

18. The method according to claim 17, wherein, The volume ratio of the first solution to the second solution is 1:0.8-1.

2.

19. The method according to any one of claims 5 to 18, wherein, The method further includes: after stirring, the obtained reaction product is sequentially freeze-dried and purified.

20. A pharmaceutical composition for antitumor treatment comprising a cyclic dinucleotide derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, wherein the tumor is breast cancer or colon cancer.

21. The pharmaceutical composition according to claim 20, wherein, The pharmaceutical composition also includes one or more pharmaceutically acceptable excipients.

22. Use of the cyclic dinucleotide derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4 in the preparation of a medicament for use against tumors, wherein the tumor is breast cancer or colon cancer.

23. The use according to claim 22, wherein, The anti-tumor drug is a drug that inhibits the growth of breast cancer cells or colon cancer cells.

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